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....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.展开更多
基金Acknowledgments This work was supported by grants from the Strategic Priority Research Program of Chinese Academy of Sciences （XDB11010500）, the External Cooperation Program of BIC, Chinese Academy of Sciences （GJHZ201305） and the National Natural Science Foundation of China （31420103918 and 31372257）.
文摘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.
