NZ716604B2 - Multiplex rna-guided genome engineering - Google Patents
Multiplex rna-guided genome engineering Download PDFInfo
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Abstract
Methods of multiplex genome engineering in cells using Cas9 is provided which includes a cycle of steps of introducing into the cell a first foreign nucleic acid encoding one or more RNAs complementary to the target DNA and which guide the enzyme to the target DNA, wherein the one or more RNAs and the enzyme are members of a co-localization complex for the target DNA, and introducing into the cell a second foreign nucleic acid encoding one or more donor nucleic acid sequences, and wherein the cycle is repeated a desired number of times to multiplex DNA engineering in cells. he enzyme are members of a co-localization complex for the target DNA, and introducing into the cell a second foreign nucleic acid encoding one or more donor nucleic acid sequences, and wherein the cycle is repeated a desired number of times to multiplex DNA engineering in cells.
Description
MULTIPLEX RNA-GUIDED GENOME ENGINEERING
RELATED APPLICATION DATA
This application claims priority to U.S. Provisional Patent Application No. 61/844,168
filed on July 9, 2013 and is hereby incorporated herein by reference in its entirety for all purposes.
STATEMENT OF GOVERNMENT INTERESTS
This invention was made with government support under DE-FG02-02ER63445 from the
Department of Energy, NSF-SynBERC from the National Science Foundation and SA5283-11210
from the National Science Foundation. The government has certain rights in the invention.
BACKGROUND
Bacterial and archaeal CRISPR-Cas systems rely on short guide RNAs in complex with
Cas proteins to direct degradation of complementary sequences present within invading foreign
nucleic acid. See Deltcheva, E. et al. CRISPR RNA maturation by trans-encoded small RNA and
host factor RNase III. Nature 471, 602-607 (2011); Gasiunas, G., Barrangou, R., Horvath, P. &
Siksnys, V. Cas9-crRNA ribonucleoprotein complex mediates specific DNA cleavage for adaptive
immunity in bacteria. Proceedings of the National Academy of Sciences of the United States of
America 109, E2579-2586 (2012); Jinek, M. et al. A programmable dual-RNA-guided DNA
endonuclease in adaptive bacterial immunity. Science 337, 816-821 (2012); Sapranauskas, R. et al.
The Streptococcus thermophilus CRISPR/Cas system provides immunity in Escherichia coli.
Nucleic acids research 39, 9275-9282 (2011); and Bhaya, D., Davison, M. & Barrangou, R.
CRISPR-Cas systems in bacteria and archaea: versatile small RNAs for adaptive defense and
regulation. Annual review of genetics 45, 273-297 (2011). A recent in vitro reconstitution of the S.
pyogenes type II CRISPR system demonstrated that crRNA (“CRISPR RNA”) fused to a normally
trans-encoded tracrRNA (“trans-activating CRISPR RNA”) is sufficient to direct Cas9 protein to
sequence-specifically cleave target DNA sequences matching the crRNA. Expressing a gRNA
homologous to a target site results in Cas9 recruitment and degradation of the target DNA. See H.
Deveau et al., Phage response to CRISPR-encoded resistance in Streptococcus thermophilus.
Journal of Bacteriology 190, 1390 (Feb, 2008).
SUMMARY
The present invention provides a method of making multiple alterations to target DNA in a
Cas9 enzyme expressing cell wherein the Cas9 enzyme forms a co-localization complex with guide
RNA complementary to the target DNA and that cleaves the target DNA in a site specific manner
comprising
(a) introducing into the Cas9 enzyme expressing cell a plurality of guide RNAs and a
plurality of donor nucleic acid sequences, wherein each of the plurality of guide RNAs and the
Cas9 enzyme are members of a co-localization complex for the target DNA,
wherein the Cas9 enzyme cleaves the target DNA and at least one of the plurality of donor
nucleic acid sequences is inserted into the target DNA to produce altered DNA in the cell, and
(b) repeating step (a) to produce multiple alterations to the DNA in the cell.
Aspects of the present disclosure are directed to the multiplex modification of DNA in a
cell using one or more guide RNAs (ribonucleic acids) to direct an enzyme having nuclease
activity expressed by the cell, such as a DNA binding protein having nuclease activity, to a target
location on the DNA (deoxyribonucleic acid) wherein the enzyme cuts the DNA and an exogenous
donor nucleic acid is inserted into the DNA, such as by homologous recombination. Aspects of the
present disclosure include cycling or repeating steps of DNA modification on a cell to create a cell
having multiple modifications of DNA within the cell. Modifications may include insertion of
exogenous donor nucleic acids.
