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

Autor:	Katrekar D; Department of Bioengineering, University of California San Diego, San Diego, United States., Xiang Y; Department of Bioengineering, University of California San Diego, San Diego, United States., Palmer N; Division of Biological Sciences, University of California San Diego, San Diego, United States., Saha A; Department of Bioengineering, University of California San Diego, San Diego, United States., Meluzzi D; Department of Bioengineering, University of California San Diego, San Diego, United States., Mali P; Department of Bioengineering, University of California San Diego, San Diego, United States.
Jazyk:	angličtina
Zdroj:	ELife [Elife] 2022 Jan 19; Vol. 11. Date of Electronic Publication: 2022 Jan 19.
DOI:	10.7554/eLife.75555
Abstrakt:	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. Towards addressing these issues, 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. As overexpression of ADAR enzymes, especially hyperactive variants, can lead to significant transcriptome-wide off-targeting, we next engineered a split-ADAR2 deaminase which resulted in >100-fold more specific RNA editing as compared to full-length deaminase overexpression. Taken together, we anticipate this systematic engineering of the ADAR2 deaminase domain will enable broader utility of the ADAR toolset for RNA biotechnology applications. Competing Interests: DK, YX 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, NP, AS, DM, PM No competing interests declared (© 2022, Katrekar et al.)
Databáze:	MEDLINE
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