Abstract 71: Cas9 protein engineering for cell cycle-specific genome editing to enhance homology directed repair

Abstract

Abstract The comprehensive and coordinated efforts of The Cancer Genome Atlas (TCGA) and the International Cancer Genome Consortium (ICGC) identified the somatic genomic alterations occurring in a broad panel of human cancers. The challenge ahead is to understand the functions of these somatically altered genes.Genome editing tools like the CRISPR/Cas9 system represent a transformative technology that allows the precise engineering of cells harboring specific (point) mutations found in specific tumor subtypes, enabling the investigation of the role of a candidate cancer gene in a stringently defined genetic context. Once recruited to its DNA target sequence in cells, Cas9 induces a DNA double strand break (DSB), which is either repaired by the non-homologous end-joining pathway (NHEJ), or by homology-directed repair (HDR). The HDR pathway allows for a precise site-specific knock-in and thus functional analysis of cancer-associated sequence variants. However, HDR activity is restricted to the late S and G2 phases of the cell cycle, while the NHEJ pathway dominates DNA repair throughout the cell cycle. Thus, the error-prone NHEJ pathway out-competes the HDR pathway, which reduces the likelihood for precise insertions, deletions or base substitutions at the DSB. Here, we devised a strategy to increase HDR by directly synchronizing the expression of Cas9 with cell cycle progression. Fusion of Cas9 to the N-terminal region of human Geminin converted this gene-editing protein into a substrate for the E3 ubiquitin ligase complex APC/Cdh1 resulting in a cell cycle tailored expression with low levels in G1, but high expression in S/G2/M. Importantly, Cas9-hGem(1/110) increased the relative rate of HDR by up to 87% at a single-copy reporter locus, and up to 42% at the endogenous MALAT1 locus compared to wild-type Cas9. In summary, our regulatory concept takes advantage of cell-autonomous pathways to enable fluctuation of Cas9 protein levels coupled to cell cycle dynamics. This resulted in higher site-specific integration events without sophisticated manipulation of cells by small molecules or additional gene targeting reagents. Future methodological developments may enable high-resolution expression of Cas9 and alternative genome engineering proteins like Cpf1 by combining cell cycle phase-specific transcriptional and post-translational regulatory elements. This might enhance HDR efficiencies even further, and will help to make sense of cancer genomic data. Citation Format: Tony Gutschner, Monika Hämmerle, Giannicola Genovese, Giulio F. Draetta, Lynda Chin. Cas9 protein engineering for cell cycle-specific genome editing to enhance homology directed repair. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 71.