Fixing Pitx3 gene Mutation with CRISPR-Cas9 Intervention in Zygotic Aphakia-Prone Mice – A Research Protocol

Abstract

Introduction: Despite the drawbacks of gene-editing, CRISPR-Cas9 remains a versatile tool for editing mutated genes. The mutated Pitx3 gene may cause aphakia in mice and Parkinsonism in humans. Pitx3 gene codes for the Pitx3 transcription factor involved in many tasks including the differentiation of dopaminergic neurons in the substantia nigra, tyrosine hydroxylase expression, dopamine transporter and lens development. We aim to edit mutated Pitx3 genes using CRISPR-Cas9 in embryonic-dopaminergic progenitor-neurons of mice and compare to wildtypes (WT) that do not undergo CRISPR-Cas9 intervention. Methods: We will use 5 female mice, 2 homozygous for mice aphakia/Pitx3 mutation (Ak) and 3 standard pseudo-pregnant females. 2-male homozygous (+/+ Ak) mice with Ak will mate with the aphakic females (+/+ Ak). Dopamine levels in both (aphakic/non-aphakic) mice will be determined. We will extract embryonic stem cells (ESC) from aphakic female’s uterus and treat with CRISPR-Cas9 and homology-directed repair (HDR). Both treated and untreated ESC will be divided between 3 pseudo-pregnant females. Post-parturition, intervention success will be assessed using presence of optical lens, dopamine levels and restriction sequence expression in treated/untreated progeny. Results: We expect to observe that untreated ESC zygotes (no CRISPR-Cas9 and HDR treatment) will not express normal Pitx3. This will result in low dopamine levels (inappropriate dopamine levels for their age group) and an aphakic mice progeny. Likewise, treated ESC zygotes will express normal Pitx3, hence have normal dopamine levels and non-aphakic phenotype. Discussion: Progeny of both groups (treated/non-treated) will be examined for aphakia, dopamine levels and restriction sequence. Dopamine levels of treated mice will be compared to dopamine levels of WT mice. Should none of the treated progeny develop lenses or have normal dopamine levels, these mice will be sacrificed to target the restriction sequence in the inserted gene — helping to evaluate why the inserted gene is unexpressed. Conclusion: This research examines CRISPR-Cas9 and HDR use on embryos with mutated genes that impair lens development and dopaminergic-neuron differentiation in humans and mice. It has potential of qualifying for clinical trials as the technique may be used to fix heritable disease-inducing genes that decrease longevity and quality of life in humans.