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

• Published in: Nature communications Vol. 9; no. 1; pp. 1133 - 11
• Main Authors: Charpentier, M., Khedher, A. H. Y., Menoret, S., Brion, A., Lamribet, K., Dardillac, E., Boix, C., Perrouault, L., Tesson, L., Geny, S., De Cian, A., Itier, J. M., Anegon, I., Lopez, B., Giovannangeli, C., Concordet, J. P.
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group UK 19.03.2018; Nature Publishing Group; Nature Portfolio
• Subjects: 13; 13/106; 13/109; 13/44; 42; 42/109; 42/41; 631/1647/1511; 631/337/4041/3196; 631/61/191; 631/61/201; Animals; Base Sequence; Carrier Proteins - chemistry; Carrier Proteins - genetics; Carrier Proteins - metabolism; Cell Line; Cell lines; CRISPR; CRISPR-Cas Systems; DNA Breaks, Double-Stranded; DNA repair; Endodeoxyribonucleases; Enhancer Elements, Genetic; Female; Genome editing; Genomes; HCT116 Cells; HEK293 Cells; Homologous Recombination; Homology; Human health and pathology; Humanities and Social Sciences; Humans; INDEL Mutation; Induced Pluripotent Stem Cells - metabolism; Integration; Life Sciences; Loci; multidisciplinary; Nuclear Proteins - chemistry; Nuclear Proteins - genetics; Nuclear Proteins - metabolism; Oocytes - metabolism; Phosphorylation; Protein Multimerization; Proteins; Rats; Recombinant Fusion Proteins - chemistry; Recombinant Fusion Proteins - genetics; Recombinant Fusion Proteins - metabolism; Recombinational DNA Repair; Repair; Ribonucleic acid; RNA; RNA, Guide, CRISPR-Cas Systems - genetics; Science; Science (multidisciplinary); Transgenes; Virus Integration - genetics; Zygote - metabolism; Zygotes
• ISSN: 2041-1723; 2041-1723
• DOI: 10.1038/s41467-018-03475-7

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. The integration of exogenous DNA into the genome using CRISPR–Cas9 often presents a challenge to researchers. Here the authors fuse CtIP to Cas9 to stimulate recombination at target loci.