Structured Review

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: 94/100, based on 21 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Average 94 stars, based on 21 article reviews
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bsmbi - by Bioz Stars, 2020-05
94/100 stars

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1) Product Images from "A modular toolbox for gRNA–Cas9 genome engineering in plants based on the GoldenBraid standard"

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

Journal: Plant Methods

doi: 10.1186/s13007-016-0101-2

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
Figure Legend 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

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

Related Articles

Clone Assay:

Article Title: Mapping person-to-person variation in viral mutations that escape polyclonal serum targeting influenza hemagglutinin
Article Snippet: .. The mutant amplicons were then cloned at high efficiency into the pHH21 vector using digestion with BsmBI, ligation with T4 DNA ligase, and electroporation into ElectroMAX DH10B competent cells (Invitrogen 18290015). ..

Article Title: Competitive endogenous RNA is an intrinsic component of EMT regulatory circuits and modulates EMT
Article Snippet: .. To generate desired vector for mutagenesis, the guide RNA vector was digested with BsmBI (Fermentas) and a pair of annealed oligos containing the sgRNA was cloned into the single guide RNA scaffold. .. The A549-Cas9 cells and MCF10A-Cas9 cells were infected with the lentivirus contained guide RNA (MOI = 0.3) and selected with 1ug/ml Puromycin for 3 days.

Amplification:

Article Title: Rapid and reliable universal cloning of influenza A virus genes by target-primed plasmid amplification
Article Snippet: .. After digestion of the PCR amplicon and recipient plasmid pHW2000 with BsmBI, the resulting fragments were ligated and an aliquot was transformed into ccd B-resistant DB 3.1™ bacteria (Invitrogen). .. Primers For amplification of influenza A virus genes, we used primers modified from ref. ( ) ( ).

Ligation:

Article Title: Mapping person-to-person variation in viral mutations that escape polyclonal serum targeting influenza hemagglutinin
Article Snippet: .. The mutant amplicons were then cloned at high efficiency into the pHH21 vector using digestion with BsmBI, ligation with T4 DNA ligase, and electroporation into ElectroMAX DH10B competent cells (Invitrogen 18290015). ..

Article Title: A multiplatform approach identifies miR-152-3p as a common epigenetically regulated onco-suppressor in prostate cancer targeting TMEM97
Article Snippet: .. Annealed oligos were diluted at 1:100 in sterile water and ligated to plasmid vector lentiCRISPRv2 (gift from Feng Zhang (Addgene plasmid #52961)) using the following parameters: 50 ng BsmBI (Fermentas) digested plasmid, 1 μl diluted oligo duplex, 1× Ligation Buffer (Roche), and 5 U T4 DNA Ligase (Roche) incubated at RT/30 min. .. The ligation reactions were used to transform highly competent Escherichia coli cells according to the manufacturer’s protocol [ ].

Mutagenesis:

Article Title: Mapping person-to-person variation in viral mutations that escape polyclonal serum targeting influenza hemagglutinin
Article Snippet: .. The mutant amplicons were then cloned at high efficiency into the pHH21 vector using digestion with BsmBI, ligation with T4 DNA ligase, and electroporation into ElectroMAX DH10B competent cells (Invitrogen 18290015). ..

Article Title: Competitive endogenous RNA is an intrinsic component of EMT regulatory circuits and modulates EMT
Article Snippet: .. To generate desired vector for mutagenesis, the guide RNA vector was digested with BsmBI (Fermentas) and a pair of annealed oligos containing the sgRNA was cloned into the single guide RNA scaffold. .. The A549-Cas9 cells and MCF10A-Cas9 cells were infected with the lentivirus contained guide RNA (MOI = 0.3) and selected with 1ug/ml Puromycin for 3 days.

Purification:

Article Title: Recovery of Murine Norovirus and Feline Calicivirus from Plasmids Encoding EMCV IRES in Stable Cell Lines Expressing T7 Polymerase
Article Snippet: .. Purified PCR fragments were treated with BsmBI and ligated into the pT7CFE1-His vector (Thermo Scientific, Rockford, IL) treated with BsmfI and SpeI enzymes. ..

Article Title: Analysis of the Histone H3.1 Interactome: A Suitable Chaperone for the Right Event
Article Snippet: .. BsmBI cut pS601x1 plasmid was filled-in with biotinylated dNTPs (Thermo Scientific), and the gel purified ScaI-BsmBI fragment nicked with Nb.BbvCI. .. Nucleosomes were assembled by salt dialysis (Extended Experimental Procedures) after which 4 molar equivalents of streptavidin (NEB) was added.

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

Incubation:

Article Title: A multiplatform approach identifies miR-152-3p as a common epigenetically regulated onco-suppressor in prostate cancer targeting TMEM97
Article Snippet: .. Annealed oligos were diluted at 1:100 in sterile water and ligated to plasmid vector lentiCRISPRv2 (gift from Feng Zhang (Addgene plasmid #52961)) using the following parameters: 50 ng BsmBI (Fermentas) digested plasmid, 1 μl diluted oligo duplex, 1× Ligation Buffer (Roche), and 5 U T4 DNA Ligase (Roche) incubated at RT/30 min. .. The ligation reactions were used to transform highly competent Escherichia coli cells according to the manufacturer’s protocol [ ].

