FAQ: Frequently asked questions

CRISPR, which stands for Clustered Regularly Interspaced Short Palindromic Repeats, is a revolutionary gene-editing technology. 
The CRISPR system includes a guide RNA (gRNA) that guides the Cas9 enzyme to a specific target DNA sequence. Cas9 then acts like molecular scissors, cutting the DNA at the targeted location. The cell’s natural repair machinery can then introduce changes (mutations) to the DNA during the repair process, leading to gene editing.

CRISPR screening is a technique that uses the CRISPR/Cas9 gene editing technology to systematically knock out or activate genes in a high-throughput manner. In drug discovery, it is employed to identify genes that are relevant to a particular disease or therapeutic target, helping researchers understand gene function and discover potential drug targets.

CRISPR screening enables researchers to perform large-scale functional genomic studies by systematically perturbing genes and observing the resulting effects on cellular phenotypes. This approach helps identify key genes involved in disease pathways, validates potential drug targets, and uncovers novel therapeutic opportunities.

Challenges include off-target effects, incomplete gene knockout or activation, and the need for careful experimental design. Additionally, the interpretation of screening results can be complex, and the validation of candidate targets requires further investigation.

CRISPR screening has contributed to the identification of novel drug targets and the elucidation of disease pathways. It has been successfully applied in various therapeutic areas, including cancer, neurodegenerative diseases, autoimmune and infectious diseases. Some studies have identified genes essential for cancer cell survival or genes associated with drug resistance, paving the way for the development of new therapeutic strategies.

Single-cell CRISPR screening involves applying CRISPR technology at the single-cell level, allowing researchers to examine the effects of gene perturbations on individual cells rather than analyzing populations. This method provides a higher resolution of cellular heterogeneity and enables the identification of rare cell populations or subtle phenotypic changes that might be missed in bulk screenings.

Advantages include the ability to capture cellular diversity, identify rare subpopulations, and uncover cell-to-cell variability. Single-cell CRISPR screening is particularly valuable for studying heterogeneous diseases, such as cancer. Challenges include the need for specialized technologies to isolate and manipulate single cells, increased complexity in data analysis, and considerations for the scalability of experiments.

Single-cell CRISPR screening has applications in drug discovery by providing insights into the genetic basis of cellular responses to treatments at a high-resolution level. It helps identify specific genetic factors contributing to drug resistance or sensitivity, paving the way for the development of more targeted and personalized therapeutic strategies. This approach is especially relevant in precision medicine, where understanding individual cellular responses is crucial for tailoring treatments to patients.

CRISPR knockout involves completely disrupting the function of a target gene by inducing a frame-shift mutation or introducing a premature stop codon, leading to the loss of gene expression.

Applications in drug discovery screening:

• Target identification: CRISPR KO is used to systematically knock out individual genes across the
  genome in a high-throughput manner.
• Phenotypic screening: By observing the resulting cellular phenotypes after gene knockout, researchers can identify genes essential for specific cellular processes or pathways relevant to a disease.
• Validation: Hits from a CRISPR KO screen can be further validated using additional assays to confirm the role of specific genes in the disease process or drug response.

CRISPR interference involves using a catalytically inactive form of the Cas9 enzyme (dCas9) to interfere with gene expression without causing a permanent knockout. It achieves this by blocking transcription or inhibiting the binding of transcriptional machinery to the target gene.

Applications in drug discovery screening:

• Fine-tuning gene expression: CRISPRi allows researchers to modulate gene expression levels,
  providing a more nuanced approach compared to complete knockout.
• Identifying druggable targets: CRISPRi can be used to identify genes that, when partially inhibited, result in a desirable phenotype, making them potential druggable targets.
• Study of essential genes: CRISPRi is particularly useful for studying essential genes where complete knockout might be lethal.

CRISPR activation involves using a modified Cas9 enzyme to activate gene expression. This can be achieved by fusing the dCas9 with transcriptional activators to promote transcription from the target gene.

Applications in drug discovery screening:

• Identification of activators: CRISPRa is used to identify genes whose increased expression leads to a desired phenotype, helping to discover potential therapeutic targets.
• Studying gain-of-function effects: CRISPRa allows researchers to mimic gain-of-function scenarios, providing insights into the consequences of increased gene expression.
• Functional genomics: By activating genes one by one or in combination, researchers can systematically explore the functional impact of different genes on cellular processes.