High frequency targeted animal transgenesis

Abstract

To provide high frequency targeted animal transgenesis.SOLUTION: This disclosure provides methods and compositions for high frequency mouse transgenesis using, for example, a Bxb1 landing pad. This disclosure demonstrates that large, single copy insertions of exogenous DNA into the mouse genome is achievable using the Bxb1 integrase system. Thus, provided herein, in some aspects, is a system that utilizes a combination of gene editing tools (e.g., CRISPR / Cas9) and a Bxb1 integrase to insert single copies of large transgenes into a specific locus.SELECTED DRAWING: None

Description

[Technical Field]

[0001] Related Applications This application claims the benefit under 35 U.S.C. §119(e) of U.S. Provisional Patent Application No. 62 / 913,092, filed October 9, 2019, which is incorporated by reference herein in its entirety.

[0002] Government-funded research This invention was made with government support under R24 ​​OD016473 and R21 OD023800 awarded by the National Institutes of Health. The government has certain rights in this invention. [Background technology]

[0003] background The genome engineering revolution continues to drive rapid modifications of the mouse genome, creating complex mutant mouse strains faster and more precisely than ever before. While small genome modifications are simple and efficient, the reliability of homologous recombination for precise insertion of large donor DNA remains problematic. The creation of humanized mice requires the integration of a gene's regulatory region to recapitulate its intended expression pattern and function. This is one reason for the use of random and essentially unregulated gene transfer, which can suffer from low efficiency, partial / incomplete integration, and multicopy concatenation. Such insertions are often placed in active loci, resulting in deleterious position effects on transgene expression and unintentional disruption of endogenous gene(s). Consequently, large transgenic projects often require substantial characterization time and extensive breeding. Summary of the Invention [Means for solving the problem]

[0004] Abstract This disclosure demonstrates that large, single-copy insertions of exogenous DNA into the mouse genome can be achieved using the Bxb1 integrase system. Accordingly, provided herein, in some aspects, is a system that utilizes a combination of gene editing tools (e.g., CRISPR / Cas9) and Bxb1 integrase to insert a single copy of a large transgene into a specific locus. In the presence of Bxb1 integrase, attP sites recombine with attB sites, converting them into attR ("right-handed") and attL ("left-handed") sites (Figure 1). In some embodiments, this system eliminates integration of plasmid / bacterial donor DNA vector sequences into the genome, which has been shown to result in transgene silencing. Herein, 30.6 kilobases (kb) of human DNA was integrated into the Rosa26 locus of C57BL / 6J mice with 11% efficiency (4 / 35). Remarkably, all four independent lines demonstrated germline transmission of the transgenic allele within 3 to 6 months from the date of microinjection, free of off-target contamination. Furthermore, the gene was properly transcribed in the liver. These results demonstrate the ability of this system to deliver intact, single-copy transgenes into defined loci in a short period of time, providing a powerful tool for precise gene transfer, i.e., rapid and reliable genetic engineering of animal models.

[0005] In some aspects, the present disclosure provides a method for targeted insertion of large transgenes (e.g., at least 20 kilobases in length) into the genome of an animal (e.g., a mammal, e.g., a rodent, e.g., a mouse) with limited off-target modifications of the genome. Surprisingly, mice genetically engineered to have the Bxb1 att (attachment site) landing pad allow for the insertion of large transgenes at high insertion frequencies. Furthermore, the Bxb1 system is not known to exhibit pseudointegration sites, particularly in the mouse genome (see, e.g., Russell JP et al. BioTechniques 2006;40:460-464).

[0006] In some embodiments, the Bxb1 landing pad mouse strain of the present disclosure is generated directly in mice through pronuclear microinjection of CRISPR / Cas9 gene editing tools comprising a polynucleotide encoding a Cas9 nuclease (or a variant or homolog thereof), a guide RNA (gRNA) targeting a genomic locus (e.g., a safe harbor locus) (e.g., the Rosa26 or Hip11 locus), and (at least one) single-stranded DNA (ssDNA) containing Bxb1 attachment site(s) flanked by homology arms to the safe harbor locus. For example, the use of these CRISPR / Cas9 gene editing tools produces mouse lines / strains with few to no off-target changes in the genome (except at the intended genomic locus). The established Bxb1 landing pad mouse line can then be used as a platform for insertion and subsequent analysis of a transgene of interest. For example, donor DNA containing a transgene of interest and the corresponding (cognate) Bxb1 attachment site(s) can be microinjected with Bxb1 integrase (or a polynucleotide encoding Bxb1 integrase) to produce transgenic mice with the transgene of interest integrated into their genome.

[0007] Some aspects of the disclosure relate to a method for detecting a Bxb1 gene that includes locating a first Bxb1 attachment site (e.g., attP or attB) and a second Bxb1 attachment site (e.g., a modified attP or attB) within its genome (e.g., a genomic locus). * or modified attB * In some embodiments, the mammal further comprises a polynucleotide encoding a Bxb1 integrase. The polynucleotide can be, for example, flanked by first and second Bxb1 attachment sites. Thus, the Bxb1 integrase can be encoded by the genome.

[0008] Another aspect of the present disclosure provides a mammalian embryo (or other animal embryo) comprising within its genome (e.g., a genomic locus) a first Bxb1 attachment site and a second Bxb1 attachment site. In some embodiments, the mammalian embryo further comprises a polynucleotide encoding a Bxb1 integrase, wherein optionally the polynucleotide is flanked by the first and second Bxb1 attachment sites.

[0009] In some embodiments, the first Bxb1 attachment site is an attP site, a modified attP * site, attB site, and modified attB * In some embodiments, the second Bxb1 attachment site is selected from an attP site, a modified attP site, * site, attB site, and modified attB * In some embodiments, the first and second Bxb1 attachment sites are heterologous to each other.

[0010] In some embodiments, the attP site comprises the sequence of SEQ ID NO: 1. In some embodiments, the modified attP * The attB site comprises the sequence of SEQ ID NO: 7. In some embodiments, the attB site comprises the sequence of SEQ ID NO: 2. In some embodiments, the modified attB * The site comprises the sequence of SEQ ID NO:8.

[0011] In some embodiments, the first and second Bxb1 attachment sites are separated from each other by between 50 and 500 nucleotide bases.

[0012] In some embodiments, the genomic locus is a safe harbor locus, optionally the Rosa26 locus. Other loci may also be targeted.

[0013] In some embodiments, the mammal is a rodent, such as a mouse or a rat. In some embodiments, the mammalian embryo is a rodent embryo, such as a mouse or a rat embryo. Other mammals, and non-mammals, may also be used.

[0014] Also provided herein is a method comprising introducing into a mammalian embryo (a) a donor polynucleotide comprising a sequence of interest flanked by a first cognate Bxb1 attachment site and a second cognate Bxb1 attachment site, and (b) a Bxb1 integrase or a polynucleotide encoding a Bxb1 integrase. Further provided herein is a method comprising introducing into the mammalian embryo a donor polynucleotide comprising a sequence of interest flanked by a first cognate Bxb1 attachment site and a second cognate Bxb1 attachment site.

[0015] In some embodiments, the method further comprises implanting the mammalian embryo into a pseudopregnant female mammal. In some embodiments, the method further comprises collecting a progeny mammal from the female mammal. In some embodiments, the method further comprises screening the progeny mammal for the presence or absence of the sequence of interest integrated into the genome of the progeny mammal.

[0016] In some embodiments, the donor polynucleotide, the Bxb1 integrase, and / or the polynucleotide encoding the Bxb1 integrase is introduced into the mammalian embryo via microinjection. Other transfection methods may also be used.

[0017] In some embodiments, the sequence of interest comprises a gene of interest.

[0018] In some embodiments, the sequence of interest has a size of at least 10 kb, at least 15 kb, at least 20 kb, at least 25 kb, or at least 30 kb.

[0019] A further aspect of the present disclosure is a method for generating a Bxb1 landing pad mammal, the method comprising: (a) introducing into a mammalian embryo: (i) a Cas9 nuclease or a polynucleotide encoding a Cas9 nuclease; (ii) a first guide RNA (gRNA) or a polynucleotide encoding a gRNA targeting a first genomic site (e.g., a locus) in the mammalian embryo; (iii) a first single-stranded DNA (ssDNA) donor comprising a first Bxb1 attachment site flanked by a left homology arm and a right homology arm; and optionally (iv) a second guide RNA (gRNA) or a polynucleotide encoding a gRNA targeting a second genomic site (e.g., a locus) in the mammalian embryo. (b) introducing a polynucleotide encoding a gRNA targeting a target gene (e.g., a gene locus), and (v) a second ssDNA comprising a second Bxb1 attachment site flanked by a left homology arm and a right homology arm; and (b) implanting the mammalian embryonic cell into a pseudopregnant female mammal, wherein the pseudopregnant female mammal is capable of giving birth to an offspring mammal.

[0020] In some embodiments, the first ssDNA further comprises a second Bxb1 attachment site upstream or downstream from the first Bxb1 attachment site, wherein both the first and second Bxb1 attachment sites are flanked by the left homology arm and the right homology arm.

[0021] In some embodiments, the mammalian embryo comprises a polynucleotide encoding Bxb1 integrase, or the method further comprises introducing into the mammalian embryo a polynucleotide encoding Bxb1 integrase.

