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The Swiss Army Knife of Gene Editing Gets New Control

The Swiss Army Knife of Gene Editing Gets New Control

Now, Caltech researchers have applied principles from the emerging field of dynamic RNA nanotechnology to exert logical control over CRISPR/Cas9 within living cells. By engineering RNA strands to interact and thereby change shape in response to an RNA trigger sequence, the group demonstrates the ability to switch CRISPR/Cas9 from on to off and from off to on. The work suggests a path to confine manipulation of a gene only to specific organs, tissues, or cell types within an organism.

The work was done in the laboratory of Niles Pierce, professor of applied and computational mathematics and bioengineering, and is described in a paper published on June 4, 2019, in the journal ACS Central Science.

An organism’s genome encodes complex biological processes that orchestrate the organism’s development, maintenance, and repair. Different genes encode instructions for different cellular behaviors, such as growing, communicating, and dying. Controlling gene activity is a fundamental way to change the cell’s behavior. Editing a gene, or, alternatively, turning it off or on in a given cell, provides biologists with a way to study the role of that gene, and likewise offers a promising avenue for doctors to treat disease.

Developed less than a decade ago, CRISPR/Cas9 technology has emerged as a game-changing tool for editing genomes and for controlling which genes are active and to what extent. The CRISPR/Cas9 complex is made up of two parts: Cas9, a protein that can edit genes; and the guide RNA (gRNA), a molecule that—as its name suggests—guides Cas9 to a target gene of choice. If desired, different variants of the Cas9 protein can be used to increase or decrease the level of activity of a target gene as well. In essence, a traditional gRNA executes the function “regulate gene Y,” where the choice of target gene “Y” is specified by the sequence of the gRNA, and the kind of regulation (activate, silence, edit, and so on) depends on the choice of Cas9 variant.

One of the remarkable features of CRISPR/Cas9 technology is that these capabilities work on many organisms across the tree of life, whether they be fungi, plants, or birds. However, the versatility of this approach is limited by the fact that the gRNA is “always on”—that is, it executes its function independent of the cell type it is in. As a result, additional measures are needed to restrict regulation of the selected target gene “Y” to specific cells in a specific state. For example, in a scenario where some cells are diseased, it would be useful to restrict gene regulation to only that subset of cells.

An important signature of cell type and state is provided by the collection of RNA molecules present inside the cell. In principle, detection of an RNA sequence “X” (where X is a marker for a specific tissue type or disease state) could serve as a trigger to induce editing, silencing, or activation of an independent target gene Y. For the last 15 years, the Pierce Lab has pursued this vision, seeking to engineer RNA molecules that can detect an RNA trigger sequence X and then change shape to target an independent gene Y for regulation. CRISPR/Cas9 is one of several naturally occurring biological pathways to which this technology could be applied.

Now, led by graduate students Mikhail Hanewich-Hollatz and Zhewei Chen, a team of researchers has engineered guide RNAs that are conditional, changing shape in response to the presence or absence of an RNA trigger to switch between inactive and active conformations. As a result, these so-called conditional guide RNAs (cgRNAs) can execute logical functions such as “if X then not Y” (i.e., if the trigger RNA X is present, then silence the gene target Y) or “if not X then not Y”. Unlike a traditional gRNA, cgRNAs are programmable at two levels, with the sequence of trigger X controlling where regulation occurs and the target-binding sequence controlling the subject of regulation (in other words, the identity of the target gene Y). In bacterial cells, the team has been able to demonstrate both ON-to-OFF logic with initially active cgRNAs that are turned off by an RNA trigger and OFF-to-ON logic with initially inactive cgRNAs that are turned on by an RNA trigger. Moreover, in work led by research scientist Lisa Hochrein, they were able to successfully port one cgRNA mechanism from bacterial to mammalian cells, leveraging the portability for which CRISPR/Cas9 is renowned.

The hope is that cgRNAs might someday be applied to the treatment of disease, with RNA X as a disease marker and target Y as a therapeutic target, enabling selective treatment of diseased cells while leaving healthy cells untouched. Alternatively, the same logic could enable biologists to study the role of a gene of interest at a specific location and developmental stage within an embryo.

