In 2019, 23,000 people died from leukemia. Twenty. Three. Thousand. We have solutions at hand, such as stem cells, and of course, CRISPR.
CRISPR Cas-9 system is a genome editing tool usually used with Cas-9, a protein (endonuclease) that is used to cut our DNA. Cas-9 acts like a homing missile scissor. It can be coded with RNA to move around and cut pieces of DNA that the scientists want. If a patient is inflicted with a disease from the mutation of a gene, such as leukemia and many other cancers, CRISPR can be used to edit the mutated gene and help the patient recover.
The CRISPR technology is named after CRISPR, the string of repeated DNA used to store viral genes. This was discovered in bacteria which were using CRISPR and Cas-9 as an immune system against viruses. First, the bacteria would snip off a little piece of the virus’s DNA to place into CRISPR. Then, the bacteria would use this to remember what the viral genetic code was. It would bind the newly made guide RNA to the enzyme Cas-9 which would search for the DNA sequence and swiftly destroy it.
In other words, CRISPR is a molecular database found in bacteria. The bacteria use this database to remember the types of viruses that attacked it, to send cas-9 to destroy the viruses’ injected DNA. To prevent further confusion, all mentions of CRISPR from here on are about the CRISPR technology, not the CRISPR in bacteria.
CRISPR and Cas-9 are used by scientists as sort of a search and replace tool, finding the DNA sequence that they are looking for in a target cell and switching it out for a new DNA piece that scientists choose. The scientists start this process by finding a piece of DNA that needs editing. Next, they design a piece of guide RNA (using CRISPR) that will lead Cas-9 towards the area of DNA that needs editing. Cas-9 cuts the DNA, and in comes the new piece of DNA to join them.
However, there are problems. Before the DNA comes, the cell will try to repair it by fusing the two ends back together in a process called NHEJ (Non-Homologous End Joining). However, if scientists place a piece of template DNA for the cell to follow the cell can be rebuilt safely in a process called HDR (Homology Directed Repair).
CRISPR seems magical doesn’t it? A universal tool to solve all disease in the world… BUT CRISPR does have issues.
CRISPR Cas-9’s double strand breaks have issues such as the activation of the p53 gene which is used for tumor suppression. If the p53 is activated from the double strand break, then the cell will die. CRISPR is also way too generalized, in terms of its editing. CRISPR disrupts the genes’ function by cutting it. Many diseases such as sickle cell anemia are caused by point mutations, a single nucleotide (the letters) off in a gene. The mutated gene needs to be rebuilt instead of disrupted by the cut.
Also, CRISPR has many ethical problems. People might not like the idea of changing something for what it would not have been naturally. I talked to some people about how they felt about CRISPR and some of them thought that it was scary for the possibility of everyone to become identical. Of course, genome editing is far from being able to change every cell of the human body to be identical to someone else’s. CRISPR has not been thoroughly tested so editing someones genes may go wrong, or editing any animal or living being’s genes can go wrong.
Base Editing
DNA is made out of little parts called bases. The four bases are A (adenine), C (Cytosine), T (Thymine), and Guanine. However in RNA, T is replaced with U (Uracil). These bases are what base editors change.
Above is a base editor which is involved in the process of base editing. It uses dCas-9 which is a version of Cas-9 which cannot cut DNA but can search and find it. The red part is a protein that changes Cs into Ts. The purple protein protects the newly created T.
The base editor would change the certain base (ex. C into T). This makes a mismatch in base pairs, and the scientists can then make a nick in the opposite strand to make the cell replace the opposite base on its own.
Base editing has the ability to change Cs into Gs and Ts into As, the two combinations for the orange part of the chart (about 14% of known diseases). However, the scientists’ aim was to make the A to T and G to C base editor but the protein that would be used to change the base didn’t exist. Dr. Liu (the inventor of base editing) and his team created a new protein to for the job. Base editing has been tested successfully on animals and plants.
In comes prime editing! Invented by scientists at Harvard and MIT they created an editor that can alter genomic mutations that create 50% of the human point mutations. Point mutations are a single change of a base in a strand of DNA. That’s a huge leap! Not only is it safer than using CRISPR alone but it can do much more than base editing!
Prime Editing
Prime editing is an improved version of CRISPR Cas-9. It features an increased range of editing, the ability to insert or delete part of genomes, while improving in efficiency and precision.
To put it in perspective, base editing can do 4 combinations, and prime editing can do… 12! Prime editing excels at precise point editing, which is basically changing one gene inside a DNA strand. Point mutations account for about 7,000 inherited genetic mutations.
Prime editing can edit non dividing cells such as muscle cells or neurons. This opens up a myriad of opportunities for fighting diseases like muscular dystrophy.
In addition to that, prime editing can insert or delete nucleotides from full strands of DNA. There has been testing for 44 strands but the scientist at MIT and Harvard believe that it can do up to 80. This shows the efficiency of prime editing.
Prime editing works with three parts: a Cas-9 nickase, a pegRNA (prime editing guide) and a reverse-transcriptase.
The pegRNA consists of three parts itself, the PBS (primer binding sequence), the template containing the edited RNA sequence, and finally the gRNA seen in the genome editors mentioned before.
Here are the steps to replacing DNA bases with prime editing:
- pegRNA leads the entire prime editor towards the target DNA sequence
2. The Cas-9 nickase cuts a nick into the strand of DNA that needs editing
3. The RNA slides into the nick, detaching the strand of DNA, binding onto the cut strand
4. The reverse transcriptase enzyme reads the RNA and produces a sequence of genetic codes from the edited part of the RNA
5. This new codes will be placed into the nick become the new part of the target DNA strand
6. However, these steps only correct for half of the strand, meaning that the other half’s bases do not match the edited bases
7. Like base editing, the solution for this problem would be to use Cas-9 to create a nick on the other side’s strand for the cell to remake the strand, with all the new bases
The main advantages of prime editing over CRISPR is the precision, the variety of targets and the safety of single strand breaks. Prime editing has been tested successfully on human and mouse cells for the Tay Sachs disease. Prime editing was a big step in genome editing and fighting diseases.
What does the future look like for prime editing?
Prime editing is still relatively new to the scientific community. There are certain limitations to prime editing but it is much more effective than traditional CRISPR. There needs to be a lot of testing before human usage as it hasn’t been tested a lot. The future looks bright for prime editing as it is such a versatile and useful tool for genome editing and even for combating genetic diseases. Something that scientists have to work on now is creating a genome editor capable of editing both strands of DNA at the same time.
Takeaways
- CRISPR Cas-9 has a bunch of issues including ehics, safety, precision and efficiency
- Base editing proved to be more safe and efficient than CRISPR for its ability to fix point mutations
- Prime editing is a more improved editor that is sort of a mix of CRISPR and base editing, more precise, safe but still taking the advantages of both
- Genome editing still has a long way to go, and prime editing was another step there
- It will be researched and experimented on but I think that scientists should keep working towards more advanced gene editors that can do more, while being safe
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