px458 plasmid vector Search Results


px458 bbs i cip treated vector plasmid  (New England Biolabs)
98
New England Biolabs px458 bbs i cip treated vector plasmid
A , CRISPR/Cas9 strategy to introduce Egr‐1 mutation. Using CRISPR/Cas9 single‐guide RNA (sgRNA) guide design platform ( http://crispr.mit.edu ), a guide was selected that gave a high score and where protospacer adjacent motif (PAM) silent mutation was possible. A target on the reverse strand (indicated by the thick black line) was chosen and annealed oligonucleotides cloned into the <t>PX458</t> (digested with Bbs I). The generated PX‐458‐Egr‐1 was transfected into human microvascular endothelial cells together with the donor nucleotide to introduce a silent PAM mutation and point mutation of serine residue in Egr‐1 at position 26 (Ser26)>Ala. This CRISPR/Cas9 system generated indels by nonhomologous end joining and the desired mutation via homology‐directed repair (HDR). Transformants were sorted by fluorescence‐activated cell sorting, and single cells with green fluorescent protein were grown and later screened. The target sequence (blue), not including the PAM site (yellow), was inserted into the guide RNA (gRNA). sgRNA contains the custom‐designed sequence fused to the scaffold RNA sequence. Sequences of the target genomic DNA and donor oligonucleotide were shown. B , Alignment of amino acid sequences of different CRISPR/Cas9 clones with that of human Egr‐1 (P18146). Multiple sequence alignment with hierarchical clustering was done using MultAlin software ( http://multalin.toulouse.inra.fr/multalin/ ). C, In silico structure prediction by trRosetta of the amino‐terminal region of Egr‐1. The amino‐terminus of the protein starts at the helix. The first 63 amino acid residues of Egr‐1 were entered into trRosetta. Ser26 (yellow) is predicted to lie in a loop region between 2 helices. Wild‐type (WT) (with Ser26) and mutant (MUT) (Ala26) Egr‐1 sequence were run through the trRosetta program, and images were generated (using Pymol software). The WT model (shown in orange) was aligned structurally with the MUT model (blue). Residue 26 is highlighted in yellow. CVM indicates cytomegalovirus; Cas9, caspase 9; DEL, deletion; EGFP, enhanced green fluorescent protein; Egr‐1, early growth response‐1; and U6, the U6 promoter.
Px458 Bbs I Cip Treated Vector Plasmid, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 98/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/px458 bbs i cip treated vector plasmid/product/New England Biolabs
Average 98 stars, based on 1 article reviews
px458 bbs i cip treated vector plasmid - by Bioz Stars, 2026-03
98/100 stars
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vector pspcas9 bb 2a gfp  (Addgene inc)
96
Addgene inc vector pspcas9 bb 2a gfp
A , CRISPR/Cas9 strategy to introduce Egr‐1 mutation. Using CRISPR/Cas9 single‐guide RNA (sgRNA) guide design platform ( http://crispr.mit.edu ), a guide was selected that gave a high score and where protospacer adjacent motif (PAM) silent mutation was possible. A target on the reverse strand (indicated by the thick black line) was chosen and annealed oligonucleotides cloned into the <t>PX458</t> (digested with Bbs I). The generated PX‐458‐Egr‐1 was transfected into human microvascular endothelial cells together with the donor nucleotide to introduce a silent PAM mutation and point mutation of serine residue in Egr‐1 at position 26 (Ser26)>Ala. This CRISPR/Cas9 system generated indels by nonhomologous end joining and the desired mutation via homology‐directed repair (HDR). Transformants were sorted by fluorescence‐activated cell sorting, and single cells with green fluorescent protein were grown and later screened. The target sequence (blue), not including the PAM site (yellow), was inserted into the guide RNA (gRNA). sgRNA contains the custom‐designed sequence fused to the scaffold RNA sequence. Sequences of the target genomic DNA and donor oligonucleotide were shown. B , Alignment of amino acid sequences of different CRISPR/Cas9 clones with that of human Egr‐1 (P18146). Multiple sequence alignment with