Details

Autor(en) / Beteiligte

• Aksoy, Yagiz A
• Nguyen, David T
• Chow, Sharron
• Chung, Roger S
• Guillemin, Gilles J
• Cole, Nicholas J
• Hesselson, Daniel

Titel

Chemical reprogramming enhances homology-directed genome editing in zebrafish embryos

Ist Teil von

• Communications biology, 2019-05, Vol.2 (1), p.198

Ort / Verlag

England

Erscheinungsjahr

2019

Quelle

EZB Electronic Journals Library

Beschreibungen/Notizen

• 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.