Different approaches: Site degeneracy data from libraries with randomized target sites (in vitro, complexity of the initial library is crucial, involves amplification of recovered sites in E. coli). Integration of episomal double stranded DNA (e.g. AAV or NIL vectors) at DSB sites (in vivo, not likely to cover all cleavable sites but shows a distribution of cleavage/insertion sites) End capture (in vitro, should detect all possible cleavage sites although it does not necessarily reflect which sites are actually cleaved in living cells) ChIP-seq (in vivo, not likely to cover all cleavable sites but potentially the best means to assess HE cleavage sites in living cells) HE site degeneracy – HE targets in genomic DNA

Isolate genomic DNA (Mix with homing site containing plasmid) (De-/methylation) Digestion with HE Capture ends generated by the HE with biotinylated oligos Cleavage with EcoP15I Binding to streptavidin beads Ligation of sequencing adapter oligos PCR amplification of the captured sequence (Cloning into pGEM-T Easy) Sequencing End capture BLAST Oligos

- 5‘ - 3‘ Exon1 Exon2 IIS 5‘ - 3‘ TTGACAGAGTGCTGCAAAAC AACTGTCTCACGACGTTTTG T G AC aggacaggGGGCTGCAAAGT c c 5‘ - - 3‘ CACTCTTCGCACC 5‘ -- 3‘ GAGACAGAGTGccttgggga A T 5‘ - - 3‘ tctgaagAGCACTTCCAACA c a 5‘ - - 3‘ Human Chr. 22 Human Chr. 5 Human Chr. 6 ? C.r. cp 23S rRNA m-CreI end capture – BLAST results hits

Transduction with lentiviral HE expression vectors Cell fixation Cell lysis and sonication ChIP Wash, elution and crosslink reversal Digestion of cellular protein and RNA DNA end repair and addition of “A” bases Ligation of sequencing adapters PCR amplification of the sequencing library Gel purification of the amplified library and QC Sequencing and data analysis ChIP-seq Map back on genome sequence

Non integrating lentiviral expression vectors with the following characteristics: - EF1  promoter - HE ORF (I-CreI, I-CreI+, m-CreI, I-MsoI, I-MsoI+, m-MsoI) - mCherry as marker (alt.: Puro, MGMT) - 2A sequence (or IRES) links the HE and the marker ORF Cell lines: ₋HT-1080 ₋U-2 OS ₋primary fibroblasts ₋hematopoietic stem cells (human/dog CD34; availability?) Antibodies: - there are no HE specific antibodies readily available. Therefore, ChIP has to rely on antibodies against existing tags (HA, myc) -positive control: methylated Histone H3 (e.g. trimethyl K4) -negative control: AB against a protein that is not associated to chromatin (e.g. GFP, mCherry?) ChIP-seq

CCCCCCCTCGACCGCAAAACGTCGTGAGACAGTTTGGTCCAGCTTGATAT GGGGGGGAGCTGGCGTTTTGCAGCACTCTGTCAAACCAGGTCGAACTATA CCCCCCCTCGACCGCAAAACGTCGTGAGACAGTTTGGTCCAGCTTGATAT GGGGGGGAGCTGGCGTTTTGCAGCACTCTGTCAAACCAGGTCGAACTATA CTGCTGCAGAAGCTTGGATCCATGA-Bio Bio-AGTACCTAGGTTCGAAGACGTCGTC NNNNGACGACGTCTTCGAACCTAGGTACT TCATGGATCCAAGCTTCTGCAGCAGNNNN CCCCCCCTCGACCGCAAAACGTCGTGA GACAGTTTGGTCCAGCTTGATAT GGGGGGGAGCTGGCGTTTTGCAG CACTCTGTCAAACCAGGTCGAACTATA CTGCTGCAGAAGCTTGGATCCATGA-Bio Bio-AGTACCTAGGTTCGAAGACGTCGTC NNNNGACGACGTCTTCGAACCTAGGTACT TCATGGATCCAAGCTTCTGCAGCAGNNNN CCCCCCCTCGACCGCAAAACGTCGTGA GACAGTTTGGTCCAGCTTGAT GGGGGAGCTGGCGTTTTGCAG CACTCTGTCAAACCAGGTCGAACTATA CTGAGCTCGGACTCTTAAGGACGTCNN GACTCGAGCCTGAGAATCCCTGCAG NNCTGCAGGAATTCTCAGGCTCGAGTC GACGTCCTTAAGAGTCCGAGCTCAG capture forw. capture rev. capture forw. capture rev. EcoP15I < < < < 68bp I-CreI cleavage Annealing/ligation of linker/capture oligos Digestion with EcoP15I Streptavidin binding of digestion products Annealing/ligation of adapter/linker oligos Wash off streptavidin beads PCR Cloning and sequencing Protocol

5’ I-CreI hs pBScapt. forw.capt. rev. 3’ I-CreI hspBS capt. forw. capt. rev. m-CreI end capture – pBSCre positive control

2/4/2 (5 ’ ): 5 ’ -CCACGCTTCTCAC-3 ’ 2/4/4 (5 ’ ): 5 ’ -TGAAACGTCGGGggacaggacc-3 ’ 2/4/5 (5 ’ ): 5 ’ -ACAACCTTCACGAgaagtctca-3 ’ 2/4/6 (3 ’ ): 5 ’ -aggggttccGTGAGACAGAGAT-3 ’ I-CreI site: 5 ’ -caaaacgtcgtgagacagtttg-3 ’ 2/4/2: no hits, sequence too short. 2/4/4: six hits on chromosome 22 (95-99% identity): ref|NT_ |Hs22_11677 Homo sapiens chromosome 22 genomi e-119 ref|NW_ |Hs22_WGA1304_36 Homo sapiens chromosome e-117 ref|NW_ |HsCraAADB02_665 Homo sapiens chromosome 22 ge e-117 ref|NT_ |Hs22_11676 Homo sapiens chromosome 22 genomi e-111 ref|NW_ |Hs22_WGA1299_36 Homo sapiens chromosome e-106 ref|NW_ |HsCraAADB02_1017 Homo sapiens genomic contig, e-105 2/4/5: three hits on chromosome 6 (97% identity): ref|NT_ |Hs6_7749 Homo sapiens chromosome 6 genomic c ref|NW_ |Hs6_WGA366_36 Homo sapiens chromosome 6 ge ref|NW_ |HsCraAADB02_247 Homo sapiens chromosome 6 gen /4/6: three hits on chromosome 5 (100% identity): ref|NT_ |Hs5_6733 Homo sapiens chromosome 5 genomic c e-93 ref|NW_ |Hs5_WGA317_36 Homo sapiens chromosome 5 ge e-93 ref|NW_ |HsCraAADB02_205 Homo sapiens chromosome 5 gen e-93 m-CreI end capture – BLAST results sites