Presentation by Eric Bennett for Copenhagen Bioscience Lecture at the Novo Nordisk Foundation on 7 December 2017

1. Eric Paul Bennett, MSc, Dr.med.
Copenhagen Center for Glycomics/Department of Odontology
Glyco meets
Genome
Targeting,,,,,,
what’s the link?
?

2. The Premise for CDO
Establish University of Copenhagen as a leading knowledge
and education center on the technological, legal and ethical
aspects of genome editing.
Establish the infrastructure for UCPH scientists to use
ZFNs/TALENs for genetic engineering of cells and organisms.
2013-2017, 16million DKr
Hans Wandall
Eric P. Bennett

3. CCG/Department of OdontologyEric P. Bennett
The ”short history ” of Precise Genome Targeting
ZFNs
CRISPR/Cas Breakthrough of the Year Science, 2015
“The Biggest Biotech Discovery
of the Century”
MIT Technology Review and Forbes magazine
December 4, 2014
TALEs target the
human genome
2011
Commercialized
open source
“democratized”

4. 4Copenhagen Center for Glycomics
ZFNsTALENs CRISPR/Cas9
Double stranded break
wt
deletion
insertion
INDEL
Cellular repair

5. Discovery & Molecular Dissection of
Diseases Caused by Defects in
Glycosylation
Glyco meets Genome Targeting:
What’s the link?

6. http://biofoundations.org/tag/glycocalyx/
Glycans are Universally found on all living Cells

7. ”Candy Floss”, restaurant ”Geist”
Glycans as a Cellular “Candy Floss”

8. Precise Gene Targeting: 13 B.C.50 ZFNs
5 TALENs
Hansen L., et al., Glycobiology, 2015, 25(2):211-24
200 CRISPRs
validated!
13 A.C.
Creating Isogenic Glycogene Engineered Cells

9. Kosicki et al. Prog Mol Biol Transl Sci. 2017 CRISPR
Pinto et al. NAR, 2017 ZFN
Lonowski et al. Nature Protocols 2017 ZFN/TALEN/CRISPR
Yang et al. NAR 2015 ZFN/TALEN/CRISPR
Duda et al. NAR, 2014 ZFN/TALEN/CRISPR
Steentoft et al., Methods Mol Biol. 2013 ZFN
Steentoft et al. Nature Methods 2011 ZFN
3X
CCG Gene Targting Publications from 2011,,,,

10. CRIPSR/Cas9 and it’s repurposed Applications
CCG/Department of Odontology
Hsu, Lander and Zhang, cell, 2014
Nuclease
(2013)
Nickase Activator
Repressor
Modulator
Epigenetic modifier
Base editor
DNA labeling Inducible regulation

11. Underlying Principle for Cas9 DNA binding
CRISPR applications:
Garneau et al, 2010, Nature, 468: 67-71.
Haurwitz et al., 2010, Science, 329: 1355-5855.
Deltcheva et al, 2011, Nature, 471: 602-9
Jinek et al., 2012. Science. 337:816.
Cong et al, 2013, Science, 339: 819-23.
Mali et al., 2013, Science, 339, 823-826.
Gilbert et al, 2014, Cell
Hilton et al., Nat Bitechnol, 2015
Komor et al., Nature, 2016
• Nuclease
• Nickase
• Activator
• Repressor
• Modulator
Epigenetic modifier
Base editor
• DNA labeling
• Inducible regulation
gRNA

12. Cas9 induced DSB Formation as a
“Proxy” for optimal Cas9 Binding
Double stranded break
wt
INDEL
Cellular repair
Nuclease
Nickase Activator
Repressor
Modulator
Epigenetic modifier
Base editor
DNA labeling

