
==== Front
Cell Rep Methods
Cell Rep Methods
Cell Reports Methods
2667-2375
Elsevier

S2667-2375(24)00206-6
10.1016/j.crmeth.2024.100833
100833
Article
Genome editing using type I-E CRISPR-Cas3 in mice and rat zygotes
Yoshimi Kazuto kyoshimi@ims.u-tokyo.ac.jp
12∗
Kuno Akihiro 3
Yamauchi Yuko 1
Hattori Kosuke 1
Taniguchi Hiromi 1
Mikamo Kouya 1
Iida Ryuya 1
Ishida Saeko 1
Goto Motohito 4
Takeshita Kohei 5
Ito Ryoji 4
Takahashi Riichi 4
Takahashi Satoru 3
Mashimo Tomoji mashimo@ims.u-tokyo.ac.jp
126∗∗
1 Division of Animal Genetics, Laboratory Animal Research Center, Institute of Medical Science, The University of Tokyo, Tokyo 108-8639, Japan
2 Division of Genome Engineering, Center for Experimental Medicine and Systems Biology, Institute of Medical Science, University of Tokyo, Tokyo 108-8639, Japan
3 Department of Anatomy and Embryology, Faculty of Medicine, University of Tsukuba, Ibaraki 305-8575, Japan
4 Central Institute for Experimental Medicine and Life Science, 3-25-12 Tonomachi, Kawasaki-ku, Kawasaki, Kanagawa 210-0821, Japan
5 Life Science Research Infrastructure Group, Advanced Photon Technology Division, RIKEN SPring-8 Center, Hyogo 679-5148, Japan
∗ Corresponding author kyoshimi@ims.u-tokyo.ac.jp
∗∗ Corresponding author mashimo@ims.u-tokyo.ac.jp
6 Lead contact

08 8 2024
19 8 2024
08 8 2024
4 8 10083321 3 2024
8 7 2024
16 7 2024
© 2024 The Authors
2024
https://creativecommons.org/licenses/by-nc-nd/4.0/ This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Summary

The type I CRISPR system has recently emerged as a promising tool, especially for large-scale genomic modification, but its application to generate model animals by editing zygotes had not been established. In this study, we demonstrate genome editing in zygotes using the type I-E CRISPR-Cas3 system, which efficiently generates deletions of several thousand base pairs at targeted loci in mice with 40%–70% editing efficiency without off-target mutations. To overcome the difficulties associated with detecting the variable deletions, we used a newly long-read sequencing-based multiplex genotyping approach. Demonstrating remarkable versatility, our Cas3-based technique was successfully extended to rats as well as mice, even by zygote electroporation methods. Knockin for SNP exchange and genomic replacement with a donor plasmid were also achieved in mice. This pioneering work with the type I CRISPR zygote editing system offers increased flexibility and broader applications in genetic engineering across different species.

Graphical abstract

Highlights

• Type I-E CRISPR-Cas3 generates large deletions in mouse and rat zygotes

• Long-read sequencing enables multiplex genotyping of variable deletion patterns

• Electroporation method can also introduce CRISPR-Cas3-mediated genome editing

• CRISPR-Cas3 with donor DNA achieves SNP and genomic replacement in mouse zygotes

Motivation

The well-established class 2 CRISPR system, including Cas9 and Cas12, has transformed genetic engineering by enabling precise zygote editing. However, these systems still pose challenges, especially in manipulating larger genomic regions for functional analysis of gene clusters and non-coding areas. To address these limitations, our study develops the use of the CRISPR-Cas3 system, known to induce large genomic deletions, in rodent zygotes. Additionally, we established the strategy of efficient and user-friendly genotyping for detecting large genomic mutations.

Yoshimi et al. introduce a zygote editing strategy using type I-E CRISPR-Cas3 for large-scale genome editing in mouse and rat zygotes. Their approach, using modified RNAs and long-read sequencing for genotyping, expands the toolkit for creating animal models and offers new possibilities for studying complex genomic regions across species.

Keywords

genome editing
CRISPR-Cas3
zygote
mouse
rat
electroporation
large deletion
replacement
multiplex genotyping
knockin
knockout
Published: August 8, 2024
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pmcIntroduction

Genetically modified laboratory animals, particularly mice and rats, are indispensable for studying human pathophysiology and gene functions.1,2 The advent of CRISPR-Cas9 genome editing technology has greatly expedited the creation of these modified organisms, including improved efficiency, increased flexibility, and simplified experimental procedures accessible without advanced skills.3,4,5,6 However, most genome editing in experimental animals still predominantly relies on Cas9, which typically induces small mutations through the non-homologous end joining repair process. While Cas12a has also been used in mice,7,8 its applications are relatively limited. Newer Cas9 variants, such as protospacer adjacent motif (PAM)-free Cas9, base editors, and prime editors, have also been developed for smaller-scale modifications.9,10,11,12 Nonetheless, challenges persist in large-scale genomic editing with multiplex single-guide RNAs, such as the risk of off-target effects, reduced efficiency compared with knockout (KO) methods, and potential errors, such as inversions or translocations.8,13

The type I CRISPR system, part of the class 1 CRISPR systems, is attracting attention in genome editing for its ability to induce large-scale deletions.14,15,16,17 The type I-E subtype in particular is noted for its unique mechanism and applications.18,19 It functions as a multi-protein cascade complex, pivotal in the CRISPR-associated antiviral defense mechanism. This complex, upon binding to target DNA, recruits and activates Cas3, a specialized endonuclease, resulting in foreign DNA cleavage—a mechanism distinctly different from those of Cas9 and Cas12 systems.20 Initially used in the microbiota, its applications have expanded to eukaryotic cells, inducing significant, ∼5-kb deletions upstream of the PAM in human cell lines. The potential PAM sequences in type I-E CRISPR-Cas3 from E. coli are known to recognize AAG, TAG, AAC, GAG, AGG, and ATG, which are different from the PAM sequences in Cas9 and Cas12a.16,21 Furthermore, type I-E CRISPR’s longer spacer sequence of 27 nt, compared with Cas9’s 20 and Cas12’s 24, may enhance target recognition specificity, potentially reducing off-target effects.

These characteristics of the type I CRISPR system, particularly its effectiveness in large-scale genomic deletions and improved target specificity, highlight its potential as a reliable tool for gene disruption in genome editing. In this study, we developed a method to produce genetically modified mice and rats using fertilized egg genome editing with the type I CRISPR system. By modifying mRNA with Cap1 and applying terminal modifications to precursor CRISPR RNA (crRNA), we enhanced expression levels and achieved protection against intracellular degradation, leading to highly efficient KO production in mice and rats. The resulting mutations, similar to those reported previously,14,16 demonstrated the induction of large-scale deletions, affirming the utility of this approach for reliable gene disruption. Additionally, we applied an automated, long-read sequencing-based analytical approach for user-friendly genotyping, the modified Determine Allele mutations and Judge Intended genotype by Nanopore sequencer (DAJIN) system.22 Finally, our efforts culminated in the successful generation of knock-in (KI) mice, marking significant progress in genome editing techniques.

