Enhancing CRISPR/Cas9 genomic editing efficiency based on optimization of sgRNA of Gossypium barbadense L.

The CRISPR/Cas9 genome editing system has been established in many crops. The advantages of its directional creation of mutants are increasingly favored by researchers. However, the CRISPR/Cas9 genome editing technology targets the editing of the target gene and also triggers off-target effects at different frequencies, which determine the reliability of the genome editing system. This study was based on the previous CRISPR/Cas9 genome editing system established in the island-cotton somatic cells. Edit the vector by constructing different codon optimization methods for Cas9, different numbers of PAM sites and different target sites, and the difference in editing efficiency and off-target effect were analyzed and compared. There was no significant difference in editing effects and off-target effects caused by Cas9-edited vectors with different optimal codons. The partially double-sgRNA had significantly higher editing efficiency and significantly lower off-target efficiency than the single sgRNA; the editing efficiency of the transformed No shift sgRNA target sequence was significantly higher than that of the Shift sgRNA and the former had a significant decrease in off-target efficiency relative to the latter. Therefore, using No shift sgRNA type target sequence can effectively improve the editing efficiency and significantly reduce the off-target efficiency, thus laying a theoretical foundation for optimizing the CRISPR/Cas9 mediated island cotton genome editing system and accurately and efficiently creating island cotton functional gene mutants in the future. [ABSTRACT FROM AUTHOR]