Virus-like particle delivery enables orthogonal genome editing in vitro and in vivo

Abstract The advent of CRISPR–Cas systems has revolutionized multiple fields, including basic science, biotechnology, and medicine. Central to this versatility is the use of programmable guide RNAs (gRNAs), which enable flexible and specific gene targeting. Building on this principle, various CRISPR-associated tools have been developed, including Cas9, base editors, prime editors, and gene-regulation platforms. However, because most CRISPR modalities share a common gRNA platform, the simultaneous use of multiple tools is constrained by interactions among different gRNAs, limiting orthogonal genome editing. This study presents a method for orthogonal multiplexed gene editing by packaging distinct CRISPR effectors and their corresponding gRNAs into separate virus-like particles (VLPs), thereby virtually eliminating the gRNA crosstalk. Furthermore, we demonstrate that this VLP-based strategy enables orthogonal, multiplexed gene editing in vivo in mouse eyes and ears. This platform expands the CRISPR toolkit by enabling simultaneous, non-interfering genetic manipulations in both cultured cells and living organisms.