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  • 99
    Thermo Fisher bsmbi
    Targeted mutagenesis using the CRISPR/Cas9 system in transient expression in N. benthamiana. a Schematic representation of the structure of Niben101Scf04205Ctg025 (XT1) and Niben101Scf04551Ctg021 (XT2) (exons in grey , introns in white ) with the sequences of the target sites. Diagnostic restriction sites are underlined and the PAM sequence is shown in bold . b Comparison of the mutation efficiency of hCas9 and pcoCas9 targeting the XT2. Red arrow shows SpeI resistant PCR fragments only visible on the gRNA and hCas9 combination. c PCR/RE assay to detect simultaneous targeted mutations on XT1 and XT2. Red arrows show <t>BsmBI</t> and SpeI resistant PCR fragments amplified from N. benthamiana genomic <t>DNA.</t> d Alignment of XT1 and XT2 sequences obtained from different clones of uncleaved bands (see c ). XT1 target site appears in blue and XT2 target site in green . Red letters and dashes indicate insertions and deletions respectively
    Bsmbi, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 376 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher esp3i sites
    Golden Gate assembly of custom TAL effector and TALEN constructs using module, array, last repeat and backbone plasmids. By using the type IIS restriction endonucleases BsaI and <t>Esp3I,</t> modules containing the desired RVDs can be released with unique cohesive ends for ordered, single-reaction assembly into array plasmids in a first step, and those arrays subsequently released and assembled in order in a second step into a backbone plasmid to create full length constructs with custom repeat arrays (see text for details). NLS, nuclear localization signal(s); AD, transcriptional activation domain; tet , tetracycline resistance; spec , spectinomycin resistance; amp , ampicillin resistance; attL1 and attL2, recombination sites for Gateway cloning; B, BamHI, and S, SphI, useful for subcloning custom repeat arrays. Unique restriction enzyme sites flanking the coding sequences, useful for subcloning the entire constructs into other vectors, are not shown but can be found in the sequence files ( Supplementary Data ).
    Esp3i Sites, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher fastdigest bsmbi
    Golden Gate assembly of custom TAL effector and TALEN constructs using module, array, last repeat and backbone plasmids. By using the type IIS restriction endonucleases BsaI and <t>Esp3I,</t> modules containing the desired RVDs can be released with unique cohesive ends for ordered, single-reaction assembly into array plasmids in a first step, and those arrays subsequently released and assembled in order in a second step into a backbone plasmid to create full length constructs with custom repeat arrays (see text for details). NLS, nuclear localization signal(s); AD, transcriptional activation domain; tet , tetracycline resistance; spec , spectinomycin resistance; amp , ampicillin resistance; attL1 and attL2, recombination sites for Gateway cloning; B, BamHI, and S, SphI, useful for subcloning custom repeat arrays. Unique restriction enzyme sites flanking the coding sequences, useful for subcloning the entire constructs into other vectors, are not shown but can be found in the sequence files ( Supplementary Data ).
    Fastdigest Bsmbi, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 93/100, based on 100 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    87
    Thermo Fisher bsmbi restriction enzyme
    Golden Gate assembly of custom TAL effector and TALEN constructs using module, array, last repeat and backbone plasmids. By using the type IIS restriction endonucleases BsaI and <t>Esp3I,</t> modules containing the desired RVDs can be released with unique cohesive ends for ordered, single-reaction assembly into array plasmids in a first step, and those arrays subsequently released and assembled in order in a second step into a backbone plasmid to create full length constructs with custom repeat arrays (see text for details). NLS, nuclear localization signal(s); AD, transcriptional activation domain; tet , tetracycline resistance; spec , spectinomycin resistance; amp , ampicillin resistance; attL1 and attL2, recombination sites for Gateway cloning; B, BamHI, and S, SphI, useful for subcloning custom repeat arrays. Unique restriction enzyme sites flanking the coding sequences, useful for subcloning the entire constructs into other vectors, are not shown but can be found in the sequence files ( Supplementary Data ).
    Bsmbi Restriction Enzyme, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 87/100, based on 31 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher fastdigest esp3i
    Golden Gate assembly of custom TAL effector and TALEN constructs using module, array, last repeat and backbone plasmids. By using the type IIS restriction endonucleases BsaI and <t>Esp3I,</t> modules containing the desired RVDs can be released with unique cohesive ends for ordered, single-reaction assembly into array plasmids in a first step, and those arrays subsequently released and assembled in order in a second step into a backbone plasmid to create full length constructs with custom repeat arrays (see text for details). NLS, nuclear localization signal(s); AD, transcriptional activation domain; tet , tetracycline resistance; spec , spectinomycin resistance; amp , ampicillin resistance; attL1 and attL2, recombination sites for Gateway cloning; B, BamHI, and S, SphI, useful for subcloning custom repeat arrays. Unique restriction enzyme sites flanking the coding sequences, useful for subcloning the entire constructs into other vectors, are not shown but can be found in the sequence files ( Supplementary Data ).
