Source 1primary paper2018
Toolkit/SpCas9
SpCas9
Also known as: S py Cas9, Streptococcus pyogenes Cas9
Taxonomy: Mechanism Branch / Component. Workflows sit above the mechanism and technique branches rather than replacing them.
Summary
SpCas9 is the Streptococcus pyogenes Cas9 CRISPR effector protein used for programmable genome editing and gene regulation. In the cited study, its activity was controlled indirectly by microRNA-dependent expression of the anti-CRISPR protein AcrIIA4, enabling cell-type-restricted activation of full-length Cas9, split-Cas9, and dCas9-VP64 variants.
Usefulness & Problems
Why this is useful
This system is useful for restricting SpCas9 activity to defined cell types by coupling endogenous microRNA signatures to anti-CRISPR regulation. The reported design enabled hepatocyte- or myocyte-specific Cas9 activation while suppressing activity in off-target cells.
Source:
We demonstrate control of genome editing and gene activation using a miR-dependent AcrIIA4 in combination with different Streptococcus pyogenes ( S py) Cas9 variants (full-length Cas9, split-Cas9, dCas9-VP64).
Source:
Here, we developed a cell type-specific Cas-ON switch based on miRNA-regulated expression of anti-CRISPR (Acr) proteins.
Problem solved
The approach addresses the problem of achieving cell-specific SpCas9 activity without changing the Cas9 protein itself, thereby reducing unwanted activity in non-target cell types. Specifically, miR-122 or miR-1 target sites in the AcrIIA4 3'UTR allowed Cas9 activation only in cells expressing the corresponding microRNA.
Source:
Here, we developed a cell type-specific Cas-ON switch based on miRNA-regulated expression of anti-CRISPR (Acr) proteins.
Published Workflows
Objective: Characterize and validate Fp2Cas9 as a cold-adapted programmable genome-editing nuclease for low-temperature applications.
Why it works: The paper combines in vitro biochemical characterization with in vivo editing to show that Fp2Cas9 is both mechanistically functional and practically usable as a low-temperature genome-editing tool.
double-stranded DNA cleavagePAM-dependent target recognitionguide RNA-supported programmable targetingnuclear-localized genome editing in vivoin vitro cleavage assayengineered sgRNA scaffold testingin vivo zebrafish mutagenesis
Stages
- 1.In vitro biochemical characterization(functional_characterization)
This stage establishes whether Fp2Cas9 functions as a programmable nuclease and whether its temperature profile supports the intended low-temperature use case before in vivo deployment.
Selection: Demonstrate DNA cleavage, define PAM requirement, confirm guide-supported programmability, and quantify low-temperature activity.
- 2.In vivo zebrafish validation(confirmatory_validation)
This stage confirms that the biochemically characterized nuclease can function in a living vertebrate model using an engineered nuclear-localized construct.
Selection: Test whether a nuclear-localized Fp2Cas9 variant can mediate mutagenesis and produce a visible phenotype in zebrafish embryos.
Steps
- 1.Measure Fp2Cas9 double-stranded DNA cleavage and PAM requirement in vitroengineered nuclease being characterized
Establish core nuclease activity and PAM dependence.
The abstract presents biochemical characterization first to determine whether Fp2Cas9 is a functional programmable nuclease before in vivo use.
- 2.Test engineered sgRNA-V2 support for programmable DNA targetingguide scaffold and nuclease pair under test
Determine whether an engineered sgRNA scaffold enables programmable targeting by Fp2Cas9.
After establishing cleavage capability, the study tests whether the nuclease can be directed programmably using the engineered guide scaffold.
- 3.Quantify temperature-dependent cleavage performancenuclease under test and comparator
Assess whether Fp2Cas9 retains activity at low temperature and compare it with SpCas9.
Temperature profiling directly tests the paper's central low-temperature objective after basic activity and programmability are established.
- 4.Evaluate nuclear-localized 2NLS-Fp2Cas9 for zebrafish slc45a2 mutagenesis in vivoengineered in vivo editing construct
Confirm that a nuclear-localized Fp2Cas9 variant can edit a vertebrate target gene and generate a visible phenotype.
