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

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 applicaUon 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.

Effective and precise adenine base editing n mouse zygotes

Dear Editor, Many human genetic diseases are caused by pathogenic single nucleotide mutations. Animal models are often used to study these diseases where the pathogenic point mutations are created and/or corrected through gene editing （e.g., the CRISPP-JCas9 system） （Komor et al., 2017; Liang et al., 2017）. CRISPR/Cas9-mediated gene editing depends on DNA double-strand breaks （DSBs）, which can be of low efficiency and lead to indels and off-target cleavage （Kim et al., 2016）. We and others have shown that base editors （BEs） may represent an attractive alternative for disease mouse model generation （Liang et al., 2017; Kim et al., 2017）. Compared to CRISPR/ Cas9, cytidine base editors （CBEs） can generate C·G to T·A mutations in mouse zygotes without activating DSB repair pathways （Liang et al., 2017; Kim et al., 2017; Komor et al., 2016）. In addition, CBEs showed much lower off-targets than CRISPR]Cas9 （Kim et al., 2017）, making the editing process potentially safer and more controllable. Recently, adenine base editors （ABEs） that were developed from the tRNA- specific adenosine deaminase （TADA） of Escherichia coli were also reported （Gaudelli et al., 2017）. As a RNA-guided programmable adenine deaminase, ABE can catalyze the conversion of A to I. Following DNA replication, base I is replaced by G, resulting in A·T to G·C conversion （Gaudelli et al., 2017; Hu et al., 2018）. The development of ABEs has clearly expanded the editing capacity and application of BEs. Here, we tested whether ABEs could effectively generate disease mouse models, and found high efficiency by ABEs in producing edited mouse zygotes and mice with single-nucleotide substitutions.