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11/13/2019
1
ETHICS AND ASSURITY OF GENOME
EDITING IN CRISPR ERA
SATU KUURE
GM-UNIT/LABORATORY ANIMAL CENTRE - HILIFE
RESEARCH PROGRAM UNIT, FACULTY OF
MEDICINE
TOPICS
•In vivo applications of CRISPR/Cas9 – animal models•Short introduction to technique•Example case: Modelling human CAKUT in mice•Current challenges
•CRISPR/Cas9 in human diseases
•Ethical concerns of CRISPR/Cas9 genome editing in humans
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IN VIVO GENOME EDITING - ANIMALS
DNA construct
huge DNA constructwith homology arms tiny guide RNA +
Cas9 mRNA/protein
IN VIVO CRISPR/Cas9 TARGETING
gRNA + Cas9 enzyme + DNA template
KnockoutConditional-allele
Precise, fast, universal
-Knockout-Conditional alleles
Cut -> NHEJ -> InDelsCut -> HDR -> Replacement
-Knockin-Point mutations-More complexgenome editing
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CRISPR/Cas9 DELIVERY IN PRODUCTION OF KO
/ KI ANIMALS
Microinjection (nuclear or cytoplasmic)
Zygote electroporation
ES cell transfection
SUCCESS RATES IN GENERATING KNOCKOUTS
Gene Injection Live pup % Editing
survival % efficiency %
Hepsin 85 56 27
Ppfia1 86 38 16
Igsf3 85 21 51
Dusp9 92 17 6*
*homozygous editing -embryonic lethality
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EXAMPLE CASE_ KNOCK-IN:
CONGENITAL ANOMALIES OF KIDNEY AND
URINARY TRACT (CAKUT)
- Prevalence: 3-6/1000 pregnancies* constitute 20-30% of all prenatal anomalies
* cause of 30 - 50% end-stage renal disease (ESRD) cases in children
* even mild forms like hypoplasia increase risk of renal & cardiovascular diseases and hypertension
- No cure
- Symptomatic treatments: dialysis & transplantation
- Mean survival time of dialysis patients 3-4years
- Lack of donor organs for transplantation (lifelong medication,
often 2nd surgery needed)
Picture_Hannu Jalanko
Helena Isoniemi - HYKS
CAKUT DERIVES FROM THE DEFECTIVE KIDNEY MORPHOGENESIS
Nephric/Wolffianduct
metanephricmesenchyme (MM)
stroma
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CAKUT GENETICS
Van der Ven et al 2018
Human CAKUT genes: ~40Mouse CAKUT genes: ~185
STRATEGY OF GENERATING KI (POINT
MUTATIONS) MICE
gRNAs
+ gRNA+ Cas9 protein+ ssDNA repair template
(containing pointmutation)
HDR
*
WT allele
KI allele P1 P2
P1 P2
Steps:1. Design gRNAs for different point mutations in mouse PlxnB2 locus2. Pronuclear injection with RNP mixture together ssDNA repair template3. Screen founders by PCR, restriction enzyme digestion and sequencing
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FOUNDER IDENTIFICATION: 1ST MUTATION
Total of zygotes injected: 432Total of zygotes transferred: 264 (60%)Pups born: 26 (9.8%)Founders: 2
Founders bred with wild type to testgermline transmission
THE USE OF ANIMALS IN RESEARCH: COURSE FOR PERSONS CARRYING OUT PROCEDURES
F1 generation
X
F0 WT
F1
Phenotyping of PlxnB2 CAKUT models begins
ASSURITY IN CRISPR/CAS9 GENOME
EDITING
istockphoto.com
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OUTCOME OF IN VIVO CRISPR/CAS9 GENOME
EDITING - GENOTYPING
• CRISPR/Cas9 versatility and ease to implement - editing outcome often unpredictable and generates mosaic
founders=> genotyping
• The type of the desired mutation directs genotyping approach - screening of the offspring depends on wanted mutagenesis
(InDel, point mutation and deletion)
=> germline transmission
CRISRP/CAS9 FLAVORS
•Unlimited Species Possibilities
Animals with ES cell method limitations can now be targeted
•Rapid In Vivo Genome Engineering
Fastest method for creation of knockout rodents (3- 6 months) and other higher eukaryotes
•Universal Tool
Move quickly from cell line proof-of-concept studies into animal models
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FLAVORS COME WITH CONCERNS
• Off-target side effects such as mutagenesis outside of desired target
• ”Too efficient”
• Shared practices and methodology missing
• Methods for efficient delivery and expression of CRISPR-Cas system need improvements
CRISPR/CAS9 APPLICATION IN HUMANS
istockphoto.com
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POTENTIAL OF CRISRP/CAS9 IN HUMAN
DISEASES
Cancer-Utilization of iPS-derived organoids originating from patient material:
1) introduction of cancer causing mutations to study pathogenesis2) characterization of cancer phenotype in organoids3) identify genes providing resistance to cancer drugs
- Correction of cancer gene mutations in patients Two clinical trials ongoing (China & USA)
Blood disorders-The first trial with beta-thalessimia &
sickle-cell anemia: correction of gene defect in bone marrow derivedstem cells
- Hemophilia in research phasedirect delivery to liver
CRISRP/CAS9 IN HUMAN – DISEASES IN
DEVELOPMENT PHASE
- Cystic fibrosis- Hereditary blindness- Muscular dystrophy- Huntington’s disease
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CRISRP/CAS9 IN HUMAN – DISEASES IN
DEVELOPMENT PHASE
- Cystic fibrosis- Hereditary blindness- Muscular dystrophy- Huntington’s disease- HIV
07/2019
11/2018
MAJOR CONCERN
The birth of first gene edited humans
- carried out without ethics oversight, review and approval- no institutional authorizations and permission from authorities- aim to inactivate the CCR5 gene in human embryos to render the resulting newborns immune to AIDS virus infection (not life threatening disease)
- leads to heritable genomic changes without information of long-term consequences
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CRISPR/CAS9 and CANCER
NEW TYPE OF BASE EDITOR – REAL HOPE FOR
THERAPY?
- Modified Cas9 – no DNA double strand breaks- Efficient & easy- Much more precise-> no need for template-DNA insertion via homologous recombination
CRISPR/Cas9 in Monogenic diseases
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QUESTIONS?
Thanks
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