CRISPR/Cas KO in HUVEC cells without selection

I am trying to use the CRISPR/Cas system to knock-out my gene of interest in HUVECs without creating a stable cell line (without a selection process) before continuing with my analysis. Could you recommend a solid protocol to reach high efficiency?


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Terracotta Harpyalmost 5 years ago

Check out this paper from Jordan Pober's lab at Yale for CRISPRizing HUVEC (PMID 25940550)

Black Manticorealmost 5 years ago

You'll need the endothelial characteristics sustained after knock-out. HUVEC is perhaps not up to this task, given its limited passage numbers before losing its endothelial identity. Editing without clonal selection will only yield heterogenous population that cannot be technically considered as 'knock-out'. Our colleagues compared WT cells with edited cells (without selection) and the target gene showed no difference on western blot.

What you need is cell with some colony-forming capacity and preferably viral vectors for high efficiency delivery, such as the endothelial colony forming cell-derived EC and lentiviral vectors in the paper Joe mentioned.

An alternative is to use TeloHAEC from ATCC, which unlike other immortalized human primary endothelial cells, seems to have maintained important endothelial markers:

https://www.atcc.org/~/media/C6E44BF2FA4E4E468110CD9FE55EDD51.ashx
Or, simply stick with siRNA. No selection needed and sometimes you can get up to 90% knock-down close to "knock-out".

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