CRISPR Cas9 + Recombinant Plasmids

What is the significance of PAM when looking at the function of CRISPR in bacteria?

1. PAM is required by Cas9 to bind to and cleave DNA.
2. This helps bacteria in distinguishing self and nonself, making it efficient as only foreign DNA contains PAM and can be cut.

Describe how the CRISPR-Cas9 system works in naturally occuring bacteria

This is a naturally occuring immune defense mechanism against bacteriophages.

1. Exposure - The bacteriophage will bind to the bacteria and inject its DNA into the cell. Cas 1 + Cas 2 enzymes will cut out a section of DNA from the virus and incorporate the section into the CRISPR gene as a spacer.

2. Expression: The CRISPR gene is transcribed and combines with tracerRNA to form guideRNA. The guideRNA binds to Cas 9 (an endonuclease) to form the CRISPR-Cas9 complex. This complex will float around the cell until it encounters a complementary viral DNA where it will bind to it and cut it.

3. Cell repair mechanisms will attempt to repair the cut, however will silence it as a result.

How can CRISPR-Cas9 be used in genetic editing?

1. Scientists would create single guide RNA (sgRNA) complementary to the target sequence (gene to be edited).
2. This would be combined with a suitable Cas9 enzyme, with a PAM suitable to the target
3. This would be delivered to the target cell
4. This complex will bind to the target sequence complementary to the guideRNA and make a cut
5. Cell repair mechanisms will attempt to repair the cut, however will be silenced instead, unless is repaired using the target gene for insertion

Remombinant Plasmids: 1st step

Making a Recombinant Plasmid:
Obtain a gene of interest and plasmid, cut with the same endonuclease  to create sticky ends.
Use DNA ligase to join the gene and plasmid.
Possible outcomes include recombinant and non-recombinant plasmids.

Recombinant Plasmids: What do we mean "transforming bacteria"?

Putting the plasmid inside the bacteria.

Recombinant plasmids: Heatshock

Uses Ca2+ ions, ice and heat to facilitate DNA uptake. 

1. The sample is heated
2. After 25-45 seconds, the sample is chilled
3. Sample is spread onto antibiotic rich agar plate.

Recombinant Plasmids: Electroporation

Passes an electric current through the solution containing bacteria and recombinant plasmids. This creates pores in the membrane, allowing for the DNA to pass through.

Recombinant Plasmids: 3rd Step

Selecting Bacteria:
Identify bacteria with recombinant plasmids using antibiotic selection and reporter genes.

Three groups: Without plasmid, with non-recombinant plasmid, and with recombinant plasmid.

Antibiotic resistance indicates transformed bacteria.

Reporter gene function changes indicates recombinant plasmids, often through colour change (NO GLOW).

Selected bacteria colonies produce the desired protein for purification.

Recombinant Plasmids: 2nd step

Transforming Bacteria:
Electroporation and heatshock methods introduce plasmids into bacteria.

DNA Profiling: How do we tell DNA samples apart and match them to people?

1. All the DNA samples from different individuals will be unique.
2. When cut with the SAME endonuclease, each sample will be CUT at different points/recognition sites
3. This will create fragments of different sizes
4. This is visualised as unique band patterns when run through a gel experiment therefore matches a person with their DNA sample.

Gel electrophoresis: Factors affecting DNA Movement

Voltage: higher voltage increases movement but may distort seperation
Gel Composition: Denser gels slow larger fragments
Buffer Concentration: affects electrical conductivity and DNA movement
Time: Longer runs allow DNA to travel further, but excessive time may cause DNA to move out of the gel.

Insulin Production

Isolation of Insulin genes: mRNA strands coding for the A and B chains of insulin are isolated and used as templates to synthesise complementary DNA (cDNA) via reverse transcription. This ensures introns are not present in the insulin genes.

Preparation of Plasmids and DNA: the plasmid and insulin DNA are cut with the same endonuclease creating sticky ends.

Formation of Recombinant plasmids: The insulin DNA sequences (A and B) are separately inserted into plasmids containing beta-galactosidase gene for detection.

Introduction: Recombinant plasmids are inserted into E.Coli bacterial cells through heatshock/electroporation transforming the bacteria. Each plasmid is introduced into separate bacterial strands to produce the A and B chains separately.

Selection of Transformed bacteria: bacteria are grown on antibiotic agar plates, allowing only recombinant plasmids to survive. Successful incorporation of the insulin genes is confirmed using beta-galactosidase activity.

Expression and Protein Extraction: Transformed bacteria are cultured, producing fusion proteins consisting of beta-galactosidase and insulin chains.

Fusion proteins are extracted and endonucleases cleave the insulin chains (A and B) from beta-galactosidase portion.

Purification and Assembly: The insulin A and B chains are purified then chemically combined.

Disulfide bonds are formed between the chains, resulting in functional human insulin ready for medical use.