The CRISPR / Cas9 technology has revolutionized the world of genetic engineering thanks to its ability to overcome the limitations of other recently developed techniques. This novel genomic editing tool significantly improves efficiency in terms of scaling processes with a reduced cost, allows to enter or eliminate more than one gene at the same time and is not species-specific, which enables its use in practically any organism.
The CRISPR / Cas9 technology has its origin in an immune response system adapted from prokaryotic organisms, which is based on using non-coding RNA as a guide to the Cas9 nuclease to induce cleavages on specific points of DNA.
This genomic editing tool consists of two key components:
1.- A non-specific endonuclease (Cas9)
2.- A short sequence of RNA (gRNA) that acts as a guide for the endonuclease to bind to a specific sequence of the genome where it will exert its action.
APPLICATIONS OF CRISPR / CAS9 TECHNOLOGY
Although initially the CRISPR / Cas technology was designed for the generation of knock-out organisms, the versatility and the possibilities of modifying the Cas9 enzyme have considerably broadened the range of applications of this tool, among which we can highlight the following:
Generation of Knock-outs
The genomic target can be any DNA sequence of about 20 nucleotides, provided that said sequence is unique compared to the rest of the genome, and that the target is found immediately after a PAM (Protospacer Adjacent Motif) motif.
Activate or inhibit target genes selectively
One of the characteristics that allows the flexibility of the CRISPR / Cas9 system is that the ability of the Cas9 enzyme to bind to the target DNA is independent of its ability to cut it. Both domains of the nuclease (RuvC- and HNH-) can be inactivated by specific mutations, giving rise to nuclease dCas9, without activity to cut the DNA but maintaining its capacity of binding to the target DNA in function of the sequence of the gRNA. This inactivation in itself may be sufficient to inhibit transcription by blocking its onset. Additionally, the dCAs9 enzyme can be coupled to specific inhibitors or activators in order to obtain more robust results.
Purify specific regions of DNA
Starting from the concept of the ChIP technique (chromatin immunoprecipitation), it has been developed in ChIP (Chromatin Immunoprecipitation mediated by DNA binding molecule), which allows the purification of any genomic sequence specified by a specific gRNA.
Visualization of DNA in living cells by fluorescence microscopy
By fusing a dCas9 to a fluorescent marker such as GFP, the enzyme can be transformed into a customizable label that allows the detection of a specific DNA sequence in living cells.
HOW TO PLAN AN EXPERIMENT WITH CRISPR / CAS9 TECHNOLOGY?
1º.- Select the genetic manipulation of interest
Different types of genetic manipulation require different CRISPR components. Then we leave a table with the components depending on the application of interest:
2º.- Select the expression system
To use a CRISPR / Cas9 system, both components (gRNA and Cas9) must be expressed in the target cells. The expression system will depend on the specific application.
3º.- Select the target sequence and design the gRNA
The steps to follow are:
- Choose the species and cell line. The genomic sequence that is used for the design of the gRNA will depend on the gene of interest as well as the species to which the cells belong.
- Select the gene and the target region. It is necessary to identify the genomic sequence of the target gene, although the exact region of said gene will depend on the specific application:
- Activate / inhibit genes: The gRNA must be directed to the expression promoter.
- Knock-outs: The gRNA targets the exons
- Genomic edition: The gRNA will be directed to a sequence close to the area that it is intended to edit.
- Select the gRNA: For the gRNA to bind to the DNA, the existence of a PAM sequence is absolutely necessary, so it will be necessary to previously identify all the PAM sequences within the target region. It is also necessary to analyze the rest of the genome in search of possible homologies of the target sequence to avoid nonspecific binding.
- Synthesize and clone the gRNA.
- Delivery of gRNA and Cas9. The delivery method will depend on the expression system that has been selected.
- Validation of the genome edition. The method for validation will depend on the specific application, although there are some general verification routes based mainly on PCR, electrophoresis and sequencing.
The CRISPR / Cas9 technology has been a real revolution in the field of genomic publishing, providing a flexible, simple, highly specific and low cost method that democratizes the use of genetic engineering in different sectors.
If you are thinking about incorporating this technology into your research line, there are specific kits with all the necessary components for the application of CRISPR / Cas technology and detailed protocols for its correct use.
Do not hesitate to contact us to resolve any questions regarding this new technique.