A method for improving the health status of pig breeds through CD163 inactivation targeting.

Abstract

This invention provides a method and composition useful for preventing porcine reproductive respiratory syndrome virus (PRRSv) in animals, including wild boars (Sus scrofa). [Solution] This invention relates to a Suidae animal in which at least one allele of the CD163 gene is inactivated, as well as a specific method and nucleic acid sequence used in gene editing to inactivate the CD163 gene. Suidae animals in which both alleles of the CD163 gene are inactivated are resistant to Porcine Reproductive Respiratory Syndrome Virus (PRRSv). Superior strains containing homozygous CD163 edited genes retain their superior characteristics.

Description

[Technical Field]

[0001] Cross-reference of related applications This application is U.S. Provisional Patent Application No. 63 / 020,128, filed on May 5, 2020. The specification, and U.S. Provisional Patent Application No. 63 / 021,3 filed on May 7, 2020. Claiming priority over Specification No. 70, Specification No. 63 / 021,370 is referred to by... Each of these elements is incorporated into this specification.

[0002] References to electronically submitted sequence listings The official copy of the sequence listing should be named RD-12-2020-US2-SEQLST. It was created on March 10, 2021, and has a size of 137,058 bytes, ASC The sequence listing in format II was submitted electronically via EFS-Web, and this specification It is submitted at the same time as the document. The sequence list contained within this ASCII format document is This is part of this specification, and the entirety of it is incorporated herein by reference.

[0003] field This disclosure relates to methods for improving the health status of pig breeds. In particular, this disclosure relates to infection To prevent and create resistant animals, animal populations, and cell lines, polynucleates of CD163 Through leotide editing targeting, wild boar (Sus scrofa) species and superior strains are targeted. Regarding methods to protect against infection by porcine reproductive respiratory syndrome virus (PRRSv) ru. [Background technology]

[0004] Viral infections are a major cause of illness and death in the livestock industry, particularly in pigs. Porcine Respiratory Disorder Syndrome (PRRS) causes significant economic losses to the pig industry worldwide. It is an epidemic infectious disease of pigs. PRRS can manifest in pigs of all ages, It primarily causes late-stage abortions and stillbirths in sows, as well as respiratory diseases in piglets. The causative agent of the disease is the positive-strand RNA PRRS virus (PRRSv). PRRS is the most economically important domesticated wild boar in North America, Europe, and Asia. It is a critical disease that places an annual burden of over $600 million on producers in North America.

[0005] Currently, there is no effective treatment program for acute PRRS. If a PRRS outbreak is detected at a pig farm, culling, thorough cleaning / decontamination, and death will be carried out. The virus must be eliminated using proper disposal of the remains. In more extreme cases... The method of culling / replacing the entire animal population eliminates PRRSv from locally infected groups. Although recorded as an effective method, this method ultimately results in significant losses.

[0006] A vaccine for PRRSv exists, but it is primarily due to the genetics within the virus's structural proteins. Due to the diversity of offspring, these vaccines have not been able to control the disease. As a result, infection prevention is currently the best control measure. (Precautionary measures) As a location, pig farms in areas or regions where PRRSv exists should replace virgin pigs and In addition to assessing the health of male pigs, the newly arrived livestock undergo 45-60 days of isolation and acclimatization. Strict control measures, including those involving mitigation, must be employed.

[0007] In recent years, there has been increasing interest in the potential role that CD163 can play in the development of PRRS. It has been paid. Despite the significant heterogeneity in the PRRSv strain, the PRRSv strain They all share a tendency towards CD163-positive cells. CD163 is a viral receptor. However, this CD163 scavenger receptor is also involved in the adhesion of monocytes to endothelial cells. To agree. For a detailed description of the function of CD163, see Onofre, Gabriela et al., ACT. This is presented in A MEDICA, 2009, 52, 57-61.

[0008] CD163 is thought to be a fusion receptor for the PRRS virus, a 130kDa 1 It is a type membrane protein that maps to chromosome 5 in pigs. The basic transcript is 10 It codes for a 76-amino acid protein. There are five isoforms for CD163. It has been reported that three of the isoforms have different cytoplasmic domains. This presents pricing patterns. However, generally speaking, the genomic molecular sequence of CD163 is peptide Signaling sequence, nine scavenger receptor cysteine-rich (SRCR) domains, Two proline / serine / threonine (PST) linker domains, cytoplasmic domain, It also contains 17 exons that encode a short cytoplasmic tail. CD163 is the PRRSv It is described as a receptor. The protein's domain 5 (SRCR5) interacts with the virus. This is the interaction site. Exon 7 of CD163 is PRR in vitro. This encodes SRCR domain 5 (SRCR5), which is used as the interaction site with Sv. Burkard (Burkard, C., PLoS Pathog. 2017, 23, 13, e1006206.) is a CD. Removal of exon 163 (number 7) conferred PRRSv resistance to porcine macrophages. This supported the finding. However, the guide used in this study (SEQ ID NOs. 272 ​​and 273) (shown as a targeting sequence including PAM) is part of a commercial breeding program. It may lack sufficient activity and specificity for gene editing as a part. Furthermore, Whitw The research by orth and his colleagues is shown in Sequence ID No. 354 and Sequence ID No. 211. Using a guide (including PAM), create a 123bp deletion within exon 7. (Whitworth, KM, Biol. Reprod., 2014, 91, 1-13). Whitworth et al. (Whitworth, KM, Nature Biotechnology, 2016, 34, 20-22) describes the function of CD163. We reported on the preparation of PRRSv-resistant pigs by knockout.

[0009] Genome editing involves deleting, inserting, or replacing specific nucleic acid sequences. This includes altering the genome. The alterations may be gene-specific or location-specific. There are also combinations. Genome editing involves Cas proteins and their cognitive polynucleotides. Site-specific nucleases such as these can be used.

[0010] CRISPR(clustered regularly interspaced (short palindromic repeat) and CRISPR-associated protein (Cas) and these constitute the CRISPR-Cas system.

[0011] Cas9 is an exemplary type II CRISPR Cas protein. Two distinct endonuclease domains (HNH domain and RuvC / RNase H Using a domain, the tracr algorithm is used to site-specifically cleave the DNA target sequence. It is an endonuclease that can be programmed by RNA / crRNA (March 2014) Refer to U.S. Patent Application Publication No. 2014-0068797, published on the 6th of the month. Also, Jinek, M., et al., Science, 337:816-821 (2012) and Karvelis et al. See also enome Biology (2015) 16:253).

[0012] The aforementioned CD163 editing demonstrates some efficiency compared to PRRSv, but is not disclosed herein. Editing cannot be done as accurately and effectively as it is intended. To reduce the amount of editing that is not done, but to provide resistance to PRRSv, use guides. Therefore, it is necessary to improve the health status of pig herds by editing the CD163 gene. It is said that... [Overview of the project]

[0013] This specification describes how to confer PRRSv resistance to pigs containing edited genes using edited CD163 It presents genes and includes these.

[0014] In some embodiments, this instruction describes how editing excise the seventh exon, and how the edited gene is distributed The restored items shown in either column numbers 426-458 or 520-555 To confer PRRSv resistance to wild boars (Sus scrofa) that may contain the genome sequence. The edited CD163 gene is presented and included. In some configurations, the edited gene is distributed It may contain repaired genome sequences selected from the group consisting of sequence numbers 426-458. In this configuration, the repaired gene sequences are sequence numbers 426, 427, and 42 8, SEQ ID NO: 429, SEQ ID NO: 430, SEQ ID NO: 431, SEQ ID NO: 432, SEQ ID NO: 43 3, SEQ ID NO: 434, SEQ ID NO: 435, SEQ ID NO: 436, SEQ ID NO: 437, SEQ ID NO: 43 8, SEQ ID NO: 439, SEQ ID NO: 440, SEQ ID NO: 441, SEQ ID NO: 442, SEQ ID NO: 44 3. Sequence ID 444, Sequence ID 445, Sequence ID 446, Sequence ID 447, Sequence ID 44 8, SEQ ID NO: 449, SEQ ID NO: 450, SEQ ID NO: 451, SEQ ID NO: 452, SEQ ID NO: 45 3. Sequence ID 454, Sequence ID 455, Sequence ID 456, Sequence ID 457, Sequence ID 45 8, SEQ ID NO: 520, SEQ ID NO: 521, SEQ ID NO: 522, SEQ ID NO: 523, SEQ ID NO: 52 4, SEQ ID NO: 525, SEQ ID NO: 526, SEQ ID NO: 527, SEQ ID NO: 528, SEQ ID NO: 52 9, SEQ ID NO: 530, SEQ ID NO: 531, SEQ ID NO: 532, SEQ ID NO: 533, SEQ ID NO: 53 4, SEQ ID NO: 535, SEQ ID NO: 536, SEQ ID NO: 537, SEQ ID NO: 538, SEQ ID NO: 53 9, SEQ ID NO: 540, SEQ ID NO: 541, SEQ ID NO: 542, SEQ ID NO: 543, SEQ ID NO: 54 4, SEQ ID NO: 545, SEQ ID NO: 546, SEQ ID NO: 547, SEQ ID NO: 548, SEQ ID NO: 54 9, SEQ ID NO: 550, SEQ ID NO: 551, SEQ ID NO: 552, SEQ ID NO: 553, SEQ ID NO: 55 4, or sequence number 555 may be included. In diverse configurations, editing is performed using gRNA pairs. It may be created, and the gRNA pair is sequence numbers 229 and 256, sequence number 230 Sequence IDs 256, 231 and 256, 237 and 256, and 24 1 and 256, Sequence IDs 229 and 258, Sequence IDs 230 and 258, Sequence ID 231 and 258, Sequence IDs 237 and 258, Sequence IDs 241 and 258, Sequence IDs Numbers 229 and 261, Sequence IDs 230 and 261, Sequence IDs 231 and 261, Sequence IDs 237 and 261, 241 and 261, 219 and 25 6, Sequence IDs 221 and 256, Sequence IDs 224 and 256, Sequence ID 227 and 256, Sequence IDs 219 and 258, Sequence IDs 221 and 258, Sequence ID 224 Sequence IDs 258, 227 and 258, 219 and 261, and 22 1 and 261, Sequence IDs 224 and 261, Sequence IDs 227 and 261, Sequence ID 249 and 256, Sequence IDs 250 and 256, Sequence IDs 249 and 258, Sequence IDs Numbers 250 and 258, sequence numbers 249 and 261, or sequence numbers 250 and 2 It could be 61. In various configurations, editing may be created using gRNA pairs. The targeting region pairs are sequence numbers 229 and 256, sequence numbers 230 and 25 6, Sequence IDs 231 and 256, Sequence IDs 241 and 256, Sequence ID 229 and 258, SEQ ID NOs. 231 and 258, SEQ ID NOs. 241 and 258, SEQ ID NOs. 219 Sequence IDs 256, 221 and 256, 224 and 256, and 22 7 and 256, Sequence IDs 227 and 258, Sequence IDs 221 and 261, Sequence ID 249 and 256, Sequence IDs 250 and 256, Sequence IDs 249 and 258, and These could be sequence numbers 249 and 261.

[0015] In various configurations, editing may be created using gRNA pairs, and gRNA pairs are These could be sequence numbers 229 and 256. In various configurations, editing uses gRNA pairs. This can occur, and the gRNA pair may be sequence numbers 230 and 256. In various configurations, editing may be created using gRNA pairs, and gRNA pairs are distributed The column numbers can be 231 and 256. In various configurations, editing is performed using gRNA pairs. It may be created, and the gRNA pair could be sequence numbers 237 and 256. In the configuration, editing may be created using gRNA pairs, and gRNA pairs are sequence numbers. These could be numbers 241 and 256. In diverse configurations, editing is created using gRNA pairs. This can occur, and the gRNA pair may be sequence numbers 229 and 258. Diverse configurations Editing can be created using gRNA pairs, and gRNA pairs are such that as shown in Sequence ID No. 2. It can be 30 or 258. In diverse configurations, editing is created using gRNA pairs. In some cases, the gRNA pair may be sequence numbers 231 and 258. Editing may be created using a gRNA pair, and the gRNA pair is SEQ ID NO: 237 And it can be 258. In diverse configurations, editing is created using gRNA pairs. There is a combination, and the gRNA pair can be sequence numbers 241 and 258. In diverse configurations, editing The cluster may be created using a gRNA pair, and the gRNA pair is sequence number 229. It can be 261. In diverse configurations, editing may be created using gRNA pairs. Yes, the gRNA pair can be sequence numbers 230 and 261. In diverse configurations, editing is It may be a gRNA pair, and the gRNA pair could be sequence numbers 231 and 261. In various configurations, editing may be created using gRNA pairs, and gRNA pairs are These could be sequence numbers 237 and 261. In various configurations, editing uses gRNA pairs. This can occur, and the gRNA pair may be sequence numbers 241 and 261. In various configurations, editing may be created using gRNA pairs, and gRNA pairs are distributed The column numbers can be 219 and 256. In various configurations, editing is performed using gRNA pairs. It may be created, and the gRNA pair could be sequence numbers 221 and 256. In the configuration, editing may be created using gRNA pairs, and gRNA pairs are sequence numbers. Possible numbers 224 and 256. In diverse configurations, editing is created using gRNA pairs. In some cases, the gRNA pair may be sequence numbers 227 and 256. Diverse configurations Editing can be created using gRNA pairs, and gRNA pairs are such that as shown in Sequence ID No. 2. It can be 19 or 258. In diverse configurations, editing is created using gRNA pairs. In some cases, the gRNA pair may be sequence numbers 221 and 258. Editing may be created using a gRNA pair, and the gRNA pair is SEQ ID NO: 224 And it can be 258. In diverse configurations, editing is created using gRNA pairs. There is a combination, and the gRNA pair can be sequence numbers 227 and 258. In diverse configurations, editing The cluster may be created using a gRNA pair, and the gRNA pair is sequence number 219. It can be 261. In diverse configurations, editing may be created using gRNA pairs. Yes, the gRNA pair can be sequence numbers 221 and 261. In diverse configurations, editing is , may be created using gRNA pairs, and gRNA pairs are SEQ ID NOs. 224 and 2 It could be 61. In various configurations, editing may be created using gRNA pairs. The gRNA pair can be sequence numbers 227 and 261. In diverse configurations, editing is performed on g It may be created using RNA pairs, and the gRNA pairs are SEQ ID NOs. 249 and 256 It is possible. In various configurations, editing may be created using gRNA pairs, g The RNA pair can be sequence numbers 250 and 256. In diverse configurations, editing occurs in the gRN. It may be created using the A pair, and the gRNA pair is sequence numbers 249 and 258. It is possible. In various configurations, editing may be created using gRNA pairs, gRN The A pair can be sequence numbers 250 and 258. In various configurations, editing occurs in the gRNA pair. It may be created using the gRNA pair, which may be sequence numbers 249 and 261. In various configurations, editing may be created using gRNA pairs, and gRNA pairs These could be sequence numbers 250 and 261. In diverse configurations, the repaired genome sequence is This may include sequence number 453, and the editing is as shown in sequence numbers 249 and 256. It can be created using columns.

[0016] In diverse configurations, this disclosure may include the CD163 gene of this instruction in wild boar (Sus sc This document presents rofa) cells and includes these. In some configurations, this instruction is for multiple species of wild boar (Su Further cell lines that may contain (s scrofa) cells are presented, and include them. In some configurations, cell lines This can be a fibroblast cell line. In diverse configurations, cells, multiple cells, or cell lines are P IC system 2, PIC system 3, PIC system 15, PIC system 19, PIC system 27, PIC This instruction may originate from strain 62 or PIC strain 65. This instruction applies to embryos that may contain multiple cells. Further images of piglets or adult pigs are provided.

[0017] This disclosure also states that the editing may be selected from the group consisting of 506-517, of the seventh exon. The wild boar (Sus scrofa) generates stop codons that result in the predicted amino acid sequence. We also present the CD163 gene edited to confer PRRSv resistance. In terms of composition, the amino acid sequences are sequence numbers 506, 507, 508, and sequence number Number 509, Sequence ID 510, Sequence ID 511, Sequence ID 512, Sequence ID 513, Sequence Number May be shown in code 514, sequence number 515, sequence number 516, or sequence number 517. In this configuration, the predicted amino acid sequence of exon 7 may be shown in SEQ ID NO: 513. In the configuration, the edits are sequence numbers 351 and 365, sequence numbers 351 and 387, sequence number Numbers 348 and 390, Sequence IDs 348 and 388, Sequence IDs 348 and 395, Distribution Column numbers 352 and 365, sequence numbers 352 and 387, sequence numbers 352 and 399 , Sequence IDs 353 and 365, Sequence IDs 353 and 387, Sequence IDs 353 and 3 99, SEQ ID NOs. 354 and 390, SEQ ID NOs. 354 and 388, SEQ ID NOs. 354 and 395, SEQ ID NOs. 358 and 361, SEQ ID NOs. 358 and 362, SEQ ID NOs. 358 and 368, SEQ ID NOs. 358 and 384, SEQ ID NOs. 358 and 394, SEQ ID NOs. 3 58 and 399, Sequence IDs 359 and 390, Sequence IDs 359 and 388, Sequence No. Numbers 359 and 395, Sequence IDs 360 and 368, Sequence IDs 360 and 384, Distribution Column numbers 360 and 389, sequence numbers 360 and 394, sequence numbers 360 and 397 , Sequence IDs 361 and 365, Sequence IDs 361 and 387, Sequence IDs 362 and 3 90, SEQ ID NOs. 362 and 388, SEQ ID NOs. 362 and 395, SEQ ID NOs. 364 and 365, SEQ ID NOs. 364 and 387, SEQ ID NOs. 364 and 399, SEQ ID NOs. 365 and 368, SEQ ID NOs. 365 and 384, SEQ ID NOs. 365 and 389, SEQ ID NOs. 3 65 and 394, Sequence IDs 365 and 397, Sequence IDs 366 and 368, Sequence Number Numbers 366 and 384, Sequence IDs 366 and 389, Sequence IDs 366 and 394, Furthermore, gRNAs selected from the group consisting of SEQ ID NOs. 366 and 397 were created. It is possible. In various configurations, the editing is done with sequence numbers 351 and 365, sequence number 348 and 390, SEQ ID NOs. 348 and 388, SEQ ID NOs. 354 and 390, SEQ ID NOs. 358 From sequence numbers 394, 362 and 390, and 366 and 394 It can be created using gRNA selected from a group. In diverse configurations, editing is created. The gRNAs used for this may be sequence numbers 351 and 365. In various configurations, The gRNAs used to create the edits may be sequence numbers 351 and 387. In such configurations, the gRNAs used to create the edits are sequence numbers 348 and 390. It is possible. In diverse configurations, the gRNA used to create the edit is SEQ ID NO: 34 It can be 8 and 388. In diverse configurations, the gRNA used to create the edit is These can be sequence numbers 348 and 395. In various configurations, they are used to create edits. The gRNAs involved can be sequence numbers 352 and 365. In diverse configurations, editing is created. The gRNAs used for this may be sequence numbers 352 and 387. The gRNAs used to create the edit could be sequence numbers 352 and 399. In various configurations, the gRNAs used to create the edits are sequence numbers 353 and 3. It can be 65. In diverse configurations, the gRNA used to create the edit is the sequence number. It can be 353 or 387. In various configurations, the gRN used to create the edit A can be sequence numbers 353 and 399. In various configurations, it is used to create edits. The gRNAs used may be sequence numbers 354 and 390. In various configurations, editing is performed. The gRNAs used to create it may be sequence numbers 354 and 388. In this case, the gRNAs used to create the edits are sequence numbers 354 and 395. In various configurations, the gRNA used to create the edit is sequence number 358. It can be 361. In diverse configurations, the gRNA used to create the edit is sequence The numbers could be 358 and 362. In various configurations, g is used to create the edit. The RNA can be sequence numbers 358 and 368. In diverse configurations, editing is created. The gRNAs used may be sequence numbers 358 and 384. In diverse configurations, editing The gRNAs used to create the cluster may be sequence numbers 358 and 394. In this configuration, the gRNAs used to create the edit are sequence numbers 358 and 399. It is possible. In diverse configurations, the gRNA used to create the edit is sequence number 359. and 390. In diverse configurations, the gRNA used to create the edit is These can be sequence numbers 359 and 388. In various configurations, they are used to create edits. The gRNAs involved can be sequence numbers 359 and 395. In diverse configurations, editing is created. The gRNAs used for this purpose may be sequence numbers 360 and 368. The gRNAs used to create the edits could be sequence numbers 360 and 384. In various configurations, the gRNAs used to create the edits are sequence numbers 360 and 38. It could be 9. In various configurations, the gRNA used to create the edit is SEQ ID NO: 3 It can be 60 or 394. In diverse configurations, the gRNA used to create the edit These can be sequence numbers 360 and 397. In various configurations, they are used to create edits. The gRNAs involved can be sequence numbers 361 and 365. In diverse configurations, editing is created. The gRNAs used for release can be sequence numbers 361 and 387. Diverse composition So, the gRNAs used to create the edit are sequence numbers 362 and 390. In various configurations, the gRNA used to create the edit is sequence number 362 and It could be 388. In diverse configurations, the gRNA used to create the edit is sequence number It could be numbers 362 and 395. In various configurations, gR is used to create the edits. NA can be sequence numbers 364 and 365. In diverse configurations, to create edits... The gRNAs used may be sequence numbers 364 and 387. In various configurations, editing The gRNAs used to create it can be sequence numbers 364 and 399. In the configuration, the gRNAs used to create the edit are sequence numbers 365 and 368. It is possible. In diverse configurations, the gRNA used to create the edit is sequence number 365. It can be 384. In diverse configurations, the gRNA used to create the edit is distributed Column numbers can be 365 and 389. In various configurations, they are used to create edits. The gRNAs can be sequence numbers 365 and 394. Diverse configurations create editing. The gRNAs used for this may be sequence numbers 365 and 397. In various configurations, The gRNAs used to create the edits could be sequence numbers 366 and 368. In such configurations, the gRNAs used to create the edits are sequence numbers 366 and 384. It is possible. In diverse configurations, the gRNA used to create the edit is SEQ ID NO: 36 It can be 6 and 389. In diverse configurations, the gRNA used to create the edit is These can be sequence numbers 366 and 394. In various configurations, they are used to create edits. The gRNAs involved could be sequence numbers 366 and 397.

[0018] In diverse configurations, the edited CD163 gene is selected from the group consisting of sequence numbers 459-504. It may have a selected nucleic acid sequence. In diverse configurations, the repaired gene is sequence number 459. 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 486, 487, 488, 489, 490, 491, 492, 493, 494, 495, 496, 497, 498, 499, It may have the nucleic acid sequences shown in 500, 501, 502, 503, or 504. In terms of composition, the repaired gene may have the sequence shown in nucleic acid sequence number 489. In terms of composition, the predicted amino acid sequence of exon 7 is the predicted amino acid sequence shown in SEQ ID NO: 513. It can be a sequence. In diverse configurations, the predicted amino acid sequence of the gene is, in the case of SEQ ID NO: 513. The repaired gene may have the nucleic acid sequence shown in SEQ ID NO: 489. Edits can be created using the sequences shown in sequence numbers 362 and 390.

[0019] This disclosure includes the CD163 variant edited to include the exogenous stop codon described above. This disclosure presents and includes wild boar (Sus scrofa) cells that may contain genes. The study includes multiple cells containing the CD163 gene edited to include an exogenous stop codon. We also present various cell lines. In some configurations, the cell line may be a fibroblast cell line. Diverse configurations So, the cells are PIC strain 2, PIC strain 3, PIC strain 15, PIC strain 19, PIC This disclosure may originate from system 27, PIC system 62, or PIC system 65. Embryo containing multiple cells, which may contain the CD163 gene edited to include a stop codon. Show, include, piglets or adult pigs.

[0020] In various embodiments, this instruction describes how to edit the wild boar (Sus scrofa) CD163 gene. gRNA pairs for sequence numbers 229 and 256, sequence numbers 230 and 256 , Sequence IDs 231 and 256, Sequence IDs 237 and 256, Sequence IDs 241 and 2 56, SEQ ID NOs. 229 and 258, SEQ ID NOs. 230 and 258, SEQ ID NOs. 231 and 258, SEQ ID NOs. 237 and 258, SEQ ID NOs. 241 and 258, SEQ ID NOs. 229 and 261, SEQ ID NOs. 230 and 261, SEQ ID NOs. 231 and 261, SEQ ID NOs. 2 37 and 261, Sequence IDs 241 and 261, Sequence IDs 219 and 256, Sequence No. Numbers 221 and 256, Sequence IDs 224 and 256, Sequence IDs 227 and 256, Distribution Column numbers 219 and 258, sequence numbers 221 and 258, sequence numbers 224 and 258 , Sequence IDs 227 and 258, Sequence IDs 219 and 261, Sequence IDs 221 and 2 61, SEQ ID NOs. 224 and 261, SEQ ID NOs. 227 and 261, SEQ ID NOs. 249 and 256, Sequence IDs 250 and 256, Sequence IDs 249 and 258, Sequence ID 250 and 258, SEQ ID NOs. 249 and 261, SEQ ID NOs. 250 and 261, SEQ ID NOs. 3 51 and 365, Sequence IDs 351 and 387, Sequence IDs 348 and 390, Sequence No. Numbers 348 and 388, Sequence IDs 348 and 395, Sequence IDs 352 and 365, Distribution Column numbers 352 and 387, sequence numbers 352 and 399, sequence numbers 353 and 365 , SEQ ID NOs. 353 and 387, SEQ ID NOs. 353 and 399, SEQ ID NOs. 354 and 3 90, SEQ ID NOs. 354 and 388, SEQ ID NOs. 354 and 395, SEQ ID NOs. 358 and 361, SEQ ID NOs. 358 and 362, SEQ ID NOs. 358 and 368, SEQ ID NOs. 358 and 384, SEQ ID NOs. 358 and 394, SEQ ID NOs. 358 and 399, SEQ ID NOs. 3 59 and 390, Sequence IDs 359 and 388, Sequence IDs 359 and 395, Sequence No. Numbers 360 and 368, Sequence IDs 360 and 384, Sequence IDs 360 and 389, Distribution Column numbers 360 and 394, sequence numbers 360 and 397, sequence numbers 361 and 365 , Sequence IDs 361 and 387, Sequence IDs 362 and 390, Sequence IDs 362 and 3 88, SEQ ID NOs. 362 and 395, SEQ ID NOs. 364 and 365, SEQ ID NOs. 364 and 387, SEQ ID NOs. 364 and 399, SEQ ID NOs. 365 and 368, SEQ ID NOs. 365 and 384, SEQ ID NOs. 365 and 389, SEQ ID NOs. 365 and 394, SEQ ID NOs. 3 65 and 397, Sequence IDs 366 and 368, Sequence IDs 366 and 384, Sequence Number Numbers 366 and 389, Sequence IDs 366 and 394, or Sequence IDs 366 and 39 The gRNA pairs having the sequences shown in 7 are presented and include these. In some configurations, gRN Pair A consists of sequence numbers 229 and 256, sequence numbers 230 and 256, and sequence number 231. Sequence IDs 256, 241 and 256, 229 and 258, and 23 1 and 258, SEQ ID NOs: 241 and 258, SEQ ID NOs: 219 and 256, SEQ ID NOs: 221 and 256, Sequence IDs 224 and 256, Sequence IDs 227 and 256, Sequence IDs Numbers 227 and 258, Sequence IDs 221 and 261, Sequence IDs 249 and 256, Sequence IDs 250 and 256, 249 and 258, 249 and 26 1. Sequence IDs 351 and 365, 348 and 390, 348 and 388, SEQ ID NOs. 354 and 390, SEQ ID NOs. 358 and 394, SEQ ID NOs. 362 It may have the sequences shown in sequence numbers 390, or sequence numbers 366 and 394. Diverse configurations Therefore, the gRNA pair may have the sequences shown in SEQ ID NOs. 249 and 256. In adulthood, the gRNA pair may have the sequences shown in SEQ ID NOs. 362 and 390.

[0021] This disclosure relates to the editing of the CD163 gene in the wild boar species (Sus scrofa) as described in this instruction. It presents a CRISPR complex containing gRNA pairs, and includes these.

[0022] This disclosure relates to a method for editing the CD163 gene in wild boar species, and the g of this instruction We present a method that includes a step using a CRISPR-CAS complex containing RNA pairs, and these Includes.

[0023] This disclosure is made possible by using a CRISPR-CAS complex containing the gRNA pair of this instruction. This paper presents methods for preparing PRSSV-resistant wild boar cells, including those described above.

[0024] This disclosure provides a method for producing PRRSv-resistant wild boar animals, a) g of this instruction Using a CRISPR complex containing RNA pairs, CD of one or more wild boar species cells 163 Steps to edit genes; b) Create an animal from one or more cells A method including steps is presented, and these are included.

[0025] This disclosure presents the use of cell lines in accordance with this instruction in the creation of PRRSv-resistant animals. , including these.

[0026] This disclosure also presents embryos, piglets, or adults containing multiple cells that comply with this instruction, and this also include.

[0027] In various embodiments, this disclosure relates to the sequence shown in SEQ ID NO: 453 and 90% or 95% A method for determining the existence or non-existence of edit sequences having % identity, wherein a) Sequence ID Differentially labeled pro b) Primer pairs shown in SEQ ID NOs: 562 and 563; and c) SEQ ID NOs: With the primer pairs shown in 556 and 557 or SEQ ID NOs. 559 and 560, A method is presented that includes the step of performing real-time PCR, and these include some components. Therefore, the edited sequence may have 100% identity with the sequence shown in sequence number 453. In this configuration, the edited sequence may have 90% identity with the sequence shown in sequence number 453. In some configurations, the edited sequence may have 95% identity with the sequence shown in sequence number 453. ru.

