Journal: eLife

Article Title: Vision-related convergent gene losses reveal SERPINE3 ’s unknown role in the eye

doi: 10.7554/eLife.77999

Figure Lengend Snippet: ( A ) UCSC genome browser visualization of the SERPINE3 genomic locus in human (hg38 assembly, top box) and zebrafish (danRer11 assembly, bottom box) shows that both species have a 1:1 ortholog with the same number of coding exons in a conserved gene order context. In the human locus, two single nucleotide polymorphisms (SNPs) are in linkage with SERPINE3 and associated with eye phenotypes. In zebrafish, we used CRISPR-Cas9 to generate two independent knockout (KO) lines. The position of guide RNAs is indicated as scissors. In the serpine3 cbg17 line, we deleted the promoter and first exon. In the serpine3 cbg18 line, we introduced a 92 bp frame-shifting deletion in exon 2 (coding exon 1) that results in three early stop codons in the original reading frame. ( B ) Relative expression of serpine3 mRNA in wild type (WT) zebrafish and serpine3 cbg17 individuals quantified by RT-qPCR relative to the expression of rpl13a. Serpine3 mRNA expression is close to zero in serpine3 cbg17 fish and significantly reduced in comparison to wild type fish (p=0.049, two-sided unequal variances t-test). Technical replicates of the qPCR are shown as individual data points; different colors represent different biological replicates. Boxplots display first quartile, median and third quartile with whiskers extending to the maximum and minimum of the three biological replicates. ( C ) In situ hybridization showing that serpine3 is expressed in the inner nuclear layer (INL) of WT zebrafish but not in the homozygous serpine3 cbg17 . Scale bar = 25 µm. ( D ) Serpine3 knockout leads to changes in eye shape in adult, homozygous knockout (KO) fish of serpine3 cbg17 and serpine3 cbg18 lines in comparison to their WT siblings (18 and 11 months, respectively). In WT, the eye shape almost perfectly corresponds to the concave shape of the iris (overlay of white and red dotted lines). In contrast, many KO individuals have alterations in eye shape, evident by notches (arrow heads) in the white line that follows the iris. Scale bar = 1 mm. ( E ) Iris solidity (ratio of eye shape/ concave eye shape) significantly differs between WT and KO siblings for both the serpine3 cbg17 (16 vs 10 eyes) and the serpine3 cbg18 (40 vs 40 eyes) line. A Wilcoxon Rank sum test was used. Boxplots display first quartile, median, and third quartile with whiskers extending to the maximum and minimum within 1.5 times interquartile range. Outliers are shown in black. Iris circularity, another quantification measure for the phenotype, is shown in . ( F ) Hematoxylin/eosin histology staining of the eye of serpine3 cbg17 fish (22 months) reveals histological differences in comparison to their WT siblings (dorsal top, ventral bottom). In comparison to WT, distance between lens and retina of serpine3 cbg17 fish is reduced (distance bars). The WT retina (top) has a distinct lamination with clear separation of the single retinal layers ( a, b ) as shown in the schematic (RPE – retinal pigment epithelium layer, POS – photoreceptor outer segment, ONL – outer nuclear layer, OPL – outer plexiform layer, INL – inner nuclear layer, IPL – inner plexiform layer, GCL – ganglion cell layer, RNFL – retinal nerve fiber layer). Although all retinal layers are present in serpine3 cbg17 fish, the layering appears distorted and the density of cells is reduced ( c–f ). Specifically, the RPE cells display an altered distribution and even local clusters (empty arrows), and displaced pigmented cells emerge in all retinal layers (yellow arrows). This was confirmed also for the cbg18 allele . Scale bar in the overviews = 200 µm, scale bar in the magnifications = 20 µm. Figure 4—source data 1. qPCR analysis of zebrafish serpine3 cbg17 homozygotes (KO) vs WT. Figure 4—source data 2. Macroscopic eye phenotype in serpine3 knockout lines. Figure 4—source data 3. Single-nucleotide polymorphism near SERPINE3 (13: 51, 341, 032–51, 362,101) are associated with human eye phenotypes. Figure 4—source data 4. Raw gel images of genotyping of the serpine3 cbg17 line with two primers (labeled image is shown in Figure 4 - figure supplement 2A). Figure 4—source data 5. Raw gel images of genotyping of the serpine3 cbg17 line with a mix of three primers (labeled image is shown in Figure 4 - figure supplement 2B). Figure 4—source data 6. Raw gel images of genotyping of the serpine3 cbg18 line with two primers (labeled image is shown in Figure 4 - figure supplement 2C).

