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 and this article on the choice of Capture Sequence and sgRNA integration site.
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
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