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

Authors: YUAN, QICHEN - Rice University; ZENG, HONGZHI - Rice University; DANIEL, TYLER - Rice University; LIU, QINGZHOUO - Children'S Nutrition Research Center (CNRC); YANG, YONGJIE - Children'S Nutrition Research Center (CNRC); OSIKPA, EMMANUEL - Rice University; YANG, QIAOCHU - Rice University; PEDDI, ADVAITH - Rice University; ABRAMSON, LILIANA - Rice University; ZHANG, BOYANG - Rice University; XU, YONG - Children'S Nutrition Research Center (CNRC); GAO, XUE - Rice University

Submitted to: Nature Communications
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 11/27/2024
Publication Date: 12/30/2024
Citation: Yuan, Q., Zeng, H., Daniel, T.C., Liu, Q., Yang, Y., Osikpa, E.C., Yang, Q., Peddi, A., Abramson, L.M., Zhang, B., Xu, Y., Gao, X. 2024. Orthogonal and multiplexable genetic perturbations with an engineered prime editor and a diverse RNA array. Nature Communications. https://doi.org/10.1038/s41467-024-55134-9.
DOI: https://doi.org/10.1038/s41467-024-55134-9

Interpretive Summary: Programmable and modular systems that can make specific changes to genes and their expressions are important for biological research and treating genetic diseases in humans. We introduce a new tool called minimal versatile genetic perturbation technology (mvGPT), which allows for flexible and precise gene editing, activation, and suppression in human cells at the same time. The mvGPT combines a small, engineered gene editor (called a prime editor), an activator that helps turn on genes, and a special array that produces various types of RNA needed for different genetic changes. With mvGPT, we can accurately edit the human genome using the prime editor along with specific guide RNAs, activate genes by using a modified RNA that recruits the activator, and silence genes through RNA interference with short-hairpin RNA. We demonstrate the flexibility of mvGPT by correcting a specific mutation in the ATP7B gene, which is associated with Wilson's disease. We also show how it can increase the expression of the PDX1 gene to potentially help treat Type I diabetes and suppress the TTR gene to help manage a condition called transthyretin amyloidosis. Besides using plasmids to deliver the mvGPT, we explored various delivery methods, showing its potential for future use in living organisms.

Technical Abstract: Programmable and modular systems capable of orthogonal genomic and transcriptomic perturbations are crucial for biological research and treating human genetic diseases. Here, we present the minimal versatile genetic perturbation technology (mvGPT), a flexible toolkit designed for simultaneous and orthogonal gene editing, activation, and repression in human cells. The mvGPT combines an engineered compact prime editor (PE), a fusion activator MS2-p65-HSF1 (MPH), and a drive-and-process multiplex array that produces RNAs tailored to different types of genetic perturbation. mvGPT can precisely edit human genome via PE coupled with a prime editing guide RNA and a nicking guide RNA, activate endogenous gene expression using PE with a truncated single guide RNA containing MPH-recruiting MS2 aptamers, and silence endogenous gene expression via RNA interference with a short-hairpin RNA. We showcase the versatility of mvGPT by simultaneously correcting a c.3207C>A mutation in the ATP7B gene linked to Wilson's disease, upregulating the PDX1 gene expression to potentially treat Type I diabetes, and suppressing the TTR gene to manage transthyretin amyloidosis. In addition to plasmid delivery, we successfully utilize various methods to deliver the mvGPT payload, demonstrating its potential for future in vivo applications.