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. 2015 Mar 24;10(11):1828-35.
doi: 10.1016/j.celrep.2015.02.040. Epub 2015 Mar 12.

Targeted Germline Modifications in Rats Using CRISPR/Cas9 and Spermatogonial Stem Cells

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Free PMC article

Targeted Germline Modifications in Rats Using CRISPR/Cas9 and Spermatogonial Stem Cells

Karen M Chapman et al. Cell Rep. .
Free PMC article

Abstract

Organisms with targeted genomic modifications are efficiently produced by gene editing in embryos using CRISPR/Cas9 RNA-guided DNA endonuclease. Here, to facilitate germline editing in rats, we used CRISPR/Cas9 to catalyze targeted genomic mutations in rat spermatogonial stem cell cultures. CRISPR/Cas9-modified spermatogonia regenerated spermatogenesis and displayed long-term sperm-forming potential following transplantation into rat testes. Targeted germline mutations in Epsti1 and Erbb3 were vertically transmitted from recipients to exclusively generate "pure," non-mosaic mutant progeny. Epsti1 mutant rats were produced with or without genetic selection of donor spermatogonia. Monoclonal enrichment of Erbb3 null germlines unmasked recessive spermatogenesis defects in culture that were buffered in recipients, yielding mutant progeny isogenic at targeted alleles. Thus, spermatogonial gene editing with CRISPR/Cas9 provided a platform for generating targeted germline mutations in rats and for studying spermatogenesis.