Article Snippet: commercial assay or kit , Alt-R CRISPR-Cas9 tracr RNA , IDT , Cat. #: 1072532 , .

Techniques: CRISPR, Knock-Out, Expressing, Quantitative RT-PCR, In Situ Hybridization, Staining, Labeling

Journal: eLife

Article Title: Vision-related convergent gene losses reveal SERPINE3 ’s unknown role in the eye

doi: 10.7554/eLife.77999

Figure Lengend Snippet:

Article Snippet: commercial assay or kit , Alt-R CRISPR-Cas9 tracr RNA , IDT , Cat. #: 1072532 , .

Techniques: Recombinant, Plasmid Preparation, Sequencing, CRISPR, Software, Expressing

Journal: eLife

Article Title: DBT is a metabolic switch for maintenance of proteostasis under proteasomal impairment

doi: 10.7554/eLife.91002

Figure Lengend Snippet: ( A ) Workflow of the CRISPR screen in retinal pigment epithelium (RPE1) cells, which were transduced with a lentiviral Genome-Scale CRISPR Knock-out (GeCKO) single guide RNA (sgRNA) library and selected for the sgRNA expression and then survival after treatments with the proteasome inhibitor MG132. Individual surviving cell colonies were collected for sequencing and subsequent analysis. ( B ) Left: The cytotoxicity analysis of wild-type (WT) and DBT knockout (KO) RPE1 cells treated with MG132 at different doses for 96 hr (n=3). Right: The time course analysis of MG132-induced cytotoxicity in the WT and DBT KO cells (n=3). ( C ) Immunoblot analysis of WT RPE1, DBT KO, and DBT’ cells. The DBT’ cells expressed an engineered DBT cDNA that resisted DBT-targeted Cas9 cleavage and rescued the DBT expression in the KO cells. ( D ) Cell viability was measured by Calcein-AM staining in WT RPE1, DBT KO, and DBT’ cells treated with MG132 (2 μM, 96 hr). Scale bar, 100 μm. ( E ) Quantification of the cell viability measured by Calcein-AM staining in ( D ) (n=9). ( F ) Left: Immunoblot analysis of RPE1 cells transfected with DBT shRNAs and non-targeting control shRNAs. Right: Quantification of the cell viability under treatment with MG132 (2 μM, 48 hr), as measured by Calcein-AM staining (n=4). ( G ) Immunoblotting and quantification of cleaved PARP as an MG132-induced cell death marker (n=4). ( H ) Immunoblotting and quantification of cleaved Caspase 3 as an MG132-induced cell death marker (n=3). Error bars represent means ± SEM. *p≤0.05; **p≤0.01; ****p≤0.0001. Figure 1—source data 1. Original and uncropped blots for . Figure 1—source data 2. Original and uncropped blots for . Figure 1—source data 3. Original and uncropped blots for . Figure 1—source data 4. Original and uncropped blots for .

Article Snippet: The specific gRNA sequences were selected by using the CRISPR design tool from Benchling, Inc The gRNAs were cloned into the gRNA/Cas9-expressing vector pLenti-CRISPR v2, conferring resistance to puromycin (Addgene 52961).

