High-frequency targeted animal gene transfer

Abstract

The present disclosure provides methods and compositions for high-frequency mouse transgenesis, for example, using Bxb1 landing pad.The present disclosure shows that the large single copy insertion of exogenous DNA into mouse genome can be achieved using the Bxb1 integrase system.Therefore, in some aspects, the present disclosure provides a system that uses 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.

Description

Technical Field

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

[0002] Research Funded by Government 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 Art

[0003] Background The revolution in genome engineering continues to drive the rapid modification of the mouse genome, creating more complex mutant mouse strains faster and more precisely than ever before. Small genome modifications are simple and efficient, but the reliability of homologous recombination for the accurate insertion of large donor DNA remains problematic. The generation of humanized mice requires the incorporation of gene regulatory regions to reproduce their intended expression patterns and functions. This is one reason for the use of random and essentially chaotic transgenesis, which can suffer from low efficiency, partial / incomplete integration, and multicopy concatenation. Such insertions are often placed at active loci, resulting in harmful position effects on transgene expression and unintended disruption of endogenous genes. In conclusion, large transgenic projects often require substantial characterization time and additional rounds of breeding.

Summary of the Invention

Means for Solving the Problems

[0004] Abstract This disclosure demonstrates that large single-copy insertion of exogenous DNA into the mouse genome is achievable using the Bxb1 integrase system. Thus, provided herein are systems that, in several aspects, utilize a combination of gene editing tools (e.g., CRISPR / Cas9) and Bxb1 integrase to insert a large single-copy transgene into a specific locus. In the presence of Bxb1 integrase, attP sites are recombined with attB sites, converting the aforementioned sites into attR ("right-handed") and attL ("left-handed") sites (Figure 1). In some embodiments, this system eliminates plasmid / bacterial donor DNA vector sequence integration into the genome (which has been shown to result in transgene silencing). Hereinafter, 30.6 kilobases (kb) of human DNA were integrated into the Rosa26 locus of C57BL / 6J mice with 11% efficiency (4 / 35). Remarkably, all four independent lines demonstrated germline transfer of transgenic alleles without off-target contamination within 3–6 months from the date of microinjection. Furthermore, the genes were properly transcribed in the liver. These results demonstrate the system's ability to deliver an intact single copy of a transgene into a designated locus in a short period of time, providing a powerful tool for precise gene transfer, i.e., rapid and reliable genetic engineering in 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) in the genome of an animal (e.g., a mammal, e.g., a rodent, e.g., a mouse) having limited off-target modification of the genome. Surprisingly, mice engineered to have a Bxb1 att (attachment site) landing pad allow insertion of large transgenes with high insertion frequencies. Further, the Bxb1 system is not known to exhibit pseudo-integration sites 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 a mouse through pronuclear microinjection of a CRISPR / Cas9 gene editing tool comprising a polynucleotide encoding 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) comprising Bxb1 attachment site(s) flanked by homology arms to the safe harbor locus. For example, use of these CRISPR / Cas9 gene editing tools results in a mouse line / strain with little 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 of a transgene of interest and subsequent analysis. For example, a donor DNA comprising the 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 having the transgene of interest integrated into the genome.

[0007] Some aspects of this disclosure relate to the presence of a first Bxb1 attachment site (e.g., attP or attB) and a second Bxb1 attachment site (e.g., a modified attP) within the genome (e.g., a genomic locus). * or a modified attB * The present invention provides a mammal (or other animal) comprising ) ). In some embodiments, the mammal further comprises a polynucleotide encoding Bxb1 integrase. The polynucleotide may be sandwiched, for example, between first and second Bxb1 attachment sites. Thus, the Bxb1 integrase may be encoded by a genome.

[0008] Other aspects of this disclosure provide a mammalian embryo (or other animal embryo) having a first Bxb1 attachment site and a second Bxb1 attachment site within its genome (e.g., a genomic locus). In some embodiments, the mammalian embryo further comprises a polynucleotide encoding Bxb1 integrase, wherein the polynucleotide is optionally 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 * The site is selected from the following locations. In some embodiments, the second Bxb1 attachment site is the attP site, modified attP * Site, attB site, and modified attB * The sites are selected. In some embodiments, the first and second Bxb1 attachment sites are heterogeneous with respect to each other.

[0010] In some embodiments, the attP site includes the sequence of sequence number 1. In some embodiments, a modified attP * The site includes the sequence of sequence number 7. In some embodiments, the attB site includes the sequence of sequence number 2. In some embodiments, a modified attB * The region includes the sequence of sequence number 8.

[0011] In some embodiments, the first and second Bxb1 attachment sites are separated from each other by only 50 to 500 nucleotides.

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

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

[0014] A method is also provided herein that includes the steps of introducing into a mammalian embryo (a) a donor polynucleotide containing a sequence of interest flanked by a first congener Bxb1 attachment site and a second congener Bxb1 attachment site, and (b) Bxb1 integrase or a polynucleotide encoding Bxb1 integrase. A method is further provided herein that includes the steps of introducing into the mammalian embryo a donor polynucleotide containing a sequence of interest flanked by a first congener Bxb1 attachment site and a second congener Bxb1 attachment site.

[0015] In some embodiments, the method further comprises the step of transplanting the mammalian embryo into a pseudopregnant female mammal. In some embodiments, the method further comprises the step of collecting offspring mammals from the female mammal. In some embodiments, the method further comprises the step of screening the offspring mammals for the presence or absence of the sequence of the objective incorporated into the genome of the offspring mammals.

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

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

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

[0019] A further aspect of the present disclosure is a method for generating a Bxb1 landing pad mammal, the method comprising: (a) a mammalian embryo containing (i) Cas9 nuclease or a polynucleotide encoding 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 containing 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 second genomic site (a second) in the mammalian embryo. (v) a polynucleotide encoding a gRNA targeting a genomic locus (e.g., a gene locus), and (b) a second ssDNA comprising a second Bxb1 attachment site sandwiched between a left homology arm and a right homology arm; and (c) a step of transplanting the mammalian embryonic cells into a pseudopregnant female mammal, wherein the pseudopregnant female mammal is capable of producing offspring mammals.

[0020] In some embodiments, the first ssDNA further includes a second Bxb1 attachment site upstream or downstream of the first Bxb1 attachment site, where both the first and second Bxb1 attachment sites are sandwiched between the left homology arm and the right homology arm.

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

[0022] In some embodiments, the above method further includes the step of collecting the offspring mammals.

[0023] A further aspect of this disclosure provides mammals (e.g., rodents, e.g., mice or rats) including mammalian embryos as described herein.

