Rationale:
Multiple studies found mutations in SCN2A, which encodes the voltage-gated sodium channel Nav1.2, to be one of the leading monogenic causes of epilepsy. However, there is no cure for SCN2A-related seizures. Since SCN2A-related seizure is a monogenic disorder, CRISPR-based genome editing to correct disease-causing mutations holds enormous promise to alleviate disease phenotypes and even cure the disease. Traditional CRISPR (e.g., CRISPR-Cas9), however, creates a double-strand DNA break in the genome. Thus, it poses some safety risks for in vivo genome editing to correct point mutations. The recent development of CRISPR Prime Editing, which employs a fusion of an engineered Cas9 with a reverse transcriptase, has been shown to achieve high accuracy in editing single nucleotides in the genome. Additionally, it only creates a single-strand DNA nick, which has fewer safety concerns and has been regarded as a more suitable genome editing strategy to correct single-point mutation. Nevertheless, one limitation of existing prime editing technology is its relatively low editing efficiency.Methods: Our lab has developed an all-RNA Prime Editing platform, which results in high (over 90%) editing efficiency in a model human induced pluripotent stem cell (iPSCs) system. We are performing experiments to use this all-RNA prime editing system to correct epilepsy-related SCN2A genetic variants.
Results: Using the all-RNA Prime editing system we optimized, we found that we can create and correct seizure-associated SCN2A-L1342P and Y84X mutation in human iPSCs with promising editing efficiency and minimal off-target effect.
Conclusions: Our results demonstrated that CRISPR Prime Editing is able to correct disease-causing SCN2A genetic mutations, laying a foundation for future clinical translation toward gene correction therapy to treat seizures.
Funding:
We would like to acknowledge the NIH R01 grants NS123154 and NS117585 for funding as well as the FamlieSCN2A Foundation Hodgkin-Huxley Award for supporting our research.