Source 1primary paper2018Nucleic Acids Research
Toolkit/dCas9*_PhlF
dCas9*_PhlF
Also known as: dCas9*_PhlF
Taxonomy: Mechanism Branch / Architecture. Workflows sit above the mechanism and technique branches rather than replacing them.
Summary
dCas9*_PhlF is a bacterial CRISPR-based transcriptional switch comprising a non-toxic dCas9* variant with the R1335K PAM-binding mutation fused to the PhlF repressor. The fusion recovered DNA-binding-dependent repression and enabled sgRNA-programmed NOT gate behavior that depends on both an sgRNA target site and a PhlF operator.
Usefulness & Problems
Why this is useful
This tool is useful for building bacterial genetic circuits with reduced dCas9-associated toxicity while retaining programmable transcriptional repression. The reported characterization of 30 orthogonal sgRNA-promoter pairs as NOT gates indicates utility for multiplexed and orthogonal circuit design.
Source:
the simultaneous use of multiple sgRNAs leads to a monotonic decline in repression and after 15 are co-expressed the dynamic range is <10-fold
Problem solved
The tool addresses the problem that dCas9 engineering to reduce toxicity can compromise DNA-binding-dependent repression in bacteria. Fusion of the non-toxic dCas9* variant to PhlF was reported to recover DNA binding and restore switch-like transcriptional control.
Source:
the simultaneous use of multiple sgRNAs leads to a monotonic decline in repression and after 15 are co-expressed the dynamic range is <10-fold
Taxonomy & Function
Primary hierarchy
Mechanism Branch
Architecture: A composed arrangement of multiple parts that instantiates one or more mechanisms.
Mechanisms
dna bindingDNA BindingmultimerizationOligomerizationRNA Bindingrna-guided target recognitiontranscriptional repressionTechniques
No technique tags yet.
Target processes
recombinationImplementation Constraints
The construct uses a dCas9* backbone carrying the R1335K PAM-binding mutation and a fusion to the PhlF repressor. Its function in bacteria requires an sgRNA, a cognate sgRNA target site, and a PhlF operator, but the supplied evidence does not provide additional construct architecture or expression details.
The supplied evidence is limited to one 2018 study in bacteria and does not report validation in other organisms or application contexts. Quantitative performance details beyond orthogonality and increased average cooperativity are not provided in the supplied evidence.
Validation
Cell-freeBacteriaMammalianMouseHumanTherapeuticIndep. Replication
Observations
successBacteriaapplication demoEscherichia coli
Inferred from claim c3 during normalization. The larger recognition region of dCas9*_PhlF mitigates toxicity in Escherichia coli, allowing substantially higher intracellular levels before growth or morphology are impacted than dCas9. Derived from claim c3. Quoted text: The larger region required for recognition mitigates toxicity in Escherichia coli, allowing up to 9600 ± 800 molecules of dCas9*_PhlF per cell before growth or morphology are impacted, as compared to 530 ± 40 molecules of dCas9.
Source:
maximum molecules per cell before growth or morphology are impacted9600 molecules per cellmaximum molecules per cell before growth or morphology are impacted530 molecules per celluncertainty800 molecules per cell(±)uncertainty40 molecules per cell(±)
mixedBacteriaapplication demo
Inferred from claim c6 during normalization. Simultaneous use of multiple sgRNAs causes a monotonic decline in repression, and when 15 are co-expressed the dynamic range falls below 10-fold. Derived from claim c6. Quoted text: the simultaneous use of multiple sgRNAs leads to a monotonic decline in repression and after 15 are co-expressed the dynamic range is <10-fold
Source:
co-expressed sgRNA count15dynamic range10 fold(<)
successBacteriaapplication demoEscherichia coli
Inferred from claim c3 during normalization. The larger recognition region of dCas9*_PhlF mitigates toxicity in Escherichia coli, allowing substantially higher intracellular levels before growth or morphology are impacted than dCas9. Derived from claim c3. Quoted text: The larger region required for recognition mitigates toxicity in Escherichia coli, allowing up to 9600 ± 800 molecules of dCas9*_PhlF per cell before growth or morphology are impacted, as compared to 530 ± 40 molecules of dCas9.