Results

Enhanced genome editing efficiency with Cap1-modified mRNAs

When exploring methods to deliver multiple factors as in CRISPR-Cas3 in preliminary experiments, experiments with all-in-one plasmids and ribonucleoprotein (RNP) complexes indicated significant challenges (Table S1). The use of all-in-one plasmids may have failed due to their large size, low expression of the six consecutive Cas components, and potential toxicity. Similarly, RNP complexes, despite their expected reduction in cellular toxicity, presented practical difficulties due to high viscosity and complications in the microinjection process, even though we could generate KO of the Rag2 gene in rats (Figure S1). Therefore, in this study, we attempted to optimize and utilize mRNA conditions.

We first evaluated the effect of modified Cap structures on mRNA translational activity in mouse zygotes. The introduction of GFP mRNA with a Cap1 structure into fertilized eggs via electroporation showed significantly increased GFP expression both 16 and 24 h after introduction compared with anti-reverse cap analog (ARCA)-modified mRNAs (Figure S2). This suggests that the Cap1 structure is more effective for early and strong translation in mouse zygotes. Further modifications to GFP mRNA, including 5-methylcytidine-5'-triphosphate (5Me-CTP), pseudouridine-5’-O-triphosphate (pseudoUTP), N1-methylpseudouridine-5'-triphosphate (N1-Me-PseudoUTP), and 5-methoxyuridine-5'-triphosphate (5Mo-UTP), were evaluated alongside Cap1. However, the fluorescence intensity observed 24 h after microinjection indicated a decrease in GFP expression with all modified mRNA patterns (Figure S2), suggesting that these chemical modifications may be unnecessary or even inhibitory during early embryonic development. To enhance KO efficiency, we modified the 3′ terminal bases of crRNA for Cas3 with 2′-O-methyl and phosphorothioate linkages. In human HEK293T cells expressing six Cas proteins (STAR Methods), transfected with modified crRNAs, we observed increased intensity of bands indicating largely deleted mutations (44.2% with modified crRNA vs. 14.9% with unmodified), demonstrating enhanced KO efficiency through crRNA stability (Figures 1A and S2).Figure 1 Enhanced genome editing efficiency by CRISPR-Cas3 with modified RNAs

(A) Cap1-modified mRNAs for CRISPR-Cas3 components (Cas3, Cas5, Cas6, Cas7, Cas8, and Cas11) and the 3′ terminal bases of crRNA for Cas3 with 2′-O-methyl and phosphorothioate linkages. See also Figure S2.

(B) Schematic of the generation of CRISPR-Cas3-mediated KO mice at the Tyr locus by the microinjection method. Several pups exhibited albino and mosaic coat colors.

(C) Electrophoresis of the PCR products with primers Tyr-F1&R1 and Tyr-F2&R2, which are shown in (B).

(D) The sequence data of the mutations around the PAM (highlighted in red) and targeted sequence (highlighted in yellow), which were validated by Sanger sequencing.

Using Cap1-modified mRNAs for CRISPR-Cas3 components (Cas3, Cas5, Cas6, Cas7, Cas8, and Cas11) and modified crRNAs, we performed genome editing in fertilized mouse eggs. Microinjection of each crRNA targeting the tyrosinase gene (Tyr) efficiently created KO pups at rates of 47.4% (9 KO pups/19 offspring) for Tyr-1 crRNA and 75.0% (9 KO pups/12 offspring) for Tyr-2 crRNA. Notably, several pups exhibited albino and mosaic coat colors (Figures 1B and S3). Genotyping with PCR and sequencing revealed various patterns of long deletions, confirming the efficacy of type I-E CRISPR-Cas3 in generating KO founders (Figures 1C and 1D). The use of unmodified crRNAs yielded significantly lower efficiency (6.7% and 6.3% for Tyr-1 and Tyr-2, respectively), underscoring the importance of crRNA modifications (Table 1). More interestingly, experiments conducted without ATP in the injected solution resulted in no KO individuals, highlighting the essential role of ATP for effective CRISPR-Cas3 activation, especially in fertilized eggs (Table 1).Table 1 Genome editing efficiencies with CRISPR-Cas3 mRNAs in zygotes

Species	Target	RNA modification	ATP	Concentration	Method	Treated embryos	Transferred embryos (%)	Pups delivered	KO/offspring (%)	
Mouse	Tyr-1	Cap1 mRNAs	+	crRNA: 100 ng/μL
CRISPR-Cas3 mRNAs: 50 ng/μL	MI	193	75 (38.9)	15	1 (6.7)	
Mouse	Tyr-1	Cap1 mRNAs, mod crRNA	+	crRNA: 100 ng/μL
CRISPR-Cas3 mRNAs: 50 ng/μL	MI	303	157 (51.8)	19	9 (47.4)	
Mouse	Tyr-1	Cap1 mRNAs, mod crRNA	+	crRNA: 200 ng/μL
CRISPR-Cas3 mRNAs: 100 ng/μL	EP	169	147 (87.0)	15	7 (46.7)	
Mouse	Tyr-1	Cap1 mRNAs, mod crRNA	–	crRNA: 100 ng/μL
CRISPR-Cas3 mRNAs: 50 ng/μL	MI	118	85 (72.0)	14	0	
Mouse	Tyr-2	Cap1 mRNAs	+	crRNA: 100 ng/μL
CRISPR-Cas3 mRNAs: 50 ng/μL	MI	245	85 (34.7)	16	1 (6.3)	
Mouse	Tyr-2	Cap1 mRNAs, mod crRNA	+	crRNA: 100 ng/μL
CRISPR-Cas3 mRNAs: 50 ng/μL	MI	88	78 (88.6)	13	9 (69.2)	
Mouse	Tyr-2	Cap1 mRNAs, mod crRNA	+	crRNA: 200 ng/μL
CRISPR-Cas3 mRNAs: 100 ng/μL	EP	85	85 (100.0)	13	3 (23.1)	
Mouse	Hbb-LCR1	Cap1 mRNAs, mod crRNA	+	crRNA: 200 ng/μL
CRISPR-Cas3 mRNAs: 100 ng/μL	EP	90	90 (100.0)	21	3 (14.3)	
Mouse	Hbb-LCR2	Cap1 mRNAs, mod crRNA	+	crRNA: 200 ng/μL
CRISPR-Cas3 mRNAs: 100 ng/μL	EP	91	89 (97.8)	28	7 (25.0)	
Rat	Il2rg	ARCA mRNAs	+	crRNA: 400 ng/μL
CRISPR-Cas3 mRNAs: 200 ng/μL	MI	153	96 (62.7)	6	0	
Rat	Il2rg	Cap1 mRNAs	+	crRNA: 100 ng/μL
CRISPR-Cas3 mRNAs: 50 ng/μL	MI	126	95 (75.4)	21	0	
Rat	Il2rg	Cap1 mRNAs, mod crRNA	+	crRNA: 100 ng/μL
CRISPR-Cas3 mRNAs: 50 ng/μL	MI	109	81 (74.3)	13	5 (38.5)	
Rat	Il2rg	Cap1 mRNAs, mod crRNA	+	crRNA: 200 ng/μL
CRISPR-Cas3 mRNAs: 100 ng/μL	EP	87	85 (97.7)	37	3 (8.1)	
mod: endo-modified, MI: microinjection, EP: electroporation

