Jennifer Doudna (UC Berkeley Professor) – The Biology of CRISPRs (2014)

Chapters

Abstract

Decoding the Revolutionary World of CRISPR-Cas9: A Glimpse into Jennifer Doudna’s Research and Its Transformative Impact

In a groundbreaking journey that reshapes our understanding of genetic manipulation, Jennifer Doudna’s extensive research on CRISPR-Cas systems unveils a transformative tool in genome editing. This research, marked by a deep dive into the intricacies of Type 1 and Type 2 CRISPR systems, collaboration with Emmanuel Charpentier, and the innovative design of chimeric RNA, has revolutionized our approach to modifying genetic material. This article delves into the key mechanisms of Cas9 – from its unique DNA targeting and cleavage to its RNA targeting capabilities – and the significant structural changes it undergoes upon nucleic acid binding, highlighting its vast potential in fields ranging from medicine to agriculture.

Jennifer Doudna’s Research on CRISPR Systems:

Jennifer Doudna, an eminent figure in RNA research, has significantly advanced our understanding of CRISPR-Cas systems, a bacterial immune defense mechanism. Her curiosity in RNA’s catalytic functions led her to explore these systems’ ability to capture and utilize sequences from invading pathogens as a means of defense. Comprising of 40 base pair repeats and intervening spacer sequences that match bacteriophage or plasmid sequences, CRISPR-Cas systems are evidence of an acquired immune response. The transcription of the CRISPR locus into a precursor RNA molecule, which is then processed into smaller RNAs, plays a crucial role. These RNAs assemble with Cas proteins to form interference complexes that recognize and degrade foreign DNA.

Exploring Type 1 and Type 2 CRISPR Systems:

Doudna’s research particularly focused on the Type 1 and Type 2 CRISPR systems. The Type 1 system, found in E. coli, features a complex Cascade mechanism for DNA recognition and degradation. In contrast, the Type 2 system, from Streptococcus pyogenes, is characterized by its simplicity and reliance on the Cas9 protein for DNA targeting.

Breakthrough in Cas9 Discovery and Genome Editing Applications:

A significant milestone in her research was the collaboration with Emmanuel Charpentier, which led to groundbreaking insights into Cas9. They discovered its programmable nature, enabling specific DNA targeting and cleavage. This pivotal finding transformed the field of genome editing by facilitating precise DNA manipulation for gene editing and correcting genetic defects.

Cas9: Protein Function and Mechanism:

Cas9 operates by using crRNA and tracrRNA for DNA targeting and can create double-stranded breaks in DNA. Detailed biochemical studies revealed that Cas9’s two distinct RNA molecules enable DNA recognition and facilitate the cleavage of both DNA strands, guided by a 20 base pair interaction between the guide RNA and the target DNA. The protospacer adjacent motif (PAM) sequence, a GG dinucleotide motif, is critical for Cas9’s functionality. Additionally, the transactivating CRISPR RNA is necessary for the stability and processing of the CRISPR RNA.

Innovative RNA Targeting and Cas9 Regulation:

Doudna’s team further explored the potential of Cas9 in RNA cleavage using a PAMmer, opening up new applications in RNA imaging and detection. Cas9’s activity is intricately regulated, with the guide RNA and PAM sequence playing pivotal roles in its DNA surveillance and cleavage functions.

Decoding Cas9’s Mechanism: Target Site Recognition and Structural Insights:

Collaborative efforts with Eric Green and Evan Ogalis shed light on Cas9’s target site recognition and the significant structural changes it undergoes upon binding to nucleic acids. Cas9 experiences substantial structural rearrangement, highlighted by a key long green helix that rotates to create a cleft for the RNA-DNA hybrid and RNA handle accommodation. This rotation is facilitated by a hinge at the base of the green helix, enabling the protein’s two lobes to adjust. In its interaction with DNA, Cas9 shows a dissociative behavior, characterized by rapid binding and unbinding. This dissociation is slowed in PAM-rich regions, particularly near cognate or nearly fully cognate target sites. The slowed dissociation in these regions leads to local unwinding of the helix adjacent to the PAM, enabling directional melting of the RNA strand and the formation of a fully competent complex for DNA cleavage.

Cas9’s Potential and Future Directions:

Cas9 technology holds immense potential, revolutionizing diverse fields with ongoing efforts aimed at refining and expanding its applications. The development of methods to redirect Cas9 for RNA targeting broadens its scope, promising new avenues in genetics, medicine, and biotechnology.

Targeting RNA with CRISPR-Cas9: Expanding the Capabilities of a Programmable Molecular Tool:

Doudna’s team, in pursuit of Cas9’s capabilities, initially focused on its DNA targeting, despite its potential for RNA targeting. Initial attempts at RNA cleavage were less efficient compared to DNA cutting due to poor kinetics. However, inspired by the activation of Cas9 for DNA cutting by a short DNA oligo containing PAM nucleotides, Doudna’s team hypothesized that a PAM oligo could similarly activate RNA cleavage. This led to successful experiments with single-stranded RNA targets and a PAMmer DNA oligo, efficiently cutting single-stranded RNA substrates. However, double-stranded RNA with PAM nucleotides in RNA form remained uncleaved, indicating the enzyme’s inability to unwind RNA duplexes.

Further experimentation demonstrated that Cas9 could be programmed to recognize and cleave specific RNA sequences with efficiency comparable to DNA targeting. This discovery paves the way for innovative applications like RNA imaging and tagless RNA detection in live cells and cell extracts. Moreover, understanding Cas9’s mechanism in DNA targeting reveals its role as a programmable double-stranded DNA endonuclease. Guide RNA binding activates Cas9 for DNA surveillance through structural rearrangement in the protein, with the PAM sequence playing a crucial role in recruiting the Cas9 complex and initiating directional DNA melting and catalytic activation. Multiple levels of regulation ensure Cas9’s engagement with bona fide targets, with the PAM sequence, guide RNA binding, and structural rearrangements contributing to its specificity and control.

Doudna also acknowledged the significant contributions of her research team, including Martin Jinek, Sam Sternberg, Cy Redding, Mitch O’Connell, and others, as well as the ongoing collaborations that continue to advance research in this exciting field.



Jennifer Doudna’s research on CRISPR-Cas9 systems represents a monumental shift in the field of genetic engineering. The discovery and elucidation of Cas9’s mechanism have opened new frontiers in precise genome editing, offering unprecedented opportunities in medical and agricultural sciences. As we continue to explore and harness the power of this technology, the future of genome manipulation looks more promising than ever, paving the way for advancements in treating genetic disorders, enhancing crop resilience, and understanding the complex fabric of life itself.

Notes by: QuantumQuest