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  1. Article: Gene editing of pigs to control influenza A virus infections.

    Kwon, Taeyong / Artiaga, Bianca L / McDowell, Chester D / Whitworth, Kristin M / Wells, Kevin D / Prather, Randall S / Delhon, Gustavo / Cigan, Mark / White, Stephen N / Retallick, Jamie / Gaudreault, Natasha N / Morozov, Igor / Richt, Juergen A

    bioRxiv : the preprint server for biology

    2024  

    Abstract: Proteolytic activation of the hemagglutinin (HA) glycoprotein by host cellular proteases is pivotal for influenza A virus (IAV) infectivity. Highly pathogenic avian influenza viruses possess the multibasic cleavage site of the HA which is cleaved by ... ...

    Abstract Proteolytic activation of the hemagglutinin (HA) glycoprotein by host cellular proteases is pivotal for influenza A virus (IAV) infectivity. Highly pathogenic avian influenza viruses possess the multibasic cleavage site of the HA which is cleaved by ubiquitous proteases, such as furin; in contrast, the monobasic HA motif is recognized and activated by trypsin-like proteases, such as the transmembrane serine protease 2 (TMPRSS2). Here, we aimed to determine the effects of TMPRSS2 on the replication of pandemic H1N1 and H3N2 subtype IAVs in the natural host, the pig. The use of the CRISPR/Cas 9 system led to the establishment of homozygous gene edited (GE)
    Language English
    Publishing date 2024-01-16
    Publishing country United States
    Document type Preprint
    DOI 10.1101/2024.01.15.575771
    Database MEDical Literature Analysis and Retrieval System OnLINE

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  2. Article ; Online: Author Correction: Mapping the genomic landscape of CRISPR-Cas9 cleavage.

    Cameron, Peter / Fuller, Chris K / Donohoue, Paul D / Jones, Brittnee N / Thompson, Matthew S / Carter, Matthew M / Gradia, Scott / Vidal, Bastien / Garner, Elizabeth / Slorach, Euan M / Lau, Elaine / Banh, Lynda M / Lied, Alexandra M / Edwards, Leslie S / Settle, Alexander H / Capurso, Daniel / Llaca, Victor / Deschamps, Stéphane / Cigan, Mark /
    Young, Joshua K / May, Andrew P

    Nature methods

    2023  Volume 20, Issue 12, Page(s) 2068

    Language English
    Publishing date 2023-11-09
    Publishing country United States
    Document type Published Erratum
    ZDB-ID 2169522-2
    ISSN 1548-7105 ; 1548-7091
    ISSN (online) 1548-7105
    ISSN 1548-7091
    DOI 10.1038/s41592-023-02114-4
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  3. Article: Rapid characterization of CRISPR-Cas9 protospacer adjacent motif sequence elements

    Karvelis, Tautvydas / Gasiunas, Giedrius / Young, Joshua / Bigelyte, Greta / Silanskas, Arunas / Cigan, Mark / Siksnys, Virginijus

    Genome biology. 2015 Dec., v. 16, no. 1

    2015  

    Abstract: To expand the repertoire of Cas9s available for genome targeting, we present a new in vitro method for the simultaneous examination of guide RNA and protospacer adjacent motif (PAM) requirements. The method relies on the in vitro cleavage of plasmid ... ...

    Abstract To expand the repertoire of Cas9s available for genome targeting, we present a new in vitro method for the simultaneous examination of guide RNA and protospacer adjacent motif (PAM) requirements. The method relies on the in vitro cleavage of plasmid libraries containing a randomized PAM as a function of Cas9-guide RNA complex concentration. Using this method, we accurately reproduce the canonical PAM preferences for Streptococcus pyogenes, Streptococcus thermophilus CRISPR3 (Sth3), and CRISPR1 (Sth1). Additionally, PAM and sgRNA solutions for a novel Cas9 protein from Brevibacillus laterosporus are provided by the assay and are demonstrated to support functional activity in vitro and in plants.
    Keywords Brevibacillus laterosporus ; RNA ; Streptococcus pyogenes ; Streptococcus thermophilus ; genome ; in vitro studies ; plasmids
    Language English
    Dates of publication 2015-12
    Size p. 253.
    Publishing place BioMed Central
    Document type Article
    ZDB-ID 2040529-7
    ISSN 1474-760X ; 1465-6906
    ISSN (online) 1474-760X
    ISSN 1465-6906
    DOI 10.1186/s13059-015-0818-7
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  4. Article ; Online: Rapid characterization of CRISPR-Cas9 protospacer adjacent motif sequence elements.

