This presentation highlights the opportunities and impacts of genome editing in poultry.

1. CSIRO HEALTH & BIOSECURITY
Genome Editing in Poultry:
Opportunities and Impacts
Dr Mark Tizard
Australian Animal Health Laboratory

2. • Poultry: major source of animal protein for future
global food security
• Impact of genetic improvements through
• Selective breeding
• Genomics and DNA markers
• Gene editing ?
• But improved intensive production can lead to an
increase in health & welfare issues
• Some key traits too complex for selective breeding
• Gene editing is set to provide game changing
interventions
• Fixation of valued alleles; deleting detrimental alleles
• Allele transplantation between strains (within species)
• However there is a caveat: the “off-target” issue
Introduction 2007 2020
CRISPR-Cas9

3. • Transgenics and gene editing in mammalian species:
the single cell zygote (injection of DNA, RNA or protein)
The challenge for precision genome engineering in birds
Images courtesy of
Dr Jacquie Jacob
University of Kentucky
• The avian ovum on the swelling yolk until its release
and fertilization (single cell zygote) at the top of the
oviduct)
• The single cell zygote is intimately linked with the yolk:
almost impossible to manipulate
• An alternative approach is required

4. Primordial Germ Cells (PGCs)
 Progenitors to ovum and sperm forming cells
 Are accessible either outside or inside of the growing chick embryo
PGCs OUTSIDE – highly skilled culture (not trivial)
 non-homologous recombination (van de Lavoir et al, 2006)
 gene targeting (Schusser et al, 2014)
 gene editing (Park et al, 2014)
PGCs INSIDE – accessing the germ cells in vivo (in ovo)
 Integrating lentiviral systems (McGrew et al, 2004)
 Direct Injection in vivo (Tyack et al, 2014)
Sperm Transfection Assisted Gene Editing (STAGE)
 Another approach which could speed up gene editing in poultry
(Cooper et al, 2017)
Methods to make a transgenic or edited chicken

5. Germ cell culture Germline Heterozygous Homozygous
chimera offspring offspring
Timeline approximations for application of the technologies
G2G1
G0 G2G1
G0 G2G1
6 months 6 months6 months
PGC via
cell culture
PGC via
Direct Injection
STAGE
Edited
hens and
roosters

6. Gene editing tools and chicken PGCs

7. Sex selection
Applications of genome engineering in our lab
Allergen free eggs The egg as a medical device
(Synthetic Biology Platform)
Improving eggs as a substrate for
vaccine production
Disease resilience

8. Genome editing using Direct Injection (DI)
• Two guide RNAs selected to remove the majority of the first exon
• PCR screen also designed
– 430 bp for the wild type or 340 bp with deletion

9. Genome editing using DI
• Using the px333 vector carrying Cas9 + 2 sgRNAs
• First functionally validated in a chicken cell line (DF1)
• 120 eggs undergo direct injection with CRISPR plasmid
 Generation of G(0) males chimeric for edited PGC (by PCR)
 Generation of G(1) heterozygous fully edited males & females
 Breeding for G(2) birds to select homozygous fully edited birds

10. • G(1) sequence analysis
G(1) characterisation – heterozygous gene deletion
• Deletion is very consistent
Wild type
Cell line
G(1) #1
G(1) #2
G(1) #3
G(1) #4
G(1) #5
G(1) #6
G(1) #7
G(1) #8
G(1) #9
G(1) #10
G(1) #11
G(1) #12
G(1) #13
NTC +ve cont
• G(1)s screened via PCR
Over all
19 positive G(1)s from 906
hatched
2.1% transmission rate

11. • Heterozygous G(1) birds were raised to sexual maturity and mated to
obtain the G(2) homozygous knockout birds
• Homozygous layer flock currently being expanded to provide eggs for trial
to assess enhanced growth of influenza vaccine virus.
G(2) – generation of homozygous gene deletion
2 eggs to make just
one dose
2 1
10x
increase
1 5
‘flu vaccine
shortage

12. • Disease impacts are significant to food production and to
human health and safety.
• Genomics and gene editing are revealing opportunities to
enhance resilience to important diseases such as:
• Avian Influenza
• Avian Leukosis Virus (J)
Disease Resilience

13. • Allergy to chicken egg affects ~ 2.5% of children
• Significant food safety issue and implications for vaccines grown in eggs
• Egg allergy is caused by 4 proteins within the egg white
• ovomucoid (Ovm)
• ovalbumin (Ova)
• ovotransferrin
• lysozyme
• Ovm is the most allergenic egg white protein, (but only 10% of total egg white
protein compared Ova at >50%)
• No clear role identified for Ovm in fertility, egg formation or nutritional value
• Ovm is our editing target (lead in to the allergenic epitopes in Ova,
ovotransferrin and lysozyme)
Improving food safety

14. Selectively hatching hens for egg laying
• Building on well-known concepts; placing a marker on the
male-determining chromosome (cf XY and XX)
• To provide an alternative to culling day-old male chicks in layer
industry
• To overcome a major ethical (and welfare) issue that impacts
egg producers
• United Egg Producers (USA) recently announced goal of
eliminating culling of day-old male chicks by 2020 (other
countries likely to follow)
• Industry feedback
 Method should screen eggs at point of lay (prior to incubation)
 Must be >98% accuracy
 Non-invasive

15. Meiosis:
separation of sex
chromosomes
Fertilisation
Ovary
Ovum Sperm
ZFP
W
Z Z
ZFP Z Z W
♂ ♀
Selectively hatching hens for egg laying
• Laser detection of “male egg”
• Removal OR Diversion (value added for vaccine production)
• Females contain
no added genes
ZZZFP W
♀ ♂
Z
• Hatchlings  hens
laying eggs for food

16. • The technology in now available to create precise, targeted
modifications to the chicken genome
• Our hope: that the impacts will lead to….
 Improved production (cost and efficiency)
• Through imporved sustainability, health & welfare
 Improved food safety
 Enhanced vaccine production
....but all of this relies on
 Safety data combined with effective regulation of the technology
 Public understanding and attitudes to gene editing (cf GMO)
 How these factors impact on industry adoption
Impacts for the poultry industry

17. Acknowledgements
GENOME ENGINEERING IMMUNOLOGY / VIROLOGY ANIMAL HUSBANDRY
• Tim Doran
• Kristie Jenkins
• Mark Tizard
•Terry Wise
• Kirsten Morris
• Terri O’Neil
• Caitlin Cooper
• Arjun Challagulla
• Shuning Shi
• Andrew Bean
• Luis Malaver
• Tamara Gough
• Matt Bruce
• Kerri Bruce
• Daniel Layton
• Sandy Matheson
• Noel Collins
• Chris Darcy
• Kelly Locke
• Susanne Wilson
• Mark Ford
CSIRO
Health & Biosecurity
Australian Animal Health Laboratory

18. Acknowledgements