CRISPR/Cas9 mediated BLOS2 knockout resulting in disappearance of yellow strips and white spots on the larval integument in Spodoptera litura.

The custom-design bacterial CRISPR/Cas9 system has been recently used in some insects, indicating a powerful technique for studies on gene function and transgenic insects. However, its use in lepidopteran pests is scarce. Here, we reported a CRISPR/Cas9 system mediated mutagenesis of biogenesis of lysosome-related organelles complex1, subunit 2 (BLOS2) gene in a noctuid pest Spodoptera litura. A fragment of SlitBLOS2 was identified by analyzing a S. litura transcriptome database by local basic BLAST, and the full length cDNA was acquired by RACE strategy. To clarify the function of SlitBLOS2, CRISPR/Cas9 based target mutagenesis of SlitBLOS2 was achieved, displaying a mosaic translucent integument in 62.3-70.6% larvae of G0 generation. Further PCR-based genotype analysis demonstrated various mutations occurred at the SlitBLOS2 specific target site. A homozygote mutant individual was obtained in G1 generation, in which the yellow strips and white spots on the larval integument completely disappeared. Our study clearly demonstrates the function of SlitBLOS2 in the integument coloration, and thus provides a useful marker gene for genome editing based gene functional study and pest control strategy in S. litura as well as other lepidopteran pests.