Gao Caixia research team made new progress in the study of high-fidelity CRISPR-Cas9 genome editing methods

is a new disruptive technology in life science, especially the gene editing tool based on CRISPR-Cas9 system has developed rapidly in recent years and has been widely used in medical, agricultural and other fields. However, Cas9 off-target phenomenon is one of the most important problems that limit its great potential, and improving the specificity of the system has always been the focus of genome editing method research.
through protein engineering, two U.S. task groups conducted targeted modifications of Cas9 proteins in the early stages, and obtained three specific and significantly improved Cas9 protein variants: eSpCas9 (1.0), eSpCas9 (1.1) and SpCas9-HF1. A recent study by Gao Caixia of the Institute of Genetics and Developmental Biology of the Chinese Academy of Sciences found that the genome editing activity of these three high-fidelity SpCas9 nucleases is strictly influenced by the length of the sgRNA guide sequence. Setting the wizard sequence to 20 bases that exactly match the target point is an important prerequisite for ensuring the activity of the three high-fidelity SpCas9 nucleases. To do this, Gao Caixia's team fused rice tRNAGlu sequences between U3 promoters and sgRNAs, using RNase P and RNase Z, the endocrine sources, to process the wizard sequences in immature sgRNAs into 20 bases that exactly match the target sequence, by This strategy keeps the activity of eSpCas9 (1.0), eSpCas9 (1.1) and SpCas9-HF1 at a level comparable to that of wild SpCas9 and maintains its specificity.
the study was published online October 11, 2017 in the journal
(DOI:10.1186/s13059-017-1325-9). Zhang Dingbo, a master's student in Gao Caixia Research Group, and Zhang Huawei, an associate researcher, are the first authors of the paper, which is supported by the Ministry of Science and Technology, the Ministry of Agriculture, the Chinese Academy of Sciences and the National Natural Resources Fund. (Source: Science.com)