[0024] Other aspects of this disclosure include providing a mammal or mammalian embryo having a single Bxb1 attachment site within its genome (e.g., a genomic locus), and providing methods for generating and using such mammal or mammalian embryo. [Brief explanation of the drawing]

[0025] [Figure 1]Figures 1A-1C: Bxb1 integrase utilizes attachment sites to integrate exogenous DNA (e.g., attP selectively positioned in the genome along with its reciprocal attB sites in the vector). In the first version (Figure 1A), a single Bxb1 attachment site in the genome works to integrate the entire donor vector into the genome. To minimize the insertion of unwanted plasmid backbone, the vector was first processed into a minicircle. The letters (A, B, C…) are shown to illustrate the orientation of the sequences. In the second version (Figure 1B), two Bxb1 attachment sites (identical except for the dinucleotide bases that confer specificity) were utilized. Using this strategy, it is unnecessary to convert the donor plasmid into a minicircle before delivery. Rather, recombination between the two heterogeneous attachment sites (e.g., manipulated / modified using alternative dinucleotide base pairs) works to exclude the plasmid backbone from integration into the landing pad. In the third version (Figure 1C), the zygote is "primed" with the transgenic Bxb1 protein to promote efficient recombination. Sandwiched between two Bxb1 attachment sites, this Bxb1 integrase transgene is eliminated after successful Bxb1-mediated replacement with the donor sequence. [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 sequence numbers 11-18. (Figure 2B) A non-restrictive enumeration of alternative dinucleotide options to enable greater design flexibility and sequential modification. Asterisks include palindromic overhangs (*), identical double base overhangs (**), and canonical overhangs (***). [Figure 3] Figure 3: Summary of screening strategies. An overview of a simple PCR-based screening strategy for identifying and confirming recombinant allele sequences, as well as for detecting off-target integration events outside the landing pad. [Figure 4]Figure 4: Long-range PCR for confirming knock-in. The example of using a long-range PCR assay to validate recombinant alleles is accurate and complete. Unlike homologous recombination, where random gene transfections or long homology arms where adjacent regions are unknown can make this type of assay difficult or impossible, candidate alleles generated using our system enable long-range PCR validation using an "In / Out" PCR strategy. In this example, almost the entire 30.6kb insertion is captured by just two PCRs, each using one primer that binds to a site in the transgene ("In") and the other primer that binds to the genome adjacent to the landing pad ("Out"). [Figure 5] Figure 5: RT-PCR for transcript confirmation. Evidence that the transgene is expressed as intended is generated using mRNA extracted from the liver (in this case, the tissue expected to express the human transgene), converted to cDNA, amplified, and sequence-validated. A schematic diagram showing alignment with a reference across the splice site is shown along with agarose gel electrophoresis of amplicons from three independent foundation lines, demonstrating that the human-derived transcript is expressed and splices correctly. [Figure 6] Figure 6: Efficiency of plasmid integration into Bxb1 mice with one landing pad (Bxb1v1) versus two landing pads (Bxb1v2). A summary of all experiments to date using Bxb1 integrase mRNA under a wide range of test conditions. Three of the four version 1 landing pad lines (single attP site) successfully generated recombinant alleles. Both version 2 (double attP site) landing pad mouse lines successfully generated recombinant alleles. In the background (B6) where most of the experiments were conducted, the dual-site (version 2) system appears to be approximately 3-4 times more efficient than the single-site version (version 1). [Modes for carrying out the invention]

[0026] Detailed explanation 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, very rarely, by CRISPR-mediated HDR). Targeted gene transfer typically relies on the use of extensive homology arms adjacent to the donor transgene described above, which results in even larger vector sizes. The use of such methods represents technical difficulties in generation and handling and requires expensive downstream work to mitigate any unintended consequences. Animals created using random gene transfer (e.g., mammalian models, e.g., rodents, e.g., mice) often suffer from locational effects, such as disruption of native genes at the site of integration and abnormal levels of transgene expression. Multicopy concatemers can result in significant overexpression or a state where numerous insertions are scattered throughout the genome, which can lead to the isolation of the transgenes during breeding, accompanied by subsequent expression changes and phenotypic instability. Furthermore, the simultaneous inclusion of elements derived from the plasmid backbone can lead to transgene silencing, potentially rendering the mammalian model unusable.

[0027] The serine recombinase encoded by the Bxb1 mycobacteriophage offers solutions to many of the challenges posed by older technologies (e.g., large-scale humanization of mouse genomes). This serine recombinase can be used to introduce any human, mouse (or any other species), or synthetic construct into mammalian genomes. Essentially, the Bxb1 integrase functions to facilitate DNA strand exchange between specific attachment sites in the phage ("attP") and its bacterial host ("attB") during its lysogenic phase. Depending on the relative orientation of the attP and attB sites, the reaction can result in sequence excision, inversion, or integration between the recognition sites, which are not reversible without the presence of further proteins, excisionases. Each attachment site is <50 nucleotide base pairs (bp) in length, making it readily usable for insertion into the host genome in molecular cloning and using now common gene editing techniques. The Bxb1 integrase described above functions in eukaryotic cells and requires no further host factors to function. Furthermore, it has been shown to function with high efficiency in cells, is unidirectional, and has no detectable pseudosites in the mouse genome. The system is also suitable for enhancement because the two central dinucleotides at the attachment site are solely responsible for the specificity of the recombination event (see, for example, Figure 2B). These combined attributes make this system useful for directly modifying mammalian (e.g., mouse) zygotes.

[0028] The present disclosure describes how to manipulate mouse strains at "landing pads" in safe harbor loci using the Bxb1 integrase system. In some embodiments, these strains allow for the introduction of a single copy of a transgene in a defined orientation without contamination by vector sequences in the recombinant allele. Knowledge of the exact location of the recombinant allele, combined with high efficiency, greatly aids in the initial identification and subsequent verification of the transgene. The present disclosure describes how mouse strains were manipulated using the Bxb1 integrase system, but it should be understood how other animals, such as other mammals (e.g., non-human mammals), can be manipulated at "landing pads" using it.

[0029] Generation of Bxb1 Landing Pad Strains A Bxb1 landing pad animal is an animal that contains (at least one) Bxb1 attachment site (e.g., attB site, Bxb1 attP site, and / or a modified version thereof) within its genome. In some embodiments, the genome of the animal contains the Bxb1 attP site (SEQ ID NO: 1) or a modified Bxb1 attP * site (SEQ ID NO: 7). In some embodiments, the genome of the animal contains the Bxb1 attB site (SEQ ID NO: 2) or a modified Bxb1 attB * site (SEQ ID NO: 8). 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., chicken), are also contemplated herein.

