Swine Comprising Modified CD163 and Associated Methods
20200045945 ยท 2020-02-13
Inventors
- Simon Geoffrey Lillico (Edinburgh Lothian, GB)
- Alan Archibald (Edinburgh Lothian, GB)
- Christopher Bruce Alexander Whitelaw (Biggar South Lanarshire, GB)
- Christine Tait-Burkard (Bo'ness, GB)
- Tahar Ait-Ali (Ivry-sur-Seine, FR)
Cpc classification
A01K67/0275
HUMAN NECESSITIES
C07K14/70596
CHEMISTRY; METALLURGY
C12N15/8509
CHEMISTRY; METALLURGY
C12N5/0645
CHEMISTRY; METALLURGY
International classification
C12N15/10
CHEMISTRY; METALLURGY
C07K14/705
CHEMISTRY; METALLURGY
Abstract
The present invention relates to genetically edited swine which produce CD163 protein in which the scavenger receptor cysteine-rich 5 (SRCR5) domain (also known as CD163 domain 5) has been deleted. Such swine have been found to be healthy and do not exhibit negative properties, and are resistant to PRRSV infection. CD163 expressed in the edited swine also demonstrates retention of the ability to function as a haemoglobin-haptoglobin scavenger. Methods of producing such swine are also provided.
Claims
1. A genetically edited swine, the swine comprising an edited genome wherein the edit results in the deletion of SRCR5 domain from the CD163 protein produced by the swine.
2. The genetically edited swine of claim 1 wherein all of the other domains of the CD163 protein are present and their amino acid sequences are unaltered.
3. The genetically edited swine of claim 1 wherein the CD163 protein produced by the genetically edited swine remains substantially functional.
4. The genetically edited swine of claim 1 wherein the CD163 protein lacks the following amino acid sequence: TABLE-US-00013 (SEQIDNO:2) HRKPRLVGGDIPCSGRVEVQHGDTWGTVCDSDFSLEAASVLCRELQCGTV VSLLGGAHFGEGSGQIWAEEFQCEGHESHLSLCPVAPRPDGTCSHSRDVG VVCS.
5. The genetically edited swine of claim 4 wherein the CD163 protein produced by the genetically edited swine has no further changes to the wild type amino acid sequence.
6. The genetically edited swine of claim 1 which is homozygous or biallelic for the genome edit that results in the deletion of the SRCR5 domain from the CD163 protein produced by the animal.
7. The genetically edited swine of claim 1 wherein all cells of the animal comprise the edited genome.
8. The genetically edited swine of claim 1 wherein the genome of the swine is edited such that the sequence which codes for SRCR5 is absent from the mature mRNA produced from the edited CD163 gene.
9. The genetically edited swine of claim 1 wherein the swine comprises an edited genome in which exon 7 of the CD163 gene has been deleted.
10. The genetically edited swine of claim 1 wherein the splice acceptor site located at the 5 of exon 7 of the CD163 gene is inactivated.
11. The genetically edited swine of claim 1 wherein exons 1 to 6 and 8 to 16 of the CD163 gene are unaltered relative to the wild type sequence.
12. The genetically edited swine of claim 11 wherein exon 7 and portions of introns 6 and 7, which flank exon 7, are deleted from the CD163 gene, but there are no other alterations in the remaining regions of the CD163 gene.
13. The genetically edited swine of claim 1 wherein the edited genome is edited such that the splice site donor sequence in intron 6 and the splice site acceptor site in intron 7 are unaltered and remain functional.
14. The genetically edited swine of claim 1 wherein the genome is edited such that at least a portion of the region of the CD163 gene extending from position 10466 to 23782 with reference to SEQ ID NO:1, is deleted.
15. The genetically edited swine of claim 1 wherein the genome is edited such that regions from positions 1 to position 10465 and from position 23783 to position 32908, with reference to SEQ ID NO:1, are unaltered.
16. The genetically edited swine of claim 1 wherein the genome is edited such that exon 7 is deleted along with up to 5000 bases, suitably up to 2000 bases, suitably up to 1000 bases, suitably up to 500 bases, suitably up to 300 bases or suitably up to 100 bases extending 5 of the 5 end of exon 7.
17. The genetically edited swine of claim 1 wherein the genome is edited such that exon 7 is deleted along with up to 75 bases extending 3 of the 3 end of exon 7.
18. The genetically edited swine of claim 1 wherein the genome is edited such that the edited genome comprises a deletion of the region extending from: a) approximately position 23060 to approximately position 23760, for example from position 23065 to position 23753, with reference to SEQ ID NO:1; b) approximately position 23260 to approximately position 23760, for example from position 23268 to position 23753, with reference to SEQ ID NO:1; or c) approximately position 23370 to approximately position 23760, for example from position 23374 to position 23753, with reference to SEQ ID NO:1.
19. The genetically edited swine of claim 1 wherein the edited genome comprises an inserted sequence.
20. The genetically edited swine of claim 1 wherein the genome is edited such that the region extending from position 23378 to position 23416, with reference to SEQ ID NO:1, is edited such that the splice acceptor site in intron 6 is inactivated.
21. The genetically edited swine of claim 1 wherein the splice acceptor site in intron 6 is partially or entirely deleted, or its sequence altered in any other suitable way so that it is no longer functional.
22. The genetically edited swine of claim 20 wherein the splice acceptor site is edited to alter the sequence from AATGCTATTTTTCAGCCCACAGGAAACCCAGG (SEQ ID NO: 3) to AATGCTATTTTTCgGCCatggGGAAACCCAGG (SEQ ID NO: 4), wherein the sequence changes are shown in lower case.
23. The genetically edited swine of claim 1 wherein the genetically edited swine has improved tolerance or resistance to PRRSV infection compared to a wild type swine, preferably wherein the animal is resistant to PRRS infection.
24. A genetically edited swine cell or embryo, wherein the edit results in the deletion of SRCR5 domain from the CD163 protein that can be produced by the swine cell or embryo.
25. A method of producing a genetically edited swine, the method comprising the steps of: a) providing a swine cell; b) editing the genome of the cell to create a genome modification which results in the deletion of SRCR5 from the CD163 protein; and c) generating an animal from said cell.
26. The method of claim 25 wherein the genome modification that results in deletion of SRCR5 from the CD163 protein is deletion of exon 7 from the CD163 gene or the inactivation of the splice acceptor site in intron 6 of the CD163 gene.
27. The method of claim 25 wherein in step a) the swine cell is a somatic cell, a gamete, a germ cell, a gametocyte, a stem cell (e.g. a totipotent stem cell or pluripotent stem cell) or a zygote.
28. The method of claim 25 wherein in step a) the swine cell is a single cell zygote and step b) of the method is at least initiated in the zygote at the single cell stage.
29. The method of claim 25 wherein in step b) comprises: introducing a site-specific nuclease to the cell, the site-specific nuclease targeting a suitable target sequence in the CD163 gene; incubating said cell under suitable conditions for said site-specific nuclease to act upon the DNA at or near to said target sequence; and thereby induce an editing event in the CD163 gene that results in deletion of SRCR5 from the CD163 protein.
30. The method of claim 29 wherein the editing event that results in deletion of SRCR5 from the CD163 protein is the deletion of exon 7 from the CD163 gene or the inactivation of the splice acceptor site in intron 6 of the CD163 gene.
31. The method of claim 29 wherein step b) comprises introducing site-specific nucleases to the cell which are targeted to target sites flanking exon 7 of the CD163 gene so as to induce double-stranded DNA cuts on either side of exon 7 and thereby cause its deletion.
32. The method of claim 31 wherein one target site is in intron 6 and the cutting site is 3 of the splice donor site at the 3 end of exon 6, and wherein another target site is in intron 7 and the cutting site is 5 of the splice acceptor site at the 5 of exon 8.
33. The method of claim 25 wherein step b) comprises introducing an upstream site-specific nuclease to the cell, the upstream site-specific nuclease targeting a target site upstream of exon 7 of the CD163, and introducing a downstream site-specific nuclease to the cell, the downstream site-specific nuclease targeting a target site downstream of exon 7 of the CD163.
34. The method of claim 29 wherein step b) comprises introducing a site-specific nuclease that targets the splice acceptor site in intron 6.
35. The method of claim 34 wherein the site-specific nuclease that targets the splice acceptor site in intron 6 creates a single double stranded cut at the desired cutting site to inactivate the splice acceptor site associated with exon 7 by non-homologous end joining (NHEJ) or by homology directed repair (HDR).
36. The method of claim 35 comprising providing an HDR template having following sequence: GAAGGAAAATATTGGAATCATATTCTCCCTCACCGAAATGCTATTTTTCgGCCatggGGAA ACCCAGGCTGGTTGGAGGGGACATTCCCTGCTCTGGTC (SEQ ID NO:16), wherein lower case letters show the changes made compared to the unaltered sequence.
37. (canceled)
38. The method of claim 25 comprising the steps of: providing a swine zygote; introducing a site-specific nuclease to the zygote, the site-specific nuclease targeting a suitable target sequence in the CD163 gene; incubating said zygote under suitable conditions for said site-specific nuclease to act upon the DNA at or near to said target sequence and thereby induce an editing event in the CD163 gene that results in deletion of SRCR5 from the CD163 protein; and generating an animal from said genetically edited zygote.
39-44. (canceled)
Description
BRIEF DESCRIPTION OF THE FIGURES
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SPECIFIC DESCRIPTION OF EMBODIMENTS OF THE INVENTION
[0123] While the making and using of various embodiments of the present invention are discussed in detail below, it should be appreciated that the present invention provides many applicable inventive concepts that can be embodied in a wide variety of specific contexts. The specific embodiments discussed herein are merely illustrative of specific ways to make and use the invention and do not delimit the scope of the invention.
[0124] To facilitate the understanding of this invention, a number of terms are defined below. Terms defined herein have meanings as commonly understood by a person of ordinary skill in the areas relevant to the present invention. Terms such as a, an and the are not intended to refer to only a singular entity, but include the general class of which a specific example may be used for illustration. The terminology herein is used to describe specific embodiments of the invention, but their usage does not delimit the invention, except as outlined in the claims.
[0125] The term swine, or variants thereof, as used herein refers to any of the animals in the Suidae family of even-toed ungulates including animals in the genus Sus and other related species, including the peccary, the babirusa, and the warthog.
[0126] The term pig or variants thereof as used herein refers to any of the animals in the genus Sus. It includes the domestic pig (Sus scrofa domesticus or Sus domesticus) and its ancestor, the common Eurasian wild boar (Sus scrofa). For the present purposes, the domestic pig is considered to be a sub-species of the species Sus scrofa. It does not include the peccary, the babirusa, and the warthog.
[0127] The term domestic pig, or variants thereof, as used herein refers to an animal of the sub-species Sus scrofa domesticus.
[0128] The term site-specific nuclease, or variants thereof, as used herein refers to engineered nucleases which can be configured to cut DNA at a desired location. Such site-specific nucleases are also known as engineered nucleases, targetable nucleases, genome editing nucleases, molecular scissors, and suchlike. Examples of site-specific nucleases include zinc finger nucleases (ZFNs), Transcription Activator-Like Effector Nucleases (TALENs), the CRISPR/Cas system (CRISPR/Cas), and meganucleases, such as hybrid meganucleases.
[0129] Genetically edited or genetically modified when used in relation to subject biological material, refers to the fact that the subject biological material has been treated to produce a genetic modification thereof compared to control, e.g. wild type, biological material.
