WO2018132936A1

WO2018132936A1 - Genetical alternation and disease modelling using cre-dependent cas9 expressing mammals

Info

Publication number: WO2018132936A1
Application number: PCT/CN2017/071367
Authority: WO
Inventors: Liangxue LAI
Original assignee: Guangzhou Institutes Of Biomedicine And Health, Chinese Academy Of Sciences
Priority date: 2017-01-17
Filing date: 2017-01-17
Publication date: 2018-07-26
Also published as: CN107739739A

Abstract

Disclosed is a method of genetical alternation and disease modeling using a cre-dependent cas9 expressing mammal. The transgenic mammal whose genome comprises a polynucleotide sequence, which comprises a polynucleotide encoding Cas9, a first pair of loxP sequences inverted in orientation to each other, and a second pair of loxP sequences inverted in orientation to each other.

Description

Genetical Alternation and Disease Modelling using Cre-Dependent Cas9 Expressing Mammals

TECHNICAL FIELD

The present disclosure relates to genetically modified mammals, and more particularly to genetical alternation and disease modelling using cre-dependent cas9 expressing mammals.

BACKGROUND

CRISPR-Cas9 system has emerged as a powerful genome editing technology to create heritable multigenic changes in prokaryotic and eukaryotic genomes combined Cas9 with multiple single-guide RNAs (sgRNAs) . This system has been improved to facilitate precise genome editing in many species of animals. The generation of gene editing animals is based on either embryo injection or somatic cell nuclear transfer approaches, which is expensive and time consuming. Direct in vivo gene editing would overcome these issues. Early efforts of in vivo gene editing were direct delivery of vectors with both Cas9 gene and sgRNAs into selected tissues of adult mice through hydrodynamic injection or orthotopic injection and successfully created specific and multiplexed genetic modifications. However, the editing efficiency was low because this approach was mediated by lentivirus or adeno-associated virus (AAV) , which is difficult to introduce such a large gene as SpCas9 endonuclease with size of 4.2 kb to the somatic cells in vivo.

SUMMARY

Embodiments of the present disclosure relate to a transgenic porcine animal whose genome comprises a polynucleotide sequence comprising a polynucleotide encoding Cas9, a first pair of loxP sequences inverted in orientation to each other, and a second pair of loxP sequences inverted in orientation to each other. In some embodiments, the first pair are loxP sequences incompatible with the second pair loxP sequences, and the polynucleotide encoding Cas9 is in an inverted transcription orientation.

Some embodiments of the present disclosure relate to a method of preparing the transgenic porcine animal as described above. The method may include providing the polynucleotide sequence, and introducing the polynucleotide sequence to the genome of the transgenic porcine animal to prepare the transgenic porcine animal.

Some embodiments of the present disclosure relate to a method of generating expression alteration of one or more gene products in cells of the transgenic porcine animal as described above, in vivo or ex vivo. The method may include delivering a vector to the cells of the porcine animal, and the vector may comprise a first polynucleotide encoding Cre recombinase and a second polynucleotide corresponding to in vivo CRISPR-Cas complex RNA (s) such that the CRISPR-Cas complex RNA (s) form a CRISPR-Cas complex that results in the expression alteration in the porcine animal.

Some embodiments of the present disclosure relate to a method of testing therapeutic efficacy of an agent on tumor cells. The method may include delivering a vector to cells of the porcine animal as described above. The vector may include a first polynucleotide encoding Cre recombinase and a second polynucleotide corresponding to in vivo CRISPR-Cas complex RNA (s) such that the CRISPR-Cas complex RNA (s) form a CRISPR-Cas complex that results in expression alteration in the porcine animal. The expression alternation may result in development of the tumor cells from the cells of the porcine animal. The method may further include applying one or more agents to be tested to the tumor cells, and determining whether physical or biochemical characteristics of the tumor cells have changed as a result of application of the one or more agents.

In some embodiments, the second pair of loxP sequences are mutated loxP sequences.

In some embodiments, the second pair of loxP sequences comprises a loxP 2272 sequence.

In some embodiments, the first pair of loxP sequences and the second pair of loxP sequences are arranged such that flipping of the first pair of loxP sequences or flipping of the second pair of loxP sequences results in excision of the sequences between a 5’ loxP sequence of the first pair of loxP sequences and a 5’ loxP sequence of the second pair of loxP sequence.

In some embodiments, the polynucleotide sequence is located in the porcine Rosa26 locus.

In some embodiments, the polynucleotide sequence is located between Exon 1 and Exon 2 of the porcine Rosa26 locus.

In some embodiments, the polynucleotide sequence comprises the nucleotide acid sequences of SEQ ID NO: 6, SEQ ID NO: 10, SEQ ID NO: 3, and SEQ ID NO: 6 in 5'-3'order.

In some embodiments, the polynucleotide sequence comprises the nucleotide acid sequences of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 10, SEQ ID NO: 3, and SEQ ID NO: 6 in 5'-3'order.

In some embodiments, the polynucleotide sequence may be introduced to the genome of the transgenic porcine animal by introducing the polynucleotide sequence to the genome of the transgenic porcine animal using transcription activator-like effector nucleases (TALENs) .

In some embodiments, the Cre recombinase is tamoxifen-induced Cre recombinase such that expression levels of Cas9 protein in cells of the porcine animal became higher with increase of tamoxifen concentrations.

In some embodiments, the vector comprises Lentivirus, AAV, or Adenovirus.

In some embodiments, the expression alteration comprises oncogenic chromosomal rearrangements.

In some embodiments, the oncogenic chromosomal rearrangements are in vivo or ex vivo chromosomal rearrangements between two genes greater than 10 Megabases (Mb) apart.

Some embodiments of the present disclosure relate to a cell of the transgenic porcine animal as described above.

Some embodiments of the present disclosure relate to progeny of the transgenic porcine animal as described above.

Some embodiments of the present disclosure relate to a targeting vector for preparing the transgenic porcine animal as described above. The targeting vector may include at least one of the polynucleotide sequence of SEQ ID NO: 11, the polynucleotide sequences of SEQ ID NO: 6, SEQ ID NO: 10, SEQ ID NO: 3, and SEQ ID NO in 5'-3'order: 6, or , the polynucleotide sequences of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 10, SEQ ID NO: 3, and SEQ ID NO: 6 in 5'-3'order, or the polynucleotide sequence of SEQ ID NO: 1.

Some embodiments of the present disclosure relate to an isolated host cell comprising the targeting vector as described above.

This Summary is provided to introduce a selection of concepts in a simplified form that are further described below in the Detailed Description. This Summary is not intended to identify key features or essential features of the claimed subject matter, nor is it intended to be used to limit the scope of the claimed subject matter.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-C, 1D-F, and 1G-H (thereafter “FIG. 1” ) illustrate generation and characterization of Cre-dependent Cas9 expressing pigs. (a) A diagram for TALEN-mediated knock-in of Cre-dependent Cas9 expressing cassette into the pRosa26 locus. Grey triangles, wild-type loxP site； white triangles, mutant loxP2272 site； SA, splice acceptor； TALEN target site and PCR primers (F1, R1, F2, R2, F, and R) are indicated. (b, c) Schematic of two alternative patterns of Cre-mediated activation SpCas9 and tdTomato: (b, left) Cre-recombinase induces inversion of both Neo and iCas9 expression cassettes flanked by two loxP sites followed by excision of Neo expression cassette flanked by two loxP2272 sites (c) ； or (b, right) Cre-recombinase induces inversion of iCas9 expression cassettes by two loxP2272 sites followed by excision of Neo expression cassette between two loxP sites (c) . (c) After inversion of iCas9 expression cassette and removal of the Neo expression cassette, SpCas9 and tdTomato expression is under control of the endogenous pRosa26 promoter. (d) Morphologically normal piglets were born from SCNT with the pRosa26-iCas9 PFFs. (e) PCR analysis confirmed the correct homologous recombination at the pRosa26 locus in of the 3/5 cloned piglets. Three positive piglets were all monoallelic modifications as detected by PCR (F2+F+R) , which is consistent with those of cells chosen as nuclear donors. Primer pairs were shown in (a) . (f) SpCas9 and tdTomato activation by Cre-recombinase in fibroblasts isolated from the ear tissues of cloned piglets shown in (d) . Cells were infected with Cre-EGFP lentivirus, and the expression of tdTomato and EGFP was observed after 48 hours under fluorescence microscope. scale bars 50 μm. (g) Flow cytometry histogram of pRosa26-iCas9 and wild-type ffibroblasts infected with Cre-EGFP lentivirus, showing SpCas9-T2A-tdTomato expression only in infected pRosa26- iCas9 fibroblasts, but not in infected wild-type or uninfected pRosa26-iCas9 fibroblasts. (h) Western blot analysis for directly verifying the expression of SpCas9 in pRosa26-iCas9 fibroblasts infected with lentivirus containing Cre. Cells not infected with Cre-lentiviruses and WT cells were used as negative control.

FIGS. 2A-B, 2C-D, 2E-F, 2G, and 2H-I (thereafter “FIG. 2” ) illustrate Ex Vivo genome editing in pRosa26-iCas9 fibroblasts by lentiviral-mediated Cre and sgRNAs expression. (a) Schematic diagram of Ex vivo genome editing experimental workflow. Firstly, pRosa 26-iCas9 fibroblasts were isolated from the ear tissues of Cre-dependent Cas9 expressing pig； Secondly, the isolated pRosa26-iCas9 fibroblasts were infected with lentivirus containing Cre, EGFP and specific sgRNAs； Finally, the genome modifications in infected cells were analyzed a week post-transduction. (b) Design of sgRNA targeting porcine GGTA1 locus and three representative Sanger sequencing reads of sub-clones into T-vector from pRosa26-iCas9 fibroblasts. (c) A diagram of lentiviral vector for Cre-recombinase, EGFP and GGTA1-sgRNA expression. (d) Sanger sequencing of PCR products containing GGTA1-sgRNA targeting site. Upper: pRosa26-iCas9 fibroblasts uninfected with lentivirus； bottom: pRosa26-iCas9 fibroblasts infected with lentivirus containing Cre-recombinase, EGFP and GGTA1-sgRNA. (e) GGTA1-sgRNA-mediated cleavage in wild-type and pRosa26-iCas9 fibroblasts infected or uninfected with lentivirus were analyzed by T7EN1 cleavage assay. (f) Western blotting analysis for verifying the expression of α-Gal epitope and SpCas9 in wild-type and pRosa26-iCas9 fibroblasts infected or uninfected with lentivirus. β-actin was used as control. (g) Design of sgRNAs targeting early exons of porcine APC, BRCA1, or BRCA2 and three representative Sanger sequencing reads of sub-clones into T-vector from pRosa26-iCas9 fibroblasts infected with lentivirus AB12. (h) A diagram of lentiviral vector AB12 containing Cre-recombinase, EGFP and APC-sgRNA, BRCA1-sgRNA, BRCA2-sgRNA. (i) Sanger sequencing results of PCR products containing APC-sgRNA, BRCA1-sgRNA, BRCA2-sgRNA targeting site, respectively.

FIGS. 3A, 3B, 3C, 3D, and 3E-F (thereafter “FIG. 3” ) illustrate induction of Eml4-Alk rearrangements in pRosa26-iCas9 fibroblasts by lentiviral-mediated Cre and sgRNAs expression. (a) Schematic representation of porcine Eml4-Alk rearrangements induced by CRISPR-Cas9. Eml4-sgRNA and Alk-sgRNA (red) were designed to target the mutation site of the porcine Eml4 gene intron 14 and porcine Alk gene intron 13. PCR primers are indicated (A, B, C, D) . (b) PCRs were performed to analyze Alk-Eml4 (primer A and D were used) and Eml4-Alk rearrangements (primer B and C were used) , and large fragment deletion (primer B and D were used) . (c) and (d) . The Alk-Eml4 (c) and Eml4-Alk (d) PCR products were sub-cloned into T-vector and the Sanger sequencing results of five independent clones and a representative chromatogram are shown in the bottom of (c) and (d) panels. (e) A diagram of Eml4-Alk mRNA fusion transcripts (upper panel) . Agarose gel electrophoresis analysis suggested that the RT-PCR products of Eml4-Alk mRNA fusion transcripts only exist in pRosa26-iCas9 fibroblasts infected with both Eml4-sgRNA and Alk-sgRNA (middle panel) . GAPDH were used as positive control (bottom panel) . (f) The Sanger sequencing results of RT-PCR products showing the sequences of Eml4-Alk mRNA fusion transcripts were identical with predicted sequences.

