If you've followed our guide to planning a successful genome editing experiment, then you'll hopefully be working in optimal conditions, and have a good idea of your guide's editing efficiency. This number should give you a rough idea of how many clones you're going to have to screen to find a targeted clones. Keep in mind the following:
- The number of clones recovered post single cell dilution is dependent on how well the cell line tolerates single cell cloning.
- This can be improved by using conditioned media or by allowing cells to recover from transfection prior to single cell plate out.
- The number of targeted clones recovered post transfection of the CRISPR reagents is dependent on the transfection efficiency of the cell line and the efficiency of the gRNA
- This can be improved by using selection during transfection, and selecting a highly active guide.
- The number of targeted clones with the desired modification is dependent on how many alleles are to be modified, the nature of the modification, and the effect of the modification on viability of the cells.
Because returning to transfection and plating cells out is time intensive and can set you back weeks, we generally recommend plating out more clones than you think you're going to need - at a minimum a 96 well plate, but you lose very little by having a few backup plates too.
Reagents and equipment required:
- 20 µL and 200 µL filter pipette tips
- Gilson pipettes or similar
- 5, 10 and 25 mL stripettes
- 100 mL sterile reagent reservoir
- LTS multichannel pipettes or similar
- Cell culture media
- TrypLE™ (cell dissociation buffer) (Life Technologies 12605010) or similar
- Class II Microbiological Safety cabinet (MSC)
- Phosphate buffered saline (PBS)
- DirectPCR Lysis Reagent (cell) (Viagen Biotech, Cat#302-C)
- Proteinase K (e.g. Sigma P4850)
- 96 well PCR plates
Step 1 - Harvesting clones
- Warm media for at least 10 minutes at 37ºC in water bath.
- Make up “direct lysis buffer” by adding 1 mL of Proteinase K (e.g. Sigma P4850) to 100 mL DirectPCR Lysis Reagent (cell) (Viagen Biotech, Cat#302-C).
- Using a multichannel pipette, transfer 25 µL/well of direct lysis buffer (from Step 2) to the required number of 96 well PCR plates. I.e. one plate for each plate of cells to be assessed.
- Transfer 96 well plates from CO2 incubator to MSC.
- Tip of the media from the 96 well plates in to a waste bucket.
- Using a multichannel pipette, wash the cells with 100 µL of PBS, then add 30 µL of TrypLE™ to each well and return the plates back to the incubator.
- Check the plates intermittently to assess if the cells have been dislodged from the bottom of the wells. Giving a little tap on the side of the plates might help to dislodge the cells quickly, but be careful to ensure no cross-contamination of wells occurs.
- Using a multichannel pipette, transfer 5µL of cells from each well of the 96 well cell culture plate into a PCR plate containing 25 µL/well of “direct lysis buffer” (from step 3).
- Seal the PCR plates and incubate the plates at 55ºC for 30min followed by 95ºC for 45 minutes to lyse the cells and denature the Proteinase K prior to PCR.
- The DNA lysis plate can be used immediately or stored at 4ºC overnight.
- The remaining cells can be used to re-seed 96 well plates for future use.
Step 2 - Characterization of derived clones
Screening to identify a clone with the required engineering event can be approached using a variety of techniques, including but not limited to:
- PCR - amplifying the target site and looking for band shifts or loss/gain of a PCR product resulting from loss/gain of a primer site.
- Surveyor Assay - this is a suitable, if not expensive, approach for screening for heterozygous clones (knockouts or knockins) - if both alleles have undergone identical modification Surveyor will not pick these up.
- Restriction digest assay (RFLP) - if your targeting strategy can be designed in such a way that a restriction site is lost or gained, this can be used to screen for targeted clones.
- Sequencing - amplifying target site and sending for sequencing will identify those clones that have one or more alleles that have been modified
- FACS - if you're knocking out a cell surface protein, then cell sorting can be used to rapidly isolate those clones that have lost the protein of interest
At Horizon we most frequently use PCR as this approach can be applied to almost any gene editing experiment, but we would encourage you to use whatever assays you have optimized in your lab. For any screen it's worth keeping in mind the following:
For a Knockout
- Modifications are likely to be different on each allele, deletions may range from one or two bases to several hundred. This can impact design of PCR primers
- Location of any SNPs – this will impact design of PCR primers. SNPs can be useful by enabling the design of an allele specific PCR using primers including the SNP.
- Size of amplicon – this should be sufficiently large to try to avoid bias towards a particular allele due to different modifications on each allele.
For a Knockin
- Allele specifc PCR primers can be designed to specific elements incorporated into the donor design, which can allow for a more specific screen.
Any clones that are identified as ‘of interest’ at the initial screen, should be validated by sequencing. Sequencing will indicate if a modification has occurred on one or multiple alleles.