Multiple exogenous nucleic acid insertions can be accomplished by a single step of
introducing into a cell, which expresses the enzyme, nucleic acids encoding a plurality of RNAs
and a plurality of exogenous donor nucleic acids, such as by co-transformation, wherein the RNAs
are expressed and wherein each RNA in the plurality guides the enzyme to a particular site of the
DNA, the enzyme cuts the DNA and one of the plurality of exogenous nucleic acids is inserted into
the DNA at the cut site. According to this aspect, many alterations or modification of the DNA in
the cell are created in a single cycle.
Multiple exogenous nucleic acid insertions can be accomplished in a cell by repeated steps
or cycles of introducing into a cell, which expresses the enzyme, one or more nucleic acids
encoding one or more RNAs or a plurality of RNAs and one or more exogenous nucleic acids or a
plurality of exogenous nucleic acids wherein the RNA is expressed and guides the enzyme to a
particular site of the DNA, the enzyme cuts the DNA and the exogenous nucleic acid is inserted
into the DNA at the cut site, so as to result in a cell having multiple alterations or insertions of
exogenous DNA into the DNA within the cell. According to one aspect, the cell expressing the
enzyme can be a cell which expresses the enzyme naturally or a cell which has been genetically
altered to express the enzyme such as by introducing into the cell a nucleic acid encoding the
enzyme and which can be expressed by the cell. In this manner, aspects of the present disclosure
include cycling the steps of introducing RNA into a cell which expresses the enzyme, introducing
exogenous donor nucleic acid into the cell, expressing the RNA, forming a co-localization complex
of the RNA, the enzyme and the DNA, enzymatic cutting of the DNA by the enzyme, and insertion
of the donor nucleic acid into the DNA. Cycling or repeating of the above steps results in
multiplexed genetic modification of a cell at multiple loci, i.e., a cell having multiple genetic
modifications.
According to certain aspects, a method of increasing rate of homologous recombination is
described by the cycling method described above. In one embodiment, genomic Cas9 directed
DNA cutting stimulates exogenous DNA via dramatically increasing the rate of homologous
recombination. According to a certain additional aspect, the exogenous donor nucleic acid includes
homology sequences or arms flanking the cut site. According to a certain additional aspect, the
exogenous donor nucleic acid includes a sequence to remove the cut sequence. According to a
certain additional aspect, the exogenous donor nucleic acid includes homology sequences or arms
flanking the cut site and a sequence to remove the cut site. In this manner, Cas9 can be used as a
negative selection against cells that do not incorporate exogenous donor DNA. Accordingly, a
negative selection method is described for identifying cells having high recombination frequency.
According to certain aspects, DNA binding proteins or enzymes within the scope of the
present disclosure include a protein that forms a complex with the guide RNA and with the guide
RNA guiding the complex to a double stranded DNA sequence wherein the complex binds to the
DNA sequence. According to one aspect, the enzyme can be an RNA guided DNA binding protein,
such as an RNA guided DNA binding protein of a Type II CRISPR System that binds to the DNA
and is guided by RNA. According to one aspect, the RNA guided DNA binding protein is a Cas9
protein.
This aspect of the present disclosure may be referred to as co-localization of the RNA and
DNA binding protein to or with the double stranded DNA. In this manner, a DNA binding protein-
guide RNA complex may be used to cut multiple sites of the double stranded DNA so as to create a
cell with multiple genetic modifications, such as multiple insertions of exogenous donor DNA.
According to certain aspects, a method of making multiple alterations to target DNA in a
cell expressing an enzyme that forms a co-localization complex with RNA complementary to the
target DNA and that cleaves the target DNA in a site specific manner is described including (a)
introducing into the cell a first foreign nucleic acid encoding one or more RNAs complementary to
the target DNA and which guide the enzyme to the target DNA, wherein the one or more RNAs
and the enzyme are members of a co-localization complex for the target DNA, introducing into the
cell a second foreign nucleic acid encoding one or more donor nucleic acid sequences, wherein the
one or more RNAs and the one or more donor nucleic acid sequences are expressed, wherein the
one or more RNAs and the enzyme co-localize to the target DNA, the enzyme cleaves the target
DNA and the donor nucleic acid is inserted into the target DNA to produce altered DNA in the cell,
and repeating step (a) multiple times to produce multiple alterations to the DNA in the cell.
