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
Insect body pigmentation and coloration are critical to adaption to the environment. To explore the mechanisms that drive pigmentation, we used the clustered regularly interspaced short palindromic repeats (CRISPR)/CR...Insect body pigmentation and coloration are critical to adaption to the environment. To explore the mechanisms that drive pigmentation, we used the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) genome editing system to target the ebony gene in the non-model insect Spodoptera litura. Ebony is crucial to melanin synthesis in insects. By directly injecting Cas9 messenger RNA and ebony-specific guide RNAs into S. litura embryos, we successfully induced a typical ebony-deficient phenotype of deep coloration of the puparium and induction of melanin formation during the pupal stage. Polymerase chain reaction-based genotype analysis demonstrated that various mutations had occurred at the sites targeted in ebony. Our study clearly demonstrates the function of ebony in the puparium coloration and also provides a potentially useful marker gene for functional studies in S. litura as well as other lepidopteran pests.展开更多
As a core member of the Hippo signaling pathway,Hpo plays a critical role in regulating growth and development.Previous studies reported that loss of function of Hpo results in increased proliferation,reduced apoptosi...As a core member of the Hippo signaling pathway,Hpo plays a critical role in regulating growth and development.Previous studies reported that loss of function of Hpo results in increased proliferation,reduced apoptosis and induction of tissue overgrowth in Drosophila.In this study,we used CRISPR/Cas9(Clustered Regularly Interspaced Short Palindromic Repeats/Casg)to study Hpo gene (BmHpo)function in the lepidopteran insect Bombyx mori,known commonly as the silkworm.Sequence analysis of BmHpo revealed an array of deletions in mutants.We found that BmHpo knockout resulted in defects in body size regnlation,in developmental defects and pigment accumulation and early death. Our data show that BmHpo is essential for regulation of insect growth and development and that CRISPR/Cas9technology can serve as a basis for functional analysis of target genes in lepidopteran insects.展开更多
Fusion of the testis occurs in most Lepidoptera insects, including Spodoptera litura, an important polyphagous pest. Testicular fusion in S. litura is advantageous for male reproduction, and the molecular mechanism of...Fusion of the testis occurs in most Lepidoptera insects, including Spodoptera litura, an important polyphagous pest. Testicular fusion in S. litura is advantageous for male reproduction, and the molecular mechanism of fusion remains unknown. Doublesex influences the formation of genitalia, the behavior of courtship, and sexually dimorphic traits in fruit-fly and silkworm, and is essential for sexual differentiation. However, its purpose in the testis of S. litura remains unknown. The doublesex gene of S. litura (Sldsx) has male-specific SldsxM and female-specific SldsF isoforms, and exhibits a higher expression level in the male testis. At the testicular fusion stage (L6D6), Sldsx attained the highest expression compared to the pre-fusion and post-fusion periods. Moreover, Sldsx had a higher expression in the peritoneal sheaths of testis than that of germ cells in the follicle. CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats/Cas9) was applied to S. litura to determine the role of Sldsx. A mixture of single guide RNA messenger RNA and Cas9 protein (300 ng/μL each) was injected into eggs within 2 h following oviposition. CRISPR/Cas9 successfully induced genomic mutagenesis of Sldsx at Go generation. The mutant males had smaller testis surrounded by less tracheae. Moreover, the mutant males had abnormal external genitalia and could not finish mating with wild-type females. Additionally, testes were fused for almost all mutant males. The results showed that Sldsx was not related to testicular fusion, and is required for both testis development and the formation and function of external genitalia in S. litura. The main roles of doublesex on the male are similar to other insects.展开更多
Ostrinia fumacalis (Lepidoptera: Pyralidae) is one of the most destructive agricultural pests in Asia. Traditional pest-management methods include sex pheromone capture, transgenic crops that produce Bacillus thuringi...Ostrinia fumacalis (Lepidoptera: Pyralidae) is one of the most destructive agricultural pests in Asia. Traditional pest-management methods include sex pheromone capture, transgenic crops that produce Bacillus thuringiensis toxin, and pesticides. Although these strategies control pest populations effectively, they also cause negative side effects, including dramatically increased pesticide resistance, severe pollution, and hazards for human health. Recently developed genome editing tools provide new prospects for pest management and have been successfully used in several species. However, few examples have been reported in the agricultural pest O. fumacalis due to a lack in genomic information. In this report, we identified only one transcript of O. fumacalis Argonaute 1 (OfAgo 1) gene from the genome and cloned the open reading frame. OfAgol presented the maximum expression at the embryo stage or in the fat body during the larval stages. To understand its function, an OfAgol mutant was constructed using the Clustered Regularly Interspaced Short Palindromic Repeat/RNA-guided Cas9 nuclease (CRISPR/Cas9). Mutagenesis of OfAgol disrupted cuticle pigmentation by dowregulating micro RNAs and pigmentation-related genes. This is the first report for the cloning and functional analysis of OfAgol, revealing a role of OfAgol in cuticle pigmentation. The current report also established a CRISPR/Cas9 system in O. fumacalisy providing a new insight for pest management.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.
