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    New England Biolabs xmai
    Transgenerational CRISPR-Cas9 activity induces new mutations in the TaGW2 and TaLpx-1 genes. NGS reads flanking the GW2T2 target site and their frequencies in (A) T 0 line GLM-2, (B) T 1 line GLM-2-9, and (C) T 2 line GLM-2-9-49 are shown. (D) Restriction enzyme digestion of polymerase chain reaction <t>(PCR)</t> amplicons to screen gw2 knockout mutations in the T 3 progenies of line GLM-2-9-49. The GW2T2 flanking region was amplified by PCR and digested with <t>XmaI;</t> non-digested PCR amplicons correspond to mutated GW2T2 target sites. The numbers on the gel image are identifiers of the GLM-2-9-49 progenies. Lanes marked with arrows are PCR products from wild-type plant not digested with XmaI and loaded as controls; the knockout mutant plant was marked with a star. BW, wild-type cultivar Bobwhite. (E) Sanger sequencing of PCR-amplified GW2T2 target sites of T 3 line GLM-2-9-49-28. Genome specific primers were used to amplify regions flanking the GW2T2 target sites. Nucleotide substitutions are marked with red rectangles, and the inserted nucleotide is shown by the red arrow. Types and frequencies of mutations at the GW2T2, LPX1T2, and MLOT1 target sites in (F) T 1 line GLM-2-5, and (G) T 2 line GLM-2-5-24 are shown. WT, wild-type alleles in wheat cultivar Bobwhite; “–” and “+” signs and numbers after them, nucleotides deleted and inserted, respectively. The frequency of each mutation type is shown on the right. The PAM sequences are underlined; the deleted nucleotides are shown with red dashed lines; the insertions and deletions are highlighted in red.
    Xmai, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "Transgenerational CRISPR-Cas9 Activity Facilitates Multiplex Gene Editing in Allopolyploid Wheat"

    Article Title: Transgenerational CRISPR-Cas9 Activity Facilitates Multiplex Gene Editing in Allopolyploid Wheat

    Journal: The Crispr Journal

    doi: 10.1089/crispr.2017.0010

    Transgenerational CRISPR-Cas9 activity induces new mutations in the TaGW2 and TaLpx-1 genes. NGS reads flanking the GW2T2 target site and their frequencies in (A) T 0 line GLM-2, (B) T 1 line GLM-2-9, and (C) T 2 line GLM-2-9-49 are shown. (D) Restriction enzyme digestion of polymerase chain reaction (PCR) amplicons to screen gw2 knockout mutations in the T 3 progenies of line GLM-2-9-49. The GW2T2 flanking region was amplified by PCR and digested with XmaI; non-digested PCR amplicons correspond to mutated GW2T2 target sites. The numbers on the gel image are identifiers of the GLM-2-9-49 progenies. Lanes marked with arrows are PCR products from wild-type plant not digested with XmaI and loaded as controls; the knockout mutant plant was marked with a star. BW, wild-type cultivar Bobwhite. (E) Sanger sequencing of PCR-amplified GW2T2 target sites of T 3 line GLM-2-9-49-28. Genome specific primers were used to amplify regions flanking the GW2T2 target sites. Nucleotide substitutions are marked with red rectangles, and the inserted nucleotide is shown by the red arrow. Types and frequencies of mutations at the GW2T2, LPX1T2, and MLOT1 target sites in (F) T 1 line GLM-2-5, and (G) T 2 line GLM-2-5-24 are shown. WT, wild-type alleles in wheat cultivar Bobwhite; “–” and “+” signs and numbers after them, nucleotides deleted and inserted, respectively. The frequency of each mutation type is shown on the right. The PAM sequences are underlined; the deleted nucleotides are shown with red dashed lines; the insertions and deletions are highlighted in red.
    Figure Legend Snippet: Transgenerational CRISPR-Cas9 activity induces new mutations in the TaGW2 and TaLpx-1 genes. NGS reads flanking the GW2T2 target site and their frequencies in (A) T 0 line GLM-2, (B) T 1 line GLM-2-9, and (C) T 2 line GLM-2-9-49 are shown. (D) Restriction enzyme digestion of polymerase chain reaction (PCR) amplicons to screen gw2 knockout mutations in the T 3 progenies of line GLM-2-9-49. The GW2T2 flanking region was amplified by PCR and digested with XmaI; non-digested PCR amplicons correspond to mutated GW2T2 target sites. The numbers on the gel image are identifiers of the GLM-2-9-49 progenies. Lanes marked with arrows are PCR products from wild-type plant not digested with XmaI and loaded as controls; the knockout mutant plant was marked with a star. BW, wild-type cultivar Bobwhite. (E) Sanger sequencing of PCR-amplified GW2T2 target sites of T 3 line GLM-2-9-49-28. Genome specific primers were used to amplify regions flanking the GW2T2 target sites. Nucleotide substitutions are marked with red rectangles, and the inserted nucleotide is shown by the red arrow. Types and frequencies of mutations at the GW2T2, LPX1T2, and MLOT1 target sites in (F) T 1 line GLM-2-5, and (G) T 2 line GLM-2-5-24 are shown. WT, wild-type alleles in wheat cultivar Bobwhite; “–” and “+” signs and numbers after them, nucleotides deleted and inserted, respectively. The frequency of each mutation type is shown on the right. The PAM sequences are underlined; the deleted nucleotides are shown with red dashed lines; the insertions and deletions are highlighted in red.

