Top Questions

I am seeing cell death after transfection in my CRISPR-Cas9 experiments. What can I do to prevent this?

If you are using transfection reagents:

Transfection reagents can be cytotoxic, and the extent of cell death can vary from one cell type to the next. If you are seeing excessive cell death, the first step should be to optimize the transfection reaction using our Alt-R® CRISPR-Cas9 HPRT Positive Control crRNA, which is available for human, mouse, and rat cells. The goal is to use the least amount of lipid transfection reagent, without significantly affecting transfection efficiency.

You could try a different transfection reagent, as minor differences in chemistry can impact toxicity. Make sure to look for transfection reagents optimized for RNA to transfect the Alt-R CRISPR-Cas9 RNAs and RNPs. We have had success using TransIT® Transfection Reagent (Mirus Bio) for transfection of the S.p. Cas9 Expression Plasmid and RNAiMAX™ Reagent (Thermo Fisher Scientific) for the Alt-R CRISPR-Cas9 RNAs in HEK-293 cells. Transfection reagents for use in other cell lines should be empirically optimized.

If you are doing electroporation:

Optimal electroporation conditions vary by cell line as well as by type, amount, and size of material (e.g., plasmid, RNA, DNA, or ribonucleoprotein) being introduced into the cells. Examples of electroporation conditions that may need optimization include number of cells, relative amounts of materials, voltage, pulse width, and number of pulses. For additional information, see Successful CRISPR genome editing in hard-to-transfect cells.

Could your targeted gene be essential for cell viability?

It is also possible that your target gene is vital to your cells and, thus, its knockout results in cell death. If the positive control (Alt-R CRISPR-Cas9 Human HPRT Positive Control crRNA) works and your target is knocked out, it is likely you are working with a vital gene. In this case, consider RNAi methods to knock down expression of your target gene [visit our Dicer-substrate short interfering RNA (DsiRNA) webpage for additional information].


Tags:
  • Cas9
  • CRISPR
  • Alt-R
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