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Sci Rep. 2017 Feb 20;7:43062. doi: 10.1038/srep43062.

Targeted RP9 ablation and mutagenesis in mouse photoreceptor cells by CRISPR-Cas9.

Lv JN1,2, Zhou GH1,2, Chen X1,2, Chen H1,2, Wu KC1,2, Xiang L1,2, Lei XL1,2, Zhang X1,2, Wu RH1,2, Jin ZB1,2.

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Lab for Stem Cell &Retinal Regeneration, Institute of Stem Cell Research, The Eye Hospital of Wenzhou Medical University, The State Key Laboratory Cultivation Base and Key Laboratory of Vision Science, Ministry of Health Wenzhou 325027, China.
Division of Ophthalmic Genetics, The Eye Hospital of Wenzhou Medical University, Wenzhou 325027, China.


Precursor messenger RNA (Pre-mRNA) splicing is an essential biological process in eukaryotic cells. Genetic mutations in many spliceosome genes confer human eye diseases. Mutations in the pre-mRNA splicing factor, RP9 (also known as PAP1), predispose autosomal dominant retinitis pigmentosa (adRP) with an early onset and severe vision loss. However, underlying molecular mechanisms of the RP9 mutation causing photoreceptor degeneration remains fully unknown. Here, we utilize the CRISPR/Cas9 system to generate both the Rp9 gene knockout (KO) and point mutation knock in (KI) (Rp9, c.A386T, P.H129L) which is analogous to the reported one in the retinitis pigmentosa patients (RP9, c.A410T, P.H137L) in 661 W retinal photoreceptor cells in vitro. We found that proliferation and migration were significantly decreased in the mutated cells. Gene expression profiling by RNA-Seq demonstrated that RP associated genes, Fscn2 and Bbs2, were down-regulated in the mutated cells. Furthermore, pre-mRNA splicing of the Fscn2 gene was markedly affected. Our findings reveal a functional relationship between the ubiquitously expressing RP9 and the disease-specific gene, thereafter provide a new insight of disease mechanism in RP9-related retinitis pigmentosa.

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