Germline transmission analysis of KO founders with large deletions confirmed that all mutations that we tested were heritable (Table S2). Additionally, to assess off-target effects, we analyzed the off-target candidate regions with five or six mismatches for the Tyr-1 target sequence in five individuals with confirmed KO mutations (Table S3). PCR and Sanger sequencing revealed no off-target mutations, indicating a safety profile comparable to that of other genome editing technologies, such as Cas9 (Figure S3).

Multiplex genotyping for Cas3-mediated large deletion by nanopore sequencing

CRISPR-Cas3-mediated genome editing often induces large deletions but lacks precise control over their position and size. Consequently, conventional PCR-based genotyping methods may lose identifying mutations, necessitating numerous Sanger sequencing reactions, resulting in a laborious process. To enhance the detection of these extensive deletions, we implemented a long-read sequencing approach using nanopore technology. This methodology is pivotal for scrutinizing sequences spanning several kilobases, characteristic of deletions identified in Cas3-based editing. Through the amplification of target regions with primers introducing adapter and barcode sequences, multiple individuals can be identified in a single sequencing run, thereby augmenting the throughput and efficiency of genotyping (STAR Methods for comprehensive details). For Tyr-KO mice, individual visualization of nanopore sequencing reads revealed reduced coverage due to large deletions in the mutant individuals, with measured deletion sizes of 12,058, 13,075, and 4,468 bases, respectively (Figure 2A). While this analysis offered an initial estimation of deletion sizes, it lacked detailed information on insertions or deletions (indels) at the deletion junctions. Consequently, we investigated the applicability of the DAJIN system, known for its high accuracy in identifying mutational polymorphisms.22 Cas3 induces mainly large deletions, but since the aligner mappy used by DAJIN2 performs local alignment, it fragments insertions within these large deletions and maps them to the reference genome, failing to identify them as a single insertion sequence. To address this issue, we added a custom script to DAJIN2 that consolidates these fragmented alignments within large deletions into a single insertion sequence. By adapting the DAJIN2 system to analyze large-scale deletions and insertions spanning multiple kilobases, we were able to obtain precise indel profiles.Figure 2 Multiplex genotyping for Cas3-mediated large deletion by nanopore sequencing

(A) Representative image of the deleted mutation at the Tyr locus by long-read sequencing using the Integrative Genomics Viewer (IGV). The targeted locus is indicated by a red arrow.

(B) Charts indicating the type of allele and the proportion of reads for that allele in each barcode sample.

(C) Automated detection of the mutation sites by DAJIN2 in each pup. The consensus sequences in HTML files for each allele showing the mutation sites are highlighted by color. The deleted, inserted, and substituted mutations are highlighted in blue, red, and green, respectively.

Visualization of the allele percentages for each pup revealed that wild-type (WT) individuals could be identified with intact control alleles, whereas KO individuals exhibited alleles with structural variations (SVs) and indels, clearly identifying them as mutant individuals (Figure 2B). Subsequent visualization of the alleles detected in each KO individual using the Integrative Genomics Viewer (IGV) demonstrated that the DAJIN2 system accurately captured the mutations confirmed by Sanger sequencing (Figure 2C). Notably, the system could accurately detect not only simple deletion mutations but also mutations involving insertions at the junction ends that occurred along with large deletions, as seen in individuals #5 and #8. Furthermore, minor alleles (5,449 bp, 3.2% in #5; 2,055 bp, 5.7% in #8), which are difficult to confirm by IGV visualization in each barcode, were detected successfully. Interestingly, the single-base deletion observed in the Sanger sequence and the 2,055-base deletion were identified as the same allele. The occurrence of multiple mutations within the same allele has also been reported in human cultured cells.16 The reason why multiple deletions occur upstream of the PAM is thought to be that the target sequence often remains after mutation induction by CRISPR-Cas3, leading to multiple events of re-recognition and re-cleavage. This study reveals that a similar situation can also occur in mouse individuals.

In the DAJIN2 analysis in this study, less common alleles were excluded due to errors likely due to nanopores or cross-contamination from other samples. Based on the findings of this research and previously reported results,22 even samples with a minimum coverage of 12 were analyzable (Table S4), so a minimum of around 1,000 coverage at a target site enabled the detection of variants as low as around 1%. Furthermore, if barcodes can be added to amplicons, then it is possible to analyze multiple individuals simultaneously. In this study, barcodes for 82 individuals, including WT, were added at once, and the DAJIN2 analysis was successful.

These results underscore the effectiveness of the nanopore-based barcoding strategy and the DAJIN2 system in facilitating the simultaneous and rational analysis of multiple samples, making it an optimal approach for high-throughput genotyping.

Cas3-mediated genome editing in rat zygotes

We extended our CRISPR-Cas3 zygote editing approach to rat embryos, targeting the rat Il2rg gene (Figure 3A). Following the microinjection of modified mRNA/crRNA components into fertilized rat eggs, we observed deletion mutations in the Il2rg gene in 5 of 13 (38.5%) of the offspring (Figure 3B; Table 1). This success demonstrates the adaptability of our CRISPR-Cas3 method across different species. Similar to our approach in mice, we used nanopore long-read sequencing and the DAJIN2 system for high-throughput genotyping of multiple rat samples, except for #5, which showed no PCR amplification.Figure 3 Cas3-mediated genome editing in rat zygotes

(A) CRISPR-Cas3 zygote editing in rat embryos, targeting the rat Il2rg gene.

(B) Genotyping of the KO rats with PCR analysis.

(C) Representative IGV image of the Il2rg locus of KO pups. The targeted locus is indicated by a red arrow.

(D) Charts indicating the type of allele and the proportion of reads for that allele in each rat.

(E) Automated detection of the mutation sites by DAJIN2 in each rat. The consensus sequences in HTML files for each allele showing the deleted mutation sites are highlighted in blue.

