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CRISPR-Cas9 gene editing technique - a boon to fixing defective genes and controlling diseases

Scientists recently were gifted a new technique in gene editing called CRISPR-Cas9 (CRISPR is an acronym for 'clustered, regularly interspaced, short palindromic repeats) and it is adopted by several laboratories worldwide because it's faster, cheaper, simple enough to use with minimal training, and allows altering of multiple genes simultaneously. This new genetic engineering technique is going to revolutionize medicine, according to scientists working in the field, because it gives humankind a powerful tool to edit, delete, add, replace, activate or suppress specific genes. Humans can, theoretically, change the genetic basis of various traits and  correct disease causing mutated genes.
Scientists have long been able to find defective genes. But fixing them has been so cumbersome that it's slowed development of genetic therapies. With gene editing, scientists home in on a piece of DNA and use molecular tools that act as scissors to snip that spot -- deleting a defective gene, repairing it or replacing it with precision.

It was initially found that these CRISPR sequences were used by bacteria to ward off predatory viruses. The mechanism was unravelled but again nothing more was thought of it then. In 2011, several scientists in the US and Europe revisited it. They found that the CRISPR mechanism could be turned around and manipulated for performing cut-and-paste functions on genomes. And the adopted techniques were fantastic. They could precisely snip off a bit of DNA from a gene and replace it with another pre-fabricated bit of DNA.

This is how precisely this is done: First, a piece of RNA is created for unzipping a DNA strand at the target site; then it is lodged in a protein called Cas9 which is the scissors part of the machinery; this complex unzips and cuts away the specific DNA bit. You can replace it with a totally new DNA bit or a corrected version, as needed.

A key ingredient in the CRISPR–Cas9 system is the DNA-cutting enzyme Cas9. But in September, synthetic biologist Feng Zhang at the Broad Institute of MIT and Harvard in Cambridge, Massachusetts, reported the discovery of a protein called Cpf1, which may make it even easier to edit genomes. (Zhang is one of those who pioneered the use of CRISPR-Cas9 for genome editing in mammalian cells).

Zhang was granted a US patent on CRISPR–Cas9 in April 2014. But several months before he filed his application in 2012, molecular biologists Jennifer Doudna at the University of California (UC), Berkeley, and Emmanuelle Charpentier, now at the Max Planck Institute for Infection Biology in Berlin, had filed their own patents. All three scientists co-founded companies that make use of CRISPR–Cas9 (1).

Researchers hope to use CRISPR for diseases like sickle cell, correcting the faulty gene in someone's own blood-producing cells rather than implanting donated ones. Doctors in Britain recently treated a 1-year-old with leukemia using donated immune cells that had been experimentally altered with an older editing method to target her cancer. A California company is testing a non-CRISPR way to make HIV patients' immune cells better resist the virus. The University of Massachusetts just reported using a CRISPR technique to switch off, rather than cut and repair, a gene in muscle cells that causes one form of muscular dystrophy. And Harvard researchers recently edited 62 spots in pig DNA, part of work to use the animals to grow organs for human transplant.
Altering genes in sperm, eggs or embryos can spread those changes to future generations, so-called germline engineering that might one day stop parents from passing inherited diseases to their children. Chinese scientists reported the first-known attempt to edit human embryos last spring, working with leftovers from fertility clinics that never could have developed into fetuses. They aimed to correct a deadly inherited gene, but uncovered problems that will require more research.
Researchers have already been tweaking the components of CRISPR-Cas9 to drive down its error rate. They have tweaked the RNAs that guide the Cas9 enzyme to a specific site in the genome, for example, and engineered the system so that researchers can easily switch it off, so that the enzyme does not have as much opportunity to make unwanted changes.
Synthetic biologist Feng Zhang at the Broad Institute of MIT and Harvard in Cambridge decided to focus on engineering the Cas9 enzyme itself. He and his team altered the enzyme so that it is less likely to act at sites with mismatches between the RNA that guides the enzyme and the DNA that it targets. They generated several versions of Cas9 that reduced off-target errors at least tenfold compared with unaltered Cas9 enzymes.
To rid families of the curse of inherited diseases, medical geneticists have dreamed about changing human DNA before birth. Their dreams are coming true with this technology!
In Britain, researchers have requested permission to gene-edit human embryos in the lab, studying early development in ways that might shed light on miscarriage.
Plant scientists have been quick to experiment with the popular CRISPR/Cas9 technique, which uses an enzyme called Cas9, guided by two RNA strands, to precisely cut segments of DNA in a genome. By disabling specific genes in wheat and rice, researchers hope to make disease-resistant strains of the crops.
Human gene editing aside, experiments are under way to force genetic changes to spread rapidly through populations of animals and plants -- changes that could wipe out invasive species or disease-carrying insects. A California team recently reported (2) a first step, hatching malaria-resistant mosquitoes that could easily spread their new protective gene to their offspring.

