For the past few months, I have been troubleshooting a CRISPR KO of human CD3e in human primary T cells. I got the KO to work one time using 1250 ng TrueCut Cas9 (Thermo) + 250 ng (or 7.5 pmol) hCD3e sgRNA (from IDT) and using electroporation parameter: 1600V/10ms/3 pulses on the Neon Transfection System (10 uL Neon Tip). I got ~75% KO of the TCR.
Since then, I have been trying to replicate those results with no luck. I have tried different T cell donors, different electroporation parameters, a new Neon kit (buffers and tips), performing the electroporation on different days after stimulation, TRCB sgRNA, increasing the Cas9+gRNA amounts...nothing is working. I have gotten this KO to work consistently in Jurkat cells with ~90% KO efficiency (same hCD3e sgRNA). Soon I will test with a new Neon Transfection machine, more T cell donors, and human sgRNAs with different target sequences.
Has anyone also experienced difficulty in performing CRISPR KO in human primary T cells, but figured out how to get it to work consistently? I would really appreciate any advice! Thanks!
We use Lonza system and get very consistent outcome. I dont have experience with your system.
Dear Karen!
You look at this thesis and protocol CRISPR TCR KNOCKOUT in it, please:
Developing a CRISPR-Mediated Knockout TCR Human T Cell Line for Use in Cloning Antigen-Specific T Cell Receptors
https://repository.asu.edu/attachments/227561/content/Hirneise_asu_0010N_20136.pdf (pages 18-22).
https://assets.fishersci.com/TFS-Assets/LSG/brochures/delivery-solutions-immune-cells-app-note.pdf
https://www.biorxiv.org/content/10.1101/2020.02.17.953463v1.full
Dear Karen, an excellent reference for successful genome editing of human primary T cells as well as moust T cells via Nucleofection is published in the Journal of Experimental Medicine:
Optimized RNP transfection for highly efficient CRISPR/Cas9-mediated gene knockout in primary T cells.
Article Optimized RNP transfection for highly efficient CRISPR/Cas9-...
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