8y0b
From Proteopedia
Crystal structure of FnCas12a in complex with pre-crRNA and 12nt target DNA
Structural highlights
FunctionCS12A_FRATN 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. CRISPR clusters are transcribed and processed into CRISPR RNA (crRNA). Has endonuclease activity on pre-crRNA and dsDNA, using different active sites. A single-RNA guided endonuclease that is also capable of guiding crRNA processing; correct processing of pre-crRNA requires only this protein and the CRISPR locus (PubMed:26422227, PubMed:27096362). pre-crRNA processing proceeds by an intramolecular nucleophilic attack on the scissile phosphate by the 2'-OH of the upstream ribonucleotide, the divalent cation (which is bound by the crRNA) is probably required for ordering the crRNA pseudoknot and/or increasing RNA binding (PubMed:28431230). RNA mutagenesis studies show pre-crRNA cleavage is highly sequence- and structure-specific (PubMed:27096362). Forms a complex with crRNA and complementary dsDNA, where the crRNA displaces the non-target DNA strand and directs endonucleolytic cleavage of both strands of the DNA (PubMed:26422227, PubMed:27096362, PubMed:28431230). Cleavage results in staggered 5-base 5' overhangs 14-18 and 21-23 bases downstream of the PAM (protospacer adjacent motif) on the non-target and target strands respectively (PubMed:26422227, PubMed:28431230, PubMed:28562584). Both target and non-target strand DNA are probably independently cleaved in the same active site (PubMed:28431230, PubMed:28562584). When this protein is expressed in E.coli it prevents plasmids homologous to the first CRISPR spacer from transforming, formally showing it is responsible for plasmid immunity (PubMed:26422227).[1] [2] [3] [4] Publication Abstract from PubMedPrecursor (pre)-CRISPR RNA (crRNA) processing can occur in both the repeat and spacer regions, leading to the removal of specific segments from the repeat and spacer sequences, thereby facilitating crRNA maturation. The processing of pre-crRNA repeat by Cas effector and ribonuclease has been observed in CRISPR-Cas9 and CRISPR-Cas12a systems. However, no evidence of pre-crRNA spacer cleavage by any enzyme has been reported in these systems. In this study, we demonstrate that DNA target binding triggers efficient cleavage of pre-crRNA spacers by type II and V Cas effectors such as Cas12a, Cas12b, Cas12i, Cas12j and Cas9. We show that the pre-crRNA spacer cleavage catalyzed by Cas12a and Cas9 has distinct characteristics. Activation of the cleavage activity in Cas12a is induced by both single-stranded DNA (ssDNA) and double-stranded DNA target binding, whereas only ssDNA target binding triggers cleavage in Cas9 toward the pre-crRNA spacer. We present a series of structures elucidating the underlying mechanisms governing conformational activation in both Cas12a and Cas9. Furthermore, leveraging the trans-cutting activity of the pre-crRNA spacer, we develop a one-step DNA detection method characterized by its simplicity, high sensitivity, and excellent specificity. DNA target binding-induced pre-crRNA processing in type II and V CRISPR-Cas systems.,Chen J, Lin X, Xiang W, Chen Y, Zhao Y, Huang L, Liu L Nucleic Acids Res. 2024 Dec 16:gkae1241. doi: 10.1093/nar/gkae1241. PMID:39676682[5] From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine. References
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