Genome-wide sgRNA library system
The Guide-it CRISPR Genome-Wide sgRNA Library System makes it easy to perform lentiviral-based, genome-wide phenotypic knockout screens using CRISPR/Cas9 in human cell lines. Each screen contains both the genome-wide sgRNA library and a Cas9 expression system in our easy-to-use Lenti-X Single Shots format to produce cell lines stably expressing both Cas9 and the sgRNA library. The library is based on the Brunello library (Doench et al. 2016; Doench, 2018), and contains >76,000 guide RNAs (sgRNAs) with four highly active guides per gene, targeting about 19,000 genes as well as non-targeting sgRNA controls.
The Guide-it CRISPR Genome-Wide sgRNA Library System makes it easy to perform lentiviral-based, genome-wide phenotypic knockout screens using CRISPR/Cas9 in human cell lines. Each screen contains both the genome-wide sgRNA library and a Cas9 expression system in our easy-to-use Lenti-X Single Shots format to produce cell lines stably expressing both Cas9 and the sgRNA library. The library is based on the Brunello library (Doench et al. 2016; Doench, 2018), and contains >76,000 guide RNAs (sgRNAs) with four highly active guides per gene, targeting about 19,000 genes as well as non-targeting sgRNA controls.
After performing a screen, use next-generation sequencing (NGS) analysis with the Guide-it CRISPR Genome-Wide sgRNA Library NGS Analysis Kit, which includes the necessary primers and kits to prepare an NGS sample from cells transduced with our sgRNA library.
This library has been carefully manufactured to ensure proper sgRNA representation. We have measured sgRNA representation at every step in the process: library synthesis, amplification, viral production, and target-cell transduction. This kit contains the validated library in a dry-down lentiviral format and Lenti-X 293T cells. To produce viral particles, simply add water to the lyophilized library mix and then add it to the 293T cells in culture. The sgRNA content is verified in every lot via NGS analysis to contain >90% of all the stated sgRNAs within a 10-fold distribution range. We have also modified the sgRNA scaffold design in the pLVXS-sgRNA-mCherry-hyg Vector to improve the Cas9-sgRNA interaction, ensuring high editing efficiency. Representation of the lentiviral sgRNA library is maintained by utilizing our Lenti-X Packaging Single Shots, a validated, high-titer packaging formulation.
Overview
- Contains >76,000 sgRNAs based on the Brunello library which maximizes on-target specificity and activity, while minimizing off-target effects (download sgRNA representation data)
- Each gene in the human genome is targeted by four highly active sgRNAs, enabling screens with a single module
- Modified sgRNA scaffold design for improved Cas9-sgRNA interaction and high editing efficiency
- Lentiviral sgRNA library is already formulated with the Lenti-X Packaging Single Shots and the Xfect Transfection Reagent, such that only water is required to produce a highly efficient transfection mix for transfecting Lenti-X 293T cells to produce high-titer sgRNA libraries
Notes
The Guide-it CRISPR Genome-Wide Library PCR Kit (Cat. # 632651) contains all the primers and reagents necessary to amplify sgRNA sequences from genomic DNA isolated from your screened cell population for NGS analysis, and allows for preparation of ten sequencing libraries (20 PCRs).
The Guide-it CRISPR Genome-Wide sgRNA Library NGS Analysis Kit (Cat. # 632647) includes the Guide-it CRISPR Genome-Wide Library PCR Kit in addition to a genomic DNA purification kit and a PCR cleanup kit.
References
Doench, J.G. Am I ready for CRISPR? A user's guide to genetic screens. Nat. Rev. Genet. 19, 67–80 (2018).
Doench, J.G. et al. Optimized sgRNA design to maximize activity and minimize off-target effects of CRISPR-Cas9. Nat. Biotechnol. 34, 184–191 (2016).
More Information
Applications
- Pooled genome-wide phenotypic screens
- Loss-of-function genetic screens
Additional product information
Please see the product's Certificate of Analysis for information about storage conditions, product components, and technical specifications. Please see the Kit Components List to determine kit components. Certificates of Analysis and Kit Components Lists are located under the Documents tab.
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