Correction of a genetic disease by CRISPR-Cas9-mediated gene editing in mouse spermatogonial stem cells

Abstract

Spermatogonial stem cells (SSCs) can produce numerous male gametes after transplantation into recipient testes, presenting a valuable approach for gene therapy and continuous production of gene-modified animals. However, successful genetic manipulation of SSCs has been limited, partially due to complexity and low efficiency of currently available genetic editing techniques. Here, we show that efficient genetic modifications can be introduced into SSCs using the CRISPR-Cas9 system. We used the CRISPR-Cas9 system to mutate an EGFP transgene or the endogenous Crygc gene in SCCs. The mutated SSCs underwent spermatogenesis after transplantation into the seminiferous tubules of infertile mouse testes. Round spermatids were generated and, after injection into mature oocytes, supported the production of heterozygous offspring displaying the corresponding mutant phenotypes. Furthermore, a disease-causing mutation in Crygc (Crygc(-/-)) that pre-existed in SSCs could be readily repaired by CRISPR-Cas9-induced nonhomologous end joining (NHEJ) or homology-directed repair (HDR), resulting in SSC lines carrying the corrected gene with no evidence of off-target modifications as shown by whole-genome sequencing. Fertilization using round spermatids generated from these lines gave rise to offspring with the corrected phenotype at an efficiency of 100%. Our results demonstrate efficient gene editing in mouse SSCs by the CRISPR-Cas9 system, and provide the proof of principle of curing a genetic disease via gene correction in SSCs.

Figure 1

Mice carrying mutant EGFP transgene generated via CRISPR-Cas9-mediated gene editing in SSCs. (A) Diagram for the generation of mice with mutant EGFP transgene through CRISPR-Cas9-mediated gene editing in SSCs. EGFP-SSCs are electroporated with RFP vector to generate YF-SSCs. The EGFP transgene is mutated by CRISPR-Cas9, resulting in RFP-SSCs. Two months after transplantation of RFP-SSCs into infertile male mice, RFP-marked RS are enriched from reconstituted testes and injected into mature oocytes to produce mice carrying mutant EGFP transgenes. RS, round spermatids; ROSI, round spermatid injection. (B) Images of YF-SSCs. Top, red fluorescence (left) and green fluorescence (right); Bottom, bright-field image (left) and overlay (right). Scale bar, 50 μm. (C) Schematic of sgRNA targeting EGFP transgene (EGFP-sgRNA). Blue line labels the sgRNA-targeting sequence. (D) FACS enrichment for RFP-SSCs after mutation of EGFP transgenes in YF-SSCs via the CRISPR-Cas9 system. Cells from P4 zone, which expressed RFP alone, were enriched for further expansion. (E) Images of RFP-SSCs. Top, red fluorescence (left) and green fluorescence (right); Bottom, bright-field image (left) and overlay (right). The EGFP transgene has been successfully mutated in YF-SSCs. Scale bar, 50 μm. (F) Methylation analysis of the DMRs of H19 and Snrpn in EGFP-SSCs and RFP-SSCs. Open and filled circles represent unmethylated and methylated CpG sites, respectively. (G) RFP-SSCs can reconstitute the testis of busulfan-treated male mice. Normal testis (left); reconstituted testis with RFP-SSCs (right). RFP-positive seminiferous tubules can be observed in reconstituted testis with red fluorescence. Scale bar, 1 mm. (H) RFP-positive cells (cells of P2 zone in left image) are sorted from reconstituted testis and haploid cells (RS) are further isolated from RFP-positive cells according to DNA content. (I) Newborn pups developed from 2-cell embryos generated after injection of RFP-positive RS into mature oocytes. (J) DNA sequencing analysis of progeny. Note that the sequence of PCR products amplified from the EGFP transgene shows 1-bp deletion in one pup.

Figure 2

Cataract mice generated via CRISPR-Cas9-mediated gene editing in SSCs. (A) Diagram for the generation of cataract mice carrying mutant Crygc genes through CRISPR-Cas9-mediated gene editing in SSCs. EGFP-SSCs are electroporated with CRISPR-Cas9 targeting the Crygc gene. One day after transfection, mCherry-positive cells, which presumably are successfully transfected with the CRISPR-Cas9 system, are enriched for further expansion. EGFP-mCrygc-SSCs are passaged for several times in vitro. Two months after transplantation of EGFP-mCrygc-SSCs into infertile male mice, EGFP-marked RS are enriched from reconstituted testes and injected into mature oocytes to produce cataract mice carrying mutant Crygc gene. (B) Schematic of sgRNA targeting endogenous Crygc gene (Crygc-sgRNA). Blue line labels the sgRNA-targeting sequence. (C) FACS enrichment for mCherry-positive cells (P4 zone), which represent the cells transfected with the CRISPR-Cas9 system, for further expansion. (D) CRISPR-Cas9 induces double strand breaks (DSB) efficiently and produces NHEJ-mediated indels in Crygc gene. Note that the sequence of PCR products amplified from the Crygc gene shows multiple peaks in SSCs. (E) Methylation analysis of the DMRs of H19 and Snrpn in EGFP-SSCs and EGFP-mCrygc-SSCs. Open and filled circles represent unmethylated and methylated CpG sites, respectively. (F) EGFP-mCrygc-SSCs can reconstitute the testis of busulfan-treated male mice. Normal testis (left); reconstituted testis with EGFP-mCrygc-SSCs (right). EGFP-positive seminiferous tubules can be observed in reconstituted testis with green fluorescence. Scale bar, 1 mm. (G) RS are sorted from cells of reconstituted testes according to DNA content. 23.3% means the total of haploid cells, including the donor-derived and recipient-derived cells. (H) DNA sequencing analysis of two pups developing from reconstructed oocytes generated after injection of RS into mature oocytes. Note that the sequences of PCR products amplified from the Crygc gene show no gene modification in one pup and 2-bp deletion in the other one. (I) The same two pups in H, after growing up to adult, show normal lenses (top) and cataract phenotype (bottom), respectively.