13. Factors affecting Cas9 targeting efficiency

14. 07-12-2017 14
/49
Gene name
(HGNC)
Protocol gRNA Sequence
Validation*
+=10-25% , ++=26-50%, +++= >50%
Amplicon
Major
indelPool
FACS
Bulk
GALNT10
BbsI/QCG ACTCTCTCAGCATCGGTCAT + +++ +1
BbsI/QCG CTCTCTCAGCATCGGTCATG + +++ +1
QCgRNA amplicon ATCTGGGAGAGAGCGATTCA + ++ -70
BbsI/QCG ATCGCTCTCTCCCAGATATC - - -
GALNT11
BbsI/QCG ATGCTTATCAGTGACCGCTT - - -
BbsI/QCG TATGCTTATCAGTGACCGCT + ++ +1
QCgRNA amplicon CGATCAAGAGTTGAGAGACT - ++ -9/-2
BbsI/QCG CATCTCTGTGGTAGCCCAAG - +++ +1
GALNT12
QCgRNA amplicon GGACACTGTAAACTGTCCGA - - -
BbsI/QCG TTCTTCTAGCAGGATATCCG + +++ +1
BbsI/QCG GGATATCCGGGGATGTCTCA + +++ -2
BbsI/QCG TGTCCGAAGGAGAGTTGACC - ++ -31
GALNT13
QCgRNA amplicon TTAATACGTGCCCGTCTTCG + ++ +1
QCgRNA amplicon ACTGTGAATGCACGTTAGGA - + +1
QCgRNA amplicon TGAATGCACGTTAGGATGGC - - -
QCgRNA amplicon AAGCAGCTGCTCCTCGAAGA + +++ +1
GALNT14
QCgRNA amplicon AGGTTAATGATATCGATCAC - - -
QCgRNA amplicon CTCCGAGGCAGACTCGATGT + ++ -35
QCgRNA amplicon CACCTACATCGAGTCTGCCT - - -
QCgRNA amplicon GGCAGACTCGATGTAGGTGA - ++ -6
QCgRNA amplicon TTGTCTTACAGGACTACACG - +++ +1
QCgRNA amplicon CTTACAGGACTACACGCGGG + +++ +1
QCgRNA amplicon GGTTAATGATATCGATCACA + +++ +1
GALNT15
QCgRNA amplicon GGATGCTGTGTACAGTCCGC - - -
QCgRNA amplicon GCTGGCTGAGGTCGTCCACG + +++ +1
QCgRNA amplicon CCTGAAGGAGATCATCCTCG - ++ +1
GALNT16
QCgRNA amplicon TGATCCGGTCCCGAGTGCGT - - -
QCgRNA amplicon CCGCCCCACGCACTCGGGAC - - -
QCgRNA amplicon ACTGCGAAGTGAACACCGAG - - -
QCgRNA amplicon ACTCGGTGTTCACTTCGCAG - - -
QCgRNA amplicon TGATGAGAAGGCCTACCTGT + +++ -12/+1
QCgRNA amplicon TCAGCTTGTCACTCTCCAGC + +++ +1
QCgRNA amplicon GTAATGGCGGGTGTCCCGGA + +++ +1
GALNT17
QCgRNA amplicon CATCCGGACCCGTCTCCTGG + +++ -1/+1
QCgRNA amplicon AGTTCACATTGACCTCGCAG + +++ -2
QCgRNA amplicon ATTCCTGGACTCCCACTGCG - + +1
GALNT18
QCgRNA amplicon ATGGCGAGATGATCCGCTTC - - -
BbsI/QCG TGAGATAGAAGAGTACCCGC - ++ -1/+1
BbsI/QCG ACCTGTACTCACCCGCATCA - ++ +1
BbsI/QCG TCCTTGATGCGGGTGAGTAC - - -
GALNT19
QCgRNA amplicon AGGACAACTTTGAGGTGCAG - - -
QCgRNA amplicon CAGCTCCCAGCTGTACCCGT - - -
QCgRNA amplicon CGTCCCACCAGTCTTTTGGG - - -
QCgRNA amplicon GTTGGTGTTGAACTTGATCG 20 60 +1
QCgRNA amplicon CGGCCCATCGCGGTGCGCAG - - -
QCgRNA amplicon AGGTTGGCCACCTCGGCGCG 15 70 +1
GALNT20
QCgRNA amplicon ATACTCTGTTCACCTCACAG + +++ +1
QCgRNA amplicon CATCAATGACATCTATCAGG - + +1
QCgRNA amplicon AGTTCCCCTTACAAGAGGAG - ++ +1
Factors Determining the gRNA Design Functionality?
L.Lonowski et al., Nat Protoc, 2017
Y. Narimatsu et al., manuscript accepted
Score 31-50
https://www.deskgen.com/landing/
22/49=45%
Gene name
(HGNC)
Protocol gRNA Sequence
Validation*
+=10-25% , ++=26-50%, +++= >50%
Amplicon
Major
indelPool
FACS
Bulk
GALNT16
QCgRNA amplicon TGATCCGGTCCCGAGTGCGT - - -
QCgRNA amplicon CCGCCCCACGCACTCGGGAC - - -
QCgRNA amplicon ACTGCGAAGTGAACACCGAG - - -
QCgRNA amplicon ACTCGGTGTTCACTTCGCAG - - -
QCgRNA amplicon TGATGAGAAGGCCTACCTGT + +++ -12/+1
QCgRNA amplicon TCAGCTTGTCACTCTCCAGC + +++ +1
QCgRNA amplicon GTAATGGCGGGTGTCCCGGA + +++ +1

