Plant genomes contain a variety of regulatory elements, functional motifs, and non-coding DNA, such as promoter cis-acting elements, miRNA coding sequences, and intergenomic regions with regulatory functions. These DNA sequences play an important role in regulating gene expression, transcriptional translation and other aspects, and are also the key target areas of gene function research and genetic improvement at present. CRISPR/Cas9 based genomic editing technology has been widely used in functional gene research and crop genetic improvement. Cas9 nuclease guided by sgRNA can produce DNA double-strand breaks at genomic target sites, and cells repair by non-homologous terminal connections, often easily forming 1~3 nucleotide insertion or deletion mutations. However, such insertion/deletion mutations are difficult to effectively disrupt the regulatory DNA functions, so the classic CRISPR/Cas9 cannot effectively manipulate these important DNA functional elements. Therefore, the development of a novel, precise and predictable polynucleotide deletion genome editing system is of great significance for the functional analysis and application of regulatory DNA sequences.

The Institute of Genetic and Developmental Biology of the Chinese Academy of Sciences is dedicated to the research and development of new technologies for plant genome editing. Recently, the research on the basis of the principle of cytosine deamination and base excision repair, first to wild type SpCas9 with cytosine deaminase APOBEC, uracil glycosylation enzyme and free of purine pyrimidine loci lyase combination, the establishment of a new polynucleotide targeted delete system, and successfully implemented in rice and wheat genome precise and predictable polynucleotide deleted. Consider the plant cell itself exists BER system, the team first use of high deamination activity APOBEC3A deaminase construct three kinds of forms of AFID system, and in the cells of rice and wheat to test multiple endogenous DNA targets, the results showed AFID - 3 mediated removal efficiency is as high as 33.1%, and produced from different between 5 '- cytosine and Cas9 cutting locus of polynucleotide deleted, predictable delete proportion is as high as 30% above. The researchers further screened the non-sibling pyrimidine deaminase and found that the truncated APOBEC3B deaminase not only had higher deaminase activity, but also had a narrower deaminase window. The researchers developed the EAFID-3 system by replacing A3A in the AFID-3 system with A3Bctd.Eafid-3 is 1.5 times more efficient at mediating predictable deletions from the deamination site that favors TC motifs to the Cas9 cleavage site. In addition, the team obtained polynucleotide deletion mutant plants by targeting the effector binding elements on the rice OsSWEET14 gene promoter using the AFID-3 system. The inoculation test of Bacterial blight showed that the polynucleotide deletion mutant produced by this system was more resistant to Bacterial blight than the insertion deletion of 1~2 bp.

Source: http://www.cas.cn/syky/202006/t20200630_4751456.shtml

Time: 2020.07.01