CRISPR-based precise gene-editing requires simultaneous delivery of multiple components into living cells, rapidly exceeding the cargo capacity of traditional viral vector systems. This challenge represents a major roadblock to genome editing applications. Delivery of complex gene editing approaches for knock-in, base editing or prime editing is nowadays typically achieved by deploying multiple viral vectors, severely limiting the rate of successful gene editing events and imposing and increasing burden on manufacturing processes.
By contrast, baculovirus vectors (BVs) are large dsDNA insect-specific viruses which can efficiently transduce a range of mammalian and human cell lines, while remaining replication and integration deficient in the mammalian hosts. BVs can accommodate >30 kb synthetic DNA cargo and, backed by a novel hybrid DNA assembly system, we demonstrate successful delivery of multifunctional CRISPR toolkits in human cells.
By encoding Cas9, sgRNA and Donor DNAs on a single, rapidly assembled baculoviral vector, we achieve with up to 30% efficacy whole-exon replacement in the intronic β-actin (ACTB) locus, including site-specific docking of very large DNA payloads. We use our approach to rescue wild-type podocin expression in steroid-resistant nephrotic syndrome (SRNS) patient derived podocytes. We demonstrate single baculovirus vectored delivery of single and multiplexed prime-editing toolkits, achieving up to 100% cleavage-free DNA search-and-replace interventions without detectable indels.
Taken together, we provide a versatile delivery platform for single base to multi-gene level genome interventions, addressing the currently unmet need for a powerful delivery system accommodating current and future CRISPR technologies without the burden of limited cargo capacity.