    Karvelis, Tautvydas / Gasiunas, Giedrius / Young, Joshua / Bigelyte, Greta / Silanskas, Arunas / Cigan, Mark / Siksnys, Virginijus

    Genome biology

    2015  Volume 16, Page(s) 253

    Abstract: To expand the repertoire of Cas9s available for genome targeting, we present a new in vitro method for the simultaneous examination of guide RNA and protospacer adjacent motif (PAM) requirements. The method relies on the in vitro cleavage of plasmid ... ...

    Abstract To expand the repertoire of Cas9s available for genome targeting, we present a new in vitro method for the simultaneous examination of guide RNA and protospacer adjacent motif (PAM) requirements. The method relies on the in vitro cleavage of plasmid libraries containing a randomized PAM as a function of Cas9-guide RNA complex concentration. Using this method, we accurately reproduce the canonical PAM preferences for Streptococcus pyogenes, Streptococcus thermophilus CRISPR3 (Sth3), and CRISPR1 (Sth1). Additionally, PAM and sgRNA solutions for a novel Cas9 protein from Brevibacillus laterosporus are provided by the assay and are demonstrated to support functional activity in vitro and in plants.
    MeSH term(s) Bacterial Proteins/genetics ; Bacterial Proteins/metabolism ; Base Sequence ; Brevibacillus/enzymology ; Brevibacillus/genetics ; CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; Endonucleases/genetics ; Endonucleases/metabolism ; Escherichia coli/genetics ; Gene Targeting/methods ; Molecular Sequence Data ; RNA, Guide, CRISPR-Cas Systems/genetics ; Streptococcus/enzymology ; Streptococcus/genetics
    Chemical Substances Bacterial Proteins ; RNA, Guide, CRISPR-Cas Systems ; Endonucleases (EC 3.1.-)
    Language English
    Publishing date 2015-11-19
    Publishing country England
    Document type Journal Article ; Research Support, Non-U.S. Gov't
    ZDB-ID 2040529-7
    ISSN 1474-760X ; 1474-760X
    ISSN (online) 1474-760X
    ISSN 1474-760X
    DOI 10.1186/s13059-015-0818-7
    Database MEDical Literature Analysis and Retrieval System OnLINE

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  5. Article ; Online: The use of cells from ANPEP knockout pigs to evaluate the role of aminopeptidase N (APN) as a receptor for porcine deltacoronavirus (PDCoV).

    Stoian, Ana / Rowland, Raymond R R / Petrovan, Vlad / Sheahan, Maureen / Samuel, Melissa S / Whitworth, Kristin M / Wells, Kevin D / Zhang, Jianqiang / Beaton, Benjamin / Cigan, Mark / Prather, Randall S

    Virology

    2019  Volume 541, Page(s) 136–140

    Abstract: The coronaviruses, porcine epidemic diarrhea virus (PEDV), transmissible gastroenteritis virus (TGEV), and porcine deltacoronavirus (PDCoV) represent important sources of neonatal diarrhea on pig farms. The requirement for aminopeptidase N (APN) as a ... ...

    Abstract The coronaviruses, porcine epidemic diarrhea virus (PEDV), transmissible gastroenteritis virus (TGEV), and porcine deltacoronavirus (PDCoV) represent important sources of neonatal diarrhea on pig farms. The requirement for aminopeptidase N (APN) as a receptor for TGEV, but not for PEDV, is well established. In this study, the biological relevance of APN as a receptor for PDCoV was tested by using CRISPR/Cas9 to knockout the APN gene, ANPEP, in pigs. Porcine alveolar macrophages (PAMs) from ANPEP knockout (KO) pigs showed resistance to PDCoV infection. However, lung fibroblast-like cells, derived from the ANPEP KO PAM cultures, supported PDCoV infection to high levels. The results suggest that APN is a receptor for PDCoV in PAMs but is not necessary for infection of lung-derived fibroblast cells. The infection of the ANPEP KO pigs with PDCoV further confirmed that APN is dispensable as a receptor for PDCoV.
    MeSH term(s) Animals ; CD13 Antigens/genetics ; CD13 Antigens/physiology ; Coronavirus Infections/etiology ; Gastroenteritis, Transmissible, of Swine/etiology ; Gene Knockout Techniques ; Porcine epidemic diarrhea virus/physiology ; Receptors, Virus/physiology ; Swine ; Swine Diseases/etiology
    Chemical Substances Receptors, Virus ; CD13 Antigens (EC 3.4.11.2)
    Keywords covid19
    Language English
    Publishing date 2019-12-24
    Publishing country United States
    Document type Journal Article ; Research Support, Non-U.S. Gov't
    ZDB-ID 200425-2
    ISSN 1096-0341 ; 0042-6822
    ISSN (online) 1096-0341
    ISSN 0042-6822
    DOI 10.1016/j.virol.2019.12.007
    Database MEDical Literature Analysis and Retrieval System OnLINE