Source:
maximum molecules per cell before growth or morphology are impacted9600 molecules per cellmaximum molecules per cell before growth or morphology are impacted530 molecules per celluncertainty800 molecules per cell(±)uncertainty40 molecules per cell(±)
mixedBacteriaapplication demo
Inferred from claim c6 during normalization. Simultaneous use of multiple sgRNAs causes a monotonic decline in repression, and when 15 are co-expressed the dynamic range falls below 10-fold. Derived from claim c6. Quoted text: the simultaneous use of multiple sgRNAs leads to a monotonic decline in repression and after 15 are co-expressed the dynamic range is <10-fold
Source:
co-expressed sgRNA count15dynamic range10 fold(<)
successBacteriaapplication demoEscherichia coli
Inferred from claim c3 during normalization. The larger recognition region of dCas9*_PhlF mitigates toxicity in Escherichia coli, allowing substantially higher intracellular levels before growth or morphology are impacted than dCas9. Derived from claim c3. Quoted text: The larger region required for recognition mitigates toxicity in Escherichia coli, allowing up to 9600 ± 800 molecules of dCas9*_PhlF per cell before growth or morphology are impacted, as compared to 530 ± 40 molecules of dCas9.
Source:
maximum molecules per cell before growth or morphology are impacted9600 molecules per cellmaximum molecules per cell before growth or morphology are impacted530 molecules per celluncertainty800 molecules per cell(±)uncertainty40 molecules per cell(±)
mixedBacteriaapplication demo
Inferred from claim c6 during normalization. Simultaneous use of multiple sgRNAs causes a monotonic decline in repression, and when 15 are co-expressed the dynamic range falls below 10-fold. Derived from claim c6. Quoted text: the simultaneous use of multiple sgRNAs leads to a monotonic decline in repression and after 15 are co-expressed the dynamic range is <10-fold
Source:
co-expressed sgRNA count15dynamic range10 fold(<)
successBacteriaapplication demoEscherichia coli
Inferred from claim c3 during normalization. The larger recognition region of dCas9*_PhlF mitigates toxicity in Escherichia coli, allowing substantially higher intracellular levels before growth or morphology are impacted than dCas9. Derived from claim c3. Quoted text: The larger region required for recognition mitigates toxicity in Escherichia coli, allowing up to 9600 ± 800 molecules of dCas9*_PhlF per cell before growth or morphology are impacted, as compared to 530 ± 40 molecules of dCas9.
Source:
maximum molecules per cell before growth or morphology are impacted9600 molecules per cellmaximum molecules per cell before growth or morphology are impacted530 molecules per celluncertainty800 molecules per cell(±)uncertainty40 molecules per cell(±)
mixedBacteriaapplication demo
Inferred from claim c6 during normalization. Simultaneous use of multiple sgRNAs causes a monotonic decline in repression, and when 15 are co-expressed the dynamic range falls below 10-fold. Derived from claim c6. Quoted text: the simultaneous use of multiple sgRNAs leads to a monotonic decline in repression and after 15 are co-expressed the dynamic range is <10-fold
Source:
co-expressed sgRNA count15dynamic range10 fold(<)
successBacteriaapplication demoEscherichia coli
Inferred from claim c3 during normalization. The larger recognition region of dCas9*_PhlF mitigates toxicity in Escherichia coli, allowing substantially higher intracellular levels before growth or morphology are impacted than dCas9. Derived from claim c3. Quoted text: The larger region required for recognition mitigates toxicity in Escherichia coli, allowing up to 9600 ± 800 molecules of dCas9*_PhlF per cell before growth or morphology are impacted, as compared to 530 ± 40 molecules of dCas9.
