Table 1.

Troubleshooting

Technique	Common Pitfalls	Possible Causes	Solutions
Electroporation of hiPSCs to deliver CRISPR-Cas9 containing plasmids (PROTOCOL 1)	Poor cell survival	Air bubbles were trapped in the tip.	Air bubbles cause a dramatic spark during electroporation. Always check the Neon Pipette Tip for bubbles before running the protocol.
		Poor hiPSC culture technique.	Feed hiPSCs daily and passage the cells before they reach confluency.
		Over handling of the hiPSCs.	Perform every step up to but not including the electroporation. If poor cell survival is observed, use gentler technique.
		dsDNA induced cellular toxicity.	Large quantities of plasmid such as >5 μg drastically decrease cell survival. If no nuclease activity is apparent with 2 μg of pX330 consider testing additional guides.
		Endotoxin contamination.	Use a kit rated to remove endotoxins.
		Laminin-521 coating issues.	If the colonies grow in “hot dog” shaped patterns, the coating is insufficient. Refer to manufacturer instructions.
		Media not properly maintained.	Refer to manufacturer’s protocol.
	Poor gene disruption efficiency	SNPs	Always use a tool such as the UCSC Genome Browser during guide sequence design. The iPSC line can also be directly sequenced.
		Air in the tip will cause a spark.	Refer to comments above.
		Genomic region refractory to genome-editing	Generate more guides upstream and downstream of the region to find a new target. Try different Cas9 orthologues.
		Transfection optimization issues	Use plasmids with reporters such as pSpCas9(BB)-2A-GFP (pX458) available from Addgene.
		Cas9 orthologue has a low activity	Try a different Cas9 nuclease.
	No observable HR	Rare HR events.	Increase the length of the homology arms. Avoid placing the selection cassette in a low complexity region. For rare events, the number of replicates must be increased.
Reagent Based Transfection of hiPSCs to Deliver CRISPR-Cas9 Containing Plasmid (ALTERNATE PROTOCOL 1).	Poor cell survival	dsDNA toxicity, Endotoxin contamination.	see above
		Improper hiPSC culture technique.	see above
	Poor gene disruption	see above	see above
Positive Selection of Genomically Modified hiPSCs (Protocol 2)	No cell survival	Increasing the drug concentration too quickly.	If proper location of selection cassette is observed by PCR, extend the length of each by a day or two. If not, perform selection for a week. Then take a heterogeneous gDNA sample and retest the PCR. This will enrich for the targeted genome-editing event. If distinct colonies are not visible, consider redesigning the pHDR. We commonly find that clones taking more then a week to emerge are due to random plasmid incorporation.
		Varying resistance from line to line.	Change the dosage escalation accordingly. Note that 0.05 μg/mL of puromycin should not cause rapid cell death even in a sensitive line. One approach is to change the media early, and observe the cells by the end of the day. For sensitive lines, 6–10 hours is sufficient to affect the cell viability. Remove the media and rinse the well. Treat the cells at a lower dosage for an additional 1–2 days.
	No cell death	Too many cells.	Overly confluent cells will render the drug dosage ineffective. However, if the selection began at an appropriate confluency, then the drug dosage needs to be escalated faster.
		Varying resistance from line to line.	For most lines 0.5 μg/mL of puromycin is sufficient for selection. We have observed that 1 μg/mL is necessary in limited cases. Include untreated cells as a control.
	Cells die after clonal section	“Herd resistance”.	Some clones seem to survive in the presence of corrected clones and do not contain the genomic modification of interest.
		User technique.	Larger colonies survive the process more often than smaller colonies.
		“Hidden” cells.	Check the margins of the well for hiPSCs. It is easier to miss these cells and assume the well is negative.
	The selection cassette is properly located but disease allele is not corrected	A design or sampling issue.	While respecting the other design considerations, the arm spanning the DSB should be oriented in this order: (most distal to center) double stranded break---> sequence modification --> selection cassette (most proximal to center). Center refers to the middle of homology sequence. Swapping the order of the DSB and modification will include HR repair that includes selection but excludes the desired modification.
	No amplification of the selection cassette at the correct location in the genome.	Random plasmid integration.	If clones emerge after 7 days, they typically do not carry the desired genomic modification. Use multiple sets of primers to rule out a false negative. Additionally, try different high fidelity polymerases.
Removal of Selection Cassette From Genomically Modified hiPSCs and Negative Selection (Protocol 4)	No cell survival	Gap junction-mediated bystander effect.	A high density of cells will prevent cell survival. Transient plasmid transfections of Cre recombinase will at best result in excision of 40%–60% of the selection cassettes. Therefore low ganciclovir concentrations and low plating densities limit the extent of the bystander effect. Alternatively, hiPSCs can be transfected with Cre recombinase an additional time before starting negative selection. Always collect a gDNA sample after Cre recombinase treatment to rule out issues with the LoxP sequences.
	No cell death	This can happen due to mutations in the viral thymidine kinase sequence.	To avoid this, take at least 3–5 clones through the entire dual selection process.