(F) Validation of the mutated sequence around the PAM (highlighted in red) and targeted sequence (highlighted in yellow) by Sanger sequencing.

Visualization of allele percentages revealed that KO individuals exhibited alleles with SVs, clearly identifying them as mutants in a manner analogous to our findings in mice (Figure 3C). Subsequent analysis using the IGV confirmed that the DAJIN2 system precisely identified the mutations in each KO individual (Figure 3D). The consensus sequences of detected SV alleles revealed deletions ranging from hundreds of bases to several kilobases upstream of the PAM sequence in all four KO rats (Figure 3E). These mutations were validated by Sanger sequencing (Figure 3F). Notably, no short indels were identified, analogous to the results obtained in mouse embryos. To assess the phenotypic impact of Il2rg KO, we performed crossbreeding with WT F344 rats. Flow cytometry analysis revealed a complete absence of the thymus and lack of T cells, B cells, and natural killer (NK) cells in the peripheral blood, consistent with previous research.23,24 This confirmed the functional KO of the Il2rg gene (Figure S4). Additionally, germline transmission analysis showed that 8 of 14 female rats successfully transmitted the mutations to their offspring (Table S2).

Delivery of mRNA by electroporation for Cas3-mediated KO in mouse and rat zygotes

Given the technical expertise required for microinjection, we explored the utility of a more accessible and simpler electroporation method for delivering CRISPR-Cas3 mRNA into zygotes.25 We used a NEPA21 electroporator to perform electroporation on mouse zygotes, not only targeting the Tyr locus but also expanding our focus to include two sites within the β-globin locus control region (Hbb-LCR). This enabled us to assess the method’s versatility across different genomic targets (Figure 4A). Electroporation resulted in successful Cas3-mediated editing at all target loci, with KO efficiencies of 14.3% for Hbb-LCR1, 25.0% for Hbb-LCR2, 46.7% for Tyr-1, and 15.4% for Tyr-2 (Table 1). Subsequent PCR and genotyping using nanopore sequencing identified several long deletions at the kilobase scale for both Hbb-LCR1 and Hbb-LCR2, as well as the Tyr locus, indicating the broad applicability and consistent efficiency of electroporation in generating KO mice (Figures 4B–4E, S5A, and S5B). Applying the same electroporation protocol to rat zygotes resulted in a KO efficiency of 8.1%, which, while lower than that observed with microinjection, confirms electroporation as a viable and effective method for generating KO in rat zygotes (Figure S5C; Table 1). These findings highlight the versatility of electroporation as a delivery method for CRISPR-Cas3-based genome editing.Figure 4 Electroporation method for genome editing in mouse zygotes

(A) Design of the targeted sequence at the Hbb-LCR locus and schematic of the generation of CRISPR-Cas3-mediated KO mice at Hbb locus by the electroporation method.

(B and D) Electrophoresis of the PCR products with primer sets (Hbb-LCR-F1&R1, Hbb-LCR-F2&R2) and IGV images of long-read sequencing data for each pup. The targeted locus is indicated by a red arrow.

(C and E) The sequence data of the mutations around the PAM (highlighted in red) and targeted sequence (highlighted in yellow), which were detected by nanopore sequencing and validated by Sanger sequencing.

The potential to generate KI mice with donor plasmids

To illustrate the versatility and accuracy of the CRISPR-Cas3 system, we performed targeted KI of the CAG-GFP-poly(A)-mCherry cassette at the Rosa26 locus via homology-directed repair (HDR) in mouse embryos (Figure 5A). We designed a crRNA targeting the Rosa26 locus alongside a donor plasmid containing a CAG-GFP-poly(A)-mCherry cassette flanked by homology arms corresponding to the target site. Considering CRISPR-Cas3’s mechanism of unwinding double-stranded DNA upstream of the PAM to introduce deletions averaging several kilobases,16 we strategically designed the replacement of the 4.8-kb Rosa26 region into the same length containing the KI sequences. The upper homology arm of 3 kb was designed to accommodate a potential Cas3-mediated large deletion, while the downstream arm was set at 1 kb. The crRNA and the donor plasmid were co-injected into mouse zygotes via microinjection. Successful KI was indicated by GFP expression, with the precise integration of the CAG-GFP-poly(A)-mCherry cassette confirmed through PCR with boundary primers and subsequent sequencing analysis (Figure 5B). This approach led to the accurate replacement of the CAG-GFP-poly(A)-mCherry reporter fragment at the Rosa26 locus in 2 of 9 (22.2%) offspring, underscoring the ability of the CRISPR-Cas3 system to achieve precise genomic editing (Figure 5C; Table 2). Furthermore, to confirm that the single-copy reporter gene cassette was functionally working, we introduced Cre mRNA into zygotes following a method reported previously26 and examined whether GFP expression would switch to mCherry expression due to recombination. As a result, the pups exhibited strong mCherry fluorescence and a loss of GFP expression with a mosaic pattern, confirming that the cassette was integrated correctly and functionally.Figure 5 Targeted replacement of the mouse Rosa26 locus with a reporter cassette using CRISPR-Cas3

(A) Targeting design of the KI of CAG-GFP-poly(A)-mCherry cassettes at the Rosa26 locus through homology-directed repair (HDR) in mouse embryos. A pup exhibited GFP fluorescence upon integration of the cassette. HA, homology arm.

(B) Precise replacement of the CAG-GFP-poly(A)-mCherry cassette was assessed through PCR with boundary primers and subsequent sequence analysis.

(C) Sequencing data at the junction of the homology arm (blue and green) and genome locus. Both pups showed precise KI of the CAG-GFP-poly(A)-mCherry cassette (red) at the Rosa26 locus.

(D) Design of Cre-dependent recombination of KI cassettes by introducing Cre mRNA into zygotes and fluorescence images of a resulting pup. Fluorescence images show pups exhibiting strong red mCherry fluorescence and mosaic loss of green GFP fluorescence.

See also Figure S6.

Table 2 KI efficiencies with CRISPR-Cas3 mRNAs in mouse zygotes

Target	KI size (bp)	KI strategy	Conc.	Background	Treated embryos	Transfered embryos (%)	Pups delivered	KI/offspring (%)	
Rosa insertion	4,531	conventional HR method	crRNA: 100 ng/μL
CRISPR-Cas3 mRNAs: 50 ng/μL
Donor plasmids: 3 ng/μL	C57BL/6	269	158 (58.7)	15	0	
Rosa replacement	4,531	conventional HR method	crRNAs: 100 ng/μL CRISPR-Cas3 mRNAs: 50 ng/μL
Donor plasmids: 3 ng/μL	C57BL/6	140	92 (65.7)	9	2 (22.2)	
Rosa insertion	4531	combi method	crRNAs: 100 ng/μL CRISPR-Cas3 mRNAs: 50 ng/μL
Donor plasmids: 3 ng/μL	C57BL/6	130	82 (63.1)	5	1a (20.0)	
c-kit-V831C	3	conventional HR method	crRNAs: 100 ng/μL CRISPR-Cas3 mRNAs: 50 ng/μL
Donor plasmids: 1 ng/μL	NOD/Shi-scid, IL-2Rγ KO (NOG)	484	284 (58.7)	25	1 (4.0)	
a Incomplete KI.