    Techniques Used: CRISPR, Activity Assay, Next-Generation Sequencing, Polymerase Chain Reaction, Knock-Out, Amplification, Mutagenesis, Sequencing

    2) Product Images from "Mechanism of efficient double-strand break repair by a long non-coding RNA"

    Article Title: Mechanism of efficient double-strand break repair by a long non-coding RNA

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkaa784

    LINP1 is able to promote DNA-PK-dependent synapsis on DNA constructs with a 6 kbp bridge. ( A ) Sketch of the molecular DNA forceps with 6 kbp bridge extended in a magnetic trap. Two ends of the construct are attached to the slide surface and magnetic bead, respectively. The bead is initially held by a 1.4 pN force (1) before the force is lowered to 0.001 pN (2) allowing the two DNA ends to meet in the presence of NHEJ components. The force is then raised again, but any synapsis prevents the bead from recovering its original position (3) until synapsis is broken (4). ( B ) Representative time-trace for Ku + DNA-PKcs + full-length LINP1 obtained upon application of the force-modulation pattern (red). DNA is prepared with blunt ends by SmaI digest. The fourth pulling cycle shows an end-interaction rupture event which can be characterized by both the change in DNA extension upon rupture, Δ l , and the duration of the synaptic event prior to rupture, t synapsis . ( C ) Histogram of DNA extension change, Δ l , upon synapsis rupture in the presence of Ku + DNA-PKcs + full-length LINP1. Green line and red line are from a fit to a double Gaussian distribution, with a peak (green) at 1737 nm and displaying 101 nm standard deviation ( n = 95 events), and a peak (red) at 387 nm and displaying 54 nm standard deviation ( n = 395 events). The entire histogram contains n = 526 events. ( D ) Lifetime distribution of the specific synaptic state for Ku + DNA-PKcs + full-length LINP1 is fit to a single-exponential distribution (green line), giving a lifetime of 4.1 ± 0.6 s (SEM, n = 95). ( E ) Representative time-trace obtained by force-cycling in the presence of T4 DNA ligase a forceps DNA prepared with sticky ends by XmaI digest. A stable, ∼1.7 μm reduction in extension is observed (down arrow), and is reversed upon introduction of Sma I (up arrow). ( F ) Histogram of ligation events per cycle in the presence of T4 DNA ligase. ( G ) Representative time-trace obtained as in (B) but in the presence of Ku + DNA-PKcs + PAXX. ( H ) Histogram of DNA extension change, Δ l , upon synapsis rupture in the presence of Ku + DNA-PKcs + PAXX. Green line and red line are from a fit to a double Gaussian distribution, with a peak (green) at 1843 nm displaying a 57 nm standard deviation ( n = 10 events), and a peak (red) at 433 nm displaying a 56 nm standard deviation ( n = 392 events). ( I ) Schematic model for the role of LINP1 from single-molecule studies.
    Figure Legend Snippet: LINP1 is able to promote DNA-PK-dependent synapsis on DNA constructs with a 6 kbp bridge. ( A ) Sketch of the molecular DNA forceps with 6 kbp bridge extended in a magnetic trap. Two ends of the construct are attached to the slide surface and magnetic bead, respectively. The bead is initially held by a 1.4 pN force (1) before the force is lowered to 0.001 pN (2) allowing the two DNA ends to meet in the presence of NHEJ components. The force is then raised again, but any synapsis prevents the bead from recovering its original position (3) until synapsis is broken (4). ( B ) Representative time-trace for Ku + DNA-PKcs + full-length LINP1 obtained upon application of the force-modulation pattern (red). DNA is prepared with blunt ends by SmaI digest. The fourth pulling cycle shows an end-interaction rupture event which can be characterized by both the change in DNA extension upon rupture, Δ l , and the duration of the synaptic event prior to rupture, t synapsis . ( C ) Histogram of DNA extension change, Δ l , upon synapsis rupture in the presence of Ku + DNA-PKcs + full-length LINP1. Green line and red line are from a fit to a double Gaussian distribution, with a peak (green) at 1737 nm and displaying 101 nm standard deviation ( n = 95 events), and a peak (red) at 387 nm and displaying 54 nm standard deviation ( n = 395 events). The entire histogram contains n = 526 events. ( D ) Lifetime distribution of the specific synaptic state for Ku + DNA-PKcs + full-length LINP1 is fit to a single-exponential distribution (green line), giving a lifetime of 4.1 ± 0.6 s (SEM, n = 95). ( E ) Representative time-trace obtained by force-cycling in the presence of T4 DNA ligase a forceps DNA prepared with sticky ends by XmaI digest. A stable, ∼1.7 μm reduction in extension is observed (down arrow), and is reversed upon introduction of Sma I (up arrow). ( F ) Histogram of ligation events per cycle in the presence of T4 DNA ligase. ( G ) Representative time-trace obtained as in (B) but in the presence of Ku + DNA-PKcs + PAXX. ( H ) Histogram of DNA extension change, Δ l , upon synapsis rupture in the presence of Ku + DNA-PKcs + PAXX. Green line and red line are from a fit to a double Gaussian distribution, with a peak (green) at 1843 nm displaying a 57 nm standard deviation ( n = 10 events), and a peak (red) at 433 nm displaying a 56 nm standard deviation ( n = 392 events). ( I ) Schematic model for the role of LINP1 from single-molecule studies.

