engen spy cas9 nls  (New England Biolabs)


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    Name:
    EnGen Spy Cas9 NLS
    Description:
    EnGen Spy Cas9 NLS 2 000 pmol
    Catalog Number:
    M0646M
    Price:
    600
    Category:
    Other Endonucleases
    Size:
    2 000 pmol
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    New England Biolabs engen spy cas9 nls
    EnGen Spy Cas9 NLS
    EnGen Spy Cas9 NLS 2 000 pmol
    https://www.bioz.com/result/engen spy cas9 nls/product/New England Biolabs
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    engen spy cas9 nls - by Bioz Stars, 2021-06
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    Images

    1) Product Images from "A streamlined CRISPR/Cas9 approach for fast genome editing in Toxoplasma gondii and Besnoitia besnoiti"

    Article Title: A streamlined CRISPR/Cas9 approach for fast genome editing in Toxoplasma gondii and Besnoitia besnoiti

    Journal: Journal of Biological Methods

    doi: 10.14440/jbm.2020.343

    Streamlined workflow for genome manipulation in T . gondii and B . besnoiti . A. Annealing of the crRNA to the tracrRNA is carried out following a temperature gradient for 30 min, resulting in the formation of the sgRNA complex. B. The sgRNA is complexed to the Cas9-NLS by an incubation step of 20 min at RT. C. RNP transfection of the parasites by electroporation can be performed within approximately 60 min.
    Figure Legend Snippet: Streamlined workflow for genome manipulation in T . gondii and B . besnoiti . A. Annealing of the crRNA to the tracrRNA is carried out following a temperature gradient for 30 min, resulting in the formation of the sgRNA complex. B. The sgRNA is complexed to the Cas9-NLS by an incubation step of 20 min at RT. C. RNP transfection of the parasites by electroporation can be performed within approximately 60 min.

    Techniques Used: Incubation, Transfection, Electroporation

    2) Product Images from "Large-scale reconstruction of cell lineages using single-cell readout of transcriptomes and CRISPRCas9 barcodes by scGESTALT"

    Article Title: Large-scale reconstruction of cell lineages using single-cell readout of transcriptomes and CRISPRCas9 barcodes by scGESTALT

    Journal: Nature protocols

    doi: 10.1038/s41596-018-0058-x

    scGESTALT barcode editing. scGESTALT barcode zebrafish were crossed to zebrafish that express heat shock-inducible Cas9 and U6-driven sgRNAs 5–9. Resulting embryos were injected with Cas9 protein and sgRNAs 1–4 at the one-cell stage. Embryos were heat shocked at 30 hpf to induce transgenic Cas9 for a late round of editing. Double transgenic (scGESTALT + , hsp:Cas9 + ; lanes 2–8, n = 7 embryos) and single transgenic (scGESTALT + , hsp:Cas9 − ; lanes 9–12, n = 4 embryos) were identified by screening for GFP expression. The gel shows PCR results of amplifying the scGESTALT barcode (unedited = ~300 bp). Large smear patterns (120–250bp) are observed in early and late edited embryos (lanes 2–8), whereas embryos that were only mutated at sites 1–4 display less editing (lanes 9–12. The band at ~200 bp in lane 12 likely represents large deletion(s) between sites 1–4 that occurred early in development and was inherited by most cells. Note that samples with such dominant large deletions should not be used for downstream experiments and analyses as they are likely to have low barcode diversity). Sample in lane 11 was likely not efficiently injected. Lane 1 represents a control embryo, which was injected with Cas9 protein only (no sgRNAs 1–4, n = 1 embryo) and was not heat shocked. As expected, the barcode is not edited in this case. This procedure was approved by the HU/FAS Committee on the Use of Animals in Research Teaching under Protocol No. 25–08.
    Figure Legend Snippet: scGESTALT barcode editing. scGESTALT barcode zebrafish were crossed to zebrafish that express heat shock-inducible Cas9 and U6-driven sgRNAs 5–9. Resulting embryos were injected with Cas9 protein and sgRNAs 1–4 at the one-cell stage. Embryos were heat shocked at 30 hpf to induce transgenic Cas9 for a late round of editing. Double transgenic (scGESTALT + , hsp:Cas9 + ; lanes 2–8, n = 7 embryos) and single transgenic (scGESTALT + , hsp:Cas9 − ; lanes 9–12, n = 4 embryos) were identified by screening for GFP expression. The gel shows PCR results of amplifying the scGESTALT barcode (unedited = ~300 bp). Large smear patterns (120–250bp) are observed in early and late edited embryos (lanes 2–8), whereas embryos that were only mutated at sites 1–4 display less editing (lanes 9–12. The band at ~200 bp in lane 12 likely represents large deletion(s) between sites 1–4 that occurred early in development and was inherited by most cells. Note that samples with such dominant large deletions should not be used for downstream experiments and analyses as they are likely to have low barcode diversity). Sample in lane 11 was likely not efficiently injected. Lane 1 represents a control embryo, which was injected with Cas9 protein only (no sgRNAs 1–4, n = 1 embryo) and was not heat shocked. As expected, the barcode is not edited in this case. This procedure was approved by the HU/FAS Committee on the Use of Animals in Research Teaching under Protocol No. 25–08.

    Techniques Used: Injection, Transgenic Assay, Expressing, Polymerase Chain Reaction

    Strategy for a CRISPR-Cas9 system that enables early and late barcode editing. .
    Figure Legend Snippet: Strategy for a CRISPR-Cas9 system that enables early and late barcode editing. .

    Techniques Used: CRISPR

    3) Product Images from "Human Semaphorin 3 Variants Link Melanocortin Circuit Development and Energy Balance"

    Article Title: Human Semaphorin 3 Variants Link Melanocortin Circuit Development and Energy Balance

    Journal: Cell

    doi: 10.1016/j.cell.2018.12.009

    Disruption of Semaphorin 3 S, Neuropilins , and Plexins Alters Energy Homeostasis in Zebrafish (A) Unrooted phylogenetic trees of Sema3, PlxnA, and Nrp genes. Genes from zebrafish (dotted lines) and mouse and human (solid lines) were used to construct the trees. Where zebrafish genes have been duplicated, a letter is used to identify paralogs. Scale bars, number of substitutions per amino acid site. (B) Heatmap showing change in length, weight, and percentage of body fat in deletion mutants relative to Cas9-only control fish; decrease (blue), increase (orange) in the phenotype of mutants relative to control fish (for the natural log fold change); ∗ genes not screened; agrp , positive control. (C) Length (mm), weight (mg) and percentage of body fat in nrp2a and nrp2b mutant fish relative to Cas9-only injected control fish. Data represented as mean ± SEM. ∗ p
    Figure Legend Snippet: Disruption of Semaphorin 3 S, Neuropilins , and Plexins Alters Energy Homeostasis in Zebrafish (A) Unrooted phylogenetic trees of Sema3, PlxnA, and Nrp genes. Genes from zebrafish (dotted lines) and mouse and human (solid lines) were used to construct the trees. Where zebrafish genes have been duplicated, a letter is used to identify paralogs. Scale bars, number of substitutions per amino acid site. (B) Heatmap showing change in length, weight, and percentage of body fat in deletion mutants relative to Cas9-only control fish; decrease (blue), increase (orange) in the phenotype of mutants relative to control fish (for the natural log fold change); ∗ genes not screened; agrp , positive control. (C) Length (mm), weight (mg) and percentage of body fat in nrp2a and nrp2b mutant fish relative to Cas9-only injected control fish. Data represented as mean ± SEM. ∗ p

