Within only a few years, research labs worldwide have adopted a new technology referred to as “CRISPR,”that facilitates making specific changes in the DNA of humans, other animals, and plants. Compared to previous techniques for modifying DNA, this new approach is much faster and easier. CRISPR allows removing a single (defective) gene from a genome and replacing it with another one, to prevent genetic diseases.
Scientists have learned how to harness CRISPR technology in the lab to make precise changes in the genes of organisms as diverse as fruit flies, fish, mice, plants and even human cells. Researchers are using crispr to knock out genes in animal models to study their function, give crops new agronomic traits, synthesize microbes that produce drugs, create gene therapies to treat disease, and to genetically correct heritable diseases in human embryos.
Jennifer Doudna, one of the pioneers of the gene-editing technique known as CRISPR, thinks the biotech tool could be an essential one for combating COVID-19 and future pandemics. Due to its capacity to be “reprogrammed” like software, CRISPR could eventually be integral to countless tests and treatments. In an interview at Disrupt 2020, Doudna was all optimism for the technique, which has already proven to be extremely useful in less immediately applicable situations.
US and China are leaders in applications of CRISPER technology. During the second biennial Department of Defense Lab Day May 18, 2017, One AFRL exhibit, called Military Applications of Gene Editing Technology, highlighted research into how geneticists and medical researchers edit parts of the genome by removing, adding or altering sections of the DNA sequence in order to remove a virus or disease caused by harmful chemical, biological or environmental agents a warfighter may have contact with.
In 2016, a Chinese group has become the first to inject a person with cells that contain genes edited using the revolutionary CRISPR–Cas9 technique. Earlier Scientists of Chinese Kunming Biomedical International and its affiliated Yunnan Key Laboratory of Primate Biomedical Research used CRISPR to create a pair of macaque monkeys with precise genetic mutations. Chinese scientists say they were among the first in using Crispr to make wheat resistant to a common fungal disease, dogs more muscular and pigs with leaner meat.
A recent breakthrough in CRISPR technology has paved the way for editing entire gene networks in a single step. While this discovery will likely shorten the timeframes required for finding cures for deadly illnesses, it can also bring us closer to threats of bioterrorism. Scientists at ETH Zurich recently published a new CRISPR technique in Nature Methods that removes one of the most significant limitations of the technology. Prior to this discovery, the process could only target a single gene for editing. The ETH scientists now managed to target 25 at once and believe that, theoretically, this method could target hundreds.
Genome editing means CRISPR to most people. Yet methods using zinc-finger nucleases, transcription activator-like effector nucleases (TALENs), and meganucleases have their own unique strengths. All of these techniques rely on cellular DNA–repair mechanisms. Options that don’t—base editing, epigenetic editing, and site-specific recombinases—offer further advantages.
“Genome editing is a juxtaposition of two discoveries,” explained panelist Philip Gregory from the gene and cell therapy company Bluebird Bio: Nucleases can make double-stranded DNA breaks (DSBs) at specific sequences, and DSBs activate repairs that can change DNA. DSB repair has two mechanisms. Nonhomologous end joining (NHEJ) links ends together, often creating insertions and deletions (indels) in the process. In genome editing, this can be used to knock out gene function. Homology-directed repair (HDR) fixes DSBs using DNA with a similar sequence. Providing cells with external homologous donor DNA introduces edits via HDR.
Many genome-editing systems work by activating DSB repair at specific sites using engineered zinc-finger nucleases (ZFNs), transcription activator-like effector-based nucleases (TALENs), or meganucleases (1). Currently, the dominant genome-editing method is CRISPR-Cas9 (clustered regularly interspaced short palindromic repeats-CRISPR-associated protein 9) (2). How do researchers choose among these systems?
“The primary consideration is the end product,” says Jon Hennebold, Oregon Health & Science University in Portland. Hennebold leads a multisite U.S. National Institutes of Health–funded program on genome-editing efficiency and safety. Companies use proprietary genome-editing systems optimized for specificity to reduce off-target effects (mutations at unintended sites). Most academic labs can get the product they want with CRISPR, which is fast and easy. “You can order the components and get started in 24 to 48 hours,” Hennebold says, “Other methods don’t have that commercial support.”
