🕒 7 min
In the previous article, we started discovering what is hidden behind the name of CRISPR-Cas9. The main idea of this series of articles is to understand the background of this technology for precise genome editing. It’s truly fascinating how one unusual feature of the bacterial genome has served as an inspiration for further discoveries and developments in biotechnology. Of course, none of this would be possible without the mutual cooperation of scientists all around the world.
A quick recap of the previous part
Last time, we learned how the CRISPR region of the genome was discovered in the first place, and what its characteristics are. The region is unique for the interchange of short palindrome sequences with spacer sequences that match parts of genomes of different phages. Also, we defined what cas genes, which are closely related with CRISPR region, are and mentioned an interesting protein, Cas9. Cas9 is a nuclease, which means it cuts nucleic acids (RNA and DNA belong to this category). In the end, we described an experiment that was crucial for discovering that the CRISPR region plays the role of acquired immunity in bacteria.
Today, we’re taking another step further: we will describe how this “bacterial immunity” actually functions at a molecular level.
Nothing without the RNA
Since bacteriophages or plasmids present a potential threat for bacterial cells, there has to be a way for the information written in the CRISPR genome region to become an active player in the fight against them. It’s important to understand that genetic information in the CRISPR region – a sequence of nucleoids – cannot do much on its own. Some other molecules that will do the active part of the work are needed: otherwise, the CRISPR region would be something like a letter no one is bound to read. Now it’s important to remember the process of transcription of DNA to RNA.
Why is RNA important in this part of the story? As we have previously discovered, inside the CRISPR region there are spacer sequences that are the same as parts of the genomes of different bacteriophages and plasmids. If the RNA is complementary to the spacer sequence’s DNA, then it will also be complementary to the DNA of the bacteriophage that attacks that cell. Remember this idea of complementarity between DNA and RNA, we’ll need it for further explanations.
And the result of transcription is…
It’s time to visit the Netherlands and meet John van der Oost, a scientist that had just started working at the Wageningen University in 1995. Initially, the goal of his project was to explore the metabolism of extremophilic archaea that live in Yellowstone. One of his cooperators on this project was Eugene Koonin, whose area of interest were the different CRISPR systems that can be found among various bacterial species, considering they can be very different across the bacterial kingdom. In general, they are split into two main classes, depending on how many Cas proteins act as molecular scissors. Last time, we encountered the CRISPR system of S. thermophilus, where only one protein, Cas9, played the nuclease role. Apparently van der Oost got really interested in that area because in 2005 he dedicated a cut of his funding precisely to CRISPR research, to discover something new.
In 2008, scientists conducted an interesting experiment on the CRISPR system of E. coli bacteria. In this experiment, they were literally watching for what would happen to the RNA molecule that gets transcribed from a CRISPR genome region. They realized that in E. coli no less than five different nucleases are needed to cut the transcribed RNA molecule into a smaller RNA molecule called CRISPR RNA or, simply, crRNA.
By sequencing the crRNA, they also discovered that their sequence starts with last eight nucleotides of those CRISPR region palindrome sequences. A complete spacer sequence follows right after, and the beginning of the next palindrome sequence. This information is extremely important because it actually tells us a bit more about the structure of crRNA: those palindrome sequences from ends of the crRNA can mutually connect and create a folded, secondary structure of the RNA molecule that’s ready to interact with other nucleic acids.
Another important accomplishment of van der Oost’s group is the first programming of the CRISPR region. They managed to literally slip genes of bacteriophage λ as spacers into the CRISPR region of a strain that normally gets infected by the that phage. Of course, the results showed that this method really gets bacteria resistant to the infection.
Small but precise
In the last article, we also talked about the experiments that were conducted by Philippe Horvath and his research team in 2010. These experiments were the first to show that the CRISPR region acts as a bacterial immune response. One of his colleagues was Sylvian Moineau, who was still interested in this area a few years after their joint discovery. This time, he wanted to discover how those very precise cuts of plasmid or bacteriophage DNA were actually made.
They worked with bacteria of the bacterial species S. thermophilus. They tried to preform a transformation of those bacteria by introducing plasmid DNA by electroporation, in order to observe how it gets destroyed inside of the cell. Unfortunately, it was almost impossible to isolate any intermediary product since the entire process happened really quickly. Still, they were fortunate enough to isolate a strain that is not as efficient in DNA degradation. Freshly cut plasmids could be found in the cytoplasm of these cells, which was perfect because it was possible to sequence them to see how they were cut.
After analyzing the results, an important discovery was made: all of these plasmids were cut exactly 3 nucleotides away from the sequence that is known as the PAM sequence (short from proto-spacer adjacent motif) today. Soon, they repeated the experiment using a bacteriophage and the result was similar: DNA is cut at the same distance. Furthermore, they showed that the number of cuts in the foreign DNA is exactly the same as the number of spacers that can be matched with it. Overall, crRNA that gets transcribed from the CRISPR region is certainly an important intermediary that provides the information of where Cas9 (or some other nuclease, depending on the species) will cut the foreign DNA.
Another kind of RNA is coming
We said that RNA molecules would be really important back at the very start. Now is the time to meet scientists Emanuelle Charpentier and Jörg Vogel, whose research area is precisely bacterial RNA. Charpentier was searching for new types of regulatory RNA molecules. She performed a bioinformatical analysis of the genome of S. pyogenes, paying close attention to the areas that do not code for proteins. As a result, she came across a region that is near the CRISPR locus from which a new type of RNA was possibly transcribed. In 2007, she started to work with Vogel, who sequenced the entire transcriptome of S. pyogenes. “Transcriptome” is a term used for all RNA molecules that get transcribed from a genome.
When they analyzed all the RNA molecules in S. pyogenes, this unusual type of RNA transcribed from region near CRISPR locus really stood out. In fact, it turned out to be the third most abundant RNA molecule in the cell. Today, this molecule is called tracrRNA. Another important discovery is that it is complementary to the palindrome sequence in 25 nucleotides. This time, complementarity points to mutual interaction between crRNA and tracrRNA. In later experiments, it was shown that tracrRNA truly is important for processing crRNA.
All the puzzle pieces
Now we have collected all pieces of the CRISPR system: we have crRNA, tracrRNA, and one or more Cas proteins (nucleases). Of course, we also need a region in the genome where information on how to make all these pieces is kept. But, why wouldn’t we try to produce all of these molecules in vitro and simply insert them into a bacterial cell?
This idea came to mind to Virginijus Siksnys, a Lithuanian chemist from Vilnius, who decided to do research on restriction enzymes. Since the most important function of the CRISPR system is cutting DNA (which is what restriction enzymes do, too), he got really interested in this area.
The goal of his team’s research was to investigate whether the functional CRISPR system of one bacterial species can be constructed in another bacterial species. In the experiment, they moved the CRISPR locus of S. thermophilus into E. coli and showed that it remains functional. Just as a reminder, E. coli usually needs five molecules to cut foreign DNA. In this experiment, they showed that only one nuclease (Cas9) is needed, even in a distinct bacterial species.
This is a very important step for the further development of CRISPR-Cas9 technology because it makes it clear that only one nuclease, Cas9, along with tracrRNA and crRNA, is all that is needed to cut foreign DNA, and that this holds true for all bacteria. Now we’re finally ready for the more exciting part – how scientists took advantage of this discovery in order to develop a new method of genetic engineering.
See you in the next part!
Image source: https://hackmd.io/@aphanotus/bi332f19