    Techniques Used: Construct, Fluorescence In Situ Hybridization, Positive Control, Mutagenesis, Injection

    4) Product Images from "A conserved role for the ALS-linked splicing factor SFPQ in repression of pathogenic cryptic last exons"

    Article Title: A conserved role for the ALS-linked splicing factor SFPQ in repression of pathogenic cryptic last exons

    Journal: Nature Communications

    doi: 10.1038/s41467-021-22098-z

    CLEs have functional impacts. a Deletion of the b4galt2 CLE using CRISPR/Cas9 rescues expression of downstream exons. Left: cut sites of the b4galt2 sgRNAs. CLE is indicated by capital letters. Center: PCR verification of Cas9 cleavage after injection of sgRNAs. Representative image; experiment performed five times. Right: RT-qPCR quantitation of the relative expression of the downstream b4galt2 exons in sfpq−/− embryos compared to siblings (±SD); n = 3 biologically independent replicates. Two-tailed unpaired t -test was performed. b In-situ hybridization of the epha4b CLE at 24 hpf, displaying strong expression in the midbrain and hindbrain of sfpq −/− embryos. c In-situ hybridization of rfng shows rhombomere boundary defects at 22ss after injection into WT embryos of the epha4b cryptic transcript or a mutated transcript with an early stop codon. Loss of boundaries seen in 8/10 embryos. d Left: in-situ hybridization of rfng shows rhombomere boundary defects of sfpq −/− embryos are rescued by injection of the epha4b cryptic splice junction morpholino but not a mismatch morpholino. Rhombomere boundaries are numbered. Right: quantification of staining in rhombomeres in three lateral view samples for each condition. Representative images; defect seen in 13/15 embryos. e Left: in-situ hybridization of DeltaA shows a loss of discrete neuronal clusters in sfpq −/− which is rescued by injection of the epha4b cryptic splice junction morpholino but not a mismatch morpholino. Right: quantification of number of DeltaA clusters in each condition. Each data point represents one embryo; embryos examined over two independent experiments. Two-tailed t -test was performed, *** p = 0.0005. c – e Upper: lateral view. Lower: dorsal view. Source data are provided as a Source Data file.
    Figure Legend Snippet: CLEs have functional impacts. a Deletion of the b4galt2 CLE using CRISPR/Cas9 rescues expression of downstream exons. Left: cut sites of the b4galt2 sgRNAs. CLE is indicated by capital letters. Center: PCR verification of Cas9 cleavage after injection of sgRNAs. Representative image; experiment performed five times. Right: RT-qPCR quantitation of the relative expression of the downstream b4galt2 exons in sfpq−/− embryos compared to siblings (±SD); n = 3 biologically independent replicates. Two-tailed unpaired t -test was performed. b In-situ hybridization of the epha4b CLE at 24 hpf, displaying strong expression in the midbrain and hindbrain of sfpq −/− embryos. c In-situ hybridization of rfng shows rhombomere boundary defects at 22ss after injection into WT embryos of the epha4b cryptic transcript or a mutated transcript with an early stop codon. Loss of boundaries seen in 8/10 embryos. d Left: in-situ hybridization of rfng shows rhombomere boundary defects of sfpq −/− embryos are rescued by injection of the epha4b cryptic splice junction morpholino but not a mismatch morpholino. Rhombomere boundaries are numbered. Right: quantification of staining in rhombomeres in three lateral view samples for each condition. Representative images; defect seen in 13/15 embryos. e Left: in-situ hybridization of DeltaA shows a loss of discrete neuronal clusters in sfpq −/− which is rescued by injection of the epha4b cryptic splice junction morpholino but not a mismatch morpholino. Right: quantification of number of DeltaA clusters in each condition. Each data point represents one embryo; embryos examined over two independent experiments. Two-tailed t -test was performed, *** p = 0.0005. c – e Upper: lateral view. Lower: dorsal view. Source data are provided as a Source Data file.

    Techniques Used: Functional Assay, CRISPR, Expressing, Polymerase Chain Reaction, Injection, Quantitative RT-PCR, Quantitation Assay, Two Tailed Test, In Situ Hybridization, Staining

    Related Articles

    Knock-Out:

    Article Title: Toxoplasma gondii co-opts the unfolded protein response to enhance migration and dissemination of infected host cells
    Article Snippet: Cultures of DCs and J774.1 macrophages were cultivated in Roswell Park Memorial Institute (RPMI) supplemented with 10% heat-inactivated fetal bovine serum (FBS) (Gibco/Invitrogen) and penicillin/streptomycin at 37°C with 5% CO2 and infected as described above. .. Generation of IRE1 knockout cells Disruption of the IRE1 gene in MEF cells was carried out using the CRISPR/Cas9 method [ ]. ..

    CRISPR:

    Article Title: Toxoplasma gondii co-opts the unfolded protein response to enhance migration and dissemination of infected host cells
    Article Snippet: Cultures of DCs and J774.1 macrophages were cultivated in Roswell Park Memorial Institute (RPMI) supplemented with 10% heat-inactivated fetal bovine serum (FBS) (Gibco/Invitrogen) and penicillin/streptomycin at 37°C with 5% CO2 and infected as described above. .. Generation of IRE1 knockout cells Disruption of the IRE1 gene in MEF cells was carried out using the CRISPR/Cas9 method [ ]. ..

    Article Title: A fast Myh super enhancer dictates adult muscle fiber phenotype through competitive interactions with the fast Myh genes
    Article Snippet: .. Mouse generation by CRISPR/Cas9 SgRNA and Cas9 purified protein were produced by the TACGENE platform. ..

    Article Title: The use of CRISPR-Cas Selective Amplicon Sequencing (CCSAS) to reveal the eukaryotic microbiome of metazoans
    Article Snippet: The reaction was incubated at 37°C for 4 h in a thermocycler, and then followed by 70°C for 10 min to deactivate the CRISPR-Cas9. .. The reaction was incubated at 37°C for 4 h in a thermocycler, and then followed by 70°C for 10 min to deactivate the CRISPR-Cas9. .. For each sample, in parallel with the CRISPR-Cas9 treatment we also prepared the reaction without CRISPR-Cas9 treatment, in which Cas9 nuclease and sgRNA were replaced with molecular grade ultrapure water (Invitrogen).

    Article Title: Genome-wide CRISPR screens of oral squamous cell carcinoma reveal fitness genes in the Hippo pathway
    Article Snippet: .. Validation sgRNA design and cloning To validate the results obtained from the screen, individual targeted genetic knockouts were generated using CRISPR/Cas9 and plasmid expressing sgRNAs targeting the gene of interest. .. Two sgRNA sequences were used for each target gene, one was selected from the CRISPR library v1 while another sequence was designed using the Genetic Perturbation Platform (GPP) web portal ( https://portals.broadinstitute.org/gpp/public/analysis-tools/sgrna-design ).

