Question: What should I consider when designing my 3' or 5' CRISPR pool?
Answer: When designing a CRISPR pool, some key factors should be taken into consideration:
The number of perturbations being included in the pool/study
The number of guides per perturbation
The percentage of non-targeting guides present in the pool/study
The number of perturbations (or targets) and the number of guides per perturbation are both largely driven by the specific research question to be answered.
If the experiment is an exploratory screen, we typically suggest incorporating 1-5 gRNA's per gene to identify guides with high on-target activity (i.e., knock-down or activation) and low off-target activity. This ensures that, when moving towards the final pool, the guides can be down-selected to only those with high on-target activity avoiding false positives or less efficient gRNAs.
It is also vital to ensure that a proportion of cells containing non-targeting guides is included in the pool to ensure that the perturbation (i.e., knock-down or activation) can be calculated.
When designing the pool, we suggest following these general principles to enable an accurate assessment of perturbation (i.e., knock-down or activation) efficiency:
- ~90% of the pool is made up of cells containing guides targeting genes of interest
- Aiming for 100-200 cells per guide to ensure a decent ratio of cells with a perturbation to cells without a perturbation
- ~10% of the pool is made up of cells containing non-targeting genes
Products: Single Cell Gene Expression, Single Cell Immune Profiling