Genetic Engineering · 2017-06-26 · Genetic Engineering (CRISPRs, Zinc Fingers, TALENS, Homologous Recombination) Transgene Injection Nuclease Injection Oocyte Transduction Sperm‐Mediated

• Genetic Engineering–CRISPRs

• Porcine Reproductive & Respiratory Syndrome Virus–SIGLEC1–CD163

• Other Applications and the Future

– Bioengineering– Cancer– Cardiovascular Disease/Atherosclerosis– Cognitive Behavior– Cutaneous Pharmacology/Wound Repair/Dermatology – Diabetes– Immunology– Lipoprotein Metabolism– Nutrition– Organ Transplantation– Orthopedics– Ophthalmology – Physiology– Regenerative Medicine

• Cancer (CAG‐Floxed‐Stop, KRASG12D, p53R167H; caCSN2‐miBRCA1‐RASSF1; APC‐/‐ )• Cardiovascular Disease (hFAT‐1+; TEK‐NOS3+; TEK‐CAT+)• Cystic Fibrosis (CFTR‐/‐; CFTRΔF508/ΔF508; CFTRΔF508/ΔF508, Tet‐on CFTR+; CFTRΔF508/ΔF508,

FABP2‐CFTR+; shRNA CFTR; MUC5AC‐/‐,MUC5B‐/‐ )• Diabetes (ssGIP‐hINS+)• Disease Resistance (SIGLEC1‐/‐; CD163‐/‐; CD163‐/Δdomain 5/HL8; CD163‐/‐, SIGLEC1‐/‐;

TMPRSS2‐/‐; CD1D‐/‐)• Enhancing Meat Quality (hFAT1+)• Muscular Dystrophy (DMD+/Δexon 46)• Pharmaceuticals (FIX+, VWF+, SERPINA1+; FVIII+, SERPINA1+, FURIN+)• Regenerative Medicine (IL2RG+/‐; RAG2‐/‐)• Retinitis Pigmentosa (P23H RHO +)• Phenylketonuria (PAH‐/‐)• Spinal Muscular Atrophy (SMN+/‐; SMN+/‐, hSMN2+)• Suicide Prodrug for Liver Transplantation (ALB‐TK1+; AFP‐CDA+) • Tracking/Tools (CAG‐eGFP+; CMV‐eGFP+; ZP3‐CRE; UBC‐Tomato+; CAG‐Tomato+;

UBC‐nls eGFP+; PSMA1‐eGFP fusion protein +)• Xenotransplantation (GGTA1‐/‐; GGTA1‐/‐, CD55+; GGTA1CD55/+; GGTA1‐/‐, CD55+,

CD59+, ENTPD1+, THBD+; SIGLEC1‐/‐)The image part with relationship ID rId3 was not found in the file.

• Metabolism• Retinitis Pigmentosa • Stargardt’s Macular Dystrophy (patient‐specific)• Liver Regeneration• Cardiac Development• Diabetes• Cardiovascular Disease• Congenital Muscular Dystrophy• Prader‐Willi Syndrome • Fetal/Maternal Interactions• Plus others that I can’t talk about‐ over 48 different

modifications already made, and over 1,080 cloned pigs.The image part with relationship ID rId3 was not found in the file.

Adapted from Prather et al 2013

Genetic Engineering(CRISPRs, Zinc Fingers, TALENS, Homologous Recombination)

Transgene InjectionNuclease InjectionOocyte Transduction

Sperm‐Mediated Gene Transfer

The image part with relationship ID rId4 was not found in the file.

Design & Build Constructs or

Guides

Or Transduction and Selection

(Lentiviruses, Adeno‐associated viruses, Replication‐defective

Retroviruses)

Karyotype/Chromosomes

Growing Cells

Comparative Physiology

Sequenced Genome

Illustration by Krista Prather

Examples• Hammer

• Handle• Head

• Drive Nails• Pull Nails

• Factory• Self-

Replicates• Makes Parts• Makes Tools

• Non-Homologous End Joining• Can result in changing a handful of

base pairs• Thus can remove exons/domains or

knockout a gene.• Swine genome is ~3,000,000,000 base

pairs.

• Engineering Somatic Cells (1 month to 2 years), or Design and Build guides (a few days)

• SCNT/ Zygote injection (1 week)• Gestation (3 months, 3 weeks & 3 days)• Puberty (6-9 months)

• The PRRS virus (PRRSv) was first detected in the U.S. in 1987 (Keffaber et al ‘89) and in Europe in 1990 (Wensvoort et al ’91)

• Type 1 are European strains, Type 2 are North American Strains

• PRRSv replicates in macrophages– induce prolonged viremia and can cause persistent infections that last for months

• It also predisposes infected pigs to other bacterial and viral pathogens The image part with relationship ID rId3 was not found in the file.

