WO2022163770A1 - Procédé d'évaluation d'outil d'édition de génome - Google Patents

Procédé d'évaluation d'outil d'édition de génome Download PDF

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WO2022163770A1
WO2022163770A1 PCT/JP2022/003152 JP2022003152W WO2022163770A1 WO 2022163770 A1 WO2022163770 A1 WO 2022163770A1 JP 2022003152 W JP2022003152 W JP 2022003152W WO 2022163770 A1 WO2022163770 A1 WO 2022163770A1
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genome editing
binding
editing tool
genome
dcas9
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康志 岡田
一穂 池田
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国立研究開発法人理化学研究所
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
    • C12Q1/44—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving esterase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/483—Physical analysis of biological material

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  • the present invention relates to methods for evaluating genome editing tools.
  • Genome editing tools represented by CRISPR/Cas9 are tools that specifically recognize specific target sequences in the genome and perform editing such as cutting. However, it is known to have off-target activity that binds to and edits other than the designed target sequence. Especially in clinical application to humans, it is necessary to quantitatively evaluate off-target activity as safety evaluation. Especially for CRISPR/Cas9, this high off-target activity is an obstacle for clinical application, and the development of improved genome editing tools with low off-target activity is underway.
  • a general method for evaluating the safety (specificity) of genome editing is to analyze the entire genome sequence after genome editing and confirm the presence or absence of mutations in sequences other than the target sequence.
  • this method cannot evaluate off-target activity below a mutation rate of about 0.5% or less due to sequence reading errors, sample-derived SNPs, mutations, etc.
  • ChIP-seq that perform genome-wide search for binding sites of genome editing tools on genomic DNA and perform sequence analysis. It is not possible to distinguish between the sites that are physically linked and the sites that are linked to cause a genome editing reaction. It is also not possible to obtain kinetic parameters of the genome editing reaction that can be used as indicators for tool improvement.
  • An object of the present invention is to provide a method for evaluating the safety (specificity) of genome editing, particularly a method for quantitative evaluation, specifically a method for quantitatively evaluating off-target activity in a genome editing tool.
  • the present inventors have conducted intensive research and found that it is possible to evaluate a genome editing tool by performing single-molecule imaging of a fluorescence-labeled genome editing tool in the nucleus of a living cell. rice field.
  • the imaging technology we succeeded in quantitatively evaluating where, with what frequency, and with what kind of kinetics the genome editing tool binds to the genome sequence, leading to the completion of the present invention.
  • rice field That is, the present invention is as follows.
  • a method for evaluating genome editing tools using fluorescence single-molecule imaging (2) The method according to (1) above, wherein the fluorescence single-molecule imaging method utilizes a binding reaction rate analysis technique. (3) The method according to (1) above, which comprises the step of analyzing the binding site of the genome editing tool to the genomic DNA by mapping the binding site in a microscope image. (4) The method according to any one of (1) to (3) above, including the step of analyzing off-target effects. (5) The method according to any one of (1) to (4) above, wherein the genome editing tool is TALE or CRISPR/dCas9. (6) The method according to any one of (1) to (5) above, wherein a Halo-tag is used to label the genome editing tool.
  • Genome-editing tools with high specificity bind specifically and repeatedly only to the target sequence, whereas genome-editing tools with low specificity repeatedly bind to multiple sites in the nucleus.
  • the presence of binding sites can be visualized.
  • mapping the binding sites in the microscope image it is possible to analyze where the binding occurs spatially. This enables comprehensive and direct safety evaluation of individual genome editing tools or individual designed target sequences to determine whether they function as highly specific genome editing tools in target cells. became possible.
  • FIG. 2 shows the results of intranuclear fluorescence single-molecule imaging of living cells (Indian barking deer cells incorporating LacO sequences) using a genome editing tool (TALE) targeting the LacO sequences.
  • TALE genome editing tool
  • FIG. 4 shows comparison of binding specificities after double staining with TALE and dCas9 targeting sequences flanking the second exon of the Muc4 gene.
  • FIG. 3 shows the results of designing three gRNAs for the second exon of the Muc4 gene and examining the difference in binding specificity among them.
  • FIG. 2 shows target sequences on genomic DNA used in Examples 2 and 3.
