WO2023177424A1 - Integration of large nucleic acids into genomes - Google Patents

Integration of large nucleic acids into genomes Download PDF

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Publication number
WO2023177424A1
WO2023177424A1 PCT/US2022/048841 US2022048841W WO2023177424A1 WO 2023177424 A1 WO2023177424 A1 WO 2023177424A1 US 2022048841 W US2022048841 W US 2022048841W WO 2023177424 A1 WO2023177424 A1 WO 2023177424A1
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Prior art keywords
polypeptide
sequence
cell
nucleic acid
genome
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PCT/US2022/048841
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English (en)
French (fr)
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Patrick Hsu
Matthew DURRANT
Alison FANTON
Chad MOON
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The Regents Of The University Of California
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Application filed by The Regents Of The University Of California filed Critical The Regents Of The University Of California
Priority to EP22932464.5A priority Critical patent/EP4493706A1/en
Priority to US18/846,744 priority patent/US20250207153A1/en
Publication of WO2023177424A1 publication Critical patent/WO2023177424A1/en

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    • C—CHEMISTRY; METALLURGY
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/90—Stable introduction of foreign DNA into chromosome
    • C12N15/902—Stable introduction of foreign DNA into chromosome using homologous recombination
    • C12N15/907—Stable introduction of foreign DNA into chromosome using homologous recombination in mammalian cells
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/102—Mutagenizing nucleic acids
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1241—Nucleotidyltransferases (2.7.7)
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1241—Nucleotidyltransferases (2.7.7)
    • C12N9/1247—DNA-directed RNA polymerase (2.7.7.6)
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1241—Nucleotidyltransferases (2.7.7)
    • C12N9/1276—RNA-directed DNA polymerase (2.7.7.49), i.e. reverse transcriptase or telomerase
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/22—Ribonucleases [RNase]; Deoxyribonucleases [DNase]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4702—Regulators; Modulating activity
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/20—Type of nucleic acid involving clustered regularly interspaced short palindromic repeats [CRISPR]
    • C—CHEMISTRY; METALLURGY
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/35—Nature of the modification
    • C12N2310/351—Conjugate
    • C12N2310/3519—Fusion with another nucleic acid

Definitions

  • compositions, methods, and systems for site-specific integration e.g., stable integration
  • a nucleic acid e.g., large nucleic acid
  • a cell e.g., a prokaryotic cell or a eukaryotic cell such as a plant cell or an animal cell.
  • compositions, methods, and systems for stably integrating one or more nucleic acids into a target site within the genome of a cell that include (a) a genome-editing system having (i) a polypeptide having a DNA binding domain and, optionally, a polymerase and (ii) a nucleic acid molecule including a guide sequence that is complementary to the target site and a nucleic acid sequence that encodes an acceptor attachment (attA) site, (b) a donor nucleic acid molecule including a nucleic acid cargo and a donor attachment (attD) site, and (c) an integrase (e.g., a large serine recombinase (LSR)) that can target the attA site and the attD site, where the integrase can facilitate recombination between the attA site and the attD site.
  • a genome-editing system having (i) a polypeptide having a DNA binding domain and, optionally, a
  • DLBs DNA double-stranded breaks
  • HR homologous recombination
  • Additional gene integration approaches such as transposase-mediated integration and lentiviral-mediated integration are not site-specific, and can result in variable gene expression, silenced gene expression, insertional mutagenesis, and/or other undesired events
  • compositions, methods, and systems for integrating e.g., stably integrating
  • nucleic acid e.g., large nucleic acid
  • a cell e.g., prokaryotic cell or a eukaryotic cell such as a plant cell or an animal cell.
  • compositions, methods, and systems for stably integrating one or more nucleic acids into a target site within the genome of a cell that include (a) a genome- editing system having (i) a polypeptide having a DNA binding domain and, optionally, a polymerase and (ii) a nucleic acid molecule including a guide sequence that is complementary to the target site and a nucleic acid sequence that encodes an attA site, (b) a donor nucleic acid molecule including a nucleic acid cargo and an attD site, and (c) an integrase (e.g., a LSR) that can target the attA site and the attD site.
  • a genome- editing system having (i) a polypeptide having a DNA binding domain and, optionally, a polymerase and (ii) a nucleic acid molecule including a guide sequence that is complementary to the target site and a nucleic acid sequence that encodes an attA site, (b) a donor
  • the genome-editing system when a genome-editing system provided herein is administered to a cell, the genome-editing system can insert the attA into the genome at the target site, and the integrase can facilitate recombination between the attA site and the attD site thereby integrating the donor nucleic acid molecule into the genome.
  • a genome-editing system e.g., a prime-editor system
  • an integrase e.g., a LSR
  • the compositions, methods, and systems provided herein not only provide precise control over the genomic integration site (thus reducing or eliminating the risk of insertional mutagenesis), but can allow the site-specific integration of large (e.g., multi-kilobase) nucleic acid cargos into the genome.
  • the compositions, methods, and systems provided herein can be applied to any appropriate gene editing application including, without limitation, gene therapy methods, gene transfer methods, production of transgenic plants, production of gene knock-out plants, and production of gene knock-out non-human animal models.
  • one aspect of this document features systems for stably integrating one or more nucleic acid sequences into a genome of a cell.
  • the systems can include, or consist essentially of, administering to a cell: (a) a genome-editing system that can insert an attA sequence into a target site within a genome of the cell; (b) a donor nucleic acid molecule comprising a nucleic acid cargo and a attD sequence; and (c) an integrase that targets the attA sequence and the attD site and can facilitate recombination between the attA site and the attD site.
  • the cell can be a mammalian cell (e.g., a human cell).
  • the cell can be a plant cell.
  • the cell can be a prokaryotic cell.
  • the genome-editing system can include (i) a polypeptide comprising a DNA binding domain and (ii) a nucleic acid comprising a guide sequence that is complementary to the target site within the genome and a sequence that encodes the attA sequence.
  • the DNA binding domain can be present in polypeptide selected from a Cas9 polypeptide, a Cas12 polypeptide, a zinc finger polypeptide, and a transcription activator-like effector (TALE) polypeptide.
  • TALE transcription activator-like effector
  • the polypeptide including the DNA binding domain can be a polymerase.
  • the polymerase can be a reverse transcriptase (RT) selected from the group consisting of a Moloney murine leukemia virus (M-MLV) RT, an avian myeloblastosis virus (AMV) RT, and a human immunodeficiency virus type 1 (HIV-1) RT.
  • RT reverse transcriptase
  • M-MLV Moloney murine leukemia virus
  • AMV avian myeloblastosis virus
  • HAV-1 human immunodeficiency virus type 1
  • the attA sequence can include from about 20 to about 100 nucleic acids.
  • the attA sequence can comprise, consist essentially of, or consist of any one of SEQ ID NOs: 11-84 and SEQ ID NO:254.
  • the attD sequence can include from about 20 to about 100 nucleic acids.
  • the attD sequence can comprise, consist essentially of, or consist of any one of SEQ ID NOs: 159-232.
  • the integrase can be a LSR.
  • the LSR can have an amino acid sequence containing a motif set forth in any one of SEQ ID NOs:233-245.
  • the LSR can have an amino acid sequence having at least 70% sequence identity to the sequence of any one of SEQ ID NOs: 85-158.
  • the LSR can have an amino acid sequence having at least 90% sequence identity to the sequence of any one of SEQ ID NOs: 85-158.
  • the LSR can comprise, consist essentially of, or consist of an amino acid sequence set forth in any one of SEQ ID NOs: 85-158.
  • the donor nucleic acid molecule can be from about 250 nt to about 30 kb.
  • this document features methods for stably integrating one or more nucleic acid sequences into a genome of a cell.
  • the methods can include, or consist essentially of, administering to a cell: (a) a genome-editing system that can insert an attA sequence into a target site within a genome of the cell; (b) a donor nucleic acid molecule comprising a nucleic acid cargo and an attD sequence; and (c) an integrase that targets the attA sequence and the attD site; where the genome- editing system integrates the attA sequence into the target site, and where the integrase facilitates recombination between the attA sequence and the attD sequence thereby integrating the donor nucleic acid molecule into the genome of the cell.
  • the cell can be a T cell, a natural killer (NK) cell, a non-human embryonic stem cell, an induced pluripotent stem cell (iPSC), a hematopoietic stem cell (HSC), a liver cell, a muscle cell, a monocytes, a B cell, a neuron, an astrocyte, or a microglial cell.
  • the cell can be a T cell and the nucleic acid sequence can encode a chimeric antigen receptor polypeptide or an engineered T cell receptor.
  • the cell is a NK cell and the nucleic acid sequence can encode a T cell receptor or an engineered natural killer cell receptor.
  • the cell can be a mammalian cell (e.g., a human cell).
  • the cell can be a plant cell.
  • the genome- editing system can include (i) a polypeptide comprising a DNA binding domain and (ii) a nucleic acid comprising a guide sequence that is complementary to the target site within the genome and a sequence that encodes the attA sequence.
  • the DNA binding domain can be present in a polypeptide selected from a Cas9 polypeptide, a Cas12 polypeptide, a zinc finger polypeptide, and a TALE polypeptide.
  • the polypeptide comprising the DNA binding domain can be a polymerase.
  • the polymerase can be an RT selected from the group consisting of a M-MLV RT, an AMV RT, and a HIV-1 RT.
  • the attA sequence can comprise, consist essentially of, or consist of any one of SEQ ID NOs: 11-84 and SEQ ID NO:254.
  • the attD sequence can comprise, consist essentially of, or consist of any one of SEQ ID NOs: 159-232.
  • the integrase can be a LSR.
  • the LSR can have an amino acid sequence containing a motif set forth in any one of SEQ ID NOs: 233 -245.
  • the LSR can have an amino acid sequence having at least 70% sequence identity to the sequence of any one of SEQ ID NOs:85-l 58.
  • the LSR can have an amino acid sequence having at least 90% sequence identity to the sequence of any one of SEQ ID NOs: 85- 158.
  • the LSR can comprise, consist essentially of, or consist of an amino acid sequence set forth in any one of SEQ ID NOs: 85- 158.
  • this document features methods for labelling a polypeptide encoded by an endogenous nucleic acid within a cell.
  • the methods can include, or consist essentially of, administering to a cell: (a) a genome-editing system that can insert an attA sequence into a target site within a genome of the cell; (b) a donor nucleic acid molecule comprising a nucleic acid cargo encoding a detectable label and an attD sequence; and (c) an integrase that targets the attA sequence and the attD site; where the genome-editing system integrates the attA sequence into the target site, and where the integrase facilitates recombination between the attA sequence and the attD sequence thereby integrating the donor nucleic acid molecule into the genome of the cell such that the cell expresses a fusion polypeptide including the polypeptide encoded by the endogenous nucleic acid fused to the detectable label.
  • the detectable label can be a HiBiT tag, a HaloTag, a Flag tag, a HA tag, a MS2/PP7 tag, a Sun/Moon tag, a poly(His) tag, a mCherry polypeptide, a green fluorescent polypeptide (GFP), a glutathione-S-transferase (GST), a luciferase, a horseradish peroxidase (HRP), an alkaline phosphatase (AP), or a apurinic/apyrimidinic endodeoxyribonuclease 2 (APEX2) polypeptide.
