2016/1/19
Recombinetics
Precision Livestock Genetics
For Biomedicine & Agriculture
Tad Sonstegard, Ph.D., CSO
A Growing Demand for Meat & Milk
Projected growth in meat
demand to 2020
Projected growth in milk production 2009 - 2018
Source:
OECD
Selective Breeding Works
Progress in the dairy industry
In 2007 U.S. Produced 34% more milk with 48% fewer dairy
cows than in 1960
Started to use 50K SNP chip in April 2008, most of the gains are
from including low heritable traits into the Selection Index.
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Narrow Genetics & Inbreeding Accumulates
Deterious Mutations
Crossbreeding Can Introgress Desirable Traits &
Dilute Congenital Mutations
Deleterious Fertility Alleles
Name
Chr
Loc
Freq
Earliest Known Ancestors
BTA
Mbase
%
Gene
HH1
5
62-68
HH2
1
93-98
4.5
Pawnee Farm Arlinda Chief
APAF1/stop gain
4.6
Willowholme Mark Anthony
HH3
8
ND
92-97
4.7
Glendell Arlinda Chief,
Gray View Skyliner
SMC2/nonsynonymous
JH1
BH1
15
11-16
23.4 Observer Chocolate Soldier
7
42-47
14.0 West Lawn Stretch Improver
CWC15/stop gain
More mutations on the horizon as selection continues
VanRaden, P.M., et al. Reporting of haplotypes with recessive effects on fertility. Proc.
Interbull Mtg., Stavanger, Norway, Aug. 26–28, 4 pp. 2011.
But Crossbreeding Dilutes Selective and
Adaptive Traits
Precision Tools for Trait Introgression
Precision Tools for Trait Introgression
Gene Editing Platforms
Clear IP and FTO?
YES
YES
NO
Kim & Kim (2014). A guide to genome engineering with programmable nucleases.
Nature Rev. Genet. 15: 321-334
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Targets for Genetic Improvement
Sustainability
Reduced environmental impact
More efficient feed conversion
Climate Adaptation
Genetic Diversity
Animal Welfare
Humane production
Product Quality
Food safety
Disease resilience
Healthfulness
Disease gene repair
Palatability
Gender selection
Tan, Carlson, Walton, Fahrenkrug, Hackett (2012) Advances in Genetics 80:37-97
Horizontal Allele Introgression
Double Muscling to Decrease
Time-to-Market and Increase RPY
• At least 7 Spontaneous Mutations of
Myostatin are known in cattle
• Results in 7% and 30% Increased RPY in
European Breeds (+/mh; mh/mh)
• Mutation not found in some breeds
• Heterozygotes come to market weight
on less feed/time.
• Can introduce by cross-breeding but
changes genetics
Tan, Carlson, Walton, Fahrenkrug, Hackett (2012) Advances in Genetics 80:37-97
WT
844Del1
282
Frame
Shift
Premature
Stop
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The SLICK hair locus derived from Senepol cattle confers
thermotolerance
Disease has Tremendous Impacts on
Global Economies
African Swine Fever
Truncation of RELA in pigs by embryo
injection with TALENs
Parturition in 4 of 7 recipients
resulting in 39 piglets, 8 of which
carried editing events (21%).
S531P
Live pigs produced from genome edited zygotes.
Lillico et al, Nature Sci Rep. 2013 Oct 10;3:2847
Polled is a Target for Introgression
Genetic Dehorning of dairy cows
• Most U.S. dairy animals are dehorned (up to $20 each)
• Hornless (Polled) genetics in some beef breeds for >500 years
• Polled at VERY low frequency in Dairy breeds (<1%), not elite
• Growing social pressure to eliminate dehorning
PC, Celtic origin (212 bp, 1,705,834–1,706,045 bp) is duplicated (and
replaces a sequence of 10 bp (1,706,051–1,706,060 bp). Angus,
Galloway, Fleckvieh, Gelbvieh and Murnau-Werdenfelser
• Traditional cross-breeding would be slow and disruptive
Medugorac, et al., (2012) Bovine
Polledness – An Autosomal
Dominant Trait with Allelic
Heterogeneity. PLoS One 7 (6)
PF, Friesian origin. P5ID (replace 7 bp (cgcatca with ttctcagaatag;
1,649,163–1,649,169) and 80,128 bp duplication (1,909,352–1,989,480
bp P80kbID, plus five point mutations at the positions (G1654405A,
C1655463T, T1671849G, T1680646C, C1768587A)
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Revolutionizing
Ethical Stewardship
by Livestock
PrecisionGenetics
Crossbreeding
Polled Allele Introgression into Holstein
b
c
Precision Crossbreeding
for Genetic Dehorning
Percentage of clones homozygous for introgression = 1-5%
Revolutionizing
Genetics
Accelerated
Genetics toLivestock
Enhance Animal
Welfare
TABLE 1:
Cell Line Parental
Cells
HP14-B6
2122
HP14- B4
2122
HP7-P4-A1
2120
HP-24.8
2120
