Jennifer Doudna (UC Berkeley Professor) – The Chemistry of CRISPR (Dec 2020)

Chapters

Abstract

CRISPR-Cas9: Revolutionizing Genome Editing and Its Ethical Implications

In a groundbreaking exploration of genetic engineering, scientists Jennifer Doudna and Emmanuelle Charpentier have unlocked the potential of CRISPR-Cas9, a revolutionary genome editing technology. Originally functioning as a natural defense mechanism in bacteria, CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) and Cas9, a “molecular scissors,” have evolved from protecting bacterial cells against viruses to enabling precise genetic modifications in various organisms. This article delves into the journey of CRISPR-Cas9, from its biological origins to its transformative role in biomedicine and agriculture, while also addressing the profound ethical concerns and challenges it brings.

Jennifer Doudna: A Pioneer in CRISPR Research

Born in 1964, Jennifer Doudna is a renowned scientist and Nobel laureate. Currently a professor at the University of California, Berkeley, Doudna’s contributions to the field of CRISPR-Cas9 technology have earned her numerous accolades. Her groundbreaking work has been instrumental in understanding the mechanisms and applications of CRISPR systems.

The Biological Roots and Mechanisms of CRISPR-Cas9

CRISPR-Cas9’s story begins in the microbial world, where bacteria utilize CRISPR sequences to fend off viral infections. This system captures snippets of viral DNA, incorporating them into the bacterial genome. The CRISPR RNAs (crRNAs) derived from these sequences then guide Cas proteins to identify and cleave the corresponding viral DNA. The key player, Cas9, is adept at cutting double-stranded DNA at targeted locations, governed by the crRNA. It’s this natural precision that scientists harnessed, transforming Cas9 into a versatile tool for genome editing.

CRISPR System Overview

CRISPR stands for Clustered Regularly Interspaced Short Palindromic Repeats, a naturally occurring adaptive immune system found in bacteria and archaea. It protects these organisms against viruses by integrating viral DNA fragments into their genome and using them to create RNA copies that guide the destruction of viral DNA or RNA.

Mechanism of CRISPR-Cas9

CRISPR-Cas9 is a specific type of CRISPR system that utilizes the Cas9 protein. It involves three key steps: Adaptation, Expression, and Interference. During adaptation, the cell acquires a small piece of foreign DNA from a virus and integrates it into the CRISPR locus. In the expression phase, an RNA copy of the integrated sequence is made and processed into individual units, each containing a viral sequence. Finally, in the interference step, these units, along with a tracer RNA and Cas9 protein, form a surveillance complex that scans the cell for matching sequences. When a match occurs, Cas9 cuts double-stranded DNA, destroying the viral DNA in the bacterial cell.

Evolution of CRISPR Systems

CRISPR systems have been operating over evolutionary time in bacteria, evolving into diverse forms that effectively protect these organisms. This diversity continues to fuel both fundamental biological research and technological advancements.

Research on CRISPR-Cas9

Jennifer Doudna’s research, particularly with her students, focused on investigating the third step of CRISPR-acquired immunity, known as interference. Their work laid the foundation for the development of CRISPR-Cas9 as a genome editing tool.

From Natural Phenomenon to Technological Marvel

The collaborative efforts of Doudna and Charpentier were pivotal in unveiling the function of CRISPR-Cas9 in cell defense, particularly in Streptococcus pyogenes. They discovered that CRISPR-Cas9 operates as a dual RNA-guided protein, capable of being programmed with a single guide RNA to target specific DNA sequences. This breakthrough paved the way for using CRISPR-Cas9 in eukaryotic cells, where it can introduce precise DNA breaks, triggering cellular repair mechanisms. These attributes of Cas9, coupled with its ability to rapidly bind and release DNA, searching for matches, mark a significant advancement in genome editing capabilities.

Discovery of the CRISPR-Cas9 System and Its Application as a DNA Editing Technology

Jennifer Doudna and Emmanuelle Charpentier began collaborating to study the CRISPR-Cas9 system in 2011, aiming to understand the function of the CRISPR-Cas9 protein. Their research revealed that CRISPR-Cas9 in nature is a dual RNA-guided protein, consisting of a CRISPR RNA molecule that directs it to a target DNA sequence and a tracer RNA that facilitates assembly with Cas9.

CRISPR-Cas9 as a Dual RNA-Guided Protein

CRISPR-Cas9 is a dual RNA-guided protein, consisting of a CRISPR RNA molecule that directs it to a target DNA sequence and a tracer RNA that facilitates assembly with Cas9. Together, these RNAs guide Cas9 to DNA sequences where it can make a cut in the double helix.

Development of a Single Guide RNA

By engineering a single guide RNA that combines targeting information and structural requirements for Cas9 assembly, it became possible to program Cas9 with a single RNA molecule. This discovery allowed for precise targeting and cleavage of DNA sequences by Cas9.

