2.12. T7 Endonuclease Assay

A T7E1 assay was performed to detect the insertion/deletion (indel) frequency after gene editing [36]. Genomic DNA was extracted from the cells 48 h after transfection with LAH5 peptide/RNP nanocomplexes using the Qiagen DNeasy Blood & Tissue Kit (Qiagen GmbH, Hilden, Germany) following the manufacturer’s instructions. PCR was performed using the primers designed for the sgRNA target locus (Table S2) using Q5^® Hot Start High-Fidelity 2X Master Mix (New England Biolabs, MA, USA). Afterward, PCR products were purified using the QIAquick PCR Purification kit (Qiagen GmbH, Hilden, Germany). PCR products were denatured at 95 °C for 10 min in the presence of NEBuffer 2 (New England Biolabs), and they were annealed by slowly lowering the temperature (95–85 °C at 2 °C per second and 85–25 °C at 1 °C per second). Subsequently, re-annealed PCR products were incubated with 5U T7E1 enzyme (New England Biolabs, MA, USA) at 37 °C for 18 min to cut heteroduplexes. DNA products were run on a 2% agarose gel in TAE buffer with a pH of 8.3 (Biorad). The indel frequency was calculated by determining the intensities of cleaved and uncleaved bands based on a densitometry analysis using ImageJ version 1.53t.