Beyond CRISPR: A guide to the many other ways to edit a genome

The popular technique has limitations that have sparked searches for alternatives.

Argonaute proteins (model pictured) are one of many potential alternatives to the CRISPR–Cas9 gene-editing system.

The CRISPR–Cas9 tool enables scientists to alter genomes practically at will. Hailed as dramatically easier, cheaper and more versatile than previous technologies, it has blazed through labs around the world, finding new applications in medicine and basic research.

But for all the devotion, CRISPR–Cas9 has its limitations. It is excellent at going to a particular location on the genome and cutting there, says bioengineer Prashant Mali at the University of California, San Diego. “But sometimes your application of interest demands a bit more.”

The zeal with which researchers jumped on a possible new gene-editing system called NgAgoearlier this year reveals an undercurrent of frustration with CRISPR–Cas9 — and a drive to find alternatives. “It’s a reminder of how fragile every new technology is,” says George Church, a geneticist at Harvard Medical School in Boston, Massachusetts.

NgAgo is just one of a growing library of gene-editing tools. Some are variations on the CRISPR theme; others offer new ways to edit genomes.

A mini-me

CRISPR–Cas9 may one day be used to rewrite the genes responsible for genetic diseases. But the components of the system — an enzyme called Cas9 and a strand of RNA to direct the enzyme to the desired sequence — are too large to stuff into the genome of the virus most commonly used in gene therapy to shuttle foreign genetic material into human cells.

A solution comes in the form of a mini-Cas9, which was plucked from the bacterium Staphylococcus aureus1. It’s small enough to squeeze into the virus used in one of the gene therapies currently on the market. Last December, two groups used the mini-me Cas9 in mice to correct the gene responsible for Duchenne muscular dystrophy2, 3.

Expanded reach

Cas9 will not cut everywhere it’s directed to — a certain DNA sequence must be nearby for that to happen. This demand is easily met in many genomes, but can be a painful limitation for some experiments. Researchers are looking to microbes to supply enzymes that have different sequence requirements so that they can expand the number of sequences they can modify.

One such enzyme, called Cpf1, may become an attractive alternative. Smaller than Cas9, it has different sequence requirements and is highly specific4, 5.

Another enzyme, called C2c2, targets RNA rather than DNA — a feature that holds potential for studying RNA and combating viruses with RNA genomes6.

True editors

Many labs use CRISPR–Cas9 only to delete sections in a gene, thereby abolishing its function. “People want to declare victory like that’s editing,” says Church. “But burning a page of the book is not editing the book.”

Those who want to swap one sequence with another face a more difficult task. When Cas9 cuts DNA, the cell often makes mistakes as it stitches together the broken ends. This creates the deletions that many researchers desire.

But researchers who want to rewrite a DNA sequence rely on a different repair pathway that can insert a new sequence — a process that occurs at a much lower frequency than the error-prone stitching. “Everyone says the future is editing many genes at a time, and I think: ‘We can’t even do one now with reasonable efficiency’,” says plant scientist Daniel Voytas of the University of Minnesota in Saint Paul.

But developments in the past few months have given Voytas hope. In April, researchers announced that they had disabled Cas9 and tethered to it an enzyme that converts one DNA letter to another. The disabled Cas9 still targeted the sequence dictated by its guide RNA, but could not cut: instead the attached enzyme switched the DNA letters, ultimately yielding a T where once there was a C7. A paper published in Science last week reports similar results8.

Voytas and others are hopeful that tethering other enzymes to the disabled Cas9 will allow different sequence changes.

Pursuing Argonautes

In May, a paper in Nature Biotechnology9 unveiled an entirely new gene-editing system. Researchers claimed that they could use a protein called NgAgo to slice DNA at a predetermined site without needing a guide RNA or a specific neighbouring genome sequence. Instead, the protein — which is made by a bacterium — is programmed using a short DNA sequence that corresponds to the target area.

The finding kicked off a wave of excitement and speculation that CRISPR–Cas9 would be unseated, but laboratories have so far failed to reproduce the results. Even so, there is still hope that proteins from the family that NgAgo belongs to — known as Ago or Argonautes — made by other bacteria could provide a way forward, says genome engineer Jin-Soo Kim at the Institute for Basic Science in Seoul.

