Efficiently Editing Dad’s Genetic Mutations

By targeting sperm stem cells rather than zygotes, researchers have developed a gene editing method to obtain healthy offspring at 100 percent efficiency.

AsianScientist (Dec. 15, 2014) – Scientists have developed a process to correct genetic mutations in single spermatogonia stem cells (SSCs) that can be used to to generate healthy progeny at 100 percent efficiency. These results have been published in Cell Research.

The clustered regularly interspaced short palindromic repeat sequences (CRISPR)-Cas9 system from bacteria has received extensive interest from scientists working on gene therapy. Previous studies have shown that the CRISPR-Cas9 system can be used to correct disease-causing mutations in mice via direct injection into the developing zygotes. Nevertheless, direct injection of CRISPR-Cas9 system into zygotes could potentially generate off-target modifications.

To circumvent these problems, researchers from the Shanghai Institutes for Biological Sciences (SIBS), Chinese Academy of Sciences and Peking University used the CRISPR-Cas9 system on SSCs instead. Led by Drs. Li Jinsong, Wu Ligang and Tang Fuchou, the research team first generated a SSC line from a male mouse that lacked the Crygc gene.

After electroporation of CRISPR-Cas9 or CRISP-Cas9 plus exogenous wildetype single-stranded DNA oligonucleotides into Crygc-/--SSCs, they derived multiple SSC lines that carried corrected Crygc gene via NHEJ (non-homologous end joining) or HDR (homology-directed repair)-mediated gene editing.

The researchers went on to analyze off-target effects in these cells by sequencing predicted potential off-targets loci and by whole-genome sequencing. They further analyzed the imprinting state of these cells by bisulfite analysis of specific imprinting genes and by whole-genome bisulfite sequencing.

Cells that were corrected and shown to be free from unwanted genetic changes were developed into spermatids and injected into eggs, giving rise to offspring with the corrected phenotype at an efficiency of 100 percent.

In addition to immediate applications in mice, the authors suggest that their SSC-based gene therapy could one day be used to treat male infertility caused by genetic defects or to avoid dominant genetic diseases carried by males.

The article can be found at: Wu et al. (2014) Correction of a Genetic Disease by CRISPR-Cas9-mediated Gene Editing in Mouse Spermatogonial Stem Cells.

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Source: Shanghai Institutes for Biological Sciences.
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