CRISPR-Cas9 gene editing experiments require the use of negative control samples in order to accurately distinguish biological effects of sgRNA-induced editing of targeted genomic DNA from non-specific effects. All Edit-R Non-targeting Controls are designed to have a minimum of three mismatches or gaps to all potential PAM-adjacent targets in human or mouse gnomes.
Ten different negative control CRISPR RNA sequences are designed and provided as glycerol stocks and as 50 µL* (2 x 25 µL) purified, concentrated lentiviral particles. Due to the biological variability inherent in different cells, time points, assays and conditions, we recommend testing at least two non-targeting sgRNAs to identify the best negative control for establishing an effective baseline for your particular experiment. Edit-R Lentiviral sgRNA Non-targeting Controls are:
- CRISPR guide RNA sequences cloned into the same optimized lentviral expression backbone as Edit-R gene targeing sgRNAs;
- available as glycerol stocks and as purified, concentrated lentiviral particles which avoids toxicity due to cellular debris and nucleases resulting from the packaging reaction and present in supernatants;
- proprietary alignment tools are used to verify at least three mismatches or gaps to any potential target in human or mouse genomes; and
- verified bioinformatically to not be homologous to any genes in the human or mouse genomes.
*Bulk volumes of lentiviral sgRNA control particles are available upon request
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 and sgRNA 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.
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.
Experimental workflow using Edit-R Lentiviral sgRNA
Experimental workflow using Edit-R Lentiviral sgRNA. Edit-R Lentiviral sgRNA are transduced into a stable cell line expressing Cas9 nuclease for efficient gene knockout, even at low MOIs.
Certificate of analysis
Validated Edit-R Lentiviral sgRNA Positive Controls and kits confirm generation of insertions and deletions (indels), and permit quantification of gene editing efficiency using DNA mismatch detection assays.
Whole genome, druggable genome, gene family and pathway libraries for knockout screening.