Surrogate reporter-based enrichment of cells containing RNA-guided Cas9 nucleaseinduced

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1 Supplementary Data Surrogate reporter-based enrichment of cells containing RNA-guided Cas9 nucleaseinduced mutations Suresh Ramakrishna 1, Seung Woo Cho 2, Sojung Kim 2, Myungjae Song 1, Ramu Gopalappa 1, Jin-Soo Kim 2,*, and Hyongbum Kim 1,* 1 Graduate School of Biomedical Science and Engineering/College of Medicine, Hanyang University, Seongdong-gu, Seoul, South Korea. 2 National Creative Research Initiatives Center for Genome Engineering and Department of Chemistry, Seoul National University, Gwanak-gu, Seoul, South Korea. Supplementary data: Supplementary Figure 1-15 Supplementary Table 1-5 1

2 Supplementary Figure 1. Expression of R and G after the cotransfection of a fluorescent reporter plasmid, plasmids expressing a RGEN. HEK293T cells were cotransfected with a fluorescent reporter plasmid, a plasmid expressing tracrrna and crrna targeting BMP1 gene, and a plasmid encoding Cas9, and observed using fluorescent microscopy at the indicated times. Scale bar =100 µm. 2

3 Supplementary Figure 2. Flow cytometric enrichment of HeLa cells containing RGENinduced mutations. Three days after cotransfection of the reporter plasmid, a plasmid encoding Cas9, and a plasmid encoding PPP1R1B targeting crrna and tracrrna (guide RNA, grna), HeLa cells were flow cytometrically sorted and analyzed. (a) Flow cytometry. The percentage of mr + eg + cells is shown. (b) The frequency of RGEN-induced mutations as determined by the T7E1 assay. Arrows indicate the expected positions of DNA bands cleaved by mismatch-sensitive T7E1. The numbers at the bottom of the gel indicate mutation percentages calculated by band intensities. 3

4 Supplementary Figure 3. Overview of the magnetic separation-mediated enrichment of cells containing RGEN-induced mutations. (a) The working mechanism of the magnetic reporter. mr is constitutively expressed by the CMV promoter (P CMV ), whereas eg and H-2K k are not expressed without RGEN activity because the eg and H-2K k sequences are out of frame and there is a stop codon before the eg and H-2K k genes. If a doublestrand break (DSB) is introduced into the target sequence by RGENs, the break is repaired by error-prone nonhomologous end-joining (NHEJ), which often results in indels. This indel formation can cause frame shifts, making the eg and H-2K k genes in frame and expressed. (b) A schematic illustrating the enrichment of mutant cells using magnetic separation. Reporter plasmids, chromosomal target loci, and sites of mutations, represented by black boxes, are shown. 4

5 Supplementary Figure 4. Expression of R and G after the cotransfection of a magnetic reporter plasmid, plasmids expressing a RGEN. HEK293T cells were cotransfected with a magnetic reporter plasmid, a plasmid expressing tracrrna and crrna targeting F13A1 gene, and a plasmid encoding Cas9, and observed using fluorescent microscopy at the indicated times. Scale bar =100 µm. 5

6 Supplementary Figure 5. Magnetic separation-mediated enrichment of HeLa cells containing RGEN-induced mutations. Magnetic separation was performed three days after cotransfection of the reporter plasmid, a plasmid encoding Cas9, and a plasmid encoding PPP1R1B-targeting crrna and tracrrna (guide RNA, grna) into HeLa cells. The frequency of RGEN-induced mutations was determined by the T7E1 assay. Arrows indicate the expected positions of DNA bands cleaved by mismatch-sensitive T7E1. The numbers at the bottom of the gel indicate mutation percentages calculated by band intensities. 6

7 Supplementary Figure 6. Overview of the hygromycin selection-mediated enrichment of cells containing RGEN-induced mutations. (a) The working mechanism of the hygromycin reporter. mr is constitutively expressed by the CMV promoter (P CMV ), whereas the hygromycin-resistance protein (Hygro R )-eg fusion protein is not expressed without RGEN activity because the Hygro R -eg sequence is out of frame and there is a stop codon before the Hygro R -eg. If a double-strand break (DSB) is introduced into the target sequence by RGENs, the break is repaired by error-prone nonhomologous end-joining (NHEJ), which often results in indels. This indel formation can cause frame shifts, making eg and Hygro R genes in frame and expressed. (b) A schematic illustrating the enrichment of mutant cells in the population of hygromycin-selected cells. Reporter plasmids, chromosomal target loci, and sites of mutations, represented by black boxes, are shown. 7

