Today I want to talk about the field of bioengineering in science, using the CRISPR-Cas9 system as an example. So let us dive in. First, we need to understand what it looks like mechanistically.

It all began way back in 1987 in Japan, when Dr. Atsuo Nakata and his team first reported the presence of clustered regularly interspaced short palindromic repeats (abbreviated CRISPR) in the genome of E. coli.

From the name, these DNA fragments are regular, repeated, and palindromic, meaning that their sequence is the same either when read from 5′ to 3′ or from 3′ to 5′. And it turns out that the unique sequences located between these palindromic repeats are foreign pieces of DNA that do not belong to the bacterium, but come from foreign genetic elements such as bacteriophages, transposons, or plasmids that have previously infected the bacterium. All of these sequences (palindromic and random) were later shown to be an important element in the adaptive immunity of bacteria. But how exactly does this work? During a viral infection, the bacterium acquires a small portion of the viral DNA and integrates it alongside the CRISPR fragment. So the non-palindromic sequences, which have been termed “spacers,” are the part of the viral material that the bacterium has already encountered in its life and has stored its footprint so as not to forget. This reminds me of how we as humans frame pictures of significant people in our lives and carefully store them in the gallery of our memory. Time may pass, but all the little details of our significant people that are important to us are kept safe. You can see the picture that illustrates the analogy between this gallery and the CRISPR loci organization. The different people in the frames in our minds are like exogenous viral material for the bacteria (they are called spacers and represent the parts of the viruses that have been excised for further storage). Between them are bacterial DNA motifs (CRISPR) that can be compared to a wooden decoration between the pictures in some designer photo frames.

During their life, bacteria make special short RNA from each of these CRISPR arrays (a spacer + a CRISPR motif itself) that can bind with a special protein with nuclease activity called Cas (Cas stands for CRISPR-associated nuclease protein). These complexes of short RNA and a Cas protein are now produced and circulate within the bacterial cell, just as antibodies circulate in the human body. Once this complex encounters free nucleic acid (which bacteria think may be a bacteriophage), the RNA of this molecular complex tries to find a complementary piece on the viral strand (it knows what the virus should look like because it already has a picture of it in its own sequence). Once the complementary strands match, the Cas protein with nuclease activity begins to make a cut in the viral DNA.

So, in essence, bacteria have several complexes consisting of a protein and a “guide” RNA that can identify a specific piece of DNA, bind to it, and make a cleavage. Of course, these amazing complexes should have been used in science, and that’s exactly what bioengineers Dr. Jennifer Doudna and Dr. Emmanuelle Charpentier did. They were the first to come up with the idea of harnessing this phenomenon for the genome editing needs of scientists. For this, they were awarded the Nobel Prize in 2020 and gave us all a great example of what bioengineering is.

In short, it is the field of science that takes advantage of many beneficial phenomena of nature to use them as specific scientific tools that enable us to achieve the desired effects. With the CRISPR-Cas9 system, we can develop certain complexes that make a cut at any desired specific location in the genome (this depends on the RNA we synthesize). Once the cut is made in the DNA, the cell tries to glue parts of the DNA back together (in the process of non-homologous end-joining), but this gene is then no longer functional. In this way, we can create a knockout of the harmful mutant gene (turn it off so it no longer functions). In addition, we can restore the function of the damaged gene by giving a cell a clue in the form of a short matrix DNA that it can use in restoring the genomic DNA after the break initiated by CRISPR-Cas9 (this process  is known as homologous recombination).

That’s how fantastic bioengineering is.  By using different functions of proteins such as DNA binding/RNA binding, nuclease activities, etc, bioengineers are able to create different tools for use in the lab and in the clinic. I find it fascinating and exciting that we can not only observe and study how nature works and operates, but also use some of its concepts to create our own. We can edit the cell’s genetic material, influence its signal transduction, produce interesting vesicles and antibodies – we can make little organs out of cells, called organoids. All this is a process of creation that can hardly be underestimated in its fascination and influence on scientific discoveries.