The Feasibility and Associated Risks of Germline

Editing

Dr. Ben Davies

University of Oxford

LEARNING OBJECTIVES

● Understand how CRISPR/Cas9 genome engineering tools works

● Describe the application of genome engineering tools within the early embryo

● Identify the problems associated with CRISPR/Cas9 genome engineering tools and thus the risks associated with their use.

At the conclusion of this presentation, participants should be able to:

DISCLOSURE

● Nothing to declare

Monogenic inherited disorders

● Over 10,000 have been identified, affecting millions of patients● Currently gene therapy is centred on delivering extra copies of

healthy genes to patients with mutations● Could repairing the actual diseased genes a better strategy?

Mutation

Mutant gene is inactivated

Mutation

Mutant gene is in repaired

Molecular Scissors

● Revolution in genetic engineering.● Changes to our DNA can now be

made using molecular scissors (nucleases).

● Genes can be deleted, mutations corrected at will.

● The CRISPR/Cas family represent the most versatile site-specific nucleases.

● These nucleases can be designed against almost any target sequence.

Wellcome Collection

CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)

● Cas9 is the nuclease which cleaves its double stranded target site● A guide-RNA (crRNA:tracrRNA) defines where the nuclease cleaves● The target site can be any 20 nt sequence followed by -NGG

----XXXXXXXXXXXXXXXXXXXXNGG--------XXXXXXXXXXXXXXXXXXXXNCC----

gRNA

template

guide RNACas9

• A source of the Cas9 nuclease• A guide-RNA to address where Cas9 cuts• Repair template• Mix together and inject

CRISPR/Cas9 Mutagenesis within the zygote

Simple Knock-outs

GeneGene Gene

• Ablating a dominant mutant gene

• Ablating genomic sequence that corrects a gene through splicing

Point mutation changes

GeneGene Gene

• Repairing a pathogenic mutation

template

Cas9

Cas9

template

12

6

39

1

2

4

57

8

10

1112

6

39

1

2

4

57

8

10

11

Electroporation is an easier (and quicker) alternative

Electroporation leads to increased embryo survival

Electroporation appears more efficient

Cas9Cas9

vs

● EP gives a higher level of mutagenesis p=0.0003

No improvement

CRISPR/Cas9 mutagenesis in human zygotes

Problems – off-target mutation

• The specificity of CRISPR is defined by only 20 nt• (near) identical sequences may be present elsewhere• …..and Cas9 nuclease tolerates certain mismatches• How real is this problem when applying CRISPR/Cas9

nucleases in the single cell embryo

● Limited off-targets reported for the available human studies

Some mixed news

● Founder mice generated with CRIPSR are almost invariably mosaic

Genome engineering problems - Mosaicism

Cas9 activeCas9

Large deletions can occur following CRISPR/Cas9

● Detectable by PCR

● Undetectable by PCR !

Complex rearrangements can occur following CRISPR/Cas9

Knock-in

*Allele 1Allele 2

Knock-in repair template*

Cas9

Knock-in template

Deletion

Biallelic modification

● Insertion/deletion mutations are the most likely outcome● Knock-in mutation on one allele, Knock-out on the other!

Predicting the repair outcomes

GeneGene Gene

----XXXXXXXXXXXXXXXXTXXXNGG--------XXXXXXXXXXXXXXXXTXXXNCC----

----XXXXXXXXXXXXXXXXTTXXXNGG--------XXXXXXXXXXXXXXXXTTXXXNCC----

Directing useful machinery to the genome

● Domains which alter DNA or activate/inhibit gene expression can be added to dCas9

● Whether this technology can be used therapeutically is yet to be determined.

Base editors – chemical modification of DNA

● dCas9 linked to enzymatic domains, chemically modified bases

● C to T ● A to G

● The machinery for base editing does less damage to DNA (no DSBs are introduced)

● No off-target mutations detectable by whole-genome sequencing

● Dr. Jiankui used CRISPR/Cas9 in an attempt disable the CCR5 receptor

● CRISPR used to introduce truncation mutation at the site of the naturally occurring CCR5 ∆32 mutation

Meanwhile in China…..

● Dr. He Jiankui presented his works at the 2nd International Summit on Human Genome Editing

● He reported using CRISPR/Cas9 to introduce a “therapeutic” mutation into a gene in human embryos

● The two embryos were implanted and twins were born – Lulu and Nana

HIV carrier HIV negativeICSI + CRISPR

guide RNACas9

Lulu

Nana

Blastocyst

Cord blood / cord / placenta DNA sequencing

PGD DNA sequencing

Dr. He Jiankui’s strategy

Cell-free DNA sequencing

● Sequence trace suggests mosaicism

● Heterozygous embryo (with mosaicism)

● Mutation is in-frame

● One intergenic off-target site identified by PGD sequencing● Subsequently not detected in cell free-DNA and placenta/cord blood DNA● Large deletions examined and gross abnormalities ruled out● Whole genome sequencing of cell free-DNA and placenta/cord blood DNA

confirmed genotypes and suggested allele frequencies of 50%

● PGD sequencing result

Data from Dr. He Jiankui’s presentation

Dangerous and nonsensical use of the technology on real embryos

● No clinical need for CCR5 gene editing● Consent from the patients doesn’t appear to have been performed by qualified

health-care professionals, but by the scientists involved in the study● Institutional ethical approval uncertain● The consequences of CCR5 knock-out throughout development and life in

humans is unclear ● CCR5 loss in mouse leads to increased susceptibility to other viral infections and

altered response to brain injury● Embryos with a mosaic genotype knowingly implanted.● Heterozygous embryo knowingly implanted – one normal copy of CCR5 remains.● Biological activity of Nana’s de novo allele (15 bp deletion = 5 amino acids

deletion) of CCR5 completely unclear.● Future medical care plan for Nana and Lulu uncertain.

This week’s

Summary

● CRISPR/Cas9 reagents can be simply introduced into the zygote for efficient targeted mutation– Pronuclear microinjection ‒ Electroporation

● Problems of CRISPR/Cas9– Off-target mutagenesis ‒ Mosaicism– Large deletions / rearrangement ‒ Biallelic modification

● Solutions– High-fidelity enzymes ‒ More predictive repair outcomes– Base editing

● What happened in China was a very premature use of the technology