[0030] The integrase encoded by mycobacteriophage Bxb1 catalyzes strand exchange between attP and attB, which are 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 with respect to 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 nine further suboptimal variations in the internal dinucleotide recognition sequence, allowing the same Bxb1 recombinase enzyme to simultaneously utilize a set 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 modified attP * The use of sites (for example, modified relative to the sequence of sequence number 1), as well as the Bxb1 attB site and modified attB * The use of a part (for example, a modified version of the sequence of sequence number 2) is intended herein.

[0031] Unless otherwise noted, it should be understood that a Bxb1 landing pad animal strain (e.g., a mammal (e.g., a rodent, e.g., a mouse)) may contain 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 contain one congener Bxb1 attachment site. Therefore, if the Bxb1 landing pad animal strain contains a Bxb1 attP site, the corresponding polynucleotide (e.g., circular donor DNA) to be inserted into the Bxb1 landing pad should contain a Bxb1 attB site; and if the Bxb1 landing pad animal strain contains a Bxb1 attB site, the corresponding polynucleotide to be inserted into the Bxb1 landing pad should contain a Bxb1 attP site.

[0032] The Bxb1 attachment site(s) described above are, in some embodiments, located at a safe harbor locus, which is an open chromatin region of the genome. A genome safe harbor (GSH) is a site in the genome that can be adapted to the integration of new genetic material in a manner that ensures the newly inserted genetic element (i) functions as expected and (ii) does not cause host genome changes that pose a risk to the host cell or organism (see, for example, Papapetrou EP and Schambach A Mol Ther 2016; 24(4): 678-684).

[0033] Non-limiting examples of safe harbor loci that may be used as provided herein include the Rosa26 locus, the Hip11 locus, the HPrt locus, and the Tigre locus. Accordingly, in some embodiments, the Rosa26 locus of the mouse (or other mammalian) strain of this disclosure may be the Bxb1 attP site or a modified attP site. *The site includes. In some embodiments, the Rosa26 locus is the Bxb1 attB site or a modified Bxb1 attB site. * This includes the site. In other embodiments, the Hip11 locus of the mouse (or other mammalian) strain of the Disclosure is the Bxb1 attP site or a modified attP site. * The site includes. In some embodiments, the Hip11 locus is the Bxb1 attB site or a modified attB site. * This includes the site. In other embodiments, the HPrt locus of the mouse (or other mammalian) strain disclosed herein is the Bxb1 attP site or a modified attP site. * Includes a site. In some embodiments, the HPrt locus is the Bxb1 attB site or a modified attB site. * This includes the site. In further embodiments, the Tigre locus of the mouse (or other mammalian) strain of the Disclosure is the Bxb1 attP site or a modified attP site. * Includes a site. In some embodiments, the Tigre locus is the Bxb1 attB site or a modified attB site. * This includes the site. 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 may include a Bxb1 attachment site near the gene's start codon, and then be "intercepted" by incorporating the gene of interest (via Bxb1 integrase) so that the 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 (or at least one) single-stranded DNA (ssDNA) donor may be used. This ssDNA donor contains one or more Bxb1 attachment sites (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 (the left homology arm), and the other homology arm is located to the right (3') of the Bxb1 attachment site (the right homology arm). The homology arms are regions of ssDNA homologous to regions of genomic DNA located at genomic (e.g., safe harbor) loci. These homology arms enable homologous recombination between the ssDNA donor and the genomic locus, resulting in the insertion of the Bxb1 attachment site(s) into the genomic locus (e.g., via CRISPR / Cas9-mediated homologous recombination repair (HDR)), as discussed below.

[0036] The homology arms can vary in length. For example, each homology arm (the left arm and the right homology arm) may have a length of 20 to 1000 nucleotides. In some embodiments, each homology arm may have 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 nucleotides. 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 NOs: 4 and SEQ ID NOs: 5, respectively. In some embodiments, the ssDNA donor contains the nucleotide sequence of SEQ ID NOs: 3 (Bxb1 attP attachment sites flanked by left and right homology arms targeting the Rosa26 locus). In some embodiments, the ssDNA donor contains the nucleotide sequence of SEQ ID NOs: 9 (modified Bxb1 attP attachment sites flanked by left and right homology arms targeting the Rosa26 locus). * (Including the attachment site.)

[0038] In some embodiments, the mice and / or mouse embryos (or other animals or animal embryos) of the present disclosure include a single Bxb1 attachment site at the genomic locus of the mouse / mouse embryo. For example, the Bxb1 attachment site may be an attP attachment site, a modified attP * Adhesion site, attB adhesion site, and modified attB * The attachment site can be selected from the available options.

[0039] In other embodiments, the mouse and / or mouse embryo (or other animal or animal embryo) of the Disclosure includes two (at least two) Bxb1 attachment sites at the genomic locus of the mouse / mouse embryo, which may be referred to herein as the first Bxb1 attachment site and the second Bxb1 attachment site. In some embodiments, the first and second Bxb1 attachment sites are attP attachment sites, modified attP * Adhesion site, attB adhesion site, and modified attB *The attachment sites are selected. The first and second Bxb1 attachment sites may be adjacent to each other (without intervening nucleotide sequences), 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 located within the same safe harbor locus (e.g., within the Rosa26 locus). Thus, in some embodiments, any two (e.g., the first and second) Bxb1 attachment sites may be 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., the first and second) Bxb1 attachment sites are separated from each other by only 1-500 bp, 1-1000 bp, 1-1500 bp, 1-2000 bp, 1-2500 bp, or 1-3000 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 Only the following can be separated from each other: ~250bp, 10~200bp, 10~150bp, 10~100bp, 10~50bp, 50~450bp, 50~400bp, 50~350bp, 50~300bp, 50~250bp, 50~200bp, 50~150bp, 50~100bp, 100~450bp, 100~400bp, 100~350bp, 100~300bp, 100~250bp, 100~200bp, or 100~150bp.

[0040] In some embodiments, the animals provided herein include a polynucleotide encoding Bxb1 integrase (e.g., a genomic polynucleotide). In such embodiments, the polynucleotide may be sandwiched between Bxb1 attachment sites such that it is removed after the expression of the integrase and the genomic integration of the gene of interest (see, for example, Figure 1C).

