A study in Nature reports the organization and mechanism of action of CasX, a recently identified RNA-guided DNA endonuclease that can be used for genome editing in human cells. This enzyme family seems to be functionally distinct to known CRISPR–Cas genome editors and offers exploitable advantages over existing systems, including its small size, high guide RNA content and minimal trans cleavage activity.

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The RNA-guided proteins Cas9 and Cas12a form part of the archaeal and bacterial adaptive immune system CRISPR, which protects against invading nucleic acids. These endonucleases have been repurposed as ‘molecular scissors’, that is, genome editing tools that can be directed to cut designated genomic locations across a range of cell types and organisms. However, existing CRISPR–Cas systems have limitations to their applicability, fuelling the search for alternative enzymes.

A previous metagenomic analysis of groundwater samples identified the protein CasX, which was found to disrupt bacterial transformation by plasmid DNA through an unknown mechanism when expressed together with RNA complementary to the plasmid. This finding suggested a probable function as a DNA endonuclease despite little sequence similarity with known Cas proteins.

Liu, Orlova, Oakes et al. combined purified wild-type CasX from Deltaproteobacteria (DpbCasX) with a single guide RNA and observed that, in vitro, CasX cleaves DNA sequences complementary to a 20-nucleotide segment of the guide RNA by generating a staggered DNA double-strand break.

To determine whether CasX can effectively manipulate genomes in vivo, the team expressed DpbCasX in Escherichia coli using a guide RNA complementary to an integrated reporter, which led to reduced cell viability in a cleavage assay. Moreover, a nuclease-deactivated DpbCasX, combined with guide RNAs targeting different sites within a reporter gene, was able to silence GFP expression in E. coli, indicating the suitability of using CasX for CRISPR interference (CRISPRi). Furthermore, catalytically active CasX was capable of inducing DNA double-strand breaks and gene editing in HEK293T cells, albeit with limited efficiency. A CasX from Planctomycetes (PlmCasX), which exhibits ~70% sequence identity to DpbCasX, showed higher efficiency than DpbCasX.

Structural data from cryo-electron microscopy (cryo-EM) images showed that the guide RNA forms a prominent scaffold that dominates the architecture of the enzyme. Taken together, this study suggests that CasX will be a useful addition to the armamentarium of RNA-programmed genome-editing platforms.