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• W2953856578 abstract "•AAV1 and AAV6 robustly transduce mouse zygotes•CRISPR-READI uses RNP electroporation and AAV donors for efficient mouse genome editing•CRISPR-READI enables complex genome engineering with HDR donor lengths up to 4.9 kb Genetically engineered mouse models harboring large sequence insertions or modifications are critical for a wide range of applications including endogenous gene tagging, conditional knockout, site-specific transgene insertion, and gene replacement; however, existing methods to generate such animals remain laborious and costly. To address this, we developed an approach called CRISPR-READI (CRISPR RNP electroporation and AAV donor infection), combining adeno-associated virus (AAV)-mediated HDR donor delivery with Cas9/sgRNA RNP electroporation to engineer large site-specific modifications in the mouse genome with high efficiency and throughput. We successfully targeted a 774 bp fluorescent reporter, a 2.1 kb CreERT2 driver, and a 3.3 kb expression cassette into endogenous loci in both embryos and live mice. CRISPR-READI is applicable to most widely used knockin schemes requiring donor lengths within the 4.9 kb AAV packaging capacity. Altogether, CRISPR-READI is an efficient, high-throughput, microinjection-free approach for sophisticated mouse genome engineering with potential applications in other mammalian species. Genetically engineered mouse models harboring large sequence insertions or modifications are critical for a wide range of applications including endogenous gene tagging, conditional knockout, site-specific transgene insertion, and gene replacement; however, existing methods to generate such animals remain laborious and costly. To address this, we developed an approach called CRISPR-READI (CRISPR RNP electroporation and AAV donor infection), combining adeno-associated virus (AAV)-mediated HDR donor delivery with Cas9/sgRNA RNP electroporation to engineer large site-specific modifications in the mouse genome with high efficiency and throughput. We successfully targeted a 774 bp fluorescent reporter, a 2.1 kb CreERT2 driver, and a 3.3 kb expression cassette into endogenous loci in both embryos and live mice. CRISPR-READI is applicable to most widely used knockin schemes requiring donor lengths within the 4.9 kb AAV packaging capacity. Altogether, CRISPR-READI is an efficient, high-throughput, microinjection-free approach for sophisticated mouse genome engineering with potential applications in other mammalian species. Genetically modified mice are invaluable assets for investigating mammalian gene function, as well as for modeling human development, physiology, and disease. In particular, knockin mice harboring large sequence insertions or substitutions are essential for a variety of applications, including endogenous gene tagging, conditional gene knockout, site-specific transgene insertion, and gene replacement. Although embryonic stem cells (ESCs) engineered by homologous recombination were classically used to generate these mouse models, the rapid adoption of CRISPR/Cas9 technology has provided an attractive alternative: direct microinjection of CRISPR components with a donor template to trigger homology-directed repair (HDR) and produce edited animals in one generation (Aida et al., 2015Aida T. Chiyo K. Usami T. Ishikubo H. Imahashi R. Wada Y. Tanaka K.F. Sakuma T. Yamamoto T. Tanaka K. Cloning-free CRISPR/Cas system facilitates functional cassette knock-in in mice.Genome Biol. 2015; 16: 87Crossref PubMed Scopus (206) Google Scholar, Cong et al., 2013Cong L. Ran F.A. Cox D. Lin S. Barretto R. Habib N. Hsu P.D. Wu X. Jiang W. Marraffini L.A. Zhang F. Multiplex genome engineering using CRISPR/Cas systems.Science. 