Cas9 Cleavage of Different DNA Sequences

Experiments using plasmid DNA and single guide RNAs demonstrated that Cas9 could be programmed to cleave different sequences of DNA. The presence of a PAM sequence adjacent to the target site is necessary for Cas9 recognition and cleavage.

Harnessing CRISPR-Cas9 as a Technology

The ability to engineer CRISPR-Cas9 as a simple two-component system for DNA double-stranded cutting opened up the possibility of harnessing it as a technology. CRISPR-Cas9 could be applied to eukaryotic cells, which repair double-strand breaks differently than bacteria.

Cas9 Mechanism for Genome Engineering

Cas9-mediated DNA repair can incorporate new genetic information at the site of a double-stranded break. Cas9, directed by single-guide RNAs, introduces double-stranded breaks, triggering DNA repair and enabling precise genome editing.

Cas9-DNA Interaction and Cleavage

Cas9 uses a guide RNA to interact with DNA at a precise position through base pairing. Cas9 undergoes conformational changes as it binds to DNA, unwinds the double helix, and catalyzes cutting. Cas9 interacts with full-length DNA molecules, holding them open for precise double-stranded cutting.

Cas9 Dynamics and DNA Searching

Cas9 is a highly dynamic protein that undergoes conformational changes during DNA binding, unwinding, and cleavage. Cas9 interacts dynamically with long pieces of DNA, quickly searching for complementary matches to the guide RNA.

Cas9’s Role in Genome Editing

Cas9-mediated genome editing is a powerful tool for introducing precise changes to the genome of cells. Cas9 has broad applications in research, medicine, and agriculture, enabling targeted gene modifications.

Expanding the Horizon: Applications and Ethical Considerations

The applications of CRISPR-Cas9 are vast, spanning fundamental research, public health, agriculture, and biomedicine. Somatic cell editing, which affects only individual cells or organisms, has shown promise in correcting mutations responsible for diseases like sickle cell anemia. However, the potential for germline editing, capable of introducing heritable changes, raises profound ethical and societal concerns. The impact of such modifications on future generations necessitates international oversight and responsible management of CRISPR technology.

Exploring New Frontiers: Diversity of CRISPR Systems and Detection Mechanisms

The diversity of CRISPR systems in nature continues to fuel both fundamental biological research and technological advancements. The discovery of various CRISPR-Cas systems, like Cas13a and Cas5, broadens the scope of genome editing and RNA manipulation. Additionally, the RNA-targeting capabilities of Cas13 and DNA cleavage properties of Cas12 have been utilized in innovative detection methods, including the rapid identification of SARS-CoV-2 in patient samples during the pandemic.

Challenges in Delivery and Future Perspectives

A significant challenge in the application of CRISPR-Cas9 technology lies in the effective delivery of these proteins into specific cell types, particularly in human patients. Research is underway to develop better delivery methods and control over the editing process. Despite these challenges, the possibilities of CRISPR-Cas technology are endless, with potential solutions to pressing issues in human health and environmental conservation.

Bacteriophage CRISPR-Cas Systems

Bacteriophages, viruses that bacteria use CRISPR to protect against, can carry their own CRISPR-Cas systems. CRISPR-Cas5, a phage-encoded protein, has RNA-guided DNA cutting capabilities like CRISPR-Cas9. Its small size offers potential advantages for delivery into eukaryotic cells.

CRISPR-Cas13 and RNA Detection

CRISPR-Cas13 proteins are naturally RNA-targeting enzymes. Cas13 can be harnessed as a detection mechanism by attaching fluorophores to RNA molecules that are cleaved upon Cas13’s recognition of an RNA sequence. This system enables highly effective detection of RNA molecules down to picomolar levels.

CRISPR-Cas12 and DNA Detection

Cas12 proteins can cleave single-stranded DNA molecules upon recognition of a double-stranded DNA target. Cas12 can be used for detection of viruses, including the human papillomavirus, in patient samples. Cas12-based detection allows for both sensitivity and specificity in identifying viral signals.

CRISPR-Cas Systems in the SARS-CoV-2 Pandemic

CRISPR-Cas detection is being used to identify SARS-CoV-2 samples in patient saliva or nasal swabs. The programmability of CRISPR-Cas enzymes makes them adaptable for future pandemic preparedness.

A Grateful Acknowledgment and Responsible Future

Jennifer Doudna’s Nobel lecture not only celebrated the remarkable achievements in CRISPR-Cas9 technology but also acknowledged the contributions of her lab members, colleagues, and the University of California, Berkeley. As CRISPR-Cas9 continues to reshape the landscape of genetic research and therapy, the scientific community and society at large must engage in a balanced dialogue, ensuring the ethical and responsible use of this transformative technology.

Notes by: Rogue_Atom