    Fastdigest Esp3i, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 91/100, based on 62 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher fastdigest bsmbi restriction enzyme
    Golden Gate assembly of custom TAL effector and TALEN constructs using module, array, last repeat and backbone plasmids. By using the type IIS restriction endonucleases BsaI and <t>Esp3I,</t> modules containing the desired RVDs can be released with unique cohesive ends for ordered, single-reaction assembly into array plasmids in a first step, and those arrays subsequently released and assembled in order in a second step into a backbone plasmid to create full length constructs with custom repeat arrays (see text for details). NLS, nuclear localization signal(s); AD, transcriptional activation domain; tet , tetracycline resistance; spec , spectinomycin resistance; amp , ampicillin resistance; attL1 and attL2, recombination sites for Gateway cloning; B, BamHI, and S, SphI, useful for subcloning custom repeat arrays. Unique restriction enzyme sites flanking the coding sequences, useful for subcloning the entire constructs into other vectors, are not shown but can be found in the sequence files ( Supplementary Data ).
    Fastdigest Bsmbi Restriction Enzyme, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 91/100, based on 18 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher type ii restriction enzyme bsmbi
    Golden Gate assembly of custom TAL effector and TALEN constructs using module, array, last repeat and backbone plasmids. By using the type IIS restriction endonucleases BsaI and <t>Esp3I,</t> modules containing the desired RVDs can be released with unique cohesive ends for ordered, single-reaction assembly into array plasmids in a first step, and those arrays subsequently released and assembled in order in a second step into a backbone plasmid to create full length constructs with custom repeat arrays (see text for details). NLS, nuclear localization signal(s); AD, transcriptional activation domain; tet , tetracycline resistance; spec , spectinomycin resistance; amp , ampicillin resistance; attL1 and attL2, recombination sites for Gateway cloning; B, BamHI, and S, SphI, useful for subcloning custom repeat arrays. Unique restriction enzyme sites flanking the coding sequences, useful for subcloning the entire constructs into other vectors, are not shown but can be found in the sequence files ( Supplementary Data ).
    Type Ii Restriction Enzyme Bsmbi, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 77/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    Thermo Fisher bsmb1
    CXCR4 gene silencing by lentivirus mediated CRISPR/SaCas9 delivery protects GHOST-X4 cells from HIV-1 infection. a A schematic diagram of lentiviral transfer vectors containing CRISPR/SaCas9 components. LentiCRISPR v2 plasmid was modified by replacing the SpCas9 with SaCas9. Then, based on the SaCas9 PAM sequence 5′-NNGRRT-3′, we designed, synthesized and cloned the CXCR4 sgRNAs into the vector using the <t>Bsmb1</t> . b CXCR4 gene disruption analysis in GHOST-X4 cells by the T7E1 cleavage assay. GHOST-X4 cells were transduced with lentiviruses (MOI of 40) in the presence of polybrene for 12 h, and the genomic DNA was extracted and used as template to amplify a CXCR4 fragment (1100 bp). Con: lentiviral vectors expressing SaCas9 only, #8 and #9: lentiviral vectors expressing SaCas9/sgRNA #8 and #9. c Flow cytometry analysis of CXCR4 expression in lentivirus transduced GHOST-X4 cells. Neg, unstained cells. Con, #8 and #9 as samples described in ( b ) stained with anti-CXCR4-PE. d DNA sequences of CXCR4 of the transduced GHOST-X4 cells. PCR products were cloned into pGEM-T Easy vector and sequenced. The PAM sequences are lined and highlighted in red; the target sequences were shown in blue; deletions are indicated with (−) and insertions with (+). N/N indicates ratio of WT or mutations to total sequenced clones. e Flow cytometry analysis of transduced GHOST-X4 cells on GFP expression 3 days post HIV-1 NL4-3 infection. Neg, no HIV-1 infection. f HIV-1 NL4-3 infection was determined by p24 level. The cultured supernatants were collected at the indicated days post infection, and HIV-1 p24 level was determined using a p24 ELISA kit. For ( c and e ), one representative out of three independent experiments is shown. For ( f ), the graph represents 3 independent infection experiments and error bars represent SEM. Statistical analysis determined using unpaired t-test (*** P
    Bsmb1, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 95/100, based on 53 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher fastdigest bsmb1
