Researchers have discovered a way to program cells to inhibit CRISPR-Cas9 activity. “Anti-CRISPR” proteins had previously been isolated from viruses that infect bacteria, but now University of Toronto and University of Massachusetts Medical School scientists report three families of proteins that turn off CRISPR systems specifically used for gene editing. The work, which appears December 15 in Cell, offers a new strategy to prevent CRISPR-Cas9 technology from making unwanted changes.
“Making CRISPR controllable allows you to have more layers of control on the system and to turn it on or off under certain conditions, such as where it works within a cell or at what point in time,” says lead author Alan Davidson, a phage biologist and bacteriologist at the University of Toronto. “The three anti-CRISPR proteins we’ve isolated seem to bind to different parts of the Cas9, and there are surely more out there.”
CRISPR inhibitors are a natural byproduct of the evolutionary arms race between viruses and bacteria. Bacteria use CRISPR-Cas complexes to target and cut up genetic material from invading viruses. In response, viruses have developed proteins that, upon infection, can quickly bind to a host bacterium’s CRISPR-Cas systems, thus nullifying their effects.
Anti-CRISPR proteins are attractive experimentally because they offer one solution for preventing potential off-target effects. Research in mice has shown that such mistakes may be rare when using CRISPR-Cas9 technology, but even the occasional error could be a serious problem when being used therapeutically in humans.
“CRISPR-Cas9 in ancillary cells, tissues, or organs is at best useless and at worst a safety risk,” says co-author and collaborator Erik J. Sontheimer, a professor in the RNA Therapeutics Institute at the University of Massachusetts Medical School. “But if you could build an off-switch that keeps Cas9 inactive everywhere except the intended target tissue, then the tissue specificity will be improved.”
“Knowing we have a safety valve will allow people to develop many more uses for CRISPR,” says co-author Karen Maxwell, an assistant professor in biochemistry who is also at the University of Toronto. “Things that may have been too risky previously might be possible now.”
While the work will be of great interest to those studying gene editing and gene drives, Davidson’s team is also curious to follow up on the biology of how bacterial CRISPRs and viral anti-CRISPRs interact.
“We didn’t set out to find anti-CRISPRs, we were just trying to understand how phages incorporate themselves into bacterial genomes and stumbled onto something that I think will be important for biotechnology,” Davidson says.
“We were being observant and following a path that we didn’t know where it could lead, and it’s just been a very fun and exciting story.”
Learn more: An anti-CRISPR for gene editing
For (probably) the first time ever, plants modified with the “genetic scissors” CRISPR-Cas9 has been cultivated, harvested and cooked.
Stefan Jansson, professor in Plant Cell and Molecular Biology at Umeå University, served pasta with “CRISPRy” vegetable fry to a radio reporter. Although the meal only fed two people, it was still the first step towards a future where science can better provide farmers and consumers across the world with healthy, beautiful and hardy plants.
CRISPR (Clustered regularly interspaced short palindromic repeats)-Cas9 is a complicated name for an easy, but targeted, way of changing the genes of an organism. The decisive discovery was published in 2012 by researchers at Umeå University, and the ”Swiss army knife of genetic engineering” has been predicted to change the world. With CRISPR-Cas9, researchers can either replace one of the billions of “letters” present in an organism’s genome (i.e. the entire gene pool consisting of DNA) or remove short segments, similar to when you edit a written text in a word processor. The technology is called “gene editing” or “genome editing”.
The first clinical applications are underway; maybe we can soon cure hereditary disease using this technology. However, the situation differs somewhat in the agricultural field. There, the issue is not IF researchers can create plants leading to a more sustainable land management, but rather if these will be allowed in farming. Will plants whose genome has been edited using CRISPR-Cas9 fall under GMO legislation or not? If they do, it makes them illegal to plant in great parts of the world. If not, they will – just like other plants – be allowed to be grown at the farmers own discretion.
Can be cultivated legally
The EU has avoided answering the question, but in November 2015 the Swedish Board of Agriculture interpreted the law as if only a segment of DNA has been removed and no “foreign DNA” has been inserted, it is not to be regarded as a genetically modified organism – a GMO. That also means that the plant can be cultivated without prior permission. In spring 2016, American authorities stated that they agreed. The organism in question there was a mushroom who had lost the part of its DNA that made it go brown. This opens up for using the technology to develop plants of the future.
