Effective gene editing by high-fidelity base editor 2 in mouse zygotes

Abstract

Targeted point mutagenesis through homologous recombination has been widely used in genetic studies and holds considerable promise for repairing disease-causing mutations in patients. However, problems such as mosaicism and low mutagenesis efficiency continue to pose challenges to clinical application of such approaches. Recently, a base editor (BE) system built on cytidine (C) deaminase and CRISPR/Cas9 technology was developed as an alternative method for targeted point mutagenesis in plant, yeast, and human cells. Base editors convert C in the deamination window to thymidine (T) efficiently, however, it remains unclear whether targeted base editing in mouse embryos is feasible. In this report, we generated a modified high-fidelity version of base editor 2 (HF2-BE2), and investigated its base editing efficacy in mouse embryos. We found that HF2-BE2 could convert C to T efficiently, with up to 100% biallelic mutation efficiency in mouse embryos. Unlike BE3, HF2-BE2 could convert C to T on both the target and non-target strand, expanding the editing scope of base editors. Surprisingly, we found HF2-BE2 could also deaminate C that was proximal to the gRNA-binding region. Taken together, our work demonstrates the feasibility of generating point mutations in mouse by base editing, and underscores the need to carefully optimize base editing systems in order to eliminate proximal-site deamination.Electronic supplementary materialThe online version of this article (doi:10.1007/s13238-017-0418-2) contains supplementary material, which is available to authorized users.

Modeling human point mutation diseases inXenopus tropicaliswith a modified CRISPR/Cas9 system

“…Recently, base editor 3 (BE3), a fusion of a clustered regularly interspaced short palindromic repeat-associated protein 9 (Cas9) nickase, a cytidine deaminase [rat apolipoprotein B mRNA editing enzyme catalytic subunit 1 (APOBEC1)], and the uracil DNA glycosylase inhibitor (UGI), has been developed as a base editor (BE3), which efficiently induces the C-G pair to T-A pair transition in living human cells without inducing double-stranded DNA breaks (17). Subsequent studies demonstrated the effectiveness of this base editing strategy in yeasts, plants, and various animal models (18)(19)(20)(21)(22)(23)(24)(25)(26)(27)(28). Its application in X. tropicalis remains to be tested.…”

The rapidly advancing Class 2 CRISPR‐Cas technologies: A customizable toolbox for molecular manipulations

“…Since the first demonstration by Komor et al, 103 base editing systems, primarily the thirdgeneration BE (BE3), have been applied in a wide range of cell types, including various cell types from human and mouse. 103,107 Successful base editing has also been achieved in living animals such as mice 108 and in zygotes to generate transgenic strains in mice 109,110 and zebrafish. 111 As most genetic diseases are caused by point mutations, BE- found to be promising for correction of more than 2,000 potential pathogenic point mutations.…”

Methods and Applications of CRISPR-Mediated Base Editing in Eukaryotic Genomes

“…There have also been successful demonstrations of base editing in vivo via delivery by adenoviral vector into mouse liver (Chadwick et al, 2017) and by RNP delivery into the mouse inner ear (Rees et al, 2017). Zebrafish (Rees et al, 2017; Zhang et al, 2017) and mouse embryos (Kim et al, 2017b; Liang et al, 2017) have also been successfully base edited, with the latter being reintroduced into surrogate mothers and giving rise to properly-edited offspring.…”