FIGS. 4A, 4B, 4C-D, 4E-F, and 4G (thereafter “FIG. 4” ) illustrate establishment and characterization of 4-OHT-inducible system in pRosa26-iCas9 fibroblasts. (a) Schematic diagram of 4-OHT-inducible system in pRosa26-iCas9 fibroblasts. Firstly, isolation of ear fibroblasts from Cre-dependent Cas9 expressing pigs； secondly, the isolated fibroblasts infected with lentivirus containing CreERT2, EGFP and sgRNAs； thirdly, the infected cells treated with or without 4-OHT； finally, analysis the SpCas9 expression and gene targeting. (b) Schematic of 4-OHT induced SpCas9 and tdTomato expression in pRosa26-iCas9 fibroblasts infected with lentivirus containing CreERT2. (c) The percentage of EGFP and tdTomato positive cells under different concentration of 4-OHT (0-10 μM) . (d) Western blot analysis for verifying the expression of SpCas9 with different concentration 4-OHT induction. (e) Western blot analysis for SpCas9 expression in pRosa26-iCas9 and wild-type fibroblasts infected/uninfected with lentivirus containing CreERT2 or treated/untreated with 4-OHT. (f) T7EN1 assays showing indels formation at GGTA1 locus in pRosa26-iCas9 infected with lentivirus containing CreERT2 and GGTA1-sgRNA and -T2A-EGFP, and simultaneously supplied with 4-OHT, while not in uninfected or untreated fibroblasts. (g) Sanger sequencing analysis the GGTA1-sgRNA targeting site. Top, pRosa26-iCas9 fibroblasts； Middle, pRosa26-iCas9 fibroblasts infected with lentivirus containing CreERT2 and GGTA1-sgRNA, but not supplied with 4-OHT； Bottom, pRosa26-iCas9 fibroblasts infected with lentivirus containing CreERT2 and GGTA1-sgRNA, simultaneously supplied with 4-OHT.

FIGS. 5A-B, 5C-D, and 5E (thereafter “FIG. 5” ) illustrate In Vivo genome editing in the ear tissues of Cre-dependent Cas9 expressing pigs. (a) Schematic diagram of stereotactic delivery of lentivirus containing Cre, EGFP and sgRNAs into the ear tissues of Cre-dependent Cas9 expressing piglets. (b) Florescence on ear tissues of Cre-dependent Cas9 expressing piglets infected with lentiviruses containing Cre, EGFP and sgRNAs was directly observed using goggles. Left, EGFP fluorescence； right, tdTomato fluorescence. (c) The EGFP and tdTomato positive cells were found under a confocal fluorescence microscope. Upper left, bright field； upper right, EGFP fluorescence； bottom left, tdTomato fluorescence； bottom right, merge, scale bars 50 μm. (d) The EGFP and tdTomato positive cells were successfully sorted out and the frequency were about 0.1％. (e) Deep sequencing of sorted EGFP and tdTomato positive cells show that all three sgRNAs could induce indels mutations near the predicted cleavage site (8.10％of APC, 20.20％of BRCA1 and 71.80％of BRCA2) , but not empty lentivirus infected cells.

FIGS. 6A, 6B-D, and 6E (thereafter “FIG. 6” ) illustrate induction primary lung tumors in Cre-dependent Cas9 expressing pigs infected with lentivirus PPK and AB12. (a) Schematic diagram of intranasal delivery of lentivirus PPK and AB12 into the lung of the Cre-dependent Cas9 expressing piglets. (b) Schematic of the lentiviral vector PPK (upper) and AB12 (bottom) . (c) and (d) The picture of lungs from sacrificed Cre-dependent Cas9 expressing pigs infected (d) or uninfected (c) with lentivirus PPK and AB12. (e) Representative lung H&E staining and immunohistochemistry images of Cre-dependent Cas9 expressing pig injected with lentivirus PPK and AB12 three-month post-transduction, tumors were stained positive for ki67 (an indicator of active cell cycle) , CK7 and TTF1 (the pneumocyte marker) . Scale bar, 50 μm.

FIGS. 7A, 7B, 7C, 7D, 7E, and 7F (thereafter “FIG. 7” ) illustrate mutation analysis in autochthonous lung tumors. (a-f) (Left) sgRNAs were designed to target porcine TP53 (a) , PTEN (b) , KRAS (c) , APC (d) , BRCA1 (e) , and BRCA2 (f) locus. In addition, five representative deep sequencing reads (r1-r5) from the lung tissues of Cre-dependent Cas9 expressing pigs infected with lentivirus PPK and AB12 showed that indels formed at the all six target sites. (Middle) Diagrams analyzed the distribution of indel length at all six sgRNAs targeting sites. (Right) Calculation of mutation efficiency and patterns (3N, 3N+1, 3N+2) in sectioned lung tumors.

FIGS. 8A-B and 8C-D (thereafter “FIG. 8” ) illustrate TALEN-mediated knock-in of Cre-dependent Cas9 expressing cassette into the porcine Rosa26 locus. (a) Schematic of TALENs targeting the exon 1 and intron 1 of the porcine albumin locus. TALEN repeats are colored differently to represent the four-repeat variable di-residue (RVD) . Each RVD recognizes one cognate-targeted DNA base (NI ＝ A, NG ＝ T, HD ＝ C, NN ＝ G) . (b) 5′-junction (1.8 Kb, F1+R1) and 3′-junction (6.0 Kb, F2+R2) PCR analysis to identify individual colonies with stable knock-in at the porcine Rosa26 locus. (c) Activation of tdTomato induced by Cre-recombinase. Selected cell colonies were infected with Cre-lentivirus and tdTomato were seen after 48 hours post-transduction. Bright field (left) and tdTomato fluorescence (right) . Scale bar, 50 μm. (d) Summary of pRosa26-TALEN-mediated albumin knock-in of Cre-dependent Cas9 expressing cassette into the porcine Rosa26 locus.

FIG. 9 shows FACS analysis of tdTomato activation induced by Cre-recombinase.

FIGS. 10A-B and 10C (thereafter “FIG. 10” ) illustrates Off-target analysis by T7EN1 cleavage assay in all three cloned Cre-dependent Cas9 expressing piglets. T7EN1 assays of the PCR products of 43 candidate off-target sites using the genome DNA of three born piglets as the template. The fragments around 43 potential off-target loci of pROSA26-TALEN were PCR amplified, then subjected to T7EN1 cleavage assay. These results suggested that no off-target in all three cloned piglets.

FIGS. 11A, 11B, and 11C (thereafter “FIG. 11” ) illustrate establishment of Cre-dependent Cas9 expressing pig colony by mating the healthy founder pig with two wild-type sows. (a) Twelve F1 Cre-dependent Cas9 expressing piglets, generated through crossbreeding male founders and wild-type sows. (b) and (c) PCR screening of individuals of F1 generations.

FIGS. 12A, 12B-D, 12E, 12F, and 12G (thereafter “FIG. 12” ) illustrate the Sanger sequencing results of PCR products around GGTA1 (a) , APC (e) , BRCA1 (f) , and BRCA2 (g) targeting sites sub-cloned to T vector. In addition, T7EN1 cleavage assays showed that APC-sgRNA (b) , BRCA1-sgRNA (c) , and BRCA2-sgRNA (d) had high cleavage efficiency in pRosa26-iCas9 fibroblasts infected with lentivirus AB12.

FIG. 13 illustrate human, murine and porcine Alk and Eml4. (a) The sequence alignment of porcine Alk exon 13, human and murine Alk exon 19. (b) The sequence alignment of porcine and murine Eml exon 14, human Eml4 exon 13. (c) The predicted porcine, murine and human Eml4–Alk proteins.

FIG. 14 shows the bright field (left) and fluorescent images (middle and right) using appropriate filters under fluorescence microscope of pRosa26-iCas9 fibroblasts (upper) , pRosa26-iCas9 fibroblasts infected with CreERT2-EGFP lentivirus without 4-OHT treatment (middle) and pRosa26-iCas9 fibroblasts infected with CreERT2-EGFP lentivirus with 4-OHT treatment (bottom) .

FIG. 15 illustrate optimization of 4-OHT treatment concentrations. Fluorescence of pRosa26 fibroblasts treated with different concentrations of 4-OHT were observed using appropriate filters under fluorescence microscope. Left, the bright field； middle, EGFP fluorescence； right, tdTomato fluorescence.

FIGS. 16AB and 16C (thereafter “FIG. 16” ) illustrate the original deep sequencing results of (a) APC (b) BRCA1 (c) BRCA2 of the ear fibroblasts from the ear tissues of Cre-dependent Cas9 expressing pigs infected with lentivirus AB12.

FIGS. 17A-B, 17C-D, and 17E-F (thereafter “FIG. 17” ) illustrate the original deep sequencing results of TP53 (a) , PTEN (b) , KRAS (c) , APC (d) , BRCA1 (e) , and BRCA2 (f) from the sectioned primary lung tumors of Cre-dependent Cas9 expressing pigs infected with lentivirus PPK and AB12.

FIG. 18 illustrates heatmap analysis of the mutation efficiency at each position (-10 bp-+10 bp) around PAM sites with different sgRNAs.

FIG. 19 shows Table 1 that includes summary of somatic cell nuclear transfer (SCNT) results for generating Cre-dependent Cas9 expressing pigs.

FIGS. 20A and 20B (thereafter “FIG. 20” ) show Table 2 that include summary of 43 potential off-target sites of pRosa26-TALEN identified by e-PCR.

DETAILED DESCRIPTION

Gene-editing pigs have been gaining increasing importance for agricultural, biomedical and pharmaceutical research. Although several gene editing pigs have been generated thanks to application of newly emerged artificial endonucleases, a Cre-dependent Cas9 expressing pig as that made in mouse would provide powerful tool to generate conditional gene modification and the specific tissue gene editing in adult pigs.

Embodiments of the present disclosure provide compositions, methods, and systems for genetical alternation and disease modelling using cre-dependent cas9 expressing mammals. By combination of somatic nuclear transfer and TALEN mediated somatic cell gene editing, applicants generate a Cre-dependent Cas9 expressing pig model. Delivery of Cre-recombinase and specific sgRNA by viral methods can induce gene editing in multiple tissues of adult pigs in vivo. Delivery of Cre and sgRNAs targeting tumor-related genes into lung tissue of the Cre-dependent Cas9 expressing pigs results in lung adenocarcinoma, enabling applicants rapidly model the dynamics of multiple mutations in tumorigenesis.

Cre-dependent Cas9-expressing mice may be used model dynamics of multiple cancer mutations； however, cancers in mice and human are biologically different. For example, basic studies on murine models often do not translate into successes in clinical trials, and only 5％of anticancer drugs developed in preclinical studies based on traditional mouse models demonstrated sufficient efficacy in phase-III testing. Moreover, pigs share many similarities with humans.

The Cre-dependent Cas9-expressing pigs with primary tumors provide clear advantages ideal for developing new diagnostic and therapeutic technologies. For example, due to the similarity of anatomy, physiology, metabolism and immunology between pigs and human, the tumorigenesis in pigs may be more similar to that in humans than to mice’s . In addition, the organ size of pigs is more similar to that of human than mice’s , and therefore the resultant tumors in pigs can be grown to large sizes similar to that in human, which is ideal for preclinical applications. Further, unlike transplantable or chemically induced cancer models, the porcine tumors induced using the methods described in the present disclosure are genetically engineered in specific tissues in vivo, which is possible to tailor tumors in a defined background and simulate human tumorigenesis.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present disclosure belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, preferred methods and materials are described. For the purposes of the present disclosure, the following terms are defined below.

The articles “a” and “an” are used herein to refer to one or to more than one (i.e. to at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element.

By “about” is meant a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length that varies by as much as 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1％to a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length.

The term "bind, " "binds, " or "interacts with" means that one molecule recognizes and adheres to a particular second molecule in a sample or organism, but does not substantially recognize or adhere to other structurally unrelated molecules in the sample.

By “coding sequence” is meant any nucleic acid sequence that contributes to the code for the polypeptide product of a gene. By contrast, the term “non-coding sequence” refers to any nucleic acid sequence that does not contribute to the code for the polypeptide product of a gene.

Throughout this specification, unless the context requires otherwise, the words “comprise” , “comprises” and “comprising” will be understood to imply the inclusion of a stated step or element or group of steps or elements but not the exclusion of any other step or element or group of steps or elements.

By “consisting of” is meant including, and limited to, whatever follows the phrase “consisting of. ” Thus, the phrase “consisting of” indicates that the listed elements are required or mandatory, and that no other elements may be present.

By “consisting essentially of” is meant including any elements listed after the phrase, and limited to other elements that do not interfere with or contribute to the activity or action specified in the disclosure for the listed elements. Thus, the phrase “consisting essentially of” indicates that the listed elements are required or mandatory, but that other elements are optional and may or may not be present depending upon whether or not they affect the activity or action of the listed elements.

The terms “complementary” and “complementarity” refer to polynucleotides (i.e., a sequence of nucleotides) related by the base-pairing rules. For example, the sequence “A-G-T, ” is complementary to the sequence “T-C-A. ” Complementarity may be “partial, ” in which only some of the nucleic acids’ bases are matched according to the base pairing rules. Or, there may be “complete” or “total” complementarity between the nucleic acids. The degree of complementarity between nucleic acid strands has significant effects on the efficiency and strength of hybridization between nucleic acid strands.

By “corresponds to” or “corresponding to” is meant (a) a polynucleotide having a nucleotide sequence that is substantially identical or complementary to all or a portion of a reference polynucleotide sequence or encoding an amino acid sequence identical to an amino acid sequence in a peptide or protein； or (b) a peptide or polypeptide having an amino acid sequence that is substantially identical to a sequence of amino acids in a reference peptide or protein.

A “decreased” or “reduced” or “lesser” amount is typically a “statistically significant” or a physiologically significant amount, and may include a decrease that is about 1.1, 1.2, 1.3, 1.4, 1.5, 1.6 1.7, 1.8, 1.9, 2, 2.5, 3, 3.5, 4, 4.5, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, or 50 or more times (e.g., 100, 500, 1000 times) (including all integers and decimal points in between and above 1, e.g., 1.5, 1.6, 1.7.1.8, etc. ) an amount or level described herein.

