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CRISPR Applications: Mouse
Lin He
UC-Berkeley
Advantages of mouse as a model organism
similar to human
Can be genetically manipulated
Isogenic and congenic genetic background
An accelerated lifespan.
Well-characterized biology
A cost-effective and efficient research tool.
Key technical advance in reverse mouse genetics
Intrauterine transfer of in vitro cultured embryo
Ann McLaren, 1959
Chimeric animal by morula aggregation and blastocyst injection (50-60s)
Andrzej Tarkowski, Beatric Mintz: morula aggregation (8C aggregates)
Richard Gardner, Ralph Brinster (blastocyst injection)
Cell culture model to study development (ES cells)
Evans, Martin, Kaufman (70s and 80s)
Homologous recombination in ES cells (late 80s)
Mario Capecchi, Olivier Smithies
Mario Capecchi and Kirk Thomas First gene-targeting in ES cells 1989
Knockout mice:
Oliver Smithies, Rudolf Jaenisch: Generation of knockout mice, beta-2
macroglobulin (1990)
Andreas Nagy: tetraploid complementation (1993)
Pre-implantation Development
Key technical advance in reverse mouse genetics
Intrauterine transfer of in vitro cultured embryo
Ann McLaren, 1959
Chimeric animal by morula aggregation and blastocyst injection (50-60s)
Andrzej Tarkowski, Beatric Mintz: morula aggregation (8C aggregates)
Richard Gardner, Ralph Brinster (blastocyst injection)
Cell culture model to study development (ES cells)
Evans, Martin, Kaufman (70s and 80s)
Homologous recombination in ES cells (late 80s)
Mario Capecchi, Olivier Smithies
Mario Capecchi and Kirk Thomas First gene-targeting in ES cells 1989
Knockout mice:
Oliver Smithies, Rudolf Jaenisch: Generation of knockout mice, beta-2
macroglobulin (1990)
Andreas Nagy: tetraploid complementation (1993)
Mouse preimplantation development
oocyte
zygote
2‐cell
4‐cell
8‐cell
morula blastocyst
Restricted potential
Totipotent
TE
TE
ICM
Totipotent and pluripotent cell fate potential
oocyte
zygote
2‐cell
4‐cell
8‐cell
morula blastocyst
Restricted potential
Totipotent
TE
TE
PE
Epiblast
mir-34a is enriched in embryonic stem cells (ESCs)
oocyte
zygote
2-cell
4-cell
8-cell
morula blastocyst
Oct4
Nanog
sox2
Embryonic stem cells
pluripotent
Key technical advance in reverse mouse genetics
Intrauterine transfer of in vitro cultured embryo
Ann McLaren, 1959
Chimeric animal by morula aggregation and blastocyst injection (50-60s)
Andrzej Tarkowski, Beatric Mintz: morula aggregation (8C aggregates)
Richard Gardner, Ralph Brinster (blastocyst injection)
Cell culture model to study development (ES cells)
Evans, Martin, Kaufman (70s and 80s)
Homologous recombination in ES cells (late 80s)
Mario Capecchi, Olivier Smithies
Mario Capecchi and Kirk Thomas First gene-targeting in ES cells 1989
Knockout mice:
Oliver Smithies, Rudolf Jaenisch: Generation of knockout mice, beta-2
macroglobulin (1990)
Andreas Nagy: tetraploid complementation (1993)
ES cell yields chimeric mouse embryos in vivo
Blastocyst injection of ES cells
Morula aggregation with ES cells
ES cell derived gametes generate normal offspring
Key technical advance in reverse mouse genetics
Intrauterine transfer of in vitro cultured embryo
Ann McLaren, 1959
Chimeric animal by morula aggregation and blastocyst injection (50-60s)
Andrzej Tarkowski, Beatric Mintz: morula aggregation (8C aggregates)
Richard Gardner, Ralph Brinster (blastocyst injection)
Cell culture model to study development (ES cells)
Evans, Martin, Kaufman (70s and 80s)
Homologous recombination in ES cells (late 80s)
Mario Capecchi, Olivier Smithies
Mario Capecchi and Kirk Thomas First gene-targeting in ES cells 1989
Knockout mice:
