CtIP fusion to Cas9 enhances transgene integration by homology-dependent repair

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.

Fig. 1

Forcing CtIP recruitment to the DNA cleavage site stimulates targeted transgene integration. a Distribution of TALEN and guide RNAs at AAVS1 safe harbor locus. gRNAs are indicated on top of their corresponding PAM motif, which is shown as lowercase in the sequence. The donor DNA used had 5′ and 3′ homology arms as indicated. b Relative HDR and indel frequencies induced by TALEN and dCas9–CtIP recruitment near the cleavage site using different guide RNAs. Human RG37 fibroblasts were transfected with the indicated plasmids and GFP transgene donor with homology arms to the targeted AAVS1 locus. HDR-mediated transgene integration was measured by FACS analysis of GFP-positive cells resulting from targeted GFP transgene integration. Indels at the cleavage site were measured by the T7E1 assay. The results are expressed as the mean of relative HDR or indel frequencies calculated by normalizing HDR or indel frequencies by that induced by TALEN transfection alone. Asterisks indicate the difference that is statistically significant when comparing cotransfection of dCas9–CtIP, guide RNA, and TALEN to TALEN-alone transfection in t-test (*P<0.05). Data are from three independent experiments. Error bars indicate standard deviation. c Relative HDR and indel frequencies induced by Cas9 and Cas9–CtIP at the cleavage site directed by T2 guide RNA. The results are expressed as the mean of relative HDR or indel frequencies calculated by normalizing HDR or indel frequencies by that induced by Cas9. Asterisks indicate that the difference is statistically significant when comparing Cas9–CtIP to Cas9 in t-test (**P<0.005). Data are from three independent experiments. Error bars indicate standard deviation. Guide RNA T2 was used because in contrast to other guides, it did not cleave the donor DNA available

Fig. 2

Identification of the “HDR-enhancer” (HE) domain of CtIP. a Schematic diagram of CtIP protein showing the different truncated CtIP proteins that have been fused to Cas9 and tested for their ability to stimulate HDR. Various sequence features of CtIP, including tetramerization and dimerization domains, and CDK phosphorylation sites S233, T245, and S276, are indicated. b Identification of a domain of CtIP, called HE, spanning aa 1 to 296, which is able to stimulate HDR when fused to Cas9. Human RG37 fibroblasts were transfected with the indicated plasmids expressing Cas9 or Cas9–CtIP derivatives, T2 guide RNA plasmid, and GFP transgene donor with homology arms to the targeted AAVS1 locus. Expression of fusion proteins was examined by western blot (Supplementary Fig. 1). Data are from four independent experiments. Error bars indicate standard deviation. c Functional analysis of HE domain. HEK293 cells were transfected with the indicated Cas9 plasmids, T2 guide RNA, and GFP transgene donor with homology arms to the AAVS1 targeted locus. HDR-mediated transgene integration was measured by FACS analysis of GFP-positive cells, resulting from targeted GFP transgene integration. Indels at the cleavage site were measured by the T7E1 assay. The results are expressed as the mean of relative HDR or indel frequencies calculated by normalizing every HDR or indel frequency by that induced by Cas9, respectively. Asterisks indicate that the difference is statistically significant when comparing Cas9–CtIP or Cas9–HE derivatives to Cas9 in nonparametric t-test (*P<0.05, **P<0.005, or ***P<0.0005). Data are from four independent experiments. Error bars indicate standard deviation. The relative expression levels of Cas9 and Cas9–HE derivatives were analyzed by western blot using anti-Cas9 and control anti-tubulin antibodies

Fig. 3

Stimulation of transgene integration by Cas9–HE and Cas9–Geminin. Relative frequencies of HDR and indels induced by Cas9 or fusion of Cas9 to HE domain, Geminin degron, or both. Human HEK293 cells were transfected with the indicated Cas9 plasmids, T2 guide RNA, and GFP transgene donor with homology arms to the AAVS1 targeted locus. HDR-mediated transgene integration was measured by FACS analysis of GFP-positive cells, resulting from targeted GFP transgene integration. Indels at the cleavage site were measured by the T7E1 assay. The results are expressed as the mean of relative HDR or indel frequency calculated by normalizing every HDR or indel frequency by that induced by Cas9. Asterisks indicate that the difference is statistically significant when comparing Cas9–HE, Cas9–HE–Geminin, and Cas9–Geminin to Cas9 in t-test (*P<0.05 or **P<0.005). Data are from three independent experiments. Error bars indicate standard deviation. Relative expression levels of Cas9 and other fusions were analyzed by western blot with anti-Cas9 and control anti-tubulin antibodies. Protein extracts were obtained with lysis buffer containing 150 mM NaCl, which resulted in inefficient solubilization of Cas9 fusions with the HE domain compared to those of Cas9 and Cas9–Geminin

Fig. 4

Stimulation of transgene integration by Cas9–HE in iPS cells. a Relative frequencies of HDR induced by Cas9–HE at DNA cleavage site directed by T2 guide RNA. Human iPS cells were transfected with the indicated Cas9 plasmids, T2 guide RNA, and puromycin transgene donor with 800-bp homology arms to the AAVS1 locus. HDR-mediated transgene integration was measured by counting puromycin-resistant clones, resulting from targeted integration at the AAVS1 locus of the puromycin resistance exon carried by the transgene. The results are expressed as the mean relative number of puromycin-resistant clones after normalizing the number of puromycin-resistant clones by that induced by Cas9. The difference between Cas9–HE and Cas9 was statistically significant (t-test, *P<0.05). Data represented are from four independent experiments. Error bars indicate standard deviation. b A clone of puromycin-resistant cells derived from puromycin selection of cells transfected with Cas9–HE, T2 guide RNA, and puromycin-resistant transgene donor was analyzed for expression of stemness markers TRA-1-60 and SSEA4 or Oct3/4 and Sox2 by FACS analysis. c After induction of cardiomyocyte differentiation in the clone analyzed in b, expression of cardiomyocyte markers alpha-actinin and MLC2-v was analyzed by immunohistochemistry. Scale bar, 50 µm

Fig. 5

HDR stimulation by the HE domain takes place at different target genes and can depend on the guide RNA used. a Relative frequencies of HDR induced by Cas9–HE were compared to those induced by Cas9 at five different target genes in HEK293 cells using previously published guide RNAs and donor plasmids. Targeted integration of the donor plasmid results in in-frame insertion of E2A-neoR cDNA. G418(neomycin)-resistant colonies were counted after Cresyl violet staining to measure HDR-mediated events and normalized by the number of colonies obtained with Cas9 to give the relative HDR frequencies indicated. Data represented are from three independent experiments for TGIF2, RAD21, and CREB genes and from four experiments for ATF4 and GABP genes and are provided in Supplementary Fig. 7. Error bars indicate standard deviation. b Relative frequencies of HDR induced by Cas9–HE were compared to those induced by Cas9 with the indicated guide RNAs, which all cleave to a small 50-bp region of the AAVS1 locus, and a common p84∆ donor plasmid, harboring ~800-bp homology arms. P84∆ was derived from the p84 donor plasmid depicted in Fig. 1a by shortening the homology arms so that they would not be cleaved by any of the guide RNAs. Asterisks indicate that the difference is statistically significant when comparing Cas9–HE to Cas9 in t-test (*P<0.05). Data represented are from five independent experiments and are provided in Supplementary Fig. 7. Error bars indicate standard deviation