    Techniques Used: Construct, Non-Homologous End Joining, Standard Deviation, Ligation

    3) Product Images from "Transgenerational CRISPR-Cas9 Activity Facilitates Multiplex Gene Editing in Allopolyploid Wheat"

    Article Title: Transgenerational CRISPR-Cas9 Activity Facilitates Multiplex Gene Editing in Allopolyploid Wheat

    Journal: The Crispr Journal

    doi: 10.1089/crispr.2017.0010

    Transgenerational CRISPR-Cas9 activity induces new mutations in the TaGW2 and TaLpx-1 genes. NGS reads flanking the GW2T2 target site and their frequencies in (A) T 0 line GLM-2, (B) T 1 line GLM-2-9, and (C) T 2 line GLM-2-9-49 are shown. (D) Restriction enzyme digestion of polymerase chain reaction (PCR) amplicons to screen gw2 knockout mutations in the T 3 progenies of line GLM-2-9-49. The GW2T2 flanking region was amplified by PCR and digested with XmaI; non-digested PCR amplicons correspond to mutated GW2T2 target sites. The numbers on the gel image are identifiers of the GLM-2-9-49 progenies. Lanes marked with arrows are PCR products from wild-type plant not digested with XmaI and loaded as controls; the knockout mutant plant was marked with a star. BW, wild-type cultivar Bobwhite. (E) Sanger sequencing of PCR-amplified GW2T2 target sites of T 3 line GLM-2-9-49-28. Genome specific primers were used to amplify regions flanking the GW2T2 target sites. Nucleotide substitutions are marked with red rectangles, and the inserted nucleotide is shown by the red arrow. Types and frequencies of mutations at the GW2T2, LPX1T2, and MLOT1 target sites in (F) T 1 line GLM-2-5, and (G) T 2 line GLM-2-5-24 are shown. WT, wild-type alleles in wheat cultivar Bobwhite; “–” and “+” signs and numbers after them, nucleotides deleted and inserted, respectively. The frequency of each mutation type is shown on the right. The PAM sequences are underlined; the deleted nucleotides are shown with red dashed lines; the insertions and deletions are highlighted in red.
    Figure Legend Snippet: Transgenerational CRISPR-Cas9 activity induces new mutations in the TaGW2 and TaLpx-1 genes. NGS reads flanking the GW2T2 target site and their frequencies in (A) T 0 line GLM-2, (B) T 1 line GLM-2-9, and (C) T 2 line GLM-2-9-49 are shown. (D) Restriction enzyme digestion of polymerase chain reaction (PCR) amplicons to screen gw2 knockout mutations in the T 3 progenies of line GLM-2-9-49. The GW2T2 flanking region was amplified by PCR and digested with XmaI; non-digested PCR amplicons correspond to mutated GW2T2 target sites. The numbers on the gel image are identifiers of the GLM-2-9-49 progenies. Lanes marked with arrows are PCR products from wild-type plant not digested with XmaI and loaded as controls; the knockout mutant plant was marked with a star. BW, wild-type cultivar Bobwhite. (E) Sanger sequencing of PCR-amplified GW2T2 target sites of T 3 line GLM-2-9-49-28. Genome specific primers were used to amplify regions flanking the GW2T2 target sites. Nucleotide substitutions are marked with red rectangles, and the inserted nucleotide is shown by the red arrow. Types and frequencies of mutations at the GW2T2, LPX1T2, and MLOT1 target sites in (F) T 1 line GLM-2-5, and (G) T 2 line GLM-2-5-24 are shown. WT, wild-type alleles in wheat cultivar Bobwhite; “–” and “+” signs and numbers after them, nucleotides deleted and inserted, respectively. The frequency of each mutation type is shown on the right. The PAM sequences are underlined; the deleted nucleotides are shown with red dashed lines; the insertions and deletions are highlighted in red.

