Kinetics and Fidelity of the Repair of Cas9-Induced Double-Strand DNA Breaks

Summary The RNA-guided DNA endonuclease Cas9 is a powerful tool for genome editing. Little is known about the kinetics and fidelity of the double-strand break (DSB) repair process that follows a Cas9 cutting event in living cells. Here, we developed a strategy to measure the kinetics of DSB repair for single loci in human cells. Quantitative modeling of repaired DNA in time series after Cas9 activation reveals variable and often slow repair rates, with half-life times up to ∼10 hr. Furthermore, repair of the DSBs tends to be error prone. Both classical and microhomology-mediated end joining pathways contribute to the erroneous repair. Estimation of their individual rate constants indicates that the balance between these two pathways changes over time and can be altered by additional ionizing radiation. Our approach provides quantitative insights into DSB repair kinetics and fidelity in single loci and indicates that Cas9-induced DSBs are repaired in an unusual manner.

[1]  B. van Steensel,et al.  Easy quantitative assessment of genome editing by sequence trace decomposition , 2014, Nucleic acids research.

[2]  George Iliakis,et al.  Biochemical evidence for Ku-independent backup pathways of NHEJ. , 2003, Nucleic acids research.

[3]  Bas van Steensel,et al.  Detection of in vivo protein–DNA interactions using DamID in mammalian cells , 2007, Nature Protocols.

[4]  T. E. Wilson,et al.  Repair of double-strand breaks by end joining. , 2013, Cold Spring Harbor perspectives in biology.

[5]  A. Seluanov,et al.  Comparison of nonhomologous end joining and homologous recombination in human cells. , 2008, DNA repair.

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

[7]  A. Knudson,et al.  Endogenous DNA double-strand breaks: Production, fidelity of repair, and induction of cancer , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[8]  M. Stratton,et al.  High burden and pervasive positive selection of somatic mutations in normal human skin , 2015, Science.

[9]  Gang Bao,et al.  Quantifying genome-editing outcomes at endogenous loci with SMRT sequencing. , 2014, Cell reports.

[10]  T Hyslop,et al.  DNA-dependent protein kinase stimulates an independently active, nonhomologous, end-joining apparatus. , 2000, Cancer research.

[11]  Peter O'Neill,et al.  Biochemical Kinetics Model of DSB Repair and Induction of γ-H2AX Foci by Non-homologous End Joining , 2008, Radiation research.

[12]  G. Iliakis,et al.  Kinetics of DNA double-strand break repair throughout the cell cycle as assayed by pulsed field gel electrophoresis in CHO cells. , 1991, International journal of radiation biology.

[13]  Anirudh Prahallad,et al.  PTPN11 Is a Central Node in Intrinsic and Acquired Resistance to Targeted Cancer Drugs. , 2014, Cell reports.

[14]  J. Guirouilh-Barbat,et al.  Defects in XRCC4 and KU80 differentially affect the joining of distal nonhomologous ends , 2007, Proceedings of the National Academy of Sciences.

[15]  R. Weichselbaum,et al.  Faster repair of DNA double-strand breaks in radioresistant human tumor cells. , 1988, International journal of radiation oncology, biology, physics.

[16]  Jennifer A. Doudna,et al.  DNA interrogation by the CRISPR RNA-guided endonuclease Cas9 , 2014, Nature.

[17]  T. Lindahl,et al.  A newly identified DNA ligase of Saccharomyces cerevisiae involved in RAD52-independent repair of DNA double-strand breaks. , 1997, Genes & development.

[18]  N. Olea,et al.  Radiation-induced DNA double-strand break rejoining in human tumour cells. , 1995, British Journal of Cancer.

[19]  George M. Church,et al.  CHOPCHOP: a CRISPR/Cas9 and TALEN web tool for genome editing , 2014, Nucleic Acids Res..

[20]  J. Haber,et al.  Increased Mutagenesis and Unique Mutation Signature Associated with Mitotic Gene Conversion , 2010, Science.

[21]  M. Bétermier,et al.  Is Non-Homologous End-Joining Really an Inherently Error-Prone Process? , 2014, PLoS genetics.

[22]  George Iliakis,et al.  PARP-1 and Ku compete for repair of DNA double strand breaks by distinct NHEJ pathways , 2006, Nucleic acids research.

[23]  Huichen Wang,et al.  Backup pathways of NHEJ are suppressed by DNA‐PK , 2004, Journal of cellular biochemistry.

[24]  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.

[25]  Toshio Ando,et al.  Real-space and real-time dynamics of CRISPR-Cas9 visualized by high-speed atomic force microscopy , 2017, Nature Communications.

[26]  Chris P. Barnes,et al.  Mechanistic Modelling and Bayesian Inference Elucidates the Variable Dynamics of Double-Strand Break Repair , 2016, bioRxiv.