[0130] Target site refers to the site having a nucleic acid sequence to which a site-specific nuclease binds. When the site-specific nuclease bind at a target site it acts to cut the DNA within or adjacent to the target site (this can be achieved by a single site-specific nuclease, or a corresponding pair or nucleases, in which case there will be two so-called half-sites, as desired), the location of the cut being referred to as the cut site or cutting site. Where a target site is defined for a site-specific nuclease above, the cut site is suitably with the target site, or adjacent to the target site. Where the target site is mentioned as being near or adjacent to a specific feature in the genome, e.g. a feature to be deleted or preserved in an editing event (such as exon 7 or a splice site), the cutting site should be located so as to achieve the desired outcome, i.e. it results in deletion or preservation of the feature, as desired. Site-specific nucleases can be designed to target any desired target site; for example, with CRISPR/Cas9 this can be achieved using a suitable sgRNA, and for ZFN or TALENs suitable proteins can be designed and obtained from commercial sources.
[0131] SRCR5 refers to an animal, typically a swine, which comprises a biallelic or homozygous CD163 SRCR5 deletion.
[0132] Unaltered with reference to a nucleic acid sequence (such as a region of the genome or a gene) means that the sequence has not been altered from the wild type sequence.
[0133] Tolerance or resistancean animal can be said to be more tolerant or resistant to PRRSV infection when the mortality rate, morbidity rate, the proportion of animals showing significant morbidity (e.g. weight loss or decreased growth rate), the level of morbidity or the duration of morbidity is reduced when animals are challenged with PRSSV infection. Any statistically significant reduction (e.g. 95% confidence, or 99% confidence using an appropriate test) in the mortality or morbidity between a population of genetically edited pigs and a population of equivalent non-edited pigs when exposed to PRRSV of the same virulence level (ideally the same isolate) demonstrates improved tolerance or resistance. Improved tolerance or resistance can be demonstrated by a reduced susceptibility to PRRSV inflection, or a lessening of the symptoms when infection occurs. Improved resistance to infection in a swine can be tested in vitro using the methodologies described below for PAM and PMM cells.
[0134] Protein and peptide, as used herein, can be used interchangeably (unless the context suggests otherwise) and mean at least two covalently attached amino acids linked by a peptidyl bond. The term protein encompasses purified natural products, or products which may be produced partially or wholly using recombinant or synthetic techniques. The terms peptide and protein may refer to an aggregate of a protein such as a dimer or other multimer, a fusion protein, a protein variant, or derivative thereof. A protein may comprise amino acids not encoded by a nucleic acid codon, i.e. non-natural amino acids.
INTRODUCTION
[0135] PRRS is one of the most economically important infectious diseases affecting pigs worldwide. The mystery swine disease was first observed almost simultaneously in North America and in Europe in the late 1980s [1,2]. The causative agent of PRRS was identified to be a virus later named PRRS virus (PRRSV). Infected pigs may present with symptoms involving inappetence, fever, lethargy, and respiratory distress. However, the most devastating effects of PRRSV infection are observed in young piglets and pregnant sows. In pregnant sows an infection with PRRSV can cause a partial displacement of the placenta, leading to full abortions or to death and mummification of fetuses in utero [3]. Late-term abortions occur in up to 30% of infected sows with litters containing up to 100% stillborn piglets. Live-born piglets from an antenatal infection are often weak and display severe respiratory symptoms, with up to 80% of them dying on a weekly basis pre-weaning [4,5]. Young piglets infected with PRRSV often display diarrhea and severe respiratory distress caused by lesions in the lung. In pre-weaned piglets the infection may be transmitted via the mammary gland secretions of an infected sow [6]. At this age the infection has a fatal outcome in up to 80% of animals. After weaning mortality rates reduce, but continued economic losses due to reduced daily gain and feed efficiency are often observed [4,7,8]. Due to reduction or loss of pregnancies, death in young piglets, and decreased growth rates in all PRRSV infected pigs it is estimated that more than $650m are lost annually to pork producers in the United States alone [9,10].
[0136] PRRSV is an enveloped, plus-strand RNA virus belonging to the Arteriviridae family in the order Nidovirales [11,12]. The PRRSV genome (15 kb) encodes at least 12 non-structural and seven structural proteins. The viral RNA is packaged by the nucleocapsid protein N, which is surrounded by the lipoprotein envelope, containing the non-glycosylated membrane proteins M and E, as well as four glycosylated glycoproteins GP2, GP3, GP4, and GP5, whereby GP2, 3, and 4 form a complex [13-17].
[0137] PRRSV has a very narrow host range, infecting only specific subsets of porcine macrophages [18-20]. It is unknown yet how widespread PRRSV infections are within the superfamily of the Suidae. Whereby European wild boars have been shown to act as a reservoir for PRRSV [21], little is known about infection in African suids, such as bushpigs and warthogs. In vitro virus replication is supported by the African Green Monkey cell line MARC-145. Entry of PRRSV into macrophages has been shown to occur via pH-dependent, receptor mediated endocytosis [22,23]. Various attachment factors and receptors have been indicated to be involved in the PRRSV entry process (reviewed in [24]). Heparan sulphate was identified early as an attachment factor of the virus [25-27]. In vitro infection of pulmonary alveolar macrophages (PAMs) but not MARC-145 cells was shown to be inhibited by an antibody targeting CD169 (sialoadhesin), a lectin expressed on the surface of macrophages [28]. Overexpression of CD169 in previously non-permissive PK-15 cells showed internalization but not productive replication of PRRSV [29]. Finally, an in vivo challenge of genetically modified pigs in which the CD169 gene had been knocked out revealed no increased resistance to PRRSV infection, suggesting that CD169 is an attachment factor that is not essential for PRRSV infection [30]. Even though cell surface protein expression is a major determinant of PRRSV binding and internalization, there appears to be a redundancy amongst cell surface attachment factors, with the potential for additional, as yet unidentified receptors, being involved [31]. The scavenger receptor CD163, also known as haptoglobin scavenger receptor or p155, is expressed on specific subtypes of macrophages and has been identified as a fusion receptor for PRRSV. The extracellular portion of CD163 forms a pearl-on-a-string structure of nine scavenger receptor cysteine-rich (SRCR) domains and is anchored by a single transmembrane segment and a short cytoplasmic domain [32]. CD163 has a variety of biological functions, including mediating systemic inflammation and the removal of hemoglobin from blood plasma (reviewed in [33,34]). Overexpression of CD163 renders non-susceptible cells permissive to PRRSV infection [35], whereby it was found that CD163 does not mediate internalization but is crucial for fusion [36]. The transmembrane anchoring and an interaction with the SRCR domain 5 (SRCR5) of CD163 were found to be essential for successful infection with PRRSV [34,35]. Recent in vivo experiments with CD163 knock-out pigs have been performed [37]. However, as CD163 has important biological functions the complete knockout could have a negative physiological impact pigs, particularly with respect to inflammation and/or infection by other pathogens.
[0138] This study aimed to generate pigs with a defined CD163 SRCR5 deletion and to assess the susceptibility of macrophages from these pigs to PRRSV infection.
[0139] Materials and Methods
[0140] All animal work was approved under UK Home Office license after review by the University of Edinburgh's Animal Ethics Committee and was carried out in accordance with the approved guidelines.
[0141] Cells and Viruses
[0142] Primary pulmonary alveolar macrophages (PAMs) for the propagation of PRRSV genotype 1, subtype 1 strain H2 (PRRSV H2) [52], subtype 2 strain DAI (PRRSV DAI) [53], and subtype 3 strain SU1-Bel (PRRSV SU1-Bel)[54] were harvested from wild type surplus research animals aged 6-9 weeks as previously described [45]. Briefly, animals were euthanized according to a schedule I method. Lungs were removed and transferred on ice to a sterile environment. PAMs were extracted from lungs by washing the lungs twice with warm PBS, massaging to release macrophages. Cells were collected by centrifugation for 10 min at 400 g. When necessary red cells were removed using red cell lysis buffer (10 mM KHCO.sub.3, 155 mM NH.sub.4Cl, 0.1 mM EDTA, pH 8.0) for 5 min before washing again with PBS. Cells were collected by centrifugation as before and frozen in 90% FBS (HI, GE Healthcare), 10% DMSO (Sigma). Cells were frozen gradually at 1 C./min in a 80 C. freezer before being transferred to 150 C.
[0143] PAMs from the animals 627, 628, 629, 630, 633, and 634 were collected at 8 weeks of age. For this the piglets were sedated using a Ketamine/Azaperone pre-medication mix and anaesthetized with Ketamine/Midazolam. Anesthesia throughout the procedure was maintained using Sevoflurane. PAMs were collected by bronchoalveolar lavage (BAL) through an intubation with an air flow access. Three lung segments were flushed in each animal using 220 ml PBS. Fluid recovery was between 60-80%. Cells were collected by centrifugation for 10 min at 400 g from the BAL fluid and frozen as above.
[0144] Peripheral blood monocytes (PBMCs) were isolated as described previously [45]. Briefly, blood was collected using EDTA coated vacuum tubes from the jugular vein of the piglets at 10 weeks of age. Blood was centrifuged at 1200 g for 15 min and buffy coat transferred to PBS. Lymphoprep (Axis-Shield) was overlaid with an equal volume of buffy coat/PBS and centrifuged for 45 min at 400 g. The mononuclear cell fraction was washed with PBS, cells collected and frozen as described above.
[0145] PAM cells were cultivated in RPMI-1640, Glutamax (Invitrogen), 10% FBS (HI, GE Healthcare), 100 IU/ml penicillin and 100 g/ml streptomycin (Invitrogen) (cRPMI). PBMCs were cultivated in cRPMI supplemented with rhCSF-1 (110.sup.4 units/ml; a gift from Chiron) for 6 days prior to infection.
[0146] PK15 cells were cultured in DMEM supplemented with Glutamax (Invitrogen), 10% FBS (HI, GE Healthcare), 100 IU/ml penicillin and 100 g/ml streptomycin (Invitrogen).
[0147] Design and In Vitro Cutting Efficiency Assessment of Guide RNAs
[0148] Three potential guide RNA sequences were selected in the 200 bp of intron 6 and one in the 97 bp long intron 7. Oligomers (Invitrogen) were ordered, hybridized as previously described [72] then ligated into the BbsI sites of plasmid pSL66 (a derivative of px458 with modifications to the sgRNA scaffold as described by [42]). The generated plasmids contain a hU6 promoter driving expression of the guide RNA sequence and a CBA promoter driving Cas9-2A-GFP with an SV40 nuclear localization signal (NLS) at the N-terminus and a nucleoplasmin NLS at the C-terminus of Cas9. Cutting efficiency of each guide was assessed by transfection of the plasmids into pig PK15 cells using a Neon transfection system (Invitrogen) set at 1400 mV with 2 pulses of 20 mS. 48 hours post-transfection GFP positive cells were collected using a FACS Aria III cell sorter (Becton Dickinson) and cultured for a further 4 days prior to preparation of genomic DNA (DNeasy Blood & Tissues Kit, Qiagen). PCR across the target sites was with oSL46 (ACCTTGATGATTGCGCTCTTSEQ ID NO:17) and oSL47 (TGTCCCAGTGAGAGTTGCAGSEQ ID NO:18) using AccuPrime Taq DNA polymerase HiFi (Life Technologies) to produce a product of 940 bp. A Cell assay (Transgenomic; Surveyor Mutation Detection Kit) was performed as previously described [73]. Co-transfection of PK15 cells with pairs of plasmids encoding guides flanking exon 7 were carried out as described above with the exception that cells were harvested at 40 hours post-transfection without enrichment for GFP expression. In this instance a truncated PCR product was observed in addition to the 940 bp fragment, indicating deletion of exon 7.
[0149] Based on both single and double cutting efficiencies guide RNAs SL26 (GAATCGGCTAAGCCCACTGTSEQ ID NO:7), located 121 bp upstream of exon 7, and SL28 (CCCATGCCATGAAGAGGGTASEQ ID NO:11), located 30 bp downstream of exon 7 were selected for in vivo experiments.