Figures

Figure 1
Figure 1. CRISPR/Cas9-Mediated Gene Targeting in Donor Stem Spermatogonia
(A) CRISPR/Cas9 cleavage of Epsti1 in rat Fibroblast and Spermatogonial (Sg d11) cultures on d11 post-transfection, and in flow sorted EGFP+ spermatogenic cells (R1661) on d56 after transplantation into the right testis of recipient R1661: transfection without (−) and with (+) Epsti1 gRNA. Arrows: predicted ~461 bp + ~226 bp Surveyor products. (B) Spermatogenesis (green fluorescence) in testis of R1661 on d56 after transplantation that developed from donor EGFP+ rat spermatogonial cultures containing CRISPR/Cas9- catalyzed Epsti1 mutations. (C) Targeted mutagenesis of Epsti1 exon 2 in rats by CRISPR/Cas9 spermatogonial gene editing. Donor spermatogonia were hemizygous for tgGCS-EGFP. FS, frame shift mutation; IF, mutation in frame, DTB, days from transplantation to first litter containing Epsti1 mutant animals. *Average value, R(n), Recipient identifier, ||Litters born 99 to 128 d post-transplantation, % of total F1 progeny/recipient. See also Figure S1. (D) Donor germline chimerism (%) in wildtype recipient rat testes. Ratio of seminiferous tubules containing EGFP+ elongating spermatids/total tubules containing elongating spermatids plotted/donor culture condition (Minus Selection and G418 Selected). See also Table S1 and Table S2. (E) Acrosome (PNA) & nuclear (Hoechst 33342) labeling marking donor-derived elongating spermatids (EGFP+) in tubule cross sections from recipient R1659. Left, 39 of 39 tubules contained elongating spermatids (EGFP+ or EGFP). Five EGFP tubules are marked with an asterisk. Scale 500 μm. Right, higher magnification image of donor spermatogenesis at stages VIII and XI in the rat seminiferous epithelial cycle [See: (Hess, 1990)]. Scale 100 μm. See also Figure S2.
Figure 2
Figure 2. Monoclonal Enrichment of Erbb3-Deficient Stem Spermatogonia
(A) CRISPR/Cas9-targeted Erbb3-mutations (exon 2) in clonally expanded rat spermatogonial lines. (B) Relative abundance of ERBB3 (upper arrowhead) and TUBA1a (lower arrowhead) in wildtype (colony D4; WT) and Erbb3-deficient (colony B9; KO) spermatogonial lines. (C) Left: Cultures of clonally expanded Wildtype (colony D4) and Erbb3 Knockout (colony B9) spermatogonia expressing tgGCS-EGFP. Scale, 50 μm. Right: Growth rates of clonally expanded Wildtype (WT) and Erbb3 Knockout (KO) spermatogonial lines. See also Figure S3. (D) NRG1-dependent development of differentiating spermatogenic cells from Wildtype and Erbb3 Knockout spermatogonial lines. (E) Spermatogenic cells from colonies D4 (Wildtype) and B9 (Erbb3 Knockout) analyzed in panel D. tgGCS-EGFP (green), ZBTB16 antibody (Red). Scale, 40 μm.
Figure 3
Figure 3. Transmission of Isogenic CRISPR/Cas9-Targeted Alleles to Rat Progeny
(A) Top: Spermatogenesis (green) derived from WT colony D4 and Erbb3 KO colony B9 in testes from R1683 at ~3.5 mo post-transplantation. Scale, 1 cm. Bottom: Seminiferous tubule cross section from R1683 illustrating spermatogenesis derived from colony B9 marked by EGFP (green). ZBTB16+ spermatogonia (red nuclei). Scale, 100 μm. See also Figure S4. (B) Genotypes of rat progeny derived from clonally expanded spermatogonial line C8. See also Figure S3. tgGCS-EGFP = hemizygous marker. (C) Recipient rat testes ~7.8 mo post-transplantation. Clonally expanded lines were transplanted into the right testis of R1686, R1702 and R1700 (EGFP+), but contralateral left testes were not transplanted. Note: right and left testes of R1697 were transplanted. Scale, 1 cm. (D) Mean testis weights from recipient rats analyzed ~7.8 mo after transplanting with wildtype (WT) spermatogonial lines (D4, n=2; B11, n=3; A6, n=2) and Erbb3-deficient (KO) spermatogonial lines (B9, n=2; C7, n=3; C8, n=3); NT, untransplanted testes, n=11 (error bars, ±SD; p values, multiple t-tests).
Figure 4
Figure 4. CRISPR/Cas9 Gene Targeting in early Embryos and Spermatogonia
(Left) Zygotes are injected with CRISPR/Cas9 constructs and transferred into surrogate female oviducts to support donor embryo development into mutant progeny. Mosaic mutant progeny born ~19–22 d post-transfer (depending on cleavage stage transferred)1 display variation in targeted alleles in various tissues, and must be crossed to wildtype stock to generate pure heterozygous mutants isogenic at respective targeted alleles in all cells of their body. (Center) Rat Pluripotent Stem Cell cultures could prospectively be genetically modified using CRISPR/Cas9. Rat ES cells with targeted mutations have been selected in culture prior to blastocyst injection (Tong et al., 2011). Injected blastocysts are transferred into uteri of surrogate females to produce mosaic/chimeric mutant animals ~19 d post-transfer1. Mosaic/chimeric animals are crossed to wildtype stock to establish pure heterozygous mutants. (Right) Spermatogonial Stem Cells can be genetically modified in culture using CRISPR/Cas9. Modified spermatogonia are injected into recipient rat testes to produce mutant spermatozoa that transmit targeted genomic modifications to heterozygous mutant progeny. Timelines for each approach listed above must consider ~75 d for rat breeder pairs to reach reproductive age, 21–23 d rat gestation time, plus a 4–5 d estrus cycle in rats (Lohmiller and Swing, 2006)2. *Includes additional 4 d to establish pseudo-pregnant female recipients by paring with vasectomized males; does not include time needed to prepare vasectomized male rats. **Remains to be determined (t.b.d) using CRISPR/Cas9; estimate based on rat ES cell lines selected following transfection with classical DNA targeting constructs (Tong et al., 2011); includes additional 4 d to establish pseudo-pregnant female recipients. ***Current study; minimum time required following delivery of CRISPR/CAS9 constructs to spermatogonia (with or without genetic selection) prior to transplantation was not studied and remains to be determined (t.b.d); includes 12 d to prepare recipient males.

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