Characterization of a retinitis pigmentosa gene slc7a14 in zebrafish by expression analysis and targeted deletion with CRISPR-Cas

Abstract

Purpose: slc7a14 is an orphan protein classified to the SLC7 subfamily of cationic amino acid transporters. Its mutations lead to autosomal recessive retinitis pigmentosa (arRP) which causes severe vision impairment and often blindness. While 2% of the Chinese patients with arRP and sporadic RP carry mutations in the slc7a14 gene. The molecular mechanism by which slc7a14 mutations causes RP is not clear. In this study, we investigated the expression dynamics of slc7a14 and knocked out slc7a14 by CRISPR-Cas system.

Methods: We conducted in situ hybridization and quantitative reverse transcription PCR (qRT-PCR) to determine the expression of slc7a14 in developing zebrafish. In situ probe was synthesized and in situ hybridization experiment was conducted on 7dpf zebrafish larvae. For time-series qRT-PCR, twenty fish embryos were obtained for each sample before 7dpf. A male adult fish was used for tissue-specific qRT-PCR. In order to generate slc7a14 mutants, we used a robust CRISPR-Cas system for zebrafish(Jao et al., (2013) PNAS. 110, 13904-13909.). Guide RNAs were designed to target the 5' end of slc7a14 gene. In vitro transcribed gRNAs and zebrafish codon-optimized Cas9 mRNAs were synthesized and co-injected into 1-cell stage embryos to knockout slc7a14. Mutation in the the slc7a14 was detected by the T7E1 assay and verified by sequencing.

Results: slc7a14 was detected during early embryogenesis and the expression level increased from 1dpf to 7dpf. In adult, High expression of slc7a14 was observed in neural tissues including brain, spinal cord and retina, but was very low in non-neural tissues. Consistent with the qRT-PCR observations in adult, slc7a14 was highly expressed in brain and retina, especially in the INL,ONL and GCL. To further investigate the function of slc7a14, we knocked out this gene using a modified CRISPR/Cas system. We conducted Visual Motor Response (VMR) experiment to analyse the vision defects. Comparing to wild-type, slc7a14 F1 mutated group had an attenuated ON and OFF responses.

Conclusions: Our findings suggest that slc7a14 broadly expresses in neural tissues including in the ONL of retina, which might explain why mutations cause retinitis pigmentosa. Disruption of slc7a14 leads to impaired vision in zebrafish. Together, our results indicate slc7a14 plays a pivotal role in vision.