CRISPR and Gene Editing: Revolutionizing Medicine and Shaping the Future of Human DNA Engineering

Rajesh Uppal April 3, 2024 BioScience, Manufacturing Comments Off on CRISPR and Gene Editing: Revolutionizing Medicine and Shaping the Future of Human DNA Engineering 1,047 Views

Introduction:

In the span of just a few years, CRISPR, an acronym for Clustered Regularly Interspaced Short Palindromic Repeat, has transformed from a novel gene-editing tool to a powerhouse technology reshaping our approach to medicine and genetics. This revolutionary system, likened to genetic scissors, has garnered attention for its ability to make precise changes to the DNA of humans, animals, and plants. Originally celebrated for its speed and efficiency in modifying DNA, CRISPR has evolved to address complex genetic issues, paving the way for groundbreaking applications in medicine and beyond.

CRISPR allows removing a single (defective) gene from a genome and replacing it with another one, to prevent genetic diseases. Compared to previous techniques for modifying DNA, this new approach is much faster and easier.

CRISPR’s Triumph: A Possible Cure for Sickle-Cell Disease

In December 2023, the FDA approved the world’s first medicine based on CRISPR technology. Developed by Vertex Pharmaceuticals, in Boston, and CRISPR Therapeutics, based in Switzerland, Casgevy is a new treatment for sickle-cell disease, a chronic blood disorder that affects about 100,000 people in the U.S., most of whom are Black.

Sickle-cell disease is caused by a genetic mutation that affects the production of hemoglobin, a protein that carries oxygen in red blood cells. Abnormal hemoglobin makes blood cells hard and shaped like a sickle. When these misshapen cells get clogged together, they block blood flow throughout the body, causing intense pain and, in some cases, deadly anemia.

The Casgevy treatment involves a complex, multipart procedure. Stem cells are collected from a patient’s bone marrow and sent to a lab. Scientists use CRISPR to knock out a gene that represses the production of “fetal hemoglobin,” which most people stop making after birth. (In 1948, scientists discovered that fetal hemoglobin doesn’t “sickle.”) The edited cells are returned to the body via infusion. After weeks or months, the body starts producing fetal hemoglobin, which reduces cell clumping and improves oxygen supply to tissues and organs.

Ideally, CRISPR will offer a one-and-done treatment. In one trial, 28 of 29 patients, who were followed for at least 18 months, were free of severe pain for at least a year. But we don’t have decades’ worth of data yet.

Casgevy is a triumph for CRISPR. But a miracle drug that’s too expensive for its intended population—or too complex to be administered where it is most needed—performs few miracles. More than 70 percent of the world’s sickle-cell patients live in sub-Saharan Africa. The sticker price for Casgevy is about $2 million, which is roughly 2,000 times larger than the GDP per capita of, say, Burkina Faso. The medical infrastructure necessary to go through with the full treatment doesn’t exist in most places. Casgevy is a wondrous invention, but as always, progress is implementation.

CRISPR 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.”

Expanding CRISPR Applications:

CRISPR-Cas9, often likened to genetic scissors, allows scientists to precisely edit genes by targeting and modifying DNA sequences. 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.

1. CRISPR’s Role in Combatting COVID-19:

The world was caught off guard when the COVID-19 pandemic struck, underscoring the urgent need for advanced tools and technologies in the realm of medicine and biotechnology. Amid the chaos, CRISPR, a revolutionary gene-editing technology, emerged as a beacon of hope. This breakthrough not only offered a novel approach to combat COVID-19 but also holds the potential to transform the landscape of medicine, allowing scientists to engineer human DNA for both therapeutic and preventive purposes.

In the context of COVID-19, researchers have explored CRISPR’s potential in developing diagnostic tools, therapeutics, and even preventive measures.

