CRISPR on the cusp

CRISPR has made the limelight in the medical news in recent months and proved the milestone of clinical efficacy and safety. Accompanied by the research of unstable and uncontrollable gene mutation sites of CRISPR, CRISPR gene editing therapy has been pushed to the forefront.

Discover

At the end of the 1980s, Japanese scientists discovered such a long series of very unique sequences in the genomes of several bacteria. In this sequence, there are multiple repetitive DNA sequences separated by a number of ever-changing non-repetitive sequences. This discovery can be said to have planted the seeds of CRISPR’s discovery.
In the early twentieth century, with the maturity of large-scale genome sequencing technology and the rise of bioinformatics, people gradually accumulated complete DNA sequence information of a large number of bacterial species. In the comparison, the researchers found that this similar strange sequence appeared in the genomes of dozens of bacteria and archaea, and the non-repetitive sequence in it was highly consistent with the genome sequence information of known phage.
This large-scale discovery proved that the existence of this strange gene is not a coincidence, so biologists began to guess and try to unravel the meaning of this gene. In 2007, scientists strictly demonstrated the function of the CRISPR sequence on the bacterial immune system in Streptococcus thermophilus. Using gene recombination technology to artificially add a CRISPR sequence to bacteria, the bacteria can produce viruses with the same genes as the CRISPR sequence. Not only that, bacteria can also integrate this CRISPR sequence into their own genome to generate “memory” to cope with the same kind of virus attack.

Mechanism

The CRISPR sequence and CRISPR associated together form the CRISPR/Cas system, which is an RNA-mediated adaptive immune system that exists in approximately 48% of bacteria and 95% of archaea, and can provide sequence-specific protection against foreign DNA Even RNA.
This defense consists of three stages:
(1) In the adaptive stage, the direct repeat sequence (repeat) is separated by a variable DNA sequence to form a CRISPR sequence. At this time, the DNA sequence is called a spacer. Cas protein cuts foreign DNA fragments, the foreign DNA fragments are called protospacer, and then integrated into the CRISPR sequence, then the foreign DNA fragments are renamed spacer;
(2) In the expression stage, the CRISPR sequence is transcribed and cut into mature crRNA;
(3) In the interference phase, crRNA recruits Cas effector protein complex to specifically cut virus homologous DNA.

Application

CRISPR can perform precise targeted editing of genes. With the deepening of research, CRISPR/Cas technology has been widely used. In addition to basic editing methods such as gene knockout and gene replacement, it can also be used for gene activation and disease model construction. , Even gene therapy.
CRISPR was first used clinically to treat β-thalassemia. Beta-thalassemia is caused by a single nucleotide error in the DNA sequence of the patient’s embryo. CRISPR/Cas can accurately find the wrong site in the DNA sequence and perform site-specific repair to achieve the goal of curing patients.
In addition to its applications in blood, CRISPR also has great potential in cancer treatment. The powerful combination of CRISPR and CAR-T cells brings new hope for the fight against cancer. In July 2016, Nature reported on the research of Professor Lu You from China. He planned to use CRISPR-edited T cells to treat patients with metastatic non-small cell lung cancer. The research progressed to November when the edited T cells were formally injected into In a patient with lung cancer. Four years later, in Nature Medicine in April this year, the team published an article stating that research supports the safety and effectiveness of gene editing. The final results showed that after completing the gene-edited cell therapy for 12 patients with advanced lung cancer who failed third-line and above treatments, the subjects were safe and tolerated, and there were no toxicities and treatment-related deaths related to cell therapy of grade 3 and above. In the proof of validity, the median progression-free survival time and median survival time data are 7.7 weeks and 42.6 weeks, respectively.
But at the same time, three recent toxicological studies have shown that CRISPR gene editing in human embryos can cause a large amount of DNA deletion and recombination. Two major factors affecting the application of gene editing are on-target efficiency and off-target efficiency. Off-target rate means that crRNA not only recognizes the target DNA, but also recognizes and cuts other DNAs with different individual bases. This results in a mismatch tolerance rate. When applied to the genome, it only needs to cut the A gene. As a result, the normal B gene is also cut off. , It will cause biological safety problems. However, recent studies have shown that CRISPR/Cas9 will also introduce unnecessary mutations near the target site, which means that part of the on-target target rate will also produce side effects in gene editing, which is more difficult to prevent and Avoided.

Watch over

Faced with unexpected editing consequences and unpredictable DNA repair, future research needs to have a clearer grasp of the mechanism of CRISPR and find ways to control CRISPR to reduce unnecessary editing and mutations that it brings to the genome. As a new drug research and development CRO, Medicilon will also pay close attention to the research progress of gene editing therapy. Watching the mature and stable application of gene editing therapy will help further improve human health!

Reference link:
https://sitn.hms.harvard.edu/flash/2014/crispr-a-game-changing-genetic-engineering-technique/
https://www.nature.com/articles/s41591-020-0840-5
https://www.nature.com/articles/d41586-020-01906-4

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