Allele-Specific CRISPR Knockout of NR2E3 G56R Mutation
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Solution Overview
Problem
Current treatments for retinitis pigmentosa, particularly those associated with the c.166G>A mutation in the NR2E3 gene, lack effective therapeutic options, and existing genome editing techniques face challenges in targeting post-mitotic photoreceptors for gene correction or knockout.
Innovation Solution
A CRISPR/Cas9 genome-editing strategy specifically designed to knockout the c.166G>A mutation in NR2E3 using guide nucleic acids and the CRISPR-associated nuclease Cas9, targeting induced pluripotent stem cells (iPSCs) to generate cells capable of differentiating into photoreceptor cells that express mature markers, thereby potentially treating autosomal dominant retinitis pigmentosa.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Manufacturing precision
If HDR pathway is used for gene correction, then precise mutation correction is achieved, but it is challenging in post-mitotic cells such as photoreceptors
Solution Approach 1:
Instead of attempting to correct the mutant allele through HDR in post-mitotic photoreceptors, the invention inverts the approach by using CRISPR/Cas9 to knockout the mutant allele through NHEJ, thereby eliminating the harmful mutation rather than correcting it through homology-directed repair
Solution Approach 2:
The invention converts the susceptibility of NHEJ to errors, which normally creates indels that may be harmful, into a beneficial outcome by intentionally inducing indels in the mutant allele to create a premature termination codon, thereby achieving selective knockout of the harmful mutant allele while preserving the wild-type allele
2Productivity
If mutant allele is knocked out using NHEJ, then efficient allele ablation is achieved, but risk of haploinsufficiency exists
Solution Approach 1:
The CRISPR guide RNA is designed to target specifically the mutant allele sequence containing the c.166G>A mutation, creating local specificity that ensures only the mutant allele is knocked out while the wild-type allele remains intact, thereby preventing haploinsufficiency
Solution Approach 2:
The invention creates asymmetry between the two alleles by designing the CRISPR target sequence to match only the mutant allele's unique sequence (containing the G>A transition), allowing differential treatment where the mutant allele is ablated while the wild-type allele is preserved
3Power
If CRISPR/Cas9 is used for genome editing, then powerful gene editing capability is achieved, but challenge in targeting post-mitotic photoreceptors remains
Solution Approach 1:
The invention performs preliminary action by using CRISPR/Cas9-mediated knockout in iPSCs before differentiation, allowing the genome editing to occur in dividing cells where NHEJ is active, and then differentiating the edited iPSCs into photoreceptors that carry the mutant allele knockout, thereby circumventing the limitation of editing post-mitotic cells directly
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
This approach allows for the specific knockout of the mutant allele, enabling the expression of wild-type NR2E3 sufficient for normal retinal development, potentially offering a clinically appealing treatment for NR2E3-related autosomal dominant retinitis pigmentosa by differentiating into functional photoreceptor cells.
Implementation Method 1
The CRISPR/Cas system comprises two elements, a Cas endonuclease and a 20-nt guide RNA (gRNA). The gRNA is situated next to a 3-nt sequence known as a protospacer adjacent motif (PAM). The most commonly used Cas is Cas9 from Streptococcus pyogenes (Sp), which recognizes an NGG PAM sequence. The combination of the PAM and gRNA molecule guides the Cas9 to the target sequence in the host DNA where it induces a double-strand break (DSB).
Implementation Method 2
The second pathway is non-homologous end joining (NHEJ) that is recruited during all phases of the cell cycle in the absence of a repair template. These indels may lead to a frameshift and a premature termination codon (PTC).
Implementation Method 3
The first pathway is homologous-directed repair (HDR), which takes place during the S/G2 phase of dividing cells. HDR is exploited for gene correction by providing a DNA sequence repair template along with the CRISPR/Cas system.
Data Source
AI summary
Retinitis pigmentosa (RP) is an inherited retinal dystrophy that causes progressive vision loss. The second most common mutation causing autosomal dominant (ad) RP is the G56R mutation in NR2E3, a transcription factor essential for photoreceptor development. The G56R variant is exclusively responsible for all cases of NR2E3-associated adRP. Currently, there is no treatment for NR2E3-related, or other, adRP, but genome editing holds promise. In this study, the inventors developed a CRISPR/Cas strategy to specifically knockout the mutant G56R allele of NR2E3 and performed a proof-of-concept study in iPSC of an adRP patient. They demonstrate allele-specific knockout of the mutant G56R allele in the absence of off-target events. Furthermore, they validated this knockout strategy in an exogenous overexpression system. They showed for the first time that G56R iPSC, as well as G56R-CRISPR iPSC, can differentiate into NR2E3-expressing retinal organoids. Overall, they demonstrate that G56R allele-specific knockout by CRISPR/Cas could be a clinically relevant approach to treat NR2E3-associated adRP.Thus, the invention refers to a site-directed genetic engineering system for specifically editing an allele containing c.166G>A mutation in NR2E3 in the genome of an individual and its use for treating autosomal dominant retinitis pigmentosa.