In vivo testing follows in vitro characterization to confirm practical editing utility in a living system.
Taxonomy & Function
Primary hierarchy
Mechanism Branch
Component: A low-level protein part used inside a larger architecture that realizes a mechanism.
Mechanisms
anti-crispr-mediated inhibitiondna bindingDNA Bindingmicrorna-dependent post-transcriptional regulationPhotocleavageTechniques
No technique tags yet.
Target processes
recombinationImplementation Constraints
The reported implementation used AcrIIA4 transgenes bearing miR-122 or miR-1 target sites in the 3'UTR to create a Cas-ON switch for SpCas9. This strategy was applied to full-length Cas9, split-Cas9, and dCas9-VP64, but the source text does not specify additional construct architecture, expression system, or cofactor requirements.
The evidence is limited to a single 2018 study centered on indirect SpCas9 control through AcrIIA4 rather than intrinsic engineering of SpCas9. The source does not provide quantitative performance metrics, delivery constraints, or independent replication data.
Validation
Cell-freeBacteriaMammalianMouseHumanTherapeuticIndep. Replication
Supporting Sources
Source 2primary paper2017Genome biology
Ranked Claims
Inserting miR-122 or miR-1 target sites into the 3’UTR of Acr transgenes enabled Acr knockdown and corresponding Cas9 activation solely in hepatocytes or myocytes, while inhibiting Cas9 in off-target cells.
We inserted target sites for miR-122 or miR-1, which are abundant specifically in liver and muscle cells, respectively, into the 3’U TR of Acr transgenes. Co-expressing these with Cas9 and sgRNAs resulted in Acr knockdown and correspondingly in Cas9 activation solely in hepatocytes or myocytes, while Cas9 was efficiently inhibited in off-target cells.
Inserting miR-122 or miR-1 target sites into the 3’UTR of Acr transgenes enabled Acr knockdown and corresponding Cas9 activation solely in hepatocytes or myocytes, while inhibiting Cas9 in off-target cells.
We inserted target sites for miR-122 or miR-1, which are abundant specifically in liver and muscle cells, respectively, into the 3’U TR of Acr transgenes. Co-expressing these with Cas9 and sgRNAs resulted in Acr knockdown and correspondingly in Cas9 activation solely in hepatocytes or myocytes, while Cas9 was efficiently inhibited in off-target cells.
Inserting miR-122 or miR-1 target sites into the 3’UTR of Acr transgenes enabled Acr knockdown and corresponding Cas9 activation solely in hepatocytes or myocytes, while inhibiting Cas9 in off-target cells.
We inserted target sites for miR-122 or miR-1, which are abundant specifically in liver and muscle cells, respectively, into the 3’U TR of Acr transgenes. Co-expressing these with Cas9 and sgRNAs resulted in Acr knockdown and correspondingly in Cas9 activation solely in hepatocytes or myocytes, while Cas9 was efficiently inhibited in off-target cells.
Inserting miR-122 or miR-1 target sites into the 3’UTR of Acr transgenes enabled Acr knockdown and corresponding Cas9 activation solely in hepatocytes or myocytes, while inhibiting Cas9 in off-target cells.
We inserted target sites for miR-122 or miR-1, which are abundant specifically in liver and muscle cells, respectively, into the 3’U TR of Acr transgenes. Co-expressing these with Cas9 and sgRNAs resulted in Acr knockdown and correspondingly in Cas9 activation solely in hepatocytes or myocytes, while Cas9 was efficiently inhibited in off-target cells.
Inserting miR-122 or miR-1 target sites into the 3’UTR of Acr transgenes enabled Acr knockdown and corresponding Cas9 activation solely in hepatocytes or myocytes, while inhibiting Cas9 in off-target cells.
