Genetic Scissors
This year the Royal Swedish Academy of Science has awarded the Nobel Prize in Chemistry to Emmanuelle Charpentier and Jennifer A. Doudna for “the development of a method for genome editing”. Let’s find out what exactly they did.
A BRIEF HISTORY OF SPACERS
In the end of the 80’s scientists have sequenced E. coli genome and have found a certain genome region that doesn’t have any code. This region consists of the repeating DNA sequences (palindromic repetitions) that’s separated by variable regions — spacers. The existence of long non-coding DNA sequences has surprised scientists because bacteria are very economic on their genetic code and commonly don’t have extra sequences. Later these repetitive “cassettes” and spacers were found in many bacteria and archaea, and was named CRISPR — clustered regularly interspaced short palindromic repeats.
Some strains have variability by the presence or absence or order of spacers in CRISPR-cassettes. This feature has become commonly used for strains typing and epidemiology analysis. Particularly, Danisco Company produces starter cultures for dairy industry, it uses this feature for classification of commercial strains.
In the beginning of 2000, different groups of scientists have compared CRISPR-spacers with DNA sequences from public databases. They found that often the spacer sequences were similar with virus sequences. At that time scientists suggested that CRISPR-cassettes can have protective function.
The results of study were interested by Danisco Company because viruses-bacteriophages inhibitis fermentation process; which may cost their business. In order to avoid it, specialists have used strong virus resistant-bacteria. It was easy to get bacteria: and make clones that can live along with viruses in a laboratory. Danisco Company made this procedure, and specialists found one small detail. CRISPRcassettes clones along with viruses have got new spacers matched the region in virus genome. They made a direct experiment by molecular genetic methods: they placed a spacer with virus DNA sequence into bacteria CRISPR-cassette. Genetic modified bacteria became virus resistant. Thus, one of the main “immune” mechanisms of bacteria directed at foreign DNA of bacteriophages and plasmids was discovered.
HOW CRISPR\CAS WORKS
In the first stage, when virus gets into a bacteria cell, part of its genome is cut out and inserted as a spacer in CRISPR. The bacterium is “immunized” with this genetic material. If the bacterium survives, it retains a “memory” of the virus. The second stage is connected with reinfection by the virus and CRISPR expression. GRISPR gene is big enough and is synthesized with RNA called — pre-crRNA. When CRISPRcassette and pre-crRNA work, then one short RNA appears. This RNA cut in certain regions to be mature. Scientists named it trans-activating CRISPR RNA, or tracrRNA.
In the third stage tracrRNA connects with CRISPR palindromic consequences. The complex connects with CAS-proteins. Nucleases cut transcript into 39–45 nucleotides of matured crRNA, it consists of a single spacer sequence, 8–9 nucleotides of palindromic repetitions on the 5’-end, and heterogeneous fragment of a repeated part on the 3’-end. These repeated parts are necessary for rod-loop structure formation (spatial configuration of RNA, including two sequences of the same line, they are complementary to each other and connect to each other by bending to one another, forming an unpaired region at the end — a loop) (Pic. 2).
In the fourth stage, interference of foreign DNA or RNA occurs due to the interaction of crRNA and CAS proteins. CrRNA complimentarily recognize the sequence of the viral genome, and causes destruction to the virus. This is the mechanism of “CRISPR immunity” against viruses and plasmids.
WHY DID THEY GET THE NOBLE PRIZE THEN
In 2012 Charpentier and Doudna groups from the Berkeley University published an article in Science. They suggested a way of re-programming CRISPR\Cas system so that it cuts DNA in areas selected by the group of researchers. In nature CRISPR RNA is coded into CRISPRcassette, binds with protein and then recognizes the target. They also found out that they can obtain artificial CRISPR RNA with chemicals and enzymatic synthesis. Then, the place of spacer in RNA will get a sequence that can be determined by scientist. Cas9 protein is able to ‘recognize’ and connect with synthetic CRISPR RNA (called “guide”) and it is programmed on recognition and cutting a certain part of DNA. Charpentier and Doudna groups explained the possibility of this approach via in vitro.
The new approach allows us to solve the following tasks:
— creating plants and animal that have new and worthy features and properties (yield, resistance to adverse environmental conditions, pests and pathogens);
— obtaining mutant model animals for study of human diseases;
— developmental methods of gene therapy, correction of genetic mutations in cultivated human stem cells;
— creating cell models for searching and preclinical study of new drugs.
A bright example of the possibilty of CRISPR\Cas application in medicine is treatment of malignant neoplasms of the blood. Red bone marrow transplantation for patients with a previous condition (myelotoxic effect of high doses of chemotherapy drugs or ionizing radiation, leading to the death of three hematopoietic lines). However, even a perfectly matched donor does not guarantee absolute compatibility. If we take the patient’s own blood stem cells and edit their genome using the CRISPR/Cas method, we will achieve a complete match of the graft and host by antigens, which means there will be no rejection reaction.
Since the discovery by Charpentier and Doudna discovered “genetic scissors” CRISPR\Cas9 in 2012, their method usage has been exponentially used. This tool has contributed to countless fundamental discoveries and gives a possibility of treating monogenetic diseases. However, many questions about CRISPR\Cas remains unsolved, and the “genetic scissors” technology is still far from being perfect.
text Filipp Novikov
edited by Olga Starkova
edited by Samraj Shiraz