[0150] Generation of Guide RNA and Quality Assessment
[0151] A DNA oligomer fragment containing the entire guide RNA scaffold and a T7 promoter was generated by PCR from the respective plasmid template as follows; a forward primer containing the T7 promoter followed by the first 18 bp of the respective guide RNA and the reverse primers oSL6 (AAAAGCACCGACTCGGTGCCSEQ ID NO:19) were used in combination with the Phusion polymerase (NEB). DNA fragments were purified on a 1.5% agarose gel using the MinElute Gel Extraction Kit (Qiagen) according to the manufacturer's instructions. DNA eluate was further treated with 200 g/ml Proteinase K (Qiagen) in 10 mM Tris-HCl pH 8.0, 0.5% SDS for 30 min at 50 C. followed by phenol/chloroform extraction. Guide RNAs were generated from the resultant DNA fragment using the MEGAshortscript Kit (Thermo Fisher) according to the manufacturer's instructions. RNA was purified using phenol/chloroform extraction followed by ethanol precipitation and resuspended in EmbryoMax Injection Buffer (Millipore). Purity and concentration of the RNA was assessed using an RNA Screen Tape (Agilent) on an Agilent TapeStation according to the manufacturer's instructions.
[0152] Zygote Injection and Transfers
[0153] Embryos were produced from Large White gilts as described previously [73]. Briefly, gilts were superovulated using a regumate/PMSG/Chorulon regime between day 11 and 15 following estrus. Following heat, the donor gilts were inseminated twice in a 6 hour interval. Zygotes were surgically recovered from mated donors into NCSU-23 HEPES base medium, then subjected to a single 2-5 pl cytoplasmic injection with an injection mix containing 50 ng/l of each guide (SL26 and SL28) and 100 ng/l Cas9 mRNA (PNA Bio or Tri-Link) in EmbryoMax Injection buffer (Millipore). Recipient females were treated identically to donor gilts but remained unmated. During surgery, the reproductive tract was exposed and 24-39 zygotes were transferred into the oviduct of recipients using a 3.5 French gauge tomcat catheter. Litter sizes ranged from 5-12 piglets.
[0154] In Vitro Assessment Genome Editing in Blastocyst
[0155] Uninjected control zygotes and injected surplus zygotes are cultivated in NCSU-23 HEPES base medium, supplemented with cysteine and BSA at 38.5 C. for 5-7 days. Blastocysts were harvested at day 7 post cultivation and the genome amplified using the REPLI-g Mini Kit (Qiagen), according to the manufacturer's instructions. Genotyping was performed as described below.
[0156] Genotyping
[0157] Genomic DNA was extracted from ear biopsy or tail clippings taken from piglets at 2 days postpartum using the DNeasy Blood and Tissue Kit (Qiagen). The region spanning intron 6 to exon 8 was amplified using primers oSL46 (ACCTTGATGATTGCGCTCTTSEQ ID NO:17) and oSL47 (TGTCCCAGTGAGAGTTGCAGSEQ ID NO:18), generating a 904 bp product from the intact allele and a 454 bp product if complete deletion of exon 7 had occurred. PCR products were analyzed by separation on a 1% agarose gel and subsequent Sanger sequencing of all truncated fragments. Fragments corresponding to the wild type length were further analyzed by T7 endonuclease I (NEB) digestion according to the manufacturer's instructions.
[0158] RNA Phenotyping
[0159] RNA was isolated from 1E6 PAM cells, isolated by BAL as described above, using the RNeasy Mini Kit (Qiagen), according to the manufacturer's instructions, including an on-column DNase digestion. First-strand cDNA was synthesized using an Oligo-dT primer in combination with SuperScript II reverse transcriptase (Invitrogen), according to the manufacturer's instructions. The cDNA was used to assess the RNA phenotype across exons 4 to 9 using primers P0083 (ATGGATCTGATTTAGAGATGAGGCSEQ ID NO:20) and P0084 (CTATGCAGGCAACACCATTTTCTSEQ ID NO:21), resulting in a PCR product of 1686 bp length for the intact allele and 1371 bp following precise deletion of exon 7. PCR products were analyzed by separation on a 1% agarose gel and subsequent Sanger sequencing of deletion fragments.
[0160] Protein Phenotype Analysis by Western Blotting
[0161] 4E5 PAM cells isolated by BAL were collected by centrifugation at 300 rcf for 10 min. The pellet was resuspended in Laemmli sample buffer containing 100 mM DTT, boiled for 10 min at 95 C. and subjected to electrophoresis on 7.5% acrylamide (Bio-Rad) gels. After transfer to a nitrocellulose membrane (Amersham), the presence of cellular proteins was probed with antibodies against CD163 (rabbit pAb, abcam, ab87099) at 1 g/ml, and -actin (HRP-tagged, mouse mAb, Sigma, A3854) at 1:2000. For CD163 the blot was subsequently incubated with HRP-labelled rabbit anti-mouse antibody (DAKO, P0260) at 1:5000. Binding of HRP-labelled antibodies was visualized using the Pierce ECL Western Blotting Substrate (Thermo Fisher), according to the manufacturer's instructions.
[0162] Quantification of CD163 mRNA by RT-qPCR
[0163] RNA was isolated from 1E6 PAMs using the RNeasy Mini Kit (Qiagen), according to the manufacturer's instructions, including an on-column DNase digestion. RNA levels were measured using the GoTaq 1-Step RT-qPCR system (Promega) according to the manufacturers' instructions on a LightCycler 480 (Roche). mRNA levels of CD163 were quantified using primers P0074 (CATGGACACGAGTCTGCTCTSEQ ID NO:22) and P0075 (GCTGCCTCCACCTTTAAGTCSEQ ID NO:23) and reference mRNA levels of -actin using primers P0081 (CCCTGGAGAAGAGCTACGAGSEQ ID NO:24) and P0082 (AAGGTAGTTTCGTGGATGCCSEQ ID NO:25).
[0164] Characterization of Macrophages by Flow Cytometry
[0165] PAMs were seeded one day prior to analysis. PBMCs were seeded seven days prior to analysis and differentiated by CSF1 stimulation to yield PBMC-derived macrophages (PMMs). Cells were harvested by scraping with a rubber policeman and fixed in 4% formaldehyde/PBS for 15 min at room temperature. Cells were incubated with blocking solution (PBS, 3% BSA) for 45 min before staining with antibodies. Cells were stained with antibodies targeting either mouse anti-pig CD14 (AbD Serotec, MGA1273F, 1:50) and mouse anti-pig CD16 (AbD Serotec, MCA2311PE, 1:200), mouse anti-pig CD169 (AbD Serotec, MCA2316F, 1:50) and mouse anti-pig CD172a (SoutherBiotech, 4525-09, 1:400), mouse anti-human CD151 (AbD Serotec, MCA1856PE, 1:50) and mouse anti-pig SWC9 (CD203a) (AbD Serotec, MCA1973F, 1:50), mouse anti-pig CD163 (AbD Serotec, MCA2311PE, 1:50), or mouse IgG1 or an IgG2b negative control (AbD Serotec, MCA928PE, MCA691F, or Sigma, F6397; same concentration as primary Ab). The cells were washed three times with PBS and resuspended in FACS buffer (2% FBS, 0.05M EDTA, 0.2% NaN.sub.3 in PBS). Gene expression determined by antibody labelling was assessed by FACS analysis on a FACS Calibur (Becton Dickinson) using FlowJo software.
[0166] High MOI Single-Round Infection Assay
[0167] PAMs were seeded one day prior to infection. PBMCs were seeded seven days prior to infection and differentiated by CSF1 stimulation to yield PBMC-derived macrophages PMMs. Cells were inoculated at MOI=1 of the respective virus strain (PRRSV H2, DAI, or SU1-Bel) in cRPMI for 3 h at 37 C. The inoculum was replaced by warm cRPMI. At 19 hpi cells were detached by using a cell scraper. Cells were fixed in 4% Formaldehyde (Sigma-Aldrich) in PBS (Gibco) for 15 min at RT, washed with PBS, and subsequently permeabilized in PBS containing 0.1% Triton-X-100 (Alfa Aesar) for 10 min. Cells were incubated with antibody against PRRSV-N (SDOW17-F, RTI, KSL0607, 1:200) and CD163 (AbD Serotec, MCA2311PE, 1:50) or mouse IgG1 negative controls, as described above, in 3% BSA in PBS. The cells were washed three times with PBS and re-suspended in FACS buffer. Infection levels, determined by antibody labelling, were assessed by FACS analysis on a FACS Calibur (Benson Dickson) using FlowJo software.
[0168] Low MOI Multiple-Round Infection Assay
[0169] PAMs were seeded one day prior to infection. PBMCs were seeded seven days prior to infection and differentiated by rhCSF1 stimulation to yield PMMs. Cells were inoculated at MOI=0.1 with the respective virus strain (PRRSV H2, DAI, or SU1-Bel) in cRPMI for 3 h at 37 C. Inoculum was removed, cells washed 1 with PBS, and infection continued. At the indicated times post inoculation samples were harvested to be assessed. All samples were frozen and processed once all samples from a time course had been collected.
[0170] Viral RNA (vRNA) was extracted from the supernatant samples using the QIAmp Viral RNA Mini Kit according to the manufacturer's instructions. The viral RNA levels were quantified by RT-qPCR using the GoTaq Probe 1-Step RT-qPCR system (Promega) for PRRSV H2 and SU1-Bel and the GoTaq 1-Step RT-qPCR system (Promega) for PRRSV DAI, according to the manufacturer's instructions. For this the following primers and probes were used: H2 fwd (GATGACRTCCGGCAYCSEQ ID NO:26), H2 rev (CAGTTCCTGCGCCTTGATSEQ ID NO:27), H2 probe (6-FAM-TGCAATCGATCCAGACGGCTT-TAMRASEQ ID NO:28), (optimal H2 primer/probe sequences obtained from JP Frossard, AHVLA), SU1-Bel fwd (TCTTTGTTTGCAATCGATCCSEQ ID NO:29), SU1-Bel rev (GGCGCACTGTATGACTGACTSEQ ID NO:30), SU1-Bel probe (6-FAM-CCGGAACTGCGCTTTCA-TAMRASEQ ID NO:31), DAI fwd (GGATACTATCACGGGCGGTASEQ ID NO:32), DAI rev (GGCACGCCATACAATTCTTASEQ ID NO:33). RNA levels were measured on a LightCycler 480 (Roche) using a standard curve generated from vRNA isolates of high titer stocks.
[0171] Infectivity of the virus produced was assessed using a TCID.sub.50 assay of selected time points on PAMs isolated from wild type surplus research animals.
[0172] mRNA and Protein Levels of Heme Oxygenase 1 Upon Hb-Hp Stimulation of PMMs
[0173] PBMCs were seeded seven days prior to analysis and differentiated by CSF1 stimulation to yield PMMs. Hemoglobin (Hb, Sigma-Aldrich, AO, H0267) and Haptoglobin (Hp, Sigma Aldrich, Phenotype 2-2, H9762) were mixed in a 1:1 wt/wt ratio in PBS for 15 min on a vertical roller before experimentation. PMMs were incubated with 100 g/ml Hb-Hp in cRPMI for 24 h at 37 C. Cells were harvested by scraping with a rubber policeman. RNA was isolated from 1E6 cells using the RNeasy Mini Kit (Qiagen), according to the manufacturer's instructions, including an on-column DNase digestion. RNA levels were measured using the GoTaq 1-Step RT-qPCR system (Promega) according to the manufacturers' instructions on a LightCycler 480 (Roche). mRNA levels of heme oxygenase 1 (HO-1) were quantified using primers P0239 (TACATGGGTGACCTGTCTGGSEQ ID NO:34) and P0240 (ACAGCTGCTTGAACTTGGTGSEQ ID NO:35) and reference mRNA levels of -actin using primers P0081 and P0082. For analysis of protein levels of HO-1 cells were collected by centrifugation at 300 rcf for 10 min. The pellet was re-suspended in Laemmli sample buffer containing 100 mM DTT, boiled for 10 min at 95 C. and subjected to electrophoresis on 12% acrylamide (Bio-Rad) gels. After transfer to a nitrocellulose membrane (Amersham), the presence of cellular proteins was probed with antibodies against HO-1 (mouse mAb, abcam, ab13248, 1:250), and calmodulin (rabbit mAb, abcam, ab45689, 1:1000). The blot was subsequently incubated with HRP-labelled goat anti-rabbit antibody (DAKO, PI-1000) at 1:5000. Binding of HRP-labelled antibodies was visualized using the Pierce ECL Western Blotting Substrate (Thermo Fisher), according to the manufacturer's instructions.
