Comprehensive interrogation of the ADAR2 deaminase domain for engineering enhanced RNA editing activity and specificity
| Autor: | Dhruva Katrekar, Yichen Xiang, Nathan Palmer, Anushka Saha, Dario Meluzzi, Prashant Mali |
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| Rok vydání: | 2022 |
| Předmět: |
RNA editing
QH301-705.5 Adenosine Deaminase Science chemical biology Protein Engineering General Biochemistry Genetics and Molecular Biology computational biology Protein Domains deep mutational scan Biochemistry and Chemical Biology ADARs Genetics Humans biochemistry human Biology (General) Nucleotide Motifs General Immunology and Microbiology General Neuroscience RNA-Binding Proteins systems biology General Medicine Tools and Resources Medicine Biochemistry and Cell Biology Transcriptome Computational and Systems Biology Human split proteins Biotechnology |
| Zdroj: | eLife, Vol 11 (2022) eLife |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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