Gene knockdown studies have been a cornerstone technique for gene function researchers for more than 20 years. CRISPRi adds a whole new layer to gene modulation – a CRISPR-based mechanism with RNAi-like effects.
We know gene knockdown. We pioneered the use of siRNA for gene knockdown, and we are proud of every one of the 60,000+ publications powered by Dharmacon siRNA reagents over the past 20 years.
With the introduction of CRISPRmod CRISPRi, the Dharmacon team delivers another tool to accomplish gene modulation. This time, by leveraging an inactive Cas9 complex to block transcription. CRISPRi is knockdown, not knockout. It’s CRISPR without the cut.
DNA to RNA to protein. It’s the central dogma of molecular biology. Researchers interrogate this fundamental principle at all stages to understand gene function and we are proud to have tools and services that span the central dogma (Figure 1). CRISPRko permanently changes the DNA or genomic sequence, RNAi inhibits mRNA expression at the translational level, and CRISPRi (and CRISPRa), as part of the CRISPRmod family, modulate mRNA transcription.
Figure 1. Diagram depicting Horizon Discovery solutions for studying gene function at multiple points of the central dogma of molecular biology.
DNA to RNA to protein - central dogma of molecular biology
DNA to RNA to protein - central dogma of molecular biology
CRISPRmod CRISPRi introduces a few important new features to those looking for a gene knockdown approach. First, we have engineered a novel, patent-pending repressor construct, dCas9-SALL1-SDS3. This bipartite repressor demonstrates dramatically improved performance compared with existing dCas9 repressor systems, such as dCas9-KRAB. Our dCas9-SALL1-SDS3 repressor knocks down genes in a wide range of cell types with greater efficacy (60-80% knockdown) over a longer time course (5-7 days).
Another advantage for our CRISPRmod CRISPRi is that we selected the repressor components based on their effective performance with synthetic single-guide RNA (sgRNA). In our hands, dCas9-KRAB was a bit uneven in its performance when used with synthetic sgRNA. Our new repressor is highly effective with synthetic sgRNA, and we offer a full array of human gene targets off the shelf. We see effective knockdown at the mRNA and protein levels, as you can see here (Figure 2) with fluorescence images.
Functional knockdown of TFRC 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 TFRC 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 TFRC and an Alexa Fluor 488 conjugated fluorescent secondary antibody. Hoechst stain was used to identify nuclei.
CRISPRi with dCas9-SALL1-SDS3 results in more robust protein level repression than CRISPRi with dCas9-KRAB
CRISPRi with dCas9-SALL1-SDS3 results in more robust protein level repression than CRISPRi with dCas9-KRAB
Application-wise, CRISPRi is an ideal platform to mimic the effects of small-molecule drugs or multiplex gene knockdowns. Those are two of the many unique features of CRISPRi we know will be of great scientific use for researchers.
CRISPRi is a new layer for CRISPR. It’s a new layer for gene modulation. Welcome to the toolbox, CRISPRi. Contact us today to learn whether it’s the right choice for your gene knockdown experiments and how to get started.
Written by Dr. Steve Smith
Dr. Smith completed a BS in Biological Sciences and a Ph.D. in Biomedical Sciences from Colorado State University in Ft. Collins, Colorado. His research in Dr. Deborah Roess's lab focused on membrane events during cellular signaling, using biochemical and optical methods. Professionally, his career has focused on tools to study gene functions as well as microscopy and imaging.