“There is still a long way to go to realize the potential of dynamic RNA nanotechnology for engineering programmable conditional regulation in living organisms, but these results with CRISPR/Cas9 in bacterial and mammalian cells provide a proof of principle that we can build on in seeking to provide biologists and doctors with powerful new tools,” says Pierce.

The paper is titled Conditional Guide RNAs: Programmable Conditional Regulation of CRISPR/Cas Function in Bacterial and Mammalian Cells via Dynamic RNA Nanotechnology.” Graduate students Mikhail Hanewich-Hollatz and Zhewei Chen are co-first authors. In addition to Pierce, co-authors are research scientist Lisa Hochrein (MS ’09, PhD ’13) and graduate student Jining Huang. Funding was provided by the Defense Advanced Research Projects Agency, the Caltech Center for Environmental Microbial Interactions (CEMI), the National Institutes of Health, the Donna and Benjamin M. Rosen Bioengineering Center at Caltech, the Natural Sciences and Engineering Research Council of Canada, the National Science Foundation Molecular Programming Project, a Professorial Fellowship at Balliol College at the University of Oxford, and the Eastman Visiting Professorship at the University of Oxford.

crispr gene art

Scientists create new CRISPR model that allows to edit several genes at once

We have seen several uses of CRISPR gene editing such as accurate cutting and pasting of specific genes in DNA. Scientists have now developed a technique which makes it possible to edit several hundreds of genes at once. This would be very beneficial to the scientists who can reprogram cells in a larger and complex manner thus assisting in the study of genetic disorders. 

CRISPR could mostly edit one gene at a time. As per the latest study, the new technique can hit 25 targets within genes at once. Biochemist Randall Platt, ETH Zurich said that this technique makes it possible to modify entire gene networks in one step which was not even thought in the past. The work has been published in the journal Nature Methods. 

The key to this process is a plasmid, a stabilised RNA structure that can hold and process several RNA molecules. These act as address labels for targeting gene sites. Along with the RNA molecules the plasmid also carries Cas9 enzyme which performs the main cutting and binding job. Cas9 has been mainly used although here Cas12a is used which improves the accuracy of CRISPR editing. Researchers were able to insert this plasmid into human cells in the laboratory. Hence it enables scientists to make gene editing at scale. It can be quite tiring to make a single change at one time as proteins, genes act in complex manners. Operations such as increasing and decreasing the gene activities can be done with greater precision. 

However, this also means that we would be moving in uncharted territories as we do not know the effects of this simultaneous change in the body of the organism. There might be secondary changes which are not expected thus increasing the risk of gene editing. 

Researchers mention that direct repeat sequences and pacers which possess the complementary sequences could create secondary RNA structures which can affect the maturation of the CRISPR RNAs present in the cells. As a result of which, it is essential to consider the complementary regions in pre-CRISPR RNA so as to improve the maturation of CRISPR RNA. Research overcoming these limitations would create several applications for genome engineering. 

CRISPR has been already used in eliminating genes responsible for diseases and killing the superbugs. There is a whole range of applications awaiting as researchers now have better toolkit at their disposal. Therefore this technique creates a platform in which different types of investigation related to genetic programs affecting the cell behaviour can be carried out. 

Journal Reference: Nature Methods

gene editing artist

Chinese scientists create safer alternative to CRISPR which avoids ethical concerns

A team of Chinese researchers have created an efficient and very safe technology for editing RNA that could avoid the side effects to a great extent and also the ethical concerns which came from the previous technologies of gene-editing. The study has been published in the journal Nature Biotechnology

This feat comes after half a year when Chinese researcher He Jiankui claimed that he had made the world’s first gene-edited twins who are immune to HIV. This announcement caused a whirlwind of condemnation all over the world. Many Chinese and international researchers condemned it as any application of gene editing was unethical if used on human embryos for reproductive purposes. He used the technology of CRISPR-Cas9, that was adapted from a genome editing system which occurred naturally in bacteria. The Cas9 enzyme, a protein that plays a very important role in the immunological defence of specific bacteria against the DNA viruses would be introduced for cutting the DNA of the viruses in the human body. 

