length human brca1 coding region  (Addgene inc)

Journal: BMC Biotechnology

Article Title: Enhancement of CRISPR-Cas9 induced precise gene editing by targeting histone H2A-K15 ubiquitination

doi: 10.1186/s12896-020-00650-x

Figure Lengend Snippet: DSB repair assays in TLR reporter cells. a Diagram of DSB repair pathway choice and ubiquitination of histone H2A at DNA double-strand breaks (DSB). Upon DSB induction, regulatory proteins bind to ubiquitin at positions K13 and K15 via ubiquitin-binding domains (UBDs). The 53BP1 and BRCA1 regulatory proteins play an important role in DSB repair pathway choice. DSB repair is executed by repair proteins and leads either to non-homologous end ligation (NHEJ) or homology-directed repair (HDR). If the NHEJ pathway is chosen, the DNA ends religate, frequently associated with nucleotide deletions or insertions. The HDR pathway enables precise sequence modifications if a DNA repair template is available. b The ‘traffic light’ reporter (TLR) system indicates the ratio of DSB repair by NHEJ or HDR. Upon induction of DSBs in the target region using CRISPR-Cas9, RFP is expressed when repair by NHEJ results in deletions that shift translation into the RFP reading frame. Venus expression reports for HDR when an intact Venus coding region is cotransfected. c Gating scheme for BFP positive cells in transfected HEK and hiPS reporter cells. Single cells were gated by using a forward scatter (FSC-H vs. FSC-A) plot. Transfected cells were gated based on expression of BFP from transfected plasmids compared to non-transfected control. d TLR assay in HEK or hiPS reporter cells. At least 10,000 cells were analyzed per sample for the Venus or RFP positive population. Double positive cells in the HEK TLR6 clone are gated using an extra window

Article Snippet: Plasmid pCAG-BRCA1 for expression of BRCA1 was constructed by amplification of 5640 bp PCR fragment, including the full length human BRCA1 coding region, using plasmid pDEST-FRT/T0-GFP-BRCA1 (Addgene ID 71116) as template and primers BRCA1-for and -rev.

Techniques: Ubiquitin Proteomics, Binding Assay, Ligation, Sequencing, CRISPR, Expressing, Transfection, Control

Journal: BMC Biotechnology

Article Title: Enhancement of CRISPR-Cas9 induced precise gene editing by targeting histone H2A-K15 ubiquitination

doi: 10.1186/s12896-020-00650-x

Figure Lengend Snippet: UBD expression vectors and transfection of TLR reporter cells. a UBD fusion proteins can compete with 53BP1 for binding at H2A-K15 and suppress NHEJ. BRCA1-UBD fusion proteins can direct this HDR factor to DSBs, while fusion with Tet repressor (TetR) or Gal4 attract the repair template molecule that include TetO or UAS binding sites, supporting HDR processing. b Plasmids constructed for expression of the Ubiquitin binding domain (UBD) of Rad18 of RNF169 in fusion with the coding region of BRCA1, Tet repressor or Gal4, driven by the CAG promoter. Plasmids include an EF1-BFP reporter gene to facilitate the FACS-based analysis

Article Snippet: Plasmid pCAG-BRCA1 for expression of BRCA1 was constructed by amplification of 5640 bp PCR fragment, including the full length human BRCA1 coding region, using plasmid pDEST-FRT/T0-GFP-BRCA1 (Addgene ID 71116) as template and primers BRCA1-for and -rev.

Techniques: Expressing, Transfection, Binding Assay, Construct, Ubiquitin Proteomics

Journal: BMC Biotechnology

Article Title: Enhancement of CRISPR-Cas9 induced precise gene editing by targeting histone H2A-K15 ubiquitination

doi: 10.1186/s12896-020-00650-x

Figure Lengend Snippet: Fluorescence-based DSB repair assay using Rad18 UBD and RNF169 UBD fusion proteins in HEK TLR6 reporter cells . Fusion constructs for BRCA1 or TetR with Rad18 UBD or RNF169 UBD were cotransfected with the TLR HDR repair template (TLR-donor-tetO), sgRNA and Cas9 into HEK TLR6 cells. The frequency of Venus + cells (green bars) and RFP + cells (red bars) within the population of BFP + cells was measured by FACS analysis 72 h after transfection and used to calculate the ratio of Venus/RFP positive cells. The X-axis shows the transfected samples and the selection of cotransfected plasmids below. Samples 1 and 2 are controls showing the basic frequency of Venus + and RFP + cells upon transfection with Cas9 and sgRNA or in combination with TLR-donor-tetO as repair template. Data from four independent experiments, each with three replicates per sample, are represented as mean values ± SD. Statistical significance of samples 3–16 in comparison to the control sample 2 was determined by two-way ANOVA and Dunnett’s multiple comparison tests with ** P < 0.01, ***P < 0.001 (HDR) and ##P < 0.01, ###P < 0.001 (NHEJ). Raw data are shown in the

Article Snippet: Plasmid pCAG-BRCA1 for expression of BRCA1 was constructed by amplification of 5640 bp PCR fragment, including the full length human BRCA1 coding region, using plasmid pDEST-FRT/T0-GFP-BRCA1 (Addgene ID 71116) as template and primers BRCA1-for and -rev.