[0022] In some embodiments, the method further comprises collecting the offspring mammal.

[0023] A further aspect of the present disclosure provides a mammal (eg, a rodent, eg, a mouse or rat) comprising the mammalian embryo described herein.

[0024] Other aspects of the present disclosure provide a mammal or mammalian embryo that includes a single Bxb1 attachment site within its genome (e.g., a genomic locus), as well as methods of producing and using the mammal or mammalian embryo. [Brief explanation of the drawings]

[0025] [Figure 1A-B]Figures 1A-1C: Bxb1 integrase utilizes attachment sites to integrate exogenous DNA (e.g., attP selectively placed in the genome with its reciprocal attB site in the vector). In the first version (Figure 1A), a single Bxb1 attachment site in the genome serves to integrate the entire donor vector into the genome. To minimize insertion of unwanted plasmid backbone, the vector was first processed into minicircles. Letters (ABC...) are shown to illustrate the sequence orientation. In the second version (Figure 1B), two Bxb1 attachment sites (identical except for a dinucleotide base that confers specificity) were utilized. Using this strategy, conversion of the donor plasmid into a minicircle prior to delivery is unnecessary. Rather, recombination between dual heterologous attachment sites (e.g., engineered / modified with alternative dinucleotide base pairs) serves to exclude the plasmid backbone from integration into the landing pad. In the third version (Fig. 1C), the zygote is "primed" with transgenic Bxb1 protein to promote efficient recombination: flanked by two Bxb1 attachment sites, the Bxb1 integrase transgene is eliminated after successful Bxb1-mediated replacement with the donor sequence. [Figure 1C] Same as above. [Figure 2] Figures 2A-2B: (Figure 2A) Bxb1 attachment site sequences shown with canonical dinucleotide (GT) 3' overhangs. The sequences, from top to bottom, are SEQ ID NOS: 11-18. (Figure 2B) A non-limiting list of alternative dinucleotide options allowing for greater design flexibility and sequential modification. Asterisks include palindromic overhangs (*), identical double-base overhangs (**), and canonical overhangs (***). [Figure 3] Figure 3: Summary of screening strategy. Overview of a simple PCR-based screening strategy to identify and confirm the sequence of recombinant alleles, as well as the detection of off-target integration events outside of the landing pad. [Figure 4]Figure 4: Long-range PCR to confirm knock-in. This example of the use of a long-range PCR assay to verify recombinant alleles is accurate and complete. Unlike random gene transfer with unknown flanking regions or homologous recombination, where long homology arms can make this type of assay difficult or impossible, candidate alleles generated using our system allow for long-range PCR verification using an "in / out" PCR strategy. In this example, nearly the entire 30.6 kb insert is captured by just two PCRs, each using one primer ("in") that binds to a site in the transgene and the other primer ("out") that binds to the genome adjacent to the landing pad. [Figure 5] Figure 5: RT-PCR to confirm transcripts. Evidence that the transgene is expressed as intended is generated using mRNA extracted from the liver (in this case, the tissue predicted for human transgene expression), converted to cDNA, amplified, and sequence-verified. A schematic showing alignment with references across splice sites is shown, along with agarose gel electrophoresis of amplicons from three independent founder lines, demonstrating that the human-derived transcript is expressed and splices properly. [Figure 6] Figure 6: Efficiency of plasmid integration into Bxb1 mice with one landing pad (Bxb1v1) versus two landing pads (Bxb1v2). Summary of all experiments to date using Bxb1 integrase mRNA under a number of test conditions. Three of the four version 1 landing pad lines (single attP site) generated successful recombinant alleles. Both version 2 (double attP site) landing pad mouse lines also generated successful recombinant alleles. In the background in which the majority of experiments were performed (B6), the double-site (version 2) system appears to be approximately 3-4 times more efficient than the single-site version (version 1). DETAILED DESCRIPTION OF THE INVENTION

[0026] Detailed Description Historically, the introduction of large (>10 kilobase) transgenes in mice has been achieved directly in zygotes by either embryonic stem cell manipulation or more commonly random gene transfer (and rarely by CRISPR-mediated HDR).Targeted gene transfer typically relies on the use of extensive homology arms flanking the donor transgene, which results in even larger vector sizes.The use of such methods presents technical difficulties in production and handling, and requires expensive downstream operations to mitigate any unintended consequences. Animal (e.g., mammalian (e.g., rodent, e.g., mouse)) models generated using random gene transfer often suffer from positional effects, such as disruption of the native gene at the site of integration, and aberrant transgene expression levels. Multicopy concatemers can result in massive overexpression, or multiple insertions can become scattered throughout the genome, which can lead to segregation of the transgene during breeding, with subsequent altered expression and the requisite phenotypic instability. Furthermore, the simultaneous inclusion of elements from the plasmid backbone can result in transgene silencing, negating the utility of the potential mammalian model.

[0027] The serine recombinase encoded by the Bxb1 mycobacteriophage offers a solution to many of the challenges posed by older technologies (e.g., large-scale humanization of the mouse genome). This serine recombinase can be used for the introduction of any human, mouse (or any other species), or synthetic construct into a mammalian genome. Essentially, the Bxb1 integrase functions during its lysogenic phase to effect DNA strand exchange between unique attachment sites in the phage ("attP") and its bacterial host ("attB"). Depending on the relative orientation of the attP and attB sites, the reaction can result in excision, inversion, or integration of sequences between the recognition sites, and is not reversible without the presence of an additional protein, the excisionase. Each attachment site is <50 nucleotide base pairs (bp) in length, making it amenable for use in molecular cloning and for insertion into a host genome using now-common gene editing techniques. The Bxb1 integrase functions in eukaryotic cells and does not require any additional host factors to function. Furthermore, it has been shown to function highly efficiently in cells, is unidirectional, and has no detectable pseudosites in the mouse genome. The system is also amenable to enhancement because only two key dinucleotides in the attachment site are responsible for the specificity of the recombination event (see, e.g., Figure 2B). These combined attributes make this system useful for directly modifying mammalian (e.g., mouse) zygotes.

[0028] This disclosure describes how to use the Bxb1 integrase system to engineer mouse strains with "landing pads" at safe harbor loci. These strains, in some embodiments, allow for the introduction of a single copy of a transgene in a defined orientation without contaminating vector sequences in the recombinant allele. Knowledge of the precise location of the recombinant allele, combined with high efficiency, greatly aids in the identification of founders and subsequent validation of the transgene. While this disclosure describes how the Bxb1 integrase system was used to engineer mouse strains, it should be understood that it can be used to engineer other animals, e.g., other mammals (e.g., non-human mammals), with "landing pads."

[0029] Generation of Bxb1 landing pad lines A Bxb1 landing pad animal is an animal that contains (at least one) Bxb1 attachment site (e.g., an attB site, a Bxb1 attP site, and / or a modified version thereof) in its genome. In some embodiments, the genome of the animal contains a Bxb1 attP site (SEQ ID NO: 1) or a modified Bxb1 attP site. * In some embodiments, the genome of the animal comprises a Bxb1 attB site (SEQ ID NO: 7). In some embodiments, the genome of the animal comprises a Bxb1 attB site (SEQ ID NO: 2) or a modified Bxb1 attB * The Bxb1 dinucleotide-modified Bxb1 attachment site (SEQ ID NO: 8) is included. Non-limiting examples of other dinucleotide-modified Bxb1 attachment sites are provided in FIG. 2B. The animal can be any animal (e.g., laboratory animal or livestock / farm animal). In some embodiments, the animal is a mammal. In some embodiments, the mammal is a rodent. The rodent can be, for example, a mouse or a rat. Other animals, such as poultry (e.g., chickens), are also contemplated herein.

[0030] The integrase encoded by mycobacteriophage Bxb1 catalyzes strand exchange between attP and attB, the attachment sites for the phage and bacterial host, respectively. Although the DNA sites are relatively small (<50 bp), the reaction is highly selective and strongly directional for these sites (see, e.g., Singh A et al. PLoS Genetics 2013;9(5):e1003490). The Bxb1 attB site exhibits at least seven unique and specific optimal variations, plus an additional nine suboptimal variations in the internal dinucleotide recognition sequence, allowing the same Bxb1 recombinase enzyme to simultaneously use a series of different constructs, each with its specific dinucleotide address (see, e.g., Ghosh P et al. J. Mol. Biol. 2006;349:331-348). Thus, the Bxb1 attP site and the modified attP site * sites (e.g., modified relative to the sequence of SEQ ID NO: 1), as well as the use of Bxb1 attB sites and modified attB * The use of sites (eg, modified relative to the sequence of SEQ ID NO:2) is contemplated herein.

[0031] Unless otherwise noted, it should be understood that a Bxb1 landing pad animal (e.g., a mammalian (e.g., rodent, e.g., mouse) strain can comprise a Bxb1 attP site, a modified Bxb1 attP site, a Bxb1 attB site, a modified Bxb1 attB site, or any combination thereof. The corresponding donor polynucleotide to be inserted into the Bxb1 landing pad should comprise cognate Bxb1 attachment site(s). Thus, if the Bxb1 landing pad animal strain comprises a Bxb1 attP site, the corresponding polynucleotide (e.g., circular donor DNA) to be inserted into the Bxb1 landing pad should comprise a Bxb1 attB site; and if the Bxb1 landing pad animal strain comprises a Bxb1 attB site, the corresponding polynucleotide to be inserted into the Bxb1 landing pad should comprise a Bxb1 attP site.