Despite these successes, attempts to insert the reporter gene through homologous recombination without adjusting the homology arm’s position yielded no KI individuals (Table 2). To improve efficiency, we used our previously established approach, Combi-CRISPR,27 which uses two crRNAs targeting genome and plasmid sequences (Figure S6). This dual crRNA strategy, along with the donor plasmid and the CRISPR-Cas3 system, facilitated the successful insertion of the CAG-GFP-poly(A)-mCherry at the Rosa26 locus in 1 of 5 (20.0%) offspring, albeit with incomplete KI, including the integration of plasmid-backbone sequences upstream of the CAG promoter (Figure S6). These findings suggest that genomic replacement with segments of identical length may be a more effective KI strategy with CRISPR-Cas3 than fragment insertion.

To demonstrate the feasibility of small-scale KIs, we attempted single-amino acid substitution (V831C) in the c-Kit gene of the non-obese diabetic/Shi-scid, interleukin-2Rγ (IL-2Rγ) KO (NOG) mouse strain through triple nucleotide substitutions, flanked by 1.5 kb of donor DNA on each side (Figure S7). This approach resulted in single-nucleotide substitution in 1 of 25 animals, with germline transmission of the allele confirmed and successful establishment of the mutant strain (Table S2). These findings indicate CRISPR-Cas3’s efficacy not only in gene transfer but also in precise single-nucleotide substitutions in zygotes. While some issues remain to be addressed, such as the need to optimize homology arm length and delivery conditions due to lower knockin efficiency compared with CRISPR-Cas9, the successful application of these KI methods underscores CRISPR-Cas3’s potential for gene function studies and the advancement of genetic engineering techniques.

Discussion

This study represents the first application of CRISPR-Cas3 for large-scale genome editing in embryos, marking a significant advance in genetic engineering. In contrast to Cas9, Cas3, via its dual nuclease and helicase activities, initiates DNA unwinding upstream from the cascade complex’s binding site, leading to progressive degradation and substantial deletions across thousands of base pairs.20,28,29 This contrasts with other CRISPR systems, such as Cas9 or Cas12, which generate double-strand breaks at specific sites without the extensive unwinding or degradation activity.30,31,32 This study demonstrates that the characteristics of CRISPR-Cas3 genome editing occur not only in cultured cells but also in zygotes. This highlights the uniqueness of Cas3 to enable the development of large genome-edited animals and underscores its utility in applications such as studying gene function, understanding the role of large gene clusters and non-coding regions (such as enhancers and silencers), and exploring the functional aspects of large genomic regions. During the submission of this paper, a study reported the generation of mice with large-scale deletions in the Y chromosome using CRISPR-Cas3.33 This study similarly demonstrated that CRISPR-Cas3 technology could introduce large-scale deletions in mouse zygotes, providing important supporting evidence for the utility of CRISPR-Cas3.

Our research demonstrates a significant increase in the efficiency of Cas3-mediated genome editing in embryos by using Cap1 mRNA and modified crRNA in the complex. As preliminary experiments in this study demonstrated, delivering all of the factors of the type I CRISPR system via plasmids or RNP requires considerable caution regarding cellular toxicity and viscosity, making it currently difficult to utilize for zygotes. In contrast, the Cap1 structure in mRNA, known to enhance mRNA stability and translation efficiency,34,35 significantly promoted the synthesis of CRISPR-Cas3 system components, especially those required for cascade complex formation. In addition, modifications at the 2′-O-methyl and phosphorothioate linkages of crRNA ends, improved stability in the complex.36,37 The synergistic effect of using Cap1-modified mRNA for Cas protein expression and modified crRNA to guide the CRISPR-Cas3 complex to the target DNA resulted in significant overall improvement in genome editing efficiency. These results highlight the importance of introducing genome editing technologies not only in fertilized eggs but also in cells and organisms.

Extending our genome editing approach to rat embryos, we successfully edited the Il2rg gene and confirmed the immunodeficiency in KO rats, consistent with the reported phenotype of X-linked severe combined immunodeficiency rats.23,24,38 Our results also demonstrate the efficacy of electroporation as an alternative method to deliver CRISPR-Cas3 components into embryos, enabling effective genome editing in both mice and rats. This method has the potential to generalize the use of CRISPR technology by making it more user friendly. In addition, the successful targeted KI of the GFP cassette at the Rosa26 locus in mouse embryos demonstrates the precision and versatility of the CRISPR-Cas3 system. Together with the observed high KO efficiency and the different deletion patterns in mice and rats, these successes underscore the system’s ability to achieve precise gene modification.

The large deletions induced by Cas3 pose a challenge to conventional genotyping methods. Our study showed that these mutations are detected inadequately by traditional Sanger sequencing, highlighting the need for more effective genotyping strategies. To address this issue, we adopted nanopore sequencing, which enables high-throughput detection of large deletions. The DAJIN2 system proved to be particularly effective in analyzing deletion patterns, especially without detecting off-target effects. The detection sensitivity of DAJIN, estimated to be approximately 1% allele detection with approximately 20 reads based on previous studies,22 may be compromised in samples with less than 500× coverage, especially for alleles with frequencies below 3%–5%. In addition, the allele frequency detection using DAJIN2 in this study suggests mosaicism in the detected mutations, highlighting the significance of selecting the appropriate tissue for DNA sampling in genotyping. It is also important to determine the extent of mosaicism, especially in F1 and F2 offspring.

In summary, we report a comprehensive method for gene modification using the CRISPR-Cas3 system in mouse and rat embryos, including genotyping analysis. This achievement not only demonstrates the flexibility and broad applicability of class 1 CRISPR systems in genetic engineering but also sets the stage for future advancements in genome editing technologies. The large-scale deletions enabled by Cas3 could be leveraged to study the functional roles of large gene clusters like the globin genes involved in hemoglobin production and remove extensive non-coding regulatory regions to understand their impact on gene expression. We are also able to generate animal models for diseases associated with large genomic rearrangements by precisely reorganizing large genomic regions, as shown in a recent paper.33 Overall, the comprehensive gene modification approach demonstrated in this study, coupled with effective genotyping strategies, positions the CRISPR-Cas3 system as a powerful tool for exploring the functional relevance of extensive genomic regions, engineering complex genetic circuits, and advancing our understanding of the intricate relationships between genome structure and biological function.

