CN112888444A

CN112888444A - Improved detection of nuclease edited sequences in automated modules and instruments via batch cell culture

Info

Publication number: CN112888444A
Application number: CN201980067901.3A
Authority: CN
Inventors: 艾琳·斯宾德勒; 艾米·海德森; 菲利普·贝尔格莱德; 查尔斯·约翰逊; 克林特·戴维斯
Original assignee: Inscripta Inc
Current assignee: Inscripta Inc
Priority date: 2018-08-14
Filing date: 2019-08-14
Publication date: 2021-06-01
Also published as: AU2019322870A1; EP3836943A4; EP3836943A1; WO2020037057A1; CA3109119A1; WO2020037051A1

Abstract

The present disclosure provides methods, automated modules, and instruments for enriching live cells edited by nucleic acid-guided nuclease genome editing. The present disclosure provides improved methods and modules (including high-throughput methods and modules) for enriching cells that have undergone editing.

Description

Improved detection of nuclease edited sequences in automated modules and instruments via batch cell culture

RELATED APPLICATIONS

The present application claims U.S. provisional application No. 62/718,449, filed on 14/8/2018; us provisional application No. 62/735,365 filed 24/9/2018, us provisional application No. 62/781,112 filed 18/12/2018, us provisional application No. 62/779,119 filed 13/12/2018, us provisional application No. 62/841,213 filed 30/4/2019. The present application is a partial continuation application of U.S. patent application No. 16/399,988 filed on 30.4.2019 and U.S. patent application No. 16/454,865 filed on 27.6.2019.

Technical Field

The present invention relates to automated modules and systems for culturing and editing live cells via batch (bulk) cell culture.

Background of the invention

In the following discussion, certain articles and methods will be described for background and introductory purposes. Nothing contained herein is to be construed as an "admission" of prior art. Applicants expressly reserve the right to demonstrate, where appropriate, that the methods recited herein do not constitute prior art under the applicable statutory provisions.

The ability to make precise, targeted changes to the genome of living cells has long been a goal in biomedical research and development. Recently, a variety of nucleases have been identified that allow manipulation of gene sequences and thus gene function. Nucleases include nucleic acid-guided nucleases that enable researchers to generate permanent editing in living cells. Editing efficiency in cell populations can be high; however, unedited cells tend to be selectively enriched in pooled (pool) or multiplexed forms due to the lack of double stranded DNA breaks that occur during editing. Double-stranded DNA breaks greatly negatively impact cell viability, leading to enhanced survival of unedited cells and making it difficult to identify edited cells in the context of unedited cells. In addition, cells with edits that confer a growth advantage or disadvantage can cause a bias in the occupancy (representation) of different edits in a population.

Accordingly, there is a need in the art of nucleic acid-guided nuclease gene editing to provide improved methods for enriching edited cells. The present invention satisfies this need.

Summary of The Invention

This summary is provided to introduce a selection of concepts in a simplified form that are further described below in the detailed description. This summary is not intended to identify key features or essential features of the claimed subject matter, nor is it intended to be used to limit the scope of the claimed subject matter. Other features, details, utilities, and advantages of the claimed subject matter will be apparent from the following written detailed description, including those aspects illustrated in the accompanying drawings and defined in the appended claims.

The present disclosure provides methods, modules, instruments, and systems for automated, high-throughput and extremely sensitive enrichment of nucleic acid-guided nuclease-edited cells. This method takes advantage of the isolation or singulation (singularization), where the term is used herein to refer to the process of isolating cells and growing them into clonal colonies. Separation (or singulation) can overcome growth bias from unedited cells, growth effects from differential editing rates, and growth bias due to adaptive effects of different edits. Indeed, it has been determined that eliminating growth rate bias via isolation or substantial isolation and growing colonies from isolated cells to saturation or final colony size (e.g., colony normalization) allows observed editing efficiencies as much as 4x or more over traditional methods. One particularly easy isolation method utilizes the batch cell culture format described in detail herein.

Thus, in one embodiment, there is provided a method for performing enrichment of cells edited by a nucleic acid-guided nuclease, the method comprising: providing a dilution of transformed cells in an appropriate liquid growth medium comprising 0.25% to 6% alginate (alginate), the dilution resulting in substantially isolated cells, wherein the cells comprise a nucleic acid-guided nuclease editing component, wherein the gRNA is optionally under the control of an inducible promoter; solidifying the alginate-containing medium with a divalent cation; allowing the isolated cells to grow for 2 to 50 doublings to establish cell colonies; optionally inducing transcription of the gRNA; allowing the cell colonies to grow to become normalized; and liquefying the alginate-containing medium with a divalent cation chelator. In some aspects, the nucleic acid-guided nuclease editing components are provided to the cell on two separate vectors, and in some aspects, the nucleic acid-guided nuclease editing components are provided to the cell on a single vector, and in some aspects, the cell is a bacterial cell, a yeast cell, or a mammalian cell.

In some aspects of this method embodiment, the percentage of alginate in the growth medium is 1% to 4%, and in some aspects the percentage of alginate in the growth medium is 2% to 3%.

In some aspects, the inducible promoter driving the gRNA is a promoter that is activated upon an increase in temperature, and in some aspects, the inducible promoter is the pL promoter, the cell is transformed with the coding sequence for the CI857 repressor, and transcription of the one or more nucleic acid-guided nuclease editing components is induced by increasing the temperature of the cell to 42 ℃.

In some aspects, with Mg removal⁺²The other divalent cation effects solidification of the alginate-containing medium, and in some embodiments, the divalent cation is Ca⁺². In some aspects, the divalent cation chelator (e.g., liquefier) is citrate, ethylenediaminetetraacetic acid (EDTA), or hexametaphosphate.

Also provided is a module for performing automated enrichment of cells edited by nucleic acid-guided nuclease editing, the module comprising: means for providing a cell transformed with one or more vectors comprising a coding sequence for a nuclease, a guide nucleic acid, and a DNA donor sequence; means for diluting the transformed cells in a medium comprising 0.25% to 6% (w/v) alginate in a container to a cell density suitable for isolating the transformed cells; means for solidifying the alginate-containing medium; means for providing a temperature at which cells are grown; and means for re-liquefying the solidified alginate-containing culture medium with a divalent cation chelator.

In some aspects, there is provided a multi-module cell editing apparatus comprising: a module for automated enrichment of cells edited by nucleic acid-guided nucleases, wherein the multi-module cell editing apparatus further comprises a liquid handling system for providing cells, diluting cells, dispensing a curing agent and dispensing a liquefying agent. Also in some aspects of the multi-module cell editing instrument that includes a module for performing automated enrichment of cells edited by nucleic acid-guided nucleases, a transformation module and/or a growth module and/or a nucleic acid assembly module and/or a reagent cartridge (reagent cartridge) is provided. In some aspects, when present, the reagent cartridge comprises CaCl₂And Na₃C₆H₅O₇. In some embodiments of the multi-module cell editing apparatus, comprising a module for performing automated enrichment of cells, a means for controlling the temperature of the spinner flask and its contents, wherein the conversion module comprises a flow-through electroporation (flow-through electroporation) device.

In other aspects, a method for performing enrichment of cells edited by a nucleic acid-guided nuclease is provided, comprising: providing a dilution of the transformed cells in a suitable liquid growth medium comprising a hydrogel in a container, the dilution resulting in isolated cells, wherein the cells comprise a gRNA under the control of an inducible promoter; solidifying the hydrogel-containing medium with a divalent cation solution; allowing the isolated cells to grow for 2 to 50 doublings to establish cell colonies; inducing transcription of the gRNA; allowing the cell colonies to grow for a time sufficient to normalize the cell colonies; and liquefying the aqueous gel-containing medium with a chelating agent for divalent cations. Also provided are modules for performing the methods, and multi-module cell editing instruments comprising the modules. In some aspects, the media containing the aqueous gel can be liquefied by agitation of the gel, e.g., magnetic beads.

In aspects of the embodiments, the inducible promoter is a temperature-inducible promoter, and the device that provides temperature to induce a nuclease or direct transcription of a nucleic acid is a peltier device. In some aspects of this embodiment, Mg is removed⁺²The other divalent cation effects solidification of the alginate-containing medium, and in some embodiments, the divalent cation is Ca⁺². In some aspects, the divalent cation chelator (e.g., liquefier) is citrate, ethylenediaminetetraacetic acid (EDTA), or hexametaphosphate.

These aspects as well as other features and advantages of the present invention are described in more detail below.

Brief Description of Drawings

FIG. 1A is a simplified flow diagram of two exemplary methods that may be performed by an automated batch cell culture module as a stand-alone instrument or as part of an automated multi-module cell processing instrument. Fig. 1B is a graph of optical density versus time showing growth curves for edited cells (dashed line) and unedited cells (solid line).

Figure 2A depicts a simplified diagram of a workflow for isolating, editing, and normalizing cells after nucleic acid-guided nuclease genome editing in a batch cell culture, wherein reversible solidification of the batch culture is utilized. Fig. 2B depicts a simplified diagram of a workflow for isolating, editing, and normalizing cells following nucleic acid-guided nuclease genome editing in batch cell culture, wherein reversible curing of batch cultures is utilized and editing is induced by induction of transcription of grnas. Fig. 2C is a photograph showing green fluorescent protein expressing e.coli (e.coli) cells of isolated colonies in solidified 2.0% alginate and medium (left panel), and 2.0% alginate after the medium has been reliquefied and e.coli cells expressing green fluorescent protein in the medium.

Figure 3A depicts an automated multi-module cell processing instrument. Figures 3B-3D depict a reagent cartridge and a flow-through electroporation device configured to be positioned in the reagent cartridge. Figures 3E-3L depict various components of one embodiment of a tangential flow filtration device that is used as a cell concentration and buffer exchange module in the automated, multi-module cell processing apparatus shown in figure 3A.

Figure 4A depicts one embodiment of a rotating growth flask for use with the cell growth modules described herein. Figure 4B shows a perspective view of one embodiment of a cell growth module including a rotating growth vial housing. Fig. 4C depicts a cross-sectional view of the cell growth module of fig. 4B. Fig. 4D shows the cell growth module of fig. 4B coupled to an LED, a detector, and a temperature regulation component.

Figure 5A is a simplified block diagram of one embodiment of an exemplary automated multi-module cell processing instrument including a batch gel separation/growth/editing/normalization module. Fig. 5B is a simplified block diagram of an alternative embodiment of an exemplary automated multi-module cell processing instrument including a batch gel separation/growth/editing/normalization module.

Fig. 6A-6C depict a process for determining whether normalization occurred when cells were cultured in a batch gel.

Fig. 7 is a photograph of an automated batch gel cell culture workflow for use in a batch gel separation/growth/editing/normalization module that utilizes a rotating growth flask as depicted in fig. 4A.

Fig. 8A, 8B and 8C depict graphs, tables and two graphs, respectively, of results obtained from an editing experiment performed as follows: liquid cell culture, without isolation or normalization but with an editing experiment with inducible editing; performing batch cell gel culture by adopting an editing experiment of separation, induction type editing and normalization; solid agar plate inoculation (SPP), using separate, inducible editing and normalized editing experiment; solid agar plate inoculation (SPP-preferred (Cherry)), using separation, inducible editing and preferred selection editing experiments; and solid agar plate inoculation (SPP), using isolation, inducible editing and normalization but not editing experiments with preferential selection and only scraping colonies from the plate and replating.

Fig. 9 depicts a recursive workflow using batch gel cell culture and processing (curing).

Detailed Description

All functions described in connection with one embodiment are intended to be applicable to the additional embodiments described herein, except where explicitly stated or where a feature or function is incompatible with the additional embodiments. For example, where a given feature or function is explicitly described in connection with one embodiment but not explicitly mentioned in connection with an alternative embodiment, it is to be understood that the feature or function may be deployed, utilized, or implemented in connection with an alternative embodiment unless the feature or function is incompatible with the alternative embodiment.

The practice of the techniques described herein may employ, unless otherwise indicated, conventional techniques and descriptions of organic chemistry, polymer technology, molecular biology (including recombinant techniques), cell biology, biochemistry, and sequencing techniques, which are within the skill of the art. Such conventional techniques include polymer array synthesis, hybridization and ligation of polynucleotides, and detection of hybridization using labels. Reference to the embodiments herein may be made in detail to a suitable technique. However, other equivalent conventional procedures may of course be used. Such conventional techniques and descriptions can be found in standard Laboratory manuals, such as, edited by Green et al, Genome Analysis: A Laboratory Manual Series (Vol.I-IV) (1999); weiner, Gabriel, Stephens editors, Genetic Variation: A Laboratory Manual (2007); dieffenbach, editors, Dveksler, PCR Primer A Laboratory Manual (2003); bowtell and Sambrook, DNA microarray: A Molecular Cloning Manual (2003); mount, Bioinformatics, Sequence and Genome Analysis (2004); a Laboratory Manual (2006); stryer, Biochemistry (fourth edition) w.h.freeman, New York n.y. (1995); gait, "Oligonucleotide Synthesis: APractcal Approach" (1984), IRL Press, London; nelson and Cox, Lehninger, Principles of Biochemistry third edition, w.h.freeman pub., New York, n.y. (2000); berg et al, Biochemistry, fifth edition, w.h.freeman pub., New York, n.y. (2002); doyle & Griffiths, ed, Cell and Tissue Culture Laboratory Procedures in Biotechnology, Doyle & Griffiths, eds., John Wiley & Sons (1998); hadlaczky editor, Mammalian Chromosome Engineering-Methods and Protocols, Humana Press (2011); and Lanza and Klimanskaya editions, Essential Stem Cell Methods, Academic Press (2011), the entire contents of the above standard laboratory manuals being incorporated herein by reference for all purposes. CRISPR-specific techniques can be found, for example, in Appasani and Church, Genome Editing and Engineering From TALENs and CRISPRs to Molecular Surgery (2018), and Lindgren and Charpentier, CRISPR: Methods and Protocols (2015); the entire contents of both of the above documents are incorporated herein by reference for all purposes.

It is noted that, as used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a cell" means one or more cells, and reference to "the system" includes reference to equivalent steps, methods and apparatus, etc., known to those skilled in the art. In addition, it should be understood that terms such as "left," "right," "top," "bottom," "front," "back," "side," "height," "length," "width," "upper," "lower," "inner," "outer," "inner," and/or "outer" as may be used herein merely describe reference points and do not necessarily limit embodiments of the present disclosure to any particular orientation or configuration. Furthermore, terms such as "first," "second," "third," and the like, disclosed herein merely denote one of a number of portions, components, steps, operations, functions, and/or reference points, and as such, do not necessarily limit embodiments of the present disclosure to any particular configuration or orientation.

In addition, the terms "about," "near," "minor," and similar terms generally refer to a range within the limits of 20%, 10%, or preferably 5% in some embodiments (including the values represented), and any values in between.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. All publications mentioned herein are incorporated herein by reference for the purpose of describing and disclosing devices, methods, and cell populations that might be used in connection with the presently described invention.

Where a range of values is provided, it is understood that each intervening value, to the extent that there is no such stated or intervening value in the stated range, is encompassed within the invention. The upper and lower values of these smaller ranges may independently be included in the smaller ranges and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges not including both of those included limits are also included in the invention.

In the following description, numerous specific details are set forth in order to provide a more thorough understanding of the present invention. It will be apparent, however, to one skilled in the art, that the present invention may be practiced without one or more of these specific details. In other instances, well-known features and processes to those skilled in the art have not been described in order to avoid obscuring the present invention.