[0032] In some embodiments, the Bxb1 attachment site(s) are located in safe harbor loci, which are open chromatin regions of the genome. Genomic safe harbors (GSHs) are sites in the genome that can accommodate the integration of new genetic material in a manner that ensures that the newly inserted genetic element (i) functions as expected and (ii) does not introduce changes to the host genome that pose a risk to the host cell or organism (see, e.g., Papapetrou EP and Schambach A Mol Ther 2016; 24(4): 678-684).

[0033] Non-limiting examples of safe harbor loci that can be used as provided herein include the Rosa26 locus, the Hip11 locus, the Hprt locus, and the Tigre locus. Thus, in some embodiments, the Rosa26 locus of a mouse (or other mammal) strain of the present disclosure contains a Bxb1 attP site or a modified attP *In some embodiments, the Rosa26 locus comprises a Bxb1 attB site or a modified Bxb1 attB site. * In other embodiments, the Hip11 locus of a mouse (or other mammal) strain of the present disclosure comprises a Bxb1 attP site or a modified attP * In some embodiments, the Hip11 locus comprises a Bxb1 attB site or a modified attB * In still other embodiments, the Hprt locus of the disclosed mouse (or other mammal) strains comprises a Bxb1 attP site or a modified attP * In some embodiments, the Hprt locus comprises a Bxb1 attB site or a modified attB * In yet other embodiments, the Tigre locus of a mouse (or other mammal) strain of the present disclosure comprises a Bxb1 attP site or a modified attP * In some embodiments, the Tigre locus comprises a Bxb1 attB site or a modified attB * Other safe harbor loci may be used as provided herein.

[0034] In some embodiments, the Bxb1 attachment site(s) are located at or near the start codon (ATG) of the endogenous gene. For example, the normal transcriptional regulatory elements of the endogenous gene can be "intercepted" by including a Bxb1 attachment site near the start codon of the gene and then integrating the gene of interest (via Bxb1 integrase) such that transcription of the gene of interest is under the control of the transcriptional regulatory elements of the endogenous gene.

[0035] To generate a Bxb1 landing pad animal, one (at least one) single-stranded DNA (ssDNA) donor can be used. This ssDNA donor contains Bxb1 attachment site(s) (e.g., a Bxb1 attP site or a Bxb1 attB site) flanked by homology arms. In some embodiments, the ssDNA contains two Bxb1 attachment sites (e.g., a Bxb1 attP site and a modified Bxb1 attP site, or a Bxb1 attB site and a modified Bxb1 attB site). One homology arm is located to the left (5') of the Bxb1 attachment site(s) (left homology arm), and the other homology arm is located to the right (3') of the Bxb1 attachment site(s) (right homology arm). A homology arm is a region of the ssDNA that is homologous to a region of genomic DNA located at a genomic (e.g., safe harbor) locus. These homology arms allow for homologous recombination between the ssDNA donor and the genomic locus, resulting in insertion of the Bxb1 attachment site(s) into the genomic locus (e.g., via CRISPR / Cas9-mediated homology-directed repair (HDR)), as discussed below.

[0036] The homology arms can vary in length. For example, each homology arm (left arm and right homology arm) can have a length of 20 to 1000 nucleotide bases. In some embodiments, each homology arm has a length of 20 to 200, 20 to 300, 20 to 400, 20 to 500, 20 to 600, 20 to 700, 20 to 800, or 20 to 900 nucleotide bases. In some embodiments, each homology arm has a length of 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, or 1000 nucleotide bases. In some embodiments, the length of one homology arm is different from the length of the other homology arm. For example, one homology arm may have a length of 20 nucleotide bases and the other homology arm may have a length of 50 nucleotide bases. In some embodiments, the donor DNA is single-stranded. In some embodiments, the donor DNA is double-stranded.

[0037] Examples of left and right homology arms targeting the Rosa26 locus are provided as SEQ ID NO: 4 and SEQ ID NO: 5, respectively. In some embodiments, the ssDNA donor comprises the nucleotide sequence of SEQ ID NO: 3 (a Bxb1 attP attachment site flanked by left and right homology arms targeting the Rosa26 locus). In some embodiments, the ssDNA donor comprises the nucleotide sequence of SEQ ID NO: 9 (a modified Bxb1 attP attachment site flanked by left and right homology arms targeting the Rosa26 locus). * attachment site).

[0038] In some embodiments, a mouse and / or mouse embryo (or other animal or animal embryo) of the present disclosure comprises a single Bxb1 attachment site in the genomic locus of the mouse / mouse embryo. For example, the Bxb1 attachment site is an attP attachment site, a modified attP * Attachment site, attB attachment site, and modified attB * The attachment site may be selected from:

[0039] In other embodiments, a mouse and / or mouse embryo (or other animal or animal embryo) of the present disclosure comprises two (at least two) Bxb1 attachment sites in the genomic locus of the mouse / mouse embryo, which may be referred to herein as a first Bxb1 attachment site and a second Bxb1 attachment site. The first and second Bxb1 attachment sites may, in some embodiments, be attP attachment sites, modified ... * Attachment site, attB attachment site, and modified attB *The first and second Bxb1 attachment sites are selected from the following: the first and second Bxb1 attachment sites may be adjacent to each other (no intervening nucleotide sequence), or they may be separated from each other by a certain number of nucleotides. The number of nucleotides separating the two Bxb1 attachment sites may vary in some embodiments, provided that each Bxb1 attachment site is within the same safe harbor locus (e.g., within the Rosa26 locus). Thus, in some embodiments, any two (e.g., first and second) Bxb1 attachment sites are separated from each other by at least 1, at least 2, at least 5, at least 10, at least 25, at least 50, at least 100, at least 150, at least 200, at least 250, at least 300, at least 350, at least 400, at least 450, at least 500, at least 1000, at least 1500, or at least 2000 nucleotide base pairs (bp). In some embodiments, any two (e.g., first and second) Bxb1 attachment sites are separated from each other by 1 to 500 bp, 1 to 1000 bp, 1 to 1500 bp, 1 to 2000 bp, 1 to 2500 bp, or 1 to 3000 nucleotide base pairs (bp). For example, any two Bxb1 attachment sites can be 1-450bp, 1-400bp, 1-350bp, 1-300bp, 1-250bp, 1-200bp, 1-150bp, 1-100bp, 1-50bp, 5-450bp, 5-400bp, 5-350bp, 5-300bp, 5-250bp, 5-200bp, 5-150bp, 5-100bp, 5-50bp, 10-450bp, 10-400bp, 10-350bp, 10-300bp, 10-450bp, 10-400bp, 10-350bp, 10-300bp, 10-50bp, 10-600bp, 10-700bp, 10-800bp, 10-900bp, 10-1100bp, 11-1200bp, 12-1400bp, 12-150bp, 12-100bp, 13-1400bp, 14-150bp, 14-100bp, 15-100bp, 16-1700bp, 16-1700bp, 17-1800bp, 18-1900bp, 19-200bp, 20-2100bp, 21-2200bp, 22-2300bp, 23-2400bp, 24-250bp, 250bp, 260bp, 270bp, 280bp, 290bp, 300bp, 310bp, 320bp, 330bp, 34 They may be separated from each other by up to 250 bp, 10 to 200 bp, 10 to 150 bp, 10 to 100 bp, 10 to 50 bp, 50 to 450 bp, 50 to 400 bp, 50 to 350 bp, 50 to 300 bp, 50 to 250 bp, 50 to 200 bp, 50 to 150 bp, 50 to 100 bp, 100 to 450 bp, 100 to 400 bp, 100 to 350 bp, 100 to 300 bp, 100 to 250 bp, 100 to 200 bp, or 100 to 150 bp.

[0040] In some embodiments, the animals provided herein comprise a polynucleotide (e.g., a genomic polynucleotide) encoding a Bxb1 integrase. In such embodiments, the polynucleotide may be flanked by Bxb1 attachment sites such that the polynucleotide is removed after expression of the integrase and genomic integration of the gene of interest (see, e.g., FIG. 1C).

[0041] In some embodiments, the insertion of the ssDNA donor containing the Bxb1 attachment site(s) is facilitated by clustered regularly interspaced short palindromic repeats (CRISPR) / Cas9 gene editing. The CRISPR-Cas9 system is a defense mechanism naturally occurring in prokaryotes that has been repurposed as an RNA-guided DNA targeting platform used for gene editing. It relies on the DNA nuclease Cas9 (CRISPR-associated protein 9) and a non-coding guide RNA (gRNA) to target DNA cleavage.

[0042] In some embodiments, the Cas9 endonuclease is selected from Streptococcus pyogenes (NGG PAM) or Staphylococcus The Cas9 nuclease is derived from SpongeBob's genus Cas9 (NNGRRT or NNGRR(N) PAM), although other Cas9 homologs, orthologs, and / or variants (e.g., evolved versions of Cas9) may be used as provided herein. Additional non-limiting examples of RNA-guided nucleases that may be used as provided herein include Cpf1 (TTN PAM); SpCas9 D1135E variant (NGG (reduced NAG binding) PAM); SpCas9 VRER variant (NGCG PAM); SpCas9 EQR variant (NGAG PAM); SpCas9 VQR variant (NGAN or NGNG PAM); Neisseria meningitidis (NM) Cas9 (NNNNGATT PAM); Streptococcus thermophilus (ST) Cas9 (NNAGAAW PAM); and Treponema denticola (TD) Cas9 (NAAAAC).

