Edit-R Lentiviral sgRNA Pooled Screening Libraries

Screening services
Dharmacon Screening libraries
Edit-R Lentiviral sgRNA Pooled Screening Libraries

Edit-R Lentiviral sgRNA Pooled Screening Libraries

Ensure library specificity with algorithm-optimized guide RNA. Efficient gene editing at single-copy integrations.

Whole genome, druggable genome, gene family and pathway libraries for knockout screening.

Please Note: Human Pooled Libraries have been updated with a new and improved vector scaffold. Please reach out to Scientific Support if you have any questions.

Perform unbiased, phenotypic knockout screens without the need for costly infrastructure

Knockout hundreds and even thousands of genes at the DNA level through pooled lentiviral library screening methods.

The ability of CRISPR guide RNA to create a double-strand break in target DNA causing functional protein disruption can vary based on the guide RNA (gRNA) sequence and position in the targeted gene. To address this, the Dharmacon reagents algorithm provides highly specific and functional RNA guides across the human genome and mouse genomes.

The Edit-R CRISPR RNA algorithm generates guide RNA sequences with:

An optimized alignment program with rapid and thorough specificity analysis, including detection of multiple mismatches and gapped alignments
Increased functional knockout of targeted genes through systematic assessment of over 1100 guide RNAs to identify characteristics critical for functional gene disruption, not just generation of indels

Highlights:

Efficient two-vector system utilizing a lentiviral vector for Cas9 expression and a gene-specific vector for sgRNA expression
Rationally designed Edit-R lentiviral sgRNAs efficient gene knockout with unparalleled specificity using a functionally validated proprietary algorithm
Deep and broad coverage of 4 or 8 sgRNAs per gene across the human genome for whole genome library or 5 -10 sgRNAs per gene across the mouse genome for increased hit confidence.
Can be combined with the promoter flexibility of Edit-R Lentiviral Cas9 Nuclease Reagents for robust editing in biologically relevant cell types

The NEW Edit-R Human Lentiviral sgRNA Pooled Screening Libraries include, per pool:

≥ 5 x 10⁸ TU/mL ( ± 20%) lentiviral particles in 10 x 100 uL pack size
Four or eight sgRNAs per gene (whole genome) and up to eight sgRNAs per gene (gene family and pathway libraries) targeting coding genes in the NCBI Reference Sequence Database
Up to 100 non-targeting sgRNA negative controls bioinformatically confirmed to not align with (target) any gene in the human genome
Gene family and pathway libraries include up to 340 gene-specific sgRNA positive controls targeting up to 34 protein-coding genes (10 sgRNA per gene); including common reference genes (ACTB, GAPDH, LAMB1, & PPIB)
A data file containing complete library information, including: gene annotations, sgRNA target sequences, complete list of controls, and counts per millions of mapped reads

The Edit-R Mouse Lentiviral sgRNA Pooled Screening Libraries include, per pool:

≥ 5 x 108 TU/mL ( ± 20%) lentiviral particles in pre-aliquoted tubes

8 x 25 µL (200 µL total) for pools ≤ 5000 constructs
16 x 25 µL (400 µL total) for pools > 5000 constructs

Five to ten sgRNAs per gene targeting coding genes in the NCBI Reference Sequence Database
100 non-targeting sgRNA negative controls bioinformatically confirmed to not align with (target) any gene in the human and mouse genomes
Up to 340 gene-specific sgRNA positive controls targeting up to 34 protein-coding genes (10 sgRNA per gene); including common reference genes (ACTB, GAPDH, LAMB1, & PPIB)
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:

Human and Mouse libraries:
- Vector-matched Edit-R lentiviral positive and non-targeting sgRNA controls for gene editing validation and experimental optimization (recommended)
- SMARTvector Non-targeting Control Particles with optimal promoter and fluorescent reporter (TurboGFP or TurboRFP) for determining relative transduction efficiency and optimizing transduction conditions in desired cells of interest. (recommended)
- Edit-R Lentiviral Cas9 Nuclease Reagents or Cas9 Stable Cell Line
Human libraries in NEW V2 backbone:
- NGS Hit Identification Primers mCMV (required)
  - Efficient PCR amplification of genomic DNA with minimal bias
  - Staggered forward primers adding diversity to NGS run
  - Addition of partial Illumina® compatible TruSeq® adapters
Mouse libraries and Human libraries in V1 backbone:
- Edit-R Mouse Pooled sgRNA Indexing PCR and Sequencing Primer Kit A for Illumina® sequencing platforms (required)
- Edit-R Mouse Pooled sgRNA Indexing PCR and Sequencing Primer Kit B for additional reverse indexing PCR primers (if experimental design requires additional multiplexing capability)
- Edit-R Lentiviral Cas9 Nuclease Reagents or Cas9 Stable Cell Line

Important Notice

The Edit-R Lentiviral sgRNA Pooled Libraries are solely for internal research use (as set forth in the Product Terms and Conditions) in laboratories where the containment measures stated below and in applicable laws and regulations are met. Products may not be used for diagnostic, therapeutic or other commercial purposes and may not to be administered to humans for any purpose or to animals for therapeutic purposes. Edit-R Lentiviral sgRNA Pooled Libraries provided as lentiviral particles are replication-incompetent, self-inactivating (SIN) and non-pathogenic (do not cause infectious human disease).

