Efficient endogenous gene repression with CRISPRi
CRISPR interference (CRISPRi) is a unique adaptation of the CRISPR-Cas9 gene editing system. The Horizon 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 trancriptional start site (TSS), it promotes transcriptional repression.
Review our CRISPRi applications page to get an overview of the technology!
Highlights of using dCas9-SALL1-SDS3 lentiviral particles
- Choose from one of three different promoters for optimum expression in your cells of interest (see options below)
- Provided as concentrated, purified lentiviral particles for immediate transduction, with a minimum ≥ 1 × 107 TU/mL functional titer, by qPCR titering
- Selection using blasticidin resistance marker (BlastR) for entire cell populations or for clonal expansion of individual cells
Not all RNA pol II promoters are equally active in different cellular environments
The activity of any given promoter controlling the transcription of dCas9-SALL1-SDS3 can differ greatly from one biological context to another, resulting in variable dCas9-SALL1-SDS3 expression levels and thus varying levels of gene repression. Choosing an optimal promoter for your cell line will be important for robust gene overexpression.
SMARTchoice promoter options for expressing dCas9-SALL1-SDS3
|hCMV||human cytomegalovirus immediate early promoter|
|mCMV||mouse cytomegalovirus immediate early promoter|
|hEF1α||human elongation factor 1 alpha promoter|
Requirements for a CRISPRi experiment using dCas9-SALL1-SDS3 lentiviral particles
- CRISPRi dCas9-SALL1-SDS3 lentiviral particles
- CRISPRi lentiviral sgRNA for your target gene (see Workflow tab)
CRISPRi workflow using synthetic sgRNA and dCas9-SALL1-SDS3 lentiviral particles
CRISPRi workflow using lentiviral sgRNA and dCas9-SALL1-SDS3 lentiviral particles
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). This system is ideal for working in difficult-to-transfect cell types.
CRISPRi with dCas9-SALL1-SDS3 results in more robust protein level repression than CRISPRi with dCas9-KRAB
Functional knockdown of FUS was assessed in U2OS cells constitutively expressing either dCas9-KRAB or dCas9-SALL1-SDS3 under the hEF1α promoter. Cells were transfected with a 25 nM sgRNA pool containing three CRISPRmod CRISPRi predesigned sgRNAs targeting FUS using DharmaFECT 4 Transfection Reagent. 72 hours post-transfection the cells were split, and at 96 hours post-transfection, cells were fixed and stained with a primary antibody targeting FUS and an Alexa Fluor 488 conjugated fluorescent secondary antibody. Hoechst stain was used to identify nuclei.
Cells expressing dCas9-SALL1-SDS3 demonstrate greater and more consistent CRISPRi gene repression compared to dCas9-KRAB
CRISPRi synthetic sgRNAs achieve maximal repression at 48-72 hours post-transfection in both dCas9-KRAB and dCas9-SALL1-SDS3-expressing cells. U2OS cells stably expressing integrated dCas9-KRAB or dCas9-SALL1-SDS3 were plated at 10,000 cells/well and transfected using DharmaFECT 4 Transfection Reagent with pools of pre-designed synthetic sgRNA (25nM) targeting CBX1, HBP1, or SEL1L. Cells were harvested at 24, 48, 72, 96, 120, and 144 hours post-transfection, total RNA was isolated, and relative gene expression was measured using RT-qPCR. The relative expression of each target gene was calculated with the Cq method using GAPDH as the housekeeping gene and normalized to a non-targeting control (NTC).
|Shipping Condition||Dry Ice|
|Storage Conditions||-80 C|
|Stability at Recommended Storage Conditions||At least 12 months|