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Comprehensive interrogation of the ADAR2 deaminase domain for engineering enhanced RNA base-editing activity, functionality and specificity

Dhruva Katrekar, Nathan Palmer, Yichen Xiang, Anushka Saha, Dario Meluzzi, Prashant Mali
doi: https://doi.org/10.1101/2020.09.08.288233
Dhruva Katrekar
1Department of Bioengineering, University of California San Diego, CA, USA
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Nathan Palmer
2Division of Biological Sciences, University of California San Diego, CA, USA
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Yichen Xiang
1Department of Bioengineering, University of California San Diego, CA, USA
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Anushka Saha
1Department of Bioengineering, University of California San Diego, CA, USA
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Dario Meluzzi
1Department of Bioengineering, University of California San Diego, CA, USA
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Prashant Mali
1Department of Bioengineering, University of California San Diego, CA, USA
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  • For correspondence: pmali@ucsd.edu
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ABSTRACT

Adenosine deaminases acting on RNA (ADARs) can be repurposed to enable programmable RNA editing, however their exogenous delivery leads to transcriptome-wide off-targeting, and additionally, enzymatic activity on certain RNA motifs, especially those flanked by a 5’ guanosine is very low thus limiting their utility as a transcriptome engineering toolset. To address this, we explored comprehensive ADAR2 protein engineering via three approaches: First, we performed a novel deep mutational scan of the deaminase domain that enabled direct coupling of variants to corresponding RNA editing activity. Experimentally measuring the impact of every amino acid substitution across 261 residues, i.e. ~5000 variants, on RNA editing, revealed intrinsic domain properties, and also several mutations that greatly enhanced RNA editing. Second, we performed a domain-wide mutagenesis screen to identify variants that increased activity at 5’-GA-3’ motifs, and discovered novel mutants that enabled robust RNA editing. Third, we engineered the domain at the fragment level to create split deaminases. Notably, compared to full-length deaminase overexpression, split-deaminases resulted in >1000 fold more specific RNA editing. Taken together, we anticipate this comprehensive deaminase engineering will enable broader utility of the ADAR toolset for RNA biotechnology and therapeutic applications.

Competing Interest Statement

D.K. and P.M. have filed patents based on this work. P.M. is a scientific co-founder of Shape Therapeutics, Seven Therapeutics, Navega Therapeutics, Boundless Biosciences, and Engine Biosciences. The terms of these arrangements have been reviewed and approved by the University of California, San Diego in accordance with its conflict of interest policies.

Copyright 
The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license.
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Posted September 09, 2020.
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Comprehensive interrogation of the ADAR2 deaminase domain for engineering enhanced RNA base-editing activity, functionality and specificity
Dhruva Katrekar, Nathan Palmer, Yichen Xiang, Anushka Saha, Dario Meluzzi, Prashant Mali
bioRxiv 2020.09.08.288233; doi: https://doi.org/10.1101/2020.09.08.288233
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Comprehensive interrogation of the ADAR2 deaminase domain for engineering enhanced RNA base-editing activity, functionality and specificity
Dhruva Katrekar, Nathan Palmer, Yichen Xiang, Anushka Saha, Dario Meluzzi, Prashant Mali
bioRxiv 2020.09.08.288233; doi: https://doi.org/10.1101/2020.09.08.288233

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