15. GlycoCRISPR: Matrix of 210 Human validated Glycogene
gRNA Designs out of >800 Designs tested
L.Lonowski et al., Nat Protoc, 2017
Y. Narimatsu et al., Glycobiology, in press
+1 as predominant
Cas9 indel event in
>60% gRNA designs
D.Paquet et al., Nature, 2016
M. van Overbeek et al., Mol. Cell, 2016

16. Cellular
DNA extraction
Days
Cell pool
Insight
CRISPR/
Cas9
Indel
Detection
Copenhagen Center for Glycomics
Indel Detection,,,, why and when?
?
Indelefficiency
days
post delivery indel detection/dynamics
100%
??
?methods for fast and sensitive
profiling of CRISPR/Cas9
induced indel dynamics is
warranted

17. M. Kosicki et al. Elsevier, 2017
Most commonly used Indel Detection Methods

18. Most Commonly used Indel Detection Methods
+ - + -
T7EI/Cel-I/Surveyor
Clone#2
K562, CRISPR/Cas9, tri-allelic
KRAS targeting
EMC
(Enzyme Mismatch Cleavage)
Cell Pool
0 1 Days
Undefined identification
of ”indel events” in cells
NGS
”deep sequencing” or Sanger
Defined identification of
individual ”indel events” in cells
0 1 Days2 3 4 5
?

19. ɸX-HaeIII
Hela-D4E
DNAcontrol
*
-1bp wt
Main Enzyme Mismatch Cleavage Assay Issue
19
• Possess background nonspecific activity
and exonucleolytic avtivity
• T7EI has preference for heteroduplex DNA
formed by deletionsY.Zhang et al., NAR, 2015
M. C. Huang et al.,. Electrophoresis 33, 2012
L. Vouillot et al., G3 5, 407–415, 2015
T. Sakurai, et al., BMC Biotechnol., 2014
T7EI
Insufficient detection
of minor indels

20. “Almost all quality improvements comes via
simplification of design,,,,layout, processes, and
procedures”
Tom Peters, American Businessman
Method for fast and sensitive profiling of
CRISPR/Cas9 induced Indel Dynamics
“Keep it simple”

