The MEFs that we have in the lab (as of 2021) are Wildtype (Purchased, ATCC), GCN2 KO (Purchased, ATCC), and ATF4 KO (using IDT CRISPR, in-house). This protocol aims to generate a step-by-step explanation on how to create a knockout MEF cell line with applicability to other cell culture lines.

The protocol here utilizes NEON electroporation with electric shock specs to match MEF cells. The NEON electroporator is located in the Cell Culture room. Before using, you but be trained by a member in Dr. Liu's lab and follow all instructions outlined in training regarding the machine's reserving and operation. The protocol also utilizes Fluorescent activated cell sorting (FACS) in the Flow Core with Wade Johnson. Before reserving a time and starting your experiment, you should talk with him to make sure that you two are on the same page when it is time to sort your CRISPR positive cells into your collection plates.

IDT required reading: https://www.idtdna.com/pages/products/crispr-genome-editing

This protocol uses tracrRNA tagged with Atto550 so that the cells can be sorted with the FACS. Thus, we also need crRNAs. There are many approaches to choosing crRNAs, which serve as the Cas nuclease guide to the specific DNA sequence that you want to cut. If two Cas nucleases with two difference guide sequences are present and working, then a section of DNA can be removed when both of the nucleases make a cut and when the DNA repair mechanism cannot use the removed section during the subsequent repair. An effective strategy (which Blaise used) utilized two sets of crRNAs. AA and AC crRNAs in his ATF4 KO experiment were located close to each other, which AB and AE were located close to each other, but the distance between the two pairs was about 400-500 base pairs. So, if in one electroporation you used AA and AB guides, you would theoretically see your PCR band decrease 400-500 base pairs (whatever the distance is between these two guides). In Blaise's successful experiment, he used AA+AB, AA+AE, AC+AB, and AC+AE for his gene of interest. Calculating the difference between the amplified length of DNA after PCR between the WT lines and the successfully active and specific CRISPR electroporated lines. It is important to consider the off-site effects and specificity of your crRNAs. Before ordering, you can assess guides for these characteristics for an educated choice on the IDT site. All the material for this experiment can be purchased through IDT.

Once the nuclease is fully assembled (referred to as the RNP complex), it must be transfected into the cells. The best method for this is either lipofection or electroporation with MEF cells. NEON electroporation is readily available and cheaper. The 10ul kit is recommended as you will use fewer reagents and expensive proteins when conducting your experiment (which is especially helpful as this process might not work the first time you do it!). 100ul kit results in a lot of electroporated cells! Both the 10ul tip and the 100ul tips are compatible with the NEON pipette under control by the Liu lab.

Once the cells have been electroporated with the RNPs, have them sit-down overnight. The next day, if your cells are alive, that is when you will FACS them with Wade into 96 well plates to grow-up from single cells. Not all of the cells will grow up, and in fact, only a few may. And of those few, some of them will be wildtype, heterogeneous, or have a successful knockout on both chromosomes. Of the successful bunch, the cells may have off-site damage that we won't be able to quantify to their genome from overly-active CRISPR, and there might be visible growth defects or high densities of suspended, dead cells. To assess the CRISPR's effectiveness in your MEF population, the MEF populations will be PCRed before FACS sorting. This will show the wildtype band and the KO band (if the CRISPR worked). PCR-ing the populations grown-up from the single cells will give you the ability to assess if your population grew-up from a cell that had the genes knocked out on both chromosomes.

Pre-protocol:

• Discuss experiment with Wade Johnson, or whoever will be doing the FACS with you. Note any sample preparation and collection.
• Design IDT crRNAs and primers. Order these, the associated Cas protein, tracrRNA, and the electroporation enhancer.
• Ensure you are trained on the NEON. Reserve a time to use the NEON. Pick up the pipette from the Liu Lab.
• Resuspend all contents ordered from IDT to their required concentrations. The crRNA and tracrRNA must be suspended to a concentration of 200uM in IDT buffer.

Protocol: Assemble the RNP.

1. Form the guide RNA by fusing the crRNA and the tracrRNA. For each 10ul reaction, combine 2.2ul crRNA suspension and 2.2ul tracrRNA suspension in 5.6ul IDT buffer in a flip-top tube.
2. Heat to 95 degrees celsius for 5 minutes.
3. Let cool to room temperature.
4. Suspend 0.3ul Cas9 in 0.2ul resuspension buffer R for each 10ul electroporation.
5. Combine 0.5ul diluted Cas9 with 0.5ul of your guide RNA.
6. Incubate at room temperature for 15 minutes.

Protocol: Perform electroporation

1. Add 0.2ul electroporation enhancer to 1.8ul IDT buffer.
2. Prepare the culture plate to receive cells after electroporation by adding media and warming in the incubator. For 10ul electroporation, 24-well plates worked well to seed in afterward.
3. Prepare the NEON by setting it up as trained to do so. Add 3ml buffer E to the NEON tube, and set the electroporation parameters. For MEF cells, 1350 V, 1 pulse, and 30ms pulse width work very well. There are alternate specifications that have also been shown to work.
4. Prepare cells for electroporation by trypsinizing and washing the cells in Hank's buffer twice. It is important to include the wash steps so that you can eliminate any matter that would interact with or negatively affect your RNP!
5. Suspend cells ar a density of 5×10^5 cells/9ul in buffer R. This is the optimal density when electroporating MEF cells.
6. Combine 9ul of the cell suspension with your diluted electroporation enhancer (2ul)
7. Add 1ul of your RNP suspension.
8. Pipette up contents into the NEON tip using the NEON pipette, place in the NEON pipette station, and electroporate.
9. After electroporation is complete, seed into the prepared wells.
10. Dilute into triplicate if you are using the 100ul tips.
11. Incubate overnight. Check if alive the next day.

Protocol: FACS

1. Prepare conditioned media by combining 2x filtered, day-old media from your stock lines with new media. Pipette into your 96-well collection plates. 100ul/well at least. Note that Blaise had the best results when collecting 3 rows of 10 cells/well and then 6 rows of single-cells. It is also important to have media in all the wells so that your plate conditions are as humid as possible!
2. Prepare your cells by washing them, then suspending them in Hank's buffer.
3. ALWAYS prepare a negative (no Atto550 transfection) control so that Wade can accurately set the gate of Atto550 positive cells.
4. Filter them through the FACS cap with a mech into the FACS tube. Ensure there is at least 2.5ml in the FACS tube.
5. Place suspension on ice.
6. Go FACS. Incubate overnight. Change media the day afterward.
7. PCR unsorted cells to see if the transfected Cas proteins were active.
8. When single-cells grow up into adequate populations, PCR them to assess whether the gene was knocked out or not.

Note that there will be many days that you will be staring through the inverted microscope assessing if your colonies are alive and/or healthy and/or if the media needs changing and/or you need to split them into bigger wells! This is no small experiment, so clear your experimental schedule!