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

[0042] In some embodiments, the Cas9 endonuclease is derived from Streptococcus pyogenes (NGG PAM) or Staphylococcus aureus (NNGRRT or NNGRR(N) PAM), but other Cas9 homologs, orthologues, and / or variants (e.g., evolved versions of Cas9) may be used as provided herein. Further non-limiting examples of RNA guide 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 guide 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 contains at least a spacer sequence that hybridizes to the target sequence (at the target site) and a CRISPR repeat sequence. In the type II system (e.g., the Streptococcus pyogenes system), the gRNA contains a tracrRNA (transactivating RNA) sequence. In the type II system, the CRISPR repeat sequence and the tracrRNA sequence hybridize to form a double helix. In the type V system, the crRNA (CRISPR RNA) sequence forms a double helix. In both systems, the double helix binds to an RNA guide nuclease (e.g., Cas9) so that the gRNA and the RNA guide nuclease form a complex. In some embodiments, the gRNA provides target specificity to the complex by association with the RNA guide nuclease. The gRNA thus directs the activity of the RNA guide nuclease. Examples of gRNA spacer regions targeting the Rosa26 locus are provided as SEQ ID NOs: 6 and SEQ ID NOs: 10.

[0044] Other genome editing techniques may be used (e.g., activator-like effector nucleases (TALENs) and / or zinc finger nucleases (ZFNs)). See, for example, 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 produced by microinjecting DNA into the pronucleus of a fertilized single-cell mouse embryo. If DNA integration occurs before the first nuclear division, some or all of the cells will contain the transgene. After injection, the oocyte is surgically transferred to the fallopian tube of a time-mated pseudopregnancy foster mother, produced by mating a female with a male that has had its vas deferens removed. The offspring produced from the injected oocyte containing the transgene are called the first generation.

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

[0047] In some embodiments, gene transfer is performed at the single-cell stage of the embryo (which may be called the zygote). In other embodiments, gene transfer is performed at the later multicellular stage of the embryo (two cells or more, also called blastomeres). In some embodiments, pronuclear microinjection is performed at the zygote stage, followed by nuclear injection at the two-cell stage (2x).

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

[0049] Any mouse can be used to generate the Bxb1 landing pad strain. Non-limiting examples of mouse strains include the C57BL / 6J mouse (664), C57BL / 6NJ (5304), FVB / NJ (1800), B6D2 (C57BL / 6 x DBA / 2J) mouse, and NGS. TM Examples include (NOD scid gamma) mice (5557) or their variants. Further 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 Bxb1 landing pad animals (e.g., mammals (e.g., rodents, e.g., mice)) may include the steps of: isolating a fertilized single-cell embryo; and microinjecting Cas9 (e.g., Cas9 protein, or DNA or mRNA encoding the Cas9 protein), gRNA (or DNA encoding gRNA), and ssDNA targeting a genomic locus (e.g., the safe harbor locus (e.g., the Rosa26 locus or other open chromatin locus)) into the pronucleus or cytoplasm of the embryo. The microinjected embryo is then transferred to a pseudopregnant female mouse. Pseudopregnancy can occur and be maintained until maturity. Pseudopregnancy explains a false pregnancy in which all signs and symptoms of pregnancy are present except for the presence of a zygote. Mice are bred when females are mated with infertile males and enter a state of pseudopregnancy after estrus resulting in infertile mating. At approximately 2-3 weeks of age, tail biopsies may be collected from the offspring, and precise 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). Primary mice with the desired integration are then bred to produce the Bxb1 landing pad mouse strain.

[0051] Targeted gene integration The above-mentioned Bxb1 landing pad animals (e.g., mammals (e.g., rodents, e.g., mice)) can, in some embodiments, be used to introduce a gene of interest at the Bxb1 attachment site in the genome of the animal. In some embodiments, the gene of interest resides on a vector. The vector is simply a DNA molecule used as a vehicle to deliver exogenous genetic material (e.g., a donor transgene) to a host cell (e.g., a mouse embryo). In some embodiments, the gene of interest resides 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 4kb) circular vector backbone in which the donor DNA is made into a >100bp-50kb circular. In some embodiments, the DNA minicircle is a plasmid derivative from which all prokaryotic vector portions have been removed (e.g., no longer containing a bacterial plasmid backbone containing antibiotic resistance markers and / or bacterial origins of replication).

[0052] Methods for generating DNA minicircles are well known in the field. For example, a parent plasmid containing a bacterial skeleton and a eukaryotic insert (containing the transgene to be expressed) can be generated in a specific E. coli strain expressing a site-directed recombinase protein. The recombination site is adjacent to the eukaryotic insert in the parent plasmid, and therefore, when the activity of the recombinase protein (non-Bxb1) is induced by methods such as arabinose induction or glucose induction, the bacterial skeleton is excised from the eukaryotic insert to produce a eukaryotic DNA minicircle and a bacterial plasmid.

[0053] The target sequence (e.g., a gene) has a length of 200 base pairs (bp) to 100 kilobases (kb) in some embodiments. The target gene has a length of at least 10 kb in some embodiments. For example, the target gene 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, the target gene 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] The donor polynucleotide(s) described above may, in some embodiments, have a length of 200 bp to 500 kb, 200 bp to 250 kb, or 200 bp to 100 kb. The donor polynucleotide may, in some embodiments, have 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 may have 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 10-100kb, 10-75kb, 10-50kb, 10-30kb, 20-100kb, 20-75kb, 20-50kb, 20-30kb, 30-100kb, 30-75kb, or 30-50kb. The donor polynucleotide may be cyclic or linear.

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

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

[0057] After introducing the above donor polynucleotides and Bxb1 integrase into the embryo, the embryo can be transplanted into a pseudopregnant female to produce genetically modified offspring animals containing the target gene (similar to the reproductive process described above).

[0058] In some embodiments, at least 10% of the genetically modified offspring animals contain the target gene 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 offspring animals contain the target gene 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 offspring animals contain the target gene integrated into the genomic locus. For example, 10% to 50%, 10% to 40%, 10% to 30%, or 10% to 20% of the genetically modified offspring animals contain the target gene integrated into the genomic locus. In some embodiments, more than 50% (e.g., 55%, 60%, 65%, or 70%) of the genetically modified offspring animals contain the target gene integrated into the genomic locus.

[0059] In some embodiments, the gene of interest (or a donor polynucleotide containing the gene of interest) has a length of at least 10 kb, and at least 10% of the genetically modified offspring animals contain the gene of interest precisely integrated into the genomic locus. For example, the gene of interest may 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 offspring animals contain the gene of interest integrated into the genomic locus.

[0060] In some embodiments, the target gene has a length of at least 10 kb, and at least 15% of the genetically modified offspring animals contain the target gene integrated into the genomic locus. In some embodiments, the target gene has a length of at least 10 kb, and at least 20% of the genetically modified offspring animals contain the target gene integrated into the genomic locus. In some embodiments, the target gene has a length of at least 10 kb, and at least 25% of the genetically modified offspring animals contain the target gene integrated into the genomic locus. In some embodiments, the target gene has a length of at least 10 kb, and at least 30% of the genetically modified offspring animals contain the target gene integrated into the genomic locus.

