Harnessing self-labeling enzymes for selective and concurrent A-to-I and C-to-U RNA base editing

Abstract

The SNAP-ADAR tool enables precise and efficient A-to-I RNA editing in a guideRNA-dependent manner by applying the self-labeling SNAP-tag enzyme to generate RNA-guided editases in cell culture. Here, we extend this platform by combining the SNAP-tagged tool with further effectors steered by the orthogonal HALO-tag. Due to their small size (ca. 2 kb), both effectors are readily integrated into one genomic locus. We demonstrate selective and concurrent recruitment of ADAR1 and ADAR2 deaminase activity for optimal editing with extended substrate scope and moderate global off-target effects. Furthermore, we combine the recruitment of ADAR1 and APOBEC1 deaminase activity to achieve selective and concurrent A-to-I and C-to-U RNA base editing of endogenous transcripts inside living cells, again with moderate global off-target effects. The platform should be readily transferable to further epitranscriptomic writers and erasers to manipulate epitranscriptomic marks in a programmable way with high molecular precision.

Functions and consequences of AID/APOBEC-mediated DNA and RNA deamination

“…Neither of these two methods was checked for off-target DNA editing, but the mAPOBEC1-SNAP system demonstrated that integration of an inducible editing enzyme reduces global off-target RNA editing, as had previously been shown for ADAR RNA base-editing technologies 149 , 150 . This method was not benchmarked against CURE, making a direct comparison difficult, but it is important to note that the reported RNA off-target activity of CURE (measured as a simple sum of sites and noting that CURE enzymes are overexpressed) is much lower than that of mAPOBEC1-SNAP (refs 147 , 149 ). Despite these recent developments, APOBEC1-based RNA-directed CBE systems still suffer from moderate levels of global off-target RNA editing and, owing to the inherent dinucleotide preference of A3A, CURE can only edit Cs present in a 5′-UC-3′ motif (Fig.…”

“…Importantly, no off-target DNA editing was detected using CURE, although a few hundred off-target RNA edits were found 147 . Two other recently reported RNA-directed CBE approaches used mAPOBEC1 or human APOBEC1 in combination with either SNAP-tagged or MS2-tagged gRNAs to target specific target mRNAs 148 , 149 (Fig. 5d , e ).…”

Precise and efficient C-to-U RNA Base Editing with SNAP-CDAR-S

“…Our initial guide RNA design was inspired from our experience with the SNAP-ADAR tool and was tested for the editing of a 5´-ACA codon in a co-transfected eGFP reporter transcript. Initial guide RNAs were 22 nt long, chemically modified by 2´-O-methylation outside the target base triplet, 26 which is the targeted cytidine plus its two closest neighboring bases, and carried a (snap) 2 self-labeling moiety 20 at the 5´-end for the recruitment of two SNAP-CDAR-S effectors per guide RNA. The exact composition, sequence and chemistry, of all guide RNAs can be found in the Supplementary Information (Supplementary Table T1a).…”

“…Furthermore, we have recently shown the concurrent and fully orthogonal usage of two independent RNA editing effectors by complementing a SNAP-tagged C-to-U editing effector with a HALO-tagged A-to-I editing tool within the same cell. 20 In the latter study, we exploited the C-to-U deaminase domain from murine APO1. Other labs have developed C-to-U RNA base editing effectors based on human APO1 or APO3A.…”

Site-directed RNA editing: recent advances and open challenges

“…The SNAP-tag was also fused to the catalytic C-terminus of murine APOBEC1 thereby allowing C-to-U editing in an RNA-guided manner. Applying the SNAP-APOBEC1 fusion, site-specific C-to-U editing with an efficiency of 20% was detected in a GAPDH transcript with no detectable A-to-I RNA editing [ 41 ].…”