基因编辑器 z-bio 2021-04-28 275

　　Michael Wiles sat with the top managers of the Jackson Laboratory in Maine, USA, and told them a new method of DNA cutting with amazing power. This laboratory, referred to as JAX, used genetic engineering technology to transform mice with the registered trademark JAXR Mice and sold them to researchers. It likes to boast: this is "the best quality and most published mouse model in the world". Wiles evaluates and develops new technologies for the laboratory. He believes that this new tool, which is cleverly adapted from the immune strategy used by bacteria and archaea to protect themselves from viruses, will revolutionize the way JAX transforms mice. "Of the dozen people present, 9 were asleep," Wiles said. "No one had heard of CRISPR at the time."

　　Nowadays, most mouse breeders know about CRISPR. For a long time, JAX and other laboratories that breed new mouse strains have relied on a laborious, multi-step process that involves using genetic engineering techniques to modify mouse embryonic stem cells, inject them into embryos, and breed several generations of mice. . Even the best team in JAX will take 2 years to successfully transform a mouse. CRISPR replaces all of this with a molecular complex that can perform targeted genetic surgery on fertilized eggs. It can produce a modified mouse within 6 months.

　　The mice whose genes have been knocked out or genetic information added through genetic modification have become a series of humans ranging from cancer and atherosclerosis to Alzheimer's disease, osteoarthritis, muscular dystrophy and Parkinson's disease. The key research model of the disease. Knockout and knock-in mice also provide a powerful tool for studying the function of specific genes.

　　"When you used to breed knockout mice, you need to have some skills." Rudolf Jaenisch from the Massachusetts Institute of Technology said, "Now, you no longer need these skills. Any fool can do it."

　　make a large impact

　　CRISPR stands for "clustered, regularly spaced short palindrome repeats". It is derived from the genetic material of prokaryotes and is a description of the latter. CRISPR uses guide RNA to deliver biological "scissors" (usually a CRISPR-related protein-Cas9) to a precise location in the genome. Once Cas9 is sheared by enzymatic hydrolysis, the cell will try to heal the injured DNA. One repair mechanism will result in gene knockout, and the other will result in gene knock-in. "All CRISPR does is to cut DNA." Wiles said, "Some other things are done by the cell repair system. This is where we can free ride."

　　The standard response of

　　 cells is to try to re-splice the double-stranded DNA at the breakpoint. This usually requires phagocytosis or addition of some bases, leading to gene insertion or deletion. In fact, this repair effort will introduce some small "typographical errors" into the DNA text, thereby rendering the gene inoperative.

　　Jaenisch demonstrated for the first time the power of CRISPR in generating knockout mice. Five months after researchers first demonstrated that CRISPR works in mammalian cells, Jaenisch and colleagues published a paper in the journal Cell in May 2013. They reported that the technology successfully disconnected five genes in a group of mouse embryonic stem cells, which was impossible before. More importantly, they demonstrated that embryonic stem cells can be completely bypassed, and two genes in single-cell mouse fertilized eggs can be knocked out at the same time. Researchers no longer need to modify embryonic stem cells and painstakingly cultivate several generations of mice to produce mice with genetic mutations in egg or sperm cells. At the same time, if researchers want to breed mice with two mutations, they no longer need to cross a single mutant and go through a similar time-consuming and cumbersome process to obtain offspring of mice with altered germ cell lines.

　　Since then, more than 500 papers have described in detail how CRISPR knocks out and knocks in mouse genes. "It has a huge impact." said Jaenisch, who bred the first genetically modified mouse in 1974. Tak Mak, a biochemist at the University of Toronto in Canada, believes that this technology has truly changed the time and efficiency of obtaining these modified animals. According to Mak's estimation, compared with the use of embryonic stem cells, the cost of using CRISPR to transform mice is about 30% cheaper, which greatly reduces his average cost.

　　not only saves costs

　　The impact of CRISPR cannot be measured solely by cost savings. The convenience and speed of this technology makes it possible to modify mice in a hurry, thus solving a specific problem recently faced by CC Hui from the Toronto Sick Children's Hospital: During the gene knockout process, the missing gene did not produce any observable The effect. Hui realized that the knocked-out gene was related to another gene that might compensate for it. He appealed to Lauryl Nutter, who oversees mouse breeding at the Toronto Phenogenomics Center. Nutter uses CRISPR to mutate interfering genes in fertilized eggs from the initial gene knockout process. "We obtained fertilized eggs and injected CRISPR-Cas9 into it. After 8 weeks, Hui got double mutants." Nutter said, "If embryonic stem cells are used, it may take several years to complete."

　　This revolution is not limited to breeding mice with germline mutations. CRISPR also allows researchers to simultaneously mutate several suspected cancer genes in adult mouse somatic cells. At the same time, CRISPR knock-in can correct disease-causing genetic defects in adult mice, such as mutations that cause hemophilia and sickle cell anemia. Several research teams plan to inject CRISPR into developing mice. The goal is to create mutations that can function as barcodes and allow scientists to track the cell lineage of cells as they differentiate.

　　Mouse breeding companies such as the Toronto Phenotype Genomics Center and JAX hope that CRISPR will greatly expand the range of mutations they produce. "Now, I can get an unusual mouse with 3 genetic modifications, and I can modify it again." Wiles said, "We can't use embryonic stem cells for continuous genetic modification. We can combine one with two genes. The modified mouse was crossed with another mouse with two genetic modifications, but it would take several years to obtain all four genetic modifications."

　　just started getting started

　　However, in some ways, the CRISPR revolution faltered. Three months after Jaenisch’s laboratory first reported on CRISPR, he and his colleagues proposed in a second paper published in the journal Cell that CRISPR can easily perform more complex genetic operations, that is, knocking in DNA fragments rather than simply Make genes lose their function. As a demonstration, they used CRISPR to knock fluorescent markers into mouse fertilized eggs. When a specific gene is turned on, the fluorescent marker will light up. Researchers have also created conditional mutants that are critical to many studies.

　　About 1/3 of mouse genes are essential for embryonic development. If these genes lose function from the beginning, mice will not be born. Therefore, scientists who use embryonic stem cells for research have cleverly designed a system called Cre-Lox recombination. The system only knocks out the gene after the mouse has developed enough to survive the loss of the above-mentioned genes. This requires the addition of additional DNA: Lox sequences flanking the target gene and a Cre gene. Among them, the Cre gene can be activated to produce an enzyme that is used to modify the DNA between Lox sites. Jaenisch's team used CRISPR to insert the same system into fertilized eggs, and reported that conditioned mice were bred with relatively high efficiency-about 16% of fertilized eggs produced mouse pups with the correct mutation.

　　Although William Skarnes, who led a team at the Sanger Institute of the Wellcome Foundation in the UK to cultivate mutant mouse embryonic stem cells, was one of the many researchers who were impressed by Jaenisch's initial report, but when he tried to bring the technology into his own experiments Shishi was disappointed. "From his paper, this technology is very simple. I am very confident that it will eliminate embryonic stem cells." Skarnes said, "but disappointingly, none of us can reproduce the efficiency reported by Jaenisch. In In the JAX laboratory, only 1% or 2% of fertilized eggs produce mouse pups with the correct mutation. At the same time, many projects are failing. Obviously, it has not proven to be a robust method."

　　Regardless of the current shortcomings of CRISPR, Wiles emphasized that its potential in transforming mice should not be ignored. "There are so many things CRISPR can do, and we are just getting started."