Supplementary Figure 1. Quantitative RT-PCR experimental validation of CRISPR/Cas9 and sgrnas expression in HEK293A transfected cells.

Transcription

1 Supplementary Figure 1. Quantitative RT-PCR experimental validation of CRISPR/Cas9 and sgrnas expression in HEK293A transfected cells. HEK293A cells were transfected with the indicated combinations of CRISPR/Cas9 expression vector and sgrnas. The level of expression was determined after 72h post-transfection by quantitative RT-PCR. The relative fold changes were normalized to control conditions. Average values from three independent experiments ± SD are shown.

2 Supplementary Figure 2. PCR analysis of 84 HEK293A selected clones transfected with E1F2-sgRNAs and CRIPSR/Cas9. PCR was performed on 84 cellular clones obtained by serial dilution from antibiotic selected E1F2 transfected HEK293A cells. Primers used to detect the breakpoint junctions are summarized in Supplementary Table 1. Almost 37% (31 out of 84) of the clones retrieved a PCR band product of the expected 900bp size. Numerals indicate the number of each clone. M, 1Kbp MW marker.

3 Supplementary Figure 3. Nucleotide sequences of the chromosomal junctions of the EWSR1/FLI1 fusion gene detected in HEK293A transfected cells. Translocation breakpoints in clones of HEK293 cells transfected with E1F2 sgrna and CRISPR/Cas9 expressing plasmids. Below the predicted sequence is summarized the output of the analysis of DNA breakpoint regions by deep sequencing performed on specific PCR products. The only two sequences obtained are depicted.

4 Supplementary Figure 4. CRISPR/Cas9 mediation of DSBs at targeted loci in hmsc transfected with sgrnas E1 and F2. SURVEYOR nuclease digestion comparing the cleavage effciciency at the indicated locus. hmsc were transfected with a plasmid expressing CRISPR/Cas9 and sgrna E1 (left), sgrna F2 (right), or without sgrna (-). Grey arrowheads indicate undigested PCR product; turquoise (EWSR1) and orange (FLI1) arrowheads indicate specific cleavage products. Numerals indicate the size of the bands.

5 Supplementary Figure 5. CRISPR/Cas9-triggered t(11:22) chromosome translocation recapitulates expression of in-frame fusion of EWSR1/FLI1 protein. Representative Western blot showing EWSR1/FLI1 protein expression in a clone of HEK293-E1F2 selected cells harboring the t(11:22) chromosome translocation. EWSR1/FLI1 (72 kda) was detected by purified mouse anti-human FLI1 (G ) antibody. A673 Ewing s sarcoma cell line was used as positive control. As a loading control anti-gapdh was used (bottom).

6 Supplementary Figure 6. Nucleotide sequences of the chromosomal junctions of the RUNX1/ETO fusion gene detected in HEK293A transfected cells. Translocation breakpoints in clones of HEK293 cells transfected with R1Et2 sgrna and CRISP/Cas9 expressing plasmids. Below the predicted sequence are shown the sequences of 5 clones.

7 Supplementary Table 1. Oligonucleotides used in this study. Name Intended Use Sequence E1 sgrna AATTTGTTTTTAGTATGCCT TGG E2 sgrna AGTGGGGAGGCAGCTATTGC AGG F1 sgrna GCAGGAGAGTCGCTTGAATG CGG F2 sgrna CTGGCCTGGGCGATAGACCG AGG R1 sgrna TCTGGAAAAGAGCGTTACTC TGG R2 sgrna AGATGTCTCCAAACCGTATC AGG Et1 sgrna GGAAGCTCCAAATTAGGCAC AGG Et2 sgrna ATCCACATAAATCGGCCAGC TGG qezh2 Rv qpcr CCCTTCTCAGATTTCTTCCCA qezh2 Fw qpcr GGACTCAGAAGGCAGTGGAG qhmga2 Rv qpcr TCTTCGGCAGACTCTTGTGA qhmga2 Fw qpcr AAAGCAGAAGCCACTGGAGA qid2 Rv qpcr TCAGCACTTAAAAGATTCCGTG qid2 Fw qpcr GACAGCAAAGCACTGTGTGG qnkx2.2 Rv qpcr GGAGCTTGAGTCCTGAGGG qnkx2.2 Fw qpcr TCTACGACAGCAGCGACAAC qnr0b1 Rv qpcr CCACTGGAGTCCCTGAATGT qnr0b1 Fw qpcr TCCAAATGCTGGAGTCTGAA qsox2 Rv qpcr GTCATTTGCTGTGGGTGATG qsox2 Fw qpcr AGAAAAACGAGGGAAATGGG qhgusb Rv qpcr AAGGATTTGGTGTGAGCGAT qhgusb Fw qpcr GAAGAAGTGGTGCGTAGGGA qgapdh Rv qpcr TGATGACCCTTTTGGCTCCC qgapdh Fw qpcr TCCAAAATCAAGTGGGGCGA ew1 Fw qpcr GTATGCCTGTTTTAGAGCTAG ft1 Fw qpcr CTTGAATGGTTTTAGAGCTAG

