Abstract:

As a nuclease, the Cas protein needs to recognize a specific protospacer adjacent motifs（PAM）sequence to exert its cleavage activity, for example SpCas9 recognizes the NGG PAM and LbCas12a recognizes the TTTV PAM. CasX protein, a new protein that can recognize the TTCN PAM sequence was discovered，which expands the editing range of genome editing technology. Using PlmCasX and DpbCasX derived from CasX, CRISPR/CasX-mediated rice gene editing system was established. The editing efficiency through transient expression of rice protoplasts mediated with PEG was analysed, and the results showed that the endogenous OsCPK16 was effectively edited by PlmCasX and DpbCasX. The subsequent rice stable genetic transformation results indicated DpbCasX showed 17.5% editing efficiency in OsCPK21 at TTCA PAM sites, while PlmCasX showed higher editing efficiency in OsCPK21 at TTCA PAM sites and in OsCPK4 at TTCG PAM sites, with editing efficiency of 66.07% and 23.21%, respectively. And the optimization of PlmCasX protein based on the MIDAS method could not improve its editing activity. The study proved that the CRISPR/CasX system has editing activity in rice, and it can recognize the characteristic of TTCR PAM, expanding the application range of gene editing technology in rice.

Key words: Oryza sativa L., genome editing, CRISPR, CasX