# US 2021/0277366 A1 · ENHANCED hAT FAMILY TRANSPOSON-MEDIATED GENE TRANSFER AND ASSOCIATED COMPOSITIONS, SYSTEMS, AND METHODS

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- Number: US 2021/0277366 A1
- Document ID: US20210277366A1
- Status: Published application
- Kind: A1
- Application number: 17/254,162
- Filed: 2019-06-21
- Published: 2021-09-09
- Earliest claimed: 2018-06-21
- Assignees: B-Mogen Biotechnologies, Inc.
- Applicants: B-Mogen Biotechnologies, Inc.
- Inventors: David Largaespada; Branden Moriarity; Beau Webber; Neil Otto; Sandeep Kumar; Leah Hogdal
- Web page: https://patentgrep.com/patent/US20210277366A1

## CPC

- C12N 9/1241: {Nucleotidyltransferases (2.7.7)}
- C12N 15/85: for animal cells
- C12N 15/90: Stable introduction of foreign DNA into chromosome
- A61K 48/00: Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
- C12N 2800/90: Vectors containing a transposable element
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D)

## Abstract

This disclosure provides various TcBuster transposases and transposons, systems, and methods of use.

## Claims

### Claim 1

A mutant TcBuster transposase comprising an amino acid sequence at least 70% identical to full-length SEQ ID NO: 1 and having one or more amino acid substitutions from Table 1.1.

### Claim 2 (depends on 1)

The mutant TcBuster transposase of claim 1, comprising an amino acid substitution that increases a net charge at a neutral pH in comparison to SEQ ID NO: 1.

### Claim 3 (depends on 2)

The mutant TcBuster transposase of claim 2, wherein the amino acid substitution that increases the net charge at the neutral pH comprises a substitution to a lysine or an arginine.

### Claim 4 (depends on 2, 3)

The mutant TcBuster transposase of claim 2 or 3, wherein amino acid substitution that increases the net charge at the neutral pH comprises a substitution of an aspartic acid or a glutamic acid to a neutral amino acid, a lysine or an arginine.

### Claim 5 (depends on 1, 2, 3, 4)

The mutant TcBuster transposase of any one of claims 1-4, comprising one or more amino acid substitutions from Table 4.1.

### Claim 6 (depends on 1, 2, 3, 4, 5)

The mutant TcBuster transposase of any one of claims 1-5, further comprising one or more amino acid substitutions from Table 4.

### Claim 7 (depends on 1, 2, 3, 4, 5, 6)

The mutant TcBuster transposase of any one of claims 1-6, comprising an amino acid substitution in a DNA Binding and Oligomerization domain; an insertion domain; a Zn-BED domain; or a combination thereof.

### Claim 8 (depends on 1, 2, 3, 4, 5, 6, 7)

The mutant TcBuster transposase of any one of claims 1-7, comprising an amino acid substitution that increases a net charge at a neutral pH within or in proximity to a catalytic domain in comparison to SEQ ID NO: 1.

### Claim 9 (depends on 1, 2, 3, 4, 5, 6, 7, 8)

The mutant TcBuster transposase of any one of claims 1-8, comprising an amino acid substitution that increases a net charge at a neutral pH in comparison to SEQ ID NO: 1, wherein the one or more amino acids are located in proximity to D223, D289, or E589, when numbered in accordance to SEQ ID NO: 1.

### Claim 10 (depends on 8, 9)

The mutant TcBuster transposase of claim 8 or 9, wherein the proximity is a distance of about 80, 75, 70, 60, 50, 40, 30, 20, 10, or 5 amino acids.

### Claim 11 (depends on 8, 9)

The mutant TcBuster transposase of claim 8 or 9, wherein the proximity is a distance of about 70 to 80 amino acids.

### Claim 12 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11)

The mutant TcBuster transposase of any one of claims 1-11, wherein the amino acid sequence of the mutant TcBuster transposase is at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% identical to full-length SEQ ID NO: 1.

