Engineered RNA polymerase variants

Abstract

The present disclosure relates to engineered RNA polymerase polypeptides and compositions thereof, and polynucleotides encoding the engineered RNA polymerase polypeptides. The present disclosure also provides methods of producing RNA using the engineered RNA polymerase polypeptides or compositions thereof.

Description

[0001] Cross-references to related applications

[0002] This application claims the benefit of U.S. Provisional Application No. 63 / 502,017, filed May 12, 2023, which is incorporated herein by reference.

[0003] References to sequence lists, tables, or computer programs The sequence list submitted at the same time with the filename CX9-243WO1_ST26.xml was created on May 10, 2024, and has a file size of 2,511,974 bytes. It is part of this specification and is incorporated herein by reference. Technical Field

[0004] This disclosure relates to engineered RNA polymerase variants, compositions thereof, and methods of using engineered RNA polymerase variants. Background Technology

[0005] RNA polymerases transcribe DNA (and in some cases RNA) into RNA transcripts. These enzymes represent the main mechanisms driving transcription. RNA polymerases have been isolated and purified to a degree sufficient for in vitro RNA production. In vitro transcription allows the synthesis of RNA molecules of any sequence, ranging in size from short oligonucleotides to several thousand bases, guided by a polynucleotide template. Typically, in vitro transcription involves engineering a template, including a promoter sequence upstream of the sequence of interest, followed by transcription using a suitable RNA polymerase. RNA transcripts can be further modified by capping, splicing, and / or adding poly-A tails. Some modifications (e.g., capping and adding poly-A tails) can occur as part of the transcription reaction, for example, by co-transcriptional capping with RNA polymerase and by including a suitable polydT sequence in the polynucleotide template. In other cases, capping and adding poly-A tails can be performed post-transcriptionally, for example, by using RNA capping enzymes (e.g., Forstovirus or vaccinia virus capping enzymes) and poly(A) polymerases (e.g., E. coli poly(A) polymerase) for poly(A) tailing. In vitro generated transcripts can be used for analytical techniques (e.g., hybridization analysis), structural studies (e.g., NMR and X-ray crystallography), biochemical and genetic studies (e.g., as antisense reagents), as functional molecules (e.g., ribozymes and aptamers), and as therapeutic agents.

[0006] RNA has become a focal point for therapeutic applications, including mRNA-based vaccines, cancer immunotherapies, genome engineering (e.g., CRISPR), and enzyme replacement therapy. For these applications, it is crucial to utilize stable RNA polymerases, enabling the production of RNA in high yields and with high fidelity, while minimizing unwanted byproducts, particularly incomplete transcripts and double-stranded RNA. Summary of the Invention

[0007] This disclosure provides engineered RNA polymerase peptides and compositions thereof, as well as polynucleotides encoding the engineered RNA polymerase peptides, wherein the engineered RNA polymerase is engineered to have improved properties, such as increased activity, increased thermostability, increased persistence, and increased yield or integrity of RNA products. This disclosure also provides methods for using engineered RNA polymerase peptides and compositions thereof for nucleic acid synthesis and other purposes.

[0008] In one aspect, this disclosure provides an engineered RNA polymerase or a functional fragment thereof, said engineered RNA polymerase or functional fragment comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity relative to the reference sequence corresponding to SEQ ID NO: 2, 4, 32, 44, 156, 234, 238, 386, 394, 588, 604, 686, 692, 712 or 800. The reference sequence of residues 8 to 890 of SEQ ID NO: 2, 4, 32, 44, 156, 234, 238, 386, 394, 588, 604, 686, 692, 712 or 800, or the reference sequence relative to the reference sequence corresponding to SEQ ID NO: 2, 4, 32, 44, 156, 234, 238, 386, 394, 588, 604, 686, 692, 712 or 800, wherein the amino acid sequence comprises one or more substitutions.

[0009] In some embodiments, the engineered RNA polymerase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 98%, 99%, or higher sequence identity with the reference sequence corresponding to SEQ ID NO: 2, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 2 or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0010] In some embodiments, the engineered RNA polymerase comprises a reference sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity relative to the reference sequence corresponding to SEQ ID NO: 4, 32, 44, 156, 234, 238, 386, 394, 588, 604, 686, 692, 712, or 800, or relative to the reference sequence corresponding to SEQ ID NO: 2, wherein the reference sequence has at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity relative to the reference sequence corresponding to SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2, 32, 44, 156, 234, 238, 386, 394, 588, 604, 686, 692, 712, or 800, or relative to the reference sequence corresponding to SEQ ID NO: 2, 32, 44, 156, 234, 238, 386, 394, 588, 604, 686, 692, 712 The reference sequence of 2, wherein the amino acid sequence contains one or more substitutions.

[0011] In some embodiments, the engineered RNA polymerase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with the even-numbered SEQ ID NO: 4-828, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 2 or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0012] In some implementations, the amino acid sequence of the engineered RNA polymerase is at amino acid positions 14, 15, 17, 19, 21, 32, 33, 38, 45, 52, 54, 55, 56, 56, 57, 60, 61, 62, 63, 67, 69, 72, 75, 78, 81, 82, 83, 84, 89, 93, 95, 100, 105, 111, 115, 122, 124, 127, 131, 133, 135. 137, 138, 139, 140, 141, 143, 150, 160, 161, 162, 164, 166, 168, 169, 170, 171, 172, 173, 179, 185, 187, 193, 196, 207, 212, 217, 218, 221, 227, 237, 239, 240, 245, 246, 250, 253, 254, 257, 261, 272, 274 275, 311, 336, 340, 342, 351, 357, 367, 375, 379, 382, ​​388, 391, 392, 394, 397, 401, 402, 403, 404, 419, 424, 437, 440, 494, 510, 514, 517, 527, 529, 530, 531, 533, 536, 589, 594, 598, 607, 609, 615, 616 The amino acid positions 617, 625, 629, 648, 650, 655, 659, 660, 663, 664, 666, 670, 672, 690, 693, 696, 718, 720, 721, 749, 759, 763, 773, 775, 779, 782, 782, 810, 854, 857, 867, 875, or 878, or combinations thereof, contain at least one substitution, wherein the amino acid position is relative to the reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 2 or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0013] In some embodiments, the amino acid sequence of the engineered RNA polymerase contains at least one substitution at the following amino acid positions: 32, 38, 56, 138, 150, 170, 171, 173, 246, 311, 336, 340, 357, 388, 394, 401, 404, 437, 514, 536, 609, 629, 659, 660, 664, 779, or 782 or combinations thereof, wherein said amino acid position is relative to the reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 2 or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0014] In some embodiments, the amino acid sequence of the engineered RNA polymerase contains at least one substitution or set of substitutions at the following amino acid positions: 401 / 404, 336, 138, 401 / 403 / 404, 336 / 510, 388, 340, 401 / 402 / 404, or 351 / 357, wherein the amino acid position is relative to the reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 2 or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0015] In some embodiments, the amino acid sequence of the engineered RNA polymerase comprises at least one substitution as described in Tables 5.1, 6.1, 7.1, 8.1, 9.1, 10.1, 11.1, 12.1, 13.1, 14.1, 15.1, 16.1, 17.1, 18.1, 19.1, 23.1, and 24.1, wherein the amino acid position is relative to the reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 2 or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0016] In some embodiments, the amino acid sequence of the engineered RNA polymerase comprises at least one substitution or set of substitutions of the engineered RNA polymerases described in Tables 5.1, 6.1, 7.1, 8.1, 9.1, 10.1, 11.1, 12.1, 13.1, 14.1, 15.1, 16.1, 17.1, 18.1, 19.1, 23.1, and 24.1, wherein the amino acid position is relative to the reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 2 or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0017] In some embodiments, the engineered RNA polymerase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 96%, 97%, 98%, 99% or higher sequence identity with a reference sequence comprising at least one substitution or set of substitutions as described in Tables 5.1, 6.1, 7.1, 8.1, 9.1, 12.1, 23.1, and 24.1, wherein the amino acid position is relative to the reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 2 or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0018] In some embodiments, the engineered RNA polymerase comprises a reference sequence of residues 8 to 890 corresponding to SEQ ID NO: 4, 32, 44, 156, 234, 238, 386, 394, 588, 604, 686, 692, 712, or 800, or an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher sequence identity with the reference sequence corresponding to SEQ ID NO: 4, 32, 44, 156, 234, 238, 386, 394, 588, 604, 686, 692, 712, or 800.

[0019] In some embodiments, the engineered RNA polymerase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 96%, 97%, 98%, 99% or higher sequence identity with the reference sequence corresponding to the even-numbered SEQ ID NOs of ...

[0020] In some embodiments, the engineered RNA polymerase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 96%, 97%, 98%, 99% or higher sequence identity with the reference sequence corresponding to SEQ ID NO: 4, 32, 44, 156, 234, 238, 386, 394, 588, 604, 686, 692, 712 or 800, wherein relative to the sequence corresponding to SEQ ID NO: The reference sequence of residues 8 to 890 of 4, 32, 44, 156, 234, 238, 386, 394, 588, 604, 686, 692, 712, or 800, or relative to the reference sequence corresponding to SEQ ID NO: 4, 32, 44, 156, 234, 238, 386, 394, 588, 604, 686, 692, 712, or 800, wherein the amino acid sequence comprises one or more substitutions.

[0021] In some embodiments, the engineered RNA polymerase comprises a reference sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with respect to the reference sequence corresponding to SEQ ID NO: 4, 32, 44, 156, 234, 238, 386, 394, 588, 604, 686, 692, 712 or 800 of the reference sequence, wherein the reference sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher of the sequence corresponding to SEQ ID NO: 4, 32, 44, 156, 234, 238, 386, 394, 588, 604, 686, 692, 712 or 800 of the reference sequence, or the reference ... A reference sequence of 4, 32, 44, 156, 234, 238, 386, 394, 588, 604, 686, 692, 712 or 800, wherein the amino acid sequence contains one or more substitutions.

[0022] In some embodiments, the amino acid sequence of the engineered RNA polymerase contains at least one substitution at the following amino acid positions: 13, 14, 15, 17, 19, 21, 32, 33, 38, 45, 52, 54, 55, 56, 56, 57, 60, 61, 62, 63, 67, 69, 72, 75, 78, 81, 82, 83, 84, 89, 93, 95, 100, 105, 111, 115, 122, 124, 12 7, 131, 133, 135, 137, 138, 139, 140, 141, 143, 150, 160, 161, 162, 164, 166, 168, 169, 170, 171, 172, 173, 179, 185, 187, 193, 196, 207, 212, 217, 218, 221, 227, 237, 239, 240, 245, 246, 250, 253, 254, 257, 2 61, 272, 274, 275, 311, 336, 340, 342, 351, 357, 367, 375, 379, 382, ​​388, 391, 392, 394, 397, 401, 402, 403, 404, 419, 424, 437, 440, 494, 510, 514, 517, 527, 529, 530, 531, 533, 536, 589, 594, 598, 607, 609 615, 616, 617, 625, 629, 648, 650, 655, 659, 660, 663, 664, 666, 670, 672, 690, 693, 696, 718, 720, 721, 749, 759, 763, 773, 775, 779, 782, 782, 810, 854, 857, 867, 875 or 878 or combinations thereof, wherein the amino acid position is relative to the position corresponding to SEQ. Reference sequences of residues 8 to 890 of SEQ ID NO: 4, 32, 44, 156, 234, 238, 386, 394, 588, 604, 686, 692, 712, or 800, or reference sequences corresponding to SEQ ID NO: 4, 32, 44, 156, 234, 238, 386, 394, 588, 604, 686, 692, 712, or 800.

[0023] In some embodiments, the amino acid sequence of the engineered RNA polymerase contains at least one substitution at the following amino acid positions: 32, 38, 56, 138, 150, 170, 171, 173, 246, 311, 336, 340, 357, 388, 394, 401, 404, 437, 514, 536, 609, 629, 659, 660, 664, 779, or 782, or combinations thereof, wherein said amino acid position is relative to the reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 4, 32, 44, 156, 234, 238, 386, 394, 588, 604, 686, 692, 712, or 800, or relative to the reference sequence corresponding to .... Reference sequences of 4, 32, 44, 156, 234, 238, 386, 394, 588, 604, 686, 692, 712 or 800.

[0024] In some embodiments, the engineered RNA polymerase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with the reference sequence corresponding to SEQ ID NO: 4, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 4 or relative to the reference sequence corresponding to SEQ ID NO: 4.

[0025] In some embodiments, the amino acid sequence of the engineered RNA polymerase contains at least one set of substitutions at the following amino acid positions: 311 / 388 / 404 / 440, 138 / 340, 138 / 311 / 336 / 340 / 388, 311 / 388 / 440, 138 / 311 / 340 / 388 / 404 / 440, 138 / 336 / 342, or 351 / 388, wherein the amino acid position is relative to the reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 4 or relative to the reference sequence corresponding to SEQ ID NO: 4.

[0026] In some embodiments, the engineered RNA polymerase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with the reference sequence corresponding to SEQ ID NO: 32, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 32 or relative to the reference sequence corresponding to SEQ ID NO: 32.

[0027] In some embodiments, the amino acid sequence of the engineered RNA polymerase contains at least one substitution or set of substitutions at the following amino acid positions: 514, 38 / 514, 38 / 394 / 514, 38 / 394 / 514 / 664, 394 / 514 / 875, 394 / 514, 38 / 124 / 394 / 514, 171 / 878, 170, 173, 172, 170 / 629, 171, 168, 166, 166 / 272 / 274 / 275, 169, 207, 168 / 217 / 218, 171 / 530, 382, ​​773, 670, 379, 392, 419, 57, 164, 810, 659, 54, 172 / 173 / 514 / 659 / 660, 170 / 171 / 172 / 514 / 659, 173 / 514 / 659 / 660, 173 / 514 / 659 / 810, 170 / 340 / 514 / 659 / 660 / 810, 170 / 171 / 173 / 336 / 340 / 514 / 659 / 660, 57 / 171 / 173 / 514 / 659 / 660 / 810, 170 / 173 / 514 / 659 / 660 / 810, 57 / 171 / 172 / 336 / 340 / 514 / 659, 173 / 514, 171 / 172 / 173 / 514 / 659 / 660, 57 / 170 / 17 1 / 173 / 514 / 659 / 660 / 810, 57 / 173 / 514, 172 / 173 / 514 / 857, 57 / 173 / 340 / 514, 170 / 171 / 173 / 336 / 514, 171 / 514, 57 / 170 / 173 / 336 / 514, 170 / 336, 173 / 514 / 659, 57 / 514 or 173 / 810, wherein the amino acid position is relative to the reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 32 or relative to the reference sequence corresponding to SEQ ID NO: 32.

[0028] In some embodiments, the engineered RNA polymerase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with the reference sequence corresponding to SEQ ID NO: 44, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 44 or relative to the reference sequence corresponding to SEQ ID NO: 44.

[0029] In some embodiments, the amino acid sequence of the engineered RNA polymerase contains at least one set of substitutions at amino acid positions 336, 143 / 336 / 340 / 782, or 143 / 336 / 340 / 357 / 514, wherein the amino acid positions are relative to a reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 44 or relative to a reference sequence corresponding to SEQ ID NO: 44.

[0030] In some embodiments, the engineered RNA polymerase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with the reference sequence corresponding to SEQ ID NO: 156, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 156 or relative to the reference sequence corresponding to SEQ ID NO: 156.

[0031] In some embodiments, the amino acid sequence of the engineered RNA polymerase contains at least one substitution or set of substitutions at the following amino acid positions: 207, 419, 207 / 336 / 340, 166 / 169, 207 / 670, 166 / 336 / 340, 207 / 419 / 670, 207 / 336 / 340 / 382 / 419 / 670, 207 / 382, 670 336 / 670, 340 / 670, 382, ​​127 / 141 / 207 / 336 / 382 / 670, 207 / 336 / 382 / 419 / 670, 419 / 670, 166 / 168 / 207 / 336 / 340 / 419 / 670, 166 / 340 or 207 / 336 / 340 / 670, wherein the amino acid position is relative to the reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 156 or relative to the reference sequence corresponding to SEQ ID NO: 156.

[0032] In some embodiments, the amino acid sequence of the engineered RNA polymerase contains at least one substitution or set of substitutions at the following amino acid positions: 38 / 437 / 779, 38 / 437, 38, 38 / 207 / 437 / 779, 517 / 779, 38 / 437 / 527 / 664, 38 / 207 / 379 / 437, 664 / 670 / 779, 38 / 779, 38 / 437 / 517, 437 / 779, 38 / 437 / 607, 38 / 379 / 779, or 517 / 670 / 779, wherein the amino acid position is relative to the reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 156 or relative to the reference sequence corresponding to SEQ ID NO: 156.

[0033] In some embodiments, the amino acid sequence of the engineered RNA polymerase contains at least one set of substitutions at amino acid positions 357 / 394 / 664 or 138 / 311 / 336 / 340 / 357 / 388 / 394 / 404 / 664, wherein the amino acid positions are relative to a reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 156 or relative to a reference sequence corresponding to SEQ ID NO: 156.

[0034] In some embodiments, the engineered RNA polymerase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with the reference sequence corresponding to SEQ ID NO: 234, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 234 or relative to the reference sequence corresponding to SEQ ID NO: 234.

[0035] In some embodiments, the amino acid sequence of the engineered RNA polymerase contains at least one substitution or set of substitutions at the following amino acid positions: 720, 196, 609, 246, 185, 594, 212, 494, 375, 721, 72, 867, 160, 718, 187, 170 / 173, 615, 137 / 138, 854, 131, 38 / 185 / 187 / 437, 185 / 187, 437 / 609, 38 / 609, 38 / 185 / 187 / 437 / 609, 437 / 609 / 720, 185 / 187 / 609, 185 / 187 / 437, 437 / 721, 609 / 720, 185 / 187 / 246 or 185 / 187 / 437 / 609 / 720, wherein the amino acid position is relative to the reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 234 or relative to the reference sequence corresponding to SEQ ID NO: 234.

[0036] In some embodiments, the engineered RNA polymerase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with the reference sequence corresponding to SEQ ID NO: 386, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 386 or relative to the reference sequence corresponding to SEQ ID NO: 386.

[0037] In some embodiments, the amino acid sequence of the engineered RNA polymerase contains at least one substitution or set of substitutions at the following amino acid positions: 170 / 609, 38 / 185 / 187 / 609, 38 / 185 / 187, 38 / 170 / 173, 38 / 173 / 185 / 187 / 720, 170 / 185 / 187 / 609, 609, 170 / 185 / 187 / 609 / 721, 38 / 609, 38 / 170 / 246 / 609, 185 / 187 / 609 / 718, 38 / 185 / 187 / 609 / 718 / 720, 38 / 170 / 246 / 609 / 718, 720, 246 / 609, 38 / 185 / 187 / 246 / 720, 38 / 609 / 721, 38 / 721, 38 / 720 / 721, 38 / 170 / 609 / 718 / 720, 38 / 718, 38 / 185 / 187 / 609 / 718 / 721, 529 / 609, 38 / 609 / 718, 185 / 187 / 609 / 718 / 720, 185 / 187 / 609 / 718 / 721, 38 / 170 / 718 / 720 / 721, 170 / 185 / 187 / 7 18 / 720, 38 / 173 / 185 / 187 / 246 / 609 / 720, 609 / 718 / 720, 170 / 185 / 187 / 718 / 720 / 721, 38 / 170 / 173 / 246 / 609 / 718, 246 / 721, 38 / 718 / 720 or 38 / 609 / 718 / 720, wherein the amino acid position is relative to the reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 386 or relative to the reference sequence corresponding to SEQ ID NO: 386.

[0038] In some embodiments, the engineered RNA polymerase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with the reference sequence corresponding to SEQ ID NO: 238, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 238 or relative to the reference sequence corresponding to SEQ ID NO: 238.

[0039] In some embodiments, the amino acid sequence of the engineered RNA polymerase contains at least one substitution or set of substitutions at the following amino acid positions: 143, 105, 775 / 779, 135, 100, 246, 779 / 782, 749, 237, 162, 83, 218, 227, 115, 274, 81, 122, 61, 32, 139, 75, 239, 257, 240, 69 / 161, 253, 14, 221, 84, 133 / 150, 60, 275, 89, 15, 254, 625, 14 / 67, 21, 93, 250, 217, 19, 95, 759, 62, or 140, wherein the amino acid position is relative to the reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 238 or relative to the reference sequence corresponding to SEQ ID NO: 238. The reference sequence for 238.

[0040] In some embodiments, the engineered RNA polymerase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with the reference sequence corresponding to SEQ ID NO: 394, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 394 or relative to the reference sequence corresponding to SEQ ID NO: 394.

[0041] In some embodiments, the amino acid sequence of the engineered RNA polymerase contains at least one substitution or set of substitutions at the following amino acid positions: 609 / 779, 246 / 609, 246, 609 / 775 / 779, 105, 143 / 609 / 775, 105 / 135 / 609 / 775, or 111 / 609, 609, wherein the amino acid position is relative to the reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 394 or relative to the reference sequence corresponding to SEQ ID NO: 394.

[0042] In some embodiments, the engineered RNA polymerase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with the reference sequence corresponding to SEQ ID NO: 588, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 588 or relative to the reference sequence corresponding to SEQ ID NO: 588.

[0043] In some embodiments, the amino acid sequence of the engineered RNA polymerase contains at least one substitution or set of substitutions at the following amino acid positions: 32, 531 / 533, 115, 83, 32 / 60 / 61, 55, 589, 357, 617, 172, 598, 56 / 193, 56, 536, 52, 17, 261, 78, 629, 63, 82, 207, 666, 150, 45, 135, 13, 33, or 245, wherein the amino acid position is relative to the reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 588 or relative to the reference sequence corresponding to SEQ ID NO: 588.

[0044] In some embodiments, the engineered RNA polymerase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with the reference sequence corresponding to SEQ ID NO: 604, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 604 or relative to the reference sequence corresponding to SEQ ID NO: 604.

[0045] In some embodiments, the amino acid sequence of the engineered RNA polymerase contains at least one substitution or set of substitutions at the following amino acid positions: 207 / 245 / 629, 17 / 56 / 150 / 598, 56 / 629, 536, 185, 56 / 150, 17 / 56 / 150 / 207 / 245 / 598 / 617 / 629, 56 / 150 / 536 / 598, 56 / 536, 185 / 666, 150, or 143 / 150 / 246 / 782, wherein the amino acid position is relative to the reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 604 or relative to the reference sequence corresponding to SEQ ID NO: 604.

[0046] In some embodiments, the engineered RNA polymerase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with the reference sequence corresponding to SEQ ID NO: 686, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 686 or relative to the reference sequence corresponding to SEQ ID NO: 686.

[0047] In some embodiments, the amino acid sequence of the engineered RNA polymerase contains at least one substitution or set of substitutions at the following amino acid positions: 143, 246 / 424 / 648 / 782, 246 / 659 / 782, 56 / 150 / 629 / 660 / 782, 721 / 782, 782, 196 / 659 / 782, or 720 / 721 / 782, wherein the amino acid position is relative to the reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 686 or relative to the reference sequence corresponding to SEQ ID NO: 686.

[0048] In some embodiments, the engineered RNA polymerase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with the reference sequence corresponding to SEQ ID NO: 692, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 692 or relative to the reference sequence corresponding to SEQ ID NO: 692.

[0049] In some embodiments, the amino acid sequence of the engineered RNA polymerase contains at least one substitution or set of substitutions at the following amino acid positions: 143 / 659 / 782, 166 / 171 / 246 / 311, 143 / 721, 782, or 143 / 171 / 659 / 782, wherein the amino acid position is relative to a reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 692 or relative to a reference sequence corresponding to SEQ ID NO: 692.

[0050] In some embodiments, the engineered RNA polymerase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with the reference sequence corresponding to SEQ ID NO: 712, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 712 or relative to the reference sequence corresponding to SEQ ID NO: 712.

[0051] In some embodiments, the amino acid sequence of the engineered RNA polymerase contains at least one substitution or set of substitutions at the following amino acid positions: 367, 382, ​​670, 672, 357, 388, 693, 397, 375, 137 / 382 / 782, 655, 137 / 382 / 392, 391, 137 / 655 / 721 / 763, 696, 663, 137 / 382, 137 / 382 / 392 / 650 / 655 / 782, 137 / 382 / 629 / 660 / 672, 616, 690, 179, 137, 357 / 382 / 670, 388 / 670 / 672, 357 / 375, 357 / 672, 629, or 655 / 670, wherein the amino acid position is relative to the corresponding SEQ ID. Reference sequence of residues 8 to 890 of NO: 712 or relative to the reference sequence corresponding to SEQ ID NO: 712.

[0052] In some embodiments, the engineered RNA polymerase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with the reference sequence corresponding to SEQ ID NO: 800, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 800 or relative to the reference sequence corresponding to SEQ ID NO: 800.

[0053] In some embodiments, the amino acid sequence of the engineered RNA polymerase contains at least one substitution or set of substitutions at the following amino acid positions: 137, 56 / 311 / 382, 56 / 137 / 143 / 721, 137 / 392, 56 / 137 / 392, 56 / 137 / 143 / 404 / 720, 56 / 143 / 246, 56 / 392, 392 / 404, 392, 56 / 137, or 56 / 196 / 392, wherein the amino acid position is relative to the reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 800 or relative to the reference sequence corresponding to SEQ ID NO: 800.

[0054] In some embodiments, the amino acid sequence of the engineered RNA polymerase comprises at least one substitution as described in Tables 5.1, 6.1, 7.1, 8.1, 9.1, 10.1, 11.1, 12.1, 13.1, 14.1, 15.1, 16.1, 17.1, 18.1, 19.1, 23.1, and 24.1, wherein the amino acid position is relative to a reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 2, 4, 32, 44, 156, 234, 238, 386, 394, 588, 604, 686, 692, 712, or 800, or relative to a reference sequence corresponding to SEQ ID NO: 2, 4, 32, 44, 156, 234, 238, 386, 394, 588, 604, 686, 692, 712, or 800.

[0055] In some embodiments, the amino acid sequence of the engineered RNA polymerase comprises at least one substitution or set of substitutions of the engineered RNA polymerases described in Tables 5.1, 6.1, 7.1, 8.1, 9.1, 10.1, 11.1, 12.1, 13.1, 14.1, 15.1, 16.1, 17.1, 18.1, 19.1, 23.1, and 24.1, wherein the amino acid position is relative to the reference sequence of residues 8 to 890 corresponding to SEQ ID NO: 2, 4, 32, 44, 156, 234, 238, 386, 394, 588, 604, 686, 692, 712, or 800, or relative to the sequence corresponding to SEQ ID NO: Reference sequences of 2, 4, 32, 44, 156, 234, 238, 386, 394, 588, 604, 686, 692, 712 or 800.

[0056] In some embodiments, the engineered RNA polymerase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 96%, 97%, 98%, 99% or higher sequence identity with a reference sequence comprising substitutions or sets of substitutions as described in Tables 5.1, 6.1, 7.1, 8.1, 9.1, 13.1, 23.1, and 24.1, wherein the amino acid position is relative to the position corresponding to SEQ ID NO: Reference sequences of residues 8 to 890 of SEQ ID NO: 2, 4, 32, 44, 156, 234, 238, 386, 394, 588, 604, 686, 692, 712, or 800, or reference sequences relative to SEQ ID NO: 2, 4, 32, 44, 156, 234, 238, 386, 394, 588, 604, 686, 692, 712, or 800.

[0057] In some embodiments, the engineered RNA polymerase comprises an amino acid sequence of residues 8 to 890 of SEQ ID NO., which includes even-numbered residues of SEQ ID NO., which includes SEQ ID NO., which includes even-numbered residues of SEQ ID NO., which optionally has 1, 2, 3, 4, 5, 6, 7, 8, 9 or up to 10 substitutions.

[0058] In some embodiments, the engineered RNA polymerase comprises residues 8 to 890 of SEQ ID NO: 4, 32, 44, 156, 234, 238, 386, 394, 588, 604, 686, 692, 712, or 800, or an amino acid sequence comprising SEQ ID NO: 4, 32, 44, 156, 234, 238, 386, 394, 588, 604, 686, 692, 712, or 800, optionally wherein the amino acid sequence has 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10 substitutions.

[0059] In some embodiments, the engineered RNA polymerase is characterized by at least one improved property compared to a reference RNA polymerase. In some embodiments, the improved property is selected from, for example, i) increased activity, ii) increased thermostability, iii) increased persistence, or iv) increased RNA product integrity, or any combination of i), ii), iii), and iv), wherein the reference RNA polymerase has a sequence of residues 8 to 890 corresponding to SEQ ID NO: 2, 4, 32, 44, 156, 234, 238, 386, 394, 588, 604, 686, 692, 712, or 800, or a sequence corresponding to SEQ ID NO: 2, 4, 32, 44, 156, 234, 238, 386, 394, 588, 604, 686, 692, 712, or 800. In some embodiments, the reference RNA polymerase has a sequence corresponding to residues 8 to 890 of SEQ ID NO: 2, or a sequence corresponding to SEQ ID NO: 2.

[0060] In some further embodiments, the engineered RNA polymerase is purified. In some embodiments, the engineered RNA polymerase is provided in solution, as a lyophilized product, or immobilized on the surface of a substrate or support medium such as a solid substrate, membrane, or particle.

[0061] On the other hand, this disclosure provides recombinant polynucleotides encoding the engineered RNA polymerase disclosed herein.

[0062] In some embodiments, the recombinant polynucleotide comprises a reference polynucleotide sequence of nucleotide residues 21 to 2670 of SEQ ID NO. corresponding to odd-numbered SEQ ID NO: 3-827, or a polynucleotide sequence having at least 70%, 75%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with SEQ ID NO. corresponding to odd-numbered SEQ ID NO: 3-827, wherein the recombinant polynucleotide encodes an RNA polymerase.

[0063] In some embodiments, the recombinant polynucleotide comprises a reference polynucleotide sequence having at least 70%, 75%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with nucleotide residues 21 to 2670 corresponding to SEQ ID NO: 3, 31, 43, 155, 233, 237, 381, 393, 587, 603, 685, 691, 711 or 799, wherein the recombinant polynucleotide encodes an RNA polymerase.

[0064] In some embodiments, the polynucleotide sequence encoding the recombinant polynucleotide of the engineered RNA polymerase is codon-optimized. In some embodiments, the polynucleotide sequence is codon-optimized for expression in bacterial, fungal, insect, or mammalian cells.

[0065] In some embodiments, the recombinant polynucleotide comprises nucleotide residues 21 to 2670 of SEQ ID NO. containing odd numbers of SEQ ID NO: 3-827 or a polynucleotide sequence containing odd numbers of SEQ ID NO: 3-827.

[0066] In another aspect, this disclosure provides an expression vector comprising at least one recombinant polynucleotide encoding the engineered RNA polymerase described herein. In some embodiments, the recombinant polynucleotide in the expression vector is operatively linked to a control sequence. In some embodiments, the control sequence comprises a promoter, particularly a heterologous promoter.

[0067] In another aspect, this disclosure also provides host cells transformed with at least one expression vector provided herein. In some embodiments, the host cell is a prokaryotic or eukaryotic cell. In some embodiments, the host cell is a bacterial cell, fungal cell, insect cell, or mammalian cell. In some preferred embodiments, the host cell is a bacterial cell, such as Escherichia coli or Bacillus subtilis.

[0068] In another aspect, this disclosure provides a method for generating an engineered RNA polymerase polypeptide, the method comprising culturing the host cells described herein under suitable culture conditions to generate at least one engineered RNA polymerase. In some embodiments, the method further includes recovering or isolating the expressed engineered RNA polymerase from the culture and / or host cells. In some embodiments, the method further includes purifying the engineered RNA polymerase.

[0069] In another embodiment, this disclosure provides a composition comprising at least one engineered RNA polymerase disclosed herein. In some embodiments, the composition further comprises at least a buffer. In some embodiments, the composition further comprises a reducing agent, such as dithiothreitol or mercaptoethanol. In some embodiments, the composition further comprises one or more nucleoside triphosphate (NTP) substrates, particularly rNTP substrates. In some embodiments, the composition further comprises a cap analog. In some embodiments, the composition further comprises a target DNA template. In some embodiments, the target DNA template comprises a promoter recognized by the engineered RNA polymerase. In some embodiments, the composition further comprises a pyrophosphatase and / or an RNase inhibitor. In some embodiments, the composition further comprises an additive, such as a molecular crowding agent.

[0070] In another aspect, this disclosure provides a method for producing RNA, the method comprising contacting a target DNA template with an engineered RNA polymerase described herein in the presence of one or more nucleoside triphosphates, under conditions suitable for transcription of a target DNA template. In some embodiments, the method further includes providing a cap analog. In some embodiments, the method further includes contacting a pyrophosphatase and / or an RNase inhibitor with the engineered RNA polymerase in the reaction. In some embodiments, suitable conditions include temperatures from about 25°C to about 40°C. In some embodiments, suitable conditions include additives, such as molecular congestors.

[0071] On the other hand, this disclosure also provides a kit comprising at least one engineered RNA polymerase disclosed herein. In some embodiments, the kit further includes a buffer, one or more rNTPs, and Mg... +2 One or more of a reducing agent and a molecular crowding agent. In some embodiments, the kit also includes a suitable DNA template. Detailed Implementation

[0072] This disclosure provides engineered RNA polymerase peptides and compositions thereof, wherein the engineered RNA polymerase exhibits one or more improved properties, particularly including increased activity, improved stability, improved thermostability, and improved product integrity. This disclosure also provides recombinant polynucleotides encoding the engineered RNA polymerase peptides and methods for producing RNA using the engineered RNA polymerase. In some embodiments, the expressed RNA-encoding peptide may be a non-coding RNA (ncRNA), such as shRNA, miRNA, or guide RNA.

[0073] Abbreviations and definitions

[0074] For the purposes of this disclosure, unless otherwise expressly defined, the technical and scientific terms used in the description herein will have the meanings commonly understood by one of ordinary skill in the art.

[0075] It should be understood that the present invention is not limited to the specific methods, schemes, and reagents described, as these may vary depending on the context in which they are used by those skilled in the art. Therefore, the terms defined below are described more fully by reference to the application as a whole.

[0076] As used herein, the singular “a / kind (a, an)” and “the / said” include plural referents unless the context clearly indicates otherwise.

[0077] As used herein, the term “includes / contains” and its cognates are used in their inclusive sense (i.e., equivalent to the term “includes” and its corresponding cognates).

[0078] It should also be understood that, where the description of the implementation scheme uses the term "comprising" and its cognates, the implementation scheme may also be described using the language "substantially composed of" or "consisting of".

[0079] Furthermore, the numerical range includes the number that defines the range. Therefore, each numerical range disclosed herein is intended to encompass every narrower numerical range falling within such a wider numerical range, as if such a narrower numerical range were explicitly stated herein. Each maximum (or minimum) numerical limit disclosed herein is also intended to include every lower (or higher) numerical limit, as if such lower (or higher) numerical limits were explicitly stated herein.

[0080] As used herein, the term “about” refers to the acceptable error for a particular value. In some cases, “about” means within 0.05%, 0.5%, 1.0%, or 2.0% of the given value range. In other cases, “about” means within 1, 2, 3, or 4 standard deviations of the given value.

[0081] Furthermore, the headings provided herein are not intended to limit the various aspects or embodiments of the invention, which can be obtained by referring to the entire application. Therefore, the terms defined below are defined more fully by reference to the entire application.

[0082] "ATCC" refers to the American Type Culture Collection, whose biobank collection includes genes and strains.

[0083] "NCBI" refers to the National Center for Biotechnology Information and the sequence database it provides.

[0084] The terms “protein,” “polypeptide,” and “peptide” are used interchangeably to refer to polymers of at least two amino acids covalently linked by amide bonds, regardless of length or post-translational modifications (e.g., glycosylation or phosphorylation).

[0085] Amino acids are referred to in this document by their commonly known three-letter symbols or by the single-letter symbols recommended by the IUPAC-IUB Committee on Biochemistry Nomenclature. The abbreviations used for gene-encoded amino acids are conventional and are as follows: alanine (Ala or A), arginine (Arg or R), asparagine (Asn or N), aspartic acid (Asp or D), cysteine ​​(Cys or C), glutamic acid (Glu or E), glycine (Gly or G), glutamine (Gln or Q), histidine (His or H), isoleucine (Ile or I), leucine (Leu or L), lysine (Lys or K), methionine (Met or M), phenylalanine (Phe or F), proline (Pro or P), serine (Ser or S), threonine (Thr or T), tryptophan (Trp or W), tyrosine (Tyr or Y), and valine (Val or V). When using three-letter abbreviations, amino acids can be either L-configured or D-configured with respect to the α-carbon (Cα) unless specifically preceded by "L" or "D" or clearly indicated from the context of the abbreviation. For example, given that "Ala" represents alanine without specifying the α-carbon configuration, "D-Ala" and "L-Ala" represent D-alanine and L-alanine, respectively. When using single-letter abbreviations, uppercase letters indicate amino acids with the L-configuration with respect to the α-carbon, and lowercase letters indicate amino acids with the D-configuration with respect to the α-carbon. For example, "A" represents L-alanine, and "a" represents D-alanine. When a polypeptide sequence is presented as a string of single-letter or three-letter abbreviations (or mixtures thereof), the sequence is conventionally presented in the order of amino (N) to carboxyl (C).

[0086] "RNA polymerase" or "RNAP" refers to an enzyme that catalyzes RNA synthesis from the 5' to 3' direction using a polynucleotide as a template. In some embodiments, RNA polymerase uses a DNA template and is referred to as DNA-directed or DNA-dependent RNA polymerase. In some embodiments, RNA polymerase uses an RNA template and is referred to as RNA-directed or RNA-dependent RNA polymerase. In some embodiments, RNA polymerase is capable of synthesizing RNA using both DNA and RNA as templates. In some embodiments, the RNA product is referred to as an "RNA transcript."

[0087] As used herein, "cap" refers to a nucleoside whose 5' carbon atom is bonded to a triphosphate group, which in turn binds to the 5' carbon of the 5' nucleotide of the RNA transcript. In some embodiments, the cap nucleoside is guanine (G). In some embodiments, the nitrogen at the 7-position of guanine in the cap is methylated and denoted as m7G. In some embodiments, the cap is a dinucleotide cap, a trinucleotide cap, or a tetranucleotide cap. The terms "capped RNA," "5'-capped RNA," and "capped mRNA" refer to the RNA and mRNA that constitute the cap, respectively.

[0088] "Fusion protein," "chimeric protein," and "chimera" refer to hybrid proteins produced by the conjugation of two or more polynucleotides that originally encode separate proteins. In some embodiments, fusion proteins are produced using recombinant technologies (e.g., molecular biology techniques known in the art).

[0089] The terms “polynucleotide,” “nucleic acid,” or “oligonucleotide” are used herein to refer to polymers comprising at least two nucleotides, wherein the nucleotides are deoxyribonucleotides or ribonucleotides or a mixture of deoxyribonucleotides and ribonucleotides. In some embodiments, the abbreviations for gene-encoding nucleosides are conventional and follow the following: adenosine (A); guanosine (G); cytidine (C); thymidine (T); and uridine (U). Unless specifically described, the abbreviation for nucleoside can be ribonucleoside or 2'-deoxyribonucleoside. Nucleosides may be designated as ribonucleoside or 2'-deoxyribonucleoside on a single or collective basis. When a polynucleotide, nucleic acid, or oligonucleotide sequence is presented as a string of single-letter abbreviations, the sequence is presented in a 5' to 3' orientation according to general convention and does not indicate phosphate. The term “DNA” refers to deoxyribonucleic acid. The term “RNA” refers to ribonucleic acid. Polynucleotides or nucleic acids may be single-stranded or double-stranded, or may include both single-stranded and double-stranded regions.

[0090] "Double strand" and "ds" refer to a double-stranded nucleic acid (e.g., DNA or RNA) molecule composed of two single-stranded polynucleotides that are complementary in sequence (A paired with T or U, C paired with G), arranged in an antiparallel 5' to 3' orientation, and held together by hydrogen bonds between nucleobases (e.g., adenine [A], guanine [G], cytosine [C], thymine [T], uridine [U]).

[0091] When used in relation to cells, polynucleotides, or peptides, “engineered,” “recombinant,” “non-natural,” and “variant” refer to a material or a material corresponding to the natural or native form of that material that has been modified in a manner not present in or similar to that found in nature, but produced or derived from synthetic materials and / or manipulated using recombinant techniques.

[0092] "Wild-type" and "naturally occurring" refer to forms found in nature. For example, wild-type polypeptide or polynucleotide sequences are sequences that exist in organisms, can be isolated from natural sources, and have not been intentionally modified by humans.

[0093] "Coding sequence" refers to the part of a nucleic acid (e.g., a gene) that encodes the amino acid sequence of a protein.

[0094] "Sequence identity percentage (%)" refers to a comparison between polynucleotides and peptides, and is determined by comparing two optimally aligned sequences within a comparison window. For optimal alignment, the portion of the polynucleotide or peptide sequence in the comparison window may contain additions or deletions (i.e., vacancies) compared to the reference sequence. The percentage can be calculated by determining the number of positions in both sequences where the same nucleic acid base or amino acid residue appears to generate a matching position number, dividing the matching position number by the total number of positions in the comparison window, and multiplying the result by 100 to obtain the sequence identity percentage. Alternatively, the percentage can be calculated by determining the number of positions in both sequences where the same nucleic acid base or amino acid residue appears, or by aligning the nucleic acid base or amino acid residue with vacancies to obtain a matching position number, dividing the matching position number by the total number of positions in the comparison window, and multiplying the result by 100 to obtain the sequence identity percentage. Those skilled in the art will understand that many established algorithms exist for aligning two sequences. The optimal alignment of sequences for comparison can be performed as is known in the art, for example, by Smith and Waterman’s local homology algorithm (Smith and Waterman, Adv. Appl. Math., 1981, 2:482), by Needleman and Wunsch’s homology alignment algorithm (Needleman and Wunsch, J. Mol. Biol., 1970, 48:443), by Pearson and Lipman’s similarity search method (Pearson and Lipman, Proc. Natl. Acad. Sci. USA, 1988, 85:2444), by computerized implementations of these algorithms (e.g., GAP, BESTFIT, FASTA, and TFASTA in the GCG Wisconsin software package), or by visual inspection. Examples of algorithms suitable for determining sequence identity percentages and sequence similarity include, but are not limited to, the BLAST and BLAST2.0 algorithms (see, for example, Altschul et al., J. Mol. Biol., 1990, 215: 403-410; and Altschul et al., Nucleic Acids Res., 1977, 3389-3402). Software for performing BLAST analysis is publicly available from the website of the National Center for Biotechnology Information. The algorithm involves first identifying high-scoring sequence pairs (HSPs) by identifying short words of length “W” in the query sequence. These short words match or satisfy a positive threshold score “T” when compared to words of the same length in a database sequence. T is called the neighborhood word score threshold (see Altschul et al., ibid.). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them.The word hit then extends in both directions along each sequence, as long as the cumulative alignment score can increase. For nucleotide sequences, the cumulative score is calculated using parameters "M" (reward score for a pair of matching residues; always >0) and "N" (penalty score for mismatched residues; always <0). For amino acid sequences, a scoring matrix is ​​used to calculate the cumulative score. The extension of the word hit in each direction stops when: the cumulative alignment score decreases from its maximum realized value by an amount "X"; the cumulative score becomes zero or lower due to the accumulation of one or more negative score residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) defaults to a word length of 11 (W), an expected value of 10 (E), M=5, N=-4, and a comparison of two strands. For amino acid sequences, the BLASTP program defaults to a word length of 3 (W), an expected value of 10 (E), and a BLOSUM62 scoring matrix (see, for example, Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA, 1989, 89:10915). Exemplary determination of sequence alignment and sequence identity percentage can be performed using the BESTFIT or GAP programs in the GCG Wisconsin software package (Accelrys, Madison WI) with the provided default parameters.

