Journal: Nature Communications

Article Title: Elevated levels of FMRP-target MAP1B impair human and mouse neuronal development and mouse social behaviors via autophagy pathway

doi: 10.1038/s41467-023-39337-0

Figure Lengend Snippet: a Population gene expression levels of MAP1B transcripts in the dorsal lateral prefrontal cortical region (BA9) among several psychiatric disorders and healthy controls collected by PsychENCODE Consortium. Y -axis: Gene expression value of MAP1B mRNA (RNA-seq TPM). ASD autism spectrum disorder ( n = 31). BPD bipolar disorder ( N = 172). SCZ schizophrenia ( N = 497). CTR, health ( N = 1166). The gene expression levels of MAP1B in ASD are significantly higher than others by one-side t -test. p < 8.66e-7 for CTR, p < 1.42e-3 for SCZ, and p < 4.83e-8 for BPD. b Experimental strategy for assessing cognitive functions of targeted Map1b gene activation in CAMK2A-expressing excitatory neurons in the PFC of mice. Activation of endogenous Map1b gene in the PFC was achieved through stereotaxic injection of AAV expressing Map1b -targeting guide RNA (AAV8- sgMap1b -hSyn1-flex-mCherry) and AAV expressing Cre driven by Camk2a promoter (AAV9- pCamK2a -GFP-Cre) into the medial PFC of dCas9Activator mice. c Representative confocal images of neurons in the prefrontal cortex (PFC) expressing GFP-Cre (green), sgRNA-mCherry (red), MAP1B (white) in AAV-injected dCas9Activator mice, assessed after behavioral tests. Scale bars: 10 μm. d Quantification of MAP1B intensity in GFP+/mCherry+ neurons in mPFC. Two-tailed, unpaired Student’s t -test, p = 0.0045. N = 4 individual mice. e Experimental scheme of testing social interest (SI) and social novelty (SN). f , g MAP1B-EE mice exhibited reduced social interest (SI, f ), p = 0.0195 and social recognition (SN, g ), p = 0.0090. Two-tailed, unpaired Student’s t -test. sgCtrl : N = 8 mice, sgMap1b : N = 7 mice. Data are presented as box plot in ( a ) (center line, median; box limits, upper and lower quartiles; whiskers, min to max). Data are presented as mean ± s.e.m in other panels. Source data are provided as a Source Data file.

Article Snippet: Five sgRNAs targeting the proximal promoter of mouse Map1b gene ( sgMap1b #1–5), between −305 bp and −24 bp relative to the TSS, were designed in Benchling based on published scoring methods (sequences are included in Supplementary Data ) and cloned to the same lentiviral sgRNA vector (Addgene #61427).

Techniques: Gene Expression, RNA Sequencing, Activation Assay, Expressing, Injection, Two Tailed Test

Journal: Nature Communications

Article Title: Elevated levels of FMRP-target MAP1B impair human and mouse neuronal development and mouse social behaviors via autophagy pathway

doi: 10.1038/s41467-023-39337-0

Figure Lengend Snippet: a Experimental scheme for assessing the autophagy in MAP1B-EE neurons. Hippocampal neurons were isolated from dCas9Activator mice and infected with LV-Cre-GFP and LV-sgRNA-mCherry. b Representative confocal images of neurons infected with either sgMap1b (MAP1B-EE) or sgCtrl (Control) expressing Cre-GFP (green), sgRNA-mCherry (red), LC3 (white). Scale bars, 10 μm. c LC3 intensity. Two-tailed, unpaired Student’s t -test with unequal variances, p = 0.0476. Ctrl: N = 3 independent biological replicates. d Sample Western blot analysis of mouse neurons with MAP1B-EE (LV- sgMap1b infected ) and controls ( sgCtrl ). e Quantitative analysis of LC3-I: p = 0.1597; LC3-II: p = 0.0297; LC3-II/I (LC3-II/LC3-I): p = 0.0434; two-tailed, unpaired Student’s t -test with unequal variances. f Sample Western blot analysis of hippocampal neurons with MAP1B-EE and controls treated with BafA1 or vehicle. g Quantitative analysis of proteins treated with BafA1 before harvest. LC3I: p = 0.1359; LC3-II: p = 0.0049; LC3-II/I: p = 0.0107; two-tailed, unpaired Student’s t -test with unequal variances. For ( e ) and ( g ), protein amounts were normalized to GAPDH and subsequently normalized to control cells. N = 3 biological replicates. h , i Experimental scheme for assessing autophagy flux. Hippocampal neurons from dCas9Activator mice were transfected with an autophagy reporter (CAG-mCherry/GFP/LC3-IRES-Cre) together with either sgCtrl or sgMap1b . j Representative confocal images of autophagy reporter-transfected cells showing autophagosomes (yellow puncta) and autophagolysosomes (red puncta) in neurons. Scale bars, 10 μm. k – n Number of total puncta, p = 0.0038 ( k ), autophagosomes, p = 0.0188 ( l ), and autophagolysosomes, p = 0.0035 ( m ), and calculated ratio of autophagolysosomes over total puncta, p = 0.3070 ( n ). Quantification of puncta was done after 3D reconstruction, two-tailed, unpaired Student’s t -test was used. N = 3 biological replicates. o Representative confocal images of p62 (white) puncta localized in hippocampal neurons. Scale bars, 10 μm. p Quantification of p62 puncta was done after 3D reconstruction. Two-tailed, unpaired Student’s t -test, p = 0.0026. N = 3 biological replicates. All data are presented as mean ± s.e.m. Source data are provided as a Source Data file.