According to one aspect, the cell is a eukaryotic cell. According to one aspect, the cell is a
yeast cell, a plant cell or an animal cell. According to one aspect, the cell is a mammalian cell.
According to one aspect, the RNA is between about 10 to about 500 nucleotides.
According to one aspect, the RNA is between about 20 to about 100 nucleotides.
According to one aspect, the one or more RNAs is a guide RNA. According to one aspect,
the one or more RNAs is a tracrRNA-crRNA fusion.
According to one aspect, the DNA is genomic DNA, mitochondrial DNA, viral DNA, or
exogenous DNA.
Further features and advantages of certain embodiments of the present invention will
become more fully apparent in the following description of embodiments and drawings thereof,
and from the claims.
BRIEF DESCRIPTION OF THE DRAWINGS
The foregoing and other features and advantages of the present embodiments will be more
fully understood from the following detailed description of illustrative embodiments taken in
conjunction with the accompanying drawings in which:
is a schematic of RNA guided genome cleavage via Cas9.
is a schematic depicting multiplexed genome engineering in yeast using Cas9.
is a schematic depicting allele replacement using oligonucleotides targeting four
loci crucial in thermotolerance in yeast.
is a graph depicting number of modifications per cell after one cycle and after two
cycles.
is a table of strains having mutations. shows thermotolerance to heat
shock for the various strains.
depicts graphical data for transformation frequency. depicts graphical
data for individual recombination frequency. depicts graphical data for co-recombination
frequency at can1 and KanMX locus.
depicts graphical data for multiplex linear cassette incorporation for two loci.
depicts graphical data for fold change in double time at 30°C. depicts
graphical data for fold change in double time at 37°C. depicts graphical data for fold
change in double time at 42°C with cells inoculated from the late stationary phase culture. depicts graphical data for fold change in double time at 42°C with cells inoculated from the late
log phase culture.
DETAILED DESCRIPTION
Embodiments of the present disclosure are based on the repeated use of exogenous DNA,
nuclease enzymes such as DNA binding proteins and guide RNAs to co-localize to DNA and digest
or cut the DNA with insertion of the exogenous DNA, such as by homologous recombination.
Such DNA binding proteins are readily known to those of skill in the art to bind to DNA for
various purposes. Such DNA binding proteins may be naturally occurring. DNA binding proteins
included within the scope of the present disclosure include those which may be guided by RNA,
referred to herein as guide RNA. According to this aspect, the guide RNA and the RNA guided
DNA binding protein form a co-localization complex at the DNA. Such DNA binding proteins
having nuclease activity are known to those of skill in the art, and include naturally occurring DNA
binding proteins having nuclease activity, such as Cas9 proteins present, for example, in Type II
CRISPR systems. Such Cas9 proteins and Type II CRISPR systems are well documented in the art.
See Makarova et al., Nature Reviews, Microbiology, Vol. 9, June 2011, pp. 467-477 including all
supplementary information hereby incorporated by reference in its entirety.
Exemplary DNA binding proteins having nuclease activity function to nick or cut double
stranded DNA. Such nuclease activity may result from the DNA binding protein having one or
more polypeptide sequences exhibiting nuclease activity. Such exemplary DNA binding proteins
may have two separate nuclease domains with each domain responsible for cutting or nicking a
particular strand of the double stranded DNA. Exemplary polypeptide sequences having nuclease
activity known to those of skill in the art include the McrA-HNH nuclease related domain and the
RuvC-like nuclease domain. Accordingly, exemplary DNA binding proteins are those that in
nature contain one or more of the McrA-HNH nuclease related domain and the RuvC-like nuclease
domain.
An exemplary DNA binding protein is an RNA guided DNA binding protein of a Type II
CRISPR System. An exemplary DNA binding protein is a Cas9 protein.