文摘Insect body pigmentation and coloration are critical to adaption to the environment. To explore the mechanisms that drive pigmentation, we used the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) genome editing system to target the ebony gene in the non-model insect Spodoptera litura. Ebony is crucial to melanin synthesis in insects. By directly injecting Cas9 messenger RNA and ebony-specific guide RNAs into S. litura embryos, we successfully induced a typical ebony-deficient phenotype of deep coloration of the puparium and induction of melanin formation during the pupal stage. Polymerase chain reaction-based genotype analysis demonstrated that various mutations had occurred at the sites targeted in ebony. Our study clearly demonstrates the function of ebony in the puparium coloration and also provides a potentially useful marker gene for functional studies in S. litura as well as other lepidopteran pests.
基金grants from the National Natural Science Foundation of China(31772517 and 31530072) Science and Technology Commission of Shanghai Municipality(No.18140902200).
文摘As a core member of the Hippo signaling pathway,Hpo plays a critical role in regulating growth and development.Previous studies reported that loss of function of Hpo results in increased proliferation,reduced apoptosis and induction of tissue overgrowth in Drosophila.In this study,we used CRISPR/Cas9(Clustered Regularly Interspaced Short Palindromic Repeats/Casg)to study Hpo gene (BmHpo)function in the lepidopteran insect Bombyx mori,known commonly as the silkworm.Sequence analysis of BmHpo revealed an array of deletions in mutants.We found that BmHpo knockout resulted in defects in body size regnlation,in developmental defects and pigment accumulation and early death. Our data show that BmHpo is essential for regulation of insect growth and development and that CRISPR/Cas9technology can serve as a basis for functional analysis of target genes in lepidopteran insects.
基金the National Natural Science Foundation of China (Grant No. 31330071,31772519)Natural Science Foundation of Guangdong Province (Grant No. S2012010010185,2017A030313210).
文摘Fusion of the testis occurs in most Lepidoptera insects, including Spodoptera litura, an important polyphagous pest. Testicular fusion in S. litura is advantageous for male reproduction, and the molecular mechanism of fusion remains unknown. Doublesex influences the formation of genitalia, the behavior of courtship, and sexually dimorphic traits in fruit-fly and silkworm, and is essential for sexual differentiation. However, its purpose in the testis of S. litura remains unknown. The doublesex gene of S. litura (Sldsx) has male-specific SldsxM and female-specific SldsF isoforms, and exhibits a higher expression level in the male testis. At the testicular fusion stage (L6D6), Sldsx attained the highest expression compared to the pre-fusion and post-fusion periods. Moreover, Sldsx had a higher expression in the peritoneal sheaths of testis than that of germ cells in the follicle. CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats/Cas9) was applied to S. litura to determine the role of Sldsx. A mixture of single guide RNA messenger RNA and Cas9 protein (300 ng/μL each) was injected into eggs within 2 h following oviposition. CRISPR/Cas9 successfully induced genomic mutagenesis of Sldsx at Go generation. The mutant males had smaller testis surrounded by less tracheae. Moreover, the mutant males had abnormal external genitalia and could not finish mating with wild-type females. Additionally, testes were fused for almost all mutant males. The results showed that Sldsx was not related to testicular fusion, and is required for both testis development and the formation and function of external genitalia in S. litura. The main roles of doublesex on the male are similar to other insects.
基金National Natural Science Foundation of China (3142010391, 31372257 and 31601903).
文摘Ostrinia fumacalis (Lepidoptera: Pyralidae) is one of the most destructive agricultural pests in Asia. Traditional pest-management methods include sex pheromone capture, transgenic crops that produce Bacillus thuringiensis toxin, and pesticides. Although these strategies control pest populations effectively, they also cause negative side effects, including dramatically increased pesticide resistance, severe pollution, and hazards for human health. Recently developed genome editing tools provide new prospects for pest management and have been successfully used in several species. However, few examples have been reported in the agricultural pest O. fumacalis due to a lack in genomic information. In this report, we identified only one transcript of O. fumacalis Argonaute 1 (OfAgo 1) gene from the genome and cloned the open reading frame. OfAgol presented the maximum expression at the embryo stage or in the fat body during the larval stages. To understand its function, an OfAgol mutant was constructed using the Clustered Regularly Interspaced Short Palindromic Repeat/RNA-guided Cas9 nuclease (CRISPR/Cas9). Mutagenesis of OfAgol disrupted cuticle pigmentation by dowregulating micro RNAs and pigmentation-related genes. This is the first report for the cloning and functional analysis of OfAgol, revealing a role of OfAgol in cuticle pigmentation. The current report also established a CRISPR/Cas9 system in O. fumacalisy providing a new insight for pest management.