Source:
maximum molecules per cell before growth or morphology are impacted9600 molecules per cellmaximum molecules per cell before growth or morphology are impacted530 molecules per celluncertainty800 molecules per cell(±)uncertainty40 molecules per cell(±)
mixedBacteriaapplication demo
Inferred from claim c6 during normalization. Simultaneous use of multiple sgRNAs causes a monotonic decline in repression, and when 15 are co-expressed the dynamic range falls below 10-fold. Derived from claim c6. Quoted text: the simultaneous use of multiple sgRNAs leads to a monotonic decline in repression and after 15 are co-expressed the dynamic range is <10-fold
Source:
co-expressed sgRNA count15dynamic range10 fold(<)
successBacteriaapplication demoEscherichia coli
Inferred from claim c3 during normalization. The larger recognition region of dCas9*_PhlF mitigates toxicity in Escherichia coli, allowing substantially higher intracellular levels before growth or morphology are impacted than dCas9. Derived from claim c3. Quoted text: The larger region required for recognition mitigates toxicity in Escherichia coli, allowing up to 9600 ± 800 molecules of dCas9*_PhlF per cell before growth or morphology are impacted, as compared to 530 ± 40 molecules of dCas9.
Source:
maximum molecules per cell before growth or morphology are impacted9600 molecules per cellmaximum molecules per cell before growth or morphology are impacted530 molecules per celluncertainty800 molecules per cell(±)uncertainty40 molecules per cell(±)
mixedBacteriaapplication demo
Inferred from claim c6 during normalization. Simultaneous use of multiple sgRNAs causes a monotonic decline in repression, and when 15 are co-expressed the dynamic range falls below 10-fold. Derived from claim c6. Quoted text: the simultaneous use of multiple sgRNAs leads to a monotonic decline in repression and after 15 are co-expressed the dynamic range is <10-fold
Source:
co-expressed sgRNA count15dynamic range10 fold(<)
successBacteriaapplication demoEscherichia coli
Inferred from claim c3 during normalization. The larger recognition region of dCas9*_PhlF mitigates toxicity in Escherichia coli, allowing substantially higher intracellular levels before growth or morphology are impacted than dCas9. Derived from claim c3. Quoted text: The larger region required for recognition mitigates toxicity in Escherichia coli, allowing up to 9600 ± 800 molecules of dCas9*_PhlF per cell before growth or morphology are impacted, as compared to 530 ± 40 molecules of dCas9.
Source:
maximum molecules per cell before growth or morphology are impacted9600 molecules per cellmaximum molecules per cell before growth or morphology are impacted530 molecules per celluncertainty800 molecules per cell(±)uncertainty40 molecules per cell(±)
mixedBacteriaapplication demo
Inferred from claim c6 during normalization. Simultaneous use of multiple sgRNAs causes a monotonic decline in repression, and when 15 are co-expressed the dynamic range falls below 10-fold. Derived from claim c6. Quoted text: the simultaneous use of multiple sgRNAs leads to a monotonic decline in repression and after 15 are co-expressed the dynamic range is <10-fold
Source:
co-expressed sgRNA count15dynamic range10 fold(<)
Supporting Sources
Ranked Claims
Thirty orthogonal sgRNA-promoter pairs were characterized as NOT gates.
A set of 30 orthogonal sgRNA-promoter pairs are characterized as NOT gates
orthogonal sgRNA-promoter pair count 30
Thirty orthogonal sgRNA-promoter pairs were characterized as NOT gates.
A set of 30 orthogonal sgRNA-promoter pairs are characterized as NOT gates
orthogonal sgRNA-promoter pair count 30
Thirty orthogonal sgRNA-promoter pairs were characterized as NOT gates.
A set of 30 orthogonal sgRNA-promoter pairs are characterized as NOT gates
orthogonal sgRNA-promoter pair count 30
Thirty orthogonal sgRNA-promoter pairs were characterized as NOT gates.
A set of 30 orthogonal sgRNA-promoter pairs are characterized as NOT gates
orthogonal sgRNA-promoter pair count 30
Thirty orthogonal sgRNA-promoter pairs were characterized as NOT gates.
