I have try to establish a CRISPR screen in primary T cells，sgRNA transduce with
lentivirus and Cas9 protein delivered by electroporation (SLICE). But in the pilot experiment, we found that the efficiency of knocking out CD45 through this system is very low. The same experiment on Jurkat also failed.
Then I tried to change the experimental conditions, for example, increasing the amount of Cas9 protein or using Cas9 mRNA, or change CD45 sgRNAs.
None of these attempts have improved CD45 knockout efficiency.
I would appreciate it if you could give me some good advice!
Try 2 gRNAs that cut at an offset from each other. Have you considered trying to knock in a selection cassette into the cut site?
Robert Adolf Brinzer
Thanks for your advice!
Because our library is a single sgRNA in lentiGuide-puro plasmid, so we just try the same backbone with a single CD45 sgRNA, but if there is a library containing 2 sgRNA per plasmid, we could try it.
For your second advice, in fact, before proceeding with electroporation Cas9 protein, we performed a drug screening with puromycin.
Thanks again for your valuable comments and look forward to your reply!
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