Wei Wensheng, a leading researcher of the technology and also a biologist at the Peking University said that this technology essentially depends on the delivery of chemically modified guide RNAs or exogenous proteins that may lead to a delivery barrier.

Zhou Zhuo, another research team member said that on the contrary, the latest technology known as Leveraging Endogenous ADAR for Programmable Editing of RNA or LEAPER uses the native proteins and hence does not alter the DNA in a direct manner. Because of these factors, it would not bring any heritable changes and is safe. It uses engineered RNA’s for recruiting native enzymes to change certain adenosine to inosine. Zhou also stated that experiments conducted at a cellular level in the last two years showed that LEAPER is capable of achieving editing efficiencies up to 80 percent. He further mentioned that the team is now conducting tests on rats. Hence we have to wait to understand if it is suitable for use in human beings.

LEAPER is active in a very broad spectrum of cell types which includes several human primary cell types. It can also restore the deficient cells of patients having Hurler syndrome without evoking any kind of innate immune responses. Being a single-molecule system, LEAPER ensures highly precise and efficient RNA editing with large scale applications for basic research and therapy. 

Journal Reference: Nature Biotechnology

CRISPR Cas9 gene editing

Parents agree to use CRISPR gene editing on their babies to avoid deafness

A Russian Scientist Dennis Rebrikov wants to use CRISPR to create more gene edited babies and has said that 5 couples have agreed for genetically editing their babies to avoid chances of deafness. The Russian biologist had planned to do gene editing to human embryos and bring them to term. Chinese scientist He Jiankui was the first ever person to produce gene edited babies and claiming that these edits would save the babies by preventing inheritance of HIV from their fathers.

Rebrikov has told scientists that five Russian parents were eager to let him edit the genes of their embryos for different and socially loaded reasons which include prevention of the offspring from inheriting parent’s deafness. The parents who are interested in the study was deaf due to mutations in the GJB2 gene. When these parents reproduce, the child is guaranteed to be born deaf. Using the CRISPR technique to edit a copy of the GJB2 gene in the embryo and he wishes to grant the parents a biological child that is not deaf. If he plans to go ahead with the CRISPR technique on human embryos then it seems there is nothing anyone can do to stop him.

The technique is clear and understandable to a common man and that each new baby would be deaf to these parents without the gene editing. Rebrikov has plans to reach out to the Russian government to seek permission for this controversial CRISPR experiment. He thinks that the use of CRISPR is more justifiable medically but it is still a controversial issue. People think that deafness is a condition that does not need to be treated at all however it is a culture that we should embrace and not consider it as a disability. Some medical devices and surgeries which give the deaf the ability to hear are perceived as genocide against the minority group in the minds of people.

In this early stage of CRISPR research, scientists think that we should not risk conducting these experiments on humans unless it is useful in saving lives and that human trials should be done only on embryos and infants where there are minimal loss and nothing to lose as said by University of Oxford bioethicist Julian Savulescu and that should not be done on an embryo which is going to lead a normal life.

Genetic Engineering Will Change Everything Forever – CRISPR

Genome editing is a group of methods that gives researchers the ability to modify the DNA of an organism. These technologies let genetic substance to be added, detached, or transformed at specific locations in the gene. Several methods to genome editing have been advanced. The latest one is recognized as CRISPR-Cas9.

The above video tells you many things right from the history of gene editing to the latest tool CRISPR. I hope you get good knowledge about CRISPR from this video.

CRISPR Cas9 and Gene Editing Explained – ScienceHook

For the scientists, the genes and its related fields have been an area of interests for research for a number of years. They have found a lot of therapies and treatments where modern technologies are used to cure various diseases as well as preventive treatments.

HIV infected T cell

Researchers eliminate HIV from infected mice with the help of CRISPR

An interdisciplinary group of researchers has claimed to have eliminated HIV from the mice genomes with the help of the gene editing tool CRISPR-Cas9 and a new drug. This is quite promising in the fight against HIV and AIDS, although a lot of work is still left before clinical trials can be started. 

