CasX was initially identified from groundwater samples via metagenomics analysis. Although it contained no similarity to other CRISPR–Cas9 enzymes, Liu et al. investigated whether CasX might act as a genome-editing enzyme. Biochemical and structural analyses of CasX from Deltaproteobacteria (DpbCasX) demonstrated that DpbCasX could cleave double-stranded DNA adjacent to a TTCN protospacer-adjacent motif with the assistance of sgRNA, and generate DNA products with staggered ends. A deactivated DpbCasX maintains RNA-guided DNA-binding ability but lacks cleavage activity for use in a CRISPR interference system. Structural analysis revealed that DpbCasX contains a unique nontarget-strand-binding (NTSB) domain and a target-strand-loading (TSL) domain. The NTSB domain is responsible for initiating DNA duplex unwinding, and the TSL domain helps to bend an RNA–DNA hybrid duplex into the RuvC domain active site for cleavage of the target DNA strand. Despite the modest genome editing efficiency of CasX compared to SpCas9 in mammalian cells, the mechanistic insights revealed in this study pave the way for using CasX for genome editing and therapeutic applications.