The “deletion” of a targeted gene may also be accomplished by targeting the mRNA of that gene, such as by using various antisense technologies (e.g., antisense oligonucleotides and siRNA) known in the art. Accordingly, targeted genes may be considered “non-functional” when the polypeptide or enzyme encoded by that gene is not expressed by the modified cell, or is expressed in negligible amounts, such that the modified cell produces or accumulates less of the polypeptide or enzyme product (e.g., albumin) than an unmodified or differently modified cell.

With regard to polynucleotides, the term “exogenous” refers to a polynucleotide sequence that does not naturally-occur in a wild-type cell or organism, but is typically introduced into the cell by molecular biological techniques. Examples of exogenous polynucleotides include vectors, plasmids, and/or man-made nucleic acid constructs encoding a desired protein. With regard to polynucleotides, the term “endogenous” or “native” refers to naturally-occurring polynucleotide sequences that may be found in a given wild-type cell or organism. Also, a particular polynucleotide sequence that is isolated from a first organism and transferred to second organism by molecular biological techniques is typically considered an “exogenous” polynucleotide with respect to the second organism. In specific embodiments, polynucleotide sequences can be “introduced” by molecular biological techniques into a microorganism that already contains such a polynucleotide sequence, for instance, to create one or more additional copies of an otherwise naturally-occurring polynucleotide sequence, and thereby facilitate overexpression of the encoded polypeptide.

As used herein, the terms “function” and “functional” and the like refer to a biological, enzymatic, or therapeutic function.

By “gene” is meant a unit of inheritance that occupies a specific locus on a chromosome and consists of transcriptional and/or translational regulatory sequences and/or a coding region and/or non-translated sequences (i.e., introns, 5’ and 3’ untranslated sequences) .

“Homology” refers to the percentage number of amino acids that are identical or constitute conservative substitutions. Homology may be determined using sequence comparison programs such as GAP (Deveraux et al., 1984, Nucleic Acids Research 12, 387-395) which is incorporated herein by reference. In this way sequences of a similar or substantially different length to those cited herein could be compared by insertion of gaps into the alignment, such gaps being determined, for example, by the comparison algorithm used by GAP.

As used herein “heterologous” refers to a combination of elements not naturally occurring. For example, heterologous DNA refers to DNA not naturally located in the cell, or in a chromosomal site of the cell. It is contemplated that the heterologous DNA includes a gene foreign to the cell. A heterologous expression regulatory element is such an element operatively associated with a different gene than the one it is operatively associated with in nature.

As used herein, the term “homologous” refers to the relationship between proteins that possess a “common evolutionary origin, ” including proteins from superfamilies (e.g., the immunoglobulin superfamily) and homologous proteins from different species (e.g., myosin light chain, etc. ) (Reeck et al., Cell 50: 667, 1987) . Such proteins (and their encoding genes) have sequence homology, as reflected by their sequence similarity, whether in terms of percent similarity or the presence of specific residues or motifs at conserved positions.

The term “host cell” includes an individual cell or cell culture which can be or has been a recipient of any recombinant vector (s) or isolated polynucleotide of the present disclosure. Host cells include progeny of a single host cell, and the progeny may not necessarily be completely identical (in morphology or in total DNA complement) to the original parent cell due to natural, accidental, or deliberate mutation and/or change. A host cell includes cells transfected or infected in vivo or in vitro with a recombinant vector or a polynucleotide of the present disclosure. A host cell which comprises a recombinant vector of the present disclosure is a recombinant host cell.

By “isolated” is meant material that is substantially or essentially free from components that normally accompany it in its native state. For example, an “isolated polynucleotide” , as used herein, refers to a polynucleotide, which has been purified from the sequences which flank it in a naturally-occurring state, e.g., a DNA fragment which has been removed from the sequences that are normally adjacent to the fragment. Alternatively, an “isolated peptide” or an “isolated polypeptide” and the like, as used herein, refer to in vitro isolation and/or purification of a peptide or polypeptide molecule from its natural cellular environment, and from association with other components of the cell.

The term "labeled, " with regard to a probe or antibody, is intended to encompass direct labeling of the probe or antibody by coupling (i.e., physically linking) a detectable substance to the probe or antibody.

The term “locus” is the specific physical location of a DNA sequence (e.g. of a gene) on a chromosome. The term “locus” usually refers to the specific physical location of a target sequence on a chromosome.

By “obtained from” is meant that a sample such as, for example, a polynucleotide or polypeptide is isolated from, or derived from, a particular source, such as a desired organism or a specific tissue within a desired organism. “Obtained from” can also refer to the situation in which a polynucleotide or polypeptide sequence is isolated from, or derived from, a particular organism or tissue within an organism. For example, a polynucleotide sequence encoding a reference polypeptide described herein may be isolated from a variety of prokaryotic or eukaryotic organisms, or from particular tissues or cells within certain eukaryotic organism.

The recitation “polynucleotide” or “nucleic acid” as used herein designates mRNA, RNA, cRNA, rRNA, cDNA or DNA. The term typically refers to polymeric form of nucleotides of at least 10 bases in length, either ribonucleotides or deoxynucleotides or a modified form of either type of nucleotide. The term includes single and double stranded forms of DNA and RNA.

The terms “polynucleotide variant” and “variant” and the like refer to polynucleotides displaying substantial sequence identity with a reference polynucleotide sequence or polynucleotides that hybridize with a reference sequence under stringent conditions that are defined hereinafter. These terms also encompass polynucleotides that are distinguished from a reference polynucleotide by the addition, deletion or substitution of at least one nucleotide. Accordingly, the terms “polynucleotide variant” and “variant” include polynucleotides in which one or more nucleotides have been added or deleted, or replaced with different nucleotides. In this regard, it is well understood in the art that certain alterations inclusive of mutations, additions, deletions and substitutions can be made to a reference polynucleotide whereby the altered polynucleotide retains the biological function or activity of the reference polynucleotide, or has increased activity in relation to the reference polynucleotide (i.e., optimized) . Polynucleotide variants include, for example, polynucleotides having at least 50％ (and at least 51％to at least 99％and all integer percentages in between, e.g., 90％, 95％, or 98％) sequence identity with a reference polynucleotide sequence described herein. The terms “polynucleotide variant” and “variant” also include naturally-occurring allelic variants and orthologs that encode these enzymes.

In certain aspects, a targeted gene may be rendered “non-functional” by changes or mutations at the nucleotide level that alter the amino acid sequence of the encoded polypeptide, such that a modified polypeptide is expressed, but which has reduced function or activity with respect to its activity (e.g., introducing transportation of albumin) , whether by modifying that polypeptide’s active site, its cellular localization, its stability, or other functional features apparent to a person skilled in the art. Such modifications to the coding sequence of a polypeptide involved in albumin expression may be accomplished according to known techniques in the art, such as site directed mutagenesis at the genomic level and/or natural selection (i.e., directed evolution) of a given cell.

“Polypeptide, ” “polypeptide fragment, ” “peptide” and “protein” are used interchangeably herein to refer to a polymer of amino acid residues and to variants and synthetic analogues of the same. Thus, these terms apply to amino acid polymers in which one or more amino acid residues are synthetic non-naturally occurring amino acids, such as a chemical analogue of a corresponding naturally occurring amino acid, as well as to naturally-occurring amino acid polymers.

The recitation polypeptide “variant” refers to polypeptides that are distinguished from a reference polypeptide sequence by the addition, deletion or substitution of at least one amino acid residue. In certain embodiments, a polypeptide variant is distinguished from a reference polypeptide by one or more substitutions, which may be conservative or non-conservative. In certain embodiments, the polypeptide variant comprises conservative substitutions and, in this regard, it is well understood in the art that some amino acids may be changed to others with broadly similar properties without changing the nature of the activity of the polypeptide. Polypeptide variants also encompass polypeptides in which one or more amino acids have been added or deleted, or replaced with different amino acid residues.

The term “reference sequence” refers generally to a nucleic acid coding sequence, or amino acid sequence, to which another sequence is being compared. All polypeptide and polynucleotide sequences described herein are included as references sequences, including those described by name and those described in the Sequence Listing.

The term “sample” is used herein in its broadest sense. A sample including polynucleotides, peptides, antibodies and the like may include a bodily fluid, a soluble fraction of a cell preparation or media in which cells were grown, genomic DNA, RNA or cDNA, a cell, a tissue, skin, hair and the like. Examples of samples include saliva, serum, biopsy specimens, blood, urine, and plasma.

The recitations “sequence identity” or, for example, comprising a “sequence 50％identical to, ” as used herein, refer to the extent that sequences are identical on a nucleotide-by-nucleotide basis or an amino acid-by-amino acid basis over a window of comparison. Thus, a “percentage of sequence identity” may be calculated by comparing two optimally aligned sequences over the window of comparison, determining the number of positions at which the identical nucleic acid base (e.g., A, T, C, G, I) or the identical amino acid residue (e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys and Met) occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison (i.e., the window size) , and multiplying the result by 100 to yield the percentage of sequence identity. Included are nucleotides and polypeptides having at least about 50％, 55％, 60％, 65％, 70％, 75％, 80％, 85％, 90％, 95％, 97％, 98％, 99％or 100％sequence identity to any of the reference sequences described herein (see, e.g., Sequence Listing) , typically where the polypeptide variant maintains at least one biological activity of the reference polypeptide.

Terms used to describe sequence relationships between two or more polynucleotides or polypeptides include “reference sequence” , “comparison window” , “sequence identity” , “percentage of sequence identity” and “substantial identity” . A “reference sequence” is at least 12 but frequently 15 to 18 and often at least 25 monomer units, inclusive of nucleotides and amino acid residues, in length. Because two polynucleotides may each comprise (1) a sequence (i.e., only a portion of the complete polynucleotide sequence) that is similar between the two polynucleotides, and (2) a sequence that is divergent between the two polynucleotides, sequence comparisons between two (or more) polynucleotides are typically performed by comparing sequences of the two polynucleotides over a “comparison window” to identify and compare local regions of sequence similarity. A “comparison window” refers to a conceptual segment of at least 6 contiguous positions, usually about 50 to about 100, more usually about 100 to about 150 in which a sequence is compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned. The comparison window may comprise additions or deletions (i.e., gaps) of about 20％or less as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. Optimal alignment of sequences for aligning a comparison window may be conducted by computerized implementations of algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package Release 7.0, Genetics Computer Group, 575 Science Drive Madison, WI, USA) or by inspection and the best alignment (i.e., resulting in the highest percentage homology over the comparison window) generated by any of the various methods selected. Reference also may be made to the BLAST family of programs as for example disclosed by Altschul et al., 1997, Nucl. Acids Res. 25: 3389. A detailed discussion of sequence analysis can be found in Unit 19.3 of Ausubel et al., “Current Protocols in Molecular Biology” , John Wiley &Sons Inc, 1994-1998, Chapter 15.

By “statistically significant, ” it is meant that the result was unlikely to have occurred by chance. Statistical significance can be determined by any method known in the art. Commonly used measures of significance include the p-value, which is the frequency or probability with which the observed event would occur, if the null hypothesis were true. If the obtained p-value is smaller than the significance level, then the null hypothesis is rejected. In simple cases, the significance level is defined at a p-value of 0.05 or less.

“Substantially” or “essentially” means nearly totally or completely, for instance, 95％, 96％, 97％, 98％, 99％or greater of some given quantity.

By “TALEN” is intended a protein comprising a Transcription Activator-like (TAL) effector binding domain and an endonuclease domain, the fusion of both domains resulting in a “monomeric TALEN” . Some monomeric TALEN can be functional per se and others require dimerization with another monomeric TALEN. The dimerization can result in a homodimeric TALEN when both monomeric TALEN are identical or can result in a heterodimeric TALEN when monomeric TALEN are different. Two monomeric TALEN are different when, for example, their RVDs numbers are different, and/or when the content (i.e. amino acid sequence) of at least one RVD is different. By “TAL effector-DNA modifying enzyme” is intended a protein comprising a Transcription Activator-Like effector binding domain and a DNA-modifying enzyme domain.

"Transformation" refers to the permanent, heritable alteration in a cell resulting from the uptake and incorporation of foreign DNA into the host-cell genome； also, the transfer of an exogenous gene from one organism into the genome of another organism.

As used herein, the term “genome” as used herein, can refer to sequences, either DNA, RNA or cDNA derived from a patient, a tissue, an organ, a single cell, a tumor, a specimen of an organic fluid taken from a patient, freely circulating nucleic acid, a fungus, a prokaryotic organism and a virus.

As used herein the terms “express” and “expression” refer to allowing or causing the information in a gene or DNA sequence to become manifest, for example producing a protein by activating the cellular functions involved in transcription and translation of a corresponding gene or DNA sequence. A DNA sequence is expressed in or by a cell to form an “expression product” such as a protein. The expression product itself, e.g. the resulting protein, may also be said to be “expressed. ” An expression product is, in various aspects, characterized as intracellular, extracellular or secreted. The term “intracellular” means inside a cell. The term “extracellular” means outside a cell, such as a transmembrane protein. A substance is “secreted” by a cell if it appears in significant measure outside the cell, from somewhere on or inside the cell.