  • the cell can be a mammalian cell (e.g., a human cell).
  • the cell can be a plant cell.
  • the genome-editing system can include (i) a polypeptide comprising a DNA binding domain and (ii) a nucleic acid comprising a guide sequence that is complementary to the target site within the genome and a sequence that encodes the attA sequence.
  • the DNA binding domain can be present in a polypeptide selected from a Cas9 polypeptide, a Cas12 polypeptide, a zinc finger polypeptide, and a TALE polypeptide.
  • the polypeptide including the DNA binding domain can be a polymerase.
  • the polymerase can be a RT selected from the group consisting of a M-MLV RT, an AMV RT, and a HIV-1 RT.
  • the attA sequence can comprise, consist essentially of, or consist of any one of SEQ ID NOs: 11-84 and SEQ ID NO:254.
  • the attD sequence can comprise, consist essentially of, or consist of any one of SEQ ID NOs: 159-232.
  • the integrase can be a LSR.
  • the LSR can have an amino acid sequence containing a motif set forth in any one of SEQ ID NOs:233-245.
  • the LSR can have an amino acid sequence having at least 70% sequence identity to the sequence of any one of SEQ ID NOs: 85- 158.
  • the LSR can have an amino acid sequence having at least 90% sequence identity to the sequence of any one of SEQ ID NOs: 85-158.
  • the LSR can comprise, consist essentially of, or consist of an amino acid sequence set forth in any one of SEQ ID NOs: 85-158.
  • this document features methods for making a non-human transgenic organism.
  • the methods can include, or consist essentially of, administering to an embryonic stem cell of a non-human organism: (a) a genome-editing system that can insert an attA sequence into a target site within a genome of the embryonic stem cell; (b) a donor nucleic acid molecule comprising a transgene and an attD sequence; and (c) an integrase that targets the attA sequence and the attD site; where the genome-editing system integrates the attA sequence into the target site, and where the integrase facilitates recombination between the attA sequence and the attD sequence thereby integrating the donor nucleic acid molecule into the genome of the cell such that the cell expresses the transgene.
  • the cell can be a non- human mammalian cell.
  • the cell can be a plant cell.
  • the transgene expressed by the plant cell can be a herbicide resistance polypeptide.
  • the genome-editing system can include (i) a polypeptide comprising a DNA binding domain and (ii) a nucleic acid comprising a guide sequence that is complementary to the target site within the genome and a sequence that encodes the attA sequence.
  • the DNA binding domain can be present in a polypeptide selected from a Cas9 polypeptide, a Cas12 polypeptide, a zinc finger polypeptide, and a TALE polypeptide.
  • the polypeptide including the DNA binding domain can be a polymerase.
  • the polymerase can be an RT selected from the group consisting of a M-MLV RT, an AMV RT, and a HIV-1 RT.
  • the attA sequence can comprise, consist essentially of, or consist of any one of SEQ ID NOs: 11-84 and SEQ ID NO:254.
  • the attD sequence can comprise, consist essentially of, or consist of any one of SEQ ID NOs: 159-232.
  • the integrase can be a LSR.
  • the LSR can have an amino acid sequence containing a motif set forth in any one of SEQ ID NOs:233-245.
  • the LSR can have an amino acid sequence having at least 70% sequence identity to the sequence of any one of SEQ ID NOs: 85-158.
  • the LSR can have an amino acid sequence having at least 90% sequence identity to the sequence of any one of SEQ ID NOs: 85-158.
  • the LSR can comprise, consist essentially of, or consist of an amino acid sequence set forth in any one of SEQ ID NOs: 83-158.
  • this document features methods for making a non-human organism having reduced or eliminated levels of a polypeptide.
  • the methods can include, or consist essentially of, administering to an embryonic cell of a non-human organism: (a) a genome- editing system that can insert an attA sequence into a target site within a genome of the cell;
  • the nucleic acid cargo can include a stop codon.
  • the nucleic acid cargo can include a nucleic acid encoding a selectable marker.
  • the nucleic acid cargo can include nucleic acid encoding a detectable label.
  • the cell can be a non-human mammalian cell.
  • the cell can be a plant cell.
  • the genome-editing system can include (i) a polypeptide comprising a DNA binding domain and (ii) a nucleic acid comprising a guide sequence that is complementary to the target site within the genome and a sequence that encodes the attA sequence.
  • the DNA binding domain can be present in a polypeptide selected from a Cas9 polypeptide, a Cas12 polypeptide, a zinc finger polypeptide, and a TALE polypeptide.
  • the polypeptide including the DNA binding domain can be a polymerase.
  • the polymerase can be an RT selected from the group consisting of a M-MLV RT, an AMV RT, and a HIV-1 RT.
  • the attA sequence can comprise, consist essentially of, or consist of any one of SEQ ID NOs: 11-84 and SEQ ID NO:254.
  • the attD sequence can comprise, consist essentially of, or consist of any one of SEQ ID NOs: 159-232.
  • the integrase can be a LSR.
  • the LSR can have an amino acid sequence containing a motif set forth in any one of SEQ ID NOs:233- 245.
  • the LSR can have an amino acid sequence having at least 70% sequence identity to the sequence of any one of SEQ ID NOs: 85-158.
  • the LSR can have an amino acid sequence having at least 90% sequence identity to the sequence of any one of SEQ ID NOs: 85-158.
  • the LSR can comprise, consist essentially of, or consist of an amino acid sequence set forth in any one of SEQ ID NOs:85-158.
  • this document features methods for treating a mammal having a disease or disorder.
  • the methods can include, or consist essentially of, administering to a mammal having a disease or disorder: (a) a genome-editing system that can insert an attA sequence into a target site within a genome of a cell within the mammal; (b) a donor nucleic acid molecule comprising a nucleic acid cargo encoding a therapeutic gene product and a attD sequence; and (c) an integrase that targets the attA sequence and the attD site; where the genome-editing system integrates the attA sequence into the target site, and where the integrase facilitates recombination between the attA sequence and the attD sequence thereby integrating the donor nucleic acid molecule into the genome of the cell such that the cell produces the therapeutic gene product.
  • the therapeutic polypeptide can be an adenosine deaminase polypeptide, an ⁇ -1 antitrypsin polypeptide, a cystic fibrosis transmembrane conductance regulator (CFTR) polypeptide, a ⁇ -hemoglobin (HBB) polypeptide, an oculocutaneous albinism II (OCA2) polypeptide, a Huntingtin (HTT) polypeptide, a dystrophia myotonica-protein kinase (DMPK) polypeptide, a low-density lipoprotein receptor (LDLR) polypeptide, an apolipoprotein B (APOB) polypeptide, a neurofibromin 1 (NF1) polypeptide, a polycystic kidney disease 1 (PKD1) polypeptide, a polycystic kidney disease 2 (PKD2) polypeptide, a coagulation factor VIII (F8) polypeptide, a dystrophin (DMD) polypeptide, a phosphate-regulating end
  • the mammal can be a human.
  • the genome-editing system can include (i) a polypeptide comprising a DNA binding domain and (ii) a nucleic acid comprising a guide sequence that is complementary to the target site within the genome and a sequence that encodes the attA sequence.
  • the DNA binding domain can be present in a polypeptide selected from a Cas9 polypeptide, a Cas12 polypeptide, a zinc finger polypeptide, and a TALE polypeptide.
  • the polypeptide including the DNA binding domain can be a polymerase.
  • the polymerase can be an RT selected from the group consisting of a M-MLV RT, an AMV RT, and a HIV-1 RT.
  • the attA sequence can comprise, consist essentially of, or consist of any one of SEQ ID NOs: 11-84 and SEQ ID NO:254.
  • the attD sequence can comprise, consist essentially of, or consist of any one of SEQ ID NOs: 159-232.
  • the integrase can be a LSR.
  • the LSR can have an amino acid sequence containing a motif set forth in any one of SEQ ID NOs:233- 245.
  • the LSR can have of an amino acid sequence having at least 70% sequence identity to the sequence of any one of SEQ ID NOs: 85- 158.
  • the LSR can have an amino acid sequence having at least 90% sequence identity to the sequence of any one of SEQ ID NOs: 85-158.
  • the LSR can comprise, consist essentially of, or consist of an amino acid sequence set forth in any one of SEQ ID NOs:85-158.
  • Figures 1A - 1C Schematic images of mechanism for using a prime editor in combination with a LSR for programmable recombination of multiple kilobase cargo into the genome.
  • Figure 1 A contains a schematic for using prime editing with a LSR supplied independently (e.g., in trans).
  • Figure IB contains a schematic for using prime editing with integrase supplied fused to a component of a prime editor complex (e.g., in cis).
  • Figure 1C contains a schematic image showing guided delivery of the prime editor to a nucleic acid target site using pegRNA & ngRNA (left) or using two twinPE pegRNAs (right).
  • Figures 2A - 2B Schematic images of mechanism for using a prime editor in combination with a LSR for programmable recombination of multiple kilobase cargo into the genome.
  • Figure 1 A contains a schematic for using prime editing with a LSR supplied independently (e.g., in trans).
  • Figure IB contains
  • FIG. 2A contains a schematic of an exemplary method for a one- step transfection to deliver a prime editing system and a LSR to cells.
  • Figure 2B contains a schematic of an exemplary method for a two-step transfection to deliver a prime editing system and a LSR to cells.
  • Sequencing results demonstrating that prime editing can be used for targeted insertion of an attA site are, from top to bottom, SEQ ID NOs:246 to 249. Sequencing results of PaOl are, from top to bottom, SEQ ID NOs:250 and 251.
  • Figure 4 PCR validation of donor integration at an attA site.
  • Figures 5A - 5B Sequencing results demonstrating site-specific donor integration.
  • Figure 5A contains results using a Bxbl LSR (SEQ ID NO:252).
  • Figure 5B contains results using a PaOl LSR (SEQ ID NO:253).
  • Figure 7 qPCR analysis showing donor integration using 1 pegRNA.
  • Figures 8A - 8B ddPCR analysis showing donor integration.
  • Figure 8A Donor integration at the LMNB1 locus using 1 pegRNA.
  • Figure 8B Donor integration at the ACTB locus using 1 pegRNA.
  • Figure 9 qPCR analysis showing donor integration using 2 pegRNAs at the AAVS1 locus.
  • Figure 10 ddPCR analysis showing donor integration at the AAVS1 locus using 2 pegRNAs and LSR delivery in trans.
  • compositions, methods, and systems for integrating e.g., stably integrating
  • nucleic acid e.g., large nucleic acid
  • a cell e.g., a prokaryotic cell or a eukaryotic cell such as a plant cell or an animal cell.
  • this document provides systems for stably integrating one or more nucleic acids into a target site within the genome of a cell that include (a) a genome- editing system having (i) a polypeptide having a DNA binding domain and, optionally, a polymerase and (ii) a nucleic acid molecule including a guide sequence that is complementary to the target site and a nucleic acid sequence that encodes an attA site, (b) a donor nucleic acid molecule including a nucleic acid cargo and an attD site, and (c) an integrase (e.g., a LSR) that can target the attA site and the attD site.
