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

Table 1 Microinjection data for floxed allele generation at seven 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
Pitx1-exon 2 floxed	93 + 862 + 91 [IvTRT]	85	76	10 (13.2)	4 (40%)^b
Ambra1-exon 4 floxed	96 + 589 + 103 [IvTRT]	67	63	8 (12.7)	6 (75%)^c
Col12a1-exon 2 floxed	55 + 527 + 55 [IvTRT]	105	79	3 (3.8)	3 (100%)^d
Ubr5-exon 58 floxed	78 + 535 + 86 [IvTRT]	20	16	2 (12.1)	2 (100%)^e
Syt1-exon 6 floxed	75 + 635 + 75 [IDT Megamer™]	51	45	8 (17.8)	1 (12.5%)^f
Syt9-exon 3 floxed	87 + 893 + 68 [IDT Megamer™]	43	41	12 (29.3)	1 (8.5%)^g
PPP2r2a-exon 3 floxed	95 + 619 + 84 [IDT Megamer™]	34	33	3 (9.1)	3 (100%)^h

^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 deletions were observed for some samples (e.g., deletions in the non-targeted alleles; Fig. 2g, h; Additional file 1: Figure S2i)
^bAnimals 3, 5, 7, and 8 were heterozygous for both 5′ and 3′ LoxP sites. Animal 5 had a floxed allele with one nucleotide insertion mutation at the intronic region, which may not affect function. Animals 2, 9, and 10 had only 5′ LoxP site, and animal 4 had only 3′ LoxP site (Fig. 1g)
^cAnimals 1, 2, 3, 5, 7, and 8 were heterozygous for both the 5′ and 3′ LoxP sites (Fig. 2g). Animal 7 had a floxed allele with 1-bp insertion mutation in the intronic region, which may not affect function
^dAnimals 1 and 2 were heterozygous for both 5′ and 3′ LoxP sites and they carried deletions in their second allele. Animal 3 was biallelic for both LoxP sites (Fig. 2h)
^eAnimals 1 and 2 were heterozygous for both 5′ and 3′ LoxP sites (Fig. 2i)
^fAnimals 4 and 7 had only 5′ LoxP insertion and the animal 6 had correctly targeted LoxP sites (Additional file 1: Figure S2g)
^gAnimal 12 had correctly targeted LoxP sites and all others were wild type (Additional file 1: Figure S2h).
^hAnimals 1 and 2 were heterozygous for both loxPs with deletions in the second allele and pup 3 was biallelic (Additional file 1: Figure S2i)

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