Engineered mobile genetic element systems

Abstract

Systems and methods for targeted insertion. Engineered nucleic acid targeting systems comprise components of non-LTR retrotransposon elements. Specifically, the recombinant mobile element systems of the present disclosure are derived from R2Tg, a non-long terminal repeat (nLTR) retrotransposon from the zebra finch Taeniopygia guttata.

Description

Attorney Docket No.: 098791-000104WOPT ENGINEERED MOBILE GENETIC ELEMENT SYSTEMS CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims benefit under 35 U.S.C. § 119(e) of U.S. Provisional Application No.63 / 608,489 filed December 11, 2023, the contents of which are incorporated herein by reference in their entirety. SEQUENCE LISTING

[0002] The instant application contains a Sequence Listing which has been submitted in XML format via Patent Center and is hereby incorporated by reference in its entirety. Said XML copy, created on December 9, 2024, is named 098791-000104WOPT_SL.xml and is 115,701bytes in size. TECHNOLOGY FIELD

[0003] The present disclosure relates to recombinant mobile element systems and uses thereof. Specifically, the recombinant mobile element systems of the present disclosure are derived from R2Tg, which is a non-long terminal repeat (nLTR) retrotransposon from the zebra finch Taeniopygia guttata. BACKGROUND

[0004] R2Tg is a non-long terminal repeat (nLTR) retrotransposon from the zebra finch Taeniopygia guttata. These mobile genetic elements (MGEs) have a single open reading frame (ORF) flanked by 5’ and 3’ UTR sequences. Like other nLTR retrotransposons, R2Tg RNA inserts itself via template-primed reverse transcription into the multicopy 28S rDNA locus. The determinants of this specificity are poorly defined, and possibly derive from both the DNA binding domains of the protein and short homologous sequences outside of either (or both) of the UTR sequences.

[0005] As an engineered therapeutic, a nLTR retrotransposon system must have a high full-length insertion efficiency at a target site. In contrast, evidence from a range of MGEs 1 4900-8642-4067.5Attorney Docket No.: 098791-000104WOPT indicates that these systems have generally not evolved to maximize one-off insertion efficiency. In addition, nLTR systems have been observed to show truncated products, suggesting that the insertion process is interrupted before reaching the 5’ terminus of the donor RNA. However, current transposition systems only find use in laboratory applications. Therapeutic uses have proven elusive. There is a need for novel and safer retrotransposon systems for this technology to find use in medicine. SUMMARY

[0006] Accordingly, this disclosure describes, in part, a recombinant mobile element system derived from R2Tg, which is a non-long terminal repeat (nLTR) retrotransposon from the zebra finch Taeniopygia guttata, having at least one mutation that increases the 3’ insertion, the 5’ insertion, or both.

[0007] In one aspect, described herein is a genome editing system comprising: i) an R2Tg enzyme comprises an amino acid sequence SEQ ID NO:1 an R2Tg enzyme comprises an amino acid sequence SEQ ID NO:1 (wt sequence) with a mutation selected from the group consisting of D944K, S870R, E638K, T1367R, G1056R, and combinations thereof; ii) a payload RNA, wherein the payload RNA comprises an insertion template and optionally one or more of a 5’ homology arm, a 3’ homology arm, and a protein binding element, wherein the insertion template comprises a sequence for a nucleic acid insertion into the genome.

[0008] In one aspect, described herein is a genome editing system comprising: i) an R2Tg enzyme comprises an amino acid sequence SEQ ID NO:2 or comprises a variant of a protein effector comprising an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 2; ii) a payload RNA, wherein the payload RNA comprises an insertion template and optionally one or more of a 5’ homology arm, a 3’ homology arm, and a protein binding element, wherein the insertion template comprises a sequence for a nucleic acid insertion into the genome.

[0009] In some embodiments of any of the aspects, the 5’ homology arm is less than or about 105 base pairs, is less than or about 50 base pairs, is less than or about 25 base pairs.

[0010] In some embodiments of any of the aspects, the 3’ homology arm is less than or about 50 base pairs, is less than or about 25 base pairs, is less than or about 10 base pairs.

[0011] In some embodiments of any of the aspects, the R2Tg element enzyme further comprises a targeting domain. 2 4900-8642-4067.5Attorney Docket No.: 098791-000104WOPT

[0012] In some embodiments of any of the aspects, the targeting domain is a natural targeting domain or an engineered targeting domain

[0013] In some embodiments of any of the aspects, the targeting domain is a natural targeting domain.

[0014] In some embodiments of any of the aspects, the targeting domain is an engineered targeting domain.

[0015] In some embodiments of any of the aspects, the nucleic acid insertion into the genome is a DNA or RNA insertion template.

[0016] In some embodiments of any of the aspects, the R2Tg element enzyme is a modified R2Tg element enzyme.

[0017] In some embodiments of any of the aspects, the coding sequence of the R2Tg element enzyme is modified.

[0018] In some embodiments of any of the aspects, the modified R2Tg element enzyme further comprises a mutation selected from S645K, E638K, V661R, G257R, G236R, I558R, D555R, S870R, T934R, C587K, E638K, V661R, L388R, E471R, L243R, D923K, S763R, I308R, T1016R, T1023K, G1056R, N1103K, Y1242K, T1367R or combinations thereof.

[0019] In some embodiments of any of the aspects, the modified R2Tg element enzyme is modified by an N-terminal or C-terminal truncation of the R2Tg element enzyme sequence.

[0020] In some embodiments of any of the aspects, the modified R2Tg element enzyme is modified by an N-terminal truncation of the R2Tg element enzyme sequence.

[0021] In some embodiments of any of the aspects, the modified R2Tg element enzyme comprises SEQ ID. NO:3.

[0022] In some embodiments of any of the aspects, the genome editing system targets a genomic locus.

[0023] In some embodiments of any of the aspects, the genome editing system targets a genomic locus other than the 28S rRNA locus.

[0024] In some embodiments of any of the aspects, an N-terminal zinc finger domain of the R2Tg element enzyme is modified to target a genomic locus other than the 28S rRNA locus.

[0025] In some embodiments of any of the aspects, the genomic locus is selected from the target site of SEQ ID NO:12 to 86.

[0026] In some embodiments of any of the aspects, a non-naturally occurring targeting region is fused to the N-terminus of the R2Tg element enzyme or inserted into the R2Tg element enzyme. 3 4900-8642-4067.5Attorney Docket No.: 098791-000104WOPT

[0027] In some embodiments of any of the aspects, the modified R2Tg element enzyme is a fusion protein.

[0028] In some embodiments of any of the aspects, the modified R2Tg element is fused to a Cas9 protein that is fully active, catalytically dead (H840A / D10A for SpCas9), or functioning as a nickase (H840A or D10A for SpCas9).

[0029] In some embodiments of any of the aspects, the modified R2Tg element is fused to a Cas12 protein that is fully active, catalytically dead, or functioning as a nickase.

[0030] In some embodiments of any of the aspects, the genome editing system further comprises a guide RNA.

[0031] In some embodiments of any of the aspects, the modified R2Tg element is fused to a TALEN protein, zinc finger protein, argonaute, or meganuclease protein.

[0032] In some embodiments of any of the aspects, the genome editing system further comprises a guide RNA.

[0033] In some embodiments of any of the aspects, the 5’ homology arm, the 3’ homology arm, or both the 5’ and 3’ homology arm of the payload RNA is engineered to target a genomic locus other than the 28 S rRNA locus.

[0034] In some embodiments of any of the aspects, the 5’ homology arm, the 3’ homology arm, or both the 5’ and 3’ homology arm target an exogenously introduced landing sequence.

[0035] In some embodiments of any of the aspects, the insertion region is introduced into the genome of a specific cell type.

[0036] In some embodiments of any of the aspects, the specific cell type is a post-mitotic cell.

[0037] In some embodiments of any of the aspects, the genome editing system functions in post-mitotic cells.

[0038] In some embodiments of any of the aspects, the genome editing system functions independently from intrinsic nucleic acid repair systems.

[0039] In some embodiments of any of the aspects, the payload RNA template further comprises a 5’ untranslated region (UTR), a 3’ UTR, or both a 5’ UTR and a 3’ UTR.

[0040] In some embodiments of any of the aspects, the 5’ homology arm and the 3’ homology arm are located between the 5’ UTR and 3’ UTR.

[0041] In some embodiments of any of the aspects, the 5’ homology arm and the 3’ homology arm are located outside the 5’ UTR and 3’ UTR. 4 4900-8642-4067.5Attorney Docket No.: 098791-000104WOPT

[0042] In some embodiments of any of the aspects, the payload RNA further comprises a 5’ untranslated region (UTR), a 3’ UTR, or both a 5’ and a 3’ UTR, wherein the UTRs are truncated.

[0043] In some embodiments of any of the aspects, the payload RNA does not comprise a 5’ UTR.

[0044] In some embodiments of any of the aspects, the payload RNA does not comprise a 3’ UTR.

[0045] In some embodiments of any of the aspects, the payload RNA further comprises a nuclear retention element.

[0046] In some embodiments of any of the aspects, the payload RNA further comprises a Cas9 or Cas12 guide RNA, and wherein the Cas9 or Cas12 guide RNA comprises an extension with a 5’ homology sequence, a 3’ homology sequence, a 5’ untranslated region (UTR), a 3’ UTR, an insertion template, or any combination thereof.

[0047] In some embodiments of any of the aspects, the nucleic acid insertion template is a sequence of greater than 1000 base pairs.

[0048] In some embodiments of any of the aspects, the R2Tg element enzyme comprises a nuclear localization signal (NLS).

[0049] In some embodiments of any of the aspects, the insertion region comprises a template for a reporter gene, a transcription factor gene, a transgene, an enzyme gene, or a therapeutic gene.

[0050] In one aspect, described herein is a method of inserting a large nucleic acid into a genome within a cell using a Cas9 or Cas12 fusion protein, wherein the method comprises supplying a Cas9 or Cas12 fusion protein to a cell, wherein the Cas9 or Cas12 fusion protein is supplied with a payload RNA template, wherein the RNA template is reverse transcribed by the Cas9 or Cas12 fusion protein prior to being inserted into the genome of the cell; and wherein the large nucleic acid is inserted into the genome of the cell.

[0051] In some embodiments of any of the aspects, the Cas9 fusion protein comprises a Cas9 portion and an R2Tg element portion.

[0052] In some embodiments of any of the aspects, the Cas9 fusion protein comprises a targeting domain, a reverse transcriptase domain, and a nickase domain.

[0053] In some embodiments of any of the aspects, the Cas12 fusion protein comprises a Cas12 portion and an R2Tg element portion.

[0054] In one aspect, described herein is a method of inserting an exogenous nucleic acid into the genome of a post-mitotic cell, wherein the method comprises subjecting the genome 5 4900-8642-4067.5Attorney Docket No.: 098791-000104WOPT of the post-mitotic cell to a modified Cas9 protein that inserts the exogenous nucleic acid into the genome of the postmitotic cell.

[0055] 48. The method of claim 47, wherein the modified Cas9 protein is fused to an R2Tg element enzyme.

[0056] 49. The method of claim 48, wherein the modified Cas9 fusion protein targets an endogenous landing site.

[0057] 50. The method of claim 48, wherein the Cas9 fusion protein targets an exogenously introduced landing site in the genome of the post-mitotic cell.

[0058] In one aspect, described herein is a method of editing a genome comprising subjecting the cell to a genome editing system as described herein.

[0059] In one aspect, described herein is a method of correcting a genetic mutation related to disease or human pathology, wherein the method comprises making small nucleotide changes or small nucleotide insertions (1-100 bp) in a human genome using a genome editing system as described herein.

[0060] In some embodiments of any of the aspects, the genome editing system is delivered via single or multi vector AAV, adenovirus, lentivirus, herpes simplex virus, PEG10 viral like particles, PNMA viral like particles, gag-like viral like particles, nanoblades, gesicles, or Friend murine leukemia virus (FMLV) viral like proteins.

[0061] In some embodiments of any of the aspects, the components of the genome editing system are delivered as all RNA in lipid nanoparticles or another RNA delivery reagent.

[0062] In some embodiments of any of the aspects, the non-LTR site specific retrotransposon is delivered as mRNA.

[0063] In some embodiments of any of the aspects, the guide RNAs are delivered as synthetic RNA.

[0064] In some embodiments of any of the aspects, the payload is delivered as mRNA.

