Journal article
Decoupling tRNA promoter and processing activities enables specific Pol-II Cas9 guide RNA expression
- Abstract:
- Spatial/temporal control of Cas9 guide RNA expression could considerably expand the utility of CRISPR-based technologies. Current approaches based on tRNA processing offer a promising strategy but suffer from high background. Here, to address this limitation, we present a screening platform which allows simultaneous measurements of the promoter strength, 5', and 3' processing efficiencies across a library of tRNA variants. This analysis reveals that the sequence determinants underlying these activities, while overlapping, are dissociable. Rational design based on the ensuing principles allowed us to engineer an improved tRNA scaffold that enables highly specific guide RNA production from a Pol-II promoter. When benchmarked against other reported systems this tRNA scaffold is superior to most alternatives, and is equivalent in function to an optimized version of the Csy4-based guide RNA release system. The results and methods described in this manuscript enable avenues of research both in genome engineering and basic tRNA biology.
- Publication status:
- Published
- Peer review status:
- Peer reviewed
Actions
Access Document
- Files:
-
-
(Preview, Version of record, 1.8MB, Terms of use)
-
- Publisher copy:
- 10.1038/s41467-019-09148-3
Authors
- Publisher:
- Springer Nature
- Journal:
- Nature Communications More from this journal
- Volume:
- 10
- Article number:
- 1490
- Place of publication:
- England
- Publication date:
- 2019-04-02
- Acceptance date:
- 2019-02-22
- DOI:
- EISSN:
-
2041-1723
- Pmid:
-
30940799
- Language:
-
English
- Keywords:
- Pubs id:
-
991313
- Local pid:
-
pubs:991313
- Deposit date:
-
2021-10-22
Terms of use
- Copyright holder:
- Knapp et al.
- Copyright date:
- 2019
- Rights statement:
- Copyright © 2021 The Author(s). 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/.
- Licence:
- CC Attribution (CC BY)
If you are the owner of this record, you can report an update to it here: Report update to this record