Introduction:
UC Davis School of Medicine launches a Distinguished Speaker Series in Research and Innovation. Dean Brashear emphasizes the importance of academic medical institutions and multidisciplinary research for groundbreaking healthcare innovations.

Introducing Dr. Jennifer Doudna:
Dr. Prasant Mahapra introduces Dr. Jennifer Doudna, a Lee Kar-Singh Chancellor’s Chair and professor at UC Berkeley. She is renowned for her co-discovery of CRISPR-Cas9 genetic engineering technology, which revolutionized genomics research. Dr. Doudna’s achievements include receiving the Nobel Prize in Chemistry in 2020 and being named one of the 100 most influential people in the world by Time Magazine.

CRISPR and Coronavirus:
Dr. Doudna’s talk focuses on CRISPR and coronavirus, divided into three parts: Fundamental science behind CRISPR genome editing. CRISPR and genome editing applications. Current work on CRISPR as a molecular diagnostic for the pandemic.

Curiosity-Driven Research:
Dr. Doudna emphasizes the importance of curiosity-driven research and discovery, leading to unpredictable and exciting outcomes. The fundamental curiosity about the world and our place in it as biological beings drives scientific exploration.

CRISPR and Genome Editing:
Dr. Doudna plans to discuss the origins of CRISPR and its potential applications in genome editing. The field is rapidly advancing, offering numerous opportunities and applications.

CRISPR as a Molecular Diagnostic:
The third part of the talk will focus on the application of CRISPR as a molecular diagnostic tool in the context of the current pandemic. Dr. Doudna highlights the power of fundamental science and the significance of collaboration in addressing real-world problems.

CRISPR: A Bacterial Defense Mechanism:
CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) is a bacterial immune system that protects against viral infections. Bacteria integrate viral DNA sequences into their CRISPR array, creating a record of past infections. CRISPR RNAs (crRNAs) are generated from the CRISPR array and form complexes with Cas proteins to target and cleave matching DNA sequences.

CRISPR-Cas9 Genome Editing:
CRISPR-Cas9 is a gene-editing tool derived from the CRISPR-Cas bacterial immune system. Cas9 is a protein that uses a guide RNA (gRNA) to target and cleave specific DNA sequences. The gRNA is designed to match the target DNA sequence, allowing precise genome editing. CRISPR-Cas9 can be used to insert, delete, or modify DNA sequences, making it a powerful tool for genetic engineering.

Discovery and Development:
The CRISPR-Cas system was initially discovered in bacteria, and its role in adaptive immunity was elucidated through research in the mid-2000s. Scientists realized that CRISPR-Cas9 could be harnessed for genome editing in eukaryotic cells. The development of a single guide RNA (sgRNA) that combines the targeting and structural elements of the natural RNAs simplified the use of CRISPR-Cas9. This breakthrough made CRISPR-Cas9 a versatile and powerful tool for precise genome editing.

Excitement for CRISPR-Cas9 Technology:
Jennifer Doudna expresses enthusiasm for CRISPR-Cas9’s potential to revolutionize genome editing in eukaryotic cells, including plants, animals, and humans. The system’s ability to target specific DNA sequences and make precise edits offers vast opportunities for research and practical applications.

CRISPR-Cas9’s Mechanism in Eukaryotic Cells:
CRISPR-Cas9 complex, guided by RNA, enters the nucleus and searches for matching DNA sequences. Upon finding a match, the DNA melts inside the protein, and the Cas9 enzyme makes a precise double-stranded cut. The cut DNA ends are then repaired by cellular machinery, allowing for targeted genome modifications.

CRISPR-Cas9 for Genetic Disease Correction:
CRISPR-Cas9 holds promise for correcting disease-causing mutations at their source. Sickle cell disease, caused by a single mutation in the beta-globin gene, is an example where CRISPR-Cas9 could potentially cure the disease by correcting the mutation. Clinical trials are ongoing, and one patient, Victoria Gray, has reportedly been successfully cured of sickle cell disease using CRISPR.

