So How did CRISPR ever come to be?
It was through the mechanism of a bacteriophage (a type of virus), aimed to inject its own DNA into the genome of the host bacterium. By doing so, the viral DNA would use the host’s own genetic processes (transcription, translation into proteins) into replicating itself, enhancing its genealogical survivability (Professor Dave Explains, 2021).
Conveniently enough, the phage genome intrinsically contains its own crRNA. If we assume that the phage genome successfully incorporates itself into the bacterial genome, and the bacteria does not die, then two important observations can be made. 1) The phage genome can replicate itself, thus enhancing its biological survivability and 2) the original bacterium can withstand such a gene capture that does not lead to the death of the bacterium outright. The second point is a vital observation here, as the phage genome cannot incorporate its own genome a second time; this is due to the discovery of what some label as “the bacterial immune system” (Biomedical and Biological Sciences, 2017). The reason for this is the bacterial genome already has an embedded sequence matching the phage, so any subsequent interaction with the phage’s DNA (assuming no mutation event has happened in between) would be redundant (Shomu’s Biology, 2020).
A more general explanation of this process includes the following:
a) Bacteriophage attaches itself to bacteria/archaea genome
b) Bacteriophage genome contains the spacer sequence, which is complementary to the host bacteria DNA or RNA target, depending on what template the host bacterium contains (Professor Dave Explains, 2021)
c) The bacteriophage DNA incorporates itself, along with the PAM site. The bacterial genome contains cas genes upstream of the incorporation event.
d) Cas9 nuclease protein recognizes the PAM site, newly placed onto the bacterial genome by the bacteriophage, attaching its catalytic domains onto the target DNA/RNA + bacteriophage genome including CRISPR sequence complex (Andrew Douch, 2022). This will form a loop that will fall off the genome, containing crRNA, trcrRNA, and the viral genome incorporated segment
e) The Cas9 undergoes processes which eventually leads into the cleavage of this complex — involving HNH rotation that is an integral part of this process (Innovative Genomics Institute, 2017), RNAse III will excise the crRNA/trcrRNA complex off. Then Cas9 cleaves the invading bacteriophage DNA off (Andrew Douch, 2022).
f) DNA repair mechanisms are then included to repair the damage (either the NHEJ or the HDR repair pathways) (Uddin et al.)
g) The bacterial cell, upon subsequent infection by the same bacteriophage, will recognize the PAM sequence à take adjacent original viral spacer DNA à form crRNA à forms CRISPR complex due to original complementary base pairing.
h) Signals Cas cascade (induce double strand break à break bacteriophage DNA). The bacteriophage cannot incorporate its genome into the bacterial genome a second time, thus relinquishing the bacteriophage’s ability to keep using the host’s genome.
It is this eventual recruitment of Cas9, followed by the DNA repair mechanisms that contribute to the development of the immune system for Archaea/bacteria. Of note, a bacterial organism that can undergo this process is Pseudomonas aeruginosa (seen in cystic fibrosis patients), as well as Streptococcus pyogenes.
(On a side note, Pseudomonas aeruginosa are these rod-shaped bacteria, which can force your body to produce snot mucus to feed themselves with your secretions via a secreted LasB toxin (Cara). I may do a post in the future regarding Cystic Fibrosis in general, related to treatment methods and applications of this technology.)
While this is a general overview of the mechanism, there were a lot of details left out. For instance, the PAM site exhibits a particular NGG sequence (N representing any nucleotide) on the genomic DNA sequence (in Streptococcus pyogenes). This is seen in the first figure below, how the PAM site is oriented on the bottom strand that is not targeted for transcription into crRNA (Palindromes, 2022).
Furthermore, the CRISPR RNA, itself is palindromic — reading the same forward as it does backwards. The palindromic sequence is complementary to a tracrRNA, subsequently cut by and RNAse III enzyme. This complex of a tracrRNA and crRNA make up a single guide RNA that recruits the cutting enzymes (Palindromes, 2022).
The figures may help to explain exactly the complex that would trigger a Cas9 excising event. The two pictures both describe how a hairpin loop, which is subsequently excised by the Cas9 enzyme, forms upon itself. The hairpin loop drops out of the picture, which signifies a gene Knockout (how genes can therefore be silenced). The palindromic DNA sequences are seen on complementary DNA strands, which consequently do not code for functional proteins. Reason being, the DNA palindromic sequences can fold onto itself very easily, and the RNA polymerase would have a difficult time for reading the DNA and transcribing a new sequence to subsequently be translated into proteins for the cell. This is shown in Figure 2 (Palindromes, 2022).
When working with CRISPR technology (infusing a guide RNA), the fundamentals of genetics become ever more important as the goal is to make sure we target 1) the correct strand and 2) the correct region. For us humans, each strand in each chromosome contains promoters for all sorts of genes that will be later transcribed and translated into proteins.
So how does the DNA repair itself after a gene has been erased?
Works Consulted
Andrew Douch. (2022, March 16). Understanding CRISPR-Cas9 [Video]. YouTube. https://www.youtube.com/watch?v=cLMo6DYdJRE
Biomedical and Biological Sciences. (2017, March 2). CRISPR System and CRISPR CAS9 Technique, The full principle (Part 1) [Video]. YouTube. https://www.youtube.com/watch?v=VtOZdThI6dM&t=1026s
Cara, E. (2022, April 29). This Bacteria Forces You to Make Snot, Then Eats It. Gizmodo. https://gizmodo.com/this-bacteria-forces-you-to-make-snot-then-eats-it-1848855824?msclkid=1e42b045cd6311ec99d97e501e14f7fd
Esvelt, K. M., Mali, P., Braff, J. L., Moosburner, M., Yaung, S. J., & Church, G. M. (2013). Orthogonal Cas9 proteins for RNA-guided gene regulation and editing. Nature methods, 10(11), 1116–1121. https://doi.org/10.1038/nmeth.2681
Integrated DNA Technologies. (2018, February 23). Optimized methods to use Cas9 nickases in genome editing [Video]. YouTube. https://www.youtube.com/watch?v=sGLdK5DloqM
Palindromes in the genome. (2022). Max-Planck-Gesellschaft. Retrieved May 6, 2022, from https://www.mpg.de/11823627/crispr-cas9-palindromes-structure
Professor Dave Explains. (2021, October 29). CRISPR-Cas9 Genome Editing Technology [Video]. YouTube. https://www.youtube.com/watch?v=IiPL5HgPehs
Shomu’s Biology. (2020, October 9). Crispr cas9 gene editing explained [Video]. YouTube. https://www.youtube.com/watch?v=OPkPwNVNLEs
Uddin, F., Rudin, C. M., & Sen, T. (2020). CRISPR Gene Therapy: Applications, Limitations, and Implications for the Future. Frontiers in oncology, 10, 1387. https://doi.org/10.3389/fonc.2020.01387