[0095] A “reference sequence” is a defined sequence used as the basis for sequence comparison. A reference sequence can be a subset of a larger sequence, such as a segment of a full-length gene or polypeptide sequence. Typically, a reference sequence is at least 20 nucleotides or amino acid residues long, at least 25 residues long, at least 50 residues long, at least 100 residues long, or the full length of a nucleic acid or polypeptide. Since each of two polynucleotides or polypeptides can (1) contain sequences similar to the two sequences (i.e., a portion of the complete sequence) and (2) also contain sequences different from the two sequences, sequence comparisons between two (or more) polynucleotides or polypeptides are typically performed by comparing the sequences of the two polynucleotides or polypeptides on a “comparison window” to identify and compare local regions of sequence similarity. In some embodiments, a “reference sequence” can be based on a primary amino acid sequence, wherein the reference sequence is a sequence that may have one or more variations in the primary sequence. For example, the phrase “reference sequence corresponding to SEQ ID NO: 2 with glycine at the residue corresponding to X38” (or “reference sequence corresponding to SEQ ID NO: 2 with glycine at the residue corresponding to position 38”) refers to a reference sequence in which the corresponding residue (e.g., arginine, R) at position X38 in SEQ ID NO: 2 has been changed to glycine (G).

[0096] A “comparison window” refers to a conceptual segment of adjacent nucleotide positions or amino acid residues, where the sequence can be compared to a reference sequence. In some embodiments, the comparison window is at least 15 to 20 consecutive nucleotides or amino acids, and wherein, for optimal alignment of the two sequences, the sequence portion within the comparison window may contain 20% or less of additions or deletions (i.e., vacancies) compared to the reference sequence (which does not contain additions or deletions). In some embodiments, the comparison window may be longer than 15-20 consecutive residues, and optionally include windows of 30, 40, 50, 100, or longer.

[0097] When used in the context of numbering a given amino acid or polynucleotide sequence, "corresponding to," "reference," and "relative to" refer to specifying the number of residues in the reference sequence when the given amino acid or polynucleotide sequence is compared to a reference sequence. In other words, the residue numbering or position of a given polymer is specified relative to the reference sequence, rather than by the actual numerical position of residues within the given amino acid or polynucleotide sequence. For example, residue matching between two sequences can be optimized by introducing vacancies, comparing a given amino acid sequence (e.g., the amino acid sequence of an engineered RNA polymerase) to a reference sequence. In these cases, the numbering of residues in the given amino acid or polynucleotide sequence is relative to the reference sequence with which it is compared, despite the presence of vacancies. In some embodiments, the reference sequence is labeled (e.g., with a histidine tag).

[0098] A "mutation" refers to an alteration in the sequence of a nucleic acid. In some embodiments, a mutation results in a change in the sequence of the encoded polypeptide (i.e., compared to the original sequence without the mutation). In some embodiments, a mutation includes substitution, resulting in a different amino acid. In some alternative embodiments, a mutation includes addition, resulting in an amino acid being added (e.g., inserted) into the original polypeptide sequence. In some further embodiments, a mutation includes deletion, resulting in an amino acid being removed from the original polypeptide sequence. Any number of mutations may be present in a given sequence.

[0099] "Amino acid difference" and "residue difference" refer to the difference of an amino acid residue at a position in a polypeptide sequence relative to the corresponding amino acid residue at a position in a reference sequence. The position of the amino acid difference is generally referred to herein as "Xn," where n refers to the corresponding position in the reference sequence on which the residue difference is based. For example, "residue difference at position X38 compared to SEQ ID NO:2" (or "residue difference at position 38 compared to SEQ ID NO:2") refers to the difference of an amino acid residue at the polypeptide position corresponding to position 38 of SEQ ID NO:2. Therefore, if the reference polypeptide of SEQ ID NO:2 has arginine at position 38, then "residue difference at position X38 compared to SEQ ID NO:2" refers to the substitution of any amino acid residue other than arginine at the polypeptide position corresponding to position 38 of SEQ ID NO:2. In some cases herein, a specific amino acid residue difference at a position is represented as "XnY," where "Xn" specifies the corresponding residue and position of the reference polypeptide (as described above), and "Y" is a single-letter identifier for the amino acid found in the engineered polypeptide (i.e., a residue different from that in the reference polypeptide). In some instances (e.g., in the tables of the embodiments), this disclosure also provides specific amino acid differences represented by the conventional symbol “AnB”, where A is a single-letter identifier of a residue in the reference sequence, “n” is the position number of the residue in the reference sequence, and B is a single-letter identifier of a residue substitution in the sequence of the engineered polypeptide. In some embodiments, amino acid differences (e.g., substitutions) are represented by the abbreviation “nB” without an identifier of a residue in the reference sequence. In some embodiments, the phrase “amino acid residue nB” indicates the presence of an amino residue in the engineered polypeptide, which may or may not be a substitution in the context of the reference sequence. In some embodiments, “substitution” includes the deletion of an amino acid and may be represented by the “-” symbol.

[0100] In some cases, the polypeptides of this disclosure may include one or more amino acid residue differences relative to a reference sequence, indicated by a list of designated positions where residue differences exist relative to the reference sequence. In some embodiments, where more than one amino acid may be used at a particular residue position of the polypeptide, the various amino acid residues that may be used are separated by " / " (e.g., X340E / X340W, X340E / W, X340E / W, or 340E / W). This disclosure includes engineered polypeptide sequences comprising one or more amino acid differences, including one or both of conserved and non-conserved amino acid substitutions and amino acid insertions and deletions in the sequence.

[0101] "Amino acid substitution set" and "substitution set" refer to a group of amino acid substitutions within a polypeptide sequence. In some embodiments, the substitution set contains 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more amino acid substitutions. In some embodiments, the substitution set refers to the set of amino acid substitutions present in any of the variant RNA polymerase polypeptides listed in any of the tables in the examples. In these substitution sets, individual substitutions are separated by semicolons (e.g., V401R; Y404L) or forward slashes (" / "; e.g., V401R / Y404L or 401R / 404L).

[0102] "Conservative amino acid substitution" refers to the substitution of a residue with a different residue having a similar side chain, and therefore generally involves the substitution of an amino acid in a polypeptide with an amino acid from the same or similar defined amino acid class. By way of example and not limitation, an amino acid with an aliphatic side chain may be substituted with another aliphatic amino acid (e.g., alanine, valine, leucine, and isoleucine); an amino acid with a hydroxyl side chain may be substituted with another amino acid with a hydroxyl side chain (e.g., serine and threonine); an amino acid with an aromatic side chain may be substituted with another amino acid with an aromatic side chain (e.g., phenylalanine, tyrosine, tryptophan, and histidine); an amino acid with a basic side chain may be substituted with another amino acid with a basic side chain (e.g., lysine and arginine); an amino acid with an acidic side chain may be substituted with another amino acid with an acidic side chain (e.g., aspartic acid or glutamic acid); and a hydrophobic or hydrophilic amino acid may be substituted with another hydrophobic or hydrophilic amino acid, respectively.

[0103] "Non-conservative substitution" refers to the substitution of an amino acid in a polypeptide with an amino acid having significantly different side chain properties. Non-conservative substitution can use amino acids between rather than within groups and affect: (a) the structure of the peptide backbone in the substituted region (e.g., proline replacing glycine); (b) charge or hydrophobicity; and / or (c) the body of the side chain. By way of example and not limitation, exemplary non-conservative substitutions include acidic amino acids substituted with basic or aliphatic amino acids; aromatic amino acids substituted with small amino acids; and hydrophilic amino acids substituted with hydrophobic amino acids.

[0104] "Deletion" refers to a modification of a peptide by removing one or more amino acids from a reference peptide. Deletions may include the removal of one or more amino acids, two or more amino acids, five or more amino acids, ten or more amino acids, fifteen or more amino acids, or twenty or more amino acids, representing up to 10% or up to 20% of the total number of amino acids constituting the reference peptide, while preserving biological activity and / or the improved properties of the engineered RNA polymerase. Deletions may target internal and / or terminal portions of the peptide. In various embodiments, deletions may include continuous segments or may be discontinuous. Deletions are indicated by "-" and may be present in a substitution set.

[0105] "Insertion" refers to the modification of a polypeptide by adding one or more amino acids to a reference polypeptide. Insertion can occur within the polypeptide itself, or at the carboxyl or amino terminus. Insertions as used herein include fusion proteins known in the art. Insertions can be continuous segments of amino acids or separated by one or more amino acids in a naturally occurring polypeptide.

[0106] The terms “functional fragment” and “bioactive fragment” are used interchangeably herein and refer to a polypeptide having an amino-terminal and / or carboxyl-terminal deletion and / or an internal deletion, but wherein the remaining amino acid sequence is identical to the corresponding position in the sequence being compared (e.g., the full-length engineered RNA polymerase of this disclosure) and substantially retains all the activities of the full-length polypeptide.

[0107] "Isolated polypeptide" refers to a polypeptide that is substantially separated from other naturally occurring contaminants, such as proteins, lipids, and polynucleotides. This term includes polypeptides that have been removed or purified from their natural environment or expression system (e.g., host cells or in vitro synthesis). Recombinant RNA polymerase polypeptides may be present intracellularly, in cell culture media, or prepared in various forms, such as lysates or isolated formulations. Therefore, in some embodiments, the recombinant RNA polymerase polypeptides provided herein are isolated polypeptides.

[0108] "Substantially pure polypeptide" refers to a composition in which the polypeptide is the dominant substance (i.e., it is more abundant than any other individual macromolecule in the composition, based on molar or weight), and is typically substantially pure when the target substance constitutes at least about 50% (in molar or weight %) of the present macromolecules. Typically, a substantially pure RNA polymerase composition will contain about 60% or more, about 70% or more, about 80% or more, about 90% or more, about 95% or more, and about 98% or more of all macromolecules present in the composition, based on molar or weight %. In some embodiments, the target substance is purified to substantially homogeneity (i.e., contaminant substances are not detectable in the composition by conventional detection methods), wherein the composition consists substantially of a single macromolecule. Solvent substances, small molecules (<500 Daltons), and elemental ions are not considered macromolecules. In some embodiments, the isolated recombinant RNA polymerase polypeptide is a substantially pure polypeptide composition.

[0109] "Improved properties" refers to an engineered RNA polymerase peptide that exhibits improved properties in any RNA polymerase property compared to a reference RNA polymerase peptide, such as a wild-type RNA polymerase peptide or another engineered RNA polymerase peptide. Improved properties include, but are not limited to, properties such as: increased protein expression, enhanced activity, improved stability, improved thermal stability, improved pH stability, improved chemical stability, improved solvent stability, improved solubility, and improved product integrity.

[0110] "Enzyme activity enhancement" and "enzyme activity improvement" refer to an improvement in the properties of an engineered RNA polymerase peptide, which can be represented by an increase in specific activity compared to a reference RNA polymerase peptide (e.g., wild-type RNA polymerase and / or other engineered RNA polymerases). Exemplary methods for determining enzyme activity are provided in the examples. An improvement in enzyme activity can be from about 1.1 times the enzyme activity of the corresponding wild-type or reference peptide to about 1.5 times, 2 times, 5 times, 10 times, 20 times, 25 times, 50 times, 75 times, 100 times, 150 times, 200 times, or more of the enzyme activity of a naturally occurring RNA polymerase from which the RNA polymerase peptide is derived or another engineered RNA polymerase.

[0111] In the context of RNA polymerase, "persistence" refers to the ability of a nucleic acid polymerase to catalyze the production of total RNA product. In some embodiments, persistence is measured by the titer of total RNA product, which refers to the concentration of total RNA, and can be measured, for example, by the methods described in the examples. In some embodiments, improved persistence of a nucleic acid polymerase refers to an increase in the titer of the full-length product compared to a reference polymerase.

[0112] In the context of RNA products, "integrity" refers to the uniformity of RNA products produced by a transcription reaction (e.g., in vitro transcription (IVT)). In some embodiments, "integrity" is defined as the amount of desired or target full-length RNA divided by the total amount of RNA produced, for example, the area of ​​the full-length RNA electrophoresis peak divided by the area of ​​the electrophoretic pattern obtained by a fragment analyzer, as described in the examples. The "integrity score" is 1 minus the reciprocal of the integrity score:

[0113] .

[0114] In the context of RNA products, an improvement in "integrity" or "integrity score" refers to an increase in the purity of the full-length product compared to a reference transcription reaction (e.g., IVT).

[0115] "Codon optimization" refers to changing the codons of polynucleotides encoding proteins to codons that are preferentially used in a particular organism, making the encoded protein more effectively expressed in that organism. Although the genetic code is degenerate, as most amino acids are represented by a few codons (called "synonyms"), codon usage in a particular organism is known to be non-random and biased towards specific codon triples. This codon bias can be even higher for a given gene, genes with a common function or ancestral origin, highly expressed proteins versus low-copy-number proteins, and aggregated protein-coding regions of an organism's genome. In some implementations, polynucleotides encoding RNA polymerase polypeptides are codon-optimized for optimal production from a selected host organism for expression.

[0116] "Control sequence" herein refers to all components that are essential or advantageous for the expression of the polynucleotides and / or polypeptides disclosed herein. Each control sequence may be native or exogenous to the nucleic acid sequence encoding the polypeptide. Such control sequences include, but are not limited to, leader sequences, polyadenylated sequences, propeptide sequences, promoter sequences, signal peptide sequences, initiation sequences, and transcription terminators. In some embodiments, the control sequence includes at least a promoter and transcription and translation termination signals. In some embodiments, the control sequence is provided with an adapter to introduce a specific restriction site that facilitates the attachment of the control sequence to the coding region of the nucleic acid sequence encoding the polypeptide.

[0117] "Operably linked" refers to a configuration in which a control sequence is appropriately placed (i.e., in a functional relationship) relative to the polynucleotide of interest, such that the control sequence directs or regulates the expression of the polynucleotide, and, where appropriate, directs or regulates the expression of the encoded polypeptide of interest.

[0118] A “promoter” or “promoter sequence” is a nucleic acid sequence, such as a coding sequence, that is recognized by a host cell for the expression of a polynucleotide of interest. A promoter sequence contains a transcriptional control sequence that mediates the expression of the polynucleotide of interest. A promoter can be any nucleic acid sequence that exhibits transcriptional activity in a selected host cell, including mutant, truncated, and heterozygous promoters, and can be obtained from a gene encoding an extracellular or intracellular polypeptide that is homologous or heterologous to that of the host cell. In some embodiments, a “promoter” or “promoter sequence” refers to a nucleic acid sequence that is recognized by an engineered RNA polymerase.

[0119] "Suitable reaction conditions" or "suitable conditions" refers to those conditions (e.g., temperature, pH, buffer, salt, solubilizer, etc.) under which the RNA polymerase of this disclosure can synthesize RNA transcripts. Exemplary "suitable conditions" are provided herein (see, for example, examples).

[0120] “Cultivation” refers to the growth of a population of cells (e.g., host cells) under suitable conditions using any suitable culture medium (e.g., liquid, gel, or solid).

[0121] A “vector” is a recombinant construct used to introduce a polynucleotide of interest into a cell. In some embodiments, a vector is an expression vector operatively linked to a suitable control sequence capable of enabling the expression of a polynucleotide or a polypeptide encoded in a polynucleotide in a suitable host. In some embodiments, the “expression vector” has a promoter sequence operatively linked to a polynucleotide (e.g., a transgene) to drive expression in a host cell, and in some embodiments, also includes a transcription terminator sequence.

[0122] "Expression" includes any step involved in peptide production, including but not limited to transcription, post-transcriptional modification, translation, and post-translational modification. In some embodiments, the term also covers peptide secretion from cells.

[0123] "Production" refers to the production of proteins and / or other compounds by cells. This term is intended to encompass any steps involved in peptide production, including but not limited to transcription, post-transcriptional modification, translation, and post-translational modification. In some embodiments, the term also encompasses the secretion of peptides from cells.

[0124] "Heterogeneous" or "recombinant" refers to a relationship between two or more nucleic acid or polypeptide sequences (e.g., promoter sequences, signal peptides, terminator sequences, etc.) that are derived from different sources and are essentially unrelated.

[0125] "Host cell" and "host strain" refer to a suitable host for an expression vector containing a polynucleotide (e.g., a polynucleotide sequence encoding at least one RNA polymerase variant) provided herein. In some embodiments, the host cell is a prokaryotic or eukaryotic cell that has been transformed or transfected with a vector constructed using recombinant DNA technology, as is known in the art.

[0126] Engineered RNA polymerase polypeptides

[0127] In one aspect, this disclosure provides RNA polymerases engineered to have improved properties, including, in particular, increased activity, improved stability, improved thermostability, and / or improved product integrity. In some embodiments, the engineered RNA polymerase variants can be used to prepare RNA and other molecular biology techniques.

[0128] In some embodiments, the engineered RNA polymerase or its functional fragment comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity relative to the reference sequence corresponding to SEQ ID NO: 2, 4, 32, 44, 156, 234, 238, 386, 394, 588, 604, 686, 692, 712 or 800. The reference sequence of residues 8 to 890 of SEQ ID NO: 2, 4, 32, 44, 156, 234, 238, 386, 394, 588, 604, 686, 692, 712, or 800, or the reference sequence relative to the reference sequence corresponding to SEQ ID NO: 2, 4, 32, 44, 156, 234, 238, 386, 394, 588, 604, 686, 692, 712, or 800, wherein the amino acid sequence comprises one or more substitutions.

[0129] In some embodiments, the engineered RNA polymerase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 98%, 99%, or higher sequence identity with the reference sequence corresponding to SEQ ID NO: 2, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 2 or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0130] In some embodiments, the engineered RNA polymerase comprises a reference sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity relative to the reference sequence corresponding to SEQ ID NO: 4, 32, 44, 156, 234, 238, 386, 394, 588, 604, 686, 692, 712, or 800, or relative to the reference sequence corresponding to SEQ ID NO: 2, wherein the reference sequence has at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity relative to the reference sequence corresponding to SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2, 32, 44, 156, 234, 238, 386, 394, 588, 604, 686, 692, 712, or 800, or relative to the reference sequence corresponding to SEQ ID NO: 2, 32, 44, 156, 234, 238, 386, 394, 588, 604, 686, 692, 712 The reference sequence of 2, wherein the amino acid sequence contains one or more substitutions.