hierarchical clustering was done using MultAlin software ( http://multalin.toulouse.inra.fr/multalin/ ). C, In silico structure prediction by trRosetta of the amino‐terminal region of Egr‐1. The amino‐terminus of the protein starts at the helix. The first 63 amino acid residues of Egr‐1 were entered into trRosetta. Ser26 (yellow) is predicted to lie in a loop region between 2 helices. Wild‐type (WT) (with Ser26) and mutant (MUT) (Ala26) Egr‐1 sequence were run through the trRosetta program, and images were generated (using Pymol software). The WT model (shown in orange) was aligned structurally with the MUT model (blue). Residue 26 is highlighted in yellow. CVM indicates cytomegalovirus; Cas9, caspase 9; DEL, deletion; EGFP, enhanced green fluorescent protein; Egr‐1, early growth response‐1; and U6, the U6 promoter.
Vector Pspcas9 Bb 2a Gfp, supplied by Addgene inc, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/vector pspcas9 bb 2a gfp/product/Addgene inc
Average 96 stars, based on 1 article reviews
vector pspcas9 bb 2a gfp - by Bioz Stars, 2026-03
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cas9 expression vector px458 pspcas9 bb 2agfp  (Addgene inc)
93
Addgene inc cas9 expression vector px458 pspcas9 bb 2agfp
A , CRISPR/Cas9 strategy to introduce Egr‐1 mutation. Using CRISPR/Cas9 single‐guide RNA (sgRNA) guide design platform ( http://crispr.mit.edu ), a guide was selected that gave a high score and where protospacer adjacent motif (PAM) silent mutation was possible. A target on the reverse strand (indicated by the thick black line) was chosen and annealed oligonucleotides cloned into the <t>PX458</t> (digested with Bbs I). The generated PX‐458‐Egr‐1 was transfected into human microvascular endothelial cells together with the donor nucleotide to introduce a silent PAM mutation and point mutation of serine residue in Egr‐1 at position 26 (Ser26)>Ala. This CRISPR/Cas9 system generated indels by nonhomologous end joining and the desired mutation via homology‐directed repair (HDR). Transformants were sorted by fluorescence‐activated cell sorting, and single cells with green fluorescent protein were grown and later screened. The target sequence (blue), not including the PAM site (yellow), was inserted into the guide RNA (gRNA). sgRNA contains the custom‐designed sequence fused to the scaffold RNA sequence. Sequences of the target genomic DNA and donor oligonucleotide were shown. B , Alignment of amino acid sequences of different CRISPR/Cas9 clones with that of human Egr‐1 (P18146). Multiple sequence alignment with hierarchical clustering was done using MultAlin software ( http://multalin.toulouse.inra.fr/multalin/ ). C, In silico structure prediction by trRosetta of the amino‐terminal region of Egr‐1. The amino‐terminus of the protein starts at the helix. The first 63 amino acid residues of Egr‐1 were entered into trRosetta. Ser26 (yellow) is predicted to lie in a loop region between 2 helices. Wild‐type (WT) (with Ser26) and mutant (MUT) (Ala26) Egr‐1 sequence were run through the trRosetta program, and images were generated (using Pymol software). The WT model (shown in orange) was aligned structurally with the MUT model (blue). Residue 26 is highlighted in yellow. CVM indicates cytomegalovirus; Cas9, caspase 9; DEL, deletion; EGFP, enhanced green fluorescent protein; Egr‐1, early growth response‐1; and U6, the U6 promoter.
Cas9 Expression Vector Px458 Pspcas9 Bb 2agfp, supplied by Addgene inc, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/cas9 expression vector px458 pspcas9 bb 2agfp/product/Addgene inc
Average 93 stars, based on 1 article reviews
cas9 expression vector px458 pspcas9 bb 2agfp - by Bioz Stars, 2026-03
93/100 stars
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vector px458  (Addgene inc)
96
Addgene inc vector px458
