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
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