Multicomponent systems for site-specific genome modifications

Abstract

Provided herein are genome editing systems comprising a reverse transcriptase and a template RNA with a translation-incapable 5' cap (TI-CAP) encoding a payload. The genome editing systems can be used to target, edit, modify, or manipulate a DNA sequence at one or more locations in a safe harbor site of the genome of a cell.

Description

MULTICOMPONENT SYSTEMS FOR SITE-SPECIFIC GENOME MODIFICATIONSCROSS-REFERENCE

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 546,897, filed November 1, 2023, which is hereby incorporated by reference in its entirety herein.SEQUENCE LISTING[0001.1] The instant application contains a Sequence Listing which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. Said XML copy, created on October 30, 2024, is named 67098-701_201_SL.xml and is 10,760 bytes in size.BACKGROUND

[0002] Genome editing is a powerful technology that enables precise, targeted changes to the genome in cells. A number of genome editing tools have been developed that allow manipulation of gene sequences, and hence gene function in cells. Genome editing tools include site directed nucleases, e.g., CRISPR-Cas systems or reverse transcriptases from nonlong terminal repeat (LTR) retrotransposons. LTRs are capable of self-amplification in their host-genome by expressing a non-LTR retrotransposon RT protein (nrRTs) which binds to and synthesizes cDNA using its own retroelement transcript RNA as template and a nick in genomic DNA catalyzed by a retroelement EN protein, as a primer for cDNA synthesis initiation (RT Primer Extension). This process, known as target-primed reverse transcription (TPRT), leads to the appearance of a new copy of a double-stranded DNA retroelement in the genome. nrRTs preferentially recognize and bind specific sequences in their cognate coding RNA. nrRTs and their cognate RNA can be engineered to reverse transcribe a template RNA encoding a payload leading to the insertion of the payload sequence into the genome.

[0003] Introduction of in vitro transcribed RNA or mRNA into a eukaryotic cell requires a 5 ’cap on the RNA in order to stabilize the RNA, prevent degradation, limit innate immune response to 5’ triphosphate, and promote translation of coding RNAs. Engineered nrRTs require the payload to be present as an RNA for the target-primed reverse transcription. However, a capped template RNA may be mistakenly translated independent of the insertion.

[0004] New methods and compositions to maintain stability of the template RNA and prevent accidental translation of the payload in the cytosol of a cell are needed.SUMMARY

[0005] In one aspect of the disclosure, a genome editing system is provided comprising an mRNA encoding a reverse transcriptase, wherein the mRNA comprises a 5’ cap (CAP); and a template RNA encoding a payload, wherein the template RNA comprises a translation- incapable 5’ cap (TI-CAP).

[0006] In one aspect of the disclosure a genome editing system is provided comprising a reverse transcriptase polypeptide; and a template RNA encoding a payload, wherein the template RNA comprises a translation-incapable 5’ cap (TI-CAP).

[0007] In some embodiments, the translation-incapable 5’ cap is a chemically modified cap. In some embodiments, the translation-incapable 5’ cap is a caged cap. In some embodiments, the caged cap is a photocaged cap.

[0008] In some embodiments, the template RNA with the translation-incapable 5’ cap is less immunogenic compared to an uncapped template RNA. In some embodiments, the payload comprises a transgene. In some embodiments, the translation-incapable 5' cap increases the stability of the template RNA compared to an uncapped template RNA. In some embodiments, the RNA comprising the translation-incapable 5’ cap is capable of being reverse transcribed by the reverse transcriptase.

[0009] In some embodiments, the template RNA further comprises a 3’ reverse transcriptase binding sequence.

[0010] In some embodiments, the 3’ reverse transcriptase recognition sequence comprises a sequence from a species selected from the group consisting of G. aculeatus, D. melanogaster, L. polyphemus, P. pungitis, N. vitripennis, G. fortis, O. latipes, Z. albicollis, T. guttata, T. castaneum, T. guttatus, D. simulans, B. mori, and A. vaga, or any combination thereof. In some embodiments, the 3’ reverse transcriptase recognition sequence comprises a sequence that is 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99 % identical to a 3’ reverse transcriptase recognition sequence from a species selected from the group consisting of G. aculeatus, D. melanogaster, L. polyphemus, P. pungitis, N. vitripennis, G. fortis, O. latipes, Z. albicollis, T. guttata, T. castaneum, T. guttatus, D. simulans, B. mori, and A. vaga, or any combination thereof.

[0011] In some embodiments, the template RNA further comprises a 5’ UTR sequence, a promoter sequence, a transcription termination sequence, and / or a Poly A sequence.

[0012] In some embodiments, the payload is at least 15 bases long.

[0013] In some embodiments, the mRNA further comprises a 5’ UTR sequence, a 3 ’UTR sequence, and / or a Poly A sequence.

[0014] In some embodiments, one or more uridine bases of the mRNA and / or the template RNA are modified uridine (U).

[0015] In some embodiments, the reverse transcriptase comprises a reverse transcriptase derived from a non-long terminal repeat (non-LTR) retroelement. In some embodiments, the reverse transcriptase comprises a reverse transcriptase from TaGu (Taeniopygia guttata) or ZoAl (Zonotrichia albicollis). In some embodiments, the reverse transcriptase is selected from the group consisting of TriCasB (Tribolium caslaneum), NaViB (Nasonia vitripennis) , OrLa (Oryzias talipes), ZoAl (Zonotrichia albicollis), TiGu (Tinamus guttatus) , TaGu (Taeniopygia guttata) , GeFo (Geospiza forlis), DroSi (Drosophila simulans), BoMo (Bombyx mori), DrMerc (Drosophila mercalorum), DrMe (Drosophila melanogasler), GaAc (G. aculealus), PuPu (P. pungilis), AdVa (Adineta vaga), HyMaA (Hydra magnipapillata) , Ciin (Ciona inleslinalis) , LiPo (Limulus polyphemus), TriCan Triops cancriformis) , LeCo (Lepidurus couesii), and any combination thereof. In some embodiments, the amino acid sequence of the reverse transcriptase comprises a sequence that is 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99 % identical to an amino acid sequence of TriCasB (Tribolium caslaneum), NaViB (Nasonia vitripennis), OrLa (Oryzias latipes), ZoAl (Zonotrichia albicollis), TiGu (Tinamus guttatus) , TaGu (Taeniopygia guttata) , GeFo (Geospiza fortis), DroSi (Drosophila simulans), BoMo Bombyx mori), DrMerc (Drosophila mercatorum), DrMe (Drosophila melanogaster) , GaAc (G. aculeatus), PuPu (P. pungitis), AdVa (didineia vaga), HyMaA (Hydra magnipapillata) , Ciin Ciona intestinalis) , LiPo (Limulus polyphemus), TriCan Triops cancriformis), LeCo (Lepidurus couesii), or any combination thereof.

[0016] In one aspect a pharmaceutical composition comprising a genome editing system of the disclosure and a pharmaceutically acceptable excipient is provided.

[0017] In one aspect a nanoparticle comprising a genome editing system of the disclosure or a pharmaceutical composition of the disclosure is provided.

[0018] In one aspect a method of inserting a payload into the genome of a cell, the method comprising contacting the cell with a genome editing system of the disclosure, a pharmaceutical composition of the disclosure, or a nanoparticle of the disclosure, wherein the transgene is inserted into the genome of the cell is provided.

[0019] In one aspect a method of inserting a payload into the genome of a cell in an organism, the method comprising contacting the cell in the organism with a genome editing system of the disclosure, a pharmaceutical composition of the disclosure, or a nanoparticle of the disclosure, wherein the transgene is inserted into the genome of the cell is provided.

[0020] In some embodiments, the insertion efficiency of a payload encoded in the translation- incapable 5’ cap (TI-CAP) RNA into the target site is increased compared to the insertion efficiency of a payload encoded in 5 ’uncapped RNA. In some embodiments, the transgene is inserted in a target site in the genome of the cell. In some embodiments, the target site is in the 28s rDNA sequence. In some embodiments, the transgene is only expressed after insertion into the genome.

[0021] In one aspect an engineered cell comprising a genome editing system of the disclosure is provided.

[0022] Each of the aspects and embodiments described herein are capable of being used together, unless excluded either explicitly or clearly from the context of the embodiment or aspect.DETAILED DESCRIPTION

[0023] Provided herein are, inter alia, compositions, systems, and methods for editing the genome in a cell (e.g., a human cell) comprising an mRNA encoding a reverse transcriptase (RT) with a 5’ cap (CAP) and a template RNA with a translation-incapable 5’ cap (TI-CAP) encoding a payload for inserting the payload into the genome of the cell.

[0024] Also provided herein are compositions, systems, and methods for editing the genome in a cell (e.g., a human cell) comprising a reverse transcriptase polypeptide and a template RNA with a translation-incapable 5’ cap (TI-CAP) encoding a payload for inserting the payload into the genome of the cell.

[0025] After delivery of the reverse transcriptase and the template RNA into the cytoplasm of the target cell the RT protein then binds to the template RNA, forming a ribonucleoprotein (RNP) complex that enters the nucleus of the target cell. Without being bound by theory, following delivery to the nucleus or nucleolus, it is currently thought that the endonuclease (EN) domain of the RT protein cleaves the bottom strand of the target genomic DNA, which provides a 3’ hydroxyl end that serves as a primer for reverse transcription of the template RNA by the reverse transcriptase (RT) domain of the RT protein. Following first strand synthesis to produce cDNA, the EN domain or a host endonuclease cleaves the opposite (e.g., the top strand) of the genomic DNA. The nick in the top strand produces another 3’ hydroxyl end that serves as a primer for second strand cDNA synthesis. It is currently unknown if second strand DNA synthesis is performed by the RT or by a cellular polymerase. The nick is then repaired, resulting in integration of the double-stranded cDNA comprising the payload sequence into the target site in the genomic DNA.