In S. pyogenes, Cas9 generates a blunt-ended double-stranded break 3bp upstream of the
protospacer-adjacent motif (PAM) via a process mediated by two catalytic domains in the protein:
an HNH domain that cleaves the complementary strand of the DNA and a RuvC-like domain that
cleaves the non-complementary strand. See Jinke et al., Science 337, 816-821 (2012) hereby
incorporated by reference in its entirety. Cas9 proteins are known to exist in many Type II
CRISPR systems including the following as identified in the supplementary information to
Makarova et al., Nature Reviews, Microbiology, Vol. 9, June 2011, pp. 467-477: Methanococcus
maripaludis C7; Corynebacterium diphtheriae; Corynebacterium efficiens YS-314;
Corynebacterium glutamicum ATCC 13032 Kitasato; Corynebacterium glutamicum ATCC 13032
Bielefeld; Corynebacterium glutamicum R; Corynebacterium kroppenstedtii DSM 44385;
Mycobacterium abscessus ATCC 19977; Nocardia farcinica IFM10152; Rhodococcus erythropolis
PR4; Rhodococcus jostii RHA1; Rhodococcus opacus B4 uid36573; Acidothermus cellulolyticus
11B; Arthrobacter chlorophenolicus A6; Kribbella flavida DSM 17836 uid43465;
Thermomonospora curvata DSM 43183; Bifidobacterium dentium Bd1; Bifidobacterium longum
DJO10A; Slackia heliotrinireducens DSM 20476; Persephonella marina EX H1; Bacteroides
fragilis NCTC 9434; Capnocytophaga ochracea DSM 7271; Flavobacterium psychrophilum JIP02
86; Akkermansia muciniphila ATCC BAA 835; Roseiflexus castenholzii DSM 13941; Roseiflexus
RS1; Synechocystis PCC6803; Elusimicrobium minutum Pei191; uncultured Termite group 1
bacterium phylotype Rs D17; Fibrobacter succinogenes S85; Bacillus cereus ATCC 10987; Listeria
innocua;Lactobacillus casei; Lactobacillus rhamnosus GG; Lactobacillus salivarius UCC118;
Streptococcus agalactiae A909; Streptococcus agalactiae NEM316; Streptococcus agalactiae 2603;
Streptococcus dysgalactiae equisimilis GGS 124; Streptococcus equi zooepidemicus MGCS10565;
Streptococcus gallolyticus UCN34 uid46061; Streptococcus gordonii Challis subst CH1;
Streptococcus mutans NN2025 uid46353; Streptococcus mutans; Streptococcus pyogenes M1 GAS;
Streptococcus pyogenes MGAS5005; Streptococcus pyogenes MGAS2096; Streptococcus
pyogenes MGAS9429; Streptococcus pyogenes MGAS10270; Streptococcus pyogenes
MGAS6180; Streptococcus pyogenes MGAS315; Streptococcus pyogenes SSI-1; Streptococcus
pyogenes MGAS10750; Streptococcus pyogenes NZ131; Streptococcus thermophiles CNRZ1066;
Streptococcus thermophiles LMD-9; Streptococcus thermophiles LMG 18311; Clostridium
botulinum A3 Loch Maree; Clostridium botulinum B Eklund 17B; Clostridium botulinum Ba4 657;
Clostridium botulinum F Langeland; Clostridium cellulolyticum H10; Finegoldia magna ATCC
29328; Eubacterium rectale ATCC 33656; Mycoplasma gallisepticum; Mycoplasma mobile 163K;
Mycoplasma penetrans; Mycoplasma synoviae 53; Streptobacillus moniliformis DSM 12112;
Bradyrhizobium BTAi1; Nitrobacter hamburgensis X14; Rhodopseudomonas palustris BisB18;
Rhodopseudomonas palustris BisB5; Parvibaculum lavamentivorans DS-1; Dinoroseobacter shibae
DFL 12; Gluconacetobacter diazotrophicus Pal 5 FAPERJ; Gluconacetobacter diazotrophicus Pal 5
JGI; Azospirillum B510 uid46085; Rhodospirillum rubrum ATCC 11170; Diaphorobacter TPSY
uid29975; Verminephrobacter eiseniae EF01-2; Neisseria meningitides 053442; Neisseria
meningitides alpha14; Neisseria meningitides Z2491; Desulfovibrio salexigens DSM 2638;
Campylobacter jejuni doylei 269 97; Campylobacter jejuni 81116; Campylobacter jejuni;
Campylobacter lari RM2100; Helicobacter hepaticus; Wolinella succinogenes; Tolumonas auensis
DSM 9187; Pseudoalteromonas atlantica T6c; Shewanella pealeana ATCC 700345; Legionella
pneumophila Paris; Actinobacillus succinogenes 130Z; Pasteurella multocida; Francisella
tularensis novicida U112; Francisella tularensis holarctica; Francisella tularensis FSC 198;
Francisella tularensis tularensis; Francisella tularensis WY96-3418; and Treponema denticola
ATCC 35405. Accordingly, aspects of the present disclosure are directed to a Cas9 protein present
in a Type II CRISPR system.