### Claim 13 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12)

The mutant TcBuster transposase of any one of claims 1-12, further comprising one or more amino acid substitutions from Table 2.

### Claim 14 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13)

The mutant TcBuster transposase of any one of claims 1-13, further comprising one or more amino acid substitutions from Table 3.

### Claim 15 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14)

The mutant TcBuster transposase of any one of claims 1-14, further comprising amino acid substitutions V377T, E469K, and D189A, when numbered in accordance with SEQ ID NO: 1.

### Claim 16 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15)

The mutant TcBuster transposase of any one of claims 1-15, further comprising amino acid substitutions K573E and E578L, when numbered in accordance with SEQ ID NO: 1.

### Claim 17 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16)

The mutant TcBuster transposase of any one of claims 1-16, further comprising amino acid substitution I452K, when numbered in accordance with SEQ ID NO: 1.

### Claim 18 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17)

The mutant TcBuster transposase of any one of claims 1-17, further comprising amino acid substitution A358K, when numbered in accordance with SEQ ID NO: 1.

### Claim 19 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18)

The mutant TcBuster transposase of any one of claims 1-18, further comprising amino acid substitution V297K, when numbered in accordance with SEQ ID NO: 1.

### Claim 20 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19)

The mutant TcBuster transposase of any one of claims 1-19, further comprising amino acid substitution N85S, when numbered in accordance with SEQ ID NO: 1.

### Claim 21 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20)

The mutant TcBuster transposase of any one of claims 1-20, further comprising amino acid substitutions I452F, V377T, E469K, and D189A, when numbered in accordance with SEQ ID NO: 1.

### Claim 22 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21)

The mutant TcBuster transposase of any one of claims 1-21, further comprising amino acid substitutions A358K, V377T, E469K, and D189A, when numbered in accordance with SEQ ID NO: 1.

### Claim 23 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22)

The mutant TcBuster transposase of any one of claims 1-22, further comprising amino acid substitutions V377T, E469K, D189A, K573E and E578L, when numbered in accordance with SEQ ID NO: 1.

### Claim 24 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23)

The mutant TcBuster transposase of any one of claims 1-23, further comprising one or more amino acid substitutions from Table 1.

### Claim 25 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24)

The mutant TcBuster transposase of any one of claims 1-24, wherein the mutant TcBuster transposase has increased transposition efficiency in comparison to a wild-type TcBuster transposase having amino acid sequence SEQ ID NO: 1.

### Claim 26 (depends on 25)

The mutant TcBuster transposase of claim 25, wherein the transposition efficiency is measured by an assay that comprises introducing the mutant TcBuster transposase or the wild-type TcBuster transposase and a TcBuster transposon containing a reporter cargo cassette into a population of cells, and detecting transposition of the reporter cargo cassette in genome of the population of cells.

### Claim 27

A fusion transposase, comprising a TcBuster transposase sequence and one or more additional Nuclear Localization Signal sequences, wherein the TcBuster transposase sequence has at least 70% identity to full-length SEQ ID NO: 1.

### Claim 28 (depends on 27)

The fusion transposase of claim 27, wherein the TcBuster transposase sequence has at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% identity to full-length SEQ ID NO: 1.

### Claim 29 (depends on 27, 28)

The fusion transposase of claim 27 or 28, wherein the TcBuster transposase sequence comprises one or more amino acid substitutions that increase a net charge at a neutral pH in comparison to SEQ ID NO: 1.

### Claim 30 (depends on 29)

The fusion transposase of claim 29, wherein the one or more amino acid substitutions comprise a substitution with a lysine or an arginine.

### Claim 31 (depends on 29, 30)

The fusion transposase of claim 29 or 30, wherein the one or more amino acid substitutions comprise a substitution of an aspartic acid or a glutamic acid with a neutral amino acid, a lysine or an arginine.

