Homology-based repair induced by CRISPRCas nucleases in mammalian embryo genome editing

Abstract

Recent advances in genome editing, especially CRISPRCas nucleases, have revolutionized both laboratory research and clinical therapeutics. CRISPR-Cas nucleases, together with the DNA damage repair pathway in cells, enable both genetic diversification by classical non-homologous end joining (c-NHEJ) and precise genome modification by homology-based repair (HBR). Genome editing in zygotes is a convenient way to edit the germline, paving the way for animal disease model generation, as well as human embryo genome editing therapy for some life-threatening and incurable diseases. HBR efficiency is highly dependent on the DNA donor that is utilized as a repair template. Here, we review recent progress in improving CRISPR-Cas nuclease-induced HBR in mammalian embryos by designing a suitable DNA donor. Moreover, we want to provide a guide for producing animal disease models and correcting genetic mutations through CRISPR-Cas nuclease-induced HBR in mammalian embryos. Finally, we discuss recent developments in precise genomemodification technology based on the CRISPR-Cas system.

Keywords

homology-based repair (HBR) / genome editing / disease modeling / embryo / precision medicine

References

[1] Adikusuma F, Piltz S, Corbett MA, Turvey M, McColl SR, Helbig KJ, Beard MR, Hughes J, Pomerantz RT, Thomas PQ (2018) Large deletions induced by Cas9 cleavage. Nature 560:E8–E9

[2] Aida T, Chiyo K, Usami T, Ishikubo H, Imahashi R, Wada Y, Tanaka KF, Sakuma T, Yamamoto T, Tanaka K (2015) Cloning-free CRISPR/Cas system facilitates functional cassette knock-in in mice. Genome Biol 16:87

[3] Aida T, Nakade S, Sakuma T, Izu Y, Oishi A, Mochida K, Ishikubo H, Usami T, Aizawa H, Yamamoto T (2016) Gene cassette knock-in in mammalian cells and zygotes by enhanced MMEJ. BMC Genomics 17:979

[4] Aird EJ, Lovendahl KN, St Martin A, Harris RS, Gordon WR (2018) Increasing Cas9-mediated homology-directed repair efficiency through covalent tethering of DNA repair template. Commun Biol 1:54

[5] Alanis-Lobato G, Zohren J, Mccarthy A, Fogarty NME, Kubikova N, Hardman E, Greco M, Wells D, Turner JMA, Niakan KK (2020) Frequent loss-of-heterozygosity in CRISPR-Cas9-edited early human embryos. bioRxiv.

[6] Anzalone AV, Randolph PB, Davis JR, Sousa AA, Koblan LW, Levy JM, Chen PJ, Wilson C, Newby GA, Raguram A (2019) Search-and-replace genome editing without double-strand breaks or donor DNA. Nature 576(7785):149–157

[7] Anzalone AV, Koblan LW, Liu DR (2020) Genome editing with CRISPR-Cas nucleases, base editors, transposases and prime editors. Nat Biotechnol 38:824–844

[8] Bak RO, Porteus MH (2017) CRISPR-Mediated Integration of Large Gene Cassettes Using AAV Donor Vectors. Cell Rep 20:750–756

[9] Baltimore D, Berg P, Botchan M, Carroll D, Charo RA, Church G, Corn JE, Daley GQ, Doudna JA, Fenner M (2015) Biotechnology. A prudent path forward for genomic engineering and germline gene modification. Science 348:36–38

[10] Bedell VM, Wang Y, Campbell JM, Poshusta TL, Starker CG, Krug RG 2nd, Tan W, Penheiter SG, Ma AC, Leung AY (2012) In vivo genome editing using a high-efficiency TALEN system. Nature 491:114–118

[11] Bennardo N, Cheng A, Huang N, Stark JM (2008) Alternative-NHEJ is a mechanistically distinct pathway of mammalian chromosome break repair. PLoS Genet 4:

[12] Bothmer A, Phadke T, Barrera LA, Margulies CM, Lee CS, Buquicchio F, Moss S, Abdulkerim HS, Selleck W, Jayaram H (2017) Characterization of the interplay between DNA repair and CRISPR/Cas9-induced DNA lesions at an endogenous locus. Nat Commun 8:13905

[13] Canaj H, Hussmann JA, Li H, Beckman KA, Goodrich L, Cho NH, Li YJ, Santos DA, McGeever A, Stewart EM (2019) Deep profiling reveals substantial heterogeneity of integration outcomes in CRISPR knock-in experiments. BioRxiv.

