Table 1 ∣.

Troubleshooting table

Step	Problem	Possible reason	Solution
2	No guide with high efficiency or low off-target scores	Selected region contains repetitive elements	Use RepeatMasker to avoid repetitive elements. Change coordinates (1 kb upstream or downstream)
9	Low DNA concentration	Not enough template or inefficient PCR	Repeat PCR with more plasmid template or pool multiple reactions
12	Smear or no band in gel	sgRNA degradation	Repeat reaction and ensure nuclease-free conditions
14	Low sgRNA concentration	Poor in vitro transcription	Repeat reaction with more DNA template or pool multiple reactions
19	No amplification of donor DNA	Not sufficient template	Repeat reaction with more template
21	Low concentration of donor DNA	Low yield	Repeat by setting up multiple reactions per donor and pool for PCR purification
35	No cells survive positive selection	Low HDR efficiency	Repeat electroporation and scale up number of targeted cells. Alternatively, extend homology arms to 160 bp using an ultramer oligo for PCR
41	Undetectable on-target cassette integration	Genotyping PCR is not optimal (more common) or cassette is present only in off-target locus	Redesign genotyping primers. Alternatively, change KI locus
45	KI cells are resistant to negative selection	Presence of untargeted cells due to incomplete positive selection	Increase concentration of positive selection. If needed, single-cell clones can be derived
49	No differential survival between negative control and targeted cells treated with negative selection	(i) Inefficient delivery of sgRNAs. (ii) sgRNAs do not efficiently create the intended DSBs	(i) Repeat with increased concentration of sgRNAs. (ii) Design new sgRNAs targeting the intended locus
57	Low frequency of cells with deletion	Negative selection not robust	Repeat deletion electroporation followed by more stringent negative selection. The cell number can be scaled accordingly to increase yield