Unmet NeedTargeted, site-specific genome editing is an important scientific tool with broad applications in basic science, the pharmaceutical industry, and translational medicine. It enables the addition of florescent or other types of gene tags for purification, visualization, or tracking. Genome editing can also introduce novel functions for endogenous genes or replace mutated, malfunctioning genes in the case of genetic disorders. The CRISPR/Cas9-based knock-in system is becoming the most-widely used methodology for targeted gene editing. This system requires two separate components, a plasmid containing the single guide RNA (sgRNA) with the Cas9 enzyme and a repair template containing the desired gene knock-in, that are delivered to the cell via electroporation or lipofection. However, this system is complex and presents some important limitations. The sgRNA and Cas9 plasmid can remain in cells for extended periods which presents a risk of unwanted targeting of non-specific genes. These component parts can be difficult to generate and often need to be adapted across various systems. Additionally, electroporation can be harsh and damaging to cells, even resulting in cell death. Consequently, a simpler, more efficient method and delivery system for targeted, site-specific genome editing is greatly needed.
Technology OverviewJohns Hopkins researchers have deigned a single-vector knock-in method for CRISPR/Cas9 based genome editing that requires one single vector that codes for the sgRNA, the Cas9 enzyme, and the repair template. This vector is packaged into a modified baculovirus-based system, which delivers the vector to the cells and genomes of interest. This is a non-integrating viral system, adaptable to most types of mammalian cells, that is capable of carrying all that is needed for CRISPR/Cas9 genome editing. The system also initiates self-digestion by the Cas9 protein it encodes in order to prevent non-specific effects.
Stage of DevelopmentThe inventors have optimized the CRISPR/Cas9 genome editing system to use only one single plasmid vector containing all the necessary parts and information needed for editing that is delivered into cells using a non-integrating viral vector. Further testing of this novel system is needed to determine its effectiveness in editing the mammalian genome, specifically in regards to replacing mutated genes in the case of genetic disorders.
