New r2 retrotransposons for gene writing

Abstract

The present disclosure describes numerous new R2 retrotransposons discovered from various species. These R2 retrotransposons exhibited good retrotransposition activities in human cells. These new retrotransposons, as well as engineered versions thereof, can be suitably used for targeted gene delivery.

Description

NEW R2 RETROTRANSPOSONS FOR GENE WRITINGCROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit under 35 U.S.C. § 1 19(e) of the United States Provisional Application Serial Nos. 63 / 569,037 and 63 / 569,042 filed March 22, 2024, the content of which is hereby incorporated by reference in its entirety.REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

[0002] The content of the electronic sequence listing (385419.xml; Size: 560,415 bytes; and Date of Creation: March 21, 2025) is herein incorporated by reference in its entirety.BACKGROUND

[0003] Unlike DNA transposons, which move via a “cut and paste” mechanism, retrotransposons move in a “copy and paste” manner, using RNA as an intermediate. Based on whether they are flanked by long terminal repeat (LTR), retrotransposons are classified as LTR type or non-LTR type. Non-LTR retrotransposons are sub-classified into two major groups, namely long interspersed nuclear elements (LINEs) and short interspersed nuclear elements (SINEs). LINEs are autonomous elements that encode proteins to mediate their own mobility, whereas SINEs are nonautonomous elements that do not encode protein and consequently require LINEs for their propagation.

[0004] Non-LTR retrotransposons are mobilized by a mechanism in which proteins translated from their open reading frame proteins combine with their own mRNA to form a ribonucleoprotein (RNP) complex. The RNP complex is inserted into the target site via a mechanism termed target-primed reverse transcription (TPRT). The TPRT process is initiated by an encoded EN domain that nicks one strand of DNA at a target site and creates a 3 ’-hydroxyl end, which is used as a primer for reverse transcription of the LINE mRNA onto the DNA target via RT.

[0005] R2 is a long interspersed element (LINE) found in a specific sequence of the 28S rDNA among a wide variety of animals. Known R2 retrotransposons include those from medaka fishOryzias latipes (R2O1) and from Bombyx mori (R2Bm). Efforts have been made to use these R2 retrotransposons for targeted gene knock-in for practical application such as gene therapy.SUMMARY

[0006] Through bioinformatic approaches with confirmative testing in human cells, the instant inventors discovered highly efficient new R2 retrotransposons from different species. These new retrotransposons, as well as engineered versions thereof, can be suitably used for targeted gene delivery.

[0007] Accordingly, one embodiment of the present disclosure provides a method for inserting an exogenous sequence to a genomic DNA of a cell, comprising introducing to the cell (a) a polypeptide or a first polynucleotide encoding the polypeptide, wherein the polypeptide comprises (i) a DNA binding domain or a CRISPR Cas protein, (ii) a reverse-transcriptase comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 6, 2, 10, 14, 18, 22, 26, 30, 34, 38, 42, 46, 50, 54, 58, 62, 66, 70, 74, 78, 82, 86, 90, 94, 98, 102, 106, 110, 114, 118, 122, 126, 130, 134, 138, 142, 146, 150, 154, 158, 162, 166, 170, 174, 178, 182, 186,190, 194, 198, 202, 206, 210, 214, 218, 222, 226, 230, 234, 238, 242, 246, 250, 254, 258, 262,266, 270, 274, 278, 282, 286, 290, 294, 298, 302, 306, 310, 314, 318, 322, 326, 330, 334, 338,342, 346, 350, 354, 358, 362, 366, 370, 374, 378, 382, 386, 390, 394, 398, 402, 406, 410, 414,418 and 422, or an amino acid sequence having at least 85% sequence identity to any of the amino acid selected from the group, and (iii) an endonuclease or a nickase, and (b) an RNA comprising (i) a template for the exogenous sequence and (ii) a 3’ UTR, or a second polynucleotide encoding the RNA.

[0008] In some embodiments, the method further comprises introducing the first polynucleotide and the second polynucleotide to the cell. In some embodiments, the first polynucleotide and the second polynucleotide are on a same vector or on separate vectors.

[0009] In some embodiments, the 3 ’UTR is an R2 retrotransposon 3’ UTR. In some embodiments, the 3 ’UTR comprise a sequence selected from the group consisting of SEQ ID NO: 8, 4, 12, 16, 20, 24, 28, 32, 36, 40, 44, 48, 52, 56, 60, 64, 68, 72, 76, 80, 84, 88, 92, 96, 100, 104, 108, 112, 116, 120, 124, 128, 132, 136, 140, 144, 148, 152, 156, 160, 164, 168, 172, 176,180, 184, 188, 192, 196, 200, 204, 208, 212, 216, 220, 224, 228, 232, 236, 240, 244, 248, 252, 256, 260, 264, 268, 272, 276, 280, 284, 288, 292, 296, 300, 304, 308, 312, 316, 320, 324, 328, 332, 336, 340, 344, 348, 352, 356, 360, 364, 368, 372, 376, 380, 384, 388, 392, 396, 400, 404, 408, 412, 416, 420 and 424.

[0010] In some embodiments, the reverse-transcriptase comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 6, 2, 10, 14, 18, 22, 26, 30, 34, 38, 42, 46, 50, 54, 58, 62, 66, 70, 74, 78, 82, 86, 90, 94, 98, 102, 106, 110, 114, 118, 122, 126, 130, 134, 138,142, 146, 150, 154, 158, 162, 166, 170, 174, 178, 182, 186, 190, 194, 198, 202, 206, 210, 214,218, 222, 226, 230, 234, 238, 242, 246, 250, 254, 258, 262, 266, 270, 274, 278, 282, 286, 290,294, 298, 302, 306, 310, 314, 318, 322, 326, 330, 334, 338, 342, 346, 350, 354, 358, 362, 366,370, 374, 378, 382, 386, 390, 394, 398, 402, 406, 410, 414, 418 and 422.

[0011] In some embodiments, the polypeptide comprises the endonuclease. In some embodiments, the polypeptide comprises the nickase, which optionally is Cas nickase. In some embodiments, the CRISPR Cas protein is selected from the group consisting of Cas9, Casl2 and Casl3.

[0012] In some embodiments, the DNA binding domain is selected from the group consisting of a transcription activator-like effector (TALE) DNA binding domain, a homeodomain protein, a zinc finger, a helix-turn-helix, a leucine zipper, and a DNA-binding domain of a homing endonuclease, a transposon or retro-transposon.

[0013] In some embodiments, the DNA binding domain is a TALE. In some embodiments, the homing endonuclease is selected from the group consisting of I-Scel, I-Crel and I-Dmol. In some embodiments, the retro-transposon is a long interspersed nuclear element (LINE), optionally selected from the group consisting of Rl, R2, R4, R5, R6, R7, R8, and R9.

[0014] In some embodiments, the polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 5, 1, 9, 13, 17, 21, 25, 29, 33, 37, 41, 45, 49, 53, 57, 61, 65,69, 73, 77, 81, 85, 89, 93, 97, 101, 105, 109, 113, 117, 121, 125, 129, 133, 137, 141, 145, 149,153, 157, 161, 165, 169, 173, 177, 181, 185, 189, 193, 197, 201, 205, 209, 213, 217, 221, 225,229, 233, 237, 241, 245, 249, 253, 257, 261, 265, 269, 273, 277, 281, 285, 289, 293, 297, 301,305, 309, 313, 317, 321, 325, 329, 333, 337, 341, 345, 349, 353, 357, 361, 365, 369, 373, 377, 381, 385, 389, 393, 397, 401, 405, 409, 413, 417 and 421, or an amino acid sequence having at least 85% sequence identity to any amino acid sequence selected from the group.

[0015] In some embodiments, the polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 5, 1, 9, 13, 17, 21, 25, 29, 33, 37, 41, 45, 49, 53, 57, 61, 65, 69, 73, 77, 81, 85, 89, 93, 97, 101, 105, 109, 113, 117, 121, 125, 129, 133, 137, 141, 145, 149,153, 157, 161, 165, 169, 173, 177, 181, 185, 189, 193, 197, 201, 205, 209, 213, 217, 221, 225,229, 233, 237, 241, 245, 249, 253, 257, 261, 265, 269, 273, 277, 281, 285, 289, 293, 297, 301,305, 309, 313, 317, 321, 325, 329, 333, 337, 341, 345, 349, 353, 357, 361, 365, 369, 373, 377,381, 385, 389, 393, 397, 401, 405, 409, 413, 417 and 421.

[0016] In some embodiments, the RNA further comprises a 5’UTR. In some embodiments, the 5’UTR comprises a sequence selected from the group consisting of SEQ ID NO: 7, 3, 11, 15, 19, 23, 27, 31, 35, 39, 43, 47, 51, 55, 59, 63, 67, 71, 75, 79, 83, 87, 91, 95, 99, 103, 107, 111, 115, 119, 123, 127, 131, 135, 139, 143, 147, 151, 155, 159, 163, 167, 171, 175, 179, 183, 187, 191,195, 199, 203, 207, 211, 215, 219, 223, 227, 231, 235, 239, 243, 247, 251, 255, 259, 263, 267,271, 275, 279, 283, 287, 291, 295, 299, 303, 307, 311, 315, 319, 323, 327, 331, 335, 339, 343,347, 351, 355, 359, 363, 367, 371, 375, 379, 383, 387, 391, 395, 399, 403, 407, 411, 415, 419 and 423.

[0017] In some embodiments, the cell is a mammalian cell, preferably a human cell. In some embodiments, the exogenous sequence encodes a protein, preferably a therapeutic protein.BRIEF DESCRIPTION OF THE DRAWINGS

[0018] FIG. 1 shows the integration activities of selected new R2 elements with digital PCR.

[0019] FIG. 2 illustrates the cis and trans configurations for R2 -based reverse transcription.

[0020] FIG. 3 shows the gene delivery rates of new R2 elements indicated by the GFP levels.

[0021] FIG. 4 shows representative GFP images and charts indicating the cargo delivery rates of selected R2 elements in a cis configuration.

[0022] FIG. 5 shows representative GFP images and charts indicating the cargo delivery rates of selected R2 elements in a trans configuration.

[0023] FIG. 6 shows the GFP cargo delivery rates of selected R2 elements in a cis configuration.

[0024] FIG. 7 shows the GFP cargo delivery rates of selected R2 elements in a trans configuration, where the RNA templates included pseudouridine-modified nucleotides.DETAILED DESCRIPTION

[0025] The following description sets forth exemplary embodiments of the present technology. It should be recognized, however, that such description is not intended as a limitation on the scope of the present disclosure but is instead provided as a description of exemplary embodiments.Newly Discovered R2 Retrotransposons

[0026] Through bioinformatic approaches with confirmative testing in human cells, the instant inventors discovered highly efficient new R2 retrotransposons. Their retrotransposition activities were confirmed in human cells in vitro.

[0027] Each of the R2 mRNA includes a 5’ untranslated region (UTR), a single open reading frame (ORF) encoding the endonuclease (EN) and reverse-transcriptase (RT) and DNA-binding domains, and a 3’ UTR. The R2 mRNA can anneal with the target site (i.e., the 28S rDNA in the human genome) and initiates the reverse transcription mechanism.

[0028] Generally, each of the R2 retrotransposons includes one or more N-terminal zinc finger domains, a SANT DNA-binding domain, a 3’ UTR binding domain, a template switching domain, a reverse-transcriptase (RT), a C-terminal zinc finger domain (C-ZnF), and an endonuclease (EN). Among them, the RT domain carries the retrotransposition activity, while the other domains are contemplated to be switchable to similar domains. For instance, the N- terminal zinc fingers can be substituted with other DNA binding domains, as discussed below, and so does the EN domain.

[0029] These new R2 retrotransposons, the species with which they are associated, and the corresponding ORF, RT and 5’ - and 3’-UTR sequences, are summarized in Tables A and B. The corresponding sequences are provided in Tables 1 and 2.Table A. Newly Discovered R2 RetrotransposonsEngineered R2 Retrotransposons

[0030] The retrotransposition of R2 retrotransposons does not require homology-directed repair (HDR) or donor DNA. Compared to HDR, which is a passive process in cells, reverse transcription of mRNA into cDNA via TPRT using the RT domain shows relatively higher processivity and fidelity, which is advantageous for synthesizing long cDNAs at the target site rather than recombination at the target site as donor DNA.

[0031] It has been demonstrated herein that the newly discovered R2 retrotransposons specifically target the 28S gene in the human genome. Based on sequence analysis, it is contemplated that the specific 28S binding is attributed to the N-terminal zinc finger (ZnF) domains, likely assisted by the SANT DNA-binding domain. Replacement of these N-terminal regions of the R2 proteins, therefore, may alternate the target specificity of the R2 retrotransposons.

[0032] Accordingly, in some embodiments, the present disclosure provides an engineered R2, in the form of a fusion protein, that includes a truncated version of an R2 ORF and one or more other heterologous elements. The truncated R2 ORF, in some embodiments, includes the reversetranscriptase (or a biological variant thereof) of one of the disclosed R2, but does not include portion of the N-terminal region of the R2, such as one or more of the N-Znfl, N-Znf2, N-Znf3, and SANT DNA-Binding Domain.

[0033] In some embodiments, the heterologous elements may be a DNA binding domain or a CRISPR Cas protein and / or an endonuclease or a nickase. It is appreciated that it is not necessary that both elements are heterologous. For instance, the fusion protein can include one or more heterologous DNA binding domains but the native endonuclease. In another example, the fusion protein can include the native DNA binding domains and a heterologous endonuclease.

[0034] Likewise, another embodiment of the present disclosure provides an engineered R2, in the form of a fusion protein, that includes a truncated version of one of the new R2 ORF and one or more other heterologous elements. The truncated R2 ORF, in some embodiments, includes the reverse-transcriptase (or a biological variant thereof) of R2, but does not include portion of the N-terminal region of R2, such as one or more of the N-Znfl, N-Znf2, N-Znf3, and SANT DNA- Binding Domain.

[0035] As used herein, a “biological variant” of a reference protein refers to a protein or fragment that has at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% sequence identity to the reference protein. In some embodiments, the biological variant retains the desired activity (e.g., reverse-transcriptase) of the reference protein. Likewise, a “biological variant” of a reference nucleic acid sequence refers to a nucleic acid sequence or fragment that has at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% sequence identity to the reference nucleic acid sequence.A. Truncated R2 ORF

[0036] As provided, an engineered R2 protein of the present disclosure can include a truncated version of the R2 ORF fused to at least one heterologous element.

[0037] In one embodiment, the truncated R2 ORF includes a reverse-transcriptase comprising the amino acid sequence of any one of SEQ ID NO: 6, 2, 10, 14, 18, 22, 26, 30, 34, 38, 42, 46, 50, 54, 58, 62, 66, 70, 74, 78, 82, 86, 90, 94, 98, 102, 106, 110, 114, 118, 122, 126, 130, 134, 138, 142, 146, 150, 154, 158, 162, 166, 170, 174, 178, 182, 186, 190, 194, 198, 202, 206, 210,214, 218, 222, 226, 230, 234, 238, 242, 246, 250, 254, 258, 262, 266, 270, 274, 278, 282, 286,290, 294, 298, 302, 306, 310, 314, 318, 322, 326, 330, 334, 338, 342, 346, 350, 354, 358, 362,366, 370, 374, 378, 382, 386, 390, 394, 398, 402, 406, 410, 414, 418 and 422 or an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% sequence identity to SEQ ID NO: 6, 2, 10, 14, 18, 22, 26, 30, 34, 38, 42, 46, 50, 54, 58, 62, 66, 70, 74, 78, 82, 86, 90, 94, 98, 102, 106, 110, 114, 118, 122, 126, 130, 134, 138, 142, 146, 150, 154, 158,162, 166, 170, 174, 178, 182, 186, 190, 194, 198, 202, 206, 210, 214, 218, 222, 226, 230, 234,238, 242, 246, 250, 254, 258, 262, 266, 270, 274, 278, 282, 286, 290, 294, 298, 302, 306, 310,314, 318, 322, 326, 330, 334, 338, 342, 346, 350, 354, 358, 362, 366, 370, 374, 378, 382, 386,390, 394, 398, 402, 406, 410, 414, 418 and 422.

