For CRISPR screens, cells must express some variation of (e.g., Cas9 nuclease, dCas9-KRAB, or dCas9-VPH) in conjunction with library sgRNA. Most CRISPR sgRNA libraries provided by Cellecta express only sgRNA so, before starting a screen, it is necessary to engineer target cells that express the appropriate Cas9 variant for the screen. After packaging the appropriate lentiviral Cas9 construct into VSV-G pseudotyped lentiviral particles, cells are transduced with the Cas9 packaged particles to generate stable cell lines to make the Cas9 cell lines. Please keep in mind the following points with regard to Cas9 construct transduction:
- Generally, the higher the expression of Cas9 (or dCas9), the greater the effect on the target gene will be (up to a point where too high expression and/or viral load become toxic for the host cell). For the standard CRISPR system, knockouts will occur more quickly and at higher frequency. For the CRISPRi and CRISPRa systems, gene repression or induction will typically be more pronounced. As a result, it is desirable to ensure strong expression of the Cas9 gene. If you have not used lentiviral vectors in your target cells before, we suggest checking which promoters will work best for the selection and marker genes. The efficacy of pol II promoters, such as CMV, EFS, EF1, UbiC, and PGK, that drive expression of cDNA genes vary significantly across cell types. Cellecta sells pre-packaged control viruses expressing different marker genes from various promoters that can be used to determine which promoter will work the best in your cells.
- Cas9 is a large protein and lentiviral constructs with this gene are close to the limit for lentiviral packaging so Cas9 constructs package poorly. The yields are more than an order of magnitude lower than usually obtained with smaller sized lentiviral constructs. Packaging reactions should be set up at a scale to ensure you produce enough packaged viral particles for your experiments.
Protocol for Making Cas9-Expressing Cells for CRISPR Screens
This protocol assumes that you are using a Cas9 vector with the blasticidin resistance marker and have determined the virus dilution that gives 50% blasticidin-resistant cells (BR-50).
- To generate the best Cas9 cell line for your screen or other experiments, follow the Individual Lentiviral Construct Packaging and Transduction of Packaged Lentiviral Viral Particles Protocols to transduce the parental cell line with increasing amounts of virus. For packaged Cas9 constructs purchased from Cellecta, use MOI below based on the transduction units of the viral lot:
- MOI = 0 (negative control, no virus)
- MOI = 1
- MOI = 3
- MOI = 10
- 72h after transduction, start appropriate antibiotic selection. Be aggressive: use an antibiotic concentration that kills 100% control cells within 3-5 days. We suggest trying the antibiotic concentration ranges below, depending on the selection marker of the Cas9 virus:
- Blasticidin: 5-20 ug/ml
- Hygromycin-B: 50-400 ug/ml
- Puromycin: 0.5-10 ug/ml
- Assay the MOI 1, 3, 10 Cas9 lines for CRISPR activity using the appropriate CRISPR-Test Assay:
- CRISPRuTest for Cas9/CRISPR-KO (Cat. # CRUTEST)
- CRISPRaTest for dCas9-VPH/dCas9-VPR/CRISPRa (Cat. # CRATEST)
- CRISPRiTest for dCas9-KRAB/CRISPRi (Cat. # CRITEST)
- Additional Cas9 activity kits are available if none of the 3 kits above are suitable for your specific cell line: CRISPR-Test Cas9 and dCas9 Activity Test Kits – Cellecta
Based on CRISPR-Test results, choose the best MOI Cas9 line for downstream applications.
- Split the selected Cas9 cell line (“8x BR-50” in this case) into 3 samples, and continue selection with 1x, 2x, and 4x increases in blasticidin concentration.
- Sample 1: Same blasticidin concentration used to calculate BR-50
- Sample 2: 2x blasticidin concentration used to calculate BR-50
- Sample 3: 4x blasticidin concentration used to calculate BR-50
- After 1 week, select the Cas9 cells that survived the highest blasticidin concentration. Expand the selected Cas9 cell sample. This is the Cas9 cell line you will use in your screen or experiment.
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