Abstract
In recent years, CRISPR-associated (Cas) nucleases have revolutionized the genome editing field. Being guided by an RNA to cleave double-stranded (ds) DNA targets near a short sequence termed a protospacer adjacent motif (PAM), Cas9 and Cas12 offer unprecedented flexibility, however, more compact versions would simplify delivery and extend application. Here, we present a collection of 10 exceptionally compact (422–603 amino acids) CRISPR–Cas12f nucleases that recognize and cleave dsDNA in a PAM dependent manner. Categorized as class 2 type V-F, they originate from the previously identified Cas14 family and distantly related type V-U3 Cas proteins found in bacteria. Using biochemical methods, we demonstrate that a 5′ T- or C-rich PAM sequence triggers dsDNA target cleavage. Based on this discovery, we evaluated whether they can protect against invading dsDNA in Escherichia coli and find that some but not all can. Altogether, our findings show that miniature Cas12f nucleases can protect against invading dsDNA like much larger class 2 CRISPR effectors and have the potential to be harnessed as programmable nucleases for genome editing.
Highlights
Clustered regularly interspaced short palindromic repeat (CRISPR) associated (Cas) microbial defense systems protect their hosts against foreign nucleic acid invasion [1,2,3]
Contrary to previous hypotheses suggesting that Cas14 and type V-U3 CRISPR systems lack the ability to defend against invading dsDNA [7, 21], we illustrate that despite their miniature size some of these nucleases have this capacity
We purified the nuclease and single guide RNAs (sgRNA) for Cas14a1 and reconstituted its nuclease activity in vitro confirming that both protospacer adjacent motif (PAM) and sgRNA recognition are required for dsDNA target cleavage
Summary
Clustered regularly interspaced short palindromic repeat (CRISPR) associated (Cas) microbial defense systems protect their hosts against foreign nucleic acid invasion [1,2,3]. Based on the number and composition of proteins involved in nucleic acid interference, CRISPR-Cas systems are categorized into distinct classes, 1-2, and types, I-VI [2, 6]. Over the past several years, these endonucleases have been adopted as robust genome editing and transcriptome manipulation tools [15,16,17,18].
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