A , CRISPR/Cas9 strategy to introduce Egr‐1 mutation. Using CRISPR/Cas9 single‐guide RNA (sgRNA) guide design platform ( http://crispr.mit.edu ), a guide was selected that gave a high score and where protospacer adjacent motif (PAM) silent mutation was possible. A target on the reverse strand (indicated by the thick black line) was chosen and annealed oligonucleotides cloned into the <t>PX458</t> (digested with Bbs I). The generated PX‐458‐Egr‐1 was transfected into human microvascular endothelial cells together with the donor nucleotide to introduce a silent PAM mutation and point mutation of serine residue in Egr‐1 at position 26 (Ser26)>Ala. This CRISPR/Cas9 system generated indels by nonhomologous end joining and the desired mutation via homology‐directed repair (HDR). Transformants were sorted by fluorescence‐activated cell sorting, and single cells with green fluorescent protein were grown and later screened. The target sequence (blue), not including the PAM site (yellow), was inserted into the guide RNA (gRNA). sgRNA contains the custom‐designed sequence fused to the scaffold RNA sequence. Sequences of the target genomic DNA and donor oligonucleotide were shown. B , Alignment of amino acid sequences of different CRISPR/Cas9 clones with that of human Egr‐1 (P18146). Multiple sequence alignment with hierarchical clustering was done using MultAlin software ( http://multalin.toulouse.inra.fr/multalin/ ). C, In silico structure prediction by trRosetta of the amino‐terminal region of Egr‐1. The amino‐terminus of the protein starts at the helix. The first 63 amino acid residues of Egr‐1 were entered into trRosetta. Ser26 (yellow) is predicted to lie in a loop region between 2 helices. Wild‐type (WT) (with Ser26) and mutant (MUT) (Ala26) Egr‐1 sequence were run through the trRosetta program, and images were generated (using Pymol software). The WT model (shown in orange) was aligned structurally with the MUT model (blue). Residue 26 is highlighted in yellow. CVM indicates cytomegalovirus; Cas9, caspase 9; DEL, deletion; EGFP, enhanced green fluorescent protein; Egr‐1, early growth response‐1; and U6, the U6 promoter.
Vector Px458, supplied by Addgene inc, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/vector px458/product/Addgene inc
Average 96 stars, based on 1 article reviews
vector px458 - by Bioz Stars, 2026-03
96/100 stars
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px458 vector  (Addgene inc)
90
Addgene inc px458 vector
A , CRISPR/Cas9 strategy to introduce Egr‐1 mutation. Using CRISPR/Cas9 single‐guide RNA (sgRNA) guide design platform ( http://crispr.mit.edu ), a guide was selected that gave a high score and where protospacer adjacent motif (PAM) silent mutation was possible. A target on the reverse strand (indicated by the thick black line) was chosen and annealed oligonucleotides cloned into the <t>PX458</t> (digested with Bbs I). The generated PX‐458‐Egr‐1 was transfected into human microvascular endothelial cells together with the donor nucleotide to introduce a silent PAM mutation and point mutation of serine residue in Egr‐1 at position 26 (Ser26)>Ala. This CRISPR/Cas9 system generated indels by nonhomologous end joining and the desired mutation via homology‐directed repair (HDR). Transformants were sorted by fluorescence‐activated cell sorting, and single cells with green fluorescent protein were grown and later screened. The target sequence (blue), not including the PAM site (yellow), was inserted into the guide RNA (gRNA). sgRNA contains the custom‐designed sequence fused to the scaffold RNA sequence. Sequences of the target genomic DNA and donor oligonucleotide were shown. B , Alignment of amino acid sequences of different CRISPR/Cas9 clones with that of human Egr‐1 (P18146). Multiple sequence alignment with hierarchical clustering was done using MultAlin software ( http://multalin.toulouse.inra.fr/multalin/ ). C, In silico structure prediction by trRosetta of the amino‐terminal region of Egr‐1. The amino‐terminus of the protein starts at the helix. The first 63 amino acid residues of Egr‐1 were entered into trRosetta. Ser26 (yellow) is predicted to lie in a loop region between 2 helices. Wild‐type (WT) (with Ser26) and mutant (MUT) (Ala26) Egr‐1 sequence were run through the trRosetta program, and images were generated (using Pymol software). The WT model (shown in orange) was aligned structurally with the MUT model (blue). Residue 26 is highlighted in yellow. CVM indicates cytomegalovirus; Cas9, caspase 9; DEL, deletion; EGFP, enhanced green fluorescent protein; Egr‐1, early growth response‐1; and U6, the U6 promoter.
