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

作者：Qiang Yan, Guosheng Liu, Yingying He, Shuang Hou, Kangli Hao, Jiale Xing, Tingting Zhang, Shutang Zhou · 发表于：Journal of Integrative Agriculture · 年份：2025 · DOI：10.1016/j.jia.2025.02.039 · 被引用次数：3 · 研究领域：Insect symbiosis and bacterial influences、Insect Resistance and Genetics、Insect and Pesticide Research

• 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 Sp Cas9. • Corazonin -/- mutants display defective phenotypes in body color, cuticle development and adaptation to low-temperature stress. 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 ( Sp Cas9), 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 Sp Cas9 could not, although xCas9 appeared to have lower activity than Sp Cas9 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 mar...