If you've followed our guide to planning a successful genome editing experiment, then you'll hopefully be working in optimal conditions for CRISPR reagent transfection and for growth from single cells. If you have a good idea of your guide RNA's editing efficiency (from a DNA mismatch assay, for example) then you can estimate how many clones to screen to find targeted clones.
For the next step, you will want to 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.
o This can be improved by using conditioned media, by allowing cells to recover from transfection prior to single cell plate out, or by dilution plating (plating multiple single cells at low density in a 10 or 20 cm plate).
• The number of targeted clones recovered post transfection is dependent on the transfection efficiency and the efficiency of the guide RNA.
o This can be improved by optimising delivery conditions for your cell line, selecting for transfected cells (if your CRISPR reagents have selection markers or reporters), and by selecting or designing a highly active guide RNA.
• The number of targeted clones with the desired modification is dependent on how many alleles need to be modified, the nature of the modification, and the effect of the modification on viability of the cells.
Returning to transfection and plating cells out is time intensive and can set you back weeks, so 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
Protocol
Part 1 - Harvesting clones
1. Warm media for at least 10 minutes at 37°C in water bath.
2. Make up direct lysis buffer by adding 1 mL of Proteinase K (e.g. Sigma P4850) to 100mL DirectPCR Lysis Reagent (Cell) (Viagen Biotech, Cat#302-C).
3. 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.
4. Transfer 96 well plates from CO2 incubator to MSC.
5. Tip off the media from the 96 well plates into a waste bucket.
6. 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.
7. 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.
8. Using a multichannel pipette, transfer 5 μL 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).
9. Seal the PCR plates and incubate the plates at 55°C for 30 minutes followed by 95°C for 45 minutes to lyse the cells and denature the Proteinase K prior to PCR.
10. The DNA lysis plate can be used immediately or stored at 4°C overnight.
11. The remaining cells can be used to re-seed 96 well plates for future use.
Part 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:
• DNA mismatch detection assay (SURVEYOR® or T7EI) - this is a relatively fast and high-throughput method for screening for heterozygous clones, either knockouts or knockins. If both alleles have undergone identical modifications, DNA mismatch detection assays will not detect these.
• Restriction Fragment Length Polymorphism (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.
• Sanger sequencing - amplifying DNA around the target site by PCR and sending it for sequencing will identify clones with one or more modified alleles.
• Phenotypic assays these are specific to the target gene or cellular pathway. For example, if you're knocking out a cell surface protein, then cell sorting can be used to rapidly isolate clones that have lost the protein of interest.
At Horizon we mostly use Sanger sequencing or Next Generation Sequencing (NGS), as these approaches can be applied to almost any gene editing experiment, but we would encourage you to use whichever assays are most suitable for your gene editing experiment. The capabilities and limitations of the most commonly used assays are summarized in this post: Strategies to detect and validate your CRISPR gene edit.
For any screen it's worth keeping in mind the following:
• Modifications can be different on each allele, and deletions may range from one or two bases to several hundred, depending on the design of your knockout strategy. This can impact the design of PCR primers.
• The location of any SNPs will also impact the design of PCR primers. Heterozygous SNPs can be useful: they may enable the design of an allele-specific PCR, using primers including the SNP.
• The PCR amplicon should be sufficiently large to avoid bias towards a particular allele due to different modifications on each allele.
For a Knock-in:
• Allele-specifc PCR primers can be designed to specific elements incorporated into the donor, which can allow for a more specific screen.
Any clones identified as "of interest" in the initial screen should be validated by sequencing to determine if a modification has occurred on one or multiple alleles.
For your next steps in validating your engineered cell line, take a look at this blog post: You've done your CRISPR transfection... what's next?
Interested in Dharmacon Edit-R CRISPR knockout products?
And as usual, if you have any questions at all, feel free to contact our team for scientific support anytime.
Learn more
More strategies to detect and validate your CRISPR gene edit – blog post
A CRISPR-Cas9 gene engineering workflow for clonal selection –application note