[0038] In one embodiment, the reverse-transcriptase includes the amino acid sequence of SEQ ID NO: 2 or an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% sequence identity to SEQ ID NO: 2. In one embodiment, the reversetranscriptase includes the amino acid sequence of SEQ ID NO: 6 or an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% sequence identity to SEQ ID NO: 6. In one embodiment, the reverse-transcriptase includes the amino acid sequence of SEQ ID NO: 10 or an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% sequence identity to SEQ ID NO: 10.

[0039] In one embodiment, the reverse-transcriptase includes the amino acid sequence of SEQ ID NO: 14 or an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% sequence identity to SEQ ID NO: 14. In one embodiment, the reversetranscriptase includes the amino acid sequence of SEQ ID NO: 18 or an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% sequence identity to SEQ ID NO: 18. In one embodiment, the reverse-transcriptase includes the amino acid sequence of SEQ ID NO: 22 or an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% sequence identity to SEQ ID NO: 22.

[0040] In one embodiment, the reverse-transcriptase includes the amino acid sequence of SEQ ID NO: 26 or an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% sequence identity to SEQ ID NO: 26. In one embodiment, the reversetranscriptase includes the amino acid sequence of SEQ ID NO: 30 or an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% sequence identity to SEQ ID NO: 30. In one embodiment, the reverse-transcriptase includes the amino acid sequence of SEQ ID NO: 34 or an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% sequence identity to SEQ ID NO: 34.

[0041] In one embodiment, the reverse-transcriptase includes the amino acid sequence of SEQ ID NO: 38 or an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% sequence identity to SEQ ID NO: 38. In one embodiment, the reversetranscriptase includes the amino acid sequence of SEQ ID NO: 42 or an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% sequence identity to SEQ ID NO: 42. In one embodiment, the reverse-transcriptase includes the amino acid sequence of SEQ ID NO: 46 or an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% sequence identity to SEQ ID NO: 46.

[0042] In one embodiment, the reverse-transcriptase includes the amino acid sequence of SEQ ID NO: 50 or an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% sequence identity to SEQ ID NO: 50. In one embodiment, the reversetranscriptase includes the amino acid sequence of SEQ ID NO: 54 or an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% sequence identity to SEQ ID NO: 54. In one embodiment, the reverse-transcriptase includes the amino acid sequence of SEQ ID NO: 58 or an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% sequence identity to SEQ ID NO: 58.

[0043] In one embodiment, the reverse-transcriptase includes the amino acid sequence of SEQ ID NO: 62 or an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% sequence identity to SEQ ID NO: 62. In one embodiment, the reversetranscriptase includes the amino acid sequence of SEQ ID NO: 66 or an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% sequence identity toSEQ ID NO: 66. In one embodiment, the reverse-transcriptase includes the amino acid sequence of SEQ ID NO: 70 or an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% sequence identity to SEQ ID NO: 70. In one embodiment, the reversetranscriptase includes the amino acid sequence of SEQ ID NO: 74 or an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% sequence identity to SEQ ID NO: 74.

[0044] In one embodiment, the reverse-transcriptase includes the amino acid sequence of SEQ ID NO: 78 or an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% sequence identity to SEQ ID NO: 78. In one embodiment, the reversetranscriptase includes the amino acid sequence of SEQ ID NO: 82 or an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% sequence identity to SEQ ID NO: 82. In one embodiment, the reverse-transcriptase includes the amino acid sequence of SEQ ID NO: 86 or an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% sequence identity to SEQ ID NO: 86.

[0045] In one embodiment, the reverse-transcriptase includes the amino acid sequence of SEQ ID NO: 90 or an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% sequence identity to SEQ ID NO: 90. In one embodiment, the reversetranscriptase includes the amino acid sequence of SEQ ID NO: 94 or an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% sequence identity to SEQ ID NO: 94. In one embodiment, the reverse-transcriptase includes the amino acid sequence of SEQ ID NO: 98 or an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% sequence identity to SEQ ID NO: 98.

[0046] In one embodiment, the reverse-transcriptase includes the amino acid sequence of SEQ ID NO: 102 or an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% sequence identity to SEQ ID NO: 102. In one embodiment, the reversetranscriptase includes the amino acid sequence of SEQ ID NO: 106 or an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% sequence identity to SEQ ID NO: 106. In one embodiment, the reverse-transcriptase includes the amino acidsequence of SEQ ID NO: 110 or an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% sequence identity to SEQ ID NO: 110.

[0047] In one embodiment, the reverse-transcriptase includes the amino acid sequence of SEQ ID NO: 114 or an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% sequence identity to SEQ ID NO: 114. In one embodiment, the reversetranscriptase includes the amino acid sequence of SEQ ID NO: 118 or an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% sequence identity to SEQ ID NO: 118. In one embodiment, the reverse-transcriptase includes the amino acid sequence of SEQ ID NO: 122 or an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% sequence identity to SEQ ID NO: 122.

[0048] In one embodiment, the reverse-transcriptase includes the amino acid sequence of SEQ ID NO: 126 or an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% sequence identity to SEQ ID NO: 126. In one embodiment, the reversetranscriptase includes the amino acid sequence of SEQ ID NO: 130 or an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% sequence identity to SEQ ID NO: 130. In one embodiment, the reverse-transcriptase includes the amino acid sequence of SEQ ID NO: 134 or an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% sequence identity to SEQ ID NO: 134.

[0049] In one embodiment, the reverse-transcriptase includes the amino acid sequence of SEQ ID NO: 138 or an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% sequence identity to SEQ ID NO: 138. In one embodiment, the reversetranscriptase includes the amino acid sequence of SEQ ID NO: 142 or an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% sequence identity to SEQ ID NO: 142. In one embodiment, the reverse-transcriptase includes the amino acid sequence of SEQ ID NO: 146 or an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% sequence identity to SEQ ID NO: 146.

[0050] In one embodiment, the reverse-transcriptase includes the amino acid sequence of SEQ ID NO: 150 or an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% sequence identity to SEQ ID NO: 150. In one embodiment, the reverse-transcriptase includes the amino acid sequence of SEQ ID NO: 154 or an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% sequence identity to SEQ ID NO: 154. In one embodiment, the reverse-transcriptase includes the amino acid sequence of SEQ ID NO: 158 or an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% sequence identity to SEQ ID NO: 158.

[0051] In one embodiment, the reverse-transcriptase includes the amino acid sequence of SEQ ID NO: 162 or an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% sequence identity to SEQ ID NO: 162. In one embodiment, the reversetranscriptase includes the amino acid sequence of SEQ ID NO: 166 or an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% sequence identity to SEQ ID NO: 166. In one embodiment, the reverse-transcriptase includes the amino acid sequence of SEQ ID NO: 170 or an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% sequence identity to SEQ ID NO: 170.

[0052] In one embodiment, the reverse-transcriptase includes the amino acid sequence of SEQ ID NO: 174 or an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% sequence identity to SEQ ID NO: 174. In one embodiment, the reversetranscriptase includes the amino acid sequence of SEQ ID NO: 178 or an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% sequence identity to SEQ ID NO: 178. In one embodiment, the reverse-transcriptase includes the amino acid sequence of SEQ ID NO: 182 or an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% sequence identity to SEQ ID NO: 182.

[0053] In one embodiment, the reverse-transcriptase includes the amino acid sequence of SEQ ID NO: 186 or an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% sequence identity to SEQ ID NO: 186. In one embodiment, the reversetranscriptase includes the amino acid sequence of SEQ ID NO: 190 or an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% sequence identity to SEQ ID NO: 190. In one embodiment, the reverse-transcriptase includes the amino acid sequence of SEQ ID NO: 194 or an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% sequence identity to SEQ ID NO: 194.

[0054] In one embodiment, the reverse-transcriptase includes the amino acid sequence of SEQ ID NO: 198 or an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% sequence identity to SEQ ID NO: 198. In one embodiment, the reversetranscriptase includes the amino acid sequence of SEQ ID NO: 202 or an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% sequence identity to SEQ ID NO: 202. In one embodiment, the reverse-transcriptase includes the amino acid sequence of SEQ ID NO: 206 or an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% sequence identity to SEQ ID NO: 206.

[0055] In one embodiment, the reverse-transcriptase includes the amino acid sequence of SEQ ID NO: 210 or an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% sequence identity to SEQ ID NO: 210. In one embodiment, the reversetranscriptase includes the amino acid sequence of SEQ ID NO: 214 or an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% sequence identity to SEQ ID NO: 214. In one embodiment, the reverse-transcriptase includes the amino acid sequence of SEQ ID NO: 218 or an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% sequence identity to SEQ ID NO: 218.

[0056] In one embodiment, the reverse-transcriptase includes the amino acid sequence of SEQ ID NO: 222 or an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% sequence identity to SEQ ID NO: 222. In one embodiment, the reversetranscriptase includes the amino acid sequence of SEQ ID NO: 226 or an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% sequence identity to SEQ ID NO: 226. In one embodiment, the reverse-transcriptase includes the amino acid sequence of SEQ ID NO: 230 or an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% sequence identity to SEQ ID NO: 230.

[0057] In one embodiment, the reverse-transcriptase includes the amino acid sequence of SEQ ID NO: 234 or an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% sequence identity to SEQ ID NO: 234. In one embodiment, the reversetranscriptase includes the amino acid sequence of SEQ ID NO: 238 or an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% sequence identity toSEQ ID NO: 238. In one embodiment, the reverse-transcriptase includes the amino acid sequence of SEQ ID NO: 242 or an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% sequence identity to SEQ ID NO: 242.

[0058] In one embodiment, the reverse-transcriptase includes the amino acid sequence of SEQ ID NO: 246 or an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% sequence identity to SEQ ID NO: 246. In one embodiment, the reversetranscriptase includes the amino acid sequence of SEQ ID NO: 250 or an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% sequence identity to SEQ ID NO: 250. In one embodiment, the reverse-transcriptase includes the amino acid sequence of SEQ ID NO: 254 or an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% sequence identity to SEQ ID NO: 254.

[0059] In one embodiment, the reverse-transcriptase includes the amino acid sequence of SEQ ID NO: 258 or an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% sequence identity to SEQ ID NO: 258. In one embodiment, the reversetranscriptase includes the amino acid sequence of SEQ ID NO: 262 or an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% sequence identity to SEQ ID NO: 262. In one embodiment, the reverse-transcriptase includes the amino acid sequence of SEQ ID NO: 266 or an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% sequence identity to SEQ ID NO: 266.

[0060] In one embodiment, the reverse-transcriptase includes the amino acid sequence of SEQ ID NO: 270 or an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% sequence identity to SEQ ID NO: 270. In one embodiment, the reversetranscriptase includes the amino acid sequence of SEQ ID NO: 274 or an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% sequence identity to SEQ ID NO: 274. In one embodiment, the reverse-transcriptase includes the amino acid sequence of SEQ ID NO: 278 or an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% sequence identity to SEQ ID NO: 278.

[0061] In one embodiment, the reverse-transcriptase includes the amino acid sequence of SEQ ID NO: 282 or an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%,95%, 98% or 99% sequence identity to SEQ ID NO: 282. In one embodiment, the reversetranscriptase includes the amino acid sequence of SEQ ID NO: 286 or an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% sequence identity to SEQ ID NO: 286. In one embodiment, the reverse-transcriptase includes the amino acid sequence of SEQ ID NO: 290 or an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% sequence identity to SEQ ID NO: 290.

[0062] In one embodiment, the reverse-transcriptase includes the amino acid sequence of SEQ ID NO: 294 or an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% sequence identity to SEQ ID NO: 294. In one embodiment, the reversetranscriptase includes the amino acid sequence of SEQ ID NO: 298 or an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% sequence identity to SEQ ID NO: 298. In one embodiment, the reverse-transcriptase includes the amino acid sequence of SEQ ID NO: 302 or an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% sequence identity to SEQ ID NO: 302.

[0063] In one embodiment, the reverse-transcriptase includes the amino acid sequence of SEQ ID NO: 306 or an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% sequence identity to SEQ ID NO: 306. In one embodiment, the reversetranscriptase includes the amino acid sequence of SEQ ID NO: 310 or an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% sequence identity to SEQ ID NO: 310. In one embodiment, the reverse-transcriptase includes the amino acid sequence of SEQ ID NO: 314 or an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% sequence identity to SEQ ID NO: 314.

[0064] In one embodiment, the reverse-transcriptase includes the amino acid sequence of SEQ ID NO: 318 or an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% sequence identity to SEQ ID NO: 318. In one embodiment, the reversetranscriptase includes the amino acid sequence of SEQ ID NO: 322 or an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% sequence identity to SEQ ID NO: 322. In one embodiment, the reverse-transcriptase includes the amino acidsequence of SEQ ID NO: 326 or an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% sequence identity to SEQ ID NO: 326.

[0065] In one embodiment, the reverse-transcriptase includes the amino acid sequence of SEQ ID NO: 330 or an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% sequence identity to SEQ ID NO: 330. In one embodiment, the reversetranscriptase includes the amino acid sequence of SEQ ID NO: 334 or an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% sequence identity to SEQ ID NO: 334. In one embodiment, the reverse-transcriptase includes the amino acid sequence of SEQ ID NO: 338 or an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% sequence identity to SEQ ID NO: 338.

[0066] In one embodiment, the reverse-transcriptase includes the amino acid sequence of SEQ ID NO: 342 or an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% sequence identity to SEQ ID NO: 342. In one embodiment, the reversetranscriptase includes the amino acid sequence of SEQ ID NO: 346 or an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% sequence identity to SEQ ID NO: 346. In one embodiment, the reverse-transcriptase includes the amino acid sequence of SEQ ID NO: 350 or an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% sequence identity to SEQ ID NO: 350.

[0067] In one embodiment, the reverse-transcriptase includes the amino acid sequence of SEQ ID NO: 354 or an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% sequence identity to SEQ ID NO: 354. In one embodiment, the reversetranscriptase includes the amino acid sequence of SEQ ID NO: 358 or an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% sequence identity to SEQ ID NO: 358. In one embodiment, the reverse-transcriptase includes the amino acid sequence of SEQ ID NO: 362 or an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% sequence identity to SEQ ID NO: 362.

[0068] In one embodiment, the reverse-transcriptase includes the amino acid sequence of SEQ ID NO: 366 or an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% sequence identity to SEQ ID NO: 366. In one embodiment, the reverse-transcriptase includes the amino acid sequence of SEQ ID NO: 370 or an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% sequence identity to SEQ ID NO: 370. In one embodiment, the reverse-transcriptase includes the amino acid sequence of SEQ ID NO: 374 or an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% sequence identity to SEQ ID NO: 374.

