González Master in Advanced Genetics Universitat Autònoma de Barcelona GENOMICS Introduction Francisco J M Mojica CRISPR 2000 Source Doudna y Charpentier 2014 Introduction ID: 930932
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Slide1
CRISPR/Cas9
Lucía Álvarez
González
Master in Advanced GeneticsUniversitat Autònoma de Barcelona
GENOMICS
Slide2Introduction
Francisco J. M. Mojica
CRISPR
2000
Source
:
Doudna
y
Charpentier
, 2014
Slide3Introduction
To
explain
the methodology of the CRISPR system,
both
as
an
adaptive
inmune
system
in
microorganisms
and as a
genome
editing
technique
.
Objective
CRISPR
as
an adaptive immune
system
in
archaea
and bacteria.
CRISPR as a
specific
and
directed genome editing technique.
Future of the technique
Slide4CRISPR in microorganism
The functioning of this mechanism is based on the ability of bacteria to recognize the genetic material of viruses and plasmids that have previously infected them (both they and their predecessors) and generate cuts in their genetic material thus preventing infection.
Clustered
Regularly Interspaced Short Palindromic
Repeats
Immune
adaptive
system
CRISPR
as
an
adaptive
immune
system
in
archaea
and bacteria.
Slide5Source
:
Doudna
y Charpentier, 2014CRISPR locus
Source
:
Horvath
y
Barrangou
, 2010
Parts of CRISPR system
CRISPR
as
an
adaptive
immune
system
in
archaea
and bacteria.
Cas 9
protein
PAM
sequence
Slide6How the system works
The insertion of a short sequence of the invading DNA as a spacer in the CRISPR cluster
ACQUISITION
EXPRESSION
New attack, the cluster is transcribed with the integrated spacer (pre-crRNA) that matures when it binds to the
tracrRNA
of the Cas9 protein
.
INTERFERENCE
The crRNA recognizes the invader thanks to the PAM sequence and the Cas9
protein,
to which it is bound by the
tracrRNA
,
cuts the invading DNA.
Source
:
Bhaya
et al., 2012
CRISPR
as
an
adaptive
immune
system
in
archaea
and bacteria.
Slide75
’
end sgRNA
crRNA3’ end sgRNA tracrRNA
CRISPR
as a
specific
and
directed
genome
editing
technique
Parts of CRISPR/Cas9 system
sgRNA
Cas9
Source
:
Xie
y Yang, 2013
Source
:
Bortesi
y Fischer, 2014
Slide8Cas9 Nickase
Cas9 Nuclease
Problem
: Off-target Modified Cas9: Cas9
N
ickase
Source
:
Schiml
y
Puchta
, 2016
CRISPR
as a
specific
and
directed
genome
editing
technique
Slide9Modified Cas9: dCas9
Source
:
Bortesi y Fischer, 2014
CRISPR
as a
specific
and
directed
genome
editing
technique
Slide10Source
:
Xie
y Yang, 2013CRISPR as a specific and directed genome editing technique
Specific Vectors
Cas9
sgRNA
RNA
polimerase
II (Pol II)
promoter
.
RNA
polimerase
III
(Pol
III
)
promoter
.
Multiplexing
Several
sgRNAs
in a vector
Multiple modifications in the genome in a single generation
Slide11Future applications
Source
:
Doudna
y
Charpentier
, 2014
Slide12Bibliography
Bhaya
, D., Davison, M.,
Barrangou, R. 2011. CRISPR-Cas System in Bacteria and Archaea: Versatile Small RNAs for Adaptative Defense and Regulation. Annual Review of Genetics. 45:273-297.Bortesi, L., and Fischer, R. 2014. The CRISPR/Cas9 system for plant genome editing and beyond. Biotechnol. Adv. 33: 41–52. Doudna, J.A., and Charpentier, E. 2014. The new frontier of genome engineering with CRISPR-Cas9. Science. 346: 1258096–1258096. Horvath,P., Barrangou,R. 2010. CRISPR/Cas, the immune System of Bacteria and Archaea. Science: 327: 167-170. Pennisi, E. 2013. The CRISPR Craze. Science. 341: 833-836.