dead cas9 dcas9  (Twist Bioscience)

Journal: Microbial Biotechnology

Article Title: A CRISPRi Gene Regulation System for Bifidobacteria

doi: 10.1111/1751-7915.70260

Figure Lengend Snippet: In vivo validation of functional CRISPR interference in bifidobacteria. (A) Schematic of B. breve UCC2003 strains with integrated CRISPRi and nanoluciferase expression systems. (B) The nanoluciferase reporter is expressed in nanoluciferase containing strains (dCas9‐nLuc) resulting in detectable luminescence as measured by luciferase assay. N = 2, * p < 0.001, two‐way ANOVA. (C) Genetic circuit containing a choline inducible dCas9 (P Bet ) directed to target nanoluciferase. (D) gRNA sequences designed to target the 5′ untranslated region (UTR) of nanoluciferase. The coding sequence is shown in orange, the RBS sequence is highlighted in blue and the dCas9 PAM sequences are underlined in blue ( Sth1 ) and purple ( Spy ). (E) Relative luminescence observed with gRNAs targeting the 5′ UTR of nanoluciferase compared to the non‐targeting gRNA NT1 when dCas9 is expressed at basal (− Choline) or induced (+ Choline) levels. N = 3 for Spy dCas9 and N = 8 for Sth1_dCas9, * p < 0.0001, two‐way ANOVA. Error bars represent standard deviation.

Article Snippet: The coding sequences of catalytically dead Cas9 (dCas9) from Streptococcus pyogenes and Streptococcus thermophilus were codon optimized for expression in B. breve and designed to exclude restriction modification motifs present in B. breve (Bottacini et al. ) prior to being chemically synthesized (Twist Biosciences, San Francisco, CA, US) and cloned into pFREM28 using AatII and XhoI (New England Biolabs).

Techniques: In Vivo, Biomarker Discovery, Functional Assay, CRISPR, Expressing, Luciferase, Sequencing, Standard Deviation

Journal: Microbial Biotechnology

Article Title: A CRISPRi Gene Regulation System for Bifidobacteria

doi: 10.1111/1751-7915.70260

Figure Lengend Snippet: CRISPRi repression of exopolysaccharide production. (A) schematic of the major EPS locus in B. breve UCC2003 and the gRNA target sequences within the Bbr_0430 open reading frame. The dCas9 PAM sequences are underlined. (B) OD measurements (OD 600nm ) of B. breve UCC2003 and a Δ430 mutant strain over an 8‐h time period. The observed drop in OD values for the Δ430 mutant strain is due to cell sedimentation. (C) OD measurements (OD 600nm ) of B. breve UCC2003 and strains containing one of three gRNAs targeting Bbr_0430 . N = 8, * p < 0.05, ** p < 0.01, *** p < 0.005, two‐way ANOVA. Error bars represent standard deviation.

Article Snippet: The coding sequences of catalytically dead Cas9 (dCas9) from Streptococcus pyogenes and Streptococcus thermophilus were codon optimized for expression in B. breve and designed to exclude restriction modification motifs present in B. breve (Bottacini et al. ) prior to being chemically synthesized (Twist Biosciences, San Francisco, CA, US) and cloned into pFREM28 using AatII and XhoI (New England Biolabs).

Techniques: Mutagenesis, Sedimentation, Standard Deviation

Journal: Microbial Biotechnology

Article Title: A CRISPRi Gene Regulation System for Bifidobacteria

doi: 10.1111/1751-7915.70260

Figure Lengend Snippet: CRISPRi repression of fucose metabolism in B. longum subsp. infantis and B. animalis subsp. animalis . (A) Schematic of the operons associated with fucose metabolism in B. longum subsp. infantis ATCC 15697. (B) Growth of B. longum subsp. infantis ATCC 15697 CRISPRi strains with dCas9 alone or in combination with gRNAs targeting fucP in media containing glucose. N = 8 Error bars represent standard deviation (C) Growth of B. infantis ATCC 15697 and CRISPRi strains with dCas9 alone and in combination with gRNAs targeting fucP in media containing fucose. N = 8 Error bars represent standard deviation. (D) Schematic of raffinose operon in B. animalis subsp. animalis ATCC 25527. (E) Growth of B. animalis subsp. animalis ATCC 25527 CRISPRi strains with dCas9 alone or in combination with gRNAs targeting rafA in media containing glucose. N = 13 Error bars represent standard deviation (F) CRISPRi strains with dCas9 alone or in combination with gRNAs targeting rafA in media containing raffinose. N = 13 Error bars represent standard deviation.

Article Snippet: The coding sequences of catalytically dead Cas9 (dCas9) from Streptococcus pyogenes and Streptococcus thermophilus were codon optimized for expression in B. breve and designed to exclude restriction modification motifs present in B. breve (Bottacini et al. ) prior to being chemically synthesized (Twist Biosciences, San Francisco, CA, US) and cloned into pFREM28 using AatII and XhoI (New England Biolabs).

Techniques: Standard Deviation

Journal: Microbial Biotechnology

Article Title: A CRISPRi Gene Regulation System for Bifidobacteria

doi: 10.1111/1751-7915.70260

Figure Lengend Snippet: CRISPRi repression of carbohydrate metabolism in B. longum subsp. longum . (A) Schematic of axu gene cluster in B. longum subsp. longum NCIMB 8809. (B) Growth of B. longum subsp. longum NCIMB 8809 and CRISPRi strains with dCas9 alone or in combination with gRNAs targeting axuA in media containing arabinose. N = 8, error bars represent standard deviation. (C) Growth of B. longum subsp. longum NCIMB 8809 and CRISPRi strains with dCas9 alone or in combination with gRNAs targeting axuA in media containing arabinoxylan from rye. N = 8, error bars represent standard deviation. (D) Growth of B. longum subsp. longum NCIMB 8809 and CRISPRi strains with dCas9 alone or in combination with gRNAs targeting axuA in media containing arabinoxylan from wheat. N = 8, error bars represent standard deviation. (E) Heatmap of genes differentially expressed following CRISPRi targeting of axuA . (F) Top ranked genes differentially expressed in B. longum subsp. longum following CRISPRi targeting of axuA .

Article Snippet: The coding sequences of catalytically dead Cas9 (dCas9) from Streptococcus pyogenes and Streptococcus thermophilus were codon optimized for expression in B. breve and designed to exclude restriction modification motifs present in B. breve (Bottacini et al. ) prior to being chemically synthesized (Twist Biosciences, San Francisco, CA, US) and cloned into pFREM28 using AatII and XhoI (New England Biolabs).

Techniques: Standard Deviation