Overview:
CRISPR Office Hours was started as a venue to facilitate communication within the genome engineering community during the COVID-19 pandemic. The weekly webinar lasted for 16 episodes, with over 2,300 attendees and 16 panelists. The production team and co-host Kevin Holden were instrumental in the success of the series. CRISPR Office Hours episodes are available on Synthego’s YouTube channel. Attendees are encouraged to interact by sending questions through the chat.

Host and Panelist Introductions:
Aditya of Empathy, VP of Marketing at Synthego, is the host. Kevin Holden, head of science at Synthego, is the co-host. Jared Carlson-Stevermer, lead cell biologist at Synthego, is a guest. Jennifer Doudna, professor at University of California, Berkeley, is the featured speaker. Jennifer’s lab was the first to describe the use of CRISPR gene editing technology. She is also president and chair of the Innovative Genomics Institute and a Howard Hughes Medical Institute investigator.

Announcement:
A special edition “Keep Calm and CRISPR On” t-shirt will be given to those who stay until the end of the episode.

Background of CRISPR:
CRISPR is a natural defense system found in bacteria. It helps bacteria combat viruses by acquiring viral DNA sequences and storing them in a distinct location called the CRISPR. The CRISPR sequence is transcribed into RNA, which combines with tracer RNA and Cas9 protein to form a guide for Cas9. The guide RNA directs Cas9 to DNA sequences matching its 20 nucleotides, allowing Cas9 to create a double-stranded break.

Harnessing CRISPR for Genome Editing:
CRISPR can be harnessed for genome editing by introducing a sequence of DNA that matches the target sequence in the genome into the CRISPR system. The guide RNA then leads Cas9 to the target sequence, where it makes a double-stranded break. Cells naturally repair double-stranded breaks, and during this process, errors can occur, leading to changes in the DNA sequence. This allows scientists to precisely edit DNA sequences, potentially fixing genetic defects or introducing beneficial changes.

CRISPR as a Potential Diagnostic Tool for Pandemics:
CRISPR technology may become a valuable diagnostic tool in the current pandemic. It can be used to rapidly detect and identify viruses by targeting specific viral sequences. CRISPR-based diagnostics can potentially be faster, more sensitive, and more accurate than traditional methods. This could be crucial for early detection and containment of infectious diseases, especially in pandemics.

Jennifer Doudna’s Career:
In 2005, Jennifer Doudna was a relatively new faculty member at UC Berkeley, where she studied RNA molecules involved in gene expression. She expanded her research to explore the biological functions of RNAs that regulate gene expression.

Introduction to CRISPR:
Jillian Banfield, a colleague at Berkeley, introduced Doudna to CRISPR systems, which protect bacteria from viruses. Doudna and her team began investigating the biochemistry of CRISPR.

Collaboration with Emmanuel Charpentier:
Doudna and Charpentier’s labs collaborated to study a specific type of CRISPR system that relied on the protein Cas9. They discovered that Cas9 uses two RNAs, CRISPR RNA, and tracer RNA, to interact with DNA and induce double-stranded breaks.

Development of the Single Guide RNA (sgRNA):
Doudna and her team realized that the CRISPR system could be re-engineered as a single guided system. They linked the CRISPR RNA and the tracer RNA into a single guide that provided target information and a structural handle for Cas9 binding.

Potential of CRISPR Technology:
The single guide RNA technology opened up new possibilities for genome editing, which could be used for applications beyond its natural function in bacteria.

Transition from Basic Science to Applied Technology:
Doudna and her team’s work on CRISPR initially focused on understanding the fundamental mechanism of the protein. However, the discovery of the sgRNA led them to realize that the technology had the potential to be used as a tool for genome editing.

Original Thinking:
Jennifer Doudna and Martin Jinek sought to simplify the CRISPR system by linking the two ends of the RNA molecules together, enabling Cas9 programming to cut any desired DNA sequence. The idea of a two-component system (one protein and one RNA) for programming Cas9 emerged, promising simplicity and ease of use.

Key Experiment:
Martin Jinek conducted a simple experiment using a single guide construct to cut DNA at any desired place, demonstrating the feasibility of a two-component CRISPR system. The discovery sparked excitement due to its potential as a powerful and easy-to-use tool for genome editing.