[0043] Guide RNA (gRNA) directs the activity of the associated RNA-guided nuclease (e.g., Cas9) to a specific target sequence within the targeted genome. See, for example, Jinek et al., Science, 337, 816-821 (2012) and Deltcheva et al., Nature, 471, 602-607 (2011). The gRNA comprises at least a spacer sequence that hybridizes to the target sequence (at the target site) and a CRISPR repeat sequence. In type II systems (e.g., Streptococcus pyogenes systems), the gRNA comprises a tracrRNA (trans-activating RNA) sequence. In the type II system, the CRISPR repeat sequence and the tracrRNA sequence hybridize to each other to form a duplex. In type V systems, the crRNA (CRISPR RNA) sequence forms a duplex. In both systems, the duplex binds to an RNA-guided nuclease (e.g., Cas9) so that the gRNA and the RNA-guided nuclease form a complex. In some embodiments, the gRNA provides target specificity to the complex through its association with the RNA-guided nuclease. The gRNA directs the activity of the RNA-guided nuclease. Examples of gRNA spacer regions targeting the Rosa26 locus are provided as SEQ ID NO:6 and SEQ ID NO:10.

[0044] Other genome editing techniques may be used (e.g., transcription activator-like effector nucleases (TALENs) and / or zinc finger nucleases (ZFNs)). See, e.g., Joung JK et al. Nat Rev Mol Cell Biol. 2013;14(1):49-55; Carroll D Genetics. 2011;188(4):773-782; and Gaj T et al. Trends Biotechnol. 2013;31(7):397-405.

[0045] Transgenic mice are most commonly generated by microinjection of DNA (pronuclear injection) into the pronuclei of fertilized single-cell (1-cell) mouse embryos. If DNA integration occurs before the first nuclear division, some or all cells will carry the transgene. After injection, eggs are surgically transferred into the oviducts of time-mated pseudopregnant foster mothers generated by mating females with vasectomized males. Offspring resulting from injected eggs carrying the transgene are referred to as founders.

[0046] Although microinjection is exemplified, other transfection systems may be used to generate the BXb1 landing pad animals of the present disclosure, such as electroporation (see, e.g., Wang W et al. J Genet Genomics 2016;43(5):319-27), embryonic stem cell-mediated gene transfer and retroviral-mediated gene transfer (see, e.g., Kumar TR et al. Methods Mol Biol. 2009;590:335-362), and viral-based gene transfer.

[0047] In some embodiments, gene transfer occurs at the single-cell stage of the embryo (which may be referred to as the zygote). In other embodiments, gene transfer occurs at a later multi-cell stage of the embryo (two or more cells, also referred to as the blastomere). In some embodiments, pronuclear microinjection occurs at the zygote stage, followed by nuclear injection at the two-cell stage (2x).

[0048] In some embodiments, animal (e.g., mammalian (e.g., rodent, e.g., mouse) strains expressing Cre recombinase-dependent Cas9 expression can be used. These mouse strains allow for in vitro CRISPR gene editing when injected with a viral vector co-expressing Cre and the gRNA. The virally expressed Cre switches on and activates Cas9 expression, which in turn edits the targeted gene(s). Furthermore, in vivo gene editing in mice can be achieved by local or systemic injection of lentivirus or adeno-associated virus expressing Cas9 and gRNA.

[0049] Any mouse can be used to generate the Bxb1 landing pad line. Non-limiting examples of mouse strains include C57BL / 6J mice (664), C57BL / 6NJ (5304), FVB / NJ (1800), B6D2 (C57BL / 6 x DBA / 2J) mice, and NGS TM (NOD scid gamma) mice (5557) or variants thereof. Additional examples include A / J (000646), 129S1 / SvImJ (002448), NOD / ShiLtJ (001976), NZO / HiLtJ (002105), CAST / EiJ (000928), PWK / PhJ (003715), WSB / EiJ (001145), DBA2 (000671), and collaborative cross (CC) strains.

[0050] In some embodiments, a method for generating a Bxb1 landing pad animal (e.g., a mammal (e.g., a rodent, e.g., a mouse)) can include isolating a fertilized single-cell embryo and microinjecting into the pronucleus or cytoplasm of the embryo a Cas9 (e.g., a Cas9 protein, or DNA or mRNA encoding a Cas9 protein), a gRNA (or DNA encoding a gRNA), and ssDNA targeting a genomic locus (e.g., a safe harbor locus (e.g., the Rosa26 locus or other open chromatin locus)). The microinjected embryos are then transferred into pseudopregnant female mice. The mice can be carried to term. Pseudopregnancy describes a false pregnancy, whereby all signs and symptoms of pregnancy are exhibited except for the presence of a zygote. Mice enter a pseudopregnant state after estrus, when females are mated with sterile males, resulting in sterile matings. At approximately 2-3 weeks of age, tail biopsies may be collected from the offspring, and correct integration into the safe harbor locus may be verified by polymerase chain reaction (PCR), sequencing, Southern blotting, and / or long-range sequencing systems (e.g., PacBio). Founder mice with the desired integration are then bred to generate the Bxb1 Landing Pad mouse line.

[0051] Targeted transgene integration The Bxb1 landing pad animal (e.g., a mammal (e.g., a rodent, e.g., a mouse)) can, in some embodiments, be used to introduce a gene of interest at a Bxb1 attachment site in the animal's genome. In some embodiments, the gene of interest is present on a vector. A vector is simply a DNA molecule used as a vehicle to carry exogenous genetic material (e.g., a donor transgene) into a host cell (e.g., a mouse embryo). In some embodiments, the gene of interest is present on a circular donor polynucleotide (e.g., a plasmid). In some embodiments, for example, when using an animal that contains only one Bxb1 attachment site in its genome, the circular donor polynucleotide is a DNA minicircle. A DNA minicircle is a small (approximately 4 kb) circular vector backbone in which donor DNA has been circularized to >100 bp to 50 kb. In some embodiments, the DNA minicircle is a plasmid derivative that has been removed from all prokaryotic vector parts (e.g., no longer contains the bacterial plasmid backbone containing antibiotic resistance markers and / or bacterial origins of replication).

[0052] Methods for generating DNA minicircles are well known in the art. For example, a parental plasmid containing a bacterial backbone and a eukaryotic insert (including the transgene to be expressed) can be generated in a specialized E. coli strain that expresses a site-specific recombinase protein. Recombination sites flank the eukaryotic insert in the parental plasmid, and thus, when the activity of the recombinase protein (non-Bxb1) is induced by methods including, but not limited to, arabinose induction, glucose induction, etc., the bacterial backbone is excised from the eukaryotic insert, resulting in a eukaryotic DNA minicircle and a bacterial plasmid.

[0053] In some embodiments, a sequence of interest (e.g., a gene) has a length of 200 base pairs (bp) to 100 kilobases (kb). In some embodiments, a gene of interest has a length of at least 10 kb. For example, a gene of interest may have a length of at least 15 kb, at least 20 kb, at least 25 kb, at least 30 kb, or at least 35 kb. In some embodiments, a gene of interest has a length of 10 to 100 kb, 10 to 75 kb, 10 to 50 kb, 10 to 30 kb, 20 to 100 kb, 20 to 75 kb, 20 to 50 kb, 20 to 30 kb, 30 to 100 kb, 30 to 75 kb, or 30 to 50 kb.

[0054] In some embodiments, the donor polynucleotide(s) have a length of 200 bp to 500 kb, 200 bp to 250 kb, or 200 bp to 100 kb. In some embodiments, the donor polynucleotide has a length of at least 10 kb. For example, the donor polynucleotide may have a length of at least 15 kb, at least 20 kb, at least 25 kb, at least 30 kb, at least 35 kb, at least 50 kb, at least 100 kb, at least 200 kb, at least 300 kb, at least 400 kb, or at least 500 kb. In some embodiments, the donor polynucleotide has a length of 10 to 500 kb, 20 to 400 kb, 10 to 300 kb, 10 to 200 kb, or 10 to 100 kb. In some embodiments, the donor polynucleotide has a length of 10 to 100 kb, 10 to 75 kb, 10 to 50 kb, 10 to 30 kb, 20 to 100 kb, 20 to 75 kb, 20 to 50 kb, 20 to 30 kb, 30 to 100 kb, 30 to 75 kb, or 30 to 50 kb. The donor polynucleotide may be circular or linear.

[0055] In some embodiments, donor polynucleotide(s) comprising the gene of interest and its corresponding (cognate) Bxb1 attachment site(s) are introduced (e.g., via microinjection) into an embryo (e.g., a single-cell embryo (zygote)). Later stage embryos or animals may also be used. Pronuclear microinjection and other gene transfer methods for use as provided herein are discussed above.

[0056] In some embodiments, the donor polynucleotide(s) is / are introduced into an embryo or animal together with a Bxb1 integrase protein, a polynucleotide encoding the Bxb1 integrase protein, or a Bxb1 integrase protein and a polynucleotide encoding the Bxb1 integrase protein. The polynucleotide may be DNA or RNA (e.g., mRNA).

