Dual-gRNA CRISPR Libraries
VectorBuilder offers the only commercially available dual-gRNA lentivirus libraries for CRISPR-based whole-genome knockout screens in human and mouse cells. Each CRISPR vector contains a pair of gRNAs targeting the same gene. When introduced into Cas9-expressing cells, each vector can produce two cuts on the same target gene. Attempts by cells to repair the broken ends of the two cut sites would typically lead to a large deletion spanning the two sites. The two cut sites are designed to flank a functionally important region of the target gene such that a deletion spanning them would most likely lead to the loss of gene function.
Dual-gRNA libraries are far more powerful than single-gRNA libraries for knockout screens because the introduction of large deletions by these libraries can have much higher efficiencies in generating loss-of-function mutations. The human and mouse dual-gRNA libraries are made in the form of ready-to-use high-titer pooled lentivirus targeting 20,048 human genes and 20,493 mouse genes, respectively. Wherever possible, each gene is targeted redundantly by 4-6 different gRNA pairs in separate vectors. These libraries can thus serve as powerful and highly cost-effective tools for performing genome-wide loss-of-function screens.
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A typical workflow for genetic screens using pooled dual-gRNA lentivirus libraries is shown in Figure 1. First, Cas9 expressing cells of interest are transduced with the lentivirus library and positively transduced cells are selected by the marker gene(s) carried on the lentiviral vector (e.g. drug-selection or fluorescence marker). Next, positively transduced cells are split into a reference population and an experimental population. Then, the experimental population is subjected to particular selective pressure (e.g. drug treatment or repeated passaging) to identify cells with the phenotype of interest. There are three major types of screening strategies: 1) viability screens that search for gRNAs enriched or depleted in surviving cells when exposed to the selective pressure; 2) reporter screens that look for gRNAs enriched in cells associated with either high or low reporter expression (e.g. gRNAs targeting transcription factors that modulate reporter gene expression); 3) behavior screens that identify gRNAs affecting genes associated with cell invasion, migration, etc. After screening, both experimental and reference cells are harvested. Enriched or depleted gRNAs in the experimental group relative to the reference group are identified using Sanger sequencing or next-generation sequencing (NGS). Candidate genes that are theoretically targeted by the enriched or depleted gRNAs can be further investigated with downstream functional studies.
Figure 1. Workflow of CRISPR-based knockout screens employing pooled dual-gRNA lentivirus libraries. Adapted from Acta Biochim Biophys Sin 44:103-112 (2012).
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 (Figure 2). 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.
Figure 2. Map of the dual-gRNA library vector.
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.Download the full list of target genes and gRNA pairs in Human Whole-Genome Dual-gRNA Library >>
Download the full list of target genes and gRNA pairs in Mouse Whole-Genome Dual-gRNA Library >>
Validation of library quality by NGS: The dual-gRNA lentivirus 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 (Figure 3).
Figure 3. Representation of gRNA pairs in different pooled libraries. gRNA read counts are normalized by NGS library size (10 million reads) and plotted in log2 scale.
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 (>108 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 (Figure 4).
Figure 4. EGFP expression in 293T cells transduced with Human Whole-Genome Dual-gRNA Lentivirus Library (MOI=10) after 4 days of puromycin selection (1.5 ug/ml). Left: bright field. Right: GFP.
|Product name||No. of genes||No. of gRNA pairs||Scale*||Catalog No.||Price (USD)|
|Human Whole-Genome Dual-gRNA Lentivirus Library||20,048||91,926||
(>1.0x 108 TU/ml, 1 ml)
(>1.0x 108 TU/ml, 5 ml)
|Mouse Whole-Genome Dual-gRNA Lentivirus Library||20,493||90,344||
(>1.0x 108 TU/ml, 1 ml)
(>1.0x 108 TU/ml, 5 ml)
* The medium scale is sufficient for >5 screens at 100x fold coverage, and the plus scale is sufficient for >25 screens at 100x fold coverage.
Not sure how to identify gRNA hits by NGS after conducting your screen? You can simply send the genomic DNA of your samples to VectorBuilder, and we will prepare the NGS libraries, perform high-throughput sequencing, analyze the data, and deliver the accurate counts of gRNAs in each sample to you in just a few weeks.
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