    Techniques Used: CRISPR, Activity Assay, Next-Generation Sequencing, Polymerase Chain Reaction, Knock-Out, Amplification, Mutagenesis, Sequencing

    4) Product Images from "Rapid modification of the pET-28 expression vector for ligation independent cloning using homologous recombination in Saccharomyces cerevisiae"

    Article Title: Rapid modification of the pET-28 expression vector for ligation independent cloning using homologous recombination in Saccharomyces cerevisiae

    Journal: Plasmid

    doi: 10.1016/j.plasmid.2014.09.005

    Construction of pGAY-28. The modification of pET-28 to replace the multiple cloning region (MCR) with a LIC cassette was accomplished in five steps. In step (1), the parent pET-28 vector is amplified in three segments: A, B, and C. Segment A contains a region homologous to the 3′-end of the linearized yeast shuttle vector YEpADH2p (Y-3′). Segment B contains the LIC cassette at its 3′-end. Segment C contains the LIC cassette at its 5′-end, and a region homologous to the 5′-end of YEpADH2p (Y-5′). In step (2), transformation of linearized YEpADH2p and the three amplified segments into competent S. cerevisiae leads to step (3), where the overlapping segments undergo homologous recombination in vivo . In step (4), two of the original primers from step (1) are used again to amplify the modified expression vector using “colony PCR”. Since these primers were originally designed to anneal upstream of a single XmaI restriction site, step (5) involves digestion of the amplicon with XmaI followed by treatment with DNA ligase, yielding the complete pGAY-28 expression vector.
    Figure Legend Snippet: Construction of pGAY-28. The modification of pET-28 to replace the multiple cloning region (MCR) with a LIC cassette was accomplished in five steps. In step (1), the parent pET-28 vector is amplified in three segments: A, B, and C. Segment A contains a region homologous to the 3′-end of the linearized yeast shuttle vector YEpADH2p (Y-3′). Segment B contains the LIC cassette at its 3′-end. Segment C contains the LIC cassette at its 5′-end, and a region homologous to the 5′-end of YEpADH2p (Y-5′). In step (2), transformation of linearized YEpADH2p and the three amplified segments into competent S. cerevisiae leads to step (3), where the overlapping segments undergo homologous recombination in vivo . In step (4), two of the original primers from step (1) are used again to amplify the modified expression vector using “colony PCR”. Since these primers were originally designed to anneal upstream of a single XmaI restriction site, step (5) involves digestion of the amplicon with XmaI followed by treatment with DNA ligase, yielding the complete pGAY-28 expression vector.

    Techniques Used: Modification, Positron Emission Tomography, Clone Assay, Plasmid Preparation, Amplification, Transformation Assay, Homologous Recombination, In Vivo, Expressing, Polymerase Chain Reaction

    5) Product Images from "Pathogenicity and Immunogenicity of Recombinant Rabies Viruses Expressing the Lagos Bat Virus Matrix and Glycoprotein: Perspectives for a Pan-Lyssavirus Vaccine"

    Article Title: Pathogenicity and Immunogenicity of Recombinant Rabies Viruses Expressing the Lagos Bat Virus Matrix and Glycoprotein: Perspectives for a Pan-Lyssavirus Vaccine

    Journal: Tropical Medicine and Infectious Disease

    doi: 10.3390/tropicalmed2030037

    Schematic representation of the construction of recombinant viruses. Restriction enzyme sites are indicated ( XmaI , PacI , AvrII , KpnI . BsiWI , AsiSI , NheI and AscI ). GAS represents the SPBN G gene with two amino acid substitutions (Asn 194 to Ser and Arg 333 to Glu). The following abbreviations were used: N, nucleoprotein; M, matrix protein; G, glycoprotein; L, RNA-dependent RNA polymerase. LBVM and LBVG represent the LBV M and G gene respectively.
    Figure Legend Snippet: Schematic representation of the construction of recombinant viruses. Restriction enzyme sites are indicated ( XmaI , PacI , AvrII , KpnI . BsiWI , AsiSI , NheI and AscI ). GAS represents the SPBN G gene with two amino acid substitutions (Asn 194 to Ser and Arg 333 to Glu). The following abbreviations were used: N, nucleoprotein; M, matrix protein; G, glycoprotein; L, RNA-dependent RNA polymerase. LBVM and LBVG represent the LBV M and G gene respectively.

    Techniques Used: Recombinant

    6) Product Images from "Rapid bacterial artificial chromosome modification for large-scale mouse transgenesis"

    Article Title: Rapid bacterial artificial chromosome modification for large-scale mouse transgenesis

    Journal: Nature protocols

    doi: 10.1038/nprot.2010.131

    Ethidium bromide–stained agarose gel pattern showing digested pLD53SC2/A-box DNA from seven different genes. DNA was prepared from PCR-positive colonies, and then digested with AscI and XmaI. Samples were analyzed on a 1.5% agarose gel. The seven genes represented include Plekha2 (lane 1), Itgb5 (lane 2), Itga7 (lane 3), Tdo2 (lane 4), Trpc6 (lane 5), Slc39a6 (lane 6) and Sostdc1 (lane 7). The last sample is pLD53SC2 alone as a vector control (C). Fragment sizes were determined by comparison with a 2-log DNA ladder. The lower bands, which range from 395 to 515 bp, are inserts of each gene. The last sample is the vector that does not contain an insert. If the cloning does not work, the lane will contain a single 3,405-bp band representing the unmodified pLD53SC2.
    Figure Legend Snippet: Ethidium bromide–stained agarose gel pattern showing digested pLD53SC2/A-box DNA from seven different genes. DNA was prepared from PCR-positive colonies, and then digested with AscI and XmaI. Samples were analyzed on a 1.5% agarose gel. The seven genes represented include Plekha2 (lane 1), Itgb5 (lane 2), Itga7 (lane 3), Tdo2 (lane 4), Trpc6 (lane 5), Slc39a6 (lane 6) and Sostdc1 (lane 7). The last sample is pLD53SC2 alone as a vector control (C). Fragment sizes were determined by comparison with a 2-log DNA ladder. The lower bands, which range from 395 to 515 bp, are inserts of each gene. The last sample is the vector that does not contain an insert. If the cloning does not work, the lane will contain a single 3,405-bp band representing the unmodified pLD53SC2.

