introduction
As an emerging targeted mutagenesis technology, base editor (BE) has been widely used in microbial cell evolution, protein engineering, metabolic engineering, and other aspects of synthetic biology. However, the existing BE in microbial cells usually has a narrow editing window (5~7 nt), corresponding to a maximum of 2~3 amino acid substitutions, which limits the potential of BE in gene diversification.
2024年4月11日，江南大学周哲敏和韩来闯共同通讯在Advanced Science 发表题为“A New-Generation Base Editor with An Expanded Editing Window for Microbial Cell Evolution In Vivo Based on CRISPR‒Cas12b Engineering”的研究论文，该研究通过对CRISPR/Cas12b进行设计，创制出“新一代碱基编辑器”，实现微生物细胞（Bacillus subtilis和Escherichia coli）的高效且大范围的碱基编辑。
It is worth noting that the "new generation base editor" has an amazing editing window of 42~64 nt in microbial cells, which is the widest BE in the current microbial cells, and can replace more than 8 amino acids at most, providing an important genetic tool for the study of targeted mutations in microorganisms and other aspects of synthetic biology.
For microbial chassis evolution, mutations usually need to occur within a large editing window, and a large number of phenotypes can be generated by diversifying target genes through iterative genome editing, combined with effective high-throughput screening techniques, to accelerate the development of chassis cells. However, the existing BE in microbial cells usually has a narrow editing window (5~7 nt), corresponding to a maximum of 2~3 amino acid substitutions, which limits the potential of BE in gene diversification. Although the editing window of BE can be expanded to a certain extent through the elongation of sgRNA and the fusion of multiple types of deaminases (Zhemin Zhou, Hao et al. Nucleic Acids Res. 2021, 49(16): 9594-9605; Hao et al. Chem. Sci. 2022, 13: 14395-14409), but its editability at the target is still limited. In order to solve the above key scientific problems, Cas12b was selected as a candidate Cas protein for the construction of BE, because its size is smaller than that of Cas9 and Cas12a, so it can be linked to deaminase to avoid the limitation of steric hindrance caused by the excessive volume of Cas protein on the range of activity of deaminase. First of all, in B. Subtilis involves the creation of a CRISPR/Cas12b-based gene editing system. Next, based on sequence alignment, molecular dynamics simulation and molecular docking strategy, the key sites affecting the activity of Cas12b DNNase were discovered, and the inactivated version of dBhCas12b was constructed for the first time, and it was used for B. Gene expression regulation in subtilis (CRISPRi). In this study, four CBE systems were designed and constructed by combining engineered dBhCas12b with cytidine deaminase (PmCDA). The construction strategy of fusing CDA to the N-terminal of dBhCas12b and adding two additional copies of UGI (CDA-dBhCas12b-UGI-UGI) can be used in B. In addition, the modified adenosine deaminase ABE8e was fused to the N-terminus of dBhCas12b to construct the ABE system, and the editing experiments showed that the system could efficiently induce A-to-G mutations with an editing window of 14 nt. Further, this study also applied a new generation of base editors to B. In the large window (15 nt) diversification of RBS+Spacer in subtilis, the RS combinatorial mutation library was constructed to maximize the regulation of gene expression. In order to test the generalizability of the new generation of BE in other microbial cells, this study was also carried out in E. coli (JM109 and BL21) was used to construct a CBE system based on temperature-sensitive plasmids for validation. Both population and monoclonal sequencing results showed that the editing window of the new generation of CBE was as high as 41~63 nt, which even exceeded the length of the protospacer (23 nt). Whole genome sequencing (WGS) showed that dBhCas12b-CBE did not have detectable off-target effects, which proved that the next-generation BE system is a high-precision genome editor, and its editing window is 8~10 times that of the currently reported BE, which is the widest BE reported in the microbial system so far.
新一代BE在E. coli中设计与功能验证（Credit: Advanced Science）
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