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〠Instrument R&D 】In the plant genome, there are a variety of regulatory elements, functional motifs and non-coding DNA, such as promoter cis-acting elements, miRNA coding sequences, and intergenic regions with regulatory functions. These DNA sequences play an important role in regulating gene expression, transcription and translation, and are currently the key target areas for gene function research and genetic improvement. The genome editing technology based on CRISPR/Cas9 has been widely used in functional gene research and crop genetic improvement. Cas9 nuclease guided by sgRNA can produce DNA double-strand breaks (DSB) at the target site of the genome. Cells are repaired by non-homologous end joining (NHEJ), and it is often easy to form insertion or deletion mutations of 1 to 3 nucleotides. . However, this insertion/deletion mutation is difficult to effectively destroy the function of regulatory DNA, so the classic CRISPR/Cas9 cannot effectively manipulate the above important DNA functional elements. Based on this, the development of a new, precise and predictable polynucleotide deletion genome editing system is of great significance for the functional analysis and application of regulatory DNA sequences.
Gao Caixia Research Group of Institute of Genetics and Developmental Biology, Chinese Academy of Sciences has long been devoted to the research and development of new technologies for plant genome editing. Recently, based on the principles of cytosine deamination and base excision repair (BER), the research team first split wild-type SpCas9 with cytosine deaminase APOBEC, uracil glycosylase (UDG) and apurinic pyrimidine sites. The combination of enzymes (AP lyase) established a new type of polynucleotide targeted deletion system (AFIDs), and successfully achieved precise and predictable polynucleotide deletion in the rice and wheat genomes. Considering that the plant cell itself has a BER system, the research team first used the APOBEC3A deaminase with high deamination activity to construct three types of AFID systems (AFID-1~3), and to identify multiple endogenous sources in rice and wheat cells. DNA targets were tested and the results showed that the deletion efficiency mediated by AFID-3 was as high as 33.1%, and polynucleotide deletions from different 5'-cytosines to Cas9 cleavage sites were generated, with a predictable deletion rate of up to 30% the above. The researchers further screened different cytosine deaminase and found that the truncated APOBEC3B deaminase (A3Bctd) not only has a higher deamination activity, but also has a narrower deamination window. The researchers replaced A3Bctd with A3A in the AFID-3 system to develop the eAFID-3 system. eAFID-3 more efficiently mediates the predictable deletion from the deamination site of the preferred TC motif to the Cas9 cleavage site, with 1.5 times the efficiency of AFID-3. In addition, the research team used the AFID-3 system to target the effector binding element on the promoter of the OsSWEET14 gene in rice to obtain mutant plants with polynucleotide deletions. The bacterial leaf blight inoculation test found that compared with the insertion deletion of 1~2 bp, the polynucleotide deletion rice mutant produced by this system is more resistant to bacterial blight.
The AFID system has the advantages of high efficiency, precision, and predictability. Therefore, the establishment of the system can provide a powerful genome editing tool for the functional research of plant genome regulatory DNA and design breeding. The research results were published online in Nature Biotechnology on June 29 (DOI: 10.1038/s41587-020-0566-4). Gao Caixia research group postdoctoral Wang Shengxing, postdoctoral Zong Yuan, doctoral student Lin Qiupeng and associate researcher Zhang Huawei are the co-first authors of the paper, and Gao Caixia is the corresponding author of the paper. The research team of Qiu Jinlong of the Institute of Microbiology, Chinese Academy of Sciences also participated in the research work. The research was supported by the Strategic Leading Science and Technology Special Project of the Chinese Academy of Sciences, the National Major Gene Technology Project of the National Transgene, and the National Natural Science Foundation of China.