### Claim 32 (depends on 27, 28, 29, 30, 31)

The fusion transposase of any one of claims 27-31, wherein the TcBuster transposase sequence comprises one or more amino acid substitutions from Table 4, Table 4.1, or both.

### Claim 33 (depends on 27, 28, 29, 30, 31, 32)

The fusion transposase of any one of claims 27-32, wherein the TcBuster transposase sequence comprises one or more amino acid substitutions in a DNA Binding and Oligomerization domain; an insertion domain; a Zn-BED domain; or a combination thereof.

### Claim 34 (depends on 27, 28, 29, 30, 31, 32, 33)

The fusion transposase of any one of claims 27-33, wherein the TcBuster transposase sequence comprises one or more amino acid substitutions from Table 1, Table 1.1, or both.

### Claim 35 (depends on 27, 28, 29, 30, 31, 32, 33, 34)

The fusion transposase of any one of claims 27-34, wherein the TcBuster transposase sequence has increased transposition efficiency in comparison to a wild-type TcBuster transposase having amino acid sequence SEQ ID NO: 1.

### Claim 36 (depends on 35)

The fusion transposase of claim 35, wherein the transposition efficiency of the TcBuster transposase sequence is measured by an assay that comprises introducing the fusion transposase or the wild-type TcBuster transposase and a TcBuster transposon containing a reporter cargo cassette into a population of cells, and detecting transposition of the reporter cargo cassette in genome of the population of cells.

### Claim 37 (depends on 27, 28, 29, 30, 31, 32, 33, 34, 35, 36)

The fusion transposase of any one of claims 27-36, wherein the TcBuster transposase sequence comprises one or more amino acid substitutions that increase a net charge at a neutral pH within or in proximity to a catalytic domain in comparison to SEQ ID NO: 1.

### Claim 38 (depends on 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37)

The fusion transposase of any one of claims 27-37, wherein the TcBuster transposase sequence comprises one or more amino acid substitutions that increase a net charge at a neutral pH in comparison to SEQ ID NO: 1, wherein the one or more amino acid substitutions are located in proximity to D223, D289, or E589, when numbered in accordance to SEQ ID NO: 1.

### Claim 39 (depends on 37, 38)

The fusion transposase of claim 37 or 38, wherein the proximity is a distance of about 80, 75, 70, 60, 50, 40, 30, 20, 10, or 5 amino acids.

### Claim 40 (depends on 37, 38)

The fusion transposase of claim 37 or 38, wherein the proximity is a distance of about 70 to 80 amino acids.

### Claim 41 (depends on 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40)

The fusion transposase of any one of claims 27-40, wherein the TcBuster transposase sequence comprises one or more amino acid substitutions from Table 2.

### Claim 42 (depends on 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41)

The fusion transposase of any one of claims 27-41, wherein the TcBuster transposase sequence comprises one or more amino acid substitutions from Table 3.

### Claim 43 (depends on 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42)

The fusion transposase of any one of claims 27-42, wherein the TcBuster transposase sequence comprises amino acid substitutions V377T, E469K, and D189A, when numbered in accordance with SEQ ID NO: 1.

### Claim 44 (depends on 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43)

The fusion transposase of any one of claims 27-43, wherein the TcBuster transposase sequence comprises amino acid substitutions K573E and E578L, when numbered in accordance with SEQ ID NO: 1.

### Claim 45 (depends on 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44)

The fusion transposase of any one of claims 27-44, wherein the TcBuster transposase sequence comprises amino acid substitution I452K, when numbered in accordance with SEQ ID NO: 1.

### Claim 46 (depends on 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45)

The fusion transposase of any one of claims 27-45, wherein the TcBuster transposase sequence comprises amino acid substitution A358K, when numbered in accordance with SEQ ID NO: 1.

### Claim 47 (depends on 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46)

The fusion transposase of any one of claims 27-46, wherein the TcBuster transposase sequence comprises amino acid substitution V297K, when numbered in accordance with SEQ ID NO: 1.