Figure 3

Correction of a genetic disease in mouse via CRISPR-Cas9-mediated gene editing in SSCs. (A) Diagram for the correction of a genetic disease in mouse through CRISPR-Cas9-mediated gene editing in SSCs. Crygc^−/−-SSCs are established from male cataract mice carrying homozygous mutant Crygc gene. Crygc^−/−-SSCs are electroporated with CRISPR-Cas9 targeting the Crygc gene with or without exogenously supplied oligonucleotide. mCherry-positive cells, which are transfected SSCs, are enriched for further expansion. Single colonies are picked and transferred into one well of 96-well plates for SSC line derivation. To derive SSC lines from singe SSCs, single mCherry-positive cells are deposited into one well of 96-well plates by BD FACSAriaII cell sorter for expansion. Clonal expansion of single SSCs or SSC colonies proceeds from 96-well plates to 6-well plates for routine culture in SSC medium. SSC lines derived from single SSCs or SSC colonies are characterized and SSC lines carrying corrected Crygc gene without off-target mutations are selected for transplantation into infertile male mice carrying homozygous EGFP transgenes. Two months later, non-EGFP-marked RS are isolated from reconstituted testes and injected into mature oocytes to produce healthy mice carrying corrected Crygc gene. (B) Crygc^−/−-4W-SSC line show typical SSC morphology. Scale bar, 50 μm. (C) DNA sequencing analysis of Crygc^−/−-SSCs shows 1-bp deletion at both alleles. (D) Clonal expansion of single SSC colony from 96-well plates to 6-well plates for routine culture in SSC medium. Left, one SSC colony in one well of 96-well plate; Right, one established SSC line from single SSC colony in one well of 6-well plate. Scale bar, 50 μm. (E) DNA sequencing analysis of SSCs from Line-NHEJ-4 and Line-NHEJ-9. Line-NHEJ-4 carries CRISPR-Cas9-mediated 3-bp deletion at the same loci of two alleles, which could not restore the correct open reading frame in Crygc gene. In contrast, Line-NHEJ-9 carries 1-bp insertion at the same loci of two alleles, thus restoring the correct open reading frame in Crygc gene. Deletions are indicated with (−). Letter marked in red represents the inserted nucleotide. (F) Schematic of sgRNA targeting mutant Crygc gene (mCrygc-sgRNA). Blue line labels the sgRNA-targeting sequence. The PAM is labeled with green line. (G) DNA sequencing analysis of SSCs from Line-HDR1–8. Note that the sequence of PCR products amplified from the Crygc gene shows that Line-HDR1-8 carries corrected Crygc gene. (H) SSC expansion of single SSC from 96-well plates to 6-well plates for routine culture in SSC medium. Left, one SSC colony formed from single SSC in one well of 96-well plate; Right, one established SSC line from single SSC in one well of 6-well plate. Scale bar, 100 μm. (I) DNA sequencing analysis of SSCs from Line-HDR1-SC-8. Line-HDR1-SC-8 carries NHEJ-mediated insertion of 1 bp (A) at 6 bp downstream of the deletion site at one allele and insertion of 1 bp (A) at 7 bp downstream of the deletion site at the other allele, which would result in the restoration of the correct open reading frame at both alleles of the Crygc gene. (J) Methylation analysis of the DMRs of H19 and Snrpn in Crygc^−/−-SSCs and Line-NHEJ-9. Open and filled circles represent unmethylated and methylated CpG sites, respectively. (K) One adult mouse from Line-NHEJ-4 shows cataract phenotype (left), and one adult mouse from Line-NHEJ-9 shows normal lenses (right). (L) Three newborn pups (left) developing from reconstructed oocytes generated after injection of RS from Line-HDR1-8 into mature oocytes. Genotyping analysis of the pups (right). Note that the sequence of PCR products amplified from the Crygc gene shows only one peak in 3 pups, indicating successful transmission of the corrected Crygc gene in repaired SSCs to progeny.