The Cas9 protein may be referred by one of skill in the art in the literature as Csn1. The S.
pyogenes Cas9 protein sequence that is the subject of experiments described herein is shown below.
See Deltcheva et al., Nature 471, 602-607 (2011) hereby incorporated by reference in its entirety.
MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAE
ATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFG
NIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSD
VDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGN
LIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAI
LLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYA
GYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELH
AILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEE
VVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFL
SGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKI
IKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWG
RLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSL
HEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRER
MKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDH
IVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNL
TKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKS
KLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVR
MIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDF
ATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVA
YSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPK
YSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVE
QHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGA
PAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD-
According to one aspect, the RNA guided DNA binding protein includes homologs and
orthologs of Cas9 which retain the ability of the protein to bind to the DNA, be guided by the RNA
and cut the DNA. According to one aspect, the Cas9 protein includes the sequence as set forth for
naturally occurring Cas9 from S. pyogenes and protein sequences having at least 30%, 40%, 50%,
60%, 70%, 80%, 90%, 95%, 98% or 99% homology thereto and being a DNA binding protein,
such as an RNA guided DNA binding protein.
According to one aspect, an engineered Cas9-gRNA system is described which enables
RNA-guided genome cutting in a site specific manner, if desired, and modification of the genome
by insertion of exogenous donor nucleic acids. The guide RNAs are complementary to target sites
or target loci on the DNA. The guide RNAs can be crRNA-tracrRNA chimeras. The Cas9 binds at
or near target genomic DNA. The one or more guide RNAs bind at or near target genomic DNA.
The Cas9 cuts the target genomic DNA and exogenous donor DNA is inserted into the DNA at the
cut site.
Accordingly, methods are directed to the use of a guide RNA with a Cas9 protein and an
exogenous donor nucleic acid to multiplex insertions of exogenous donor nucleic acids into DNA
within a cell expressing Cas9 by cycling the insertion of nucleic acid encoding the RNA and
exogenous donor nucleic acid, expressing the RNA, colocalizing the RNA, Cas9 and DNA in a
manner to cut the DNA, and insertion of the exogenous donor nucleic acid. The method steps can
be cycled in any desired number to result in any desired number of DNA modifications. Methods
of the present disclosure are accordingly directed to editing target genes using the Cas9 proteins
and guide RNAs described herein to provide multiplex genetic and epigenetic engineering of cells.
Further aspects of the present disclosure are directed to the use of DNA binding proteins or
systems in general for the multiplex insertion of exogenous donor nucleic acids into the DNA, such
as genomic DNA, of a cell, such as a human cell. One of skill in the art will readily identify
exemplary DNA binding systems based on the present disclosure.
Cells according to the present disclosure include any cell into which foreign nucleic acids
can be introduced and expressed as described herein. It is to be understood that the basic concepts
of the present disclosure described herein are not limited by cell type. Cells according to the
present disclosure include eukaryotic cells, prokaryotic cells, animal cells, plant cells, fungal cells,
archael cells, eubacterial cells and the like. Cells include eukaryotic cells such as yeast cells, plant
cells, and animal cells. Particular cells include mammalian cells, such as human cells. Further,
cells include any in which it would be beneficial or desirable to modify DNA.
Target nucleic acids include any nucleic acid sequence to which a co-localization complex
as described herein can be useful to nick or cut. Target nucleic acids include genes. For purposes
of the present disclosure, DNA, such as double stranded DNA, can include the target nucleic acid
and a co-localization complex can bind to or otherwise co-localize with the DNA at or adjacent or
near the target nucleic acid and in a manner in which the co-localization complex may have a
desired effect on the target nucleic acid. Such target nucleic acids can include endogenous (or
naturally occurring) nucleic acids and exogenous (or foreign) nucleic acids. One of skill based on
the present disclosure will readily be able to identify or design guide RNAs and Cas9 proteins
which co-localize to a DNA including a target nucleic acid. One of skill will further be able to
identify transcriptional regulator proteins or domains which likewise co-localize to a DNA
including a target nucleic acid. DNA includes genomic DNA, mitochondrial DNA, viral DNA or
exogenous DNA. According to one aspect, materials and methods useful in the practice of the
present disclosure include those described in Di Carlo, et al., Nucleic Acids Research, 2013, vol. 41,
No. 7 4336-4343 hereby incorporated by reference in its entirety for all purposes including
exemplary strains and media, plasmid construction, transformation of plasmids, electroporation of
transcient gRNA cassette and donor nucleic acids, transformation of gRNA plasmid with donor
DNA into Cas9-expressing cells, galactose induction of Cas9, identification of CRISPR-Cas targets
in yeast genome, etc. Additional references including information, materials and methods useful to
one of skill in carrying out the invention are provided in Mali,P., Yang,L., Esvelt,K.M., Aach,J.,
Guell,M., DiCarlo,J.E., Norville,J.E. and Church,G.M. (2013) RNA-Guided human genome
engineering via Cas9. Science, 10.1126fscience.1232033; Storici,F., Durham,C.L., Gordenin,D.A.
