Recombineering-based no-cleavage gene-editing toolkit for large-scale genome engineering and functional screening

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Project Abstract Recombineering-based no-cleavage gene-editing toolkit for large-scale genome engineering and functional screening Exemplified by the CRISPR-Cas9 system, gene-editing technology is a powerful collection of tools for probing the hidden mechanisms of human diseases by understanding and controlling the functions of human genome variants. Limitations of existing CRISPR tools stem from two sources: 1) Cas9 cutting causes uncontrollable DNA damage at on/off-target sites, leading to toxicity and stress response. Recent studies confirmed that cutting-induced damages lead to significant gene expression changes and enrichment of p53-mutant cells, thus confounding some CRISPR screens; 2) CRISPR enzymes do not repair the target DNA, thus relying on endogenous DNA repair to complete editing. This results in low efficiency and high variability for Cas9-mediated homology-directed repair (HDR) across cell types and models. To overcome these limitations, we have identified a recombineering-based gene-editing tool, termed RecE/T- induced Editing via Designer-Cas9-Initiated Targeting (REDIT). REDIT uses deactivated Cas9 (dCas9) and generates minimal DNA break and near-zero toxicity. REDIT uses phage recombineering proteins RecE/T for gene-editing, bypassing the dependence on endogenous repair mechanisms. Our proof-of-concept demonstration showed that REDIT achieved efficient kb-scale editing without DNA cutting. We will focus on technology development and validation with well-characterized models using gold-standard assays. The proposed RecE/T-like recombineering proteins present new opportunities as they promote strand invasion/exchange without cleavage when genome sites become transiently accessible via dCas9 DNA-unwinding. Our goal is to develop a safe, scalable toolkit with up to 80% HDR efficiency for kilobase gene- editing and pooled knock-in screening.