CRISPR GENOME EDITING
Pubblicato un nuovo metodo di Gene editing CRISPR che consente di modificare le singole lettere di DNA
28 Aprile 2016
I ricercatori della Harvard University hanno sviluppato un nuovo metodo di utilizzo di CRISPR per modificare singole lettere nel codice del DNA. Questo apre la possibilità di invertire mutazioni causate dalla modifica di una sola lettera, che rappresenta circa due terzi di tutte le mutazioni genetiche.
CRISPR ha rivoluzionato il mondo del gene editing, permettendo agli scienziati di condurre una ricerca avanzata sulle malattie e non è costoso o troppo complicato. Ma una cosa è sempre mancata, la specificità.
In un articolo sulla rivista Nature, i ricercatori hanno descritto il nuovo metodo, che non ha bisogno di tagliare entrambi i filamenti di DNA a doppia elica per alterare il codice genetico. E' possibile convertire direttamente una singola lettera di DNA in un'altra, senza eliminare e l'inserire una serie di lettere casuali.
In particolare, i ricercatori hanno deciso di incollare due proteine ad un tipo di enzima Cas9 che non provoca rotture del DNA. Le due proteine hanno consentito a Cas9 di convertire direttamente una lettera specifica nel codice genetico a quattro lettere.
I test attuali sono stati condotti su cellule murine ed umane e su una mutazione che causa la malattia ad insorgenza tardiva di Alzheimer ed il cancro al seno. In un tentativo, sono stati in grado di cambiare in modo efficace il 58% delle cellule senza effetti collaterali indesiderati, mentre in altri test sono arrivati fino al 75%.
Questo livello di specificità consente un gene editing di alta precisione. Anche se ancora in fase iniziale e ancora evidenzia una serie di limitazioni, il nuovo metodo potrebbe un giorno consentire il trattamento di malattie senza alterare in modo permanente il patrimonio genetico umano.
Fonte: The Verge
Genome editing: 7 facts about a revolutionary technology
What everyone should know about cut-and-paste genetics.
The ethics of human-genome editing is in the spotlight again as a large international meeting on the topic is poised to kick off in Washington DC. Ahead of the summit, which is being jointly organized by the US National Academy of Sciences, the US National Academy of Medicine, the Chinese Academy of Sciences and Britain’s Royal Society and held on 1–3 December, we bring you seven key genome-editing facts.
Human embryos are at the centre of a debate over the ethics of genome editing.
1. Just one published study describes genome editing of human germ cells.
In April, a group led by Junjiu Huang at Sun Yat-sen University in Guangzhou, China, described their use of the popular CRISPR–Cas9 technology to edit the genomes of human embryos. Only weeks before the researchers’ paper appeared in Protein & Cell1, rumours about the work had prompted fresh debate over the ethics of tinkering with the genomes of human eggs, sperm or embryos, known collectively as germ cells. Huang and colleagues used non-viable embryos, which could not result in a live birth. But in principle, edits to germ cells could be passed to future generations.
2. The law on editing human germ cells varies wildly across the world.
Germany strictly limits experimentation on human embryos, and violations can be a criminal offence. By contrast, in China, Japan, Ireland and India, only unenforceable guidelines restrict genome editing in human embryos. Many researchers long for international guidelines, and some hope that the upcoming summit in Washington DC could be the start of the process to create them.
The CRISPR-Cas9 system makes editing genomes cheap and easy.
3. You don’t have to be a pro to hack genomes.
The CRISPR–Cas9 technology has made modifying DNA so cheap and easy that amateur biologists working in converted garages or community laboratories are starting to dabble.
4. Cas9 is not the only enzyme in town.
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 genomes2. (Zhang is one of those who pioneered the use of CRISPR-Cas9 for genome editing in mammalian cells).
5. Pigs are on the front line of genome-editing experiments.
Dogs, goats and monkeys are all part of the growing CRISPR zoo. But pigs in particular have been at the heart of several eye-catching announcements — from micropigs that weigh about six times less than many farm pigs, to super-muscly pigs, to a pig whose genome has been edited in 62 places (the aim being to produce a suitable non-human organ donor).
6. Gates, Google and DuPont want a piece of the genome-editing action.
In August, several high-profile investors, including the Bill & Melinda Gates Foundation and Google Ventures, pumped US$120 million into the genome-editing firm Editas Medicine of Cambridge, Massachusetts. Big Agriculture is following suit: DuPont forged an alliance with the genome-editing firm Caribou Biosciences of Berkeley, California, in October, and announced its intention to use CRISPR–Cas9 technology to engineer crops.
7. The CRISPR–Cas9 system is at the centre of a patent row.
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 patent. UC Berkeley has since requested that the United States Patent and Trademark Office determine who should get credit for harnessing the CRISPR–Cas9 system, in particular for its application in human cells. And a similar debate is playing out in Europe, where oppositions to a patent that Zhang and his colleagues won in February have been filed. All three scientists co-founded companies that make use of CRISPR–Cas9.
Gene-editing summit supports some research in human embryos
Three-day meeting calls for further discussions on modifications to the gene pool.
Gene-editing technology should not be used to modify human embryos that are intended for use in establishing a pregnancy, an international summit declared in a statement issued on 3 December.
The International Summit on Human Gene Editing also called for cautious development of medical applications that cannot be passed on to offspring — such as correction of the mutations that cause sickle-cell disease or modification of immune cells to target cancer.
