Introduction:
During the explorers program we learned complicated concepts such as CRISPR I never thought I would understand. CRISPR is a technology that allows scientists to edit genes using
a protein called Cas-9 which traverses through DNA sequences in order to bind a specific gRNA into a gene. CAS-9 traverses through DNA sequences until it finds a PAM sequence (nucleotide+GG). Once it finds PAM, Cas9 unwinds the DNA to see if the gRNA and the unwound DNA partially matches. If it matches, Cas9 makes a double stranded cut into the DNA 3 spaces downstream and binds the gRNA to the unwound DNA. The cut DNA heals after time and forms a mutation. We put our knowledge to use by conducting an experiment.
Experiment:
The objective of our experiment was to observe the changes in a gene after it undergoes a Cas9 reaction. To do this we added PCR grade water, Cas9 buffer, Cas9 nuclease in two microtubes.
Tubes of Cas9
In each microtubes we put two different gRNAs, mixed the microtubes and incubated them for 10 minutes.
Microfuge used to mix microtubes
This created the perfect environment for a Cas9 reaction to occur. We then added B2M PCR product (a DNA sample that was cloned) to each tube and set both to incubate for 15 minutes at 37 celsius on a heat block. This starts the Cas-9 reaction in which the DNA is unwound, cut and changed. We add proteinase K and incubate the tubes for 10 minutes which helps degrade proteins like Cas9. The next step of the experiment was to make 1% agarose gel and load it into a gel box. We mixed 50 ml of buffer and 0.05 g of agarose and microwaved it. We poured the gel onto a gel tray, placed the gel tray in a casting system, placed a comb into the gel to create wells and waited 15 minutes for it to solidify.
Placing gel box in casting system
The casting system allows us to observe the differences in our samples as they travel through the gel. We then loaded the 4 wells with 100bp DNA Ladder both Cas-9/gRNA samples and B2M PCR-product as a control. To make the B2M PCR product you need to mix pcr water b2m pcr product (a sample of cloned DNA)and dye. Once the wells were loaded, we turned on the machine, waited 25 minutes and recorded our results.
Results/Reflection:
(Referring to second photo)The first well with many bands is the DNA ladder, the second well with one band is the original B2M PCR product and the last two wells have the altered DNA. The altered PCR product has one more band than the original meaning that the gRNA was bonded into the DNA and the experiment was successful.
My group’s results
Another group’s results
I really enjoyed this experiment as it helped me understand the process scientists use to make CRISPR and how they use it.
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