    CXCR4 gene silencing by lentivirus mediated CRISPR/SaCas9 delivery protects GHOST-X4 cells from HIV-1 infection. a A schematic diagram of lentiviral transfer vectors containing CRISPR/SaCas9 components. LentiCRISPR v2 plasmid was modified by replacing the SpCas9 with SaCas9. Then, based on the SaCas9 PAM sequence 5′-NNGRRT-3′, we designed, synthesized and cloned the CXCR4 sgRNAs into the vector using the <t>Bsmb1</t> . b CXCR4 gene disruption analysis in GHOST-X4 cells by the T7E1 cleavage assay. GHOST-X4 cells were transduced with lentiviruses (MOI of 40) in the presence of polybrene for 12 h, and the genomic DNA was extracted and used as template to amplify a CXCR4 fragment (1100 bp). Con: lentiviral vectors expressing SaCas9 only, #8 and #9: lentiviral vectors expressing SaCas9/sgRNA #8 and #9. c Flow cytometry analysis of CXCR4 expression in lentivirus transduced GHOST-X4 cells. Neg, unstained cells. Con, #8 and #9 as samples described in ( b ) stained with anti-CXCR4-PE. d DNA sequences of CXCR4 of the transduced GHOST-X4 cells. PCR products were cloned into pGEM-T Easy vector and sequenced. The PAM sequences are lined and highlighted in red; the target sequences were shown in blue; deletions are indicated with (−) and insertions with (+). N/N indicates ratio of WT or mutations to total sequenced clones. e Flow cytometry analysis of transduced GHOST-X4 cells on GFP expression 3 days post HIV-1 NL4-3 infection. Neg, no HIV-1 infection. f HIV-1 NL4-3 infection was determined by p24 level. The cultured supernatants were collected at the indicated days post infection, and HIV-1 p24 level was determined using a p24 ELISA kit. For ( c and e ), one representative out of three independent experiments is shown. For ( f ), the graph represents 3 independent infection experiments and error bars represent SEM. Statistical analysis determined using unpaired t-test (*** P
    Fastdigest Bsmb1, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 82/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    80
    Thermo Fisher bsmbi fast digest
    CXCR4 gene silencing by lentivirus mediated CRISPR/SaCas9 delivery protects GHOST-X4 cells from HIV-1 infection. a A schematic diagram of lentiviral transfer vectors containing CRISPR/SaCas9 components. LentiCRISPR v2 plasmid was modified by replacing the SpCas9 with SaCas9. Then, based on the SaCas9 PAM sequence 5′-NNGRRT-3′, we designed, synthesized and cloned the CXCR4 sgRNAs into the vector using the <t>Bsmb1</t> . b CXCR4 gene disruption analysis in GHOST-X4 cells by the T7E1 cleavage assay. GHOST-X4 cells were transduced with lentiviruses (MOI of 40) in the presence of polybrene for 12 h, and the genomic DNA was extracted and used as template to amplify a CXCR4 fragment (1100 bp). Con: lentiviral vectors expressing SaCas9 only, #8 and #9: lentiviral vectors expressing SaCas9/sgRNA #8 and #9. c Flow cytometry analysis of CXCR4 expression in lentivirus transduced GHOST-X4 cells. Neg, unstained cells. Con, #8 and #9 as samples described in ( b ) stained with anti-CXCR4-PE. d DNA sequences of CXCR4 of the transduced GHOST-X4 cells. PCR products were cloned into pGEM-T Easy vector and sequenced. The PAM sequences are lined and highlighted in red; the target sequences were shown in blue; deletions are indicated with (−) and insertions with (+). N/N indicates ratio of WT or mutations to total sequenced clones. e Flow cytometry analysis of transduced GHOST-X4 cells on GFP expression 3 days post HIV-1 NL4-3 infection. Neg, no HIV-1 infection. f HIV-1 NL4-3 infection was determined by p24 level. The cultured supernatants were collected at the indicated days post infection, and HIV-1 p24 level was determined using a p24 ELISA kit. For ( c and e ), one representative out of three independent experiments is shown. For ( f ), the graph represents 3 independent infection experiments and error bars represent SEM. Statistical analysis determined using unpaired t-test (*** P
    Bsmbi Fast Digest, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 80/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher bsmbi digestion enzyme
    CRISPR vector design for knockout of A 2A R in K562 Cells. ( a ) Map of human chromosomes, with chromosome 22q11.23 highlighted, showing the location of the A 2A R gene. Additional arrowheads indicate genes with similar regions to the A 2A R gene, including the A 2B R gene on chromosome 17. ( b ) Left, guide-RNAs found for the A 2A R gene. Right, guide sequence #1 was selected for insertion into the <t>plentiCRISPR</t> loxP vector plasmid DNA. <t>BsmBI</t> cut sites were added to gRNA #1 to facilitate insertion. ( c ) Top, Cas9 cut site in the A 2A R gene. Bottom, map of the plentiCRISPR construct. ( d ) T7 E1 INDEL assay for CRISPR targeting efficiency. Lanes 1 and 5, universal marker. Lane 2, PCR product. Lanes 3 and 4, PCR product after denaturing and renaturing, without (3) and with (4) T7 EI enzyme. Arrowheads indicate T7 E1 cutting.
    Bsmbi Digestion Enzyme, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 80/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Targeted mutagenesis using the CRISPR/Cas9 system in transient expression in N. benthamiana. a Schematic representation of the structure of Niben101Scf04205Ctg025 (XT1) and Niben101Scf04551Ctg021 (XT2) (exons in grey , introns in white ) with the sequences of the target sites. Diagnostic restriction sites are underlined and the PAM sequence is shown in bold . b Comparison of the mutation efficiency of hCas9 and pcoCas9 targeting the XT2. Red arrow shows SpeI resistant PCR fragments only visible on the gRNA and hCas9 combination. c PCR/RE assay to detect simultaneous targeted mutations on XT1 and XT2. Red arrows show BsmBI and SpeI resistant PCR fragments amplified from N. benthamiana genomic DNA. d Alignment of XT1 and XT2 sequences obtained from different clones of uncleaved bands (see c ). XT1 target site appears in blue and XT2 target site in green . Red letters and dashes indicate insertions and deletions respectively