Any investigator who purchases lentiviral particle products is responsible for consulting with their institution's health and biosafety personnel for specific guidelines on the handling of lentiviral vector particles. Furthermore, each investigator is fully responsible for obtaining the required permissions for research using and the acceptance of replication-incompetent SIN lentiviral vectors and replication-defective lentiviral particles into their local jurisdiction and institution.

** Please contact Scientific Support for more information

Number of targeted genes and number of lentiviral pools provided with respective human or mouse Edit-R Lentiviral sgRNA pooled libraries


	Human		Mouse
Edit-R Lentiviral sgRNA Pooled Library¹	Number of targeted genes	Number of pools	Number of targeted genes	Number of pools x number of constructs per pool^2,3
Whole Genome	18890	1 pool	19683	19 pools x 10600 constructs
Druggable Genome	6411	1 pool	9827	10 pools x 10170 constructs
GPCR	470	1 pool	498	1 pool x 5311 constructs
Ion Channel	581	1 pool	340	1 pool x 3818 constructs
Protein Kinase	1139	1 pool	708	1 pool x 7451 constructs
Phosphatase	282	1 pool	269	1 pool x 3089 constructs
Protease	689	1 pool	533	1 pool x 5712 constructs
Ubiquitin Conjugate	564	1 pool	517	1 pool x 5564 constructs

Volume of lentiviral particles required for screening with Edit-R Human Whole Genome library

Volume of lentiviral particles at 5 × 108 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. 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	0.07 mL
200	2	8	0.13 mL
400	2	4	0.13 mL
400	2	8	0.25 mL

Gene knockout screening workflow using the Edit-R Lentiviral sgRNA Pooled Screening Library platform

A Cas9-expressing stable cell line (mixed or clonal cell population) is first generated with Edit-R Lentiviral Cas9 Nuclease particles by selection with blasticidin. These cells are then transduced with an Edit-R lentiviral sgRNA 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 Edit-R Lentiviral sgRNAs in additional phenotypic and/or biochemical assays.

Schematic maps of the Edit-R Lentiviral Cas9 Nuclease and sgRNA vectors

Schematic maps and table of vector elements of the Edit-R Lentiviral Cas9 Nuclease (A) and sgRNA (B) vectors. The Edit-R Lentiviral CRISPR-Cas9 platform is a two-vector system that utilizes a lentiviral vector with multiple Pol II promoter options for maximal Cas9 expression and a gene-specific vector for sgRNA expression designed to the target site of interest.

High quality pooled screening begins with rigorous lentiviral sgRNA pooled library production

High quality pooled screening begins with rigorous lentiviral sgRNA pooled library production. A pooled library comprised of 7519 lentiviral sgRNAs targeting human kinases 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 percent-recovery of input sgRNAs (99.5%) and the distribution of 90% and 70% of the sgRNAs in the pool are within 3.6- and 2.1- fold of each other, respectively (Panel A.). The pooled plasmid DNA library was then packaged into lentiviral particles and transduced at MOI 0.3 into U2OS cells from which genomic DNA was isolated at 24 hours post-transduction (T0), PCR-amplified and prepped for NGS. Distribution of lentiviral sgRNAs was not substantially affected, indicating rigorous production and maximized retention of constructs from production into pooled screening (Panel B.).

Pooled lentiviral sgRNA human kinase library screen identifies high-confidence viability hits

Pooled lentiviral sgRNA human kinase library screen identifies high-confidence viability hits. A pooled lentiviral library comprised of 7079 Edit-R sgRNAs targeting 708 human kinases, 340 positive control sgRNAs targeting 34 essential genes and 100 non-targeting control sgRNAs was transduced into a Cas9-expressing U2OS cell line at MOI 0.3 and 1000-fold sgRNA representation with two biological replicates. At 24 hours post-transduction, the reference (T0) cell populations were harvested and the experimental (T1) cell populations were subjected to 2 ug/mL puromycin treatment (selective pressure). At 8 days post-selection T1 cell populations were harvested. Genomic DNA was harvested from both T0 and T1 samples and PCR-amplified with Edit-R Pooled sgRNA Forward and Reverse Index PCR primers for high-throughput sequencing on an Illumina platform. sgRNA abundance is shown as an MA plot of normalized count data. The sgRNAs that were significantly (adjusted p-value ≤ 0.05) higher and lower abundance and also showed > 2-fold abundance change are in red and blue, respectively. The green line indicates zero fold abundance change.