2013; 339: 819-823Crossref PubMed Scopus (9981) Google Scholar, Fujii et al., 2013Fujii W. Kawasaki K. Sugiura K. Naito K. Efficient generation of large-scale genome-modified mice using gRNA and CAS9 endonuclease.Nucleic Acids Res. 2013; 41: e187Crossref PubMed Scopus (166) Google Scholar, Jinek et al., 2012Jinek M. Chylinski K. Fonfara I. Hauer M. Doudna J.A. Charpentier E. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity.Science. 2012; 337: 816-821Crossref PubMed Scopus (9327) Google Scholar, Li et al., 2013Li D. Qiu Z. Shao Y. Chen Y. Guan Y. Liu M. Li Y. Gao N. Wang L. Lu X. et al.Heritable gene targeting in the mouse and rat using a CRISPR-Cas system.Nat. Biotechnol. 2013; 31: 681-683Crossref PubMed Scopus (534) Google Scholar, Nakagawa et al., 2016Nakagawa Y. Sakuma T. Nishimichi N. Yokosaki Y. Yanaka N. Takeo T. Nakagata N. Yamamoto T. Ultra-superovulation for the CRISPR-Cas9-mediated production of gene-knockout, single-amino-acid-substituted, and floxed mice.Biol. Open. 2016; 5: 1142-1148Crossref PubMed Scopus (25) Google Scholar, Wang et al., 2013Wang H. Yang H. Shivalila C.S. Dawlaty M.M. Cheng A.W. Zhang F. Jaenisch R. One-step generation of mice carrying mutations in multiple genes by CRISPR/Cas-mediated genome engineering.Cell. 2013; 153: 910-918Abstract Full Text Full Text PDF PubMed Scopus (2585) Google Scholar, Yang et al., 2013Yang H. Wang H. Shivalila C.S. Cheng A.W. Shi L. Jaenisch R. One-step generation of mice carrying reporter and conditional alleles by CRISPR/Cas-mediated genome engineering.Cell. 2013; 154: 1370-1379Abstract Full Text Full Text PDF PubMed Scopus (1185) Google Scholar). Multiple technical refinements have also been developed to increase the rate of HDR in mouse zygotes through the use of single-stranded DNA (ssDNA), linearized doubled-stranded DNA (dsDNA), or chemically modified dsDNA donor templates, co-injection of HDR-stimulating compounds, and timed microinjection in two-cell-stage embryos (Gu et al., 2018Gu B. Posfai E. Rossant J. Efficient generation of targeted large insertions by microinjection into two-cell-stage mouse embryos.Nat. Biotechnol. 2018; 36: 632-637Crossref PubMed Scopus (143) Google Scholar, Maruyama et al., 2015Maruyama T. Dougan S.K. Truttmann M.C. Bilate A.M. Ingram J.R. Ploegh H.L. Increasing the efficiency of precise genome editing with CRISPR-Cas9 by inhibition of nonhomologous end joining.Nat. Biotechnol. 2015; 33: 538-542Crossref PubMed Scopus (766) Google Scholar, Miura et al., 2018Miura H. Quadros R.M. Gurumurthy C.B. Ohtsuka M. Easi-CRISPR for creating knock-in and conditional knockout mouse models using long ssDNA donors.Nat. Protoc. 2018; 13: 195-215Crossref PubMed Scopus (125) Google Scholar, Quadros et al., 2017Quadros R.M. Miura H. Harms D.W. Akatsuka H. Sato T. Aida T. Redder R. Richardson G.P. Inagaki Y. Sakai D. et al.Easi-CRISPR: a robust method for one-step generation of mice carrying conditional and insertion alleles using long ssDNA donors and CRISPR ribonucleoproteins.Genome Biol. 2017; 18: 92Crossref PubMed Scopus (224) Google Scholar, Yao et al., 2018Yao X. Zhang M. Wang X. Ying W. Hu X. Dai P. Meng F. Shi L. Sun Y. Yao N. et al.Tild-CRISPR Allows for Efficient and Precise Gene Knockin in Mouse and Human Cells.Dev. Cell. 2018; 45: 526-536.e5Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar, Yoshimi et al., 2016Yoshimi K. Kunihiro Y. Kaneko T. Nagahora H. Voigt B. Mashimo T. ssODN-mediated knock-in with CRISPR-Cas for large genomic regions in zygotes.Nat. Commun. 