  • Genome editing tool generally refers to a technology in which a DNA cleavage domain is fused to a system that recognizes the base sequence of target DNA, but in the present invention, a system that recognizes and binds to the base sequence of target DNA is intended, regardless of the presence or absence of DNA cleavage.
  • ZFN zinc-finger nuclease
  • TALEN transcription activator-like effector nuclease
  • CRISPR clustered regularly interspaced short palindromic repeats
  • Cas CRISPR-associated proteins
  • ZFNs are artificial nucleases with zinc fingers as DNA-binding domains, and since one zinc finger recognizes 3 nucleotides, ZFNs with 3-6 zinc fingers are specific for 9-18 base pairs (bp). It binds and introduces a DNA double-strand break with a pairwise specificity of 18-36 bp.
  • TALENs are artificial nucleases that have TALEs of the phytopathogenic bacterium Xanthomonas as a DNA-binding domain.
  • the DNA-binding domain of TALE consists of 34 amino acids that recognize one base, and TALENs with 15 to 20 units of 34 amino acids are created for each of the sense strand and antisense strand, and the DNA double strand is attached to the target site. Introduce cutting.
  • the CRISPR/Cas system introduces a DNA double-strand break at the target base sequence using a complex of a Cas nuclease that has DNA double-strand breaking activity and a target-sequence-specific single-stranded guide RNA.
  • the CRISPR/Cas system has become a standard technology for genome editing used around the world due to its convenience, high efficiency, and versatility.
  • CasX and the like preferably Cas9.
  • Cas9 includes Streptococcus pyogenes-derived Cas9 (SpCas9), Staphylococcus aureus-derived Cas9 (SaCas9), Francisella novicida-derived Cas9 (FnCas9), Campylobacter jejuni-derived Cas9 (CjCas9), and Streptococcus thermophilus-derived Cas9 (St1Cas9, St3Cas9).
  • Cpf1 includes Acidaminococcus sp.-derived Cpf1 (AsCpf1) and Lachnospiraceae bacterium-derived Cpf1 (LbCpf1).
  • the CRISPR/Cas system can also use mutants (subspecies) in which the amino acid sequence of Cas nuclease is mutated.
  • dCas9 (dead Cas9) is a nuclease-deficient form of Cas9 that binds to DNA sequences but does not cleave them.
  • nCas9 (nickase Cas9) introduces a single-stranded nick, and xCas9 (Hu, JH et al., Nature, 556, 57-63. (2018)) has broad PAM compatibility and high DNA specificity.
  • any genome editing tool may be used, but since the binding of the genome editing tool to the genome sequence is evaluated, the one that does not cut the DNA sequence, such as dCas9, is preferred.
  • Single-molecule imaging is an imaging technique that tracks the behavior of molecules at the single-molecule level.
  • Fluorescent single-molecule imaging is an imaging technique that visualizes the dynamics of fluorescently-labeled molecules of interest in solutions or cells using a microscope.
  • molecules in solutions and in cells usually exhibit diffusional motion (Brownian motion).
  • diffusional motion is constrained and halted.
  • Kinetic analysis of molecular binding and dissociation is performed using this dynamic change as an index.
  • mapping the binding sites in the microscope image it is possible to analyze where the binding occurs spatially.
  • the genome editing tool is fluorescently labeled, the binding of the fluorescently labeled genome editing tool and the target sequence on the genomic DNA is visualized, and quantitatively evaluated by reaction rate analysis.
  • the fluorescent label can be added to any position of the genome editing tool, but when using dCas9 as the genome editing tool, for example, it is preferably added to the C-terminus of dCas9. Furthermore, it is preferred for localization to the nucleus that a nuclear localization signal sequence is added to the C-terminus.
  • fluorescent labels examples include green fluorescent protein (e.g., green fluorescent protein; GFP, enhanced green fluorescent protein; EGFP, monomeric enhanced green fluorescent protein; mEGFP), rhodamine dyes (e.g., tetramethylrhodamine; TMR, tetramethylrhodamine methylester; TMRM), silicon A rhodamine dye (eg, SaraFluor), JF646, etc. can be used, or a tag sequence (eg, HaloTag, SNAP-tag, CLIP-tag) can be added to covalently bind a fluorescent label.