[0057] After introducing the donor polynucleotide and the Bxb1 integrase into the embryo, the embryo can be implanted into a pseudopregnant female (similar to the breeding process described above) to generate a genetically modified offspring animal containing the gene of interest.

[0058] In some embodiments, at least 10% of the genetically modified progeny animals comprise the gene of interest integrated into the genomic locus. In some embodiments, at least 11%, at least 12%, at least 13%, at least 14%, or at least 15% of the genetically modified progeny animals comprise the gene of interest integrated into the genomic locus. In some embodiments, at least 15%, at least 20%, at least 30%, at least 35%, at least 40%, at least 45%, or at least 50% of the genetically modified progeny animals comprise the gene of interest integrated into the genomic locus. For example, between 10% and 50%, between 10% and 40%, between 10% and 30%, or between 10% and 20% of the genetically modified progeny animals comprise the gene of interest integrated into the genomic locus. In some embodiments, more than 50% (e.g., 55%, 60%, 65%, or 70%) of the genetically modified progeny animals comprise the gene of interest integrated into the genomic locus.

[0059] In some embodiments, the gene of interest (or donor polynucleotide comprising the gene of interest) has a length of at least 10 kb, and at least 10% of the genetically modified progeny animals contain the gene of interest precisely integrated into the genomic locus. For example, the gene of interest can have a length of at least 10 kb, at least 15 kb, at least 20 kb, at least 25 kb, at least 30 kb, at least 35 kb, at least 40 kb, at least 45 kb, or at least 50 kb, and at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, or at least 50% of the genetically modified progeny animals contain the gene of interest integrated into the genomic locus.

[0060] In some embodiments, the gene of interest is at least 10 kb in length, and at least 15% of the genetically modified progeny animals contain the gene of interest integrated into the genomic locus. In some embodiments, the gene of interest is at least 10 kb in length, and at least 20% of the genetically modified progeny animals contain the gene of interest integrated into the genomic locus. In some embodiments, the gene of interest is at least 10 kb in length, and at least 25% of the genetically modified progeny animals contain the gene of interest integrated into the genomic locus. In some embodiments, the gene of interest is at least 10 kb in length, and at least 30% of the genetically modified progeny animals contain the gene of interest integrated into the genomic locus.

[0061] In some embodiments, the gene of interest is at least 15 kb in length, and at least 15% of the genetically modified progeny animals contain the gene of interest integrated into the genomic locus. In some embodiments, the gene of interest is at least 15 kb in length, and at least 20% of the genetically modified progeny animals contain the gene of interest integrated into the genomic locus. In some embodiments, the gene of interest is at least 15 kb in length, and at least 25% of the genetically modified progeny animals contain the gene of interest integrated into the genomic locus. In some embodiments, the gene of interest is at least 15 kb in length, and at least 30% of the genetically modified progeny animals contain the gene of interest integrated into the genomic locus.

[0062] In some embodiments, the gene of interest is at least 20 kb in length, and at least 15% of the genetically modified progeny animals contain the gene of interest integrated into the genomic locus. In some embodiments, the gene of interest is at least 20 kb in length, and at least 20% of the genetically modified progeny animals contain the gene of interest integrated into the genomic locus. In some embodiments, the gene of interest is at least 20 kb in length, and at least 25% of the genetically modified progeny animals contain the gene of interest integrated into the genomic locus. In some embodiments, the gene of interest is at least 20 kb in length, and at least 30% of the genetically modified progeny animals contain the gene of interest integrated into the genomic locus.

[0063] In some embodiments, the gene of interest is at least 25 kb in length, and at least 15% of the genetically modified progeny animals contain the gene of interest integrated into the genomic locus. In some embodiments, the gene of interest is at least 25 kb in length, and at least 20% of the genetically modified progeny animals contain the gene of interest integrated into the genomic locus. In some embodiments, the gene of interest is at least 25 kb in length, and at least 25% of the genetically modified progeny animals contain the gene of interest integrated into the genomic locus. In some embodiments, the gene of interest is at least 25 kb in length, and at least 30% of the genetically modified progeny animals contain the gene of interest integrated into the genomic locus.

[0064] In some embodiments, the gene of interest is at least 30 kb in length, and at least 15% of the genetically modified progeny animals comprise the gene of interest integrated into the genomic locus. In some embodiments, the gene of interest is at least 30 kb in length, and at least 20% of the genetically modified progeny animals comprise the gene of interest integrated into the genomic locus. In some embodiments, the gene of interest is at least 30 kb in length, and at least 25% of the genetically modified progeny animals comprise the gene of interest integrated into the genomic locus. In some embodiments, the gene of interest is at least 30 kb in length, and at least 30% of the genetically modified progeny animals comprise the gene of interest integrated into the genomic locus.

[0065] In some embodiments, the gene of interest is at least 35 kb in length, and at least 15% of the genetically modified progeny animals contain the gene of interest integrated into the genomic locus. In some embodiments, the gene of interest is at least 35 kb in length, and at least 20% of the genetically modified progeny animals contain the gene of interest integrated into the genomic locus. In some embodiments, the gene of interest is at least 35 kb in length, and at least 25% of the genetically modified progeny animals contain the gene of interest integrated into the genomic locus. In some embodiments, the gene of interest is at least 35 kb in length, and at least 30% of the genetically modified progeny animals contain the gene of interest integrated into the genomic locus.

[0066] Further embodiments Further embodiments are encompassed by the following numbered paragraphs: 1. A mammal comprising within its genome a first Bxb1 attachment site and a second Bxb1 attachment site.

[0067] 2. The mammal of paragraph 1, further comprising a polynucleotide encoding a Bxb1 integrase, optionally wherein said polynucleotide is flanked by said first and second Bxb1 attachment sites.

[0068] 3. The first Bxb1 attachment site may be an attP site, a modified attP * site, attB site, and modified attB * Selected from the site; The second Bxb1 attachment site may be an attP site, a modified attP * site, attB site, and modified attB * Selected from the site; Optionally, the first and second Bxb1 attachment sites are heterologous to each other, and optionally the genome does not contain attR and / or attL sites, and are separated from each other by, e.g., 50 to 500 nucleotide bases. 3. The mammal of paragraph 1 or 2.

[0069] 4. The attP site comprises the sequence of SEQ ID NO: 1, and the modified attP * The attB site comprises the sequence of SEQ ID NO: 7, the attB site comprises the sequence of SEQ ID NO: 2, and / or the modified attB * 4. The mammal of paragraph 3, wherein the site comprises the sequence of SEQ ID NO:8.

[0070] 5. A mammal described in any one of paragraphs 1 to 4, wherein the first and second Bxb1 attachment sites are separated from each other by 50 to 500 nucleotide bases.

[0071] 6. The mammal of any one of the preceding paragraphs, wherein the first and second Bxb1 attachment sites are within a safe harbor locus, optionally the Rosa26 locus.

[0072] 7. A mammal described in any one of the preceding paragraphs, wherein the mammal is a rodent, optionally a mouse.

[0073] 8. A mammalian embryo comprising within its genome a first Bxb1 attachment site and a second Bxb1 attachment site.

[0074] 9. The mammalian embryo of paragraph 8, further comprising (a) a Bxb1 integrase or (b) a polynucleotide encoding a Bxb1 integrase, wherein optionally the polynucleotide is flanked by the first and second Bxb1 attachment sites.

[0075] 10. The first Bxb1 attachment site may be an attP site, a modified attP * site, attB site, and modified attB * Selected from the site; The second Bxb1 attachment site may be an attP site, a modified attP * site, attB site, and modified attB * Selected from the site; Optionally, the first and second Bxb1 attachment sites are heterologous to each other, and optionally the genome does not contain attR and / or attL sites, and are separated from each other by, e.g., 50 to 500 nucleotide bases. 10. The mammalian embryo of paragraph 8 or 9.

[0076] 11. The attP site comprises the sequence of SEQ ID NO: 1, and the modified attP * The attB site comprises the sequence of SEQ ID NO: 7, the attB site comprises the sequence of SEQ ID NO: 2, and / or the modified attB * 11. The mammalian embryo of paragraph 10, wherein the site comprises the sequence of SEQ ID NO:8.

[0077] 12. The mammalian embryo of any one of paragraphs 8 to 11, wherein the first and second Bxb1 attachment sites are separated from each other by 50 to 500 nucleotide base pairs.

[0078] 13. The mammalian embryo of any one of the preceding paragraphs, wherein the first and second Bxb1 attachment sites are within a safe harbor locus, optionally the Rosa26 locus.

[0079] 14. The mammalian embryo of any one of the preceding paragraphs, wherein the mammalian embryo is a single-cell embryo or a multicellular embryo.

[0080] 15. The mammalian embryo of any one of the preceding paragraphs, wherein the mammalian embryo is a rodent embryo, optionally a mouse embryo.

[0081] 16. A method comprising introducing into the mammalian embryo described in any one of the preceding paragraphs: (a) a donor polynucleotide comprising a sequence of interest flanked by a first cognate Bxb1 attachment site and a second cognate Bxb1 attachment site; and (b) Bxb1 integrase or a polynucleotide encoding Bxb1 integrase.

[0082] 17. A method comprising introducing into the mammalian embryo described in any one of the preceding paragraphs a donor polynucleotide comprising a sequence of interest flanked by a first cognate Bxb1 attachment site and a second cognate Bxb1 attachment site.

