Table 1.

Description of assays applied at different generational stages when producing gene-edited animals. The expected outcomes at G0 and G1 stages are detailed for correctly edited animals with the assays applied. Refer to Figure 2 for visual representation of the described assays.

Generation	Method	Allele type
		Deletion	Indel and point mutation	Tag/Large KI	Floxed
G0	On target PCR and Sanger sequencing PCR spanning target region. If required, sequence confirmation of allele in both directions.	Reduced amplicon size compared to WT control. Allele sequence to be confirmed by sequencing if specific segment to be excised/deletion too small to be visualized.	Amplicons of equivalent size to WT control. Allele sequence to be confirmed by sequencing.	Larger amplicons compared to WT control. PCR assays can also be designed to: • identify donor insertion (light green primer pair) • Specifically amplify on-target donor insertion (paired light and dark green primers) Allele sequence to be confirmed by sequencing.	Amplicon compared to WT control, depending on relative size of floxed region size difference may or may not be discernible by agarose gel electrophoresis. PCR assays can also be designed to identify: • donor insertion and presence of both LoxP sites in cis (light green primer pair) • on-target donor insertion (paired light and dark green primers) Allele sequence to be confirmed by sequencing.
	Long read sequencing Deep interrogation of allele sequence using large number of single reads across large interval. Detection of upstream/downstream events/re-arrangements.a	N/Aa	N/Aa	Identification of discrete alleles in mosaic animals, including discrimination of features (e.g. LoxP sites) in cis or trans of one another. Confirmation of repetitive regions of allele sequence that cannot be resolved using Sanger sequencing.	Identification of discrete alleles in mosaic animals, including discrimination of features (e.g. LoxP sites) in cis or trans of one another. Confirmation of repetitive regions of allele sequence that cannot be resolved using Sanger sequencing.
G1 (G0 × WT)	On-target PCR and Sanger sequencing PCR spanning target region. Sequence confirmation of allele in both directions.	Same PCR assays as for G0. Transmitted allele sequence confirmed by sequencing.	Same PCR assays as for G0. Transmitted allele sequence to be confirmed by sequencing.	Same PCR assays as for G0. Transmitted allele sequence to be confirmed by sequencing.	Same PCR assays as for G0. Transmitted allele sequence to be confirmed by sequencing.
	Off-target PCR and Sanger sequencing	PCR spanning off-target site(s). Sequence confirmation of allele in both directions. No evidence of off-target activity detected, i.e. no difference in amplicon size and sequence is WT.
	ddPCR copy counting Copy counting of excised region/insertion sites and HDR donor templates if used to check for random integrations.	Copy counting of the excised region shows copy number of 1.	Copy counting of correct G1 should give copy numbers: Mutant = 1 copy, WT = 1 copy	Copy counting of correct G1 should give copy numbers: Mutant = 1 copy, WT (insertion point) = 1 copy	Copy counting of correct G1 should give copy numbers: 5′ LoxP site = 1, Floxed exon = 2, 3′ LoxP site = 1
	Long read sequencing Deep interrogation of allele sequence using large number of single reads across large interval. Detection of upstream/downstream events/re-arrangements.a			Confirmation of repetitive regions of allele sequence that cannot be resolved using Sanger sequencing. Entire interval should be covered without error.	Confirmation of repetitive regions of allele sequence that cannot be resolved using Sanger sequencing. Entire interval should be covered without error.

HDR, homology directed repair; PCR, polymerase chain reaction; ddPCR, droplet digital PCR; WT, wild-type;

^aShort-read next generation sequencing can be employed.