[0028] In various embodiments, this teaching applies to sequence numbers 556, 557, 559, A PC selected from the group consisting of sequence numbers 560, 562, and 563. The R primers are presented and include these. In some configurations, the PCR primers are SEQ ID NOs. It may have the sequence shown in 556. In various configurations, the PCR primer is sequence number 556. It may have the sequence shown in 7. In various configurations, the PCR primer is for sequence number 559. The sequence shown may be present. In various configurations, the PCR primer is shown in SEQ ID NO: 560. It may have a sequence. In various configurations, the PCR primer is shown in SEQ ID NO: 562. It may have a sequence. In various configurations, PCR primers may have the sequence shown in SEQ ID NO: 563. It may have.

[0029] In some embodiments, this disclosure relates to SEQ ID NO: 558, SEQ ID NO: 561, and SEQ ID NO: 5 We present real-time PCR probes selected from a group of 64, including these. In the configuration of the part, the probe has the sequence shown in sequence number 558. In various configurations, The probe has the sequence shown in sequence number 561. In various configurations, the probe is arranged It has the sequence shown in column number 564.

[0030] In various embodiments, this instruction also addresses the presence of the edited genome sequence shown in SEQ ID NO: 453. a) Sequence IDs 556 and 557, and Sequence ID 562, are used to determine non-existence. and 563, or b) Sequence IDs 559 and 560, and Sequence ID 562 and The use of PCR primers shown in 563 is presented, and includes these.

[0031] In various embodiments, this instruction also addresses the presence of the edited genome sequence shown in SEQ ID NO: 453. To determine non-existence, a) Sequence IDs 558 and 564, or b) Sequence ID 56 The use of PCR probes shown in 1 and 564 is presented, and includes these.

[0032] In various embodiments, this instruction is a method for creating PRRSv-resistant pigs, and the sequence The method includes a step of editing the pig genome to include the genome sequence shown in number 453. The law is presented and includes these. In some configurations, the step of editing the pig genome is sequence number This may include administering gRNA having sequences shown in numbers 249 and 256. In this configuration, administration involves a pre-formed substance containing gRNA and CAS protein. This involves injecting the RNP complex into a zygote, embryo, or MII-stage oocyte. In terms of composition, the pig is PIC (Pig Improvement Company, Ltd). , Basingstoke, UK) (Trade Mark) Line 2, PIC (Trademark) Line 3, PIC (Trademark) Line 3, PIC (trademark) line 15, PIC (trademark) line 19, PIC (trademark) line 27, PIC (trademark) line It is a pig of lineage 62, or PIC(trademark) lineage 65.

[0033] In various embodiments, this disclosure describes how editing exects the seventh exon, and the edited gene is distributed The restored genome sequence shown in column number 453 was used in the wild boar (Sus scrofa), and PR We present a wild boar (Sus scrofa) species containing an edited gene that confers RSv resistance, and This includes. In some configurations, the edit has the sequences shown in sequence numbers 249 and 256. This can be done by guide RNA (gRNA). In diverse configurations, animals are PIC (trademark). Line 2, PIC (trademark) line 3, PIC (trademark) line 15, PIC (trademark) line 19, P Editing of IC(trademark) system 27, PIC(trademark) system 62, or PIC(trademark) system 65 It may be an animal. In diverse configurations, this disclosure presents cells prepared from the animals of this instruction. , including this. In various configurations, this disclosure provides cell lines prepared from cells in accordance with this instruction. This includes, and in some configurations, the cell line may be a fibroblast cell line.

[0034] In some embodiments, this instruction describes how editing excise the seventh exon, and how the edited gene is distributed The restored genome sequence shown in column number 453 was used in the wild boar (Sus scrofa), and PR This document presents and includes the CD163 gene edited to confer RSv resistance. In this configuration, the edit is created using the sequences shown in sequence numbers 249 and 256. In various configurations, this disclosure describes wild boars (Sus scro) containing the CD163 gene according to this instruction. fa) Present cells. In some configurations, this disclosure includes multiple types of cells that comply with this instruction. Cell lines are presented. In some configurations, the cell line may be a fibroblast cell line. In diverse configurations, , the cells are PIC line 2, PIC line 3, PIC line 15, PIC line 19, PIC line 27. It can be isolated from PIC system 62 or PIC system 65.

[0035] In various embodiments, this disclosure describes editing the wild boar (Sus scrofa) CD163 gene. A gRNA pair for which the guide sequences shown in SEQ ID NOs. 249 and 256 are included. It presents RNA pairs and includes them.

[0036] In various embodiments, this instruction is a method for creating PRRSv-resistant pigs, and the sequence The method includes a step of editing the pig genome to include the genome sequence shown in number 453. The law is presented and includes these. In some configurations, the step of editing the pig genome is sequence number This may include administering gRNA having the sequences shown in numbers 249 and 256. In this configuration, the administered substance is a pre-formed substance containing gRNA and CAS protein. This may include injecting the RNP complex into a zygote, embryo, or MII-stage oocyte. In such a configuration, the pigs are PIC(trademark) system 2, PIC(trademark) system 3, PIC(trademark) Line 15, PIC (trademark) line 19, PIC (trademark) line 27, PIC (trademark) line 62 , or it could be a pig of PIC(trademark) lineage 65. [Modes for carrying out the invention]

[0037] The aspect of this instruction is to improve the health status of a specific breed of pig by inactivating the target CD163. Methods that may vary and include methods understood by those skilled in the art, but which are not limited to these. Not limited to. All terms used herein refer to specific aspects only. The purpose is to limit the scope in any form and to any extent. It should be further understood that this is not intended. For example, this specification and its appendices The singular forms "a," "an," and "so" used in the patent claims. "no" can refer to multiple objects unless it is explicitly indicated otherwise. Furthermore, all units, prefixes, and symbols may be displayed in their SI-approved forms. Numerical ranges enumerated herein include the numbers that define the range, and within the defined range Includes each integer.

[0038] To make this application easier to understand, certain terms are first defined. Unless otherwise specified, all technical terms and Scientific terms are defined as having the same meaning as those generally understood by those skilled in the art in the field relating to the aspects of this application. It has the same meaning. In the embodiment of this application, without excessive experimentation, the description herein is Many similar methods and materials, many modified methods and materials, Many equivalent methods and materials may be used, but in this specification, preferred methods and The materials are described. In the description of the aspects of this application and in the claims, the following conditions are set forth below. In accordance with the rules, the following terminology is used.

[0039] As used herein, "animal cells" refers to somatic cells, cultured cells, gamete cells, and blood cells. This refers to cells including, but not limited to, cysts, zygotes, and embryonic cells. These animals The cells may be reproductive cells or non-reproductive cells. As used herein, Cells can be isolated from animals or embryos and maintained in tissue cultures. This may include gene-edited cells and non-gene-edited supporting or feeder cells. This also includes mixed cultures.

[0040] As used herein, the terms "gene edited" and "gene" are used to mean "genetic edited." "Genetically edited" and "Genome-edited" The term is "homing endonuclease," or "targeting endonuclease." Often referred to as "rease," these are naturally occurring endonucleases or artificially modified types. This refers to the use of homing technology using endonucleases. "Genome editing" and "gene editing" "Editing" refers to the process of deleting, inserting, or substituting specific nucleic acid sequences. This refers to altering the genome. These alterations can be gene-specific or site-specific. In some cases, however, modification of the gene sequence itself is not required. Genome editing involves cutting nucleic acids, This creates sites for modification, such as endonucleases in the CRISPR system. It can be used. Other endonucleases are also available and suitable for use, but outside the target site. Cleavage and specificity can be important issues. In systems such as CRISPR, nucleases... This involves introducing site-specific DSBs, and in this specification, a polynucleus referred to as the "target sequence". By complexing with rheotide, it can be directed towards the target site. (Not limited to theory) As described above, the DSB is then repaired by the endogenous non-homologous end joining (NHEJ) mechanism. Shut up.

[0041] Numerous endonucleases suitable for gene editing and adapted for this purpose are known. In the field of technology, CRISPR (clustered regularly intercepted aced short palindromic repeat) system (for example, CRIS) PR / Cas9 system), TALEN (transcription activator- (like effector nuclease), and zinc finger nuclease Gene editing methods, including those involving gene editing nucleases such as (ZFN), are publicly known. Animals that gather exogenous DNA sequences, particularly in transgenic animals, originate from alien species. Transgenic animals can be distinguished from those created by the incorporation of sequences that have identity with the given sequence. In this specification, the term "gene editing" refers to the introduction of more than 10 nucleotides. This includes small deletions and insertions, and results from them. The term "gene editing" is... Furthermore, progeny created from early gene-edited animals through sexual crossbreeding or asexual reproduction. This also includes later animals such as those mentioned above.

[0042] As used herein, the terms “restorative template” or “restorative array” refer to a method for guiding a restoration. To ensure accurate and selective editing, the nuclei within the genome of cells undergoing DSB repair This refers to polynucleotides introduced at the rease target site. The repair template is a nucleotide having a double-segment break (DSB). It may be used to selectively alter or delete the arrangement of the constellations, and generally, 5 It includes a 'side genome hybridization region' and a '3' side genome hybridization region. In one embodiment, the 5' genome hybridizing region and the 3' genome hybridizing region The region may share at least 80% homology. In some embodiments, the 5' side genome high The hybridization region and the 3' genome hybridization region may share identity. Further description of the repair mold in this disclosure is presented below. In the missing mold, adjacent Genomic regions can be separated by the deletion of one or more nucleotides. The target editing involves adding one or more stop codons, provided that include adjacent genomic regions. It can be prepared using a repair template with base changes. The identity percentage is between 80% and 100% using gap-free alignment. This is possible. As presented herein, the core identity region within the repair template is the target chromosome region. By franking homology regions that can share 80% to 100% homology with the region It can be trapped. To put it without being limited to theory, core domain identity is the fidelity of the repair process. While it can be increased, the expansion of homology regions on both sides increases efficiency and for editing. It allows for genotype variations between cells that are targeted.

[0043] As used herein, the terms “wild type” or “WT” refer to a natural population of pigs or This refers to the dominant phenotype, genotype, or gene in a population of pig strains. Used to compare the phenotype and genotype of gene-edited cells and gene-edited animals. In this case, the term "wild type" refers to non-gene-edited cells and non-gene-edited animals of the same lineage. This refers to similar breeding with similar genetic backgrounds. In some embodiments, this disclosure relates to similar breeding with similar genetic backgrounds. This document presents a comparison of a certain edited pig with an unedited pig. In one aspect, this disclosure is presented for the same breeding line This paper presents a comparison between edited pigs (the standard) and unedited pigs.

[0044] As used herein, the term "knockout" refers to a reduction or elimination of its expression. This refers to the disruption of gene function. Knockout is the disruption of double-strand breaks (DSBs) within chromosomes. Created through creation, then non-homologous end joining (NHEJ), or homology-oriented repair ( Using homologous recombination of DNA repair templates or targeting vectors via HDR. And it can be repaired. Speaking without being limited to theory, HDR knockouts can also be genomically modified. MMEJ(micr It can also be prepared by (ohomology-mediated end joining). Knockout also refers to the complete, partial, or conditioned loss of gene function. The resulting substitution vector, or hit-end-run vector, or gene vector It can also be produced through the random insertion of wrap vectors.

[0045] References to deletions within a range of nucleotide sequences in this specification are enumerated. It includes all nucleotides within the specified range. For example, nucleotide "a" ~ nucleotide The deletion of five base pairs in Otid "e" refers to the deletion of the nucleotides "a", "b", and "c". Each of "d" and "e" (where "b", "c", and "d" are "a" and " This means that the element between "e" and the next element is missing.

[0046] As used herein, the term “editing” refers, for example, to genes, coding DNA sequences. The field of polynucleotide sequences, such as CDS or non-coding DNA sequences. Includes changes compared to the raw sequence. The term "editing" includes insertions, deletions, and splice donors. This may include editing body parts, editing points, etc.

[0047] The term "CRISPR" used herein refers to "clustered regular y interspaced short palindromic repeat”) Alternatively, the term "CRISPR" refers to the CRISPR segment of genetic material and RNA segment. By utilizing the ligation and the enzymes produced by the CRISPR segment, within the genome of other organisms This refers to a gene editing system that identifies and modifies specific DNA sequences. The CRISPR system is type I. This includes Type II and Type III CRISPR systems. The term "CRISPR" as used herein refers to CRISPR systems. The terms "PR-associated protein" or "Cas" are strictly defined as CRISPR elements. This refers to a family of proteins that can associate, and is always a CRISPR segment. It occurs in the vicinity of repeat clusters. For example, Cas proteins have Cas9 f Family member proteins, Cas6 family member proteins (e.g., Csy This may include Cas4 and Cas6, and Cas5 family member proteins, but these It is not limited to the following. Examples of the Cas protein family and methods for identifying them are found in Haft. This is disclosed in Daniel H., et al., PLoS Comput. Bio., 2005, 1, e60.

[0048] Without being limited to any particular academic theory, CRISPR nucleases are complementary. It forms a complex with a guide RNA (gRNA) that hybridizes with the target nucleic acid molecule, and This allows CRISPR nucleases to be guided to target nucleic acid molecules. crRNA is an invading agent. In the pathogen, the specific protospacer sequence and the repeat sequence that may be complementary to it It includes spacer sequences. It is designed to be complementary to target sequences within the eukaryotic genome. The spacer sequence is what can be used. CRISPR nucleases are active in these sequences. In this specification, they can associate with their respective crRNAs. This specification refers to Sequence IDs 22-27 RNA spacers containing the first 20 nucleotides of each of 1 and 347-425 The potentially contained crRNAs are listed, and these include those used herein. SEQ ID NOs. 22-27 1, and each of 347-425, have thymidine replaced with uridine and deoxyribose This document presents and includes the corresponding RNA sequence in which the is replaced with ribose. The above sequence numbers also include PAMs (see below) present within the targeted genome sequence. (See reference) This also includes. Those skilled in the art will know that the gRNA targeting this sequence is PAM Recognizing that it does not contain, and therefore gRNA having the sequences shown in these sequence numbers However, it only includes the first 20 nucleotides, and this allows us to recognize that PAM is excluded. It is likely.

[0049] Some CRISPR nucleases, such as CasX and Cas9, exhibit functional activity. However, there is another non-coding RN called trans-activated crRNA (tracrRNA). A component may be required. The first 20 nucleotides of each of sequence numbers 22-271, Selected from the group consisting of the first 20 nucleotides of each of sequence numbers 347-425. crRNAs containing spacer sequences are described in Jinek, et al., Science, 337, 2012, 816-821. As described, it is covalently linked to the 5' end of tracrRNA, and this specification In the book, it is called "single guide RNA" (sgRNA), which is covalently bonded to a single nucleic acid molecule. It can be linked to. The tracrRNA used herein is also a "guide RNA skeleton". Also referred to as a sequence or "skeleton" sequence. The gRNA used herein is a single guide R Spacer for use with separate tracrRNA within NA and crRNA It includes both columns and separate molecules. The gRNA is complementary to the spacer sequence within the crRNA. It is possible to induce an active Cas complex at the sexual site as the target site, in this case Ca s-nucleases can cleave target sites. crRNAs target potential DNA target sequences. A region having complementarity with tracrRNA forms an interbase-pair hydrogen bond, and a secondary structure The structure may have a second region that can typically form at least one stem structure. Regions that have complementarity with the NA target sequence may be spacers or spacer sequences. TracrRNA and crRNA are two types of RNA that form the secondary structure of RNA, with many cells. They can interact via base-pair hydrogen bonds. tracrRNA / crRNA and Cas9 The formation of a complex with the protein facilitates the binding of the Cas9 protein to DNA. Information changes, Cas9 protein endonuclease activity, and endonuclease As a result, the crease Cas9 induces crRNA-induced site-directed DNA cleavage. It brings about (Svitashev et al., Nature Communications, 2016, 7, 113274). In reality, 2 The 0 nucleotide and guide RNA backbone are DNA (in vivo (in cells) or It is expressed from the promoter and transcribed in vitro (via T7 polymerase). (It may be formed to create an RNA guide), or the guide RNA backbone may be chemically synthesized. It can be a dual (crRNA and trRNA) guide, or a single guide RNA. .

[0050] The Cas9 protein / tracrRNA / crRNA complex targets double-stranded DNA sequences. To cleave the DNA target sequence, the protospacer adjacent motif (P) of the cognitive sequence It may be adjacent to AM. Design the crRNA to have an appropriate spacer sequence. By injecting the complex, it targets the desired gene locus, for example, the gene whose sequence modification is desired. It can be targeted to cut off in the constellation Leo.

[0051] In this technology field, various type II CRISPR-Cas-related crRNA sequences and tracr RNA sequences and their associated predicted secondary structures are publicly known (e.g., Ran, FA, et al., Nature, 2015, 520, 186-191; Fonfara et al., Nucleic Acids Research, 2014 See 42, 2577-2590). In certain aspects of this specification, Ran et al.'s Type II C The RISPR-Cas system is presented.

[0052] The spacer for the type II CRISPR-Cas system depends on the Cas protein used. In some cases, the target nucleic acid sequence located at the 5' end of the PAM may hybridize with the PAM. It may also hybridize with the target nucleic acid sequence, which is located on the 3' side. PAM is used It can vary depending on the Cas polypeptide being synthesized. For example, Streptococcus pyogenes. When Cas9 derived from is used, PAM is sequence: 5'-NRR-3'[in sequence, R can be A or G, N is any nucleotide, and N is the target. [Includes a target nucleic acid sequence located immediately 3' to the target nucleic acid sequence targeted by the binding nucleic acid sequence] It may be a sequence within the target nucleic acid sequence. Preferably, the PAM of Streptococcus pyogenes. It contains 5'-NGG-3'. Another example is S. thermophilus. When Cas9 derived from CRISPR3 (Sther CR3) is used, PAM is , sequence: 5'-nGGnG-3', which may be a sequence within the target nucleic acid sequence (Sapranausk See as et al., Nucleic Acids Research, 2011, 39, 9275-9282.

[0053] Other Cas proteins recognize other PAMs, and those skilled in the art can recognize any particular Cas protein The PAM for quality can be determined. In the art, the targeting sequence described herein is used. An increasing number of CAS proteins and systems suitable for use are known. Shah et al., See RNA Biol., 2013, 10, 891-899. Regarding the CAS system and its PAM sequences... A representative example is shown in Table 1 below.

[0054] [Table 1-1]

[0055] [Table 1-2]

[0056] Methods relying on any CRISPR / CAS system are used to control the distribution of Streptococcus pyogenes. For the column, the PAM sequences of NRR (e.g., NGG and NAA) presented below are It may also be present, and the sequence of S. thermophilus is presented. It may also have the PAM sequence of NGGNG. Table 3 and the sequence listing show the targeting sequence. The columns and chromosomal positions are presented. Those skilled in the art will recognize and utilize the target sequences disclosed herein. It will also be understood that known CAS systems with different PAM requirements can be engineered to recognize and utilize the target sequences disclosed herein. Examples of modifications are presented in Table 1.

[0057] The term "CRISPR / CasN" or "CRISPR / CasN system" refers to a programmable nuclease system for gene editing that includes a CasN (e.g., Cas2, Cas5, Cas6, Cas9, etc.) protein or its derivative and one or more non-coding RNAs that can provide the functions of CRISPR RNA (crRNA) and trans-activating RNA (tracrRNA) for CasN. The crRNA and tracrRNA may be used individually or combined to generate a "guide RNA" (gRNA). The crRNA or gRNA can provide a sequence complementary to the genomic target. The term "Cpf1" or "CAS12" refers to another programmable RNA-guided endonuclease (Zetsche et al., Cell, 163:759-771, 2015) of class 2 CRISPR-Cas systems that has been described and used for gene editing. This system can use a non-specific endonuclease unit derived from the Cpf1 protein family, and the specificity of cleavage is conferred by a single crRNA (lacking tracrRNA). Similar to Cas9, the Cpf1 coding sequence can be fused to the UTR sequences described herein to improve its stability and thereby improve the efficiency of the resulting gene editing method. The crRNA and tracrRNA may be used individually or combined to generate a "guide RNA" (gRNA). The crRNA or gRNA can provide a sequence complementary to the genomic target. The crRNA or gRNA can provide a sequence complementary to the genomic target.

[0058] The term "Cpf1" or "CAS12" refers to another programmable RNA-guided endonuclease (Zetsche et al., Cell, 163:759-771, 2015) of class 2 CRISPR-Cas systems that has been described and used for gene editing. This system can use a non-specific endonuclease unit derived from the Cpf1 protein family, and the specificity of cleavage is conferred by a single crRNA (lacking tracrRNA). Similar to Cas9, the Cpf1 coding sequence can be fused to the UTR sequences described herein to improve its stability and thereby improve the efficiency of the resulting gene editing method. This system can use a non-specific endonuclease unit derived from the Cpf1 protein family, and the specificity of cleavage is conferred by a single crRNA (lacking tracrRNA). [[ID=3�]]Similar to Cas9, the Cpf1 coding sequence can be fused to the UTR sequences described herein to improve its stability and thereby improve the efficiency of the resulting gene editing method. Similar to Cas9, the Cpf1 coding sequence can be fused to the UTR sequences described herein to improve its stability and thereby improve the efficiency of the resulting gene editing method. Similar to Cas9, the Cpf1 coding sequence can be fused to the UTR sequences described herein to improve its stability and thereby improve the efficiency of the resulting gene editing method. [[ID=X]] Similar to Cas9, the Cpf1 coding sequence can be fused to the UTR sequences described herein to improve its stability and thereby improve the efficiency of the resulting gene editing method.

[0059] As used herein, the term "TALEN (transcription activat or-like effector nuclease)" or "TALEN" refers to a nuclease engineered to enable targeted modification of a given DNA sequence. A TALEN can include a non-specific DNA cleavage nuclease fused to a TALE DNA binding domain that can be engineered to enable targeted gene editing. A "TA LE DNA binding domain" or "TALE" can be a polypeptide that includes one or more TALE repeat domains / TALE repeat units. The repeat domain can be involved in the binding of the TALE to its cognate target DNA sequence. A single "repeat unit" (also referred to as a "repeat") can be 33-35 amino acids in length and can exhibit at least partial sequence homology with other TALE repeat sequences within a naturally occurring TALE protein. A "designed" DNA binding protein can be a non-naturally occurring protein, the design / composition of which is obtained in principle from rational criteria. TALENs are discussed and disclosed in Joung et al., Nat. Rev. Mol. Cell. Biol., 20 13, 14, 49-55. A "selected TALE" can be a non-naturally found protein, the production of which is obtained primarily from empirical processes such as phage display, interaction trap ping, or hybrid selection. This specification presents the use of TALENs in pigs described herein and includes these. The single "repeat unit" (also called "repeat") can be 33-35 amino acids in length and can exhibit at least partial sequence homology with other TALE repeat sequences within a naturally occurring TALE protein. A "designed" DNA binding protein can be a non-naturally occurring protein, the design / composition of which is obtained in principle from rational criteria. and can exhibit at least partial sequence homology with other TALE repeat sequences within a naturally occurring TALE protein. A "designed" DNA binding protein can be a non-naturally occurring protein, the design / composition of which is obtained in principle from rational criteria. "Designed" DNA-binding proteins can be non-naturally occurring proteins, the design / composition of which is obtained in principle from rational criteria. A "designed" DNA-binding protein can be a non-naturally occurring protein, the design / composition of which is obtained in principle from rational criteria. For TALENs, see Joung et al., Nat. Rev. Mol. Cell. Biol., 20 13, 14, 49-55, where they are discussed and disclosed. A "selected TALE" can be a protein not found in nature, the production of which is obtained primarily from empirical processes such as phage display, interaction trap ping, or hybrid selection. This specification presents the use of TALENs in pigs described herein and includes these. ping, or hybrid selection. This specification presents the use of TALENs in pigs described herein and includes these. This specification presents the use of TALENs in pigs described herein and includes these.

[0060] "Resistance" or "disease resistance" refers to the ability of an organism to defend itself against attacks by pathogens. Alternatively, it refers to the degree to which the organism can resist and remain free from the disease. This organism demonstrates complete resistance. This means that the organism maintains a de facto immunity from the pathogen. Alternatively, the organism is a pathogen The degree of non-infection of an organism due to this may be less than that of an equivalent organism without resistance, resulting in partial resistance. This can be supported. Resistance allows organisms to avoid the consequences of organism-pathogen interactions. Resistance can arise from specific characteristics of an organism. Resistance is the ability of an organism to resist disease symptoms associated with a pathogen. Can it be avoided, or can the incidence / severity of clinical signs associated with the pathogen be reduced? This can be supported by the extent to which it can alleviate clinical symptoms associated with the pathogen.

[0061] The terms "increased resistance" and "decreased susceptibility" refer to infection by a given pathogen and related factors. A statistically significant decrease in the incidence and / or severity of related clinical signs or symptoms. It refers to a decrease. For example, "increased resistance" or "decreased sensitivity" refers to having an unmodified chromosomal sequence. Compared to control animals, animals with deletions or inactivation of chromosomal sequences within the CD163 gene The incidence of clinical signs or symptoms associated with PRRSv infection in materials and / Alternatively, it refers to a statistically significant reduction in severity. "A statistically significant reduction in clinical symptoms." The term means the incidence of at least one clinical symptom in the modified group, Without limiting to these, in some cases, clinical symptoms appear after a challenge by an infectious agent. Therefore, it can be statistically reduced by at least 80% compared to the unmodified control group.

[0062] As used herein, "reduction of the incidence and / or severity of clinical signs" or "clinical The term "reduction of bed symptoms" is used to describe the difference between wild-type infections with similar genetic backgrounds in other respects and The comparison involved a reduction in the number of infected individuals within the group, a reduction in the number of individuals exhibiting clinical signs of infection, or a decrease in the number of individuals exhibiting clinical signs of infection. This means loss, or reduction in the severity of any clinical sign present in one or more subjects. For example, these terms are used when compared to those with a similar background in other respects. Infection, pulmonary pathology, viremia, any clinical signs, antibody production, reduction of pathogen burden, disease Encompassing pathogen shedding, reducing pathogen transmission, or alleviating any symptomatic clinical signs associated with PRRS. Includes. Comparison of clinical signs between unedited CD163 pigs and CD163-edited pigs is performed individually. This can occur in some cases, and also between groups. In some instances, individual pigs may not present clinical signs. In some embodiments, the CD163-edited pig population exhibits a reduction in clinical signs. The animal group may consist of at least 100 animals. In one embodiment, CD16 of this specification 3. Edited pigs may have reduced clinical signs of reproductive syndrome. In some embodiments, CD163 edited pigs In this animal group, the number of immature births may be reduced, resulting in stillborn piglets or mummified piglets. The number may be reduced, and the number of PRRSv-positive piglets may be reduced, and female piglets In some cases, the delay in the return of the ta to breeding behavior may be reduced. In one embodiment, CD1 of this specification 63 Edited: Pigs show reduced loss of appetite, reduced fever, reduced drowsiness, reduced pneumonia, and reduced agalactia. , as well as the reduction of subcutaneous edema and hind limb edema, including but not limited to female pigs and It may show a reduction in clinical signs in nulliparous pigs. In one embodiment, CD163 of this specification In grouped pigs, including heifers and sows, red / blue discoloration of the ears and vulva may be reduced. In one embodiment, CD163 edited sows exhibited a low delay in returning to estrus after weaning. can exhibit a decrease. This specification presents a reduction in deaths in a group of CD163 - unedited piglets and CD163 - edited female pigs, and includes these. This specification also includes a reduction in clinical symptoms in fattening pigs, and presents these as well. In one aspect, the group of CD163 - edited fattening female pigs can have reduced respiratory clinical symptoms selected from the group consisting of fever, sneezing, tachypnea, dyspnea, coughing, pneumonia, lethargy, periorbital edema, and ocular and nasal discharge. Preferably, in one or more animals of the present teachings, these clinical symptoms have no modification within the CD163 gene, have a similar background, and can be reduced by at least 10% compared to infected subjects with a similar background. In one aspect, the clinical symptoms can be reduced by at least 80% in the subjects of the present teachings. In another aspect, the clinical symptoms can be reduced by at least 85% in the pigs of the present teachings. In a further aspect, the clinical symptoms can be reduced by at least 90% in the pigs of the present teachings. In yet another aspect, the clinical symptoms can be reduced by at least 95% in the pigs of the present teachings. In one aspect according to the present disclosure, the clinical symptoms can be reduced by 80% - 100% compared to unedited CD163 pigs. As used herein, the term "breeding" refers to a process that includes the use of superior male animals and superior female animals to create selected offspring of the next generation. This process further includes the fusion of male gametes and female gametes such that fertilization occurs. Such fusion can be brought about by mating (copulation), or can be brought about by artificial methods in vitro or in vivo. Such artificial methods include artificial insemination, surgically assisted artificial insemination, in vitro fertilization, intracytoplasmic sperm injection, zona pellucida opening, in vitro culture of fertilized oocytes,

[0063] ​ This may include, but is not limited to, nourishment, ovarian introduction, and ovarian division. The term "breeding" also refers to the process of enabling more offspring from a particular superior female. This may also include the introduction of fertilized oocytes into the reproductive tract of female animals.

[0064] As used herein, "reference sequence" refers to the sequence used as the basis for sequence comparison. It is a specified array. The reference array may be a specific subset or the entirety of an array; for example, It may also be a reference sequence as a segment of the full-length promoter sequence, and the complete promoter sequence In some cases, the reference array may be a column. It could be a reference genome (GenBank accession number: GCA_000003025.6).

