#323 - CRISPR and the future of gene editing: scientific advances, genetic therapies, disease treatment potential, and ethical considerations | Feng Zhang, Ph.D.
Feng Zhang, a professor of neuroscience at MIT and a pioneering figure in gene editing, discusses his groundbreaking work in CRISPR technology and optogenetics. He explores CRISPR's applications for treating genetic diseases, delivery challenges, ethical implications, and the future of genetic medicine.
Deep Dive Analysis
14 Topic Outline
Feng Zhang's Journey and the Origins of Optogenetics
The Discovery and Naming of CRISPR Sequences
How Bacteria Use CRISPR as an Adaptive Immune System
Limitations of Pre-CRISPR Gene Editing Technologies
CRISPR's Revolution in Gene Editing and its Mechanisms
Challenges and Progress in Gene Editing Delivery Methods
CRISPR's Role in Treating Sickle Cell Anemia
Base Editing and the Role of AI in Protein Engineering
Accelerating Biomedical Research with Transgenic Mice
Cas13: The RNA-Targeting CRISPR System and Diagnostics
Therapeutic Applications of CRISPR in the Liver and Eye
Ethical Implications and Debates of Germline Gene Editing
Feng Zhang's Early Life and Inspiration for Science
Optimism for the Future of Science and Technology
8 Key Concepts
Optogenetics
A technology developed to study brain cells by inserting a light-sensing gene from green algae into neurons. This allows scientists to control brain activity with light, enabling high-resolution study of neural circuits and their role in behaviors like sleep, memory, and motivation.
CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)
Repetitive DNA sequences found in bacterial genomes, interspaced with unique segments. These unique segments are snippets of viral DNA, forming a 'memory' that allows bacteria to recognize and defend against future viral infections, acting as an adaptive immune system.
Cas Proteins (CRISPR-associated proteins)
Enzymes, primarily nucleases, that work with CRISPR RNA. They bind to the guide RNA and search for matching viral DNA sequences. Upon finding a match, the Cas protein cleaves the viral DNA, neutralizing the threat to the bacteria.
PAM Sequence (Protospacer Adjacent Motif)
A short, specific nucleotide sequence (typically three letters) found in viral genomes but not in the bacterial host's genome. Cas9 requires the presence of a PAM sequence next to its target DNA to activate cleavage, preventing the CRISPR system from cutting the bacteria's own DNA.
Zinc Finger Nucleases
An earlier gene-editing technology that used protein domains, where each 'finger' recognized three DNA letters. Multiple fingers were tethered together to achieve specificity, but engineering these proteins was cumbersome and often ineffective due to complex protein design.
TALENs (Transcription Activator-Like Effector Nucleases)
Another gene-editing technology derived from bacteria, where proteins recognized DNA in a programmable fashion. Individual protein domains recognized single DNA letters, making them easier to design than zinc fingers, but still challenging to engineer due to their repetitive nature and propensity for recombination.
Base Editing
A gene-editing methodology developed by David Liu that uses a modified Cas9 (without its DNA-cleaving activity) as a guide. This guided Cas9 directs a deaminase enzyme to chemically modify a single DNA base, allowing for precise single-letter changes without creating a double-stranded DNA break.
Cas13
An RNA-targeting CRISPR-associated protein, analogous to Cas9 but for RNA viruses. It uses guide RNA to recognize and cleave viral RNA genomes. Uniquely, once Cas13 recognizes a target RNA, it activates a 'suicidal' function, cleaving any other RNA in the cell, which is useful for diagnostics like rapid viral detection.
9 Questions Answered
Feng Zhang's work in optogenetics was bottlenecked by the inability to precisely insert genes into specific locations in the genome to target particular brain cell types. This challenge motivated him to seek easier and more precise gene-editing methods.
Initially, scientists observed repetitive DNA sequences in bacteria. Later, a Spanish researcher, Francisco Mojica, discovered that the non-repeating segments between these repeats matched viral DNA sequences, suggesting that bacteria acquired these snippets to defend against viruses.
When a virus infects a bacterium, the CRISPR system can acquire a small segment of the viral DNA and insert it into its own genome. During subsequent infections, Cas9, guided by RNA derived from these viral snippets, recognizes and cleaves the incoming viral DNA, neutralizing the threat.
Previous technologies required complex and cumbersome protein engineering for each specific gene target, akin to building new hardware for every function. CRISPR, on the other hand, is like a 'smartphone' where the Cas9 protein is the hardware, and easily synthesized guide RNA acts as 'software' to target different genes, making it much more versatile and accessible.
Cells repair DNA either through non-homologous end joining (NHEJ), which often introduces small mistakes that can inactivate a gene (useful for silencing deleterious mutations), or through homology-directed repair (HDR), which uses a provided template DNA to precisely insert or correct sequences at the cut site.
The biggest challenge is delivery: getting the gene-editing machinery (the 'payload') into the correct cells and tissues in the body efficiently and safely. While payload technologies like Cas9 and base editing have advanced significantly, targeted delivery remains a major bottleneck.
The approved CRISPR therapy for sickle cell anemia works by modifying a different gene in the patient's bone marrow stem cells to turn on the production of fetal hemoglobin. This approach leverages a simple DNA cut rather than a precise single-base correction to alleviate symptoms.
