Action mechanisms of CRISPR/Cas system and its application in genetic improvement of crops

CRISPR/Cas system is an emerging gene-editing technology, which can knock out multiple genes at multiple specific loci. Compared with other gene editing systems including ZFNs (zinc finger nucleases) and TALENs (transcription activator-like effector nucleases), CRISPR/Cas system possesses distinguishing advantages including higher targeting efficiency, easier to design and operate, and less time and labor, and thereby is widely applied in microbial, animal and plant genome editing researches. The CRISPR/Cas system was initially found in eubacteria and archaebacteria to be an adaptive immune system that is used to degrade exogenous viruses or plasmids. Multiple CRISPR/Cas systems have been so far reported. CRISPR is a DNA fragment with clustered regularly interspaced short palindromic repeats, which interacts with Cas protein and consequently edits the genome. The main action mechanism of CRISPR/Cas system is that matured crRNA (CRISPR-derived RNA) is hybridized with tracrRNA (trans-activating RNA) to form a single guide RNA (sgRNA), and thereby guides Cas endonuclease to cut double-strand DNA within a 20-nt short sequence, which is complementary with crRNA. Such targeted double-strand breaks activate two distinct DNA repair mechanisms including non-homologous end joining (NHEJ) and homology-directed repair (HDR). The NHEJ mechanism is easy to induce deletion or insertion mutation at the cleavage site. Thus, different sgRNAs can be designed for different target sites to perform gene editing at specific sites. This review mainly focuses on the progress and action mechanisms of CRISPR/Cas9 system and its application in a variety of important crops, and finally makes some prospects about low frequency off-target phenomenon in CRISPR/Cas9 operation and its future applications.