[0069] In one embodiment, the reverse-transcriptase includes the amino acid sequence of SEQ ID NO: 378 or an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% sequence identity to SEQ ID NO: 378. In one embodiment, the reversetranscriptase includes the amino acid sequence of SEQ ID NO: 382 or an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% sequence identity to SEQ ID NO: 382. In one embodiment, the reverse-transcriptase includes the amino acid sequence of SEQ ID NO: 386 or an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% sequence identity to SEQ ID NO: 386.

[0070] In one embodiment, the reverse-transcriptase includes the amino acid sequence of SEQ ID NO: 390 or an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% sequence identity to SEQ ID NO: 390. In one embodiment, the reversetranscriptase includes the amino acid sequence of SEQ ID NO: 394 or an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% sequence identity to SEQ ID NO: 394. In one embodiment, the reverse-transcriptase includes the amino acid sequence of SEQ ID NO: 398 or an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% sequence identity to SEQ ID NO: 398.

[0071] In one embodiment, the reverse-transcriptase includes the amino acid sequence of SEQ ID NO: 402 or an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% sequence identity to SEQ ID NO: 402. In one embodiment, the reversetranscriptase includes the amino acid sequence of SEQ ID NO: 406 or an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% sequence identity to SEQ ID NO: 406. In one embodiment, the reverse-transcriptase includes the amino acid sequence of SEQ ID NO: 410 or an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% sequence identity to SEQ ID NO: 410.

[0072] In one embodiment, the reverse-transcriptase includes the amino acid sequence of SEQ ID NO: 414 or an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% sequence identity to SEQ ID NO: 414. In one embodiment, the reversetranscriptase includes the amino acid sequence of SEQ ID NO: 418 or an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% sequence identity to SEQ ID NO: 418. In one embodiment, the reverse-transcriptase includes the amino acid sequence of SEQ ID NO: 422 or an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% sequence identity to SEQ ID NO: 422.

[0073] In some embodiments, the truncated R2 ORF further includes the 3’ UTR binding domain of the R2 ORF. In some embodiments, the truncated R2 ORF further includes the template switching domain of the R2 ORF. In some embodiments, the truncated R2 ORF further includes the C-terminal ZnF. In some embodiments, the truncated R2 ORF further includes the endonuclease domain. In some embodiments, the truncated R2 ORF includes all elements of the wild-type R2 ORF, except those that are specifically substituted with a corresponding heterologous element (e.g., a heterologous endonuclease).B. Heterologous Targeting Domain

[0074] In some embodiments, one or more of the N-terminal ZnF domains is replaced with a heterologous targeting domain (e.g., a DNA-binding domain or a CRISPR Cas protein).

[0075] A “DNA-binding domain” refers to a protein domain capable of binding a nucleic acid sequence of interest, preferably a double strand nucleic acid molecule. The DNA binding domain recognizes and binds nucleic acid at specific polynucleotide sequences, further referred to as “nucleic acid target sequence.” The DNA-binding domain comprises any of the DNA-binding domain known in the art, such as transcription activator-like effector (TALE), homeodomain protein, a zinc finger, a helix-turn-helix, a leucine zipper, and a DNA-binding domain of a homing endonuclease, a transposon or retro-transposon. In certain embodiments, the DNA- binding domain recognizes a target DNA sequence in the genome.

[0076] A “homeodomain protein” consists of three linked alpha helices (helices 1, 2 and 3). Helices 2 and 3 are arranged in a conspicuous helix turn helix motif. A 60 amino acid longregion (homeodomain) within helix 3 binds specifically to DNA segments that contain the sequence 5’ATTA3’. In animals, there are 16 major classes of homeodomain protein, ANTP, PRD, PRD-LIKE, POU, HNF, CUT (with four subclasses: ONECUT, CUX, SATB, and CMP), LIM, ZF, CERS, PROS, SIX / SO, plus the TALE superclass with the classes IRO, MKX, TGIF, PBC, and MEIS. Further homeodomain proteins include but not limited to HEX motif, EH1 motif, Octapeptide / Hep / EHl / TN / GEH motif, WRPW motif, TUP1, OAR motif, CUT domain, such as ONECUT, CUX, SATB, CMP domains, COMPASS domain, HNF1A (LFB1) transcription factor, POU, OCT-1, OCT-2, Pit 1, LIM domain, PROSPERO (PROS), SIX / SO and CERS (LASS).

[0077] The “zinc finger” (ZF) includes at least nine types, C2H2, C2HC, C3H, C4, C6, C3HC4, C2HC5, C4HC3, and C8, in which C and H represent cysteine and histidine, respectively. C2H2 zinc finger is a loop of 12 amino acids with two cysteines and two histidines at the base of the loop (Cys2His2 zinc finger motif consisting of an a helix and an antiparallel sheet) that tetrahedrally coordinate a zinc ion. C2H2 zinc finger is represented by TFIIIA and further comprises Adnp, Tshz, Zeb, Zfhx and Zhx. C2HC-typezinc finger domains, also referred to as retroviral -type (RT) zinc finger sequences, are required for viral genome packaging and RNA or single-stranded DNA binding in eukaryotes. The C3H family proteins are divided into 18 groups based on the different amino acid spacing numbers between C and H in zinc finger motif. C4 finger has one alpha helix and contains a zinc atom bound to four cysteine amino acids. The C4 zinc finger contains a 70 amino acid long region near the zinc atom that binds specifically to DNA segments. The C4 family includes seven types: GAT A, FYVE, TimlO / DDP, LSD1, A20, TFIIB, and Zn-finger in Ran-binding protein. C3HC4-type finger, also termed RING finger, can be categorized into seven types with different conserved motifs, such as RING- H2, RING-HC, RING-v, RING-D, RING-S / T, RING-G and RING-C2. The C2HC5 motif, also referred to as LIM. More information on the zinc finger can be found in the prior art publication, such as Li et al., International Journal of Molecular Sciences, (2020) 21(4): 1361. Further examples of the zinc finger proteins include Spl, Glucocorticoid receptor, estrogen receptor, progesterone receptor, thyroid hormone receptor (erbA), retinoid acid receptor, and the vitamin D3 receptor.

[0078] A “leucine zipper” consists of an alpha helix that contains a region in which every seventh amino acid is leucine, which has the effect of lining up all the leucine residues on one side of the alpha helix. The leucine residues allow for the dimerization of the two lecine zipper proteins and formation of Y shaped dimer. Dimerization may occur between two of the same proteins (homodimers, e.g., Jun-Jun) or two different proteins (heterodimers, e.g., Fos-Jun). A leucine zipper contains a 20 amino acid long region that binds specifically to DNA segments. Examples of the Leucine Zipper proteins include but not limited to CCAAT / enhancer binding protein (C / EBP), Cyclic AMP response element binding protein (CREB), Finkel osteogeneic sarcoma virus (Fos) protein, Jun, GCN4, and HSF.

[0079] A “helix loop helix” (HLH) or “helix-turn-helix” consists of a short alpha helix connected by a loop to a loner alpha helix. The loop allows for dimerization of two HLH proteins and formation of Y shaped dimer. The dimerization may occur between two of the same proteins (homo dimers) or two different proteins (heterodimers). Example of HLH domains include but not limited to MyoD, Myc, Pho4, SREBP-la, and Max-Mad.

[0080] A “Transcription Activator like Effector” (TALE) are proteins that are encoded by phytopathogenic bacteria of the genus Xanthomonas and Ralstonia to influence the gene expression of host plant cells during bacterial infection. These proteins are helix-loop-helix- containing transcriptional factors that comprise a DNA binding region and an N-terminal domain that appears to interact with the bacterial transport machinery for introducing the protein into the plant cell. The C-terminal domain of the TALE protein seems to interact with the plant host's transcriptional machinery to induce expression of sets of plant genes that are beneficial to the invading bacteria. The DNA binding portion of the proteins is found in the middle section of the protein and is made of an array of repeat units, each approximately 33-35 amino acids in length, which have been shown to be responsible for interacting with the target DNA. In a preferred embodiment, said DNA-binding domain is derived from a TALE engineered to bind a specific nucleic acid target sequence. Unlimited examples of the TALE include KNOX and BEL, such as PBC, MEIS, PREP, IRO, MKX and TGIF.

[0081] A “homing endonuclease (HEs)” or meganuleases (MNs), are sequence-specific endonucleases with large cleavage sites (14-25 bp) that can create double-stranded breaks atspecific locations. The homing endonucleases are encoded by mobile genetic elements that induce recombination in a process called homing. The homing endonucleases genes (HEGs) have invaded many genomic niches including group I and group II introns. Some HEGs can move from an ORF-containing intron to an “ORF-less” intron. There are four major families of homing endonucleases (HEs), naming is based on conserved amino acid motifs: the H-N-H, HIS- CYS, LAGLID ADG, and GIY-YIG families of HEs. The LAGLIDADG family of HEs is the most frequently encountered among group I introns. However GIY-YIG endonucleases have been identified within numerous group I introns, and LAGLIDADG HEs and the H-N-H domain are present within the ORFs of some group II introns. Additional HE-like proteins have been described, the PD-(D / E)XK HEs are found in bacterial tRNA group I introns, the very-short patch repair (Vsr) endonucleases (a predicted family of phage HEs based on metagenomic), and the Holliday junction resolvase-like HEs found in some phage introns. In certain embodiments of the present disclosure, the homing endonuclease comprises Pl-Scel, Pl-Pful, I-Crel, I-Ceul, I- Dmol, F-TevI, F-TevII, I-TevI, I-TevII, I-Ppol, I-Dirl, I-Njal, I-NanI, I-Nitl, I-SceV, I-SceVI and I-Llal, I-Hmul, I-HmuII, LTevIII and I-Cmoel. In a particular embodiment, the homing endonuclease is I-Ppol (Intron-Encoded Endonuclease).

[0082] Retrotransposon is a class of eucaryotic genes capable of replicating to new locations within their own genome through an RNA intermediate. Retrotransposons are widespread in metazoan genomes. The proportion of the genome that contains retrotransposons often exceeds that containing DNA transposons, particularly in higher vertebrates such as humans. Most retrotransposons integrate into random sites of the host genome, but some have a sequence preference. In particular, a few non-LTR subclades integrate into the genome in a highly sequence-specific manner. By leaving their 5’ and / or 3’ ends of genomic copies (usually with a poly (A) stretch and target-site duplication (TSD)), the site-specific non-LTR retrotransposons can be searched throughout the DNA database.

[0083] There are five clades of restriction enzyme-like endonuclease (RLE)-encoding elements based on their RT sequence similarity. Most of three clades (NeSL, R2, and R4) are target specific and two clades (HERO and CRE) have some site-specific elements. R2 (R2 clade) was found at a specific sequence in the 28S rDNA of many invertebrate and vertebrate species. R4 inAscaris lumbricoides and Dong (R4 clade) were found at another site of the 28S rDNA and microsatellite TAA repeats, respectively.

[0084] In contrast to RLE-encoding elements, most of the apurinic / apyrimidinic endonuclease (APE)-encoding non-LTR elements do not insert themselves in a sequence-specific manner, but do have weak target-site specificity, e.g., human LI for TAAA repeats. However, two clades of APE-encoding non-LTR elements, Txl and Rl, are known to be sequence-specific.

[0085] Among the site-specific non-LTR retrotransposons, Rl clade (R1 / R6 / R7 / RT), R2 clade (R2 / R8 / R9), R4 clade (R4) and NeSl clade (R5) target the rRNA genes, whereas R7 and R8 target the 18S rDNA, and Rl, R2, R4, R5, R6, R9, and RT target the 28S rDNA. All R-element target sites within 28S and 18S rDNA are highly conserved among organisms (see Fujiwara et al., Microbiology Spectrum, (2015) Vol. 3, Issue 2).

[0086] In a preferred embodiment, the retrotransposon is rDNA specific and comprises Rl, R2, R4, R5, R6, R7, R8, R9 and RT. In certain embodiments, the heterologous DNA-binding domain provided herein comprises the DNA-binding domain of any one of the Rl, R2, R4, R5, R6, R7, R8, R9 and RT.

[0087] In certain embodiments, the one or more N-terminal ZnF domains is replaced with a CRISPR Cas protein. In certain embodiments, the CRISPR Cas protein comprises Cas9, Casl2a / Cpfl and Casl3. The clustered regularly interspaced short palindromic repeat (CRISPR) / Cas mediated genome editing technology has seen wild use since its invention due to its simplicity and efficiency, leading to a new era of genetic engineering and gene therapy. The targeting and cleavage are achieved by a single CRISPR RNA (“crRNA”)-bound Cas protein in Class 2 CRISPR-Cas systems. Class 2 systems can be subdivided into Type II Cas9 and Type V Cas 12 that target DNA, as well as Type VI Cas 13 that target RNA. Cas9 and Casl2a / Cpfl have been well studied in the past and broadly harnessed for gene editing in various cell types and organisms in prokaryotes and eukaryotes. Both Cas9 and Casl2 systems utilize guide RNA to recognize the target site and protospacer adjacent motifs (PAMs) to determine the cleavage site and generate double strand breaks. However, the binding and cleavage of DNA by Cas9 and Casl2 are quite different. Cas9 recognizes a 3’-G-rich PAM and produces blunt ends cleaved by the RuvC and HNH domains, whereas Cas 12 recognizes a 5’ T-rich PAM and producesstaggered ends cleaved solely by the RuvC domain. Pre-assembled Cas 13 and crRNA recognizes target RNAs. Upon RNA-binding, Cas 13 will undergo a conformational change and induce the catalytic activity of its nuclease domains, resulting in the cleavage of target transcripts. In certain embodiments, the gRNA or the crRNA can be designed to target a DNA sequence in the genome. In certain embodiments, the Cas protein comprises an inactive nuclease domain. In certain embodiments, the Cas protein does not comprise an active nuclease domain.

[0088] In certain embodiments, the guide RNA (“gRNA”) comprises two short, non-coding RNA species referred to as crRNA and trans-acting RNA (“tracrRNA”). In an exemplary system, the gRNA forms a complex with a Cas protein of the present disclosure. The gRNA:Cas protein complex binds a target polynucleotide sequence in the genome. The gRNA can be supplied separately that works with the Cas protein in guiding the Cas protein (or the fusion) to a target location on a genomic sequence.

[0089] In one aspect of the present disclosure, a fusion protein provided herein that comprises a first fragment that is a truncated R2 protein and a second fragment that can be a DNA-binding domain or a CRISPR Cas protein. In certain embodiments, the second fragment is N-terminal to the first fragment. In certain embodiments, the second fragment can be alternatively C-terminal to the first fragment. The first and second fragments can be fused directly or via a linker sequence.C. Heterologous Endonuclease or Nickase

[0090] In some embodiments, the native endonuclease of the R2 ORF can be replaced with a biological variant (e. , a sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% sequence identity to the native endonuclease) or a heterologous endonuclease or a nickase.

[0091] An endonuclease is an enzyme that cleaves internal phosphodiester bonds of polynucleotides. These enzymes are either specific or non-specific to the sequences being cleaved. The endonucleases that are specific to a particular sequence are termed restriction endonucleases, which cleave large DNA molecules at specific sequences of four to six nucleotides.

[0092] In some embodiments the endonuclease is a eukaryotic endonuclease. Non-limiting examples include Neurospora endonuclease, SI nuclease, Pl -nuclease, Mung bean nuclease I, Ustilago nuclease, DNase I, AP endonuclease and Endo R. Other examples include Fokl nuclease, type-II restriction 1 -like endonucleases (RLE-type nuclease), and RLE-type endonucleases (REL).