[0174] Quantification and Visualization of Hemoglobin-Haptoglobin Uptake
[0175] PBMCs were seeded seven days prior to analysis and differentiated by CSF1 stimulation to yield PMMs. For fluorescence microscopy, cells were seeded on glass cover slips. Hemoglobin (Sigma-Aldrich, AO, H0267) was labeled with Alexa Fluor 488 (AF-488) using a protein labelling kit (Molecular Probes) according to the manufacturer's instructions. Hb.sub.AF488 and Hp were mixed in a 1:1 wt/wt ratio in PBS for 15 min on a vertical roller before experimentation. PMMs were incubated with 10 g/ml Hb.sub.AF488-Hp in cRPMI for 30 min at 37 C.
[0176] For quantification by FACS the cells were collected with a rubber policeman and washed three times with Ca.sup.2+/Mg.sup.2+-free PBS to remove surface bound Hb.sub.AF488-Hp as described previously [60]. Cells were fixed in 4% (wt/v) formaldehyde (Sigma-Aldrich) in PBS (Gibco) for 15 min at RT, washed with PBS, and subsequently permeabilized in PBS containing 0.1% Triton-X-100 (Alfa Aesar) for 10 min. Cells were stained with mouse anti pig CD163 antibody (AbD Serotec, MCA2311PE, 1:50) as described above then washed three times with PBS and re-suspended in FACS buffer. Gene expression determined by antibody labelling was assessed by analysis on a FACS Calibur (Becton Dickinson) using FlowJo software.
[0177] For immunofluorescence imaging cells were washed three times with Ca.sup.2+/Me-free PBS and fixed in 4% formaldehyde (Sigma-Aldrich) in PBS (Gibco) for 15 min at RT, washed with PBS, then permeabilized in PBS containing 0.1% Triton-X-100 (Alfa Aesar) for 10 min. Cells were washed with PBS and incubated with antibody against CD163 (rabbit pAb, abcam, ab87099, 5 g/ml) in blocking buffer (PBS, 3% FBS) for 1 h, washed, and incubated with secondary goat anti-rabbit AF594 antibody (A11037, 1:100), AF647 phalloidin (A22287, 1:100), and DAPI (1:10,000; all Life Technologies). The samples were analyzed using a confocal laser-scanning microscope (Zeiss LSM-710).
[0178] Immunofluorescence Analysis of RTC Formation in Infected PAMs
[0179] PAMs were seeded onto coverslips one day prior to infection. Cells were inoculated at MOI=2 of the respective virus strain (PRRSV H2, DAI, or SU1-Bel) in cRPMI for 3 h at 37 C. The inoculum was replaced by warm cRPMI. At 19 hpi cells were fixed in 4% formaldehyde (Sigma-Aldrich) in PBS (Gibco) for 15 min at RT, washed with PBS, and permeabilized as described above. Cells were washed with PBS and incubated with antibody against PRRSV nsp2 (A gift from Ying Fang, South Dakota State University, [74], 1:400) in blocking buffer for 1 h, washed, and incubated with secondary goat anti-mouse AF488 antibody (A11029, 1:100), AF568 phalloidin (A12380, 1:100), and DAPI (1:10,000; all Life Technologies). The samples were analyzed using a confocal laser-scanning microscope (Zeiss LSM-710).
[0180] Results
[0181] Generation of Live CD163 SRCR5 Deletion Pigs by CRISPR/Cas9 Editing in Zygotes
[0182] The CD163 gene is not correctly represented in the current pig reference genome sequence (Sscrofa10.2) [38]. Through targeted sequencing we have established a detailed model of the porcine CD163 locus (unpublished results L. Zen/A. Archibald/T. Ait-Ali)the genomic sequence of the CD163 gene is set out below as SEQ ID NO:1. Briefly, CD163 is encoded by 16 exons with exons 2-13 predicted to encode the SRCR domains of the protein [39]. Interestingly, SRCR5 is predicted to be encoded by one single exon, namely exon 7 (
[0183] All four sequences were assessed in vitro for cutting efficiency by transfection of porcine kidney PK15 cells with a plasmid based on px458 [42] encoding the complete single guide sequence (sgRNA), driven by the hU6 promoter, and a CAG promoter driving NLS-Cas9-2A-GFP. Transfected cells were isolated by fluorescence activated cell sorting (FACS) for GFP and cutting efficiency at the target site was assessed using a Cell surveyor assay. Three out of four guides were shown to direct cutting of DNA as anticipated (2 upstream and one downstream of exon 7). Following double transfection assay and subsequent PCR analysis it was found that the combination of guides SL26 and 51_28 effectively generated the exon 7 deletion in the CD163 gene (
[0184] sgRNAs SL26 and SL28 were microinjected together with mRNA encoding the Cas9 nuclease into the cytosol of zygotes. Editing efficiency was assessed in a small number of injected zygotes by in vitro culture to the blastocyst stage, genomic DNA extraction, whole genome amplification and PCR amplification across exon 7. The analysis revealed that two out of 17 blastocysts contained a deletion of the intended size and Sanger sequencing confirmed the deletion of exon 7. Edited blastocyst B2 showed a clean deletion and subsequent re-ligation at the cutting sites of sgSL26 and sgSL28, whilst edited blastocyst B14 showed that in addition to the intended deletion there was also a random insertion of 25 nucleotides at the target site. None of the full length PCR products showed nucleotide mismatches at either cutting site in a T7 endonuclease assay. The editing rate in the blastocysts corresponds to an overall editing rate of 11.7%.
[0185] To generate live pigs, 24-39 zygotes injected with sgSL26, sgSL28, and Cas9 mRNA were transferred into the oviduct of recipient gilts. A total of 32 live piglets were born and genotyping of ear and tail biopsies revealed that four of the piglets had an exon 7 deletion, corresponding to 12.5% of the total. In addition to the intended deletion of exon 7, three out of the four animals showed insertion of new DNA at the target site probably as a consequence of non-homologous end joining repair. Pig 347 showed a 2 bp truncation at the sgSL26 cutting site and a 66 bp insertion between the cutting sites, pig 346 showed a deletion of 304 bp after the cutting site of sgSL26, and pig 310 showed a short 9 bp insertion (having the sequence TCAGTCACT) at the cutting sites. Pig 345 was found to have a precise deletion of exon 7 without insertion or deletion of random nucleotides at the cut sites (
[0186] Genotype and Phenotype of F1 Generation Pigs
[0187] To generate fully homozygous and heterozygous pigs, 310 was mated with 345. This mating yielded a litter of 6 heterozygous, 2 biallelic/homozygous CD163 SRCR5 deletion (SRCR5), and 4 wild type CD163 piglets (
TABLE-US-00006 TABLE 1 Genotypes and growth of assessed F1 animals. Animal ID Gender Birth weight 60 day weight Type 628 male 1.2 kg 25 kg wild type 633 female 1.6 kg 26 kg wild type 627 male 1.6 kg 25 kg heterozygous 634 female 1.3 kg 27 kg heterozygous 629 male 1.4 kg 25 kg biallelic 630 male 1.6 kg 27 kg homozygous
[0188] Animals 627, 628, 629, 630, 633, and 634 were selected for further analysis, representing the various genotypes (wild type, heterozygous, and biallelic/homozygous) and genders. Growth rates of both SRCR5 and heterozygous animals were comparable to wild type animals (Table 1). Blood samples were taken from all six animals at 10 weeks of age and analyzed by a full blood count conducted by the diagnostics laboratory at the Royal (Dick) School of Veterinary Studies, University of Edinburgh. The blood counts of all animals were within reference values (Table 1). Size, stature and other morphological features of SRCR5 and heterozygous pigs were comparable to their wild type siblings (
[0189] At 8 weeks of age, pulmonary alveolar macrophages (PAMs) were isolated from all six animals by bronchoalveolar lavage (BAL). DNA was extracted from the PAMs and analyzed by PCR and Sanger sequencing. The PAM genotype confirmed the results obtained from the ear biopsies; 628 and 633 were wild type, 627 and 633 heterozygous, and 629 and 630 SRCR5, respectively. Sequencing of PCR products confirmed that all editing events had resulted in complete deletion of exon 7. Whilst pigs 627 and 633 had a clean deletion of exon 7 with precise re-ligation at the sgSL26 and sgSL28 cutting sites in one allele, 629 had one allele with a clean deletion and one allele with a 9 bp insertion between the sites, and pig 630 had both alleles with the 9 bp insertion. RNA was extracted from the PAMs, converted into cDNA using oligo(dT) primed reverse transcription, amplified by PCR and analyzed by Sanger sequencing. PCR products spanning exons 4 to 9 showed the expected 315 bp deletion in both heterozygous and SRCR5 animals (
[0190] Pulmonary Alveolar Macrophages of SRCR5 Pigs are Fully Differentiated and Express Macrophage-Specific Surface Proteins
[0191] PAMs isolated by BAL were characterized for the expression of macrophage-specific surface proteins. CD14 and CD16 are not expressed on monocytes but levels increase upon maturation into macrophages. In PAMs CD14 is found at moderate levels, whilst CD16 is strongly expressed [44]. CD14/CD16 staining of the PAMs from the SRCR5, heterozygous, and wild type animals were all within the previously observed and documented levels [45], with difference being observed between the various genotypes (data not shown). CD172a, or also known as SIRP, is expressed at high levels on both monocytes and macrophages [46] and was expressed at high levels in cells from all animals. CD169, described as an attachment factor for PRRSV [29], is not expressed in monocytes but is highly expressed in tissue macrophages [47] and was expressed at expected levels in cells from our animals (data not shown). As in humans, expression of CD163 in pigs is restricted to monocytes and macrophages. CD163 is expressed at high levels in tissue macrophages, but at low levels in blood monocytes and in bone marrow-derived macrophages [48] (porcine macrophage markers are reviewed in [49]). Both the wild type and the SRCR5 deletion CD163 were recognized on the surface of the PAMs (
[0192] SRCR5 Pulmonary Alveolar Macrophages are not Susceptible to Infection with PRRSV Genotype 1
[0193] PRRSV has two different genotypes with distinct geographic distribution, with genotype 1 being found primarily in Europe and Asia and genotype 2 in the Americas and Asia. The two genotypes show differences in both antigenicity and severity of pathology and have >15% genome divergence between them (reviewed in [50]). Genotype 1 can be further divided into three subtypes, based on the ORF7 sequence and geographical distribution, whereby subtype 1 is pan-European whilst subtypes 2 and 3 are currently limited to Eastern Europe [51]. Here we tested all genotype 1 subtypes of PRRSV, represented by subtype 1 strain H2 (PRRSV H2) [52], subtype 2 strain DAI (PRRSV DAI) [53], and subtype 3 strain SU1-Bel (PRRSV SU1-Bel) [54], originally isolated from the UK, Lithuania, and Belarus, respectively.
