CRISPR: the genetic scissors

What is CRISPR technology and how can change our lives?

CRISPR/Cas9 technology is a molecular tool used for “editing” or “correcting” the genome of any cell, including human cells. It would be like genetic scissors able to cut any DNA molecule in a very precise and fully controlled way. This ability to cut DNA is what allows its sequence to be modified, removing or inserting new DNA. In this post, we are going to see how classic CRISPR editing come about and works. Previous posts about basic notions of molecular genetics and gene editing would help you to understand it.

History of CRISPR

The acronym CRISPR/Cas9 comes from Clustered Regularly Interspaced Short Palindromic Repeats. Cas is the name of a series of proteins, mainly nucleases, which were named the CRISPR-associated system.

It all began in 1987 with the publication of a paper outlining the defense mechanisms used by some bacteria (Streptococcus pyogenes) against viral infections. These bacteria have enzymes that can distinguish between their own genetic material and the genetic material of viruses, and once the distinction has been made, they destroy the genetic material of the virus.

But it wasn't until much later, after the genomes of several bacteria and other microbes had been mapped, that the fundamentals of this mechanism became clear. A certain area of the genome of many microorganisms, especially archaea, was found to be full of palindromic repeats (which read the same forwards and backwards) with no apparent function. These repeats were separated from each other by sequences called "spacers" that resembled others in viruses and plasmids. A sequence known as the "leader" comes directly before those repeats and "spacers". These sequences were referred to as CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats). Some genes called cas genes that encoded a specific kind of nuclease could be located very near to this cluster. Nucleases are enzymes able to cleave the phosphodiester bonds between nucleotides of nucleic acids and effect single and double-stranded breaks.

When a virus enters the bacterium, it takes control of the cellular machinery and for this, it interacts with different cellular components. But the bacteria that have this defense system have a complex made up of a Cas protein linked to RNA produced from the CRISPR sequences. Then the genetic material of the virus can interact with this complex. If that happens, the viral genetic material is inactivated and subsequently degraded. But the system goes further. A little portion of the viral DNA can be taken by cas proteins, modified, and then integrated into the collection of CRISPR sequences. That way, if that bacterium (or its offspring) encounters that same virus, it will now much more efficiently inactivate the viral genetic material. Consequently, it is a true bacterial immune system.

During the following years, research on this system continued, but it wasn't until 2012 that the crucial step was taken to turn this discovery—this biological observation—into a useful molecular tool in the laboratory. In August of this year, a team of researchers led by Drs Emmanuelle Charpentier at Umeå University and Jennifer Doudna at the University of California at Berkeley published an article in the journal Science demonstrating how to turn that natural machinery into a "programmable" editing tool, which was used to cut any strand of DNA in vitro. In other words, they managed to program the system so that it would go to a specific position of any DNA (not only viral) and cut it. To achieve this, some RNAs were used to direct the system toward the DNA to be cut.

How is DNA edited with this technology?

It all begins with the design of an RNA molecule (guide RNA) specific to a DNA sequence, and according to the principles of nucleotide complementarity, it means that it will only bind that sequence, the one to be edited. Then both the guide RNA and the Cas 9 enzyme are injected into a cell. Once inside, the guide RNA recognizes the exact site of the genome, and the Cas9, an endonuclease, cut both strands of DNA. In essence, we may say that the guide RNA serves as a guide dog, bringing the executor Cas9 to the location where it needs to carry out its task.

Then, the cell is forced to fix this DNA damage. Why forced? Because DNA breaks make it impossible for regular cells to survive. Due to this "duty," the cell even possesses defenses that stop it from surviving or proliferating in the event of DNA damage. The demand for repair in the cell is so great that it will usually try to fix the damaged area, even at the risk of introducing mutations (small insertions or deletions - INDELS). CRISPR/Cas9 gene editing system takes advantage of these cellular DNA repair processes, and scientists can exploit this in two different ways:

• First, in the attempt to restore DNA damage by joining the two extremities of the broken DNA, the natural mechanisms result in either a hole or the addition of a few nucleotides following the cut spot (the precise DNA sequence where the guide RNA was bound). The original purpose of the snipped DNA fragment is lost as a result.
• A second alternative enables the precise insertion of a particular sequence at the original cleavage site. For this, logically, we have to give the cell the sequence that we want to integrate into the DNA. This way we can introduce point mutations in a specific DNA region to eliminate the expression of a gene, or we can even supply the cell with DNA “templates” that present desired alterations to correct gene mutations responsible for genetic diseases.

But look, we always depend on the cell’s own material and let it be said, a bit of chance.

What is it for?

In a molecular way we can say that this tool can be used to regulate gene expression, label specific sites of the genome in living cells, identify and modify gene functions, and correct defective genes. It is also already being used to create animal models to study complex diseases such as Parkinson's or Duchenne Muscular Dystrophy (DMD).

By Miriam Martínez

Miriam is a Human Biologist expert in neuropharmacology. After a master’s degree in Pharmaceutical and Biotech Industry, she obtained her PhD in Biomedicine from Pompeu Fabra University (Barcelona). During her doctorate, she focused her research on the behavioral analysis of animal models for neurophenotypical characterization. Since then, she has been working in the healthcare marketing and publicity sector, where she has contributed to developing marketing campaigns for several pharmaceutical brands. In 2021, she joined ZeClinics with a branding and marketing strategy focus.