ArticleNucleic acids research2024
CRISPR-AsCas12f1 couples out-of-protospacer DNA unwinding with exonuclease activity in the sequential target cleavage.
Article in Nucleic acids research, 2024. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 5 papers.
What it found
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Who cites it
5 citing papers in PubMed.
- Transgene-free plant genome editing via viral delivery of miniature CRISPR-Cas12f.Functional & integrative genomics · 2026Article
- Noncanonical target-strand cytosine base editing via engineered Un1Cas12f1 platform.Nature communications · 2025Article
- Gene disruption via a transient hypercompact CRISPR-AsCas12f1 system in Kluyveromyces marxianus.Biotechnology letters · 2025Article
- Structural and mechanistic insights into the sequential dsDNA cleavage by SpCas12f1.Nucleic acids research · 2025Article
- Application of the transposon-associated TnpB system of CRISPR-Cas in bacteria:Frontiers in microbiology · 2025Review
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Authors and funding
14 authors.
Funding
Abstract
Type V-F CRISPR-Cas12f is a group of hypercompact RNA-guided nucleases that present a versatile in vivo delivery platform for gene therapy. Upon target recognition, Acidibacillus sulfuroxidans Cas12f (AsCas12f1) distinctively engenders three DNA break sites, two of which are located outside the protospacer. Combining ensemble and single-molecule approaches, we elucidate the molecular details underlying AsCas12f1-mediated DNA cleavages. We find that following the protospacer DNA unwinding and non-target strand (NTS) DNA nicking, AsCas12f1 surprisingly carries out bidirectional exonucleolytic cleavage from the nick. Subsequently, DNA unwinding is extended to the out-of-protospacer region, and AsCas12f1 gradually digests the unwound DNA beyond the protospacer. Eventually, the single endonucleolytic target-strand DNA cleavage at 3 nt downstream of the protospacer readily dissociates the ternary AsCas12f1-sgRNA-DNA complex from the protospacer adjacent motif-distal end, leaving a staggered double-strand DNA break. The coupling between the unwinding and cleavage of both protospacer and out-of-protospacer DNA is promoted by Mg2+. Kinetic analysis on the engineered AsCas12f1-v5.1 variant identifies the only accelerated step of the protospacer NTS DNA trimming within the sequential DNA cleavage. Our findings provide a dynamic view of AsCas12f1 catalyzing DNA unwinding-coupled nucleolytic cleavage and help with practical improvements of Cas12f-based genome editing tools.
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Registered trials
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