Mapping the genetic landscape of DNA double-strand break repair

Cells repair DNA double-strand breaks (DSBs) through a complex set of pathways that are critical for maintaining genomic integrity. Here we present Repair-seq, a high-throughput screening approach that measures the effects of thousands of genetic perturbations on the distribution of mutations introduced at targeted DNA lesions. Using Repair-seq, we profiled DSB repair outcomes induced by two programmable nucleases (Cas9 and Cas12a) after knockdown of 476 genes involved in DSB repair or associated processes in the presence or absence of oligonucleotides for homology-directed repair (HDR). The resulting data enabled principled, data-driven inference of DSB end joining and HDR pathways and demonstrated that repair outcomes with superficially similar sequence architectures can have markedly different genetic dependencies. Systematic interrogation of these dependencies then uncovered unexpected relationships among DSB repair genes and isolated incompletely characterized repair mechanisms. This work provides a foundation for understanding the complex pathways of DSB repair and for optimizing genome editing across modalities.

[1]  David R. Liu,et al.  Enhanced prime editing systems by manipulating cellular determinants of editing outcomes , 2021, Cell.

[2]  G. Gupta,et al.  Mechanism, cellular functions and cancer roles of polymerase-theta-mediated DNA end joining , 2021, Nature Reviews Molecular Cell Biology.

[3]  T. Paull.  Reconsidering pathway choice: a sequential model of mammalian DNA double-strand break pathway decisions. , 2021, Current opinion in genetics & development.

[4]  Sara B. Linker,et al.  Incorporation of a nucleoside analog maps genome repair sites in postmitotic human neurons , 2021, Science.

[5]  Yufeng Shen,et al.  Functional interrogation of DNA damage response variants with base editing screens , 2021, Cell.

[6]  M. Lieber,et al.  The molecular basis and disease relevance of non-homologous DNA end joining , 2020, Nature Reviews Molecular Cell Biology.

[7]  Cheng-Zhong Zhang,et al.  Chromothripsis as an on-target consequence of CRISPR-Cas9 genome editing , 2020, Nature Genetics.

[8]  David R. Liu,et al.  Genome editing with CRISPR–Cas nucleases, base editors, transposases and prime editors , 2020, Nature Biotechnology.

[9]  S. Tsang,et al.  Allele-Specific Chromosome Removal after Cas9 Cleavage in Human Embryos , 2020, Cell.

[10]  Katie Weiner.  Impact of chromatin context on Cas9-induced DNA double-strand break repair pathway balance , 2020 .

[11]  V. Myer,et al.  Detection and Modulation of DNA Translocations During Multi-Gene Genome Editing in T Cells. , 2020, The CRISPR journal.

[12]  R. Medema,et al.  Impact of chromatin context on Cas9-induced DNA double-strand break repair pathway balance , 2020, bioRxiv.

[13]  Thomas M. Norman,et al.  Combinatorial single-cell CRISPR screens by direct guide RNA capture and targeted sequencing , 2020, Nature Biotechnology.

[14]  J. Haber,et al.  A Rad51-independent pathway promotes single-strand template repair in gene editing , 2020, bioRxiv.

[15]  J. Sekelsky,et al.  Mechanistic basis for microhomology identification and genome scarring by polymerase theta , 2019, Proceedings of the National Academy of Sciences.

[16]  Kejiao Li,et al.  A Genetic Map of the Response to DNA Damage in Human Cells , 2019, Cell.

[17]  J. Loparo,et al.  A Mechanism to Minimize Errors during Non-homologous End Joining. , 2019, Molecular cell.

[18]  Judith L. Campbell,et al.  Multiple roles of DNA2 nuclease/helicase in DNA metabolism, genome stability and human diseases , 2019, Nucleic acids research.

[19]  Bo Huang,et al.  Deep profiling reveals substantial heterogeneity of integration outcomes in CRISPR knock-in experiments , 2019, bioRxiv.

[20]  David R. Liu,et al.  Search-and-replace genome editing without double-strand breaks or donor DNA , 2019, Nature.

[21]  Lisle E. Mose,et al.  Genetic determinants of cellular addiction to DNA polymerase theta , 2019, Nature Communications.