    Article Title: Rotavirus induces intercellular calcium waves through ADP signaling
    Article Snippet: MA104-GCaMP6s cells (pLVX-IRES-Hygro) were transduced with the P2Y1 CRISPR/Cas9-expressing construct and at 72 hrs post-transduction the cells were passaged in the presence of 50 μg/mL hygromycin B and 10 μg/mL puromycin to select for co-expression of GCaMP6s and CRISPR/Cas9. .. The J2-derived jHIE-GCaMP6s(hygro) enteroid cultures described above were transduced with the CRISPR/Cas9-expressing constructs to make two separate enteroid lines and grown with 50 μg/mL hygromycin B and 1 μg/mL puromycin to select for co-expression of GCaMP6s and CRISPR/Cas9. .. Genotyping was performed on whole genomic DNA prepared using DirectPCR (Viagen) with Proteinase K (Qiagen) according to manufacturer’s instructions.

    Article Title: Genetic variation among wMel strains of Wolbachia pipientis differentially rescues a bag of marbles partial loss of function mutant in Drosophila melanogaster
    Article Snippet: We backcrossed females of all CRISPR/Cas9 mutants to w1118 isogenic males for three generations, and then crossed the mutants to the w1118 ; TM2/TM6 line to maintain the bam mutants over the TM6 balancer. .. All CRISPR/Cas9 edits in the lines were confirmed by Sanger sequencing (Cornell BRC Genomics Facility). .. PCR assays to detect Wolbachia To test for the presence of Wolbachia , we used the Zymo quick DNA miniprep kit to prepare DNA from three replicate samples each with three female flies.

    Article Title: Rotavirus induces intercellular calcium waves through ADP signaling
    Article Snippet: .. MA104-GCaMP6s cells (pLVX-IRES-Hygro) were transduced with the P2Y1 CRISPR/Cas9-expressing construct and at 72 hrs post-transduction the cells were passaged in the presence of 50 μg/mL hygromycin B and 10 μg/mL puromycin to select for co-expression of GCaMP6s and CRISPR/Cas9. .. The J2-derived jHIE-GCaMP6s(hygro) enteroid cultures described above were transduced with the CRISPR/Cas9-expressing constructs to make two separate enteroid lines and grown with 50 μg/mL hygromycin B and 1 μg/mL puromycin to select for co-expression of GCaMP6s and CRISPR/Cas9.

    Purification:

    Article Title: A fast Myh super enhancer dictates adult muscle fiber phenotype through competitive interactions with the fast Myh genes
    Article Snippet: .. Mouse generation by CRISPR/Cas9 SgRNA and Cas9 purified protein were produced by the TACGENE platform. ..

    Produced:

    Article Title: A fast Myh super enhancer dictates adult muscle fiber phenotype through competitive interactions with the fast Myh genes
    Article Snippet: .. Mouse generation by CRISPR/Cas9 SgRNA and Cas9 purified protein were produced by the TACGENE platform. ..

    Incubation:

    Article Title: The use of CRISPR-Cas Selective Amplicon Sequencing (CCSAS) to reveal the eukaryotic microbiome of metazoans
    Article Snippet: The reaction was incubated at 37°C for 4 h in a thermocycler, and then followed by 70°C for 10 min to deactivate the CRISPR-Cas9. .. The reaction was incubated at 37°C for 4 h in a thermocycler, and then followed by 70°C for 10 min to deactivate the CRISPR-Cas9. .. For each sample, in parallel with the CRISPR-Cas9 treatment we also prepared the reaction without CRISPR-Cas9 treatment, in which Cas9 nuclease and sgRNA were replaced with molecular grade ultrapure water (Invitrogen).

    Clone Assay:

    Article Title: Genome-wide CRISPR screens of oral squamous cell carcinoma reveal fitness genes in the Hippo pathway
    Article Snippet: .. Validation sgRNA design and cloning To validate the results obtained from the screen, individual targeted genetic knockouts were generated using CRISPR/Cas9 and plasmid expressing sgRNAs targeting the gene of interest. .. Two sgRNA sequences were used for each target gene, one was selected from the CRISPR library v1 while another sequence was designed using the Genetic Perturbation Platform (GPP) web portal ( https://portals.broadinstitute.org/gpp/public/analysis-tools/sgrna-design ).

    Generated:

    Article Title: Genome-wide CRISPR screens of oral squamous cell carcinoma reveal fitness genes in the Hippo pathway
    Article Snippet: .. Validation sgRNA design and cloning To validate the results obtained from the screen, individual targeted genetic knockouts were generated using CRISPR/Cas9 and plasmid expressing sgRNAs targeting the gene of interest. .. Two sgRNA sequences were used for each target gene, one was selected from the CRISPR library v1 while another sequence was designed using the Genetic Perturbation Platform (GPP) web portal ( https://portals.broadinstitute.org/gpp/public/analysis-tools/sgrna-design ).

    Plasmid Preparation:

    Article Title: Genome-wide CRISPR screens of oral squamous cell carcinoma reveal fitness genes in the Hippo pathway
    Article Snippet: .. Validation sgRNA design and cloning To validate the results obtained from the screen, individual targeted genetic knockouts were generated using CRISPR/Cas9 and plasmid expressing sgRNAs targeting the gene of interest. .. Two sgRNA sequences were used for each target gene, one was selected from the CRISPR library v1 while another sequence was designed using the Genetic Perturbation Platform (GPP) web portal ( https://portals.broadinstitute.org/gpp/public/analysis-tools/sgrna-design ).

    Expressing:

    Article Title: Genome-wide CRISPR screens of oral squamous cell carcinoma reveal fitness genes in the Hippo pathway
    Article Snippet: .. Validation sgRNA design and cloning To validate the results obtained from the screen, individual targeted genetic knockouts were generated using CRISPR/Cas9 and plasmid expressing sgRNAs targeting the gene of interest. .. Two sgRNA sequences were used for each target gene, one was selected from the CRISPR library v1 while another sequence was designed using the Genetic Perturbation Platform (GPP) web portal ( https://portals.broadinstitute.org/gpp/public/analysis-tools/sgrna-design ).

    Transduction:

    Article Title: Rotavirus induces intercellular calcium waves through ADP signaling
    Article Snippet: MA104-GCaMP6s cells (pLVX-IRES-Hygro) were transduced with the P2Y1 CRISPR/Cas9-expressing construct and at 72 hrs post-transduction the cells were passaged in the presence of 50 μg/mL hygromycin B and 10 μg/mL puromycin to select for co-expression of GCaMP6s and CRISPR/Cas9. .. The J2-derived jHIE-GCaMP6s(hygro) enteroid cultures described above were transduced with the CRISPR/Cas9-expressing constructs to make two separate enteroid lines and grown with 50 μg/mL hygromycin B and 1 μg/mL puromycin to select for co-expression of GCaMP6s and CRISPR/Cas9. .. Genotyping was performed on whole genomic DNA prepared using DirectPCR (Viagen) with Proteinase K (Qiagen) according to manufacturer’s instructions.

    Article Title: Rotavirus induces intercellular calcium waves through ADP signaling
    Article Snippet: .. MA104-GCaMP6s cells (pLVX-IRES-Hygro) were transduced with the P2Y1 CRISPR/Cas9-expressing construct and at 72 hrs post-transduction the cells were passaged in the presence of 50 μg/mL hygromycin B and 10 μg/mL puromycin to select for co-expression of GCaMP6s and CRISPR/Cas9. .. The J2-derived jHIE-GCaMP6s(hygro) enteroid cultures described above were transduced with the CRISPR/Cas9-expressing constructs to make two separate enteroid lines and grown with 50 μg/mL hygromycin B and 1 μg/mL puromycin to select for co-expression of GCaMP6s and CRISPR/Cas9.