  • a genome- editing system having (i) a polypeptide having a DNA binding domain and, optionally, a polymerase and (ii) a nucleic acid molecule including a guide sequence that is complementary to the target site and a nucleic acid sequence that encodes an attA site, (b) a donor nucleic
  • the genome- editing system can insert the attA into the genome at the target site, and the integrase can facilitate recombination between the attA site and the attD site thereby integrating the donor nucleic acid molecule into the genome.
  • compositions, methods, and systems provided herein e.g., a system for stably integrating one or more nucleic acids into a target site within the genome of a cell including (a) a genome- editing system that can insert an attA into a target site within a genome, (b) a donor nucleic acid molecule including a nucleic acid cargo and an attD site, and (c) an integrase (e.g., a LSR) that can target the attA site and the attD site) can be used to integrate (e.g., stably integrate) a nucleic acid into a genomes of any appropriate type of cell.
  • a genome- editing system that can insert an attA into a target site within a genome
  • a donor nucleic acid molecule including a nucleic acid cargo and an attD site
  • an integrase e.g., a LSR
  • compositions, methods, and systems provided herein can be used to integrate nucleic acid (e.g., large nucleic acid) into a prokaryotic cell. In some cases, the compositions, methods, and systems provided herein can be used to integrate nucleic acid (e.g., large nucleic acid) into a eukaryotic cell.
  • Examples of cell types that can have a nucleic acid stably integrated within the genome as described herein include, without limitation, stem cells (e.g., non-human embryonic stem cells, induced pluripotent stem cells (iPSCs), and hematopoietic stem cells (HSCs)), immune cells (e.g., T cells, macrophages, monocytes, B cells, and natural killer (NK) cells), liver cells, muscle cells, and brain cells (e.g., neurons, astrocytes, and microglia).
  • stem cells e.g., non-human embryonic stem cells, induced pluripotent stem cells (iPSCs), and hematopoietic stem cells (HSCs)
  • immune cells e.g., T cells, macrophages, monocytes, B cells, and natural killer (NK) cells
  • liver cells e.g., muscle cells, and brain cells (e.g., neurons, astrocytes, and microglia).
  • a system including (a) a genome-editing system that can insert an attA into a target site within a genome, (b) a donor nucleic acid molecule including a nucleic acid cargo and an attD site, and (c) an integrase (e.g., a LSR) that can target the attA site and the attD site) can be used to integrate (e.g., stably integrate) a nucleic acid into a plant cell or a mammalian cell.
  • a genome-editing system that can insert an attA into a target site within a genome
  • a donor nucleic acid molecule including a nucleic acid cargo and an attD site
  • an integrase e.g., a LSR
  • Examples of plants whose cells can have a nucleic acid stably integrated into a target site within the genome as described herein include, without limitation, wheat, corn, soy, rice, tobacco, Arabidopsis thaliana, cacao, banana, and sunflower.
  • Examples of mammals whose cells can have a nucleic acid stably integrated into a target site within the genome as described herein include, without limitation, humans, non- human primates such as chimpanzees and monkeys, dogs, cats, horses, cows, pigs, sheep, mice, rats, rabbits, guinea pigs, birds, fish (e.g., zebrafish (Danio rerio), medaka (Oryzias talipes), and turquoise killifish (Nothobranchius furzeri)), nematodes (e.g., Caenorhabditis elegans), and flies (e.g., Drosophila melanogaster).
  • fish e.g.
  • a genome-editing system in a system for stably integrating one or more nucleic acids into a target site within the genome of a cell can include (i) a polypeptide having a DNA binding domain and, optionally, a polymerase and (ii) a nucleic acid molecule including a guide sequence that is complementary to the target site and a nucleic acid sequence that encodes an attA site.
  • a polypeptide having a DNA binding domain and, optionally, a polymerase can include any appropriate DNA binding domain.
  • a DNA binding domain can be included in a polypeptide including a DNA binding domain.
  • a DNA binding domain can be included in a polypeptide including a DNA binding domain and including nuclease activity.
  • a DNA binding domain can be included in a polypeptide including a DNA binding domain and including nickase activity.
  • a DNA binding domain can be included in any appropriate polypeptide having nuclease activity.
  • nucleases include, without limitation, clustered regularly interspaced short palindromic repeat (CRISPR)-associated (Cas) polypeptides, zinc-finger nucleases (ZFNs), and transcription activator- like effector (TALE) polypeptides.
  • CRISPR clustered regularly interspaced short palindromic repeat
  • ZFNs zinc-finger nucleases
  • TALE transcription activator- like effector
  • a nuclease can be as described elsewhere (see, e.g., Urnov and Rebar, Biochem. Pharmacol., 64(5-6): 919-23 (2002); and Miller et al., Nat. Biotechnol., 29(2): 143-8 (2011)).
  • a DNA binding domain can be included a Cas polypeptide.
  • a Cas polypeptide can be any appropriate Cas polypeptide.
  • a Cas polypeptide can be isolated from an organism (e.g., a bacterium).
  • a Cas polypeptide can be a recombinant polypeptide.
  • a Cas polypeptide can be a synthetic polypeptide.
  • Cas polypeptides include, without limitation, Cas9 polypeptides (e.g., a Cas9 nuclease or a Cas9 nickase) such as Cas9 polypeptides from Streptococcus pyogenes (SpCas9 polypeptides) and Cas9 polypeptides from Staphylococcus aureus (SaCas9 polypeptides), Cas12 polypeptides (e.g., a Cas12 nuclease or a Cas12 nickase).
  • Cas9 polypeptides e.g., a Cas9 nuclease or a Cas9 nickase
  • a Cas polypeptide having a DNA binding domain can have any appropriate amino acid sequence.
  • Cas polypeptide sequences include, without limitation, amino acid sequences set forth in SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, or SEQ ID NO:6.
  • a Cas polypeptide having a DNA binding domain can have one or more amino acid modifications (e.g., one or more insertions, one or more deletions, and/or one or more substitutions) relative to a Cas polypeptide described herein (e.g., SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO: 5, and SEQ ID NO:6), provided the Cas polypeptide maintains the ability to cleave nucleic acid (e.g., maintains its nuclease activity and/or its nickase activity).
  • a Cas polypeptide having a DNA binding domain can have at least 70% (e.g., 70%, 75%, 80%, 85%, 90%, 93%, 95%, 96%, 97%, 98%, or 99%) sequence identity to any one of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6, provided the Cas polypeptide maintains the ability to cleave nucleic acid (e.g., maintains its nuclease activity and/or its nickase activity).
  • a Cas polypeptide having a DNA binding domain can include one or more additional polypeptides (e.g., a subcellular localization signal such as a nuclear localization signal (NLS)).
  • additional polypeptides e.g., a subcellular localization signal such as a nuclear localization signal (NLS)
  • a Cas polypeptide having a DNA binding domain can be as described elsewhere (see, e.g., Cong et al., Science 339(6121):819-23 (2013); Hsu et al., Nat. Biotechnol., 31:827-832 (2013); Jinek et al., Science, 337(6096): 816-21 (2012); Mali et al., Science, 339(6121):823-6 (2013); Nishimasu et al., Cell, 156(5):935-49 (2014); and Friedland et al., Genome Biol., 16:257 (2015)).
  • the polymerase can be any appropriate polymerase.
  • the polymerase can be a transcriptase (e.g., reverse transcriptase).
  • examples of polymerases include, without limitation, reverse transcriptases from a Moloney murine leukemia virus (M-MLV RTs), reverse transcriptases from an avian myeloblastosis virus (AMV RTs), and reverse transcriptases from a human immunodeficiency virus type 1 (HIV-1 RTs).
  • a polymerase can be as described elsewhere (see, e.g., Gao et al., bioRxiv doi.org/10.1101/2021.11.05.467423 (2021)).
  • a polymerase e.g., a reverse transcriptase
  • a polymerase can have any appropriate amino acid sequence. Examples of polymerase sequences include, without limitation, amino acid sequences set forth in SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, or SEQ ID NO:10.
  • a polymerase can have one or more amino acid modifications (e.g., one or more insertions, one or more deletions, and/or one or more substitutions) relative to a polymerase described herein (e.g, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO: 10), provided the polymerase maintains the ability to synthesize nucleic acid (e.g, maintains its polymerase activity).
  • amino acid modifications e.g., one or more insertions, one or more deletions, and/or one or more substitutions
  • a polymerase can have at least 70% (e.g, 70%, 75%, 80%, 85%, 90%, 93%, 95%, 96%, 97%, 98%, or 99%) sequence identity to any one of SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO: 10, provided the polymerase maintains the ability to synthesize nucleic acid (e.g, maintains its polymerase activity).
  • a polymerase e.g, a reverse transcriptase
  • a polymerase can include one or more additional polypeptides (e.g, a subcellular localization signal such as a NLS).
  • a polymerase e.g, a reverse transcriptase
  • a polymerase can be as described elsewhere (see, e.g, Baranauskas et al. Protein Eng. Des. Sei., 25(10):657-68 (2012); Anzalone et al. Nature, 576(7785): 149-157 (2019); loannidi et al, BioRxiv, DOI 10.1101/2021.11.01.466786 (2021); Perbal et al, Retrovirology, 5:49 (2008); Komshi et al, Biotechnol. Lett., 34(7): 1209-15 (2012); Hu et al. Cold Spring Harb. Perspect. Med, 2(10):a006882 (2012); UniProt Accession No. Q9WJQ2; and Japanese Patent Application Publication JP2012120506A).
  • a nucleic acid molecule including a guide sequence that is complementary to a target site and a nucleic acid sequence that encodes an attA site in a genome editing system provided herein can include any appropriate guide sequence.
  • a guide sequence can be a guide RNA (gRNA).
  • gRNA guide RNA
  • a guide sequence can be complementary to (e.g, can be designed to be complementary to) any appropriate target site.
  • a target site within a genome can be designed specifically for the desired outcome of the stably integrated nucleic acid. For example, when a stably integrated nucleic acid is designed to express a transgene, the target site can be designed such that expression of any endogenous nucleic acid is not disrupted.
  • the target site can be designed to be within the endogenous nucleic acid encoding the polypeptide (e.g., a coding sequence within that endogenous nucleic acid or a non-coding sequence within that endogenous nucleic acid).
  • a nucleic acid molecule including a guide sequence that is complementary to a target site and a nucleic acid sequence that encodes an attA site in a genome editing system provided herein can include any appropriate nucleic acid sequence that encodes an attA site.
  • An attA site is an attachment site for an integrase described herein.
  • an attA site can be an acceptor attachment site derived from a bacterial target sequence (e.g., an attB site).
  • an attA site can be acceptor attachment site derived from a phage target sequence (e.g., an attP site).
  • nucleic acid molecule including a guide sequence that is complementary to a target site and a nucleic acid sequence that encodes an attA site in a genome editing system can be engineered to include a nucleic acid sequence that encodes an attA site.
  • a nucleic acid sequence that encodes an attA site can be inserted into a nucleic acid using standard cloning or oligo capture techniques.