Genome Editing Animation:
CRISPR editing involves Cas9 guided by a single guide RNA searching for a specific DNA sequence within the nucleus. Upon finding the target sequence, the DNA melts apart, and the RNA forms a hybrid with one strand. Cas9 cuts the DNA, creating a double-stranded break that is repaired by cellular enzymes, leading to sequence changes or insertions. This double-stranded break repair model was first proposed by Jack Szostak in the 1980s and further developed by Maria Jason.

CRISPR’s Simplicity and Advantages:
CRISPR stands out as a simple and powerful technology for genome editing compared to earlier methods. Its simplicity has facilitated its widespread adoption for various fundamental and applied research purposes.

Applications of CRISPR:
CRISPR has found applications across biological research, healthcare, agriculture, therapeutics, and diagnostics. The technology has transformed the way scientists manipulate genomes and has opened up new avenues for research and innovation.

Complementary Technological Advances:
The development of CRISPR coincided with other technological advancements, such as DNA sequencing and genome sequencing, which further enhanced its potential and impact.

CRISPR-Based Gene Editing: A Powerful Toolkit for Biological Research and Therapeutics:
CRISPR-based gene editing technologies have revolutionized the study of genetics and enabled advanced computational tools for understanding genetic systems. This toolkit offers immense opportunities for biologists and researchers to explore and manipulate biological systems.

Correcting Disease-Causing Mutations: A New Era of Therapeutics:
With CRISPR-based gene editing, scientists can now correct disease-causing mutations that were previously untreatable. This technology holds the promise of curing genetic diseases like sickle cell disease by precisely correcting the underlying genetic defects.

Case Study: CRISPR Therapeutics’ Success in Curing Sickle Cell Disease:
CRISPR Therapeutics, co-founded by Emmanuel Charpentier, has demonstrated the successful use of CRISPR to cure a patient with sickle cell disease. By activating a form of hemoglobin called fetoglobin, CRISPR restored the patient’s blood cells to normal function.

The Promise of Accessible and Affordable Genetic Therapies:
Jennifer Doudna emphasizes the need to make CRISPR-based genetic therapies accessible and affordable to all patients. The goal is to transform these therapies into standard care for individuals suffering from genetic diseases.

Ongoing Research and Future Directions:
Doudna’s research focuses on developing strategies to make CRISPR-based gene editing more accessible and affordable. She aims to ensure that these therapies can benefit a wider population and improve the lives of those affected by genetic diseases.

Impact of CRISPR-Cas9 Beyond Molecular Biology:
CRISPR-Cas9 technology has the potential to transform the world and improve people’s lives by curing diseases and addressing global challenges.

Jennifer Doudna’s Excitement for the Future:
Doudna expresses her enthusiasm for the future of CRISPR-Cas9 technology and acknowledges the role of researchers in advancing its applications.

Continued Investigation of CRISPR-Cas Mechanisms:
Research efforts are ongoing to explore the fundamental mechanisms of CRISPR-Cas proteins and discover new variants.

Discovery of Novel CRISPR Nuclease in Phage:
A recent collaboration with Jill Banfield’s lab revealed the existence of large phage carrying compact CRISPR systems with a single Cas5 protein and a CRISPR array.

Potential Advantages of Phage-Derived Cas5:
Cas5 is a compact and potentially easier to deliver into cells compared to Cas9, making it attractive for gene editing applications.

Microbiological and Technological Implications:
Researchers are investigating the role of Cas5 in phage biology and exploring its potential use in gene editing delivery vehicles.

Origin of Phage-Derived CRISPR Systems:
Doudna suggests that these systems may have been acquired by phage from bacteria, providing a selective advantage in phage-phage interactions or gene regulation within hosts.

Collaboration with Melanie Ott on CRISPR-Based COVID Diagnostics:
Doudna highlights a collaboration with Melanie Ott to develop CRISPR-based diagnostic tools for COVID-19.

Discovery of Cas13’s Diagnostic Potential:
Alexandra East-Seletsky’s research revealed that Cas13 proteins can be utilized for diagnostics by targeting RNA sequences.

