6gtc

Transition state structure of Cpf1(Cas12a) I1 conformationTransition state structure of Cpf1(Cas12a) I1 conformation

6gtc, resolution 3.91Å

Structural highlights

6gtc is a 4 chain structure with sequence from Fratn. Full crystallographic information is available from OCA. For a guided tour on the structure components use FirstGlance.
Gene:	cas12a, cpf1, FTN_1397 (FRATN)
Experimental data:	Check
Resources:	FirstGlance, OCA, PDBe, RCSB, PDBsum, ProSAT

Function

[CS12A_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 PubMed

Cas12a, also known as Cpf1, is a type V-A CRISPR-Cas RNA-guided endonuclease that is used for genome editing based on its ability to generate specific dsDNA breaks. Here, we show cryo-EM structures of intermediates of the cleavage reaction, thus visualizing three protein regions that sense the crRNA-DNA hybrid assembly triggering the catalytic activation of Cas12a. Single-molecule FRET provides the thermodynamics and kinetics of the conformational activation leading to phosphodiester bond hydrolysis. These findings illustrate why Cas12a cuts its target DNA and unleashes unspecific cleavage activity, degrading ssDNA molecules after activation. In addition, we show that other crRNAs are able to displace the R-loop inside the protein after target DNA cleavage, terminating indiscriminate ssDNA degradation. We propose a model whereby the conformational activation of the enzyme results in indiscriminate ssDNA cleavage. The displacement of the R-loop by a new crRNA molecule will reset Cas12a specificity, targeting new DNAs.

Conformational Activation Promotes CRISPR-Cas12a Catalysis and Resetting of the Endonuclease Activity.,Stella S, Mesa P, Thomsen J, Paul B, Alcon P, Jensen SB, Saligram B, Moses ME, Hatzakis NS, Montoya G Cell. 2018 Dec 13;175(7):1856-1871.e21. doi: 10.1016/j.cell.2018.10.045. Epub, 2018 Nov 29. PMID:30503205^[5]

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.

References

↑ Zetsche B, Gootenberg JS, Abudayyeh OO, Slaymaker IM, Makarova KS, Essletzbichler P, Volz SE, Joung J, van der Oost J, Regev A, Koonin EV, Zhang F. Cpf1 is a single RNA-guided endonuclease of a class 2 CRISPR-Cas system. Cell. 2015 Oct 22;163(3):759-71. doi: 10.1016/j.cell.2015.09.038. Epub 2015 Sep, 25. PMID:26422227 doi:http://dx.doi.org/10.1016/j.cell.2015.09.038
↑ Fonfara I, Richter H, Bratovic M, Le Rhun A, Charpentier E. The CRISPR-associated DNA-cleaving enzyme Cpf1 also processes precursor CRISPR RNA. Nature. 2016 Apr 28;532(7600):517-21. doi: 10.1038/nature17945. Epub 2016 Apr 20. PMID:27096362 doi:http://dx.doi.org/10.1038/nature17945
↑ Swarts DC, van der Oost J, Jinek M. Structural Basis for Guide RNA Processing and Seed-Dependent DNA Targeting by CRISPR-Cas12a. Mol Cell. 2017 Apr 20;66(2):221-233.e4. doi: 10.1016/j.molcel.2017.03.016. PMID:28431230 doi:http://dx.doi.org/10.1016/j.molcel.2017.03.016
↑ Stella S, Alcon P, Montoya G. Structure of the Cpf1 endonuclease R-loop complex after target DNA cleavage. Nature. 2017 Jun 22;546(7659):559-563. doi: 10.1038/nature22398. Epub 2017 May, 31. PMID:28562584 doi:http://dx.doi.org/10.1038/nature22398
↑ Stella S, Mesa P, Thomsen J, Paul B, Alcon P, Jensen SB, Saligram B, Moses ME, Hatzakis NS, Montoya G. Conformational Activation Promotes CRISPR-Cas12a Catalysis and Resetting of the Endonuclease Activity. Cell. 2018 Dec 13;175(7):1856-1871.e21. doi: 10.1016/j.cell.2018.10.045. Epub, 2018 Nov 29. PMID:30503205 doi:http://dx.doi.org/10.1016/j.cell.2018.10.045

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OCA

[1] Zetsche B, Gootenberg JS, Abudayyeh OO, Slaymaker IM, Makarova KS, Essletzbichler P, Volz SE, Joung J, van der Oost J, Regev A, Koonin EV, Zhang F. Cpf1 is a single RNA-guided endonuclease of a class 2 CRISPR-Cas system. Cell. 2015 Oct 22;163(3):759-71. doi: 10.1016/j.cell.2015.09.038. Epub 2015 Sep, 25. PMID:26422227 doi:http://dx.doi.org/10.1016/j.cell.2015.09.038

[2] Fonfara I, Richter H, Bratovic M, Le Rhun A, Charpentier E. The CRISPR-associated DNA-cleaving enzyme Cpf1 also processes precursor CRISPR RNA. Nature. 2016 Apr 28;532(7600):517-21. doi: 10.1038/nature17945. Epub 2016 Apr 20. PMID:27096362 doi:http://dx.doi.org/10.1038/nature17945

[3] Swarts DC, van der Oost J, Jinek M. Structural Basis for Guide RNA Processing and Seed-Dependent DNA Targeting by CRISPR-Cas12a. Mol Cell. 2017 Apr 20;66(2):221-233.e4. doi: 10.1016/j.molcel.2017.03.016. PMID:28431230 doi:http://dx.doi.org/10.1016/j.molcel.2017.03.016

[4] Stella S, Alcon P, Montoya G. Structure of the Cpf1 endonuclease R-loop complex after target DNA cleavage. Nature. 2017 Jun 22;546(7659):559-563. doi: 10.1038/nature22398. Epub 2017 May, 31. PMID:28562584 doi:http://dx.doi.org/10.1038/nature22398

[5] Stella S, Mesa P, Thomsen J, Paul B, Alcon P, Jensen SB, Saligram B, Moses ME, Hatzakis NS, Montoya G. Conformational Activation Promotes CRISPR-Cas12a Catalysis and Resetting of the Endonuclease Activity. Cell. 2018 Dec 13;175(7):1856-1871.e21. doi: 10.1016/j.cell.2018.10.045. Epub, 2018 Nov 29. PMID:30503205 doi:http://dx.doi.org/10.1016/j.cell.2018.10.045

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