How many total transduced cells do I need for a whole genome sgRNA-library screen and why?
How many total transduced cells do I need for a whole genome sgRNA-library screen and why?
We recommend performing each screen using a minimum of 7.65 x 107 cells transduced at 40% efficiency when using the Guide-it CRISPR Genome-Wide sgRNA Library System. See the calculations below:
Total number of guides:19,114 genes targeted by 4 sgRNAs each = 76,456 sgRNAs (plus 156 control sgRNAs) = 76,612 sgRNAs
Number of cells transduced per guide:At a low sgRNA representation in the transduced cells (<400 for each sgRNA), the library will have more sgRNAs falling outside 2 standard deviations (SD) of the initial distribution, leading to erroneous interpretation of dropout or enrichment. This noise can be greatly mitigated by achieving fold coverage of 400X or greater (400 cells carrying the same sgRNA).Total cells = 76,612 x 400 = 3.06 x 107
Transduction efficiency needed:To ensure that the majority of the cells have only one guide RNA, the cells should be transduced at an efficiency of 20–60% (using Poisson distribution). We choose 30–40%.
Total cell number at the time of transduction:3.06 x 107/0.4 (40% transduction efficiency) = 7.65 x 107 cells.