5.3. In Vitro Cleavage Assay

The 5 Kb DNA fragments for selected target genes containing the target site were amplified using PCR, purified, and eluted with RNase-free water. The RNP complex was prepared according to manufacturer’s instructions and 1 μM RNP was mixed with the purified target DNA (100–150  ng) in 10 X Cas9 reaction buffer (20 mM HEPES, pH 7.5, 150  mM KCl, 10  mM MgCl₂, 0.5  mM DTT) in a total volume of 10 μL, followed by digestion at 37  °C for 60 min. Further, to release the DNA substrate from the complex, 1 μL proteinase K was added to reaction and incubated for 10 min at 56 °C (https://sg.idtdna.com/pages/support/guides-and-protocols). The digested DNA products for respective samples were separated on a 2% agarose gel immediately after the digestion process, and cleavage activity was measured by the number of digested products over the total amount of input target DNA. The obtained DNA bands were quantified using Gel quantification software (ChemiDoc imaging system, BIO-RAD, Hercules, CA, USA). The in vitro digestion assay was performed before every transformation to confirm that the RNP complex was functional and capable of editing the respective genes.