This summer has been the first time that plants that have been gene-edited using CRISPR-Cas9 – in a way that does not classify the plant as GMO – have been allowed to be cultivated outside of the lab. This is definitely the first time in Europe, and even if it been done before in other parts of the world, it has been kept secret. This time, it was a cabbage plant and the Radio Sweden gardening show “Odla med P1” took part in the harvest leading to the probably first-ever meal of CRISPR-Cas9 genome-edited plants. The first CRISPR meal to have been enjoyed was “Tagliatelle with CRISPRy fried vegetables”.
“The CRISPR-plants in question grew in a pallet collar in a garden outside of Umeå in the north of Sweden and were neither particularly different nor nicer looking than anything else,” says plant scientist Stefan Jansson. But they represent both a new phase of agriculture where scientific advances will be implemented in new plant species and that to a small or large extent will be made available to farmers across the world. In other words: a meal for the future.
Duke engineers use CRISPR to generate neuronal cells from connective tissue
Researchers have used CRISPR—a revolutionary new genetic engineering technique—to convert cells isolated from mouse connective tissue directly into neuronal cells.
In 2006, Shinya Yamanaka, a professor at the Institute for Frontier Medical Sciences at Kyoto University at the time, discovered how to revert adult connective tissue cells, called fibroblasts, back into immature stem cells that could differentiate into any cell type. These so-called induced pluripotent stem cells won Yamanaka the Nobel Prize in medicine just six years later for their promise in research and medicine.
Since then, researchers have discovered other ways to convert cells between different types. This is mostly done by introducing many extra copies of “master switch” genes that produce proteins that turn on entire genetic networks responsible for producing a particular cell type.
Now, researchers at Duke University have developed a strategy that avoids the need for the extra gene copies. Instead, a modification of the CRISPR genetic engineering technique is used to directly turn on the natural copies already present in the genome.
These early results indicate that the newly converted neuronal cells show a more complete and persistent conversion than the method where new genes are permanently added to the genome. These cells could be used for modeling neurological disorders, discovering new therapeutics, developing personalized medicines and, perhaps in the future, implementing cell therapy.
The study was published on August 11, 2016, in the journal Cell Stem Cell.
“This technique has many applications for science and medicine. For example, we might have a general idea of how most people’s neurons will respond to a drug, but we don’t know how your particular neurons with your particular genetics will respond,” said Charles Gersbach, the Rooney Family Associate Professor of Biomedical Engineering and director for the Center for Biomolecular and Tissue Engineering at Duke. “Taking biopsies of your brain to test your neurons is not an option. But if we could take a skin cell from your arm, turn it into a neuron, and then treat it with various drug combinations, we could determine an optimal personalized therapy.”
“The challenge is efficiently generating neurons that are stable and have a genetic programming that looks like your real neurons,” says Joshua Black, the graduate student in Gersbach’s lab who led the work. “That has been a major obstacle in this area.”
In the 1950s, Professor Conrad Waddington, a British developmental biologist who laid the foundations for developmental biology, suggested that immature stem cells differentiating into specific types of adult cells can be thought of as rolling down the side of a ridged mountain into one of many valleys. With each path a cell takes down a particular slope, its options for its final destination become more limited.
If you want to change that destination, one option is to push the cell vertically back up the mountain—that’s the idea behind reprogramming cells to be induced pluripotent stem cells. Another option is to push it horizontally up and over a hill and directly into another valley.
“If you have the ability to specifically turn on all the neuron genes, maybe you don’t have to go back up the hill,” said Gersbach.
Previous methods have accomplished this by introducing viruses that inject extra copies of genes to produce a large number of proteins called master transcription factors. Unique to each cell type, these proteins bind to thousands of places in the genome, turning on that cell type’s particular gene network. This method, however, has some drawbacks.
“Rather than using a virus to permanently introduce new copies of existing genes, it would be desirable to provide a temporary signal that changes the cell type in a stable way,” said Black. “However, doing so in an efficient manner might require making very specific changes to the genetic program of the cell.”
In the new study, Black, Gersbach, and colleagues used CRISPR to precisely activate the three genes that naturally produce the master transcription factors that control the neuronal gene network, rather than having a virus introduce extra copies of those genes.
CRISPR is a modified version of a bacterial defense system that targets and slices apart the DNA of familiar invading viruses. In this case, however, the system has been tweaked so that no slicing is involved. Instead, the machinery that identifies specific stretches of DNA has been left intact, and it has been hitched to a gene activator.
