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"CRISPR the new
Bio-revolution in the bench”
Pereyra-Bonnet Federico, PhD
Investigador Asistente CONICET
Instituto Universitario
Hospital Italiano, Argentina.
CRISPR-Cas9 are molecular
scissors for gene editing
“Molecular scissors to cut and modify
the DNA with unprecedented efficiency”
DNA
Ledford, 2016, Nature (531) 156-159.
Why CRISPR is a bio-revolution?
Low cost
Specificity
Efficient
Easy
Gene correction
Transgenic organisms Gene
function
Model diseases
Genetic engineering
“CRISPR would make reality all
the promises given by the GENE
THERAPY in a more easy,
efficient and economic way "
The impact of CRISPR
technology in the Labs
You can see the
impact of CRISPR
in the rapid
increase of papers
published around
the world.
0
200
400
600
800
1000
1200
1400
Year 2011 2012 2013 2014 2015
Papers 1 3 78 315 717 1267
Papers 1 3 78 315 717 1267
www.ncbi.nlm.nih.gov/pubmed/?term=crispr+cas9
CRISPR Papers
The impact of CRISPR
technology in the web
Inputs and Outputs from Google Search as
a measure of social interest in the web!
CRISPR Gene Therapy Stem Cells
4.860.000 7.710.000 66.100.000
Wen Xue, a postdoc student spent one year
and U$S 20.000 to make a transgenic
mouse. After CRISPR Xue said “We had the
mouse in one month”.
Dr Rost from the Univ Medical Center in
Netherlands said “The new tool (CRISPR)
have democratized the field”… (It’s low cost
and easy to use).
“It’s just so fun” said Dr Parnham from UCLA.
Comments extracted from “Riding the
CRISPR wave” (Ledford, 2016, Nature (531) 156-159).
The impact of CRISPR
technology in the researchers
Luz Brillante Luz Azul Superposición
CBA
At that time gene editing
was hard work!!!
CRISPR-Cas9 for gene editing is
just the beginning!
Epigenome=Software
K9ac
EPIGENETICS
DNA
Genome=Hardware
K9ac
EPIGENETICS
Question 1: Can we regulate the expression of one gene
changing only its EPIGENETIC marks?
DISEASES
CELLULAR
FUNCTIONS IMPRINTING
X INACTIVATION
PHENOTYPES DEVELOMPMENT
Question 2: Can CRISPR change these EPIGENETIC marks
without modifying the DNA sequence?
CRISPR-Cas9 with broken scissors
CRISPR-ON: to Epigenetic Editing There are two chief ingredients in
the CRISPR–Cas9 system: a Cas9
enzyme that cut the DNA like a
molecular scissors, and a small
RNA molecule that directs the
scissors to a specific sequence.
Gilbert et al., 2013.Cell.154(2):442-51.
Cheng et al., 2013. Cell Research. 23:1163-1171.
In CRISPR-ON the Cas9 endonuclease is inactived.
CRISPR-ON does not
cut the DNA, but the
complex can still
bind to specific
targets and carries
an activator protein.
Activating a gene with
CRISPR-ON system
RNA-guide
(directs the CRISPR-ON
to a specific sequence)
Plasmid
Guide design program Zhang, MIT. http://crispr.mit.edu
Vectors: Addgene #47108; #48226.
OFF ON
Target gene
Activating a gene with
CRISPR-ON system
PROGRAM Zhang, MIT http://crispr.mit.edu
Target gene
Activating a gene with
CRISPR-ON system
CRISPR-ON to modify the
Epigenetic
Modify epigenetics marks to
regulate gene transcription.
Epigenetic
Editing
Epigenetic
Therapy
Modify epigenetics marks in
vivo to treate a disease.
=
=
Our Goal: find new strategies to generate bonafide insulin-
producing cells for Diabetes treatment.
1Biopsy from patient
with T1D 2In vitro reprogramming
to insulin-producing
cells
3Autologous transplantation
of reprogrammed cells
Protocols and Informed Consent were approved by the Institutional Ethics Committee 1672.
PATENT presented:
INPI Nº 20130101884
C+ R1 R2 R1 R2
R1 R2 R1 R2
HF3 HF6 HF3 HF6
Control Cells CRISPR-on
C-
HEK293T
HeLa
HFs
INS
ACTB
INS
ACTB
INS
ACTB
RT-PCR
A
HUMAN
PANCREAS
HEK293T
CRISPR-on
B
C
81 34 63 25
2780
0
1000
2000
3000
4000
mR
NA
ex
press
ion
INS
ACTB
CRISPR- on
D
*
CRISPR- on
INS
ACTB
a b
c d
RT-qPCR
By CRISPR-ON we activated the Insulin gene in
fibroblasts from patients with Diabetes.
