Global quantification exposes abundant low-level off-target activity by base editors

Buchumenski, Ilana; Roth, Shalom Hillel; Kopel, Eli; Katsman, Efrat; Feiglin, Ariel; Levanon, Erez Y.; Eisenberg, Eli

doi:10.1101/gr.275770.121

Cited by 16 publications

(18 citation statements)

References 52 publications

(89 reference statements)

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“…4b). As seen for ADAR-based effectors before, 10,12,32 A-to-I off-target editing was a combination of enhanced editing at known sites and editing at novel sites, whereas the large majority of C-to-U off-target editing were novel sites. We further analyzed the outcomes of the editing reactions and found that only a moderate number of all editing events resulted in missense mutations (Fig.…”

Section: Both Tools Show Moderate Global A-to-i and C-to-u Off-target...mentioning

confidence: 83%

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

Latifi

Mack

Tellioglu

et al. 2023

Preprint

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Site-directed RNA base editing enables the transient and dosable change of genetic information and represents a recent strategy to manipulate cellular processes, paving ways to novel therapeutic modalities. While tools to introduce adenosine-to-inosine changes have been explored quite intensively, the engineering of precise and programmable tools for cytidine-to-uridine editing is somewhat lacking behind. Here we demonstrate that the cytidine deaminase domain evolved from the ADAR2 adenosine deaminase, taken from the RESCUE-S tool, provides very efficient and highly programmable editing when changing the RNA targeting mechanism from Cas13-based to SNAP-tag-based. Optimization of the guide RNA chemistry further allowed to dramatically improve editing yields in the difficult-to-edit 5prime-CCN sequence context thus improving the substrate scope of the tool. Regarding editing efficiency, SNAP-CDAR-S outcompeted the RESCUE-S tool clearly on all tested targets, and was highly superior in perturbing the beta-catenin pathway. NGS analysis showed similar, moderate global off-target A-to-I and C-to-U editing for both tools.

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Section: Both Tools Show Moderate Global A-to-i and C-to-u Off-target...mentioning

confidence: 83%

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

Latifi

Mack

Tellioglu

et al. 2023

Preprint

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“…Subsequently, other linkages have been used, such as the MS2 system (Azad et al 2017;Montiel-Gonzalez et al 2018). A common problem with delivering the naked DD with a gRNA is that it tends to create many off-target edits, not only within the targeted message but also in unrelated messages across the transcriptome (Vallecillo-Viejo et al 2018;Buchumenski et al 2021). Approaches such as localizing the DD to the nucleus, or splitting it into two units that only form a functional deaminase enzyme when they come together, have significantly reduced unwanted edits (Vallecillo-Viejo et al 2018;Katrekar et al 2022a).…”

Section: Site-directed Rna Editing Systemsmentioning

confidence: 99%

“…For example, editing by the N peptide-BoxB system was shown to persist for over a month when delivered by AAV to mice (Sinnamon et al 2022(Sinnamon et al , 2020. A significant drawback to the engineered systems, however, is their propensity to generate off-target edits, most likely due to the delivery of additional catalytic activity uncoupled to ADAR's natural targeting architecture (Buchumenski et al 2021;Vallecillo-Viejo et al 2018). A good compromise for precise, longer duration editing may be to deliver gRNAs for endogenous recruitment via AAV.…”

Section: Site-directed Rna Editing Systemsmentioning

confidence: 99%

Site-directed A → I RNA editing as a therapeutic tool: moving beyond genetic mutations

Quiroz

Siskel

Rosenthal

2023

RNA

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Adenosine deamination by the ADAR family of enzymes is a natural process that edits genetic information as it passes through messenger RNA. Adenosine is converted to inosine in mRNAs, and this base is interpreted as guanosine during translation. Realizing the potential of this activity for therapeutics, a number of researchers have developed systems that redirect ADAR activity to new targets, ones that aren’t normally edited. These site-directed RNA editing (SDRE) systems can be broadly classified into two categories: ones that deliver an antisense RNA oligonucleotide to bind opposite a target adenosine, creating an editable structure that endogenously expressed ADARs recognize, and ones that tether the catalytic domain of recombinant ADAR to an antisense RNA oligonucleotide that serves as a targeting mechanism, much like with CRISPR-Cas or RNAi. To date, SDRE has been used mostly to try and correct genetic mutations. Here we argue that these applications are not ideal SDRE, mostly because RNA edits are transient and genetic mutations are not. Instead, we suggest that SDRE could be used to tune cell physiology to achieve temporary outcomes that are therapeutically advantageous, particularly in the nervous system. These include manipulating excitability in nociceptive neural circuits, abolishing specific phosphorylation events to reduce protein aggregation related to neurodegeneration or reduce the glial scarring that inhibits nerve regeneration, or enhancing G protein-coupled receptor signaling to increase nerve proliferation for the treatment of sensory disorders like blindness and deafness.

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“…However, other strategies use RNA oligos supplemented by engineered ADAR-like proteins [27][28][29][30]. A major challenge in this field is achieving high on-target editing efficiency [31]. It is therefore of much interest to identify highly potent natural ADARs that could be used to induce high-level editing.…”

Section: Introductionmentioning

confidence: 99%

Identification of exceptionally potent adenosine deaminases RNA editors from high body temperature organisms

et al. 2023

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The most abundant form of RNA editing in metazoa is the deamination of adenosines into inosines (A-to-I), catalyzed by ADAR enzymes. Inosines are read as guanosines by the translation machinery, and thus A-to-I may lead to protein recoding. The ability of ADARs to recode at the mRNA level makes them attractive therapeutic tools. Several approaches for Site-Directed RNA Editing (SDRE) are currently under development. A major challenge in this field is achieving high on-target editing efficiency, and thus it is of much interest to identify highly potent ADARs. To address this, we used the baker yeast Saccharomyces cerevisiae as an editing-naïve system. We exogenously expressed a range of heterologous ADARs and identified the hummingbird and primarily mallard-duck ADARs, which evolved at 40–42°C, as two exceptionally potent editors. ADARs bind to double-stranded RNA structures (dsRNAs), which in turn are temperature sensitive. Our results indicate that species evolved to live with higher core body temperatures have developed ADAR enzymes that target weaker dsRNA structures and would therefore be more effective than other ADARs. Further studies may use this approach to isolate additional ADARs with an editing profile of choice to meet specific requirements, thus broadening the applicability of SDRE.

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Global quantification exposes abundant low-level off-target activity by base editors

Cited by 16 publications

References 52 publications

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

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

Site-directed A → I RNA editing as a therapeutic tool: moving beyond genetic mutations

Identification of exceptionally potent adenosine deaminases RNA editors from high body temperature organisms

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