[0093] Many known transposons and retrotransposons also include related endonucleases. In some embodiments, the heterologous endonuclease is one from Rl, R2, R4, R5, R6, R7, R8, R9 or RT.

[0094] In some embodiments, the R2 endonuclease is substituted with a nickase. A nikcase (or nicking endonuclease) is an enzyme that cuts one strand of a double-stranded DNA at a specific recognition nucleotide sequence, to produce DNA molecules that are nicked. Nickases can be prepared from Cas enzyme with site mutations (e. , D10A Cas9).

[0095] The heterologous DNA-binding domain / Cas and / or the heterologous endonuclease can be used to the truncated R2 ORF through a linker sequence. The term “linker sequence” refers to an innocuous length of nucleic acid or protein that joins two other sections of nucleic acid or protein.

[0096] Linkers within the scope of the present disclosure are characterized in terms of amino acid content, length, rigidity and secondary structure. Linkers within the scope of the present disclosure separate the first fragment and the second fragment and allow proper folding and functioning of each domain. In this manner, a linker can be tailored to the particular first and second fragments. According to one aspect, functional independence of the structural and fused (heterologous) domains is maximized by a suitable linker to limit steric interference between domains during the export and assembly processes of the bacterial cell. According to an additional aspect, cell stress is minimized by limiting the overall length of the fusion protein. Longer linker sequences and higher induction levels stress the biosynthetic machinery of the cells, inhibiting cell growth and leading to cell lysis in extreme cases.

[0097] Linkers within the scope of the present disclosure facilitate functioning of the first and second fragments. Linkers within the scope of the present disclosure allow efficient proteinprocessing and export through the bacterial curli secretion machinery as well as provide the proper spatial and physicochemical separation of the polypeptides to retain their respective functions.

[0098] Linkers within the scope of the present disclosure include amino acid residues. The amino acid residues may be any of the naturally occurring amino acid residues. Amino acid residues may also be synthetic amino acids known to those of skill in the art. Representative amino acids which may be used in linkers include Glycine, Alanine, Valine, Leucine, Isoleucine, Serine, Cysteine, Selenocysteine, Threonine, Methionine, Proline, Phenylalanine, Tyrosine, Tryptophan, Histidine, Lysine, Arginine, Aspartate, Glutamate, Asparagine, and Glutamine.

[0099] According to one aspect, a linker sequence is a polypeptide sequence of at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 24, 48 or more amino acids. In some embodiments, the linker sequence comprises from about 3 amino acids to about 50 amino acids. In some embodiments, the linker sequence comprises from about 3 amino acids to about 40 amino acids. In some embodiments, the linker sequence comprises from about 5 amino acids to about 30 amino acids. In some embodiments, the linker sequence comprises from about 5 amino acids to about 25 amino acids. In some embodiments, the linker sequence comprises from about 5 amino acids to about 24 amino acids. In some embodiments, the linker sequence comprises from about 5 amino acids to about 23 amino acids. In some embodiments, the linker sequence comprises from about 5 amino acids to about 22 amino acids. In some embodiments, the linker sequence comprises from about 5 amino acids to about 21 amino acids. In some embodiments, the linker sequence comprises from about 5 amino acids to about 20 amino acids. In some embodiments, the linker sequence comprises from about 5 amino acids to about 19 amino acids. In some embodiments, the linker sequence comprises from about 5 amino acids to about 18 amino acids. In some embodiments, the linker sequence comprises from about 5 amino acids to about 17 amino acids. In some embodiments, the linker sequence comprises from about 5 amino acids to about 16 amino acids. In some embodiments, the linker sequence comprises from about 5 amino acids to about 15 amino acids. In some embodiments, the linker sequence comprises from about 5 amino acids to about 14 amino acids. In some embodiments, the linker sequence comprises from about 5 amino acids to about 13 amino acids. In some embodiments, the linker sequence comprises from about 5 amino acids to about 12 amino acids. In some embodiments, the linker sequence comprisesfrom about 5 amino acids to about 11 amino acids. In some embodiments, the linker sequence comprises from about 5 amino acids to about 10 amino acids.

[0100] In some embodiments, the linker sequence comprises a flexible polypeptide, e.g., a polypeptide not having a rigid secondary and / or tertiary structure. In some embodiments, the linker sequence comprises glycine and serine residues. In some embodiments at least 50% of the amino acids comprised by the linker sequence are glycine or serine residues, e.g. at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or more are glycine or serine residues. In some embodiments, the linker sequence consists of glycine and serine residues. In some embodiments, the linker sequence comprises a rigid polypeptide.

[0101] In some embodiments, the truncated R2 ORF and the one or more heterologous elements are not delivered and expressed together. Instead, they can be delivered and expressed separately, or are conjugated or bound to one another.

[0102] Therefore, in one aspect, the present disclosure provides a complex comprising a first polypeptide comprising the truncated R2 ORF and a second polypeptide comprising the targeting domain (e.g., a DNA-binding domain or a CRISPR Cas protein) and / or the endonuclease / nickase, wherein the first polypeptide is conjugated or bound to the second polypeptide. The term “conjugate” or “bind” as used herein refers to conjugation of a first conjugation moiety and a second conjugation moiety which form a complex and stabilized by chemical reactions with few of several available chemical groups (chemical conjugation) or via one or more covalent bonds (biological conjugation).

[0103] In certain embodiments, the first polypeptide includes a first conjugation moiety and the second polypeptide comprises a second conjugation moiety, wherein the first polypeptide and the second polypeptide are brought together by the conjugation moi eties and form a stable complex.

[0104] In certain embodiments, the first polypeptide and the second polypeptide may respectfully but regardless of order, further include (a) a receptor and a corresponding ligand, (b) a peptide and a corresponding antibody, or (c) an enzyme and a corresponding substrate.Protein-Coding Sequence for Delivery

[0105] In one embodiment, the R2 ORF, or an engineered version thereof, can be provided as a mRNA. The mRNA can further include the corresponding 5’UTR and / or 3’UTR of the native R2.

[0106] In some embodiments, to insert a different sequence (e.g., a second protein-coding sequence) to a target genome, the mRNA can further include that second protein-coding sequence. In some embodiments, the second protein-coding sequence is 3’ to the R2 ORF. In some embodiments, the second protein-coding sequence is included in the mRNA in a reversecomplement orientation such that it cannot be directly translated by the mRNA. Rather it is translated after integration to the target genome. In some embodiments, the second proteincoding sequence is inserted at 3’ to the R2 ORF via a self-cleaving peptide sequence, such as 2A self-cleaving peptide.

[0107] Alternatively, in some embodiments, the second protein-coding sequence is provided on a separate mRNA that flanks the second protein-coding sequence with the 5’UTR and / or 3’UTR of the native R2.

[0108] In some embodiments, the second protein is a therapeutic protein that can be helpful in treating a disease or condition in a patient. Examples include:- Insulin - used to treat diabetes- Phenylalanine hydroxylase - used to treat PKU (Phenylketonuria) disease- Erythropoietin - used to treat anemia- Filgrastim (G-CSF) - used to treat neutropenia- Interferons - used to treat cancer, multiple sclerosis, and viral infections- Tumor necrosis factor (TNF) inhibitors - used to treat autoimmune diseases, such as rheumatoid arthritis and Crohn's disease- Monoclonal antibodies - used to treat cancer, autoimmune diseases, and some viral infections- Follitropin alfa - used to treat infertility- Coagulation factor - used to treat bleeding disorders, such as Hemophilia- Enzyme replacement therapy enzymes - used to treat lysosomal storage disorders, such as Gaucher's disease- Growth hormone - used to treat growth hormone deficiency.

[0109] In some embodiments, the polynucleotide (c. ., mRNA) further includes a downstream flanking sequence that is 3’ to the protein-coding sequence. Addition of a downstream flanking sequence can improve the retrotransposition efficiency. In certain embodiments, the proteincoding sequence may comprise, at the 3’ end of the gene encoding sequence, one or more consecutive nucleotides (rDNA sequences) of the “downstream sequence” starting from the EN cleavage site. The protein-coding sequence may comprise, at the 3’ end of the gene encoding sequence, different lengths of the downstream sequence depending on the type of polynucleotide or vector used for delivery. In certain embodiments, a length of the downstream sequence of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 200, 250 nt can be included to the 3’ mRNA of the fusion protein-encoding sequence. In some embodiments, the length can be 1-250, 1-200, 4-180, 10-150, 15-150, 40-90, 50-90, 70-90 or 71-90 nt, without limitation.

[0110] In some embodiments, the polynucleotide (e.g., mRNA) further includes an upstream flanking sequence that is 5’ to the protein-coding sequence. Addition of an upstream flanking sequence may also improve the retrotransposition efficiency. In certain embodiments, the protein-coding sequence may comprise, at the 5’ end of the gene encoding sequence, one or more consecutive nucleotides (rDNA sequences) of the “upstream sequence” starting from the EN cleavage site. The protein-coding sequence may comprise, at the 5’ end of the gene encoding sequence, different lengths of the downstream sequence depending on the type of polynucleotide or vector used for delivery. In certain embodiments, a length of the downstream sequence of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 200, 250 nt can be included to the 5’ mRNA of the fusion protein-encoding sequence. In some embodiments, the length can be 1-250, 1-200, 4-180, 10-150, 15-150, 40-90, 50-90, 70-90 or 71-90 nt, without limitation.[oni] In some embodiments, the R2 3’UTR has the nucleic acid sequence of any one of SEQ ID NO: 8, 4, 12, 16, 20, 24, 28, 32, 36, 40, 44, 48, 52, 56, 60, 64, 68, 72, 76, 80, 84, 88, 92, 96,100, 104, 108, 1 12, 1 16, 120, 124, 128, 132, 136, 140, 144, 148, 152, 156, 160, 164, 168, 172,176, 180, 184, 188, 192, 196, 200, 204, 208, 212, 216, 220, 224, 228, 232, 236, 240, 244, 248,252, 256, 260, 264, 268, 272, 276, 280, 284, 288, 292, 296, 300, 304, 308, 312, 316, 320, 324,328, 332, 336, 340, 344, 348, 352, 356, 360, 364, 368, 372, 376, 380, 384, 388, 392, 396, 400,404, 408, 412, 416, 420 and 424. In some embodiments, the 3’UTR has at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% sequence identity to any one of SEQ ID NO: 8, 4, 12, 16, 20, 24, 28, 32, 36, 40, 44, 48, 52, 56, 60, 64, 68, 72, 76, 80, 84, 88, 92, 96, 100, 104, 108, 112, 116, 120, 124, 128, 132, 136, 140, 144, 148, 152, 156, 160, 164, 168, 172, 176,180, 184, 188, 192, 196, 200, 204, 208, 212, 216, 220, 224, 228, 232, 236, 240, 244, 248, 252,256, 260, 264, 268, 272, 276, 280, 284, 288, 292, 296, 300, 304, 308, 312, 316, 320, 324, 328,332, 336, 340, 344, 348, 352, 356, 360, 364, 368, 372, 376, 380, 384, 388, 392, 396, 400, 404,408, 412, 416, 420 and 424. In some embodiments, the 3’UTR is directly connected to the downstream flanking sequence.

[0112] In some embodiments, the polynucleotide (e.g., mRNA) further includes a 5’ untranslated region (UTR) 5’ to the protein-coding sequence. When an upstream flanking sequence is also included, preferably the upstream flanking sequence is 5’ to the 5’ UTR.

[0113] In some embodiments, the R2 5 ’UTR has the nucleic acid sequence of any one of SEQ ID NO: 7, 3, 1 1, 15, 19, 23, 27, 31, 35, 39, 43, 47, 51, 55, 59, 63, 67, 71, 75, 79, 83, 87, 91 , 95, 99, 103, 107, 111, 115, 119, 123, 127, 131, 135, 139, 143, 147, 151, 155, 159, 163, 167, 171,175, 179, 183, 187, 191, 195, 199, 203, 207, 211, 215, 219, 223, 227, 231, 235, 239, 243, 247,251, 255, 259, 263, 267, 271, 275, 279, 283, 287, 291, 295, 299, 303, 307, 311, 315, 319, 323,327, 331, 335, 339, 343, 347, 351, 355, 359, 363, 367, 371, 375, 379, 383, 387, 391, 395, 399,403, 407, 411, 415, 419 and 423. In some embodiments, the 5 ’UTR has at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% sequence identity to any one of SEQ ID NO: 7, 3, 11, 15, 19, 23, 27, 31, 35, 39, 43, 47, 51, 55, 59, 63, 67, 71, 75, 79, 83, 87, 91, 95, 99, 103, 107, 111, 115, 119, 123, 127, 131, 135, 139, 143, 147, 151, 155, 159, 163, 167, 171, 175,179, 183, 187, 191, 195, 199, 203, 207, 211, 215, 219, 223, 227, 231, 235, 239, 243, 247, 251,255, 259, 263, 267, 271, 275, 279, 283, 287, 291, 295, 299, 303, 307, 311, 315, 319, 323, 327,331, 335, 339, 343, 347, 351, 355, 359, 363, 367, 371, 375, 379, 383, 387, 391, 395, 399, 403,407, 411, 415, 419 and 423.

[0114] In some embodiments, the R2 ORF includes an RT that includes SEQ ID NO:2 or a biological variant, or the R2 ORF includes SEQ ID NO: 1 or a biological variant, and the corresponding R2 3’UTR includes SEQ ID NO: 4 or a biological variant. In some embodiments, a corresponding R2 5’UTR is used, which includes SEQ ID NO: 3 or a biological variant. In some embodiments, the R2 ORF includes an RT that includes SEQ ID NO: 6 or a biological variant, or the R2 ORF includes SEQ ID NO: 5 or a biological variant, and the corresponding R2 3’UTR includes SEQ ID NO: 8 or a biological variant. In some embodiments, a corresponding R2 5’UTR is used, which includes SEQ ID NO: 7 or a biological variant. In some embodiments, the R2 ORF includes an RT that includes SEQ ID NOTO or a biological variant, or the R2 ORF includes SEQ ID NO: 9 or a biological variant, and the corresponding R2 3’UTR includes SEQ ID NO: 12 or a biological variant. In some embodiments, a corresponding R2 5’UTR is used, which includes SEQ ID NO: 11 or a biological variant.

[0115] In some embodiments, the R2 ORF includes an RT that includes SEQ ID NO: 14 or a biological variant, or the R2 ORF includes SEQ ID NO: 13 or a biological variant, and the corresponding R2 3’UTR includes SEQ ID NO: 16 or a biological variant. In some embodiments, a corresponding R2 5’UTR is used, which includes SEQ ID NO: 15 or a biological variant. In some embodiments, the R2 ORF includes an RT that includes SEQ ID NO: 18 or a biological variant, or the R2 ORF includes SEQ ID NO: 17 or a biological variant, and the corresponding R2 3’UTR includes SEQ ID NO: 20 or a biological variant. In some embodiments, a corresponding R2 5’UTR is used, which includes SEQ ID NO: 19 or a biological variant. In some embodiments, the R2 ORF includes an RT that includes SEQ ID NO:22 or a biological variant, or the R2 ORF includes SEQ ID NO: 21 or a biological variant, and the corresponding R2 3’UTR includes SEQ ID NO: 24 or a biological variant. In some embodiments, a corresponding R2 5’UTR is used, which includes SEQ ID NO: 23 or a biological variant.

