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| | ==Crystal structure of Cas9 bound to PAM-containing DNA target== | | ==Crystal structure of Cas9 bound to PAM-containing DNA target== |
| - | <StructureSection load='4un3' size='340' side='right' caption='[[4un3]], [[Resolution|resolution]] 2.59Å' scene=''> | + | <StructureSection load='4un3' size='340' side='right'caption='[[4un3]], [[Resolution|resolution]] 2.59Å' scene=''> |
| | == Structural highlights == | | == Structural highlights == |
| - | <table><tr><td colspan='2'>[[4un3]] is a 4 chain structure. Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=4UN3 OCA]. For a <b>guided tour on the structure components</b> use [http://oca.weizmann.ac.il/oca-docs/fgij/fg.htm?mol=4UN3 FirstGlance]. <br> | + | <table><tr><td colspan='2'>[[4un3]] is a 4 chain structure with sequence from [https://en.wikipedia.org/wiki/Streptococcus_pyogenes Streptococcus pyogenes] and [https://en.wikipedia.org/wiki/Synthetic_construct Synthetic construct]. The January 2015 RCSB PDB [https://pdb.rcsb.org/pdb/static.do?p=education_discussion/molecule_of_the_month/index.html Molecule of the Month] feature on ''Cascade and CRISPR'' by David Goodsell is [https://dx.doi.org/10.2210/rcsb_pdb/mom_2015_1 10.2210/rcsb_pdb/mom_2015_1]. Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=4UN3 OCA]. For a <b>guided tour on the structure components</b> use [https://proteopedia.org/fgij/fg.htm?mol=4UN3 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]] 2.593Å</td></tr> |
| - | <tr id='related'><td class="sblockLbl"><b>[[Related_structure|Related:]]</b></td><td class="sblockDat">[[4un4|4un4]], [[4un5|4un5]]</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=4un3 FirstGlance], [http://oca.weizmann.ac.il/oca-bin/ocaids?id=4un3 OCA], [http://www.rcsb.org/pdb/explore.do?structureId=4un3 RCSB], [http://www.ebi.ac.uk/pdbsum/4un3 PDBsum]</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=4un3 FirstGlance], [http://oca.weizmann.ac.il/oca-bin/ocaids?id=4un3 OCA], [https://pdbe.org/4un3 PDBe], [https://www.rcsb.org/pdb/explore.do?structureId=4un3 RCSB], [https://www.ebi.ac.uk/pdbsum/4un3 PDBsum], [https://prosat.h-its.org/prosat/prosatexe?pdbcode=4un3 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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| | From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.<br> | | From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.<br> |
| | </div> | | </div> |
| | + | <div class="pdbe-citations 4un3" style="background-color:#fffaf0;"></div> |
| | | | |
| | ==See Also== | | ==See Also== |
| - | *[[Anisotropic refinement|Anisotropic refinement]] | + | *[[CRISPR-Cas9|CRISPR-Cas9]] |
| - | *[[Cas9|Cas9]] | + | *[[CRISPR-Cas9 Part II|CRISPR-Cas9 Part II]] |
| - | *[[Endonuclease|Endonuclease]] | + | *[[Endonuclease 3D structures|Endonuclease 3D structures]] |
| | == References == | | == References == |
| | <references/> | | <references/> |
| | __TOC__ | | __TOC__ |
| | </StructureSection> | | </StructureSection> |
| - | [[Category: Anders, C]] | + | [[Category: Cascade and CRISPR]] |
| - | [[Category: Duerst, A]] | + | [[Category: Large Structures]] |
| - | [[Category: Jinek, M]] | + | [[Category: RCSB PDB Molecule of the Month]] |
| - | [[Category: Niewoehner, O]] | + | [[Category: Streptococcus pyogenes]] |
| - | [[Category: Crispr]] | + | [[Category: Synthetic construct]] |
| - | [[Category: Genome editing]] | + | [[Category: Anders C]] |
| - | [[Category: Hydrolase-dna-rna complex]] | + | [[Category: Duerst A]] |
| - | [[Category: Protein-rna complex]] | + | [[Category: Jinek M]] |
| - | [[Category: Rnp]] | + | [[Category: Niewoehner O]] |
| 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
The CRISPR-associated protein Cas9 is an RNA-guided endonuclease that cleaves double-stranded DNA bearing sequences complementary to a 20-nucleotide segment in the guide RNA. Cas9 has emerged as a versatile molecular tool for genome editing and gene expression control. RNA-guided DNA recognition and cleavage strictly require the presence of a protospacer adjacent motif (PAM) in the target DNA. Here we report a crystal structure of Streptococcus pyogenes Cas9 in complex with a single-molecule guide RNA and a target DNA containing a canonical 5'-NGG-3' PAM. The structure reveals that the PAM motif resides in a base-paired DNA duplex. The non-complementary strand GG dinucleotide is read out via major-groove interactions with conserved arginine residues from the carboxy-terminal domain of Cas9. Interactions with the minor groove of the PAM duplex and the phosphodiester group at the +1 position in the target DNA strand contribute to local strand separation immediately upstream of the PAM. These observations suggest a mechanism for PAM-dependent target DNA melting and RNA-DNA hybrid formation. Furthermore, this study establishes a framework for the rational engineering of Cas9 enzymes with novel PAM specificities.
Structural basis of PAM-dependent target DNA recognition by the Cas9 endonuclease.,Anders C, Niewoehner O, Duerst A, Jinek M Nature. 2014 Jul 27. doi: 10.1038/nature13579. PMID:25079318[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
- ↑ Anders C, Niewoehner O, Duerst A, Jinek M. Structural basis of PAM-dependent target DNA recognition by the Cas9 endonuclease. Nature. 2014 Jul 27. doi: 10.1038/nature13579. PMID:25079318 doi:http://dx.doi.org/10.1038/nature13579
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