    Techniques Used: Staining, Agarose Gel Electrophoresis, Polymerase Chain Reaction, Plasmid Preparation, Clone Assay

    7) Product Images from "Pathogenicity and Immunogenicity of Recombinant Rabies Viruses Expressing the Lagos Bat Virus Matrix and Glycoprotein: Perspectives for a Pan-Lyssavirus Vaccine"

    Article Title: Pathogenicity and Immunogenicity of Recombinant Rabies Viruses Expressing the Lagos Bat Virus Matrix and Glycoprotein: Perspectives for a Pan-Lyssavirus Vaccine

    Journal: Tropical Medicine and Infectious Disease

    doi: 10.3390/tropicalmed2030037

    Schematic representation of the construction of recombinant viruses. Restriction enzyme sites are indicated ( XmaI , PacI , AvrII , KpnI . BsiWI , AsiSI , NheI and AscI ). GAS represents the SPBN G gene with two amino acid substitutions (Asn 194 to Ser and Arg 333 to Glu). The following abbreviations were used: N, nucleoprotein; M, matrix protein; G, glycoprotein; L, RNA-dependent RNA polymerase. LBVM and LBVG represent the LBV M and G gene respectively.
    Figure Legend Snippet: Schematic representation of the construction of recombinant viruses. Restriction enzyme sites are indicated ( XmaI , PacI , AvrII , KpnI . BsiWI , AsiSI , NheI and AscI ). GAS represents the SPBN G gene with two amino acid substitutions (Asn 194 to Ser and Arg 333 to Glu). The following abbreviations were used: N, nucleoprotein; M, matrix protein; G, glycoprotein; L, RNA-dependent RNA polymerase. LBVM and LBVG represent the LBV M and G gene respectively.

    Techniques Used: Recombinant

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    New England Biolabs xmai
    Transgenerational CRISPR-Cas9 activity induces new mutations in the TaGW2 and TaLpx-1 genes. NGS reads flanking the GW2T2 target site and their frequencies in (A) T 0 line GLM-2, (B) T 1 line GLM-2-9, and (C) T 2 line GLM-2-9-49 are shown. (D) Restriction enzyme digestion of polymerase chain reaction <t>(PCR)</t> amplicons to screen gw2 knockout mutations in the T 3 progenies of line GLM-2-9-49. The GW2T2 flanking region was amplified by PCR and digested with <t>XmaI;</t> non-digested PCR amplicons correspond to mutated GW2T2 target sites. The numbers on the gel image are identifiers of the GLM-2-9-49 progenies. Lanes marked with arrows are PCR products from wild-type plant not digested with XmaI and loaded as controls; the knockout mutant plant was marked with a star. BW, wild-type cultivar Bobwhite. (E) Sanger sequencing of PCR-amplified GW2T2 target sites of T 3 line GLM-2-9-49-28. Genome specific primers were used to amplify regions flanking the GW2T2 target sites. Nucleotide substitutions are marked with red rectangles, and the inserted nucleotide is shown by the red arrow. Types and frequencies of mutations at the GW2T2, LPX1T2, and MLOT1 target sites in (F) T 1 line GLM-2-5, and (G) T 2 line GLM-2-5-24 are shown. WT, wild-type alleles in wheat cultivar Bobwhite; “–” and “+” signs and numbers after them, nucleotides deleted and inserted, respectively. The frequency of each mutation type is shown on the right. The PAM sequences are underlined; the deleted nucleotides are shown with red dashed lines; the insertions and deletions are highlighted in red.
    Xmai, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Transgenerational CRISPR-Cas9 activity induces new mutations in the TaGW2 and TaLpx-1 genes. NGS reads flanking the GW2T2 target site and their frequencies in (A) T 0 line GLM-2, (B) T 1 line GLM-2-9, and (C) T 2 line GLM-2-9-49 are shown. (D) Restriction enzyme digestion of polymerase chain reaction (PCR) amplicons to screen gw2 knockout mutations in the T 3 progenies of line GLM-2-9-49. The GW2T2 flanking region was amplified by PCR and digested with XmaI; non-digested PCR amplicons correspond to mutated GW2T2 target sites. The numbers on the gel image are identifiers of the GLM-2-9-49 progenies. Lanes marked with arrows are PCR products from wild-type plant not digested with XmaI and loaded as controls; the knockout mutant plant was marked with a star. BW, wild-type cultivar Bobwhite. (E) Sanger sequencing of PCR-amplified GW2T2 target sites of T 3 line GLM-2-9-49-28. Genome specific primers were used to amplify regions flanking the GW2T2 target sites. Nucleotide substitutions are marked with red rectangles, and the inserted nucleotide is shown by the red arrow. Types and frequencies of mutations at the GW2T2, LPX1T2, and MLOT1 target sites in (F) T 1 line GLM-2-5, and (G) T 2 line GLM-2-5-24 are shown. WT, wild-type alleles in wheat cultivar Bobwhite; “–” and “+” signs and numbers after them, nucleotides deleted and inserted, respectively. The frequency of each mutation type is shown on the right. The PAM sequences are underlined; the deleted nucleotides are shown with red dashed lines; the insertions and deletions are highlighted in red.

