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Functional and specific targeting for high-confidence gene knockout results
Edit-R Lentiviral sgRNAs express RNA for guiding Cas9 nuclease to create double-strand breaks in the target DNA. In the Edit-R lentiviral sgRNA vector backbone, the gene-specific guide RNA is expressed under the control of a human U6 promoter, while expression of the puromycin resistance marker (PuroR) is driven from the mouse CMV promoter and allows for rapid selection of cells with integrated sgRNA.
Each Edit-R Lentiviral sgRNA is specific to the gene and genomic site of interest. Generating knockouts in difficult-to-transfect cells or following up hits from a pooled lentiviral sgRNA library screen are key applications of this guide format.
While CRISPR-Cas9 is a highly effective tool for interrogating gene function, not all guide RNAs are effective in attaining functional protein knockout. To address this problem, Horizon developed an algorithm that is trained to select guides that give the highest likelihood of generating a functional gene knockout, not just creating an insertion or deletion.
Experimental workflow using Edit-R Lentiviral sgRNA
The Edit-R Lentiviral Gene Engineering system utilizes Cas9 nuclease and the single guide RNA in a two-step process. First, Edit-R Lentiviral Cas9 Nuclease Expression particles are utilized to generate cell lines stably expressing Cas9 nuclease. These cells can subsequently be transduced with Edit-R Lentiviral sgRNA particles to achieve efficient gene editing - even at low MOIs - for phenotypic analyses in a population of cells or in isolated clonal cell lines.
Testing gene editing with Cas9 under control of different promoters show that high levels of gene editing are achieved with Edit-R Lentiviral sgRNA in multiple cell lines.
Testing gene editing with Cas9 under control of different promoters show that high levels of gene editing are achieved with Edit-R Lentiviral sgRNA in multiple cell lines. A recombinant U2OS ubiquitin-EGFP proteasome cell line (Ubi[G76V]-EGFP) and HEK293T cells were stably transduced with lentiviral particles containing Cas9 and a blasticidin resistance gene. A population of stably integrated cells were selected with blasticidin for a minimum of 10 days before transduction with sgRNAs. Cells were transduced with sgRNA lentiviral particles at low MOI to obtain cells with one integrant and selected with puromycin for seven days prior to analysis. The relative frequency of gene editing in the puromycin-selected cells was calculated from a DNA mismatch detection assay using T7 Endonuclease I.
Schematic map of the plasmid vector elements of the Edit-R Lentiviral sgRNA vector.
In the Edit-R Lentiviral sgRNA vector backbone, the gene-specific crRNA and the tracrRNA are expressed under the control of a human U6 promoter, while expression of the puromycin resistance marker (PuroR) is driven from the mouse CMV promoter and allows for rapid selection of cells with integrated sgRNA. The plasmid contains the AmpR resistance marker for growth and selection in E. coli.
Algorithm applies to both synthetic crRNAs and expressed sgRNAs
Here we are targeting the gene PSMD11 at 12 different sites and measuring functionality by EGFP signal to indicate disruption of the proteasome. Cells were either transfected with crRNA:tracrRNAs, or transduced with lentiviral particles for expression of a sgRNA of the same design. There is no significant difference between the two guide RNA formats when targeting the same genomic site; a good design for crRNA will translate into a good design for sgRNA.
Designs with gaps & mismatches can cause off-target cleavage
T7EI mismatch analysis for a crRNA showing the intended target site (GGTCATCTGGGAGAAAAGCG) and a predicted off-target site that was identified by the Edit-R alignment tool but no other online tool, containing one gap and one mismatch (GGT-ATCTGGGAGAAAAGCa) Many commonly used web-based crRNA specificity tools do not fully account for gaps when performing alignments.
Successful CRISPR-Cas9 gene editing utilizing co-delivery of Edit-R Lentiviral sgRNA and Cas9 expression plasmid using DharmaFECT kb transfection reagent
U2OS cells were transfected with equal amounts of Edit-R hCMV-mKate2-Cas9 Expression plasmid (Cat #U-004100-120) and Edit-R Human PPIB Lentiviral sgRNA plasmid in a 96-well format. Transient transfections were done with increasing amounts of total DNA (50 to 200 ng) and DharmaFECT kb transfection reagent (Cat #T-2006-01), 0.2 to 0.8 µL per well. The percentage of gene editing was estimated 72 hours after transfection by DNA mismatch detection assay using T7EI with gel densitometry.
The Edit-R Predesigned Guide RNA Guarantee
We guarantee that EVERY predesigned guide RNA 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.
|Shipping Condition||Dry Ice, Frozen Gel Packs|
|Storage Conditions||-80 C|
|Stability at Recommended Storage Conditions||At least 12 months|