[0194] PAMs were infected at an MOI=1 in a single-round infection. 19 hours post inoculation (hpi) the cells were harvested and stained with a FITC-labelled antibody against PRRSV-N protein. Infection levels were assessed by FACS analysis. All three virus subtypes resulted in infection levels of 40-60% in wild type and heterozygous animals, with more than 98% of infected cells being classified as CD163 positive. A slightly higher, statistically significant infection was observed in heterozygous animals infected with PRRSV H2 and DAI. The reason for this is unclear, but may reflect either altered CD163 protein expression profile in heterozygous animals or other, as yet unidentified, genetic properties. By contrast, cells from both SRCR5 animals (629 and 630) were found to be highly resistant to infection in this assay (
[0195] Peripheral Blood Monocytes from SRCR5 Pigs Differentiate into CD163-Expressing Macrophages Upon CSF1-Induction and Express Macrophage-Specific Markers
[0196] To assess the differentiation potential of monocytes into CD163-expressing macrophages we isolated peripheral blood monocytes (PBMCs) from whole blood then were differentiated them into macrophages by CSF1-induction for seven days. Expression of macrophage specific markers was assessed by immunofluorescence labelling and FACS analysis. CD14 and CD16 levels are clear indicators of the differentiation of peripheral blood monocytes with levels of both increasing significantly upon differentiation [44,46]. In addition to CD172a, CD169, and CD163, whose roles as macrophage markers are discussed above, we included a PBMC differentiation marker, SWC9, also known as CD203a, and the putative PRRSV attachment factor CD151 [55,56].
[0197] CD14/CD16 staining of the PMBC-derived macrophages (PMMs) from the SRCR5, heterozygous, and wild type animals were all within the previously observed and documented levels, with no difference being observed between the genotypes (
[0198] SRCR5 Peripheral Blood Monocyte-Derived Macrophages Still Function as CD163-Dependent Hemoglobin-Haptoglobin Scavengers.
[0199] In addition to its contribution to PRRSV susceptibility, CD163 has been described to have a variety of important biological functions. CD163 is an erythroblast binding factor, enhancing the survival, proliferation and differentiation of immature erythroblasts, through association with SRCR domain 2 and CD163 expressing macrophages also clear senescent and malformed erythroblasts. SRCR domain 3 plays a crucial role as a haemoglobin (Hb)-haptoglobin (Hp) scavenger receptor. Free Hb is oxidative and toxic; once complexed with Hp is cleared through binding to SRCR3 on the surface of macrophages and subsequent endocytosis. This prevents oxidative damage, maintains homeostasis, and aids the recycling of iron. CD163 expressing macrophages were also found to be involved in the clearance of a cytokine named TNF-like weak inducer of apoptosis (TWEAK), with all SRCRs apart from SRCR5 being involved in this process [57]. Soluble CD163 can be found at a high concentration in blood plasma but its function in this niche is still unknown (reviewed in [34,58]). Maintaining these biological functions is likely to be important to the production of healthy, genetically edited animals. Interestingly, none of the biological functions assigned to CD163 have yet been linked to SRCR5. In order to confirm whether SRCR5 macrophages were still able to take up Hb-Hp complexes we performed a variety of in vitro experiments. Hb-Hp complex uptake in PMMs in vitro has been investigated extensively in the past, with PMMs able to take up both Hb and Hb-Hp complexes in a CD163-dependent manner and the inducible form of heme oxygenase, heme oxygenase 1 (HO-1), being upregulated upon Hb-Hp uptake [59,60].
[0200] PBMCs were differentiated into PMMs by CSF1-induction for seven days, following which PMMs were incubated in the presence of Hb-Hp for 24 h to stimulate HO-1 upregulation. The HO-1 mRNA upregulation, assessed by RT-qPCR, increased 2- to 6-fold in the PMMs from all animals (
[0201] Peripheral Blood Monocyte-Derived Macrophages from SRCR5 Animals are not Susceptible to Infection with PRRSV Genotype 1
[0202] To explore the possibility that PMMs could be a suitable alternative to monitor PRRSV infection and investigate whether SRCR5 PMMs, like PAMs, are resistant to PRRSV infection we tested infectivity with all three genotype 1 subtypes of PRRSV, represented by the strains described above.
[0203] PMMs were infected at an MOI=1 in a single-round infection. 19 hpi cells were harvested and stained with a FITC-labelled antibody against PRRSV-N protein, with infection levels assessed by FACS. All three subtypes showed infection levels of 35-80% in wild type and heterozygous animals. As observed in PAMs, a slightly higher, statistically significant infection was observed in heterozygous animals infected with PRRSV H2, whilst no significant infection was observed in the cells from SRCR5 animals (
[0204] The Arrest in Infection of SRCR5 Pulmonary Alveolar Macrophages (PAMs) Occurs Prior to the Formation of the Replication/Transcription Complex.
[0205] In the porcine kidney cell line PK-15, lacking CD163 expression, transfected with the PRRSV attachment factor CD169 the virus was found to be internalized but not to undergo uncoating [36]. This indicates that CD163, in a close interplay with attachment/internalization factors, plays a major role in the entry process of PRRSV. To assess whether the infection process in SRCR5 macrophages is arrested prior to replication we inoculated PAM cells with all three PRRSV genotype 1 subtypes, represented by the strains described above, at MOI=2. The inoculum was removed 3 hpi and infection allowed to continue up to 22 hpi. Cells were fixed and stained for the replication-transcription complexes (RTC) formed by PRRSV upon replication initiation. PRRSV nsp2 protein, involved in the formation of double membrane vesicles (reviewed in [61]) was chosen as a representative marker for the RTC. The cells were permeabilized and stained for the presence of PRRSV nsp2. We found that macrophages from both the wild type and the heterozygous animals infected with PRRSV formed RTCs, independent of the subtype. However, in the macrophages from SRCR5 animals no RTC formation was observed. This underlines the involvement of CD163 in the entry and uncoating process of PRRSV infection. It also supports the deletion of SRCR5 as an effective method to abrogate PRRSV infection before the virus or viral proteins are amplified (
DISCUSSION
[0206] The results of this study show that live pigs carrying a CD163 SRCR5 deletion are healthy and maintain the main biological functions of the protein, whilst the deletion renders target cells of PRRSV resistant to infection with the virus. By using two sgRNAs flanking exon 7 of CD163 in CRISPR/Cas9 editing in zygotes we achieved excision of said exon from the genome of pigs yielding a CD163 SRCR5 genotype. The expression of the truncated gene was confirmed by PCR of cDNA, RT-qPCR and western blotting against CD163. Macrophages isolated from the lungs of wild type CD163, heterozygous and SRCR5 animals showed full differentiation and expression of macrophage surface markers characteristic of macrophages isolated from the pulmonary alveolar areas. PAMs are the primary target cells of PRRSV infection. Assessing infection of PAMs from the different genotype animals in both high dose, single-round infections and low dose, multiple-round infections showed PAMs from SRCR5 pigs to be resistant to infection in vitro. The differentiation ability of cells of the monocytes/macrophages lineage from genetically edited CD163 animals was further confirmed by isolation and differentiation of PBMCs. PMMs from SRCR5 pigs were also shown to be resistant to PRRSV infection. PMMs have a crucial biological role, serving as scavengers for Hb-Hp complexes in the blood. Using uptake experiments of fluorescently labelled Hb-Hp complexes as well as gene upregulation assays to monitor the increase of HO-1 upon Hb-Hp stimulation we confirmed that this important biological function is maintained in macrophages isolated from SRCR5 animals.
[0207] Using CRISPR/Cas9 editing in zygotes generated live pigs with exon 7 CD163 deletions. Editing efficiency was highly variable, dependent on surgery days, in both in vitro cultivated blastocysts as well as born animals, whereby it needs to be considered that overall numbers are low. The reagents used on the various surgery days were the same and both insemination and surgery times were kept consistent. However, there are many elements in the genome editing process that rely on highly skilled personnel and technical reproducibility. Recent developments in nucleic acid delivery methods for genome editing in zygotes may offer possible solutions to standardize the genome editing process. Various groups recently reported successful genome editing by in vitro electroporation of CRISPR/Cas9 regents into zygotes isolated from mice and rats without removing the zona pellucida [62-64]. Using electroporation to deliver genome editing reagents in vivo Takahasi et al. showed high success with this method in mouse embryos after 1.6 days of gestation [65]. Use of in vitro electroporation could standardize the injection process and reduce the requirement for highly trained personnel. As an alternative, in vivo electroporation would remove both the requirement for donor animals and the long handling process of zygotes prior to re-implantation, however this procedure has currently only been developed for mice (reviewed in [66]). Three out of four of the founder animals were found to be edited in a mosaic pattern. In animal 310 the mosaicism seems to result from a delayed activity of the CRISPR/Cas9 complex, resulting in an edit of one allele in a single cells at the 4- or 8-cell stage. In animals 345 and 347 an initial editing event appears to occur in one allele at the 1-cell stage and a second editing event, modifying the second allele in one of the cells at the 2-cell stage, resulting in homozygous/heterozygous mosaic animals. Mosaicism has been observed in various studies employing injection of genome editors into porcine zygotes [67-69]. Asymmetric spreading of introduced mRNA seems unlikely following results of Sato et al., who performed in vitro EGFP mRNA injections using parthenogenetically activated porcine oocytes, whereby a relatively homogenous fluorescence pattern could be observed [69]. Rather, mosaicism seems to result from Cas9 protein/sgRNA complexes remaining active throughout several cell divisions or delayed mRNA expression possibly triggered by cell division. The former theory is supported by the genotype of 345 and 347, which very likely have developed from an initial editing step in one allele at the one cell stage and editing of the second allele in one of the 2-cell or 4-cell stage cells. To generate more biallelic animals by direct injection of zygotes, a more active reagent set may be beneficial. Recent studies indicate that injection of Cas9/sgRNA ribonucleoproteins (RNPs) is more efficient than mRNA injection. Also, RNP injection can be combined with in vitro electroporation [70].
[0208] The mating of the F0 generation animals 310 and 345 resulted in wild type, heterozygous and biallelic edited animals. This showed that despite mosaicism both animals are germline heterozygous. None of the offspring showed any adverse effect from the genome editing under standard husbandry conditions. Interestingly, one of the animals, 630, displayed a putative gene conversion event. Based on the mechanism of interallelic gene conversion we assume that a homologous recombination occurred in this animal between one allele showing the edited genotype of 345 and the other allele the edited genotype of 310. The gene conversion appears to have occurred at the zygote stage, rendering 630 homozygous for the genotype of 310 (reviewed in [71]).
[0209] PRRSV shows a very narrow host cell tropism, only infecting specific porcine macrophage subsets. Isolating these cells from our genetically edited animals and their wild type siblings we showed that removal of the CD163 SRCR5 domain results in complete resistance of the macrophages towards PRRSV infection. We further demonstrated that SRCR5 animals are resistant to infection with all European subtypes of genotype 1. This shows that a targeted removal of SRCR5 is sufficient to achieve complete resistance to PRRSV infection in vitro. PRRSV attachment factors CD151 and CD169 are still expressed on SRCR5 macrophages underlining that these proteins are not sufficient for PRRSV infection. PRRSV infection on macrophages from the SRCR5 animals was halted before the formation of replication transcription complexes proving CD163 to be involved in the entry or uncoating stage of the PRRSV replication cycle. The SRCR5 macrophages will provide a new tool to study this process in detail in a true-to-life system.
[0210] As there could be a genetic variation of CD163 within the Suidae superfamily we performed an in vitro control experiment to assess the susceptibility of warthog (Phacocherus africanus) PMMs to PRRSV infection. Interestingly, warthog PMMs were found to be as susceptible to infection with all PRRSV genotype 1 subtypes as the pig PMMs. They all replicated the virus at a similar rate and to comparable titers (data not shown). This indicates that genetic variation of CD163 within the Suidae superfamily is probably very limited and PRRSV infection may be widespread. This also shows that the virus poses a threat to African pig breeding countries. The SRCR5 animals have several advantages over previously described genome edited animals resistant to PRRSV infection. Whitworth et al. generated animals with a premature stop codon in exon 3 of the CD163 gene, resulting in an ablation of CD163 expression [37]. In contrast to this we have demonstrated that specific application of genome editing tools in vivo can be used to efficiently generate animals with precise deletion of exon 7 of CD163, and that these animals retain expression of the remainder of the CD163 protein on the surface of specific differentiated macrophages in a native conformation. We further showed that the macrophages from these SRCR5 animals retain full differentiation potential, both in PAMs as well as PBMCs stimulated to differentiate by CSF-1 addition, and that macrophages from edited animals retain the ability to perform crucial biological functions associated with CD163 expression, such as hemoglobin/haptoglobin uptake. Overall, this study demonstrates that it is possible to utilize a targeted genome editing approach to render swine resistant to PRRSV infection, whilst retaining biological function of the targeted gene. Introduction of CD163 SRCR5 deletion animals in pig breeding could significantly reduce the economic losses associated with PRRSV infection.