    Journal: The Crispr Journal

    Article Title: Transgenerational CRISPR-Cas9 Activity Facilitates Multiplex Gene Editing in Allopolyploid Wheat

    doi: 10.1089/crispr.2017.0010

    Figure Lengend Snippet: Transgenerational CRISPR-Cas9 activity induces new mutations in the TaGW2 and TaLpx-1 genes. NGS reads flanking the GW2T2 target site and their frequencies in (A) T 0 line GLM-2, (B) T 1 line GLM-2-9, and (C) T 2 line GLM-2-9-49 are shown. (D) Restriction enzyme digestion of polymerase chain reaction (PCR) amplicons to screen gw2 knockout mutations in the T 3 progenies of line GLM-2-9-49. The GW2T2 flanking region was amplified by PCR and digested with XmaI; non-digested PCR amplicons correspond to mutated GW2T2 target sites. The numbers on the gel image are identifiers of the GLM-2-9-49 progenies. Lanes marked with arrows are PCR products from wild-type plant not digested with XmaI and loaded as controls; the knockout mutant plant was marked with a star. BW, wild-type cultivar Bobwhite. (E) Sanger sequencing of PCR-amplified GW2T2 target sites of T 3 line GLM-2-9-49-28. Genome specific primers were used to amplify regions flanking the GW2T2 target sites. Nucleotide substitutions are marked with red rectangles, and the inserted nucleotide is shown by the red arrow. Types and frequencies of mutations at the GW2T2, LPX1T2, and MLOT1 target sites in (F) T 1 line GLM-2-5, and (G) T 2 line GLM-2-5-24 are shown. WT, wild-type alleles in wheat cultivar Bobwhite; “–” and “+” signs and numbers after them, nucleotides deleted and inserted, respectively. The frequency of each mutation type is shown on the right. The PAM sequences are underlined; the deleted nucleotides are shown with red dashed lines; the insertions and deletions are highlighted in red.

    Article Snippet: To screen the gw2 knockout mutants, the GW2T2 target region from all three homoeologs was amplified, and PCR products were digested with XmaI (NEB).

    Techniques: CRISPR, Activity Assay, Next-Generation Sequencing, Polymerase Chain Reaction, Knock-Out, Amplification, Mutagenesis, Sequencing

    LINP1 is able to promote DNA-PK-dependent synapsis on DNA constructs with a 6 kbp bridge. ( A ) Sketch of the molecular DNA forceps with 6 kbp bridge extended in a magnetic trap. Two ends of the construct are attached to the slide surface and magnetic bead, respectively. The bead is initially held by a 1.4 pN force (1) before the force is lowered to 0.001 pN (2) allowing the two DNA ends to meet in the presence of NHEJ components. The force is then raised again, but any synapsis prevents the bead from recovering its original position (3) until synapsis is broken (4). ( B ) Representative time-trace for Ku + DNA-PKcs + full-length LINP1 obtained upon application of the force-modulation pattern (red). DNA is prepared with blunt ends by SmaI digest. The fourth pulling cycle shows an end-interaction rupture event which can be characterized by both the change in DNA extension upon rupture, Δ l , and the duration of the synaptic event prior to rupture, t synapsis . ( C ) Histogram of DNA extension change, Δ l , upon synapsis rupture in the presence of Ku + DNA-PKcs + full-length LINP1. Green line and red line are from a fit to a double Gaussian distribution, with a peak (green) at 1737 nm and displaying 101 nm standard deviation ( n = 95 events), and a peak (red) at 387 nm and displaying 54 nm standard deviation ( n = 395 events). The entire histogram contains n = 526 events. ( D ) Lifetime distribution of the specific synaptic state for Ku + DNA-PKcs + full-length LINP1 is fit to a single-exponential distribution (green line), giving a lifetime of 4.1 ± 0.6 s (SEM, n = 95). ( E ) Representative time-trace obtained by force-cycling in the presence of T4 DNA ligase a forceps DNA prepared with sticky ends by XmaI digest. A stable, ∼1.7 μm reduction in extension is observed (down arrow), and is reversed upon introduction of Sma I (up arrow). ( F ) Histogram of ligation events per cycle in the presence of T4 DNA ligase. ( G ) Representative time-trace obtained as in (B) but in the presence of Ku + DNA-PKcs + PAXX. ( H ) Histogram of DNA extension change, Δ l , upon synapsis rupture in the presence of Ku + DNA-PKcs + PAXX. Green line and red line are from a fit to a double Gaussian distribution, with a peak (green) at 1843 nm displaying a 57 nm standard deviation ( n = 10 events), and a peak (red) at 433 nm displaying a 56 nm standard deviation ( n = 392 events). ( I ) Schematic model for the role of LINP1 from single-molecule studies.