[0083] 18. The method of paragraph 16 or 17, further comprising the step of implanting said mammalian embryo into a pseudopregnant female mammal.

[0084] 19. The method of paragraph 18, further comprising the step of collecting an offspring mammal from said female mammal.

[0085] 20. The method of paragraph 19, further comprising screening the progeny mammal for the presence or absence of the sequence of interest integrated into the genome of the progeny mammal.

[0086] 21. The method of any one of the preceding paragraphs, wherein the donor polynucleotide, the Bxb1 integrase, and / or the polynucleotide encoding Bxb1 integrase are introduced into the mammalian embryo via microinjection.

[0087] 22. The method of any one of the preceding paragraphs, wherein the sequence of interest comprises a gene of interest.

[0088] 23. The method of any one of the preceding paragraphs, wherein the sequence of interest has a size of at least 10 kb, at least 15 kb, at least 20 kb, at least 25 kb, or at least 30 kb.

[0089] 24. A method for generating a Bxb1 landing pad mammal, said method comprising: (a) introducing into a mammalian embryo (i) a Cas9 nuclease or a polynucleotide encoding a Cas9 nuclease, (ii) a first guide RNA (gRNA) or a polynucleotide encoding a gRNA that targets a first genomic site in the mammalian embryo, (iii) a first single-stranded DNA (ssDNA) donor comprising a first Bxb1 attachment site flanked by a left homology arm and a right homology arm; optionally, (iv) a second guide RNA (gRNA) or a polynucleotide encoding a gRNA that targets a second genomic site in the mammalian embryo, and (v) a second ssDNA comprising a second Bxb1 attachment site flanked by a left homology arm and a right homology arm; and (b) implanting said mammalian embryonic cell into a pseudopregnant female mammal, wherein said pseudopregnant female mammal is capable of giving birth to an offspring mammal.

[0090] 25. The method of paragraph 24, wherein the first ssDNA further comprises a second Bxb1 attachment site upstream or downstream from the first Bxb1 attachment site, and wherein both the first and second Bxb1 attachment sites are flanked by the left homology arm and the right homology arm.

[0091] 26. The method of paragraph 24 or 25, wherein the mammalian embryo comprises Bxb1 integrase or a polynucleotide encoding Bxb1 integrase, or step (a) further comprises introducing Bxb1 integrase or a polynucleotide encoding Bxb1 integrase into the mammalian embryo.

[0092] 27. The method of any one of paragraphs 24 to 26, further comprising collecting the offspring mammals.

[0093] 28. The method of any one of paragraphs 24 to 27, wherein the mammalian embryo is a rodent embryo, optionally a mouse embryo.

[0094] 29. A mammal, comprising a mammalian embryo according to paragraphs 8-14.

[0095] 30. The mammal of paragraph 29, wherein the mammal is a rodent, optionally a mouse.

[0096] 31. A mammal containing a Bxb1 attachment site within its genome.

[0097] 32. The mammal of paragraph 31, further comprising Bxb1 integrase or a polynucleotide encoding Bxb1 integrase.

[0098] 33. The Bxb1 attachment site is an attP site, a modified attP * site, attB site, or modified attB * 33. The mammal of paragraph 31 or 32, which is a site.

[0099] 34. The attP site comprises the sequence of SEQ ID NO: 1, and the modified attP * The attB site comprises the sequence of SEQ ID NO: 7, the attB site comprises the sequence of SEQ ID NO: 2, and / or the modified attB * 34. The mammal of paragraph 33, wherein the site comprises the sequence of SEQ ID NO:8.

[0100] 35. The mammal of any one of the preceding paragraphs, wherein the Bxb1 attachment site is within a safe harbor locus, optionally the Rosa26 locus.

[0101] 36. The mammal of any one of the preceding paragraphs, wherein the mammal is a rodent, optionally a mouse.

[0102] 37. A mammalian embryo containing Bxb1 attachment sites within its genome.

[0103] 38. The mammalian embryo of paragraph 37, further comprising Bxb1 integrase or a polynucleotide encoding Bxb1 integrase.

[0104] 39. The Bxb1 attachment site is an attP site, a modified attP * site, attB site, or modified attB * 39. The mammalian embryo of paragraph 37 or 38, which is a site.

[0105] 40. The attP site comprises the sequence of SEQ ID NO: 1, and the modified attP * The attB site comprises the sequence of SEQ ID NO: 7, the attB site comprises the sequence of SEQ ID NO: 2, and / or the modified attB * 40. The mammalian embryo of paragraph 39, wherein the site comprises the sequence of SEQ ID NO:8.

[0106] 41. The mammalian embryo of any one of the preceding paragraphs, wherein the Bxb1 attachment site is within a safe harbor locus, optionally the Rosa26 locus.

[0107] 42. The mammalian embryo of any one of the preceding paragraphs, wherein the mammalian embryo is a single-cell embryo or a multicellular embryo.

[0108] 43. The mammalian embryo of any one of the preceding paragraphs, wherein the mammalian embryo is a rodent embryo, optionally a mouse embryo.

[0109] 44. A method comprising introducing into the mammalian embryo described in any one of paragraphs 37 to 43: (a) a donor polynucleotide comprising a sequence of interest and a cognate Bxb1 attachment site, and (b) Bxb1 integrase or a polynucleotide encoding Bxb1 integrase.

[0110] 45. A method comprising introducing into the mammalian embryo of any one of paragraphs 37 to 43 a donor polynucleotide comprising a sequence of interest and a cognate Bxb1 attachment site.

[0111] 46. ​​The method of paragraph 44 or 45, further comprising the step of implanting the mammalian embryo into a pseudopregnant female mammal.

[0112] 47. The method of paragraph 46, further comprising the step of collecting an offspring mammal from said female mammal.

[0113] 48. The method of paragraph 47, further comprising screening the progeny mammal for the presence or absence of the sequence of interest integrated into the genome of the progeny mammal.

[0114] 49. The method of any one of the preceding paragraphs, wherein the donor polynucleotide, the Bxb1 integrase, and / or the polynucleotide encoding Bxb1 integrase are introduced into the mammalian embryo via microinjection.

[0115] 50. The method of any one of the preceding paragraphs, wherein the donor polynucleotide is a minicircle.

[0116] 51. The method of any one of the preceding paragraphs, wherein the sequence of interest comprises a gene of interest.

[0117] 52. The method of any one of the preceding paragraphs, wherein the sequence of interest has a size of at least 3 kb, at least 4 kb, at least 5 kb, at least 6 kb, at least 7 kb, at least 8 kb, at least 9 kb, or at least 10 kb.

[0118] 53. A method for generating a Bxb1 landing pad mammal, the method comprising: (a) In mammalian embryos, (i) a Cas9 nuclease or a polynucleotide encoding a Cas9 nuclease; (ii) a guide RNA (gRNA) or a polynucleotide encoding a gRNA that targets a genomic locus in the mammalian embryo; and (iii) a single-stranded DNA (ssDNA) donor containing a Bxb1 attachment site flanked by a left homology arm and a right homology arm; introducing (b) implanting said mammalian embryonic cell into a pseudopregnant female mammal, wherein said pseudopregnant female mammal is capable of giving birth to an offspring mammal; A method that encompasses

[0119] 54. The method of paragraph 53, wherein the mammalian embryo comprises a polynucleotide encoding Bxb1 integrase or wherein step (a) further comprises introducing a polynucleotide encoding Bxb1 integrase into the mammalian embryo.

[0120] 55. The method of paragraph 54, further comprising collecting the offspring mammal.

[0121] 56. The method of paragraph 55, wherein the mammalian embryo is a rodent embryo, optionally a mouse embryo.

[0122] 57. A mammal, comprising a mammalian embryo according to paragraphs 37-43.

[0123] 58. The mammal of paragraph 57, wherein the mammal is a rodent, optionally a mouse. [Example]

[0124] Example Example 1. Bxb1 Mouse - One (attP) Landing Pad Using CRISPR / Cas9 and oligonucleotide donors, we engineered a single attP site into the genome of the above mice (C57BL / 6J, NSG TM The vector was inserted into the Rosa26 locus of mouse strains (PWK / PhJ, DBA / 2J, A / J, 129S1 / SvImJ, or FVB / NJ).

[0125] Fertilized zygotes were isolated from C57BL / 6J mice. The pronuclei of these zygotes were microinjected with (1) Cas9 mRNA, protein, or both mRNA and protein (concentrations ranged from 60 to 100 ng / μL for mRNA and 30 to 60 ng / μL for protein), (2) gRNA (concentrations ranged from 30 to 50 ng / μL), and (3) an approximately 200-bp ssDNA oligo (SEQ ID NO: 3) targeting the Rosa26 locus. This ssDNA oligo contains 152 bases of homology (SEQ ID NOs: 4 and 5) flanking the 48-base-pair Bxb1 attP site (SEQ ID NO: 2). Microinjected zygotes were transferred into pseudopregnant mice and carried to term. At approximately 2 to 3 weeks of age, tail biopsies were collected from the offspring and tested for correct integration of the attP site by PCR and sequencing. Mice carrying the Bxb1 attP locus were generated by mating the Bxb1 attP mouse strain.