8 Supplementary Figure 7. Expression of R and G after the cotransfection of a hygromycin reporter plasmid, plasmids expressing a RGEN. HEK293T cells were cotransfected with a hygromycin reporter plasmid, a plasmid expressing tracrrna and crrna targeting PPP1R1B gene, and a plasmid encoding Cas9, and observed using fluorescent microscopy at the indicated times. Scale bar =100 µm. 8

9 Supplementary Figure 8. Hygromycin selection-mediated enrichment of HeLa cells containing RGEN-induced mutations. HeLa cells were treated with hygromycin for two days starting two days after the cotransfection of the reporter plasmid, a plasmid encoding Cas9, and a plasmid encoding PPP1R1B-targeting crrna and tracrrna (guide RNA, grna). The frequency of RGEN-induced mutations was determined by the T7E1 assay. Arrows indicate the expected positions of DNA bands cleaved by mismatch-sensitive T7E1. The numbers at the bottom of the gel indicate mutation percentages calculated by band intensities. 9

10 Supplementary Figure 9. The effect of transfection efficiency on the RGEN-induced mutation frequency. HEK293T cells were transfected with a reporter plasmid and plasmids encoding Cas9 and F13A1-targeting guide RNA (grna). Three days after transfection, R + G -, R + G +, R dim, R medium, and R bright cells were sorted using flow cytometry and subjected to the T7E1 assay. (a) Flow cytometry. Areas marked with boxes represent the gates used for sorting. (b) Mutation frequencies were calculated by the T7E1 assay. Arrows indicate the expected positions of DNA bands cleaved by mismatch-sensitive T7E1. The numbers at the bottom of the gel indicate mutation percentages calculated by band intensities. 10

11 Supplementary Figure 10. The effects of reporter cotransfection on RGEN-induced genetic modification. HEK293T cells were cotransfected with a plasmid expressing tracrrna and crrna targeting the PPP1R1B gene, a plasmid encoding Cas9, and various amounts of a fluorescent reporter plasmid (PPP1R1B reporter). To make the total amount of transfected DNA constant, the cells were cotransfected with compensating amounts of a reporter plasmid with a different target sequence (BMP1 reporter). Three days after the cotransfection, the frequency of RGEN-induced mutations were determined by the T7E1 assay. Arrows indicate the expected positions of DNA bands cleaved by mismatch-sensitive T7E1. The numbers at the bottom of the gel indicate mutation percentages calculated by band intensities. 11

12 Supplementary Figure 11. No off-target mutations in flow cytometrically sorted and unsorted cells. Flow cytometric sorting was performed three days after cotransfection of the reporter plasmid, a plasmid encoding Cas9, and a plasmid encoding BMP1-targeting crrna and tracrrna (guide RNA, grna) into HEK293T cells. (a) Potential off-target sites were selected. (b-c) The frequency of RGEN-induced mutations was determined by the T7E1 assay at the on-target (b) and the potential off-target sites (c). No mutations were detected (assay sensitivity, ~0.5%) at potential off-target sites, whereas a high frequency of on-target mutations was observed in flow cytometrically sorted cells. The numbers at the bottom of the gel indicate mutation percentages calculated by band intensities. 12

13 Supplementary Figure 12. No off-target mutations in magnetically separated and unseparated cells. Magnetic separation was performed three days after cotransfection of the reporter plasmid, a plasmid encoding Cas9, and a plasmid encoding F13A1-targeting crrna and tracrrna (guide RNA, grna) into HEK293T cells. (a) Potential off-target sites were selected. (b-c) The frequency of RGEN-induced mutations was determined by the T7E1 assay at the on-target (b) and the potential off-target sites (c). No mutations were detected (assay sensitivity, ~0.5%) at potential off-target sites, whereas a high frequency of on-target mutations was observed in magnetically separated cells. The numbers at the bottom of the gel indicate mutation percentages calculated by band intensities. 13

