Mouse Whole-Genome Dual-gRNA Library

Amerigo Scientific offers high-quality, premade dual-gRNA lentivirus libraries for CRISPR-based whole-genome knockout screens in human and mouse cells. Wherever possible, each gene is targeted redundantly by 4-6 different gRNA pairs in separate vectors. These libraries can serve as powerful and highly cost-effective tools for performing genome-wide loss-of-function screens. Additionally, we can design and construct custom pooled gRNA libraries for your target gene list.

Types of dual-gRNA CRISPR libraries offered
Ready-to-use, pooled, lentivirus libraries targeting the entire human genome (20,048 human genes)
Ready-to-use, pooled, lentivirus libraries targeting the entire mouse genome (20,493 mouse genes)

• Unique dual-gRNA lentiviral vector design
As the only commercially available whole genome dual-gRNA lentivirus libraries, each lentiviral vector in the libraries contains a pair of gRNA expression cassettes and an EGFP/puromycin dual marker cassette. The paired gRNAs from the same vector are expressed simultaneously when the vector is introduced into cells, targeting two separate sites on the same gene, resulting in two CRISPR cut sites that then lead to large loss-of-function deletions for the knockout screen. The two gRNAs in each vector are coupled with two different U6 promoters (human U6 and macaque U6) and two different gRNA scaffolds (described in Nat Methods. 14:573 (2017)) that are distinct in sequence but equivalent in function. This reduces unwanted recombination between the two gRNA cassettes and also allows the PCR amplification and sequencing by NGS of either the upstream or the downstream gRNA, or both, from cells transduced with the libraries.

• Whole genome and high-coverage targeting
The human and mouse dual-gRNA lentivirus libraries target 20,048 human genes and 20,493 mouse genes, respectively. On average, each gene is targeted by 4-6 different gRNA pairs selected by a set of parameters, including 1) off-target scores to minimize off-target effect; 2) locations of the cut sites to ensure that deletions spanning the two sites would most likely lead to the loss of gene function; 3) distances between the cut sites to bias the resulting mutations toward large deletions across the cut sites rather than local mutations at each cut site, and 4) number of alternative transcripts affected in each gene to maximize the number of transcripts being knocked out. All the gRNA cut sites are located within annotated exons. Therefore, even if large deletions fail to be created between sites, any local mutations within a cut site (typically small deletions) can also generate mutations that are likely to disrupt gene function. Detailed lists of target genes and gRNA can be found under “Documents”.

• Validation of library quality by NGS
The dual-gRNA lentivirus CRISPR libraries have been validated by NGS in which both gRNAs of the pairs have been fully sequenced. This showed that >85% and >75% of the total sequencing reads matched the designed gRNA pairs for human and mouse libraries, respectively. Furthermore >99% and >97% of the designed gRNA pairs for human and mouse libraries were detected by NGS, respectively. Thus, both libraries have excellent coverage of the designed gRNA pairs with low error rate. To our knowledge, these are the highest-quality dual-gRNA libraries that have been reported.

• High uniformity
The representation of gRNA pairs in both libraries are highly uniform

• Well-established lentiviral system and ready-to-use high-titer lentivirus
The pooled CRISPR libraries are expressed in the third-generation lentiviral vector system, which is a highly efficient expression system in a wide variety of cells. This lentiviral vector system is ideal for in vitro genetic screens since it can introduce gRNAs into cells permanently, efficiently and relatively uniformly. All pooled CRISPR libraries are provided as ready-to-use lentivirus with high functional titer (>10^8 TU/ml), which saves you time in virus packaging and titer measurement. The third-generation lentiviral vector system is optimized for improved biosafety given its incompetent self-replication feature.

• Dual marker expression for efficient and versatile selection or tracking of positively transduced cells
A dual-marker expression cassette of EGFP and puromycin resistance gene (Puro) is expressed from the lentiviral vector, allowing for selection of positively transduced cells by puromycin and visual tracking by green fluorescence.

Please contact us at for specific academic pricing.