T7 endonuclease I (T7E1) assay to detect indels in Creb5

The T7E1 assay was used to detect on-target CRISPR/Cas9 induced insertions and deletions (indels) in cultured cells. cDNA derived from mRNA isolated from either lentiCRISPRv2 or lentiCRISPRCreb5gRNA infected bovine superficial zone chondrocytes was employed for the T7E1 assay. A 210 bp cDNA fragment, which flanks the Creb5 gRNA cleavage site, was amplified using Creb5 RT-qPCR primers (Supplementary Table 1; Creb5 primers 1) in a 25 μl PCR reaction (12.5 μl Q5 High-Fidelity 2X Master Mix, 1.25 μl Forward Primer, 1.25 μl Reverse Primer, cDNA, nuclease-free H₂O to a final volume of 25 μl). After denaturation of the cDNA at 98 °C 30 s; the PCR machine was programed to cycle 35 times at: 98 °C 10 s, 60 °C 15 s, 72 °C 15 s; 72 °C 2 min; 4 °C hold. The PCR product was denatured and annealed in an 18 μl reaction (15 μl PCR product, 2 μl NEB Buffer2 (10X), 1 μl nuclease-free H₂O) with the following the cycling conditions: 95 °C 10 min; 95–85 °C (ramp rate of −2 °C/s); 85–25 °C (ramp rate of −2 °C/s). After denaturing and reannealing the PCR products, T7E1 (2 μl) was added, and the mixture was incubated at 37 °C for 60 min. Cleavage of the PCR products by T7 endonuclease was assayed by agarose gel electrophoresis.