SYSTEMS AND METHODS FOR TRANSPOSING CARGO NUCLEOTIDE SEQUENCES
The present disclosure provides systems and methods for transposing a cargo nucleotide sequence to a target nucleic acid site. These systems and methods may comprise a first double-stranded nucleic acid comprising the cargo nucleotide sequence, wherein the cargo nucleotide sequence is configured to interact with a transposase, and the transposase, wherein said transposase is configured to transpose the cargo nucleotide sequence to the target nucleic acid site.
1 .- 156 . (canceled)
157 . An engineered transposase system, comprising:
(a) a double-stranded nucleic acid comprising a heterologous cargo nucleotide sequence, wherein said heterologous cargo nucleotide sequence is configured to interact with a transposase; and
(b) said transposase, wherein:
(i) said transposase is configured to transpose said heterologous cargo nucleotide sequence to a target nucleic acid locus; and
(ii) said transposase comprises a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 7, 8, 5, 6, 1-4, or 9-349.
158 . The engineered transposase system of claim 157 , wherein said transposase comprises a sequence having at least 90% sequence identity to SEQ ID NO: 7.
159 . The engineered transposase system of claim 157 , wherein said transposase comprises a sequence having at least 90% sequence identity to SEQ ID NO: 8.
160 . The engineered transposase system of claim 157 , wherein said transposase comprises a sequence having at least 90% sequence identity to SEQ ID NO: 5.
161 . The engineered transposase system of claim 157 , wherein said transposase comprises a sequence having at least 90% sequence identity to SEQ ID NO: 6.
162 . The engineered transposase system of claim 157 , wherein said transposase comprises one or more nuclear localization sequences (NLSs) proximal to an N-terminus or a C-terminus of said transposase.
163 . The engineered transposase system of claim 162 , wherein said one or more NLSs comprise a sequence having at least 80% sequence identity to a sequence from the group consisting of SEQ ID NOs: 455-470
164 . The engineered transposase system of claim 157 , wherein said double-stranded nucleic acid comprising a flanking sequence flanking said heterologous cargo nucleotide sequence, wherein said flanking sequence comprises a sequence having at least 70% sequence identity to at least 90 consecutive nucleotides of any one of SEQ ID NOs: 356, 357, 355, 354, 350-353, or 358-454.
165 . The engineered transposase system of claim 157 , wherein said transposase is configured to recognize an insertion motif adjacent to said target nucleic acid locus.
166 . The engineered transposase system of claim 165 , wherein said insertion motif comprises at least three consecutive nucleotides of the sequence AATGAC.
167 . The engineered transposase system of claim 157 , wherein said double-stranded nucleic acid comprising said heterologous cargo nucleotide sequence and said transposase are in a cell, and wherein said target nucleic acid locus is in said cell.
168 . The engineered transposase system of claim 167 , wherein said cell is a eukaryotic cell.
169 . A method for modifying a target nucleic acid locus, comprising:
contacting said target nucleic acid locus with:
a transposase comprising a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 7, 8, 5, 6, 1-4, or 9-349,
wherein said transposase is complexed with a double-stranded deoxyribonucleic acid polynucleotide comprising a heterologous cargo nucleotide sequence.
170 . The method of claim 169 , wherein said transposase comprises a sequence having at least 90% sequence identity to SEQ ID NO: 7.
171 . The method of claim 169 , wherein said transposase comprises a sequence having at least 90% sequence identity to SEQ ID NO: 8.
172 . The method of claim 169 , wherein said transposase comprises a sequence having at least 90% sequence identity to SEQ ID NO: 5.
173 . The method of claim 169 , wherein said transposase comprises a sequence having at least 90% sequence identity to SEQ ID NO: 6.
174 . The method of claim 169 , wherein said target nucleic acid locus is in a cell.
175 . The method of claim 169 , wherein said transposase inserts said heterologous cargo nucleotide sequence into said target nucleic acid locus.
176 . An engineered transposase system, comprising:
(a) a double-stranded nucleic acid comprising a heterologous cargo nucleotide sequence, wherein said heterologous cargo nucleotide sequence is configured to interact with a transposase; and
(b) said transposase,
wherein said transposase is configured to transpose said heterologous cargo nucleotide sequence to a target nucleic acid locus, and
wherein said double-stranded nucleic acid comprises a flanking sequence flanking said heterologous cargo nucleotide sequence, wherein said flanking sequence has at least 70% sequence identity to at least 90 consecutive nucleotides of any one of SEQ ID NOs: 356, 357, 355, 354, 350-353, or 358-454.