As used herein “transfection” refers to the introduction of a foreign nucleic acid into a cell. The term “transformation” refers to the introduction of a “foreign” (i.e. exogenous, heterologous, extrinsic or extracellular) gene, DNA or RNA sequence to an embryonic stem (ES) cell or pronucleus, so that the cell will express the introduced gene or sequence to produce a desired substance in a transgenic animal.

As used herein a “promoter” or “promoter sequence” is a DNA regulatory region capable of binding RNA polymerase in a cell and initiating transcription of a coding sequence. In one aspect, the promoter sequence is bound at its 3′terminus by a transcription initiation site and extends upstream (5′direction) to include the minimum number of bases or elements necessary to initiate transcription at levels detectable above background. In a related aspect, within the promoter sequence is found a transcription initiation site (conveniently defined for example, by mapping with nuclease S 1) , as well as protein binding domains (consensus sequences) responsible for the binding of RNA polymerase. The promoter may be operatively associated with other expression control sequences, including enhancer and repressor sequences.

In one aspect, promoters used to control gene expression include, but are not limited to, cytomegalovirus (CMV) promoter (U.S. Pat. No. 5,385,839 and No. 5,168,062) , the SV40 early promoter region (Benoist and Chambon, Nature 290: 304-3101981) , the promoter contained in the 3′long terminal repeat of Rous sarcoma virus (Yamamoto, et al., Cell 22: 787-797, 1980) , the herpes thymidine kinase promoter (Wagner et al., Proc. Natl. Acad. Sci. USA 78: 1441-1445, 1981) , the regulatory sequences of the metallothionein gene (Brinster et al., Nature 296: 39-42, 1982) ； promoter elements from yeast or other fungi such as the Gal 4 promoter, the ADC (alcoho) dehydrogenase) promoter, PGK (phosphoglycerol kinase) promoter, alkaline phosphatase promoter； and transcriptional control regions that exhibit neuronal or brain specific expression, such as the gonadotropic releasing hormone gene control region which is active in the hypothalamus (Mason et al., Science 234: 1372-1378, 1986) , the Thy1.2 “pan-neuronal” promoter, and synapsin I promoter (Howland et al., Brain Neurobiol Aging 16: 685-699, (995) , active in neurons. It is also contemplated that the promoter is an endogenous blood clotting factor promoter. The worker of ordinary skill in the art will understand that any promoter known in the art is useful, and that the cell type in which expression is desired can dictate use of a particular promoter.

As used herein a coding sequence is “under the control of, ” “operably linked to” or “operatively associated with” transcriptional and translational control sequences in a cell when RNA polymerase transcribes the coding sequence into RNA, which is then trans-RNA spliced (if it contains introns) and translated, in the case of mRNA, into the protein encoded by the coding sequence.

By “vector” is meant a polynucleotide molecule, preferably a DNA molecule derived, for example, from a plasmid, bacteriophage, yeast or virus, into which a polynucleotide can be inserted or cloned. A vector preferably contains one or more unique restriction sites and can be capable of autonomous replication in a defined host cell including a target cell or tissue or a progenitor cell or tissue thereof, or be integrable with the genome of the defined host such that the cloned sequence is reproducible. Accordingly, the vector can be an autonomously replicating vector, i.e., a vector that exists as an extra-chromosomal entity, the replication of which is independent of chromosomal replication, e.g., a linear or closed circular plasmid, an extra-chromosomal element, a mini-chromosome, or an artificial chromosome. The vector can contain any means for assuring self-replication. Alternatively, the vector can be one which, when introduced into the host cell, is integrated into the genome and replicated together with the chromosome (s) into which it has been integrated. Such a vector may comprise specific sequences that allow recombination into a particular, desired site of the host chromosome. A vector system can comprise a single vector or plasmid, two or more vectors or plasmids, which together contain the total DNA to be introduced into the genome of the host cell, or a transposon. The choice of the vector will typically depend on the compatibility of the vector with the host cell into which the vector is to be introduced. In the present case, the vector is preferably one which is operably functional in a host cell, such as a plasmid. The vector can include a reporter gene, such as a green fluorescent protein (GFP) , which can be either fused in frame to one or more of the encoded polypeptides, or expressed separately. The vector can also include a selection marker such as an antibiotic resistance gene that can be used for selection of suitable transformants.

The term “wild-type” refers to a gene or gene product that has the characteristics of that gene or gene product when isolated from a naturally-occurring source. A wild-type gene or gene product (e.g., a polypeptide) is that which is most frequently observed in a population and is thus arbitrarily designed the “normal” or “wild-type” form of the gene.

As used herein “selectable marker” refers to a gene encoding an enzyme or other protein that confers upon the cell or organism in which it is expressed an identifiable phenotypic change such as resistance to a drug, antibiotic or other agent, such that expression or activity of the marker is selected for (for example, but without limitation, a positive marker, such as the neo gene) or against (for example, and without limitation, a negative marker, such as the dipteheria gene) . A heterologous selectable marker refers to a selectable marker gene that has been inserted into the genome of an animal in which it would not normally be found. Examples of selectable markers include, but are not limited to, an antibiotic resistance gene such as neomycin (neo) , puromycin (Puro) , diphtheria toxin, phosphotransferase, hygromycin phosphotransferase, xanthineguanine phosphoribosyl transferase, the Herpes simplex virus type 1 thymidine kinase, adenine phosphoribosyltransferase and hypoxanthine phosphoribosyltransferase. The worker of ordinary skill in the art will understand any selectable marker known in the art is useful in the method.

As used herein, “tolerance” refers to the lack of an antigen-recipient's immune response which would otherwise occur, e.g., in response to the introduction of a non-self MHC antigen into the recipient. Tolerance involves, in various aspects, humoral, cellular, or both humoral and cellular responses. Tolerance, as used herein, refers not only to complete immunologic tolerance to an antigen or compound, i.e., no immune response, but also to partial immunologic tolerance, i.e., a limited immune response which does not completely eliminate, inhibit, or otherwise suppress the response to the compound. For instance, in some aspects, a tolerant subject exhibits a detectable immune response to a compound, but it is significantly less than, or decreased compared to, a non-tolerant subject's immune response when exposed to the same compound.

As used herein, a “transgenic animal” is a non-human animal in which one or more, and preferably essentially all, of the cells of the animal contain a transgene introduced by way of human intervention, such as by transgenic techniques known in the art. The transgene can be introduced into the cell, directly or indirectly by introduction into a precursor of the cell, by way of deliberate genetic manipulation, such as by microinjection or by infection with a recombinant virus. A “transgene” is a gene or genetic material that has been transferred from one organism to another. Typically, the term describes a segment of DNA containing a gene sequence that has been isolated from one organism and is introduced into a different organism. This non-native segment of DNA may retain the ability to produce RNA or protein in the transgenic organism, or it may alter the normal function of the transgenic organism's genetic code.

Embodiments of the present disclosure relate to a transgenic porcine animal whose genome comprises a polynucleotide sequence comprising a polynucleotide encoding Cas9, a first pair of loxP sequences inverted in orientation to each other, and a second pair of loxP sequences inverted in orientation to each other.

Genetically modified pigs have many applications in agriculture and biomedicine. Production of genetically modified pigs, however, is still inefficient, expensive due to unavailability of authentic germline-competent pluripotent stem cells. In some embodiments, the Cre-dependent Cas9-expressing pigs may be generated via somatic cell nuclear transfer, providing a versatile large animal tool model to circumvent this bottleneck and expanding the existing CRISPR-Cas9 toolbox to facilitate powerful in vivo genome editing applications in pigs.

Some embodiments of the present disclosure relate to a method of preparing the transgenic porcine animal as described above. The method may include providing the polynucleotide sequence, and introducing the polynucleotide sequence to the genome of the transgenic porcine animal thereby preparing the transgenic porcine animal.

In some embodiments, for vector construction, to make consistent and high level expression of Cas9 expression, the Cre-dependent Cas9 expressing cassette may be introduced into the porcine Rosa26, which is a safe harbor locus used for constitutive, ubiquitous gene expression. To avoid potential problems associated with leaky transcription, applicants reversely orients SpCas9-encoding cDNA relative to Rosa26 transcription without using the stop codons. Thus, the Cre-dependent Cas9 expressing pigs obtained have no SpCas9 leaky expression, ensuring faithful activation of SpCas9 and tdTomato, which is more suitable for sophisticated in vivo gene modification and lineage-tracing experiments. Also, the resulted in constitutive Cas9-expressing pigs are healthy and fertile.

Some embodiments of the present disclosure relate to a method of generating expression alteration of one or more gene products in cells of the transgenic porcine animal as described above, in vivo or ex vivo. The method may include delivering a vector to the cells of the porcine animal, the vector comprising a first polynucleotide encoding Cre recombinase and a second polynucleotide corresponding to in vivo CRISPR-Cas complex RNA (s) such that the CRISPR-Cas complex RNA (s) form a CRISPR-Cas complex that results in the expression alteration in the porcine animal.

In some embodiments, when the primary porcine fibroblasts isolated from the ear tissue of the Cre-dependent Cas9 expressing piglets are infected with lentivirus containing Cre-recombinase and U6-sgRNA loci, a single or multiple genes disruption may be generated efficiently. Thus, Cre-dependent Cas9 expressing construct may provide functional levels of SpCas9 expression. In addition, when pRosa26-iCas9 fibroblasts are used to conduct an oncogenic chromosomal rearrangement in fibroblasts, modeling human EML4-ALK variant, which is one of the most frequent rearrangements in solid human cancers, both Eml4–Alk inversion and large deletion of the region in cells expressing Cre-recombinase may be found. Thus, the pRosa26-iCas9 fibroblasts can be adapted to engineer large deletions, inversions, and chromosomal translocations in the porcine genome by Cre-mediated Cas9 expression. When a vector containing CreERT2 and EGFP is introduced to pRosa26-iCas9 fibroblasts by lentivirus, Cas9 protein expression may be induced by 4-OHT and result in mutation in endogenous genes. Thus, expression of spCas9 may be tightly controlled with by a small molecule and led to efficient endogenous gene editing in the pRosa26-iCas9 system as described in the present disclosure.

In some embodiments, Cre-dependent Cas9 expressing pigs may be used to introduce indels for multigene in vivo using lentivirus-mediated Cre-recombinase and sgRNA expression. The use of Cre-dependent Cas9 expressing pigs in conjunction with multiplex sgRNA delivery enable introducing of multiple genetic lesions in the same animal to more closely recapitulate the nature of mutation accumulation in evolving tumors.

Although the Cre-dependent Cas9 expressing mice can be used to model dynamics of multiple mutations in cancer, there are clear differences in cancer biology between mice and humans. As a consequence, basic studies in murine models often do not translate into success in clinical trials. Only 5％of anticancer agents developed in preclinical studies on the basis of traditional mouse models demonstrate sufficient efficacy in phase-III trials. Pigs share many similarities with humans.

The Cre-dependent Cas9 expressing pigs carrying primary tumors have a number of clear advantages that make them ideal for development new diagnostic and therapeutic technology. First, the tumorigenesis in pigs may be more similar to the process in humans. Second, the resultant tumors in pigs could be grown to very large sizes, ideal for a number of preclinical applications. Third, unlike transplantable or chemically induced cancer models, the obtained porcine tumors were genetically engineered in specific tissue in vivo, and hence it is possible to tailor-make tumors of a defined background and more similar to the process of tumorigenesis in humans. Fourth, the entire procedure is quite simple, fast and rather inexpensive.

As multigene interactions play a critical role in virtually any biological process, multiplex genetic mutation using the Cre-dependent Cas9 expressing pigs will likely find many applications beyond cancer biology, enabling the investigation of multigenic effects in large animal. The lentivirus and AAV adeno-associated virus (AAV) are modular and can be easily modified to target many tissue types and virtually any gene of interest with combinations of loss-of-function and/or gain-of-function mutations. The Cre-dependent Cas9 expressing pigs provide a unique early-stage screening platform for determine consequence of a candidate mutation, which can be further validated by conventional genetic modification.

In some embodiments, the first pair are loxP sequences incompatible with the second pair loxP sequences, and the polynucleotide encoding Cas9 is in an inverted transcription orientation. For example, the second pair of loxP sequences are mutated loxP sequences (e.g., a loxP 2272 sequence) .

In some embodiments, the polynucleotide sequence is located in the porcine Rosa26 locus. For example, the polynucleotide sequence is located between Exon 1 and Exon 2 of the porcine Rosa26 locus.

In some embodiments, the vector comprises Lentivirus, AAV, or Adenovirus.

Some embodiments of the present disclosure relate to a targeting vector for preparing the transgenic porcine animal as described above. The targeting vector may include at least one of the polynucleotide sequence of SEQ ID NO: 11, the polynucleotide sequences of SEQ ID NO: 6, SEQ ID NO: 10, SEQ ID NO: 3, and SEQ ID NO: 6 in 5'-3'order, or the polynucleotide sequences of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 10, SEQ ID NO: 3, and SEQ ID NO: 6 in 5'-3'order, or the polynucleotide sequence of SEQ ID NO: 1.

