DNA repair after Cas9 cutting can result in deletions/insertions, genomic rearrangements, and rare nucleotide substitutions. However, most work has only focused on deletions/insertions resulting from repair after CRISPR-Cas9 action. Here, we comprehensively analyzed the editing outcomes induced by CRISPR-Cas9 treatment in yeast Xanthophyllomyces dendrorhous by Sanger and Illumina sequencing and identified diverse DNA repair patterns, including DNA deletions, interchromosomal translocations, and on-target nucleotide substitutions (point mutations). Some deletions were observed repeatedly, and others, especially large deletions, varied in size. Genome sequencing and structural variation analysis showed that the interchromosomal translocations happened between Cas9 target sites and the endogenous ADH4 promoter. In contrast to previous studies, analysis revealed that the on-target point mutations were not random. Importantly, these point mutations showed strong sequence dependence that is not consistent with previous work in Hela cells, where CRISPR-mediated substitutions were found to lack sequence dependence and conversion preferences. Finally, we found that the non-homologous end joining components Ku70, Ku80, Mre11, or RAD50, and the overlapping roles of non-essential DNA polymerases were necessary for the production of both point mutations and deletions. This work expands our knowledge of CRISPR-Cas9 mediated DNA repair.
Get full access to this article
View all access options for this article.
References
1.
MaliP, YangLH, EsveltKM, et al.RNA-guided human genome engineering via Cas9. Science. 2013; 339:823–826. DOI: 10.1126/science.1232033.
2.
CubbonA, Ivancic-BaceI, BoltEL. CRISPR-Cas immunity, DNA repair and genome stability. Biosci Rep. 2018; 38. DOI: 10.1042/BSR20180457.
3.
van OverbeekM, CapursoD, CarterMM, et al.DNA repair profiling reveals nonrandom outcomes at Cas9-mediated breaks. Mol Cell. 2016; 63:633–646. DOI: 10.1016/j.molcel.2016.06.037.
4.
LemosBR, KaplanAC, BaeJE, et al.CRISPR/Cas9 cleavages in budding yeast reveal templated insertions and strand-specific insertion/deletion profiles. Proc Natl Acad Sci U S A. 2018; 115:E2040–E2047. DOI: 10.1073/pnas.1716855115.
5.
PannunzioNR, WatanabeG, LieberMR. Nonhomologous DNA end-joining for repair of DNA double-strand breaks. J Biol Chem. 2018; 293:10512–10523. DOI: 10.1074/jbc.TM117.000374.
6.
AllenF, CrepaldiL, AlsinetC, et al.Predicting the mutations generated by repair of Cas9-induced double-strand breaks. Nat Biotechnol. 2019; 37:64–72. DOI: 10.1038/nbt.4317.
7.
FilippoJS, SungP, KleinH. Mechanism of eukaryotic homologous recombination. Annu Rev Biochem. 2008; 77:229–257. DOI: 10.1146/annurev.biochem.77.061306.125255.
8.
ChuVT, WeberT, WefersB, et al.Increasing the efficiency of homology-directed repair for CRISPR-Cas9-induced precise gene editing in mammalian cells. Nat Biotechnol. 2015; 33:543-U160. DOI: 10.1038/nbt.3198.
9.
MaruyamaT, DouganSK, TruttmannMC, et al.Increasing the efficiency of precise genome editing with CRISPR-Cas9 by inhibition of nonhomologous end joining. Nat Biotechnol. 2015; 33:538–542. DOI: 10.1038/nbt.3190.
10.
CannyMD, MoattiN, WanLCK, et al.Inhibition of 53BP1 favors homology-dependent DNA repair and increases CRISPR-Cas9 genome-editing efficiency. Nat Biotechnol. 2018; 36:95–102. DOI: 10.1038/nbt.4021.
11.
Kato-InuiT, TakahashiG, HsuS, et al.Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 with improved proof-reading enhances homology-directed repair. Nucleic Acids Res. 2018; 46:4677–4688. DOI: 10.1093/nar/gky264.
12.
RichardsonCD, RayGJ, DeWittMA, et al.Enhancing homology-directed genome editing by catalytically active and inactive CRISPR-Cas9 using asymmetric donor DNA. Nat Biotechnol. 2016; 34:339–344. DOI: 10.1038/nbt.3481.
13.
ShenMW, ArbabM, HsuJY, et al.Predictable and precise template-free CRISPR editing of pathogenic variants. Nature. 2018; 563:646–651. DOI: 10.1038/s41586-018-0686-x.