[22]  J. D. den Dunnen,et al.  Templated Insertions: A Smoking Gun for Polymerase Theta-Mediated End Joining. , 2019, Trends in genetics : TIG.

[23]  Ryan T. Leenay,et al.  Large dataset enables prediction of repair after CRISPR–Cas9 editing in primary T cells , 2019, Nature Biotechnology.

[24]  James R. Rybarski,et al.  Massively parallel kinetic profiling of natural and engineered CRISPR nucleases , 2019, bioRxiv.

[25]  N. Willis,et al.  DNA double-strand break repair-pathway choice in somatic mammalian cells , 2019, Nature Reviews Molecular Cell Biology.

[26]  J. Kanno,et al.  Exosome-mediated horizontal gene transfer occurs in double-strand break repair during genome editing , 2019, Communications Biology.

[27]  Haiyan Jiang,et al.  Development of a gene-editing approach to restore vision loss in Leber congenital amaurosis type 10 , 2019, Nature Medicine.

[28]  William Stafford Noble,et al.  Massively parallel profiling and predictive modeling of the outcomes of CRISPR/Cas9-mediated double-strand break repair , 2018, bioRxiv.

[29]  Andrew R. Bassett,et al.  Predicting the mutations generated by repair of Cas9-induced double-strand breaks , 2018, Nature Biotechnology.

[30]  David K. Gifford,et al.  Predictable and precise template-free CRISPR editing of pathogenic variants , 2018, Nature.

[31]  G. Peng,et al.  Dna2 nuclease deficiency results in large and complex DNA insertions at chromosomal breaks , 2018, Nature.

[32]  Anob M. Chakrabarti,et al.  Target-Specific Precision of CRISPR-Mediated Genome Editing , 2018, bioRxiv.

[33]  Qiang Wu,et al.  Precise and Predictable CRISPR Chromosomal Rearrangements Reveal Principles of Cas9-Mediated Nucleotide Insertion. , 2018, Molecular cell.

[34]  Jacob E. Corn,et al.  CRISPR–Cas9 genome editing in human cells occurs via the Fanconi anemia pathway , 2018, Nature Genetics.

[35]  Anne-Claude Gingras,et al.  The Shieldin complex mediates 53BP1-dependent DNA repair , 2018, Nature.

[36]  A. Bradley,et al.  Repair of double-strand breaks induced by CRISPR–Cas9 leads to large deletions and complex rearrangements , 2018, Nature Biotechnology.

[37]  James R. Rybarski,et al.  Kinetic basis for DNA target specificity of CRISPR-Cas12a , 2018, bioRxiv.

[38]  Martin A. M. Reijns,et al.  CRISPR screens identify genomic ribonucleotides as a source of PARP-trapping lesions , 2018, Nature.

[39]  Chunaram Choudhary,et al.  DNA Repair Network Analysis Reveals Shieldin as a Key Regulator of NHEJ and PARP Inhibitor Sensitivity , 2018, Cell.

[40]  H. te Riele,et al.  DNA mismatch repair and oligonucleotide end-protection promote base-pair substitution distal from a CRISPR/Cas9-induced DNA break , 2018, Nucleic acids research.

[41]  Leland McInnes,et al.  UMAP: Uniform Manifold Approximation and Projection for Dimension Reduction , 2018, ArXiv.

[42]  Fabian J Theis,et al.  SCANPY: large-scale single-cell gene expression data analysis , 2018, Genome Biology.

[43]  John G Doench,et al.  Am I ready for CRISPR? A user's guide to genetic screens , 2017, Nature Reviews Genetics.

[44]  D. Durocher,et al.  Inhibition of 53BP1 favors homology-dependent DNA repair and increases CRISPR-Cas9 genome-editing efficiency , 2017, Nature Biotechnology.

[45]  J. Haber,et al.  CRISPR/Cas9 cleavages in budding yeast reveal templated insertions and strand-specific insertion/deletion profiles , 2017, Proceedings of the National Academy of Sciences.

[46]  Steven A. Roberts,et al.  Secondary structure forming sequences drive SD-MMEJ repair of DNA double-strand breaks , 2017, Nucleic acids research.

[47]  Max A. Horlbeck,et al.  Combined CRISPRi/a-Based Chemical Genetic Screens Reveal that Rigosertib Is a Microtubule-Destabilizing Agent , 2017, Molecular cell.

