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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