[0131] In some embodiments, the engineered RNA polymerase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 96%, 97%, 98%, 99% or higher sequence identity with the reference sequence corresponding to the even-numbered residues of SEQ ID NO: 2, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to the residues of SEQ ID NO: 2 or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0132] In some implementations, the amino acid sequence of the engineered RNA polymerase is located at amino acid positions 13, 14, 15, 17, 19, 21, 32, 33, 38, 45, 52, 54, 55, 56, 56, 57, 60, 61, 62, 63, 67, 69, 72, 75, 78, 81, 82, 83, 84, 89, 93, 95, 100, 105, 111, 115, 122, 124, 127, 131, 133, 1 35, 137, 138, 139, 140, 141, 143, 150, 160, 161, 162, 164, 166, 168, 169, 170, 171, 172, 173, 179, 185, 187, 193, 196, 207, 212, 217, 218, 221, 227, 237, 239, 240, 245, 246, 250, 253, 254, 257, 261, 272, 27 4, 275, 311, 336, 340, 342, 351, 357, 367, 375, 379, 382, ​​388, 391, 392, 394, 397, 401, 402, 403, 404, 419, 424, 437, 440, 494, 510, 514, 517, 527, 529, 530, 531, 533, 536, 589, 594, 598, 607, 609, 615, 616 The amino acid positions 617, 625, 629, 648, 650, 655, 659, 660, 663, 664, 666, 670, 672, 690, 693, 696, 718, 720, 721, 749, 759, 763, 773, 775, 779, 782, 782, 810, 854, 857, 867, 875, or 878, or combinations thereof, contain at least one substitution, wherein the amino acid position is relative to the reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 2 or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0133] In some embodiments, the amino acid sequence of the engineered RNA polymerase comprises at least one substituted or amino acid residue: 13N, 14Q / Y, 15T, 17N, 19E, 21G, 32S / T / V, 33E, 38G, 45K, 52T, 54H, 55D / S / W, 56E / G / N / T / V, 57G, 60G / L / M / R, 61W / Y, 62T, 63S, 67R, 69S, 72R, 75S / Y, 78S, 81C / L, 82S, 83K, 84A, 89Q, 93Y, 95Y, 100R, 105R, 111K, 115L / V, 122I, 124F, 127G, 131T, 133C, 135G / K, 1 37G, 138R, 139R, 140R, 141A, 143N / Q / R / V, 150Q / Y, 160F / L, 161R, 162V, 164 W, 166D / R / T / V, 168K / L / R / S / T / V / W, 169G / L, 170L / Q / R / S, 171E / F / L / Y, 172A / E / G / K / R / V, 173D / H / L / Y, 179N, 185I / L / P, 187G / H / L, 193G, 196A / C / G / S / V , 207S, 212R / Y, 217P / V, 218Q / T, 221C, 227V, 237L, 239L / R, 240M, 245C, 246K / R / V, 250R, 253Y, 254G / S, 257L / V, 261M, 272N, 274G / L, 275A / Y, 311G, 336L / M / S / V, 340E / W, L342Q, 351L, 357E / P, 367F / P / T, 375F / P, 379A / S, 382G / L / R, 388K / R, 391F / S, 392A / M / R, 394K, 397S, 401R, 402T, 403V, ​​404I / L / S / Y, 4 19R / S, 424A, 437P, 440K, 494G, 510V, 514I / S, 517R, 527E, 529R, 530N, 531Q, 533S, 536E / V, 589C, 594G, 598A / K / L / R / V, 607Y, 609C / E / F / P / W, 615A, 616A , 617R, 625C, 629D / K / R, 648F, 650M, 655M / R, 659R, 660H / W / Y, 663L, 664K / P, 666M, 670Q / R, 672G / P / R / S / T, 690S, 693R, 696L, 718F / N / S / V / Y, 720A / G / I / P / S, 721A / E / M / S / T / Y, 749R, 759M, 763S, 773A, 775K / R, 779R, 782R / V, 810K,854T, 857V, 867Q, 875C, or 878M, or combinations thereof, wherein the amino acid position is relative to the reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 2 or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0134] In some embodiments, the amino acid sequence of the engineered RNA polymerase includes at least one substitution of I13N, A14Q / Y, K15T, D17N, S19E, I21G, A32S / T / V, D33E, R38G, A45K, E52T, G54H, E55D / S / W, A56E / G / N / T / V, R57G, K60G / L / M / R, M61W / Y, F62T, E63S, K67R, G69S, A72R, A75S / Y, K78S, I81C / L, T82S, T83K, L84A, I89Q, N93Y, W95Y, K100R, K105R, Q111K, E115L / V, A122 I, I124F, K127G, A131T, L133C, S135G / K, D137G, N138R, T139R, T140R, V141 A. A143N / Q / R / V, R150Q / Y, R160F / L, I161R, R162V, L164W, A166D / R / T / V, H1 68K / L / R / S / T / V / W, F169G / L, K170L / Q / R / S, K171E / F / L / Y, N172A / E / G / K / R / V, V173D / H / L / Y, K179N, Y185I / L / P, K187G / H / L, V193G, D196A / C / G / S / V, A20 7S, H212R / Y, I217P / V, H218Q / T, V221C, L227V, H237L, Q239L / R, N240M, G24 5C, Q246K / R / V, T250R, L253Y, A254G / S, Y257L / V, I261M, S272N, M274G / L, F 275A / Y, A311G, K336L / M / S / V, K340E / W, L342Q, W351L, R357E / P, L367F / P / T , D375F / P, E379A / S, T382G / L / R, A388K / R, V391F / S, Y392A / M / R, R394K, A397 S, V401R, R402T, I403V, ​​L404I / S / Y, K419R / S, P424A, S437P, N440K, E494G, A510V, L514I / S, C517R, G527E, Q529R, H530N, H531Q, L533S, N536E / V, L589C , I594G, D598A / K / L / R / V, E607Y, T609C / E / F / P / W, K615A, V616A, K617R, G62 5C, A629D / K / R, S648F, E650M, Q655M / R, E659R, D660H / W / Y, Q663L, W664K / P,I666M, K670Q / R, L672G / P / R / S / T, E690S, S693R, V696L, K718F / N / S / V / Y, K720A / G / I / P / S, K721A / E / M / S / T / Y, P749R, L759M, R763S, D773A, E775K / R, H779R, E782R / V, G810K, D854T, A857V, K867Q, G875C, or N878M or combinations thereof, wherein the amino acid position is relative to the reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 2 or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0135] In some embodiments, the amino acid sequence of the engineered RNA polymerase contains at least one substitution at the following amino acid positions: 32, 38, 56, 138, 150, 170, 171, 173, 246, 311, 336, 340, 357, 388, 394, 401, 404, 437, 514, 536, 609, 629, 659, 660, 664, 779, or 782 or combinations thereof, wherein said amino acid position is relative to the reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 2 or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0136] In some embodiments, the amino acid sequence of the engineered RNA polymerase comprises at least one substituted or amino acid residue 32V, 38G, 56T, 138R / N, 150Y, 170Q, 171E, 173Y, 246K, 311A / G, 336K / S, 340K / W, 357E, 388A / K, 394K, 401R, 404L / S, 437P, 514S, 536V, 609E, 629A, 659R, 660H / W, 664P, 779R, or 782V or a combination thereof, wherein the amino acid position is relative to the reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 2 or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0137] In some embodiments, the amino acid sequence of the engineered RNA polymerase comprises at least one substitution of A32V, R38G, A56T, N138R / N, R150Y, K170Q, K171E, V173Y, Q246K, A311G, K336K / S, K340E / W, R357E, A388K / R, R394K, V401R, Y404L / S, S437P, L514S, N536V, T609E, R629A, E659R, D660H / W, W664P, H779R, or E782V or a combination thereof, wherein the amino acid position is relative to the reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 2 or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0138] In some embodiments, the amino acid sequence of the engineered RNA polymerase contains at least one substitution or set of substitutions at the following amino acid positions: 401 / 404, 138 / 311 / 336 / 340 / 388, 38 / 514, 170 / 171 / 173 / 336 / 340 / 514 / 659 / 660, 38 / 437 / 779, 357 / 394 / 664, 38, 138 / 311 / 336 / 340 / 357 / 388 / 394 / 404 / 664, 246 / 609, 32, 536, 56 / 150, 56 / 150 / 629 / 660 / 782 or 629, wherein the amino acid position is relative to the reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 2 or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0139] In some embodiments, the amino acid sequence of the engineered RNA polymerase contains at least one substitution or set of substitutions or amino acid residues such as 401R / 404L, 138R / 311G / 336S / 340W / 388K, 38G / 514S, 170Q / 171E / 173Y / 336K / 340K / 514S / 659R / 660H, or 38G / 437P / 779R. 357E / 394K / 664P, 38G, 138N / 311A / 336K / 340K / 357E / 388A / 394K / 404S / 664P, 246K / 609E, 32V, 536V, 56T / 150Y, 56T / 150Y / 629A / 660W / 782V or 629K, wherein the amino acid position is relative to the reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 2 or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0140] In some embodiments, the amino acid sequence of the engineered RNA polymerase includes at least one substitution or set of substitutions: V401R / Y404L, N138R / A311G / K336S / K340W / A388K, R38G / L514S, K170Q / K171E / V173Y / S336K / W340K / L514S / E659R / D660H, R38G / S437P / H779R, R357E / R394K / W664P, R38G, R138N / G311A / S336K / W340K / R357E / K388A / R394K / L404S / W664P, Q246K / T609E, A32V, N536V, A56T / R150Y, E56T / R150Y / R629A / H660W / E782V or A629K, wherein the amino acid position is relative to the reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 2 or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0141] In some embodiments, the amino acid sequence of the engineered RNA polymerase contains at least one substitution or set of substitutions at the following amino acid positions: 401 / 404, 336, 138, 401 / 403 / 404, 336 / 510, 388, 340, 401 / 402 / 404, or 351 / 357, wherein the amino acid position is relative to the reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 2 or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0142] In some embodiments, the amino acid sequence of the engineered RNA polymerase comprises at least one substitution or set of substitutions or amino acid residues 401R / 404L, 336S, 138R, 336V, 401R / 403V / 404S, 336M / 510V, 336L, 388K, 340E, 401R / 402T / 404S, 351L / 357E, or 401R / 404I, wherein the amino acid position is relative to the reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 2 or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0143] In some embodiments, the amino acid sequence of the engineered RNA polymerase comprises at least one substitution or set of substitutions V401R / Y404L, K336S, N138R, K336V, V401R / I403V / Y404S, K336M / A510V, K336L, A388K, K340E, V401R / R402T / Y404S, W351L / R357E, or V401R / Y404I, wherein the amino acid position is relative to the reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 2 or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0144] In some embodiments, the amino acid sequence of the engineered RNA polymerase contains at least one set of substitutions at the following amino acid positions: 311 / 388 / 401 / 440, 138 / 340 / 401 / 404, 138 / 311 / 336 / 340 / 388 / 401 / 404, 311 / 388 / 401 / 404 / 440, 138 / 311 / 340 / 388 / 401 / 440, 138 / 336 / 342 / 401 / 404, or 351 / 388 / 401 / 404, wherein the amino acid position is relative to the reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 2 or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0145] In some embodiments, the amino acid sequence of the engineered RNA polymerase comprises at least one set of substitutions or amino acid residues 311G / 388K / 401R / 440K, 138R / 340W / 401R / 404L, 138R / 311G / 336S / 340W / 388K / 401R / 404L, 311G / 388K / 401R / 404L / 440K, 138R / 311G / 340E / 388K / 401R / 440K, 138R / 336M / 342Q / 401R / 404L, or 351L / 388K / 401R / 404L, wherein the amino acid position is relative to the reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 2 or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0146] In some embodiments, the amino acid sequence of the engineered RNA polymerase contains at least one set of substitutions at the following amino acid positions: 138 / 311 / 336 / 340 / 388 / 401 / 404 / 514, 38 / 138 / 311 / 336 / 340 / 388 / 401 / 404 / 514, 38 / 138 / 311 / 336 / 340 / 388 / 394 / 401 / 404 / 514, 38 / 138 / 311 / 336 / 340 / 388 / 394 / 401 / 404 / 514 / 664, 138 / 311 / 336 / 340 / 388 / 394 / 401 / 404 / 514 / 875, 138 / 311 / 336 / 340 / 388 / 394 / 401 / 404 / 514, 38 / 124 / 138 / 311 / 336 / 340 / 388 / 394 / 401 / 404 / 514, 138 / 171 / 311 / 336 / 340 / 388 / 401 / 404 / 878, 138 / 170 / 311 / 336 / 340 / 388 / 401 / 404, 138 / 173 / 311 / 336 / 340 / 388 / 401 / 404, 138 / 172 / 311 / 336 / 340 / 388 / 401 / 404, 138 / 170 / 311 / 336 / 340 / 388 / 401 / 404 / 629, 138 / 171 / 311 / 336 / 340 / 388 / 401 / 404, 138 / 168 / 311 / 336 / 340 / 388 / 401 / 404, 138 / 166 / 311 / 336 / 340 / 388 / 401 / 404, 138 / 166 / 272 / 274 / 275 / 311 / 336 / 340 / 388 / 40 1 / 404, 138 / 169 / 311 / 336 / 340 / 388 / 401 / 404, 138 / 207 / 311 / 336 / 340 / 388 / 401 / 404, 138 / 168 / 217 / 218 / 311 / 336 / 340 / 388 / 401 / 404, 138 / 171 / 311 / 3 36 / 340 / 388 / 401 / 404 / 530, 138 / 311 / 336 / 340 / 382 / 388 / 401 / 404, 138 / 311 / 336 / 340 / 388 / 401 / 404 / 773, 138 / 311 / 336 / 340 / 388 / 401 / 404 / 670, 138 / 311 / 336 / 340 / 379 / 388 / 401 / 404, 138 / 311 / 336 / 340 / 388 / 392 / 401 / 404, 138 / 311 / 336 / 340 / 388 / 401 / 404 / 419, 57 / 138 / 311 / 336 / 340 / 388 / 401 / 404,138 / 164 / 311 / 336 / 340 / 388 / 401 / 404、138 / 311 / 336 / 340 / 388 / 401 / 404 / 810、138 / 311 / 336 / 340 / 388 / 401 / 404 / 659、54 / 138 / 311 / 336 / 340 / 388 / 401 / 404、138 / 172 / 173 / 311 / 336 / 340 / 388 / 401 / 404 / 514 / 659 / 660、138 / 170 / 171 / 172 / 311 / 336 / 340 / 388 / 401 / 404 / 514 / 659、138 / 173 / 311 / 336 / 340 / 388 / 401 / 404 / 514 / 659 / 660、138 / 173 / 311 / 336 / 340 / 388 / 401 / 404 / 514 / 659 / 810、138 / 170 / 311 / 336 / 388 / 401 / 404 / 514 / 659 / 660 / 810、138 / 170 / 171 / 173 / 311 / 388 / 401 / 404 / 514 / 659 / 660、57 / 138 / 171 / 173 / 311 / 336 / 340 / 388 / 401 / 404 / 514 / 659 / 660 / 810、138 / 170 / 173 / 311 / 336 / 340 / 388 / 401 / 404 / 514 / 659 / 660 / 810、57 / 138 / 171 / 172 / 311 / 388 / 401 / 404 / 514 / 659、138 / 173 / 311 / 336 / 340 / 388 / 401 / 404 / 514、138 / 171 / 172 / 173 / 311 / 336 / 340 / 388 / 401 / 404 / 514 / 659 / 660、57 / 138 / 170 / 171 / 173 / 311 / 336 / 340 / 388 / 401 / 404 / 514 / 659 / 660 / 810、57 / 138 / 173 / 311 / 336 / 340 / 388 / 401 / 404 / 514、138 / 172 / 173 / 311 / 336 / 340 / 388 / 401 / 404 / 514 / 857、57 / 138 / 173 / 311 / 336 / 388 / 401 / 404 / 514、138 / 170 / 171 / 173 / 311 / 340 / 388 / 401 / 404 / 514、138 / 171 / 311 / 336 / 340 / 388 / 401 / 404 / 514、57 / 138 / 170 / 173 / 311 / 340 / 388 / 401 / 404 / 514、138 / 170 / 311 / 340 / 388 / 401 / 404、138 / 173 / 311 / 336 / 340 / 388 / 401 / 404 / 514 / 659、57 / 138 / 311 / 336 / 340 / 388 / 401 / 404 / 514 or 138 / 173 / 311 / 336 / 340 / 388 / 401 / 404 / 810, wherein the amino acid position is relative to the reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 2 or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0147] In some embodiments, the amino acid sequence of the engineered RNA polymerase comprises at least one set of substitutions or amino acid residues, such as 138R / 311G / 336S / 340W / 388K / 401R / 404L / 514S, 38G / 138R / 311G / 336S / 340W / 388K / 401R / 404L / 514S, 38G / 138R / 311G / 336S / 340W / 388K / 394K / 401R / 404L / 514S / 664P, ... 6S / 340W / 388K / 394K / 401R / 404L / 514S / 875C, 138R / 311G / 336S / 340W / 38 8K / 394K / 401R / 404L / 514S, 38G / 124F / 138R / 311G / 336S / 340W / 388K / 394 K / 401R / 404L / 514I, 138R / 171E / 311G / 336S / 340W / 388K / 401R / 404L / 878 M、138R / 170Q / 311G / 336S / 340W / 388K / 401R / 404L、138R / 173Y / 311G / 336S / 340W / 388K / 401R / 404L, 138R / 172V / 311G / 336S / 340W / 388K / 401R / 404L ,138R / 173L / 311G / 336S / 340W / 388K / 401R / 404L, 138R / 172E / 311G / 336S / 340W / 388K / 401R / 404L, 138R / 170S / 311G / 336S / 340W / 388K / 401R / 404L / 629D、138R / 171F / 311G / 336S / 340W / 388K / 401R / 404L、138R / 168V / 311G / 336S / 340W / 388K / 401R / 404L、138R / 172K / 311G / 336S / 340W / 388K / 401R / 404L、138R / 166R / 311G / 336S / 340W / 388K / 401R / 404L、138R / 168K / 311G / 336S / 340W / 388K / 401R / 404L、138R / 166V / 272N / 274L / 275Y / 311G / 336S / 340W / 388K / 401R / 404L, 138R / 170R / 311G / 336S / 340W / 388K / 401R / 404L,138R / 169L / 311G / 336S / 340W / 388K / 401R / 404L, 138R / 207S / 311G / 336S / 340W / 388K / 401R / 404L, 138R / 169G / 311G / 336S / 340W / 388K / 401R / 404L, 1 38R / 168L / 217P / 218Q / 311G / 336S / 340W / 388K / 401R / 404L, 138R / 168W / 311G / 336S / 340W / 388K / 401R / 404L, 138R / 172R / 311G / 336S / 340W / 388K / 401 R / 404L, 138R / 166D / 311G / 336S / 340W / 388K / 401R / 404L, 138R / 171L / 311G / 336S / 340W / 388K / 401R / 404L, 138R / 171Y / 311G / 336S / 340W / 388K / 401R / 404L / 530N, 138R / 172A / 311G / 336S / 340W / 388K / 401R / 404L, 138R / 173H / 311G / 336S / 340W / 388K / 401R / 404L, 138R / 311G / 336S / 340W / 382R / 388K / 40 1R / 404L, 138R / 311G / 336S / 340W / 388K / 401R / 404L / 773A, 138R / 168R / 311G / 336S / 340W / 388K / 401R / 404L, 138R / 311G / 336S / 340W / 388K / 401R / 404 L / 670R, 138R / 168T / 311G / 336S / 340W / 388K / 401R / 404L, 138R / 172G / 311G / 336S / 340W / 388K / 401R / 404L, 138R / 311G / 336S / 340W / 379A / 388K / 401R / 404L, 138R / 311G / 336S / 340W / 388K / 392M / 401R / 404L, 138R / 311G / 336S / 340W / 388K / 401R / 404L / 419R, 138R / 311G / 336S / 340W / 388K / 392R / 401R / 40 4L、138R / 311G / 336S / 340W / 388K / 392A / 401R / 404L、57G / 138R / 311G / 336S / 340W / 388K / 401R / 404L、138R / 168S / 311G / 336S / 340W / 388K / 401R / 404L、138R / 164W / 311G / 336S / 340W / 388K / 401R / 404L、138R / 311G / 336S / 340W / 388K / 401R / 404L / 670Q、138R / 311G / 336S / 340W / 388K / 401R / 404L / 810K、138R / 311G / 336S / 340W / 382G / 388K / 401R / 404L、138R / 311G / 336S / 340W / 388K / 401R / 404L / 659R、54H / 138R / 311G / 336S / 340W / 388K / 401R / 404L、138R / 311G / 336S / 340W / 388K / 401R / 404L / 419S、138R / 172E / 173Y / 311G / 336S / 340W / 388K / 401R / 404L / 514S / 659R / 660H、138R / 170Q / 171E / 172E / 311G / 336S / 340W / 388K / 401R / 404L / 514S / 659R、138R / 173Y / 311G / 336S / 340W / 388K / 401R / 404L / 514S / 659R / 660H、138R / 173Y / 311G / 336S / 340W / 388K / 401R / 404L / 514S / 659R / 810K、138R / 170Q / 311G / 336S / 388K / 401R / 404L / 514S / 659R / 660H / 810K、138R / 170Q / 171E / 173Y / 311G / 388K / 401R / 404L / 514S / 659R / 660H、57G / 138R / 171E / 173Y / 311G / 336S / 340W / 388K / 401R / 404L / 514S / 659R / 660H / 810K、138R / 170Q / 173Y / 311G / 336S / 340W / 388K / 401R / 404L / 514S / 659R / 660H / 810K、57G / 138R / 171E / 172K / 311G / 388K / 401R / 404L / 514S / 659R、138R / 173Y / 311G / 336S / 340W / 388K / 401R / 404L / 514S、138R / 171E / 172E / 173Y / 311G / 336S / 340W / 388K / 401R / 404L / 514S / 659R / 660H、57G / 138R / 170Q / 171E / 173Y / 311G / 336S / 340W / 388K / 401R / 404L / 514S / 659R / 660H / 810K、57G / 138R / 173Y / 311G / 336S / 340W / 388K / 401R / 404L / 514S, 138R / 172K / 1 73D / 311G / 336S / 340W / 388K / 401R / 404L / 514S / 857V, 57G / 138R / 173Y / 311 G / 336S / 388K / 401R / 404L / 514S, 138R / 170Q / 171E / 173Y / 311G / 340W / 388 K / 401R / 404L / 514S, 138R / 171E / 311G / 336S / 340W / 388K / 401R / 404L / 514S 57G / 138R / 170Q / 173Y / 311G / 340W / 388K / 401R / 404L / 514S, 138R / 170Q / 311G / 340W / 388K / 401R / 404L, 138R / 173Y / 311G / 336S / 340W / 388K / 401R / 404L / 514S / 659R, 57G / 138R / 311G / 336S / 340W / 388K / 401R / 404L / 514S or 138R / 173Y / 311G / 336S / 340W / 388K / 401R / 404L / 810K, wherein the amino acid position is relative to the corresponding SEQ Reference sequence of residues 8 to 890 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0148] In some embodiments, the amino acid sequence of the engineered RNA polymerase contains at least one set of substitutions at the following amino acid positions: 38 / 138 / 311 / 340 / 388 / 401 / 404 / 514, 38 / 138 / 143 / 311 / 388 / 401 / 404 / 514 / 782, or 38 / 138 / 143 / 311 / 357 / 388 / 401 / 404, wherein the amino acid position is relative to the reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 2 or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0149] In some embodiments, the amino acid sequence of the engineered RNA polymerase comprises at least one set of substitutions or amino acid residues 38G / 138R / 311G / 340W / 388K / 401R / 404L / 514S, 38G / 138R / 143V / 311G / 388K / 401R / 404L / 514S / 782V or 38G / 138R / 143V / 311G / 357E / 388K / 401R / 404L, wherein the amino acid position is relative to the reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 2 or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0150] In some embodiments, the amino acid sequence of the engineered RNA polymerase contains at least one set of substitutions at the following amino acid positions: 138 / 173 / 207 / 311 / 336 / 340 / 388 / 401 / 404 / 514 / 659 / 660, 138 / 173 / 311 / 336 / 340 / 388 / 401 / 404 / 419 / 514 / 659 / 660, 138 / 173 / 207 / 311 / 388 / 401 / 404 / 514 / 659 / 660, 138 / 166 / 169 / 173 / 311 / 336 / 340 / 388 / 401 / 404 / 514 / 659 / 660, 138 / 173 / 207 / 311 / 336 / 340 / 388 / 401 / 404 / 514 / 659 / 660 / 670, 138 / 166 / 173 / 311 / 388 / 401 / 404 / 514 / 659 / 660, 138 / 173 / 207 / 311 / 336 / 340 / 388 / 401 / 404 / 419 / 51 4 / 659 / 660 / 670, 138 / 173 / 207 / 311 / 382 / 388 / 401 / 404 / 419 / 514 / 659 / 660 / 670, 138 / 173 / 207 / 311 / 336 / 340 / 382 / 388 / 401 / 404 / 514 / 659 / 660, 138 / 1 73 / 311 / 336 / 340 / 388 / 401 / 404 / 514 / 659 / 660 / 670, 138 / 173 / 311 / 340 / 388 / 401 / 404 / 514 / 659 / 660 / 670, 138 / 173 / 311 / 336 / 388 / 401 / 404 / 514 / 659 / 660 / 670, 138 / 173 / 311 / 336 / 340 / 382 / 388 / 401 / 404 / 514 / 659 / 660, 127 / 138 / 141 / 173 / 207 / 311 / 340 / 382 / 388 / 401 / 404 / 514 / 659 / 660 / 670, 138 / 17 3 / 207 / 311 / 340 / 382 / 388 / 401 / 404 / 419 / 514 / 659 / 660 / 670, 138 / 173 / 311 / 336 / 340 / 388 / 401 / 404 / 419 / 514 / 659 / 660 / 670, 138 / 166 / 168 / 173 / 207 / 3 11 / 388 / 401 / 404 / 419 / 514 / 659 / 660 / 670, 138 / 166 / 173 / 311 / 336 / 388 / 401 / 404 / 514 / 659 / 660 or 138 / 173 / 207 / 311 / 388 / 401 / 404 / 514 / 659 / 660 / 670,The amino acid positions are relative to the reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 2 or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0151] In some embodiments, the amino acid sequence of the engineered RNA polymerase comprises at least one set of substitutions or amino acid residues, such as 138R / 173Y / 207S / 311G / 336S / 340W / 388K / 401R / 404L / 514S / 659R / 660H, 138R / 173Y / 311G / 336S / 340W / 388K / 401R / 404L / 419R / 514S / 659R / 660H, 138R / 173Y / 207S / 311G / 388K / 401R / 404L / 514S / 659R / 660H, 138R / 166R / 169L / 173Y / 311G / 336S / 340W / 388K / 401R / 404L / 514S / 659R / 660H, 138R / 173Y / 207S / 311G / 336S / 340W / 388K / 401R / 404L / 514S / 659R / 660H / 670R、138R / 166R / 173Y / 311G / 3 88K / 401R / 404L / 514S / 659R / 660H, 138R / 173Y / 207S / 311G / 336S / 340W / 3 88K / 401R / 404L / 419R / 514S / 659R / 660H / 670R、138R / 173Y / 207S / 311G / 38 2G / 388K / 401R / 404L / 419R / 514S / 659R / 660H / 670R, 138R / 173Y / 207S / 31 1G / 336S / 340W / 382G / 388K / 401R / 404L / 514S / 659R / 660H, 138R / 173Y / 31 1G / 336S / 340W / 388K / 401R / 404L / 514S / 659R / 660H / 670R、138R / 173Y / 31 1G / 340W / 388K / 401R / 404L / 514S / 659R / 660H / 670R、138R / 173Y / 311G / 336 S / 388K / 401R / 404L / 514S / 659R / 660H / 670R, 138R / 173Y / 311G / 336S / 340 W / 382G / 388K / 401R / 404L / 514S / 659R / 660H, 127G / 138R / 141A / 173Y / 207S / 311G / 340W / 382G / 388K / 401R / 404L / 514S / 659R / 660H / 670R、138R / 173Y / 207S / 311G / 340W / 382G / 388K / 401R / 404L / 419R / 514S / 659R / 660H / 670R、138R / 173Y / 311G / 336S / 340W / 388K / 401R / 404L / 419R / 514S / 659R / 660H / 670R, 1 38R / 166R / 168V / 173Y / 207S / 311G / 388K / 401R / 404L / 419R / 514S / 659R / 660H / 67 0R, 138R / 166V / 173Y / 311G / 336S / 388K / 401R / 404L / 514S / 659R / 660H or 138R / 173Y / 207S / 311G / 388K / 401R / 404L / 514S / 659R / 660H / 670R, wherein the amino acid position is relative to the reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 2 or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0152] In some embodiments, the amino acid sequence of the engineered RNA polymerase contains at least one set of substitutions at the following amino acid positions: 38 / 138 / 173 / 311 / 336 / 340 / 388 / 401 / 404 / 437 / 514 / 659 / 660 / 779, 38 / 138 / 173 / 311 / 336 / 340 / 388 / 401 / 404 / 437 / 514 / 659 / 660, 38 / 138 / 173 / 311 / 336 / 340 / 388 / 401 / 404 / 514 / 659 / 660, 38 / 138 / 1 73 / 207 / 311 / 336 / 340 / 388 / 401 / 404 / 437 / 514 / 659 / 660 / 779, 138 / 173 / 311 / 336 / 340 / 388 / 401 / 404 / 514 / 517 / 659 / 660 / 779, 38 / 138 / 173 / 311 / 336 / 340 / 388 / 401 / 404 / 437 / 514 / 527 / 659 / 660 / 664, 38 / 138 / 173 / 207 / 311 / 336 / 340 / 379 / 388 / 401 / 404 / 43 7 / 514 / 659 / 660, 138 / 173 / 311 / 336 / 340 / 388 / 401 / 404 / 514 / 659 / 660 / 664 / 670 / 779, 38 / 138 / 173 / 311 / 336 / 340 / 388 / 401 / 404 / 514 / 659 / 660 / 779, 38 / 138 / 173 / 311 / 336 / 340 / 388 / 401 / 404 / 437 / 514 / 517 / 659 / 660, 138 / 173 / 311 / 336 / 340 / 388 / 401 / 40 4 / 437 / 514 / 659 / 660 / 779, 38 / 138 / 173 / 311 / 336 / 340 / 388 / 401 / 404 / 437 / 514 / 607 / 659 / 660, 38 / 138 / 173 / 311 / 336 / 340 / 379 / 388 / 401 / 404 / 514 / 659 / 660 / 779 or 138 / 173 / 311 / 336 / 340 / 388 / 401 / 404 / 514 / 517 / 659 / 660 / 670 / 779, wherein the amino acid position is relative to the reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 2 or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0153] In some embodiments, the amino acid sequence of the engineered RNA polymerase comprises at least one set of substitutions or amino acid residues, such as 38G / 138R / 173Y / 311G / 336S / 340W / 388K / 401R / 404L / 437P / 514S / 659R / 660H / 779R, 38G / 138R / 173Y / 311G / 336S / 340W / 388K / 401R / 404L / 437P / 514S / 659R / 660H, 38G / 138R / 173Y / 311G / 336S / 340W / 388K / 401R / 404L / 514S / 659R / 660H, 38G / 138R / 173Y / 20 7S / 311G / 336S / 340W / 388K / 401R / 404L / 437P / 514S / 659R / 660H / 779R, 138R / 173Y / 311G / 336S / 340W / 388K / 401R / 404L / 514S / 517R / 659R / 660H / 779R, 3 8G / 138R / 173Y / 311G / 336S / 340W / 388K / 401R / 404L / 437P / 514S / 527E / 659R / 660H / 664K、38G / 138R / 173Y / 207S / 311G / 336S / 340W / 379S / 388K / 401R / 404 L / 437P / 514S / 659R / 660H, 138R / 173Y / 311G / 336S / 340W / 388K / 401R / 404L / 514S / 659R / 660H / 664K / 670R / 779R、38G / 138R / 173Y / 311G / 336S / 340W / 388 K / 401R / 404L / 514S / 659R / 660H / 779R、38G / 138R / 173Y / 311G / 336S / 340W / 3 88K / 401R / 404L / 437P / 514S / 517R / 659R / 660H, 138R / 173Y / 311G / 336S / 340W / 388K / 401R / 404L / 437P / 514S / 659R / 660H / 779R、38G / 138R / 173Y / 311G / 33 6S / 340W / 388K / 401R / 404L / 437P / 514S / 607Y / 659R / 660H, 38G / 138R / 173Y / 311G / 336S / 340W / 379S / 388K / 401R / 404L / 514S / 659R / 660H / 779R、138R / 17 3Y / 311G / 336S / 340W / 388K / 401R / 404L / 514S / 517R / 659R / 660H / 670R / 779R、38G / 138R / 173Y / 311G / 336S / 340W / 388K / 401R / 404L / 437P / 514S / 659R / 660H / 779R、38G / 138R / 173Y / 311G / 336S / 340W / 388K / 401R / 404L / 437P / 514S / 659R / 660H、38G / 138R / 173Y / 311G / 336S / 340W / 388K / 401R / 404L / 514S / 659R / 660H、38G / 138R / 173 Y / 207S / 311G / 336S / 340W / 388K / 401R / 404L / 437P / 514S / 659R / 660H / 779R、138R / 173Y / 311G / 336S / 340W / 388K / 401R / 404L / 514S / 517R / 659R / 660H / 779R、38G / 138R / 173Y / 311G / 336S / 340W / 388K / 401R / 404L / 437P / 514S / 527E / 659R / 660H / 664K、38G 138R / 173Y / 207S / 311G / 336S / 340W / 379S / 388K / 401R / 404L / 437P / 514S / 659R / 660H 138R / 173Y / 311G / 336S / 340W / 388K / 401R / 404L / 514S / 659R / 660H / 664K / 670R / 779R 38G / 138R / 173Y / 311G / 336S / 340W / 388K / 401R / 404L / 514S / 659R / 660H / 779 R, 38G / 138R / 173Y / 311G / 336S / 340W / 388K / 401R / 404L / 437P / 514S / 517R / 659R / 660H, 138R / 173Y / 311G / 336S / 340W / 388K / 401R / 404L / 437P / 514S / 659R / 660H / 779R, 38G / 138R / 173Y / 311G / 336S / 340W / 388K / 401R / 404L / 437P / 514S / 607Y / 659R / 660H38G / 138R / 173Y / 311G / 336S / 340W / 379S / 388K / 401R / 404L / 514S / 659R / 660H / 779R or 138R / 173Y / 311G / 336S / 340W / 388K / 401R / 404L / 514S / 517R / 659R / 660H / 670R / 779R, wherein the amino acid position is relative to the reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 2 or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0154] In some embodiments, the amino acid sequence of the engineered RNA polymerase contains at least one set of substitutions at the following amino acid positions: 138 / 173 / 311 / 336 / 340 / 357 / 388 / 394 / 401 / 404 / 514 / 659 / 660 / 664 or 173 / 357 / 394 / 401 / 404 / 514 / 659 / 660 / 664, wherein the amino acid position is relative to the reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 2 or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0155] In some embodiments, the amino acid sequence of the engineered RNA polymerase comprises at least one set of substitutions or amino acid residues 138R / 173Y / 311G / 336S / 340W / 357E / 388K / 394K / 401R / 404L / 514S / 659R / 660H / 664P or 173Y / 357E / 394K / 401R / 404S / 514S / 659R / 660H / 664P, wherein the amino acid position is relative to the reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 2 or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0156] In some embodiments, the amino acid sequence of the engineered RNA polymerase contains at least one set of substitutions at the following amino acid positions: 38 / 138 / 173 / 311 / 336 / 340 / 388 / 401 / 404 / 437 / 514 / 659 / 660 / 720 / 779, 38 / 138 / 173 / 196 / 311 / 336 / 340 / 388 / 401 / 404 / 437 / 514 / 659 / 660 / 779, 38 / 138 / 173 / 311 / 336 / 340 / 388 / 401 / 404 / 437 / 514 / 609 / 659 / 660 / 779, 38 / 138 / 173 / 246 / 311 / 336 / 3 40 / 388 / 401 / 404 / 437 / 514 / 659 / 660 / 779, 38 / 138 / 173 / 185 / 311 / 336 / 340 / 388 / 401 / 404 / 437 / 514 / 659 / 660 / 779, 38 / 138 / 173 / 311 / 336 / 340 / 388 / 40 1 / 404 / 437 / 514 / 594 / 659 / 660 / 779, 38 / 138 / 173 / 212 / 311 / 336 / 340 / 388 / 401 / 404 / 437 / 514 / 659 / 660 / 779, 38 / 138 / 173 / 311 / 336 / 340 / 388 / 401 / 404 / 437 / 494 / 514 / 659 / 660 / 779、38 / 138 / 173 / 311 / 336 / 340 / 375 / 388 / 401 / 404 / 437 / 514 / 659 / 660 / 779、38 / 138 / 173 / 311 / 336 / 340 / 388 / 401 / 404 / 437 / 514 / 659 / 660 / 721 / 779, 38 / 72 / 138 / 173 / 311 / 336 / 340 / 388 / 401 / 404 / 437 / 514 / 659 / 660 / 779, 38 / 138 / 173 / 311 / 336 / 340 / 388 / 401 / 404 / 437 / 514 / 65 9 / 660 / 779 / 867, 38 / 138 / 160 / 173 / 311 / 336 / 340 / 388 / 401 / 404 / 437 / 514 / 659 / 660 / 779, 38 / 138 / 173 / 311 / 336 / 340 / 388 / 401 / 404 / 437 / 514 / 659 / 660 / 718 / 779, 38 / 138 / 173 / 187 / 311 / 336 / 340 / 388 / 401 / 404 / 437 / 514 / 659 / 660 / 779, 38 / 138 / 170 / 311 / 336 / 340 / 388 / 401 / 404 / 437 / 514 / 659 / 660 / 779,38 / 138 / 173 / 311 / 336 / 340 / 388 / 401 / 404 / 437 / 514 / 615 / 659 / 660 / 779、38 / 137 / 173 / 311 / 336 / 340 / 388 / 401 / 404 / 437 / 514 / 659 / 660 / 779、38 / 138 / 173 / 311 / 336 / 340 / 388 / 401 / 404 / 437 / 514 / 659 / 660 / 779 / 854、38 / 131 / 138 / 173 / 311 / 336 / 340 / 388 / 401 / 404 / 437 / 514 / 659 / 660 / 779、138 / 173 / 185 / 187 / 311 / 336 / 340 / 388 / 401 / 404 / 514 / 659 / 660 / 779、38 / 138 / 173 / 185 / 187 / 311 / 336 / 340 / 388 / 401 / 404 / 437 / 514 / 659 / 660 / 779、38 / 138 / 173 / 311 / 336 / 340 / 388 / 401 / 404 / 514 / 609 / 659 / 660 / 779、138 / 173 / 311 / 336 / 340 / 388 / 401 / 404 / 437 / 514 / 609 / 659 / 660 / 779、138 / 173 / 185 / 187 / 311 / 336 / 340 / 388 / 401 / 404 / 514 / 609 / 659 / 660 / 779、38 / 138 / 173 / 311 / 336 / 340 / 388 / 401 / 404 / 514 / 609 / 659 / 660 / 720 / 779、38 / 138 / 173 / 185 / 187 / 311 / 336 / 340 / 388 / 401 / 404 / 437 / 514 / 609 / 659 / 660 / 779、38 / 138 / 173 / 185 / 187 / 311 / 336 / 340 / 388 / 401 / 404 / 514 / 659 / 660 / 779、38 / 138 / 173 / 311 / 336 / 340 / 388 / 401 / 404 / 514 / 659 / 660 / 721 / 779、38 / 138 / 173 / 311 / 336 / 340 / 388 / 401 / 404 / 437 / 514 / 609 / 659 / 660 / 720 / 779、38 / 138 / 173 / 185 / 187 / 246 / 311 / 336 / 340 / 388 / 401 / 404 / 437 / 514 / 659 / 660 / 779 or 38 / 138 / 173 / 185 / 187 / 311 / 336 / 340 / 388 / 401 / 404 / 514 / 609 / 659 / 660 / 720 / 779, wherein the amino acid position is relative to the reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 2 or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0157] In some embodiments, the amino acid sequence of the engineered RNA polymerase comprises at least one set of substitutions or amino acid residues, such as 38G / 138R / 173Y / 311G / 336S / 340W / 388K / 401R / 404L / 437P / 514S / 659R / 660H / 720A / 779R, 38G / 138R / 173Y / 196C / 311G / 336S / 340W / 388K / 401R / 404L / 437P / 514S / 659R / 660H / 779R, 38G / 138R / 173Y / 311G / 336S / 340W / 388K / 401R / 404L. / 437P / 514S / 609W / 659R / 660H / 779R、38G / 138R / 173Y / 196V / 311G / 336S / 340W / 388K / 401R / 404L / 437P / 514S / 659R / 660H / 779R、38G / 138R / 17 3Y / 246V / 311G / 336S / 340W / 388K / 401R / 404L / 437P / 514S / 659R / 660H / 779R、38G / 138R / 173Y / 185L / 311G / 336S / 340W / 388K / 401R / 404L / 437P / 514S / 659R / 660H / 779R、38G / 138R / 173Y / 311G / 336S / 340W / 388K / 401R / 404L / 437P / 514S / 594G / 659R / 660H / 779R、38G / 138R / 173Y / 212Y / 31 1G / 336S / 340W / 388K / 401R / 404L / 437P / 514S / 659R / 660H / 779R、38G / 1 38R / 173Y / 311G / 336S / 340W / 388K / 401R / 404L / 437P / 494G / 514S / 659R / 660H / 779R、38G / 138R / 173Y / 196A / 311G / 336S / 340W / 388K / 401R / 404L / 437P / 514S / 659R / 660H / 779R、38G / 138R / 173Y / 212R / 311G / 336S / 34 0W / 388K / 401R / 404L / 437P / 514S / 659R / 660H / 779R、38G / 138R / 173Y / 3 11G / 336S / 340W / 375F / 388K / 401R / 404L / 437P / 514S / 659R / 660H / 779R,38G / 138R / 173Y / 311G / 336S / 340W / 388K / 401R / 404L / 437P / 514S / 659R / 660H / 721T / 779R、38G / 138R / 173Y / 311G / 336S / 340W / 388K / 401R / 404L / 43 7P / 514S / 659R / 660H / 721A / 779R, 38G / 72R / 138R / 173Y / 311G / 336S / 340W / 388K / 401R / 404L / 437P / 514S / 659R / 660H / 779R, 38G / 138R / 173Y / 185I / 311G / 336S / 340W / 388K / 401R / 404L / 437P / 514S / 659R / 660H / 779R, 38G / 138R / 173Y / 311G / 336S / 340W / 388K / 401R / 404L / 437P / 514S / 659R / 660H / 7 21S / 779R, 38G / 138R / 173Y / 311G / 336S / 340W / 388K / 401R / 404L / 437P / 514S / 659R / 660H / 779R / 867Q, 38G / 138R / 173Y / 311G / 336S / 340W / 388K / 401 R / 404L / 437P / 514S / 659R / 660H / 721Y / 779R、38G / 138R / 160F / 173Y / 311G / 336S / 340W / 388K / 401R / 404L / 437P / 514S / 659R / 660H / 779R、38G / 138R / 173Y / 311G / 336S / 340W / 388K / 401R / 404L / 437P / 514S / 659R / 660H / 718F / 779R、38G / 138R / 173Y / 311G336S / K340W / 388K / 401R / 404L / 437P / 514S / 6 09F / 659R / 660H / 779R, 38G / 138R / 173Y / 311G / 336S / 340W / 388K / 401R / 404L / 437P / 514S / 659R / 660H / 720S / 779R, 38G / 138R / 173Y / 187L / 311G / 336 S / 340W / 388K / 401R / 404L / 437P / 514S / 659R / 660H / 779R、38G / 138R / 173Y / 311G / 336S / 340W / 388K / 401R / 404L / 437P / 514S / 659R / 660H / 718V / 779R、38G / 138R / 170L / 311G / 336S / 340W / 388K / 401R / 404L / 437P / 514S / 659R / 660H / 779R、38G / 138R / 173Y / 311G / 336S / 340W / 388K / 401R / 404L / 437P / 514S / 609E / 659R / 660H / 779R、38G / 138R / 173Y / 311G / 336S / 340W / 388K / 401R / 404L / 437P / 514S / 659R / 660H / 718S / 779R、38G / 138R / 173Y / 311G / 33 6S / 340W / 388K / 401R / 404L / 437P / 514S / 609C / 659R / 660H / 779R、38G / 13 8R / 173Y / 311G / 336S / 340W / 388K / 401R / 404L / 437P / 514S / 609P / 659R / 66 0H / 779R、38G / 138R / 173Y / 311G / 336S / 340W / 388K / 401R / 404L / 437P / 51 4S / 659R / 660H / 721M / 779R、38G / 138R / 173Y / 311G / 336S / 340W / 388K / 401 R / 404L / 437P / 514S / 615A / 659R / 660H / 779R、38G / 138R / 173Y / 187H / 311G / 336S / 340W / 388K / 401R / 404L / 437P / 514S / 659R / 660H / 779R、38G / 138R / 173Y / 196S / 311G / 336S / 340W / 388K / 401R / 404L / 437P / 514S / 659R / 660H / 779R、38G / 138R / 173Y / 311G / 336S / 340W / 388K / 401R / 404L / 437P / 514S / 659R / 660H / 721E / 779R、38G / 138R / 173Y / 311G / 336S / 340W / 388K / 401R / 404L / 437P / 514S / 659R / 660H / 718Y / 779R、38G / 137G / 173Y / 311G / 336S / 340W / 388K / 401R / 404L / 437P / 514S / 659R / 660H / 779R, 38G / 138R / 173Y / 187G / 311G / 336S / 340W / 388K / 401R / 404L / 437P / 514S / 659R / 660H / 779R38G / 138R / 173Y / 311G / 336S / 340W / 388K / 401R / 404L / 437P / 514S / 659R / 660H / 720G / 779R、38G / 138R / 173Y / 311G / 336S / 340W / 388K / 401R / 404L / 43 7P / 514S / 659R / 660H / 720P / 779R, 38G / 138R / 173Y / 311G / 336S / 340W / 388K / 401R / 404L / 437P / 514S / 659R / 660H / 779R / 854T, 38G / 138R / 173Y / 311G / 336S / 340W / 388K / 401R / 404L / 437P / 514S / 659R / 660H / 720I / 779R、38G / 138R / 160L / 173Y / 311G / 336S / 340W / 388K / 401R / 404L / 437P / 514S / 659R / 660H / 779R, 38G / 131T / 138R / 173Y / 311G / 336S / 340W / 388K / 401R / 404L / 437P / 514S / 659R / 660H / 779R, 138R / 173Y / 185L / 187H / 311G / 336S / 340W / 3 88K / 401R / 404L / 514S / 659R / 660H / 779R, 38G / 138R / 173Y / 185L / 187H / 311G / 336S / 340W / 388K / 401R / 404L / 437P / 514S / 659R / 660H / 779R, 38G / 138 R / 173Y / 185L / 187L / 311G / 336S / 340W / 388K / 401R / 404L / 437P / 514S / 659R / 660H / 779R、38G / 138R / 173Y / 311G / 336S / 340W / 388K / 401R / 404L / 514S / 609C / 659R / 660H / 779R、138R / 173Y / 311G / 336S / 340W / 388K / 401R / 404L / 437P / 514S / 609P / 659R / 660H / 779R、138R / 173Y / 185L / 187H / 311G / 336S 340W / 388K / 401R / 404L / 514S / 609C / 659R / 660H / 779R, 38G / 138R / 173Y / 311G / 336S / 340W / 388K / 401R / 404L / 514S / 609P / 659R / 660H / 720A / 779R38G / 138R / 173Y / 185L / 187L / 311G / 336S / 340W / 388K / 401R / 404L / 437P / 514S / 609P / 659R / 660H / 779R、38G / 13 8R / 173Y / 185L / 187L / 311G / 336S / 340W / 388K / 401R / 404L / 514S / 659R / 660H / 779R、38G / 138R / 173Y / 185L / 187 H / 311G / 336S / 340W / 388K / 401R / 404L / 514S / 659R / 660H / 779R, 38G / 138R / 173Y / 311G / 336S / 340W / 388K / 401R / 404L / 514S / 609P / 659R / 660H / 779R、38G / 138R / 173Y / 311G / 336S / 340W / 388K / 401R / 404L / 514S / 659R / 660H / 721S / 779R、38G / 138R / 173Y / 185L / 187H / 311G / 336S / 340W / 388K / 401R / 404L / 437P / 514S / 609E / 659R / 660H / 7 79R、38G / 138R / 173Y / 311G / 336S / 340W / 388K / 401R / 404L / 437P / 514S / 609P / 659R / 660H / 720A / 779R、38G / 138 R / 173Y / 185L / 187L / 246V / 311G / 336S / 340W / 388K / 401R / 404L / 437P / 514S / 659R / 660H / 779R or 38G / 138R / 173Y / 185L / 187H / 311G / 336S / 340W / 388K / 401R / 404L / 514S / 609P / 659R / 660H / 720A / 779R, wherein the amino acid position is relative to the reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 2 or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0158] In some embodiments, the amino acid sequence of the engineered RNA polymerase contains at least one set of substitutions at the following amino acid positions: 138 / 173 / 311 / 336 / 340 / 357 / 388 / 394 / 401 / 404 / 514 / 659 / 660 / 664, 138 / 170 / 173 / 311 / 336 / 340 / 357 / 388 / 394 / 401 / 404 / 514 / 609 / 659 / 660 / 664, 38 / 138 / 173 / 185 / 187 / 311 / 336 / 340 / 357 / 388 / 394 / 401 / 404 / 514 / 609 / 659 / 660 / 664, 38 / 138 / 173 / 18 5 / 187 / 311 / 336 / 340 / 357 / 388 / 394 / 401 / 404 / 514 / 659 / 660 / 664, 38 / 138 / 170 / 311 / 336 / 340 / 357 / 388 / 394 / 401 / 404 / 514 / 659 / 660 / 664, 38 / 138 / 185 / 187 / 311 / 336 / 340 / 357 / 388 / 394 / 401 / 404 / 514 / 659 / 660 / 664 / 720, 138 / 170 / 173 / 185 / 187 / 311 / 336 / 340 / 357 / 388 / 394 / 401 / 404 / 514 / 609 / 659 / 660 / 6 64, 138 / 173 / 311 / 336 / 340 / 357 / 388 / 394 / 401 / 404 / 514 / 609 / 659 / 660 / 664, 138 / 170 / 173 / 185 / 187 / 311 / 336 / 340 / 357 / 388 / 394 / 401 / 404 / 514 / 609 / 6 59 / 660 / 664 / 721, 38 / 138 / 173 / 311 / 336 / 340 / 357 / 388 / 394 / 401 / 404 / 514 / 609 / 659 / 660 / 664, 38 / 138 / 170 / 173 / 246 / 311 / 336 / 340 / 357 / 388 / 394 / 401 / 404 / 514 / 609 / 659 / 660 / 664, 138 / 173 / 185 / 187 / 311 / 336 / 340 / 357 / 388 / 394 / 401 / 404 / 514 / 609 / 659 / 660 / 664 / 718, 38 / 138 / 173 / 185 / 187 / 311 / 336 / 34 0 / 357 / 388 / 394 / 401 / 404 / 514 / 609 / 659 / 660 / 664 / 718 / 720, 38 / 138 / 170 / 173 / 246 / 311 / 336 / 340 / 357 / 388 / 394 / 401 / 404 / 514 / 609 / 659 / 660 / 664 / 718,138 / 173 / 311 / 336 / 340 / 357 / 388 / 394 / 401 / 404 / 514 / 659 / 660 / 664 / 720、138 / 173 / 246 / 311 / 336 / 340 / 357 / 388 / 394 / 401 / 404 / 514 / 609 / 659 / 660 / 664、38 / 138 / 173 / 185 / 187 / 246 / 311 / 336 / 340 / 357 / 388 / 394 / 401 / 404 / 514 / 659 / 660 / 664 / 720、38 / 138 / 173 / 311 / 336 / 340 / 357 / 388 / 394 / 401 / 404 / 514 / 609 / 659 / 660 / 664 / 721、38 / 138 / 173 / 311 / 336 / 340 / 357 / 388 / 394 / 401 / 404 / 514 / 659 / 660 / 664 / 721、38 / 138 / 173 / 311 / 336 / 340 / 357 / 388 / 394 / 401 / 404 / 514 / 659 / 660 / 664 / 720 / 721、38 / 138 / 170 / 173 / 311 / 336 / 340 / 357 / 388 / 394 / 401 / 404 / 514 / 609 / 659 / 660 / 664 / 718 / 720、38 / 138 / 173 / 311 / 336 / 340 / 357 / 388 / 394 / 401 / 404 / 514 / 659 / 660 / 664 / 718、38 / 138 / 173 / 185 / 187 / 311 / 336 / 340 / 357 / 388 / 394 / 401 / 404 / 514 / 609 / 659 / 660 / 664 / 718 / 721、138 / 173 / 311 / 336 / 340 / 357 / 388 / 394 / 401 / 404 / 514 / 529 / 609 / 659 / 660 / 664、38 / 138 / 173 / 311 / 336 / 340 / 357 / 388 / 394 / 401 / 404 / 514 / 609 / 659 / 660 / 664 / 718、138 / 173 / 185 / 187 / 311 / 336 / 340 / 357 / 388 / 394 / 401 / 404 / 514 / 609 / 659 / 660 / 664 / 718 / 720、138 / 173 / 185 / 187 / 311 / 336 / 340 / 357 / 388 / 394 / 401 / 404 / 514 / 609 / 659 / 660 / 664 / 718 / 721、38 / 138 / 170 / 173 / 311 / 336 / 340 / 357 / 388 / 394 / 401 / 404 / 514 / 659 / 660 / 664 / 718 / 720 / 721、138 / 170 / 173 / 185 / 187 / 311 / 336 / 340 / 357 / 388 / 394 / 401 / 404 / 514 / 659 / 660 / 664 / 718 / 720, 38 / 138 / 185 / 187 / 246 / 311 / 336 / 340 / 357 / 388 / 394 / 401 / 404 / 514 / 609 / 659 / 660 / 664 / 720, 138 / 173 / 311 / 336 / 340 / 357 / 388 / 394 / 401 / 404 / 514 / 609 / 659 / 660 / 664 / 718 / 720, 138 / 170 / 173 / 185 / 187 / 311 / 336 / 340 / 357 / 388 / 394 / 401 / 404 / 514 / 659 / 660 / 664 / 718 / 720 / 721, 38 / 138 / 170 / 246 / 311 / 336 / 340 / 357 / 388 / 394 / 401 / 404 / 514 / 609 / 659 / 660 / 664 / 718, 138 / 173 / 246 / 311 / 336 / 340 / 357 / 388 / 394 / 401 / 404 / 514 / 659 / 660 / 664 / 721, 38 / 138 / 17 3 / 311 / 336 / 340 / 357 / 388 / 394 / 401 / 404 / 514 / 659 / 660 / 664 / 718 / 720 or 38 / 138 / 173 / 311 / 336 / 340 / 357 / 388 / 394 / 401 / 404 / 514 / 609 / 659 / 660 / 664 / 718 / 720, wherein the amino acid position is relative to the reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 2 or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0159] In some embodiments, the amino acid sequence of the engineered RNA polymerase comprises at least one set of substitutions or amino acid residues, such as 138R / 173Y / 311G / 336S / 340W / 357E / 388K / 394K / 401R / 404L / 514S / 659R / 660H / 664P, 138R / 170L / 173Y / 311G / 336S / 340W / 357E / 388K / 394K / 401R / 404L / 514S / 609P / 659R / 660H / 664P, or 38G / 138R / 173Y / 185L / 187L / 311G / 336S / 340W / 357E / 38 8K / 394K / 401R / 404L / 514S / 609C / 659R / 660H / 664P, 38G / 138R / 173Y / 18 5L / 187H / 311G / 336S / 340W / 357E / 388K / 394K / 401R / 404L / 514S / 659R / 66 0H / 664P、38G / 138R / 170L / 311G / 336S / 340W / 357E / 388K / 394K / 401R / 40 4L / 514S / 659R / 660H / 664P, 38G / 138R / 185L / 187L / 311G / 336S / 340W / 357 E / 388K / 394K / 401R / 404L / 514S / 659R / 660H / 664P / 720A, 138R / 170L / 17 3Y / 185L / 187H / 311G / 336S / 340W / 357E / 388K / 394K / 401R / 404L / 514S / 60 9C / 659R / 660H / 664P, 138R / 173Y / 311G / 336S / 340W / 357E / 388K / 394K / 4 01R / 404L / 514S / 609E / 659R / 660H / 664P、138R / 170L / 173Y / 185L / 187H / 3 11G / 336S / 340W / 357E / 388K / 394K / 401R / 404L / 514S / 609C / 659R / 660H / 664P / 721S, 38G / 138R / 173Y / 311G / 336S / 340W / 357E / 388K / 394K / 401R / 4 04L / 514S / 609E / 659R / 660H / 664P、138R / 170L / 173Y / 185L / 187L / 311G / 336S / 340W / 357E / 388K / 394K / 401R / 404L / 514S / 609E / 659R / 660H / 664P,38G / 138R / 170L / 173Y / 246V / 311G / 336S / 340W / 357E / 388K / 394K / 401R / 404L / 514S / 609P / 659R / 660H / 664P、138R / 173Y / 185L / 187H / 311G / 336S / 340W / 357E / 388K / 394K / 401R / 404L / 514S / 609E / 659R / 660H / 664P / 718S、38G / 138R / 173Y / 185L / 187L / 311G / 336S / 340W / 357E / 388K / 394K / 401R / 40 4L / 514S / 609W / 659R / 660H / 664P / 718N / 720A、38G / 138R / 170L / 173Y / 246 V / 311G / 336S / 340W / 357E / 388K / 394K / 401R / 404L / 514S / 609C / 659R / 660 H / 664P / 718S、138R / 173Y / 311G / 336S / 340W / 357E / 388K / 394K / 401R / 404 L / 514S / 659R / 660H / 664P / 720A、138R / 173Y / 246V / 311G / 336S / 340W / 357E / 388K / 394K / 401R / 404L / 514S / 609E / 659R / 660H / 664P、38G / 138R / 173Y / 185L / 187L / 246V / 311G / 336S / 340W / 357E / 388K / 394K / 401R / 404L / 514S / 659R / 660H / 664P / 720A, 38G / 138R / 173Y / 311G / 336S / 340W / 357E / 388K / 394K / 401R / 404L / 514S / 609C / 659R / 660H / 664P / 721S, 38G / 138R / 173Y / 31 1G / 336S / 340W / 357E / 388K / 394K / 401R / 404L / 514S / 659R / 660H / 664P / 721S, 38G / 138R / 173Y / 311G / 336S / 340W / 357E / 388K / 394K / 401R / 404L / 514 S / 659R / 660H / 664P / 720A / 721S、38G / 138R / 170L / 173Y / 311G / 336S / 340W / 357E / 388K / 394K / 401R / 404L / 514S / 609C / 659R / 660H / 664P / 718S / 720A、38G / 138R / 173Y / 311G / 336S / 340W / 357E / 388K / 394K / 401R / 404L / 514S / 659R / 660H / 664P / 718S、38G / 138R / 170L / 173Y / 246V / 311G / 336S / 340W / 357E / 3 88K / 394K / 401R / 404L / 514S / 609P / 659R / 660H / 664P / 718S、38G / 138R / 173Y / 185L / 187L / 311G / 336S / 340W / 357E / 388K / 394K / 401R / 404L / 514S / 609P / 6 59R / 660H / 664P / 718S / 721S, 138R / 173Y / 311G / 336S / 340W / 357E / 388K / 394K / 401R / 404L / 514S / 529R / 609E / 659R / 660H / 664P, 38G / 138R / 173Y / 311G / 3 36S / 340W / 357E / 388K / 394K / 401R / 404L / 514S / 609P / 659R / 660H / 664P / 718S, 138R / 173Y / 185L / 187L / 311G / 336S / 340W / 357E / 388K / 394K / 401R / 404L / 5 14S / 609C / 659R / 660H / 664P / 718S / 720A、138R / 173Y / 185L / 187H / 311G / 336 S / 340W / 357E / 388K / 394K / 401R / 404L / 514S / 609C / 659R / 660H / 664P / 718S / 721S, 38G / 138R / 170L / 173Y / 311G / 336S / 340W / 357E / 388K / 394K / 401R / 404L / 514S / 659R / 660H / 664P / 718S / 720A / 721S, 138R / 170L / 173Y / 185L / 187L / 311G / 336S / 340W / 357E / 388K / 394K / 401R / 404L / 514S / 659R / 660H / 664P / 718S / 720A, 38G / 138R / 185L / 187L / 246V / 311G / 336S / 340W / 357E / 388K / 394K / 401R / 404L / 514S / 609E / 659R / 660H / 664P / 720A、138R / 173Y / 311G / 336S / 34 0W / 357E / 388K / 394K / 401R / 404L / 514S / 609E / 659R / 660H / 664P / 718S / 720A、138R / 170L / 173Y / 185L / 187H / 311G / 336S / 340W / 357E / 388K / 394K / 401R / 404L / 514S / 659R / 660H / 664P / 718S / 720A / 721S, 38G / 138R / 170L / 2 46V / 311G / 336S / 340W / 357E / 388K / 394K / 401R / 404L / 514S / 609E / 659R / 660H / 664P / 718S、138R / 173Y / 246V / 311G / 336S / 340W / 357E / 388K / 39 4K / 401R / 404L / 514S / 659R / 660H / 664P / 721S, 38G / 138R / 173Y / 311G / 336S / 340W / 357E / 388K / 394K / 401R / 404L / 514S / 659R / 660H / 664P / 718S / 720A or 38G / 138R / 173Y / 311G / 336S / 340W / 357E / 388K / 394K / 401R / 404L / 514S / 609E / 659R / 660H / 664P / 718S / 720A, wherein the amino acid position is relative to the corresponding SEQ ID Reference sequence of residues 8 to 890 of NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0160] In some embodiments, the amino acid sequence of the engineered RNA polymerase contains at least one set of substitutions at the following amino acid positions: 38 / 138 / 143 / 173 / 311 / 336 / 340 / 388 / 401 / 404 / 514 / 659 / 660, 38 / 105 / 138 / 173 / 311 / 336 / 340 / 388 / 401 / 404 / 514 / 659 / 660, 38 / 138 / 173 / 311 / 336 / 340 / 388 / 401 / 404 / 514 / 659 / 660 / 775 / 779, 38 / 135 / 138 / 173 / 311 / 336 / 340 / 388 / 401 / 404 / 5 14 / 659 / 660, 38 / 100 / 138 / 173 / 311 / 336 / 340 / 388 / 401 / 404 / 514 / 659 / 660, 38 / 138 / 173 / 246 / 311 / 336 / 340 / 388 / 401 / 404 / 514 / 659 / 660, 38 / 138 / 173 / 311 / 336 / 340 / 388 / 401 / 404 / 514 / 659 / 660 / 779 / 782, 38 / 138 / 173 / 311 / 336 / 340 / 388 / 401 / 404 / 514 / 659 / 660 / 749, 38 / 138 / 173 / 237 / 311 / 336 / 340 / 388 / 401 / 404 / 514 / 659 / 660, 38 / 138 / 162 / 173 / 311 / 336 / 340 / 388 / 401 / 404 / 514 / 659 / 660, 38 / 83 / 138 / 173 / 311 / 336 / 340 / 388 / 401 / 404 / 514 / 659 / 660, 38 / 138 / 173 / 218 / 311 / 336 / 340 / 388 / 401 / 404 / 514 / 659 / 660, 38 / 138 / 173 / 227 / 311 / 336 / 340 / 388 / 401 / 404 / 514 / 659 / 660, 38 / 115 / 138 / 173 / 311 / 336 / 340 / 388 / 401 / 404 / 514 / 659 / 660, 38 / 138 / 173 / 274 / 311 / 336 / 340 / 388 / 401 / 404 / 514 / 659 / 660, 38 / 81 / 138 / 173 / 311 / 336 / 340 / 388 / 401 / 404 / 514 / 659 / 660, 38 / 122 / 138 / 173 / 311 / 336 / 340 / 388 / 401 / 404 / 514 / 659 / 660, 38 / 61 / 138 / 173 / 311 / 336 / 340 / 388 / 401 / 404 / 514 / 659 / 660,32 / 38 / 138 / 173 / 311 / 336 / 340 / 388 / 401 / 404 / 514 / 659 / 660、38 / 138 / 139 / 173 / 311 / 336 / 340 / 388 / 401 / 404 / 514 / 659 / 660、38 / 75 / 138 / 173 / 311 / 336 / 340 / 388 / 401 / 404 / 514 / 659 / 660、38 / 138 / 173 / 239 / 311 / 336 / 340 / 388 / 401 / 404 / 514 / 659 / 660、38 / 138 / 173 / 257 / 311 / 336 / 340 / 388 / 401 / 404 / 514 / 659 / 660、38 / 138 / 173 / 240 / 311 / 336 / 340 / 388 / 401 / 404 / 514 / 659 / 660、38 / 69 / 138 / 161 / 173 / 311 / 336 / 340 / 388 / 401 / 404 / 514 / 659 / 660、38 / 138 / 173 / 253 / 311 / 336 / 340 / 388 / 401 / 404 / 514 / 659 / 660、14 / 38 / 138 / 173 / 311 / 336 / 340 / 388 / 401 / 404 / 514 / 659 / 660、38 / 138 / 173 / 221 / 311 / 336 / 340 / 388 / 401 / 404 / 514 / 659 / 660、38 / 84 / 138 / 173 / 311 / 336 / 340 / 388 / 401 / 404 / 514 / 659 / 660、38 / 133 / 138 / 150 / 173 / 311 / 336 / 340 / 388 / 401 / 404 / 514 / 659 / 660、38 / 60 / 138 / 173 / 311 / 336 / 340 / 388 / 401 / 404 / 514 / 659 / 660、38 / 138 / 173 / 275 / 311 / 336 / 340 / 388 / 401 / 404 / 514 / 659 / 660、38 / 89 / 138 / 173 / 311 / 336 / 340 / 388 / 401 / 404 / 514 / 659 / 660、15 / 38 / 138 / 173 / 311 / 336 / 340 / 388 / 401 / 404 / 514 / 659 / 660、38 / 138 / 173 / 254 / 311 / 336 / 340 / 388 / 401 / 404 / 514 / 659 / 660、38 / 138 / 173 / 311 / 336 / 340 / 388 / 401 / 404 / 514 / 625 / 659 / 660、14 / 38 / 67 / 138 / 173 / 311 / 336 / 340 / 388 / 401 / 404 / 514 / 659 / 660、21 / 38 / 138 / 173 / 311 / 336 / 340 / 388 / 401 / 404 / 514 / 659 / 660, 38 / 93 / 138 / 173 / 311 / 336 / 340 / 388 / 401 / 404 / 514 / 659 / 660, 38 / 138 / 173 / 250 / 311 / 336 / 340 / 388 / 401 / 404 / 514 / 659 / 660, 38 / 138 / 173 / 217 / 311 / 336 / 340 / 388 / 401 / 404 / 514 / 659 / 660, 19 / 38 / 138 / 173 / 311 / 336 / 340 / 388 / 401 / 404 / 514 / 659 / 660, 38 / 95 / 138 / 173 / 311 / 336 / 340 / 388 / 401 / 404 / 514 / 659 / 660, 38 / 138 / 173 / 311 / 336 / 340 / 388 / 401 / 404 / 514 / 659 / 660 / 759, 38 / 62 / 138 / 173 / 311 / 336 / 340 / 388 / 401 / 404 / 514 / 659 / 660 or 38 / 138 / 140 / 173 / 311 / 336 / 340 / 388 / 401 / 404 / 514 / 659 / 660, wherein the amino acid position is relative to the corresponding SEQ Reference sequence of residues 8 to 890 of IDNO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0161] In some embodiments, the amino acid sequence of the engineered RNA polymerase comprises at least one set of substitutions or amino acid residues: 38G / 138R / 143N / 173Y / 311G / 336S / 340W / 388K / 401R / 404L / 514S / 659R / 660H, 38G / 138R / 143Q / 173Y / 311G / 336S / 340W / 388K / 401R / 404L / 514S / 659R / 660H, 38G / 138R / 143R / 173Y / 311G / 336S / 340W / 388K / 401R / 404L / 514S / 659R / 660H, 38G / 105R / 138R / 173Y / 311G / 336S / 340W / 388K / 401R / 404L / 514S / 659R / 660H ,38G / 138R / 173Y / 311G / 336S / 340W / 388K / 401R / 404L / 514S / 659R / 660H / 775R / 779R, 38G / 135K / 138R / 173Y / 311G / 336S / 340W / 388K / 401R / 404L / 514S / 659R / 660H, 38G / 100R / 138R / 173Y / 311G / 336S / 340W / 388K / 401R / 4 04L / 514S / 659R / 660H, 38G / 138R / 173Y / 311G / 336S / 340W / 388K / 401R / 4 04L / 514S / 659R / 660H / 775K / 779R、38G / 138R / 173Y / 246K / 311G / 336S / 34 0W / 388K / 401R / 404L / 514S / 659R / 660H、38G / 138R / 173Y / 311G / 336S / 34 0W / 388K / 401R / 404L / 514S / 659R / 660H / 779R / 782R、38G / 138R / 173Y / 311 G / 336S / 340W / 388K / 401R / 404L / 514S / 659R / 660H / 749R、38G / 138R / 173 Y / 237L / 311G / 336S / 340W / 388K / 401R / 404L / 514S / 659R / 660H, 38G / 138R / 162V / 173Y / 311G / 336S / 340W / 388K / 401R / 404L / 514S / 659R / 660H, 38G / 83K / 138R / 173Y / 311G / 336S / 340W / 388K / 401R / 404L / 514S / 659R / 660H、38G / 138R / 173Y / 218T / 311G / 336S / 340W / 388K / 401R / 404L / 514S / 659R / 660H、38G / 138R / 173Y / 227V / 311G / 336S / 340W / 388K / 401R / 404L / 514S / 65 9R / 660H, 38G / 115L / 138R / 173Y / 311G / 336S / 340W / 388K / 401R / 404L / 514S / 659R / 660H, 38G / 138R / 173Y / 274G / 311G / 336S / 340W / 388K / 401R / 404L / 514S / 659R / 660H、38G / 81C / 138R / 173Y / 311G / 336S / 340W / 388K / 401R / 404L / 514S / 659R / 660H、38G / 122I / 138R / 173Y / 311G / 336S / 340W / 388K / 40 1R / 404L / 514S / 659R / 660H、38G / 61Y / 138R / 173Y / 311G / 336S / 340W / 388K / 401R / 404L / 514S / 659R / 660H、32S / 38G / 138R / 173Y / 311G / 336S / 340W / 3 88K / 401R / 404L / 514S / 659R / 660H, 38G / 138R / 139R / 173Y / 311G / 336S / 340W / 388K / 401R / 404L / 514S / 659R / 660H, 38G / 75Y / 138R / 173Y / 311G / 336S 340W / 388K / 401R / 404L / 514S / 659R / 660H, 38G / 138R / 173Y / 239R / 311G / 336S / 340W / 388K / 401R / 404L / 514S / 659R / 660H, 38G / 138R / 173Y / 257L / 3 11G / 336S / 340W / 388K / 401R / 404L / 514S / 659R / 660H、38G / 138R / 173Y / 24 0M / 311G / 336S / 340W / 388K / 401R / 404L / 514S / 659R / 660H、38G / 69S / 138R / 161R / 173Y / 311G / 336S / 340W / 388K / 401R / 404L / 514S / 659R / 660H、38G / 138R / 173Y / 253Y / 311G / 336S / 340W / 388K / 401R / 404L / 514S / 659R / 660H、14Y / 38G / 138R / 173Y / 311G / 336S / 340W / 388K / 401R / 404L / 514S / 659R / 6 60H、38G / 138R / 173Y / 221C / 311G / 336S / 340W / 388K / 401R / 404L / 514S / 65 9R / 660H, 38G / 84A / 138R / 173Y / 311G / 336S / 340W / 388K / 401R / 404L / 514S / 659R / 660H, 38G / 115V / 138R / 173Y / 311G / 336S / 340W / 388K / 401R / 404L / 514S / 659R / 660H、38G / 133C / 138R / 150Q / 173Y / 311G / 336S / 340W / 388K / 401R / 404L / 514S / 659R / 660H、38G / 60L / 138R / 173Y / 311G / 336S / 340W / 38 8K / 401R / 404L / 514S / 659R / 660H、38G / 60G / 138R / 173Y / 311G / 336S / 340W / 388K / 401R / 404L / 514S / 659R / 660H、32V / 38G / 138R / 173Y / 311G / 336S / 3 40W / 388K / 401R / 404L / 514S / 659R / 660H、38G / 138R / 173Y / 275A / 311G / 3 36S / 340W / 388K / 401R / 404L / 514S / 659R / 660H、38G / 89Q / 138R / 173Y / 311 G / 336S / 340W / 388K / 401R / 404L / 514S / 659R / 660H、38G / 138R / 173Y / 257V / 311G / 336S / 340W / 388K / 401R / 404L / 514S / 659R / 660H、38G / 138R / 173Y / 274L / 311G / 336S / 340W / 388K / 401R / 404L / 514S / 659R / 660H, 15T / 38G / 138R / 173Y / 311G / 336S / 340W / 388K / 401R / 404L / 514S / 659R / 660H, 38G / 138 R / 173Y / 254S / 311G / 336S / 340W / 388K / 401R / 404L / 514S / 659R / 660H、38G / 61W / 138R / 173Y / 311G / 336S / 340W / 388K / 401R / 404L / 514S / 659R / 660H、38G / 138R / 173Y / 311G / 336S / 340W / 388K / 401R / 404L / 514S / 625C / 659R / 660H, 14Q / 38G / 67R / 138R / 173Y / 311G / 336S / 340W / 388K / 401R / 404L / 514S / 659R / 660H, 21G / 38G / 138R / 173Y / 311G / 336S / 340W / 388K / 401R / 404L / 514S / 659R / 660H, 38G / 138R / 173Y / 254G / 311G / 336S / 340W / 388K / 401R / 404L / 514S / 659R / 660H, 38G / 93Y / 138R / 173Y / 311G / 336S / 340W / 388K / 401R / 404L / 514S / 659R / 660H, 38G / 138R / 173Y / 250R / 311G / 336S / 340W / 38 8K / 401R / 404L / 514S / 659R / 660H, 38G / 138R / 173Y / 217V / 311G / 336S / 340W / 388K / 401R / 404L / 514S / 659R / 660H, 38G / 60M / 138R / 173Y / 311G / 336S / 340W / 388K / 401R / 404L / 514S / 659R / 660H, 38G / 138R / 173Y / 239L / 311G / 336S / 340W / 388K / 401R / 404L / 514S / 659R / 660H, 38G / 75S / 138R / 173Y / 31 1G / 336S / 340W / 388K / 401R / 404L / 514S / 659R / 660H、32T / 38G / 138R / 173Y / 311G / 336S / 340W / 388K / 401R / 404L / 514S / 659R / 660H、19E / 38G / 138R / 173Y / 311G / 336S / 340W / 388K / 401R / 404L / 514S / 659R / 660H、38G / 95Y / 138R / 173Y / 311G / 336S / 340W / 388K / 401R / 404L / 514S / 659R / 660H、38G / 81 L / 138R / 173Y / 311G / 336S / 340W / 388K / 401R / 404L / 514S / 659R / 660H、38G / 60R / 138R / 173Y / 311G / 336S / 340W / 388K / 401R / 404L / 514S / 659R / 660H、38G / 138R / 173Y / 311G / 336S / 340W / 388K / 401R / 404L / 514S / 659R / 660H / 759M, 38G / 62T / 138R / 173Y / 311G / 336S / 340W / 388K / 401R / 404L / 514S / 659R / 660H, or 38G / 138R / 140R / 173Y / 311G / 336S / 340W / 388K / 401R / 404L / 514S / 659R / 660H, wherein the amino acid position is relative to the reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 2 or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0162] In some embodiments, the amino acid sequence of the engineered RNA polymerase contains at least one set of substitutions at the following amino acid positions: 38 / 138 / 170 / 311 / 336 / 340 / 357 / 388 / 394 / 401 / 404 / 514 / 609 / 659 / 660 / 664 / 779, 38 / 138 / 170 / 246 / 311 / 336 / 340 / 357 / 388 / 394 / 401 / 404 / 514 / 609 / 659 / 660 / 664, 38 / 138 / 170 / 246 / 311 / 336 / 340 / 357 / 388 / 394 / 401 / 404 / 514 / 659 / 660 / 664, 38 / 138 / 170 / 311 / 336 / 340 / 357 / 388 / 394 / 401 / 404 / 514 / 609 / 659 / 660 / 664 / 775 / 779, 38 / 105 / 138 / 170 / 311 / 336 / 340 / 357 / 388 / 394 / 401 / 404 / 514 / 659 / 660 / 664、38 / 138 / 143 / 170 / 311 / 336 / 340 / 357 / 388 / 394 / 401 / 404 / 514 / 609 / 659 / 660 / 664 / 775、38 / 105 / 135 / 138 / 170 / 311 / 336 / 340 / 357 / 388 / 394 / 401 / 404 / 514 / 6 09 / 659 / 660 / 664 / 775, 38 / 111 / 138 / 170 / 311 / 336 / 340 / 357 / 388 / 394 / 401 / 404 / 514 / 609 / 659 / 660 / 664 or 38 / 138 / 170 / 311 / 336 / 340 / 357 / 388 / 394 / 401 / 404 / 514 / 609 / 659 / 660 / 664, wherein the amino acid position is relative to the reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 2 or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0163] In some embodiments, the amino acid sequence of the engineered RNA polymerase contains at least one set of substitutions or amino acid residues: 38G / 138R / 170L / 311G / 336S / 340W / 357E / 388K / 394K / 401R / 404L / 514S / 609E / 659R / 660H / 664P / 779R, 38G / 138R / 170L / 246K / 311G / 336S / 340W / 357E / 388K / 394K / 401R / 404L / 514S / 609E / 659R / 660H / 6 64P、38G / 138R / 170L / 246K / 311G / 336S / 340W / 357E / 388K / 394K / 401R / 404L / 514S / 659R / 660H / 664P、38G / 138R / 170L / 311G / 336S / 340W / 357E / 388K / 394K / 401R / 404L / 514S / 609E / 659R / 660H / 664P / 775K / 779R, 38G / 105R / 138R / 170L / 311G / 336S / 340W / 357E / 388K / 394K / 401R / 404L / 514S / 659R / 660H / 664P、38G / 138R / 143N / 170L / 311G / 336S / 340W / 357E / 388K / 394K / 401R / 40 4L / 514S / 609E / 659R / 660H / 664P / 775K, 38G / 105R / 135K / 138R / 170L / 311G / 336S / 340W / 357E / 388K / 394K / 401R / 404L / 514S / 6 09E / 659R / 660H / 664P / 775K, 38G / 111K / 138R / 170L / 311G / 336S / 340W / 357E / 388K / 394K / 401R / 404L / 514S / 609E / 659R / 660H / 664P or 38G / 138R / 170L / 311G / 336S / 340W / 357E / 388K / 394K / 401R / 404L / 514S / 609E / 659R / 660H / 664P, wherein the amino acid position is relative to the reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 2 or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0164] In some embodiments, the amino acid sequence of the engineered RNA polymerase contains at least one set of substitutions at the following amino acid positions: 32 / 38 / 138 / 170 / 246 / 311 / 336 / 340 / 357 / 388 / 394 / 401 / 404 / 514 / 609 / 659 / 660 / 664, 38 / 138 / 170 / 246 / 311 / 336 / 340 / 357 / 388 / 394 / 401 / 404 / 514 / 531 / 533 / 609 / 659 / 660 / 664, 38 / 115 / 138 / 170 / 246 / 311 / 336 / 340 / 357 / 388 / 394 / 401 / 404 / 51 4 / 609 / 659 / 660 / 664, 38 / 83 / 138 / 170 / 246 / 311 / 336 / 340 / 357 / 388 / 394 / 401 / 404 / 514 / 609 / 659 / 660 / 664, 32 / 38 / 60 / 61 / 138 / 170 / 246 / 311 / 336 / 340 / 357 / 388 / 394 / 401 / 404 / 514 / 609 / 659 / 660 / 664, 38 / 55 / 138 / 170 / 246 / 311 / 336 / 340 / 357 / 388 / 394 / 401 / 404 / 514 / 609 / 659 / 660 / 664, 38 / 138 / 17 0 / 246 / 311 / 336 / 340 / 357 / 388 / 394 / 401 / 404 / 514 / 589 / 609 / 659 / 660 / 664, 38 / 138 / 170 / 246 / 311 / 336 / 340 / 357 / 388 / 394 / 401 / 404 / 514 / 609 / 659 / 660 / 664, 38 / 138 / 170 / 246 / 311 / 336 / 340 / 357 / 388 / 394 / 401 / 404 / 514 / 609 / 617 / 659 / 660 / 664, 38 / 138 / 170 / 172 / 246 / 311 / 336 / 340 / 357 / 388 / 394 / 401 / 404 / 514 / 609 / 659 / 660 / 664, 38 / 138 / 170 / 246 / 311 / 336 / 340 / 357 / 388 / 394 / 401 / 404 / 514 / 598 / 609 / 659 / 660 / 664, 38 / 56 / 138 / 170 / 193 / 246 / 311 / 336 / 340 / 357 / 388 / 394 / 401 / 404 / 514 / 609 / 659 / 660 / 664, 38 / 56 / 138 / 170 / 246 / 311 / 336 / 340 / 357 / 388 / 394 / 401 / 404 / 514 / 609 / 659 / 660 / 664,38 / 138 / 170 / 246 / 311 / 336 / 340 / 357 / 388 / 394 / 401 / 404 / 514 / 536 / 609 / 659 / 660 / 664、38 / 52 / 138 / 170 / 246 / 311 / 336 / 340 / 357 / 388 / 394 / 401 / 404 / 514 / 609 / 659 / 660 / 664、17 / 38 / 138 / 170 / 246 / 311 / 336 / 340 / 357 / 388 / 394 / 401 / 404 / 514 / 609 / 659 / 660 / 664、38 / 138 / 170 / 246 / 261 / 311 / 336 / 340 / 357 / 388 / 394 / 401 / 404 / 514 / 609 / 659 / 660 / 664、38 / 78 / 138 / 170 / 246 / 311 / 336 / 340 / 357 / 388 / 394 / 401 / 404 / 514 / 609 / 659 / 660 / 664、38 / 138 / 170 / 246 / 311 / 336 / 340 / 357 / 388 / 394 / 401 / 404 / 514 / 609 / 629 / 659 / 660 / 664、38 / 63 / 138 / 170 / 246 / 311 / 336 / 340 / 357 / 388 / 394 / 401 / 404 / 514 / 609 / 659 / 660 / 664、38 / 82 / 138 / 170 / 246 / 311 / 336 / 340 / 357 / 388 / 394 / 401 / 404 / 514 / 609 / 659 / 660 / 664、38 / 138 / 170 / 207 / 246 / 311 / 336 / 340 / 357 / 388 / 394 / 401 / 404 / 514 / 609 / 659 / 660 / 664、38 / 138 / 170 / 246 / 311 / 336 / 340 / 357 / 388 / 394 / 401 / 404 / 514 / 609 / 659 / 660 / 664 / 666、38 / 138 / 150 / 170 / 246 / 311 / 336 / 340 / 357 / 388 / 394 / 401 / 404 / 514 / 609 / 659 / 660 / 664、38 / 45 / 138 / 170 / 246 / 311 / 336 / 340 / 357 / 388 / 394 / 401 / 404 / 514 / 609 / 659 / 660 / 664、38 / 135 / 138 / 170 / 246 / 311 / 336 / 340 / 357 / 388 / 394 / 401 / 404 / 514 / 609 / 659 / 660 / 664、13 / 38 / 138 / 170 / 246 / 311 / 336 / 340 / 357 / 388 / 394 / 401 / 404 / 514 / 609 / 659 / 660 / 664、33 / 38 / 138 / 170 / 246 / 311 / 336 / 340 / 357 / 388 / 394 / 401 / 404 / 514 / 609 / 659 / 660 / 664 or 38 / 138 / 170 / 245 / 246 / 311 / 336 / 340 / 357 / 388 / 394 / 401 / 404 / 514 / 609 / 659 / 660 / 664, wherein the amino acid position is relative to the reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 2 or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0165] In some embodiments, the amino acid sequence of the engineered RNA polymerase contains at least one set of substitutions or amino acid residues, such as 32V / 38G / 138R / 170L / 246K / 311G / 336S / 340W / 357E / 388K / 394K / 401R / 404L / 514S / 609E / 659R / 660H / 664P, 38G / 138R / 170L / 246K / 311G / 336S / 340W / 357E / 388K / 394K / 401R / 404L / 514S / 531Q / 533S / 609E / 659R / 660H / 664P, 38G / 115L / 1 38R / 170L / 246K / 311G / 336S / 340W / 357E / 388K / 394K / 401R / 404L / 514S / 609E / 659R / 660H / 664P、38G / 83K / 138R / 170L / 246K / 311G / 336S / 340W / 357E / 388K / 394K / 401R / 404L / 514S / 609E / 659R / 660H / 664P, 32V / 38G / 60M / 61W / 138R / 170L / 246K / 311G / 336S / 340W / 357E / 388K / 394K / 401R / 4 04L / 514S / 609E / 659R / 660H / 664P, 38G / 55D / 138R / 170L / 246K / 311G / 3 36S / 340W / 357E / 388K / 394K / 401R / 404L / 514S / 609E / 659R / 660H / 664P, 38G / 138R / 170L / 246K / 311G / 336S / 340W / 357E / 388K / 394K / 401R / 404L / 514S / 589C / 609E / 659R / 660H / 664P、38G / 138R / 170L / 246K / 311G / 336S / 340W / 357P / 388K / 394K / 401R / 404L / 514S / 609E / 659R / 660H / 664P, 38 G / 138R / 170L / 246K / 311G / 336S / 340W / 357E / 388K / 394K / 401R / 404L / 51 4S / 609E / 617R / 659R / 660H / 664P, 38G / 138R / 170L / 172G / 246K / 311G / 3 36S / 340W / 357E / 388K / 394K / 401R / 404L / 514S / 609E / 659R / 660H / 664P,38G / 138R / 170L / 246K / 311G / 336S / 340W / 357E / 388K / 394K / 401R / 404L / 51 4S / 598A / 609E / 659R / 660H / 664P、38G / 56G / 138R / 170L / 193G / 246K / 311G / 336S / 340W / 357E / 388K / 394K / 401R / 404L / 514S / 609E / 659R / 660H / 664P, 38G / 56T / 138R / 170L / 246K / 311G / 336S / 340W / 357E / 388K / 394K / 401R / 404L / 514S / 609E / 659R / 660H / 664P、38G / 138R / 170L / 246K / 311G / 336S / 340W / 3 57E / 388K / 394K / 401R / 404L / 514S / 536V / 609E / 659R / 660H / 664P、38G / 52T / 138R / 170L / 246K / 311G / 336S / 340W / 357E / 388K / 394K / 401R / 404L / 514S / 609E / 659R / 660H / 664P、17N / 38G / 138R / 170L / 246K / 311G / 336S / 340W / 357 E / 388K / 394K / 401R / 404L / 514S / 609E / 659R / 660H / 664P, 38G / 56N / 138R / 170L / 246K / 311G / 336S / 340W / 357E / 388K / 394K / 401R / 404L / 514S / 609E / 65 9R / 660H / 664P、38G / 138R / 170L / 246K / 311G / 336S / 340W / 357E / 388K / 394K / 401R / 404L / 514S / 598K / 609E / 659R / 660H / 664P、38G / 138R / 170L / 246K / 2 61M / 311G / 336S / 340W / 357E / 388K / 394K / 401R / 404L / 514S / 609E / 659R / 660H / 664P, 38G / 56E / 138R / 170L / 246K / 311G / 336S / 340W / 357E / 388K / 394K / 401R / 404L / 514S / 609E / 659R / 660H / 664P、38G / 55S / 138R / 170L / 246K / 311 G / 336S / 340W / 357E / 388K / 394K / 401R / 404L / 514S / 609E / 659R / 660H / 664P、38G / 138R / 170L / 246K / 311G / 336S / 340W / 357E / 388K / 394K / 401R / 404L / 514S / 536E / 609E / 659R / 660H / 664P、38G / 138R / 170L / 246K / 311G / 336S / 340 W / 357E / 388K / 394K / 401R / 404L / 514S / 598L / 609E / 659R / 660H / 664P, 38G / 78S / 138R / 170L / 246K / 311G / 336S / 340W / 357E / 388K / 394K / 401R / 404L / 51 4S / 609E / 659R / 660H / 664P、38G / 56V / 138R / 170L / 246K / 311G / 336S / 340W / 357E / 388K / 394K / 401R / 404L / 514S / 609E / 659R / 660H / 664P、38G / 138R / 17 0L / 246K / 311G / 336S / 340W / 357E / 388K / 394K / 401R / 404L / 514S / 609E / 629R / 659R / 660H / 664P、38G / 138R / 170L / 246K / 311G / 336S / 340W / 357E / 388K / 394K / 401R / 404L / 514S / 598R / 609E / 659R / 660H / 664P, 38G / 63S / 138R / 170L / 246K / 311G / 336S / 340W / 357E / 388K / 394K / 401R / 404L / 514S / 609E / 659R / 660H / 664P、38G / 82S / 138R / 170L / 246K / 311G / 336S / 340W / 357E / 388K / 39 4K / 401R / 404L / 514S / 609E / 659R / 660H / 664P、38G / 138R / 170L / 207S / 246K / 311G / 336S / 340W / 357E / 388K / 394K / 401R / 404L / 514S / 609E / 659R / 660H / 664P、38G / 138R / 170L / 246K / 311G / 336S / 340W / 357E / 388K / 394K / 401R / 40 4L / 514S / 598V / 609E / 659R / 660H / 664P、38G / 138R / 170L / 246K / 311G / 336S / 340W / 357E / 388K / 394K / 401R / 404L / 514S / 609E / 659R / 660H / 664P / 666M、38G / 138R / 150Y / 170L / 246K / 311G / 336S / 340W / 357E / 388K / 394K / 401R / 404L / 514S / 609E / 659R / 660H / 664P, 38G / 45K / 138R / 170L / 246K / 311G / 336S / 340W / 357E / 388K / 394K / 401R / 404L / 514S / 60 9E / 659R / 660H / 664P, 38G / 55W / 138R / 170L / 246K / 311G / 336S / 340W / 357E / 388K / 394K / 401R / 404L / 514S / 609E / 659R / 660H / 664P、38G / 135G / 138R / 170L / 246K / 311G / 336S / 340W / 357E / 388K / 394K / 401R / 404L / 514S / 609E / 659R / 660H / 664P, 13N / 38G / 138R / 170L / 246K / 311G / 336S / 340W / 357E / 3 88K / 394K / 401R / 404L / 514S / 609E / 659R / 660H / 664P, 33E / 38G / 138R / 170L / 246K / 311G / 336S / 340 W / 357E / 388K / 394K / 401R / 404L / 514S / 609E / 659R / 660H / 664P or 38G / 138R / 170L / 245C / 246K / 311G / 336S / 340W / 357E / 388K / 394K / 401R / 404L / 514S / 609E / 659R / 660H / 664P, wherein the amino acid position is relative to the reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 2 or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0166] In some implementation schemes,The amino acid sequence of the engineered RNA polymerase contains at least one set of substitutions at the following amino acid positions: 32 / 38 / 138 / 170 / 207 / 245 / 246 / 311 / 336 / 340 / 357 / 388 / 394 / 401 / 404 / 514 / 609 / 629 / 659 / 660 / 664, 17 / 32 / 38 / 56 / 138 / 150 / 170 / 246 / 311 / 336 / 340 / 357 / 388 / 394 / 401 / 404 / 514 / 598 / 609 / 659 / 660 / 664, 32 / 38 / 56 / 138 / 170 / 246 / 311 / 336 / 340 / 357 / 388 / 394 / 401 / 404 / 514 / 609 / 629 / 659 / 660 / 664、32 / 38 / 138 / 170 / 246 / 311 / 336 / 340 / 357 / 388 / 394 / 401 / 404 / 514 / 536 / 609 / 659 / 660 / 664、32 / 38 / 138 / 170 / 185 / 246 / 311 / 336 / 340 / 357 / 388 / 394 / 401 / 404 / 514 / 609 / 659 / 660 / 664, 32 / 38 / 56 / 138 / 150 / 170 / 246 / 311 / 336 / 340 / 357 / 388 / 394 / 401 / 404 / 514 / 609 / 659 / 660 / 664, 17 / 32 / 38 / 56 / 138 / 150 / 170 / 207 / 245 / 246 / 311 / 336 / 340 / 357 / 388 / 394 / 401 / 404 / 514 / 598 / 609 / 617 / 629 / 659 / 660 / 664, 32 / 38 / 56 / 138 / 150 / 170 / 246 / 311 / 336 / 340 / 357 / 388 / 394 / 401 / 404 / 514 / 536 / 598 / 609 / 659 / 660 / 664、32 / 38 / 56 / 138 / 170 / 246 / 311 / 336 / 340 / 357 / 388 / 394 / 401 / 404 / 514 / 536 / 609 / 659 / 660 / 664, 32 / 38 / 138 / 170 / 185 / 246 / 311 / 336 / 340 / 357 / 388 / 394 / 401 / 404 / 514 / 609 / 659 / 660 / 664 / 666, 32 / 38 / 138 / 150 / 170 / 246 / 311 / 336 / 3 40 / 357 / 388 / 394 / 401 / 404 / 514 / 609 / 659 / 660 / 664 or 32 / 38 / 138 / 143 / 150 / 170 / 246 / 311 / 336 / 340 / 357 / 388 / 394 / 401 / 404 / 514 / 609 / 659 / 660 / 664 / 782,The amino acid positions are relative to the reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 2 or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0167] In some embodiments, the amino acid sequence of the engineered RNA polymerase contains at least one set of substitutions or amino acid residues: 32V / 38G / 138R / 170L / 207S / 245C / 246K / 311G / 336S / 340W / 357E / 388K / 394K / 401R / 404L / 514S / 609E / 629R / 659R / 660H / 664P, 17N / 32V / 38G / 56E / 138R / 150Y / 170L / 246K / 311G / 336S / 340W / 357E / 388K / 394K / 401R / 404L / 514S / 598A / 609E / 659R / 660H / 664P, 32V / 38G / 56E / 138R / 170L / 246K / 311G / 336S / 340W / 357E / 388 K / 394K / 401R / 404L / 514S / 609E / 629R / 659R / 660H / 664P, 32V / 38G / 138R / 170L / 246K / 311G / 336S / 340W / 357E / 388K / 394K / 401R / 404L / 514S / 536V / 609E / 659R / 660H / 664P, 32V / 38G / 138R / 170L / 185P / 246K / 311G / 336S / 34 0W / 357E / 388K / 394K / 401R / 404L / 514S / 609E / 659R / 660H / 664P, 32V / 38G / 56T / 138R / 150Y / 170L / 246K / 311G / 336S / 340W / 357E / 388K / 394K / 401R / 404L / 514S / 609E / 659R / 660H / 664P, 17N / 32V / 38G / 56E / 138R / 150Y / 170L / 207S / 245C / 246K / 311G / 336S / 340W / 357E / 388K / 394K / 401R / 404L / 514S / 598A / 609E / 617R / 629R / 659R / 660H / 664P、32V / 38G / 56E / 138R / 150Y / 17 0L / 246K / 311G / 336S / 340W / 357E / 388K / 394K / 401R / 404L / 514S / 536V / 59 8K / 609E / 659R / 660H / 664P, 32V / 38G / 56V / 138R / 170L / 246K / 311G / 336S / 340W / 357E / 388K / 394K / 401R / 404L / 514S / 536V / 609E / 659R / 660H / 664P,32V / 38G / 138R / 170L / 185P / 246K / 311G / 336S / 340W / 357E / 388K / 394K / 401R / 404L / 514S / 609E / 659R / 660H / 664P / 666M, 32V / 38G / 138R / 150Y / 170L / 246K / 311G / 336S / 340W / 357E / 388K / 394K / 401R / 404L / 514S / 609E / 659R / 660H / 664P or 32V / 38G / 138R / 143N / 150Y / 170L / 246V / 311G / 336S / 340W / 357E / 388K / 394K / 401R / 404L / 514S / 609E / 659R / 660H / 664P / 782V, wherein the amino acid position is relative to the reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 2 or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0168] In some embodiments, the amino acid sequence of the engineered RNA polymerase contains at least one set of substitutions at the following amino acid positions: 32 / 38 / 56 / 138 / 143 / 170 / 246 / 311 / 336 / 340 / 357 / 388 / 394 / 401 / 404 / 514 / 609 / 629 / 659 / 660 / 664, 32 / 38 / 56 / 138 / 170 / 246 / 311 / 336 / 340 / 357 / 388 / 394 / 401 / 404 / 424 / 514 / 609 / 629 / 648 / 659 / 660 / 664 / 782、32 / 38 / 56 / 138 / 170 / 246 / 311 / 336 / 340 / 357 / 388 / 394 / 401 / 404 / 514 / 609 / 629 / 660 / 664 / 782、32 / 38 / 56 / 138 / 150 / 170 / 246 / 311 / 336 / 340 / 357 / 388 / 394 / 401 / 404 / 514 / 609 / 659 / 660 / 664 / 782, 32 / 38 / 56 / 138 / 170 / 246 / 311 / 336 / 340 / 357 / 388 / 394 / 401 / 404 / 514 / 609 / 629 / 659 / 660 / 664 / 721 / 782, 32 / 38 / 56 / 138 / 170 / 246 / 311 / 336 / 340 / 357 / 388 / 394 / 401 / 404 / 514 / 609 / 629 / 659 / 660 / 664 / 782, 32 / 38 / 5 6 / 138 / 170 / 196 / 246 / 311 / 336 / 340 / 357 / 388 / 394 / 401 / 404 / 514 / 609 / 629 / 660 / 664 / 782 or 32 / 38 / 56 / 138 / 170 / 246 / 311 / 336 / 340 / 357 / 388 / 394 / 401 / 404 / 514 / 609 / 629 / 659 / 660 / 664 / 720 / 721 / 782, wherein the amino acid position is relative to the reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 2 or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0169] In some embodiments, the amino acid sequence of the engineered RNA polymerase contains at least one set of substitutions or amino acid residues: 32V / 38G / 56E / 138R / 143N / 170L / 246K / 311G / 336S / 340W / 357E / 388K / 394K / 401R / 404L / 514S / 609E / 629R / 659R / 660H / 664P, 32V / 38G / 56E / 138R / 170L / 246R / 311G / 336S / 340W / 357E / 388K / 394K / 401R / 404L / 424A / 514S / 609E / 629R / 648F / 659R / 660H / 664P / 782V, 32V / 38G / 56E / 138R / 170L / 246R / 311G / 336S / 340W / 357E / 388K / 394K / 401R / 404L / 514S / 609E / 629R / 660H / 664P / 782V, 32V / 38G / 56T / 138R / 150Y / 170L / 246K / 311G / 336S / 340W / 357E / 388K / 394K / 401R / 404L / 514S / 609E / 659R / 660W / 664P / 782V、32V / 38G / 56E / 138R / 170L / 246K / 311G / 336S / 340W / 357E / 388K / 394K / 401R / 404L / 514S / 609E / 629R / 659R / 660H / 664P / 721T / 782V , 32V / 38G / 56E / 138R / 170L / 246K / 311G / 336S / 340W / 357E / 388K / 394K / 401R / 404L / 514S / 609E / 629R / 659R / 660H / 664P / 782V, 32V / 38G / 5 6E / 138R / 170L / 196G / 246K / 311G / 336S / 340W / 357E / 388K / 394K / 401R / 404L / 514S / 609E / 629R / 660H / 664P / 782V or 32V / 38G / 56E / 138R / 170L / 246K / 311G / 336S / 340W / 357E / 388K / 394K / 401R / 404L / 514S / 609E / 629R / 659R / 660H / 664P / 720S / 721A / 782V, wherein the amino acid position is relative to the reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 2 or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0170] In some embodiments, the amino acid sequence of the engineered RNA polymerase contains at least one set of substitutions at the following amino acid positions: 32 / 38 / 56 / 138 / 143 / 150 / 170 / 246 / 311 / 336 / 340 / 357 / 388 / 394 / 401 / 404 / 514 / 609 / 660 / 664 / 782, 32 / 38 / 56 / 138 / 150 / 166 / 170 / 171 / 246 / 336 / 340 / 357 / 388 / 394 / 401 / 404 / 514 / 609 / 659 / 660 / 664, 32 / 38 / 56 / 138 / 143 / 150 / 170 / 246 / 31 1 / 336 / 340 / 357 / 388 / 394 / 401 / 404 / 514 / 609 / 659 / 660 / 664 / 721, 32 / 38 / 56 / 138 / 150 / 170 / 246 / 311 / 336 / 340 / 357 / 388 / 394 / 401 / 404 / 514 / 609 / 659 / 660 / 664 / 782, or 32 / 38 / 56 / 138 / 143 / 150 / 170 / 171 / 246 / 311 / 336 / 340 / 357 / 388 / 394 / 401 / 404 / 514 / 609 / 660 / 664 / 782, wherein the amino acid position is relative to the position corresponding to SEQ Reference sequence of residues 8 to 890 of SEQ ID NO: 2 or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0171] In some embodiments, the amino acid sequence of the engineered RNA polymerase contains at least one set of substitutions or amino acid residues 32V / 38G / 56T / 138R / 143N / 150Y / 170L / 246K / 311G / 336S / 340W / 357E / 388K / 394K / 401R / 404L / 514S / 609E / 660H / 664P / 782V, 32 V / 38G / 56T / 138R / 150Y / 166T / 170L / 171E / 246R / 336S / 340W / 357E / 388K / 394K / 401R / 40 4L / 514S / 609E / 659R / 660H / 664P, 32V / 38G / 56T / 138R / 143N / 150Y / 170L / 246K / 311G / 336 S / 340W / 357E / 388K / 394K / 401R / 404L / 514S / 609E / 659R / 660H / 664P / 721T, 32V / 38G / 56 T / 138R / 150Y / 170L / 246K / 311G / 336S / 340W / 357E / 388K / 394K / 401R / 404L / 514S / 609E / 6 59R / 660H / 664P / 782V or 32V / 38G / 56T / 138R / 143N / 150Y / 170L / 171E / 246K / 311G / 336S / 340W / 357E / 388K / 394K / 401R / 404L / 514S / 609E / 660H / 664P / 782V, wherein the amino acid position is relative to the reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 2 or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0172] In some embodiments, the amino acid sequence of the engineered RNA polymerase contains at least one set of substitutions at the following amino acid positions: 32 / 38 / 56 / 138 / 150 / 170 / 246 / 311 / 336 / 340 / 357 / 367 / 388 / 394 / 401 / 404 / 514 / 609 / 659 / 660 / 664 / 782, 32 / 38 / 56 / 138 / 150 / 170 / 246 / 311 / 336 / 340 / 357 / 382 / 388 / 394 / 401 / 404 / 514 / 609 / 659 / 660 / 664 / 782, 32 / 38 / 56 / 138 / 150 / 170 / 246 / 311 / 33 6 / 340 / 357 / 388 / 394 / 401 / 404 / 514 / 609 / 659 / 660 / 664 / 670 / 782、32 / 38 / 56 / 138 / 150 / 170 / 246 / 311 / 336 / 340 / 357 / 388 / 394 / 401 / 404 / 514 / 609 / 659 / 660 / 664 / 672 / 782, 32 / 38 / 56 / 138 / 150 / 170 / 246 / 311 / 336 / 340 / 357 / 388 / 394 / 401 / 404 / 514 / 609 / 659 / 660 / 664 / 782, 32 / 38 / 56 / 138 / 150 / 170 / 246 / 3 11 / 336 / 340 / 357 / 388 / 394 / 401 / 404 / 514 / 609 / 659 / 660 / 664 / 693 / 782、32 / 38 / 56 / 138 / 150 / 170 / 246 / 311 / 336 / 340 / 357 / 388 / 394 / 397 / 401 / 404 / 514 / 609 / 659 / 660 / 664 / 782、32 / 38 / 56 / 138 / 150 / 170 / 246 / 311 / 336 / 340 / 357 / 375 / 388 / 394 / 401 / 404 / 514 / 609 / 659 / 660 / 664 / 782、32 / 38 / 56 / 137 / 138 / 150 / 170 / 246 / 311 / 336 / 340 / 357 / 382 / 388 / 394 / 401 / 404 / 514 / 609 / 659 / 660 / 664, 32 / 38 / 56 / 138 / 150 / 170 / 246 / 311 / 336 / 340 / 357 / 388 / 394 / 401 / 40 4 / 514 / 609 / 655 / 659 / 660 / 664 / 782, 32 / 38 / 56 / 137 / 138 / 150 / 170 / 246 / 311 / 336 / 340 / 357 / 382 / 388 / 392 / 394 / 401 / 404 / 514 / 609 / 659 / 660 / 664 / 782,32 / 38 / 56 / 138 / 150 / 170 / 246 / 311 / 336 / 340 / 357 / 388 / 391 / 394 / 401 / 404 / 514 / 609 / 659 / 660 / 664 / 782、32 / 38 / 56 / 137 / 138 / 150 / 170 / 246 / 311 / 336 / 340 / 357 / 388 / 394 / 401 / 404 / 514 / 609 / 655 / 659 / 660 / 664 / 721 / 763 / 782、32 / 38 / 56 / 138 / 150 / 170 / 246 / 311 / 336 / 340 / 357 / 388 / 394 / 401 / 404 / 514 / 609 / 659 / 660 / 664 / 696 / 782、32 / 38 / 56 / 138 / 150 / 170 / 246 / 311 / 336 / 340 / 357 / 388 / 394 / 401 / 404 / 514 / 609 / 659 / 660 / 663 / 664 / 782、32 / 38 / 56 / 137 / 138 / 150 / 170 / 246 / 311 / 336 / 340 / 357 / 382 / 388 / 394 / 401 / 404 / 514 / 609 / 659 / 660 / 664 / 782、32 / 38 / 56 / 137 / 138 / 150 / 170 / 246 / 311 / 336 / 340 / 357 / 382 / 388 / 392 / 394 / 401 / 404 / 514 / 609 / 650 / 655 / 659 / 660 / 664、32 / 38 / 56 / 137 / 138 / 150 / 170 / 246 / 311 / 336 / 340 / 357 / 382 / 388 / 394 / 401 / 404 / 514 / 609 / 629 / 659 / 660 / 664 / 672 / 782、32 / 38 / 56 / 138 / 150 / 170 / 246 / 311 / 336 / 340 / 357 / 388 / 394 / 401 / 404 / 514 / 609 / 616 / 659 / 660 / 664 / 782、32 / 38 / 56 / 138 / 150 / 170 / 246 / 311 / 336 / 340 / 357 / 388 / 394 / 401 / 404 / 514 / 609 / 659 / 660 / 664 / 690 / 782、32 / 38 / 56 / 138 / 150 / 170 / 179 / 246 / 311 / 336 / 340 / 357 / 388 / 394 / 401 / 404 / 514 / 609 / 659 / 660 / 664 / 782、32 / 38 / 56 / 137 / 138 / 150 / 170 / 246 / 311 / 336 / 340 / 357 / 388 / 394 / 401 / 404 / 514 / 609 / 659 / 660 / 664 / 782、32 / 38 / 56 / 138 / 150 / 170 / 246 / 311 / 336 / 340 / 357 / 382 / 388 / 394 / 401 / 404 / 514 / 609 / 659 / 660 / 664 / 670 / 782, 32 / 38 / 56 / 138 / 150 / 170 / 246 / 311 / 336 / 340 / 357 / 388 / 394 / 401 / 404 / 514 / 609 / 659 / 660 / 664 / 670 / 672 / 782, 32 / 38 / 5 6 / 138 / 150 / 170 / 246 / 311 / 336 / 340 / 357 / 388 / 394 / 401 / 404 / 514 / 609 / 629 / 659 / 660 / 664 / 782 or 32 / 38 / 56 / 138 / 150 / 170 / 246 / 311 / 336 / 340 / 357 / 388 / 394 / 401 / 404 / 514 / 609 / 655 / 659 / 660 / 664 / 670 / 782, wherein the amino acid position is relative to the reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 2 or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0173] In some embodiments, the amino acid sequence of the engineered RNA polymerase contains at least one set of substitutions or amino acid residues: 32V / 38G / 56T / 138R / 150Y / 170L / 246K / 311G / 336S / 340W / 357E / 367P / 388K / 394K / 401R / 404L / 514S / 609E / 659R / 660W / 664P / 782V, 32V / 38G / 56T / 138R / 150Y / 170L / 246K / 311G / 336S / 340W / 357E / 382R / 388K / 394K / 401R / 404L / 514S / 609E / 659R / 660W / 664 P / 782V, 32V / 38G / 56T / 138R / 150Y / 170L / 246K / 311G / 336S / 340W / 357E / 388 K / 394K / 401R / 404L / 514S / 609E / 659R / 660W / 664P / 670R / 782V, 32V / 38G / 56 T / 138R / 150Y / 170L / 246K / 311G / 336S / 340W / 357E / 382L / 388K / 394K / 401R / 404L / 514S / 609E / 659R / 660W / 664P / 782V, 32V / 38G / 56T / 138R / 150Y / 170L / 2 46K / 311G / 336S / 340W / 357E / 388K / 394K / 401R / 404L / 514S / 609E / 659R / 660 W / 664P / 672P / 782V, 32V / 38G / 56T / 138R / 150Y / 170L / 246K / 311G / 336S / 340 W / 357P / 388K / 394K / 401R / 404L / 514S / 609E / 659R / 660W / 664P / 782V, 32V / 3 8G / 56T / 138R / 150Y / 170L / 246K / 311G / 336S / 340W / 357E / 388R / 394K / 401R / 404L / 514S / 609E / 659R / 660W / 664P / 782V, 32V / 38G / 56T / 138R / 150Y / 170L / 246K / 311G / 336S / 340W / 357E / 388K / 394K / 401R / 404L / 514S / 609E / 659R / 66 0W / 664P / 693R / 782V, 32V / 38G / 56T / 138R / 150Y / 170L / 246K / 311G / 336S / 34 0W / 357E / 388K / 394K / 401R / 404L / 514S / 609E / 659R / 660W / 664P / 672S / 782V、32V / 38G / 56T / 138R / 150Y / 170L / 246K / 311G / 336S / 340W / 357E / 388K / 394K / 397S / 401R / 404L / 514S / 609E / 659R / 660W / 664P / 782V, 32V / 38G / 56T / 138R / 150Y / 170L / 246K / 311G / 336S / 340W / 357E / 375P / 388K / 394K / 401R / 404L / 5 14S / 609E / 659R / 660W / 664P / 782V、32V / 38G / 56T / 138R / 150Y / 170L / 246K / 3 11G / 336S / 340W / 357E / 388K / 394K / 401R / 404L / 514S / 609E / 659R / 660W / 66 4P / 672G / 782V、32V / 38G / 56T / 137G / 138R / 150Y / 170L / 246K / 311G / 336S / 34 0W / 357E / 382R / 388K / 394K / 401R / 404L / 514S / 609E / 659R / 660W / 664P, 32V / 38G / 56T / 138R / 150Y / 170L / 246K / 311G / 336S / 340W / 357E / 388K / 394K / 401R / 404L / 514S / 609E / 655R / 659R / 660W / 664P / 782V, 32V / 38G / 56T / 137G / 138R / 150Y / 170L / 246K / 311G / 336S / 340W / 357E / 382R / 388K / 392A / 394K / 401R / 404L / 514S / 609E / 659R / 660W / 664P / 782V、32V / 38G / 56T / 138R / 150Y / 170L / 246K / 311G / 336S / 340W / 357E / 388K / 391F / 394K / 401R / 404L / 514S / 609E / 65 9R / 660W / 664P / 782V, 32V / 38G / 56T / 137G / 138R / 150Y / 170L / 246K / 311G / 336S / 340W / 357E / 388K / 394K / 401R / 404L / 514S / 609E / 655M / 659R / 660W / 664 P / 721T / 763S / 782V、32V / 38G / 56T / 138R / 150Y / 170L / 246K / 311G / 336S / 340 W / 357E / 388K / 394K / 401R / 404L / 514S / 609E / 659R / 660W / 664P / 696L / 782V、32V / 38G / 56T / 138R / 150Y / 170L / 246K / 311G / 336S / 340W / 357E / 388K / 394K / 401R / 404L / 514S / 609E / 659R / 660W / 664P / 672R / 782V、32V / 38G / 56T / 138R / 150Y / 170L / 246K / 311G / 336S / 340W / 357E / 388K / 394K / 401R / 404L / 514S / 60 9E / 659R / 660W / 663L / 664P / 782V、32V / 38G / 56T / 138R / 150Y / 170L / 246K / 311 G / 336S / 340W / 357E / 367T / 388K / 394K / 401R / 404L / 514S / 609E / 659R / 660W / 664P / 782V、32V / 38G / 56T / 138R / 150Y / 170L / 246K / 311G / 336S / 340W / 357E / 3 67F / 388K / 394K / 401R / 404L / 514S / 609E / 659R / 660W / 664P / 782V, 32V / 38G / 56T / 137G / 138R / 150Y / 170L / 246K / 311G / 336S / 340W / 357E / 382G / 388K / 394K / 401R / 404L / 514S / 609E / 659R / 660W / 664P / 782V、32V / 38G / 56T / 137G / 138R / 150Y / 170L / 246K / 311G / 336S / 340W / 357E / 382G / 388K / 392R / 394K / 401R / 4 04L / 514S / 609E / 650M / 655M / 659R / 660W / 664P、32V / 38G / 56T / 137G / 138R / 1 50Y / 170L / 246K / 311G / 336S / 340W / 357E / 382R / 388K / 394K / 401R / 404L / 514S / 609E / 629R / 659R / 660Y / 664P / 672T / 782V, 32V / 38G / 56T / 138R / 150Y / 170L / 246K / 311G / 336S / 340W / 357E / 388K / 391S / 394K / 401R / 404L / 514S / 609E / 65 9R / 660W / 664P / 782V、32V / 38G / 56T / 138R / 150Y / 170L / 246K / 311G / 336S / 34 0W / 357E / 388K / 394K / 401R / 404L / 514S / 609E / 616A / 659R / 660W / 664P / 782V、32V / 38G / 56T / 138R / 150Y / 170L / 246K / 311G / 336S / 340W / 357E / 388K / 394K / 401R / 404L / 514S / 609E / 659R / 660W / 664P / 690S / 782V、32V / 38G / 56T / 138R / 150Y / 170L / 179N / 246K / 311G / 336S / 340W / 357E / 388K / 394K / 401R / 404L / 514S / 609E / 659R / 660W / 664P / 782V、32V / 38G / 56T / 137G / 138R / 150Y / 170L / 246K / 311G / 336S / 340W / 357E / 388K / 394K / 401R / 404L / 514S / 609E / 659R / 660W / 664P / 782V、32V / 38G / 56T / 138R / 150Y / 170L / 246K / 311G / 336S / 340W / 357P / 382R / 388K / 394K / 401R / 404L / 514S / 609E / 659R / 660W / 664P / 670R / 782V、32V / 38G / 56T / 138R / 150Y / 170L / 246K / 311G / 336S / 340W / 357E / 388R / 394K / 401R / 404L / 514S / 609E / 659R / 660W / 664P / 670R / 672P / 782V、32V / 38G / 56T / 138R / 150Y / 170L / 246K / 311G / 336S / 340W / 357P / 375P / 388K / 394K / 401R / 404L / 514S / 609E / 659R / 660W / 664P / 782V、32V / 38G / 56T / 138R / 150Y / 170L / 246K / 311G / 336S / 340W / 357P / 388K / 394K / 401R / 404L / 514S / 609E / 659R / 660W / 664P / 672G / 782V、32V / 38G / 56T / 138R / 150Y / 170L / 246K / 311G / 336S / 340W / 357E / 388K / 394K / 401R / 404L / 514S / 609E / 629K / 659R / 660W / 664P / 782V or 32V / 38G / 56T / 138R / 150Y / 170L / 246K / 311G / 336S / 340W / 357E / 388K / 394K / 401R / 404L / 514S / 609E / 655R / 659R / 660W / 664P / 670R / 782V, wherein the amino acid position is relative to the reference sequence corresponding to residues 8 to 890 of SEQ ID NO:2 or relative to the sequence corresponding to SEQ ID NO:2. Reference sequence for NO: 2.

