CRISPRmod CRISPRa All-in-one Lentiviral sgRNA Whole Genome Pooled Library

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CRISPRmod CRISPRa All-in-one Lentiviral sgRNA Whole Genome Pooled Library

CRISPRmod CRISPRa All-in-one Lentiviral sgRNA Whole Genome Pooled Library

Perform overexpression of thousands of genes at the transcriptional level with a single transduction of CRISPRa all-in-one pooled lentiviral library eliminating the need to perform sequential transductions.

This library format is beneficial for performing gene activation in difficult-to-transfect or primary cells allowing for efficient genomic screening facilitating advancements in genetic research and therapeutic discovery.

The choice of promoters (mCMV and hEF1α) allows for optimal dCas9-VPR expression in the cells of interest.

CRISPRmod CRISPRa system requires two components to operate: a CRISPRa sgRNA and a catalytically deactivated Cas9 (dCas9) fused to transcriptional activators (VPR). Our CRISPRa all-in-one libraries provide a straightforward method to study genomic function by overexpression in its native context.

The CRISPRmod CRISPRa guide RNA designs use a published CRISPRa v2 algorithm (Horlbeck et al 2016). CRISPRa guide RNAs target sequences upstream of the target gene's promoter region or the transcriptional start site (TSS) to result in activation.

Schematic map of the CRISPRmod CRISPRa All-in-one lentiviral vector

Highlights:

Efficient All-in-one vector system utilizing single lentiviral vectors for both dCas9-VPR and gene-specific sgRNA expression
Rationally designed lentiviral sgRNAs mediate efficient and specific gene activation
Deep and broad coverage with 4 or 8 sgRNAs per gene across the human genome for increased hit confidence
Multiple promoter options for robust activation in biologically relevant cell types
High quality, concentrated, purified lentiviral particles for direct transduction with minimal cytotoxicity; delivered at titers of ≥ 2 x 10⁷ TU/mL ( ± 20%)

Each CRISPRmod CRISPRa All-in-one lentiviral pooled screening library includes:

≥ 2 x 10⁷ TU/mL (± 20%) lentiviral particles in 10 x 100 uL aliquots with mCMV or hEF1α promoter options
Up to 100 non-targeting gRNA negative controls bioinformatically confirmed to not align with (target) any gene in the human genome
A data file containing complete library information, including: gene annotations, sgRNA target sequences, complete list of controls, and counts per millions of mapped reads

Products recommended in our validated protocol:

Vector-matched CRISPRa EGFP delivery controls for transduction optimization and promoter selection
NGS Hit Identification primers for mCMV or hEF1α vectors for:

Efficient PCR amplification of genomic DNA with minimal bias
Staggered forward primers adding diversity to NGS run
Addition of partial Illumina® compatible TruSeq® adapters

Information on Edit-R Lentiviral sgRNA pooled library coverage

The number of genes and constructs are subject to change at any time without notice. Clone and gene counts are available upon request. Contact Scientific Support

Volume of lentiviral particles required for whole genome screening

Volume of lentiviral particles at 2 × 107 TU/mL in HEK293T cells required for the indicated fold representation, biological replicates, and sgRNAs/gene, assuming experimental cells have same relative transduction efficiency as HEK293T cells. The volume of lentiviral particles required for your screen can be calculated using the CRISPR pooled screening protocol tracking worksheet.
Fold representation	Replicates	sgRNAs per gene	Volume of lentiviral particles
200	2	4	1.7 mL
200	2	8	3.3 mL
400	2	4	3.3 mL
400	2	8	6.6 mL

Gene overexpression screening workflow using the CRISPRmod CRISPRa All-in-one Lentiviral Pooled Library platform

Cells are transduced at a low multiplicity of infection (MOI) with a CRISPRmod CRISPRa All-in-one Lentiviral Pooled Library and selected with puromycin. Transduced cells are split into reference and experimental populations for application of a selective pressure and/or phenotypic selection. Genomic DNA is then isolated from the reference and experimental populations of transduced cells and sgRNA constructs within the isolated gDNA are amplified using vector-specific NGS Hit Identification Primers. Amplicons can then be indexed and prepared for high-throughput sequencing on an Illumina platform using commercially available Illumina TruSeq® prep kits. The integrated sgRNA sequences in both reference and experimental samples are identified and relative abundance compared. sgRNA constructs that are enriched or depleted are identified as hits to be confirmed and studied further using individual CRISPRmod CRISPRa All-in-one Lentiviral sgRNAs in additional phenotypic and/or biochemical assays.

High quality pooled screening begins with rigorous lentiviral pooled library production

High quality pooled screening begins with rigorous lentiviral pooled library production. A CRISPRmod CRISPRa Human All-in-one Lentiviral Whole Genome Pooled Library comprising 4 lentiviral sgRNAs per gene was produced and the quality of the plasmid DNA library was verified by next-generation sequencing (NGS). Counts per million mapped reads were obtained to determine the percent-recovery of input sgRNAs (98.8%) and that the distribution of 90% and 70% of the sgRNAs in the pool are within 4.3- and 2.4- fold of each other, respectively, indicating high sgRNA recovery and uniform distribution of sgRNAs.

Matched fluorescent delivery controls enable rapid functional titering and selection of optimal promoter prior to screening

Matched fluorescent delivery controls enable rapid functional titering and selection of optimal promoter prior to screening. DU145 (A) and Jurkat (B) cells were transduced with 5-fold dilution series of CRISPRmod CRISPRa All-in-one GFP Delivery controls mCMV (green) and hEF1α (purple) with functional titering units (TU) derived in HEK293 cells. At 72 hours post-transduction, the percentages of EGFP-expressing cells were quantified by flow cytometry. Dilutions exhibiting between 5 to 25% EGFP-expressing cells can be used to calculate the relative functional titer in the cell line of interest and the relative transduction efficiency of the cell line. The percentages of EGFP-expressing cells or mean fluorescence intensity of EGFP-expressing cells at a given dilution (exhibiting 5-25% EGFP-expressing cells) can be used to compare promoter activity in the cell line. While both promoters are similarly active in Jurkat cells, the mCMV promoter is substantially more active in DU145 cells than the hEF1α promoter.

Highly concordant hits between whole-genome CRISPRa screens performed using the CRISPRmod All-in-one dCas9-VPR and Synergistic Activation Mediator (SAM) systems

Highly concordant resistance hits using a CRISPRmod CRISPRa Human All-in-one lentiviral sgRNA Whole Genome Pooled Library (A) or the three vector Synergistic Activation Mediator System (B) in A375 melanoma cells treated with vemurafenib (PLX-4032) for 16 days. DrugZ screen analysis showing log2 fold enrichment of each gene and associated p-values; significantly enriched genes (positive Log2 Fold Change) drive resistance to the drug treatment indicating these genes potentially act as drug antagonists. Highlighted hits were identified with both technologies and have been previously validated by CRISPRa screening (Konermann et al., 2015).