• Vaccinations to date have been ineffective– Non‐ neutralizing antibodies actually enhance viral replication in alveolar macrophages

– Antibody dependent enhancement (ADE)• Sows/Gilts

– severe reproductive failure and a high rate of late abortion – Early farrowing– Decreased litter size, Increased number of mummies

• Boars– Low libido in males– Fever, low sperm count

• Young and growing pigs– Pneumonia, Diarrhea, mortality of 12‐15%

http://www.thepigsite.com/diseaseinfo/97/porcine‐reproductive‐respiratory‐syndrome‐prrs


• Costs $660,000,000 annually in North America (Holtkamp et al ‘13)

• Costs €1,500,000,000 annually in Europe (European PRRSpecive ‘15)

• Converts to ~$6,000,000 each day!• Doesn’t include Asia.


• Sustainability costs• Threat to food security• Animal welfare• Psychological & emotional costs

– Initial binding with heparan sulfate– Binding/Internalization by Sialoadhesin– Internalization/uncoating of the virus by CD163

Van Breedam et al., 2010

1. Binding to Heparan Sulphate

2. Binding and Internalization

by Sialoadhesin

(SIGLEC1)

3. CD163- and Low pH-

Dependent Release of GenomeFrom the

Endosome


SIGLEC1+/- pigsBorn 1/16-18/2011 Patent Pending

Prather et al ‘13 J Virology


• SIGLEC1-/- is not embryonic lethal.• SIGLEC1-/- did not affect susceptibility

to PRRSv.• Could cells from these pigs be useful

diagnostic tools?


• Conceive Idea – June ‘02.• Search for $$$• Founder male heterozygotes – Born Jan ‘11.• Founders reach puberty Sept-Oct ’11.• F1’s born (heterozygous males and females)

Nov ’11-Jan ‘12.• F1’s reach puberty summer ‘12.• Homozygous Knockout animals born Oct ‘12.• Challenge experiments began Nov ‘12.• Publication ’13 almost 11 years later.


• A series of experiments similar to that for SIGLEC1 provided evidence that CD163 is not only an entry mediator but also facilitates infection (Calvert et al ’07, and as

outline by Welsh & Calvert ’10). • Other candidates include CD151, VIM

and CD209.

Van Gorp et al ‘10 J Vir

Human CD163

Human CD163L

• Traditional knockout by homologous recombination and addition of a stop codon

• Domain Swap– Remove extracellular domain SRCR5 from

CD163– Replace with extracellular domain 8 from

hCD163L

• Haptoglobin-Hemaglobin – SRCR #3• Erythroblast – SRCR #2• TNFSF12 (aka TWEAK) – SRCR #1-4 &

#6-9 • Bacteria – SRCR #2• PRRSv- SRCR #5• ASFV #2 – No (Popescu et al ’16 Vir.)

• CRISPR/Cas9 mediated editing in fetal-derived fibroblast cells

• Zygote injection of CRISPRs and Cas9

Using the CRISPR/Cas9 system to edit specific genes in fetal fibroblast cell line and then clone the pigs

Using the CRISPR/Cas9 system to edit specific genes in pig zygotes

• Engineering Somatic Cells (1 month to 2 years)

• SCNT/ Zygote injection (1 week)• Gestation (3 months, 3 weeks & 3 days)• Puberty (6-9 months)

• In the case of CD163 it was less than 6 months from the day we implemented the CRISPR/Cas9 system until we had gene edited piglets.

PGK

LoxP LoxPmNeo & poly(A)pSRCR5

EncodinghSRCR8

exogenousintron

Short ArmCD163 exons 8‐101578 bp

Long ArmCD163 intron3469 bp

WT exons7 8 9

8 9

Wild Type CD163

Domain Swap Targeting Vector for CD163

A

B1230

7775

3752 7765

8791

DS

Difficult to obtain Domain Swap Targeting EventImproved targeting vector 3 times, single stranded, double stranded, linearized with different enzymes, removed targeting vector backbone, keep it intact, male cells, female cells…..Performed 38 transfectionsScreened 3,399 colonies= 0 targeting events

CRISPRs!!!!

