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Table 2 Microinjection data for knock-in allele generation at six 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
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%)
  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 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))
  2. bAnimal 4 had bi-allelic insertions of the knock-in cassette (Fig. 3c)
  3. 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)
  4. 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)