title: Characterization of the interplay between DNA repair and CRISPR/Cas9-induced DNA lesions at an endogenous locus.
abstract: 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.
authors.affiliation: Editas Medicine, 11 Hurley Street, Cambridge, Massachusetts 02141, USA.
authors.forename: Anne
authors.initials: A
authors.lastname: Bothmer
authors.affiliation: Editas Medicine, 11 Hurley Street, Cambridge, Massachusetts 02141, USA.
authors.forename: Tanushree
authors.initials: T
authors.lastname: Phadke
authors.affiliation: Editas Medicine, 11 Hurley Street, Cambridge, Massachusetts 02141, USA.
authors.forename: Luis A
authors.initials: LA
authors.lastname: Barrera
authors.affiliation: Editas Medicine, 11 Hurley Street, Cambridge, Massachusetts 02141, USA.
authors.forename: Carrie M
authors.initials: CM
authors.lastname: Margulies
authors.affiliation: Editas Medicine, 11 Hurley Street, Cambridge, Massachusetts 02141, USA.
authors.forename: Christina S
authors.initials: CS
authors.lastname: Lee
authors.affiliation: Editas Medicine, 11 Hurley Street, Cambridge, Massachusetts 02141, USA.
authors.forename: Frank
authors.initials: F
authors.lastname: Buquicchio
authors.affiliation: Editas Medicine, 11 Hurley Street, Cambridge, Massachusetts 02141, USA.
authors.forename: Sean
authors.initials: S
authors.lastname: Moss
authors.affiliation: Editas Medicine, 11 Hurley Street, Cambridge, Massachusetts 02141, USA.
authors.forename: Hayat S
authors.initials: HS
authors.lastname: Abdulkerim
authors.affiliation: Editas Medicine, 11 Hurley Street, Cambridge, Massachusetts 02141, USA.
authors.forename: William
authors.initials: W
authors.lastname: Selleck
authors.affiliation: Editas Medicine, 11 Hurley Street, Cambridge, Massachusetts 02141, USA.
authors.forename: Hariharan
authors.initials: H
authors.lastname: Jayaram
authors.affiliation: Editas Medicine, 11 Hurley Street, Cambridge, Massachusetts 02141, USA.
authors.forename: Vic E
authors.initials: VE
authors.lastname: Myer
authors.affiliation: Editas Medicine, 11 Hurley Street, Cambridge, Massachusetts 02141, USA.
authors.forename: Cecilia
authors.initials: C
authors.lastname: Cotta-Ramusino
mesh_terms: D024682:BRCA2 Protein / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D000076987:CRISPR-Associated Protein 9; D064113:CRISPR-Cas Systems; D045744:Cell Line, Tumor; D064112:Clustered Regularly Interspaced Short Palindromic Repeats; D004247:DNA / Q000235:genetics* / Q000378:metabolism; D053903:DNA Breaks, Double-Stranded; D004720:Endonucleases / Q000235:genetics / Q000378:metabolism; D000072669:Gene Editing / Q000379:methods*; D015894:Genome, Human; D057809:HEK293 Cells; D006801:Humans; D020014:K562 Cells; D010006:Osteoblasts / Q000166:cytology / Q000378:metabolism; D000094704:RNA, Guide, CRISPR-Cas Systems / Q000235:genetics / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism; D051135:Rad51 Recombinase / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism; D059767:Recombinational DNA Repair
chemical_list: D024682:BRCA2 Protein; C551750:BRCA2 protein, human; D001426:Bacterial Proteins; D000094704:RNA, Guide, CRISPR-Cas Systems; D034741:RNA, Small Interfering; D004247:DNA; C495153:RAD51 protein, human; D051135:Rad51 Recombinase; D000076987:CRISPR-Associated Protein 9; C000606107:Cas9 endonuclease Streptococcus pyogenes; D004720:Endonucleases