The Central Dogma of Biology:
Jennifer Doudna’s journey into science began with her father introducing her to Jim Watson’s book, The Double Helix, which sparked her interest in the field. In college, Doudna studied chemistry but always aimed to apply it to understanding living systems. Biology classes introduced her to the central dogma, which emphasizes the flow of genetic information from DNA to RNA to proteins, with DNA as the code of life and proteins performing various functions in cells.

RNA’s Intriguing Role:
Initially, Doudna perceived RNA as a boring intermediary molecule, but her graduate studies with Jack Szostak revealed the fascinating world of non-coding RNAs with diverse functions. This interest in RNA eventually led her to explore CRISPR.

CRISPR: A Bacterial Defense System:
Jill Banfield, a colleague at the University of California, Berkeley, shared her observations about repetitive DNA sequences called CRISPRs found in bacteria and archaea. These CRISPR arrays contained unique DNA sequences flanked by repetitive elements and often matched viral sequences, suggesting a role in protecting cells from viral infection. CRISPR-associated genes (Cas genes) were also identified near these arrays, but their functions were unknown.

Doudna’s Involvement in CRISPR Research:
Jill Banfield’s hypothesis that CRISPR sequences might be deployed as RNA molecules to target viral DNA intrigued Doudna. She began a series of experiments with postdoc Blake Wiedenheft to understand how this protective mechanism worked. The research focused on understanding the fundamental mechanism of CRISPR, not with the intention of developing a genome editing tool.

Unexpected Connection to Genome Editing:
The work on CRISPR’s protective mechanism unexpectedly proved incredibly useful for genome editing, although this connection was not initially apparent. Doudna’s presentation would delve into this connection and explain how CRISPR’s ability to target and modify DNA led to its revolutionary potential in genome editing.

Introduction to CRISPR Systems:
CRISPR systems are a recently discovered defense mechanism found in bacteria. They protect bacteria from viral infections by acquiring and storing fragments of viral DNA in a CRISPR array. These fragments are used to recognize and target subsequent infections by the same virus.

Function of CRISPR Systems:
Upon viral infection, the CRISPR system is activated, and the stored viral DNA fragments are used to guide the Cas genes. Cas genes encode proteins that form a complex that targets and cleaves the viral DNA, preventing the virus from replicating and infecting the cell. The CRISPR system provides bacteria with a rapid and specific defense against viral infections.

Discovery of CRISPR Systems:
CRISPR systems were initially discovered in bacteria that are not commonly studied in the laboratory. These bacteria are often found in extreme environments and have not been cultured in the lab. The discovery of CRISPR systems in these bacteria suggests that they are widespread and play a significant role in the ecology of bacteria.

Research on CRISPR Systems:
In recent years, research on CRISPR systems has intensified, with scientists using genetic and biochemical tools to study their structure, function, and evolution. This research has led to a greater understanding of how CRISPR systems work and their potential applications in various fields, including medicine, agriculture, and biotechnology.

Applications of CRISPR Systems:
CRISPR systems have the potential to be used as a powerful tool for genome editing, allowing scientists to make precise changes to DNA. This technology has applications in treating genetic diseases, developing new therapies, and creating genetically modified organisms. CRISPR systems are also being explored for use in diagnostics and as a potential treatment for infectious diseases.

CRISPR-Cas9 Overview:
CRISPR-Cas9 is a gene-editing technology that utilizes a protein called Cas9 and a guide RNA (gRNA) to make targeted changes to DNA. Cas9 acts like a pair of scissors, cutting the DNA double helix at a specific location determined by the gRNA sequence. This cut triggers cellular repair mechanisms, leading to either small alterations or larger changes to the DNA sequence at the cut site.

CRISPR-Cas9 Mechanism:
CRISPR arrays in bacteria store sequences from invading viruses. The cell generates an RNA copy of the CRISPR array, which is processed into individual units, each containing a viral sequence. These RNA units, along with a tracer RNA, bind to Cas9, forming an RNA-guided protein complex. The complex searches for DNA sequences matching the viral sequence in the gRNA. Upon finding a match, Cas9 unwinds the DNA and forms an RNA-DNA hybrid, leading to a double-stranded break in the DNA.

Targeted Genome Editing:
By designing the gRNA sequence, researchers can program Cas9 to cut DNA at a specific location in the genome. This targeted DNA break triggers cellular repair mechanisms, allowing scientists to make precise changes to the DNA sequence. These changes can include small alterations, such as single base pair changes, or larger modifications, such as insertions or deletions of genetic material.

Applications of CRISPR-Cas9:
CRISPR-Cas9 has revolutionized genetic research, enabling scientists to study gene function, disease mechanisms, and develop new treatments. It has potential applications in medicine, agriculture, and biotechnology. For example, CRISPR-Cas9 can be used to correct genetic defects, develop disease-resistant crops, and create microorganisms for biofuel production.

3D Structure of Cas9:
A 3D model of Cas9 reveals its structure and mechanism of action. The protein unwinds the DNA double helix, forming an RNA-DNA hybrid, and then makes a double-stranded break in the DNA. This detailed understanding of Cas9’s structure and function has facilitated the development of more efficient and accurate gene-editing tools.

How Cas9 Unwinds DNA:
Cas9 unwinds DNA to generate a cut, then passes the broken ends to repair enzymes for repair. Controlling the repair process is an ongoing challenge.

Structural Rearrangements of Cas9:
Cas9 undergoes large structural rearrangements when assembling with nucleic acid. A movie showing the morphing between Cas9 states highlights these rearrangements. Conformational changes are supported by experimental evidence.