and Resnick,M.A. (2003) Chromosomal site-specific double-strand breaks are efficiently targeted
for repair by oligonucleotides in yeast. PNAS, 100, 14994-14999 and Jinek,M., Chylinski,K.,
Fonfara,l., Hauer,M., Doudna,J.A. and Charpentier,E. (2012) A programmable dual-RNA-Guided
DNA endonuclease in adaptive bacterial immunity. Science, 337, 816-821 each of which are
hereby incorporated by reference in their entireties for all purposes.
Foreign nucleic acids (i.e. those which are not part of a cell’s natural nucleic acid
composition) may be introduced into a cell using any method known to those skilled in the art for
such introduction. Such methods include transfection, transduction, viral transduction,
microinjection, lipofection, nucleofection, nanoparticle bombardment, transformation, conjugation
and the like. One of skill in the art will readily understand and adapt such methods using readily
identifiable literature sources.
The following examples are set forth as being representative of the present disclosure.
These examples are not to be construed as limiting the scope of the present disclosure as these and
other equivalent embodiments will be apparent in view of the present disclosure, figures and
accompanying claims.
EXAMPLE I
General Process for Multiplexed Gene Editing Using CRISPR-Cas9 in Yeast
Cas9 from the CRISPR immune system of Streptococcous pyogenes is used to stimulate
homologous recombination and to select against cells that do not recombine transformed DNA in
Saccharaomyces cerevisiae. A general method of RNA-guided DNA cleavage using Cas9 is
presented in A co-localization complex is formed between Cas9, a guide RNA and the
target DNA. A double stranded break is created in the target DNA by Cas9. Donor DNA is then
inserted into the DNA by homologous recombination. The donor DNA includes flanking
sequences on either side of the cut site and a sequence that removes the Cas9 cleavage site. The
result is integration of the donor DNA into the DNA, which may be genomic DNA.
A general method for high frequency donor DNA recombination using multiplexed DNA
engineering in yeast using Cas9 is described as follows and with reference to Cells not
having a naturally present Cas9 RNA guided endonuclease may be transformed with DNA to allow
the cell to express a Cas9 RNA guided endonuclease. Cells are grown that express a Cas9 RNA-
guided endonuclease. A plasmid including one or more nucleic acids encoding one or more guide
RNAs and a selection marker known to those of skill in the art is created for introduction into a cell
and expression of the one or more guide RNAs. As shown in a pool of plasmids is shown
each with a nucleic acid encoding a guide RNA to be used for a different gene to be inserted into
the genomic DNA of the cell, i.e. gene A, gene B, gene C, gene D and gene E. A pool of donor
DNA is also described including double stranded donor DNA for gene A, gene B, gene C, gene D
and gene E.
Cells are washed and conditioned with lithium acetate. Cells may be further washed and
mixed with a pool of exogenous donor nucleic acids, such as double stranded oligonucleotides, for
example a DNA cassette, and the plasmids including the nucleic acids encoding the guide RNAs.
As shown in the cells are transformed with the exogenous donor nucleic acids and the
plasmids using PEG 3350 and lithium acetate.
As shown in cells are selected for the one or more guide RNAs using the selection
marker. The selected cells express the one or more guide RNAs. One or more co-localization
complexes are formed between a guide RNA, a Cas9 RNA-guided endonuclease and DNA in the
cell. The endonuclease cuts the DNA and a donor nucleic acid is inserted into the cell by
recombination, such as homologous recombination. The cells are then cured for the plasmid and
the cells are then optionally subjected to one or additional cycles of the above steps. A plurality of
cycles may be performed. A cell subjected to a plurality of cycles exhibits high recombination
frequency. Alternatively, the cells are deselected for plasmid maintenance or otherwise the cells
are placed in media to select against cells with the plasmid. The process is then repeated beginning
with the cell growth step. Accordingly, methods include cycling of cells already modified by a
prior cycle or selecting cells from a prior cycle which have not been modified and further cycling
the unmodified cells to effect modification of DNA as described herein.