Diagnostic Tools: CRISPR-based diagnostic tools have been designed for the rapid and accurate detection of the SARS-CoV-2 virus, which causes COVID-19. These tests offer advantages in terms of speed and specificity, aiding in efficient and reliable screening.
Therapeutics: The adaptability of CRISPR technology has paved the way for potential therapeutics. Researchers have explored using CRISPR to target and disable specific viral genes, hindering the virus’s ability to replicate. This avenue could lead to innovative antiviral treatments.
Vaccine Development: CRISPR has played a role in vaccine development by enabling the creation of viral vectors for vaccine delivery. This has expedited the vaccine development process and provided a platform for responding rapidly to emerging viral threats.

2. Diverse Genetic Modifications:

Initially utilized in a laboratory setting, CRISPR has found applications across a spectrum of organisms. Scientists have employed CRISPR to knock out genes in animal models, develop new agronomic traits in crops, synthesize microbes for drug production, and even correct heritable diseases in human embryos. This versatility underscores CRISPR’s adaptability to various genetic editing needs.

3. Shaping the Future of Human DNA Engineering:

Beyond the immediate battle against COVID-19, CRISPR holds immense promise in the broader field of human DNA engineering. Here are key areas where CRISPR is making groundbreaking contributions:

Genetic Diseases: CRISPR has shown potential in treating genetic disorders by correcting or mitigating the effects of disease-causing mutations. Clinical trials are underway to explore CRISPR’s efficacy in diseases such as sickle cell anemia and muscular dystrophy.
Cancer Therapies: In the realm of oncology, CRISPR is being investigated as a tool to edit the genetic code of cancer cells, making them more susceptible to treatments or even triggering their self-destruction.
Enhancing Human Traits: The ability to edit the human genome raises ethical questions but also opens the door to enhancing certain traits. While discussions around “designer babies” are complex and controversial, CRISPR’s capabilities allow for the exploration of genetic modifications.
Immunity Against Future Pandemics: The lessons learned from the COVID-19 pandemic have spurred research into creating a sort of genetic immunity. Scientists are exploring ways to use CRISPR to engineer human cells for enhanced resistance against a broad spectrum of viruses, potentially averting future pandemics.

The CRISPR family, known for its genomic editing prowess, has expanded its utility with the discovery that Cas13a can effectively bind to and cleave RNA within human cells, positioning it as a potential diagnostic tool for detecting various pathogens. Researchers from Harvard and MIT’s Broad Institute have taken this a step further by leveraging Cas13’s antiviral capability to create the Cas13-Assisted Restriction of Viral Expression and Readout (CARVER) system. CARVER not only detects RNA viruses but also demonstrates the ability to combat them. In experiments, CRISPR-Cas13 efficiently fought against human-pathogenic viruses like lymphocytic choriomeningitis virus (LCMV), influenza A virus (IAV), and vesicular stomatitis virus (VSV) within infected human cells. The integration of the Cas13-based nucleic acid detection technology SHERLOCK enhances CARVER’s diagnostic capabilities, making it a dual-function system for detecting and combating RNA viruses.

Scientists have achieved a significant breakthrough by delivering CRISPR genome editing components directly into human sperm cells, targeting a gene associated with male infertility. Using a brief electrical pulse, the CRISPR components penetrated the tough outer layer of sperm cells without compromising their viability. The sperm cells remained healthy and retained the potential to fertilize an egg. While in the early stages, this research holds promise for preventing inherited diseases transmitted by fathers, potentially addressing more than 10,000 genetic disorders caused by single gene mutations, including sickle cell disease, cystic fibrosis, and muscular dystrophy. This innovative approach offers a potential alternative to correcting faulty genes in fertilized embryos, which may exhibit mosaicism as cells rapidly divide.

Jennifer Doudna, a trailblazer in CRISPR research, envisions CRISPR’s reprogrammable nature as an integral tool in tests and treatments for present and future pandemics. Its direct method of detection, adaptability to target different virus sections, and potential for rapid manufacturing position CRISPR as a formidable ally against viral threats.

