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5b2s

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Current revision

Crystal structure of the Streptococcus pyogenes Cas9 EQR variant in complex with sgRNA and target DNA (TGAG PAM)

Structural highlights

5b2s is a 4 chain structure with sequence from Streptococcus pyogenes and Streptococcus pyogenes serotype M1. Full crystallographic information is available from OCA. For a guided tour on the structure components use FirstGlance.
Method:	X-ray diffraction, Resolution 2.2Å
Ligands:	, , ,
Resources:	FirstGlance, OCA, PDBe, RCSB, PDBsum, ProSAT

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 RNA-guided endonuclease Cas9 cleaves double-stranded DNA targets bearing a PAM (protospacer adjacent motif) and complementarity to the guide RNA. A recent study showed that, whereas wild-type Streptococcus pyogenes Cas9 (SpCas9) recognizes the 5'-NGG-3' PAM, the engineered VQR, EQR, and VRER SpCas9 variants recognize the 5'-NGA-3', 5'-NGAG-3', and 5'-NGCG-3' PAMs, respectively, thus expanding the targetable sequences in Cas9-mediated genome editing applications. Here, we present the high-resolution crystal structures of the three SpCas9 variants in complexes with a single-guide RNA and its altered PAM-containing, partially double-stranded DNA targets. A structural comparison of the three SpCas9 variants with wild-type SpCas9 revealed that the multiple mutations synergistically induce an unexpected displacement in the phosphodiester backbone of the PAM duplex, thereby allowing the SpCas9 variants to directly recognize the altered PAM nucleotides. Our findings explain the altered PAM specificities of the SpCas9 variants and establish a framework for further rational engineering of CRISPR-Cas9.

Structural Basis for the Altered PAM Specificities of Engineered CRISPR-Cas9.,Hirano S, Nishimasu H, Ishitani R, Nureki O Mol Cell. 2016 Mar 17;61(6):886-94. doi: 10.1016/j.molcel.2016.02.018. PMID:26990991^[3]

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.

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
↑ Hirano S, Nishimasu H, Ishitani R, Nureki O. Structural Basis for the Altered PAM Specificities of Engineered CRISPR-Cas9. Mol Cell. 2016 Mar 17;61(6):886-94. doi: 10.1016/j.molcel.2016.02.018. PMID:26990991 doi:http://dx.doi.org/10.1016/j.molcel.2016.02.018

1 Structural highlights
2 Function
3 Publication Abstract from PubMed
4 See Also
5 References

Proteopedia Page Contributors and Editors (what is this?)

OCA

Retrieved from "http://52.214.119.220/wiki/index.php/5b2s"

@@ Line 4: / Line 4: @@
 == Structural highlights ==
 <table><tr><td colspan='2'>[[5b2s]] 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=5B2S OCA]. For a <b>guided tour on the structure components</b> use [https://proteopedia.org/fgij/fg.htm?mol=5B2S FirstGlance]. <br>
-</td></tr><tr id='ligand'><td class="sblockLbl"><b>[[Ligand|Ligands:]]</b></td><td class="sblockDat" id="ligandDat"><scene name='pdbligand=ACT:ACETATE+ION'>ACT</scene>, <scene name='pdbligand=EDO:1,2-ETHANEDIOL'>EDO</scene>, <scene name='pdbligand=K:POTASSIUM+ION'>K</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]] 2.2&#8491;</td></tr>
+<tr id='ligand'><td class="sblockLbl"><b>[[Ligand|Ligands:]]</b></td><td class="sblockDat" id="ligandDat"><scene name='pdbligand=ACT:ACETATE+ION'>ACT</scene>, <scene name='pdbligand=EDO:1,2-ETHANEDIOL'>EDO</scene>, <scene name='pdbligand=K:POTASSIUM+ION'>K</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'>[https://proteopedia.org/fgij/fg.htm?mol=5b2s FirstGlance], [http://oca.weizmann.ac.il/oca-bin/ocaids?id=5b2s OCA], [https://pdbe.org/5b2s PDBe], [https://www.rcsb.org/pdb/explore.do?structureId=5b2s RCSB], [https://www.ebi.ac.uk/pdbsum/5b2s PDBsum], [https://prosat.h-its.org/prosat/prosatexe?pdbcode=5b2s ProSAT]</span></td></tr>
 </table>

5b2s

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Current revision

Crystal structure of the Streptococcus pyogenes Cas9 EQR variant in complex with sgRNA and target DNA (TGAG PAM)

Structural highlights

Function

Publication Abstract from PubMed

See Also

References

Contents

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