EXAMPLE II
Detailed Cycling Protocol
Cells are grown (uracil auxotrophs, with constitutive Cas9 expression) to an optical density
of 0.8 to 1.0 in 5 ml SC yeast media or of SC + FOA (100 µg/ml). The cells are spun at 2250 x g
for 3 minutes, and are washed once with 10 ml water. the cells are sun and resuspended in 1 ml of
100 mM lithium acetate. The cells are pelleted and resuspended in 500 µl 100 mM lithium acetate.
A transformation mixture is created by adding in the following order, 50 µl of cells; DNA mixture
including 1 nmol of double stranded oligonucleotide pool, 5 µg each of guide RNA (p426 vector,
with uracil marker) and fill to 70 µl with water to achieve desired final volume; 240 µl 50% PEG
3350; and 36 µl 1 M lithium acetate. The mixture is incubated at 30°C for 30 minutes. The
mixture is then vortexed and the cells are heat shocked by incubating the mixture at 42°C for 20
minutes. The cells are then pelleted and the supernatant is removed. The cells are inoculated with
ml SC-uracil to select for uracil gene containing gRNA plasmid. The cells are allowed to recover
for 2 days. After two days, 100 µl of the cell culture is inoculated into 5 ml fresh SC and allowed
to grow for 12 hours to deselect for plasmid maintenance. 100 µl of the SC culture cells are then
inoculated into 5 ml of SC + FOA (100µg/mL) media to select against cells with the plasmid. This
completes one cycle of the process. The process is repeated for any number of desired cycles. The
total process may include 1 cycle, 2 cycles, 3 cycles, 4 cycles, 5 cycles, 6 cycles, 7 cycles, 8 cycles,
9 cycles, 10 cycles, 15 cycles, 20, cycles, 25 cycles, etc.
EXAMPLE III
Thermotolerance to Heat Shock in Select Mutants
Using the methods described herein, thermotolerance to heat shock in select mutants has
been shown. Genes that have been shown to increase thermotolerance in yeast upon knockout or
point mutation were targeted by the guide RNA-Cas9 system described herein. Four genes were
selected for mutation: UBC1, SCH9, TFS1, and RAS2. SCH9 is a protein kinase that regulates
osmostress, nutrient and environmental stress genes. TFS1 inhibits carboxypeptidase Y and Ira2p,
inhibits Ras GAP activity and responds to DNA replicative stress. RAS2 is a GTP binding protein
that regulates nitrogen starvation and is involved in stress response pathways. For each of SCH9,
TFS1 and RAS2, a donor DNA was created which is an allele containing a serine to alanine
mutation in the coding region. UBC1-E2 is a ubiquitin-conjugating enzyme. A donor DNA
including a point mutation that removes a phosphorylation site resulting in thermotolerance was
created.
Using the methods described herein the genes were targeted using guide RNA designed to
direct Cas9 cleavage to the loci of the genes along with double stranded oligonucleotide to impart
the changes. As shown in allele replacement was achieved using oligonucleotides targeting
four loci responsible for thermotolerance in yeast. According to the schematic, four plasmids each
incorporating a nucleic acid encoding a guide RNA for one of the genes were created: UBC1
gRNS plasmid, TFS1 gRNA plasmid, SCH9 gRNA plasmid and RAS2 gRNA plasmid. Each
plasmid had a corresponding double stranded donor oligonucleotide: ubc1 (S97A) double stranded
oligonucleotide, tfs1 (tag) double stranded oligonucleotide, sch9 (tag) double stranded
oligonucleotide and ras (tag) double stranded oligonucleotide. The plasmids and the corresponding
double stranded donor oligonucleotides were co-transformed into yeast as a pool. Two cycles were
performed and the number of modifications per cell as a function of percentage of cells in the cell
population is shown at A significant number of cells included one and two modifications
after cycle 2. One triple mutant was able to be isolated (data not shown.)
is a table of the strains resulting from the methods described herein showing
strains transformed with one donor oligonucleotide, strains transformed with two donor
oligonucleotides and a strain transformed with three donor oligonucleotides. shows the
effect of incubation at 42°C for three hours compared to no incubation and s slight decrease in wild
type cell number. also shows the effect of incubation at 55°C for two hours compared to
no incubation. The mutants most tolerant to heat shock at 55°C were sch9, sch9 tfs1 and tfs1
ubc1(s97a).