[0065] In this specification, two or more nucleotide sequences or protein sequences are referred to. The terms used, “identity percentage” or “~ percent identical,” are ( i) Two optimally aligned sequences (nucleotide sequences or protein sequences) (ii) In both sequences, the same nucleic acid bases (nucleotides) are compared across the comparison region. Determine the number of positions where a sequence (or amino acid residue) exists (for proteins). (iii) Then, find the number of matching positions, and the number of matching positions within the comparison domain (iv) Divide by the total number, and then multiply this quotient by 100% to create the identity percentage. It is calculated by doing so. The "identity percentage" is calculated without specifying a particular comparison range. When calculated for a matching array, the identity percentage matches across the alignment region. The number of positions can be determined by dividing by the total length of the reference array. For the purpose of the request, the two sequences (query sequence and target sequence) are optimally aligned. If you want to allow gaps in those alignments, then the query array will be the same "Uniformity Percentage" is the number of identical positions between two arrays, multiplied by their total length in the query array. It is equal to the number obtained by dividing it by the total number of positions across the comparison domain, and then multiplying it by 100%. It is possible. When the percentage of sequence identity is used for proteins, non-identical residue positions The position can often differ due to conservative amino acid substitutions, in which case the amino acid residues are the same Substitution with other amino acid residues having different chemical properties (e.g., charge or hydrophobicity) There is a combination, and it is recognized that this does not change the functional properties of the molecule. The sequence is preserved. In cases where there are differences in the persistence of a permutation, percent sequence identity corrects for the conservative nature of the permutation. It can be adjusted upwards in this way. Different sequences can be created by such conservative substitutions. It is said to have "similarity" or "similarity".

[0066] For optimal alignment of sequences for calculating identity percentages, in the art This refers to sequence identity between two or more nucleotide or protein sequences. Alternatively, ClustalW or Basic Lo can be used to compare sequence similarities. cal Alignment Search Tool (BLAST (registered trademark), Na (National Library of Medicine, Bethesda, MD) Various pairwise array alignment algorithms / programs or multiple array alignments Alignment algorithms / programs are publicly known. In this art, other alignment methods And comparison methods are also known, but the alignment and identity percentage between the two sequences ( The range of identity percentages described above (including the range of identity percentages) is the ClustalW algorithm. This can be determined by (for example, Chenna, R., et al., Nucleic Acids Research, 2003, 3) 1, 3497-3500; Thompson, JD, et al., Nucleic Acids Research, 1994, 22, 4673-468 0; Larkin MA et al., Bioinformatics, 2007, 23, 2947-2948; and Altschul, SF, et al. See al., J. Mol. Biol., 1990, 215, 403-410.

[0067] The identity of the sequences used herein is determined according to the alignment of the two sequences. This is expressed in terms of the percentage of identity between two sequences. This specification refers to adjacent genome regions. A restoration template array is provided that can have at least 80% identity with respect to the region. In one embodiment, The repair template can share at least 80% identity with the adjacent genomic region, 5 Identity of the 'region and region 3' with respect to the chromosomal sequence flanking the intended gene editing site. It may have a core domain.

[0068] This specification is selected from the group consisting of Sequence IDs 22-271 and 347-425. We present target sequences and target sequences that are 100% identical to them, and include, but are not limited to, these. Not done. These target sequences may include PAM sequences, as listed in Table 1. However, in a certain embodiment, when incorporated into guide RNA, the spacer region becomes part of the sequence. The first 20 nucleotides of each of numbers 22-271, and each of sequence numbers 347-425 They share 100% identity with sequences selected from the first 20 nucleotides of each group. Also, 15 to 20 nucleotides from each of sequence numbers 22 to 271, or sequence numbers. Spacer sequences that may contain 15 to 20 nucleotides from each of the numbers 347 to 425. The remaining sequence may include PAM sequences that can exist within the genome, but not gRNA molecules. (Does not include). Also, guide R may have one or more mismatches with the target sequence. NA spacer arrays are also included and are presented. In one embodiment, the mismatch is To ensure that identity is maintained at the donuclease cleavage site, the target sequence This can be a mismatch at the distal end. Typically, the mismatch is a nucleus at the 3' end. Compared to the crease site, it may be located at the 5' end of the target sequence. In one embodiment, The target sequence, or the spacer sequence of the guide RNA prepared therefrom, is a single mismatch. It may have. In another embodiment, the target sequence, or the space of the guide RNA prepared therefrom The sequence may have two mismatches. In another embodiment, the sequence, or prepared therefrom The spacer sequence of the guide RNA may have three mismatches. In another embodiment, 4 A sequence that may have fewer than one mismatch, or a spacer for guide RNA prepared therefrom. - Includes a sequence. In some embodiments, the mismatch region is relative to the PAM sequence and the cleavage site. This can be limited to terminal nucleotides located distally.

[0069] In one embodiment, the target sequence and the spacer sequence of the gRNA guide are sequence numbers 22-2 The first 20 nucleotides of each of 71, and the first 2 of each of sequence numbers 347-425 It may have at least 90% identity with sequences selected from the group consisting of 0 nucleotides. In a further embodiment, the RNA guide has 15 nucleotides at the 3' end of the sequence and at least 9 It may have 0% identity and 100% identity. In one embodiment, the target sequence and the RNA molecule The id may have at least 95% identity. In a further embodiment, the RNA guide may At least 95% or 100% identity with the 15 nucleotides at the 3' end of the sequence. It may have. In one embodiment, the target sequence and the RNA guide have at least 96% identity. It may be. In a further embodiment, the RNA guide has 15 nucleotides at the 3' end of the sequence and a small At the very least, it can have 96% or 100% identity. In one embodiment, the target sequence and RNA guides can have at least 97% identity. In a further embodiment, RNA guides The id has at least 97% identity or 100% identity with the 15 nucleotides at the 3' end of the sequence. They may have the same identity. In one embodiment, the target sequence and the RNA guide are at least 98% identical. It may have identity. In a further embodiment, the RNA guide is the 15 nucleus at the 3' end of the sequence. It may have at least 98% or 100% identity with Ochido. In some embodiments, The target sequence and the RNA guide may have at least 99% identity. In a further embodiment, The RNA guide has at least 99% identity with the 15 nucleotides at the 3' end of the sequence. They may have 100% identity. In some embodiments, the target sequence and the RNA guide are less than At least 100% identity can be achieved. This specification also applies to each of Sequence IDs 22 to 271. The first 15 nucleotides of, or the first 15 nucleos of each of sequence numbers 347-425. Spacer arrays that may include cyds are also included and are presented. In one embodiment, spacer - The sequence is the first 16 nucleotides in each of sequence numbers 22-271, or sequence number Each of numbers 347-425 may contain the first 16 nucleotides. In another embodiment, spe The sequence is the first 17 nucleotides in each of sequence numbers 22-271, or the sequence It may include the first 17 nucleotides in each of the sequence numbers 347 to 425. In one embodiment, The spacer sequence consists of the first 18 nucleotides in each of sequence numbers 22-271, This may include the first 18 nucleotides in each of sequence numbers 347-425. In another embodiment, the spacer array is the first 19 nuclei in each of sequence numbers 22 to 271. Reotide, or the first 19 nucleotides in each of SEQ ID NOs. 347-425 Shut up.

[0070] In this specification, the preferred meaning of "fertilization" as used to refer to the breeding of pigs is viable. This includes any technique for generating embryos. Fertilization involves insemination of sows and in vitro fertilization. This can include both fertilization or ex vivo fertilization. Generally, it is beneficial for inseminating sows. There are three available methods: conventional artificial insemination (AI), intrauterine insemination (IUI), and There is also deep intrauterine insemination (DIUI). All of these techniques involve the amount of fertilization, typically It relies on freshly collected, unfrozen semen. In vitro fertilization (IVF) involves the collection of unfertilized oocytes and Following this, these oocytes, as with standard ET, in vivo (i.e., Living animals; an alternative to in vitro (i.e., laboratory) fertilization (as discussed above) Fertilization can occur through semen. Subsequently, fertilized oocytes derived from an oocyte donor may also The embryo can be transferred to another female (embryonic recipient). Embryo transfer (ET) is performed by transferring the embryo to one female (embryonic recipient). The collection of fertilized oocytes or embryos from the donor, and the reproductive status of these embryos, are determined by the donor. This could involve transplantation into another female (embryonic recipient) synchronized with the breeding process.

[0071] As described by Cameron et. al. (Nat Methods, 2017, 14, 600-606), CRIS The PR-Cas9 system is used in both basic research and biotechnology, and in genome editing. It can be used for gene or genome editing using CRISPR technology and related technologies. Application to [unspecified area] may result in undesirable off-target cleavage activity, as well as potential pathogenicity and other [unspecified]. This can result in negative phenotypic outcomes. In recent years, various approaches have been developed to minimize off-target cleavage. Although various genome-wide experimental methods have been developed, some methods are limited to intracellular contexts. This is potentially biased due to inefficiencies in the proper recovery of the amputation site. (Cameron et al.) The SITE-SEQ® assay (Caribou Biosc) disclosed in al. The method developed by iences, Inc. (Berkeley CA) shows that Cass in the sample genome The nine cleavage sites are comprehensively listed, followed by (1) extraction and purification of high molecular weight genomic DNA. (2) In vitro ribonucleoprotein (RNP) mediated by Cas9 (3) Disconnection, fragmentation, adapter ligation, and Cas9 disconnection fragments Affinity purification for concentration, and (4) ILLUMINA (registered trademark) (ILLUMINA®, San Diego, CA) For sequencing Tracking via amplification and indexing of the SITE-SEQ® library. In the experiment, these sites can be explored for intracellular off-target editing (Ca See meron et al., Nat Methods, 2017, 14, 600-606. Genome editing technology So, what other diverse methods are there to discover potential off-target edits and reduce their number? It is publicly known. Such bioinformatics tools identify appropriate target sequences. Therefore, it can be extremely beneficial, but the error rate is generally higher than desired, and the system has a wide distribution. If column data is missing, the method may be limited. Furthermore, the fidelity of DSB disconnection repair may vary. This can hinder the ability to predict and prevent undesirable off-target effects. Furthermore, testing of computer-selected target sequences showed high efficiency and a small number of Selected sequences with fluate target cleavage can be identified. High-throughput sequencing In combination with the spectroscopy method, in-cell culture testing is used to identify low-performing target constructs. It can be used.

[0072] For example, Streptococcus pyogenes (Streptococcus pyogenes) The type II CRISPR system, derived from Cas9, interacts with the skeletal sequence and sequence within mammalian cells. The first 20 nucleotides of each of numbers 22-271, and sequence numbers 347-425 Each includes a sequence selected from a group consisting of the first 20 nucleotides, a specificity-determining type CRISPR RNA (crRNA), and auxiliary transactivating RNA (trac It can be reconstituted using rRNA. The term "off-target effect" is broadly used to mean off The results of the target treatment or procedure differ from those intended, and are not arbitrary. This refers to the effect of off-target editing. An example of off-target editing is when unintended actions occur in an unintended location. A double helix, resulting in the insertion, deletion, or rearrangement of a nucleotide or repair template DNA. This may include cleavage. Some systems, for example, Streptococcus pyogenes and S. thermoph In the CRISPR system of S. thermophilus, crRNA double strands and tracrR NA double helix can be fused to produce a single guide RNA (sgRNA). The first 20 nucleotides of RNA can be complementary to the target DNA sequence. It can be a spacer region, and at the 3' end of these 20 nucleotides Then, a protospacer-adjacent motif (PAM) within the genome (not within the guide) follows. In one embodiment, the first 20 nucleotides and PAM sequence of the sgRNA are sequence number The sequence may be selected from the groups consisting of numbers 22-271 and 347-425. Therefore, as shown in Table 2 below, and sequence numbers 22-271 and 347-425 As you can see, it is either Streptococcus pyogenes or S. thermophilus. Specific targeting by Cas nucleases derived from either of these targets crRNA in bone. This can be achieved by combining it with a specific sequence. The 20-nucleotide guide of sgRNA The d-sequence + PAM sequence (e.g., 23-25 ​​nucleotides) is a tightly controlled targeting This can result in cleavage and breakage of the distal portion of the sgRNA guide sequence relative to PAM. In this case, off-target cleavage activity involving mismatches between 3-5 base pairs is observed in the DNA sequence. It has been found that this can still occur in [location]. Furthermore, different types of guide RNA The structure actually increases or decreases cleavage in off-target areas. This affects cutting accuracy. In addition to various techniques for off-target cutting, further notes... For more information, see Zhang et al., Mol Ther Nucleic Acids, 2015, 4, e264. It is being done. The mechanism and operation of off-target cutting are still not well understood, however This means that it may be difficult to predict and compensate for its effects. The consequences can be serious: off-target cuts can ultimately lead to genomic instability. This can disrupt the functionality of other normal genes.

[0073] This specification describes the location in which both alleles of the CD163 gene may have inactivation editing. This provides pigs that may be resistant to infection by PRRSv, and includes these The CD163 pigs disclosed herein further comprise novel sequences or polypeptides. First, it also includes non-natural amino acids resulting from frameshift mutations or missense mutations. No.

[0074] The gene editing disclosed herein affects CD163-positive fetuses (e.g., one or two This enables the protection of fetuses that have the wild-type CD163 allele. CD163-positive fetuses As long as the mother possesses inactivation editing in both alleles of the CD163 gene In this case, protection from intrauterine PRRSv infection may be possible (PCT / US2018 / 0 (Specification No. 27944). For example, inactivation of both alleles of the CD163 gene. The parent with editing is crossed with a male that has two wild-type CD163 alleles. There is a risk of this occurring, and the resulting heterozygous fetus is affected by PRRSv infection in the uterus. They will be protected.

[0075] In one embodiment, the method described herein is used to inactivate one allele of CD163 Pigs with editing can be produced. Heterozygous CD163 allele (one of which is edited) Pigs that possess (one of which is wild type) also have heterozygous CD163 antagonists. By crossbreeding with other pigs that have the gene or a homozygous edited CD163 allele. In some cases, offspring derived from this breeding program may have a homozygous edited CD163 allele. Grandchildren may be selected for resistance to infection by PRRSv. This disclosure also concerns animals However, if homozygous for one or more specific genetic markers or alleles This may involve creating or improving pig animals and pig populations, as well as creating or improving pig animals and pig populations. Methods for doing so are also presented, and these are included. In various aspects, this disclosure includes one or more To create animals that can be heterozygous for a specific genetic marker or allele, The aforementioned animals were subjected to one or more of a specific genetic marker or allele. By crossbreeding with other animals that are terozygous, one or more specific genetic markers are identified. Alternatively, by creating animals that can be homozygous for alleles, we can create pig animals and This invention provides animals and methods for creating or improving the breeding of pig populations, including these. In some embodiments, homozygosity for each of the specific genetic markers and alleles. Multiple rounds of mating and / or backcrossing may be required to obtain the desired sex. In this configuration, a single mating may be sufficient to obtain homozygosity.

[0076] Various techniques known in this field inactivate genes to create knockout animals. and / or edited genes are introduced into animals to create pioneering kinetics, and knockout or Nucleic acid constructs can be used to create animal lineages that can be incorporated into the genome. In terms of techniques, to put it without limitation, there is prenuclear microinjection (USA 4,8 Specification No. 73,191), retrovirus-mediated gene transfer into germline cells (Van de Putten et al., Proc. Natl. Acad. Sci. USA, 1985, 82, 6148-1652), to embryonic stem cells Gene targeting (Thompson et al., Cell, 1989, 56, 313-321), electroporation of embryos pore (Lo, Mol. Cell. Biol., 1983, 3, 1803-1814), sperm-mediated gene transfer (Lavitrano et al., Proc. Natl. Acad. Sci. USA, 2002, 99, 14230-14235; Lavitrano et al., Rep rod. Fert. Develop., 2006, 18, 19-23), and cumulus cells in vitro as well. Alternatively, this may involve the transformation of somatic cells such as mammary gland cells, or adult, fetal, or embryonic stem cells. , nuclear transfer (Wilmut et al., Nature, 1997, 385, 810-813 and Wakayama et al., Nature, This may include (1998, 394, 369-374). Pronuclear microinjection, sperm-mediated inheritance. Transfusion and transfusion of somatic cells may be other useful techniques. Animals have all of their cells It can be genetically edited in the sense that it involves editing of germline cells. If a method is used to create an animal that is a mosaic, the animal is a mosaic animal. In addition, in the case of inbred animals, the offspring that are genetically edited are produced using standard methods. They can be produced and selected. For example, if the cells are edited at the blastocyst stage, they can form mosaic animals. Cloning may be used to create it, and if a single cell is edited, the genome Modifications may also be made. In some embodiments, inactivation knockout editing is homozygous. It may be possible.

[0077] In embryo / zygote microinjection, nucleic acid constructs, mRNA, proteins, Polynucleotides, or combinations thereof, can be introduced into a fertilized egg. In one embodiment, One or two-cell fertilized eggs can be used. This results in a visible nuclear structure containing genetic material derived from the sperm head and egg cell within the protoplasm. It is possible. In some embodiments, pronuclear stage fertilized eggs may be obtained in vitro. In some cases, eggs can be obtained in vivo (i.e., by surgery, from donor animals). (It is recovered from the tube). In another embodiment, the in vitro fertilized egg is, for example, obtained by a method known in the art. It can be produced from the ovaries of recovered wild pigs using the follicles aspirated. Agung et al., J Reprod Dev., 2013, 59, 103-106 and Appeltant et al., J Repro See d Dev., 2016, 62, 439-449. In one embodiment, mature oocytes are used for in vitro fertilization. It may be made available for use in law. In another embodiment, mature oocytes are used in the CR of this specification. The ISPR / Cas gene editing system may be injected.

[0078] This specification further presents in vitro fertilization of mature oocytes. In one embodiment, the oocytes are As presented above, it can be matured in vitro. In another embodiment, Mature oocytes can be retrieved from nulliparous pigs. In vitro fertilization is performed according to established methods. Appeltant et al., J Reprod Dev., 2011, 57, 9-16; and Fowler et al., Repr See od Biol., 2018, 18, 203-211.

[0079] The zygote or embryo is obtained by artificial insemination and flushing for germline editing. It is possible. Then, using the method presented herein, the recovered zygote or embryo is It can be edited. In some aspects, linear nucleic acid constructs, mRNA, proteins, polynucleotides A cyd, or a combination thereof, is one of the pronuclei, or a cell in the zygote or embryo after fertilization. It may also be injected into spores or pre-fertilized gamete cells, or by other methods (e.g.) For example, it may be introduced by electroporation. In one embodiment, Cas nucleazetane Protein, and the skeleton of Sequence ID No. 19, and Sequence ID Nos. 22-271, and 347-425 Includes a guide RNA containing a targeting sequence selected from one of the following, The resulting RNP complex can be prepared and injected into an embryo, zygote, or oocyte. In another embodiment, the Cas nuclease and the skeleton of Sequence ID No. 19 and Sequence ID Nos. 22-27 From RNA sequences selected from the group consisting of the first 20 nucleotides of each of 1 A pre-formed RNP complex containing a guide RNA with a selected spacer sequence is The prepared repair template, along with sequence numbers 1-13 listed in Table 6, is used with the embryo, zygote, and Or it can be injected into oocytes. This specification also describes spacer sequences and the ones listed in Table 6. This also includes combinations with repair templates that may have sequences of sequence numbers 1 to 13, and these are also presented. In one embodiment, the injected zygote or embryo is transferred to the recipient female (for example, the recipient female). It is implanted (into the oviduct of a female ent) to create a transgenic animal or a gene-edited animal. In this manner, it can be developed in the recipient female. In one embodiment, the method involves precipitation of lipids. To allow visualization of the pronuclei, the samples were centrifuged at 15,000 × g for 5 minutes. The zygotes can be separated, fertilized in vitro or in vivo. The embryo is further provided. The zygote or embryo is EPPENDORF® FEMT. OJET® (EPPENDORF® AG, Germany) injection It can be injected using a container and cultured until blastocyst formation. The rate of embryonic division, and the blastocyst The formation and quality of the product can be recorded.

[0080] In one embodiment, the CRISPR / Cas editing system and gRNA are presented as nucleic acid constructs. It is possible. In another embodiment, Cas nucleases have a skeletal sequence and sequence numbers 22-271 The first 20 nucleotides of each, and the first 20 nucleotides of each of sequence numbers 347-425 m It can be presented as RNA. In another embodiment, Cas nucleases consist of a skeletal sequence and a sequence number. A targeting molecule selected from the group consisting of the first 20 nucleotides of each of numbers 22-271. Along with the guide RNA containing the sequence, the group consisting of sequence numbers 1 to 13 is listed in Table 6. It can be presented as mRNA in combination with a selected repair template. In a further embodiment, gene tags The nasal nuclease and skeletal sequence, along with sequence numbers 22-271 The first 20 nucleotides of each of the sequence numbers 347-425 It encodes a guide RNA containing a targeting sequence selected from the group consisting of rheosides. It can be presented by microinjection of transcriptable DNA. In this case, the skeletal sequence may be the sequence of sequence number 19. Furthermore, this specification also refers to the targeting A repair template that may have a ping sequence and one of the sequences listed in sequence numbers 1 to 13. Combinations with the above are also included and are provided. By those skilled in the art, the objectives of this method can be achieved. In the oocyte, various combinations of nucleases, gRNAs, and arbitrarily selected repair templates are found in the oocyte. It will be understood that it can be introduced into the blastula, zygote, blastula, and embryo. Other skeletal arrangements For use with other Cas nucleases, see Sequence IDs 22-271 and Sequence IDs. It is further understood that it can be used in conjunction with targeting sequences 347-425. Any Cas nuclease having the PAM sequence of NGG or NGGNG is this Suitable for preparing skeletal sequences for combination with targeting sequences of the specification. Various embodiments Therefore, the desired edit may have the final genome sequence of sequence numbers 426-505. Partial composition Therefore, the desired edit lacks the 7th exon and the final nucleus shown in sequence numbers 426-458. It may contain a rheotide sequence. In diverse configurations, the final genome sequence may include an exogenous stop codon. It is possible to have the final genome sequence shown in sequence number genome ~504. In this configuration, these partial CD163 genes are shown in SEQ ID NOs. 506-517. It may have the amino acid sequence of exon 7.

[0081] In one embodiment, the injected zygote or embryo is transferred to the recipient female (for example, the recipient female It is implanted (into the oviduct of a female) to create a transgenic animal or a gene-edited animal. Thus, it can be developed in the recipient female. In one embodiment, the method involves depositing lipids To enable visualization of the pronuclei, the sample was centrifuged at 15,000 × g for 5 minutes. A zygote that can be separated, fertilized in vitro or in vivo It further provides embryos. The zygote or embryo is EPPENDORF® FEMTO It can be injected using the JET® injector and cultured until blastocyst formation. The rate of development, as well as the formation and quality of the blastocyst, can be recorded.

[0082] This specification describes the possibility of injecting CRISPR / Cas / gRNA combinations in accordance with this disclosure. A recipient sow having one or more embryos is provided, and these are included in one embodiment. So, the Cas nuclease and the skeleton of Sequence ID No. 19, and each of Sequence IDs No. 22-271 The first 20 nucleotides and the first 20 nucleotides of each of the sequence numbers 347-425 A pre-formed guide RNA containing a spacer sequence selected from the group consisting of D An RNP complex may be provided. In various embodiments, Cas nuclease and SEQ ID NO: 19 skeletons, each consisting of the first 20 nucleotides of sequence numbers 22-271 and their respective sequences. Includes sequences selected from the group consisting of the first 20 nucleotides of each number from 347 to 425. Two pre-formed RNP complexes containing guide RNA with different spacer sequences. Combination may be provided. Also, Cas nuclease and the first of each of sequence numbers 22-271 From the first 20 nucleotides of each of the sequence numbers 347-425 A guide RNA containing a targeting sequence selected from a group of pre-formed elements may be included. Recipient sows that may have one or more embryos injected with the RNP complex are also offered. These are also provided. In one embodiment, a pre-formed RNP complex is prepared and presented. In conjunction with the repair templates listed in 6 (SEQ ID NOs: 1-13), the embryo, zygote, or oocyte is used. It can be injected into. In one embodiment, the recipient sow is a female of a different lineage than the donor embryo. It could be a pig.

[0083] The embryo or zygote can be surgically implanted into the uterus of a synchronizing recipient. Typically, , 100-200 (or 150-200) embryos or zygotes, using a catheter It can be deposited at the ampulla-isthmus junction of the fallopian tube. After surgery, regarding pregnancy, Real-time ultrasound examination may be performed. In some embodiments, this specification is 1 to 100, More typically, embryos treated with a combination of 30-60 CRISPR / Cas / gRNAs It may include recipient sows having embryos, in which case the embryos are sequence numbers 1- It may contain the gene-edited CD163 gene, which includes sequences 18 or 426-505. In some embodiments, the transplanted embryo may contain a mosaic of edited cells. Alternatively, it may be non-mosaic. This also includes CD163 gene-edited embryos.

[0084] Methods to improve the health status of livestock animals or herds of livestock include targeting within the genome of animal cells. The procedure may include modifying the columns to form a cleavage-type CD163 polypeptide. Improved animals that may possess fragmented CD163 polypeptides have also been presented, and these are also included. In this embodiment, the predicted cleaved polypeptide does not contain non-natural amino acids. Theoretically, To put it simply, cleaved gene products, in vivo, are intracellular proteas It can be rapidly digested by -se. In some aspects of this specification, cleaved CD16 3 results in the complete disappearance of the protein. In a further embodiment, cleaved CD1 63 may be undetectable in vivo. In one embodiment, cleaved CD16 3 may be undetectable by immunofluorescence labeling and FACS analysis. In yet another embodiment... The cleaved form of CD163 is one of the expression analysis methods presented herein, It may be undetectable when using expression analysis methods, including the expression analysis method presented in Example 5. In yet another embodiment, cleaved CD163 is one of the expression analysis methods presented herein. In either case, when using an expression analysis method including the expression analysis method presented in Example 8 It may be undetectable.

[0085] In another embodiment, CD163 gene-edited pigs have cleavage-type CD163 with 25 or fewer non-natural amino acids. It can contain 163 genes. Speaking without being limited to theory, the cleaved CD163 protein is Therefore, it can be destabilized and targeted for degradation within cells. CD163 protein sequences and CD163 polypeptides containing amino acids are undetectable. It is possible. In another embodiment, the predicted cleaved CD163 protein is the same as the native protein. Ours may contain 211 or fewer amino acids. In one embodiment, cleaved CD163 protein It is predicted that it will contain 144 or fewer amino acids from natural proteins. In this case, the cleaved CD163 protein is one of the amino acids of the natural protein, with 133 or fewer amino acids. It is predicted that it will contain acid. In yet another embodiment, the cleaved CD163 protein is natural It is predicted that the natural protein will contain 129 or fewer amino acids. In one embodiment, The cleaved CD163 protein contains 113 or fewer amino acids compared to the natural protein. It can be predicted that... In another aspect, the cleaved CD163 protein is a natural tan It is predicted that the protein will contain 108 or fewer amino acids. In a further embodiment, The cleaved CD163 protein contains 93 or fewer amino acids compared to natural proteins. This can be predicted. In yet another aspect, the cleaved CD163 protein is a natural protein It is predicted that the content will contain 74 or fewer amino acids, as presented herein. The cleaved CD163 protein is derived from 32-2 of the natural CD163 polypeptide sequence. It can contain 11 amino acids and 25 or fewer unnatural amino acids, and intracellular It may be undetectable in cell extracts.

[0086] In other embodiments, the modified CD163 protein may have cleavage by a single amino acid substitution. In some embodiments, the modified CD163 protein has a predicted amino acid count of 1,010 amino acids or less. It may have an acid sequence.

[0087] This specification describes the cleavage of the CD163 protein (e.g., the CD163 reference sequence, NP). We present a CD163 gene-edited pig that may have amino acids 1-40 of _999141, These include these. To put it without being limited to theory, signal peptides (and also signal sequences and (Even if it is publicly known) cleavage within the cell results in impaired translocation of the protein to the cell membrane. Furthermore, it is thought that the polypeptide is subsequently broken down within the cell. Therefore, CD163 protein with cleavage of the nal peptide is resistant to infection by PRRSv. This results in a CD163 null animal that is resistant. In one embodiment, within the signal sequence, the protein In CD163 gene-edited animals, the quality is cleaved, and of the non-natural amino acid sequences, 25 The following amino acids may be included. In one embodiment, the gene-edited pig contains the natural CD163 protein. The quality may include the first 39 or fewer amino acids. In one embodiment, gene-edited pigs The first 39 or fewer amino acids of the natural CD163 protein, and 15 or fewer non-natural amino acids. May contain amino acids. This specification also refers to the first of the natural CD163 proteins. Genetically edited pigs, which are expected to contain 38 or fewer amino acids, are also included and are presented. In one embodiment, gene-edited pigs are the first of the natural CD163 proteins. It may contain 38 or fewer amino acids and 15 or fewer non-natural amino acids. Furthermore, according to this specification, It is predicted that the natural CD163 protein will contain the first 36 or fewer amino acids. This also includes gene-edited pigs, which are also presented. In one embodiment, gene-edited pigs are The first 36 or fewer amino acids of the natural CD163 protein, and 15 or fewer non-natural amino acids. It may contain amino acids. The cleaved proteins are undetectable even by methods known to those skilled in the art. It's possible.

[0088] In one embodiment, gene-edited pigs have the first 34 of the natural CD163 protein It is predicted that it will contain amino acids that do not exceed a certain value, and non-natural amino acids that do not exceed 15. In one embodiment, gene-edited pigs have more than the first 32 of the natural CD163 proteins. It is predicted to contain natural amino acids and no more than 15 non-natural amino acids. The protein may be undetectable even by methods known to those skilled in the art.

[0089] Furthermore, this disclosure indicates that the exogenous stop codon is located in exon 7 (SEQ ID NOs: 459-504). The introduced sequence may include a deletion in exon 7 (sequences 426-458), C This shows the cleavage of the D163 protein, including these cleavages. There are more than 1,010 of these cleavages. It may contain CD163 protein, which includes amino acids that are not present.

[0090] The breeding method uses animals that are homozygous for inactivated genes, as well as early homozygous founder plants. It is used to create heterozygous wound agents or other heterozygous animals. Homozygous animals can be produced. Gene-edited pigs described herein are edited In order to ultimately produce pigs that are homozygous or heterozygous for genes, other Edited pigs or wild-type pigs of the intent can be crossbred. In some embodiments, homozygous animals PIC (trademark) superior pig strains 2, 3, 15, 19, 27, 62, 65, and these It can be any one of the animal strains produced by crossbreeding with a combination. So, homozygous animals are produced by crossing animals from lineage 2 with animals from lineage 3. It could be a hybrid animal.