As used herein, the term "complementary" refers to watson-crick base pairing between nucleotides, and specifically refers to nucleotides that are hydrogen bonded to each other, wherein a thymine residue or a uracil residue is linked to an adenine residue by two hydrogen bonds, and a cytosine residue and a guanine residue are linked by three hydrogen bonds. In general, a nucleic acid includes a nucleotide sequence that is described as having a "percent complementarity" or a "percent homology" with a specified second nucleotide sequence. For example, the nucleotide sequence may have 80%, 90%, or 100% complementarity to a specified second nucleotide sequence, indicating that 8 nucleotides of 10 nucleotides of the sequence, 9 nucleotides of 10 nucleotides of the sequence, and 10 nucleotides of the sequence are complementary to the specified second nucleotide sequence. For example, the nucleotide sequence 3'-TCGA-5' is 100% complementary to the nucleotide sequence 5 '-AGCT-3'; the nucleotide sequence 3'-TCGA-5' is 100% complementary to a region of the nucleotide sequence 5 '-TTAGCTGG-3'.

The term DNA "control sequences" collectively refers to promoter sequences, polyadenylation signals, transcription termination sequences, upstream regulatory domains, origins of replication, internal ribosome entry sites, nuclear localization sequences, enhancers, and the like, which collectively provide replication, transcription, and translation of a coding sequence in a recipient cell. These types of control sequences need not all be present, provided that the selected coding sequence is capable of replication, transcription and, for some components, translation in an appropriate host cell.

As used herein, the term "donor DNA" or "donor nucleic acid" refers to a nucleic acid designed to introduce DNA sequence modifications (insertions, deletions, substitutions) into a locus by homologous recombination using a nucleic acid-guided nuclease. For homology-directed repair, the donor DNA must be sufficiently homologous to the region of the genomic target sequence flanking the "cleavage site" or site to be edited. The length of the homology arms will depend, for example, on the type and size of modification being made. For example, the donor DNA will have at least one region of sequence homology (e.g., one homology arm) with the genomic target locus. In many cases and preferably, the donor DNA will have two regions of sequence homology (e.g., two homology arms) with the genomic target locus. Preferably, an "insertion" region or "DNA sequence modification" region (one desires to introduce nucleic acid modifications at a genomic target locus in a cell) will be located between the two regions of homology. The DNA sequence modification may change one or more bases at a particular site or at more than one particular site of the target genomic DNA sequence. Changes may include changes to 1,2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 50, 75, 100, 150, 200, 300, 400, or 500 or more base pairs of the target sequence. Deletions or insertions may be of 1,2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 50, 75, 100, 150, 200, 300, 400, or 500 or more base pairs of the target sequence. The donor DNA optionally further comprises alterations to the target sequence, such as PAM mutations that prevent binding of the post-editing nuclease at the PAM or spacer sequence in the target sequence.

As used herein, "enrichment" refers to enrichment of edited cells by isolation or substantial isolation of the cells, initial growth of the cells into cell colonies (e.g., culture), editing (optionally induced, particularly in a bacterial system), and growing the cell colonies into final-sized colonies (e.g., saturation or normalization of colony growth).

The term "guide nucleic acid" or "guide RNA" or "gRNA" refers to a polynucleotide comprising: 1) a guide sequence capable of hybridizing to a genomic target locus, and 2) a scaffold sequence capable of interacting or complexing with a nucleic acid-guided nuclease.

"homology" or "identity" or "similarity" refers to sequence similarity between two peptides, or more generally, in the context of the present disclosure, to sequence similarity between two nucleic acid molecules. The term "homologous region" or "homology arm" refers to a region of the donor DNA that has a degree of homology to the target genomic DNA sequence. Homology can be determined by comparing the position in each sequence, which can be aligned for comparison purposes. When a position in the compared sequences is occupied by the same base or amino acid, then the molecules are homologous at that position. The degree of homology between sequences is a function of the number of matched or homologous positions shared by the sequences.

As used herein, the term "isolation" or "isolating" refers to isolating individual cells such that each cell (and colonies formed by each cell) will be separated from other cells; e.g., a single cell in a single microwell, or 100 single cells in their respective microwells. In one embodiment, an "isolated" or "isolated cell" is produced by a poisson distribution of arrayed cells. The terms "substantially isolated", "largely isolated" and "substantially isolated" refer to cells in a panel or batch that are largely separated from each other. That is, when 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 25, 26, 27, 28, 29, 30 or up to 50 (but preferably 10 or fewer cells) cells are delivered into a microwell. In one embodiment, "substantially isolated" or "largely isolated" results from a "substantially poisson distribution" of the arrayed cells. For more complex editing libraries, or for libraries that may contain lethal edits or have greatly varying adaptive effects, the cells are preferably isolated via poisson distribution.

"operably linked" refers to an arrangement of elements that are described such that the elements are configured to perform their usual function. Thus, a control sequence operably linked to a coding sequence is capable of effecting the transcription, and in some cases, translation, of the coding sequence. Control sequences need not be contiguous with the coding sequence, provided that they function to direct expression of the coding sequence. Thus, for example, intervening untranslated yet transcribed sequences can be present between a promoter sequence and a coding sequence, and the promoter sequence can still be considered "operably linked" to the coding sequence. Indeed, such sequences need not be located on the same contiguous DNA molecule (i.e., chromosome) and may still have interactions leading to regulatory changes.

A "promoter" or "promoter sequence" is a DNA regulatory region capable of binding RNA polymerase and initiating transcription of a polynucleotide or polypeptide coding sequence (such as messenger RNA, ribosomal RNA, small or nucleolar RNA, guide RNA, or any kind of RNA) transcribed by any RNA polymerase I, II or III of any class. Promoters may be constitutive or inducible. In the methods described herein, the promoter that drives transcription of the gRNA is optionally inducible.

As used herein, the term "selectable marker" refers to a gene introduced into a cell that confers a trait suitable for artificial selection. Commonly used selectable markers are well known to those of ordinary skill in the art. Drug-selective markers such as ampicillin/carbenicillin, kanamycin, chloramphenicol, erythromycin, tetracycline, gentamicin, bleomycin, streptomycin, puromycin, hygromycin, blasticidin and G418 may be used. In other embodiments, selectable markers include, but are not limited to, human nerve growth factor receptor (detected with MAb, such as described in U.S. Pat. No. 6,365,373), truncated human growth factor receptor (detected with MAb), mutant human dihydrofolate reductase (DHFR; available fluorescent MTX substrate), secreted alkaline phosphatase (SEAP; available fluorescent substrate); human thymidylate synthase (TS; conferring resistance to the anticancer agent fluorodeoxyuridine), human glutathione S-transferase alpha (GSTA 1; conjugation of glutathione to the stem cell selective alkylating agent busulfan; chemoprotectant selective marker in CD34+ cells), CD24 cell surface antigen in hematopoietic stem cells, rhamnose, human CAD genes conferring resistance to N-phosphonoacetyl-L-aspartic acid (PALA), human multidrug resistance protein 1(human multi-drug resistance-1, MDR-1; P glycoprotein surface protein that can be selected by increasing resistance or enriched by FACS), human CD25 (IL-2. alpha.; detectable by MAb-FITC), methylguanine-DNA methyltransferase (MGMT; selectable by carmustine), and cytidine deaminase (CD; selectable by Ara-C). As used herein, "selective medium" refers to a cell growth medium to which a selective marker or a compound or biological moiety that selects for a selective marker has been added.

The term "target genomic DNA sequence", "target sequence", or "genomic target locus" refers to any locus in a nucleic acid (e.g., a genome) of a cell or population of cells in vitro or in vivo, wherein it is desired to alter at least one nucleotide using a nucleic acid-guided nuclease editing system. The target sequence may be a genomic locus or an extrachromosomal locus.

A "vector" is any of a variety of nucleic acids that comprise a desired sequence or sequences to be delivered to and/or expressed in a cell. The vector is usually composed of DNA, although RNA vectors are also useful. Vectors include, but are not limited to, plasmids, F cosmids (fosmid), phagemids (phagemid), viral genomes, YACs, BACs, mammalian synthetic chromosomes, and the like. As used herein, the phrase "engine vector" comprises the coding sequence of a nuclease to be used in the nucleic acid-guided nuclease systems and methods of the present disclosure (optionally under the control of an inducible promoter). In bacterial systems, the engine vector may further comprise a λ Red recombinant engineered system or an equivalent thereof, and a selectable marker. As used herein, the phrase "editing vector" comprises coding sequences for a donor nucleic acid and a gRNA, the donor nucleic acid comprising an alteration to the target sequence that prevents binding of a nuclease after editing at a PAM or spacer sequence in the target sequence; the coding sequence for the gRNA is optionally under the control of an inducible promoter (and in bacterial systems, preferably under the control of an inducible promoter). The editing carrier may also comprise a selectable marker and/or a barcode. In some embodiments, the engine carrier and the editing carrier may be combined; that is, the contents of the engine carrier may be present on the editing carrier.

Inducible editing of a common nucleic acid-guided genomic nuclease System

The present disclosure provides instruments, modules, and methods for nucleic acid guided nuclease genome editing that provide 1) enhanced editing efficiency of observed nucleic acid guided nuclease editing methods, and 2) improvements in the enrichment of cells whose genomes have been correctly edited, including high throughput screening techniques. Methods are provided herein that take advantage of the advantages of isolation (cells are isolated and grown into clonal colonies) and normalization. Isolation or substantial isolation, culture then editing (optionally with grnas under the control of an inducible promoter) and normalization overcome growth bias from unedited cells. The apparatus, modules and methods can be applied to all cell types, including archaeal cells, prokaryotic cells and eukaryotic (e.g., yeast, fungal, plant and animal) cells. Most normal mammalian tissue-derived cells, except those derived from the hematopoietic system, are anchorage dependent (anchorage dependent) and require surface or cell culture support for normal proliferation. Adherent cells can be grown on magnetic beads separated in a batch gel. Cell culture beads or scaffolds suitable for this purpose typically have a diameter of 100-300 μm and have a density slightly higher than that of the medium (here liquefied medium, to facilitate easy separation of the cells and medium, e.g. medium exchange), but also low enough to allow complete suspension of the carrier at a minimum agitation rate, in order to avoid hydrodynamic damage to the cells. Many different types of microcarriers can be used and different microcarriers are optimized for different types of cells. Positively charged carriers such as Cytodex 1 (dextran-based, GE Healthcare), DE-52 (cellulose-based, Sigma-Aldrich Labware), DE-53 (cellulose-based, Sigma-Aldrich Labware), HLX 11-170 (polystyrene-based); collagen or ECM (extracellular matrix) coated carriers such as Cytodex 3 (dextran based, GE Healthcare) or HyQ-sphere Pro-F102-4 (polystyrene based, Thermo Scientific); uncharged carriers, such as HyQspheres P102-4 (Thermo Scientific); or gelatin-based macroporous carriers (Cultisphere, Percell Biolytica) or cellulose (Cytopore, GE Healthcare).

The instruments, modules, and methods described herein employ editing cassettes comprising a guide rna (gRNA) sequence covalently linked to a donor DNA sequence, wherein the gRNA is optionally under the control of an inducible promoter, particularly in bacterial systems (e.g., the editing cassette is a CREATE cassette; see USPN 9/982,278 published on day 29 of 2019, month 5, and USPN10/240,167 published on day 26 of 2019, USPN 10/266,849 published on day 23 of 2019, and U.S. publication No. 15/948,785 filed on day 9 of 2018, month 4, us 16/275,439 filed on day 14 of 2019, and U.S. publication No. 16/275,465 filed on day 14 of 2019, month 2, the entire contents of which are incorporated by reference in their entirety). The disclosed methods allow cells to be transformed, substantially isolated, grown for several doublings (e.g., in culture), and then editing induced in the cells. The isolation procedure effectively eliminates the predominant effects of unedited cells in the cell population. The combination of substantially isolating the cells, then allowing initial growth, followed by optional induction of gRNA (and optionally nuclease) transcription, and normalization of the cell colonies, allows for an increase of 2-250x, 10-225x, 25-200x, 40-175x, 50-150x, 60-100x, or 50-100x in identifying edited cells over prior art methods, and allows for the generation of a cell library comprising the edited cells with the edited genome arrayed (arrayed) or pooled. In addition, iterative editing systems can be created using these methods to generate combinatorial cell libraries with two to many edits in each cell genome.

The apparatus, compositions, and methods described herein improve editing systems in which nucleic acid-guided nucleases (e.g., RNA-guided nucleases) are used to edit specific target regions in the genome of an organism. A nucleic acid-guided nuclease complexed with a suitable synthetic guide nucleic acid in a cell can cleave the genome of the cell at a desired location. The guide nucleic acid helps the nucleic acid-guided nuclease recognize and cleave DNA at a particular target sequence. By manipulating the nucleotide sequence of the guide nucleic acid, the nucleic acid guided nuclease can be programmed to target any DNA sequence for cleavage, provided that there is a suitable promiscuous sequence adjacent motif (PAM) in the vicinity. In certain aspects, a nucleic acid-guided nuclease editing system can use two separate guide nucleic acid molecules that combine to function as a guide nucleic acid, e.g., CRISPR RNA (crRNA) and trans-activation CRISPR RNA (tracrRNA). In other aspects, the guide nucleic acid may be a single guide nucleic acid comprising both a crRNA sequence and a tracrRNA sequence, or a single guide nucleic acid that does not require a tracrRNA.

Typically, a guide nucleic acid (e.g., a gRNA) is complexed with a compatible nucleic acid-guided nuclease, and can then hybridize to a target sequence, thereby guiding the nuclease to the target sequence. The guide nucleic acid may be DNA or RNA; alternatively, the guide nucleic acid may comprise both DNA and RNA. In some embodiments, the guide nucleic acid may comprise modified or non-naturally occurring nucleotides. In the case where the guide nucleic acid comprises an RNA, the gRNA is encoded by a DNA sequence on a polynucleotide molecule (such as a plasmid, linear construct) or located in an editing cassette, and optionally (particularly in bacterial systems) under the control of an inducible promoter.

The guide nucleic acid comprises a guide sequence, wherein the guide sequence is a polynucleotide sequence that is sufficiently complementary to the target sequence to hybridize to the target sequence and that directs the sequence-specific binding of the complexed nucleic acid-guided nuclease to the target sequence. When optimally aligned using a suitable alignment algorithm, the degree of complementarity between the leader sequence and the corresponding target sequence is about or greater than about 50%, 60%, 75%, 80%, 85%, 90%, 95%, 97.5%, 99% or greater. Any suitable algorithm for aligning sequences can be used to determine the optimal alignment. In some embodiments, the length of the guide sequence (the portion of the guide nucleic acid that hybridizes to the target sequence) is about or greater than about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 75, or more nucleotides. In some embodiments, the leader sequence is less than about 75, 50, 45, 40, 35, 30, 25, 20 nucleotides in length. Preferably, the leader sequence is 10-30 or 15-20 nucleotides in length, or 15, 16, 17, 18, 19 or 20 nucleotides in length.

In the methods and compositions of the invention, the guide nucleic acid is provided as a sequence expressed from a plasmid or vector and comprises both the guide sequence and the scaffold sequence as a single transcript. Alternatively, the guide nucleic acid may be transcribed from two separate sequences, at least one of which is under the control of an inducible promoter. The guide nucleic acid can be engineered to target a desired target DNA sequence by altering the guide sequence such that the guide sequence is complementary to the target DNA sequence, thereby allowing hybridization between the guide sequence and the target DNA sequence. Typically, to produce edits in a target DNA sequence, the gRNA/nuclease complex binds to the target sequence defined by the guide RNA, and the nuclease recognizes a Protospacer Adjacent Motif (PAM) sequence adjacent to the target sequence. The target sequence may be any polynucleotide (DNA or RNA) endogenous or exogenous to or in vitro in a prokaryotic or eukaryotic cell. For example, the target sequence may be a polynucleotide located in the nucleus of a eukaryotic cell. The target sequence may be a sequence encoding a gene product (e.g., a protein) and/or a non-coding sequence (e.g., a regulatory polynucleotide, intron, PAM or "junk" DNA).