Polymerase Chain Reaction:

Article Title: Rapid and reliable universal cloning of influenza A virus genes by target-primed plasmid amplification
Article Snippet: .. After digestion of the PCR amplicon and recipient plasmid pHW2000 with BsmBI, the resulting fragments were ligated and an aliquot was transformed into ccd B-resistant DB 3.1™ bacteria (Invitrogen). .. Primers For amplification of influenza A virus genes, we used primers modified from ref. ( ) ( ).

Article Title: Recovery of Murine Norovirus and Feline Calicivirus from Plasmids Encoding EMCV IRES in Stable Cell Lines Expressing T7 Polymerase
Article Snippet: .. Purified PCR fragments were treated with BsmBI and ligated into the pT7CFE1-His vector (Thermo Scientific, Rockford, IL) treated with BsmfI and SpeI enzymes. ..

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

Electroporation:

Article Title: Mapping person-to-person variation in viral mutations that escape polyclonal serum targeting influenza hemagglutinin
Article Snippet: .. The mutant amplicons were then cloned at high efficiency into the pHH21 vector using digestion with BsmBI, ligation with T4 DNA ligase, and electroporation into ElectroMAX DH10B competent cells (Invitrogen 18290015). ..

Transformation Assay:

Article Title: Rapid and reliable universal cloning of influenza A virus genes by target-primed plasmid amplification
Article Snippet: .. After digestion of the PCR amplicon and recipient plasmid pHW2000 with BsmBI, the resulting fragments were ligated and an aliquot was transformed into ccd B-resistant DB 3.1™ bacteria (Invitrogen). .. Primers For amplification of influenza A virus genes, we used primers modified from ref. ( ) ( ).

Plasmid Preparation:

Article Title: Rapid and reliable universal cloning of influenza A virus genes by target-primed plasmid amplification
Article Snippet: .. After digestion of the PCR amplicon and recipient plasmid pHW2000 with BsmBI, the resulting fragments were ligated and an aliquot was transformed into ccd B-resistant DB 3.1™ bacteria (Invitrogen). .. Primers For amplification of influenza A virus genes, we used primers modified from ref. ( ) ( ).

Article Title: Mapping person-to-person variation in viral mutations that escape polyclonal serum targeting influenza hemagglutinin
Article Snippet: .. The mutant amplicons were then cloned at high efficiency into the pHH21 vector using digestion with BsmBI, ligation with T4 DNA ligase, and electroporation into ElectroMAX DH10B competent cells (Invitrogen 18290015). ..

Article Title: Recovery of Murine Norovirus and Feline Calicivirus from Plasmids Encoding EMCV IRES in Stable Cell Lines Expressing T7 Polymerase
Article Snippet: .. Purified PCR fragments were treated with BsmBI and ligated into the pT7CFE1-His vector (Thermo Scientific, Rockford, IL) treated with BsmfI and SpeI enzymes. ..

Article Title: Analysis of the Histone H3.1 Interactome: A Suitable Chaperone for the Right Event
Article Snippet: .. BsmBI cut pS601x1 plasmid was filled-in with biotinylated dNTPs (Thermo Scientific), and the gel purified ScaI-BsmBI fragment nicked with Nb.BbvCI. .. Nucleosomes were assembled by salt dialysis (Extended Experimental Procedures) after which 4 molar equivalents of streptavidin (NEB) was added.

Article Title: Competitive endogenous RNA is an intrinsic component of EMT regulatory circuits and modulates EMT
Article Snippet: .. To generate desired vector for mutagenesis, the guide RNA vector was digested with BsmBI (Fermentas) and a pair of annealed oligos containing the sgRNA was cloned into the single guide RNA scaffold. .. The A549-Cas9 cells and MCF10A-Cas9 cells were infected with the lentivirus contained guide RNA (MOI = 0.3) and selected with 1ug/ml Puromycin for 3 days.

Article Title: A multiplatform approach identifies miR-152-3p as a common epigenetically regulated onco-suppressor in prostate cancer targeting TMEM97
Article Snippet: .. Annealed oligos were diluted at 1:100 in sterile water and ligated to plasmid vector lentiCRISPRv2 (gift from Feng Zhang (Addgene plasmid #52961)) using the following parameters: 50 ng BsmBI (Fermentas) digested plasmid, 1 μl diluted oligo duplex, 1× Ligation Buffer (Roche), and 5 U T4 DNA Ligase (Roche) incubated at RT/30 min. .. The ligation reactions were used to transform highly competent Escherichia coli cells according to the manufacturer’s protocol [ ].

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  • 94
    Thermo Fisher bsmbi digestion
    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 <t>U6</t> expression cassette containing two inverted <t>BsmbI</t> 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
    Bsmbi Digestion, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/bsmbi digestion/product/Thermo Fisher
    Average 94 stars, based on 2 article reviews
    Price from $9.99 to $1999.99
    bsmbi digestion - by Bioz Stars, 2020-05
    94/100 stars
      Buy from Supplier

    91
    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: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/bsmbi digestion enzyme/product/Thermo Fisher
    Average 91 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    bsmbi digestion enzyme - by Bioz Stars, 2020-05
    91/100 stars
      Buy from Supplier

    96
    Thermo Fisher bsm bi
    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.
    Bsm Bi, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 96/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/bsm bi/product/Thermo Fisher
    Average 96 stars, based on 2 article reviews
    Price from $9.99 to $1999.99
    bsm bi - by Bioz Stars, 2020-05
    96/100 stars
      Buy from Supplier

    Image Search Results


    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

    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

    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