[14] Carlson-Stevermer J, Abdeen AA, Kohlenberg L, Goedland M, Molugu K, Lou M, Saha K (2017) Assembly of CRISPR ribonucleoproteins with biotinylated oligonucleotides via an RNA aptamer for precise gene editing. Nat Commun 8:1711

[15] Ceccaldi R, Rondinelli B, D’Andrea AD (2016) Repair pathway choices and consequences at the double-strand break. Trends Cell Biol 26:52–64

[16] Chang HHY, Pannunzio NR, Adachi N, Lieber MR (2017) Nonhomologous DNA end joining and alternative pathways to double-strand break repair. Nat Rev Mol Cell Biol 18:495–506

[17] Chen F, Pruett-Miller SM, Huang Y, Gjoka M, Duda K, Taunton J, Collingwood TN, Frodin M, Davis GD (2011) High-frequency genome editing using ssDNA oligonucleotides with zinc-finger nucleases. Nat Methods 8:753–755

[18] Chen JS, Dagdas YS, Kleinstiver BP, Welch MM, Sousa AA, Harrington LB, Sternberg SH, Joung JK, Yildiz A, Doudna JA (2017) Enhanced proofreading governs CRISPR-Cas9 targeting accuracy. Nature 550:407–410

[19] Chen S, Sun S, Moonen D, Lee C, Lee AY, Schaffer DV, He L (2019) CRISPR-READI: Efficient Generation of Knockin Mice by CRISPR RNP Electroporation and AAV Donor Infection. Cell Rep 27(3780–3789):

[20] Chen Y, Zhi S, Liu W, Wen J, Hu S, Cao T, Sun H, Li Y, Huang L, Liu Y (2020) Development of highly efficient dual-AAV split adenosine base editor for in vivo gene therapy. Small Methods 4(9):2000309

[21] Choulika A, Perrin A, Dujon B, Nicolas JF (1995) Induction of homologous recombination in mammalian chromosomes by using the I-SceI system of Saccharomyces cerevisiae. Mol Cell Biol 15:1968–1973

[22] Chu VT, Weber T, Wefers B, Wurst W, Sander S, Rajewsky K, Kuhn R (2015) Increasing the efficiency of homology-directed repair for CRISPR-Cas9-induced precise gene editing in mammalian cells. Nat Biotechnol 33:543–548

[23] Codner GF, Mianne J, Caulder A, Loeffler J, Fell R, King R, Allan AJ, Mackenzie M, Pike FJ, McCabe CV (2018) Application of long single-stranded DNA donors in genome editing: generation and validation of mouse mutants. BMC Biol 16:70

[24] Cong L, Ran FA, Cox D, Lin S, Barretto R, Habib N, Hsu PD, Wu X, Jiang W, Marraffini LA (2013) Multiplex genome engineering using CRISPR/Cas systems. Science 339:819–823

[25] Cornu TI, Mussolino C, Cathomen T (2017) Refining strategies to translate genome editing to the clinic. Nat Med 23:415–423

[26] De Ravin SS, Li L, Wu X, Choi U, Allen C, Koontz S, Lee J, Theobald-Whiting N, Chu J, Garofalo M (2017) CRISPRCas9 gene repair of hematopoietic stem cells from patients with X-linked chronic granulomatous disease. Sci Transl Med 9: eaah3480

[27] Dever DP, Bak RO, Reinisch A, Camarena J, Washington G, Nicolas CE, Pavel-Dinu M, Saxena N, Wilkens AB, Mantri S (2016) CRISPR/Cas9 beta-globin gene targeting in human haematopoietic stem cells. Nature 539:384–389

[28] DeWitt MA, Magis W, Bray NL, Wang T, Berman JR, Urbinati F, Heo SJ, Mitros T, Munoz DP, Boffelli D (2016) Selection-free genome editing of the sickle mutation in human adult hematopoietic stem/progenitor cells. Sci Transl Med 8:

[29] Doman JL, Raguram A, Newby GA, Liu DR (2020) Evaluation and minimization of Cas9-independent off-target DNA editing by cytosine base editors. Nat Biotechnol 38:620–628

[30] Doudna JA (2020) The promise and challenge of therapeutic genome editing. Nature 578:229–236

[31] Egli D, Zuccaro MV, Kosicki M, Church GM, Bradley A, Jasin M (2018) Inter-homologue repair in fertilized human eggs? Nature 560:E5–E7

[32] Gaj T, Gersbach CA, Barbas CF 3rd (2013) ZFN, TALEN, and CRISPR/Cas-based methods for genome engineering. Trends Biotechnol 31:397–405