It may seem odd that a gene editing tool is used for removing infectious disease, however, HIV being a retrovirus embeds itself in DNA for replication. ART which stands for Antiretroviral therapy can help in suppressing HIV replication however it is not effective for eliminating the disease completely. The reason being it is incapable of purging cells where the virus has been dormant. The study has been published in the Nature Communications journal. When CRISPR-Cas9 is used with a newer form of ART, it eliminated the virus from the genome, a feat achieved for the first time. 

Experiments were carried out on genetically modified mice which had similarities with human beings. The team led by Kamel Khalili, Lewis Katz School of Medicine, Temple University was successful in eliminating every single trace of HIV in about 30 percent of infected mice. Though not perfect, it provides hope to be optimistic. Khalili said that they can now proceed for trials in non-human primates, with clinical trials in humans within a year. 

Khalili is also the founder and lead scientific advisor of Excision BioTherapeutics. This company uses CRISPR for treating viral diseases. It also possesses an exclusive license for the commercial application of the therapy. However, researchers have to proceed with caution as they have to prove that it is free of long term side effects such as cancer. 

Antiretroviral therapy for the treatment of AIDS has benefitted many people all over the world, but it is not a cure technically. Patients have to be on a regular dosage of medicines for keeping the HIV virus in check. We are in a dire need of a therapy that eliminates HIV from the body completely. 

Khalili took the help Howard Gendelman, professor of infectious diseases at the University of Nebraska Medical Center for the new study. He has been working on a new form of ART known as LASER. It stands for long-acting slow-effective release. It essentially targets the cells where HIV hides and suppresses its replication for long periods of time. For achieving this the drug is wrapped in nanocrystals, through which it spreads to tissues where HIV is supposedly dormant. It then slowly releases the drug. 

LASER ART caught Khalili’s attention. It was incorporated with CRISPR-Cas9 and was successful in eliminating HIV from one-third of infected mice. Scientists have to proceed with caution as CRISPR has the risk of causing cancer in the body. Since it stays in the body for a long time it could cut other sites in an uncontrolled way, hence being cancerous. 

CRISPR Cas9

Using human genome, scientists build CRISPR for RNA to open pathways for medicine

Less than a decade ago, biology underwent one of those once-in-a-generation events that shakes up a scientific field, when the discovery of gene editing technology called CRISPR/Cas-9 made it possible to precisely alter the sequence of DNA in a living being.

But while DNA may be the raw blueprints for life, RNA is the architect—translating those ideas into reality for the cell through proteins and regulation. While CRISPR systems that target RNA have recently been discovered, none offers a single clear solution.

A group of scientists from the University of Chicago has announced a breakthrough method to alter RNA—and instead of using a protein from bacteria, like CRISPR, the new system is built out of parts from the human genome. Announced June 20 in Cell, the discovery could open new pathways for treating diseases or injuries by temporarily altering how the genetic template is carried out in the cell.

“People had delayed targeting RNA for a long time because it’s so complex in how it works,” said study author Bryan Dickinson, an associate professor of chemistry at UChicago. “But I think now we’re realizing that complexity is an opportunity to figure out how to exploit and change those pathways. In principle, you could make even more dramatic changes to the cell than with DNA, and now we finally have the tools to do so.”

Even as DNA-targeting CRISPR methods begin their initial clinical trials in humans, scientists have become increasingly interested in equivalent systems for RNA. An RNA-targeting method that can safely be applied to humans would be a valuable complement to CRISPR, Dickinson said.

“If you imagine the universe of diseases that CRISPR is going to correct, it’ll be really important ones, but only those that are based off of one single mutation in your DNA,” said Dickinson, whose work tries to create functional molecules that lead to biological breakthroughs. “There are many more diseases out there with multiple causes in the cell, which may be much more difficult to understand—and there will also be those where the risks associated with changing someone’s DNA permanently are just too high.”

