The Cas9-eGFP-NLS sequence was PCR-amplified from the L1 cassette (see above) using forward and reverse oligonucleotides with flanking Nde I and Xho I restriction enzyme recognition sites, respectively, using Q5 Hot Start High-Fidelity DNA Polymerase (New England Biolabs). Corresponding restriction enzyme digestion of the amplicons was followed by ligation (T4 Ligase, New England Biolabs) into the pET-21a+ vector, yielding a construct encoding Cas9-eGFP-NLS with a C-terminal His6 tag. Chemical transformation of ArcticExpress (DE3) RIL competent cells (Agilent Technologies) and selection on Luria agar plates containing gentamicin (20 mg/liter) and ampicillin (100 mg/liter) was used to select for plasmid-containing colonies. Plasmid integrity was confirmed following purification (GeneJET Plasmid Miniprep Kit, Thermo Fisher Scientific) and Sanger sequencing using vector- and fragment-specific oligonucleotides. Protein expression was induced by adding 1 mM isopropyl-β-d-thiogalactopyranoside to the cultures, which were then incubated at 18°C for 16 hours. Bacterial cells were lysed using a microfluidizer (Microfluidics), and affinity purification was performed using the ÄKTA pure 25 L System with a HisTrap HP nickel-Sepharose column (GE Healthcare Life Sciences). The in vitro assay that determines cleavage efficiency was performed as previously described (57). This protocol uses the purified Cas9-eGFP-NLS protein, a double-stranded DNA (dsDNA) PCR fragment containing the sgRNA target sites, and in vitro–transcribed sgRNA. Reaction products were analyzed by agarose gel electrophoresis.

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