Maximizing the Efficacy of CRISPR/Cas Homology-Directed Repair Gene Targeting

Abstract

Clustered regularly interspaced short palindromic repeats/CRISPR-associated system (CRISPR/Cas) is a powerful gene editing tool that can introduce double-strand breaks (DSBs) at precise target sites in genomic DNA. In mammalian cells, the CRISPR/Cas-generated DSBs can be repaired by either template-free error-prone end joining (e.g., non-homologous end joining/microhomology-mediated end joining [NHEJ]/[MMEJ]) or templated error-free homology-directed repair (HDR) pathways. CRISPR/Cas with NHEJ/MMEJ DNA repair results in various length insertions/deletion mutations (indels), which can cause frameshift mutations leading to a stop codon and subsequent gene-specific knockout (i.e., loss of function). In contrast, CRISPR/Cas with HDR DNA repair, utilizing an exogenous repair template harboring specific nucleotide (nt) changes, can be employed to intentionally edit out or introduce mutations or insertions at specific genomic sites (i.e., targeted gene knock-in). This review provides an overview of HDR-based gene-targeting strategies to facilitate the knock-in process, including improving gRNA cleavage efficiency, optimizing HDR efficacy, decreasing off-target effects, suppressing NHEJ/MMEJ activity, and thus expediting the screening of CRISPR/Cas-edited clonal cells.