[0174] In some embodiments, the amino acid sequence of the engineered RNA polymerase contains at least one set of substitutions at the following amino acid positions: 32 / 38 / 56 / 137 / 138 / 150 / 170 / 246 / 311 / 336 / 340 / 357 / 388 / 394 / 401 / 404 / 514 / 609 / 629 / 659 / 660 / 664 / 782, 32 / 38 / 56 / 138 / 150 / 170 / 246 / 336 / 340 / 357 / 382 / 388 / 394 / 401 / 404 / 514 / 609 / 629 / 659 / 660 / 664 / 782 32 / 38 / 56 / 137 / 138 / 143 / 150 / 170 / 246 / 311 / 336 / 340 / 357 / 388 / 394 / 401 / 404 / 514 / 609 / 629 / 659 / 660 / 664 / 721 / 782, 32 / 38 / 56 / 137 / 138 / 150 / 170 / 246 / 311 / 336 / 340 / 357 / 388 / 392 / 394 / 401 / 404 / 514 / 609 / 629 / 659 / 660 / 664 / 782, 32 / 38 / 137 / 138 / 143 / 150 / 170 / 24 6 / 311 / 336 / 340 / 357 / 388 / 394 / 401 / 514 / 609 / 629 / 659 / 660 / 664 / 720 / 782, 32 / 38 / 56 / 138 / 143 / 150 / 170 / 246 / 311 / 336 / 340 / 357 / 388 / 394 / 401 / 404 / 514 / 609 / 629 / 659 / 660 / 664 / 782, 32 / 38 / 56 / 138 / 150 / 170 / 246 / 311 / 336 / 340 / 357 / 388 / 392 / 394 / 401 / 404 / 514 / 60 9 / 629 / 659 / 660 / 664 / 782, 32 / 38 / 56 / 138 / 150 / 170 / 246 / 311 / 336 / 340 / 357 / 388 / 392 / 394 / 401 / 514 / 609 / 629 / 659 / 660 / 664 / 782 or 32 / 38 / 56 / 138 / 150 / 170 / 196 / 246 / 311 / 336 / 340 / 357 / 388 / 392 / 394 / 401 / 404 / 514 / 609 / 629 / 659 / 660 / 664 / 782, wherein the amino acid position is relative to the reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 2 or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0175] In some embodiments, the amino acid sequence of the engineered RNA polymerase contains at least one set of substitutions or amino acid residues: 32V / 38G / 56T / 137G / 138R / 150Y / 170L / 246K / 311G / 336S / 340W / 357E / 388K / 394K / 401R / 404L / 514S / 609E / 629K / 659R / 660W / 664P / 782V, 32V / 38G / 56E / 138R / 150Y / 170L / 246K / 336S / 340W / 357E / 382R / 388K / 394K / 401R / 404L / 514S / 609E / 629 K / 659R / 660W / 664P / 782V, 32V / 38G / 56E / 137G / 138R / 143N / 150Y / 170L / 246K / 311G / 336S / 340W / 357E / 388K / 394K / 401R / 404L / 514S / 609E / 629K / 659R / 660W / 664P / 721A / 782V, 32V / 38G / 56T / 137G / 138R / 150Y / 170L / 24 6K / 311G / 336S / 340W / 357E / 388K / 392A / 394K / 401R / 404L / 514S / 609E / 62 9K / 659R / 660W / 664P / 782V, 32V / 38G / 56E / 137G / 138R / 150Y / 170L / 246K / 311G / 336S / 340W / 357E / 388K / 392A / 394K / 401R / 404L / 514S / 609E / 629K / 659R / 660W / 664P / 782V、32V / 38G / 137G / 138R / 143N / 150Y / 170L / 246K / 311G / 336S / 340W / 357E / 388K / 394K / 401R / 514S / 609E / 629K / 659R / 660W / 664P / 720S / 782V, 32V / 38G / 56E / 138R / 143N / 150Y / 170L / 246R / 311G / 33 6S / 340W / 357E / 388K / 394K / 401R / 404L / 514S / 609E / 629K / 659R / 660W / 66 4P / 782V, 32V / 38G / 56E / 138R / 150Y / 170L / 246K / 311G / 336S / 340W / 357E / 388K / 392R / 394K / 401R / 404L / 514S / 609E / 629K / 659R / 660W / 664P / 782V,32V / 38G / 56T / 138R / 150Y / 170L / 246K / 311G / 336S / 340W / 357E / 388K / 392R / 394K / 401R / 5 14S / 609E / 629K / 659R / 660W / 664P / 782V, 32V / 38G / 56T / 138R / 150Y / 170L / 246K / 311G / 33 6S / 340W / 357E / 388K / 392R / 394K / 401R / 404L / 514S / 609E / 629K / 659R / 660W / 664P / 782V, 32V / 38G / 56T / 137G / 138R / 150Y / 170L / 246K / 311G / 336S / 340W / 357E / 388K / 392R / 394K / 40 1R / 404L / 514S / 609E / 629K / 659R / 660W / 664P / 782V, 32V / 38G / 56E / 137G / 138R / 150Y / 170 L / 246K / 311G / 336S / 340W / 357E / 388K / 394K / 401R / 404L / 514S / 609E / 629K / 659R / 660W / 6 64P / 782V or 32V / 38G / 56E / 138R / 150Y / 170L / 196S / 246K / 311G / 336S / 340W / 357E / 388K / 392A / 394K / 401R / 404L / 514S / 609E / 629K / 659R / 660W / 664P / 782V, wherein the amino acid position is relative to the reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 2 or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0176] In some embodiments, the amino acid sequence of the engineered RNA polymerase contains at least one substitution at the amino acid positions described in Tables 5.1, 6.1, 7.1, 8.1, 9.1, 10.1, 11.1, 12.1, 14.1, 15.1, 16.1, 17.1, 18.1, 19.1, 23.1, and 24.1, wherein the amino acid position is relative to the reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 2 or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0177] In some embodiments, the amino acid sequence of the engineered RNA polymerase comprises at least one substitution as described in Tables 5.1, 6.1, 7.1, 8.1, 9.1, 10.1, 11.1, 12.1, 14.1, 15.1, 16.1, 17.1, 18.1, 19.1, 23.1, and 24.1, wherein the amino acid position is relative to the reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 2 or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0178] In some embodiments, the amino acid sequence of the engineered RNA polymerase contains at least one substitution or set of substitutions at the amino acid positions described in Tables 5.1, 6.1, 7.1, 8.1, 9.1, 10.1, 11.1, 12.1, 14.1, 15.1, 16.1, 17.1, 18.1, 19.1, 23.1, and 24.1, wherein the amino acid position is relative to the reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 2 or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0179] In some embodiments, the amino acid sequence of the engineered RNA polymerase comprises at least one substitution or set of substitutions of the engineered RNA polymerase variants described in Tables 5.1, 6.1, 7.1, 8.1, 9.1, 10.1, 11.1, 12.1, 14.1, 15.1, 16.1, 17.1, 18.1, 19.1, 23.1, and 24.1, wherein the amino acid position is relative to the reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 2 or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0180] In some embodiments, the engineered RNA polymerase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 96%, 97%, 98%, 99% or higher sequence identity with a reference sequence comprising at least one substitution or set of substitutions as described in Tables 5.1, 6.1, 7.1, 8.1, 9.1, 12.1, 23.1, and 24.1, wherein the amino acid position is relative to the reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 2 or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0181] In some embodiments, the engineered RNA polymerase comprises a reference sequence of residues 8 to 890 corresponding to SEQ ID NO: 4, 32, 44, 156, 234, 238, 386, 394, 588, 604, 686, 692, 712, or 800, or an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher sequence identity with the reference sequence corresponding to SEQ ID NO: 4, 32, 44, 156, 234, 238, 386, 394, 588, 604, 686, 692, 712, or 800.