Techniques: CRISPR, Transduction, Knock-Out, Expressing, Sequencing, Western Blot, Staining, Transfection, Control, Marker

Journal: bioRxiv

Article Title: The Mechanism of MICU-Dependent Gating of the Mitochondrial Ca 2+ Uniporter

doi: 10.1101/2020.04.04.025833

Figure Lengend Snippet: ( A ) A schematic arrangement of various subunits in the MCU complex. Four MCU and four EMRE subunits form the pore of the MCU complex (only two MCU and two EMRE subunits are shown for simplicity). EMRE also tethers MICU1 subunit to the pore on the cytosolic side of the IMM (i.e., in the mitochondrial intermembrane space, IMS). MICU1 forms homodimers or hetero-dimerizes with MICU2 or MICU3 (not shown). Each MICU subunit has two EF hands that bind cytosolic Ca 2+ . ( B to F ) CRISPR-mediated indels in various MCU subunit genes and the resulting mutant alleles. The CRISPR binding sites (for sgRNA) are highlighted in yellow , and their PAM sequences are highlighted in green . The translational initiation codon (ATG) is shown in bold where applicable. (B) Overview of the MCU gene and indels in the knockout. A sgRNA was used to target exon 3. The sequence of targeted region in MCU gene is shown; exon 3 is underlined. Targeted sequencing indicates frame-shift indels ( red ) in both alleles ( Al- 1 and Al- 2). (C) Overview of the EMRE gene and truncated region in the knockout. Two sgRNAs were used for CRISPR-Cas9–mediated deletion in the exon-2 ( underlined ) and the flanking region. Targeted sequencing indicates same 259-bp deletion ( red ) in both alleles. (D) Overview of the MICU1 gene and truncated region in the knockout. Two sgRNAs were used for CRISPR-Cas9– mediated deletion in the exon-3 ( underlined ) and the flanking region. Targeted sequencing indicates that almost all of exon-3 is deleted along with a portion of the flanking region ( red ) in both alleles ( Al- 1 and A l- 2). (E) Overview of the MICU2 gene and truncated region in the knockout. Two sgRNAs were used for CRISPR-Cas9–mediated deletion in the exon-1 ( underlined ) and the flanking region. Targeted sequencing indicates that almost all of exon-1 is deleted ( red ) in both alleles. (F) Overview of the MICU3 gene and truncated region in the knockout. Two sgRNAs were used for CRISPR-Cas9–mediated deletion in the exon-1 ( underlined ). Targeted sequencing indicates a 73-bp deletion in the expected cut area ( red ) in both alleles.

Article Snippet: MEFs were transfected with the Cas9 gRNA vector (Addgene: PX459) via electroporation (Invitrogen Neon transfection system) using the following parameters: 1×10 6 cells and 1 μg of two different gRNA-Cas9 plasmids.

Techniques: CRISPR, Mutagenesis, Binding Assay, Knock-Out, Sequencing

Journal: FEBS Open Bio

Article Title: Tollip‐deficient zebrafish display no abnormalities in development, organ morphology or gene expression in response to lipopolysaccharide

doi: 10.1002/2211-5463.13423

Figure Lengend Snippet: CRISPR/Cas9‐based genome editing allows for the generation of a Tollip‐deficient zebrafish line. (A) Schemes of the zebrafish tollip transcript variants 1 and 2 (v1 and v2), based on the Ensembl database, showing exons (E), translated sequences (gray), and UTR regions (white). (B) Schematic illustration of the structure of Tollip protein isoforms with the C2 and CUE domains indicated. (C) Partial DNA sequence of the target site within exon 2 of the tollip gene in wild‐type tollip +/+ fish (left) and homozygous tollip −/− knockout fish (right). Deletion of eight nucleotides observed in the mutant line is shadowed in dark gray. There is an additional nucleotide change flanking the deletion (double peak marked R in the chromatogram, corresponding to A or G, with a predicted amino acid change D to G in the truncated protein product), indicating mosaicism of the generated line. (D) Schematic illustration of the predicted structure of Tollip protein isoforms synthesized from the mutated tollip gene. (E) Western blot of the 5 dpf protein lysates from the wild‐type ( tollip +/+ ) line and tollip −/− siblings. Top panel shows Tollip (~ 35 kDa) and a bottom panel shows α‐tubulin (~ 55 kDa) signal. (F) qPCR analysis of the expression of tollip transcripts during early zebrafish development (1–5 dpf). Bars represent the means ± SEM from 3–4 independent experiments (encompassing a pool of 10 larvae/condition). Mann–Whitney U test, * P < 0.05, ****P < 0.0001.

