CRISPR/Cas9-mediated targeted gene mutagenesis in Spodoptera litura

Custom-designed nuclease technologies such as the clustered regularly in- terspaced short palindromic repeat （CRISPR）-associated （Cas） system provide attractive genome editing tools for insect functional genetics. The targeted gene mutagenesis me- diated by the CR1SPR/Cas9 system has been achieved in several insect orders including Diptera, Lepidoptera and Coleoptera. However, little success has been reported in agricul- tural pests due to the lack of genomic information and embryonic microinjection techniques in these insect species. Here we report that the CRISPR/Cas9 system induced efficient gene mutagenesis in an important Lepidopteran pest Spodoptera litura. We targeted the S. litura Abdominal-A （Slabd-A） gene which is an important embryonic development gene and plays a significant role in determining the identities of the abdominal segments of in- sects. Direct injection of Cas9 messenger RNA and Slabd-A-specific single guide RNA （sgRNA） into S. litura embryos successfully induced the typical abd-A deficient pheno- type, which shows anomalous segmentation and ectopic pigmentation during the larval stage. A polymerase chain reaction-based analysis revealed that the Cas9/sgRNA complex effectively induced a targeted mutagenesis in S. litura. These results demonstrate that the CRISPR/Cas9 system is a powerful tool for genome manipulation in Lepidopteran pests such as S. litura.