CRISPR-Cas9 system functioning in living organisms. Illustrator: Stephen Dixon
The so-called gene editing technology refers to the deletion and insertion of DNA nucleotide sequence. In other words, gene editing technology enables people to rewrite DNA according to their own will, a book of life written by deoxynucleotides. However, for a long time, DNA editing can only be done by physical and chemical mutagenesis and homologous recombination. However, these methods either edit at random or require a lot of manpower and material resources to operate. Therefore, it is a long-term dream for researchers to edit DNA and nucleotide sequences conveniently and accurately. The birth and maturity of CRISPR-Cas9 system marks the gradual realization of this dream. In addition, this technology also shows great application prospects not only in the field of scientific research, but also in the research fields of medical care, agriculture and animal husbandry. Therefore, the patent of this technology is a milestone in its application.
From bacterial immune system to DNA editing tools
CRISPR-Cas9 system is not made for human use. Its essence is actually a defense system against foreign DNA such as viruses in bacteria. There are a series of DNA sequences arranged in clusters in some bacterial genomes, which are called "short palindrome repeats at regular intervals" (CRISPR). It is found that the interval sequence of these repetitive sequences is the same as that of many phage DNA sequences that can invade bacteria. Further research found that after these sequences were transcribed into RNA, they could form a complex with a protein called Cas produced by bacteria to guide Cas protein, so this RNA was also called gRNA. When the complex detects that the invading DNA is consistent with gRNA sequence, Cas protein can cut the invading DNA to achieve the purpose of defense.
Note: Strictly speaking, gRNA in bacteria consists of two parts: one part is tracrRNA needed to activate Cas protein, and the other part is crRNA from spacer to recognize invading DNA. In the artificially constructed CRISPR-Cas9 system vector, these two RNA fragments can be fused into one.
CRISPR-Cas system is a sequence-specific DNA cleavage mechanism, which quickly attracted people's interest. Because the system can cut DNA and its sequence specificity is determined by the sequence of crRNA, it has become an ideal tool for DNA editing. CRISPR-Cas system in bacteria is extremely diverse, and the system from Streptococcus pyogenes in which Cas9 protein participates has been studied thoroughly. Therefore, it was modified, and the sequence encoding Cas9 protein and its auxiliary element * * * were made into a single vector. At the same time, in order to let these components enter the nucleus of eukaryotic cells, a nuclear entry signal element is added. In this way, researchers only need to synthesize a DNA sequence for the DNA sequence to be edited and insert it into a specific part of this vector. After the artificially constructed gRNA is transferred into the host cell, it can guide Cas9 protein to cut the specific DNA sequence of the host cell, thus playing the role of gene editing.
Schematic diagram of CRISPR-Cas9 gene editing system. Image source: Progress in biochemistry and biophysics
The broad prospect of DNA editing
CRISPR-Cas9 system is called the third generation gene editing technology. Compared with its two predecessors, ZFN system and Tarun system, it has some incomparable advantages. First of all, CRISPR-Cas9 system has more available locations. Theoretically, every eight bases in the genome can find a position that can be edited by CRISPR-Cas9. It can be said that this technology can operate on any gene, while Taran and ZFN systems can only find one available position among hundreds or even thousands of bases, which greatly limits the scope of use. Secondly, CRISPR-Cas9 system is more scalable. For example, Cas9 protein can be modified so that it does not cut the DNA double strand, but only cuts the single strand, which can greatly reduce the risk of chromosome variation caused by non-homologous end connection after cutting the double strand. In addition, Cas9 protein can be linked with other functional proteins to study the effects of these proteins on cell-specific DNA sequences. Third, more importantly, the CRISPR-Cas9 system is very convenient to use and can be completed in a few simple steps. Almost any laboratory can work without the help of commercial companies like ZFN and Talon. Because of the above characteristics, CRISPR-Cas9 was rated as one of the biological breakthroughs in 20 13 10. It is worth noting that many important studies on CRISPR-Cas9 system in eukaryotic cells were conducted by China scholar Zhang Feng.
Because the CRISPR-Cas9 system derived from bacteria can also work well in eukaryotic cells, it shows its great application potential. For example, in the field of basic scientific research, CRISPR-Cas9 system is often used to knock out some genes at fixed points, so as to study the biological functions of these genes. At the same time, the commercial application potential of CRISPR-Cas9 system should not be underestimated. For example, in the field of biotherapy, combined with the technology of inducing pluripotent stem cells (iPS), people can redevelop iPS cells repaired by gene editing into normal tissues and organs for patients to use. In livestock breeding, editing some key trait genes can greatly speed up the breeding of improved varieties.
Because of many excellent features and broad application prospects, CRISPR-Cas9 has become a hot spot in patent application. Although there have been many patents on the application of CRISPR sequence or Cas protein, this extensive research was approved as the first patent including a complete set of CRISPR-Cas9 system vectors and operation methods. This means that the gene editing operation using this technology in the future will involve the content protected by this patent. So, will patent approval hinder the use of this technology? At present, basic research is unlikely to be affected, because Eric Lander, director of the Broad Institute, said in the press release of the patent announcement: "Considering that the mission of the Broad Institute is to accelerate our understanding and treatment of diseases, we promise to authorize research teams around the world to use this technology." However, it remains to be seen whether the emergence of this patent will have an impact on enterprises that use this technology in more profitable commercial applications. After all, in addition to the above statement, Yuanda has another sentence: "Enjoy the right to restrict this patent."