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 retrotransposase, and the retrotransposase, wherein said retrotransposase is configured to transpose the cargo nucleotide sequence to the target nucleic acid site.
1 .- 91 . (canceled)
92 . An engineered retrotransposase system comprising:
(a) a double-stranded nucleic acid comprising a cargo nucleotide sequence, wherein said cargo nucleotide sequence is configured to interact with a heterologous retrotransposase; and
(b) said heterologous retrotransposase, wherein:
(i) said heterologous retrotransposase is configured to transpose said cargo nucleotide sequence to a target nucleic acid locus; and
(ii) said heterologous retrotransposase comprises a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 1-16, 32-41, 61, or 42-45.
93 . The engineered retrotransposase system of claim 92 , wherein said heterologous retrotransposase comprises a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 1-16.
94 . The engineered retrotransposase system of claim 92 , wherein said heterologous retrotransposase comprises a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 32-41 or 61.
95 . The engineered retrotransposase system of claim 92 , wherein said heterologous retrotransposase comprises a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 42-44.
96 . The engineered retrotransposase system of claim 92 , wherein said heterologous retrotransposase comprises a sequence having at least 80% sequence identity to SEQ ID NO: 45.
97 . The engineered retrotransposase system of claim 92 , wherein said cargo nucleotide sequence is flanked by a 3′ untranslated region (UTR) and a 5′ untranslated region (UTR).
98 . The engineered retrotransposase system of claim 92 , wherein said engineered retrotransposase system is comprised in a cell, wherein said cell further comprises a deoxyribonucleic acid polynucleotide encoding said heterologous retrotransposase.
99 . The engineered retrotransposase system of claim 92 , wherein said heterologous retrotransposase comprises a nuclear localization sequence (NLS).
100 . The engineered retrotransposase system of claim 99 , wherein said NLS comprises a sequence having at least 80% sequence identity to a sequence of any one of SEQ ID NOs: 17-32.
101 . The engineered retrotransposase system of claim 98 , wherein said cell is a eukaryotic cell.
102 . A method for synthesizing complementary deoxyribonucleic acid (cDNA), the method comprising:
(a) providing a ribonucleic acid (RNA) template in a cell, wherein said RNA template comprises a cargo nucleotide sequence and said cell comprises a nucleic acid primer to initiate cDNA synthesis from said RNA template; and
(b) synthesizing cDNA initiated by said nucleic acid primer using said RNA template and a heterologous retrotransposase, wherein said heterologous retrotransposase comprises a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 1-16, 32-41, 61, or 42-45.
103 . The method of claim 102 , wherein said heterologous retrotransposase comprises a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 1-16.
104 . The method of claim 102 , wherein said heterologous retrotransposase comprises a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 32-41 or 61.
105 . The method of claim 102 , wherein said heterologous retrotransposase comprises a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 42-44.
106 . The method of claim 102 , wherein said heterologous retrotransposase comprises a sequence having at least 80% sequence identity to SEQ ID NO: 45.
107 . The method of claim 102 , wherein said cargo nucleotide sequence is flanked by a 3′ untranslated region (UTR) and a 5′ untranslated region (UTR).
108 . The method of claim 102 , wherein said RNA template and said heterologous retrotransposase are encoded by a deoxyribonucleic acid polynucleotide in said cell.
109 . The method of claim 102 , wherein said heterologous retrotransposase comprises a nuclear localization sequence (NLS).
110 . The method of claim 109 , wherein said NLS comprises a sequence having at least 80% sequence identity to a sequence of any one of SEQ ID NOs: 17-32.
111 . The method of claim 102 , wherein said cell is a eukaryotic cell.