From DNA break repair pathways to CRISPR/Cas-mediated gene knock-in methods

Abstract

Various DNA breaks created via programmable CRISPR/Cas9 nuclease activity results in different intracellular DNA break repair pathways. Based on the cellular repair pathways, CRISPR-based gene knock-in methods can be categorized into two major strategies: 1) Homology-independent strategies which are targeted insertion events based on non-homologous end joining, and 2) Homology-dependent strategies which are targeted insertion events based on the homology-directed repair. This review elaborates on various gene knock-in methods in mammalian cells using the CRISPR/Cas9 system and in sync with DNA-break repair pathways. Gene knock-in methods are applied in functional genomics and gene therapy. To compensate or correct genetic defects, different CRISPR-based gene knock-in strategies can be used. Thus, researchers need to make a conscious decision about the most suitable knock-in method. For a successful gene-targeted insertion, some determinant factors should be considered like cell cycle, dominant DNA repair pathway, size of insertions, and donor properties. In this review, different aspects of each gene knock-in strategy are discussed to provide a framework for choosing the most appropriate gene knock-in method in different applications.

Keywords: CRISPR/Cas9; DNA repair; Gene knock-in; Genome targeting; Homology-directed repair; Non-homologous end-joining.