7ox7: Difference between revisions
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==== | ==Target-bound SpCas9 complex with TRAC chimeric RNA-DNA guide== | ||
<StructureSection load='7ox7' size='340' side='right'caption='[[7ox7]]' scene=''> | <StructureSection load='7ox7' size='340' side='right'caption='[[7ox7]], [[Resolution|resolution]] 2.60Å' scene=''> | ||
== Structural highlights == | == Structural highlights == | ||
<table><tr><td colspan='2'>Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id= OCA]. For a <b>guided tour on the structure components</b> use [https://proteopedia.org/fgij/fg.htm?mol= FirstGlance]. <br> | <table><tr><td colspan='2'>[[7ox7]] is a 4 chain structure with sequence from [https://en.wikipedia.org/wiki/Streptococcus_pyogenes_serotype_M1 Streptococcus pyogenes serotype M1] 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=7OX7 OCA]. For a <b>guided tour on the structure components</b> use [https://proteopedia.org/fgij/fg.htm?mol=7OX7 FirstGlance]. <br> | ||
</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=7ox7 FirstGlance], [http://oca.weizmann.ac.il/oca-bin/ocaids?id=7ox7 OCA], [https://pdbe.org/7ox7 PDBe], [https://www.rcsb.org/pdb/explore.do?structureId=7ox7 RCSB], [https://www.ebi.ac.uk/pdbsum/7ox7 PDBsum], [https://prosat.h-its.org/prosat/prosatexe?pdbcode=7ox7 ProSAT]</span></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]] 2.6Å</td></tr> | ||
<tr id='ligand'><td class="sblockLbl"><b>[[Ligand|Ligands:]]</b></td><td class="sblockDat" id="ligandDat"><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=7ox7 FirstGlance], [http://oca.weizmann.ac.il/oca-bin/ocaids?id=7ox7 OCA], [https://pdbe.org/7ox7 PDBe], [https://www.rcsb.org/pdb/explore.do?structureId=7ox7 RCSB], [https://www.ebi.ac.uk/pdbsum/7ox7 PDBsum], [https://prosat.h-its.org/prosat/prosatexe?pdbcode=7ox7 ProSAT]</span></td></tr> | |||
</table> | </table> | ||
== Function == | |||
[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;"> | |||
== Publication Abstract from PubMed == | |||
The off-target activity of the CRISPR-associated nuclease Cas9 is a potential concern for therapeutic genome editing applications. Although high-fidelity Cas9 variants have been engineered, they exhibit varying efficiencies and have residual off-target effects, limiting their applicability. Here, we show that CRISPR hybrid RNA-DNA (chRDNA) guides provide an effective approach to increase Cas9 specificity while preserving on-target editing activity. Across multiple genomic targets in primary human T cells, we show that 2'-deoxynucleotide (dnt) positioning affects guide activity and specificity in a target-dependent manner and that this can be used to engineer chRDNA guides with substantially reduced off-target effects. Crystal structures of DNA-bound Cas9-chRDNA complexes reveal distorted guide-target duplex geometry and allosteric modulation of Cas9 conformation. These structural effects increase specificity by perturbing DNA hybridization and modulating Cas9 activation kinetics to disfavor binding and cleavage of off-target substrates. Overall, these results pave the way for utilizing customized chRDNAs in clinical applications. | |||
Conformational control of Cas9 by CRISPR hybrid RNA-DNA guides mitigates off-target activity in T cells.,Donohoue PD, Pacesa M, Lau E, Vidal B, Irby MJ, Nyer DB, Rotstein T, Banh L, Toh MS, Gibson J, Kohrs B, Baek K, Owen ALG, Slorach EM, van Overbeek M, Fuller CK, May AP, Jinek M, Cameron P Mol Cell. 2021 Sep 2;81(17):3637-3649.e5. doi: 10.1016/j.molcel.2021.07.035. PMID:34478654<ref>PMID:34478654</ref> | |||
From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.<br> | |||
</div> | |||
<div class="pdbe-citations 7ox7" style="background-color:#fffaf0;"></div> | |||
==See Also== | |||
*[[Endonuclease 3D structures|Endonuclease 3D structures]] | |||
== References == | |||
<references/> | |||
__TOC__ | __TOC__ | ||
</StructureSection> | </StructureSection> | ||
[[Category: Large Structures]] | [[Category: Large Structures]] | ||
[[Category: | [[Category: Streptococcus pyogenes serotype M1]] | ||
[[Category: Synthetic construct]] | |||
[[Category: Baek K]] | |||
[[Category: Banh L]] | |||
[[Category: Cameron P]] | |||
[[Category: Donohoue P]] | |||
[[Category: Fuller CK]] | |||
[[Category: Gibson J]] | |||
[[Category: Irby MJ]] | |||
[[Category: Jinek M]] | |||
[[Category: Kohrs B]] | |||
[[Category: Lau E]] | |||
[[Category: May AP]] | |||
[[Category: Nyer DB]] | |||
[[Category: Owen ALG]] | |||
[[Category: Pacesa M]] | |||
[[Category: Rotstein T]] | |||
[[Category: Slorach EM]] | |||
[[Category: Toh MT]] | |||
[[Category: Vidal B]] | |||
[[Category: Van Overbeek M]] | |||