    Construct:

    Article Title: Rotavirus induces intercellular calcium waves through ADP signaling
    Article Snippet: MA104-GCaMP6s cells (pLVX-IRES-Hygro) were transduced with the P2Y1 CRISPR/Cas9-expressing construct and at 72 hrs post-transduction the cells were passaged in the presence of 50 μg/mL hygromycin B and 10 μg/mL puromycin to select for co-expression of GCaMP6s and CRISPR/Cas9. .. The J2-derived jHIE-GCaMP6s(hygro) enteroid cultures described above were transduced with the CRISPR/Cas9-expressing constructs to make two separate enteroid lines and grown with 50 μg/mL hygromycin B and 1 μg/mL puromycin to select for co-expression of GCaMP6s and CRISPR/Cas9. .. Genotyping was performed on whole genomic DNA prepared using DirectPCR (Viagen) with Proteinase K (Qiagen) according to manufacturer’s instructions.

    Article Title: Rotavirus induces intercellular calcium waves through ADP signaling
    Article Snippet: .. MA104-GCaMP6s cells (pLVX-IRES-Hygro) were transduced with the P2Y1 CRISPR/Cas9-expressing construct and at 72 hrs post-transduction the cells were passaged in the presence of 50 μg/mL hygromycin B and 10 μg/mL puromycin to select for co-expression of GCaMP6s and CRISPR/Cas9. .. The J2-derived jHIE-GCaMP6s(hygro) enteroid cultures described above were transduced with the CRISPR/Cas9-expressing constructs to make two separate enteroid lines and grown with 50 μg/mL hygromycin B and 1 μg/mL puromycin to select for co-expression of GCaMP6s and CRISPR/Cas9.

    Sequencing:

    Article Title: Genetic variation among wMel strains of Wolbachia pipientis differentially rescues a bag of marbles partial loss of function mutant in Drosophila melanogaster
    Article Snippet: We backcrossed females of all CRISPR/Cas9 mutants to w1118 isogenic males for three generations, and then crossed the mutants to the w1118 ; TM2/TM6 line to maintain the bam mutants over the TM6 balancer. .. All CRISPR/Cas9 edits in the lines were confirmed by Sanger sequencing (Cornell BRC Genomics Facility). .. PCR assays to detect Wolbachia To test for the presence of Wolbachia , we used the Zymo quick DNA miniprep kit to prepare DNA from three replicate samples each with three female flies.

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    New England Biolabs crispr cas9 sgrna
    Competition of the f Myh gene promoters for the shared SE. (A) Diagram showing the strategy for in vivo deletion of Myh1 and Myh4 by <t>CRISPR/Cas9.</t> (B) PCR used for the screening of mutant animals and sequence of the deleted allele. (C) Diagram showing the strategy for in vivo inversion of Myh1 and Myh4 by CRISPR/Cas9. (D) PCR used for the screening of mutant animals and sequence of the inverted allele. (E) Immunostaining against MYH2, MYH4 and MYH7 proteins of adult distal hindlimb sections of 2-3-month-old WT, Myh(1-4) Del/+ , Myh(1-4) Inv/+, and of Myh(1-4) Inv3’/+ animals. (F) Quantification of Myh2, Myh1, Myh4, Myh8 and Myh13 mRNAs by RT-qPCR from adult Soleus of WT, Myh(1-4) Del/+ , and Myh(1-4) Del/Del animals. (G) Quantification of Myh2, Myh1, Myh4, Myh8 and Myh13 mRNAs by RT-qPCR from adult Soleus of WT, Myh(1-4) Inv/+ and Myh(1-4) Inv/Inv animals. (H) Quantification of Myh2, Myh1, Myh4, Myh8 and Myh13 mRNAs by RT-qPCR from adult Soleus of WT, Myh(1-4) Inv3’/+ and of Myh(1-4) Inv3’/Inv3’ animals. (I) Quantification of Myh4 premRNA from adult TA of WT, Myh(1-4) Inv/Inv and of Myh(1-4) Inv3’/Inv3’ animals. (J) Quantification of Myh1 premRNA from adult TA of WT, Myh(1-4) Inv/Inv and of Myh(1- 4) Inv3’/Inv3’ animals. Numerical data are presented as mean ± S.E.M. * P
    Crispr Cas9 Sgrna, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/crispr cas9 sgrna/product/New England Biolabs
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    crispr cas9 sgrna - by Bioz Stars, 2021-06
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    Competition of the f Myh gene promoters for the shared SE. (A) Diagram showing the strategy for in vivo deletion of Myh1 and Myh4 by CRISPR/Cas9. (B) PCR used for the screening of mutant animals and sequence of the deleted allele. (C) Diagram showing the strategy for in vivo inversion of Myh1 and Myh4 by CRISPR/Cas9. (D) PCR used for the screening of mutant animals and sequence of the inverted allele. (E) Immunostaining against MYH2, MYH4 and MYH7 proteins of adult distal hindlimb sections of 2-3-month-old WT, Myh(1-4) Del/+ , Myh(1-4) Inv/+, and of Myh(1-4) Inv3’/+ animals. (F) Quantification of Myh2, Myh1, Myh4, Myh8 and Myh13 mRNAs by RT-qPCR from adult Soleus of WT, Myh(1-4) Del/+ , and Myh(1-4) Del/Del animals. (G) Quantification of Myh2, Myh1, Myh4, Myh8 and Myh13 mRNAs by RT-qPCR from adult Soleus of WT, Myh(1-4) Inv/+ and Myh(1-4) Inv/Inv animals. (H) Quantification of Myh2, Myh1, Myh4, Myh8 and Myh13 mRNAs by RT-qPCR from adult Soleus of WT, Myh(1-4) Inv3’/+ and of Myh(1-4) Inv3’/Inv3’ animals. (I) Quantification of Myh4 premRNA from adult TA of WT, Myh(1-4) Inv/Inv and of Myh(1-4) Inv3’/Inv3’ animals. (J) Quantification of Myh1 premRNA from adult TA of WT, Myh(1-4) Inv/Inv and of Myh(1- 4) Inv3’/Inv3’ animals. Numerical data are presented as mean ± S.E.M. * P