Challenges of CRISPR-Cas9 for Clinical Use:
Accessibility and affordability of CRISPR-Cas9 therapy remain significant challenges. Efficient and targeted delivery of genome editing molecules into specific cell types and tissues is crucial for successful therapeutic applications. Safety and efficacy of CRISPR-Cas9 interventions need to be thoroughly evaluated to minimize potential risks.

Ethical Considerations for Human Embryo Editing:
The possibility of using CRISPR-Cas9 to edit human embryos raises ethical concerns regarding safety, efficacy, and responsible use. Transparency mechanisms are being developed to ensure open communication and public accessibility of research involving human embryo gene editing.

CRISPR-Cas Systems in Bacteria:
CRISPR-Cas systems exhibit diverse functions, biochemistry, and recognition mechanisms across different bacterial species. Both bacteria and phage can encode CRISPR-Cas systems, with phage using them to target other phage or control gene expression in their hosts.

Discovery of Cas13’s Second Enzymatic Activity:
Research by Alexandra Isletsky revealed that Cas13 proteins possess a second enzymatic activity that allows them to cleave nonspecific RNA molecules upon target sequence binding. This activity could be beneficial in bacteria during infections, triggering RNA degradation and potentially leading to cell attenuation or death.

CRISPR-Cas Detection of Viral Targets:
CRISPR-Cas systems can be used to detect viral targets. Cas13 proteins can be programmed to recognize different viral targets. Cas13 proteins can cleave RNA molecules that are labeled with a quenched fluorophore pair, releasing a fluorescent signal. This RNAse alert assay can be used to detect viral targets down to picomolar levels.

Cas12 Protein Activities:
Another class of CRISPR proteins called Cas12 also has similar RNA-cleaving activity. Janice Chen discovered this activity in Cas12 proteins. Cas12 proteins can also be used to detect viral targets.

Implications:
These findings suggest that CRISPR-Cas systems could be used to develop new diagnostic tools for viral infections. CRISPR-Cas systems could also be used to develop new antiviral therapies.

CRISPR-Cas12a’s Unique Features:
CRISPR-Cas12a, similar to Cas9, employs an RNA-guided mechanism for double-stranded DNA recognition and cleavage. However, CRISPR-Cas12a possesses the additional ability to cleave single-stranded DNA nonspecifically upon target sequence recognition. This single-stranded DNA cleavage activity is absent in CRISPR-Cas9 systems.

Target-Activated Single-Stranded DNA Cleavage:
CRISPR-Cas12a can recognize and cleave single-stranded DNA substrates after target sequence activation. This activity is independent of double-stranded DNA cleavage and is specific to single-stranded DNA.

Mechanism of Action:
CRISPR-Cas12a cleaves double-stranded DNA targets, resulting in the expected plasmid cleavage observed in agarose gels. Upon activation, the enzyme can also cleave nonspecific single-stranded DNA present in the environment, suggesting a potential role in eliminating viral single-stranded phage genomes.

Application in Viral Detection:
CRISPR-Cas12a’s unique features make it a promising tool for viral detection. Researchers designed CRISPR RNAs to uniquely detect specific viral strains, such as the two most common strains of human papillomavirus. Human samples were collected, treated, and amplified, followed by CRISPR-Cas12a-based detection using fluoro-labeled single-stranded DNAs. The system successfully detected and distinguished between the viral strains, comparable to the results obtained through polymerase chain reaction.

Future Directions:
Janice Chen, inspired by this research, decided to focus her career on developing CRISPR-Cas12a technology as a robust diagnostic tool for viruses and other nucleic acid sequences, enabling the differentiation between various types of sequences.

Jennifer Doudna’s Lab’s Pivot to Focus on Testing During the SARS-CoV-2 Pandemic:
Jennifer Doudna and her lab members at Berkeley shifted their research focus from their ongoing projects to address the urgent need for better testing methods during the SARS-CoV-2 pandemic.