[0091] Gene editing is being used to address a variety of diseases in the Suidae family, including PRRS. It is being used. Over the past 15 years, several gene editing technologies have been developed. Generally, these platforms introduce double-strand breaks into specific regions of the genome. It can be designed in such a way. Then, the introduced cleavage can be repaired by the cell's own mechanisms. ru.

[0092] One repair pathway, non-homologous end joining (NHEJ), has evolved throughout the entire biological world. It is a conserved and major double-strand break repair pathway in mammalian cells. The process results in random insertions or deletions of nucleotides across the cleavage site. This results in the DNA repair template. As a practical problem, this imperfect fidelity is used in trans. It can be used to bring about advantages by bringing about a target site, Alternative alleles (e.g., single nucleotide polymorphisms, or single or multiple bases) DNA repair templates containing insertions or base deletions are co-delivered with gene editing reagents. In some cases, DNA repair templates may be used to repair new DNA through the use of homologous recombination mechanisms in cells. It can be directed to produce alleles. In one embodiment according to this specification, DN A repair mold allows for high-efficiency CRISPR / CAS cutting positions (e.g., according to Table 3, high editing frequency). Introducing deletions that result in cleaved proteins containing only the wild-type sequence, (degrees), and The repair template can be designed and selected based on its ability to perform gene editing. This can increase the value. DNA repair templates suitable for the method of this application may include SEQ ID NOs: 1 to 13. In one embodiment, the DNA repair template is matched with the guide sequences described in Table 6 below. Unlike gene transfer, genome editing as described herein involves the addition of foreign DNA sequences to the genome. It does not lead to implementation as a result.

[0093] However, these exogenously added DNA repair templates are randomly combined within the genome. Since there is a possibility of being included, the junction end spans the junction end, in frame translation end The resulting deletion of the DNA sequence leads to the creation of a stop codon. Identifying and using id pairs is also advantageous; two guides, by NHEJ, end When repaired using the terminal method, this new DNA sequence is transcribed into mRNA and then used to produce proteins. If translated into quality, it may terminate the production of the CD163 protein. The function of the CD163 protein is lost, but not always, often, in protein synthesis Immature terminations involve the formation of unstable polypeptides that can be degraded but are undetectable by standard methods. This results in the CD163 gene forming an exogenous stop codon at the junction end. The guide pair for cleaving the 7th exon is sequence numbers 351 and 365, sequence number 3 51 and 387, Sequence IDs 348 and 390, Sequence IDs 348 and 388, Sequence No. Numbers 348 and 395, Sequence IDs 352 and 365, Sequence IDs 352 and 387, Distribution Column numbers 352 and 399, sequence numbers 353 and 365, sequence numbers 353 and 387 , SEQ ID NOs. 353 and 399, SEQ ID NOs. 354 and 390, SEQ ID NOs. 354 and 3 88, SEQ ID NOs. 354 and 395, SEQ ID NOs. 358 and 361, SEQ ID NOs. 358 and 362, SEQ ID NOs. 358 and 368, SEQ ID NOs. 358 and 384, SEQ ID NOs. 358 and 394, SEQ ID NOs. 358 and 399, SEQ ID NOs. 359 and 390, SEQ ID NOs. 3 59 and 388, Sequence IDs 359 and 395, Sequence IDs 360 and 368, Sequence No. Numbers 360 and 384, Sequence IDs 360 and 389, Sequence IDs 360 and 394, Distribution Column numbers 360 and 397, sequence numbers 361 and 365, sequence numbers 361 and 387 , Sequence IDs 362 and 390, Sequence IDs 362 and 388, Sequence IDs 362 and 3 95, SEQ ID NOs. 364 and 365, SEQ ID NOs. 364 and 387, SEQ ID NOs. 364 and 399, SEQ ID NOs. 365 and 368, SEQ ID NOs. 365 and 384, SEQ ID NOs. 365 and 389, SEQ ID NOs. 365 and 394, SEQ ID NOs. 365 and 397, SEQ ID NOs. 3 66 and 368, Sequence IDs 366 and 384, Sequence IDs 366 and 389, Sequence Number This may include numbers 366 and 394, as well as sequence numbers 366 and 397.

[0094] In one embodiment, this instruction is intended to ensure that editing occurs early within the CD163 gene, thereby improving the quality of the gene. This may include a simple, accurate, and reproducible single CD163 loss-of-function edit in the context of the system. Several considerations and methods: In tissue cultures, near the 5' end of the CD163 gene Efficient cutting in the porcine genome, identifying guides with a small number of mismatches in the porcine genome. On-target cleavage determined by quantatical analysis and biochemical pre-screening. Its high specificity and the ability to perform targeted gene editing at intended sites within porcine embryo-like cells Based on its ability to guide, a combination of single guide RNA-Cas proteins, and DNA repair templates may be selected to direct specific gene editing in superior pigs. Examples of specific gene editing of CD163 are discussed in detail below. In this specification, specific gene editing is performed according to Sequence IDs 1-18 and 426-505. The CD163 gene may include a sequence selected from the group consisting of the following: Cells containing the CD163 gene were selected from the group consisting of sequence numbers 1-18 and 426-505. It may include at least one allele having a selected sequence. In one embodiment, CD Cells containing two gene-editing alleles for 163 genes are affected by either allele. However, it may include sequences selected from the group consisting of sequence numbers 1-18 and 426-505. In one embodiment, the gene-edited CD163 gene is used in sequence numbers 1-18 and 4. Selected from the group consisting of 26 to 505, the same sequence (for example, two opposite sequences of sequence number 1) It may include genes, or two alleles of sequence number 2. In one embodiment, the cell Nomu is each of the sequences selected from the group consisting of sequence numbers 1-18 and 426-505. One by one (for example, the allele of sequence number 1 and the allele of sequence number 2, and these) This may include the gene-edited CD163 gene, including all combinations.

[0095] Furthermore, a cell mixture that may include CD163-edited cells and non-gene-edited cells has also been presented. These are also included. In one embodiment, the mixture may be an embryo. In another embodiment, the mixture This may be a cell culture. In a further embodiment, the cell mixture does not contain proliferating cells. In some embodiments, this specification describes tissue cultures of CD163-edited cells and such cultures Methods for preparation are presented and may include these. Cultures according to this specification are CD163 A mixture of edited cells (for example, cells having the allele of SEQ ID NO: 1 and the allele of SEQ ID NO: 2) A mixture of cells containing the gene, cells containing the allele of SEQ ID NO: 1 and the allele of SEQ ID NO: 3. This may include a mixture of cells containing genes, and mixtures of all combinations thereof. In one embodiment, the tissue culture of non-reproductive cells may contain cells containing a single CD163 edit. In a further embodiment, cells having a single CD163 edit are present in the culture. It may contain one or both alleles.

[0096] Endonucleases and guide R that may be used in the products and methods described herein. Specific examples of NA skeletal arrangements are listed in Table 2.

[0097] [Table 2]

[0098] The core strain of superior pigs is sequenced against a publicly available reference sequence for the CD163 gene. These can be oriented and aligned. These data are for the PRRS resistance project. These sequences can be used for the development of gene editing reagents. Each of these sequences is a 3-nucleus nGG (AGG, CGG, TGG, GG) is a motif of ocidal or 5-nucleotide molecules. G), nGGnG(AGGTG, CGGTG, TGGTG, GGGTG, AGGGG, C GGGG, TGGGG, GGGGG, AGGAG, CGGAG, TGGAG, GGGAG Conservative RNA induction (which can consist of AGGCG, CGGCG, TGGCG, GGGCG) The presence of a type CRISPR-Cas9 recognition site can be scanned. This is called the PAM sequence. This motif has a 20-nucleotide space that can be used for base pairing with RNA. It can be located adjacent to the sihr sequence, on the 3' side. The appropriate site can be identified, c In some cases, an rRNA guide sequence may be prepared, while in other cases, a single guide sequence may be prepared. This is shown in Table 3 below.

[0099] Without being limited to any particular theory, when complexed, guide RNA-Ca The s9 protein is capable of recognizing DNA sites for cleavage, and then non-phase End-to-end joining (random repair) or DNA template repair pathway (homologous-directed repair: HDR) It can then be repaired by intracellular components.

[0100] Guide RNA (gRNA) is produced across exons 1 to 7 of CD163. Editing within the virus-binding domain (domain 5) of the CD163 protein is possible. This inhibits the virus's ability to bind to this protein, thereby preventing the virus from reaching the pig's lung. It may prevent uptake by macrophages. Editing within CD163 is homozygous in pigs. If the animal is of the sex, it may be resistant to PRRS infection. This specification refers to sequence numbers. The group consisting of the first 20 nucleotides of each of numbers 22-271 and 347-425. The following gRNA sequences, including the crRNA sequences listed in Table 3, can be selected from these. This includes sequences 22-271 and 347-425, which are shown as DNA sequences. The corresponding RNA equivalent, and generally, PAM mochi, which is not contained within gRNA. The existence of the o will be obvious to those skilled in the art. This specification also refers to Sequence IDs 22-271, and a sequence selected from the group consisting of the first 20 nucleotides of each of 347-425 The RNA sequences that may be included are also presented. Sequence IDs 22-271 and 347-425 are g Combined with an RNA backbone sequence, it is expressed as RNA for use in gRNA preparation. It is then mixed with Cas nuclease to form an active ribonucleotide protein complex. The sgRNP complex can then be prepared. The sgRNP complex can then be used alone or as part of a sequence of events. Along with a repair template selected from the group consisting of 1 to 13, mature oocytes, zygotes, or early It can be injected into the embryo. After cutting and template-directed repair, the CD163 gene acts as a repair template. The sequence may be edited to incorporate the editing process in CD163 gene-edited cells. NOM may contain sequences selected from the group consisting of sequence numbers 1 to 13.

[0101] In one embodiment, this disclosure describes editing within the CD163 gene (exons 1-7) As present early, in superior pigs, a simple, accurate, and reproducible single CD163 may include loss of function editing. In one embodiment, the pig is sequence numbers 1-18 and 4 It may possess the CD163 gene containing a sequence selected from the group consisting of 26 to 505. Within the genomic region containing the markers in Table 8, SEQ ID NOs. 1-18 and 425-505 are included. This also includes pigs, and these are also presented. Furthermore, within the genomic region containing the markers in Table 8, This also includes pigs that may contain column number 2, and these are also presented. Additionally, the markers in Table 8 are included. The NOM region also includes pigs that may contain sequence numbers 426-458, and these are also presented. Furthermore, some pigs may contain sequence numbers 459-504 within the genomic region containing the markers listed in Table 8. These are included and also presented. Using porcine embryonic fibroblast cell lines derived from these superior pigs We will use this to identify gene editing pairs that efficiently cleave and edit the CD163 gene. The combination of sea urchin, 250 guide RNA, and Cas9 protein can be investigated. The specific guide and the found combination with the endonuclease protein are located at the target site. , their endonuclease activity (average editing frequency), and the editing location on CD163 Both are listed in Table 3. Because the effective editing activity is high, combinations with a high average editing frequency are preferred. It is desirable that the average editing frequency is greater than 1 for each combination. In one embodiment, the combination is average The editing frequency may exceed 5. In another aspect, the combination has an average editing frequency of 10. It may exceed this. In another aspect, the combination may exceed 15 if the average editing frequency is greater than 15. This disclosure presents and includes combinations in which the average edit frequency may exceed 20. In one embodiment, the average editing frequency of a combination may exceed 25. In yet another embodiment, The average editing frequency for each combination can exceed 30.

[0102] As shown in Table 3, the average editing frequency varied considerably among target sequences. Furthermore, Multiple target sequences result in different editing frequencies depending on the Cas protein source. For example, sequence number 24 and sequence number 25 are targeted for overlapping positions and edited. The difference in collection frequency was 10 times. In this region, regarding these sequences, Streptococcus pyogenes ( S. pyogenes yielded an editing frequency nearly 20 times higher. In contrast, sequence number 33 RNPs incorporating 34 bind indistinguishable average editing frequencies among Cas proteins. This resulted in the development of Cas9 nuclease derived from Streptococcus pyogenes. While incorporating RNP can lead to a better average edit frequency, this is not always the case. No. As shown for SEQ ID NOs. 60 and 61, S. thermophilus (S. thermop RNPs incorporating the Cas9 protein derived from hilus have excellent editing frequency (for example, This resulted in a ratio of 3.9 to 22.0. Therefore, this specification refers to CRISPR / Efficient guide RNA distribution for targeting CD163 using the Cas system. Presents a sequence or an efficient targeting sequence. In one embodiment, the RNP is the sequence number. From the group consisting of the first 20 nucleotides of each of the ranges 22-271 and 347-425 It may include the selected array.

[0103] All positional indications in this specification refer to the Sscrofa 11.1 reference genome (GenB This refers to the ANK accession number: GCA_000003025.6).

[0104] The target site sequences, SEQ ID NOs. 22-271 and 347-425, are porcine CD16 It can be an editing sequence of the target sequence within 3 genes. Within each target site sequence, the first 20 nuclei Otid may correspond to the guide RNA spacer, and the remaining 3 or 5 nucleotids These are, respectively, Streptococcus pyogenes or S. thermophilus. These could be PAM sequences for ilus. Sequence numbers 22-271 and 347-425 The first 20 bases correspond to the guide RNA sequence, which is equivalent to DNA, and R as a guide. DNA bases can also be used instead of NA bases, but the appropriate ribonuclease base is deo Substitution with xyribonuclease base. Guide RNA sequence is DNA deletion and / or Alternatively, the CD163 gene in the pig genome results in the insertion of DNA nucleotides. To direct a DNA cut either within the coding region or the non-coding region, Endonuclei of Streptococcus pyogenes or S. thermophilus It can be paired with argase. Depending on the location of the target site disclosed in Table 3, guide / end The pair of nuclease combinations is CD16 between the guide / endonuclease combination pair. The region of the three genes can be selected for deletion.

[0105] The target site sequences, sequence numbers 212-271 and 347-425, are CD163 A target that can be used to design a guide for deleting the seventh exon of a gene. This is a targeting sequence. Using Streptococcus pyogenes, the sixth intron target... The seventh index is a ting guide, sequence numbers 212-253 and 262-271. The correspondence with sequence numbers 254-261, which are the targeting guide, is for exon VII. This results in two double-strand breaks on the CD163 gene, which can lead to excision. The matching of sequence numbers 249 and 256, which serve as a targeting guide, is also important. This results in the extraction of exon VII. SEQ ID NOs: 351 and 365, SEQ ID NO: 3 51 and 387, Sequence IDs 348 and 390, Sequence IDs 348 and 388, Sequence No. Numbers 348 and 498, Sequence IDs 352 and 365, Sequence IDs 352 and 387, Distribution Column numbers 352 and 399, sequence numbers 353 and 365, sequence numbers 353 and 387 , SEQ ID NOs. 353 and 399, SEQ ID NOs. 354 and 390, SEQ ID NOs. 354 and 3 88, SEQ ID NOs. 354 and 395, SEQ ID NOs. 358 and 361, SEQ ID NOs. 358 and 362, SEQ ID NOs. 358 and 368, SEQ ID NOs. 358 and 384, SEQ ID NOs. 358 and 394, SEQ ID NOs. 358 and 399, SEQ ID NOs. 359 and 390, SEQ ID NOs. 3 59 and 388, Sequence IDs 359 and 395, Sequence IDs 360 and 368, Sequence No. Numbers 360 and 384, Sequence IDs 360 and 389, Sequence IDs 360 and 394, Distribution Column numbers 360 and 397, sequence numbers 361 and 365, sequence numbers 361 and 387 , Sequence IDs 362 and 390, Sequence IDs 362 and 388, Sequence IDs 362 and 3 95, SEQ ID NOs. 364 and 365, SEQ ID NOs. 364 and 387, SEQ ID NOs. 364 and 399, SEQ ID NOs. 365 and 368, SEQ ID NOs. 365 and 384, SEQ ID NOs. 365 and 389, SEQ ID NOs. 365 and 394, SEQ ID NOs. 365 and 397, SEQ ID NOs. 3 66 and 368, Sequence IDs 366 and 384, Sequence IDs 366 and 389, Sequence Number Correspondence of some guides, including numbers 366 and 394, or sequence numbers 366 and 397. This leads to the introduction of a stop codon within exon 7.

[0106] For each targeting sequence, Table 3 lists its sequence number and the CAS9 nuclea used. - A species of homing arm, targeting the wild boar (Sus scrofa) genome containing the PAM sequence. In fetal fibroblast assays, the position of the ting sequence and the average size of a specific guide were observed. Editing efficiency measured by aggregation rate, and the exons on CD163 that are targeted List them.

[0107] [Table 3-1]

[0108] [Table 3-2]

[0109] [Table 3-3]

[0110] Table 3-4

[0111] Table 3-5

[0112] Table 3-6

[0113] Table 3-7

[0114] Table 3-8

[0115] Table 3-9

[0116] Table 3-10

[0117] Table 3-11

[0118] Table 3-12

[0119] Table 3-13

[0120] Table 3-14

[0121] [Table 3-15]

[0122] [Table 3-16]

[0123] [Table 3-17]

[0124] An efficient guide that cuts the initial region (exons 1-4) of the CD163 gene. A pair of doRNA-Cas9 was selected, and potential off-target bindings were identified within the porcine genome. Then, it was screened further.

[0125] In some cases, the use of guide RNA and endonucleases is intentional within the genome. It has been observed that this results in DNA breaks at locations where DNA has not been previously broken. dsDN The repair process for A-cuts can be random, so off-target cut events This results in unwanted changes to coding or regulatory regions within the genome. Therefore, a large number of single guides or paired guides are intended to be edited with similar frequency. If editing is possible, in selecting a guide or guide pair for editing experiments, Considering off-target cutting is advantageous.

[0126] Numerous computer and biochemical methods have been developed to elucidate off-target factors. The computer method is Cas-OFFinder (Bae, S., et al., Bioinform). atics, 2014, 30, 1473-1475), CRISPR-offinder(Zhao, C., et al., Int. J. Biol. Sci., 2017, 13, 1470-1478), and CRISPR-OFF (Alkan, F. This may include, but is not limited to, the following (e.g., Genome Biol., 2018, 19, 177). Other computer-based methods are readily available. For biochemical methods, GUIDE-Seq (Tsai, SQ, et al., Nat Biotechnol. 2015, 33, 187-197), SITE-SEQ (registered (Registered trademark) (Cameron, P., et al., Nat Methods 2017, 14, 600-606), and CIRCL This may include E-seq (Tsai, SQ, et al., Nat. Methods, 2017, 14, 607-614). These are not the only methods available. Other biochemical methods are readily available to those skilled in the art. The o-ter method is considerably faster and cheaper compared to the biochemical method, whereas the biochemical method is It has been shown to excel in identifying validated off-target edits.

[0127] As described above, in porcine fetal fibroblasts, high frequency of intentional editing A subset of guide RNAs that supported this were identified using SITE-SEQ®. Assays were performed for opposite sexes. In vitro, naked gDNA and RN were used. When using P-editing reagents, SITE-SEQ® is used to reduce the risk of cleavage. Off-target cleavage in the intracellular environment can lead to problems in vitro. Therefore, it can be more complex than off-target cleavage simulated biochemically. In particular, Factors such as effective RNP concentration and target availability due to chromatin state , which contributes to the number of off-target edits realized in porcine cells or edited pigs. Fortunately, biochemical methods such as SITE-SEQ (registered trademark) allow researchers to carefully examine the results. This can result in a list of sites to be addressed. These sites are TOPO cloning and TO Sequencing using PO clones, ILLUMINA® unit replication sequence sequencing Sequencing, Nanopore sequencing, other NGS sequencing methods, and distribution This includes column capture (Gnirke, A., et al., Nat. Biotechnol., 2009, 27, 182-189). It may be examined by methods that are not limited to these.

[0128] The screening was performed in edited porcine fibroblasts, and then biochemically in injected embryos. The procedure was performed on designated off-target areas. The validated off-target editing guide was used. Therefore, it was removed from the priority list for use in creating edited pigs. Using the strategies outlined above Therefore, animals created using guides with known off-targets are considered off-target. The presence of editing can be examined closely. Animals that certainly contain off-target editing can be identified. It can also be excluded from the breeding program, and off-target editing can be removed through breeding. This screening can also be done within the wild boar (Sus scrofa) genome, using guide R. It contains NA and 1 to 5 mismatches, which allows for off-target binding. To identify sequences, bioinformatics methods such as BLAST® search are used. It may include. In computer algorithms, when these mismatches are permissible. The number of potential off-target binding sites within the genome is detailed in Table 4 below.

[0129] [Table 4]

[0130] One of the factors contributing to cleavage at these off-target sites is gene editing. Are the components delivered as a DNA vector or as an RNA-protein complex? How is it delivered? Cas9 and guide RNA are delivered, rather than via a DNA vector. However, when delivered as a single guide ribonucleotide protein complex (sgRNP) In addition, there are sites that may have a small number of mismatches, such as 1 or 2 bases, away from the intended target site. The question is whether it can be discriminated against in a reliable manner.

[0131] The second consideration for maximizing the specificity of the Cas9 guide-RNA reagent is, in Prescreen guide RNA-protein pairs using in vitro biochemical methods. It is possible. Several laboratories screen for off-target editing sites. The methods have been published. These biochemical methods identify potential off-targets within purified genomic DNA. These assays can identify Cas9 cleavage sites. Using these assays, genomic DNA can be limited Digestion by sgRNP in the range of baseline to saturation concentration is possible, thereby enabling high cleavage sensitivity. It is possible to restore both off-target sites related to sex and off-target sites with low resection sensitivity. For example, Cameron et al., "SITE-SEQ(R): A Genome-wide Method to Measure Ca See "s9 Cleavage," Protocol Exchange (2017).

[0132] Off-target sites identified through off-target screening are possible within cells. To guide a careful and comprehensive examination of off-target areas, and the editing frequency and It can be used to measure both functional intracellular results. Selected, several gases Screening of id RNA-Cas protein pairs is possible, and the screening is 1 )Cutting that is more efficient the closer you get to the 5' end of the CD163 gene, and 2) Bioinf This can support the small number of mismatched sequences determined by the omatics method.

[0133] Using these guide selection criteria, several studies have been conducted on the cleavage activity in porcine parthenogenetic embryos. A guide RNA-Cas protein pair can be selected. Pig oocytes use electric current to select the appropriate RNA-Cas protein pair. The mixture is doubled, injected with guide RNA-Cas protein, and grown for 7 days. It is possible to collect and sequence DNA across the intended target site. Several guide RNA-Cas proteins frequently confer editing within parthenogenetic embryos. The identification of the qualitative pair is possible, and further occurrences can be selected.

[0134] This specification describes optimal target sites for CRISPR / Cas-mediated cleavage and gene editing. Methods for identification and selection are presented and include. In one embodiment, the method is editing The steps include identifying the target genomic region and all 20 nucleotide sequences within the target region. The steps are to identify the pig genome and to perform bioinformatics screening. A sequence that matches a non-target site within, containing 1 to 4 mismatches, and Casta The steps include identifying and removing sequences with appropriate PAM sequences based on protein; and Prepare a CRISPR / Cas RNP complex containing a doRNA backbone and target sequence, and CR The ISPR / Cas RNP complex was introduced into porcine cells in culture, and guide RNA / C This may include the step of determining the average editing frequency of the combination of as. In one embodiment, Guide R The step of determining the average editing frequency of NA / Cas combinations is performed by PCR at the target site. The surrounding region is amplified, and deep sequencing of the unit replication sequence (amplicon) is performed. This may include comparing it to untreated cells. In one embodiment, the method includes at least 15 We present a step for selecting a preferred target sequence that may have an average editing frequency of . Identifying and selecting optimal target sites for RISPR / Cas-mediated cleavage and gene editing. A method for doing so was also presented, in which case the guide RNA and Cas protein are expressed vector It may be presented as part of a ter(s). Suitable cells are known to those skilled in the art, Such cells may include primary fetal fibroblasts of the intended pig breeding bloodline. These are not the only options.

[0135] Multiple restoration templates can be designed to produce in-frame stop codons. (List) The repair template is then used in conjunction with the appropriate guide RNA within the endonuclease system. After synthesis and repair, in-frame termination codons (TAA, TGA, TAG) can be created. Restoration templates of different lengths, having sequence identity on one side of the edited area, can be used. In one embodiment, the repair mold is located on one side of the CRISPR / Cas endonuclease cleavage site. On the side of, at least 15 nucleotides (for example, 15 nucleotides...15 nucleotides) They may share rheotide. In other embodiments, the restoration mold has 100 on both sides of the edited area. Nucleotides exceeding a certain number (for example, >100 nucleotides...>100 nucleotides) It can be shared. The repair template uses end overhangs, even in the case of a single strand of complementary DNA. Yes, they can be double-stranded or sticky strands. In addition, these templates are not limited to DNA. It is not fixed, and it may be RNA, or it may be a modified nucleotide (e.g., inosine). Yes, and there may also be a mixture of these bases, and the 5' and / or 3' ends may decompose. It is protected because there is an exonuclease that cleaves nucleotides from the ends. End protection by modified bases is essential for both DNA and RNA, preventing the formation of exonuclear cells. Digestion by enzymes can be prevented. To put it without being limited to theory, on both sides of the editing area, Increasing the length of the identity region (e.g., homology arm) improves the efficiency and specificity of the repair process. This can increase the amount of at least 25 nucleotides on both sides of the editing site. This includes restoration molds that can have 100% identity, and these are presented. The unified domain allows for accurate and efficient editing of desired sequences (e.g., deletions of sequences 1-13). Ensure that this is the case. In one embodiment, the core identity region is at least the area flanking the edited portion. It can be 40 nucleotides. In yet another embodiment, the core identity region is on both sides of the editing site. It may be at least 50 nucleotides in the region. In one embodiment, the flanking core region The region may contain 60 nucleotides with 100% identity. The embodiment also includes the editorial department. This also includes repair templates that may have 70 nucleotides identical to the chromosomal regions flanking the position. Therefore, the core identity region is the 75 nuclei that have identity with respect to the chromosomal region surrounding the edited site. It may include Otid. In one embodiment, the core identity region is 25-40 flanking nuclei. It may include Otid. In a further embodiment, the core identity region is 40-75 frankings. May contain creotides.

[0136] In some embodiments, the core identity region is a further region having homology to the target site. It may be further sandwiched by ("Flanking homology regions"). As presented herein, In one embodiment, the flanking homology region includes 100% identity with respect to the target site. For example, sequence numbers 1-13 are from lines 2, 3, 15, 19, 27, 62. Alternatively, as found in 65, on one side of the target editing site, the CD163 region Shared 100% homology to (e.g., intra-core region and flanking homology) (100% homology within the region). Also includes repair templates that may contain sequence numbers 1-13. In rare cases, the core identity region is located on one side of the desired editing site, within the genome. Each of the 25 nucleotides (for example, 50-100 bases) contains 100% identity. It is possible to obtain at least 80% identity for one side of the genome in the core region. It can be sandwiched by (for example, nucleotides 1-49 and 101 of sequence numbers 1-18) ~150). In another embodiment, the core region can have 100% homology, The Flanking homology region may share 85% homology. In a further embodiment, the core region It is possible to have 100% homology, and the franking homology region is 90% They may share homology. In yet another embodiment, the core region may have 100% homology. This is possible, and the flanking homology region can share 95% homology. , core regions with 100% homology and flanking homology sharing 97% homology This also includes repair molds that may have regions. In some embodiments, the flanking homology region is 9 They may share 9% homology. Speaking without being limited to theory, the length of the Flanking homology domain... The increased potential allows for the introduction of desired CD163 editing into related animals without further genome modification. It is thought that polymorphisms or changes are incorporated into the CD163 genome of other pigs. The specific repair sequences to be included are specifically included and presented.

[0137] This specification describes a furan that may have more than 50 nucleotides on each side of the editing site. This includes King homology regions, which are presented here. In one embodiment, the Flanking homology region is Each side of the editing site may have more than 75 nucleotides. In one embodiment, The ping homology region may have more than 100 nucleotides on each side of the editing site. Furthermore, each side of the editing site may have more than 200 nucleotides, frankincense The franking homology region is also included. In some embodiments, the franking homology region is on each side of the edit area. In this case, it may have between 30 and 1000 nucleotides.

[0138] This specification includes further modifications to the 5' and 3' ends of the repair mold, The following is presented. Repair templates are not limited to DNA, and can also be RNA, and modified nucleic acids It may be a rheotide (e.g., inosine), or a mixture of these bases, 5' The terminal and / or 3' ends may be protected from degradation. The nucleotide is cleaved from the ends. Because exonucleases exist, end protection by modified bases is necessary for DNA and RN This specification can prevent digestion by intracellular exonucleases in both A's. As presented in the book, the 5' and 3' ends of the restoration template include the flanking homology region. The ends may be modified to prevent decomposition.

[0139] This specification may include a guide polynucleotide and a Cas protein, ribonucleotide It presents and contains a protein (RNP) complex. In one embodiment, Cas protein The quality can be Cas9 of S. thermophilus. In another embodiment, The Cas protein may be Cas9 derived from Streptococcus pyogenes. In the technical field, other suitable Cas proteins are known, and for exemplary Cas systems... This is described in Table 1 above. The selection of the appropriate Cas protein depends on the required PAM formulation. Methods for identifying Cas proteins and modifying Pam sequences, depending on the combination of sequences. The law is publicly known. Similarly, the Cas system uses the skeletal sequence of the guide RNA (e.g., tracrR Those requirements for the NA sequence differ. In one embodiment, the RNP complex is Casta The protein and the first 20 nucleotides of each of the sequence numbers 22-271 and 347-425. It has at least 98% sequence identity with an RNA sequence selected from the group consisting of D. It may include guide nucleotides. In another embodiment, the RNP complex may include SEQ ID NOs: 22-27 A sequence selected from the groups consisting of the first 20 nucleotides of each of the following sequences: 1 and 347-425. It may include guide RNA containing . In one embodiment, the RNP complex may include SEQ ID NOs: 22-271 and sequences selected from the group consisting of the first 20 nucleotides of each of 347-425 It may contain a guide polynucleotide having 99% identity with the corresponding RN. The guide polynucleotides of the P complex are, respectively, SEQ ID NOs. 22-271 and 347-425. The sequence may include a sequence selected from the group consisting of the first 20 nucleotides of each, as presented herein. As described above, the RNP complex is combined with the RNA backbone as part of the sgRNA. It may contain one of the sequences from sequence numbers 22-271 and 347-425. In some embodiments, the RNP complex may be formed before injection into target cells, It may also be injected or introduced separately.