    Journal: bioRxiv

    Article Title: A fast Myh super enhancer dictates adult muscle fiber phenotype through competitive interactions with the fast Myh genes

    doi: 10.1101/2021.04.17.438406

    Figure Lengend Snippet: Competition of the f Myh gene promoters for the shared SE. (A) Diagram showing the strategy for in vivo deletion of Myh1 and Myh4 by CRISPR/Cas9. (B) PCR used for the screening of mutant animals and sequence of the deleted allele. (C) Diagram showing the strategy for in vivo inversion of Myh1 and Myh4 by CRISPR/Cas9. (D) PCR used for the screening of mutant animals and sequence of the inverted allele. (E) Immunostaining against MYH2, MYH4 and MYH7 proteins of adult distal hindlimb sections of 2-3-month-old WT, Myh(1-4) Del/+ , Myh(1-4) Inv/+, and of Myh(1-4) Inv3’/+ animals. (F) Quantification of Myh2, Myh1, Myh4, Myh8 and Myh13 mRNAs by RT-qPCR from adult Soleus of WT, Myh(1-4) Del/+ , and Myh(1-4) Del/Del animals. (G) Quantification of Myh2, Myh1, Myh4, Myh8 and Myh13 mRNAs by RT-qPCR from adult Soleus of WT, Myh(1-4) Inv/+ and Myh(1-4) Inv/Inv animals. (H) Quantification of Myh2, Myh1, Myh4, Myh8 and Myh13 mRNAs by RT-qPCR from adult Soleus of WT, Myh(1-4) Inv3’/+ and of Myh(1-4) Inv3’/Inv3’ animals. (I) Quantification of Myh4 premRNA from adult TA of WT, Myh(1-4) Inv/Inv and of Myh(1-4) Inv3’/Inv3’ animals. (J) Quantification of Myh1 premRNA from adult TA of WT, Myh(1-4) Inv/Inv and of Myh(1- 4) Inv3’/Inv3’ animals. Numerical data are presented as mean ± S.E.M. * P

    Article Snippet: Mouse generation by CRISPR/Cas9 SgRNA and Cas9 purified protein were produced by the TACGENE platform.

    Techniques: In Vivo, CRISPR, Polymerase Chain Reaction, Mutagenesis, Sequencing, Immunostaining, Quantitative RT-PCR

    The f Myh -SE is required for adult f Myh and neonatal Myh8 genes expression. (A) A mouse line deleted for the f Myh -SE element was generated by injecting specific sgRNAs and Cas9 protein into mouse oocytes. (B) f Myh- SE -/- mice died at birth (P0) without breathing and air in their lungs. (C) f Myh- SE -/- E18.5 fetuses did not present severe visible malformations. (D) Quantification of Myh mRNAs by RT-qPCR in control and f Myh- SE -/- E18.5 forelimb skeletal muscles. Mutant muscles showed decreased Myh2 , Myh1 , Myh4 and Myh8 mRNAs levels. (E) RNascope experiments against Myh3 and Myh8 mRNAs on isolated E18.5 forelimb fibers of control and mutant mice. (F) Same as (E) showing a decreased accumulation of Myh2 and Myh4 mRNAs in mutant mice compared to their littermate controls. (G) Immunostaining at the distal hindlimb level of E18.5 control and mutant fetuses revealing MYH3 and MYH8 positive myofibers. (H) Same as (G), zoom in the EDL of control and mutant. (I) In toto immunostaining of diaphragms from E18.5 mutant and control fetuses showing in red Actin filaments (phalloidine), in green AchR (alpha-bungarotoxin), and in pink neurofilaments showing altered repartition of NMJ and punctated Actin aggregates in mutant diaphragms. (J) Myofibers from mutant diaphragm showed defects in sarcomeres organization as shown by phalloidine staining. (K) Electronic microscopy pictures of the sarcomeres defects present in mutant E18.5 fetuses compared to their littermate controls. For D (n=3). For E and F, scale bar: 50 μm. For G, scale bar: 500 μm. For H, scale bar: 100 μm. Numerical data are presented as mean ± s.e.m. * P

    Journal: bioRxiv

    Article Title: A fast Myh super enhancer dictates adult muscle fiber phenotype through competitive interactions with the fast Myh genes

    doi: 10.1101/2021.04.17.438406

    Figure Lengend Snippet: The f Myh -SE is required for adult f Myh and neonatal Myh8 genes expression. (A) A mouse line deleted for the f Myh -SE element was generated by injecting specific sgRNAs and Cas9 protein into mouse oocytes. (B) f Myh- SE -/- mice died at birth (P0) without breathing and air in their lungs. (C) f Myh- SE -/- E18.5 fetuses did not present severe visible malformations. (D) Quantification of Myh mRNAs by RT-qPCR in control and f Myh- SE -/- E18.5 forelimb skeletal muscles. Mutant muscles showed decreased Myh2 , Myh1 , Myh4 and Myh8 mRNAs levels. (E) RNascope experiments against Myh3 and Myh8 mRNAs on isolated E18.5 forelimb fibers of control and mutant mice. (F) Same as (E) showing a decreased accumulation of Myh2 and Myh4 mRNAs in mutant mice compared to their littermate controls. (G) Immunostaining at the distal hindlimb level of E18.5 control and mutant fetuses revealing MYH3 and MYH8 positive myofibers. (H) Same as (G), zoom in the EDL of control and mutant. (I) In toto immunostaining of diaphragms from E18.5 mutant and control fetuses showing in red Actin filaments (phalloidine), in green AchR (alpha-bungarotoxin), and in pink neurofilaments showing altered repartition of NMJ and punctated Actin aggregates in mutant diaphragms. (J) Myofibers from mutant diaphragm showed defects in sarcomeres organization as shown by phalloidine staining. (K) Electronic microscopy pictures of the sarcomeres defects present in mutant E18.5 fetuses compared to their littermate controls. For D (n=3). For E and F, scale bar: 50 μm. For G, scale bar: 500 μm. For H, scale bar: 100 μm. Numerical data are presented as mean ± s.e.m. * P

    Article Snippet: Mouse generation by CRISPR/Cas9 SgRNA and Cas9 purified protein were produced by the TACGENE platform.

    Techniques: Expressing, Generated, Mouse Assay, Quantitative RT-PCR, Mutagenesis, Isolation, Immunostaining, Staining, Microscopy

    Muscle phenotype of enhancers A and B deletion. (A) Diagram showing the strategies for in vivo deletions of enhancer A or B by CRISPR/Cas9. (B) PCR showing the DNA fragment amplified after EnhA deletion, and sequence of the deleted allele. (C) Sequence of the deleted EnhB allele. (D) Immunostaining against MYH1 on adult distal hindlimb sections in control and EnhA -/- . (E) Quantification of Myh3, Myh2, Myh1, Myh4, Myh8 and Myh13 mRNAs and of Linc-Myh by RT-qPCR in Soleus of control, EnhA and EnhB mutants. (F) Schema of the transgene used to generate a transgenic mouse line expressing nuclear beta- galactosidase under the control of the 1.8kb EnhB DNA element. (G) Beta-galactosidase positive nuclei are detected in the fast tibialis posterior but not in the slow soleus. For E, n=3. Numerical data are presented as mean ± S.E.M. * P

    Journal: bioRxiv

    Article Title: A fast Myh super enhancer dictates adult muscle fiber phenotype through competitive interactions with the fast Myh genes

    doi: 10.1101/2021.04.17.438406

    Figure Lengend Snippet: Muscle phenotype of enhancers A and B deletion. (A) Diagram showing the strategies for in vivo deletions of enhancer A or B by CRISPR/Cas9. (B) PCR showing the DNA fragment amplified after EnhA deletion, and sequence of the deleted allele. (C) Sequence of the deleted EnhB allele. (D) Immunostaining against MYH1 on adult distal hindlimb sections in control and EnhA -/- . (E) Quantification of Myh3, Myh2, Myh1, Myh4, Myh8 and Myh13 mRNAs and of Linc-Myh by RT-qPCR in Soleus of control, EnhA and EnhB mutants. (F) Schema of the transgene used to generate a transgenic mouse line expressing nuclear beta- galactosidase under the control of the 1.8kb EnhB DNA element. (G) Beta-galactosidase positive nuclei are detected in the fast tibialis posterior but not in the slow soleus. For E, n=3. Numerical data are presented as mean ± S.E.M. * P

    Article Snippet: Mouse generation by CRISPR/Cas9 SgRNA and Cas9 purified protein were produced by the TACGENE platform.

