CRISPR lentiviral library titration

[Sebastian Widholz]

Dear all,

I'm preparing my genome wide CRISPR screen and did lentiviral titration of the library to determine the best MOI. I stick to the protocol by Joung, Konermann et al, bioRxiv (2016): Protocol: Genome-scale CRISPR-Cas9 Knockout and Transcriptional Activation Screening. They suggest that for titration, 3x10^6 cells per 12-well should be used in combination with different amounts of viral supernatant. I did this experiment, but found out after seeding that I miscalculated the number of infected cells to 1x10^6. So my question is, whether this affects the calculation of the MOI or if this is negligible as the surviving fraction of cells is determined by CellTiterGlo assay as relation from the cells with puromycin and the ones without.

I'd really appreciate your help as this would save me the time to repeat this experiment!

Thanks!

Best,
Sebastian

[Julia Joung]

Hi Sebastian,

I'm happy to hear that you are using our protocol for CRISPR screening.

Altering the number of infected cells to 1x10^6 can potentially alter the efficiency of viral integration and thus the calculation of the MOI (e.g., the exposed surface area of the cell is higher in the case of 1x10^6 cells). In this case, you can either proceed with your screening transduction with 1x10^6 cells per 12-well using the MOI you have calculated from this virus titer experiment, which will be a lot of wells to transduce, or you can redo the virus titer with 3x10^6 cells per 12-well.

By the way, we finally published the protocol online - the newest version has some additional clarifications that you might find helpful.

Best,

Julia

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[Sebastian Widholz]

Dear Julia,

thanks for your quick reply and your suggestions.

Best
Sebastian

[Sebastian Widholz]

Dear Julia,

another problem occured. I repeated the experiment with 3x10^6 cells and performed Cell Titer Glo assay. However, for some cell lines, the maximum surviving fraction for the maximum amount of lentiviral supernatant I used (400 µl) is around 10%. I see a nice linear relationship between surviving fraction and amount of lentivirus used, but I did not reach the intended surviving fraction of 30%. Furthermore, I do not see any saturation. Is it possible to extrapolate the amount of lentivirus accordingly to reach a surviving fraction of 30%, although not particularly tested? Or would you recommend repeating the experiment with higher amounts of supernatant?

Your help would be greatly appreciated!

Thanks!
Best,
Sebastian

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[Julia Joung]

Hi Sebastian,

The maximum amount of virus that I have used with the spinfection protocol is 1 mL virus supernatant in a 12-well, and generally the titer scales linearly at this lower range, so you can extrapolate linearly to obtain an MOI of 0.25 at 1 mL. For screening, we recommend screening at a maximum MOI of 0.3 to ensure that most cells receive only 1 lentiviral construct, which means you can screen with MOI of 0.25, as long as you ensure that you will have 500 cells/sgRNA in your library after spinfection. For instance, if you have 100,000 sgRNAs in your library, you will need to spinfect at least 100,000*500/0.25 = 200,000,000 cells using 1 mL virus per well.

Best,

Julia

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