@article{9fdcd87f47b4452781b083842dead6ee,
title = "Precision genome editing using synthesis-dependent repair of Cas9-induced DNA breaks",
abstract = "The RNA-guided DNA endonuclease Cas9 has emerged as a powerful tool for genome engineering. Cas9 creates targeted double-stranded breaks (DSBs) in the genome. Knockin of specific mutations (precision genome editing) requires homology-directed repair (HDR) of the DSB by synthetic donor DNAs containing the desired edits, but HDR has been reported to be variably efficient. Here, we report that linear DNAs (single and double stranded) engage in a high-efficiency HDR mechanism that requires only ∼35 nucleotides of homology with the targeted locus to introduce edits ranging from 1 to 1,000 nucleotides. We demonstrate the utility of linear donors by introducing fluorescent protein tags in human cells and mouse embryos using PCR fragments. We find that repair is local, polarity sensitive, and prone to template switching, characteristics that are consistent with gene conversion by synthesis-dependent strand annealing. Our findings enable rational design of synthetic donor DNAs for efficient genome editing.",
keywords = "CRISPR, HDR, PCR repair template, SDSA, Short homology arms",
author = "Alexandre Paix and Andrew Folkmann and Goldman, {Daniel H.} and Heather Kulaga and Grzelak, {Michael J.} and Dominique Rasoloson and Supriya Paidemarry and Rachel Green and Reed, {Randall R.} and Geraldine Seydoux",
note = "Funding Information: We thank the Johns Hopkins University (JHU) Genetic Resources Core Facility{\textquoteright}s Sequencing Facility, the JHU Transgenic Facility, and the JHU Ross Flow Cytometry Core Facility for expert support; Dr. Jonathan Weissman for the gift of HEK293T GFP1–10 cells; Andrew Holland and Tyler Moyer for tissue culture help; and Boris Zinshteyn for assistance with Illumina sequencing and data analysis. This work was supported by NIH Grants R01HD37047 (to G.S.), R01DC004553 (to R.R.R.), and F32GM117814 (to A.F.). G.S. and R.G. are investigators of the Howard Hughes Medical Institute. D.H.G. is a Damon Runyon Fellow supported by the Damon Runyon Cancer Research Foundation (DRG-2280‐16). A.P. dedicates this work to Marcel Bodelet. Funding Information: ACKNOWLEDGMENTS. We thank the Johns Hopkins University (JHU) Genetic Resources Core Facility{\textquoteright}s Sequencing Facility, the JHU Transgenic Facility, and the JHU Ross Flow Cytometry Core Facility for expert support; Dr. Jonathan Weissman for the gift of HEK293T GFP1–10 cells; Andrew Holland and Tyler Moyer for tissue culture help; and Boris Zinshteyn for assistance with Illu-mina sequencing and data analysis. This work was supported by NIH Grants R01HD37047 (to G.S.), R01DC004553 (to R.R.R.), and F32GM117814 (to A.F.). G.S. and R.G. are investigators of the Howard Hughes Medical Institute. D.H.G. is a Damon Runyon Fellow supported by the Damon Runyon Cancer Research Foundation (DRG-2280‐16). A.P. dedicates this work to Marcel Bodelet.",
year = "2017",
month = dec,
day = "12",
doi = "10.1073/pnas.1711979114",
language = "English (US)",
volume = "114",
pages = "E10745--E10754",
journal = "Proceedings of the National Academy of Sciences of the United States of America",
issn = "0027-8424",
publisher = "National Academy of Sciences",
number = "50",
}