[48]  M. Lieber,et al.  Non-homologous DNA end joining and alternative pathways to double-strand break repair , 2017, Nature Reviews Molecular Cell Biology.

[49]  I. Weissman,et al.  Index switching causes “spreading-of-signal” among multiplexed samples in Illumina HiSeq 4000 DNA sequencing , 2017, bioRxiv.

[50]  Eric A. Hendrickson,et al.  Mechanisms of precise genome editing using oligonucleotide donors. , 2017, Genome research.

[51]  S. Bell,et al.  Mcm10 regulates DNA replication elongation by stimulating the CMG replicative helicase , 2017, Genes & development.

[52]  V. Myer,et al.  Characterization of the interplay between DNA repair and CRISPR/Cas9-induced DNA lesions at an endogenous locus , 2017, Nature Communications.

[53]  Daniel Durocher,et al.  The control of DNA repair by the cell cycle , 2016, Nature Cell Biology.

[54]  Max A. Horlbeck,et al.  Compact and highly active next-generation libraries for CRISPR-mediated gene repression and activation , 2016, eLife.

[55]  David W. Wyatt,et al.  Essential Roles for Polymerase θ-Mediated End Joining in the Repair of Chromosome Breaks. , 2016, Molecular cell.

[56]  A. May,et al.  DNA Repair Profiling Reveals Nonrandom Outcomes at Cas9-Mediated Breaks. , 2016, Molecular cell.

[57]  James E Haber,et al.  The democratization of gene editing: Insights from site-specific cleavage and double-strand break repair. , 2016, DNA repair.

[58]  David R. Liu,et al.  Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage , 2016, Nature.

[59]  Charles H. Yoon,et al.  Dissecting the multicellular ecosystem of metastatic melanoma by single-cell RNA-seq , 2016, Science.

[60]  Sham Sunder,et al.  Overhang polarity of chromosomal double-strand breaks impacts kinetics and fidelity of yeast non-homologous end joining , 2016, Nucleic acids research.

[61]  L. Symington,et al.  Microhomology-Mediated End Joining: A Back-up Survival Mechanism or Dedicated Pathway? , 2015, Trends in biochemical sciences.

[62]  A. Regev,et al.  Cpf1 Is a Single RNA-Guided Endonuclease of a Class 2 CRISPR-Cas System , 2015, Cell.

[63]  H. Ploegh,et al.  Inhibition of non-homologous end joining increases the efficiency of CRISPR/Cas9-mediated precise [TM: inserted] genome editing , 2016 .

[64]  Jeremy M. Stark,et al.  DNA Damage Response Factors from Diverse Pathways, Including DNA Crosslink Repair, Mediate Alternative End Joining , 2015, PLoS genetics.

[65]  C. Schild-Poulter,et al.  The Ku heterodimer: function in DNA repair and beyond. , 2015, Mutation research. Reviews in mutation research.

[66]  A. Carr,et al.  TopBP1: A BRCT-scaffold protein functioning in multiple cellular pathways. , 2014, DNA repair.

[67]  Max A. Horlbeck,et al.  Genome-Scale CRISPR-Mediated Control of Gene Repression and Activation , 2014, Cell.

[68]  David W. Wyatt,et al.  Mechanism of Suppression of Chromosomal Instability by DNA Polymerase POLQ , 2014, PLoS genetics.

[69]  P. L. Bergsagel,et al.  Repair of DNA double-strand breaks by templated nucleotide sequence insertions derived from distant regions of the genome , 2014, Proceedings of the National Academy of Sciences.

[70]  K. Caldecott,et al.  One ring to bring them all--the role of Ku in mammalian non-homologous end joining. , 2014, DNA repair.

[71]  David J. Chen,et al.  DNA-PK: a dynamic enzyme in a versatile DSB repair pathway. , 2014, DNA repair.

[72]  Neville E. Sanjana,et al.  Genome-Scale CRISPR-Cas9 Knockout Screening in Human Cells , 2014, Science.

[73]  E. Lander,et al.  Genetic Screens in Human Cells Using the CRISPR-Cas9 System , 2013, Science.

[74]  Luke A. Gilbert,et al.  CRISPR-Mediated Modular RNA-Guided Regulation of Transcription in Eukaryotes , 2013, Cell.

