#323 - CRISPR and the future of gene editing: scientific advances, genetic therapies, disease treatment potential, and ethical considerations | Feng Zhang, Ph.D.

CRISPR gene editing technology has revolutionized modern science, stemming from its discovery in bacterial DNA sequences. CRISPR systems were not only a fascinating molecular phenomenon but also laid the groundwork for significant advances in genetic engineering.

CRISPR systems were first discovered in bacterial DNA sequences in the 1980s

A group of Japanese researchers studying E. coli DNA sequences discovered regions within the bacterial genomes that had repetitive DNA sequences not typical of gene-encoding sequences. These repetitions were grouped together and interspaced by short, fixed-length gaps, forming a pattern. Feng Zhang explains that the most obvious signals when observing bacterial DNA are the CRISPR repeats, due to their conserved sequences that repeat many times. The sequences were palindromic, which means reading the repeat sequences from the top strand and in reverse on the bottom, they appeared almost the same. These sequences, later named CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats), describe the appearance of these patterns.

Researchers observed repeating DNA sequences with short, unique segments in between

Feng Zhang discusses Francisco Mojica looking at the repeating sequences within the bacterial genome. It was Mojica’s work in the early 2000s that found that the non-repeating sequences between CRISPR repeats matched sequences from viruses, suggesting a role in bacterial-viral interactions. Mojica's discovery set the stage for the development of CRISPR as a tool for gene editing, as it indicated that these sequences were derived from viruses and foreign to the bacteria.

The function of CRISPR as a bacterial immune system was deciphered in the early 2000s

Researchers found the unique spacer segments matched viral DNA sequences, indicating CRISPR's role in fighting off viruses

Zhang reflects on the realization that the snippets of genetic information of the virus acquired by the bacteria, usually about 30 letters long, were sufficient for the bacteria to uniquely recognize the virus. Once a bacterium integrated a piece of the virus’s DNA into their CRISPR system, they effectively acquired immunity against that virus.

CRISPR systems use guide RNA to direct Cas proteins to cleave matching viral DNA, provid ...

The episode dives into the potential of CRISPR technology, highlighting its revolutionizing role in treating genetic diseases through precise gene editing.

CRISPR allows for precise, programmable gene editing by delivering a Cas9 enzyme and guide RNA

The Cas9 enzyme can be directed to specific DNA sequences by the guide RNA

Dr. Feng Zhang speaks about gene editing as a two-part system: the delivery technology and the medicine or 'payload' technology. CRISPR uses guide RNA to direct the enzyme Cas9 to specific DNA sequences for editing. This RNA-guided process is more precise compared to older systems like zinc finger nucleases or TALENs that used proteins for DNA recognition. Cas9 can open host DNA and find its match without its own helicase and then make cuts where it matches the guide RNA.

This enables targeted disruption, correction, or insertion of genetic material

CRISPR's precision allows for the modification of specific genes, with transformative effects in various fields, particularly by creating transgenic mice in biomedical research. Zhang uses the analogy of smartphone apps to describe CRISPR's efficiency, where different software (guide RNA) can be loaded onto a single device (Cas9) to target varying genes. CRISPR RNA is easy to synthesize, and after the completion of the Human Genome Project, it is now possible to target almost any gene to perform cuts, edits, or insertions, as needed.

CRISPR has shown promise for treating monogenic genetic disorders

Diseases like sickle cell anemia, Huntington's, and metabolic disorders are caused by single gene mutations

Zhang underscores the capability of CRISPR to treat genetic diseases where pathogenic mutations cause the production of a mutant protein detrimental to the patient. For instance, Huntington's disease involves an expanded sequence of DNA repeats; CRISPR could potentially shorten these. Likewise, sickle cell anemia, caused by a single point mutation, is more prevalent in black populations due to its protective effect against malaria.

CRISPR can be used to inactivate or correct the causative mutations in these diseases

For diseases like sickle cell anemia, CRISPR targets the mutation causing the disease, possibly providing a lasting solution by expressing the fetal version of hemoglobin to lessen the disease's symptoms. Zhang explains that gene editing therapy, involving cutting DNA rather than making a point mutation, can potentially treat other diseases like Huntington's and metabolic liver disorders by inactivating genes to stop the production of toxic proteins.

CRISPR-based therapies are in clinical trials for some genetic diseases

Approaches include ex vivo gene editing of patient cells, followed by re-introduction

CRISPR's promise in treating sickle cell disease involves a potential one-time cure by ex vivo gene editing. Doctors can harvest the patient's bone marrow cells, modify them in the lab with mRNA for Cas9 and guide RNA, then reintroduce these edited cells. This process aims to correct the sickle ...

Feng Zhang, Peter Attia, and others delve into the complexities of CRISPR-Cas9 gene editing, including its delivery methods, ethical controversies, and limitations.

Efficient in vivo delivery of CRISPR components remains a key challenge

The podcast opens with discussions on the importance of delivery methods for the success of CRISPR-based therapies. They talk about the success and limitations in targeting cells, mentioning specific cells in the liver and eye. Zhang acknowledges that although CRISPR components like Cas9 can already be delivered to some cells in vivo, the large size of proteins like Cas9 and Cas-13 poses substantial challenges. Nanoparticle formulations and various viral vectors are being developed to improve the efficiency of in vivo delivery, yet no method has achieved complete efficiency. For instance, even though lipid nanoparticles can deliver Cas9 and guide RNA to the liver, when it comes to gene editing for repairing vision, the combined inefficiencies of the delivery system with gene editing result in incomplete restoration.

The large size of Cas9 protein makes it difficult to package into viral vectors for delivery

Zhang discusses how the size of the Cas9 protein, which is 1,300 amino acids long, and Cas-13, which is roughly 1,000 amino acids, complicate their packaging into viral vectors. This issue has led to the exploration for new, more compact proteins. Although some have been discovered, they often lack the specificity or activity of Cas9.

Nanoparticle and other formulations are being developed to improve in vivo delivery

Attia talks about the lipid nanoparticle delivery system used in the liver, and Zhang indicates that large-scale mRNA production and the formulation of nanoparticles are steps toward improving in vivo delivery and reducing costs over time.

The long-term safety and potential for unintended effects of germline editing are ethical concerns

The podcast addresses the ethical considerations of gene editing, including the debate surrounding germline modification. A controversial case from 2018 involved editing the embryos of a couple where the father was HIV positive, resulting in the births of mosaic baby girls, meaning the editing was not entirely successful and introduced unintended effects. Multinational working groups are now tasked to tackle these ethical discussions, with a general consensus against pursuing germline editing at the moment.

There is ongoing debate about whether germline editing should be pursued, even for severe genetic diseases

Zhang states the technological capability exists to treat disorders of inborn errors of metabolism or other genetic mutations with gene editing. There's a debate and ongoing discussions about the suitability of germline editing for conditions with severe implications, such as Huntington's disease and inborn errors of metabolism. One alternative solution offered is pre-implantation genetic testing to screen out embryos with mutations.

Regulations vary globally, with some countries banning or restricting germline modifications

When discussing the regulation of gene editing, Attia implies a global variation in how such technologies are received and governed. Zhang refers to legal regulations in the US that prevent germ ...