Processing
CRISPR screening
Overview
CRISPR screening is an experimental approach used to find a small number of important genes or genetic sequences within a massive number of genetic sequences (e.g., within the entire genome). CRISPR screening can identify genes that influence drug resistance and many other physiological effects.
What is CRISPR screening?
CRISPR screening is an experimental method that uses CRISPR genome editing to identify a small number of genes (out of the whole genome) involved in a certain physiological effect within cells.
Applications of CRISPR screening include identifying genes associated with a disease, that cause drug resistance in pathogenic organisms or in cancer cells, that cause susceptibility to environmental toxins, and many other uses in basic research on cells.
The CRISPR basics handbook
Everything you need to know about CRISPR, from A to Z, from theory to practice, for beginners as well as advanced users.
How do I screen with CRISPR methods?
CRISPR screening works because of the ease with which insertion or deletion mutations (indels) can be introduced at any targeted site in the genome by CRISPR genome editing. These targeted gene knockout events are the key for most CRISPR experiments[1].
Most CRISPR screening is done in cell culture, although some methods have been devised for use in animal models. In most CRISPR discovery experiments, scientists knock out every gene that could possibly be important for a certain phenotype, knocking out only one gene per cell. In the resulting population of cells, some cells die, while others survive and may even be able to grow better, becoming the predominant cell type. Then, the scientists do next-generation sequencing (NGS) on the final population of cells to determine the gene responsible for the observed phenotype.
Some CRISPR screens use individually plated (arrayed) CRISPR guide RNA (gRNA), which can be used in ribonucleoproteins for CRISPR screening on cells in multiwell plates. This approach does not use lentiviruses. However, most CRISPR screens start with a pool of DNA oligos which are used to make a pool (library) of lentiviruses. Both the non-viral (arrayed) and viral (pooled) approaches to CRISPR screening have pros and cons. Selecting the type of screen should take into consideration many factors. For more detailed information on this choice and for more explanation of how both types of screens are used, see our DECODED article, Overview: What is CRISPR screening?
CRISPR vs. RNAi screens
RNA interference (RNAi) can also be used to screen the genome for genes of interest, but the RNAi approach is limited by its relatively low efficiency and precision. CRISPR screens circumvent these limitations and can be used to target almost every gene in any genome systematically, to determine the roles genes play in physiological effects. However, RNAi screens still have value when scientists wish to screen for genes that have different effects when partially knocked down. The choice of method therefore depends on the kind of results needed in the experiment.
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References
- Poirier JT. CRISPR libraries and screening. In: Torres-Ruiz R, Roguez-Perales S, eds. Progress in Molecular Biology and Translational Science. Vol 152. Cambridge: Academic Press, an imprint of Elsevier; 2017:69-82.
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