CRISPR interference (CRISPRi) is a powerful technique derived from the CRISPR-Cas9 system, which originally evolved in bacteria as a defense mechanism against invading viruses. CRISPRi allows for precise control over gene expression by using a modified Cas protein (usually Cas9) to target specific DNA sequences and inhibit gene transcription without inducing double-stranded breaks. CRISPRi is being used in a greater number of applications, including to understand the role of specific genes and their function, in particular where residual gene product levels or activity produces differing phenotypes to those of a complete loss of expression. This includes many genes identified by genome-wide association studies (GWASs) as contributing to multifactorial diseases such as PD, as well as certain incompletely penetrant disease-causing mutations. Here are some key applications of CRISPR interference:

  1. Gene Function Studies: CRISPRi enables researchers to study the function of genes by selectively repressing their expression. By targeting specific genes and observing the resulting phenotypic changes, scientists can elucidate the roles of those genes in various biological processes.
  2. Gene Regulation: CRISPRi can be used to modulate gene expression levels in a reversible and tunable manner. This capability is valuable for investigating the effects of gene dosage on cellular functions and for studying regulatory networks.
  3. Genome-Wide Screens: CRISPRi screens allow for large-scale interrogation of gene function by systematically targeting and repressing individual genes across the genome. These screens can identify genes involved in specific biological pathways, disease processes, or cellular phenotypes.
  4. Epigenome Editing: CRISPRi can be used to target specific regions of the genome for epigenetic modifications, such as DNA methylation or histone modifications, without altering the underlying DNA sequence. This capability has implications for studying epigenetic regulation and potentially treating diseases associated with aberrant epigenetic marks.
  5. Synthetic Biology: CRISPRi enables precise control over gene expression for synthetic biology applications, such as metabolic engineering, biosensor development, and the construction of gene circuits. It allows researchers to fine-tune cellular behavior and engineer complex biological systems.
  6. Therapeutic Applications: CRISPRi holds promise for therapeutic interventions by selectively repressing the expression of disease-causing genes. Unlike CRISPR-Cas9, which introduces permanent genetic changes, CRISPRi offers a reversible and potentially safer approach for modulating gene expression in therapeutic contexts.
  7. Functional Genomics: CRISPRi technology complements other genomic tools, such as RNA interference (RNAi) and CRISPR knockout (CRISPRko), by offering additional control and specificity for studying gene function and regulation.
    Perspectives
    • Understanding the cellular effects of disease-causing mutations in neurodegeneration is a vital tool for understanding disease mechanisms and therapies. Modulation of gene expression using CRISPR is a key tool for understanding the relationship between genetic variants and the functional impact on cells.
    • CRISPRi allows robust and sustained knockdown of genes in disease-relevant neuronal and glial cell types and can identify novel biology using pooled genome-wide screens. CRISPRi may also be used to knock down disease-causing mutations in relevant cell types to phenocopy patient neurons.
    • Multiplexing CRISPR guides with imaging/selection markers will enable innovative applications of CRISPRi. These applications include GI mapping and -omics readouts at scale in addition to specific applications such as cell painting. As well as identifying genes that modify the risk conferred by mutations with incomplete penetrance.

Overall, CRISPR interference is a versatile tool with diverse applications in basic research, biotechnology, and potential clinical interventions. Its precise targeting and ability to modulate gene expression make it a valuable asset for understanding and manipulating biological systems.
https://www.news-medical.net/life-sciences/CRISPR-Interference-(CRISPRi)-for-Gene-Suppression.aspx

Dr. Md. Monirul Islam
Senior Scientist
ASRBC, ACI Seed

Fig: An overview of the utility and applications of CRISPRi in patient and control iPSC-derived cells. CRISPRi can be integrated with various cell models such as control and patient-derived neurons, glia, and organoids. This approach can be used to create new disease models from control neurons and understand the effects of disease-causing mutations (or risk modifiers) using patient neurons. In addition, these cell models apply to various single-readout screens, such as survival or FACS-based screens, and screens with more complex readouts such as genetic interactions, cell painting, and -omics. Additionally, CRISPRi can be integrated with drug discovery methods such as small molecule screening and validation of screening hits