"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
DNA
“Molecular scissors to cut and modify
the DNA with unprecedented efficiency”
Ledford, 2016, Nature (531) 156-159.
Why CRISPR is a bio-revolution?
Low cost
Specificity
Efficient
Easy
Gene
correction
Model
diseases
“CRISPR would make reality all
the promises given by the GENE
THERAPY in a more easy,
efficient and economic way "
Transgenic
organisms
Genetic
engineering
Gene
function
The impact of CRISPR
technology in the Labs
Papers 1 3 78 315 717 1267
1400
1200
CRISPR Papers
1000
You can see the
impact of CRISPR
Papers
3 78 rapid
315 717
in 1the
1267
increase
of papers
published around
the world.
800
600
400
200
0
Year
2011
2012
2013
2014
2015
www.ncbi.nlm.nih.gov/pubmed/?term=crispr+cas9
The impact of CRISPR
technology in the web
CRISPR
Gene Therapy
Stem Cells
4.860.000
7.710.000
66.100.000
Inputs and Outputs from Google Search as
a measure of social interest in the web!
Comments extracted from “Riding the
CRISPR wave” (Ledford, 2016, Nature (531) 156-159).
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.
The impact of CRISPR
technology in the researchers
Luz Brillante
A
Luz Azul
B
Superposición
C
At that time gene editing
was hard work!!!
CRISPR-Cas9 for gene editing is
just the beginning!
EPIGENETICS
K9ac
DNA
Genome=Hardware
Epigenome=Software
CELLULAR
FUNCTIONS
DISEASES
IMPRINTING
X INACTIVATION
EPIGENETICS
PHENOTYPES
DEVELOMPMENT
Question 1: Can we regulate the expression of one gene
changing only its EPIGENETIC marks?
Question 2: Can CRISPR change these EPIGENETIC marks
without modifying the DNA sequence?
CRISPR-Cas9 with broken scissors
CRISPR-ON: to Epigenetic Editing
CRISPR-ON does not
cut the DNA, but the
complex can still
bind to specific
targets and carries
an activator protein.
In CRISPR-ON the Cas9 endonuclease is inactived.
Gilbert et al., 2013.Cell.154(2):442-51.
Cheng et al., 2013. Cell Research. 23:1163-1171.
Activating a gene with
CRISPR-ON system
Plasmid
Vectors: Addgene #47108; #48226.
RNA-guide
(directs the CRISPR-ON
to a specific sequence)
Guide design program
Zhang, MIT. http://crispr.mit.edu
Activating a gene with
CRISPR-ON system
OFF
ON
Target gene
Activating a gene with
CRISPR-ON system
Target gene
PROGRAM
Zhang, MIT
http://crispr.mit.edu
CRISPR-ON to modify the
Epigenetic
Epigenetic
Editing
=
Modify epigenetics marks to
regulate gene transcription.
Epigenetic
Therapy
=
Modify epigenetics marks in
vivo to treate a disease.
Our Goal: find new strategies to generate bonafide insulinproducing cells for Diabetes treatment.
1 Biopsy from patient
with T1D
2
In vitro reprogramming
to insulin-producing
cells
PATENT presented:
INPI Nº 20130101884
3 Autologous transplantation
of reprogrammed cells
Protocols and Informed Consent were approved by the Institutional Ethics Committee 1672.
0,42
0,41
0,40
By CRISPR-ON we activated the Insulin gene in
a
b
A
B
fibroblasts from patients with
Diabetes.
0,39
0,38
0,37
0,36
CRISPR- on
0,35
0,34
HUMAN
PANCREAS
RT-PCR
0,33
0,32
RT-qPCR
INS
0,31
ACTB
HEK293T
CRISPR-on
0,30
0,29
Control
Cells CRISPR-on
R1
0,27
R2
R1
C
R2
c
CINS
ACTB
0,25
0,24
0,23
R1
R2
R1
R2
0,22
HeLa
INS
ACTB
0,21
0,20
6
HF6
CRISPR- on
INS
2000
INS
1000
81
34
63
ACTB
25
HF3 HF6
0,18
INS
ACTB
0,17
5
3000
dD
0
0,19
HF3
HFs
*
4000
0,26
HEK293T
2780
mRNA expression
0,28
C+
0,16
0,15
4
0,14
0,10
a
30
0,03
0,03
Ctrol cells
GAPDH
IgG
H3K9ac
IgG
H3K9ac
IgG
H3K4me3
IgG
b
CRISPR-on
Ctrol cells
0,02
0,02
H3K9ac
H3K9ac
0,04
0,04
H3K4me3
% Input
0
40
H3K4me3
0,05
0,00
0,05
60
IgG control
*
CRISPR-on
Ctrol cells
INS
IgG
0,07
0,50
0,50
0,06
H3K4me3
1
70
IgG
%Input
0,08
1,00
1,00
80
50
H3K9ac
1,50
1,50
H3K9ac
Ctrol cells
CRISPR-on
Co-IP
H3K4me3
0,09
2
90
0,11
H3K4me3
promoter
00
K9ac
0,12
B
H3K9ac
3
0,13
b
H3K4me3
ChiP of INS
CRISPR-on
Sat2
0,01
20
0,01
Ctrol cells
CRISPR-on
GAPDH
GAPDH
Ctrol cells
INS
INS
CRISPR-on
IgG
H3K9ac
H3K4me3
IgG
H3K9ac
Control CRISPR
cells
H3K4me3
IgG
H3K9ac
IgG
H3K9ac
H3K4me3
IgG
Control CRISPR
cells
H3K4me3
H3K9ac
IgG
H3K4me3
-69
-102
-135
-180
-206
-234
-357
-345
Cytosine position relative to TSS
H3K9ac
0,00
0
H3K4me3
0,00
10
Control CRISPR
cells
Ctrol cells
CRISPR-on
Sat2
Sat2
Carla Giménez et al., 2016 Gene Therapy.
Activating INS via CRISPR-ON
Conclusions
1
Nowadays, CRISPR-Cas9 is the most effective
tool for gene editing.
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.
As all new technology, CRISPR must be still
4 restricted to research to be deeply tested,
before it becomes a real option to treat human
diseases.
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”.
email: [email protected]
twitter @ICBMEdiabetes
The cellular alchemists …
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!
Financial
supports