The guide nucleic acid can be part of an editing cassette encoding a donor nucleic acid; that is, the editing cartridge may be a CREATE cartridge (see, e.g., USPN 9/982,278 published on day 29 in 2019, USPN10/240,167 published on day 26 in month 3 in 2019, USPN 10/266,849 published on day 23 in month 4 in 2019, and U.S. publication No. 15/948,785 filed on day 9 in month 4 in 2018, U.S. publication No. 16/275,439 filed on day 14 in month 2 in 2019, and U.S. publication No. 16/275,465 filed on day 14 in month 2 in 2019, the entire contents of which are incorporated by reference in their entirety). The guide nucleic acid and donor nucleic acid may be under the control of a single (optionally inducible) promoter and typically under the control of a single (optionally inducible) promoter. Alternatively, the guide nucleic acid may not be part of the editing cassette, but may be encoded on the backbone of the engine vector or editing vector. For example, a sequence encoding a guide nucleic acid can be first assembled or inserted into a vector backbone, followed by insertion into a donor nucleic acid. In other cases, the donor nucleic acid may be inserted or assembled into the vector backbone first, followed by insertion of the sequence encoding the guide nucleic acid. In yet other cases, sequences encoding the guide nucleic acid and the donor nucleic acid (inserted, e.g., in an editing cassette) are inserted or assembled into the vector simultaneously but separately. In yet other embodiments, and preferably, the editing cassette comprises both a sequence encoding a guide nucleic acid and a sequence encoding a donor nucleic acid.

The target sequence is associated with PAM, a short nucleotide sequence recognized by the gRNA/nuclease complex. Different nucleic acid-guided nucleases have different requirements on the precise sequence and length of PAM; however, a PAM is typically a 2-7 base pair sequence adjacent or proximal to a target sequence and may be 5 'or 3' relative to the target sequence, depending on the nuclease. Engineering the PAM interaction domain of a nucleic acid-guided nuclease may allow for alteration of PAM specificity, improve target site recognition accuracy, reduce target site recognition accuracy, and increase versatility of the nucleic acid-guided nuclease. In certain embodiments, genome editing of a target sequence both introduces a desired DNA alteration into the target sequence (e.g., genomic DNA of a cell), and removes, mutates or inactivates a pro-spacer (PAM) region in the target sequence; that is, the donor DNA typically contains alterations to the target sequence to prevent binding of the nuclease at the PAM in the target sequence after editing has occurred. Inactivating the PAM at the target sequence prevents additional editing of the cellular genome at the target sequence, for example, when subsequently exposed to a nucleic acid-guided nuclease complexed with a synthetic guide nucleic acid in a subsequent round of editing. Thus, a nucleic acid guided nuclease complexed with a synthetic guide nucleic acid complementary to the target sequence can be used to select cells with PAM with the desired target sequence editing and alteration. Cells that have not undergone the first editing event will be cut, causing double stranded DNA breaks, and will therefore not continue to survive. Cells containing the desired target sequence editing and PAM alteration will not be cut because these edited cells no longer contain the necessary PAM site and will continue to grow and proliferate.

The range of target sequences that a nucleic acid guided nuclease can recognize is limited by the need for a particular PAM to be located in the vicinity of the desired target sequence. Thus, it is often difficult to target editing with the precision necessary for genome editing. It has been found that nucleases can recognize some PAMs well (e.g., classical PAMs), while recognizing other PAMs less well or poorly (e.g., non-classical PAMs). Because the methods disclosed herein allow for the identification of edited cells in a large background of unedited cells, the methods allow for the identification of edited cells with PAM less than optimal; that is, the methods for identifying edited cells herein allow for the identification of edited cells even if the editing efficiency is very low. In addition, the methods of the invention expand the range of target sequences that can be edited, as it is easier to identify cells in which edits, including genome edits, are associated with less functional PAMs.

For the nuclease component of a nucleic acid-guided nuclease editing system, a polynucleotide sequence encoding a nucleic acid-guided nuclease can be codon optimized for expression in a particular cell, such as an archaeal cell, a prokaryotic cell, or a eukaryotic cell. The eukaryotic cell can be a yeast cell, a fungal cell, an algal cell, a plant cell, an animal cell, or a human cell. The eukaryotic cell can be a eukaryotic cell of or derived from a particular organism, such as a mammal, including but not limited to a human, mouse, rat, rabbit, dog, or non-human mammal, including a non-human primate. The choice of nucleic acid guided nuclease to be used depends on many factors, such as what type of editing will be done in the target sequence and whether the appropriate PAM is located in the vicinity of the desired target sequence. Nucleases for use in the methods described herein include, but are not limited to, Cas 9, Cas 12/CpfI, MAD2 or MAD7 or other MAD enzymes. Like the guide nucleic acid, the nuclease may be encoded by a DNA sequence on a vector (e.g., an engine vector) and under the control of a constitutive promoter or an inducible promoter. Again, at least one of the nuclease and the guide nucleic acid, and preferably both, are under the control of an inducible promoter.

Another component of the nucleic acid-guided nuclease system is a donor nucleic acid. In some embodiments, the donor nucleic acid is on the same polynucleotide (e.g., a vector or editing (CREATE) cassette) as the guide nucleic acid. The donor nucleic acid is designed to serve as a template for homologous recombination with the target sequence (a nuclease nick (nick) or cleavage guided by the nucleic acid as part of the gRNA/nuclease complex). The donor nucleic acid polynucleotide can be any suitable length, such as about or greater than about 30, 35, 40, 45, 50, 75, 100, 150, 200, 500, 1000, 2500, 5000 nucleotides in length or longer. In certain preferred aspects, the donor nucleic acid may be provided as an oligonucleotide of between 40-300 nucleotides, more preferably between 50-250 nucleotides. The donor nucleic acid comprises a region (e.g., homology arm) that is complementary to a portion of the target sequence. When optimally aligned, for example, about 10, 15, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90 or more nucleotides of the donor nucleic acid overlap (are complementary) with the target sequence. In many embodiments, the donor nucleic acid comprises two homology arms (regions complementary to the target sequence) flanking a mutation or difference between the donor nucleic acid and the target template. The donor nucleic acid comprises at least one mutation or alteration compared to the target sequence, such as having an insertion, deletion, modification, or any combination thereof compared to the target sequence.

The donor nucleic acid is typically provided as an editing cassette that is inserted into a vector backbone, where the vector backbone can comprise a promoter that drives transcription of the gRNA and the donor nucleic acid. In addition, more than one, e.g., two, three, four, or more guide/donor nucleic acid cassettes can be inserted into the engine vector, where the guide nucleic acids are under the control of separate, different promoters, under the control of separate, similar promoters, or where all guide/donor nucleic acid pairs are under the control of a single promoter. (see, e.g., USSN 16/275,465 filed on 2/14/2019, incorporated into a multiple-yield cassette.) the promoter driving transcription of the gRNA and donor nucleic acid (or driving more than one gRNA/donor nucleic acid pair) is optionally an inducible promoter (and in bacterial systems is preferably an inducible promoter), and the promoter driving transcription of the nuclease is also optionally an inducible promoter.

The advantage of inducible editing is that substantially or largely isolated cells can be grown several to many cell doublings before editing is initiated, which increases the likelihood of survival of cells with editing because the double-stranded cleavage caused by active editing is highly toxic to the cells. This toxicity not only leads to cell death of the edited colonies, but also to growth lag of the edited cells that do survive but must be repaired and restored after editing. However, when the edited cells have a chance to recover, the size of the colonies of the edited cells will eventually catch up to the size of the colonies of the unedited cells (e.g., a process of "normalizing" or growing the colonies to a "final size"; see, e.g., FIG. 1B described below).

In addition to the donor nucleic acid, the editing cassette may comprise one or more primer sites. The primer sites can be used to amplify the editing cassettes by using oligonucleotide primers; for example, if the primer site flanks one or more other components of the editing cassette.

Also, as described above, the donor nucleic acid may comprise (in addition to at least one mutation relative to the target sequence) one or more PAM sequence alterations that mutate, delete or inactivate the PAM site in the target sequence. If the same nuclease is used, the PAM sequence alteration in the target sequence "immunizes" the PAM site against the nucleic acid-guided nuclease and protects the target sequence from further editing in subsequent rounds of editing.

The editing box may also contain a bar code. Barcodes are unique DNA sequences that correspond to donor DNA sequences such that the barcode can identify edits made to the corresponding target sequence. Barcodes may contain more than four nucleotides. In some embodiments, the editing cassette comprises a collection of donor nucleic acids that represents, for example, a gene-wide or genome-wide library of donor nucleic acids. The library of editing cassettes is cloned into a vector backbone, where, for example, each different donor nucleic acid design is associated with a different barcode or, alternatively, each different cassette molecule is associated with a different barcode.

In addition, in some embodiments, the expression vector or cassette encoding component of the nucleic acid-guided nuclease system further encodes a nucleic acid-guided nuclease that comprises one or more Nuclear Localization Sequences (NLSs), such as about or more than about 1,2, 3, 4, 5, 6, 7,8, 9, 10, or more NLSs. In some embodiments, the engineered nuclease comprises an NLS at or near the amino terminus, an NLS at or near the carboxy terminus, or a combination thereof.

The engine vector and editing vector comprise control sequences operably linked to the component sequences to be transcribed. As described above, the promoter that drives transcription of one or more components of the nucleic acid-guided nuclease editing system can be inducible. A number of gene regulatory control systems have been developed for the controlled expression of genes in plant cells, microbial cells and animal cells, including mammalian cells, including the pL promoter (induced by heat inactivation of the CI857 repressor), the pBAD promoter (induced by the addition of arabinose to the cell growth medium) and the rhamnose inducible promoter (induced by the addition of rhamnose to the cell growth medium). Other systems include the tetracycline-controlled transcriptional activation system (Tet-On/Tet-Off, Clontech, Inc. (Palo Alto, CA); Bujard and Gossen, PNAS,89(12):5547-5551(1992)), the Lac Switch induced system (Wyborski et al, Environ Mol Mutagen,28(4):447-58 (1996); DuCoeur et al, stratages 5(3):70-72 (1992); U.S. Pat. No. 4,833,080), the ecdysone-Inducible gene expression system (No. et al, PNAS,93 (8): 3346-3351(1996)), the cuumemate gene conversion system (Mullick et al, BMC Biotechnology,6:43 (548)) and the tacrine-Inducible gene expression system (Zhan et al, 1996)) as well as other Inducible genes. In the present methods used in the modules and instruments described herein, it is preferred that at least one of the nucleic acid-guided nuclease editing components (e.g., a nuclease or a gRNA) is under the control of a promoter that is activated by a temperature increase, as such a promoter allows the promoter to be activated by a temperature increase and inactivated by a temperature decrease, thereby "turning off" the editing process. Thus, in case the promoter is inactivated by a decrease in temperature, the editing in the cell can be switched off without the need to change the medium or to add other genetic elements, e.g. in other genetic elements (on an existing plasmid, on another plasmid or integrated into the genome); to remove, for example, the inducer used to induce editing in the medium.

A simplified flow diagram of two alternative

exemplary methods

100a and 100b for isolating or substantially isolating cells and normalizing cell colony size is shown in fig. 1A, where method 100a does not employ an inducible editing mechanism, and method 100b employs an inducible promoter that drives transcription of a gRNA. Referring to fig. 1A, the method 100a begins by transforming a cell 110 with components necessary to perform nucleic acid-guided nuclease editing. For example, cells can be transformed simultaneously with separate engine vectors and editing vectors; the cell may have expressed the nuclease (e.g., the cell may have been transformed with an engine vector, or the coding sequence for the nuclease may have stably integrated into the genome of the cell), such that only the editing vector needs to be transformed into the cell; alternatively, the cell can be transformed with a single vector that contains all the components necessary to perform nucleic acid-guided nuclease genome editing.

Various delivery systems can be used to introduce (e.g., transform or transfect) nucleic acid-guided nuclease editing system components into a host cell 110. These delivery systems include the use of yeast systems, lipofection systems, microinjection systems, biolistic systems, virosomes (virosomes), liposomes, immunoliposomes, polycations, lipids: nucleic acid conjugates, viral particles, artificial viral particles, viral vectors, electroporation, cell permeable peptides, nanoparticles, nanowires, exosomes (exosomes). Alternatively, molecular trojan horse liposomes (molecular trojan horse liposomes) can be used to deliver nucleic acid guided nuclease components across the blood brain barrier. Of interest, especially in the case of multi-module cell editing instruments, is the use of electroporation, especially flow-through electroporation (either as a stand-alone instrument or as a module in an automated multi-module system), as described in, for example, USSN16/147,120 filed on 9/28/2018; USSN16/147,353 filed on 28/9/2018; USSN16/147,865 filed on 30/9/2018; and USSN 16/426,310 filed on 30/5/2019; and USPN10,323,258 published on 18 th 6 th 2019. If the separation/growth/editing/normalization module is one module in an automated multi-module cell editing instrument, cells can be transformed in an automated cell transformation module.

Isolating or substantially isolating the cell 120 after transforming the cell with the components required to perform nucleic acid-guided nuclease editing; that is, the cells are diluted in liquid medium (if necessary) to isolate or separate the cells from each other in the liquid. Separation can then be performed by solidifying or "gelling" the liquid medium, wherein the cells are separated from each other in a three-dimensional gel; that is, for example, cells are suspended in a liquid, and then the liquid is solidified into a gel, thereby fixing the separated or singulated cells in a three-dimensional space.

After isolation of the cells in 100a, the cells are actively edited due to the editing mechanism under the control of a constitutive promoter. As the cells are edited, they grow into final-sized colonies 130; that is, colonies produced by the isolated cells grow into colonies to the extent that cell growth peaks and is normalized or saturated for both edited and unedited cells. Normalization occurs when the nutrients in the medium surrounding the growing cell colony are depleted. Also, in the embodiment 100a shown in fig. 1A, the editing components are under the control of a constitutive promoter; thus, editing begins immediately (or almost immediately) after transformation. However, in other embodiments, such as the one shown in 100b, at least the guide nucleic acid (and e.g. the lambda Red recombination system component in a bacterial system) may be under the control of an inducible promoter, in which case editing may be induced after, for example, a desired number of cell doublings. At this point in the method 100a, final size colonies 140 are pooled by liquefying the solidified medium and, for example, vortexing the liquid to mix the cells from the normalized colonies. Also, since isolation overcomes growth bias from unedited cells or cells that exhibit adaptive effects due to editing performed, isolation/normalization alone enriches the total cell population with edited cells; that is, the combination of isolation and normalization (e.g., growing colonies to final size) allows for high throughput enrichment of edited cells.

The method 100b shown in fig. 1A is similar to the method 100a in that the cell of interest is transformed 110 with components required for nucleic acid-guided nuclease editing. As described above, a cell can be transformed with both an engine vector and an editing vector, can already express a nuclease (e.g., a cell can already be transformed with an engine vector, or the coding sequence for a nuclease can be stably integrated into the genome of a cell), such that only the editing vector needs to be transformed into the cell, or can be transformed with a single vector that contains all the components necessary to perform nucleic acid-guided nuclease genome editing. Furthermore, if the separation/growth/editing/normalization module is one of the automated multi-module cell editing instruments, cell transformation can be performed in an automated transformation module using a flow-through electroporation module, as described below with respect to fig. 3A-3D.

