Recommendations for Positive Selection Viability Screens (a.k.a. Survival or Rescue Screens)

Rescue screens allow for identification of genes that are functionally required for cell sensitivity to a treatment or to understand underlying drug mechanisms. Positive screens are also known as enrichment screens. Many positive screens use FACS to look for modulators of signaling molecules such as NF-κB, p53, c-myc, HSF-1, and HIF-1α using fluorescent reporter cell lines, or cells expressing specific antibody-detectable markers, such as specific receptors.

Length of the Screen

A positive screen involves a selection that eliminates most of the cells. With this sort of screen, the goal is to isolate a small population of cells with sgRNAs that enable the cells to pass through the selection step. The critical factor here is the nature of the selection, which ultimately determines the screen procedure.

NOTE: In most cases, it is advisable to wait 1-2 weeks after sgRNA library transduction before carrying out the selection step. The wait period is needed to allow knockout of the alleles of the target gene in most transduced cells, and the development of the resistant phenotype before applying selection. Cells should then be harvested as soon as positive selection is completed. Growing and expanding clones after positive selection is not advised.

MOI of Transduction

A positive screen involves isolation of a small population of cells with sgRNA sequences that will be over-represented or enriched when compared to the starting library sgRNA counts. As with any screen, to ensure reproducible and reliable results, it is critical that you transduce enough cells to maintain sufficient representation of each sgRNA construct present in the library. The number of cells stably transduced with the sgRNA library at the time of transduction should exceed the complexity of the sgRNA library by at least 500-fold.

Example: The starting population should be at least 25 million infected cells to start a screen with a library with 50,000 sgRNAs.

Antibiotic Selection of Transduced Cells

In order to prevent loss of transduced cells and enrichment for multiple integrants, it is recommended that antibiotic selection of transduced cells based on library lentivector selection marker (usually puromycin) is started no sooner than 72 hours after library transduction. Also, it is recommended to use the appropriate antibiotic concentration that removes the non-transduced cells with minimal killing of transduced cells. To determine this concentration, please follow the procedure described in “Antibiotic Selection of Library Transduced Cells”.

Maintenance of the Cells

A positive selection screen often involves the comparison of two types of samples: selected and unselected (control) samples. After transduction and before selection, it is best practice not to discard any cells. However, this is often not practical. If cells have to be discarded or split before beginning the selection, the number of remaining cells in each sample should always exceed the complexity of the library by at least 1,000-fold (e.g., keep at least 5.0 × 107 cells after every splitting step, for a 50K library).

Baseline Controls for Positive Selection Screens

In order to calculate fold-enrichment of the sgRNA sequences present in the selected population, a baseline control is needed. Depending on the screen, the plasmid library itself, the pre-selection cells, or the mock-selected cells can be used as baseline.


Recommendations for Negative Selection Viability Screens (a.k.a. Dropout Viability Screens)

A standard dropout viability screen (negative selection screen) relies on the fact that some of the gRNAs in the screen are either cytotoxic or cytostatic (presumably by having knocked out an essential target gene). Cells with gRNAs that do not inhibit growth grow normally, populating the culture; cells with lethal gRNA do not propagate.

The endpoint analysis involves looking for gRNA sequences that are underrepresented in the sample population relative to the original library.

Length of the Screen

For a CRISPR dropout viability screen to work, the cells need to be cultured long enough for knockouts to develop in all the alleles of each gRNA’s target gene, and then for cells with unaffected growth to significantly increase their proportion relative to the affected cells.

NOTE: The length of any particular screen may need to be altered depending on the specifics (e.g., cell growth rates, types of targets of interest, use of additional compounds, etc.).

  • Typically, we find allowing for 3 weeks after transduction to be a good starting point.

If the screen is not run long enough, all the gRNA counts will be in a narrow range and it will be difficult to identify significantly depleted gRNA sequences from background variability. If the screen is run too long, the range of representation of gRNA sequences may become broader due to the natural growth variance in different cells in the population. This phenomenon, often referred to as genetic drift, can increase the background variance of the screen and make it difficult to identify significantly depleted gRNAs from background variability.

Antibiotic Selection of Transduced Cells

In order to prevent loss of transduced cells and enrichment for multiple integrants, it is recommended that antibiotic selection of transduced cells based on library lentivector selection marker (usually puromycin) is started no sooner than 72 hours after library transduction. Also, it is recommended to use the appropriate antibiotic concentration that removes the non-transduced cells with minimal killing of transduced cells. To determine this concentration, please follow the procedure described in “Antibiotic Selection of Library Transduced Cells”.

Maintaining Library Representation throughout Screen

As mentioned above, the number of cells stably transduced with the sgRNA library at the time of transduction should exceed the complexity of the sgRNA library by at least 500-fold. For a library with 50,000 sgRNAs, the starting population should be at least 25 million infected cells. The MOI of transduction should be kept at or below 0.5 to ensure that most transduced cells carry only one integrated provirus.

After transduction, the ideal is to never discard any cells at any time during the experiment (e.g., at treatment, harvesting, DNA purification, etc.). However, this is often not practical—especially for a negative screen where most of the cells propagate normally. If the number of cells becomes too large and you are forced to discard a fraction, the number of remaining cells should always exceed the complexity of the library by at least 1,000-fold (e.g., for a 50K library screen, keep at least 50 million cells after every splitting step). Similarly, when amplifying gRNAs from isolated DNA, you should always use all of the genomic DNA recovered from cell samples, up to a number of cells corresponding to 1000x the library size.

Baseline Controls for Negative Screens

In a simple screen aimed at identifying gRNAs which are cytotoxic in a given cell line, we typically use the library itself as the baseline control since the sgRNA frequency distributions in both plasmid and packaged lentiviral libraries are virtually identical. The plasmid library has already been sequenced as part of the QC when we made the library, so it is not necessary to re-sequence the library at this point. If you would also like to use transduced cells as a baseline control, we typically recommend harvesting and sequencing genomic DNA from cells by about 18 hours post-transduction.

In more complex experiments, aiming at identifying differential toxicity between isogenic cell lines or between compound-treated and non-treated cells, other baselines will be needed.

Last modified: May 04, 2019

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