Effective gene editing by high-fidelity base editor 2 in mouse zygotes

Abstract

Targeted point mutagenesis through homologous recombination has been widely used in genetic studies and holds considerable promise for repairing diseasecausing mutations in patients. However, problems such as mosaicism and low mutagenesis efficiency continue to pose challenges to clinical application of such approaches. Recently, a base editor (BE) system built on cytidine (C) deaminase and CRISPR/Cas9 technology was developed as an alternative method for targeted point mutagenesis in plant, yeast, and human cells. Base editors convert C in the deamination window to thymidine (T) efficiently, however, it remains unclear whether targeted base editing in mouse embryos is feasible. In this report, we generated a modified highfidelity version of base editor 2 (HF2-BE2), and investigated its base editing efficacy in mouse embryos. We found that HF2-BE2 could convert C to T efficiently, with up to 100% biallelic mutation efficiency in mouse embryos. Unlike BE3, HF2-BE2 could convert C to T on both the target and non-target strand, expanding the editing scope of base editors. Surprisingly, we found HF2-BE2 could also deaminate C that was proximal to the gRNA-binding region. Taken together, our work demonstrates the feasibility of generating point mutations in mouse by base editing, and underscores the need to carefully optimize base editing systems in order to eliminate proximal-site deamination.

Keywords

base editor / high-fidelity / mouse embryos / proximal-site deamination / whole-genome sequencing

References

[1] AndersC, NiewoehnerO, DuerstA, JinekM (2014) Structural basis of PAM-dependent target DNA recognition by the Cas9 endonuclease.Nature513:569–573

[2] BansalV, LibigerO (2011) A probabilistic method for the detection and genotyping of small indels from population-scale sequence data.Bioinformatics27:2047–2053

[3] CapecchiMR (2005) Gene targeting in mice: functional analysis of the mammalian genome for the twenty-first century.Nat Rev Genet6:507–512

[4] ChoSW, KimS, KimJM, KimJS (2013) Targeted genome engineering in human cells with the Cas9 RNA-guided endonuclease.Nat Biotechnol31:230–232

[5] CongL, RanFA, CoxD, LinS, BarrettoR, HabibN, HsuPD, WuX, JiangW, MarraffiniLA (2013) Multiplex genome engineering using CRISPR/Cas systems.Science339:819–823

[6] ConticelloSG (2008) The AID/APOBEC family of nucleic acid mutators.Genome Biol9:229

[7] FuY, ReyonD, JoungJK (2014a) Targeted genome editing in human cells using CRISPR/Cas nucleases and truncated guide RNAs.Methods Enzymol546:21–45

[8] FuY, SanderJD, ReyonD, CascioVM, JoungJK (2014b) Improving CRISPR-Cas nuclease specificity using truncated guide RNAs.Nat Biotechnol32:279–284

[9] GajT, GersbachCA, BarbasCF 3rd (2013) ZFN, TALEN, and CRISPR/Cas-based methods for genome engineering.Trends Biotechnol31:397–405

[10] HarrisRS, Petersen-MahrtSK, NeubergerMS (2002) RNA editing enzyme APOBEC1 and some of its homologs can act as DNA mutators.Mol Cell10:1247–1253

[11] HsuPD, LanderES, ZhangF (2014) Development and applications of CRISPR-Cas9 for genome engineering.Cell157:1262–1278

[12] InuiM, MiyadoM, IgarashiM, TamanoM, KuboA, YamashitaS, AsaharaH, FukamiM, TakadaS (2014) Rapid generation of mouse models with defined point mutations by the CRISPR/Cas9 system.Sci Rep4:5396

[13] JiangW, BikardD, CoxD, ZhangF, MarraffiniLA (2013) RNAguided editing of bacterial genomes using CRISPR-Cas systems.Nat Biotechnol31:233–239

[14] JinekM, ChylinskiK, FonfaraI, HauerM, DoudnaJA, CharpentierE (2012) A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity.Science337:816–821

[15] JinekM, EastA, ChengA, LinS, MaE, DoudnaJ (2013) RNAprogrammed genome editing in human cells.Elife2:e00471

[16] KidaneD, MurphyDL, SweasyJB (2014) Accumulation of abasic sites induces genomic instability in normal human gastric epithelial cells during Helicobacter pylori infection.Oncogenesis3:e128

[17] KimH, KimJS (2014) A guide to genome engineering with programmable nucleases.Nat Rev Genet15:321–334

[18] KimK, RyuSM, KimST, BaekG, KimD, LimK, ChungE, KimS, KimJS (2017a) Highly efficient RNA-guided base editing in mouse embryos.Nat Biotechnol

[19] KimYB, KomorAC, LevyJM, PackerMS, ZhaoKT, LiuDR (2017b) Increasing the genome-targeting scope and precision of base editing with engineered Cas9-cytidine deaminase fusions.Nat Biotechnol

[20] KingmaPS, CorbettAH, BurchamPC, MarnettLJ, OsheroffN (1995) Abasic sites stimulate double-stranded DNA cleavage mediated by topoisomerase II. DNA lesions as endogenous topoisomerase II poisons.J Biol Chem270:21441–21444

[21] KleinstiverBP, PrewMS, TsaiSQ, TopkarVV, NguyenNT, ZhengZ, GonzalesAP, LiZ, PetersonRT, YehJR (2015) Engineered CRISPR-Cas9 nucleases with altered PAM specificities.Nature523:481–485

