Saturation profiling of drug-resistant genetic variants using prime editing

The sequence read data pertaining to the PEER-seqs 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