After transforming the cells with components necessary for performing nucleic acid-guided nuclease editing, the cells are isolated 120; that is, the cells are diluted in a liquid medium, if necessary, to separate the cells from each other. After the cells are appropriately diluted in liquid medium, the liquid medium is allowed to solidify or "gel" thereby immobilizing and sequestering the diluted cells in three-dimensional space.

After the cells are isolated and isolated 120, the cells are grown, for example, between 2 and 50 doublings, or between 5 and 40 doublings, or between 10 and 30 doublings, to establish clonal colonies 150. After colony establishment, in this embodiment 100b, induced editing 160 is performed by, for example, activating an inducible promoter that controls transcription of one or more components required for nucleic acid-guided nuclease editing (such as, for example, transcription of a gRNA, donor DNA, nuclease, or, in the case of bacteria, a recombinantly engineered system). After the induction of editing 160, many edited cells in the clonal colony die due to double stranded DNA breaks that occur during the editing process; however, in a proportion of the edited cells, the genome is edited, the double-stranded break is repaired correctly, and the cells survive and continue to grow. These edited cells then start to grow and re-establish colonies. After induction of editing and sufficient time to complete the process, the induced cells were grown to a final size of 170 (normalized colony size). The normalized colonies 140 are then pooled as in embodiment 100a by liquefying the solidified medium and, for example, vortexing the liquid to mix the cells from the normalized colonies.

Fig. 1B is a graph of OD versus time for unedited cells (solid line) and edited cells (dashed line). Note that the OD (e.g., growth) of the edited cells initially lags behind the unedited cells, but eventually catches up due to, for example, the unedited cells depleting the medium of nutrients and exiting the logarithmic growth phase. The colonies were grown for a sufficient time to allow the growth of the edited colonies to catch up to the unedited colonies (close to the size of the unedited colonies, e.g. the number of cells in the unedited colonies).

Exemplary workflow for editing and enriching edited cells

The methods described herein provide enhanced observed editing efficiency of nucleic acid-guided nuclease editing methods, which is a result of isolation/growth/editing/normalization. The combination of the separation and normalization processes overcomes the growth bias towards unedited cells and the adaptive effects of editing (including differential editing rates) so that all cells can be "billed" equally to each other. As a result of the methods described herein, even in nucleic acid-guided nuclease systems where editing is not optimal (such as in systems targeting non-classical PAM), the observed editing efficiency is improved; that is, edited cells can be identified even in the context of large unedited cells. The observed editing efficiency can be improved up to 80% or more.

Fig. 2A is an exemplary workflow 200 for optimizing the presence of edited cells observed following nucleic acid guided nuclease genome editing, which workflow 200 may be performed in an automated separation/growth/editing/normalization module, and may optionally be part of an automated multi-module cell editing instrument. First, the transformed cells 204 are suspended at a predetermined density in medium plus alginate (solidifying agent) in a container 202, which container 202 optionally contains an antibiotic or other selective compound, to allow only cells that have been transformed with both the engine vector and the editing vector (if both vectors are used) or a combined engine/editing vector to grow. Also, in some embodiments, two vectors are used, an engine vector and an editing vector, and in some embodiments, a single vector is used that contains all the necessary exogenous components for nucleic acid-guided nuclease editing. Of course, the media used together depend on the type of cell being edited, e.g., bacteria, yeast or mammals. For example, media for bacterial growth include LB, SOC, M9 basal medium (minimal medium), and Magic medium (Magic medium); media for yeast cell growth include TPD, YPG, YPAD, and synthetic basal media; and media for mammalian cell growth include MEM, DMEM, IMDM, RPMI, and Hanks.

Natural polymers and proteins capable of forming hydrogels are alginate, chitosan, hyaluronic acid, dextran, collagen and fibrin; synthesis of polymers and proteins capable of forming hydrogelsExamples include polyethylene glycol, poly (hydroxyethyl methacrylate), polyvinyl alcohol and polycaprolactone. Alginate has been used as a preferred solidifying agent in the methods described herein due to a number of advantageous properties. Alginates are polysaccharides consisting of residues of the linear (straight-chain) 1, 4-linked β -D-mannuronic acid and its C5-epimer α -D-guluronic acid. Alginate has high affinity for alkaline earth metals and can be used for removing Mg⁺²Other divalent cations form ionic hydrogels. The chelation of gel-forming ions occurs between two consecutive residues in the alginate chains and an intermolecular gel network is formed due to the co-association of consecutive residues in different alginate chains. Advantageously, the ionotropic gelling alginate may be solubilised by treatment with a divalent cation chelating agent such as citrate and ethylenediaminetetraacetic acid (EDTA) or hexametaphosphate. It was found that 2% (w/v) alginate in the culture medium properly separated the cells; however, suitable ranges for the percentage of alginate in the growth medium include 0.25% to 6% (w/v) alginate, or 0.5% to 5% (w/v) alginate, or 1% to 4% (w/v) alginate, or 2% to 3% (w/v) alginate. In addition, neither the alginate/culture medium solidification nor re-liquefaction processes (described in more detail below) affect cell viability. Furthermore, induced editing by raising the temperature of the batch gel to 42 ℃ (described in more detail below) did not affect the integrity of the solidified medium or the isolation of the isolated clonal cell colonies.

The culture of mammalian cells using hydrogels has been performed to mimic the 3D cellular environment present in tissues, allowing for a more biologically relevant cellular environment. As with tissue mimetics, in the case of mammalian cell editing, alginates can be chemically functionalized to alter physicochemical and biological characteristics and properties to better bind and promote mammalian cell growth after cells are isolated in solidified alginate media. Since cells do not have receptors that recognize alginate, the proliferation and differentiation of some mammalian cells in alginate hydrogels requires signaling molecule and matrix interactions. For example, cell attachment peptides, in particular the sequence RGD (arginine-glycine-aspartic acid) have been shown to improve the adaptation of cells to the matrix, also for alginate. Using aqueous carbodiimide chemistry, alginates can be modified by covalently grafting peptide sequences onto alginate molecules. (for a thorough discussion of 3D cell culture in alginate hydrogels, see Andersen et al, microarray, 4:133-61 (2015)), alternatively, mammalian cells can be grown on beads as described above, wherein the beads are then suspended in alginate media.

After suspending the cells at the appropriate density, by e.g. adding CaCl₂The alginate in the medium was allowed to solidify 201 (described below with respect to example 5). Note that some areas of solidified alginate have no cells 206 and some areas have one cell 204. Next, the cells are grown 203 to obtain a predetermined approximate number of doublings. Because the cells are immobilized in three-dimensional space, the resulting colonies 208 are immobilized in three-dimensional space. The colonies were grown to final size 207 (that is, edited and unedited cell colony growth was normalized), sodium citrate 209 was added to the medium so that the solidified medium/alginate was re-liquefied and the cells from colonies 214, 216 (including edited and unedited cells, respectively) were again suspended in liquid medium. After the medium is reliquefied, the cells are recovered and analyzed 211 or used for a second round of editing 213. Also, since the combination of the separation and normalization processes overcomes the growth bias from unedited cells or cells that exhibit adaptive effects due to editing performed, the combination of the separation and normalization processes alone enriches the total cell population with edited cells; that is, isolation and normalization (e.g., growing colonies to final size) allows for high throughput enrichment of edited cells.

Fig. 2B depicts a simplified diagram of a workflow 250 for isolating, editing, and normalizing cells following nucleic acid-guided nuclease genome editing in batch cell culture, wherein reversible immobilization of batch cultures is utilized and editing is induced by inducing transcription of grnas. Cells 204 are first suspended in a container at an appropriate density and then at step 201By adding, for example, CaCl₂The alginate in the medium was solidified (described below with respect to example 5). Note that some areas of solidified alginate have no cells 206 and some areas have one cell 204. Next, the cells are grown 203 to obtain a predetermined approximate number of doublings. Because the cells are immobilized in three-dimensional space, the resulting colonies 208 are immobilized in three-dimensional space. Editing is then induced by inducing transcription of the gRNA 205. After the induction of editing, many of the cells in the edited colony 212 die due to the toxicity of the double strand break caused by editing. The growth of cells in unedited colony 210 was unaffected by the double strand break and continued to thrive. In step 207, colonies of both edited and unedited cells are grown to final size (that is, edited cell colonies 212 and unedited cell colonies 210 are grown to normalization), then sodium citrate 209 is added to the medium so that the solidified medium/alginate is re-liquefied and the cells from colonies 214, 216 (including edited and unedited cells, respectively) are again suspended in liquid medium. After the medium is reliquefied, the cells are recovered and analyzed 211 or used for a second round of editing 213. Also, since the combination of the separation and normalization processes overcomes the growth bias from unedited cells or cells that exhibit adaptive effects due to editing performed, the combination of the separation and normalization processes alone enriches the total cell population with edited cells; that is, isolation and normalization (e.g., growing colonies to final size) allows for high throughput enrichment of edited cells.

FIG. 2C is a photograph of E.coli cells expressing green fluorescent protein showing isolated colonies in solidified 2.0% alginate and medium (left panel), and E.coli cells expressing green fluorescent protein in 2.0% alginate and medium after the medium has been reliquefied.

Automated system including a separation/growth/editing/normalization module

Fig. 3A depicts an exemplary automated multi-module cell processing instrument 300 that includes a batch cell culture separation/growth/editing/normalization module 340 to, for example, perform the exemplary workflow described above with respect to fig. 2A and 2B, as well as additional exemplary modules. A gantry (gantry)302 is shown that provides an automated mechanical motion system (actuator) (not shown) that provides XYZ axis motion control to, for example, a module of an automated multi-module cell processing instrument 300, including, for example, an air displacement pipettor 332. In some automated multi-module cell processing instruments, the air displacement pipettor is moved by a gantry, and the various modules and reagent cartridges remain stationary; however, in other embodiments, the pipetting system may remain stationary while the various modules move. Also included in the automated multi-module cell processing instrument 300 is a wash or reagent cartridge 304, including a reservoir 306. As described below with respect to fig. 3B, the wash or reagent cartridge 304 may be configured to hold a large tube, such as a wash solution or a solution that is often used throughout an iterative process. In one example, the wash or reagent cartridge 304 may be configured to remain in place when two or more reagent cartridges 310 are used and replaced sequentially. Although the reagent cartridge 310 and the wash or reagent cartridge 304 are shown as separate cassettes in fig. 3A, the contents of the wash cassette 304 may be incorporated into the reagent cartridge 310.

The exemplary automated multi-module cell processing instrument 300 of fig. 3A also includes a cell growth module 334. In the embodiment shown in fig. 3A, the cell growth module 334 includes two rotating growth flasks (RGVs) 318, 320 (described in detail below with respect to fig. 3E) and a cell concentration module 322. In some embodiments, there is a separate separation/growth/editing and normalization module 340; however, in some embodiments, the separation/growth/editing and normalization module may be a separate RGV in the growth module 334, as certain embodiments of the separation/growth/editing/normalization module utilize a spinner flask similar or identical to the spinner flask used to culture cells prior to transformation. In addition, after growth, editing, and normalization have occurred, the edited cells can be prepared (e.g., the edited cells are concentrated and rendered electrically competent) for another transformation for another round of editing using cell concentration procedures for, e.g., media exchange and cell concentration. Here, the cell concentration module 322 is part of the cell growth module 334; however, in some embodiments, the cell concentration module 322 may be separate from the cell growth module 334, such as in a separate dedicated module.

Also shown is a separation/growth/editing/normalization module 340 separate from the growth module 334 as part of the automated multi-module cell processing instrument 300 of fig. 3A, where the module 340 is serviced by, for example, the air displacement pipettor 332. The separation/growth/editing/normalization module implementing the workflow described in fig. 2A and 2B and shown in fig. 3A may employ an "off the shelf" liquid handling instrument, such as those commercially available from: openstrons (OT-2)^TMSystem, Brooklyn, NY); ThermoFisher Scientific (Versette)^TMSystem, carlsbad, california); labcell (Access)^TMSystem, Carlsbad, CA); perkin Elmer (Janus)^TMSystem, San Jose, CA); agilent Inc (Bravo)^TMSystem, Santa Clara, CA); BioTek inc. (Winoosky, VT); hudson corporation (Solo)^TMSystem, Springfield, NJ); andrew Alliance (Andrew)^TMSystem, Waltham, MA); and Hamilton Robotics (tool set, Reno, NV). Also visible in fig. 3A is a waste reservoir 326, and a nucleic acid assembly/desalting module 314, the nucleic acid assembly/desalting module 314 including a reaction chamber or tube receptacle (not shown), and further including a magnet 316 to allow purification of nucleic acids using, for example, magnetic Solid Phase Reversible Immobilization (SPRI) beads (Applied Biological Materials inc., Richmond, BC). The reagent cartridge, transformation module, and cell growth module are described in more detail below.

Fig. 3B depicts an exemplary combined reagent cartridge and electroporation device 310 ("cartridge") that can be used with the separation/growth/editing/normalization module in an automated multi-module cell processing instrument. In certain embodiments, the material used to make the cartridge is thermally conductive in that, in certain embodiments, the cartridge 310 contacts a thermal device (not shown), such as a peltier device or a thermoelectric cooler, that heats or cools the reagent in the reagent receptacle or reservoir 312. The reagent receptacle or reservoir 312 may be a receptacle into which individual reagent tubes are inserted as shown in FIG. 3B, or the reagent receptacle may contain reagent without an inserted tube. In addition, the receptacle in the reagent cartridge may be configured for any combination of direct filling of tubing, connected tubing, and reagent.

In one embodiment, the reagent receptacle or reservoir 312 of the reagent cartridge 310 is configured to accommodate tubes of various sizes, including, for example, 250ml tubes, 25ml tubes, 10ml tubes, 5ml tubes, and Eppendorf tubes or microcentrifuge tubes. In yet another embodiment, all receptacles may be configured to accommodate tubes of the same size, e.g., 5ml tubes, and the reservoir insert may be used to accommodate smaller tubes in a reagent reservoir (not shown). In yet another embodiment, particularly in embodiments where the reagent cartridge is disposable, the reagent reservoir contains the reagent without the need for an inserted tube. In this disposable embodiment, the reagent cartridge may be part of a kit, wherein the reagent cartridge is pre-loaded with reagents and the receptacle or reservoir is sealed with, for example, foil, heat seal acrylic, etc., and provided to the consumer, which can then be used in an automated multi-module cell processing instrument. As will be understood by those skilled in the art in view of this disclosure, the reagent contained in the reagent cartridge will vary according to the workflow; that is, the reagents will vary depending on the process to which the cells are subjected in the automated multi-module cell processing instrument.

Reagents, e.g. cell samples, culture media, CaCl₂、Na₃C₆H₅O₇(sodium citrate), enzymes, buffers, nucleic acid vectors, expression cassettes, proteins or peptides, reaction components (such as for example MgCl)₂Dntps, nucleic acid assembly reagents, gap repair reagents, etc.), a washing solution, ethanol, magnetic beads for nucleic acid purification and separation, etc. may be placed at known positions in the reagent cartridge. In some embodiments of the cartridge 310, the cartridge includes a script (not shown) that is readable by a processor (not shown) to dispense reagents. Further, the cartridge 310, which is a component of the automated multi-module cell processing instrument, may include a script that specifies two, three, four, five, ten, or more passes to be performed by the automated multi-module cell processing instrumentThe process. In certain embodiments, the reagent cartridge is disposable and prepackaged with reagents tailored to perform a particular cell processing protocol (e.g., genome editing or protein production). Because reagent cartridges vary in content, and the components/modules of an automated multi-module cell processing instrument or system may not change, the scripts associated with a particular reagent cartridge may be matched to the reagents used and the cell process being performed. Thus, for example, a reagent cartridge may be prepackaged with reagents for genome editing and scripts specifying the processing steps to perform single or recursive genome editing in an automated multi-module cell processing instrument.

