br Methods and materials br Acknowledgements We

Methods and materials
Acknowledgements We are grateful to Lin Lin, Yong Liu, Trine Skov Petersen and Bettina Hansen (Department of Biomedicine, Aarhus University) for their kind technical advice and support. This project is supported by grants from Det Frie Forskningsråd (Danish Council for Independent Research) - DFF-1337-00128, DFF-1335-00763 [Y.L.], Lundbeckfonden (Lundbeck Foundation) - R173-2014-1105 [Y.L.], R151-2013-14439 [L.B.], European Union Seventh Framework Programme (PIAP-GA-2012-324451-STEMMAD) and Innovation Fund Denmark “Brainstem” (4108-00008B). K.X. and Y.Z. are financially supported by the China Scholarship Council. Y.Z. has financial support from a PhD scholarship from the HEALTH faculty of Aarhus University. K.X. 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 table. Resource details H1 Abcc8-A2 cell line heterozygous mutation for Abcc8 was generated by targeting exon2 of abcc8 using CRISPR/Cas9 gene edit system (Fig. 1.A). H1 purchase NVP-BGJ398 were transfected with Cas9-G418 and 2 sgRNAs plasmids targeting two sites on exon2 for double nickase activity. G418 resistance cells were plated onto matrigel-coated 96-well plates for single cell selection according to 90 cells per plate. Thirty individual colonies were picked and expanded for confirmation. Genomic DNA was isolated and analyzed by high fidelity PCR amplification followed by sequencing of exon2 of abcc8 at the targeted region. Three heterozygous ES clones were obtained and one of them, Abcc8-A2, was chosen for further characterization. Genome sequencing showed that Abcc8-A2 has heterozygous mutation with1 base pair insertion (Fig. 1.B). Abcc8-A2 cell line has classical stem cell morphology with a high nuclear to cytoplasmic ratio (Fig. 1.C). Abcc8-A2 was pluripotent identified by immunostaining for Oct4 and Nanog (Fig. 1.D) and has normal karyotype (Fig. 1.E). Furthermore this cell retained the potential to differentiate into three germ layers upon embryo body formation in vitro, as showed by RNA expression analysis (Fig. 1.F). To test whether off-target effect occurred in Abcc8-A2, a total of 8 potential off-target sites (POTs) for each sgRNA were predicted by the CRISPR design online tool. All POTs were PCR amplified and then sequenced. Sequencing results showed that no mutation was detected in these POTs (Fig. S1.A, B), indicating that the Cas9/sgRNA system did not induce undesirable off-target effect in the abcc8 deficient ES cells.
Materials and methods
Conflict of interest
Acknowledgements We thank all the other members in the lab of Prof. Yin-xiong Li. This work was financially supported by Haizhu District Science and Technology Plan (2011-ZD-02, 2014-CG-05), Ministry of Science and Technology 973 Program (2015CB964700) and Guangdong Province Science and Technology Plan (2016B030301007).