Mechanism of Cas13 for Diagnostics:
Cas13 interacts with RNA target sequences, leading to collateral cleavage of reporter RNA molecules, enabling signal detection.

CRISPR-Based Detection of RNA and DNA Viruses:
CRISPR-Cas systems can be used for detection of RNA and DNA viruses by cutting fluorophore-labeled RNA or DNA molecules after interacting with the target sequence. This interaction triggers a detectable signal, allowing for the identification of the target virus. This approach is programmable, enabling detection of various viruses by reprogramming the CRISPR-Cas system.

CRISPR-Based Detection in COVID-19:
The need for rapid and accurate testing during the COVID-19 pandemic accelerated the development of CRISPR-based detection methods. Companies like Sherlock and Mammoth, and academic labs, including Melanie Ott’s team at the Gladstone, are working towards developing point-of-care testing for coronavirus using CRISPR.

IGI’s Contribution to COVID-19 Testing:
Jennifer Doudna and her team at the Innovative Genomics Institute (IGI) recognized the urgent need for testing in March 2020. Abby Stahl, Connor Sucheta, Enrique Lenshao, and Jennifer Hamilton, along with collaborators, established a clinical testing laboratory at IGI within three weeks. The lab received approval from the University of California Office of the President to accept patient samples and test for coronavirus using polymerase chain reaction (PCR).

Clinical Lab at Berkeley:
The lab has been running a routine clinical test for coronavirus for several months and has partnered with organizations to provide testing to underserved communities. They are also working to open up the Berkeley campus safely for undergraduate students’ return in the fall. A saliva test is now being run in addition to the nasal swab test. Collaborating with companies and academic groups developing CRISPR to validate a point-of-care test for rapid coronavirus detection.

Cold Spring Harbor Genome Engineering Meeting:
The annual meeting will be held virtually this year due to COVID-19. It will feature outstanding science, panel discussions, and a seminar celebrating Rosalind Franklin’s 100th birth anniversary. Walter Isaacson will chair a panel discussion on coronavirus, CRISPR, and science changes during the pandemic.

Advice for Female Junior Faculty:
Pursue your biggest and most exciting ideas, even if others are skeptical. Don’t let anyone tell you that you can’t do something. Find mentors and role models who support and encourage you. Take advantage of opportunities for collaboration and networking.

Trust Your Judgment and Take Risks:
Jennifer Doudna encourages aspiring scientists, especially women, to trust their judgment and passion when pursuing ideas. She emphasizes the importance of taking risks to do interesting work, as not every idea will pan out. Doudna advises against letting others derail you from your goals and encourages embracing risks to make groundbreaking discoveries.

Breakthroughs in Genome Editing and Delivery:
Doudna highlights the potential for further advancements in genome editing technologies, such as the ability to insert large pieces of DNA. She sees opportunities for breakthrough science, discoveries, and transformative engineering in this area. Doudna also emphasizes the need to address the challenge of delivering genome editing molecules into cells and tissues, particularly in the human body and plants.

Biotechnology Applications for Sustainable Living:
Doudna identifies climate change as the biggest challenge humanity faces in the coming centuries. She proposes a two-pronged approach to address this challenge: Developing plants resistant to drought, pests, and other effects of climate change through agriculture. Manipulating microbes to contribute to agriculture, environmental applications, and the human microbiome.

World CRISPR Day and Keynote Address:
Synthego announces the organization of a virtual scientific symposium called World CRISPR Day in October. Jennifer Doudna will be the keynote speaker at this event. The symposium aims to bring together leading researchers and pioneers in CRISPR to discuss the latest trends in research for a wide range of genome engineering applications.

Gratitude to Synthego Employees:
Synthego expresses its appreciation to its workers, particularly those who have been working on-site during the pandemic. The company acknowledges their contribution to providing RNA molecules for COVID-19 testing, engineering cell lines for COVID-19 research, and supporting ongoing cancer and disease research.

Limited Edition Keep Calm and CRISPR On Shirt:
Synthego offers a limited edition Keep Calm and CRISPR On shirt to its audience. The shirt has been popular among lab workers and is available for purchase at synthego.com/calm.