[27]  E. Greene.  DNA Sequence Alignment during Homologous Recombination* , 2016, The Journal of Biological Chemistry.

[28]  S. Jackson,et al.  A High-Throughput, Flow Cytometry-Based Method to Quantify DNA-End Resection in Mammalian Cells , 2012, Cytometry. Part A : the journal of the International Society for Analytical Cytology.

[29]  M. Lieber,et al.  The mechanism of double-strand DNA break repair by the nonhomologous DNA end-joining pathway. , 2010, Annual review of biochemistry.

[30]  Markus Löbrich,et al.  Factors determining DNA double‐strand break repair pathway choice in G2 phase , 2011, The EMBO journal.

[31]  James E. DiCarlo,et al.  RNA-Guided Human Genome Engineering via Cas9 , 2013, Science.

[32]  L. Banaszynski,et al.  A Rapid, Reversible, and Tunable Method to Regulate Protein Function in Living Cells Using Synthetic Small Molecules , 2006, Cell.

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

[34]  F. Fan,et al.  Evaluation of the Comet Assay for Assessing the Dose-Response Relationship of DNA Damage Induced by Ionizing Radiation , 2013, International journal of molecular sciences.

[35]  M. Calos,et al.  In vivo blunt-end cloning through CRISPR/Cas9-facilitated non-homologous end-joining , 2015, bioRxiv.

[36]  John C. Rose,et al.  Rapidly inducible Cas9 and DSB-ddPCR to probe editing kinetics , 2017, Nature Methods.

[37]  S. Raghavan,et al.  Homology and enzymatic requirements of microhomology-dependent alternative end joining , 2015, Cell Death and Disease.

[38]  Shondra M. Pruett-Miller,et al.  Nuclease-mediated gene editing by homologous recombination of the human globin locus , 2013, Nucleic acids research.

[39]  E. Soutoglou,et al.  Nuclear compartmentalization of DNA repair. , 2016, Current opinion in genetics & development.

[40]  Jennifer Doudna,et al.  RNA-programmed genome editing in human cells , 2013, eLife.

[41]  R. Tjian,et al.  Dynamics of CRISPR-Cas9 genome interrogation in living cells , 2015, Science.

[42]  David W. Hall,et al.  Precise estimates of mutation rate and spectrum in yeast , 2014, Proceedings of the National Academy of Sciences.

[43]  M. Jasin,et al.  Distinct genetic control of homologous recombination repair of Cas9-induced double-strand breaks, nicks and paired nicks , 2016, Nucleic acids research.

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

[45]  R. Ferrell,et al.  Mutational inactivation of the p53 gene in the human erythroid leukemic K562 cell line. , 1993, Leukemia research.

[46]  Daesik Kim,et al.  Highly efficient RNA-guided genome editing in human cells via delivery of purified Cas9 ribonucleoproteins , 2014, Genome research.

[47]  Le Cong,et al.  Multiplex Genome Engineering Using CRISPR/Cas Systems , 2013, Science.

[48]  B. Wold,et al.  Effects of different DNA polymerases in ligation-mediated PCR: enhanced genomic sequencing and in vivo footprinting. , 1992, Proceedings of the National Academy of Sciences of the United States of America.

[49]  M. McVey,et al.  MMEJ repair of double-strand breaks (director's cut): deleted sequences and alternative endings. , 2008, Trends in genetics : TIG.

[50]  A. Riggs,et al.  Ligation-mediated PCR for quantitative in vivo footprinting , 2000, Nature Biotechnology.

[51]  Bo Stenerlöw,et al.  Measurement of Prompt DNA Double-Strand Breaks in Mammalian Cells without Including Heat-Labile Sites: Results for Cells Deficient in Nonhomologous End Joining , 2002, Radiation research.

[52]  J. Harper,et al.  Induction and quantification of γ-H2AX foci following low and high LET-irradiation , 2006 .

[53]  Jacob E Corn,et al.  Enhancing homology-directed genome editing by catalytically active and inactive CRISPR-Cas9 using asymmetric donor DNA , 2016, Nature Biotechnology.

[54]  Marian Thomson,et al.  Analysis of the genome sequences of three Drosophila melanogaster spontaneous mutation accumulation lines. , 2009, Genome research.

[55]  Shaojie Zhang,et al.  CRISPR-Cas9 nuclear dynamics and target recognition in living cells , 2016, The Journal of cell biology.

[56]  Jeffry D. Sander,et al.  CRISPR-Cas systems for editing, regulating and targeting genomes , 2014, Nature Biotechnology.

[57]  P. Jeggo,et al.  The rate of X-ray-induced DNA double-strand break repair in the embryonic mouse brain is unaffected by exposure to 50 Hz magnetic fields , 2015, International journal of radiation biology.

[58]  D. Schatz,et al.  V(D)J recombination: mechanisms of initiation. , 2011, Annual review of genetics.