14 Supplementary Figure 13. No off-target mutations in hygromycin-selected and unselected cells. HEK293T cells were treated with hygromycin for two days starting two days after the cotransfection of the reporter plasmid, a plasmid encoding Cas9, and a plasmid encoding PPP1R1B-targeting crrna and tracrrna (guide RNA, grna). (a) Potential off-target sites were selected. (b-c) The frequency of RGEN-induced mutations was determined by the T7E1 assay at the on-target (b) and the potential off-target sites (c). No mutations were detected (assay sensitivity, ~0.5%) at potential off-target sites, whereas a high frequency of on-target mutations was observed in hygromycin-selected cells. The numbers at the bottom of the gel indicate mutation percentages calculated by band intensities. 14

15 TTTCCCATGATTCCTTCATATTTGCATATACGATACAAGGCTGTTAGAGAGATAATTGGAATTAATTT GACTGTAAACACAAAGATATTAGTACAAAATACGTGACGTAGAAAGTAATAATTTCTTGGGTAGTTTG CAGTTTTAAAATTATGTTTTAAAATGGACTATCATATGCTTACCGTAACTTGAAAGTATTTCGATTTC TTGGCTTTATATATCTTGTGGAAAGGACGAAACACCGGAACCATTCAAAACAGCATAGCAAGTTAAAA TAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTTGAATTGTCGATTAG TGAACGGATCTCGACGGTATCGATCACGAGACTAGCCTCGAGCGGCCGCCCCCTTCACCGAGGGCCTA TTTCCCATGATTCCTTCATATTTGCATATACGATACAAGGCTGTTAGAGAGATAATTGGAATTAATTT GACTGTAAACACAAAGATATTAGTACAAAATACGTGACGTAGAAAGTAATAATTTCTTGGGTAGTTTG CAGTTTTAAAATTATGTTTTAAAATGGACTATCATATGCTTACCGTAACTTGAAAGTATTTCGATTTC TTGGCTTTATATATCTTGTGGAAAGGACGAAACACCGGAGAGCGTGTGTGGCCCGATGGTTTTAGAGC TATGCTGTTTTGTTTTTT Supplementary Figure 14. The sequence of the prg2-ct plasmid encoding tracrrna and BMP1-targeting crrna. The tracrrna and crrna sequences are highlighted in red and blue, respectively, and the BMP1 target sequence is shown in bold. 15

16 Supplementary Figure 15. prg2-ct plasmid map CRISPR RNA (crrna) and trans-activating crrna (tracrrna) are separately transcribed by the U6 promoter (pu6). The ampicillin resistance gene (Amp) enables the selection of the transformed bacterial cells. 16

17 Supplementary Table 1. The frequency of out-of-frame mutations Target gene BMP1 F13A1 PPP1R1B Out-of-frame mutations among total mutations(%) Before enrichment 100 (3/3) 67 (2/3) 100 (3/3) 92 (11/12) 100 (2/2) 67 (2/3) 100 (3/3) 100 (3/3) 100 (2/2) After enrichment 100 (7/7) 57 (4/7) 67 (4/6) 100 (12/12) 67 (4/6) 100 (7/7) 60 (3/5) 80 (4/5) 67 (4/6) Out-of-frame mutations among total sequenced alleles (%) Before After enrichment enrichment (3/67) (7/21) (2/95) (4/22) (3/101) (4/30) (11/387) (12/37) (2/72) (4/19) (2/88) (7/28) (3/57) (3/22) (3/42) (4/19) 3.0 (2/67) 15 (4/26) Fold enrichment of out-of-frame mutations Enrichment method Flow cytometry Magnetic separation Hygromycin selection Flow cytometry Magnetic separation Hygromycin selection Flow cytometry Magnetic separation Hygromycin selection The mutation frequency was calculated by sequencing the genomic DNA containing the RGEN target sites after PCR amplification. The ratios in the parentheses represent the number of out-of-frame mutation sequences/the total number of sequenced amplicons. 17

18 Supplementary Table 2. Oligonucleotides used for reporter plasmid construction Gene Direction Sequence (5 to 3 ) BMP1 F13A1 PPP1R1B AATTCCAGAGCGTGTGTGGCCCGATGGGGAGGAG GATCCTCCTCCCCATCGGGCCACACACGCTCTGG AATTCAACAAGCTGATTGTCCGCAGAGGGAGGAG GATCCTCCTCCCTCTGCGGACAATCAGCTTGTTG AATTCTTGGGTCTCTTCGACTTGAGATGGAGGAG GATCCTCCTCCATCTCAAGTCGAAGAGACCCAAG Target sequences are in bold and PAM sequences are underlined. 18