In the present disclosure, applicants establish a Cre-dependent Cas9 expressing pig for provide a tool model to broadly enable the application of Cas9 in pigs. In some embodiments, ex vivo genome editing (e.g., single gene knockout, multi-gene knockout, chromosome rearrangement and large fragment deletion) may be performed using lentivirus-mediated delivery of Cre-recombinase and sgRNAs in pRosa26-iCas9 fibroblasts. Using these Cre-dependent Cas9 expressing pigs, delivery of Cre-recombinase and specific sgRNA by viral methods may be performed efficiently to facilitate genome editing in multiple tissues of pigs in vivo. For example, intranasal inoculation method may be used to deliver lentivirus to the lung that simultaneously target multiple tumor suppressor genes (e.g., TP53, PTEN, APC, BRCA1 and BRCA2) and one oncogene (KRAS) . CRISPR-Cas9-mediated these genes mutation in somatic cells may lead to rapid lung tumor growth, enabling rapidly modeling of the dynamics of multiple mutations in tumorigenesis.

The various embodiments described above can be combined to provide further embodiments. Aspects of some embodiments can be modified, if necessary to employ concepts of the various patents, applications and publications to provide yet further embodiments.

These and other changes can be made to some embodiments in light of the above-detailed description. In general, in the following claims, the terms used should not be construed to limit the claims to the specific embodiments disclosed in the specification and the claims, but should be construed to include all possible embodiments along with the full scope of equivalents to which such claims are entitled. Accordingly, the claims are not limited by the disclosure.

Examples

Example 1: Generation of Cre-dependent Cas9 expressing pigs

To generate transgenic pigs with conditional expression of Cas9, applicants plan to insert a Cre-dependent Cas9 expressing cassette into the porcine Rosa26 (pRosa26) locus through homologous recombination. The targeting vector for homologous recombination repair (HDR) contains a 5′short arm (1.2 Kb) and a 3′long arm (5.7 Kb) , which together span ～6.9 kb of the pRosa26 locus. The expression cassette containing a pair of loxP sites, a pair of mutant loxP2272 sites, a viral splice acceptor, a promoterless neomycin-resistance (Neo) gene, and an inverted SpCas9 (iCas9) , was inserted between the two homologous arms. To facilitate visualization of SpCas9-expressing cells, a tdTomato Fluorescent protein was inserted into downstream of SpCas9 via a self-cleaving T2A peptide. The loxP and mutant loxP2272 sites were arranged to flank the Neo and iCas9 genes as indicated in FIG. 1 (a) . Both loxP and loxP2272 sites are recognized by Cre-recombinase, but incompatible with one another in recombination reactions. Due to the specific position and orientation of pairs of loxP and loxP2272 sites, Cre-recombinase-mediated recombination would first induce inversion of the intervening DNA at either the loxP or the loxP2272 sites, thus yielding a direct repeat of either two loxP or two loxP2272 sites (FIG. 1 (b) . 1b) . A further Cre-mediated excision will then irreversiblely remove the neomycin resistance cassette along with its neighboring loxP or loxP2272 site and place the SpCas9 expression cassette under control of the endogenous pRosa26 promoter (FIG. 1 (c) ) .

For gene targeting, Primary porcine fetal fibroblasts (PFFs) derived from a 35-day-old fetus were electroporated with the linear targeting donor and pRosa26-TALENs (FIG. 8 (a) ) . After selection with G418 (1 mg/ml from day 10 to day 14) , 101 surviving individual cell colonies were expanded and screened by 5′-and 3′-arm PCR analysis. A total of 75 out of 101 colonies (75/101, 74.3％) were correctly targeted based on 5′-and 3′-arm PCR analysis and tdTomato expression induced by Cre-recombinase-lentivirus infection (FIG. 8 (b, c, d) ) . To examine the modification patterns in both pRosa26 alleles among these 75 correctly targeted cell colonies, applicants then sequenced PCR-amplified fragments covering the target locus. Applicants found that all colonies had knock-in mutation in one allele and NHEJ-mediated mutation or intact in the other, while no homozygous knock-in colonies were identified (date not shown) . The cell colonies with correctly targeted genotypes were infected with the Cre-recombinase-lentivirus. These cells expressed a high level of tdTomato (FIG. 8 (c) , indirectly validating the expression of SpCas9.

Nineteen correctly targeted PFF cell colonies were chosen as donor cells for somatic cell nuclear transfer (SCNT) . Applicants usually pooled 8 or 10 colonies for one nuclear transfer and the reconstructed embryos from the pooled cells were transferred to a surrogate. A total of 2214 cloned embryos were generated and transferred into ten surrogate mothers (Table 1) . Three surrogates were confirmed pregnant by ultrasound examination one month after the embryo transfer. These pregnant surrogates all developed to full term and gave birth to 5 male cloned piglets after 120-130 days of gestation (FIG. 1 (d) ) . PCR analysis of genomic DNA extracted from ear tissue of each piglet showed that 3 piglets (Cre-dependent Cas9 expressing pigs) were confirmed to have one allele with predetermined Cre-dependent Cas9 expressing cassette knock-in at pRosa26 locus and the other that remained intact, which is exactly consistent with that of cells used for nuclear donors (FIG. 1 (e) ) . Applicants next examined whether the cells of cloned piglets could express SpCas9 and tdTomato after Cre-recombinase-mediated recombination. Fibroblasts isolated from the ear tissues of the three live piglets (pRosa26-iCas9 fibroblasts) were infected with lentiviruses containing Cre-recombinase and EGFP expression cassette, in which, EGFP is the indirect indicator for Cre-recombinase expression. Forty-eight hours after infection, tdTomato expression in green cells was confirmed by both fluorescence microscope and flow cytometry (FIG. 1 (f) , (g) , FIG. 9) . tdTomato positive and EGFP positive cells were then sorted by fluorescence-activated cell sorting (FACS) and analyzed by Western blotting for SpCas9 expression. The Western blotting results showed that expression of Cre-recombinase in pRosa26-iCas9 fibroblasts induced recombination at either the loxP or the loxP2272 sites and led to expression of SpCas9 protein (FIG. 1 (h) ) .

To determine the specificity of pRosa26-TALENs, applicants computationally predicted potential off-target sites employing the e-PCR program (www. ncbi. nlm. nih. gov/sutils/e-pcr) in scanning the porcine genomic sequence. Using previously reported criteria, applicants identified 43 potential off-target sites (Table 2) . Genomic DNA extracted from all three cloned piglets was used as a PCR template to amplify the potential off-target regions. T7EN1 cleavage assay and DNA sequencing results suggested that no mutations occurred in any of the potential off-target sites in all of the three cloned piglets (FIG. 10 (a-c) ) .

The Cre-dependent Cas9 expressing pig population was established by mating the founder pig with two wild-type sows. One delivered 12 piglets, and the other delivered 9. Consistent with that of the founders, twelve (eight males and four female) of the 21-offspring harbored the Cre-dependent Cas9 expressing cassette, as confirmed by PCR analysis (FIG. 11 (a-c) ) .

Example 2: Ex Vivo genome editing in pRosa26-iCas9 fibroblasts by lentiviral- mediated sgRNAs expression

To determine whether the Cre-dependent Cas9 expressing construct could provide functional levels of SpCas9 expression, the primary porcine fibroblasts isolated from the ear tissue of the Cre-dependent Cas9 expressing piglets were infected with lentivirus containing Cre-recombinase and U6-sgRNA locus (FIG. 2 (a) ) . Applicants first had a trial on a single gene disruption in pRosa26-iCas9 fibroblasts. The endogenous a-galactotransferase (GGTA1) gene, which is related to hyper acute rejection in xenotransplation with pig organs to human, was used as first gene of interest. The pRosa26-iCas9 fibroblasts were infected with a Cre-U6-GGTA1-sgRNA lentivirus encoding Cre-recombinase and one sgRNA targeting the exon 1 of porcine GGTA1 locus (FIG. 2 (b, c) ) . Seven days post-transduction, Genomic DNA was isolated and screened for the presence of site-specific gene modification by PCR amplification of regions surrounding the target sites as well as T7EN1 cleavage assay. Sanger sequencing of PCR products showed that GGTA1 was successfully mutated in pRosa26-iCas9 fibroblasts (FIG. 2 (d) ) . The PCR products were sub-cloned into T vector and further subjected to Sanger sequencing. Sixteen of 20 sub-clone subjected to analysis (80.0％) exhibited the intended mutant alleles (FIG. 2 (b) , FIG. 12 (a) ) . The Western blotting showed that Gal-α-1, 3-Gal expression in the collected fibroblasts dramatically decreased (FIG. 2 (f) ) . The T7EN1 cleavage bands also were visible in target GGTA1 locus (FIG. 2€) .

Applicants next examined whether multiple genetic alterations could be introduced simultaneously into these pRosa26-iCas9 fibroblasts after Cre-mediated recombination. A lentivirus vector (lentiviral AB12 vector) that expresses Cre-recombinase, EGFP and three sgRNAs targeting exons of porcine APC, BRCA1, and BRCA2 locus (FIG. 2 (g, h) ) was constructed. pRosa26-iCas9 fibroblasts were infected with AB12 vector and collected 7 days after transduction. The analysis of PCR products of the three loci by T7EN1 cleavage assay and sequencing showed that all three sgRNAs produced effective cleavage at target loci (FIG. 2 (i) , Supplementary FIG. 5 (b-d) ) . Sanger sequencing of the amplified products sub-cloned into T-vector further showed that the cleavage efficiency in all the three loci appeared very high, as 18/20 (90.0％) of APC, 17/20 (85.0％) of BRCA1 and 17/20 (85.0％) of BRCA2 harbored indels mutation (FIG. 2 (g) , FIG. 5 (e-g) ) .

Example 3: Ex Vivo oncogenic chromosomal rearrangements in pRosa26-iCas9 fibroblasts by lentiviral-mediated sgRNAs expression

Chromosomal rearrangements between the genes echinoderm microtubule-associated protein like 4 (EML4) and anaplastic lymphoma kinase (ALK) were associated with the pathogenesis of human non-small cell lung cancers (NSCLC) and is one of the most frequent rearrangements in solid human cancers. Modelling such genetic events in animal models has proven challenging and requires complex manipulation of the germline. Although recent reports described an efficient method to induce specific chromosomal rearrangements using viral-mediated delivery of the CRISPR-Cas9 system to somatic cells of adult mice. Modelling such genetic events in large animals has not been reported. Applicants used the pRosa26-iCas9 fibroblasts to model this most common human EML4-ALK variant. In the porcine genome, Eml4 and Alk are located on chromosome 3, approximately 11 megabases (Mb) apart, in a region that is syntonic to human chromosome 2 (p21–p23) (FIG. 2 (a) ) . Applicants engineered lentiviral vectors expressing Cre-recombinase, EGFP, and sgRNAs specific targeting intron 14 of porcine Eml4 gene (which corresponds to intron 13 of the human EML4 gene and intron 14 of the mouse Eml4 gene) or intron 13 of porcine Alk gene (which corresponds to intron 19 of the human ALK gene and the mouse Alk gene) (FIG. 2 (a) , FIG. 13 (a-c) ) . pRosa26-iCas9 fibroblasts were transduced with single (Eml4 or Alk sgRNA) or both lentiviruses (Eml4 and Alk sgRNAs) . One week post-infection, PCR analysis confirmed that both Eml4–Alk inversion (A-D and B-C primers) and large deletion of the region between the two cut sites (B-D primers) occurred in cells expressing Cre-recombinase along with the appropriate pair of sgRNAs, while not in those only expressing a single targeting sgRNA (FIG. 3 (b, c) ) . The PCR products were then sub-cloned into T vector subjected to Sanger sequence, confirming that either accurate DNA-end junctions by direct repair of the predicted DNA cleavage sites (three bases on the 5’ end of the PAM sequence) or junctions with base insertions or deletions by NHEJ had been generated (FIG. 3 (c, d) ) . As predicted by the chromosomal inversion, such engineered Eml4-Alk rearrangements should produce in-frame fusion Eml4-Alk mRNA transcripts joining coding exons 1–14 of the Eml4 gene and exons 14–23 of the Alk gene. Indeed, the Eml4-Alk mRNA fusion transcripts encoding for an in-frame EML4-ALK chimeric protein identical to that found in human NSCLC were confirmed by sequencing the RT-PCR products (FIG. 3 (e, f) , FIG. 13 (c) ) . These results confirmed that the pRosa26-iCas9 fibroblasts can be adapted to engineer large deletions, inversions, and chromosomal translocations in the porcine genome by Cre-mediated Cas9 expression.

