CRISPR-Cas9 is a powerful new gene editing technique with the promise of widespread applications in research, medicine, and industry — as well as to provoke political and moral controversy.
Open thread: CRISPR-Cas9
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CRISPR-Cas9 editing has been developed from a bacterial defence system that shreds the DNA of invading viruses. CRISPR stands for “clustered regularly interspaced short palindromic repeats”. These are short strings of RNA, a molecule similar to DNA, each designed to fix onto a particular segment of a virus’s DNA. Cas9 is an enzyme which, guided by CRISPRs, cuts the DNA at the specified point.
Modifying this arrangement for the purposes of genetic engineering is simple, at least in theory. Since DNA and RNA work in essentially the same ways in all living organisms, designing appropriately customised CRISPR guide molecules can induce Cas9 to cut any cell’s DNA wherever the designers choose, eliminating undesirable sequences of genetic “letters”. Since cells will then try to repair this sort of damage, genetic engineers can, by providing corrected versions of the DNA that has been deleted for use as templates which a cell can copy, encourage the repair mechanism to fix the problem in the way they had intended.
The hope was that, by being given such templates, embryos could be purged of nascent genetic disease. That hope appeared fulfilled, at least in part. By the end of the experiment, 72% of the embryos were free of mutant versions of MYBPC3, an improvement on the 50% that would have escaped HCM had no editing taken place.
In achieving this, Dr Ma and her colleagues overcame two problems often encountered by practitioners of CRISPR-Cas9 editing. One is that the guidance system may go awry, with the CRISPR molecules leading the enzyme to parts of the genome that are similar, but not quite identical, to the intended target. Happily, they found no evidence of such off-target editing.
A second problem is that, even if the edits happen in the right places, they might not reach every cell. Many previous experiments, including some on embryos, have led to mosaicism, a condition in which the result of the editing process is an individual composed of a mixture of modified and unmodified cells. If the aim of an edit is to fix a genetic disease, such mosaicism risks nullifying the effect.
The technology along these lines that has got furthest is called CAR-T, where CAR stands for “Chimeric antigen receptor”. These CARs are produced by splicing together the gene for an antibody that recognises a tumour antigen and the gene for a receptor that sits on the surface of the T-cells; put this new gene into a T-cell and it will be precisely targeted at the tumour. The small clinical trials undertaken to date suggest that this could be extremely effective. A trial of 31 patients with acute lymphoblastic leukaemia brought a complete, and unprecedented, remission in 93% of cases. A CAR-T therapy called Kymriah (tisagenlecleucel), made by the Swiss firm Novartis to treat B-cell acute lymphoblastic leukaemia, was approved for use in America on August 30th.
There are two main limitations to CAR-T. One is that so far the T-cells have been programmed to target a molecule, CD19, which is only common to the surface of a few blood cancers. The other is that CAR-T has been known to trigger immune reactions which can prove fatal. Neither problem is obviously insoluble. Editing genes has been made much easier by a new technology known as CRISPR-Cas9, which has already been used to improve the way that CAR-T cells are engineered in mice. It may well eventually allow the receptors used in such therapies to be personalised to the specifics of the patient’s cancer. And more precision, as well as experience, should lead to immune responses less likely to run away with themselves.
What such advances will not do, though, is make such treatments cheaper. Novartis’s new therapy costs $475,000. Genome-editing treatments seem likely to be the most expensive cancer treatments the world has yet seen. And that is saying quite a lot, since many of the newer cancer treatments are eye-wateringly pricey (see chart).
Gene editing takes another step forward
It can now edit individual genetic letters
The first human test in the U.S. involving the gene-editing tool CRISPR could begin at any time and will employ the DNA cutting technique in a bid to battle deadly cancers. Doctors at the University of Pennsylvania say they will use CRISPR to modify human immune cells so that they become expert cancer killers, according to plans posted this week to a directory of ongoing clinical trials. The study will enroll up to 18 patients fighting three different types of cancer — multiple myeloma, sarcoma, and melanoma — in what could become the first medical use of CRISPR outside China, where similar studies have been under way. An advisory group to the National Institutes of Health initially gave a green light to the Penn researchers in June 2016, but until now it was not known whether the trial would proceed.