[0061] In some embodiments, the target gene has a length of at least 15 kb, and at least 15% of the genetically modified offspring animals contain the target gene integrated into the genomic locus. In some embodiments, the target gene has a length of at least 15 kb, and at least 20% of the genetically modified offspring animals contain the target gene integrated into the genomic locus. In some embodiments, the target gene has a length of at least 15 kb, and at least 25% of the genetically modified offspring animals contain the target gene integrated into the genomic locus. In some embodiments, the target gene has a length of at least 15 kb, and at least 30% of the genetically modified offspring animals contain the target gene integrated into the genomic locus.

[0062] In some embodiments, the target gene has a length of at least 20 kb, and at least 15% of the genetically modified offspring animals contain the target gene integrated into the genomic locus. In some embodiments, the target gene has a length of at least 20 kb, and at least 20% of the genetically modified offspring animals contain the target gene integrated into the genomic locus. In some embodiments, the target gene has a length of at least 20 kb, and at least 25% of the genetically modified offspring animals contain the target gene integrated into the genomic locus. In some embodiments, the target gene has a length of at least 20 kb, and at least 30% of the genetically modified offspring animals contain the target gene integrated into the genomic locus.

[0063] In some embodiments, the target gene has a length of at least 25 kb, and at least 15% of the genetically modified offspring animals contain the target gene integrated into the genomic locus. In some embodiments, the target gene has a length of at least 25 kb, and at least 20% of the genetically modified offspring animals contain the target gene integrated into the genomic locus. In some embodiments, the target gene has a length of at least 25 kb, and at least 25% of the genetically modified offspring animals contain the target gene integrated into the genomic locus. In some embodiments, the target gene has a length of at least 25 kb, and at least 30% of the genetically modified offspring animals contain the target gene integrated into the genomic locus.

[0064] In some embodiments, the target gene has a length of at least 30 kb, and at least 15% of the genetically modified offspring animals contain the target gene integrated into the genomic locus. In some embodiments, the target gene has a length of at least 30 kb, and at least 20% of the genetically modified offspring animals contain the target gene integrated into the genomic locus. In some embodiments, the target gene has a length of at least 30 kb, and at least 25% of the genetically modified offspring animals contain the target gene integrated into the genomic locus. In some embodiments, the target gene has a length of at least 30 kb, and at least 30% of the genetically modified offspring animals contain the target gene integrated into the genomic locus.

[0065] In some embodiments, the target gene has a length of at least 35 kb, and at least 15% of the genetically modified offspring animals contain the target gene integrated into the genomic locus. In some embodiments, the target gene has a length of at least 35 kb, and at least 20% of the genetically modified offspring animals contain the target gene integrated into the genomic locus. In some embodiments, the target gene has a length of at least 35 kb, and at least 25% of the genetically modified offspring animals contain the target gene integrated into the genomic locus. In some embodiments, the target gene has a length of at least 35 kb, and at least 30% of the genetically modified offspring animals contain the target gene integrated into the genomic locus.

[0066] Further embodiments Further embodiments are covered by the numbered paragraphs below. 1. A mammal whose genome contains a first Bxb1 attachment site and a second Bxb1 attachment site.

[0067] 2. The mammal according to paragraph 1, further comprising a polynucleotide encoding Bxb1 integrase, wherein the polynucleotide is optionally sandwiched between the first and second Bxb1 attachment sites.

[0068] 3. The first Bxb1 attachment site is the attP site, modified attP * Site, attB site, and modified attB * Selected from the body parts; The second Bxb1 attachment site is the attP site, modified attP * Site, attB site, and modified attB * Selected from the body parts; If necessary, the first and second Bxb1 attachment sites are heterogeneous to each other, and if necessary, the genome does not contain attR and / or attL sites, and is separated from each other by, for example, only 50 to 500 nucleotides. Mammals as described in paragraph 1 or 2.

[0069] 4. The attP region includes the sequence of sequence number 1, and the modified attP * The part includes the sequence of sequence number 7, the attB part includes the sequence of sequence number 2, and / or the modified attB * The part is the mammal described in paragraph 3, which includes the sequence of sequence number 8.

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

[0071] 6. The mammal described in any one of the preceding paragraphs, wherein the first and second Bxb1 attachment sites are located within the Safe Harbor locus, and optionally within the Rosa26 locus.

[0072] 7. The mammals described in any one of the paragraphs are rodents, and optionally mice.

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

[0074] 9. A mammalian embryo according to paragraph 8, further comprising (a) Bxb1 integrase or (b) a polynucleotide encoding Bxb1 integrase, wherein the polynucleotide is optionally sandwiched between the first and second Bxb1 attachment sites.

[0075] 10. The first Bxb1 attachment site is the attP site, modified attP * Site, attB site, and modified attB * Selected from the body parts; The second Bxb1 attachment site is the attP site, modified attP * Site, attB site, and modified attB * Selected from the body parts; If necessary, the first and second Bxb1 attachment sites are heterogeneous to each other, and if necessary, the genome does not contain attR and / or attL sites, and is separated from each other by, for example, only 50 to 500 nucleotides. Mammalian embryos as described in paragraph 8 or 9.

[0076] 11. The attP region includes the sequence of sequence number 1, and the modified attP * The part includes the sequence of sequence number 7, the attB part includes the sequence of sequence number 2, and / or the modified attB * The site is the mammalian embryo described in paragraph 10, containing the sequence of sequence number 8.

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

[0078] 13. The mammalian embryo described in any one of the preceding paragraphs, wherein the first and second Bxb1 attachment sites are located within the Safe Harbor locus, and optionally within the Rosa26 locus.

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

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

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

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

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

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

[0085] 20. The method according to paragraph 19, further comprising the step of screening the offspring mammals for the presence or absence of the sequence of the choice incorporated into the genome of the offspring mammals.

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

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

[0088] 23. The method according to any one of the paragraphs, wherein the desired array has a size of at least 10kb, at least 15kb, at least 20kb, at least 25kb, or at least 30kb.

[0089] 24. A method for generating a Bxb1 landing pad mammal, wherein the method is (a) The step of 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 region in the mammalian embryo, (iii) a first single-stranded DNA (ssDNA) donor comprising a first Bxb1 attachment site flanked by the left homology arm and the right homology arm; optionally (iv) a second guide RNA (gRNA) or a polynucleotide encoding a gRNA targeting a second genomic region in the mammalian embryo, and (v) a second ssDNA comprising a second Bxb1 attachment site flanked by the left homology arm and the right homology arm; and (b) A method comprising the step of transplanting the mammalian embryo cells into a pseudopregnant female mammal, wherein the pseudopregnant female mammal is capable of producing offspring mammals.