[22] KleinstiverBP, PattanayakV, PrewMS, TsaiSQ, NguyenNT, ZhengZ, JoungJK (2016) High-fidelity CRISPR-Cas9 nucleases with no detectable genome-wide off-target effects.Nature529:490–495

[23] KomorAC, KimYB, PackerMS, ZurisJA, LiuDR (2016) Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage.Nature533:420–424

[24] KomorAC, BadranAH, LiuDR (2017) CRISPR-based technologies for the manipulation of eukaryotic genomes.Cell168:20–36

[25] LiH, DurbinR (2010) Fast and accurate long-read alignment with Burrows-Wheeler transform.Bioinformatics26:589–595

[26] LiJ, SunY, DuJ, ZhaoY, XiaL (2016) Generation of targeted point mutations in rice by a modified CRISPR/Cas9 system.Mol Plant

[27] LuY, ZhuJK (2016) Precise editing of a target base in the rice genome using a modified CRISPR/Cas9 system.Mol Plant

[28] MaW, PanduriV, SterlingJF, Van HoutenB, GordeninDA, ResnickMA (2009) The transition of closely opposed lesions to doublestrand breaks during long-patch base excision repair is prevented by the coordinated action of DNA polymerase delta and Rad27/Fen1.Mol Cell Biol29:1212–1221

[29] MaY, ZhangJ, YinW, ZhangZ, SongY, ChangX (2016) Targeted AID-mediated mutagenesis (TAM) enables efficient genomic diversification in mammalian cells.Nat Methods13:1029–1035

[30] MaliP, AachJ, StrangesPB, EsveltKM, MoosburnerM, KosuriS, YangL, ChurchGM (2013) CAS9 transcriptional activators for target specificity screening and paired nickases for cooperative genome engineering.Nat Biotechnol31:833–838

[31] McKennaA, HannaM, BanksE, SivachenkoA, CibulskisK, KernytskyA, GarimellaK, AltshulerD, GabrielS, DalyM (2010) The Genome Analysis Toolkit: a MapReduce framework for analyzing next-generation DNA sequencing data.Genome Res20:1297–1303

[32] NishidaK, ArazoeT, YachieN, BannoS, KakimotoM, TabataM, MochizukiM, MiyabeA, ArakiM, HaraKY, (2016) Targeted nucleotide editing using hybrid prokaryotic and vertebrate adaptive immune systems.Science353

[33] NishimasuH, RanFA, HsuPD, KonermannS, ShehataSI, DohmaeN, IshitaniR, ZhangF, NurekiO (2014) Crystal structure of Cas9 in complex with guide RNA and target DNA.Cell156:935–949

[34] PorteusMH (2006) Mammalian gene targeting with designed zinc finger nucleases.Mol Ther13:438–446

[35] PorteusMH, CarrollD (2005) Gene targeting using zinc finger nucleases.Nat Biotechnol23:967–973

[36] QiLS, LarsonMH, GilbertLA, DoudnaJA, WeissmanJS, ArkinAP, LimWA (2013) Repurposing CRISPR as an RNA-guided platform for sequence-specific control of gene expression.Cell152:1173–1183

[37] SaraconiG, SeveriF, SalaC, MattiuzG, ConticelloSG (2014) The RNA editing enzyme APOBEC1 induces somatic mutations and a compatible mutational signature is present in esophageal adenocarcinomas.Genome Biol15:417

[38] SuzukiK, TsunekawaY, Hernandez-BenitezR, WuJ, ZhuJ, KimEJ, HatanakaF, YamamotoM, AraokaT, LiZ (2016) In vivo genome editing via CRISPR/Cas9 mediated homology-independent targeted integration.Nature540:144–149

[39] TessonL, UsalC, MenoretS, LeungE, NilesBJ, RemyS, SantiagoY, VincentAI, MengX, ZhangL (2011) Knockout rats generated by embryo microinjection of TALENs.Nat Biotechnol29:695–696

[40] ThomasKR, CapecchiMR (1987) Site-directed mutagenesis by gene targeting in mouse embryo-derived stem cells.Cell51:503–512

[41] WuY, LiangD, WangY, BaiM, TangW, BaoS, YanZ, LiD, LiJ (2013) Correction of a genetic disease in mouse via use of CRISPR-Cas9.Cell stem cell13:659–662

[42] YangH, WangH, ShivalilaCS, ChengAW, ShiL, JaenischR (2013) One-step generation of mice carrying reporter and conditional alleles by CRISPR/Cas-mediated genome engineering.Cell154:1370–1379

[43] YangL, BriggsAW, ChewWL, MaliP, GuellM, AachJ, GoodmanDB, CoxD, KanY, LeshaE (2016) Engineering and optimising deaminase fusions for genome editing.Nat Commun7:13330

[44] ZhangX, LiangP, DingC, ZhangZ, ZhouJ, XieX, HuangR, SunY, SunH, ZhangJ(2016) Efficient production of gene-modified mice using Staphylococcus aureus Cas9.Sci Rep6:32565

[45] ZongY, WangY, LiC, ZhangR, ChenK, RanY, QiuJL, WangD, GaoC (2017) Precise base editing in rice, wheat and maize with a Cas9- cytidine deaminase fusion.Nat Biotechnol

RIGHTS & PERMISSIONS

2017 The Author(s) 2017. This article is an open access publication