Abstract 5607: A highly efficient donor system for CRISPR knock-in editing

Abstract

Abstract CRISPR is a potent tool widely used for genome editing. While CRISPR knock-out, achieved through in-del mutations via cellular repair mechanisms, has proven remarkably effective, the knock-in system for exogenous fragment insertion encounters challenges due to limited specificity and efficiency. Two primary methods for exogenous fragment insertion, namely NHEJ (Non-Homologous End Joining) and HR (Homologous Recombination), exist. HR allows for the construction of DNA insertions with precise junctions but is comparatively less efficient than NHEJ. To address these limitations, our system focuses on enhancing the efficiency of HR-based genome editing. Primarily, we have optimized the Lenti All-in-one CRISPR system to augment the delivery and targeting efficiency of the Cas9-gRNA complex. The system has been successfully employed to knock out Glutamine Synthetase (GLUL) in HEK293T cells, exhibiting superior efficiency compared to commercially available tools. Secondly, we have implemented a pseudo-lenti system to amplify the entry capacity of donor DNA with homology arms into target cells. This technique has shown an improved HR effect in the experiment to tag a GFP at the C terminals of CDC25A. Lastly, we leverage proximity effects by fusing Cas9 with a capture protein that can grasp a bait sequence attached to the donor DNA, thereby facilitating its transfer to the target sites. Our system has demonstrated exceptional efficacy as a CRISPR knock-out platform and a powerful tool for DNA tagging functionality on target genes. Citation Format: Lipeng Wu, Dezhong Yin, Hua Su, Justin Fellows, Mao Fu, Julian Heller, Brian Park, Alex Juminaga. A highly efficient donor system for CRISPR knock-in editing [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5607.