    Techniques: In Vivo, CRISPR, Polymerase Chain Reaction, Amplification, Sequencing, Immunostaining, Quantitative RT-PCR, Transgenic Assay, Expressing

    Phenotype of f Myh -SE deletion in E18.5 mutant fetuses. (A) Diagram showing the strategy for in vivo deletion of the f Myh-SE by CRISPR/Cas9. (B) Sequence of the deleted allele. (C) Immunostaining of distal hindlimb sections of control and fMyh-SE -/- showing MYH8 (green), MYH7 (purple) and Laminin (white) expression. (D) RNascope experiments against Myh2 and Myh4 mRNAs on isolated E18.5 fibers of control and mutant fetuses. (E) Myofibers from mutant diaphragm showed defects in sarcomeres organization as revealed by phalloidine staining. For E, scale bar: 500 μm.

    Journal: bioRxiv

    Article Title: A fast Myh super enhancer dictates adult muscle fiber phenotype through competitive interactions with the fast Myh genes

    doi: 10.1101/2021.04.17.438406

    Figure Lengend Snippet: Phenotype of f Myh -SE deletion in E18.5 mutant fetuses. (A) Diagram showing the strategy for in vivo deletion of the f Myh-SE by CRISPR/Cas9. (B) Sequence of the deleted allele. (C) Immunostaining of distal hindlimb sections of control and fMyh-SE -/- showing MYH8 (green), MYH7 (purple) and Laminin (white) expression. (D) RNascope experiments against Myh2 and Myh4 mRNAs on isolated E18.5 fibers of control and mutant fetuses. (E) Myofibers from mutant diaphragm showed defects in sarcomeres organization as revealed by phalloidine staining. For E, scale bar: 500 μm.

    Article Snippet: Mouse generation by CRISPR/Cas9 SgRNA and Cas9 purified protein were produced by the TACGENE platform.

    Techniques: Mutagenesis, In Vivo, CRISPR, Sequencing, Immunostaining, Expressing, Isolation, Staining

    Role of the different enhancers composing the SE. (A) Schematic representation of the snATAC-seq peaks along the 42kb SE and the enhancers A and B deleted by CRISPR/Cas9 editing. (B) Immunostaining against fast MYH2, MYH4 and slow MYH7 on adult leg sections of 2-3-month-old mouse female deleted for enhancer A or B. (C) Same as (B), zoom in Tibialis posterior and FHL muscle of WT and EnhA -/- mutant. (D) Immunostaining against fast MYH1 in Tibialis posterior and FHL muscle of WT and EnhA -/- mutant. The absence of EnhA induced an increased number of MYH1 positive fibers. (E) Quantification of f Myh mRNA and of Linc-Myh in adult TA of control and EnhA and EnhB mutant by RT-qPCR experiments. Schema of the interactions identified. For E, n=3. Numerical data are presented as mean ± S.E.M. * P

    Journal: bioRxiv

    Article Title: A fast Myh super enhancer dictates adult muscle fiber phenotype through competitive interactions with the fast Myh genes

    doi: 10.1101/2021.04.17.438406

    Figure Lengend Snippet: Role of the different enhancers composing the SE. (A) Schematic representation of the snATAC-seq peaks along the 42kb SE and the enhancers A and B deleted by CRISPR/Cas9 editing. (B) Immunostaining against fast MYH2, MYH4 and slow MYH7 on adult leg sections of 2-3-month-old mouse female deleted for enhancer A or B. (C) Same as (B), zoom in Tibialis posterior and FHL muscle of WT and EnhA -/- mutant. (D) Immunostaining against fast MYH1 in Tibialis posterior and FHL muscle of WT and EnhA -/- mutant. The absence of EnhA induced an increased number of MYH1 positive fibers. (E) Quantification of f Myh mRNA and of Linc-Myh in adult TA of control and EnhA and EnhB mutant by RT-qPCR experiments. Schema of the interactions identified. For E, n=3. Numerical data are presented as mean ± S.E.M. * P

    Article Snippet: Mouse generation by CRISPR/Cas9 SgRNA and Cas9 purified protein were produced by the TACGENE platform.

    Techniques: CRISPR, Immunostaining, Mutagenesis, Quantitative RT-PCR

    The promoters of f Myh genes compete for the shared SE (A) Schema of the distinct f Myh alleles generated by CRISPR/Cas9 editing. (B) Immunostaining against MYH4 (blue), MYH2 (green) and slow MYH7 (red) of adult distal hindlimb sections of wt, Myh(1-4) Del/Del , Myh(1-4) Inv/Inv and of Myh(1-4) Inv3’/Inv3’ mutants. (C) Immunostaining against neonatal MYH8 of adult leg sections in wt, Myh(1-4) Del/Del , Myh(1- 4) Inv/Inv and of Myh(1-4) Inv3’/Inv3’ . (D) Same as (C) against extraocular MYH13. (E) Quantification of Myh2, Myh1, Myh4, Myh8 and Myh13 mRNAs of adult wt, Myh(1-4) Del/+ and Myh(1-4) Del/Del TA by RT-qPCR experiments. (F) Quantification of Myh2, Myh1, Myh4, Myh8 and Myh13 mRNAs of adult wt, Myh(1-4) Inv/+ and Myh(1-4) Inv/Inv TA by RT-qPCR experiments. (G) Quantification of Myh2, Myh1, Myh4, Myh8 and Myh13 mRNAs of adult wt, Myh(1-4) Inv3’/+ and Myh(1-4) Inv3’/Inv3’ TA by RT-qPCR. For E, F, G (n=3). Numerical data are presented as mean ± S.E.M. * P

    Journal: bioRxiv

    Article Title: A fast Myh super enhancer dictates adult muscle fiber phenotype through competitive interactions with the fast Myh genes

    doi: 10.1101/2021.04.17.438406

    Figure Lengend Snippet: The promoters of f Myh genes compete for the shared SE (A) Schema of the distinct f Myh alleles generated by CRISPR/Cas9 editing. (B) Immunostaining against MYH4 (blue), MYH2 (green) and slow MYH7 (red) of adult distal hindlimb sections of wt, Myh(1-4) Del/Del , Myh(1-4) Inv/Inv and of Myh(1-4) Inv3’/Inv3’ mutants. (C) Immunostaining against neonatal MYH8 of adult leg sections in wt, Myh(1-4) Del/Del , Myh(1- 4) Inv/Inv and of Myh(1-4) Inv3’/Inv3’ . (D) Same as (C) against extraocular MYH13. (E) Quantification of Myh2, Myh1, Myh4, Myh8 and Myh13 mRNAs of adult wt, Myh(1-4) Del/+ and Myh(1-4) Del/Del TA by RT-qPCR experiments. (F) Quantification of Myh2, Myh1, Myh4, Myh8 and Myh13 mRNAs of adult wt, Myh(1-4) Inv/+ and Myh(1-4) Inv/Inv TA by RT-qPCR experiments. (G) Quantification of Myh2, Myh1, Myh4, Myh8 and Myh13 mRNAs of adult wt, Myh(1-4) Inv3’/+ and Myh(1-4) Inv3’/Inv3’ TA by RT-qPCR. For E, F, G (n=3). Numerical data are presented as mean ± S.E.M. * P

    Article Snippet: Mouse generation by CRISPR/Cas9 SgRNA and Cas9 purified protein were produced by the TACGENE platform.