But as always there would be dooms-day predictors. And they are already sounding alarm bells. The ethical debate that has erupted around CRISPR is because it can also be used to edit germline cells or pluripotent stem cells. Germline cells are eggs and sperm cells. Any changes in these will naturally be inherited by subsequent generations. Pluripotent stem cells can develop into any kind of tissue, and changes in these would affect large number of cells.

The International Society for Stem Cell Research and other groups that are very optimistic about the technique argue that Scientists should be permitted to conduct basic research on human germ line modification.

Like I always keep saying, you can use any technology in any way you want. The choice is definitely yours. Just because some people can misuse some technology, completely stopping work on it is not a sensible thing when there are many benefits and we can use the technology for our advantage.

CRISPR is the most powerful genome-editing tool that scientists have. We need to explore its potential to avert the horrors of genetic diseases but do so without jeopardizing our values or harming generations of human lives.

There are people who are suffering and need new technologies that can give them hope of living a normal life like healthy people around them. How can we deny them their hopes, dreams, aspirations and in the end life itself?





Tweaking the human genome with current and future gene-editing tools could lead to sophisticated treatments and prevention strategies for disease. The promise of those applications is reason enough to move forward with such work in the lab and clinic, albeit cautiously, the dozen scientists and bioethicists who organized the International Summit on Human Gene Editing said on Dec., 3rd, 2015, after three days of deliberation and presentations in Washington, D.C. The international summit on gene editing, sponsored by Britain’s Royal Society, the Chinese Academy of Sciences, and the US National Academies, has grappled with thorny questions related to gene editing during three days of sometimes-heated discussion on editing the human genome. What Scientists discussed this week is when and how to apply gene editing for research and clinical applications in humans. Gene editing could include altering genes in one person—say to treat leukemia in one patient or make a cosmetic change—but, more controversially, it could also include making changes to the germ line that would then alter the genome for an individual’s children, grandchildren and the following generations, with potentially unknown repercussions.

Altering the human germ line, such as eggs, sperm and embryos—considered more controversial than altering somatic cells to treat diseases like cancer—should for now proceed only in the laboratory, they said. "If, in the process of research, early human embryos or germ line cells undergo gene editing, the modified cells should not be used to establish a pregnancy," they said in the offiical statement. Overall, scientists should steer clear of applying it in the clinic—that is, using it to treat actual patients (something they could not have done anyway until it was proved safe and effective in animal trials), until scientists learn more about it and there is "broad societal consensus about the appropriateness of the proposed application." The issue, however, should be revisited on a regular basis, the group said.


CRISPR gene-editing tested in a person for the first time

A Chinese group has become the first to inject a person with cells that contain genes edited using the revolutionary CRISPR–Cas9 technique.

On October 28th 2016, a team led by oncologist Lu You at Sichuan University in Chengdu delivered the modified cells into a patient with aggressive lung cancer as part of a clinical trial at the West China Hospital, also in Chengdu.

The researchers removed immune cells from the recipient’s blood and then disabled a gene in them using CRISPR–Cas9, which combines a DNA-cutting enzyme with a molecular guide that can be programmed to tell the enzyme precisely where to cut. The disabled gene codes for the protein PD-1, which normally puts the brakes on a cell’s immune response: cancers take advantage of that function to proliferate.

Lu’s team then cultured the edited cells, increasing their number, and injected them back into the patient, who has metastatic non-small-cell lung cancer. The hope is that, without PD-1, the edited cells will attack and defeat the cancer.

