Supplemental Information. CRISPR/Cas9-loxP-Mediated Gene Editing. as a Novel Site-Specific Genetic Manipulation Tool
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1 OMTN, Volume 7 Supplemental Information CRISPR/Cas9-loxP-Mediated Gene Editing as a Novel Site-Specific Genetic Manipulation Tool Fayu Yang, Changbao Liu, Ding Chen, Mengjun Tu, Haihua Xie, Huihui Sun, Xianglian Ge, Lianchao Tang, Jin Li, Jiayong Zheng, Zongming Song, Jia Qu, and Feng Gu
2 Figure S1. Schematic of the combination of CRISPR/Cas9 and traditional site-specific genetic manipulation tools.
3 Figure S2. Performance of CRISPR/SpCas9-eloxP mediated site-specific genome editing examined with flow cytometry (FCM) HEK-293 cells were co-transfected with plasmids (pmtmg and CRISPR/SpCas9-eloxP,0.75ug each) and flow cytometry were performed at 60h post transfection (Red indicates mt; Green indicates mg)..
4 Figure S3. Genotyping of 293-AAVS-mTmG at AAVS1 locus. A. PCR products of GAPDH gene as control (Primers: F4/R4). B. Genotyping of the AAVS1 gene in 293-AAVS-mTmG and parental cells (Primers: F5/R5). The unlabelled bands are the primer dimer. Figure S4. Optimization of transfection conditions of CRISPR/SpCas9 -eloxp plasmid to inactivate mt. 293-AAVS-mTmG cells were transfected with different amounts of CRISPR/SpCas9-eloxP plasmid. The cells were saturated with 2.0 mg of CRISPR/SpCas9-eloxP plasmids.
5 Figure S5. Genotyping of CRISPR/SpCas9-eloxP system mediated excision at human AAVS1 locus. CRISPR/SpCas9-eloxP mediated excision of mtomoto between two loxp sites in 293-AAVS-mTmG. Excision bands were detected by PCR (Primers: F1/R2). Figure S6. Non-canonical PAM for CRISPR/SpCas9-loxP to inactivate mt. A. Targeting sequence and corresponding PAMs for CRISPR /SpCas9. B. NGA or NAG PAM and the results showed that CRISPR/SpCas9-loxP with non-canonical PAM NGA but not NAG could achieve relative highly efficient excision of a specific gene between two loxp sites.
6 Figure S7. Efficiency comparison of CRISPR/SaCas9-eloxP and SpCas9-eloxP mediated gene editing. px330 and px601 represent CRISPR /SpCas9 and CRISPR/SaCas9, respectively. Error bars are the standard deviation (SD, n=3). Figure S8. CRISPR/ SaCas9-eloxP mediated NHEJ pattern at the human AAVS1 locus. CRISPR/SpCas9-eloxP mediated excision of mtomoto between two loxp sites in 293-AAVS-mTmG. NHEJ pattern were detected by sequencing. Pink nucleotides indicate sgrna, blue nucleotides indicate indels.
7 Table S1 Target sequence Target sites for CRISPR/Cas9 System with PAMs Target site ID Target sequence (5`-3`) PAM eloxp-1(px330) GTATGCTATACGAAGTTATT AGG + eloxp-2(px601) TACATTATACGAAGTTATATT AAGGGT - Strand AAVS1 TCACCAATCCTGTCCCTAG TGG + LoxP-3(pX330) CGTATAGCATACATTATACG NAG + LoxP-4(pX330) TTCGTATAATGTATGCTATA NGA - Oligonucleotide sequences for sgrna architecture Primer Name Primer sequence (5`-3`) eloxp-1-f CACCGGTATGCTATACGAAGTTATT eloxp-1-r AAACAATAACTTCGTATAGCATACC eloxp-2-f CACCGTACATTATACGAAGTTATATT eloxp-2-r AAACAATATAACTTCGTATAATGTAC AAVS1-F CACCGTCACCAATCCTGTCCCTAG AAVS1-R AAACCTAGGGACAGGATTGGTGAC LoxP-3-F CACCGCGTATAGCATACATTATACG LoxP-3-R AAACCGTATAATGTATGCTATACGC LoxP-4-F CACCGTTCGTATAATGTATGCTATA LoxP-4-R