CRISPR-CasX is an RNA-dominated enzyme active for human genome editing

The RNA-guided CRISPR-associated (Cas) proteins Cas9 and Cas12a provide adaptive immunity against bacteriophage and function as powerful tools for genome editing in wide-ranging cell types. Here we present a third and fundamentally distinct RNA-guided platform, CRISPR-CasX, which uses a unique structure and mechanism for programmable double-stranded DNA cleavage. Biochemical and in vivo data demonstrate that CasX is active for E. coli and human genome modification. Eight cryo-EM structures of CasX in different states of assembly with its guide RNA and double-stranded DNA substrates reveal an extensive RNA scaffold and an unanticipated domain required for DNA unwinding. These data demonstrate how CasX activity arose through convergent evolution to establish an enzyme family that is functionally separate from both Cas9 and Cas12a.