Journal: Frontiers in Bioengineering and Biotechnology

Article Title: GenoMine: a CRISPR-Cas9-based kill switch for biocontainment of Pseudomonas putida

doi: 10.3389/fbioe.2024.1426107

Figure Lengend Snippet: Scheme of GenoMine, a CRISPR-Cas9-based kill switch in Pseudomonas putida (A) The genome of Pseudomonas putida KT2440 contains abundant repetitive sequences. These repetitive regions can easily be used as CRISPR-Cas9 targets leading to a chain reaction of cleavages in the bacterial chromosome (B) GenoMine cassette introduced downstream the PP_5322 locus of the genome of Pseudomonas putida KT2440 containing a CRISPR array with two constitutively expressed spacers (REP and ISPpu9) that target repetitive regions of the genome itself. DR stands for direct repeat (C) When ScCas9 is transformed into cells containing the GenoMine cassette, a chain reaction of cleavages starts resulting in cell death (D) Experimental validation of the hypothesis depicting the targeting efficiency associated with cell death of Pseudomonas putida “GenoMine” relative to that obtained in the wild type strain when transformed with a pSEVAb62 plasmid constitutively expressing ScCas9 or with an empty plasmid (mean ± s.d., n = 3 biological).

Article Snippet: Plasmids pSEVAb23 containing these constructs , alongside an empty pSEVAb23 vector were transformed in both wild type and GenoMine P. putida strains, which were subsequently plated on media supplemented with the different inducers to quantify the cell population survival to the CRISPR-Cas9 lethal response.

Techniques: CRISPR, Transformation Assay, Plasmid Preparation, Expressing

Journal: Frontiers in Bioengineering and Biotechnology

Article Title: GenoMine: a CRISPR-Cas9-based kill switch for biocontainment of Pseudomonas putida

doi: 10.3389/fbioe.2024.1426107

Figure Lengend Snippet: Different circuits controlling the expression of ScCas9 in Pseudomonas putida GenoMine (A) Schematic representation of the riboregulator circuits (conventional or toehold) controlling the expression of ScCas9 (B) Schematic representation of the xylS/pM expression system endowed with the ON/OFF digitaliser module and controlling the expression of ScCas9 (C) Designs of AND YES gates according to the American National Standards Institute (ANSI), in this case corresponding to the riboregulators and the ON/OFF DM, respectively. Following binary logic, there are only two states allowed, 1 and 0 or “on and off”. A and B represent the inputs 3MB and rhamnose, and X represents the output of the circuit, in this case the activation of Cas9 (D) Cleavage survival after transformation of ScCas9 under the control of conventional RR10 (E) Cleavage survival after transformation of ScCas9 under the control of conventional RR12 (F) Cleavage survival after transformation of ScCas9 under the control of Toehold 2.1 (G) Cleavage survival after transformation of ScCas9 under the control of a digitalised version of the xylS/pM system. Relative cleavage survival was calculated separately for each strain in each figure as the ratio between CFU growing upon different induction conditions (purple bars represent single induction with 3MB, pink bars single induction with rhamnose and red bars double 3MB + rhamnose induction) and total CFU growing on plates without the addition of inducers (grey bars) and expressed in percentage. Only significant values are indicated for a parametric two-tailed t-test between two groups, where *p < 0.05; **p < 0.01; ***p < 0.001; and ****p < 0.0001; non-significant values were not depicted (mean ± s.d., n ≥ 3 biological).

Article Snippet: Plasmids pSEVAb23 containing these constructs , alongside an empty pSEVAb23 vector were transformed in both wild type and GenoMine P. putida strains, which were subsequently plated on media supplemented with the different inducers to quantify the cell population survival to the CRISPR-Cas9 lethal response.

Techniques: Expressing, Activation Assay, Transformation Assay, Control, Two Tailed Test

Journal: Frontiers in Bioengineering and Biotechnology

Article Title: GenoMine: a CRISPR-Cas9-based kill switch for biocontainment of Pseudomonas putida

doi: 10.3389/fbioe.2024.1426107

Figure Lengend Snippet: Different circuits controlling the expression of TetR, which works as a transcriptional repressor of ScCas9 in Pseudomonas putida GenoMine (A) Schematic representation of the riboregulator circuits (conventional or toehold) controlling the expression of TetR (B) Schematic representation of the xylS/pM expression system endowed with the ON/OFF digitaliser module and controlling the expression of TetR (C) ScCas9 located on a separated plasmid under the control of the TetR/pLtetO expression system. ScCas9 can be repressed in the presence of TetR, which, in turn, gets expressed upon full induction of the genetic circuits depicted in either (A) or (B) (D) Designs of NAND (NOT + AND) and NOT gates according to the American National Standards Institute (ANSI), in this case corresponding to the riboregulators and the ON/OFF DM, respectively. Following binary logic, there are only two states allowed, 1 and 0 or ‘on and off’. A and B represent the inputs 3MB and rhamnose, and X represents the output of the circuit, in this case the activation of TetR and, correspondingly, the repression of ScCas9 (E) Cleavage survival of cells bearing TetR under the control of RR10 after transformation with pLtetO-ScCas9 (F) Cleavage survival of cells bearing TetR under the control of conventional RR12 after transformation with pLtetO-ScCas9 (G) Cleavage survival of cells bearing TetR under the control of Toehold 2.1 after transformation with pLtetO-ScCas9 (H) Cleavage survival of cells bearing TetR under the control of a digitalized version of the xylS/pM expression system after transformation with pLtetO-ScCas9. Relative cleavage survival was calculated separately for each strain in each figure as the ratio between CFUs growing upon different induction conditions (purple bars represent single induction with 3MB, pink bars single induction with rhamnose and red bars double 3MB + rhamnose induction) and total CFUs growing on plates without the addition of inducers (grey bars) and expressed in percentage. Only significant values are indicated for a parametric two-tailed t-test between two groups, where *p < 0.05; **p < 0.01; ***p < 0.001; and ****p < 0.0001; non-significant values were not depicted (mean ± s.d., n = 3 biological).

Article Snippet: Plasmids pSEVAb23 containing these constructs , alongside an empty pSEVAb23 vector were transformed in both wild type and GenoMine P. putida strains, which were subsequently plated on media supplemented with the different inducers to quantify the cell population survival to the CRISPR-Cas9 lethal response.

Techniques: Expressing, Plasmid Preparation, Control, Activation Assay, Transformation Assay, Two Tailed Test

Journal: Frontiers in Bioengineering and Biotechnology

Article Title: GenoMine: a CRISPR-Cas9-based kill switch for biocontainment of Pseudomonas putida

doi: 10.3389/fbioe.2024.1426107

Figure Lengend Snippet: AcrIIA4 as a repressor of ScCas9 in Pseudomonas putida GenoMine (A) Schematic representation of the inhibition design (B) AcrIIA4 does not inhibit ScCas9’s cleavage ability in the GenoMine strain. Targeting efficiency was calculated in Pseudomonas putida GenoMine using the ratio of CFU obtained when transformed with either pSEVAb62-ScCas9 (targeting conditions) or an empty pSEVAb62 (non-targeting conditions), both in the presence and in the absence of a constitutively expressed AcrIIA4 (mean ± s.d., n = 2 biological).

Article Snippet: Plasmids pSEVAb23 containing these constructs , alongside an empty pSEVAb23 vector were transformed in both wild type and GenoMine P. putida strains, which were subsequently plated on media supplemented with the different inducers to quantify the cell population survival to the CRISPR-Cas9 lethal response.

Techniques: Inhibition, Transformation Assay