  • an attA site can be any appropriate length (e.g., can include any number of nucleotides).
  • an attA site can include from about 20 nucleotides to about 100 nucleotides (e.g., from about 20 nucleotides to about 90 nucleotides, from about 20 nucleotides to about 80 nucleotides, from about 20 nucleotides to about 70 nucleotides, from about 20 nucleotides to about 60 nucleotides, from about 20 nucleotides to about 50 nucleotides, from about 20 nucleotides to about 40 nucleotides, from about 20 nucleotides to about 30 nucleotides, from about 30 nucleotides to about 100 nucleotides, from about 40 nucleotides to about 100 nucleotides, from about 50 nucleotides to about 100 nucleotides, from about 60 nucleotides to about 100 nucleotides, from about 70 nucleotides
  • An attA site can include any appropriate nucleic acid sequence.
  • attA sequences include, without limitation, nucleic acid sequences set forth in SEQ ID NOs: 11-84 and SEQ ID NO:254.
  • an attA site can have one or more amino acid modifications (e.g., one or more insertions, one or more deletions, and/or one or more substitutions) relative to an attA site described herein (e.g., SEQ ID NOs: 11-84 and SEQ ID NO:254), provided the attA site maintains the ability to be recognized and recombined by an integrase (e.g., a LSR).
  • an integrase e.g., a LSR
  • an attA site can have at least 50% (e.g., 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 93%, 95%, 96%, 97%, 98%, or 99%) sequence identity to a sequence set forth in any one of SEQ ID NOs: 11-84 and SEQ ID NO:254, provided that the attA site maintains the ability to be recognized and recombined by an integrase (e.g., a LSR).
  • an integrase e.g., a LSR
  • an attA sequence can be as described elsewhere (see, e.g., U.S. Serial No. 63/275,288, filed on November 3, 2021).
  • a system for stably integrating one or more nucleic acids into a target site within the genome of a cell can include any appropriate integrase.
  • integrase refers to a polypeptide that can recognize an attA site and an attD site and can meditate nucleic acid recombination between the attA site and the attD site.
  • an integrase can be a serine recombinase such as a large serine recombinase (LSR).
  • an integrase can be a landing pad integrase.
  • an integrase can be a genome-targeting integrase.
  • an integrase can be a multi-targeting integrase.
  • an integrase can be linked (e.g., covalently linked) to a polypeptide comprising a DNA binding domain and, optionally, a polymerase.
  • a polymerase e.g., a polymerase for converting DNA to DNA to DNA.
  • an integrase and a polypeptide comprising a DNA binding domain and, optionally, a polymerase can be provided together (e.g., as a fusion polypeptide comprising both the integrase and the polypeptide comprising a DNA binding domain and, optionally, a polymerase).
  • the integrase when an integrase is linked to a polypeptide comprising a DNA binding domain and, optionally, a polymerase, the integrase can be linked directly to the polypeptide comprising a DNA binding domain and, optionally, a polymerase. In some cases when an integrase is linked to a polypeptide comprising a DNA binding domain and, optionally, a polymerase, the integrase can be linked to the polypeptide comprising a DNA binding domain and, optionally, a polymerase via a linker (e.g., a peptide linker).
  • a linker e.g., a peptide linker
  • an integrase e.g., serine recombinase such as a LSR
  • an integrase can include any appropriate amino acid sequence.
  • an integrase can have an amino acid sequence that includes one or more of the motifs set forth in SEQ ID NOs:233-245 (written in the common Prosite format).
  • Examples of integrase sequences include, without limitation, amino acid sequences set forth in SEQ ID NOs:85-158.
  • an integrase can have one or more amino acid modifications (e.g., one or more insertions, one or more deletions, and/or one or more substitutions) relative to an integrase described herein (e.g., SEQ ID NOs: 85-158), provided the integrase maintains the ability to recognize and recombine an attA site and an attD site.
  • one or more amino acid modifications e.g., one or more insertions, one or more deletions, and/or one or more substitutions
  • an integrase can have at least 70% (e.g., 70%, 75%, 80%, 85%, 90%, 93%, 95%, 96%, 97%, 98%, or 99%) sequence identity to a sequence set forth in any one of SEQ ID NOs: 85- 158, provided that the integrase site maintains the ability to recognize and recombine an attA site and an attD site.
  • 70% e.g., 70%, 75%, 80%, 85%, 90%, 93%, 95%, 96%, 97%, 98%, or 99%
  • an integrase e.g., serine recombinase such as a LSR
  • a LSR serine recombinase
  • an integrase can be as described elsewhere (see, e.g., U.S. Serial No. 63/275,288, filed on November 3, 2021).
  • a donor nucleic acid molecule including a nucleic acid cargo and an attD site in a system for stably integrating one or more nucleic acids into a target site within the genome of a cell can be any appropriate donor nucleic acid molecule.
  • a donor nucleic acid molecule can be a linear nucleic acid molecule.
  • a donor nucleic acid molecule can be a circular nucleic acid molecule (e.g., a plasmid or a minicircle).
  • a donor nucleic acid molecule can be any appropriate size (e.g., can include any number of nucleotides).
  • a donor nucleic acid molecule is from about 0.25 kb (250 nucleotides (nt)) to about 30 kb (e.g., from about 0.5 kb to about 30 kb, from about 1 kb to about 30 kb, from about 2 kb to about 30 kb, from about 5 kb to about 30 kb, from about 7 kb to about 30 kb, from about 10 kb to about 30 kb, from about 12 kb to about 30 kb, from about 15 kb to about 30 kb, from about 18 kb to about 30 kb, from about 20 kb to about 30 kb, from about 22 kb to about 30 kb, from about 25 kb to about 30 kb, from about 27 kb to about 30 kb, from about 0.25 kb to about 30
  • a donor nucleic acid molecule can include any appropriate nucleic acid cargo.
  • a nucleic acid cargo can be any polynucleotide sequence that can be delivered to and inserted into a target site within the genome of a cell using a system for stably integrating one or more nucleic acids into a target site within the genome of a cell provided herein.
  • a nucleic acid cargo can include a nucleic acid encodes a gene product (e.g., a polypeptide or a non-coding RNA).
  • a nucleic acid cargo in a donor nucleic acid molecule of a system for stably integrating one or more nucleic acids into a target site within the genome of a cell can encode a polypeptide.
  • polypeptides that can be encoded by a nucleic acid cargo in a donor nucleic acid molecule include, without limitation, detectable labels (e.g., peptide tags, fluorescent polypeptides, and enzymes), therapeutic polypeptides and biologically active fragments thereof (e.g., polypeptides useful for treating a diseases and/or condition) such as transcription factors, genome engineering systems, and polypeptides for eliciting an immune response, antibodies.
  • a nucleic acid cargo in a donor nucleic acid molecule of a system for stably integrating one or more nucleic acids into a target site within the genome of a cell can encode a RNA (e.g., a non- coding RNA).
  • RNA examples include, without limitation, tRNA, rRNA, inhibitory RNAs (e.g., antisense RNAs, microRNAs (miRNAs), small interfering RNAs (siRNAs), short hairpin RNAs (shRNAs), and agomiRs), antagomiRs, aptamers, and long non-coding RNAs (IncRN As).
  • inhibitory RNAs e.g., antisense RNAs, microRNAs (miRNAs), small interfering RNAs (siRNAs), short hairpin RNAs (shRNAs), and agomiRs
  • antagomiRs aptamers
  • aptamers examples include, without limitation, tRNA, rRNA, inhibitory RNAs (e.g., antisense RNAs, microRNAs (miRNAs), small interfering RNAs (siRNAs), short hairpin RNAs (shRNAs), and agomiRs),
  • the donor nucleic acid also can include one or more regulatory elements operably linked to the nucleic acid encoding the gene product.
  • regulatory elements can include promoter sequences, enhancer sequences, response elements, signal peptides, internal ribosome entry sequences, polyadenylation signals, terminators, and inducible elements that modulate expression (e.g., transcription or translation) of a nucleic acid.
  • the choice of regulatory element(s) can depend on several factors, including, without limitation, inducibility, targeting, and the level of expression desired.
  • a promoter can be included in a donor nucleic acid molecule to facilitate transcription of a nucleic acid cargo encoding a gene product.
  • a promoter can be a naturally occurring promoter or a recombinant promoter.
  • a promoter can be ubiquitous or inducible (e.g., in the presence of tetracycline), and can affect the expression of a nucleic acid encoding a gene product in a general or tissue-specific manner.
  • promoters include, without limitation, human ubiquitin C promoters, human synapsin 1 gene promoters, human glial fibrillary acidic protein promoters, promoters with tetracycline response elements, human elongation factor- 1 alpha promoters, cytomegalovirus promoters, CAG promoters, simian vacuolating virus 40 promoters, phosphoglycerate kinase gene promoters, and Ca 2+ /calmodulin-dependent protein kinase II promoters.
  • a donor nucleic acid molecule can contain a promoter and nucleic acid encoding a polypeptide.
  • the promoter is operably linked to a nucleic acid encoding a polypeptide such that it drives expression of the polypeptide in cells.
  • a donor nucleic acid molecule can contain a promoter and nucleic acid encoding a non-coding RNA.
  • a donor nucleic acid molecule can include one or more additional nucleic acid elements.
  • a donor nucleic acid molecule can be flanked by inverted terminal repeats (ITRs; e.g., AAV ITRs).
  • a donor nucleic acid molecule can include an attD site and, optionally, nucleic acid cargo that can encode a gene product, and can lack any other nucleic acid elements.
  • bacterial elements such as an origin of replication (Ori) site can be removed from the plasmid.
  • other coding sequences such as nucleic acid encoding a selectable marker such as an antibiotic resistance gene can be removed from the plasmid.
  • a donor nucleic acid molecule can include any appropriate attD site.
  • an attD site can be donor attachment site derived from a phage donor sequence (e.g., an attP site).
  • an attD site can be any appropriate length (e.g., can include any number of nucleotides).
  • an attD site can include from about 20 nucleotides to about 100 nucleotides (e.g., from about 20 nucleotides to about 90 nucleotides, from about 20 nucleotides to about 80 nucleotides, from about 20 nucleotides to about 70 nucleotides, from about 20 nucleotides to about 60 nucleotides, from about 20 nucleotides to about 50 nucleotides, from about 20 nucleotides to about 40 nucleotides, from about 20 nucleotides to about 30 nucleotides, from about 30 nucleotides to about 100 nucleotides, from about 40 nucleotides to about 100 nucleotides, from about 50 nucleotides to about 100 nucleotides, from about 60 nucleotides to about 100 nucleotides, from about 70 nucleotides
  • an attD site can include from about 25 nucleotides to about 45 nucleotides.
  • An attD site can include any appropriate nucleic acid sequence.
  • Examples of attD sequences include, without limitation, nucleic acid sequences set forth in SEQ ID NOs: 159- 232.
  • an attD site can have one or more amino acid modifications (e.g., one or more insertions, one or more deletions, and/or one or more substitutions) relative to an attD site described herein (e.g., SEQ ID NOs: 159-232), provided the attD site maintains the ability to be recognized and recombined by an integrase (e.g., an LSR).
