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CRISPR/Cas9-mediated mutation in Xenopus laevis to study the cell biology of hereditary forms of blindness

University of British Columbia

CRISPR/Cas9-mediated mutation of the Xenopus laevis genome has enabled the modeling of autosomal recessive (AR) retinal disease for the first time in this species. Here, knockout (KO) methodology specific to X. laevis rhodopsin1 has been optimized for the targeted mutation of three X. laevis genes orthologous to human genes responsible for AR forms of blindness: cdhr1, rpe65, and gnat1. At 14 days post-fertilization (dpf), these KO mutations did not cause retinal degeneration (RD). However, PCDH21 KO led to disorganization of rod outer segment (ROS) discs. RPE65 KO caused a significant reduction in ROS length by 44 dpf (p=8.6E-8) despite normal rod opsin localization, yet no cone death despite cone opsin mislocalization. Electroretinography (ERG) revealed a significant reduction in photoreceptor responses to light in RPE65 KOs (p=1.889E-13). GNAT1 KO also caused a significant reduction in ROS length by 33 dpf (p=0.014). Further characterization of KO phenotypes is needed to determine whether they can serve as models of AR retinal dystrophy. KO technology was applied to transgenic X. laevis lines expressing human T4K (hT4K) or bovine P23H (bP23H) rhodopsin to characterize components of the respective cell death mechanisms. In animals expressing hT4K rhodopsin, RPE65 KO prevented RD (p=1.02E-10) whereas GNAT1 KO exacerbated RD (p=2.21E-4), suggesting that cell death induced by hT4K rhodopsin requires light and bound chromophore, yet is mitigated by heterotrimeric transducin. In contrast, RPE65 KO in animals expressing bP23H rhodopsin exacerbated RD (p=1.0E-3), suggesting that cell death induced by bP23H rhodopsin requires light but is mitigated by bound chromophore. RPE65 KO was applied to transgenic X. laevis expressing XOP-eGFP-mRFP-LC3, which allows for the quantification of autophagic structures in rods, to characterize the mechanism by which light regulates changes in autophagy. RPE65 KO prevented up-regulation of light-induced autophagy in rods (p=2.5E-7), suggesting that this increase cannot be attributed to an increased misfolding of rhodopsin due to a lack of chromophore in the light. Here we show that CRISPR/Cas9-mediated KO in established X. laevis transgenic lines can be used to test hypotheses about disease mechanisms. These results offer an opportunity for new investigations into the cell biology of inherited retinal disorders.

Text

2018-04-16

Attribution-NonCommercial-NoDerivatives 4.0 International

http://hdl.handle.net/2429/65400

Neuroscience

University of British Columbia

UBCV

http://creativecommons.org/licenses/by-nc-nd/4.0/