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| | <SX load='5vzl' size='340' side='right' viewer='molstar' caption='[[5vzl]], [[Resolution|resolution]] 3.90Å' scene=''> | | <SX load='5vzl' size='340' side='right' viewer='molstar' caption='[[5vzl]], [[Resolution|resolution]] 3.90Å' scene=''> |
| | == Structural highlights == | | == Structural highlights == |
| - | <table><tr><td colspan='2'>[[5vzl]] is a 3 chain structure with sequence from [http://en.wikipedia.org/wiki/ ], [http://en.wikipedia.org/wiki/Bacteriophage_sp. Bacteriophage sp.] and [http://en.wikipedia.org/wiki/Streptococcus_pyogenes_(serotype_1_/_m1_gas) Streptococcus pyogenes (serotype 1 / m1 gas)]. Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=5VZL OCA]. For a <b>guided tour on the structure components</b> use [http://proteopedia.org/fgij/fg.htm?mol=5VZL FirstGlance]. <br> | + | <table><tr><td colspan='2'>[[5vzl]] is a 3 chain structure with sequence from [https://en.wikipedia.org/wiki/Bacteriophage_sp. Bacteriophage sp.] and [https://en.wikipedia.org/wiki/Streptococcus_pyogenes_M1_GAS Streptococcus pyogenes M1 GAS]. Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=5VZL OCA]. For a <b>guided tour on the structure components</b> use [https://proteopedia.org/fgij/fg.htm?mol=5VZL FirstGlance]. <br> |
| - | </td></tr><tr id='NonStdRes'><td class="sblockLbl"><b>[[Non-Standard_Residue|NonStd Res:]]</b></td><td class="sblockDat"><scene name='pdbligand=GTP:GUANOSINE-5-TRIPHOSPHATE'>GTP</scene></td></tr> | + | </td></tr><tr id='method'><td class="sblockLbl"><b>[[Empirical_models|Method:]]</b></td><td class="sblockDat" id="methodDat">Electron Microscopy, [[Resolution|Resolution]] 3.9Å</td></tr> |
| - | <tr id='gene'><td class="sblockLbl"><b>[[Gene|Gene:]]</b></td><td class="sblockDat">csn1, cas9, ERS445054_00848 ([http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&srchmode=5&id=160490 Streptococcus pyogenes (serotype 1 / M1 GAS)])</td></tr> | + | <tr id='ligand'><td class="sblockLbl"><b>[[Ligand|Ligands:]]</b></td><td class="sblockDat" id="ligandDat"><scene name='pdbligand=GTP:GUANOSINE-5-TRIPHOSPHATE'>GTP</scene></td></tr> |
| - | <tr id='resources'><td class="sblockLbl"><b>Resources:</b></td><td class="sblockDat"><span class='plainlinks'>[http://proteopedia.org/fgij/fg.htm?mol=5vzl FirstGlance], [http://oca.weizmann.ac.il/oca-bin/ocaids?id=5vzl OCA], [http://pdbe.org/5vzl PDBe], [http://www.rcsb.org/pdb/explore.do?structureId=5vzl RCSB], [http://www.ebi.ac.uk/pdbsum/5vzl PDBsum], [http://prosat.h-its.org/prosat/prosatexe?pdbcode=5vzl 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=5vzl FirstGlance], [http://oca.weizmann.ac.il/oca-bin/ocaids?id=5vzl OCA], [https://pdbe.org/5vzl PDBe], [https://www.rcsb.org/pdb/explore.do?structureId=5vzl RCSB], [https://www.ebi.ac.uk/pdbsum/5vzl PDBsum], [https://prosat.h-its.org/prosat/prosatexe?pdbcode=5vzl ProSAT]</span></td></tr> |
| | </table> | | </table> |
| | == Function == | | == Function == |
| - | [[http://www.uniprot.org/uniprot/A0A0C6FZC2_STRPY A0A0C6FZC2_STRPY]] 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] | + | [https://www.uniprot.org/uniprot/CAS9_STRP1 CAS9_STRP1] 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) (Probable). 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. The target strand not complementary to crRNA is first cut endonucleolytically, then trimmed by 3'-5' exonucleolytically. DNA-binding requires protein and both RNA species. Cas9 probably recognizes a short motif in the CRISPR repeat sequences (the PAM or protospacer adjacent motif) to help distinguish self versus nonself.<ref>PMID:21455174</ref> <ref>PMID:22745249</ref> |
| | <div style="background-color:#fffaf0;"> | | <div style="background-color:#fffaf0;"> |
| | == Publication Abstract from PubMed == | | == Publication Abstract from PubMed == |
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| | [[Category: Bacteriophage sp]] | | [[Category: Bacteriophage sp]] |
