Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
Filters applied. Clear all
. 2018 Aug 13;8(1):12059.
doi: 10.1038/s41598-018-30137-x.

i-GONAD (Improved Genome-Editing via Oviductal Nucleic Acids Delivery), a Convenient in Vivo Tool to Produce Genome-Edited Rats

Affiliations
Free PMC article

i-GONAD (Improved Genome-Editing via Oviductal Nucleic Acids Delivery), a Convenient in Vivo Tool to Produce Genome-Edited Rats

Shuji Takabayashi et al. Sci Rep. .
Free PMC article

Abstract

Zygote-microinjection or in vitro electroporation of isolated zygotes are now widely used methods to produce genome-edited mice. However, these technologies require laborious and time-consuming ex vivo handling of fertilized eggs, including zygote isolation, gene delivery into zygotes and embryo transfer into recipients. We recently developed an alternative method called improved genome-editing via oviductal nucleic acids delivery (i-GONAD), which does not require the above-mentioned ex vivo handing of zygotes, but instead involves intraoviductal instillation of genome-editing components, Cas9 protein and synthetic gRNAs, into the oviducts of pregnant females at the late 1-cell embryo stage under a dissecting microscope and subsequent electroporation. With this method, we succeeded in generating genome-edited mice at relatively high efficiencies (for example, knockout alleles were produced at ~97% efficiency). Here, we extended this improved technology to rats, and found that i-GONAD can create genome-edited rats in various strains, including Sprague Dawley and Lewis, and F1 hybrids (between Sprague Dawley and Brown Norway), with efficiencies of ~62% for indel mutations and ~9% for knock-ins. Thus, i-GONAD will be especially useful for the production of genome-edited rats in small laboratories where expensive micromanipulator systems and highly skilled personnel for embryo manipulation are unavailable.

Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1
Figure 1
(A) One-cell rat embryos isolated from 0.7 dpc female SD rats. Note the presence of cumulus cell-free 1-cell embryos. Bar = 100 μm. (B) The i-GONAD procedure. Under appropriate anesthesia, ovary/oviduct/uterus of a pregnant rat at 0.7 dpc was exposed under a dissecting microscope. After positioning the oviduct, it was gently grasped in forceps (a). A micropipette was then inserted into the lumen of the oviduct by piercing the oviduct wall and (controlled by a mouthpiece) approximately ~1.5 µL solution was immediately expelled into the oviduct (b). The injected substances can be easily identified by the co-injected Fast Green (c,d). After instillation, the entire oviduct was covered with a piece of wet Kimwipe towel and then subjected to in vivo electroporation using tweezer-type electrodes (e). After removal of electrodes, the electroporated area remains intact (f). (C) Detection of fluorescent 2-cell embryos after i-GONAD. When pregnant (0.7 dpc) SD rats were subjected to in vivo electroporation after intraoviductal instillation of Rhodamine + EGFP mRNA-containing solution (i-GONAD), some of the recovered 2-cell embryos exhibited distinct red and/or green fluorescence (a–c). In contrast, intraoviductal instillation of Rhodamine alone (without electroporation) (Control) resulted in embryos showing no fluorescence (d,e). Bar = 100 μm. (D) Flowchart of experiments to test the feasibility of i-GONAD for the production of mid-gestational fetuses carrying indels at a single target locus (Tyr). In Exp-1, BN females were mated to SD males to obtain pregnant BN rats, while in Exp-2, SD females were mated to BN males to obtain pregnant SD rats. (E) Schematic illustration of the wild-type Tyr locus. The target sequence of Tyr exon 2 (ex2) recognized by gRNA is overlined and the PAM sequence is marked in red. The target nucleotide “G” marked in green is a key nucleotide for tyrosinase activity; nucleotide replacement at this position often causes an albino phenotype. Primers for amplification 598 bp of exon 2, Rat Tyr-F and -R, are shown. (F) Offspring obtained after i-GONAD in Exp-1 or -2. The embryos numbered BSF1-#7, BSF1-#10 and SBF1-#24 exhibited non-pigmented eyes. The fetus numbered SBF1-#15 had pigmented eyes (shown by an arrow), probably as a result of unsuccessful genome editing at the Tyr locus. (G) Direct sequencing of PCR products derived from the embryos shown in (F). Red arrow in BSF1-#10, which showed eye non-pigmentation, indicate heterozygous bi-allelic KO at the Tyr target (nucleotide “G” marked in green in E) because one allele (called allele-A) has the nucleotide “A” (shown in brown) and the other allele (called wild-type allele or allele-G) lacks 6-bp sequence above the PAM. In contrast, the red arrow in SBF1-#15 indicates heterozygous mono-allelic KO, because one allele (called allele-A) has the nucleotide “A” (shown in brown), whereas the wild-type nucleotide “G” exists at the same position as allele-G.
Figure 2
Figure 2
(A) Flowchart of the experiments used to test the feasibility of i-GONAD for the production of rats with ssODN-mediated KI at a single target locus (Tyr). In Exp-3, LEW females were mated to LEW males to obtain pregnant LEW rats. LEW is an albino strain with non-pigmented eyes. In Exp-4 and 5, SD females were mated to SD males to obtain pregnant SD rats. SD is an albino strain with non-pigmented eyes and coat. NHEJ-inhibitor, SCR7, was co-injected with RNP + ssODN in Exp-5. (B) Schematic illustration of the mutated Tyr locus. The target sequence of Tyr exon 2 (ex2) recognized by gRNA is overlined and the PAM sequence is marked in red. The nucleotide “A” marked in brown is a mutated nucleotide that causes the albino phenotype. Primers for amplification 598 bp of exon 2, Rat Tyr-F and -R, are shown. In the ssODN, the wild-type nucleotide “G” that corresponds to the mutated nucleotide “A” is shown in green. (C) Offspring obtained after i-GONAD of pregnant LEW females. The fetuses numbered LEW-#6 had albino eyes, while the fetus numbered LEW-#16 had pigmented eyes (shown by an arrow), probably as a result of successful KI of the ssODN into the Tyr locus. (D) Direct sequencing of PCR products derived from the embryos shown in (C). Red arrows in LEW-#6, which has non-pigmented eyes, indicate heterozygous bi-allelic mutations at the Tyr target, because one allele (called allele-A) has the nucleotide “A” (shown in brown) and the other allele (called allele-A’) has one nucleotide insertion above the PAM in allele-A. In contrast, the red arrow in LEW-#16 indicates a mosaic pattern of electrophoretograms, in which at least three nucleotides are present at the target site, including the wild-type G (shown in green) in one allele (called allele-G). Nucleotide(s) enclosed by boxes are those inserted. (E) Offspring obtained after i-GONAD of pregnant SD females. The two offspring numbered SD-#54 and -#55 showed pigmented coat color, but the other rats had albino coats. (F) Direct sequencing of PCR products derived from the animals shown in (E). Red arrows in SD-#54, which has pigmented eyes and/or coat, indicate the presence of a nucleotide “G” (shown in green) at the Tyr target in one allele (called allele-G).
Figure 3
Figure 3
(A) F1 offspring (SD-#75-1 to -8) of SD-#75 F0 female (showing pigmentation) after mating with unedited F0 male SD littermates. (B) Direct sequencing of PCR products derived from the animals is shown in (A). Red arrows indicate the position that is important for tyrosinase function. Note that the modified traits in the Tyr locus found in the F0 mouse (SD-#75) are successfully transmitted to the F1 offspring (including SD-#75-1 to -5 and -7).
Figure 4
Figure 4
(A) Schematic illustration of the wild-type Pax6 locus. Exons (ex1 to ex3) and introns are indicated by black boxes and a black line, respectively. Dual guide RNAs were used on the Pax6 locus: namely, the two target sequences recognized by gRNA are overlined and the PAM sequences are shown in red. The position of primers (Rat Pax6-F and -R) used to identify indels around the target sequences is indicated by arrowheads. (B) Fetal offspring (Pax6-#1 to #8) at 16.5 dpc obtained after i-GONAD targeted towards the paired domain of Pax6. Various abnormal phenotypes such as deformity of the facial structure together with loss of eye cup (as exemplified by Pax6-#1, #2 and #8) are discernible. (C) Representative 16.5 dpc fetuses exhibiting various types of craniofacial structure. Fetus Pax6-#5 and -#3 exhibited normal facial structure, and was wild-type and heterozygous for Pax6, respectively. Fetus Pax6-#8 completely lacked eyes and the lateral nasal prominence, and was homozygous knockout for Pax6. (D) Genotyping of the fetuses shown in (B). Fetuses Pax6-#1 to #3 and #8 have heterozygous PCR products (*allele-1of Pax6-#1, **allele-2 of Pax6-#1, allele-1of Pax6-#2, ††allele-2 of Pax6-#2, +allele-1of Pax6-#3, ++allele-2 of Pax6-#3, $allele-1of Pax6-#8 and $$allele-2 of Pax6-#8). Fetuses Pax6-#4 to #7 have wild-type PCR products (1,489 bp). (E) Direct sequencing of PCR products derived from the animals shown in (B). Fetus Pax6-#5, which was judged as wild-type through morphological inspection (shown in B and C) and genotyping (shown in D), exhibited normal sequence for Pax6. In contrast, fetus Pax6-#1, which was judged as KO through morphological inspection (shown in B) and genotyping (shown in D), exhibited a 7 bp sequence deletion (underlined) in front of the PAM (shown in red). (F) Comparison of PAX6 amino acid sequences from fetuses derived from SD females subjected to i-GONAD-mediated induction of indels at the Pax6 locus. The control amino acid sequence of wild-type PAX6 protein is shown on the top. The amino acid sequences of the mutated PAX6 proteins (derived from Pax6-#1 to #3 and #8) are shown. Predicted consequences of the mutation on amino acid sequences are highlighted in red. Stop codons are shown as x. The green bar indicates the paired domain.

Similar articles

See all similar articles

Cited by 5 articles

References

    1. Horvath P, Barrangou R. CRISPR/Cas, the immune system of bacteria and archaea. Science. 2010;327:167–170. doi: 10.1126/science.1179555. - DOI - PubMed
    1. Wiedenheft B, Sternberg SH, Doudna JA. RNA-guided genetic silencing systems in bacteria and archaea. Nature. 2012;482:331–338. doi: 10.1038/nature10886. - DOI - PubMed
    1. Sander JD, Joung JK. CRISPR-Cas systems for editing, regulating and targeting genomes. Nat Biotechnol. 2014;32:347–355. doi: 10.1038/nbt.2842. - DOI - PMC - PubMed
    1. Aida T, et al. Cloning-free CRISPR/Cas system facilitates functional cassette knock-in in mice. Genome Biol. 2015;16:87. doi: 10.1186/s13059-015-0653-x. - DOI - PMC - PubMed
    1. Yang H, et al. One-step generation of mice carrying reporter and conditional alleles by CRISPR/Cas-mediated genome engineering. Cell. 2013;154:1370–1379. doi: 10.1016/j.cell.2013.08.022. - DOI - PMC - PubMed

Publication types

Feedback