    Journal: Nucleic Acids Research

    Article Title: Mechanism of efficient double-strand break repair by a long non-coding RNA

    doi: 10.1093/nar/gkaa784

    Figure Lengend Snippet: LINP1 is able to promote DNA-PK-dependent synapsis on DNA constructs with a 6 kbp bridge. ( A ) Sketch of the molecular DNA forceps with 6 kbp bridge extended in a magnetic trap. Two ends of the construct are attached to the slide surface and magnetic bead, respectively. The bead is initially held by a 1.4 pN force (1) before the force is lowered to 0.001 pN (2) allowing the two DNA ends to meet in the presence of NHEJ components. The force is then raised again, but any synapsis prevents the bead from recovering its original position (3) until synapsis is broken (4). ( B ) Representative time-trace for Ku + DNA-PKcs + full-length LINP1 obtained upon application of the force-modulation pattern (red). DNA is prepared with blunt ends by SmaI digest. The fourth pulling cycle shows an end-interaction rupture event which can be characterized by both the change in DNA extension upon rupture, Δ l , and the duration of the synaptic event prior to rupture, t synapsis . ( C ) Histogram of DNA extension change, Δ l , upon synapsis rupture in the presence of Ku + DNA-PKcs + full-length LINP1. Green line and red line are from a fit to a double Gaussian distribution, with a peak (green) at 1737 nm and displaying 101 nm standard deviation ( n = 95 events), and a peak (red) at 387 nm and displaying 54 nm standard deviation ( n = 395 events). The entire histogram contains n = 526 events. ( D ) Lifetime distribution of the specific synaptic state for Ku + DNA-PKcs + full-length LINP1 is fit to a single-exponential distribution (green line), giving a lifetime of 4.1 ± 0.6 s (SEM, n = 95). ( E ) Representative time-trace obtained by force-cycling in the presence of T4 DNA ligase a forceps DNA prepared with sticky ends by XmaI digest. A stable, ∼1.7 μm reduction in extension is observed (down arrow), and is reversed upon introduction of Sma I (up arrow). ( F ) Histogram of ligation events per cycle in the presence of T4 DNA ligase. ( G ) Representative time-trace obtained as in (B) but in the presence of Ku + DNA-PKcs + PAXX. ( H ) Histogram of DNA extension change, Δ l , upon synapsis rupture in the presence of Ku + DNA-PKcs + PAXX. Green line and red line are from a fit to a double Gaussian distribution, with a peak (green) at 1843 nm displaying a 57 nm standard deviation ( n = 10 events), and a peak (red) at 433 nm displaying a 56 nm standard deviation ( n = 392 events). ( I ) Schematic model for the role of LINP1 from single-molecule studies.

    Article Snippet: To test the ligation of DNA molecules, 0.1 U/μl of T4 DNA ligase was added, after which the ligated DNA was cleaved using either 0.2 U/μl XmaI or 0.1 U/μl of SmaI (New England Biolabs).

    Techniques: Construct, Non-Homologous End Joining, Standard Deviation, Ligation

    Transgenerational CRISPR-Cas9 activity induces new mutations in the TaGW2 and TaLpx-1 genes. NGS reads flanking the GW2T2 target site and their frequencies in (A) T 0 line GLM-2, (B) T 1 line GLM-2-9, and (C) T 2 line GLM-2-9-49 are shown. (D) Restriction enzyme digestion of polymerase chain reaction (PCR) amplicons to screen gw2 knockout mutations in the T 3 progenies of line GLM-2-9-49. The GW2T2 flanking region was amplified by PCR and digested with XmaI; non-digested PCR amplicons correspond to mutated GW2T2 target sites. The numbers on the gel image are identifiers of the GLM-2-9-49 progenies. Lanes marked with arrows are PCR products from wild-type plant not digested with XmaI and loaded as controls; the knockout mutant plant was marked with a star. BW, wild-type cultivar Bobwhite. (E) Sanger sequencing of PCR-amplified GW2T2 target sites of T 3 line GLM-2-9-49-28. Genome specific primers were used to amplify regions flanking the GW2T2 target sites. Nucleotide substitutions are marked with red rectangles, and the inserted nucleotide is shown by the red arrow. Types and frequencies of mutations at the GW2T2, LPX1T2, and MLOT1 target sites in (F) T 1 line GLM-2-5, and (G) T 2 line GLM-2-5-24 are shown. WT, wild-type alleles in wheat cultivar Bobwhite; “–” and “+” signs and numbers after them, nucleotides deleted and inserted, respectively. The frequency of each mutation type is shown on the right. The PAM sequences are underlined; the deleted nucleotides are shown with red dashed lines; the insertions and deletions are highlighted in red.