[0182] In some embodiments, the engineered RNA polymerase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 96%, 97%, 98%, 99% or higher sequence identity with the sequence corresponding to the even-numbered SEQ ID NOs of ...

[0183] In some embodiments, the engineered RNA polymerase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 96%, 97%, 98%, 99% or higher sequence identity with the reference sequence corresponding to SEQ ID NO: 4, 32, 44, 156, 234, 238, 386, 394, 588, 604, 686, 692, 712 or 800, wherein relative to the sequence corresponding to SEQ ID NO: The reference sequence of residues 8 to 890 of 4, 32, 44, 156, 234, 238, 386, 394, 588, 604, 686, 692, 712, or 800, or relative to the reference sequence corresponding to SEQ ID NO: 4, 32, 44, 156, 234, 238, 386, 394, 588, 604, 686, 692, 712, or 800, wherein the amino acid sequence comprises one or more substitutions.

[0184] In some embodiments, the engineered RNA polymerase comprises a reference sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with respect to the reference sequence corresponding to SEQ ID NO: 4, 32, 44, 156, 234, 238, 386, 394, 588, 604, 686, 692, 712 or 800 of the reference sequence, wherein the reference sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher of the sequence corresponding to SEQ ID NO: 4, 32, 44, 156, 234, 238, 386, 394, 588, 604, 686, 692, 712 or 800 of the reference sequence, or the reference ... A reference sequence of 4, 32, 44, 156, 234, 238, 386, 394, 588, 604, 686, 692, 712 or 800, wherein the amino acid sequence contains one or more substitutions.

[0185] In some embodiments, the amino acid sequence of the engineered RNA polymerase contains at least one substitution at the following amino acid positions: 13, 14, 15, 17, 19, 21, 32, 33, 38, 45, 52, 54, 55, 56, 56, 57, 60, 61, 62, 63, 67, 69, 72, 75, 78, 81, 82, 83, 84, 89, 93, 95, 100, 105, 111, 115, 122, 124, 1 27, 131, 133, 135, 137, 138, 139, 140, 141, 143, 150, 160, 161, 162, 164, 166, 168, 169, 170, 171, 172, 173, 179, 185, 187, 193, 196, 207, 212, 217, 218, 221, 227, 237, 239, 240, 245, 246, 250, 253, 254, 257 261, 272, 274, 275, 311, 336, 340, 342, 351, 357, 367, 375, 379, 382, ​​388, 391, 392, 394, 397, 401, 402, 403, 404, 419, 424, 437, 440, 494, 510, 514, 517, 527, 529, 530, 531, 533, 536, 589, 594, 598, 607, 6 09, 615, 616, 617, 625, 629, 648, 650, 655, 659, 660, 663, 664, 666, 670, 672, 690, 693, 696, 718, 720, 721, 749, 759, 763, 773, 775, 779, 782, 782, 810, 854, 857, 867, 875 or 878 or combinations thereof, wherein the amino acid position is relative to the position corresponding to SEQ. Reference sequences of residues 8 to 890 of SEQ ID NO: 4, 32, 44, 156, 234, 238, 386, 394, 588, 604, 686, 692, 712, or 800, or reference sequences corresponding to SEQ ID NO: 4, 32, 44, 156, 234, 238, 386, 394, 588, 604, 686, 692, 712, or 800.

[0186] In some embodiments, the amino acid sequence of the engineered RNA polymerase comprises at least one substituted amino acid residue: 13N, 14Q / Y, 15T, 17N, 19E, 21G, 32S / T / V, 33E, 38G / R, 45K, 52T, 54H, 55D / S / W, 56A / E / G / N / T / V, 57G, 60G / L / M / R, 61W / Y, 62T, 63S, 67R, 69S, 72R, 75S / Y, 78S, 81C / L, 82S, 83K, 84A, 89Q, 93Y, 95Y, 100R, 105R, 111K, 115L / V, 122I, 124F, 127G, 131T, 133C, 135G. / K, 137G, 138N / R, 139R, 140R, 141A, 143N / Q / R / V, 150Q / Y, 160F / L, 161R, 16 2V, 164W, 166D / R / T / V, 168K / L / R / S / T / V / W, 169G / L, 170L / Q / R / S, 171E / F / L / Y、172A / E / G / K / R / V、173D / H / L / Y / V、179N、185I / L / P、187G / H / L、193G、196 A / C / G / S / V, 207S, 212R / Y, 217P / V, 218Q / T, 221C, 227V, 237L, 239L / R, 240M, 245C, 246K / R / V, 250R, 253Y, 254G / S, 257L / V, 261M, 272N, 274G / L, 275A / Y, 311A / G, 336K / L / M / S / V, 340E / K / W, 342Q, 351L, 357E / P, 367F / P / T, 375F / P, 379A / S, 382G / L / R, 388A / K / R, 391F / S, 392A / M / R, 394K, 397S, 401R, 402T, 4 03V, 404I / L / S / Y, 419R / S, 424A, 437P / S, 440K, 494G, 510V, 514I / L / S, 517R, 527E, 529R, 530N, 531Q, 533S, 536E / V, 589C, 594G, 598A / K / L / R / V, 607Y, 60 9C / E / F / P / W, 615A, 616A, 617R, 625C, 629A / D / K / R, 648F, 650M, 655M / R, 659E / R, 660H / W / Y, 663L, 664K / P, 666M, 670Q / R, 672G / P / R / S / T, 690S, 693R, 696 L, 718F / N / S / V / Y, 720A / G / I / P / S, 721A / E / M / S / T / Y, 749R, 759M, 763S, 773A,775K / R, 779R, 782E / R, 782V, 810K, 854T, 857V, 867Q, 875C, or 878M, or combinations thereof, wherein the amino acid position is relative to reference sequences corresponding to residues 8 to 890 of SEQ ID NO: 4, 32, 44, 156, 234, 238, 386, 394, 588, 604, 686, 692, 712, or 800, or relative to reference sequences corresponding to SEQ ID NO: 4, 32, 44, 156, 234, 238, 386, 394, 588, 604, 686, 692, 712, or 800.

[0187] In some embodiments, the amino acid sequence of the engineered RNA polymerase contains at least one substitution at the following amino acid positions: 32, 38, 56, 138, 150, 170, 171, 173, 246, 311, 336, 340, 357, 388, 394, 401, 404, 437, 514, 536, 609, 629, 659, 660, 664, 779, or 782, or combinations thereof, wherein said amino acid position is relative to the reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 4, 32, 44, 156, 234, 238, 386, 394, 588, 604, 686, 692, 712, or 800, or relative to the reference sequence corresponding to .... Reference sequences of 4, 32, 44, 156, 234, 238, 386, 394, 588, 604, 686, 692, 712 or 800.

[0188] In some embodiments, the amino acid sequence of the engineered RNA polymerase comprises at least one substitution or amino acid residue 32V, 38G, 56T, 138R / N, 150Y, 170Q, 171E, 173Y, 246K, 311A / G, 336K / S, 340K / W, 357E, 388A / K, 394K, 401R, 404L / S, 437P, 514S, 536V, 609E, 629A, 659R, 660H / W, 664P, 779R, or 782V, or a combination thereof, wherein the amino acid position is relative to the reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 4, 32, 44, 156, 234, 238, 386, 394, 588, 604, 686, 692, 712, or 800, or relative to the reference sequence corresponding to ... Reference sequences of 4, 32, 44, 156, 234, 238, 386, 394, 588, 604, 686, 692, 712 or 800.

[0189] In some embodiments, the engineered RNA polymerase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with the reference sequence corresponding to SEQ ID NO: 4, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 4 or relative to the reference sequence corresponding to SEQ ID NO: 4.

[0190] In some embodiments, the engineered RNA polymerase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 96%, 97%, 98%, 99% or higher sequence identity with the reference sequence corresponding to the even-numbered residues of SEQ ID NO: 28-40, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues of SEQ ID NO: 4 or relative to the reference sequence corresponding to SEQ ID NO: 4.

[0191] In some embodiments, the amino acid sequence of the engineered RNA polymerase contains at least one substitution at the following amino acid positions: 311 / 388 / 404 / 440, 138 / 340, 138 / 311 / 336 / 340 / 388, 311 / 388 / 440, 138 / 311 / 340 / 388 / 404 / 440, 138 / 336 / 342, or 351 / 388, wherein the amino acid position is relative to the reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 4 or relative to the reference sequence corresponding to SEQ ID NO: 4.

[0192] In some embodiments, the amino acid sequence of the engineered RNA polymerase comprises at least one set of substitutions or amino acid residues 311G / 388K / 404Y / 440K, 138R / 340W, 138R / 311G / 336S / 340W / 388K, 311G / 388K / 440K, 138R / 311G / 340E / 388K / 404Y / 440K, 138R / 336M / 342Q, or 351L / 388K, wherein the amino acid position is relative to the reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 4 or relative to the reference sequence corresponding to SEQ ID NO: 4.

[0193] In some embodiments, the amino acid sequence of the engineered RNA polymerase comprises at least one set of substitutions: A311G / A388K / L404Y / N440K, N138R / K340W, N138R / A311G / K336S / K340W / A388K, A311G / A388K / N440K, N138R / A311G / K340E / A388K / L404Y / N440K, N138R / K336M / L342Q, or W351L / A388K, wherein the amino acid position is relative to the reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 4 or relative to the reference sequence corresponding to SEQ ID NO: 4.

[0194] In some embodiments, the engineered RNA polymerase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with the reference sequence corresponding to SEQ ID NO: 32, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 32 or relative to the reference sequence corresponding to SEQ ID NO: 32.

[0195] In some embodiments, the engineered RNA polymerase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 96%, 97%, 98%, 99% or higher sequence identity with the reference sequence corresponding to the even-numbered residues of SEQ ID NO: 42-188, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 32 or relative to the reference sequence corresponding to SEQ ID NO: 32.

[0196] In some embodiments, the amino acid sequence of the engineered RNA polymerase contains at least one substitution or set of substitutions at the following amino acid positions: 514, 38 / 514, 38 / 394 / 514, 38 / 394 / 514 / 664, 394 / 514 / 875, 394 / 514, 38 / 124 / 394 / 514, 171 / 878, 170, 173, 172, 170 / 629, 171, 168, 166, 166 / 272 / 274 / 275, 169, 207, 168 / 217 / 218, 171 / 530, 382, ​​773, 670, 379, 392, 419, 57, 164, 810, 659, 54, 172 / 173 / 514 / 659 / 660, 170 / 171 / 172 / 514 / 659, 173 / 514 / 659 / 660, 173 / 514 / 659 / 810, 170 / 340 / 514 / 659 / 660 / 810, 170 / 171 / 173 / 336 / 340 / 514 / 659 / 660, 57 / 171 / 173 / 514 / 659 / 660 / 810, 170 / 173 / 514 / 659 / 660 / 810, 57 / 171 / 172 / 336 / 340 / 514 / 659, 173 / 514, 171 / 172 / 173 / 514 / 659 / 660, 57 / 170 / 17 1 / 173 / 514 / 659 / 660 / 810, 57 / 173 / 514, 172 / 173 / 514 / 857, 57 / 173 / 340 / 514, 170 / 171 / 173 / 336 / 514, 171 / 514, 57 / 170 / 173 / 336 / 514, 170 / 336, 173 / 514 / 659, 57 / 514 or 173 / 810, wherein the amino acid position is relative to the reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 32 or relative to the reference sequence corresponding to SEQ ID NO: 32.

[0197] In some embodiments, the amino acid sequence of the engineered RNA polymerase contains at least one substitution or set of substitutions or amino acid residues such as 514S, 38G / 514S, 38G / 394K / 514S, 38G / 394K / 514S / 664P, 394K / 514S / 875C, 394K / 514S, 38G / 124F / 394K / 514I, 171E / 878M, 170Q, 173Y, 172V, 173L, 172E, 170S / 629D, 171F, 168V, 172K, 166R, 168K, 166V / 272N / 274L / 275Y, 17 0R、169L、207S、169G、168L / 217P / 218Q、168W、172R、166D、171L、171Y / 530N、172A、173H、382R、773A、168R、670R、168T、172G、379A、392M、4 19R, 392R, 392A, 57G, 168S, 164W, 670Q, 810K, 382G, 659R, 54H, 419S, 172E / 173Y / 514S / 659R / 660H, 170Q / 171E / 172E / 514S / 659R, 173Y / 51 4S / 659R / 660H, 173Y / 514S / 659R / 810K, 170Q / 340K / 514S / 659R / 660H / 810K, 170Q / 171E / 173Y / 336K / 340K / 514S / 659R / 660H, 57G / 171E / 1 73Y / 514S / 659R / 660H / 810K, 170Q / 173Y / 514S / 659R / 660H / 810K, 57G / 171E / 172K / 336K / 340K / 514S / 659R, 173Y / 514S, 171E / 172E / 173Y / 5 14S / 659R / 660H, 57G / 170Q / 171E / 173Y / 514S / 659R / 660H / 810K, 57G / 173Y / 514S, 172K / 173D / 514S / 857V, 57G / 173Y / 340K / 514S, 170Q / 171E / 173Y / 336K / 514S, 171E / 514S, 57G / 170Q / 173Y / 336K / 514S, 170Q / 336K, 173Y / 514S / 659R, 57G / 514S or 173Y / 810K, wherein the amino acid position is relative to the corresponding SEQ Reference sequence of residues 8 to 890 of SEQ ID NO: 32 or relative to the reference sequence corresponding to SEQ ID NO: 32.

[0198] In some embodiments, the amino acid sequence of the engineered RNA polymerase includes at least one substitution or set of substitutions: L514S, R38G / L514S, R38G / R394K / L514S, R38G / R394K / L514S / W664P, R394K / L514S / G875C, R394K / L514S, R38G / I124F / R394K / L514I, K171E / N878M, K170Q, V173Y, N172V, V173L, N172E, K170S / A629D, K171F, H168V, N172K, A166R, H168K, A166V / S272N / M274L / F275Y, K170R, F169L, A207S, F169G, H168L / I217P / H218Q, H168W, N172R, A166D, K171L, K171Y / H530N, N172A, V173H, T382R, D773A, H168R, K 670R, H168T, N172G, E379A, Y392M, K419R, Y392R, Y392A, R57G, H168S, L16 4W, K670Q, G810K, T382G, E659R, G54H, K419S, N172E / V173Y / L514S / E659R / D660H、K170Q / K171E / N172E / L514S / E659R、V173Y / L514S / E659R / D660H、 V173Y / L514S / E659R / G810K, K170Q / W340K / L514S / E659R / D660H / G810K, K 170Q / K171E / V173Y / S336K / W340K / L514S / E659R / D660H, R57G / K171E / V17 3Y / L514S / E659R / D660H / G810K, K170Q / V173Y / L514S / E659R / D660H / G810 K、R57G / K171E / N172K / S336K / W340K / L514S / E659R、V173Y / L514S、K171E / N172E / V173Y / L514S / E659R / D660H, R57G / K170Q / K171E / V173Y / L514S / E6 59R / D660H / G810K, R57G / V173Y / L514S, N172K / V173D / L514S / A857V, R57G / V173Y / W340K / L514S, K170Q / K171E / V173Y / S336K / L514S, K171E / L514S,R57G / K170Q / V173Y / S336K / L514S, K170Q / S336K, V173Y / L514S / E659R, R57G / L514S, or V173Y / G810K, wherein the amino acid position is relative to the reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 32 or relative to the reference sequence corresponding to SEQ ID NO: 32.

[0199] In some embodiments, the engineered RNA polymerase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with the reference sequence corresponding to SEQ ID NO: 44, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 44 or relative to the reference sequence corresponding to SEQ ID NO: 44.

[0200] In some embodiments, the engineered RNA polymerase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 96%, 97%, 98%, 99% or higher sequence identity with the reference sequence corresponding to the even-numbered residues of SEQ ID NO: 190-194, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues of SEQ ID NO: 44 or relative to the reference sequence corresponding to SEQ ID NO: 44.

[0201] In some embodiments, the amino acid sequence of the engineered RNA polymerase contains at least one substitution or set of substitutions at amino acid positions 336, 143 / 336 / 340 / 782, or 143 / 336 / 340 / 357 / 514, wherein the amino acid position is relative to a reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 44 or relative to a reference sequence corresponding to SEQ ID NO: 44.

[0202] In some embodiments, the amino acid sequence of the engineered RNA polymerase comprises at least one substitution or set of substitutions or amino acid residues 336K, 143V / 336K / 340K / 782V, or 143V / 336K / 340K / 357E / 514L, wherein the amino acid position is relative to a reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 44 or relative to a reference sequence corresponding to SEQ ID NO: 44.

[0203] In some embodiments, the amino acid sequence of the engineered RNA polymerase comprises at least one substitution or set of substitutions S336K, A143V / S336K / W340K / E782V, or A143V / S336K / W340K / R357E / S514L, wherein the amino acid position is relative to a reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 44 or relative to a reference sequence corresponding to SEQ ID NO: 44.

[0204] In some embodiments, the engineered RNA polymerase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with the reference sequence corresponding to SEQ ID NO: 156, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 156 or relative to the reference sequence corresponding to SEQ ID NO: 156.

[0205] In some embodiments, the engineered RNA polymerase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 96%, 97%, 98%, 99% or higher sequence identity with the reference sequence corresponding to the even-numbered residues of SEQ ID NO: 196-260, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 156 or relative to the reference sequence corresponding to SEQ ID NO: 156.

[0206] In some embodiments, the amino acid sequence of the engineered RNA polymerase contains at least one substitution or set of substitutions at the following amino acid positions: 207, 419, 207 / 336 / 340, 166 / 169, 207 / 670, 166 / 336 / 340, 207 / 419 / 670, 207 / 336 / 340 / 382 / 419 / 670, 207 / 382, 670 336 / 670, 340 / 670, 382, ​​127 / 141 / 207 / 336 / 382 / 670, 207 / 336 / 382 / 419 / 670, 419 / 670, 166 / 168 / 207 / 336 / 340 / 419 / 670, 166 / 340 or 207 / 336 / 340 / 670, wherein the amino acid position is relative to the reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 156 or relative to the reference sequence corresponding to SEQ ID NO: 156.

[0207] In some embodiments, the amino acid sequence of the engineered RNA polymerase contains at least one substitution or set of substitutions or amino acid residues 207S, 419R, 207S / 336K / 340K, 166R / 169L, 207S / 670R, 166R / 336K / 340K, 207S / 419R / 670R, 207S / 336K / 340K / 382G / 419R / 670R, 207S / 382G, 670R, 336K / 670R, 340K / 670R, 382G, 127G / 141A / 207S / 336K / 382G / 670R, 207S / 336K / 382G / 419R / 670R, 419R / 670R, 166R / 168V / 207S / 336K / 340K / 419R / 670R, 166V / 340K or 207S / 336K / 340K / 670R, wherein the amino acid position is relative to the reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 156 or relative to the reference sequence corresponding to SEQ ID NO: 156.

[0208] In some embodiments, the amino acid sequence of the engineered RNA polymerase includes at least one substitution or set of substitutions: A207S, K419R, A207S / S336K / W340K, A166R / F169L, A207S / K670R, A166R / S336K / W340K, A207S / K419R / K670R, A207S / S336K / W340K / T382G / K419R / K670R, A207S / T382G, K670R, S336K / K670R. The amino acid positions are relative to the reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 156 or relative to the reference sequence corresponding to SEQ ID NO: 156.

[0209] In some embodiments, the amino acid sequence of the engineered RNA polymerase contains at least one substitution or set of substitutions at the following amino acid positions: 38 / 437 / 779, 38 / 437, 38, 38 / 207 / 437 / 779, 517 / 779, 38 / 437 / 527 / 664, 38 / 207 / 379 / 437, 664 / 670 / 779, 38 / 779, 38 / 437 / 517, 437 / 779, 38 / 437 / 607, 38 / 379 / 779, or 517 / 670 / 779, wherein the amino acid position is relative to the reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 156 or relative to the reference sequence corresponding to SEQ ID NO: 156.

[0210] In some embodiments, the amino acid sequence of the engineered RNA polymerase comprises at least one substitution or set of substitutions or amino acid residues 38G / 437P / 779R, 38G / 437P, 38G, 38G / 207S / 437P / 779R, 517R / 779R, 38G / 437P / 527E / 664K, 38G / 207S / 379S / 437P, 664K / 670R / 779R, 38G / 779R, 38G / 437P / 517R, 437P / 779R, 38G / 437P / 607Y, 38G / 379S / 779R, or 517R / 670R / 779R, wherein the amino acid position is relative to the reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 156 or relative to the reference sequence corresponding to SEQ ID NO: 156.

[0211] In some embodiments, the amino acid sequence of the engineered RNA polymerase contains at least one substitution or set of substitutions: R38G / S437P / H779R, R38G / S437P, R38G, R38G / A207S / S437P / H779R, C517R / H779R, R38G / S437P / G527E / W664K, R38G / A207S / E379S / S437P, W664K / K670R / H779R, R38G / H779R, R38G / S437P / C517R, S437P / H779R, R38G / S437P / E607Y, R38G / E379S / H779R or C517R / K670R / H779R, wherein the amino acid position is relative to the reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 156 or relative to the reference sequence corresponding to SEQ ID NO: 156.

[0212] In some embodiments, the amino acid sequence of the engineered RNA polymerase contains at least one set of substitutions at amino acid positions 357 / 394 / 664 or 138 / 311 / 336 / 340 / 357 / 388 / 394 / 404 / 664, wherein the amino acid positions are relative to a reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 156 or relative to a reference sequence corresponding to SEQ ID NO: 156.

[0213] In some embodiments, the amino acid sequence of the engineered RNA polymerase comprises at least one set of substitutions or amino acid residues 357E / 394K / 664P or 138N / 311A / 336K / 340K / 357E / 388A / 394K / 404S / 664P, wherein the amino acid position is relative to a reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 156 or relative to a reference sequence corresponding to SEQ ID NO: 156.

[0214] In some embodiments, the amino acid sequence of the engineered RNA polymerase comprises at least one set of substitutions R357E / R394K / W664P or R138N / G311A / S336K / W340K / R357E / K388A / R394K / L404S / W664P, wherein the amino acid position is relative to a reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 156 or relative to a reference sequence corresponding to SEQ ID NO: 156.

[0215] In some embodiments, the engineered RNA polymerase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with the reference sequence corresponding to SEQ ID NO: 234, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 234 or relative to the reference sequence corresponding to SEQ ID NO: 234.

[0216] In some embodiments, the engineered RNA polymerase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 96%, 97%, 98%, 99% or higher sequence identity with the reference sequence corresponding to the even-numbered residues of SEQ ID NO: 262-384, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 234 or relative to the reference sequence corresponding to SEQ ID NO: 234.

[0217] In some embodiments, the amino acid sequence of the engineered RNA polymerase contains at least one substitution or set of substitutions at the following amino acid positions: 720, 196, 609, 246, 185, 594, 212, 494, 375, 721, 72, 867, 160, 718, 187, 170 / 173, 615, 137 / 138, 854, 131, 38 / 185 / 187 / 437, 185 / 187, 437 / 609, 38 / 609, 38 / 185 / 187 / 437 / 609, 437 / 609 / 720, 185 / 187 / 609, 185 / 187 / 437, 437 / 721, 609 / 720, 185 / 187 / 246 or 185 / 187 / 437 / 609 / 720, wherein the amino acid position is relative to the reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 234 or relative to the reference sequence corresponding to SEQ ID NO: 234.

[0218] In some embodiments, the amino acid sequence of the engineered RNA polymerase contains at least one substitution or set of substitutions or amino acid residues: 720A, 196C, 609W, 196V, 246V, 185L, 594G, 212Y, 494G, 196A, 212R, 375F, 721T, 721A, 72R, 185I, 721S, 867Q, 721Y, 160F, 718F, 609F, 720S, 187L, 718V, 170L / 173V, 609E, 718S, 609C, 609P, 721M, 615A, 187H, 196S, 721E, 718Y, 137G / 138N, 187G, 720G, 720P, 854T, 7 20I, 160L, 131T, 38R / 185L / 187H / 437S, 185L / 187H, 185L / 187L, 437S / 609C, 38 R / 609P, 38R / 185L / 187H / 437S / 609C, 437S / 609P / 720A, 185L / 187L / 609P, 185L / 187L / 437S, 185L / 187H / 437S, 437S / 609P, 437S / 721S, 185L / 187H / 609E, 609P / 720A, 185L / 187L / 246V or 185L / 187H / 437S / 609P / 720A, wherein the amino acid position is relative to the reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 234 or relative to the reference sequence corresponding to SEQ ID NO: 234.

[0219] In some embodiments, the amino acid sequence of the engineered RNA polymerase includes at least one substitution or set of substitutions: K720A, D196C, T609W, D196V, Q246V, Y185L, I594G, H212Y, E494G, D196A, H212R, D375F, K721T, K721A, A72R, Y185I, K721S, K867Q, K721Y. R160F, K718F, T609F, K720S, K187L, K718V, K170L / Y173V, T609E, K718S, T609C, T609P, K72 1M, K615A, K187H, D196S, K721E, K718Y, D137G / R138N, K187G, K720G, K720P, D854T, K720I, R 160L, A131T, G38R / Y185L / K187H / P437S, Y185L / K187H, Y185L / K187L, P437S / T609C, G38R / T609P, G38R / Y185L / K187H / P437S / T609C, P437S / T609P / K720A, Y185L / K187L / T609P, Y185L / K187L / P437S, Y185L / K187H / P437S, P437S / T609P, P437S / K721S, Y185L / K187H / T609E, T609P / K720A, Y185L / K187L / Q246V or Y185L / K187H / P437S / T609P / K720A, wherein the amino acid position is relative to the reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 234 or relative to the reference sequence corresponding to SEQ ID NO: 234.