Academic labs have no reason to work with other methods,” says Charles Gersbach, a biomedical engineer at Duke University in Durham, North Carolina. “For plain-vanilla genome editing, Cas9 and a gRNA will get the job done.” Cas9, an enzyme from bacterial antiviral systems, makes DSBs at DNA sites that are complementary to a guide RNA (gRNA) and also have a nearby protospacer-adjacent motif (PAM) sequence. CRISPR repeats aren’t needed for editing, so Cas9 plus a gRNA can knock out genes by NHEJ. Providing a DNA fragment promotes HDR-mediated edits.
Ru Gunawardane, director of Stem Cells and Gene Editing at Seattle’s Allen Institute for Cell Science and an LSINW panelist, says CRISPR has been “a game changer” in fulfilling the institute’s mission of understanding how cells act in normal, disease, and treatment conditions. Researchers at the institute use CRISPR to tag organelle markers in stem cells with fluorescent proteins, then track these fusion proteins and their interactions under different situations. Currently, their work includes differentiating tagged cells into cardiomyocytes.
Caixia Gao, a plant biologist at the Chinese Academy of Sciences in Beijing, says CRISPR is also common in her field. “All methods are very efficient at making site-specific mutations,” she says, “but CRISPR takes the least time and has the lowest costs.”
CRISPER gene editing technology
CRISPR is an acronym for Clustered Regularly Interspaced Short Palindromic Repeat. This name refers to the unique organization of short, partially palindromic repeated DNA sequences found in the genomes of bacteria and other microorganisms. If a viral infection threatens a bacterial cell, the CRISPR immune system can thwart the attack by destroying the genome of the invading virus. The genome of the virus includes genetic material that is necessary for the virus to continue replicating. Thus, by destroying the viral genome, the CRISPR immune system protects bacteria from ongoing viral infection.
CRISPR RNA sequences are copied from the viral DNA sequences which then guide bacterial molecular machinery to destroy the viral material. Because CRISPR RNA sequences are copied from the viral DNA sequences acquired during adaptation, they are exact matches to the viral genome and thus serve as excellent guides.
Rather than relying on bacteria to generate CRISPR RNAs, scientists first design and synthesize short RNA molecules that match a specific DNA sequence—for example, in a human cell. Then, like in the targeting step of the bacterial system, this ‘guide RNA’ shuttles molecular machinery to the intended DNA target. Once localized to the DNA region of interest, the molecular machinery can silence a gene or even change the sequence of a gene.
The advantages CRISPR offers are threefold, Doudna explained: first, it’s a “direct” method of detection. Current tests rely on enzymes and proteins that are indirect evidence of infection, which limits their reliability and timing — you can’t, for instance, detect the virus before it starts producing that secondary evidence. CRISPR detects RNA from the viral genome itself. “We’re finding in the laboratory that that means that you can get a signal faster, and you can also get a signal that is more directly correlated to the level of the virus,” she said.
Second, the sequence that the CRISPR complex searches for can easily be changed. “That means that scientists can reprogram the CRISPR system trivially, to target different sections of the coronavirus to make sure that we’re not missing viruses that have mutated,” Doudna said. “We’re already working on a strategy to co-detect influenza and coronavirus; as you know, it’s really important to be able to do that, but also to pivot very quickly to detect new viruses that are emerging.” “I don’t think any of us thinks that viral pandemics are going away,” she continued. “The current pandemic is a call to arms; we have to make sure that scientifically we’re ready for the next attack by a new virus.” And third, a CRISPR-based test wouldn’t be manufactured with the same materials as other tests, making it easier to manufacture alongside them. Managing supply chains effectively will be crucial for getting vaccines, tests and treatments to people as quickly as possible.
