CRISPR-associated endodeoxyribonuclease Cas12f1

UniProtKB accession:  A0A482D308

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UniProtKB description:  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 (in vitro) (Probable). Upon expression in E.coli of this protein, a mini CRISPR array and the probable tracrRNA, the protein associates with both RNAs (PubMed:30337455). The mini system is not active in E.coli against phiX174 phage, nor is it active in protection against transformation by foreign plasmids (PubMed:30337455, PubMed:32246713). In vitro the purified protein-tracrRNA-crRNA complex cleaves ssDNA complementary to the crRNA; target cleavage requires both tracrRNA and crRNA, but not a protospacer adjacent motif (PAM). The tracrRNA-crRNA can be replaced by a single guide RNA (sgRNA). 2-nucleotide mismatches in the middle of the crRNA:DNA heteroduplex decrease cleavage. Cleavage occurs just downstream of the heteroduplex (PubMed:30337455). Activation of this protein results in non-specific ssDNA degradation in vitro (PubMed:30337455, PubMed:32246713). In vitro and in E.coli (coexpressed with sgRNA) has dsDNA endonuclease activity, recognizing the 5' PAM sequence TTTR; both sgRNA and a PAM are required for activity. Cleaves the target strand 24 and the nontarget strand 22 bases upstream of the PAM (respectively), resulting in 5' overhangs (PubMed:32246713, PubMed:33333018). The 2 monomers interact differently with the sgRNA and target DNA. Mutagenesis of a dimeric construct shows that one of the RuvC monomers probably cleaves both DNA strands (PubMed:33333018).