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  6. Article ; Online: In Silico and Fluorescence In Situ Hybridization Mapping Reveals Collinearity between the Pennisetum squamulatum Apomixis Carrier-Chromosome and Chromosome 2 of Sorghum and Foxtail Millet.

    Sapkota, Sirjan / Conner, Joann A / Hanna, Wayne W / Simon, Bindu / Fengler, Kevin / Deschamps, Stéphane / Cigan, Mark / Ozias-Akins, Peggy

    PloS one

    2016  Volume 11, Issue 3, Page(s) e0152411

    Abstract: Apomixis, or clonal propagation through seed, is a trait identified within multiple species of the grass family (Poaceae). The genetic locus controlling apomixis in Pennisetum squamulatum (syn Cenchrus squamulatus) and Cenchrus ciliaris (syn Pennisetum ... ...

    Abstract Apomixis, or clonal propagation through seed, is a trait identified within multiple species of the grass family (Poaceae). The genetic locus controlling apomixis in Pennisetum squamulatum (syn Cenchrus squamulatus) and Cenchrus ciliaris (syn Pennisetum ciliare, buffelgrass) is the apospory-specific genomic region (ASGR). Previously, the ASGR was shown to be highly conserved but inverted in marker order between P. squamulatum and C. ciliaris based on fluorescence in situ hybridization (FISH) and varied in both karyotype and position of the ASGR on the ASGR-carrier chromosome among other apomictic Cenchrus/Pennisetum species. Using in silico transcript mapping and verification of physical positions of some of the transcripts via FISH, we discovered that the ASGR-carrier chromosome from P. squamulatum is collinear with chromosome 2 of foxtail millet and sorghum outside of the ASGR. The in silico ordering of the ASGR-carrier chromosome markers, previously unmapped in P. squamulatum, allowed for the identification of a backcross line with structural changes to the P. squamulatum ASGR-carrier chromosome derived from gamma irradiated pollen.
    MeSH term(s) Apomixis/genetics ; Chromosomes, Artificial, Bacterial ; Chromosomes, Plant ; Contig Mapping ; Genetic Linkage ; In Situ Hybridization, Fluorescence ; Pennisetum/genetics ; Setaria Plant/genetics ; Sorghum/genetics
    Language English
    Publishing date 2016-03-31
    Publishing country United States
    Document type Journal Article ; Research Support, Non-U.S. Gov't
    ISSN 1932-6203
    ISSN (online) 1932-6203
    DOI 10.1371/journal.pone.0152411
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  7. Article ; Online: A catalogue of biochemically diverse CRISPR-Cas9 orthologs.

    Gasiunas, Giedrius / Young, Joshua K / Karvelis, Tautvydas / Kazlauskas, Darius / Urbaitis, Tomas / Jasnauskaite, Monika / Grusyte, Mantvyda M / Paulraj, Sushmitha / Wang, Po-Hao / Hou, Zhenglin / Dooley, Shane K / Cigan, Mark / Alarcon, Clara / Chilcoat, N Doane / Bigelyte, Greta / Curcuru, Jennifer L / Mabuchi, Megumu / Sun, Zhiyi / Fuchs, Ryan T /
    Schildkraut, Ezra / Weigele, Peter R / Jack, William E / Robb, G Brett / Venclovas, Česlovas / Siksnys, Virginijus

    Nature communications

    2020  Volume 11, Issue 1, Page(s) 5512

    Abstract: Bacterial Cas9 nucleases from type II CRISPR-Cas antiviral defence systems have been repurposed as genome editing tools. Although these proteins are found in many microbes, only a handful of variants are used for these applications. Here, we use ... ...

