CRISPR-Cas Mechanism

• Cas1 and Cas2 search DNA for the sequence NGG, known as a PAM.
• Cas1 and Cas2 cut a 20 base pair piece of DNA upstream of the PAM, this piece is called a protospacer.
• Cas1 and Cas2 insert the protospacer into the CRISPR array at the 5' end.
• RNA polymerase transcribes the CRISPR array into pre-CRISPR RNA.
• Tracer RNA binds to the repeat regions of the pre-CRISPR RNA.
• RNAase cuts the repeat region and the tracer RNA, forming gRNA.
• Cas9 picks up the gRNA, forming a CRISPR-Cas complex.
• gRNA guides Cas9 to the correct location on the DNA.

Defense Mechanism

• If a cell is reinfected, Cas9 searches for a PAM.
• Upon finding a PAM sequence, Cas9 unwinds the DNA and compares it to the bases upstream of the PAM with its gRNA.
• If the sequences are complementary, it cuts the viral DNA 4 bases upstream of the PAM within the protospacer region.
• Enzymes within the bacterium repair the cut viral DNA.
• Repair mechanisms can introduce errors like nucleotide additions, deletions, or insertions in the middle of the viral gene.
• These mutations often render viral genes non-functional, which is beneficial in bacteriophage infiltration.
• If no mutation occurs after the cut, the gRNA repeats the process until DNA repair mechanisms induce a mutation, inactivating the virus.

Gene Editing Process

• Synthetic sgRNA with a complementary spacer is created in a lab to target DNA.
• A Cas9 enzyme with an appropriate target PAM sequence is obtained.
• Cas9 and sgRNA are mixed, creating the CRISPR-Cas9 complex.
• The sgRNA-Cas9 mixture is injected into a specific cell, such as a zygote.
• Cas9 identifies the target PAM sequence and verifies the alignment of sgRNA with the DNA.
• Cas9 cuts the selected DNA sequence, creating a blunt end cut.
• The cell attempts to repair the blunt end cut; new nucleotides may be introduced at this site.
• Scientists may inject specific nucleotide sequences into the cell to ligate into the gap.

PCR (Polymerase Chain Reaction)

• The purpose of PCR is to amplify DNA.
• Step 1: Identify the DNA region of interest to be amplified.
• Step 2: Identify a 25 base sequence at the 3' end of each strand that flanks the region of interest.
• DNA primers, complementary to the 25 base sequences, are synthesized.

PCR Machine Temperature Steps

• 95 Degrees Celsius: DNA is denatured, breaking hydrogen bonds between complementary nucleobases and creating single strands.
• 50 Degrees Celsius: Short primers quickly anneal to the region of interest before the two DNA strands can re-anneal.
• 72 Degrees Celsius: Taq polymerase attaches to both primers, adding complementary base pairs to create a complementary DNA strand, Taq polymerase optimal temperature, extends from 5' to 3' direction.
• This amplification process is repeated 25-30 times to produce many copies of the DNA region of interest.

Gel Electrophoresis

• Agarose is melted and poured onto a tray to create wells at one end of the gel.
• As the gel sets, it forms a porous structure that allows DNA to move through it.
• The gel tray is placed in an electrophoresis chamber with a buffer solution.
• The buffer allows for the conduction of electricity.
• The gel is placed in an electrophoresis chamber and covered with buffer.
• Connect a negative electrode to the side with the DNA samples (wells) and a positive electrode on the opposite side.
• The gel needs to be stained to make the DNA visible.
• Smaller DNA fragments migrate faster and further away from the negative electrode.
• Larger DNA fragments migrate slower and remain closer to the wells.

Recombination

• Recombination joins DNA from different sources.

Transformation

• Transformation changes bacteria by introducing a plasmid.