[0065] In some embodiments of any of the aspects, the genome editing system targets and edits the genome at more than one site. BRIEF DESCRIPTION OF THE DRAWINGS

[0066] An understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention may be utilized, and the accompanying drawings of which: 6 4900-8642-4067.5Attorney Docket No.: 098791-000104WOPT

[0067] FIGS.1A and 1B depict the insertion activities across the range of cargo sizes with different homology arms for R2Tg wild type sequence.

[0068] FIGS.2A and 2B illustrate the insertion activities across the range of cargo sizes with different homology arms for R2Tg sequence with 184 nucleotide truncations at the N- terminus.

[0069] FIG.3 shows the mRNA payload insertion of R2Tg wild type sequence against the R2Tg dead sequence.

[0070] FIGS.4A and 4B illustrate the R2Tg mutant screen for full-length insertion with for R2Tg sequence with 184 nucleotide truncations at the N-terminus. DETAILED DESCRIPTION OF THE INVENTION

[0071] In some embodiments, the invention disclosed herein is a genome editing system comprising: i) an R2Tg enzyme comprises an amino acid sequence SEQ ID NO:2, or comprises a variant of a protein effector comprising an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 2; ii) a payload RNA, wherein the payload RNA comprises an insertion template and optionally one or more of a 5’ homology arm, a 3’ homology arm, and a protein binding element, wherein the insertion template comprises a sequence for a nucleic acid insertion into the genome.

[0072] In some embodiments, the invention disclosed herein is a genome editing system comprising: i) an R2Tg enzyme comprises an amino acid sequence SEQ ID NO:3, or comprises a variant of a protein effector comprising an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 3; ii) a payload RNA, wherein the payload RNA comprises an insertion template and optionally one or more of a 5’ homology arm, a 3’ homology arm, and a protein binding element, wherein the insertion template comprises a sequence for a nucleic acid insertion into the genome.

[0073] In some embodiments SEQ ID NO: 2 comprises a mutation selected from S645K, E638K, V661R, G257R, G236R, I558R, D555R, S870R, T934R, C587K, E638K, V661R, L388R, E471R, L243R, D923K, S763R, I308R, T1016R, T1023K, G1056R, N1103K, Y1242K, T1367R or combinations thereof.

[0074] In some embodiments SEQ ID NO: 2 comprises a mutation selected from S645K, E638K, V661R, G257R, G236R, I558R, D555R, S870R, T934R, C587K, E638K, V661R, 7 4900-8642-4067.5Attorney Docket No.: 098791-000104WOPT L388R, E471R, L243R, D923K, S763R, I308R, T1016R, T1023K, G1056R, N1103K, Y1242K, T1367R or combinations thereof; and wherein SEQ ID NO:2 is modified by an N- terminal truncation. In one embodiment, the N-terminal truncation is at least 50 nucleotides, at least 100 nucleotides, at least 150 nucleotides, at least 180 nucleotides.

[0075] In some embodiments, the invention disclosed herein is a genome editing system comprising: i) an R2Tg enzyme comprises an amino acid sequence SEQ ID NO:4, or comprises a variant of a protein effector comprising an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 4; ii) a payload RNA, wherein the payload RNA comprises an insertion template and optionally one or more of a 5’ homology arm, a 3’ homology arm, and a protein binding element, wherein the insertion template comprises a sequence for a nucleic acid insertion into the genome.

[0076] In some embodiments, the invention disclosed herein is a genome editing system comprising: i) an R2Tg enzyme comprises an amino acid sequence SEQ ID NO:5, or comprises a variant of a protein effector comprising an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% identity to an amino acid sequence selected from the group consisting of SEQ ID NO:5; ii) a payload RNA, wherein the payload RNA comprises an insertion template and optionally one or more of a 5’ homology arm, a 3’ homology arm, and a protein binding element, wherein the insertion template comprises a sequence for a nucleic acid insertion into the genome.

[0077] In some embodiments SEQ ID NO:4 comprises a mutation selected from D944K, S645K, E638K, V661R, G257R, G236R, I558R, D555R, T934R, C587K, E638K, V661R, L388R, E471R, L243R, D923K, S763R, I308R, T1016R, T1023K, G1056R, N1103K, Y1242K, T1367R or combinations thereof.

[0078] In some embodiments SEQ ID NO:4 comprises a mutation selected from D944K, S645K, E638K, V661R, G257R, G236R, I558R, D555R, T934R, C587K, E638K, V661R, L388R, E471R, L243R, D923K, S763R, I308R, T1016R, T1023K, G1056R, N1103K, Y1242K, T1367R or combinations thereof; and wherein SEQ ID NO:4 is modified by an N- terminal truncation. In one embodiment, the N-terminal truncation is at least 50 nucleotides, at least 100 nucleotides, at least 150 nucleotides, at least 180 nucleotides.

[0079] In some embodiments, the invention disclosed herein is a genome editing system comprising: i) an R2Tg enzyme comprises an amino acid sequence SEQ ID NO:6, or comprises a variant of a protein effector comprising an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% identity to an amino acid sequence selected 8 4900-8642-4067.5Attorney Docket No.: 098791-000104WOPT from the group consisting of SEQ ID NO: 6; ii) a payload RNA, wherein the payload RNA comprises an insertion template and optionally one or more of a 5’ homology arm, a 3’ homology arm, and a protein binding element, wherein the insertion template comprises a sequence for a nucleic acid insertion into the genome.

[0080] In some embodiments, the invention disclosed herein is a genome editing system comprising: i) an R2Tg enzyme comprises an amino acid sequence SEQ ID NO:7, or comprises a variant of a protein effector comprising an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 7; ii) a payload RNA, wherein the payload RNA comprises an insertion template and optionally one or more of a 5’ homology arm, a 3’ homology arm, and a protein binding element, wherein the insertion template comprises a sequence for a nucleic acid insertion into the genome.

[0081] In some embodiments SEQ ID NO: 6 comprises a mutation selected from D944K, S645K, V661R, G257R, G236R, I558R, D555R, S870R, T934R, C587K, E638K, V661R, L388R, E471R, L243R, D923K, S763R, I308R, T1016R, T1023K, G1056R, N1103K, Y1242K, T1367R or combinations thereof.

[0082] In some embodiments SEQ ID NO: 6 comprises a mutation selected from D944K, S645K, V661R, G257R, G236R, I558R, D555R, S870R, T934R, C587K, E638K, V661R, L388R, E471R, L243R, D923K, S763R, I308R, T1016R, T1023K, G1056R, N1103K, Y1242K, T1367R or combinations thereof; and wherein SEQ ID NO:6 is modified by an N- terminal truncation. In one embodiment, the N-terminal truncation is at least 50 nucleotides, at least 100 nucleotides, at least 150 nucleotides, at least 180 nucleotides.

[0083] In some embodiments, the invention disclosed herein is a genome editing system comprising: i) an R2Tg enzyme comprises an amino acid sequence SEQ ID NO:8, or comprises a variant of a protein effector comprising an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 8; ii) a payload RNA, wherein the payload RNA comprises an insertion template and optionally one or more of a 5’ homology arm, a 3’ homology arm, and a protein binding element, wherein the insertion template comprises a sequence for a nucleic acid insertion into the genome.

[0084] In some embodiments, the invention disclosed herein is a genome editing system comprising: i) an R2Tg enzyme comprises an amino acid sequence SEQ ID NO:9, or comprises a variant of a protein effector comprising an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% identity to an amino acid sequence selected 9 4900-8642-4067.5Attorney Docket No.: 098791-000104WOPT from the group consisting of SEQ ID NO: 9; ii) a payload RNA, wherein the payload RNA comprises an insertion template and optionally one or more of a 5’ homology arm, a 3’ homology arm, and a protein binding element, wherein the insertion template comprises a sequence for a nucleic acid insertion into the genome.

[0085] In some embodiments SEQ ID NO: 8 comprises a mutation selected from D944K, S645K, E638K, V661R, G257R, G236R, I558R, D555R, S870R, T934R, C587K, E638K, V661R, L388R, E471R, L243R, D923K, S763R, I308R, T1016R, T1023K, G1056R, N1103K, Y1242K or combinations thereof.

[0086] In some embodiments SEQ ID NO: 8 comprises a mutation selected from D944K, S645K, E638K, V661R, G257R, G236R, I558R, D555R, S870R, T934R, C587K, E638K, V661R, L388R, E471R, L243R, D923K, S763R, I308R, T1016R, T1023K, G1056R, N1103K, Y1242K or combinations thereof; and wherein SEQ ID NO:8 is modified by an N- terminal truncation. In one embodiment, the N-terminal truncation is at least 50 nucleotides, at least 100 nucleotides, at least 150 nucleotides, at least 180 nucleotides.

[0087] In some embodiments, the invention disclosed herein is a genome editing system comprising: i) an R2Tg enzyme comprises an amino acid sequence SEQ ID NO:10, or comprises a variant of a protein effector comprising an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 10; ii) a payload RNA, wherein the payload RNA comprises an insertion template and optionally one or more of a 5’ homology arm, a 3’ homology arm, and a protein binding element, wherein the insertion template comprises a sequence for a nucleic acid insertion into the genome.

[0088] In some embodiments, the invention disclosed herein is a genome editing system comprising: i) an R2Tg enzyme comprises an amino acid sequence SEQ ID NO:11, or comprises a variant of a protein effector comprising an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 11; ii) a payload RNA, wherein the payload RNA comprises an insertion template and optionally one or more of a 5’ homology arm, a 3’ homology arm, and a protein binding element, wherein the insertion template comprises a sequence for a nucleic acid insertion into the genome.

[0089] In some embodiments SEQ ID NO: 10 comprises a mutation selected from D944K, S645K, E638K, V661R, G257R, G236R, I558R, D555R, S870R, T934R, C587K, E638K, V661R, L388R, E471R, L243R, D923K, S763R, I308R, T1016R, T1023K, N1103K, Y1242K, T1367R or combinations thereof. 10 4900-8642-4067.5Attorney Docket No.: 098791-000104WOPT

[0090] In some embodiments SEQ ID NO: 10 comprises a mutation selected from D944K, S645K, E638K, V661R, G257R, G236R, I558R, D555R, S870R, T934R, C587K, E638K, V661R, L388R, E471R, L243R, D923K, S763R, I308R, T1016R, T1023K, N1103K, Y1242K, T1367R or combinations thereof; and wherein SEQ ID NO:10 is modified by an N- terminal truncation. In one embodiment, the N-terminal truncation is at least 50 nucleotides, at least 100 nucleotides, at least 150 nucleotides, at least 180 nucleotides.

[0091] TABLE 1 MASCPKPGPPVSAGAMSLESGLTTHSVLAIERGPNSLANSGSDFGGGGLG D  1 11 4900-8642-4067.5Attorney Docket No.: 098791-000104WOPT ID  2 12 4900-8642-4067.5Attorney Docket No.: 098791-000104WOPT ID  3 13 4900-8642-4067.5Attorney Docket No.: 098791-000104WOPT ID  4 14 4900-8642-4067.5Attorney Docket No.: 098791-000104WOPT ID  5 15 4900-8642-4067.5Attorney Docket No.: 098791-000104WOPT ID  6 16 4900-8642-4067.5Attorney Docket No.: 098791-000104WOPT ID  7 17 4900-8642-4067.5Attorney Docket No.: 098791-000104WOPT ID  8 18 4900-8642-4067.5Attorney Docket No.: 098791-000104WOPT ID  9 19 4900-8642-4067.5Attorney Docket No.: 098791-000104WOPT ID  10 20 4900-8642-4067.5Attorney Docket No.: 098791-000104WOPT D  11

[0092] Unless otherwise defined, all technical and / or scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of the invention, exemplary methods and / or materials are described below. In case of conflict, the patent specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and are not intended to be necessarily limiting. General Definitions

[0093] In the discussion unless otherwise stated, adjectives such as “substantially” and “about” modifying a condition or relationship characteristic of a feature or features of an embodiment of the invention, are understood to mean that the condition or characteristic is defined to within tolerances that are acceptable for operation of the embodiment for an application for which it is intended. Unless otherwise indicated, the word “or” in the 21 4900-8642-4067.5Attorney Docket No.: 098791-000104WOPT specification and claims is considered to be the inclusive “or” rather than the exclusive or, and indicates at least one of and any combination of items it conjoins.

[0094] It should be understood that the terms “a” and “an” as used above and elsewhere herein refer to “one or more” of the enumerated components. It will be clear to one of ordinary skill in the art that the use of the singular includes the plural unless specifically stated otherwise. Therefore, the terms “a,” “an” and “at least one” are used interchangeably in this application.