[0026] The following descriptions and examples illustrate embodiments of the present disclosure in detail. Although the present disclosure has been described in some details by way of illustration and example for purposes of clarity and understanding, it will be apparent that certain changes and modifications can be practiced within the scope of the appended claims.

[0027] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0028] Although various features of the disclosure can be described in the context of a single embodiment, the features can also be provided separately or in any suitable combination. Conversely, although the present disclosure can be described herein in the context of separate embodiments for clarity, the present disclosure can also be implemented in a single embodiment. It is to be understood that the present disclosure is not limited to the particular embodiments described herein and as such can vary. Those of skill in the art will recognize that there are variations and modifications of the present disclosure, which are encompassed within its scope.

[0029] It is intended that every maximum numerical limitation given throughout this specification includes every lower numerical limitation, as if such lower numerical limitations were expressly written herein. Every minimum numerical limitation given throughout this specification will include every higher numerical limitation, as if such higher numerical limitations were expressly written herein. Every numerical range given throughout this specification will include every narrower numerical range that falls within such broader numerical range, as if such narrower numerical ranges were all expressly written herein.

[0030] All patent filings, websites, other publications, accession numbers and the like cited above or below are incorporated by reference in their entirety for all purposes to the same extent as if each individual item were specifically and individually indicated to be so incorporated by reference. If different versions of a sequence are associated with an accession number at different times, the version associated with the accession number at the effective filing date of this application is meant. The effective filing date means the earlier of the actual filing date or filing date of a priority application referring to the accession number if applicable. Likewise, if different versions of a publication, website or the like are published at different times, the version most recently published at the effective filing date of the application is meant unless otherwise indicated. Any feature, step, element, embodiment, or aspect of the disclosure can be used in combination with any other unless specifically indicated otherwise.Definitions

[0031] All terms are intended to be understood as they would be understood by a person skilled in the art. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the disclosure pertains.

[0032] The following definitions supplement those in the art and are directed to the current application and are not to be imputed to any related or unrelated cases, e.g., to any commonly owned patent or application. Although any methods and materials similar or equivalent to those described herein can be used in the practice for testing of the present disclosure, the preferred materials and methods are described herein. Accordingly the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

[0033] In this application, the use of the singular includes the plural unless specifically stated otherwise. It must be noted that, as used in the specification, the singular forms “a”, “an”, and “the” include plural referents unless the context clearly dictates otherwise.

[0034] In this application, the use of “or” means “and / or” unless stated otherwise. The terms “and / or” and “any combination thereof’ and their grammatical equivalents as used herein, can be used interchangeably. These terms can convey that any combination is specifically contemplated. Solely for illustrative purposes, the following phrases “A, B, and / or C” or “A, B, C, or any combination thereof’ can mean “A individually; B individually; C individually; A and B; B and C; A and C; and A, B, and C”. The term “or” can be used conjunctively or disjunctively, unless the context specifically refers to a disjunctive use.

[0035] Furthermore, the use of the term “including” as well as other forms, such as “include”, “includes” and “included”, is not limiting.

[0036] Reference in the specification to “some embodiments”, “an embodiment”, “one embodiment” or “other embodiments” means that a particular feature, structure, or characteristic described in connection with the embodiments is included in at least some embodiments, but not necessarily all embodiments, of the present disclosures.

[0037] As used in this specification and claim(s), the words “comprising” (and any form of comprising, such as “comprise” and “comprises”), “having” (and any form of having, such as “have” and “has”), “including” (and any form of including, such as “includes” and “include”) or “containing” (and any form of containing, such as “contains” and “contain”) are inclusive or open-ended and do not exclude additional, unrecited elements or method steps. It is contemplated that any embodiment discussed in this specification can be implemented withrespect to any method or composition of the disclosure, and vice versa. Furthermore, compositions of the present disclosure can be used to achieve methods of the present disclosure.

[0038] The term “about” or “approximately” means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, z.e., the limitations of the measurement system. For example, “about” can mean within 1 or more than 1 standard deviation, per the practice in the art. Alternatively, “about” can mean a range of up to 20%, up to 10%, up to 5%, or up to 1% of a given value. In another example, the amount “about 10” includes 10 and any amounts from 9 to 11. In yet another example, the term “about” in relation to a reference numerical value can also include a range of values plus or minus 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% from that value. Alternatively, particularly with respect to biological systems or processes, the term “about” can mean within an order of magnitude, preferably within 5-fold, and more preferably within 2-fold, of a value. Where particular values are described in the application and claims, unless otherwise stated the term “about” meaning within an acceptable error range for the particular value should be assumed.

[0039] As may be used herein, the terms “nucleic acid,” “nucleic acid molecule,” “nucleic acid oligomer,” “oligonucleotide,” “nucleic acid sequence,” “nucleic acid fragment” and “polynucleotide” are used interchangeably and are intended to include, but are not limited to, a polymeric form of nucleotides covalently linked together that may have various lengths, either deoxyribonucleotides or ribonucleotides, or analogs, derivatives or modifications thereof. Different polynucleotides may have different three-dimensional structures, and may perform various functions, known or unknown. Non-limiting examples of polynucleotides include a gene, a gene fragment, an exon, an intron, intergenic DNA (including, without limitation, heterochromatic DNA), messenger RNA (mRNA), transfer RNA, ribosomal RNA, a ribozyme, cDNA, a recombinant polynucleotide, a branched polynucleotide, a plasmid, a vector, isolated DNA of a sequence, isolated RNA of a sequence, a nucleic acid probe, and a primer. Polynucleotides useful in the methods of the disclosure may comprise natural nucleic acid sequences and variants thereof, artificial nucleic acid sequences, or a combination of such sequences.

[0040] The term “identical” or percent “identity,” in the context of two or more nucleic acid or polypeptide sequences, refers to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same, when compared and aligned for maximum correspondence. Methods of alignment of sequences forcomparison are well known in the art. Once aligned, the number of matches is determined by counting the number of positions where an identical nucleotide or amino acid residue is present in both sequences. The percent sequence identity is determined by dividing the number of matches in the alignment by the length of the reference sequence, followed by multiplying the resulting value by 100. For example, a peptide sequence that has 1166 matches when aligned with a test sequence having 1554 amino acids is 75.0 percent identical to the test sequence (1166=1554 * 100=75.0). As the terms are used herein, gaps in the alignment do not decrease the percent sequence identity. Unless otherwise specified, optimal alignment of sequences for comparison is conducted by the global alignment algorithm of Needleman and Wunsch, Mol. Biol. 48:443 (1970) as implemented by EMBOSS Needle (on the World Wide Web at ebi.ac.uk / Tools / psa / emboss_needle / ) (Madeira et al. Nucleic Acids Res. 50(Wl):W276-W279 (2022)). In embodiments, other alignment methods may be used, including without limitation those described in Devereux, et al., Nucleic Acids Res.12:387-95 (1984) ; Altschul et al., J. Mol. Biol. 215:403-10 (1990) (BLAST); Carrillo and Lipman Siam J. Appl. Math. 48(5) (1988); Computational Molecular Biology (Lesk, AM, ed., 1989); Biocomputing Informatics and Genome Projects, (Smith, DW, ed., 1993); Computer Analysis of Sequence Data, Part I, (Griffin and Griffin, eds., 1994); Sequence Analysis in Molecular Biology (von Heijne, 2012); Sequence Analysis Primer (Gribskov and Devereux, J., eds. 1993). Sequence identity is calculated using the implementation of the Needleman-Wunsch algorithm provided by the National Library of Medicine (on the World Wide Web at blast. ncbi.nlm.nih.gov / Blast.cgi?PAGE_TYPE=BlastSearch&BLAST_SPEC=Global Ain)

[0041] For example, sequence identity can be determined by standard methods that are commonly used to compare the similarity of two polypeptide or two polynucleotide sequences. Using a computer program such as EMBOSS Needle or BLAST, two polypeptide or two polynucleotide sequences are aligned for optimal matching of their respective residues (either along the full length of one or both sequences, or along a predetermined portion of one or both sequences). The programs provide a default opening penalty and a default gap penalty, and a scoring matrix such as PAM 250 (a standard scoring matrix; see Dayhoff et al., in Atlas of Protein Sequence and Structure, vol. 5, supp. 3 (1978)) that can be used in conjunction with the computer program.

[0042] By "binding" is meant attaching by a covalent bond or a non-covalent bond. Non- covalent bonds include those formed by van der Waals forces, hydrogen bonds, ionic bonds, entrapment or physical encapsulation, absorption, adsorption, and / or other intermolecularforces. Binding can be effectuated by any useful means, such as by enzymatic binding (e.g., enzymatic ligation) or by chemical binding (e.g., chemical ligation).

[0043] The term “promoter,” as used herein, generally refers to the regulatory DNA region which controls transcription or expression of a gene and which may be located adjacent to or overlapping a nucleotide or region of nucleotides at which RNA transcription is initiated. A promoter may contain specific DNA sequences which bind protein factors, often referred to as transcription factors, which facilitate binding of RNA polymerase to the DNA leading to gene transcription. A ‘basal promoter’, also referred to as a ‘core promoter’, may generally refer to a promoter that contains all the basic elements to promote transcriptional expression of an operably linked polynucleotide. Eukaryotic basal promoters can contain a TATA-box or a CAAT box.

[0044] The term “expression,” as used herein, generally refers to the process by which a nucleic acid sequence or a polynucleotide is transcribed from a DNA template (such as into mRNA or other RNA transcript) or the process by which a transcribed mRNA is subsequently translated into peptides, polypeptides, or proteins. Transcripts and encoded polypeptides may be collectively referred to as “gene product.” If the polynucleotide is derived from genomic DNA, expression may include splicing of the mRNA in a eukaryotic cell.

