br Materials and methods br Author

Materials and methods
Author disclosure statement
Acknowledgements We are grateful to A. Raducanu and A. Böttcher for comments and discussions and to A. Theis, B. Vogel and K. Diemer for their technical support. This work was funded in part by the German Center for Diabetes Research (DZD e.V.) and has received funding for the HumEn project from the European Union\'s Seventh Framework Programme for Research, Technological Development and Demonstration under grant agreement no. 602587 (http://www.hum-en.eu/).
Resource table Resource details The study was approved by the “De Videnskabsetiske Komiteer for Region Hovedstaden” (protocol number H-4-2011-157), Copenhagen, Denmark and written informed consent was obtained in all cases. To protect the patient family privacy, no personal patient information is presented here. Mutations in presenilin 1 (PSEN1) gene are the most common known causes of inherited Alzheimer\'s disease (AD). A human induced pluripotent stem cell (hiPSC) line was previously generated from a skin biopsy obtained from a 48-year-old woman carrying a heterozygous mutation in exon 4 of the PSEN1 gene, which causes a change in amino PCI32765 A79V. Episomal plasmids carrying gene sequences for hOCT4, hSOX2, hKLF4, hL-MYC, hLIN28 and a short hairpin against TP53 (Okita et al., 2011) were used to reprogram the fibroblasts into iPSCs, successfully establishing integration and feeder-free iPSCs. This cell line has been described previously as a bona fide iPSC line with normal karyotype (Li et al., 2016). The A79V-hiPSC (c.236 C>T) was gene-corrected with the CRISPR/Cas9 system (Ran et al., 2013) by replacing the point mutation “T” with the wild-type nucleotide “C” (Fig. 1A). The nucleotide substitution/correction was validated by sequencing (Fig. 1B). Gene-integrity was also checked by confirming that the DNA sequence surrounding the mutation and the CRISPR cutting site were not altered, with no frameshifts or other mutation(s) being generated upon gene editing, confirming successful gene correction of A79V-hiPSC, named A79V-GC-hiPSC (“GC”=gene corrected). The sequencing of the isogenic line confirmed the c.236 T>C replacement in exon 4 of the PSEN1 gene corresponding to the wild-type (healthy) sequence (Fig. 1B). Immunocytochemical (ICC) analysis of the pluripotency markers OCT4, NANOG, SSEA3, SSEA4, TRA-1-60 and TRA-1-81 (Fig. 1C) confirmed the gene-edited line maintained its pluripotency like the mother line previously published. Finally, karyotyping was performed to confirm that no chromosomal aberrations were introduced in the isogenic line upon gene correction (Fig. 1D).
Materials and methods
Verification and authentication Karyotyping was performed by Institute of Medical Genetics and Applied Genomics, University of Tübingen (Tübingen, Germany) and a minimum of 20 metaphases were analyzed. The results showed a normal 46, XX karyotype, without any detectable abnormalities (Fig. 1D). A79V-GC-hiPSC line identity and purity was confirmed by sequencing of PSEN1 (Fig. 1B) and ICC for pluripotency genes expression (Fig. 1C).
Acknowledgements We would like to thank Ulla Poulsen and Ida Jørring for excellent technical assistance. We thank the following agencies for financial support: the European Union 7th Framework Program (PIAP-GA-2012-324451-STEMMAD), Innovation Fund Denmark “Brainstem” (4108-00008B). Carlota Pires is further supported by the Programme of Excellence 2016 (Copenhagen as the next leader in precise genetic engineering CDO2016: 2016CDO04210) from the University of Copenhagen.
Resource details The human iPS cell line TSD-01-hiPSC was generated from fibroblasts of a patient with infantile Tay-Sachs diseases (TSD), which is an autosomal recessive neurodegenerative disorder caused by deficiency of the HEXA gene. The patient fibroblasts, deposited at the Coriell Institute for Medical Research (GM00502), carry two different mutant HEXA alleles. One allele has a 4-bp insertion at nucleotide 1278 in exon 11 (1278insTATC) and the other has a G>C transversion that abolishes the splice site in intron 12 (IVS12+1G>C).