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19 September 2015 Research proposal for Hai Zhou, Ph.D. Background My 2014 Cell Research paper: As a Master's student in the laboratory of Professor Jinsong Li at the Chinese Academy of Sciences in Shanghai, I established SSC culture, SSC transplantation, SSC electrotransfection and FACS of haploid gametes. My colleagues and I then applied the newly developed CRISPR-Cas9 system for genome editing in SSCs. Our results show that genetic modifications can be induced efficiently by CRISPR-Cas9 system in mouse SSCs: CRISPR-Cas9 can not only specifically mutate an EGFP transgene but also the endogenous Crygc gene in SCCs. The mutated SSCs were transplanted into the seminiferous tubules of the testes of mice, which were made infertile by treatment with busulfan. By FACS we found that round spermatids were generated from the transplanted SSCs. By injecting these round spermatids into oocytes, we obtained heterozygous offspring displaying the expected mutant phenotype. A disease-causing mutation in the Crygc gene (Crygc-/-) existed already in mouse. We next derived SSCs of Crygc gene (Crygc-/-) mice. Using CRISPR-Cas9, SSCs could be repaired via nonhomologous end joining (NHEJ)-mediated indels or homology-directed repair (HDR). By single cell expansion we derived multiple SSC lines carrying the corrected Crygc gene with no evidence of off-target effects and no mosaicism. These cells led to the production of offspring with the repaired disease phenotype at an efficiency of 100%. Thus, CRISPR-Cas9 can be efficiently applied in mouse SSCs and that there was no mosaic phenomenon. March 2015 Cell Reports paper Chapman et al. This paper entitled "Targeted Germline Modifications in Rats Using CRISPR/Cas9 and Spermatogonial Stem Cells" extends our 2014 paper to rat. This paper shows that CRISPR/Cas9-modified spermatogonia can regenerate spermatogenesis and display long-term sperm-forming potential following transplantation into rat testes. They obtained genetically modified rats by combining rat SSCs with CRISPR-Cas9. The degu (Octodon degus) is a small caviomorph rodent that is endemic central Chile.
 Elderly degus develop in captivity a disease that has remarkably similarity to human Alzheimer's disease. Scope of the research plan The subject of the research is develop degu SSCs and apply CRISPR-Cas9 to obtain genetically modified degu strains, with mutations in the Amyloid Precusor Protein (APP) gene, the tau gene, and other genes. 
Step 1: Establish SSCs from genetically unmodified degus The spermatogonial stem cell is an undifferentiated male germ cell, originating in a seminiferous tubule and dividing into two primary spermatocytes in the production of spermatozoa. SSCs have been established in human, mouse, rat and so on. Establishing degu SSCs is the first, and critical, step of my project. Step 2: Apply CRISPR-Cas9 for genome editing of degu SSCs Next I will mutate selected genes (one at the time) in degu SSCs using CRISPR-Cas9. The obvious target genes are APP and tau, but other genes could be considered as well. There is reason to doubt that genome editing with CRISPR-Cas9 is not possible in degu. Step 3: Develop mutant degus Mutant degu SSCs will be transplanted into the tested of male degus, which have been made infertile by treatment with busulfan. I plan to rely on natural mating systerm to obtain genetically modified degu offspring. Step 4: Characterize the mutant phenotype I will characterize the brains of mutant degus mainly by histological techniques, to determine if the naturally occurring, Alzheimer's-like disease develops faster, more severely, and in more mutant animals. The subject of the research is develop degu SSCs and apply CRISPR-Cas9 to obtain genetically modified degu strains, with mutations in the Amyloid Precusor Protein (APP) gene, the tau gene, and other genes. 
Step 1: Establish SSCs from genetically unmodified degus The spermatogonial stem cell is an undifferentiated male germ cell, originating in a seminiferous tubule and dividing into two primary spermatocytes in the production of spermatozoa. SSCs have been established in human, mouse, rat and so on. Establishing degu SSCs is the first, and critical, step of my project. Step 2: Apply CRISPR-Cas9 for genome editing of degu SSCs Next I will mutate selected genes (one at the time) in degu SSCs using CRISPR-Cas9. The obvious target genes are APP and tau, but other genes could be considered as well. There is reason to doubt that genome editing with CRISPR-Cas9 is not possible in degu. Step 3: Develop mutant degus Mutant degu SSCs will be transplanted into the tested of male degus, which have been made infertile by treatment with busulfan. I plan to rely on natural mating systerm to obtain genetically modified degu offspring. Step 4: Characterize the mutant phenotype I will characterize the brains of mutant degus mainly by histological techniques, to determine if the naturally occurring, Alzheimer's-like disease develops faster, more severely, and in more mutant animals.