Px458 Vector, supplied by Addgene inc, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/px458 vector/product/Addgene inc
Average 90 stars, based on 1 article reviews
px458 vector - by Bioz Stars, 2026-03
90/100 stars
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px458  (Addgene inc)
94
Addgene inc px458
A , CRISPR/Cas9 strategy to introduce Egr‐1 mutation. Using CRISPR/Cas9 single‐guide RNA (sgRNA) guide design platform ( http://crispr.mit.edu ), a guide was selected that gave a high score and where protospacer adjacent motif (PAM) silent mutation was possible. A target on the reverse strand (indicated by the thick black line) was chosen and annealed oligonucleotides cloned into the <t>PX458</t> (digested with Bbs I). The generated PX‐458‐Egr‐1 was transfected into human microvascular endothelial cells together with the donor nucleotide to introduce a silent PAM mutation and point mutation of serine residue in Egr‐1 at position 26 (Ser26)>Ala. This CRISPR/Cas9 system generated indels by nonhomologous end joining and the desired mutation via homology‐directed repair (HDR). Transformants were sorted by fluorescence‐activated cell sorting, and single cells with green fluorescent protein were grown and later screened. The target sequence (blue), not including the PAM site (yellow), was inserted into the guide RNA (gRNA). sgRNA contains the custom‐designed sequence fused to the scaffold RNA sequence. Sequences of the target genomic DNA and donor oligonucleotide were shown. B , Alignment of amino acid sequences of different CRISPR/Cas9 clones with that of human Egr‐1 (P18146). Multiple sequence alignment with hierarchical clustering was done using MultAlin software ( http://multalin.toulouse.inra.fr/multalin/ ). C, In silico structure prediction by trRosetta of the amino‐terminal region of Egr‐1. The amino‐terminus of the protein starts at the helix. The first 63 amino acid residues of Egr‐1 were entered into trRosetta. Ser26 (yellow) is predicted to lie in a loop region between 2 helices. Wild‐type (WT) (with Ser26) and mutant (MUT) (Ala26) Egr‐1 sequence were run through the trRosetta program, and images were generated (using Pymol software). The WT model (shown in orange) was aligned structurally with the MUT model (blue). Residue 26 is highlighted in yellow. CVM indicates cytomegalovirus; Cas9, caspase 9; DEL, deletion; EGFP, enhanced green fluorescent protein; Egr‐1, early growth response‐1; and U6, the U6 promoter.
Px458, supplied by Addgene inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/px458/product/Addgene inc
Average 94 stars, based on 1 article reviews
px458 - by Bioz Stars, 2026-03
94/100 stars
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plasmid vectors expressing hspcas9  (Addgene inc)
95
Addgene inc plasmid vectors expressing hspcas9
A , CRISPR/Cas9 strategy to introduce Egr‐1 mutation. Using CRISPR/Cas9 single‐guide RNA (sgRNA) guide design platform ( http://crispr.mit.edu ), a guide was selected that gave a high score and where protospacer adjacent motif (PAM) silent mutation was possible. A target on the reverse strand (indicated by the thick black line) was chosen and annealed oligonucleotides cloned into the <t>PX458</t> (digested with Bbs I). The generated PX‐458‐Egr‐1 was transfected into human microvascular endothelial cells together with the donor nucleotide to introduce a silent PAM mutation and point mutation of serine residue in Egr‐1 at position 26 (Ser26)>Ala. This CRISPR/Cas9 system generated indels by nonhomologous end joining and the desired mutation via homology‐directed repair (HDR). Transformants were sorted by fluorescence‐activated cell sorting, and single cells with green fluorescent protein were grown and later screened. The target sequence (blue), not including the PAM site (yellow), was inserted into the guide RNA (gRNA). sgRNA contains the