TY - JOUR
T1 - Chemical reprogramming enhances homology-directed genome editing in zebrafish embryos
AU - Aksoy, Yagiz A.
AU - Nguyen, David T.
AU - Chow, Sharron
AU - Chung, Roger S.
AU - Guillemin, Gilles J.
AU - Cole, Nicholas J.
AU - Hesselson, Daniel
N1 - Copyright the Author(s) 2019. Version archived for private and non-commercial use with the permission of the author/s and according to publisher conditions. For further rights please contact the publisher.
PY - 2019/5/23
Y1 - 2019/5/23
N2 - 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.
AB - 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.
UR - http://purl.org/au-research/grants/nhmrc/1095215
UR - http://purl.org/au-research/grants/nhmrc/1063981
UR - http://purl.org/au-research/grants/arc/DP140103233
UR - http://www.scopus.com/inward/record.url?scp=85070998170&partnerID=8YFLogxK
U2 - 10.1038/s42003-019-0444-0
DO - 10.1038/s42003-019-0444-0
M3 - Article
C2 - 31149642
VL - 2
SP - 1
EP - 9
JO - Communications Biology
JF - Communications Biology
SN - 2399-3642
M1 - 198
ER -