Collaboration and Formation of a Five Lab Academic Consortium:
Doudna and her lab collaborated with other labs at UC Berkeley, Gladstone, and UCSF to form a five lab academic consortium, fostering team science and pooling expertise to develop CRISPR-based diagnostic tests. The consortium, dubbed the “lab without walls,” brought together close to 50 individuals who dropped their original projects to work on this critical initiative.

Mammoth Biosciences’ Progress in CRISPR-based Diagnostics:
Doudna, as a co-founder of Mammoth Biosciences, highlighted the company’s advancements in CRISPR-based diagnostics. Mammoth Biosciences’ data demonstrated promising sensitivity results for their CRISPR-based test compared to commercially available SARS-CoV-2 tests.

Establishment of a Clinical Testing Laboratory with CLIA License:
Doudna acknowledged the crucial role of Ralph Green and colleagues at UC Davis in establishing a pop-up clinical testing laboratory at UC Berkeley early in the pandemic. The lab obtained CLIA (Clinical Laboratory Improvement Amendments) certification, allowing them to conduct testing in the Berkeley community and with healthcare partners. Recently, the lab received its independent CLIA certification from the state of California, and Dr. Petros Jancopoulos took over as medical director.

Future Plans for Testing CRISPR Diagnostics and Collaborations:
The lab’s access to the medical laboratory facility enables them to run FDA tests, report results to individuals and physicians, and compare the CRISPR diagnostic with robust PCR assays. The consortium is specifically focused on developing CRISPR-based diagnostics using the Cas13 protein, which detects RNA, directly targeting the SARS-CoV-2 RNA virus.

Background and Motivation:
Jennifer Doudna describes a collaborative effort to develop CRISPR-based tests for COVID-19 surveillance testing. The goal is to provide a fast, quantitative, and simple method for detecting SARS-CoV-2 RNA, especially in asymptomatic individuals.

Direct Detection with Cas13:
Cas13 proteins programmed with different CRISPR RNAs target multiple sequences within the SARS-CoV-2 genome. Activation of Cas13 leads to reporter cleavage and release of a detectable signal. Combining multiple guide RNAs improves speed and sensitivity. Sensitivity is lower than PCR tests but may be suitable for detecting highly infectious individuals.

LAMP-Cas13 Amplification:
LAMP (loop-mediated isothermal amplification) is used to amplify the target RNA sequence. The amplified DNA is converted back to RNA using T7 RNA polymerase. Cas13 is used to detect only the RNA containing the targeted viral sequence, improving specificity. Sensitivity is slightly better than direct detection with Cas13.

Nuclease Chain Reaction (NCR):
NCR amplifies the activity of Cas13 by utilizing the natural mechanism of CSM6 ribonuclease in bacteria. A secondary activator is cleaved by Cas13, which activates CSM6. CSM6 then cuts more RNA, producing more secondary activators, leading to a snowball effect. NCR provides fast detection and relatively sensitive limit of detection. Background signal is a challenge but can be mitigated.

Implementation and Future Directions:
The different test formats are being integrated into point-of-care devices for on-site testing. The goal is to have prototype devices operational by the end of 2020 or early 2021. Studies will be conducted at Berkeley and Gladstone under IRB approval.

Overview:
Jennifer Doudna, a renowned scientist, presented her work on CRISPR diagnostics, highlighting the contributions of her team and collaborators. She discussed the use of donated saliva and nasal swabs for testing, comparing the results with PCR assays conducted in a clinical laboratory.

Team Collaboration:
Doudna expressed gratitude to her team members, recognizing their dedication and flexibility in adapting to new projects. She acknowledged Gavin Knott, Brittany Thornton, Dylan Thomas, Jenny, Connor, Abby, and Enrique for their contributions to the CRISPR diagnostic development.

CRISPR Diagnostic Development:
Doudna mentioned the involvement of Shruti, Tina, and Noam, three talented postdocs, who worked under the guidance of Dave Savage and others to develop the nuclease chain reaction. She also recognized the contributions of her entire lab, including undergraduate researchers, who were unable to participate due to density restrictions.