    Techniques: Generated, CRISPR, Immunostaining, Quantitative RT-PCR

    Optimization of Cas9 nuclear targeting and delivery into B . cinerea protoplasts. (A-D) Subcellular localization of genetically delivered Cas9-GFP constructs fused to different NLS. Fluorescence microscopy images of 18 h old germlings on glass slides. Arrowheads depict nuclei. Only in (C) and (D), fluorescence is concentrated in the nuclei. Scale bars: 5 μm. (E) Transformation rates (NHEJ-mediated, Ipr R Bos1 k.o. transformants) obtained in B . cinerea with different Cas9 delivery strategies. Cas9 was expressed from a chromosomally integrated gene (Cas9-SV40 x4 -NLS; stable), transiently from a gene on a telomere vector (Cas9-GFP-SV40 x4 -NLS; transient) or added as a protein (Cas9-Stu x2 -NLS; RNP) together with Bos1-T2 sgRNA to B . cinerea protoplasts. The p values by one-way ANOVA followed by Tukey’s multiple comparisons post hoc test, relative to stable Cas9 expression, are indicated. * p ≤ 0.05; stable (n = 4), transient (n = 4), RNP (n = 11). (F) Comparison of different NLS arrangements on genome editing efficiency of Cas9-sgRNA RNPs targeting Bos1 . Values are relative to transformation rate with SV40-Cas9-SV40. In (E) and (F), no Ipr R colonies were obtained without Cas9. The p values by one-way ANOVA followed by Dunnett’s multiple comparisons post hoc test are indicated. *** p value ≤ 0.001; n = 4.

    Journal: PLoS Pathogens

    Article Title: CRISPR/Cas with ribonucleoprotein complexes and transiently selected telomere vectors allows highly efficient marker-free and multiple genome editing in Botrytis cinerea

    doi: 10.1371/journal.ppat.1008326

    Figure Lengend Snippet: Optimization of Cas9 nuclear targeting and delivery into B . cinerea protoplasts. (A-D) Subcellular localization of genetically delivered Cas9-GFP constructs fused to different NLS. Fluorescence microscopy images of 18 h old germlings on glass slides. Arrowheads depict nuclei. Only in (C) and (D), fluorescence is concentrated in the nuclei. Scale bars: 5 μm. (E) Transformation rates (NHEJ-mediated, Ipr R Bos1 k.o. transformants) obtained in B . cinerea with different Cas9 delivery strategies. Cas9 was expressed from a chromosomally integrated gene (Cas9-SV40 x4 -NLS; stable), transiently from a gene on a telomere vector (Cas9-GFP-SV40 x4 -NLS; transient) or added as a protein (Cas9-Stu x2 -NLS; RNP) together with Bos1-T2 sgRNA to B . cinerea protoplasts. The p values by one-way ANOVA followed by Tukey’s multiple comparisons post hoc test, relative to stable Cas9 expression, are indicated. * p ≤ 0.05; stable (n = 4), transient (n = 4), RNP (n = 11). (F) Comparison of different NLS arrangements on genome editing efficiency of Cas9-sgRNA RNPs targeting Bos1 . Values are relative to transformation rate with SV40-Cas9-SV40. In (E) and (F), no Ipr R colonies were obtained without Cas9. The p values by one-way ANOVA followed by Dunnett’s multiple comparisons post hoc test are indicated. *** p value ≤ 0.001; n = 4.

    Article Snippet: Cas9-NLS, containing N- and C-terminal SV40 NLS, was purchased from NEB.

    Techniques: Construct, Fluorescence, Microscopy, Transformation Assay, Non-Homologous End Joining, Plasmid Preparation, Expressing

    IRE1 facilitates migration of infected DCs in vivo . (A-B) IRE1 was depleted in bone marrow-derived DCs by CRISPR/Cas9 and loss of IRE1 expression was assayed by RT- qPCR and immunoblot analyses. (C) WT or ire1 (-) DCs were infected for 18 h and the percentage of infection was determined by counting the number of parasites in 100 cells. (D) Infected WT and ire1 (-) cells were inoculated into mice by i.p. injection (10 6 infected cells). At the indicated hpi, the spleen of each mouse was harvested, and the number of parasites was determined by PCR. ***p

    Journal: bioRxiv

    Article Title: Toxoplasma gondii co-opts the unfolded protein response to enhance migration and dissemination of infected host cells

    doi: 10.1101/2020.04.14.042069

    Figure Lengend Snippet: IRE1 facilitates migration of infected DCs in vivo . (A-B) IRE1 was depleted in bone marrow-derived DCs by CRISPR/Cas9 and loss of IRE1 expression was assayed by RT- qPCR and immunoblot analyses. (C) WT or ire1 (-) DCs were infected for 18 h and the percentage of infection was determined by counting the number of parasites in 100 cells. (D) Infected WT and ire1 (-) cells were inoculated into mice by i.p. injection (10 6 infected cells). At the indicated hpi, the spleen of each mouse was harvested, and the number of parasites was determined by PCR. ***p

    Article Snippet: Generation of IRE1 knockout cells Disruption of the IRE1 gene in MEF cells was carried out using the CRISPR/Cas9 method [ ].

    Techniques: Migration, Infection, In Vivo, Derivative Assay, CRISPR, Expressing, Quantitative RT-PCR, Mouse Assay, Injection, Polymerase Chain Reaction

    MEF cells were transfected with Cas9 bound to sgRNA targeted to IRE1 as described in the methods section; lowered IRE1 expression resulting from the CRISPR/Cas9 gene editing was evaluated by (A) RT-qPCR and (B) immunoblot analyses. GAPDH was included as a loading control for the immunoblot analyses. (C) WT and IRE1 -/- MEF cells were treated with 1 µM thapsigargin (TG), or no stress agent, for 6 h. Cells were then harvested and the XBP1s mRNA levels were measured by RT-qPCR. The values of XBP1s mRNAs were normalized to values of total XBP1 transcripts. (±SD, n=3) ***p

    Journal: bioRxiv

    Article Title: Toxoplasma gondii co-opts the unfolded protein response to enhance migration and dissemination of infected host cells

    doi: 10.1101/2020.04.14.042069

    Figure Lengend Snippet: MEF cells were transfected with Cas9 bound to sgRNA targeted to IRE1 as described in the methods section; lowered IRE1 expression resulting from the CRISPR/Cas9 gene editing was evaluated by (A) RT-qPCR and (B) immunoblot analyses. GAPDH was included as a loading control for the immunoblot analyses. (C) WT and IRE1 -/- MEF cells were treated with 1 µM thapsigargin (TG), or no stress agent, for 6 h. Cells were then harvested and the XBP1s mRNA levels were measured by RT-qPCR. The values of XBP1s mRNAs were normalized to values of total XBP1 transcripts. (±SD, n=3) ***p

    Article Snippet: Generation of IRE1 knockout cells Disruption of the IRE1 gene in MEF cells was carried out using the CRISPR/Cas9 method [ ].