[0140] This specification also includes the first 20 nucleotides of each of sequence numbers 22-271, and Selected from the group consisting of the first 20 nucleotides of each of sequence numbers 347-425, Isolation guide RNAs, which may include spacers, are also presented and are included. In one embodiment, The Cas protein may be a protein containing sequence number 20 or 21.

[0141] This specification also describes how to prepare guide RNA for CRISPR / Cas RNPs. This also includes and presents DNA vectors that can encode guide RNA. The vectors are the first of each of the sequence numbers 22-271 or 347-425. A sequence selected from a group consisting of 20 nucleotides, and a cis-form as part of a single transcription unit. It may contain a guide RNA backbone arranged in cis for appropriate use. The DNA vector may yield the sgRNA of this specification. Promoter, selection ma Appropriate expression vector skeletons, including the vector and origin of replication, are well known to those skilled in the art. In practice The 20 nucleotides and guide RNA backbone are DNA (in vivo (in cells) Alternatively, it is expressed from the promoter in vitro (via T7 polymerase). (It may be transcribed to form an RNA guide) or the skeleton may be a chemically synthesized double (crRNA and trRNA) can be guides or single guide RNAs.

[0142] Specific guide RNAs cause deletion of gene compartments located between these guides. By using a guide RNA that can be paired with it, a portion of the CD163 gene can be excised. They can be paired in this way. Example pairing of guide RNAs (listed as DNA sequences) This is shown in Table 5. The listed guide pairs that do not have the seventh exon amino acid sequence are all Remove the entire exon. The complete amino acid sequence for these deletions is shown in SEQ ID NO: 5 As shown in 53. All guides in Table 5 are for DNA excision between two guides. Furthermore, by NHEJ repair between cleavage sites without using DNA repair templates, the desired sequence can be created. It can produce the amino acid sequences shown in SEQ ID NOs. 506-517 as a guide. The pair introduces an exogenous stop codon that spans the cleavage end of the DNA introduced by the guide. ru.

[0143] [Table 5-1]

[0144] [Table 5-2]

[0145] [Table 5-3]

[0146] In one embodiment, the guide RNA-Cas protein pair further comprises a DNA repair template. Exemplary guides and restorative templates for creating stop codons are listed in Table 6. These guides, and other guides, describe the disruption of CD163 mRNA translation or processing. Examples of destruction include creating in-frame stop codons or splicing exons. Disrupt, interfere with, or destroy the other restoration molds They can be paired. Other examples include the disappearance of the ATG start codon, or the paired Nu Clease is responsible for the exons within CD163 (for example, the 7th exon corresponding to domain 5). When removing a deletion, the repair template is a single repair template rather than random non-homologous end joins. When using pairing guide RNA that can promote Tocam, the program of repair results This may include but is not limited to these. To put it without theoretical limitations, the analysis is P Bioinformatics mismanagement through the incorporation of additional nG (nGGnG) within AM In addition to consistent reduction of the titch, in vitro and in vivo studies have shown that of the titch. This reveals a reduction in DNA cleavage. The reduction in mismatch is due to alignment (DNA sequence is This may include mismatches due to whether or not there is a mismatch with the RNA guide sequence. See section 4. Mismatches may not always occur in off-target editing (in VIT). The presence of cleavage or indels, respectively, in or in vivo, is a sign of a problem. Get editing is likely due to a mismatch. In the most preferred embodiment, open The method described herein does not result in off-target editing in pigs and is disclosed herein. These pigs do not have off-target editing in their genomes.

[0147] [Table 6]

[0148] Stochastic repair processes in native cells, such as non-homologous end joining (NHEJ) repair, are sometimes... This involves the insertion or deletion of one or more nucleotides at the double-strand break. If this repair process results in a double-strand break within the gene's coding region, the protein The translation of the encoded mRNA up to the in-frame stop codon, which terminates the quality of the translation. Shifts can occur within the reading frame. This is due to a naturally occurring, accidental mutation in the gene. Translation can result in shortened proteins compared to the unmodified protein product, The amino acid sequence of this newly encoded polypeptide is unique and likely to be targeted. It will reduce or even improve the cellular fitness within the material. In some embodiments, To exclude the formation of frame shift mutations and the translation of unwanted polypeptide sets Therefore, editing pigs are the result of endonuclease cleavage, which is the result of separate, naturally occurring NHEJ repair. Regarding the formation of in-frame translation termination codons at or near the site of the fracture, It can be leaned.

[0149] Genomic DNA using two intron guide RNAs together with endonucleases. Sequence excision includes, but is not limited to, terminal junctions of nuclease cleavage sites, N This can be achieved by HEJ repair. Introns are non-coding sequences, therefore nuclease NHEJ repair results (outcomes) that include indels near the cleavage site are intron sequences. And / or it may be possible to excavate the intended DNA region, which may include an exon sequence. Because the NHEJ repair outcome in this region occurs more frequently than other outcomes, gene editing When selecting guide RNA pairs for use in experiments, regarding the frequency of repair outcomes It is advantageous to consider this. Sequence IDs 520-555 are observed in blastocysts. The repair outcomes of exon 7 extraction from the guide pair shown in 7 are illustrated. The restoration outcomes designed for the guide pairs are listed in Table 5.

[0150] [Table 7]

[0151] In another embodiment, a DNA repair template is incorporated together with a guide RNA-Cas protein pair. This allows for gene editing without resulting in the addition of new amino acids. A stop codon of the 'nframe' can be created. In one embodiment, the DNA repair template is dsDNA It is possible. In another embodiment, the DNA repair template may be ssDNA, double-stranded or single-stranded. Co-introduction of the main-strand DNA repair template is initiated by endonucleases at double-strand break sites. In or near this location, the translation termination codon (TAA, TGA, Used to delete or insert DNA nucleotides to form a TAG. It can be used. DNA repair templates involve several nucleotide changes compared to the natural sequence. A polynucleotide modification template containing which the target DNA sequence can be directly edited. It is possible to create the edited CD163 gene along with an in-frame stop codon. Polynucleotide modifications that can be cotransfected together with endonuclease editing reagents. The templates may further include. In one embodiment, these gene-edited CD163 genes encode The protein is transcribed into mRNA, and then translated into protein, C It is possible to synthesize only the shortened, non-functional forms of the D163 polypeptide. Therefore, DNA repair templates with longer sequence homology regions may be more efficient. In the application method, the DNA repair template involves a sequence change, ranging from less than 50 nucleotides to 1000 nucleotides. Within the DNA repair template, which can extend beyond the rheotide, there is a sequence identity region (homology area). It may contain (mu).

[0152] Regarding the combination of Cas9 protein / guide RNA in conjunction with the DNA repair template, Several embodiments are shown in Table 6. As shown in Table 6, several Cas9 proteins The combination of quality / guide RNA determines the sequence homology on each side of the targeted deletion. It has 75 bases (flanking homology of 50 bases and 50 bases of core homology on one side). It can be paired with DNA repair templates that have, but are not limited to, sexual regions. In this case, the combination of the protein-guide RNA complex and the template is transferred to the cell. It can be done. To put it without being limited by theory, double-strand breaks using DNA repair templates. Repair involves the repair of one or more nucleotides at the cleavage site or adjacent sites. The deletion can direct the formation of in-frame translation termination codons. The animals and cells used to co-introduce the porcine CD163 gene using the co-introduced DNA repair template. This supports the idea that double-strand breaks directed by endonucleases can be repaired. As presented herein, the repair mold having sequence numbers 1 to 13 is second E An in-frame translation termination codon is introduced within the xxon, thereby enabling a shortened, non-functional C It is possible to produce the D163 protein.

[0153] The use of DNA repair templates as described herein is not limited to the loss of CD163 function. The specification may also describe repair templates that direct the removal or addition of nucleotides from genes. This further includes and presents the types. In one embodiment, the encoded CD163 mRNA The stability or half-life modulation is modulated by editing according to this method. In yet another embodiment, the amino of the mature protein responsible for binding to the PRRS virus The DNA sequences that code for acids may be removed or replaced. In some embodiments, the expression and / or activity of CD163 may be reduced by at least 90%, It does not disappear. In some aspects of this instruction, the removal and introduction of bases, or The combination of these methods is described to enable the formation of in-frame stop codons. Those skilled in the art may also foresee the use of the spacer array in conjunction with other repair molds. cormorant.

[0154] This specification includes pigs derived from superior pig lines containing the edited CD163 gene. This is presented. In one embodiment, the superior pig lineage is PIC(trademark) lineage 15, PIC (trademark) line 17, PIC (trademark) line 27, PIC (trademark) line 65, PIC (trademark) Line 14, PIC (trademark) line 62, PIC337, PIC800, PIC280, PI C327, PIC408, PIC(trademark)399, PIC410, PIC415, PIC 359, PIC380, PIC837, PIC260, PIC265, PIC210, P IC (trademark) system 2, PIC (trademark) system 3, PIC (trademark) system 4, PIC (trademark) system Line 5, PIC (trademark) line 18, PIC (trademark) line 19, PIC (trademark) line 92, P IC95, PIC (trademark) CAMBOROUGH (registered trademark) (Pig Improve ment Company, Limited, Basingstoke, UK), PIC 1070, PIC (trademark), CAMBOROUGH (registered trademark), 40, PIC (trademark), CA MBOROUGH (registered trademark) 22, PIC1050, PIC (trademark) CAMBOROU GH(registered trademark)29, PIC(trademark)CAMBOROUGH(registered trademark)48, or PIC(trademark) CAMBOROUGH(registered trademark) x54 may be. In various forms, Superior pig strains are PIC (trademark) superior pig strains 2, 3, 15, 19, 27, 62, or It could be 65. In another embodiment, the pig is derived from a superior pig lineage, with edited CD163 genes. It may include offspring. In one embodiment, the pig has the genotypes shown in Tables 8 to 14, CD Edited CD163 may be included within the 163 genome region. Tables 8-14 show the presence of single nucleotide polymorphisms. The following shows the various locations on chromosome 5 near the CD163 gene.

[0155] Each table presents the alleles that are homozygous in that lineage, and the genetic signature of the lineage ( In addition to identifying genome signatures, it also displays their unique genome editing regions. This is based on the following. Therefore, the propagation of the edited CD163 lineage is traceable and from the lineage. Animals containing the resulting edited CD163 gene can be identified. Other pig strains are shown in Table 8. When compared to the lines in Table 14, whether multiple alleles are heterozygous or not These are opposing genotypes. Thus, the combination of gene signatures in each lineage is specific. It can be used to distinguish between pigs belonging to a particular lineage and pigs that do not belong to a specific lineage. In one embodiment, gene signature is a method for breeding and tracking CD163-edited genomes across generations. It brings about. In one embodiment, the CD163 edited gene having the gene signatures listed in Tables 8 to 14 Later generations containing "mu" can be prepared.

[0156] [Table 8-1]

[0157] [Table 8-2]

[0158] [Table 9]

[0159] [Table 10]

[0160] [Table 11]

[0161] [Table 12]

[0162] [Table 13]

[0163] [Table 14]

[0164] Excellent PIC (trademark) systems 2, 3, 15, 19, 27, 62, Line 65 is a line selected for its superior commercial phenotype. In one embodiment, C D163 gene-edited cells and CD163 gene-edited animals are found in wild populations and many It may be free of harmful alleles present in commercial animal populations. In some embodiments, CD 163 gene-edited cells and CD163 gene-edited animals have been shown to have epithelial hypoplasia, cutaneous melanoma, Proliferative dermatitis, mammary gland abnormalities, spinal shortening, caudal dysplasia, caudal agenesis, alopecia, alopecia (2), wool Hair follicles, hydrocephalus, tufted hair follicles, leglessness, tridactyly, syndactyly, polydactyly, Plavusca factor, iridacea chromasia, type AIII congenital tremor, type AIV congenital tremor, congenital ataxia, hindlimb paralysis, leg curvature Curvature, leg thickening, malignant hyperthermia, hemophilia (von Willebrand's disease), leukemia, hemolytic disease Edema, acute respiratory distress ("Barker's disease"), rickets, renal hypoplasia, polycystic kidney disease, uterine aplasia. , porcine stress syndrome (PSS), halothane (HAL) sensitivity, steep shoulder (back mass, back A group consisting of (partial dysplasia, kyphosis), hyperossification, mammary gland hypoplasia, and breast underdevelopment is selected. One or more of the harmful alleles presented herein may be absent. As described above, the improved method for preparing CD163 gene-edited animals involves a new method for editing harmful gene loci. The introduction of mutations, or novel aphenotypic, hypophenotypic, hyperphenotypic, novel mutations in non-target sites. The occurrence of trait and anti-trait mutations can be avoided. In superior lines, these latter changes are in the population. Within the gene, multiple loci can control desired traits in a continuous, quantitative manner. Because it can interfere with genes, it may not be desirable in some cases. Many such quantitative trait genes The constellation QTL is well known and typically has a bell-shaped curve relative to the number of animals observed. It can be characterized by linear quality values: back fat, average daily feed intake, lifetime daily weight gain, and - Among commercially important traits, such as depth, but not limited to these. Therefore, polygenic inheritance of such traits is common.

[0165] Similarly, traits associated with productivity can be multiplexed, and multiple QTLs can be found. These traits can be controlled by [methods / methods]. These traits were typically measured visually, but today, The method involves measuring back fat thickness (bfp), loin depth (ldp), and intramuscular fat (uip). This may include ultrasonic methods. As shown in Table 15, superior strains have a high lifetime increase in price. While possessing body mass, it may also have desired phenotypic traits, including high levels of back fat and loin depth. Similarly, superior sows are prolific breeders, have large litter sizes, few stillbirths, and produce piglets. They may have nipples sufficient for weaning and raising.

[0166] [Table 15]

[0167] This specification is a CD163 knockout edit (CD163 - / - ) homozygous Possible selected superior strains of gene-edited pigs are presented, and these are included. In one embodiment, the strain The lineage described herein contains the edited CD163 gene, lineage 2 of PIC®, Lineage 3, Lineage 15, Lineage 19, Lineage 27, Lineage 62, Lineage 65, and their descendants The following groups can be selected: Gene-edited strains 2, 3, 15, 19, and Lineages 27, 62, and 65 represent the CD163 genomic region shown in Tables 8-14. They may include and can be easily distinguished from each other, from non-improved lines, and from other superior lines. In some embodiments relating to this specification, heterozygous pigs and homozygous pigs as defined herein are This specification may not contain mutations outside the target site. This specification refers to Sequence IDs 1-18 and Including 426-505, at least 90% of the genotype markers in Tables 8-14 This document presents pigs and cells that may share a genetic signature and may possess an edited CD163 gene. and these are included. This specification includes Sequence ID No. 2 and the genotype markers in Tables 8 to 14. Our group has an edited CD163 gene that may share a genetic signature with at least 90% of us. The presenter presents, including, pigs and cells. In one embodiment, the CD163 edited pig also The gene signature of CD163-edited cells is less than one of the genotype markers listed in Tables 8-14. They can share a genetic signature that includes at least 95%. Also, the genotype markers in Tables 8 to 14 Our CD163-edited pigs or C163-edited pigs may share a genetic signature containing at least 97% of ours. CD163-edited cells are also included. In one embodiment, CD163-edited pigs or CD163-edited The cells contain at least 98% of the genotype markers listed in Tables 8-14, indicating a genetic signature. They can share. Another embodiment is at least 99 of the genotype markers in Tables 8 to 14. Presents CD163-edited pigs or CD163-edited cells that may share a genetic signature containing %. Perform.

[0168] As presented in this specification, for lines 2, 3, 15, 19, 27, 6 2, and 65, gene-edited CD163 - / - Animals and gene-edited CD163 - / - The cells can maintain the desired commercial traits and can be free of harmful off-target mutations and can contain an edited CD163 gene containing any one of SEQ ID NOs: 1-18 and 426-505. In certain embodiments, for the lines 2, 3, 15, 19, 27, 62, and 65 of the present disclosure, the gene-edited CD1 63 63 - / - Animals and gene-edited CD163 - / - The cells can contain at least 90% of the low depth of the non-edited pig line and can contain an edited CD163 gene containing any one of SEQ ID NOs: 1-18 and 426-505. In certain embodiments, for the lines 2, 3, 15, 19, 27, 62, and 65 of the present disclosure, the gene-edited CD163 Animals and gene-edited CD163 gene-edited CD163 - / - Animals and gene-edited CD163 - / - The cells can contain at least 90% of the lifetime daily weight gain of the non-edited pig line. In certain embodiments, for the lines 2, 3, 15, 19, 27, 62, and 65 of the present disclosure, the gene-edited CD16 3​​​​​​​​​Pigs and cells that may share a genetic signature containing at least 90% of the markers. Present and include these.

[0169] As presented herein, systems 2, 3, 15, 19, 27, and 6 2, and gene-edited CD163 of line 65 - / - Animals and gene editing CD163 - / - The cells may retain desired reproductive traits. In some embodiments, Sequence IDs 1-18 of this disclosure may be used. Or lineages 2, 3, 15, and 15 containing edited CD163 genes 426-505. 19, lineage 27, lineage 62, and lineage 65, gene-edited CD163 - / - Animals and Gene-edited CD163 - / - The cells represent at least 90% of all littermates of the unedited pig lineage. This may constitute an edit of Sequence IDs 1-18 or 426-505 of the present disclosure. Lineages 2, 3, 15, 19, 27, and 62, which contain the CD163 gene. and line 65, gene-edited CD163 - / - Animals and gene editing CD163 - / - The cells can constitute a number of stillborn piglets no more than 110% compared to unedited pig lines. In one embodiment, the edited CD163 gene of sequence numbers 1-18 or 426-505 of the present disclosure. This includes systems 2, 3, 15, 19, 27, 62, and 65. gene editing CD163 - / - Animals and gene editing CD163 - / - Cells are unedited Compared to the Ta lineage, it can achieve at least 90% of the average number of teats.

[0170] In one embodiment, the edited CD163 gene has sequence numbers 1-18 or 426-505. Line 2, gene editing CD163 - / - Animals and gene editing CD163 - / - Cells , may have back fat that is at least 90% of the amount found in unedited strain 2 animals. In one embodiment, the gene-edited CD163 of lineage 2. - / - Animals and gene editing CD1 63 - / - The cells may have back fat, which is at least 97% of that of unedited strain 2 animals.

[0171] In one embodiment, the edited CD163 gene has sequence numbers 1-18 or 426-505. Line 3, gene editing CD163 - / - Animals and gene editing CD163 - / - Cells , may have back fat that is at least 90% of the amount found in unedited strain 3 animals. In one embodiment, the gene-edited CD163 of lineage 3. - / - Animals and gene editing CD1 63 - / - The cells may have back fat, which is present in at least 95% of non-edited strains of animals. In one embodiment, lineage 3, gene-edited CD163 - / - Animals and gene editing CD163 - / - The cells may have back fat, which is at least 97% of that of unedited strain 2 animals.

[0172] In one embodiment, the edited CD163 gene has sequence numbers 1-18 or 426-505. Line 15, gene editing CD163 - / - Animals and gene editing CD163 - / - cell It has back fat that is at least 90% of the amount found in the 15 unedited strains of animals. It is possible. In one embodiment, the gene-edited CD163 of lineage 15. - / - Animals and gene editing CD163 - / -The cells contain back fat, which is present in at least 95% of the unedited strains of 15 animals. It is possible. In one embodiment, the gene-edited CD163 of lineage 15. - / - Animals and gene editing CD163 - / - The cells have back fat, which is at least 97% of that of non-edited strain 2 animals. Shut up.

[0173] In one embodiment, the edited CD163 gene has sequence numbers 1-18 or 426-505. Line 19, gene editing CD163 - / - Animals and gene editing CD163 - / - cell It has back fat that is at least 90% of the amount found in the 19 unedited strains of animals. It is possible. In one embodiment, the gene-edited CD163 of lineage 19. - / - Animals and gene editing CD163 - / - The cells have back fat, which is present in at least 95% of the non-edited strains of 19 animals. It is possible. In one embodiment, the gene-edited CD163 of lineage 19. - / - Animals and gene editing CD163 - / - The cells have back fat, which is at least 97% of that of non-edited strain 2 animals. Shut up.

[0174] In one embodiment, the edited CD163 gene has sequence numbers 1-18 or 426-505. Line 27, gene editing CD163 - / - Animals and gene editing CD163 - / - cell It has back fat that is at least 90% of the amount found in the 27 unedited strains of animals. It is possible. In one embodiment, the gene-edited CD163 of lineage 27. - / - Animals and gene editing CD163 - / - The cells have back fat, which is present in at least 95% of the 27 non-edited strains of animals. It is possible. In one embodiment, the gene-edited CD163 of lineage 27. - / - Animals and gene editing CD163 - / - The cells have back fat, which is at least 97% of that of non-edited strain 2 animals. Shut up.

[0175] In one embodiment, the edited CD163 gene has sequence numbers 1-18 or 426-505. Line 62, gene editing CD163 - / - Animals and gene editing CD163 - / - cell It has back fat that is at least 90% of the amount found in the 62 unedited strains of animals. It is possible. In one embodiment, the gene-edited CD163 of lineage 62. - / - Animals and gene editing CD163 - / - The cells have back fat, which is present in at least 95% of the unedited strains of 62 animals. It is possible. In one embodiment, the gene of line 62 having the edited CD163 gene of sequence number 2 Editing CD163 - / - Animals and gene editing CD163 - / - The cells were from the unedited lineage 62. It may have back fat that is at least 95% of the amount found in animals. In one embodiment, , lineage 62, gene editing CD163 - / - Animals and gene editing CD163 - / - cell It may have back fat, which is at least 97% of that of an unedited strain 2 animal.

[0176] In one embodiment, the edited CD163 gene has sequence numbers 1-18 or 426-505. Line 65, gene editing CD163 - / - Animals and gene editing CD163 - / - cell It has back fat that is at least 90% of the amount found in the unedited strain of 65 animals. It is possible. In one embodiment, the gene-edited CD163 of lineage 65.- / - Animals and gene editing CD163 - / - The cells have back fat, which is present in at least 95% of the unedited strain 65 animals. It is possible. In one embodiment, the gene-edited CD163 of lineage 65. - / - Animals and gene editing CD163 - / - The cells have back fat, which is at least 97% of that of non-edited strain 2 animals. Shut up.

[0177] In one embodiment, the edited CD163 gene has sequence numbers 1-18 or 426-505. Line 2, gene editing CD163 - / - Animals and gene editing CD163 - / - Cells , the number of all littermates is at least 90% of the amount found in unedited lineage 2 animals. It is possible. In one embodiment, the gene-edited CD163 of lineage 2. - / - Animals and gene editing CD163 - / - The cells represent at least 95% of the total litter number of animals from the unedited lineage 2. It may have. In one embodiment, lineage 2, gene editing CD163 - / - Animals and Genetics Collection CD163 - / - The cells represent at least 97% of all littermates of non-edited lineage 2 animals. It can have a number.

[0178] In one embodiment, the edited CD163 gene has sequence numbers 1-18 or 426-505. Line 3, gene editing CD163 - / - Animals and gene editing CD163 - / - Cells , the number of all littermates is at least 90% of the amount found in the unedited three lineages It is possible. In one embodiment, the gene-edited CD163 of lineage 3. - / - Animals and gene editing CD163 - / -The cells represent at least 95% of the total litter number of animals from the unedited lineage 3. It may have. In one embodiment, lineage 3, gene editing CD163 - / - Animals and Genetics Collection CD163 - / - The cells represent at least 97% of all littermates of non-edited lineage 3 animals. It can have a number.

[0179] In one embodiment, the edited CD163 gene has sequence numbers 1-18 or 426-505. Line 15, gene editing CD163 - / - Animals and gene editing CD163 - / - cell This represents at least 90% of the amount found in the unedited lineage of 15 animals, for all littermates. It may have a number. In one embodiment, line 15, gene-edited CD163 - / - Animals and Genetics Sub-edit CD163 - / - The cells represent at least 95% of the entire population of 15 non-edited strains of animals. It may have littermates. In one embodiment, line 15 has gene-edited CD163 - / - Animals Gene editing CD163 - / - The cells were found in at least 97% of the 15 non-edited animal strains. It may have a total number of littermates.

[0180] In one embodiment, the edited CD163 gene has sequence numbers 1-18 or 426-505. Line 19, gene editing CD163 - / - Animals and gene editing CD163 - / - cell This represents at least 90% of the amount found in the unedited lineage of 19 animals, for all littermates. It may have a number. In one embodiment, line 19, gene-edited CD163 - / - Animals and Genetics Sub-edit CD163 - / - The cells represent at least 95% of the entire population of 19 non-edited strains of animals. It may have a litter size. In certain embodiments, the gene-edited CD163 of strain 19 - / - animals and gene-edited CD163 - / - cells may have a litter size that is at least 97% of that of non-edited strain 19 animals.

[0181] In certain embodiments, the gene-edited CD163 of strain 27 having the edited CD163 gene of SEQ ID NO: 1-18 or 426-505 animals and gene-edited CD163 - / - cells - / - may have a litter size that is at least 90% of the amount found in non-edited strain 27 animals. In certain embodiments, the gene-edited CD163 of strain 27 animals and - / - gene- edited CD163 - / - cells may have a litter size that is at least 95% of that of non-edited strain 27 animals. In certain embodiments, the gene-edited CD163 of strain 27 animals and - / - gene- edited CD163 - / - cells may have a litter size that is at least 97% of that of non-edited strain 27 animals.

[0182] In certain embodiments, the gene-edited CD163 of strain 62 having the edited CD163 gene of SEQ ID NO: 1-18 or 426-505 animals and gene-edited CD163 - / - cells - / - may have a litter size that is at least 90% of the amount found in non-edited strain 62 animals. In certain embodiments, the gene-edited CD163 of strain 62 animals and - / - gene- edited CD163 - / - cells may have a litter size that is at least 95% of that of non-edited strain 62 animals. In certain embodiments, the gene-edited CD163 of strain 62 animals and- / - Animal and gene - edited CD163 - / - cells may have at least 97% of the total number of full - sib offspring of non - edited strain 62 animals.