Article Snippet: The Cas9 RNA was synthesized from pCS2‐nCas9n (a gift from Wenbiao Chen, Addgene plasmid, #47929 [ ]) using mMESSAGE mMACHINE T7 Transcription Kit (Life Technologies, #AM1344M).

Techniques: CRISPR, Sequencing, Knock-Out, Mutagenesis, Generated, Synthesized, Western Blot, Expressing, MANN-WHITNEY

Journal: Scientific Reports

Article Title: Efficient generation of transgenic cattle using the DNA transposon and their analysis by next-generation sequencing

doi: 10.1038/srep27185

Figure Lengend Snippet: ( a ) After 45 days of embryo transfer, pregnancy was confirmed by ultrasonography. ( b ) The calf was delivered without assistant. ( c ) When ultraviolet light was exposed to nose of tg cattle, GFP expression was strongly observed. And the tg cattle grew up to 12 months old without any healthy issue ( d ). To determine GFP or RFP expression in a piece of tissue or primary skin cells via recombination, the tissue and cells were cultured and transfected with Dre recombinase mRNA by nucleofection (( e ) a piece of tissue from tg cattle-brightness, ( e` ) before Dre recombinase transfection (GFP), ( e`` ) after Dre recombinase transfection (RFP)). The primary skin cells from the tg cattle were isolated, cultured and transfected with Dre recombinase mRNA. Before transfection, only GFP expression was observed, RFP expression were observed via GFP gene excision by recombination (( f – f`` ) before transfection brightness, fluorescence, and merged, respectively; ( g – g`` ) after transfection brightness, fluorescence, and merged, respectively). The transgene integration and recombination were confirmed by genomic DNA PCR (( h ) 1: Molecular maker, 2: Wild type cattle, 3: Blood from tg cattle, 4: Positive control (DNAs), 5: Negative control) and RT-PCR (( i ) 1: Wild type cattle, 2: cDNA from tg cattle, 3: Negative control). After Dre recombinase transfection, GFP excision was confirmed by genomic DNA PCR (( j ) 1: Molecular marker, 2: Before transfection, 3: After transfection, 4: Negative control). Gel image was cropped and original image was seen in .

Article Snippet: As briefly, primary cells from a transgenic cattle (SNU-PB-2) were transfected with plasmid DNAs (Cas9 with CMV promoter, single guide RNA for GFP with U6 promoter (Toolgen, Seoul, Republic of Korea), donor DNAs for Knock-In; ) using Nucleofactor technology (Neon ® , Invitrogen; program #16).

Techniques: Expressing, Cell Culture, Transfection, Isolation, Fluorescence, Positive Control, Negative Control, Reverse Transcription Polymerase Chain Reaction, Marker

Journal: Scientific Reports

Article Title: Efficient generation of transgenic cattle using the DNA transposon and their analysis by next-generation sequencing

doi: 10.1038/srep27185

Figure Lengend Snippet: ( a ) After 45 days of embryo transfer, pregnancy was confirmed by ultrasonography. ( b ) The calf was delivered without any assistance and grew up to 2 months. Analyzing the calf without ultraviolet light, GFP expression was observed in the eyes ( c ) and nose ( d ). The tg cattle have been grown to 5 months old without any health issue ( e ). When ultraviolet light was exposed to the head, GFP expression was strongly observed ( f ). To know GFP in skin cells, the primary skin cells from the tg cattle were isolated and cultured. In over 99% of cells, GFP expression were observed (( g ) brightness; ( g` ) fluorescence). The transgene integration was confirmed by genomic DNA PCR (( h ) 1: Molecular maker, 2: Wild type cattle, 3: Blood from tg cattle, 4: Positive control (DNAs), 5: Negative control) and RT-PCR using primary cells (( i ) 1: cDNA from Wild type cattle, 2: cDNA from tg cattle, 3: Negative control). Gel image was cropped and original image was seen in .

