Mapping the sugar dependency for rational generation of a DNA-RNA hybrid-guided Cas9 endonuclease

Abstract

The CRISPR–Cas9 RNA-guided endonuclease system allows precise and efficient modification of complex genomes and is continuously developed to enhance specificity, alter targeting and add new functional moieties. However, one area yet to be explored is the base chemistry of the associated RNA molecules. Here we show the design and optimisation of hybrid DNA–RNA CRISPR and tracr molecules based on structure-guided approaches. Through careful mapping of the ribose requirements of Cas9, we develop hybrid versions possessing minimal RNA residues, which are sufficient to direct specific nuclease activity in vitro and in vivo with reduced off-target activity. We identify critical regions within these molecules that require ribose nucleotides and show a direct correlation between binding affinity/stability and cellular activity. This is the first demonstration of a non-RNA-guided Cas9 endonuclease and first step towards eliminating the ribose dependency of Cas9 to develop a XNA-programmable endonuclease.

Metadata

Item Type:	Article
Authors/Creators:	Rueda, F.O. https://orcid.org/0000-0001-5929-5294 Bista, M. Newton, M.D. https://orcid.org/0000-0002-9453-6791 Goeppert, A.U. Cuomo, M.E. Gordon, E. Kröner, F. Read, J.A. Wrigley, J.D. Rueda, D. https://orcid.org/0000-0003-4657-6323 Taylor, B.J.M. https://orcid.org/0000-0001-6101-3786
Copyright, Publisher and Additional Information:	© The Author(s) 2017. This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/ licenses/by/4.0/.
Keywords:	Bacterial Proteins; Biocatalysis; CRISPR-Associated Protein 9; Clustered Regularly Interspaced Short Palindromic Repeats; DNA; Endonucleases; Nucleic Acid Conformation; RNA; RNA, Guide, CRISPR-Cas Systems
Dates:	Accepted: 12 October 2017 Published (online): 20 November 2017 Published: 20 November 2017
Institution:	The University of Sheffield
Academic Units:	The University of Sheffield > Faculty of Science (Sheffield) > School of Biosciences (Sheffield)
Date Deposited:	17 Oct 2025 11:30
Last Modified:	17 Oct 2025 11:30
Status:	Published
Publisher:	Springer Science and Business Media LLC
Refereed:	Yes
Identification Number:	10.1038/s41467-017-01732-9
Related URLs:	PubMed URL
Open Archives Initiative ID (OAI ID):	oai:eprints.whiterose.ac.uk:232919

CORE (COnnecting REpositories)

Mapping the sugar dependency for rational generation of a DNA-RNA hybrid-guided Cas9 endonuclease

Abstract

Metadata

Download

Published Version

Export

Statistics