Domain Swap Targeting Vector

Whitworth et al ‘14, Biol Reprod

• CRISPR‐Cas is a microbial adaptive immune system that uses RNA‐guided nucleases to cleave invading foreign genetic elements (Sorek et al., 2008; Mkarova et al., 2011)

• This system has been repurposed for mammalian genome engineering using SpCas9 along with a fusion of the tracrRNA and mature crRNA to create a chimeric single guide RNA (sgRNA)

Ran et al, 2013

• Genomic Sequence Confirmed and CRISPRs designed (1 day, if good EST data available)

– 7-18-13 • CRISPRs pairs ordered from IDT (1 day)

– 7-19-13• CRISPRs pairs annealed and ligated

and transformed• Bacterial Colonies picked, propagated

and sequence confirmed• Positive colonies re-propagated in

larger preps for transfections– 7/22/13-7/26/13 (1 week)

• Design Assays for smaller and large deletions

– This project had large assays– Small assay 435 bp was developed

(2 days)

• Cells ready for transfection 8-7-14• First Transfection with CRISPRs (14

days)– 8/9/13-8/23/13

• Colonies picked and genotyped (2 days)– 8/23/14-8/24/14

• Positive Colonies were propagated and sequence confirmed

– 8-26-13 (this took along time)• Nuclear Transfer

– 9/19/13• Embryo Transfer

– 9/20/13• First Pigs modified by CRISPRS 1/13/14

Total Time by SCNT: 5.5 ‐ 6 months


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Weaned wildtype

and CD163edited

piglets prior to transport to Kansas

State University.

Patent Pending

Whitworth et al, ‘16Nature Biotechnology


• 3 piglets predicted to be null (CD163-/- )• 8 WT piglets

Piglet ID

Predicted Translation

Maternal Allele

Paternal Allele

#43 CD163-/- 7 bp addition in exon 7

2 bp deletion in exon 7 + 377 bp intron deletion in the preceding intron


2 bp deletion in exon 7 + 377 bp intron deletion in the preceding intron


11 bp deletion in exon 7

• Identity blinded to the crew at KSU• Housed in the K-State BL-2 LARC facility• Allowed to acclimate for 3 days after arrival at facility• Challenge IM and IN with 105 TCID50 of NVSL 97-7985.

(Standard Type 2 lab isolate from 1997, relatively “hot” in terms of replication and pathogenesis)

• Pigs were maintained in the same pen; therefore, all pigs constantly exposed to virus

• Monitored daily for clinical signs• Blood collected on days 0,4,7,11,14,21,28,35 and weights

collected at least weekly• Study terminated 35 days after infection• Lung lavage for PAMs• Lungs and tissues removed for histopathology


http://www.scuba-diving-smiles.com/palm-tree-clipart.html


Whitworth et al, ’16Nature Biotechnology

Data provided by Maureen Kerrigan

Over 3000 pigs challenged at Kansas State with a response like these.


Viral RNA IgG

• CD163+/+ pigs had interstitial edema with the infiltration of mononuclear cells and the mononuclear infiltrate consisted mainly of lymphocytes and plasma cells and lesser numbers of macrophages. In contrast there was no evidence for pulmonary changes in the CD163‐/‐pigs.


Arginine 561 located in domain 5 of CD163 is important to maintain in vitro infectivity of PRRSv

In vitro studies showed that CD163 lacking domain 5 do not become infected with PRRSv

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Source of PAMSType 1 WT CD163-HL11m Null13-15 56 +/-9 0 0Lelystad 62 +/-15 0 003-1059 50 +/-18 0 003-1060 61 +/-12 0 001-08 64 +/-20 0 04353-PZ 62 +/-15 0 0Type 2NVSL 97 59 +/-15 8 +/-8 0KS-06 56 +/-20 12 +/-9 0P129 64 +/-11 8 +/-6 0VR2332 54 +/-5 6 +/-3 0CO 10-90 43 +/-18 8 +/- 8 0CO 10-84 51 +/-22 7 +/-4 0MLV-ResP 55 +/-12 3 +/-1 0KS62 49 +/-3 10 +/-11 0KS483 55 +/-23 6 +/-3 0*Results are presented as percent infected PAMs (n=3: mean±S.D.)

WT (circles), HL8 (squares) and Null (triangles) CD163 modified pigs were infected with a contemporary Type 1 isolate, SD-1305 or a Type 2 isolate, NVSL.

The open box shows viremia for the HL11m pig#101. The number pigs in each group for the Type 1: WT n=4, HL11 n=5 and null n=3; and for the Type 2: n=4 for WT, n=4 for HL11 and n=3 for the null pigs.

