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Background Most single nucleotide variants (SNVs) occur in noncoding sequence where millions of transcription factor binding sites (TFBS) reside. Here, a comparative analysis of CRISPR-mediated homology-directed repair (HDR) versus the recently reported prime editing 2 (PE2) system was carried out in mice over a TFBS called a CArG box in the Tspan2 promoter. Results Quantitative RT-PCR showed loss of Tspan2 mRNA in aorta and bladder, but not heart or brain, of mice homozygous for an HDR-mediated three base pair substitution in the Tspan2 CArG box. Using the same protospacer, mice homozygous for a PE2-mediated single-base substitution in the Tspan2 CArG box displayed similar cell-specific loss of Tspan2 mRNA; expression of an overlapping long noncoding RNA was also nearly abolished in aorta and bladder. Immuno-RNA fluorescence in situ hybridization validated loss of Tspan2 in vascular smooth muscle cells of HDR and PE2 CArG box mutant mice. Targeted sequencing demonstrated variable frequencies of on-target editing in all PE2 and HDR founders. However, whereas no on-target indels were detected in any of the PE2 founders, all HDR founders showed varying levels of on-target indels. Off-target analysis by targeted sequencing revealed mutations in many HDR founders, but none in PE2 founders. Conclusions PE2 directs high-fidelity editing of a single base in a TFBS leading to cell-specific loss in expression of an mRNA/long noncoding RNA gene pair. The PE2 platform expands the genome editing toolbox for modeling and correcting relevant noncoding SNVs in the mouse.

Background Epiretinal prostheses have been greatly successful in helping restore the vision of patients blinded by retinal degenerative diseases. The design of stimulating electrodes plays a crucial role in the performance of epiretinal prostheses. The objective of this study was to investigate, through computational modeling analysis, the effects on the excitation of retinal ganglion cells (RGCs) when different three-dimensional (3-D) electrodes were placed in the epiretinal space. Methods 3-D finite element models of retinal electrical stimulation were created in COMSOL using a platinum microelectrode, a vitreous body, multi-layered retinal tissue, and retinal pigment epithelium (RPE). Disk and non-planar electrodes with different 3-D structures were used in the epiretinal electrical stimulation. In addition, a multi-RGC model including ionic mechanisms was constructed in NEURON to study the excitability of RGCs in response to epiretinal electrical stimulation by different types of electrodes. Threshold current, threshold charge density, and the activated RGC area were the three key factors used to evaluate the stimulating electrode’s performance. Results As the electrode-retina distance increased, both threshold current and threshold charge density showed an approximately linear relationship. Increasing the disk electrode’s diameter resulted in an increase in threshold current and a decrease in threshold charge density. Non-planar electrodes evoked different activation responses in RGCs than the disk electrode. Concave electrodes produced superior stimulation localization and electrode safety while convex electrodes performed relatively poorly. Conclusions Investigation of epiretinal electrical stimulation using different 3-D electrodes would further the optimization of electrode design and help improve the performance of epiretinal prostheses. The combination of finite element analysis in COMSOL and NEURON software provides an efficient way to evaluate the influences of various 3-D electrodes on epiretinal electrical stimulation. Non-planar electrodes had larger threshold currents than disk electrodes. Of the five types of electrodes, concave hemispherical electrodes may be the ideal option, considering their superior stimulation localization and electrode safety.