1. Ingeniería

Mouse Network meeting 2015
Mouse genome engineering via
CRISPR/Cas9 system
Medical Research Council Harwell
Mary Lyon Centre
Joffrey MIANNÉ
CRISPR/Cas9 to engineer the mouse
genome
• Origin of the CRISPR/Cas9 system:
 Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)CRISPR Associated (Cas) (CRISPR/Cas)
 RNA-based adaptive immune system found in prokaryotes
 Specific recognition and cleavage of invading nucleic acids
 Modified to be used as a genome engineering tool
PAM
Protospacer
Adjacent Motif (-NGG)
sgRNA
Adapted from www.sigmaaldrich.com
CRISPR/Cas9 to engineer the mouse
genome
Non-Homologous End Joining
NHEJ
Homology Directed Repair
HDR
Adapted from Shan & al, 2014
• Principle:
 Cas9 mRNA
 sgRNA(s)
 DNA donor template
Pronuclear injection in 1
cell stage embryos
CRISPR/Cas9 to engineer the mouse
genome
• Indel(s) / Knock-out(s)
 Use of Cas9 nuclease + 1 sgRNA
 Outcomes: small insertions or deletions
(NHEJ alleles) which can lead to
frameshifts and KO
• Point mutation(s)
 Use of Cas9 nuclease + 1 sgRNA + ssODN
donor template
 Outcomes: NHEJ alleles / HDR alleles
WT
WT / HDR
• Tailored deletion(s)
 Use of Cas9 nuclease + 2 sgRNAs
 In-vivo deletions: 30 bp to >50 kbp
 Outcomes: NHEJ alleles / Alleles with
tailored deletion
• Gene targeting
 Use of Cas9 nuclease + 1 or 2 sgRNAs +
donor plasmid
 In-vivo knock-in(s): 1 bp up to >2 kbp
 Outcomes: NHEJ alleles / Alleles with
tailored deletion / HDR alleles
Ex: Flox
Adapted from Zheng & al, 2014
g1
g2
MRC Harwell CRISPR/Cas9 projects
underway
• Indel(s) / Knock-Out(s)
 Alleles with indel(s) found in
every project (14/14) carried
out.
• Point Mutation(s)
 HDR
alleles
with
point
mutation(s)
found
in
4
projects from 8 performed.
Type of project
# projects
# projects with
NHEJ alleles
# projects with HDR alleles
Indel(s) / Knock-Out
2
2
/
Point mutation(s)
8
8
4
Gene targeting “LoxP site(s)”
4
4
0 (before optimisation)
Total
14
14
4*
* 4 projects were found successful for HDR out of 12 possible
• Tailored deletion(s)
 Not
tested
in-vivo,
but
successfully tested in-vitro in 3
different projects with 49, 52
and 521 bp deletions.
• Gene targeting
 Few unsuccessful experiments
were
performed
before
optimisation of the system to
introduce LoxP sites.
Mosaicism and illegitimate repair in
F0 animals
• Target 1, animal #4 genotyping results:
sgRNA_#1
sgRNA_#2
Reference
Allele 1
Allele 2
Allele 3
CG  GC



15 nt duplication
29 nt duplication
3 nt deletion
Allele 1 : Illegitimate repair  Correct repair at the target + 15 nt insertion (duplication)
Allele 2 : NHEJ repair  29 nt insertion (duplication)
Allele 3 : NHEJ repair  3 nt deletion + 29 nt insertion (duplication)
Hidden mosaicism
• Cdh23 target, F0 #30 ear clip genotyping results:
sgRNA_#1
sgRNA_#2
Reference
Allele 1
Allele 2
AG


CT
24 nt deletion
24 nt deletion
Allele 1 = NHEJ repair  24 nt deletion
Allele 2 = Illegitimate repair  Correct repair at the target + 24 nt deletion
• Cdh23 target, alleles found in F0 #30 offspring:
sgRNA_#1
sgRNA_#2
Reference
Allele 3
Allele 1
Allele 2
Allele 4
24 nt deletion
AG




Allele
Allele
Allele
Allele
1
2
3
4
=
=
=
=
CT
24 nt deletion
NHEJ repair  24 nt deletion
Illegitimate repair  Correct repair at the target + 24 nt deletion
WT
HDR  Correctly repaired
Characterisation of CRISPR/Cas9
mutant mice
F0
F1
X
• Sequencing of both F0 and F1 animals is
required to assess the genotype
Optimisation of Micro-injection
settings
• Procedure:
 Cas9 mRNA
 sgRNA(s)
 DNA donor template
Pronuclear injection in 1
cell stage embryos
In-vitro culture of
embryos (7 days)
DNA extraction, PCR and
Sanger sequencing
• Parameters assessed:
 place of injection (cytoplasmic vs pronuclear)
 delivery pressure
 concentration
Optimisation of Micro-Injection
settings
• Results:
Crb1 target mutation rate (%)
100
85.7
90
81.2
81.8
81.8
80
70
71.4
66.7
60
60
50
43.6
40
30
19
20
10
0
7.3
0
Before optimisation
0
Embryos
Live mice
After optimisation
CRISPR/Cas9 IMPC pilot
• IMPC pilot:
 Cas9 mRNA
 2 sgRNA(s)
 Plasmid donor template
Pronuclear injection in 1
cell stage embryos
IKMC tm1a allele
Cas9 + 2 sgRNAs
+ vector
Cas9 + 2 sgRNAs
+ vector
lacZ
Knockout-first (tm1a)
Cas9 + 2 sgRNAs
+ vector
lacZ
Conditional (tm1c)
Vectors provided by Sanger
LacZ deletion (tm1b)
CRISPR/Cas9 ES cells pilot
• Pilot work in ES cells:
 Cas9 + sgRNAs plasmids
 Plasmid donor (IKMC)
Co-transfection into
mouse ES cells
IKMC tm1a allele
loxP
Ex n-1
lacZ
loxP
Neo
loxP
Critical Ex (n)
Ex n+1
Summary
• Knock-out alleles and introduction of point mutations are
now generated at high efficiency.
• Micro-Injection settings have been optimised. More tests
are currently being processed to optimise others
parameters (protein vs mRNA, ssODN donor size, plasmid
donors, NHEJ inhibitor…).
• Try new targets and more complex modifications (Flox,
IMPC pilot).
• ES cell trials to introduce larger constructs.
• The quality control of the alleles obtained
CRISPR/Cas9 tools will be essential and complex.
through
Acknowledgment
•
Molecular Biology Group
Lydia Teboul
Joffrey Mianné
Adam Caulder
Gemma Codner
Jorik Loeffler
•
•
Collaborators
Bill Skarnes, WTSI
Vivek Iyer, WTSI
Jason Heaney, BCM
Micro-Injection Team
Deb Bogani
Martin Fray
Wendy Gardiner
Michael Walker
•
The Mary Lyon Centre husbandry staff
Sara Wells
Questions?
IKMC tm1a allele
Cas9 + 2 sgRNAs
+ vector
Cas9 + 2 sgRNAs
+ vector
lacZ
Knockout-first (tm1a)
Cas9 + 2 sgRNAs
+ vector
lacZ
Conditional (tm1c)
LacZ deletion (tm1b)