### Claim 48 (depends on 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47)

The fusion transposase of any one of claims 27-47, wherein the TcBuster transposase sequence comprises amino acid substitution N85S, when numbered in accordance with SEQ ID NO: 1.

### Claim 49 (depends on 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48)

The fusion transposase of any one of claims 27-48, wherein the TcBuster transposase sequence comprises amino acid substitutions I452F, V377T, E469K, and D189A, when numbered in accordance with SEQ ID NO: 1.

### Claim 50 (depends on 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49)

The fusion transposase of any one of claims 27-49, wherein the TcBuster transposase sequence comprises amino acid substitutions A358K, V377T, E469K, and D189A, when numbered in accordance with SEQ ID NO: 1.

### Claim 51 (depends on 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50)

The fusion transposase of any one of claims 27-50, wherein the TcBuster transposase sequence comprises amino acid substitutions V377T, E469K, D189A, K573E and E578L, when numbered in accordance with SEQ ID NO: 1.

### Claim 52 (depends on 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51)

The fusion transposase of any one of claims 27-51, wherein the TcBuster transposase sequence has 100% identity to full-length SEQ ID NO: 1.

### Claim 53 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26)

A fusion transposase comprising a TcBuster transposase sequence and a DNA sequence specific binding domain, wherein the TcBuster transposase sequence has an amino acid sequence of the mutant TcBuster of any one of claims 1-26.

### Claim 54 (depends on 53)

The fusion transposase of claim 53, wherein the DNA sequence specific binding domain comprises a TALE domain, zinc finger domain, AAV Rep DNA-binding domain, or any combination thereof.

### Claim 55 (depends on 53, 54)

The fusion transposase of claim 53 or 54, wherein the DNA sequence specific binding domain comprises a TALE domain.

### Claim 56 (depends on 53, 54, 55)

The fusion transposase of any one of claims 53-55, wherein the TcBuster transposase sequence and the DNA sequence specific binding domain are separated by a linker.

### Claim 57 (depends on 56)

The fusion transposase of claim 56, wherein the linker comprises at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, or at least 50 amino acids.

### Claim 58 (depends on 56, 57)

The fusion transposase of claim 56 or 57, wherein the linker comprises SEQ ID NO: 9.

### Claim 59

A polynucleotide comprising a nucleic acid sequence at least about 80%, 85%, 90%, 95%, or 98% identical to or complementary with full length SEQ ID NO: 204 or 207.

### Claim 60 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26)

A polynucleotide that codes for the mutant TcBuster transposase of any one of claims 1-26.

### Claim 61 (depends on 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58)

A polynucleotide that codes for the fusion transposase of any one of claims 27-58.

### Claim 62 (depends on 59, 60, 61)

The polynucleotide of any one of claims 59-61, wherein the polynucleotide comprises DNA that encodes the mutant TcBuster transposase or the fusion transposase.

### Claim 63 (depends on 59, 60, 61, 62)

The polynucleotide of any one of claims 59-62, wherein the polynucleotide comprises messenger RNA (mRNA) that encodes the mutant TcBuster transposase or the fusion transposase.

### Claim 64 (depends on 63)

The polynucleotide of claim 63, wherein the mRNA is chemically modified.

### Claim 65 (depends on 59, 60, 61, 62, 63, 64)

The polynucleotide of any one of claims 59-64, wherein the polynucleotide comprises nucleic acid sequence encoding for a transposon recognizable by the mutant TcBuster transposase or the fusion transposase.

### Claim 66 (depends on 59, 60, 61, 62, 63, 64, 65)

The polynucleotide of any one of claims 59-65, wherein the polynucleotide is present in a DNA vector.