[0183] In certain embodiments, gene - edited CD163 animals and gene - edited CD163 cells of strain 65 having the edited CD163 gene of SEQ ID NO: 1 - 18 or 426 - 505 - / - Animal and gene - edited CD163 - / - cells may have at least 90% of the total number of full - sib offspring found in non - edited strain 65 animals. In certain embodiments, gene - edited CD163 animals and gene - edited CD163 - / - Animal and gene - edited CD163 - / - cells may have at least 95% of the total number of full - sib offspring of non - edited strain 65 animals. In certain embodiments, gene - edited CD163 animals and gene - edited CD163 - / - Animal and gene - edited CD163<00​​​​​​​​​​​​​​​​​​​​OUGH (registered trademark), PIC1070, PIC (trademark), CAMBOROUGH (registered trademark) (Mark) 40, PIC (Trademark) CAMBOROUGH (Registered Trademark) 22, PIC1050, P IC (trademark) CAMBOROUGH (registered trademark) 29, PIC (trademark) CAMBOROUGH GH(registered trademark)48, or PIC(trademark)CAMBOROUGH(registered trademark)x54 CD163 edited animals containing germplasm are presented and included. The term germplasm refers to the intact genome, which is present within cells or the nucleus and includes chromosomes. The term "progenitor" refers to any gametes, germ cells, or any other cells from which an animal can be cloned. It may contain somatic cells. The edited germplasm is one of the edits from sequence numbers 426-505. This may include edits with aggregated genome sequences. The edited germplasm is sequence numbers 426, 427, and 428. ,429,430,431,432,433,434,435,436,437,438 ,439,440,441,442,443,444,445,446,447,448 ,449,450,451,452,453,454,455,456,457,458 ,459,460,461,462,463,464,465,466,467,468 ,469,470,471,472,473,474,475,476,477,478 ,479,480,481,482,483,484,485,486,487,488 ,489,490,491,492,493,494,495,496,497,498 , 499, 500, 501, 502, 503, 504, or 505 The edit may include having two edited genome sequences. The edited germplasm is PIC(trademark) strain 15 and , may contain genomes that are 80%, 85%, 90%, or 95% similar or identical. Edited germplasm It is 80%, 85%, 90%, and 95% similar or identical to PIC(trademark) system 17. May contain genome. Edited germplasm is PIC(trademark) strain 27 and 80%, 85%, 90%. It may contain genomes that are 95% similar or identical. The edited germplasm is PIC(trademark) strain 6. May include genomes that are 80%, 85%, 90%, or 95% similar or identical. The reproductive quality is 80%, 85%, 90%, and 95% similar or identical to PIC (trademark) strain 14. It may contain a certain genome. The edited germplasm is PIC(trademark) strain 62 and 80%, 85%, 9 It may contain genomes that are 0%, 95%, similar, or identical. The edited germplasm is PIC337 and It may contain genomes that are 80%, 85%, 90%, or 95% similar or identical. Edited germplasm PIC800 and includes genomes that are 80%, 85%, 90%, and 95% similar or identical. The edited germplasm is 80%, 85%, 90%, and 95% similar or identical to that of PIC280. It may contain genomes that are PIC327 and 80%, 85%, 90%, 9%. May contain 5% similar or identical genomes. Edited germplasm is PIC408 and 80% It may contain genomes that are 85%, 90%, or 95% similar or identical. Edited germplasm is PIC (Trademark) 399 and contains genomes that are 80%, 85%, 90%, or 95% similar or identical. The edited germplasm is 80%, 85%, 90%, and 95% similar or identical to that of PIC410. It may contain genomes that are PIC415 and 80%, 85%, 90%, 9%. May contain 5% similar or identical genomes. Edited germplasm is PIC359 and 80% It may contain genomes that are 85%, 90%, or 95% similar or identical. Edited germplasm is PIC 380 may include genomes that are 80%, 85%, 90%, or 95% similar or identical. The germplasm is 80%, 85%, 90%, and 95% similar to or identical to PIC837. May contain nom. Edited germplasm is similar to PIC260, with 80%, 85%, 90%, and 95%. Or it may contain an identical genome. The edited germplasm is PIC265 and 80%, 85%, It may contain genomes that are 90%, 95%, similar, or identical. Edited germplasm is PIC210 and , may contain genomes that are 80%, 85%, 90%, or 95% similar or identical. Edited germplasm This is a trademark that is 80%, 85%, 90%, and 95% similar or identical to PIC (trademark) system 2. May contain nom. The edited germplasm is PIC(trademark) lineage 3 and 80%, 85%, 90%, 9 May contain 5% similar or identical genomes. The edited germplasm is from PIC(trademark) strain 4, It may contain genomes that are 80%, 85%, 90%, or 95% similar or identical. Edited germplasm PIC (trademark) System 5 and genotypes that are 80%, 85%, 90%, and 95% similar or identical. May contain mu. The edited germplasm is PIC(trademark) lineage 18 and 80%, 85%, 90%, 9 May contain 5% similar or identical genomes. The edited germplasm is PIC(trademark) strain 19 and , may contain genomes that are 80%, 85%, 90%, or 95% similar or identical. Edited germplasm It is 80%, 85%, 90%, and 95% similar or identical to PIC(trademark) system 92. It may contain a genome. The edited germplasm is similar to PIC95, with 80%, 85%, 90%, and 95% of the content being the same. Or it may contain an identical genome. Edited germplasm is PIC(trademark) CAMBOROUG H (registered trademark) and containing genomes that are 80%, 85%, 90%, or 95% similar or identical. The edited germplasm is similar to or identical to PIC1070, with 80%, 85%, 90%, and 95% of the germplasm. It may contain a single genome. Edited germplasm is PIC(trademark) CAMBOROUGH(registered trademark). (Trademark) 40 may include genomes that are 80%, 85%, 90%, or 95% similar or identical. The edited germplasm is PIC(trademark) CAMBOROUGH(registered trademark)22, and 80%, 8 It may contain genomes that are 5%, 90%, or 95% similar or identical. Edited germplasm is PIC1 This may include genomes that are 80%, 85%, 90%, or 95% similar or identical to O50. The germplasm is PIC(trademark)CAMBOROUGH(registered trademark)29, and 80%, 85% , may contain genomes that are 90% or 95% similar or identical. Edited germplasm is PIC (trademark). )CAMBOROUGH(registered trademark)48 and 80%, 85%, 90%, 95% similarly It may contain identical genomes. Edited germplasm is PIC(trademark) CAMBOROUGH( (Registered trademark) x54 and genomes that are 80%, 85%, 90%, or 95% similar or identical. I can see it.

[0185] This specification applies to PIC(trademark) systems 2, 3, 15, 19, 27, and CD163 edited animals containing germplasm of lineage 62 or lineage 65 are presented and included. The germplasm contains one of the edited genome sequences from sequence numbers 426 to 505. May contain aggregates. Edited germplasm includes sequence numbers 426, 427, 428, 429, 430, 43 1, 432, 433, 434, 435, 436, 437, 438, 439, 440, 44 1, 442, 443, 444, 445, 446, 447, 448, 449, 450, 45 1, 452, 453, 454, 455, 456, 457, 458, 459, 460, 46 1, 462, 463, 464, 465, 466, 467, 468, 469, 470, 47 1, 472, 473, 474, 475, 476, 477, 478, 479, 480, 48 1, 482, 483, 484, 485, 486, 487, 488, 489, 490, 49 1, 492, 493, 494, 495, 496, 497, 498, 499, 500, 50 It has one of the edited genome sequences 1, 502, 503, 504, or 505. This may include editing. The edited germplasm has the edited genome sequence shown in SEQ ID NO: 453. May include editing. The edited germplasm contains the CD163 edit sequence, including sequence number 453, P IC (trademark) system 2 and genomes that are 80%, 85%, 90%, and 95% similar or identical. It may contain. The edited germplasm contains the CD163 edit sequence, including sequence number 453, PIC( (Trademark) System 3 and includes genomes that are 80%, 85%, 90%, and 95% similar or identical. The edited germplasm contains the CD163 edit sequence, including sequence number 453, and is PIC(trademark) Lineage 15 may include genomes that are 80%, 85%, 90%, or 95% similar or identical. The edited germplasm contains the CD163 edit sequence, including sequence number 453, of the PIC® strain. 19 may include genomes that are 80%, 85%, 90%, or 95% similar or identical. The germplasm contains the CD163 edit sequence, including sequence number 453, of PIC® lineage 27. This may include genomes that are 80%, 85%, 90%, or 95% similar or identical. (Edited reproduction) The quality is PIC(trademark) system 62, which includes sequence number 453 and CD163 edit sequence, It may contain genomes that are 80%, 85%, 90%, or 95% similar or identical. Edited germplasm , including sequence number 453, with CD163 edit sequence, PIC(trademark) system 65 and 80 It may contain genomes that are 85%, 90%, or 95% similar or identical.

[0186] Edited germplasm may contain edits having the edited genome sequence shown in SEQ ID NO: 489. The germplasm contains the CD163 edit sequence, including sequence number 489, of PIC® lineage 2. This may include genomes that are 80%, 85%, 90%, or 95% similar or identical. (Edited reproduction) The quality is PIC(trademark) system 3, with CD163 edit sequence including sequence number 489, and 8 It may contain genomes that are 0%, 85%, 90%, or 95% similar or identical. Edited germplasm is PIC(trademark) system 15, with CD163 edit sequence including sequence number 489, and 80% It may contain genomes that are 85%, 90%, or 95% similar or identical. Edited germplasm is sequence PIC(trademark) system 19, with CD163 editing sequence including number 489, and 80%, 8 It may contain genomes that are 5%, 90%, or 95% similar or identical. Edited germplasm is sequence number PIC(trademark) system 27, including 489, with CD163 edit sequence, and 80%, 85% It may contain genomes that are 90% or 95% similar or identical. Edited germplasm is sequence number 48. PIC(trademark) system 62, with CD163 editing sequence including 9, and 80%, 85%, 9 It may contain genomes that are 0%, 95%, similar, or identical. The edited germplasm contains sequence number 489. Includes, with CD163 editing sequence, PIC(trademark) system 65, and 80%, 85%, 90% , may contain genomes that are 95% similar or identical. Edited germplasm includes sequence numbers 506-51. It may have a predicted amino acid sequence for exon 7 derived from any one of the seven. The germplasm may have the predicted amino acid sequence of exon 7, as shown in Sequence ID No. 513.

[0187] Edited germplasm may contain edits having the edited genome sequence shown in Sequence ID No. 505. The germplasm contains the CD163 edit sequence, including sequence number 505, and is classified as PIC® lineage 2. This may include genomes that are 80%, 85%, 90%, or 95% similar or identical. (Edited reproduction) The quality is PIC(trademark) system 3, with CD163 edit sequence including sequence number 505, and 8 It may contain genomes that are 0%, 85%, 90%, or 95% similar or identical. Edited germplasm is PIC(trademark) system 15, with CD163 edit sequence including sequence number 505, and 80% It may contain genomes that are 85%, 90%, or 95% similar or identical. Edited germplasm is sequence PIC(trademark) system 19, with CD163 editing sequence including number 505, and 80%, 8 It may contain genomes that are 5%, 90%, or 95% similar or identical. Edited germplasm is sequence number PIC(trademark) system 27, with CD163 editing sequence including 505, and 80%, 85% It may contain genomes that are 90% or 95% similar or identical. Edited germplasm is sequence number 50 PIC(trademark) system 62, with CD163 editing sequence including 5, and 80%, 85%, 9 May contain genomes that are 0%, 95%, similar, or identical. Edited germplasm may contain sequence number 505. Includes, with CD163 editing sequence, PIC(trademark) system 65, and 80%, 85%, 90% It may contain genomes that are 95% similar or identical. Edited germplasm is shown in SEQ ID NO: 518. It may have the predicted amino acid sequence of exon 7.

[0188] This specification also refers to PIC(trademark) systems 2, 3, 15, 19, and 27. Cells of lineage 62 or lineage 65 are also presented and included. In some embodiments, PIC( Cells of strain 2 (trademark) have one of the edited genome sequences from sequence numbers 426 to 505. This may include. Cells of PIC(trademark) lineage 2 have the genome sequence shown in Sequence ID No. 453 edited. It may include a collection. In some embodiments, cells of PIC(trademark) lineage 3 are sequence numbers 426~ May contain any one of the 505 edited genome sequences. PIC® strain 3 cells. This may include editing of the genome sequence shown in Sequence ID No. 453. In some embodiments, PI Cells of strain C(trademark) 15 are edited genotypes of any one of sequence numbers 426-505. May contain an edited sequence. Cells of PIC(trademark) strain 15 may contain an edited sequence as shown in sequence number 453. May contain nom sequences. In some embodiments, cells of PIC(trademark) strain 19 may contain sequence numbers. May contain one of the edited genome sequences from 426 to 505. PIC (trademark) lineage. The 19 cells may include the genome sequence editing shown in SEQ ID NO: 453. Some embodiments So, the cells of PIC(trademark) lineage 27 are one of the sequence numbers 426-505. It may contain edited genome sequences. Cells of PIC® strain 27 are shown in SEQ ID NO: 453. This may include editing of the genome sequence. In some embodiments, cells of PIC® strain 62 This may contain one of the edited genome sequences from sequence numbers 426 to 505. PIC Cells of (trademark) strain 62 may include the genome sequence editing shown in SEQ ID NO: 453. In one embodiment, cells of PIC(trademark) strain 65 are selected from among sequence numbers 426-505. It may contain any one of the edited genome sequences. Cells of PIC® strain 65 contain the sequence number This may include editing of the genome sequence shown in 453. The edited germplasm is shown in SEQ ID NO: 513. Furthermore, it may have a predicted amino acid sequence for exon 7.

[0189] This specification is characterized by the CD16 series, as indicated by Sequence IDs 1-18 and 426-505. We present and include hybrid animals that may involve editing of three genes. In some configurations, Gene editing may include sequence number 453. In one embodiment, the hybrid animal is CAM BOROUGH(R) (PIC(TM) UK Limited, Basingst CD163 (from the UK) - / - It could be a hybrid animal. CAMBORO UGH (registered trademark) pigs can be produced by crossbreeding line 2 and line 3. It is a hybrid. In one embodiment, the hybrid animal has the edited CD163 gene of Sequence ID No. 2. CD163, which is part of the CAMBOROUGH (registered trademark) lineage. - / - Hybrid It can be an object. In one aspect, a hybrid animal is an edited CD of sequence numbers 426-458. CD163, a strain of the CAMBOROUGH® line possessing 163 genes. - / - Ha It can be a hybrid animal. In various forms, hybrid animals are the same as sequence number 453. CD163 of the CAMBOROUGH® line, which possesses the CD163 gene. - / - It may be a hybrid animal. In one embodiment, a hybrid animal is sequence number 459 CAMBOROUGH® lineage possessing ~504 edited CD163 genes, CD163 - / - It can be a hybrid animal. In various forms, hybrid animals are, CAMBOROUGH (registered trademark) has the edited CD163 gene sequence of SEQ ID NO: 489. ) System, CD163 - / - It could be a hybrid animal. CAMBOROUGH (Registered (Trademark) Hybrid pigs are pigs that produce large litter sizes and uniform, robust piglets. Yes. CAMBOROUGH® hybrid pigs have a long productive lifespan and a low mortality rate. It is low. CAMBOROUGH(registered trademark)CD163 - / - Hybrid pigs are this To retain the desired commercial characteristics. In one embodiment, CAMBOROUGH® C D163 - / - Hybrid pigs have a litter size that is not genetically edited (CAMBOROUGH). (Registered Trademark) Indistinguishable from hybrid pigs. In certain embodiments as described herein, The heterozygous and homozygous pigs do not contain extra-target mutations.

[0190] This specification further proposes methods for preparing CD163 gene-edited hybrid animals. This includes, and in some embodiments, the first parent is used for crossbreeding with the second parent. PIC (trademark) systems 2, 3, 15, 19, 27, 62, or system One of the 65 strains: CD163 gene-edited boar, CD163 gene-edited heifer , or may include CD163 gene-edited sows. In one embodiment, gene-edited, The genome of parent 1 is selected from the group consisting of sequence numbers 1-18 or 426-505. It may include sequences. In some embodiments, the genome may include sequence number 453. In various embodiments, The genome may contain sequence number 489. In various embodiments, the genome may contain sequence number 505. It may include. In some embodiments, the method may include PIC(trademark) systems 2, 3, 15, and 1 9. CD163 gene-edited boar from one of the following lines: line 27, line 62, or line 65. From a group consisting of CD163 gene-edited heifers or CD163 gene-edited sows A second parent may be selected. This specification also describes how to prepare CD163-edited animals. Methods, which are PIC(trademark) systems 2, 3, 15, 19, 27, and This may include a step of crossbreeding a progeny from either line 62 or line 65. Methods are also presented, and these are included.

[0191] In one embodiment, this specification describes a successful knockout editing of CD163 Lines 2, 3, 15, and 65 (each) may have at least one copy of the gene. Here, we present heterozygous pigs derived from Tables 8, 9, 10, and 14. These edited pigs may exhibit a healthy phenotype without significant adverse effects derived from the editing process. In some aspects, this specification discloses how to produce edited pigs with a healthy phenotype. The methods used to edit are shown in Table 11 for lines 19, 27, and 62 (each line in Table 11). We present pigs derived from Tables 12 and 13). Heterozygous pigs are F1 heterozygotes. To produce conjugable pigs, they can be crossbred with unedited animals of the corresponding strain. In this embodiment, heterozygous pigs of lines 2, 3, 15, and 65 are line 2, 3, 15 , and 65, may be crossbred with a second heterozygous pig, homozygous CD163-edited pigs can be produced in Mendelian ratios. In particular, gene editing is unique. In some cases, identification may be possible using sequence numbers 1-18 and 426-505, as shown in Tables 8-8. Genetic information containing at least 90% of the 14 genotype markers By sharing the name, this enables the detection of CD163 gene-edited genomes in any subsequent generation. and enable breeding. In one embodiment, the gene signature is the genotype marker in Tables 8 to 14. —of which, 95% or more of the genotype markers may be shared. In another embodiment, Genetic signature is determined by 97% or more of the genotype markers listed in Tables 8-14. They share genetic markers. Also, 98% of the genotype markers in Tables 8 to 14 This also includes genetic signatures that may share genotype markers beyond these. The important point is, The genomic regions that can be identified using the genotype markers in Tables 8-14 of the CD163 compilation. The relationship between the preparation of any progeny with the desired editing and editing in any progeny is significant. The objective is to enable tracking of a concentrated area. In certain embodiments according to this specification, the following Terrorzygous and homozygous pigs may not contain extra-target mutations. .

[0192] This specification presents embryos or zygotes that can be obtained from superior strains of pigs, and includes these. In one embodiment, the embryo or zygote is of lineage 2, lineage 3, lineage 15, lineage 15 of PIC(trademark). A superior pig lineage selected from the group consisting of lineage 19, lineage 27, lineage 62, or lineage 65. These can be obtained. In some embodiments, the embryo or zygote may be a frozen embryo or frozen zygote. In another embodiment, the embryo or zygote may be a frozen blastocyst. As presented herein, The embryos were prepared from in vitro mature oocytes recovered from estrus-synchronized heifers. It is possible. The surrounding cumulus cells are removed from the in vitro mature oocyte, and the washed male cells The sperm were incubated together and incubated. After incubation, the prognostic contact The combination involves CRISPR-Cas endonuclease and sequence numbers 22- listed in Table 3. With a guide RNA containing the first 20 nucleotides of 271 or 347-425 RNP mixtures containing combinations can be microinjected. The injected embryos are 1- It can be transplanted into a female surrogate mother at the 4-cell stage. In one embodiment, the RNP mixture is shown in Table 6. The enumerated repair molds (SEQ ID NOs: 1-13) may further be included. In one embodiment, the injected The juveniles were bred in a way that synchronized estrus, via midline laparotomy under general anesthesia. The embryos can be surgically implanted into the fallopian tubes of a female surrogate mother (each surrogate mother can have 40-60 embryos). (It is injected.)

[0193] This specification is CD163 - / - We present a selection of superior gene-edited pig lines that may be such, These include. In one embodiment, the systems are PIC(trademark) system 15, PIC(trademark) system 1 7, PIC (trademark) system 27, PIC (trademark) system 65, PIC (trademark) system 14, PI C (trademark) line 62, PIC337, PIC800, PIC280, PIC327, PI C408, PIC(trademark)399, PIC410, PIC415, PIC359, PIC 380, PIC837, PIC260, PIC265, PIC210, PIC (trademark) series system 2, PIC (trademark) system 3, PIC (trademark) system 4, PIC (trademark) system 5, PIC ( Trademark) line 18, PIC (trademark) line 19, PIC (trademark) line 92, PIC95, PI ©CAMBOROUGH (trademark), PIC1070, PIC (trademark)CAM BOROUGH (registered trademark) 40, PIC (trademark), CAMBOROUGH (registered trademark) 2 2. PIC1050, PIC(trademark) CAMBOROUGH(registered trademark) 29, PIC( (Trademark) CAMBOROUGH (Registered Trademark) 48, or PIC (Trademark) CAMBOROUGH It may be GH(registered trademark)x54. In some embodiments, the system is as described herein. The PIC(trademark) strains 2, 3, 15, 19, and 19, which contain the CD163 gene. The group can be selected from lineage 27, lineage 62, lineage 65, and their progeny. Edited: System 2, System 3, System 15, System 19, System 27, System 62, and System 6 5 includes the CD163 genomic region presented in Tables 8 to 14 above, and is a non-refined system. It can be easily distinguished from the main lineage and other superior lines. Similarly, as shown in Tables 8 to 14. CD163 - / - Progeny including genomic regions can also be identified. Therefore, this specification, CD163 - / - We present a progeny of pigs that may possess a specific genomic region.

[0194] This specification presents a hybrid pig line containing the edited CD163 gene of this instruction. These include. In some embodiments, the hybrid pig lineage is the edited PIC(trademark) lineage, It may be produced by crossbreeding with at least one other edited PIC(trademark) lineage. In some embodiments, the pig lineage is derived from germplasm of three or more pig lines. It can be introduced and produced by sequential crossbreeding. In one embodiment, the lineage is PIC (trademark) Line 15, PIC (trademark) line 17, PIC (trademark) line 27, PIC (trademark) line 65 , PIC (trademark) series 14, PIC (trademark) series 62, PIC337, PIC800, P IC280, PIC327, PIC408, PIC(trademark)399, PIC410, PI C415, PIC359, PIC380, PIC837, PIC260, PIC265, PIC210, PIC (trademark) system 2, PIC (trademark) system 3, PIC (trademark) system 4, PIC (trademark) system 5, PIC (trademark) system 18, PIC (trademark) system 19, PIC (trademark) system (Mark) System 92, PIC95, PIC(trademark), CAMBOROUGH(registered trademark), PIC 1070, PIC (trademark), CAMBOROUGH (registered trademark), 40, PIC (trademark), CA MBOROUGH (registered trademark) 22, PIC1050, PIC (trademark) CAMBOROU GH(registered trademark)29, PIC(trademark)CAMBOROUGH(registered trademark)48, or It could be PIC(trademark) CAMBOROUGH(registered trademark) x54.

[0195] In various forms, PIC(trademark) series 65 is sold under the trademark name PIC337. In various forms, the PIC(trademark) series 62 is sold under the trademark name PIC408. In various forms, this involves crossbreeding PIC (trademark) lineage 15 with lineage 17. Hybrid pigs created by this company are sold under the trademark names PIC800 or PIC280. In various forms, PIC(trademark) system 27 is sold under the trademark name PIC327. It is being sold. In various forms, PIC(trademark) system 65 and PIC(trademark) system 62 and Hybrids created by crossbreeding different species include PIC399 and PIC410. It is sold under the trademark name PIC415. In various forms, PIC(trademark) system 6 The hybrid created by crossbreeding 5 with PIC line 27 is PIC It is sold under the trademark name 359. In various forms, PIC (trademark) system 800 pig ( (A hybrid of PIC(trademark) system 15 and PIC(trademark) system 17), P The hybrid, prepared by crossbreeding the IC (trademark) strain 65 pig, is PI Sold under the trademark names C380 or PIC837. In various forms, PIC (trademark) (Trademark) System 14 is sold under the trademark name PIC260. In various forms, PIC( High (trademark) Lineage 14 and PIC (trademark) Lineage 65 were created by crossbreeding them. The hybrid form is sold under the trademark name PIC265. In various forms, PIC (trademark) A hybrid created by crossbreeding (Trademark) Line 2 and PIC (Trademark) Line 3. The chipset is PIC210, PIC(trademark), CAMBOROUGH(registered trademark), and P It is sold under the trademark name IC1050. In various forms, it is part of the PIC(trademark) system 3, Hybrid versions with PIC(trademark) system 92 are sold under the trademark name PIC95. In the diverse configuration, PIC(trademark) lineage 19 and PIC(trademark) lineage 3 were crossbred. Hybrid bodies made from these materials are sold under the trademark name PIC1070. In such a manner, crossbreeding PIC(trademark) lineage 18 and PIC(trademark) lineage 3 is performed. The hybrid body created by this is PIC(trademark) CAMBOROUGH(registered trademark). It is sold under the trademark names of 40. In various forms, it is sold under the PIC(trademark) system 19 and PI C system 1050 (This itself is a hybrid of PIC (trademark) system 2 and system 3) The hybrid created by crossbreeding (a certain species) is called PIC(trademark)CAM. Sold under the trademark name BOROUGH(registered trademark)22. In various forms, PIC The (trademark) system and PIC1070 (which itself is the PIC (trademark) system 19 and system 3 Hybrids created by crossbreeding (which are hybrids of) Sold under the trademark names PIC (trademark) and CAMBOROUGH (registered trademark) 29. In such a manner, PIC(trademark) system 18 and PIC system 1050 (which itself is PIC( Created by crossbreeding a hybrid of lineage 2 (trademark) and lineage 3. The hybrid body is a trademark of PIC (trademark) CAMBOROUGH (registered trademark) 48. It is sold under the name of PIC(trademark) system 4 and PIC(trademark) system The hybrid created by crossbreeding 5 is PIC(trademark)CAMBO This instruction is sold under the trademark name ROUGH(registered trademark)x54. This instruction is included on CD1 of this instruction. Present one of the aforementioned strains or hybrids of pig, including 63 edits. These include...

[0196] In various embodiments, this instruction is based on the edited versions shown in Sequence IDs 453, 489, or 505. A pig containing the edited CD163 gene, including the row, PIC (trademark) line 15, PIC (trademark) line 17, PIC (trademark) line 27, PIC (trademark) line 65, PIC (trademark) Line 14, PIC (trademark) line 62, PIC (trademark) line 2, PIC (trademark) line 3, P IC (trademark) system 4, PIC (trademark) system 5, PIC (trademark) system 18, PIC (trademark) System 19, PIC (trademark) System 92, or two or more of these systems. This document presents and includes pigs that could be hybrids of the L lineage. In various forms, The hybrid pig is a cross between PIC(trademark) strain 15 and PIC(trademark) strain 17. A hybrid of PIC(trademark) lineage 65 and PIC(trademark) lineage 62, PIC(trademark) A hybrid of (Trademark) lineage 65 and PIC (Trademark) lineage 27, and PIC (Trademark) lineage 15, A successive hybrid of PIC(trademark) system 17, in which the hybrid offspring are successive So, PIC(trademark) system 65, PIC(trademark) system 14, and PIC(trademark) system 65 A hybrid of PIC(trademark) lineage 2 and PIC(trademark) lineage 3, PIC( A hybrid of (trademark) lineage 3 and PIC (trademark) lineage 92, and PIC (trademark) lineage 19, A hybrid of PIC(trademark) lineage 3, PIC(trademark) lineage 18, and PIC(trademark) lineage A successive hybrid, PIC (trademark) lineage 2, which is crossbred with a hybrid of lineage 3. And, a hybrid pig with PIC(trademark) lineage 3 and a pig with PIC(trademark) lineage 19 Sequential hybrid, PIC(trademark) system 19 and PIC(trademark) system 3 hybrid A successive hybrid of a PIC(trademark) pig and a PIC(trademark) pig of type 2, PIC(trademark) A hybrid of the PIC (trademark) strain 2 was crossbred with a pig of the PIC (trademark) strain 18. A successive hybrid with (trademark) system 3, or with PIC(trademark) system 4 and PIC(trademark) It may be a hybrid with lineage 5.

[0197] In various forms, hybrid lines containing the edited CD163 gene are lines 2, 3, and 15. Crossbreed two or more lines from among 19, 27, 62, or 65 It can be produced by doing so. In some embodiments, a hybrid containing the edited CD163 gene is produced. The lineage may be the CAMBOROUGH(registered trademark) lineage. (Registered Trademark) The pig is a hybrid that can be prepared by crossbreeding lineage 2 and lineage 3. In various forms, hybrid lines contain the edited CD163 gene, PIC (trademark). ) May include 837 hybrid lines. PIC(trademark)837 pig is PIC(trademark) line A hybrid can be prepared by crossbreeding the 800 pig with the PIC (trademark) 65 pig. It is a hybrid.

[0198] This specification may be obtained from lines 2, 3, 15, 19, 27, 62, or 65. CD163 - / - Genome region and lineages 2, 3, 15, 19, 27, 62, or 65 A second CD163, which can be obtained from a different lineage, is selected from among them. - / - Includes the genomic region CD163 - / - This presents and includes hybrid successor lines. In one embodiment, CD1 63 - / - The hybrid progeny lines have CD163 within the genomic region as shown in Table 8. - / - conflict The gene and the CD163 within the genomic region according to Table 10. - / - It may include alleles. In this manner, CD163 - / - Hybrid progeny lines have C within the genomic region as shown in Table 8. D163 - / - Alleles and CD163 within the genomic region according to Table 14 - / - Alleles It may include children. In some cases, CD163 - / - The hybrid successor lineage follows Table 9. CD163 within the genomic region - / - Alleles and CD1 within the genome region according to Table 10 63 - / - It may include alleles. In one embodiment, CD163 - / - Later generations of hybrids The lineage is defined as CD163 within the genomic region according to Table 9. - / - Alleles and genotypes according to Table 14 CD163 within the area - / - It may include alleles.

[0199] This specification is characterized by the CD16 series, as indicated by Sequence IDs 1-18 and 426-505. We present and include hybrid animals that may include editing of 3 genes. In one embodiment, C Hybrid animals containing edited D163 genes can be characterized by Sequence ID No. 2. In one embodiment, hybrid animals including editing of the CD163 gene are sequence numbers 426-4 It can be characterized by 58. In one embodiment, a hybrid including editing of the CD163 gene The animal can be characterized by sequence number 453. In one embodiment, the CD163 gene Hybrid animals, including edits, can be characterized by sequences 459-504. In one embodiment, a hybrid animal containing editing of the CD163 gene is characterized by SEQ ID NO: 489. It can be marked. In some cases, hybrid animals are CAMBOROUGH (registered trademark). ) System, CD163 - / - It could be a hybrid animal. CAMBOROUGH (Registered (Trademark) Pig is a hybrid that can be prepared by crossbreeding strain 2 and strain 3. .

[0200] In various forms, hybrid pigs have sequence number 2 in the genomic region according to Table 8, and The genomic region may include Sequence ID No. 2 as shown in Table 10. In one embodiment, the hybrid pig is Sequence ID 453 within the genome region according to Table 8, and sequence number within the genome region according to Table 10. This may include item 453. In one embodiment, the hybrid pig has a distribution within the genomic region according to Table 8. This may include column number 489 and sequence number 489 within the genomic region according to Table 10. Now, CD163 - / - Hybrid progeny lines are sequence number 2 within the genomic region according to Table 8. , and may include SEQ ID NO: 2 within the genomic region according to Table 14. In one embodiment, CD163 - / -Hybrid progeny lines are identified by sequence number 453 in the genomic region according to Table 8, and Table 1 This may include sequence number 453 within the genomic region according to 4. In one embodiment, CD163 - / - Ha The hybrid progeny lines follow sequence number 489 in the genomic region according to Table 8, and according to Table 14. It may include sequence number 489 within the genomic region. In one embodiment, CD163 - / - Hybrid The descendant lineage is defined as sequence number 2 within the genomic region according to Table 9, and within the genomic region according to Table 10. This may include sequence number 2 of CD163. - / - The later hybrid lineage is shown in the table. Sequence ID 453 in the genome region according to 9, and Sequence ID 4 in the genome region according to Table 10. May include 53. In some embodiments, CD163 - / - The hybrid successor lineage follows Table 9. Includes sequence number 489 within the genomic region, and sequence number 489 within the genomic region according to Table 10. It can be seen. In one embodiment, the hybrid pig has sequence number 2 in the genomic region according to Table 8, This may include sequence number 2 within the genomic region according to Table 10. In one embodiment, hybrid The data points are sequence number 453 within the genome region according to Table 8, and the data points within the genome region according to Table 10. This may include column number 453. In one embodiment, the hybrid pig has a genomic region according to Table 8. This may include sequence number 489 and sequence number 489 within the genomic region according to Table 10. In this configuration, CD163 - / - Hybrid progeny lines are sequence numbers within the genomic region according to Table 8. This may include sequence number 2 and sequence number 2 within the genomic region according to Table 14. In one embodiment, CD1 63 - / - Hybrid progeny lines are sequence number 453 in the genomic region according to Table 8, and This may include sequence number 453 within the genomic region according to Table 14. In one embodiment, CD163- / - Hybrid progeny lines are identified by sequence number 489 in the genome region according to Table 8, and in Table 14. This may include sequence number 489 within the corresponding genomic region. In one embodiment, CD163 - / - Hive The Lid progeny lineage has Sequence ID No. 2 within the genomic region according to Table 9, and the genomic region according to Table 10. May include sequence number 2 within the region. In one embodiment, CD163 - / - The later generation of hybrids Sequence ID 453 within the genome region according to Table 9, and sequence number within the genome region according to Table 10. May include item 453. In some embodiments, CD163 - / - The hybrid successor lineage is shown in Table 9. Sequence ID 489 in the genomic region that follows, and Sequence ID 489 in the genomic region that follows Table 10 This may include. In some embodiments according to this specification, heterozygous pigs and homozygous pigs as specified herein. Competitive pigs may not contain mutations outside the target site.

