Background of CRISPR:
Research in microbiology laboratories revealed a possible adaptive immune system in bacteria. CRISPR stands for Clustered Regularly Interspaced Short Palindromic Repeats. Bacteria and archaeal cells integrate viral DNA into their genome, providing immunity against viral infections. CRISPR-associated (Cas) proteins use RNA copies of the integrated viral DNA to detect and cleave viral DNA or RNA.

CRISPR Mechanism in Bacteria:
Bacteria acquire small pieces of viral DNA into a specific locus in their genome. CRISPR locus contains repeated sequences flanking integrated viral DNA, called spacers. RNA copies of the integrated viral sequences are made and processed into individual units. These RNA units combine with tracer RNA and Cas9 protein to form a surveillance complex. The surveillance complex searches for sequences matching the guide RNA. Cas9 cuts double-stranded DNA upon sequence match, triggering destruction of the DNA.

CRISPR-Cas System Using Cas9:
One type of CRISPR-Cas system uses Cas9 protein for DNA interference. CRISPR-acquired immunity involves three steps: adaptation, expression, and interference. Adaptation: Integration of viral DNA into CRISPR locus. Expression: Production of RNA copies of the integrated viral sequences. Interference: Cleavage of viral DNA or RNA using Cas9 and guide RNA.

Background:
Jennifer Doudna and her colleagues investigated the third step of the CRISPR pathway called interference, which involves RNA-guided detection of viral DNA. In collaboration with Emmanuelle Charpentier, they aimed to understand the function of the CRISPR-Cas9 protein in protecting cells from viral infection.

Dual RNA-Guided System:
CRISPR-Cas9 was found to be a dual RNA-guided protein, requiring both a CRISPR RNA for target DNA recognition and a tracer RNA for assembly with Cas9. This dual RNA system naturally guides Cas9 to target DNA sequences and make a cut in the double helix of DNA.

Engineering Single Guide RNA:
Researchers engineered a single guide RNA that combined both the targeting information and the structural requirement for Cas9 assembly into a single RNA molecule. This single guide RNA allowed Cas9 to be programmed and directed to cleave double-stranded DNA at a desired sequence.

Key Experiment:
Martin Jinek performed an experiment to test the single-guided Cas9 system. Guide RNAs were designed to recognize different sequences in a plasmid DNA, and Cas9 was added to cleave the DNA at those sites. The cleaved DNA products were separated using an agarose gel system, revealing fragments of DNA at different positions representing the different sizes of the cleaved DNA.

Harnessing CRISPR-Cas9 for Technology:
The ability to engineer Cas9 as a two-component system for DNA double-stranded cutting opened up possibilities for harnessing it as a technology. CRISPR-Cas9 could be used in eukaryotic cells to introduce double-stranded breaks in the genome, triggering repair pathways that could lead to changes in the genome.

Double-Strand Break Repair in Eukaryotic Cells:
In eukaryotic cells, double-stranded breaks in the genome are detected and repaired, either by non-homologous end joining or by integrating DNA homologous to the sequence flanking the break. CRISPR-Cas9’s ability to induce double-stranded breaks could be used to trigger these repair pathways and introduce changes to the genome at precise locations.

Genome Engineering:
The precise double-stranded break generated by Cas9, coupled with the repair pathways in eukaryotic cells, allows for the introduction of changes to the genome at specific locations. This process, known as genome engineering, enables scientists to make targeted modifications to the DNA sequence of an organism.

Cas9 Structure and Mechanism:
Cas9 protein holds orange guide RNA and a blue double helical DNA molecule. Cas9 uses an RNA guide to interact with DNA, forming an RNA-DNA helix inside the protein. Cas9 undergoes a large conformational change as it holds on to DNA and catalyzes cutting.

Cas9 Dynamics:
Cas9 is a highly dynamic protein that can bind and release DNA very quickly. Cas9 can search through large stretches of DNA to find a sequence that matches the guide RNA.

Somatic and Germline Editing:
Genome editing can be conducted in somatic cells or germ cells. Somatic cell edits are not heritable, while germline edits can be passed on to offspring. Germline editing raises ethical and societal issues and requires careful consideration.

Applications in Human Health:
CRISPR-Cas9 can correct disease-causing mutations in somatic cells, offering potential treatments for genetic diseases. Germline editing could advance future applications, but it must be managed carefully and transparently.

Diversity of CRISPR Systems:
Nature offers a wide variety of CRISPR systems, driving fundamental biology research and potential technological applications. New CRISPR-Cas systems like CRISPR-Cas5 have been discovered, offering unique properties and possibilities.

Other Biochemical Activities of CRISPR-Cas Proteins:
CRISPR-Cas proteins have diverse biochemical activities beyond DNA cutting, such as RNA editing and epigenetic regulation. These activities could lead to new genome editing strategies and applications.

CRISPR-Cas for RNA and DNA Detection:
Researchers discovered that Cas13 proteins can detect RNA molecules at picomolar levels by cleaving small pieces of RNA upon recognition of an RNA sequence. Cas12 proteins can cleave single-stranded DNA molecules upon recognition of a double-stranded DNA target.

Cas12 Activity for Human Papillomavirus Detection:
Cas12 activity was used to detect the human papillomavirus in human patient samples and distinguish between different strains of the virus.

CRISPR-Cas Detection in SARS-CoV-2 Pandemic:
CRISPR-Cas detection methods are being used to identify SARS-CoV-2 samples in patient saliva or nasal swabs, with the potential for a simple readout mechanism using cell phones.

Programmability of CRISPR-Cas Enzymes:
The programmability of CRISPR-Cas enzymes allows for easy adaptation to detect different viruses and pathogens, making it a valuable tool for future pandemic preparedness.

Fundamental Research Driving CRISPR-Cas Technology:
Delivery and control of CRISPR-Cas proteins into specific cell types are key areas for future research, along with fundamental studies to further advance the field.

Endless Possibilities and Real-World Applications:
CRISPR-Cas technology has the potential to solve real-world problems in human health and the environment, with endless possibilities for future applications.