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 |
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 with the All-in-one Lentiviral sgRNA using a mixed popultion (left side) or clonal cell line (right side) experimental approach.