The corn genome editing team of the Beijing Academy of Agricultural and Forthan sciences has made new progress in gene editing technology

Original title: Beijing Academy of Agricultural and Forthan sciences corn genome editing team in the gene editing technology has made new progress
December 16, Beijing Academy of Agricultural and Forthology
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editorial team presented a research paper entitled "Genome engineering in plant using an efficient CRISPR-xCas9 toolset with an expanded PAM compatibility" in the new frontiers series of journal Fro
ntiers in Genome Editing. The study established a gene knockout system based on SpCas9 variant xCas9 and a cytosine base editing system, successfully extending the range of gene-edited pre-region sequence proximity base sequence (PAM) to GD (D-A, T, G) and relaxation-type NG, providing the possibility of selecting gene editing targets within a broader genome range.
expanding the CRISPR/Cas9 nuclease PAM range is key to making it more widely used in plants. In 2018, the David Liu team reported on Nature a SpCas9 variant (xCas9) that identifies NG, GAA, and GATHAM. In 2019, several laboratories reported on the performance of xCas9 on plants and found that it was able to expand genome editing to NGPAM site, but for GAA and GAT PAM site, only one GATPAM site implementation in rice T0 seedlings was reported for editing. Another SpCas9 variant (SpCas9-NG), which currently has a larger PAM range, does not recognize GAA and GATPAM in plants or animals. Therefore, in order to expand the scope of genome editing to GAA and GATPAM bits, the xCas9 gene editing system in plants needs to be optimized and improved.
study developed an xCas9-based gene editing system (CRISPR-xCas9) by strengthening sgRNA by using tRNA connections to form an efficient tRNA-esgRNA system, and for the first time found CRISPR-xCas 9 in the plant can effectively cut GAAPAM site and multiple GATPAM site, at the same time found that it can also effectively cut multiple GAGPAM site (no reported in animals), and still maintain the ability to edit the relaxation type of NGPAM site. The study further developed an xCas9-based cytosine base editing system (xCas9n-CBE) using a similar carrier structure, and found that xCas9n-CBE can be used in plants to effectively base edit the genomic targets of GA and slack NG as PAM. The team successfully developed another new base editor (xCas9-pBE) based on xCas9 in 2019, and compared to the xCas9n-CBE developed in this study, it not only expands the NGCPAM bits that edit xCas9-pBE cannot edit, but also shows higher average base editing efficiency and wider editing windows. In addition to the two new base editors mentioned above, the team has developed several new base editors based on SpCas9 and its variant VQR, as well as SaCas9 variant SaKKH, in 2019, enabling C-to-T base replacement for PAM targets NAG, NGA, NNMRRT and NNKRGT. The comprehensive application of these base editors with different characteristics can expand the target area of genome editing on the crop genome on a wider scale, and has great potential for application.
Zhang Chengwei of the National Corn Center is the first author of this article and Yang Jinxiao is the first author of this paper. This study was supported by the Beijing Scholars (BSP041) Project and the Beijing Natural Science Foundation (6204041). The Corn Genome Editing Team was formed in 2017 and currently focuses on base editing, precise replacement, histological editing and its efficient use in crops such as corn and rice. This is the team's 8th high-level research paper published in an international academic journal since its inception.
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