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Efficient Generation of hiPSC Neural Lineage Specific Knockin Reporters Using the CRISPR/Cas9 and Cas9 Double Nickase System
Published on: May 28, 2015
Generation of a BEST1 Pr-EGFP reporter human embryonic stem cell line via CRISPR/Cas9 editing
Shuaiyan Lu1, Ming Chen2, Xiaoyu Liu2
1National Engineering Research Center of Ophthalmology and Optometry, Eye Hospital, Wenzhou Medical University, Wenzhou, China; Oujiang Laboratory (Zhejiang Lab for Regenerative Medicine, Vision and Brain Health), Wenzhou, China; State Key Laboratory of Ophthalmology, Optometry and Visual Science, Eye Hospital, Wenzhou Medical University, Wenzhou, China.
Abstract:
The retinal pigment epithelium (RPE) cell, located between the neural retina and choriocapillaris, is vital for retinal maintenance and photoreceptor function. Human embryonic stem cells (hESCs) provide a limitless source of RPE cells for transplantation. Using CRISPR/Cas9, we inserted a fusion of the BEST1 promoter (an RPE-specific marker) and the EGFP gene into the AAVS1 locus to track differentiation in hESC-induced RPE (hESC-iRPE). The resulting gene-edited line, WAe009-A-2 M, maintained a normal karyotype, expressed pluripotency markers, and demonstrated differentiation potential, making it invaluable for RPE development and therapeutic research.