21. Amplicon electrohoresis
Detection
Single-base Size discrimination
power of the Sequenator!

22. 22
DSB
deletion
insertion
*
*
*
*
Tri-primer PCR
wt
INDELs
RFU
25%
6%
0.5%
11%
0.2%
10%
-16 -7 -5 -3 -1 +10
insertionsdeletions
360 370 380 390 400
3600
2800
2000
1200
400
0
indel size/bp
wt size/bp
Zhang Yang
Indel Detection by Amplicon Analysis (IDAA)
Z. Yang et al., Nucleic Acids Res. 2015;43(9):e59
Precise Gene Targeting
PCT/DK2015/050405

23. IDAATM for Genome Targeting Indel Profiling
Copenhagen Center for Glycomics
*
*
*
*
Tri-primer PCR
IDAA
TM
Z. Yang et al., Nucleic Acids Res. 2015
K562 CRISPR/Cas9, tri-
allelic KRAS Targeting
Pool
T7EI
Clone#2
306 314
Clone#2
WT
316
Cell pool
IDAA
+ - + -
345
345344
-1bp/Hela- D4E
WT
WT
-1bp
RFU
335 345 355
872
ɸX
-1 WT
HeLa COSMC-ZFN Targeting
IDAA
T7EI
IDAA
2h PCR
+
2h CE

24. Slide 24
NGS/MiSeq indel profile
IDAATM Indel Profile & Detection Sensitivity similar to NGS
F R+
6-FAM
Indel Detection by Amplicon Analysis
IDAA
IDAA indel profile
Cell Pool
DNA lysate
d0 d2
+
gRNA2
1X106 cells
vs

25. IDAA amplicon size/bp345315285
30000
20000
15000
10000
5000
25000
0
1
2
345
RFU
ABI3500xl
-15 -11 -8 -4 +1 indel size/bp
Fragments/reads
0
50000
100000
150000
200000
250000
300000
350000
400000
-60
-57
-54
-51
-48
-45
-42
-39
-36
-33
-30
-27
-24
-21
-18
-15
-12
-9
-6
-3
0
3
6
9
12
15
18
21
24
27
30
33
36
39
42
45
48
0
1
2
3
45
NGS
L.Lonowski, Nat Protoc, 2017
amplicon size/bp345315285
300
400
200
100
345 012
6
-15-23-25-26
7
8
indel size/bp0 +1-10-20-30
Zoom in
0
1000
2000
3000
4000
5000
-60
-57
-54
-51
-48
-45
-42
-39
-36
-33
-30
-27
-24
-21
-18
-15
-12
-9
-6
-3
0
3
6
9
12
15
18
21
24
27
30
33
36
39
42
45
48
8
Fragments/reads
Zoom in
7
6
45 0123
IDAA Indel Profile & Detection Sensitivity similar to NGS

26. L.Lonowski, Nat Protoc, 2017
Reliable Sanger detection limit 17%
S. Jamuar et al., N ENGL J MED, 2014
Reliable T7EI (EMC) detection limit 2-5%
Y. Fu , J. Sander, Nat. Biotech, 2013
NGS detection limit as low as 0.12%
S. Jamuar et al., N ENGL J MED; 2014, Tsai, Nat Biotech, 2015
%relativetowtpeak
log scale
Peak number
26,5%
11,5%
0,23%
0,09%
0,13%
19,6%
10,5%
0,59%
0,22% 0,31%
0,01
0,1
1
10
100
1 2 3 4 5 6 7 8
IDAA%/wt
MiSeq%/wt
IDAA Indel Profile & Detection Sensitivity similar to NGS
≈0.1%

27. IDAA Indel “Finger Print” of a given gRNA Design
Copenhagen Center for Glycomics
K562
HEK293
L.Lonowski, Nat Protoc, 2017

28. Copenhagen Center for Glycomics
Dynamics of Indel Profiles Induced by
Various CRISPR/Cas9 Delivery Methods
Emmanouil
Metzakopian
SangerInstitute

