A novel toolkit for the efficient delivery of Cas9/sgRNA complexes to chromosomes in cells

Abstract

Introductory paragraphThe application of gene editing technology is currently limited by the lack of safe and efficient methods to deliver RNA-guided endonucleases to target cells. We engineered lentivirus-based nanoparticles to copackage the U6-sgRNA template and the CRISPR-associated protein 9 (Cas9) fused with a virion-targeted protein Vpr (Vpr.Prot.Cas9), for simultaneous delivery to cells. In addition to Vpr, equal spatiotemporal control of thevpr.prot.cas9andgag/polgene expression of was required for efficient packaging of the fusion protein into virus particles. Transduction of the unconcentrated, Vpr.Prot.Cas9-containing vectors resulted in >98% disruption of theEGFPgene in reporter HEK293-EGFP cells with minimal cytotoxicity. Furthermore, we detected indels in the targeted endogenous loci at frequencies of up to 100% in cell lines derived from lymphocytes and monocytes and up to 15% in primary CD4+ T cells by high-throughput sequencing. This approach may provide a platform for the efficient, safe and selective delivery of genome editing enzymes to cells and it may be suitable for simultaneous gene disruption and transgene delivery.