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CSIRO HEALTH & BIOSECURITY
Genome Editing in Poultry:
Opportunities and Impacts
Dr Mark Tizard
Australian Animal Health La...
• Poultry: major source of animal protein for future
global food security
• Impact of genetic improvements through
• Selec...
• Transgenics and gene editing in mammalian species:
the single cell zygote (injection of DNA, RNA or protein)
The challen...
Primordial Germ Cells (PGCs)
 Progenitors to ovum and sperm forming cells
 Are accessible either outside or inside of th...
Germ cell culture Germline Heterozygous Homozygous
chimera offspring offspring
Timeline approximations for application of ...
Gene editing tools and chicken PGCs
Sex selection
Applications of genome engineering in our lab
Allergen free eggs The egg as a medical device
(Synthetic Biol...
Genome editing using Direct Injection (DI)
• Two guide RNAs selected to remove the majority of the first exon
• PCR screen...
Genome editing using DI
• Using the px333 vector carrying Cas9 + 2 sgRNAs
• First functionally validated in a chicken cell...
• G(1) sequence analysis
G(1) characterisation – heterozygous gene deletion
• Deletion is very consistent
Wild type
Cell l...
• Heterozygous G(1) birds were raised to sexual maturity and mated to
obtain the G(2) homozygous knockout birds
• Homozygo...
• Disease impacts are significant to food production and to
human health and safety.
• Genomics and gene editing are revea...
• Allergy to chicken egg affects ~ 2.5% of children
• Significant food safety issue and implications for vaccines grown in...
Selectively hatching hens for egg laying
• Building on well-known concepts; placing a marker on the
male-determining chrom...
Meiosis:
separation of sex
chromosomes
Fertilisation
Ovary
Ovum Sperm
ZFP
W
Z Z
ZFP Z Z W
♂ ♀
Selectively hatching hens fo...
• The technology in now available to create precise, targeted
modifications to the chicken genome
• Our hope: that the imp...
Acknowledgements
GENOME ENGINEERING IMMUNOLOGY / VIROLOGY ANIMAL HUSBANDRY
• Tim Doran
• Kristie Jenkins
• Mark Tizard
•Te...
Acknowledgements
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Genome Editing in Poultry - Mark Tizard

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

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  • DOWNLOAD FULL BOOKS, INTO AVAILABLE FORMAT ......................................................................................................................... ......................................................................................................................... 1.DOWNLOAD FULL. PDF EBOOK here { https://tinyurl.com/y6a5rkg5 } ......................................................................................................................... 1.DOWNLOAD FULL. EPUB Ebook here { https://tinyurl.com/y6a5rkg5 } ......................................................................................................................... 1.DOWNLOAD FULL. doc Ebook here { https://tinyurl.com/y6a5rkg5 } ......................................................................................................................... 1.DOWNLOAD FULL. PDF EBOOK here { https://tinyurl.com/y6a5rkg5 } ......................................................................................................................... 1.DOWNLOAD FULL. EPUB Ebook here { https://tinyurl.com/y6a5rkg5 } ......................................................................................................................... 1.DOWNLOAD FULL. doc Ebook here { https://tinyurl.com/y6a5rkg5 } ......................................................................................................................... ......................................................................................................................... ......................................................................................................................... .............. Browse by Genre Available eBooks ......................................................................................................................... Art, Biography, Business, Chick Lit, Children's, Christian, Classics, Comics, Contemporary, Cookbooks, Crime, Ebooks, Fantasy, Fiction, Graphic Novels, Historical Fiction, History, Horror, Humor And Comedy, Manga, Memoir, Music, Mystery, Non Fiction, Paranormal, Philosophy, Poetry, Psychology, Religion, Romance, Science, Science Fiction, Self Help, Suspense, Spirituality, Sports, Thriller, Travel, Young Adult,
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Genome Editing in Poultry - Mark Tizard

  1. 1. CSIRO HEALTH & BIOSECURITY Genome Editing in Poultry: Opportunities and Impacts Dr Mark Tizard Australian Animal Health Laboratory
  2. 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. 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. 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. 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. 6. Gene editing tools and chicken PGCs
  7. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 18. Acknowledgements

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