A novel tool for monitoring endogenous alpha-synuclein transcription by NanoLuciferase
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1 A novel tool for monitoring endogenous alpha-synuclein transcription by NanoLuciferase tag insertion at the 3 end using CRISPR-Cas9 genome editing technique Sambuddha Basu 1, 3, Levi Adams 1, 3, Subhrangshu Guhathakurta 1*, Yoon-Seong Kim 1,2 Figure Legends Supplementary Figure 1 Sequence alignment of wild type SNCA against 293T-SNCA-3 NL genomic DNA sequence. Part of the sequence alignment is shown depicting the 3 end of the SNCA of 293T-SNCA-3 NL. The wild type (WT) SNCA genomic DNA sequence (reference sequence: GRCh38.p7) was aligned with the sequencing data from 293T-SNCA-3 NL to check for the proper incorporation of the NanoLuc construct at the end of the coding sequence of SNCA. Sequence 1 is expected SNCA genomic DNA, sequence 2 is 293T-SNCA-3 NL sequence. The last exon of SNCA is highlighted in blue. The start and stop codons of NanoLuc are highlighted by yellow and red colors respectively. The aligned bases between both the sequences are marked by star (*). The in-frame incorporation of the NanoLuc can be seen in between the two restriction enzyme sites, SacI and HindIII (underlined) in 293T-SNCA-3 NL cells (2). Sequence alignment was done using Clustal Omega program. Supplementary Figure 2 293T-SNCA-3 NL cdna indicates correct insertion of NanoLuc sequence. a. PCR of SNCA from 293T-SNCA-3 NL cdna using cdna sequencing primers (Table 1) produces two bands, one matching expected wild-type size and one matching expected size for wild-type with NanoLuc insertion (NanoLuc Tagged α-syn). b. Part of the sequence alignment is shown depicting the coding region of the gene and incorporation of the Nanoluc sequence in 293T-SNCA-3 NL cell line. The WT SNCA mrna sequence (reference sequence:
2 NM_ , transcript variant 4) was aligned with the sequencing data from 293T-SNCA- 3 NL to check for the proper incorporation of the NanoLuc construct at the end of the coding sequence of SNCA. Sequence (1) is WT SNCA mrna transcript, sequence (2) is 293T-SNCA- 3 NL sequence. The start and stop codons in the WT SNCA mrna sequence are highlighted by green and red colors respectively. The aligned bases between both the sequences are marked by star (*). The in-frame incorporation of the NanoLuc can be seen in between the two restriction enzyme sites, SacI and HindIII (underlined) in 293T-SNCA-3 NL cells (2). The NanoLuc insert is unique to 293T-SNCA-3 NL cell line and not found in WT SNCA mrna sequence. The new stop codon after the NanoLuc sequence in 293T-SNCA-3 NL cell is marked by yellow. N denotes unread base in the sequence. Sequence alignment was done using Clustal Omega program. Supplementary Figure 3 Full western blot of 293T-SNCA-3 NL cells showing NanoLuc tagged and WT α-synuclein. Full western image from Fig. 2a. -SYN was detected using polyclonal rabbit antibody (Santa Cruz, SC-7011-R). Wild-type -SYN is shown at approximately 15 kda and is present in both wild-type 293T cells and 293T-SNCA-3 NL cells, but absent from 293T-SNCA-knockout cells. NanoLuc-fused -SYN is shown at approximately 34 kda, and only appears in 293T-SNCA-3 NL cells. The unmarked bands present in the blot are non-specific bands that commonly appear with this particular polyclonal antibody. Supplementary Figure 4 Comparable increase of endogenous and NanoLuc-tagged SNCA levels following dopamine treatment. a. Schematic representation of PCR strategy to avoid
3 preferential amplification issues from heterozygous allele sizes. For WT amplification, primers used were SNCA Exon 4 Forward and Insert Confirmation Reverse (Table 1) to give an amplification product size of 338 bp. For NanoLuc specific amplification, primer pair used were NanoLuc Internal Forward and Insert confirmation reverse to get a product size of 356 bp. Elongation time was restricted for NanoLuc specific amplification to prevent competitive wildtype amplification. b, c. Qualitative image of RT-PCR and western blot analyses of 293T- SNCA-3 NL cdna and protein under vehicle and dopamine (100 M) treated condition show comparable increasing trend for both wild-type and NanoLuc tagged -SYN.
