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.
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.
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.
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.
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.
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.
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.
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 (Cat #GSGH11829) 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. Lanes shown are representatives of triplicate samples. UT, untransfected control.