Journal: Scientific Reports

Article Title: Transiently expressed CRISPR/Cas9 induces wild-type dystrophin in vitro in DMD patient myoblasts carrying duplications

doi: 10.1038/s41598-022-07671-w

Figure Lengend Snippet: Dystrophin correction in immortalized DUPmyo cells electroporated with CRISPR/Cas9-expressing plasmids. ( a ) Schematic of the plasmid where CR0 and CR2 were cloned. ( b ) T7E1 assay performed on HEK293T transfected with the LCR2 plasmid (lentiviral vector expressing sgRNA2), CR0 (negative control plasmid) and CR2 (plasmid expressing sgRNA2). Arrows indicated cleaved bands of expected molecular size in cells expressing the nuclease. LCR2 was used as a reference to evaluate the targeting efficiency of CR2. CR0 did not show cleaved bands as LCR2 and CR2, proving it is a valid negative control for monitoring the targeting effect of CRISPR/Cas9. NT = untreated cells. ( c ) FACS analysis of immortalized DUPmyo-i myoblasts electroporated by NEON. ( d ) T7E1 assay performed on the total pool of electroporated DUPmyo-i cells expressing CR0 and CR2. ( e ) Efficiency of genomic targeting evaluated in T7E1 replicates (n = 3). ( f ) Mutated dystrophin transcript in cells expressing CR0 and CR2 (Kruskall-Wallis test, p = 0.0552) (n = 3 technical replicates/sample). ( g ) Western blot showing dystrophin correction (427 kDa band) in cells expressing CR2. Vinculin (116 KDa band) and meta-vinculin (124 kDa band) were probed as a loading control and a measure of myogenic differentiation, respectively. ( h ) Percentages of mutated dystrophin assessed in electroporated DUPmyo-i (Kruskall-Wallis test, p = 0.0679) (n = 3). i) Comparison of mutated dystrophin observed in patient-myoblasts following LCR2-transduction (n = 4) and CR2 electroporation (n = 3) (Mann–Whitney test, p = 0.4). NT = untreated cells. TotCR0/TotCR2 = total pool of cells expressing CR0/CR2. WT = protein derived from the immortalized murine H2K 2B4 cells, expressing wild-type dystrophin (positive control), LCR2 = transduced cells.

Article Snippet: The best sgRNA (sgRNA2) was also cloned into the integrating pL-CRISPR.EFS.GFP plasmid from Benjamin Ebert’s laboratory (Addgene #57818), following the specified protocol.

Techniques: CRISPR, Expressing, Plasmid Preparation, Clone Assay, Transfection, Negative Control, Western Blot, Control, Comparison, Transduction, Electroporation, MANN-WHITNEY, Derivative Assay, Positive Control

Journal: Autophagy

Article Title: The Epstein-Barr virus deubiquitinase BPLF1 regulates stress-induced ribosome UFMylation and reticulophagy

doi: 10.1080/15548627.2024.2440846

Figure Lengend Snippet: Reagents used in this paper.

Article Snippet: px330-UFSP2 sgRNA2 , Addgene; gift from Yihong Ye , 134637.

Techniques: Recombinant, Diagnostic Assay, Transfection, Mutagenesis, Cloning, cDNA Synthesis, SYBR Green Assay, DC Protein Assay, Protein Purification, Knock-Out, Plasmid Preparation

Journal: bioRxiv

Article Title: Importin 13-dependent Axon Diameter Growth Regulates Conduction Speeds along Myelinated CNS Axons

