Source 1primary paper2018Nucleic Acids Research
Toolkit/dCas9*
dCas9*
Also known as: non-toxic version of dCas9, R1335K mutant dCas9
Taxonomy: Mechanism Branch / Component. Workflows sit above the mechanism and technique branches rather than replacing them.
Summary
dCas9* is a Streptococcus pyogenes dCas9 variant carrying the PAM-binding mutation R1335K, engineered to eliminate PAM recognition and reduce toxicity in bacteria. In the cited study, dCas9* was also fused to the PhlF repressor to recover targetable transcriptional repression through a combined sgRNA target site and PhlF operator requirement.
Usefulness & Problems
Why this is useful
This tool is useful for bacterial genetic circuit design because it provides a non-toxic dCas9-derived scaffold while retaining programmability through sgRNAs in the reported system. The PhlF fusion format further enables orthogonal NOT-gate construction and increased cooperativity relative to dCas9 alone.
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 reported engineering addresses the toxicity associated with dCas9 in bacteria by eliminating PAM binding through the R1335K mutation. It also addresses the loss of effective targetable repression from PAM-binding disruption by restoring repression through fusion to the PhlF repressor.
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
Component: A low-level protein part used inside a larger architecture that realizes a mechanism.
Mechanisms
DNA Bindingloss of pam recognition through pam-binding mutationmultimerization-enhanced cooperativitysequence-specific dna binding via sgrna-guided dcas9 targetingsgrna-guided sequence-specific dna bindingtranscriptional repression via dual-site requirement for sgrna target site and phlf operatorTechniques
No technique tags yet.
Target processes
No target processes tagged yet.
Implementation Constraints
The core engineering change is an R1335K mutation in Streptococcus pyogenes dCas9 that eliminates PAM binding. In the reported implementation, dCas9* was fused to the PhlF repressor, and repression depended on both an sgRNA binding site and a PhlF operator in the promoter architecture.
The supplied evidence is limited to a single 2018 study in bacteria and does not provide broader validation across organisms, delivery formats, or application classes. Quantitative performance details, off-target behavior, and standalone repression capability of dCas9* without the PhlF fusion are not described in the provided evidence.
Validation
Cell-freeBacteriaMammalianMouseHumanTherapeuticIndep. Replication
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
3 sources5 linked approval claimsfirst-pass slug dcas9
The engineering of switchable or activatable dCas9 proteins would benefit from a single system for both positive and negative selection of dCas9 activity.
Source:
we construct a non-toxic version of dCas9 by eliminating PAM (protospacer adjacent motif) binding with a R1335K mutation (dCas9*)
Source:
Here, we review the use of dCas9 as a novel and versatile tool for fundamental studies on epigenetic landscapes, chromatin structure and transcription regulation
Source:
intended applicationsupports
The system has potential utility in directed evolution experiments to create switchable dCas9 proteins.
possesses potential utility in directed evolution experiments to create switchable dCas9 proteins
Source:
selection outcomesupports
In the positive selection system, E. coli expressing active dCas9 variants are isolated by growth in the presence of ampicillin.
E. coli expressing active dCas9 variants are isolated in the positive selection system through growth in the presence of ampicillin.
Source:
selection outcomesupports
The negative selection can isolate cells lacking dCas9 activity through growth in M9 minimal media or growth in media containing streptomycin.
The negative selection can isolate cells lacking dCas9 activity through two separate mechanisms: growth in M9 minimal media or growth in media containing streptomycin.
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:
tool scopesupports
dCas9 is described as a novel and versatile tool for fundamental studies of epigenetic landscapes, chromatin structure, and transcription regulation.
Here, we review the use of dCas9 as a novel and versatile tool for fundamental studies on epigenetic landscapes, chromatin structure and transcription regulation
Source:
Comparisons
Source-backed strengths
The cited work reports that the R1335K PAM-binding mutation produced a non-toxic dCas9* variant. It further reports that dCas9*_PhlF recovered DNA binding, that 30 orthogonal sgRNA-promoter pairs were characterized as NOT gates, and that PhlF multimerization increased average cooperativity relative to dCas9 alone.
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.