For example, the reagent cartridge may include a script to remove competent cells from a reservoir, transfer the cells to a transformation module (such as the flow-through electroporation device 330 in the reagent cartridge 310), remove a nucleic acid solution comprising a vector with an expression cassette from another reservoir in the reagent cartridge, transfer the nucleic acid solution to the transformation module, initiate the transformation process for a specified time, and then move the transformed cells to yet another reservoir in the reagent cartridge or to another module, such as a cell separation, editing, and growth module in an automated multi-module cell processing instrument. In another example, the reagent cartridge may include a script to transfer the nucleic acid solution comprising the vector from a reservoir in the reagent cartridge, the nucleic acid solution comprising the editing oligonucleotide cassette in a reservoir in the reagent cartridge, and the nucleic acid assembly mixture from another reservoir to the nucleic acid assembly/desalting module (314 of fig. 3A). The script may also specify process steps to be performed by other modules in the automated multi-module cell processing instrument. For example, the script may specify heating the nucleic acid assembly/desalting reservoir to 50 ℃ for 30min to generate an assembly product; and desalting and resuspending the assembled product via magnetic bead-based nucleic acid purification (including a series of magnetic beads, ethanol washes, and pipetting and mixing of buffers). These processes are described in more detail below.

As described below with respect to fig. 3C and 3D, an exemplary reagent cartridge 310 for use in an automated multi-module cell processing instrument may include one or more electroporation devices 330, preferably flow-through electroporation devices. The electroporation is carried outA widely used method for permeabilization of cell membranes, which method works by temporarily creating pores in the cell membrane with electrical stimulation. Applications of electroporation include the delivery of DNA, RNA, siRNA, peptides, proteins, antibodies, drugs, or other substances to various cells, such as mammalian cells (including human cells), plant cells, archaeal cells, yeast cells, other eukaryotic cells, bacteria, and other cell types. Electrical stimulation may also be used for cell fusion in the generation of hybridomas or other fused cells. In a typical electroporation procedure, cells are suspended in a buffer or medium that facilitates cell survival. For bacterial cell electroporation, low conductivity media, such as water, glycerol solutions, etc., are typically used to reduce the heat generated by transient large currents. In a conventional electroporation device, cells and material to be electroporated into the cells (collectively referred to as a "cell sample") are placed in a cup (cuvette) embedded with two flat electrodes for electrical discharge. For example, Bio-Rad (Hercules, Calif.) produces GENE PULSER XCELL for electroporating cells in cuvettes^TMA series of products. Traditionally, electroporation requires high field strengths; however, flow-through electroporation devices included in reagent cartridges (such as those shown in fig. 3B-3D) achieve efficient cell electroporation and are low in toxicity. The reagent cartridge of the present disclosure allows for particularly easy integration with robotic liquid handling instruments (such as air displacement pipettes) typically used in automated instruments and systems. Such automated instruments include, but are not limited to, off-the-shelf automated liquid handling systems from Tecan (mannidorf, Switzerland), Hamilton (Reno, NV), Beckman Coulter (Fort Collins, CO), and the like, as described above.

Fig. 3C and 3D are top and bottom perspective views, respectively, of an exemplary flow-through electroporation device 350, which flow-through electroporation device 350 may be part of the reagent cartridge 300 in fig. 3B or may be contained in a separate module (e.g., a transformation/transfection module). Fig. 3C depicts a flow-through electroporation cell 350. The flow-through electroporation cell 350 has an aperture defining a cell sample inlet 352 and a cell sample outlet 354. Fig. 3D is a bottom perspective view of the flow-through electroporation device 350 of fig. 3C. The inlet and

outlet apertures

352, 354 are visible in this view. Also visible in fig. 3D are the bottom of the inlet 362 corresponding to the aperture 352, the bottom of the outlet 364 corresponding to the outlet aperture 354, the bottom of the defined flow channel 366, and the bottoms of the two electrodes 368 on either side of the flow channel 366. In addition, flow-through electroporation devices may include push-suction pneumatics to allow for multiple pass (multi-pass) electroporation procedures; that is, cells to be electroporated can be "sucked" from an inlet to an outlet for one-pass electroporation and then "pushed" from the outlet end to the inlet end of the flow-through electroporation device to pass again between the electrodes for another pass-through electroporation. Exemplary flow-through electroporation devices for use in the automated multi-module cell processing instruments disclosed herein include electroporation devices described in: USSN16/147,120 filed on 28/9/2018; USSN16/147,353 filed on 28/9/2018; USSN16/147,865 filed on 30/9/2018; and USSN 16/426,310 filed on 30/5/2019; and USPN10,323,258, published 2019 on day 6, month 18, all of which are incorporated herein by reference in their entirety. Furthermore, the process may be repeated from one to many times. In addition, other embodiments of the reagent cartridge may provide or accommodate electroporation devices that are not configured as flow-through devices, such as the electroporation devices described in USSN 16/109,156 filed on 22/8/2018.

The exemplary automated multi-module cell processing instrument 300 of fig. 3A also includes a nucleic acid assembly module. The nucleic acid assembly module 314 is configured to perform, for example, isothermal nucleic acid assembly. Isothermal nucleic acid assembly links more than one DNA fragment in a single isothermal reaction, requiring fewer components and process manipulations. For example, isothermal nucleic acid assembly can combine up to 20 or more nucleic acid fragments simultaneously based on sequence identity. The assembly method requires that the nucleic acid to be assembled comprises an overlap of at least 15 bases with adjacent nucleic acid fragments. These fragments were mixed with a mixture of three enzymes (an exonuclease, a polymerase and a ligase) and buffer components. Because in some embodiments, the process is isothermal and can be performed in a 1-step or 2-step process using a single reaction vessel, isothermal nucleic acid assembly methods are suitable for use in automated multi-module cell processing instruments. The 1-step method allows the assembly of up to five different fragments using a one-step isothermal process. The master mix of fragments and enzyme was combined and incubated at 50 ℃ for up to one hour. To create more complex constructs or to incorporate fragments of 100bp to 10kb, a 2-step method is generally used, wherein a 2-step reaction requires two separate additions of the master mix; once for the exonuclease and annealing steps and a second time for the polymerase and ligation steps.

In one embodiment of the exemplary automated multi-module cell processing instrument 300 of fig. 3A, an aliquot of the vector, the oligonucleotides (e.g., genes or editing sequences of interest) to be inserted into the vector, and the nucleic acid assembly mixture may be withdrawn from three of sixteen reagent reservoirs 312 disposed within the reagent cartridge 310. The vector, oligonucleotide and reaction mixture are combined in a reaction chamber or tube located in a tube receptacle (not shown) in the nucleic acid assembly module and the module is heated to 50 ℃. After the nucleic acid assembly reaction has occurred, the magnetic beads may be removed from one of the reagent reservoirs 312 disposed within the reagent cartridge 310 and added to the nucleic acid assembly mixture in the reaction chamber of the nucleic acid module 314. As can be seen in fig. 3A, a magnet 316 (such as a solenoid magnet) is adjacent or proximal to the nucleic acid assembly module 314. After adding the magnetic beads to the nucleic acid assembly reactants, the nucleic acid products bind to the magnetic beads, and after a certain period of incubation, the magnet 316 is engaged, thereby separating the magnetic beads bound to the nucleic acids in the reaction chamber. The reaction solution (supernatant) in the nucleic acid assembly module 314 may be removed by the air displacement pipettor 332 and the wash solution and/or ethanol may be removed from the reagent reservoir 312 in the reagent cartridge 310 or from the wash solution reservoir 306 in the wash cassette 304 and used to wash the bead-bound nucleic acids. In washing beads and bound nucleic acids, the magnet can be disengaged and then the magnet is re-engaged to remove the wash solution from the nucleic acid assembly module. Alternatively, the magnet may not detach upon washing the beads and bound nucleic acids. The desalted assembled vector + oligonucleotides can then be moved to a flow-through electroporation device (transformation/transfection module), such as described with respect to fig. 3B-3D.

Fig. 3E is a model of tangential flow filtration used in the TFF module described below. The TFF device is part of a module in an automated multi-module cell processing instrument. After growth in the cell growth module, TFF was used to concentrate and render the cells electrocompetent. The cells may be cells loaded into the spinner flask for a first round of editing, or the cells may be cells that have undergone a round of editing, recovered from the liquefied alginate medium, re-grown in the spinner flask and prepared for a second round of editing. The TFF device is designed taking into account two main design considerations. First, the geometry of the TFF device results in filtration of the cell culture over a large surface area, thereby minimizing processing time. Second, the design of the TFF device is configured to minimize filter fouling (fouling). Fig. 3E is a general model 30 of tangential flow filtration. The TFF device operates using tangential flow filtration (also known as cross-flow filtration). Fig. 3E shows the cells flowing over the membrane 34, wherein the feed stream of cells 32 in culture medium or buffer is parallel to the membrane 34. TFF is different from dead-end filtration (dead-end filtration), in which both the feed stream and the pressure drop are perpendicular to the membrane or filter.

Fig. 3F-7L depict embodiments of Tangential Flow Filtration (TFF) devices/modules. Fig. 3F depicts the configuration of a retentate member (cell) 3022 (on the left), a membrane or filter 3024 (middle), and a permeate member 3020 (on the right). In fig. 3F, the retentate member 3022 comprises a tangential flow channel 3002, the tangential flow channel 3002 having a serpentine configuration that starts at one lower corner of the retentate member 3022 (specifically at the retentate port 3028), crosses over and up and then down and over the retentate member 3022, and ends at a second retentate port 3028 at the other lower corner of the retentate member 3022. Also visible on retentate member 3022 is an energy director 3091, the energy director 3091 surrounding the area where membrane or filter 3024 is located. In this embodiment, the energy director 3091 cooperates with the retentate member 3022 and the permeate member 3020 via energy director components on the permeate member 3020 and serves to facilitate ultrasonic welding or bonding of the retentate member 3022 to the permeate member 3020. Membrane or filter 3024 has a through-hole for retentate port 3028 and is configured to be located within the enclosure of energy director 3091 between retentate member 3022 and permeate member 3020. In addition to the energy director 3091, the permeate member 3020 also includes through holes for the retentate port 3028 at each bottom corner (each bottom corner mating with the through holes for the retentate port 3028 at the bottom corner of the membrane 3024 and the retentate port 3028 in the retentate member 3022) as well as tangential flow channels 3002 and a single permeate port 3026 located at the top and center of the permeate member 3020. The tangential flow channel 3002 structure in this embodiment has a serpentine configuration and an undulating geometry, although other geometries may be used. In some aspects, the length of the tangential flow channel is 10mm to 1000mm, 60mm to 200mm, or 80mm to 100 mm. In some aspects, the channel structure has a width of 10mm to 120mm, 40mm to 70mm or 50mm to 60 mm. In some aspects, the tangential flow channel 1202 is rectangular in cross-section. In some aspects, the cross-section of the tangential flow channel 1202 has a width of 5 μm to 1000 μm and a height of 5 μm to 1000 μm, a width of 300 μm to 700 μm and a height of 300 μm to 700 μm, or a width of 400 μm to 600 μm and a height of 400 μm to 600 μm. In other aspects, the tangential flow channel 1202 has a circular, elliptical, trapezoidal, or rectangular cross-section and a hydraulic radius of 100 μm to 1000 μm, a hydraulic radius of 300 μm to 700 μm, or a hydraulic radius of 400mm to 600 μm.

Fig. 3G is a side perspective view of the reservoir assembly 3050. The reservoir assembly 3050 includes retentate reservoirs 3052 on either side of a single permeate reservoir 3054. During cell concentration, the retentate reservoir 3052 is used to contain the cells and media as the cells are transferred through the TFF device or module and into the retentate reservoir. The permeate reservoir 3054 is used to collect filtrate fluid removed from the cell culture during cell concentration or old buffer or culture medium during cell growth. In the embodiment depicted in fig. 3F-3L, the buffer or culture medium is supplied to the permeate member from a reagent reservoir separate from the device module. Also visible in fig. 3G are recess 3032 for receiving a pneumatic port (not shown), permeate port 3026 and retentate port throughbore 3028. The retentate reservoir is fluidly coupled to a retentate port 3028, which retentate port 3028 in turn is fluidly coupled to a portion of a tangential flow channel (not shown) disposed in the retentate member. The permeate reservoir is fluidly coupled to a permeate port 3026, which in turn fluidly couples to a portion of a tangential flow channel (not shown) disposed in the permeate member, wherein the portion of the tangential flow channel is divided into two portions by a membrane (not shown). In embodiments including this embodiment, up to 120mL of cell culture may be grown and/or filtered, or up to 100mL, 90mL, 80mL, 70mL, 60mL, 50mL, 40mL, 30mL, or 20mL of cell culture may be grown and/or concentrated.

Fig. 3H depicts a top view of the reservoir assembly 3050 shown in fig. 3G, fig. 3I depicts a lid 3044 of the reservoir assembly 3050 shown in fig. 3G, and fig. 3J depicts a gasket 3045 shown in fig. 3G disposed on the lid 3044 of the reservoir assembly 3050 in operation. Fig. 3H is a top view of the reservoir assembly 3050, showing two retentate reservoirs 3052, one on each side of the permeate reservoir 3054. It can also be seen that a recess 3032 that will mate with a pneumatic port (not shown), and a fluid channel 3034 located at the bottom of the retentate reservoir 3052, the fluid channel 3034 fluidly couples the retentate reservoir 3052 with the retentate port 3028 (not shown) via the permeate member 3220 and a through hole in the membrane 3024 for the retentate port (also not shown). Fig. 3I depicts a lid 3044, the lid 3044 configured to be disposed on top of a reservoir assembly 3050. The lid 3044 has circular openings (cut-out) at the top of the retentate reservoir 3052 and the permeate reservoir 3054. Likewise, a fluid channel 3034 is visible at the bottom of the retentate reservoir 3052, wherein the fluid channel 3034 fluidly couples the retentate reservoir 3052 with a retentate port 3028 (not shown). Three pneumatic ports 3030 are also shown for each retentate reservoir 3052 and permeate reservoir 3054. Fig. 3J depicts a gasket 3045, the gasket 3045 configured to be disposed on a lid 3044 of a reservoir assembly 3050. Three fluid transfer ports 3042 are visible for each of the retentate reservoir 3052 and the permeate reservoir 3054. Likewise, three pneumatic ports 3030 are shown for each retentate reservoir 3052 and permeate reservoir 3054.

Fig. 3K depicts an exploded view of TFF module 3000. A visible component reservoir assembly 3050, a lid 3044 to be disposed on the reservoir assembly 3050, a gasket 3045 to be disposed on the lid 3044, a retentate member 3022, a membrane or filter 3024, and a permeate member 3020. Also visible is a permeate port 3026, which mates with the permeate port 3026 on the permeate reservoir 3054; and two retentate ports 3028 that mate with the retentate port 3028 on the retentate reservoir 3052 (with only one retentate reservoir 3052 being clearly visible in this fig. 3K). The membrane 3024 and the through-hole of the retentate port 3028 in the permeate member 3020 are also seen.

