CRISPR-Mediated Gene Integration Quantification

The measurement of HR activity by ASHRA was described previously (17 (link)). In brief, cells grown in 3.5 cm dishes were transfected with siRNA against BRCA1 and the HA-tagged BRCA1 expression vector. On the next day, the donor vector (Addgene ID: #169798) and the expression vector for gRNA and Cas9 (Addgene ID: #169795 and #169796; 0.5 μg each) were cotransfected into the cells. After 48 hours of incubation, genomic DNA was extracted using the Blood Genomic DNA Extraction Mini Kit (Favorgen). Quantitative PCR was performed on a CFX96 Touch Real-time PCR detection system (Bio-Rad) using GoTaq qPCR master mix (Promega). Quantification of the knocked-in allele and control allele by qPCR was performed using the following primer sets: 5′-GTCCTGCTGGAGTTCGTGACCG-3′ and 5′-GTGCAATCAAAGTCCTCGGC-3′ for the knocked-in allele, and 5′-AGTTGCGTTACACCCTTTCTTG-3′ and 5′-GTGCAATCAAAGTCCTCGGC-3′ for the control allele. The relative quantity of the knocked-in allele was calculated by the ΔΔC_t method.