Jennifer Doudna (UC Berkeley Professor) – Remarks at Rosalind Franklin Society (Jan 2015)

Chapters

Abstract

The Discovery of CRISPR-Cas9: Unveiling a Revolution in Genome Editing

Introduction

In the field of molecular biology, few discoveries have sparked as much excitement and potential as the CRISPR-Cas9 system. Jennifer Doudna, a name synonymous with this groundbreaking research, has pioneered the exploration and application of this novel RNA-guided DNA editing system. Originating from the study of bacterial defense mechanisms against viruses, CRISPR-Cas9 has transcended its biological roots to become a versatile tool, revolutionizing genetic research and holding immense promise for disease treatment and understanding of biology.

CRISPR-Cas9: From Bacterial Defense to Biotechnological Breakthrough

The journey into the world of CRISPR-Cas9 began with a curious observation in bacterial genomes. Renowned scientists, including Doudna and her collaborator Emmanuelle Charpentier, discovered that bacteria utilize CRISPR-Cas systems as a form of adaptive immune defense. These systems capture viral DNA fragments and integrate them into the bacterial genome, creating a ‘memory’ of past infections. When faced with future attacks, bacteria use these snippets to recognize and destroy the invading virus. This fascinating mechanism provided the initial spark for Doudna’s groundbreaking research.

Discovery of CRISPR Loci and Their Potential Role in Bacterial Immunity:

In 2005, three research papers highlighted the presence of CRISPR loci in bacterial genomic sequences. These loci contained repetitive sequences interspersed with sequences matching those found in viruses or plasmids. This observation sparked the idea that CRISPR loci might be involved in an acquired immune system in bacteria.

Collaboration with Jill Banfield:

Jennifer Doudna received a phone call from Jill Banfield, a scientist studying bacterial communities. Banfield shared her findings on CRISPR loci and suggested their potential role in RNA-mediated protection against viral infections.

CRISPR-Cas System: A Multifaceted Defense Mechanism:

CRISPR loci and associated Cas genes form a sophisticated defense system in bacteria. Foreign DNA from viruses is detected and integrated into the CRISPR locus, creating new spacers flanked by repeats. These CRISPR sequences are transcribed into RNA molecules and broken down into smaller RNAs containing viral sequences. RNA-protein complexes, guided by these viral sequences, recognize and degrade matching viral DNA, providing protection against infection.

Research at Denisco:

Philippe Horvath and Rodolf Barango, scientists at Denisco, a yogurt company, investigated CRISPR’s role in protecting bacterial cultures from viral infections. Their research provided genetic evidence supporting the CRISPR-Cas system’s function in bacterial immunity.

Unraveling the Mysteries of CRISPR-Cas9

Doudna’s lab, in collaboration with Charpentier’s, embarked on a detailed study of the molecular workings of CRISPR-Cas9. They uncovered the system’s reliance on two critical RNA molecules: CRISPR RNA (crRNA) and trans-activating CRISPR RNA (tracrRNA). These RNAs bind to the Cas9 protein, guiding it to specific DNA sequences for cleavage. The team’s eureka moment arrived when they realized that these RNAs could be fused into a single, programmable guide RNA (sgRNA). This innovation transformed Cas9 from a bacterial defense mechanism into a programmable tool for genome editing.

Invitation to the American Society of Microbiology Conference:

Doudna received an unexpected invitation to an American Society of Microbiology conference in 2011.

Doudna’s Collaboration with Charpentier to Study CRISPR-Cas9:

Doudna attended a conference where she met Charpentier, a microbiologist who had recently published a paper on CRISPR in Streptococcus pyogenes. Charpentier’s research revealed a second RNA critical for CRISPR molecule production in Streptococcus pyogenes. Doudna’s interest in RNA led to discussions with Charpentier about studying the function of the Cas9 protein in the CRISPR system.

Cas9 as a Dual RNA-Guided DNA Endonuclease:

Their collaboration involved Doudna’s lab in Berkeley, Charpentier’s lab in Sweden, and a graduate student in Vienna. Martin Jinek and Christoph Chylinski, researchers from Doudna’s and Charpentier’s labs, respectively, made significant contributions to the research. Cas9, represented by a blue blob in the cartoon, binds to two separate RNA molecules. One RNA, derived from the CRISPR locus, includes a sequence that can base pair with DNA. The other RNA, called tracr, interacts with the first RNA and helps Cas9 bind to DNA.

