Table 1 ∣.

Troubleshooting table

Step	Problem	Possible reason	Solution
8	Smearing in the digested product	Digestion time is too long	Do not digest >30 min or the product will degrade
18	No colony on LB plates (including the negative control)	The ligation or transformation efficiency is low	Check T4 DNA ligase or competent cells
	Too many colonies in the negative control	The digestion is not complete	Redigest the vector with BsmBI
29	The 293FT cells detach during lentiviral packaging	The cells are not healthy	Obtain a new batch of 293FT cells
		The plate is not well coated	Double the coating time, reseed the cells and perform gently
54	HIEs aggregate together after digestion	The trypsin-EDTA is expired or not working well	Use freshly prepared trypsin-EDTA
			Wash HIEs with 1× PBS before digestion to remove FBS in the well
73	All HIEs are dead after selection	The titer of lentiviruses is too low	Remake lentivirus with a titer determination step
	Few HIEs die after selection in the nontransduced well	The puromycin or hygromycin-B antibiotic is expired	Prepare fresh antibiotic solution before use
79	Hard to inoculate Matrigel in 96-well plates	The tip is not optimal	Use desired nonsticky P200 tips for inoculation
		Too many bubbles in the Matrigel	Try not to generate bubbles in Matrigel
			Put the tube on ice to remove bubbles
92	The single-cell clone does not grow after passaging the cells	The gene may be an essential gene	Check that the KO is viable
		The stem cell is dying	Increase the screening size to get some surviving single-cell clones. All cells grow differently, so the initial screening size is important. We have found obtaining more growing enteroid clones is critical since the clones that are not growing or expanding cannot be maintained or passaged even if they are KO clones
109	No PCR product or nonspecific bands	The concentration of genomic DNA may be too low	Check the DNA concentration by Nanodrop
		The primers are not specific enough to amplify the genomic DNA	Redesign the primer set with similar annealing temperatures
			Increase primer length and annealing temperature
109	Obtain too many wild-type single-cell clones without indels	The gene may be an essential gene	Use knockdown or conditional KO strategy
		The puromycin selection does not work well to kill nontransduced clones	Extend the puromycin selection time for an extra 2 weeks with freshly prepared >2 μg/mL puromycin
		The sgRNA efficiency is not ideal	Switch to another sgRNA sequence, and repeat the experiment