TL;DR
No, probably not. But i possibly could make my eyes glow-in-the-dark. Although that might not be a good idea.
Intro into gene editing with CRISPR-CAS9
Marta de Menezes is a bio-artist who workes with CRISPR-CAS9 gene editing. Inspired by her lecture today at the Bio-Hack Academy, i want to learn more about CRISPR-CAS9, its possibilities, and learn how to use it.
What is CRISPR-CAS9?
CRISPR-CAS9 is a method used for gene editing which allows us to relatively easy alter characteristics in living organisms. This method is originally found in bacteria to protect them from enemy DNA injected by bacteriophages.
Performing CRISPR-CAS9 in the lab
Prior to performing the CRISPR/Cas9 process i practiced with pipetting. Because we have to work with samples as small as 2 μL it is important to able to pipet those quantities.
Part 1: Synthesis of sgRNA
First we synthesized the sgRNA:
We prepared the Diluted Ribonuclease Inhibitor on day one.
Because something went wrong during purification i performed the experiment twice. For the second experiment i didn’t executed part one, but got the sgRNA (unpurified) from another group.
Experiment 1: sgHL3 & sgHL4
On day two we performed the other steps in part 1 of the protocol. I added both the sgHL3 and sgHL4. The total volume of my synthesized sgRNA (before purifying) is 55 μL.
Experiment 2: sgHL3
The sgRNA for experiment two was made following all the steps from the protocol part 1, using sgHL3
Part 2: Purification of sgRNA
Experiment 1: Monarch kit
Instead of following the protocol, we used the monarch kit and it’s description.
Because the initial sample i made was 55 μL i changed the measurements used in the Monarch protocol:
Step 1: 110 μL RNA cleanup binding buffer
Step 2: 165 μL ethanol
Step 3: As stated in protocol
Step 4: As stated in protocol.
Step 5: As stated in protocol.
!! Then i discovered a mistake !!
The RNA cleanup wash buffer should have been diluted with ethanol as stated on the front of the Monarch protocol.
I missed this step. My RNA cleanup wash buffer is 4x as strong as needed. This probably made the cleaning too strong or too weak. Because i didn’t wanted to discard the project i continued.
Step 6: As stated in protocol.
Step 7: 20 μL Nuclease-free H20 (Kit T2040)
I stored the mixture in ice until ready for part 3.
Experiment 2: Zymo Research kit
I started with 50 μL unpurified RNA to which i added 150 μL RNA cleanup binding buffer (from the previous used Monarch kit) instead of TRI Reagent.
I followed the RNA cleanup protocol as shown above.
Part 3: In vitro cleavage of DNA using CRISPR/Cas9
I executed the same process in part 3 for both the samples i made.
I followed the protocol i started with:
After adding the Proteinase K and incubating the samples i stored them in the freezer for later evaluation.
The tube marked with :( is the Monarch kit.
The tube marked with :) is the Zymo research kit.
Results
Soon more.
