Large-Scale Engineering of Extracellular Nanoparticles for Genome Editing with CRISPR-Cas9 Ribonucleoproteins

By Peter Gee and Caroline Beckett

Background: Despite advances in human genetics, patients with rare diseases such as Duchenne muscular dystrophy (DMD) still have limited therapeutic options and a short life expectancy. With the advent of CRISPR-Cas9 technology, scientists can now target specific regions of the genome to make precise changes to disease-causing mutations in human cells. In a clinical setting the functional CRISPR components, Cas9 nuclease and single-guide RNA (sgRNA), must be delivered efficiently and directly to the patient’s body. Previous studies in mice have demonstrated that adeno-associated viruses deliver CRISPR-Cas9 effectively, but there is still concern that long-term expression from CRISPR-Cas9 DNA vectors could lead to adverse effects such as non-specific cleavage of important genes involved in cellular function or unintended mutation in oncogenes, which could cause tumors. Moreover, immunological responses to AAV could reduce its efficacy for delivery. For patients to realize the full benefit of recent developments in genomics, clinicians need an alternate delivery system that can transport CRISPR-Cas9 components, transiently, allowing CRISPR-Cas9 to act on a target DNA sequence, make the therapeutic change, and then degrade quickly afterward to limit the chance of adverse events.

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