Example 4: Tamoxifen induced genome editing in pRosa26-iCas9 fibroblasts by lentiviral-mediated sgRNAs expression

Applicants next examined whether the expression of SpCas9 could be tightly controlled in the pRosa26-iCas9 fibroblasts by an external 4-hydroxytamoxifen (4-OHT) induction (FIG. 4 (a, b) ) . A vector containing CreERT2 and EGFP was introduced to pRosa26-iCas9 by lentivirus. Seven days post-transduction, cells were treated with 4-OHT for 48 hours and red fluorescence was found under fluorescence microscope, but not in uninfected cells or infected cells without 4-OHT treatment (FIG. (7) ) . The activation efficacy of CreERT2 with different 4-OHT concentration was examined by flow cytometry (for tdTomato expression) and Western blotting analysis (for SpCas9 expression) . The ratio of cells expressing tdTomato and expression level of Cas9 protein became higher with the increase of 4-OHT concentrations, reaching peak at 2 μM (FIG. 4 (c, d) , FIG. 15) . To assess if the Cas9 protein expression induced 4-OHT by is able to induce mutation in endogenous genes, applicants next infected pRosa26-iCas9 fibroblasts with lentivirus expressing CreERT2 and a sgRNA targeting porcine GGTA1 gene. Twenty-four hours post-lentiviral-infection, cells were treated with 4-OHT with concentration of 2 μM for 12 hours. Six days after withdrawing 4-OHT, PCR products for GGTA1 locus in cells were cleaved with T7EN1, followed by Sanger sequencing of. The T7EN1 cleavage bands were visible. Indels were seen in the infected pRosa26-iCas9 cells, but not in the cells only infected with CreERT2 or treated with 4-OHT (FIG. 4 (f, g) ) . Above data indicated that induction of spCas9 with tamoxifen led to efficient endogenous gene editing in the pRosa26-iCas9 system.

Example 5: In Vivo genome editing in the ear of Cre-dependent Cas9 expressing pigs

To demonstrate direct genome editing in vivo in the Cre-dependent Cas9 expressing pigs, applicants applied lentivirus-mediated expression of Cre-recombinase and sgRNA in the porcine ear tissues. The lentivirus AB12 vector that expresses Cre-recombinase, EGFP and three sgRNAs targeting APC, BRCA1, and BRCA2 locus (FIG. 2 (g, h) ) was delivered via stereotactic injection into the subcutaneous tissue of the Cre-dependent Cas9 expressing porcine ears (FIG. 5 (a, b) ) . After three weeks of lentiviral inoculation, applicants first evaluated fluorescent protein expression in living piglets using goggles. With appropriate excitation and emission filters, high levels of tdTomato and EGFP fluorescence emitted in the injected region of the Cre-dependent Cas9 expressing piglets, but not in the control piglet (FIG.

) . The ear fibroblasts were then isolated from the ear tissue with tdTomato and EG FP fluorescence. As shown in FIG. 5 (d) , tdTomato positive and EGFP positive cells were found under a confocal microscope, meaning that in vivo Cas9 expression was successfully induce. The frequency of cells carrying tdTomato and EGFP were then determined by flow cytometry. The cells with fluorescence were successfully sorted out and the frequency was about 0.10％ (FIG. 5€) . Genome DNA of FACS-sorted positive cells was isolated to investigate the presence of multiple genes disruption in vivo. PCR products from the targeting sites of the APC, BRCA1 and BRCA2 loci were amplified and analyzed by deep sequence. Indels mutations were found near the predicted cleavage sites of all the three genes. (8.10％of APC, 20.20％of BRCA1 and 71.80％of BRCA2) (FIG. 5 (e) , FIG. 16) , while not in cells infected with empty lentivirus.

Example 6: Induction of lung cancer in Cre-dependent Cas9 expressing pigs through somatic genome editing

Applicants next investigated whether the Cre-dependent Cas9 expressing pigs could been used for rapid cancer modelling by targeting multiple genes related to tumorigenesis in vivo. Applicants attempted to induce multi-lesion lung cancer by targeting the most frequently mutated tumor-related genes including the oncogene KRAS and the tumor suppressor genes TP53, PTEN, APC, BRCA1 and BRCA2 (FIG. 6 (a) ) . To model the dynamics of mutations in these six genes, in addition to lentiviral vector AB12 expressing sgRNAs targeting APC, BRCA1, and BRCA2 locus, applicants also constructed another lentiviral vector PPK capable of simultaneously generating TP53, PTEN, KRAS mutations (FIG. 6 (b) ) . Multiple sgRNAs were designed targeting the fourth exon in TP53, the fifth exon in PTEN and the second exon in KRAS, respectively (FIG. 7 (a-c) ) . The lentivirus PPK and AB12 were produced in HEK293T cells and purified by ultracentrifugation. The lentiviral particles (PPK and AB12) were introduced to lungs of the Cre-dependent Cas9 expressing pigs by intranasal delivery method (FIG. 6 (a) ) . Three month after PPK and AB12 infection, the Cre-dependent Cas9 expressing pigs presented lung cancer symptoms including cough, breathing difficulty and weight loss. The Cre-dependent Cas9 pigs infected with or without lentivirus PPK and AB12 were then sacrificed to retrieve the lungs. By macroscopic observation at necropsy, large tumors were easily visible on the surface of the lung (FIG. 6 (c, d) ) . The tumors with large size were then sectioned for hematoxylin and eosin (H&E) staining and immunohistochemistry (IHC) analysis. Alveolar adenomas were found in tumor sections. A large amount of cells stained positive for Ki67, an indicator of active cell cycle, indicating stronger proliferative ability of these cells than that of normal alveolar cells. In addition, two pulmonary adenocarcinoma markers, cytokeratin 7 (CK7) and thyroid transcription factor-1 (TTF1) were stained positive in many cells of tumors (FIG. 6 (e) ) .

To confirm Cas9-mediated editing of the alleles and precisely characterize the events at single-nucleotide resolution, applicants then dissected the tumors and adjacent tissues without visible tumors from the lungs of PPK and AB12-treated pigs and performed captured Illumina deep sequencing of TP53, PTEN, KRAS, APC, BRCA1 and BRCA2 target loci. Applicants identified indels in TP53, PTEN, KRAS, APC, BRCA1 and BRCA2 at the predicted cutting sites, with 8.0％TP53 indels, 15.8％PTEN indels, 8.7％KRAS indels, 15.1％APC indels, 16.6％BRCA1 indels and 15.5％BRCA2 indels in the dissected tumors (FIG. 7 (a-f) , FIG. 17) . A large fraction of these indels potentially disrupted the endogenous gene function because they were mostly out of frame (i.e., 3n+1bp or 3n+2bp in length) (FIG. 7 (a-f) ) . Furthermore, using the control samples as a background model to analyze the mutational rate revealed that sgTarget samples were enriched for mutations within 7 bp upstream of the PAM sequences in predicted cutting sites, strongly suggesting that they are not secondary consequences of tumor progression (FIG. 18) . These data suggest that delivery of lentiviral PPK and AB12 into the lungs of Cre-dependent Cas9 expressing pigs is able to generate multi-gene putative loss-of-function mutations and can rapid modelling lung cancer.

Example 7: Animals

A local strain of Chinese Bama mini-pigs from Southern China was used as experimental animals of gene targeting in this works. The used pigs were maintained under conventional housing conditions in the Animal Center of Guangzhou Institutes of Biomedicine and Health. All the protocols involving the use of animals complied with the guidelines of the Institutional Animal Care and Use Committee at Guangzhou Institute of Biomedicine and Health, Chinese Academy of Sciences (Animal Welfare Assurance #A5748-01) . All surgical procedures were performed under anesthesia using propofol (2 mg/Kg) or under anesthesia machines for further anesthesia (O₂ flux: 3 L/min, the concentration of isoflurance: 3％) . All efforts were made to minimize animal suffering.

Example 8: Construction of pROSA26-TALENs and targeting vector

TALENs targeting porcine ROSA26 locus were designed and constructed through Golden Gate TALEN Assembly as previously described. pFlexibleDT-ROSA26-iCas9 targeting vector was constructed based on the reported pFlexibleDT-ROSA26-iEGFP targeting vector. Briefly, applicants removed the EGFP sequence and added multiple clone sites (MCS) including SalI, NotI and MluI into the pFlexibleDT-ROSA26-iEGFP, and obtained new intermediate vector named pFlexibleDT-ROSA26-LN. Next, the SpCas9-T2A-tdTomato cassette were digested with SalI and NotI from the plasmid pCAG-SpCas9-T2A-tdTomato and inserted into the SalI and NotI digested pFlexibleDT-ROSA26-LN vector. In this way, applicants obtained the final pFlexibleDT-ROSA26-iCas9 targeting vector. In summary, the targeting vector pFlexibleDT-ROSA26-iCas9 contains a 1.2 Kb 5’ arm and a 5.6 Kb 3’ arm of pROSA26, a viral splice acceptor (SA) , a promoterless neomycin-resistance (Neo) gene with a SV40 polyA signal sequence, and an inverted SpCas9-T2A-tdTomato with a SV40 polyA signal sequence. The different direction loxP and mutant loxP2272 sites were arranged to flank the Neo and inverted SpCas9-T2A-tdTomato expression cassette, which could result in removal of the Neo–gene expression cassette and inversion of the inverted SpCas9-T2A-tdTomato expression cassette after Cre-mediated recombination. This Cre-mediated recombination would place SpCas9-T2A-tdTomato expression cassette directly under the control of the porcine endogenous ROSA26 promoter.

Example 9: Isolation of porcine fetal and ear fibroblasts

Porcine fetal fibroblasts (PFFs) were isolated from 35-day-old fetuses of the Chinese Bama mini-pigs. The fetuses, removed limbs， head, tails and viscera, were digested. The head, limbs, tails and viscera were removed and the remaining tissues were cut into small pieces with sterile scissors and then digested in PFF culture medium containing 0.5 mg/mL collagenase IV (Life Technology) and 2500 IU/mL DNase (Sigma) for 4–6 hours at 37℃. The PFF culture medium contained Dulbecco’s modified Eagles medium (DMEM, Hyclone) , 10％fetal bovine serum (FBS, Gibco) , 1％Non-Essential Amino Acids (NEAA, Gibco) , 2 mM GlutaMAX (Gibco) , 1 mM sodium pyruvate (Gibco) , and 2％penicillin-streptomycin (Hyclone) . Dissociated cells were centrifuged at 250 g for 5 min and then plated on 10 cm culture dishes. Isolated PFFs were cultured overnight and frozen in FBS containing 10％dimethylsulfoxide for future use.

Porcine ear fibroblasts (PEFs) were isolated from the ear tissues of the newborn piglets. Ear tissues were treated with 75％ethanol for 5 minutes and washed three times with PBS containing 2％penicillin-streptomycin. The ear tissues were then chopped into small pieces, and digested in PFF culture medium containing 0.5 mg/mL collagenase IV and 2500 IU/mL DNase for 4–6 hours at 37℃. The isolated PEFs were cultured by the same as PFFs as described above.

Example 10: Generation and identification of pROSA26-iCas9 targeted PFF colonies.

One day before electroporation, PFFs were thawed and grown in 10 cm culture dishes until 90％confluent. Then, approximately 1×10⁶ cells were electroporated using the Neon^TM transfection system (Life technology) at 1350 V with 1 pulse of 30 ms duration in 100 μL of Buffer B containing 15 μg of linearized (using ApaLI restriction enzyme (Thermo Scientific) ) targeting donors and 7 μg of each TALEN. The transfected cells were divided into twenty 10 cm culture dishes and then recovered for 24 hours. After cell recovery, 1 mg/mL G418 (Merck) was added to the PFF culture medium. After 8–12 days of selection, G418-resistant colonies were picked and cultured in 24-well plates by using cloning cylinders. When 70％–80％confluency was reached, the cell colonies were sub-cultured and 10％of each colony was lysed individually in 10 μL of NP-40 lysis buffer (0.45％NP-40 plus 0.6％Proteinase K) for 60 min at 56 ℃ and then for 10 min at 95 ℃. The lysate was used as template for PCR screening. PCR screening was performed using Long PCR Enzyme Mix (Thermo Scientific) in accordance with the manufacturer’s instructions. PCR analysis was used to confirm the HDR with the 5’ junctions primers (F1: SEQ ID NO: 16, R1: SEQ ID NO: 17) , the 3’ junctions primers (F2: SEQ ID NO: 18, R2: SEQ ID NO: 19) . The PCR conditions were 95 ℃ for 5 min； 98 ℃ for 10 s, 68 ℃ for 30 s (-0.6℃/cycle) , 68 ℃ for 2 min (5-arm) /6 min (3-arm) , for 35 cycles； 72 ℃ for 10 min； hold at 12℃. To determine the occurrence of monoallelic or biallelic targeting, competitive PCR was performed using the primers F2+F+R (F2: SEQ ID NO: 18, F: SEQ ID NO: 20, R: SEQ ID NO: 21) (monoallelic targeting: 892 bp and 590 bp； biallelic targeting: 892 bp； wildtype: 590 bp) . The PCR conditions were 95 ℃ for 5 min； 98 ℃ for 10 s, 68 ℃ for 30 s (-0.6℃/cycle) , 68 ℃ for 1 min, for 35 cycles； 72 ℃ for 5 min； hold at 12 ℃. The positive cell colonies were expanded and then cryopreserved in liquid nitrogen for further SCNT.

Example 11: Somatic cell nuclear transfer and generation of Cre-dependent Cas9

expressing pigs

The protocol of SCNT was performed as previously described. Before embryo transfer was performed, the reconstructed embryos were maintained in an embryo-development medium covered with mineral oil at 38.5℃ for 20 h. The reconstructed embryos were then surgically transferred into the oviducts of surrogates the day after the observed estrus. An ultrasound scanner was used to monitor the pregnancy status of the surrogates weekly after a month of implantation, and the cloned piglets were delivered through natural birth. The genomic DNA extracted from the ear tissue of newborn piglets was used as a PCR template. The primers used for PCR genotyping were similar to those for cell colony genotyping.