[0220] In some embodiments, the engineered RNA polymerase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with the reference sequence corresponding to SEQ ID NO: 386, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 386 or relative to the reference sequence corresponding to SEQ ID NO: 386.

[0221] In some embodiments, the engineered RNA polymerase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 96%, 97%, 98%, 99% or higher sequence identity with the reference sequence corresponding to the even-numbered residues of SEQ ID NO: 386, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 386 or relative to the reference sequence corresponding to SEQ ID NO: 386.

[0222] In some embodiments, the amino acid sequence of the engineered RNA polymerase contains at least one substitution or set of substitutions at the following amino acid positions: 170 / 609, 38 / 185 / 187 / 609, 38 / 185 / 187, 38 / 170 / 173, 38 / 173 / 185 / 187 / 720, 170 / 185 / 187 / 609, 609, 170 / 185 / 187 / 609 / 721, 38 / 609, 38 / 170 / 246 / 609, 185 / 187 / 609 / 718, 38 / 185 / 187 / 609 / 718 / 720, 38 / 170 / 246 / 609 / 718, 720, 246 / 609, 38 / 185 / 187 / 246 / 720, 38 / 609 / 721, 38 / 721, 38 / 720 / 721, 38 / 170 / 609 / 718 / 720, 38 / 718, 38 / 185 / 187 / 609 / 718 / 721, 529 / 609, 38 / 609 / 718, 185 / 187 / 609 / 718 / 720, 185 / 187 / 609 / 718 / 721, 38 / 170 / 718 / 720 / 721, 170 / 185 / 187 / 7 18 / 720, 38 / 173 / 185 / 187 / 246 / 609 / 720, 609 / 718 / 720, 170 / 185 / 187 / 718 / 720 / 721, 38 / 170 / 173 / 246 / 609 / 718, 246 / 721, 38 / 718 / 720 or 38 / 609 / 718 / 720, wherein the amino acid position is relative to the reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 386 or relative to the reference sequence corresponding to SEQ ID NO: 386.

[0223] In some embodiments, the amino acid sequence of the engineered RNA polymerase contains at least one substitution or set of substitutions or amino acid residues: 170L / 609P, 38G / 185L / 187L / 609C, 38G / 185L / 187H, 38G / 170L / 173V, 38G / 173V / 185L / 187L / 720A, 170L / 185L / 187H / 609C, 609E, 170L / 185L / 187H / 609C / 721S, 38G / 609E, 170 L / 185L / 187L / 609E, 38G / 170L / 246V / 609P, 185L / 187H / 609E / 718S, 38G / 185L / 187L / 609W / 718N / 720A, 38G / 170L / 246V / 609C / 718S, 720A, 246V / 609E, 38G / 185L / 187L / 246V / 720A, 38G / 609C / 721S, 38G / 721S, 38G / 720A / 721S, 38G / 170L / 609C / 718S / 720A, 38G / 718S, 38G / 170L / 246V / 609P / 718S, 38G / 185L / 187L / 609P / 718S / 721S, 529R / 609E, 3 8G / 609P / 718S, 185L / 187L / 609C / 718S / 720A, 185L / 187H / 609C / 718S / 721S, 38G / 170L / 718S / 720A / 721S, 170L / 18 5L / 187L / 718S / 720A, 38G / 173V / 185L / 187L / 246V / 609E / 720A, 609E / 718S / 720A, 170L / 185L / 187H / 718S / 720A / 721S, 38G / 170L / 173V / 246V / 609E / 718S, 246V / 721S, 38G / 718S / 720A or 38G / 609E / 718S / 720A, wherein the amino acid position is relative to the reference sequence corresponding to residues 8 to 890 of SEQ ID NO:386 or relative to the reference sequence corresponding to SEQ ID NO:386.

[0224] In some embodiments, the amino acid sequence of the engineered RNA polymerase includes at least one substitution or set of substitutions: K170L / T609P, R38G / Y185L / K187L / T609C, R38G / Y185L / K187H, R38G / K170L / Y173V, R38G / Y173V / Y185L / K187L / K720A, K170L / Y185L / K187H / T609C, T609E, K170L / Y185L / K187H / T609C / K721S, R38G / T609E, K170L / Y185L / K1 87L / T609E, R38G / K170L / Q246V / T609P, Y185L / K187H / T609E / K718S, R38G / Y185L / K187L / T609W / K718N / K720A, R38G / K170L / Q246V / T6 09C / K718S, K720A, Q246V / T609E, R38G / Y185L / K187L / Q246V / K720A, R38G / T609C / K721S, R38G / K721S, R38G / K720A / K721S, R38G / K170 L / T609C / K718S / K720A, R38G / K718S, R38G / K170L / Q246V / T609P / K718S, R38G / Y185L / K187L / T609P / K718S / K721S, Q529R / T609E, R38G / T609P / K718S, Y185L / K187L / T609C / K718S / K720A, Y185L / K187H / T609C / K718S / K721S, R38G / K170L / K718S / K720A / K721S, K170L / Y18 5L / K187L / K718S / K720A, R38G / Y173V / Y185L / K187L / Q246V / T609E / K720A, T609E / K718S / K720A, K170L / Y185L / K187H / K718S / K720A / K721S, R38G / K170L / Y173V / Q246V / T609E / K718S, Q246V / K721S, R38G / K718S / K720A or R38G / T609E / K718S / K720A, wherein the amino acid position is relative to the reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 386 or relative to the reference sequence corresponding to SEQ ID NO: 386.

[0225] In some embodiments, the engineered RNA polymerase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with the reference sequence corresponding to SEQ ID NO: 238, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 238 or relative to the reference sequence corresponding to SEQ ID NO: 238.

[0226] In some embodiments, the engineered RNA polymerase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 96%, 97%, 98%, 99% or higher sequence identity with the reference sequence corresponding to the even-numbered residues of SEQ ID NO: 462-584, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 238 or relative to the reference sequence corresponding to SEQ ID NO: 238.

[0227] In some embodiments, the amino acid sequence of the engineered RNA polymerase contains at least one substitution or set of substitutions at the following amino acid positions: 143, 105, 775 / 779, 135, 100, 246, 779 / 782, 749, 237, 162, 83, 218, 227, 115, 274, 81, 122, 61, 32, 139, 75, 239, 257, 240, 69 / 161, 253, 14, 221, 84, 133 / 150, 60, 275, 89, 15, 254, 625, 14 / 67, 21, 93, 250, 217, 19, 95, 759, 62, or 140, wherein the amino acid position is relative to the reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 238 or relative to the reference sequence corresponding to SEQ ID NO: 238. The reference sequence for 238.

[0228] In some embodiments, the amino acid sequence of the engineered RNA polymerase contains at least one substitution or set of substitutions or amino acid residues such as 143N, 143Q, 143R, 105R, 775R / 779R, 135K, 100R, 775K / 779R, 246K, 779R / 782R, 749R, 237L, 162V, 83K, 218T, 227V, 115L, 274G, 81C, 122I, 61Y, 32S, 139R, 75Y, 239R, 257L, 240M. The amino acid positions are 69S / 161R, 253Y, 14Y, 221C, 84A, 115V, 133C / 150Q, 60L, 60G, 32V, 275A, 89Q, 257V, 274L, 15T, 254S, 61W, 625C, 14Q / 67R, 21G, 254G, 93Y, 250R, 217V, 60M, 239L, 75S, 32T, 19E, 95Y, 81L, 60R, 759M, 62T, or 140R, wherein the amino acid positions are relative to the reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 238 or relative to the reference sequence corresponding to SEQ ID NO: 238.

[0229] In some embodiments, the amino acid sequence of the engineered RNA polymerase includes at least one substitution or set of substitutions: A143N, A143Q, A143R, K105R, E775R / H779R, S135K, K100R, E775K / H779R, Q246K, H779R / E782R, P749R, H237L, R162V, T83K, H218T, L227V, E115L, M274G, I81C, A122I, M61Y, A32S, T139R, A75Y, Q239R, Y257L, N240M, G69S / I. 161R, L253Y, A14Y, V221C, L84A, E115V, L133C / R150Q, K60L, K60G, A32V, F275A, I89Q, Y257V, M274L, K15T, A254S, M61W, G625C, A14Q / K67R, I21G, A254G, N93Y, T250R, I217V, K60M, Q239L, A75S, A32T, S19E, W95Y, I81L, K60R, L759M, F62T, or T140R, wherein the amino acid position is relative to the reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 238 or relative to the reference sequence corresponding to SEQ ID NO: 238.

[0230] In some embodiments, the engineered RNA polymerase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with the reference sequence corresponding to SEQ ID NO: 394, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 394 or relative to the reference sequence corresponding to SEQ ID NO: 394.

[0231] In some embodiments, the engineered RNA polymerase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 97%, 98%, 99% or higher sequence identity with the reference sequence corresponding to the even-numbered residues of SEQ ID NO: 586-602, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 394 or relative to the reference sequence corresponding to SEQ ID NO: 394.

[0232] In some embodiments, the amino acid sequence of the engineered RNA polymerase contains at least one substitution or set of substitutions at the following amino acid positions: 609 / 779, 246 / 609, 246, 609 / 775 / 779, 105, 143 / 609 / 775, 105 / 135 / 609 / 775, or 111 / 609, 609, wherein the amino acid position is relative to the reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 394 or relative to the reference sequence corresponding to SEQ ID NO: 394.

[0233] In some embodiments, the amino acid sequence of the engineered RNA polymerase comprises at least one substitution or set of substitutions or amino acid residues 609E / 779R, 246K / 609E, 246K, 609E / 775K / 779R, 105R, 143N / 609E / 775K, 105R / 135K / 609E / 775K, 111K / 609E or 609E, wherein the amino acid position is relative to a reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 394 or relative to a reference sequence corresponding to SEQ ID NO: 394.

[0234] In some embodiments, the amino acid sequence of the engineered RNA polymerase comprises at least one substitution or set of substitutions: T609E / H779R, Q246K / T609E, Q246K, T609E / E775K / H779R, K105R, A143N / T609E / E775K, K105R / S135K / T609E / E775K, Q111K / T609E, or T609E, wherein the amino acid position is relative to a reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 394 or relative to a reference sequence corresponding to SEQ ID NO: 394.

[0235] In some embodiments, the engineered RNA polymerase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with the reference sequence corresponding to SEQ ID NO: 588, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 588 or relative to the reference sequence corresponding to SEQ ID NO: 588.

[0236] In some embodiments, the engineered RNA polymerase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 96%, 97%, 98%, 99% or higher sequence identity with the reference sequence corresponding to the even-numbered residues of SEQ ID NO: 604-680, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 588 or relative to the reference sequence corresponding to SEQ ID NO: 588.

[0237] In some embodiments, the amino acid sequence of the engineered RNA polymerase contains at least one substitution or set of substitutions at the following amino acid positions: 32, 531 / 533, 115, 83, 32 / 60 / 61, 55, 589, 357, 617, 172, 598, 56 / 193, 56, 536, 52, 17, 261, 78, 629, 63, 82, 207, 666, 150, 45, 135, 13, 33, or 245, wherein the amino acid position is relative to the reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 588 or relative to the reference sequence corresponding to SEQ ID NO: 588.

[0238] In some embodiments, the amino acid sequence of the engineered RNA polymerase comprises at least one substitution or set of substitutions or amino acid residues 32V, 531Q / 533S, 115L, 83K, 32V / 60M / 61W, 55D, 589C, 357P, 617R, 172G, 598A, 56G / 193G, 56T, 536V, 52T, 17N, 56N, 598K, 261M, 56E, 55S, 536E, 598L, 78S, 56V, 629R, 598R, 63S, 82S, 207S, 598V, 666M, 150Y, 45K, 55W, 135G, 13N, 33E, or 245C, wherein the amino acid position is relative to the reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 588 or relative to the sequence corresponding to SEQ ID NO: 588. Reference sequence for NO:588.

[0239] In some embodiments, the amino acid sequence of the engineered RNA polymerase includes at least one substitution or set of substitutions: A32V, H531Q / L533S, E115L, T83K, A32V / K60M / M61W, E55D, L589C, E357P, K617R, N172G, D598A, A56G / V193G, A56T, N536V, E52T, D17N. The amino acid positions are A56N, D598K, I261M, A56E, E55S, N536E, D598L, K78S, A56V, A629R, D598R, E63S, T82S, A207S, D598V, I666M, R150Y, A45K, E55W, S135G, I13N, D33E, or G245C, wherein the amino acid positions are relative to the reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 588 or relative to the reference sequence corresponding to SEQ ID NO: 588.

[0240] In some embodiments, the engineered RNA polymerase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with the reference sequence corresponding to SEQ ID NO: 604, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 604 or relative to the reference sequence corresponding to SEQ ID NO: 604.

[0241] In some embodiments, the engineered RNA polymerase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 96%, 98%, 99% or higher sequence identity with the reference sequence corresponding to the even-numbered residues of SEQ ID NO: 604, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 604 or relative to the reference sequence corresponding to SEQ ID NO: 604.

[0242] In some embodiments, the amino acid sequence of the engineered RNA polymerase contains at least one substitution or set of substitutions at the following amino acid positions: 207 / 245 / 629, 17 / 56 / 150 / 598, 56 / 629, 536, 185, 56 / 150, 17 / 56 / 150 / 207 / 245 / 598 / 617 / 629, 56 / 150 / 536 / 598, 56 / 536, 185 / 666, 150, or 143 / 150 / 246 / 782, wherein the amino acid position is relative to the reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 604 or relative to the reference sequence corresponding to SEQ ID NO: 604.

[0243] In some embodiments, the amino acid sequence of the engineered RNA polymerase comprises at least one substitution or set of substitutions or amino acid residues 207S / 245C / 629R, 17N / 56E / 150Y / 598A, 56E / 629R, 536V, 185P, 56T / 150Y, 17N / 56E / 150Y / 207S / 245C / 598A / 617R / 629R, 56E / 150Y / 536V / 598K, 56V / 536V, 185P / 666M, 150Y, or 143N / 150Y / 246V / 782V, wherein the amino acid position is relative to a reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 604 or relative to a reference sequence corresponding to SEQ ID NO: 604.

[0244] In some embodiments, the amino acid sequence of the engineered RNA polymerase comprises at least one substitution or set of substitutions: A207S / G245C / A629R, D17N / A56E / R150Y / D598A, A56E / A629R, N536V, Y185P, A56T / R150Y, D17N / A56E / R150Y / A207S / G245C / D598A / K617R / A629R, A56E / R150Y / N536V / D598K, A56V / N536V, Y185P / I666M, R150Y, or A143N / R150Y / K246V / E782V, wherein the amino acid position is relative to the reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 604 or relative to the reference sequence corresponding to SEQ ID NO: 604. Reference sequence for 604.

[0245] In some embodiments, the engineered RNA polymerase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with the reference sequence corresponding to SEQ ID NO: 686, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 686 or relative to the reference sequence corresponding to SEQ ID NO: 686.

[0246] In some embodiments, the engineered RNA polymerase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 96%, 97%, 98%, 99% or higher sequence identity with the reference sequence corresponding to the even-numbered residues of SEQ ID NO: 706-720, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 686 or relative to the reference sequence corresponding to SEQ ID NO: 686.

[0247] In some embodiments, the amino acid sequence of the engineered RNA polymerase contains at least one substitution or set of substitutions at the following amino acid positions: 143, 246 / 424 / 648 / 782, 246 / 659 / 782, 56 / 150 / 629 / 660 / 782, 721 / 782, 782, 196 / 659 / 782, or 720 / 721 / 782, wherein the amino acid position is relative to the reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 686 or relative to the reference sequence corresponding to SEQ ID NO: 686.

[0248] In some embodiments, the amino acid sequence of the engineered RNA polymerase comprises at least one substitution or set of substitutions or amino acid residues 143N, 246R / 424A / 648F / 782V, 246R / 659E / 782V, 56T / 150Y / 629A / 660W / 782V, 721T / 782V, 782V, 196G / 659E / 782V, or 720S / 721A / 782V, wherein the amino acid position is relative to a reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 686 or relative to a reference sequence corresponding to SEQ ID NO: 686.

[0249] In some embodiments, the amino acid sequence of the engineered RNA polymerase comprises at least one substitution or set of substitutions: A143N, K246R / P424A / S648F / E782V, K246R / R659E / E782V, E56T / R150Y / R629A / H660W / E782V, K721T / E782V, E782V, D196G / R659E / E782V, or K720S / K721A / E782V, wherein the amino acid position is relative to a reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 686 or relative to a reference sequence corresponding to SEQ ID NO: 686.

[0250] In some embodiments, the engineered RNA polymerase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with the reference sequence corresponding to SEQ ID NO: 692, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 692 or relative to the reference sequence corresponding to SEQ ID NO: 692.

[0251] In some embodiments, the engineered RNA polymerase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 96%, 97%, 98%, 99% or higher sequence identity with the reference sequence corresponding to the even-numbered residues of SEQ ID NO: 722-730, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 692 or relative to the reference sequence corresponding to SEQ ID NO: 692.

[0252] In some embodiments, the amino acid sequence of the engineered RNA polymerase contains at least one substitution or set of substitutions at the following amino acid positions: 143 / 659 / 782, 166 / 171 / 246 / 311, 143 / 721, 782, or 143 / 171 / 659 / 782, wherein the amino acid position is relative to a reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 692 or relative to a reference sequence corresponding to SEQ ID NO: 692.

[0253] In some embodiments, the amino acid sequence of the engineered RNA polymerase comprises at least one substitution or set of substitutions or amino acid residues 143N / 659E / 782V, 166T / 171E / 246R / 311A, 143N / 721T, 782V or 143N / 171E / 659E / 782V, wherein the amino acid position is relative to a reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 692 or relative to a reference sequence corresponding to SEQ ID NO: 692.

[0254] In some embodiments, the amino acid sequence of the engineered RNA polymerase comprises at least one substitution or set of substitutions A143N / R659E / E782V, A166T / K171E / K246R / G311A, A143N / K721T, E782V, or A143N / K171E / R659E / E782V, wherein the amino acid position is relative to a reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 692 or relative to a reference sequence corresponding to SEQ ID NO: 692.

[0255] In some embodiments, the engineered RNA polymerase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with the reference sequence corresponding to SEQ ID NO: 712, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 712 or relative to the reference sequence corresponding to SEQ ID NO: 712.

[0256] In some embodiments, the engineered RNA polymerase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 96%, 97%, 98%, 99% or higher sequence identity with the reference sequence corresponding to the even-numbered residues of SEQ ID NO: 732-802, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 712 or relative to the reference sequence corresponding to SEQ ID NO: 712.

[0257] In some embodiments, the amino acid sequence of the engineered RNA polymerase contains at least one substitution or set of substitutions at the following amino acid positions: 367, 382, ​​670, 672, 357, 388, 693, 397, 375, 137 / 382 / 782, 655, 137 / 382 / 392, 391, 137 / 655 / 721 / 763, 696, 663, 137 / 382, 137 / 382 / 392 / 650 / 655 / 782, 137 / 382 / 629 / 660 / 672, 616, 690, 179, 137, 357 / 382 / 670, 388 / 670 / 672, 357 / 375, 357 / 672, 629, or 655 / 670, wherein the amino acid position is relative to the corresponding SEQ ID. Reference sequence of residues 8 to 890 of NO: 712 or relative to the reference sequence corresponding to SEQ ID NO: 712.

[0258] In some embodiments, the amino acid sequence of the engineered RNA polymerase contains at least one substitution or set of substitutions or amino acid residues 367P, 382R, 670R, 382L, 672P, 357P, 388R, 693R, 672S, 397S, 375P, 672G, 137G / 382R / 782E, 655R, 137G / 382R / 392A, 391F, 137G / 655M / 721T / 763S, 696L, 672R, 663L, 36 7T, 367F, 137G / 382G, 137G / 382G / 392R / 650M / 655M / 782E, 137G / 382R / 629R / 660Y / 672T, 391S, 616A, 690S, 179N, 137G, 357P / 382R / 670R, 388R / 670R / 672P, 357P / 375P, 357P / 672G, 629K, or 655R / 670R, wherein the amino acid position is relative to the reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 712 or relative to the reference sequence corresponding to SEQ ID NO: 712.

[0259] In some embodiments, the amino acid sequence of the engineered RNA polymerase includes at least one substitution or set of substitutions: L367P, T382R, K670R, T382L, L672P, E357P, K388R, S693R, L672S, A397S, D375P, L672G, D137G / T382R / V782E, Q655R, D137G / T382R / Y392A, V391F, D137G / Q655M / K721T / R763S, V696L, L672R, Q663L, L367T, L3 67F, D137G / T382G, D137G / T382G / Y392R / E650M / Q655M / V782E, D137G / T382R / A629R / W660Y / L672T, V391S, V616A, E690S, K179N, D137G, E357P / T382R / K670R, K388R / K670R / L672P, E357P / D375P, E357P / L672G, A629K, or Q655R / K670R, wherein the amino acid position is relative to the reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 712 or relative to the reference sequence corresponding to SEQ ID NO: 712.

[0260] In some embodiments, the engineered RNA polymerase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with the reference sequence corresponding to SEQ ID NO: 800, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 800 or relative to the reference sequence corresponding to SEQ ID NO: 800.

[0261] In some embodiments, the engineered RNA polymerase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 96%, 97%, 98%, 99% or higher sequence identity with the reference sequence corresponding to the even-numbered residues of SEQ ID NO: 804-828, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 800 or relative to the reference sequence corresponding to SEQ ID NO: 800.

[0262] In some embodiments, the amino acid sequence of the engineered RNA polymerase contains at least one substitution or set of substitutions at the following amino acid positions: 137, 56 / 311 / 382, 56 / 137 / 143 / 721, 137 / 392, 56 / 137 / 392, 56 / 137 / 143 / 404 / 720, 56 / 143 / 246, 56 / 392, 392 / 404, 392, 56 / 137, or 56 / 196 / 392, wherein the amino acid position is relative to the reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 800 or relative to the reference sequence corresponding to SEQ ID NO: 800.

[0263] In some embodiments, the amino acid sequence of the engineered RNA polymerase comprises at least one substitution or set of substitutions or amino acid residues 137G, 56E / 311A / 382R, 56E / 137G / 143N / 721A, 137G / 392A, 56E / 137G / 392A, 56A / 137G / 143N / 404Y / 720S, 56E / 143N / 246R, 56E / 392R, 392R / 404Y, 392R, 137G / 392R, 56E / 137G, or 56E / 196S / 392A, wherein the amino acid position is relative to a reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 800 or relative to a reference sequence corresponding to SEQ ID NO: 800.

[0264] In some embodiments, the amino acid sequence of the engineered RNA polymerase comprises at least one substitution or set of substitutions: D137G, T56E / G311A / T382R, T56E / D137G / A143N / K721A, D137G / Y392A, T56E / D137G / Y392A, T56A / D137G / A143N / L404Y / K720S, T56E / A143N / K246R, T56E / Y392R, Y392R / L404Y, Y392R, D137G / Y392R, T56E / D137G, or T56E / D196S / Y392A, wherein the amino acid position is relative to the reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 800 or relative to the reference sequence corresponding to SEQ ID NO: 800. Reference sequence for 800.

[0265] In some embodiments, the amino acid sequence of the engineered RNA polymerase contains at least one substitution at the amino acid positions described in Tables 5.1, 6.1, 7.1, 8.1, 9.1, 10.1, 11.1, 12.1, 13.1, 14.1, 15.1, 16.1, 17.1, 18.1, 19.1, 23.1, and 24.1, wherein the amino acid position is relative to a reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 2, 4, 32, 44, 156, 234, 238, 386, 394, 588, 604, 686, 692, 712, or 800, or relative to a reference sequence corresponding to SEQ ID NO: 2, 4, 32, 44, 156, 234, 238, 386, 394, 588, 604, 686, 692, 712, or 800.

[0266] In some embodiments, the amino acid sequence of the engineered RNA polymerase comprises at least one substitution as described in Tables 5.1, 6.1, 7.1, 8.1, 9.1, 10.1, 11.1, 12.1, 13.1, 14.1, 15.1, 16.1, 17.1, 18.1, 19.1, 23.1, and 24.1, wherein the amino acid position is relative to a reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 2, 4, 32, 44, 156, 234, 238, 386, 394, 588, 604, 686, 692, 712, or 800, or relative to a reference sequence corresponding to SEQ ID NO: 2, 4, 32, 44, 156, 234, 238, 386, 394, 588, 604, 686, 692, 712, or 800.

[0267] In some embodiments, the amino acid sequence of the engineered RNA polymerase contains substitutions or sets of substitutions at the amino acid positions described in Tables 5.1, 6.1, 7.1, 8.1, 9.1, 10.1, 11.1, 12.1, 13.1, 14.1, 15.1, 16.1, 17.1, 18.1, 19.1, 23.1, and 24.1, wherein the amino acid positions are reference sequences relative to residues 8 to 890 corresponding to SEQ ID NO: 2, 4, 32, 44, 156, 234, 238, 386, 394, 588, 604, 686, 692, 712, or 800, or relative to reference sequences corresponding to SEQ ID NO: 2, 4, 32, 44, 156, 234, 238, 386, 394, 588, 604, 686, 692, 712, or 800.

[0268] In some embodiments, the amino acid sequence of the engineered RNA polymerase comprises at least one substitution or set of substitutions of the engineered RNA polymerases described in Tables 5.1, 6.1, 7.1, 8.1, 9.1, 10.1, 11.1, 12.1, 13.1, 14.1, 15.1, 16.1, 17.1, 18.1, 19.1, 23.1, and 24.1, wherein the amino acid position is relative to the reference sequence of residues 8 to 890 corresponding to SEQ ID NO: 2, 4, 32, 44, 156, 234, 238, 386, 394, 588, 604, 686, 692, 712, or 800, or relative to the sequence corresponding to SEQ ID NO: Reference sequences of 2, 4, 32, 44, 156, 234, 238, 386, 394, 588, 604, 686, 692, 712 or 800.

[0269] In some embodiments, the engineered RNA polymerase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 96%, 97%, 98%, 99% or higher sequence identity with a reference sequence comprising substitutions or sets of substitutions as described in Tables 5.1, 6.1, 7.1, 8.1, 9.1, 13.1, 23.1, and 24.1, wherein the amino acid position is relative to the position corresponding to SEQ ID NO: Reference sequences of residues 8 to 890 of SEQ ID NO: 2, 4, 32, 44, 156, 234, 238, 386, 394, 588, 604, 686, 692, 712, or 800, or reference sequences relative to SEQ ID NO: 2, 4, 32, 44, 156, 234, 238, 386, 394, 588, 604, 686, 692, 712, or 800.

[0270] In some embodiments, the amino acid sequence of the engineered RNA polymerase comprises residues 8 to 890 of the amino acid sequences described in Tables 5.1, 6.1, 7.1, 8.1, 9.1, 10.1, 11.1, 12.1, 13.1, 14.1, 15.1, 16.1, 17.1, 18.1, 19.1, 23.1, and 24.1, or comprises the amino acid sequences described in Tables 5.1, 6.1, 7.1, 8.1, 9.1, 10.1, 11.1, 12.1, 13.1, 14.1, 15.1, 16.1, 17.1, 18.1, 19.1, 23.1, and 24.1.

[0271] In some embodiments, the engineered RNA polymerase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with the sequence corresponding to residues 8 to 890: SEQ ID NO: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 1 34, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236, 238 240, 242, 244, 246, 248, 250, 252, 254, 256, 258, 260, 262, 264, 266, 268, 270, 272, 274, 276, 278, 280, 282, 284, 286, 288, 290, 292, 294, 296, 298, 300, 302, 304, 306, 308, 310, 312, 314, 316, 318, 320, 322, 324, 326, 328, 330, 332, 334, 336, 338, 340, 342, 3 44, 346, 348, 350, 352, 354, 356, 358, 360, 362, 364, 366, 368, 370, 372, 374, 376, 378, 380, 382, ​​384, 386, 388, 390, 392, 394, 396, 398, 400, 402, 404, 406, 408, 410, 412, 414, 416, 418, 420, 422, 424, 426, 428, 430, 432, 434, 436, 438, 440, 442, 444, 446, 448450, 452, 454, 456, 458, 460, 462, 464, 466, 468, 470, 472, 474, 476, 478, 480, 482, 484, 486, 488, 490, 492, 494, 496, 498, 400, 402, 404, 406, 408, 410, 412, 414, 416, 418, 420, 422, 424, 426, 428, 430, 432, 434, 436, 438, 440, 442, 444, 446, 448, 450, 452, 454, 456, 458, 460, 462, 464, 466, 468, 470, 472, 474, 476, 478, 480, 482, 484, 486, 488, 490, 492, 494, 496, 498, 500, 502, 504, 506, 508, 510, 512, 514, 516, 518, 520, 522, 524, 526, 528, 530, 532, 534, 536, 538, 540, 542, 544, 546, 548, 550, 552, 554, 556, 558, 560, 562, 564, 566, 568, 570, 572, 574, 576, 578, 580, 582, 584, 586, 588 590, 592, 594, 596, 598, 600, 602, 604, 606, 608, 610, 612, 614, 616, 618, 620, 622, 624, 626, 628, 630, 632, 634, 636, 638, 640, 642, 644, 646, 648, 650, 652, 654, 656, 658, 660, 662, 664, 666, 668, 670, 672, 674, 676, 678, 680, 682, 684, 686, 688, 690, 692, 694, 696, 698, 700, 702, 704, 706, 708 710, 712, 714, 716, 718, 720, 722, 724, 276, 728, 730, 732, 734, 736, 738, 740, 742, 744, 746, 748, 750, 752, 754, 756, 758, 760, 762, 764, 766, 768, 770, 772, 774, 776, 778, 780, 782, 784, 786, 788, 790, 792, 794, 796, 798, 800, 802, 804, 806, 808, 810, 812, 814, 816, 818, 820, 822, 824, 826 or 828.

[0272] In some embodiments, the engineered RNA polymerase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with the sequence corresponding to: SEQ ID NO: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236, 238, 240 242, 244, 246, 248, 250, 252, 254, 256, 258, 260, 262, 264, 266, 268, 270, 272, 274, 276, 278, 280, 282, 284, 286, 288, 290, 292, 294, 296, 298, 300, 302, 304, 306, 308, 310, 312, 314, 316, 318, 320, 322, 324, 326, 328, 330, 332, 334, 336, 338, 340, 342, 344, 346 348, 350, 352, 354, 356, 358, 360, 362, 364, 366, 368, 370, 372, 374, 376, 378, 380, 382, ​​384, 386, 388, 390, 392, 394, 396, 398, 400, 402, 404, 406, 408, 410, 412, 414, 416, 418, 420, 422, 424, 426, 428, 430, 432, 434, 436, 438, 440, 442, 444, 446, 448, 450, 452,454, 456, 458, 460, 462, 464, 466, 468, 470, 472, 474, 476, 478, 480, 482, 484, 486, 488, 490, 492, 494, 496, 498, 400, 402, 404, 406, 408, 410, 412, 414, 416, 418, 420, 422, 424, 426, 428, 430, 432, 434, 436, 438, 440, 442, 444, 446, 448, 450, 452, 454, 456, 458, 460, 462, 464, 466, 468, 470, 47 2, 474, 476, 478, 480, 482, 484, 486, 488, 490, 492, 494, 496, 498, 500, 502, 504, 506, 508, 510, 512, 514, 516, 518, 520, 522, 524, 526, 528, 530, 532, 534, 536, 538, 540, 542, 544, 546, 548, 550, 552, 554, 556, 558, 560, 562, 564, 566, 568, 570, 572, 574, 576, 578, 580, 582, 584, 586, 588, 590, 592, 594, 596, 598, 600, 602, 604, 606, 608, 610, 612, 614, 616, 618, 620, 622, 624, 626, 628, 630, 632, 634, 636, 638, 640, 642, 644, 646, 648, 650, 652, 654, 656, 658, 660, 662, 664, 666, 668, 670, 672, 674, 676, 678, 680, 682, 684, 686, 688, 690, 692, 694, 696, 698, 700, 702, 704, 706, 708, 71 0, 712, 714, 716, 718, 720, 722, 724, 276, 728, 730, 732, 734, 736, 738, 740, 742, 744, 746, 748, 750, 752, 754, 756, 758, 760, 762, 764, 766, 768, 770, 772, 774, 776, 778, 780, 782, 784, 786, 788, 790, 792, 794, 796, 798, 800, 802, 804, 806, 808, 810, 812, 814, 816, 818, 820, 822, 824, 826 or 828.

[0273] In some embodiments, the amino acid sequence of the engineered RNA polymerase comprises residues 8 to 890 of SEQ ID NO. with even numbers from SEQ ID NO: 4-828, or comprises SEQ ID NO. with even numbers from SEQ ID NO: 4-828. In some embodiments, the amino acid sequence optionally has 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10 amino acid insertions, deletions, or substitutions. In some embodiments, the amino acid sequence optionally has 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10 amino acid substitutions. In some embodiments, the amino acid sequence optionally has 1, 2, 3, 4, or up to 5 amino acid insertions, deletions, or substitutions. In some embodiments, the amino acid sequence optionally has 1, 2, 3, 4, or up to 5 amino acid substitutions. In some embodiments, the amino acid substitutions comprise non-conserved or conserved substitutions. In some embodiments, the amino acid substitutions comprise conserved substitutions.

[0274] In some embodiments, the engineered RNA polymerase comprises an amino acid sequence containing residues 8 to 890 of the following: SEQ ID NO: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250, 252, 254, 256, 258, 260, 262, 264 266, 268, 270, 272, 274, 276, 278, 280, 282, 284, 286, 288, 290, 292, 294, 296, 298, 300, 302, 304, 306, 308, 310, 312, 314, 316, 318, 320, 322, 324, 326, 328, 330, 332, 334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360, 362, 364, 366, 368, 370, 372, 374, 376, 378, 380, 382 384, 386, 388, 390, 392, 394, 396, 398, 400, 402, 404, 406, 408, 410, 412, 414, 416, 418, 420, 422, 424, 426, 428, 430, 432, 434, 436, 438, 440, 442, 444, 446, 448, 450, 452, 454, 456, 458, 460, 462, 464, 466, 468, 470, 472, 474, 476, 478, 480, 482, 484, 486, 488, 490, 492, 494, 496, 498, 400,402, 404, 406, 408, 410, 412, 414, 416, 418, 420, 422, 424, 426, 428, 430, 432, 434, 436, 438, 440, 442, 444, 446, 448, 450, 452, 454, 456, 458, 460, 462, 464, 466, 468, 470, 472, 474, 476, 478, 480, 482, 484, 486, 488, 490, 492, 494, 496, 498, 500, 502, 504, 506, 50 8, 510, 512, 514, 516, 518, 520, 522, 524, 526, 528, 530, 532, 534, 536, 538, 540, 542, 544, 546, 548, 550, 552, 554, 556, 558, 560, 562, 564, 566, 568, 570, 572, 574, 576, 578, 580, 582, 584, 586, 588, 590, 592, 594, 596, 598, 600, 602, 604, 606, 608, 610, 612, 614, 616, 618, 620, 622, 624, 626, 628, 630, 632, 634, 636, 638, 640, 642, 644, 646, 648, 650, 652, 654, 656, 658, 660, 662, 664, 666, 668, 670, 672, 674, 676, 678, 680, 682, 684, 686, 688, 690, 692, 694, 696, 698, 700, 702, 704, 706, 708, 710, 712, 714, 716, 718, 720, 72 2, 724, 276, 728, 730, 732, 734, 736, 738, 740, 742, 744, 746, 748, 750, 752, 754, 756, 758, 760, 762, 764, 766, 768, 770, 772, 774, 776, 778, 780, 782, 784, 786, 788, 790, 792, 794, 796, 798, 800, 802, 804, 806, 808, 810, 812, 814, 816, 818, 820, 822, 824, 826, or 828. In some embodiments, the amino acid sequence optionally has 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10 amino acid insertions, deletions, or substitutions. In some embodiments, the amino acid sequence optionally has 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10 amino acid substitutions. In some embodiments, the amino acid sequence optionally has 1, 2, 3, 4, or up to 5 amino acid insertions, deletions, or substitutions.Deletion or substitution. In some embodiments, the amino acid sequence optionally has 1, 2, 3, 4, or up to 5 amino acid substitutions. In some embodiments, the amino acid substitutions include non-conservative or conserved substitutions. In some embodiments, the amino acid substitutions include conserved substitutions.

[0275] In some embodiments, the engineered RNA polymerase comprises an amino acid sequence including the following: SEQ ID NO: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 14 8, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250, 252, 254, 256, 258, 260, 262, 264, 266, 2 68, 270, 272, 274, 276, 278, 280, 282, 284, 286, 288, 290, 292, 294, 296, 298, 300, 302, 304, 306, 308, 310, 312, 314, 316, 318, 320, 322, 324, 326, 328, 330, 332, 334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360, 362, 364, 366, 368, 370, 372, 374, 376, 378, 380, 382, ​​384, 386, 388, 390, 392, 394, 396, 398, 400, 402, 404, 406, 408, 410, 412, 414, 416, 418, 420, 422, 424, 426, 428, 430, 432, 434, 436, 438, 440, 442, 444, 446, 448, 450, 452, 454, 456, 458, 460, 462, 464, 466, 468, 470, 472, 474, 476, 478, 480, 482, 484, 486, 488, 490, 492, 494, 496, 498, 400, 402, 404, 406,408, 410, 412, 414, 416, 418, 420, 422, 424, 426, 428, 430, 432, 434, 436, 438, 440, 442, 444, 446, 448, 450, 452, 454, 456, 458, 460, 462, 464, 466, 468, 470, 472, 474, 476, 478, 480, 482, 484, 486, 488, 490, 492, 494, 496, 498, 500, 502, 504, 506, 508, 510, 512 514, 516, 518, 520, 522, 524, 526, 528, 530, 532, 534, 536, 538, 540, 542, 544, 546, 548, 550, 552, 554, 556, 558, 560, 562, 564, 566, 568, 570, 572, 574, 576, 578, 580, 582, 584, 586, 588, 590, 592, 594, 596, 598, 600, 602, 604, 606, 608, 610, 612, 614, 616, 61 8, 620, 622, 624, 626, 628, 630, 632, 634, 636, 638, 640, 642, 644, 646, 648, 650, 652, 654, 656, 658, 660, 662, 664, 666, 668, 670, 672, 674, 676, 678, 680, 682, 684, 686, 688, 690, 692, 694, 696, 698, 700, 702, 704, 706, 708, 710, 712, 714, 716, 718, 720, 722, 7 24, 276, 728, 730, 732, 734, 736, 738, 740, 742, 744, 746, 748, 750, 752, 754, 756, 758, 760, 762, 764, 766, 768, 770, 772, 774, 776, 778, 780, 782, 784, 786, 788, 790, 792, 794, 796, 798, 800, 802, 804, 806, 808, 810, 812, 814, 816, 818, 820, 822, 824, 826 or 828. In some embodiments, the amino acid sequence optionally has 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10 amino acid insertions, deletions, or substitutions. In some embodiments, the amino acid sequence optionally has 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10 amino acid substitutions. In some embodiments, the amino acid sequence optionally has 1, 2, 3, 4, or up to 5 amino acid insertions, deletions, or substitutions.Deletion or substitution. In some embodiments, the amino acid sequence optionally has 1, 2, 3, 4, or up to 5 amino acid substitutions. In some embodiments, the amino acid substitutions include non-conservative or conserved substitutions. In some embodiments, the amino acid substitutions include conserved substitutions.

[0276] In some embodiments, the engineered RNA polymerase of this disclosure has RNA polymerase activity. In some embodiments, the engineered RNA polymerase has RNA polymerase activity and at least one improved or enhanced property compared to a reference RNA polymerase.

[0277] In some embodiments, the engineered RNA polymerase has increased RNA polymerase activity compared to a reference RNA polymerase. In some embodiments, the engineered RNA polymerase exhibits activity that is 1.1, 1.2, 1.3, 1.4, 1.5, 2, 2.5, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, or 40 times or more compared to a reference RNA polymerase. In some embodiments, the activity increase is described as a sustained increase.

[0278] In some embodiments, the engineered RNA polymerase exhibits improved thermal stability compared to a reference RNA polymerase. Specifically, in some embodiments, the engineered RNA polymerase exhibits improved thermal stability at 33°C or higher compared to a reference RNA polymerase.

[0279] In some embodiments, the engineered RNA polymerase has improved product integrity compared to a reference RNA polymerase. In some embodiments, product integrity refers to the percentage of the desired RNA product relative to the total RNA produced in a transcription reaction (e.g., an in vitro transcription reaction). In the method of the reference examples, product integrity is the area of ​​the desired full-length RNA electrophoretic peak divided by the total area of ​​the electrophoretic traces measured by a fragment analyzer. In some embodiments, improved product integrity is characterized by an increase in the purity of the full-length RNA product produced in an in vitro transcription reaction compared to a reference in vitro transcription reaction (e.g., under different conditions and / or with different RNA polymerases). In some embodiments, the engineered RNA polymerase has at least a 1.1, 1.2, 1.3, 1.4, 1.5, 2, 2.5, 3, 3.5, or 4-fold improvement in product integrity compared to a reference RNA polymerase.

[0280] In some embodiments, the reference RNA polymerase has a sequence of residues 8 to 890 corresponding to SEQ ID NO: 2, 4, 32, 44, 156, 234, 238, 386, 394, 588, 604, 686, 692, 712, or 800, or a sequence corresponding to SEQ ID NO: 2, 4, 32, 44, 156, 234, 238, 386, 394, 588, 604, 686, 692, 712, or 800. In some embodiments, the reference RNA polymerase has a sequence of residues 8 to 890 corresponding to SEQ ID NO: 2, or a sequence corresponding to SEQ ID NO: 2.