doi: 10.1101/2023.05.19.541431

Figure Lengend Snippet: (A) Brightfield images of control and ue57 mutant zebrafish at 5 dpf, depicting normal growth and gross morphology. (B) The Mauthner axon (somite 15) labelled using Tg(hspGFF62A:Gal4); Tg(UAS:mRFP) in control and ue57 mutant zebrafish illustrates the smaller axon diameter in mutants at 5dpf. Labelling of myelin with Tg(mbp:eGFPCAAX) shows that the Mauthner axon is myelinated (white arrows). Magnification of the mutant Mauthner axon (region within dashed square) is shown in (B’). (C) Mauthner axon diameter measured at somite 15 using the Tg(hspGFF62A:Gal4); Tg(UAS:mRFP) reporter (Two-tailed unpaired t-test, ****p<0.0001). (D) In a 20 min open field test at 5 dpf, ue57 mutant zebrafish spend significantly less time swimming than controls (Two-tailed Mann-Whitney test, ****P<0.0001, n= 144 ctrl, n = 31 ue57 , from 4 different clutches of fish). (E) A region of exon 21 (last exon) of the ipo13b gene where a C>T base pair change (highlighted in red) was identified in ue57 mutants. (F) This base pair change results in the introduction of a premature stop codon in the highly conserved C-terminal region of importin 13b, predicted to result in a truncated protein missing the last 30 amino acids. (G) Overview of a region in exon 3 of the ipo13b gene indicating the site targeted with an sgRNA for cas9-mediated DNA cleavage (PAM sequence highlighted in red), and the resulting mutations in the ue76 and ue77 mutant lines. (H) The mutations disrupt key residues previously shown to bind Ran-GTP (asterisks) , and result in frame shifts followed by premature stop codons. (I) Representative images of a lateral view of the Mauthner axon (somite 15) labeled using the Tg(hspGFF62A:Gal4); Tg(UAS:GFP) reporter in a 4 dpf control, ipo13b ue , ipo13b ue and ipo13b ue zebrafish and 5 dpf ipo13b ue /ue and ipo13b ue /ue zebrafish, with quantification of axon diameter shown in (J-M) (****p<0.0001 Two-tailed unpaired t-test (J-L) or one-way ANOVA with Tukey’s multiple comparisons test (M)). For all bar graphs, each point represents an individual animal. Scale bars: 300 µm (A), 20 µm (B,B’), 10 µm (I) .

Article Snippet: The annealed oligos were then ligated into U6 promotor-based cassettes: ipo13b sgRNA1 into pU6a:sgRNA#1 (Addgene #64245), ipo13b sgRNA2 into pU6a:sgRNA#2 (Addgene #64246), and ipo13b sgRNA3 into pU6b:sgRNA#3 (Addgene #64247).

Techniques: Control, Mutagenesis, Two Tailed Test, MANN-WHITNEY, Sequencing, Labeling

Journal: bioRxiv

Article Title: Importin 13-dependent Axon Diameter Growth Regulates Conduction Speeds along Myelinated CNS Axons

doi: 10.1101/2023.05.19.541431

Figure Lengend Snippet: (A) Representative live-imaging time course of the Mauthner axon (somite 15) from 2 dpf – 7 dpf in a control and ipo13b ue zebrafish labeled using Tg(hspGFF62A:Gal4); Tg(UAS:GFP). (B) Quantification of Mauthner axon diameter growth followed for the same axons at somite 15 from 2 – 7 dpf (n=7 wildtype, 17 heterozygous, 6 mutants, 2-way ANOVA with Tukey’s multiple comparisons test, **p<0.01, ***p<0.001, ****p<0.0001, wt and het are not significantly different from one another). (C) Representative live-images of the entire Mauthner neuron in control and ipo13b ue57 zebrafish at 3 dpf, which were used to measure axon length in D. Area boxed in magenta is enlarged in (C’). (D) Quantification of the entire length of the Mauthner axon at 3 dpf (Two-tailed unpaired t-test with Welch’s correction, p=0.5992). (E) Representative live-imaging of the Mauthner neuron at 4 dpf in control and ipo13b ue zebrafish labelled using the transgenic line Tg(hspGFF62A:Gal4); Tg(UAS:mem-Scarlet). Bracket indicates the position of the cell body and arrow points to the axon. (F) Quantification of the Mauthner cell body area at 4 dpf (Two-tailed unpaired t-test, p=0.0638). (G) Schematic overview of the electrophysiological set-up for measuring conduction velocity along the Mauthner axon. (H) Example traces of Mauthner whole-cell current clamp recording showing action potentials generated in response to Mauthner axon extracellular stimulation. There is a longer latency between the stimulus artifact and action potential peak in the mutants indicating a slower conduction velocity. (I) Conduction velocity measurements along Mauthner axon from 2 dpf to 5 dpf in ipo13b ue animals and control siblings (2-way ANOVA with Sidak’s multiple comparisons test, **p=0.0025 (3dpf), 0.0071 (4dpf), ****p<0.0001). MiMi (J) or Mid3i (K) neuron (top panel) and its axon (somite 15) in control (middle panel) and ipo13b ue (bottom panel) animals labelled using the transgenic reporter Tg(hspGFF62A:Gal4); Tg(UAS:mem-Scarlet). (L) Quantification of MiMi axon diameter at 5 dpf (Two-tailed unpaired t-test, ***p<0.0003). (M) Quantification of Mid3i axon diameter at 5 dpf (Two-tailed unpaired t-test, *p<0.016). For all bar graphs, each point represents an individual animal. Scale bars: 10 µm (A, E, J, K), 100 µm (C), 50 µm (C’).

