The democratization of gene editing: Insights from site-specific cleavage and double-strand break repair

Abstract

DNA double-strand breaks (DSBs) are dangerous lesions that if not properly repaired can lead to genomic change or cell death. Organisms have developed several pathways and have many factors devoted to repairing DSBs, which broadly occurs by homologous recombination, which relies on an identical or homologous sequence to template repair, or nonhomologous end-joining. Much of our understanding of these repair mechanisms has come from the study of induced DNA cleavage by site-specific endonucleases. In addition to their biological role, these cellular pathways can be co-opted for gene editing to study gene function or for gene therapy or other applications. While the first gene editing experiments were done more than 20 years ago, the recent discovery of RNA-guided endonucleases has simplified approaches developed over the years to make gene editing an approach that is available to the entire biomedical research community. Here, we review DSB repair mechanisms and site-specific cleavage systems that have provided insight into these mechanisms and led to the current gene editing revolution.

Keywords: CRISPR-Cas9; Double-strand break repair; Gene editing; HO endonuclease; Homologous recombination; Homology-directed repair; I-SceI endonuclease; Nonhomologous end-joining.

Figure 1

Gene targeting. A. Ends-out gene targeting. A DNA fragment carrying a selectable marker (neo) recombines by homologous recombination with an intact chromosome. B. A DSB introduced into the target site on a chromosome can incorporate a linear DNA fragment by annealing of 5′ to 3′ resected ends of both the DSB and the fragment. Incorporation of the fragment often proceeds by gene conversion as illustrated in Fig. 2A. C. Single-strand template repair of a chromosomal DSB.

Figure 2

Homologous recombination of chromosomal DSBs. A. Noncrossover gene conversion by synthesis-dependent strand annealing (SDSA). B. Crossover-associated gene conversion through a double Holliday junction (dHJ) intermediate. The dissolution of the dHJ intermediate can also lead to a noncrossover outcome. C. Break-induced replication occurs when only one end of a DSB shares homology with a genomic template.