Programming enzymes

Other gene-editing systems are also in the pipeline, although some have lingered there for years. For an extensive project that aimed to edit genes in bacteria, Church’s lab did not reach for CRISPR at all. Instead, the team relied heavily on a system called lambda Red, which can be programmed to alter DNA sequences without the need for a guide RNA. But despite 13 years of study in Church’s lab, lambda Red works only in bacteria.

Church and Feng Zhang, a bioengineer at the Broad Institute of MIT and Harvard in Cambridge, Massachusetts, say that their labs are also working on developing enzymes called integrases and recombinases for use as gene editors. “By exploring the diversity of enzymes, we can make the genome-editing toolbox even more powerful,” says Zhang. “We have to continue to explore the unknown.”



One of developmental biology’s most perplexing questions concerns what signals transform masses of undifferentiated cells into tremendously complex organisms, a process called ontogeny.

New research by University at Buffalo scientists, published last week in PLOS ONE, provides evidence that it all begins with a single “master” growth factor receptor that regulates the entire genome.

“The finding provides a new level of understanding of the fundamental aspects of how organisms develop,” says senior author Michal K. Stachowiak, PhD, professor in the Department of Pathology and Anatomical Sciences in the UB School of Medicine and Biomedical Sciences and senior author. He also directs the Stem Cell Engraftment and In Vivo Analysis Facility and the Stem Cell Culture and Training Facility at the Western New York Stem Cell Culture and Analysis Center at UB.

“Our research shows how a single growth factor receptor protein moves directly to the nucleus in order to program the entire genome,” he said.

The research challenges a long-held supposition in biology that specific types of growth factors only functioned at a cell’s surface. For two decades, Stachowiak’s team has been intrigued by the possibility that growth factors function from within the nucleus, a point, he says, this current paper finally proves.

A more advanced understanding of how organisms form, based on this work, has the potential to significantly enhance the understanding and treatment of cancers, which result from uncontrolled development as well as congenital diseases, the researchers say. The new research also will contribute to the understanding of how stem cells work.

This work was conducted on mouse embryonic stem cells, not human cells.

Organizing ‘this cacophony of genes’

“We’ve known that the human body has almost 30,000 genes that must be controlled by thousands of transcription factors that bind to those genes,” Stachowiak said, “yet we didn’t understand how the activities of genes were coordinated so that they properly develop into an organism.

“Now we think we have discovered what may be the most important player, which organizes this cacophony of genes into a symphony of biological development with logical pathways and circuits,” he said.

At the center of the discovery is a single protein called nuclear Fibroblast Growth Factor Receptor 1 (nFGFR1). “FGFR1 occupies a position at the top of the gene hierarchy that directs the development of multicellular animals,” said Stachowiak.

The FGFR1 gene is known to govern gastrulation, occurring in early development, where the three-layered embryonic structure forms. It also plays a major role in the development of the central and peripheral nervous systems and the development of the body’s major systems, including muscles and bones.

To study how nuclear FGFR1 worked, the UB team used genome-wide sequencing of mouse embryonic stem cells programmed to develop cells of the nervous system, with additional experiments in which nuclear FGFR1 was either introduced or blocked. The researchers found that the protein was responsible, either alone or with so-called partner nuclear receptors, for ensuring that embryonic stem cells develop into differentiated cells. By targeting thousands of genes, it controls the development of the major points of growth in the body (known as axes) as well as neuronal and muscle development.

The research shows that nuclear FGFR1 binds to promoters of genes that encode transcription factors, the proteins that control which genes are turned on or off in the genome.

“We found that this protein works as a kind of ‘orchestration factor,’ preferably targeting certain gene promoters and enhancers. The idea that a single protein could bind thousands of genes and then organize them into a hierarchy, that was unknown,” Stachowiak said. “Nobody predicted it.”

Sequencing advances

The discovery that a single protein can exert such a global genomic function stems from recent advances in DNA sequencing technologies, which allow for the sequencing of a complex genome in just hours.

