Plasmid Preparation:Article Title: Applications of recombined ScCas9 enzymes for PAM-free DNA modification
Article Snippet: To generate SpRYc, the N-terminal ORF of Sc++ (Addgene Plasmid #155011), corresponding to residues (1-1119) was PCR amplified and assembled using Gibson Assembly into the pCMV-T7-SpRY-P2A-EGFP backbone (Addgene Plasmid #139989), preserving residues 1111-1368 of SpRY's ORF. pCMV-T7-SpCas9-P2A-EGFP (Addgene Plasmid #139987) was used for SpCas9, and Sc++ was similarly integrated within the backbone. .. Analogously, the ORFs of SpCas9, SpRY, and SpRYc were integrated within the ABE8e (Addgene Plasmid #138489) and AncBE4Max (Addgene Plasmid #112094) backbones. sgRNA plasmids were constructed by annealing oligonucleotides coding for crRNA sequences (Tables 1 and 2) as well as 4 bp overhangs, and subsequently performing a T4 DNA Ligase-mediated ligation reaction into a plasmid backbone immediately downstream of the human U6 promoter sequence. ..
Article Title: The Protein Tyrosine Phosphatase CD45 promotes PMN Transepithelial Migration, Antimicrobial Function and Colonic Mucosal Repair
Article Snippet: .. The SpCas9 of lentiCRISPR V2 (Addgene plasmid # 52961) was modified to generate a plasmid containing the high fidelity eSpCas9 1.1 ( ) termed lentiCRISPR eSpCAS9. ..
Article Title: The BRCA1-A complex restricts replication fork reversal-dependent DNA repair in ATM deficient cells
Article Snippet: .. For SpCas9, stable cell lines expressing SpCas9 were first generated by lentiviral transduction with lenti-SpCas9 hygro (Addgene, plasmid #104995), and subsequent selection with hygromycin (500 μg/ml). sgRNA guides were cloned into either lentiGuide-Puro (Addgene, plasmid #52963) or lentiGuide-neo (U6-sgRNA-P2A-Neo, reconstructed from Addgene plasmid #52963) vector digested with BsmBI-v2 (New England Biolabs) following the protocol described previously . sgRNA plasmids (2.7 μg) were packaged into lentivirus particles by co-transfecting with packaging plasmids psPAX2 (2 μg, Addgene, plasmid #12260) and pMD2.G (1.3 μg, Addgene, plasmid #12259) into HEK293T cells grown in a 6-well plate using lipofectamine 2000 (Invitrogen). .. Lentivirus supernatant was collected 48 h post-transfection, filtered through a 0.45-μm filter (Millipore) and subsequently used to transduce the target cells using polybrene (8 μg/ml, Sigma-Aldrich) by spin infection at 2000 rpm for 90 min.
Article Title: Compositions for use in treating autosomal dominant BEST1-related retinopathies
Article Snippet: .. In addition, the px458 plasmid carrying the sgRNA 581G-17nt sequence (SEQ ID NO: 13) expresses both, the SpCas9 and the red fluorescent protein mcherry (Addgene #64324), while the px458 plasmid carrying sgRNA 749A-20nt (SEQ ID NO: 55) expresses the SpCas9 as well as the green fluorescent protein GFP (Addgene #48138). ..
Construct:Article Title: Applications of recombined ScCas9 enzymes for PAM-free DNA modification
Article Snippet: To generate SpRYc, the N-terminal ORF of Sc++ (Addgene Plasmid #155011), corresponding to residues (1-1119) was PCR amplified and assembled using Gibson Assembly into the pCMV-T7-SpRY-P2A-EGFP backbone (Addgene Plasmid #139989), preserving residues 1111-1368 of SpRY's ORF. pCMV-T7-SpCas9-P2A-EGFP (Addgene Plasmid #139987) was used for SpCas9, and Sc++ was similarly integrated within the backbone. .. Analogously, the ORFs of SpCas9, SpRY, and SpRYc were integrated within the ABE8e (Addgene Plasmid #138489) and AncBE4Max (Addgene Plasmid #112094) backbones. sgRNA plasmids were constructed by annealing oligonucleotides coding for crRNA sequences (Tables 1 and 2) as well as 4 bp overhangs, and subsequently performing a T4 DNA Ligase-mediated ligation reaction into a plasmid backbone immediately downstream of the human U6 promoter sequence. ..
Ligation:Article Title: Applications of recombined ScCas9 enzymes for PAM-free DNA modification
Article Snippet: To generate SpRYc, the N-terminal ORF of Sc++ (Addgene Plasmid #155011), corresponding to residues (1-1119) was PCR amplified and assembled using Gibson Assembly into the pCMV-T7-SpRY-P2A-EGFP backbone (Addgene Plasmid #139989), preserving residues 1111-1368 of SpRY's ORF. pCMV-T7-SpCas9-P2A-EGFP (Addgene Plasmid #139987) was used for SpCas9, and Sc++ was similarly integrated within the backbone. .. Analogously, the ORFs of SpCas9, SpRY, and SpRYc were integrated within the ABE8e (Addgene Plasmid #138489) and AncBE4Max (Addgene Plasmid #112094) backbones. sgRNA plasmids were constructed by annealing oligonucleotides coding for crRNA sequences (Tables 1 and 2) as well as 4 bp overhangs, and subsequently performing a T4 DNA Ligase-mediated ligation reaction into a plasmid backbone immediately downstream of the human U6 promoter sequence. ..
