CRISPR-Cas9

1. How the CRISPR-Cas9 works?

CRISPR-Cas9 is a 3rd generation genome editing system which is discovered from prokaryotic self-defense system such as bacteria. This self-defense system can be broken down into 3 stages. First, adaptation, second, expression, third, interference.

The word CRISPR is related to first stage, adaptation. This stage is conducted when CRISPR has no invaded DNA information. CRISPR is a specific sequence and it stands for Clustered Regularly Interspaced Short Palindromic Repeats. Once viruses such as bacteriophages infect bacteria, enzymes in bacteria called Cas1 and Cas2 then excises the invaded DNA from viruses into pieces consist of 21 nucleotides which are called ‘spacer’, and attach them to its own DNA’s CRISPR loci. The regions of invaded DNA that were excised is not random but instead were found adjacent to sequence called protospacer adjacent motif (PAM).

During expression, the caught sequences are transcribed into CRISPR-RNA (crRNA) and recruit proteins to form CRISPR-Cas complex. Before recruiting proteins, usually, though it depends on the type of CRISPR-Cas system, a small fragment of RNA called tracrRNA pairs with crRNA. Now, the RNA that are used in base pairing with target DNA is called guide RNA (gRNA) and the complex has such gRNA is called CRISPR-Cas9 complex.

The last step is interference. During interference, the CRISPR-Cas9 complex detect and degrade foreign DNA. The complex is double-RNA-guided dsDNA endonuclease so that it causes DSB. The broken foreign DNA then repaired by NHEJ or HDR. NHEJ repairing process are error prone, so the process cause the gene to be knock-out. HDR repairing process can change the gene into what we want by introducing sequence intentionally (called host RNA or donor DNA). To cause DSB, the complex first have to find out the sequence complementary to gRNA. It recognizes PAMs just like adaptation step. Once it find out complementary sequence among PAMs, it cuts DNA, resulting in self-defense system or genome editing as described above.

 

 

2. Pros and Cons of CRISPR-Cas9

Pros

1. It is much simpler than previous genome editing technique. CRISPR can generate DSB wherever we want by using only single protein. Also the part that need to be modified is RNA, not protein such as 1^st and 2end generation genome editing technique
2. It is much cheaper.
3. It is much faster.
4. It is much more precise
5. Because RNA doesn’t combine with other components in the cell, it is able to inject several CRISPR at the same time.

           Cons

1. It causes DSB, which means abnormal DNA structure and error possibility.
2. It causes off-target binding. Actually, according to the corresponding paper published on May 30th in nature method, it has been revealed that CRISPR-Cas9 system can cause unexpected mutation after editing in vivo. Researchers treated two mice with CRISPR and compared them to one uncorrected mouse about a mutation in the Pde6b gene. They found that several indels and single nucleotide variant which are unexpected and among them, 5 indels and 24 single nucleotide variants caused mutations in protein coding sequences. Furthermore, the 5 indels and 1 single nucleotide variant are turned out to be deleterious.
3. Its appearance is so sudden that researchers haven’t built philosophy to use it yet. So it should be cautious in using it to human.

 

3. Application of CRISPR-Cas9

HIV cure

Cancer cure-T cell modification

Eye disease cure

Sickle cell disease cure

Hemophilia cure

This part is going to be filled soon

 

4. Further research

CasX & CasY

Cpf1

ABPs

This part is going to be filled soon

5. Questions

What would happen if we are able to modify telomerase gene with CRISPR system?

-  Telomere is a kind of cap for DNA that protect its content. The telomere is located on the both ends of double strands and elongated by the enzyme ‘telomerase’. If there is not enough telomere, the content of DNA would be attacked or scattered. Theoretically, it looks like telomere can be infinitely long but it doesn’t. Especially in somatic cell, lack of telomerase induces shortening of telomere and cause cell apoptosis in the end. Therefore, telomere is a clock that control the life span of DNA. But in germ line cell and cancer cell, telomerase keeps telomere long enough so that the cell would live forever (in aspect of telomere). Such feature is critical for germ line cell to deliver intact information to daughter cells. But in case of cancer cell, it is a pain for patient if the cell doesn’t die even it has to be died. Therefore, if we are able to knock-out telomerase gene in the cancer cell with CRISPR, then we can lead the cancer cell to die and cure the cancer. I think it is efficient way because using CRISPR is cheap and easy. Also it doesn’t go with pain. And it doesn’t depends on the type of cancer because telomere is a general feature of DNA. Also telomerase gene is easier to modify compare to telomere because many proteins are bind to telomere and those make hard for Cas9 to act.