# Durable HTT silencing using non-evolved dCas9 epigenome editors in patient-derived cells

## TL;DR

Researchers used a modified CRISPR system to silence the huntingtin gene in patient-derived cells, offering a potential new treatment for Huntington's disease.

## Contribution

A novel epigenetic editing approach using SpdCas9 achieves durable and selective silencing of HTT in patient-derived neuronal stem cells.

## Key findings

- SpdCas9 fused to repressive epigenetic domains significantly downregulates HTT expression.
- HTT silencing is mitotically stable for up to six weeks in rapidly dividing cells.
- Targeted DNA methylation and repressive histone marks are responsible for the silencing effect.

## Abstract

Huntington’s disease (HD) is an autosomal dominant neurodegenerative disorder caused by a trinucleotide repeat expansion in exon 1 of the huntingtin (HTT) gene. Nuclease-deficient Cas9 protein (dCas9) epigenetic editing for targeted gene regulation is a promising therapeutic approach for HD through downregulation of the causative gene, HTT. A screen of several dCas9 variants with expanded PAM recognition was fused to KRAB and DNMT3A/L to assess the ability to downregulate total HTT. Surprisingly, only SpdCas9 could significantly downregulate HTT, while expanded PAM recognition variants dxCas9 and dCas9-VQR were less efficient or unable to reduce HTT expression. Using our lead construct with SpdCas9, DNA methylation changes were assessed through reduced representation bisulfite sequencing, showing high on-target increases in DNA methylation and few off-targets. In addition, HTT silencing was mitotically stable for up to 6 weeks in a rapidly dividing cell line. Finally, significant downregulation of HTT was achieved in patient-derived neuronal stem cells, showing the efficacy of this system in a disease-relevant cell type. This approach represents a novel therapeutic pathway for the treatment of HD.

Targeting the huntingtin promoter using CRISPRoff results in significant silencing of HTT across multiple cell types including patient-derived neuronal stem cells. This effect is driven mechanistically through targeted promoter methylation and accumulation of repressive histone marks. The silencing of HTT was proven to be highly selective and durable.