Sequencing:Article Title: Applications of recombined ScCas9 enzymes for PAM-free DNA modification
Article Snippet: To generate SpRYc, the N-terminal ORF of Sc++ (Addgene Plasmid #155011), corresponding to residues (1-1119) was PCR amplified and assembled using Gibson Assembly into the pCMV-T7-SpRY-P2A-EGFP backbone (Addgene Plasmid #139989), preserving residues 1111-1368 of SpRY's ORF. pCMV-T7-SpCas9-P2A-EGFP (Addgene Plasmid #139987) was used for SpCas9, and Sc++ was similarly integrated within the backbone. .. Analogously, the ORFs of SpCas9, SpRY, and SpRYc were integrated within the ABE8e (Addgene Plasmid #138489) and AncBE4Max (Addgene Plasmid #112094) backbones. sgRNA plasmids were constructed by annealing oligonucleotides coding for crRNA sequences (Tables 1 and 2) as well as 4 bp overhangs, and subsequently performing a T4 DNA Ligase-mediated ligation reaction into a plasmid backbone immediately downstream of the human U6 promoter sequence. ..
Article Title: Compositions for use in treating autosomal dominant BEST1-related retinopathies
Article Snippet: .. In addition, the px458 plasmid carrying the sgRNA 581G-17nt sequence (SEQ ID NO: 13) expresses both, the SpCas9 and the red fluorescent protein mcherry (Addgene #64324), while the px458 plasmid carrying sgRNA 749A-20nt (SEQ ID NO: 55) expresses the SpCas9 as well as the green fluorescent protein GFP (Addgene #48138). ..
Modification:Article Title: The Protein Tyrosine Phosphatase CD45 promotes PMN Transepithelial Migration, Antimicrobial Function and Colonic Mucosal Repair
Article Snippet: .. The SpCas9 of lentiCRISPR V2 (Addgene plasmid # 52961) was modified to generate a plasmid containing the high fidelity eSpCas9 1.1 ( ) termed lentiCRISPR eSpCAS9. ..
Clone Assay:
Article Title: The BRCA1-A complex restricts replication fork reversal-dependent DNA repair in ATM deficient cells
Article Snippet: .. For SpCas9, stable cell lines expressing SpCas9 were first generated by lentiviral transduction with lenti-SpCas9 hygro (Addgene, plasmid #104995), and subsequent selection with hygromycin (500 μg/ml). sgRNA guides were cloned into either lentiGuide-Puro (Addgene, plasmid #52963) or lentiGuide-neo (U6-sgRNA-P2A-Neo, reconstructed from Addgene plasmid #52963) vector digested with BsmBI-v2 (New England Biolabs) following the protocol described previously . sgRNA plasmids (2.7 μg) were packaged into lentivirus particles by co-transfecting with packaging plasmids psPAX2 (2 μg, Addgene, plasmid #12260) and pMD2.G (1.3 μg, Addgene, plasmid #12259) into HEK293T cells grown in a 6-well plate using lipofectamine 2000 (Invitrogen). .. Lentivirus supernatant was collected 48 h post-transfection, filtered through a 0.45-μm filter (Millipore) and subsequently used to transduce the target cells using polybrene (8 μg/ml, Sigma-Aldrich) by spin infection at 2000 rpm for 90 min.
Expressing:
Article Title: The BRCA1-A complex restricts replication fork reversal-dependent DNA repair in ATM deficient cells
Article Snippet: .. For SpCas9, stable cell lines expressing SpCas9 were first generated by lentiviral transduction with lenti-SpCas9 hygro (Addgene, plasmid #104995), and subsequent selection with hygromycin (500 μg/ml). sgRNA guides were cloned into either lentiGuide-Puro (Addgene, plasmid #52963) or lentiGuide-neo (U6-sgRNA-P2A-Neo, reconstructed from Addgene plasmid #52963) vector digested with BsmBI-v2 (New England Biolabs) following the protocol described previously . sgRNA plasmids (2.7 μg) were packaged into lentivirus particles by co-transfecting with packaging plasmids psPAX2 (2 μg, Addgene, plasmid #12260) and pMD2.G (1.3 μg, Addgene, plasmid #12259) into HEK293T cells grown in a 6-well plate using lipofectamine 2000 (Invitrogen). .. Lentivirus supernatant was collected 48 h post-transfection, filtered through a 0.45-μm filter (Millipore) and subsequently used to transduce the target cells using polybrene (8 μg/ml, Sigma-Aldrich) by spin infection at 2000 rpm for 90 min.