[0281] In some embodiments, the engineered RNA polymerase has improved properties selected from the following, compared to a reference RNA polymerase: i) increased activity, ii) increased thermostability, iii) increased persistence, or iv) increased RNA product integrity, or any combination of i), ii), iii), and iv). In some embodiments, the reference RNA polymerase has a sequence of residues 8 to 890 corresponding to SEQ ID NO: 2, 4, 32, 44, 156, 234, 238, 386, 394, 588, 604, 686, 692, 712, or 800, or a sequence corresponding to SEQ ID NO: 2, 4, 32, 44, 156, 234, 238, 386, 394, 588, 604, 686, 692, 712, or 800. In some embodiments, the reference RNA polymerase has a sequence of residues 8 to 890 corresponding to SEQ ID NO: 2, or a sequence corresponding to SEQ ID NO: 2.

[0282] In some embodiments, the engineered RNA polymerase is expressed as a fusion protein. In some embodiments, the engineered RNA polymerase described herein may be fused to a variety of polypeptide sequences (e.g., polypeptide tags, by way of example but not limited to, that can be used for detection and / or purification). In some embodiments, the fusion protein of the engineered RNA polymerase comprises a glycine-histidine or histidine-tag (His-tag). In some embodiments, the engineered RNA polymerase comprises polylysine, such as 2-12 lysine units, for example, to conjugate with a support medium. In some embodiments, the fusion protein of the engineered RNA polymerase comprises an epitope tag, such as c-myc, FLAG, V5, or hemagglutinin (HA). In some embodiments, the fusion protein of the engineered RNA polymerase comprises a GST, SUMO, Strep, MBP, or GFP tag. In some embodiments, the fusion is fused to the amino (N-) terminus of the engineered RNA polymerase polypeptide. In some embodiments, the fusion is fused to the carboxyl (C-) terminus of the engineered RNA polymerase polypeptide.

[0283] In some embodiments, the engineered RNA polymerase peptide described herein is an isolated composition. In some embodiments, as further discussed herein, the engineered RNA polymerase peptide is substantially purified or purified.

[0284] In some embodiments, this disclosure also provides functional or bioactive fragments of the engineered RNA polymerase polypeptides described herein. Therefore, for each embodiment of the engineered RNA polymerase herein, a functional or bioactive fragment of the engineered RNA polymerase (e.g., the engineered RNA polymerases provided in Tables 5.1, 6.1, 7.1, 8.1, 9.1, 10.1, 11.1, 12.1, 13.1, 14.1, 15.1, 16.1, 17.1, 18.1, 19.1, 23.1, and 24.1) is also provided herein. In some embodiments, the functional or bioactive fragment of the engineered RNA polymerase comprises at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% of the activity of the RNA polymerase polypeptide from which it is derived (i.e., the parental RNA polymerase). In some embodiments, the functional or bioactive fragment comprises at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the parental sequence of the RNA polymerase. In some embodiments, the functional fragment will be truncated to less than 5, 10, 15, 25, 30, 35, 40, 45, 50, 55, 60, 65, or 70 amino acids.

[0285] In some embodiments, the functional or bioactive fragment of the engineered RNA polymerase polypeptide described herein includes at least a mutation or set of mutations in the amino acid sequence of the engineered RNA polymerase described herein. Therefore, in some embodiments, the functional or bioactive fragment of the engineered RNA polymerase exhibits enhancing or improved properties associated with the mutation or set of mutations in the parental RNA polymerase.

[0286] Polynucleotides encoding engineered polypeptides, expression vectors, and host cells

[0287] In another aspect, this disclosure provides recombinant polynucleotides encoding the engineered RNA polymerase described herein. In some embodiments, the recombinant polynucleotide is operatively linked to one or more heterologous regulatory sequences controlling gene expression to produce a recombinant polynucleotide construct capable of expressing the engineered RNA polymerase. In some embodiments, an expression construct containing at least one heterologous polynucleotide encoding an engineered RNA polymerase polypeptide is introduced into a suitable host cell to express the corresponding RNA polymerase polypeptide.

[0288] As will be apparent to those skilled in the art, the availability of protein sequences and knowledge of codons corresponding to various amino acids provide a description of all polynucleotides capable of encoding the subject polypeptide. The degeneracy of the genetic code (where the same amino acid is encoded by substitutional or synonymous codons) allows for the preparation of extremely large quantities of nucleic acids, all of which encode the engineered RNA polymerase of this disclosure. Therefore, this disclosure provides methods and compositions for producing each and every possible variant of the polynucleotide encoding the engineered RNA polymerase polypeptide described herein, which can be prepared by selecting combinations based on possible codon selection, and all such polynucleotide variants should be considered to be specifically disclosed for any polypeptide described herein, including the examples (e.g., Tables 5.1, 6.1, 7.1, 8.1, 9.1, 10.1, 11.1, 12.1, 13.1, 14.1, 15.1, 16.1, 17.1, 18.1, 19.1, 23.1, and 24.1) and the amino acid sequences presented in the sequence listing.

[0289] In some embodiments, the codons are preferably optimized for use by the selected host cell in protein production. In some embodiments, preferred codons in bacteria are used for expression in bacteria. In some embodiments, preferred codons in fungal cells are used for expression in fungal cells. In some embodiments, preferred codons in insect cells are used for expression in insect cells. In some embodiments, preferred codons in mammalian cells are used for expression in mammalian cells. In some embodiments, the codon-optimized polynucleotide encoding the engineered RNA polymerase polypeptide described herein contains preferred codons at approximately 40%, 50%, 60%, 70%, 80%, 90%, or greater than 90% of the codon positions in the full-length coding region.

[0290] Therefore, in some embodiments, the recombinant polynucleotide of this disclosure comprises a polynucleotide sequence encoding the engineered RNA polymerase polypeptide described herein. In some embodiments, the polynucleotide sequence of the recombinant polynucleotide is codon-optimized. In some embodiments, the polynucleotide sequence of the recombinant polynucleotide is codon-optimized for expression in bacterial cells.

[0291] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered RNA polymerase comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity relative to the sequence corresponding to SEQ ID NO: 2, 4, 32, 44, 156, 234, 238, 386, 394, 588, 604, 686, 692, 712, or 800. The reference sequence of residues 8 to 890 of SEQ ID NO: 2, 4, 32, 44, 156, 234, 238, 386, 394, 588, 604, 686, 692, 712, or 800, or the reference sequence relative to the reference sequence corresponding to SEQ ID NO: 2, 4, 32, 44, 156, 234, 238, 386, 394, 588, 604, 686, 692, 712, or 800, wherein the amino acid sequence comprises one or more substitutions.

[0292] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered RNA polymerase comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with the reference sequence corresponding to SEQ ID NO: 2, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 2 or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0293] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered RNA polymerase, said engineered RNA polymerase comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity relative to the sequence corresponding to SEQ ID NO: 4, 32, 44, 156, 234, 238, 386, 394, 588, 604, 686, 692, 712, or 800. The reference sequence of residues 8 to 890 of SEQ ID NO: 2, or relative to the reference sequence corresponding to SEQ ID NO: 2, contains one or more substitutions.

[0294] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered RNA polymerase comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with the even-numbered sequences of SEQ ID NO: 4-828, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 2 or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0295] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered RNA polymerase, said engineered RNA polymerase comprising an amino acid sequence containing at least one substituted amino acid at the following amino acid positions: 13, 14, 15, 17, 19, 21, 32, 33, 38, 45, 52, 54, 55, 56, 56, 57, 60, 61, 62, 63, 67, 69, 72, 75, 78, 81, 82, 83, 84, 89, 93, 95, 1 00, 105, 111, 115, 122, 124, 127, 131, 133, 135, 137, 138, 139, 140, 141, 143, 150, 160, 161, 162, 164, 166, 168, 169, 170, 171, 172, 173, 179, 185, 187, 193, 196, 207, 212, 217, 218, 221, 227, 237, 239, 240, 245, 246 250, 253, 254, 257, 261, 272, 274, 275, 311, 336, 340, 342, 351, 357, 367, 375, 379, 382, ​​388, 391, 392, 394, 397, 401, 402, 403, 404, 419, 424, 437, 440, 494, 510, 514, 517, 527, 529, 530, 531, 533, 536, 589, 594, 5 98, 607, 609, 615, 616, 617, 625, 629, 648, 650, 655, 659, 660, 663, 664, 666, 670, 672, 690, 693, 696, 718, 720, 721, 749, 759, 763, 773, 775, 779, 782, 782, 810, 854, 857, 867, 875 or 878 or combinations thereof, wherein the amino acid position is relative to the reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 2 or relative to the reference sequence corresponding to SEQ ID NO: 2.

[0296] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered RNA polymerase comprising an amino acid sequence containing at least one substituted amino acid at the following amino acid positions: 32, 38, 56, 138, 150, 170, 171, 173, 246, 311, 336, 340, 357, 388, 394, 401, 404, 437, 514, 536, 609, 629, 659, 660, 664, 779, or 782, or combinations thereof, wherein the amino acid position is relative to a reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 2 or relative to a reference sequence corresponding to SEQ ID NO: 2.

[0297] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered RNA polymerase comprising an amino acid sequence containing at least one substitution or set of substitutions at the following amino acid positions: 401 / 404, 336, 138, 401 / 403 / 404, 336 / 510, 388, 340, 401 / 402 / 404, or 351 / 357, wherein the amino acid position is relative to a reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 2 or relative to a reference sequence corresponding to SEQ ID NO: 2.

[0298] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an RNA polymerase comprising an amino acid sequence contained at the amino acid positions described in Tables 5.1, 6.1, 7.1, 8.1, 9.1, 10.1, 11.1, 12.1, 13.1, 14.1, 15.1, 16.1, 17.1, 18.1, 19.1, 23.1, and 24.1, wherein the amino acid positions are relative to a reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 2 or relative to a reference sequence corresponding to SEQ ID NO: 2.

[0299] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an RNA polymerase comprising at least one substituted amino acid sequence as described in Tables 5.1, 6.1, 7.1, 8.1, 9.1, 10.1, 11.1, 12.1, 13.1, 14.1, 15.1, 16.1, 17.1, 18.1, 19.1, 23.1, and 24.1, wherein the amino acid position is relative to a reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 2 or relative to a reference sequence corresponding to SEQ ID NO: 2.

[0300] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an RNA polymerase comprising an amino acid sequence containing at least one substitution or set of substitutions at the amino acid positions described in Tables 5.1, 6.1, 7.1, 8.1, 9.1, 10.1, 11.1, 12.1, 13.1, 14.1, 15.1, 16.1, 17.1, 18.1, 19.1, 23.1, and 24.1, wherein the amino acid positions are relative to a reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 2 or relative to a reference sequence corresponding to SEQ ID NO: 2.

[0301] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an RNA polymerase comprising an amino acid sequence comprising at least one substitution or set of substitutions of the engineered RNA polymerases described in Tables 5.1, 6.1, 7.1, 8.1, 9.1, 10.1, 11.1, 12.1, 13.1, 14.1, 15.1, 16.1, 17.1, 18.1, 19.1, 23.1, and 24.1, wherein the amino acid position is relative to a reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 2 or relative to a reference sequence corresponding to SEQ ID NO: 2.

[0302] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered RNA polymerase comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 94%, 95%, 96%, 98%, 99%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with the reference sequence corresponding to SEQ ID NO: 4, 32, 44, 156, 234, 238, 386, 394, 588, 604, 686, 692, 712, or 800.

[0303] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered RNA polymerase comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with the sequence corresponding to the even-numbered SEQ ID NOs of ...

[0304] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered RNA polymerase, said engineered RNA polymerase comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity relative to the sequence corresponding to SEQ ID NO: 4, 32, 44, 156, 234, 238, 386, 394, 588, 604, 686, 692, 712, or 800. The reference sequence of residues 8 to 890 of 4, 32, 44, 156, 234, 238, 386, 394, 588, 604, 686, 692, 712, or 800, or relative to the reference sequence corresponding to SEQ ID NO: 4, 32, 44, 156, 234, 238, 386, 394, 588, 604, 686, 692, 712, or 800, wherein the amino acid sequence comprises one or more substitutions.

[0305] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered RNA polymerase comprising a reference sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with respect to the reference sequence corresponding to SEQ ID NO: 4, 32, 44, 156, 234, 238, 386, 394, 588, 604, 686, 692, 712 or 800 of the reference sequence, wherein the reference sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 83%, 84%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with respect to the reference sequence corresponding to SEQ ID NO: 4, 32, 44, 156, 234, 238, 386, 394, 588, 604, 686, 692, 712 or 800 of the reference sequence, or ... A reference sequence of 4, 32, 44, 156, 234, 238, 386, 394, 588, 604, 686, 692, 712 or 800, wherein the amino acid sequence contains one or more substitutions.

[0306] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered RNA polymerase, said engineered RNA polymerase comprising an amino acid sequence containing at least one substituted amino acid at the following amino acid positions: 13, 14, 15, 17, 19, 21, 32, 33, 38, 45, 52, 54, 55, 56, 56, 57, 60, 61, 62, 63, 67, 69, 72, 75, 78, 81, 82, 83, 84, 89, 93, 95, 1 00, 105, 111, 115, 122, 124, 127, 131, 133, 135, 137, 138, 139, 140, 141, 143, 150, 160, 161, 162, 164, 166, 168, 169, 170, 171, 172, 173, 179, 185, 187, 193, 196, 207, 212, 217, 218, 221, 227, 237, 239, 240, 245, 246 250, 253, 254, 257, 261, 272, 274, 275, 311, 336, 340, 342, 351, 357, 367, 375, 379, 382, ​​388, 391, 392, 394, 397, 401, 402, 403, 404, 419, 424, 437, 440, 494, 510, 514, 517, 527, 529, 530, 531, 533, 536, 589, 594, 5 98, 607, 609, 615, 616, 617, 625, 629, 648, 650, 655, 659, 660, 663, 664, 666, 670, 672, 690, 693, 696, 718, 720, 721, 749, 759, 763, 773, 775, 779, 782, 782, 810, 854, 857, 867, 875, or 878, or combinations thereof, wherein the amino acid position is relative to the position corresponding to SEQ. Reference sequences of residues 8 to 890 of SEQ ID NO: 4, 32, 44, 156, 234, 238, 386, 394, 588, 604, 686, 692, 712, or 800, or reference sequences corresponding to SEQ ID NO: 4, 32, 44, 156, 234, 238, 386, 394, 588, 604, 686, 692, 712, or 800.

[0307] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered RNA polymerase comprising an amino acid sequence containing at least one substituted amino acid sequence at the following amino acid positions: 32, 38, 56, 138, 150, 170, 171, 173, 246, 311, 336, 340, 357, 388, 394, 401, 404, 437, 514, 536, 609, 629, 659, 660, 664, 779, or 782, or combinations thereof, wherein the amino acid position is relative to a reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 4, 32, 44, 156, 234, 238, 386, 394, 588, 604, 686, 692, 712, or 800, or relative to the sequence corresponding to SEQ ID NO: Reference sequences of 4, 32, 44, 156, 234, 238, 386, 394, 588, 604, 686, 692, 712 or 800.

[0308] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered RNA polymerase comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with respect to the reference sequence corresponding to SEQ ID NO: 4, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 4 or relative to the reference sequence corresponding to SEQ ID NO: 4.

[0309] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered RNA polymerase comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with the reference sequence corresponding to the even-numbered residues of SEQ ID NO: 28-40, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 4 or relative to the reference sequence corresponding to SEQ ID NO: 4.

[0310] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered RNA polymerase comprising an amino acid sequence containing at least one set of substitutions at the following amino acid positions: 311 / 388 / 404 / 440, 138 / 340, 138 / 311 / 336 / 340 / 388, 311 / 388 / 440, 138 / 311 / 340 / 388 / 404 / 440, 138 / 336 / 342, or 351 / 388, wherein the amino acid positions are relative to a reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 4 or relative to a reference sequence corresponding to SEQ ID NO: 4.

[0311] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered RNA polymerase comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with the reference sequence corresponding to SEQ ID NO: 32, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 32 or relative to the reference sequence corresponding to SEQ ID NO: 32.

[0312] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered RNA polymerase comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 98%, 99% or higher sequence identity with the reference sequence corresponding to the even-numbered residues of SEQ ID NO: 42-188, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 32 or relative to the reference sequence corresponding to SEQ ID NO: 32.

[0313] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered RNA polymerase, said engineered RNA polymerase comprising an amino acid sequence containing at least one substitution or set of substitutions at the following amino acid positions: 514, 38 / 514, 38 / 394 / 514, 38 / 394 / 514 / 664, 394 / 514 / 875, 394 / 514, 38 / 124 / 394 / 514, 171 / 878, 170, 173, 172. 170 / 629, 171, 168, 166, 166 / 272 / 274 / 275, 169, 207, 168 / 217 / 218, 171 / 530, 382, ​​773, 670, 379, 392, 419, 57, 164, 810, 659, 54, 172 / 173 / 514 / 659 / 660, 170 / 171 / 172 / 514 / 659, 173 / 514 / 659 / 660, 173 / 514 / 65 9 / 810, 170 / 340 / 514 / 659 / 660 / 810, 170 / 171 / 173 / 336 / 340 / 514 / 659 / 660, 57 / 171 / 173 / 514 / 659 / 660 / 810, 170 / 173 / 514 / 659 / 660 / 810, 57 / 171 / 172 / 336 / 340 / 514 / 659, 173 / 514, 171 / 172 / 173 / 514 / 659 / 660, 5 7 / 170 / 171 / 173 / 514 / 659 / 660 / 810, 57 / 173 / 514, 172 / 173 / 514 / 857, 57 / 173 / 340 / 514, 170 / 171 / 173 / 336 / 514, 171 / 514, 57 / 170 / 173 / 336 / 514, 170 / 336, 173 / 514 / 659, 57 / 514 or 173 / 810, wherein the amino acid position is relative to the reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 32 or relative to the reference sequence corresponding to SEQ ID NO: 32.

[0314] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered RNA polymerase comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with the reference sequence corresponding to SEQ ID NO: 44, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 44 or relative to the reference sequence corresponding to SEQ ID NO: 44.

[0315] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered RNA polymerase comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 96%, 97%, 98%, 99% or higher sequence identity with respect to the reference sequence corresponding to the even-numbered residues of SEQ ID NO: 190-194, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to the residues of SEQ ID NO: 44 or relative to the reference sequence corresponding to SEQ ID NO: 44.

[0316] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered RNA polymerase comprising an amino acid sequence containing at least one substitution or set of substitutions at the following amino acid positions: 336, 143 / 336 / 340 / 782, or 143 / 336 / 340 / 357 / 514, wherein the amino acid position is relative to a reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 44 or relative to a reference sequence corresponding to SEQ ID NO: 44.

[0317] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered RNA polymerase comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with the reference sequence corresponding to SEQ ID NO: 156, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 156 or relative to the reference sequence corresponding to SEQ ID NO: 156.

[0318] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered RNA polymerase comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 96%, 97%, 98%, 99% or higher sequence identity with the reference sequence corresponding to the even-numbered residues of SEQ ID NO: 196-260, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 156 or relative to the reference sequence corresponding to SEQ ID NO: 156.

[0319] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered RNA polymerase, said engineered RNA polymerase comprising an amino acid sequence containing at least one substitution or set of substitutions at the following amino acid positions: 207, 419, 207 / 336 / 340, 166 / 169, 207 / 670, 166 / 336 / 340, 207 / 419 / 670, 207 / 336 / 340 / 382 / 419 / 670, 207 / 382, 670, 336 / 670, 340 / 670, 382, ​​127 / 141 / 207 / 336 / 382 / 670, 207 / 336 / 382 / 419 / 670, 419 / 670, 166 / 168 / 207 / 336 / 340 / 419 / 670, 166 / 340 or 207 / 336 / 340 / 670, wherein the amino acid position is relative to the reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 156 or relative to the reference sequence corresponding to SEQ ID NO: 156.

[0320] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered RNA polymerase comprising an amino acid sequence containing at least one substitution or set of substitutions at the following amino acid positions: 38 / 437 / 779, 38 / 437, 38, 38 / 207 / 437 / 779, 517 / 779, 38 / 437 / 527 / 664, 38 / 207 / 379 / 437, 664 / 670 / 779, 38 / 779, 38 / 437 / 517, 437 / 779, 38 / 437 / 607, 38 / 379 / 779, or 517 / 670 / 779, wherein the amino acid position is relative to a reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 156 or relative to a reference sequence corresponding to SEQ ID NO: 156.

[0321] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered RNA polymerase comprising an amino acid sequence containing at least one set of substitutions at amino acid positions 357 / 394 / 664 or 138 / 311 / 336 / 340 / 357 / 388 / 394 / 404 / 664, wherein the amino acid positions are relative to a reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 156 or relative to a reference sequence corresponding to SEQ ID NO: 156.

[0322] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered RNA polymerase comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with the reference sequence corresponding to SEQ ID NO: 234, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 234 or relative to the reference sequence corresponding to SEQ ID NO: 234.

[0323] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered RNA polymerase comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 96%, 97%, 98%, 99% or higher sequence identity with the reference sequence corresponding to the even-numbered residues of SEQ ID NO: 262-384, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 234 or relative to the reference sequence corresponding to SEQ ID NO: 234.

[0324] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered RNA polymerase, said engineered RNA polymerase comprising an amino acid sequence containing at least one substitution or set of substitutions at the following amino acid positions: 720, 196, 609, 246, 185, 594, 212, 494, 375, 721, 72, 867, 160, 718, 187, 170 / 173, 615, 137 / 138, 854, 131, 38 / 185 / 187 / 437, 185 / 187, 437 / 609, 38 / 609, 38 / 185 / 187 / 437 / 609, 437 / 609 / 720, 185 / 187 / 609, 185 / 187 / 437, 437 / 721, 609 / 720, 185 / 187 / 246 or 185 / 187 / 437 / 609 / 720, wherein the amino acid position is relative to the reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 234 or relative to the reference sequence corresponding to SEQ ID NO: 234.

[0325] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered RNA polymerase comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with the reference sequence corresponding to SEQ ID NO: 386, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 386 or relative to the reference sequence corresponding to SEQ ID NO: 386.

[0326] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered RNA polymerase comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 96%, 97%, 98%, 99% or higher sequence identity with the reference sequence corresponding to the even-numbered residues of SEQ ID NO: 388-460, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 386 or relative to the reference sequence corresponding to SEQ ID NO: 386.

[0327] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered RNA polymerase, said engineered RNA polymerase comprising an amino acid sequence containing at least one substitution or set of substitutions at the following amino acid positions: 170 / 609, 38 / 185 / 187 / 609, 38 / 185 / 187, 38 / 170 / 173, 38 / 173 / 185 / 187 / 720, 170 / 185 / 187 / 609, 609, 170 / 185 / 187 / 609 / 721, 38 / 609, 38 / 170 / 246 / 609, 185 / 187 / 609 / 718, 38 / 185 / 187 / 609 / 718 / 720, 38 / 170 / 246 / 609 / 718, 720, 246 / 609, 38 / 185 / 187 / 246 / 720, 38 / 609 / 72 1, 38 / 721, 38 / 720 / 721, 38 / 170 / 609 / 718 / 720, 38 / 718, 38 / 185 / 187 / 609 / 718 / 721, 529 / 609, 38 / 609 / 718, 185 / 187 / 609 / 718 / 720, 185 / 187 / 609 / 718 / 721, 38 / 170 / 718 / 720 / 721, 170 / 18 5 / 187 / 718 / 720, 38 / 173 / 185 / 187 / 246 / 609 / 720, 609 / 718 / 720, 170 / 185 / 187 / 718 / 720 / 721, 38 / 170 / 173 / 246 / 609 / 718, 246 / 721, 38 / 718 / 720 or 38 / 609 / 718 / 720, wherein the amino acid position is relative to the reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 386 or relative to the reference sequence corresponding to SEQ ID NO: 386.

[0328] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered RNA polymerase comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with the reference sequence corresponding to SEQ ID NO: 238, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 238 or relative to the reference sequence corresponding to SEQ ID NO: 238.

[0329] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered RNA polymerase comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 98%, 99% or higher sequence identity with the reference sequence corresponding to the even-numbered residues of SEQ ID NO: 462-584, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 238 or relative to the reference sequence corresponding to SEQ ID NO: 238.

[0330] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered RNA polymerase comprising an amino acid sequence containing at least one substitution or set of substitutions at the following amino acid positions: 143, 105, 775 / 779, 135, 100, 246, 779 / 782, 749, 237, 162, 83, 218, 227, 115, 274, 81, 122, 61, 32, 139, 75, 239, 257, 240, 69 / 161, 253, 14, 221, 84, 133 / 150, 60, 275, 89, 15, 254, 625, 14 / 67, 21, 93, 250, 217, 19, 95, 759, 62, or 140, wherein the amino acid positions are relative to the corresponding SEQ ID NO: The reference sequence of residues 8 to 890 of SEQ ID NO: 238 or the reference sequence corresponding to SEQ ID NO: 238.

[0331] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered RNA polymerase comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with the reference sequence corresponding to SEQ ID NO: 394, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 394 or relative to the reference sequence corresponding to SEQ ID NO: 394.

[0332] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered RNA polymerase comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 97%, 98%, 99% or higher sequence identity with the reference sequence corresponding to the even-numbered residues of SEQ ID NO: 586-602, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 394 or relative to the reference sequence corresponding to SEQ ID NO: 394.

[0333] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered RNA polymerase comprising an amino acid sequence containing at least one substitution or set of substitutions at the following amino acid positions: 609 / 779, 246 / 609, 246, 609 / 775 / 779, 105, 143 / 609 / 775, 105 / 135 / 609 / 775, or 111 / 609, 609, wherein the amino acid position is relative to a reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 394 or relative to a reference sequence corresponding to SEQ ID NO: 394.

[0334] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered RNA polymerase comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with the reference sequence corresponding to SEQ ID NO: 588, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 588 or relative to the reference sequence corresponding to SEQ ID NO: 588.

[0335] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered RNA polymerase comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 98%, 99%, or higher sequence identity with respect to the reference sequence corresponding to the even-numbered residues of SEQ ID NO: 588, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to the residues of SEQ ID NO: 588 or relative to the reference sequence corresponding to SEQ ID NO: 588.

[0336] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered RNA polymerase comprising an amino acid sequence containing at least one substitution or set of substitutions at the following amino acid positions: 32, 531 / 533, 115, 83, 32 / 60 / 61, 55, 589, 357, 617, 172, 598, 56 / 193, 56, 536, 52, 17, 261, 78, 629, 63, 82, 207, 666, 150, 45, 135, 13, 33, or 245, wherein the amino acid position is relative to a reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 588 or relative to a reference sequence corresponding to SEQ ID NO: 588.

[0337] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered RNA polymerase comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with the reference sequence corresponding to SEQ ID NO: 604, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 604 or relative to the reference sequence corresponding to SEQ ID NO: 604.

[0338] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered RNA polymerase comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 98%, 99% or higher sequence identity with respect to the reference sequence corresponding to the even-numbered residues of SEQ ID NO: 604, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 604 or relative to the reference sequence corresponding to SEQ ID NO: 604.

[0339] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered RNA polymerase comprising an amino acid sequence containing at least one substitution or set of substitutions at the following amino acid positions: 207 / 245 / 629, 17 / 56 / 150 / 598, 56 / 629, 536, 185, 56 / 150, 17 / 56 / 150 / 207 / 245 / 598 / 617 / 629, 56 / 150 / 536 / 598, 56 / 536, 185 / 666, 150, or 143 / 150 / 246 / 782, wherein the amino acid position is relative to a reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 604 or relative to a reference sequence corresponding to SEQ ID NO: 604.

[0340] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered RNA polymerase comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with the reference sequence corresponding to SEQ ID NO: 686, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 686 or relative to the reference sequence corresponding to SEQ ID NO: 686.

[0341] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered RNA polymerase comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 98%, 99% or higher sequence identity with respect to the reference sequence corresponding to the even-numbered residues of SEQ ID NO: 706-720, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to the residues of SEQ ID NO: 686 or relative to the reference sequence corresponding to SEQ ID NO: 686.

[0342] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered RNA polymerase comprising an amino acid sequence containing at least one substitution or set of substitutions at the following amino acid positions: 143, 246 / 424 / 648 / 782, 246 / 659 / 782, 56 / 150 / 629 / 660 / 782, 721 / 782, 782, 196 / 659 / 782, or 720 / 721 / 782, wherein the amino acid position is relative to a reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 686 or relative to a reference sequence corresponding to SEQ ID NO: 686.

[0343] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered RNA polymerase comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with the reference sequence corresponding to SEQ ID NO: 692, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 692 or relative to the reference sequence corresponding to SEQ ID NO: 692.

[0344] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered RNA polymerase comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 96%, 97%, 98%, 99% or higher sequence identity with the reference sequence corresponding to the even-numbered residues of SEQ ID NO: 722-730, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 692 or relative to the reference sequence corresponding to SEQ ID NO: 692.

[0345] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered RNA polymerase comprising an amino acid sequence containing at least one substitution or set of substitutions at the following amino acid positions: 143 / 659 / 782, 166 / 171 / 246 / 311, 143 / 721, 782, or 143 / 171 / 659 / 782, wherein the amino acid position is relative to a reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 692 or relative to a reference sequence corresponding to SEQ ID NO: 692.

[0346] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered RNA polymerase comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with the reference sequence corresponding to SEQ ID NO: 712, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 712 or relative to the reference sequence corresponding to SEQ ID NO: 712.

[0347] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered RNA polymerase comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with respect to the reference sequence corresponding to the even-numbered residues of SEQ ID NO: 732-802, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to the residues of SEQ ID NO: 712 or relative to the reference sequence corresponding to SEQ ID NO: 712.

[0348] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered RNA polymerase, said engineered RNA polymerase comprising an amino acid sequence containing at least one substitution or set of substitutions at the following amino acid positions: 367, 382, ​​670, 672, 357, 388, 693, 397, 375, 137 / 382 / 782, 655, 137 / 382 / 392, 391, 137 / 655. / 721 / 763, 696, 663, 137 / 382, 137 / 382 / 392 / 650 / 655 / 782, 137 / 382 / 629 / 660 / 672, 616, 690, 179, 137, 357 / 382 / 670, 388 / 670 / 672, 357 / 375, 357 / 672, 629 or 655 / 670, wherein the amino acid position is relative to the reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 712 or relative to the reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 712 or relative to the reference sequence corresponding to SEQ ID NO: 712.

[0349] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered RNA polymerase comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with the reference sequence corresponding to SEQ ID NO: 800, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 800 or relative to the reference sequence corresponding to SEQ ID NO: 800.

[0350] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered RNA polymerase comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 96%, 97%, 98%, 99% or higher sequence identity with the reference sequence corresponding to the even-numbered residues of SEQ ID NO: 804-828, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 800 or relative to the reference sequence corresponding to SEQ ID NO: 800.

[0351] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered RNA polymerase comprising an amino acid sequence containing at least one substitution or set of substitutions at the following amino acid positions: 137, 56 / 311 / 382, 56 / 137 / 143 / 721, 137 / 392, 56 / 137 / 392, 56 / 137 / 143 / 404 / 720, 56 / 143 / 246, 56 / 392, 392 / 404, 392, 56 / 137, or 56 / 196 / 392, wherein the amino acid position is relative to a reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 800 or relative to a reference sequence corresponding to SEQ ID NO: 800.

[0352] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered RNA polymerase, said engineered RNA polymerase comprising a substituted amino acid sequence at an amino acid position described in Tables 5.1, 6.1, 7.1, 8.1, 9.1, 10.1, 11.1, 12.1, 13.1, 14.1, 15.1, 16.1, 17.1, 18.1, 19.1, 23.1, and 24.1, wherein said amino acid position is a reference sequence relative to residues 8 to 890 corresponding to SEQ ID NO: 2, 4, 32, 44, 156, 234, 238, 386, 394, 588, 604, 686, 692, 712, or 800, or relative to the sequence corresponding to SEQ ID NO: Reference sequences of residues 8 to 890 of 2, 4, 32, 44, 156, 234, 238, 386, 394, 588, 604, 686, 692, 712 or 800.

[0353] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered RNA polymerase, said engineered RNA polymerase comprising at least one substituted amino acid sequence as described in Tables 5.1, 6.1, 7.1, 8.1, 9.1, 10.1, 11.1, 12.1, 13.1, 14.1, 15.1, 16.1, 17.1, 18.1, 19.1, 23.1, and 24.1, wherein said amino acid position is relative to a reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 2, 4, 32, 44, 156, 234, 238, 386, 394, 588, 604, 686, 692, 712, or 800, or relative to the sequence corresponding to SEQ ID NO: Reference sequences of residues 8 to 890 of 2, 4, 32, 44, 156, 234, 238, 386, 394, 588, 604, 686, 692, 712 or 800.

[0354] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered RNA polymerase, said engineered RNA polymerase comprising an amino acid sequence containing at least one substitution or set of substitutions at an amino acid position of the engineered RNA polymerase described in Tables 5.1, 6.1, 7.1, 8.1, 9.1, 10.1, 11.1, 12.1, 13.1, 14.1, 15.1, 16.1, 17.1, 18.1, 19.1, 23.1, and 24.1, wherein said amino acid position is relative to a reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 2, 4, 32, 44, 156, 234, 238, 386, 394, 588, 604, 686, 692, 712, or 800, or relative to the sequence corresponding to SEQ ID NO: Reference sequences of residues 8 to 890 of 2, 4, 32, 44, 156, 234, 238, 386, 394, 588, 604, 686, 692, 712 or 800.

[0355] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered RNA polymerase comprising an amino acid sequence comprising at least one substitution or set of substitutions of an engineered RNA polymerase variant described in Tables 5.1, 6.1, 7.1, 8.1, 9.1, 10.1, 11.1, 12.1, 13.1, 14.1, 15.1, 16.1, 17.1, 18.1, 19.1, 23.1, and 24.1, wherein the amino acid position is relative to a reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 2, 4, 32, 44, 156, 234, 238, 386, 394, 588, 604, 686, 692, 712, or 800, or relative to a reference sequence corresponding to SEQ ID NO: Reference sequences of residues 8 to 890 of 2, 4, 32, 44, 156, 234, 238, 386, 394, 588, 604, 686, 692, 712 or 800.

[0356] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered RNA polymerase, said engineered RNA polymerase comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 96%, 97%, 98%, 99% or higher sequence identity with a reference sequence comprising at least one substitution or set of substitutions as described in Tables 5.1, 6.1, 7.1, 8.1, 9.1, 10.1, 11.1, 12.1, 13.1, 14.1, 15.1, 16.1, 17.1, 18.1, 19.1, 23.1, and 24.1, wherein said amino acid position is relative to the sequence corresponding to SEQ ID NO: Reference sequences of residues 8 to 890 of SEQ ID NO: 2, 4, 32, 44, 156, 234, 238, 386, 394, 588, 604, 686, 692, 712, or 800, or reference sequences relative to SEQ ID NO: 2, 4, 32, 44, 156, 234, 238, 386, 394, 588, 604, 686, 692, 712, or 800.

[0357] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered RNA polymerase comprising residues 8 to 890 of SEQ ID NO. containing even numbers from SEQ ID NO: 4-828 or an amino acid sequence containing even numbers from SEQ ID NO: 4-828.

[0358] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered RNA polymerase, said engineered RNA polymerase comprising an amino acid sequence containing residues 8 to 890: SEQ ID NO:4,6,8,10,12,14,16,18,20,22,24,26,28,30,32,34,36,38,40,42,44,46,48,50,52,54,56,58,60,62,64,66,68,70,72,74,76,78,8 0, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 14 2, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250, 252, 254, 256, 258, 260, 262, 264, 266, 268, 270, 272, 274, 276, 278, 280, 282, 284, 286, 288, 290, 292, 294, 296, 298, 300, 302, 304, 306, 308, 310, 312, 314, 316, 318, 320, 322, 324, 326, 328, 330, 332, 334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360, 362, 364, 366, 368, 370, 37 2, 374, 376, 378, 380, 382, ​​384, 386, 388, 390, 392, 394, 396, 398, 400, 402, 404, 406, 408, 410, 412, 414, 416, 418, 420, 422, 424, 426, 428, 430, 432, 434, 436, 438, 440, 442, 444, 446, 448, 450, 452, 454, 456, 458, 460, 462, 464, 466, 468, 470, 472, 474, 476, 478, 480, 482, 484, 486,488, 490, 492, 494, 496, 498, 400, 402, 404, 406, 408, 410, 412, 414, 416, 418, 420, 422, 424, 426, 428, 430, 432, 434, 436, 438, 440, 442, 444, 446, 448, 450, 452, 454, 456, 458, 460, 462, 464, 466, 468, 470, 472, 474, 476, 478, 480, 482, 484, 486, 488, 490, 492, 494, 496, 4 98, 500, 502, 504, 506, 508, 510, 512, 514, 516, 518, 520, 522, 524, 526, 528, 530, 532, 534, 536, 538, 540, 542, 544, 546, 548, 550, 552, 554, 556, 558, 560, 562, 564, 566, 568, 570, 572, 574, 576, 578, 580, 582, 584, 586, 588, 590, 592, 594, 596, 598, 600, 602, 604, 606, 60 8, 610, 612, 614, 616, 618, 620, 622, 624, 626, 628, 630, 632, 634, 636, 638, 640, 642, 644, 646, 648, 650, 652, 654, 656, 658, 660, 662, 664, 666, 668, 670, 672, 674, 676, 678, 680, 682, 684, 686, 688, 690, 692, 694, 696, 698, 700, 702, 704, 706, 708, 710, 712, 714, 716, 718 720, 722, 724, 276, 728, 730, 732, 734, 736, 738, 740, 742, 744, 746, 748, 750, 752, 754, 756, 758, 760, 762, 764, 766, 768, 770, 772, 774, 776, 778, 780, 782, 784, 786, 788, 790, 792, 794, 796, 798, 800, 802, 804, 806, 808, 810, 812, 814, 816, 818, 820, 822, 824, 826 or 828.

[0359] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered RNA polymerase, said engineered RNA polymerase comprising an amino acid sequence including: SEQ ID NO: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 14 4, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250, 252, 254, 256, 258, 2 60, 262, 264, 266, 268, 270, 272, 274, 276, 278, 280, 282, 284, 286, 288, 290, 292, 294, 296, 298, 300, 302, 304, 306, 308, 310, 312, 314, 316, 318, 320, 322, 324, 326, 328, 330, 332, 334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360, 362, 364, 366, 368, 370, 372, 374 376, 378, 380, 382, ​​384, 386, 388, 390, 392, 394, 396, 398, 400, 402, 404, 406, 408, 410, 412, 414, 416, 418, 420, 422, 424, 426, 428, 430, 432, 434, 436, 438, 440, 442, 444, 446, 448, 450, 452, 454, 456, 458, 460, 462, 464, 466, 468, 470, 472, 474, 476, 478, 480, 482, 484, 486, 488, 490,492, 494, 496, 498, 400, 402, 404, 406, 408, 410, 412, 414, 416, 418, 420, 422, 424, 426, 428, 430, 432, 434, 436, 438, 440, 442, 444, 446, 448, 450, 452, 454, 456, 458, 460, 462, 464, 466, 468, 470, 472, 474, 476, 478, 480, 482, 484, 486, 488, 490, 492, 494, 496, 498, 500 502, 504, 506, 508, 510, 512, 514, 516, 518, 520, 522, 524, 526, 528, 530, 532, 534, 536, 538, 540, 542, 544, 546, 548, 550, 552, 554, 556, 558, 560, 562, 564, 566, 568, 570, 572, 574, 576, 578, 580, 582, 584, 586, 588, 590, 592, 594, 596, 598, 600, 602, 604, 606, 608, 61 0, 612, 614, 616, 618, 620, 622, 624, 626, 628, 630, 632, 634, 636, 638, 640, 642, 644, 646, 648, 650, 652, 654, 656, 658, 660, 662, 664, 666, 668, 670, 672, 674, 676, 678, 680, 682, 684, 686, 688, 690, 692, 694, 696, 698, 700, 702, 704, 706, 708, 710, 712, 714, 716, 718, 7 20, 722, 724, 276, 728, 730, 732, 734, 736, 738, 740, 742, 744, 746, 748, 750, 752, 754, 756, 758, 760, 762, 764, 766, 768, 770, 772, 774, 776, 778, 780, 782, 784, 786, 788, 790, 792, 794, 796, 798, 800, 802, 804, 806, 808, 810, 812, 814, 816, 818, 820, 822, 824, 826 or 828.

[0360] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence having at least 70%, 75%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with a reference polynucleotide sequence corresponding to an odd-numbered SEQ ID NO. of SEQ ID NO: 3-827, wherein the recombinant polynucleotide encodes an RNA polymerase.

[0361] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence having at least 70%, 75%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with a reference polynucleotide sequence corresponding to SEQ ID NO: 3, 31, 43, 155, 233, 237, 381, 393, 587, 603, 685, 691, 711 or 799, wherein the recombinant polynucleotide encodes an RNA polymerase.

[0362] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with a reference polynucleotide sequence corresponding to nucleotide residues 21 to 2670: SEQ ID NO: 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 1 33, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175, 177, 179, 181, 183, 185, 187, 189, 191, 193, 195, 197, 199, 201, 203, 205, 207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 23 7, 239, 241, 243, 245, 247, 249, 251, 253, 255, 257, 259, 261, 263, 265, 267, 269, 271, 273, 275, 277, 279, 281, 283, 285, 287, 289, 291, 293, 295, 297, 299, 301, 303, 305, 307, 309, 311, 313, 315, 317, 319, 321, 323, 325, 327, 329, 331, 333, 335, 337, 339, 341 343, 345, 347, 349, 351, 353, 355, 357, 359, 361, 363, 365, 367, 369, 371, 373, 375, 377, 379, 381, 383, 385, 387, 389, 391, 393, 395, 397, 399, 401, 403, 405, 407, 409, 411, 413, 415, 417, 419, 421, 423, 425, 427, 429, 431, 433, 435, 437, 439, 441, 443, 445447, 449, 451, 453, 455, 457, 459, 461, 463, 465, 467, 469, 471, 473, 475, 477, 479, 481, 483, 485, 487, 489, 491, 493, 495, 497, 399, 401, 403, 405, 40 7, 409, 411, 413, 415, 417, 419, 421, 423, 425, 427, 429, 431, 433, 435, 437, 439, 441, 443, 445, 447, 449, 451, 453, 455, 457, 459, 461, 463, 465, 467, 4 69, 471, 473, 475, 477, 479, 481, 483, 485, 487, 489, 491, 493, 495, 497, 499, 501, 503, 505, 507, 509, 511, 513, 515, 517, 519, 521, 523, 525, 527, 529 531, 533, 535, 537, 539, 541, 543, 545, 547, 549, 551, 553, 555, 557, 559, 561, 563, 565, 567, 569, 571, 573, 575, 577, 579, 581, 583, 585, 587, 589, 591 593, 595, 597, 599, 601, 603, 605, 607, 609, 611, 613, 615, 617, 619, 621, 623, 625, 627, 629, 631, 633, 635, 637, 639, 641, 643, 645, 647, 649, 651, 65 3, 655, 657, 659, 661, 663, 665, 667, 669, 671, 673, 675, 677, 679, 681, 683, 685, 687, 689, 691, 693, 695, 697, 699, 701, 703, 705, 707, 709, 711, 713, 7 15, 717, 719, 721, 723, 275, 727, 729, 731, 733, 735, 737, 739, 741, 743, 745, 747, 749, 751, 753, 755, 757, 759, 761, 763, 765, 767, 769, 771, 773, 775, 777, 779, 781, 783, 785, 787, 789, 791, 793, 795, 797, 799, 801, 803, 805, 807, 809, 811, 813, 815, 817, 819, 821, 823, 825, or 827, wherein the recombinant polynucleotide encodes an RNA polymerase.

