323 - CRISPR and the future of gene editing: scientific advances, genetic therapies, & more

Optogenetics involved inserting an algal gene into specific brain cells to control their activity with light. However, precisely targeting diverse brain cell types required accurate gene insertion into specific genomic locations, which was a significant bottleneck. This motivated Feng Zhang to pivot towards making gene editing more accessible and precise.

In the 1980s, Japanese researchers observed unusual repetitive DNA sequences in bacterial genomes, specifically E.coli. These 'clustered regularly interspaced short palindromic repeats' (CRISPR) were later understood to be a bacterial immune system, incorporating snippets of viral DNA to defend against future infections.

Cas (CRISPR-associated) proteins, primarily Cas9, are a type of nuclease that work with guide RNA. The guide RNA, which contains a sequence matching the target viral DNA, directs the Cas protein to the specific viral sequence. Once a match is found, the Cas protein activates its nuclease activity to cut and inactivate the viral DNA.

ZFNs were difficult to use because they required sophisticated protein engineering to recognize specific DNA sequences. TALENs were easier but still cumbersome due to their repetitive protein structure, making it time-consuming and often inefficient to engineer them for new targets. CRISPR, specifically Cas9, offered a 'smartphone' like programmability, being much easier and more efficient to design.

When CRISPR creates a double-stranded DNA break, cells use two main repair pathways. The first is non-homologous end joining (NHEJ), which can often inactivate a gene. The second is homology-directed repair (HDR), which uses a template DNA to copy desired sequences into the break site, allowing precise DNA sequence changes.

For Sickle Cell Anemia, current gene therapy approaches modify stem cells from the patient's bone marrow. Instead of directly correcting the single point mutation, the therapy uses CRISPR to activate the expression of fetal hemoglobin, which can alleviate disease symptoms by compensating for the defective adult hemoglobin.

The main bottleneck in advanced gene therapy is delivery. While the 'payload' technologies like Cas9, base editing, and prime editing are highly sophisticated, getting these powerful gene-editing tools into the right cells and tissues in the human body with sufficient efficiency remains a major hurdle.

AI, particularly systems like AlphaFold 2 by DeepMind, rapidly predicts protein structures from their amino acid sequences. This dramatically accelerates protein engineering, which is crucial for designing new gene-editing tools, understanding their function, and potentially developing novel therapeutic payloads and delivery systems.

Before CRISPR, creating a transgenic mouse could take over a year through complex stem cell modification and breeding. With CRISPR, gene-editing components can be directly injected into a single-cell embryo, resulting in a transgenic mouse within the 21-day gestation period. This has dramatically accelerated biomedical research by reducing the time to develop animal models.

The Holy Grail is to engineer a Cas protein that is significantly smaller than Cas9 (ideally 300-500 amino acids) while still maintaining the efficiency of a small guide RNA. Smaller proteins are essential to fit within compact viral delivery systems, overcoming a key obstacle in administering gene therapies.

In the liver, scientists are targeting the PCSK9 gene using lipid nanoparticles to reduce cholesterol for cardiovascular disease. In the eye, gene therapies like Luxturna (for LCA2) deliver missing genes, and CRISPR strategies for LCA10 are being developed to inactivate mutant genes that cause degeneration.

In 2018, a Chinese scientist controversially edited human embryos to remove the CCR5 gene, aiming for HIV immunity. Despite the father being HIV positive, the medical need for germline editing was debatable, the edits were incomplete (mosaicism), and it sparked widespread ethical concerns about 'designer babies' and the irreversible nature of changes passed to future generations.

The debate centers on whether germline editing, which makes heritable changes, should be permitted. Advocates point to treating severe monogenic diseases with no other options, while opponents raise concerns about the 'slippery slope' to altering non-medical traits (like intelligence), potential unforeseen biological consequences, and societal implications like inequality.

Zhang credits an inspiring 7th-grade molecular biology teacher, Ed Pelkington, and a volunteer opportunity at Iowa Methodist Medical Center's gene therapy lab (secured by Pelkington) during high school. These experiences, including seeing glowing human cancer cells after gene insertion, ignited his passion for experimental science and rational medicine design.