RS-1 enhances CRISPR/Cas9- and TALEN-mediated knock-in efficiency.

Jun Song,Y Eugene Chen,Jifeng Zhang,Dongshan Yang,Jie Xu,Tianqing Zhu

doi:10.1038/ncomms10548

Abstract

Zinc-finger nuclease, transcription activator-like effector nuclease and CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 (CRISPR-associated protein 9) are becoming major tools for genome editing. Importantly, knock-in in several non-rodent species has been finally achieved thanks to these customizable nucleases; yet the rates remain to be further improved. We hypothesize that inhibiting non-homologous end joining (NHEJ) or enhancing homology-directed repair (HDR) will improve the nuclease-mediated knock-in efficiency. Here we show that the in vitro application of an HDR enhancer, RS-1, increases the knock-in efficiency by two- to five-fold at different loci, whereas NHEJ inhibitor SCR7 has minimal effects. We then apply RS-1 for animal production and have achieved multifold improvement on the knock-in rates as well. Our work presents tools to nuclease-mediated knock-in animal production, and sheds light on improving gene-targeting efficiencies on pluripotent stem cells.

Highlights

Zinc-finger nuclease, transcription activator-like effector nuclease and CRISPR/Cas[9] (CRISPR-associated protein 9) are becoming major tools for genome editing
Non-homologous end joining (NHEJ) and homology-directed repair (HDR) are the two main mechanisms responsible for DNA repair after nucleases generate double-strand breaks (DSB) at the target site[9], where non-homologous end joining (NHEJ) would lead to KO characterized by unpredictable insertions or deletions, whereas HDR results in knock-in events, when a donor vector is co-introduced
We have examined the effects of a potent NHEJ inhibitor, SCR7, and an HDR enhancer, RS-1, on improving the efficiency of Cas9- or transcription activator-like effector nuclease (TALEN)-mediated knock-in in rabbits

Summary

Introduction

Zinc-finger nuclease, transcription activator-like effector nuclease and CRISPR (clustered regularly interspaced short palindromic repeats)/Cas[9] (CRISPR-associated protein 9) are becoming major tools for genome editing. The advent of zinc-finger nuclease (ZFN), transcription activator-like effector nuclease (TALEN) and CRISPR (clustered regularly interspaced short palindromic repeats)/Cas[9] (CRISPR-associated protein 9) technologies has changed the landscape of gene targeting These customizable nucleases are efficient in generating double-strand breaks (DSB) in the genome that can lead to a functional knockout (KO) of the targeted gene or be used to knock-in a DNA sequence at a specific locus in the genome in a number of species[1,2]. Our experience with the rabbit models confirmed these findings: the knock-in rates are below 1% when calculated as the ratio of total knock-in kits over total embryos transferred or 0–10% when calculated by ratio of total knock-in kits over total kits born This low efficiency has become a rate-limiting factor for a broader application of nuclease-mediated gene modifications for transgenic animal production, as well as in pluripotent stem cells. Our work adds a useful tool to the transgenic animal research community and sheds light on regenerative medicine, as a substantial percentage of animal modelling and stem cell-based research and therapies will require targeted gene modifications

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Results

Conclusion