2016; 7: 10431Crossref PubMed Scopus (231) Google Scholar). Despite these improvements, complex editing in mice remains challenging because of the need for microinjection, a costly procedure with a high technical barrier and low throughput (Brinster et al., 1985Brinster R.L. Chen H.Y. Trumbauer M.E. Yagle M.K. Palmiter R.D. Factors affecting the efficiency of introducing foreign DNA into mice by microinjecting eggs.Proc. Natl. Acad. Sci. U S A. 1985; 82: 4438-4442Crossref PubMed Scopus (776) Google Scholar, Nagy et al., 2003Nagy A. Gertenstein M. Vintersten K. Behringer R. Manipulating the Mouse Embryo: A Laboratory Manual..3rd. Cold Spring Harbor Laboratory Press, 2003Google Scholar). Recently, we and others developed electroporation-based methods to deliver CRISPR reagents into zygotes for highly efficient genome engineering; these strategies have garnered popularity because of improved throughput, technical ease, and cost-effectiveness across multiple mammalian species (Chen et al., 2016Chen S. Lee B. Lee A.Y.-F. Modzelewski A.J. He L. highly efficient mouse genome editing by CRISPR ribonucleoprotein electroporation of zygotes.J. Biol. Chem. 2016; 291: 14457-14467Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar, Hashimoto et al., 2016Hashimoto M. Yamashita Y. Takemoto T. Electroporation of Cas9 protein/sgRNA into early pronuclear zygotes generates non-mosaic mutants in the mouse.Dev. Biol. 2016; 418: 1-9Crossref PubMed Scopus (142) Google Scholar, Hashimoto and Takemoto, 2015Hashimoto M. Takemoto T. Electroporation enables the efficient mRNA delivery into the mouse zygotes and facilitates CRISPR/Cas9-based genome editing.Sci. Rep. 2015; 5: 11315Crossref PubMed Scopus (201) Google Scholar, Hur et al., 2016Hur J.K. Kim K. Been K.W. Baek G. Ye S. Hur J.W. Ryu S.-M. Lee Y.S. Kim J.-S. Targeted mutagenesis in mice by electroporation of Cpf1 ribonucleoproteins.Nat. Biotechnol. 2016; 34: 807-808Crossref PubMed Scopus (152) Google Scholar, Miyasaka et al., 2018Miyasaka Y. Uno Y. Yoshimi K. Kunihiro Y. Yoshimura T. Tanaka T. Ishikubo H. Hiraoka Y. Takemoto N. Tanaka T. et al.CLICK: one-step generation of conditional knockout mice.BMC Genomics. 2018; 19: 318Crossref PubMed Scopus (50) Google Scholar, Ohtsuka et al., 2018Ohtsuka M. Sato M. Miura H. Takabayashi S. Matsuyama M. Koyano T. Arifin N. Nakamura S. Wada K. Gurumurthy C.B. i-GONAD: a robust method for in situ germline genome engineering using CRISPR nucleases.Genome Biol. 2018; 19: 25Crossref PubMed Scopus (78) Google Scholar, Remy et al., 2017Remy S. Chenouard V. Tesson L. Usal C. Ménoret S. Brusselle L. Heslan J.-M. Nguyen T.H. Bellien J. Merot J. et al.Generation of gene-edited rats by delivery of CRISPR/Cas9 protein and donor DNA into intact zygotes using electroporation.Sci. Rep. 2017; 7: 16554Crossref PubMed Scopus (50) Google Scholar, Takahashi et al., 2015Takahashi G. Gurumurthy C.B. Wada K. Miura H. Sato M. Ohtsuka M. GONAD: Genome-Editing via Oviductal Nucleic Acids Delivery system: a novel microinjection independent genome engineering method in mice.Sci. Rep. 2015; 5: 11406Crossref PubMed Scopus (69) Google Scholar, Tanihara et al., 2016Tanihara F. Takemoto T. Kitagawa E. Rao S. Do L.T.K. Onishi A. Yamashita Y. Kosugi C. Suzuki H. Sembon S. et al.Somatic cell reprogramming-free generation of genetically modified pigs.Sci. Adv. 2016; 2: e1600803Crossref PubMed Scopus (65) Google Scholar, Wang et al., 2016Wang W. Kutny P.M. Byers S.L. Longstaff C.J. DaCosta M.J. Pang C. Zhang Y. Taft R.A. Buaas F.W. Wang H. Delivery of Cas9 protein into mouse zygotes through a series of electroporation dramatically increased the efficiency of model creation.J. Genet. Genomics. 