Enhanced hAT Family Transposon-Mediated Gene Transfer and Associated Compositions, Systems, and Methods

Abstract

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

What is claimed is: 
     
         1 . A mutant TcBuster transposase comprising an amino acid sequence at least 70% identical to full-length SEQ ID NO: 1 and amino acid substitutions V377T, E469K, D189A,K573E, E578L, or any combination thereof, when numbered in accordance with SEQ ID NO: 1. 
     
     
         2 . The mutant TcBuster transposase of  claim 1 , comprising amino acid substitutions V377T, E469K, and D189A, when numbered in accordance with SEQ ID NO: 1. 
     
     
         3 . The mutant TcBuster transposase of  claim 1 , comprising amino acid substitutions K573E and E578L, when numbered in accordance with SEQ ID NO: 1. 
     
     
         4 . The mutant TcBuster transposase of  claim 1 , 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 when measured by an assay that comprises introducing the mutant 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 the population of cells. 
     
     
         5 . The mutant TcBuster transposase of  claim 1 , comprising the amino acid sequence at least 90% identical to SEQ ID NO: 1. 
     
     
         6 . The mutant TcBuster transposase of  claim 1 , further comprising amino acid substitution I452F, when numbered in accordance with SEQ ID NO: 1. 
     
     
         7 . The mutant TcBuster transposase of  claim 1 , further comprising amino acid substitution N85S, when numbered in accordance with SEQ ID NO: 1. 
     
     
         8 . The mutant TcBuster transposase of  claim 1 , further comprising amino acid substitution A358K, when numbered in accordance with SEQ ID NO: 1. 
     
     
         9 . The mutant TcBuster transposase of  claim 1 , further comprising amino acid substitution K573E, E578L, or K573E and E578L, when numbered in accordance with SEQ ID NO: 1. 
     
     
         10 . The mutant TcBuster transposase of  claim 1 , further comprising amino acid substitution V297K, when numbered in accordance with SEQ ID NO: 1. 
     
     
         11 . A polynucleotide encoding a mutant TcBuster transposase comprising an amino acid sequence at least 70% identical to full-length SEQ ID NO: 1 and amino acid substitutions V377T, E469K, D189A,K573E, E578L, or any combination thereof, when numbered in accordance with SEQ ID NO: 1. 
     
     
         12 . A method of genome editing, comprising: introducing into a cell a mutant TcBuster transposase or a polynucleotide encoding the mutant TcBuster transposase, and a transposon recognizable by the mutant TcBuster transposase, wherein the mutant TcBuster transposase comprises an amino acid sequence at least 70% identical to full-length SEQ ID NO: 1 and amino acid substitutions V377T, E469K, D189A, K573E, E578L, or any combination thereof, when numbered in accordance with SEQ ID NO: 1. 
     
     
         13 . The method of  claim 12 , comprising amino acid substitutions V377T, E469K, and D189A, when numbered in accordance with SEQ ID NO: 1. 
     
     
         14 . The method of  claim 12 , comprising amino acid substitutions K573E and E578L, when numbered in accordance with SEQ ID NO: 1. 
     
     
         15 . The method of  claim 12 , 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 when measured by an assay that comprises introducing the mutant 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 the population of cells. 
     
     
         16 . The method of  claim 12 , wherein the introducing comprises electroporating the cell. 
     
     
         17 . The method of  claim 12 , wherein the cell comprises a primary cell isolated from a subject. 
     
     
         18 . The method of  claim 17 , wherein the subject has a cancer or a tumor. 
     
     
         19 . The method of  claim 17 , wherein the cell comprises a primary immune cell. 
     
     
         20 . A system for genome editing, comprising: a mutant TcBuster transposase or a polynucleotide encoding the mutant TcBuster transposase, and a transposon recognizable by the mutant TcBuster transposase or the fusion transposase, wherein the mutant TcBuster transposase comprises an amino acid sequence at least 70% identical to full-length SEQ ID NO: 1 and amino acid substitutions V377T, E469K, D189A, K573E, E578L, or any combination thereof, when numbered in accordance with SEQ ID NO: 1. 
     
     
         21 . The system of  claim 20 , wherein the mutant TcBuster transposase comprises amino acid substitutions V377T, E469K, and D189A, when numbered in accordance with SEQ ID NO: 1. 
     
     
         22 . The system of  claim 20 , wherein the mutant TcBuster transposase comprises amino acid substitutions K573E and E578L, when numbered in accordance with SEQ ID NO: 1. 
     
     
         23 . The system of  claim 20 , comprising the polynucleotide encoding the mutant TcBuster transposase or the fusion transposase that is messenger RNA (mRNA). 
     
     
         24 . The system of  claim 23 , wherein the mRNA is chemically modified. 
     
     
         25 . The system of  claim 20 , wherein the transposon is present in a mini-circle plasmid. 
     
     
         26 . The system of  claim 20 , wherein the transposon comprises a cargo cassette positioned between two inverted repeats. 
     
     
         27 . The system of  claim 26 , wherein a left inverted repeat of the two inverted repeats comprises a sequence having at least 80% identity to SEQ ID NO: 3. 
     
     
         28 . The system of  claim 26 , wherein a right inverted repeat of the two inverted repeats comprises a sequence having at least at least 80%identity to SEQ ID NO: 4. 
     
     
         29 . The system of  claim 26 , wherein the cargo cassette is in a reverse direction. 
     
     
         30 . The system of  claim 26 , wherein the cargo cassette comprises a transgene coding 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.