[0045] As used herein, “operably linked,” “operable linkage,” “operatively linked”, or grammatical equivalents thereof generally refer to juxtaposition of genetic elements, e.g., a promoter, an enhancer, a polyadenylation sequence, etc., wherein the elements are in a relationship permitting them to operate in the expected manner. For instance, a regulatory element, which may comprise promoter or enhancer sequences, is operatively linked to a coding region if the regulatory element helps initiate transcription of the coding sequence. There may be intervening residues between the regulatory element and coding region so long as this functional relationship is maintained.

[0046] A “vector” as used herein, generally refers to a macromolecule or association of macromolecules that comprises or associates with a polynucleotide and which may be used to mediate delivery of the polynucleotide to a cell. Examples of vectors include plasmids, viral vectors, liposomes, and other gene delivery vehicles. The vector generally comprises genetic elements, e.g., regulatory elements, operatively linked to a gene to facilitate expression of the gene in a target.

[0047] As used herein, “an expression cassette” and “a nucleic acid cassette” are used interchangeably generally to refer to a combination of nucleic acid sequences or elements that are expressed together or are operably linked for expression. In some cases, anexpression cassette refers to the combination of regulatory elements and a gene or genes to which they are operably linked for expression.

[0048] As used herein, an “engineered” object generally indicates that the object has been modified by human intervention. According to non-limiting examples: a nucleic acid may be modified by changing its sequence to a sequence that does not occur in nature; a nucleic acid may be modified by ligating it to a nucleic acid that it does not associate with in nature such that the ligated product possesses a function not present in the original nucleic acid; an engineered nucleic acid may synthesized in vitro with a sequence that does not exist in nature; a protein may be modified by changing its amino acid sequence to a sequence that does not exist in nature; an engineered protein may acquire a new function or property. An “engineered” system comprises at least one engineered component.

[0049] Included in the current disclosure are variants of any of the reverse transcriptases described herein with one or more conservative amino acid substitutions. Such conservative substitutions can be made in the amino acid sequence of a polypeptide without disrupting the three-dimensional structure or function of the polypeptide. Conservative substitutions can be accomplished by substituting amino acids with similar hydrophobicity, polarity, and R chain length for one another. Additionally, or alternatively, by comparing aligned sequences of homologous proteins from different species, conservative substitutions can be identified by locating amino acid residues that have been mutated between species (e.g., non-conserved residues) without altering the basic functions of the encoded proteins. Such conservatively substituted variants may include variants with at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% identity to any one of the reverse transcriptase sequences described herein. In some embodiments, such conservatively substituted variants are functional variants.

[0050] Conservative substitution tables providing functionally similar amino acids are available from a variety of references (see, for e.g., Creighton, Proteins: Structures and Molecular Properties (W H Freeman & Co.; 2nd edition (December 1993)). The following eight groups each contain amino acids that are conservative substitutions for one another:1) Alanine (A), Glycine (G);2) Aspartic acid (D), Glutamic acid (E);3) Asparagine (N), Glutamine (Q);4) Arginine (R), Lysine (K);5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V);6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W);7) Serine (S), Threonine (T); and8) Cysteine (C), Methionine (M)

[0051] The terms “target site” or “target sites”, as used herein, refer to, with respect to the present invention in particular, a position in a gene that when targeted with a nuclease will be bound and / or cleaved by the reverse transcriptase.

[0052] The terms “target” or “targets” or “to target” or “targeting”, as used herein, refer to, with respect to the inventions of the instant application, aiming or directing a nuclease to a particular, selected DNA sequence using, for example, a selected or engineered DNA binding domain or guide RNA.

[0053] The term “treating”, “treatment”, or any grammatical variant thereof of a condition as used herein includes preventing or alleviating a condition, slowing the onset or rate of development of a condition, reducing the risk of developing a condition, preventing or delaying the development of symptoms associated with a condition, reducing or ending symptoms associated with a condition, generating a complete or partial regression of a condition, curing a condition, or some combination thereof.

[0054] A “subject” or “individual” can be a vertebrate, a mammal, or a human. Mammals include, but are not limited to, farm animals, sport animals, pets, primates, rodents, mice and rats. In one aspect, a subject is a human.

[0055] It is appreciated that certain features of the disclosure, which are, for clarity, described in the context of separate embodiments, can also be provided in combination in a single embodiment. Conversely, various features of the disclosure, which are, for brevity, described in the context of a single embodiment, can also be provided separately or in any suitable sub-combination. All combinations of the embodiments pertaining to the disclosure are specifically embraced by the present disclosure and are disclosed herein just as if each and every combination was individually and explicitly disclosed. In addition, all subcombinations of the various embodiments and elements thereof are also specifically embraced by the present disclosure and are disclosed herein just as if each and every such sub combination was individually and explicitly disclosed herein.Genome Editing Systems

[0056] Provided herein are, inter alia, genome editing systems comprising a reverse transcriptase and a template RNA with a translation-incapable 5’ cap (TI-CAP) encoding a payload. The genome editing systems can be used to target, edit, modify, or manipulate a DNA sequence at one or more locations in the genome of a cell.

[0057] In some embodiments, the genome editing system can be an mRNA encoding a reverse transcriptase and a 5’ cap (CAP), and a template RNA encoding a payload and a translation- incapable 5’ cap (TI-CAP).

[0058] In some embodiments, the genome editing system can be a genome editing system comprising (a) a reverse transcriptase polypeptide and (b) a template RNA encoding a payload comprises a translation-incapable 5’ cap (TI-CAP).Template RNA

[0059] In some aspects, the disclosure provides for a template RNA with a translation-incapable 5’ cap (TI-CAP). RNAs with a translation-incapable 5’ cap have no or reduced translation into a polypeptide in a cell compared to capped RNA.

[0060] In some embodiments, the translation-incapable 5’ cap can be a chemically modified cap. In some embodiments, the translation-incapable 5’ cap can be a chemically caged cap. In some embodiments, the caged cap is a photocaged cap. In some embodiments, the caged cap is photocleavable.

[0061] In some embodiments, the template RNA with the translation-incapable 5’ cap is less immunogenic compared to an uncapped template RNA. In some embodiments, the template RNA with the translation-incapable 5’ cap has increased stability compared to an uncapped template RNA.

[0062] In some embodiments, the translation-incapable 5' cap increases the stability of the template RNA compared to an uncapped template RNA. In some embodiments, the translation incapable 5' cap decreases the immunogenicity of the template RNA compared to an uncapped template RNA.

[0063] In some embodiments, the RNA with the translation-incapable 5’ cap is capable of being reverse transcribed by a reverse transcriptase.

[0064] In some embodiments, the payload encoded by the template RNA can be a heterologous polynucleotide. In some embodiments, the payload encoded by the template RNA can be a transgene. In some embodiments, the payload encoded by the template RNA can be an ORF for a transgene. In some embodiments, the payload encoded by the template RNA can be apolypeptide or a protein. In some embodiments, the polypeptide or protein is a therapeutic polypeptide or protein. In some embodiments, the payload encoded by the template RNA encodes for a repair template of an endogenous gene. In some embodiments, the repair template includes an insertion, a deletion, or a mutation compared to the endogenous gene. In some embodiments, the payload sequence encodes a reporter protein such as GFP or luciferase. In some embodiments, the payload sequence encodes a therapeutic protein that replaces or complements a defective gene or protein. In some embodiments, the therapeutic protein is used to treat a disease or condition in a subject or patient.

[0065] In some embodiments, the payload sequence encodes a regulatory RNA. In some embodiments, the regulatory RNA is selected from a ligand-binding riboswitch, such as a ligand-activated riboswitch or an allosteric ribozyme (aptazyme), a small RNA (sRNA), a small interfering RNA (siRNA) or a short hairpin RNA (shRNA). In some embodiments, the payload encoded by the template RNA can be an siRNA. In some embodiments, the siRNA or shRNA can inhibit a therapeutic target.

[0066] In some embodiments, the payload is about 15 bases in length. In some embodiments, the payload is about 20 bases in length. In some embodiments, the payload is about 25 bases in length. In some embodiments, the payload is about 30 bases in length. In some embodiments, the payload is about 40 bases in length. In some embodiments, the payload is about 50 bases in length. In some embodiments, the payload is about 60 bases in length. In some embodiments, the payload is about 70 bases in length. In some embodiments, the payload is about 80 bases in length. In some embodiments, the payload is about 90 bases in length. In some embodiments, the payload is about 100 bases in length. In some embodiments, the payload is about 150 bases in length. In some embodiments, the payload is about 200 bases in length. In some embodiments, the payload is about 350 bases in length. In some embodiments, the payload is about 400 bases in length. In some embodiments, the payload is about 450 bases in length. In some embodiments, the payload is about 500 bases in length. In some embodiments, the payload is about 550 bases in length. In some embodiments, the payload is about 600 bases in length. In some embodiments, the payload is about 650 bases in length. In some embodiments, the payload is about 700 bases in length. In some embodiments, the payload is about 750 bases in length. In some embodiments, the payload is about 800 bases in length. In some embodiments, the payload is about 850 bases in length. In some embodiments, the payload is about 900 bases in length. In some embodiments, the payload is about 950 bases in length. In some embodiments, the payload is about 1000 bases in length. In some embodiments, the payload is about 1500 bases inlength. In some embodiments, the payload is about 2000 bases in length. In some embodiments, the payload is about 2500 bases in length. In some embodiments, the payload is about 3000 bases in length. In some embodiments, the payload is about 3500 bases in length. In some embodiments, the payload is about 4000 bases in length. In some embodiments, the payload is about 5000 bases in length. In some embodiments, the payload is about 6000 bases in length. In some embodiments, the payload is about 7000 bases in length. In some embodiments, the payload is about 8000 bases in length. In some embodiments, the payload is about 9000 bases in length. In some embodiments, the payload is about 10000 bases in length. In some embodiments, the payload is more than 10000 bases in length.