Article Snippet: As briefly, primary cells from a transgenic cattle (SNU-PB-2) were transfected with plasmid DNAs (Cas9 with CMV promoter, single guide RNA for GFP with U6 promoter (Toolgen, Seoul, Republic of Korea), donor DNAs for Knock-In; ) using Nucleofactor technology (Neon ® , Invitrogen; program #16).

Techniques: Expressing, Isolation, Cell Culture, Fluorescence, Positive Control, Negative Control, Reverse Transcription Polymerase Chain Reaction

Journal: The Journal of Cell Biology

Article Title: Hook3 is a scaffold for the opposite-polarity microtubule-based motors cytoplasmic dynein-1 and KIF1C

doi: 10.1083/jcb.201812170

Figure Lengend Snippet: Endogenous Hook3 and KIF1C interact specifically. (A) Domain organization of KIF1C and Hook3. KIF1C contains an amino-terminal kinesin motor domain and regions of predicted coiled coil (CC), a forkhead-associated domain (FHA), and a proline-rich (P-rich) region in its carboxy-terminal tail. Hook3 is largely made up of regions of predicted CC and contains dynein/dynactin and KIF1C-binding regions ( ; ). The Hook domain, which is also involved in dynein binding , is indicated. (B) 293T cells were transfected with control CRISPR/Cas9 (CTRL) or with CRISPR/Cas9-gRNA specific for KIF1C . KIF1C knockout (KIF1C KO ) was confirmed in two different clones by immunoblotting with an anti-KIF1C antibody. Clone #1 was selected for further assays. β-Actin provided a loading control. (C) KIF1C KO cells were infected with viral particles encoding MSCV-driven KIF1C-BioID-3xFLAG plasmid to obtain near-endogenous KIF1C-BioID protein expression levels. Immunoblots were performed using the indicated antibodies. β-Actin provided a loading control. (D) A volcano plot showing enrichment versus significance of proteins identified in KIF1C-BioID experiments relative to control (BioID alone) experiments. Proteins not present in the BioID control or with an enrichment ratio greater than threefold and a P value >0.05 relative to the control (dashed red lines) were considered significant hits. KIF1C, Hook3, and Tc-Tex-1 (DYNLT1, a dynein light chain) are marked in red. (E) Immunoprecipitation (IP) of endogenous Hook3 and KIF1C with the indicated antibodies from 293T cells. Immunoblots were performed with anti-Hook3 or KIF1C antibodies. (F) Human Hook1, Hook2, and Hook3 tagged with the HaloTag on their amino termini and 3xFLAG on their carboxy termini were transiently transfected into 293T cells and immunoprecipitated with FLAG affinity resin (FLAG-IP). Immunoblots were performed with anti-KIF1C and anti-FLAG antibodies. 3xFLAG-sfGFP provided a control. Protein molecular weight markers are shown in kilodaltons on the anti-FLAG immunoblot. (G) Human KIF1A, KIF1B, KIF1C, KIF5A, KIF5B, and KIF5C were each tagged with BioID-3xFLAG on their carboxy termini and stably expressed in 293T cells. Tagged proteins were immunoprecipitated with FLAG affinity resin (FLAG-IP), and immunoblots were performed with anti-Hook3 and anti-FLAG antibodies. BioID-3xFLAG provided a control. Protein molecular weight markers are shown in kilodaltons on the anti-FLAG immunoblot.

