Protocol for examining and eliminating base editor-induced genome-wide and transcriptome-wide off-target mutations

Fusion of CRISPR-Cas9 with cytidine deaminases leads to base editors (BEs) for programmable C-to-T editing, which holds potentials in clinical applications but suffers from off-target (OT) mutations. Here, we applied a cleavable deoxycytidine deaminase inhibitor (dCDI) domain to construct a transformer BE (tBE) system that induces efficient editing with only background levels of genome-wide and transcriptome-wide OT mutations. This step-by-step protocol describes the plasmid construction of tBE system, determination of genome/transcriptome-wide OT mutations and tBE-mediated base editing in vivo.

A Broadly Conserved Deoxycytidine Deaminase Protects Bacteria from Phage Infection

SUMMARYThe El Tor biotype of Vibrio cholerae is responsible for perpetuating the longest cholera pandemic in recorded history (1961-current). The genomic islands VSP-1 and -2 are two understudied genetic features that distinguish El Tor from previous pandemics. To understand their utility, we calculated the co-occurrence of VSP genes across bacterial genomes. This analysis predicted the previously uncharacterized vc0175, herein renamed deoxycytidylate deaminase Vibrio (dcdV), is in a gene network with dncV, a cyclic GMP-AMP synthase involved in phage defense. DcdV consists of two domains, a P-loop kinase and a deoxycytidylate deaminase, that are required for the deamination of dCTP and dCMP, inhibiting phage predation by corrupting cellular nucleotide concentrations. Additionally, DcdV is post-translationally inhibited by a unique noncoding RNA encoded 5’ of the dcdV locus. DcdV homologs are conserved in bacteria and eukaryotes and our results identify V. cholerae DcdV as the founding member of a previously undescribed bacterial phage defense system.

HIV restriction factor APOBEC3G binds in multiple steps and conformations to search and deaminate single-stranded DNA

APOBEC3G (A3G), an enzyme expressed in primates with the potential to inhibit human immunodeficiency virus type 1 (HIV-1) infectivity, is a single-stranded DNA (ssDNA) deoxycytidine deaminase with two domains, a catalytically active, weakly ssDNA binding C-terminal domain (CTD) and a catalytically inactive, strongly ssDNA binding N-terminal domain (NTD). Using optical tweezers, we measure A3G binding a single, long ssDNA substrate under various applied forces to characterize the binding interaction. A3G binds ssDNA in multiple steps and in two distinct conformations, distinguished by degree of ssDNA contraction. A3G stabilizes formation of ssDNA loops, an ability inhibited by A3G oligomerization. Our data suggests A3G securely binds ssDNA through the NTD, while the CTD samples and potentially deaminates the substrate. Oligomerization of A3G stabilizes ssDNA binding but inhibits the CTD’s search function. These processes explain A3G’s ability to efficiently deaminate numerous sites across a 10,000 base viral genome during the reverse transcription process.