[0095] For purposes of better understanding the present teachings and in no way limiting the scope of the teachings, unless otherwise indicated, all numbers expressing quantities, percentages or proportions, and other numerical values used in the specification and claims, are to be understood as being modified in all instances by the term “about.” Accordingly, unless indicated to the contrary, the numerical parameters set forth in the following specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained. At the very least, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.

[0096] The terms "one or more" or "at least one" or "X or more", where X is a number and understand to mean X or increases one by one of X, such as one or more or at least one member(s) or "X or more" of a group of members, is clear per se, by means of further exemplification, the term encompasses inter alia a reference to any one of said members, or to any two or more of said members, such as, e.g., any >3, >4, >5, >6 or >7 etc. of said members, and up to all said members.

[0097] It is understood that where a numerical range is recited herein, the present invention contemplates each integer between, and including, the upper and lower limits, unless otherwise stated.

[0098] In the description and claims of the present application, each of the verbs, “comprise,” “include” and “have” and conjugates thereof, are used to indicate that the object or objects of the verb are not necessarily a complete listing of components, elements or parts of the subject or subjects of the verb. Other terms as used herein are meant to be defined by their well-known meanings in the art.

[0099] As used herein, the term “targeting sequence” or “targeting molecule” refers a nucleotide sequence or molecule comprising a nucleotide sequence that is capable of hybridizing to a specific target sequence, e.g., the targeting sequence has a nucleotide sequence which is at least partially complementary to the sequence being targeted along the 22 4900-8642-4067.5Attorney Docket No.: 098791-000104WOPT length of the targeting sequence. For example, the targeting sequence or targeting molecule may be part of a RNA guide molecule that can form a complex with a CRISPR nuclease. When the RNA guide molecule comprising the targeting sequence is present contemporaneously with the CRISPR nuclease, the RNA guide molecule is capable of targeting the CRISPR nuclease to the specific target sequence. In another example, the targeting sequence or targeting molecule may comprise a homology arm that targets a RNA template molecule to a target site for insertion or copying of an insert template sequence at the target site. Each possibility represents a separate embodiment.

[0100] As used herein, “cell-specific,” or “cell-type specific,” would be understood by one of skill in the art to mean occurring or being expressed at a higher frequency or existing at an increased level in one cell type in contrast to other cell types.

[0101] The term “nucleic acid” is understood to refer to both ribonucleic acid (RNA) and deoxyribonucleic acid (DNA) molecules. This may include chemically synthesized nucleic acid molecules, single stranded or double stranded nucleic acid molecules, linearized nucleic acid molecules, circularized nucleic acid molecules, chemically modified nucleic acid molecules, and nucleic acids with biochemical modifications. “Nucleic acid” refers to both RNA and DNA molecules including, without limitation, complementary DNA (“cDNA”), genomic DNA (“gDNA”), and messenger RNA (“mRNA”), and also includes synthetic nucleic acid molecules, such as those that are chemically synthesized or recombinantly produced, such as RNA templates, as described herein. The nucleic acid molecule can be double-stranded or single-stranded, circular, or linear.\

[0102] As used herein, “insertion” of a sequence into a target site refers to the net addition of DNA sequence at the target site, e.g., where there are new nucleotides in the heterologous object sequence with no cognate positions in the unedited target site. In some embodiments, a nucleotide alignment of the PBS sequence and heterologous object sequence to the target nucleic acid sequence would result in an alignment gap in the target nucleic acid sequence.

[0103] As used herein, a “deletion” generated by a heterologous object sequence in a target site refers to the net deletion of DNA sequence at the target site, e.g., where there are nucleotides in the unedited target site with no cognate positions in the heterologous object sequence. In some the target nucleic acid sequence would result in an alignment gap in the molecule comprising the PBS sequence and heterologous object sequence.

[0104] The term “mutation region,” as used herein, refers to a region in a template nucleic acid having one or more sequence difference relative to the corresponding sequence 23 4900-8642-4067.5Attorney Docket No.: 098791-000104WOPT in a target nucleic acid. The sequence difference may comprise, for example, a substitution, insertion, frameshift, or deletion.

[0105] The term “mutated” when applied to nucleic acid sequences means that nucleotides in a nucleic acid sequence are inserted, deleted, or changed compared to a reference (e.g., native) nucleic acid sequence. A single alteration may be made at a locus (a point mutation), or multiple nucleotides may be inserted, deleted, or changed at a single locus. In addition, one or more alterations may be made at any number of loci within a nucleic acid sequence. A nucleic acid sequence may be mutated by any method known in the art.

[0106] The term “fusion protein” is used herein to refer to protein construction comprising the site-specific nuclease connected to the non-LTR retrotransposon polypeptide for example by a polypeptide linker or other suitable linker. It should be understood that the term “fusion protein” includes embodiments where the composition comprises a site-specific nuclease and a non-LTR retrotransposon already connected to one another, or embodiments where the site-specific and non-LTR retrotransposon comprise two separate components that may come together to form a single complex, for example, through the use of engineered domains on each polypeptide that functions as binding partners to bring the site-specific and non-LTR retrotransposon together.

[0107] The terms "non-naturally occurring" or "engineered" are used interchangeably and indicate human manipulation. The terms, when referring to nucleic acid molecules or polypeptides may mean that the nucleic acid molecule or the polypeptide is at least substantially free from at least one other component with which they are naturally associated in nature and as found in nature.

[0108] As used herein, “genomic DNA” refers to linear and / or chromosomal DNA and / or to plasmid or other extrachromosomal DNA sequences present in the cell or cells of interest. In some embodiments, the cell of interest is a eukaryotic cell. In some embodiments, the cell of interest is a prokaryotic cell. In some embodiments, the methods produce double-stranded breaks (DSBs) at pre-determined target sites in a genomic DNA sequence, resulting in mutation, insertion, and / or deletion of DNA sequences at the target site(s) in a genome.

[0109] The terms "nuclear localization sequence" and "NLS" are used interchangeably to indicate an amino acid sequence / peptide that directs the transport of a protein with which it is associated from the cytoplasm of a cell across the nuclear envelope barrier. The term "NLS" is intended to encompass not only the nuclear localization sequence of a particular peptide, but also derivatives thereof that are capable of directing translocation of a cytoplasmic 24 4900-8642-4067.5Attorney Docket No.: 098791-000104WOPT polypeptide across the nuclear envelope barrier. NLSs are capable of directing nuclear translocation of a polypeptide when attached to the N-terminus, the C-terminus, or both the N- and C-termini of the polypeptide. In addition, a polypeptide having an NLS coupled by its N-terminus or C-terminus to amino acid side chains located randomly along the amino acid sequence of the polypeptide will be translocated. Typically, an NLS consists of one or more short sequences of positively charged lysines or arginines exposed on the protein surface, but other types of NLS are known.

[0110] The RNA template may further comprise additional portions. For example, the RNA template may comprise binding sites for proteins having reverse transcriptase activity to facilitate reverse transcription of the RNA template. For example, the RNA template may comprise a portion, e.g., of one or more nucleotides, that are complimentary to a target nucleic acid site. Such a portion of an RNA template is referred to as a “homology arm.” A homology arm may be 4-10, 10-20, 20-50, 50-100, 100-200, 200-400 nucleotides in length or longer.

[0111] An RNA template molecule may be designed, for example, for correction of a mutant gene or for increased expression of a wild-type gene. It will be readily apparent that an insert template portion of an RNA template molecule is typically not identical to a sequence of a genomic target site that the RNA insert template will replace. For example, an RNA template may contain a non-homologous insert template sequence flanked by one or two homology arms, or regions that share homology to a target site, in order to facilitate introduction of the non-homologous insert template sequence at the target site.

[0112] An insert template portion of an RNA template molecule may comprise a sequence selected from the group consisting of a gene encoding a protein (e.g., a coding sequence encoding a protein that is lacking in the cell or in the individual or an alternate version of a gene encoding a protein), a regulatory sequence and / or a sequence that encodes a structural nucleic acid such as a microRNA or siRNA. An insert template generally contains at least one sequence difference relative to the target site sequence. Accordingly, the at least one sequence difference is an alteration intended to be introduced into the target site sequence. The at least one difference results in an introduction of a new sequence, deletion of the original target site sequence, or substitution of the target site sequence for a different sequence, or any combination of the above.

[0113] An RNA molecule comprising an RNA template portion may further comprise a RNA guide portion. The RNA guide portion may include a scaffold region that binds to a CRISPR nuclease portion of the inventive fusion protein described herein. The RNA guide 25 4900-8642-4067.5Attorney Docket No.: 098791-000104WOPT portion may be a single guide RNA (sgRNA). An RNA molecule comprising an RNA template portion and a RNA guide portion may be arranged in any conformation, e.g. the RNA template portion may be upstream or downstream of the RNA guide portion. Furthermore, the RNA template portion and the RNA guide portion may be connected to each other by a linker portion. The linker portion may be 1-10, 10-20, 20-50, 50-100 nucleotides in length or longer.

[0114] In some embodiments, the site specific nuclease may comprise a paired nickase in which each site-specific nuclease in the pair is fused with a non-LTR retrotransposon protein and creates a nick on opposing strands of a targeted insertion site and whereby the corresponding R2Tg non-LTR retrotransposons facilitate insertion of the donor polynucleotide from the donor construct.

[0115] In some embodiments, the site-specific nuclease is a Cas polypeptide and the composition further comprises a guide molecule capable of forming a complex with the Cas polypeptide and directing the Cas polypeptide-non-LTR retrotransposon polypeptide to a target site adjacent to the targeted insertion site.

[0116] In some embodiments, the guide directs the polypeptides (e.g., a complex or fusion protein of the Cas and non-LTR retrotransposon polypeptide) to a target sequence 5’ or 3’ of the targeted insertion site, and wherein the Cas polypeptide generates a double-strand break at the targeted insertion site.

[0117] In some embodiments, the R2Tg element naturally targets the 28S rRNA locus. The instant disclosure contemplates the insertion of payloads into either the 28 S rRNA locus or into other genomic loci. In some embodiments, the insertion site is a targeted genomic insertion site. In some embodiments, the insertion site is targeted by a targeting domain in a fusion protein. In some embodiments, the insertion site is targeted by a targeting domain in a landing pad. In some embodiments, the insertion site has been exogenously introduced to the genome (i.e. via lentivirus, or recombinase, and the like).

[0118] In some embodiments, the instant disclosure also contemplates additional R2Tg non-LTR site-specific retrotransposons for use in or as part of the genome editing system described herein that do not target the 28S rRNA locus. In some embodiments, the genome is targeted for a large genetic insertion. In some embodiments, the insertion site is a targeted genomic insertion site. In some embodiments, the insertion site is targeted by a targeting domain in a fusion protein. In some embodiments, the insertion site has been exogenously introduced to the genome (i.e. via lentivirus, or recombinase, and the like). 26 4900-8642-4067.5Attorney Docket No.: 098791-000104WOPT

[0119] In some embodiments, targeted genomic insertion site is selected from the target site in Table 2 or Table 3.

[0120] TABLE 2 iSEQ ID NO:Attorney Docket No.: 098791-000104WOPT

[0121] TABLE 3 Safe harbor region  SEQ ID NO: Attorney Docket No.: 098791-000104WOPT

[0122] The term “payload” as used herein means at least a nucleic acid that may be integrated into a host genome. Thus, “payload RNA” will be understood to comprise an RNA molecule comprising at least an insertion region, wherein the insertion region can be integrated into a host genome. Payloads of the instant disclosure may encode proteins, such as enzymes. In some embodiments, the payload may act as a regulatory element. Thus, if an embodiment of the disclosure states, by way of example, that “the payload comprises a therapeutic protein,” it is generally understood that the payload comprises a template that, upon insertion, will lead to expression of a therapeutic protein encoded by the template.

[0123] In some embodiments, the insertion region comprises a template for a reporter gene. In some embodiments, the reporter gene encodes a fluorescent protein. In some embodiments, the reporter gene encodes a green fluorescent protein. In some embodiments, the reporter gene encodes eGFP.

[0124] In some embodiments, the insertion region comprises a template for a transcription factor gene.

[0125] In some embodiments, the insertion region comprises a template for a transgene.

[0126] In some embodiments, the insertion region comprises a template for an enzyme gene, or a therapeutic gene. In some embodiments, the therapeutic protein can be used in conjunction with another therapeutic.

