What are the kit components of the Guide-it CRISPR Genome-Wide sgRNA Library System?
What are the kit components of the Guide-it CRISPR Genome-Wide sgRNA Library System?
There are five components to the library system as described below:
632646 Guide-it CRISPR Genome-Wide sgRNA Library System, 5 Screens
-Guide-it Genome-Wide sgRNA Library Transfection Mix, 2 x 5 vials
-Guide-it Cas9 Lentiviral Transfection Mix, 5 vials
-pLVXS-sgRNA-mCherry-hyg Vector, 5ug
-Lenti-X 293T Cell Line, 2 x 106 cells
-Lenti-X GoStix Plus (Sample), 3 tests
1. The Guide-it Genome-Wide sgRNA Library Transfection Mix is the library itself in our easy-to-use lyophilized Lenti-X Single Shots format and is used for performing phenotypic knockout screens in human cells. There is no need to amplify the library, as each vial contains all the necessary plasmids (library and lentiviral packaging plasmids) and transfection reagents to generate self-inactivating lentiviral particles containing a complete sgRNA library targeting the entire human genome (>19,000 genes). Simply resuspend the lyophilized transfection mix in water and add the mixture to Lenti -X 293T cells to produce lentiviral particles. The sgRNA library is expressed from the U6 promoter and includes four highly active guides per gene, with guide design based on the Brunello library (Doench et al. 2016). The library backbone is the pLVXS-sgRNA-mCherry-hyg Vector. In addition to expressing sgRNA, the sgRNA lentiviral particles also code for the mCherry fluorescent protein to facilitate simple, functional titer assays, as well as hygromycin resistance to enable selection for stably transduced cells. Representation of the guide RNAs has been verified by NGS in both the plasmid library as well as in transduced target cell populations to be >90% within a 10-fold range to improve the power of your screens. Use two vials per library screen (one vial for the treatment population, and the other for the untreated control population).
2. The Guide-it Cas9 Lentiviral Transfection Mix contains the pLVXS-EF1a-Cas9-PGK-Puro Vector in our easy-to-use Lenti-X Single Shots format. This Cas9 transfection mix is used to create your Cas9-expressing target cell line. Each vial includes all the necessary plasmids and transfection reagents to generate self-inactivating lentiviral particles that express Cas9 under the EF1-alpha promoter in transduced target cells. Simply add water to the vial, mix well, and then add the transfection mix to Lenti-X 293T cells to produce lentiviral particles ready to transduce your target cells. The vector contains a puromycin resistance gene to enable positive selection for stably transduced cells. Cell lines expressing Cas9 produced using this system can be used for gene editing experiments, including phenotypic knockout screens.
3. The pLVXS-sgRNA-mCherry-hyg Vector is the empty version of the vector used for manufacturing the library; it can be used to clone specific sgRNAs and confirm hits after performing screens. The cloning site features a BsmBI restriction site to enable insertion of candidate single-guide RNAs (sgRNAs) for downstream assays. Once the sgRNAs are re-cloned, the researcher can produce virus using the Lenti-X Packaging Single Shots (not included).
4. Our Lenti-X 293T Cell Line was clonally selected because it is easily transfected and supports a high-level expression of viral proteins, allowing you to produce the highest possible lentiviral titers (up to 108 IFU/ml) required for library screens.
5. We provide a sample kit of Lenti-X GoStix Plus, which is the simplest method ever to measure lentiviral titer. The test involves applying 20 µl of supernatant to a GoStix cassette and waiting 10 minutes for the appearance of test and control bands that indicate the presence of lentiviral p24. The results on the cassette can then be analyzed using a free smartphone app, which quantifies lentivirus titer by comparing the intensities of the test and control bands to an internal control curve. Learn more about Lenti-X GoStix Plus.