CRISPR Screenowanie i Wykrywanie Celów

CRISPR-Cas9 System Components

Cas9 protein – a DNA endonuclease that cleaves both strands of the target DNA molecule.

Guide RNA (gRNA) – a short RNA sequence that directs Cas9 to the specific genomic location for cleavage.

Protospacer – the DNA sequence targeted by gRNA.

CRISPR-Cas9 Screening Protocol

The goal of this protocol is to identify genes that regulate the expression of a target gene using CRISPR-Cas9 technology. This approach, known as CRISPR screening, allows researchers to systematically assess the impact of genetic perturbations on cellular processes.

First, guide RNAs (gRNAs) are designed to target different regions of the genome. A key consideration is to select gRNAs that have minimal off-target effects – meaning they don’t unintentionally cut at other locations in the DNA sequence. The number of gRNAs used depends on the size of the gene being screened and the desired level of stringency.

Next, a library of gRNAs is generated, typically using a chemical synthesis method. This library contains many different gRNAs that can be tested simultaneously. Each gRNA directs the Cas9 protein to a specific location in the genome where it can cut both strands of DNA.

The cells are then subjected to CRISPR-Cas9 mediated gene editing. The resulting cells will have mutations at the target sites identified by the gRNAs. These mutations can disrupt the function of the targeted genes, allowing researchers to identify those that influence the expression of the target gene.

Following gene editing, a phenotypic screen is performed to assess the impact of the mutations on cellular characteristics. This could involve measuring changes in cell growth, viability, or other relevant parameters. The goal is to identify cells with altered phenotypes – i.e., cells where the targeted genes have had an effect on the target gene's expression.

Hits identified during the phenotypic screen are then validated using more specific techniques, such as quantitative PCR (qPCR) and Western blotting. These methods confirm that the mutations at the target sites are indeed responsible for the observed changes in cellular characteristics. Secondary validation may also involve functional assays to assess the direct impact of the targeted genes on the expression of the target gene.

Finally, pathway analysis is performed to understand how the identified hits contribute to the overall regulation of the target gene. This can provide insights into the complex networks of interactions that govern gene expression.

Ekranowanie CRISPR i Identyfikacja Celów

Przykład: Ekranowanie syntetycznie śmiertelne (Synthetic Lethal Screen)

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