Question: What resources are available for my CRISPR experimental design?
Answer: Ultimately, optimizing experimental design prior to sequencing can help guarantee accurate sgRNA detection and a successful CRISPR screen. There are a few considerations and resources available when designing a Single Cell CRISPR experiment.
Experimental Design Considerations
Traditionally, cell lines are transduced with a pooled lentiviral library containing guide RNAs (gRNAs) targeting tens to hundreds to thousands of genes in a given genome. Some considerations during experimental design are:
1) sgRNA design strategy - please see Technical Note: Guide RNA Specifications Compatible with Feature Barcoding technology for CRISPR Screening (for both 3' and 5' CRISPR screening) and this article on the choice of Capture Sequence and sgRNA integration site (for 3' CRISPR screening).
2) Pool design strategy - please see the article How should I design my CRISPR pool?
3) Cell transduction
Some factors to consider:
- Transduction efficiency: the proportion of cells that are successfully transduced with the CRISPR/dCas9 constructs and sgRNA.
- Modification: Proportion of those cells that are subsequently modified at the target locus by dCas9
- Modification coverage: Proportion of those cells that contain desired modification in all copies of the gene.
- The multiplicity of infection (MOI): Proportion of infective particles relative to the number of cells in the sample. For tips on determining MOI, please see the article: What is MOI and how do I assess it?
4) Selection: To enable the selection of cells containing sgRNA, we recommend will need to go through rounds of passages and selection to ensure the population is enriched for the cells containing sgRNAs. A double selection strategy is recommended (Puromycin/blasticydin selection and Flow sort for GFP/BFP marker). For more information, please see the article: Do you recommend enriching for transduced cells?
5) Cell lines: If working with culturable cells (e.g. suspension or adherent), it may be worth considering establishing a stable Cas9-expressing cell line. Then, gRNAs for different targets can be introduced to the Cas9-expressing cells through transient transfection.
Many different suspension and adherent cell lines have previously been used to establish Cas9-expressing cell lines with common examples including A375, HEK293T, Jurkat, and K562.
Experimental Design Resources
- We have more information about getting started with CRISPR in our 3' CRISPR webinar.
- See "Direct capture of CRISPR guides enables scalable, multiplexed, and multi-omic Perturb-seq" (https://www.biorxiv.org/content/10.1101/503367v1) as a reference.
- Millipore has numerous online resources available for CRISPR experiment design.
Products: Single Cell Gene Expression, Single Cell Immune Profiling