HEK293.clover
cell line
The HEK293.clover cell line was designed to test the cleavage efficiency of Cas12a nucleases with the TTTN PAM sequence by a large target disruption assay. HEK293.clover cells were generated as follows: a modified clover sequence was inserted after the ATG of human prion protein coding sequence with a self-cleaving donor plasmid by NHEJ-knock-in in HEK293 cells (293 H, Gibco).
Modified clover sequence
Three consecutive thymidines or adenines are marked by yellow or blue, respectively.
Disruption assay
Cells were plated one day prior to transfection on 48-well plates at a density of approximately 25,000-30,000 cells/well. Cells were co-transfected with two types of plasmids: Cas12a variant expression plasmid (137 ng) and crRNA and mCherry coding plasmid (97 ng) using 1 µL Turbofect reagent per well in 48-well plates. Transfected cells were analysed six days post-transfection by flow cytometry. For control an inactive LbCas12a expressing nuclease vector was used. Disruption activities of the nucleases were calculated as follows:
1 – (sample GFP % / average GFP % of negative controls)
Transfection efficiency was calculated via mCherry expressing cells. Transfections were performed in triplicate.
Reference
Tóth, Eszter ; Varga, Éva ; Kulcsár, Péter István ; Kocsis-Jutka, Virág ; Krausz, Sarah Laura ; Nyeste, Antal ; Welker, Zsombor ; Huszár, Krisztina ; Ligeti, Zoltán ; Tálas, András ; Welker, Ervin
Improved LbCas12a variants with altered PAM specificities further broaden the genome targeting range of Cas12a nucleases
NUCLEIC ACIDS RESEARCH 48 : 7 pp. 3722-3733. , 12 p. (2020)