We inserted target sites for miR-122 or miR-1, which are abundant specifically in liver and muscle cells, respectively, into the 3’U TR of Acr transgenes. Co-expressing these with Cas9 and sgRNAs resulted in Acr knockdown and correspondingly in Cas9 activation solely in hepatocytes or myocytes, while Cas9 was efficiently inhibited in off-target cells.
Inserting miR-122 or miR-1 target sites into the 3’UTR of Acr transgenes enabled Acr knockdown and corresponding Cas9 activation solely in hepatocytes or myocytes, while inhibiting Cas9 in off-target cells.
We inserted target sites for miR-122 or miR-1, which are abundant specifically in liver and muscle cells, respectively, into the 3’U TR of Acr transgenes. Co-expressing these with Cas9 and sgRNAs resulted in Acr knockdown and correspondingly in Cas9 activation solely in hepatocytes or myocytes, while Cas9 was efficiently inhibited in off-target cells.
Inserting miR-122 or miR-1 target sites into the 3’UTR of Acr transgenes enabled Acr knockdown and corresponding Cas9 activation solely in hepatocytes or myocytes, while inhibiting Cas9 in off-target cells.
We inserted target sites for miR-122 or miR-1, which are abundant specifically in liver and muscle cells, respectively, into the 3’U TR of Acr transgenes. Co-expressing these with Cas9 and sgRNAs resulted in Acr knockdown and correspondingly in Cas9 activation solely in hepatocytes or myocytes, while Cas9 was efficiently inhibited in off-target cells.
A miR-dependent AcrIIA4 enabled control of genome editing and gene activation with different Streptococcus pyogenes Cas9 variants including full-length Cas9, split-Cas9, and dCas9-VP64.
We demonstrate control of genome editing and gene activation using a miR-dependent AcrIIA4 in combination with different Streptococcus pyogenes ( S py) Cas9 variants (full-length Cas9, split-Cas9, dCas9-VP64).
A miR-dependent AcrIIA4 enabled control of genome editing and gene activation with different Streptococcus pyogenes Cas9 variants including full-length Cas9, split-Cas9, and dCas9-VP64.
We demonstrate control of genome editing and gene activation using a miR-dependent AcrIIA4 in combination with different Streptococcus pyogenes ( S py) Cas9 variants (full-length Cas9, split-Cas9, dCas9-VP64).
A miR-dependent AcrIIA4 enabled control of genome editing and gene activation with different Streptococcus pyogenes Cas9 variants including full-length Cas9, split-Cas9, and dCas9-VP64.
We demonstrate control of genome editing and gene activation using a miR-dependent AcrIIA4 in combination with different Streptococcus pyogenes ( S py) Cas9 variants (full-length Cas9, split-Cas9, dCas9-VP64).
A miR-dependent AcrIIA4 enabled control of genome editing and gene activation with different Streptococcus pyogenes Cas9 variants including full-length Cas9, split-Cas9, and dCas9-VP64.
We demonstrate control of genome editing and gene activation using a miR-dependent AcrIIA4 in combination with different Streptococcus pyogenes ( S py) Cas9 variants (full-length Cas9, split-Cas9, dCas9-VP64).
A miR-dependent AcrIIA4 enabled control of genome editing and gene activation with different Streptococcus pyogenes Cas9 variants including full-length Cas9, split-Cas9, and dCas9-VP64.
We demonstrate control of genome editing and gene activation using a miR-dependent AcrIIA4 in combination with different Streptococcus pyogenes ( S py) Cas9 variants (full-length Cas9, split-Cas9, dCas9-VP64).
A miR-dependent AcrIIA4 enabled control of genome editing and gene activation with different Streptococcus pyogenes Cas9 variants including full-length Cas9, split-Cas9, and dCas9-VP64.
We demonstrate control of genome editing and gene activation using a miR-dependent AcrIIA4 in combination with different Streptococcus pyogenes ( S py) Cas9 variants (full-length Cas9, split-Cas9, dCas9-VP64).
A miR-dependent AcrIIA4 enabled control of genome editing and gene activation with different Streptococcus pyogenes Cas9 variants including full-length Cas9, split-Cas9, and dCas9-VP64.