[0067] In some embodiments, the payload is between 15 bases and 20 bases in length. In some embodiments, the payload is between 20 bases and 30 bases in length. In some embodiments, the payload is between 30 bases and 40 bases in length. In some embodiments, the payload is between 40 bases and 50 bases in length. In some embodiments, the payload is between 50 bases and 60 bases in length. In some embodiments, the payload is between 60 bases and 70 bases in length. In some embodiments, the payload is between 70 bases and 80 bases in length. In some embodiments, the payload is between 80 bases and 90 bases in length. In some embodiments, the payload is between 90 bases and 100 bases in length. In some embodiments, the payload is between 100 bases and 200 bases in length. In some embodiments, the payload is between 200 bases and 300 bases in length. In some embodiments, the payload is between 300 bases and 400 bases in length. In some embodiments, the payload is between 400 bases and 500 bases in length. In some embodiments, the payload is between 500 bases and 600 bases in length. In some embodiments, the payload is between 600 bases and 700 bases in length. In some embodiments, the payload is between 700 bases and 800 bases in length. In some embodiments, the payload is between 800 bases and 900 bases in length. In some embodiments, the payload is between 900 bases and 1000 bases in length. In some embodiments, the payload is between 1000 bases and 2000 bases in length. In some embodiments, the payload is between 2000 bases and 3000 bases in length. In some embodiments, the payload is between 3000 bases and 4000 bases in length. In some embodiments, the payload is between 4000 bases and 5000 bases in length, n some embodiments, the payload is between 5000 bases and 6000 bases in length. In some embodiments, the payload is between 6000 bases and 7000 bases in length. In some embodiments, the payload is between 7000 bases and 8000 bases in length. In someembodiments, the payload is between 8000 bases and 9000 bases in length, n some embodiments, the payload is between 9000 bases and 10000 bases in length. In some embodiments, the payload is between 15 bases and 10000 bases in length. In some embodiments, the payload is between 100 bases and 10000 bases in length.

[0068] In some embodiments, the template RNA further comprises regulatory elements. In some embodiments, the template RNA further comprises a 5’ UTR sequence. In some embodiments, the template RNA further comprises a promoter sequence. In some embodiments, the template RNA further comprises a Poly A sequence. In some embodiments, the template RNA further comprises a transcription termination sequence. In some embodiments, the template RNA further comprises a 5’ UTR sequence, a promoter sequence, a Poly A sequence, and / or a transcription termination sequence.

[0069] In some embodiments, the regulatory elements of the template RNA are operably linked to each other. It will be understood that the relative positions of the individual elements in the template RNA can vary in the 5’ to 3’ direction. For example, in some embodiments, the template RNA comprises, in a 5’ to 3’ direction a 5’ UTR sequence, a promoter sequence, a Poly A sequence, and / or a transcription termination sequence. It will be further understood that the individual elements in the template RNA can vary in their 5’ to 3’ orientation relative to other elements. In some embodiments, the direction of transcription of the payload sequence in the template can be reversed, such that in one orientation the promoter is closest to the 5’ end of the template RNA, or in a second orientation the promoter is closest to the 3’ end of the template RNA.

[0070] In some embodiments, the promoter is an RNA polymerase (Pol) II promoter. In some embodiments, the promoter is selected from an EFS promoter, ABPnat mini promoter, CRNM-TTR enhancer promoter, AAV-rDNA TTR promoter, or CBh promoter.

[0071] In some embodiments, the promoter is an immediate early cytomegalovirus (CMV) promoter, an Elongation Growth Factor-la (EF-la)promoter, a simian virus 40 (SV40) early promoter, a mouse mammary tumor virus (MMTV), a human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, a MoMuLV promoter, an avian leukemia virus promoter, an Epstein-Barr virus immediate early promoter, a Rous sarcoma virus promoter, a U6 promoter, as well as human gene promoters such as, but not limited to, the actin promoter, the myosin promoter, the hemoglobin promoter, and the creatine kinase promoter. Other suitable promoters include an MND promoter or an SSFV promoter. In some embodiments, the promoter is a liver specific promoter. In some embodiments, the liver specific promoter is a thyroxine binding globulin (TBG) promoter, a cytomegalovirusimmediate-early enhancer / chicken beta actin promoter (CAG) promoter, or a al -antitrypsin promoter.

[0072] In some embodiments, the polyA sequence is selected from a short SV40 poly, SNRP1 poly A, a synthetic polyA, a BGH polyA, or a BGH polyA min. In some embodiments, the template RNA includes a WPRE3 3’ enhancer.

[0073] In some embodiments, the template RNA further comprises a 3’ reverse transcriptase (RT) binding sequence. In some embodiments, the RT binding sequence comprises a sequence isolated from the 3’ region of a natural non-LTR retroelement or an organism comprising a non-LTR retroelement. In some embodiments, the RT binding sequence comprises a 3’UTR sequence. In some embodiments, the 3’UTR sequence is isolated from an organism comprising a non-LTR retroelement. In some embodiments, the 3’UTR sequence is isolated from an organism selected from the group consisting of G. aculeatus, D. melanogaster, L. polyphemus, P. pungitis, N. vitripennis, G. fortis, O. latipes, Z. albicollis, T. guttata, T. castaneum, T. guttatus, D. simulans, B. mori, and A. vaga. In some embodiments, the RT binding sequence comprises a sequence having greater than or equal to 60% sequence identity (e.g., greater than or equal to 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% identity) to a sequence isolated from G. aculeatus, D. melanogaster, L. polyphemus, P. pungitis, N. vitripennis, G. fortis, O. latipes, Z. albicollis, T. guttata, T. castaneum, T. guttatus, D. simulans, B. mori, or A. vaga.

[0074] In some embodiments, the RT binding sequence comprises a modified (non-natural) sequence. For example, the RT binding sequence can be modified to increase or decrease binding to an RT protein of the disclosure.

[0075] In some embodiments, the RT binding sequence is about 20 bases in length. In some embodiments, the RT binding sequence is about 30 bases in length. In some embodiments, the RT binding sequence is about 40 bases in length. In some embodiments, the RT binding sequence is about 50 bases in length. In some embodiments, the RT binding sequence is about 60 bases in length. In some embodiments, the RT binding sequence is about 65 bases in length. In some embodiments, the RT binding sequence is about 70 bases in length. In some embodiments, the RT binding sequence is about 75 bases in length. In some embodiments, the RT binding sequence is about 80 bases in length. In some embodiments, the RT binding sequence is about 90 bases in length. In some embodiments, the RT binding sequence is about 100 bases in length. In some embodiments, the RT binding sequence is about 150 bases in length. In some embodiments, the RT binding sequence is about 200 bases in length. In some embodiments, the RT binding sequence is about 350 bases in length.In some embodiments, the RT binding sequence is about 400 bases in length. In some embodiments, the RT binding sequence is about 450 bases in length. In some embodiments, the RT binding sequence is about 500 bases in length.

[0076] In some embodiments, the RT binding sequence is between about 20 bases and about 500 bases in length. In some embodiments, the RT binding sequence is between about 30 bases and about 500 bases in length. In some embodiments, the RT binding sequence is between about 40 bases and about 500 bases in length. In some embodiments, the RT binding sequence is between about 50 bases and about 500 bases in length. In some embodiments, the RT binding sequence is between about 60 bases and about 500 bases in length. In some embodiments, the RT binding sequence is between about 70 bases and about 500 bases in length. In some embodiments, the RT binding sequence is between about 80 bases and about 500 bases in length. In some embodiments, the RT binding sequence is between about 90 bases and about 500 bases in length. In some embodiments, the RT binding sequence is between about 100 bases and about 500 bases in length. In some embodiments, the RT binding sequence is between about 150 bases and about 500 bases in length. In some embodiments, the RT binding sequence is between about 200 bases and about 500 bases in length. In some embodiments, the RT binding sequence is between about 300 bases and about 500 bases in length. In some embodiments, the RT binding sequence is between about 400 bases and about 500 bases in length. In some embodiments, the RT binding sequence is between about 20 bases and about 100 bases in length. In some embodiments, the RT binding sequence is between about 100 bases and about 200 bases in length. In some embodiments, the RT binding sequence is between about 100 bases and about 500 bases in length. In some embodiments, the RT binding sequence is between about 20 bases and about 200 bases in length.Additional Components

[0077] In some embodiments, the template RNA, further comprises 5’ and 3’ elements that regulate transcription, translation, and / or insertion of the payload sequence at a target site in the host cell genome. Non-limiting examples of these elements are described below.

[0078] In some embodiments, the template RNA comprises a Kozak consensus translation start site upstream or 5’ of the payload sequence. In some embodiments, the Kozak sequence comprises the sequence 5’-GCCACC-3’).

[0079] In some embodiments, the template RNA comprises an RNA polymerase (RNAP) terminator sequence located 5’ of the promoter sequence. The RNAP terminator sequence functions to stop RNA polymerase read-through from genes at the target insertion site. Insome embodiments, the RNAP terminator sequence comprises the sequence 5’- AGGTCGACCAGATGTCCGAGGTCGACCAGTTGTCCG-3’ (SEQ ID N0:2).

[0080] In some embodiments, the template RNA includes a 5’ sequence that promotes sitespecific insertion of the heterologous polynucleotide into a target site in the eukaryotic genome.

[0081] In some embodiments, the template RNA comprises a 3’ sequence that promotes sitespecific insertion of the heterologous polynucleotide into the eukaryotic genome. In some embodiments, the template RNA comprises a 3’ sequence that enhances the efficiency and fidelity of target-primed reverse transcription.

[0082] In some embodiments, the template RNA comprises a sequence useful for purification of the template RNA. In some embodiments, the sequence useful for purification of the template RNA comprises a hairpin structure that binds to the PP7 coat protein or a truncated version thereof. See, for example, Hogg, J.R. & Collins, K. RNA-based affinity purification reveals 7SK RNPs with distinct composition and regulation. RNA 13, 868-880 (2007).

