We would like to knock out several genes (independently) using the CRISPR/Cas9 system. We tend to use HeLa cells for our work for their ease of transfection for siRNA and for microscopy. But I am concerned about their karyotype and wonder if knocking out a gene in HeLa by the CRISPR method is feasible. Is there a recommended cell line for CRISPR? We would like it to be easy to transfect and suitable for imaging subcellular compartments by microscopy. We have considered HFF cells but they are apparently difficult to transfect. Thanks.
Hello!
I believe you can use CRISPR methodology for genome editing in any cell line (theoretically), including HeLa cells. And indeed, in a quick search I`ve seen there are several papers claiming to have used CRISPR/Cas9 system in HeLa cells (Ebina et al, 2013; Chen et al, 2013; Cheng et al, 2013).
HEK293 cells are most commonly used in papers that used CRISPR/Cas9 system for knocking out genes. It's somewhat recommended in CRISPR/Cas9 nuclease kits (as seen in the GeneArt CRISPR Nuclease Vector kit). It seems the transfection in these cells can be done easily by Lipofection methods. Also, this cell type is suitable for microscopy imaging.
Best regards.
References
Ebina, Hirotaka, et al. "Harnessing the CRISPR/Cas9 system to disrupt latent HIV-1 provirus." Scientific reports 3 (2013).
Chen, Baohui, et al. "Dynamic imaging of genomic loci in living human cells by an optimized CRISPR/Cas system." Cell 155.7 (2013): 1479-1491.
Cheng, Albert W., et al. "Multiplexed activation of endogenous genes by CRISPR-on, an RNA-guided transcriptional activator system." Cell research 23.10 (2013): 1163-1171.
Dear Michael,
I think it is more important to ask yourself - why are you doing these gene disruption experiments? If you are knocking out components of say cancer signaling pathways then likely a tumorigenic cell line is appropriate. Alternatively a non-tumorigenic cell line might be better suited if you are looking more at other genes and karyotype is important. Similarly, if you were interested in looking at genes involved in cytoskeleton then an epithelial cell line might be preferred.
If you are looking at a range of genes then going with a "model" cell line may be easiest in terms of transfection efficiency and survival of knock out clones. Ideally doing these KO's in more than one cell type should give a better indication of the consequences of cell-type on your results!
Good luck!
Dear Michael,
I've been using CRISPR/Cas9 system to knock out genes in HEK293FT cells and they work very efficiently. If your aim is to knock out a human gene and see its adverse effects, you can first try if your construct is working by testing it in HEK293FT cells. They are very easy to transfect and transduce, whichever way you prefer to check. It is recommended to use transfection method for transient expression of Cas9 and you can start seeing effects 24-48hr post-transfection. Best of luck!
Any cell line should work, including transformed and primary. Optimization of plasmid delivery will be required.
Horizon have tried to establish several model systems, including human HAP1 that is nearly haploid. Although we found a bit higher than expected levels of genomic instability in "WT" HAP1 cells, and there is a good chance that haploid lines will be spontaneously diploid in several weeks:
https://www.horizondiscovery.com/cell-lines/all-products/isogenic-cell-lines/hap1-cells
Michael Sacher I hope you have solved your problem. I just wanted to share my experience with HeLa.
I have been working with CRISPR/Cas9 technology for the past three years and knockout out several genes in different cell lines. For the first time, I have selected HeLa and started the stable knockout experiment.
Initial validation had been performed by using T7E1 assay. Later, I sequenced to check the mutation. Out of 20 clones, 3 of them showed wild-type and other showed frameshit mutation. Unfortunately, my western blot results showed no reduction in the protein level.
Do you have anything to tell me how to sort out this problem?
TIA.
We are trying to make a CRSPR ko of a very large protien, HEKT cell line was successfully made. We are using the same guides for the HELA's and it is proving difficult. I have tried 3 times with transfection, selection in puro, then single colony selecting- by western blot- all clones are still positive for the gene. I'm going to try sequential transfections- has anyone tried this approach?
As in, transfect with the two guides, select in puromycin. Then plate and transfect again with the guides? Before single colony plating.
Thanks for any input!
Katie Boucher have you had any results with the new approach?
I have also been having trouble trying to generate Hela KO for some genes. No one of the KOs has been successful, although the cells have been growing under selection. Have you had any success ?
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