Journal: Frontiers in Genetics

Article Title: A CRISPR-Cas13a Based Strategy That Tracks and Degrades Toxic RNA in Myotonic Dystrophy Type 1

doi: 10.3389/fgene.2020.594576

Figure Lengend Snippet: Tracking the expanded CUG RNA foci and stress granules in COS-M6 cells. (A) Mammalian expression vector pX330 encoding crRNA under a pU6 promoter and d Lsh Cas13a ( Lsh Cas13a R1278A -eGFP) under a pCMV promoter. The crRNA contains the direct repeat hairpin (DR, gray box) and the spacer (black box). The d Lsh Cas13a is flanked by an N-terminal nucleoplasmin NLS and a C-terminal c-Myc NLS . (B) Structure of the crDM1 with DR sequence highlighted in gray, spacer in black and target CUG repeats in blue. The 3′-PFS uracil is highlighted in red. (C) Without the expression of crRNA, d Lsh Cas13a primarily localizes in the nucleus. (D) Comparison of non-targeting (crλNT) and CUG-targeting (crDM1) d Lsh Cas13a tracking nuclear CUG foci in COS-M6 cells. The expression of the interrupted CUG x960 repeats is driven by a CMV promoter in DT960 plasmid. (E) Comparison of non-targeting (crλNT) and ACTB -targeting (crACTB) d Lsh Cas13a tracking cytoplasmic stress granules in COS-M6 cells. The stress condition was induced by adding 200 μM sodium arsenite for 1 h 30 min before fixation. Stress granules are labeled with anti-G3BP1 Alexa 594 antibodies by immunofluorescence (IF).

Article Snippet: Wildtype Lsh Cas13a was cloned from pC001 (Addgene #79150, gift from Feng Zhang) into pAcGFP-C1 (Clonetech) and subsequently underwent site-directed mutagenesis to create the pAcGFP-C1- Lsh C2c2R1278A plasmid.

Techniques: Expressing, Plasmid Preparation, Sequencing, Comparison, Labeling, Immunofluorescence

Journal: Frontiers in Genetics

Article Title: A CRISPR-Cas13a Based Strategy That Tracks and Degrades Toxic RNA in Myotonic Dystrophy Type 1

doi: 10.3389/fgene.2020.594576

Figure Lengend Snippet: Degradation of CUG repeats in biochemical assays and in DM1 patient-derived myoblasts. (A) Domain organization of Lsh Cas13a purified in E. coli (not drawn to scale). NTD stands for N-terminal domain. The (His) x6 and maltose binding protein (MBP) tags were removed after purification by thrombin cleavage (dotted box). (B) Cleavage assay of CUG x30 RNA targets using purified Lsh Cas13a with or without in vitro transcribed crDM1. The RNA cleavage products were separated on a 10% TBE-Urea gel and quantified by Fiji ImageJ. The amount of uncut RNA at each time point was expressed as a percentage of the quantity at 0 h (100%). Error bars were calculated from four independent experiments using the same protein and RNA preparations. (C) Lentiviral construct for Lsh Cas13a-crRNA delivery. The EF-1α-driven Lsh Cas13a was conjugated to a nucleoplasmin NLS, an SV40 NLS, a P2A cleavage signal and an in-frame puromycin-resistant (puromycin R ) gene. (D) Comparison of CUG RNA foci in normal myoblasts, untreated DM1 myoblasts, non-targeted DM1 myoblasts (DM1 myoblast NT), and CUG-targeted DM1 myoblasts (DM1 myoblast targeting) by FISH. (E) Quantification of foci counts of cell lines created in panel (D) . The number of cells used for quantification was gathered from two experiments each with two technical replicates.

Article Snippet: Wildtype Lsh Cas13a was cloned from pC001 (Addgene #79150, gift from Feng Zhang) into pAcGFP-C1 (Clonetech) and subsequently underwent site-directed mutagenesis to create the pAcGFP-C1- Lsh C2c2R1278A plasmid.

Techniques: Derivative Assay, Purification, Binding Assay, Cleavage Assay, In Vitro, Construct, Comparison

Journal: Frontiers in Genetics

Article Title: A CRISPR-Cas13a Based Strategy That Tracks and Degrades Toxic RNA in Myotonic Dystrophy Type 1

doi: 10.3389/fgene.2020.594576

Figure Lengend Snippet: Lsh Cas13a reverses downstream missplicing events and knocks down DMPK mRNA quantity in transduced DM1 myoblasts. (A) Splicing patterns from six MBNL1-regulated genes were analyzed in normal myoblasts, untreated DM1 myoblasts, DM1 myoblasts NT and DM1 myoblasts targeting. The ratios of top and bottom bands (as indicated by arrows) were quantified with standard deviation (±SD) in Fiji ImagJ from two separate experiments. (B) The DMPK mRNA level was measured using qRT-PCR from the four cell lines. The relative DMPK mRNA level from each cell line was normalized to that of the normal myoblast (white bar). Three biological replicates each with three technical replicates were used in this assay.

Article Snippet: Wildtype Lsh Cas13a was cloned from pC001 (Addgene #79150, gift from Feng Zhang) into pAcGFP-C1 (Clonetech) and subsequently underwent site-directed mutagenesis to create the pAcGFP-C1- Lsh C2c2R1278A plasmid.

Techniques: Standard Deviation, Quantitative RT-PCR