“NextGen DNA sequencing allows us to analyze millions of DNA sequences selected by the interacting protein,” Stachowiak said.

In the UB research, the DNA sequencing data were processed by the supercomputer at the university’s Center for Computational Research (CCR). Stachowiak and his colleagues then spent weeks aligning these data to the genome and conducting further analyses.

“We imposed nuclear FGFR1 on every little corner of genome,” he said. “The computer spit out which genes are affected by nuclear FGFR1: it was an enormously complex network of genome activity.”

They found that the protein binds to genes that make neurons and muscles as well as to an important oncogene, TP53, which is involved in a number of common cancers.

Other studies in Stachowiak’s laboratory demonstrate that these interactions also take place in the human genome, controlling function and possibly underlying diseases like schizophrenia. Targeting of the nuclear FGFR1 allows for the reactivation of neural development in the adult brain in preclinical studies and thus, Stachowiak says, may offer unprecedented opportunity for regenerative medicine. Nuclear accumulation of nuclear FGFR1 may be altered in some cancer cells, and thus could become a focus in cancer therapy, he added.

Stachowiak concluded: “This seminal discovery lends new perspectives to the origin, nature and treatment of a variety of human diseases.”

Too Much Information? Geneticist Mark Robson Discusses Accidental Genetic Findings.

Genetic testing of tumors is becoming increasingly common in cancer care. The molecular alterations found in a tumor can provide critical information for making an accurate diagnosis and determining the best treatment.

Although current clinical testing usually focuses on a panel of specific mutations, cancer centers are developing programs to analyze entire cancer genomes routinely — an approach made possible by cheaper sequencing costs — in order to individualize care. This process raises a thorny issue: What happens when a genome analysis of a person’s tumor reveals that he or she is at risk for developing a different type of cancer or other disease?

Recently, Memorial Sloan-Kettering Clinical Genetics Service Chief Kenneth Offit, Clinical Genetics Service Clinic Director Mark E. Robson, and researcher Yvonne Bombardpublished a viewpoint in the Journal of the American Medical Association regarding this question of incidental genetic findings, which cancer researchers have dubbed the “incidentalome.”

We asked Dr. Robson to discuss some of the issues surrounding accidental genetic findings and what Memorial Sloan-Kettering is doing to address them.

What is an example of a genetic variation that might be discovered by accident while sequencing the genome of a patient’s tumor?

For instance, you could be sequencing a lung cancer tumor in search of an EGFR mutation to target with an anticancer drug, and find a mutation in BRCA1, which is associated with increased risk for breast and ovarian cancer. Since most of a tumor’s DNA sequence is identical to the sequence of a normal cell from that same patient, this additional variation is probably inherited — and is what is called a germline mutation.

In that situation, are you obligated to inform the patient? It’s a very complex question. There are many variables to consider, such as individual preference, whether anything can be done to control risk, and whether other people — such as close relatives — may be affected.

Has this actually become a problem for doctors and researchers, or is it still a hypothetical situation for now?

Right now, most clinical testing of tumors is for a relatively limited number of specific mutations, not the full genome. But soon we’re going to be testing for a much broader panel of genes, increasing the chances of incidental findings.

On the research side, it’s quickly becoming an issue. Many tumor samples that have been stored in tissue banks for years or decades are now being fully sequenced. If incidental discoveries are made during that process, is there an obligation to try to find those patients and inform them? This has not been established, and there are obvious practical barriers. We need to lay the intellectual groundwork now for how we’re going to respond to these questions.

What steps have been taken at Memorial Sloan-Kettering to address the issue?

This summer, our Institutional Review Board (IRB), which oversees all of our patient-related research, updated part of our patient consent policy. When patients agree to have a tissue sample taken, they are asked whether they are open to being re-contacted if an investigator finds something that might affect their health.

Under the new procedure, if a researcher finds something that might be important to communicate to the patient, the specific question will be put before the IRB and carefully considered. If there is agreement the information should be conveyed, and the patient has indicated that he or she wants to be re-contacted, we’ll reach out to that person. We think this protects the people participating in our studies without restricting important research.

