Introduction:
Jennifer Doudna, a renowned scientist, expresses her gratitude to the Royal Swedish Academy of Sciences, her family, colleagues, and lab members for their support. She highlights the honor of sharing her research on CRISPR-Cas9, a revolutionary genome editing technology, with the audience.

Origin of CRISPR Ideas:
Doudna traces the origins of CRISPR research back two decades, when studies revealed the presence of an adaptive immune system in bacteria. This system, called CRISPR, enables bacteria and archaeal cells to protect themselves against viral infections. CRISPR works by integrating small pieces of viral DNA into the organism’s genome and using that information to detect and cleave viral DNA or RNA using CRISPR-associated (Cas) proteins.

CRISPR Mechanism in Bacteria:
Doudna explains the process of CRISPR operation in bacteria, where viruses inject their DNA into bacterial cells. The bacteria acquire small pieces of viral DNA into the CRISPR locus, a specific site in the genome containing repeated sequences flanking integrated sequences from viruses called spacers. An RNA copy of the integrated sequence is generated, processed into individual units, and combined with a tracer RNA and Cas9 protein to form a surveillance complex. This complex searches the cell for sequences matching the guide RNA sequence, and upon a match, Cas9 cuts double-stranded DNA, leading to the destruction of the DNA in the bacterial cell.

Evolution of CRISPR Systems:
CRISPR has evolved over time in bacteria, diversifying into various forms that operate effectively in different organisms. Doudna focuses on one particular type of CRISPR-Cas system that utilizes the Cas9 protein.

Three Steps of CRISPR-Acquired Immunity:
Doudna outlines the three steps involved in CRISPR-acquired immunity: adaptation, expression, and interference. Adaptation: The bacteria acquire a small piece of foreign DNA from a virus and integrate it into the CRISPR locus. Expression: The integrated viral DNA sequence is transcribed into RNA and processed into individual units called guide RNAs. Interference: The guide RNAs, along with tracer RNA and Cas9 protein, form a surveillance complex that searches for and cleaves DNA sequences matching the guide RNA sequence, leading to viral DNA destruction.

Conclusion:
Doudna concludes her introduction by highlighting the significance of CRISPR-Cas9 technology and its potential to revolutionize various fields, including medicine, agriculture, and basic biology.

Background:
CRISPR-Cas9 is a protein system found in bacteria that provides protection against viral infections. In 2011, Jennifer Doudna and Emmanuelle Charpentier began collaborating to study the function of CRISPR-Cas9.

CRISPR-Cas9 Function:
CRISPR-Cas9 is a dual RNA-guided protein. One RNA molecule (CRISPR RNA) directs Cas9 to a specific DNA sequence. A second RNA molecule (tracer RNA) interacts with CRISPR RNA and is required for Cas9 assembly. Together, these two RNAs guide Cas9 to DNA sequences, where it makes a cut in the double helix of DNA.

Significance:
Understanding how CRISPR-Cas9 works in nature led to the development of a single guide RNA that combines the targeting information from both CRISPR RNA and tracer RNA. This simplified system opened up new possibilities for engineering CRISPR-Cas9 for various applications.

Protein Engineering and Cleavage Activity:
Cas9 protein can be programmed with single guide RNAs to cleave double-stranded DNA at a desired sequence. Single-guide Cas9 can cleave different sequences of DNA, allowing targeted DNA double-stranded cutting.

CRISPR-Cas9 Technology for Eukaryotic Cells:
Double-stranded breaks introduced by Cas9 in eukaryotic cells trigger DNA repair pathways. Non-homologous end joining can introduce small disruptions, while integration of homologous DNA can incorporate new genetic information. Cas9-mediated double-stranded breaks enable precise genome editing in eukaryotic cells.

Molecular Mechanism of Cas9:
Cas9 holds onto its guide RNA and the target DNA molecule, forming an RNA-DNA helix. Cas9 undergoes a large conformational change as it binds to DNA and catalyzes cutting. The dynamic nature of Cas9 allows it to search for and recognize the target DNA sequence.

Cas9’s Interactions with DNA:
Cas9 can bind and release DNA quickly, allowing it to search through large stretches of DNA for a match with the guide RNA. When Cas9 recognizes a match, it melts apart the DNA strands to form an RNA-DNA helix, triggering DNA cleavage.

