Sökning: onr:"swepub:oai:DiVA.org:umu-169087" >
Bridge helix argini...
Bridge helix arginines play a critical role in Cas9 sensitivity to mismatches
-
Bratovic, Majda (författare)
-
- Fonfara, Ines (författare)
- Umeå universitet,Molekylär Infektionsmedicin, Sverige (MIMS),Institutionen för molekylärbiologi (Medicinska fakulteten),Umeå Centre for Microbial Research (UCMR),Department of Regulation in Infection Biology, Max Planck Institute for Infection Biology, Berlin, Germany; Department of Regulation in Infection Biology, Helmholtz Centre for Infection Research, Braunschweig, Germany
-
- Chylinski, Krzysztof (författare)
- Umeå universitet,Institutionen för molekylärbiologi (Medicinska fakulteten),Molekylär Infektionsmedicin, Sverige (MIMS),Umeå Centre for Microbial Research (UCMR),Max F. Perutz Laboratories, University of Vienna, Vienna, Austria; Protein Technologies Facility, The Vienna Biocenter Core Facilities GmbH (VBCF), Vienna, Austria
-
visa fler...
-
Galvez, Eric J. C. (författare)
-
Sullivan, Timothy J. (författare)
-
Boerno, Stefan (författare)
-
Timmermann, Bernd (författare)
-
Boettcher, Michael (författare)
-
- Charpentier, Emmanuelle, 1968- (författare)
- Umeå universitet,Molekylär Infektionsmedicin, Sverige (MIMS),Umeå Centre for Microbial Research (UCMR),Institutionen för molekylärbiologi (Medicinska fakulteten),Max Planck Unit for the Science of Pathogens, Berlin, Germany; Department of Regulation in Infection Biology, Max Planck Institute for Infection Biology, Berlin, Germany; Institute for Biology, Humboldt University, Berlin, Germany; Department of Regulation in Infection Biology, Helmholtz Centre for Infection Research, Braunschweig, Germany
-
visa färre...
-
(creator_code:org_t)
- 2020-03-02
- 2020
- Engelska.
-
Ingår i: Nature Chemical Biology. - : Nature Publishing Group. - 1552-4450 .- 1552-4469. ; 16:5, s. 587-595
- Relaterad länk:
-
https://pure.mpg.de/...
-
visa fler...
-
https://urn.kb.se/re...
-
https://doi.org/10.1...
-
visa färre...
Abstract
Ämnesord
Stäng
- The RNA-programmable DNA-endonuclease Cas9 is widely used for genome engineering, where a high degree of specificity is required. To investigate which features of Cas9 determine the sensitivity to mismatches along the target DNA, we performed in vitro biochemical assays and bacterial survival assays in Escherichia coli. We demonstrate that arginines in the Cas9 bridge helix influence guide RNA, and target DNA binding and cleavage. They cluster in two groups that either increase or decrease the Cas9 sensitivity to mismatches. We show that the bridge helix is essential for R-loop formation and that R63 and R66 reduce Cas9 specificity by stabilizing the R-loop in the presence of mismatches. Additionally, we identify Q768 that reduces sensitivity of Cas9 to protospacer adjacent motif-distal mismatches. The Cas9_R63A/Q768A variant showed increased specificity in human cells. Our results provide a firm basis for function- and structure-guided mutagenesis to increase Cas9 specificity for genome engineering. Tuning CRISPR-Cas9 nuclease specificity enables precision genome engineering. Identifying arginine residues along the bridge helix of SpCas9 that mediate Cas9 mismatch sensitivity enabled engineering of Cas9 with increased specificity in human cells.
Ämnesord
- NATURVETENSKAP -- Biologi -- Biokemi och molekylärbiologi (hsv//swe)
- NATURAL SCIENCES -- Biological Sciences -- Biochemistry and Molecular Biology (hsv//eng)
Publikations- och innehållstyp
- ref (ämneskategori)
- art (ämneskategori)
Hitta via bibliotek
Till lärosätets databas