CRISPR “has transformed labs around the world,” says Jing-Ruey Joanna Yeh, a chemical biologist at Massachusetts General Hospital’s Cardiovascular Research Center, in Charlestown, who contributed to the development of the technology. “Because this system is so simple and efficient, any lab can do it.” Editing with CRISPR is like placing a cursor between two letters in a word processing document and hitting “delete” or clicking “paste.” And the tool can cost less than US $50 to assemble.
CCR5 is a coreceptor of HIV, via which the virus targets CD4 T cells to suppress their immune response. It has already been demonstrated that CCR5 mutations that lead to nonfunctional proteins could make cells resistant to HIV. Chinese scientist Hongkui Deng and coworkers have started to use the CRISPR-Cas9 system to render the CCR5 gene nonfunctional in stem cells and they have transplanted these stem cells into an HIV patient diagnosed two years prior. Recently, they published a case report in the New England Journal of Medicine revealing that although the patient’s HIV infection was not cured, no unintended genetic alterations were detected, indicating that the therapy is likely safe.
The work Hongkui Deng and his team have done is ethically different from what Jiankui He did last year. Of note, Jiankui He used CRISPR to edit the CCR5 gene in human embryos, attempting to make unborn twins immune to HIV. However, any off-target genome edits could have lead to detrimental effects in the babies. This is in stark contrast to Deng’s study using CRISPR to edit CCR5 in adult stem cells that were then transplanted into a patient, as the CCR5 gene would remain functional in non-blood cells.
The barrier to CRISPR, however, is not theoretical but practical: It’s still more or less lab-bound because therapies using the technology are still very much under review. It is in clinical trials in some forms and COVID-19-related applications could be fast-tracked, but its novelty means it will be slower to reach those who need it. Not to mention the cost.
“This underscores what I think is one of the key challenges that we face in this age of advancing biotechnologies,” said Doudna. “That is, how do we make a technology like CRISPR affordable and accessible to a lot of people? I’d like to see a day when CRISPR is the standard of care for treating a rare genetic disease, and it’s going to take some real R&D to get there.”
Perhaps one of the avenues for advancement will be the newly discovered sibling technique, CRISPR Cas-Φ (that’s a “phi”), which works similarly but is much more compact, owing to its origin as, apparently, a countermeasure by viruses that invade CRISPR-bearing bacteria. “Who knew they carried around their own form of CRISPR?” mused Doudna. “But they do, and it’s a very interesting protein, because it’s very small compared to the original formats for CRISPR that allows a much, much smaller protein to be able to do [this] kind of editing.”
CRISPER is not perfect
But CRISPR is not perfect. Base editors (think of them as gene-editing pencils) can rewrite individual DNA letters. They home in on specific areas of DNA and swap out certain bases — A, C, T, or G — for others. But after the swap, base editors—like the cytosine base editor that converts C•G to T•A — perform unwanted off-target edits. Until now, even the best CRISPR tool, SpCas9, could only bind to about one in 16 locations along DNA, leaving many genetic mutations out of reach. “That type of off-target editing can occur at random locations in the genome,” said Liu. “When you run the experiment 10 times, you get 10 different answers. That makes it so challenging to study.”
Now, in two papers published in Nature Biotechnology, researchers at Harvard University, the Broad Institute, and the Howard Hughes Medical Institute have invented new CRISPR tools that address both issues. The first paper describes newly designed cytosine base editors that reduce an elusive type of off-target editing by 10- to 100-fold, making new variants that are especially promising for treating human disease. The second describes a new generation of all-star CRISPR-Cas9 proteins the team evolved that are capable of targeting a much larger fraction of pathogenic mutations, including the one responsible for sickle cell anemia, which was prohibitively difficult to access with previous CRISPR methods.
“Since the era of human genome editing is in its fragile beginnings, it’s important that we do everything we can to minimize the risk of any adverse effects when we start to introduce these into people,” said David Liu, the lead author on the papers. “Minimizing this kind of elusive off-target editing is an important step toward achieving that goal.”
