Is it possible to inactivate GFP after knockout of my target gene by a second round of CRISPR/Cas9 genome editing?

Gene-editing Discussion
1

Hi,
in my lab we have introduced knockouts of different target genes in AICS-0036-006 iPSC using CRISPR/Cas9. Now we also need knock-out cells without GFP tag as controls. Is it possible to use a GFP-specific crRNA to inactivate the GFP tag in a second round of CRISPR/Cas9 genome editing?
Best wishes
mra

2

Hi mra,
Yes, that is indeed possible by disrupting the GFP coding sequence. Many possible sequences should be able to accomplish that, but here is one that is commonly used for either disrupting GFP or introducing a single base pair change via HDR to change its color-Genome editing via delivery of Cas9 ribonucleoprotein - PMC.

Best,
Janani