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Elimination of Toxic Microsatellite Repeat Expansion RNA by RNA-Targeting Cas9.

Ranjan Batra, David A Nelles, Elaine Pirie, Steven M Blue, Ryan J Marina, Harrison Wang, Isaac A Chaim, James D Thomas, Nigel Zhang, Vu Nguyen, Stefan Aigner, Sebastian Markmiller, Guangbin Xia, Kevin D Corbett, Maurice S Swanson, Gene W Yeo
Cell · 2017 · Vol. 170 (5) · pp. 899-912.e10
PubMed DOI PMC Full Text

Abstract

Microsatellite repeat expansions in DNA produce pathogenic RNA species that cause dominantly inherited diseases such as myotonic dystrophy type 1 and 2 (DM1/2), Huntington's disease, and C9orf72-linked amyotrophic lateral sclerosis (C9-ALS). Means to target these repetitive RNAs are required for diagnostic and therapeutic purposes. Here, we describe the development of a programmable CRISPR system capable of specifically visualizing and eliminating these toxic RNAs. We observe specific targeting and efficient elimination of microsatellite repeat expansion RNAs both when exogenously expressed and in patient cells. Importantly, RNA-targeting Cas9 (RCas9) reverses hallmark features of disease including elimination of RNA foci among all conditions studied (DM1, DM2, C9-ALS, polyglutamine diseases), reduction of polyglutamine protein products, relocalization of repeat-bound proteins to resemble healthy controls, and efficient reversal of DM1-associated splicing abnormalities in patient myotubes. Finally, we report a truncated RCas9 system compatible with adeno-associated viral packaging. This effort highlights the potential of RCas9 for human therapeutics.

Publication Types

["Journal Article"]

Keywords

["ALS", "CRISPR", "Huntington\u2019s disease", "RNA-targeting Cas9", "adeno-associated virus", "gene therapy", "microsatellite repeat expansion", "minimal Cas9", "myotonic dystrophy"]

MeSH Terms

["Animals", "COS Cells", "Cell Line", "Cells, Cultured", "Chlorocebus aethiops", "Clustered Regularly Interspaced Short Palindromic Repeats", "Genetic Therapy", "Microsatellite Repeats", "Oligonucleotides, Antisense", "RNA Splicing", "Trinucleotide Repeat Expansion"]

Funding

R01 HG004659 NHGRI NIH HHS (United States)
R01 NS103172 NINDS NIH HHS (United States)
T32 GM008666 NIGMS NIH HHS (United States)
K12 GM068524 NIGMS NIH HHS (United States)
R01 NS075449 NINDS NIH HHS (United States)

Linked Datasets (1)

GSE100943 GSE via ncbi_elink
GEO

Microsatellite expansion RNA visualization, elimination, and reversal of molecular pathology by RNA-targeting Cas9

Homo sapiens
36 data files
FileTypeSize
Myoblast1_S1_L001_R1_001.fastq.gz RNA-Seq 1.4 GB
Myoblast1_S1_L001_R1_001.fastq.gz RNA-Seq 1.4 GB
Myoblast1_S1_L002_R1_001.fastq.gz RNA-Seq 1.5 GB
Myoblast1_S1_L002_R1_001.fastq.gz RNA-Seq 1.5 GB
Myoblast2_S2_L001_R1_001.fastq.gz RNA-Seq 2.4 GB
Myoblast2_S2_L001_R1_001.fastq.gz RNA-Seq 2.4 GB
Myoblast2_S2_L002_R1_001.fastq.gz RNA-Seq 2.4 GB
Myoblast2_S2_L002_R1_001.fastq.gz RNA-Seq 2.4 GB
Myoblast3_S3_L001_R1_001.fastq.gz RNA-Seq 2.4 GB
Myoblast3_S3_L001_R1_001.fastq.gz RNA-Seq 2.4 GB
Myoblast3_S3_L002_R1_001.fastq.gz RNA-Seq 2.4 GB
Myoblast3_S3_L002_R1_001.fastq.gz RNA-Seq 2.4 GB
Myoblast4_S4_L001_R1_001.fastq.gz RNA-Seq 1.4 GB
Myoblast4_S4_L001_R1_001.fastq.gz RNA-Seq 1.4 GB
Myoblast4_S4_L002_R1_001.fastq.gz RNA-Seq 1.4 GB
Myoblast4_S4_L002_R1_001.fastq.gz RNA-Seq 1.4 GB
Myoblast5_S5_L001_R1_001.fastq.gz RNA-Seq 1.8 GB
Myoblast5_S5_L001_R1_001.fastq.gz RNA-Seq 1.8 GB
Myoblast5_S5_L002_R1_001.fastq.gz RNA-Seq 1.8 GB
Myoblast5_S5_L002_R1_001.fastq.gz RNA-Seq 1.8 GB
Myotube1_S6_L001_R1_001.fastq.gz RNA-Seq 2.3 GB
Myotube1_S6_L001_R1_001.fastq.gz RNA-Seq 2.3 GB
Myotube1_S6_L002_R1_001.fastq.gz RNA-Seq 2.3 GB
Myotube1_S6_L002_R1_001.fastq.gz RNA-Seq 2.3 GB
Myotube2_S7_L001_R1_001.fastq.gz RNA-Seq 1.2 GB
Myotube2_S7_L001_R1_001.fastq.gz RNA-Seq 1.2 GB
Myotube2_S7_L002_R1_001.fastq.gz RNA-Seq 2.5 GB
Myotube2_S7_L002_R1_001.fastq.gz RNA-Seq 2.5 GB
Myotube3_S8_L001_R1_001.fastq.gz RNA-Seq 1.9 GB
Myotube3_S8_L001_R1_001.fastq.gz RNA-Seq 1.9 GB
Myotube3_S8_L002_R1_001.fastq.gz RNA-Seq 2.0 GB
Myotube3_S8_L002_R1_001.fastq.gz RNA-Seq 2.0 GB
Myotube4_S9_L001_R1_001.fastq.gz RNA-Seq 2.2 GB
Myotube4_S9_L001_R1_001.fastq.gz RNA-Seq 2.2 GB
Myotube4_S9_L002_R1_001.fastq.gz RNA-Seq 2.3 GB
Myotube4_S9_L002_R1_001.fastq.gz RNA-Seq 2.3 GB

Analysis Pipelines (1)

GSE100943 RNA-seq Data Processing 7 steps
RNA-seq geo_data_processing GSE100943