Recent advances in the development of genome editing technologies based on programmable nucleases have substantially improved our ability to make precise changes in the genomes of eukaryotic cells. Genome editing is already broadening our ability to elucidate the contribution of genetics to disease by facilitating the creation of more accurate cellular and animal models of pathological processes. A particularly tantalizing application of programmable nucleases is the potential to directly correct genetic mutations in affected tissues and cells to treat diseases that are refractory to traditional therapies. Here we discuss current progress toward developing programmable nuclease–based therapies as well as future prospects and challenges.

Therapeutic genome editing: prospects and challenges

A hexanucleotide repeat expansion (HRE), (GGGGCC)n, in C9orf72 is the most common genetic cause of the neurodegenerative diseases amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Here we identify a molecular mechanism by which structural polymorphism of the HRE leads to ALS/FTD pathology and defects. The HRE forms DNA and RNA G-quadruplexes with distinct structures and promotes RNA•DNA hybrids (R-loops). The structural polymorphism causes a repeat-length-dependent accumulation of transcripts aborted in the HRE region. These transcribed repeats bind to ribonucleoproteins in a conformation-dependent manner. Specifically, nucleolin, an essential nucleolar protein, preferentially binds the HRE G-quadruplex, and patient cells show evidence of nucleolar stress. Our results demonstrate that distinct C9orf72 HRE structural polymorphism at both DNA and RNA levels initiates molecular cascades leading to ALS/FTD pathologies, and provide the basis for a mechanistic model for repeat-associated neurodegenerative diseases.

C9orf72 nucleotide repeat structures initiate molecular cascades of disease

RNA Toxicity from the ALS/FTD C9ORF72 Expansion Is Mitigated by Antisense Intervention

The finding that a GGGGCC (G4C2) hexanucleotide repeat expansion in the chromosome 9 ORF 72 (C9ORF72) gene is a common cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) links ALS/FTD to a large group of unstable microsatellite diseases. Previously, we showed that microsatellite expansion mutations can be bidirectionally transcribed and that these mutations express unexpected proteins by a unique mechanism, repeat-associated non-ATG (RAN) translation. In this study, we show that C9ORF72 antisense transcripts are elevated in the brains of C9ORF72 expansion-positive [C9(+)] patients, and antisense GGCCCC (G2C4) repeat-expansion RNAs accumulate in nuclear foci in brain. Additionally, sense and antisense foci accumulate in blood and are potential biomarkers of the disease. Furthermore, we show that RAN translation occurs from both sense and antisense expansion transcripts, resulting in the expression of six RAN proteins (antisense: Pro-Arg, Pro-Ala, Gly-Pro; and sense: Gly-Ala, Gly-Arg, Gly-Pro). These proteins accumulate in cytoplasmic aggregates in affected brain regions, including the frontal and motor cortex, hippocampus, and spinal cord neurons, with some brain regions showing dramatic RAN protein accumulation and clustering. The finding that unique antisense G2C4 RNA foci and three unique antisense RAN proteins accumulate in patient tissues indicates that bidirectional transcription of expanded alleles is a fundamental pathologic feature of C9ORF72 ALS/FTD. Additionally, these findings suggest the need to test therapeutic strategies that target both sense and antisense RNAs and RAN proteins in C9ORF72 ALS/FTD, and to more broadly consider the role of antisense expression and RAN translation across microsatellite expansion diseases.

https://www.pnas.org/content/pnas/110/51/E4968.full.pdf

RAN proteins and RNA foci from antisense transcripts in C9ORF72 ALS and frontotemporal dementia

Expansions of short nucleotide repeats produce several neurological and neuromuscular disorders including Huntington disease, muscular dystrophy, and amyotrophic lateral sclerosis. A common pathological feature of these diseases is the accumulation of the repeat-containing transcripts into aberrant foci in the nucleus. RNA foci, as well as the disease symptoms, only manifest above a critical number of nucleotide repeats, but the molecular mechanism governing foci formation above this characteristic threshold remains unresolved. Here we show that repeat expansions create templates for multivalent base-pairing, which causes purified RNA to undergo a sol-gel transition in vitro at a similar critical repeat number as observed in the diseases. In human cells, RNA foci form by phase separation of the repeat-containing RNA and can be dissolved by agents that disrupt RNA gelation in vitro. Analogous to protein aggregation disorders, our results suggest that the sequence-specific gelation of RNAs could be a contributing factor to neurological disease.