Genotype
NT
Blast
Session Rate (%)
Homozygous
polled
Homozygous
polled
Homozygous
polled
Heterozygous
polled
Summary
1
1
Embryos/ Preg
Recips
D40
Preg
D90
27/64 (42)
9/9
6
0
Revolutionizing
Genetics
Accelerated
Genetics toLivestock
Enhance Animal
Welfare
Off Target Analysis
• Sequenced 2 animals and 2 progenitor cell lines to 20X
• No mis-targeting of the Polled allele
• No evidence of NHEJ based INDELs near Degenerate Sites
Liveborn 60
days
0
0
3/15 (20)
1/1
1
1
1*
0
2
25/82 (30)
9/9
2
2
2
2**
3
35/151 (23)
7/7
5
2
2***
0
70/295
(24%)
26/26
14/26
(54%)
5/26
(19%)
5/26
(19%)
2/26
(7%)
* RCI1001
** Spotigy (RCI1002) and Buri (RCI1003)
*** RCI1004
•
•
Populations segregate alleles with a range of
phenotypic effect
Rare
alleles
causing
Mendelian
traits
Lowfrequency
alleles with
intermediate
effect
Indels ID, groomed to remove noise, breed and animal specific polymorphisms
identical in progenitor cells and edited animals.
Indel assessed for proximity to degenerate TALEN off-targets;
• up to 10 bp mismatches/TALEN pair, 5 bp/monomer.
• allowed spacer length to vary from 12 bp to 25 bp (norm 17)
• considered targets for both TALEN hetero- and homodimers
• 1 site (POLLED) of 49 bp in length, to 61,751 sites totaling 3,036,765 bp.
Multiple genes are implicted in complex traits
High-effect
common
variants
influencing
common traits
Common
alleles in
common
traits
Courtesy Tad Sonstegard
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Non-Meiotic Allele Introgression for
Accelerated Breeding
•
Precision Crossbreeding
•
Resolving bad “moves”
•
Resolve antagonistic alleles
•
Break linkage disequilibrium
•
Traits/alleles hard to select for
•
Novel Alleles
Precision Breeding with Selection & Direction
Genetic Selection and Direction
Genet Sel Evol. 2015 Jul 2;47(1):55. Potential of
promotion of alleles by genome editing to improve
quantitative traits in livestock breeding programs. Jenko
J1, Gorjanc G2, Cleveland MA3, Varshney RK4, Whitelaw
CB5, Woolliams JA6, Hickey JM7.
FDA Industry Guidance
Generally Recognized as Safe: GRAS
Regulation of Genetically Engineered Animals Containing Heritable
Recombinant DNA Constructs (1/15/2009)
Assertion: Gene-edited animals contain DNA that is IDENTICAL to what is widely
consumed and generally recognized as safe (GRAS)
“…FDA defines 'genetically engineered (GE) animals' as those animals modified by
rDNA techniques, including the entire lineage of animals that contain the
modification. The term 'GE animal' can refer to animals with heritable rDNA
constructs…”
GRAS exemptions are granted for substances that are generally recognized,
among experts qualified by scientific training and experience to evaluate their
safety, as having been adequately shown through scientific procedures (or, in the
case of a substance used in food prior to January 1, 1958, through either scientific
procedures or through experience based on common use in food) to be safe
under the conditions of their intended use.
The rDNA construct (an article) in a GE animal that is intended to affect the
structure or function of the body of the GE animal, regardless of the intended use
of products that may be produced by the GE animal, meets the FFDCA (Federal
Food, Drug, and Cosmetic Act) drug definition.
Recombinetics' gene-edited animals DO NOT contain rDNA constructs
Science Based Regulation
Self-affirmed. The manufacturer of this chemical or substance had performed all
necessary research, including the formation of an expert panel to review safety
concerns, and is prepared to use these findings to defend its product's GRAS
status.
Regulation in a Rational World
SAFE Labelling Act – Safe & Accurate Food Labeling Act
DARK ACT – Denying Americans the Right to Know
DEFINITION OF GMO PRODUCT :
1. rDNA/in vitro AND
2. The outcome not achievable by traditional breeding
By this logic, animals derived from native-allele introgression should NOT be
defined as a GMO. CORRECT!
>Current or heirloom alleles should be
regulated only by post-market
surveillance
>Novel or rationally designed alleles
should require minimum pre-market
safety assessment
BUT : The Bill exclusively refers to the regulation of GMO plants
PASSED the House, in the Senate. Lobby your Senators!
Nature’s Bounty, Nature’s Way
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