MACBETH, a multi-automated genome editing method, has been developed in the microbial glutamate bacillus.

The CRISPR/Cas9 system greatly enriches the genome editing method of pronuclear organisms.
Due to the efficient lethal screening capacity of CRISPR/Cas9 system and the low homologous recombination efficiency of pronuclear organisms, multi-target and automated genome editing is still difficult to achieve, which severely limits the genetic modification efficiency of strains.
, Zheng Ping, a researcher at the Tianjin Institute of Industrial Biotechnology of the Chinese Academy of Sciences, led a team of systems and synthetic biotechnology, researcher Sun Yibin, and researcher Wang Ma led a team of high-throughput new molecular biosynthesis team that developed multiple automated genome editing methods MACBETH (Multiplex Automated Coryne Corebase Editing Method) in the major industrial platforms of microbial glutamate barbacify.
MACBETH genome editing diagram.
a, different Cas9-AID-gRNA complex and target gene action relationship diagram; b, different Cas9-AID-gRNA complex to target gene editing efficiency; c, upp gene edited genotype analysis; d, rfp gene edited genotype analysis; e, rfp gene edited phetype analysis.
the method combined with the positioning function of crispR/Cas9 system and the base editing function of cytosine deaminase (AID), the editing efficiency from C to T can be achieved at the chromosome target site, with editing efficiency of up to 90%. MACBETH can simultaneously generate an early termination cryptin in multiple genes to inactivate the target gene.
the automation platform of Tianjin Engineering Institute enables full-process automation from plasmid construction, genome editing, acquisition of correct mutant strains and phenotype verification, with editing capacity of thousands of mutants per month.
as an example, MACBETH is used to build a single inactivated strain bank of 94 regulatory factors at once, with a success rate of 100%. Because no additional DNA templates are needed, the method can reduce the difficulty and cost of genome editing, and can quickly build a genome-wide monogene inactive strain bank without affecting the genome structure, promising to accelerate the basic and application studies of glutamate bacillus, and provide technical support for the transformation of glutamate sticks into a universal microbial chassis.
, the method also provides a reference for multi-target and automated genome editing in other pronuclear organisms.
the research results were published in the publishing of The Publishing of The Book of Financial, with Assistant Researcher Wang Wei and Research Intern Liu Ye as co-authors of the paper.
the research was supported by the National Natural Science Foundation of China, the Key Research Project of Frontier Science of the Chinese Academy of Sciences, the Key Deployment Project of the Chinese Academy of Sciences, the "100-Person Plan" of the Chinese Academy of Sciences and the Tianjin Special Branch Project.
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