We demonstrate control of genome editing and gene activation using a miR-dependent AcrIIA4 in combination with different Streptococcus pyogenes ( S py) Cas9 variants (full-length Cas9, split-Cas9, dCas9-VP64).
The Cas-ON switch should facilitate cell-specific activation of CRISPR/Cas orthologues for which a potent anti-CRISPR protein is known.
Our Cas-ON switch should facilitate cell-specific activation of any CRISPR/Cas orthologue, for which a potent anti-CRISPR protein is known.
The Cas-ON switch should facilitate cell-specific activation of CRISPR/Cas orthologues for which a potent anti-CRISPR protein is known.
Our Cas-ON switch should facilitate cell-specific activation of any CRISPR/Cas orthologue, for which a potent anti-CRISPR protein is known.
The Cas-ON switch should facilitate cell-specific activation of CRISPR/Cas orthologues for which a potent anti-CRISPR protein is known.
Our Cas-ON switch should facilitate cell-specific activation of any CRISPR/Cas orthologue, for which a potent anti-CRISPR protein is known.
The Cas-ON switch should facilitate cell-specific activation of CRISPR/Cas orthologues for which a potent anti-CRISPR protein is known.
Our Cas-ON switch should facilitate cell-specific activation of any CRISPR/Cas orthologue, for which a potent anti-CRISPR protein is known.
The Cas-ON switch should facilitate cell-specific activation of CRISPR/Cas orthologues for which a potent anti-CRISPR protein is known.
Our Cas-ON switch should facilitate cell-specific activation of any CRISPR/Cas orthologue, for which a potent anti-CRISPR protein is known.
The Cas-ON switch should facilitate cell-specific activation of CRISPR/Cas orthologues for which a potent anti-CRISPR protein is known.
Our Cas-ON switch should facilitate cell-specific activation of any CRISPR/Cas orthologue, for which a potent anti-CRISPR protein is known.
The Cas-ON switch should facilitate cell-specific activation of CRISPR/Cas orthologues for which a potent anti-CRISPR protein is known.
Our Cas-ON switch should facilitate cell-specific activation of any CRISPR/Cas orthologue, for which a potent anti-CRISPR protein is known.
The Cas-ON system was adapted to Neisseria meningitidis Cas9 using its cognate inhibitors AcrIIC1 and AcrIIC3.
Finally, to showcase its modularity, we adapted our Cas-ON system to the smaller and more target-specific Neisseria meningitidis (Nme) Cas9 orthologue and its cognate inhibitors AcrIIC1 and AcrIIC3.
The Cas-ON system was adapted to Neisseria meningitidis Cas9 using its cognate inhibitors AcrIIC1 and AcrIIC3.
Finally, to showcase its modularity, we adapted our Cas-ON system to the smaller and more target-specific Neisseria meningitidis (Nme) Cas9 orthologue and its cognate inhibitors AcrIIC1 and AcrIIC3.
The Cas-ON system was adapted to Neisseria meningitidis Cas9 using its cognate inhibitors AcrIIC1 and AcrIIC3.
Finally, to showcase its modularity, we adapted our Cas-ON system to the smaller and more target-specific Neisseria meningitidis (Nme) Cas9 orthologue and its cognate inhibitors AcrIIC1 and AcrIIC3.
The Cas-ON system was adapted to Neisseria meningitidis Cas9 using its cognate inhibitors AcrIIC1 and AcrIIC3.
Finally, to showcase its modularity, we adapted our Cas-ON system to the smaller and more target-specific Neisseria meningitidis (Nme) Cas9 orthologue and its cognate inhibitors AcrIIC1 and AcrIIC3.
The Cas-ON system was adapted to Neisseria meningitidis Cas9 using its cognate inhibitors AcrIIC1 and AcrIIC3.
Finally, to showcase its modularity, we adapted our Cas-ON system to the smaller and more target-specific Neisseria meningitidis (Nme) Cas9 orthologue and its cognate inhibitors AcrIIC1 and AcrIIC3.
