CRISPR-mediated mutagenesis of RNA editing cis-regulatory elements

We used CRISPR KI technology to mutagenize the cis-regulatory elements including the editing stem and the editing complementary sequence of three RNA editing substrates-NEIL1, TTYH2, AJUBA. We designed single nucleotide variants, double-nucleotide variants and other mutant isoforms to interrogate the primary sequence and the secondary structures around the endogenous RNA editing sites. Gene specific primers were used to make the NGS library for each locus. The unique mutation of each tested isoform can be used as barcode and RNA editing level can be measured for each isoform from the pooled library. We found that RNA sequence and structure features synergistically determine the editing levels. Several features, such as mutation number, free energy, and probability of active conformation, play an important role in determining editing efficiency. This study systematically investigated the contribution of cis-regulatory elements to ADAR1 RNA editing by combining CRISPR-based saturation mutagenesis and deep sequencing technology. Overall design: Examination of the RNA editing level of three endogenous substrates whose cis-elements have been subject to saturation mutagenesis.