[33] Gaj T, Staahl BT, Rodrigues GMC, Limsirichai P, Ekman FK, Doudna JA, Schaffer DV (2017) Targeted gene knock-in by homologydirected genome editing using Cas9 ribonucleoprotein and AAV donor delivery. Nucleic Acids Res 45:

[34] Gaudelli NM, Komor AC, Rees HA, Packer MS, Badran AH, Bryson DI, Liu DR (2017) Programmable base editing of A*T to G*C in genomic DNA without DNA cleavage. Nature 551:464–471

[35] Ge XA, Hunter CP (2019) Efficient homologous recombination in mice using long single stranded DNA and CRISPR Cas9 nickase. G3 (Bethesda) 9:281–286

[36] Grunewald J, Zhou R, Garcia SP, Iyer S, Lareau CA, Aryee MJ, Joung JK (2019a) Transcriptome-wide off-target RNA editing induced by CRISPR-guided DNA base editors. Nature 569:433–437

[37] Grunewald J, Zhou R, Iyer S, Lareau CA, Garcia SP, Aryee MJ, Joung JK (2019b) CRISPR DNA base editors with reduced RNA off-target and self-editing activities. Nat Biotechnol 37:1041–1048

[38] Gu B, Posfai E, Rossant J (2018) Efficient generation of targeted large insertions by microinjection into two-cell-stage mouse embryos. Nat Biotechnol 36:632–637

[39] Gurumurthy CB, O’Brien AR, Quadros RM, Adams J Jr, Alcaide P, Ayabe S, Ballard J, Batra SK, Beauchamp MC, Becker KA (2019) Reproducibility of CRISPR-Cas9 methods for generation of conditional mouse alleles: a multi-center evaluation. Genome Biol 20:171

[40] Hendel A, Kildebeck EJ, Fine EJ, Clark J, Punjya N, Sebastiano V, Bao G, Porteus MH (2014) Quantifying genome-editing outcomes at endogenous loci with SMRT sequencing. Cell Rep 7:293–305

[41] Hisano Y, Sakuma T, Nakade S, Ohga R, Ota S, Okamoto H, Yamamoto T, Kawahara A (2015) Precise in-frame integration of exogenous DNA mediated by CRISPR/Cas9 system in zebrafish. Sci Rep 5:8841

[42] Iyer S, Mir A, Vega-Badillo J, Roscoe BP, Ibraheim R, Zhu LHJ, Lee JY, Liu PP, Luk K, Mintzer E (2019) Efficient homologydirected repair with circular ssDNA donors. bioRxiv.

[43] Jiang F, Taylor DW, Chen JS, Kornfeld JE, Zhou K, Thompson AJ, Nogales E, Doudna JA (2016) Structures of a CRISPR-Cas9 R-loop complex primed for DNA cleavage. Science 351:867–871

[44] Jin S, Zong Y, Gao Q, Zhu Z, Wang Y, Qin P, Liang C, Wang D, Qiu JL, Zhang F (2019) Cytosine, but not adenine, base editors induce genome-wide off-target mutations in rice. Science 364:292–295

[45] Jinek M, Chylinski K, Fonfara I, Hauer M, Doudna JA, Charpentier E (2012) A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 337:816–821

[46] Kan Y, Ruis B, Takasugi T, Hendrickson EA (2017) Mechanisms of precise genome editing using oligonucleotide donors. Genome Res 27:1099–1111

[47] Kim D, Lim K, Kim ST, Yoon SH, Kim K, Ryu SM, Kim JS (2017a) Genome-wide target specificities of CRISPR RNA-guided programmable deaminases. Nat Biotechnol 35:475–480

[48] Kim K, Ryu SM, Kim ST, Baek G, Kim D, Lim K, Chung E, Kim S, Kim JS (2017b) Highly efficient RNA-guided base editing in mouse embryos. Nat Biotechnol 35:435–437

[49] Kim YB, Komor AC, Levy JM, Packer MS, Zhao KT, Liu DR (2017c) Increasing the genome-targeting scope and precision of base editing with engineered Cas9-cytidine deaminase fusions. Nat Biotechnol 35:371–376

[50] Kim D, Kim DE, Lee G, Cho SI, Kim JS (2019) Genome-wide target specificity of CRISPR RNA-guided adenine base editors. Nat Biotechnol 37:430–435

[51] Klompe SE, Vo PLH, Halpin-Healy TS, Sternberg SH (2019) Transposon-encoded CRISPR-Cas systems direct RNA-guided DNA integration. Nature 571:219–225