    Journal: Plant Methods

    Article Title: A modular toolbox for gRNA–Cas9 genome engineering in plants based on the GoldenBraid standard

    doi: 10.1186/s13007-016-0101-2

    Figure Lengend Snippet: Targeted mutagenesis using the CRISPR/Cas9 system in transient expression in N. benthamiana. a Schematic representation of the structure of Niben101Scf04205Ctg025 (XT1) and Niben101Scf04551Ctg021 (XT2) (exons in grey , introns in white ) with the sequences of the target sites. Diagnostic restriction sites are underlined and the PAM sequence is shown in bold . b Comparison of the mutation efficiency of hCas9 and pcoCas9 targeting the XT2. Red arrow shows SpeI resistant PCR fragments only visible on the gRNA and hCas9 combination. c PCR/RE assay to detect simultaneous targeted mutations on XT1 and XT2. Red arrows show BsmBI and SpeI resistant PCR fragments amplified from N. benthamiana genomic DNA. d Alignment of XT1 and XT2 sequences obtained from different clones of uncleaved bands (see c ). XT1 target site appears in blue and XT2 target site in green . Red letters and dashes indicate insertions and deletions respectively

    Article Snippet: The resulting PCR products were purified with the QIAquick PCR purification kit (QIAGEN) following the manufacturer’s protocol and restriction reactions were set up with 500 ng of purified DNA and the corresponding restriction enzyme; BsmBI (Fermentas) for XT1 and SpeI (Fermentas) for XT2.

