Orthogonal and multiplexable genetic perturbations with an engineered prime editor and a diverse RNA array

Area of Science:

• Molecular Biology
• Genetics
• Biotechnology

Background:

• Orthogonal genomic and transcriptomic perturbations are vital for biological research and genetic disease therapies.
• Existing technologies often lack the flexibility for simultaneous, multi-faceted genetic manipulation.

Purpose of the Study:

• To introduce the minimal versatile genetic perturbation technology (mvGPT), a novel toolkit for simultaneous and orthogonal gene editing, activation, and repression in human cells.
• To demonstrate the capability of mvGPT to perform diverse genetic modifications concurrently.

Main Methods:

• mvGPT integrates a compact prime editor (PE), MS2-p65-HSF1 (MPH) fusion activator, and a multiplex RNA production system.
• Gene editing is achieved using PE with guide RNAs; gene activation utilizes PE with MPH-recruiting aptamers; gene repression employs RNA interference via short-hairpin RNA.

Main Results:

• mvGPT successfully performed simultaneous genome editing (ATP7B gene), gene activation (PDX1), and gene repression (TTR) in human cells.
• The toolkit demonstrated versatility through various delivery methods, including plasmid delivery.
• Proof-of-concept applications included correcting a Wilson's disease mutation, upregulating a gene for diabetes, and downregulating a gene for amyloidosis.

Conclusions:

• mvGPT offers a flexible and powerful platform for simultaneous orthogonal genetic and transcriptomic perturbations.
• The technology holds significant potential for advancing biological research and developing novel therapeutic strategies for genetic disorders.
• Successful delivery methods suggest future applicability for in vivo applications.