The GFP-fused recombinant SpyCas9 used in this study carries two NLS peptides between Cas9 and GFP, which is followed by another NLS at its C terminus. The Cas9 protein with an N-terminal 6His tag and maltose-binding protein was expressed in Escherichia coli Rosetta 2 cells (EMD Millipore). TEV protease was used to cleave the His tag and maltose-binding protein. The GFP-fused Cas9 was purified according to the protocols described previously (42). After purification, the GFP-fused Cas9 was stored in a protein buffer that comprised 150 mM KCl, 10% glycerol, 20 mM Hepes at pH 7.5, and 1 mM tris(2-chloroethyl) phosphate (TCEP) at −80°C.

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