8 ew2 Fw qpcr GCTATTGCGTTTTA AGCTAG ft2 Fw qpcr ATAGACCGGTTTTAGAGCTAG sgrna Rv qpcr TCAAGTTGATAACGGACTAGCC qcas9 Rv qpcr CCCTTATCCACGACTTCCTC qcas9 Fw qpcr CCTCACATTTCGGATACCCTAC AML1 RT2 S RT PCR GTTTGTCGGTCGAAGTGGAA ETO RT2 AS RT PCR GTGAGTCTGGCATTGTGGA EWSFLI-RT-S RT PCR TCCTACAGCCAAGCTCCAAGTC EWSFLI-RT- AS RT PCR GGACTTTTGTTGAGGCCAGA EWSFLI T Fw genomic PCR CCAACAGAGCAGCAGCTAC Eto Rv 1 genomic PCR AGAGAGAGAGAGAGAGAGAGAGA Eto Rv 2 genomic PCR CAAACAACTGGCAATACCTACAC Eto Rv3 genomic PCR GATTAGGATGGACGAGACACATC Eto Fw1 genomic PCR CTCCTGTCATCCTCCACATTTC Eto Fw2 genomic PCR CAGAGGTGTGATTGGAAGGTAG AML Rv1 genomic PCR GGTGGGCATGGTGATTATTTG AML Rv2 genomic PCR CAGGCACACAGCATCATTTG AML Fw1 genomic PCR GGACCATGTCTCAGATTCCTTG AML Fw2 genomic PCR GATAGAGAGTGGGAGGGTAGAA EWSR_1 Fw genomic PCR AGGCTGGTCTCGAACTCCTG EWSR_1 Rv genomic PCR CCTAAGATGTCTCCAAAAGCAGC EWSR_2 Fw genomic PCR AGTCATGAGCCACTGCGCCC EWSR_2 Rv genomic PCR TTAGCACACTGCTAGGGGCTG FLI_1 Fw genomic PCR GTGCTTGTTCTTCACACCTGG FLI_1 Rv genomic PCR GGTTTGGCTTAATCGGCCTCC FLI_2 Fw genomic PCR CACCACAGGAAATGCAAGGAGG FLI_2 Rv genomic PCR AAATGCTGTGGGGGAAGTGGC

9 OTe1 Fw Off targets EWSR1 TGCTTTCCAGCCCTAGAGGA OTe1 Rv Off targets EWSR1 TGCTCACGTTTGCCATGAGA OTe 2 Fw Off targets EWSR1 GGAGACACTCATTGGCTCTTAC OTe 2 Rv Off targets EWSR1 CCAGACACATCATACAGCTATCC OTe3 Fw Off targets EWSR1 CCTCATTTACTCCCAATCAAGCC OTe3 Rv Off targets EWSR1 GAAATGTGTCTCCCTCCTAACTC OTe4 Fw Off targets EWSR1 ACTGATGTGTGCCATTGTGTTG OTe4 Rv Off targets EWSR1 TTGGCAGTGGTGTGCTTAT OTe5 Fw Off targets EWSR1 GATCACAAGGTCAGGAGATCAA OTe5 Rv Off targets EWSR1 ACCACACACACAAACACACCAAAG OTe6 Fw Off targets EWSR1 CCTGTTGGCTCATTCCTATGT OTe6 Rv Off targets EWSR1 AGCTAAGAAATGGGAAGGAAGAG OTf1 Fw Off targets FLI1 AGCACACTATGCAACCAGAGA OTf1 Rv Off targets FLI1 GGCCTGGGATATTCAGGGTG OTf2 Fw Off targets FLI1 GGAGTGCATGGGAGTTGAGT OTf2 Rv Off targets FLI1 GCAGAGGAAGTGAGAAGCGT OTf3 Fw Off targets FLI1 GCTGGGCTGTCTGAATGTCT OTf3 Rv Off targets FLI1 GAGGGTTTTGGGGGAATTTGC OTf4 Fw Off targets FLI1 CATGTAGCGAGGGTAGCAGG OTf4 Rv Off targets FLI1 CTAACTGTGTGGCTGGGGAC

10 Supplementary Table 2. Translocation frequency in cultures transfected with CRISPR/Cas9 and pairwise sgrna EWS/FLI1 Selected HEK293A assay #1 assay #2 assay #3 total avg cell nuclei t(11;22) cell nuclei t(11;22) cell nuclei t(11;22) cell nuclei t(11;22) SD E1F n.a. E1F E2F n.a. E2F n.a. Non-selected HEK293A assay #1 assay #2 assay #3 total avg cell nuclei t(11;22) cell nuclei t(11;22) cell nuclei t(11;22) cell nuclei t(11;22) SD E1F n.a. E1F E2F n.a. E2F n.a. MSCs assay #1 assay #2 assay #3 total avg SD cell nuclei t(11;22) cell nuclei t(11;22) cell nuclei t(11;22) cell nuclei t(11;22) E1F

11 AML-1/ETO Non-selected HEK293A assay #1 assay #2 assay #3 total avg cell nuclei t(8;21) cell nuclei t(8;21) cell nuclei t(8;21) cell nuclei t(8;21) SD R1Et R1Et R2Et R2Et CD34+ cells assay #1 assay #2 assay #3 total avg SD cell nuclei t(8;21) cell nuclei t(8;21) cell nuclei t(8;21) cell nuclei t(8;21) R1Et