How to Make Recombinant Plasmids

• Endonuclease enzymes cut DNA at specific restriction sites.
• Every restriction enzyme recognizes a specific restriction site and typically involves a 6 base palindrome sequence that requires sticky ends.
• Step 1: Choose an endonuclease to cut both upstream and downstream of the gene, resulting in sticky ends.
• Step 2: The plasmid should have two genes that encode for easily observable traits and must contain the same restriction site that the endonuclease uses.
• Step 3: Using Restriction Endonuclease: Create cDNA to remove introns from DNA using reverse transcriptase to convert mRNA into DNA.
• Both the plasmid and the human gene (from cDNA) are cut, creating complementary sticky ends that are likely to join.
• DNA ligase creates a sugar-phosphate bond along the DNA backbone if joined.
• Only some plasmids incorporate the human gene.
• Step 4: Mix both recombinant and normal plasmids with bacteria; bacteria naturally uptake plasmids.
• Bacteria that uptake a plasmid are now transformed bacteria.
• Step 5: Culture bacteria on an agar plate that expresses the observable trait without the restriction site.
• Bacteria that don't express the trait do not contain a plasmid.
• Transfer the remaining bacteria to another agar plate with an environment that encodes for the second observable gene with a restriction site.
• Bacteria that don't express this second trait have the recombinant plasmid.
• These bacteria now have the recombinant plasmid.
• Culture the selected bacteria in a fresh agar plate.

Bioethics Approaches

• Consequence-Based: Aims to maximize positive outcomes and minimize negative effects by considering the consequences. Focus is on achieving the greatest good for the greatest number of shareholders.
• Duty/Rule-Based: Focuses on acting in accordance with a set of rules and responsibilities, with less regard for the consequences. Driven by a fundamental duty to act in a certain way.
• Virtues-Based: Driven by the character of the person instead of rules and consequences; emphasizes the moral nature of the individual. Provides guidance to behave in the the way a morally good person would hope to.

Ethical Concepts

• Integrity: Commitment to knowledge by encouraging individuals to act truthfully and honestly when finding and presenting results.
• Justice: Commitment to fairness, involving consideration of different people's opinions and positions, especially those directly affected or marginalized.
• Beneficence: Commitment to maximizing benefits, acting in a way that benefits others, promotes personal well-being, and the good of other persons."
• Minimizing harm as much as possible, acting in a way to remove as much harm as possible.
• Respect: Commitment to consideration and considering the values of others, including personal welfare, beliefs, and freedom by prioritizing the the freedom of others to make their own decisions.

RNA Types

• mRNA (messenger RNA): Carries genetic information from DNA in the nucleus to the ribosomes in the cytoplasm and acts as the blueprint for protein synthesis.
• rRNA (ribosomal RNA): Makes up the structure of ribosomes, where proteins are synthesized, helps bind mRNA and tRNA together during protein synthesis, and catalyzes the formation of peptide bonds between amino acids.
• tRNA (transfer RNA): Brings amino acids to the ribosome during protein synthesis. Also has an anticodon that matches to the codons on mRNA, helping match the correct amino acids to the growing protein chain.

Universal Triplet Code

• Universal triplet code is the genetic code is consists of sequences of three nucleotides, called codons, on the mRNA that specifies a particular amino acid or a stop signal during protein synthesis.
• The genetic code is the same in almost all living organisms.
• The code is degenerate because multiple codons can code for the same amino acid and helps protect against mutations.

Protein Structure

• Primary Structure: the sequence of amino acids in a polypeptide chain.
• The order of amino acids is determined by the gene encoding the protein
• Peptide bonds hold the structure together
• Secondary Structure: chain folds into alpha-helices and beta-pleated sheets by hydrogen bonds of backbone structures
• Tertiary Structure: 3D shape of a protein through interactions between the side chains, including hydrophobic interactions, ionic bonds, hydrogen bonds, and disulphide bridges and determines the protein's specific function.
• Quaternary Structure: some proteins made up of more than one polypeptide chain, and the structure refers to the arrangement of these chains to create a functional protein complex.