Question: Do you recommend enriching for transduced cells?
Answer: In general, we do suggest enriching for transduced cells as there are many factors that can impact the proportion of cells that contain a guide (these will provide template for our CRISPR Screening Assay) and cells lacking a guide (these cells can provide useful Gene Expression information but cannot provide functional material/template for our CRISPR Screening Assay).
For most typical pooled library screens, cells are infected at a very low multiplicity of infection (MOI) to increase the chances that an infected cell receives only one guide RNA. However, this naturally results in a significant proportion of the cells lacking a guide RNA.
A positive screening strategy can be employed which will result in the loss of many of the transduced cells, however those that survive the selection conditions (most commonly antibiotic) will be enriched for cells containing guide RNAs. It is worth noting that the cell type (i.e. cell lines or primary cells) being utilized in the screen may impact whether a positive screening strategy can be implemented.
Cells can also be enriched by using FACS to sort cells expressing fluorescent proteins (i.e. GFP, mCherry etc).
Combining both strategies (positive selection and FACs) can result in a population of cells where ~90% of all cells contain a single guide. This yields a final sgRNA library trace in line with our Gene Expression and CRISPR Screening User Guide:
If the final sgRNA library trace does not have this distribution, possible causes can be:
- A high proportion of the cells lacking a guide RNA which can result from
- The cells being utilized in the screen not having a stable/consistent level of Cas9 expression
- Very low transduction efficiency
- Inefficient or ineffective selection
- Problems with the vector or sgRNA
An example of an enrichment strategy that has been shown to be compatible with Single Cell CRISPR Screening protocols is outlined below:
- Cells stably expressing Cas9 are transduced at a low to moderate MOI (0.1 - 0.5)
- Post transduction, cells are allowed to recover for ~2 days, then selected to purity using puromycin for ~3 days
- After another period of recovery, ~2 days, GFP levels are recorded via flow cytometry, using BFP expression to gate for transduced cells
- For estimating the level of knock-down, GFP levels from normal (GFP-) cells were subtracted
- Reference: https://www.biorxiv.org/content/10.1101/503367v1