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From Proteopedia
Cryo-EM structure of Acidibacillus sulfuroxidans Cas12f in complex with sgRNA and target DNA
Structural highlights
FunctionCS12F_SULT2 CRISPR (clustered regularly interspaced short palindromic repeat), is an adaptive immune system that provides protection against mobile genetic elements (viruses, transposable elements and conjugative plasmids). CRISPR clusters contain sequences complementary to antecedent mobile elements and target invading nucleic acids (Probable). CRISPR clusters are transcribed and processed into CRISPR RNA (crRNA), which requires a trans-encoded small RNA (tracrRNA), but not this protein (By similarity). Recognizes a short motif in the CRISPR repeat sequences (the 5' PAM or protospacer adjacent motif, YTT in this organism) to help distinguish self versus nonself, as targets within the CRISPR locus do not have PAMs. Has dsDNA endonuclease activity upon expression in E.coli of this protein, a mini CRISPR array and the probable tracrRNA. Plasmid cleavage is centered around positions 19-24 base pairs 3' of PAM. The mini system protects E.coli against transformation by foreign plasmids (PubMed:32246713).[UniProtKB:A0A482D308][1] [2] Publication Abstract from PubMedCompact CRISPR-Cas systems offer versatile treatment options for genetic disorders, but their application is often limited by modest gene-editing activity. Here we present enAsCas12f, an engineered RNA-guided DNA endonuclease up to 11.3-fold more potent than its parent protein, AsCas12f, and one-third of the size of SpCas9. enAsCas12f shows higher DNA cleavage activity than wild-type AsCas12f in vitro and functions broadly in human cells, delivering up to 69.8% insertions and deletions at user-specified genomic loci. Minimal off-target editing is observed with enAsCas12f, suggesting that boosted on-target activity does not impair genome-wide specificity. We determine the cryo-electron microscopy (cryo-EM) structure of the AsCas12f-sgRNA-DNA complex at a resolution of 2.9 A, which reveals dimerization-mediated substrate recognition and cleavage. Structure-guided single guide RNA (sgRNA) engineering leads to sgRNA-v2, which is 33% shorter than the full-length sgRNA, but with on par activity. Together, the engineered hypercompact AsCas12f system enables robust and faithful gene editing in mammalian cells. An engineered hypercompact CRISPR-Cas12f system with boosted gene-editing activity.,Wu T, Liu C, Zou S, Lyu R, Yang B, Yan H, Zhao M, Tang W Nat Chem Biol. 2023 Nov;19(11):1384-1393. doi: 10.1038/s41589-023-01380-9. Epub , 2023 Jul 3. PMID:37400536[3] From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine. References
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