CRISPER is not perfect

CRISPR-mediated genome editing, while revolutionary, is not without its limitations. The PAM requirement restricts target sequences, and the large size of Cas9 poses challenges for effective delivery to cells, especially via commonly used vectors like adeno-associated viruses in gene therapy. Concerns about off-target effects persist, though some of these worries may arise from studies deliberately conducted under low-specificity conditions to monitor progress, leading to potential inaccuracies.

To address these concerns and ensure the utmost confidence in their products, companies are investing in custom genome-editing methods that prioritize efficiency and specificity. Despite initial investments, industry scientists argue that the payoffs, in terms of preventing problems later in development, justify the resources devoted to these alternative editing technologies.

Advances in CRISPR Technology:

Researchers at Harvard University, the Broad Institute, and the Howard Hughes Medical Institute have addressed limitations in CRISPR technology through the development of new tools, as outlined in two papers published in Nature Biotechnology. The first paper introduces redesigned cytosine base editors that significantly reduce an elusive type of off-target editing by 10- to 100-fold, holding promise for more effective treatment of human diseases. The second paper details a new generation of CRISPR-Cas9 proteins capable of targeting a larger fraction of pathogenic mutations, including those responsible for conditions like sickle cell anemia. These advancements aim to minimize the risk of adverse effects in human genome editing, marking a crucial step in enhancing the precision and safety of CRISPR-based technologies.

A groundbreaking study led by George Church from MIT achieved a record-breaking 13,000 edits in a single cell using CRISPR technology, a significant improvement compared to previous efforts. The researchers employed a set of dead-Cas9 base editor (dBEs) variants, avoiding excessive DNA cleavage that leads to cell death. The new dBEs enabled the editing of thousands of loci in each cell, showcasing the potential of CRISPR in making precise and extensive genomic modifications.

Another advancement in CRISPR technology comes from the Liu Lab, introducing the prime editing system. This innovative approach allows the deletion, insertion, and correction of various point mutations in cellular DNA with enhanced efficiency and accuracy. The prime editing complex includes an engineered Cas9, a prime editing guide RNA (pegRNA), and a reverse transcriptase, enabling targeted DNA modifications. The system exhibits the potential to correct around 89% of human pathogenic variants, presenting a powerful tool for addressing disease-related genetic mutations.

1. Beyond Single-Gene Editing:

A recent breakthrough in CRISPR technology allows scientists to edit entire gene networks in a single step. This development not only accelerates the search for cures but also raises concerns about potential bioterrorism threats. Scientists at ETH Zurich have demonstrated the ability to target 25 genes simultaneously, marking a significant stride in the efficiency of genetic editing.

2. Genome Editing Landscape:

CRISPR has become synonymous with genome editing, yet alternative methods like zinc-finger nucleases, TALENs, and meganucleases present unique strengths. Researchers weigh the end product and the advantages of each method. CRISPR’s dominance in simplicity and efficiency makes it a preferred choice, particularly in academic labs and commercial applications.

Moreover, scientists are expanding the applications of CRISPR beyond gene editing. A recent study by researchers from the Keck Graduate Institute and the University of California, Berkeley, utilized CRISPR-Cas9 and a graphene chip for highly sensitive digital detection of DNA. By combining CRISPR’s genome-searching capability with graphene’s high sensitivity, the conventional amplification step is eliminated, offering a versatile tool for DNA detection in various fields. The CRISPR-Chip approach holds promise for streamlined and efficient DNA detection processes.

Global Leadership in CRISPR Technology:

The United States and China have emerged as leaders in the application of CRISPR technology. Military applications of gene-editing technology underscore its potential in addressing threats from harmful agents. Chinese researchers, in particular, have showcased pioneering achievements, from creating genetically modified monkeys to developing disease-resistant wheat, reinforcing their commitment to advancing CRISPR applications.