INS
K9ac
0
10
20
30
40
50
60
70
80
90
100
-35
7
-34
5
-23
4
-20
6
-18
0
-13
5
-10
2
-69
% D
NA
met
hy
lati
on
Cytosine position relative to TSS
Ctrol cells
CRISPR-on
0,00
0,01
0,02
0,03
0,04
0,05
0,06
0,07
0,08
0,09
0,10
0,11
0,12
0,13
0,14
0,15
0,16
0,17
0,18
0,19
0,20
0,21
0,22
0,23
0,24
0,25
0,26
0,27
0,28
0,29
0,30
0,31
0,32
0,33
0,34
0,35
0,36
0,37
0,38
0,39
0,40
0,41
0,42
0,43
0,44
0,45
0,46
0,47
0,48
0,49
0,50
0,51
0,52
0,53
0,54
0,55
0,56
0,57
0,58
0,59
0,60
0,61
0,62
0,63
0,64
0,65
0,66
0,67
0,68
0,69
0,70
0,71
0,72
0,73
0,74
0,75
0,76
0,77
0,78
0,79
0,80
0,81
0,82
0,83
0,84
0,85
0,86
0,87
0,88
0,89
0,90
0,91
0,92
0,93
0,94
0,95
0,96
0,97
0,98
0,99
1,00
1,01
1,02
1,03
1,04
1,05
1,06
1,07
1,08
H3
K4
me
3
H3
K9
ac
IgG
H3
K4
me
3
H3
K9
ac
IgG
H3
K4
me
3
H3
K9
ac
IgG
H3
K4
me
3
H3
K9
ac
IgG
H3
K4
me
3
H3
K9
ac
IgG
H3
K4
me
3
H3
K9
ac
IgG
Ctrol cells CRISPR-on Ctrol cells CRISPR-on Ctrol cells CRISPR-on
GAPDH INS Sat2
%In
pu
t
0,00
0,50
1,00
1,50
H3
K4
me
3
H3
K9
ac
IgG
H3
K4
me
3
H3
K9
ac
IgG
H3
K4
me
3
H3
K9
ac
IgG
H3
K4
me
3
H3
K9
ac
IgG
H3
K4
me
3
H3
K9
ac
IgG
H3
K4
me
3
H3
K9
ac
IgG
Ctrol cells CRISPR-on Ctrol cells CRISPR-on Ctrol cells CRISPR-on
GAPDH INS Sat2
%In
pu
t
1,50
1,00
0,50
0,05
0,04
0,03
0,02
0,01
0,00Control
cells
Control
cells
Control
cells
CRISPR CRISPR CRISPR
GAPDH INS Sat2
0
1
2
3
4
5
6
a b
H3K4me3
H3K9ac
IgG control
% I
np
ut
BA
*
a bChiP of INS
promoter
Co-IP
Carla Giménez et al., 2016 Gene Therapy.
Conclusions
2
CRISPR-ON does not cause genetic
modifications. This is a clear advantage if we
are thinking about translational medicine.
3 We and others show that the CRISPR-ON system
could be used for future epigenetic therapies.
4
As all new technology, CRISPR must be still
restricted to research to be deeply tested,
before it becomes a real option to treat human
diseases.
Nowadays, CRISPR-Cas9 is the most effective
tool for gene editing.
1
STATEMENT
“The International Society for Stem Cell Research calls
for a moratorium on attempts at clinical application of
nuclear genome editing of the human germ line to enable
more extensive scientific analysis of the potential risks of
genome editing and broader public discussion of the
social and ethical implications”.
Financial
supports
Group
Dr Pereyra-Bonnet F
Dr Hyon Sung-Ho
Dr Grosembacher Luis
Dr De Santibañes Martín
Lic Barbich Mariana
Dra Mónica Loresi
Bioq Ielpi Marcelo
Post-Doctoral Fellow
Dra Mejias Pilar
Doctoral Fellow
Lic Giménez Carla
Magister Fellow
Lic Rueda Nelson
Undergraduate student
Srta Curti Lucia
Collaborators
Dr Fernandez-Rafael (AGRO-UBA)
Dra Fainstein-Day Patricia
Qui Balzarretti Marta
Dr Litwak Leon
Dr García-Rivello
Dr Mutto Adrián (UNSAM)
Thanks!
em
ail: fe
de
ric
o.p
ere
yra
@h
osp
ita
lita
lin
o.o
rg
.a
r
tw
itte
r @
IC
BM
Ed
ia
betes
The cellular alchemists …