  • an integrase e.g., an LSR
  • an attD site can have at least 50% (e.g., 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 93%, 95%, 96%, 97%, 98%, or 99%) sequence identity to a sequence set forth in any one of SEQ ID NOs: 159-232, provided that the attD site maintains the ability to be recognized and recombined by an integrase (e.g., a LSR).
  • an integrase e.g., a LSR
  • an attD sequence can be as described elsewhere (see, e.g., U.S. Serial No. 63/275,288, filed on November 3, 2021).
  • Also provided herein are methods for using systems for stably integrating one or more nucleic acids into a target site within the genome of a cell e.g., systems including (a) a genome-editing system that can insert an attA into a target site within a genome, (b) a donor nucleic acid molecule including a nucleic acid cargo and an attD site, and (c) an integrase (e.g., a LSR) that can target the attA site and the attD site).
  • systems including (a) a genome-editing system that can insert an attA into a target site within a genome, (b) a donor nucleic acid molecule including a nucleic acid cargo and an attD site, and (c) an integrase (e.g., a LSR) that can target the attA site and the attD site).
  • a genome-editing system that can insert an attA into a target site within a genome
  • a system for stably integrating one or more nucleic acids into a target site within the genome of a cell can be delivered to a cell to stably integrate a nucleic acid into the genome of the cell.
  • a system including (a) a genome-editing system that can insert an attA into a target site within a genome, (b) a donor nucleic acid molecule including a nucleic acid cargo and an attD site, and (c) an integrase (e.g., a LSR) that can target the attA site and the attD site can be delivered to a cell to stably integrate the nucleic acid cargo into the genome of the cell.
  • an integrase e.g., a LSR
  • the components of a system for stably integrating one or more nucleic acids into a target site within the genome of a cell provided herein can be delivered to a cell in vitro. In some cases, the components of a system for stably integrating one or more nucleic acids into a target site within the genome of a cell provided herein can be delivered to a cell ex vivo. In some cases, the components of a system for stably integrating one or more nucleic acids into a target site within the genome of a cell provided herein can be delivered to a cell in vivo.
  • any appropriate method can be used to deliver components of a system for stably integrating one or more nucleic acids into a target site within the genome of a cell provided herein (e.g., systems including (a) a genome-editing system that can insert an attA into a target site within a genome, (b) a donor nucleic acid molecule including a nucleic acid cargo and an attD site, and (c) an integrase (e.g., a LSR) that can target the attA site and the attD site) to cells (e.g., cells within a living mammal).
  • a genome-editing system that can insert an attA into a target site within a genome
  • a donor nucleic acid molecule including a nucleic acid cargo and an attD site
  • an integrase e.g., a LSR
  • a genome-editing system that can insert an attA into a target site within a genome can be delivered to a cell as a complex including (i) a polypeptide having a DNA binding domain and, optionally, a polymerase and (ii) a nucleic acid molecule including a guide sequence that is complementary to the target site and a nucleic acid sequence that encodes an attA site.
  • a genome-editing system that can insert an attA into a target site within a genome can be delivered to a cell as a nucleic acid encoding the genome-editing system (e.g., a vector designed to express the genome-editing system) such that a complex including (i) a polypeptide having a DNA binding domain and, optionally, a polymerase and (ii) a nucleic acid molecule including a guide sequence that is complementary to the target site and a nucleic acid sequence that encodes an attA site is formed within the cell.
  • an integrase that can target the attA site and the attD site can be delivered to a cell as a polypeptide.
  • an integrase that can target the attA site and the attD site can be delivered to a cell as a nucleic acid encoding the integrase (e.g., a vector designed to express the integrase).
  • a donor nucleic acid molecule including a nucleic acid cargo and an attD site can be delivered to a cell as a linear nucleic acid molecule.
  • a donor nucleic acid molecule including a nucleic acid cargo and an attD site can be delivered to a cell as a circular nucleic acid (e.g., a vector).
  • a genome-editing system that can insert an attA into a target site within a genome and an integrase that can target the attA site and the attD site can be delivered to a cell as polypeptides, and a donor nucleic acid molecule including a nucleic acid cargo and an attD site are administered to cell can be delivered to the cell in the form of a vector (e.g., a non-viral vector).
  • a vector e.g., a non-viral vector
  • nucleic acid encoding a genome-editing system that can insert an attA into a target site within a genome, nucleic acid encoding an integrase that can target the attA site and the attD site, and a donor nucleic acid molecule including a nucleic acid cargo and an attD site can be delivered to a cell in the form of one or more vectors (e.g., one or more viral vectors and/or one or more non- viral vectors).
  • vectors e.g., one or more viral vectors and/or one or more non- viral vectors.
  • nucleic acid encoding an integrase that can target the attA site and the attD site, and/or a donor nucleic acid molecule including a nucleic acid cargo and an attD site is a viral vector
  • any appropriate viral vector can be used.
  • a viral vector can be derived from a positive-strand virus or a negative-strand virus.
  • a viral vector can be derived from a virus with a DNA genome or a RNA genome.
  • a viral vector can be a chimeric viral vector.
  • a viral vector can infect dividing cells.
  • a viral vector can infect non-dividing cells.
  • virus-based vectors that can be used to deliver nucleic acid encoding a genome-editing system that can insert an attA into a target site within a genome, nucleic acid encoding an integrase that can target the attA site and the attD site, and/or a donor nucleic acid molecule including a nucleic acid cargo and an attD site include, without limitation, virus-based vectors based on adenoviruses, adeno-associated viruses (AAVs), Sendai viruses, retroviruses, or lentiviruses.
  • AAVs adeno-associated viruses
  • Sendai viruses retroviruses
  • lentiviruses Sendai viruses
  • nucleic acid encoding an integrase that can target the attA site and the attD site, and/or a donor nucleic acid molecule including a nucleic acid cargo and an attD site is a non-viral vector
  • any appropriate non-viral vector can be used.
  • a non-viral vector can be an expression plasmid (e.g., a cDNA expression vector).
  • nucleic acid encoding a genome-editing system that can insert an attA into a target site within a genome and/or nucleic acid encoding an integrase is delivered to a cell
  • the nucleic acid can be used for transient expression of a genome-editing system and/or an integrase or for stable expression of a genome-editing system and/or an integrase.
  • nucleic acid in cases where a nucleic acid encoding a genome-editing system that can insert an attA into a target site within a genome and/or nucleic acid encoding an integrase is used to deliver a genome-editing system and/or an integrase to a cell, the nucleic acid also can include one or more regulatory elements operably linked to the nucleic acid encoding the genome-editing system and/or the integrase.
  • regulatory elements can include promoter sequences, enhancer sequences, response elements, signal peptides, internal ribosome entry sequences, polyadenylation signals, terminators, and inducible elements that modulate expression (e.g., transcription or translation) of a nucleic acid.
  • a promoter can be included in a nucleic acid encoding a genome-editing system that can insert an attA into a target site within a genome and/or nucleic acid encoding an integrase to facilitate transcription of the genome- editing system and/or the integrase.
  • a promoter can be a naturally occurring promoter or a recombinant promoter.
  • a promoter can be ubiquitous or inducible (e.g., in the presence of tetracycline), and can affect the expression of a nucleic acid encoding a gene product in a general or tissue-specific manner.
  • promoters include, without limitation, human ubiquitin C promoters, human synapsin 1 gene promoters, human glial fibrillary acidic protein promoters, promoters with tetracycline response elements, human elongation factor- 1 alpha promoters, cytomegalovirus promoters, CAG promoters, simian vacuolating virus 40 promoters, phosphoglycerate kinase gene promoters, and Ca 2+ /calmodulin-dependent protein kinase II promoters.
  • operably linked refers to positioning of a regulatory element in a donor nucleic acid molecule relative to a nucleic acid encoding a genome- editing system that can insert an attA into a target site within a genome and/or nucleic acid encoding an integrase in such a way as to permit or facilitate expression of the encoded genome-editing system and/or the encoded integrase.
  • a nucleic acid encoding a genome-editing system that can insert an attA into a target site within a genome can contain a promoter and nucleic acid encoding a genome- editing system.
  • the promoter is operably linked to a nucleic acid encoding a genome-editing system that can insert an attA into a target site within a genome such that it drives expression of the genome- editing system in cells.
  • a nucleic acid encoding an integrase can contain a promoter and nucleic acid encoding the integrase.
  • the promoter is operably linked to a nucleic acid encoding an integrase such that it drives expression of the integrase in cells.
  • the components of a system for stably integrating one or more nucleic acids into a target site within the genome of a cell e.g., systems including (a) a genome- editing system that can insert an attA into a target site within a genome, (b) a donor nucleic acid molecule including a nucleic acid cargo and an attD site, and (c) an integrase (e.g., a LSR) that can target the attA site and the attD site) can be delivered to cells (e.g., cells within a living mammal) at the same time.
  • a genome- editing system that can insert an attA into a target site within a genome
  • a donor nucleic acid molecule including a nucleic acid cargo and an attD site e.g., a LSR
  • an integrase e.g., a LSR
  • a system for stably integrating one or more nucleic acids into a target site within the genome of a cell can be delivered to a cell in a single composition containing (a) a genome-editing system that can insert an attA into a target site within a genome (or nucleic acid encoding such a genome-editing system), (b) a donor nucleic acid molecule including a nucleic acid cargo and an attD site, and (c) an integrase (e.g., a LSR) that can target the attA site and the attD site (or nucleic acid encoding such an integrase).
  • a genome-editing system that can insert an attA into a target site within a genome (or nucleic acid encoding such a genome-editing system)
  • a donor nucleic acid molecule including a nucleic acid cargo and an attD site e.g., a LSR
  • a system for stably integrating one or more nucleic acids into a target site within the genome of a cell can be delivered to a cell in a single composition containing (a) a genome-editing system that can insert an attA into a target site within a genome linked (e.g., covalently linked as a fusion polypeptide) to (b) a donor nucleic acid molecule including a nucleic acid cargo and an attD site, and containing (c) an integrase (e.g., a LSR) that can target the attA site and the attD site.
  • a genome-editing system that can insert an attA into a target site within a genome linked (e.g., covalently linked as a fusion polypeptide) to (b) a donor nucleic acid molecule including a nucleic acid cargo and an attD site, and containing (c) an integrase (e.g., a LSR) that can target the
  • a system for stably integrating one or more nucleic acids into a target site within the genome of a cell can be delivered to a cell in a single composition containing a nucleic acid encoding a polypeptide (e.g., a fusion polypeptide) including both a genome-editing system that can insert an attA into a target site within a genome linked and an integrase (e.g., a LSR) that can target the attA site and an attD site, and a donor nucleic acid molecule including a nucleic acid cargo and the attD site.
  • a polypeptide e.g., a fusion polypeptide
  • a genome-editing system that can insert an attA into a target site within a genome linked and an integrase (e.g., a LSR) that can target the attA site and an attD site
  • a donor nucleic acid molecule including a nucleic acid cargo and the at
  • the components of a system for stably integrating one or more nucleic acids into a target site within the genome of a cell provided herein (e.g., systems including (a) a genome- editing system that can insert an attA into a target site within a genome, (b) a donor nucleic acid molecule including a nucleic acid cargo and an attD site, and (c) an integrase (e.g., a LSR) that can target the attA site and the attD site) can be delivered to cells (e.g., cells within a living mammal) independently.