[0126] These mice were then used as recipients for integrase-mediated recombination with donors containing compatible cognate attB sites (Figures 2A-2B). To avoid insertion of DNA from the vector backbone, plasmids were converted into minicircles (System Biosciences, LLC) prior to microinjection.

[0127] Fertilized zygotes were isolated from the Bxb1 attP C57BL / 6J mouse strain. The pronuclei of these zygotes were microinjected with 100 ng / μL of mRNA encoding Bxb1 integrase and 1–10 ng / μL of donor DNA. The donor DNA contained a Bxb1 attB site compatible with the host embryo's Bxb1 attP site. The donor DNA was a bacterial vector-free minicircle, using techniques well known and established in the art. The microinjected zygotes were transferred into pseudopregnant mice and carried to term. At >2–3 weeks of age, tail biopsies were collected from the offspring and tested for correct integration of the DNA by PCR and sequencing.

[0128] The results for this version are outlined in Table 1. [Table 1]

[0129] Example 2. Bxb1 Mouse - Two (attP) Landing Pads We generated our version 2 recipient mice through sequential modification of the version 1 line. Again, we used CRISPR / Cas9 and oligonucleotide donors to create a second attP *The modified attP site was inserted approximately 240 bp away from the original attP site. At the modified site, the dinucleotide pairing was changed from GT to GA. The addition of the second site allows for the elimination of the vector backbone without first having to convert the donor construct into a minicircle (Figure 1B). Note that in this example, two attachment sites were inserted sequentially; however, they could have been inserted simultaneously, for example, using a single donor polynucleotide. The version 2 mice also allow for the insertion of even larger tracts of DNA, because the essential attB sites can be placed into any vector (including BACs) simply by flanking the desired region to be integrated.

[0130] Screening and validation are among the greatest challenges when generating mice with large knock-ins. However, knowing the exact location and orientation of the transgene allows for rapid and easy identification by PCR. A general strategy for screening is outlined in Figure 3.

[0131] We tested version 2 mice using a recombineered BAC (total size 33,939 bp) to insert a 30,570 bp tract of human genomic DNA, the results of which are summarized in Table 2. [Table 2]

[0132] The present inventors tested version 2 mice by inserting nucleic acids of various lengths (ranging from 1.5 kb to 30.6 kb). The results are summarized in Table 3. [Table 3-1] [Table 3-2]

[0133] Four founder candidates were backcrossed to wild-type mice, and their N1 progeny were evaluated for germline transmission of both the desired recombination allele (REC) and any off-target insertion (OTI) events. Offspring from two of the four founder lines also carried unwanted random transgenic alleles. The OTI and REC alleles were segregated, indicating that the insertion events were unrelated. Long-range PCR was performed to confirm that the integrated alleles were intact (Figure 4).

[0134] Because the transgene is known to have high expression in human liver, RNA was isolated from mouse livers from three of the four colonies (a colony from the fourth primary was not included in these studies because it had not yet reached sufficient size). cDNA was prepared from total RNA, and a 1,270-bp PCR product was generated from each line (Figure 5). Sanger sequencing of this product was then used to confirm that the transcript was indeed the humanized allele.

[0135] [ka] [ka]

[0136] [ka] [ka]

[0137] All references, patents, and patent applications disclosed herein are each cited and, in some cases, incorporated by reference with respect to the subject matter which may include the entire document.

[0138] The indefinite articles "a" and "an," as used herein and in the claims, unless expressly indicated to the contrary, should be understood to mean "at least one."

[0139] Unless expressly stated to the contrary, it should also be understood that in any method claimed herein that includes more than one step or act, the order of the method steps or acts is not necessarily limited to the order in which the method steps or acts are recited.

[0140] In the claims and the above specification, all transitional phrases such as "comprising," "including," "carrying," "having," "containing," "involving," "holding," "composed of," and the like, are to be understood to mean open ended, i.e., including but not limited to. Only the transitional phrases "consisting of" and "consisting essentially of" shall be closed or semi-closed transitional phrases, respectively, as set forth in the United States Patent Office Manual of Patent Examining Procedures, Section 2111.03.

[0141] The terms "about" and "substantially" before a numerical value mean ±10% of the stated numerical value.