### Claim 67 (depends on 66)

The polynucleotide of claim 66, wherein the DNA vector comprises a mini-circle plasmid. WO 2019/246486 PCT/US2019/038410

### Claim 68 (depends on 59, 60, 61, 62, 63, 64, 65, 66, 67)

The polynucleotide of any one of claims 59-67, wherein the polynucleotide is codon optimized for expression in human cells.

### Claim 69 (depends on 60, 61, 62, 63, 64, 65, 66, 67, 68)

The polynucleotide of any one of claims 60-68, comprising a nucleic acid sequence at least about 70%, 75%, 80%, 85%, 90%, 95%, or 98% identical to or complementary with full length SEQ ID NO: 204 or 207.

### Claim 70 (depends on 60, 61, 62, 63, 64, 65, 66, 67, 68)

The polynucleotide of any one of claims 60-68, comprising a nucleic acid sequence at least about 80%, 85%, 90%, 95%, or 98% identical to or complementary with full length SEQ ID NO: 204 or 207.

### Claim 71 (depends on 59, 60, 61, 62, 63, 64, 65, 66, 67, 68)

The polynucleotide of any one of claims 59-68, comprising a nucleic acid sequence at least about 95% identical to or complementary with full length SEQ ID NO: 204 or 207.

### Claim 72 (depends on 59, 60, 61, 62, 63, 64, 65, 66, 67, 68)

The polynucleotide of any one of claims 59-68, comprising a nucleic acid sequence 100% identical to or complementary with full length SEQ ID NO: 204 or 207.

### Claim 73 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58)

A cell producing the mutant TcBuster transposase or fusion transposase of any one of claims 1-58.

### Claim 74 (depends on 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72)

A cell containing the polynucleotide of any one of claims 59-72.

### Claim 75 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26)

A method comprising: introducing into a cell the mutant TcBuster transposase of any one of claims 1-26 and a transposon recognizable by the mutant TcBuster transposase.

### Claim 76 (depends on 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58)

A method comprising: introducing into a cell the fusion transposase of any one of claims 27-58 and a transposon recognizable by the fusion transposase.

### Claim 77 (depends on 75, 76)

The method of claim 75 or 76, wherein the introducing comprises contacting the cell with a polynucleotide encoding the mutant TcBuster transposase or the fusion transposase.

### Claim 78 (depends on 77)

The method of claim 77, wherein the polynucleotide comprises DNA that encodes the mutant TcBuster transposase or the fusion transposase.

### Claim 79 (depends on 77)

The method of claim 77, wherein the polynucleotide comprises messenger RNA (mRNA) that encodes the mutant TcBuster transposase or the fusion transposase.

### Claim 80 (depends on 79)

The method of claim 79, wherein the mRNA is chemically modified.

### Claim 81 (depends on 75, 76, 77, 78, 79, 80)

The method of any one of claims 75-80, wherein the introducing comprises contacting the cell with a DNA vector that contains the transposon.

### Claim 82 (depends on 81)

The method of claim 81, wherein the DNA vector comprises a mini-circle plasmid.

### Claim 83 (depends on 77, 78, 79, 80, 81, 82)

The method of any one of claims 77-82, wherein the introducing comprises contacting the cell with a plasmid vector that contains both the transposon and the polynucleotide encoding the mutant TcBuster transposase or the fusion transposase.

### Claim 84 (depends on 75, 76, 77, 78, 79, 80, 81, 82, 83)

The method of any one of claims 75-83, wherein the introducing comprises contacting the cell with the mutant TcBuster transposase or the fusion transposase as a purified protein.

### Claim 85 (depends on 75, 76, 77, 78, 79, 80, 81, 82, 83, 84)

The method of any one of claims 75-84, wherein the transposon comprises a cargo cassette positioned between two inverted repeats.

### Claim 86 (depends on 85)

The method of claim 85, wherein a left inverted repeat of the two inverted repeats comprises a sequence having at least 50%, at least 60%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% identity to SEQ ID NO: 3.

### Claim 87 (depends on 85)

The method of claim 85, wherein a left inverted repeat of the two inverted repeats comprises SEQ ID NO: 3.