With all the genetic research taking place at Memorial Sloan-Kettering, is the IRB facing a deluge of these cases?

So far, no. The way the analyses are being conducted is that the computer looks for mutations in specific spots and subtracts all other information about the inherited genetic sequence before the investigator sees it. In other words, if you have genetic variants present in the tumor that are also in the normal cells, they are being filtered out by the software. The investigator ends up seeing variants that are only in the tumor.

As we pointed out in the JAMA paper, this is one way of limiting potential incidentalome issues.

But some researchers don’t have the germline DNA sequence available for comparison purposes, so while sequencing the tumor they see potentially relevant variations. For example, they could be sequencing a prostate cancer genome and see a mutation in theBRCA1 gene, which increases risk of other cancers.

The question becomes, under what circumstances do you tell the patient, and what about the patient’s siblings or children who may carry the mutation as well? In addition, sometimes multiple variants associated with disease risk may be found — and how do we provide counseling for all of them at once?

Have you gotten a sense from patients about what their preference usually is regarding being informed of these incidental genetic discoveries?

Commonly, people say, “I want to know everything,” but the devil’s in the details when you start considering the risk for diseases that can’t be prevented or treated. We are setting up focus groups of patients and unaffected people to try to understand how people think when they are confronted with these situations and how they prioritize different types of genetic information. We also have an active IRB protocol in which we are giving people who had their sequence determined as part of research studies the opportunity to learn their results.

Right now, it’s not clear what the dividing lines are. We want to reach a point where mutations are sorted into different categories, where certain incidental findings are nearly always appropriate to communicate to patients, others almost never so, and some require more context to determine.

We’re moving from the traditional model of asking patients if they would like to hear the results of a specific test before that test is performed, to this brave new world where we’re trying to help people make decisions about genetic information revealed by accident that is not possible to fully anticipate. It’s a very complicated issue, but it also offers a tremendous opportunity to benefit patients.

If you are interested in participating in the focus group, call 646-888-4867. Everyone is welcome, including patients, relatives, Memorial Sloan-Kettering employees, and the general public. No sequencing is provided.

Source: MSKCC




Faces are sculpted by ‘junk DNA’

Scientists have identified thousands of regions in the genome that control the activity of genes for facial features.

Smiling child

‘Transcriptional enhancers‘ switch genes on or off in different parts of the face. Photograph: Rex Features

Researchers have started to figure out how DNA fine-tunes faces. In experiments on mice, they have identified thousands of regions in the genome that act like dimmer switches for the many genes that code for facial features, such as the shape of the skull or size of the nose.

Specific mutations in genes are already known to cause conditions such as cleft lips or palates. But in the latest study, a team of researchers led by Axel Visel of the Lawrence Berkeley National Laboratory in Berkeley, California, wanted to find out how variations seen across the normal range of faces are controlled.

Though everybody’s face is unique, the actual differences are relatively subtle. What distinguishes us is the exact size and position of things like the nose, forehead or lips. Scientists know that our DNA contains instructions on how to build our faces, but until now they have not known exactly how it accomplishes this.

Visel’s team was particularly interested in the portion of the genome that does not encode for proteins – until recently nicknamed “junk” DNA – but which comprises around 98% of our genomes. In experiments using embryonic tissue from mice, where the structures that make up the face are in active development, Visel’s team identified more than 4,300 regions of the genome that regulate the behaviour of the specific genes that code for facial features.

The results of the analysis are published on Thursday in Science.

These “transcriptional enhancers” tweak the function of hundreds of genes involved in building a face. Some of them switch genes on or off in different parts of the face, others work together to create, for example, the different proportions of a skull, the length of the nose or how much bone there is around the eyes.

“If you think about face development, a gene that is important for both development of the nose and the mouth might have two different enhancers and one of them activates the gene in the nose and the other just in the mouth,” said Visel.

“Certainly, one evolutionary advantage that is associated with this is that you can now change the sequence of the nose or mouth enhancers and, independently, affect the activity of the gene in just one structure or the other. It may be a way a way that nature has evolved in which you can fine-tune the expression of genes in complex ways without having to mess with the gene itself. If you destroy the protein itself that usually has much more severe consequences.”

