Prime editing enables precise installation of genomic substitutions, insertions and deletions in living systems. Efficient in vitro and in vivo delivery of prime editing components, however, remains a challenge.
Here we report prime editor engineered virus-like particles that deliver prime editor proteins, prime editing guide RNAs and nicking single guide RNAs as transient ribonucleoprotein complexes. We systematically engineered v3 and v3b PE-eVLPs with 65- to 170-fold higher editing efficiency in human cells compared to a PE-eVLP construct based on our previously reported base editor eVLP architecture.
In this Article, we report the development of a PE-eVLP system that delivers complete PE systems including pegRNAs and nicking sgRNAs as RNPs.
Standard and test gRNAs extracted from PE-eVLPs were serially diluted and reverse-transcribed to generate cDNA using SuperScript IV Reverse Transcriptase following manufacturer’s protocol. Briefly, a sequence-specific reverse primer that binds the 3′ end of gRNAs was annealed to the template RNA upon incubation at 65 °C for 5 min. RT mix was then added to the annealed RNA, and the reaction was incubated at 65 °C for 20 min, followed by 80 °C for 10 min.
Levy, J. M. et al. Cytosine and adenine base editing of the brain, liver, retina, heart and skeletal muscle of mice via adeno-associated viruses.Golczak, M., Kiser, P. D., Lodowski, D. T., Maeda, A. & Palczewski, K. Importance of membrane structural integrity for RPE65 retinoid isomerization activity.This work was supported by NIH UG3AI150551, U01AI142756, R35GM118062 and RM1HG009490; the Bill and Melinda Gates Foundation; and HHMI.
, Representative western blot comparing expression of the gag-PE fusion protein from v3 PE-eVLPs versus the P4–PE fusion protein from v3b PE-eVLPs in producer cells transfected with the corresponding fusion proteins., Prime editing efficiencies of v3b PE-eVLPs with Gag–P3–Pol or Gag–MCP–P3–Pol. The Gag–MCP–P3–Pol fusion construct is not compatible with the efficient production of PE-eVLPs.locus in N2A cells with the Com aptamer inserted at various locations in the epegRNAs.
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