Table 1.

Troubleshooting

Technique	Problem	Possible Cause	Solution
Transformation of gRNA cloning vector	No gRNA sequence in vector	Inefficient cloning	Screen additional clones or repeat
Cleavage efficiency of Cas9 gRNA	Poor to undetectable cleavage	SNPs	Sequence target region in cells to be edited to confirm matching sequence
		Refractory chromosomal region	Design guides in another region
		Lethal mutation	None
ssODN Design	Cannot create unique restriction site with silent mutations	Limited sequence availability	A unique restriction site can be removed with silent mutations, but gRNA cleavage sometimes creates false positives when screening
Plasmid transfections	Poor cell survival	Toxic load of DNA	Decrease concentrations of plasmid/ssODN
	Low efficiency	Cell density	Determine the cell number for 80–85% confluency
		Plasmid ratios incorrect	Test multiple plasmid concentrations to achieve optimal GFP expression
		Lipofectamine Stem volume	Optimize volume needed for transfection
Clone screening	Heterogeneity of clones	Clone proximity too close upon manual isolation	Plate sorted cells at a higher dilution (no more than 15,000 sorted cells per 10 cm2 dish)
	No PCR product or dirty PCR product	Proteinase K minipreps may not be adequately clean for PCR reaction	Prepare purified genomic DNA