    Techniques: Mutagenesis, CRISPR, Expressing, Diagnostic Assay, Sequencing, Polymerase Chain Reaction, Amplification, Clone Assay

    Cpf1–Flip–Cre-inducible sequential mutagenesis by a single crRNA FlipArray. a Schematic of vectors used in the study. The Cpf1-Flip construct contains an EFS promoter driving expression of Cpf1 and puromycin resistance, and a U6 expression cassette containing two inverted BsmbI restriction sites, flanked by a lox66 sequence and an inverted lox71 sequence. After BsmbI digestion, a crRNA FlipArray is cloned in. The FlipArray inverts upon Cre recombination, thereby switching the crRNA that is expressed. b Schematic of experimental design. Cells were first infected with lentivirus containing EFS-Cpf1-puro; U6-FlipArray. After 7 days, cells were then infected with lentivirus containing EFS-Cre to induce inversion of the FlipArray. Prior to Cre recombination, only crNf1 is expressed; following Cre recombination, crPten becomes expressed. c Sequences of the FlipArray construct before and after Cre recombination. Red boxes denote mutants from wild-type loxP . Prior to Cre, single mutant lox66 and lox71 sites are present. After Cre recombination, a wild-type loxP site and a double mutant lox72 site are generated

    Journal: Nature Communications

    Article Title: Programmable sequential mutagenesis by inducible Cpf1 crRNA array inversion

    doi: 10.1038/s41467-018-04158-z

    Figure Lengend Snippet: Cpf1–Flip–Cre-inducible sequential mutagenesis by a single crRNA FlipArray. a Schematic of vectors used in the study. The Cpf1-Flip construct contains an EFS promoter driving expression of Cpf1 and puromycin resistance, and a U6 expression cassette containing two inverted BsmbI restriction sites, flanked by a lox66 sequence and an inverted lox71 sequence. After BsmbI digestion, a crRNA FlipArray is cloned in. The FlipArray inverts upon Cre recombination, thereby switching the crRNA that is expressed. b Schematic of experimental design. Cells were first infected with lentivirus containing EFS-Cpf1-puro; U6-FlipArray. After 7 days, cells were then infected with lentivirus containing EFS-Cre to induce inversion of the FlipArray. Prior to Cre recombination, only crNf1 is expressed; following Cre recombination, crPten becomes expressed. c Sequences of the FlipArray construct before and after Cre recombination. Red boxes denote mutants from wild-type loxP . Prior to Cre, single mutant lox66 and lox71 sites are present. After Cre recombination, a wild-type loxP site and a double mutant lox72 site are generated

    Article Snippet: After BsmbI digestion (FastDigest Esp3I, ThermoScientific) to linearize the U6 crRNA expression cassette, oligo cloning was performed to insert a lox66 sequence, a DR, two BsmbI sites, and an inverted lox71 .

    Techniques: Mutagenesis, Construct, Expressing, Sequencing, Clone Assay, Infection, Generated

    Construction strategy of the low-copy pMKP ccd B reverse genetic vector to clone unstable HA segments of IAV. The resulting vector is a hybrid of pSMART-LC-Kan and the Pol-I/ ccd B/Pol-II cassette of pMP ccd B with either AarI or Bsa I or BsmB I sites for restriction/cloning of the desired insert. Briefly, the sequence representing the Pol-I/ ccd B/Pol-II cassette excised from pMP ccd B by digestion with BsrB I were blunt end phosphorylated, and ligated into the EcoR V linearized pSMART-LC-Kan plasmid to generate pMKP ccd B. The pMKP ccd B was suitable to clone and maintain the genetically unstable RT-PCR product of H9N2/SA- and H5N1/VSVRI-HA. Transcriptional terminator (T); Repressor of Primer (ROP); low copy origin of replication (Ori); kanamycin resistance gene (Kanr), ampicillin resistance gene (Ampr).

    Journal: PLoS ONE

    Article Title: Efficient Generation of Recombinant Influenza A Viruses Employing a New Approach to Overcome the Genetic Instability of HA Segments

    doi: 10.1371/journal.pone.0116917

    Figure Lengend Snippet: Construction strategy of the low-copy pMKP ccd B reverse genetic vector to clone unstable HA segments of IAV. The resulting vector is a hybrid of pSMART-LC-Kan and the Pol-I/ ccd B/Pol-II cassette of pMP ccd B with either AarI or Bsa I or BsmB I sites for restriction/cloning of the desired insert. Briefly, the sequence representing the Pol-I/ ccd B/Pol-II cassette excised from pMP ccd B by digestion with BsrB I were blunt end phosphorylated, and ligated into the EcoR V linearized pSMART-LC-Kan plasmid to generate pMKP ccd B. The pMKP ccd B was suitable to clone and maintain the genetically unstable RT-PCR product of H9N2/SA- and H5N1/VSVRI-HA. Transcriptional terminator (T); Repressor of Primer (ROP); low copy origin of replication (Ori); kanamycin resistance gene (Kanr), ampicillin resistance gene (Ampr).

