GeneCopoeia offers CRISPR sgRNA libraries for high-throughput knockout of human genes in specific or custom gene groups or pathways. Loss-of-function screening by gene knockout is a powerful tool for systematic genetic analysis in mammalian cells, facilitating gene discovery, genome-scale functional interrogation (e.g., signal transduction pathways) and drug discovery (e.g., target identification and drug mechanism studies).
The GeneHero™ human single guide RNA (sgRNA) libraries are individually cloned into lentiviral vectors and sequence-verified for dual-use (transfection or transduction) delivery methods designed for large-scale functional screens. For each targeted gene, a minimum of 2 barcoded sgRNAs targeting different regions are created, optimized and sequence-verified to ensure efficient gene knockout. Further, each sgRNA-expressing plasmid is individually cultured in E. coli before pooling, providing the best possible representation of each sgRNA in the pools. The libraries can be ordered as pools of sgRNAs in pre-defined gene families or as custom sets, and are available as lentiviral particles, transfection-ready plasmid DNA or bacterial stocks. They can be transfected or transduced into cell lines, including those stably expressing Cas9 nuclease.
Figure 1. Illustration of large-scale screening with sgRNA library
Applications
High-throughput knockout screening with many sgRNAs, either individually or in pools.
Drug target discovery (Figure 2).
Drug target validation.
Phenotypic screens.
Reporter assays.
Figure 2. Workflow for CRISPR sgRNA libraries. A. Pooled screen. Cells infected with each sgRNA library pool are screened for the desired readout. Pooled cells are subjected to Sanger sequencing for individual sgRNAs, or deep sequencing to look for over- or under-representation of individual sgRNAs. B. Knockout screen using arrayed sgRNAs. Cells are infected with individual sgRNA lentiviruses. Wells are screened for the readout of interest. Individual sgRNAs corresponding to the phenotype of interest are already known without sequencing.
The GeneHero™ CRISPR human sgRNA libraries consist of sequence-verified sgRNA target sites cloned into a lentiviral vector. The clones do not carry the Cas9 nuclease. You will need to either purchase a lentiviral Cas9 clone or a cell line stably expressing Cas9. For lentiviral transduction, we recommend using a cell line stably expressing Cas9 before transduction with the libraries.
Human sgRNA libraries
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Each sublibrary is a tube containing ~1 ml of glycerol stock.
Each sublibrary is a tube containing 10-15 μg of transfection-ready DNA.
Each sublibrary is a tube containing 2.5 x 106 TU lentiviral particles in 25 μL.
Answer: Yes. Currently, our sgRNA libraries are available standard as pools. However, if you wish to receive your libraries as individually arrayed clones, simply contact us for a custom quote.
Answer: Yes. However, we strongly recommend that you first establish a cell line that is stably expressing Cas9, in order to best maintain consistent representation of each sgRNA in the library. First, transduce your cells with the Cas9 lentiviral clone. Once you have established the stable Cas9-expressing cell line, use the sgRNA libraries for transduction.
Answer: You can’t completely ensure that every cell is infected with one sgRNA, or that all sgRNAs are represented in the pool of infected cells. However, these libraries are small, so if the number of infected cells was 100 × the number of sgRNAs in the library, then you should have close to complete representation.
Answer: Yes. The lentiviral plasmids are “dual-use”, so that they can either be packaged into lentiviral particles or transfected into cells by standard transfection methods.