  • a genome- editing system that can insert an attA into a target site within a genome
  • a donor nucleic acid molecule including a nucleic acid cargo and an attD site
  • an integrase e.g., a LSR
  • a system for stably integrating one or more nucleic acids into a target site within the genome of a cell can be delivered to a cell as in a first composition containing (a) a genome-editing system that can insert an attA into a target site within a genome (or nucleic acid encoding such a genome-editing system), and a second composition containing (b) a donor nucleic acid molecule including a nucleic acid cargo and an attD site and (c) an integrase (e.g., a LSR) that can target the attA site and the attD site (or nucleic acid encoding such an integrase).
  • a first composition containing (a) a genome-editing system that can insert an attA into a target site within a genome (or nucleic acid encoding such a genome-editing system), and a second composition containing (b) a donor nucleic acid molecule including a nucleic acid cargo and an at
  • a system for stably integrating one or more nucleic acids into a target site within the genome of a cell can be delivered to a cell as in a first composition containing (a) a genome-editing system that can insert an attA into a target site within a genome (or nucleic acid encoding such a genome- editing system) and (b) a donor nucleic acid molecule including a nucleic acid cargo and an attD site, and a second composition containing (c)an integrase (e.g., a LSR) that can target the attA site and the attD site (or nucleic acid encoding such an integrase).
  • a first composition containing (a) a genome-editing system that can insert an attA into a target site within a genome (or nucleic acid encoding such a genome- editing system) and (b) a donor nucleic acid molecule including a nucleic acid cargo and an attD site, and a
  • a system for stably integrating one or more nucleic acids into a target site within the genome of a cell can be delivered to a cell as in a first composition containing (a) a genome-editing system that can insert an attA into a target site within a genome (or nucleic acid encoding such a genome-editing system), a second composition containing (b) a donor nucleic acid molecule including a nucleic acid cargo and an attD site, and a third composition containing (c) an integrase (e.g., a LSR) that can target the attA site and the attD site (or nucleic acid encoding such an integrase).
  • a first composition containing (a) a genome-editing system that can insert an attA into a target site within a genome (or nucleic acid encoding such a genome-editing system), a second composition containing (b) a donor nucleic acid molecule including a nucleic
  • the methods and materials provided herein e.g., systems including (a) a genome- editing system that can insert an attA into a target site within a genome, (b) a donor nucleic acid molecule including a nucleic acid cargo and an attD site, and (c) an integrase (e.g., a LSR) that can target the attA site and the attD site) can be used for labelling a gene product (e.g., a polypeptide or a non-coding RNA) within a cell (e.g., a plant cell or a mammalian cell).
  • a gene product e.g., a polypeptide or a non-coding RNA
  • the methods and materials provided herein can be used to label a gene product encoded by an endogenous nucleic acid within a cell (e.g., a prokaryotic cell or a eukaryotic cell such as a plant cell or an animal cell).
  • a cell e.g., a prokaryotic cell or a eukaryotic cell such as a plant cell or an animal cell.
  • a gene product within a cell can be labeled by delivering a system for stably integrating one or more nucleic acids into a target site within the genome of a cell provided herein (e.g., a system including (a) a genome- editing system that can insert an attA into a target site within a genome, (b) a donor nucleic acid molecule including a nucleic acid cargo and an attD site, and (c) an integrase (e.g., a LSR) that can target the attA site and the attD site) to a cell (e.g., a plant cell or a mammalian cell) to stably integrate a nucleic acid encoding a detectable label in-frame with an endogenous nucleic acid encoding a target gene product such that the encoded target gene product is fused to the detectable label.
  • a genome-editing system that can insert an attA into a target site within a genome that is in-frame with an endogenous nucleic acid encoding a target gene product
  • a donor nucleic acid molecule including a nucleic acid cargo encoding a detectable label and an attD site
  • an integrase that can target the attA site and the attD site can be delivered to a cell to stably integrate the nucleic acid cargo encoding the detectable label into the genome such that the encoded target gene product is fused to the detectable label.
  • a nucleic acid cargo encoding a detectable label is stably integrated into the genome of a cell (e.g., a plant cell or a mammalian cell) to label a target polypeptide within the cell
  • a detectable label can be used.
  • detectable labels include, without limitation, luminescent tags (e.g., HiBiT), peptide tags (e.g., HaloTag, Flag tags, HA tags, MS2/PP7 tags, Sun/Moon tags, and poly(His) tags), fluorescent polypeptides (e.g., mCherry and green fluorescent polypeptides (GFPs; e.g., mNeonGreen)), and enzymes (e.g., glutathione-S-transferases (GSTs), luciferases, horseradish peroxidases (HRPs), alkaline phosphatases (APs), and apurinic/apyrimidinic endodeoxyribonuclease 2 (APEX2) polypeptides).
  • luminescent tags e.g., HiBiT
  • peptide tags e.g., HaloTag, Flag tags, HA tags, MS2/PP7 tags, Sun/Moon tags, and poly(His) tags
  • a nucleic acid cargo encoding a detectable label can be integrated into the genome upstream of an endogenous nucleic acid encoding a target polypeptide such that the detectable label is fused to the N-terminus of the target polypeptide.
  • a nucleic acid cargo encoding a detectable label can be integrated into the genome downstream of an endogenous nucleic acid encoding a target polypeptide such that the detectable label is fused to the C-terminus of the target polypeptide.
  • the methods and materials provided herein e.g., systems including (a) a genome- editing system that can insert an attA into a target site within a genome, (b) a donor nucleic acid molecule including a nucleic acid cargo and an attD site, and (c) an integrase (e.g., a LSR) that can target the attA site and the attD site) can be used to increase expression of a polypeptide within a cell (e.g., a plant cell or a mammalian cell).
  • a cell e.g., a plant cell or a mammalian cell.
  • the methods and materials provided herein can be used to increase expression of a polypeptide encoded by an endogenous nucleic acid within a cell (e.g., a prokaryotic cell or a eukaryotic cell such as a plant cell or an animal cell).
  • a cell e.g., a prokaryotic cell or a eukaryotic cell such as a plant cell or an animal cell.
  • expression of a polypeptide within a cell can be increased by delivering a system for stably integrating one or more nucleic acids into a target site within the genome of a cell provided herein (e.g., a system including (a) a genome-editing system that can insert an attA into a target site within a genome, (b) a donor nucleic acid molecule including a nucleic acid cargo and an attD site, and (c) an integrase (e.g., a LSR) that can target the attA site and the attD site) to a cell (a plant cell or a mammalian cell) to stably integrate a regulatory element (e.g., a promoter sequence) near (e.g., upstream of) an endogenous nucleic acid encoding a target polypeptide such that the regulatory element is operably linked to and increases expression of the encoded target polypeptide.
  • a regulatory element e.g., a promoter sequence
  • a genome-editing system that can insert an attA into a target site within a genome near an endogenous nucleic acid encoding a target polypeptide
  • a donor nucleic acid molecule including a nucleic acid cargo containing a promoter sequence and an attD site
  • an integrase that can target the attA site and the attD site can be delivered to a cell to stably integrate the promoter sequence into the genome such that the expression of the encoded target polypeptide is increased.
  • the methods and materials provided herein e.g., systems including (a) a genome- editing system that can insert an attA into a target site within a genome, (b) a donor nucleic acid molecule including a nucleic acid cargo and an attD site, and (c) an integrase (e.g., a LSR) that can target the attA site and the attD site) can be used for making a transgenic organism (e.g., a non-human transgenic organism).
  • a transgenic organism e.g., a non-human transgenic organism.
  • the methods and materials provided herein can be used to express an exogenous polypeptide within a cell such as a eukaryotic cell.
  • the methods and materials provided herein can be used to stably integrate a transgene (e.g., a transgene encoding an exogenous polypeptide) into the genome of a cell (e.g., an embryonic stem cell) that can give rise to an animal (e.g., a non- human animal).
  • a transgene e.g., a transgene encoding an exogenous polypeptide
  • the methods and materials provided herein can be used to stably integrate a transgene (e.g., a transgene encoding an exogenous polypeptide) into the genome of a cell (e.g., a plant cell) that can give rise to a plant.
  • a transgenic organism e.g., a non- human transgenic organism
  • a system for stably integrating one or more nucleic acids into a target site within the genome of a cell e.g., a system including (a) a genome- editing system that can insert an attA into a target site within a genome, (b) a donor nucleic acid molecule including a nucleic acid cargo and an attD site, and (c) an integrase (e.g., a LSR) that can target the attA site and the attD site) to a cell (e.g., a plant cell or a non-human embryonic stem cell) to stably integrate a transgene (e.g., a transgene encoding a polypeptide of interest) into the genome such that the transgene is expressed by the cell.
  • a transgene e.g., a transgene encoding a polypeptide of interest
  • a genome- editing system that can insert an attA into a target site within a genome
  • a donor nucleic acid molecule including a transgene and an attD site
  • an integrase that can target the attA site and the attD site can be delivered to a cell to stably integrate the transgene into the genome such that the transgene is expressed by the cell.
  • the methods and materials provided herein e.g., systems including (a) a genome- editing system that can insert an attA into a target site within a genome, (b) a donor nucleic acid molecule including a nucleic acid cargo and an attD site, and (c) an integrase (e.g., a LSR) that can target the attA site and the attD site) can be used for making a transgenic cell (e.g., a transgenic immune cell such as a transgenic T cell, a transgenic NK cell, or a transgenic macrophage) having (e.g., engineered to have) a receptor (e.g., a T cell receptor (TCR); a NK cell receptor (NKR), or a chimeric antigen receptor (CAR)).
  • a transgenic cell e.g., a transgenic immune cell such as a transgenic T cell, a transgenic NK cell, or a transgenic macrophage
  • a genome-editing system that can insert an attA into a target site within a genome
  • an integrase that can target the attA site and the attD site can be delivered to a T cell (e.g., an ex vivo human T cell) to stably integrate the transgene into the genome of the T cell such that the CAR is expressed by the T cell (e.g., to generate a CAR T cell).
  • a T cell e.g., an ex vivo human T cell
  • a genome-editing system that can insert an attA into a target site within a genome
  • a donor nucleic acid molecule including a transgene encoding a TCR e.g., a wild type TCR or an engineered TCR
  • an integrase that can target the attA site and the attD site can be delivered to an NK cell (e.g., an ex vivo human NK cell) to stably integrate the transgene into the genome of the NK cell such that the TCR is expressed by the NK cell (e.g., to generate an NK cell expressing the TCR).
  • an NK cell e.g., an ex vivo human NK cell
  • a genome- editing system that can insert an attA into a target site within a genome
  • a donor nucleic acid molecule including a transgene encoding a NKR e.g., a wild type NKR or an engineered NKR
  • an integrase that can target the attA site and the attD site can be delivered to an NK cell (e.g., an ex vivo human NK cell) to stably integrate the transgene into the genome of the NK cell such that the NKR is expressed by the NK cell (e.g., to generate an NK cell expressing the NKR).