[0127] In some embodiments, the payload comprises a 5’UTR. In some embodiments, the payload comprises a 3’UTR. In some embodiments, the payload comprises a 5’UTR and a 3’ UTR. In some embodiments, the payload consists of a 5’UTR. In some embodiments, the payload consists of a 3’UTR. In some embodiments, the payload comprises a 5’UTR and a 5’ homology arm. In some embodiments, the payload comprises a 3’UTR and a 3’ homology arm. In some embodiments, the payload comprises a 5’UTR, a 5’ homology arm, a 3’UTR and a 3’ homology arm. In some embodiments, the payload comprises a 5’ homology arm, a 3’UTR and a 3’ homology arm. In some embodiments, the payload comprises a 5’UTR, a 5’ homology arm, and a 3’ homology arm. In some embodiments, the payload comprises a 5’ homology arm and a 3’ homology arm. In some embodiments, the 3’ homology arm comprises less than 30 base pairs. In some embodiments the 3’ homology arm comprises less than 20 base pairs. In some embodiments, the 3’ homology arm comprises less than 10 base pairs. In some embodiments, the 3’ homology arm comprises less than 5 base pairs.

[0128] In some embodiments, the polypeptides herein (e.g., site-specific nuclease polypeptides, the R2Tg non-LTR retrotransposon polypeptide, or fusion protein thereof) may 29 4900-8642-4067.5Attorney Docket No.: 098791-000104WOPT further comprise (e.g., fused to) one or more nuclear localization sequences (NLSs), such as about or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more NLSs. In some embodiments, the polypeptides and proteins comprise about or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more NLSs at or near the amino-terminus, about or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more NLSs at or near the carboxy-terminus, or a combination of these (e.g. zero or at least one or more NLS at the amino-terminus and zero or at one or more NLS at the carboxy terminus). The NLS(s) may be at an internal location of the protein, i.e., not at the C-terminus or N-terminus. When more than one NLS is present, each may be selected independently of the others, such that a single NLS may be present in more than one copy and / or in combination with one or more other NLSs present in one or more copies. In a preferred embodiment of the invention, the polypeptides comprise at most 6 NLSs. In some embodiments, an NLS is considered near the N- or C-terminus when the nearest amino acid of the NLS is within about 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 40, 50, or more amino acids along the polypeptide chain from the N- or C-terminus.

[0129] In some embodiments, at least one nuclear localization signal (NLS) is attached to the nucleic acid sequences encoding the polypeptides. In preferred embodiments at least one or more C-terminal or N-terminal NLSs are attached (and hence nucleic acid molecule(s) coding for the Cas protein can include coding for NLS(s) so that the expressed product has the NLS(s) attached or connected). In a preferred embodiment a C-terminal NLS is attached for optimal expression and nuclear targeting in eukaryotic cells, preferably human cells. The invention also encompasses methods for delivering multiple nucleic acid components, wherein each nucleic acid component is specific for a different target locus of interest thereby modifying multiple target loci of interest. The nucleic acid component of the complex may comprise one or more protein-binding RNA aptamers. The one or more aptamers may be capable of binding a bacteriophage coat protein.

[0130] In some embodiments, an NLS comprises the amino acid sequence MDSLLMNRRKFLYQFKNVRWAKGRRETYLC (SEQ ID NO: 87), PKKRKVEGADKRTADGSEFESPKKKRKV (SEQ ID NO: 88), RKSGKIAAIWKRPRKPKKKRKV (SEQ ID NO: 89), KRTADGSEFESPKKKRKV (SEQ ID NO: 90), KKTELQTTNAENKTKKL (SEQ ID NO: 91), KRGINDRNFWRGENGRKTR (SEQ ID NO: 92), KRPAATKKAGQAKKKK (SEQ ID NO: 93), or a functional fragment or variant thereof. Exemplary NLS sequences are also described in PCT / EP2000 / 011690, the contents of which are incorporated herein by reference for their disclosure of exemplary nuclear localization sequences. In some embodiments, an NLS comprises an amino acid 30 4900-8642-4067.5Attorney Docket No.: 098791-000104WOPT sequence as disclosed in Table 8. An NLS of this table may be utilized with one or more copies in a polypeptide in one or more locations in a polypeptide, e.g., 1, 2, 3 or more copies of an NLS in an N-terminal domain, between peptide domains, in a C-terminal domain, or in a combination of locations, in order to improve subcellular localization to the nucleus. Multiple unique sequences may be used within a single polypeptide. Sequences may be naturally monopartite or bipartite, e.g., having one or two stretches of basic amino acids, or may be used as chimeric bipartite sequences. Sequence references correspond to UniProt accession numbers, except where indicated as SeqNLS for sequences mined using a subcellular localization prediction algorithm (Lin et al BMC Bioinformat 13:157 (2012), incorporated herein by reference in its entirety).

[0131] In some embodiments, the NLS is a bipartite NLS. A bipartite NLS typically comprises two basic amino acid clusters separated by a spacer sequence (which may be, e.g., about 10 amino acids in length). A monopartite NLS typically lacks a spacer. An example of a bipartite NLS is the nucleoplasmin NLS, having the sequence KR[PAATKKAGQA]KKKK (SEQ ID NO: 93), wherein the spacer is bracketed (SEQ ID NO: 94). Another exemplary bipartite NLS has the sequence PKKKRKVEGADKRTADGSEFESPKKKRKV (SEQ ID NO: 95). Exemplary NLSs are described in International Application WO2020051561, which is herein incorporated by reference in its entirety, including for its disclosures regarding nuclear localization sequences.

[0132] In some embodiments, the NLS modification, UTRs modifications and various other components are provided in International Application No. WO2022198014, WO2023039407, WO2020252361, WO2021102042, WO2023069972, WO2023069948, and WO2022173830, each of which is herein incorporated by reference in its entirety. Delivery

[0133] The present disclosure also provides delivery systems for introducing components of the systems and compositions herein to cells, tissues, organs, or organisms. A delivery system may comprise one or more delivery vehicles and / or cargos. Exemplary delivery systems and methods include those described in paragraphs

[0117] to

[0278] of Feng Zhang et al., (WO2016106236A1), and pages 1241-1251 and Table 1 of Lino CA et al., Delivering CRISPR: a review of the challenges and approaches, DRUG DELIVERY, 2018, VOL.25, NO.1, 1234-1257, which are incorporated by reference herein in their entireties.

[0134] In some embodiments, the delivery systems may be used to introduce the components of the systems and compositions to plant cells. For example, the components 31 4900-8642-4067.5Attorney Docket No.: 098791-000104WOPT may be delivered to plant using electroporation, microinjection, aerosol beam injection of plant cell protoplasts, biolistic methods, DNA particle bombardment, and / or Agrobacterium- mediated transformation. Examples of methods and delivery systems for plants include those described in Fu et al., Transgenic Res.2000 Feb;9(l):l l-9; Klein RM, et al., Biotechnology.

[0135] 1992;24:384-6; Casas AM et al., Proc Natl Acad Sci U S A.1993 Dec 1; 90(23): 11212-11216; and U.S. Pat. No.5,563,055, Davey MR et al., Plant Mol Biol.1989 Sep; 13(3):273-85, which are incorporated by reference herein in their entireties.

[0136] The delivery systems may comprise one or more cargos. The cargos may comprise one or more components of the systems and compositions herein. A cargo may comprise one or more of the following: i) one or more plasmids encoding the engineered proteins; (ii) mRNA molecules encoding the engineered proteins; (iii) the engineered proteins. In some examples, a cargo may comprise a plasmid encoding one or more engineered proteins herein. Physical delivery

[0137] In some embodiments, the cargos may be introduced to cells by physical delivery methods. Examples of physical methods include microinjection, electroporation, and hydrodynamic delivery. Both nucleic acid and proteins may be delivered using such methods. For example, the engineered protein or mRNA thereof may be prepared in vitro , isolated, (refolded, purified if needed), and introduced to cells. Microinjection

[0138] Microinjection of the cargo directly to cells can achieve high efficiency, e.g., above 90% or about 100%. In some embodiments, microinjection may be performed using a

[0139] microscope and a needle (e.g., with 0.5-5.0 pm in diameter) to pierce a cell membrane and deliver the cargo directly to a target site within the cell. Microinjection may be used for in vitro and ex vivo delivery.

[0140] Plasmids comprising coding sequences for the engineered proteins may be microinjected. In some cases, microinjection may be used i) to deliver DNA directly to a cell nucleus, and / or ii) to deliver mRNA (e.g., in vitro transcribed) to a cell nucleus or cytoplasm.

[0141] Microinjection may be used to generate genetically modified animals. For example, gene editing cargos may be injected into zygotes to allow for efficient germline modification. Such approach can yield normal embryos and full-term mouse pups harboring the desired modification(s). 32 4900-8642-4067.5Attorney Docket No.: 098791-000104WOPT Electroporation

[0142] In some embodiments, the cargos and / or delivery vehicles may be delivered by electroporation. Electroporation may use pulsed high-voltage electrical currents to transiently open nanometer-sized pores within the cellular membrane of cells suspended in buffer, allowing for components with hydrodynamic diameters of tens of nanometers to flow into the cell. In some cases, electroporation may be used on various cell types and efficiently transfer cargo into cells. Electroporation may be used for in vitro and ex vivo delivery.

[0143] Electroporation may also be used to deliver the cargo to into the nuclei of mammalian cells by applying specific voltage and reagents, e.g., by nucleofection. Such approaches include those described in Wu Y, et al. (2015). Cell Res 25:67-79; Ye L, et al. (2014). Proc Natl Acad Sci USA 111:9591-6; Choi PS, Meyerson M. (2014). Nat Commun 5:3728; Wang J, Quake SR. (2014). Proc Natl Acad Sci 111:13157-62. Electroporation may also be used to deliver the cargo in vivo , e.g., with methods described in Zuckermann M, et al. (2015). Nat Commun 6:7391. Hydrodynamic delivery

[0144] Hydrodynamic delivery may also be used for delivering the cargos, e.g., for in vivo delivery. In some examples, hydrodynamic delivery may be performed by rapidly pushing a large volume (8-10% body weight) solution containing the gene editing cargo into the bloodstream of a subject (e.g., an animal or human), e.g., for mice, via the tail vein. As blood is incompressible, the large bolus of liquid may result in an increase in hydrodynamic pressure that temporarily enhances permeability into endothelial and parenchymal cells, allowing for cargo not normally capable of crossing a cellular membrane to pass into cells. This approach may be used for delivering naked DNA plasmids and proteins. The delivered cargos may be enriched in liver, kidney, lung, muscle, and / or heart. Transfection

[0145] The cargos, e.g., nucleic acids, may be introduced to cells by transfection methods for introducing nucleic acids into cells. Examples of transfection methods include calcium phosphate-mediated transfection, cationic transfection, liposome transfection, dendrimer transfection, heat shock transfection, magnetofection, lipofection, impalefection, optical transfection, proprietary agent-enhanced uptake of nucleic acid. 33 4900-8642-4067.5Attorney Docket No.: 098791-000104WOPT Delivery vehicles

[0146] The delivery systems may comprise one or more delivery vehicles. The delivery vehicles may deliver the cargo into cells, tissues, organs, or organisms (e.g., animals or plants). The cargos may be packaged, carried, or otherwise associated with the delivery vehicles. The delivery vehicles may be selected based on the types of cargo to be delivered, and / or the delivery is in vitro and / or in vivo. Examples of delivery vehicles include vectors, viruses, non-viral vehicles, and other delivery reagents described herein.

[0147] The delivery vehicles in accordance with the present invention may a greatest dimension (e.g. diameter) of less than 100 microns (pm). In some embodiments, the delivery vehicles have a greatest dimension of less than 10 pm. In some embodiments, the delivery vehicles may have a greatest dimension of less than 2000 nanometers (nm). In some embodiments, the delivery vehicles may have a greatest dimension of less than 1000 nanometers (nm). In some embodiments, the delivery vehicles may have a greatest dimension (e.g., diameter) of less than 900 nm, less than 800 nm, less than 700 nm, less than 600 nm, less than 500 nm, less than 400 nm, less than 300 nm, less than 200 nm, less than 150nm, or less than 100nm, less than 50nm. In some embodiments, the delivery vehicles may have a greatest dimension ranging between 25 nm and 200 nm.

[0148] In some embodiments, the delivery vehicles may be or comprise particles. For example, the delivery vehicle may be or comprise nanoparticles (e.g., particles with a greatest dimension (e.g., diameter) no greater than 1000nm. The particles may be provided in different forms, e.g., as solid particles (e.g., metal such as silver, gold, iron, titanium), non- metal, lipid-based solids, polymers), suspensions of particles, or combinations thereof. Metal, dielectric, and semiconductor particles may be prepared, as well as hybrid structures (e.g., core-shell particles).

