Key result
All-in-one virus-like particles utilizing the com/Com aptamer and ABP pair efficiently co-delivered Cas9 mRNA and sgRNA, achieving 30% insertions and deletions at the target locus in vitro.
Why the study?
Generating high-titer normal integrating lentiviral vectors is challenging, and previous virus-like particles were inefficient for co-delivering Cas9 mRNA and sgRNA because modifying Gag impaired particle assembly.
The novel design of all-in-one VLPs and aptamer-containing lentiviral vectors improves the efficiency of Cas9/sgRNA co-delivery and overall gene expression.
Lentiviral vectors (LVs) are widely used for delivering foreign genes for long-term expression. Normal LVs contain the RNA genome, which is reverse transcribed into DNA and integrates into the host genome to mediate long-term gene expression. Recently, virus-like particles (VLPs) were developed for mRNA delivery. In these VLPs, packaging mRNA into the particles is achieved via interactions between the aptamer and aptamer-binding protein (ABP), and mRNA is not reverse transcribed or integrated. These VLPs are useful for delivering Cas9 mRNA for short-term endonuclease expression. Generating high-titer normal integrating LVs is challenging. Until recently, VLPs were not efficient for co-delivering Cas9 mRNA and single guide RNA (sgRNA). By fusing ABP to the N-terminus of HIV Gag protein, hybrid particles were developed to co-deliver Cas9 mRNA and sgRNA. But the method for modifying Gag impaired particle assembly. Previously we found that adding an ABP after the second zinc finger domain of nucleocapsid (NC) protein had minimal effects on particle assembly. Based on this observation, we developed hybrid particles to co-deliver Cas9 mRNA and sgRNA. We further improved LVs for integrated gene expression by including an aptamer sequence in lentiviral transfer plasmids, which improved lentiviral particle production and enhanced LV genomic RNA packaging. In summary, we describe development of new all-in-one VLPs for co-delivery of Cas9 mRNA and sgRNA and new LVs for enhanced vector production and gene expression.
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Yadav et al. (2022) studied Gene editing (in vitro). All-in-one virus-like particles (VLPs) with com/Com aptamer/ABP pair vs. Cas9 RNP VLPs or MS2/MCP VLPs was evaluated on Genome editing activity (insertions and deletions). All-in-one virus-like particles utilizing the com/Com aptamer and ABP pair efficiently co-delivered Cas9 mRNA and sgRNA, achieving 30% insertions and deletions at the target locus in vitro.
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