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

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.

Fig. 1. Engineering a compact and efficient prime editing system.

a Schematic of the drive-and-process (DAP) array. b Schematic of the BFP fluorescent reporter. On-target 1-locus MPE converts BFP to GFP and can be used as an indicator of prime editing efficiency. EFS, elongation factor 1α short promoter. c Evaluation of PE variants with different C-terminal (left) and N-terminal NLSs using the BFP-to-GFP reporter. Dashed lines show the highest GFP conversion achieved by the best variants: C-terminal SV40 and N-terminal VirD2. X-axes lists the PE variants tested. n = 3 biological replicates d. Integration of engineered pegRNA (epegRNA) into the DAP array. n = 4 (left) and n = 3 (right) biological replicates e Performance of PE variants with truncated MMLV-RT, as indicated by BFP-to-GFP conversion rates. n = 3 biological replicates. f Rational engineering of 451 aa MMLV-RT by introducing previously reported beneficial mutations. Upper dash line indicates the D200C mutant and lower dash line indicates the 451 aa MMLV-RT without additional mutations. n = 3 biological replicates. g Comparison among top-performing 451 aa MMLV-RT variants. n = 8 biological replicates. h Comparison of engineered prime editors and PE2 targeting the endogenous HEK3 locus in HEK293T cell. n = 3 biological replicates. Bars represent the mean ± S.D. for all plots. NC represents a non-transfected control for all relevant plots. Source data are provided as a Source Data file.

Fig. 2. Multiplex and orthogonal gene activation with PEAK.

a Schematic of gene activation using either the Cas9 nickase variant or a prime editor. TSS transcription start site, GOI gene of interest. b Plasmid fluorescent reporters 1–3 with 8 × target protospacer, a miniCMV promoter, and green fluorescent proteins of varying half-lives. CL1 and PEST are degrons that can destabilize their fused proteins and half lives of EGFPs follows the hierarchy: EGFP-CL1-PEST < EGFP-PEST < EGFP. c Representative fluorescence microscope images showcasing the activation of Reporter 1 in HEK293T cells by Cas9 variants and prime editor from n = 3 biological replicates. Scale bar indicates 100 μm. d Flow cytometry analysis of gene activation across Reporters 1-3 in HEK293T, K562, and Hela cells, quantified by mean fluorescent intensity. e Activation of endogenous genes in HEK293T cells using dCas9 + MPH + agRNA, comparing agRNAs generated by either the hCtRNA promoter or the human U6 promoter. f Multiplex endogenous gene activation using dCas9, MPH, and DAP arrays encoding multiple agRNAs. g Comparison between multiplex gene activation by SAM + DAP and by PEAK + MPH + DAP. h Schematic of a full-length agRNA and a truncated spacer agRNA. i, j Activation of endogenous gene IL1B (i) and RHOXF2 (j) in HEK293T cells via PEAK, MPH, and truncated spacer agRNAs. k Endogenous IL1B and RHOXF2 gene activation using SAM system or PEAK with MPH and truncated agRNAs. A 19-nt-spacer and an 11-nt-spacer agRNAs were coupled with PEAK to activate endogenous IL1B and RHOXF2, respectively. Bars represent the mean ± s.d. from n = 3 independent biological replicates. Source data are provided as a Source Data file.

Fig. 3. Multiplex and orthogonal gene repression with DAP shRNAs.

a Illustration of gene repression using PE2-mediated CRISPRi strategy targeting an EGFP reporter gene. b EGFP reporter repression with 33 different sgRNAs that span the EGFP gene. c Schematic of gene silencing achieved via shRNAs produced by the DAP array. d Comparative analysis of gene repression efficiencies using three different methods: PE2-mediated CRISPRi, dCas9-KRAB-MECP2 fusion protein, and DAP shRNA-mediated RNAi. GPP: Broad Institute GPP web portal. e Repression of the endogenous MLH1 gene using shRNAs designed by different web tools and expressed from DAP arrays. GEN GenScript siRNA design tool, INV InvivoGen siRNA Wizard. f Multiplex gene repression with DAP-shRNA array. FWD and REV are two DAP arrays encoding the same set of shRNAs in opposite order. Experiments were performed in HEK293T cells and analyzed using reverse transcription-quantitative polymerase chain reaction (RT-qPCR) or flow cytometry. Bars represent mean ± s.d. from n = 4 (b, d) and n = 3 (e, f) independent biological replicates. Source data are provided as a Source Data file.

Fig. 4. Complex genetic diseases study and combinatorial delivery approaches using DAP array, PEAK and MPH.

a Schematic of a hypothetical complex genetic disease model involving Wilson’s disease, Type I diabetes, and Transthyretin amyloidosis. Treatment of the disease model requires orthogonal editing of the ATP7B gene, activation of the PDX1 gene, and repression of the TTR gene. b Design of a DAP array encoding a shRNA for gene silencing, a truncated agRNA for gene activation, and a ngRNA and a epegRNA for gene editing. c, d Therapeutic genetic perturbation in HepG2 disease cell line transfected by plasmids encoding the DAP array, PEAK, and MPH. REV: the direction of DAP array was reversed as compared to FWD DAP array. e Genetic perturbation in HEK293T cells transfected with plasmids encoding the DAP array, PEAK, and MPH to install the c.3207C>A mutation in the ATP7B gene, upregulate the expression of RHOXF2 gene, and silence the MLH1 gene. f, g Combinatorial delivery of the DAP array (AAV), PEAK (mRNA), and MPH (mRNA) into HEK293T cells. h, i Combinatorial delivery using plasmids for the DAP array and MPH, and lentivirus for PEAK. Controls were untreated cells. A stable cell line expressing PEAK was established before introducing the DAP array and MPH via plasmid transfection. Gene editing outcomes were analyzed by Sanger sequencing and transcriptional regulations were analyzed by RT-qPCR. Error bars represent mean ± s.d. from n = 3 independent biological replicates. Source data are provided as a Source Data file. c, f, h Created in BioRender. Yuan, Q. (2023) BioRender.com/b09r397.