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Efficient endogenous gene repression with CRISPRi
CRISPR interference (CRISPRi) is a unique adaptation of the CRISPR-Cas9 gene editing system. The Horizon CRISPRmod CRISPRi system utilizes a catalytically deactivated Cas9 (dCas9) fused to our proprietary transcriptional repressors (SALL1 and SDS3). When paired with an algorithm-designed guide RNA that targets a gene immediately downstream of the transcriptional start site (TSS), it promotes transcriptional repression.
Review our CRISPRi applications page to get an overview of the technology!
Highlights of CRISPRi lentiviral sgRNA reagents
- Available as glycerol stocks or high-titer purified lentivirus particles
- Designed with a published algorithm by Horlbeck, et. al. (2016) that has demonstrated strong levels of gene repression from optimized designs
- Purified, concentrated high-titer lentivirus particles can be directly transduced into cells, including difficult-to-transfect cells and lines
Requirements for a CRISPRi experiment using lentiviral sgRNA
- CRISPRi lentiviral sgRNA for your target gene
- CRISPRi dCas9-SALL1-SDS3 lentiviral particles (see Workflow tab)
Schematic diagram of the CRISPRi lentiviral sgRNA vectors
In the CRISPRi 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 gene (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.
CRISPRi lentiviral sgRNA workflow
Workflow using CRISPRi dCas9-SALL1-SDS3 lentiviral particles to establish a stable dCas9-SALL1-SDS3 expressing cell line, then delivering CRISPRi lentiviral sgRNA particles (left) or plasmid sgRNA (right).
CRISPRi lentiviral sgRNA controls
- CRISPRi sgRNAs targeting well-characterized genes to determine the effectiveness of your experimental conditions for maximum repression.
- CRISPRi non-targeting controls to evaluate baseline cellular responses to CRISPRi components in the absence of target-specific sgRNA.
- Lentiviral particles or mRNA that express nuclease-deactivated Cas9 fused to our proprietary transcriptional repressors for CRISPRi experiments.
CRISPRi lentiviral sgRNA is well suited for long term repression
U2OS and A549 cells stably expressing integrated dCas9-KRAB or dCas9-SALL1-SDS3 were plated at 50,000 cells/well in 24 well plates and transduced with CRISPRi sgRNA expressing lentiviral particles targeting PPIB, SEL1L, BRCA1 or CANX at a MOI of 0.3 to obtain cells with a single integrant. Cells were selected with 2 µg/mL puromycin for 5 days, split into two populations, and allowed to recover for an additional day. At 7 days post-transduction one population was harvested for RT-qPCR analysis. The replicate populations of cells were cultured for 7 additional days before harvest. Total RNA was isolated from harvested plates and relative gene expression was calculated with the Cq method using GAPDH as the housekeeping gene and normalized to a non-targeting control.
- M. A. Horlbeck et al., Compact and highly active next-generation libraries for CRISPR-mediated gene repression and activation. eLife. 5, e19760 (2016).