    Techniques: Transfection, Expressing, CRISPR, Quantitative RT-PCR

    CRISPR/Cas9 knockout of P2Y1 receptor reduces intercellular calcium waves. (A-B) MA104-GCaMP cells infected with rotavirus (RV SA114F) at MOI 0.01 and mock- or treated with apyrase grade VI or grade VII, imaged ~3–25 hpi. (A) Ca 2+ spikes per RV-infected cell and neighboring (NB) +3 and +5 cells and (B) the average magnitude of Ca 2+ spikes/cell in RV-infected and NB cells (data combined from N =3 independent experiments). (C) Normalized GCaMP fluorescence increase in parental or P2Y1 knockout (KO) MA104-GCaMP cells treated with 1 nM ADP (n=27 cells, data combined from N =3 independent experiments, Mann-Whitney statistical test). (D) Parental or P2Y1 knockout MA104-GCaMP monolayers mock- or RV-infected, fixed at 24 hpi, and immunostained for RV antigen (pink). Scale bar = 100 μm. ( E ) Maximum % increase in GCaMP fluorescence of MA104-GCaMP cells (+/− 10 μM BPTU) after addition of supernatant from mock- or RV (SA114F)-infected parental (Par) or P2Y1 KO MA104-GCaMP cells (MOI 5, harvested ~5–6 hpi). (n≥210 cells per condition, data combined from N =3 independent experiments). (F) Representative Ca 2+ traces of parental or P2Y1 knockout cells infected with RV (SA11cl3-mRuby3). (G) Ca 2+ spikes/cell in RV-infected or NB+3 or NB+5 cell and (H) average magnitude of Ca 2+ spikes/cell (data combined from N =3 independent experiments). (A-B, E, G-H) Kruskal-Wallis with Dunn’s multiple comparisons test used. Data represented as mean ± SD, (*p

    Journal: Science (New York, N.Y.)

    Article Title: Rotavirus induces intercellular calcium waves through ADP signaling

    doi: 10.1126/science.abc3621

    Figure Lengend Snippet: CRISPR/Cas9 knockout of P2Y1 receptor reduces intercellular calcium waves. (A-B) MA104-GCaMP cells infected with rotavirus (RV SA114F) at MOI 0.01 and mock- or treated with apyrase grade VI or grade VII, imaged ~3–25 hpi. (A) Ca 2+ spikes per RV-infected cell and neighboring (NB) +3 and +5 cells and (B) the average magnitude of Ca 2+ spikes/cell in RV-infected and NB cells (data combined from N =3 independent experiments). (C) Normalized GCaMP fluorescence increase in parental or P2Y1 knockout (KO) MA104-GCaMP cells treated with 1 nM ADP (n=27 cells, data combined from N =3 independent experiments, Mann-Whitney statistical test). (D) Parental or P2Y1 knockout MA104-GCaMP monolayers mock- or RV-infected, fixed at 24 hpi, and immunostained for RV antigen (pink). Scale bar = 100 μm. ( E ) Maximum % increase in GCaMP fluorescence of MA104-GCaMP cells (+/− 10 μM BPTU) after addition of supernatant from mock- or RV (SA114F)-infected parental (Par) or P2Y1 KO MA104-GCaMP cells (MOI 5, harvested ~5–6 hpi). (n≥210 cells per condition, data combined from N =3 independent experiments). (F) Representative Ca 2+ traces of parental or P2Y1 knockout cells infected with RV (SA11cl3-mRuby3). (G) Ca 2+ spikes/cell in RV-infected or NB+3 or NB+5 cell and (H) average magnitude of Ca 2+ spikes/cell (data combined from N =3 independent experiments). (A-B, E, G-H) Kruskal-Wallis with Dunn’s multiple comparisons test used. Data represented as mean ± SD, (*p

    Article Snippet: MA104-GCaMP6s cells (pLVX-IRES-Hygro) were transduced with the P2Y1 CRISPR/Cas9-expressing construct and at 72 hrs post-transduction the cells were passaged in the presence of 50 μg/mL hygromycin B and 10 μg/mL puromycin to select for co-expression of GCaMP6s and CRISPR/Cas9.

    Techniques: CRISPR, Knock-Out, Infection, Fluorescence, MANN-WHITNEY

    CRISPR/Cas9 knockout of P2Y1 receptor in jHIEs reduces intercellular calcium waves. (A-B) Normalized GCaMP fluorescence increase in (A) parental or P2Y1 knockout (KO) (J2)HIE-GCaMP6s monolayers treated with 10 nM ADP (n=75 cells, data combined from N =3 independent experiments) and (B) parental or P2Y2 KO (J2)HIE-GCaMP6s monolayers treated with 10 μM ATP-γS (n=30 cells, data representative of N =3 independent experiments). (C-D) Parental or P2Y1 KO jHIE-GCaMP6s or parental or P2Y2 KO jHIE-GCaMP6s monolayers mock- or RV-infected, fixed at 24 hpi, and immunostained for RV antigen (pink) and counterstained with DAPI (gray). (E) Representative Ca 2+ traces per field-of-view (FOV) of mock- or RV-infected parental, P2Y1 KO jHIE-GCaMP6s, or P2Y2 KO jHIE-GCaMP6s monolayers. (F) Ca 2+ spikes/FOV and (G) average magnitude of Ca 2+ spikes/FOV. (A-B) Mann-Whitney test or (F-G) Kruskal-Wallis with Dunn’s multiple corrections test used. Scale bar = 50 μm. Data represented as mean ± SD, (***p

    Journal: Science (New York, N.Y.)

    Article Title: Rotavirus induces intercellular calcium waves through ADP signaling

    doi: 10.1126/science.abc3621

    Figure Lengend Snippet: CRISPR/Cas9 knockout of P2Y1 receptor in jHIEs reduces intercellular calcium waves. (A-B) Normalized GCaMP fluorescence increase in (A) parental or P2Y1 knockout (KO) (J2)HIE-GCaMP6s monolayers treated with 10 nM ADP (n=75 cells, data combined from N =3 independent experiments) and (B) parental or P2Y2 KO (J2)HIE-GCaMP6s monolayers treated with 10 μM ATP-γS (n=30 cells, data representative of N =3 independent experiments). (C-D) Parental or P2Y1 KO jHIE-GCaMP6s or parental or P2Y2 KO jHIE-GCaMP6s monolayers mock- or RV-infected, fixed at 24 hpi, and immunostained for RV antigen (pink) and counterstained with DAPI (gray). (E) Representative Ca 2+ traces per field-of-view (FOV) of mock- or RV-infected parental, P2Y1 KO jHIE-GCaMP6s, or P2Y2 KO jHIE-GCaMP6s monolayers. (F) Ca 2+ spikes/FOV and (G) average magnitude of Ca 2+ spikes/FOV. (A-B) Mann-Whitney test or (F-G) Kruskal-Wallis with Dunn’s multiple corrections test used. Scale bar = 50 μm. Data represented as mean ± SD, (***p

    Article Snippet: MA104-GCaMP6s cells (pLVX-IRES-Hygro) were transduced with the P2Y1 CRISPR/Cas9-expressing construct and at 72 hrs post-transduction the cells were passaged in the presence of 50 μg/mL hygromycin B and 10 μg/mL puromycin to select for co-expression of GCaMP6s and CRISPR/Cas9.

    Techniques: CRISPR, Knock-Out, Fluorescence, Infection, MANN-WHITNEY