Chimeric transposases and uses thereof
AssigneePoseida Therapeutics, Inc.
InventorsJoseph S. LUCAS, Blair B. MADISON, Olga BATALOV
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Abstract
Provided herein are chimeric transposases and chimeric site-specific fusion proteins, polynucleotides encoding the chimeric transposases and chimeric site-specific fusion proteins, and vectors and transposons comprising the polynucleotides. Also provided are methods of making the chimeric transposases and chimeric fusion proteins, cells that are modified using the chimeric transposases or chimeric site-specific fusion proteins provided herein and methods using such cells.
Claims31
A chimeric transposase comprising, in N-terminal to C-terminal order:
- (i) a target-specific DNA binding domain, (ii) a truncated Super piggyBac (SPB) transposase comprising a C-terminal Cysteine Rich Domain (CRD) deletion within amino acid residues 535-594 of the sequence of the SPB comprising the sequence set forth in SEQ ID NO:
- 1, and (iii) one or more MosI DNA binding domain(s).
A chimeric transposon inverted terminal repeat (ITR) polynucleotide, comprising in the 5′ to 3′ direction:
- (i) a polynucleotide comprising nucleotides 1-16 of the nucleic acid sequence set forth in SEQ ID NO:
- 12 and (ii) a polynucleotide comprising two MosI DNA binding sites.
A chimeric transposon ITR polynucleotide, comprising in the 5′ to 3′ direction:
- (i) a polynucleotide comprising nucleotides 1-16 of the nucleic acid sequence set forth in SEQ ID NO:
- 65 and (ii) a polynucleotide comprising two MosI DNA binding sites.
A LINE1-targeting chimeric site-specific transposase fusion protein comprising, in the N-terminal to C-terminal direction, (i) a left or a right LINE1 TAL-targeting DNA binding domain, (ii) a linker sequence, and (iii) a chimeric PB:MosI transposase.
A transposon, comprising (i) a chimeric LE PB:MosI ITR polynucleotide and (ii) a chimeric RE PB:MosI ITR polynucleotide, wherein the LE PB:MosI ITR polynucleotide and an RE PB:MosI ITR polynucleotide comprise the same nucleic acid sequence.
A transposon, comprising (i) a chimeric LE PB:MosI ITR polynucleotide comprising the nucleic acid sequence of SEQ ID NO:
- 54 and (ii) a chimeric RE PB:MosI ITR polynucleotide comprising the nucleic acid sequence of SEQ ID NO:
- 55.
A transposon comprising the nucleic acid sequence of SEQ ID NO:
- 56.
A method for site-specific transposition of a DNA molecule into the genome of a cell, comprising introducing into the cell:
- a) a nucleic acid encoding a chimeric site-specific transposase fusion protein comprising a DNA binding domain and a chimeric transposase comprising, in N-terminal to C-terminal order:
- a piggyBac transposase comprising a C-terminal Cysteine Rich Domain (CRD) deletion and one or more MosI DNA binding domain(s);
- b) wherein the fusion protein is expressed in the cell;
- and
- c) a DNA molecule comprising a transposon comprising a chimeric LE transposon ITR polynucleotide and a chimeric RE transposon ITR polynucleotide;
- wherein the expressed chimeric site-specific transposase fusion protein integrates the transposon by site-specific transposition into the TTAA sequence of cellular genome.
A method for generating an engineered cell by site-specific transposition, comprising introducing into the cell:
- a) a nucleic acid encoding a chimeric site-specific transposase fusion protein comprising a DNA binding domain and a chimeric transposase comprising, in N-terminal to C-terminal order:
- a piggyBac transposase comprising a C-terminal Cysteine Rich Domain (CRD) deletion and one or more MosI DNA binding domain(s);
- b) wherein the chimeric site-specific transposase fusion protein is expressed in the cell;
- and
- c) a DNA molecule comprising a transposon comprising a chimeric LE transposon ITR polynucleotide and a chimeric RE transposon ITR polynucleotide;
- wherein the expressed chimeric site-specific transposase fusion protein integrates the transposon by site-specific transposition into the TTAA sequence into the genome of the cell thereby generating the engineered cell.
Dependencies are inferred from the claim text.