    Article Snippet: Meanwhile, the pHH21, pHW2000, pMPccd B and pMKPccd B were linearized with the “FastDigest ” BsmB I (Thermo Scientific, USA) at 37°C for 15 min.

    Techniques: Plasmid Preparation, Clone Assay, Sequencing, Reverse Transcription Polymerase Chain Reaction

    Golden Gate assembly of custom TAL effector and TALEN constructs using module, array, last repeat and backbone plasmids. By using the type IIS restriction endonucleases BsaI and Esp3I, modules containing the desired RVDs can be released with unique cohesive ends for ordered, single-reaction assembly into array plasmids in a first step, and those arrays subsequently released and assembled in order in a second step into a backbone plasmid to create full length constructs with custom repeat arrays (see text for details). NLS, nuclear localization signal(s); AD, transcriptional activation domain; tet , tetracycline resistance; spec , spectinomycin resistance; amp , ampicillin resistance; attL1 and attL2, recombination sites for Gateway cloning; B, BamHI, and S, SphI, useful for subcloning custom repeat arrays. Unique restriction enzyme sites flanking the coding sequences, useful for subcloning the entire constructs into other vectors, are not shown but can be found in the sequence files ( Supplementary Data ).

    Journal: Nucleic Acids Research

    Article Title: Efficient design and assembly of custom TALEN and other TAL effector-based constructs for DNA targeting

    doi: 10.1093/nar/gkr218

    Figure Lengend Snippet: Golden Gate assembly of custom TAL effector and TALEN constructs using module, array, last repeat and backbone plasmids. By using the type IIS restriction endonucleases BsaI and Esp3I, modules containing the desired RVDs can be released with unique cohesive ends for ordered, single-reaction assembly into array plasmids in a first step, and those arrays subsequently released and assembled in order in a second step into a backbone plasmid to create full length constructs with custom repeat arrays (see text for details). NLS, nuclear localization signal(s); AD, transcriptional activation domain; tet , tetracycline resistance; spec , spectinomycin resistance; amp , ampicillin resistance; attL1 and attL2, recombination sites for Gateway cloning; B, BamHI, and S, SphI, useful for subcloning custom repeat arrays. Unique restriction enzyme sites flanking the coding sequences, useful for subcloning the entire constructs into other vectors, are not shown but can be found in the sequence files ( Supplementary Data ).

    Article Snippet: Finally, into this plasmid, the XbaI–SacI fragment of pTAL3 containing the TALEN backbone construct was introduced at the corresponding sites. pTAL1 was created by replacing the SphI fragment of tal1C in pCS691 with the corresponding SphI fragment of pTAL3, containing the lacZ gene and the Esp3I sites and flanking sequences for accepting final arrays. pCS691 is a derivative of Gateway entry vector pENTR-D (Invitrogen) containing between the attL sites, the complete tal1c gene preceded by both Kozak and Shine–Dalgarno consensus sequences for efficient translation in eukaryotic or bacterial cells, respectively.

    Techniques: Construct, Plasmid Preparation, Activation Assay, Clone Assay, Subcloning, Sequencing

    CXCR4 gene silencing by lentivirus mediated CRISPR/SaCas9 delivery protects GHOST-X4 cells from HIV-1 infection. a A schematic diagram of lentiviral transfer vectors containing CRISPR/SaCas9 components. LentiCRISPR v2 plasmid was modified by replacing the SpCas9 with SaCas9. Then, based on the SaCas9 PAM sequence 5′-NNGRRT-3′, we designed, synthesized and cloned the CXCR4 sgRNAs into the vector using the Bsmb1 . b CXCR4 gene disruption analysis in GHOST-X4 cells by the T7E1 cleavage assay. GHOST-X4 cells were transduced with lentiviruses (MOI of 40) in the presence of polybrene for 12 h, and the genomic DNA was extracted and used as template to amplify a CXCR4 fragment (1100 bp). Con: lentiviral vectors expressing SaCas9 only, #8 and #9: lentiviral vectors expressing SaCas9/sgRNA #8 and #9. c Flow cytometry analysis of CXCR4 expression in lentivirus transduced GHOST-X4 cells. Neg, unstained cells. Con, #8 and #9 as samples described in ( b ) stained with anti-CXCR4-PE. d DNA sequences of CXCR4 of the transduced GHOST-X4 cells. PCR products were cloned into pGEM-T Easy vector and sequenced. The PAM sequences are lined and highlighted in red; the target sequences were shown in blue; deletions are indicated with (−) and insertions with (+). N/N indicates ratio of WT or mutations to total sequenced clones. e Flow cytometry analysis of transduced GHOST-X4 cells on GFP expression 3 days post HIV-1 NL4-3 infection. Neg, no HIV-1 infection. f HIV-1 NL4-3 infection was determined by p24 level. The cultured supernatants were collected at the indicated days post infection, and HIV-1 p24 level was determined using a p24 ELISA kit. For ( c and e ), one representative out of three independent experiments is shown. For ( f ), the graph represents 3 independent infection experiments and error bars represent SEM. Statistical analysis determined using unpaired t-test (*** P

