Systematic identification of CRISPR off-target effects by CROss-seq

DearEditor,The CRISPR-mediated genome editing tools,including nucleases,base editors(ABE/CBE),transposases/recombinases,and prime editor(PE),have been extensively applied in basic and clinical researches,although the off-target effect remains a major concern(Anzalone et al.,2020).Recently,various methods have been developed to assess the specificity and accuracy of different tools(Zhang et al.,2021),yet each method is designed for limited editing systems,and none of them can simultaneously detect off-target sites in vivo and in vitro.A versatile method for profiling genome-wide off-target effects of various tools remains lacking.

Cost-effective generation of A-to-G mutant mice by zygote electroporation of adenine base editor ribonucleoproteins

More than 32,000 pathogenic single nucleotide polymorphisms(SNPs)have been identified in the human genome(Gaudelli et al.,2017).Genetically modified mice with pathogenic SNPs are good models for studies of disease pathogenesis and the development of new therapeutics.Accordingly,an efficient,high-throughput method for the generation of mouse models with SNPs is needed.

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.