Methods and compositions for modulating a genome
Methods and compositions for modulating a target genome are disclosed. The composition may comprise a first RNA encoding a polypeptide comprising a retrotransposase reverse transcriptase domain and a retrotransposase endonuclease domain. The composition may also comprise a second RNA comprising a sequence that binds the polypeptide and a heterologous object sequence. The composition may insert the sequence of the heterologous object sequence into a target DNA.
1. A composition for modifying DNA, the composition comprising a first engineered RNA and a second engineered RNA, wherein:
(a) the first engineered RNA encodes a polypeptide, wherein the polypeptide comprises (i) a reverse transcriptase domain and (ii) a endonuclease domain, wherein the polypeptide comprises an amino acid sequence according to any of SEQ ID NOs: 1016-1021; and
(b) the second engineered RNA comprises a template RNA, wherein the template RNA comprises (i) a sequence according to SEQ ID NO: 1140 that binds the encoded polypeptide and (ii) a heterologous object sequence, wherein (a) and (b) are separate RNAs.
2. The composition of claim 1 , wherein the template RNA further comprises a sequence comprising at least 20 nucleotides of at least 80% identity to a target DNA.
3. The composition of claim 2 , wherein the target DNA is a genomic safe harbor (GSH) site.
4. The composition of claim 1 , wherein the template RNA comprises: (iii) at least 10 bases of identity to a target DNA at the 3′ end of the template RNA, and (iv) at least 10 bases of identity to the target DNA at the 5′ end of the template RNA.
5. The composition of claim 1 , wherein the template RNA comprises: (iii) at least 3 bases of identity to a target DNA at the 3′ end of the template RNA, and (iv) at least 3 bases of identity to the target DNA at the 5′ end of the template RNA.
6. The composition of claim 1 , wherein the sequence that binds the polypeptide comprises one or both of a 3′ untranslated region and a 5′ untranslated region.
7. The composition of claim 1 , wherein the heterologous object sequence encodes an enzyme, a membrane protein, a blood factor, an intracellular protein, an extracellular protein, a structural protein, a signaling protein, a regulatory protein, a transport protein, or a motor protein.
8. The composition of claim 1 , wherein the heterologous object sequence comprises a non-coding sequence or a regulatory sequence.
9. The composition of claim 1 , wherein the heterologous object sequence encodes a therapeutic polypeptide or fragment thereof.
10. The pharmaceutical composition of claim 8 , wherein the regulatory sequence is a promoter.
11. The composition of claim 1 , wherein the polypeptide further comprises one or both of a nuclear localization signal and a nucleolar localization signal.
12. The composition of claim 1 , which comprises only RNA, or comprises more RNA than DNA by an RNA:DNA ratio of at least 10:1, 20:1, 30:1, 40:1, 50:1, 60:1, 70:1, 80:1, 90:1, or 100:1.
13. The composition of claim 1 , which comprises no more than 1%, 0.5%, 0.2%, 0.1%, 0.05%, 0.02%, or 0.01% DNA by mass or by molar amount of nucleic acid.
14. The composition of claim 1 , which is capable of modifying DNA in the absence of homologous recombination activity.
15. The composition of claim 1 , wherein the template RNA does not encode a reverse transcriptase.
16. The composition of claim 1 , wherein the template RNA does not encode an endonuclease.