Recently, the Crop Pest Functional Genomics Research Innovation Team of the Institute of Plant Protection, Chinese Academy of Agricultural Sciences published an online publication titled”SpRY greatly expands the internationally renowned academic journal Genome Biology” Genome editing scope in rice with highly flexible PAM recognition” research paper. In this study, SpCas9 mutants SpG and SpRY were applied to plants for the first time, and the targeted operation of NRN PAM and NYN PAM was realized in rice. The series of tools developed have greatly expanded the application range of the CRISPR system in plant genomes.
In recent years, genome editing technology has developed rapidly and is widely used in human gene therapy, animal and plant molecular breeding and other fields. Since the traditional Streptococcus pyogenes Cas9 (SpCas9) and the Cas proteins excavated later are typical G-PAM recognition types, they are somewhat limited in application. Researchers have been looking for a variety of new CRISPR/Cas systems in order to break through the restriction on the recognition specificity of G-PAM and expand the target range of various gene editing tools in the organism’s genome.
This study focuses on two new mutants of SpCas9, SpG and SpRY. The study found that SpG has a preference for NG PAM, but its activity is lower than that previously discovered by the team SpCas9-NG. SpRY has achieved high-efficiency editing on a large number of genomic target sites, and has a preference for both G-PAM and A-PAM. The cytosine base editor rBE66, developed based on SpRY nickase, realizes targeted editing of target bases by identifying NAG PAM to OsCOI2 and BSR-K1. The editing efficiency is 26.00%and 4.26%, respectively. The adenine base editor rBE62, developed by fusion of SpRY nickase and adenosine deaminase TadA8e, can effectively recognize NAA, NAT and NAC PAM. The editing efficiency of OsMPK13, OsGS and OsGSK4 is as high as 29.79%, 93.75%and 51.28, respectively.%. In addition, this study revealed that SpRY has high-frequency self-editing events (that is, editing the sgRNA sequence on T-DNA), while the self-editing events of SpRY nickase-mediated base editing occur less frequently. These results indicate that SpRY can be used for site-specific editing of the rice genome, especially single-base editing, and broadens the scope of CRISPR technology in plant genome editing, for the development of subsequent genome editing derivatives, and future plant gene function research materials and new germplasm The customization of materials provides strong theoretical guidance and technical support.
Master student Xu Ziyan (Zhou Xueping’s research group) and doctoral student Kuang Yongjie (Zhou Huanbin’s research group) and postdoctoral Ren Bin (co-supervisor:Researcher Zhou Huanbin and Researcher Wang Guirong) are the co-first authors. Researcher Zhou Huanbin and Professor Zhou Xueping are the co-corresponding authors. Many members of the team worked together to complete the work. Professor Sun Wenxian from China Agricultural University and Carl Spetz from the Norwegian Institute of Bioeconomics also participated in the research work. It has been supported by the National Natural Science Foundation of China, the Science and Technology Innovation Project of the Chinese Academy of Agricultural Sciences, the technological innovation task of improving the quality and efficiency of the base platform, and the National Genetically Modified Science and Technology Program of China.
link to thesis:https://genomebiology.biomedcentral.com/articles/10.1186/s13059-020-02231-9