[52] Kohama Y, Higo S, Masumura Y, Shiba M, Kondo T, Ishizu T, Higo T, Nakamura S, Kameda S, Tabata T (2020) Adeno-associated virus-mediated gene delivery promotes S-phase entry-independent precise targeted integration in cardiomyocytes. Sci Rep 10:15348

[53] Komor AC, Kim YB, Packer MS, Zuris JA, Liu DR (2016) Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage. Nature 533:420–424

[54] Krooss SA, Dai Z, Schmidt F, Rovai A, Fakhiri J, Dhingra A, Yuan Q, Yang T, Balakrishnan A, Steinbruck L (2020) Ex vivo/in vivo gene editing in hepatocytes using “All-in-One” CRISPR-Adeno-Associated Virus vectors with a self-linearizing repair template. iScience 23:

[55] Kurt IC, Zhou R, Iyer S, Garcia SP, Miller BR, Langner LM, Grunewald J, Joung JK (2020) CRISPR C-to-G base editors for inducing targeted DNA transversions in human cells. Nat Biotechnol 39(1):41–46

[56] Kwart D, Paquet D, Teo S, Tessier-Lavigne M (2017) Precise and efficient scarless genome editing in stem cells using CORRECT. Nat Protoc 12:329–354

[57] Lanza DG, Gaspero A, Lorenzo I, Liao L, Zheng P, Wang Y, Deng Y, Cheng C, Zhang C, Seavitt JR (2018) Comparative analysis of single-stranded DNA donors to generate conditional null mouse alleles. BMC Biol 16:69

[58] Lee AY, Lloyd KC (2014) Conditional targeting of Ispd using paired Cas9 nickase and a single DNA template in mice. FEBS Open Bio 4:637–642

[59] Lee K, Mackley VA, Rao A, Chong AT, Dewitt MA, Corn JE, Murthy N (2017) Synthetically modified guide RNA and donor DNA are a versatile platform for CRISPR-Cas9 engineering. Elife 6:

[60] Li H, Beckman KA, Pessino V, Huang B, Weissman JS, Leonetti MD (2019) Design and specificity of long ssDNA donors for CRISPRbased knock-in. BioRxiv.

[61] Liang P, Huang J (2019) Off-target challenge for base editormediated genome editing. Cell Biol Toxicol 35:185–187

[62] Liang P, Xu Y, Zhang X, Ding C, Huang R, Zhang Z, Lv J, Xie X, Chen Y, Li Y (2015) CRISPR/Cas9-mediated gene editing in human tripronuclear zygotes. Protein Cell 6:363–372

[63] Liang P, Sun H, Sun Y, Zhang X, Xie X, Zhang J, Zhang Z, Chen Y, Ding C, Xiong Y (2017) Effective gene editing by high-fidelity base editor 2 in mouse zygotes. Protein Cell 8:601–611

[64] Liang P, Sun H, Zhang X, Xie X, Zhang J, Bai Y, Ouyang X, Zhi S, Xiong Y, Ma W (2018) Effective and precise adenine base editing in mouse zygotes. Protein Cell 9:808–813

[65] Liang P, Huang JJCB, Toxicology (2019a) Off-target challenge for base editor-mediated genome editing. Cell Biol Toxicol 35:185–187

[66] Liang P, Xie X, Zhi S, Sun H, Zhang X, Chen Y, Chen Y, Xiong Y, Ma W, Liu D (2019b) Genome-wide profiling of adenine base editor specificity by EndoV-seq. Nat Commun 10:67

[67] Ling X, Xie B, Gao X, Chang L, Zheng W, Chen H, Huang Y, Tan L, Li M, Liu T (2020). Improving the efficiency of precise genome editing with site-specific Cas9-oligonucleotide conjugates. Sci Adv 6:eaaz0051

[68] Liskay RM, Letsou A, Stachelek JL (1987) Homology requirement for efficient gene conversion between duplicated chromosomal sequences in mammalian cells. Genetics 115:161–167

[69] Liu M, Rehman S, Tang X, Gu K, Fan Q, Chen D, Ma W (2018a) Methodologies for improving HDR efficiency. Front Genet 9:691

[70] Liu Z, Chen M, Chen S, Deng J, Song Y, Lai L, Li Z (2018b) Highly efficient RNA-guided base editing in rabbit. Nat Commun 9:2717

[71] Liu Z, Lu Z, Yang G, Huang S, Li G, Feng S, Liu Y, Li J, Yu W, Zhang Y (2018c) Efficient generation of mouse models of human diseases via ABE- and BE-mediated base editing. Nat Commun 9:2338