19 Supplementary Table 3. Oligonucleotides used for crrna construction Gene Direction Sequence (5 to 3 ) BMP1 F13A1 PPP1R1B CCGGAGAGCGTGTGTGGCCCGATG AAACCATCGGGCCACACACGCTCT CCGGACAAGCTGATTGTCCGCAGA AAACTCTGCGGACAATCAGCTTGT CCGGTGGGTCTCTTCGACTTGAGA AAACTCTCAAGTCGAAGAGACCCA Target sequences are in bold. 19

20 Supplementary Table 4. Primers used in the T7E1 assay Gene Direction Sequence (5 to 3 ) BMP1 F13A1 PPP1R1B AACTCATGCCTCGAAGAAGC CTAGGGCTTCCTGCTCACC ATGCATGTTGGGTGACTGAA GAAAGGAATGTGTGGGCACT GAATGTCCCCTTGAGGCTTT CATTTTCAGCACGAGACCAA GTGAGTGCCTTGTGGTCTGA ACTGTGCCTGTACCCACCTC GCTTGGTTCTGGTTCGGTT AGGGAAGCAGGAAAGAGGAG CTGCAGTGACAACCAACCAG CTCCCCACTGACAAAAGCTC 20

21 Supplementary Table 5. Primers used in the T7E1 assay for Off-target analysis Gene Off-Targets Direction Sequence (5 to 3 ) TGCTCAGGAAGCCAGTGAAT OT TAGCCCAGGGCATAAGGGAT TCACCAAGTTTGCCCAGGTG CCAGGTCATCCACAGGGAAG CCAGTTCTTGCCACCAGAGG BMP1 OT-2 OT-3 OT-4 OT AGATCTCCATTGCCTGGCTC CCGTGCTGACTAACACTGGA TTGGTCATTGGTTGCCCACT TGATCACGAGACTAGCCAGC TGGCAAAAGCGAGAAGGGTA AAGTGTGAGGATGCTGGTGA ACAGGAGAGGCTACTTGGCT CCGCTTCTTTGCCTCTCTGA TCCCTGGAGAAGTCCCGAAT ACAGAACTGTTTGGTGGGCA ATGCATATTACCCAGGGCCG TATTTTGCAAGGGGCCACAGT ATGACTAGTCGGGGAGAAGGA CCGTCCATCTATTGCTCCCC CAGGCACCCCACTACTTCAC AGACAGGCTAGGCCATTTGA OT AGGCCTGGGCTTATCCTGTA ACTGGTGCTGAGAACAAGACA CCCTCAACCCTGAGAACCAC GCTCTGTGCTGACTGGTAGA F13A1 PPP1R1B OT-2 OT-3 OT-4 OT GGGAGATGAATTCTGCTTTCAACC GGCAAAGGCTGAGTGGTGTA TCAACCCTTCAACTTTGGTTTTGA GCCCAATTTCCTTCACTCCG GTTTCAGTCCTCCATGTCCCT ATCTGATTGTGGCGTCGCTT TGGGGAAGGGGATGGGTTAG TGCATGCCATCTCAGTGTCC TGGAATCTGCCAGGCCTATG ACGGGCAAGTTCTTTCAGCA TCCAGGGGTTGGCAAATTGT GAGAGGCATTCCTCCCCAAA 21

22 PPP1R1B OT-2 OT-3 OT GCATGTTGGCCATGCTGTAT CGAACCCAATCCAGGTCCTC CATCTGTACCCACCCATGCC TAACTGACACCTGCCCTAGC CAGCCTACCCTCTCTGCTTG AGCAAACTGCAATGACAGGC GTAGGGTAGGACTTGGCTGG CCTGTCAGGCTCTGAACCAA ACCAAGTTCAGTGTGTGGGA GCAAGCAGGGTGCAAATGTT GTGATCACGTGCGTCTCTGA TGCTTGGTTGTGTGCATTGG GCTGTGTAGCGATTGCACTG 22