[0211] Inactivation of Splice Acceptor Site in Intron 6
[0212] An alternative strategy to delete the SRCR5 domain of CD163 is to inactivate the splice acceptor site located at the 5 end of exon 7 in the CD163 gene.
[0213] Inactivation of the splice acceptor site in exon 7 can be achieved in a number of ways, and two suitable strategies are discussed briefly below, one involving creating a double stranded cut followed by non-homologous end joining (NHEJ), and the other using homology directed repair (HDR). The first option suitably uses a single guide RNA and NHEJ by the target cell. Using the second approach, HDR, a template is provided which is used by the cell's double strand break repair machinery to introduce a sequence modification. Thereby some nucleotides will be replaced in order to destroy the splice acceptor site in a targeted manner, whilst introducing a restriction site (in the example NcoI) which allows for convenient confirmation that the HDR event has taken place.
[0214] Suitable methodologies for achieving editing events in pig embryos and generation of animals from edited embryos are discussed above, and are also extensively discussed in the literature, and thus for conciseness they will not be repeated here.
[0215] In the case of CRISPR/Cas9 mediated gene editing, suitable guide RNA sequences to target the splice acceptor site are as follows:
TABLE-US-00007 sgRNA1: (SEQIDNO:12) AACCAGCCTGGGTTTCCTGT sgRNA2: (SEQIDNO:13) CAACCAGCCTGGGTTTCCTG
[0216] These two guide sequences result in the induction of double stranded cut sites at the following sequences at the 5 end of exon 7 by Cas9:
TABLE-US-00008 (SEQIDNO:14) ACA|GGAAACCCAGGCTGGTT-usingsgRNA1 (SEQIDNO:15) CAG|GAAACCCAGGCTGGTTG-usingsgRNA2
[0217] Approach 1NHEJ
[0218] An RNP complex of sgRNA1 or 2 with Cas9 binds to the target site in the CD163 gene and causes a double-strand break. Where the break occurs NHEJ events arise, commonly resulting in and insertion of deletion event. It is highly likely that either insertion or deletion events will result in the inactivation of the intron 6 splice acceptor site. It is thereafter simply a matter of identifying embryos having the requisite disabling of the splice acceptor site.
[0219] Approach 2HDR
[0220] Again, an RNP complex of sgRNA1 or 2 with Cas9 binds to the target site in the CD163 gene and causes a double-strand break. In this case, however, an HDR template is provided, for example a single or double stranded DNA molecule, which comprises a sequence which results in a change of the sequence in the CD163 gene from:
TABLE-US-00009 (SEQIDNO:3) AATGCTATTTTTCAGCCCACAGGAAACCCAGG to: (SEQIDNO:4) AATGCTATTTTTCgGCCatggGGAAACCCAGG
[0221] A suitable HDR template has the following sequence: GAAGGAAAATATTGGAATCATATTCTCCCTCACCGAAATGCTATTTTTCgGCCatggGGAAAC CCAGGCTGGTTGGAGGGGACATTCCCTGCTCTGGTC (SEQ ID NO:16lower case letters show the changes made compared to the unaltered sequence).
[0222] The converted sequence in the context of CD163 results in inactivation of the splice acceptor site and the introduction of the NcoI restriction site. The presence of the NcoI site facilitates identification of embryos/animals in which the desired HDR edit has been achieved.
[0223] Further Experimental Work
[0224] Genome Editing in Zygotes for SRCR5 CD163 Pigs and Breeding for a Genotypically Uniform F2 Generation
[0225] Founder generation F0 animals carrying a deletion of exon 7 in the CD163 gene, which encodes the scavenger receptor cysteine-rich domain 5 (SRCR5) of the protein, were generated by CRISPR/Cas9 gene editing as described above (see also 75). Therefore, zygotes were microinjected with two guide RNAs, sgSL26 and sgSL28, in combination with Cas9 mRNA to achieve CRISPR/Cas9-mediated double-strand breaks (DSBs) flanking exon 7. Subsequent DSB repair lead to a deletion of exon 7 (
[0226] As previously described, SRCR5 animals express the SRCR5 CD163 mRNA and protein at equivalent levels to wildtype siblings. Furthermore, native-structure SRCR5 CD163 is recognized on the surface of pulmonary alveolar macrophages (PAMs) by a respective antibody. We have further analyzed whether template-based protein structure prediction using RaptorX confirms these findings towards proper folding of the subdomains and the complete SRCR5 CD163 protein (39). As seen in
[0227] Previously, we have shown that PAMs and in vitro differentiated peripheral blood monocytes are resistant to infection with both, porcine reproductive and respiratory syndrome virus 1 (PRRSV-1) and PRRSV-2. Now, we aimed to confirm the in vitro results by assessing resistance towards PRRSV-1 infection in vivo. Therefore, we selected four homozygous SRCR5 F2 animals and four wildtype siblings and semi-siblings. The animals were co-housed from weaning. At 6 weeks of age they were transferred to the specific pathogen-free (SPF) unit and co-housed for the duration of the challenge (
[0228] SRCR5 Pigs Show Normal Whole Blood Counts and Soluble CD163 Serum Levels
[0229] Prior to being moved to the SPF unit blood samples were taken from all eight pigs and analyzed by a full blood count conducted by the diagnostics laboratory at the Royal (Dick) School of Veterinary Studies, University of Edinburgh. The blood counts of all animals were within reference values indicating good general health and the absence of infection or inflammation. Furthermore, the hemoglobin levels of all animals were within reference values, indicating normal function of the hemoglobin/haptoglobin scavenging activity of CD163 (Table 2).
[0230] Serum was collected from all animals prior to movement to the SPF unit and on day 0 prior to challenge with PRRSV-1. The soluble CD163 (sCD163) serum levels were assessed using a commercially available enzyme-linked immunosorbent assay (ELISA) recognizing soluble porcine CD163. Serum CD163 levels were found to be 463.568.99 ng/ml in SRCR5 pigs and 433.269.57 ng/ml in wildtype pigs (
TABLE-US-00010 TABLE 2 Whole blood count results of SRCR5 & wildtype piglets at 5.5 weeks of age. 4-7 SRCR5, 8-11 wildtype pigs. Ref Indicator 4 5 6 7 8 9 10 11 Unit Values WBC 22.5 24 14 15.1 12.4 19.6 26.1 14.4 10.sup.9/l 11-22 Neutrophils 5.85 4.8 4.62 5.889 4.34 7.252 7.83 4.32 10.sup.9/l 2-15 (segmented) Neutrophils 26 20 33 39 35 37 30 30 % 20-70 (segmented) Neutrophils 0 0 0 0 0 0 0 0 10.sup.9/l 0-0.8 (non- segmented) Neutrophils 0 0 0 0 0 0 0 0 % 0-4 (non- segmented) Lymphocytes 15.3 18.72 8.82 8.305 7.564 11.76 16.182 9.36 10.sup.9/l 3.8-16.5 Lymphocytes 68 78 63 55 61 60 62 65 % 35-75 Monocytes 0.675 0.48 0.42 0.755 0.496 0.588 1.044 0.576 10.sup.9/l 0-1 Monocytes 3 2 3 5 4 3 4 4 % 0-10 Eosinophils 0.675 0 0 0.151 0 0 1.044 0.144 10.sup.9/l 0-1.5 Eosinophils 3 0 0 1 0 0 4 1 % 0-15 Basophils 0 0 0.14 0 0 0 0 0 10.sup.9/l 0-0.5 Basophils 0 0 1 0 0 0 0 0 % 0-3 RBC 6.03 6.64 6.99 6.58 6.3 6.53 7.52 6.97 10.sup.12/l 5-9 PCV/ 0.384 0.391 0.383 0.388 0.382 0.39 0.429 0.421 0.36-0.43 Hematocrit Hb 11.5 11.9 10.9 11.8 11.6 12 13.8 12.3 g/dl 10-16 MCV 63.7 58.9 54.8 58.9 60.7 59.8 57.1 60.5 fL 50-62 MCHC 29.9 30.4 28.3 30.5 30.3 30.9 32.1 29.1 g/dl 30-36 Platelets 219 230 605 397 483 519 219 606 120-720 RDW 20.9 23.1 28.9 20.6 21 18 17 22.6
[0231] SRCR5 Pigs Show No Signs of PRRSV-1 Infection
[0232] At 7-8 weeks of age the pigs were inoculated intranasally with the PRRSV-1, subtype 2 strain BOR-57 (77). Generally, infections with PRRSV-1, subtype2 strains are associated with mild respiratory symptoms, elevated body temperature, extensive lung pathology and high viremia. The challenge was conducted for a period of 14 days following inoculation at day 0 and day 1 with 5E6 TCID.sub.50 of the virus each. Rectal temperature, respiratory and other potential symptoms, and demeanor were recorded each day and serum samples were collected on day 0 (prior to challenge), 3, 7, 10, and 14 (prior to euthanasia). Weights were recorded on day 0, 7, and 14 (prior to euthanasia). People conducting the challenge and analyzing the pathology were blind to the genotype of the animals.
[0233] The rectal temperature showed significant elevations on days 6-9 of the challenge in the wildtype animals, whereas no body temperature increase was observed in the SRCR5 animals (
[0234] Overall, no signs of infection were detected in SRCR5 animals despite the high inoculation volume and exposure to infected and shedding wildtype animals showing that SRCR5 animals are resistant to PRRSV-1 infection, confirming the results found in vitro with both PRRSV-1 and PRRSV-2.
[0235] SRCR5 Pigs Show No Cytokine Response to PRRSV-1 Infection and Generally Normal Cytokine Levels
[0236] To assess the inflammation and infection response following PRRSV-1 infection a panel of 20 cytokines were analyzed towards their level in the serum of the pigs. Therefore, we used commercial quantitative antibody arrays and serum samples collected on day 0 (prior to challenge), 3, 7, 10, and 14 of the challenge. Overall, cytokine levels on day 0, considered a baseline, were similar between SRCR5 and wildtype pigs. The monokine induced by gamma interferon (MIG, also known as CXCL9) was found to show consistently higher levels in wildtype pigs until day 14, when no significant difference was detected anymore. MIG is a T-cell chemoattractant to inflammation sites and involved in repair of tissue damage. In wildtype animals MIG was strongly upregulated on days 7 and 10 of the challenge (80) (
[0237] Otherwise we could see a sequence of cytokine response, with early increase of interferon (IFN) and interleukin-17A (IL-17A), and the interleukin 1 receptor antagonist (IL-1ra) (
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[0322] Nucleic Acid Sequences:
[0323] CD163 Guide Sequences:
TABLE-US-00011 sgSL25 (SEQIDNO:5) TGAAAAATAGCATTTCGGTG CD163genecutlocation: (SEQIDNO:6) CAC|CGAAATGCTATTTTTCA sgSL26 (SEQIDNO:7) GAATCGGCTAAGCCCACTGT CD163genecutlocation: (SEQIDNO:8) GAATCGGCTAAGCCCAC|TGT sgSL27 (SEQIDNO:9) GTCCTCCATTTACTGTAATC CD163genecutlocation: (SEQIDNO:10) GAT|TACAGTAAATGGAGGAC sgSL28 (SEQIDNO:11) CCCATGCCATGAAGAGGGTA CD163genecutlocation: (SEQIDNO:11) CCCATGCCATGAAGAGG|GTA Cutlocationsareshownbythe|symbol.