The treatment went smoothly, and that the participant will get a second injection, but the researchers declined to give details because of patient confidentiality. The team plans to treat a total of ten people, who will each receive either two, three or four injections. It is primarily a safety trial, and participants will be monitored for six months to determine whether the injections are causing serious adverse effects. Lu’s team will also watch them beyond that time to see if they seem to be benefiting from the treatment.

Other oncologists are excited about CRISPR’s entry onto the cancer scene.


In 2016 alone, dozens of scientific papers from researchers around the world have detailed the results of studies — some promising, some critical — that used CRISPR to snip out and replace unwanted DNA to develop treatments for cancer, HIV, blindness, chronic pain, muscular dystrophy and Huntington's disease, to name a few. 

Although it will be a few more years before any CRISPR-based treatments could be tested in people, "hardly a day goes by without numerous new publications outlining new findings about human health and human genetics that took advantage" of this new tool (3)


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A new genetic tool may help eradicate Africa’s main malaria-carrying mosquitoes. A self-propagating cut-and-paste system known as a gene drive can sterilize female Anopheles gambiae mosquitoes, researchers report December 7 in Nature Biotechnology. This is the second gene drive aimed at eliminating malaria. The first, published November 23 in the Proceedings of the National Academy of Sciences (SN Online: 11/23/15), would stop mosquitoes from transmitting the parasite. The new gene drive would eliminate the mosquitoes themselves by making it impossible for females to reproduce. Gene drives are engineered pieces of DNA designed to slice a target gene and insert themselves. Like Star Trek’s Borg, gene drives assimilate every unaltered target gene they encounter. These ambitious bits of DNA break standard inheritance rules to get passed on to more than 50 percent of an altered animal’s offspring, “driving” themselves quickly through populations (SN: 12/12/15, p. 16). Evolutionary geneticist Austin Burt of Imperial College London first conceived the idea of gene drives in 2003. For more than a decade, the drives remained mostly theoretical, but thanks to precision molecular scissors called CRISPR/Cas9, four gene drives, including the two in mosquitoes, were described this year. “They all work terrifically,” says George Church, a geneticist at Harvard University. Cas9 is a DNA-cutting enzyme borrowed from bacteria. Researchers can design RNA molecules to guide the enzyme to particular genes. Church is pleased to see that this latest mosquito gene drive works, but says further modifications may be needed before it is ready for release in the field. Researchers may also want to combine approaches, first releasing a gene drive that would prevent mosquitoes from carrying malaria, then later releasing one to control mosquito populations, Church suggests. In the new study, Burt and colleagues first used CRISPR/Cas9 and another type of gene editor known as TALENs to disrupt each of three genes that are active at high levels in mosquito ovaries. Females carrying two copies of any one of the three disrupted genes were sterile. Once the researchers had confirmed that messing with the genes would make the mosquitoes sterile, the team built gene drives to insert into the genes. Under normal circumstances, only 50 percent of progeny would inherit any given gene. With the gene drives, 76.1 percent to 99.6 percent of the offspring inherited the drive, the researchers found. This gene drive had some technical glitches, so it won’t be the final version that researchers would release to control wild mosquito populations. But the researchers are hopeful that future gene drives could curb populations of A. gambiae, says study coauthor Tony Nolan, a molecular biologist at Imperial College London. “We need new approaches for vector control, and this is a promising one.”

UK scientists just got approval to edit human embryos
Scientists in Britain just got approval to conduct research that involves editing the genetic material of healthy human embryos.

This is a big deal: The UK's Human Fertilisation and Embryology Authority is the first government agency in the world to endorse research that involves altering the human genome for research — a move that could signal broader acceptance for a promising (but controversial) new area of science.

The research team, led by Dr. Kathy Niakan at the UK's Francis Crick Institute, is trying to better understand which genes allow a healthy human embryo to develop. Niakan’s team will use a promising new technique, known as CRISPR/Cas9, to edit genes that are active following conception. They'll then stop the experiments at day seven and destroy the embryos (so that they can't be used to start a pregnancy).

The hope is that this gene hacking could help researchers better understand what causes miscarriages and infertility — and perhaps one day lead to better treatments for infertility.




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