[72] Liu Y, Li X, He S, Huang S, Li C, Chen Y, Liu Z, Huang X, Wang X (2020) Efficient generation of mouse models with the prime editing system. Cell Discov 6:27

[73] Ma Y, Zhang X, Shen B, Lu Y, Chen W, Ma J, Bai L, Huang X, Zhang L (2014) Generating rats with conditional alleles using CRISPR/Cas9. Cell research 24:122–125

[74] Ma H, Marti-Gutierrez N, Park SW, Wu J, Lee Y, Suzuki K, Koski A, Ji D, Hayama T, Ahmed R (2017a) Correction of a pathogenic gene mutation in human embryos. Nature 548:413–419

[75] Ma M, Zhuang F, Hu X, Wang B, Wen XZ, Ji JF, Xi JJ (2017b) Efficient generation of mice carrying homozygous double-floxp alleles using the Cas9-Avidin/Biotin-donor DNA system. Cell Res 27:578–581

[76] Ma H, Marti-Gutierrez N, Park SW, Wu J, Hayama T, Darby H, Van Dyken C, Li Y, Koski A, Liang D (2018) Ma et al. reply. Nature 560:E10–E23.

[77] Macintosh KL (2019) Heritable Genome Editing and the Downsides of a Global Moratorium. CRISPR J 2:272–279

[78] Mali P, Yang L, Esvelt KM, Aach J, Guell M, DiCarlo JE, Norville JE, Church GM (2013) RNA-guided human genome engineering via Cas9. Science 339:823–826

[79] Menoret S, De Cian A, Tesson L, Remy S, Usal C, Boule JB, Boix C, Fontaniere S, Creneguy A, Nguyen TH (2015) Homologydirected repair in rodent zygotes using Cas9 and TALEN engineered proteins. Sci Rep 5:14410

[80] Miura H, Gurumurthy CB, Sato T, Sato M, Ohtsuka M (2015) CRISPR/Cas9-based generation of knockdown mice by intronic insertion of artificial microRNA using longer single-stranded DNA. Sci Rep 5:12799

[81] Miyasaka Y, Uno Y, Yoshimi K, Kunihiro Y, Yoshimura T, Tanaka T, Ishikubo H, Hiraoka Y, Takemoto N, Tanaka T (2018) CLICK: one-step generation of conditional knockout mice. BMC Genomics 19:318

[82] Mohr S, Ghanem E, Smith W, Sheeter D, Qin Y, King O, Polioudakis D, Iyer VR, Hunicke-Smith S, Swamy S (2013) Thermostable group II intron reverse transcriptase fusion proteins and their use in cDNA synthesis and next-generation RNA sequencing. RNA 19:958–970

[83] Murgha YE, Rouillard JM, Gulari E (2014) Methods for the preparation of large quantities of complex single-stranded oligonucleotide libraries. PLoS ONE 9:

[84] Nakade S, Tsubota T, Sakane Y, Kume S, Sakamoto N, Obara M, Daimon T, Sezutsu H, Yamamoto T, Sakuma T (2014) Microhomology-mediated end-joining-dependent integration of donor DNA in cells and animals using TALENs and CRISPR/Cas9. Nat Commun 5:5560

[85] Nakao H, Harada T, Nakao K, Kiyonari H, Inoue K, Furuta Y, Aiba A (2016) A possible aid in targeted insertion of large DNA elements by CRISPR/Cas in mouse zygotes. Genesis 54:65–77

[86] Ohtsuka M, Sato M, Miura H, Takabayashi S, Matsuyama M, Koyano T, Arifin N, Nakamura S, Wada K, Gurumurthy CB (2018) i-GONAD: a robust method for in situ germline genome engineering using CRISPR nucleases. Genome Biol 19:25

[87] Okamoto S, Amaishi Y, Maki I, Enoki T, Mineno J (2019) Highly efficient genome editing for single-base substitutions using optimized ssODNs with Cas9-RNPs. Sci Rep 9:4811

[88] Paix A, Schmidt H, Seydoux G (2016) Cas9-assisted recombineering in C. elegans: genome editing using in vivo assembly of linear DNAs. Nucleic Acids Res 44:e128

[89] Palermo G, Miao Y, Walker RC, Jinek M, McCammon JA (2016) Striking Plasticity of CRISPR-Cas9 and Key Role of Non-target DNA, as Revealed by Molecular Simulations. ACS Cent Sci 2:756–763