Article Snippet: Five sgRNAs targeting the proximal promoter of mouse Map1b gene ( sgMap1b #1–5), between −305 bp and −24 bp relative to the TSS, were designed in Benchling based on published scoring methods (sequences are included in Supplementary Data ) and cloned to the same lentiviral sgRNA vector (Addgene #61427).

Techniques: Isolation, Infection, Control, Expressing, Two Tailed Test, Western Blot, Transfection

Journal: Nature Communications

Article Title: Elevated levels of FMRP-target MAP1B impair human and mouse neuronal development and mouse social behaviors via autophagy pathway

doi: 10.1038/s41467-023-39337-0

Figure Lengend Snippet: a , b Analysis of LC3 intensity of human neurons with MAP1B-EE. Representative confocal images of human neurons ( a ) stained with dCas9-GFP (green), sgRNA-mCherry (red), and LC3 (white). Scale bars, 10 μm. b Two-tailed, unpaired Student’s t -test, p = 0.0155. n = 3 independent neuronal differentiations, N = 1. c Representative confocal images of neurons in ex vivo human cortical slices infected with LV-shRNA-mCherry (red) and stained with DAPI (blue) and LC3 (white). Scale bars: 20 μm. d Quantification of LC3 intensity in mCherry+ neurons in human cortical slices. Two-tailed, unpaired Student’s t -test, p < 0.0001. N = 3 individual cortices. e Representative confocal images of neurons in the rhesus macaque cortex expressing shRNA-mCherry (red), LC3 (white) in lentivirus-infected ex vivo macaque cortical slices. Scale bars: 20 μm. f Quantification of LC3 intensity in mCherry+ neurons in macaque cortical slices. Two-tailed, unpaired Student’s t -test, p = 0.0238. N = 3 individual cortices. g , h Analysis of LC3 intensity of 5q13.2trip ASD neurons with or without MAP1B knockdown. Representative confocal images ( g ) of neurons stained with DAPI (blue), shRNA-GFP (green) and LC3 (red). Scale bars, 10 μm. h Two-tailed, unpaired Student’s t -test, p = 0.0434. n = 3 independent neuronal differentiation, N = 1. Data are presented as mean ± s.e.m. Source data are provided as a Source Data file.

Article Snippet: Five sgRNAs targeting the proximal promoter of mouse Map1b gene ( sgMap1b #1–5), between −305 bp and −24 bp relative to the TSS, were designed in Benchling based on published scoring methods (sequences are included in Supplementary Data ) and cloned to the same lentiviral sgRNA vector (Addgene #61427).

