In vitro CRISPR-mediated gamma globin gene activation in HEK293

Blood disorders like B-thalassemia or sickle cell anaemia obligate patients to require constant blood transfusions. These, among others, make blood a highly demanded resource in the medical field. Blood has been obtained from in-vitro erythroid differentiation from embryonic stem cells or induced pluripotent stem cells, but this blood tends to produce embryonic haemoglobin. In this project, we aim to use a CRISPR/Cas9 modified version and an aptamer approach to alter the expression of the β-globin locus upregulating the gamma globin gene to produce foetal haemoglobin. Guide vectors were created targeting previously known sequences in the gamma haemoglobin gene. HEK cells were transfected with different combinations of CRISPR/Cas9 elements, as well as different combinations of aptamers/effectors, to test the amount of gene activation achieved. Gene expression analysis was performed through RTq/PCR. It was found that HbG activation can be achieved to some extent, however, the levels of gamma haemoglobin attained are very low compared with the normal levels of HbG expressed in other cell lines like K562. From the different grouping of vectors (dCas9 protein + aptamer/coating protein + activation effectors) tested, the combination of dCas9ES + PP7/PCP + VPH presented the best performance. References Basak, Anindita, and Vijay G. Sankaran. 2016. “Regulation of the Fetal Hemoglobin Silencing Factor BCL11A.” Annals of the New York Academy of Sciences 1368 (1): 25–30. doi:10.1111/nyas.13024. Borg, Joseph, Petros Papadopoulos, Marianthi Georgitsi, Laura Gutiérrez, Godfrey Grech, Pavlos Fanis, Marios Phylactides, et al. 2010. “Haploinsufficiency for the Erythroid Transcription Factor KLF1 Causes Hereditary Persistence of Fetal Hemoglobin.” Nature Genetics 42 (9): 801–5. doi:10.1038/ng.630. MOL2NET, 2019, 5, ISSN: 2624-5078 2 http://sciforum.net/conference/mol2net-05 Chavez, Alejandro, Marcelle Tuttle, Benjamin W. Pruitt, Ben Ewen-Campen, Raj Chari, Dmitry TerOvanesyan, Sabina J. Haque, et al. 2016. “Comparison of Cas9 Activators in Multiple Species.” Nature Methods 13 (7): 563–67. doi:10.1038/nmeth.3871. Chen, Baohui, Luke A. Gilbert, Beth A. Cimini, Joerg Schnitzbauer, Wei Zhang, Gene-Wei Li, Jason Park, et al. 2013. “Dynamic Imaging of Genomic Loci in Living Human Cells by an Optimized CRISPR/Cas System.” Cell 155 (7): 1479–91. doi:10.1016/j.cell.2013.12.001. Dominguez, Antonia A., Wendell A. Lim, and Lei S. Qi. 2016. “Beyond Editing: Repurposing CRISPR–Cas9 for Precision Genome Regulation and Interrogation.” Nature Reviews. Molecular Cell Biology 17 (1): 5–15. doi:10.1038/nrm.2015.2. Forget, Bernard G., and H. Franklin Bunn. 2013. “Classification of the Disorders of Hemoglobin.” Cold Spring Harbor Perspectives in Medicine 3 (2). doi:10.1101/cshperspect.a011684. Gilbert, Luke A., Matthew H. Larson, Leonardo Morsut, Zairan Liu, Gloria A. Brar, Sandra E. Torres, Noam Stern-Ginossar, et al. 2013. “CRISPR-Mediated Modular RNA-Guided Regulation of Transcription in Eukaryotes.” Cell 154 (2): 442–51. doi:10.1016/j.cell.2013.06.044. Hardwick, J. M., L. Tse, N. Applegren, J. Nicholas, and M. A. Veliuona. 1992. “The Epstein-Barr Virus R Transactivator (Rta) Contains a Complex, Potent Activation Domain with Properties Different from Those of VP16.” Journal of Virology 66 (9): 5500–5508. Konermann, Silvana, Mark D. Brigham, Alexandro E. Trevino, Julia Joung, Omar O. Abudayyeh, Clea Barcena, Patrick D. Hsu, et al. 2014a. “Genome-Scale Transcriptional Activation by an Engineered CRISPR-Cas9 Complex.” Nature 517 (7536). Nature Publishing Group: 583–88. doi:10.1038/nature14136. ———. 2014b. “Genome-Scale Transcriptional Activation by an Engineered CRISPR-Cas9 Complex.” Nature 517 (7536): 583–88. doi:10.1038/nature14136. Ma, Hanhui, Li-Chun Tu, Ardalan Naseri, Maximiliaan Huisman, Shaojie Zhang, David Grunwald, and Thoru Pederson. 2016. “Multiplexed Labeling of Genomic Loci with DCas9 and Engineered SgRNAs Using CRISPRainbow.” Nature Biotechnology 34 (5): 528–30. doi:10.1038/nbt.3526. Sander, Jeffry D., and J. Keith Joung. 2014. “CRISPR-Cas Systems for Editing, Regulating and Targeting Genomes.” Nature Biotechnology 32 (4): 347–55. doi:10.1038/nbt.2842. Slaymaker, Ian M., Linyi Gao, Bernd Zetsche, David A. Scott, Winston X. Yan, and Feng Zhang. 2016. “Rationally Engineered Cas9 Nucleases with Improved Specificity.” Science (New York, N.Y.) 351 (6268): 84–88. doi:10.1126/science.aad5227. Vora, Suhani, Marcelle Tuttle, Jenny Cheng, and George Church. 2016. “Next Stop for the CRISPR Revolution: RNA-Guided Epigenetic Regulators.” The FEBS Journal 283 (17): 3181–93. doi:10.1111/febs.13768. Wilber, Andrew, Arthur W. Nienhuis, and Derek A. Persons. 2011. “Transcriptional Regulation of Fetal to Adult Hemoglobin Switching: New Therapeutic Opportunities.” Blood 117 (15): 3945–53. doi:10.1182/blood-2010-11-316893. Wiles, Michael V., Wenning Qin, Albert W. Cheng, and Haoyi Wang. 2015. “CRISPR-Cas9-Mediated Genome Editing and Guide RNA Design.” Mammalian Genome: Official Journal of the International Mammalian Genome Society 26 (9–10): 501–10. doi:10.1007/s00335-015-9565-z. Xu, Jian, Vijay G. Sankaran, Min Ni, Tobias F. Menne, Rishi V. Puram, Woojin Kim, and Stuart H. Orkin. 2010. “Transcriptional Silencing of γ-Globin by BCL11A Involves Long-Range Interactions and Cooperation with SOX6.” Genes & Development 24 (8): 783–98. doi:10.1101/gad.1897310.