The invention discloses an efficient, simple and convenient whole
genome CRISPR / Cas9 knockout
plasmid library for erencigia parviflora as well as construction and application of the whole
genome CRISPR / Cas9 knockout
plasmid library, and belongs to the technical field of
biology. On the basis of the Eriphaga parviflora T2T
genome sequence, a
bioinformatics tool is utilized to accurately predict a knockout target spot, double sgRNA is designed to improve the knockout accuracy and efficiency, and meanwhile, the off-target risk is reduced; the method comprises the following steps: synthesizing a double sgRNA-
pool chip, amplifying, recovering, connecting a carrier, transforming and the like, so that the phytoneum parvum
CRISPR-Cas9 knockout
plasmid library which is simple and convenient to operate and high in
throughput is successfully constructed. In the double sgRNA
design process, advanced tools are adopted to improve the prediction accuracy; after the library is constructed, a strict quality inspection step is also included, so that the library quality is ensured. The invention not only provides a new way for the
gene editing of the erencephalium leptosphaerium, but also provides reference for the
gene editing of other multi-copy and high-repetitive-sequence biological genes, has obvious effects in solving the off-target problem and improving the editing efficiency and accuracy, and has important scientific significance and application value for the research of
functional genomics.