Example 12: Design and construction sgRNA vectors.

U6-sgRNA cloning vector was purchased from Addgene. In this vector, two BbsI restriction sites were located at the downstream region of U6 promoter. GGTA1-sgRNA, APC-sgRNA, BRCA1-sgRNA, BRCA2-sgRNA, EML4-sgRNA, ALK-sgRNA, TP53-sgRNA, PTEN-sgRNA and KRAS-sgRNA were designed by G-N19-NGG rule. A pair of complementary oligonucleotides of sgRNA were synthesized and annealed at 98 ℃ for 5 min and ramped down to 4 ℃ to generate the double-strand DNA, which was then cloned into the BbsI-digested U6-sgRNA cloning vector. These constructed plasmids were further confirmed by Sanger sequence analysis. The primers used in this study are listed in Table 4.

Example 13: Lentivirus vector design, production and purification.

The U6-sgRNA expression cassettes were PCR amplified from the constructed U6-sgRNA vectors and cloned into the lentiviral vector FUGW-Cre-T2A-EGFP. Lentiviral vectors PPL and AB12 were constructed through Golden Gate Assembly as previously described. To produce the lentiviruses, HEK293T cells were seeded at 5×10⁶ cells per 10 cm culture dish the day before transfection in HEK293T culture media (DMEM supplemented with 10％FBS) . For each dish, 12.5 μg of lentiviral vectors and 12.5 μg of auxiliary packaging vectors (7.5 μg of psPAX2 and 5 μg of pMD2. G) were cotransfected into HEK293T cells using a calcium phosphate transfection method following the previously reported protocol. Lentiviruses were harvested after 48 hours transfection and concentrated by ultracentrifugation at 50,000×g for 2.5 hours at 4 ℃. After centrifugation, the supernatant was carefully aspirated, and the pellet was resuspended in 200 μL of sterile PBS (Gibco) or

reduced serum medium (Gibco) . Aliquots were then stored at -80 ℃ for future use.

Example 14: Cell infection and FACS.

For infection of pROSA26-iCas9 fibroblasts with purified lentiviruses, applicants seeded 5×10⁴ fibroblasts into 6-well dishes. After 24 hours, pROSA26-iCas9 fibroblasts were incubated 6 hours with a diluted viral stock. Polybrene (8 μg/mL, Sigma) was added to increase infection efficiency. Seven days post-infection, fibroblasts were analyzed by fluorescence microscope and flow cytometry. For FACS, cells were trypsinized, washed twice with PBS. EGFP and tdTomato expression were then analyzed by flow cytometry.

Example 15: Genomic DNA extraction and indels analysis

Genomic DNA from both cells and tissues was extracted using TIANGEN genomic DNA extraction kit following the manufacturer’s recommended protocol. pROSA26-iCas9 fibroblasts and tissues infected by lentivirus were used as templates for PCR for both captured sequencing and T7EN1 cleavage assay using high-fidelity polymerases (KOD-Plus-Neo, TOYOBO) . Some PCR products were selected to ligate into pMD18-T vector (Takara) and further sequenced to determine the exact mutant sequences.

Example 16: Western blotting

Cells were lysed in RIPA buffer supplemented with protease and phosphatase inhibitors (Roche) on ice for 15 minutes. The protein lysates were mixed with SDS loading buffer (62.6 mM Tris-HCl, 10％glycerol, 0.01％bromophenol blue, 2％SDS, pH 6.8) after determination of the protein concentration according to Lowry method (Bio-Rad) . The samples were then boiled for 15 minutes, loaded onto SDS-PAGE gels for separation and transferred to nitrocellulose membrane. The membranes were blocked with 5％skim milk in TBST for 2 hours. The PVDF were then incubated with the primary antibody for 2 hours (rabbit anti-CRISPR-Cas9 antibody, Hangzhou HuaAn Biotechnology Company； mouse anti-a-Gal Epitope antibody, M86, Enzo) . After rinsing three times TBST, the membrane were incubated for 1 hours at room temperature with HRP-conjugated secondary antibodies, and then washed with TBST three times again. The signal was visualized with ECL plus (Amersham) in accordance with the manufacturer’s instructions.

Example 17: T7EN1 cleavage assay

Lentivirus infected cells were harvested and genomic DNA was extracted using TIANGEN genomic DNA extraction kit. The T7EN1 cleavage assay was performed as previously reported. Briefly, PCR products around the target sites were amplified using the primer pairs listed in Table 4 and purified by TIANGEN PCR cleanup kit following the manufacturer’s recommended protocol. purified PCR products were denatured, and annealed to form heteroduplex DNA in NEBuffer2 (New England Biolab) using the thermocycler with the following protocol: 95 ℃, 5 min； 95-75 ℃ at -2 ℃/s； 75-16 ℃ at -0.1 ℃/s； hold at 4 ℃. Hybridized PCR products were treated with 5 U of T7 Endonuclease I at 37 ℃ for 15 min. Products were separated by 2％agarose gel and detected by ethidium bromide staining.

Example 18: RT-PCR

RT-PCR was conducted to investigate the expression profiles of ALK and EML4. Total RNAs were extracted from EML4-sgRNA or/and ALK-sgRNA lentivirus infected fibroblasts using TRIzol Reagent (Life technologies) . cDNAs were prepared using PrimeScript^TM II Reverse Transcriptase kit (Takara) , before that genomic DNA was digested with DNaseI. The primers used to amplify EML4-ALK fusion transcript were EML4-F: SEQ ID NO: 58 and ALK-R: SEQ ID NO: 59. The porcine glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as the control gene (forward GAPDH-F: SEQ ID NO: 147； reverse GAPDH-R: SEQ ID NO: 147) . The RT-PCR products were predicted to be 279 bp (EML4-ALK fusion transcript) and 234 bp (GAPDH) and sequenced by BGI.

Example 19: In vivo lentivirus transduction.

Intranasal delivery and stereotactic injection of lentivirus were performed accordingly to previously reported protocol. In brief, 1-week-old piglets were anesthetized using isoflurane. For intranasal delivery, previously purified lentiviruses solution were pipetted directly over the opening of one nostril the piglets and dispense the virus dropwise until the entire volume of virus has been inhaled. For stereotactic delivery into the ear, the packaged lentiviruses were delivered into the subcutaneous tissue of the Cre-dependent Cas9 expressing porcine ears using the 30G needle and syringe. The postoperative piglets were housed in a temperature-controlled environment (37℃) until achieving ambulatory recovery.

Example 20: Captured illumina sequencing and indels analysis.

Genomic DNA was extracted from both cells and cancer-like tissues after infected by lentivirus carrying Cre-recombinase, EGFP and sgRNAs targeting specific locus using TIANGEN genomic DNA extraction kit following the recommended manufacturer’s instructions protocol. The extracted genomic DNA were used as a template for PCR for captured sequencing. Genomic PCR products were subjected to library preparation using the Nextera XT DNA Sample Prep Kit (Illumina) or using customized barcoding methods. Briefly, low-cycle, first-round PCR was performed to amplify the target site. Second-round PCR was performed to add generic adapters, which were then used for a third round of PCR for sample barcoding. Samples were pooled in equal amounts and purified using QiaQuick PCR Cleanup (QIAGEN) , quantified using Qubit (Life Technologies) . Mixed barcoded library was sequenced on an Illumina MiSeq System.

Example 21: H&E and immunohistochemistry staining

The lung tissues obtained from the sacrificed wild-type and Cre-dependent Cas9 expressing piglets were fixed in 4％paraformaldehyde for 2 days. The fixed tissues were subsequently dissected, embedded in paraffin wax, and then cross-sectioned at 3 μM. The sections were deparaffinized with xylene and then rehydrated with a graded series of alcohol (100％, 90％, 80％, 70％, and 50％) , followed by H₂O. For H&E staining, the rehydrated sections were stained with hematoxylin and eosin, differentiated, and then cover-slipped. For immunohistochemistry staining, sections were stained using standard immunohistochemistry staining (IHC) protocols as previously described. The following antibodies were used for IHC: anti-Ki67 (Novus NB500-170, 1: 100) , anti-CK7 (ZSJB-BIO, ZA-0573) , anti-TTF (ZSJB-BIO, ZM-0250) .

Example 22: Off-target analysis

For pROSA26-TALENs, the electronic-PCR program downloaded from the National Center for Biotechnology Information website (http: //www. ncbi. nlm. nih. gov/sutils/e-pcr/) was used to identify potential off-target sites in the porcine genome. The criteria for identifying off-target sites were up to 10 mismatches, 3-bp gaps in the two effector binding elements EBEs, and <100 bp between the two putative off-target sites. The sites with the spacer region within the range of 39–60 bp for a total of 43 sites were amplified and sequenced.

Sequence identifiers for various constructs are provided in Tables 3 and 4, and the sequence listing thereof are provided below.

Table 3 Sequences

Table 4. Primers used in the embodiments

SEQ ID NO	Site Label	PCR Primers (5’-3’)
SEQ ID NO: 16	F1	TGCGTGAGTCTCTGAGCGCAG
SEQ ID NO: 17	R1	GGCATCAGAGCAGCCGATTGT
SEQ ID NO: 18	F2	GATGCTGTGCCGGTCGGTGTT
SEQ ID NO: 19	R2	GGTCAAACAGTGGCTCACATCT
SEQ ID NO: 20	F	CTCGTCATCGCCTCCATGTCAG
SEQ ID NO: 21	R	GTTGGGCCTATGCTCAAGATGG
SEQ ID NO: 22	GGTA1-gRNA-F	CACCGAGAAAATAATGAATGTCAA
SEQ ID NO: 23	GGTA1-gRNA-R	AAACTTGACATTCATTATTTTCTC
SEQ ID NO: 24	TP53-gRNA-F	CACCGCAGCTATGATTTCCGTCTA
SEQ ID NO: 25	TP53-gRNA-R	AAACTAGACGGAAATCATAGCTGC
SEQ ID NO: 26	PTEN-gRNA-F	CACCGCAGCAATTCACTGTAAAGC
SEQ ID NO: 27	PTEN-gRNA-R	AAACGCTTTACAGTGAATTGCTGC
SEQ ID NO: 28	KRAS-gRNA-F	CACCGTAGTTGGAGCTGGTGGCGT
SEQ ID NO: 29	KRAS-gRNA-F	AAACACGCCACCAGCTCCAACTAC
SEQ ID NO: 30	APC-gRNA-F	CACCGGCAACTTCGGGTAACGGTC
SEQ ID NO: 31	APC-gRNA-R	AAACGACCGTTACCCGAAGTTGCC
SEQ ID NO: 32	BRCA1-gRNA-F	CACCGGAAGAAATGGATTTATCTG
SEQ ID NO: 33	BRCA1-gRNA-R	AAACCAGATAAATCCATTTCTTCC
SEQ ID NO: 34	BRCA2-gRNA-F	CACCGGCACAGAAGGTTTATGTGC
SEQ ID NO: 35	BRCA2-gRNA-R	AAACGCACATAAACCTTCTGTGCC
SEQ ID NO: 36	EML4-gRNA-F	CACCGTGAAGTGCCAGAGCATACA
SEQ ID NO: 37	EML4-gRNA-R	AAACTGTATGCTCTGGCACTTCAC
SEQ ID NO: 38	ALK-gRNA-F	CACCGGATTAGAACACAAGTCCTC
SEQ ID NO: 39	ALK-gRNA-R	AAACGAGGACTTGTGTTCTAATCC
SEQ ID NO: 40	GGTA1-F	ATCCTTCCCAACCCAGACGG
SEQ ID NO: 41	GGTA1-R	ACAGCAATGCCAGATCCGAGC
SEQ ID NO: 42	TP53-F	TCCATCCGCAGTCCTCTGAGCT
SEQ ID NO: 43	TP53-R	GATGAGAGGCCAAGGTCAAGTG
SEQ ID NO: 44	PTEN-F	TGGCCTCCCTATCTAATGGGGA
SEQ ID NO: 45	PTEN-R	AGAAACAAGGGTTACCAACTAGC
SEQ ID NO: 46	KRAS-F	GCACATCTGTGGTCAACGGGC
SEQ ID NO: 47	KRAS-R	CTCCCCAGAGAAGACTGAAGAC
SEQ ID NO: 48	APC-F	GCTCCATTAAATGCCAGAGCCA
SEQ ID NO: 49	APC-R	CCGATTGTTCTGGAGATACCCA
SEQ ID NO: 50	BRCA1-F	GCTGTTTTACAGTGTTCTGTCA
SEQ ID NO: 51	BRCA1-R	AGAGCTACAGCTTCTGCGAC
SEQ ID NO: 52	BRCA2-F	GCAGATCCTTGAATGTACAGGTC

