Correction of a genetic disease by CRISPR-Cas9-mediated gene editing in mouse spermatogonial stem cells
Authors:
Wu Y, Zhou H, Fan X, Zhang Y, Zhang M, Wang Y, Xie Z, Bai M, Yin Q, Liang D, Tang W, Liao J, Zhou C, Liu W, Zhu P, Guo H, Pan H, Wu C, Shi H, Wu L, Tang F, Li J
In:
Source:
Cell Res
Publication Date:
(
2015
)
Issue:
25(1)
:
67-79
Research Area:
Stem Cells
Platform:
Nucleofector® I/II/2b
Experiment
Electroporation of SSCs: The pX330-mcherry plasmids harboring corresponding sgRNAs and oligo DNA were transfected into SSCs with Amaxa Cell Line Nucleofector Kit L (Lonza) using Amaxa Nucleofector according to the manufacturer’s instruction. For mutation of multiple endogenous genes, the pX330-mcherry plasmids harboring corresponding sgRNAs were simultaneously transfected into SSCs. 24 h after transfection, the SSCs expressing mCherry were separated with flow cytometry and plated on MEF feeders. Single colonies were picked and expanded for genotyping or transplantation. For the establishment of YF-SSC line, the EGFP-SSCs were nucleofected with PB-mRFP and pBase (PB transposase enzyme). After sorting of SSCs harboring red fluorescent protein with flow cytometry for 2 rounds, SSC line stably expressing EGFP and mRFP was established. For deriving SSC lines from single SSCs, FACS using a BD FACSAriaII cell sorter was adopted to deposit one mCherry-positive cell into each well of the 96-well plates. Lonza short summary: shows the proof of principle of CRISPR based gene therapy on mouse spermatogonial stem cells leading to genetically corrected progeny.
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.
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