Recent Chinese Breakthrough

Chinese scientists at the Chinese Academy of Sciences Institute of Genetics and Developmental Biology have developed a new gene-editing tool named CyDENT, presenting a more efficient alternative to CRISPR technology. Unlike CRISPR/Cas9, which cuts double-strand DNA to make edits, CyDENT performs strand-specific gene editing without any cuts, offering precision in the editing process. The modular design of CyDENT utilizes different modules for various editing stages, allowing researchers to tailor applications for specific variations. The system employs a protein-based approach, bypassing the need for a guide RNA, which is challenging in editing DNA in human mitochondria or plant chloroplasts. This innovation is particularly crucial as the US contemplates potential export restrictions on China’s biotech sector.

CyDENT’s application extends to the mitochondria, where single-nucleotide variations associated with diseases can be therapeutically addressed with precise base conversion. In chloroplasts, responsible for photosynthesis, CyDENT’s precision could enhance the efficiency of converting sunlight to energy in crops. The tool’s capability to selectively edit one DNA strand at a time offers more controlled and efficient research into cell genomes. As the biotech sector in China faces potential US restrictions, developments like CyDENT underscore the nation’s efforts to build its biotech potential, emphasizing the need for research and innovation in the field of gene editing.

CRISPR’s Future Landscape:

1. Precision Advancements:

Recent innovations, including base editing, prime editing, and CRISPR-Cas13, address CRISPR’s imperfections. These advancements offer more precise edits, reducing off-target effects and expanding the scope of targetable genetic mutations. The ongoing evolution of CRISPR technologies brings us closer to realizing its full potential in treating diseases.

3. Ethical Considerations:

With the powerful capabilities of CRISPR, ethical considerations become paramount. As CRISPR advances toward applications in human sperm cells, questions of inheritable genetic alterations arise.

As CRISPR technology progresses, ethical considerations surrounding human DNA engineering become paramount. Striking a balance between scientific advancement and ethical responsibility is crucial. Robust regulatory frameworks must be in place to ensure the responsible use of gene-editing technologies, preventing misuse or unintended consequences.

2. Safety Measures and Responsible Use:

The Defense Advanced Research Projects Agency (DARPA) initiated the Safe Genes program in 2017 to tackle security and safety challenges linked to the rapid evolution of gene-editing technologies. The program seeks to establish a comprehensive biosafety and biosecurity toolkit, facilitating the ethical use of gene editing while minimizing the risks of misuse or unintended consequences. The program’s objectives include understanding gene-editing technologies, safeguarding against misuse, and protecting against biological threats. Structured in two phases, it has funded seven research teams focusing on detecting and correcting unwanted genetic changes, preventing the spread of engineered constructs, and mitigating risks in complex biological systems. The program’s ongoing efforts aim to yield practical tools for biosafety, biosecurity, and international norms, ensuring the responsible application of gene-editing technologies across diverse fields like medicine, agriculture, and environmental science. Overall, DARPA’s Safe Genes program is pivotal in navigating the balance between innovation and risk management in the dynamic landscape of gene editing.

Initiatives like DARPA’s Safe Genes program highlight the need for caution in the widespread use of gene-editing technologies. As CRISPR continues to advance, establishing safety measures and protocols to mitigate risks of unintentional consequences or misuse becomes imperative.

Conclusion:

CRISPR and gene editing have emerged as transformative forces in the fight against COVID-19 and hold immense potential for reshaping the future of medicine. CRISPR’s journey from a laboratory tool to a transformative force in genetics and medicine is awe-inspiring. Its applications, from combatting pandemics to editing entire gene networks, showcase the immense potential it holds.

As CRISPR technology advances, the scientific community faces the challenge of ensuring its responsible use, navigating ethical dilemmas, and addressing safety concerns. While the technology’s applications in treating diseases and enhancing human traits offer unprecedented possibilities, ethical considerations and regulatory measures are essential to guide its responsible use.

The future promises not just precision in genetic editing but also a profound impact on human health, agriculture, and our understanding of the genetic code. As we navigate the intricate landscape of genetic engineering, CRISPR stands as both a powerful tool for scientific progress and a call for thoughtful reflection on the ethical dimensions of manipulating the building blocks of life.