HE site degeneracy – HE targets in genomic DNA
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)
End capture
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
BLAST
Oligos
m-CreI end capture – BLAST results
?
5‘ - C C A C G C T T C T C A C - 3‘
Human Chr. 22 5‘ - T G A A A C G T C G G G g g a c a g g a c c - 3‘
+10
+9
+11
+8
+7
+6
+5
+4
+3
+2
+1
-2
-1
-4
-3
-5
-6
-7
-8
5‘ - a g g g g t t c c G T G A G A C A G A G A T - 3‘
-9
Human Chr. 6
-10
5‘ - A C A A C C T T C A C G A g a a g t c t c a - 3‘
-11
Human Chr. 5
5‘ - C A A A A C G T C G T G A G A C A G T T T G - 3‘
C.r. cp 23S rRNA
3‘ - G T T T T G C A G C A C T C T G T C A A A C - 5‘
Exon2
Exon1
IIS
hits
ChIP-seq
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
Map back on genome sequence
ChIP-seq
• Non integrating lentiviral expression vectors with the following characteristics:
- EF1a 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?)
CCCCCCCTCGACCGCAAAACGTCGTGA GACAGTTTGGTCCAGCTTGATAT
GGGGGGGAGCTGGCGTTTTGCAGCACTCTGTCAAACCAGGTCGAACTATA
I-CreI cleavage
CCCCCCCTCGACCGCAAAACGTCGTGA
GACAGTTTGGTCCAGCTTGATAT
GGGGGGGAGCTGGCGTTTTGCAG
CACTCTGTCAAACCAGGTCGAACTATA
Annealing/ligation of linker/capture oligos
<
EcoP15I
TCATGGATCCAAGCTTCTGCAGCAGNNNNGACAGTTTGGTCCAGCTTGATAT
Bio-AGTACCTAGGTTCGAAGACGTCGTCCACTCTGTCAAACCAGGTCGAACTATA
<
<
<
CCCCCCCTCGACCGCAAAACGTCGTGACTGCTGCAGAAGCTTGGATCCATGA-Bio
GGGGGGGAGCTGGCGTTTTGCAG NNNNGACGACGTCTTCGAACCTAGGTACT
EcoP15I
Digestion with EcoP15I
Streptavidin binding of digestion products
Annealing/ligation of adapter/linker oligos
Wash off streptavidin beads
PCR
capture rev.
TCATGGATCCAAGCTTCTGCAGCAGNNNN GACAGTTTGGTCCAGCTTGATNNCTGCAGGAATTCTCAGGCTCGAGTC
Bio-AGTACCTAGGTTCGAAGACGTCGTCCACTCTGTCAAACCAGGTCGAACTATAGACGTCCTTAAGAGTCCGAGCTCAG
68bp
capture forw.
capture forw.
GACTCGAGCCTGAGAATCCCTGCAG CCCCCCCTCGACCGCAAAACGTCGTGACTGCTGCAGAAGCTTGGATCCATGA-Bio
CTGAGCTCGGACTCTTAAGGACGTCNN GGGGGAGCTGGCGTTTTGCAG NNNNGACGACGTCTTCGAACCTAGGTACT
capture rev.
68bp
Cloning and sequencing
Protocol
m-CreI end capture – pBSCre positive control
capt. forw.
capt. rev.
pBS
3’ I-CreI hs
5’ I-CreI hs
pBS
capt. rev.
capt. forw.
m-CreI end capture – BLAST results
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...
ref|NW_001838745.1|Hs22_WGA1304_36 Homo sapiens chromosome 22...
ref|NW_927628.1|HsCraAADB02_665 Homo sapiens chromosome 22 ge...
ref|NT_011519.10|Hs22_11676 Homo sapiens chromosome 22 genomi...
ref|NW_001838740.2|Hs22_WGA1299_36 Homo sapiens chromosome 22...
ref|NW_921371.1|HsCraAADB02_1017 Homo sapiens genomic contig,...
435
429
429
407
392
387
2e-119
1e-117
1e-117
5e-111
1e-106
7e-105
2/4/5: three hits on chromosome 6 (97% identity):
ref|NT_007592.14|Hs6_7749 Homo sapiens chromosome 6 genomic c...
ref|NW_001838973.1|Hs6_WGA366_36 Homo sapiens chromosome 6 ge...
ref|NW_922984.1|HsCraAADB02_247 Homo sapiens chromosome 6 gen...
1184
1184
1184
0.0
0.0
0.0
2/4/6: three hits on chromosome 5 (100% identity):
ref|NT_006576.15|Hs5_6733 Homo sapiens chromosome 5 genomic c...
ref|NW_001838924.2|Hs5_WGA317_36 Homo sapiens chromosome 5 ge...
ref|NW_922518.1|HsCraAADB02_205 Homo sapiens chromosome 5 gen...
348
348
348
4e-93
4e-93
4e-93
sites