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Dropout Viability Screens

Cellecta RNAi viability screen flowchart

Cellecta RNAi viability screen flowchart

Identify Genes Essential for Growth and Proliferation

  • Identify genes essential for cell viability
  • Find targets that increase cell sensitivity to compounds or treatments
  • Look for synthetically lethal interactions with known genetic lesions

Using complex pooled lentiviral-based shRNA and CRISPR libraries, Cellecta can perform an unbiased genome-wide or focused loss of function screen to identify gene functions which are required for cell viability. Many oncology groups utilize this sort of screen to find potential therapeutic targets to disrupt cell growth or proliferation.

A Typical Dropout Screen

A typical dropout screen to identify genes essential for cell viability involves an RNAi screening of 2 or 4 cell lines over a period of 6 to 12 cell divisions. CRISPR screens typically require more doublings in order to show the phenotype. Target cells are infected with a complex lentiviral shRNA expression library for which the shRNA sequence distribution has been characterized by Next-Gen Sequencing (NGS). After a period of growth producing several cell doublings, the distribution of shRNA sequences in the cultured cell population are assayed again by NGS of the uniquely identifiable barcodes present in the genomic DNA of the infected population.

By comparing the number of each shRNA sequence remaining in the cells cultured for a period of time under optimal conditions with the shRNA in the starting library, it is evident which shRNA inhibit cell growth since they are underrepresented or depleted (i.e., they drop out), in the assay population. This is typically because these shRNA target genes essential for cell growth and continued cell viability, so cells containing shRNA interfering with these essential genes are selected against in the population over time.

As there are many essential genes in any cells, multiple cell lines are often necessary to identify which set of essential genes are associated with a specific biology. For example, determination of essential genes for cells containing a specific gene lesion (e.g., a RAS inactivation mutation or a MYC translocation) would require, at minimum, comparison of essential genes in cells that are normal for the lesion with cells containing the lesion; thus, two cell lines. The remaining genotype of the two cell lines should be as close as possible to help ensure that any variations in the responses is a result of the genetic difference of interest between the lines. Ideally, isogenic lines engineered to have a single genetic variation between them are optimal for these sort of studies. Results also improve when multiple cell lines containing the lesion are compared with more than one line that is wild-type for the genetic variation.

For broad-based or genome-wide dropout screening, the Human Genome-Wide shRNA Library is the best choice. We also offer the option of doing a screen with a custom library targeting any set of genes.

An example of an RNAi prostate cancer viability screen with a custom focused shRNA library is provided in a recent Application Note published in BioTechniques (PDF).

Chronic Myelogenous Leukemia K562 Lethality Screen Scatter Plot

Chronic Myelogenous Leukemia K562 Lethality Screen Scatter Plot

How Many Cells Are Needed for a Dropout Viability Screen?

With dropout viability screens, it is critical to infect enough cells so that each shRNA sequence appears in enough cells to stably grow in a mixed population for several passages. The negative effect on growth of select shRNA sequences is only visible in the background of a vigorously grown culture—these sequences are depleted in representation after several passages.

To generate robust data and differentiate true positives from genetic drift in the growing population, it is essential to tightly control the number of screening parameters. We routinely infect at minimum 200-400 times more cells than shRNA sequences in the library—so, for a library of ~55,000 shRNA sequences, over 10 million cells should be transduced. In addition, biological triplicates are necessary to eliminate spontaneous dropouts. We have optimized these parameters from experience to help ensure that we generate robust data. However, they do practically limit the size of the library we can effectively and routinely screen. This is the reason most of our predefined libraries have ~55,000 shRNA.

How Long Does a Dropout Viability Screen Take?

An RNAi screen itself takes 2-3 months, while a CRISPR screen may take slightly longer. If you are interested in having Cellecta perform an RNAi or CRISPR screen to identify genes critical for the response of interest in your biological system of interest, please contact us at sales@cellecta.com.