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Method for improving pig health by targeted inactivation of CD163

A technique for pairing, editing pigs, applied to other methods of inserting foreign genetic material, microbe-based methods, botanical equipment and methods, etc., can solve problems such as inability to be precise and efficient

Pending Publication Date: 2021-07-23
GENUS PLC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0014] While the aforementioned CD163 edits demonstrated some demonstration of utility against PRRSv, they were not as precise and efficient as the edits disclosed in the present invention

Method used

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  • Method for improving pig health by targeted inactivation of CD163
  • Method for improving pig health by targeted inactivation of CD163
  • Method for improving pig health by targeted inactivation of CD163

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0217] This example illustrates the selection of target sites for knockout of the porcine CD163 gene in porcine cells.

[0218]Single or paired delivery of guide RNAs by generating DNA sequence insertions, deletions, and combinations thereof in the porcine CD163 gene (Sscrofa11.1, GenBank Accession: GCA_000003025.6) using Streptococcus Cas9 / gRNA RNA-guided DNA endonuclease In combination with the protein, the sequence change in the CD163 gene reduces or abolishes the function, stability or expression of the CD163 messenger RNA or protein. The CD163 gene sequence from 120 nucleotides upstream of the translation initiation site (chr5:63300192) to 59 nucleotides downstream of CD163 exon 7 (chr5:63323390) was screened for genes with adjacent nGG or nGGnG Guide RNA binding sites for PAM sequences required for cleavage by the Cas9 protein from Streptococcus pyogenes or Streptococcus thermophilus CRISPR3 (Streptococcus thermophilus CR3), respectively, the sequences of which are prese...

Embodiment 2

[0220] This example demonstrates nucleofection to deliver a guide RNA / Cas9 endonuclease to porcine fetal fibroblasts.

[0221] To test the DNA cleavage activity of the edited porcine CD163 alleles generated in living cells, CRISPR-Cas endonucleases and guide RNAs targeting the sequences listed in Table 3 were nucleoffected into porcine fetal fibroblasts . To prepare porcine fetal fibroblast (PFF) cell lines from 28-35-day-old fetuses, mix 3.2 µg of Cas9 protein (S. The volume was 2.23 µl, which was then nucleoffected into PFF cells using a Lonza electroporator. In preparation for nucleofection, harvest PFF cells using trypsin expression (recombinant trypsin). After this, remove the medium from the cells, wash the medium once with HBSS or DPBS, and incubate at 38.5°C with trypsin for 3–5 min. Cells were then harvested with complete medium. Cells were pelleted by centrifugation (300 x g for five minutes at room temperature), the supernatant was discarded, and the cells were ...

Embodiment 3

[0224] This example illustrates the editing frequency of a guide RNA / Cas9 combination directed to porcine CD163.

[0225] Nucleotide sequence changes were introduced into the porcine CD163 gene by delivering Cas9 protein complexed with guide RNAs to fetal fibroblasts, as described in Example 2.

[0226] To assess DNA double-strand cleavage at the porcine CD163 genomic target site mediated by the guide RNA / Cas endonuclease system, a region of genomic DNA approximately 250 bp around the target site was amplified by PCR and then passed through the amplicon Deep sequencing checks PCR products for edits. After 3 transfections, PFF genomic DNA was extracted as described in Example 2. The region surrounding the expected target site is PCR amplified with NEB Q5 polymerase and the sequence required for amplicon-specific barcoding is added, by two rounds of PCR, using tailed primers for ILLUMINA ® (ILLUMINA ® , San Diego, CA) sequenced. at ILLUMINA ® MISEQ ® Personal Sequencer (I...

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Abstract

The present invention relates to methods and compositions for preventing porcine reproductive and respiratory syndrome (PRRSv) in animals, including animals of a population of pigs. The present teachings relate to pigs in which at least one allele of the CD163 gene has been inactivated, and specific methods and nucleic acid sequences for inactivating the CD163 gene in gene editing. The pig of which the two alleles of the CD163 gene are inactivated has resistance to porcine reproductive and respiratory syndrome (PRRSv). The superior lines comprising the homozygous CD163 editing gene retain their superior characteristics.

Description

[0001] Cross References to Related Applications [0002] This application claims the benefit and priority of U.S. Provisional Patent Application No. 63 / 020,128, filed May 5, 2020, and U.S. Provisional Patent Application No. 63 / 021,370, filed May 7, 2020. Applications Nos. 63 / 020,128 and 63 / 021,370 are hereby incorporated by reference in their entirety, respectively. [0003] References to Sequence Listings Submitted Electronically [0004] An official copy of the sequence listing is submitted electronically in ASCII format via EFS-Web, the file has the file name "RD-12-2020-US2-SEQLST", the creation date is March 10, 2021, the size is 137058 bytes, and the The patent specification is submitted at the same time. The sequence listing contained in the ASCII format file is a part of the patent specification and is hereby fully incorporated into this application by reference. technical field [0005] The present invention relates to methods of improving the health of swine. Spe...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/12C12N5/10C12N15/113C12N9/22C12N15/873C12N15/90C12Q1/6888C12N15/11A01K67/027C12R1/91
CPCC07K14/70596C12N15/1138C12N9/22C12N15/873A01K67/0276C12N2310/20C12N2510/00A01K2227/108A01K2267/02A01K2217/075C12N15/8509C12N5/0609A01K2207/15C12R2001/91C12N15/8778C12N15/90C12Q2600/124C12Q1/6876C12N5/0656C12N15/907C12Q1/6888C12N2320/34C12Q2600/156A01K67/0275A01K2217/052C12N15/113
Inventor 本杰明·比顿布莱恩·伯格迪伦·巴恩斯马修·坎贝尔安德鲁·马克·奇根乔纳森·爱德华·莱特纳马修·斯科特·库尔伯森威廉·托马斯·克里斯蒂安森
Owner GENUS PLC
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