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

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.