Article Snippet: Briefly, in vitro–transcribed 20-nucleotide Alt-R CRISPR RNA (CrRNA, Hs.Cas9.KIF1C.1.AD) and Alt-R CRISPR/Cas9 trans-activating CRISPR RNA (tracrRNA) were purchased from Integrated DNA Technologies.

Techniques: Binding Assay, Transfection, Control, CRISPR, Knock-Out, Clone Assay, Western Blot, Infection, Plasmid Preparation, Expressing, Immunoprecipitation, Molecular Weight, Stable Transfection

Journal: The Journal of Cell Biology

Article Title: Hook3 is a scaffold for the opposite-polarity microtubule-based motors cytoplasmic dynein-1 and KIF1C

doi: 10.1083/jcb.201812170

Figure Lengend Snippet: KIF1C recruits Hook3 to the cell periphery. (A) 293T KIF1C KO cells (KO) were infected with viral particles encoding MSCV-driven KIF1C-tagRFP-3xFLAG or KIF1C Δ794-807 -tagRFP-3xFLAG plasmids. Immunoblots were performed with the indicated antibodies. Low and high exposures with the KIF1C antibody are shown. β-Actin provided a loading control. 293T cells transfected with CRISPR-Cas9 (CTRL) were used as control cells. (B) Confocal microscopy of KIF1C and Hook3 localization in stable 293T cell lines expressing KIF1C-tagRFP-3xFLAG or KIF1C Δ794-807 -tagRFP-3xFLAG. Cells were grown on glass coverslips, fixed, and stained for endogenous Hook3 (Endo-Hook3). The tagRFP and Hook3 signals are shown in representative maximum intensity projections. The overlap of intensity profiles (AU) generated from drawing a 15-µm line segment across individual z-sections is shown to the right of the images. (C) The mean normalized Hook3 intensity within KIF1C foci for KIF1C-tagRFP-3xFLAG ( n = 25) or KIF1C Δ794-807 -tagRFP-3xFLAG ( n = 24). Foci were determined by thresholding the KIF1C image, and masks of these foci were used to measure the Hook3 intensity in the corresponding regions in maximum projection images. Box plots represent the maximum and minimum values. Statistical significance was calculated with an unpaired t test. ****, P < 0.0001. Representative data from three independent experiments is shown. (D) Confocal microscopy of KIF1C and Hook3 in U2OS cells. Cells were grown on glass coverslips and transiently cotransfected with the indicated sfGFP-tagged Hook3 (full-length Hook3, Hook3 NT [aa 1–552], and Hook3 CT [aa 553–718]) or control sfGFP constructs, and KIF1C-V5. 24 h after transfections, cells were fixed and stained with V5-specific antibody. The V5 and sfGFP signals are shown in representative maximum intensity projections. The overlap of intensity profiles (AU) generated from drawing a 15-µm line segment across an individual z-section is shown to the right of the images. (E) The mean normalized Hook3 intensity within KIF1C foci for cells transfected with different Hook3 constructs (CTRL, n = 27; Hook3, n = 28; Hook3 NT , n = 33; Hook3 CT , n = 29). Foci were determined by thresholding the KIF1C image, and masks of these foci were used to measure the Hook3 intensity in these corresponding regions in maximum projection images. Box plots represent maximum and minimum values. Statistical significance was calculated with one-way ANOVA with Tukey post-test, ****, P < 0.0001. Representative data from three independent experiments is shown. ns, not significant.

Article Snippet: Briefly, in vitro–transcribed 20-nucleotide Alt-R CRISPR RNA (CrRNA, Hs.Cas9.KIF1C.1.AD) and Alt-R CRISPR/Cas9 trans-activating CRISPR RNA (tracrRNA) were purchased from Integrated DNA Technologies.

Techniques: Infection, Western Blot, Control, Transfection, CRISPR, Confocal Microscopy, Expressing, Staining, Generated, Construct