title: CRISPR/Cas9-AAV Mediated Knock-in at NRL Locus in Human Embryonic Stem Cells.
abstract: Clustered interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9)-mediated genome engineering technologies are sparking a new revolution in biological research. This technology efficiently induces DNA double strand breaks at the targeted genomic sequence and results in indel mutations by the error-prone process of nonhomologous end joining DNA repair or homologous recombination with a DNA repair template. The efficiency of genome editing with CRISPR/Cas9 alone in human embryonic stem cells is still low. Gene targeting with adeno-associated virus (AAV) vectors has been demonstrated in multiple human cell types with maximal targeting frequencies without engineered nucleases. However, whether CRISPR/Cas9-mediated double strand breaks and AAV based donor DNA mediated homologous recombination approaches could be combined to create a novel CRISPR/Cas9-AAV genetic tool for highly specific gene editing is not clear. Here we demonstrate that using CRISPR/Cas9-AAV, we could successfully knock-in a DsRed reporter gene at the basic motifleucine zipper transcription factor (NRL) locus in human embryonic stem cells. For the first time, this study provides the proof of principle that these two technologies can be used together. CRISPR/Cas9-AAV, a new genome editing tool, offers a platform for the manipulation of human genome.
authors.affiliation: School of Ophthalmology and Optometry, Eye Hospital, Wenzhou Medical University, State Key Laboratory Cultivation Base and Key Laboratory of Vision Science, Ministry of Health and Zhejiang Provincial Key Laboratory of Ophthalmology and Optometry, Wenzhou, Zhejiang, China.
authors.forename: Xianglian
authors.initials: X
authors.lastname: Ge
authors.affiliation: Reproductive Medicine Center, The Second Affiliated Hospital, Wenzhou Medical University, Wenzhou, China.
authors.forename: Haitao
authors.initials: H
authors.lastname: Xi
authors.affiliation: School of Ophthalmology and Optometry, Eye Hospital, Wenzhou Medical University, State Key Laboratory Cultivation Base and Key Laboratory of Vision Science, Ministry of Health and Zhejiang Provincial Key Laboratory of Ophthalmology and Optometry, Wenzhou, Zhejiang, China.
authors.forename: Fayu
authors.initials: F
authors.lastname: Yang
authors.affiliation: School of Ophthalmology and Optometry, Eye Hospital, Wenzhou Medical University, State Key Laboratory Cultivation Base and Key Laboratory of Vision Science, Ministry of Health and Zhejiang Provincial Key Laboratory of Ophthalmology and Optometry, Wenzhou, Zhejiang, China.
authors.forename: Xiao
authors.initials: X
authors.lastname: Zhi
authors.affiliation: Reproductive Medicine Center, The First Affiliated Hospital, Wenzhou Medical University, Wenzhou, China.
authors.forename: Yanghua
authors.initials: Y
authors.lastname: Fu
authors.affiliation: School of Ophthalmology and Optometry, Eye Hospital, Wenzhou Medical University, State Key Laboratory Cultivation Base and Key Laboratory of Vision Science, Ministry of Health and Zhejiang Provincial Key Laboratory of Ophthalmology and Optometry, Wenzhou, Zhejiang, China.
authors.forename: Ding
authors.initials: D
authors.lastname: Chen
authors.affiliation: Faculty of Health Sciences, University of Macau, Macau, China.
authors.forename: Ren-He
authors.initials: RH
authors.lastname: Xu
authors.affiliation: Institute of Reproductive and Stem Cell Engineering, Central South University, Changsha, China.
authors.forename: Ge
authors.initials: G
authors.lastname: Lin
authors.affiliation: School of Ophthalmology and Optometry, Eye Hospital, Wenzhou Medical University, State Key Laboratory Cultivation Base and Key Laboratory of Vision Science, Ministry of Health and Zhejiang Provincial Key Laboratory of Ophthalmology and Optometry, Wenzhou, Zhejiang, China.
authors.forename: Jia
authors.initials: J
authors.lastname: Qu
authors.affiliation: Reproductive Medicine Center, The First Affiliated Hospital, Wenzhou Medical University, Wenzhou, China.
authors.forename: Junzhao
authors.initials: J
authors.lastname: Zhao
authors.affiliation: School of Ophthalmology and Optometry, Eye Hospital, Wenzhou Medical University, State Key Laboratory Cultivation Base and Key Laboratory of Vision Science, Ministry of Health and Zhejiang Provincial Key Laboratory of Ophthalmology and Optometry, Wenzhou, Zhejiang, China.
authors.forename: Feng
authors.initials: F
authors.lastname: Gu