With CRISPR-Cas9 I have conducted a targeted knockout of a DNA region encoding a certain protein (working with leukocytes). My question is, how long does it take until this protein is not detectable any more (e.g. via surface staining + flow cytometry) after the DNA has been manipulated? Or in other terms: how often are transcription, translation etc. taking place?

  • $\begingroup$ What cell line are you working with? Different celltypes can have fast differences in turnover rates of (DNA/)RNA/proteins. $\endgroup$ – Nicolai Mar 26 at 12:42
  • $\begingroup$ Thank you! Actually I am working with fresh human PBMCs (and thereof with CD4+ cells) $\endgroup$ – Mina Mar 26 at 14:17

To answer your question multiple parameters which one should be taken into account.

There are two main sets of parameters: gene specific and cell line specific.

Parameters related to your specific gene of interest :

  • Do you know the half-life of the RNA of your gene of interest? Most of the time it is not known but there are several tools and resources which can predict it based on a lot of different features such lengths and stability of 3'UTR, 5'UTR, number of exons introns, etc. Another indication is if there are already people who have either perform a genetic perturbation (siRNA, CRISPR, etc) or a degradation assay. In this case you might be able to guess what is the relative half-life of the RNA or the protein.
  • Which leads us to the half-life of the protein of interest. Proteins which are soluble tends generally to have a smaller half-life than membrane proteins (not necessary true there are exceptions). For example doing a complete functional knockout of a transcription factor might be easier than a GPCR receptor. If it is a membrane protein, to which organelle does it localize? Is the organelle recycling the membrane a lot or is the cell dividing? In this case you might have chances that the viable proteins present before the genetic perturbation get diluted. In these examples, you might get ride of a soluble protein in less than 24-48 hours, whereas a very stable membrane protein might take 7-10 days.
  • The number of RNA copies is important as well as the number bound to ribosomes (less important) and their localization in the cell (less important)
  • The abundance of the protein in general.

Parameters related to your cell line of interest:

  • The lineage of the cell line you selected: different cell lines have different global transcription and translation rates.
  • The rate of division of the cell line (extremely important parameter): the faster the cell line will divide the faster the mRNA copies which were not perturbed (wild-type) and the proteins present before the genetic perturbation will be diluted between daughter cells.
  • Number of copies in the cell line: if there are multiple copies of your gene of interest, you would need to make sure that each copy of the gene has been perturbed to lead to a functional KO.
  • Genomic stability of the cell line: some cells lines like HeLa are quite known for genomic instability where a lot of the genome can be rearranged can perturbed copies can be fix using viable copies

Last but not least, it depends technically how you do the CRISPR-Cas9 genetic perturbation: transfection of the gRNA, electroporation of the Cas9 with the gRNA, lentiviral mediated delivery etc. The time I have provided are after all the components have been delivered and are present in large amount in the nucleus.

I hope that helps!

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