in general provides graphical information on the optimization of multiplex
oligonucleotide incorporation for two loci. depicts the transformation frequency versus
the amount of each plasmid transformed (µg). depicts the individual recombination
frequency versus the amount of each plasmid transformed (µg). depicts the co-
recombination frequency at can1 and KanMX locus versus the amount of each plasmid
transformed (µg).
in general provides graphical information on the multiplex linear cassette
incorporation for two loci. The graph charts for the first left most bar, transformation frequency for
p426 gRNA ADE2 + HygR Cassette; for the next bar, transformation frequency for p426 gRNA
CAN1 + G418R cassette, for the next three bars, transformation frequency for p426 gRNA +
ADE2 p426 gRNA CAN1 + HygR Cassette + G418R cassette.
in general is a growth rate analysis showing double time in exponential growth in
elevated temperatures for select mutants. graphs the fold change in double time at 30°C
for the wild type and the mutants identified. graphs the fold change in double time at
37°C for the wild type and the mutants identified. graphs the fold change in double time
at 42°C for the wild type and the mutants identified as inoculated from the late stationary phase
culture. graphs the fold change in double time at 42°C for the wild type and the mutants
identified as inoculated from the late log phase culture. The graphical data shows a lower doubling
time at 37°C for sch9 tfs1 and tfs1 ubc1(S97A). The graphical data shows lower doubling time at
42°C for ras2 tfs1, sch9 ubc1(S97A), tfs1 ubc1(S97A) and ras2 tfs1 ubc1(S97A).
The term “comprising” as used in this specification and claims means “consisting at least
in part of”. When interpreting statements in this specification, and claims which include the term
“comprising”, it is to be understood that other features that are additional to the features prefaced
by this term in each statement or claim may also be present. Related terms such as “comprise” and
“comprised” are to be interpreted in similar manner.
In this specification where reference has been made to patent specifications, other external
documents, or other sources of information, this is generally for the purpose of providing a context
for discussing the features of the invention. Unless specifically stated otherwise, reference to such
external documents is not to be construed as an admission that such documents, or such sources of
information, in any jurisdiction, are prior art, or form part of the common general knowledge in the
art.
In the description in this specification reference may be made to subject matter that is not
within the scope of the claims of the current application. That subject matter should be readily
identifiable by a person skilled in the art and may assist in putting into practice the invention as
defined in the claims of this application.
Claims (14)
1. A method of making multiple alterations to target DNA in a Cas9 enzyme expressing cell wherein the Cas9 enzyme forms a co-localization complex with guide RNA complementary to the target DNA and that cleaves the target DNA in a site specific manner 5 comprising (a) introducing into the Cas9 enzyme expressing cell a plurality of guide RNAs and a plurality of donor nucleic acid sequences, wherein each of the plurality of guide RNAs and the Cas9 enzyme are members of a co-localization complex for the target DNA, wherein the Cas9 enzyme cleaves the target DNA and at least one of the plurality of donor 10 nucleic acid sequences is inserted into the target DNA to produce altered DNA in the cell, and (b) repeating step (a) to produce multiple alterations to the DNA in the cell.
2. The method of claim 1 wherein the cell is a eukaryotic cell.
3. The method of claim 1 wherein the cell is a yeast cell, a plant cell or an animal cell.
4. The method of claim 1 wherein each of the plurality of guide RNAs is between 15 about 10 to about 500 nucleotides.
5. The method of claim 1 wherein each of the plurality of guide RNAs is between about 20 to about 100 nucleotides.
6. The method of claim 1 wherein each of the plurality of guide RNAs is a tracrRNA- crRNA fusion. 20
7. The method of claim 1 wherein the DNA is genomic DNA, mitochondrial DNA, viral DNA, or exogenous DNA.
8. The method of claim 1 wherein the donor nucleic acid sequence is inserted by recombination.
9. The method of claim 1 wherein the donor nucleic acid sequence is inserted by 25 homologous recombination.
10. The method of claim 1 wherein the plurality of guide RNAs and the plurality of donor nucleic acid sequences are present on one or more plasmids.
11. The method of claim 1 wherein each of the plurality of donor nucleic acids includes homology sequences or arms flanking the site of cleavage. 30
12. The method of claim 1 wherein each of the plurality of donor nucleic acids includes a sequence to remove the site of cleavage.
13. The method of claim 1 wherein step (a) is repeated multiple times.
14. A method as claimed in any one of claims 1-13 substantially as herein described and with reference to any example thereof.
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