### Claim 88 (depends on 85, 86, 87)

The method of any one of claims 85-87, wherein a right inverted repeat of the two inverted repeats comprises a sequence having at least 50%, at least 60%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% identity to SEQ ID NO: 4.

### Claim 89 (depends on 85, 86, 87)

The method of any one of claims 85-87, wherein a right inverted repeat of the two inverted repeats comprises SEQ ID NO: 4.

### Claim 90 (depends on 85)

The method of claim 85, wherein a left inverted repeat of the two inverted repeats comprises a sequence having at least 50%, at least 60%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% identity to SEQ ID NO: 5.

### Claim 91 (depends on 85)

The method of claim 85, wherein a left inverted repeat of the two inverted repeats comprises SEQ ID NO: 5.

### Claim 92 (depends on 85, 90)

The method of claim 85, 90, or 91, wherein a right inverted repeat of the two inverted repeats comprises a sequence having at least 50%, at least 60%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% identity to SEQ ID NO: 6.

### Claim 93 (depends on 85, 90)

The method of claim 85, 90, or 91, wherein a right inverted repeat of the two inverted repeats comprises SEQ ID NO: 6.

### Claim 94 (depends on 85)

The method of claim 85, wherein a left inverted repeat of the two inverted repeats comprises a sequence having at least 50%, at least 60%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% identity to SEQ ID NO: 205.

### Claim 95 (depends on 85)

The method of claim 85, wherein a left inverted repeat of the two inverted repeats comprises SEQ ID NO: 205.

### Claim 96 (depends on 85, 94)

The method of claim 85, 94, or 95, wherein a right inverted repeat of the two inverted repeats comprises a sequence having at least 50%, at least 60%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% identity to SEQ ID NO: 206.

### Claim 97 (depends on 85, 94)

The method of claim 85, 94, or 95, wherein a right inverted repeat of the two inverted repeats comprises SEQ ID NO: 206.

### Claim 98 (depends on 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97)

The method of any one of claims 85-97, wherein the cargo cassette comprises a promoter selected from the group consisting of: CMV, EFS, MND, EF1α, CAGCs, PGK, UBC, U6, H1, and Cumate.

### Claim 99 (depends on 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98)

The method of any one of claims 85-98, wherein the cargo cassette comprises a CMV promoter.

### Claim 100 (depends on 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99)

The method of any one of claims 85-99, wherein the cargo cassette is present in a forward direction.

### Claim 101 (depends on 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99)

The method of any one of claims 85-99, wherein the cargo cassette is present in a reverse direction.

### Claim 102 (depends on 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101)

The method of any one of claims 75-101, wherein the introducing comprises transfecting the cell with the aid of electroporation, microinjection, calcium phosphate precipitation, cationic polymers, dendrimers, liposome, microprojectile bombardment, fugene, direct sonic loading, cell squeezing, optical transfection, protoplast fusion, impalefection, magnetofection, nucleofection, or any combination thereof.

### Claim 103 (depends on 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102)

The method of any one of claims 75-102, wherein the introducing comprises electroporating the cell.

### Claim 104 (depends on 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103)

The method of any one of claims 75-103, wherein the cell is a primary cell isolated from a subject.

### Claim 105 (depends on 104)

The method of claim 104, wherein the subject is a human.

### Claim 106 (depends on 104, 105)

The method of claim 104 or 105, wherein the subject is a patient with a disease.

### Claim 107 (depends on 104, 105, 106)

The method of any one of claims 104-106, wherein the subject has been diagnosed with cancer or tumor.

### Claim 108 (depends on 104, 105, 106, 107)

The method of any one of claims 104-107, wherein the cell is isolated from blood of the subject.

### Claim 109 (depends on 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108)

The method of any one of claims 75-108, wherein the cell comprises a primary immune cell.