Fig. 3L depicts one embodiment of an assembled TFF module 3000. The retentate member 3022, the membrane member 3024, and the permeate member 3020 are coupled side-by-side (side-to-side) with the reservoir assembly 3050. Two retentate ports 3028, which couple the tangential flow channels 3002 in the retentate member 3022 to two retentate reservoirs (not shown), and one permeate port 3026, which couple the tangential flow channels 3002 in the permeate/filtrate member 3020 to a permeate reservoir (not shown), also see tangential flow channels 3002, which are formed by the cooperation of the retentate member 3022 and the permeate member 3020, with the membrane 3024 sandwiched between the retentate member 3022 and the permeate member 3020 and dividing the tangential flow channels 3002 into two parts, also see energy director 3091, in this fig. 3L energy director 3091 has been used to ultrasonically weld or couple the retentate member 3022 and the permeate member 3020 around the membrane 3024, a cover 3044 can be seen on top of the reservoir assembly 3050, and a gasket 3045 disposed on the cover 3044. gasket 3045 engages and provides fluid transfer ports 3042 and pneumatic ports 3030, respectively, and seals with fluid transfer ports 3032 and pneumatic ports 3030, respectively And (4) connecting. Fig. 3L also shows the length, height and width dimensions of TFF module 3000. The assembled TFF device 3000 is typically 50mm to 175mm in height, or 75mm to 150mm in height, or 90mm to 120mm in height; a length of 50mm to 175mm, or a length of 75mm to 150mm, or a length of 90mm to 120 mm; and a depth of 30mm to 90mm, or a depth of 40mm to 75mm, or a depth of 50mm to 60 mm. An exemplary TFF device has a height of 110mm, a length of 120mm, and a depth of 55 mm. Additional information and alternative embodiments regarding TFF, see, e.g., USSN62/728,365 filed on 7/9/2018; 62/857,599 filed on 6/5/2019; and 62/867,415 filed on 27/6/2019.

Batch gel culture using growth modules in automated instruments

FIG. 4A depicts one embodiment of a rotary growing bottle (RGV) that can be used for: 1) growing the cells to an OD suitable for transformation, and 2) using as a container the batch cell culture procedure depicted in fig. 1A, fig. 2A, and fig. 2B. In embodiments where RGVs are used in cell growth for transformation, the RGVs can constantly measure the optical density of the growing cell culture. One advantage of the cell growth module is that optical density can be measured continuously (dynamic monitoring) or at specific time intervals (e.g., every 5 seconds, 10 seconds, 15 seconds, 20 seconds, 30 seconds, 45 seconds, or 60 seconds, or every 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, or 10 minutes, etc.). Alternatively, OD may be measured at specific time intervals early in the cell growth cycle and continuously after the OD of the cell culture reaches the set-point OD. The cell growth module is controlled by a processor that can be programmed to measure the OD constantly or at user-defined intervals. For example, a script on the reagent cartridge may also specify the frequency of reading the OD, as well as the target OD and target time. In addition, the user may manually set a target time at which the user desires the cell culture to reach the target OD. To achieve the target OD at the target time, the processor measures the OD of the growing cells, calculates the cell growth rate in real time, and predicts the time at which the target OD will be achieved. The processor then automatically adjusts the temperature of the RGVs (and cell culture) as needed. Lower temperatures slow growth, while higher temperatures increase growth.

In the RGV embodiment depicted in fig. 4A, RGV 400 is a transparent container having an open end 424 for receiving liquid media and cells, a central vial region 406 defining a main reservoir for growing cells, a tapered to constricted region 418 defining at least one light path 410, a closed end 416, and a drive engagement mechanism 412. The RGV has a central longitudinal axis 420 about which the vial rotates, and the light path 410 is generally perpendicular to the longitudinal axis of the vial. The first light path 410 is located in the lower constriction of the tapered-to-constriction region 408. Optionally, some embodiments of the RGV 400 have a second light path 418 in the tapered region that tapers to the constriction region 408. In this embodiment, both optical paths are located in the region of the RGV that is always filled with cell culture (cells + growth medium) and are not affected by the rotational speed of the RGV. The first optical path 410 is shorter than the second optical path 418 when the OD value of the cell culture in the vial is at a high level (e.g., later in the cell growth process), thereby allowing sensitive measurement of the OD value when the OD value of the cell culture in the vial is at a high level (e.g., later in the cell growth process), and the second optical path 418 allows sensitive measurement of the OD value when the OD value of the cell culture in the vial is at a lower level (e.g., early in the cell growth process). The drive engagement mechanism 412 engages a motor (not shown) to rotate the bottle.

The RGV 400 may be reusable or, preferably, like the reagent cartridge, the RGV is a consumable. In some embodiments, the RGV is a consumable and is pre-filled with growth media to provide the user, wherein the vial is sealed with a foil seal at the open end 424. The growth module depicted in fig. 4A-4D comprising a rotating growth flask may also be employed as a container for batch cell culture, separation, editing and pooling (see, e.g., fig. 7).

When a rotating growth flask (RGV) is used as the separation module, cells in a medium containing 0.25% to 6% alginate are transferred to the rotating growth flask by, for example, a liquid handling system, wherein the cells are first diluted appropriately to allow each cell to separate or substantially separate from the other cells when the medium gels; and secondly, cell colonies grown from the isolated cells in the gelled or solidified medium are separated from other cell colonies. After loading the suitably diluted cells into the RGV, the cells are loaded by slowly adding dropwise an appropriate amount of e.g. CaCl to the RGV₂(preferably while rotating the RGV at a low speed) to trigger the solidification or gelation of the medium. After the medium is solidified, the cells can be grown to final size colonies (e.g., normalized) (see, e.g., fig. 2A, where no editing induction occurs) or can be allowed to growCells are grown, e.g., 2-50 doublings, then editing is induced, e.g., by raising the temperature of the RGV to 42 ℃ for a period of time to induce the pL promoter to drive gRNA transcription, then the temperature is lowered and the cells are grown to a final size or desired cell concentration (see, e.g., fig. 2B). After the cells have grown to a final size (e.g., the cells are in a senescent state and the size of the cell colonies no longer increases), the gelled or solidified medium is liquefied by dropwise addition of an appropriate amount of, for example, sodium citrate to the solidified medium (preferably while rotating the RGV at a low speed). The cells and media can then be removed from the RGV by the liquid handling system and filtered in, for example, a filtration module (such as an FTT device), as described with respect to fig. 3F-3L.

FIG. 4B is a perspective view of one embodiment of a cell growth device 430. FIG. 4C depicts a cross-sectional view of the cell growth device 430 of FIG. 4B. In both figures, the rotary growth bottle 400 is seen to be located within the main housing 436, with the extended lip 402 of the rotary growth bottle 400 extending above the main housing 436. Additionally, the end housing 452, lower housing 432, and flange 434 are shown in both figures. Flange 434 is used to attach cell growth device 430 to a heating/cooling device or other structure (not shown). Fig. 4C depicts additional details. In fig. 4C, upper seat 442 and lower seat 440 are shown within main housing 636. The upper and

lower shoes

442, 440 support the vertical load of the rotating growth bottle 400. The lower housing 432 contains a drive motor 438. The cell growth apparatus 430 of fig. 4C includes two optical paths: a primary optical path 444 and a secondary optical path 450. The optical path 444 corresponds to the optical path 410 in the taper-to-constriction portion of the rotating growth flask 400, and the optical path 450 corresponds to the optical path 408 in the taper-to-constriction portion of the rotating growth flask 400. Light path 410 and light path 408 are not shown in fig. 4C, but can be seen in fig. 4A. In addition to the optical path 444 and the optical path 440, there is an emitter plate 448 to illuminate the optical path and a detector plate 446 to detect light after it has passed through the cell culture fluid in the rotating growth flask 400.

The motor 438 is engaged with the drive mechanism 412 and is used to rotate the rotary growth bottle 400. In some embodiments, the motor 438 is a brushless DC type drive motor with a built-in drive controller that can be set to maintain a constant Revolutions Per Minute (RPM) between 0 and about 3000 RPM. Alternatively, other motor types may be used, such as stepper motors, servo motors, brushed DC motors, and the like. Optionally, the motor 438 may also have a directional controller to allow for reversing the direction of rotation, and a tachometer to sense and report the actual RPM. The motor is controlled by a processor (not shown) according to standard protocols programmed into the processor and/or user input, for example, and the motor may be configured to vary the RPM to induce axial precession (axial advancement) of the cell culture to enhance mixing, for example, to prevent cell aggregation, increase ventilation, and optimize cell respiration.

The main housing 436, end housing 452, and lower housing 432 of the cell growth device 430 can be made of any suitable, robust material, including aluminum, stainless steel, and other thermally conductive materials, including plastics. These structures, or portions thereof, may be formed by various techniques, such as metal fabrication, injection molding, construction of fused structural layers, and the like. While the rotating growth vial 400 is contemplated as being reusable in some embodiments, it is preferably a consumable, and the other components of the cell growth device 430 are preferably reusable and used as a stand-alone desktop device or as a module in a multi-module cell processing system.

The processor (not shown) of cell growth apparatus 430 may be programmed with information to be used as a "blank" or control for growing cell cultures. A "blank" or control is a container containing only cell growth medium that yields 100% transmission and an OD of 0, while a cell sample will deflect light and will have a lower percentage of transmission and a higher OD. As the cells grow and become denser in the medium, the transmittance will decrease and the OD will increase. The processor (not shown) of cell growth device 430 can be programmed to use a blank wavelength value corresponding to growth media typically used for cell culture (e.g., mammalian cells, bacterial cells, animal cells, yeast cells, etc.). Optionally, a second spectrophotometer and container may be included in cell growth apparatus 430, wherein the second spectrophotometer is used to read the blank at specified intervals.

Fig. 4D shows cell growth device 430 as part of an assembly that includes cell growth device 430 of fig. 4B coupled to light source 490, detector 492, and thermal component 494. The rotating growth flask 400 is inserted into the cell growth apparatus. Components of the light source 490 and detector 492 (e.g., such as photodiodes with gain control to cover 5-log) are coupled to the main housing of the cell growth apparatus. A lower housing 432 containing a motor to rotate the spinner flask 400 is shown, as well as one of the flanges 434 securing the cell growth apparatus 430 to the assembly. Further, the thermal component 494 is shown as a peltier device or a thermoelectric cooler. In this embodiment, thermal control is achieved by attaching and electrically integrating cell growth device 430 to thermal component 494 via flange 434 on the base of lower housing 432. Thermoelectric coolers can "pump" heat to either side of the junction to cool or heat the surface depending on the direction of current flow. In one embodiment, a thermistor is used to measure the temperature of the main housing and then the rotating growth flask 400 is controlled to about +/-0.5 ℃ by a standard electronic proportional-integral-derivative (PID) controller loop.

In use, cells are seeded (e.g., cells may be removed from an automated liquid handling system or by a user) into the pre-filled growth medium of the rotating growth vial 400 by piercing the foil seal or membrane. The programming software of cell growth apparatus 430 sets a controlled temperature for growth, typically 30 ℃, and then slowly starts spinning the spinning growth flask 400. The cell/growth medium mixture slowly moves vertically up the walls due to centrifugal forces, allowing the rotating growth flask 400 to expose a large surface area of the mixture to a normal oxygen environment. The growth monitoring system continuously reads OD or OD measurements at preset or preprogrammed time intervals. These measurements are stored in internal memory and, if necessary, the software plots the measurements against time to show the growth curve. If enhanced mixing is required, for example to optimise growth conditions, the speed of rotation of the bottle may be varied to cause axial precession of the liquid and/or a complete change of direction may be made at programmed intervals. Growth monitoring can be programmed to automatically terminate the growth phase at a predetermined OD, and then the mixture is rapidly cooled to a lower temperature to inhibit further growth.

One application of cell growth apparatus 430 is to constantly measure the optical density of a growing cell culture. One advantage of the cell growth apparatus is that the optical density can be measured continuously (dynamic monitoring) or at specific time intervals (e.g., every 5 seconds, 10 seconds, 15 seconds, 20 seconds, 30 seconds, 45 seconds, or 60 seconds, or every 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 4 minutes, 7 minutes, 8 minutes, 9 minutes, or 10 minutes). Although the cell growth device 430 has been described in the context of measuring the Optical Density (OD) of a growing cell culture, the skilled artisan, in view of the teachings of the present specification, will appreciate that other cell growth parameters may be measured in addition to, or instead of, the OD of the cell culture. As with the optional measurements described above with respect to cell growth of the solid wall device or module, spectroscopy using visible, UV or Near Infrared (NIR) light allows the concentration of nutrients and/or waste products in the cell culture to be monitored and other spectroscopic measurements to be made; that is, other spectral characteristics may be measured via, for example, dielectric impedance spectroscopy, visible fluorescence, fluorescence polarization, or luminescence. In addition, cell growth device 430 may include other sensors for measuring, for example, dissolved oxygen, carbon dioxide, pH, conductivity, and the like.

Method for editing cells in a batch of gel using an instrument

Fig. 5A is a simplified block diagram of an embodiment of an exemplary automated multi-module cell processing instrument including a separation/growth/editing/normalization module for enriching edited cells. Cell processing apparatus 500 may include a housing 544, a reservoir 502 of cells to be transformed or transfected, and a growth module (cell growth device) 504. Cells to be transformed are transferred from the reservoir to the growth module for culture until the cells reach the target OD. After the cells reach the target OD, the growth module may cool or freeze the cells for later processing, or the cells may be transferred to an optional filtration module 530 where the cells are rendered electrically receptive and concentrated to a volume most suitable for cell transformation. After concentration, the cells are then transferred to an electroporation device 608 (e.g., transformation/transfection module).

In addition to a reservoir for storing cells, system 500 may include a reservoir 516 for storing editing oligonucleotide cassettes and a reservoir 518 for storing expression vector backbones. Both the editing oligonucleotide cassette and the expression vector backbone are transferred from the reagent cartridge into the nucleic acid assembly module 520, wherein the editing oligonucleotide cassette is inserted into the expression vector backbone. The assembled nucleic acids can be transferred to an optional purification module 522 for desalting and/or other purification and/or concentration procedures as needed to prepare the assembled nucleic acids for transformation. Optionally, pre-assembled nucleic acids, such as editing vectors, may be stored in reservoir 516 or reservoir 518. After the processing performed by the purification module 522 is complete, the assembled nucleic acids are transferred to, for example, an electroporation device 508, which electroporation device 508 already contains a cell culture grown to a target OD and electrically competent via the filtration module 530. In the electroporation device 508, the assembled nucleic acids are introduced into the cells. Following electroporation, the cells are transferred to the combined recovery/selection module 510. See USPN10,253,316 published on 9/4/2019 for an example of a multi-module cell editing apparatus; USPN10,329,559 published on 25.6.2019; USPN10,323,242 published on 18 th 6 th 2019; and USSN 16/412,175 filed on day 14, month 5, 2019; USSN 16/412,195 filed on 14 th 6 th 2019; and USSN 16/423,289 filed on 29/5/2019, all of which are incorporated herein by reference in their entirety.

After recovery and optional selection, the cells are transferred to a separation, editing and growth module 540 where the cells are diluted and partitioned such that there is an average of one cell per compartment in module 540. After isolation, cells are grown to final size (e.g., to normalize colonies). After colonies were grown to final size, the colonies were pooled. Also, the separation overcomes growth bias from unedited cells and growth bias caused by adaptive effects of different edits.

The restore, select, and separate, edit, and grow modules may all be separate, may be arranged and combined as shown in fig. 5A, or may be arranged or combined in other configurations. In certain embodiments, such as those described with respect to the rotating growth flask shown in fig. 4A, all recovery, selection, separation, editing, and normalization are performed in a single container/module (e.g., rotating growth flask 400 in growth module 430 of fig. 4B).