New CRISPR Methods
Scientists at ETH Zurich recently published a new CRISPR technique in Nature Methods that removes one of the most significant limitations of the technology. Prior to this discovery, the process could only target a single gene for editing. The ETH scientists now managed to target 25 at once and believe that, theoretically, this method could target hundreds. Here’s how they describe the process:
[W]e demonstrate that both Cas12a and a clustered regularly interspaced short palindromic repeat (CRISPR) array can be encoded in a single transcript by adding a stabilizer tertiary RNA structure. By leveraging this system, we illustrate constitutive, conditional, inducible, orthogonal and multiplexed genome engineering of endogenous targets using up to 25 individual CRISPR RNAs delivered on a single plasmid. Our method provides a powerful platform to investigate and orchestrate the sophisticated genetic programs underlying complex cell behaviors.
While the method only increases CRISPR’s efficiency, time plays a significant role in genetic editing. Complex genetic conditions occur through the interaction of genes in a cell. Targeting each gene individually to test a different configuration takes a long time and that process can require significant repetition in order to discover the desired genetic variation. Reducing the time required to produce each variant makes the discovery process significantly more efficient. That may lead to finding important gene therapies that can cure morbid conditions with daily death tolls. With antibiotic resistance on the rise, many expect CRISPR will offer a viable alternative to a problem the Center for Disease Control (CDC) calls “one of the most urgent threats to the public’s health.”
Researchers make record-breaking 13,000 edits in a single cell by CRISPR:
A study led by George Church from MIT succeeded in making 13,000 edits by CRISPR in a single cell, which is approximately 2,000-fold higher than that achieved by the same group collaborating with Luhan Yang’s group in 2015, wherein they mutated every single PERV gene in a porcine primary cell line. PERVs are viruses that incorporate their genetic materials into pig genomes. In this new study, the researchers designed a set of dead-Cas9 base editor (dBEs) variants that enable the editing of thousands of loci in each cell without causing cell death (too much DNA cleavage always leads to cell death). Their dBEs allow up to 13,200 cleavages in 293T cells.
Prime editing makes CRISPR more powerful and precise:
Recently, researchers from the Liu Lab have developed a new CRISPR editing system called prime editing that enables the deletion and insertion of specific sequences, as well as correction of any type of point mutations in cellular DNA. The prime editor complex is composed of an engineered Cas9 that creates a single nick on one DNA strand, a prime editing guide RNA (or pegRNA, which is comprised of “guide sequences” that guide the whole complex to the target and the edited sequence that is designed to replace the old target sequence), and a reverse transcriptase than can make new DNA by reverse transcription of the edited sequence of pegRNA. Besides its ability to insert and delete specific sequences, prime editing also has enhanced efficiency and accuracy compared to the other engineered Cas9 variants. This study also indicates that prime editing has the potential to correct roughly 89% of human pathogenic variants, which increase susceptibility or predisposition to a certain disease or disorder.
CRISPR-Chip enables highly sensitive digital detection of DNA
Scientists are now turning the CRISPR technique into a Swiss Army knife by exploring its application in various fields other than gene editing. Recently, a study conducted by researchers from the Keck Graduate Institute and the University of California, Berkeley, used CRISPR-Cas9 and a graphene chip to detect DNA. By combining the two, the conventional amplification step is no longer necessary to detect DNA due to CRISPR’s genome-searching capability and graphene’s high sensitivity. According to their paper published in Nature Biomedical Engineering, CRISPR complexes were immobilized on graphene-based transistors.
CRISPR-Cas13 is programmed to kill and detect RNA viruses in human cells:
The CRISPR family has been expanding rapidly since the discovery of its ability to edit the human genome. Researchers at Zhang Lab originally revealed that Cas13a could bind and cleave RNA in human cells, which makes it a potential diagnostic tool to detect viruses, bacteria, or other targets.
