Characterization of the interplay between DNA repair and CRISPR/Cas9-induced DNA lesions at an endogenous locus

The CRISPR–Cas9 system provides a versatile toolkit for genome engineering that can introduce various DNA lesions at specific genomic locations. However, a better understanding of the nature of these lesions and the repair pathways engaged is critical to realizing the full potential of this technology. Here we characterize the different lesions arising from each Cas9 variant and the resulting repair pathway engagement. We demonstrate that the presence and polarity of the overhang structure is a critical determinant of double-strand break repair pathway choice. Similarly, single nicks deriving from different Cas9 variants differentially activate repair: D10A but not N863A-induced nicks are repaired by homologous recombination. Finally, we demonstrate that homologous recombination is required for repairing lesions using double-stranded, but not single-stranded DNA as a template. This detailed characterization of repair pathway choice in response to CRISPR–Cas9 enables a more deterministic approach for designing research and therapeutic genome engineering strategies.

[1]  Jennifer A. Doudna,et al.  Conformational control of DNA target cleavage by CRISPR–Cas9 , 2015, Nature.

[2]  D. Geschwind,et al.  Advances in autism genetics: on the threshold of a new neurobiology , 2008, Nature Reviews Genetics.

[3]  Feng Zhang,et al.  Crystal Structure of Cas9 in Complex with Guide RNA and Target DNA , 2014, Cell.

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

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

[6]  M. Jasin,et al.  Assaying break and nick-induced homologous recombination in mammalian cells using the DR-GFP reporter and Cas9 nucleases. , 2014, Methods in enzymology.

[7]  X. Chen,et al.  Small RNA existed in commercial reverse transcriptase: primary evidence of functional small RNAs , 2014, Protein & Cell.

[8]  Ying Sun,et al.  CRISPR/Cas9-mediated gene editing in human tripronuclear zygotes , 2015, Protein & Cell.

[9]  L. Symington.  End resection at double-strand breaks: mechanism and regulation. , 2014, Cold Spring Harbor perspectives in biology.

[10]  Jiajie Zhang,et al.  PEAR: a fast and accurate Illumina Paired-End reAd mergeR , 2013, Bioinform..

[11]  T. Helleday,et al.  Spontaneous Homologous Recombination Is Induced by Collapsed Replication Forks That Are Caused by Endogenous DNA Single-Strand Breaks , 2005, Molecular and Cellular Biology.

[12]  A. D’Andrea,et al.  Repair Pathway Choices and Consequences at the Double-Strand Break. , 2016, Trends in cell biology.

[13]  I. Longden,et al.  EMBOSS: the European Molecular Biology Open Software Suite. , 2000, Trends in genetics : TIG.

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

[15]  L. Galluzzi,et al.  Autophagy Mediates Tumor Suppression via Cellular Senescence. , 2016, Trends in cell biology.

[16]  R. Barrangou,et al.  Cas9–crRNA ribonucleoprotein complex mediates specific DNA cleavage for adaptive immunity in bacteria , 2012, Proceedings of the National Academy of Sciences.

[17]  D. Roth,et al.  Modernizing the nonhomologous end-joining repertoire: alternative and classical NHEJ share the stage. , 2013, Annual review of genetics.

[18]  C. Shaw,et al.  Mus81 and converging forks limit the mutagenicity of replication fork breakage , 2015, Science.

[19]  Eli J. Fine,et al.  DNA targeting specificity of RNA-guided Cas9 nucleases , 2013, Nature Biotechnology.

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

[21]  Pharmacological inhibition of DNA-PK stimulates Cas9-mediated genome editing , 2015, Genome Medicine.

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

[23]  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.

[24]  Keith W. Caldecott,et al.  Single-strand break repair and genetic disease , 2008, Nature Reviews Genetics.

[25]  J. Doudna,et al.  A Programmable Dual-RNA–Guided DNA Endonuclease in Adaptive Bacterial Immunity , 2012, Science.

[26]  N. Maizels,et al.  Homology-directed repair of DNA nicks via pathways distinct from canonical double-strand break repair , 2014, Proceedings of the National Academy of Sciences.

[27]  Hong Wu,et al.  A distinct replication fork protection pathway connects Fanconi anemia tumor suppressors to RAD51-BRCA1/2. , 2012, Cancer cell.

[28]  I. Ial,et al.  Nature Communications , 2010, Nature Cell Biology.

[29]  Jeremy M. Stark,et al.  An RNF168 fragment defective for focal accumulation at DNA damage is proficient for inhibition of homologous recombination in BRCA1 deficient cells , 2014, Nucleic acids research.

[30]  David A. Scott,et al.  Double Nicking by RNA-Guided CRISPR Cas9 for Enhanced Genome Editing Specificity , 2013, Cell.

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

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

[33]  J. Keith Joung,et al.  Improving CRISPR-Cas nuclease specificity using truncated guide RNAs , 2014, Nature Biotechnology.

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

[35]  E. Lander,et al.  Development and Applications of CRISPR-Cas9 for Genome Engineering , 2014, Cell.

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

[37]  Raquel Herrador,et al.  Rad51-mediated replication fork reversal is a global response to genotoxic treatments in human cells , 2015, The Journal of cell biology.

[38]  A. Egashira,et al.  Double-Strand Break Repair-Independent Role for BRCA2 in Blocking Stalled Replication Fork Degradation by MRE11 , 2011, Cell.

[39]  Gonçalo R. Abecasis,et al.  The Sequence Alignment/Map format and SAMtools , 2009, Bioinform..

[40]  M. Jasin,et al.  Homologous recombination and human health: the roles of BRCA1, BRCA2, and associated proteins. , 2015, Cold Spring Harbor perspectives in biology.

[41]  G. Church,et al.  CAS9 transcriptional activators for target specificity screening and paired nickases for cooperative genome engineering , 2013, Nature Biotechnology.

[42]  J. Doudna,et al.  The new frontier of genome engineering with CRISPR-Cas9 , 2014, Science.

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

[44]  M. Lopes,et al.  Rad51 protects nascent DNA from Mre11 dependent degradation and promotes continuous DNA synthesis , 2010, Nature Structural &Molecular Biology.

[45]  M. Lopes,et al.  Replication fork reversal in eukaryotes: from dead end to dynamic response , 2015, Nature Reviews Molecular Cell Biology.

[46]  Philippe Horvath,et al.  The Streptococcus thermophilus CRISPR/Cas system provides immunity in Escherichia coli , 2011, Nucleic acids research.

[47]  James E Haber,et al.  Sources of DNA double-strand breaks and models of recombinational DNA repair. , 2014, Cold Spring Harbor perspectives in biology.

[48]  Sarah K. Baxter,et al.  Coupling endonucleases with DNA end–processing enzymes to drive gene disruption , 2012, Nature Methods.