But the summit statement, authored by a 12-member organizing committee, cautioned that many technical and ethical issues should be settled before anyone attempts ‘germline’ editing — the deletion of a gene prenatally in an effort to erase an inherited disease from an embryo and prevent it from being passed on to future generations.
“It would be irresponsible to proceed with any clinical use of germline editing unless and until (i) the relevant safety and efficacy issues have been resolved … and (ii) there is broad societal consensus about the appropriateness of the proposed application,” the statement said.
The organizing committee stopped short of calling for a ban on editing human embryos and germ cells for basic research. “We don't want to slam the door on this idea forever,” said biochemist Jennifer Doudna of the University of California, Berkeley.
The three-day international summit took place at the the US National Academy of Sciences in Washington DC. The meeting was jointly hosted by the US science and medicine academies, the UK Royal Society and the Chinese Academy of Sciences.
Some ethicists and a few scientists at the summit expressed fear that even altering embryos that are not intended for implantation will pave the way for germline editing. And some worried that public opposition to embryo research could cause a backlash against use of genome editing as therapy. “I don’t want even a shadow of a shadow of a concern about the germline stuff to fall on efforts to treat HIV or [ß-thalassaemia] or sickle cell,” said Fyodor Urnov, a scientist at Sangamo BioSciences in Richmond, California.
Others saw a complete ban on embryo research as unrealistic: even if some researchers agree to abstain from editing embryos, or if some countries ban it outright, such work will inevitably be done in places with less oversight, argued George Church, a geneticist at Harvard Medical School in Boston, Massachusetts. “You create a nexus for your worst nightmares,” he said.
The work now continues: over the next year, scientists and ethicists from the three hosting countries will convene to examine issues raised at the meeting. Their consensus report is scheduled to be released in late 2016.
Enzyme tweak boosts precision of CRISPR genome edits
Engineered enzyme drives genome-editing errors below detection limit.
A powerful technique for editing genomes is now more precise. By tweaking an enzyme, researchers have reduced the error rate for the technique, known as CRISPR–Cas9 — in some cases to undetectable levels, they report on 6 January in Nature1.
Researchers use CRISPR–Cas9 to make precise changes to genomes that remove or edit a faulty gene. It has worked on nearly every creature on which they have tested it, including human embryos.
The technique relies on an enzyme called Cas9, that uses a 'guide RNA' molecule to home in on its target DNA. Cas9 cuts the DNA at that site, and the cell's natural DNA repair machinery then takes over to mend the cut — deleting a short fragment of DNA or stitching in a new sequence in the process.
But the technology is not infallible: sometimes the Cas9 enzyme creates unwanted mutations. As CRISPR inches out of the laboratory and towards the clinic — with debates raging over whether it should be deployed in embryos — researchers have pushed to reduce the error rate.
The latest study moves the field closer to that goal, says lead author Keith Joung, a pathologist at Massachusetts General Hospital in Boston. “This is a significant move forward,” he says. “We can very much reduce the probability of off-targets.”
Some researchers argue that the error rate does not have to be zero for CRISPR to be clinically useful. “At some point everyone needs to decide how specific is specific enough,” says Charles Gersbach, a bioengineer at Duke University in Durham, North Carolina. “The idea that you would make a tool that has absolutely no off-target effects is a little too utopian.”
Previous work has shown that using a shorter strand of ‘guide RNA’ to direct the Cas9 enzyme to the targeted DNA could cut down on some errors2. And in December, synthetic biologist Feng Zhang of the Broad Institute of MIT and Harvard in Cambridge, Massachusetts, and his colleagues announced that they had engineered Cas9 to make it less error-prone3.
For the latest study, Joung and his colleagues tackled a different region of the Cas9 enzyme, altering the part of the protein that makes contact with the DNA target. The team also used a more sensitive method for detecting errors.
They tested their high-fidelity enzyme, called SpCas9-HF1, with eight different guide RNAs. The engineered enzyme cut its target DNA nearly as well as the unaltered form, and made only one mistake with one of the guide RNAs. The unaltered Cas9 enzyme, by contrast, made mistakes when guided by seven of the eight RNAs.
Cas9's mistakes have been a focus in many discussions about genome editing — including in the debate over using the technique in human embryos. But that focus may be misplaced, says George Church, a geneticist at the Wyss Institute in Boston. With careful design of the guide RNA, Church says that researchers could already avoid most off-target cuts.
And although the work is important given the speed with which CRISPR–Cas9 is moving into therapeutics, says Gersbach, the system will need extra safety checks before it is deemed safe for use in humans.
Pursuit of perfection
In December, Gersbach and his colleagues announced that they had used CRISPR–Cas9 to repair the genetic mutation that causes Duchenne muscular dystrophy in mice4. To do so, his team used a virus to carry Cas9 into muscle cells. That virus can continue to express the enzyme for much longer than it was in Joung’s experiments, leaving more opportunity for off-target cuts.
The US Food and Drug Administration has not outlined its requirements for approving a CRISPR–Cas9 clinical trial, but Sangamo BioSciences of Richmond, California, has already used another genome-editing tool, called zinc finger nucleases, in clinical trials in more than 80 patients.
For those trials, regulators wanted safety data on how well the modified cells performed, in addition to information about off-target mutations, says Fyodor Urnov, a senior scientist at the Sangamo. The company was required to show that altered immune cells called T cells still behaved like normal T cells, for example, or that edited liver cells continued to function without showing signs of toxicity.
“This study is a solid advance for the Cas9 field,” says Urnov. “But when you think about deploying editing in the clinical space, we have a healthy sense of how long the road ahead is.”