The Cas-ON system was adapted to Neisseria meningitidis Cas9 using its cognate inhibitors AcrIIC1 and AcrIIC3.
Finally, to showcase its modularity, we adapted our Cas-ON system to the smaller and more target-specific Neisseria meningitidis (Nme) Cas9 orthologue and its cognate inhibitors AcrIIC1 and AcrIIC3.
The Cas-ON system was adapted to Neisseria meningitidis Cas9 using its cognate inhibitors AcrIIC1 and AcrIIC3.
Finally, to showcase its modularity, we adapted our Cas-ON system to the smaller and more target-specific Neisseria meningitidis (Nme) Cas9 orthologue and its cognate inhibitors AcrIIC1 and AcrIIC3.
The authors developed a cell type-specific Cas-ON switch based on miRNA-regulated expression of anti-CRISPR proteins.
Here, we developed a cell type-specific Cas-ON switch based on miRNA-regulated expression of anti-CRISPR (Acr) proteins.
The authors developed a cell type-specific Cas-ON switch based on miRNA-regulated expression of anti-CRISPR proteins.
Here, we developed a cell type-specific Cas-ON switch based on miRNA-regulated expression of anti-CRISPR (Acr) proteins.
The authors developed a cell type-specific Cas-ON switch based on miRNA-regulated expression of anti-CRISPR proteins.
Here, we developed a cell type-specific Cas-ON switch based on miRNA-regulated expression of anti-CRISPR (Acr) proteins.
The authors developed a cell type-specific Cas-ON switch based on miRNA-regulated expression of anti-CRISPR proteins.
Here, we developed a cell type-specific Cas-ON switch based on miRNA-regulated expression of anti-CRISPR (Acr) proteins.
The authors developed a cell type-specific Cas-ON switch based on miRNA-regulated expression of anti-CRISPR proteins.
Here, we developed a cell type-specific Cas-ON switch based on miRNA-regulated expression of anti-CRISPR (Acr) proteins.
The authors developed a cell type-specific Cas-ON switch based on miRNA-regulated expression of anti-CRISPR proteins.
Here, we developed a cell type-specific Cas-ON switch based on miRNA-regulated expression of anti-CRISPR (Acr) proteins.
The authors developed a cell type-specific Cas-ON switch based on miRNA-regulated expression of anti-CRISPR proteins.
Here, we developed a cell type-specific Cas-ON switch based on miRNA-regulated expression of anti-CRISPR (Acr) proteins.
Mutations in the increased-fidelity SpCas9 variants may reduce cleavage without reducing DNA binding.
the mutations in these variants may diminish the cleavage, but not the DNA-binding, of SpCas9s
Approval Evidence
2 sources2 linked approval claimsfirst-pass slugs spcas9, streptococcus-pyogenes-cas9
different Streptococcus pyogenes ( S py) Cas9 variants
Source:
Streptococcus pyogenes Cas9 (SpCas9)
Source:
functional scopesupports
A miR-dependent AcrIIA4 enabled control of genome editing and gene activation with different Streptococcus pyogenes Cas9 variants including full-length Cas9, split-Cas9, and dCas9-VP64.
We demonstrate control of genome editing and gene activation using a miR-dependent AcrIIA4 in combination with different Streptococcus pyogenes ( S py) Cas9 variants (full-length Cas9, split-Cas9, dCas9-VP64).
Source:
mechanistic effectsupports
Mutations in the increased-fidelity SpCas9 variants may reduce cleavage without reducing DNA binding.
the mutations in these variants may diminish the cleavage, but not the DNA-binding, of SpCas9s
Source:
Comparisons
Source-backed strengths
The cited work demonstrated functional control across multiple Streptococcus pyogenes Cas9 formats, including full-length Cas9, split-Cas9, and dCas9-VP64. It also showed cell-type-restricted control in hepatocyte and myocyte contexts through microRNA-responsive AcrIIA4 knockdown.
Ranked Citations
- 1.