Jennifer Doudna (UC Berkeley Professor) – Creator Space Science Symposium | BASF (Sep 2015)

Chapters

Abstract

Revolutionizing Biology: The Emergence of CRISPR-Cas9 Gene Editing

In the field of molecular biology, few discoveries have sparked as much excitement and potential as the CRISPR-Cas9 gene-editing technology. Curiosity-driven experiments on bacterial viral infection led to the discovery of CRISPR technology. DNA, the fundamental chemical of life, encodes the information necessary for cells to function and develop. The sequencing of various genomes, including human genomes, has provided valuable information about the genetic code. Developed through a combination of serendipitous discovery and dedicated research, this technology stands at the forefront of genetic engineering, offering unparalleled precision and ease in DNA modification. From its roots in understanding bacterial immune systems to its application in wide-ranging fields including human health, agriculture, and ethics, CRISPR-Cas9 has revolutionized our approach to genetic manipulation. This article delves into the journey of this groundbreaking discovery, its mechanisms, applications, and the profound ethical considerations it raises.

The Discovery and Science Behind CRISPR-Cas9

Background Science

The roots of CRISPR-Cas9 lie in the bacterial defense mechanism against viruses. Bacteria utilize CRISPR loci, consisting of repeated and unique DNA sequences, in conjunction with CRISPR-associated (Cas) genes to combat viral infections. This system, initially an enigma, was later identified as a form of adaptive immunity in bacteria. CRISPR loci are often accompanied by CRISPR-associated (Cas) genes, suggesting a genetic signature of a bacterial system. CRISPR-Cas systems employ a variety of mechanisms to protect bacteria from viral infection.

CRISPR-Cas System in Bacteria

CRISPR loci in bacteria contain genetic information stolen from viruses and integrated into the bacterial genome. These CRISPR systems function as adaptive immune systems, allowing bacteria to acquire immunity to viruses. Bacteria detect foreign DNA from viruses and integrate small pieces of viral sequence into the CRISPR locus. The CRISPR sequences are transcribed into RNA, which is broken up into short pieces that include viral sequences. These RNA pieces assemble with Cas proteins to form RNA-protein complexes that use the viral sequence information to base pair with matching DNA sequences, leading to the destruction of the viral DNA.

Precision Genome Engineering with CRISPR-Cas

CRISPR-Cas technology allows for exact genetic modifications. It employs a guide RNA to direct the Cas9 enzyme to specific DNA sequences, enabling targeted cutting and editing of DNA. This ability to precisely manipulate genetic material heralded a new era in molecular biology, offering solutions for correcting mutations and introducing desirable traits.

The Path to Discovery: Jennifer Doudna and Collaborations

Serendipitous Discovery and Doudna’s Research

Jennifer Doudna’s lab has a longstanding interest in understanding how cells control genetic information, especially the role of RNA molecules. Cells have different types of molecules that act on genetic information encoded in DNA, including RNA molecules that can be transcribed from DNA and then translated into proteins or operate as RNA itself.

Collaboration Leading to Breakthrough

Jennifer Doudna’s lab investigated the assembly of RNA protein complexes that protect cells. At a conference, she met Emmanuel Charpentier, who studied a bacteria’s CRISPR immune system, crucial for preventing viral infection. They collaborated to study the Cas9 gene and its function. Understanding the CRISPR immune system, Doudna’s collaboration with Emmanuel Charpentier and others was pivotal in uncovering the potential of Cas9. Their discovery that Cas9, when combined with a guide RNA, could act as programmable “molecular scissors,” revolutionized the field. They simplified the system by combining the RNA components, making it a versatile tool for genome editing.

Practical Applications and Demonstrations

Doudna’s collaboration with Russell Vance led to practical demonstrations of CRISPR-Cas9’s capabilities. During a conversation with her colleague, an immunologist at Berkeley, Jennifer Doudna discussed testing the CRISPR-Cas9 system in mice. They injected the system into a fertilized mouse egg, targeting a gene responsible for the black coat color in black mice. Six out of eight healthy pups were born white instead of black. DNA sequencing revealed a change in the genome at the site where Cas9 triggered a break in the gene responsible for the black coat color, confirming the precision and effectiveness of CRISPR-Cas9.

Mechanisms and Behavior of Cas9

Target Site Discovery and Cas9 Dynamics

Understanding Cas9’s mechanism involves exploring its DNA binding and cutting process. Cas9 locates specific DNA sequences through a guided RNA, with structural changes enabling the unwinding and cutting of DNA. Recent studies have provided deeper insights into this process, highlighting the protein’s rapid movement and efficient targeting within cells.

Cas9 in Living Cells

Research into Cas9’s behavior in live cells, using fluorescent markers and 3D tracking, has revealed its dynamic nature and fast kinetics. Ongoing studies aim to quantify Cas9’s target search time and behavior at off-target sites.

Applications, Ethics, and Future Prospects

Doudna’s Presentation: Applications and Ethical Considerations

Jennifer Doudna’s presentation on gene editing technology extends beyond scientific applications. She emphasizes the ethical and safety implications of CRISPR-Cas9, especially in human germline editing. This technology’s potential to correct genetic diseases is juxtaposed with the risks of creating “designer babies.” Doudna advocates for public engagement and international discussions to responsibly navigate these ethical dilemmas.

Current Applications of CRISPR:

– Research and applications have expanded beyond bacteria to include plants, fungi, and animals.

– Companies are engineering pigs with humanized organs for transplantation using CRISPR-Cas9 technology.

– Scientists are engineering stem cells and animal models to study human diseases using CRISPR.

– Clinical trials are underway to cure genetic diseases by editing genes in patients.

Germline Editing and Ethical Implications:

– CRISPR technology can be used to edit the germline of animals, including humans.

– A conference was held to discuss the potential of human germline editing and propose a prudent path forward.

– Companies have a role in educating the public about CRISPR technology and its benefits and limitations.

Societal and Media Response:

– Media attention has focused on the potential of CRISPR to engineer perfect babies.

– The ethical and safety implications of germline editing sparked public and scientific discussions.

Upcoming International Meeting:

– The National Academies and Royal Society are collaborating to organize an international meeting.

– Scientists from around the world will gather to discuss the ethical and scientific aspects of CRISPR technology, particularly in the context of human germline editing.

Conclusion

CRISPR-Cas9 stands as a beacon of innovation in molecular biology, symbolizing the power of scientific curiosity and collaboration. While it offers remarkable capabilities in genetic engineering, it also presents complex ethical challenges. As we venture further into this era of genetic manipulation, the balance between technological advancement and ethical responsibility remains paramount.

Notes by: MatrixKarma