Easi-CRISPR: a robust method for one-step generation of mice carrying conditional and insertion alleles using long ssDNA donors and CRISPR ribonucleoproteins

Table 2 Microinjection data for knock-in allele generation at six loci

Gene-insertion cassette	ssDNA length Left Arm-Cassette-Right Arm (bases) [source of ssDNA]	Zygotes injected	Zygotes transferred	Live-born animals (percentage of transferred zygotes)	Targeted animals (%)^a
Fgf8-P2A-FlpO	105 + 1368 + 98 [IDT Megamer™]	22	13	4 (30.8)	1 (25%)^b
Slc26a5-P2A-FlpO	99 + 1368 + 72 [IDT Megamer™]	28	22	3 (13.6)	1 (33%)^c
Mafb-P2A-FlpO	85 + 1368 + 96 [IDT Megamer™]	58	53	8 (15.1)	2 (25%)
Otoa-rtTA	96 + 1220 + 98 [IDT Megamer™]	19	18	2 (11.1)	1 (50%)
Mmp9-T2A-mCitrine	60 + 782 + 60 [IvTRT]	52	50	12 (24)	8 (67%)^d
Mmp13-T2A-mCherry	60 + 779 + 60 [IvTRT]	55	52	10 (19.2)	4 (40%)

^aThe alleles that did not contain the inserts were not analyzed for the presence of indels because genotyping assays were mainly designed to identify the targeted-insertion alleles. However, noticeable sequence additions or deletions were observed for some samples in these assays (e.g., additions in Slc26a5 animal 1 (Additional file 1: Figure S11), in Mmp9 animal 10 (Additional file 1: Figure S14), and deletion in Mmp9 animal 4 (Additional file 1: Figure S14))
^bAnimal 4 had bi-allelic insertions of the knock-in cassette (Fig. 3c)
^cAnimal 1 had additional sequences at the 3′ junction (sequence not fully characterized and pup 3 had a precise insertion at both junctions (Additional file 1: Figure S11)
^dAnimal 4 appeared to be mosaic containing both a correctly targeted allele and a deletion in the 3′ junction (sequence not fully characterized) (Additional file 1: Figure S14)

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