KosickiM, TombergK, BradleyA. Repair of double-strand breaks induced by CRISPR-Cas9 leads to large deletions and complex rearrangements. Nat Biotechnol. 2018; 36:765–771. DOI: 10.1038/nbt.4192.
16.
BassettAR, TibbitC, PontingCP, et al.Highly efficient targeted mutagenesis of Drosophila with the CRISPR/Cas9 system. Cell Rep. 2013; 4:220–228. DOI: 10.1016/j.celrep.2013.06.020.
17.
WangT, WeiJJ, SabatiniDMet al.Genetic screens in human cells using the CRISPR-Cas9 system. Science. 2014; 343:80–84. DOI: 10.1126/science.1246981.
18.
HwangGH, YuJ, YangS, et al.CRISPR-sub: Analysis of DNA substitution mutations caused by CRISPR-Cas9 in human cells. Comput Struct Biotec. 2020; 18:1686–1694. DOI: 10.1016/j.csbj.2020.06.026.
19.
MannazzuI, LandolfoS, da SilvaTL, et al.Red yeasts and carotenoid production: Outlining a future for non-conventional yeasts of biotechnological interest. World J Microbiol Biot. 2015; 31:1665–1673. DOI: 10.1007/s11274-015-1927-x.
20.
WeryJ, VerdoesJC, van OoyenAJJ. Efficient transformation of the astaxanthin-producing yeast Phaffia rhodozyma. Biotechnol Tech. 1998; 12:399–405. DOI: 10.1023/A:1008834600770.
21.
GaoSL, TongYY, WenZQ, et al.Multiplex gene editing of the Yarrowia lipolytica genome using the CRISPR-Cas9 system. J Ind Microbiol Biotechnol. 2016; 43:1085–1093. DOI: 10.1007/s10295-016-1789-8.
BlinK, PedersenLE, WeberT, et al.CRISPy-web: An online resource to design sgRNAs for CRISPR applications. Synth Syst Biotechnol. 2016; 1:118–121. DOI: 10.1016/j.synbio.2016.01.003.
24.
WaterhouseA, BertoniM, BienertS, et al.SWISS-MODEL: Homology modelling of protein structures and complexes. Nucleic Acids Res. 2018; 46:W296–W303. DOI: 10.1093/nar/gky427.
25.
KavanaghKL, DunfordJE, BunkocziG, et al.The crystal structure of human geranylgeranyl pyrophosphate synthase reveals a novel hexameric arrangement and inhibitory product binding. J Biol Chem. 2006; 281:22004–22012. DOI: 10.1074/jbc.M602603200.
26.
Martinez-MoyaP, WattSA, NiehausK, et al.Proteomic analysis of the carotenogenic yeast Xanthophyllomyces dendrorhous. BMC Microbiol, 2011; 11. DOI: 10.1186/1471-2180-11-131.
27.
DiCarloJE, NorvilleJE, MaliP, et al.Genome engineering in Saccharomyces cerevisiae using CRISPR-Cas systems. Nucleic Acids Res. 2013; 41:4336–4343. DOI: 10.1093/nar/gkt135.
28.
SharmaR, GasselS, SteigerS, et al.The genome of the basal agaricomycete Xanthophyllomyces dendrorhous provides insights into the organization of its acetyl-CoA derived pathways and the evolution of Agaricomycotina. BMC Genomics. 2015; 16. DOI: 10.1186/s12864-015-1380-0.
29.
ChenK, WallisJW, McLellanMD, et al.BreakDancer: An algorithm for high-resolution mapping of genomic structural variation. Nat Methods. 2009; 6:677–681. DOI: 10.1038/Nmeth.1363.
ZhangJJ, KobertK, FlouriT, et al.PEAR: A fast and accurate Illumina Paired-End reAd mergeR. Bioinformatics. 2014; 30:614–620. DOI: 10.1093/bioinformatics/btt593
32.
LangmeadB, SalzbergSL. Fast gapped-read alignment with Bowtie 2. Nat Methods. 2012; 9:357–359. DOI: 10.1038/Nmeth.1923.
33.
NagyA, GaramszegiN, VagvolgyiC, et al.Electrophoretic karyotypes of Phaffia rhodozyma strains. FEMS Microbiol Lett. 1994; 123:315–318. DOI: org/10.1111/j.1574–6968.1994.tb07241.x.
34.
MeyerD, FuBXH, HeyerarWD. DNA polymerases delta and lambda cooperate in repairing double-strand breaks by microhomology-mediated end-joining in Saccharomyces cerevisiae. Proc Natl Acad Sci USA. 2015; 112:E6907–E6916. DOI: 10.1073/pnas.1507833112.
