Improved cytosine base editors generated from TadA variants

Abstract

Cytosine base editors (CBEs) enable programmable genomic C·G-to-T·A transition mutations and typically comprise a modified CRISPR–Cas enzyme, a naturally occurring cytidine deaminase, and an inhibitor of uracil repair. Previous studies have shown that CBEs utilizing naturally occurring cytidine deaminases may cause unguided, genome-wide cytosine deamination. While improved CBEs that decrease stochastic genome-wide off-targets have subsequently been reported, these editors can suffer from suboptimal on-target performance. Here, we report the generation and characterization of CBEs that use engineered variants of TadA (CBE-T) that enable high on-target C·G to T·A across a sequence-diverse set of genomic loci, demonstrate robust activity in primary cells and cause no detectable elevation in genome-wide mutation. Additionally, we report cytosine and adenine base editors (CABEs) catalyzing both A-to-I and C-to-U editing (CABE-Ts). Together with ABEs, CBE-Ts and CABE-Ts enable the programmable installation of all transition mutations using laboratory-evolved TadA variants with improved properties relative to previously reported CBEs.

In the business of base editors: Evolution from bench to bedside

“…To reduce DNA off-target editing events, researchers have mutated the rAPOBEC1 protein to reduce its catalytic activity, as well as identified APOBEC homologs that naturally have lower gRNA-independent off-target editing activities [44,45]. Additionally, researchers have leveraged the previously undesired cytidine deamination activity observed with ABEs to engineer TadA-derived CBEs that have no gRNA-independent off-target DNA editing activity, like their ABE counterparts [46][47][48].…”

Beyond knockouts: fine‐tuning regulation of gene expression in plants with CRISPR‐Cas‐based promoter editing

“…However, its inherent editing properties, including a preference for G‐C rich sequences, a high rate of 1 bp or small insertions and deletions (InDels), a risk of nonspecific editing due to 3 bp mismatches, and blunt end DNA cleavage, fall short of fulfilling the requirements of plant promoter editing. Although various Cas9 editing tools have been developed, such as eSpCas9 (Slaymaker et al ., 2016) and HypaCas9 (Chen et al ., 2017), which enhance specificity and reduce off‐target effects, and expanded PAM versions of SpCas9, like SpyCas9‐NG (Nishimasu et al ., 2018; Zhong et al ., 2019), xCas9 (Hu et al ., 2018; Zhong et al ., 2019), and SpRY (Walton et al ., 2020; Ren et al ., 2021b), and various base editors for precise base conversions (Ren et al ., 2021a; Wu et al ., 2022; Lam et al ., 2023), personalized editing tools, and strategies for plant promoter editing are still elusive in achieving effective ‘non‐silent’ editing events that achieve precise target gene expression regulation using Cas9 nuclease and its derivatives.…”

“…Moreover, it is theoretically possible to edit plant promoters using base editing for key nucleotide conversion (Fig. 3f; Lam et al ., 2023) and prime editing for reverse transcription template‐directed modification (Fig. 3g; Chen & Liu, 2023).…”

Recognizing CRISPR as the new age disease‐modifying drug: Strategies to bioengineer CRISPR/Cas for direct in vivo delivery

“…The system demonstrated a 30-folds higher enhanced HDR even at lower RNP delivered concentrations. 27 CRISPR base editing techniques utilize base editors such as TadA, ADAR, and APOBEC enzymes 28,29,30 capable of catalyzing the conversion of one nucleobase to another (point mutation) at specific loci of genes using the CRISPR's "guide" functionality. Base editors coupled with CRISPR's genome search capabilities created the means of forming precise base mutations (transversions and transitions) at any selected gene.…”