In further experiments to test their findings, the scientists genetically engineered mice to lack three of the enhancers they had identified. They then used CT (computed tomography) scanning to build 3D images of the resulting mouse skulls at the age of eight weeks.

Compared with normal mice, the skulls of the modified mice had microscopic, but consistent, changes in the length and width of the faces, as expected. Importantly, all of the modified mice only showed subtle changes in their faces, and there were no serious harmful results such as cleft lips or palates.

Though the work was done in mice, Visel said that the lessons transfer across to humans very well. “When you look at the anatomy and development of the mouse versus the human, we find that the faces are actually very similar. Both are mammals and they have, essentially, all the same major bones and structures in their skulls, they just have a somewhat different shape in the mouse. The same genes that are important for mouse face development are important in humans.”

Visel said that the primary use of this information, beyond basic genetic knowledge, would be as part of a diagnostic tool, for clinicians who might be able to advise parents if they are likely to pass on particular mutations to their children.

Peter Hammond, a professor of computational biology at University College London‘s Institute of Child Health, who researches genetic effects on facial development, said understanding how faces develop can be important for health.

“There are many genetic conditions where the face is a first clue to diagnosis, and even though the facial differences are not necessarily severe the condition may involve significant intellectual impairment or adverse behavioural traits, as well as many other effects,” he said. “Diagnosis is important for parents as it reduces the stress of not knowing what is wrong, but also can be important for prognosis.”

The technology to go beyond diagnosis and make precise corrections of the genome does not yet exist and, even if it did, it is not clear that changing genes or enhancers to create “designer” faces would be worthwhile. “I don’t think it would be desirable to even attempt that. It’s certainly not something that motivates me to work on this,” said Visel. “And I don’t think anyone working in this field would seriously view this as a possible motivation.”

Researchers identify key proteins that help establish cell function

Researchers at the University of California, San Diego School of Medicine have developed a new way to parse and understand how special proteins called “master regulators” read the genome, and consequently turn genes on and off.

Writing in the October 13, 2013 Advance Online Publication of Nature, the scientists say their approach could make it quicker and easier to identify specific gene associated with increased – an essential step toward developing future targeted treatments, preventions and cures for conditions ranging from diabetes to neurodegenerative disease.

“Given the emerging ability to sequence the genomes of individual patients, a major goal is to be able to interpret that DNA sequence with respect to disease risk. What diseases is a person genetically predisposed to?” said principal investigator Christopher Glass, MD, PhD, a professor in the departments of Medicine and Cellular and Molecular Medicine at UC San Diego.

“Mutations that occur in protein-coding regions of the genome are relatively straight forward, but most mutations associated with disease risk actually occur in regions of the genome that do not code for proteins,” said Glass. “A central challenge has been developing a strategy that assesses the potential functional impact of these non-coding mutations. This paper lays the foundation for doing so by examining how natural genetic variation alters the function of genomic regions controlling gene expression in a cell specific-manner.”

Cells use hundreds of different proteins called transcription factors to “read” the genome, employing those instructions to turn genes on and off. These factors tend to be bound close together on the genome, forming functional units called “enhancers.” Glass and colleagues hypothesized that while each cell has tens of thousands of enhancers consisting of myriad combinations of factors, most enhancers are established by just a handful of special transcription factors called “master regulators.” These master regulators play crucial, even disproportional, roles in defining each cell’s identity and function, such as whether it will be a muscle, skin or heart cell.

“Our main idea was that the binding of these master regulators is necessary for the co-binding of the other transcription factors that together enable enhancers to regulate the expression of nearby genes,” Glass said.

The scientists tested and validated their hypothesis by looking at the effects of approximately 4 million DNA sequence differences affecting master regulators in macrophage cells in two strains of mice. Macrophages are a type of immune response cell. They found that DNA sequence mutations deciphered by master regulators not only affected how they bound to the genome, but also impacted neighboring needed to make functional .

