Characterization of Cme and Yme thermostable Cas12a orthologs

Author:

Fuchs Ryan T.ORCID,Curcuru Jennifer L.ORCID,Mabuchi Megumu,Noireterre AudreyORCID,Weigele Peter R.ORCID,Sun ZhiyiORCID,Robb G. BrettORCID

Abstract

AbstractCRISPR-Cas12a proteins are RNA-guided endonucleases that cleave invading DNA containing target sequences adjacent to protospacer adjacent motifs (PAM). Cas12a orthologs have been repurposed for genome editing in non-native organisms by reprogramming them with guide RNAs to target specific sites in genomic DNA. After single-turnover dsDNA target cleavage, multiple-turnover, non-specific single-stranded DNA cleavage in trans is activated. This property has been utilized to develop in vitro assays to detect the presence of specific DNA target sequences. Most applications of Cas12a use one of three well-studied enzymes. Here, we characterize the in vitro activity of two previously unknown Cas12a orthologs. These enzymes are active at higher temperatures than widely used orthologs and have subtle differences in PAM preference, on-target cleavage, and trans nuclease activity. Together, our results enable refinement of Cas12a-based in vitro assays especially when elevated temperature is desirable.

Publisher

Springer Science and Business Media LLC

Subject

General Agricultural and Biological Sciences,General Biochemistry, Genetics and Molecular Biology,Medicine (miscellaneous)

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