  • green fluorescent protein e.g., green fluorescent protein; GFP, enhanced green fluorescent protein
  • EGFP monomeric enhanced green fluorescent protein
  • mEGFP rhodamine dyes
  • TMR tetramethylrhodamine methylester
  • TMRM silicon A rhodamine dye
  • JF646, etc. can be used, or a tag sequence (e
  • dCas9-gRNA fluorescently labeled A complex of dCas9 and guide RNA (hereinafter also referred to as dCas9-gRNA) is produced. Since dCas9-gRNA needs to bind to genomic DNA, a nuclear localization signal is added. The obtained dCas9-gRNA is brought into contact with the cells to be measured.
  • the diffusion dynamics (diffusion movement) of dCas9-gRNA in the cell nucleus is determined.
  • the diffusion kinetics is determined using the fluorescent label added to dCas9 as an index.
  • HaloTag is used as a fluorescent label, it is stained with HaloTag TMR ligand (Promega), STELLA Fluo 650 HaloTag ligand (Goryo Chemical), or the like. Any known method can be used to determine the diffusion dynamics of dCas9-gRNA, but for the purposes of the present invention, it is necessary to track the diffusion dynamics of dCas9 at the level of a single molecule. method is used.
  • single-molecule imaging may be performed by highly inclined and laminated optical sheet microscopy, HILO microscopy; HILO method) that illuminates with light thinner than the thickness of cells under a microscope. Physics 49(6), 318-321 (2009)).
  • HILO method samples such as cells are illuminated in a thin layer of light, brightly illuminating only the desired areas and not illuminating unnecessary areas, so higher image quality can be expected compared to conventional methods.
  • the annular HILO method which is an improved version of the HILO method.
  • the laser beam is divided into 72 parts on the circumference by a transmissive diffractive optical element, magnified by an axicon lens, and then focused on the rear exit exit pupil plane of a high numerical aperture (NA) objective lens.
  • NA numerical aperture
  • TIRF Ordinary total internal reflection illumination
  • microscopes provide illumination from only one direction, but annular illumination is preferable because illumination from all directions makes it possible to uniformly illuminate a wider field of view.
  • the diffusion kinetics of the genome editing tool is determined from the captured fluorescence images.
  • the position and brightness information of the bright spots of the genome editing tool are obtained using a bright spot detection algorithm for the captured fluorescence image. Furthermore, information on the trajectory of each bright spot is acquired by connecting the bright spots located closest to each other under a certain threshold value in the time-series images.
  • the bright spot detection algorithm an existing algorithm can be used as long as it can estimate the position of a single fluorescent molecule, preferably at high speed and with high accuracy. Specific examples include a two-dimensional Gaussian fitting method. .
  • an algorithm based on radial symmetry Optics Letters Vol. 37, Issue 13, pp. 2481-2483 (2012)
  • various other estimation methods have been proposed.
  • the slow diffusion kinetics of genome-editing tools in the cell nucleus implies a long duration of binding of genome-editing tools to genomic DNA, in which case the binding of genome-editing tools to their target sites is specific.
  • slow diffusion kinetics means that dCas9-gRNA has a long binding duration to genomic DNA.
  • the binding of dCas9-gRNA to the target site can be specific.
  • the fast diffusion dynamics of the genome editing tool in the cell nucleus means that the binding duration of the genome editing tool to the genomic DNA is short, and in this case, the binding of the genome editing tool to the target site is not selective. means no.
  • fast diffusion kinetics means that the duration of binding of dCas9-gRNA to genomic DNA is short.
  • the binding of dCas9-gRNA to the target site may be non-specific.
  • the method for measuring the duration of binding is not particularly limited, but it can be measured quantitatively by, for example, reaction rate analysis.
  • a short binding duration means, for example, in the case of TALE, a time constant of 0.1 to 1 second, preferably about 0.2 to 0.5 seconds, and in the case of CRISPR/Cas9, A time constant of 0.1 to 1 second, preferably about 0.1 to 0.5 seconds is intended. In the case of /Cas9, the time constant is intended to be 1 to 5 seconds, preferably about 1 to 3 seconds.
