How many non-targeting gRNAs should be included in a CRISPRi/a library?

I am planing to design and generate a small pooled CRISPRi/a library with around 50-80 genes (3-5 gRNAs per gene) that enable screens with single-cell RNA-seq readouts. I am wondering does anyone have any idea how many non-targeting gRNAs as negative controls should I include in the library? Is it necessary to also include any positive controls in the library. Thank you so much.


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Fuchsia Kelpieabout 3 years ago

I think including something like 5-10 non-targeting sgRNAs should be sufficient for your screen. It's not necessary to include positive controls in your library, but if you do know a few positive control genes, it would be nice to include them to see if your screening parameters are ideal.

Orchid Pegasusabout 3 years ago

I think it sounds great! Thank you so much for your reply.

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