Expanding C\u2013T base editing toolkit with diversified cytidine deaminases

Tian-Lin Cheng,Zilong Qiu,Wenhao Zhou,Shuo Li,Xiaolin Wang,Bo Yuan

doi:10.1038/s41467-019-11562-6

Abstract

Base editing tools for cytosine to thymine (C–T) conversion enable genome manipulation at single base-pair resolution with high efficiency. Available base editors (BEs) for C–T conversion (CBEs) have restricted editing scopes and nonnegligible off-target effects, which limit their applications. Here, by screening diversified lamprey cytidine deaminases, we establish various CBEs with expanded and diversified editing scopes, which could be further refined by various fusing strategies, fusing at either N-terminus or C–terminus of nCas9. Furthermore, off-target analysis reveals that several CBEs display improved fidelity. Our study expands the toolkits for C–T conversion, serves as guidance for appropriate choice and offers a framework for benchmarking future improvement of base editing tools.

Highlights

Base editing tools for cytosine to thymine (C–T) conversion enable genome manipulation at single base-pair resolution with high efficiency
clustered regularly interspaced short palindromic repeat (CRISPR)-associated (Cas) nucleases are recruited by a single guide RNA onto specific genome target(s) for DNA cleavage, and induced double-stranded DNA breaks (DSBs) are repaired mainly through non-homologous end-joining (NHEJ) pathway, which often results in insertions and deletions, leading to gene inactivation[2,3]
We selected rAPOBEC1, human activationinduced deaminase (hAID), Petromyzon marinus cytidine deaminase 1 (PmCDA1) and 13 divergent lamprey CDAs and CDAL deaminases to evaluate their applicability for CBEs24, and their sequence divergence was analyzed by sequence alignment (Supplementary Fig. 1)

Summary

Introduction

Base editing tools for cytosine to thymine (C–T) conversion enable genome manipulation at single base-pair resolution with high efficiency. Quantification analysis revealed that 10/13 NT-CBEs containing CDA/CDALs displayed ≥40% C–T conversion at specific C site for ≥2/4 sgRNAs (-A/B/C/D), and their highest editing efficiencies were comparable to NT-CBEs containing rAPOBEC1, hAID and PmCDA1 (Fig. 2b).

Results

Conclusion