The findings have practical importance for scientists and doctors investigating the genetic underpinnings of disease, said Glass. “Without actual knowledge of where the master regulator binds, there is relatively little predictive value of the DNA sequence for non-coding variants. Our work shows that by collecting a focused set of data for the master regulators of a particular cell type, one can greatly reduce the ‘search space’ of the in a particular cell type that would be susceptible to the effects of mutations. This allows prioritization of mutations for subsequent analysis, which can lead to new discoveries and real-world benefits.”

Source:  University of California – San Diego

Scientist who mapped human genome says we will be able to ‘print’ alien life from Mars

J. Craig Venter says the next revolution in genetics will come from synthetic biology, as we learn to design and ‘print’ organisms with computers.

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Scientists will soon be able to design and print simple organisms using biological 3D printers says J. Craig Venter, the scientist who led the private-sector’s mapping of the human genome.

Venter predicts that new methods of digital design and manufacture will provide the next revolution in genetic with synthetic cells and organism tailor-made to tackle humanity’s problems: a toolkit of sequenced genes will be used to create disease-resistant animals; higher yielding crops; and drugs that extend human life and boost our brain power.

These ideas have been outlined in Venter’s latest book ‘Life at the Speed of Light: From the Double Helix to the Dawn of Digital Life’, in which the geneticists asks the age-old question ‘what is life?’ before detailing the history – and future – of creating the stuff from scratch.

For Venter life can be reduced to “protein robots” and “DNA machines” but he also believes that technology will unlock far more exotic opportunities for creating life. The title of the publication refers to the idea that we may be able to transmit DNA sequences found on Mars back to Earth (at the speed of light) to be replicated at home by biological printers.

“I am confident that life once thrived on Mars and may well still exist there today,” writes Venter. “The day is not far off when we will be able to send a robotically controlled genome-sequencing unit in a probe to other planets to read the DNA sequence of any alien microbe life that may be there.”

Venter’s ideas may sound like science fiction but he has achieved comparable feats in the past. Frustrated by what he viewed as slow government-led efforts to sequence the human genome in the 90s, Venter raised private capital to create a rival effort under the company name of Celera

Fears that Venter and his backers would attempt to patent the genome spurred the US-led effort into action and global genes-race was sparked, with both sides eventually agreeing to announce their result one day apart in February 2001.

Venter parted ways with Celera in 2002 and founded the J.Craig Venter institute in 2006. In 2010 he and his colleagues at the institute announced that they had created the world’s first synthetic organism.

The team creating a bacterium genome from scratch and ‘watermarked’ it with custom DNA strings (these included an encoded email address) before transplanting it into another cell. The cell then began to reproduce, making it the first living species created by humanity.

Although such pioneering work frequently raises ethical questions over the danger of humanity ‘playing God’, Venter writes that he is not concerned with such concerns. In ‘Life at the Speed of Light’ he writes: “My greatest fear is not the abuse of technology but that we will not use it at all.”

The ENCODE Project: ENCyclopedia Of DNA Elements.

ENCODE Overview

The National Human Genome Research Institute (NHGRI) launched a public research consortium named ENCODE, the Encyclopedia Of DNA Elements, in September 2003, to carry out a project to identify all functional elements in the human genome sequence. The project started with two components – a pilot phase and a technology development phase.

The pilot phase tested and compared existing methods to rigorously analyze a defined portion of the human genome sequence (See: ENCODE Pilot Project). The conclusions from this pilot project were published in June 2007 in Nature and Genome Research []. The findings highlighted the success of the project to identify and characterize functional elements in the human genome. The technology development phase also has been a success with the promotion of several new technologies to generate high throughput data on functional elements.

With the success of the initial phases of the ENCODE Project, NHGRI funded new awards in September 2007 to scale the ENCODE Project to a production phase on the entire genome along with additional pilot-scale studies. Like the pilot project, the ENCODE production effort is organized as an open consortium and includes investigators with diverse backgrounds and expertise in the production and analysis of data (See: ENCODE Participants and Projects). This production phase also includes a Data Coordination Center [] to track, store and display ENCODE data along with a Data Analysis Center to assist in integrated analyses of the data. All data generated by ENCODE participants will be rapidly released into public databases (See: Accessing ENCODE Data) and available through the project’s Data Coordination Center.