Techniques: Staining, Two Tailed Test, Ex Vivo, Infection, shRNA, Expressing, Knockdown

Journal: Nature Communications

Article Title: A role for Biofoundries in rapid development and validation of automated SARS-CoV-2 clinical diagnostics

doi: 10.1038/s41467-020-18130-3

Figure Lengend Snippet: a Schematic of the genetic construct of the engineered MS2-SARS-CoV-2 N-gene VLP encompassing the MS2 maturation protein (MP) in green and coat proteins (CP) in orange, linked via His-tag (yellow), under transcriptional control of the T7 promoter and T7 terminator sequences. The SARS-CoV-2 N protein RNA is packaged using a downstream pac site . Schematic created using DNAplotlib . b The VLP constructs were expressed in E. coli and purified using HiTrap® TALON® Crude and HiTrap® Heparin columns. SDS-PAGE analysis of the purification steps includes a protein marker (M) followed by pellet (P) and soluble fraction (S) of the cell lysate, followed by the column flow through (FT) and protein elution fractions, where the CP-His-CP dimer (~28 kDa) is indicated by an arrow. c Purified VLPs were analysed by dynamic light scattering (DLS), which showed a uniform particle population of ~27 nm. Error bars represent the SD of three technical replicates. d Reverse-transcriptase droplet digital PCR (RT-ddPCR) was performed for absolute quantification of the purified VLPs. Serial dilutions of 1, 10, and 100 thousand-fold of the purified VLPs in the presence and absence of a reverse transcription (RT) enzyme were analysed. Droplets were clustered using a threshold determined using a python implementation of an online tool ( http://definetherain.org.uk ). Dotted lines represent the cut offs for the positive and negative clusters. Any data points between the two dotted lines are considered droplet “rain”. Source data are available in the Source Data file.

Article Snippet: The N-gene was cloned into a MS2 VLP expression plasmid backbone (Addgene #128233) using Type IIs assembly.

Techniques: Construct, Control, Purification, SDS Page, Marker, Reverse Transcription, Digital PCR, Quantitative Proteomics

Journal: Nature Communications

Article Title: A role for Biofoundries in rapid development and validation of automated SARS-CoV-2 clinical diagnostics

doi: 10.1038/s41467-020-18130-3

Figure Lengend Snippet: a Schematic of the CRISPR-Cas13a nucleic acid detection workflow from patient samples. Viral RNA is amplified using target-specific primers and detected with target-specific crRNA activating Cas13a to collaterally cleave a fluorescent probe. b CDC N1, N2, and N3 primer sets were employed to amplify the N-gene RNA released from MS2-SARS-CoV-2 VLPs at 2.5, 25, and 250 copies per reaction. The CRISPR-Cas13a detection time course was analysed using a fluorescence microplate reader. Error bars represent the SEM of n = 3 biologically independent amplification reactions and four CRISPR detection technical replicates. c Schematic of the RT-loop-mediated isothermal amplification (RT-LAMP) diagnostic workflow using target-specific LAMP primers. The isothermal amplification of a target results in the acidification of the RT-LAMP master mix and a subsequent pH-associated colour change that is detected using a microplate reader. d Time-course detection of three replicate RT-LAMP reactions using the MS2-SARS-CoV-2 VLPs at 0, 5, 10, 20, 30, and 40 copies per reaction, performed at 65 °C using the BMG CLARIOstar Plus microplate reader. Source data are available in the Source Data file.

Article Snippet: The N-gene was cloned into a MS2 VLP expression plasmid backbone (Addgene #128233) using Type IIs assembly.

Techniques: CRISPR, Amplification, Fluorescence, Diagnostic Assay

Journal: Nature structural & molecular biology

Article Title: Spatiotemporal allele organization by allele-specific CRISPR live-cell imaging: SNP-CLING