[0149] Nanoparticles may also be used to deliver the compositions and systems to plant cells, e.g., as described in WO 2008042156, US 20130185823, and WO2015089419. Vectors

[0150] The systems, compositions, and / or delivery systems may comprise one or more vectors. The present disclosure also includes vector systems. A vector system may comprise one or more vectors. In some embodiments, a vector refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. Vectors include nucleic acid molecules that are single-stranded, double-stranded, or partially double-stranded; nucleic acid molecules that comprise one or more free ends, no free ends (e.g., circular); nucleic acid 34 4900-8642-4067.5Attorney Docket No.: 098791-000104WOPT molecules that comprise DNA, RNA, or both; and other varieties of polynucleotides known in the art. A vector may be a plasmid, e.g., a circular double stranded DNA loop into which additional DNA segments can be inserted, such as by standard molecular cloning techniques. Certain vectors may be capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Some vectors (e.g., non-episomal mammalian vectors) are integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. In certain examples, vectors may be expression vectors, e.g., capable of directing the expression of genes to which they are operatively-linked. In some cases, the expression vectors may be for expression in eukaryotic cells. Common expression vectors of utility in recombinant DNA techniques are often in the form of plasmids.

[0151] Examples of vectors include pGEX, pMAL, pRIT5, E. coli expression vectors (e.g., pTrc, pET l id, yeast expression vectors (e.g., pYepSecl, pMFa, pJRY88, pYES2, and picZ, Baculovirus vectors (e.g., for expression in insect cells such as SF9 cells) (e.g., pAc series and the pVL series), mammalian expression vectors (e.g., pCDM8 and pMT2PC.

[0152] In a single vector there can be a promoter for each RNA coding sequence. Alternatively or additionally, in a single vector, there may be a promoter controlling (e.g., driving transcription and / or expression) multiple RNA encoding sequences. Regulatory elements

[0153] A vector may comprise one or more regulatory elements. The regulatory element(s) may be operably linked to coding sequences of the engineered proteins. The term “operably linked” is intended to mean that the nucleotide sequence of interest is linked to the regulatory element(s) in a manner that allows for expression of the nucleotide sequence (e.g. in an in vitro transcription / translation system or in a host cell when the vector is introduced into the host cell).

[0154] Examples of regulatory elements include promoters, enhancers, internal ribosomal entry sites (IRES), and other expression control elements (e.g., transcription termination signals, such as polyadenylation signals and poly-U sequences). Such regulatory elements are described, for example, in Goeddel, GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990). Regulatory elements include those that direct constitutive expression of a nucleotide sequence in many types of host cell and those that direct expression of the nucleotide sequence only in certain host cells (e.g., tissue-specific regulatory sequences). A tissue-specific promoter may direct 35 4900-8642-4067.5Attorney Docket No.: 098791-000104WOPT expression primarily in a desired tissue of interest, such as muscle, neuron, bone, skin, blood, specific organs (e.g., liver, pancreas), or particular cell types (e.g., lymphocytes). Regulatory elements may also direct expression in a temporal-dependent manner, such as in a cell-cycle dependent or developmental stage-dependent manner, which may or may not also be tissue or cell-type specific.

[0155] Examples of promoters include one or more pol III promoter (e.g., 1, 2, 3, 4, 5, or more pol III promoters), one or more pol II promoters (e.g., 1, 2, 3, 4, 5, or more pol II promoters), one or more pol I promoters (e.g., 1, 2, 3, 4, 5, or more pol I promoters), or combinations thereof. Examples of pol III promoters include, but are not limited to, U6 and HI promoters. Examples of pol II promoters include, but are not limited to, the retroviral Rous sarcoma virus (RSV) LTR promoter (optionally with the RSV enhancer), the cytomegalovirus (CMV) promoter (optionally with the CMV enhancer), the SV40 promoter, the dihydrofolate reductase promoter, the b-actin promoter, the phosphoglycerol kinase (PGK) promoter, and the EF1a promoter. Viral vectors

[0156] The cargos may be delivered by viruses. In some embodiments, viral vectors are used. A viral vector may comprise virally-derived DNA or RNA sequences for packaging into a virus (e.g., retroviruses, replication defective retroviruses, adenoviruses, replication defective adenoviruses, and adeno-associated viruses). Viral vectors also include polynucleotides carried by a virus for transfection into a host cell. Viruses and viral vectors may be used for in vitro , ex vivo , and / or in vivo deliveries. Adeno associated virus (AAV)

[0157] The systems and compositions herein may be delivered by adeno associated virus (AAV). AAV vectors may be used for such delivery. AAV, of the Dependovirus genus and Parvoviridae family, is a single stranded DNA virus. In some embodiments, AAV may provide a persistent source of the provided DNA, as AAV delivered genomic material can exist indefinitely in cells, e.g., either as exogenous DNA or, with some modification, be directly integrated into the host DNA. In some embodiments, AAV do not cause or relate with any diseases in humans. The virus itself is able to efficiently infect cells while provoking little to no innate or adaptive immune response or associated toxicity.

[0158] Examples of AAV that can be used herein include AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-8, and AAV-9. The type of AAV may be selected with 36 4900-8642-4067.5Attorney Docket No.: 098791-000104WOPT regard to the cells to be targeted; e.g., one can select AAV serotypes 1, 2, 5 or a hybrid capsid AAV1, AAV2, AAV5 or any combination thereof for targeting brain or neuronal cells; and one can select AAV4 for targeting cardiac tissue. AAV8 is useful for delivery to the liver. AAV-2-based vectors were originally proposed for CFTR delivery to CF airways, other serotypes such as AAV-1, AAV-5, AAV-6, and AAV-9 exhibit improved gene transfer efficiency in a variety of models of the lung epithelium. Examples of cell types targeted by AAV are described in Grimm, D. et al, J. Virol.82: 5887-5911 (2008)). In some examples, AAV particles may be created in HEK 293 T cells. Once particles with specific tropism have been created, they are used to infect the target cell line much in the same way that native viral particles do. This may allow for persistent presence of engineered proteins in the infected cell type, and what makes this version of delivery particularly suited to cases where long-term expression is desirable. Examples of doses and formulations for AAV that can be used include those describe in US Patent Nos.8,454,972 and 8,404,658.

[0159] Various strategies may be used for delivery the systems and compositions herein with AAVs. In some examples, coding sequences of engineered proteins may be packaged directly onto one DNA plasmid vector and delivered via one AAV particle. In some examples, AAVs may be used to deliver gRNAs into cells that have been previously engineered to express the engineered protein. In some examples, coding sequences of two or more engineered proteins may be made into two separate AAV particles, which are used for co-transfection of target cells. Lentiviruses

[0160] The systems and compositions herein may be delivered by lentiviruses. Lentiviral vectors may be used for such delivery. Lentiviruses are complex retroviruses that have the ability to infect and express their genes in both mitotic and post-mitotic cells.

[0161] Examples of lentiviruses include human immunodeficiency vims (HIV), which may use its envelope glycoproteins of other viruses to target a broad range of cell types; minimal non-primate lentiviral vectors based on the equine infectious anemia virus (EIAV), which may be used for ocular therapies. In certain embodiments, self-inactivating lentiviral vectors with an siRNA targeting a common exon shared by HIV tat / rev, a nucleolar- localizing TAR decoy, and an anti-CCR5-specific hammerhead ribozyme (see, e.g., DiGiusto et al.(2010) Sci Transl Med 2:36ra43) may be used / and or adapted to the nucleic acid- targeting system herein. 37 4900-8642-4067.5Attorney Docket No.: 098791-000104WOPT

[0162] Lentiviruses may be pseudo-typed with other viral proteins, such as the G protein of vesicular stomatitis virus. In doing so, the cellular tropism of the lentiviruses can be altered to be as broad or narrow as desired. In some cases, to improve safety, second- and third- generation lentiviral systems may split essential genes across three plasmids, which may reduce the likelihood of accidental reconstitution of viable viral particles within cells.

[0163] In some examples, leveraging the integration ability, lentiviruses may be used to create libraries of cells comprising various genetic modifications, e.g., for screening and / or studying genes and signaling pathways. Adenoviruses

[0164] The systems and compositions herein may be delivered by adenoviruses. Adenoviral vectors may be used for such delivery. Adenoviruses include nonenveloped viruses with an icosahedral nucleocapsid containing a double stranded DNA genome. Adenoviruses may infect dividing and non-dividing cells. Non-viral vehicles

[0165] The delivery vehicles may comprise non-viral vehicles. In general, methods and vehicles capable of delivering nucleic acids and / or proteins may be used for delivering the systems compositions herein. Examples of non-viral vehicles include lipid nanoparticles, cell- penetrating peptides (CPPs), DNA nanoclews, gold nanoparticles, streptolysin O, multifunctional envelope-type nanodevices (MENDs), lipid-coated mesoporous silica particles, and other inorganic nanoparticles. Lipid particles

[0166] The delivery vehicles may comprise lipid particles, e.g., lipid nanoparticles (LNPs) and liposomes. Lipid nanoparticles (LNPs)

[0167] LNPs may encapsulate nucleic acids within cationic lipid particles (e.g., liposomes), and may be delivered to cells with relative ease. In some examples, lipid nanoparticles do not contain any viral components, which helps minimize safety and immunogenicity concerns. Lipid particles may be used for in vitro, ex vivo , and in vivo deliveries. Lipid particles may be used for various scales of cell populations. 38 4900-8642-4067.5Attorney Docket No.: 098791-000104WOPT

[0168] In some examples. LNPs may be used for delivering DNA molecules and / or RNA molecules. In certain cases, LNPs may be use for delivering RNP complexes.

[0169] Components in LNPs may comprise cationic lipids 1,2- dilineoyl-3- dimethylammonium -propane (DLinDAP), l,2-dilinoleyloxy-3-N,N- dimethylaminopropane (DLinDMA), l,2-dilinoleyloxyketo-N,N-dimethyl-3-aminopropane (DLinK-DMA), 1,2- dilinoleyl-4-(2-dimethylaminoethyl)-[l,3]-dioxolane (DLinKC2-DMA), (3- o-[2"- (methoxypolyethyleneglycol 2000) succinoyl]-l,2-dimyristoyl-sn-glycol (PEG-S-DMG), R-3- [(ω-methoxy-poly(ethylene glycol)2000) carbamoyl]-l,2-dimyristyloxlpropyl-3-amine (PEG- C-DOMG), and any combination thereof. Preparation of LNPs and encapsulation may be adapted from Rosin et al, Molecular Therapy, vol.19, no.12, pages 1286-2200, Dec.2011. Liposomes

[0170] In some embodiments, a lipid particle may be liposome. Liposomes are spherical vesicle structures composed of a uni- or multilamellar lipid bilayer surrounding internal aqueous compartments and a relatively impermeable outer lipophilic phospholipid bilayer. In some embodiments, liposomes are biocompatible, nontoxic, can deliver both hydrophilic and lipophilic drug molecules, protect their cargo from degradation by plasma enzymes, and transport their load across biological membranes and the blood brain barrier (BBB).

[0171] Liposomes can be made from several different types of lipids, e.g., phospholipids. A liposome may comprise natural phospholipids and lipids such as 1,2-distearoryl-sn- glycero-3 -phosphatidyl choline (DSPC), sphingomyelin, egg phosphatidylcholines, monosialoganglioside, or any combination thereof.