[0324] Genomic Sequence of the CD163 Gene Locus in Large White Pigs (SEQ ID NO 1)
[0325] Bold=exons
[0326] Single underlined and dashed underline=splice acceptor site predictions
[0327] Double underlined=splice donor site predictions
[0328] sgRNA binding locations and cutting sites are indicated in lowercase italics, and the particular sgRNA binding to the sites is also indicated.
TABLE-US-00012 1 TCTTCATCCTATTAGAGACACTGCTATACAGCAGAAATTGACACAACATTGTAAATCAAC 61 TATACTTTAATAAAATAAAAAAAAGAAATACAAGTGCTTTCTACAGACAATCTGCACAAG 121 TTATTTGTTAGACATATTTGATTATAGAATTAATATTAAAAGGGGTTATAACAATCAAGC 181 ATTGATAATTTAATTATGTTTGCCTATTTTACTTTAGTTTTTTGACATAACTGTGTAACT 241 ATTGCGATTTTTTTATTCCTAATGTAATTAGTTCAAAACAAAGTGCAGAAATTTAAAATA 301 TTCAATTCAACAACAGTATATAAGTCAATATTCCCCCCTTAAATTTTTACAAATCTTTAG 361 GGAGTGTTTCTCAATTTCTCAATTTCTTTGGTTGTTTCATGTCCCATATGGAAGAAAACA 421 TGGGTGTGAAAGGGAAGCTTACTCTTTTGATTACTTCCCTTTTCTGGTTGACTCCACCTC 481 CATTATGAAGCCTTTCTGTATTTTTGTGGAAGTGAAATGATTTTTAGAATTCTTAGTGGT 541 TCTCTTCTTCAGGAGAACATTTCTAGGTAATAATACAAGAAGATTTAAATGGCATAAAAC 601 CTTGGAATGGACAAACTCAGAATGGTGCTACATGAAAACTCTGGATCTGCAGGTAAAATC 661 TTCTCATTTATTCTATATTTACCTTTTAATAGAGTGTAGCAATATTCCGACAGTCAATCA 721 ATCTGATTTAATAGTGATTGGCATCTGGAGAAGAAGTAACAGGGAAAAGGCAATAAGCTT 781 ATAAGGGGAACTTTTATCTTCCATAGAATCAAAATTGAAGACGTGACTAGAAGAAGGATT 841 AGATTTGGCATCAGTTTTGTAAAATTGCTGAGGTGAAATTAAGTAAGGGATGAAAATTAA 901 CTAAATTGTGTTGAGTATGAAACTAGTAGTTGTTAGAAAAGATAGAACATGAAGGAATGA 961 ATATTGATTGAAAGTTGATGACCTAGAGGACATTTAGACTAACACCTCTGAGTGTCAAAG 1021 TCTAATTTATGATTTACATCGATGCGTTAAACTCATTTAACATTCTTACTTTTTTCCCCT 1081 CAAGCATTTAAGCTGAAGTATAACATTTCACATGAAAGCCTGGATTATAAATGCACAGTT 1141 CAGTGACCTATCTCAGAGGAGTGACTGCCATAGCATTTTTTTTGTCTTTTTGCCTTCAGA 1201 GCCACAGCAACGCGGGATCCGAAGCCGCGTCTGCGACCCACACCACAGCTCACGGCAATG 1261 CCGGATCTTTAACCCACTGAGCGAGGCCGGGGATCGAACCCGCAGTCTCATGGTTCCTAG 1321 TAGGATTCGTTAACCACTGCGCCACGACGGGAACTCCTACCATAGCATTTTTACTTTTAA 1381 GTTACTGTTGGTTTAGAGTAAGAAGGAGAAATGAGAGTGATGGAGCGTTTGCTATATTTG 1441 GAGACAAGGTCCTATATTGGAGGTTCTCAAATATAAATTTTGTCGCTTTTTCCTCCAATG 1501 TATTGTTCAACTACTATTTAGCAGGCCACTGTGCCAGGTACTGGTGAAACTGGTGAACAT 1561 GATAGATGTAATTCATTCCCTCATGGAACTTTCCATCTAACAATGTGGATCAGGTAGGCT 1621 TGGAGATGAGAATGCCAGTGGTTGACTATGACTCTGTGGCTGAAGGGAGAGCTACTCACT 1681 TCGTAGTTTCATCAATGTCTTTTTGGTTTTCCAGGTTTTAAGCCCTGCTCTTGCAATTCT 1741 TTTCCCTTCTCCAACTTTCTTCTAATTTCTCACCCCTAGGATGCCTATAAACATGAGTAT 1801 TTTCAAAGCTACTTCACTGAGGTTATATGATCCTCGTGTGAATTTTTCCTGCCTGCCTTG 1861 CCATTTAGAAGGAAGTGTTTCCTGGAATTTCCATTGTGGCTTGGTGGTTAAAGACCCTGC 1921 ATTGTCTCTGTGAGGATGTGGGTTCAATCTCTGGCCTCATTCAGTGAGTGGGTTAAGGAT 1981 CTGGTGTCGCTGCAAGCTGTGGCTAAGATCCCACATTGCCATGGCTGTGGTGTAGACTGG 2041 CACCTGGAGCTCTGATTTGACCACAATCCTAGGAACTTCAGATGTTGCCATAAAAAGAAA 2101 AAAAAAGTTAGGAAGGGTTTTCTGTCTTGTTTTGACCTTTGTTAATCTCAAACCTTTGGA 2161 ACCATCTCTCCTCCAAAACCTCCTTTGGGTAAGACTGTATGTTTGCCCTCTCTCTTCTTT 2221 TCGCAGACTTTAGAAGATGTTCTGCCCATTTAAGTTCCTTCACTTTTGCTGTAGTCGCTG 2281 TTCTCAGTGCCTGCTTGGTCACTAGTTCTCTTGGTGAGTACTTTGACAAATTTACTTGTA 2341 ACCTAGCCCACTGTGACAAGAAACACTGAAAAGCAAATAATTCTCCTGAAGTCTAGATAG 2401 CATCTAAAAACATGCTTCATGGTTTCAAAGGATCAGATATTAAAAACCCCAAATAGGTAC 2461 AGAACCATGTGGCTCTCTCCCCCCAAACAAATAAAACGTTAGCATGGTTTTCAAAAAAAT 2521 AAAATAACCTTCACAGGAAAAATGGATTTTACTTAAGATTTGAAATAATATCTAACTAAA 2581 AAATAGGGAATAATGCAGAAGAGGAGAAACCTCAGAATTGTTGGGATGAAGGAATTTTTA 2641 GTAACACTAAAAATTCAAGTGCCAAAATTTGTCTAAAATTGTATTCAGGGAAGCCAGATA 2701 TATATCAGTGAAATCGCCAGTTCCTATATTAGCTAAAATAATCACAAGGCTGTAGCAGAG 2761 ACAGTTCAGAGAGAGGTGGAGATGAGATTTTTTTTTTTTAAGTATAATTGATTTACAATG 2821 TTGTGGCAATTTCTGTTGTATAGCAAGAGATAGAATTATTTTATGGTGGAAGATAATAGA 2881 AAAATATATCCATATCAATTTCCATTTGAGTAGATAAATTTCAATTAGAGTTCAACTAGC 2941 AATTAGTAGTTTTGCATACATGGTGAAATATATTCATGGTATTTTGCATATATGTGTGAA 3001 ATAGGTACTAAATTCCTCATAACTGTTCTTTTTAGTCTCACCATCAGCCTCTACTGATCT 3061 TAGGATTTTGGAGAAACATACATAGTTCATCCCTATAAAATGCCATAAAATCTCATTTTT 3121 ACATTAAACCATCCAAGAGATTATATAAATTGACCTTATAAAGAATATCAGCCATAAAAT 3181 AAAGGTATCATAGTATGGGATTATTTAGCTTTATTGGTTCTATGTCACTGCTTAATTTGA 3241 AACCTGTGATATTGCTGTTTGTTTTTGAACTCCTATGAAATAACATTCTCCCATTGTACC 3301 ATGGATGGGTCCAGAAACATTTCTCAAATCTGGCTTTGAAAAATAAATAAGTAATCTAAA 3361 GAATAATAATTCTCTACTTGCTCTTTGAATCTTGACCAATTGCTGCATTTACCTATTGTT 3421 ACAGGAGGAAAAGACAAGGAGCTGAGGCTAACGGGTGGTGAAAACAAGTGCTCTGGAAGA 3481 GTGGAGGTGAAAGTGCAGGAGGAGTGGGGAACTGTGTGTAATAATGGCTGGGACATGGAT 3541 GTGGTCTCTGTTGTTTGTAGGCAGCTGGGATGTCCAACTGCTATCAAAGCCACTGGATGG 3601 GCTAATTTTAGTGCAGGTTCTGGACGCATTTGGATGGATCATGTTTCTTGTCGAGGGAAT 3661 GAGTCAGCTCTCTGGGACTGCAAACATGATGGATGGGGAAAGCATAACTGTACTCACCAA 3721 CAGGATGCTGGAGTAACCTGCTCAGGTAAGACATACACAAATAAGTCAAGCCTATACATG 3781 AAATGCTTTGTGGGAAAAAATGTATAGATGAGTTAAAAACAAAAAGGAACCAGTTTTCTA 3841 TAAGTCATCTAGTCCATGTATAAAATTACCCAATCCATTACTAAAAGACCACTTCTGGTA 3901 TTTTACACATGACAAAGCCCATATTAAAAAAAAAAAATTCAGAAGAGATTCTGAATGCTA 3961 TAATAAATGAGCAAGTGACTAGCTTCAATTTTATATTAGGTCATTCTACCTTCTACTTCT 4021 ACATGAAAATATCATAATGTCTAAGTTAATTCCTTGTCCCCTTTCCCAATAAAGCACTGC 4081 TTTCATGCACTGGCCTATGAATCATGAACTTTTTGCCCTTTAACTGATGATCAACTTACC 4141 AAATCAAGAAATAAATATTCTTAGCACTGATCCTTTTTTGTTGTTGTTGGAGGAAGAATG 4201 TTTTGCAAAGTAGAATTGCTTTTTTCTGTTTAACAGTGCTATTCATTTCATTTACATGGT 4261 CGTTTTAATTTATAAAACATTTCATAAGTTTCACCTCATATGCCCTTACAATAACTCAGG 4321 AAGTTATATGTTAGACCTTTCTGCTGACAAATCCCAGAGTCATGTTTCTGACCCAGTTCA 4381 GATTCCTTGGCTTCCCATTTCTCTTTGCTCATGTCATTGACCTTTATGCAGCCCTCTTAC 4441 CTCCCACCTTTCTATTACAGACCATCTCCTCCATAGGACTGGTGTTAGAAAGTACTAATC 4501 TCTACCCAGGCATTGTGGTGCAATGTGGGCAGCACAGGCTGGTATCTAGAAAAATGCTGA 4561 AGTGAATTCCAGCTCAGCTGCTCGTTAATACTATTGTTTTAAGTAAGCTGTTCAATCCTT 4621 TGAAATTCACTTTCTGAGCACTCAGTGATATAATAAATGTAGAGTTACTGGTACACTGTC 4681 TGGTATGTAATAGGTGTTAAAAATTAACCTTAGTTTCCTCATGGGTCACTGCTTCTCATT 4741 ACCTAGACAACTCATTTCTCTTTCTTCCTCTTTCTCTTTCTCCATTCTCCTCCTCCTTCT 4801 TCCTCTTCTTCTTGTCTTTTATTGTTATTCATTTTGCTGAGAAAGTTAAGAAATAACAAC 4861 