/pdf/rna-phase-transitions-in-repeat-expansion-disorders-1bmjn74rin.pdf

RNA phase transitions in repeat expansion disorders

http://www.jbc.org/content/288/14/9860.full.pdf

The Disease-associated r(GGGGCC)n Repeat from the C9orf72 Gene Forms Tract Length-dependent Uni- and Multimolecular RNA G-quadruplex Structures

Background: The expanded C9orf72 (GGGGCC)n repeat causes amyotrophic lateral sclerosis and frontotemporal dementia. Results: The r(GGGGCC)n repeat forms stable, tract length-dependent unimolecular and multimolecular RNA G-quadruplexes. Conclusion: G-quadruplex formation by the r(GGGGCC)n repeats may contribute to normal and pathogenic functions of C9orf72. Significance: Understanding the RNA structures formed by this repeat may have implications for how repeat expansion causes disease. Certain DNA and RNA sequences can form G-quadruplexes, which can affect promoter activity, genetic instability, RNA splicing, translation, and neurite mRNA localization. Amyotrophic lateral sclerosis and frontotemporal dementia were recently shown to be caused by expansion of a (GGGGCC)n·(GGCCCC)n repeat in the C9orf72 gene. Mutant r(GGGGCC)n-containing transcripts aggregate in nuclear foci possibly sequestering repeat-binding proteins, suggesting a toxic RNA pathogenesis. We demonstrate that the r(GGGGCC)n RNA but not the C-rich r(GGCCCC)n RNA forms extremely stable uni- and multimolecular parallel G-quadruplex structures (up to 95 °C). Multimolecular G-quadruplex formation is influenced by repeat number and RNA concentration. MBNL1, a splicing factor that is sequestered in myotonic dystrophy patients by binding to expanded r(CUG)n repeat hairpins, does not bind the C9orf72 repeats, but the splicing factor ASF/SF2 can bind the r(GGGGCC)n repeat. Because multimolecular G-quadruplexes are enhanced by repeat length, RNA-RNA interactions facilitated by G-quadruplex formation at expanded repeats might influence transcript aggregation and foci formation in amyotrophic lateral sclerosis-frontotemporal dementia cells. Tract length-dependent G-quadruplex formation by the C9orf72 RNA should be considered when assessing the role of this repeat in C9orf72 gene activity, protein binding, transcript foci formation, and translation of the C9orf72 product, including the noncanonical repeat-associated non-ATG translation (RAN translation) into pathologic dipeptide repeats, as well as any oligonucleotide repeat-based therapy.

The Disease-associated r(GGGGCC) n Repeat from the C9orf72 Gene Forms Tract Length-dependent Uni- and Multimolecular RNA

The battle for survival between bacteria and bacteriophages (phages) is an arms race where bacteria develop defenses to protect themselves from phages and phages evolve counterstrategies to bypass these defenses CRISPR-Cas adaptive immune systems represent a widespread mechanism by which bacteria protect themselves from phage infection In response to CRISPR-Cas, phages have evolved protein inhibitors known as anti-CRISPRs Here, we describe the discovery and mechanisms of action of anti-CRISPR proteins We discuss the potential impact of anti-CRISPRs on bacterial evolution, speculate on their evolutionary origins, and contemplate the possible next steps in the CRISPR-Cas evolutionary arms race We also touch on the impact of anti-CRISPRs on the development of CRISPR-Cas-based biotechnological tools

Phage-Encoded Anti-CRISPR Defenses

Anti-CRISPR-Associated Proteins Are Crucial Repressors of Anti-CRISPR Transcription

Clustered regularly interspaced short palindromic repeats (CRISPR) together with their accompanying cas (CRISPR-associated) genes are found frequently in bacteria and archaea, serving to defend against invading foreign DNA, such as viral genomes. CRISPR-Cas systems provide a uniquely powerful defense because they can adapt to newly encountered genomes. The adaptive ability of these systems has been exploited, leading to their development as highly effective tools for genome editing. The widespread use of CRISPR-Cas systems has driven a need for methods to control their activity. This review focuses on anti-CRISPRs (Acrs), proteins produced by viruses and other mobile genetic elements that can potently inhibit CRISPR-Cas systems. Discovered in 2013, there are now 54 distinct families of these proteins described, and the functional mechanisms of more than a dozen have been characterized in molecular detail. The investigation of Acrs is leading to a variety of practical applications and is providing exciting new insight into the biology of CRISPR-Cas systems.

Sabrina Y. Stanley

Papers

The Disease-associated r(GGGGCC)n Repeat from the C9orf72 Gene Forms Tract Length-dependent Uni- and Multimolecular RNA G-quadruplex Structures

The Disease-associated r(GGGGCC) n Repeat from the C9orf72 Gene Forms Tract Length-dependent Uni- and Multimolecular RNA

Phage-Encoded Anti-CRISPR Defenses

Anti-CRISPR-Associated Proteins Are Crucial Repressors of Anti-CRISPR Transcription

Anti-CRISPRs: Protein Inhibitors of CRISPR-Cas Systems.