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| | <StructureSection load='6k4u' size='340' side='right'caption='[[6k4u]], [[Resolution|resolution]] 3.20Å' scene=''> | | <StructureSection load='6k4u' size='340' side='right'caption='[[6k4u]], [[Resolution|resolution]] 3.20Å' scene=''> |
| | == Structural highlights == | | == Structural highlights == |
| - | <table><tr><td colspan='2'>[[6k4u]] is a 4 chain structure with sequence from [http://en.wikipedia.org/wiki/Strp1 Strp1]. Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=6K4U OCA]. For a <b>guided tour on the structure components</b> use [http://oca.weizmann.ac.il/oca-docs/fgij/fg.htm?mol=6K4U FirstGlance]. <br> | + | <table><tr><td colspan='2'>[[6k4u]] is a 4 chain structure with sequence from [https://en.wikipedia.org/wiki/Streptococcus_pyogenes Streptococcus pyogenes] and [https://en.wikipedia.org/wiki/Streptococcus_pyogenes_serotype_M1 Streptococcus pyogenes serotype M1]. Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=6K4U OCA]. For a <b>guided tour on the structure components</b> use [https://proteopedia.org/fgij/fg.htm?mol=6K4U FirstGlance]. <br> |
| - | </td></tr><tr id='gene'><td class="sblockLbl"><b>[[Gene|Gene:]]</b></td><td class="sblockDat">cas9 ([http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&srchmode=5&id=301447 STRP1])</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]] 3.2Å</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=6k4u FirstGlance], [http://oca.weizmann.ac.il/oca-bin/ocaids?id=6k4u OCA], [http://pdbe.org/6k4u PDBe], [http://www.rcsb.org/pdb/explore.do?structureId=6k4u RCSB], [http://www.ebi.ac.uk/pdbsum/6k4u PDBsum], [http://prosat.h-its.org/prosat/prosatexe?pdbcode=6k4u 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=6k4u FirstGlance], [http://oca.weizmann.ac.il/oca-bin/ocaids?id=6k4u OCA], [https://pdbe.org/6k4u PDBe], [https://www.rcsb.org/pdb/explore.do?structureId=6k4u RCSB], [https://www.ebi.ac.uk/pdbsum/6k4u PDBsum], [https://prosat.h-its.org/prosat/prosatexe?pdbcode=6k4u ProSAT]</span></td></tr> |
| | </table> | | </table> |
| | == Function == | | == Function == |
| - | [[http://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> | + | [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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| | </div> | | </div> |
| | <div class="pdbe-citations 6k4u" style="background-color:#fffaf0;"></div> | | <div class="pdbe-citations 6k4u" style="background-color:#fffaf0;"></div> |
| | + | |
| | + | ==See Also== |
| | + | *[[Endonuclease 3D structures|Endonuclease 3D structures]] |
| | == References == | | == References == |
| | <references/> | | <references/> |
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| | </StructureSection> | | </StructureSection> |
| | [[Category: Large Structures]] | | [[Category: Large Structures]] |
| - | [[Category: Strp1]] | + | [[Category: Streptococcus pyogenes]] |
| - | [[Category: Chen, W]] | + | [[Category: Streptococcus pyogenes serotype M1]] |
| - | [[Category: Gan, J]] | + | [[Category: Chen W]] |
| - | [[Category: Ji, Q]] | + | [[Category: Gan J]] |
| - | [[Category: Wang, Y]] | + | [[Category: Ji Q]] |
| - | [[Category: Zhang, H]] | + | [[Category: Wang Y]] |
| - | [[Category: Zhang, Y]] | + | [[Category: Zhang H]] |
| - | [[Category: Endonuclease]]
| + | [[Category: Zhang Y]] |
| - | [[Category: Hydrolase-dna complex]]
| + | |
| 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
Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas systems have been harnessed as powerful genome editing tools in diverse organisms. However, the off-target effects and the protospacer adjacent motif (PAM) compatibility restrict the therapeutic applications of these systems. Recently, a Streptococcus pyogenes Cas9 (SpCas9) variant, xCas9, was evolved to possess both broad PAM compatibility and high DNA fidelity. Through determination of multiple xCas9 structures, which are all in complex with single-guide RNA (sgRNA) and double-stranded DNA containing different PAM sequences (TGG, CGG, TGA, and TGC), we decipher the molecular mechanisms of the PAM expansion and fidelity enhancement of xCas9. xCas9 follows a unique two-mode PAM recognition mechanism. For non-NGG PAM recognition, xCas9 triggers a notable structural rearrangement in the DNA recognition domains and a rotation in the key PAM-interacting residue R1335; such mechanism has not been observed in the wild-type (WT) SpCas9. For NGG PAM recognition, xCas9 applies a strategy similar to WT SpCas9. Moreover, biochemical and cell-based genome editing experiments pinpointed the critical roles of the E1219V mutation for PAM expansion and the R324L, S409I, and M694I mutations for fidelity enhancement. The molecular-level characterizations of the xCas9 nuclease provide critical insights into the mechanisms of the PAM expansion and fidelity enhancement of xCas9 and could further facilitate the engineering of SpCas9 and other Cas9 orthologs.
Molecular basis for the PAM expansion and fidelity enhancement of an evolved Cas9 nuclease.,Chen W, Zhang H, Zhang Y, Wang Y, Gan J, Ji Q PLoS Biol. 2019 Oct 11;17(10):e3000496. doi: 10.1371/journal.pbio.3000496., eCollection 2019 Oct. PMID:31603896[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
- ↑ Chen W, Zhang H, Zhang Y, Wang Y, Gan J, Ji Q. Molecular basis for the PAM expansion and fidelity enhancement of an evolved Cas9 nuclease. PLoS Biol. 2019 Oct 11;17(10):e3000496. doi: 10.1371/journal.pbio.3000496., eCollection 2019 Oct. PMID:31603896 doi:http://dx.doi.org/10.1371/journal.pbio.3000496
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