Characterization of a novel type III CRISPR-Cas effector provides new insights into the allosteric activation and suppression of the Cas10 DNase
Open Access
- 12 May 2020
- journal article
- research article
- Published by Springer Science and Business Media LLC in Cell Discovery
- Vol. 6 (1), 1-16
- https://doi.org/10.1038/s41421-020-0160-4
Abstract
Antiviral defense by type III CRISPR-Cas systems relies on two distinct activities of their effectors: the RNA-activated DNA cleavage and synthesis of cyclic oligoadenylate. Both activities are featured as indiscriminate nucleic acid cleavage and subjected to the spatiotemporal regulation. To yield further insights into the involved mechanisms, we reconstituted LdCsm, a lactobacilli III-A system in Escherichia coli. Upon activation by target RNA, this immune system mediates robust DNA degradation but lacks the synthesis of cyclic oligoadenylates. Mutagenesis of the Csm3 and Cas10 conserved residues revealed that Csm3 and multiple structural domains in Cas10 function in the allosteric regulation to yield an active enzyme. Target RNAs carrying various truncations in the 3ʹ anti-tag were designed and tested for their influence on DNA binding and DNA cleavage of LdCsm. Three distinct states of ternary LdCsm complexes were identified. In particular, binding of target RNAs carrying a single nucleotide in the 3ʹ anti-tag to LdCsm yielded an active LdCsm DNase regardless whether the nucleotide shows a mismatch, as in the cognate target RNA (CTR), or a match, as in the noncognate target RNA (NTR), to the 5′ tag of crRNA. In addition, further increasing the number of 3ʹ anti-tag in CTR facilitated the substrate binding and enhanced the substrate degradation whereas doing the same as in NTR gradually decreased the substrate binding and eventually shut off the DNA cleavage by the enzyme. Together, these results provide the mechanistic insights into the allosteric activation and repression of LdCsm enzymes.Keywords
Funding Information
- Det Frie Forskningsråd (DFF-4181-00274)
- National Natural Science Foundation of China (31771380)
- Shandong University
This publication has 70 references indexed in Scilit:
- Structure and Mechanism of the CMR Complex for CRISPR-Mediated Antiviral ImmunityMolecular Cell, 2012
- Essential Features and Rational Design of CRISPR RNAs that Function with the Cas RAMP Module Complex to Cleave RNAsMolecular Cell, 2012
- Modulation of CRISPR locus transcription by the repeat-binding protein Cbp1 in SulfolobusNucleic Acids Research, 2011
- Self versus non-self discrimination during CRISPR RNA-directed immunityNature, 2010
- CRISPR/Cas, the Immune System of Bacteria and ArchaeaScience, 2010
- RNA-Guided RNA Cleavage by a CRISPR RNA-Cas Protein ComplexCell, 2009
- CRISPR Interference Limits Horizontal Gene Transfer in Staphylococci by Targeting DNAScience, 2008
- CRISPR Provides Acquired Resistance Against Viruses in ProkaryotesScience, 2007
- FACS-optimized mutants of the green fluorescent protein (GFP)Gene, 1996
- High- and low-copy-number Lactococcus shuttle cloning vectors with features for clone screeningGene, 1993