Mar 13, 2025
CRISPRi Knockdown in NGN2 iPSCs
- 1Gandhi Lab
Protocol Citation: Alexis Penverne 2025. CRISPRi Knockdown in NGN2 iPSCs. protocols.io https://dx.doi.org/10.17504/protocols.io.rm7vzk268vx1/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: January 22, 2025
Last Modified: March 13, 2025
Protocol Integer ID: 118874
Abstract
CRISPRi Knockdown in NGN2 iPSCs with dCas9 using sgRNAs
Doxycycline-inducible NGN2 iPSCs are maintained on Geltrex in mTeSR (STEMCELL) , and passaged using 0.5 micromolar (µM) ) EDTA. iPSCs should be between 50-70% confluent to increase effectiveness of transduction.
Prepare lentiviral stocks with sgRNAs targeting the genes for Knockdown.
Dilute aliquot of lentivirus preparation in enough media to cover cells in 6-well plate. We diluted our stocks 1:100 in 1mL of mTeSR.
Note
Using 1mL of media to save lentiviral stock, we transduced the cells overnight and changed the media early the next day to ensure cell survival.
Change media with 2 mL of mTesR per well of a 6-well plate. Leave the cells to recover for 2 days, changing the media daily.
Once cells look healthy after transduction step, add 0.5ug/mL of Puromycin to kill untransduced cells. Keep puromycin as long as control untransduced cells are not dead.
Note
Include antibiotic to which lentiviral construct presents the relevant resistance. Keep one well of untransduced cells to control whether the antibiotic accurately gets rid of cells that haven't taken up the plasmid. It is possible to increase the antibiotic concentration to 1ug/mL if cells sare staying alive.