SEQ ID NO: 53	BRCA2-R	GGGATTGAACTCGAAACCTCA
SEQ ID NO: 54	A	GCCCCCTCCCCATGAATCAT
SEQ ID NO: 55	B	TAGGGGCCAAAGTCAGCCATC
SEQ ID NO: 56	C	GCCTTACTCCTGCTCAAGCA
SEQ ID NO: 57	D	CCAACACCACACAGTTAGCTAG
SEQ ID NO: 58	EML4-F	TGGGGAATGGAGATGTGCTT
SEQ ID NO: 59	ALK-R	TGAGGGTGATGTTTTTCCGAG
SEQ ID NO: 60	OT1-F	GGCACTCTGTCTCCAAAGCT
SEQ ID NO: 61	OT1-R	TTCCTGAACCTAAGGCAATAC
SEQ ID NO: 62	OT2-F	CATTCTACTGCTGGTTTCAG
SEQ ID NO: 63	OT2-R	TACAAACAGGCTTCTTGCTC
SEQ ID NO: 64	OT3-F	ACACTGGCTTTGGATGACCT
SEQ ID NO: 65	OT3-R	GCAAGAGTGTCAGAAGTGGC
SEQ ID NO: 66	OT4-F	AGCAATATCTGATTTAACCCTC
SEQ ID NO: 67	OT4-R	CTGCCCTGAATGCCCTTACT
SEQ ID NO: 68	OT5-F	CTGGGAACCTGACTGCCTCG
SEQ ID NO: 69	OT5-R	TAGCCAAGACATGCCAGAAC
SEQ ID NO: 70	OT6-F	TACATCAGAGCCTCAACAACT
SEQ ID NO: 71	OT6-R	CCCTGGGATCTAAGATACTG
SEQ ID NO: 72	OT7-F	GTGAGAACACGTTTGGGATT
SEQ ID NO: 73	OT7-R	GTGGATTAGCCACATCAAAC
SEQ ID NO: 74	OT8-F	GAGTGATAAATCCAGGCAGAG
SEQ ID NO: 75	OT8-R	GTGGATTAGCCACATCAAAC
SEQ ID NO: 76	OT9-F	CCTCCTTATCAGACCAGTCAT
SEQ ID NO: 77	OT9-R	TGCTCCTGTTTCATACCCT
SEQ ID NO: 78	OT10-F	GCTGGATGGGAGGTTAGAT
SEQ ID NO: 79	OT10-R	CCAGGTCAGGAGAAAGGAT
SEQ ID NO: 80	OT11-F	AAGAACTCAAGCTGTGGCC
SEQ ID NO: 81	OT11-R	TCTTGGTTGTTCTTGGACC
SEQ ID NO: 82	OT12-F	ACGGGTTCATCCCAGTTCT
SEQ ID NO: 83	OT12-R	GGACAGTGGAGCAGGCAGT
SEQ ID NO: 84	OT13-F	GGCTTCGACTAGATGCTGT
SEQ ID NO: 85	OT13-R	GCCATCCTGGAAAGAGTGC
SEQ ID NO: 86	OT14-F	CTTCTTGACAGAACTGGGTG
SEQ ID NO: 87	OT14-R	CTGATGGTTGTTTTGGTCTG
SEQ ID NO: 88	OT15-F	ATGGGATTGGCAAATGGTC
SEQ ID NO: 89	OT15-R	GCTAGGGATTAAATCGGAGC
SEQ ID NO: 90	OT16-F	GAAGCAAAGAATTGGAGGTC
SEQ ID NO: 91	OT16-R	GGTGGGTTCAGATGGTATG

SEQ ID NO: 92	OT17-F	GCTCTTTCCAGCACCAACC
SEQ ID NO: 93	OT17-R	ATCATCCCCTTCACAGAGC
SEQ ID NO: 94	OT18-F	GCTGAGCACTGACACCAAAAT
SEQ ID NO: 95	OT18-R	CAGACATCAATCCAACCGA
SEQ ID NO: 96	OT19-F	AATTAGGCGAAGACGAAACG
SEQ ID NO: 97	OT19-R	AGACTCAGTGGCTTGAACAG
SEQ ID NO: 98	OT20-F	AAATCTTGGGTCTGAGGCT
SEQ ID NO: 99	OT20-R	GTTCAAGGATGAAGGTCTCC
SEQ ID NO: 100	OT21-F	ATGCTACCTACATGGGAGTTC
SEQ ID NO: 101	OT21-R	AGGGATGAGAGAAGCACTGT
SEQ ID NO: 102	OT22-F	CTTCTGAACAGCCTCCCATC
SEQ ID NO: 103	OT22-R	TCACCCAAGGAGATGACACT
SEQ ID NO: 104	OT23-F	CTATTCCTTCACCAAGCACTC
SEQ ID NO: 105	OT23-R	GCGAAATAGGAGGGAAAGA
SEQ ID NO: 106	OT24-F	GCCCATATTTCCACCAGAC
SEQ ID NO: 107	OT24-R	AGGGAGGGAGACAGGAGTG
SEQ ID NO: 108	OT25-F	TGCTCAGTGGGTTAAGGAT
SEQ ID NO: 109	OT25-R	CAGGTACTGACCTGCTGGATA
SEQ ID NO: 110	OT26-F	CCAAACACCACCTACAGAG
SEQ ID NO: 111	OT26-R	TTCTCATAATGACTCATTTAC
SEQ ID NO: 112	OT27-F	GAGGCATGGATTACTACAAGG
SEQ ID NO: 113	OT27-R	TTGTGAGCCCAATTCCTTC
SEQ ID NO: 114	OT28-F	TTTGGTAAAGTGCGAGATT
SEQ ID NO: 115	OT28-R	ATCTATCCAGGGACCAACG
SEQ ID NO: 116	OT29-F	AATAACTGTCAAATGGCAAGG
SEQ ID NO: 117	OT29-R	AGGAGAAATAGCGGCAGAG
SEQ ID NO: 118	OT30-F	AATAACTGTCAAATGGCAAGG
SEQ ID NO: 119	OT30-R	AGGAGAAATAGCGGCAGAG
SEQ ID NO: 120	OT31-F	ACTGATAATGCTGGTGAGG
SEQ ID NO: 121	OT31-R	GATAAAGGAGAAAGACATGG
SEQ ID NO: 123	OT32-F	ACCGTGAGCCAAGGAGTGT
SEQ ID NO: 124	OT32-R	GCGTATGTAGGTCCGTTGT
SEQ ID NO: 125	OT33-F	TTTTGCTGCGTCTGGAATG
SEQ ID NO: 126	OT33-R	AGGACGGTTTTCTCAGGCT
SEQ ID NO: 127	OT34-F	GCCAGGCTTCTATGATTTC
SEQ ID NO: 128	OT34-R	AAGGACCCAGTGTTGTCAGT
SEQ ID NO: 129	OT35-F	AGGGTCCTGTTTGGCTGAT
SEQ ID NO: 130	OT35-R	CGTGAGGAAGAGTCAGAGGC
SEQ ID NO: 131	OT36-F	CCCGCTCTGCTTTCTGTCT

SEQ ID NO: 132	OT36-R	GCAGCGAGACCACTGAGAA
SEQ ID NO: 133	OT37-F	CCAAAATACGAACCCAGTAG
SEQ ID NO: 134	OT37-R	CTCAACTTCACATCTGGCTC
SEQ ID NO: 135	OT38-F	CCTTTCCTCCCTATAACTTGC
SEQ ID NO: 136	OT38-R	GGATTTGATGCTTCTGGTCTC
SEQ ID NO: 137	OT39-F	TCAGACTCGTAGCCACCTT
SEQ ID NO: 138	OT39-R	TGAGCTACGATGGGAACTT
SEQ ID NO: 139	OT40-F	GACTGACCCAAAGATATGACC
SEQ ID NO: 140	OT40-R	CTGGGAACTTCTCCGTGTT
SEQ ID NO: 141	OT41-F	ATAACAGCAGCAGGTGCAA
SEQ ID NO: 142	OT41-R	GGACAGAGGTGGTAAATGACT
SEQ ID NO: 143	OT42-F	GGGCAACTCAAACTTCCAT
SEQ ID NO: 144	OT42-R	TGAACCACGACAAGAACTCC
SEQ ID NO: 145	OT43-F	GTGCCACAGAGAGAAACTTG
SEQ ID NO: 146	OT43-R	GATGTGATGGTAAACGGGAT
SEQ ID NO: 147	forward GAPDH-F	GATGGCCCCTCTGGGAAACTGTG
SEQ ID NO: 148	reverse GAPDH-R	GGACGCCTGCTTCACCACCTTCT

Although the subject matter has been described in language specific to structural features and/or methodological acts, it is to be understood that the subject matter defined in the appended claims is not necessarily limited to the specific features or acts described above. Rather, the specific features and acts are disclosed as example forms of implementing the claims.

Claims

A transgenic porcine animal whose genome comprises a polynucleotide sequence comprising a polynucleotide encoding Cas9, a first pair of loxP sequences inverted in orientation to each other, and a second pair of loxP sequences inverted in orientation to each other, wherein the first pair are loxP sequences incompatible with the second pair loxP sequences, and the polynucleotide encoding Cas9 is in an inverted transcription orientation.
A method of preparing the transgenic porcine animal of claim 1, the method comprising:

providing the polynucleotide sequence； and

introducing the polynucleotide sequence to the genome of the transgenic porcine animal thereby preparing the transgenic porcine animal.
A method of generating expression alteration of one or more gene products in cells of the transgenic porcine animal of claim 1, in vivo or ex vivo, the method comprising:

delivering a vector to the cells of the porcine animal, the vector comprising a first polynucleotide encoding Cre recombinase and a second polynucleotide corresponding to in vivo CRISPR-Cas complex RNA (s) such that the CRISPR-Cas complex RNA (s) form a CRISPR-Cas complex that results in the expression alteration in the porcine animal.
A method of testing therapeutic efficacy of an agent on tumor cells, the method comprising:

delivering a vector to cells of the porcine animal of claim 1, the vector comprising a first polynucleotide encoding Cre recombinase and a second polynucleotide corresponding to in vivo CRISPR-Cas complex RNA (s) such that the CRISPR-Cas complex RNA (s) form a CRISPR-Cas complex that results in expression alteration in the porcine animal, the expression alternation resulting in development of the tumor cells from the cells of the porcine animal；

applying one or more agents to be tested to the tumor cells； and

determining whether physical or biochemical characteristics of the tumor cells have changed as a result of application of the one or more agents.
The transgenic porcine animal of any of claims 1-4, wherein the second pair of loxP sequences are mutated loxP sequences.
The transgenic porcine animal of any of claims 1-4, wherein the second pair of loxP sequences comprises a loxP 2272 sequence.
The transgenic porcine animal of any of claims 1-4, wherein the first pair of loxP sequences and the second pair of loxP sequences are arranged such that flipping of the first pair of loxP sequences or flipping of the second pair of loxP sequences results in excision of the sequences between a 5’ loxP sequence of the first pair of loxP sequences and a 5’ loxP sequence of the second pair of loxP sequence.
The transgenic porcine animal of any of claims 1-4, wherein the polynucleotide sequence is located in the porcine Rosa26 locus.
The transgenic porcine animal of any of claims 1-4, wherein the polynucleotide sequence is located between Exon 1 and Exon 2 of the porcine Rosa26 locus.
The transgenic porcine animal of any of claims 1-4, wherein the polynucleotide sequence comprises the nucleotide acid sequences of SEQ ID NO: 6, SEQ ID NO: 10, SEQ ID NO: 3, and SEQ ID NO: 6 in 5'-3'order.
The transgenic porcine animal of any of claims 1-4, wherein the polynucleotide sequence comprises the nucleotide acid sequences of SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 3, and SEQ ID NO: 6 in 5'-3'order.
The transgenic porcine animal of any of claims 1-4, wherein the polynucleotide sequence comprises the nucleotide acid sequences of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 3, and SEQ ID NO: 6 in 5'-3'order.
The transgenic porcine animal of any of claims 1-4, wherein the polynucleotide sequence comprises the nucleotide acid sequences of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 10, SEQ ID NO: 3, and SEQ ID NO: 6 in 5'-3'order.
The method of claim 2, wherein the introducing the polynucleotide sequence to the genome of the transgenic porcine animal comprising:

introducing the polynucleotide sequence to the genome of the transgenic porcine animal using transcription activator-like effector nucleases (TALENs) .
The method of any of claims 3 and 4, wherein the Cre recombinase is tamoxifen-induced Cre recombinase such that expression levels of Cas9 protein in cells of the porcine animal became higher with increase of tamoxifen concentrations.
The method of any of claims 3 and 4, wherein the vector comprises Lentivirus, AAV, or Adenovirus.
The method of any of claims 3 and 4, wherein the expression alteration comprises oncogenic chromosomal rearrangements.
The method of 17, wherein the oncogenic chromosomal rearrangements are in vivo or ex vivo chromosomal rearrangements between two genes greater than 10 Megabases (Mb) apart.
A cell of the transgenic porcine animal of claim 1.
Progeny of the transgenic porcine animal of claim 1.
A targeting vector for preparing the transgenic porcine animal of claim 1, the targeting vector comprising at least one of the polynucleotide sequence of SEQ ID NO: 11, the polynucleotide sequences of SEQ ID NO: 6, SEQ ID NO: 10, SEQ ID NO: 3, and SEQ ID NO: 6 in 5'-3'order, or the polynucleotide sequences of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 10, SEQ ID NO: 3, and SEQ ID NO: 6 in 5'-3'order, or the polynucleotide sequence of SEQ ID NO: 1.
The targeting vector of claim 21, wherein the targeting vector comprises the polynucleotide sequence of SEQ ID NO: 15.
An isolated host cell comprising the targeting vector of claim 21.