Fig 1.
Cas9/CRISPR-mediated loxp sequence insertion adjacent to miR-34c.
(a) Schematic representation of DNA sequence structure and genotyping primer design. Primers are indicated as orientation-specific arrows. The loxp insertion adds 46bp to the 288bp WT external amplicon size (334bp total, white box). miR-34c is indicated by the light grey box (77bp). The sgRNA target site is indicated by the dark grey box (20bp). The sgRNA was designed to target the wild-type sequence, with minimal effect on the HDR construct, or edited genomic sequence. (b) Agarose gel images showing amplicons using external forward (Fw) and revers (Rv) primers. WT bands are marked with black arrows, whereas the “loxp shift” bands are indicated using red arrows. Note that successful loxp insertion led to band shifts ranging from 288bp to 334bp. Germline genotyping assays were performed using the testes from Founder 3 (3t) and Founder 9 (9t) (t = testis). Founder 9 showed much greater abundance of the loxp shift bands than Founder 3. (c) Tail DNA PCR genotyping for F1 progeny of Founder 3, which was bred with WT females, showed no germline transmission of the loxp allele (Pups 13–21). (d) Tail DNA PCR genotyping results of F1 pups from Founder 9, which was crossed with WT females, showing successful germline transmission of the loxp allele. (e) Sequencing results of Founder 9 tail DNA, showing the successful loxp insertion, flanked by two engineered Kpn1 sites. (f) Sequencing results of tail DNA from Founder 9’s F1 progeny (Pup 22).
Fig 2.
Cas9/CRISPR-mediated deletion in Ubqlnl.
(a) Schematic representation of DNA sequence structure and genotyping primer design. Primers are indicated as orientation-specific arrows. The wild-type external amplicon is 376bp, while the deletion alleles vary in size, relative to the wild-type amplicon. The deletion forward (Fwd) primer, paired with the external reverse (Rev) primer, amplifies a 192bp amplicon in wild type. Deletion alleles that cover this region do not amplify. sgRNA binding sites are indicated by the dark grey boxes. The approximate regions of the deletions in Founders 7 and 10 are depicted by the red line. (b) The founder pups (1–17) were genotyped using the external primer set. The WT allele band is indicated by the black arrow. Any band, above or below, represents either an insertion, or deletion, respectively (*). (c) The founder generation was likewise genotyped with the deletion internal primer amplicon. (d) Founders 7 and 10 were germline genotyped, using the external primer set. (e) Founders 7 and 10 were germline genotyped, using deletion primer set (t = testis; s = sperm; L = left; R = right). (f-g) The F1 progeny of Founder 7 (deletion/deletion based on tail lysate genotyping) was likewise genotyped, and the results suggest mosaicism in Founder 7’s germline because some pups received the deletion allele from Founder 7, while others were homozygous wild type. (h) Representative sequencing results for Founder 7 using tail DNA. (i) Representative sequencing results for Founder 7’s F1 progeny using tail DNA. Sequences of the amplicons using external primers for Founder 7 and one of Founder 7’s progeny (F1 Pup 49) displayed a large deletion.
Fig 3.
Cas9/CRISPR-mediated deletion of miR-741 shows mosaicism in multiple forms.
(a) Schematic representation of DNA sequence structure and genotyping primer design. Primers are indicated as orientation-specific arrows. The wild-type amplicon is 484bp, while the deletion alleles vary in size, depending on the NHEJ. sgRNA binding sites are indicated by dark grey boxes. miR-741 is depicted as a light grey box. The approximate deletion in Founder 5 is depicted by the green line, whereas that of Founder 15 is depicted by the red line. (b) Agarose gel images showing tail DNA PCR genotyping for founder mice using the external primer set. The approximate band size of the WT allele amplicon is indicated by the black arrow. The deletion/insertion alleles were variable in size, depending on the founder mice; Founder 5 and 15 had a 132bp and a 30bp deletion, respectively. Founders 5 and 6 represent mosaic males, showing both wild-type allele amplification (upper band) and deletion allele amplification (lower band). (c) PCR germline genotyping results of Founders 5 and 15, showing the presence of the multiple alleles (t = testis, s = sperm). (d) PCR tail genotyping of F1 pups (F1 Pups 16–22) fathered by Founder 5. (e) PCR tail genotyping of F1 pups derived from Founder 15 (F1 Pups 23–29). (f) Representative sequencing results for wild-type miR741 (upper), Founder 5 (lower) and Founder 15 (middle). (g) Representative sequencing results for wild-type miR741 (upper) and F1 Pup 23 fathered by Founder 15.