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

Qiang Yan1*, Guosheng Liu1*, Yingying He2, 3*, Shuang Hou1, Kangli Hao2, 3, Jiale Xing2, 3, Tingting Zhang2, Shutang Zhou1#

1 State Key Laboratory of Cotton Bio-breeding and Integrated Utilization, School of Life Sciences, Henan University, Kaifeng 475004, China

2 Research Institute of Applied Biology, Shanxi University, Taiyuan 030006, China

3 School of Life Sciences, Shanxi University, Taiyuan 030006, China

 Highlight

· xCas9 shows effective gene-editing capabilities at target sites with a non-canonical AG PAM.

· xCas9 can cleave target sites with canonical NGG PAMs, but with lower activity than SpCas9.

· Corazonin-/- mutants display defective phenotypes in body color, cuticle development and adaptation to low-temperature stress.

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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Cas9SpCas9)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(corazoninCrz)为靶标,探索了xCas9在不同PAM靶位点基因编辑活性。结果表明,尽管xCas9在经典NGG PAM靶位点的活性低于SpCas9,但其能够效切割SpCas9无法编辑的AG PAM靶位点。应用xCas9成功构建了Crz-/-蝗虫品系。Crz-/-品系表现出白化体色PaleVermilionCinnabarWhite β-carotene-binding protein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 showbroad 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 (Crzand 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 rolein 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.

Keywords:  CRISPR/Cas9       xCas9             corazonin             migratory locust             cuticle             environmental adaptation  
Online: 20 February 2025  
Fund: 

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).

About author:  #Correspondence Shutang Zhou, Mobile: +86-13837179891, E-mail: szhou@henu.edu.cn *These authors contributed equally to this study.

Cite this article: 

Qiang Yan, Guosheng Liu, Yingying He, Shuang Hou, Kangli Hao, Jiale Xing, Tingting Zhang, Shutang Zhou. 2025. CRISPR/xCas9-mediated corazonin knockout reveals the effectiveness of xCas9 editing and the crucial role of corazonin in insect cuticle development. Journal of Integrative Agriculture, Doi:10.1016/j.jia.2025.02.039

Andreatta G, Broyart C, Borghgraef C, Vadiwala K, Kozin V, Polo A, Bileck A, Beets I, Schoofs L, Gerner C, Raible F. 2020. Corazonin signaling integrates energy homeostasis and lunar phase to regulate aspects of growth and sexual maturation in Platynereis. Proceedings of the National Academy of Sciences of the United States of America, 117, 1097-1106.

Boerjan B, Verleyen P, Huybrechts J, Schoofs L, De Loof A. 2010. In search for a common denominator for the diverse functions of arthropod corazonin: A role in the physiology of stress? General and Comparative Endocrinology, 166, 222-233.

Brinkman E K, Chen T, Amendola M, van Steensel B. 2014. Easy quantitative assessment of genome editing by sequence trace decomposition. Nucleic Acids Research, 42, e168.

Bustin S A, Benes V, Garson J A, Hellemans J, Huggett J, Kubista M, Mueller R, Nolan T, Pfaffl M W, Shipley G L, Vandesompele J, Wittwer C T. 2009. The MIQE guidelines: Minimum information for publication of quantitative real-time PCR experiments. Clinical Chemistry, 55, 611-622.

Canny M D, Moatti N, Wan L C K, Fradet-Turcotte A, Krasner D, Mateos-Gomez P A, Zimmermann M, Orthwein A, Juang Y C, Zhang W, Noordermeer S M, Seclen E, Wilson M D, Vorobyov A, Munro M, Ernst A, Ng T F, Cho T, Cannon P M, Sidhu S S,  et al. 2018. Inhibition of 53BP1 favors homology-dependent DNA repair and increases CRISPR-Cas9 genome-editing efficiency. Nature Biotechnology, 36, 95-102.

Chang H, Cassau S, Krieger J, Guo X, Knaden M, Kang L, Hansson B S. 2023. A chemical defense deters cannibalism in migratory locusts. Science, 380, 537-543.

Chen D, Tang J X, Li B, Hou L, Wang X, Kang L. 2018. CRISPR/Cas9-mediated genome editing induces exon skipping by complete or stochastic altering splicing in the migratory locust. BMC Biotechnology, 18, 60.

Doudna J A, Charpentier E. 2014. Genome editing. The new frontier of genome engineering with CRISPR-Cas9. Science, 346, 1258096.

Foquet B, Song H. 2021. The role of the neuropeptide [His7]-corazonin on phase-related characteristics in the Central American locust. Journal of Insect Physiology, 131, 104244.

