1. CRISPR-Cas9 in development and genetic disease
Abigail Allen

2. CRISPR-Cas9
Pairing of CRISPR/Cas9 complex makes up a prokaryotic defense mechanism that can degrade viral DNA
CRISPR sequences contain portions of viral DNA sequences
Cas9 can be programmed to target specific DNA sequences for cleavage

3. CRISPR-Cas9 in correction of “bad” genes
Non-homologous end joining
Homology directed repair
Ma et al., 2017

4. HBB gene editing in human tripronuclear (3PN) zygotes
3PN zygotes can develop into blastocysts, but won’t fully develop (Liang et al., 2015)
Helps with ethical issues
Error-prone NHEJ was preferred in hemoglobin (HBB), which introduced off-target effects
Endogenous templates could be used as HDR templates with higher efficiency, but expected off- target mutations
Edited embryos were mosaic
Could not determine gene profile from pre-implantation genetic diagnosis (PGD)
Base mispairing represented in red

5. CCR5 gene editing in human tripronuclear (3PN) zygotes
3PN commonly found during in vitro fertilization process (Kang et al., 2016)
C-C chemokine receptor type 5 (CCR5)
Heterozygous/homozygous individuals for CCR5△32 have slower progression of HIV infection
Attempted genetic integration of CCR5△32 by HDR, though this was less efficient than NHEJ
CCR5△32 can be introduced, though not without off-target mutations

6. Genome editing in embryogenesis
POU5F1 targeted to determine effect on Oct4 expression (Fogarty et al., 2017)
OCT4 deficient embryos affected expression of embryonic genes, such as PAX6
Suggest OCT4 downregulation negatively affects embryonic development
Gave a successful method of genome editing during embryogenesis

7. Germline correction of genetic disease using CRISPR-Cas9
Germline genetic mutation tested in mouse embryos, using various reporter genes (Yang et al., 2013)
Used methods to reduce off target effects
Germline correction tested in mice with the successful deletion of cataracts (Wu et al., 2013)
Used specific oligos, Cas9 mRNA, and sgRNAs, which successfully and specifically targeted mutant Crygc gene
Modified mutant allele in less than 50% of pups
Between 18-31% of total pups had no cataracts via CRISPR-Cas9 correction

8. Correction of human genetic disease using CRISPR-Cas9 in development
Gene correction performed in hypertrophic cardiomyopathy (HCM) due to MYBPC3 mutation (Ma et al., 2017)
CRISPR-Cas9 can reduce/prevent mosaicism in M-phase oocytes
Helps to reduce mutant embryos, and those that may grow with a mosaic of wild type and diseased genes
3PN zygotes were used before in study of disease, though both had issues of mosaicism (Kang et al., 2016, Liang et al., 2015)
Ma et al. found improved efficiency of HDR repair, though NHEJ still occurred

9. Conclusions and ethical concerns
Improved recombination techniques have been developed to produce embryos without disease-carrying alleles
Still, is imperfect in correcting genetic disease, as CRISPR- Cas9 does not have 100% efficiency
Some ethical concerns, as it opens the door for germline editing of embryos for superficial alteration
Further research is required for improving efficiency, as well as consideration of how bioethical concerns will be addressed

10. References
Fogarty, N. M. E., McCarthy, A., Snijders, K. E., Powell, B. E., Kubikova, N., Blakeley, P., … Niakan, K. K. (2017). Genome editing reveals a role for OCT4 in human embryogenesis. Nature, 550, 67–73.
Kang, X., He, W., Huang, Y., Yu, Q., Chen, Y., Gao, X., … Fan, Y. (2016). Introducing precise genetic modifications into human 3PN embryos by CRISPR/Cas-mediated genome editing. Journal of Assisted Reproduction and Genetics, 33, 581–
Liang, P., Xu, Y., Zhang, X., Ding, C., Huang, R., Zhang, Z., … Huang, J. (2015). CRISPR/Cas9-mediated gene editing in humantripronuclear zygotes. Protein & Cell, 6(5), 363–372.
Ma, H., Marti-Gutierrez, N., Park, S.-W., Wu, J., Lee, Y., Suzuki, K., … Mitalipov, S. (2017). Correction of a pathogenic gene mutation in human embryos. Nature, 548, 413–
Wu, Y., Liang, D., Wang, Y., Bai, M., Tang, W., Bao, S., … Li, J. (2013). Correction of a genetic disease in mouse via use of CRISPR-Cas9. Cell Stem Cell, 13, 659–662. Retrieved from 
Yang, H., Wang, H., Shivalila, C. S., Cheng, A. W., Linyu, S., & Jaenisch, R. (2013). One- step generation of mice carrying reporter and conditional alleles by CRISPR/Cas mediated genome engineering. Cell, 154(6), 1370–