4 ms·
Looking at that paper, the 80% viability doesn't refer to after CRISPR/Cas9 had time to do the selection, it refers to passage through their chip and puromycin
by nonbel 10y ago
Looking at that paper, the 80% viability doesn't refer to after CRISPR/Cas9 had time to do the selection, it refers to passage through their chip and puromycin selection (which just indicates the plasmid got in the cell):
"Plasmids encoding Cas9 and sgRNA targeting phosphatase and tensin homolog (Pten) (fig. S6A) were delivered into MCF7 cells, followed by culture for 48 hours and puromycin selection. More than 80% of the cells survived the selection process, indicating the high delivery efficiency of our method.
[...]
Similar to Pten knockout, more than 80% of 53BP1 knockout cells survived the
selection process."
You have to look at figure 5B, where the caption reads:
"Cells (5 × 10^4) from (A) were seeded in 60-mm dishes in complete medium and cultured for 7 days. Cells were trypsinized and collected for cell count in a Countess II FL Automated Cell Counter (Life Technologies) daily for 7 days."
So they started with 5 x 10^4 cells but if you look at the first day after being in culture, it is much less than that. I can't tell how low from the chart which shows it as basically indistinguishable from zero. They do not seem to report those values anywhere.
Also, you can look at figure S5 and see that they saw GFP get knocked out in >2% of control cells (Cas9 only). They don't do a similar quantification for the Pten, but if they had, I am pretty sure some low percentage of cells would be shown as already knocked out for Pten.
- nonbel 10y ago>"knocked out in >2% of control cells" That should be 1%.