Limitations of the study

In this study, we optimized the mRNA expression vector for the CRISPR-Cas3 system and attempted to establish a platform for generating genetically modified animals, including KOs and KIs, by applying it to mouse and rat zygotes. As a result, we demonstrated the feasibility of using CRISPR-Cas3 in vivo but also identified several technical challenges.

First, because CRISPR-Cas3 is a large protein complex composed of six subunits, delivery to zygotes was difficult, necessitating the use of mRNA in this study. A large all-in-one plasmid encoding all subunits failed to function. We also attempted delivery via RNP complexes, but their high viscosity made microinjection challenging. In contrast, RNPs are commonly used with Cas9 for genome editing in various cells and zygotes. Further optimization, such as stabilizing CRISPR-Cas3 RNPs or using glycerol-free buffers, may enable future applications.

Second, for genotyping using long-read sequencing, detailed optimization of primer conditions was required to obtain PCR products exceeding 10 kilobase pairs, depending on the target sequence. Additionally, when no PCR amplification was observed, it was difficult to distinguish between WT and large deletions extending beyond the primer-binding regions, necessitating appropriate primer design and condition evaluation. Furthermore, DAJIN2 allele frequency analysis is dependent on PCR amplification efficiency, potentially overestimating allele frequencies for large deletions. Therefore, F0 data should serve as a guide, and mosaic states should be confirmed in subsequent generations like F1.

Finally, for KI applications, we demonstrated two examples using plasmid donor DNA. However, KI efficiency largely depends on the target sequence size and location, leaving room for further optimization. As more knowledge accumulates on factors like homology arm length, donor DNA amount, and the use of single-stranded DNA, common improvements for CRISPR-Cas3-mediated KI methods are expected to be identified.

STAR★Methods

Key resources table

REAGENT or RESOURCE	SOURCE	IDENTIFIER	
Bacterial and virus strains	
	
E.coli:Bacterial Strain JM109(DE3)	Promega	Cat#P9801	
E. coli: MAX Efficiency™ DH5α Competent Cells	Thermo Fisher Scientific	Cat#18258012	
	
Chemicals, peptides, and recombinant proteins	
	
ATP	TaKaRa	Cat#4041	
KSOM	ARK Resource	https://www.ark-resource.co.jp/product_re/142/	
M2	ARK Resource	https://www.ark-resource.co.jp/product_re/142/	
PMSG	ASKA Animal Health Co., Ltd.	https://aska-animal.co.jp/products/detail/husbandry/sero.html	
hCG	ASKA Pharmaceutical Co., Ltd.	https://www.aska-pharma.co.jp/iryouiyaku/item/detail.php?id=302	
Rat KSOM	ARK Resource	https://www.ark-resource.co.jp/product_re/142/	
Tks Gflex DNA Polymerase	TaKaRa	Cat#R060A	
KOD One PCR Master Mix -Blue-	TOYOBO	Cat#KMM-201	
KOD -Multi & Epi-	TOYOBO	Cat#KME-101	
	
Critical commercial assays	
	
mMESSAGE mMACHINE T7 Transcription Kit	Invitrogen	Cat#AM1344	
MEGAshortscript T7 Transcription Kit	Invitrogen	Cat#AM1354	
CleanCap Reagent AG	TriLink	Cat#N-7113-1	
Monarch RNA Cleanup Kit	NEB	Cat#T2040L	
MagMAX™ DNA Multi-Sample Ultra 2.0 Kit	Applied Biosystems	Cat#A36570	
KOD -Multi & Epi-	TOYOBO	Cat#KME-101	
Ligation Sequencing Kit V14	Oxford Nanopore technologies	Cat#SQK-LSK-114	
	
Deposited data	
	
Raw data of Nanopore sequencing	This paper	PRJNA1114408	
Mouse reference genome NCBI GRCm39, mm39	Genome Reference Consortium	https://www.ncbi.nlm.nih.gov/datasets/genome/GCF_000001635.27/	
Rat reference genome NCBI mRatBN7.2, rn7	Genome Reference Consortium	https://www.ncbi.nlm.nih.gov/datasets/genome/GCF_015227675.2/	
	
Experimental models: cell lines	
	
Human: HEK293T cells	ATCC	Cat#CRL-3216	
	
Experimental models: organisms/strains	
	
Mouse: C57BL/6JJcl	CLEA Japan Inc.	https://www.clea-japan.com/products/inbred/item_a0420	
Mouse: Jcl:ICR	CLEA Japan Inc.	https://www.clea-japan.com/products/outbred/item_a0340	
Mouse: NOD.Cg-PrkdcscidIl2rgtm1Sug/ShiJic	the Central Institute for Experimental Medicine and Life Science	https://www.ciea.or.jp/en/laboratory_animal/nog.html	
Rat: F344/Jcl	CLEA Japan Inc.	https://www.clea-japan.com/products/inbred/item_a0470	
Rat: Iar:Wistar-Imamichi	Institute for Animal Reproduction	https://www.iar.or.jp/gp_rat/wi_rat/index_wi.html	
	
Oligonucleotides	
	
Primers for genotyping, see Table S5	This paper	N/A	
	
Recombinant DNA	
	
pDonor: Rosa26-GFP-mCherry-Replacement, see Table S5	This paper	N/A	
pDonor: Rosa26-GFP-mCherry-Integration, see Table S5	This paper	N/A	
pDonor: mc-Kit-V831C, see Table S5	This paper	N/A	
	
Software and algorithms	
	
Prism 8	GraphPad	https://www.graphpad.com/scientific-software/prism	
ImageJ	Schneider et al., 2012	https://imagej.nih.gov/ij/	
DAJIN2	Kuno et al.22	https://github.com/akikuno/DAJIN2	
cstag	Kuno43	https://github.com/akikuno/cstag	
mappy	Li et al., 2018	https://github.com/lh3/minimap2/tree/master/python	
pysam	Li et al.41	https://github.com/pysam-developers/pysam	
IGV	Robinson et al., 2011	https://igv.org/	
GGGenome	Provided by DBCLS in Japan	https://GGGenome.dbcls.jp/	
UCSC Genome Browser	Karolchik et al.42	https://genome.ucsc.edu/	
	
Other	
	
Microscope	OLYMPUS	SZ61	
Microscope	OLYMPUS	IX73	
Manipulator	NARISHIGE	MMO-4	
Manipulator	NARISHIGE	MM-94	
Microinjector	NARISHIGE	IM-12	
Microinjector	Eppendorf	FemtoJet 4i	
Electroporator	Nepa Gene Co., Ltd.	NEPA21 Type II	
KingFisher Duo Prime Purification System	Thermo Scientific	5400110	
T100 Thermal Cycler	Bio-Rad	T100	
GelDoc Go Imaging System	Bio-Rad	https://www.bio-rad.com/ja-jp/category/geldoc-go-gel-imaging-system?ID=O494SO15	
MinION Mk1C	Oxford Nanopore technologies	SLW12M-M1C	

Resource availability

Lead contact

Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact Tomoji Mashimo (mashimo@ims.u-tokyo.ac.jp).

