The 2020 Nobel Prize in Chemistry was awarded to Jennifer Doudna and Emmanuelle Charpentier for their research achievements in gene editing technology CRISPR. Before the development of CRISPER-Cas9 technology, the first generation zinc finger nucleases (ZFNs) and the second generation transcription activator effector nucleases (TALENs) have been widely used. The principle of the three is to induce double strand breaks (DSB) in the genome sequence, and then correct them through endogenous repair mechanism, so as to achieve the purpose of gene editing such as gene fragment deletion, insertion and mutation.
It has been known for decades that endogenous genomic sequences are exchanged with exogenous donor DNA molecules by homologous recombination (HR). The late oliver smithies explained for the first time how homologous DNA molecules were recombined and correctly inserted into specific positions of mammalian chromosomes. For this reason, Smith won the 2007 Nobel Prize in Physiology and Medicine together with mario capecchi and Martin Evans. ?
In 2009, scientists used ZFNs technology for the first time to create the world's first gene knockout rat. 1996, vigorously develop ZFNs technology. By transforming the domain of ZFN, ZFN can be designed artificially to recognize specific DNA and combine it with the target DNA sequence. Then, endonuclease FOKI can cut DNA double-stranded to form DSB, and finally complete DNA self-repair. This technology has the characteristics of simple design and high efficiency in the development process, but with the development of science, people find that it has a long cycle and is easy to miss the target. And high cytotoxicity.
TALEN, the second generation gene editing technology, as an alternative product of ZFNs, entered a rapid development period on 202 1. In 20 12, science magazine listed talun technology as one of the top ten scientific breakthroughs of the year. TALEN is the full name of transcription activator-like effector, which comes from plant pathogenic bacteria. The main principle of Talen technology is to identify the sequences on both sides of the target through two Tales. Each TALE fuses a FokI endonuclease domain; FokI forms dimer cleavage target through TALE, induces double strand breaks, promotes DNA self-repair process, and finally achieves the purpose of gene editing. TALEN has the advantages of flexible technical design and strong identification specificity.
ZFNs uses 30 amino acids to form a DNA recognition domain corresponding to three bases, while TALE protein uses 34 amino acids to form a DNA recognition domain corresponding to just one base. In addition, compared with ZFNs technology, TALE has a decisive advantage that it can be modularized. By deleting, adding and freely combining different TALE proteins, DNA fragments can be easily located and the gene editing cycle can be shortened. However, the DNA sequence that the virus can transport is limited by liposome transfection or electroporation. When the virus infects and delivers foreign aid DNA for gene therapy, the transfection efficiency is inevitably inversely proportional to the size of protein, so too large TALE will undoubtedly lead to the reduction of DNA cutting efficiency. In addition, this technology also has the same shortcomings as ZFNs, such as high missed detection rate and high cytotoxicity.
However, scientists quickly developed a new generation of gene editing technology, which is more efficient and faster than the previous two generations. Accurate and low price, which is also known as CRISPR/Cas9 technology, is mainly composed of clustered short palindrome repeat sequence CRISPR and accounting endonuclease Cas9. 20 1 1 reveals the molecular mechanism of crispr/cass9 system. On 20 14, Jennifer, an American biochemist, first expounded the working principle of crispr/cass9 system, and proved that it can find the corresponding DNA sequence under the guidance of a guiding RNA(gRNA) and cut it. The working principle of CRISPR/Cas9 system is that crRNA combines with TRAC RNA through base pairing to form a TRAC RNA/crRNA complex, which guides the nuclease Cas9 protein to cut double-stranded DNA at the sequence target site paired with crRNA. However, two kinds of RNA, crRNA and tracrRNA, were artificially designed and transformed into sgRNA with guiding function, thus guiding Cas9 to cut DNA at fixed sites. Soon after, Zhang Feng, a biologist in China, Massachusetts Institute of Technology, proved that this system can also be used in mammalian cells. CRISPR/Cas9 system is a unique natural defense system for bacteria and archaea, which is used to resist the invasion of viruses or foreign particles. When foreign genes invade, the defense system's CRISPR sequence will express RNA different from the invading genome sequence, and then CRISPR-related enzymes will cut the foreign genome DNA at the sequence recognition, thus achieving the defense purpose. ?
Principle of CRISPR/Cas9 technology
1.sgRNA combines with Cas9 protein to form RNP complex.
2.RNP complex is located at the target site of genome under the guidance of sgRNA.
3.Cas9 protein cleaves DNA double strand at the target site, resulting in double strand breaks (DSB).
4.4. Mechanism of cell emergency repair. DSB: Non-homologous terminal ligation (NHEJ) repair or homologous recombination repair (HDR).
5. In most cases (> 80%), cells use the NHEJ repair pathway to randomly delete or insert a single base at the target site to obtain gene knockout Indel.
6. In rare cases (
7. By introducing foreign gene fragments or mutant bases into homologous fragments, a gene site-directed insertion model or a gene site-directed mutation model can be obtained.
In recent years, CRISPR/Cas gene editing technology has developed rapidly, involving applications in biology, medicine, agriculture and environment. 20 17 The CRISPR/Cas9 gene editing technology was applied to CAR-T therapy. Yang Luhan published an article in the journal Science, and knocked out the endogenous retrovirus (PERV) sequence in the pig genome by CRISPR/Cas9 technology. In the same year, Academician Yuan Longping, the father of hybrid rice, announced the use of CRISPR/Cas9 technology to knock out the genes related to cadmium absorption and accumulation in rice.
Up to now, CRISPR/Cas9 technology has made a new breakthrough. Compared with the previous two generations of gene editing technology, CRISPR/Cas9 technology has high cutting efficiency and convenience. ZFNs and TALENs need hundreds of bases to complete the assembly of the localization system, while CRISPR only needs a gRNA corresponding to the target gene one by one, and Cas9 protein itself has the activity of endonuclease, and no additional endonuclease is needed. It provides great convenience for large-scale treatment of point mutation genetic diseases in the future. In addition, this technology has the advantage of simple design and can target almost any cell sequence.
Through continuous exploration, starfish has developed a virus-free technology to transfect plasmids into cells by constructing transposon system plasmids. Under the action of transposase, the high-copy Cas9 protein and sgRNA expression elements were integrated into the genome, which saved 3-4 weeks compared with the traditional virus method and saved about 40% in price. With the development of gene editing technology, Starfish will follow the pace of scientific and technological development and escort your scientific research. ?
refer to
Knodt GJ. CRISPR-Cas leads the future of genetic engineering. Science. 20 18 Aug 3 1； 36 1(6405):866-869.doi: 10. 1 126/science . aat 50 1 1。
Becquero, Gomez-ospina N, Porteous MH. The gene editor is in the center of the stage. Trend gene. 2065 438+08 Aug； 34(8):600-6 1 1.doi: 10. 10 16/j . TIG . 20 18.05 . 004。
The history of mammalian genome editing. Mammalian genome. 20 17 August; 28(7-8):237-246.doi: 10. 1007/s 00335-0 17-9699-2。