COMMUNICATION 1700400 (1 of 4) © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.small-methods.com A CRISPR-Based Selective Gene Inhibition Method Reveals Dynamic Features of a Cell Nucleus Nanobody Related to the Disease Myotonic Dystrophy Hanhui Ma,* Pablo Reyes-Gutierrez, and Thoru Pederson Dr. H. Ma, Dr. P. Reyes-Gutierrez, Dr. T. Pederson Department of Biochemistry and Molecular Pharmacology University of Massachusetts Medical School Worcester, MA 01605, USA E-mail: hanhui.ma@umassmed.edu DOI: 10.1002/smtd.201700400 mRNA processing at play in these sites. These findings about these small nuclear bodies have new ramifications for how the pathogenesis of this muscle disease can be envisioned. This study also demonstrates a “Small Method”, in which the binding of CRISPR complexes to a specific gene can be used to determine the cell biological consequences of a site-specific roadblock of the RNA transcription process at the target DNA site. The wild-type dmpk gene contains a run of 5–30 copies of CTG repeats down- stream from translational stop cite in exon 15 (Figure 1A), which is expanded to as many as 3000 copies in the mutant gene. We developed a novel clustered regularly interspaced short palindromic repeats (CRISPR)-based labeling strategy to detect the dmpk CTG repeat expansion in myotonic dystrophy patient cells. This method was based on our previous work using CRISPR technology to label-specific genomic sites. [5–7] A non- DNA-cutting form of Cas9 (dCas9) was fused to green fluores- cent protein (GFP) and a guide RNA was designed to target the CTG repeat (Figure 1B; see Experimental Section). When dCas9-GFP was expressed together with the appropriate guide RNA in myotonic dystrophy patient-derived cells, a single, bright fluorescent signal was observed, whereas no signal was observed in normal cells (Figure 2). We know from other results that this labeling method does not have the sensitivity to detect the nonexpanded allele. CRISPR-based inhibition of transcription of specific gene loci has been achieved by tethering a transcriptional initiation repressor protein to dCas9. [8] However, in our case we reasoned that the expanded CTG repeat in the mutant dmpk allele was so long that a great many dCas9-guide RNA complexes would be bound, as is compatible with the bright signal detected in the dCas9-GFP labeling (Figure 2). Unlike tethering a tran- scriptional initiation repressor to dCas9 with the guide RNA designed for a sequence at the RNA polymerase initiation site, our thought was that the large number of dCas9-CTG repeat tar- geting guide RNA complexes all along the expanded allele would constitute an extensive array of roadblocks to RNA polymerase elongation and thus would freeze transcript production, unlike initiation inhibition would likely allow nascent transcripts to be completed and thus perhaps not constitute such a complete and instantaneous shutdown of the transcript pipeline. A major form of the severe pediatric muscle disease myotonic dystrophy is caused by a CTG repeat expansion in the gene (dmpk) for the enzyme DM protein kinase 1. As a consequence, this mutant allele produces aberrant tran- scripts that have elongated tracts of CUG repeats. These RNAs appear to stall within the cell nucleus, congressing in small, discrete bodies situated away from their site of gene transcription. Here, clustered regularly interspaced short palin- dromic repeats (CRISPR)-based approach is deployed that reveals that these nuclear nanobodies are not static structures. When the transcription of the dmpk gene is halted by a targeted CRISPR-based inhibition method, the preex- isting transcripts delocalize from the nuclear bodies, showing that they are con- tinuously trafficking through and are not stalled there in an accretion mode. This report is an example of how a novel method can address a nanoscale dimension of molecular cell biology in the nucleus, one with direct medical relevance. Gene Expression There are 14 human diseases that are caused by expansion of a repeated sequence in a particular gene, thought to arise from stochastic errors in DNA replication. [1] One of these is a form of myotonic dystrophy, in which a CTG repeated sequence in exon 15 of one allele of the dmpk gene has undergone an expansion from the normal number of 5–30 copies to as many as 3000. [2] The expansion in this region of the gene leads to the production of aberrantly long RNAs that coalesce into nuclear foci. [3,4] It is thought that this congression may not only impair the correct processing of these elongated dmpk mRNAs, but that perhaps these foci accrete such a high concentration mRNA processing factors as to globally impair processing of all other mRNAs throughout the nucleus as a possible basis for the pathogenesis of this disease. In this scenario the nuclear foci are envisioned as sites where the elongated dmpk transcripts are stalled, thus sequestering an ever-increasing proportion of all the mRNA processing factors in the nucleus. Despite the plausibility of this hypothesis, here we show that the elongated dmpk tran- scripts are in fact dynamically moving through these nuclear nanobodies, implying that there is no impairment of normal Small Methods 2018, 1700400