Article Snippet: The annealed oligos were then ligated into U6 promotor-based cassettes: ipo13b sgRNA1 into pU6a:sgRNA#1 (Addgene #64245), ipo13b sgRNA2 into pU6a:sgRNA#2 (Addgene #64246), and ipo13b sgRNA3 into pU6b:sgRNA#3 (Addgene #64247).

Techniques: Imaging, Control, Labeling, Two Tailed Test, Transgenic Assay, Generated

Journal: bioRxiv

Article Title: Importin 13-dependent Axon Diameter Growth Regulates Conduction Speeds along Myelinated CNS Axons

doi: 10.1101/2023.05.19.541431

Figure Lengend Snippet: (A) Representative electron micrographs of cross sections of the ventral spinal cord at 7 dpf in control and (B) ipo13b ue animals. The Mauthner axon is labelled ‘M’. (C) Quantification of Mauthner axon diameter from 7 dpf electron micrographs (Two-tailed unpaired t-test with Welch’s correction, ****p<0.0001). (D) Mean diameter for the 30 largest axons in each hemi ventral spinal cord at 7 dpf, excluding Mauthner (Two-tailed unpaired t-test with Welch’s correction, **p=0.0061). (E) Representative electron micrographs of cross sections of the dorsal spinal cord at 7 dpf in control and (F) ipo13b ue zebrafish. (G) Number of myelinated axons in the dorsal and ventral tracts of each hemi spinal cord at 7 dpf (Two-tailed unpaired t-test, ****p<0.0001). (H) Distribution of axon diameters for the 30 largest axons in each hemi ventral spinal cord at 7 dpf, excluding Mauthner (n=7 control and 8 mutants, the distributions are significantly different, Kolmogorov-Smirnov test, ****p<0.0001). (I) Distribution of axon diameters for the 30 largest axons in each hemi dorsal spinal cord at 7 dpf (n=7 control and 8 mutants, distributions are significantly different, Kolmogorov-Smirnov test, **p=0.0030). Scale bars = 1 µm.

Article Snippet: The annealed oligos were then ligated into U6 promotor-based cassettes: ipo13b sgRNA1 into pU6a:sgRNA#1 (Addgene #64245), ipo13b sgRNA2 into pU6a:sgRNA#2 (Addgene #64246), and ipo13b sgRNA3 into pU6b:sgRNA#3 (Addgene #64247).

Techniques: Control, Two Tailed Test

Journal: bioRxiv

Article Title: Importin 13-dependent Axon Diameter Growth Regulates Conduction Speeds along Myelinated CNS Axons

doi: 10.1101/2023.05.19.541431

Figure Lengend Snippet: (A-B) Representative electron micrographs of a cross section of the Mauthner axon in the ventral spinal cord at 7 dpf in control and ipo13b ue57 zebrafish. A region of each axon is magnified in A’ and B’ to allow visualization of the distribution of neurofilaments. (C) Density of neurofilaments in control and ipo13b ue mutant Mauthner axons at 7 dpf (Two-tailed unpaired t-test, ***p=0.0006). (D) Nearest neighbour distribution of neurofilaments in the Mauthner axon at 7 dpf, showing a shift to closer nearest neighbours in the ipo13b ue mutants. For all graphs, each point represents an individual animal. Scale bars: 500nm (A), 100nm (B).