Recently, scientists from Harvard and MIT’s Broad Institute explored CAS13’s ability to detect and kill RNA viruses, many of which are detrimental and incurable human pathogens such as Ebola and Zika. The researchers combined Cas13’s antiviral activity with its diagnostic capability to create a system called Cas13-Assisted Restriction of Viral Expression and Readout (CARVER) to detect and kill RNA viruses. The scientists demonstrated the antiviral efficiency of CRISPR-Cas13 in human cells infected with lymphocytic choriomeningitis virus (LCMV), influenza A virus (IAV), or vesicular stomatitis virus (VSV). They also incorporated the Cas13-based nucleic acid detection technology SHERLOCK (Specific High Sensitivity Enzymatic Reporter UnLOCKing) to enable the system to serve as a “detective” as well.
Scientists use CRISPR in human sperm cells
US scientists have successfully delivered CRISPR genome editing components inside human sperm cells for the first time. In the current study, presented at the annual meeting of the European Society of Human Reproduction and Embryology, the scientists used genome editing to directly target a gene associated with male infertility in mature sperm cells. The team found a single 1100-volt electrical pulse, a fiftieth of second long, allowed the CRISPR components to break through the tough outer exterior of the sperm cells without killing them.
According to the team behind the study, the sperm cells remained relatively healthy and still had the potential to fertilise an egg. Although the research is at an early stage, it could lead to a new way of preventing inherited diseases passed on by fathers. ‘In theory all single gene disorders transmittable by the male can be treated if we are able to successfully use CRISPR/Cas9 on sperm,’ lead researcher Dr Diane Choi at Weill Cornell Medicine in New York City, New York, told New Scientist magazine.
There are more than 10,000 genetic disorders that are “single gene” conditions, which means they are passed onto the child through a mutation in a single piece of either parent’s DNA. These disorders include sickle cell disease, cystic fibrosis and muscular dystrophy
To date, a handful of groups have used genome editing to correct faulty genes in fertilised embryos. One concern with this approach is that as the cells of embryos rapidly divide, DNA may be repaired in some cells but not others, a phenomenon known as mosaicism.
CRISPR-mediated genome editing has drawbacks, though. The PAM requirement limits target sequences. Cas9 is large, so its gene is difficult to deliver to cells via vectors such as adeno-associated viruses commonly used in gene therapy. Scientists worry about off-target effects, although experts note that concerns about unintended mutations are often based on calculations from studies on improving editing. These studies may deliberately use low-specificity conditions to facilitate monitoring progress.
To ensure the highest confidence in their products, companies invest time and money in custom genome-editing methods focused on efficiency and specificity. Initial investments pay off, industry scientists say, by preventing problems later in development.
ZFNs are the genome-editing reagents used by the genomic medicine company Sangamo, based in Brisbane, California. Chief Technology Officer Ed Rebar explains that Sangamo’s core editing reagent is a ZFN dimer. The typical target site is 36 basepairs. Each ZFN is a chimeric protein of the nuclease domain from the FokI restriction enzyme and an array of zinc-finger DNA-binding domains built by “mixing and matching” from Sangamo’s archive of thousands of two-finger subunits. Strategies for diversifying the ZFN architecture for high targeting capability include attaching the FokI domain to the N- or C-terminus of the zinc-finger array and inserting base-skipping linkers between fingers. With Sangamo’s high-throughput, automated process for generating ZFNs, Rebar says, “Starting from a target gene name, we can generate an initial set of editing reagents within two weeks.”
In a demonstration study, Rebar and colleagues designed ZFNs that introduced indels at 25 of 28 bases in a promoter relevant to studying hemoglobinopathies. Despite this precision and the advantage of being smaller than Cas9, ZFNs are not as commonly used as CRISPR-based methods. Sangamo provides ZFNs via industry and academic partnerships but holds the modules, expertise—and patents—for making them. TALENs attach FokI to arrays of DNA-binding modules, originally from plant pathogens, that each target a single basepair. TALENs are smaller than Cas9, but larger than ZFNs. The modules have high DNA-binding affinity but include repeated sequences that create cloning challenges.