[0083] In some embodiments, the template RNA comprises a sequence that binds to a DNA binding protein, which allows for enrichment of the inserted double strand sequences in the target DNA by purifying fragments of the genomic DNA comprising the sequence that bind the DNA binding protein. In some embodiments, the payload sequence is flanked by sequences that bind to a DNA binding protein, such that one sequence is located 5’ of the payload sequence (e.g., upstream of the promoter sequence), and another sequence is located 3’ of the payload sequence (e.g., downstream of the polyA sequence). In some embodiments, the template RNA comprises a lacO operator sequence that binds to the LacI protein. In some embodiments, the template RNA comprises a first lacO operator sequence located 5’ of the payload sequence and a second lacO operator sequence located 3 ’ of the payload sequence.

[0084] In some embodiments, the template RNA comprises a) a 5’ sequence that is homologous to a DNA sequence located 5’ to a target insertion site in the eukaryotic genome; or (b) a 3’ sequence that is homologous to a DNA sequence located 3’ to a target insertion site in the eukaryotic genome; or both (a) and (b). In some embodiments, the 5’ homologous sequence comprises about 1 to 36 nucleotides of homologous sequence that base pairs with a complementary sequence at the target site. In some embodiments, the 3’ homologous sequence comprises about 1 to 30 nucleotides of homologous sequence that base pairs with a complementary sequence at the target site.Reverse Transcriptases

[0085] In some aspects, the genome editing systems of the disclosure comprise a reverse transcriptase (RT). In some aspects, the genome editing systems of the disclosure comprise an mRNA encoding for a reverse transcriptase.

[0086] In some embodiments, the reverse transcriptase is derived from a non-long terminal repeat retroelement.

[0087] Exemplary non-LTR retroelement reverse transcriptase (RT) are described in WO2022 / 155055 which is incorporated by reference herein in its entirety (and for the reverse transcriptases disclosed in particular). As used herein, the term “Reverse Transcriptase (RT)” is used in its broadest sense to refer to any biopolymer with reverse transcription activity. In some embodiments, an RT may be derived from a non-LTR RT from the Zonotrichia albicollis, Taeniopygia guttata, Tinamus guttatus, Geospiza fortis, Pungitis pungitis, Oryzias latipes, Danio rerio, Oryzias melastigma, Petromyzon marinus, Salmo trutta, Salmo salar, or Gasterosteus aculeatus, Drosophila mercatorum, Drosophila melanogaster, Nasonia vitripennis, Tribolium castaneum, Drosophila simulans, Apis cerana, Bombyx mori, Lepidurus couesii, Triops cancriformis, Limulus polyphemus, Hydra magnipapillata, Adineta vaga, Ciona intestinalis, other birds, other arthropods, other fish, other tunicates, other animals (including mammals and humans) or the like’s genomes.

[0088] In some embodiments, the mRNA encodes an amino acid sequence that is substantially identical to an RT protein isolated from Zonotrichia albicollis, Taeniopygia guttata, Tinamus guttatus, Geospiza fortis, Pungitis pungitis, Oryzias latipes, Daniorerio, Oryzias melastigma, Petromyzon marinus, Salmo trutta, Salmo salar, Gasterosteus aculeatus, Drosophila mercatorum, Drosophila melanogaster, Nasonia vitripennis, Tribolium castaneum, Drosophila simulans, Apis cerana, Bombyx mori, Lepidurus couesii, Triops cancriformis, Limulus polyphemus, Hydra magnipapillata, Adineta vaga, o r Ciona intestinalis. In some embodiments, the mRNA encodes an amino acid sequence having at least 60% sequence identity (e.g., greater than or equal to 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% identity) to an RT protein isolated from Zonotrichia albicollis, Taeniopygia guttata, Tinamus guttatus, Geospiza fortis, Pungitis pungitis, Oryzias latipes, Daniorerio, Oryzias melastigma, Petromyzon marinus, Salmo trutta, Salmo salar, Gasterosteus aculeatus, Drosophila mercatorum, Drosophila melanogaster, Nasonia vitripennis, Tribolium castaneum, Drosophila simulans, Apis cerana, Bombyx mori, Lepidurus couesii, Triops cancriformis, Limulus polyphemus, Hydra magnipapillata, Adineta vaga, o r Cionaintestinalis. In some embodiments, the RT protein comprises an RT protein isolated from other animals.

[0089] In some embodiments, the RNA encoding the reverse transcriptase is an mRNA. In some embodiments, the mRNA further comprises regulatory elements. In some embodiments, the template RNA further comprises a 5’ UTR sequence. In some embodiments, the mRNA further comprises a Poly A sequence. In some embodiments, the template RNA further comprises a 3 ’UTR sequence. In some embodiments, the mRNA further comprises a 5’ UTR sequence, a Poly A sequence, and / or a 3 ’UTR sequence.

[0090] In some embodiments, the mRNA further comprises a 5’ cap. In some embodiments, the mRNA further comprises a translation capable 5’ cap.

[0091] In some embodiments, the mRNA comprises nucleic acid mimetics selected from the group of peptide nucleic acid (PNA), morpholino nucleic acid, cyclohexenyl nucleic acid (CeNAs), and locked nucleic acid (LNA). In some embodiments, the mRNA comprises modified sugar moieties, optionally wherein the modified sugar moiety is selected from the group of N1 -methylpseudouridine, 9-Methyladenine, 2'-O-(2 -methoxy ethyl), 2'- dimethylaminooxyethoxy, 2'-dimethylaminoethoxyethoxy, 2'-O-methyl, and 2'-fluoro. In some embodiments, the mRNA comprises a modified nucleobase, optionally wherein the modified nucleobase is selected from the group of a 5-methylcytosine; a 5 -hydroxymethyl cytosine; a xanthine; a hypoxanthine; a 2-aminoadenine; a 6-methyl derivative of adenine; a 6-methyl derivative of guanine; a 2-propyl derivative of adenine; a 2-propyl derivative of guanine; a 2-thiouracil; a 2-thiothymine; a 2-thiocytosine; a 5-halouracil; a 5-halocytosine; a 5-propynyl uracil; a 5-propynyl cytosine; a 6-azo uracil; a 6-azo cytosine; a 6-azo thymine; a pseudouracil; a 4-thiouracil; an 8-halo; an 8-amino; an 8-thiol; an 8-thioalkyl; an 8-hydroxyl; a 5-halo; a 5-bromo; a 5 -trifluoromethyl; a 5-substituted uracil; a 5-substituted cytosine; a 7- methylguanine; a 7-methyladenine; a 2-F-adenine; a 2-amino-adenine; an 8-azaguanine; an 8-azaadenine; a 7-deazaguanine; a 7-deazaadenine; a 3 -deazaguanine; a 3 -deazaadenine; a tricyclic pyrimidine; a phenoxazine cytidine; a phenothiazine cytidine; a substituted phenoxazine cytidine; a carbazole cytidine; a pyridoindole cytidine; a 7-deaza-adenine; a 7- deazaguanosine; a 2-aminopyridine; a 2-pyridone; a 5-substituted pyrimidine; a 6- azapyrimidine; an N-2, N-6 or O-6 substituted purine; a 2-aminopropyladenine; a 5- propynyluracil; or a 5-propynylcytosine. In some embodiments, the mRNA comprises a non-naturally occurring or a non-natural internucleoside linkage selected from the group of a phosphorothioate, a phosphoramidate, a non-phosphodiester, a heteroatom, a chiral phosphorothioate, a phosphorodithioate, a phosphotriester, an aminoalkylphosphotriester, a3'-alkylene phosphonates, a 5'-alkylene phosphonate, a chiral phosphonate, a phosphinate, a 3'-amino phosphoramidate, an aminoalkylphosphoramidate, a phosphorodiamidate, a thionophosphoramidate, a thionoalkylphosphonate, a thionoalkylphosphotriester, a selenophosphate, or a boranophosphate.Modified Uridines

[0092] In some embodiments, the mRNA encoding the RT and / or the template RNA comprises one or more modified uridine (U) nucleosides. RNAs containing unmodified uridines can activate the innate immune response and are less stable in cells. Modified uridines can provide the following advantages: i) they reduce the innate immune response in a host organism when cells are transfected with the RT mRNA and template RNA of the disclosure, ii) increase RNA stability, iii) increase the amount of protein produced when the RNAs are translated and iv) improve the payload insertion efficiency relative to an template RNA without modified uridines.

[0093] In some embodiments, the mRNA encoding the RT protein or the template RNA comprises one or more modified uridine (U) nucleosides, selected from the group consisting of Nl-methyl-pseudouridine (Nlm'PU), pseudouridine (TU), 5-methyluridine (5meU), 5- methyoxyuridine (5moU), and mixtures thereof. In some embodiments, the mRNA encoding the RT protein or the template RNA comprises Nl-methyl-pseudouridine (NlmTU). In some embodiments, the mRNA encoding the RT protein or the template RNA comprises a mixture or combination of unmodified uridines and modified uridines selected from the group consisting of NlmTU, U, 5meU, and 5moU.

[0094] In some embodiments, the template RNA comprises one or more modified uridine nucleosides.

[0095] In some embodiments, the template RNA comprises one or more modified uridines selected from the group consisting of Nl-methyl-pseudouridine (Nlm'PU), pseudouridine (TU), 5-methyluridine (5meU), 5-methyoxyuridine (5moU), and mixtures thereof. In some embodiments, the template RNA comprises a single type of modified uridine selected from one of the following: Nl-methyl-pseudouridine (NlmT'U), pseudouridine (TU), 5- methyluridine (5meU), or 5-methyoxyuridine (5moU). In some embodiments, the template RNA comprises Nl-methyl-pseudouridine (NlmT'U). In some embodiments, the template RNA comprises a mixture or combination of unmodified uridines and modified uridines selected from the group consisting of Nlm'PU, U, 5meU, and 5moU.