[0116] In some embodiments, the R2 ORF includes an RT that includes SEQ ID NO: 26 or a biological variant, or the R2 ORF includes SEQ ID NO: 25 or a biological variant, and the corresponding R2 3’UTR includes SEQ ID NO: 28 or a biological variant. In some embodiments, a corresponding R2 5’UTR is used, which includes SEQ ID NO: 27 or a biological variant. In some embodiments, the R2 ORF includes an RT that includes SEQ IDNO:30 or a biological variant, or the R2 ORF includes SEQ ID NO: 29 or a biological variant, and the corresponding R2 3’UTR includes SEQ ID NO: 32 or a biological variant. In some embodiments, a corresponding R2 5’UTR is used, which includes SEQ ID NO: 31 or a biological variant. In some embodiments, the R2 ORF includes an RT that includes SEQ ID NO:34 or a biological variant, or the R2 ORF includes SEQ ID NO: 33 or a biological variant, and the corresponding R2 3’UTR includes SEQ ID NO: 36 or a biological variant. In some embodiments, a corresponding R2 5’UTR is used, which includes SEQ ID NO: 35 or a biological variant.

[0117] In some embodiments, the R2 ORF includes an RT that includes SEQ ID NO:38 or a biological variant, or the R2 ORF includes SEQ ID NO: 37 or a biological variant, and the corresponding R2 3’UTR includes SEQ ID NO: 40 or a biological variant. In some embodiments, a corresponding R2 5’UTR is used, which includes SEQ ID NO: 39 or a biological variant. In some embodiments, the R2 ORF includes an RT that includes SEQ ID NO:42 or a biological variant, or the R2 ORF includes SEQ ID NO: 41 or a biological variant, and the corresponding R2 3’UTR includes SEQ ID NO: 44 or a biological variant. In some embodiments, a corresponding R2 5’UTR is used, which includes SEQ ID NO: 43 or a biological variant. In some embodiments, the R2 ORF includes an RT that includes SEQ ID NO:46 or a biological variant, or the R2 ORF includes SEQ ID NO: 45 or a biological variant, and the corresponding R2 3’UTR includes SEQ ID NO: 48 or a biological variant. In some embodiments, a corresponding R2 5’UTR is used, which includes SEQ ID NO: 47 or a biological variant.

[0118] In some embodiments, the R2 ORF includes an RT that includes SEQ ID NO:50 or a biological variant, or the R2 ORF includes SEQ ID NO: 49 or a biological variant, and the corresponding R2 3’UTR includes SEQ ID NO: 52 or a biological variant. In some embodiments, a corresponding R2 5’UTR is used, which includes SEQ ID NO: 51 or a biological variant. In some embodiments, the R2 ORF includes an RT that includes SEQ ID NO:54 or a biological variant, or the R2 ORF includes SEQ ID NO: 53 or a biological variant, and the corresponding R2 3’UTR includes SEQ ID NO: 56 or a biological variant. In some embodiments, a corresponding R2 5’UTR is used, which includes SEQ ID NO: 55 or a biological variant. In some embodiments, the R2 ORF includes an RT that includes SEQ IDNO:58 or a biological variant, or the R2 ORF includes SEQ ID NO: 57 or a biological variant, and the corresponding R2 3’UTR includes SEQ ID NO: 60 or a biological variant. In some embodiments, a corresponding R2 5’UTR is used, which includes SEQ ID NO: 59 or a biological variant.

[0119] In some embodiments, the R2 ORF includes an RT that includes SEQ ID NO: 62 or a biological variant, or the R2 ORF includes SEQ ID NO: 61 or a biological variant, and the corresponding R2 3’UTR includes SEQ ID NO: 64 or a biological variant. In some embodiments, a corresponding R2 5’UTR is used, which includes SEQ ID NO: 63 or a biological variant. In some embodiments, the R2 ORF includes an RT that includes SEQ ID NO:66 or a biological variant, or the R2 ORF includes SEQ ID NO: 65 or a biological variant, and the corresponding R2 3’UTR includes SEQ ID NO: 68 or a biological variant. In some embodiments, a corresponding R2 5’UTR is used, which includes SEQ ID NO: 67 or a biological variant. In some embodiments, the R2 ORF includes an RT that includes SEQ ID NO:70 or a biological variant, or the R2 ORF includes SEQ ID NO: 69 or a biological variant, and the corresponding R2 3’UTR includes SEQ ID NO: 72 or a biological variant. In some embodiments, a corresponding R2 5’UTR is used, which includes SEQ ID NO: 71 or a biological variant.

[0120] In some embodiments, the R2 ORF includes an RT that includes SEQ ID NO:74 or a biological variant, or the R2 ORF includes SEQ ID NO: 73 or a biological variant, and the corresponding R2 3’UTR includes SEQ ID NO: 76 or a biological variant. In some embodiments, a corresponding R2 5’UTR is used, which includes SEQ ID NO: 75 or a biological variant. In some embodiments, the R2 ORF includes an RT that includes SEQ ID NO:78 or a biological variant, or the R2 ORF includes SEQ ID NO: 77 or a biological variant, and the corresponding R2 3’UTR includes SEQ ID NO: 80 or a biological variant. In some embodiments, a corresponding R2 5’UTR is used, which includes SEQ ID NO: 79 or a biological variant. In some embodiments, the R2 ORF includes an RT that includes SEQ ID NO: 82 or a biological variant, or the R2 ORF includes SEQ ID NO: 81 or a biological variant, and the corresponding R2 3’UTR includes SEQ ID NO: 84 or a biological variant. In some embodiments, a corresponding R2 5’UTR is used, which includes SEQ ID NO: 83 or a biological variant.

[0121] In some embodiments, the R2 ORF includes an RT that includes SEQ ID NO:86 or a biological variant, or the R2 ORF includes SEQ ID NO: 85 or a biological variant, and the corresponding R2 3’UTR includes SEQ ID NO: 88 or a biological variant. In some embodiments, a corresponding R2 5’UTR is used, which includes SEQ ID NO: 87 or a biological variant. In some embodiments, the R2 ORF includes an RT that includes SEQ ID NO:90 or a biological variant, or the R2 ORF includes SEQ ID NO: 89 or a biological variant, and the corresponding R2 3’UTR includes SEQ ID NO: 92 or a biological variant. In some embodiments, a corresponding R2 5’UTR is used, which includes SEQ ID NO: 91 or a biological variant. In some embodiments, the R2 ORF includes an RT that includes SEQ ID NO:94 or a biological variant, or the R2 ORF includes SEQ ID NO: 93 or a biological variant, and the corresponding R2 3’UTR includes SEQ ID NO: 96 or a biological variant. In some embodiments, a corresponding R2 5’UTR is used, which includes SEQ ID NO: 95 or a biological variant.

[0122] In some embodiments, the R2 ORF includes an RT that includes SEQ ID NO: 98 or a biological variant, or the R2 ORF includes SEQ ID NO: 97 or a biological variant, and the corresponding R2 3’UTR includes SEQ LD NO: 100 or a biological variant. In some embodiments, a corresponding R2 5’UTR is used, which includes SEQ ID NO: 99 or a biological variant. In some embodiments, the R2 ORF includes an RT that includes SEQ ID NO: 102 or a biological variant, or the R2 ORF includes SEQ ID NO: 101 or a biological variant, and the corresponding R2 3’UTR includes SEQ ID NO: 104 or a biological variant. In some embodiments, a corresponding R2 5’UTR is used, which includes SEQ ID NO: 103 or a biological variant. In some embodiments, the R2 ORF includes an RT that includes SEQ ID NO: 106 or a biological variant, or the R2 ORF includes SEQ ID NO: 105 or a biological variant, and the corresponding R2 3’UTR includes SEQ ID NO: 108 or a biological variant. In some embodiments, a corresponding R2 5’UTR is used, which includes SEQ ID NO: 107 or a biological variant.

[0123] In some embodiments, the R2 ORF includes an RT that includes SEQ ID NO: 110 or a biological variant, or the R2 ORF includes SEQ ID NO: 109 or a biological variant, and the corresponding R2 3’UTR includes SEQ ID NO: 112 or a biological variant. In some embodiments, a corresponding R2 5’UTR is used, which includes SEQ ID NO: 111 or abiological variant. In some embodiments, the R2 ORF includes an RT that includes SEQ ID NO: 114 or a biological variant, or the R2 ORF includes SEQ ID NO: 113 or a biological variant, and the corresponding R2 3’UTR includes SEQ ID NO: 116 or a biological variant. In some embodiments, a corresponding R2 5’UTR is used, which includes SEQ ID NO: 115 or a biological variant. In some embodiments, the R2 ORF includes an RT that includes SEQ ID NO: 118 or a biological variant, or the R2 ORF includes SEQ ID NO: 117 or a biological variant, and the corresponding R2 3’UTR includes SEQ ID NO: 120 or a biological variant. In some embodiments, a corresponding R2 5’UTR is used, which includes SEQ ID NO: 119 or a biological variant.

[0124] In some embodiments, the R2 ORF includes an RT that includes SEQ ID NO: 122 or a biological variant, or the R2 ORF includes SEQ ID NO: 121 or a biological variant, and the corresponding R2 3’UTR includes SEQ ID NO: 124 or a biological variant. In some embodiments, a corresponding R2 5’UTR is used, which includes SEQ ID NO: 123 or a biological variant. In some embodiments, the R2 ORF includes an RT that includes SEQ ID NO: 126 or a biological variant, or the R2 ORF includes SEQ ID NO: 125 or a biological variant, and the corresponding R2 3’UTR includes SEQ ID NO: 128 or a biological variant. In some embodiments, a corresponding R2 5’UTR is used, which includes SEQ ID NO: 127 or a biological variant. In some embodiments, the R2 ORF includes an RT that includes SEQ ID NO: 130 or a biological variant, or the R2 ORF includes SEQ ID NO: 129 or a biological variant, and the corresponding R2 3’UTR includes SEQ ID NO: 132 or a biological variant. In some embodiments, a corresponding R2 5’UTR is used, which includes SEQ ID NO: 131 or a biological variant.

[0125] In some embodiments, the R2 ORF includes an RT that includes SEQ ID NO: 134 or a biological variant, or the R2 ORF includes SEQ ID NO: 133 or a biological variant, and the corresponding R2 3’UTR includes SEQ ID NO: 136 or a biological variant. In some embodiments, a corresponding R2 5’UTR is used, which includes SEQ ID NO: 135 or a biological variant. In some embodiments, the R2 ORF includes an RT that includes SEQ ID NO: 138 or a biological variant, or the R2 ORF includes SEQ ID NO: 137 or a biological variant, and the corresponding R2 3’UTR includes SEQ ID NO: 140 or a biological variant. In some embodiments, a corresponding R2 5’UTR is used, which includes SEQ ID NO: 139 or abiological variant. In some embodiments, the R2 ORF includes an RT that includes SEQ ID NO: 142 or a biological variant, or the R2 ORF includes SEQ ID NO: 141 or a biological variant, and the corresponding R2 3’UTR includes SEQ ID NO: 144 or a biological variant. In some embodiments, a corresponding R2 5’UTR is used, which includes SEQ ID NO: 143 or a biological variant.

[0126] In some embodiments, the R2 ORF includes an RT that includes SEQ ID NO: 146 or a biological variant, or the R2 ORF includes SEQ ID NO: 145 or a biological variant, and the corresponding R2 3’UTR includes SEQ ID NO: 148 or a biological variant. In some embodiments, a corresponding R2 5’UTR is used, which includes SEQ ID NO: 147 or a biological variant. In some embodiments, the R2 ORF includes an RT that includes SEQ ID NO: 150 or a biological variant, or the R2 ORF includes SEQ ID NO: 149 or a biological variant, and the corresponding R2 3’UTR includes SEQ ID NO: 152 or a biological variant. In some embodiments, a corresponding R2 5’UTR is used, which includes SEQ ID NO: 151 or a biological variant. In some embodiments, the R2 ORF includes an RT that includes SEQ ID NO:154 or a biological variant, or the R2 ORF includes SEQ ID NO: 153 or a biological variant, and the corresponding R2 3’UTR includes SEQ ID NO: 156 or a biological variant. In some embodiments, a corresponding R2 5’UTR is used, which includes SEQ ID NO: 155 or a biological variant.

[0127] In some embodiments, the R2 ORF includes an RT that includes SEQ ID NO: 158 or a biological variant, or the R2 ORF includes SEQ ID NO: 157 or a biological variant, and the corresponding R2 3’UTR includes SEQ ID NO: 160 or a biological variant. In some embodiments, a corresponding R2 5’UTR is used, which includes SEQ ID NO: 159 or a biological variant. In some embodiments, the R2 ORF includes an RT that includes SEQ ID NO: 162 or a biological variant, or the R2 ORF includes SEQ ID NO: 161 or a biological variant, and the corresponding R2 3’UTR includes SEQ ID NO: 164 or a biological variant. In some embodiments, a corresponding R2 5’UTR is used, which includes SEQ ID NO: 163 or a biological variant. In some embodiments, the R2 ORF includes an RT that includes SEQ ID NO: 166 or a biological variant, or the R2 ORF includes SEQ ID NO: 165 or a biological variant, and the corresponding R2 3’UTR includes SEQ ID NO: 168 or a biological variant. In someembodiments, a corresponding R2 5’UTR is used, which includes SEQ ID NO: 167 or a biological variant.

[0128] In some embodiments, the R2 ORF includes an RT that includes SEQ ID NO: 170 or a biological variant, or the R2 ORF includes SEQ ID NO: 169 or a biological variant, and the corresponding R2 3’UTR includes SEQ ID NO: 172 or a biological variant. In some embodiments, a corresponding R2 5’UTR is used, which includes SEQ ID NO: 171 or a biological variant. In some embodiments, the R2 ORF includes an RT that includes SEQ ID NO: 174 or a biological variant, or the R2 ORF includes SEQ ID NO: 173 or a biological variant, and the corresponding R2 3’UTR includes SEQ ID NO: 176 or a biological variant. In some embodiments, a corresponding R2 5’UTR is used, which includes SEQ ID NO: 175 or a biological variant. In some embodiments, the R2 ORF includes an RT that includes SEQ ID NO:178 or a biological variant, or the R2 ORF includes SEQ ID NO: 177 or a biological variant, and the corresponding R2 3’UTR includes SEQ ID NO: 180 or a biological variant. In some embodiments, a corresponding R2 5’UTR is used, which includes SEQ ID NO: 179 or a biological variant.

[0129] In some embodiments, the R2 ORF includes an RT that includes SEQ ID NO: 182 or a biological variant, or the R2 ORF includes SEQ ID NO: 181 or a biological variant, and the corresponding R2 3’UTR includes SEQ ID NO: 184 or a biological variant. In some embodiments, a corresponding R2 5’UTR is used, which includes SEQ ID NO: 183 or a biological variant. In some embodiments, the R2 ORF includes an RT that includes SEQ ID NO: 186 or a biological variant, or the R2 ORF includes SEQ ID NO: 185 or a biological variant, and the corresponding R2 3’UTR includes SEQ ID NO: 188 or a biological variant. In some embodiments, a corresponding R2 5’UTR is used, which includes SEQ ID NO: 187 or a biological variant. In some embodiments, the R2 ORF includes an RT that includes SEQ ID NO: 190 or a biological variant, or the R2 ORF includes SEQ ID NO: 189 or a biological variant, and the corresponding R2 3’UTR includes SEQ ID NO: 192 or a biological variant. In some embodiments, a corresponding R2 5’UTR is used, which includes SEQ ID NO: 191 or a biological variant.