custom‐designed sequence fused to the scaffold RNA sequence. Sequences of the target genomic DNA and donor oligonucleotide were shown. B , Alignment of amino acid sequences of different CRISPR/Cas9 clones with that of human Egr‐1 (P18146). Multiple sequence alignment with hierarchical clustering was done using MultAlin software ( http://multalin.toulouse.inra.fr/multalin/ ). C, In silico structure prediction by trRosetta of the amino‐terminal region of Egr‐1. The amino‐terminus of the protein starts at the helix. The first 63 amino acid residues of Egr‐1 were entered into trRosetta. Ser26 (yellow) is predicted to lie in a loop region between 2 helices. Wild‐type (WT) (with Ser26) and mutant (MUT) (Ala26) Egr‐1 sequence were run through the trRosetta program, and images were generated (using Pymol software). The WT model (shown in orange) was aligned structurally with the MUT model (blue). Residue 26 is highlighted in yellow. CVM indicates cytomegalovirus; Cas9, caspase 9; DEL, deletion; EGFP, enhanced green fluorescent protein; Egr‐1, early growth response‐1; and U6, the U6 promoter.
Plasmid Vectors Expressing Hspcas9, supplied by Addgene inc, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/plasmid vectors expressing hspcas9/product/Addgene inc
Average 95 stars, based on 1 article reviews
plasmid vectors expressing hspcas9 - by Bioz Stars, 2026-03
95/100 stars
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cas9 t2a gfp expression vector  (Addgene inc)
93
Addgene inc cas9 t2a gfp expression vector
A , CRISPR/Cas9 strategy to introduce Egr‐1 mutation. Using CRISPR/Cas9 single‐guide RNA (sgRNA) guide design platform ( http://crispr.mit.edu ), a guide was selected that gave a high score and where protospacer adjacent motif (PAM) silent mutation was possible. A target on the reverse strand (indicated by the thick black line) was chosen and annealed oligonucleotides cloned into the <t>PX458</t> (digested with Bbs I). The generated PX‐458‐Egr‐1 was transfected into human microvascular endothelial cells together with the donor nucleotide to introduce a silent PAM mutation and point mutation of serine residue in Egr‐1 at position 26 (Ser26)>Ala. This CRISPR/Cas9 system generated indels by nonhomologous end joining and the desired mutation via homology‐directed repair (HDR). Transformants were sorted by fluorescence‐activated cell sorting, and single cells with green fluorescent protein were grown and later screened. The target sequence (blue), not including the PAM site (yellow), was inserted into the guide RNA (gRNA). sgRNA contains the custom‐designed sequence fused to the scaffold RNA sequence. Sequences of the target genomic DNA and donor oligonucleotide were shown. B , Alignment of amino acid sequences of different CRISPR/Cas9 clones with that of human Egr‐1 (P18146). Multiple sequence alignment with hierarchical clustering was done using MultAlin software ( http://multalin.toulouse.inra.fr/multalin/ ). C, In silico structure prediction by trRosetta of the amino‐terminal region of Egr‐1. The amino‐terminus of the protein starts at the helix. The first 63 amino acid residues of Egr‐1 were entered into trRosetta. Ser26 (yellow) is predicted to lie in a loop region between 2 helices. Wild‐type (WT) (with Ser26) and mutant (MUT) (Ala26) Egr‐1 sequence were run through the trRosetta program, and images were generated (using Pymol software). The WT model (shown in orange) was aligned structurally with the MUT model (blue). Residue 26 is highlighted in yellow. CVM indicates cytomegalovirus; Cas9, caspase 9; DEL, deletion; EGFP, enhanced green fluorescent protein; Egr‐1, early growth response‐1; and U6, the U6 promoter.
Cas9 T2a Gfp Expression Vector, supplied by Addgene inc, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/cas9 t2a gfp expression vector/product/Addgene inc
Average 93 stars, based on 1 article reviews
cas9 t2a gfp expression vector - by Bioz Stars, 2026-03
93/100 stars
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cas9 expressing vector px458 pwh464  (Addgene inc)
97
Addgene inc cas9 expressing vector px458 pwh464

