Faculty Opinions recommendation of CasX enzymes comprise a distinct family of RNA-guided genome editors.
Year: 2019
Type: peer-review
Abstract: 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.Archaea and bacteria utilize CRISPR-Cas systems (clustered regularly interspaced short palindromic repeats and CRISPR-associated proteins) for adaptive immunity against invading nucleic acids 1,2 .CRISPR arrays, consisting of repeated sequences interleaved with sequences acquired from foreign DNA, are templates for CRISPR RNAs (crRNAs) that guide a Cas nuclease to cleave complementary DNA sequences.In addition to their microbial functions, RNA-guided DNA binding and cutting have proven to be transformative tools for genome and epigenome editing across wide-ranging cell types and organisms [3][4][5] .Despite extensive effort, just two kinds of CRISPR-Cas nucleases, Cas9 and Cas12a (Cpf1), provide the foundation for this revolutionary technology 6,7 .Metagenomic analysis of microbial DNA from groundwater samples revealed a new protein, CasX (a placeholder name pending re-analysis of the class 2 CRISPR-Cas phylogeny) which is also referred as Cas12e 5 , that prevented bacterial transformation by plasmid DNA when expressed with cognate crRNAs targeting the plasmid 8 .Sequence analysis of CasX revealed no similarity to other CRISPR-Cas enzymes, except for the presence of a RuvC nuclease domain similar to that found in both Cas9 and Cas12a enzyme families as well as transposases and recombinases 8 .Phylogenetic analysis suggests that CasX arose from a TnpB-type transposase by an independent insertion event into ancestral CRISPR loci, distinct from Cas12a and the remaining type V effectors (Extended Data Fig. 1a).Consistent with this hypothesis, the CasX RuvC domain shares less than 16% identity to RuvC domains in either Cas9 or Cas12a (Extended Data Fig. 1b).This evolutionary ambiguity of CasX hinted that CasX may have a structure and molecular mechanism distinct from other CRISPR-Cas enzymes.However, without full reconstitution of the CasX enzyme, it was not possible to determine the basis of the previously reported plasmid interference activity.We demonstrate here that CasX is an RNA-guided DNA endonuclease that generates a staggered double-stranded break in DNA at sequences complementary to a 20-nucleotide segment of its guide RNA.We further find that CasX induces programmable, site-specific genome repression in E. coli and genome editing in human cells.Biochemical data shows that CasX is a hybrid enzyme containing elements of both Cas9 and Cas12a as well as novel RNA folds and protein domains, establishing this enzyme family as the third CRISPR-Cas system effective for genetic manipulation.The small size of CasX (<1000 amino acids), DNA cleavage characteristics and derivation from non-pathogenic microorganisms, offer important advantages over other CRISPR-Cas genome editing enzymes.Eight molecular
Authors: Zucai Suo, Adarsh Kumar
Institution: Florida State University
Language: English
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: green
OpenAlex ID: W4229906255