Wells et al, Submitted

SD-1305 NVSL

• How does a fetus get infected?• Reproductive PRRS accounts for 45% of the

losses (abortions, mummified/dead fetuses, weak-born neonates).

• Reproductive PRRS results from infection ~Day 90-114.

• Replication in maternal macrophages. • Virus then crosses placenta to infect

individual fetuses.• Fetus to fetus transmission• Primary site of infection in the fetus is the

thymus

Prather et al, in prep.

Will knocking out CD163 in the sow protect a wild type fetus?

Pregnant Gilt/Sow Challenge

Treatment 1CD163 KO sowCD163 KO fetus

Treatment 3CD163 KO sowCD163 HET fetus

Treatment 2WT SowWT Fetus

CD163 KOCD163 KO

Wild TypeWild Type

CD163 HETCD163 KO

• Gilts were infected with 105 TCID50 of NVSL 97-7895 diluted in 5 ml of culture medium.

• ½ of the inoculum was administered by intramuscular injection and ½ administered intranasally.

• Co-Housed.


• CD163-/- gilts. No clinical signs• CD163+/+ gilts. One aborted on day 106.

Viremia = 5.5 log10.• Days 17-19 post infection gilts were

euthanized.


The numbers on the left identify each dam. Below each number in parentheses is the result for PRRS PCR in serum, measured as log10 templates per reaction.

Asterisks identify samples obtained from abdominal fluid. “N” is negative for PRRSv nucleic acid (Ct>39).

The number within each circle refers to anatomical pathology: 1) normal fetus; 2) small fetus; 3) placental changes, such as detached or necrotic; 4) meconium staining; 5) For the dead and necrotic. ND, not determined.

Prather et al, submitted.



• Most of the WT fetuses from WT gilts were positive for PRRSv.

• Wide variation between fetuses.• Viremia level was not always

correlated with gross pathology, e.g. Fetus #5, Gilt 138

• None of the fetuses on the CD163-/-

gilts were infected, and morphology was ‘normal’


• SIGLEC1-/- pigs are not resistant to Type 2 PRRSv.

• CD163 -/- pigs are resistant to BOTH Type 1 and Type 2 PRRSv.

• CD163 -/- gilts provide protection to fetuses in utero.

• Domain 5 of CD163 is a contributor to infection: Type 1 vs Type 2

1. Binding to Heparan Sulphate

2. Binding and Internalization

by Sialoadhesin

(SIGLEC1)

3. CD163- and Low pH-

Dependent Release of GenomeFrom the

Endosome


CD163 is a Gatekeeper!

• Are all gene knockouts bad?– Embryonic Lethal– Neonatal Lethal

• CFTR, RAG2, IL2RG– Detrimental

• DMD, PAH, APC– Infertile– No general impact

• GGTA1, B4GALNT2, TMPRSS2, CMAH, CD163, SIGLEC1

• CD163– Show there are no unintended

consequences (health, growth, feed efficiency, etc.)

– FDA approval

• Edits in Production Agriculture??– 3,000,000,000 bp genome– Disease Resistance

• African Swine Fever (RELA)• Bovine Respiratory Disease (Mannheimia

haemolytica: CD18)• TMPRSS2-/- in pigs for influenza?

– Animal Welfare Issues (Polled Dairy Cattle)– Productivity– Alter the Composition of the Carcass

• Answer Basic and Applied Questions about Human Medicine and Domestic Animal Biology

Zygote InjectionsKiho LeeLee Spate

CRISPR and Targeting Vector DesignKevin WellsKristin Whitworth

Transfection and GenotypingMykel AndersonMariah ThomasJoshua Benne

Nuclear Transfer and Embryo TransferJoshua BenneStephanie MurphyJennifer TesonJiude MaoClifton Murphy

Surrogate and Piglet CareMelissa SamuelJason DowellTricia MeyerEntire Prather Lab

Funding SourcesGenus plcThe Christopher Columbus Fellowship FoundationFood for the 21st CenturyUSDA ARS

Genus plcAlan MilehamDave McLarenJon Lightner

SIGLEC1-/-

ProjectJon GreenTina EgenKristin WhitworthKansas State UniversityBob RowlandRachel BardotBenjamin TribleMaureen KerriganCatherine EwenAda Cino-OzunaBhupinder Bawa

Genetic Engineering · 2017-06-26 · Genetic Engineering (CRISPRs, Zinc Fingers, TALENS, Homologous Recombination) Transgene Injection Nuclease Injection Oocyte Transduction Sperm‐Mediated

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