    Abstract Bacterial Cas9 nucleases from type II CRISPR-Cas antiviral defence systems have been repurposed as genome editing tools. Although these proteins are found in many microbes, only a handful of variants are used for these applications. Here, we use bioinformatic and biochemical analyses to explore this largely uncharacterized diversity. We apply cell-free biochemical screens to assess the protospacer adjacent motif (PAM) and guide RNA (gRNA) requirements of 79 Cas9 proteins, thus identifying at least 7 distinct gRNA classes and 50 different PAM sequence requirements. PAM recognition spans the entire spectrum of T-, A-, C-, and G-rich nucleotides, from single nucleotide recognition to sequence strings longer than 4 nucleotides. Characterization of a subset of Cas9 orthologs using purified components reveals additional biochemical diversity, including both narrow and broad ranges of temperature dependence, staggered-end DNA target cleavage, and a requirement for long stretches of homology between gRNA and DNA target. Our results expand the available toolset of RNA-programmable CRISPR-associated nucleases.
    MeSH term(s) Base Sequence ; CRISPR-Associated Protein 9/genetics ; CRISPR-Associated Protein 9/metabolism ; CRISPR-Cas Systems/genetics ; Computational Biology ; DNA Cleavage ; Gene Editing/methods ; RNA, Guide, CRISPR-Cas Systems/genetics ; RNA, Guide, CRISPR-Cas Systems/metabolism ; Sequence Homology, Nucleic Acid
    Chemical Substances RNA, Guide, CRISPR-Cas Systems ; CRISPR-Associated Protein 9 (EC 3.1.-)
    Language English
    Publishing date 2020-11-02
    Publishing country England
    Document type Journal Article
    ZDB-ID 2553671-0
    ISSN 2041-1723 ; 2041-1723
    ISSN (online) 2041-1723
    ISSN 2041-1723
    DOI 10.1038/s41467-020-19344-1
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  8. Article ; Online: Mapping the genomic landscape of CRISPR-Cas9 cleavage.

    Cameron, Peter / Fuller, Chris K / Donohoue, Paul D / Jones, Brittnee N / Thompson, Matthew S / Carter, Matthew M / Gradia, Scott / Vidal, Bastien / Garner, Elizabeth / Slorach, Euan M / Lau, Elaine / Banh, Lynda M / Lied, Alexandra M / Edwards, Leslie S / Settle, Alexander H / Capurso, Daniel / Llaca, Victor / Deschamps, Stéphane / Cigan, Mark /
    Young, Joshua K / May, Andrew P

    Nature methods

    2017  Volume 14, Issue 6, Page(s) 600–606

    Abstract: RNA-guided CRISPR-Cas9 endonucleases are widely used for genome engineering, but our understanding of Cas9 specificity remains incomplete. Here, we developed a biochemical method (SITE-Seq), using Cas9 programmed with single-guide RNAs (sgRNAs), to ... ...

    Abstract RNA-guided CRISPR-Cas9 endonucleases are widely used for genome engineering, but our understanding of Cas9 specificity remains incomplete. Here, we developed a biochemical method (SITE-Seq), using Cas9 programmed with single-guide RNAs (sgRNAs), to identify the sequence of cut sites within genomic DNA. Cells edited with the same Cas9-sgRNA complexes are then assayed for mutations at each cut site using amplicon sequencing. We used SITE-Seq to examine Cas9 specificity with sgRNAs targeting the human genome. The number of sites identified depended on sgRNA sequence and nuclease concentration. Sites identified at lower concentrations showed a higher propensity for off-target mutations in cells. The list of off-target sites showing activity in cells was influenced by sgRNP delivery, cell type and duration of exposure to the nuclease. Collectively, our results underscore the utility of combining comprehensive biochemical identification of off-target sites with independent cell-based measurements of activity at those sites when assessing nuclease activity and specificity.
    MeSH term(s) CRISPR-Cas Systems/genetics ; Chromosome Mapping/methods ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Genome/genetics ; High-Throughput Nucleotide Sequencing ; Sequence Analysis, DNA
    Language English
    Publishing date 2017-05-01
    Publishing country United States
    Document type Journal Article
    ZDB-ID 2169522-2
    ISSN 1548-7105 ; 1548-7091
    ISSN (online) 1548-7105
    ISSN 1548-7091
    DOI 10.1038/nmeth.4284
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