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 All-in-one Non-targeting Controls are designed to have a minimum of three mismatches or gaps to all potential PAM-adjacent targets in human, mouse, and rat 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 All-in-one Lentiviral sgRNA Non-targeting Controls are:
- CRISPR guide RNA sequences and Cas9 cloned into the same optimized lentviral expression backbone as Edit-R all-in-one gene targeting sgRNAs;
- available as glycerol stocks and as purified, concentrated lentiviral particles which avoid 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, mouse, or rat genomes; and
- verified bioinformatically to not be homologous to any genes in the human, mouse and rat genomes.
*Bulk volumes of all-in-one lentiviral sgRNA control particles are available upon request
|Shipping Condition||Dry Ice, Frozen Gel Packs|
|Storage Conditions||-80 C|
|Stability at Recommended Storage Conditions||At least 24 months|
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.
|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.