[0130] In some embodiments, the R2 ORF includes an RT that includes SEQ ID NO: 194 or a biological variant, or the R2 ORF includes SEQ ID NO: 193 or a biological variant, and the corresponding R2 3’UTR includes SEQ ID NO: 196 or a biological variant. In some embodiments, a corresponding R2 5’UTR is used, which includes SEQ ID NO: 195 or a biological variant. In some embodiments, the R2 ORF includes an RT that includes SEQ ID NO: 198 or a biological variant, or the R2 ORF includes SEQ ID NO: 197 or a biological variant, and the corresponding R2 3’UTR includes SEQ ID NO: 200 or a biological variant. In some embodiments, a corresponding R2 5’UTR is used, which includes SEQ ID NO: 199 or a biological variant. In some embodiments, the R2 ORF includes an RT that includes SEQ ID NO:202 or a biological variant, or the R2 ORF includes SEQ ID NO: 201 or a biological variant, and the corresponding R2 3’UTR includes SEQ ID NO: 204 or a biological variant. In some embodiments, a corresponding R2 5’UTR is used, which includes SEQ ID NO: 203 or a biological variant.

[0131] In some embodiments, the R2 ORF includes an RT that includes SEQ ID NO:206 or a biological variant, or the R2 ORF includes SEQ ID NO: 205 or a biological variant, and the corresponding R2 3’UTR includes SEQ ID NO: 208 or a biological variant. In some embodiments, a corresponding R2 5’UTR is used, which includes SEQ ID NO: 207 or a biological variant. In some embodiments, the R2 ORF includes an RT that includes SEQ ID NO:210 or a biological variant, or the R2 ORF includes SEQ ID NO: 209 or a biological variant, and the corresponding R2 3’UTR includes SEQ ID NO: 212 or a biological variant. In some embodiments, a corresponding R2 5’UTR is used, which includes SEQ ID NO: 211 or a biological variant. In some embodiments, the R2 ORF includes an RT that includes SEQ ID NO:214 or a biological variant, or the R2 ORF includes SEQ ID NO: 213 or a biological variant, and the corresponding R2 3’UTR includes SEQ ID NO: 216 or a biological variant. In some embodiments, a corresponding R2 5’UTR is used, which includes SEQ ID NO: 215 or a biological variant.

[0132] In some embodiments, the R2 ORF includes an RT that includes SEQ ID NO:218 or a biological variant, or the R2 ORF includes SEQ ID NO: 217 or a biological variant, and the corresponding R2 3’UTR includes SEQ ID NO: 220 or a biological variant. In some embodiments, a corresponding R2 5’UTR is used, which includes SEQ ID NO: 219 or abiological variant. In some embodiments, the R2 ORF includes an RT that includes SEQ ID NO:222 or a biological variant, or the R2 ORF includes SEQ ID NO: 221 or a biological variant, and the corresponding R2 3’UTR includes SEQ ID NO: 224 or a biological variant. In some embodiments, a corresponding R2 5’UTR is used, which includes SEQ ID NO: 223 or a biological variant. In some embodiments, the R2 ORF includes an RT that includes SEQ ID NO:226 or a biological variant, or the R2 ORF includes SEQ ID NO: 225 or a biological variant, and the corresponding R2 3’UTR includes SEQ ID NO: 228 or a biological variant. In some embodiments, a corresponding R2 5’UTR is used, which includes SEQ ID NO: 227 or a biological variant.

[0133] In some embodiments, the R2 ORF includes an RT that includes SEQ ID NO:230 or a biological variant, or the R2 ORF includes SEQ ID NO: 229 or a biological variant, and the corresponding R2 3’UTR includes SEQ ID NO: 232 or a biological variant. In some embodiments, a corresponding R2 5’UTR is used, which includes SEQ ID NO: 231 or a biological variant. In some embodiments, the R2 ORF includes an RT that includes SEQ ID NO:234 or a biological variant, or the R2 ORF includes SEQ ID NO: 233 or a biological variant, and the corresponding R2 3’UTR includes SEQ ID NO: 236 or a biological variant. In some embodiments, a corresponding R2 5’UTR is used, which includes SEQ ID NO: 235 or a biological variant. In some embodiments, the R2 ORF includes an RT that includes SEQ ID NO:238 or a biological variant, or the R2 ORF includes SEQ ID NO: 237 or a biological variant, and the corresponding R2 3’UTR includes SEQ ID NO: 240 or a biological variant. In some embodiments, a corresponding R2 5’UTR is used, which includes SEQ ID NO: 239 or a biological variant.

[0134] In some embodiments, the R2 ORF includes an RT that includes SEQ ID NO:242 or a biological variant, or the R2 ORF includes SEQ ID NO: 241 or a biological variant, and the corresponding R2 3’UTR includes SEQ ID NO: 244 or a biological variant. In some embodiments, a corresponding R2 5’UTR is used, which includes SEQ ID NO: 243 or a biological variant. In some embodiments, the R2 ORF includes an RT that includes SEQ ID NO:246 or a biological variant, or the R2 ORF includes SEQ ID NO: 245 or a biological variant, and the corresponding R2 3’UTR includes SEQ ID NO: 248 or a biological variant. In some embodiments, a corresponding R2 5’UTR is used, which includes SEQ ID NO: 247 or abiological variant. In some embodiments, the R2 ORF includes an RT that includes SEQ ID NO:250 or a biological variant, or the R2 ORF includes SEQ ID NO: 249 or a biological variant, and the corresponding R2 3’UTR includes SEQ ID NO: 252 or a biological variant. In some embodiments, a corresponding R2 5’UTR is used, which includes SEQ ID NO: 251 or a biological variant.

[0135] In some embodiments, the R2 ORF includes an RT that includes SEQ ID NO:254 or a biological variant, or the R2 ORF includes SEQ ID NO: 253 or a biological variant, and the corresponding R2 3’UTR includes SEQ ID NO: 256 or a biological variant. In some embodiments, a corresponding R2 5’UTR is used, which includes SEQ ID NO: 255 or a biological variant. In some embodiments, the R2 ORF includes an RT that includes SEQ ID NO:258 or a biological variant, or the R2 ORF includes SEQ ID NO: 257 or a biological variant, and the corresponding R2 3’UTR includes SEQ ID NO: 260 or a biological variant. In some embodiments, a corresponding R2 5’UTR is used, which includes SEQ ID NO: 259 or a biological variant. In some embodiments, the R2 ORF includes an RT that includes SEQ ID NO:262 or a biological variant, or the R2 ORF includes SEQ ID NO: 261 or a biological variant, and the corresponding R2 3’UTR includes SEQ ID NO: 264 or a biological variant. In some embodiments, a corresponding R2 5’UTR is used, which includes SEQ ID NO: 263 or a biological variant.

[0136] In some embodiments, the R2 ORF includes an RT that includes SEQ ID NO:266 or a biological variant, or the R2 ORF includes SEQ ID NO: 265 or a biological variant, and the corresponding R2 3’UTR includes SEQ ID NO: 268 or a biological variant. In some embodiments, a corresponding R2 5’UTR is used, which includes SEQ ID NO: 267 or a biological variant. In some embodiments, the R2 ORF includes an RT that includes SEQ ID NO:270 or a biological variant, or the R2 ORF includes SEQ ID NO: 269 or a biological variant, and the corresponding R2 3’UTR includes SEQ ID NO: 272 or a biological variant. In some embodiments, a corresponding R2 5’UTR is used, which includes SEQ ID NO: 271 or a biological variant. In some embodiments, the R2 ORF includes an RT that includes SEQ ID NO:274 or a biological variant, or the R2 ORF includes SEQ ID NO: 273 or a biological variant, and the corresponding R2 3’UTR includes SEQ ID NO: 276 or a biological variant. In someembodiments, a corresponding R2 5’UTR is used, which includes SEQ ID NO: 275 or a biological variant.

[0137] In some embodiments, the R2 ORF includes an RT that includes SEQ ID NO:278 or a biological variant, or the R2 ORF includes SEQ ID NO: 277 or a biological variant, and the corresponding R2 3’UTR includes SEQ ID NO: 280 or a biological variant. In some embodiments, a corresponding R2 5’UTR is used, which includes SEQ ID NO: 279 or a biological variant. In some embodiments, the R2 ORF includes an RT that includes SEQ ID NO:282 or a biological variant, or the R2 ORF includes SEQ ID NO: 281 or a biological variant, and the corresponding R2 3’UTR includes SEQ ID NO: 284 or a biological variant. In some embodiments, a corresponding R2 5’UTR is used, which includes SEQ ID NO: 283 or a biological variant. In some embodiments, the R2 ORF includes an RT that includes SEQ ID NO:286 or a biological variant, or the R2 ORF includes SEQ ID NO: 285 or a biological variant, and the corresponding R2 3’UTR includes SEQ ID NO: 288 or a biological variant. In some embodiments, a corresponding R2 5’UTR is used, which includes SEQ ID NO: 287 or a biological variant.

[0138] In some embodiments, the R2 ORF includes an RT that includes SEQ ID NO:290 or a biological variant, or the R2 ORF includes SEQ ID NO: 289 or a biological variant, and the corresponding R2 3’UTR includes SEQ ID NO: 292 or a biological variant. In some embodiments, a corresponding R2 5’UTR is used, which includes SEQ ID NO: 291 or a biological variant. In some embodiments, the R2 ORF includes an RT that includes SEQ ID NO:294 or a biological variant, or the R2 ORF includes SEQ ID NO: 293 or a biological variant, and the corresponding R2 3’UTR includes SEQ ID NO: 296 or a biological variant. In some embodiments, a corresponding R2 5’UTR is used, which includes SEQ ID NO: 295 or a biological variant. In some embodiments, the R2 ORF includes an RT that includes SEQ ID NO:298 or a biological variant, or the R2 ORF includes SEQ ID NO: 297 or a biological variant, and the corresponding R2 3’UTR includes SEQ ID NO: 300 or a biological variant. In some embodiments, a corresponding R2 5’UTR is used, which includes SEQ ID NO: 299 or a biological variant.

[0139] In some embodiments, the R2 ORF includes an RT that includes SEQ ID NO:302 or a biological variant, or the R2 ORF includes SEQ ID NO: 301 or a biological variant, and the corresponding R2 3’UTR includes SEQ ID NO: 304 or a biological variant. In some embodiments, a corresponding R2 5’UTR is used, which includes SEQ ID NO: 303 or a biological variant. In some embodiments, the R2 ORF includes an RT that includes SEQ ID NO:306 or a biological variant, or the R2 ORF includes SEQ ID NO: 305 or a biological variant, and the corresponding R2 3’UTR includes SEQ ID NO: 308 or a biological variant. In some embodiments, a corresponding R2 5’UTR is used, which includes SEQ ID NO: 307 or a biological variant. In some embodiments, the R2 ORF includes an RT that includes SEQ ID NO:310 or a biological variant, or the R2 ORF includes SEQ ID NO: 309 or a biological variant, and the corresponding R2 3’UTR includes SEQ ID NO: 312 or a biological variant. In some embodiments, a corresponding R2 5’UTR is used, which includes SEQ ID NO: 311 or a biological variant.

[0140] In some embodiments, the R2 ORF includes an RT that includes SEQ ID NO:314 or a biological variant, or the R2 ORF includes SEQ ID NO: 313 or a biological variant, and the corresponding R2 3’UTR includes SEQ ID NO: 316 or a biological variant. In some embodiments, a corresponding R2 5’UTR is used, which includes SEQ ID NO: 315 or a biological variant. In some embodiments, the R2 ORF includes an RT that includes SEQ ID NO:318 or a biological variant, or the R2 ORF includes SEQ ID NO: 317 or a biological variant, and the corresponding R2 3’UTR includes SEQ ID NO: 320 or a biological variant. In some embodiments, a corresponding R2 5’UTR is used, which includes SEQ ID NO: 319 or a biological variant. In some embodiments, the R2 ORF includes an RT that includes SEQ ID NO:322 or a biological variant, or the R2 ORF includes SEQ ID NO: 321 or a biological variant, and the corresponding R2 3’UTR includes SEQ ID NO: 324 or a biological variant. In some embodiments, a corresponding R2 5’UTR is used, which includes SEQ ID NO: 323 or a biological variant.

[0141] In some embodiments, the R2 ORF includes an RT that includes SEQ ID NO:326 or a biological variant, or the R2 ORF includes SEQ ID NO: 325 or a biological variant, and the corresponding R2 3’UTR includes SEQ ID NO: 328 or a biological variant. In some embodiments, a corresponding R2 5’UTR is used, which includes SEQ ID NO: 327 or abiological variant. In some embodiments, the R2 ORF includes an RT that includes SEQ ID NO:330 or a biological variant, or the R2 ORF includes SEQ ID NO: 329 or a biological variant, and the corresponding R2 3’UTR includes SEQ ID NO: 332 or a biological variant. In some embodiments, a corresponding R2 5’UTR is used, which includes SEQ ID NO: 331 or a biological variant. In some embodiments, the R2 ORF includes an RT that includes SEQ ID NO:334 or a biological variant, or the R2 ORF includes SEQ ID NO: 333 or a biological variant, and the corresponding R2 3’UTR includes SEQ ID NO: 336 or a biological variant. In some embodiments, a corresponding R2 5’UTR is used, which includes SEQ ID NO: 335 or a biological variant.

[0142] In some embodiments, the R2 ORF includes an RT that includes SEQ ID NO:338 or a biological variant, or the R2 ORF includes SEQ ID NO: 337 or a biological variant, and the corresponding R2 3’UTR includes SEQ ID NO: 340 or a biological variant. In some embodiments, a corresponding R2 5’UTR is used, which includes SEQ ID NO: 339 or a biological variant. In some embodiments, the R2 ORF includes an RT that includes SEQ ID NO:342 or a biological variant, or the R2 ORF includes SEQ ID NO: 341 or a biological variant, and the corresponding R2 3’UTR includes SEQ ID NO: 344 or a biological variant. In some embodiments, a corresponding R2 5’UTR is used, which includes SEQ ID NO: 343 or a biological variant. In some embodiments, the R2 ORF includes an RT that includes SEQ ID NO:346 or a biological variant, or the R2 ORF includes SEQ ID NO: 345 or a biological variant, and the corresponding R2 3’UTR includes SEQ ID NO: 348 or a biological variant. In some embodiments, a corresponding R2 5’UTR is used, which includes SEQ ID NO: 347 or a biological variant.

[0143] In some embodiments, the R2 ORF includes an RT that includes SEQ ID NO:350 or a biological variant, or the R2 ORF includes SEQ ID NO: 349 or a biological variant, and the corresponding R2 3’UTR includes SEQ ID NO: 352 or a biological variant. In some embodiments, a corresponding R2 5’UTR is used, which includes SEQ ID NO: 351 or a biological variant. In some embodiments, the R2 ORF includes an RT that includes SEQ ID NO:354 or a biological variant, or the R2 ORF includes SEQ ID NO: 353 or a biological variant, and the corresponding R2 3’UTR includes SEQ ID NO: 356 or a biological variant. In some embodiments, a corresponding R2 5’UTR is used, which includes SEQ ID NO: 355 or abiological variant. In some embodiments, the R2 ORF includes an RT that includes SEQ ID NO:358 or a biological variant, or the R2 ORF includes SEQ ID NO: 357 or a biological variant, and the corresponding R2 3’UTR includes SEQ ID NO: 360 or a biological variant. In some embodiments, a corresponding R2 5’UTR is used, which includes SEQ ID NO: 359 or a biological variant.

