Download Using CRISPR for genetic alteration_Joffrey Maine

Introduction to Mouse Genetics & Phenotyping
Using CRISPR for genetic alteration
Joffrey Mianné
Mary Lyon Centre
CRISPR/Cas origins
• Origin of the CRISPR/Cas system:
 Clustered-Regularly Interspaced Short Palindromic Repeats
(CRISPR)-CRISPR Associated (Cas) protein
 RNA-based adaptive immune system found in procaryotes
− Specific recognition and cleavage of invading nucleic acids
 Type II CRISPR/Cas system from Streptococcus Pyogenes
is one of the simplest
− crRNA
− tracrRNA
− Cas9 protein
Introduction to Mouse Genetics & Phenotyping
CRISPR/Cas origins
• From bacterial immune system to genome
engineering tool:
Jinek & al, 2012
Introduction to Mouse Genetics & Phenotyping
CRISPR/Cas for genome engineering
• From bacterial immune system to genome
engineering tool:
PAM
Protospacer
Adjacent Motif (-NGG)
sgRNA
Adapted from www.sigmaaldrich.com
• Nickase
Introduction to Mouse Genetics & Phenotyping
Ran & al, 2013. C ell
CRISPR/Cas for genome engineering
Adapted from Shan & al, 2014
Non-Homologous End Joining
NHEJ
Homology Directed Repair
HDR
- Knock-Out by introduction of an indel
- Introduction of point mutations (ssODN donor)
- Tailored deletion
- Introduction of tags/LoxP sites/complex
sequences (ssODN [short] or plasmid [long]
Introduction to Mouse Genetics & Phenotyping
donor)  is proving difficult
CRISPR/Cas for genome engineering
• Point mutation project design with Cas9:
 sgRNA selection = compromise
− As close as possible from the point mutation to introduce
− As specific as possible (low number and low probability of
potential off-target sites, especially on the targeted
chromosome)
− Possibility to mutate the PAM (silent mutation) in order to
prevent re-processing of correctly mutated allele (if not
possible, try to introduce silent mutations within the seed
region)
 ssODN design
− 60nt homology arms
− Contain the intended modification + silent mutation(s)
 Keep the genotyping step in mind while designing
Introduction to Mouse Genetics & Phenotyping
CRISPR/Cas for genome engineering
• Example:
 60nt HA
Additional silent point mutations G→C to
kill the PAM sequence of sgRNA_#1
(5’-CTG → 5’-CTC = Leu)
60nt HA 
Targeted amino acid:
5’-CGG (Arg)→ 5’-TAG (Stop)
Additional silent point mutation needed within the PAM sequence of the guide
used. This additional modification will prevent the system to “re-process/recut” the allele repair through Homology Directed Repair (HDR) with the donor
oligo (= expected allele).
Introduction to Mouse Genetics & Phenotyping
Genotype complexity and mosaicism
−
Cas9 mRNA
−
sgRNA(s)
−
DNA donor template
Pronuclear injection in 1
cell stage embryos
= Mutagenesis event
Mutant heterozygous
(2 alleles)
Introduction to Mouse Genetics & Phenotyping
Mutant mosaic
(3 alleles)
Mutant mosaic
(5 alleles)
Genotype complexity and mosaicism
• PCR product subcloning:
Transformation
Subcloning
Each colony
contains 1 plasmid
Mixed PCR
products
Sequencing of each
individual product
Each plasmid
contains 1 PCR
product
Introduction to Mouse Genetics & Phenotyping
Genotype complexity and mosaicism
• Germ line transmission assessment:
X
Mutant
founder (F0)
WT
F1 population
=
Heterozygous: WT / To determine
X
Allele not
transmitted
Ex:
WT : A C T A G T T A C A G
Mut: A C T A C A G G G A T
New allele
Introduction to Mouse Genetics & Phenotyping
Correct mutation
Real life example
• Project design: Introducing a point mutation in FTO
CGT  GCT
(ArgAla)
Sequencing orientation
Introduction to Mouse Genetics & Phenotyping
Real life example
• PCR product subcloning:
Subcloning
Allele 1
sgRNA #4
Allele 2
Reference
Al l ele 1
Al l ele 2
Al l ele 3
Targeted mutation
Allele 3
Introduction to Mouse Genetics & Phenotyping
Real life example
• Germ line transmission assessment:
X
WT
sgRNA #4
Reference
Al l ele 1
Al l ele 2
Al l ele 3
Targeted mutation
F1 population
WT/WT (Allele 3)
Introduction to Mouse Genetics & Phenotyping
WT/Correct point
mutations (Allele 1)
Targeted mutation
Breeding and phenotyping
Introduction to Mouse Genetics & Phenotyping
• Summary
 CRISPR/Cas9 technology can be used to efficiently
introduce indels and point mutations through direct
injection into zygotes
 The outcome is unpredictable. Quality control of the alleles
obtained through CRISPR/Cas9 mutagenesis is essential
and complex
 F0 should be treated as mosaic
 Phenotype data should be acquired from F1 onwards, once
the alleles have been segregated and fully characterised
Introduction to Mouse Genetics & Phenotyping
• Interesting websites
 CRISPR design tools
http://crispr.mit.edu/
http://www.sanger.ac.uk/htgt/wge/
http://tefor.net/crispor/crispor.cgi
 Poly Peak Parser:
http://yosttools.genetics.utah.edu/PolyPeakParser /
• Interesting papers

Jinek & al, Science. 2012

Singh & al, Genetics. 2014

Yang & al, Cell. 2013

Yang & al, Nature Protocols. 2014