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Chemical reprogramming enhances homology-directed genome editing in zebrafish embryos

Abstract

Precise genome editing is limited by the inefficiency of homology-directed repair (HDR) compared to the non-homologous end-joining (NHEJ) of double strand breaks (DSBs). The CRISPR (clustered regularly interspaced short palindromic repeat)/Cas9 system generates precise, locus-specific DSBs that can serve as substrates for HDR. We developed an in vivo visual reporter assay to quantify HDR-mediated events at single-cell resolution in zebrafish and used this system to identify small-molecule modulators that shift the DNA repair equilibrium in favor of HDR. By further optimizing the reaction environment and repair template, we achieved dramatic enhancement of HDR-mediated repair efficiency in zebrafish. Accordingly, under optimized conditions, inhibition of NHEJ with NU7441 enhanced HDR-mediated repair up to 13.4-fold. Importantly, we demonstrate that the increase in somatic HDR events correlates directly with germline transmission, permitting the efficient recovery of large seamlessly integrated DNA fragments in zebrafish.

Type Journal
ISBN 2399-3642 (Electronic) 2399-3642 (Linking)
Authors Aksoy, Y. A.; Nguyen, D. T.; Chow, S.; Chung, R. S.; Guillemin, G. J.; Cole, N. J.; Hesselson, D.
Responsible Garvan Author Dr Daniel Hesselson
Publisher Name Communications Biology
Published Date 2019-05-23
Published Volume 2
Published Issue 198
Published Pages 1
Status Always Electronic
DOI 10.1038/s42003-019-0444-0
URL link to publisher's version https://www.ncbi.nlm.nih.gov/pubmed/31149642