doi: 10.1038/s41594-017-0015-3

Figure Lengend Snippet: (a) sgRNAs harboring internal protein-binding RNA-motifs (MS2, or PP7, or Puf1) direct non-catalytic dCas9 to each targeted locus. The corresponding RNA-binding proteins (RBP) MS2, or PP7, or PUM1, are fused to mVenus, or mCherry, or iRFP670, and fluorescently label up to three different loci. For allele-specific labelling, either the 2 nd or 3 rd nucleotide in the dCas9 PAM-motif 5′-NRG-3′ was substituted by a heterozygous SNP to a non-specific dCas9-motif (IUPAC code: ‘Y’ = ‘C’ or ‘T’; ‘H’ = ‘A’, ‘C’ or ‘T’), thereby preventing dCas9-binding to either the 129S1 or CAST alleles in mouse hybrid cells. Sanger sequencing of selected SNPs confirmed heterozygosity. (b) Allele-specific visualization of 129S1- Ypel4 (yellow = MS2-mVenus) and CAST- Ypel4 (red = PP7-mCherry) in male 129S1/CAST mESCs (scale bar = 5 μm, n = 4, 35 nuclei, dashed line = mESC nucleus, arrowheads = SNP-CLING foci of maternal and paternal Ypel4 alleles). (c) Three sgRNAs in MS2 and another set of three sgRNAs in PP7 targeted the CISTR-ACT locus (sgRNA-pool 1 and 2) in RPE-1 cells to elucidate specificity. (d) All measurable foci exhibited two-color co-localization indicating locus-specificity in female RPE-1 cells (edges of foci: < 1 voxel distance = < 50 nm 3 , scale bar = 500 nm, n = 4, 35 nuclei, arrowheads = CLING foci of CISTR-ACT ). (e) To address resolution, two sgRNAs pools targeted XIST (yellow = MS2-mVenus) and TSIX (red = PP7-mCherry), separated by a topological associated domain (TAD) boundary; genomic linear distance ~69 kb. (f) Distinct, non-co-localized signals occurred in 6 out of 10 cells between XIST and TSIX , with spatial displacements of ∼163-638 nm in all three dimensions in RPE-1 cells (scale bar = 100 nm, arrowheads = CLING foci of XIST and TSIX ).

Article Snippet: Flanking BbsI restriction sites were used to ligate sgRNAs with a backbone expressing either three MS2, or three PP7, or six Puf1 motifs (Addgene #68426, #68424), . mCherry or mVenus fluorescent proteins were expressed as fusion proteins with either MS2 or PP7 binding proteins (Addgene #68420), independent of dCas9 (Addgene #68416).

Techniques: Protein Binding, RNA Binding Assay, Binding Assay, Sequencing

Journal: Nature structural & molecular biology

Article Title: Spatiotemporal allele organization by allele-specific CRISPR live-cell imaging: SNP-CLING

doi: 10.1038/s41594-017-0015-3

Figure Lengend Snippet: (a) Inter -allelic distances of Hdac4 [CI = 2.31-3.59] on largest chromosome 1 compared to a locus on small chromosome 18 (44.21 Mb, [CI = 2.14-3.19 μm]) in female 129S1/CAST MEFs were similar (each 80 nuclei, scale bars = 5 μm, arrowheads = SNP-CLING foci of maternal and paternal Hdac4 alleles). (b) Inter -allelic distances between 129S1 and CAST alleles of loci on gene dense chromosomes (chr.7:99.55 Mb [CI = 2.48-3.96 μm], chr. 11 – Sox9 [CI = 1.82-2.75 μm]) or gene-poor chromosome 15 ( Cistr-act [CI = 2.05-3.03 μm]). Chr. 11 distances were different to chr. 7 (two-tailed Mann Whitney rank sum test, * p = 0.02, each 80 nuclei, arrowheads = SNP-CLING foci of maternal and paternal Sox9 alleles). (c) Inter -allelic distances of the SNP-CLING-labeled alleles on chr.7 (arrowheads: yellow = paternal MS2-mVenus, red = maternal PP7-mCherry) relative to inter -genic distances of a CLING-labeled locus (non-allele-specific) on chr.18 (arrowheads: purple = Puf1-PUM1-iRFP670, Pearson's r 2 = 0.71, significance of Pearson's: *** p < 0.0001, 80 nuclei).

Article Snippet: Flanking BbsI restriction sites were used to ligate sgRNAs with a backbone expressing either three MS2, or three PP7, or six Puf1 motifs (Addgene #68426, #68424), . mCherry or mVenus fluorescent proteins were expressed as fusion proteins with either MS2 or PP7 binding proteins (Addgene #68420), independent of dCas9 (Addgene #68416).

Techniques: Two Tailed Test, MANN-WHITNEY, Labeling

Journal: Nature structural & molecular biology

Article Title: Spatiotemporal allele organization by allele-specific CRISPR live-cell imaging: SNP-CLING

