Journal: Microbial Cell Factories

Article Title: Development of a CRISPR/Cas9 genome editing toolbox for Corynebacterium glutamicum

doi: 10.1186/s12934-017-0815-5

Figure Lengend Snippet: Gene deletion using CRISPR/Cas9 and plasmid-borne editing template in C. glutamicum SL4. a Schematic representation of pCas9, pgRNA3, and gene deletion event. b Transformants harboring pCas9 and pgRNA3 on a SGY plate supplemented with Km, Cm, and IPTG (1 mM). pCas9 and pgRNA3 were co-transformed into strain SL4 simultaneously and cells were spread on SGY plates with Km, Cm, and IPTG (1 mM) immediately after recovery. Colonies marked in red cycles were the so-called abnormally large colonies. c Schematic representation of different patterns of pCas9 mutation. Gene deletion, cas9 gene was removed from pCas9. Nonsense mutation, a T465G mutation occurred, generating a stop codon (TAT to TAG) in cas9 gene. Insertional inactivation, a transposase encoding gene from E. coli was inserted into cas9 gene, which deactivated cas9 . d Colony counting and editing efficiency calculation of C. glutamicum SL4. Colonies on SGY plates supplemented with Km, Cm, and IPTG (1 mM) were counted and verified using colony PCR. e PCR verification of ldhA deletion in C. glutamicum SL4 using the primer pair ( ldhA -up and ldhA -down) shown in a . M, DNA marker; –, wild-type control; 1–8, eight small colonies. This displayed the result of Replicate 1 in c . The results of Replicate 2 and Replicate 3 were shown in Additional file : Figure S2

Article Snippet: After 2–3 days cultivation, colonies were picked for verification, revealing that ldhA deletion efficiencies reached 33.3 ± 2.5 and 60.0% in strains ATCC 13969 and ATCC 13032, respectively (Table ).

Techniques: CRISPR, Plasmid Preparation, Transformation Assay, Mutagenesis, Marker, Control

Journal: Microbial Cell Factories

Article Title: Development of a CRISPR/Cas9 genome editing toolbox for Corynebacterium glutamicum

doi: 10.1186/s12934-017-0815-5

Figure Lengend Snippet: Gene insertion using CRISPR/Cas9 and plasmid-borne editing template in C. glutamicum SL4. a Schematic representation of pCas9, pgRNA4, and gene insertion event. b Colony counting and editing efficiency calculation of C. glutamicum SL4. pCas9 and pgRNA4 were co-transformed into C. glutamicum SL4 simultaneously and cells were spread on SGY plates supplemented with Km, Cm, and IPTG (1 mM) immediately after recovery. Colonies on the selective plates were counted and verified using colony PCR. c PCR verification of rfp insertion in C. glutamicum SL4 using the primer pair ( rfp -up and ldhA -down) shown in a . M, DNA marker; –, wild-type control; 1–10, ten small colonies. This displayed the result of Replicate 1 in b . The results of Replicate 2 and Replicate 3 were shown in Additional file : Figure S4

Article Snippet: After 2–3 days cultivation, colonies were picked for verification, revealing that ldhA deletion efficiencies reached 33.3 ± 2.5 and 60.0% in strains ATCC 13969 and ATCC 13032, respectively (Table ).

Techniques: CRISPR, Plasmid Preparation, Transformation Assay, Marker, Control

Journal: Microbial Cell Factories

Article Title: Development of a CRISPR/Cas9 genome editing toolbox for Corynebacterium glutamicum

doi: 10.1186/s12934-017-0815-5

Figure Lengend Snippet: ssDNA-directed recombineering using CRISPR/Cas9 and RecT in C. glutamicum SL4Δ ldhA :: rfp . a Schematic representation of pCas9 and pgRNA5 which were used for ssDNA-directed recombineering. b Operation scheme of ssDNA-directed recombineering using CRISPR/Cas9 and RecT for rfp deactivation. A 90mer ssDNA targeted to the lagging strand ( rfp -off1, Additional file : Table S1) is designed to introduce two successive stop codons (highlighted in red) in rfp gene. PAM sequence of the gRNA is shaded grey. Plasmid pgRNA5 was first transformed into C. glutamicum SL4Δ ldhA :: rfp . The resultant strain SL4Δ ldhA :: rfp (pgRNA5) was cultivated in SGY medium supplemented with Km and sodium propionate to induce RecT expression. Electrocompetent cells were then prepared and transformed with pCas9 and ssDNA. After recovery, cells were spread on SGY plates supplemented with Km, Cm, and IPTG (1 mM) to induce Cas9 expression for counter-selection. Colonies were picked randomly and verified by measuring their fluorescence outputs. c Fluorescence output detection of candidate mutants of C. glutamicum SL4Δ ldhA :: rfp off1 . –, wild-type C. glutamicum SL4 control; +, C. glutamicum SL4Δ ldhA :: rfp ; 1–10, ten colonies. This displayed the result of Replicate 1. The results of Replicate 2 and Replicate 3 were shown in Additional file : Figure S6. d rfp gene sequencing of C. glutamicum SL4Δ ldhA :: rfp off1 mutants. Nucleotides in red box represents the two successive stop codons (TAG TAA) introduced by ssDNA-directed recombineering. e Str R phenotype test of C. glutamicum SL4 rpsL K43R mutants. Ten colonies were picked randomly from SGY plates supplemented with Km, Cm, and IPTG (1 mM) and patched onto SGY plates (Str − ) and SGY plates supplemented with streptomycin (Str + ). Seven out of ten colonies were Str R . f rpsL gene sequencing of C. glutamicum SL4 rpsL K43R mutants. Nucleotides in red box represents the mutated codon (AAG to CGC) introduced by ssDNA-directed recombineering

Article Snippet: After 2–3 days cultivation, colonies were picked for verification, revealing that ldhA deletion efficiencies reached 33.3 ± 2.5 and 60.0% in strains ATCC 13969 and ATCC 13032, respectively (Table ).

Techniques: CRISPR, Introduce, Sequencing, Plasmid Preparation, Transformation Assay, Expressing, Selection, Fluorescence, Control

Journal: Microbial Cell Factories

Article Title: Development of a CRISPR/Cas9 genome editing toolbox for Corynebacterium glutamicum

doi: 10.1186/s12934-017-0815-5

Figure Lengend Snippet: Strains and plasmids used in this study

Article Snippet: After 2–3 days cultivation, colonies were picked for verification, revealing that ldhA deletion efficiencies reached 33.3 ± 2.5 and 60.0% in strains ATCC 13969 and ATCC 13032, respectively (Table ).

Techniques: Plasmid Preparation, Cloning, Mutagenesis, Construct, Expressing