[90] Palermo G, Miao Y, Walker RC, Jinek M, McCammon JA (2017) CRISPR-Cas9 conformational activation as elucidated from enhanced molecular simulations. Proc Natl Acad Sci USA 114:7260–7265

[91] Papaioannou I, Disterer P, Owen JS (2009) Use of internally nuclease-protected single-strand DNA oligonucleotides and silencing of the mismatch repair protein, MSH2, enhances the replication of corrected cells following gene editing. J Gene Med 11:267–274

[92] Paquet D, Kwart D, Chen A, Sproul A, Jacob S, Teo S, Olsen KM, Gregg A, Noggle S, Tessier-Lavigne M (2016) Efficient introduction of specific homozygous and heterozygous mutations using CRISPR/Cas9. Nature 533:125–129

[93] Pavel-Dinu M, Wiebking V, Dejene BT, Srifa W, Mantri S, Nicolas CE, Lee C, Bao G, Kildebeck EJ, Punjya N (2019) Gene correction for SCID-X1 in long-term hematopoietic stem cells. Nat Commun 10:1634

[94] Pritchard CEJ, Kroese LJ, Huijbers IJ (2017) Direct generation of conditional alleles using CRISPR/Cas9 in mouse zygotes. Methods Mol Biol 1642:21–35

[95] Quadros RM, Miura H, Harms DW, Akatsuka H, Sato T, Aida T, Redder R, Richardson GP, Inagaki Y, Sakai D (2017) 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 18:92

[96] Ran FA, Hsu PD, Wright J, Agarwala V, Scott DA, Zhang F (2013) Genome engineering using the CRISPR-Cas9 system. Nat Protoc 8:2281–2308

[97] Rees HA, Wilson C, Doman JL, Liu DR (2019) Analysis and minimization of cellular RNA editing by DNA adenine base editors. Sci Adv 5:eaax5717

[98] Reichmann J, Nijmeijer B, Hossain MJ, Eguren M, Schneider I, Politi AZ, Roberti MJ, Hufnagel L, Hiiragi T, Ellenberg J (2018) Dualspindle formation in zygotes keeps parental genomes apart in early mammalian embryos. Science 361:189–193

[99] Remy S, Chenouard V, Tesson L, Usal C, Menoret S, Brusselle L, Heslan JM, Nguyen TH, Bellien J, Merot J (2017) Generation of gene-edited rats by delivery of CRISPR/Cas9 protein and donor DNA into intact zygotes using electroporation. Sci Rep 7:16554

[100] Renaud JB, Boix C, Charpentier M, De Cian A, Cochennec J, Duvernois-Berthet E, Perrouault L, Tesson L, Edouard J, Thinard R (2016) Improved genome editing efficiency and flexibility using modified oligonucleotides with TALEN and CRISPR-Cas9 nucleases. Cell Rep 14:2263–2272

[101] Richardson CD, Ray GJ, DeWitt MA, Curie GL, Corn JE (2016) Enhancing homology-directed genome editing by catalytically active and inactive CRISPR-Cas9 using asymmetric donor DNA. Nat Biotechnol 34:339–344

[102] Richardson CD, Kazane KR, Feng SJ, Zelin E, Bray NL, Schafer AJ, Floor SN, Corn JE (2018) CRISPR-Cas9 genome editing in human cells occurs via the Fanconi anemia pathway. Nat Genet 50:1132–1139

[103] Roche PJR, Gytz H, Hussain F, Cameron CJF, Paquette D, Blanchette M, Dostie J, Nagar B, Akavia UD (2018) Doublestranded biotinylated donor enhances homology-directed repair in combination with Cas9 Monoavidin in Mammalian cells. CRISPR J 1:414–430

[104] Rossant J (2018) Gene editing in human development: ethical concerns and practical applications. Development 145

[105] Rouet P, Smih F, Jasin M (1994) Introduction of double-strand breaks into the genome of mouse cells by expression of a rarecutting endonuclease. Mol Cell Biol 14:8096–8106

[106] Ryu SM, Koo T, Kim K, Lim K, Baek G, Kim ST, Kim HS, Kim DE, Lee H, Chung E (2018) Adenine base editing in mouse embryos and an adult mouse model of Duchenne muscular dystrophy. Nat Biotechnol 36:536–539

[107] Sakuma T, Yamamoto T (2017) Magic wands of CRISPR-lots of choices for gene knock-in. Cell Biol Toxicol 33:501–505