Techniques: Fluorescence, Construct, Transfection, Selection, Comparison, Control

Journal: BMC Biotechnology

Article Title: Enhancement of CRISPR-Cas9 induced precise gene editing by targeting histone H2A-K15 ubiquitination

doi: 10.1186/s12896-020-00650-x

Figure Lengend Snippet: Knockin of a triple FLAG Tag into endogenous genes in HEK cells. a Schematic drawing of the targeting strategy at the endogenous LMNA, GABPA, CREB1, and AAVS1 loci. HEK cells were transfected with expression vectors for Cas9, sgRNA and a donor vector with tetO elements for introduction of a triple FLAG sequence into the first or last exon of the GABPA (exon 10), CREB1 (exon 9) or LMNA (exon 1) gene and into the AAVS1 site of the PPP1RC12C (first intron) gene, respectively. Three days after transfection, genomic DNA was isolated and the target region was amplified by a two-step PCR reaction using the indicated primers (arrows, green: Illumina adapter). The secondary PCR products were sequenced by amplicon sequencing. b , c , d , e . DSB repair events were quantified by deep sequencing reads for each target gene. The fraction of reads showing HDR (green bars) or Indel events (red bars) is shown on the Y-axis in relation to the total number of reads showing wildtype or gene editing events and was used to calculate the ratio of HDR/NHEJ DSB repair. The table shows the selection of cotransfected plasmids of each sample for the expression of Cas9, sgRNA and BRCA1- and TetR- with Rad18 UBD or RNF169 UBD fusion proteins. Data are presented as mean values ± SD from two independent experiments. * P < 0.05, **P < 0.01 (HDR) and #P < 0.05, ##P < 0.01 (NHEJ); t-test. Raw data are shown in the

Article Snippet: Plasmid pCAG-BRCA1 for expression of BRCA1 was constructed by amplification of 5640 bp PCR fragment, including the full length human BRCA1 coding region, using plasmid pDEST-FRT/T0-GFP-BRCA1 (Addgene ID 71116) as template and primers BRCA1-for and -rev.

Techniques: Knock-In, FLAG-tag, Transfection, Expressing, Plasmid Preparation, Sequencing, Isolation, Amplification, Selection

Journal: BMC Biotechnology

Article Title: Enhancement of CRISPR-Cas9 induced precise gene editing by targeting histone H2A-K15 ubiquitination

doi: 10.1186/s12896-020-00650-x

Figure Lengend Snippet: Knockin of GFP into the LMNB1 gene. a Schema of the human LMNB1 gene exon 1, Cas9 target site and the donor vector for the insertion of the GFP coding region downstream of the LMNB1 start codon, flanked by 5′- and 3′- homology arms. Upon homologous recombination (HDR) a GFP/LMNB1 fusion protein is produced. b HEK cells were transfected either with the tetO modified LMNB1 targeting vector (LMNB1-donor-tetO) alone or together with an expression vector for LMNB1-sgRNA and Cas9 (sgLMNB1/Cas9) or with expression vectors for BRCA1, BRCA1-Rad18 UBD , BRCA-RNF169 UBD , TetR-Rad18 UBD or TetR-RNF169 UBD as shown in the table. The HDR frequency was determined as the number of GFP positive cells using FACS analysis 10 days after transfection and its relative increase in comparison to the control sample 2 is given as HDR score. Statistical significance of samples 3–10 in comparison to the control sample 2 was determined by ordinary one-way ANOVA and Dunnett’s multiple comparison tests with *P < 0.05, **P < 0.01, *** P < 0.001. Data from three independent experiments, each with three replicates per sample, are presented as mean values ± S.D. Raw data are shown in the

Article Snippet: Plasmid pCAG-BRCA1 for expression of BRCA1 was constructed by amplification of 5640 bp PCR fragment, including the full length human BRCA1 coding region, using plasmid pDEST-FRT/T0-GFP-BRCA1 (Addgene ID 71116) as template and primers BRCA1-for and -rev.

Techniques: Knock-In, Plasmid Preparation, Homologous Recombination, Produced, Transfection, Modification, Expressing, Comparison, Control