After the normalized cell colonies are pooled, the cells can be stored, for example, in storage block 512, where the cells can be maintained, for example, at 4 ℃ until the cells are removed for further study. Alternatively, these cells can be used for another round of editing. The multi-module cell processing instrument is controlled by a processor 542, the processor 542 being configured to run the instrument based on user input (as indicated by one or more scripts, or as a combination of user input or scripts). The processor 542 may control the timing, duration, temperature and operation of the various modules of the system 600 as well as the dispensing of reagents. For example, the processor 542 may cool the cells after transformation until editing is desired, at which point the temperature may be raised to a temperature that facilitates genome editing and cell growth. The processor may be programmed with standard protocol parameters from which a user may select, the user may manually specify one or more parameters, or one or more scripts associated with the reagent cartridge may specify one or more operational and/or reaction parameters. In addition, the processor may notify the user (e.g., via an application to a smartphone or other device) that the cells have reached the target OD, and update the user regarding the progress of the cells in the various modules in the multi-module system.

The automated multi-module cell processing instrument 500 is a nuclease-guided genome editing system and can be used in a single editing system (e.g., one or more edits are introduced into a cell genome in a single editing process). The system of fig. 5B described below is configured to perform sequential editing, e.g., two or more genome edits are provided sequentially in a cell using different nuclease-guided systems; and/or recursive editing, e.g., the sequential introduction of two or more genome edits in a cell using a single nuclease-guided system.

Figure 5B shows another embodiment of a multi-module cell processing instrument. This embodiment depicts an exemplary system for recursive genetic editing of a population of cells. As with the embodiment shown in fig. 5A, cell processing instrument 550 may include a housing 544, a reservoir 502 for storing cells to be transformed or transfected, and a cell growth module 504 including a rotating growth flask. Cells to be transformed are transferred from the reservoir to the cell growth module for culture until the cells reach the target OD. After the cells reach the target OD, the growth module may cool or freeze the cells for later processing or transfer the cells to the TFF module 530 where the cells undergo buffer exchange and become electrically receptive and the volume of the cells may be substantially reduced. After concentrating the cells to an appropriate volume, the cells are transferred to an electroporation device 508. In addition to a reservoir for storing cells, the multi-module cell processing instrument further comprises a reservoir 506 for storing a carrier pre-assembled with editing oligonucleotide cartridges. The pre-assembled nucleic acid vector is transferred to the electroporation device 508, the electroporation device 508 already containing a culture of cells grown to the target OD. In the electroporation device 508, nucleic acids are electroporated into cells. After electroporation, the cells are transferred to an optional recovery module 556 where the cells are briefly recovered after transformation.

After recovery, the cells may be transferred to storage module 512, where the cells may be stored, for example, at 4 ℃ for later processing, or the cells may be transferred to selection/separation/editing/normalization module 558. In the separate/edit/grow module 558, the cells are diluted such that the cells are separated from each other in three-dimensional space. The arrayed cells are in selection medium to select for cells that have been transformed or transfected with the editing vector. After isolation, the liquid medium is solidified and the cells continue to grow to form clonal colonies in three-dimensional space. Optionally, editing is induced by providing conditions (e.g., temperature, addition of an inductive or repressive chemical) to induce editing. After editing, the cells are grown to a final size or desired cell concentration or optical density (e.g., normalization of colonies), and then the cells are pooled and transferred to storage unit 514, or may be transferred to growth module 504 for another round of growth, transformation, and editing. Between pooling and transfer to the growth module, there may be one or more additional steps, such as media exchange by, for example, filtration through TFF, cell concentration, and the like. Note that the selection/separation/growth/editing and normalization modules may be the same module, with all processes being performed in the same container (such as the rotating growth flask of fig. 4A-4D). After the putatively edited cells are pooled, they may be subjected to another round of editing, starting with growth, concentration and processing of the cells to become electrocompetent, and transformed via electroporation module 508 with yet another donor nucleic acid in another editing cassette.

In the electroporation device 508, cells from the first round of editing are transformed with a second set of editing oligonucleotides (or other types of oligonucleotides), and the cycle is repeated until the cells are transformed and edited with the desired number of, e.g., editing cassettes. The multi-module cell processing instrument illustrated in fig. 5B is controlled by processor 542, and processor 542 is configured to run the instrument based on user input, or is controlled by one or more scripts, including at least one script associated with a reagent cartridge. The processor 542 may control the timing, duration, and temperature of the various processes of the system 550, the dispensing of reagents, and other operations of the various modules. For example, a script or processor may control the dispensing of cells, reagents, vectors, and editing oligonucleotides; which editing oligonucleotides are used for cell editing and in which order; time, temperature and other conditions used in the recovery and expression modules, the wavelength at which the OD is read in the cell growth module, the target OD to which the cell is grown, and the target time at which the cell will reach the target OD. Additionally, the processor can be programmed to notify a user (e.g., via an application) of the progress of the cells in the automated multi-module cell processing instrument.

Examples

The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention, and are not intended to limit the scope of what the inventors regard as their invention nor are they intended to represent that the experiments below are all or the only experiments performed. Other equivalent methods, procedures and compositions are intended to be included within the scope of the present invention. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperature, etc.) but some experimental error and deviation should be accounted for. Unless otherwise specified, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees celsius, and pressure is at or near atmospheric.

Example 1: editing cassette and skeleton augmentation and assembly

Preparing an editing box:the 5nM oligonucleotide synthesized on the chip was amplified in 50. mu.L volume using Q5 polymerase. PCR conditions were 95 ℃ for 1 min; 8 cycles at 95 ℃ for 30 seconds/at 60 ℃ for 30 seconds/at 72 ℃ for 2.5 minutes; finally at 72 ℃ for 5 minutes. After amplification, the PCR products were subjected to SPRI bead clean-up, where 30 μ Ι _ SPRI mix was added to 50 μ Ι _ PCR reaction and incubated for 2 min. The tube was subjected to a magnetic field for 2 minutes, the liquid was removed, and the beads were washed 2 times with 80% ethanol with 1 minute intervals between washes. After the last wash, the beads were dried for 2 minutes, 50 μ Ι _ 0.5 × TE pH 8.0 was added to the tube, and the beads were vortexed to mix. The slurry was incubated at room temperature for 2 minutes and then subjected to a magnetic field for 2 minutes. The eluate was removed and the DNA quantified.

After quantification, a second amplification procedure was performed using dilutions of the eluate from the SPRI clean-up. PCR was performed under the following conditions: at 95 ℃ for 1 minute; 18 cycles at 95 ℃ for 30 seconds/at 72 ℃ for 2.5 minutes; finally at 72 ℃ for 5 minutes. Amplicons were checked on a 2% agarose gel and pools with the cleanest products were identified. Amplification products that appear to have heterodimers or chimeras are not used.

Preparing a framework:the purified scaffolds were subjected to a series of 10-fold serial dilutions and each dilution scaffold series was amplified under the following conditions: at 95 ℃ for 1 minute; then the30 cycles at 95 ℃ for 30 seconds/at 60 ℃ for 1.5 minutes/at 72 ℃ for 2.5 minutes; finally at 72 ℃ for 5 minutes. Following amplification, the amplified scaffolds were subjected to SPRI clean-up as described above for the cassettes. Elute the backbone to 100. mu.L ddH₂And O, quantifying, and then carrying out nucleic acid assembly.

Isothermal nucleic acid assembly:150ng of backbone DNA was combined with 100ng of cassette DNA. An equal volume of 2x gibson master mix was added and the reaction was incubated at 50 ℃ for 45 minutes. After assembly, the assembled backbone and cassette were SPRI cleaned as described above.

Example 2: creation of cell lines transformed with Engine vectors

And (3) transformation:mu.L of engine vector DNA (containing the coding sequence for MAD7 nuclease under the control of pL inducible promoter, chloramphenicol resistance gene and lambda Red recombinant engineering system) was added to 50. mu.L of E.coli EC1 strain cells. Transformed cells were plated on LB plates containing 25. mu.g/mL chloramphenicol (chlorine) and cultured overnight to accumulate clonal isolates. The next day, colonies were picked, grown overnight in LB + 25. mu.g/mL chlorine, and glycerol stocks were prepared from saturated overnight cultures by adding 500. mu.L of 50% glycerol to 1000. mu.L of culture. The EC1 stock solution containing the engine vehicle was frozen at-80 ℃.

Example 3: preparation of competent cells

A1 mL aliquot of a fresh overnight grown culture of EC1 cells transformed with the engine vector was added to a 250mL flask containing 100mL LB/SOB + 25. mu.g/mL chlorine medium. Cells were grown to 0.4-0.7OD and stopped by transferring the culture to ice for 10 min. Cells were centrifuged for 5 min at 8000x g in JA-18 rotor and 50mL ice-cold ddH₂O or 10% glycerol wash 3X and centrifuge in JA-18 rotor at 8000x g for 5 min. The washed cells were resuspended in 5mL of ice-cold 10% glycerol and aliquoted into 200. mu.L fractions. Optionally, the glycerol stock solution may be stored at-80 ℃ at this point for later use.

Example 4: batch cell 3D isolation, colony normalization and processing in a rotating growth flask

Editing batch cell culture:this protocol describes a standard batch culture protocol using alginate as the solidifying agent. By adding CaCl₂Alginate was solidified and liquefied after chelating agent addition, and both processes can be performed at temperatures suitable for enrichment of nucleic acid guided nuclease edited bacterial and yeast cells by isolation, growth, editing and normalization. This protocol was used for an inducible system that utilized both nucleases and grnas to make colonies phenotypically different. Alginate (alginate, a1112Sigma-Aldrich (st. louis, MO), low viscosity sodium alginate salt from brown algae).

Solution:

table 1: LB alginate

The desired amounts of LB and DI H as set forth in Table 1₂O was combined into the flask. The stir bar was added to the vial and the alginate was added slowly while the LB/alginate mixture was stirred on the stir plate. The LB/alginate mixture is then sterilized by autoclaving using standard conditions (e.g. 121 ℃, 20min, liquid circulation). After autoclaving, the solution was immediately cooled on ice. The cells and the desired antibiotic are added to the appropriate concentration prior to use of the LB/alginate solution.

Table 2: LB alginate, fraction for arabinose induction (1% final concentration)

The desired amounts of LB and DI H as set forth in Table 2₂O were combined into one bottle. The stir bar was added to the vial and the alginate was added slowly while the LB/alginate mixture was stirred on the stir plate. The LB/alginate mixture is then sterilized by autoclaving using standard conditions (e.g. 121 ℃, 20min, liquid circulation). After autoclaving, the solution was immediately cooled on ice. In the use of LB/alginatePrior to the solution, the cells and the required antibiotics were added to the appropriate concentration and 1ml of 20% arabinose was also added to 19ml of LB alginate solution to obtain a final arabinose concentration of 1%. Next, a calcium chloride (1M) solution was prepared using calcium chloride dihydrate (MW. 147.01g/mol), and the calcium chloride solution was filter-sterilized. In addition, a 1M sodium citrate solution was prepared using trisodium citrate dihydrate (MW 294.10g/mol), which was also filter sterilized.

Editing was performed according to the protocol described above to prepare LB alginate (25 ml per sample) and LB alginate + 1% arabinose (25 ml per sample). 10ml alginate + 1% arabinose solution was added to each 50ml conical tube. The conical tube was maintained at 30 ℃ for use after the conversion protocol was complete. Transformation was performed using a Nepagene electroporator set-up for E.coli, using 500ng of the nucleic acid module (vector + editing cassette library) transformed into ec83 (recombinant engineered competent cells). Cells were allowed to recover in 3000. mu.L of SOB in a 15ml conical tube while shaking at 30 ℃ for 3 hours. After 3 hours, the alginate tubes and transformation tubes were removed from the 30 ℃ incubator and 250 μ L of cells were added to each tube containing 25ml of alginate solution (1: 10). By slowly transferring 20ml of alginate + cell solution to 30ml of 100mM CaCl₂The alginate solution is allowed to solidify in solution. The alginate slurry was then centrifuged at 4000x g for 10 min. The supernatant was decanted and the batch gel incubated at 30 ℃ for 9 hours. After incubation at 30 ℃ for 9 hours, the temperature was moved to 42 ℃ for 2 hours to induce editing.

After editing, the temperature was moved back to 30 ℃ for overnight growth. To re-liquefy (dissolve) the alginate, 40ml of DI water was added to each conical tube and 10ml of 1M sodium citrate was added. The tube was then shaken at 30 ℃ for 30-45 minutes. For singleplex recovery, the library was recovered by diluting the cells and plating the cells on selective culture plates. Different dilutions were plated and the plates were also observed (spot) to obtain colony counts. Cells were grown on selective media for 12-24 hours and colonies were picked into 96-well plates, each containing 750ml of LB. Selected colonies were allowed to grow for 24 hours and each sample was prepared for DNA extraction and next generation sequencing. For amplicon recovery, cells were spun at 5,000x g for 10 minutes. The supernatant was removed and the cells were resuspended in 500. mu.L of 0.8 NaCl. Use of Zyppy^TMPlasmid Miniprep kit (Zymo Research, Bath, UK) extracts plasmid DNA from the library and prepares samples for PCR of the inserted sequences and analysis of the amplicons via next generation sequencing.

Figures 6A-6C are depictions of experiments performed to demonstrate that normalization was achieved in batch culture comparing the amount of wild type (inert) plasmid and editing plasmid (GalK) in batch gels with liquid cell cultures (see example 7 below). In the first step (shown in fig. 6A), the wild-type plasmid was used to transform the e.coli cell line, and additionally (separately), the editing plasmid was used to transform the e.coli cell line. After transformation, pools of transformed cells were combined in the following proportions: 50:50, 10:1 and 1:10 (wild type and editing cells, respectively) and were distributed between batch and liquid cultures, where six replicates were prepared for each group. Controls included 100% wild type and 100% edited cells, as well as standard plating controls. In FIG. 6B, batch and liquid cultures (experimental and control) were grown at 30 ℃ for 6 hours, at 42 ℃ for 2 hours, and at 30 ℃ overnight. Next, viable cells were recovered from each culture (e.g., six experimental cultures and controls for each batch culture and liquid culture). Figure 6C depicts plasmid extraction and isolation of cells recovered from bulk gel cultures and from liquid cultures (shown) and controls (not shown). Phenotypic evaluation was used to determine if normalization occurred in the batch gel cultures. Phenotypic readings include a red/white screen on MacConkey agar. The results obtained show that the cells edited in the bulk gel are closest compared to the cell mixture at 25% edited in alginate and at 50:50 loading ratio edited in liquid and 7% on plate.

FIG. 7 depicts a batch of alginate in a module comprising a rotating growth flask (as shown in FIG. 4E and described above) that can be used in a multi-module cell editing system (as shown in FIG. 3A and described above)Separation, growth, induction, editing and normalization workflows. In a first step, 10ml of LB medium containing alginate was added to a spinner flask already containing the transformed cells to be edited. In addition to alginate, the medium also contains antibiotics to select for properly transformed cells. Then slowly adding 1.5ml of 1MCaCL to the LB alginate cell culture in the rotating growth flask₂The medium is allowed to solidify. Cells were grown at 30 ℃ for 6 hours to establish cell colonies, at 42 ℃ for 2 hours (which induces editing), and then grown overnight at 30 ℃ to normalize edited and unedited cell colonies. After normalization, the solidified LB alginate medium was liquefied by adding 10ml of 1M sodium citrate to the solidified medium and the liquefied normalized cell culture was filtered in a filtration module for buffer exchange, cell concentration and, if necessary, to render the cells electrocompetent for another round of editing. Liquefaction dispersed all cells throughout the culture.

