Global quantification exposes abundant low-level off-target activity by base editors
- Ilana Buchumenski1,
- Shalom Hillel Roth1,
- Eli Kopel1,
- Efrat Katsman1,
- Ariel Feiglin1,
- Erez Y. Levanon1,2,4 and
- Eli Eisenberg3,4
- 1The Mina and Everard Goodman Faculty of Life Sciences, Bar-Ilan University, Ramat Gan 5290002, Israel;
- 2The Institute of Nanotechnology and Advanced Materials, Bar-Ilan University, Ramat Gan, 5290002, Israel;
- 3Raymond and Beverly Sackler School of Physics and Astronomy and Sagol School of Neuroscience, Tel Aviv University, Tel Aviv, 69978, Israel
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↵4 These authors contributed equally to this work.
Abstract
Base editors are dedicated engineered deaminases that enable directed conversion of specific bases in the genome or transcriptome in a precise and efficient manner, and hold promise for correcting pathogenic mutations. A major concern limiting application of this powerful approach is the issue of off-target edits. Several recent studies have shown substantial off-target RNA activity induced by base editors and demonstrated that off-target mutations may be suppressed by improved deaminases versions or optimized guide RNAs. Here, we describe a new class of off-target events that are invisible to the established methods for detection of genomic variations and were thus far overlooked. We show that nonspecific, seemingly stochastic, off-target events affect a large number of sites throughout the genome or the transcriptome, and account for the majority of off-target activity. We develop and employ a different, complementary approach that is sensitive to the stochastic off-target activity and use it to quantify the abundant off-target RNA mutations due to current, optimized deaminase editors. We provide a computational tool to quantify global off-target activity, which can be used to optimize future base editors. Engineered base editors enable directed manipulation of the genome or transcriptome at single-base resolution. We believe that implementation of this computational approach would facilitate design of more specific base editors.
Footnotes
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[Supplemental material is available for this article.]
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Article published online before print. Article, supplemental material, and publication date are at https://www.genome.org/cgi/doi/10.1101/gr.275770.121.
- Received May 13, 2021.
- Accepted October 14, 2021.
This article is distributed exclusively by Cold Spring Harbor Laboratory Press for the first six months after the full-issue publication date (see https://genome.cshlp.org/site/misc/terms.xhtml). After six months, it is available under a Creative Commons License (Attribution-NonCommercial 4.0 International), as described at http://creativecommons.org/licenses/by-nc/4.0/.