Oliver Smithies, Rudolf Jaenisch: Generation of knockout mice, beta-2
macroglobulin (1990)
Andreas Nagy: tetraploid complementation (1993)
Tetraploid complementation- All ES cell mouse
Tetraploid embryo
ES cells
Gene targeting using ESCs
Construction the targeting vectors
Homologous recombination in ESCs
Screening edited ESCs by southern
3-6 months
Bastocyst injection of ESCs
Generate viable, fertile chimeras
3 months
This step is often efficient
Germline transmission
Generate heterozygous mice
3 months
Transgenic mice
Zygote pronuclear injection
Holding
pipette
Fast genome editing (3-4 months)
Germline transmission is easy
limited editing capacity
Pronucleus injection
Phenotype can be evident in founders
Gene targeting using ESCs
Transgenics
3-6 m
3m
3m
<1m
3m
Application of CRISPR editing in mice
Germline mouse models
Transmittable genetic alleles
Multiple genetic manipulations
Simple design and easy manipulation
One-step CRISPR editing of mouse zygotes (simple editing)
CRISPR editing of ES cells (complex editing)
Somatic mouse models
Recapitulate the somatic nature of some diseases (cancer)
Bypass the embryonic lethality caused by whole-body knockout
Tissue specific, inducible CRISPR editing
Tissue specific delivery of the CRISPR system
Inducible Cas9 mouse models enable somatic editing.
Application of CRISPR editing in mice
Gene knockout / simple modifications
Genomic structural variations
large deletion (up to 1.6 Mb)
duplication
translocation
inversion
CRISPR genome editing in mouse ES cells
Targeting ESCs for multiple genes.
(up to 5 genes simultaneously,
2 are Y-linked)
20/96 are bi-allelicly edited on all
3 genes
Delivery: plasmids transfection
Wang et. al., Cell, 2013
The first attempt for CRISPR genome editing in mice
Cas9 mRNA + sgRNA; Targeting Oct4-IRES-GFP/+ mice
Zygote injection. No pronucleus injection!!
1/5 was edited by NHEJ
Shen et. al., Cell Research, 2013
Wang et. al., Cell, 2013
Major considerations for CRISPR editing in mice
Cas9 delivery (mRNA vs. DNA)
Efficiency of editing
Toxicity of Cas9 to mouse embryos
Germline transmission
Off-target effects
CRISPR editing of single or multiple genes in vivo
Cas9 mRNA + sgRNA zygote injection
Live birth rate 10-20% (low toxicity)
Hiighly efficient NHEJ editing
Wang et. al., Cell, 2013
Multiplexed precise HDR-mediated genome editing in vivo
20% bi-allelicly HDR edited
~90% HDR edited on one gene
This is an simplified HDR!
Wang et. al., Cell, 2013
Applications of HDR-editing in mouse genetics
I. Insertion of a small fragment (ssDNA donor)
Donor: 42bp V5 tag, 60bp flanking homology
~30% efficiency
Yang et al., Cell, 2013
Applications of HDR-editing in mouse genetics
II. Insertion of a large fragment (double-stranded circular donor vector)
10-20% editing
Simultaneous injection of cas9 mRNA, sgRNA and DNA donor into zygote cytoplasm.
Donor DNA: 2kb+3kb homology arms.
Yang et al., Cell, 2013
Applications of HDR-editing in mouse genetics
III. Generation of conditional allele (two ssDNA donors)
Two LoxP in one allele: 20% efficiency
However, deletion is a major complicating issue for this strategy
Yang et al., Cell, 2013
Delivery methods for CRISPR editing in germline models
Li et al., NBT, 2013
mRNA+sgRNA injection into cytoplasm, 90% NHEJ editing efficiency
Linearized DNA injection into pronucleus, 9% NHEJ editing efficiency
Germline transmission is not affected by CRISPR editing
Sung et al., Genome Research, 2014
Cas9 RNP injection into zygote cytoplasm, 90% NHEJ editing efficiency
The key challenging step is microinjection
Chen et al., JBC, 2016
Wang et al., J Genet Genomics, 2016
Cas9 RNP electroporation into mouse zygotes.