2016; 43: 319-327Crossref PubMed Scopus (60) Google Scholar). Although electroporation-based approaches are highly effective in introducing indel mutations, large deletions, and small insertions or substitutions, the use of short (typically <200 nt) single-stranded oligodeoxynucleotides (ssODNs) as HDR donors renders these techniques unsuitable for editing schemes involving targeted insertion of multi-kilobase sequences. Alternatively, long ssODNs (lssODNs) can be applied for more complex editing, and several lssODN synthesis methods, including reverse transcription, plasmid nicking followed by gel extraction, and selective strand phosphorylation or degradation, have been reported (Chen et al., 2011Chen F. Pruett-Miller S.M. Huang Y. Gjoka M. Duda K. Taunton J. Collingwood T.N. Frodin M. Davis G.D. High-frequency genome editing using ssDNA oligonucleotides with zinc-finger nucleases.Nat. Methods. 2011; 8: 753-755Crossref PubMed Scopus (350) Google Scholar, Li et al., 2017Li H. Beckman K.A. Pessino V. Huang B. Weissman J.S. Leonetti M.D. Design and specificity of long ssDNA donors for CRISPR-based knock-in.bioRxiv. 2017; https://doi.org/10.1101/178905Crossref Scopus (0) Google Scholar, Miura et al., 2018Miura H. Quadros R.M. Gurumurthy C.B. Ohtsuka M. Easi-CRISPR for creating knock-in and conditional knockout mouse models using long ssDNA donors.Nat. Protoc. 2018; 13: 195-215Crossref PubMed Scopus (125) Google Scholar, Miyasaka et al., 2018Miyasaka Y. Uno Y. Yoshimi K. Kunihiro Y. Yoshimura T. Tanaka T. Ishikubo H. Hiraoka Y. Takemoto N. Tanaka T. et al.CLICK: one-step generation of conditional knockout mice.BMC Genomics. 2018; 19: 318Crossref PubMed Scopus (50) Google Scholar, Ohtsuka et al., 2018Ohtsuka M. Sato M. Miura H. Takabayashi S. Matsuyama M. Koyano T. Arifin N. Nakamura S. Wada K. Gurumurthy C.B. i-GONAD: a robust method for in situ germline genome engineering using CRISPR nucleases.Genome Biol. 2018; 19: 25Crossref PubMed Scopus (78) Google Scholar, Quadros et al., 2017Quadros R.M. Miura H. Harms D.W. Akatsuka H. Sato T. Aida T. Redder R. Richardson G.P. Inagaki Y. Sakai D. et al.Easi-CRISPR: a robust method for one-step generation of mice carrying conditional and insertion alleles using long ssDNA donors and CRISPR ribonucleoproteins.Genome Biol. 2017; 18: 92Crossref PubMed Scopus (224) Google Scholar, Remy et al., 2017Remy S. Chenouard V. Tesson L. Usal C. Ménoret S. Brusselle L. Heslan J.-M. Nguyen T.H. Bellien J. Merot J. et al.Generation of gene-edited rats by delivery of CRISPR/Cas9 protein and donor DNA into intact zygotes using electroporation.Sci. Rep. 2017; 7: 16554Crossref PubMed Scopus (50) Google Scholar, Takahashi et al., 2015Takahashi G. Gurumurthy C.B. Wada K. Miura H. Sato M. Ohtsuka M. GONAD: Genome-Editing via Oviductal Nucleic Acids Delivery system: a novel microinjection independent genome engineering method in mice.Sci. Rep. 2015; 5: 11406Crossref PubMed Scopus (69) Google Scholar, Yoshimi et al., 2016Yoshimi K. Kunihiro Y. Kaneko T. Nagahora H. Voigt B. Mashimo T. ssODN-mediated knock-in with CRISPR-Cas for large genomic regions in zygotes.Nat. Commun. 2016; 7: 10431Crossref PubMed Scopus (231) Google Scholar). However, these protocols carry several notable drawbacks, including limited length (typically ≤2000 nt), GC-content and other sequence constraints, suboptimal yield, and costly reagents (Chen et al., 2011Chen F. Pruett-Miller S.M. Huang Y. Gjoka M. Duda K. Taunton J. Collingwood T.N. Frodin M. Davis G.D. High-frequency genome editing using ssDNA oligonucleotides with zinc-finger nucleases.Nat. Methods. 2011; 8: 753-755Crossref PubMed Scopus (350) Google Scholar, Li et al., 2017Li H. Beckman K.A. Pessino V. Huang B. Weissman J.S. Leonetti M.D. Design and specificity of long ssDNA donors for CRISPR-based knock-in.bioRxiv. 2017; https://doi.org/10.1101/178905Crossref Scopus (0) Google Scholar, Miura et al., 2018Miura H. Quadros R.M. Gurumurthy C.B. Ohtsuka M. Easi-CRISPR for creating knock-in and conditional knockout mouse models using long ssDNA donors.Nat. Protoc. 2018; 13: 195-215Crossref PubMed Scopus (125) Google Scholar, Miyasaka et al., 2018Miyasaka Y. Uno Y. Yoshimi K. Kunihiro Y. Yoshimura T. Tanaka T. Ishikubo H. Hiraoka Y. Takemoto N. Tanaka T. et al.CLICK: one-step generation of conditional knockout mice.BMC Genomics. 