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

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

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

Repress thousands of genes at the transcriptional level with CRISPRi all-in-one pooled lentiviral libraries, eliminating the need to create a dCas9 cell line and with just a single round of enrichment.

The All-in-one format CRISPRmod CRISPRi libraries provide enhanced target gene repression for efficient whole genome screening, facilitating advancements in genetic research and therapeutic discovery

The CRISPRmod CRISPRi system requires two components to operate: a CRISPRi sgRNA and a catalytically deactivated Cas9 (dCAS9) fused to transcriptional repressors SALL1-SDS3.

dCas9-SALL1-SDS3 was developed to mediate more robust and consistent gene repression across the genome than the commonly used dCas9-KRAB system.

The choice of promoters (mCMV and hEF1α) allows for optimal dCas9-SALL1-SDS3 expression in the cells of interest.

The CRISPRmod CRISPRi guide RNA designs use a published CRISPRi v2 algorithm (Horlbeck et al 2016). CRISPRi guide RNAs bind sequences immediately downstream of the target gene's transcriptional start site (TSS) to repress transcription.

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

Highlights:

Efficient All-in-one vector system utilizing single lentiviral vectors for both dCas9-SALL1-SDS3 and gene-specific sgRNA repression
Rationally designed lentiviral sgRNAs mediate efficient and specific gene repression
Deep and broad coverage with 4 or 8 sgRNAs per gene across the human genome for increased hit confidence
Multiple promoter options for robust inactivation 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 CRISPRi 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 CRISPRi 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 repression screening workflow using the CRISPRmod CRISPRi All-in-one Lentiviral Pooled Library platform

Cells are transduced at a low multiplicity of infection (MOI) with a CRISPRmod CRISPRi 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 CRISPRi All-in-one Lentiviral sgRNAs in additional phenotypic and/or biochemical assays.

Timeline of primary screen using All-in-on Lentiviral Pooled Libraries

High quality pooled screening begins with rigorous lentiviral pooled library production

High quality pooled screening begins with rigorous lentiviral pooled library production. A CRISPRmod CRISPRi 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 (98.2%) and that the distribution of 90% and 70% of the sgRNAs in the pool are within 11.2- and 3.7- 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 CRISPRi 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.

Enhanced essential gene dropout with the All-in-one dCas9-SALL1-SDS3 CRISPRi system

Enhanced essential gene dropout with the CRISPRmod CRISPRi Human All-in-one lentiviral sgRNA Whole Genome Pooled Library (dark purple/ green) compared to a single vector dCas9-KRAB whole genome pooled library (light purple/ light green) in A375 melanoma cells. Group level analysis of published set of 684 core essential genes (HART_CEGv2, purple) and non-essential genes (HART_NEG, green) at screening endpoint compared to T0 (Hart et al., 2017). Guides targeting non-essential genes exhibit neutral behavior while those targeting essential genes dropout during the screen as cells expressing these guides should not be able to survive.

Highly concordant hits between whole-genome CRISPRi screens performed using the CRISPRmod All-in-one dCas9-SALL1-SDS3 and dCas9-KRAB systems

Highly concordant resistance hits using a CRISPRmod CRISPRi Human All-in-one lentiviral sgRNA Whole Genome Pooled Library (A) or a single vector dCas9-KRAB whole genome pooled library (B) in A375 melanoma cells treated with vemurafenib (PLX-4032). 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.