[0096] In some embodiments, the template RNA comprising modified uridines is not cleavable by a ribozyme. In some embodiments, a template RNA comprising the modified uridines Nl-methyl-pseudouridine (NlmTU) or pseudouridine ( U) is not cleavable by a ribozyme. In some embodiments, a template RNA comprising a modified uridine increases the efficiency of insertion into the eukaryotic genome compared to template RNA comprising an unmodified uridine.

[0097] In some embodiments, cellular toxicity is decreased when the template RNA comprises a modified uridine.

[0098] It will be understood by a person of skill in the art that modified uridines are distributed throughout the template RNA sequence, and that in some embodiments, all the uridines comprise the same modified uridine (e.g., all the uridines are Nl-methyl-pseudouridine (NlmTU) or all the modified uridines are pseudouridine (TU)).Engineered Cells

[0099] In one aspect, the present disclosure provides engineered cells comprising the genome editing systems described herein.

[0100] In some embodiments, the cell is a eukaryotic cell. In some embodiments, the cell is a human cell. In some embodiments, the cell is a diseased cell. In some embodiments, the cell is derived from a human subject with a disease. In some embodiments, the cell is an autologous cell. In some embodiments, the cell is an allogeneic cell (from a donor).

[0101] In some embodiments, a heterologous polynucleotide is inserted in the cellular genome by the genome editing system. In some embodiments, the heterologous polynucleotide is a transgene. In some embodiments, the heterologous polynucleotide is a siRNA.

[0102] In some embodiments, the heterologous polynucleotide is inserted at a so-called “safe harbor” site in the host cell genome, which does not alter normal cellular physiology or metabolism. Examples of safe harbor sites include regions of the genome with high copy numbers of repeated genes, such that disruption of one gene will not significantly alter normal cellular physiology or metabolism. Examples of high copy number regions include rDNA genes that encode rRNA. Thus, in some embodiments, the target insertion site is located in a ribosomal RNA gene or ribosomal DNA (rDNA). In some embodiments, the heterologous polynucleotide is inserted in genomic DNA that encodes a ribosomal RNA (rRNA). In some embodiments, the heterologous polynucleotide is inserted in a 5S, 8S, 18S, or 28S rDNA sequence. In some embodiments, the heterologous polynucleotide is inserted in a 28S rDNA sequence in the genome.

[0103] In some embodiments, the cell expresses the heterologous polynucleotide. In some embodiments, cell expresses a polypeptide or a protein encoded by the heterologous polynucleotide.Pharmaceutical Compositions

[0104] Also provided herein are pharmaceutical compositions comprising the genome editing systems described herein and a pharmaceutically acceptable carrier or a pharmaceutically acceptable excipient.

[0105] In some embodiments, the pharmaceutical composition comprises an mRNA encoding the RT protein and the template RNA described herein. In some embodiments, the pharmaceutical composition comprises an RT protein and the template RNA described herein.

[0106] In some embodiments, the pharmaceutical composition comprises a lipid nanoformulation, such as a liposome or a lipid nanoparticle (LNP). In some embodiments, the pharmaceutical composition comprises a pharmaceutically acceptable excipient or salt. Examples of pharmaceutically acceptable excipients are described in the United States Pharmacopoeia (USP), the European Pharmacopoeia (EP), the British Pharmacopoeia, and the International Pharmacopoeia.

[0107] As used herein, a “pharmaceutically acceptable carrier” or a “pharmaceutically acceptable excipient” or a “pharmaceutically acceptable salt” according to the present disclosure is a component such as a carrier, diluent, or excipient of a composition that is compatible with the other ingredients of the composition in that it can be combined with the agents and / or compositions of the present disclosure without eliminating the biological activity of the agents or the compositions and is suitable for use in subjects as provided herein without undue adverse side effects (such as toxicity, irritation, allergic response, and death). Side effects are “undue” when their risk outweighs the benefit provided by the pharmaceutical composition. Non-limiting examples of pharmaceutically acceptable components include, without limitation, any of the standard pharmaceutical carriers such as phosphate buffered saline solutions, water, sterile water, polyethylene glycol, polyvinyl pyrrolidone, lecithin, arachis oil, sesame oil, emulsions such as oil / water emulsions or water / oil emulsions, microemulsions, nanocarriers and various types of wetting agents. Additives such as water, alcohols, oils, glycols, preservatives, flavoring agents, coloring agents, suspending agents, and the like may also be included in the composition along with the carrier, diluent, or excipient. In one embodiment, a pharmaceutically acceptable carrier appropriate for use in the compositions disclosed herein is sterile, pathogen free, and / orotherwise safe for administration to a subject without risk of associated infection and other undue adverse side effects.

[0108] Any of the pharmaceutical compositions disclosed herein can be formulated for administration using any number of administrative methods available in the art. Administration can be by a variety of routes including pump, patch, catheter, stent, oral, rectal, transdermal, subcutaneous, intravenous, intramuscular, intranasal, and the like.Delivery Methods

[0109] The compositions and genome editing systems of the disclosure can be introduced into target cells using a method compatible with RNA delivery or RNP delivery.

[0110] In some embodiments, a genome editing system may be formulated in delivery vehicles. In general, delivery vehicles may facilitate in vivo or in vitro transfection of subject cells by protecting genome editing system components from degradation in the extracellular environment, facilitating uptake by subject cells, enhancing endosomal escape, and any combination thereof. Delivery vehicles may include but are not limited to nanoparticles including lipid-based nanoparticles (e.g., lipid nanoparticles (LNPs), liposomes, and micelles) and non-lipid nanoparticles (e.g., virus like particles (VLPs) and polymeric delivery particles).[oni] In some embodiments, mRNA encoding the RT protein and the template RNA are introduced into the target cell using a lipid nanoformulation, such as a liposome or lipid nanoparticle (LNP), a lipofection reagent, or by electroporation. In some embodiments, the RT protein and the template RNA are introduced into the target cell using a lipid nanoformulation, such as a liposome or lipid nanoparticle (LNP), a lipofection reagent, or by electroporation.

[0112] LNPs useful herein are known in the art and generally comprise an ionizable (cationic) lipid, a phospholipid, cholesterol, and a polymer-conjugated lipid. Without wishing to be bound by theory, cholesterol promotes membrane fusion and aids in LNP stability, a phospholipids may aid in endosomal escape and provide structure to the LNP bilayer, polymer-conjugated lipids reduce LNP aggregation and “protects” the LNP from nonspecific endocytosis by immune cells, and the ionizable (cationic) lipid enhances endosomal escape and complexes negatively charged cargo (such as mRNA encoding the RT protein or the template RNA).

[0113] In some embodiments, the target cell is not transduced with a virus. Virus transduction is associated with various undesirable effects on cells, including mutations in the host cellchromosomes, random integration, and the presence of double-strand breaks that can cause cellular toxicity.Methods of Using Genome Editing Systems

[0114] In one aspect of the disclosure, methods for editing the genome of a cell with the genome editing systems described herein are provided.

[0115] In some embodiments, the method comprises contacting a cell with a genome editing system of the disclosure. In some embodiments, the method comprises contacting a cell with an LNP comprising a genome editing system of the disclosure.

[0116] In some embodiments, the disclosure provides methods for inserting a heterologous polynucleotide at a target site into a eukaryotic genome. In some embodiments, the disclosure provides methods for inserting a heterologous therapeutic polynucleotide at a target site into a eukaryotic genome. In some embodiments, the polynucleotide encodes for a polypeptide or a protein. In some embodiments, the polypeptide or protein is a therapeutic polypeptide or protein. In some embodiments, the payload encoded by the template RNA encodes for a repair template of an endogenous gene. In some embodiments, the repair template includes an insertion, a deletion, or a mutation compared to the endogenous gene. In some embodiments, the payload sequence encodes a reporter protein such as GFP or luciferase. In some embodiments, the payload sequence encodes a therapeutic protein that replaces or complements a defective gene or protein. In some embodiments, the therapeutic protein is used to treat a disease or condition in a subject or patient. In some embodiments, the disclosure provides methods for inserting a heterologous siRNA at a target site into a eukaryotic genome. In some embodiments, the disclosure provides methods for deleting a genomic sequence at a target site in a eukaryotic genome. In some embodiments, the disclosure provides methods for exchanging a heterologous polynucleotide at a target site in a eukaryotic genome.

[0117] In some embodiments, the method comprises transfecting a eukaryotic cell with: (a) an mRNA encoding a reverse transcriptase (RT) and (b) a template RNA with a translation- incapable 5’ cap (TI-CAP) encoding a payload polynucleotide.

[0118] In some aspects, the method is an in vivo method. In some embodiments, the method is an ex vivo method.

[0119] In some embodiments, the insertion efficiency of the payload encoded in the translation- incapable 5’ cap (TI-CAP) RNA into the target site is increased compared to the insertion efficiency of the payload encoded in 5 ’uncapped RNA.Methods of Treatment

[0120] Also provided are methods of treating a subject or patient with the genome editing systems and pharmaceutical compositions described herein. The methods can be used to treat a disease associated with a defective or mutated gene in a subject, such as but not limited to diseases caused by single-gene defects (monogenic disorders).

[0121] In some embodiments, the methods can be used to treat polygenic disorders.

[0122] In some aspects, the method is an in vivo method. In some embodiments, the method is an ex vivo method.

[0123] In some embodiments, the methods comprise administering an effective dose of a pharmaceutical composition of the disclosure to a patient in need of treatment. The pharmaceutical composition can be administered via any suitable method that results in targeted integration of the payload sequence into one or more cells of the subject. In some embodiments, the pharmaceutical composition is administered intravenously, intramuscularly, subcutaneously, intraocularly, intraretinally, within the CNS or other neural tissue, or intranasally.

