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| | ==Crystal structure of catalytically inactive FnCas12a in complex with a crRNA guide and a dsDNA target== | | ==Crystal structure of catalytically inactive FnCas12a in complex with a crRNA guide and a dsDNA target== |
| - | <StructureSection load='6i1k' size='340' side='right' caption='[[6i1k]], [[Resolution|resolution]] 2.65Å' scene=''> | + | <StructureSection load='6i1k' size='340' side='right'caption='[[6i1k]], [[Resolution|resolution]] 2.65Å' scene=''> |
| | == Structural highlights == | | == Structural highlights == |
| - | <table><tr><td colspan='2'>[[6i1k]] is a 4 chain structure with sequence from [http://en.wikipedia.org/wiki/Fratn Fratn]. Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=6I1K OCA]. For a <b>guided tour on the structure components</b> use [http://oca.weizmann.ac.il/oca-docs/fgij/fg.htm?mol=6I1K FirstGlance]. <br> | + | <table><tr><td colspan='2'>[[6i1k]] is a 4 chain structure with sequence from [https://en.wikipedia.org/wiki/Francisella_tularensis_subsp._novicida_U112 Francisella tularensis subsp. novicida U112] and [https://en.wikipedia.org/wiki/Synthetic_construct Synthetic construct]. Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=6I1K OCA]. For a <b>guided tour on the structure components</b> use [https://proteopedia.org/fgij/fg.htm?mol=6I1K FirstGlance]. <br> |
| - | </td></tr><tr id='ligand'><td class="sblockLbl"><b>[[Ligand|Ligands:]]</b></td><td class="sblockDat"><scene name='pdbligand=EDO:1,2-ETHANEDIOL'>EDO</scene>, <scene name='pdbligand=MG:MAGNESIUM+ION'>MG</scene></td></tr> | + | </td></tr><tr id='method'><td class="sblockLbl"><b>[[Empirical_models|Method:]]</b></td><td class="sblockDat" id="methodDat">X-ray diffraction, [[Resolution|Resolution]] 2.65Å</td></tr> |
| - | <tr id='gene'><td class="sblockLbl"><b>[[Gene|Gene:]]</b></td><td class="sblockDat">cas12a, cpf1, FTN_1397 ([http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&srchmode=5&id=401614 FRATN])</td></tr> | + | <tr id='ligand'><td class="sblockLbl"><b>[[Ligand|Ligands:]]</b></td><td class="sblockDat" id="ligandDat"><scene name='pdbligand=EDO:1,2-ETHANEDIOL'>EDO</scene>, <scene name='pdbligand=MG:MAGNESIUM+ION'>MG</scene></td></tr> |
| - | <tr id='resources'><td class="sblockLbl"><b>Resources:</b></td><td class="sblockDat"><span class='plainlinks'>[http://oca.weizmann.ac.il/oca-docs/fgij/fg.htm?mol=6i1k FirstGlance], [http://oca.weizmann.ac.il/oca-bin/ocaids?id=6i1k OCA], [http://pdbe.org/6i1k PDBe], [http://www.rcsb.org/pdb/explore.do?structureId=6i1k RCSB], [http://www.ebi.ac.uk/pdbsum/6i1k PDBsum], [http://prosat.h-its.org/prosat/prosatexe?pdbcode=6i1k ProSAT]</span></td></tr> | + | <tr id='resources'><td class="sblockLbl"><b>Resources:</b></td><td class="sblockDat"><span class='plainlinks'>[https://proteopedia.org/fgij/fg.htm?mol=6i1k FirstGlance], [http://oca.weizmann.ac.il/oca-bin/ocaids?id=6i1k OCA], [https://pdbe.org/6i1k PDBe], [https://www.rcsb.org/pdb/explore.do?structureId=6i1k RCSB], [https://www.ebi.ac.uk/pdbsum/6i1k PDBsum], [https://prosat.h-its.org/prosat/prosatexe?pdbcode=6i1k ProSAT]</span></td></tr> |
| | </table> | | </table> |
| | == Function == | | == Function == |
| - | [[http://www.uniprot.org/uniprot/CS12A_FRATN 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).<ref>PMID:26422227</ref> <ref>PMID:27096362</ref> <ref>PMID:28431230</ref> <ref>PMID:28562584</ref> | + | [https://www.uniprot.org/uniprot/CS12A_FRATN 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).<ref>PMID:26422227</ref> <ref>PMID:27096362</ref> <ref>PMID:28431230</ref> <ref>PMID:28562584</ref> |
| | <div style="background-color:#fffaf0;"> | | <div style="background-color:#fffaf0;"> |
| | == Publication Abstract from PubMed == | | == Publication Abstract from PubMed == |
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| | </div> | | </div> |
| | <div class="pdbe-citations 6i1k" style="background-color:#fffaf0;"></div> | | <div class="pdbe-citations 6i1k" style="background-color:#fffaf0;"></div> |
| | + | |
| | + | ==See Also== |
| | + | *[[Endonuclease 3D structures|Endonuclease 3D structures]] |
| | == References == | | == References == |
| | <references/> | | <references/> |
| | __TOC__ | | __TOC__ |
| | </StructureSection> | | </StructureSection> |
| - | [[Category: Fratn]] | + | [[Category: Francisella tularensis subsp. novicida U112]] |
| - | [[Category: Jinek, M]] | + | [[Category: Large Structures]] |
| - | [[Category: Swarts, D C]] | + | [[Category: Synthetic construct]] |
| - | [[Category: Crispr-cas12a]] | + | [[Category: Jinek M]] |
| - | [[Category: Crispr-cpf1]] | + | [[Category: Swarts DC]] |
| - | [[Category: Fncas12a]]
| + | |
| - | [[Category: Fncpf1]]
| + | |
| - | [[Category: Hydrolase]]
| + | |
| Structural highlights
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
CRISPR-Cas12a (Cpf1) is an RNA-guided DNA-cutting nuclease that has been repurposed for genome editing. Upon target DNA binding, Cas12a cleaves both the target DNA in cis and non-target single-stranded DNAs (ssDNAs) in trans. To elucidate the molecular basis for both DNase cleavage modes, we performed structural and biochemical studies on Francisella novicida Cas12a. We show that guide RNA-target strand DNA hybridization conformationally activates Cas12a, triggering its trans-acting, non-specific, single-stranded DNase activity. In turn, cis cleavage of double-stranded DNA targets is a result of protospacer adjacent motif (PAM)-dependent DNA duplex unwinding, electrostatic stabilization of the displaced non-target DNA strand, and ordered sequential cleavage of the non-target and target DNA strands. Cas12a releases the PAM-distal DNA cleavage product and remains bound to the PAM-proximal DNA cleavage product in a catalytically competent, trans-active state. Together, these results provide a revised model for the molecular mechanisms of both the cis- and the trans-acting DNase activities of Cas12a enzymes, enabling their further exploitation as genome editing tools.
Mechanistic Insights into the cis- and trans-Acting DNase Activities of Cas12a.,Swarts DC, Jinek M Mol Cell. 2019 Feb 7;73(3):589-600.e4. doi: 10.1016/j.molcel.2018.11.021. Epub, 2019 Jan 10. PMID:30639240[5]
From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.
See Also
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
- ↑ Swarts DC, Jinek M. Mechanistic Insights into the cis- and trans-Acting DNase Activities of Cas12a. Mol Cell. 2019 Feb 7;73(3):589-600.e4. doi: 10.1016/j.molcel.2018.11.021. Epub, 2019 Jan 10. PMID:30639240 doi:http://dx.doi.org/10.1016/j.molcel.2018.11.021
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