  • the binding site of the genome editing tool to the genomic DNA can be analyzed by mapping the binding site of the genome editing tool to the genomic DNA in the microscope image. Furthermore, by extracting such binding events and plotting the binding frequency at each location (pixel) in the nucleus, it is possible to investigate whether the binding of the genome editing tool to genomic DNA is selective. For example, if the binding of dCas9-gRNA in the nucleus occurs only at a specific site, it means that the binding of dCas9-gRNA to genomic DNA is selective, thus the gRNA used has off-target activity. It can be judged that the safety is low and the safety is high.
  • the binding in the nucleus of dCas9-gRNA repeatedly binds to multiple sites in the nucleus, it means that the binding of dCas9-gRNA to genomic DNA is non-selective, therefore, The gRNA used can be judged to have high off-target activity and low safety. Since the genome editing tool is designed based on the target sequence to be bound, it is possible to predict the site (number) on the genomic DNA to which the genome editing tool binds. Therefore, if the number of binding sites significantly exceeds the predicted number, it can be determined to have non-specific binding, that is, to have high off-target activity and low safety.
  • Example 1 Locus-targeted evaluation Each genome editing tool was evaluated with loci as targets.
  • TALE genome editing tools
  • LacO lac operator
  • DM cells Indian barking deer fibroblasts
  • TMR tetramethylrhodamine
  • EGFP Enhanced Green Fluorescent Protein
  • LacI-EGFP specifically binds to the LacO sequence incorporated into cells, and fluorescently stains a specific site on the chromosome. After that, LacI was dissociated from LacO by the addition of isopropyl ⁇ -D-thiogalactopyranoside (IPTG), and the binding of TALE to the site was observed and analyzed. Similarly, we evaluated a genome editing tool (CRISPR/dCas9) targeting major satellite sequences in mice. Mouse 3T3 cells (CL5611) (living cells) were used for the measurement. Target sequence: CAAGAAAACTGAAAATCA (NGG) (SEQ ID NO: 1) (NGG) is the PAM sequence, dCas9 is dSpCas9.
  • Halo-tag was fused to dCas9 for fluorescent dye labeling.
  • Halo-tag fused dCas9 was expressed in guide RNA and animal cells, and fluorescent labeling was performed by binding TMR to Halo-tag.
  • Single-molecule imaging in the nucleus was carried out by observing the prepared sample (encapsulated with a cover glass) using a microscope using annular illumination and oblique thin-layer illumination. Equivalent observation is possible with a general total internal reflection illumination microscope on the market, but it is possible to obtain high-contrast images by devising the shape of the light source and making it ring-shaped (annular illumination method). Become. The fluorescence signal from the sample was photographed with an scMOS camera and analyzed for the position and duration of bright spots.
  • the illumination light enters the cell at an angle to the sample, and the incident angle is adjusted so that the inside of the nucleus on the side closer to the coverslip can be observed with high contrast. It was adjusted.
  • the expression level of dCas9 and fluorescent labeling with Halo-tag were adjusted to a level suitable for single-molecule observation (specifically, one molecule or less labeled molecule per 1 cubic ⁇ m).
  • Figure 1 shows the kinetics of binding and dissociation of TALE to its target site. As shown in FIG. 1, we were able to visualize the kinetics of binding and dissociation of TALE to genomic DNA at the single-molecule level.
  • Fluorescently-labeled TALE selectively bound to the LacO sequence that was artificially integrated into the genome at one site (Fig. 2, left panel).
  • fluorescently labeled dCas9 was found to bind to many sites in the nucleus (Fig. 2, right panel). Binding kinetics analysis was performed from the results of fluorescent single-molecule imaging in the nucleus using TALE and dCas9. The results are shown in FIG. 3 (left: TALE, right: dCas9).
  • TALE binding time analysis showed that 65% of binding events were as short as 0.3 seconds in duration and 35% of binding events were as short as 12 seconds in duration. The former is considered to be weak binding during searching for the target site, and the latter is specific binding to the target site.
  • 75% of binding events had a duration of 0.2 seconds and 25% had a duration of 2 seconds.
  • Example 2 Off-target evaluation Double staining with TALE and dCas9 was performed to compare binding specificity.
