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Table 1 Microinjection data for floxed allele generation at seven loci

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

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
  1. 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)
  2. 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)
  3. 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
  4. 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)
  5. eAnimals 1 and 2 were heterozygous for both 5′ and 3′ LoxP sites (Fig. 2i)
  6. fAnimals 4 and 7 had only 5′ LoxP insertion and the animal 6 had correctly targeted LoxP sites (Additional file 1: Figure S2g)
  7. gAnimal 12 had correctly targeted LoxP sites and all others were wild type (Additional file 1: Figure S2h).
  8. 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)