Dataset Information

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.

SUBMITTER: Wu Y

PROVIDER: S-EPMC4650588 | biostudies-literature | 2015 Jan

REPOSITORIES: biostudies-literature

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

Wu Yuxuan Y Zhou Hai H Fan Xiaoying X Zhang Ying Y Zhang Man M Wang Yinghua Y Xie Zhenfei Z Bai Meizhu M Yin Qi Q Liang Dan D Tang Wei W Liao Jiaoyang J Zhou Chikai C Liu Wujuan W Zhu Ping P Guo Hongshan H Pan Hong H Wu Chunlian C Shi Huijuan H Wu Ligang L Tang Fuchou F Li Jinsong J

Cell research 20141205 1

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 CR ...[more]

PMID: 25475058

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Project description:Genetic and molecular modifications of the large dsDNA chloroviruses, with genomes of 290 to 370 kb, would expedite studies to elucidate the functions of both identified and unidentified virus-encoded proteins. These plaque-forming viruses replicate in certain unicellular, eukaryotic chlorella-like green algae. However, to date, only a few of these algal species and virtually none of their viruses have been genetically manipulated due to lack of practical methods for genetic transformation and genome editing. Attempts at using Agrobacterium-mediated transfection of chlorovirus host Chlorella variabilis NC64A with a specially-designed binary vector resulted in successful transgenic cell selection based on expression of a hygromycin-resistance gene, initial expression of a green fluorescence gene and demonstration of integration of Agrobacterium T-DNA. However, expression of the integrated genes was soon lost. To develop gene editing tools for modifying specific chlorovirus CA-4B genes using preassembled Cas9 protein-sgRNA ribonucleoproteins (RNPs), we tested multiple methods for delivery of Cas9/sgRNA RNP complexes into infected cells including cell wall-degrading enzymes, electroporation, silicon carbide (SiC) whiskers, and cell-penetrating peptides (CPPs). In one experiment two independent virus mutants were isolated from macerozyme-treated NC64A cells incubated with Cas9/sgRNA RNPs targeting virus CA-4B-encoded gene 034r, which encodes a glycosyltransferase. Analysis of DNA sequences from the two mutant viruses showed highly targeted nucleotide sequence modifications in the 034r gene of each virus that were fully consistent with Cas9/RNP-directed gene editing. However, in ten subsequent experiments, we were unable to duplicate these results and therefore unable to achieve a reliable system to genetically edit chloroviruses. Nonetheless, these observations provide strong initial suggestions that Cas9/RNPs may function to promote editing of the chlorovirus genome, and that further experimentation is warranted and worthwhile.

Dataset Information

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

Publications

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

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