CRISPR/Cas12a mediated genome editing to introduce amino acid substitutions into the mechanosensitive channel MscCG of Corynebacterium glutamicum

Against the background of a growing demand for the implementation of environmentally friendly production processes, microorganisms are engineered for the large-scale biosynthesis of chemicals, fuels or food and feed additives from sustainable resources. Since strain development is expensive and time-consuming, continuous improvement of molecular tools for the genetic modification of the microbial production hosts is absolutely vital. Recently, the CRISPR/Cas12a technology for engineering of Corynebacterium glutamicum as important platform organism for industrial amino acid production has been introduced. Here, this system was advanced by designing an easy-to-construct crRNA delivery vector using simple oligonucleotides. In combination with a C. glutamicum strain engineered for the chromosomal expression of the β-galactosidase-encoding lacZ gene, this new plasmid was used to investigate CRISPR/Cas12a targeting and editing at various positions relative to the PAM site.Finally, we used this system to perform...