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 DNAHE 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 captureEnd capture

BLAST

Oligos

- 5‘- 3‘

Exon1 Exon2IIS

5‘ -3‘ -

-1-2-3-4-5-6-11

-10 -9 -8 -7 +1 +2 +3 +4 +5 +6 +7 +8 +9 +11

+10

TTGACAGAGTGCTGCAAAACAACTGTCTCACGACGTTTTGT GA C

aggacaggGGGCTGCAAAGT c c5‘ - - 3‘

CACTCTTCGCACC5‘ - - 3‘

GAGACAGAGTGccttgggga A T5‘ - - 3‘

tctgaagAGCACTTCCAACA c a5‘ - - 3‘

Human Chr. 22

Human Chr. 5

Human Chr. 6

?

C.r. cp 23S rRNA

m-CreI end capture – BLAST resultsm-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-seqChIP-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-seqChIP-seq

CCCCCCCTCGACCGCAAAACGTCGTGA GACAGTTTGGTCCAGCTTGATATGGGGGGGAGCTGGCGTTTTGCAGCACTCTGTCAAACCAGGTCGAACTATA

CCCCCCCTCGACCGCAAAACGTCGTGA GACAGTTTGGTCCAGCTTGATATGGGGGGGAGCTGGCGTTTTGCAG CACTCTGTCAAACCAGGTCGAACTATA

CTGCTGCAGAAGCTTGGATCCATGA-Bio

Bio-AGTACCTAGGTTCGAAGACGTCGTC

NNNNGACGACGTCTTCGAACCTAGGTACT

TCATGGATCCAAGCTTCTGCAGCAGNNNN

CCCCCCCTCGACCGCAAAACGTCGTGA

GACAGTTTGGTCCAGCTTGATAT

GGGGGGGAGCTGGCGTTTTGCAG

CACTCTGTCAAACCAGGTCGAACTATA

CTGCTGCAGAAGCTTGGATCCATGA-Bio

Bio-AGTACCTAGGTTCGAAGACGTCGTC

NNNNGACGACGTCTTCGAACCTAGGTACT

TCATGGATCCAAGCTTCTGCAGCAGNNNN

CCCCCCCTCGACCGCAAAACGTCGTGA

GACAGTTTGGTCCAGCTTGAT

GGGGGAGCTGGCGTTTTGCAG

CACTCTGTCAAACCAGGTCGAACTATA

CTGAGCTCGGACTCTTAAGGACGTCNNGACTCGAGCCTGAGAATCCCTGCAG

NNCTGCAGGAATTCTCAGGCTCGAGTCGACGTCCTTAAGAGTCCGAGCTCAG

capture forw.

capture rev.

capture forw.

capture rev.

EcoP15I

EcoP15I

<

<

<

<

68bp

68bp

I-CreI cleavage

Annealing/ligation of linker/capture oligos

Digestion with EcoP15IStreptavidin binding of digestion productsAnnealing/ligation of adapter/linker oligosWash off streptavidin beadsPCR

Cloning and sequencing Protocol

5’ I-CreI hspBScapt. forw. capt. rev.

3’ I-CreI hs pBS capt. forw.capt. rev.

m-CreI end capture – pBSCre positive controlm-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_011520.11|Hs22_11677 Homo sapiens chromosome 22 genomi... 435 2e-119ref|NW_001838745.1|Hs22_WGA1304_36 Homo sapiens chromosome 22... 429 1e-117ref|NW_927628.1|HsCraAADB02_665 Homo sapiens chromosome 22 ge... 429 1e-117ref|NT_011519.10|Hs22_11676 Homo sapiens chromosome 22 genomi... 407 5e-111ref|NW_001838740.2|Hs22_WGA1299_36 Homo sapiens chromosome 22... 392 1e-106ref|NW_921371.1|HsCraAADB02_1017 Homo sapiens genomic contig,... 387 7e-105

2/4/5: three hits on chromosome 6 (97% identity): ref|NT_007592.14|Hs6_7749 Homo sapiens chromosome 6 genomic c... 1184 0.0 ref|NW_001838973.1|Hs6_WGA366_36 Homo sapiens chromosome 6 ge... 1184 0.0 ref|NW_922984.1|HsCraAADB02_247 Homo sapiens chromosome 6 gen... 1184 0.0

2/4/6: three hits on chromosome 5 (100% identity): ref|NT_006576.15|Hs5_6733 Homo sapiens chromosome 5 genomic c... 348 4e-93ref|NW_001838924.2|Hs5_WGA317_36 Homo sapiens chromosome 5 ge... 348 4e-93ref|NW_922518.1|HsCraAADB02_205 Homo sapiens chromosome 5 gen... 348 4e-93

m-CreI end capture – BLAST resultsm-CreI end capture – BLAST results

sites