[75]  Linda Z. Shi,et al.  Microhomology-mediated End Joining and Homologous Recombination share the initial end resection step to repair DNA double-strand breaks in mammalian cells , 2013, Proceedings of the National Academy of Sciences.

[76]  K. Cimprich,et al.  A role for the MRN complex in ATR activation via TOPBP1 recruitment. , 2013, Molecular cell.

[77]  Luke A. Gilbert,et al.  Repurposing CRISPR as an RNA-Guided Platform for Sequence-Specific Control of Gene Expression , 2013, Cell.

[78]  Jesse J. Salk,et al.  Detection of ultra-rare mutations by next-generation sequencing , 2012, Proceedings of the National Academy of Sciences.

[79]  Stephen J. Elledge,et al.  A genome-wide homologous recombination screen identifies the RNA-binding protein RBMX as a component of the DNA damage response , 2012, Nature Cell Biology.

[80]  Tony Z. Jia,et al.  Digital RNA sequencing minimizes sequence-dependent bias and amplification noise with optimized single-molecule barcodes , 2012, Proceedings of the National Academy of Sciences.

[81]  J Wade Harper,et al.  A genome-wide camptothecin sensitivity screen identifies a mammalian MMS22L-NFKBIL2 complex required for genomic stability. , 2010, Molecular cell.

[82]  S. Elledge,et al.  The DNA damage response: making it safe to play with knives. , 2010, Molecular cell.

[83]  S. Elledge,et al.  A genetic screen identifies the Triple T complex required for DNA damage signaling and ATM and ATR stability. , 2010, Genes & development.

[84]  J Wade Harper,et al.  A genetic screen identifies FAN1, a Fanconi anemia-associated nuclease necessary for DNA interstrand crosslink repair. , 2010, Molecular cell.

[85]  M. McVey,et al.  Dual Roles for DNA Polymerase Theta in Alternative End-Joining Repair of Double-Strand Breaks in Drosophila , 2010, PLoS genetics.

[86]  M. McVey,et al.  Synthesis-dependent microhomology-mediated end joining accounts for multiple types of repair junctions , 2010, Nucleic acids research.

[87]  Jeremy M. Stark,et al.  53BP1 Inhibits Homologous Recombination in Brca1-Deficient Cells by Blocking Resection of DNA Breaks , 2010, Cell.

[88]  M. Nussenzweig,et al.  53BP1 regulates DNA resection and the choice between classical and alternative end joining during class switch recombination , 2010, The Journal of experimental medicine.

[89]  R. Bambara,et al.  Dna2 is a structure-specific nuclease, with affinity for 5′-flap intermediates , 2009, Nucleic acids research.

[90]  R. Wollman,et al.  A genome-wide siRNA screen reveals diverse cellular processes and pathways that mediate genome stability. , 2009, Molecular cell.

[91]  A. Shevchenko,et al.  The Mre11-Rad50-Nbs1 complex mediates activation of TopBP1 by ATM. , 2009, Molecular biology of the cell.

[92]  Jeremy M. Stark,et al.  Alternative-NHEJ Is a Mechanistically Distinct Pathway of Mammalian Chromosome Break Repair , 2008, PLoS genetics.

[93]  Jean Gautier,et al.  A forward chemical genetic screen reveals an inhibitor of the Mre11-Rad50-Nbs1 complex. , 2008, Nature chemical biology.

[94]  B. A. Ballif,et al.  ATM and ATR Substrate Analysis Reveals Extensive Protein Networks Responsive to DNA Damage , 2007, Science.

[95]  X. Yu,et al.  Patching broken chromosomes with extranuclear cellular DNA. , 1999, Molecular cell.

[96]  L. Thompson,et al.  XRCC3 promotes homology-directed repair of DNA damage in mammalian cells. , 1999, Genes & development.

[97]  S. Boulton,et al.  Saccharomyces cerevisiae Ku70 potentiates illegitimate DNA double‐strand break repair and serves as a barrier to error‐prone DNA repair pathways. , 1996, The EMBO journal.

[98]  P. Rouet,et al.  Introduction of double-strand breaks into the genome of mouse cells by expression of a rare-cutting endonuclease. , 1994, Molecular and cellular biology.