Article Snippet: The annealed oligos were then ligated into U6 promotor-based cassettes: ipo13b sgRNA1 into pU6a:sgRNA#1 (Addgene #64245), ipo13b sgRNA2 into pU6a:sgRNA#2 (Addgene #64246), and ipo13b sgRNA3 into pU6b:sgRNA#3 (Addgene #64247).

Techniques: Control, Mutagenesis, Two Tailed Test

Journal: bioRxiv

Article Title: Importin 13-dependent Axon Diameter Growth Regulates Conduction Speeds along Myelinated CNS Axons

doi: 10.1101/2023.05.19.541431

Figure Lengend Snippet: (A) Schematic overview of the transgenic CRISPR/Cas9 strategy used to generate neuron specific ipo13b mutants. (B) Representative images of the Mauthner axon (somite 15) in the neuron specific ipo13b mutants (cas9+sgRNA) and control animals (cas9 or sgRNA only) labelled using Tg(hspGFF62A:Gal4); Tg(UAS:mRFP). (C) Quantification of Mauthner axon diameter in the neuron specific ipo13b mutants (Kruskal-Wallis test with Dunn’s multiple comparisons test, ****p<0.0001). Each point represents an individual Mauthner axon. Scale bars = 10 µm.

Article Snippet: The annealed oligos were then ligated into U6 promotor-based cassettes: ipo13b sgRNA1 into pU6a:sgRNA#1 (Addgene #64245), ipo13b sgRNA2 into pU6a:sgRNA#2 (Addgene #64246), and ipo13b sgRNA3 into pU6b:sgRNA#3 (Addgene #64247).

Techniques: Transgenic Assay, CRISPR, Control

Journal: bioRxiv

Article Title: Importin 13-dependent Axon Diameter Growth Regulates Conduction Speeds along Myelinated CNS Axons

doi: 10.1101/2023.05.19.541431

Figure Lengend Snippet: (A) Super-resolution imaging of Mauthner axons labelled using the transgenic line Tg(hspGFF62A:Gal4); Tg(UAS:RFP) alongside whole-cell patch clamp traces showing action potentials generated in response to extracellular stimulation of the same axons (B). (C) Conduction velocity along Mauthner axon plotted against axon diameter for controls axons (3-5dpf) and neuronal specific ipo13b mutant axons (5 dpf). Control points are fitted with a linear regression line (R=0.4959 for control, neuronal specific ipo13 mutants are not significantly different from controls by simple linear regression test). (D) Conduction velocity measurements along Mauthner axon at 5 dpf in neuronal specific ipo13b mutant animals and control siblings (Mann-Whitney Test, ***p=0.0002). (E) Axon diameter measurements for the same axons as in (D) (Mann-Whitney Test, ****p<0.0001). (F) The success rate of action potential firing by the Mauthner neuron in response to 10 stimulations at 300 Hz, 500 Hz and 1000 Hz (no significant differences using mixed-effects analysis with Sidak’s multiple comparisons test, n=5 3dpf controls, 5 4dpf controls, 6 5dpf controls, 10 ipo13b ue mutants, 11 neuronal specific ipo13b mutants). (G) Resting membrane potential in control and neuronal specific ipo13b mutant Mauthner neurons (no significant differences, Two-tailed unpaired t-test). (H) Depiction of three consecutive action potentials, which have slight variations in their latency of arrival. This variation is referred to as jitter. (I) The precision of action potential arrival (jitter) along Mauthner axon (no significant differences, one-way ANOVA with Tukey’s multiple comparisons test). For all bar graphs, and C, each point represents an individual animal. Scale bars = 10 µm.

Article Snippet: The annealed oligos were then ligated into U6 promotor-based cassettes: ipo13b sgRNA1 into pU6a:sgRNA#1 (Addgene #64245), ipo13b sgRNA2 into pU6a:sgRNA#2 (Addgene #64246), and ipo13b sgRNA3 into pU6b:sgRNA#3 (Addgene #64247).

Techniques: Imaging, Transgenic Assay, Patch Clamp, Generated, Mutagenesis, Control, MANN-WHITNEY, Membrane, Two Tailed Test