CRISPR/Cas9 system and twin virus vector system integrate in rice to achieve directed knock-in of foreign genes.

On March 19, the journal Molecular Plant published online a research paper entitled Gene targeting by homology-directed repair in rice using a geminivirus-base CRISPR/Cas9 system at the Center for Plant Adversity Biology, Shanghai Institute of Life Sciences, Chinese Academy of Sciences.
this work integrates CRISPR/Cas9 system and twin virus vector system to achieve directed entry of extrogenic genes in rice, with an efficiency of up to 19%.
the study provides a new method for simple and efficient omniogenic recombination or fixed-point tapping of exogenetation fragments in the plant genome.
CRISPR/Cas9 system has been widely used in gene editing, using non-dominant homolog directed repair (HDR) in the repair pathway to achieve gene homolog recombination (homolog recombination, HR) or directional insertion (knock in), but the efficiency of the repair is very low, greatly affecting crispr/Cas9 gene editing.
one of the great challenges of HDR in the plant world, as opposed to animal and human cells, is how to deliver enough of the supply molecules to the cells.
have made a number of attempts to break through this bottleneck, and Voytas and others have recently developed new ways to use geminivirus for HDR.
Geminivirus is a group of plant viruses with many members that infect most single-leaf and geminid plants, and their genomes consist of 2.5-3kb of single-stranded ring DNA that can be transformed into the supply molecules needed for HDR.
when Geminivirus infected plant cells, the virus's large replication properties allowed the cell to produce large numbers of supply molecules, up to hundreds to thousands of copies, greatly increasing the risk of HDR.
zhu health research team modified a member of Geminivirus, the wheat dwarf virus (WDV), and tests in rice-injured tissue found that it provided hundreds of times as many provider molecules as traditional T-DNA.
The study further integrated CRISPR/Cas9 system and WDV system, supplemented by directional screening, inserted GFP markers into the ACT1 and GST sites of rice endoens, and the identification of the transformation plant showed that the efficiency of directed tapping of the external gene could reach up to 19%.
the study provides a simple and efficient method for omniogenic recombination or targeted tapping of exogenetation fragments in the plant genome, which will play an important role in gene functional research and crop fine breeding.
the work has received financial support from the Chinese Academy of Sciences.
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