  • the target sequence used (SEQ ID NO: 2) is shown in FIG.
  • gRNAs targeting sequences flanking the second exon of the MUC4 gene of human cultured cells (HeLa cells) were designed and used (1 to 3 in FIG. 6).
  • analysis was performed using gRNA No. 2.
  • TALE was also analyzed in the same manner (the target sequence of TALE is indicated by the arrow).
  • EGFP was fused to TALE and Halo-tag was fused to dCas9 for fluorescence labeling.
  • a dCas9-Halo plasmid, a gRNA plasmid, and a TALE-EGFP plasmid were transfected using X-treme GENE TM .
  • TMR Halo-ligand was added and cultured for about 20 hours. After that, they were re-sewn on a glass-bottom dish, and after several hours, they were observed by thin-layer oblique illumination using a microscope using ring-shaped illumination. The results are shown in FIG. The upper row shows the results of dCas9, and the lower row shows the results of TALE. Co-stained sites were detected and confirmed to specifically bind to target sites.
  • Example 3 Differences due to gRNA (target sequence) Using dCas9, comparison of binding specificities due to differences in gRNA type was performed. The target sequences used are shown in FIG. Using CRISPR/dCas9, gRNAs targeting sequences flanking the second exon of the MUC4 gene of human cultured cells (HeLa cells) were designed and used (1 to 3 in FIG. 6). In this example, gRNAs Nos. 1 to 3 were used for analysis. Halo-tag was fused to dCas9 for fluorescence labeling. The dCas9-Halo plasmid and the gRNA plasmid were transfected with X-treme GENE TM .
  • TMR Halo-ligand was added and cultured for about 20 hours. After that, they were re-sewn on a glass-bottom dish, and after several hours, they were observed by thin-layer oblique illumination using a microscope using ring-shaped illumination. The results are shown in FIG. The left two columns are the results using the first gRNA, the middle two columns are the results using the second gRNA, and the right two columns are the results using the third gRNA. Selective binding was observed with the second and third gRNAs, especially the third gRNA, indicating low off-target activity of genome editing tools using these gRNAs.
  • results can be given as numerical values such as binding time or the number of binding sites, enabling quantitative evaluation of genome editing tools.
  • Genome-editing tools with high specificity bind specifically and repeatedly only to the target sequence, whereas genome-editing tools with low specificity repeatedly bind to multiple sites in the nucleus. The presence of binding sites can be visualized. Further, by mapping the binding sites in the microscope image, it is possible to analyze where the binding occurs spatially. This enables comprehensive and direct safety evaluation of individual genome editing tools or individual designed target sequences to determine whether they function as highly specific genome editing tools in target cells. became possible.
  • This application is based on Japanese Patent Application No. 2021-012474 filed on January 28, 2021, the entire contents of which are incorporated herein by reference.

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Abstract

La présente invention concerne un procédé d'évaluation d'outil d'édition de génome comprenant les étapes suivantes : une étape consistant à mettre en contact des cellules et des outils d'édition de génome marqués par fluorescence; et une étape consistant à déterminer le comportement des outils d'édition de génome dans les noyaux cellulaires en utilisant un procédé d'imagerie moléculaire par fluorescence. Ledit procédé d'évaluation permet d'évaluer quantitativement la sécurité (spécificité) de l'édition de génome et d'évaluer quantitativement les activités hors cible des outils d'édition de génome.
PCT/JP2022/003152 2021-01-28 2022-01-27 Procédé d'évaluation d'outil d'édition de génome WO2022163770A1 (fr)

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NAGASHIMA RYOSUKE, HIBINO KAYO, ASHWIN S.S., BABOKHOV MICHAEL, FUJISHIRO SHIN, IMAI RYOSUKE, NOZAKI TADASU, TAMURA SACHIKO, TANI T: "Single nucleosome imaging reveals loose genome chromatin networks via active RNA polymerase II", THE JOURNAL OF CELL BIOLOGY, THE ROCKEFELLER UNIVERSITY PRESS, US, vol. 218, no. 5, 6 May 2019 (2019-05-06), US , pages 1511 - 1530, XP055954868, ISSN: 0021-9525, DOI: 10.1083/jcb.201811090 *

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