Materials availability

Among the genetically modified animals generated in this study, one strain of CAG-GFP-pA-mCherry KI mice, and two strains of severely immunodeficient rat by Il2rg-KO and Rag2-KO have been deposited at RIKEN BioResource Research Center and National BioResource Project-Rat, respectively and will be available for future use (C57BL/6J-Gt(ROSA)26Sorem1(CAG-EGFP,-mCherry)Larc: Cat# RBRC12313, F344-Il2rgem1Larc: Cat#1030, F344-Rag2em1Larc: Cat#1031). The KI strain on the NOG genetic background is planned to be provided by the Central Institute for Experimental Medicine and Life Science in Japan.

Data and code availability

• DAJIN2 has been deposited at GitHub (https://github.com/akikuno/DAJIN2). The version 0.4.0 used in this study can be available from https://doi.org/10.5281/zenodo.10537120. Identifiers are listed in the key resources table.

• Sequencing data by Nanopore is deposited in the SRA at NCBI and the bioproject accession ID is PRJNA1114408.

• Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request.

Experimental model and study participant details

Jcl:ICR pseudopregnant female mice, C57BL/6JJcl cryopreserved zygotes, and F344/Jcl rats for collecting zygotes were purchased from CLEA Japan Inc. (Tokyo, Japan). Iar:Wistar-Imamichi female rats for surrogates were purchased from Japan SLC, Inc. (Hamamatsu, Japan). Sex was not selected for the zygotes used in these experiments, but available sex genotyping information obtained from nanopore sequencing is reported in Table S4. All animals were housed and maintained under conditions of 50% humidity and a 12:12-h light:dark cycle. They were fed a standard pellet diet (CE2; Oriental Yeast Co., Tokyo, Japan) and tap water ad libitum. NOG mice used in this study were maintained in the Central Institute for Experimental Medicine and Life Science (CIEM) under specific pathogen-free conditions. The Animal Experiment Committee of the Institute of Medical Science, University of Tokyo, and the Animal Experimentation Committee of CIEM approved the animal experiments.

Method details

Preparation of plasmids, RNAs, and RNPs

CRISPR-Cas3-expressing plasmids used for zygotes and human cells were previously deposited in Addgene (#204619 and #134921). All targeting crRNA-expressing plasmids were created by the insertion of 32 bp double-stranded oligonucleotides for target sequences at the BbsI restriction site in the crRNA expression plasmid, which was previously constructed.16 All of the targeted sequences are listed in Table S5.

All RNAs of the EGFP and CRISPR-Cas3 components (Cas3, Cas5, Cas6, Cas7, Cas8, and Cas11) with a bipartite nuclear localization signal (bpNLS) with the ARCA capping structure were transcribed in vitro from synthetic double-stranded DNA templates obtained from Integrated DNA Technologies (IA, USA) using a MEGAshortscript T7 Transcription Kit (Thermo Fisher Scientific, MA, USA). All mRNAs with the Cap1 structure were purchased from Elixirgen Scientific, Inc. (Kawasaki, Japan). The custom crRNAs with or without modification of the three bases at both ends were obtained from IDT or Agilent Technologies (CA, USA). All sequences for RNAs are listed in Table S5.

EcoCas3 proteins were generated with Sf9 cells, which were previously reported.20,39 Briefly, the Sf9 cells were infected with baculovirus incorporating EcoCas3 gene with a His-tag and TEV recognition site at a multiplicity of infection (MOI) of 2 for 24 h at 28°C and cultured for 4 days at 20°C for sufficient expression of the EcoCas3 protein. The expressed EcoCas3 protein was purified using nickel affinity resin (Ni-NTA; Qiagen, Hilden, Düsseldorf, Germany). The eluted protein from Ni-NTA was digested with TEV protease for removal His-tag. Finally, the protein was purified by size-exclusion chromatography (Superdex 200 Increase 10/300 GL,Thermo Fisher Scientific) in 0.2 M NaCl, 10% glycerol, 1 mM DTT, and 20 mM HEPES-Na (pH 7.0).

Cascade and crRNA complexes (EcoCascade/crRNA) from E. coli were also prepared as previously described.20,39 Briefly, EcoCas11 with a His-tag and HRV3C protease recognition site, EcoCascade operon, and pre-crRNA were transformed into JM109(DE3) strain to express the recombinant EcoCascade/crRNA protein complex. Expressed recombinant EcoCascade/crRNA was purified using Ni-NTA resin; after removal of the His-tag with HRV3C protease, EcoCascade/crRNA was purified in 350 mM NaCl, 1 mM DTT, and 20 mM HEPES-Na (pH 7.0) by size exclusion chromatography (Superdex 200 Increase 10/300 GL, Thermo Fisher Scientific).

Detection of large deletion mutations in human cells

To evaluate the modified crRNAs in human cells, we used HEK293T cells with CRISPR-Cas3 expressing plasmids as previously reported.16 Briefly, Cas3, Cas5, Cas6, Cas7, Cas8, Cas11 and crRNA expression vectors were transfected into HEK293T cells. Two days after the transfection, whole DNA was extracted from collected cells with the Maxwell RSC Genomic DNA Kit (Promega Corp, WI, USA), in accordance with the manufacturer’s protocol. The targeted regions were amplified using Quick Taq HS DyeMix (Toyobo) or Gflex (Takara Bio) and electrophoresed on an agarose gels. The intensity of bands indicating largely deleted mutations were evaluated by ImageJ software.

Microinjection and electroporation into mouse and rat embryos

Pronuclear-stage mouse embryos were prepared by thawing frozen embryos (CLEA Japan Inc.) in KSOM medium (ARK Resource, Kumamoto, Japan) and incubating them for 2–3 h before microinjection. Pronuclear-stage rat embryos were collected from naturally mated female rats that were superovulated by injection with pregnant mare serum gonadotropin (PMSG; Aska Pharmaceutical Co., Tokyo, Japan) and human chorionic gonadotropin (HCG; Aska Pharmaceutical Co.). Those embryos were cultured in a modified Rat KSOM medium (ARK Resource).