Collaborations and Acknowledgements:
Doudna acknowledged the collaboration with Jill Banfield, an accomplished scientist, on fundamental CRISPR biology. She also extended gratitude to Emmanuel, with whom she initiated the CRISPR-Cas9 work many years ago. Doudna expressed appreciation for the funding sources and Adgene’s role in distributing CRISPR reagents at cost to nonprofits worldwide, facilitating access to the technology.

Questions and Discussion:
The presentation concluded with an introduction to Dr. Angela Haxu, who moderated the discussion and answer session. Dr. Green was the first to ask questions, expressing excitement and enthusiasm for the presentation.

Conclusion:
The presentation showcased the collaborative efforts of Jennifer Doudna’s team and highlighted the development of CRISPR diagnostics. The session concluded with a moderated discussion, allowing for questions and further insights from the audience.

Public Perception of Genetic Engineering:
Medical uses of genetic engineering may be more readily accepted than agricultural applications due to emotional reactions and mistrust associated with food manipulation. Biomedical uses, such as treatments for children’s diseases, evoke more trust and support.

Advice for Young Women in STEM:
Pursue your passions and interests in STEM fields. Seek supportive mentors who can guide and encourage you during challenging times. Perseverance is key as things may not always go as planned.

CRISPR-Cas Technology Applications:
CRISPR-Cas technology has potential for diagnostic capabilities, but its use in treatment, especially for infectious diseases like COVID-19, faces challenges. Delivery of CRISPR molecules into target cells remains a significant obstacle for therapeutic applications. Detection using CRISPR may be a more feasible option for this pandemic. Advancements in delivery strategies may enable effective and efficient cell delivery in the future.

CRISPR-Cas12a for Rapid and Accurate Pathogen Detection:
CRISPR-Cas12a has the potential to revolutionize diagnostic methods for various pathogens. It takes advantage of CRISPR’s natural ability for detection and multiplexing. This allows for rapid, specific, and sensitive detection of pathogens, including viruses like COVID-19. CRISPR-Cas12a can detect multiple viruses simultaneously in a single sample.

CRISPR-Cas12a for Gene Editing in Monogenic and Polygenic Diseases:
CRISPR-Cas12a can be used for gene editing in both monogenic and polygenic diseases. In monogenic diseases, CRISPR-Cas12a can precisely target and correct genetic defects. For polygenic diseases, CRISPR-Cas12a can potentially be used to make multiple edits simultaneously, though it is more challenging. Ongoing research is exploring the use of CRISPR-Cas12a for CAR T cell therapy.

Addressing Off-Target Effects and Safety Concerns:
Off-target editing can occur if guide RNAs have close matches to undesired positions in the genome. Careful design of guide RNAs and limiting the amount of genome-editing molecules can minimize off-target effects. CRISPR-Cas12a can be used safely if proper attention is paid to off-target effects, especially in clinical use.

CRISPR-Cas12a in Embryos:
Editing embryos using CRISPR-Cas12a can be challenging and requires further research. Undesired outcomes and limited understanding of DNA repair mechanisms in embryos need to be addressed.

Potential for Other Gene Editing Mechanisms:
CRISPR-Cas12a is part of a larger toolbox of gene editing mechanisms found in prokaryotic cells. Cas12a and Cas13 are examples of other Cas proteins with unique capabilities. Ongoing research is exploring the potential of these mechanisms for various applications.

CRISPR-Based Gene Editing Toolbox:
Bacterial genomes are being explored for diverse gene editing systems and enzymes. Academic labs are also contributing to the study and engineering of gene editing mechanisms. The goal is to create a virtual toolbox of gene editing tools for various genetic manipulation needs.

CRISPR DNA Integration in Bacteria:
The mechanism of CRISPR DNA integration into the bacterial genome before phage lysis is not fully understood. CRISPR integrase’s reaction mechanism is known, but its role in the viral life cycle is still unclear. Some defective bacteriophage may allow bacteria to acquire functional spacer sequences in the CRISPR array, providing protection to those cells. This phenomenon is thought to occur in natural settings, where some cells survive phage infection and pass on their immunity.