Assay Development
The majority of the work that goes into a screen occurs beforehand in the assay development phase. Several experiments must be conducted to ensure proper setup and execution of the downstream screen, and we recommend following the steps outlined below:
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Identify the appropriate lentiviral vector(s):
- For guidance on vector choice, please review Vectors for Assay Development.
- For complete customization of a new vector, see our modular vector assembly tool, Fragmid. If what you are looking for is not available in the Fragmid toolkit, designing a new fragment is straightforward: Adding a New pF Fragment to Fragmid.
- To order a small DNA aliquot of a vector, use this form: Individual_Vector_Order_Form.xlsx.
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Review the basics of lentiviral transduction:
- For those new to working with lentivirus or seeking tips on lentiviral transduction, refer to the optimization protocols for a Lentiviral Spinfection
- or a Lentiviral No-Spin Infection.
- For difficult-to-transduce cells, consider adding VPX Viral-Like Particles to improve transduction efficiency.
- Identify the proper concentration of the relevant selection drug(s) in the cells of interest: Puromycin, Blasticidin and Hygromycin Titration.
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Confirm Cas activity:
- We maintain a suite of positive control vectors to confirm Cas activity: Vectors for Assay Development. Example protocols: CRISPRko, CRISPRko all-in-one, CRISPRa/i or CRISPRbe.
- Cas expression can be confirmed using the Cas9 Detection Using Flow Cytometry protocol, but an activity-based assay is much preferred.
- To order an aliquot of virus of a control, Cas, or activity assay vector, use this form: Individual_Vector_Order_Form.xlsx.
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Optimize the transduction conditions of the library virus to ensure one integration per cell:
- Empirically test a range of virus volumes by performing the Pooled Screen Viral Titration protocol.
- Take Coverage Considerations In Pooled Genetic Screens into account at this stage to achieve the most accurate screening results.
- To order a small aliquot of library virus to test in the cells of interest, review Ordering Pooled Libraries.
FAQs:
Q: Can individual reagents be ordered for an assay development experiment?
GPP maintains individual (arrayed) reagents that can be provided as bacterial streaks, plasmid DNA and/or lentivirus:
- sgRNAs
- Largely for CRISPR knockout
- shRNAs
- Our collection of RNAi reagents
- ORFs
- Human Open Reading Frames
Q: Can individual customized reagents be generated for a screen?
GPP can produce custom sgRNA or shRNA constructs in 96-well plate format.
For small numbers of constructs, our recommended protocol is: Cloning of sgRNA or shRNA Constructs. To then prep the constructs as a glycerol or DNA stock, review our shRNA/sgRNA/ORF Glycerol and Plasmid DNA Preparation protocol.
Q: Can library plasmid DNA be amplified to make lentivirus?
GPP can amplify existing libraries to create more pDNA and/or lentivirus, following this protocol: pDNA Library Amplification.
Q: What protocol does GPP use to make lentivirus?
We use the following protocols for lentivirus production in varying formats:
For experiments that require a more concentrated form of lentivirus, review our Lentiviral Concentration protocol. To determine lentiviral titer, we recommend performing our Functional Viral Titering Assay. To produce VPX viral-like particles to improve transduction efficiency in challenging cell models, follow our protocol: Production of VPX Viral-Like Particles.
Q: If thinking about an in vivo screen, are there additional resources?
Indeed. Maintaining coverage in an in vivo setting often requires specific experimentation. Cell Counter Libraries can be used for this purpose.