MACBETH: Multiplex automated Corynebacterium glutamicum base editing method.

CRISPR/Cas9 or Cpf1-introduced double strand break dramatically decreases bacterial cell survival rate, which hampers multiplex genome editing in bacteria. In addition, the requirement of a foreign DNA template for each target locus is labor demanding and may encounter more GMO related regulatory hurdle in industrial applications. Herein, we developed a multiplex automated Corynebacterium glutamicum base editing method (MACBETH) using CRISPR/Cas9 and activation-induced cytidine deaminase (AID), without foreign DNA templates, achieving single-, double-, and triple-locus editing with efficiencies up to 100%, 87.2% and 23.3%, respectively. In addition, MACBETH was applied to generate a combinatorial gene inactivation library for improving glutamate production, and pyk&ldhA double inactivation strain was found to improve glutamate production by 3-fold. Finally, MACBETH was automated with an integrated robotic system, which would enable us to generate thousands of rationally engineered strains per month for metabolic engineering of C. glutamicum. As a proof of concept demonstration, the automation platform was used to construct an arrayed genome-scale gene inactivation library of 94 transcription factors with 100% success rate. Therefore, MACBETH would be a powerful tool for multiplex and automated bacterial genome editing in future studies and industrial applications.