Hypervac vc2124

Double-strand DNA (dsDNA) templates were synthesized with forward template DNA including T7 promoter and 20 bp crRNA sequence, reverse template DNA, Power-Pfu (500 U/µL, NanoHelix), and dNTPs (10 mM, Thermo Scientific). sgRNAs were synthesized by in vitro transcription using the dsDNA template, T7 RNA polymerase (50,000 units/mL, NEB), 50 mM MgCl₂, 0.1 M DTT, rNTPs (100 mM, Jena Bioscience), and RNase inhibitor murine (40,000 units/mL, NEB) at 37 ℃ for 16 h, precipitated with isopropanol (Sigma) and purified with GeneAll ExpinTM PCR SV kit (GeneAll Biotechnology). sgRNA concentration was measured using NanoDrop 2000 (Thermo Scientific), and characterized by 1.0% denaturing formaldehyde gel electrophoresis with MOPS buffer (Biosolution). sgRNA was freeze-dried using HyperVAC VC2124 (Hanil Scientific) for long-term storage.

sgRNA targeting the RAD52 gene was prepared using DNA templates consisting of a T7 promoter, crRNA, and tracrRNA, by PCR amplification with Power‐pfu (500 U µL⁻¹) in Power‐pfu buffer (NanoHelix) added with dNTP (10 mM, Qiagen) and primers (50 pmol µL⁻¹). Then, in vitro transcription was performed by adding the synthesized DNA templates, NTPs (100 mM, Jena Bioscience), to buffer including T7 RNA polymerase (50 000 units mL⁻¹), 50 mM MgCl₂, 0.1 M dithiothreitol, and RNase inhibitor murine (New England Biolabs), and incubating at 37 °C for 16 h. The synthesized sgRNA was purified with GeneAll Expin PCR SV kit (GeneAll Biotechnology), the concentration was measured using NanoDrop 2000, and the product was vacuum‐dried for 2 h using HyperVAC VC2124 (Hanil Scientific). RNPs were formed by adding Cas9‐AzF or Cas9 conjugates (500 nM) with sgRNA (500 nM) at a molar ratio of 1:1, and incubation at room temperature for 10 min.