### Claim 110 (depends on 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109)

The method of any one of claims 75-109, wherein the cell comprises a primary leukocyte.

### Claim 111 (depends on 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110)

The method of any one of claims 75-110, wherein the cell comprises a primary T cell.

### Claim 112 (depends on 111)

The method of claim 111, wherein the primary T cell comprises a gamma delta T cell, a helper T cell, a memory T cell, a natural killer T cell, an effector T cell, or any combination thereof

### Claim 113 (depends on 109, 110, 111, 112)

The method of any one of claims 109-112, wherein the primary immune cell comprises a CD3+ cell.

### Claim 114 (depends on 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113)

The method of any one of claims 75-113, wherein the cell comprises a stem cell.

### Claim 115 (depends on 114)

The method of claim 114, wherein the stem cell is selected from the group consisting of:
embryonic stem cell, hematopoietic stem cell, epidermal stem cell, epithelial stem cell, bronchoalveolar stem cell, mammary stem cell, mesenchymal stem cell, intestine stem cell, endothelial stem cell, neural stem cell, olfactory adult stem cell, neural crest stem cell, testicular cell, and any combination thereof.

### Claim 116 (depends on 114)

The method of claim 114, wherein the stem cell comprises induced pluripotent stem cell.

### Claim 117 (depends on 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116)

The method of any one of claims 85-116, wherein the cargo cassette comprises a transgene.

### Claim 118 (depends on 117)

The method of claim 117, wherein the transgene codes for a protein selected from the group consisting of: a cellular receptor, an immunological checkpoint protein, a cytokine, and any combination thereof.

### Claim 119 (depends on 117, 118)

The method of claim 117 or 118, wherein the transgene codes for a cellular receptor selected from the group consisting of: a T cell receptor (TCR), a B cell receptor (BCR), a chimeric antigen receptor (CAR), or any combination thereof

### Claim 120 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58)

A method of treatment, comprising:
(a) introducing into a cell a transposon and the mutant TcBuster transposase or the fusion transposase of any one of claims 1-58, which recognize the transposon, thereby generating a genetically modified cell; and
(b) administering the genetically modified cell to a patient in need of the treatment.

### Claim 121 (depends on 120)

The method of claim 120, wherein the genetically modified cell comprises a transgene introduced by the transposon.

### Claim 122 (depends on 120, 121)

The method of claim 120 or 121, wherein the patient has been diagnosed with cancer or tumor.

### Claim 123 (depends on 120, 121, 122)

The method of any one of claims 120-122, wherein the administering comprises transfusing the genetically modified cell into blood vessels of the patient.

### Claim 124 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58)

A system for genome editing, comprising: the mutant TcBuster transposase or the fusion transposase of any one of claims 1-58, and a transposon recognizable by the mutant TcBuster transposase or the fusion transposase.

### Claim 125 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58)

A system for genome editing, comprising: the polynucleotide encoding a mutant TcBuster transposase or the fusion transposase of any one of claims 1-58, and a transposon recognizable by the mutant TcBuster transposase or the fusion transposase.

### Claim 126 (depends on 125)

The system of claim 125, wherein the polynucleotide comprises a nucleic acid sequence at least 80% identical to or complementary with SEQ ID NO: 204 or 207.

### Claim 127 (depends on 125)

The system of claim 125, wherein the polynucleotide comprises DNA that encodes the mutant TcBuster transposase or the fusion transposase.

### Claim 128 (depends on 125, 127)

The system of claim 125 or 127, wherein the polynucleotide comprises messenger RNA (mRNA) that encodes the mutant TcBuster transposase or the fusion transposase.

### Claim 129 (depends on 128)

The system of claim 128, wherein the mRNA is chemically modified.

### Claim 130 (depends on 124, 125, 126, 127, 128, 129)

The system of any one of claims 124-129, wherein the transposon is present in a DNA vector.

### Claim 131 (depends on 130)

The system of claim 130, wherein the DNA vector comprises a mini-circle plasmid.

