Saturday, May 11, 2024
5/11/2024
Abstract Genetic disorders take a significant toll on individuals, families, and communities. The cause of many of these diseases is a single point mutation in the genome. Despite advances in the diagnosis and underlying genetic foundation for these disorders, curative treatments have remained elusive. The discovery of the CRISPR-Cas9 system and its ability to edit human genomes has brought renewed hope for curative therapies. However, correcting point mutations in the genome requires precise repair of the DNA break induced by Cas9 through the homology-directed repair (HDR) pathway. Unfortunately, this repair pathway is inefficient, leading to low genome correction frequencies. Acrigen Biosciences, Inc. is pioneering the use of anti-CRISPR (Acr) proteins to enhance genome correction by increasing the efficacy of HDR. This Phase I project will use a recently discovered Acr to transiently tether repair DNA to the Cas9 nuclease, increasing the local concentration of repair template to the Cas9 DNA cleavage site. This will increase the efficiency of HDR and subsequent correction of the disease causing mutation. The Phase I proposal has the following Aims: 1) Establish a human cell reporter system to assess the efficiency of homologous recombination. Acrigen will design CRISPR-Cas9 guide RNAs and donor DNA constructs to convert the fluorescent reporter EGFP to BFP through HDR. The reporter system will be validated in human cells. 2) Increase reporter HDR efficiency using Acr technology. Acrigen will validate binding of Acr to both Cas9 and the HDR DNA donor. Acrigen will then use Acr to transiently tether the donor DNA to Cas9 and assess the efficiency of homologous recombination in the fluorescent reporter system. 3) Validate Acr- enhanced HDR against SMN2. Acrigen will design Cas9 guide RNAs and donor DNA templates to convert truncated SMN protein to full-length SMN by editing a single point transition in the SMN2 gene. Finally, Acrigen will then use Acr to increase HDR efficiency and SMN conversion in spinal muscular atrophy (SMA) patient- derived cells. At the end of Phase I, we will have developed a new approach to increase HDR efficiency in cells and will have validated this technology to enhance conversion of SMN as a potential cure for SMA.
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