[0144] In some embodiments, the R2 ORF includes an RT that includes SEQ ID NO: 362 or a biological variant, or the R2 ORF includes SEQ ID NO: 361 or a biological variant, and the corresponding R2 3’UTR includes SEQ ID NO: 364 or a biological variant. In some embodiments, a corresponding R2 5’UTR is used, which includes SEQ ID NO: 363 or a biological variant. In some embodiments, the R2 ORF includes an RT that includes SEQ ID NO:366 or a biological variant, or the R2 ORF includes SEQ ID NO: 365 or a biological variant, and the corresponding R2 3’UTR includes SEQ ID NO: 368 or a biological variant. In some embodiments, a corresponding R2 5’UTR is used, which includes SEQ ID NO: 367 or a biological variant. In some embodiments, the R2 ORF includes an RT that includes SEQ ID NO:370 or a biological variant, or the R2 ORF includes SEQ ID NO: 369 or a biological variant, and the corresponding R2 3’UTR includes SEQ ID NO: 372 or a biological variant. In some embodiments, a corresponding R2 5’UTR is used, which includes SEQ ID NO: 371 or a biological variant.

[0145] In some embodiments, the R2 ORF includes an RT that includes SEQ ID NO:374 or a biological variant, or the R2 ORF includes SEQ ID NO: 373 or a biological variant, and the corresponding R2 3’UTR includes SEQ ID NO: 376 or a biological variant. In some embodiments, a corresponding R2 5’UTR is used, which includes SEQ ID NO: 375 or a biological variant. In some embodiments, the R2 ORF includes an RT that includes SEQ ID NO:378 or a biological variant, or the R2 ORF includes SEQ ID NO: 377 or a biological variant, and the corresponding R2 3’UTR includes SEQ ID NO: 380 or a biological variant. In some embodiments, a corresponding R2 5’UTR is used, which includes SEQ ID NO: 379 or a biological variant. In some embodiments, the R2 ORF includes an RT that includes SEQ ID NO:382 or a biological variant, or the R2 ORF includes SEQ ID NO: 381 or a biological variant, and the corresponding R2 3’UTR includes SEQ ID NO: 384 or a biological variant. In someembodiments, a corresponding R2 5’UTR is used, which includes SEQ ID NO: 383 or a biological variant.

[0146] In some embodiments, the R2 ORF includes an RT that includes SEQ ID NO:386 or a biological variant, or the R2 ORF includes SEQ ID NO: 385 or a biological variant, and the corresponding R2 3’UTR includes SEQ ID NO: 388 or a biological variant. In some embodiments, a corresponding R2 5’UTR is used, which includes SEQ ID NO: 387 or a biological variant. In some embodiments, the R2 ORF includes an RT that includes SEQ ID NO:390 or a biological variant, or the R2 ORF includes SEQ ID NO: 389 or a biological variant, and the corresponding R2 3’UTR includes SEQ ID NO: 392 or a biological variant. In some embodiments, a corresponding R2 5’UTR is used, which includes SEQ ID NO: 391 or a biological variant. In some embodiments, the R2 ORF includes an RT that includes SEQ ID NO:394 or a biological variant, or the R2 ORF includes SEQ ID NO: 393 or a biological variant, and the corresponding R2 3’UTR includes SEQ ID NO: 396 or a biological variant. In some embodiments, a corresponding R2 5’UTR is used, which includes SEQ ID NO: 395 or a biological variant.

[0147] In some embodiments, the R2 ORF includes an RT that includes SEQ ID NO:398 or a biological variant, or the R2 ORF includes SEQ ID NO: 397 or a biological variant, and the corresponding R2 3’UTR includes SEQ ID NO: 400 or a biological variant. In some embodiments, a corresponding R2 5’UTR is used, which includes SEQ ID NO: 399 or a biological variant. In some embodiments, the R2 ORF includes an RT that includes SEQ ID NO:402 or a biological variant, or the R2 ORF includes SEQ ID NO: 401 or a biological variant, and the corresponding R2 3’UTR includes SEQ ID NO: 404 or a biological variant. In some embodiments, a corresponding R2 5’UTR is used, which includes SEQ ID NO: 403 or a biological variant. In some embodiments, the R2 ORF includes an RT that includes SEQ ID NO:406 or a biological variant, or the R2 ORF includes SEQ ID NO: 405 or a biological variant, and the corresponding R2 3’UTR includes SEQ ID NO: 408 or a biological variant. In some embodiments, a corresponding R2 5’UTR is used, which includes SEQ ID NO: 407 or a biological variant.

[0148] In some embodiments, the R2 ORF includes an RT that includes SEQ ID NO:410 or a biological variant, or the R2 ORF includes SEQ ID NO: 409 or a biological variant, and the corresponding R2 3’UTR includes SEQ ID NO: 412 or a biological variant. In some embodiments, a corresponding R2 5’UTR is used, which includes SEQ ID NO: 411 or a biological variant. In some embodiments, the R2 ORF includes an RT that includes SEQ ID NOAM or a biological variant, or the R2 ORF includes SEQ ID NO: 413 or a biological variant, and the corresponding R2 3’UTR includes SEQ ID NO: 416 or a biological variant. In some embodiments, a corresponding R2 5’UTR is used, which includes SEQ ID NO: 415 or a biological variant. In some embodiments, the R2 ORF includes an RT that includes SEQ ID NO:418 or a biological variant, or the R2 ORF includes SEQ ID NO: 417 or a biological variant, and the corresponding R2 3’UTR includes SEQ ID NO: 420 or a biological variant. In some embodiments, a corresponding R2 5’UTR is used, which includes SEQ ID NO: 419 or a biological variant.

[0149] In some embodiments, the R2 ORF includes an RT that includes SEQ ID NO:422 or a biological variant, or the R2 ORF includes SEQ ID NO: 421 or a biological variant, and the corresponding R2 3’UTR includes SEQ ID NO: 424 or a biological variant. In some embodiments, a corresponding R2 5’UTR is used, which includes SEQ ID NO: 423 or a biological variant.Gene Delivery and Treatments

[0150] The R2 retrotransposition systems disclosed here are suitable for delivering exogenous genes to a target cell, such as a mammalian cell.

[0151] Accordingly, in some embodiments, the present disclosure provides a method for integrating an exogenous polynucleotide to the genome of a target cell. In one embodiment, the method entails introducing to the cell the polynucleotide comprising an R2 retrotransposon of the instant disclosure. In some embodiments, the retrotransposon is delivered to the target cell as an mRNA molecule directly.

[0152] In some embodiments, when the polynucleotide comprises a CRISPR Cas protein encoding sequence, the method further comprises introducing to the cell a guide RNA (gRNA) that binds to the CRISPR Cas protein and recognizes a target DNA sequence in the genome.

[0153] mRNAs may be synthesized according to any of a variety of known methods. For example, the mRNAs may be synthesized via in vitro transcription (IVT). Briefly, IVT is typically performed with a linear or circular DNA template containing a promoter, a pool of ribonucleotide triphosphates, a buffer system that may include DTT and magnesium ions, and an appropriate RNA polymerase (e.g., T3, T7 or SP6 RNA polymerase), DNase I, pyrophosphatase, and / or RNase inhibitor. The exact conditions will vary according to the specific application.

[0154] The mRNA may be synthesized as unmodified or modified mRNA. Typically, mRNAs are modified to enhance stability. Modifications of mRNA can include, for example, modifications of the nucleotides of the RNA. A modified mRNA can thus include, for example, backbone modifications, sugar modifications or base modifications. In some embodiments, antibody encoding mRNAs (e. ., heavy chain and light chain encoding mRNAs) may be synthesized from naturally occurring nucleotides and / or nucleotide analogues (modified nucleotides) including, but not limited to, purines (adenine (A), guanine (G)) or pyrimidines (thymine (T), cytosine (C), uracil (U)), and as modified nucleotides analogues or derivatives of purines and pyrimidines, such as e.g. 1 -methyl -adenine, 2-methyl-adenine, 2-methylthio-N-6- isopentenyl -adenine, N6-methyl-adenine, N6-isopentenyl-adenine, 2-thio-cytosine, 3-methyl- cytosine, 4-acetyl-cytosine, 5-methyl-cytosine, 2,6-diaminopurine, 1-methyl-guanine, 2-methyl- guanine, 2,2-dimethyl-guanine, 7-methyl-guanine, inosine, 1 -methyl -inosine, pseudouracil (5- uracil), dihydro-uracil, 2-thio-uracil, 4-thio-uracil, 5-carboxymethylaminomethyl-2-thio-uracil, 5-(carboxyhydroxymethyl)-uracil, 5 -fluoro-uracil, 5-bromo-uracil, 5- carboxymethylaminomethyl-uracil, 5-methyl-2 -thio-uracil, 5-methyl-uracil, N-uracil-5-oxyacetic acid methyl ester, 5-methylaminomethyl-uracil, 5-methoxyaminomethyl -2-thio-uracil, 5’- methoxycarbonylmethyl-uracil, 5-methoxy-uracil, uracil-5-oxyacetic acid methyl ester, uracil-5- oxyacetic acid (v), 1-methyl-pseudouracil, queosine, 13-D-mannosyl-queosine, wybutoxosine, and phosphoramidates, phosphorothioates, peptide nucleotides, methylphosphonates, 7- deazaguanosine, 5 -methylcytosine and inosine. The preparation of such analogues is known to a person skilled in the art e.g. from the U.S. Pat. Nos. 4,373,071, 4,401,796, 4,415,732, 4,458,066,4,500,707, 4,668,777, 4,973,679, 5,047,524, 5,132,418, 5,153,319, 5,262,530 and 5,700,642, the disclosure of which is included here in its full scope by reference.

[0155] In some embodiments, the mRNAs may contain RNA backbone modifications. Typically, a backbone modification is a modification in which the phosphates of the backbone of the nucleotides contained in the RNA are modified chemically. Exemplary backbone modifications typically include, but are not limited to, modifications from the group consisting of methylphosphonates, methylphosphoramidates, phosphoramidates, phosphorothioates (e.g. cytidine 5 ’-O-(l -thiophosphate)), boranophosphates, positively charged guanidinium groups etc., which means by replacing the phosphodiester linkage by other anionic, cationic or neutral groups.

[0156] In some embodiments, the mRNAs may contain sugar modifications. A typical sugar modification is a chemical modification of the sugar of the nucleotides it contains including, but not limited to, sugar modifications chosen from the group consisting of 2’-deoxy-2’-fluoro- oligoribonucleotide (2’ -fluoro-2’ -deoxy cytidine 5 ’ -triphosphate, 2’-fluoro-2’-deoxyuridine 5’- triphosphate), 2’-deoxy-2’-deamine-oligoribonucleotide (2’-amino-2’-deoxycytidine 5’- triphosphate, 2’-amino-2’-deoxyuridine 5 ’-triphosphate), 2’-O-alkyloligoribonucleotide, 2’- deoxy-2’-C-alkyloligoribonucleotide (2’-O-methylcytidine 5 ’-triphosphate, 2’-methyluridine 5’- triphosphate), 2’-C-alkyloligoribonucleotide, and isomers thereof (2’-aracytidine 5 ’-triphosphate, 2’-arauridine 5 ’-triphosphate), or azidotriphosphates (2’ -azido-2’ -deoxy cytidine 5 ’ -triphosphate, 2’ -azido-2’ -deoxyuridine 5 ’ -triphosphate).

[0157] In some embodiments, the mRNAs may contain modifications of the bases of the nucleotides (base modifications). A modified nucleotide which contains a base modification is also called a base-modified nucleotide. Examples of such base-modified nucleotides include, but are not limited to, 2-amino-6-chloropurine riboside 5 ’ -triphosphate, 2-aminoadenosine 5’- triphosphate, 2-thiocytidine 5 ’-triphosphate, 2-thiouridine 5 ’-triphosphate, 4-thiouridine 5’- triphosphate, 5-aminoallylcytidine 5 ’ -triphosphate, 5-aminoallyluridine 5 ’-triphosphate, 5- bromocytidine 5 ’-triphosphate, 5-bromouridine 5 ’-triphosphate, 5-iodocytidine 5 ’-triphosphate, 5-iodouridine 5 ’-triphosphate, 5-methylcytidine 5 ’-triphosphate, 5-methyluridine 5’- triphosphate, 6-azacytidine 5 ’-triphosphate, 6-azauridine 5 ’-triphosphate, 6-chloropurine riboside5 ’-triphosphate, 7-deazaadenosine 5’-triphosphate, 7-deazaguanosine 5 ’-triphosphate, 8- azaadenosine 5 ’-triphosphate, 8-azidoadenosine 5 ’-triphosphate, benzimidazole riboside 5’- triphosphate, N1 -methyladenosine 5 ’-triphosphate, N1 -methylguanosine 5 ’-triphosphate, N6- m ethyladenosine 5 ’ -triphosphate, 06-methylguanosine 5 ’-triphosphate, pseudouridine 5’- triphosphate, puromycin 5 ’-triphosphate or xanthosine 5 ’-triphosphate. In some embodiments, the RNA contains pseudouridine-modified nucleotides.

[0158] In some embodiments, the mRNAs include a 5’ cap structure. A 5’ cap is typically added as follows: first, an RNA terminal phosphatase removes one of the terminal phosphate groups from the 5’ nucleotide, leaving two terminal phosphates; guanosine triphosphate (GTP) is then added to the terminal phosphates via a guanylyl transferase, producing a 5’5’5 triphosphate linkage; and the 7-nitrogen of guanine is then methylated by a methyltransferase. Examples of cap structures include, but are not limited to, m7G(5’)ppp (5’(A,G(5’)ppp(5)A and G(5)ppp(5’)G.

[0159] In some embodiments, the retrotransposon of the present disclosure is delivered by a vector such as a plasmid vector and a viral vector (e.g., adenoviral vector).

[0160] In some embodiments, the target mammalian cell is a human cell. In some embodiments, the human cell is an induced pluripotent stem cell (iPSC). When an mRNA is directly introduced to the target stem cell, in a preferred embodiment, the downstream flanking sequence of the retrotransposon has a length of at least 5 nt, such as 5 to 50 nt, 7 to 30 nt, or 8 to 20 nt.

[0161] In some embodiments, the R2 ORF and the exogenous gene are included in the same retrotransposon polynucleotide. In some embodiments, only the exogenous gene is included in the retrotransposon polynucleotide, while the R2 ORF is delivered separately on a different polynucleotide.

[0162] In some embodiments, the delivery is in vitro. In some embodiments, the delivery is in vivo or ex vivo. In some embodiments, the retrotransposon polynucleotide is delivered by parenteral means, or locally.

[0163] The term “parenteral” as used herein refers to modes of administration which include intravenous, intramuscular, intraperitoneal, intrasternal, subcutaneous and intra-articular injection and infusion.

[0164] Delivery can be systemic or local. In addition, it may be desirable to introduce the retrotransposon polynucleotide of the disclosure into the central nervous system by any suitable route, including intraventricular and intrathecal injection; intraventricular injection may be facilitated by an intraventricular catheter, for example, attached to a reservoir, such as an Ommaya reservoir. Pulmonary administration can also be employed, e.g., by use of an inhaler or nebulizer, and formulation with an aerosolizing agent.EXAMPLES

[0165] The following examples are included to demonstrate specific embodiments of the disclosure. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques to function well in the practice of the disclosure, and thus can be considered to constitute specific modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the disclosure.Example 1: Discovery and Testing of New R2 Retrotransposons

[0166] This example describes the discovery and testing of new R2 retrotransposons from various species.