29. Dynamics of Indel Profiles Induced by Various
CRISPR/Cas9 Delivery Methods
CCG/Department of OdontologyEric P. Bennett
Human ST6GALNACI
+1

30. Genome Editing Workflow
based on FACS and IDAA
+
gRNA
A
B
L.Lonowski et al., Nat Protoc, 2017
Z.Yang et al., Nat Biotech, 2015
BRIC, KU
MortenFrödin

31. Dynamics of Indel Profiles Induced by Various
CRISPR/Cas9 Delivery Methods
CCG/Department of OdontologyEric P. Bennett
Human ST6GALNACI
+1

32. Copenhagen Center for Glycomics
Dynamics of Indel Profiles Induced by Cas9-sygRNA
(RNP) Delivery
WT
+1
ST6GALNACI
M. Kosicki, et al., Progress in Molecular Biology and Translational Science, Elsevier, 2017

33. Copenhagen Center for Glycomics
Dynamics of Indel Profiles Induced by
Various CRISPR/Cas9 Delivery Methods
M. Kosicki, et al., Progress in Molecular Biology and Translational Science, Elsevier, 2017

34. CCG/Department of OdontologyEric P. Bennett
1
2
3
Therapeutic issues:
• Point of care setup
• Gene targeting tool (CRISPR/ZFN/TALEN)?
• Delivery/LV/non-viral/RNP/RNA issue?
• Post editing stemness of cells?
• Safety?
• Gene targeting dynamics and efficacy?
Human
disease targeted
not targeted
?

35. Therapeutic haemapoetic Target Cell Type:
CD34+ Stemcell (≈ 0.03-0.09%)
Copenhagen Center for Glycomics
L. Eidenschink et al., Clinical Cytometry, (2012)
G.E. Tjrannfjord et al., Blood, Vol 84, (1994)
Blood
Plasma
Erythrocytes + Granulocytes
Peripheral Blood Mononuclear Cells
Cox, Platt, Zhang, 2015, Nat Medicin?
Quantify the targeting
efficacy/profile?

36. Sarah B. Laskey& Robert F. Siliciano, Nature Reviews, Microbiology, 2014
The Infectious HIV Cycle
Copenhagen Center for Glycomics

37. IDAATM for Therapeutic Genome
Targeting Profiling
Cox, Platt, Zhang, 2015, Nat Medicine
Copenhagen Center for Glycomics37
Toni Cathomen Claudio Mussolino
CCR5
?
Quantify the targeting
efficacy/profile?

38. 38
IDAA Reagents and Services
https://coboscientific.com/
info@coboscientific.com
https://tagc.dk/
Ship Amplicons

39. GlycoDisplay HEK293 Glycan-optimization Platform
Screening
Functional testing
Glyco-optimized
product
PCT 62/193,403

40. Henrik Claus Yoshiki Eric
Copenhagen Center for Glycomics
Spin OutSpin Out
Yang

41. Spin OutSpin Out
Glyco meets Genome Targeting

42. Financial support
U. of Copenhagen
Danish Research Councils
The Carlsberg Foundation
The Alfred Benzons
Foundation
The AP Møller Foundation
The Novo Nordisk
Foundation
EU FP7, EU Marie Curie
NIH-NCI
Henrik Clausen
Hans Wandall
Zhang Yang
Yoshiki Narimatsu
Weihua Tian
Claus Kristensen
Lars Hansen
Catharina Steentoft
Rita Pinto
Flaminia Lorenzetti
Copenhagen Center for Glycomics
Malene Ambjørn
Lundbeck, DK
Sofia Håkansson Buch
Shamim H.Rahman
Mark Behlke
Greg Davids
MilliporeMerck, US
Gurpreet Balrey PhD
Merck, UK
Jon Chesnut
Jason Potter
Morten Frødin
BRIC, KU
Toni Cathomen
Claudio Mussolino
University of Freiburg, DE
Emmanouil Metzakopian
Sanger Institute, UK