2018; 19: 318Crossref PubMed Scopus (50) Google Scholar, Quadros et al., 2017Quadros R.M. Miura H. Harms D.W. Akatsuka H. Sato T. Aida T. Redder R. Richardson G.P. Inagaki Y. Sakai D. et al.Easi-CRISPR: a robust method for one-step generation of mice carrying conditional and insertion alleles using long ssDNA donors and CRISPR ribonucleoproteins.Genome Biol. 2017; 18: 92Crossref PubMed Scopus (224) Google Scholar). Furthermore, electroporation of lssODNs typically yields much lower knockin efficiencies than that of short ssODNs, likely because of inefficient lssODN delivery into zygotes (Ohtsuka et al., 2018Ohtsuka M. Sato M. Miura H. Takabayashi S. Matsuyama M. Koyano T. Arifin N. Nakamura S. Wada K. Gurumurthy C.B. i-GONAD: a robust method for in situ germline genome engineering using CRISPR nucleases.Genome Biol. 2018; 19: 25Crossref PubMed Scopus (78) Google Scholar, Yoshimi et al., 2016Yoshimi K. Kunihiro Y. Kaneko T. Nagahora H. Voigt B. Mashimo T. ssODN-mediated knock-in with CRISPR-Cas for large genomic regions in zygotes.Nat. Commun. 2016; 7: 10431Crossref PubMed Scopus (231) Google Scholar). Due to these restrictions, targeted knockins are predominantly performed using microinjection or ESC-based approaches with plasmids or linearized dsDNA as HDR donors. Here, we developed a strategy for complex mouse genome engineering that leverages the simplicity and throughput of CRISPR electroporation while overcoming the current limitations of large HDR donor delivery. Recombinant adeno-associated virus (rAAV) has emerged as a safe and efficient gene delivery vector with the innate ability to transduce mammalian cells and stimulate gene targeting by promoting homologous recombination (Hiramoto et al., 2018Hiramoto T. Li L.B. Funk S.E. Hirata R.K. Russell D.W. Nuclease-free adeno-associated virus-mediated Il2rg gene editing in X-SCID mice.Mol. Ther. 2018; 26: 1255-1265Abstract Full Text Full Text PDF PubMed Scopus (11) Google Scholar, Hirata et al., 2002Hirata R. Chamberlain J. Dong R. Russell D.W. Targeted transgene insertion into human chromosomes by adeno-associated virus vectors.Nat. Biotechnol. 2002; 20: 735-738Crossref PubMed Scopus (145) Google Scholar, Khan et al., 2011Khan I.F. Hirata R.K. Russell D.W. AAV-mediated gene targeting methods for human cells.Nat. Protoc. 2011; 6: 482-501Crossref PubMed Scopus (138) Google Scholar, Kotterman et al., 2015Kotterman M.A. Chalberg T.W. Schaffer D.V. Viral vectors for gene therapy: translational and clinical outlook.Annu. Rev. Biomed. Eng. 2015; 17: 63-89Crossref PubMed Scopus (299) Google Scholar, Russell and Hirata, 1998Russell D.W. Hirata R.K. Human gene targeting by viral vectors.Nat. Genet. 1998; 18: 325-330Crossref PubMed Scopus (266) Google Scholar). In particular, rAAV donors packaged with ssDNA genomes have successfully served as repair templates for HDR upon Cas9-mediated cleavage in mammalian cell lines, enhancing the efficiency of site-specific gene integration by >10-fold relative to plasmid donor nucleofection (Gaj et al., 2017Gaj T. Staahl B.T. Rodrigues G.M.C. Limsirichai P. Ekman F.K. Doudna J.A. Schaffer D.V. Targeted gene knock-in by homology-directed genome editing using Cas9 ribonucleoprotein and AAV donor delivery.Nucleic Acids Res. 2017; 45: e98Crossref PubMed Scopus (52) Google Scholar). More recently, three separate rAAV6 vectors were used to deliver Cas9, single-guide RNA (sgRNA), and donor components into mouse zygotes to mediate gene targeting, demonstrating targeted knockin of up to ∼700 bp (Yoon et al., 2018Yoon Y. Wang D. Tai P.W.L. Riley J. Gao G. Rivera-Pérez J.A. Streamlined ex vivo and in vivo genome editing in mouse embryos using recombinant adeno-associated viruses.Nat. Commun. 