AAACTATAGCATACATTATACGAAC Table S2 Primers sequences PCR primers Primer Name Primer sequence (5`-3`) Product(bp) Anneaing( ) F1 GGGACTTCCTTTGTCCCAAATC 740bp 58 R1 GGGAAGGACAGCTTCTTGTAATC F2 CAGGCATAGAGTGTCTGCTATT 725bp 58 R2 GATGAACTTCAGGGTCAGCTT F3 GGGCTATGAACTAATGACGGA 966bp 60 R3 GTCCAGGCCAAGTAGGTG F4 CTGGCACCCTATGGACACG 367bp 60 R4 GTCTTCTGGGTGGCAGTGAT F5 ACCTCTCACTCCTTTCATTTGG 1299bp 60 R5 TGAGTTTGCCAAGCAGTCA F6 CTCTCGTTTCTTAGGATGGCC R6 GAAAGCAAGAGGATGGAGAGG Sequencing primers Primer Name U6-F Jet-F Primer sequence (5`-3`) GAGGGCCTATTTCCCATGATTC TGAACACCATATCCATCCGGCGTA
8 Table S3 Off-target analysis for CRISPR/SpCas9-loxP System PCR primers Primer Name Primer sequence (5`-3`) Product(bp) Anneaing( ) F7 GACAGAGAGGCACAAAGTCATA 305bp 58 R7 AGAATACTGGTCCCTGGAAATG F8 GAGACGGAGTCTTGTTCCATAG 499bp 58 R8 GTGTTGTTAGTTGGAAGAGTCATTAG F9 GCCATGATGACTTCACCTGAT 455bp 58 R9 TTGGTTTGGGCAATACCTTCT F10 AGCTGACTTCTCACCACAAAC 374bp 58 R10 GCAATGACACAATTGCCTAACC F11 TGGAGATTAAGAGACAAGGGAATG 458bp 58 R11 GTAATATCACAGCTACTTGGAGAGG F12 GGCAGAGAAGGGCAAGTAAG 348bp 58 R12 GCCACGTCAGTAGTGGTATTT F13 GCATCAGAAAGCACAGAGACTA 441bp 58 R13 GCTTCCTTTACTCTCACCCTTTA F14 ACAGTGGTAAAGACCAATCAGG 300bp 58 R14 CTGCTGTGTACCTATGTCAGAAG F15 TGGAGATTAAGAGACAAGGGAATG 458bp 58 R15 GTAATATCACAGCTACTTGGAGAGG F16 TGGTGCGTGCCTGTAATC 420bp 58 F16 CCAGTTGCCTTTGAAGTCATTC Analysis of off-target induced by CRISPR/SpCas9-loxP System Chromosome Target sequence (5`-3`) PAM Off-target eloxp-1(px330) GTATGCTATACGAAGTTATT AGG Chr 1 GTAGGGTAAAGGAAGTTATT AGG No Chr 3 CTATGACATACTAAGTTATT AGG No Chr 6 GTAT ACTGTTTGAAGTTATT TGG No Chr 8 AAATGCTAACCGAAGTTATT TGG No Chr 9 AAATGCTAAAGGAAGTTATT TAG No Chr 10 AAATGCTTTAGGAAGTTATT CGG No Chr 12 AAATGCTAAAAGAAGTTATT CAG No Chr 13 GTAATGTATACAAAGTTATT TGG No Chr 13 ATATTCTCTAAGAAGTTATT AGG No Chr 18 TAATGCTAAAGGAAGTTATT TGG No
9 Table S4 Off-target analysis for CRISPR/SaCas9-loxP System PCR primers Primer Name Primer sequence (5`-3`) Product(bp) Anneaing( ) F17 GCATCACAGTCACAGCATTT 263bp 58 R17 GAAAGGGCAGACCTAGGATATAA F18 CATTAGGCTTTATCGACCTAGTGA 373bp 58 R18 CATGTCAATTTAGGCAGGGTTT F19 GTAGCCTCTAGCCACATGTTT 320bp 58 R19 TCACCACCTTTGGTGTTCTC F20 CCTGTTCCACTCTCCCTTTATAC 386bp 58 R20 ACAGAAGAGTAGACCTATTTGTAAG F21 CCTCCTGAATAGCTGGGATTAC 391bp 58 R21 CCCTCCCTGGTTAGAAGAAAC F22 GGCAGAATCTGAAAGTGGATACT 551bp 58 R22 GCCTCAATGAGCTCCCAAATA F23 AACCCAATTCCCATCAACTAAATC 446bp 58 R23 AGGGAAGAGTCATTGGTAAAGTC F24 GTGCCTGGCCAACATTTATC 459bp 58 R24 CCCACAAGAGTGGAGGATTATT F25 CTGTTGAGCATTTAATTTCGGTGA 436bp 58 R25 TCACACATGTCTTTGGAGATTGA F26 TCCAAGGTAGTGGCATTGATAC 410bp 58 R26 AAGAAAGGAGGGAACTCTTGATAC Analysis of off-target induced by CRISPR/SaCas9-loxP System Chromosome Target sequence (5`-3`) PAM Off-target eloxp-2(px601) TACATTATACGAAGTTATATT AAGGGT Chr 4 TATATTATACAAACATATATT AAGAAT No Chr 4 TTCATTATATGAAGTTCAATT CAGGAT No Chr 7 CACTATCTACGAGGTTATATT AAGTGA No Chr 7 ACTACTTTTAGAAGTTATATT AAGGCC No Chr 8 TTCTTTATAAGAAGATATATT CTGAGT No Chr 11 TACAATCTATGAAGTGATATT TAGGAT No Chr 13 ACATTATACGATGTTACATT ATATGA No Chr 19 TAAACCCAAAGAAGTTATATT AAGATT No Chr X TACTTTATATAAACTTATATT TGGAAT No Chr X TCACTTCAAGAATGTTATATT AAGGGA No
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