Cas9 Expressing Vector Px458 Pwh464, supplied by Addgene inc, used in various techniques. Bioz Stars score: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/cas9 expressing vector px458 pwh464/product/Addgene inc
Average 97 stars, based on 1 article reviews
cas9 expressing vector px458 pwh464 - by Bioz Stars, 2026-03
97/100 stars
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px458 vector encoding cas9 nuclease  (Addgene inc)
92
Addgene inc px458 vector encoding cas9 nuclease

Px458 Vector Encoding Cas9 Nuclease, supplied by Addgene inc, used in various techniques. Bioz Stars score: 92/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/px458 vector encoding cas9 nuclease/product/Addgene inc
Average 92 stars, based on 1 article reviews
px458 vector encoding cas9 nuclease - by Bioz Stars, 2026-03
92/100 stars
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Image Search Results


A , CRISPR/Cas9 strategy to introduce Egr‐1 mutation. Using CRISPR/Cas9 single‐guide RNA (sgRNA) guide design platform ( http://crispr.mit.edu ), a guide was selected that gave a high score and where protospacer adjacent motif (PAM) silent mutation was possible. A target on the reverse strand (indicated by the thick black line) was chosen and annealed oligonucleotides cloned into the PX458 (digested with Bbs I). The generated PX‐458‐Egr‐1 was transfected into human microvascular endothelial cells together with the donor nucleotide to introduce a silent PAM mutation and point mutation of serine residue in Egr‐1 at position 26 (Ser26)>Ala. This CRISPR/Cas9 system generated indels by nonhomologous end joining and the desired mutation via homology‐directed repair (HDR). Transformants were sorted by fluorescence‐activated cell sorting, and single cells with green fluorescent protein were grown and later screened. The target sequence (blue), not including the PAM site (yellow), was inserted into the guide RNA (gRNA). sgRNA contains the custom‐designed sequence fused to the scaffold RNA sequence. Sequences of the target genomic DNA and donor oligonucleotide were shown. B , Alignment of amino acid sequences of different CRISPR/Cas9 clones with that of human Egr‐1 (P18146). Multiple sequence alignment with hierarchical clustering was done using MultAlin software ( http://multalin.toulouse.inra.fr/multalin/ ). C, In silico structure prediction by trRosetta of the amino‐terminal region of Egr‐1. The amino‐terminus of the protein starts at the helix. The first 63 amino acid residues of Egr‐1 were entered into trRosetta. Ser26 (yellow) is predicted to lie in a loop region between 2 helices. Wild‐type (WT) (with Ser26) and mutant (MUT) (Ala26) Egr‐1 sequence were run through the trRosetta program, and images were generated (using Pymol software). The WT model (shown in orange) was aligned structurally with the MUT model (blue). Residue 26 is highlighted in yellow. CVM indicates cytomegalovirus; Cas9, caspase 9; DEL, deletion; EGFP, enhanced green fluorescent protein; Egr‐1, early growth response‐1; and U6, the U6 promoter.

Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: Serine 26 in Early Growth Response‐1 Is Critical for Endothelial Proliferation, Migration, and Network Formation

doi: 10.1161/JAHA.120.020521

Figure Lengend Snippet: A , CRISPR/Cas9 strategy to introduce Egr‐1 mutation. Using CRISPR/Cas9 single‐guide RNA (sgRNA) guide design platform ( http://crispr.mit.edu ), a guide was selected that gave a high score and where protospacer adjacent motif (PAM) silent mutation was possible. A target on the reverse strand (indicated by the thick black line) was chosen and annealed oligonucleotides cloned into the PX458 (digested with Bbs I). The generated PX‐458‐Egr‐1 was transfected into human microvascular endothelial cells together with the donor nucleotide to introduce a silent PAM mutation and point mutation of serine residue in Egr‐1 at position 26 (Ser26)>Ala. This CRISPR/Cas9 system generated indels by nonhomologous end joining and the desired mutation via homology‐directed repair (HDR). Transformants were sorted by fluorescence‐activated cell sorting, and single cells with green fluorescent protein were grown and later screened. The target sequence (blue), not including the PAM site (yellow), was inserted into the guide RNA (gRNA). sgRNA contains the custom‐designed sequence fused to the scaffold RNA sequence. Sequences of the target genomic DNA and donor oligonucleotide were shown. B , Alignment of amino acid sequences of different CRISPR/Cas9 clones with that of human Egr‐1 (P18146). Multiple sequence alignment with hierarchical clustering was done using MultAlin software ( http://multalin.toulouse.inra.fr/multalin/ ). C, In silico structure prediction by trRosetta of the amino‐terminal region of Egr‐1. The amino‐terminus of the protein starts at the helix. The first 63 amino acid residues of Egr‐1 were entered into trRosetta. Ser26 (yellow) is predicted to lie in a loop region between 2 helices. Wild‐type (WT) (with Ser26) and mutant (MUT) (Ala26) Egr‐1 sequence were run through the trRosetta program, and images were generated (using Pymol software). The WT model (shown in orange) was aligned structurally with the MUT model (blue). Residue 26 is highlighted in yellow. CVM indicates cytomegalovirus; Cas9, caspase 9; DEL, deletion; EGFP, enhanced green fluorescent protein; Egr‐1, early growth response‐1; and U6, the U6 promoter.

Article Snippet: The annealed oligonucleotide was ligated into the PX458/ Bbs I/CIP‐treated vector plasmid using the Quick T4 DNA ligase (New England Biolabs) and then transformed into XL‐10 Gold ultracompetent cells (Agilent, Santa Clara, CA).

Techniques: CRISPR, Introduce, Mutagenesis, Clone Assay, Generated, Transfection, Fluorescence, FACS, Sequencing, Software, In Silico

Journal: eLife

Article Title: microRNA-mediated regulation of microRNA machinery controls cell fate decisions

doi: 10.7554/eLife.72289

Figure Lengend Snippet:

Article Snippet: Recombinant DNA reagent , sgRNA and Cas9 expressing vector (pX458) pWH464 , Addgene , Cat# 48138 , .

Techniques: Transduction, Reverse Transcription, SYBR Green Assay, Control, ALP Assay, Bicinchoninic Acid Protein Assay, Mutagenesis, Recombinant, Expressing, Plasmid Preparation