2018; 9: 412Crossref PubMed Scopus (41) Google Scholar). Of note, AAV’s naturally occurring ssDNA genomes can be engineered into a duplexed form, termed self-complementary AAV (scAAV), which can also facilitate efficient gene targeting (Hirsch et al., 2010Hirsch M.L. Green L. Porteus M.H. Samulski R.J. Self-complementary AAV mediates gene targeting and enhances endonuclease delivery for double-strand break repair.Gene Ther. 2010; 17: 1175-1180Crossref PubMed Scopus (32) Google Scholar). In this study, we identified AAV1 as an optimal naturally occurring serotype for highly efficient transduction of mouse zygotes. We then developed CRISPR-READI (CRISPR RNP electroporation and AAV donor infection), a highly efficient method that combines AAV-mediated HDR donor delivery with Cas9/sgRNA RNP electroporation to generate complex genome modifications in mice. Using CRISPR-READI, we successfully inserted a 774 bp fluorescent reporter or a 2.1 kb CreERT2 cassette into the endogenous Sox2 locus, as well as a 3.3 kb gene expression cassette into the Rosa26 locus, in both preimplantation stage embryos and mice. CRISPR-READI permits the use of HDR donors up to 4.9 kb, the packaging capacity of recombinant AAVs (Dong et al., 1996Dong J.-Y. Fan P.-D. Frizzell R.A. Quantitative analysis of the packaging capacity of recombinant adeno-associated virus.Hum. Gene Ther. 1996; 7: 2101-2112Crossref PubMed Scopus (395) Google Scholar), and thus enables a broad range of complex genome modifications, including site-specific integration of reporters, Cre drivers, and expression cassettes. Altogether, CRISPR-READI produces genetically edited animals harboring multi-kilobase modifications with unparalleled efficiency and throughput. To characterize the native capacity of rAAV vectors to penetrate the zona pellucida and deliver the viral genome into mouse zygotes, we infected C57BL/6J mouse zygotes with a panel of eight natural AAV serotypes packaged with a self-complementary CMV-eGFP reporter (scAAV-CMV-eGFP) at a dose of 2 × 108 genome copies (GCs) per culture droplet (Figure 1A; Figure S1). Strong fluorescent signal was detected only for serotypes 1 and 6 (scAAV1 and scAAV6), which yielded comparable signal intensity by the four- to eight-cell stage, suggesting that efficient rAAV-mediated DNA delivery can be achieved in mouse zygotes (Figure 1A; Figure S1). While ∼20% of scAAV1- or scAAV6-transduced embryos exhibited bright eGFP fluorescence, all treated embryos displayed signal above background (Figure S1). Although a previous study used rAAV6 for mouse editing (Yoon et al., 2018Yoon Y. Wang D. Tai P.W.L. Riley J. Gao G. Rivera-Pérez J.A. Streamlined ex vivo and in vivo genome editing in mouse embryos using recombinant adeno-associated viruses.Nat. Commun. 2018; 9: 412Crossref PubMed Scopus (41) Google Scholar), we selected rAAV1 for knockin experiments with CRISPR-READI because of the superior viral yield of rAAV1 compared with rAAV6 when produced in HEK293T cells (Vandenberghe et al., 2010Vandenberghe L.H. Xiao R. Lock M. Lin J. Korn M. Wilson J.M. Efficient serotype-dependent release of functional vector into the culture medium during adeno-associated virus manufacturing.Hum. Gene Ther. 2010; 21: 1251-1257Crossref PubMed Scopus (87) Google Scholar). To generate edited animals with CRISPR-READI, we devised a workflow wherein mouse zygotes are harvested from superovulated females, infected with rAAV1 donors, electroporated with pre-assembled Cas9/sgRNA RNPs, and cultured to the two-cell stage before transferring to the oviducts of pseudopregnant females (Figure 1B). In a proof-of-principle CRISPR-READI experiment, we designed a strategy to insert an EcoRI restriction site into Tyrosinase (Tyr) exon 1 using a ∼960 bp donor (including ∼480 bp homology arms) that was packaged into an scAAV1 vector (scAAV1-Tyr) (Figure 1C). As intracellular trafficking, nuclear localization, and capsid uncoating precede AAV-mediated HDR editing (Nonnenmacher and Weber, 2012Nonnenmacher M. Weber T. Intracellular transport of recombinant adeno-associated virus vectors.Gene Ther. 2012; 19: 649-658Crossref PubMed Scopus (179) Google Scholar), we pre-incubated the zygotes for 5 h with three doses of scAAV1-Tyr (1.1 × 108, 4.3 × 108, and 1.7 × 109 GCs), introduced Cas9/sgRNA RNP by electroporation, and then continued viral incubation for a total of 24 h (Figure 1D). Treated embryos were grown for another 48 h post-transduction until the morula or early blastocyst stage, and editing efficiency was assessed by screening for the engineered EcoRI site using restriction fragment-length polymorphism (RFLP) analysis (Chen et al., 2016Chen S. Lee B. Lee A.Y.-F. Modzelewski A.J. He L. highly efficient mouse genome editing by CRISPR ribonucleoprotein electroporation of zygotes.J. Biol. Chem. 2016; 291: 14457-14467Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar). As anticipated, HDR editing efficiency was dependent on viral titer. The highest AAV dose, 1.7 × 109 GCs, resulted in 48% (9 of 19) embryos harboring the precise sequence substitution, with 11% (2 of 19) showing bi-allelic HDR-mediated editing, while the lower doses of 1.1 × 108 and 4.3 × 108 GCs yielded HDR rates of 33% and 21%, respectively (Figure 1D; Figure S2A; Table S1). Although increased AAV dosage elicited a higher HDR rate, it was also correlated with a moderate reduction in embryo viability, as 41% (19 of 46) embryos developed to the morula stage in culture when treated with 1.7 × 109 GCs, while 63% (25 of 43) embryos did so with 1.1 × 108 GCs (Figure 1D; Table S1). Altogether, these results suggest that the optimal AAV dose for CRISPR-READI ranges from 108 to 109 GCs, and a balance between HDR rate and embryo viability should be considered when determining AAV dosage for each CRISPR-READI experiment. HDR requires nuclear co-localization of Cas9, sgRNA, and the donor template. Because multiple intracellular trafficking steps must occur prior to nuclear import of the AAV genome (Nonnenmacher and Weber, 2012Nonnenmacher M. Weber T. Intracellular transport of recombinant adeno-associated virus vectors.Gene Ther. 2012; 19: 649-658Crossref PubMed Scopus (179) Google Scholar), we reasoned that optimizing the timing of RNP delivery relative to rAAV transduction would enhance HDR editing efficiency. Using the Tyr editing scheme described above (Figure 1C), we pre-incubated zygotes with the rAAV donor vector for 0, 2, 4, 6, 8, or 10 h prior to RNP electroporation (Figure 1E). In all conditions, we achieved a minimum of 40% HDR-mediated editing (Figure 1E; Figure S2B; Table S1). We found that RNP electroporation at 6 h after rAAV transduction resulted in maximal editing, with 77% (10 of 13) assayed embryos harboring the precise sequence modification and 15% (2 of 13) showing bi-allelic editing. Notably, this exceeds the HDR frequencies we previously achieved with CRISPR-EZ, using electroporation of short ssODNs (46%) (Chen et al., 2016Chen S. Lee B. Lee A.Y.-F. Modzelewski A.J. He L. highly efficient mouse genome editing by CRISPR ribonucleoprotein electroporation of zygotes.J. Biol. Chem. 2016; 291: 14457-14467Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar). Henceforth, we settled on 6 h of rAAV pre-incubation prior to RNP electroporation for subsequent CRISPR-READI experiments. As double-strand breaks induced by Cas9 can be repaired either by non-homologous end joining (NHEJ) or HDR, we next sought to characterize the extent of NHEJ in this system. To quantitatively measure the frequency of HDR versus NHEJ-mediated