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


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.

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This study was partly supported by the DARPA Living Foundries initiative. The authors would like to thank Dr. Brian Chaikind, Dr. Carrie Cizauskas, Michael Flashman, Dr. Mike Hamady, Dr. Sharon Hoover, Dr. Stefan de Kok, Michael Martyn III, Dr. Aaron Miller, and Dr. Solomon Stonebloom for their assistance with the project.

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Correspondence to Jed Dean.

Electronic supplementary material

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Supplementary material 1 Transformation efficiency for 100 bp sequence swaps (S1A) and SNPs (S1B) is higher for control plasmids and editing constructs transformed into the cas9-integrated strain of NRRL-B11474 C. glutamicum (blue points) as compared to the wild-type reference strain (black points). Data points are jittered. Transformation efficiency is broken down by target locus, plasmid origin of replication, and edit fragment homology arm length. The gRNA control plasmids contain a gRNA targeted to the respective test locus, but no edit fragment. Each point represents data from a unique transformation event (EPS 35 kb)

Supplementary material 2 (EPS 32 kb)


Supplementary material 3 Differential editing efficiency with CASE1 and CG1 plasmid origins of replication at various edit fragment homology arm lengths. We analyzed combined editing efficiency data to examine the effect of plasmid origin of replication on percent colonies edited. Data is broken out by 100 bp sequence swaps (top panel) and SNPs (bottom panel), by edit fragment homology arm length, and by plasmid origin of replication. Error bars represent 95% confidence intervals around the mean. At every homology arm length except 2000 bp for sequence swaps, the mean editing efficiency resulting from plasmids containing the CASE1 origin of replication was greater than or equal to the mean editing efficiency of CG1-containing plasmids. A two-tailed t test revealed significant differences in editing efficiency for sequence swaps at 500 bp homology arm lengths (CASE1: M = 91.8%, SD= 10.1%; CG1: M = 46.2%, SD= 18.8%; t(7.68) = − 5.24, p = 0.0004), and at 125 bp homology arm lengths (CASE1: M = 27.6%, SD= 22.6%; CG1: M = 0.0%, SD= 0.0%; t(4) = − 2.74, p = 0.0261) (EPS 11 kb)


Supplementary material 4 Coverage plots showing representative analysis of NGS-sequencing data for a successful 100 bp sequence swap edit at the cg3031 test locus (S3A), an unsuccessful SNP edit at the cg0167 test locus (S3B), and a partial SNP edit at the cg3404 test locus (S3C). In every case, the same library of sequencing reads is aligned to a WT reference sequence (bottom alignments) and an edited reference sequence (top alignments). The coverage plots displayed are truncated segments of the full alignments, zoomed-in on the region of interest. Figure S3A displays a successful edit in which the edit alignment shows no mismatches or gaps in coverage depth, while the WT alignment shows a gap in coverage, where the sample’s parental WT sequence has been replaced by the 100 bp edit sequence. S3B displays an unsuccessfully edited SNP, where the WT alignment shows no mismatches or gaps in coverage depth, and the edit alignment shows two mismatches, where the intended SNPs failed to be incorporated and WT sequence remains. Figure S3C displays a partial edit, where the edit alignment is correct at the site, where the first SNP was incorporated, but there is a mismatch at the site, where the second SNP failed to be incorporated, and the wild-type alignment inversely shows a mismatch at the site of the first successful SNP and a correct alignment at the site of the second unsuccessful SNP (EPS 62 kb)

Supplementary material 5 (EPS 53 kb)

Supplementary material 6 (EPS 53 kb)

Supplementary material 7 (XLSX 12 kb)

Supplementary material 8 (XLSX 12 kb)

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Cameron Coates, R., Blaskowski, S., Szyjka, S. et al. Systematic investigation of CRISPR–Cas9 configurations for flexible and efficient genome editing in Corynebacterium glutamicum NRRL-B11474. J Ind Microbiol Biotechnol 46, 187–201 (2019).

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  • CRISPR–Cas9
  • Corynebacterium glutamicum
  • Genetic engineering
  • Genome editing