A set of 30 orthogonal sgRNA-promoter pairs are characterized as NOT gates
orthogonal sgRNA-promoter pair count 30
Thirty orthogonal sgRNA-promoter pairs were characterized as NOT gates.
A set of 30 orthogonal sgRNA-promoter pairs are characterized as NOT gates
orthogonal sgRNA-promoter pair count 30
Thirty orthogonal sgRNA-promoter pairs were characterized as NOT gates.
A set of 30 orthogonal sgRNA-promoter pairs are characterized as NOT gates
orthogonal sgRNA-promoter pair count 30
PhlF multimerization increases average cooperativity relative to dCas9 alone.
PhlF multimerization leads to an increase in average cooperativity from n = 0.9 (dCas9) to 1.6 (dCas9*_PhlF)
average cooperativity 0.9average cooperativity 1.6
PhlF multimerization increases average cooperativity relative to dCas9 alone.
PhlF multimerization leads to an increase in average cooperativity from n = 0.9 (dCas9) to 1.6 (dCas9*_PhlF)
average cooperativity 0.9average cooperativity 1.6
PhlF multimerization increases average cooperativity relative to dCas9 alone.
PhlF multimerization leads to an increase in average cooperativity from n = 0.9 (dCas9) to 1.6 (dCas9*_PhlF)
average cooperativity 0.9average cooperativity 1.6
PhlF multimerization increases average cooperativity relative to dCas9 alone.
PhlF multimerization leads to an increase in average cooperativity from n = 0.9 (dCas9) to 1.6 (dCas9*_PhlF)
average cooperativity 0.9average cooperativity 1.6
PhlF multimerization increases average cooperativity relative to dCas9 alone.
PhlF multimerization leads to an increase in average cooperativity from n = 0.9 (dCas9) to 1.6 (dCas9*_PhlF)
average cooperativity 0.9average cooperativity 1.6
PhlF multimerization increases average cooperativity relative to dCas9 alone.
PhlF multimerization leads to an increase in average cooperativity from n = 0.9 (dCas9) to 1.6 (dCas9*_PhlF)
average cooperativity 0.9average cooperativity 1.6
PhlF multimerization increases average cooperativity relative to dCas9 alone.
PhlF multimerization leads to an increase in average cooperativity from n = 0.9 (dCas9) to 1.6 (dCas9*_PhlF)
average cooperativity 0.9average cooperativity 1.6
An R1335K PAM-binding mutation in dCas9 produced a non-toxic dCas9* variant, and fusion to the PhlF repressor recovered DNA binding in dCas9*_PhlF.
we construct a non-toxic version of dCas9 by eliminating PAM (protospacer adjacent motif) binding with a R1335K mutation (dCas9*) and recovering DNA binding by fusing it to the PhlF repressor (dCas9*_PhlF)
An R1335K PAM-binding mutation in dCas9 produced a non-toxic dCas9* variant, and fusion to the PhlF repressor recovered DNA binding in dCas9*_PhlF.
we construct a non-toxic version of dCas9 by eliminating PAM (protospacer adjacent motif) binding with a R1335K mutation (dCas9*) and recovering DNA binding by fusing it to the PhlF repressor (dCas9*_PhlF)
An R1335K PAM-binding mutation in dCas9 produced a non-toxic dCas9* variant, and fusion to the PhlF repressor recovered DNA binding in dCas9*_PhlF.
we construct a non-toxic version of dCas9 by eliminating PAM (protospacer adjacent motif) binding with a R1335K mutation (dCas9*) and recovering DNA binding by fusing it to the PhlF repressor (dCas9*_PhlF)
An R1335K PAM-binding mutation in dCas9 produced a non-toxic dCas9* variant, and fusion to the PhlF repressor recovered DNA binding in dCas9*_PhlF.