[0167] The amino acid sequence of the open reading frame (ORF) of known R2 sequences, in particular those of the main functional domains, was used to search for analogs in the GenBank database. The genomic and amino acid sequences of the hits were examined with respect to domains / motif common to R2 retrotransposons, including zinc finger domains, SANT / Myb domains, reverse-transcriptase domain, and endonuclease domains.

[0168] R2 retrotransposons selected based on such sequence analysis (Table 1) were subjected to in vitro retrotransposition activity assessment. For each R2 retrotransposon, the entiresequence of the open reading frame (ORF), its reverse-transcriptase (RT) domain (underlined in the ORF sequence and also separated listed), and the corresponding 5’UTR and 3’UTR sequences are provided.

[0169] The retrotransposition activities of these newly discovered R2 retrotransposons were tested with cultured human cell line, 293T. Cells were grown in Dulbecco’s minimal essential medium (DMEM) supplemented with 10% fetal bovine serum (Gibco) and 1% penicillin / streptomycin (Invitrogen) in 5% CO2 at 37 °C. Cells were grown as adherent cell cultures and passaged every 72 h at a 1 :5 dilution (volume of cells: final volume of medium) until confluency was reached, in order to maintain log-phase growth.

[0170] Cells were seeded at 60% confluence in 24-well cluster plates one day before transfection. For transfection, OptiMEM medium (Invitrogen) was pre-warmed to room temperature. Transfection of mRNA into cells was performed with Lipofectamine® MessengerMAX™ (Invitrogen). mRNA Lipofectamine® MessengerMAX™ Reagent (1.5 pL of each) were mixed with 25 pl OptiMEM medium and incubated for 10 min at temperature. 0.5 pg of mRNA was mixed with OptiMEM medium in total volume 25 pL. Then, mixed 25 pL of lipofectamine reagent mixture with 25 pL mRNA mixture and incubated for 5 min at temperature. Mixtures were applied to each well. Helper virus infected the plasmid transfected cells at the same time and continue to incubate for 72 h. Integration of the donor sequence into the human genome was measured with quantitative PCR (qPCR) or or digital PCR (dPCR) in isolated genomic DNA.Table 1. R2 SequencesExample 2. PCR Verification of Integration

[0171] This example used PCT to confirm that the newly identified R2 elements were capable of integrating themselves to the human genome. The tested R2 elements included R2AspMar (No. 23 in Table A), R2ATLH (No. 39), R2AspTig (No. 2), R2Vila (No. 45), R2PoEx (No. 12), R2Hal (No. 14), R2Age (benchmark), R2MelGeo (No. 8), R2MelCri (No. 35), R2PoAt (No. 37), R2TyAl (No. 44), R2HarHar (No. 32), R2ChPi (No. 38), R2Boja (No. 47), R2CapEur (No. 17), R2Bofu (No. 48), R2ColStr (No. 5), R2CroAda (No. 28), R2Tral (No. 46), R2CucCan (No. 29), R2CarCar (No. 10).

[0172] The mRNA templates derived from the full-length wild type R2 constructs were transfected into human HEK293T cell lines. Junctions at the 28S rDNA target site were analyzed by dPCR to assess genome integration activities. Both 5’ and 3’ junctions were measured. The Y axis reflects integrated R2 copy numbers per haploid genome (FIG. 1). As shown in FIG. 1, all of these constructed successfully inserted themselves to the ITEK293T genome.Example 3. Testing of new R2 Sequences

[0173] This example tested a few newly identified R2 retrotransposons for their ability in delivering transgene to a target cell, via trans or cis configurations.

[0174] In a cis configuration (FIG. 2, panel above), a transgene (e.g., GFP) is inserted downstream of the R2 ORF within the R2 retrotransposon. Upon insertion into the target genome along with the R2 ORF, the transgene can be expressed by the host. In a trans configuration(FIG. 2, panel below), the transgene is flanked by the UTR sequences of the R2 (forming a “donor mRNA”) for insertion into the target genome, while the R2 ORF is provided as a separate mRNA (“helper” mRNA) which is not inserted.

[0175] In a first batch, R2AtLH (No. 39), R2AspMar (No. 23), and R2AspTig (No. 2), were tested in the cis configuration and R2AtLH (No. 39), R2AspTig (No. 2), R2AspMar (No. 23), R2Vila (No. 45), and R2Boja (No. 47) were tested in the trans configuration. As shown in FIG. 3, all of them exhibited high integration efficiency.

[0176] In a second batch, R2AspTig (No. 2) and R2AtLH (No. 39) were tested. HEK cells were transfected with the mRNA in cis with a CMV-GFP-PolyA cassette carrying a cargo, or in trans with a CMV-GFP-PolyA cassette carrying a cargo and an R2 cassette encoding the R2 protein.

[0177] Cells were imaged and subjected to flow cytometry analysis at Day 3 post transfection, with no observed reduction in viability relative to transfection of GFP mRNA. As shown in FIG.4 (cis) and FIG. 5 trans), all tested R2 elements exhibited excellent gene delivery efficiency, in particular R2AtLH and R2MicRuf.

[0178] In a third batch, 14 new identified R2 elements, including R2ATLH (No. 39), R2MicRuf (No. 74), R2FasGig2 (No. 61), R2MobBir (No. 104), R2AriEle (No. 22), R2FasGigl (No. 60), R2MasLat (No. 54), R2VipLat (No. 89), R2LiaOli (No. 53), R2OpiVivl (No. 65), R2SchSol (No. 71), R2FasGig3 (No. 62), R2FasHep (No. 64), R2MyxAsil (No. 99), and R2Ptesp (No. 106), were tested for gene delivery. Cis constructs with GFP cargo were tested, and %GFP+ cells were measured by flow cytometry. As shown in FIG. 6, these R2 elements would effectively deliver GFP to the target cells.Example 4. Gene Delivery with Templates Including Pseudouridine-Modified Nucleotides

[0179] In this example, six additional novel R2 elements were tested for gene delivery, including R2MicRuf (No. 74), R2ATLH (No. 39), R2FasGig2 (No. 61), R2FasGigl (No. 60), R2MobBir (No. 104), R2VipLat (No. 89), and R2AriEle (No. 22). Trans constructs with GFP cargo were tested, and %GFP+ cells were measured by flow cytometry.

[0180] Here, all the RNA templates by in vitro transcription used pseudouridine-modified nucleotides. With such modifications, it appeared that all of the tested trans R2 constructs had excellent gene delivery efficiencies (FIG. 7).* * *

[0181] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0182] The inventions illustratively described herein may suitably be practiced in the absence of any element or elements, limitation or limitations, not specifically disclosed herein. Thus, for example, the terms “comprising”, “including,” “containing”, etc. shall be read expansively and without limitation. Additionally, the terms and expressions employed herein have been used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed.

[0183] Thus, it should be understood that although the present invention has been specifically disclosed by preferred embodiments and optional features, modification, improvement and variation of the inventions embodied therein herein disclosed may be resorted to by those skilled in the art, and that such modifications, improvements and variations are considered to be within the scope of this invention. The materials, methods, and examples provided here are representative of preferred embodiments, are exemplary, and are not intended as limitations on the scope of the invention.

[0184] The invention has been described broadly and generically herein. Each of the narrower species and subgeneric groupings falling within the generic disclosure also form part of the invention. This includes the generic description of the invention with a proviso or negative limitation removing any subject matter from the genus, regardless of whether or not the excised material is specifically recited herein.

[0185] In addition, where features or aspects of the invention are described in terms of Markush groups, those skilled in the art will recognize that the invention is also thereby described in terms of any individual member or subgroup of members of the Markush group.

[0186] All publications, patent applications, patents, and other references mentioned herein are expressly incorporated by reference in their entirety, to the same extent as if each were incorporated by reference individually. In case of conflict, the present specification, including definitions, will control.

[0187] It is to be understood that while the disclosure has been described in conjunction with the above embodiments, that the foregoing description and examples are intended to illustrate and not limit the scope of the disclosure. Other aspects, advantages and modifications within the scope of the disclosure will be apparent to those skilled in the art to which the disclosure pertains.

Claims

CLAIMS:

1. A method for inserting an exogenous sequence to a genomic DNA of a cell, comprising introducing to the cell(a) a polypeptide or a first polynucleotide encoding the polypeptide, wherein the polypeptide comprises(i) a DNA binding domain or a CRISPR Cas protein,(ii) a reverse-transcriptase comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 6, 2, 10, 14, 18, 22, 26, 30, 34, 38, 42, 46, 50, 54, 58, 62, 66, 70, 74, 78, 82, 86, 90, 94, 98, 102, 106, 110, 114, 118, 122, 126, 130, 134, 138, 142, 146, 150, 154, 158, 162, 166, 170, 174,178, 182, 186, 190, 194, 198, 202, 206, 210, 214, 218, 222, 226, 230, 234,238, 242, 246, 250, 254, 258, 262, 266, 270, 274, 278, 282, 286, 290, 294,298, 302, 306, 310, 314, 318, 322, 326, 330, 334, 338, 342, 346, 350, 354,358, 362, 366, 370, 374, 378, 382, 386, 390, 394, 398, 402, 406, 410, 414,418 and 422, or an amino acid sequence having at least 85% sequence identity to any of the amino acid selected from the group, and(iii) an endonuclease or a nickase, and(b) an RNA comprising (i) a template for the exogenous sequence and (ii) a 3’ UTR, or a second polynucleotide encoding the RNA.

2. The method of claim 1, which comprises introducing the first polynucleotide and the second polynucleotide to the cell.

3. The method of claim 2, wherein the first polynucleotide and the second polynucleotide are on a same vector or on separate vectors.

4. The method of any preceding claim, wherein the 3 ’UTR is an R2 retrotransposon 3’ UTR.

5. The method of claim 4, wherein the 3 ’UTR comprise a sequence selected from the group consisting of SEQ ID NO: 8, 4, 12, 16, 20, 24, 28, 32, 36, 40, 44, 48, 52, 56, 60, 64, 68, 72, 76,80, 84, 88, 92, 96, 100, 104, 108, 1 12, 116, 120, 124, 128, 132, 136, 140, 144, 148, 152, 156, 160, 164, 168, 172, 176, 180, 184, 188, 192, 196, 200, 204, 208, 212, 216, 220, 224, 228, 232,236, 240, 244, 248, 252, 256, 260, 264, 268, 272, 276, 280, 284, 288, 292, 296, 300, 304, 308,312, 316, 320, 324, 328, 332, 336, 340, 344, 348, 352, 356, 360, 364, 368, 372, 376, 380, 384,388, 392, 396, 400, 404, 408, 412, 416, 420 and 424.

6. The method of any preceding claim, wherein the reverse-transcriptase comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 6, 2, 10, 14, 18, 22, 26, 30, 34, 38, 42, 46, 50, 54, 58, 62, 66, 70, 74, 78, 82, 86, 90, 94, 98, 102, 106, 110, 114, 118, 122, 126, 130, 134, 138, 142, 146, 150, 154, 158, 162, 166, 170, 174, 178, 182, 186, 190, 194, 198,202, 206, 210, 214, 218, 222, 226, 230, 234, 238, 242, 246, 250, 254, 258, 262, 266, 270, 274,278, 282, 286, 290, 294, 298, 302, 306, 310, 314, 318, 322, 326, 330, 334, 338, 342, 346, 350,354, 358, 362, 366, 370, 374, 378, 382, 386, 390, 394, 398, 402, 406, 410, 414, 418 and 422.

7. The method of any preceding claim, wherein the polypeptide comprises the endonuclease.

8. The method of any one of claims 1-7, wherein the polypeptide comprises the nickase, which optionally is Cas nickase.

9. The method of any preceding claim, wherein the CRISPR Cas protein is selected from the group consisting of Cas9, Cas 12 and Cas 13.

10. The method of any one of claims 1-8, wherein the DNA binding domain is selected from the group consisting of a transcription activator-like effector (TALE) DNA binding domain, a homeodomain protein, a zinc finger, a helix-tum-helix, a leucine zipper, and a DNA-binding domain of a homing endonuclease, a transposon or retro-transposon.

11. The method protein of claim 10, wherein the DNA binding domain is a TALE.

12. The method of claim 10, wherein the homing endonuclease is selected from the group consisting of I-Scel, LCrel and I-Dmol.

13. The method of claim 10, wherein the retro-transposon is a long interspersed nuclear element (LINE), optionally selected from the group consisting of Rl, R2, R4, R5, R6, R7, R8, and R9.

14. The method of any one of claims 1-5, wherein the polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 5, 1, 9, 13, 17, 21, 25, 29, 33, 37, 41, 45, 49, 53, 57, 61, 65, 69, 73, 77, 81, 85, 89, 93, 97, 101, 105, 109, 113, 117, 121, 125, 129, 133, 137, 141, 145, 149, 153, 157, 161, 165, 169, 173, 177, 181, 185, 189, 193, 197, 201, 205,209, 213, 217, 221, 225, 229, 233, 237, 241, 245, 249, 253, 257, 261, 265, 269, 273, 277, 281,285, 289, 293, 297, 301, 305, 309, 313, 317, 321, 325, 329, 333, 337, 341, 345, 349, 353, 357,361, 365, 369, 373, 377, 381, 385, 389, 393, 397, 401, 405, 409, 413, 417 and 421, or an amino acid sequence having at least 85% sequence identity to any amino acid sequence selected from the group.

15. The method of claim 14, wherein the polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 5, 1, 9, 13, 17, 21, 25, 29, 33, 37, 41, 45, 49, 53, 57, 61, 65, 69, 73, 77, 81, 85, 89, 93, 97, 101, 105, 109, 113, 117, 121, 125, 129, 133, 137,141, 145, 149, 153, 157, 161, 165, 169, 173, 177, 181, 185, 189, 193, 197, 201, 205, 209, 213,217, 221, 225, 229, 233, 237, 241, 245, 249, 253, 257, 261, 265, 269, 273, 277, 281, 285, 289,293, 297, 301, 305, 309, 313, 317, 321, 325, 329, 333, 337, 341, 345, 349, 353, 357, 361, 365,369, 373, 377, 381, 385, 389, 393, 397, 401, 405, 409, 413, 417 and 421.

16. The method of any preceding claim, wherein the RNA further comprises a 5’UTR.

17. The method of claim 16, wherein the 5’UTR comprises a sequence selected from the group consisting of SEQ ID NO: 7, 3, 11, 15, 19, 23, 27, 31, 35, 39, 43, 47, 51, 55, 59, 63, 67, 71, 75, 79, 83, 87, 91, 95, 99, 103, 107, 111, 115, 119, 123, 127, 131, 135, 139, 143, 147, 151, 155, 159, 163, 167, 171, 175, 179, 183, 187, 191, 195, 199, 203, 207, 211, 215, 219, 223, 227,231 , 235, 239, 243, 247, 251, 255, 259, 263, 267, 271, 275, 279, 283, 287, 291, 295, 299, 303,307, 311, 315, 319, 323, 327, 331, 335, 339, 343, 347, 351, 355, 359, 363, 367, 371, 375, 379,383, 387, 391, 395, 399, 403, 407, 411, 415, 419 and 423.

18. The method of any preceding claim, wherein the cell is a mammalian cell, preferably a human cell.

19. The method of any preceding claim, wherein the exogenous sequence encodes a protein, preferably a therapeutic protein.

20. The method of any preceding claim, wherein the RNA comprises pseudouridine-modified nucleotides.

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