Parameter	Original Protocol	Modified Protocol
Heat shock prior to electroporation	40 °C, 350 rpm, 30 min in microtube Eppendorf Thermomixer	Same
sgRNA expression vector plasmid. pPH331	1 µg	1 µg or 2 µg
Cas9 expression vector plasmid, pPH187	2 µg	2 µg or 4 µg
Electroporator	NEPA21, Complex waveform Fig. 1(A)	Bio-Rad Gene Pulser II, simple exponential pulsesFig. 1(B)
Post electroporation recovery	500 µL TAP+Arg 24-well plate Wrap in aluminum	Same
Recovery temp, 1st & 2nd day	1st day 33 °C 2nd day 22 °C	1st day 30–32 °C 2nd day 21–25 °C
Recovery Plates	1.5% agar, TAP + 100 mg/L-arginine + 3 mg/mL yeast extract + 2 mg/mL tryptone + 10 mg/mL paromomycin	Same. Plates without yeast extract and tryptone were also tested; three 100 mm plates per electroporation
Wait time to count colonies	7–10 days	19–25 days