8y0d
Crystal structure of SauCas9 in complex with sgRNA and 20nt ssDNA target
Structural highlights
FunctionCAS9_STAAU 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 spacers, sequences complementary to antecedent mobile elements, and target invading nucleic acids. CRISPR clusters are transcribed and processed into CRISPR RNA (crRNA). In type II CRISPR systems correct processing of pre-crRNA requires a trans-encoded small RNA (tracrRNA), endogenous ribonuclease 3 (rnc) and this protein. The tracrRNA serves as a guide for ribonuclease 3-aided processing of pre-crRNA. Subsequently Cas9/crRNA/tracrRNA endonucleolytically cleaves linear or circular dsDNA target complementary to the spacer; Cas9 is inactive in the absence of the 2 guide RNAs (gRNA). Cas9 recognizes the protospacer adjacent motif (PAM) in the CRISPR repeat sequences to help distinguish self versus nonself, as targets within the bacterial CRISPR locus do not have PAMs. PAM recognition is also required for catalytic activity.[HAMAP-Rule:MF_01480][1] [2] 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[3] From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine. References
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