TCTAACCTCTACATCGACCACCTAGAGCAAAGTTAAAAATAATAATAAACCTTGCCAGAC 4921 TCTTACTATAATTGTTGCTGTCTATAGAGTTGACTGTTTAAGTTAAGACATCAGTATAGT 4981 ATTTTTAATTTTTGTGTTTTTTTTTTCATACTTTTACATGAGGATCCTTTATATAAGGAT 5041 GAGTTAAACAAACTTGATTTTTGAAGTTTATACCCCTGAGGCTCAACTGCATAATAATAG 5101 AAAGGGATCCATAGCCTCTCAAGGACTTAACTAGTTTCATGAGTTTTCAGAATCTGAATT 5161 TCTGAGATTCTCCACCCCAATTAAAGCTCAAGCCTCAGAACATATATCCTTCTCTTGGTA 5221 AATTCTATTCTTATCACATGCGTAATAATAAAAAAGAGAGATGTTGGAGACAGATTTTTT 5281 TCCTCACATTCTGTCTCTACTGTTTTCTAGGTGTTTGATTCTGTGTTATTTAACCTCAGT 5341 TTGCTTATCTGTGAAGTAGGGATTATGGTAATAACATATAATGCTTAATGTTGTAAAGAC 5401 TAAAGAAGATAGCATATGTAACACATTTGGAACAGGGAATGCATATTTTGATTGTGAGCT 5461 CTTATTATTATTACCAATCAGCCATAATAAAAATCTTGTTAAGTGGAGGTCTTTGGATTT 5521 CAGAGCTTTTAAAATCTAATTACTTTTTCAAAAAAGAGCTTCTTAGTGTTTTTTTTTTTT 5581 AACCACAAAGTGTTTCTATTTTTTAGGTGTCCCAAAATTTCATTCCAAATATCTTTTTCT 5641 CAGATATTTTAGTCCTCATAGAACACCTAGGGATAGTGTATAGAGAAAATTTTCTTTATT 5701 AAAAAGCTGTTCTTTGCTAAAAATTGTAGCAGGTACTTTTGGGAGGGGGGAAAACTTTGA 5761 TTCAGAAACTGCTAAGACATGGAGTGTTTTGACTAATTTTTCCTCAATTTTTAATGTTTT 5821 TTATACCATAGGGTACTTTTGCAAACTATTATGCATACTTATATATTTTTACTTTTTTCC 5881 TGTCTTTTAACTTCCAAATTCAACTTCAGACAATTATTCATGCACTAAACTGTTGTAGTA 5941 AGAAAGATTAAAATTAAAAAATTAACCATTCAACAAATGACTGGTTTGCCATTTTTACTA 6001 CTTTGTTGTATGAACAATTTTTTTTTCTACAAATGAATACTTTGAGTCTGATTTATCCAT 6061 TCCTACATAAAAGTTTTTACTATATCTTAGTATTGGAAGGAAACAAAACAAAACACAATG 6121 TAAATTTTAATCTATAAATTTTGGGGGGGGGTAAATATACATAGATGAAAGTCTTAACCA 6181 TTAATTAGAGTCAAAAGATTAAAATTCTCCAATATGTGAACTTAGGCTGCATCCAAAATG 6241 AAGCATCATTTTTAAGGACAGCATCAAAAGTGACCAGAGGAATTTTACTTTCTTTCTTTT 6301 TTTTTTTTTTTTTGAATTTTAGTTTCTAAACTCACTTCTGAATAAATACAACTTCTAAAT 6361 TCTCGTCTTTTCTCTACTCTAGATGGATCTGATTTAGAGATGAGGCTGGTGAATGGAGGA 6421 AACCGGTGCTTAGGAAGAATAGAAGTCAAATTTCAAGGACGGTGGGGAACAGTGTGTGAT 6481 GATAACTTCAACATAAATCATGCTTCTGTGGTTTGTAAACAACTTGAATGTGGAAGTGCT 6541 GTCAGTTTCTCTGGTTCAGCTAATTTTGGAGAAGGTTCTGGACCAATCTGGTTTGATGAT 6601 CTTGTATGCAATGGAAATGAGTCAGCTCTCTGGAACTGCAAACATGAAGGATGGGGAAAG 6661 CACAATTGCGATCATGCTGAGGATGCTGGAGTGATTTGCTTAAGTAAGGACTGACCTGGG 6721 TTTGTTCTGTTCTCCATGAGAGGGCAAAAAAAGGGGAGTAAAAGTCTTAAAAGCTCAAAC 6781 TGTTAAAAACATAATGATGATTGCTTCTTTTATCATCTTATTATTATCTAATTTCAGGTC 6841 GAAATTCTAGTACCTGTGCAGTTTTTTACCTTAACTGAAATTAAGATAAATAGGATAGGG 6901 AGGAAGGATGAGCAGTGACATTTAGGTCCAAGTCATGAGGTTAGAAGGAAATGTTCAGAG 6961 AATAGCCCATTCCCTCAGCCCTCAAAGAAAGAAAGAAAGAAAAAGAAAAAAAAAAAGAAA 7021 GCTTAACTAGAAAATTTTGTTCTCTGGATGTTTTAGAGGCAAACCATCCCTTTTATCATT 7081 CCTTACCTACAAAGCCCTTCTCTTTAATCACATTGACCCACCCTTTCCTAAACTATTAGT 7141 TCAAATTCACATAATTGAATGCTTTTAAAACTTGGTTTCCTCTTATAATTATATTTATGT 7201 TGTAAGGAGGCACTGTGTCTTGTCTAGAGACTTTCATGTTCTATGCTTGATTATGGGACA 7261 GGGACATGGCTTTGTCTGCTCCAGGATGTCACTCTCCTTTTTTCACTTGAGCTCCTAGTT 7321 TGAAGAAGACCTAGTAAGTCTTGAACTCCAGGGAGTCTTTAGGAAACTATCCCTAGAGCA 7381 AAACTGTCCCTGAATTCACCCAGTGTCTTTTTTTTTTTTTTCAAATGAAGGAACTTTAGT 7441 TCAAACTAAATTTAAAATAAGGGAATTCTAATTCAGAATACTGGGAAATCCAGGAGATTA 7501 CAATTGGCTTCATGTGTGATTGGATTCAGCACTTCACCAATGTCATCAGGGTTCTGGTTC 7561 TTTTTTTATTTCTTGAATTGGCTTTTTTTTTTTTTTCCTTGTTGAACAATATGACTATCT 7621 ATACTTTGAACCACAAAGAAAGTGATTCCTACAGAAAAGACAGAATGTGTTAGCTGAAGG 7681 AAGGGAATGGGACTTGGGGTAGAAAAAAACACCTTCCGTATTCCTTAACCTATCAAAAAT 7741 TTCTAGGTACCCCTAACTAAAATCCTAATTCAAGCATATTGGAGGAACTTGACAAATCCA 7801 GGAATAATATTATCCGTTATCAAATACATGCACATCATTTACATTTCTCCATGTCTCTGC 7861 TCATGCAGTTCCCGGCCCTAACTCTACCAAAGTATTACTCTCCATCTCCCTCTTTTTTTT 7921 TTTAATGATTTTTATTTTTTCTGTTATGACTGGTTTACAGTGTTCTGTCAATTTTCTACT 7981 GTACAGCAAAGTGACCCAGTCACACATTCATATATACATTCTTTTTCTCACATTATCCTC 8041 CATCAGGCTCCATCACAAGTGACTAGACATAGTTCCCAGAGCTATGCAGCAGGATCTCAT 8101 TGCTGCTCCATTCCAAAGGCAACAGTTCACATCTATTAACCCCAGATTCCCAGTCCACCC 8161 CACTCCCTTCCCCTCCCTCTTGGCAACCACAAGTCTGTTCTCCAAGTTCATGAGTTTATT 8221 TTCTGTGGAAAGTTTTATTTGTGCAGTATGTTAGATTCCAGATATAAGTGCTATCATATG 8281 GTATTTGTCCTTCTCTTTCTGACTGACTTCACAAAGTATGAGAGTCTCTAGTTCCATCCA 8341 TGTTACTGCAAATGGCATTATTAATCTCCATCTTTTTTTGTTCATGTATATGTTACCCAG 8401 ATTCCTTGACTTTTCTACATCATCAAGATATTGTTGATCACTTCTTTGTAGTGATTTCTG 8461 CCCTTCTCTGATGTCCTGTGACACTAGTCTGGATTATTCATTTACCTGAAACCACATGTC 8521 TCTTATAATGTGTATCCCAAATTAAATATGTCTATTGTAATGTGTATCCCAAATTAAATA 8581 TTTATCTTTCTAAAAAAAAAAATTTCTAGGCCCCCAATCAGCATGTTTCTTCTCAGTGTG 8641 TTTTATACATGCTGCAGAATCATAATAGACAGCATAATAGACAGCATAACAAAAACTAAA 8701 AATGCCAGGGGAAAAAAGCAATTTACTGATTACAACATATTACTCAGAATCAAGTTCTGT 8761 TCTTTGAGGAATATTGATTGGGGGAAAATGAAAATAATGATGGGGAGGTCCCTTTTCTCT 8821 TTGCTTTGCTTTTAAACTACGGAAGTAGTCAGAAAGGGGTCAGGAATGTAATATAAACCA 8881 GGTAGTCCTGGTAGGTAACGCAGCCGGAGGCAAAAGTGAGTGTTGAGTATTGAGGCAAAC 8941 TGGAGGGCATGGATACCACCTAGACAGATGCAAATATATATTTAACAGGGAAAAAAGAAC 9001 CAAACAATTTCAACAAAAAACCAAACAATTCCAACAAAATTGGTCCAATAAGCAAACCTC 9061 TAGATAAATTTCAGTCCCTGGATGTTTTGTTAGGAACTCTTCCTACAATGCGTGCTTTCC 9121 ATTCTGAAAAGTCCTATCTACTTGCCTGATCCACTTCTCCTTCCATCCTAAACGATTTTC 9181 AGTGGTAGTATATTACTGTTGTCTCTGTCTCTACTTATATATCTTCCCCTTTTCACTCAC 9241 TCCTCTCAGGTACAGCTCTTCAGTTTGCCCTTATTCTTGTTTCCTTGTCAATGACTTGTT 9301 TTGTGTCCCTCTTACAGATGGAGCAGACCTGAAACTGAGAGTGGTAGATGGAGTCACTGA 9361 ATGTTCAGGAAGATTGGAAGTGAAATTCCAAGGAGAATGGGGAACAATCTGTGATGATGG 9421 CTGGGATAGTGATGATGCCGCTGTGGCATGTAAGCAACTGGGATGTCCAACTGCTGTCAC 9481 TGCCATTGGTCGAGTTAACGCCAGTGAGGGAACTGGACACATTTGGCTTGACAGTGTTTC 9541 TTGCCATGGACACGAGTCTGCTCTCTGGCAGTGTAGACACCATGAATGGGGAAAGCATTA 9601 TTGCAATCATAATGAAGATGCTGGTGTGACATGTTCTGGTAAGTGAAAACAAAACACCGG 9661 AAGGACCTGTGTTCTTCAGGATTAGGAATGGATATGAGATAGGAGAAAAATTGTATCTAA 9721 TATTTTCTTTGTTGGGAATTCTTTTACAGTTGTGACAAATCTTTAACATATTCTTCATTT 9781 GAGTAGTTTGGAGGGTTGTCTGACTGTTTTCTATAATAAATGTCCCAAGTGCTATGAGGT 9841 ACCACATTTCAAATTCTAATTCTACCTGAAGCTCCAAAAAGACAAAATGTTATAGGTCTT 9901 TTCTTTATATCTAATTTGCTTATGGTTTTTAGCCATTGACAATTTTTTTTTTCTTAACTC 9961 TTGAAACTATAATCCTATTTCTAACCAAATTCATGTTCTATACTGGCTCTTCAAAAACCC 10021 AGGAGATGGGAAAGCCAGAATCTCCAGTGTTTCAGCTTCTGGGAAGGAGCAAGTTTTTAA 10081