we construct a non-toxic version of dCas9 by eliminating PAM (protospacer adjacent motif) binding with a R1335K mutation (dCas9*) and recovering DNA binding by fusing it to the PhlF repressor (dCas9*_PhlF)
An R1335K PAM-binding mutation in dCas9 produced a non-toxic dCas9* variant, and fusion to the PhlF repressor recovered DNA binding in dCas9*_PhlF.
we construct a non-toxic version of dCas9 by eliminating PAM (protospacer adjacent motif) binding with a R1335K mutation (dCas9*) and recovering DNA binding by fusing it to the PhlF repressor (dCas9*_PhlF)
An R1335K PAM-binding mutation in dCas9 produced a non-toxic dCas9* variant, and fusion to the PhlF repressor recovered DNA binding in dCas9*_PhlF.
we construct a non-toxic version of dCas9 by eliminating PAM (protospacer adjacent motif) binding with a R1335K mutation (dCas9*) and recovering DNA binding by fusing it to the PhlF repressor (dCas9*_PhlF)
An R1335K PAM-binding mutation in dCas9 produced a non-toxic dCas9* variant, and fusion to the PhlF repressor recovered DNA binding in dCas9*_PhlF.
we construct a non-toxic version of dCas9 by eliminating PAM (protospacer adjacent motif) binding with a R1335K mutation (dCas9*) and recovering DNA binding by fusing it to the PhlF repressor (dCas9*_PhlF)
Repression by dCas9*_PhlF requires both the 30 bp PhlF operator and the 20 bp sgRNA binding site.
Both the 30 bp PhlF operator and 20 bp sgRNA binding site are required to repress a promoter.
Repression by dCas9*_PhlF requires both the 30 bp PhlF operator and the 20 bp sgRNA binding site.
Both the 30 bp PhlF operator and 20 bp sgRNA binding site are required to repress a promoter.
Repression by dCas9*_PhlF requires both the 30 bp PhlF operator and the 20 bp sgRNA binding site.
Both the 30 bp PhlF operator and 20 bp sgRNA binding site are required to repress a promoter.
Repression by dCas9*_PhlF requires both the 30 bp PhlF operator and the 20 bp sgRNA binding site.
Both the 30 bp PhlF operator and 20 bp sgRNA binding site are required to repress a promoter.
Repression by dCas9*_PhlF requires both the 30 bp PhlF operator and the 20 bp sgRNA binding site.
Both the 30 bp PhlF operator and 20 bp sgRNA binding site are required to repress a promoter.
Repression by dCas9*_PhlF requires both the 30 bp PhlF operator and the 20 bp sgRNA binding site.
Both the 30 bp PhlF operator and 20 bp sgRNA binding site are required to repress a promoter.
Repression by dCas9*_PhlF requires both the 30 bp PhlF operator and the 20 bp sgRNA binding site.
Both the 30 bp PhlF operator and 20 bp sgRNA binding site are required to repress a promoter.
Simultaneous use of multiple sgRNAs causes a monotonic decline in repression, and when 15 are co-expressed the dynamic range falls below 10-fold.
the simultaneous use of multiple sgRNAs leads to a monotonic decline in repression and after 15 are co-expressed the dynamic range is <10-fold
co-expressed sgRNA count 15dynamic range 10 fold
Simultaneous use of multiple sgRNAs causes a monotonic decline in repression, and when 15 are co-expressed the dynamic range falls below 10-fold.
the simultaneous use of multiple sgRNAs leads to a monotonic decline in repression and after 15 are co-expressed the dynamic range is <10-fold
co-expressed sgRNA count 15dynamic range 10 fold
Simultaneous use of multiple sgRNAs causes a monotonic decline in repression, and when 15 are co-expressed the dynamic range falls below 10-fold.
the simultaneous use of multiple sgRNAs leads to a monotonic decline in repression and after 15 are co-expressed the dynamic range is <10-fold
co-expressed sgRNA count 15dynamic range 10 fold
Simultaneous use of multiple sgRNAs causes a monotonic decline in repression, and when 15 are co-expressed the dynamic range falls below 10-fold.
