CRISPR/Cas9 –From Yoghurt to Designer Babiesinbios10/PDF/2017/Kuchka_11152017.pdf · •Patients...
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Source: nextbigfuture.com CRISPR/Cas9 – From Yoghurt to Designer Babies
Transcript of CRISPR/Cas9 –From Yoghurt to Designer Babiesinbios10/PDF/2017/Kuchka_11152017.pdf · •Patients...
Source:nextbigfuture.com
CRISPR/Cas9– FromYoghurttoDesignerBabies
OBJECTIVES
1. RelatethefunctioningofbacterialCRISPR/Cas systemstoacquiredimmunity
2. DescribehowCRISPR/Cas9 cutsDNA
3. ExplainhowCRISPR/Cas9 isusedingenomeediting
4. ProvideexamplesofCRISPR/Cas9 genomeediting
Source:www.artstation.com
Source:Barrangou etal.(2007)Science
Streptococcusthermophiluschromosome
~1.8millionbasepairs~1900genes
CRISPR/Cas Locus
CRISPR LOCUS
(Clustered Regularly Interspersed Short Palindromic Repeats)
=PALINDROMES- Borroworrob?WasitacaroracatIsaw?Yo,bananaboy!Madam,I’mAdam.Aman,aplan,acanal,Panama!
=SPACERS
PALINDROMICSEQUENCESINDNA CANFOLDINTODIFFERENTSTRUCTURES
CRISPR LOCUS
(Clustered Regularly Interspersed Short Palindromic Repeats)
=PALINDROMICREPEATS
=SPACERS
Source:EnvironmentalHealthPerspectives
Bacteriophage(virusesthatinfectbacteria)
Source:oprah.com
HOUSEGUESTS FROM HELL
BACTERIOPHAGE LIFECYCLE(LYTIC)
“PROPHAGE”
Source:MolecularBiology,Craigetal.
BACTERIUM
injectionofbacteriophageDNAintobacterium
expressionofbacteriophagegenes– synthesisofbacteriophageproteinsandreplicationbacteriophageDNA
assemblyofnewbacteriophageandlysis ofinfectedcell
CRISPR LOCUS
(Clustered Regularly Interspersed Short Palindromic Repeats)
=PALINDROMICREPEATS
=SPACERS
SPACER SEQUENCESINCRISPRLOCUSHAVEHOMOLOGYTOBACTERIOPHAGEDNA
AdaptedfromBarrangou etal.(2007)Science
BACTERIOPHAGEGENES
CRISPR/Cas (CRISPRAssociatedLoci)
Cas GENESENCODEPROTEINSANDRNAINVOLVEDINCRISPR FUNCTION
AdaptedfromBarrangou etal.(2007)Science
EXPERIMENTALRESULTS
AdaptedfromBarrangou etal.(2007)Science
CRISPRLOCUSANDGENESPROVIDEPROTECTIONAGAINSTVIRALINFECTION!!!!!
STAGESOFCRISPR/Cas9FUNCTION1. AcquisitionofForeignDNA2. CRISPRRNAProcessing3. RNA-GuidedTargetingofForeignDNA
Source:Doudna LabWebsite
STAGESOFCRISPR/Cas9FUNCTION1. AcquisitionofForeignDNA2. CRISPRRNAProcessing3. RNA-GuidedTargetingofForeignDNA
Source:Doudna LabWebsite
Source:ScientificAmerican
• Cas9isadouble-strandedendonuclease(anenzymethatcleavesbothstrandsofDNA).
• Cas9’scleavagesiteisdeterminedbyRNAsequencesderivedfromtheCRISPRlocus.
Source:Doudna andCharpentier (2014)Science,346.
Cas9CANFUNCTIONASANRNA-PROGRAMMABLEENDONUCLEASEGUIDEDBYASYNTHETICGUIDERNA(sgRNA)
CONSEQUENCESOFCRISPR-Cas9DNACLEAVAGE
Source:MolecularBiology,Craigetal.
Δ
Longetal.(2014)Science,345;1184– 1188.
POTENTIALPROBLEMSWITHCRISPR/CAS9
1. Mosaicism
2.Off-TargetEffects
Longetal.(2014)Science,345;1184– 1188.
• 11mdx-C(corrected)miceanalyzed• Highdegreeofmosaicism– 2to100%correction• Musclerescueexceededefficiencyofgenecorrection• Nodetectedoff-targeteffects
BODYWEIGHT/TIME MARKERSOFLIVERDAMAGE
EXPERIMENTALSET-UP
• Twogenestargetedatonce
• Genes– Ppar-γ;Rag-1
• ExperimentalSet-up– InjectCas9mRNAandsgRNAs intoone-cellembryostomutategenes
• Transferembryostosurrogatemothers
Cas9/sgRNAinducedMutations
Nodetectedoff-targeteffects
Mosaicismnotassayedattimeofpublication
• PatientsundergoingIVFtreatmentatCenterforReproductiveMedicine(Guangzhou,China)
• Normaldiploidzygotesusedforstudy(informedconsentofpatients)
• AttemptsweremadetocorrecttwomutationsusingCRISPR/Cas9
• Mutationinβ-globin genethatcausesthalassemia• MutationinG6PD genethatcausesanemia
• Spermcarryingmutationsinjectedintooocytestogeneratezygotes
• Cas9protein,sgRNAs,andssDNAwereinjectedintozygotes
• Embryoswereculturedfortwodaysandthen“harvested”foranalyses
RESULTS• Samplesizeverylow(10embryosforβ-globinstudy,only2forG6PD)
• Forβ-globinstudy,editingefficiencywas50%,genecorrectionefficiencywas50%• Additionalmutationsweredetectedinuncorrectedβ-globingenes• Nomentionofmosaicismoroff-targeteffects
• ForG6PDstudy,only2embryoswerestudied,bothhadcorrectedgenes• Oneoftwoembryoswasmosaic(50%cellscorrected,50%cellshadanadditional
mutation)• Nooff-targeteffectsweredetected
Source:Tangetal.(2017)Mol.Genet.Genomics,292:525-533.
Source:Doudna andCharpentier (2014)Science,346.
Source:Doudna andCharpentier (2014)Science,346.
SUMMARY
• CRISPR/Cas systemsfunctionasatypeofacquiredimmunityinbacteria
• Cas9 isanRNA-programmabledouble-strandedendonuclease
• Double-strandbreakscreatedbyCas9/sgRNAs canberepaired,changed,correctedbyhomologousrecombination
• GeneeditingbyCas9/sgRNAs hasbeenaccomplishedinmanyanimalsincludinghumans
