Homologous Recombination-based CRISPR Precision Gene Editing Technology and Its Application in Crop Breeding

Abstract

With the advent of CRISPR technologies, almost at any specific locus in any genome of animal or plant, large-size DNA fragment can be inserted or replaced seamlessly following the occurrence of CRISPR nuclease-produced double-strand break (DSB) and donor DNA, and the induction of homology-directed repair (HDR) can be realized. At present, the homologous recombination (HR)-based CRISPR precision gene editing plays increasingly important roles in crop gene functional analysis and new techniques breeding. In this review, aiming at effective realization of HR-based CRISPR precision gene editing in plant cells, we briefly review the two HR-based mechanisms underlying CRISPR precision gene editing, i.e., synthesis-dependent strand annealing (SDSA) and non-homologous end joining (NHEJ) assisted single strand annealing (SSA). Based on this, we elaborate the DSB-producing and HDR-inducing CRISPR nucleases and DNA/RNA donors, mainly including Cas9/12 and fusion proteins, sgRNA/crRNA and modifiers, and donor DNA/RNA and modifiers. Furthermore, we summarize the adopted delivery systems of CRISPR components and donor DNA/RNA during plant genetic transformation, in light of keeping spatiotemporal consistency of DSB and donor DNA/RNA to improve HR efficiency. Finally, we provide an outlook for the promising applications in crop functional genomics study and new techniques breeding of HR-based CRISPR precision gene editing.

Keywords

• crispr
• dna double-strand break
• homologous recombination
• homology-directed repair
• gene editing