CRISPR Editing Efficiency Dropping Suddenly

Organic Chemistry
1

Hi there

can anyone help!!

My CRISPR-Cas9 knockout efficiency dropped from ~80% to ~40% after switching to a new cell batch.

Could this be due to cell-cycle differences, my sgRNA degrading faster, or innate immune activation in the new line?

Thanks

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2

Sarah Canon

Yes—all three are plausible, but they differ a lot in likelihood and how fast you can rule them in or out. A drop from ~80% → ~40% after a cell batch change most often points to cell-state differences (especially cell cycle and fitness), with sgRNA stability and innate immune sensing as secondary possibilities. Suggestions for you to circumvent this :

Transfect at lower confluency (30–50%)

Synchronize briefly (e.g., thymidine block → release)

Increase Cas9 exposure window (in our case, RNP > plasmid helped us)

Let me know if you require any specific details

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