Dibenzocyclooctyne-Branched Primer Assembled Gene Nanovector and Its Potential Applications in Genome Editing.

The CRISPR/Cas9 system has been widely used as an efficient genome editing toolkit for gene therapy. The delivery of vectors encoding the full CRISPR/Cas9 components including Cas9 gene and gRNA expression element into cells is the crucial step to effective genome editing. However, the cargo gene sequence for genome editing is usually large, which reduces the cargo encapsulation efficiency and affects the vector size. To obtain a nanovector with high cargo gene loading capacity and biocompatible size, we report the construction of a gene nanovector from branch-PCR with a dibenzocyclooctyne (DBCO)-branched primer and establish the correlation mapping between gene length and nanovector size. The results show that the size of nanovectors can be tuned according to the gene length. According to the findings, we constructed nanovectors carrying the full CRISPR/Cas9 components in 100-200 nm and validated their application in genome editing. The results show that this kind of nanovector exhibits higher serum stability than plasmids and can reach comparable genome editing efficiency with plasmids. Hence, this type of gene nanovector obtained through branch-PCR can carry large gene cargos and maintain a biocompatible nanoscale size, which we envisage will expand its medical applications in gene therapy.