In MI, CRISPR-Cas3 mRNAs and crRNAs, plasmids or RNPs were injected into the male pronuclei of embryos using a micromanipulator (Narishige, Tokyo, Japan). For EL, they were electroporated into embryos using a NEPA21 Super Electroporator (Nepa Gene, Chiba, Japan). The poring pulses for the electroporation were as follows: voltage 225 V, pulse width 1.0 ms (for mice) or 2.0 ms (for rats), pulse interval 50 ms, and number of pulses +4. The first and second transfer pulses were as follows: voltage 20 V, pulse width 50 ms, pulse interval 50 ms, and number of pulses +5. The concentrations of each construct for introduction are listed in Table 1. Mouse zygotes were cultured in KSOM medium overnight and divided two-cell embryos were transferred into the oviducts of pseudopregnant females anesthetized with isoflurane (DS Pharma Animal Health Co., Ltd., Osaka, Japan). Rat zygotes at the pronuclear stage were transferred into pseudopregnant females on the same day.

To measure the expression level of GFP in embryos, we captured images using an All-in-One fluorescence microscope (BZ-X800; Keyence, Osaka, Japan). Data were analyzed by two-way ANOVA followed by Tukey’s multiple comparisons test.

Genotyping analysis

For PCR and sequence analysis, the genomic DNA from a piece of the tail was extracted with the Maxwell RSC Genomic DNA Kit (Promega Corp, WI, USA) or MagMAX DNA Multi-Sample Ultra Kit with KingFisher Duo Prime (Thermo Fisher Scientific, MA, USA), in accordance with the manufacturer’s protocol. The targeted region, 5′ genome-donor boundary, inside of knock-in donor, and donor-3′ genome boundary were amplified by PCR with Tks Gflex DNA Polymerase (Takara Bio Inc., Kusatsu, Japan). These PCR amplicons were then directly sequenced using the BigDye Terminator v3.1 (Thermo Fisher Scientific) cycle sequencing mix and the standard protocol. To detect the potential off-target sites of the target sites at Tyr in mice, we detected lower mismatched regions to 32 bases of the on-target sequence except for multiples of six with potential PAM sequences AAG, TAG, AAC, GAG, AGG and ATG as following the previous reports.16 We used the GGGenome (https://GGGenome.dbcls.jp/) for search them. All primer sets used for genotyping analysis are shown in Table S5.

Multiplex genotyping with nanopore sequences

The purified genomic DNA was amplified by PCR using KOD Multi & Epi (Toyobo, Osaka, Japan) and target amplicon primers with the universal sequence (22-mer) located on the 5′ side. 5-fold dilutions of the PCR products were used as templates for nested PCR performed using KOD Multi & Epi and barcode attachment primers. Equal amounts of barcoded PCR products were mixed and column-purified, and the amount of PCR product was adjusted to 20–30 ng/μL. The library was prepared using Ligation Sequencing kit (SQK-LSK109 or SQK-LSK114; ONT, Oxford, UK) and NEBNext End repair/dA-tailing Module NEB Blunt/TA Ligase Master Mix (New England Biolabs, MA, USA), in accordance with the manufacturer’s instructions. The prepared library was loaded into the Flow Cell (R9.4.1 or R10.4.1) and run with MinION Mk1C for 24 h. After base calling, we demultiplexed the barcoding libraries, performed alignment with the default parameter settings of the device, and visualized the results using the Integrative Genomics Viewer (IGV ver. 2.16.2).

Genetic analysis by DAJIN2 system

DAJIN2 (version 0.4.0) generates BAM files to visualize the DAJIN2-reported alleles in a genome browser, as previously reported.22 DAJIN2 classifies obtained nanopore sequencing reads according to their mutation profiles. First, DAJIN2 performs alignment of the sequences in a user-inputted FASTA file using minimap2 via mappy (version 2.26).40 Then, it converts the sequences into the MIDSV format to obtain the mutation profile for each base in the sequence. Then, the mutation rate of the sample is subtracted from the control mutation rate, and the resulting matrix is clustered. The labels assigned through clustering are used as the alleles of the sample. Then, pysam (version 0.21)41 generates sorted BAM files of each allele. Next, the target genome coordinates and chromosome length are obtained from the UCSC Genome Browser,42 in accordance with the user-inputted FASTA file and genome assembly ID. Then, DAJIN2 replaces the chromosome number and chromosome length in SN and LN headers of BAM files. The consensus sequence for each allele is output as a FASTA file and an HTML file using cstag (version 1.0.5).43 In the HTML file, the mutated nucleotides are colored. To generate the consensus sequence, we compared FASTA alleles and compressed MIDSV sequences.

Quantification and statistical analysis

Details of data analysis and quantification are described in each subsection of the STAR Methods section. Data plotting and statistical analyses were performed using Prism 8.4.3 (GraphPad Software). Results involving multiple comparisons are presented as mean ± SD, and statistical significance was determined using one-way or two-way ANOVA followed by Tukey’s multiple comparisons test. Statistical significance was defined as p < 0.001.

Supplemental information

Document S1. Figures S1‒S7 and Tables S1, S2, and S4

Table S3. Off-target candidates for the targets in the mouse genome, related to STAR Methods

Table S5. Sequences used in the study, related to STAR Methods

Document S2. Article plus supplemental information

Acknowledgments

We thank Tomoko Saito and Yoshikazu Totsuka at Institute of Immunology Co., Ltd., for expert technical support with microinjection. We also thank the IMSUT FACS Core laboratory for assistance with flow cytometry analysis. We are also grateful to Edanz (https://jp.edanz.com/ac) for editing a draft of this manuscript. This study was supported in part by 10.13039/501100001691 JSPS 10.13039/501100001691 KAKENHI from the 10.13039/501100001700 Ministry of Education, Culture, Sports, Science, and Technology of Japan (MEXT) (18H03974 , 19KK0401 , 22K19238 , 23H00367 , and 24K02010 ) and 22H04922 (AdAMS), the 10.13039/501100020953 COI-NEXT project from the 10.13039/501100002241 Japan Science and Technology Agency (JPMJPF2010 ), and a grant from the 10.13039/100009619 Japan Agency for Medical Research and Development (24bm12230009 ). The KO and KI strains will be deposited in the National BioResource Project (NBRP)-Mouse and Rat in Japan.

Author contributions

K.Y. and T.M. conceived the study, analyzed the data, and wrote the paper. K.Y. and Y.Y. performed microinjection and electroporation of CRISPR-Cas3 and donor templates into mouse embryos. K.H. performed microinjection of CRISPR-Cas3 into rat embryos. Y.Y., H.T., and K.M. performed the mouse experiments, PCR, and sequence analyses. K.H., R. Iida., and S.I. performed the rat experiments, PCR, and sequence analyses. M.G., R. Ito., and R.T. performed the generation of KI mice of the NOG strain. A.K. and S.T. developed the DAJIN2 system and analyzed the sequencing data. All authors have read and approved the manuscript before submission.

Declaration of interests

K.Y. and T.M. are cofounders of C4U Corporation. T.M. is an outside board member of and K.Y. and K.T. are scientific advisors to C4U.

Supplemental information can be found online at https://doi.org/10.1016/j.crmeth.2024.100833.
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