crispr mouse igi - innovative genomics institute (igi) · crispr editing of es cells (complex...

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

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

ES cell yields chimeric mouse embryos in vivoBlastocyst injection of ES cells

Morula aggregation with ES cells

ES cell derived gametes generate normal offspring

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.


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


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


Multiplexed precise HDR-mediated genome editing in vivo

20% bi-allelicly HDR edited

~90% HDR edited on one gene


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)


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)


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


88% bi-allelic editing and 46% HDR editing

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 viabilityEasy, economic and high-throughput

CRISPR-EZ Challenges

Large, circular plasmid donor delivery is difficultOther Cas9 variantsOther mammals (cat, cow, pig, ect.)


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


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

crispr mouse igi - innovative genomics institute (igi) · crispr editing of es cells (complex...

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