![]() ![]() This is because ADAR catalytic domains are used for their deaminase activity and these domains can react with RNA substrates in the absence of a targeting domain ( Zheng et al., 2017 Montiel-Gonzalez et al., 2013 Eifler et al., 2013 Hanswillemenke et al., 2015 Wang and Beal, 2016 Matthews et al., 2016 Phelps et al., 2015). ![]() However, in each of the reported directed RNA editing systems, off target activity is observed ( Cox et al., 2017 Montiel-Gonzalez et al., 2013 Montiel-Gonzalez et al., 2016 Vallecillo-Viejo et al., 2018 Wettengel et al., 2017 Vogel et al., 2018). The ability of ADARs to convert A to I has spurred efforts to develop new proteins capable of directing ADAR-catalyzed deamination to specific adenosines present in mRNAs because of disease-associated G to A mutations in the genome ( Cox et al., 2017 Montiel-Gonzalez et al., 2013 Vallecillo-Viejo et al., 2018). Among its many consequences on RNA function, ADAR-mediated A to I editing can alter miRNA recognition sites, redirect splicing and change the meaning of specific codons ( Wang et al., 2013 Rueter et al., 1999 Yeo et al., 2010). Since I base pairs with C, it functions like G in cellular processes such as splicing, translation and reverse transcription ( Figure 1A) ( Bass, 2002 Nishikura, 2010). ADARs (adenosine deaminases that act on RNA) are known to convert A to inosine (I) in duplex RNA ( Bass, 2002 Bass and Weintraub, 1988 Goodman et al., 2012). This and other systems designed to direct RNA editing reactions use deaminase domains from the ADAR enzymes ( Hanswillemenke et al., 2015 Montiel-Gonzalez et al., 2013 Montiel-Gonzalez et al., 2016 Cox et al., 2017 Stafforst and Schneider, 2012). Methods for directing the deamination of specific adenosines in RNA have also been described including a recent report of Cas13 fusion proteins capable of RNA editing for Programmable A to I replacement (REPAIR) ( Cox et al., 2017 Hanswillemenke et al., 2015 Montiel-Gonzalez et al., 2016). In complex with the appropriate single guide RNAs (sgRNAs), these proteins are capable of directing specific single nucleotide changes (C to T or A to G) in the genomes of bacteria, mammalian cells and mice ( Gaudelli et al., 2017 Gao et al., 2018 Kim et al., 2017 Komor et al., 2016). Nucleotide-specific editors have been reported recently that use cleavage inactive Cas mutants fused to either cytidine or adenosine deaminase domains ( Gaudelli et al., 2017 Komor et al., 2016). CRISPR-Cas-mediated selective cleavage of duplex DNA coupled with homology-directed repair with appropriately designed donor DNA fragments has become a popular approach for introducing specific sequence changes in genomes ( Nunez et al., 2016 Ran et al., 2013 Hsu et al., 2014). ![]() Finally, we demonstrate directed RNA editing in vitro and in human cells using mutant ADAR2 proteins and modified guide RNAs with reduced off target activity.Ī variety of systems have been developed for directing reactions that change nucleic acid sequence, either in DNA (genome editing) or in RNA (transcriptome editing) ( Montiel-Gonzalez et al., 2013 Montiel-Gonzalez et al., 2016 Nunez et al., 2016 Ran et al., 2013 Hsu et al., 2014 Cox et al., 2017 Gaudelli et al., 2017 Stafforst and Schneider, 2012). ![]() A crystal structure of the E488Y mutant bound to abasic site-containing RNA shows the accommodation of the tyrosine side chain. Replacing this nucleobase with a hydrogen atom removes the clash and restores editing activity. This residue is juxtaposed to the nucleobase that pairs with the editing site adenine, suggesting a steric clash for the bulky mutants. Mutations in human ADAR2 (adenosine deaminase acting on RNA 2) that introduce aromatic amino acids at position 488 reduce background RNA editing. Here we report selective combinations of mutant editing enzyme and directing oligonucleotide. However, unwanted reactions at off target sites complicate their use. Molecules capable of directing changes to nucleic acid sequences are powerful tools for molecular biology and promising candidates for the therapeutic correction of disease-causing mutations. ![]()
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