[0172] Several other additives may be added to liposomes in order to modify their structure and properties. For instance, liposomes may further comprise cholesterol, sphingomyelin, and / or l,2-dioleoyl-sn-glycero-3- phosphoethanolamine (DOPE), e.g., to increase stability and / or to prevent the leakage of the liposomal inner cargo. Stable nucleic-acid-lipid particles (SNALPs)

[0173] In some embodiments, the lipid particles may be stable nucleic acid lipid particles (SNALPs). SNALPs may comprise an ionizable lipid (DLinDMA) (e.g., cationic at low pH), a neutral helper lipid, cholesterol, a diffusible polyethylene glycol (PEG)-lipid, or any combination thereof. In some examples, SNALPs may comprise synthetic cholesterol, dipalmitoylphosphatidylcholine, 3 -N-[(w-m ethoxy polyethylene glycol)2000)carbamoyl]- l,2-dimyrestyloxypropylamine, and cationic l,2-dilinoleyloxy-3-N,Ndimethylaminopropane. 39 4900-8642-4067.5Attorney Docket No.: 098791-000104WOPT In some examples, SNALPs may comprise synthetic cholesterol, l,2-distearoyl-sn-glycero-3- phosphocholine, PEG- cDMA, and l,2-dilinoleyloxy-3-(N;N-dimethyl)aminopropane (DLinDMA). Other lipids

[0174] The lipid particles may also comprise one or more other types of lipids, e.g., cationic lipids, such as amino lipid 2,2-dilinoleyl-4-dimethylaminoethyl-[l,3]- dioxolane (DLin-KC2-DMA), DLin-KC2-DMA4, C12- 200 and colipids disteroylphosphatidyl choline, cholesterol, and PEG-DMG. Lipoplexes / polyplexes

[0175] In some embodiments, the delivery vehicles comprise lipoplexes and / or polyplexes. Lipoplexes may bind to negatively charged cell membrane and induce endocytosis into the cells. Examples of lipoplexes may be complexes comprising lipid(s) and non-lipid components. Examples of lipoplexes and polyplexes include FuGENE-6 reagent, a non-liposomal solution containing lipids and other components, zwitterionic amino lipids (ZALs), Ca2J) (e.g., forming DNA / Ca2+microcomplexes), polyethylenimine (PEI) (e.g., branched PEI), and poly(L-lysine) (PLL). Cell penetrating peptides

[0176] In some embodiments, the delivery vehicles comprise cell penetrating peptides (CPPs). CPPs are short peptides that facilitate cellular uptake of various molecular cargo (e.g., from nanosized particles to small chemical molecules and large fragments of DNA).

[0177] CPPs may be of different sizes, amino acid sequences, and charges. In some examples, CPPs can translocate the plasma membrane and facilitate the delivery of various molecular cargoes to the cytoplasm or an organelle. CPPs may be introduced into cells via different mechanisms, e.g., direct penetration in the membrane, endocytosis-mediated entry, and translocation through the formation of a transitory structure.

[0178] CPPs may have an amino acid composition that either contains a high relative abundance of positively charged amino acids such as lysine or arginine or has sequences that contain an alternating pattern of polar / charged amino acids and non-polar, hydrophobic amino acids. These two types of structures are referred to as polycationic or amphipathic, respectively. A third class of CPPs are the hydrophobic peptides, containing only apolar residues, with low net charge or have hydrophobic amino acid groups that are crucial for 40 4900-8642-4067.5Attorney Docket No.: 098791-000104WOPT cellular uptake. Another type of CPPs is the trans-activating transcriptional activator (Tat) from Human Immunodeficiency Virus 1 (HIV-1). Examples of CPPs include to Penetratin, Tat (48-60), Transportan, and (R-AhX-R4) (Ahx refers to aminohexanoyl), Kaposi fibroblast growth factor (FGF) signal peptide sequence, integrin b3 signal peptide sequence, polyarginine peptide Args sequence, Guanine rich-molecular transporters, and sweet arrow peptide. Examples of CPPs and related applications also include those described in US Patent 8,372,951.

[0179] CPPs can be used for in vitro and ex vivo work quite readily, and extensive optimization for each cargo and cell type is usually required. In some examples, CPPs may be covalently attached to the engineered protein directly, which is then complexed with the gRNA and delivered to cells. CPP may also be used to delivery RNPs.

[0180] CPPs may be used to deliver the compositions and systems to plants. In some examples, CPPs may be used to deliver the components to plant protoplasts, which are then regenerated to plant cells and further to plants. DNA nanoclews

[0181] In some embodiments, the delivery vehicles comprise DNA nanoclews. A DNA nanoclew refers to a sphere-like structure of DNA (e.g., with a shape of a ball of yarn). The nanoclew may be synthesized by rolling circle amplification with palindromic sequences that aide in the self-assembly of the structure. The sphere may then be loaded with a payload. An example of DNA nanoclew is described in Sun W et al, J Am Chem Soc.2014 Oct 22; 136(42): 14722-5; and Sun W et al, Angew Chem Int Ed Engl.2015 Oct 5;54(41):12029-33. A DNA nanoclew may be coated, e.g., coated with PEI to induce endosomal escape. Gold nanoparticles

[0182] In some embodiments, the delivery vehicles comprise gold nanoparticles (also referred to AuNPs or colloidal gold). Gold nanoparticles may form complex with cargos. Gold nanoparticles may be coated, e.g., coated in a silicate and an endosomal disruptive polymer, PAsp(DET). Examples of gold nanoparticles include AuraSense Therapeutics' Spherical Nucleic Acid (SNA™) constructs, and those described in Mout R, et al. (2017). ACS Nano 11:2452-8; Lee K, et al. (2017). Nat Biomed Eng 1:889-901. iTOP 41 4900-8642-4067.5Attorney Docket No.: 098791-000104WOPT

[0183] In some embodiments, the delivery vehicles comprise iTOP. iTOP refers to a combination of small molecules drives the highly efficient intracellular delivery of native proteins, independent of any transduction peptide. iTOP may be used for induced transduction by osmocytosis and propanebetaine, using NaCl-mediated hyperosmolality together with a transduction compound (propanebetaine) to trigger macropinocytotic uptake into cells of extracellular macromolecules. Examples of iTOP methods and reagents include those described in D'Astolfo DS, Pagliero RJ, Pras A, et al. (2015). Cell 161:674-690. Polymer-based particles

[0184] In some embodiments, the delivery vehicles may comprise polymer-based particles (e.g., nanoparticles). In some embodiments, the polymer-based particles may mimic a viral mechanism of membrane fusion. The polymer-based particles may be a synthetic copy of Influenza virus machinery and form transfection complexes with various types of nucleic acids ((siRNA, miRNA, plasmid DNA or shRNA, mRNA) that cells take up via the endocytosis pathway, a process that involves the formation of an acidic compartment. The low pH in late endosomes acts as a chemical switch that renders the particle surface hydrophobic and facilitates membrane crossing. Once in the cytosol, the particle releases its payload for cellular action. This Active Endosome Escape technology is safe and maximizes transfection efficiency as it is using a natural uptake pathway. Streptolysin O (SLO)

[0185] The delivery vehicles may be streptolysin O (SLO). SLO is a toxin produced by Group A streptococci that works by creating pores in mammalian cell membranes. SLO may act in a reversible manner, which allows for the delivery of proteins (e.g., up to 100 kDa) to the cytosol of cells without compromising overall viability. Examples of SLO include those described in Sierig G, et al. (2003). Infect Immun 71 :446-55; Walev I, et al. (2001). Proc Natl Acad Sci U S A 98:3185-90; Teng KW, et al. (2017). Elife 6:e25460. Multifunctional envelope-type nanodevice (MEND)

[0186] The delivery vehicles may comprise multifunctional envelope-type nanodevice (MENDs). MENDs may comprise condensed plasmid DNA, a PLL core, and a lipid film shell. A MEND may further comprise cell-penetrating peptide (e.g., stearyl octaarginine). The cell penetrating peptide may be in the lipid shell. The lipid envelope may be modified 42 4900-8642-4067.5Attorney Docket No.: 098791-000104WOPT with one or more functional components, e.g., one or more of: polyethylene glycol (e.g., to increase vascular circulation time), ligands for targeting of specific tissues / cells, additional cell- penetrating peptides (e.g., for greater cellular delivery), lipids to enhance endosomal escape, and nuclear delivery tags. In some examples, the MEND may be a tetra-lamellar MEND (T- MEND), which may target the cellular nucleus and mitochondria. In certain examples, a MEND may be a PEG-peptide-DOPE-conjugated MEND (PPD-MEND), which may target bladder cancer cells. Examples of MENDs include those described in Kogure K, et al. (2004). J Control Release 98:317-23; Nakamura T, et al. (2012). Acc Chem Res 45:1113-21. Lipid-coated mesoporous silica particles

[0187] The delivery vehicles may comprise lipid-coated mesoporous silica particles. Lipid-coated mesoporous silica particles may comprise a mesoporous silica nanoparticle core and a lipid membrane shell. The silica core may have a large internal surface area, leading to high cargo loading capacities. In some embodiments, pore sizes, pore chemistry, and overall particle sizes may be modified for loading different types of cargos. The lipid coating of the particle may also be modified to maximize cargo loading, increase circulation times, and provide precise targeting and cargo release. Examples of lipid-coated mesoporous silica particles include those described in Du X, et al. (2014). Biomaterials 35:5580-90; Durfee PN, et al. (2016). ACS Nano 10:8325-45. Inorganic nanoparticles

[0188] The delivery vehicles may comprise inorganic nanoparticles. Examples of inorganic nanoparticles include carbon nanotubes (CNTs) (e.g., as described in Bates K and Kostarelos K. (2013). Adv Drug Deliv Rev 65:2023-33.), bare mesoporous silica nanoparticles (MSNPs) (e.g., as described in Luo GF, et al. (2014). Sci Rep 4:6064), and dense silica nanoparticles (SiNPs) (as described in Luo D and Saltzman WM. (2000). Nat Biotechnol 18:893-5). Exosomes

[0189] The delivery vehicles may comprise exosomes. Exosomes include membrane bound extracellular vesicles, which can be used to contain and delivery various types of biomolecules, such as proteins, carbohydrates, lipids, and nucleic acids, and complexes thereof (e.g., RNPs). Examples of exosomes include those described in Schroeder A, et al., J 43 4900-8642-4067.5Attorney Docket No.: 098791-000104WOPT Intern Med.2010 Jan;267(l):9-21; El-Andaloussi S, et al., Nat Protoc.2012 Dec;7(12):2112- 26; Uno Y, et al., Hum Gene Ther.2011 Jun;22(6):711-9; Zou W, et al., Hum Gene Ther. 2011 Apr;22(4):465-75.

[0190] In some examples, the exosome may form a complex (e.g., by binding directly or indirectly) to one or more components of the cargo. In certain examples, a molecule of an exosome may be fused with first adapter protein and a component of the cargo may be fused with a second adapter protein. The first and the second adapter protein may specifically bind each other, thus associating the cargo with the exosome. Examples of such exosomes include those described in Ye Y, et al., Biomater Sci.2020 Apr 28. doi: 10.1039 / d0bm00427h.