the simultaneous use of multiple sgRNAs leads to a monotonic decline in repression and after 15 are co-expressed the dynamic range is <10-fold
co-expressed sgRNA count 15dynamic range 10 fold
Simultaneous use of multiple sgRNAs causes a monotonic decline in repression, and when 15 are co-expressed the dynamic range falls below 10-fold.
the simultaneous use of multiple sgRNAs leads to a monotonic decline in repression and after 15 are co-expressed the dynamic range is <10-fold
co-expressed sgRNA count 15dynamic range 10 fold
Simultaneous use of multiple sgRNAs causes a monotonic decline in repression, and when 15 are co-expressed the dynamic range falls below 10-fold.
the simultaneous use of multiple sgRNAs leads to a monotonic decline in repression and after 15 are co-expressed the dynamic range is <10-fold
co-expressed sgRNA count 15dynamic range 10 fold
Simultaneous use of multiple sgRNAs causes a monotonic decline in repression, and when 15 are co-expressed the dynamic range falls below 10-fold.
the simultaneous use of multiple sgRNAs leads to a monotonic decline in repression and after 15 are co-expressed the dynamic range is <10-fold
co-expressed sgRNA count 15dynamic range 10 fold
The larger recognition region of dCas9*_PhlF mitigates toxicity in Escherichia coli, allowing substantially higher intracellular levels before growth or morphology are impacted than dCas9.
The larger region required for recognition mitigates toxicity in Escherichia coli, allowing up to 9600 ± 800 molecules of dCas9*_PhlF per cell before growth or morphology are impacted, as compared to 530 ± 40 molecules of dCas9.
maximum molecules per cell before growth or morphology are impacted 9600 molecules per cellmaximum molecules per cell before growth or morphology are impacted 530 molecules per celluncertainty 800 molecules per celluncertainty 40 molecules per cell
The larger recognition region of dCas9*_PhlF mitigates toxicity in Escherichia coli, allowing substantially higher intracellular levels before growth or morphology are impacted than dCas9.
The larger region required for recognition mitigates toxicity in Escherichia coli, allowing up to 9600 ± 800 molecules of dCas9*_PhlF per cell before growth or morphology are impacted, as compared to 530 ± 40 molecules of dCas9.
maximum molecules per cell before growth or morphology are impacted 9600 molecules per cellmaximum molecules per cell before growth or morphology are impacted 530 molecules per celluncertainty 800 molecules per celluncertainty 40 molecules per cell
The larger recognition region of dCas9*_PhlF mitigates toxicity in Escherichia coli, allowing substantially higher intracellular levels before growth or morphology are impacted than dCas9.
The larger region required for recognition mitigates toxicity in Escherichia coli, allowing up to 9600 ± 800 molecules of dCas9*_PhlF per cell before growth or morphology are impacted, as compared to 530 ± 40 molecules of dCas9.
maximum molecules per cell before growth or morphology are impacted 9600 molecules per cellmaximum molecules per cell before growth or morphology are impacted 530 molecules per celluncertainty 800 molecules per celluncertainty 40 molecules per cell
The larger recognition region of dCas9*_PhlF mitigates toxicity in Escherichia coli, allowing substantially higher intracellular levels before growth or morphology are impacted than dCas9.
The larger region required for recognition mitigates toxicity in Escherichia coli, allowing up to 9600 ± 800 molecules of dCas9*_PhlF per cell before growth or morphology are impacted, as compared to 530 ± 40 molecules of dCas9.
maximum molecules per cell before growth or morphology are impacted 9600 molecules per cellmaximum molecules per cell before growth or morphology are impacted 530 molecules per celluncertainty 800 molecules per celluncertainty 40 molecules per cell
The larger recognition region of dCas9*_PhlF mitigates toxicity in Escherichia coli, allowing substantially higher intracellular levels before growth or morphology are impacted than dCas9.