[0201] In one embodiment, the hybrid animal is of the CAMBOROUGH® lineage, CD 163 - / - It can be a hybrid animal. CAMBOROUGH® pigs are This is a hybrid that can be prepared by crossbreeding lineage 2 and lineage 3. In one embodiment, Hybrid pigs have sequence numbers 426-505 in the genomic region according to Table 8, and Table 1 This may include sequence numbers 426-505 within the genomic region following 0. In one embodiment, hybrid Dobuta has sequence number 453 in the genomic region according to Table 8, and in the genomic region according to Table 10. This may include sequence number 453. In one embodiment, the hybrid pig has a genome region according to Table 8. This may include SEQ ID NO: 489 within the region and SEQ ID NO: 489 within the genomic region according to Table 10. In one embodiment, CD163 - / - Hybrid progeny lines have a distribution within the genomic region as shown in Table 8. Includes column numbers 426-505 and sequence numbers 426-505 within the genomic region according to Table 14. It can be seen. In one aspect, CD163 - / - Hybrid progeny lines follow the genome regions shown in Table 8. This may include SEQ ID NO: 453 within the region and SEQ ID NO: 489 within the genomic region according to Table 14. In one embodiment, CD163 - / - Hybrid progeny lines have a distribution within the genomic region as shown in Table 8. This may include column number 489 and sequence number 453 within the genomic region according to Table 14. Now, CD163 - / - Hybrid progeny lines are sequence number 4 within the genomic region according to Table 9. This may include sequences 26-505 and sequences 426-505 within the genomic region according to Table 10. In one embodiment, CD163 - / - Hybrid progeny lines have a distribution within the genomic region as shown in Table 9. This may include column number 453 and sequence number 453 within the genomic region according to Table 10. Now, CD163 - / - Hybrid progeny lines are sequence number 4 within the genomic region according to Table 9. This may include sequence 89 and sequence number 489 within the genomic region according to Table 10.

[0202] In various forms, PIC(trademark) system 2, PIC(trademark) system 3, PIC(trademark) system 15, PIC (trademark) system 19, PIC (trademark) system 27, PIC (trademark) system 62, or The PIC(trademark) strain 65 is an edited pig, as shown in Sequence ID No. 518, with a predicted amino acid distribution. The CD163 gene may contain a sequence. In various forms, the edited CD163 gene may contain a sequence. It may have a 123bp deletion, as shown in number 505. In various configurations, this deletion is A HRKPRLV--------------------------------- --------TVVSLLGGAHFGEGSGQIWAEEFQCEGHESHL Exon 7 of SLCPVAPRPDGTCSHSRDVGVVCS (Sequence ID 518) It may have an amino acid sequence. In various embodiments, it may include the amino acid sequence shown in SEQ ID NO: 518. Pigs possessing the edited CD163 gene are hybrids between two PIC(trademark) lines. They can be offspring. In various forms, pigs have the nucleotide sequence shown in Sequence ID No. 505. It may have an edited sequence of CD163 exon 7, including a column. In various embodiments, the pig may have P It may be a descendant of crossbreeding between IC (trademark) lineage 2 and PIC (trademark) lineage 3. In some embodiments relating to this specification, heterozygous pigs and homozygous pigs as defined herein are: It may not contain mutations outside the target site.

[0203] Importantly, the CD163-edited pigs and CD163-edited cells described herein are their The goal is to retain the desired commercial phenotype. Edited pigs exhibit significant acute adverse effects derived from the editing process. It may present with a normal phenotype without any associated effects. In some cases, lineages 2, 3, 15, and lineages... Pigs of lineage 19, lineage 27, lineage 62, and lineage 65 are commercially available as shown in Table 15. It can retain the desired phenotype.

[0204] Methods to improve the health of existing livestock herds are to use the methods described above, C The method may include a step of modifying the D163 locus. In one embodiment, the method involves transforming porcine cells into , endonuclease or polynucleotide encoding the endonuclease, and the first 20 nucleotides of each of sequence numbers 22-271, or sequence number 347- A guidepo containing a sequence selected from each of the 425 groups consisting of the first 20 nucleotides. The step of introducing a renucleotide, and the endonuclease, at the target sequence, In this vicinity, under conditions that allow it to act on DNA, the cells are incubated This allows for recombinant, homologous directive repair at or near the target site. The steps of inducing double or non-homologous end joining and the target sequence having modifications The steps include identifying at least one cell and creating an animal from the animal cell. This may include. In some aspects of this specification, endonuclease is S. thermo Guide RNA and Cas protein derived from S. thermophilus It may be an RNP complex. In another embodiment, the endonuclease is a Streptococcus pyogenes (S. pyo). It is an RNP complex consisting of guide RNA and Cas protein derived from genes. ru.

[0205] In one embodiment, the method involves transferring nucleotides 50-100 of sequence numbers 1-13 to cells. The step may further include providing a repair guide that includes sequences selected from a group. Another embodiment The repair guide is selected from a group consisting of 50 to 100 nucleotides from sequence numbers 1 to 13. It is possible to include sequences such as nucleotides 1-49 of sequence numbers 1-13, and For 101-150, it may further include 85% homology. In one embodiment, the restoration guide This may include sequences selected from the group consisting of sequence numbers 1 to 13. Therefore, methods for improving the health status of existing animal populations are those discussed above, and the desired commercial This demonstrates the maintenance of phenotype. In one embodiment, the desired commercial phenotype is similar to the genetic background. Of the phenotypes observed in groups of unedited pigs with land, at least 90% It's possible.

[0206] The CD163 gene editing locus can be edited using conventional breeding methods or by incorporating artificial insemination. They can then be introduced into animal populations. To prepare homozygous animals, use SEQ ID NOs. 1-18 or It has at least one gene-editing CD163 locus containing sequences 426-505. Crossbreeding between these parents is possible, and homozygous progeny (existing in a Mendelian ratio of 1:4) ( may be selected.) As presented herein, the parent CD163 locus and one gene Combining it with the sub-edited CD163 locus may be suitable for improving animal populations. Furthermore, CD163 Breeding animals with gene editing loci requires that all animals in the animal group have the described CD16 Up to 3 gene editing loci, the health status of the animal population can be improved. In particular, the health status of the animal population Health status was evident long before the animal group was crossbred with the CD163 gene-edited animal group. This can be improved. Specifically, and as discussed above, PRRSv resistance C Pig fetuses from D163 gene-edited sows are protected from PRRSv. Furthermore, as those skilled in the art will know, as the number of homozygous CD163-edited animals increases, PRRSv Knowing that the number of suitable vector pigs for transmission will decrease (for example, as immunity develops in the animal population) Therefore, this method improves the animal population by introducing herd immunity. It could lead to this.

[0207] One useful method for detecting desired editing is to use PCR primers that sandwich the region of interest, By using real-time PCR with a probe that specifically anneals to the target region, The probe is labeled with both a fluorophore and a quencher. PCR reaction Then, the primers and probes are sequence-dependently applied to the complementary DNA strand of the region of interest. Hybridize. The probe is intact, so the fluorophore and the quencher are close together. Upon contact, the quencher absorbs the fluorescence emitted by the fluorophores. Polymerase then... —It extends from there and begins DNA synthesis. When polymerase reaches the probe, The exonuclease activity of merase cleaves the hybridized probe. As a result, the fluorophores are separated from the quencher and emit fluorescence. This fluorescence is Detected by an real-time measuring instrument. For each PCR cycle, these steps are reversed. This allows for the detection of specific products.

[0208] In this application, three separate sets of primers and probes were designed. Immer's first set (sequences 556 and 557) is unedited, including sequence number 249. The probe (SEQ ID NO: 558) sandwiches the genome sequence and the unedited genome DN between the primers. It binds to A. The second set of primers (SEQ ID NOs. 559 and 560) is SEQ ID NOs. The probe (sequence number 561) sandwiches the unedited genome sequence containing 256 between the primers. It binds to the unedited sequence. The final set of primers (sequences 562 and 563) is The wound was created by excising the sequence between the cut site of sequence number 249 and the cut site of sequence number 256. The desired seventh exon deletion edit sequence is inserted. The probe (sequence number 564) is... These primers were designed to bond to the desired edit between them. Then, commercially available Using a real-time PCR kit, we probed a variety of animals for the desired editing. To put it without limitation, PRIMETIME®, Applied by IDT TAQMAN (registered trademark) by Biosystem (Roche Molecular Systems, Inc. (Pleasonton, CA), and Qiagen A variety of commercially available real-time PCR kits, including various kits from Bio-Rad. Such kits exist. Those skilled in the art will know that any such kit can achieve similar results as taught in this instruction. It will be recognized that it can be used in conjunction with the primers and methods. [Examples]

[0209] [Example 1] This example illustrates the selection of target sites for knockout of the porcine CD163 gene within porcine cells. To show.

[0210] Directional navigation by Streptococcus Cas9 / gRNA Using DNA endonuclease, the porcine CD163 gene (Sscrofa11) was analyzed. 1. Insertion of DNA sequence within GenBank accession number: GCA_000003025.6) This involves creating additions, deletions, and combinations thereof, resulting in sequence changes within the CD163 gene. However, the function, stability, or expression of the CD163 messenger RNA or protein To reduce or eliminate it, the combination of guide RNA and protein is simple Delivered individually or as a pair. 120 nucleotides upstream of the translation initiation site (chr5:6 3300192)~CD163 Exon 7 (chr5:63323390) 59 nuclei The sequence of the CD163 gene downstream of rheotide is listed in Table 2 and filed together with this specification. The sequence listing includes Streptococcus pyogenes, or Stre Streptococcus thermophilus CRISPR3 (S. The Cas9 protein derived from each of the following species of S. thermophilus (CR3) The cleavage requires a gamma having either an nGG or nGGnG adjacent PAM sequence. We screened for idRNA binding sites. DNA sequences of target sites for editing. , the location of the target site on the CD163 gene, and the editing activity (as described in Example 3) The measured values ​​are shown in Table 3. The guide RNA molecule is the corresponding RNA nucleotide. However, the PAM sequence (nGG for Streptococcus pyogenes, and S. terumo) is associated with this, but the PAM sequence (nGG for Streptococcus pyogenes, and S. terumo) is associated with this. It lacked the nGGnG for S. thermophilus and had the same sequence as the target. Guide polynucleotide molecules may also consist of DNA bases, and DNA bases and It may also consist of a mixture with RNA bases. The target site sequence is filed together with this specification. The sequence listing will include sequences 22-271 and 347-425. The indicated target The site sequence is conserved across the porcine germplasm and can be screened by DNA sequencing. It will be done.

[0211] [Example 2] This example demonstrates the interaction of guide RNA / Cas9 endonuclease with porcine fetal fibroblasts. Nucleofection for delivery is illustrated.

[0212] DNA cleavage activity in living cells for the creation of edited porcine CD163 alleles. To investigate, we examined the CRISPR-Cas endonucleases and GYS listed in Table 3. The targeting sequence of the doRNA was nucleofected into pig fetal fibroblasts. Fetal fibroblast (PFF) cell lines were prepared from fetuses aged 28-35 days, and 3.2 μg of C was added. as9 protein (Streptococcus pyogenes or S. thermophilus) philus)) and 2.2 μg of in vitro transcription-type single guide RNA are mixed in water for 2 Combined to a total volume of 0.23 μl, then, using a Lonza electroporator, P Nucleofect was applied to FF cells. TrypL was used to prepare for nucleofection. PFF cells were collected using E express (recombinant trypsin), and at this time, Remove the culture medium from the cells, wash once with HBSS or DPBS, and then use TrypLE. In the presence of [the substance], the cells were incubated at 38.5°C for 3-5 minutes. Then, complete medium and Cells were collected from both subjects. They were then separated by centrifugation (300 × g, at room temperature for 5 minutes). The cells were pelleted, the supernatant was discarded, and then the cells were sampled using trypan blue staining. To obtain a single cell suspension, the cells were resuspended in 10 ml of LPBS. .

[0213] The appropriate amount of cells is pelleted by centrifugation (300 × g, at room temperature for 5 minutes). Discard the supernatant and add 7 cells per 1 ml to P3, which is nucleofection buffer. 5 x 10 6 The cells were resuspended at the final concentration. Using a multichannel pipette, 20 μl of cell suspension is placed in each nucleofection plate containing the RNP mixture. The RNP / cell mixture was added to the wells and then gently mixed with the resuspended cells. Transfer to a cleofection plate and run the CM138 program (supplied by the manufacturer). Nucleofect was performed using 80 μl of warmed Embryonic Fibrobla. st Medium, EFM (2.77 mM glucose, 1.99 mM L-glutamic acid) It contains 0.5 mM sodium pyruvate and 100 μM 2-mercaptoyl ester. Tanol, non-essential amino acid solution in 1x concentration Eagle Minimum Essential Medium (MEM NEA) A) 100 μg / mL penicillin-streptomycin and 12% fetal bovine blood Dulbecco's modified Eagle medium (DMEM) supplemented with phosphate is used after nucleofection. It was added to each well. The suspension was gently mixed by pipetting, and then 10 0 μl contains 900 μl of EFM, which has been pre-incubated at 38.5°C. The sample was then transferred to a 2-well plate. Next, the plate was incubated at 5% CO2, 38.5°C, for 48 hours. It was incubated for an extended period. 48 hours after nucleofection, the genomic DNA was... Prepared from transfected PFF cells and control PFF cells, 15 μl QUICKE XTRACT(TM) DNA Extraction Solution (Lucige n, Madison, WI) are added to the pelleted cells, and then incubated at 37°C for 10 minutes. Dissolve by incubating at 65°C for 8 minutes and then at 95°C for 5 minutes. Next, the lysate was kept at 4°C until it was ready for use in DNA sequencing.

[0214] [Example 3] This example shows a guide RNA / Cas9 combination directed to porcine CD163. The editing frequency of "se" is illustrated as an example.

[0215] As described in Example 2, the Cas9 protein, which was complexed with the guide RNA, By delivering it to fetal fibroblasts, it alters the nucleotide sequence of porcine CD163 It was introduced into the transmission system.

[0216] The porcine CD163 genome is mediated by the guide RNA / Cas endonuclease system. To assess DNA double-strand breaks at the target site, PCR is used to examine the area around the target site. The surrounding genomic DNA region of approximately 250 bp is amplified, and then the PCR product is used to create a unit replication sequence. Deep sequencing using (AMPLICON) was used to investigate the presence of editing. After triple transfection, the PFF genomic DNA is transfected as described in Example 2. The necessary sequences were added to the unit replication sequence-specific barcode, and the NEB Q5 polycode was extracted. Melase is used to PCR amplify the region surrounding the intended target site, and a tailed primer is used. Using ILLUMINA (registered trademark), via two rounds of PCR, A(registered trademark), San Diego, CA) performed sequencing. The result obtained The resulting PCR amplified product is ILLUMINA(registered trademark) MISEQ(registered trademark) Perso nal Sequencer (ILLUMINA®, San Diego, C A) Deep sequencing was performed on the above. Cas9 protein and guide RNA were traced. By comparison with control experiments that were excluded from the infection, or by comparison with a reference genome. Therefore, the resulting reads are examined for the presence of edits at the predicted cleavage site. Discussed the combination of target site, Cas9 protein, and guide RNA, as edited by the NHEJ. To calculate the frequency, edited reads (DNA sequences derived from the control treatment or reference genome) When compared to the total number of unit replicate sequences containing insertions or deletions, the barcode and Leads of appropriate length (wild-type leads) containing a perfect match with the forward primer. The total number of reads (reads + edited reads) was divided by the total number of reads. On average, the total read count per sample is approximately The value is 7000, and the NHEJ activity is shown in Table 3 as the average (n=3) edit rate. As stated, 0-58.2% of the leads contain edits, and the average across all combinations is... The editing frequency was approximately 16%. In this example, various guide RNAs were used in fetal fibroblasts. A complex of Streptococcus pyogenes or S. thermophilus (S. thermo) It is mediated by transfecting the Cas9 protein derived from philus. The nucleotide sequence of the porcine CD163 gene was edited via double-strand break stimulation. support.

[0217] [Example 4] This example illustrates the formation of in-frame stop codons using a DNA repair template. .

[0218] When co-delivered with Cas9 protein and guide RNA, use a DNA repair template. The stop codon was then introduced into the porcine CD163 gene. The endonuclide is disclosed in Table 6. It forms a rease-guide complex and repair template, and edits the CD163 gene in the blastocyst. It was used for this purpose. Base deletions were verified by the method of Example 5.

[0219] [Example 5] This example illustrates the molecular characterization of an edited animal genome.

[0220] Tissue samples were collected from genome-edited animals according to the examples described herein. These included tail samples and ear samples. The section sample or blood sample was of the appropriate tissue type. The tissue sample contained DNA. To preserve its integrity, the samples were frozen at -20°C within one hour of collection.

[0221] DNA was extracted from the tissue sample after digestion with proteinase K in a lysis buffer. Characterization was performed on two different sequencing platforms, and short sequence reads were identified using ILLUM. Using the INA® platform, long sequence reads are available at Oxford NA. NOPORE (trademark) platform (Oxford NANOPORE (trademark) Te The study was conducted on a computer at Technologies, Oxford, UK.

[0222] For short sequence reads, a two-step PCR is used to amplify the target region, and then... The first step was to combine the gene with the manufacturer's proprietary primer. Locus-specific primers are used to amplify the target gene locus from a DNA sample. It was a specific PCR. The second step is to allow sequencing to occur. The index array and adapter array for the single are replicated from the first step. It was joined to it.

[0223] The locus-specific primers for the first step of PCR are ILLUMINA (registered (Trademark) Paired-end sequencing leads are designed to span across the amplified fragment, <300bp The region was selected to amplify the protein. In the event that a deletion or naturally occurring point mutation occurs, Multiple unit replication sequences are preferred because they can lead to redundancy if proper binding of imagers is prevented. It was good. The sequence data for the unit replication sequence is from ILLUMINA®. Control platform (MISEQ®, ILLUMINA®, Generated using San Diego, CA. Sequence reads were analyzed and edited. This characterizes the outcome.

[0224] For long sequence reads, a two-step PCR is used to amplify the target region, and then... The first step was to combine the genes with the vendor's proprietary adapter. Locus-specific primers are used to amplify the target gene locus from a DNA sample. This is a specific PCR. The second step of PCR is for the preparation of the sequencing library. Therefore, as if the DNA is prepared, the sequencing index is used in the first step. The PCR product was joined to the unit replication sequence from PCR. The PCR product from step 2 had the ends of the DNA attached. Polish and ligate the adapter containing motor protein, DN To allow access to pores for A-chain-based sequencing, the distributor's key It underwent a series of chemical reactions caused by the bot.

[0225] To amplify different regions of the CD163 gene, for the range of first-step PCR We designed locus-specific primers and amplified regions of varying lengths. Then, we performed the normalization. Mix the lysated DNA with the loading buffer supplied by the distributor. Then, it was loaded into the NANOPORE(trademark) flow cell.

[0226] Long sequence reads have lower precision per base than short reads, but the target region This is extremely useful for observing the extensive spatial relationships of sequences in the vicinity of a given position.

[0227] [Example 6] This example aims to produce pigs with a CD163 editing gene that confers PRRSv resistance. Let's illustrate the method.

[0228] Porcine oocytes are isolated, fertilized, and then the resulting zygotes are edited to create genetic material. I created a genetically modified pig.

[0229] The CD163 RNP complex is fertilized in vivo or in vitro. It was then microinjected into the cytoplasm of a single-cell zygote of a pig. The zygotes were incubated to create edited multicellular embryos, and gene-edited pigs were born. The embryo was introduced into the surrogate mother heifer via a standard procedure. To that end, derived from PIC(trademark) systems 2, 3, 15, and 65, Heifers in the spring puberty stage were given a 0.22% altrenost solution (20-30ml per animal). The subjects were subjected to estrus synchronization by a 14-day treatment with 6 mg of the matrix. Administration of PMSG 36 hours after the final dose induces follicular proliferation, and PMSG Ovulation was induced by administering hCG 82 hours after the initial administration. Subsequently, in viv To produce zygotes, estrous females are selected from the corresponding PIC(trademark) strain. Artificial insemination (AI) was performed using male pig semen. The conjugates obtained in vivo were... Sterile TL-HE, replenished with 0.3% BSA (w / v) 12-24 hours after AI. The fertilized zygotes were surgically retrieved by retrograde flushing of the fallopian tubes using PES medium. , a Cas9 protein and guide RNA complex that targets CD163 (2 2-50 picoliters (5-50 ng / μl and 12.5-35 ng / μl) (pl) was subjected to single intracytoplasmic injection and cultured in PZM5 medium (Yoshioka, K., et al., Biol. Reprod., 2002, 60: 112-119; Suzuki, C., et al., Reprod. Fertil. Dev., 2006 18, 789-795; Yoshioka, K., J. Reprod. Dev. 2008, 54, 208-213). Injected joints The body was obtained by midline laparotomy under general anesthesia, and was not bred in a manner synchronized with estrus. The embryos were surgically implanted into the fallopian tubes of female surrogate mothers (each surrogate mother received 20 to 60 embryos). .

[0230] In vitro fertilized embryos for gene editing were derived from unfertilized PIC(trademark) oocytes. Estrus Synchronized PIC(trademark) immature oocytes derived from heifers are medium-sized (3-6 mm). It was collected from the follicle. Next, the oocyte was selected, which had dark cytoplasm and intact surrounding cumulus cells. The cells were selected for maturation. The cumulus / oocyte complex was placed in 38.5°C and humidified air. 500 μl of mature medium TCM-199 is incubated with 5% CO2 for 42-44 hours. (Invitrogen) (3.05 mM glucose, 0.91 mM sodium pyruvate) Thorium, 0.57 mM cysteine, 10 ng / ml EGF, 0.5 μg / ml yellow Body-forming hormone (LH), 0.5 μg / ml FSH, 10 ng / ml gentamicin (Sigma) and wells supplemented with 10% follicular fluid were placed in the wells. At the end, vortexing for 3 minutes in the presence of 0.1% hyaluronidase Then, the surrounding cumulus cells were removed from the oocyte. Next, the in vitro matured oocyte was extracted. In groups of 25-30 oocytes, IVF medium (113.1 mM NaCl, 3 mM KCl, 7.5 mM CaCl2, 11 mM glucose, 20 mM Tris, 2 mM Caffeine, 5 mM sodium pyruvate, and 2 mg / ml bovine serum albumin Place in a 100 μl droplet of modified Tris buffer medium (containing BSA) and add fresh E An established protocol (Abeyde) is used, which involves using extended boar semen. Fertilization was performed according to era, Biol. Reprod., 57:729-734, 1997. Extended testing was performed. 1 ml of used semen is mixed with Dulbecco's phosphate-buffered saline containing 1 mg / ml of BSA. Mix with DPBS to a final volume of 10 ml, and bake at 1000 × g, 25°C for 4 minutes. The sperm were then centrifuged and washed three times in DPBS. After the final wash, the sperm The sperm were resuspended in mTBM medium, and the sperm count was 1 × 10⁶ per 1 ml. 5 At the final concentration of each egg, The solution was added to the parent cells and co-incubated at 38.5°C and 5% CO2 for 4-5 hours. The procedure was performed. Five hours after IVF, the estimated zygote was microinjected, and the procedure was performed between 18 and 42 hours. After a certain period (1-4 cell stage), the embryos were transplanted into female surrogate mothers. Each surrogate mother was injected with 20-60 embryos. Pregnancy is confirmed by the absence of a return to estrus (after 21 days), and 28 days after embryo transfer, We performed an ultrasound test.

[0231] To establish the frequency of Cas9-guided RNA-targeted gene editing in pig embryos. To achieve this, the non-injected control conjugates and the surplus injected conjugates produced by in vitro fertilization are used in PZ The cells were cultured in M3 medium or PZM5 medium at 38.5°C for 5 to 7 days. On day 7, the blastocyst was collected, and the genomic DNA was isolated for next-generation sequencing. .

[0232] [Example 7] This example illustrates the creation and characterization of gene-edited pigs.

[0233] Animals derived from PIC(trademark) strains 2, 3, 15, and 65 are described in Example 6. The cells were edited using the method described above. The success of the editing was confirmed using the method of Example 5. Fibroblasts The strain was grown from collagenase-treated ear-cutting samples extracted from edited animals, and Ameri can Type Culture Collection (ATCC (registered trademark)) It was deposited. The address of ATCC (registered trademark) is 10801 University Bo Ulevard, Manassas, VA 20110-2209. CD163 edition. A representative sample of the PIC(trademark) System 2 was deposited with ATCC on April 3, 2019, and is classified as A TTC (Registered Trademark) Patent Accession Number: PTA-125814 was assigned. CD163 A representative sample of PIC(trademark) System 3 was deposited with ATCC on April 3, 2019. ATTC (registered trademark) Patent Accession Number: PTA-125815 was assigned. CD16 A representative sample of PIC(trademark) system 15 was deposited with ATCC on April 3, 2019. And it was assigned the ATTC (registered trademark) patent application number: PTA-125816. CD 163 Edited PIC(trademark) System 65 representative sample was certified by ATCC on April 3, 2019. The patent was deposited with (registered trademark) and the ATTC (registered trademark) patent application number: PTA-125813 was assigned. They were identified. Each deposit was made in accordance with the Budapest Convention. Representative animals from each lineage are We confirmed that the heterozygous editing specified in Table 16 was present.

[0234] [Table 16] Each of the animals in Table 16 has nucleotides 63301999-633 from chromosome 5. A healthy phenotype with the deletion of nucleotide 02005 (in the second exon) was presented.

[0235] Collagener derived from unedited animals of PIC (trademark) strains 19, 27, and 62. Further cell lines were propagated from the treated ear-cut samples and deposited with ATCC. PIC (trademark) A representative sample of lineage 19 was deposited with ATCC (registered trademark) on April 3, 2019, and A TTC (Registered Trademark) Patent Accession Number: PTA-125811 was assigned. PIC (Trademark) A representative sample of lineage 27 was deposited with ATCC on April 3, 2019, and registered with ATCC (Registered (Registered trademark) Patent application number: PTA-125907 was assigned. PIC (trademark) system 6 A representative sample of 2 was deposited with ATCC on April 3, 2019, and is registered under the ATTC® trademark. Authorization number: PTA-125812 has been assigned. Each deposit is in accordance with the Budapest Convention. I created them. Using standard cloning methods, I created PTA-125811 and PTA-125 Animals were created and edited from cell lines deposited as 812 and PTA-125907. The strain was edited using the method of Example 6 to create the new lineage.

[0236] Single nucleotide polymorphisms (SNPs) in the vicinity of the CD163 gene are used in the deposited PIC (trademark) strain. Analyze each of the following: 2, 3, 15, 19, 27, 62, and 65) and identify each strain. We selected SNP profiles for each lineage that were available. We defined the lineage signature. The starting dataset for this purpose consists of 7 deposited PIC(trademark) strains and 330 whole genome samples. It was a collection of microbial animals. It focused on the CD163 gene on chromosome 5. A 6Mb area was extracted for signing purposes, and a signature regarding the lineage was formed along with it for comparison. To identify a small number of SNPs, intraphylogenetic and interphylogenetic variations are considered within each region. We examined nucleotides.

[0237] In order to be a candidate signature to be incorporated into the signature of a given lineage, for each chromosome position The following criteria must be met: sequence coverage must be present for 90% of the animals in the strain. The above must be true for at least 5 of the 7 systems. Not to be found; and all animals with data within the target strain must have the same homozygous genotype. It was imposed that they must possess this genotype. For each of the other lineages, the gene for this genotype. Type frequencies were calculated across all sequencing animals in this lineage. Cut off at least 30% of the difference between the highest and lowest genotype frequencies per individual. The condition was imposed (that is, a spread of genotype frequencies must be observed within the six lines). stomach).

[0238] Each system is defined using a combination of measurements of discriminative ability and measurements of uniform distribution. A subset of determinable genotypes was selected. Tables 8-14 show the homozygous genotypes within each pig strain. The locations on chromosome 5 of SNPs in which the sex allele is fixed are shown. The set of homozygous alleles distinguishes each pig strain from other strains. As indicated in Tables 8 to 14, the genotypes are such that which allele is present at each position. This indicates whether or not it is molten.

[0239] [Example 8] This example shows different levels of immune cells isolated from wild-type pigs and gene-edited pigs. We will compare the PRRSv resistance of the two.