doi: 10.1038/s41594-017-0015-3

Figure Lengend Snippet: (a) Examples of merged images demonstrate positioning of the studied alleles (arrowheads: red = PP7-mCherry, yellow = MS2-mVenus), and the nucleoli (rRNA-GFP) in 129S1/CAST MEFs (scale bars = 5 μm, n = 5). (b) Similar allelic distances and distributions of loci to the nucleoli (means ± SD), between 129S1 and CAST alleles in 129S1/CAST MEFs (normalized to nuclei sizes, each combination > 80 nuclei). (c) Examples of loci-nuclear periphery measurements (see panel a, n = 6, arrowheads = SNP-CLING foci). (d) Allelic distances and distributions of loci to the nuclear periphery were similar between 129S1 or CAST alleles (each combination > 80 nuclei). Allelic distances of the studied loci to the nucleoli (means ± SD) were significantly smaller, than their distances to the nuclear periphery (two-tailed Mann Whitney rank sum testing, * p < 0.05, *** p < 0.0001). (e) Spearman correlations of all allelic distances to the nucleoli (n = 620 alleles, r 2 = 0.329, *** p < 0.0001) or to the nuclear periphery (r 2 = 0.512, *** p < 0.0001). 61 % of 129S1 or 64 % CAST inherited loci were closer than 1 μm to the nucleolus. 35 % of 129S1 or 30 % of CAST loci were closer than 1 μm to the periphery. (f) Number of nucleoli counted in human RPE-1 or MEFs was higher in MEFs (two-tailed Mann Whitney rank sum testing, *** p < 0.0001). (g) Similar inter -nucleoli distances in human RPE-1 or MEFs (Kruskal-Wallis multiple comparisons testing [not adjusted], ns = not significant, medians with 25 th to 75 th percentiles, min/max, and sample sizes [n] are depicted, RPE-1 CI lower = 3.02-3.31 μm, CI upper = 3.67-3.99 μm, MEFs CI lower = 3.06-3.35 μm, CI upper = 3.63-4.7 μm).

Article Snippet: Flanking BbsI restriction sites were used to ligate sgRNAs with a backbone expressing either three MS2, or three PP7, or six Puf1 motifs (Addgene #68426, #68424), . mCherry or mVenus fluorescent proteins were expressed as fusion proteins with either MS2 or PP7 binding proteins (Addgene #68420), independent of dCas9 (Addgene #68416).

Techniques: Two Tailed Test, MANN-WHITNEY

Journal: Nature structural & molecular biology

Article Title: Spatiotemporal allele organization by allele-specific CRISPR live-cell imaging: SNP-CLING

doi: 10.1038/s41594-017-0015-3

Figure Lengend Snippet: (a) Example of CLING-labeled FIRRE loci (arrowheads: red = PP7-mCherry, 108 nuclei) and their distances to the closest nucleolus (rRNA-GFP) in female RPE-1 cells (scale bars = 5 μm). Loci-nucleoli distances revealed that FIRRE was closer to the perinucleolar space than GAPDH (107 nuclei, two-tailed Mann-Whitney rank sum test, *** p < 0.0001). (b) Example of maternal and paternal Firre alleles (arrowheads) in female 129S1 (yellow = MS2-mVenus), CAST (red = PP7-mCherry) MEFs. Firre's allelic distances to the nucleoli (129S1 CI = 0.77-1.32 μm, CAST CI = 0.81-1.38 μm) or to the nuclear periphery (80 nuclei, means ± SD, 129S1 CI = 1.38-1.84 μm, CAST CI = 1.48-1.89 μm). Firre was closer to the nucleoli than to the nuclear periphery (two-tailed Mann Whitney rank sum test, *** p < 0.0001). (c) Scheme of interacting loci between two non-homologous chromosomes in spatial proximity, targeted by SNP-CLING or CLING. (d) SNP-CLING of maternal 129S1-derived Firre (yellow = MS2-mVenus) with either Ypel4 -129S1 or Ypel4 -CAST (red = PP7-mCherry) in male mESCs (n = 8, arrowheads = SNP-CLING foci). (e) Allele-biased interaction of Firre with the paternal Ypel4 -CAST locus (** p = 0.0012, Χ 2 -test, each quantification > 130 nuclei, n = 5) in male 129S1/CAST mESCs.

Article Snippet: Flanking BbsI restriction sites were used to ligate sgRNAs with a backbone expressing either three MS2, or three PP7, or six Puf1 motifs (Addgene #68426, #68424), . mCherry or mVenus fluorescent proteins were expressed as fusion proteins with either MS2 or PP7 binding proteins (Addgene #68420), independent of dCas9 (Addgene #68416).

Techniques: Labeling, Two Tailed Test, MANN-WHITNEY, Derivative Assay