Futahashi R, Osanai-Futahashi M. 2021. Pigments in insects. In: Hashimoto H, Goda M, Futahashi R, Kelsh R, Akiyama T, eds., Pigments, Pigment Cells and Pigment Patterns. Springer, Singapore. pp. 3-43.

Geurts M H, de Poel E, Amatngalim G D, Oka R, Meijers F M, Kruisselbrink E, van Mourik P, Berkers G, de Winter-de Groot K M, Michel S, Muilwijk D, Aalbers B L, Mullenders J, Boj S F, Suen S W F, Brunsveld J E, Janssens H M, Mall M A, Graeber S Y, van Boxtel R, et al. 2020. CRISPR-based adenine editors correct nonsense mutations in a cystic fibrosis organoid biobank. Cell Stem Cell, 26, 503-510.e507.

Gospocic J, Shields E J, Glastad K M, Lin Y, Penick C A, Yan H, Mikheyev A S, Linksvayer T A, Garcia B A, Berger S L, Liebig J, Reinberg D, Bonasio R. 2017. The neuropeptide corazonin controls social behavior and caste identity in ants. Cell, 170, 748-759.e712.

Guo X, Yu Q, Chen D, Wei J, Yang P, Yu J, Wang X, Kang L. 2020. 4-Vinylanisole is an aggregation pheromone in locusts. Nature, 584, 584-588.

Heu C C, McCullough F M, Luan J, Rasgon J L. 2020. CRISPR-Cas9-Based genome editing in the silverleaf  whitefly (Bemisia tabaci). The CRISPR Journal, 3, 89-96.

Hou L, Guo S, Wang Y, Nie X, Yang P, Ding D, Li B, Kang L, Wang X. 2021. Neuropeptide ACP facilitates lipid oxidation and utilization during long-term flight in locusts. Elife, 10, e65279.

Hou L, Jiang F, Yang P, Wang X, Kang L. 2015. Molecular characterization and expression profiles of neuropeptide precursors in the migratory locust. Insect Biochemistry and Molecular Biology, 63, 63-71.

Hu J H, Miller S M, Geurts M H, Tang W, Chen L, Sun N, Zeina C M, Gao X, Rees H A, Lin Z, Liu D R. 2018. Evolved Cas9 variants with broad PAM compatibility and high DNA specificity. Nature, 556, 57-63.

Hua K, Tao X, Han P, Wang R, Zhu J K. 2019. Genome engineering in rice using Cas9 variants that recognize NG PAM sequences. Molecular Plant, 12, 1003-1014.

Imura E, Shimada-Niwa Y, Nishimura T, Huckesfeld S, Schlegel P, Ohhara Y, Kondo S, Tanimoto H, Cardona A, Pankratz M J, Niwa R. 2020. The Corazonin-PTTH neuronal axis controls systemic body growth by regulating basal ecdysteroid biosynthesis in Drosophila melanogaster. Current Biology, 30, 2156-2165.e2155.

Jiang X, Dimitriou E, Grabe V, Sun R, Chang H, Zhang Y, Gershenzon J, Rybak J, Hansson B S, Sachse S. 2024. Ring-shaped odor coding in the antennal lobe of migratory locusts. Cell, 187, 3973-3991.e3924.

Kapan N, Lushchak O V, Luo J, Nassel D R. 2012. Identified peptidergic neurons in the Drosophila brain regulate insulin-producing cells, stress responses and metabolism by coexpressed short neuropeptide F and corazonin. Cellular and Molecular Life Sciences, 69, 4051-4066.

Kim Y J, Spalovska-Valachova I, Cho K H, Zitnanova I, Park Y, Adams M E, Zitnan D. 2004. Corazonin receptor signaling in ecdysis initiation. Proceedings of the National Academy of Sciences of the United States of America, 101, 6704-6709.

Kistler K E, Vosshall L B, Matthews B J. 2015. Genome engineering with CRISPR-Cas9 in the mosquito Aedes aegypti. Cell Reports, 11, 51-60.

Kubrak O I, Lushchak O V, Zandawala M, Nassel D R. 2016. Systemic corazonin signalling modulates stress responses and metabolism in Drosophila. Open Biology, 6, 160152.

Li X, Fan D, Zhang W, Liu G, Zhang L, Zhao L, Fang X, Chen L, Dong Y, Chen Y, Ding Y, Zhao R, Feng M, Zhu Y, Feng Y, Jiang X, Zhu D, Xiang H, Feng X, Li S, et al. 2015. Outbred genome sequencing and CRISPR/Cas9 gene editing in butterflies. Nature Communications, 6, 8212.