Summary:
Cas9, engineered as a single-guide RNA-guided DNA cleaver, has revolutionized genome editing technology. Its ability to introduce precise double-stranded breaks in DNA enables targeted changes to the genome, offering potential applications in various fields, including medicine, agriculture, and biotechnology.

Genome Editing Applications:
Genome editing can be applied across various fields, including fundamental research, public health, agriculture, and biomedicine. Genome editing can be conducted in somatic cells (fully differentiated cells) and germ cells (cells with pluripotency).

Impact of Genome Editing:
Somatic cell editing: Changes are not heritable and affect only one cell, tissue type, or individual organism. Germ cell editing: Changes have the potential to be heritable and affect not only the individual but also their progeny.

Ethical Considerations:
Germline editing in humans raises significant ethical and societal issues due to its potential to introduce permanent changes that affect future generations. Responsible use of CRISPR-Cas9, particularly in the human germline, requires transparency and careful consideration.

Somatic Cell Editing Opportunities:
Correction of disease-causing mutations: CRISPR-Cas9 technology has been successfully used to correct disease-causing mutations in humans, such as the mutation responsible for sickle cell anemia. Treating genetic diseases: CRISPR-Cas9 technology offers promising avenues for treating genetic diseases by correcting disease-causing mutations in patients.

CRISPR-Cas9 Germline Editing:
CRISPR-Cas9 technology has the potential to impact human health significantly. Germline editing using CRISPR-Cas9 raises ethical and safety concerns. International efforts are underway to control the use of CRISPR-Cas9 in germline editing. A recent report discusses the science, technology, and criteria for using CRISPR-Cas9 in the human germline.

Diversity of CRISPR Systems in Nature:
CRISPR systems exhibit incredible diversity in nature. Studying these systems enhances our understanding of fundamental biology. It also enables the harnessing of CRISPR technologies in various organisms.

Discovery of CRISPR-Cas5:
Phages, the viruses that bacteria use CRISPR against, can carry their own CRISPR-Cas systems. CRISPR-Cas5 is a tiny phage-encoded protein with RNA-guided DNA cutting capabilities. This protein’s small size and easy delivery into eukaryotic cells make it promising for future applications.

Biochemical Activities of CRISPR-Cas Proteins:
CRISPR-Cas proteins have diverse biochemical activities beyond DNA editing. Cas13 is a class of CRISPR proteins that naturally target RNA. Cas13’s RNA-targeting activity can be harnessed as a detection mechanism. Researchers have developed a method to use Cas13 to detect specific RNA sequences by attaching fluorophores to cleaved RNA fragments.

Detection of RNA and DNA Molecules:
CRISPR-Cas systems can be utilized for the detection of RNA and DNA molecules with high sensitivity. RNAse Alert, an off-the-shelf kit, was used for RNA detection at picomolar levels. Cas12, another CRISPR-Cas enzyme, can cleave single-stranded DNA molecules upon recognition of a double-stranded DNA target. Cas12 activity has been applied for the detection of human papillomavirus in patient samples and distinguishing between different strains.

Specificity and Future Pandemic Preparedness:
CRISPR-Cas systems offer specificity in identifying viral signals in patient samples. CRISPR-Cas detection has been used for identifying SARS-CoV-2 in patient saliva and nasal swabs during the COVID-19 pandemic. The programmability of CRISPR-Cas enzymes enables future pandemic preparedness by adapting to new viral threats.

RNA-Guided Gene Regulation:
RNA-guided gene regulation is fundamental to the function of CRISPR-Cas systems in bacteria. This mechanism also underlies the use of CRISPR-Cas technology for genome editing and other applications.

Delivery and Control:
Delivery and control of CRISPR-Cas9 and related proteins into cells are crucial for genome editing applications, including in human patients. Ongoing research focuses on developing better delivery methods to target specific cell types.

Future Possibilities:
The potential applications of CRISPR-Cas systems are vast and hold promise for solving real-world problems in human health and the environment. Fundamental research will continue to drive advancements in the field, leading to new discoveries and applications.