The larger region required for recognition mitigates toxicity in Escherichia coli, allowing up to 9600 ± 800 molecules of dCas9*_PhlF per cell before growth or morphology are impacted, as compared to 530 ± 40 molecules of dCas9.
maximum molecules per cell before growth or morphology are impacted 9600 molecules per cellmaximum molecules per cell before growth or morphology are impacted 530 molecules per celluncertainty 800 molecules per celluncertainty 40 molecules per cell
The larger recognition region of dCas9*_PhlF mitigates toxicity in Escherichia coli, allowing substantially higher intracellular levels before growth or morphology are impacted than dCas9.
The larger region required for recognition mitigates toxicity in Escherichia coli, allowing up to 9600 ± 800 molecules of dCas9*_PhlF per cell before growth or morphology are impacted, as compared to 530 ± 40 molecules of dCas9.
maximum molecules per cell before growth or morphology are impacted 9600 molecules per cellmaximum molecules per cell before growth or morphology are impacted 530 molecules per celluncertainty 800 molecules per celluncertainty 40 molecules per cell
The larger recognition region of dCas9*_PhlF mitigates toxicity in Escherichia coli, allowing substantially higher intracellular levels before growth or morphology are impacted than dCas9.
The larger region required for recognition mitigates toxicity in Escherichia coli, allowing up to 9600 ± 800 molecules of dCas9*_PhlF per cell before growth or morphology are impacted, as compared to 530 ± 40 molecules of dCas9.
maximum molecules per cell before growth or morphology are impacted 9600 molecules per cellmaximum molecules per cell before growth or morphology are impacted 530 molecules per celluncertainty 800 molecules per celluncertainty 40 molecules per cell
Approval Evidence
1 source5 linked approval claimsfirst-pass slug dcas9-phlf
recovering DNA binding by fusing it to the PhlF repressor (dCas9*_PhlF)
Source:
cooperativity changesupports
PhlF multimerization increases average cooperativity relative to dCas9 alone.
PhlF multimerization leads to an increase in average cooperativity from n = 0.9 (dCas9) to 1.6 (dCas9*_PhlF)
Source:
engineering resultsupports
An R1335K PAM-binding mutation in dCas9 produced a non-toxic dCas9* variant, and fusion to the PhlF repressor recovered DNA binding in dCas9*_PhlF.
we construct a non-toxic version of dCas9 by eliminating PAM (protospacer adjacent motif) binding with a R1335K mutation (dCas9*) and recovering DNA binding by fusing it to the PhlF repressor (dCas9*_PhlF)
Source:
mechanismsupports
Repression by dCas9*_PhlF requires both the 30 bp PhlF operator and the 20 bp sgRNA binding site.
Both the 30 bp PhlF operator and 20 bp sgRNA binding site are required to repress a promoter.
Source:
resource limitationsupports
Simultaneous use of multiple sgRNAs causes a monotonic decline in repression, and when 15 are co-expressed the dynamic range falls below 10-fold.
the simultaneous use of multiple sgRNAs leads to a monotonic decline in repression and after 15 are co-expressed the dynamic range is <10-fold
Source:
toxicity reductionsupports
The larger recognition region of dCas9*_PhlF mitigates toxicity in Escherichia coli, allowing substantially higher intracellular levels before growth or morphology are impacted than dCas9.
The larger region required for recognition mitigates toxicity in Escherichia coli, allowing up to 9600 ± 800 molecules of dCas9*_PhlF per cell before growth or morphology are impacted, as compared to 530 ± 40 molecules of dCas9.
Source:
Comparisons
Source-backed strengths
An R1335K PAM-binding mutation generated a non-toxic dCas9* variant, and PhlF fusion recovered DNA binding in the resulting dCas9*_PhlF construct. PhlF multimerization increased average cooperativity relative to dCas9 alone, and 30 orthogonal sgRNA-promoter pairs were characterized as NOT gates.
Source:
we construct a non-toxic version of dCas9 by eliminating PAM (protospacer adjacent motif) binding with a R1335K mutation (dCas9*) and recovering DNA binding by fusing it to the PhlF repressor (dCas9*_PhlF)
Ranked Citations
- 1.