Li Y, Zhang J, Chen D, Yang P, Jiang F, Wang X, Kang L. 2016. CRISPR/Cas9 in locusts: Successful establishment of an olfactory deficiency line by targeting the mutagenesis of an odorant receptor co-receptor (Orco). Insect Biochemistry and Molecular Biology, 79, 27-35.

Liu K, Petree C, Requena T, Varshney P, Varshney G K. 2019. Expanding the CRISPR toolbox in zebrafish for studying development and disease. Expanding the CRISPR Toolbox in Zebrafish for Studying Development and Disease, 7, 13. 

Liu X, Li G, Zhou X, Qiao Y, Wang R, Tang S, Liu J, Wang L, Huang X. 2019. Improving editing efficiency for the sequences with NGH PAM using xCas9-Derived base editors. Molecular Therapy-Nucleic Acids, 17, 626-635.

Lu Y, Tian Y, Shen R, Yao Q, Wang M, Chen M, Dong J, Zhang T, Li F, Lei M, Zhu J K. 2020. Targeted, efficient sequence insertion and replacement in rice. Nature Biotechnology, 38, 1402-1407.

Ma Z, Guo W, Guo X, Wang X, Kang L. 2011. Modulation of behavioral phase changes of the migratory locust by the catecholamine metabolic pathway. Proceedings of the National Academy of Sciences of the United States of America, 108, 3882-3887.

Mabashi-Asazuma H, Kuo C W, Khoo K H, Jarvis D L. 2015. Modifying an insect cell N-Glycan processing pathway using CRISPR-Cas technology. Acs Chemical Biology, 10, 2199-2208.

McClure K D, Heberlein U. 2013. A small group of neurosecretory cells expressing the transcriptional regulator apontic and the neuropeptide corazonin mediate ethanol sedation in Drosophila. Journal of Neuroscience, 33, 4044-4054.

Ni X Y, Zhou Z D, Huang J, Qiao X. 2020. Targeted gene disruption by CRISPR/xCas9 system in Drosophila melanogaster. Archives of Insect Biochemistry and Physiology, 104, e21662.

Pan Y, Fang G, Wang Z, Cao Y, Liu Y, Li G, Liu X, Xiao Q, Zhan S. 2021. Chromosome-level genome reference and genome editing of the tea geometrid. Molecular Ecology Resources, 21, 2034-2049.

Port F, Strein C, Stricker M, Rauscher B, Heigwer F, Zhou J, Beyersdorffer C, Frei J, Hess A, Kern K, Lange L, Langner N, Malamud R, Pavlovic B, Radecke K, Schmitt L, Voos L, Valentini E, Boutros M. 2020. A large-scale resource for tissue-specific CRISPR mutagenesis in Drosophila. Elife, 9, e53865.

Ragionieri L, Verdonck R, Verlinden H, Marchal E, Vanden Broeck J, Predel R. 2022. Schistocerca neuropeptides - An update. Journal of Insect Physiology, 136, 104326.

Ren X, Sun J, Housden B E, Hu Y, Roesel C, Lin S, Liu L P, Yang Z, Mao D, Sun L, Wu Q, Ji J Y, Xi J, Mohr S E, Xu J, Perrimon N, Ni J Q. 2013. Optimized gene editing technology for Drosophila melanogaster using germ line-specific Cas9. Proceedings of the National Academy of Sciences of the United States of America, 110, 19012-19017.

Sha K, Choi S H, Im J, Lee G G, Loeffler F, Park J H. 2014. Regulation of ethanol-related behavior and ethanol metabolism by the Corazonin neurons and Corazonin receptor in Drosophila melanogaster. PLoS ONE, 9, e87062.

Shi Z, Jiang H, Liu G, Shi S, Zhang X, Chen Y. 2022. Expanding the CRISPR/Cas genome-editing scope in Xenopus tropicalis. Cell and Bioscience, 12, 104.

Song J, Li W, Zhao H, Gao L, Fan Y, Zhou S. 2018. The microRNAs let-7 and miR-278 regulate insect metamorphosis and oogenesis by targeting the juvenile hormone early-response gene Krüppel-homolog 1. Development, 145, dev170670.

Sugahara R, Saeki S, Jouraku A, Shiotsuki T, Tanaka S. 2015. Knockdown of the corazonin gene reveals its critical role in the control of gregarious characteristics in the desert locust. Journal of Insect Physiology, 79, 80-87.