  • an NK cell e.g., an ex vivo human NK cell
  • Any appropriate receptor e.g., any appropriate TCR, any appropriate NKR, or any appropriate CAR
  • a cell e.g., an immune cell such as a T cell or a NK cell
  • a CAR can be as described elsewhere (e.g., De Bousser et al., Cancers (Basel), 13(23):6067 (2021); Eyquem et al., Nature, 543(7643):113-117 (2017); and Larson et al., Nat. Rev. Cancer, 21(3): 145-161 (2021)).
  • the methods and materials provided herein e.g., systems including (a) a genome- editing system that can insert an attA into a target site within a genome, (b) a donor nucleic acid molecule including a nucleic acid cargo and an attD site, and (c) an integrase (e.g., a LSR) that can target the attA site and the attD site) can be used for making a transgenic plant having (e.g., engineered to have) pathogen resistance (e.g., bacterial resistance or viral resistance).
  • pathogen resistance e.g., bacterial resistance or viral resistance
  • a genome-editing system that can insert an attA into a target site within a genome
  • a donor nucleic acid molecule including a transgene encoding a pathogen resistance polypeptide and an attD site can be delivered to a plant cell to stably integrate the transgene into the genome such that the pathogen resistance polypeptide is expressed by the cell.
  • Any appropriate pathogen resistance polypeptide can be integrated into a plant cell genome to create a pathogen resistant transgenic plant as described herein.
  • a pathogen resistance polypeptide can be as described elsewhere (e.g., Dong et al., Plant Physiol., 180(l):26-38 (2019)).
  • the methods and materials provided herein e.g., systems including (a) a genome- editing system that can insert an attA into a target site within a genome, (b) a donor nucleic acid molecule including a nucleic acid cargo and an attD site, and (c) an integrase (e.g., a LSR) that can target the attA site and the attD site) can be used for making a transgenic plant having (e.g., engineered to have) herbicide resistance.
  • a genome- editing system that can insert an attA into a target site within a genome
  • a donor nucleic acid molecule including a nucleic acid cargo and an attD site
  • an integrase e.g., a LSR
  • a genome-editing system that can insert an attA into a target site within a genome
  • a donor nucleic acid molecule including a transgene encoding a herbicide resistance polypeptide and an attD site can be delivered to a plant cell to stably integrate the transgene into the genome such that the herbicide resistance polypeptide is expressed by the cell.
  • Any appropriate herbicide resistance polypeptide can be integrated into a plant cell genome to create an herbicide resistant transgenic plant as described herein.
  • an herbicide resistance polypeptide can be as described elsewhere (e.g., Sun et al., Molecular Plant, 9.4:628-631 (2016); Li et al., Nature Plants, 2: 16139 (2016); Tatsis et al., Curr. Opin. Biotech., 42: 126-132 (2016); Ducat et al., Curr. Opin. Chem. Biol., 16(3-4):337-344 (2012); Sanghera et al., Curr. Genomics., 12(l):30-43 (2011); Dong et al., Nat. Commun., 11: 1178 (2020); and Lu et al., Nat.
  • the methods and materials provided herein e.g., systems including (a) a genome- editing system that can insert an attA into a target site within a genome, (b) a donor nucleic acid molecule including a nucleic acid cargo and an attD site, and (c) an integrase (e.g., a LSR) that can target the attA site and the attD site) can be used for making an organism (e.g., a non- human organism) having reduced or eliminated levels of a polypeptide (e.g., a non-human knock-out organism).
  • a genome- editing system that can insert an attA into a target site within a genome
  • a donor nucleic acid molecule including a nucleic acid cargo and an attD site e.g., a LSR
  • an integrase e.g., a LSR
  • the methods and materials provided herein can be used to disrupt and/or replace an endogenous nucleic acid encoding a target polypeptide within a cell such as a eukaryotic cell.
  • the methods and materials provided herein can be used to stably integrate a nucleic acid molecule (e.g., knock-out cassette) into the genome of a cell (e.g., an embryonic stem cell) that can give rise to an organism (e.g., a non-human animal) to disrupt and/or replace an endogenous nucleic acid encoding a target polypeptide.
  • a nucleic acid molecule e.g., knock-out cassette
  • an organism e.g., a non-human animal
  • the methods and materials provided herein can be used to stably integrate a nucleic acid molecule (e.g., knock-out cassette) into the genome of a cell (e.g., a plant cell) that can give rise to a plant to disrupt and/or replace an endogenous nucleic acid encoding a target polypeptide.
  • a nucleic acid molecule e.g., knock-out cassette
  • an endogenous nucleic acid encoding a target polypeptide within a cell can be disrupted and/or replaced by delivering a system for stably integrating one or more nucleic acids into a target site within the genome of a cell provided herein (e.g., a system including (a) a genome-editing system that can insert an attA into a target site within a genome, (b) a donor nucleic acid molecule including a nucleic acid cargo and an attD site, and (c) an integrase (e.g., a LSR) that can target the attA site and the attD site) to a cell (a plant cell or a mammalian cell) to stably integrate a nucleic acid molecule within an endogenous nucleic acid encoding a target polypeptide such that the nucleic acid molecule disrupts and/or replaces the endogenous nucleic acid encoding a target polypeptide and expression of the endogen
  • a genome-editing system that can insert an attA into a target site within a genome that is in-frame with an endogenous nucleic acid encoding a target polypeptide
  • a donor nucleic acid molecule including a nucleic acid cargo and an attD site and
  • an integrase that can target the attA site and the attD site can be delivered to a cell to stably integrate the nucleic acid cargo into the genome such that the nucleic acid cargo disrupts and/or replaces an endogenous nucleic acid encoding a target polypeptide such that the nucleic acid molecule disrupts and/or replaces the endogenous nucleic acid encoding a target polypeptide and expression of the encoded target polypeptide is reduced or eliminated.
  • a nucleic acid cargo that can be stably integrated into a genome of a cell (e.g., a non-human animal cell or a plant cell) to disrupt and/or replace an endogenous nucleic acid encoding a target polypeptide such that expression of the encoded the target polypeptide is reduced or eliminated can include a stop codon.
  • a nucleic acid cargo that can be stably integrated into a genome of a cell (e.g., a non-human animal cell or a plant cell) to disrupt and/or replace an endogenous nucleic acid encoding a target polypeptide such that expression of the encoded the target polypeptide is reduced or eliminated can include a splice acceptor site.
  • a nucleic acid cargo that can be stably integrated into a genome of a cell e.g., a non-human animal cell or a plant cell
  • a nucleic acid cargo that can be stably integrated into a genome of a cell to disrupt and/or replace an endogenous nucleic acid encoding a target polypeptide such that expression of the encoded the target polypeptide is reduced or eliminated
  • a nucleic acid cargo can be stably integrated into a genome of a cell such that the selectable marker is under the control of the regulatory elements for the disrupted and/or replaced endogenous nucleic acid encoding a target polypeptide.
  • a nucleic acid cargo that can be stably integrated into a genome of a cell e.g., a non-human animal cell or a plant cell
  • a detectable label such that the detectable label is expressed by the cell.
  • a nucleic acid cargo can be stably integrated into a genome of a cell such that the detectable label is under the control of the regulatory elements for the disrupted and/or replaced endogenous nucleic acid encoding a target polypeptide.
  • the methods and materials provided herein e.g., systems including (a) a genome- editing system that can insert an attA into a target site within a genome, (b) a donor nucleic acid molecule including a nucleic acid cargo and an attD site, and (c) an integrase (e.g., a LSR) that can target the attA site and the attD site) can be used for treating a mammal (e.g., a human) having a disease or disorder.
  • a mammal e.g., a human
  • a genome-editing system that can insert an attA into a target site within a genome
  • a donor nucleic acid molecule including a transgene encoding a therapeutic gene product and an attD site
  • an integrase that can target the attA site and the attD site can be delivered to a cell to stably integrate the transgene into the genome such that the therapeutic gene product is expressed by the cell.
  • the methods and materials provided herein can be used to treat a mammal (e.g., a human) have a disease or disorder associated with reduced or eliminated levels of a gene product (e.g., reduced or eliminated levels of a polypeptide or reduced or eliminated levels of a non-coding RNA).
  • a mammal e.g., a human
  • a disease or disorder associated with a mutated gene product e.g., a mutated polypeptide or a mutated non-coding RNA.
  • the mammal can be any appropriate mammal.
  • mammals that can be treated as described herein include, without limitation, humans, non-human primates such as chimpanzees and monkeys, dogs, cats, horses, cows, pigs, sheep, mice, rats, rabbits, guinea pigs, birds, fish, (e.g., zebrafish (Danio rerio), medaka (Oryzias Latipes), and turquoise killifish (Nothobranchius fii zeri)).
  • nematodes e.g., Caenorhabditis elegans
  • flies e.g., Drosophila melanogaster.
  • the components of a system for stably integrating one or more nucleic acids into a target site within the genome of a cell provided herein (e.g., systems including (a) a genome-editing system that can insert an attA into a target site within a genome, (b) a donor nucleic acid molecule including a nucleic acid cargo and an attD site, and (c) an integrase (e.g., a LSR) that can target the attA site and the attD site) can be delivered to cells within a living mammal (e.g., can be delivered to in vivo cells).
  • a genome-editing system that can insert an attA into a target site within a genome
  • a donor nucleic acid molecule including a nucleic acid cargo and an attD site e.g., a LSR
  • an integrase e.g., a LSR
  • the components of a system for stably integrating one or more nucleic acids into a target site within the genome of a cell provided herein (e.g., systems including (a) a genome-editing system that can insert an attA into a target site within a genome, (b) a donor nucleic acid molecule including a nucleic acid cargo and an attD site, and (c) an integrase (e.g., a LSR) that can target the attA site and the attD site) can be delivered to cells obtained from a mammal (e.g., can be delivered to ex vivo cells), and then the cells containing the stably integrated nucleic acid can be administered to the mammal to be treated.
  • systems including (a) a genome-editing system that can insert an attA into a target site within a genome, (b) a donor nucleic acid molecule including a nucleic acid cargo and an attD site, and (c) an integras
  • the components of a system for stably integrating one or more nucleic acids into a target site within the genome of a cell provided herein are delivered ex vivo to cell obtained from the mammal to be treated (e.g., an autologous cell). In some cases, the components of a system for stably integrating one or more nucleic acids into a target site within the genome of a cell provided herein are delivered ex vivo to cell obtained from a donor mammal (e.g., an allogeneic cell).
  • a donor mammal e.g., an allogeneic cell
  • any appropriate transgene encoding a therapeutic gene product can be integrated into a cell genome to treat a mammal as described herein.