### Claim 132 (depends on 125, 126, 127, 128, 129, 130, 131)

The system of any one of claims 125-131, wherein the polynucleotide and the transposon are present in a same plasmid.

### Claim 133 (depends on 124, 125, 126, 127, 128, 129, 130, 131, 132)

The system of any one of claims 124-132, wherein the transposon comprises a cargo cassette positioned between two inverted repeats.

### Claim 134 (depends on 133)

The system of claim 133, wherein a left inverted repeat of the two inverted repeats comprises a sequence having at least 50%, at least 60%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% identity to SEQ ID NO: 3.

### Claim 135 (depends on 133)

The system of claim 133, wherein a left inverted repeat of the two inverted repeats comprises SEQ ID NO: 3.

### Claim 136 (depends on 133, 134, 135)

The system of any one of claims 133-135, wherein a right inverted repeat of the two inverted repeats comprises a sequence having at least 50%, at least 60%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% identity to SEQ ID NO: 4.

### Claim 137 (depends on 133, 134, 135)

The system of any one of claims 133-135, wherein a right inverted repeat of the two inverted repeats comprises SEQ ID NO: 4.

### Claim 138 (depends on 133)

The system of claim 133, wherein a left inverted repeat of the two inverted repeats comprises a sequence having at least 50%, at least 60%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% identity to SEQ ID NO: 5.

### Claim 139 (depends on 133)

The system of claim 133, wherein a left inverted repeat of the two inverted repeats comprises SEQ ID NO: 5.

### Claim 140 (depends on 133, 138)

The system of claim 133, 138, or 139, wherein a right inverted repeat of the two inverted repeats comprises a sequence having at least 50%, at least 60%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% identity to SEQ ID NO: 6.

### Claim 141 (depends on 133, 138)

The system of claim 133, 138, or 139, wherein a right inverted repeat of the two inverted repeats comprises SEQ ID NO: 6.

### Claim 142 (depends on 133)

The system of claim 133, wherein a left inverted repeat of the two inverted repeats comprises a sequence having at least 50%, at least 60%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% identity to SEQ ID NO: 205.

### Claim 143 (depends on 133)

The system of claim 133, wherein a left inverted repeat of the two inverted repeats comprises SEQ ID NO: 205.

### Claim 144 (depends on 133, 142)

The system of any one of claim 133, 142, or 143, wherein a right inverted repeat of the two inverted repeats comprises a sequence having at least 50%, at least 60%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% identity to SEQ ID NO: 206.

### Claim 145 (depends on 133, 142)

The system of any one of claim 133, 142, or 143, wherein a right inverted repeat of the two inverted repeats comprises SEQ ID NO: 206.

### Claim 146 (depends on 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145)

The system of any one of claims 133-145, wherein the cargo cassette comprises a promoter selected from the group consisting of: CMV, EFS, MND, EF1α, CAGCs, PGK, UBC, U6, H1, and Cumate.

### Claim 147 (depends on 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145)

The system of any one of claims 133-145, wherein the cargo cassette comprises a CMV promoter.

### Claim 148 (depends on 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147)

The system of any one of claims 133-147, wherein the cargo cassette comprises a transgene.

### Claim 149 (depends on 148)

The system of claim 148, wherein the transgene codes for a protein selected from the group consisting of: a cellular receptor, an immunological checkpoint protein, a cytokine, and any combination thereof.

### Claim 150 (depends on 148, 149)

The system of claim 148 or 149, wherein the transgene codes for a cellular receptor selected from the group consisting of: a T cell receptor (TCR), a B cell receptor (BCR), a chimeric antigen receptor (CAR), or any combination thereof.

### Claim 151 (depends on 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150)

The system of any one of claims 133-150, wherein the cargo cassette is present in a forward direction.

### Claim 152 (depends on 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150)

The system of any one of claims 133-150, wherein the cargo cassette is present in a reverse direction.