[108] Sather BD, Romano Ibarra GS, Sommer K, Curinga G, Hale M, Khan IF, Singh S, Song Y, Gwiazda K, Sahni J (2015) Efficient modification of CCR5 in primary human hematopoietic cells using a megaTAL nuclease and AAV donor template. Sci Transl Med 7:

[109] Savic N, Ringnalda FC, Lindsay H, Berk C, Bargsten K, Li Y, Neri D, Robinson MD, Ciaudo C, Hall J (2018) Covalent linkage of the DNA repair template to the CRISPR-Cas9 nuclease enhances homology-directed repair. Elife 7:

[110] Shen B, Zhang X, Du Y, Wang J, Gong J, Zhang X, Tate PH, Li H, Huang X, Zhang W (2013) Efficient knockin mouse generation by ssDNA oligonucleotides and zinc-finger nuclease assisted homologous recombination in zygotes. PLoS ONE 8:

[111] Shen MW, Arbab M, Hsu JY, Worstell D, Culbertson SJ, Krabbe O, Cassa CA, Liu DR, Gifford DK, Sherwood RI (2018) Predictable and precise template-free CRISPR editing of pathogenic variants. Nature 563:646–651

[112] Stahl S, Hultman T, Olsson A, Moks T, Uhlen M (1988) Solid phase DNA sequencing using the biotin-avidin system. Nucleic Acids Res 16:3025–3038

[113] Sternberg SH, LaFrance B, Kaplan M, Doudna JA (2015) Conformational control of DNA target cleavage by CRISPR-Cas9. Nature 527:110–113

[114] Strecker J, Ladha A, Gardner Z, Schmid-Burgk JL, Makarova KS, Koonin EV, Zhang F (2019) RNA-guided DNA insertion with CRISPR-associated transposases. Science 365:48–53

[115] Surun D, Schneider A, Mircetic J, Neumann K, Lansing F, Paszkowski-Rogacz M, Hanchen V, Lee-Kirsch MA, Buchholz F (2020) Efficient generation and correction of mutations in human iPS cells utilizing mRNAs of CRISPR base editors and prime editors. Genes (Basel) 11(5):511

[116] Suzuki K, Tsunekawa Y, Hernandez-Benitez R, Wu J, Zhu J, Kim EJ, Hatanaka F, Yamamoto M, Araoka T, Li Z (2016) In vivo genome editing via CRISPR/Cas9 mediated homology-independent targeted integration. Nature 540:144–149

[117] Urnov FD, Rebar EJ, Holmes MC, Zhang HS, Gregory PD (2010) Genome editing with engineered zinc finger nucleases. Nat Rev Genet 11:636–646

[118] Wang J, Exline CM, DeClercq JJ, Llewellyn GN, Hayward SB, Li PW, Shivak DA, Surosky RT, Gregory PD, Holmes MC (2015) Homology-driven genome editing in hematopoietic stem and progenitor cells using ZFN mRNA and AAV6 donors. Nat Biotechnol 33:1256–1263

[119] Wang Y, Liu KI, Sutrisnoh NB, Srinivasan H, Zhang J, Li J, Zhang F, Lalith CRJ, Xing H, Shanmugam R (2018) Systematic evaluation of CRISPR-Cas systems reveals design principles for genome editing in human cells. Genome Biol 19:62

[120] Wilde JJ, Aida T, Wienisch M, Zhang Q, Qi P, Feng G (2018) Efficient zygotic genome editing via RAD51-enhanced interhomolog repair. bioRxiv.

[121] Wossidlo M, Nakamura T, Lepikhov K, Marques CJ, Zakhartchenko V, Boiani M, Arand J, Nakano T, Reik W, Walter J (2011) 5-Hydroxymethylcytosine in the mammalian zygote is linked with epigenetic reprogramming. Nat Commun 2:241

[122] Wu Y, Liang D, Wang Y, Bai M, Tang W, Bao S, Yan Z, Li D, Li J (2013) Correction of a genetic disease in mouse via use of CRISPR-Cas9. Cell Stem Cell 13:659–662

[123] Yang H, Wang H, Shivalila CS, Cheng AW, Shi L, Jaenisch R (2013) One-step generation of mice carrying reporter and conditional alleles by CRISPR/Cas-mediated genome engineering. Cell 154:1370–1379

[124] Yang Y, Wang L, Bell P, McMenamin D, He Z, White J, Yu H, Xu C, Morizono H, Musunuru K (2016) A dual AAV system enables the Cas9-mediated correction of a metabolic liver disease in newborn mice. Nat Biotechnol 34:334–338

