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This study details a reliable and efficient method for CRISPR–Cas9 genome engineering in the high amino acid-producing strain of Corynebacterium glutamicum, NRRL-B11474. Our investigation demonstrates that a plasmid-encoded single-guide RNA paired with different edit-encoding fragments is sufficient to generate edits without the addition of an exogenous recombinase. This approach leverages a genome-integrated copy of the cas9 gene for reduced toxicity, in combination with a single plasmid carrying the targeting guide RNA and matching edit fragment. Our study systematically investigated the impact of homology arm length on editing efficiency and demonstrates genome editing with homology arm lengths as small as 25 bp for single-nucleotide polymorphisms and 75 bp for 100 bp sequence swaps. These homology arm lengths are smaller than previously reported for other strains of C. glutamicum. Our study finds that C. glutamicum NRRL-B11474 is not amenable to efficient transformation with plasmids containing the BL1, NG2, or CC1 origins of replication. This finding differs from all previously reported approaches to plasmid-based CRISPR–Cas9 or Cpf1 editing in other strains of C. glutamicum. Two alternative origins of replication (CG1 and CASE1) can be used to successfully introduce genome edits; furthermore, our data demonstrate improved editing efficiency when guide RNAs and edit fragments are encoded on plasmids carrying the CASE1 origin of replication (compared to plasmids carrying CG1). In addition, this study demonstrates that efficient editing can be done using an integrated Cas9 without the need for a recombinase. We demonstrate that the specifics of CRISPR–Cas9 editing configurations may need to be tailored to enable different edit types in a particular strain background. Refining configuration parameters such as edit type, homology arm length, and plasmid origin of replication enables robust, flexible, and efficient CRISPR–Cas9 editing in differing genetic strain contexts.

Systematic investigation of CRISPR–Cas9 configurations for flexible and efficient genome editing in Corynebacterium glutamicum NRRL-B11474