The genome editing technology, CRISPR/Cas9, is well known for assisting researchers in manipulating the genome of target cells and has already been discussed in a few posts.
This article is about repurposing the typical genome editing technology to interfere with gene expression, known as CRISPR interference (CRISPRi). The purpose of CRISPRi is regulation of the genome instead of modification. Using this system you can either activate or repress gene expression.
From CRISPR to CRISPRi
The usual CRISPR system is composed of 2 components, a “guide” (gRNA) and a CRISPR associated endonuclease enzyme (Cas 9).
In the CRISPRi system, you co-express a catalytically dead Cas 9 (dCas9), lacking endonuclease activity, with the gRNA. The gRNA is complementary to the region of the gene of interest you wish to repress or activate.
Here are a few key features of CRISPRi system:
1. Unlike the genome modifications induced by the typical nuclease active Cas9, the dCas9-mediated gene interference is reversible and does not permanently modify genomic DNA.
2. The dCas9 molecule retains the ability to bind to target DNA, based on the gRNA targeting sequence, but it cannot cleave the target DNA.
3. You can tag the dCas9 with transcriptional repressors or activators to regulate expression of the ‘target gene’. The concept is schematically shown in Figure 1.
4. The simplest dCas9-based activators and repressors consist of dCas9 fused directly to a single transcriptional activator (e.g., dCas9-VP64 or dCas9-VP16) or transcriptional repressor (e.g. dCas9-KRAB).
5. The CRISPR-mediated gene activation, termed CRISPRa, is currently limited to gene activation in bacteria only.
How to Get Started
Once you’ve decided on the desired genetic manipulation for your CRISPRi experiment (repression or activation), select an expression system as outlined on the addgene website.
You use the following variables to help you choose:
- Species of Cas9 and gRNA
- Species of promoter and expression pattern of promoter for Cas9 and gRNA
- Presence of a selectable marker (drug or fluorophore to quantify)
- Delivery method
- Detection method
After successful delivery of the gRNA and dCas9 (CRISPRi system) to your target cells, you will need to determine if your desired interference has occurred. Your detection method will depend on your specific application. Generally, after 3-5 days of transfection, you can dissociate CRISPRi cells and use flow cytometry to examine expression at the single-cell level. The system can now handle up to five independent operations (activate/inhibit genes at 5 different sites) at once. However, the process will be limited by the number of guide RNA and proteins that can be inserted in to a cell at a given time point, for the intended gene regulation.
The CRISPRi system goes beyond editing to repurpose the well-known CRISPR Cas9 system for precise genome regulation. The CRISPRi system holds great promise as a genetic programming platform, with potential applications in biomedical and clinical research.
- Dominguez AA, et. al. (2016) Beyond editing: repurposing CRISPR-Cas9 for precision genome regulation and interrogation. Nat Rev Mol Cell Biol. 17(1): 5-15.
- Gilbert LA et. al. (2013) CRISPR-Mediated Modular RNA-Guided Regulation of Transcription in Eukaryotes. Cell. 154(2): 442-51.
- Larson MH et. al. (2013) CRISPR interference (CRISPRi) for sequence-specific control of gene expression. Nat. Protocols. 8(11): 2180-96.
- Qi LS et. al. (2013) Repurposing CRISPR as an RNA-Guided Platform for Sequence-Specific Control of Gene Expression. Cell. 152(5): 1173-83.
- CRISPR/Cas9 Guide: Web site https://www.addgene.org/crispr/guide/
- Qi and Weissman Labs CRISPR plasmids: Website https://www.addgene.org/crispr/qi-weissman/