Co-CRISPR: a valuable toolkit for mutation enrichment in the gene editing of Spodoptera frugiperda

Insect Sci. 2022 Sep 28. doi: 10.1111/1744-7917.13122. Online ahead of print.

ABSTRACT

CRISPR/Cas9 system has been successfully applied in dozens of diverse species. While the screening of successful CRISPR/Cas9 editing events remains particularly laborious, especially for which occur at relatively low frequency. Recently, co-CRISPR strategy was proved to enrich the desired CRISPR events. Here, the co-CRISPR strategy was developed in the fall armyworm (FAW), Spodoptera frugiperda, with kynurenine 3-monooxygenase gene (kmo) as a marker. The kmo mosaics induced by sgRNAs/Cas9 displayed the darker green color phenotype in larvae compared to wild type (brown), and the mosaic-eye adults were significantly acquired from the mosaic larvae group. In kmo knockout strain, no significant difference was observed in the larval development and adult reproduction. The acetylcholinesterase 2 (ace2) and Wnt1 were selected as target genes to construct the co-CRISPR strategy using kmo marker. By co-injection of kmo and ace2 sgRNAs, the mutant efficiency of ace2 was significantly increased in the kmo mosaic (larvae or adults) groups. Similarly, more malformed pupae with Wnt1 mutations were observed in the darker green larvae group. Taken together, this study demonstrated that kmo was a suitable visible marker gene for the application and extension of co-CRISPR strategy in FAW. Using darker green color in larvae or mosaic-eye in adults from kmo knockout as a marker, the mutant efficiency of a target gene could be enriched in FAW group consisting of marked individuals. The co-CRISPR strategy is helpful for the gene function studies by the knockout technique with no or lethal phenotypes. This article is protected by copyright. All rights reserved.

PMID:36169087 | DOI:10.1111/1744-7917.13122