[125] Yang L, Zhang X, Wang L, Yin S, Zhu B, Xie L, Duan Q, Hu H, Zheng R, Wei Y (2018) Increasing targeting scope of adenosine base editors in mouse and rat embryos through fusion of TadA deaminase with Cas9 variants. Protein Cell 9:814–819

[126] Yao X, Wang X, Hu X, Liu Z, Liu J, Zhou H, Shen X, Wei Y, Huang Z, Ying W (2017) Homology-mediated end joining-based targeted integration using CRISPR/Cas9. Cell Res 27:801–814

[127] Yao X, Liu Z, Wang X, Wang Y, Nie YH, Lai L, Sun R, Shi L, Sun Q, Yang H (2018a) Generation of knock-in cynomolgus monkey via CRISPR/Cas9 editing. Cell research 28:379–382

[128] Yao X, Zhang M, Wang X, Ying W, Hu X, Dai P, Meng F, Shi L, Sun Y, Yao N (2018b) Tild-CRISPR allows for efficient and precise gene Knockin in mouse and human cells. Dev Cell 45(526–536):

[129] Yeh CD, Richardson CD, Corn JE (2019) Advances in genome editing through control of DNA repair pathways. Nat Cell Biol 21:1468–1478

[130] Yin H, Song CQ, Dorkin JR, Zhu LJ, Li Y, Wu Q, Park A, Yang J, Suresh S, Bizhanova A (2016) Therapeutic genome editing by combined viral and non-viral delivery of CRISPR system components in vivo. Nat Biotechnol 34:328–333

[131] Yoon Y, Wang D, Tai PWL, Riley J, Gao G, Rivera-Perez JA (2018) Streamlined ex vivo and in vivo genome editing in mouse embryos using recombinant adeno-associated viruses. Nat Commun 9:412

[132] Yoshimi K, Kaneko T, Voigt B, Mashimo T (2014) Allele-specific genome editing and correction of disease-associated phenotypes in rats using the CRISPR-Cas platform. Nat Commun 5:4240

[133] Yoshimi K, Kunihiro Y, Kaneko T, Nagahora H, Voigt B, Mashimo T (2016) ssODN-mediated knock-in with CRISPR-Cas for large genomic regions in zygotes. Nat Commun 7:10431

[134] Yu Y, Leete TC, Born DA, Young L, Barrera LA, Lee SJ, Rees HA, Ciaramella G, Gaudelli NM (2020) Cytosine base editors with minimized unguided DNA and RNA off-target events and high ontarget activity. Nat Commun 11:2052

[135] Zhang WW, Matlashewski G (2019) Single-strand annealing plays a major role in double-strand DNA break repair following CRISPRCas9 Cleavage in Leishmania. mSphere 4

[136] Zhang JP, Li XL, Li GH, Chen W, Arakaki C, Botimer GD, Baylink D, Zhang L, Wen W, Fu YW (2017) Efficient precise knockin with a double cut HDR donor after CRISPR/Cas9-mediated double-stranded DNA cleavage. Genome Biol 18:35

[137] Zhao D, Li J, Li S, Xin X, Hu M, Price MA, Rosser SJ, Bi C, aZhang X (2020a) Glycosylase base editors enable C-to-A and C-to-G base changes. Nat Biotechnol

[138] Zhao D, Li J, Li S, Xin X, Hu M, Price MA, Rosser SJ, Bi C, Zhang X (2020b) New base editors change C to A in bacteria and C to G in mammalian cells. Nat Biotechnol

[139] Zhou C, Sun Y, Yan R, Liu Y, Zuo E, Gu C, Han L, Wei Y, Hu X, Zeng R (2019) Off-target RNA mutation induced by DNA base editing and its elimination by mutagenesis. Nature 571:275–278

[140] Zuccaro MV, Xu J, Mitchell C, Marin D, Zimmerman R, Rana B, Weinstein E, King RT, Palmerola KL, Smith ME (2020) Allele-specific chromosome removal after Cas9 cleavage in human embryos. Cell 183(6):1650–1664

[141] Zuo E, Sun Y, Wei W, Yuan T, Ying W, Sun H, Yuan L, Steinmetz LM, Li Y, Yang H (2019) Cytosine base editor generates substantial off-target single-nucleotide variants in mouse embryos. Science 364:289–292

[142] Zuo E, Sun Y, Yuan T, He B, Zhou C, Ying W, Liu J, Wei W, Zeng R, Li Y (2020) A rationally engineered cytosine base editor retains high on-target activity while reducing both DNA and RNA off-target effects. Nat Methods 17:600–604

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