PAM recognition by miniature CRISPR–Cas12f nucleases triggers programmable double-stranded DNA target cleavage

Karvelis et al. (2020). Nucleic Acids Res. DOI: 10.1093/nar/gkaa208 Citations: 362

Miniature Cas12f nucleases, half the size of Cas9, were assumed too small for active dsDNA cleavage. Biochemical assays showed ten Cas12f proteins recognize 5' T- or C-rich PAMs and cleave dsDNA. Two 422-amino-acid variants protected E. coli against invading plasmids.

Key findings

  • Biochemical screening of 10 compact Cas12f nucleases (422-603 amino acids) from Cas14 and type V-U3 families demonstrated PAM-dependent dsDNA cleavage using adapter ligation and deep sequencing. All nucleases cleaved targets 21-24 bp downstream of the PAM, with Cas12f2 cutting after position 21 and Cas12f1 after position 24. Cas14 family members recognized T-rich PAM sequences (TTTA) while type V-U3 nucleases exhibited broader specificity with both T-rich and C-rich motifs.

  • Purified Un1Cas12f1 protein combined with in vitro transcribed sgRNA demonstrated robust dsDNA cleavage activity, contradicting previous reports of exclusive ssDNA targeting. The nuclease achieved complete plasmid conversion to linear form under optimized conditions (46°C, 5-25 mM NaCl, 10 mM Mg2+, 20-nt spacer). Run-off sequencing revealed staggered 5' overhangs centered at positions 20-24 bp from the PAM, with RuvC domain alanine substitutions (D326A, D510A) completely abolishing activity.

  • Plasmid interference assays in E. coli revealed divergent in vivo functionality between Cas12f families despite similar in vitro dsDNA cleavage capabilities. Type V-U3 bacterial nucleases AsCas12f1 and SpCas12f1 efficiently blocked low-copy plasmid transformation in heterologous hosts, while all five tested Cas14 family members (Un1Cas12f1, Un2Cas12f1, Mi1Cas12f2, Mi2Cas12f2, AuCas12f2) failed to interfere. This demonstrated that miniature type V-U3 effectors (422-497 aa) function as bona fide CRISPR-Cas systems.

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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.