A high-fidelity CRISPR-Cas13 system improves abnormalities associated with C9ORF72-linked ALS/FTD

Abstract

An abnormal expansion of a GGGGCC hexanucleotide repeat in the C9ORF72 gene is the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), two debilitating neurodegenerative disorders driven in part by gain-of-function mechanisms involving transcribed forms of the repeat expansion. By utilizing a Cas13 variant with reduced collateral effects, we developed a high-fidelity RNA-targeting CRISPR-based system for C9ORF72-linked ALS/FTD. When delivered to the brain of a transgenic rodent model, this Cas13-based platform effectively curbed the expression of the GGGGCC repeat-containing RNA without affecting normal C9ORF72 levels, which in turn decreased the formation of RNA foci and reversed transcriptional deficits. This high-fidelity Cas13 variant possessed improved transcriptome-wide specificity compared to its native form and mediated efficient targeting in motor neuron-like cells derived from a patient with ALS. Our results lay the foundation for the implementation of RNA-targeting CRISPR technologies for C9ORF72-linked ALS/FTD.

Low copy CRISPR-Cas13d mitigates collateral RNA cleavage

“…Due to increasing interest in high-fidelity variants of Rfx Cas13d for transcriptome engineering 16,17 , we wanted to evaluate the activity of a recently described high-fidelity variant (hfCas13d) 8 using low-copy number lentiviral delivery. We generated a doxycycline-inducible hfCas13d HAP1 cell line via lentiviral transduction and, using it, targeted 4,890 genes with eight gRNAs each to compare the nuclease activity of hfCas13d to Rfx Cas13d 13 ( Fig.…”