The CRISPR system was administered to mouse fibroblasts in the laboratory. The tests showed that, once activated by CRISPR, the three neuronal master transcription factor genes robustly activated neuronal genes. This caused the fibroblasts to conduct electrical signals—a hallmark of neuronal cells. And even after the CRISPR activators went away, the cells retained their neuronal properties.
“When blasting cells with master transcription factors made by viruses, it’s possible to make cells that behave like neurons,” said Gersbach. “But if they truly have become autonomously functioning neurons, then they shouldn’t require the continuous presence of that external stimulus.”
The experiments showed that the new CRISPR technique produced neuronal cells with an epigenetic program at the target genes matching the neuronal markings naturally found in mouse brain tissue.
“The method that introduces extra genetic copies with the virus produces a lot of the transcription factors, but very little is being made from the native copies of these genes,” explained Black. “In contrast, the CRISPR approach isn’t making as many transcription factors overall, but they’re all being produced from the normal chromosomal position, which is a powerful difference since they are stably activated. We’re flipping the epigenetic switch to convert cell types rather than driving them to do so synthetically.”
The next steps, according to Black, are to extend the method to human cells, raise the efficiency of the technique and try to clear other epigenetic hurdles so that it could be applied to model particular diseases.
“In the future, you can imagine making neurons and implanting them in the brain to treat Parkinson’s disease or other neurodegenerative conditions,” said Gersbach. “But even if we don’t get that far, you can do a lot with these in the lab to help develop better therapies.”
Gene-editing technique to treat lung cancer is due to be tested in people in August
Chinese scientists are on the verge of being first in the world to inject people with cells modified using the CRISPR–Cas9 gene-editing technique.
A team led by Lu You, an oncologist at Sichuan University’s West China Hospital in Chengdu, plans to start testing such cells in people with lung cancer next month. The clinical trial received ethical approval from the hospital’s review board on July 6.
“It’s an exciting step forward,” says Carl June, a clinical researcher in immunotherapy at the University of Pennsylvania in Philadelphia.
UMMS scientists develop multicolored labeling system to track genomic locations in live cells
“Most people are using CRISPR for editing genomes. We are using it to label DNA and track the movement of DNA in live cells,” said research specialist Hanhui Ma, PhD, who coauthored the study with Thoru Pederson, PhD, the Vitold Arnett professor of cell biology and professor of biochemistry and molecular pharmacology.
Knowing the precise location of genomic elements in live cells is critical to understanding chromosome dynamics because the genes that control our biology and health do so according to their location in 3-dimensional space, said Drs. Pederson and Ma. For a gene to be transcribed and expressed, it must be accessible on the chromosome. Where DNA is positioned in the crowded nucleus plays an important role in everything from embryonic development to cancer.
Current technologies, however, are only capable of following, at most, three genomic locations at a time in live cells. Labeling more sites requires that cells be fixed by bathing them in formaldehyde, thus killing them and making it impossible to observe how the chromosome’s structure changes over time or in response to stimuli.
To overcome this technological hurdle, Pederson and Ma turned to CRISPR/Cas9. To tag specific locations along the genome using the CRISPR/Cas9 complex, they created a Cas9 mutation that makes the nuclease inactive so it only binds to the DNA and doesn’t cut the genome. Once deactivated, the CRISPR/Cas9 element is ferried to a specific location on the genome by a guide RNA that can be programmed by the researchers.
New delivery method boosts efficiency of CRISPR genome-editing system
The genome-editing technique known as CRISPR allows scientists to clip a specific DNA sequence and replace it with a new one, offering the potential to cure diseases caused by defective genes. For this potential to be realized, however, scientists must find a way to safely deliver the CRISPR machinery and a corrected copy of the DNA into the diseased cells.
MIT researchers have now developed a way to deliver the CRISPR genome repair components more efficiently than previously possible, and they also believe it may be safer for human use. In a study of mice, they found that they could correct the mutated gene that causes a rare liver disorder, in 6 percent of liver cells — enough to cure the mice of the disease, known as tyrosinemia.
“This finding really excites us because it makes us think that this is a gene repair system that could be used to treat a range of diseases — not just tyrosinemia but others as well,” says Daniel Anderson, associate professor in MIT’s Department of Chemical Engineering and a member of MIT’s Koch Institute for Integrative Cancer Research and Institute for Medical Engineering and Science (IMES).