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, Horizon uses an algorithm for genome-wide design of highly specific and functional RNA guides across the human, mouse and rat 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
Using the Edit-R CRISPR RNA algorithm, we have designed lentiviral sgRNAs showing highly efficient gene knockout at single-copy integration levels required for pooled lentiviral screens.
- 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 5 -10 sgRNAs per gene across the human, mouse and rat genomes 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
Each Edit-R Lentiviral sgRNA Pooled Screening Library includes:
- ≥ 5 x 108 TU/mL ( ± 20%) lentiviral particles in pre-aliquoted tubes
- 8 x 25 µL (200 µL total) for libraries ≤ 5000 constructs
- 16 x 25 µL (400 µL total) for libraries > 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, mouse and rat 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
Edit-R Lentiviral sgRNA libraries are available for defined sub-libraries of gene families up to the entire genome for human and mouse; rat libraries and custom Edit-R Lentiviral sgRNA collections are available upon request.
- Request a custom collection of human, mouse, or rat gene-targeting sgRNAs
- Available in pool sizes between 50 to 10,000 constructs
- Pools are provided as purified, concentrated lentiviral particles ≥ 5 x 108 TU/mL
Products recommended in our validated protocol:
- Vector-matched Edit-R Lentiviral sgRNA Non-targeting Control for transduction optimization
- Edit-R Pooled sgRNA Indexing PCR and Sequencing Primer Kits (A & B) with 12 unique indexing primers each, optimized and experimentally validated for:
- Efficient PCR amplification of genomic DNA with minimal bias
- High-throughput multiplexed Next Gen Sequencing on an Illumina flow cell for hit identification
Information on Edit-R Lentiviral sgRNA pooled library coverageThe number of genes and constructs per pool are subject to change at any time without notice.Clone and gene counts available upon request.
|Edit-R Lentiviral sgRNA Pooled Library||# targeted genes||Number of pools and average constructs per pool||# targeted genes||Number of pools and average constructs per pool|
|Whole Genome||18,525||18 pools of 10,450 constructs||19,683||20 pools of 10,300 constructs|
|Druggable Genome||7359||7 pools of 10,730 constructs||9827||10 pools of 10,170 constructs|
|GPCR||378||1 pool of 4127 constructs||498||1 pool of 5311 constructs|
|Ion Channels||345||1 pool of 3829 constructs||340||1 pool of 3818 constructs|
|Protein Kinases||700||1 pool of 7262 constructs||708||1 pool of 7451 constructs|
|Phosphatases||247||1 pool of 2846 constructs||269||1 pool of 3089 constructs|
|Proteases||473||1 pool of 5080 constructs||533||1 pool of 5712 constructs|
|Ubiquitin Conjugation||564||1 pool of 5642 constructs||517||1 pool of 5564 constructs|
|Cell Cycle Regulation||**|
|DNA Damage Response||**|
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 technical support for more information
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 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. Edit-R Pooled sgRNA Indexing PCR primers are used to PCR amplify integrated constructs and add Illumina flow cell binding sequences. The resulting amplicons are sequenced on Illumina platform sequencers, using the Edit-R Pooled sgRNA Read 1 and Index Read Sequencing primers. 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 and table of vector elements 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.