| | [[Category: Large Structures]] | | [[Category: Large Structures]] |
| - | [[Category: Doudna, J A]] | + | [[Category: Streptococcus pyogenes M1 GAS]] |
| - | [[Category: Jiang, F]] | + | [[Category: Doudna JA]] |
| - | [[Category: Liu, J J]] | + | [[Category: Jiang F]] |
| - | [[Category: Nogales, E]] | + | [[Category: Liu JJ]] |
| - | [[Category: Anti-crispr]] | + | [[Category: Nogales E]] |
| - | [[Category: Cas9]]
| + | |
| - | [[Category: Crispr]]
| + | |
| - | [[Category: Cryo-em]]
| + | |
| - | [[Category: Gene editing]]
| + | |
| - | [[Category: Immune system-rna complex]]
| + | |
| - | [[Category: Off-target]]
| + | |
| - | [[Category: On-target]]
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| Structural highlights
Function
CAS9_STRP1 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) (Probable). 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. The target strand not complementary to crRNA is first cut endonucleolytically, then trimmed by 3'-5' exonucleolytically. DNA-binding requires protein and both RNA species. Cas9 probably recognizes a short motif in the CRISPR repeat sequences (the PAM or protospacer adjacent motif) to help distinguish self versus nonself.[1] [2]
Publication Abstract from PubMed
CRISPR (clustered regularly interspaced short palindromic repeats)-Cas9 gene editing technology is derived from a microbial adaptive immune system, where bacteriophages are often the intended target. Natural inhibitors of CRISPR-Cas9 enable phages to evade immunity and show promise in controlling Cas9-mediated gene editing in human cells. However, the mechanism of CRISPR-Cas9 inhibition is not known, and the potential applications for Cas9 inhibitor proteins in mammalian cells have not been fully established. We show that the anti-CRISPR protein AcrIIA4 binds only to assembled Cas9-single-guide RNA (sgRNA) complexes and not to Cas9 protein alone. A 3.9 A resolution cryo-electron microscopy structure of the Cas9-sgRNA-AcrIIA4 complex revealed that the surface of AcrIIA4 is highly acidic and binds with a 1:1 stoichiometry to a region of Cas9 that normally engages the DNA protospacer adjacent motif. Consistent with this binding mode, order-of-addition experiments showed that AcrIIA4 interferes with DNA recognition but has no effect on preformed Cas9-sgRNA-DNA complexes. Timed delivery of AcrIIA4 into human cells as either protein or expression plasmid allows on-target Cas9-mediated gene editing while reducing off-target edits. These results provide a mechanistic understanding of AcrIIA4 function and demonstrate that inhibitors can modulate the extent and outcomes of Cas9-mediated gene editing.
Disabling Cas9 by an anti-CRISPR DNA mimic.,Shin J, Jiang F, Liu JJ, Bray NL, Rauch BJ, Baik SH, Nogales E, Bondy-Denomy J, Corn JE, Doudna JA Sci Adv. 2017 Jul 12;3(7):e1701620. doi: 10.1126/sciadv.1701620. eCollection 2017, Jul. PMID:28706995[3]
From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.
See Also
References
- ↑ Deltcheva E, Chylinski K, Sharma CM, Gonzales K, Chao Y, Pirzada ZA, Eckert MR, Vogel J, Charpentier E. CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III. Nature. 2011 Mar 31;471(7340):602-7. doi: 10.1038/nature09886. PMID:21455174 doi:http://dx.doi.org/10.1038/nature09886
- ↑ Jinek M, Chylinski K, Fonfara I, Hauer M, Doudna JA, Charpentier E. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science. 2012 Aug 17;337(6096):816-21. doi: 10.1126/science.1225829. Epub 2012, Jun 28. PMID:22745249 doi:http://dx.doi.org/10.1126/science.1225829
- ↑ Shin J, Jiang F, Liu JJ, Bray NL, Rauch BJ, Baik SH, Nogales E, Bondy-Denomy J, Corn JE, Doudna JA. Disabling Cas9 by an anti-CRISPR DNA mimic. Sci Adv. 2017 Jul 12;3(7):e1701620. doi: 10.1126/sciadv.1701620. eCollection 2017, Jul. PMID:28706995 doi:http://dx.doi.org/10.1126/sciadv.1701620
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