editing, we treated embryos using scAAV1-Tyr at a dosage of 1.7 × 109 GCs, amplified the edited region using PCR, and clonally analyzed editing events by Sanger sequencing (10 clones from each of 10 embryos). As expected, NHEJ occurs frequently, as 90% (9 of 10) of embryos carried indels. In comparison, 70% (7 of 10) harbored the desired HDR-mediated point mutation, most of which exhibited both HDR- and NHEJ-mediated editing events (Figure S3A). HDR frequencies of each edited embryo ranged from 10% to 67%, revealing a degree of editing mosaicism. AAV vectors can be packaged in either single-stranded (ssAAV) or self-complementary (scAAV) forms, the latter of which bypasses the necessity of second-strand synthesis and in turn enhances transduction efficiency (McCarty et al., 2003McCarty D.M. Fu H. Monahan P.E. Toulson C.E. Naik P. Samulski R.J. Adeno-associated virus terminal repeat (TR) mutant generates self-complementary vectors to overcome the rate-limiting step to transduction in vivo.Gene Ther. 2003; 10: 2112-2118Crossref PubMed Scopus (416) Google Scholar). In a previous study, scAAV vectors were also shown to modestly enhance gene correction in mammalian cells in vitro relative to their single-stranded equivalents, possibly because of their increased stability, their two copies of the donor template in opposite polarities, or their ability to undergo double crossover events (Hirsch et al., 2010Hirsch M.L. Green L. Porteus M.H. Samulski R.J. Self-complementary AAV mediates gene targeting and enhances endonuclease delivery for double-strand break repair.Gene Ther. 2010; 17: 1175-1180Crossref PubMed Scopus (32) Google Scholar). Using the same Tyr editing scheme, we found that scAAV1-Tyr moderately outperformed ssAAV1-Tyr by ∼17% in HDR efficiency, demonstrating that scAAV vectors promote HDR-mediated editing in mouse embryos (Figure S3B; Table S1). However, it is important to note that scAAV vectors possess half the packaging capacity of ssAAV and thus limit the length of HDR donors to ∼2.4 kb (McCarty et al., 2003McCarty D.M. Fu H. Monahan P.E. Toulson C.E. Naik P. Samulski R.J. Adeno-associated virus terminal repeat (TR) mutant generates self-complementary vectors to overcome the rate-limiting step to transduction in vivo.Gene Ther. 2003; 10: 2112-2118Crossref PubMed Scopus (416) Google Scholar). A common strategy to characterize the expression pattern of a gene is to insert a fluorescent reporter under the control of its endogenous promoter. We designed a 1.7 kb scAAV1 donor vector consisting of a 774 bp P2A-mStrawberry insert flanked by ∼480 bp homology arms (scAAV1-Sox2-mStr) to knock in the P2A-mStrawberry cassette immediately downstream of the 3′ terminus of the Sox2 open reading frame (ORF) (Figure 2A). Mouse zygotes were treated with CRISPR-READI using scAAV1-Sox2-mStr at three doses (3.2 × 107, 1.3 × 108, and 5.0 × 108 GCs) and cultured to the blastocyst stage. Robust red fluorescence was detected in the inner cell mass (ICM) of a subset of the resulting blastocysts, recapitulating endogenous Sox2 expression (Figure 2B). The scAAV1-Sox2-mStr donor at a dose of 1.3 × 108 GCs produced the highest number of correctly targeted embryos, with 33% (6 of 18) of blastocysts showing ICM-specific fluorescent signal (Figure 2B; Figure S4A; Table S1). Because many widely use" @default.
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• W2953856578 date "2019-06-01" @default.
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• W2953856578 title "CRISPR-READI: Efficient Generation of Knockin Mice by CRISPR RNP Electroporation and AAV Donor Infection" @default.
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• W2953856578 doi "https://doi.org/10.1016/j.celrep.2019.05.103" @default.
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