CRISPR/xCas9介导的corazonin基因敲除揭示了xCas9编辑的有效性以及 corazonin在昆虫表皮发育中的关键作用

doi:10.1016/j.jia.2025.02.039

Journal of Integrative Agriculture ›› 2025, Vol. 24 ›› Issue (10): 3953-3965.DOI: 10.1016/j.jia.2025.02.039

CRISPR/xCas9介导的corazonin基因敲除揭示了xCas9编辑的有效性以及 corazonin在昆虫表皮发育中的关键作用

收稿日期:2024-10-10 修回日期:2025-02-20 接受日期:2024-12-20 出版日期:2025-10-20 发布日期:2025-09-24

CRISPR/xCas9-mediated corazonin knockout reveals the effectiveness of xCas9 editing and the crucial role of corazonin in insect cuticle development

Qiang Yan^1*, Guosheng Liu^1*, Yingying He^{2, 3*}, Shuang Hou¹, Kangli Hao^{2, 3}, Jiale Xing^{2, 3}, Tingting Zhang², Shutang Zhou^1#

¹State Key Laboratory of Cotton Bio-breeding and Integrated Utilization, School of Life Sciences, Henan University, Kaifeng 475004, China
²Research Institute of Applied Biology, Shanxi University, Taiyuan 030006, China
³School of Life Sciences, Shanxi University, Taiyuan 030006, China

Received:2024-10-10 Revised:2025-02-20 Accepted:2024-12-20 Online:2025-10-20 Published:2025-09-24
About author:#Correspondence Shutang Zhou, Mobile: +86-13837179891, E-mail: szhou@henu.edu.cn * These authors contributed equally to this study.
Supported by:
This work was supported by the Key Research and Development Project of Henan Province, China (221111112200), the National Natural Science Foundation of China (32070502 and 32072419), the Fundamental Research Program of Shanxi Province, China (202303021224005), and the Natural Science Foundation of Henan Province, China (232300420185).

摘要/Abstract

摘要：

基于CRISPR/Cas9的基因编辑技术极大地推动了生命科学领域的研究，并展现出广泛的实际应用潜力。然而，Cas9系统往往受到靶位点附近间隔序列邻近基序（PAM）需求的限制。xCas9是从化脓性链球菌Cas9（SpCas9）衍生的一种变体，已知其可以识别更广泛的PAM范围。然而，目前xCas9在非模式昆虫中的应用研究仍较为匮乏。本研究以具有全球性危害的飞蝗（Locusta migratoria）为研究对象，选择黑化诱导神经肽（corazonin，Crz）为靶标，探索了xCas9在不同PAM靶位点的基因编辑活性。结果表明，尽管xCas9在经典NGG PAM靶位点的活性低于SpCas9，但其能够有效切割SpCas9无法编辑的AG PAM靶位点。应用xCas9成功构建了Crz^-/-型蝗虫品系。该Crz^-/-品系表现出白化体色，Pale、Vermilion、Cinnabar、White和 β-carotene-binding protein等体色相关基因的表达显著下调。此外，Crz^-/-突变体的几丁质合成酶1（Chitin synthase 1）表达量显著降低，表皮中几丁质含量明显减少，且角质层更为紧密和坚硬。更进一步地，Crz^-/-突变体在低温胁迫下表现出适应能力下降，包括生命周期延长、体重降低以及体型变小。这些研究结果表明，xCas9能够有效实现昆虫基因组编辑，且Crz在昆虫体色、表皮发育及适应低温胁迫中发挥关键作用。本研究拓展了xCas9在非模式昆虫中的应用，并为深入理解昆虫表皮发育及环境适应的调控机制提供了新见解。

Abstract:

CRISPR/Cas9-based gene editing research has advanced greatly and shows broad potential for practical application in life sciences, but the Cas9 system is often constrained by the requirement of a protospacer adjacent motif (PAM) at the target site. While xCas9, a variant derived from Streptococcus pyogenes Cas9 (SpCas9), can recognize a broader range of PAMs, its application in non-model insects is lacking. In this study, we explored xCas9 activity in gene editing by selecting corazonin (Crz) and the target sites with various PAMs in Locusta migratoria, a destructive insect pest worldwide. We found that xCas9 could cleave the target site with AG PAM while SpCas9 could not, although xCas9 appeared to have lower activity than SpCas9 at the canonical NGG PAMs. The heritable homozygous Crz^–/– locust strain was generated by the application of xCas9. The Crz^–/– strain showed an albino body color, with significantly downregulated expression of several body color-related genes including Pale, Vermilion, Cinnabar, White and β-carotene-binding protein. In addition, Crz^–/– mutants exhibited significantly reduced expression of Chitin synthase 1, along with a markedly lower chitin content as well as compact and rigid cuticles. Furthermore, Crz^–/– mutants displayed impaired performance under low-temperature stress, including prolonged lifespan, reduced body weight and smaller body size. Our results suggest that xCas9 is effective for insect genome editing, and Crz plays essential roles in insect body color, cuticle development and adaptation to low-temperature stress. The findings of this study extend the application of xCas9 in non-model insects and provide new insights into our understanding of the regulation of insect cuticle development and environmental adaptation.

Key words: CRISPR/Cas9 , xCas9 , corazonin , migratory locust , cuticle , environmental adaptation

Qiang Yan, Guosheng Liu, Yingying He, Shuang Hou, Kangli Hao, Jiale Xing, Tingting Zhang, Shutang Zhou. CRISPR/xCas9介导的corazonin基因敲除揭示了xCas9编辑的有效性以及 corazonin在昆虫表皮发育中的关键作用[J]. Journal of Integrative Agriculture, 2025, 24(10): 3953-3965.

Qiang Yan, Guosheng Liu, Yingying He, Shuang Hou, Kangli Hao, Jiale Xing, Tingting Zhang, Shutang Zhou. CRISPR/xCas9-mediated corazonin knockout reveals the effectiveness of xCas9 editing and the crucial role of corazonin in insect cuticle development[J]. Journal of Integrative Agriculture, 2025, 24(10): 3953-3965.

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CRISPR/xCas9介导的corazonin基因敲除揭示了xCas9编辑的有效性以及 corazonin在昆虫表皮发育中的关键作用

CRISPR/xCas9-mediated corazonin knockout reveals the effectiveness of xCas9 editing and the crucial role of corazonin in insect cuticle development

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