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.
Highlights
Cytosine base editors (CBEs), combining cytidine deaminases with the Cas[9] nickase, enable targeted C-to-T conversions in genomic DNA and are powerful genomeediting tools used in biotechnology and medicine
We first selected seven Ades, including EBV-BORF217,18, KSHV-ORF6117,19, HIV-1-Vif[20], SIVmac239-Vif[21,22], HSV-1-ICP619, EV71-2C23 and HBV-HBx24, which originate from different viruses and have been reported to antagonize APOBEC3 deaminases in vitro (Table 1)
We found that Ades were safer off switches for CBEs than conventional Acrs because they directly inhibit the activities of cytidine deaminase, the effector domain in CBEs
Summary
Cytosine base editors (CBEs), combining cytidine deaminases with the Cas[9] nickase (nCas9), enable targeted C-to-T conversions in genomic DNA and are powerful genomeediting tools used in biotechnology and medicine. We report the creation of a cell type-specific CBE-ON switch based on a microRNA-responsive Ade vector, showing its practicality These natural deaminase-specific Ades are tools that can be used to regulate the genome-engineering functions of BEs. CRISPR-guided DNA base editors, which precisely install targeted point mutations without requiring DNA double strand breaks (DSBs) or donor templates, have exhibited a powerful genome manipulation capability in various organisms[1,2]. Ade[1] was used to generate a cell type-specific CBE-ON switch based on a microRNAresponsive Ade vector These Ades, together with existing inhibitors, strengthen the inhibitor toolbox for efficient regulation of BE activity in gene modification and therapeutic applications
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