[0142] Where a range of values ​​is provided, each value between the upper and lower limits of that range is specifically contemplated and described herein. In certain embodiments, for example, the following items are provided: (Item 1) A mammal comprising within its genome a first Bxb1 attachment site and a second Bxb1 attachment site. (Item 2) 2. The mammal of claim 1, further comprising a polynucleotide encoding a Bxb1 integrase, optionally wherein the polynucleotide is flanked by the first and second Bxb1 attachment sites. (Item 3) The first Bxb1 attachment site may be an attP site, a modified attP * site, attB site, and modified attB * Selected from the site; The second Bxb1 attachment site may be an attP site, a modified attP * site, attB site, and modified attB * Selected from the site; 2. The mammal of claim 1, wherein optionally the first and second Bxb1 attachment sites are heterologous to each other. (Item 4) The attP site comprises the sequence of SEQ ID NO: 1, and the modified attP * The attB site comprises the sequence of SEQ ID NO: 7, the attB site comprises the sequence of SEQ ID NO: 2, and / or the modified attB * 4. The mammal of item 3, wherein the site comprises the sequence of SEQ ID NO:8. (Item 5) 2. The mammal of item 1, wherein the first and second Bxb1 attachment sites are separated from each other by 50 to 500 nucleotide bases. (Item 6) 2. The mammal of item 1, wherein the first and second Bxb1 attachment sites are within a safe harbor locus, optionally the Rosa26 locus. (Item 7) 2. The mammal according to item 1, wherein the mammal is a rodent, optionally a mouse. (Item 8) A mammalian embryo comprising within its genome a first Bxb1 attachment site and a second Bxb1 attachment site. (Item 9) 9. The mammalian embryo of item 8, further comprising a polynucleotide encoding a Bxb1 integrase, wherein optionally the polynucleotide is flanked by the first and second Bxb1 attachment sites. (Item 10) The first Bxb1 attachment site may be an attP site, a modified attP * site, attB site, and modified attB * Selected from the site; The second Bxb1 attachment site may be an attP site, a modified attP * site, attB site, and modified attB * Selected from the site; 9. The mammalian embryo of item 8, wherein optionally the first and second Bxb1 attachment sites are heterologous to each other. (Item 11) The attP site comprises the sequence of SEQ ID NO: 1, and the modified attP * The attB site comprises the sequence of SEQ ID NO: 7, the attB site comprises the sequence of SEQ ID NO: 2, and / or the modified attB * Item 11. The mammalian embryo of item 10, wherein the site comprises the sequence of SEQ ID NO:8. (Item 12) 9. The mammalian embryo of item 8, wherein the first and second Bxb1 attachment sites are separated from each other by 50 to 500 nucleotide base pairs. (Item 13) 9. The mammalian embryo of item 8, wherein the first and second Bxb1 attachment sites are within a safe harbor locus, optionally the Rosa26 locus. (Item 14) 9. The mammalian embryo of item 8, wherein the mammalian embryo is a single-cell embryo or a multicellular embryo. (Item 15) 9. The mammalian embryo according to item 8, wherein the mammalian embryo is a rodent embryo, optionally a mouse embryo. (Item 16) A method comprising introducing into a mammalian embryo described in any one of the preceding items (a) a donor polynucleotide comprising a sequence of interest flanked by a first cognate Bxb1 attachment site and a second cognate Bxb1 attachment site, and (b) Bxb1 integrase or a polynucleotide encoding Bxb1 integrase. (Item 17) A method comprising introducing into a mammalian embryo described in any one of the preceding items a donor polynucleotide comprising a sequence of interest flanked by a first cognate Bxb1 attachment site and a second cognate Bxb1 attachment site. (Item 18) 17. The method of claim 16, further comprising implanting the mammalian embryo into a pseudopregnant female mammal. (Item 19) 20. The method of claim 18, further comprising the step of collecting an offspring mammal from said female mammal. (Item 20) 20. The method of claim 19, further comprising screening the progeny mammal for the presence or absence of the sequence of interest integrated into the genome of the progeny mammal. (Item 21) 17. The method of claim 16, wherein the donor polynucleotide, the Bxb1 integrase, and / or the polynucleotide encoding Bxb1 integrase is introduced into the mammalian embryo via microinjection. (Item 22) 17. The method of item 16, wherein the sequence of interest comprises a gene of interest. (Item 23) 17. The method of item 16, wherein the sequence of interest has a size of at least 10 kb, at least 15 kb, at least 20 kb, at least 25 kb, or at least 30 kb. (Item 24) 1. A method for generating a Bxb1 landing pad mammal, the method comprising: (a) introducing into a mammalian embryo (i) a Cas9 nuclease or a polynucleotide encoding a Cas9 nuclease, (ii) a first guide RNA (gRNA) or a polynucleotide encoding a gRNA that targets a first genomic site in the mammalian embryo, (iii) a first single-stranded DNA (ssDNA) donor comprising a first Bxb1 attachment site flanked by a left homology arm and a right homology arm; optionally, (iv) a second guide RNA (gRNA) or a polynucleotide encoding a gRNA that targets a second genomic site in the mammalian embryo, and (v) a second ssDNA comprising a second Bxb1 attachment site flanked by a left homology arm and a right homology arm; and (b) implanting said mammalian embryonic cell into a pseudopregnant female mammal, wherein said pseudopregnant female mammal is capable of giving birth to an offspring mammal; A method that encompasses (Item 25) 25. The method of claim 24, wherein the first ssDNA further comprises a second Bxb1 attachment site upstream or downstream from the first Bxb1 attachment site, wherein both the first and second Bxb1 attachment sites are flanked by the left homology arm and the right homology arm. (Item 26) 25. The method of claim 24, wherein the mammalian embryo comprises a polynucleotide encoding Bxb1 integrase, or step (a) further comprises introducing a polynucleotide encoding Bxb1 integrase into the mammalian embryo. (Item 27) 25. The method of claim 24, further comprising collecting the offspring mammals. (Item 28) 25. The method of item 24, wherein the mammalian embryo is a rodent embryo, optionally a mouse embryo. (Item 29) A mammal, comprising the mammalian embryo described in item 8. (Item 30) 30. The mammal according to item 29, wherein the mammal is a rodent, optionally a mouse. (Item 31) A mammal containing a Bxb1 attachment site within its genome. (Item 32) 32. The mammal of item 31, further comprising a polynucleotide encoding Bxb1 integrase. (Item 33) The Bxb1 attachment site can be an attP site, a modified attP * site, attB site, or modified attB * 32. The mammal according to item 31, which is a site. (Item 34) The attP site comprises the sequence of SEQ ID NO: 1, and the modified attP * The attB site comprises the sequence of SEQ ID NO: 7, the attB site comprises the sequence of SEQ ID NO: 2, and / or the modified attB * 34. The mammal of item 33, wherein the site comprises the sequence of SEQ ID NO:8. (Item 35) 32. The mammal of item 31, wherein the Bxb1 attachment site is within a safe harbor locus, optionally the Rosa26 locus. (Item 36) 32. The mammal according to item 31, wherein the mammal is a rodent, optionally a mouse. (Item 37) Mammalian embryos containing Bxb1 attachment sites within their genomes. (Item 38) 38. The mammalian embryo of item 37, further comprising a polynucleotide encoding Bxb1 integrase. (Item 39) The Bxb1 attachment site can be an attP site, a modified attP * site, attB site, or modified attB * 38. The mammalian embryo of item 37, which is a site. (Item 40) The attP site comprises the sequence of SEQ ID NO: 1, and the modified attP * The attB site comprises the sequence of SEQ ID NO: 7, the attB site comprises the sequence of SEQ ID NO: 2, and / or the modified attB *40. The mammalian embryo of item 39, wherein the site comprises the sequence of SEQ ID NO:8. (Item 41) 38. The mammalian embryo of item 37, wherein the Bxb1 attachment site is within a safe harbor locus, optionally the Rosa26 locus. (Item 42) 38. The mammalian embryo of item 37, wherein the mammalian embryo is a single-cell embryo or a multicellular embryo. (Item 43) 38. The mammalian embryo of item 37, wherein the mammalian embryo is a rodent embryo, optionally a mouse embryo. (Item 44) 44. A method comprising introducing into the mammalian embryo of any one of items 37 to 43: (a) a donor polynucleotide comprising a sequence of interest and a cognate Bxb1 attachment site, and (b) Bxb1 integrase or a polynucleotide encoding Bxb1 integrase. (Item 45) 44. A method comprising introducing into the mammalian embryo of any one of items 37 to 43 a donor polynucleotide comprising a sequence of interest and a cognate Bxb1 attachment site. (Item 46) 45. The method of claim 44, further comprising the step of implanting the mammalian embryo into a pseudopregnant female mammal. (Item 47) 47. The method of claim 46, further comprising the step of collecting progeny mammals from said female mammal. (Item 48) 48. The method of claim 47, further comprising screening the progeny mammal for the presence or absence of the sequence of interest integrated into the genome of the progeny mammal. (Item 49) 45. The method of claim 44, wherein the donor polynucleotide, the Bxb1 integrase, and / or the polynucleotide encoding Bxb1 integrase is introduced into the mammalian embryo via microinjection. (Item 50) 45. The method of claim 44, wherein the donor polynucleotide is a minicircle. (Item 51) 45. The method of item 44, wherein the sequence of interest comprises a gene of interest. (Item 52) 45. The method of item 44, wherein the sequence of interest has a size of at least 3 kb, at least 4 kb, at least 5 kb, at least 6 kb, at least 7 kb, at least 8 kb, at least 9 kb, or at least 10 kb. (Item 53) 1. A method for generating a Bxb1 landing pad mammal, the method comprising: (a) In mammalian embryos, (i) a Cas9 nuclease or a polynucleotide encoding a Cas9 nuclease; (ii) a guide RNA (gRNA) or a polynucleotide encoding a gRNA that targets a genomic locus in the mammalian embryo; and (iii) a single-stranded DNA (ssDNA) donor containing a Bxb1 attachment site flanked by a left homology arm and a right homology arm; introducing (b) implanting said mammalian embryonic cell into a pseudopregnant female mammal, wherein said pseudopregnant female mammal is capable of giving birth to an offspring mammal; A method that encompasses (Item 54) 54. The method of claim 53, wherein the mammalian embryo comprises a polynucleotide encoding Bxb1 integrase, or step (a) comprises introducing a polynucleotide encoding Bxb1 integrase into the mammalian embryo. (Item 55) 55. The method of claim 54, further comprising the step of collecting the offspring mammals. (Item 56) 56. The method of item 55, wherein the mammalian embryo is a rodent embryo, optionally a mouse embryo. (Item 57) 38. A mammal comprising the mammalian embryo of item 37. (Item 58) 58. The mammal according to item 57, wherein the mammal is a rodent, optionally a mouse.

Claims

1. The genome contains a first Bxb1 attB attachment site and a second Bxb1 attB within the Rosa26 locus. ＊ a mouse comprising an attachment site, wherein the first Bxb1 attB attachment site comprises the sequence of SEQ ID NO: 2, and the second Bxb1 attB ＊ The attachment site comprises the sequence of SEQ ID NO: 8, mouse.

2. and a polynucleotide encoding a Bxb1 integrase, wherein the polynucleotide optionally contains the first Bxb1 attB attachment site and the second Bxb1 attB attachment site. ＊ The mouse of claim 1 , wherein the mouse is sandwiched between attachment sites.

3. The first Bxb1 attB attachment site and the second Bxb1 attB ＊ 2. The mouse of claim 1, wherein the attachment sites are separated from each other by 50 to 500 nucleotide bases.

4. Mouse embryos were transfected with (a) a first Bxb1 attB attachment site and a second Bxb1 attB ＊ 1. A method comprising introducing (a) a donor polynucleotide comprising a sequence of interest flanked by attachment sites, and (b) Bxb1 integrase or a polynucleotide encoding Bxb1 integrase, The mouse embryo contains a first Bxb1 attP attachment site and a second Bxb1 attP within the Rosa26 locus of its genome. ＊ including an attachment site, The first Bxb1 attB attachment site comprises the sequence of SEQ ID NO: 2, and the second Bxb1 attB ＊ The method, wherein the attachment site comprises the sequence of SEQ ID NO:

8.

5. 5. The method of claim 4, further comprising implanting the mouse embryo into a pseudopregnant female mouse.

6. The method of claim 5, further comprising the step of collecting offspring mice from the female mice.

7. 7. The method of claim 6, further comprising screening the offspring mice for the presence or absence of the sequence of interest integrated into the genome of the offspring mice.

8. The method of claim 4, wherein the donor polynucleotide, the Bxb1 integrase, and / or the polynucleotide encoding Bxb1 integrase is introduced into the mouse embryo via microinjection.

9. The method of claim 4 , wherein the sequence of interest comprises a gene of interest.

10. 5. The method of claim 4, wherein the sequence of interest has a size of at least 10 kb, at least 15 kb, at least 20 kb, at least 25 kb, or at least 30 kb.

11. 10. A method for generating a mouse according to claim 1, said method comprising: (a) Into a mouse embryo, (i) a Cas9 nuclease or a polynucleotide encoding a Cas9 nuclease; (ii) a first guide RNA (gRNA) or a polynucleotide encoding the first gRNA that targets a first genomic site in the Rosa26 locus of the genome of the mouse embryo; (iii) a first single-stranded DNA (ssDNA) donor comprising a first Bxb1 attB attachment site flanked by a left homology arm and a right homology arm; (iv) a second guide RNA (gRNA) or a polynucleotide encoding the second gRNA that targets a second genomic site in the Rosa26 locus of the genome of the mouse embryo; (v) a second Bxb1 attB flanked by the left and right homology arms ＊ a second ssDNA containing an attachment site; introducing (b) implanting the mouse embryonic cells into a pseudopregnant female mouse, wherein the pseudopregnant female mouse is capable of giving birth to offspring mice; It encompasses The first Bxb1 attB attachment site comprises the sequence of SEQ ID NO: 2, and the second Bxb1 attB ＊ The method, wherein the attachment site comprises the sequence of SEQ ID NO:

8.

12. The method of claim 11, wherein the mouse embryo comprises a polynucleotide encoding Bxb1 integrase, or step (a) further comprises introducing a polynucleotide encoding Bxb1 integrase into the mouse embryo.

13. The method of claim 11, further comprising collecting the offspring mice.

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