Functional and specific targeting for high-confidence gene knockout results
CRISPR-Cas9 is a highly effective tool for interrogating gene function, yet not all guide RNAs (gRNA) are effective in attaining functional protein knockout. To address this problem, Dharmacon developed an algorithm that is trained and validated for functional gene knockout, not just the ability to create insertions and deletions (indels). Edit-R All-in-one lentiviral single guide RNA (sgRNA) are CRISPR reagents for generating functional gene knockouts in difficult-to-transfect cells or for following up hits from a pooled lentiviral sgRNA library screen.
Edit-R All-in-one Lentiviral sgRNAs express a chimeric structure comprised of a crRNA and tracrRNA fused through a short linker for the programming of Cas9 nuclease and creation of DNA double-strand breaks (DSBs). The Cas9 transcript is expressed from another promoter on the same lentiviral vector; reducing the need to perform sequential transductions. In the Edit-R lentiviral sgRNA vector backbone, the gene-specific crRNA and tracrRNA are expressed under the control of a human U6 promoter, while expression of the Cas9 and puromycin resistance marker (PuroR) are driven from either the human EF1a or mouse CMV promoter and allow for rapid selection of cells with integrated sgRNA. Each Edit-R All-in-one Lentiviral sgRNA is specific to the gene or genomic site of choice.
The Edit-R All-in-one Lentiviral sgRNA is an integrated vector delivering stable expression of both the Cas9 nuclease and the single guide RNA. The use of a single vector makes the system ideal for cells that are not readily amenable to multiple transduction/transfection events, are difficult to obtain or culture, and do not rapidly divide.
High quality, concentrated, purified lentiviral particles for direct transduction with minimal cytotoxicity; delivered at titers of 107 TU/mL
Glycerol stocks for isolation and delivery of plasmid DNA or for packaging your own particles
Predesigned sgRNA generated using the Edit-R CRISPR RNA algorithm for unparalleled specificity and functionality
100% of Edit-R predesigned guide RNA are guaranteed to edit!
Edit-R predesigned crRNA and lentiviral sgRNAs are guaranteed to edit your target, or we will replace it! No restrictions on Cas9 nuclease formats – if your Edit-R positive control works, so will your gene-specific guide RNA. Click on the Specifications tab to learn more about our guarantee.
Edit-R All-in-one Lentiviral sgRNAs are supplied as glycerol stocks and as concentrated high-titer particles for straightforward delivery into Cas9-expressing cells for efficient knockout without need for cloning or packaging. Purified, concentrated lentiviral particles can be directly transduced into difficult-to-transfect cells without the toxic cellular debris and contaminants found in supernatant preparations. Glycerol stocks can be grown and the expression plasmid purified for immediate transfection or packaging into particles.
The Edit-R Predesigned Guide RNA Guarantee
We guarantee that EVERY predesigned Edit-R CRISPR-Cas9 crRNA, Edit-R lentiviral sgRNA and All-in-one Lentiviral sgRNAs (guide RNAs) will provide successful editing at the target site when delivered as described in the Edit-R Technical Manuals.
The Edit-R guide RNA guarantee is valid when used with any wild type S. pyogenes Cas9 nuclease, including mRNA, expression plasmid, protein, or stable Cas9 expression, and Edit-R crRNAs must be used with Edit-R tracrRNA for the guarantee to apply.
Analysis of editing of the treated cell population must be shown using a T7EI or Surveyor mismatch detection assay. If successful editing is not observed for a predesigned Edit-R guide RNA while an appropriate side-by-side Edit-R positive control is successful, a one-time replacement of a different predesigned Edit-R guide RNA of the same format and quantity will be provided at no cost.
A replacement will only be approved upon discussion with our Technical Support team.
Successful editing at the DNA level does not always lead to functional gene knockout; it is recommended to test multiple guide RNAs to determine the most effective guide RNA for knockout of your target gene.
This guarantee does not extend to any accompanying experimental costs, does not apply to guide RNAs ordered via the CRISPR Design Tool, and will not be extended to the replacement guide RNA.
Schematic diagram of the Edit-R All-in-one Lentiviral sgRNA vector
In the Edit-R All-in-one Lentiviral sgRNA vector backbone, the gene-specific guide RNA is expressed under the control of a human U6 promoter, while expression of the Cas9 and puromycin resistance marker (PuroR) is driven from either the human EF1α or the mouse CMV promoter. The plasmid contains the AmpR resistance marker for growth and selection in E. coli.
| Vector Element ||Utility |
|Cas9 || Human codon-optimized S. pyogenes Cas9 nuclease for cleavage of targeted DNA when programmed with a sgRNA |
|T2A || Self-cleaving peptide allows for simultaneous expression of puromycin resistance and Cas9 protein from a single transcript |
|PuroR || Puromycin resistance marker permits antibiotic selection of transduced mammalian cells |
|mCMV || Mouse cytomegalovirus immediate early promoter |
|hEF1α || Human elongation factor 1 alpha short promoter |
|U6 || Human RNA polymerase III promoter U6 |
|sgRNA || Optimized single guide RNA, a fusion of gene-specific crRNA with the tracrRNA scaffold |
|5' LTR || 5' Long Terminal Repeat necessary for lentiviral particle production and integration of the construct into the host cell genome |
|Ψ || Psi packaging sequence allows lentiviral genome packaging using lentiviral packaging systems |
|RRE || Rev Response Element enhances titer by increasing packaging efficiency of full-length lentiviral genomes |
|WPRE || Woodchuck Hepatitis Post-transcriptional Regulatory Element enhances transgene expression in target cells |
|3' SIN LTR || 3' Self-inactivating Long Terminal Repeat for generation of replication-incompetent lentiviral particles |
|SV40 pA || Simian virus 40 polyadenylation signal |
|pUC ori || pUC origin of replication |
| SV40 ori ||Simian virus 40 origin of replication |
|AmpR || Ampicillin resistance gene for vector propagation in E. coli cultures |
Comparison of Edit-R™ two-vector and all-in-one system
For the two-vector system, cells were previously transduced with a constitutive hEF1α- or mCMV-Cas9 expression lentiviral particles at an MOI of 0.3, and selected with 10 µg/mL blasticidin for 10 days, followed by transduction with Non-targeting control (NTC), DNMT3B- or PPIB-sgRNA lentiviral particles at an MOI of 0.3. For the all-in-one system, wild-type cells were transduced with the all-in-one were selected with 2 µg/mL puromycin for 3 days. For the all-in-one system, wild-type cells were transduced with the NTC, DNMT3B or PPIB all-in-one lentiviral particles that also expresses a constitutive hEF1α- or mCMV-Cas9 nuclease at an MOI of 0.3. All transduced cells were selected with 2 µg/mL puromycin for 3 days. The cells were then lysed and analyzed for indels using a DNA mismatch detection assay with T7EI.
Gene knockout workflow using the Edit-R All-in-one Lentiviral sgRNA system
Gene knockout workflow with the All-in-one Lentiviral sgRNA using a mixed popultion (left side) or clonal cell line (right side) experimental approach.
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