Dan Carlson is chief scientific officer at Recombinetics, a St. Paul, Minnesota–based biotechnology company that uses TALENs and CRISPR to generate animals and cell lines for clinical research models and agriculture. Using these methods, he says, “we can target almost any site in a genome.” With in-house resources, even TALENs take only “a few hundred bucks and about a week” to generate, Carlson adds, so scientists choose the method that is most reproducible, consistent, and specific, based on pilot studies. These initial investments ensure the company is responsible with resources, he says. “It costs too much to sort out problems on the back end.”
Meganucleases, also called homing endonucleases, are smaller than Cas9, despite their name, which refers to recognition sequences that can be up to 40 basepairs in length. Hybrid megaTALs combine the simple assembly of TALENS with the DNA-cleavage specificity of meganucleases. Two biotech companies that use meganuclease-based methods are Bluebird Bio in Cambridge, Massachusetts, and Precision BioSciences in Durham, North Carolina.
Barry Stoddard, a structural biologist at Fred Hutchinson Cancer Research Center, Seattle, has a panel of 50–60 meganucleases that his lab engineers to recognize specific sequences. “It takes one day to make CRISPR to target a gene,” he says, “and 100 days to make a meganuclease.” Still, Stoddard gets many requests for engineered meganucleases, because their precision is highly valued for applications such as developing therapeutics for which “100 days is nothing.”
Gene editing breakthrough allows precise fixes of humans and could destroy thousands of most deadly diseases
A new gene editing breakthrough allows scientists to easily snip out problems in genetic code, potentially removing thousands of deadly inherited diseases. The “base editor” is a molecular machine that directly converts one building block of DNA into another. DNA sequences contain four “base” chemicals that pair up on the molecule’s twin-stranded double helix in specific ways. Together guanine (G), adenine (A), thymine (T) and cytosine (C) make up the letters of the genetic code. The new system converts the DNA base-pair A-T to G-C, a microscopically small effect that has massive implications for science and medicine.
Professor Liu, from Harvard University said: “We developed a new base editor, a molecular machine, that in a programmable, irreversible, efficient and clean manner can correct these mutations in the genome of living cells. “When targeted to certain sites in human genomic DNA, this conversion reverses the mutation that is associated with a particular disease.” Roughly half the 32,000 single-letter changes in the genetic code known to be associated with human disease involve a change the other way, from G-C to A-T.
The “machine”, called an Adenine Base Editor (ABE), was tested in the laboratory by correcting the mutation responsible for hereditary haemochromatosis (HHC), a disease that causes iron overload in the body. ABE was also used to install a beneficial mutation that protects against blood diseases including sickle cell anaemia.
Professor Robin Lovell-Badge, group leader at The Francis Crick Institute, London, said: “Many genetic diseases are due to alterations (mutations) where a single base pair has been substituted for another. “This makes these new base editing methods of great value in both basic research to make disease models and, in theory, to correct genetic disease.
As such, edits made to the DNA using the new tools are far more precise than the leading and most famous technology, CRISPR. “CRISPR is like scissors, and base editors are like pencils,” said David Liu, the chemical and molecular biologist who led the study.
DARPA’s Safe Genes program
Yet without careful precautions, a gene drive released into the wild could spread or change in unexpected ways. The U.S. Defense Advanced Research Projects Agency (DARPA) has awarded a combined $65 million over four years to seven research teams toward projects designed to improve the safety and accuracy of gene editing.
The DARPA’s Safe Genes program aims to deliver novel biological capabilities to facilitate the safe and expedient pursuit of advanced genome editing applications, while also providing the tools and methodologies to mitigate the risk of unintentional consequences or intentional misuse of these technologies.
Kevin Esvelt, head of the Sculpting Evolution lab at MIT Media Lab, which is applying for Safe Genes funding in collaboration with eight other research groups, predicts that eventually, perhaps around 15 years from now, an accident will allow a drive with potential to spread globally to escape laboratory controls. “It’s not going to be bioterror,” he says, “it’s going to be ‘bioerror.’”
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