    Journal: Retrovirology

    Article Title: Genome modification of CXCR4 by Staphylococcus aureus Cas9 renders cells resistance to HIV-1 infection

    doi: 10.1186/s12977-017-0375-0

    Figure Lengend Snippet: CXCR4 gene silencing by lentivirus mediated CRISPR/SaCas9 delivery protects GHOST-X4 cells from HIV-1 infection. a A schematic diagram of lentiviral transfer vectors containing CRISPR/SaCas9 components. LentiCRISPR v2 plasmid was modified by replacing the SpCas9 with SaCas9. Then, based on the SaCas9 PAM sequence 5′-NNGRRT-3′, we designed, synthesized and cloned the CXCR4 sgRNAs into the vector using the Bsmb1 . b CXCR4 gene disruption analysis in GHOST-X4 cells by the T7E1 cleavage assay. GHOST-X4 cells were transduced with lentiviruses (MOI of 40) in the presence of polybrene for 12 h, and the genomic DNA was extracted and used as template to amplify a CXCR4 fragment (1100 bp). Con: lentiviral vectors expressing SaCas9 only, #8 and #9: lentiviral vectors expressing SaCas9/sgRNA #8 and #9. c Flow cytometry analysis of CXCR4 expression in lentivirus transduced GHOST-X4 cells. Neg, unstained cells. Con, #8 and #9 as samples described in ( b ) stained with anti-CXCR4-PE. d DNA sequences of CXCR4 of the transduced GHOST-X4 cells. PCR products were cloned into pGEM-T Easy vector and sequenced. The PAM sequences are lined and highlighted in red; the target sequences were shown in blue; deletions are indicated with (−) and insertions with (+). N/N indicates ratio of WT or mutations to total sequenced clones. e Flow cytometry analysis of transduced GHOST-X4 cells on GFP expression 3 days post HIV-1 NL4-3 infection. Neg, no HIV-1 infection. f HIV-1 NL4-3 infection was determined by p24 level. The cultured supernatants were collected at the indicated days post infection, and HIV-1 p24 level was determined using a p24 ELISA kit. For ( c and e ), one representative out of three independent experiments is shown. For ( f ), the graph represents 3 independent infection experiments and error bars represent SEM. Statistical analysis determined using unpaired t-test (*** P

    Article Snippet: For lenti-SaCas9-CXCR4-gRNA plasmid, we modified the lentiCRISPR v2 plasmid (Addgene #52961) by replacing the SpCas9 with SaCas9, and cloned the sgRNAs into the vector using the Bsmb1 (Fermentas).

    Techniques: CRISPR, Infection, Plasmid Preparation, Modification, Sequencing, Synthesized, Clone Assay, Cleavage Assay, Transduction, Expressing, Flow Cytometry, Cytometry, Staining, Polymerase Chain Reaction, Cell Culture, Enzyme-linked Immunosorbent Assay

    CRISPR vector design for knockout of A 2A R in K562 Cells. ( a ) Map of human chromosomes, with chromosome 22q11.23 highlighted, showing the location of the A 2A R gene. Additional arrowheads indicate genes with similar regions to the A 2A R gene, including the A 2B R gene on chromosome 17. ( b ) Left, guide-RNAs found for the A 2A R gene. Right, guide sequence #1 was selected for insertion into the plentiCRISPR loxP vector plasmid DNA. BsmBI cut sites were added to gRNA #1 to facilitate insertion. ( c ) Top, Cas9 cut site in the A 2A R gene. Bottom, map of the plentiCRISPR construct. ( d ) T7 E1 INDEL assay for CRISPR targeting efficiency. Lanes 1 and 5, universal marker. Lane 2, PCR product. Lanes 3 and 4, PCR product after denaturing and renaturing, without (3) and with (4) T7 EI enzyme. Arrowheads indicate T7 E1 cutting.

