Comprehensive interrogation of the ADAR2 deaminase domain for engineering enhanced RNA editing activity and specificity

Abstract
Data availability
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Abstract

Adenosine deaminases acting on RNA (ADARs) can be repurposed to enable programmable RNA editing, however their enzymatic activity on adenosines flanked by a 5' guanosine is very low, thus limiting their utility as a transcriptome engineering toolset. To address this issue, we first performed a novel deep mutational scan of the ADAR2 deaminase domain, directly measuring the impact of every amino acid substitution across 261 residues, on RNA editing. This enabled us to create a domain wide mutagenesis map while also revealing a novel hyperactive variant with improved enzymatic activity at 5'-GAN-3' motifs. However, exogenous delivery of ADAR enzymes, especially hyperactive variants, leads to significant transcriptome wide off-targeting. To solve this problem, we engineered a split ADAR2 deaminase which resulted in 1000-fold more specific RNA editing as compared to full-length deaminase overexpression. We anticipate that this systematic engineering of the ADAR2 deaminase domain will enable broader utility of the ADAR toolset for RNA biotechnology and therapeutic applications.

Data availability

Sequencing data will be accessible via NCBI GEO under accession GSE158656. Source data has been made available with the submission.

The following data sets were generated

1. Katrekar D
(2022) ADAR Protein Engineering
NCBI Gene Expression Omnibus, GSE158656.

https://www.ncbi.nlm.nih.gov/geo/

Article and author information

Author details

Dhruva Katrekar

Bioengineering, University of California, San Diego, La Jolla, United States

Competing interests
Dhruva Katrekar, has filed a patent pertaining to the screening methodology, novel mutants and splitting of the ADAR2-DD (application number: 63/075,717). Is now an employee of Shape Therapeutics..

"This ORCID iD identifies the author of this article:" 0000-0002-8028-3244
Yichen Xiang

Department of Bioengineering, University of California, San Diego, La Jolla, United States

Competing interests
No competing interests declared.
Nathan Palmer

Division of Biological Sciences, University of California, San Diego, La Jolla, United States

Competing interests
No competing interests declared.

"This ORCID iD identifies the author of this article:" 0000-0001-6347-9379
Anushka Saha

Department of Bioengineering, University of California, San Diego, La Jolla, United States

Competing interests
No competing interests declared.
Dario Meluzzi

Department of Bioengineering, University of California, San Diego, La Jolla, United States

Competing interests
No competing interests declared.
Prashant Mali

Department of Bioengineering, University of California, San Diego, La Jolla, United States

For correspondence
pmali@ucsd.edu

Competing interests
Prashant Mali, has filed a patent pertaining to the screening methodology, novel mutants and splitting of the ADAR2-DD (application number: 63/075,717). Is a scientific co-founder of Shape Therapeutics, Navega Therapeutics, Boundless Biosciences, and Engine Biosciences..

"This ORCID iD identifies the author of this article:" 0000-0002-3383-1287

Funding

National Human Genome Research Institute (R01HG009285)

Prashant Mali

National Cancer Institute (R01CA222826)

Prashant Mali

National Institute of General Medical Sciences (R01GM123313)

Prashant Mali

U.S. Department of Defense (PR210085)

Prashant Mali

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Copyright

This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.