What is CRISPR/Cas?

What is CRISPR/Cas? why is it so revolutionary and can it be used by anyone to easily alter the genome of anything? About a great tool in genetic engineering, what it can and cannot do.

When the Nobel Prize in Chemistry went to Jennifer Doudna and Emmanuelle Charpentier in 2020, nobody was really surprised. When I saw Emmanuelle Charpentier at an award ceremony in 2016, it was clear to most that the Nobel Committee was just waiting for an agreement in a patent dispute to nominate the two scientists. The work that has made them known worldwide is the article “A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity” which appeared in the journal Science in 2012. It describes the use of a bacterial defense system, now known as CRISPR/Cas, in genetic engineering.

Emmanuelle Charpentier, one of the developers of CRISPR/Cas, at the Gairdner Award ceremony in the Canadian Embassy in Berlin on September 6th, 2016, own picture

What is CRISPR/Cas

But what is CRISPR/Cas and why is it such a giant leap in genetic engineering? CRISPR describes certain repetitive motifs found in the genomes of a wide variety of bacteria. In the early 2000s, it was discovered that these motifs are related to a defense mechanism in these bacteria that protects them from viral attacks. If a virus infects a cell, it injects its genetic material and thus reprograms the cell for its own purposes. The cell now produces new viruses according to this blueprint and in most cases destroys itself. Viruses are a huge threat to bacteria, which do not have specialized immune cells like we do. So they developed many strategies to counter it.

One of the most widespread of these defense systems is CRISPR/Cas. Certain CRISPR-associated (Cas) proteins bind the DNA introduced by a virus, cut out small pieces of it and insert them between repeat sequences in the bacterium’s genome (CRISPR). If the bacterium is attacked again by this or a similar virus, another Cas protein can now immediately recognize the viral DNA with the help of these stored recognition sequences and cut it before it can multiply.

Revolution in genetic engineering

The two Nobel Prize winners have deciphered this part of the adaptive immune response of the bacteria: The programmable cutting of DNA sequences. And they developed a method to change this programming at will. The trick is that the Cas protein cuts DNA whose sequence matches an RNA sequence to which it is attached. This so-called guide RNA can be produced relatively easily and thus the exact target at which the DNA is to be cut can be set.

Guide RNA and CRISPR-associated protein Cas9 (white) from Staphylococcus aureus, CC von Thomas Splettstoesser (www.scistyle.com)

Since this target programming is very precise, these molecular scissors (in contrast to the tools that were previously available for genetic engineering) can cut the genetic material in a living cell at exactly one point. Today other similarly precise gene scissors have been developed but they are not nearly as easy to program.

Cuts or breaks in DNA often occur naturally. So there are repair mechanisms in each cell to close them. The simplest way to rejoin the two ends of a cut DNA strand is non-homologous end joining. This links two DNA ends without any regard to errors or wether they fit or not. Unfortunately this does usually lead to a few individual letters (base pairs) lost from the DNA sequence or a few new, random ones added.

Small error in the code, big effect – the knock-out

If this sequence is a gene, i.e. the blueprint for a protein, this is devastating for this blueprint in two out of three cases. The reason for this lies in the language of DNA, the code. Every three letters (bases) in DNA form a word. The words are amino acids, i.e. the basic building blocks of proteins, which in turn are the whole sentence that is in a gene.

The problem with writing this way is that there are no spaces that determine where one word ends and the next begins. As a result, there are three possible places to start reading, which lead to completely different words and phrases. A special three-letter word, the start codon, defines the start of reading, i.e. translating the DNA into a protein. The end is again determined by a special word, one of three possible stop codons.

This type of code means that if a single letter or two is added or omitted the entire meaning of the rest of the sentence changes because the reading frame shifts. As a result the protein encoded by the gene is no longer produced correctly. So it will be turned off.

In genetic engineering this is called a knock-out.

Adding new code – the knock-in

If you don’t just want to destroy a gene, but want to insert new information, you take advantage of the cell’s other repair method: Homology directed repair. This is actually the better way for the cell to repair breaks in the DNA, as it is less prone to errors. If the cell has a piece of DNA available that has the same (or similar) sequence as the pieces before and after the cut, the cell incorporates that piece and swaps out the broken piece.

This method can be used to insert a new sequence into the genome of a cell. I have explained how to do this here.

With both methods, the sequence originally recognized and cut by the Cas protein is removed and the genome is not cut again after the repair.

Applications from medicine to agriculture

So what can we do with CRISPR/Cas? Since it made targeted changes in the genome of a wide variety of organisms significantly easier, the progress in genetic engineering was vastly accelerated: In research, the function of a gene can be examined quickly by switching it off. In agriculture, plants can be made more resistant to drought and pests by incorporating resistances from other plants. The first genetically modified animals are also being developed, such as a catfish that is supposed to become more resistant to diseases with the help of a gene from the alligator.

In medicine, the first gene therapies will probably heal regular patients in 2023. The first therapies are mostly carried out outside the body. For example, blood stem cells are isolated from patients with congenital sickle cell anemia, a defective gene is repaired and these stem cells are then injected back into the patient. These can then make functioning red blood cells in the patient, which they were previously unable to do.

Principle of CAR-T-cell therapy, CC by NIH

Another important group of gene therapies that work in a similar way are CAR-T-cells. These are the patient’s own immune cells, which have been taught by (by introducing the gene for a special receptor) to recognize and destroy cancer cells more effectively. Several thousand CRISPR/Cas-based gene therapies are currently in the clinical trials.

So CRISPR/Cas is just beginning to change many areas of our lives.

Can everyone change genes with CRISPR/Cas?

CRISPR/Cas is often described as an extremely simple tool that anyone can use in their kitchen to alter the genes of humans and other organisms. Of course it’s not that simple. On the one hand, the “simple” of a molecular biologist is often anything but simple and in addition to a lot of knowledge still requires special equipment and chemicals that cannot be found in a normal kitchen.

Second, it’s one thing to change a unicellular organism or germ cell of a mouse. However, it is quite another to specifically change a group of cells or even all cells in a finished multicellular organism such as a human being. The only tissues in the human body that we can currently access reasonably effectively with gene therapies are the liver, bone marrow, and the eye.

To illustrate that it is not that simple, this diagram shows what the workflow for a single genetic modification in the very simple unicellular baker’s yeast Saccharomyces cerevisiae looks like. The system and also various images of the Ellis Lab are used here.

Schematic workflow of a CRISPR/Cas experiment in yeast, modified from Ellis Lab

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