基于CRISPR/Cas9靶向小菜蛾常染色体基因和性染色体定点敲入外源序列

doi:10.1016/j.jia.2024.04.029

Journal of Integrative Agriculture ›› 2024, Vol. 23 ›› Issue (9): 3089-3103.DOI: 10.1016/j.jia.2024.04.029

基于CRISPR/Cas9靶向小菜蛾常染色体基因和性染色体定点敲入外源序列

收稿日期:2023-10-18 接受日期:2024-03-06 出版日期:2024-09-20 发布日期:2024-08-21

Knock-in of exogenous sequences based on CRISPR/Cas9 targeting autosomal genes and sex chromosomes in the diamondback moth, Plutella xylostella

Shanyu Li^1*, Guifang Lin^1*, Haoqi Wen¹, Haiyan Lu¹, Anyuan Yin¹, Chanqin Zheng¹, Feifei Li¹, Qingxuan Qiao¹, Lu Jiao¹, Ling Lin¹, Yi Yan¹, Xiujuan Xiang¹, Huang Liao¹, Huiting Feng¹, Yussuf Mohamed Salum¹, Minsheng You¹, Wei Chen^2#, Weiyi He^1#

¹ State Key Laboratory for Ecological Pest Control of Fujian and Taiwan Crops/Ministerial and Provincial Joint Innovation Centre for Safety Production of Cross-Strait Crops/International Joint Research Laboratory of Ecological Pest Control, Ministry of Education, Institute of Applied Ecology, Fujian Agriculture and Forestry University, Fuzhou 350002, China
² Ganzhou Key Laboratory of Greenhouse Vegetable, Gannan Normal University, Ganzhou 341000, China

Received:2023-10-18 Accepted:2024-03-06 Online:2024-09-20 Published:2024-08-21
About author:#Correspondence Wei Chen, E-mail: weichen@gnnu.edu.cn; Weiyi He, Mobile: +86-15005010958, E-mail: wy.he@fafu.edu.cn * These authors contributed equally to this study.
Supported by:
This work is supported by the National Natural Science Foundation of China (32172503 and 32260721), the Natural Science Foundation of Fujian Province, China (2023J01069), the State Key Laboratory of Ecological Pest Control for Fujian and Taiwan Crops, China (SKL2022001), the Innovation Fund of Fujan Agriculture and Forestry University, China (KFB23014A), and the Undergraduate Training Program for Innovation and Entrepreneurship of Fujian Province, China (S202210389101).

摘要/Abstract

摘要：

通过位点特异性的基因组编辑，可以实现基于精确性别分离和只释放不育雄性的害虫遗传控制策略。本研究发现，Pxfl(2)d除了在雌成虫卵巢发育和卵孵化方面的已知功能外，其单等位基因的突变还能显著影响十字花科害虫小菜蛾雄成虫的正常交配行为和睾丸发育。基于CRISPR/Cas9的基因敲入结果表明，外源绿色荧光蛋白（GFP）基因可被定点整合到位于常染色体的Pxfl(2)d中，用于标记突变体。但由于Pxfl(2)d突变体生殖能力严重下降，该基因并不适合作为靶点来建立稳定遗传的基因敲入品系。本研究还进一步筛选了W染色体和Z染色体连锁区域，检测CRISPR/Cas9介导的基因敲入效率。结果证实，这两种类型的染色体都可以作为外源序列定点插入的靶标。本研究最终获得了在小菜蛾Z染色体连锁区域同时整合了Cas9和青色荧光蛋白（CFP）表达盒的纯合敲入品系（Z-Cas9-CFP），也可用于早期性别鉴定。通过将靶向Pxfl(2)d的sgRNA单独注射到Z-Cas9-CFP品系雌成虫产下的卵中，基因编辑效率达到29.73 %，证实成功表达了具有功能的Cas9基因。综上所述，本研究证明了外源基因在小菜蛾不同基因组区域敲入的可行性，而遗传品系的建立则需要选择合适的位点。研究结果为进一步开发害虫遗传防控策略提供了重要的工作基础和技术支持。

Abstract:

Genetic pest control strategies based on precise sex separation and only releasing sterile males can be accomplished by site-specific genome editing. In the current study, we showed that the mutation of single-allele Pxfl(2)d can significantly impair the normal mating behavior and testis development in male adults of the notorious cruciferous insect pest Plutella xylostella, in addition to its known functions in the ovarian development in female adults and egg hatching. Subsequent CRISPR/Cas9-based knock-in experiments revealed that site-specific integration of an exogenous green fluorescent protein (GFP) gene into autosomal Pxfl(2)d for labelling mutants could be achieved. However, this gene is not a suitable target for GFP insertion to establish a genetically stable knock-in strain because of the severe decline in reproductive capacity. We further screened for the W-chromosome-linked and Z-chromosome-linked regions to test the knock-in efficiency mediated by CRISPR/Cas9. The results verified that both types of chromosomes can be targeted for the site-specific insertion of exogenous sequences. We ultimately obtained a homozygous knock-in strain with the integration of both Cas9 and cyan fluorescent protein (CFP) expression cassettes on a Z-linked region in P. xylostella, which can also be used for early sex detection. By injecting the sgRNA targeting Pxfl(2)d alone into the eggs laid by female adults of the Z-Cas9-CFP strain, the gene editing efficiency reached 29.73%, confirming the success of expressing a functional Cas9 gene. Taken together, we demonstrated the feasibility of the knock-in of an exogenous gene to different genomic regions in P. xylostella, while the establishment of a heritable strain required the positioning of appropriate sites. This study provides an important working basis and technical support for further developing genetic strategies for insect pest control.

Key words: cruciferous specialist , fl(2)d , CRISPR/Cas9 , knock-in , sex chromosome

Shanyu Li, Guifang Lin, Haoqi Wen, Haiyan Lu, Anyuan Yin, Chanqin Zheng, Feifei Li, Qingxuan Qiao, Lu Jiao, Ling Lin, Yi Yan, Xiujuan Xiang, Huang Liao, Huiting Feng, Yussuf Mohamed Salum, Minsheng You, Wei Chen, Weiyi He. 基于CRISPR/Cas9靶向小菜蛾常染色体基因和性染色体定点敲入外源序列[J]. Journal of Integrative Agriculture, 2024, 23(9): 3089-3103.

Shanyu Li, Guifang Lin, Haoqi Wen, Haiyan Lu, Anyuan Yin, Chanqin Zheng, Feifei Li, Qingxuan Qiao, Lu Jiao, Ling Lin, Yi Yan, Xiujuan Xiang, Huang Liao, Huiting Feng, Yussuf Mohamed Salum, Minsheng You, Wei Chen, Weiyi He. Knock-in of exogenous sequences based on CRISPR/Cas9 targeting autosomal genes and sex chromosomes in the diamondback moth, Plutella xylostella[J]. Journal of Integrative Agriculture, 2024, 23(9): 3089-3103.

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基于CRISPR/Cas9靶向小菜蛾常染色体基因和性染色体定点敲入外源序列

Knock-in of exogenous sequences based on CRISPR/Cas9 targeting autosomal genes and sex chromosomes in the diamondback moth, Plutella xylostella

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