    Journal: Scientific Reports

    Article Title: A Novel Method for Screening Adenosine Receptor Specific Agonists for Use in Adenosine Drug Development

    doi: 10.1038/srep44816

    Figure Lengend Snippet: CRISPR vector design for knockout of A 2A R in K562 Cells. ( a ) Map of human chromosomes, with chromosome 22q11.23 highlighted, showing the location of the A 2A R gene. Additional arrowheads indicate genes with similar regions to the A 2A R gene, including the A 2B R gene on chromosome 17. ( b ) Left, guide-RNAs found for the A 2A R gene. Right, guide sequence #1 was selected for insertion into the plentiCRISPR loxP vector plasmid DNA. BsmBI cut sites were added to gRNA #1 to facilitate insertion. ( c ) Top, Cas9 cut site in the A 2A R gene. Bottom, map of the plentiCRISPR construct. ( d ) T7 E1 INDEL assay for CRISPR targeting efficiency. Lanes 1 and 5, universal marker. Lane 2, PCR product. Lanes 3 and 4, PCR product after denaturing and renaturing, without (3) and with (4) T7 EI enzyme. Arrowheads indicate T7 E1 cutting.

    Article Snippet: Using the BsmBI digestion enzyme (Thermo Fisher Scientific), the plentiCRISPR loxP plasmid was linearized and eluted for insertion of the A2A R guide RNA (gRNA).

    Techniques: CRISPR, Plasmid Preparation, Knock-Out, Sequencing, Construct, Marker, Polymerase Chain Reaction

    Construction strategy of the low-copy pMKP ccd B reverse genetic vector to clone unstable HA segments of IAV. The resulting vector is a hybrid of pSMART-LC-Kan and the Pol-I/ ccd B/Pol-II cassette of pMP ccd B with either AarI or Bsa I or BsmB I sites for restriction/cloning of the desired insert. Briefly, the sequence representing the Pol-I/ ccd B/Pol-II cassette excised from pMP ccd B by digestion with BsrB I were blunt end phosphorylated, and ligated into the EcoR V linearized pSMART-LC-Kan plasmid to generate pMKP ccd B. The pMKP ccd B was suitable to clone and maintain the genetically unstable RT-PCR product of H9N2/SA- and H5N1/VSVRI-HA. Transcriptional terminator (T); Repressor of Primer (ROP); low copy origin of replication (Ori); kanamycin resistance gene (Kanr), ampicillin resistance gene (Ampr).

    Journal: PLoS ONE

    Article Title: Efficient Generation of Recombinant Influenza A Viruses Employing a New Approach to Overcome the Genetic Instability of HA Segments

    doi: 10.1371/journal.pone.0116917

    Figure Lengend Snippet: Construction strategy of the low-copy pMKP ccd B reverse genetic vector to clone unstable HA segments of IAV. The resulting vector is a hybrid of pSMART-LC-Kan and the Pol-I/ ccd B/Pol-II cassette of pMP ccd B with either AarI or Bsa I or BsmB I sites for restriction/cloning of the desired insert. Briefly, the sequence representing the Pol-I/ ccd B/Pol-II cassette excised from pMP ccd B by digestion with BsrB I were blunt end phosphorylated, and ligated into the EcoR V linearized pSMART-LC-Kan plasmid to generate pMKP ccd B. The pMKP ccd B was suitable to clone and maintain the genetically unstable RT-PCR product of H9N2/SA- and H5N1/VSVRI-HA. Transcriptional terminator (T); Repressor of Primer (ROP); low copy origin of replication (Ori); kanamycin resistance gene (Kanr), ampicillin resistance gene (Ampr).

    Article Snippet: The pCR2.1 constructs carrying the cloned NA-segments, proved by enzymatic digestion and subsequent sequencing, were digested by “FastDigest” BsmB I enzyme (Thermo Scientific, USA) at 37°C for 15 min, and then the NA-cDNAs were ligated into BsmB I-linearized pHW2000 using T4 ligase (Thermo Scientific, USA).

    Techniques: Plasmid Preparation, Clone Assay, Sequencing, Reverse Transcription Polymerase Chain Reaction