CRISPR base editing of cis-regulatory elements enables target gene perturbations

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 inducing single-base substitutions in DNA, can be harnessed to perturb target gene expression via the 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-?B led to a marked reduction in HTT gene expression. We find that these gene perturbations were persistent and specific, as a transcriptome-wide RNA-seq analysis revealed virtually no off-target effects. We further show that this base-editing platform could lower HTT in vivo, as its delivery to a mouse model of Huntington's disease decreased HTT expression in striatal neurons, an outcome that we show also increased survival. Finally, we use this approach to target the amyloid-beta precursor protein, demonstrating that multiplexed editing of its promoter region could significantly perturb its expression, supporting the applicability of this method. These findings thus demonstrate the potential for base editors to regulate target gene expression. Overall design: Differentially expressed gene analysis of RNA-seq data of RNA extracted from HEK293T cells transfected with a third generation base editor targeting the HTT promoter vs a non-targeting third generation base editor.