A New‐Generation Base Editor with an Expanded Editing Window for Microbial Cell Evolution In Vivo Based on CRISPR‒Cas12b Engineering

Abstract

Abstract Base editors (BEs) are widely used as revolutionary genome manipulation tools for cell evolution. To screen the targeted individuals, it is often necessary to expand the editing window to ensure highly diverse variant library. However, current BEs suffer from a limited editing window of 5–6 bases, corresponding to only 2–3 amino acids. Here, by engineering the CRISPR‒Cas12b, the study develops dCas12b‐based CRISPRi system, which can efficiently repress gene expression by blocking the initiation and elongation of gene transcription. Further, based on dCas12b, a new‐generation of BEs with an expanded editing window is established, covering the entire protospacer or more. The expanded editing window results from the smaller steric hindrance compared with other Cas proteins. The universality of the new BE is successfully validated in Bacillus subtilis and Escherichia coli. As a proof of concept, a spectinomycin‐resistant E. coli strain (BL21) and a 6.49‐fold increased protein secretion efficiency in E. coli JM109 are successfully obtained by using the new BE. The study, by tremendously expanding the editing window of BEs, increased the capacity of the variant library exponentially, greatly increasing the screening efficiency for microbial cell evolution.

Keywords

• base editor
• CRISPR‒Cas12b
• editing window
• protein evolution
• synthetic biology