title: DNA Repair Profiling Reveals Nonrandom Outcomes at Cas9-Mediated Breaks.
abstract: The repair outcomes at site-specific DNA double-strand breaks (DSBs) generated by the RNA-guided DNA endonuclease Cas9 determine how gene function is altered. Despite the widespread adoption of CRISPR-Cas9 technology to induce DSBs for genome engineering, the resulting repair products have not been examined in depth. Here, the DNA repair profiles of 223 sites in the human genome demonstrate that the pattern of DNA repair following Cas9 cutting at each site is nonrandom and consistent across experimental replicates, cell lines, and reagent delivery methods. Furthermore, the repair outcomes are determined by the protospacer sequence rather than genomic context, indicating that DNA repair profiling in cell lines can be used to anticipate repair outcomes in primary cells. Chemical inhibition of DNA-PK enabled dissection of the DNA repair profiles into contributions from c-NHEJ and MMEJ. Finally, this work elucidates a strategy for using "error-prone" DNA-repair machinery to generate precise edits.
authors: Caribou Biosciences, Inc., Berkeley, CA 94710, USA.
authors: Megan
authors: M
authors: van Overbeek
authors: Caribou Biosciences, Inc., Berkeley, CA 94710, USA.
authors: Daniel
authors: D
authors: Capurso
authors: Caribou Biosciences, Inc., Berkeley, CA 94710, USA.
authors: Matthew M
authors: MM
authors: Carter
authors: Caribou Biosciences, Inc., Berkeley, CA 94710, USA.
authors: Matthew S
authors: MS
authors: Thompson
authors: Development and Molecular Pathways Department, Novartis Institutes for BioMedical Research, Cambridge, MA 02139, USA.
authors: Elizabeth
authors: E
authors: Frias
authors: Development and Molecular Pathways Department, Novartis Institutes for BioMedical Research, Cambridge, MA 02139, USA.
authors: Carsten
authors: C
authors: Russ
authors: Development and Molecular Pathways Department, Novartis Institutes for BioMedical Research, Cambridge, MA 02139, USA.
authors: John S
authors: JS
authors: Reece-Hoyes
authors: Caribou Biosciences, Inc., Berkeley, CA 94710, USA.
authors: Christopher
authors: C
authors: Nye
authors: Caribou Biosciences, Inc., Berkeley, CA 94710, USA.
authors: Scott
authors: S
authors: Gradia
authors: Caribou Biosciences, Inc., Berkeley, CA 94710, USA.
authors: Bastien
authors: B
authors: Vidal
authors: Caribou Biosciences, Inc., Berkeley, CA 94710, USA.
authors: Jiashun
authors: J
authors: Zheng
authors: Development and Molecular Pathways Department, Novartis Institutes for BioMedical Research, Cambridge, MA 02139, USA.
authors: Gregory R
authors: GR
authors: Hoffman
authors: Caribou Biosciences, Inc., Berkeley, CA 94710, USA.
authors: Christopher K
authors: CK
authors: Fuller
authors: Caribou Biosciences, Inc., Berkeley, CA 94710, USA. Electronic address: andy.may@cariboubio.com.
authors: Andrew P
authors: AP
authors: May
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D000076987:CRISPR-Associated Protein 9; D064113:CRISPR-Cas Systems; D053903:DNA Breaks, Double-Stranded; D059766:DNA End-Joining Repair; D004720:Endonucleases / Q000235:genetics / Q000378:metabolism*; D000072669:Gene Editing; D020869:Gene Expression Profiling / Q000379:methods*; D045325:HCT116 Cells; D057809:HEK293 Cells; D006801:Humans; D020014:K562 Cells; D034622:RNA Interference; D000094704:RNA, Guide, CRISPR-Cas Systems / Q000235:genetics / Q000378:metabolism; D013997:Time Factors; D014162:Transfection
chemical_list: D001426:Bacterial Proteins; D000094704:RNA, Guide, CRISPR-Cas Systems; D000076987:CRISPR-Associated Protein 9; C000606107:Cas9 endonuclease Streptococcus pyogenes; D004720:Endonucleases