View
3
Download
0
Category
Preview:
Citation preview
CRISPR Applications: Mouse
Lin He
UC-Berkeley
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.
Advantages of mouse as a model organism
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 SmithiesMario 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)
Key technical advance in reverse mouse genetics
Pre-implantation Development
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 SmithiesMario 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)
Key technical advance in reverse mouse genetics
TE
oocyte zygote 2‐cell 4‐cell 8‐cell morula blastocyst
Mouse preimplantation development
Restricted potential
TE
ICM
Totipotent
TE
oocyte zygote 2‐cell 4‐cell 8‐cell morula blastocyst
Restricted potential
TE
PEEpiblast
Totipotent
Totipotent and pluripotent cell fate potential
oocyte zygote 2-cell 4-cell 8-cell morula blastocyst
mir-34a is enriched in embryonic stem cells (ESCs)
Embryonic stem cells
pluripotent
Oct4Nanogsox2
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 SmithiesMario 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)
Key technical advance in reverse mouse genetics
ES cell yields chimeric mouse embryos in vivoBlastocyst injection of ES cells
Morula aggregation with ES cells
ES cell derived gametes generate normal offspring
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 SmithiesMario 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)
Key technical advance in reverse mouse genetics
ES cells
Tetraploid embryo
Tetraploid complementation- All ES cell mouse
Gene targeting using ESCs
Construction the targeting vectorsHomologous recombination in ESCsScreening edited ESCs by southern3-6 months
Bastocyst injection of ESCsGenerate viable, fertile chimeras3 months
This step is often efficient
Germline transmissionGenerate heterozygous mice3 months
Transgenic mice
Zygote pronuclear injection
Holdingpipette 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
3 m
3 m
3m
<1m
Application of CRISPR editing in mice
Germline mouse modelsTransmittable genetic allelesMultiple 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 modelsRecapitulate the somatic nature of some diseases (cancer) Bypass the embryonic lethality caused by whole-body knockoutTissue specific, inducible CRISPR editing
Tissue specific delivery of the CRISPR systemInducible Cas9 mouse models enable somatic editing.
Application of CRISPR editing in mice
Gene knockout / simple modifications
Genomic structural variationslarge deletion (up to 1.6 Mb)duplicationtranslocationinversion
CRISPR genome editing in mouse ES cells
Wang et. al., Cell, 2013
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
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
Cas9 delivery (mRNA vs. DNA)
Efficiency of editing
Toxicity of Cas9 to mouse embryos
Germline transmission
Off-target effects
Major considerations for CRISPR editing in mice
CRISPR editing of single or multiple genes in vivo
Cas9 mRNA + sgRNA zygote injectionLive 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
Wang et. al., Cell, 2013
This is an simplified HDR!
Applications of HDR-editing in mouse genetics
I. Insertion of a small fragment (ssDNA donor)
Yang et al., Cell, 2013
~30% efficiency
Donor: 42bp V5 tag, 60bp flanking homology
Applications of HDR-editing in mouse genetics
II. Insertion of a large fragment (double-stranded circular donor vector)
Yang et al., Cell, 2013
10-20% editing
Simultaneous injection of cas9 mRNA, sgRNA and DNA donor into zygote cytoplasm.Donor DNA: 2kb+3kb homology arms.
Applications of HDR-editing in mouse geneticsIII. Generation of conditional allele (two ssDNA donors)
Yang et al., Cell, 2013
Two LoxP in one allele: 20% efficiencyHowever, deletion is a major complicating issue for this strategy
Delivery methods for CRISPR editing in germline models
Li et al., NBT, 2013
mRNA+sgRNA injection into cytoplasm, 90% NHEJ editing efficiencyLinearized DNA injection into pronucleus, 9% NHEJ editing efficiencyGermline transmission is not affected by CRISPR editing
Sung et al., Genome Research, 2014
Cas9 RNP injection into zygote cytoplasm, 90% NHEJ editing efficiency
Chen et al., JBC, 2016 Wang et al., J Genet Genomics, 2016
Cas9 RNP electroporation into mouse zygotes. Efficient NHEJ and HDR editing3x increase in embryo survival (standard birth rate is 10-20%)
The key challenging step is microinjection
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
Chen et al., JBC, 2016
88% bi-allelic editing and 46% HDR editing
CRISPR-EZ: CRISPR- RNP Electroporation of Zygotes
Chen et al., JBC, 2016
CRISPR-EZ Advantages
100% Cas9 RNP delivery Highly efficient NHEJ and HDR editing
indel, point mutation, deletion, insertion>3x increase in embryo viabilityEasy, economic and high-throughput
CRISPR-EZ Challenges
Large, circular plasmid donor delivery is difficultOther Cas9 variantsOther mammals (cat, cow, pig, ect.)
Application of CRISPR editing in mice
Gene knockout / modificationOne step CRISPR editing in zygotes
Genomic structural variationslarge deletionduplicationtranslocationinversion
CRISPR editing in ESCs or somatic cells.
A large chromosomal deletion by CRISPR editing in vivo
A large intragenic LAF4 deletion detected in a patientDeletion of laf4 has no phenotype. The ~500kb deletion could lead to a truncated Laf4 protein, givingrise to malformation of limbs, shortened femur, triangular tibia
ES cell editing
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
Madallo et al., Nature, 2014
A low efficiency editing events amplified by selective growth advantage
Chromosomal rearrangement by CRISPR editing in vivo
Application of CRISPR editing in mice
Germline mouse modelsTransmittable genetic alleles
One-step CRISPR editing of mouse zygotesCRISPR editing of ES cells (complex editing)
Somatic mouse modelsNon-transmittable genetic modificationsTissue specific, inducible CRISPR editingLow editing efficiency can be compensated by selective advantages
Tissue specific delivery of CRISPR/Cas9 system
Live: Hydrodynamic injection, iv injectionPlasmid DNA, Adenovirus
Lung: Intratracheal injection / intranasal intubationAdenovirus, AAV, lentivirus
Hematopoietic cells: ex vivo engineeringLentivirus, retrovirus, DNA electroporation
Brain: Stereotactic deliveryAAV
Tissue specific CRISPR editing in mice
Inducible CRISPR/Cas9 mice
Xue et al., Nature, 2014
CRISPR-mediated direct mutation of cancer genes in the mouse liver
DNA Plasmid
Hydrodynamic inj
20-30% cells affected
Interrogation of gene function in adult brain using CRISPR-Cas9
70% reduction of MeCP2 positive cells in DG
Swiech et al., NBT, 2014
A Cre-dependent, Cas9 expressing miceOvercome the difficulty to deliver Cas9 to somatic cells
Platt et al., Cell, 2014
CRISPR-Cas9 knock-in 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
CRISPR-Cas9 knockin mice for inducible genome editing
Dow et al., NBT, 2014
CRISPR editing in mice, remaining challenges
Somatic mouse modelsRapid, 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
Germline mouse modelsSimple design, easy manipulation, rapid and multiplex editing
More reliable sgRNA design (particularly for desirable HDR editing)Complex genome editing still requires ESCsPrecise genotyping in mouse embryos
Recommended