[0191] Features of the compositions or methods can include one or more of the following enumerated embodiments. 1. Embodiment 1. A genome editing system comprising: i) an R2Tg enzyme comprises an amino acid sequence SEQ ID NO:1 an R2Tg enzyme comprises an amino acid sequence SEQ ID NO:1 with a mutation selected from the group consisting of D944K, S870R, E638K, T1367R, G1056R, and combinations thereof; ii) a payload RNA, wherein the payload RNA comprises an insertion template and optionally one or more of a 5’ homology arm, a 3’ homology arm, and a protein binding element, wherein the insertion template comprises a sequence for a nucleic acid insertion into the genome. 2. Embodiment 2. A genome editing system comprising: i) an R2Tg enzyme comprises an amino acid sequence SEQ ID NO:2 or comprises a variant of a protein effector comprising an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 2; ii) a payload RNA, wherein the payload RNA comprises an insertion template and optionally one or more of a 5’ homology arm, a 3’ homology arm, and a protein binding element, wherein the insertion template comprises a sequence for a nucleic acid insertion into the genome. 44 4900-8642-4067.5Attorney Docket No.: 098791-000104WOPT 3. Embodiment 3. The genome editing system of Embodiment 1 or 2, wherein the 5’ homology arm is less than or about 105 base pairs, is less than or about 50 base pairs, is less than or about 25 base pairs. 4. Embodiment 4. The genome editing system of Embodiment 1 or 2, wherein the 3’ homology arm is less than or about 50 base pairs, is less than or about 25 base pairs, is less than or about 10 base pairs. 5. Embodiment 5. The genome editing system of Embodiment 1 or 2, wherein the R2Tg element enzyme further comprises a targeting domain. 6. Embodiment 6. The genome editing system of Embodiment 5, wherein the targeting domain is a natural targeting domain. 7. Embodiment 7. The genome editing system of Embodiment 5, wherein the targeting domain is an engineered targeting domain. 8. Embodiment 8. The genome editing system of Embodiment 1 or 2, wherein the nucleic acid insertion into the genome is a DNA or RNA insertion template. 9. Embodiment 9. The genome editing system of Embodiment 1 or 2, wherein the R2Tg element enzyme is a modified R2Tg element enzyme. 10. Embodiment 10. The genome editing system of Embodiment 9, wherein the coding sequence of the R2Tg element enzyme is modified. 11. Embodiment 11. The genome editing system of Embodiments 2 to 10, wherein the modified R2Tg element enzyme further comprises a mutation selected from S645K, E638K, V661R, G257R, G236R, I558R, D555R, S870R, T934R, C587K, E638K, V661R, L388R, E471R, L243R, D923K, S763R, I308R, T1016R, T1023K, G1056R, N1103K, Y1242K, T1367R or combinations thereof. 12. Embodiment 12. The genome editing system of Embodiment 9, wherein the modified R2Tg element enzyme is modified by an N-terminal or C-terminal truncation of the R2Tg element enzyme sequence. 45 4900-8642-4067.5Attorney Docket No.: 098791-000104WOPT 13. Embodiment 13. The genome editing system of Embodiment 12, wherein the modified R2Tg element enzyme is modified by an N-terminal truncation of the R2Tg element enzyme sequence. 14. Embodiment 14. The genome editing system of Embodiment 12 or 13, wherein the modified R2Tg element enzyme comprises SEQ ID. NO:3. 15. Embodiment 15. The genome editing system of Embodiment 1 or 2, wherein the genome editing system targets a genomic locus. 16. Embodiment 16. The genome editing system of Embodiment 1 or 2, wherein the genome editing system targets a genomic locus other than the 28S rRNA locus. 17. Embodiment 17. The genome editing system of Embodiment 16, wherein an N- terminal zinc finger domain of the R2Tg element enzyme is modified to target a genomic locus other than the 28S rRNA locus. 18. Embodiment 18. The genome editing system of Embodiment 16, wherein the genomic locus is selected from the target site listed the in Tables 2 and 3, e.g., SEQ ID NO:12-86. 19. Embodiment 19. The genome editing system of Embodiment 16, wherein a non- naturally occurring targeting region is fused to the N-terminus of the R2Tg element enzyme or inserted into the R2Tg element enzyme. 20. Embodiment 20. The genome editing system of Embodiment 9, wherein the modified R2Tg element enzyme is a fusion protein. 21. Embodiment 21. The genome editing system of Embodiment 9, wherein the modified R2Tg element is fused to a Cas9 protein that is fully active, catalytically dead (H840A / D10A for SpCas9), or functioning as a nickase (H840A or D10A for SpCas9). 22. Embodiment 22. The genome editing system of Embodiment 9, wherein the modified R2Tg element is fused to a Cas12 protein that is fully active, catalytically dead, or functioning as a nickase. 46 4900-8642-4067.5Attorney Docket No.: 098791-000104WOPT 23. Embodiment 23. The genome editing system of Embodiment 21, further comprising a guide RNA. 24. Embodiment 24. The genome editing system of Embodiment 9, wherein the modified R2Tg element is fused to a TALEN protein, zinc finger protein, argonaute, or meganuclease protein. 25. Embodiment 25. The genome editing system of Embodiment 22, further comprising a guide RNA. 26. Embodiment 26. The genome editing system of Embodiment 16, wherein the 5’ homology arm, the 3’ homology arm, or both the 5’ and 3’ homology arm of the payload RNA is engineered to target a genomic locus other than the 28 S rRNA locus. 27. Embodiment 27. The genome editing system of Embodiment 1 or 2, wherein the 5’ homology arm, the 3’ homology arm, or both the 5’ and 3’ homology arm target an exogenously introduced landing sequence. 28. Embodiment 28. The genome editing system of Embodiment 1 or 2, wherein the insertion region is introduced into the genome of a specific cell type. 29. Embodiment 29. The genome editing system of Embodiment 28, wherein the specific cell type is a post-mitotic cell. 30. Embodiment 30. The genome editing system of Embodiment 1or 2, wherein the genome editing system functions in post-mitotic cells. 31. Embodiment 31. The genome editing system of Embodiment 1 or 2, wherein the genome editing system functions independently from intrinsic nucleic acid repair systems. 32. Embodiment 32. The genome editing system of Embodiment 1 or 2, wherein the payload RNA template further comprises a 5’ untranslated region (UTR), a 3’ UTR, or both a 5’ UTR and a 3’ UTR. 33. Embodiment 33. The genome editing system of Embodiment 32, wherein the 5’ homology arm and the 3’ homology arm are located between the 5’ UTR and 3’ UTR. 47 4900-8642-4067.5Attorney Docket No.: 098791-000104WOPT 34. Embodiment 34. The genome editing system of Embodiment 32, wherein the 5’ homology arm and the 3’ homology arm are located outside the 5’ UTR and 3’ UTR. 35. Embodiment 35. The genome editing system of Embodiment 1 or 2, wherein the payload RNA further comprises a 5’ untranslated region (UTR), a 3’ UTR, or both a 5’ and a 3’ UTR, wherein the UTRs are truncated. 36. Embodiment 36. The genome editing system of Embodiment 1 or 2, wherein the payload RNA does not comprise a 5’ UTR. 37. Embodiment 37. The genome editing system of Embodiment 1 or 2, wherein the payload RNA does not comprise a 3’ UTR. 38. Embodiment 38. The genome editing system of Embodiment 1 or 2, wherein the payload RNA further comprises a nuclear retention element. 39. Embodiment 39. The genome editing system of Embodiment 1 or 2, wherein the payload RNA further comprises a Cas9 or Cas12 guide RNA, and wherein the Cas9 or Cas12 guide RNA comprises an extension with a 5’ homology sequence, a 3’ homology sequence, a 5’ untranslated region (UTR), a 3’ UTR, an insertion template, or any combination thereof. 40. Embodiment 40. The genome editing system of Embodiment 1 or 2, wherein the nucleic acid insertion template is a sequence of greater than 1000 base pairs. 41. Embodiment 41. The genome editing system of Embodiment 1 or 2, wherein the R2Tg element enzyme comprises a nuclear localization signal (NLS). 42. Embodiment 42. The genome editing system of Embodiment 1 or 2, wherein the insertion region comprises a template for a reporter gene, a transcription factor gene, a transgene, an enzyme gene, or a therapeutic gene. 43. Embodiment 43. A method of inserting a large nucleic acid into a genome within a cell using a Cas9 or Cas12 fusion protein, wherein the method comprises supplying a Cas9 or Cas12 fusion protein to a cell, wherein the Cas9 or Cas12 fusion protein is supplied with a payload RNA template, wherein the RNA template is reverse transcribed by the Cas9 or Cas12 fusion protein prior to being inserted into the 48 4900-8642-4067.5Attorney Docket No.: 098791-000104WOPT genome of the cell; and wherein the large nucleic acid is inserted into the genome of the cell. 44. Embodiment 44. The method of Embodiment 43, wherein the Cas9 fusion protein comprises a Cas9 portion and an R2Tg element portion. 45. Embodiment 45. The method of Embodiment 44, wherein the Cas9 fusion protein comprises a targeting domain, a reverse transcriptase domain, and a nickase domain. 46. Embodiment 46. The method of Embodiment 43, wherein the Cas12 fusion protein comprises a Cas12 portion and an R2Tg element portion. 47. Embodiment 47. A method of inserting an exogenous nucleic acid into the genome of a post-mitotic cell, wherein the method comprises subjecting the genome of the post- mitotic cell to a modified Cas9 protein that inserts the exogenous nucleic acid into the genome of the postmitotic cell. 48. Embodiment 48. The method of Embodiment 47, wherein the modified Cas9 protein is fused to an R2Tg element enzyme. 49. Embodiment 49. The method of Embodiment 48, wherein the modified Cas9 fusion protein targets an endogenous landing site. 50. Embodiment 50. The method of Embodiment 48, wherein the Cas9 fusion protein targets an exogenously introduced landing site in the genome of the post-mitotic cell. 51. Embodiment 51. A method of editing a genome comprising subjecting the cell to the genome editing system of Embodiment 1 or 2. 52. Embodiment 52. A method of correcting a genetic mutation related to disease or human pathology, wherein the method comprises making small nucleotide changes or small nucleotide insertions (1-100 bp) in a human genome using the genome editing system of Embodiment 1 or 2. 53. Embodiment 53. The method of Embodiment 52 or the genome editing system of any one of Embodiments above, wherein the genome editing system is delivered via single or multi vector AAV, adenovirus, lentivirus, herpes simplex virus, PEG10 viral 49 4900-8642-4067.5Attorney Docket No.: 098791-000104WOPT like particles, PNMA viral like particles, gag-like viral like particles, nanoblades, gesicles, or Friend murine leukemia virus (FMLV) viral like proteins. 54. Embodiment 54. The method of Embodiment 52 or Embodiment 53, or the genome editing system of any one of Embodiments above, wherein the components of the genome editing system are delivered as all RNA in lipid nanoparticles or another RNA delivery reagent. 55. Embodiment 55. The method of Embodiment 54, wherein the non-LTR site specific retrotransposon is delivered as mRNA. 56. Embodiment 56. The method of any Embodiments above, wherein the guide RNAs are delivered as synthetic RNA. 57. Embodiment 57. The method of any Embodiments above, wherein the payload is delivered as mRNA. 58. Embodiment 58. The genome editing system of Embodiment 1 or 2, wherein the genome editing system targets and edits the genome at more than one site.

[0192] The technology described herein is further illustrated by the following examples which in no way should be construed as being further limiting. EXPERIMENTAL DESIGN

[0193] Developed a workflow for arrayed mutant screening that captures both the initiation of insertion at the 3’ of the cargo and its completion.

[0194] Developed an evolutionary approach for semi-rational R2Tg protein mutagenesis based on amino acid usage across related orthologs.

[0195] Identified mutant variants with either increased initial 3’ insertion, full-length 5’ insertion, or both. Example 1: Identification of starting R2Tg variant and donor sequence

[0196] In AlphaFold-predictions of the structure of R2Tg, a significant stretch of the N- terminal region appears to be completely unstructured. We reasoned that this may relate to 50 4900-8642-4067.5Attorney Docket No.: 098791-000104WOPT non-canonical translation initiation playing a role in the wild-type R2Tg; the observed start codon (ATG) may not be the true start of the polypeptide and so the true N-terminal start position may exclude these unstructured residues. We therefore designed experiments comparing full-length R2Tg to an N-terminally truncated variant in which the residues up to and including E184 were removed. In these experiment, the R2Tg protein was delivered as an mRNA with a consensus Kozak sequence and AUG start codon, both of which drive canonical translation initiation mechanisms.

[0197] For each R2Tg protein variant, we compared a range of donor mRNA to assess the dependence of insertion efficiency on cargo size and sequence. These mRNA were produced in vitro and consisted of a 5’ 7-methyl guanosine (m7G) cap structure, a stretch of RNA derived from the plasmid vector backbone, a homology arm sequence (each of 25, 50, or 105bp) complementary to the human 28S rDNA sequence upstream of the canonical R2 target site, the 5’ UTR, the cargo, the 3’ UTR, a homology arm sequence (10bp) complementary to the human 28S rDNA sequence downstream of the canonical R2 target site, and a hard-coded poly(A) tail.

[0198] For the R2Tg mutagenesis screen, we selected an mRNA donor of intermediate length containing a synthetic antisense expression cassette to represent an artificial (i.e. non- R2 ORF) sequence. The total insertion length would therefore be 1,598bp including 5’ (175bp) and 3’ (325bp) UTRs, with a 50bp homology arm upstream of the 5’ UTR.