Efficient NHEJ and HDR editing
3x increase in embryo survival (standard birth rate is 10-20%)
CRISPR-EZ: CRISPR- RNP Electroporation of Zygotes
Chen et al., JBC, 2016
CRISPR-EZ  a highly accessible technology
CRISPR-EZ  An efficient genome editing tool in vivo
88% bi-allelic editing and 46% HDR editing
Chen et al., JBC, 2016
CRISPR-EZ: CRISPR- RNP Electroporation of Zygotes
CRISPR-EZ Advantages
100% Cas9 RNP delivery
Highly efficient NHEJ and HDR editing
indel, point mutation, deletion, insertion
>3x increase in embryo viability
Easy, economic and high-throughput
CRISPR-EZ Challenges
Large, circular plasmid donor delivery is difficult
Other Cas9 variants
Other mammals (cat, cow, pig, ect.)
Chen et al., JBC, 2016
Application of CRISPR editing in mice
Gene knockout / modification
One step CRISPR editing in zygotes
Genomic structural variations
large deletion
duplication
translocation
inversion
CRISPR editing in ESCs or somatic cells.
A large chromosomal deletion by CRISPR editing in vivo
ES cell editing
A large intragenic LAF4 deletion detected in a patient
Deletion of laf4 has no phenotype.
The ~500kb deletion could lead to a truncated Laf4 protein, giving
rise to malformation of limbs, shortened femur, triangular tibia
Kraft et al., Cell Reports, 2015
Chromosomal rearrangement by CRISPR editing in vitro
Translocation
Inversion
Choi et al., Nat Commun, 2014
Chromosomal rearrangement by CRISPR editing in vivo
Eml4–Alk inversion, express the Eml4–Alk fusion gene, display histopathological
and molecular features typical of ALK1 human NSCLCs.
Madallo et al., Nature, 2014
Chromosomal rearrangement by CRISPR editing in vivo
A low efficiency editing events amplified by selective growth advantage
Madallo et al., Nature, 2014
Application of CRISPR editing in mice
Germline mouse models
Transmittable genetic alleles
One-step CRISPR editing of mouse zygotes
CRISPR editing of ES cells (complex editing)
Somatic mouse models
Non-transmittable genetic modifications
Tissue specific, inducible CRISPR editing
Low editing efficiency can be compensated by selective advantages
Tissue specific CRISPR editing in mice
Tissue specific delivery of CRISPR/Cas9 system
Live:
Hydrodynamic injection, iv injection
Plasmid DNA, Adenovirus
Lung: Intratracheal injection / intranasal intubation
Adenovirus, AAV, lentivirus
Hematopoietic cells: ex vivo engineering
Lentivirus, retrovirus, DNA electroporation
Brain: Stereotactic delivery
AAV
Inducible CRISPR/Cas9 mice
CRISPR-mediated direct mutation of cancer genes
in the mouse liver
DNA Plasmid
Hydrodynamic inj
20-30% cells affected
Xue et al., Nature, 2014
Interrogation of gene function in adult brain using
CRISPR-Cas9
70% reduction of MeCP2 positive cells in DG
Swiech et al., NBT, 2014
CRISPR-Cas9 knock-in mice for inducible genome editing
A Cre-dependent, Cas9 expressing mice
Overcome the difficulty to deliver Cas9 to somatic cells
Platt et al., Cell, 2014
CRISPR-Cas9 knockin mice for inducible genome editing
Expansion of desired editing events in cancer models
Platt et al., Cell, 2014
CRISPR-Cas9 knockin mice for inducible genome editing
Dow et al., NBT, 2014
CRISPR editing in mice, remaining challenges
Germline mouse models
Simple design, easy manipulation, rapid and multiplex editing
More reliable sgRNA design (particularly for desirable HDR editing)
Complex genome editing still requires ESCs
Precise genotyping in mouse embryos
Somatic mouse models
Rapid, easy, tissue specific, inducible, multiplex genome editing.
Delivery of Cas9 for building somatic mouse models. (improved viral gene
delivery, improved Cas9 RNP delivery, smaller Cas9 variants, improved
Cas9 mouse models)
Off target effects and precise genotyping of targeted cells
The combination of CRISPR with traditional Cre-LoxP methods could leads
to more precise modeling of human disease