Journal: Scientific Reports

Article Title: A novel tool for monitoring endogenous alpha-synuclein transcription by NanoLuciferase tag insertion at the 3′end using CRISPR-Cas9 genome editing technique

doi: 10.1038/srep45883

Figure Lengend Snippet: ( a ) Schematic representation of cloning strategy. SNCA gene map showing exons (1a and 1b non-coding, 2–6 coding) and the 3′UTR. Transfection of sgRNA targeting the 3′end of exon 6 induces a DSB near the stop codon (TAA). Donor vector design contains 5′ homology arm of 790 bp encompassing part of intron 5 and exon 6 upstream from the stop codon and the NanoLuc-3′ homology arm of 800 bp downstream of the stop codon containing part of the 3′UTR. Co-transfection of donor vector with the CRISPR/Cas9 construct precisely incorporated the NanoLuc right before the stop codon by HDR of the SNCA gene. ( b ) Following puromycin selection and single cell dilution, genomic DNA from all surviving isogenic colonies were screened for the NanoLuc insert with pNL1.1 NanoLuc vector and HEK293T LVX cells as controls. From 15 colonies recovered, two were positive for the NanoLuc insertion. ( c ) Gene specific PCR with primers in the intron 5 (A) and the 3′UTR of SNCA showed colony 9 had a heterozygous insertion in 293T-SNCA-3′NL cells (Lane 1); PCR with forward primer on the NanoLuc (B or NanoLuc Internal Forward Primer) and the same 3′UTR reverse primer (cDNA sequencing Reverse Primer) showed comparable amplification of the NanoLuc tagged allele (Lane 2); PCR of the wild-type α-SYN and NanoLuc from the HEK293T LVX as controls (Insertion Confirmation Forward Primer and cDNA sequencing Reverse Primer) (Lanes 3 and 4) ( d ) Excerpt of Sanger sequencing results showing insertion of the NanoLuc sequence with restriction sites precisely before the stop codon of SNCA and with correct continuation of the 3′UTR after the NanoLuc sequence.

Article Snippet: Upstream homology arm and the NanoLuc were ligated together and cloned into NotI and AatII digested pGEM -T Easy vector (Promega Corporation; cat no. A137A) and transformed into competent DH5α cells.

Techniques: Cloning, Transfection, Plasmid Preparation, Cotransfection, CRISPR, Construct, Selection, Sequencing, Amplification

Journal: Scientific Reports

Article Title: A novel tool for monitoring endogenous alpha-synuclein transcription by NanoLuciferase tag insertion at the 3′end using CRISPR-Cas9 genome editing technique

doi: 10.1038/srep45883

Figure Lengend Snippet: Sequences of the oligo used for the generation and confirmation of 293T- SNCA -3′NL cells.

Article Snippet: Upstream homology arm and the NanoLuc were ligated together and cloned into NotI and AatII digested pGEM -T Easy vector (Promega Corporation; cat no. A137A) and transformed into competent DH5α cells.

Techniques: Sequencing

Journal: Scientific Reports

Article Title: A novel tool for monitoring endogenous alpha-synuclein transcription by NanoLuciferase tag insertion at the 3′end using CRISPR-Cas9 genome editing technique

doi: 10.1038/srep45883

Figure Lengend Snippet: ( a ) 293T- SNCA -3′NL cells treated with 10 μM 5-AzadC for 72 hours which induced a significant increase in the NanoLuc activity as compared to the control. The NanoLuc activity was corroborated by increase in α-SYN transcript as shown in the RT-PCR. ( b ) Methylation status of 23 CpG sites on the SNCA intron1 was determined by bisulfite sequencing. Amplified PCR products were cloned into pGEM-T Easy vector and 10 clones were sequenced. (Left) Comparison of vehicle and 5-AzadC treatment (10 μM, 72 hours) showing unmethylated (open circles) and methylated (closed circles) cytosines for all 10 clones (y-axis) at each of the 23 CpGs in intron1 (x-axis). (Right) Scatter plot showing overall decrease in methylation by 31.7% compared to the control. ( c ) Similarly, 293T- SNCA -3′NL cells treated with dopamine at 100 μM concentration for 48 hours, increased NanoLuc activity significantly. Increase in the NanoLuc activity was confirmed by RT-PCR after dopamine treatment. ( d ) Following HDAC inhibitor (sodium butyrate) treatment at concentrations of 2.5 mM and 5.0 mM for 24 hours, the 293T- SNCA -3′NL cells showed a significant dose dependent increase in the NanoLuc activity. This dose-dependent increase in the NanoLuc activity was also confirmed by RT-PCR following same treatment paradigm. β-actin amplification was used as an internal control for all the PCRs. Error bars show the mean from three technical repeats. p values are given for t-test (5-AzadC, dopamine), one-way ANOVA (Sodium butyrate) where *represents p < 0.05, **represents p < 0.01, ***represents p < 0.0001.

Article Snippet: Upstream homology arm and the NanoLuc were ligated together and cloned into NotI and AatII digested pGEM -T Easy vector (Promega Corporation; cat no. A137A) and transformed into competent DH5α cells.

Techniques: Activity Assay, Control, Reverse Transcription Polymerase Chain Reaction, Methylation, Methylation Sequencing, Amplification, Clone Assay, Plasmid Preparation, Comparison, Concentration Assay