CRISPR has dramatically accelerated the process of creating transgenic mice. Instead of taking over a year through traditional methods involving stem cell modification and breeding mosaics, Cas9 and guide RNA can now be directly injected into a single-cell embryo, producing a transgenic mouse in just 2-3 months.
The primary concerns include the lack of medical justification for some edits (like the CCR5 case), the 'slippery slope' argument that allowing disease treatment could lead to 'designer babies' for non-medical enhancements (like intelligence or height), and the current imprecision and inefficiency of the technology for permanent, heritable changes.
7 Actionable Insights
1. Nurture Youth STEM Curiosity
Actively support educational systems that excite children about science and technology, providing opportunities for them to explore their curiosities and maintain their natural optimism and energy, as this is crucial for attracting the best talent to STEM fields.
2. Prioritize Foundational Science
When pursuing rapidly evolving fields like biology, build a strong scientific foundation by studying established disciplines such as chemistry and physics, while simultaneously engaging in practical lab work and staying current with the latest literature.
3. Practice Open Science & Mentorship
Strive to be a good contributing member of the scientific community by actively sharing information, reagents, and knowledge, and by providing opportunities for others to explore and develop, mirroring the actions of effective mentors.
4. Seek Mentorship and Nurture Others
Actively seek out teachers and mentors who are dedicated to student development and provide opportunities for growth, and in turn, contribute to nurturing the next generation in your own field.
5. Cultivate Optimism for Progress
Maintain an optimistic outlook, as it is presented as the only effective way to approach challenges and drive progress, thereby avoiding the inherent downsides of pessimism.
6. Consider Pre-implantation Genetic Testing
For couples undergoing IVF, consider utilizing pre-implantation genetic testing to screen embryos for known genetic mutations, which can potentially prevent the inheritance of certain diseases.
7. Rapid RNA Virus Detection
Utilize Cas13-based diagnostic methods for simple and rapid detection of RNA viruses, similar to an at-home antigen test, by mixing a sample with a buffer and applying it to a paper strip to check for a band.
6 Key Quotes
If you wanted to study sleep, you can put this gene into different groups of cells in the brain and stimulate them. And you can find out which ones of these are controlling wakefulness or which ones are causing the mouse to become more sleepy. So if you do this systematically one by one from one type of cell to another type of cell, you can gradually start to put together a picture of how the brain is wired together.
Feng Zhang
CRISPR is really a brilliant acronym. And so C-R-I-S-P-R stands for exactly how these repeats look. Clustered, regularly, interspaced, short, palindromic repeat. So CRISPR. It's really brilliant and it's very catchy.
Feng Zhang
With CRISPR, the promise is that CRISPR is like the smartphone. You can load software onto it to recognize different genes. And the software is the CRISPR RNA.
Feng Zhang
Genetic medicine is very powerful. CRISPR is part of it, but it's really a two component system. There is the medicine itself, and then there's also the delivery technology. So you need to have the right vehicle for delivery and the right payload to be able to treat the disease in the right cell.
Feng Zhang
I think I've been very fortunate. And I think there are a couple of things that I over time have developed more and more gratefulness and appreciation for. Number one, I think is the importance of teachers.
Feng Zhang
I am an optimist. I think that's the only way to be, because if you're not optimistic, then there's only downside when you are like that. But I am optimistic about science.
Feng Zhang
4 Protocols
CRISPR-Cas9 System for Bacterial Viral Defense (Reinfection)
Feng Zhang- Virus injects genetic information into the bacteria.
- The CRISPR repeat area in the bacteria's genome is turned on, producing CRISPR RNA (guide RNA) that carries a 20-30 base pair sequence matching the incoming virus.
- Cas protein binds to this CRISPR RNA, forming a complex.
- The Cas-guide RNA complex searches along all DNA sequences in the bacteria.
- When it finds a match in the virus's DNA (adjacent to a PAM sequence), it activates the nuclease.
- The nuclease cuts the viral DNA, inactivating the virus within minutes.
Sickle Cell Anemia Gene Therapy (Approved Method)
Feng Zhang- Doctors mobilize and harvest bone marrow stem cells from the patient.
- In the laboratory, these harvested cells are incubated in a bath of messenger RNA (mRNA) for Cas9 and guide RNA.
- Electroporation (zapping with electrical current) is used to rupture cell membranes, allowing the mRNA and guide RNA to enter the cells.
- Inside the cells, the Cas9 mRNA is translated into Cas9 protein, which then binds to the guide RNA.
- The Cas9-guide RNA complex modifies a specific gene (not the sickle cell mutation directly) to turn on the expression of fetal hemoglobin.
- The modified stem cells are then re-infused into the patient, providing a potential cure.
Creating Transgenic Mice with CRISPR
Feng Zhang- Inject the gene-editing Cas9 protein and guide RNA directly into a single-cell mouse embryo.
- The Cas9-guide RNA complex modifies the desired gene in the embryo.
- The embryo develops and is born as a transgenic mouse after a 21-day gestation period.
CRISPR-Cas13 for Rapid Viral Diagnostics
Feng Zhang- Take a sample (e.g., swab) from the patient.
- Mix the sample into a buffer containing Cas13 and its guide RNA.
- If the target virus RNA sequence is present, Cas13 will recognize it and activate its 'suicidal' function, cleaving any other RNA in the buffer.
- Load the buffer onto a paper strip.
- Observe for a band to appear, indicating the presence of the virus (similar to an antigen test).