35.
OwensDDG, CaulderA, FronteraV, et al.Microhomologies are prevalent at Cas9-induced larger deletions. Nucleic Acids Res. 2019; 47:7402–7417. DOI: 10.1093/nar/gkz459.
36.
GuptaD, HeinenCD. The mismatch repair-dependent DNA damage response: Mechanisms and implications. DNA Repair. 2019; 78:60–69. DOI: 10.1016/j.dnarep.2019.03.009.
37.
PaullTT. 20 years of Mre11 biology: No end in sight. Mol Cell. 2018; 71:419–427. DOI: 10.1016/j.molcel.2018.06.033.
38.
MeyerD, FuBXH, ChavezM, et al.Cooperation between non-essential DNA polymerases contributes to genome stability in Saccharomyces cerevisiae. DNA Repair. 2019; 76:40–49. DOI: 10.1016/j.dnarep.2019.02.004.
39.
KimHS, JeongYK, HurJK, et al.Adenine base editors catalyze cytosine conversions in human cells. Nat Biotechnol. 2019; 37:1145–1148. DOI: 10.1038/s41587-019-0254-4.
40.
FuBXH, SmithJD, FuchsRT, et al.Target-dependent nickase activities of the CRISPR-Cas nucleases Cpf1 and Cas9. Nat Microbiol. 2019; 4:888–897. DOI: 10.1038/s41564-019-0382-0.
41.
JinekM, ChylinskiK, FonfaraI, et al.A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science. 2012; 337:816–821. DOI: 10.1126/science.1225829.
42.
LiP, LiJ, LiM, et al.Multiple end joining mechanisms repair a chromosomal DNA break in fission yeast. DNA Repair. 2012; 11:120–130. DOI: 10.1016/j.dnarep.2011.10.011.
43.
ChangHHY, PannunzioNR, AdachiN, et al.Non-homologous DNA end joining and alternative pathways to double-strand break repair. Nat Rev Mol Cell Bio. 2017; 18:495–506. DOI: 10.1038/nrm.2017.48.
44.
RoidosP, SungaleeS, BenfattoS, et al.A scalable CRISPR/Cas9-based fluorescent reporter assay to study DNA double-strand break repair choice. Nat Commun. 2020; 11. DOI: 10.1038/s41467-020-17962-3.
45.
GaudelliNM, KomorAC, ReesHA, et al. Programmable base editing of A. T to G. C in genomic DNA without DNA cleavage. Nature. 2017;551:464–471. DOI: 10.1038/nature24644.
46.
KomorAC, KimYB, PackerMS, et al.Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage. Nature. 2016; 533:420–424. DOI: 10.1038/nature17946.
47.
DoudnaJA. The promise and challenge of therapeutic genome editing. Nature. 2020; 578:229–236. DOI: 10.1038/s41586-020-1978-5.
48.
LaugheryMF, MayesHC, PedrozaIK, et al.R-loop formation by dCas9 is mutagenic in Saccharomyces cerevisiae. Nucleic Acids Res. 2019; 47:2389–2401. DOI: 10.1093/nar/gky1278.
49.
DoiG, OkadaS, YasukawaT, et al.Catalytically inactive Cas9 impairs DNA replication fork progression to induce focal genomic instability. Nucleic Acids Res. 2021; 49. DOI: 10.1093/nar/gkaa1241.
50.
ShivramH, CressBF, KnottGJ, et al.Controlling and enhancing CRISPR systems. Nat Chem Biol. 2021; 17:10–19. DOI: 10.1038/s41589-020-00700-7.
51.
LangeSS, TakataK, WoodRD. DNA polymerases and cancer. Nat Rev Cancer. 2011; 11:96–110. DOI: 10.1038/nrc2998.
52.
ReijnsMAM, KempH, DingJ, et al.Lagging-strand replication shapes the mutational landscape of the genome. Nature. 2015; 518:502–506. DOI: 10.1038/nature14183.
53.
EmersonCH, BertuchAA. Consider the workhorse: Nonhomologous end-joining in budding yeast. Biochem Cell Biol. 2016; 94:396–406. DOI: 10.1139/bcb-2016-0001.
Supplementary Material
Please find the following supplemental material available below.
For Open Access articles published under a Creative Commons License, all supplemental material carries the same license as the article it is associated with.
For non-Open Access articles published, all supplemental material carries a non-exclusive license, and permission requests for re-use of supplemental material or any part of supplemental material shall be sent directly to the copyright owner as specified in the copyright notice associated with the article.
