Fig. 1: Cas9 Protein [Photo credit: BMJ Journal)
Each strand of DNA that every human has is unique to them by the time of egg fertilization. Unlike the clothes we put on our bodies or the ways in which we interact with our peers or the activities we choose to spend our time doing, this DNA is said to be unmodifiable.
Well, up until recently, that is.
Molecular biologists since the 1960s have been making observations about the repeating clusters found in strands of DNA called palindromic repeats. For instance, around two-thirds of human DNA is composed of repetitive elements (Broad Institute, 2020). This discovery makes up the basis of the modern, incredibly intricate gene-editing technology: CRISPR.
CRISPR stands for Clustered Regularly Interspaced Palindromic Repeats― a term coined by researchers Francisco Mojica and Ruud Jansen in 2001. The repeats are 20-40 nucleotides in length, read from left to right (palindromically). Between the repeats, there are unique segments of spacer DNA as the interspaces. According to Bozeman Science, the spacer DNA is fundamentally just the DNA strand’s history of priorly fought off infections, allowing it to perfectly match up with the viral DNA that once infected it. Cas9 is the second necessary component in the CRISPR process; this is a 160 kilodalton protein, “Cas'' meaning “CRISPR-associated.” This protein may act as both helicases, which unwinds DNA for replication, as well as a nuclease, which cuts DNA as if it’s a pair of molecular scissors. Scientists discovered that this is a way for bacteria to fight off bacteriophages, which are essentially viruses that infect bacteria. When a bacterium is invaded by a bacteriophage by the latter injecting its DNA through the cell membrane, the bacteriophage will take the cas proteins through the process of transcription and translation. The DNA is transcribed to make CRISPR RNA (crRNA), which can fit into a cas protein as it prepares to fight off any DNA injected from the bacteriophage by breaking it into pieces. This allows the cas protein to destroy the infection before it actually infects and reproduces in the bacteriophage, eventually killing the bacteria. In the circumstance that there isn’t a matching spacer DNA, a class 1 cas protein is formed, and not only breaks apart the viral DNA, but also copies it into the CRISPR system. This copy of viral DNA would become spacer DNA between the repeats, for these segments are simply the history of previous infections, as stated earlier (Bozeman Science, 2016). Essentially, this is an immune system for bacteria. However, scientists soon discovered that this technology has the potential to be used in more than just bacteria.
Nobel Prize winners Jennifer Doudna and Emmanuelle Charpentier looked further into the idea of using the CRISPR-Cas9 system to both edit and create new DNA. According to Bozeman Science, they used one Cas9 complex, as seen in Fig. 1, which first and foremost contained a nuclease section to cut DNA. crRNA fits into the cas, in addition to tracer RNA (tracrRNA). The spacer segment is also in the cas protein, which is there in an effort to match up to any viral DNA that may invade the cell. Once matched, this complex will easily break it down before it has the chance to invade the cell. The two scientists soon realized that they could use this system to change any sequence of non-viral DNA in which they pleased by replacing the spacer DNA with their own carefully-formulated sequence. By connecting their own sequence with the tracrRNA, they created a new simple system, coining the term tracrRNA-crRNA chimera. A chimera is essentially a single organism made up of two sets of DNA with codes for two separate organisms, hence why the new RNA that they invented as a mix of two separate RNA types is considered to be one (Bozeman Science, 2016). The simple system that Doudna and Chapentier created has two parts, the first of which being Cas9 and the second as the tracrRNA-crRNA chimera, which is also known as the guide RNA (gRNA). These parts are displayed in Fig. 1, where the gRNA has the information as to where the scientists want to cut, while the Cas9 protein does the cutting. A gRNA is made to correspond with the segment of DNA that the Cas9 wants to cut, allowing the DNA to feed through. The protospacer adjacent motif (PAM) is a DNA sequence from 2-6 base pairs in length that follows the targeted DNA region; without it, the Cas9 would be unable to bind to the target DNA sequence. It distinguishes bacterial self from non-self DNA, which prevents the location of the CRISPR repeats from being destroyed by the Cas9 nuclease (McDade, 2020). The Cas9 cuts the DNA segment once it’s in between the nuclease, breaking the gene and causing it to become inactive. When the cell tries to fix this, many mutations may arise, which is where both Doudna and Charpentier chose to inactivate the genes before the gene itself makes any attempt to. Since this is crafted carefully by scientists, said scientists can choose exactly the nucleotides by which the Cas9 may cut and put it into the gRNA. To insert another gene in place, not only are the Cas9 and gRNA required but also is the host DNA which will be inserted. As the Cas9 breaks the DNA, the host DNA is added, allowing the DNA to fix it. Essentially, this adds a new gene to any organism’s genome that’s fully able to be formulated by scientists (Bozeman Science, 2016).
From lessening the symptoms of diseases such as sickle cell anemia to growing the agriculture industry, CRISPR-Cas9 technology is undeniably the future of genetics. New technology like this will continue to advance and modernize the fields of chemistry and genetics forever.
 Bozeman Science. (2016, February 18). What is CRISPR? [Video]. YouTube.
 Broad Institute Writers. (2020, September 21). Research Highlights: CRISPR. Broad Institute.
 Richardson, M. W. (2019, July 15). What Is CRISPR Currently Being Used For? BrainFacts.org.