Super mix taqman

The ddPCR assays were performed with the PrimePCR™ ddPCR™ Mutation Detection Assay kit and PrimePCR™ ddPCR™ EGFR Exon 19 Deletions Screening Kit (Bio-Rad Laboratories) (Additional file 1: Table S1). We used cfDNA from Multiplex I cfDNA Reference Standards (Horizon Discovery) that included wild-type cfDNA with mutant allele frequencies of 5%, 1%, and 0.1%. cfDNA Reference Standards (Horizon Discovery) with 0.1% mutant allele was serially diluted to wild-type cfDNA for analytical sensitivity of the ddPCR assay (Additional file 1: Table S3). Healthy control samples and DNA-free samples were also analyzed (Additional file 1: Table S2) [25 (link), 26 (link)]. Amplifications were carried out in a reaction volume of 20 μL on a QX100 Droplet Digital PCR System (Bio-Rad). The 20 μL PCR mix was composed of 10 μL Bio-Rad Super mix TaqMan, 1–2 μL of each amplification primer/probe mix, and 8–9 μL NAs. Thermal cycling comprised an initial denaturing and polymerase hot-start activating step of 10 min at 95 °C, followed by 40 cycles of 95 °C for 30 s and 55 °C for 60 s. Results were analyzed with QuantaSoft v.1.7.2 software (Bio-Rad) and reported as copies per milliliter of plasma.

The number of T790M mutant copies in cfDNA samples before and after CRISPR-CPPC was quantified using ddPCR with the PrimePCR™ ddPCR™ Mutation Detection Assay kit (Bio-Rad, Hercules, CA, USA) according to the manufacturer’s instructions. Amplification was performed in a reaction volume of 20 µL using a QX100 Droplet Digital PCR System (Bio-Rad, Hercules, CA, USA). The PCR mix was composed of 10 µL of Bio-Rad Super mix TaqMan, 2 µL of T790M primer/probe mix, and 8 µL of post-CRISPR-CPPC cfDNA. The post-CRISPR-CPPC product was diluted 100-times for the optimal separation of false-positive and true-positive events. Thermal cycling conditions were as follows: 10 min at 95 °C, followed by 40 cycles of 95 °C for 30 s and 55 °C for 60 s. Results were analyzed with Quantasoft v.1.7.2 software (Bio-Rad, Hercules, CA, USA).
The methods of qPCR and NGS are written in Additional file 1.

The ddPCR assays were performed using the PrimePCR™ ddPCR™ Mutation Detection Assay kit, and the PrimePCR™ ddPCR™ EGFR Exon 19 Deletions Screening Kit (Bio-Rad Laboratories, Hercules, CA, USA) [11 (link)]. Tumor-derived nucleic acids (cfDNA and exoTNA) were extracted from 1 to 2 mL of plasma or pleural fluid. The ddPCR assay for detecting EGFR mutations was validated using the Multiplex I cfDNA Reference Standards (Horizon Discovery, Cambridge, UK) and healthy control samples from a previous study [11 (link)]. Briefly, the amplifications were performed in a reaction volume of 20 μL on a QX100 Droplet Digital PCR System (Bio-Rad). The 20 μL of the PCR mixture was composed of 10 μL Bio-Rad Super mix TaqMan, 1–2 μL of each amplification primer/probe mix, and 8–9 μL of nucleic acids (NAs). Thermal cycling comprised an initial denaturing and polymerase hot-start activating step of 10 min at 95 °C, followed by 40 cycles of 95 °C for 30 s and 55 °C for 60 s. The results were analyzed with QuantaSoft v.1.7.2 software (Bio-Rad) and reported as copies per milliliter of plasma. The ddPCR assay was validated for detecting EGFR mutations and determining the limit of detection (LOD) in a previous study [11 (link)]. The assays were considered “positive” if the measured event rate was ≥ 2 events/assay and “negative” if the event rate within a gated region was < 2 events/assay.