CRISPR first became a business with yogurt.
The dairy industry uses the bacterium Streptococcus thermophilus to convert lactose into lactic acid, which gels milk. Viruses called bacteriophages can attack S. thermophilus, spoiling the yogurt culture. In 2007, Rodolphe Barrangou and Philippe Horvath were working at Danisco, one of the world’s leading makers of yogurt cultures, when they found that the S. thermophilus genome contains odd chunks of repeated DNA sequences—so-called clustered regularly interspaced short palindromic repeats (CRISPR), which Spain’s Francisco Mojica had first described in 1993 in the genome of the salt-loving microbe Haloferax mediterranei. The Danisco team found that the CRISPR sequences match the phage DNA, enabling S. thermophilus to recognize and fight off infections.
DuPont, which acquired Danisco in 2011, began using the insights to create bacteriophage-resistant S. thermophilus for yogurt and cheese production. Today, “whether you’ve had yogurt in Tel Aviv or nachos in California, you’ve eaten a CRISPR-enhanced dairy product,” says Barrangou, who is now a food scientist at North Carolina State University in Raleigh.
Further ahead, there is hope that CRISPR-Cas9 will help treat diseases such as AIDS, cystic fibrosis, Huntington’s chorea and Duchenne muscular dystrophy.
However, a study just published in Nature Biotechnology has found that when CRISPR-Cas9 is used to edit genomes, off-target DNA damage is more common than had previously been appreciated. This piece of research, co-ordinated by Allan Bradley of the Wellcome Sanger Institute, in Cambridge, England, looked at genetic changes in mouse and human cells across large stretches of the genome. In around 20% of cells examined, the use of CRISPR-Cas9 had caused unintended deletions or rearrangements of strings of DNA more than 100 base pairs long—and some as long as several thousand DNA base pairs. This raises the possibility that non-target genes or regulatory sequences could be affected by the editing process, a discovery which comes in the wake of other recent work which raised concerns that CRISPR-Cas9 gene-editing might trigger cancers. Cue investor panic and nosedives in the share prices of gene-editing companies.
Although Dr Bradley’s study certainly adds to concerns over the accuracy and safety of CRISPR-Cas9, it is by no means a show stopper. There are a number of caveats which may, in time, turn out to mean the findings are less concerning than they seem today.
For one thing, as Robin Lovell-Badge, an expert in the area who works at the Francis Crick Institute in London, observes, the study focuses on a form of genome-editing called “non-homology end joining”. This, he says, is known to be an untrustworthy approach compared with other ways of using CRISPR-Cas9. Moreover, the actual impact of the technique (and, indeed, of any gene-editing tool) will depend on the types of cells being edited and the nature of the changes being made.
https://www.3dmoleculardesigns.com/Custom-3D-Print-Molecular-Models/CRISPR-Cas9.htm
In 1960 George Craig, an American entomologist, suggested that such subversive genes might be a way of controlling the populations of disease-carrying mosquitoes, for example by making them more likely to have male offspring than female ones. In 2003 Austin Burt, at Imperial College, described how a gene drive that could cut a place for itself in a chromosome and copy itself into the resulting gap could, in the right circumstances, drive a species to extinction.
A fascinating idea, but one hard to put into practice—until, in 2012, a powerful new gene-editing tool called crispr-Cas9 became available. Gene drives based on crispr-Cas9 could easily be engineered to target specific bits of the chromosome and insert themselves seamlessly into the gap, thus ensuring that every gamete gets a copy (see diagram). By 2016, gene drives had been created in yeast, fruitflies and two species of mosquito. In work published in the journal Nature Biotechnology in September, Andrea Crisanti, Mr Burt and colleagues at Imperial showed that one of their gene drives could drive a small, caged population of the mosquito Anopheles gambiae to extinction—the first time a gene drive had shown itself capable of doing this. The next step is to try this in a larger caged population.
CRISPR-Cas genome-editing systems, often just known as CRISPR, are molecular machines that can be programmed to home in on specific sections of dna in the genome and cut both strands of the double helix molecule. This system allows genes to be knocked out or, in some cases, added.