Because the effects of RNA alteration are temporary rather than permanent, an RNA-CRISPR is inherently less risky, because doctors can simply stop the treatment if there are intolerable side effects. It could also be used for things like briefly boosting a person’s system to accelerate wound healing: “We know what to do for that—you would encourage processes for cell growth and proliferation,” Dickinson said. “But those are the same things that cause cancer, so you could never do that at the DNA level.”

But translating these microbial systems into therapeutics is going to be challenging, he said. “RNA-targeting drugs need to be continually administered, so the foreign nature of CRISPR/Cas systems it going to create an immune backlash when applied to humans.”

This presents key roadblocks for natural CRISPR systems, which Dickinson’s team realized it had an opportunity to correct by reengineering the whole system from scratch.

“In principle, you could make even more dramatic changes to the cell than with DNA, and now we finally have the tools to do so.”

—Assoc. Prof. Bryan Dickinson on targeting RNA

Because it’s a very large protein, CRISPR is generally too big to use the most common delivery system to insert genetic material into cells—“phages,” which originate from tiny viruses. This is a problem, especially if you need to deliver them continually. More critically, because CRISPR comes from a microbe, there are significant concerns about the human immune system reacting to it.

Instead, the team broke down CRISPR into its components based on what each part does, and looked for human versions of those proteins that did equivalent tasks. Then they cobbled those together into a cohesive whole—which is smaller than CRISPR, and made out of human material.

“Although there’s still a lot of work to do, the crazy thing is it actually works,” Dickinson said.

Their system succeeded in altering RNA in tests in the lab. The scientists plan to improve the system at a few points where the performance is not as good as CRISPR, they said, but they’re encouraged by the early results.

“As we learn more, you could imagine targeting multiple RNAs in different ways, and doing more complex reprogramming of the cell at the RNA level,” Dickinson said. “It’s a really exciting field right now.”

The first author was graduate student Simone Rauch; other co-authors were visiting scholar Michael Srienc, postdoctoral fellow Huiqing Zhou, high school student Emily He and graduate student Zijie Zhang.

The scientists are working with the Polsky Center for Entrepreneurship and Innovation at the University of Chicago to advance this discovery.

Materials provided by University of Chicago

Red reporter cells showing the change in edited cells on the right.

Scientists demonstrate genetic editing of stem cells inside the body of mice

Researchers have been successful in genetic modification of stem cells inside the body of mice for the first time ever. This feat can unlock various avenues in stem cell therapy, which have not been explored so far. The study has been published in Cell Reports.

Stem cells can grow into all other types of body cells and the body uses this ability for its growth and repair in several parts. Because of the potential in stem cells, researchers are always looking to incorporate stem cells in the medical treatments but it has not been easy so far. For example in the case of a bone marrow transplant, the stem cells which produce blood have to be first removed from the human body, genetically modified and only then be transfused back into the human body.

From the results of this experiment on mice, it is likely that the complicated extraction step can be bypassed. The genetic edits which are needed can be performed in vivo, which is estimated to be much faster and effective than the techniques which are currently used.

Amy Wagers from Harvard University who is the principal researcher behind the study commented that when stem cells are taken out of the body, they are being removed from the circumstances which provide nourishment and helps to sustain them. As a result of this change, they go into shock. The researchers wanted to make the genetic changes in the cells without isolating or transplanting them as it changes them altogether.

Researchers did this by using adeno-associated virus(AAV). This virus is able to enter the body for infecting and altering the cells without generating any sort of disease. In the tests which were conducted on mice, AAVs packed with CRISPR gene editing technology were released in various types of skin, blood, muscle stem cells, progenitor cells.

With the help of activated reporter genes which turn to fluorescent red inside cells, researchers could observe the genetic changes, upto 60 percent of stem cells in skeletal muscles, 38 percent in bone marrow and 27 percent of progenitor cells.

Sharif Tabebordbar from Broad Institute, Massachusetts who is a member of the team said that till now delivering genes to stem cells using AAV was not possible as the cells get divided very fast in living bodies. But the team was successful in modifying the genome of the stem cells within the body itself.

However, the job is not yet done as this has to be successfully demonstrated in the human bodies but the combination of AAV and CRISPR has turned out to be quite promising for tackling various problems.