UseofGeneEditingTechnologies

inRodents
CarlisleP.Landel,Ph.D.

TheMouseasAModel
Mammal
Small,easytomaintain,fecund
Wellunderstoodgenetics
Similaritytohumans>90%
Availabilityofinbredstrains
Highlyamenabletogenomemodification
Thousandsofstrainsofgeneticallymodifiedmouse
strainsexist
BanksofEScellswithmutationsinalmostallmouse
genesexist

MilestonesinMouseGenetics
1903
1920s
1930s
1940s

Mendels laws verified in mammals using mouse coat colors

Genetic basis for cancer: tumor susceptible mouse strains
Extrachromosomal transmission of tumors (virus, MMTV)
Histocompatibility antigens mapped (8 linkage groups)
Effect of uterine environment on birth defects established
Mouse mutants used to model human congenital defects
1950s Biological effects of radiation and chemical mutagens
1980
Transgenic mouse technology: pronuclear injection
1984
Interspecies homologies in linkages evident
1986
Embryonic stem cell technology: blastocyst injection
1990s ENU mutagenesis projects
2000
>10,000 genes, >25,000 markers in mouse
2003
Mouse genome draft sequence available
2007
Knock-Out Mouse Project (KOMP)
2009
Targeted Nucleases come online commercially

TheRatasaModel
Larger
Moreexpensivetomaintain,slightlylonger
generationtime
Morematerialperanimal

Preferredfortoxicologicalandbehavioralstudies
Somegeneticphenotypesmorecloselymimicthe
humanconditioncomparedtomice
Availabilityofgenomemodificationwas
traditionallymuchmorelimitedthanwithmice
Initialtransgenesisattemptswereinefficient
NoEScellsuntil2009

ESCellMediatedTargeted
Mutagenesis
(Obtain/produceESCells)
(thebadolddays)
ManipulateEScellsinvitro
Isolatetargetedclones
Timeline:48weeks

Inserttargetedclonesintoblastocysts
Transferinjectedblaststofostermothers
Screenforchimericoffspring
carryingtargetedmutation
Timeline:48weeks

Matetoproduceheterozygousgermlinefounders
Timeline:8weeks
Matetoproducehomozygousmutantanimals
Timeline:8weeks
TotalTimelineToHomozygousMutants:8monthsminimum

TheTargetedNucleaseFamily
ZFNs
Firstappliedtoratsin2009andtomicein2010
Disadvantages:expensive,notsoeasytoconstruct,
limitedtargetavailability,onerousIPreachthrough

TALENs(XTNs)
Rodentapplicationsbeganin2011
EasiertomakethanZFNs,cantheoreticallytargetany
sequence

CRISPR/CAS9
Rodentapplicationsbeganin2013
Extremelyeasytoengineer,amenabletomultiplex
targeting
Morepronetoofftargetmutagenesis,somewhattarget
restricted

DoubleStrandBreakRepair
NonHomologous
EndJoining

Homologous
RecombinationRepair

ErrorProne
Introducessmalldeletions/insertions
ErrorFree
7

TargetedNucleaseMutagenesis
DNE

DNE

DNE

coding seekwence

coding sequence
DNA break

DNA break

coding se ekwence

coding se quence
mutagenic
repair by
NHEJ
codingquence

DNA break

cargo

coding sequence
plasmid DNA

plasmid DNA

homologous
recombination
cargo

homologous
recombination
coding sequence

TargetedNucleaseMediated
MutagenesisProcess
Treat1cellembryoswithreagent
Transfertreatedembryostofostermothers
Screenformutantoffspring
Timeline:68weeks

MouseEmbryoMicroinjection

DebutofCRISPR/CAS9

ResultsofWangetal.
CRISPRtargetingisextremelyefficient
Ashighas100%targetedpupsand95%
homozygoustargetingforsomealleles/conditions
80%ofpupsfromdoublegenetargetingwere
doublehomozygotes

ImplicationsofWangetal.
Genetargetinginrodentsisnowextremely
easy
IsEScellmediatedtargetedmutagenesis
obsolete?

Simultaneoustargetingofmultiplegenes
promisestremendoussavingsoftimeand
animals

TraditionalProductionofLines
CarryingTwoMutations
Mut 1
wt

Mut 1
wt

Mut 2
wt

Mut 2
wt

Mut 1
wt

Mut 1
wt

Mut 2
wt

25%ofanimals
8weekprocess

Mut 2
wt

Mut 1
Mut 1

Mut 2
Mut 2

6.25%ofanimals
8weekprocess

TraditionalProductionofLines
CarryingThreeMutations
Mut 1
wt

Mut 3
wt

Mut 2
wt

Mut 1
wt

Mut 2
wt

Mut 3
wt

12.5%ofanimals
8weekprocess
Mut 1
wt

Mut 2
wt

Mut 3
wt
Mut 1
Mut 1

Mut 1
wt

Mut 2
wt

Mut 3
wt

Mut 2
Mut 2

Mut 3
Mut 3

1.6%ofanimals
8weekprocess

TargetedNucleaseMutagenesisHas
BeenUsedToGenerate:
Knockoutmutations,i.e.,inactivatespecific
genes
Knockinmutations,i.e.,insertgeneticmaterial
atadefinedgenomicsite
Singlebasechangesatspecificsites
Smallinsertionsofspecificsequence,e.g.
recombinasetargetslikeloxP,etc.
Transgeneinsertionsatsafesitesorunderthe
controlofendogenouspromoters

Largerscaledeletionsorinsertions

RemainingIssues
Offtargetmutagenesis
Systemmodificationforincreasedfidelity
Intheanimalmodelworld,notsuchanissue:Well
justbreedthemout.

Convertingarttoaroutinemethodology
Whatdefinesagoodtarget?Notalllociareequal.
Howdowebiasthesystemindelsvs insertions?
Whatarethebestreagents/conditions/practicesfor
success?

TargetedNucleasesHave
RevolutionizedMouseGenome
Engineering
NolongerlimitedbyEScellavailability
Anystraincan(potentially)betargeted
Any(rodent)speciescanbetargeted

Timelinestomodelgenerationhavebeen
significantlyshortened
Animaluseformodelproductionhas
decreased

Questions?

CarlisleP.Landel,Ph.D clandel@mac.com