Sugahara R, Tanaka S, Jouraku A, Shiotsuki T. 2016. Functional characterization of the corazonin-encoding gene in phase polyphenism of the migratory locust, Locusta migratoria (Orthoptera: Acrididae). Applied Entomology and Zoology, 51, 225-232.

Sugahara R, Tanaka S, Jouraku A, Shiotsuki T. 2017. Two types of albino mutants in desert and migratory locusts are caused by gene defects in the same signaling pathway. Gene, 608, 41-48.

Sugahara R, Tanaka S, Jouraku A, Shiotsuki T. 2018. Identification of a transcription factor that functions downstream of corazonin in the control of desert locust gregarious body coloration. Insect Biochemistry and Molecular Biology, 97, 10-18.

Sun L, Zhang T, Lan X, Zhang N, Wang R, Ma S, Zhao P, Xia Q. 2024. High-throughput screening of PAM-Flexible Cas9 variants for expanded genome editing in the silkworm (Bombyx mori). Insects, 15, 241.

Tanaka Y, Hua Y, Roller L, Tanaka S. 2002. Corazonin reduces the spinning rate in the silkworm, Bombyx mori. Journal of Insect Physiology, 48, 707-714.

Tawfik A I, Tanaka S, De Loof A, Schoofs L, Baggerman G, Waelkens E, Derua R, Milner Y, Yerushalmi Y, Pener M P. 1999. Identification of the gregarization-associated dark-pigmentotropin in locusts through an albino mutant. Proceedings of the National Academy of Sciences of the United States of America, 96, 7083-7087.

Thakkar N, Hejzlarova A, Brabec V, Dolezel D. 2023. Germline editing of Drosophila using CRISPR-Cas9-Based cytosine and adenine base editors. The CRISPR Journal, 6, 557-569.

Tsuchiya R, Kaneshima A, Kobayashi M, Yamazaki M, Takasu Y, Sezutsu H, Tanaka Y, Mizoguchi A, Shiomi K. 2021. Maternal GABAergic and GnRH/corazonin pathway modulates egg diapause phenotype of the silkworm Bombyx mori. Proceedings of the National Academy of Sciences of the United States of America, 118, e2020028118.

Veenstra J A. 1989. Isolation and structure of corazonin, a cardioactive peptide from the American cockroach. FEBS Letters, 250, 231-234.

Xiao A, Cheng Z, Kong L, Zhu Z, Lin S, Gao G, Zhang B. 2014. CasOT: A genome-wide Cas9/gRNA off-target searching tool. Bioinformatics, 30, 1180-1182.

Yan Q, He Y, Yue Y, Jie L, Wen T, Zhao Y, Zhang M, Zhang T. 2022. Construction of homozygous mutants of migratory locust using CRISPR/Cas9 technology. Journal of Visualized Experiments, 181, e63629.

Yang H, Ren S, Yu S, Pan H, Li T, Ge S, Zhang J, Xia N. 2020. Methods favoring homology-directed repair choice in response to CRISPR/Cas9 induced-double strand breaks. International Journal of Molecular Sciences, 21, 6461.

Yang M, Wang Y, Liu Q, Liu Z, Jiang F, Wang H, Guo X, Zhang J, Kang L. 2019. A beta-carotene-binding protein carrying a red pigment regulates body-color transition between green and black in locusts. Elife, 8, e41362.

Zandawala M, Nguyen T, Balanya Segura M, Johard H A D, Amcoff M, Wegener C, Paluzzi J P, Nassel D R. 2021. A neuroendocrine pathway modulating osmotic stress in Drosophila. Plos Genetics, 17, e1009425.

Zer-Krispil S, Zak H, Shao L, Ben-Shaanan S, Tordjman L, Bentzur A, Shmueli A, Shohat-Ophir G. 2018. Ejaculation induced by the activation of Crz neurons is rewarding to Drosophila males. Current Biology, 28, 1445-1452.e1443.

Zhang S, Li J, Zhang D, Zhang Z, Meng S, Li Z, Liu X. 2023. miR-252 targeting temperature receptor CcTRPM to mediate the transition from summer-form to winter-form of Cacopsylla chinensis. Elife, 12, RP88744.

Zhang T, Wen T, Yue Y, Yan Q, Du E, Fan S, Roth S, Li S, Zhang J, Zhang X, Zhang M. 2021. Egg tanning improves the efficiency of CRISPR/Cas9-mediated mutant locust production by enhancing defense ability after microinjection. Journal of Integrative Agriculture, 20, 2716-2726.

 

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