4 Supplementary Figure gggagccatttcctatctcattggctgtcagtgctgatgcgtaattga 2 TTAGTGTAAGTGGGGAGCCATTTCCTATCTCATTGGCTGTCAGTGCTGATGCGTAATTGA ************************************************ 1 aacttatactaacagtgtgtgctgtctttttgatttttctaatattaggaagggtatcaa 2 AACTTATACTAACAGTGTGTGCTGCCTTTTTGATTTTTCTAATATTAGGAAGGGTATCAA ************************ *********************************** 1 gactacgaacctgaagccgagctcatggtcttcacactcgaagatttcgttggggactgg 2 GACTACGAACCTGAAGCCGAGCTCATGGTCTTCACACTCGAAGATTTCGTTGGGGACTGG 1 CGACAGACAGCCGGCTACAACCTGGACCAAGTCCTTGAACAGGGAGGTGTGTCCAGTTTG 2 CGACAGACAGCCGGCTACAACCTGGACCAAGTCCTTGAACAGGGAGGTGTGTCCAGTTTG 1 TTTCAGAATCTCGGGGTGTCCGTAACTCCGATCCAAAGGATTGTCCTGAGCGGTGAAAAT 2 TTTCAGAATCTCGGGGTGTCCGTAACTCCGATCCAAAGGATTGTCCTGAGCGGTGAAAAT 1 GGGCTGAAGATCGACATCCATGTCATCATCCCGTATGAAGGTCTGAGCGGCGACCAAATG 2 GGGCTGAAGATCGACATCCATGTCATCATCCCGTATGAAGGTCTGAGCGGCGACCAAATG 1 GGCCAGATCGAAAAAATTTTTAAGGTGGTGTACCCTGTGGATGATCATCACTTTAAGGTG 2 GGCCAGATCGAAAAAATTTTTAAGGTGGTGTACCCTGTGGATGATCATCACTTTAAGGTG 1 ATCCTGCACTATGGCACACTGGTAATCGACGGGGTTACGCCGAACATGATCGACTATTTC 2 ATCCTGCACTATGGCACACTGGTAATCGACGGGGTTACGCCGAACATGATCGACTATTTC 1 GGACGGCCGTATGAAGGCATCGCCGTGTTCGACGGCAAAAAGATCACTGTAACAGGGACC 2 GGACGGCCGTATGAAGGCATCGCCGTGTTCGACGGCAAAAAGATCACTGTAACAGGGACC 1 CTGTGGAACGGCAACAAAATTATCGACGAGCGCCTGATCAACCCCGACGGCTCCCTGCTG 2 CTGTGGAACGGCAACAAAATTATCGACGAGCGCCTGATCAACCCCGACGGCTCCCTGCTG 1 TTCCGAGTAACCATCAACGGAGTGACCGGCTGGCGGCTGTGCGAACGCATTCTGGCGTAA 2 TTCCGAGTAACCATCAACGGAGTGACCGGCTGGCGGCTGTGCGAACGCATTCTGGCGTAA 1 AAGCTTaaatatctttgctcccagtttcttgagatctgctgacagatgttccatcctgta 2 AAGCTTAAATATCTTTGCTCCCAGTTTCTTGAGATCTGCTGACAGATGTTCCATCCTGTA 1 caagtgctcagttccaatgtgcccagtcatgacatttctcaaagtttttacagtgtatct 2 CAAGTGCTCAGTTCCAATGTACCCAGTCATGACATTTCTCAAAGTTTTTACAGTGTATCT ******************** *************************************** 1 cgaagtcttccatcagcagtgattgaagtatctgtacctgcccccactcagcattt CGAAGTCTTCCATCAGCAGTGATTGAAGTATCTGTACCTGCCCCCACTCAGCATTTCGGT ********************************************************
5 Supplementary Figure 2 a b 1 TGGTGTAA-AGGAATTCATTAGCCATGGATGTATTCATGAAAGGACTTTCAAAGGCCAAG 2 TGGTGTAAAGGAATTACATTAGCCATGGATGTATTCATGAAAGGACTTTCAAAGGCCAAG ******** * * * ******************************************** 1 GAGGGAGTTGTGGCTGCTGCTGAGAAAACCAAACAGGGTGTGGCAGAAGCAGCAGGAAAG 2 GAGGGAGTTGTGGCTGCTGCTGAGAAAACCAAACAGGGTGTGGCAGAAGCAGCAGGAAAG 1 ACAAAAGAGGGTGTTCTCTATGTAGGCTCCAAAACCAAGGAGGGAGTGGTGCATGGTGTG 2 ACAAAAGAGGGTGTTCTCTATGTAGGCTCCAAAACCAAGGAGGGAGTGGTGCATGGTGTG 1 GCAACAGTGGCTGAGAAGACCAAAGAGCAAGTGACAAATGTTGGAGGAGCAGTGGTGACG 2 GCAACAGTGGCTGAGAAGACCAAAGAGCAAGTGACAAATGTTGGAGGAGCAGTGGTGACG 1 GGTGTGACAGCAGTAGCCCAGAAGACAGTGGAGGGAGCAGGGAGCATTGCAGCAGCCACT 2 GGTGTGACAGCAGTAGCCCAGAAGACAGTGGAGGGAGCAGGGAGCATTGCAGCAGCCACT 1 GGCTTTGTCAAAAAGGACCAGTTGGGCAAGAATGAAGAAGGAGCCCCACAGGAAGGAATT 2 GGCTTTGTCAAAAAGGACCAGTTGGGCAAGAATGAAGAAGGAGCCCCACAGGAAGGAATT 1 CTGGAAGATATGCCTGTGGATCCTGACAATGAGGCTTATGAAATGCCTTCTGAGGAAGGG 2 CTGGAAGATATGCCTGTGGATCCTGACAATGAGGCTTATGAAATGCCTTCTGAGGAAGGG 1 TATCAAGACTACGAACCTGAAGCCTAA TATCAAGACTACGAACCTGAAGCCGAGCTCATGGTCTTCACACTCGAAGATTTCGTT