[0124] In some embodiments, the cell is removed from the subject or patient before being transfected ex vivo with the genome editing systems of the disclosure. In some embodiments, the subject or patient is a human, a cell is removed from the human and transfected with mRNA encoding an RT protein and a template RNA of the disclosure. In some embodiments, the subject or patient is a human, a cell is removed from the human and transfected with a RT protein and a template RNA of the disclosure. Following ex vivo transfection, correct insertion of the heterologous polynucleotide comprising the payload sequence can be determined, for example by amplifying sequences at the 5’ and / or 3’ insertion junctions, and / or amplifying the payload sequence. A correctly targeted insertion can also be determined by sequencing the genomic target site. Expression of the payload sequence can also be determined, for example, by detecting expression of a product encoded by the payload sequence, such as a protein or regulatory RNA by methods known in the art such as RT-PCR or immunohistochemistry. After correct integration and / or expression of the payload sequence is determined, the correctly targeted cells are administered to the subject (autologous therapy).Kits and Articles of Manufacture

[0125] The disclosure provides kits and articles of manufacture comprising the genome editing systems described herein, and engineered cells comprising the genome editing systemsdescribed herein. In some embodiments, the kit comprises articles such as vials, syringes, and instructions for use.

[0126] In some embodiments, the kit comprises a reverse transcriptase and a template RNA of the disclosure.

[0127] In some embodiments, the kit comprises a plurality of engineered cells comprising the genome editing systems described herein. In some embodiments, the plurality of immune cells comprises a plurality of T cells. In some embodiments, the plurality of immune cells comprises a plurality of NK cells.

[0128] In some embodiments, the kit further comprises instructions for useEXAMPLES

[0129] These examples are provided for illustrative purposes only and not to limit the scope of the claims provided herein.EXAMPLE 1. Design and production of modified cap template RNA

[0130] This example provides a method for producing template RNA comprising modified translation-incapable (TI) cap.In vitro transcription to produce RNA with cap modifications

[0131] Briefly, the template RNA is designed with a payload, for example containing the ORF of the gene encoding fumarylacetoacetate hydrolase (FAH, Uniprot ID P16930)). The DNA sequence corresponding to the template RNA with the payload is cloned into a plasmid. Template RNA encoding plasmids for in vitro transcription (IVT) are linearized with restriction enzymes Pvul and Bbsl. The linearized plasmid is purified using phenol chloroform isoamyl alcohol (PCI) extraction and quantified using Nanodrop.

[0132] The IVT reaction is performed using HiScribe T7 High Yield RNA Synthesis Kit (NEB, Cat# E2040S). The IVT reaction is assembled based on the manufacturer’s recommendation with a few modifications, UTP is replaced with 100% N1 Methyl Pseudo UTP (TriLink, N- 1081) and a translation-incapable cap, for example a photo-activated caged cap is added to a final concentration of 8mM. The reaction mixture is incubated at 37°C for 3 h followed by precipitating the RNA with 3 M lithium chloride to remove the unincorporated NTPs. The precipitated RNA is resuspended and treated with DNase I (NEB: M0303S) and rSAP (shrimp alkaline phosphate (NEB: M0371S)) to remove the DNA template and 5’ triphosphate from uncapped RNA, respectively. POROS GoPure Oligo (dT)25 Pre-packed Column (Thermo Scientific: A48352) ("(dT)25" disclosed as SEQ ID NO: 3) is used to purify the RNA on AKTA purifier FPLC system (GE Healthcare). The column is pre-equilibrated with 20 mM Tris pH 7.5, 500 mM NaCl, and 1 mM EDTA before loading the RNA. A low-salt buffer (4 mM Tris pH 7.5, 100 mM NaCl, and 0.2 mM EDTA) is used to wash the column and the RNA is eluted from the column with nuclease free water. Both the Reverse Transcriptase (RT)-mRNA and the Template RNA containing an ORF encoding a designated gene are produced using the same protocol.EXAMPLE 2. Transfection and protein expression of template RNA with or without cap

[0133] This example describes the transfection and protein expression of template RNA comprising modified translation-incapable (TI) cap.Transfection ofRNAs into RPE-1 cells

[0134] Briefly, template RNA with different 5’ ends are produced as described in Example 1.RNA 5’ ends comprise CleanCap 3’OMe, no cap, and translation-incapable (TI) cap (for example a photocaged cap).

[0135] hTERT RPE-1 cells are aliquoted into a 6-well plate at a density of 250 - 500 thousand cells per well and incubated at 37°C in a 5% CO2 incubator for 18 - 24 h.

[0136] For the transfection, 10 pl of Messenger Max (Invitrogen Lipofectamine MessengerMAX, LMRNA003) are diluted by adding 250 pl of Opti-MEM, mixed well and incubated at room temperature for 10 min.

[0137] Transfections are performed with either the template RNA alone or with two RNAs where the template RNA is transfected together with the RT-mRNA. For template RNA-only transfection, a total of 1 pg of template RNA is added to 250 pl of Opti-MEM. In case of two RNA transfection, a total of 5 pg of RT-mRNA and the template RNA mixed at a desired molar ratio is diluted in 250 pl of Opti-MEM.

[0138] The diluted RNA in Opti-MEM is then mixed with diluted and incubated Messenger Max and incubated for 5 more minutes at room temperature. The resulting mixture is then added into two wells (250 pl each) seeded with hTERT RPE-1 cells.

[0139] Transfected cells are placed in 37°C incubator with 5% CO2. Cells are imaged at Day 1 and Day 2 post transfection to assess cell health and transfection efficiency via image analysis.

[0140] On Day 2, cells are washed with 1 ml of lx PBS followed by addition of 500 pl of Trypsin-EDTA (0.25%) and incubated for 3 min in 37°C incubator with 5% CO2.Analysis of protein expression from the Template RNA

[0141] This section provides a representative method for analyzing the expression of both 5’ capped as well as uncapped template RNA in hTERt RPE-1 cells.

[0142] Template RNA containing the ORF of the FAH gene encoding the fumarylacetoacetate hydrolase (Uniprot ID P16930) capped with CleanCap 3’0Me (TriLink, N-7413), photo caged or other translation-incapable (Tl-cap) are produced using the method described in Example 1, and transfected separately into RPE-1 cells as described above. The cells are collected 24 h post transfection.

[0143] The expression of FAH protein in the RPE-1 cells is analyzed by immunocytochemistry (ICC) in which the target protein is detected and visualized in cells using antibodies specifically recognizing fumarylacetoacetate hydrolase. The cells are fixed for 10 min with 4% (v / v) formaldehyde followed by wash twice with lx PBS for 5 min each time. The cells are blocked with 10% goat or donkey serum followed by incubation with primary antibody (targeted against the fumarylacetoacetate hydrolase) for 1 h. The cells are washed three times with lx PBS for 5 min each followed by incubation with secondary antibody conjugated with Alexa Fluor 555. Cells are counter stained with DAPI for nuclei envision. The cells are mounted in cover glass and the images are acquired with a fluorescence microscope. The results are expected to show that different levels of FAH protein expression are observed depending on the cap used on the template RNA. The highest expression is expected with CleanCap 3’0Me. Moderate expression may be observed when no cap is used due to leaky expression. No expression is expected when the Tl-cap is used.EXAMPLE 3. Analysis of immunogenicity of Template RNA with or without cap

[0144] This example describes analysis of immunogenicity of the template RNA produced with no cap or a translation-incapable cap.

[0145] Briefly, the hTERT RPE-1 cells are aliquoted into a 96-well plate at a density of 1440 cells per well and incubated at 37°C in a 5% CO2 incubator for 18 - 24 h.

[0146] For the transfection, 10 pl of Messenger Max (Invitrogen Lipofectamine MessengerMAX, LMRNA003) is diluted by adding 250 pl of Opti-MEM, mixed well and incubated at room temperature for 10 min.

[0147] For RNA transfection, a total of 150 ng of template RNA are added to 250 pl of Opti- MEM. In case of two RNA transfection, a total of 5 pg of RT-mRNA and uncapped Template RNA or translation-incapable cap Template RNA are mixed together at a molar ratio of 1 :6 and is diluted in 250 pl of Opti-MEM.

[0148] The diluted RNA in Opti-MEM is then mixed with diluted and incubated Messenger Max and incubated for 5 more minutes at room temperature. The resulting mixture is then added into two wells (250 pl each) seeded with hTERT RPE-1 cells.

[0149] Transfected cells are placed in 37°C incubator with 5% CO2. After 24 h, approximately 90 pl of the culture supernatant is collected and the quantities of interferon-P (IFN-P) are analyzed using Meso scale discovery (MSD) S-PLEX Human IFN-P kit (K151ADRS).EXAMPLE 4. ddPCR analysis of full-length transgene insertion efficiency

[0150] This example describes how the efficiency of full-length transgene insertion at the genomic site is determined and compared between Template RNA with no cap and with a translation-incapable cap (for example a photocaged cap).

[0151] The corresponding uncapped Template RNA or translation-incapable capped Template RNA are transfected together with the RT-mRNA as described in Example 1. After 24 h cells are washed with lx PBS and pelleted by centrifugation. Cells are resuspended with Cell Lysis Buffer (10 mM Tris pH 7.5, 0.5% (w / v) SDS, 0.1 M EDTA and 0.2 mg / ml RNaseA) and lysed by incubation at 56°C for 10 min followed by incubation at 37°C for 1 h. Equal volumes of phenol chlorofom isoamyl alcohol (PCI) is added, followed by brief vortex mixing for 10s and centrifuged at 21,000x g for 15 min at room temperature.