Multiple Functions of Cas9:

In bacteria, Cas9 protects cells from viruses by recognizing multiple RNA sequences, allowing for multiplexing. Scientists can program Cas9 to recognize multiple different sites in a single cell, enabling precise genome manipulation and editing.

Transforming Genetic Research and Therapeutic Possibilities

The simplicity and versatility of the CRISPR-Cas9 system have catapulted it to the forefront of genetic research. Its ability to facilitate targeted DNA breaks and leverage cellular repair mechanisms has opened new avenues in gene function study, therapy development, and disease treatment. The potential applications are vast, ranging from gene therapy for inherited disorders to novel approaches in cancer treatment.

Engineering a Single Guide RNA:

Researchers explored the sequence requirements of the two RNA molecules involved in CRISPR-Cas9. By trimming and linking the RNA molecules together, they created a single guide RNA (sgRNA). The sgRNA contains both the targeting information and the structural information needed for Cas9 binding.

Testing the Programmable DNA Endonuclease:

Experiments were conducted to test the sgRNA-guided Cas9 system on a plasmid DNA molecule. Five different sgRNAs were designed to target specific sequences on the plasmid. Cleavage reactions were performed, and the resulting DNA fragments were analyzed. The results confirmed that Cas9 could make precise cuts in the DNA at the intended locations.

Excitement and Potential Applications:

The programmable nature of CRISPR-Cas9 generated excitement due to its potential as a powerful technology. Researchers recognized the opportunity to harness natural DNA repair pathways to manipulate genomic DNA sequences. Non-homologous end joining and homology-directed repair are two major DNA repair pathways that can be utilized for genetic engineering. Compared to other protein-based DNA editing methods, CRISPR-Cas9 offers advantages in terms of simplicity and ease of use.

Broader Implications in Genomics and Genome Editing:

The availability of CRISPR-Cas9 as a programmable DNA editing tool has fueled an explosion of research in genomics and genome editing. Researchers can now make targeted changes to genomes, enabling the study of gene function and the development of new therapies. The potential applications of CRISPR-Cas9 span a wide range of fields, including medicine, agriculture, and biotechnology.

Rapid Adoption and Growth of CRISPR-Cas9:

The initial publication of Cas9 research in 2012 led to a flurry of publications in 2013 showcasing its versatility in genome engineering across various organisms. The technology has seen exponential growth in applications and is being used in labs worldwide.

Diverse Applications of CRISPR-Cas9:

Model organisms and biotechnology applications in plants and fungi are being engineered using CRISPR-Cas9. Researchers are exploring its potential in systems biology and single-celled organisms. Biomedical applications, including gene therapy, cancer drug resistance screening, and pathogen targeting, are actively being pursued.

The Future of Genome Editing

Jennifer Doudna’s work on CRISPR-Cas9 represents more than a scientific breakthrough; it embodies the power of curiosity-driven research. Her journey from observing a unique bacterial phenomenon to revolutionizing genome editing underscores the importance of fundamental science. As we look to the future, CRISPR-Cas9 stands not only as a testament to human ingenuity but also as a beacon of hope for untold advancements in medical science and beyond.

Curiosity-Driven Research Leads to Unexpected Discoveries:

CRISPR-Cas9 emerged from basic science research, highlighting the importance of funding curiosity-driven projects. The project team included key individuals like Emmanuel Charpentier, Martin Jinek, Christoph, Inez Fonfara, and Mickey Hauer.

Funding and Support:

– National Science Foundation (NSF) provided essential funding for initial experiments leading to the CRISPR-Cas9 discovery.

– Howard Hughes Medical Institute (HHMI) supported the research through project-based funding, allowing flexibility to explore new avenues.



CRISPR-Cas9 is a revolutionary genome editing technology with broad applications in various fields, ranging from basic research to clinical medicine. Its discovery emphasizes the importance of curiosity-driven research and funding for such projects.

Notes by: Flaneur