The Guide-it CRISPR Genome-Wide Library PCR Kit is designed for NGS library preparation from cell populations screened using the Guide-it CRISPR Genome-Wide sgRNA Library System (Cat. # 632646), and is also sold as part of the Guide-it CRISPR Genome-Wide sgRNA Library NGS Analysis Kit (Cat. # 632647). The kit contains all the primers and reagents necessary for amplification of proviral sgRNA sequences from screened cell populations for analysis by NGS. This kit includes enough reagents for performing 20 PCRs.
The Guide-it CRISPR Genome-Wide sgRNA Library NGS Analysis Kit enables preparation of next-generation sequencing (NGS) libraries from cell populations screened using the Guide-it CRISPR Genome-Wide sgRNA Library System (Cat. # 632646), allowing for identification of sgRNA sequences and corresponding gene knockouts that have increased or decreased in frequency under the conditions of the screen. The kit provides all the components necessary to prepare ten sequencing libraries (20 PCRs), including a genomic DNA purification kit, primers, PCR enzyme and buffers, and a PCR cleanup kit.
The Guide-it CRISPR Genome-Wide sgRNA Library System includes all of the components necessary to perform five lentiviral-based, genome-wide CRISPR/Cas9 knockout screens in human cells. Provided with the kit are lentiviral vectors for expression of Cas9 and >76,000 sgRNAs targeting >19,000 human genes (four guides per gene) in lyophilized, ready-to-transfect formats that simplify the production of high-titer lentivirus. The sgRNA library included with this kit is based on the Brunello library (Doench et al. 2016), and incorporates design features intended to maximize on-target efficacy and minimize off-target effects. To ensure balanced guide representation, we analyze the sgRNA library by NGS to confirm that >90% of the included sgRNAs fall within a 10-fold distribution range. Enrichment or depletion of sgRNAs in screened cells can be analyzed by NGS with the Guide-it CRISPR Genome-Wide sgRNA Library NGS Analysis Kit (Cat. # 632647).
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
Applications
- Pooled genome-wide phenotypic screens
- Loss-of-function genetic screens