[0240] CD163 surface expression analysis was performed using monocyte-derived macromolecules recovered from edited and wild-type animals. A lophage (MoMφ), each of which follows the method described in the above-mentioned example. This was performed on MoMφ with editing of the CD163 gene and MoMφ without editing. The edits tested are shown in Table 17. The four edits are available in various sizes, as shown. The fifth edit includes the deletion of , and the fifth edit includes the insertion of two base pairs; all edits in Table 15 are the This is a 2-exon edit. All deletions or insertions are truncated CD163 polypeptides. This results in CD163 expression being linked to mouse anti-porcine CD163 monoclonal Using the antibody clone 2A10 / 11, for immunofluorescence labeling and FACS analysis. Further evaluation was performed. MoMφ, a CD163-edited cell, was clearly shown in cell surface expression analysis. As a result, the cells lacked functional epitopes on their surface.

[0241] [Table 17]

[0242] Homozygous editing involves two base pair deletion edits of CD163 in cells, PRRSv To investigate viral infections, MoMφ was infected with type 1 PRRSv and type 2 PRRSv. The cells were stained. Cells were counted within the microscopic field 24 hours after infection to determine the replication of PRRS. The number of cells containing v was determined. CD163 homozygous edited MoMφ cells were type 1 PR It was found that neither RSv nor Type 2 PRRSv were in an acceptable state.

[0243] [Example 9] This example shows the seventh e from wild boar (S. scrofa) CD163 in porcine fibroblasts. This supports the use of two guides for removing the kuson.

[0244] DN of CD163 exon 7 encoding SRCR5 of mature CD163 polypeptide To remove the A sequence, from 450bp upstream to 59bp downstream of the 7th exon of CD163 The DNA sequence located within the intron region of *Streptococcus pyog enes)(NGG) and Streptococcus thermophilus Recognition sites of Cas9 protein and guide RNA by ilus)(NGGNG) We investigated. 48 sites were identified within 450 bp of the 6th intron, and 10 sites were identified within the 7th intron. These 58 sites were identified within a 59bp region of the intron. These 58 sites are related to Cas9 and guide The ability to bind to RNA and direct gene editing was first developed in porcine fetal fibroblasts. We investigated using a single guide. These single guide RNA-Cas9 proteins (spacers) - A subset of guide RNAs that resulted in high-frequency editing across the recognition site, In contrast, we further investigated their ability to remove the seventh exon of CD163. Nucleofection as described in Example 2 allows the guide to be transferred to porcine embryonic fibroblasts. Implemented: Each guide was prepared as an RNP, and then, before transfection, 2 Two complex sets were combined in a total volume of 2.23 μl. Editing for guide pairs. The frequency was determined as described in Example 3. The deletion of exon 7 of CD163 was combined This results in NHEJ-mediated repair, which provides endonuclease cleavage sites. The frequency is shown in Table 18.

[0245] [Table 18-1]

[0246] [Table 18-2]

[0247] The frequency of editing the cutout guides for the desired edit varied widely.

[0248] [Example 10] This example demonstrates the excision of exon 7 in a porcine blastocyst using dual guide RNA. Let's give an example.

[0249] In addition, a subset of guides screened in porcine fetal fibroblasts was used in porcine embryos. We also investigated their ability to remove exon 7 of CD163 within the cyst. A subset of the test guide within the embryo resulted in a deletion of exon 7 in porcine fibroblasts. These efficiency improvements, along with the reduction in off-target edits for each internal guide, Based on the combination of these efficiencies (see "Modes for Carrying Out the Invention"), they are selected. Edited pig embryos were created as described above. In short, they were recovered from slaughterhouse ovaries. The oocytes were fertilized in vitro as described in Example 6. pi = 200 hPa. FEMTOJET(registered trademark) 4i, set to ti=0.25 seconds and pc=15hPa. microinjector(Eppendorf; Hamburg, German By using a single pulse from y), the cells of the putative zygote were detected 4-5 hours after fertilization. The sgRNP solution was injected into the material. The outer diameter was set to 1.2 mm and the inner diameter to 0.94 mm. Glass capillary pipettes (Sutter Instrument, Navato, CA, The material (USA) is stretched to an extremely fine point of <0.5 μm. Microinjection is N arishige (Narishige International USA, Ami Tyville, NY) Inverted microscope equipped with a micromanipulator A TL-Hep, supplemented with 3 mg / ml BSA (Proliant), is placed on a heated platform. The procedure was performed in es(ABT360,LLC). After injection, the estimated conjugate w was subjected to 5% CO2, 5 In an incubator environment with %O2 and 90%N2, PZM5 (Japan, Tokyo, Cosmo Bio Inc.) The embryos were cultured for 7 days in (a company). The editing frequency of the blastocysts was as described in Example 3. It was decided that the terminal NHEJ would result in the deletion of exon 7 of CD163. The frequency of repairs is shown in Table 19.

[0250] [Table 19]

[0251] In this embodiment, multiple guide pairs are used to delete the seventh exon of CD163. However, this supports the idea that efficiency can vary significantly between guide pairs and between cell types.

[0252] [Example 11] In this example, the immature stop codon is identified as the seventh exon of wild boar (S. scrofa) CD163. This supports the use of two guides to introduce this concept.

[0253] Bioinformatics methods were used to identify the guides within the seventh exon of CD163. When the cut created by the guide is ligated as a whole during NHEJ, We screened their ability to create in-frame stop codons. Oenometric predictions were made using pig fetal fibroblasts, as described in Example 2. Verified internally: Before transfection, the guides were individually complexed, then 2 The mixture was combined in a total volume of 0.23 μl. The editing frequency was determined as described in Example 3. This results in the introduction of an immature stop codon within the seventh exon of CD163, at the terminal. The frequency of inter-NHEJ repair is shown in Table 20.

[0254] [Table 20-1]

[0255] [Table 20-2]

[0256] This embodiment demonstrates the editing efficiency of guide pairs designed to introduce stop codons, which is a pig This illustrates widespread variation within fibroblasts.

[0257] [Example 12] This example illustrates the editing efficiency of a guide for introducing stop codons into pig blastocysts.

[0258] A subset of guides screened in porcine fetal fibroblasts was used in porcine embryos, CD We also investigated their ability to introduce immature stop codons into the 7th exon of 163. In pig embryos, a subset of the test guides was found in pig fibroblasts with CD163 at level 7. Introducing immature stop codons within the thon, their efficiency, and each of the intrapairs described above Selected based on the combination of the absence of off-target factors observed in the id. Edit frequency This was determined as described in Example 3. Immature termination in the 7th exon of CD163 Table 21 shows the frequency of terminal NHEJ repair, which results in codon introduction.

[0259] [Table 21]

[0260] This embodiment is used to introduce an immature stop codon into exon 7 of CD163. This confirms that the editing efficiency of possible guide pairs varies widely.

[0261] [Example 13] This embodiment illustrates variable repair outcomes for NHEJ repair.

[0262] Subsequence of guide RNA designed to delete exon 7 of CD163 The ret was placed in the coding region of exon 7 within the porcine blastocyst, as described in Example 9. We investigated their ability to delete flanking regions. The editing frequency of blastocysts was The determination was made as described in Example 3. In this example, as a result of NHEJ-mediated repair Table 7 shows a subset of DNA sequences observed in vivo in porcine blastocysts. The following describes five guide RNA pairs in this context. In addition to simple nucleotide deletions, deletions are also shown. More complex NHEJ media include insertion, rearrangement, inversion, and any combination thereof. It is also known that intercavitary repair DNA sequences can occur. Without being limited by theory, These variability in repair outcomes also occurred in single-guide and paired-guide within porcine blastocysts. It can also result from DNA cleavage by endonucleases used. Table 7 also shows The frequency of observation of DNA sequences associated with each repair outcome varies among guide RNA pairs. This also shows that, in some cases, but not all, the cleavage site of the pairing guide RNA The frequency of DNA sequences associated with end-to-end junction was the most highly representative repair outcome. For some guide RNA pairs, a single major DNA repair outcome was observed, For other guide RNA pairs, multiple DNA repair outcomes are observed occurring at similar frequencies. The majority of DNA repair outcomes were shown for these five guide RNA pairs. This involves a deletion in the DNA sequence of CD163 exon 7, which corresponds to the SRCR 5 domain. This resulted in the following: Therefore, to create edited pigs, which guide RNA pair should be used? The decision of whether or not to use it depended heavily on multiple alleles within the population.

[0263] This example uses two intron guide RNAs to delete genomic DNA. In combination, the resulting NHEJ-mediated repair outcome is that the DNA break is resolved. Not only the method, but also the frequency of these resolutions varies between guide RNA pairs. To support this, the DN of single or double guide RNA for the creation of edited animals. To observe the A repair outcome, guide RNA pairs are screened within fibroblast cells or embryos. It is advantageous to do so.

[0264] [Example 14] This example demonstrates the presence of the spacer sequence shown in Sequence ID No. 249 within the cell, and / or Alternatively, a real-time PCR assay can be used to identify the desired excision and editing of the 7th exon. To give an example.

[0265] For this assay, we designed two primer sets and two probes. One of the primer sets is a spacer shown in SEQ ID NO: 249 within the unedited genome. The sequence was designed to sandwich the sequence. The sequences of these primers are sequence numbers 556 and 5 This is shown in 57. The sequence of sequence number 558 is labeled with a HEX fluorescent moiety. The sequence was designed to anneal with the unedited genome between the PCR primers. Other seprimets having the sequences shown in numbers 562 and 563 are available for the desired editing. The sequence was designed to sandwich the other elements. It has the sequence shown in sequence number 564, and the FAM fluorescence portion The labeled probe anneals with nucleotides across the editing junction region. The design was used. Real-time PCR was performed using both primer sets to determine the alleles. Tail samples and / or / ma samples of pigs whose state (wild type, homozygous, or heterozygous) is known. Alternatively, the procedure was performed on genomic DNA extracted from ear samples. 5 μl of 2×PRIMETI ME (Registered Trademark) Master Mix (Integrated DNA Technology) Use 0.5 μl of each primer (10 μl) of ologies, Coralville, IA. M) 0.5 μl of each probe (2.5 μM) and 2 μl of genomic DNA were mixed together. PCR is performed with initial denaturation at 95°C for 3 minutes, followed by 15 seconds at 95°C and 3 minutes at 64°C. The experiment was conducted using 35 cycles of 0 seconds and 30 seconds at 72°C. Final elongation was performed at 72°C. The reaction was carried out for 2 minutes, and the reactants were then kept in a cycler at 4°C. Fluorescence was measured. As shown in the diagram, as predicted, the homozygote is on the y-axis (representing the wavelength of the FAM portion) Heterozygotes are close together, forming a group near the center of the graph, while wild-type pigs are located along the X-axis (HEX section). They clustered in close proximity to the wavelength (represented by minutes). Therefore, the assay is shown in Sequence ID No. 249. We successfully detected edits based on the modified spacer array.

[0266] [Example 15] This embodiment demonstrates the presence of the spacer sequence shown in Sequence ID No. 256 within the cell, and / or Alternatively, a real-time PCR assay can be used to identify the desired excision and editing of the 7th exon. To give an example.

[0267] For this assay, we designed two primer sets and two probes. One primer set sandwiched the spacer sequence shown in Sequence ID No. 256. The sequences of their primers are shown in SEQ ID NOs. 559 and 560. SEQ ID NOs. 561 The probe, which has the following sequence and is labeled with a HEX fluorescent moiety, has an unedited spacer sequence and Designed for annealing. Other primer sets and probes are available for desired editing. Designed to target the specified target, as described in Example 14 (SEQ ID NO: 56) 2-564). Real-time PCR was performed as described in Example 14, but in reality The fluorescence was obtained using the primers and probes in the example. As predicted, homo The zygotes are close to the y-axis (representing the FAM region), while the heterozygotes are grouped near the center of the graph. The wild-type pigs formed groups close to the X-axis (representing the hexagonal region). Therefore, The assay successfully detected edits based on the spacer sequence shown in SEQ ID NO: 256.

[0268] [Example 16] This example demonstrates the previously published method for measuring intracellular CD163 CRISPR-CAS gRNA activity. A comparison between the previously provided guide pair and the guide pair in this instruction is illustrated.

[0269] Each pair of guides was examined in porcine fibroblasts as described in Example 2. Example 1 As described in section 0, each pair was further examined within pig blastocysts. The results are shown in Table 22 below. This will be shown.

[0270] [Table 22]

[0271] These data indicate that the guide in this disclosure provides a percentage of cells with the desired editing. This demonstrates at least a twofold improvement compared to the guidelines already disclosed in the references. Therefore, the guide in this disclosure is more efficient than the guides already disclosed in the literature.

[0272] [Example 17] This embodiment illustrates a CD163 edited pig challenged with type I PRRSv.

[0273] A pig derived from PIC(trademark) strain 2, as described in Example 6, SEQ ID NO: 249 Edited according to the guide shown in 256. Editing was carried out as described in Example 5. They acknowledged this. Next, they crossbred the edited pigs with other species to create pigs that were homozygous for editing. These homozygous edited pigs were given billions of 10 4 ~10 5 (4~5 log T CID 50 ) was administered 3 ml of type I PRSSv(SD13-15). 1.5 ml was administered intramuscularly using a 21-gauge needle. The remaining 1.5 ml was administered intranasally. Serum samples were collected on day 0 (before vaccination on the day of vaccination), day 3, day 5, day 10, and day 14. And obtained on day 21. Following the manufacturer's instructions, TETRACORE® EZ- PRRSV MPX 4.0 Master Mix, and Enzyme with ROX (TETRACORE®, Rockville, MD) is used with blood Real-time PCR to determine the presence of virus in a clean sample. EU Real-time PCR The corrected counts are shown in Table 23. Using standard methods known in the art, the data is further refined. To make it intuitively understandable (a higher number indicates that more viruses have been detected) The count was reciprocated.

[0274] [Table 23]

[0275] Throughout the experiment, PRRSv was not detected in the serum of the edited animals.

[0276] Serum samples are also used in ELISA using the IDEXX PRRS X3 antibody test kit. The test is conducted at an authorized Veterinary Diagnostic Laboratory. This was performed by ory. The results are shown as "Sample:Positive Rate". A ratio of 0.40 or higher is considered positive. The result was considered positive. The ratios for each sample are shown in Table 24.

[0277] [Table 24]

[0278] Edited pigs do not show a positive rate. In contrast, all wild-type pigs, by day 21, This shows the positive rate. This further indicates that PRRSv is not circulating in the serum of edited pigs. Here are some examples.

[0279] In this embodiment, the pigs edited in the guides of Sequence IDs 249 and 256 are Type I PRRS This demonstrates resistance to V virus infection.

[0280] [Example 18] This embodiment illustrates a CD163 edited pig challenged with Type II PRRSv.

[0281] Pigs derived from PIC (trademark) strains 2 and 3 were used as described in Example 6. Edited according to the guide shown in Sequence IDs 249 and 256. The editing is described in Example 5. I confirmed it as described. Next, I crossbred the edited pigs with other species to determine if they were homozygous for editing. A certain type of pig was created. Then, these homozygous edited pigs were given billions of 10 4 ~10 5 Individual (4-5 log TCID) 50 ) containing 3 ml of type II PRRSv (NVSL9 The drug 7-7895) was administered. 1.5 ml was given intramuscularly using a 21-gauge needle. The remaining 1.5 ml was administered intranasally. Serum samples were collected on day 0 (before vaccination on the day of administration) and day 3. Obtained on day 1, day 5, day 10, day 14, and day 21. TE was used according to the manufacturer's instructions. TRACORE(R) EZ-PRRSV MPX 4.0 Master Mix , and using Enzyme with ROX, the presence of the virus in serum samples is determined. To determine the results, real-time PCR was performed. Regarding NA correction for real-time PCR... The numbers are shown in Table 25. The data can be understood more intuitively (the larger the number, the more...). The count was reciprocated to represent the number of viruses present.

[0282] [Table 25]

[0283] The virus count in edited pigs is extremely low to zero compared to wild-type pigs.

[0284] Serum samples are also used in ELISA using the IDEXX PRRS X3 antibody test kit. The test is conducted at an authorized Veterinary Diagnostic Laboratory. This was performed by ory. The results are shown as "Sample:Positive Rate". A ratio of 0.40 or higher is considered positive. The result was considered positive. The ratios for each sample are shown in Table 26.

[0285] [Table 26]

[0286] Edited pigs showed no positive rate, while all wild-type pigs tested positive by day 10. The rate is shown. In summary, these data show that in edited pig serum, the virus is circulating. To illustrate the absence of something, give an example.

[0287] This example shows that pigs with CD163 gene editing according to SEQ ID NOs. 249 and 256 are This demonstrates resistance to type II PRRSv infection.

[0288] Each of the aforementioned references and applications is incorporated herein by reference in its entirety. This disclosure has been described in detail, but the teachings set forth in the attached claims are... It is clear that modifications and changes are permitted as long as they do not deviate from the indicated scope. cormorant.

Claims

1. CD16 edited to confer PRRSv resistance to wild boar species (Sus scrofa) The three genes, wherein the editing cuts out the seventh exon, and the edited gene is sequence number A repaired genome sequence selected from the groups consisting of 426-458 and 520-555 Includes edited CD163 gene.

2. The aforementioned edits include sequence numbers 229 and 256, sequence numbers 230 and 256, and sequence number 2 31 and 256, Sequence IDs 237 and 256, Sequence IDs 241 and 256, Sequence Number Numbers 229 and 258, Sequence IDs 230 and 258, Sequence IDs 231 and 258, Distribution Column numbers 237 and 258, sequence numbers 241 and 258, sequence numbers 229 and 261 , Sequence IDs 230 and 261, Sequence IDs 231 and 261, Sequence IDs 237 and 2 61, SEQ ID NOs. 241 and 261, SEQ ID NOs. 219 and 256, SEQ ID NOs. 221 and Sequence IDs 256, 224 and 256, 227 and 256, and 219 and 258, Sequence IDs 221 and 258, Sequence IDs 224 and 258, Sequence ID 2 27 and 258, Sequence IDs 219 and 261, Sequence IDs 221 and 261, Sequence Number Numbers 224 and 261, Sequence IDs 227 and 261, Sequence IDs 249 and 256, Distribution Column numbers 250 and 256, sequence numbers 249 and 258, sequence numbers 250 and 258 Selected from the group consisting of sequence numbers 249 and 261, and sequence numbers 250 and 261. Using a guide RNA (gRNA) pair for the selected excision site of exon 7, The edited CD163 gene described in claim 1 is provided.

3. The aforementioned edits include sequence numbers 229 and 256, sequence numbers 230 and 256, and sequence number 2 31 and 256, Sequence IDs 241 and 256, Sequence IDs 229 and 258, Sequence Number Numbers 231 and 258, Sequence IDs 241 and 258, Sequence IDs 219 and 256, Distribution Column numbers 221 and 256, sequence numbers 224 and 256, sequence numbers 227 and 256 , Sequence IDs 227 and 258, Sequence IDs 221 and 261, Sequence IDs 249 and 2 56, Sequence IDs 250 and 256, Sequence IDs 249 and 258, and Sequence ID 2 gRN for the excision site of exon VII, selected from the group consisting of 49 and 261 The edited CD163 gene according to claim 1, which is created using an A pair.

4. The edited CD1 according to claim 1, wherein the repaired genome sequence is shown in SEQ ID NO:

453. 63 genes.

5. The editing is created using the sequences shown in sequence numbers 249 and 256, claim The edited CD163 gene described in 1.

6. The repaired genome sequence is shown in SEQ ID NO: 453, and the editing is shown in SEQ ID NO: 249 and The edited CD163 gene according to claim 1, created using the sequence shown in 256. 。

7. A wild boar species cell containing the edited CD163 gene according to any one of claims 1 to 6.

8. A cell line comprising a plurality of cells as described in claim 7.

9. The cell line according to claim 8, which is a fibroblast cell line.

10. PIC system 2, PIC system 3, PIC system 15, PIC system 19, PIC system 27, P The cell according to claim 7, derived from IC lineage 62 or PIC lineage 65.

11. CD16 edited to confer PRRSv resistance to wild boar species (Sus scrofa) The three genes, wherein the editing is selected from the group consisting of sequence numbers 506 to 517. Edited CD163 to create stop codons that result in a predicted amino acid sequence of 7 exons. gene.

12. The aforementioned edits include sequence numbers 351 and 365, sequence numbers 351 and 387, and sequence number 3 48 and 390, Sequence IDs 348 and 388, Sequence IDs 348 and 395, Sequence Number Numbers 352 and 365, Sequence IDs 352 and 387, Sequence IDs 352 and 399, Distribution Column numbers 353 and 365, sequence numbers 353 and 387, sequence numbers 353 and 399 , Sequence IDs 354 and 390, Sequence IDs 354 and 388, Sequence IDs 354 and 3 95, Sequence IDs 358 and 361, Sequence IDs 358 and 362, Sequence ID 358 and 368, Sequence IDs 358 and 384, Sequence IDs 358 and 394, Sequence ID 358 and 399, Sequence IDs 359 and 390, Sequence IDs 359 and 388, Sequence ID 3 59 and 395, Sequence IDs 360 and 368, Sequence IDs 360 and 384, Sequence Number Numbers 360 and 389, Sequence IDs 360 and 394, Sequence IDs 360 and 397, Distribution Column numbers 361 and 365, sequence numbers 361 and 387, sequence numbers 362 and 390 , Sequence IDs 362 and 388, Sequence IDs 362 and 395, Sequence IDs 364 and 3 65, Sequence IDs 364 and 387, Sequence IDs 364 and 399, Sequence ID 365 and Sequence IDs 368, 365 and 384, 365 and 389, and 365 and 394, Sequence IDs 365 and 397, Sequence IDs 366 and 368, Sequence ID 3 66 and 384, sequence numbers 366 and 389, sequence numbers 366 and 394, and It is created using gRNA selected from the group consisting of SEQ ID NOs: 366 and 397. The CD163 gene according to claim 11.

13. The aforementioned edits include sequence numbers 351 and 365, sequence numbers 348 and 390, and sequence number 3 48 and 388, Sequence IDs 354 and 390, Sequence IDs 358 and 394, Sequence Number Selected from the group consisting of numbers 362 and 390, and sequence numbers 366 and 394. The CD163 gene according to claim 11, which is created using gRNA.

14. The repaired gene contains a nucleic acid sequence selected from the group consisting of sequence numbers 459-504. The CD163 gene as described in claim 11.

15. Claim that the predicted amino acid sequence of CD163 exon 7 is shown in SEQ ID NO: 513 The CD163 gene described in 11.

16. The repaired gene has the nucleic acid sequence shown in SEQ ID NO: 489, according to claim 11. CD163 gene.

17. The aforementioned edit is created using the sequences shown in sequence numbers 362 and 390, The CD163 gene described in item 11.

18. The predicted amino acid sequence of the gene is shown in Sequence ID No. 513, and the repaired gene is distributed The nucleic acid sequence is shown in sequence number 489, and the editing is shown in sequence numbers 362 and 390. The CD163 gene according to claim 11, which is created using the sequence described above.

19. A wild boar species cell containing the CD163 gene according to any one of claims 11 to 18.

20. A cell line comprising a plurality of cells as described in claim 19.

21. The cell line according to claim 20, which is a fibroblast cell line.

22. PIC system 2, PIC system 3, PIC system 15, PIC system 19, PIC system 27, P The cell according to claim 19, derived from IC lineage 62 or PIC lineage 65.

23. A pair of gRNAs for editing the CD163 gene of the wild boar species (Sus scrofa), Column numbers 229 and 256, sequence numbers 230 and 256, sequence numbers 231 and 256 , Sequence IDs 237 and 256, Sequence IDs 241 and 256, Sequence IDs 229 and 2 58, Sequence IDs 230 and 258, Sequence IDs 231 and 258, Sequence ID 237 and Sequence IDs 258, 241 and 258, 229 and 261, and 230 and 261, SEQ ID NOs. 231 and 261, SEQ ID NOs. 237 and 261, SEQ ID NOs. 2 41 and 261, Sequence IDs 219 and 256, Sequence IDs 221 and 256, Sequence Number Sequence numbers 224 and 256, Sequence numbers 227 and 256, Sequence numbers 219 and 258, Distribution Column numbers 221 and 258, sequence numbers 224 and 258, sequence numbers 227 and 258 , SEQ ID NOs. 219 and 261, SEQ ID NOs. 221 and 261, SEQ ID NOs. 224 and 2 61, Sequence IDs 227 and 261, Sequence IDs 249 and 256, Sequence ID 250 and 256, Sequence IDs 249 and 258, Sequence IDs 250 and 258, Sequence ID 249 and 261, SEQ ID NOs. 250 and 261, SEQ ID NOs. 351 and 365, SEQ ID NOs. 3 51 and 387, Sequence IDs 348 and 390, Sequence IDs 348 and 388, Sequence No. Numbers 348 and 395, Sequence IDs 352 and 365, Sequence IDs 352 and 387, Distribution Column numbers 352 and 399, sequence numbers 353 and 365, sequence numbers 353 and 387 , Sequence IDs 353 and 399, Sequence IDs 354 and 390, Sequence IDs 354 and 3 88, Sequence IDs 354 and 395, Sequence IDs 358 and 361, Sequence ID 358 and 362, Sequence IDs 358 and 368, Sequence IDs 358 and 384, Sequence ID 358 and 394, Sequence IDs 358 and 399, Sequence IDs 359 and 390, Sequence ID 3 59 and 388, Sequence IDs 359 and 395, Sequence IDs 360 and 368, Sequence No. Numbers 360 and 384, Sequence IDs 360 and 389, Sequence IDs 360 and 394, Distribution Column numbers 360 and 397, sequence numbers 361 and 365, sequence numbers 361 and 387 , Sequence IDs 362 and 390, Sequence IDs 362 and 388, Sequence IDs 362 and 3 95, Sequence IDs 364 and 365, Sequence IDs 364 and 387, Sequence ID 364 and 399, Sequence IDs 365 and 368, Sequence IDs 365 and 384, Sequence ID 365 and 389, Sequence IDs 365 and 394, Sequence IDs 365 and 397, Sequence ID 3 66 and 368, Sequence numbers 366 and 384, Sequence numbers 366 and 389, Sequence number Selected from the group consisting of numbers 366 and 394, and sequence numbers 366 and 397. gRNA pair.

24. Sequence IDs 229 and 256, 230 and 256, 231 and 2 56, Sequence IDs 241 and 256, Sequence IDs 229 and 258, Sequence ID 231 and Sequence IDs 258, 241 and 258, 219 and 256, and 221 and 256, Sequence IDs 224 and 256, Sequence IDs 227 and 256, Sequence ID 2 27 and 258, Sequence IDs 221 and 261, Sequence IDs 249 and 256, Sequence No. Numbers 250 and 256, Sequence IDs 249 and 258, Sequence IDs 249 and 261, Distribution Column numbers 351 and 365, sequence numbers 348 and 390, sequence numbers 348 and 388 , Sequence IDs 354 and 390, Sequence IDs 358 and 394, Sequence IDs 362 and 3 Selected from the group consisting of 90 and sequence numbers 366 and 394, as described in claim 23. gRNA pairs.

25. The CRISPR complex for editing the CD163 gene in wild boar (Sus scrofa) A CRISPR complex comprising the gRNA pair described in any one of claims 23 to 24. 。

26. A method for editing the CD163 gene of the wild boar species (Sus scrofa), according to the claim. Use a CRISPR-CAS complex containing the gRNA pair described in any one of items 23-24. A method that includes the steps to do so.

27. CRISPR-CAS complex containing the gRNA pair described in any one of claims 23 to 24 To prepare PRSSv-resistant wild boar (Sus scrofa) cells using human remains Methods.

28. A method for producing PRRSv-resistant wild boar species (Sus scrofa), a) A CRISPR complex comprising the gRNA pair described in any one of claims 23 to 24 The steps include: using to edit the CD163 gene in one or more wild boar species cells; b) A step of producing an animal from the cells or a plurality of cells. A method that includes this.

29. Use of the cell line according to claim 8 in the production of PRRSv-resistant animals.

30. Use of the cell line according to claim 19 in the production of PRRSv-resistant animals.

31. An embryo, piglet, comprising a plurality of cells according to any one of claims 7 to 10 or 19 to 22 Or adult.

32. Presence or absence of an edited sequence having 90% identity with the sequence shown in Sequence ID No. 453 A method of determination, a) Differentially differentiated the sequences shown in Sequence ID No. 564 and Sequence ID No. 558 or 561. Identified probes; b) Primer pairs shown in Sequence ID Nos. 562 and 563; and c) The ply shown in Sequence IDs 556 and 557 or Sequence IDs 559 and 560 Mar vs A method comprising the step of performing real-time PCR.

33. Claim 3, wherein the edited sequence has 100% identity with the sequence shown in sequence number 453. The method described in 2.

34. Sequence ID 556, Sequence ID 557, Sequence ID 559, Sequence ID 560, Sequence ID 562 PCR primers selected from the group consisting of , and SEQ ID NO:

563.

35. Selected from the group consisting of Sequence ID No. 558, Sequence ID No. 561, and Sequence ID No.

564. Altime PCR probe.

36. a) To determine the presence or absence of the edited genome sequence shown in Sequence ID No. 453 Sequence IDs 556 and 557, and sequence IDs 562 and 563, or b) Sequence ID number PCR primers shown in numbers 559 and 560, and sequence numbers 562 and 563 - Use.

37. a) To determine the presence or absence of the edited genome sequence shown in Sequence ID No. 453 b) PCR shown in SEQ ID NOs. 558 and 564, or b) SEQ ID NOs. 561 and 564 Use of a probe.

38. A method for producing PRRSv-resistant pigs, wherein the genome sequence shown in Sequence ID No. 453 A method comprising the step of editing a pig genome to include the following.

39. The step of editing the pig genome involves the sequences shown in sequence numbers 249 and 256. The method according to claim 38, comprising the step of administering the gRNA having the gRNA.

40. The administration step involves a pre-formed RNP containing the gRNA and CAS protein. Claim 39 comprises injecting the complex into a zygote, embryo, or MII-stage oocyte. Method of description.

41. The pigs are PIC strain 2, PIC strain 3, PIC strain 15, PIC strain 19, PIC strain The pig according to claim 38, which is of lineage 27, PIC lineage 62, or PIC lineage 65. Law.

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