[0090] 25. The method according to paragraph 24, wherein the first ssDNA further includes a second Bxb1 attachment site upstream or downstream of the first Bxb1 attachment site, wherein both the first and second Bxb1 attachment sites are sandwiched between the left homology arm and the right homology arm.

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

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

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

[0094] 29. Mammals, including the mammalian embryos described in paragraphs 8-14.

[0095] 30. The mammals mentioned above are rodents, and optionally mice, as described in paragraph 29.

[0096] 31. Mammals that contain a Bxb1 attachment site within their genome.

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

[0098] 33. The Bxb1 attachment site is the attP site, modified attP * Part, attB part, or modified attB * The mammals described in paragraph 31 or 32, which are the body parts.

[0099] 34. The attP site includes the sequence of sequence number 1, and the modified attP * The part includes the sequence of sequence number 7, the attB part includes the sequence of sequence number 2, and / or the modified attB * The part is the mammal described in paragraph 33, which includes the sequence of sequence number 8.

[0100] 35. The mammal described in any one of the preceding paragraphs, wherein the Bxb1 attachment site is located within the Safe Harbor locus, or optionally within the Rosa26 locus.

[0101] 36. The mammals described in any one of the preceding paragraphs are rodents, and optionally mice.

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

[0103] 38. A mammalian embryo as described in paragraph 37, further comprising Bxb1 integrase or a polynucleotide encoding Bxb1 integrase.

[0104] 39. The Bxb1 attachment site is the attP site, modified attP * Part, attB part, or modified attB * The mammalian embryo, as described in paragraph 37 or 38.

[0105] 40. The attP site includes the sequence of sequence number 1, and the modified attP * The part includes the sequence of sequence number 7, the attB part includes the sequence of sequence number 2, and / or the modified attB * The site is the mammalian embryo described in paragraph 39, containing the sequence of sequence number 8.

[0106] 41. The mammalian embryo described in any one of the preceding paragraphs, wherein the Bxb1 attachment site is located within the Safe Harbor locus, or optionally within the Rosa26 locus.

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

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

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

[0110] 45. A method comprising the step of introducing a donor polynucleotide containing the sequence of interest and a congener Bxb1 attachment site into a mammalian embryo as described in any one of paragraphs 37-43.

[0111] 46. ​​The method according to 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 offspring mammals from the female mammal.

[0113] 48. The method according to paragraph 47, further comprising the step of screening the offspring mammals for the presence or absence of the sequence of interest incorporated into the genome of the offspring mammals.

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

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

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

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

[0118] 53. A method for generating a Bxb1 landing pad mammal, wherein the method is (a) In mammalian embryos, (i) Cas9 nuclease or polynucleotide encoding Cas9 nuclease, (ii) A guide RNA (gRNA) or a polynucleotide encoding a gRNA that targets a genomic locus in the mammalian embryo, and (iii) Single-stranded DNA (ssDNA) donors containing the Bxb1 attachment site sandwiched between the left homology arm and the right homology arm, The process of introducing; and (b) A step of transplanting the mammalian embryo cells into a pseudopregnant female mammal, wherein the pseudopregnant female mammal is capable of producing offspring mammals, A method of including.

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

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

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

[0122] 57. Mammals, including the mammalian embryos described in paragraphs 37-43.

[0123] 58. The mammals mentioned above are rodents, and optionally mice, as described in paragraph 57. [Examples]

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

[0125] Fertilized zygotes were isolated from C57BL / 6J mice. The pronuclei of these zygotes were microinjected with (1) Cas9 as mRNA, or protein, or both (concentrations ranging from 60–100 ng / μL for mRNA and 30–60 ng / μL for protein), (2) gRNA (concentration ranging from 30–50 ng / μL), and (3) an approximately 200 bp ssDNA oligo (SEQ ID NO: 3) targeting the Rosa26 locus. This ssDNA oligo contains 152 homologous base pairs (SEQ ID NOs: 4 and 5) adjacent to a 48-base pair Bxb1 attP site (SEQ ID NO: 2). The microinjected zygotes were transferred to pseudopregnant mice and retained until maturity. At approximately 2–3 weeks of age, tail biopsies were collected from the offspring and tested for accurate incorporation of the attP site by PCR and sequencing. Mice possessing the aforementioned Bxb1 attP site were created by mating Bxb1 attP mouse strains.

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

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

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

[0129] Example 2. Bxb1 Mouse - Two (attP) Landing Pads The inventors generated their version 2 recipient mice through sequential modification of the version 1 strain. To reiterate, the inventors used CRISPR / Cas9 and oligonucleotide donors to generate a second attP *The (modified) site was inserted approximately 240 bp away from the original attP site. At this modified site, dinucleotide pairing was changed from GT to GA. The addition of the second site allows for the elimination of the vector skeleton without first having to convert the donor construct into a minicircle (Figure 1B). Note that in this example, the two attachment sites were inserted sequentially, but they could also be inserted simultaneously using, for example, a single donor polynucleotide. The version 2 mouse described above also allows for the insertion of even larger tracts of DNA, because the essential attB site can be positioned in any vector (including BAC) simply by placing it adjacent to the desired region to be incorporated.

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

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

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

[0133] Four primary candidates were backcrossed to wild-type mice, and their N1 offspring were evaluated for germline transmission of both desired recombinant alleles (RECs) and optional off-target insertion (OTI) events. Offspring from two of the four primary lines also possessed undesirable random transgenic alleles. The OTI and REC alleles were isolated, indicating that they were not associated with the insertion events. Long-range PCR was performed to confirm that the integrated alleles were intact (Figure 4).

[0134] Since the above-mentioned transgene is known to be highly expressed in the human liver, RNA was isolated from the livers of mice from three of the four colonies (the colony from the fourth primary generation was not included in these tests as it had not yet reached a sufficient size). cDNA was prepared from the total RNA, and 1,270 bp PCR products were generated from each system (Figure 5). Then, Sanger sequencing of these products was used to confirm that the transcripts were indeed humanized alleles.

[0135] [ka] [ka]

[0136] [ka] [ka]

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

[0138] When used herein and in the claims, the indefinite articles “a” and “an” should be understood to mean “at least one” unless the opposite is expressly indicated.

[0139] Unless otherwise expressly indicated, it should also be understood that in any method claimed herein that includes more than one step or action, the order of the steps or actions of the Method is not necessarily limited to the order in which the steps or actions of the Method are described.

[0140] In the claims and the above specification, all transitional phrases such as “comprising,” “including,” “carrying,” “having,” “containing,” “involving,” “holding,” and “composed of” should be understood to mean “including but not limited to.” Only the transitional phrases “consisting of” and “consisting essentially of” are closed or semi-closed transitional phrases, respectively, as shown in Section 2111.03 of the U.S. Patent and Trademark Examination Manual.

