Genetic disorders take a significant toll on individuals, families, and communities. The cause of many of thesediseases is a single point mutation in the genome. Despite advances in the diagnosis and underlying geneticfoundation for these disorders, curative treatments have remained elusive. The discovery of the CRISPR-Cas9system 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 thehomology-directed repair (HDR) pathway. Unfortunately, this repair pathway is inefficient, leading to low genomecorrection frequencies. Acrigen Biosciences, Inc. is pioneering the use of anti-CRISPR (Acr) proteins to enhancegenome correction by increasing the efficacy of HDR. This Phase I project will use a recently discovered Acr totransiently tether repair DNA to the Cas9 nuclease, increasing the local concentration of repair template to theCas9 DNA cleavage site. This will increase the efficiency of HDR and subsequent correction of the diseasecausing mutation. The Phase I proposal has the following Aims: 1) Establish a human cell reporter system toassess the efficiency of homologous recombination. Acrigen will design CRISPR-Cas9 guide RNAs and donorDNA constructs to convert the fluorescent reporter EGFP to BFP through HDR. The reporter system will bevalidated in human cells. 2) Increase reporter HDR efficiency using Acr technology. Acrigen will validate bindingof Acr to both Cas9 and the HDR DNA donor. Acrigen will then use Acr to transiently tether the donor DNA toCas9 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 converttruncated SMN protein to full-length SMN by editing a single point transition in the SMN2 gene. Finally, Acrigenwill 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 incells and will have validated this technology to enhance conversion of SMN as a potential cure for SMA.
Public Health Relevance Statement: Narrative
Many genetic disorders are caused by single point mutations in the genome. While precise correction of these
mutations is possible using CRISPR-mediated homology-directed repair, current techniques are insufficient for
effective therapies. Acrigen Biosciences, Inc. is commercializing technology to enhance CRISPR gene
correction by increasing the efficiency of homology-directed gene repair.
Project Terms:
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