Dcas9-krab-mecp2
Dcas9-krab-mecp2 - Related Videos
07:44
An Electrochemiluminescence-Based Assay for MeCP2 Protein Variants
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Cited by 2 •
2020
The electrochemiluminescence immunoassay (ECLIA) is a novel approach for quantitative detection of endogenous and exogenously applied MeCP2 protein variants, which produces highly quantitative, accurate and reproducible measurements with low intra- and inter-assay error over a wide working range. Here, the protocol for the MeCP2-ECLIA in a 96-well format is described.
08:27
A Non-random Mouse Model for Pharmacological Reactivation of Mecp2 on the Inactive X Chromosome
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2019
Here, we describe a protocol to generate a viable female murine model with non-random X chromosome inactivation, i.e., the maternally-inherited X chromosome is inactive in 100% of the cells. We also describe a protocol to test feasibility, tolerability, and safety of pharmacological reactivation of the inactive X chromosome in vivo.
11:15
Pooled shRNA Screen for Reactivation of MeCP2 on the Inactive X Chromosome
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Cited by 2 •
2018
We report a small hairpin RNA (shRNA) and next generation sequencing-based protocol for identifying regulators of X-chromosome inactivation in a murine cell line with firefly luciferase and hygromycin resistance genes fused to the methyl CpG binding protein 2 (MeCP2) gene on the inactive X chromosome.
09:56
Epigenetic Engineering of K562 Cells: Dual-Vector Episomal Strategy for Stable Targeted DNA Methylation using dCas9-DNMT3A and -HDAC1 Fusion Proteins
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2025
This protocol describes a non-integrative, episomal CRISPR/dCas9-based system for targeted epigenetic editing in K562 cells, combining dCas9-DNMT3A and dCas9-HDAC1 effectors with specific sgRNAs to induce locus-specific DNA methylation with precision and reduced off-target effects.
07:27
CRISPR Interference-Based Gene Silencing: A Technique for Targeted Repression of Gene Function in Pathogenic Leptospira
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2025
This video demonstrates a CRISPR interference-based gene silencing in pathogenic Leptospira. Targeted repression of gene expression using a CRISPR/Cas system that blocks mRNA formation helps identify candidate genes for pathological and pharmacological studies.
FAQs
The system separates transcriptional repression from DNA cleavage. When a single-guide RNA directs catalytically inactive Cas9 to a complementary sequence, the attached KRAB and MeCP2 domains recruit corepressor proteins and promote compact, transcriptionally inactive chromatin. Gene output can therefore decrease through chromatin regulation rather than mutation, allowing comparison with the original DNA sequence.
KRAB and MeCP2 contribute to repression through coordinated chromatin effects. These domains recruit corepressor proteins and promote a compact chromatin state that is less compatible with transcription. Their combined action helps explain why the observed reduction in gene expression reflects an epigenetic regulatory mechanism rather than guide-RNA binding alone.
Promoters and enhancers represent different regulatory locations that can be tested with the same programmable repression strategy. Directing the system to either type of element helps researchers investigate whether that sequence contributes to gene expression. This makes targeted repression useful for assigning functional roles to regulatory DNA while leaving the sequence itself unchanged.
dCas9-KRAB-MeCP2 changes gene activity through recruited repression and chromatin compaction instead of cutting or rewriting the targeted DNA. That distinction is valuable in genetics because researchers can examine the consequence of reduced expression while preserving the underlying sequence. The preserved sequence supports studies focused on regulatory function rather than permanent DNA alteration.
A conceptual workflow begins by selecting a gene or regulatory element for study, then using a single-guide RNA designed to complement the chosen DNA sequence. The guide directs catalytically inactive Cas9 to that location, where KRAB and MeCP2 establish repression. Researchers can then examine whether targeting the selected site reduces gene expression or reveals regulatory function.
A reduction in expression after targeting a promoter or enhancer supports a functional connection between that element and the regulated gene. The approach therefore provides a way to test regulatory contributions directly, rather than relying only on sequence inspection. In genetic studies, these results can help connect specific regulatory regions with gene-control mechanisms.
Its programmable targeting allows many selected genes or regulatory elements to be examined for effects on gene expression. Because repression occurs without cutting DNA, experiments can focus on gene function and regulatory control while preserving sequence information. This supports functional genomics and genetic screens designed to identify elements whose reduced activity produces informative outcomes.
Researchers can use targeted repression to study regulatory regions associated with disease and ask whether their activity influences gene expression. Since the underlying DNA sequence remains intact, the method is suited to examining regulatory consequences without converting the experiment into a sequence-editing study. This provides genetic context for interpreting how noncoding regulatory elements may contribute to disease-related mechanisms.