  • therapeutic gene products include, without limitation, adenosine deaminase (e.g., to treat a mammal having severe combined immunodeficiency (SCID)), ⁇ -1 antitrypsin (e.g., to treat a mammal having liver damage such as cirrhosis), cystic fibrosis transmembrane conductance regulator (CFTR; e.g., to treat a mammal having cystic fibrosis (CF)), ⁇ -hemoglobin (HBB; e.g., to treat a mammal having thalassemia), oculocutaneous albinism II (0CA2; e.g., to treat a mammal having oculocutaneous albinism (OCA), Huntingtin (HTT; e.g., to treat a mammal having Huntington's disease), dys
  • a therapeutic gene product can be as described elsewhere (e.g., Suzuki et al., Mol. Then, 28.7:1684-1695 (2020); Pierce et al., Cold Spring Harbor Perspect. Med. 5:9 a017285 (2015); Urnov et al., Nature, 435.7042:646-651 (2005); Phelps et al., Human Mol. Gen., 4.8:1251-1258 (1995); and Ellerby et al., Neurotherapeutics, 16(4): 924-927 (2019)).
  • Example 1 Stable Integration of Multi-Kilobase DNA Cargos Into Eukaryotic Cell Genomes
  • LSRs Large serine recombinases
  • This Example describes the utilization of a prime editor in combination with a LSR for programmable recombination of multiple kilobase cargo into the genome.
  • a prime editor can be used to insert an attA site into a desired genomic context, and a LSR can integrate a nucleic acid cargo into the target site.
  • Schematic images of exemplary methods of using a prime editor in combination with a LSR for programmable recombination of multiple kilobase cargo into the genome are shown in Figure 1.
  • spacer sequences, extension templates, and SpCas9 sgRNA scaffold sequences were synthesized (Integrated DNA Technologies) and cloned via ligation of annealed oligonucleotides into BsmBI digested acceptor vector (pU6-pegRNA-GG-acceptor, Addgene plasmid no. 132777).
  • BsmBI digested acceptor vector pU6-pegRNA-GG-acceptor, Addgene plasmid no. 132777.
  • spacers were synthesized (Integrated DNA Technologies) and cloned via ligation of annealed oligonucleotides into BbsI digested acceptor vector (pCB007 SpCas9_sgRNA_cloning_Backbone).
  • HEK-293FT cells were grown in DMEM (Gibco) media supplemented with 10% FBS (Hyclone), penicillin (10,000 I.U./mL), and streptomycin (10,000 ug/mL).
  • 20,000 HEK293FT cells were plated into poly-D-lysine coated 96 well plates.
  • 250ng prime editor plasmid pCMV-PE2-P2A-GFP Addgene plasmid #132776)
  • 83 ng pegRNA plasmid 83 ng pegRNA plasmid
  • 27.6 ng ngRNA plasmid were transfected into the cells using Lipofectamine 2000 (Thermo).
  • cells were extracted with DNA QuickExtract (Lucigen). Edits were verified via PCR (Platinum Superfi PCR Master Mix, Thermo) across the edited locus. Sanger sequencing was analyzed with ICE analysis (Synthego) to determine the percentage of cells containing the edit.
  • Prime editing and LSR mediated donor integration were confirmed using PCR (Platinum Superfi PCR Master Mix, Thermo Fisher) across the insertion junction.
  • PCR Platinum Superfi PCR Master Mix, Thermo Fisher
  • the same quantities of Prime editor, ngRNA, pegRNA, LSR, and donor plasmid were co-transfected on day 0, and cells were harvested on day 5 for PCR.
  • Prime editing plasmid (pCMV-PE2, Addgene Plasmid #132775) was modified with gibson cloning to include an XTEN 48 linker, a L139P mutation in the MMuLV RT, and either a (GGS)6 (for cis LSR delivery) or a self-cleavable P2A (for trans LSR delivery) linker and BsmbI golden gate landing pad at the C terminus of the RT.
  • Human codon optimized LSRs were cloned into the BsmBI landing pad via golden gate assembly.
  • HEK293FT mammalian cells
  • 20,000 HEK293FT cells were plated into poly-D-lysine coated 96 well plates.
  • 375ng effector plasmid, lOOng pegRNA, and 50ng ngRNA were transfected into the cells using Lipofectamine 2000 (Thermo).
  • media was removed and cells were resuspended in 40uL DNA QuickExtract (Lucigen).
  • the cells were transferred to a PCR plate, and incubated at 65°C for 15 minutes, 68°C for 15 minutes, and 98°C for 10 minutes.
  • samples were purified with 0.9X Ampure XP beads (Beckman Coulter).
  • IOUL qPCR reactions were performed with 5uL Taqman Fast Advanced 2x Master Mix, 250nM of each primer, 200nM of each probe, and luL of extracted genomic DNA. qPCR was run on the 480 LightCycler (Roche), which calculated Ct values. Delta Ct indicates the difference between the Ct of the integration and reference probe Ct values. ddPCR of donor integration
  • gDNA was extracted and PCR was performed on target locus (HEK3).
  • Sanger sequencing and ICE analysis confirmed that the attA for Bxbl and PaOl, which is encoded on the pegRNA, can be integrated into the target locus (Figure 3).
  • Absolute integration efficiency was determined utilizing a single pegRNA by performing ddPCR of the integration junction and normalizing to an unedited locus (Figure 8A, 8B). All LSRs tested had detected LSR-mediated integration at the ACTB and LMNB1 locus, and no integration was seen in the PE-LSR-Donor and Donor only controls. Consistent with qPCR, trans delivery was slightly more efficient than cis delivery in all cases. qPCR of donor integration, 1 step delivery, 2 pegRNAs
  • Example 2 Exemplary Sequences
  • Example 3 Transgenic Animals
  • a system for stably integrating one or more nucleic acid sequences into a genome of a cell as provided herein is delivered to an embryonic stem cell of a non-human mammal (e.g., a mouse) to integrate a donor nucleic molecule containing a desired transgene into the genome of the embryonic stem cell.
  • a non-human mammal e.g., a mouse
  • a genome-editing system comprising (i) a polypeptide comprising a DNA binding domain and, optionally, a polymerase and (ii) a nucleic acid comprising a guide sequence that is complementary to a target site within said genome and a sequence that encodes an attA sequence; (b) a donor nucleic acid molecule comprising a transgene and an attD sequence; and (c) an integrase that targets said attA sequence and said attD site and can facilitate recombination between said attA site and said attD site are delivered to an embryonic stem cell of a non-human mammal (e.g., a mouse) to integrate the donor nucleic molecule containing the desired transgene into the genome of the embryonic stem cell.
  • a non-human mammal e.g., a mouse
  • the embryonic stem cell containing the transgene is injected into an inner cell mass of a blastocyst, and the blastocyst is then implanted into the uterus of female non-human mammal (e.g., a female mouse).
  • Transgenic mice are selected from the offspring.
  • a system for stably integrating one or more nucleic acid sequences into a genome of a cell as provided herein is delivered to a non-human animal model (e.g., an adult mouse having a particular disease) to integrate a donor nucleic molecule containing a knock-out cassette into the genome of one or more cells within the non-human animal model.
  • a non-human animal model e.g., an adult mouse having a particular disease
  • a genome-editing system comprising (i) a polypeptide comprising a DNA binding domain and, optionally, a polymerase and (ii) a nucleic acid comprising a guide sequence that is complementary to a target site within said genome and a sequence that encodes an attA sequence; (b) a donor nucleic acid molecule comprising a knock-out cassette and an attD sequence; and (c) an integrase that targets said attA sequence and said attD site and can facilitate recombination between said attA site and said attD site are delivered to a non-human mammal (e.g., a mouse) to integrate the donor nucleic molecule containing the knock-out cassette into one or more cells within the non-human animal model.
  • a non-human mammal e.g., a mouse
  • a system for stably integrating one or more nucleic acid sequences into a genome of a cell as provided herein is delivered to T cells to generate engineered T cells such as CAR T cells.
  • a genome-editing system comprising (i) a polypeptide comprising a DNA binding domain and, optionally, a polymerase and (ii) a nucleic acid comprising a guide sequence that is complementary to a target site within said genome and a sequence that encodes an attA sequence;
  • a donor nucleic acid molecule comprising a transgene encoding a particular receptor (e.g., a TCR or a CAR) and an attD sequence; and
  • an integrase that targets said attA sequence and said attD site and can facilitate recombination between said attA site and said attD site are delivered to T cells (e.g., T cells obtained from the mammal to be treated) to integrate the donor nucleic molecule containing the transgene encoding the particular receptor (e.g., the TCR or the CAR) into the T cells such that the particular receptor is expressed by the T cell (e.g., to generate
  • a system for stably integrating one or more nucleic acid sequences into a genome of a cell as provided herein is delivered to T cells (e.g., T cells obtained from a mammal (e.g., a human) having cancer).
  • T cells e.g., T cells obtained from a mammal (e.g., a human) having cancer.
  • a genome-editing system comprising (i) a polypeptide comprising a DNA binding domain and, optionally, a polymerase and (ii) a nucleic acid comprising a guide sequence that is complementary to a target site within said genome and a sequence that encodes an attA sequence;
  • a donor nucleic acid molecule comprising a transgene encoding a receptor (e.g., a TCR or a CAR that can target an antigen expressed by cancer cells within a mammal) and an attD sequence; and
  • an integrase that targets said attA sequence and said attD site and can facilitate recombination between said attA site and said attD site are delivered to T cells (e.g., T cells obtained from the mammal to be treated) to integrate the donor nucleic molecule containing the transgene encoding the particular receptor (e.g., the TCR or the CAR) into the T cells such that the particular
  • the generated engineered T cells are administered to the mammal (e.g., a human) having cancer to treat the mammal.
  • the mammal e.g., a human
  • a system for stably integrating one or more nucleic acid sequences into a genome of a cell as provided herein is delivered to a mammal (e.g., a human) having a disease associated with nucleotide repeats (e.g., C9orf72 amyotrophic lateral sclerosis and frontotemporal dementia (C9 ALS/FTD)) to integrate a donor nucleic molecule containing a nucleic acid encoding a therapeutic gene product (e.g., a wild type C9orf72 polypeptide) to treat the mammal.
  • a mammal e.g., a human
  • a disease associated with nucleotide repeats e.g., C9orf72 amyotrophic lateral sclerosis and frontotemporal dementia (C9 ALS/FTD)
  • C9 ALS/FTD frontotemporal dementia
  • a genome-editing system comprising (i) a polypeptide comprising a DNA binding domain and, optionally, a polymerase and (ii) a nucleic acid comprising a guide sequence that is complementary to a target site upstream of a G4C2 repeat within said genome and a sequence that encodes an attA sequence; (b) a donor nucleic acid molecule comprising a splice acceptor, at least a portion of a wild type C9orf72 gene, and transcription termination signal and an attD sequence; and (c) an integrase that targets said attA sequence and said attD site and can facilitate recombination between said attA site and said attD site are delivered to cells within the mammal to integrate the donor nucleic molecule containing the splice acceptor, the at least a portion of a wild type C9orf72 gene, and the transcription termination signal into the cells such that a wild type C9orf72 polypeptide

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US20190390189A1 (en) * 2017-02-15 2019-12-26 Bluebird Bio, Inc. Donor repair templates multiplex genome editing
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WO2020165901A1 (en) * 2019-02-11 2020-08-20 Ramot At Tel-Aviv University Ltd. Site specific recombinase integrase variants and uses thereof in gene editing in eukaryotic cells
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