Saturation resistance profiling of EGFR variants against tyrosine kinase inhibitors using prime editing
The sequence read data pertaining to the SynPRIME development initiative encompasses the identification of Single Nucleotide Variants (SNVs) within the EGFR tyrosine kinase domain, specifically spanning exons 18 to 24. PC-9 cells, which had been transduced with the SynPRIME library spanning exons 18 to 24, were harvested on day 10 of cultivation. These cells were subsequently divided into two cohorts: a drug-treatment group and an untreated group. The cohorts were subjected to a 10-day treatment regimen with either afatinib or osimertinib. Deep sequencing was performed on day 10 and day 21 for both the drug-treated and untreated groups. The same experimental protocol was applied to T790M-PC-9 cells with osimertinib. Additionally, deep sequencing was conducted on unedited PC-9 cells and T790M-PC-9 cells, focusing on exons 18 to 24.For the essential RPL-15 library investigation, PC-9 cells were transduced with the RPL-15 exon 2 SynPRIME library and harvested at both day 5 and day 14 for the purpose of analyzing the depleting effect. Deep sequencing was also carried out on unedited PC-9 cells, specifically targeting exon 2 of RPL-15.In vivo small library of exon 20-targeting constructs was synthesized based on in vitro data and subsequently cloned into the luciferin-crRNA expression vector. PC-9 cells were transduced with the resulting lentiviral library and underwent selection via puromycin. Following a 10-day selection period, approximately 1x10^7 cells were subcutaneously injected into NOG mice. This study featured three replicates for each of the two groups: the drug-challenge group and the untreated group. All mice used in this study were six weeks of age at the time of cell implantation. Ten days post-transplantation, the presence of tumors was confirmed in all mice through the use of the IVIS (in vivo imaging system). Subsequently, a regimen of oral administration of osimertinib at a dosage of 2.5 mg/kg was initiated within the drug-challenge group and continued for a duration of two weeks. Following the completion of the treatment phase, all mice from both experimental groups were humanely euthanized, and tumors were meticulously dissected for subsequent deep sequencing analysis