Inhibition of base editors with anti-deaminases derived from viruses

Abstract

Cytosine base editors (CBEs), combining cytidine deaminases with the Cas9 nickase (nCas9), enable targeted C-to-T conversions in genomic DNA and are powerful genome-editing tools used in biotechnology and medicine. However, the overexpression of cytidine deaminases in vivo leads to unexpected potential safety risks, such as Cas9-independent off-target effects. This risk makes the development of deaminase off switches for modulating CBE activity an urgent need. Here, we report the repurpose of four virus-derived anti-deaminases (Ades) that efficiently inhibit APOBEC3 deaminase-CBEs. We demonstrate that they antagonize CBEs by inhibiting the APOBEC3 catalytic domain, relocating the deaminases to the extranuclear region or degrading the whole CBE complex. By rationally engineering the deaminase domain, other frequently used base editors, such as CGBE, A&CBE, A&CGBE, rA1-CBE and ABE8e, can be moderately inhibited by Ades, expanding the scope of their applications. As a proof of concept, the Ades in this study dramatically decrease both Cas9-dependent and Cas9-independent off-target effects of CBEs better than traditional anti-CRISPRs (Acrs). Finally, we report the creation of a cell type-specific CBE-ON switch based on a microRNA-responsive Ade vector, showing its practicality. In summary, these natural deaminase-specific Ades are tools that can be used to regulate the genome-engineering functions of BEs.

Unveiling neural network potential in forecasting CRISPR effects and off-target prophecies for gene editing

“… Base Editing: Demonstrates excellent predictive performance, achieving an AUROC of 0.991 and a notable AUPRC of 0.571. The high AUROC indicates effective discrimination between true positives and false positives, while the substantial AUPRC underscores the precision of the predictions [28].  Prime Editing: Displays strong predictive power with an AUROC of 0.983 and a respectable AUPRC of 0.392.…”

Genome-edited rabbits: Unleashing the potential of a promising experimental animal model across diverse diseases

“…Strategies such as protein engineering have also been employed to introduce mutations or deletions in the protein domains of the editing tools, thereby enhancing their safety and efficacy ( Doman et al, 2020 ; Qian et al, 2023 ; Zhao et al, 2023 ; Zuo et al, 2019 ). Notably, Liu et al ( 2022 ) developed the virus-derived anti-deaminase (Ade) system and demonstrated its capacity to inhibit both Cas9- and deaminase-dependent off-target effects. Moreover, low efficiency in the context of large gene insertions is a significant barrier to the application of CRISPR.…”

Gene editing innovations and their applications in cardiomyopathy research

“…Similarly to Acrs, Ades are deaminases produced by viruses to defend themselves against the antiviral activity of the bacterial APOBEC3 ( Uriu et al, 2021 ). Of note, a recent study described four virus-derived deaminases as being more efficient CBE-inactivating tools than Acrs ( Liu et al, 2022b ). Acrs or Ades hold promise for future therapeutic applications, as they offer the possibility to control the timing and duration of CRISPR activity, thus enhancing safety of the gene editing therapeutic approaches.…”