[0141] The terms "about" and "substantially" preceding a number mean ±10% of the stated number.

[0142] Where a range of values ​​is provided, each value between the upper and lower limits of the above range is specifically intended and described herein. In certain embodiments, for example, the following items are provided: (Item 1) A mammal whose genome contains a first Bxb1 attachment site and a second Bxb1 attachment site. (Item 2) The mammal according to item 1, further comprising a polynucleotide encoding Bxb1 integrase, wherein the polynucleotide is optionally sandwiched between the first and second Bxb1 attachment sites. (Item 3) The first Bxb1 attachment site is the attP site, modified attP * Site, attB site, and modified attB * Selected from the body parts; The second Bxb1 attachment site is the attP site, modified attP * Site, attB site, and modified attB * Selected from the body parts; The mammals described in item 1, wherein the first and second Bxb1 attachment sites are heterogeneous with respect to each other as necessary. (Item 4) The attP region includes the sequence of sequence number 1, and the modified attP * The part includes the sequence of sequence number 7, the attB part includes the sequence of sequence number 2, and / or the modified attB * The part is the mammal described in item 3, which contains the sequence of sequence number 8. (Item 5) The first and second Bxb1 attachment sites are separated from each other by only 50 to 500 nucleotides, as described in item 1, in the mammal. (Item 6) The first and second Bxb1 attachment sites are located within the Safeharbour locus, and optionally the Rosa26 locus, in the mammals described in item 1. (Item 7) The aforementioned mammals are rodents, and, if necessary, mice, as listed in item 1. (Item 8) A mammalian embryo containing a first Bxb1 attachment site and a second Bxb1 attachment site within its genome. (Item 9) The mammalian embryo according to item 8, further comprising a polynucleotide encoding Bxb1 integrase, wherein the polynucleotide is optionally sandwiched between the first and second Bxb1 attachment sites. (Item 10) The first Bxb1 attachment site is the attP site, modified attP * Site, attB site, and modified attB * Selected from the body parts; The second Bxb1 attachment site is the attP site, modified attP * Site, attB site, and modified attB * Selected from the body parts; The mammalian embryo described in item 8, wherein the first and second Bxb1 attachment sites are heterogeneous with respect to each other, as necessary. (Item 11) The attP region includes the sequence of sequence number 1, and the modified attP * The part includes the sequence of sequence number 7, the attB part includes the sequence of sequence number 2, and / or the modified attB * The site is the mammalian embryo described in item 10, containing the sequence of sequence number 8. (Item 12) The mammalian embryo described in item 8, wherein the first and second Bxb1 attachment sites are separated from each other by only 50 to 500 nucleotide base pairs. (Item 13) The first and second Bxb1 attachment sites are located within the Safeharbour locus, and optionally within the Rosa26 locus, in the mammalian embryo described in item 8. (Item 14) The mammalian embryo described above is a single-cell embryo or a multicellular embryo, as described in item 8. (Item 15) The mammalian embryos mentioned above are rodent embryos, and, if necessary, mouse embryos, as described in item 8. (Item 16) A method comprising the steps of introducing (a) a donor polynucleotide containing a sequence of interest flanked by a first congener Bxb1 attachment site and a second congener Bxb1 attachment site, and (b) Bxb1 integrase or a polynucleotide encoding Bxb1 integrase into a mammalian embryo as described in any one of the preceding items. (Item 17) A method comprising the step of introducing a donor polynucleotide containing a target sequence flanked by a first congener Bxb1 attachment site and a second congener Bxb1 attachment site into a mammalian embryo as described in any one of the preceding items. (Item 18) The method according to item 16, further comprising the step of implanting the mammalian embryo into a pseudopregnant female mammal. (Item 19) The method of item 18, further comprising the step of collecting offspring mammals from the aforementioned female mammal. (Item 20) The method according to item 19, further comprising the step of screening the offspring mammals for the presence or absence of the sequence of the objective incorporated into the genome of the offspring mammals. (Item 21) The method according to item 16, wherein the donor polynucleotide, the Bxb1 integrase, and / or the polynucleotide encoding the Bxb1 integrase are introduced into the mammalian embryo via microinjection. (Item 22) The aforementioned target sequence is the method described in item 16, comprising the target gene. (Item 23) The method according to item 16, wherein the desired sequence has a size of at least 10kb, at least 15kb, at least 20kb, at least 25kb, or at least 30kb. (Item 24) A method for generating a Bxb1 landing pad mammal, wherein the method is (a) The step of introducing into a mammalian embryo a first single-stranded DNA (ssDNA) donor comprising (i) Cas9 nuclease or a polynucleotide encoding Cas9 nuclease, (ii) a first guide RNA (gRNA) or a polynucleotide encoding a gRNA targeting a first genomic region in the mammalian embryo, (iii) a first Bxb1 attachment site flanked by the left homology arm and the right homology arm; optionally (iv) a second guide RNA (gRNA) or a polynucleotide encoding a gRNA targeting a second genomic region in the mammalian embryo, and (v) a second ssDNA comprising a second Bxb1 attachment site flanked by the left homology arm and the right homology arm; and (b) A step of transplanting the mammalian embryo cells into a pseudopregnant female mammal, wherein the pseudopregnant female mammal is capable of producing offspring mammals, A method of including. (Item 25) The method according to item 24, wherein the first ssDNA further includes a second Bxb1 attachment site upstream or downstream of the first Bxb1 attachment site, where both the first and second Bxb1 attachment sites are sandwiched between the left homology arm and the right homology arm. (Item 26) The method according to item 24, wherein the mammalian embryo contains a polynucleotide encoding Bxb1 integrase, or step (a) further comprises the step of introducing a polynucleotide encoding Bxb1 integrase into the mammalian embryo. (Item 27) The method of item 24, further comprising the step of collecting the offspring mammals. (Item 28) The mammalian embryos are rodent embryos, and optionally mouse embryos, as described in item 24. (Item 29) Mammals, including mammalian embryos as described in item 8. (Item 30) The aforementioned mammals are rodents, and, if necessary, mice, as listed in item 29. (Item 31) Mammals that contain a Bxb1 attachment site within their genome. (Item 32) The mammal described in item 31, further comprising a polynucleotide encoding Bxb1 integrase. (Item 33) The Bxb1 attachment site is the attP site, modified attP * Part, attB part, or modified attB * The mammal described in item 31, which is the body part. (Item 34) The attP region includes the sequence of sequence number 1, and the modified attP * The part includes the sequence of sequence number 7, the attB part includes the sequence of sequence number 2, and / or the modified attB * The part is the mammal described in item 33, which contains the sequence of sequence number 8. (Item 35) The Bxb1 attachment site is located within the Safeharbour locus, or optionally the Rosa26 locus, in the mammals described in item 31. (Item 36) The aforementioned mammals are rodents, and, if necessary, mice, as listed in item 31. (Item 37) A mammalian embryo that contains a Bxb1 attachment site within its genome. (Item 38) A mammalian embryo as described in item 37, further comprising a polynucleotide encoding Bxb1 integrase. (Item 39) The Bxb1 attachment site is the attP site, modified attP * Part, attB part, or modified attB * The part in question is the mammalian embryo described in item 37. (Item 40) The attP region includes the sequence of sequence number 1, and the modified attP * The part includes the sequence of sequence number 7, the attB part includes the sequence of sequence number 2, and / or the modified attB * The site is the mammalian embryo described in item 39, containing the sequence of sequence number 8. (Item 41) The Bxb1 attachment site is located within the Safe Harbor locus, or optionally the Rosa26 locus, in the mammalian embryo described in item 37. (Item 42) The mammalian embryo described above is a single-cell embryo or a multicellular embryo, as described in item 37. (Item 43) The mammalian embryos mentioned above are rodent embryos, mouse embryos as appropriate, and mammalian embryos as described in item 37. (Item 44) A method comprising the steps of introducing (a) a donor polynucleotide containing the sequence of interest and a congener Bxb1 attachment site, and (b) Bxb1 integrase or a polynucleotide encoding Bxb1 integrase into a mammalian embryo as described in any one of items 37 to 43. (Item 45) A method comprising the step of introducing a donor polynucleotide containing the target sequence and a congener Bxb1 attachment site into a mammalian embryo as described in any one of items 37 to 43. (Item 46) The method according to item 44, further comprising the step of implanting the aforementioned mammalian embryo into a pseudopregnant female mammal. (Item 47) The method of item 46, further comprising the step of collecting offspring mammals from the female mammal. (Item 48) The method according to item 47, further comprising the step of screening the offspring mammals for the presence or absence of the sequence of the objective incorporated into the genome of the offspring mammals. (Item 49) The method according to item 44, wherein the donor polynucleotide, the Bxb1 integrase, and / or the polynucleotide encoding the Bxb1 integrase are introduced into the mammalian embryo via microinjection. (Item 50) The donor polynucleotide is a minicircle, as described in item 44. (Item 51) The aforementioned target sequence is the method described in item 44, comprising the target gene. (Item 52) The method according to item 44, wherein the desired sequence has a size of at least 3kb, at least 4kb, at least 5kb, at least 6kb, at least 7kb, at least 8kb, at least 9kb, or at least 10kb. (Item 53) A method for generating a Bxb1 landing pad mammal, wherein the method is (a) In mammalian embryos, (i) Cas9 nuclease or polynucleotide encoding Cas9 nuclease, (ii) A guide RNA (gRNA) or a polynucleotide encoding a gRNA that targets a genomic locus in the mammalian embryo, and (iii) Single-stranded DNA (ssDNA) donors containing the Bxb1 attachment site sandwiched between the left homology arm and the right homology arm, The process of introducing; and (b) A step of transplanting the mammalian embryo cells into a pseudopregnant female mammal, wherein the pseudopregnant female mammal is capable of producing offspring mammals, A method of including. (Item 54) The method according to item 53, wherein the mammalian embryo contains a polynucleotide encoding Bxb1 integrase, or step (a) comprises the step of introducing a polynucleotide encoding Bxb1 integrase into the mammalian embryo. (Item 55) The method of item 54, further comprising the step of collecting the offspring mammals. (Item 56) The mammalian embryos are rodent embryos, and optionally mouse embryos, as described in item 55. (Item 57) Mammals, including mammalian embryos as described in item 37. (Item 58) The aforementioned mammals are rodents, and, if necessary, mice, as listed in item 57.

