Hi,
I performed CRISPR/CAS9 genome editing in HL-60 cell line (suspension culture) to modify a gene of interest for a desired mutation (I used the sgRNA for my gene and a single strand ultramers as donor DNA).
By PCR screening, the mutation was introduced at very low percentage, estimated below 1%.
To avoid too many plates for the isolation of a pure clone with the mutation of interest, I decided to prepare a 10 cell/well dilution in a 96 well plate.
I isolated the positive well in which the mutation is now around 2-3% of the total amount of the cells (a little bit of enrichment).
To isolate single clone from cells in suspension it is indicated to dilute 0.5 cell/well or less, but should have CRISPR/CAS9 efficiency much higher than 2-3%.
What is your experience? should I try a single cell dilution with this positive percentage (2-3%) or should it be better to enrich again, for instance with around 5 cell/well to increase the clone with the desired mutation of interest before that step?
Dear Abdul
At this stage (2-3%) I would probably use single cell sorting and seed cells at 1 cell/well in mw96 plates. If you make a couple of plates (even 3 accounting for the fact the some cells will die after sorting), you should make sure to have at least 4-6 + clones. Screening 200 clones by PCR is doable, you don't have to expand them massively and there are good protocols for extracting DNA from mw96 confluent wells while keeping a backup replica plate to expand them in case they are positive.
In a couple of weeks you should have your positive clone to expand.
Hope it helps
Francesco
Abdul Waheed Khan
Dear Abdul
I have trouble in raising the single clone of HL60 cell line, I can only get less than 5 singe clones for a 96 well plate, which is a very low clone formation rate.
Do you have any tips in HL-60 single clone formation?
I using FACS to sorting a single cell at a well.
Hi Abdul,
if you need a single clone there is no way out of single cell seeding/limited dilution at 0,5 cells per well. Additional upside potential of this is, that your population is more stable, you avoid phenotypic drifts of you cell population.
Cheers
Axel
Something that has worked for us in the past with a cell line that does not grow out from single cells per cell (albeit, they are adherent cells):
Assuming you are using antibiotic selection to kill off cells not taking up the guide encoding plasmid, after single cell sorting in 100 ul in 96 well plate, let the cells sit overnight. The next day, add 1500 of the parental cell line (which is sensitive to your antibiotic) and let them grow for 10 days (may need to be longer or shorter depending on your cell line) without antibiotics. In this time, your single colony will hopefully grow in the presence of other cells. After the 10 days, we selected again for 15 days with the antibiotic to kill off all of the parental cells. After continued passage, we had over 1/3 of the wells still growing out from the single colony.
Not sure if this will help or work in your suspension cell format, but thought I would share something that has worked from us.
- Badges
- Science topic
- Similar topics
- Physical Sciences
- Materials Science
- Plating