    Journal: The Crispr Journal

    Article Title: Transgenerational CRISPR-Cas9 Activity Facilitates Multiplex Gene Editing in Allopolyploid Wheat

    doi: 10.1089/crispr.2017.0010

    Figure Lengend Snippet: Transgenerational CRISPR-Cas9 activity induces new mutations in the TaGW2 and TaLpx-1 genes. NGS reads flanking the GW2T2 target site and their frequencies in (A) T 0 line GLM-2, (B) T 1 line GLM-2-9, and (C) T 2 line GLM-2-9-49 are shown. (D) Restriction enzyme digestion of polymerase chain reaction (PCR) amplicons to screen gw2 knockout mutations in the T 3 progenies of line GLM-2-9-49. The GW2T2 flanking region was amplified by PCR and digested with XmaI; non-digested PCR amplicons correspond to mutated GW2T2 target sites. The numbers on the gel image are identifiers of the GLM-2-9-49 progenies. Lanes marked with arrows are PCR products from wild-type plant not digested with XmaI and loaded as controls; the knockout mutant plant was marked with a star. BW, wild-type cultivar Bobwhite. (E) Sanger sequencing of PCR-amplified GW2T2 target sites of T 3 line GLM-2-9-49-28. Genome specific primers were used to amplify regions flanking the GW2T2 target sites. Nucleotide substitutions are marked with red rectangles, and the inserted nucleotide is shown by the red arrow. Types and frequencies of mutations at the GW2T2, LPX1T2, and MLOT1 target sites in (F) T 1 line GLM-2-5, and (G) T 2 line GLM-2-5-24 are shown. WT, wild-type alleles in wheat cultivar Bobwhite; “–” and “+” signs and numbers after them, nucleotides deleted and inserted, respectively. The frequency of each mutation type is shown on the right. The PAM sequences are underlined; the deleted nucleotides are shown with red dashed lines; the insertions and deletions are highlighted in red.

    Article Snippet: Screening of gw2 knockout mutants To screen the gw2 knockout mutants, the GW2T2 target region from all three homoeologs was amplified, and PCR products were digested with XmaI (NEB).

    Techniques: CRISPR, Activity Assay, Next-Generation Sequencing, Polymerase Chain Reaction, Knock-Out, Amplification, Mutagenesis, Sequencing

    Construction of pGAY-28. The modification of pET-28 to replace the multiple cloning region (MCR) with a LIC cassette was accomplished in five steps. In step (1), the parent pET-28 vector is amplified in three segments: A, B, and C. Segment A contains a region homologous to the 3′-end of the linearized yeast shuttle vector YEpADH2p (Y-3′). Segment B contains the LIC cassette at its 3′-end. Segment C contains the LIC cassette at its 5′-end, and a region homologous to the 5′-end of YEpADH2p (Y-5′). In step (2), transformation of linearized YEpADH2p and the three amplified segments into competent S. cerevisiae leads to step (3), where the overlapping segments undergo homologous recombination in vivo . In step (4), two of the original primers from step (1) are used again to amplify the modified expression vector using “colony PCR”. Since these primers were originally designed to anneal upstream of a single XmaI restriction site, step (5) involves digestion of the amplicon with XmaI followed by treatment with DNA ligase, yielding the complete pGAY-28 expression vector.

    Journal: Plasmid

    Article Title: Rapid modification of the pET-28 expression vector for ligation independent cloning using homologous recombination in Saccharomyces cerevisiae

    doi: 10.1016/j.plasmid.2014.09.005

    Figure Lengend Snippet: Construction of pGAY-28. The modification of pET-28 to replace the multiple cloning region (MCR) with a LIC cassette was accomplished in five steps. In step (1), the parent pET-28 vector is amplified in three segments: A, B, and C. Segment A contains a region homologous to the 3′-end of the linearized yeast shuttle vector YEpADH2p (Y-3′). Segment B contains the LIC cassette at its 3′-end. Segment C contains the LIC cassette at its 5′-end, and a region homologous to the 5′-end of YEpADH2p (Y-5′). In step (2), transformation of linearized YEpADH2p and the three amplified segments into competent S. cerevisiae leads to step (3), where the overlapping segments undergo homologous recombination in vivo . In step (4), two of the original primers from step (1) are used again to amplify the modified expression vector using “colony PCR”. Since these primers were originally designed to anneal upstream of a single XmaI restriction site, step (5) involves digestion of the amplicon with XmaI followed by treatment with DNA ligase, yielding the complete pGAY-28 expression vector.

    Article Snippet: The 5′ and 3′ ends of this amplicon contained the XmaI restriction site native to the original pET-28b(+) vector, and therefore restriction with XmaI followed by ligation with T4 DNA ligase (NEB) yielded the complete pGAY-28 expression vector that was transformed into competent E. coli TOP10 cells.

    Techniques: Modification, Positron Emission Tomography, Clone Assay, Plasmid Preparation, Amplification, Transformation Assay, Homologous Recombination, In Vivo, Expressing, Polymerase Chain Reaction