EMX1 knockout in HEK293 with CRISPR/Cas9
Introduction
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Purpose: To knock-out the EMX1 gene in Human Embryonic Kidney (HEK293) cell lines using CRISPR/Cas9
Skills learned during this module:
- Culturing HEK293 cells
- Designing guide RNAs using Benchling's CRISPR design tools
- Assembling guide RNAs into a SpCas9 plasmid
- Transfecting plasmids into HEK293 cells
- Assaying for indels
Note: For more information on the EMX1 gene and assaying for identification of its knockout, please reference
(A. Ran, 2013). The guide RNA cloning protocol is
from information from the Zhang Lab made available on Addgene. Also see the CRISPR forum for any help needed.
To edit this protocol, sign up/sign in with Benchling, click the clock icon on the top right, and click the Clone From Version button.
Materials
Procedure
Aspirate media from your cells using a Pasteur pipette
Wash cells with 10 mL of PBS and aspirate with a fresh pipette
Add 3 mL of trypsin and incubate in 37°C for 3 minutes
Add 7 mL of media and titurate up and down
Centrifuge cells (make sure it's balanced!) for 5 minutes at 1.2 rpm
Resuspend cells with 5 mL of media
Add 9 mL of fresh media to a new T75, and then add 1 mL of resuspended cells to the flask
Titurate and incubate. Passage your cells every 2-4 days depending on confluency!
Click the + on the left side panel, and then CRISPR and then CRISPR Guides
Search for the EMX1 gene. Ensure all settings (including the hg38 genome) are the same as the image below and save in your class project and click Next
Select Single guide and ensure that the guide length is set to 20, the genome is hg38, and that the PAM is NGG, and click Finish. You can also input your own custom PAM site by typing into the bar.
Use the Annotations tab on the right side panel, and click on Exon 3 to select the third exon's amino acids.
On the Design CRISPR tab, click on the + to set the target region as the selected Exon 3
Sort all sequences using the off-target score by clicking on the off-target-score column. Select the one shown below:
When choosing guide RNAs, look at both the on-target and off-target scores. The higher, the better! (see http://crispr.mit.edu/about#score)
Go to Save above your list of guides and select oligos on Benchling. Save the guides in the Training project. Go to the top of the Design CRISPR tab and save the analysis as “EXM1 Exon 3 CRISPR Analysis”
Click Assemble and then choose the PX458 (WT-2A-EGFP) plasmid. The 2A will cause the plasmid to break into two parts so that SpCas9 will go into the nucleus and EGFP will remain in the cytosol.
After saving your assembly to your project's folder, click "copy the primer list" to order your primers for next week!
Digest and dephosphorylate 5 μg of SpCas9 plasmid with BbsI for 30 min at 37°C.
Gel purify digested plasmid using QIAquick Gel Extraction Kit and elute in EB. (See
protocol.)
Phosphorylate and anneal each pair of oligos:
Please use the T4 Ligation Buffer since the buffer supplied with the T4 PNK enzyme does not include ATP (or supplement to 1mM ATP).
Put the phosphorylation/annealing reaction in a thermocycler using the following parameters:
Dilute annealed oligos from Step 3 at a 1:200 dilution into sterile water or EB.
Set up ligation reaction and incubate at room temperature for 10 min:
(optional) Treat ligation reaction with PlasmidSafe exonuclease to prevent unwanted recombination products.
Plate HEK293 cells at 1.3 x 105 cells/well in a total volume of 500 uL on a 24-well plate
Cells are ready for transfection at 70-90% confluency. Using Lipofectamine, transfect 500 ng of the assembled plasmid according manufacturer's instructions
Check cells 24 hours after transfection for efficiency by estimating fluorescence percentage under a fluorescent microscope
Add an additional 500 uL of warm media gently
Incubate cells for 48-72 hours after transfection before indel analysis