[0152] The aqueous layer containing genomic DNA is collected into a fresh tube and mixed with 300 mM sodium acetate pH 5.5 and equal volume of 100% isopropanol. This sample is centrifuged at 21,000x g for 10 min to precipitate genomic DNA. The supernatant is discarded, and the pellet is washed with 1 ml of 70% (v / v) ethanol and centrifuged at 21,000x g for 1 min. The supernatant is discarded, and the pellet is air dried for 5 min followed by suspension in nuclease free water.

[0153] The genomic DNA concentration is quantified using the IX DNA HS quantitative Assay kit (Invitrogen: Q33231) according to manufacturer’s instructions.

[0154] ddPCR analysis is performed using ddPCR Supermix for Probes (Bio-Rad Cat#1863010) on Bio-Rad QX200 Digital Droplet System. Five nanograms of genomic DNA are used as template in each reaction. The primers and probe are 900nM and 250nM, respectively. The primers and probe sequences are listed in the Table 1. The amplification protocol follows the manufacturer’s recommendation for the supermix used. Tbp, a single copy gene, is used as the control to verify that the genomic DNA samples do not contain any inhibitors that may affect ddPCR quantitation accuracy. Data analyses are performed using the QX Manager software and the absolute copy numbers for the 5’ and 3’ junction is determined, from which the 5’-to-3’ junction ratio is calculated. Because the translation-incapable cap is expected to protect the RNA from degradation caused by 5 ’-3’ exo nucleases, the ratio of 5’-to-3’ junction with the TI cap should be higher than that without cap.Table 1. Primers and probes used in ddPCR analysis.EXAMPLE 5. A photocaged Cap that can be used as the Tl-Cap

[0155] This example describes an RNA with a translation-incapable cap and determination of translational inhibition in a cell. The example further describes selection of additional modifications to the cap structure than can inhibit translation in a cell.

[0156] Shown below is an exemplary structure of a translational-incapable cap where the N2 position of the guanosine residue is modified to interfere with the binding of the translation initiation factor eIF4E to the capped RNA, thus inhibiting translation of the RNA carrying the modified cap. In this particular example, the R group used to modify the N2 position of guanosine has the property of being photocleaved, which will render the cap in Cap 0 state,thus translational active (Klbcker et al, Nature Chemistry, vol 14, 2022).DMNB-Cap 1

[0157] To verify the translation inhibition abilities of the N2 modified cap, mRNA for reporter gene eGFP is produced through in vitro transcription (IVT) using the conditions as described in Example 1.

[0158] As a positive control, FAH mRNA is produced with translation-capable mCAP (TriLink, Cat# N-7001). Initially, the efficiency of mRNA production using both these caps is measured by analyzing the final yields. Next, both mRNAs are transfected, and the corresponding FAH protein expression is analyzed as described in Example 2. The mRNA capped with DMNB-cap without being exposed to the photocleavage condition inhibits GFP expression whereas that with the mCAP modification results in higher level of GFP expression.

[0159] Additionally to the photocaged cap with an R group that is photocleavable other modified caps can be used to inhibit RNA translation. Because the N2 group is involved in eIF4E binding, which is essential for translation initiation, any R group, especially ones that are bulky in size, will likely lead to translation inhibition. In fact, caps modified with different R groups can be screened for translation inhibition by monitoring the efficiency of reporter gene expression in a cell or in an in vitro translation reaction as a way to select the R group or groups that have the highest translation inhibition ability.

[0160] While the disclosure has been particularly shown and described with reference to specific embodiments (some of which are preferred embodiments), it should be understood by those having skill in the art that various changes in form and detail may be made therein without departing from the spirit and scope of the present disclosure as disclosed herein.

Claims

CLAIMS1. A genome editing system comprising:(i) an mRNA encoding a reverse transcriptase, wherein the mRNA comprises a 5’ cap (CAP); and(ii) a template RNA encoding a payload, wherein the template RNA comprises a translation-incapable 5’ cap (TI-CAP).

2. A genome editing system comprising:(i) a reverse transcriptase polypeptide; and(ii) a template RNA encoding a payload, wherein the template RNA comprises a translation-incapable 5’ cap (TI-CAP).

3. The genome editing system of claim 1 or claim 2, wherein the translation-incapable 5’ cap is a chemically modified cap.

4. The genome editing system of claim 1 or claim 2, wherein the translation-incapable 5’ cap is a caged cap.

5. The genome editing system of claim 4, wherein the caged cap is a photocaged cap.

6. The genome editing system of claim 1 or claim 2, wherein the template RNA with the translation-incapable 5’ cap is less immunogenic compared to an uncapped template RNA.

7. The genome editing system of claim 1 or claim 2, wherein the payload comprises a transgene.

8. The genome editing system of claim 1 or claim 2, wherein the translation-incapable 5' cap increases the stability of the template RNA compared to an uncapped template RNA.

9. The genome editing system of claim 1 or claim 2, wherein the RNA comprising the translation-incapable 5’ cap is capable of being reverse transcribed by the reverse transcriptase.

10. The genome editing system of claim 1 or claim 2, wherein the template RNA further comprises a 3’ reverse transcriptase binding sequence.

11. The genome editing system of claim 10, wherein the 3’ reverse transcriptase recognition sequence comprises a sequence from a species selected from the group consisting of G. aculeatus, D. melanogaster, L. polyphemus, P. pungitis, N. vitripennis, G. fortis, O. latipes, Z. albicollis, T. guttata, T. castaneum, T. guttatus, D. simulans, B. mori, and A. vaga, or any combination thereof.

12. The genome editing system of claim 11, wherein the 3’ reverse transcriptase recognition sequence comprises a sequence that is 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99 % identical to a 3’ reverse transcriptase recognition sequence from a species selected from the group consisting of G. aculeatus, D. melanogaster, L. polyphemus, P. pungitis, N. vitripennis, G. fortis, O. latipes, Z. albicollis, T. guttata, T. castaneum, T. guttatus, D. simulans, B. mori, and A. vaga, or any combination thereof.

13. The genome editing system of claim 1 or claim 2, wherein the template RNA further comprises a 5’ UTR sequence, a promoter sequence, a transcription termination sequence, and / or a Poly A sequence.

14. The genome editing system of claim 1 or claim 2, wherein the payload is at least 15 bases long.

15. The genome editing system of claim 1, wherein the mRNA further comprises a 5’ UTR sequence, a 3 ’UTR sequence, and / or a Poly A sequence.

16. The genome editing system of claim 1 or claim 2, wherein one or more uridine bases of the mRNA and / or the template RNA are modified uridine (U).

17. The genome editing system of claim 1 or claim 2, wherein the reverse transcriptase comprises a reverse transcriptase derived from a non-long terminal repeat (non-LTR) retroelement.

18. The genome editing system of claim 1 or claim 2, wherein the reverse transcriptase comprises a reverse transcriptase from TaGu (Taeniopygia guttata) or ZoAl (Zonotrichia albicollis).

19. The genome editing system of claim 17, wherein the reverse transcriptase is selected from the group consisting of TriCasB (Tribolium castaneum), NaViB (Nasonia vitripennis), OrLa (Oryzias latipes), ZoAl (Zonotrichia albicollis), TiGu (Tinamus guttatus), TaGu (Taeniopygia guttata) , GeFo (Geospiza fortis), DroSi (Drosophila simulans), BoMo (Bombyx mori), DrMerc (Drosophila mercatorum), DrMe (Drosophila melanogaster), GaAc (G. aculeatus), PuPu (P. pungitis), AdVa (Adineta vaga), HyMaA (Hydra magnipapillata) , Ciin (Ciona intestinalis) , LiPo (Limulus polyphemus), TriCan Triops cancriformis) , LeCo (Lepidurus couesii), and any combination thereof.

20. The genome editing system of claim 17, wherein the amino acid sequence of the reverse transcriptase comprises a sequence that is 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99 % identical to an amino acid sequence of TriCasB (Tribolium castaneum), NaViB (Nasonia vitripennis) , OrLa (Oryzias latipes), ZoAl (Zonotrichia albicollis), TiGu (Tinamus guttatus) , TaGu (Taeniopygia guttata) , GeFo (Geospizafortis), DroSi (Drosophila simulans), BoMo (Bombyx mori), DrMerc Drosophila mercaloriim). DrMe (Drosophila melanogaster) , GaAc (G. aculealus). PuPu (P. pungilis). AdVa (Adineta vaga), HyMaA (Hydra magnipapillata) , Ciin (Ciona intestinalis) , LiPo (Limulus polyphemus), TriCan Triops cancriformis) , LeCo (Lepidurus couesii). or any combination thereof.

21. A pharmaceutical composition comprising the genome editing system of claim 1 or claim 2 and a pharmaceutically acceptable excipient.

22. A nanoparticle comprising the genome editing system of claim 1 or claim 2 or the pharmaceutical composition of claim 21.

23. A method of inserting a payload into the genome of a cell, the method comprising contacting the cell with a genome editing system of claim 1 or claim 2, a pharmaceutical composition of claim 21, or a nanoparticle of claim 22, wherein the transgene is inserted into the genome of the cell.

24. A method of inserting a payload into the genome of a cell in an organism, the method comprising contacting the cell in the organism with a genome editing system of claim 1 or claim 2, a pharmaceutical composition of claim 21, or a nanoparticle of claim 22, wherein the transgene is inserted into the genome of the cell.

25. The method of claim 23, wherein the insertion efficiency of a payload encoded in the translation-incapable 5’ cap (TI-CAP) RNA into the target site is increased compared to the insertion efficiency of a payload encoded in 5 ’uncapped RNA.

26. The method of claim 23, wherein the transgene is inserted in a target site in the genome of the cell.

27. The method of claim 23, wherein the target site is in the 28s rDNA sequence.

28. The method of claim 23, wherein the transgene is only expressed after insertion into the genome.

29. An engineered cell comprising a genome editing system of claim 1 or claim 2.

Images