In view of this disclosure, it should be apparent to those of ordinary skill in the art that the described process may be recursive; that is, the cells may undergo the workflow described with respect to fig. 7, and the resulting edited culture may then undergo another round (or several to many rounds) of additional editing (e.g., recursive editing) with a different editing carrier. For example, cells from a first round of editing may be diluted and an aliquot of edited cells edited by editing carrier a may be combined with editing carrier B, an aliquot of edited cells edited by editing carrier a may be combined with editing carrier C, an aliquot of edited cells edited by editing carrier a may be combined with editing carrier D, and so on for a second round of editing. After the second round, a third round of editing can be performed on an aliquot of each double edited cell, where, for example, an aliquot of each of the AB edited, AC edited, AD edited cells is combined with additional editing carriers such as editing carriers X, Y and Z. That is, double edited cell AB can be combined with and edited by vectors X, Y and Z to produce triple edited cells ABX, ABY, and ABZ; the double edited cell AC can be combined with and edited by vectors X, Y and Z to produce triple edited cells ACX, ACY and ACZ; and double edited cellular AD can be combined with and edited by vectors X, Y and Z to produce triple edited cells ADX, ADY and ADZ, and so on. In this process, many permutations and combinations of edits can be performed, resulting in a very diverse population of cells and cell libraries. In any recursive process, "processing" (cure) "the previous engine carrier and the editing carrier (or single engine + editing carrier in a single carrier system) is advantageous. "processing" is a process in which one or more vectors used in a previous round of editing are eliminated from the transformed cells. The processing may be achieved by: for example, the vector is cleaved using a processing plasmid to render the editing vector and/or the engine vector (or single, combined vectors) nonfunctional; dilution of the vector in the cell population via cell growth (that is, the more growth cycles the cell undergoes, the fewer daughter cells will retain the editing or engine vector), or by, for example, utilizing a heat-sensitive origin of replication on the editing or engine vector (or combined vectors). The treatment conditions will depend on the mechanism used for the treatment; that is, in this example, how the plasmid cleaves the editing and/or engine plasmid is addressed. Processing in the context of the isolation, growth, editing and normalization reactions described herein is described in example 8.

Example 5: standard plating for comparison with batch culture

This protocol describes a standard plating protocol for enrichment of nucleic acid-guided nuclease edited bacterial cells by isolation, growth, editing and normalization. This protocol was used for an inducible system that utilized both nucleases and grnas to make colonies phenotypically different. From the resulting agar plates, edited cells can be selected with high (-80%) confidence. Although it is clear that this protocol can be used to enrich for edited cells, in the experiments described herein, this "standard plating protocol" or "SPP" was used to compare the efficiency of isolation, editing and normalization of batch cell cultures. The protocol described in example 7 for liquid cell culture was used for the same purpose.

Materials: in addition to standard molecular biology tools, the following would be required:

TABLE 3

The scheme is as follows: the input to this protocol was frozen electrocompetent cells and purified nucleic acid assembly products. Immediately after electroporation, the cell/DNA mixture was transferred to a culture tube containing 2.7mL of SOB medium. Prepare a 2.7mL aliquot in a 14mL culture tube prior to electroporation to allow for faster recovery of cells from the electroporation cuvette; the final volume recovered was 3 mL. All culture tubes were placed in a shaking incubator set at 250RPM and 30 ℃ for 3 hours. While the culture was restored, the necessary number of LB agar plates containing chloramphenicol and carbenicillin + 1% arabinose were removed from the refrigerator and warmed to room temperature. More than one dilution was used for each plate to have countable and isolated colonies on the plate. And (3) paving suggestion:

TABLE 4

Type of sequencing	Suggested dilution	Volume of the decking
			Singleweight
	10^-1To 10^-3	300μL
			Amplicons	Is free of	300 μ L (═ 1/10 recovery rate)

After 3 hours, the culture tube was taken out from the shaking incubator. First, the plating for amplicon sequencing was performed as per the table above. The cultures were evenly distributed on agar using plated beads. The magnetic beads were removed from the plate, which was then dried in a laminar flow hood without covering. When the plate was dried, the remaining culture was used to perform serial dilutions, with standard dilutions being 50 μ L of culture added to 450 μ L of sterile 0.8% NaCl. The plates/tubes for these dilutions (as well as the original culture) were maintained at 4 ℃ in preparation for additional dilutions needed based on colony counts. Plating for whole genome sequencing was performed according to table 4. Additional or fewer dilutions may be used based on library/competent cell knowledge. Cultures were spread evenly throughout the agar using sterile plated beads. The beads were then removed from the plate and the plate was allowed to dry uncovered in a laminar flow hood. While the plates were dry, the incubator was programmed according to the following settings: at 30 ℃ for 9 hours → at 42 ℃ for 2 hours → at 30 ℃ for 9 hours. The agar plates were placed in a pre-set incubator and after completion of the temperature cycle (-21 hours), the agar plates were removed from the incubator. If the editing induction was successful, the size difference of the colonies will be visible.

Example 6: liquid cell culture procedure for comparison with batch culture

Liquid culture procedure for control:the library of editing cassettes was transformed via electroporation into a specific strain of e.coli expressing Mad7 (nuclease) and λ Red (recombinant) proteins. In addition to editing cassette libraries, transformation of process control vectors (and editing cassette libraries) is crucial for calculating transformation efficiency and editing efficiency (sgRNA efficiency). Immediately after transformation, the electroporated cells were transferred to medium for recovery.

Table 5: related QC assay/M-tool overview

After electroporation and recovery, cells from these process control transformations were plated on LB agar plates containing the appropriate antibiotics. After overnight growth on the plates, cells were scraped and then plated on selective MacConkey phenotype agar plates for sugar editing (sugar edits) tested: xylose, galactose or lactose, or the cells were scraped and replated on LB agar to determine clonality (clonality) of individual cells from the plate. In more detail, after 3 hours of incubation (recovery), the culture tube was taken out from the shaking incubator. Upon incubation of the culture tubes, 250mL baffled shake flasks containing 25mL LB +100ug/mL carbenicillin and 25ug/mL chloramphenicol and 1% arabinose were prepared. After incubation, 250. mu.L of undiluted culture of each transformation was transferred to a 250mL shake flask prepared. The incubator was set to the following temperature settings: at 30 ℃ for 9 hours → at 42 ℃ for 2 hours → at 30 ℃ for 9 hours. This temperature protocol was used for additional recovery during the first 9 hours, followed by nuclease induction during the two hour step. Lambda induction was triggered by arabinose in the medium (recombinant engineered system). The vial was incubated/shaken at 250 RPM. After the temperature cycle was complete (-21 hours), the flask was removed from the incubator/shaker.

Serial dilutions of each culture were made with 0.8% NaCl, with standard dilutions being made by adding 50. mu.L of culture to 450. mu.L of sterile 0.8% NaCl and making 10^-5To 10^-7The solution was diluted to produce isolated colonies. 300uL of each dilution of each culture was plated on LB agar plates containing standard concentrations of chloramphenicol and carbenicillin. Arabinose was not used in the agar plates, as all editing should occur during incubation/shaking. Plates were placed in an incubator at 30 ℃ for overnight growth, colonies were formed in the incubator overnight, and colonies were picked the next day for whole genome next generation sequencing using 250 μ L of culture as the input plasmid extraction protocol.

Example 7: results

FIGS. 8A, 8B, and 8C show the results of editing rates and colony forming capabilities obtained by performing editing experiments as follows: liquid cell culture, without isolation or normalization but with inducible editing; performing batch cell gel culture by adopting an editing experiment of separation, induction type editing and normalization; solid agar plate inoculation (SPP), adopting separation, induction type editing and normalization editing experiment; solid agar plate inoculation (SPP-preferred), adopting separation, induction type editing and selection editing experiment; and solid agar plate inoculation (SPP), editing experiments with isolation and induction editing only and scraping colonies only from plates and replating. In this context, "preferentially" refers to preferentially picking small colonies, where, when cells are plated and grown into colonies, it has been determined that small colonies are likely colonies of edited cells, where large rapidly growing colonies are typically unedited cells (e.g., evacuees). See, e.g., USPN10,253,316 filed on 30/6/2018; USPN10,329,559 filed on 7/2/2019; and USPN10,323,242 filed on day 2, month 7, 2019; and USSN 16/412,175 filed on day 14, month 5, 2019; USSN 16/412,195 filed on 5/14/2019; USSN 16/454,865 filed on 6.6.2019; and USSN 16/423,289 filed on 28.5.2019, all of which are incorporated herein by reference in their entirety.

Fig. 8A shows that liquid culture produced a very low% of the observed editing rate, about 1% -2%; standard Plating Procedures (SPP) resulted in an observed editing rate of about 75% in the cell population; the batch alginate cell culture protocol produced an observed editing rate of about 50%; standard plating procedure plus selection-by-preference (SPP-preference) (e.g., manually picking only small colonies in the plated cells, where it is assumed that small colonies represent colonies of the edited cells) procedure resulted in an observed editing rate of about 95%; and the Standard Plating Procedure (SPP) but not normalized or preferred picked resulted in an observed edit rate of about 8%. Thus, it is evident that SPP + preferential selection produces the highest observed editing rate, but requires manual intervention to select colonies. Furthermore, SPP was not selected preferentially but included isolation, induced editing and normalization to produce a high (75%) observed editing rate, whereas the easily automated batch gel cell culture process produced an observed editing rate of about 50%.

Figure 8B provides the observed clonogenic capacity of the Standard Plating Procedure (SPP), standard plating procedure + selection (SPP preferred), standard plating procedure + scraping plates containing colonies that were induced to edit (but also including unedited cells), and batch procedure. The first column gives the scores of colonies examined that have more than half of the reads as the called edit reads. The second column gives the fraction of colonies with more than 90% of reads being called edit reads. If some colonies were examined to be between the cut-off values of 50% and 90% (indicating that not all cells in the selected colonies were edited), a higher score here indicates how complete the editing was. That is, when one cell hits the plate and begins to grow into a clonal colony (e.g., about 100 cells), then editing is induced, some but not all cells are edited, while unedited cells cause incomplete editing (e.g., less than 100% clonality) of such a colony. The third column provides unique edit numbers of colonies in > 50% of the clonal colonies. Note that SPP-preference provided the highest clonality and unique edit numbers, but bulk gel cell cultures provided good clonality (44/95 at > 50%), and a high proportion of clonal colonies consisted of unique edits (42/44).

Finally, FIG. 8C provides a diagram of the data in FIG. 8B. This figure indicates the extent of incomplete editing.

Example 8: recursive editing and processing

After transformation, recovery, separation, editing and lysis (as described above), the cells are then pelleted (in a centrifuge or using a filter) and resuspended in fresh medium. Cells were then grown in bulk gels at 42 ℃, which induced processing of plasmids (e.g., combined engine/editing plasmids). When the cells reached the optimal growth stage, the batch of gel was redissolved and the cells were made competent by washing and concentrating the cells in 10% glycerol using a centrifuge or filtration. These cells were then transformed with a second combined engine/editing plasmid and isolated, grown, induced edited and normalized as described above. This recursive process results in 33% of all cells having edits at both target sites. The recursive process is depicted in fig. 9.

While this invention is satisfied by embodiments in many different forms, as described in detail in connection with preferred embodiments of the invention, it is to be understood that this disclosure is to be considered as an example of the principles of the invention and is not intended to limit the invention to the specific embodiments illustrated and described herein. Many variations may be made by those skilled in the art without departing from the spirit of the invention. The scope of the invention is to be determined by the appended claims and their equivalents. The abstract and headings should not be construed as limiting the scope of the invention as they are intended to enable the appropriate authorities and the public to quickly ascertain the general nature of the invention. In the appended claims, unless the term "device" is used, the device may, according to 35 u.s.c. § 112,

neither the features nor the elements enumerated therein should be interpreted as device-plus-function limitations.

Claims

1. A method for performing enrichment of cells edited by a nucleic acid-guided nuclease, the method comprising:

providing a dilution of transformed cells in a suitable liquid growth medium comprising 0.5% -6% alginate, the dilution resulting in isolated cells, wherein the cells comprise one or more nucleic acid-guided nuclease editing components under the control of an inducible promoter;

solidifying the medium comprising alginate with a solution of divalent cations;

providing conditions to allow nucleic acid-guided editing;

allowing the cell colonies to grow to become normalized; and

liquefying the medium comprising alginate with a solution comprising a divalent cation chelator.

2. The enrichment method of claim 1, wherein the nucleic acid-guided nuclease editing module is provided to the cells on a single vector.

3. The enrichment method of claim 1, wherein the coding sequence for the nuclease is provided on an engine vector and the editing cassette comprising the sequences of the gRNA and the donor DNA is provided on an editing vector.

4. The enrichment method of claim 1, wherein the cells are bacterial cells, yeast cells, or mammalian cells.

5. The enrichment method of claim 1, wherein the percentage of alginate in the growth medium is 1% -4%.

6. The enrichment method of claim 4, wherein the percentage of alginate in the growth medium is 2% -3%.

7. The enrichment method of claim 1, wherein editing is induced and transcription of the gRNA is under the control of an inducible promoter.

8. The enrichment method according to claim 7, wherein the inducible promoter is a pL promoter.

9. The enrichment method of claim 7, wherein transcription is induced by raising the temperature of the cells to 42 ℃.

10. The enrichment method of claim 7, wherein the isolated cells are grown at 30 ℃ for 6-12 hours to achieve 2-50 doublings before inducing transcription of the grnas.

11. An automated multi-module cell processing instrument for performing automated enrichment of cells edited by nucleic acid-guided nuclease editing, the automated multi-module cell processing instrument comprising:

a cell receptacle configured to receive a cell;

a nucleic acid receptacle configured to receive an editing carrier;

a reagent cartridge configured to contain a reagent;

a growing module;

a filtration module;

a conversion module;

a dilution module;

a separation module comprising a temperature controlled vessel configured to perform the solidifying, permitting, providing, permitting, and liquefying steps of claim 1; and

a liquid handling system configured to transfer liquid from the cell receptacle to the growth module, transfer liquid from the growth module to the filtration module, transfer liquid from the filtration module to the transformation module, transfer liquid from the nucleic acid receptacle to the transformation module, transfer liquid from the transformation module to the dilution module, and transfer liquid from the dilution module to the separation module without user intervention.

12. The instrument for performing automated enrichment of cells according to claim 11, wherein the gRNA is under the transcriptional control of an inducible promoter, and the inducible promoter is a temperature-inducible promoter.

13. The instrument for performing automated enrichment of cells according to claim 11, wherein the reagent cartridge contains a culture medium, a solution of divalent cations and a solution of a chelator of divalent cations.

14. According to claim 13The automated multi-module cell processing apparatus of (1), wherein the divalent cation is CaCl₂And the chelating agent is Na₃C₆H₅O₇。

15. The automated multi-module cell processing instrument of claim 11, wherein the transformation module comprises a flow-through electroporation device.

16. The automated multi-module cell processing instrument of claim 11, wherein the filtration module comprises a tangential flow filtration device.

17. A method for performing enrichment of cells edited by a nucleic acid-guided nuclease, the method comprising:

providing a dilution of transformed cells in a suitable liquid growth medium comprising a hydrogel in a container, the dilution resulting in isolated cells, wherein the cells comprise a nucleic acid-guided nuclease editing component under the control of an inducible promoter;

solidifying the hydrogel-containing medium with a solution of divalent cations;

allowing the isolated cells to grow for 2 to 50 doublings to establish cell colonies;

inducing transcription of the nucleic acid-guided nuclease editing component;

growing the cells for a period of time sufficient to allow the cell colonies to become normalized; and

liquefying the hydrogel-containing medium with a chelating agent for the divalent cation.