[0199] Table 4 Element  SEQ ID NO T7 promoter  taatacgactcactataag  96 51 4900-8642-4067.5Attorney Docket No.: 098791-000104WOPT52 4900-8642-4067.5Attorney Docket No.: 098791-000104WOPT 'Attorney Docket No.: 098791-000104WOPT54 4900-8642-4067.5Attorney Docket No.: 098791-000104WOPT

[0200] Given that the R2Tg enzyme interacts with multiple negatively-charged substrates (DNA and RNA), we prioritized amino acid substitutions to the positively charged polar residues arginine (R), lysine (K), and asparagine (N). We then used an evolutionary approach to select residue positions that would be amenable to substitution with R, K and N amino acids. Multiple sequence alignments (MSAs) for R2Tg were obtained either through BLAST or from a curated list of full-length R2 family nLTR retrotransposons. We then selected aligned positions at which some or all orthologs in the MSA had either an R, K or N but at which R2Tg did not. We reasoned that given the relatively close evolutionary relationships within the alignments, the presence of these amino acids at the orthologous positions would make mutations to R2Tg at the corresponding sites more likely to retain and improve functionality. 55 4900-8642-4067.5Attorney Docket No.: 098791-000104WOPT Example 3: Arrayed mutant screen and ddPCR readout

[0201] Mutant variant mRNAs of the truncated R2Tg were generated through in vitro transcription, using a sequence-verified plasmid template and / or a linear dsDNA template generated through overlap-extension PCR. R2Tg mRNAs were co-transfected with the donor mRNA in HEK293T cells in 96 well plate format, and after three days genomic DNA was harvested. To quantify the frequency of both 3’ UTR insertion (indicative of insertion initiation) and 5’ UTR insertion (indicative of complete, full-length insertion), we developed corresponding junction ddPCR primer-probe sets. Junction amplicon detection was normalized to a reference primer-probe set targeting an established autosomal reference gene (ApoB), allowing us to calculate the relative copy number of the R2Tg cargo insertions. To reduce the possibility for non-specific primer amplification and facilitate gDNA loading into ddPCR droplets, we pre-digested gDNA samples with two parallel restriction enzyme sets corresponding to the 5’ UTR (NcoI & BamHI) and 3’ UTR (BbsI & BsrGI) detection assays. Results:

[0202] R2Tg ∆184N appears to have higher full-length insertion activity across a range of cargo sizes compared to wild type (wt) R2Tg as shown in FIGS.1 and 2.

[0203] Point mutants of R2Tg ∆184N have increased 3’ insertion frequencies, 5’ insertion frequencies, or both relative to “wild-type” R2Tg ∆184N as shown in FIGS.3 and 4.

[0204] TABLE 5 ddPCR data for truncated R2Tg ∆184aa with mutations Pre-cloned; 3' Pre-cloned; 5' OE-PCR; 3' UTR OE-PCR; 5' UTR UTR UTRAttorney Docket No.: 098791-000104WOPTAttorney Docket No.: 098791-000104WOPTAttorney Docket No.: 098791-000104WOPT 35 35 34Attorney Docket No.: 098791-000104WOPT 35 94 68 61 63 50 08 15 81 27 74 41 49 66 33 82 37 98 01 89 35 09 89 46 67 55 484900-8642-4067.5Attorney Docket No.: 098791-000104WOPT 08 29 48 46 06 52 42 81 81 20 91 46 13 29 70 07 51 73 88 57 56 21 65 19 27 43 53 68 25Attorney Docket No.: 098791-000104WOPT 51 54 22 28 05 67 04 83 74 89 01 60 09 73 75 24 55 23 70 97 52 80 56 03 81 45 14 934900-8642-4067.5Attorney Docket No.: 098791-000104WOPT 84 38 38 68 31 14

[0205] While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby.

[0206] All references, including patents and patent applications, are hereby incorporated by reference. 63 4900-8642-4067.5

Claims

Attorney Docket No.: 098791-000104WOPT CLAIMS We claim:

1. A genome editing system comprising: i) an R2Tg enzyme comprises an amino acid sequence SEQ ID NO:1 an R2Tg enzyme comprises an amino acid sequence SEQ ID NO:1 (wt sequence) with a mutation selected from the group consisting of D944K, S870R, E638K, T1367R, G1056R, and combinations thereof; ii) a payload RNA, wherein the payload RNA comprises an insertion template and optionally one or more of a 5’ homology arm, a 3’ homology arm, and a protein binding element, wherein the insertion template comprises a sequence for a nucleic acid insertion into the genome.

2. A genome editing system comprising: i) an R2Tg enzyme comprises an amino acid sequence SEQ ID NO:2 or comprises a variant of a protein effector comprising an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 2; ii) a payload RNA, wherein the payload RNA comprises an insertion template and optionally one or more of a 5’ homology arm, a 3’ homology arm, and a protein binding element, wherein the insertion template comprises a sequence for a nucleic acid insertion into the genome.

3. The genome editing system of claim 1 or 2, wherein the 5’ homology arm is less than or about 105 base pairs, is less than or about 50 base pairs, is less than or about 25 base pairs.

4. The genome editing system of claim 1 or 2, wherein the 3’ homology arm is less than or about 50 base pairs, is less than or about 25 base pairs, is less than or about 10 base pairs.

5. The genome editing system of claim 1 or 2, wherein the R2Tg element enzyme further comprises a targeting domain.

6. The genome editing system of claim 5, wherein the targeting domain is a natural targeting domain. 64 4900-8642-4067.5Attorney Docket No.: 098791-000104WOPT 7. The genome editing system of claim 5, wherein the targeting domain is an engineered targeting domain.

8. The genome editing system of claim 1 or 2, wherein the nucleic acid insertion into the genome is a DNA or RNA insertion template.

9. The genome editing system of claim 1 or 2, wherein the R2Tg element enzyme is a modified R2Tg element enzyme.

10. The genome editing system of claim 9, wherein the coding sequence of the R2Tg element enzyme is modified.

11. The genome editing system of claims 2 to 10, wherein the modified R2Tg element enzyme further comprises a mutation selected from S645K, E638K, V661R, G257R, G236R, I558R, D555R, S870R, T934R, C587K, E638K, V661R, L388R, E471R, L243R, D923K, S763R, I308R, T1016R, T1023K, G1056R, N1103K, Y1242K, T1367R or combinations thereof.

12. The genome editing system of claim 9, wherein the modified R2Tg element enzyme is modified by an N-terminal or C-terminal truncation of the R2Tg element enzyme sequence.

13. The genome editing system of claim 12, wherein the modified R2Tg element enzyme is modified by an N-terminal truncation of the R2Tg element enzyme sequence.

14. The genome editing system of claim 12 or 13, wherein the modified R2Tg element enzyme comprises SEQ ID. NO:

3.

15. The genome editing system of claim 1 or 2, wherein the genome editing system targets a genomic locus.

16. The genome editing system of claim 1 or 2, wherein the genome editing system targets a genomic locus other than the 28S rRNA locus.

17. The genome editing system of claim 16, wherein an N-terminal zinc finger domain of the R2Tg element enzyme is modified to target a genomic locus other than the 28S rRNA locus. 65 4900-8642-4067.5Attorney Docket No.: 098791-000104WOPT 18. The genome editing system of claim 16, wherein the genomic locus is selected from the target site of SEQ ID NO:12 to 86.

19. The genome editing system of claim 16, wherein a non-naturally occurring targeting region is fused to the N-terminus of the R2Tg element enzyme or inserted into the R2Tg element enzyme.

20. The genome editing system of claim 9, wherein the modified R2Tg element enzyme is a fusion protein.

21. The genome editing system of claim 9, wherein the modified R2Tg element is fused to a Cas9 protein that is fully active, catalytically dead (H840A / D10A for SpCas9), or functioning as a nickase (H840A or D10A for SpCas9).

22. The genome editing system of claim 9, wherein the modified R2Tg element is fused to a Cas12 protein that is fully active, catalytically dead, or functioning as a nickase.

23. The genome editing system of claim 21, further comprising a guide RNA.

24. The genome editing system of claim 9, wherein the modified R2Tg element is fused to a TALEN protein, zinc finger protein, argonaute, or meganuclease protein.

25. The genome editing system of claim 22, further comprising a guide RNA.

26. The genome editing system of claim 16, wherein the 5’ homology arm, the 3’ homology arm, or both the 5’ and 3’ homology arm of the payload RNA is engineered to target a genomic locus other than the 28 S rRNA locus.

27. The genome editing system of claim 1 or 2, wherein the 5’ homology arm, the 3’ homology arm, or both the 5’ and 3’ homology arm target an exogenously introduced landing sequence.

28. The genome editing system of claim 1 or 2, wherein the insertion region is introduced into the genome of a specific cell type.

29. The genome editing system of claim 28, wherein the specific cell type is a post-mitotic cell. 66 4900-8642-4067.5Attorney Docket No.: 098791-000104WOPT 30. The genome editing system of claim 1or 2, wherein the genome editing system functions in post-mitotic cells.

31. The genome editing system of claim 1 or 2, wherein the genome editing system functions independently from intrinsic nucleic acid repair systems.

32. The genome editing system of claim 1 or 2, wherein the payload RNA template further comprises a 5’ untranslated region (UTR), a 3’ UTR, or both a 5’ UTR and a 3’ UTR.

33. The genome editing system of claim 32, wherein the 5’ homology arm and the 3’ homology arm are located between the 5’ UTR and 3’ UTR.

34. The genome editing system of claim 32, wherein the 5’ homology arm and the 3’ homology arm are located outside the 5’ UTR and 3’ UTR.

35. The genome editing system of claim 1 or 2, wherein the payload RNA further comprises a 5’ untranslated region (UTR), a 3’ UTR, or both a 5’ and a 3’ UTR, wherein the UTRs are truncated.

36. The genome editing system of claim 1 or 2, wherein the payload RNA does not comprise a 5’ UTR.

37. The genome editing system of claim 1 or 2, wherein the payload RNA does not comprise a 3’ UTR.

38. The genome editing system of claim 1 or 2, wherein the payload RNA further comprises a nuclear retention element.

39. The genome editing system of claim 1 or 2, wherein the payload RNA further comprises a Cas9 or Cas12 guide RNA, and wherein the Cas9 or Cas12 guide RNA comprises an extension with a 5’ homology sequence, a 3’ homology sequence, a 5’ untranslated region (UTR), a 3’ UTR, an insertion template, or any combination thereof.

40. The genome editing system of claim 1 or 2, wherein the nucleic acid insertion template is a sequence of greater than 1000 base pairs.

41. The genome editing system of claim 1 or 2, wherein the R2Tg element enzyme comprises a nuclear localization signal (NLS). 67 4900-8642-4067.5Attorney Docket No.: 098791-000104WOPT 42. The genome editing system of claim 1 or 2, wherein the insertion region comprises a template for a reporter gene, a transcription factor gene, a transgene, an enzyme gene, or a therapeutic gene.

43. A method of inserting a large nucleic acid into a genome within a cell using a Cas9 or Cas12 fusion protein, wherein the method comprises supplying a Cas9 or Cas12 fusion protein to a cell, wherein the Cas9 or Cas12 fusion protein is supplied with a payload RNA template, wherein the RNA template is reverse transcribed by the Cas9 or Cas12 fusion protein prior to being inserted into the genome of the cell; and wherein the large nucleic acid is inserted into the genome of the cell.

44. The method of claim 43, wherein the Cas9 fusion protein comprises a Cas9 portion and an R2Tg element portion.

45. The method of claim 44, wherein the Cas9 fusion protein comprises a targeting domain, a reverse transcriptase domain, and a nickase domain.

46. The method of claim 43, wherein the Cas12 fusion protein comprises a Cas12 portion and an R2Tg element portion.

47. A method of inserting an exogenous nucleic acid into the genome of a post-mitotic cell, wherein the method comprises subjecting the genome of the post-mitotic cell to a modified Cas9 protein that inserts the exogenous nucleic acid into the genome of the postmitotic cell.

48. The method of claim 47, wherein the modified Cas9 protein is fused to an R2Tg element enzyme.

49. The method of claim 48, wherein the modified Cas9 fusion protein targets an endogenous landing site.

50. The method of claim 48, wherein the Cas9 fusion protein targets an exogenously introduced landing site in the genome of the post-mitotic cell.

51. A method of editing a genome comprising subjecting the cell to the genome editing system of claim 1 or 2. 68 4900-8642-4067.5Attorney Docket No.: 098791-000104WOPT 52. A method of correcting a genetic mutation related to disease or human pathology, wherein the method comprises making small nucleotide changes or small nucleotide insertions (1-100 bp) in a human genome using the genome editing system of claim 1 or 2.

53. The method of claim 52 or the genome editing system of any one of claims above, wherein the genome editing system is delivered via single or multi vector AAV, adenovirus, lentivirus, herpes simplex virus, PEG10 viral like particles, PNMA viral like particles, gag- like viral like particles, nanoblades, gesicles, or Friend murine leukemia virus (FMLV) viral like proteins.

54. The method of claim 52 or claim 53, or the genome editing system of any one of claims above, wherein the components of the genome editing system are delivered as all RNA in lipid nanoparticles or another RNA delivery reagent.

55. The method of claim 54, wherein the non-LTR site specific retrotransposon is delivered as mRNA.

56. The method of any claims above, wherein the guide RNAs are delivered as synthetic RNA.

57. The method of any claims above, wherein the payload is delivered as mRNA.

58. The genome editing system of claim 1 or 2, wherein the genome editing system targets and edits the genome at more than one site. 69 4900-8642-4067.5

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