It is not a perfect mechanism. One concern, for example, is that editing can alter DNA in places it isn’t supposed to and that these “off-target” effects could trigger cancers. A second worry is that the cell can fill gaps with random dna when it is making repairs. These could silence genes that the organism may need. A third concern is that although CRISPR successfully hunts down and cuts out faulty dna, it is harder to get it to insert the right new genes.
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Both teams made use of “jumping genes” or transposons (often called selfish genes), which are pieces of dna that seem to hop around genomes with little more purpose than to proliferate. They were thought to do so aimlessly but, in 2017, it was discovered that some contained gene-editing systems that were very good at recognising specific dna sequences. These were able to control where the jumping genes landed. That, in turn, led to the idea, says Dr Sternberg, that it might be possible to harness jumping genes in gene editing.
CRISPR Now Cuts and Splices Whole Chromosomes
CRISPR Gene Editing In Human Embryos Wreaks Chromosomal Mayhem
The first preprint was posted online on June 5 by developmental biologist Kathy Niakan of the Francis Crick Institute in London and her colleagues. In that study, the researchers used CRISPR-Cas9 to create mutations in the POU5F1 gene, which is important for embryonic development. Of 18 genome-edited embryos, about 22% contained unwanted changes affecting large swathes of the DNA surrounding POU5F1. They included DNA rearrangements and large deletions of several thousand DNA letters — much greater than typically intended by researchers using this approach. Another group, led by stem-cell biologist Dieter Egli of Columbia University in New York City, studied embryos created with sperm carrying a blindness-causing mutation in a gene called EYS2. The team used CRISPR-Cas9 to try to correct that mutation, but about half of the embryos tested lost large segments of the chromosome — and sometimes the entire chromosome — on which EYS is situated. And a third group, led by reproductive biologist Shoukhrat Mitalipov of Oregon Health & Science University in Portland, studied embryos made using sperm with a mutation that causes a heart condition. This team also found signs that editing affected large regions of the chromosome containing the mutated gene.
The three studies offered different explanations for how the DNA changes arose. Egli and Niakan’s teams attributed the bulk of the changes observed in their embryos to large deletions and rearrangements. Mitalipov’s group instead said that up to 40% of the changes it found were caused by a phenomenon called gene conversion, in which DNA-repair processes copy a sequence from one chromosome in a pair to heal the other. Mitalipov and his colleagues reported similar findings in 2017, but some researchers were skeptical that frequent gene conversions could occur in embryos. They noted that the maternal and paternal chromosomes are not next to each other at the time the gene conversion is postulated to occur, and that the assays the team used to identify gene conversions could have been picking up other chromosomal changes, including deletions. Egli and his colleagues directly tested for gene conversions in their latest preprint and failed to find them, and Burgio points out that the assays used in the Mitalipov preprint are similar to those the team used in 2017. One possibility is that DNA breaks are healed differently at various positions along the chromosome, says Jin-Soo Kim, a geneticist at Seoul National University and a co-author of the Mitalipov preprint.
The Perlman mice are, however, not quite perfect for the job. Though their genome has had human ace2 added, the mouse variety has not been subtracted. This means they make both versions of the protein. Also, the human version of the gene is in the wrong place in the cell nucleus. The ace2 gene’s proper home is the x chromosome, one of the sex chromosomes. In Perlman mice it is elsewhere. That could change its activity.
Help may be on its way, though, from Wang Youchun at the National Institutes for Food and Drug Control in Beijing. As they describe in this week’s Cell Host & Microbe, Dr Wang and his colleagues have excised the murine version of the ace2 gene completely from their own mice and substituted the human version in exactly the same place. They did this using crispr/Cas9 gene-editing technology, a technique unavailable to Dr Perlman back in 2007.
First successful CRISPR gene-editing directly in the blood of a live patient
Patrick Doherty was a healthy, active 65-year-old living County Donegal, Ireland when he was diagnosed with transthyretin amyloidosis, the same rare genetic disease that killed his father. When he visited a doctor, he learned of a new experimental treatment for the condition—with an emphasis on the “experimental” side.