Using modified DNA fragments as donors, the Center of Excellence in Molecular Plant Science of Chinese Academy of Sciences (CAS) has established an efficient fragment targeting knockout and replacement method on rice. The efficiency of this method is up to 47.3%, which will greatly facilitate plant research and breeding.

In recent years, CRISPR/Cas mediated site-directed editing of plant genome has played an important role in the study of gene function and precision breeding of crops, showing broad development potential and application prospects. However, CRISPR/Cas mediated site-specific knockout of plant genomes can only produce random insertions and deletions at specific sites in the genome, and the efficiency of precise fragment insertion and replacement has been very low, which limits its application in plant research and breeding. There is an urgent need to establish more efficient systems for inserting and replacing fragments of plant genomes. 

The researchers found that simultaneous thiomodification and phosphorylation of donor fragments improved the efficiency of targeted knockouts. We targeted various DNA fragments, such as translation enhancers, transcriptional regulatory elements and promoters, at 14 loci in various T0-generation gene editing rice plants. Through the analysis of 1393 edited plants, it was found that the knockout efficiency of this method was up to 47.3%, 25% on average. The new method can even achieve multiple gene targeting at four sites at the same time.

On this basis, the researchers proposed a method of homologous recombination mediated by repetitive fragments. Using this method, the researchers achieved precise fusion of fragment replacement and in-situ tagging proteins at five gene loci with a maximum efficiency of 11.4 percent.

Time:2020.07.10

Source: http://www.xinhuanet.com/science/2020-07/09/c_139198989.htm