Page 68 - Read Online

Ghaseminejad et al. J Transl Genet Genom 2022;6:111-25 https://dx.doi.org/10.20517/jtgg.2021.49 Page 113

with adRP RHO alleles.

Therefore, a gene-based treatment strategy for autosomal dominant disease involving genes such as RHO
that do not have an associated haploinsufficiency disorder is to develop allele-specific therapeutics that
[18]
knock out the mutant allele, leaving the endogenous WT allele unaffected . Another potential solution is
to replace the mutant gene with an exogenous WT copy. In this paper, we introduce and characterize an
X. laevis model of adRP caused by a mutation in the Rho.L gene developed by our research group, and
subsequently use it to model treatments of these types. We explore three different CRISPR-based gene-
editing strategies to prevent RD, in which CRISPR/Cas9 can efficiently induce double-stranded breaks
resulting in pan-retinal genomic alterations. First, we use a single sgRNA to target and introduce indels into
the mutant Rho.L allele. Second, we use a pair of sgRNAs targeting the mutant Rho.L allele and an upstream
sequence to introduce large inactivating deletions in the mutant allele. Third, we use a single sgRNA
targeting the mutant Rho.L allele and a single-stranded DNA oligonucleotide repair template to restore the
mutant allele. Each approach is designed to have minimal effects on the remaining WT allele, ideally
converting the phenotype to that of a heterozygous null (1 and 2) or WT (3). Our system may provide
insight into the potential of these therapeutic approaches by illustrating an idealized scenario of very high
editing efficiency combined with very early treatment.

METHODS
In-vitro transcription of sgRNA
[19]
Procedures were essential, as previously described . Briefly, oligonucleotides corresponding to sgRNA
variable regions were cloned into the PDR274 vector (Addgene 42250, gift of Keith Joung). SgRNAs were in
vitro transcribed from plasmids containing inserts of interest using the HiScribe in vitro transcription kit
(New England Biolabs), purified using the miRNeasy kit (Qiagen), quantified using a Nanodrop
spectrophotometer, assessed for integrity by agarose gel electrophoresis, and stored at -80 °C prior to use.

Gene editing
[12]
Microinjections were performed according to the methods described by Feehan et al. , except that Cas9
protein (New England Biolabs) was used in place of Cas9 mRNA. Briefly, a WT/ Rho.LΔ11Δ1 female
X. laevis was injected with human chorionic gonadotropin (HCG) to induce ovulation. Oocytes were
fertilized in vitro using sperm from an HCG-primed WT male. Utilizing a Hamilton syringe pump with
continuous flow (36 μL/h), plates of ~125 embryos were injected with mixtures of one or two sgRNAs (0.5 μ
L of each guide at 875 ng/μL), Cas9 protein (0.5 μL at 20 μM), 0.25 μL eGFP (enhanced green fluorescent
protein) mRNA and nuclease-free H O to make a final volume of 2.5 μL. Each embryo was injected for 1 s,
2
equating to 20 nL of editing reagent mixture. At 36 h post-fertilization, successfully injected embryos were
identified by eGFP fluorescence using an epifluorescence-equipped Leica MZ16F microscope.

Animal husbandry
Embryos were housed in glass dishes in an 18 °C incubator on a 12 h dark: 12 h light cycle. Embryos and
tadpoles were reared in10 mM NaCl, 0.2 mM KCl, 0.1 mM MgCl , 0.2 mM CaCl . Post-metamorphic frogs
2
2
were raised in 10 mM NaCl.
Dot blot assay, immunohistochemical labeling and confocal microscopy
At 14 days post fertilization (dpf) (developmental stage 48) normally developed tadpoles were sacrificed by
pithing. The left eye was solubilized in 100 μL of a 1:1 mixture of phosphate-buffered saline and SDS-PAGE
loading buffer containing 1 mM ethylenediaminetetraacetic acid and 100 μg/mL phenylmethylsulfonyl
fluoride, while the contralateral eye was fixed in 4% paraformaldehyde buffered with 0.1 M sodium
[20]
phosphate pH 7.4. Dot blot assays were performed on solubilized eyes as described by Tam et al. and