Diversity of CRISPR Systems:
CRISPR systems are diverse in nature, allowing scientists to test different proteins and compare them. Different forms of these enzymes are used for genome editing and other applications.

Additional Family of Proteins:
A new family of proteins is being studied, showing similar mechanisms to Cas9.

Cas12a: A Different Type of CRISPR System:
Cas12a, also known as CPF1, is an RNA-guided protein found in some bacteria with a CRISPR system that protects cells from foreign DNA. Unlike Cas9, Cas12a has a single active site called RUV-C and naturally possesses a single RNA molecule for guiding it to matching DNA sequences.

Janice Chen’s Experiment and Unexpected Result:
Janice Chen, a student in Jennifer Doudna’s lab, set out to understand how substrate recognition triggers DNA cutting by Cas12a. During her experiment, she encountered an unexpected result: Cas12a caused total DNA degradation when exposed to a single-stranded piece of DNA (M13 phage), even when the guide RNA did not match the phage sequence.

Cas12a’s Unique Activity: Cutting Single-Stranded DNA:
Further experiments revealed that Cas12a has an activity distinct from Cas9: it can cut single-stranded DNA molecules after contacting a target strand. This unique property allows Cas12a to degrade single-stranded DNA molecules, even those that do not match the guide RNA sequence, once it is activated by a matching target strand.

Activation of Cas12a for Single-Stranded DNA Cleavage:
Cas12a requires activation by a single-stranded DNA molecule called the activator, which base pairs with the guide RNA. Once activated, Cas12a can then cleave unrelated single-stranded DNA molecules, demonstrating its specificity for single-stranded DNA substrates.

Cas12a’s Double-Stranded DNA Cleavage and Activation:
Cas12a can also cleave double-stranded DNA molecules when presented with a matching guide RNA sequence. This double-stranded DNA cleavage activates Cas12a for single-stranded DNA cleavage, resulting in the degradation of single-stranded DNA molecules that do not match the guide RNA sequence.

Biochemical Properties of Cas12a:
Cas12a is an RNA-guided enzyme that exhibits targeted double-stranded DNA cleavage, similar to Cas9. It possesses unique single-stranded DNA cleavage activity, which is rapidly activated upon binding to a target sequence. This single-stranded DNA cutting activity is reminiscent of the Tasmanian devil’s destructive behavior.

Harnessing Cas12a for DNA Detection:
Cas12a’s single-stranded DNA cleavage activity can be harnessed for DNA detection purposes. A simple assay was developed to detect human papillomavirus (HPV) in human anal swabs using Cas12a. The assay showed high accuracy in detecting HPV16 and HPV18 serotypes. This detection method is rapid, requiring less than an hour to perform.

Cas12a Applications Beyond Detection:
Cas12a’s single-stranded DNA cutting activity may have implications for genome editing applications. Researchers are investigating the potential undesirable changes that may occur in genomes due to this activity. Cas12a’s single-stranded DNA cleavage property could be utilized to detect single-stranded DNA transiently available in cells. This could have applications in studying DNA replication, transcription, and other cellular processes.

Cas13: RNA-Guided RNA-Binding Proteins:
Cas13 proteins are RNA-guided RNA-binding proteins, initially known as C2C2. Cas13 enzymes exhibit RNA cleavage activity upon interacting with an RNA sequence. This activity can be harnessed for RNA detection, similar to Cas12a for DNA detection.

The Future of Simple Technologies for Genome Editing:
Jennifer Doudna reflects on the potential of simple technologies for genome editing. She emphasizes the need to consider the ethical, societal, and global implications of these technologies. Doudna believes that such technologies have the potential to revolutionize medicine, agriculture, and energy production.

CRISPR-Cas9 Adoption and Research Explosion:
Remarkable adoption of CRISPR-Cas9 for research and applications 8,400 PubMed entries on CRISPR-Cas9 as of February 2018 CRISPR-Cas9 editing has been successfully performed in a wide variety of organisms, including plants, animals, and humans

CRISPR-Cas9 for Neurological Disorders:
Ongoing research to deliver CRISPR-Cas9 into the brain for targeted gene editing Chemical modifications to Cas9 allow it to enter cells and penetrate the blood-brain barrier Selective editing of neurons in a mouse model of Huntington’s disease

CRISPR-Cas9 for Organ Transplantation:
Efforts to engineer pigs with more humanized organs for organ transplantation Removal of endogenous retroviruses in the pig genome to improve compatibility with human recipients

CRISPR-Cas9 in Agriculture and Plant Biology:
Targeted gene editing in tomato plants to increase fruit yields Potential for CRISPR-Cas9 to revolutionize agriculture and improve crop production

Somatic vs. Germline Editing:
Distinction between editing somatic cells and germ cells (leading to heritable changes) Precise gene editing in frog embryos demonstrates the targeted nature of CRISPR-Cas9

Ethical Considerations of Germline Editing:
Experiment in monkeys highlighted the ethical challenges of germline editing Public discussions and reports on the implications of human germline editing Ongoing debate and research on the responsible use of CRISPR-Cas9 technology

Public Outreach and Education:
The Innovative Genomics Institute (Berkeley-UCSF partnership) promotes ethics and outreach Engaging with high school students and the general public to explain CRISPR-Cas9 technology Collaboration with funding agencies to support research and public understanding

Gratitude and Acknowledgements:
Appreciation for collaborators, colleagues, and funding agencies that have supported CRISPR-Cas9 research Recognition of the team members who have contributed to the advancements in CRISPR-Cas9 technology