Abstract
In genome editing with CRISPR–Cas9, transgene integration often remains challenging. Here, we present an approach for increasing the efficiency of transgene integration by homology-dependent repair (HDR). CtIP, a key protein in early steps of homologous recombination, is fused to Cas9 and stimulates transgene integration by HDR at the human AAVS1 safe harbor locus. A minimal N-terminal fragment of CtIP, designated HE for HDR enhancer, is sufficient to stimulate HDR and this depends on CDK phosphorylation sites and the multimerization domain essential for CtIP activity in homologous recombination. HDR stimulation by Cas9–HE, however, depends on the guide RNA used, a limitation that may be overcome by testing multiple guides to the locus of interest. The Cas9–HE fusion is simple to use and allows obtaining twofold or more efficient transgene integration than that with Cas9 in several experimental systems, including human cell lines, iPS cells, and rat zygotes.
Highlights
In genome editing with CRISPR–Cas9, transgene integration often remains challenging
CtIP is a key protein in the initial step of homologous recombination that acts as a cofactor for MRE11 endonuclease in triggering DNA end resection13–16
We first targeted DNA cleavage at the AAVS1 locus with TALENs and coexpressed dCas9–CtIP with guide RNAs specific to sequences next to the TALEN cleavage site (Fig. 1a) in RG37DR cells, human fibroblasts transformed by SV4017
Summary
In genome editing with CRISPR–Cas, transgene integration often remains challenging. Here, we present an approach for increasing the efficiency of transgene integration by homologydependent repair (HDR). NHEJ inhibition, for instance, following Ligase 4 inactivation, can increase HDR10 Another approach has been to fuse the Geminin degron to Cas in order to induce its degradation in G1 and restrict target DNA cleavage to S/G2 phases. We report a simple approach to increase HDR using Cas nuclease fused to an N-terminal domain of CtIP, a key protein in early steps of homologous recombination This approach forces CtIP to the cleavage site and increases transgene integration by HDR. HDR stimulation by the Cas9–HE fusion depends on the guide RNA used, and this limitation may be overcome by testing multiple guide RNAs. In the absence of donor DNA, Cas9–HE induced a pattern of indels different from Cas, and deletions between short stretches of homologous sequences were favored, suggesting that cNHEJ was partially inhibited and MMEJ was enhanced, likely due to the stimulation of DNA resection by the HE domain. Using Cas9–HE is straightforward, does not require using genetically modified cells or pharmacological reagents, and our results suggest that DNA repair pathways can be biased locally, at the site of DNA cleavage, to favor HDR and precise genome editing
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