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| | ==Crystal structure of FnCas12a bound to a crRNA== | | ==Crystal structure of FnCas12a bound to a crRNA== |
| - | <StructureSection load='5ng6' size='340' side='right' caption='[[5ng6]], [[Resolution|resolution]] 3.34Å' scene=''> | + | <StructureSection load='5ng6' size='340' side='right'caption='[[5ng6]], [[Resolution|resolution]] 3.34Å' scene=''> |
| | == Structural highlights == | | == Structural highlights == |
| - | <table><tr><td colspan='2'>[[5ng6]] is a 8 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=5NG6 OCA]. For a <b>guided tour on the structure components</b> use [http://oca.weizmann.ac.il/oca-docs/fgij/fg.htm?mol=5NG6 FirstGlance]. <br> | + | <table><tr><td colspan='2'>[[5ng6]] is a 8 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=5NG6 OCA]. For a <b>guided tour on the structure components</b> use [https://proteopedia.org/fgij/fg.htm?mol=5NG6 FirstGlance]. <br> |
| - | </td></tr><tr id='ligand'><td class="sblockLbl"><b>[[Ligand|Ligands:]]</b></td><td class="sblockDat"><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]] 3.342Å</td></tr> |
| - | <tr id='gene'><td class="sblockLbl"><b>[[Gene|Gene:]]</b></td><td class="sblockDat">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=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=5ng6 FirstGlance], [http://oca.weizmann.ac.il/oca-bin/ocaids?id=5ng6 OCA], [http://pdbe.org/5ng6 PDBe], [http://www.rcsb.org/pdb/explore.do?structureId=5ng6 RCSB], [http://www.ebi.ac.uk/pdbsum/5ng6 PDBsum], [http://prosat.h-its.org/prosat/prosatexe?pdbcode=5ng6 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=5ng6 FirstGlance], [http://oca.weizmann.ac.il/oca-bin/ocaids?id=5ng6 OCA], [https://pdbe.org/5ng6 PDBe], [https://www.rcsb.org/pdb/explore.do?structureId=5ng6 RCSB], [https://www.ebi.ac.uk/pdbsum/5ng6 PDBsum], [https://prosat.h-its.org/prosat/prosatexe?pdbcode=5ng6 ProSAT]</span></td></tr> |
| | </table> | | </table> |
| | == Function == | | == Function == |
| - | [[http://www.uniprot.org/uniprot/CPF1_FRATN CPF1_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). This protein is 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). When this protein is expressed in E.coli it prevents plasmids homologous to the first CRISPR spacer from transforming, showing it is responsible for plasmid immunity (PubMed:26422227). Has dsDNA endonuclease activity, results in staggered 5-base 5' overhangs 18 and 23 bases downstream of the PAM (protospacer adjacent motif) on the non-targeted and targeted strands respectively (PubMed:26422227).<ref>PMID:26422227</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 5ng6" style="background-color:#fffaf0;"></div> | | <div class="pdbe-citations 5ng6" 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: Oost, J van der]] | + | [[Category: Synthetic construct]] |
| - | [[Category: Swarts, D C]] | + | [[Category: Jinek M]] |
| - | [[Category: Ca]] | + | [[Category: Swarts DC]] |
| - | [[Category: Cas12a]] | + | [[Category: Van der Oost J]] |
| - | [[Category: Cpf1]]
| + | |
| - | [[Category: Crispr]]
| + | |
| - | [[Category: Crrna]]
| + | |
| - | [[Category: Genome editing]]
| + | |
| - | [[Category: Hydrolase]]
| + | |
| - | [[Category: Nuclease]]
| + | |
| - | [[Category: R-loop]]
| + | |
| - | [[Category: Ruvc]]
| + | |
| 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
The CRISPR-associated protein Cas12a (Cpf1), which has been repurposed for genome editing, possesses two distinct nuclease activities: endoribonuclease activity for processing its own guide RNAs and RNA-guided DNase activity for target DNA cleavage. To elucidate the molecular basis of both activities, we determined crystal structures of Francisella novicida Cas12a bound to guide RNA and in complex with an R-loop formed by a non-cleavable guide RNA precursor and a full-length target DNA. Corroborated by biochemical experiments, these structures reveal the mechanisms of guide RNA processing and pre-ordering of the seed sequence in the guide RNA that primes Cas12a for target DNA binding. Furthermore, the R-loop complex structure reveals the strand displacement mechanism that facilitates guide-target hybridization and suggests a mechanism for double-stranded DNA cleavage involving a single active site. Together, these insights advance our mechanistic understanding of Cas12a enzymes and may contribute to further development of genome editing technologies.
Structural Basis for Guide RNA Processing and Seed-Dependent DNA Targeting by CRISPR-Cas12a.,Swarts DC, van der Oost J, Jinek M Mol Cell. 2017 Apr 20;66(2):221-233.e4. doi: 10.1016/j.molcel.2017.03.016. PMID:28431230[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, 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
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