br Materials and methods br Author disclosure statement br A

Materials and methods
Author disclosure statement
Acknowledgments This study was supported by fund from National Medical Research Council (NMRC) of China to SZ (fund number: 30400447) and HW (fund number: 30500548).
Resource table. Resource details To engineer a biallelic mutation in the Keap1 gene, we introduced preassembled Cas9 protein/single-chain guide RNA (sgRNA) ribonucleoproteins (RNP) targeting the cysteine-rich N-terminal intervening region (IVR) domain of Keap1 (Fig. 1A). The sgRNA-induced indel frequency was measured 3days post-transfection using T7 endonuclease I (T7E1), and the identity of the mutations was confirmed by deep sequencing (Fig. 1B, C). After single-cell seeding, individual clones were picked and expanded. We confirmed the targeted disruption of Keap1 by deep sequencing, which showed a high rate of mutation (66%), with mao inhibitors list deletions occurring in 13% of the clones. For further characterization, we selected the H9Keap1KO-A31 line, with insertion of a biallelic single nucleotide (A) (Fig. 1D). The H9Keap1KO-A31 line grew well, showed normal morphology (Fig. 1E), and expressed the pluripotency markers OCT4, SSEA4, TRA-1-60, and TRA-1-80 (Fig. 1F). The cell line had a stable karyotype (46, XX) (Fig. 1G). It also showed differentiation potential which formed teratomas with tissues derived from all germlines, including gut (endoderm), osteoid (mesoderm), and neuronal epithelia and pigmented cells (ectoderm) (Fig. 1H). Additionally, mRNA expression of the three germ layer markers was detected in 14-day cultured embryonic bodies (EB) (Table 1). To confirm the safety of the cell line, we tested for mycoplasma (data not shown), virus, and bacterial infections (Fig. S1).
Materials and methods
Conflict of interest
Acknowledgments This work was supported by Korea National Institute of Health funds (2016-NG61004-00).