6 ************************ 2 GGGGACTGGCGACAGACAGCCGGCTACAACCTGGACCAAGTCCTTGAACAGGGAGGTGTG 2 TCCAGTTTGTTTCAGAATCTCGGGGTGTCCGTAACTCCGATCCAAAGGATTGTCCTGAGC 2 GGTGAAAATGGGCTGAAGATCGACATCCATGTCATCATCCCGTATGAAGGTCTGAGCGGC 2 GACCAAATGGGCCAGATCGAAAAAATTTTTAAGGTGGTGTACCCTGTGGATGATCATCAC 2 TTTAAGGTGATCCTGCACTATGGCACACTGGTAATCGACGGGGTTACGCCGAACATGATC 2 GACTATTTCGGACGGCCGTATGAAGGCATCGCCGTGTTCGACGGCAAAAAGATCACTGTA 2 ACAGGGACCCTGTGGAACGGCAACAAAATTATCGACGAGCGCCTGATCAACCCCGACGGC 2 TCCCTGCTGTTCCGAGTAACCATCAACGGAGTGACCGGCTGGCGGCTGTGCGAACGCATT GAATATCTTTGCTCCCAGTTTCTTGAGATCTGCTGACAGATGTTC 2 CTGGCGTAAAAGCTTAAATATCTTTGCTCCCAGTTTCTTGAANTCTGCTGACAGATGTTC ************************* ***************** 1 CATCCTGTACAAGTGCTCAGTTCCAATGTGCCCAGTCATGACATTTCTCAA 2 CATCCTGTACAAGTGCTCANTTCCAATGTGCCCAGTCATGACATTTCTCAA ******************* *******************************
7 Supplementary Figure 3
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Gene Forward Primer Reverse Primer GAPDH ATCATCCCTGCCTCTACTGG GTCAGGTCCACCACTGACAC SSB1 AACTTCAGTGAGCCAAACCC GTTCTCAGAGGCTGGAGAGG
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Symbol Accession Number Sense-primer (5-3 ) Antisense-primer (5-3 ) T a C ACTB NM_001101.3 CCAGAGGCGTACAGGGATAG CCAACCGCGAGAAGATGA 57 HSD3B2 NM_000198.3 CTTGGACAAGGCCTTCAGAC TCAAGTACAGTCAGCTTGGTCCT 60
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Supplementary Figure 1
Supplementary Figure 1 Product purity of cytosine base editing for the wheat genomic loci tested. Product distributions and indels frequencies at four representative wheat genomic DNA sites in wheat protoplasts
MIT Department of Biology 7.013: Introductory Biology - Spring 2005 Instructors: Professor Hazel Sive, Professor Tyler Jacks, Dr.
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Page 1 of 7 Page 1 of 7 Return to Web Version ZFN Technology Biowire Volume 10 Article 1 Have your genomic work cut out for you The genomes of several organisms, including humans, have been sequenced,
PrimePCR Assay Validation Report
Gene Information Gene Name Gene Symbol Organism Gene Summary Gene Aliases RefSeq Accession No. UniGene ID Ensembl Gene ID peptidylglycine alpha-amidating monooxygenase PAM Human This gene encodes a multifunctional
PrimePCR Assay Validation Report
Gene Information Gene Name 3-hydroxybutyrate dehydrogenase, type 1 Gene Symbol Organism Gene Summary Gene Aliases RefSeq Accession No. UniGene ID Ensembl Gene ID BDH1 Human This gene encodes a member of
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PrimePCR Assay Validation Report
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P-Box Dsf -----------------MG-TAG--DRLLD-IPCKVCGDRSSGKHYGIYSCDGCSGFFKR 39 NR2E1 -----------------MSKPAGSTSRILD-IPCKVCGDRSSGKHYGVYACDGCSGFFKR 42 Tll MQSSEGSPDMMDQKYNSVRLSPAASSRILYHVPCKVCRDHSSGKHYGIYACDGCAGFFKR
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