Abstract
The CRISPR-Cas9 system is used for genome editing in mammalian cells by introducing double-strand breaks (DSBs) which are predominantly repaired via non-homologous end joining (NHEJ) or to lesser extent by homology-directed repair (HDR). To enhance HDR for improving the introduction of precise genetic modifications, we tested fusion proteins of Cas9 nuclease with HDR effectors to enforce their localization at DSBs. Using a traffic-light DSB repair reporter (TLR) system for the quantitative detection of HDR and NHEJ events in human HEK cells we found that Cas9 fusions with CtIP, Rad52, and Mre11, but not Rad51C promote HDR up to twofold in human cells and significantly reduce NHEJ events. We further compared, as an alternative to the direct fusion with Cas9, two components configurations that associate CtIP fusion proteins with a Cas9-SunTag fusion or with guide RNA that includes MS2 binding loops. We found that the Cas9-CtIP fusion and the MS2-CtIP system, but not the SunTag approach increase the ratio of HDR/NHEJ 4.5–6-fold. Optimal results are obtained by the combined use of Cas9-CtIP and MS2-CtIP, shifting the HDR/NHEJ ratio by a factor of 14.9. Thus, our findings provide a simple and effective tool to promote precise gene modifications in mammalian cells.
Highlights
The RNA guided Cas9 nuclease is used to create targeted double-strand breaks (DSBs) in the genome of mammalian cells and represents a versatile tool for genome editing (Barrangou and Doudna, 2016; Komor et al, 2016)
We tested the ability of Cas9/homology-directed repair (HDR) effector fusion proteins and found that CtIP and Rad52 and MRE11A are effective for HDR stimulation, counteracting the inhibition of end resection by 53BP1 without strong suppression of nonhomologous end joining (NHEJ)
Our results show that the performance of the single chain MS2diCtIP fusion was comparable to the Cas9-CtIP direct fusion and that the combination of both proteins had an additive effect on NHEJ suppression, shifting the HDR/NHEJ repair of the reporter construct by a factor of 14.9
Summary
The RNA guided Cas nuclease is used to create targeted double-strand breaks (DSBs) in the genome of mammalian cells and represents a versatile tool for genome editing (Barrangou and Doudna, 2016; Komor et al, 2016). Since HDR requires the presence of a repair template and is restricted to the S and G2 phases of the cell cycle it occurs less frequently than NHEJ. This presents a barrier to applications that rely on precise sequence modifications, such as the correction of mutations in somatic gene therapy or the modeling of disease mutations. To reinforce precise gene editing at Cas induced DSBs, tools for shifting the repair pathway choice in favor of HDR must be developed.
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