Claims

1. Within the Rosa26 locus of that genome, there is a first Bxb1 attP attachment site and a second Bxb1 attP ＊ A mouse including an attachment site, wherein the first Bxb1 attP attachment site includes the sequence of sequence number 1, and the second Bxb1 attP* attachment site includes the sequence of sequence number 7.

2. The present invention further comprises a polynucleotide encoding Bxb1 integrase, wherein the polynucleotide optionally comprises the first Bxb1 attP attachment site and the second Bxb1 attP ＊ A mouse as described in claim 1, which is held between attachment sites.

3. The first Bxb1 attP attachment site and the second Bxb1 attP ＊ The mouse according to claim 1, wherein the attachment sites are separated from each other by only 50 to 500 nucleotides.

4. In the mouse embryo, (a) the first Bxb1 attB attachment site and the second Bxb1 attB ＊ A method comprising the step of introducing a donor polynucleotide containing the target sequence sandwiched between attachment sites, and (b) Bxb1 integrase or a polynucleotide encoding Bxb1 integrase, The mouse embryo has a first Bxb1 attP attachment site and a second Bxb1 attP within the Rosa26 locus of its genome. ＊ Including the attachment site, A method wherein the first Bxb1 attP attachment site includes the sequence of sequence number 1, and the second Bxb1 attP* attachment site includes the sequence of sequence number 7.

5. The method according to claim 4, further comprising the step of transplanting the mouse embryo into a pseudopregnant female mouse.

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

7. The method according to claim 6, further comprising the step of screening the offspring mice for the presence or absence of the desired sequence incorporated into the genome of the offspring mice.

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

9. The method according to claim 4, wherein the target sequence includes the target gene.

10. The method according to claim 4, wherein the desired array 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. A method for producing the mouse described in claim 1, wherein the method is: (a) a mouse embryo containing (i) Cas9 nuclease or a polynucleotide encoding Cas9 nuclease, (ii) a first guide RNA (gRNA) or a polynucleotide encoding a first gRNA targeting a first genomic region at the Rosa26 locus of the mouse embryo's genome, (iii) a first single-stranded DNA (ssDNA) donor including a first Bxb1 attP attachment site flanked by the left homology arm and the right homology arm; (iv) a second guide RNA (gRNA) or a polynucleotide encoding a second gRNA targeting a second genomic region at the Rosa26 locus of the mouse embryo's genome, and (v) a second Bxb1 attP attachment site flanked by the left homology arm and the right homology arm. ＊ A step of introducing a second ssDNA including the attachment site; and (b) A step of transplanting the mouse embryonic cells into a pseudopregnant female mouse, wherein the pseudopregnant female mouse is capable of producing offspring mice, It includes, A method wherein the first Bxb1 attP attachment site includes the sequence of sequence number 1, and the second Bxb1 attP* attachment site includes the sequence of sequence number 7.

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

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

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