Stable Transfection:Article Title: The BRCA1-A complex restricts replication fork reversal-dependent DNA repair in ATM deficient cells
Article Snippet: .. For SpCas9, stable cell lines expressing SpCas9 were first generated by lentiviral transduction with lenti-SpCas9 hygro (Addgene, plasmid #104995), and subsequent selection with hygromycin (500 μg/ml). sgRNA guides were cloned into either lentiGuide-Puro (Addgene, plasmid #52963) or lentiGuide-neo (U6-sgRNA-P2A-Neo, reconstructed from Addgene plasmid #52963) vector digested with BsmBI-v2 (New England Biolabs) following the protocol described previously . sgRNA plasmids (2.7 μg) were packaged into lentivirus particles by co-transfecting with packaging plasmids psPAX2 (2 μg, Addgene, plasmid #12260) and pMD2.G (1.3 μg, Addgene, plasmid #12259) into HEK293T cells grown in a 6-well plate using lipofectamine 2000 (Invitrogen). .. Lentivirus supernatant was collected 48 h post-transfection, filtered through a 0.45-μm filter (Millipore) and subsequently used to transduce the target cells using polybrene (8 μg/ml, Sigma-Aldrich) by spin infection at 2000 rpm for 90 min.
Generated:Article Title: The BRCA1-A complex restricts replication fork reversal-dependent DNA repair in ATM deficient cells
Article Snippet: .. For SpCas9, stable cell lines expressing SpCas9 were first generated by lentiviral transduction with lenti-SpCas9 hygro (Addgene, plasmid #104995), and subsequent selection with hygromycin (500 μg/ml). sgRNA guides were cloned into either lentiGuide-Puro (Addgene, plasmid #52963) or lentiGuide-neo (U6-sgRNA-P2A-Neo, reconstructed from Addgene plasmid #52963) vector digested with BsmBI-v2 (New England Biolabs) following the protocol described previously . sgRNA plasmids (2.7 μg) were packaged into lentivirus particles by co-transfecting with packaging plasmids psPAX2 (2 μg, Addgene, plasmid #12260) and pMD2.G (1.3 μg, Addgene, plasmid #12259) into HEK293T cells grown in a 6-well plate using lipofectamine 2000 (Invitrogen). .. Lentivirus supernatant was collected 48 h post-transfection, filtered through a 0.45-μm filter (Millipore) and subsequently used to transduce the target cells using polybrene (8 μg/ml, Sigma-Aldrich) by spin infection at 2000 rpm for 90 min.
Transduction:Article Title: The BRCA1-A complex restricts replication fork reversal-dependent DNA repair in ATM deficient cells
Article Snippet: .. For SpCas9, stable cell lines expressing SpCas9 were first generated by lentiviral transduction with lenti-SpCas9 hygro (Addgene, plasmid #104995), and subsequent selection with hygromycin (500 μg/ml). sgRNA guides were cloned into either lentiGuide-Puro (Addgene, plasmid #52963) or lentiGuide-neo (U6-sgRNA-P2A-Neo, reconstructed from Addgene plasmid #52963) vector digested with BsmBI-v2 (New England Biolabs) following the protocol described previously . sgRNA plasmids (2.7 μg) were packaged into lentivirus particles by co-transfecting with packaging plasmids psPAX2 (2 μg, Addgene, plasmid #12260) and pMD2.G (1.3 μg, Addgene, plasmid #12259) into HEK293T cells grown in a 6-well plate using lipofectamine 2000 (Invitrogen). .. Lentivirus supernatant was collected 48 h post-transfection, filtered through a 0.45-μm filter (Millipore) and subsequently used to transduce the target cells using polybrene (8 μg/ml, Sigma-Aldrich) by spin infection at 2000 rpm for 90 min.
Selection:Article Title: The BRCA1-A complex restricts replication fork reversal-dependent DNA repair in ATM deficient cells
Article Snippet: .. For SpCas9, stable cell lines expressing SpCas9 were first generated by lentiviral transduction with lenti-SpCas9 hygro (Addgene, plasmid #104995), and subsequent selection with hygromycin (500 μg/ml). sgRNA guides were cloned into either lentiGuide-Puro (Addgene, plasmid #52963) or lentiGuide-neo (U6-sgRNA-P2A-Neo, reconstructed from Addgene plasmid #52963) vector digested with BsmBI-v2 (New England Biolabs) following the protocol described previously . sgRNA plasmids (2.7 μg) were packaged into lentivirus particles by co-transfecting with packaging plasmids psPAX2 (2 μg, Addgene, plasmid #12260) and pMD2.G (1.3 μg, Addgene, plasmid #12259) into HEK293T cells grown in a 6-well plate using lipofectamine 2000 (Invitrogen). .. Lentivirus supernatant was collected 48 h post-transfection, filtered through a 0.45-μm filter (Millipore) and subsequently used to transduce the target cells using polybrene (8 μg/ml, Sigma-Aldrich) by spin infection at 2000 rpm for 90 min.
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