CRISPR base editing of cis-regulatory elements enables the perturbation of neurodegeneration-linked genes

Abstract

CRISPR technology has demonstrated broad utility for controlling target gene expression; however, there remains a need for strategies capable of modulating expression via the precise editing of non-coding regulatory elements. Here, we demonstrate that CRISPR base editors, a class of gene-modifying proteins capable of creating single-base substitutions in DNA, can be used to perturb gene expression via their targeted mutagenesis of cis-acting sequences. Using the promoter region of the human huntingtin (HTT) gene as an initial target, we show that editing of the binding site for the transcription factor NF-κB led to a marked reduction in HTT gene expression in base-edited cell populations. We found that these gene perturbations were persistent and specific, as a transcriptome-wide RNA analysis revealed minimal off-target effects resulting from the action of the base editor protein. We further demonstrate that this base-editing platform could influence gene expression in vivo as its delivery to a mouse model of Huntington's disease led to a potent decrease in HTT mRNA in striatal neurons. Finally, to illustrate the applicability of this concept, we target the amyloid precursor protein, showing that multiplex editing of its promoter region significantly perturbed its expression. These findings demonstrate the potential for base editors to regulate target gene expression.

Keywords: AAV; CRISPR; base editing; cis-regulatory elements; gene regulation.

Graphical abstract

Figure 1

CRISPR interference can identify actionable elements in the human HTT promoter (A) The positions of the binding sites for the transcription factors (TFs) NF-κB, AP2, SP1, AP4, and AML1 in the promoter region of the human HTT gene. The approximate locations of the sgRNA binding sites for the CRISPR interference (CRISPRi) tiling screen are indicated by purple bars. (B) Normalized Renilla luciferase expression in HEK293T cells 3 days after transfection with the pHTT-RLuc reporter and expression vectors encoding dCas9 and each sgRNA. Renilla expression in each transfection was normalized to firefly luciferase, and all relative values were normalized to those from cells transfected with pHTT-RLuc and dCas9 with a non-targeted sgRNA. (C) Sequence of the human HTT promoter and the NF-κB binding site, with the CBE binding sites indicated by purple arrows. Underlined bases denote the target cytosines for each CBE, with the numbering specifying their position in relation to the translation start site. The NF-κB consensus motif is shown in the logo illustration, with black arrowheads indicating the target cytosines for CBE-3. (D) Heatmap showing the mean editing frequency for each base by the candidate CBEs in HEK293T cells, as determined by deep sequencing (n = 3). Numbering indicates the relative position of the base to the translation start codon. (E) Base conversion frequencies within edited reads at positions −142C and −141C in the HTT promoter in HEK293T cells 3 days after transfection with plasmids encoding CBE-3, as determined by deep sequencing (n = 3). Bars indicate means and error bars indicate SEMs. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001; 1-tailed unpaired t test.

Figure 2

Base editing of the NF-κB binding site can lower HTT expression (A) Relative HTT mRNA in HEK293T cells 7 days after transfection, with plasmids encoding a targeted or non-targeted CBE and enriched by FACS using a transient reporter (n = 3). (B) Relative HTT mRNA in bulk unsorted HEK293T cells 7 days after transfection with plasmids encoding the CBE-3 or a deactivated CBE-3 (dCBE-3). (A and B) All of the data were normalized to HTT mRNA in cells transfected with a non-targeted CBE (n = 3). (C) Sanger sequencing of the NF-κB binding site in expanded HEK293T clones originally transfected with plasmids encoding CBE-3. Purple boxes indicate the bases edited by CBE-3, while the orange box indicates an indel mutation. The sequence above the top trace indicates the wild-type HTT sequence, while the numbering indicates the positions of the first and last nucleotides within this window relative to the translation start codon. Purple bases represent the target bases. (D) Heatmap showing (top) relative HTT protein and (bottom) relative HTT mRNA in the expanded HEK293T clones. Values are relative to the measured HTT protein and mRNA from unedited HEK293T clones. (E) Volcano plot of RNA-seq data comparing bulk unsorted HEK293T cells 7 days after transfection with plasmids encoding CBE-3 to cells transfected with a non-targeted CBE (n = 3). The red circle indicates the HTT gene, while the colored circles indicate non-target differentially expressed genes (>1.25-fold change [FC], false discovery rate [FDR]-adjusted p < 0.1]. Bars represent means and error bars indicate SEMs. ∗p < 0.05, ∗∗p < 0.01; 1-tailed unpaired t test.

Figure 3

Targeting the HTT promoter in vivo can lower HTT expression in a mouse model of HD (A) Schematic of the AAV vectors used in this study. Abbreviations are as follows: ITR, inverted terminal repeat; CAG, cytomegalovirus early enhancer/chicken β-actin promoter; NLS, nuclear localization signal; FLAG, FLAG epitope tag; 3x HA, three repeats of the human influenza hemagglutinin (HA) epitope tag. (B) Overview of the intrastriatal injections conducted on R6/2 mice. (C) Representative immunofluorescence staining of the striatum 4 weeks after mice were injected with 3 × 10¹⁰ particles each of dual AAV1 particles encoding the N- or C-terminal split-intein CBE domains with sgRNAs targeting the human HTT promoter (AAV1-CBE-hHTT) or the mouse Rosa26 locus (AAV1-CBE-mRosa26) and 3 × 10¹⁰ particles of AAV1-EGFP-KASH. Scale bar, 30 μm. (D and E) Relative HTT mRNA (n = 4) (D) and (E) heatmap showing the mean editing frequencies at positions −142C and −141C in the human HTT promoter (n = 3) in FACS-enriched EGFP⁺ nuclei from treated (AAV1-CBE-HTT) or untreated (AAV1-CBE-mRosa26) R6/2 mice co-injected with 3 × 10¹⁰ particles of AAV1-EGFP-KASH. (D) Data were normalized to HTT mRNA in EGFP⁺ nuclei from mice injected with AAV1-CBE-mRosa26. (F and G) Mean survival (F) and a (G) Kaplan-Meier analysis of R6/2 mice injected with 3 × 10¹⁰ particles each of the AAV1-CBE-hHTT or AAV1-CBE-mRosa26 vectors (n = 14). (D and F) Bars represent means and error bars indicate SEMs. ∗p < 0.05, ∗∗p < 0.01; (D) 1-tailed unpaired t test; (F) 2-tailed unpaired t test; (G) log rank Mantel-Cox test.

Figure 4

Base editing of the human APP promoter can reduce the expression of the APP gene (A) Top; The positions of the binding sites for the TFs AP1, SP1, CTCF, GSF1, and AP1, and GGGCGC boxes. CBE binding sites are indicated by purple arrows. The HSF1 consensus motif is shown in the logo illustration, with black arrowheads indicating the target cytosine for CBE-1. Bottom: Sequences targeted by the CBEs. (B) Sanger sequencing traces showing the editing frequencies for the candidate CBEs in HEK293T cells, as determined by EditR. (C) Relative APP mRNA in unsorted bulk HEK293T cells 7 days after transfection with plasmids encoding the CBEs. (D) Relative APP mRNA in unsorted bulk HEK293T cells 7 days after transfection with combinations of plasmids encoding the CBEs or dCBEs. (C and D) Data were normalized to the relative APP mRNA in cells transfected with a non-targeted CBE (n = 3). Bars represent means and error bars indicate SEMs. ∗∗p < 0.01; 1-tailed unpaired t test.