Methods and compositions for genomic integration

A composition comprising a genome editing system comprising (i) one or more synthetic or recombinant RNA molecules comprising:

• (I)(a) a sequence encoding a Cas endonuclease, wherein the Cas endonuclease is Cas9 nickase capable of cleavage of a strand of genomic DNA at a target site;
• and
• (b) a guide RNA, wherein the guide RNA comprises a sequence capable of guiding the Cas endonuclease to the target site of the genomic DNA to create a 5′-P terminus and a 3′-OH terminus at the target site,
• (II) (a) a cassette comprising
• (i) a sequence encoding human LINE1 ORF2p, wherein the human LINE1 ORF2p comprises a reverse transcriptase (RT) domain capable of target-primed reverse transcription (TPRT) at the 3′-OH terminus at the target site and an endonuclease (EN) domain:
• wherein the sequence encoding human LINE1 ORF2p is fused to a first sequence encoding a nuclear localization sequence (NLS) that is N-terminal to the sequence encoding the human LINE1 ORF2p and a second sequence encoding an NLS sequence that is C-terminal to the sequence encoding the human LINE1 ORF2p to facilitate nuclear import, wherein the human LINE1 ORF2p comprises a sequence with at least 95% sequence identity to SEQ ID NO:
• 55;
• and wherein the LINE1 ORF2p comprises a D205 mutation that abrogates endonuclease activity;
• and
• (ii) a sequence encoding human LINE1 ORF1p;
• (b) an insert sequence, wherein the insert sequence comprises a sequence encoding an exogenous therapeutic polypeptide;
• (c) a 5′ nucleic acid sequence flanking the insert sequence and upstream of the insert sequence, wherein the 5′ nucleic acid sequence is an untranslated region (UTR), and
• (d) a 3′ nucleic acid sequence flanking the insert sequence and downstream of the insert sequence, wherein the 3′ nucleic acid sequence is an untranslated region (UTR) and comprises:
• (i) a genomic DNA priming sequence capable of binding to and priming the genomic DNA at the target site cleaved by the Cas endonuclease, and
• (ii) binding site for the human LINE1 ORF2p, wherein the genomic DNA priming sequence and the binding site for the human LINE1 ORF2p are capable of starting TPRT at the 3′-OH terminus at the target site by the human LINE1 ORF2p for incorporating the insert sequence.