Combined Theoretical, Bioinformatic, and Biochemical Analyses of RNA Editing by Adenine Base Editors

Rallapalli, Kartik L.; Ranzau, Brodie L.; Ganapathy, Kaushik R.; Paesani, Francesco; Komor, Alexis C.

doi:10.1089/crispr.2021.0131

Cited by 5 publications

(17 citation statements)

References 74 publications

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“…In fact, at all six mRNA sites that were analyzed, ABE0.1 displayed equivalent or higher editing than ABE7.10. [12] These observations suggested to us that perhaps ABE0.1 could also deaminate adenosines in DNA that are embedded in this motif. We found that A • T to G • C DNA editing activity by ABE0.1 was previously evaluated at only non-TACG motifs (Figure 2A).…”

Section: Abe01 Can Edit Dna At Tacg Motifsmentioning

confidence: 86%

“…As mentioned previously, we have shown that wtTadA can deaminate UACG motifs in mRNA, independent of Cas9 targeting. [12] Therefore, to avoid EGFP fluorescence due to editing of the EGFP mRNA, we incorporated the target adenosine into the non-coding strand of the reporter. With this in mind, we developed two reporters in which a G • C to A • T mutation within a TGCG motif resulted in a non-functional EGFP protein.…”

Section: A Gfp Reporter Assay Enhances Editing Detectionmentioning

confidence: 99%

“…In our previous simulation studies of ABEs, we used minimalistic models (specifically, we simulated only the TadA variant bound to either its substrate ssDNA or tRNA). [11,12] However, in this current study, we were able to expand upon these minimal models by using the recently published cryo-EM structure of the ABE8e R-loop complex (PDB ID: 6VPC). [21] We will note that in this structure, ABE8e consists of two TadA subunits, only one of which is complexed with the DNA substrate 5'-GTTCCACTTT-3'.…”

Section: Computational Simulations Reveal the Molecular Basis Of Edit...mentioning

confidence: 99%

“…[12,[15][16][17] We subsequently observed that the wtTadA enzyme also efficiently deaminates adenosines in these motifs transcriptome-wide, which was quite surprising given wtTadA was previously thought to discriminately target the tRNA Arg ACG from other RNAs in the cell. [12] While initial experiments concluded that ABE0.1 was incapable of editing DNA, none of the tested protospacers contained a TACG target (Figure 2A). [2] Given wtTadA's natural affinity for the UACG sequence, even outside of its tRNA anticodon loop structure, we sought to evaluate if ABE0.1 could edit DNA in these sequence contexts.…”

Section: Introductionmentioning

confidence: 98%

“…To better inform the creation of new base editors, we have sought to better understand the evolution of ABEs, particularly the first-round mutation, D108N (Figure 1C-D), which is sufficient and imperative for imparting TadA with detectable DNA editing activity. [11,12] In nature, tRNA editing enzymes have evolved to recognize specific RNA structures or sequences. [13] As a result, they modify only a specific nucleotide on one or multiple tRNAs within the cell.…”

Section: Introductionmentioning

confidence: 99%

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The Wild‐Type tRNA Adenosine Deaminase Enzyme TadA Is Capable of Sequence‐Specific DNA Base Editing

et al. 2023

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Base editors are genome editing tools that enable site‐specific base conversions through the chemical modification of nucleobases in DNA. Adenine base editors (ABEs) convert A ⋅ T to G ⋅ C base pairs in DNA by using an adenosine deaminase enzyme to modify target adenosines to inosine intermediates. Due to the lack of a naturally occurring adenosine deaminase that can modify DNA, ABEs were evolved from a tRNA‐deaminating enzyme, TadA. Previous experiments with an ABE comprising a wild‐type (wt) TadA showed no detectable activity on DNA, and directed evolution was therefore required to enable this enzyme to accept DNA as a substrate. Here we show that wtTadA can perform base editing in DNA in both bacterial and mammalian cells, with a strict sequence motif requirement of TAC. We leveraged this discovery to optimize a reporter assay to detect base editing levels as low as 0.01 %. Finally, we used this assay along with molecular dynamics simulations of full ABE:DNA complexes to better understand how the sequence recognition of mutant TadA variants change as they accumulate mutations to better edit DNA substrates.

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Section: Abe01 Can Edit Dna At Tacg Motifsmentioning

confidence: 86%

Section: A Gfp Reporter Assay Enhances Editing Detectionmentioning

confidence: 99%

Section: Computational Simulations Reveal the Molecular Basis Of Edit...mentioning

confidence: 99%

Section: Introductionmentioning

confidence: 98%

Section: Introductionmentioning

confidence: 99%

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The Wild‐Type tRNA Adenosine Deaminase Enzyme TadA Is Capable of Sequence‐Specific DNA Base Editing

et al. 2023

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Development of multiplexed orthogonal base editor (MOBE) systems

Cowan,

Gu,

et al. 2024

Nat Biotechnol

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Unlocking the secrets of ABEs: the molecular mechanism behind their specificity

Chen

McAndrew

Lapinaitė

2023

Biochemical Society Transactions

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CRISPR–Cas, the bacterial immune systems, have transformed the field of genome editing by providing efficient, easily programmable, and accessible tools for targeted genome editing. DNA base editors (BE) are state-of-the-art CRISPR-based technology, allowing for targeted modifications of individual nucleobases within the genome. Among the BEs, adenine base editors (ABEs) have shown great potential due to their ability to convert A-to-G with high efficiency. However, current ABEs have limitations in terms of their specificity and targeting range. In this review, we provide an overview of the molecular mechanism of ABEs, with a focus on the mechanism of deoxyadenosine deamination by evolved tRNA-specific adenosine deaminase (TadA). We discuss how mutations and adjustments introduced via both directed evolution as well as rational design have improved ABE efficiency and specificity. This review offers insights into the molecular mechanism of ABEs, providing a roadmap for future developments in the precision genome editing field.

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Combined Theoretical, Bioinformatic, and Biochemical Analyses of RNA Editing by Adenine Base Editors

Cited by 5 publications

References 74 publications

The Wild‐Type tRNA Adenosine Deaminase Enzyme TadA Is Capable of Sequence‐Specific DNA Base Editing

The Wild‐Type tRNA Adenosine Deaminase Enzyme TadA Is Capable of Sequence‐Specific DNA Base Editing

Development of multiplexed orthogonal base editor (MOBE) systems

Unlocking the secrets of ABEs: the molecular mechanism behind their specificity

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