[0363] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with a reference polynucleotide sequence corresponding to the following: SEQ ID NO: 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175, 177, 179, 181, 183, 185, 187, 189, 191, 193, 195, 197, 199, 201, 203, 205, 207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 239 241, 243, 245, 247, 249, 251, 253, 255, 257, 259, 261, 263, 265, 267, 269, 271, 273, 275, 277, 279, 281, 283, 285, 287, 289, 291, 293, 295, 297, 299, 301, 303, 305, 307, 309, 311, 313, 315, 317, 319, 321, 323, 325, 327, 329, 331, 333, 335, 337, 339, 341, 343, 345, 347, 349, 351, 353, 355, 357, 359, 361, 363, 365, 367, 369, 371, 373, 375, 377, 379, 381, 383, 385, 387, 389, 391, 393, 395, 397, 399, 401, 403, 405, 407, 409, 411, 413, 415, 417, 419, 421, 423, 425, 427, 429, 431, 433, 435, 437, 439, 441, 443, 445, 447, 449, 451,453, 455, 457, 459, 461, 463, 465, 467, 469, 471, 473, 475, 477, 479, 481, 483, 485, 487, 489, 491, 493, 495, 497, 399, 401, 403, 405, 407, 409, 411, 413, 415, 417, 419, 421, 423, 425, 427, 429, 431, 433, 435, 437, 439, 441, 443, 445, 447, 449, 451, 453, 455, 457, 459, 461, 463, 465, 467, 469, 471, 47 3, 475, 477, 479, 481, 483, 485, 487, 489, 491, 493, 495, 497, 499, 501, 503, 505, 507, 509, 511, 513, 515, 517, 519, 521, 523, 525, 527, 529, 531, 533, 535, 537, 539, 541, 543, 545, 547, 549, 551, 553, 555, 557, 559, 561, 563, 565, 567, 569, 571, 573, 575, 577, 579, 581, 583, 585, 587, 589, 591, 593, 5 95, 597, 599, 601, 603, 605, 607, 609, 611, 613, 615, 617, 619, 621, 623, 625, 627, 629, 631, 633, 635, 637, 639, 641, 643, 645, 647, 649, 651, 653, 655, 657, 659, 661, 663, 665, 667, 669, 671, 673, 675, 677, 679, 681, 683, 685, 687, 689, 691, 693, 695, 697, 699, 701, 703, 705, 707, 709, 711, 713, 715, 717, 719, 721, 723, 275, 727, 729, 731, 733, 735, 737, 739, 741, 743, 745, 747, 749, 751, 753, 755, 757, 759, 761, 763, 765, 767, 769, 771, 773, 775, 777, 779, 781, 783, 785, 787, 789, 791, 793, 795, 797, 799, 801, 803, 805, 807, 809, 811, 813, 815, 817, 819, 821, 823, 825, or 827, wherein the recombinant polynucleotide encodes an RNA polymerase.

[0364] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence containing nucleotide residues 21 to 2670: SEQ ID NO: 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 1 47, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175, 177, 179, 181, 183, 185, 187, 189, 191, 193, 195, 197, 199, 201, 203, 205, 207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 239, 241, 243, 245, 247, 249, 251, 253, 255, 257, 259, 261, 263, 26 5, 267, 269, 271, 273, 275, 277, 279, 281, 283, 285, 287, 289, 291, 293, 295, 297, 299, 301, 303, 305, 307, 309, 311, 313, 315, 317, 319, 321, 323, 325, 327, 329, 331, 333, 335, 337, 339, 341, 343, 345, 347, 349, 351, 353, 355, 357, 359, 361, 363, 365, 367, 369, 371, 373, 375, 377, 379, 381, 383 385, 387, 389, 391, 393, 395, 397, 399, 401, 403, 405, 407, 409, 411, 413, 415, 417, 419, 421, 423, 425, 427, 429, 431, 433, 435, 437, 439, 441, 443, 445, 447, 449, 451, 453, 455, 457, 459, 461, 463, 465, 467, 469, 471, 473, 475, 477, 479, 481, 483, 485, 487, 489, 491, 493, 495, 497, 399, 401403, 405, 407, 409, 411, 413, 415, 417, 419, 421, 423, 425, 427, 429, 431, 433, 435, 437, 439, 441, 443, 445, 447, 449, 451, 453, 455, 457, 459, 461, 463, 465, 467, 469, 471, 473, 475, 477, 479, 481, 483, 485, 487, 489, 491, 493, 495, 497, 499, 501, 503, 505, 507, 5 09, 511, 513, 515, 517, 519, 521, 523, 525, 527, 529, 531, 533, 535, 537, 539, 541, 543, 545, 547, 549, 551, 553, 555, 557, 559, 561, 563, 565, 567, 569, 571, 573, 575, 577, 579, 581, 583, 585, 587, 589, 591, 593, 595, 597, 599, 601, 603, 605, 607, 609, 611, 613, 61 5, 617, 619, 621, 623, 625, 627, 629, 631, 633, 635, 637, 639, 641, 643, 645, 647, 649, 651, 653, 655, 657, 659, 661, 663, 665, 667, 669, 671, 673, 675, 677, 679, 681, 683, 685, 687, 689, 691, 693, 695, 697, 699, 701, 703, 705, 707, 709, 711, 713, 715, 717, 719, 721 723, 275, 727, 729, 731, 733, 735, 737, 739, 741, 743, 745, 747, 749, 751, 753, 755, 757, 759, 761, 763, 765, 767, 769, 771, 773, 775, 777, 779, 781, 783, 785, 787, 789, 791, 793, 795, 797, 799, 801, 803, 805, 807, 809, 811, 813, 815, 817, 819, 821, 823, 825 or 827.

[0365] In some embodiments, the engineered RNA polymerase comprises an amino acid sequence including the following: SEQ ID NO: 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175, 177, 179, 181, 183, 185, 187, 189, 191, 193, 195, 197, 199, 201, 203, 205, 207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 239, 241, 243, 245, 247, 249, 251, 253, 255, 257, 259, 261, 263, 265, 26 7, 269, 271, 273, 275, 277, 279, 281, 283, 285, 287, 289, 291, 293, 295, 297, 299, 301, 303, 305, 307, 309, 311, 313, 315, 317, 319, 321, 323, 325, 327, 329, 331, 333, 335, 337, 339, 341, 343, 345, 347, 349, 351, 353, 355, 357, 359, 361, 363, 365, 367, 369, 371, 373, 375, 377, 379, 381, 383, 385, 3 87, 389, 391, 393, 395, 397, 399, 401, 403, 405, 407, 409, 411, 413, 415, 417, 419, 421, 423, 425, 427, 429, 431, 433, 435, 437, 439, 441, 443, 445, 447, 449, 451, 453, 455, 457, 459, 461, 463, 465, 467, 469, 471, 473, 475, 477, 479, 481, 483, 485, 487, 489, 491, 493, 495, 497, 399, 401, 403, 405,407, 409, 411, 413, 415, 417, 419, 421, 423, 425, 427, 429, 431, 433, 435, 437, 439, 441, 443, 445, 447, 449, 451, 453, 455, 457, 459, 461, 463, 465, 467, 469, 471, 473, 475, 477, 479, 481, 483, 485, 487, 489, 491, 493, 495, 497, 499, 501, 503, 505, 507, 509, 511 513, 515, 517, 519, 521, 523, 525, 527, 529, 531, 533, 535, 537, 539, 541, 543, 545, 547, 549, 551, 553, 555, 557, 559, 561, 563, 565, 567, 569, 571, 573, 575, 577, 579, 581, 583, 585, 587, 589, 591, 593, 595, 597, 599, 601, 603, 605, 607, 609, 611, 613, 615, 61 7, 619, 621, 623, 625, 627, 629, 631, 633, 635, 637, 639, 641, 643, 645, 647, 649, 651, 653, 655, 657, 659, 661, 663, 665, 667, 669, 671, 673, 675, 677, 679, 681, 683, 685, 687, 689, 691, 693, 695, 697, 699, 701, 703, 705, 707, 709, 711, 713, 715, 717, 719, 721, 7 23, 275, 727, 729, 731, 733, 735, 737, 739, 741, 743, 745, 747, 749, 751, 753, 755, 757, 759, 761, 763, 765, 767, 769, 771, 773, 775, 777, 779, 781, 783, 785, 787, 789, 791, 793, 795, 797, 799, 801, 803, 805, 807, 809, 811, 813, 815, 817, 819, 821, 823, 825 or 827.

[0366] In some embodiments, the recombinant polynucleotide encoding any of the RNA polymerases described herein is manipulated in a variety of ways to promote the expression of the RNA polymerase polypeptide. Therefore, in some embodiments, this disclosure provides an expression vector comprising a recombinant polynucleotide encoding the engineered RNA polymerase described herein. In some embodiments, the expression vector comprises one or more control sequences operatively linked to the recombinant polynucleotide to regulate the expression of the RNA polymerase encoding the polynucleotide and / or the expression of the corresponding polypeptide. In some embodiments, the control sequences particularly include a leader sequence, a polyadenylated sequence, a propeptide sequence, a signal peptide sequence, and a transcription terminator. In some embodiments, the choice of control sequences depends on the host type to which the expression vector is to be introduced.

[0367] In some embodiments, suitable promoters are selected based on the host cell. For bacterial host cells, suitable promoters for directing the transcription of the nucleic acid constructs of this disclosure include, but are not limited to, promoters derived from the *Escherichia coli* lac operon, the *Streptomyces agarase* gene (dagA), the *Bacillus subtilis* fructan sucrase gene (sacB), the *Bacillus licheniformis* α-amylase gene (amyL), the *Bacillus thermophilus* maltose amylase gene (amyM), the *Bacillus amyloliquefaciens* α-amylase gene (amyQ), the *Bacillus licheniformis* penicillinase gene (penP), the *Bacillus subtilis* xylA and xylB genes, and prokaryotic β-lactamase genes (see, for example, Villa-Kamaroff et al., Proc. Natl Acad. Sci. USA, 1978, 75:3727-3731), and the tac promoter (see, for example, DeBoer et al., Proc. Natl Acad. Sci. USA, 1983, 80:21-25). Exemplary promoters for filamentous fungal host cells include, but are not limited to, promoters derived from the genes of Aspergillus oryzae TAKA amylase, Rhizopus oryzae aspartic protease, Aspergillus niger neutral α-amylase, Aspergillus niger acid-stable α-amylase, Aspergillus niger or Aspergillus avocado glucosylase (glaA), Rhizopus oryzae lipase, Aspergillus oryzae alkaline protease, Aspergillus oryzae triose phosphate isomerase, Aspergillus nidulans acetamipridase, and Fusarium oxysporum trypsin-like protease (see, for example, WO 96 / 00787), as well as the NA2-tpi promoter (a hybrid of the promoters of the Aspergillus niger neutral α-amylase and Aspergillus oryzae triose phosphate isomerase genes), and their mutant, truncated, and hybrid promoters. Exemplary yeast cell promoters may be derived from the genes of *Saccharomyces cerevisiae* enolase (ENO-1), *Saccharomyces cerevisiae* galactokinase (GAL1), *Saccharomyces cerevisiae* alcohol dehydrogenase / glyceraldehyde-3-phosphate dehydrogenase (ADH2 / GAP), and *Saccharomyces cerevisiae* 3-phosphate glycerate kinase. Other useful promoters for yeast host cells are known in the art (see, for example, Romanos et al., Yeast, 1992, 8:423-488). Exemplary promoters for insect cells include, but are not limited to, polyhedroprotein, p10, ELT, OpIE2, and hr5 / ie1 promoters. Exemplary promoters for mammalian cells include, but are not limited to, promoters derived from cytomegalovirus (CMV), chicken β-actin fused with a CMV enhancer, simian virus 40 (SV40), phosphoglycerate kinase from *Homo sapiens*, β-actin, elongation factor-1a, or glyceraldehyde-3-phosphate dehydrogenase, or promoters derived from chicken β-actin.

[0368] In some embodiments, the control sequence is a suitable transcription terminator sequence (i.e., a sequence recognized by the host cell to terminate transcription). In some embodiments, the terminator sequence is operatively linked to the 3' end of a nucleic acid sequence encoding an RNA polymerase polypeptide. Any suitable terminator that is functional in the selected host cell can be used in this invention. For bacterial expression, the transcription terminator can be a Rho-dependent terminator that depends on the Rho transcription factor, or a Rho-independent or intrinsic terminator that does not require the transcription factor. Exemplary bacterial transcription terminators are described in Peters et al., J Mol Biol., 2011, 412(5):793-813. Exemplary transcription terminators for filamentous fungal host cells can be obtained from the genes of Aspergillus oryzae TAKA amylase, Aspergillus niger glucosylase, Aspergillus nidulans anthranilate synthase, Aspergillus niger α-glucosidase, and Fusarium oxysporum trypsin-like protease. Exemplary terminators for yeast host cells can be obtained from the genes of Saccharomyces cerevisiae enolase, Saccharomyces cerevisiae cytochrome C (CYC1), and Saccharomyces cerevisiae glyceraldehyde-3-phosphate dehydrogenase. Other useful terminators for yeast host cells are known in the art (see, for example, Romanos et al., ibid.). Exemplary terminators for insect and mammalian cells include, but are not limited to, terminators derived from cytomegalovirus (CMV), simian virus 40 (SV40), human growth hormone hGH, bovine growth hormone BGH, and human or rabbit β-globulin.

[0369] In some embodiments, the control sequence is a suitable leader sequence, i.e., an untranslated region of mRNA important for translation in the host cell. In some embodiments, the leader sequence is operatively linked to the 5' end of a nucleic acid sequence encoding an RNA polymerase polypeptide. Any suitable leader sequence functional in the selected host cell may be used in this disclosure. Exemplary leader sequences for filamentous fungal host cells are obtained from the genes of Aspergillus oryzae TAKA amylase and Aspergillus nidulans triose phosphate isomerase. Suitable leader sequences for yeast host cells are obtained from the genes of Saccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae 3-phosphoglycerate kinase, Saccharomyces cerevisiae α-factor, and Saccharomyces cerevisiae alcohol dehydrogenase / glyceraldehyde-3-phosphate dehydrogenase (ADH2 / GAP). Suitable leader sequences for mammalian host cells include, but are not limited to, 5'-UTR elements present in orthopoxvirus mRNA.

[0370] In some embodiments, the control sequence is a polyadenylated sequence (i.e., a sequence operatively linked to the 3' end of a nucleic acid sequence and recognized by the host cell during transcription as a signal to add polyadenylated residues to the transcribed mRNA). Any suitable polyadenylated sequence functional in a selected host cell can be used in this invention. Exemplary polyadenylated sequences for filamentous fungal host cells include, but are not limited to, genes for Aspergillus oryzae TAKA amylase, Aspergillus niger glucosylase, Aspergillus nidulans anthranilate synthase, Fusarium oxysporum trypsin-like protease, and Aspergillus niger α-glucosidase. Useful polyadenylated sequences for yeast host cells are known (see, for example, Guo and Sherman, Mol. Cell. Biol., 1995, 15:5983-5990). Useful polyadenylated and 3' UTR sequences for mammalian host cells include, but are not limited to, the 3'-UTR of α- and β-globin mRNAs, which contain several sequence elements that increase mRNA stability and translation.

[0371] In some embodiments, the control sequence is also a signal peptide (i.e., a coding region encoding an amino acid sequence linked to the amino terminus of a polypeptide and directing the encoded polypeptide into the cellular secretion pathway). In some embodiments, the 5' end of the coding sequence of the nucleic acid sequence inherently contains a signal peptide coding region naturally linked in the translation reading frame to a segment encoding the coding region of the secretory polypeptide. Alternatively, in some embodiments, the 5' end of the coding sequence contains a signal peptide coding region that is exogenous to the coding sequence. Any suitable signal peptide coding region directing the expressed polypeptide into the secretion pathway of a selected host cell can be used to express the engineered polypeptide. Effective signal peptide coding regions of bacterial host cells are signal peptide coding regions, including but not limited to those obtained from the genes of Bacillus NClB 11837 maltose amylase, Bacillus stearothermophilus α-amylase, Bacillus subtilis protease, Bacillus licheniformis β-lactamase, Bacillus stearothermophilus neutral proteases (nprT, nprS, nprM), and Bacillus subtilis prsA. Other signal peptides are known in the art (see, for example, Simonen and Palva, Microbiol. Rev., 1993, 57:109-137). In some embodiments, the effective signal peptide coding regions of filamentous fungal host cells include, but are not limited to, signal peptide coding regions derived from the genes of *Aspergillus oryzae* TAKA amylase, *Aspergillus niger* neutral amylase, *Aspergillus niger* glucosylamylase, *Rhizopus oryzae* aspartic protease, *Polytrichum spp.* cellulase, and *Polytrichum spp.* lipase. Useful signal peptides of yeast host cells include, but are not limited to, those derived from the genes of *Saccharomyces cerevisiae* α-factor and *Saccharomyces cerevisiae* invertase. Useful signal peptides of mammalian host cells include, but are not limited to, signal peptides derived from the genes of immunoglobulin γ (IgG) or other human secreted proteins (e.g., human β-galactosidase polypeptide).

[0372] In some embodiments, the control sequence is a propeptide coding region encoding an amino acid sequence located at the N-terminus of the polypeptide. The resulting polypeptide is referred to as a "proenzyme," "propeptide," or "zymogen." The propeptide can be converted from a propeptide into a mature, active polypeptide by catalytic or autocatalytic cleavage of the propeptide. The propeptide coding region can be obtained from any suitable source, including but not limited to the genes for Bacillus subtilis alkaline protease (aprE), Bacillus subtilis neutral protease (nprT), Saccharomyces cerevisiae α-factor, Rhizopus oryzae aspartic protease, and Myceliophthora thermophila lactase (see, for example, WO95 / 33836). When both the signal peptide and the propeptide region are present at the N-terminus of the polypeptide, the propeptide region is located near the N-terminus of the polypeptide, and the signal peptide region is located near the N-terminus of the propeptide region.

[0373] In some implementations, the control sequences include one or more regulatory sequences that facilitate the regulation of polynucleotide and / or corresponding encoded polypeptide expression in response to the growth status of the host cell. Examples of regulatory systems are those that turn gene expression on or off in response to chemical or physical stimuli, including the presence of regulatory compounds. In prokaryotic host cells, suitable regulatory sequences include, but are not limited to, the lac, tac, and trp operon systems. In yeast host cells, suitable regulatory systems include, but are not limited to, the ADH2 system or the GAL1 system. In filamentous fungi, suitable regulatory sequences include, but are not limited to, the TAKA α-amylase promoter, the Aspergillus niger glucosylase promoter, and the Aspergillus oryzae glucosylase promoter. Exemplary inducible promoters regulated by exogenous factors include the zinc-inducible sheep metallothionein (MT) promoter, the dexamethasone (Dex) inducible promoter, the mouse mammary tumor virus (MMTV) promoter, the ecdysone insect promoter, the tetracycline inducible promoter system, the RU486 inducible promoter system, and the rapamycin inducible promoter system.

[0374] Recombi...

Claims

1. An engineered RNA polymerase or a functional fragment thereof, said engineered RNA polymerase or functional fragment comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity relative to the reference sequence corresponding to SEQ ID NO: 2, 4, 32, 44, 156, 234, 238, 386, 394, 588, 604, 686, 692, 712 or 800. The reference sequence of residues 8 to 890 of SEQ ID NO: 2, 4, 32, 44, 156, 234, 238, 386, 394, 588, 604, 686, 692, 712, or 800, or the reference sequence relative to the reference sequence corresponding to SEQ ID NO: 2, 4, 32, 44, 156, 234, 238, 386, 394, 588, 604, 686, 692, 712, or 800, wherein the amino acid sequence contains one or more substitutions.

2. The engineered RNA polymerase of claim 1, comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with respect to the reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 2 or with respect to the reference sequence corresponding to SEQ ID NO: 2, wherein the amino acid sequence comprises one or more substitutions.

3. The engineered RNA polymerase of claim 1, comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with respect to the reference sequence corresponding to SEQ ID NO: 4, 32, 44, 156, 234, 238, 386, 394, 588, 604, 686, 692, 712, or 800, or with respect to the reference sequence corresponding to SEQ ID NO: 2, wherein the amino acid sequence is at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 99%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher, relative to the reference sequence corresponding to SEQ ID NO: 2, or with respect to the reference sequence corresponding to SEQ ID NO: 2, 712, 800, 89 ... The reference sequence for ID NO: 2, wherein the amino acid sequence contains one or more substitutions.

4. The engineered RNA polymerase of claim 1, comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with respect to the reference sequence corresponding to SEQ ID NO: 2, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to SEQ ID NO: 2 or relative to the reference sequence corresponding to SEQ ID NO:

2.

5. The engineered RNA polymerase according to any one of claims 1-4, wherein the amino acid sequence of the engineered RNA polymerase contains at least one substitution at the following amino acid positions: 13, 14, 15, 17, 19, 21, 32, 33, 38, 45, 52, 54, 55, 56, 56, 57, 60, 61, 62, 63, 67, 69, 72, 75, 78, 81, 82, 83, 84, 89, 93, 95, 100, 105, 111, 115, 122, 124, 127, 131, 133, 135, 137, 138, 139, 140, 141, 143, 150, 160, 161, 162, 164, 166, 168, 169, 170, 171, 172, 173, 179, 185, 187, 193, 196, 207, 212, 217, 218, 221, 227, 237, 239, 240, 245, 246, 250 253, 254, 257, 261, 272, 274, 275, 311, 336, 340, 342, 351, 357, 367, 375, 379, 382, ​​388, 391, 392, 394, 397, 401, 402, 403, 404, 419, 424, 437, 440, 494, 510, 514, 517, 527, 529, 530, 531, 533, 536, 589, 594, 598 607, 609, 615, 616, 617, 625, 629, 648, 650, 655, 659, 660, 663, 664, 666, 670, 672, 690, 693, 696, 718, 720, 721, 749, 759, 763, 773, 775, 779, 782, 782, 810, 854, 857, 867, 875, or 878 or combinations thereof, wherein the amino acid position is relative to the reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 2 or relative to the reference sequence corresponding to SEQ ID NO:

2.

6. The engineered RNA polymerase according to any one of claims 1-5, wherein the amino acid sequence of the engineered RNA polymerase comprises at least one substituted amino acid residue: 13N, 14Q / Y, 15T, 17N, 19E, 21G, 32S / T / V, 33E, 38G, 45K, 52T, 54H, 55D / S / W, 56E / G / N / T / V, 57G, 60G / L / M / R, 61W / Y, 62T, 63S, 67R, 69S, 72R, 75S / Y, 78S, 81C / L, 82S, 83K, 84A, 89Q, 93Y, 95Y, 100R, 105R, 111K, 115L / V, 122I, 124F. , 127G, 131T, 133C, 135G / K, 137G, 138R, 139R, 140R, 141A, 143N / Q / R / V, 150 Q / Y, 160F / L, 161R, 162V, 164W, 166D / R / T / V, 168K / L / R / S / T / V / W, 169G / L, 17 0L / Q / R / S, 171E / F / L / Y, 172A / E / G / K / R / V, 173D / H / L / Y, 179N, 185I / L / P, 18 7G / H / L, 193G, 196A / C / G / S / V, 207S, 212R / Y, 217P / V, 218Q / T, 221C, 227V, 23 7L, 239L / R, 240M, 245C, 246K / R / V, 250R, 253Y, 254G / S, 257L / V, 261M, 272N , 274G / L, 275A / Y, 311G, 336L / M / S / V, 340E / W, L342Q, 351L, 357E / P, 367F / P / T, 375F / P, 379A / S, 382G / L / R, 388K / R, 391F / S, 392A / M / R, 394K, 397S, 401 R, 402T, 403V, ​​404I / L / S / Y, 419R / S, 424A, 437P, 440K, 494G, 510V, 514I / S, 5 17R, 527E, 529R, 530N, 531Q, 533S, 536E / V, 589C, 594G, 598A / K / L / R / V, 607 Y, 609C / E / F / P / W, 615A, 616A, 617R, 625C, 629D / K / R, 648F, 650M, 655M / R, 65 9R, 660H / W / Y, 663L, 664K / P, 666M, 670Q / R, 672G / P / R / S / T, 690S, 693R, 696 L, 718F / N / S / V / Y, 720A / G / I / P / S, 721A / E / M / S / T / Y, 749R, 759M, 763S, 773A,775K / R, H779R, 782R / V, 810K, 854T, 857V, 867Q, 875C, or 878M or combinations thereof, wherein the amino acid position is relative to the reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 2 or relative to the reference sequence corresponding to SEQ ID NO:

2.

7. The engineered RNA polymerase according to any one of claims 1-5, wherein the amino acid sequence of the engineered RNA polymerase comprises at least one substitution at the following amino acid positions: 32, 38, 56, 138, 150, 170, 171, 173, 246, 311, 336, 340, 357, 388, 394, 401, 404, 437, 514, 536, 609, 629, 659, 660, 664, 779, or 782 or combinations thereof, wherein the amino acid position is relative to the reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 2 or relative to the reference sequence corresponding to SEQ ID NO:

2.

8. The engineered RNA polymerase according to any one of claims 1-5 and 7, wherein the amino acid sequence of the engineered RNA polymerase comprises at least one substitution or amino acid residue 32V, 38G, 56T, 138R / N, 150Y, 170Q, 171E, 173Y, 246K, 311A / G, 336K / S, 340K / W, 357E, 388A / K, 394K, 401R, 404L / S, 437P, 514S, 536V, 609E, 629A, 659R, 660H / W, 664P, 779R, or 782V or a combination thereof, wherein the amino acid position is relative to the reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 2 or relative to the reference sequence corresponding to SEQ ID NO:

2.

9. The engineered RNA polymerase according to any one of claims 1-5, wherein the amino acid sequence of the engineered RNA polymerase comprises at least one substitution or set of substitutions at the following amino acid positions: 401 / 404, 336, 138, 401 / 403 / 404, 336 / 510, 388, 340, 401 / 402 / 404, or 351 / 357, wherein the amino acid position is relative to the reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 2 or relative to the reference sequence corresponding to SEQ ID NO:

2.

10. The engineered RNA polymerase according to any one of claims 1-5 and 9, wherein the amino acid sequence of the engineered RNA polymerase comprises at least one substitution or set of substitutions or amino acid residues 401R / 404L, 336S, 138R, 336V, 401R / 403V / 404S, 336M / 510V, 336L, 388K, 340E, 401R / 402T / 404S, 351L / 357E or 401R / 404I, wherein the amino acid position is relative to the reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 2 or relative to the reference sequence corresponding to SEQ ID NO:

2.

11. The engineered RNA polymerase of claim 1, wherein the amino acid sequence of the engineered RNA polymerase comprises at least one substitution as described in Tables 5.1, 6.1, 7.1, 8.1, 9.1, 10.1, 11.1, 12.1, 13.1, 14.1, 15.1, 16.1, 17.1, 18.1, 19.1, 23.1, and 24.1, wherein the amino acid position is relative to the reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 2 or relative to the reference sequence corresponding to SEQ ID NO:

2.

12. The engineered RNA polymerase of claim 1, wherein the amino acid sequence of the engineered RNA polymerase comprises at least one substitution or set of substitutions of the engineered RNA polymerases described in Tables 5.1, 6.1, 7.1, 8.1, 9.1, 10.1, 11.1, 12.1, 13.1, 14.1, 15.1, 16.1, 17.1, 18.1, 19.1, 23.1, and 24.1, wherein the amino acid position is relative to the reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 2 or relative to the reference sequence corresponding to SEQ ID NO:

2.

13. The engineered RNA polymerase of claim 1, comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 94%, 95%, 96%, 98%, 99%, 90%, 91%, 92%, 93%, 96%, 97%, 98%, 99%, or higher sequence identity with the reference sequence corresponding to SEQ ID NO: 4, 32, 44, 156, 234, 238, 386, 394, 588, 604, 686, 692, 712, or 800.

14. The engineered RNA polymerase of claim 1, comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 98%, 99%, or higher sequence identity with the reference sequence of the even-numbered SEQ ID NOs corresponding to SEQ ID NOs: 4-828.

15. The engineered RNA polymerase of claim 1, comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity relative to the sequence corresponding to SEQ ID NO: 4, 32, 44, 156, 234, 238, 386, 394, 588, 604, 686, 692, 712, or 800. The reference sequence of residues 8 to 890 of SEQ ID NO: 4, 32, 44, 156, 234, 238, 386, 394, 588, 604, 686, 692, 712 or 800, or the reference sequence corresponding to SEQ ID NO: 4, 32, 44, 156, 234, 238, 386, 394, 588, 604, 686, 692, 712 or 800, wherein the amino acid sequence contains one or more substitutions.

16. The engineered RNA polymerase of claim 1, comprising a reference sequence comprising residues 8 to 890 of SEQ ID NO. corresponding to even-numbered SEQ ID NO: 4-828 or a reference sequence comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with respect to the reference sequence corresponding to SEQ ID NO: 4, 32, 44, 156, 234, 238, 386, 394, 588, 604, 686, 692, 712 or 800 of ... A reference sequence of 4, 32, 44, 156, 234, 238, 386, 394, 588, 604, 686, 692, 712 or 800, wherein the amino acid sequence contains one or more substitutions.

17. The engineered RNA polymerase of claim 16, wherein the amino acid sequence of the engineered RNA polymerase comprises at least one substitution at the following amino acid positions: 13, 14, 15, 17, 19, 21, 32, 33, 38, 45, 52, 54, 55, 56, 56, 57, 60, 61, 62, 63, 67, 69, 72, 75, 78, 81, 82, 83, 84, 89, 93, 95, 100, 105, 111, 115, 122, 124, 127, 131, 133, 135, 137, 138, 139, 140, 141, 143, 150, 160, 161, 162, 164, 166, 168, 169, 170, 171, 172, 173, 179, 185, 187, 193, 196, 207, 212, 217, 218, 221, 227, 237, 239, 240, 245, 246, 250, 253 254, 257, 261, 272, 274, 275, 311, 336, 340, 342, 351, 357, 367, 375, 379, 382, ​​388, 391, 392, 394, 397, 401, 402, 403, 404, 419, 424, 437, 440, 494, 510, 514, 517, 527, 529, 530, 531, 533, 536, 589, 594, 598, 60 7, 609, 615, 616, 617, 625, 629, 648, 650, 655, 659, 660, 663, 664, 666, 670, 672, 690, 693, 696, 718, 720, 721, 749, 759, 763, 773, 775, 779, 782, 782, 810, 854, 857, 867, 875, or 878 or combinations thereof, wherein the amino acid position is relative to the position corresponding to SEQ. Reference sequences of residues 8 to 890 of SEQ ID NO: 4, 32, 44, 156, 234, 238, 386, 394, 588, 604, 686, 692, 712, or 800, or reference sequences corresponding to SEQ ID NO: 4, 32, 44, 156, 234, 238, 386, 394, 588, 604, 686, 692, 712, or 800.

18. The engineered RNA polymerase according to any one of claims 15-17, wherein the amino acid sequence of the engineered RNA polymerase comprises at least one substituted or amino acid residue: 13N, 14Q / Y, 15T, 17N, 19E, 21G, 32S / T / V, 33E, 38G / R, 45K, 52T, 54H, 55D / S / W, 56A / E / G / N / T / V, 57G, 60G / L / M / R, 61W / Y, 62T, 63S, 67R, 69S, 72R, 75S / Y, 78S, 81C / L, 82S, 83K, 84A, 89Q, 93Y, 95Y, 100R, 105R, 111K, 115L / V, 1 22I, 124F, 127G, 131T, 133C, 135G / K, 137G, 138N / R, 139R, 140R, 141A, 143 N / Q / R / V, 150Q / Y, 160F / L, 161R, 162V, 164W, 166D / R / T / V, 168K / L / R / S / T / V / W, 169G / L, 170L / Q / R / S, 171E / F / L / Y, 172A / E / G / K / R / V, 173D / H / L / Y / V, 17 9N, 185I / L / P, 187G / H / L, 193G, 196A / C / G / S / V, 207S, 212R / Y, 217P / V, 218Q / T, 221C, 227V, 237L, 239L / R, 240M, 245C, 246K / R / V, 250R, 253Y, 254G / S, 257L / V, 261M, 272N, 274G / L, 275A / Y, 311A / G, 336K / L / M / S / V, 340E / K / W, 34 2Q, 351L, 357E / P, 367F / P / T, 375F / P, 379A / S, 382G / L / R, 388A / K / R, 391F / S , 392A / M / R, 394K, 397S, 401R, 402T, 403V, ​​404I / L / S / Y, 419R / S, 424A, 437P / S, 440K, 494G, 510V, 514I / L / S, 517R, 527E, 529R, 530N, 531Q, 533S, 536E / V, 589C, 594G, 598A / K / L / R / V, 607Y, 609C / E / F / P / W, 615A, 616A, 617R, 625 C, 629A / D / K / R, 648F, 650M, 655M / R, 659E / R, 660H / W / Y, 663L, 664K / P, 666M , 670Q / R, 672G / P / R / S / T, 690S, 693R, 696L, 718F / N / S / V / Y, 720A / G / I / P / S,721A / E / M / S / T / Y, 749R, 759M, 763S, 773A, 775K / R, 779R, 782E / R, 782V, 810K, 854T, 857V, 867Q, 875C, or 878M or combinations thereof, wherein the amino acid position is relative to the reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 4, 32, 44, 156, 234, 238, 386, 394, 588, 604, 686, 692, 712, or 800, or relative to the reference sequence corresponding to SEQ ID NO: 4, 32, 44, 156, 234, 238, 386, 394, 588, 604, 686, 692, 712, or 800.

19. The engineered RNA polymerase according to any one of claims 15-17, wherein the amino acid sequence of the engineered RNA polymerase comprises at least one substitution at the following amino acid positions: 32, 38, 56, 138, 150, 170, 171, 173, 246, 311, 336, 340, 357, 388, 394, 401, 404, 437, 514, 536, 609, 629, 659, 660, 664, 779, or 782, or combinations thereof, wherein the amino acid position is relative to the reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 4, 32, 44, 156, 234, 238, 386, 394, 588, 604, 686, 692, 712, or 800, or relative to the amino acid sequence corresponding to SEQ ID NO: Reference sequences of 4, 32, 44, 156, 234, 238, 386, 394, 588, 604, 686, 692, 712 or 800.

20. The engineered RNA polymerase according to any one of claims 15-19, wherein the amino acid sequence of the engineered RNA polymerase comprises at least one substituted amino acid residue 32V, 38G, 56T, 138R / N, 150Y, 170Q, 171E, 173Y, 246K, 311A / G, 336K / S, 340K / W, 357E, 388A / K, 394K, 401R, 404L / S, 437P, 514S, 536V, 609E, 629A, 659R, 660H / W, 664P, 779R, or 782V or a combination thereof, wherein the amino acid position is relative to the position corresponding to SEQ ID NO: Reference sequences of residues 8 to 890 of SEQ ID NO: 4, 32, 44, 156, 234, 238, 386, 394, 588, 604, 686, 692, 712, or 800, or reference sequences relative to the reference sequences corresponding to SEQ ID NO: 4, 32, 44, 156, 234, 238, 386, 394, 588, 604, 686, 692, 712, or 800.

21. The engineered RNA polymerase of claim 1 or 15, comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with respect to the reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 4 or with respect to the reference sequence corresponding to SEQ ID NO: 4, wherein the amino acid sequence comprises one or more substitutions.

22. The engineered RNA polymerase of claim 16 or 21, wherein the amino acid sequence of the engineered RNA polymerase comprises at least one substitution or set of substitutions at the following amino acid positions: 311 / 388 / 404 / 440, 138 / 340, 138 / 311 / 336 / 340 / 388, 311 / 388 / 440, 138 / 311 / 340 / 388 / 404 / 440, 138 / 336 / 342, or 351 / 388, wherein the amino acid position is relative to the reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 4 or relative to the reference sequence corresponding to SEQ ID NO:

4.

23. The engineered RNA polymerase of claim 21 or 22, comprising an amino acid sequence of the engineered RNA polymerase containing at least one set of substitutions or amino acid residues 311G / 388K / 404Y / 440K, 138R / 340W, 138R / 311G / 336S / 340W / 388K, 311G / 388K / 440K, 138R / 311G / 340E / 388K / 404Y / 440K, 138R / 336M / 342Q, or 351L / 388K, wherein the amino acid position is relative to the reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 4 or relative to the reference sequence corresponding to SEQ ID NO:

4.

24. The engineered RNA polymerase of claim 1 or 15, comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with respect to the reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 32 or with respect to the reference sequence corresponding to SEQ ID NO: 32, wherein the amino acid sequence comprises one or more substitutions.

25. The engineered RNA polymerase of claim 16 or 24, wherein the amino acid sequence of the engineered RNA polymerase contains at least one substitution or set of substitutions at the following amino acid positions: 514, 38 / 514, 38 / 394 / 514, 38 / 394 / 514 / 664, 394 / 514 / 875, 394 / 514, 38 / 124 / 394 / 514, 171 / 878, 170, 173, 172, 170 / 629, 171, 168, 166, 166 / 272 / 274 / 275, 169, 207, 168 / 217 / 218, 171 / 530, 382, ​​773, 670, 379, 392, 419, 57, 164, 810, 659, 54, 172 / 173 / 514 / 659 / 660, 170 / 171 / 172 / 514 / 659, 173 / 514 / 659 / 660, 173 / 514 / 659 / 810, 1 70 / 340 / 514 / 659 / 660 / 810, 170 / 171 / 173 / 336 / 340 / 514 / 659 / 660, 57 / 171 / 173 / 514 / 659 / 660 / 810, 170 / 173 / 514 / 659 / 660 / 810, 57 / 171 / 172 / 336 / 340 / 514 / 659, 173 / 514, 171 / 172 / 173 / 514 / 659 / 660, 57 / 17 0 / 171 / 173 / 514 / 659 / 660 / 810, 57 / 173 / 514, 172 / 173 / 514 / 857, 57 / 173 / 340 / 514, 170 / 171 / 173 / 336 / 514, 171 / 514, 57 / 170 / 173 / 336 / 514, 170 / 336, 173 / 514 / 659, 57 / 514 or 173 / 810, wherein the amino acid position is relative to the reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 32 or relative to the reference sequence corresponding to SEQ ID NO:

32.

26. The engineered RNA polymerase of claim 24 or 25, wherein the amino acid sequence of the engineered RNA polymerase comprises at least one substitution or set of substitutions or amino acid residues 514S, 38G / 514S, 38G / 394K / 514S, 38G / 394K / 514S / 664P, 394K / 514S / 875C, 394K / 514S, 38G / 124F / 394K / 514I, 171E / 878M, 170Q, 173Y, 172V, 173L, 172E, 170S / 629D, 171F, 168V, 172K, 166R, 168K, 166V / 2 72N / 274L / 275Y, 170R, 169L, 207S, 169G, 168L / 217P / 218Q, 168W, 172R , 166D, 171L, 171Y / 530N, 172A, 173H, 382R, 773A, 168R, 670R, 168T, 172 G, 379A, 392M, 419R, 392R, 392A, 57G, 168S, 164W, 670Q, 810K, 382G, 65 9R, 54H, 419S, 172E / 173Y / 514S / 659R / 660H, 170Q / 171E / 172E / 514S / 65 9R、173Y / 514S / 659R / 660H、173Y / 514S / 659R / 810K、170Q / 340K / 514S / 659R / 660H / 810K, 170Q / 171E / 173Y / 336K / 340K / 514S / 659R / 660H, 57G / 171E / 173Y / 514S / 659R / 660H / 810K、170Q / 173Y / 514S / 659R / 660H / 81 0K、57G / 171E / 172K / 336K / 340K / 514S / 659R、173Y / 514S、171E / 172E / 17 3Y / 514S / 659R / 660H, 57G / 170Q / 171E / 173Y / 514S / 659R / 660H / 810K, 57G / 173Y / 514S, 172K / 173D / 514S / 857V, 57G / 173Y / 340K / 514S, 170Q / 171E / 173Y / 336K / 514S, 171E / 514S, 57G / 170Q / 173Y / 336K / 514S, 170Q / 336K, 173Y / 514S / 659R, 57G / 514S or 173Y / 810K, wherein the amino acid position is relative to the corresponding SEQ Reference sequence of residues 8 to 890 of SEQ ID NO: 32 or relative to the reference sequence corresponding to SEQ ID NO:

32.

27. The engineered RNA polymerase of claim 1 or 15, comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with respect to the reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 44 or the reference sequence corresponding to SEQ ID NO: 44, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 44 or relative to the reference sequence corresponding to SEQ ID NO:

44.

28. The engineered RNA polymerase of claim 16 or 27, wherein the amino acid sequence of the engineered RNA polymerase contains at least one substitution or set of substitutions at amino acid positions 336, 143 / 336 / 340 / 782 or 143 / 336 / 340 / 357 / 514, wherein the amino acid position is relative to a reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 44 or relative to a reference sequence corresponding to SEQ ID NO:

44.

29. The engineered RNA polymerase of claim 27 or 28, wherein the amino acid sequence of the engineered RNA polymerase comprises at least one substitution or set of substitutions or amino acid residues 336K, 143V / 336K / 340K / 782V, or 143V / 336K / 340K / 357E / 514L, wherein the amino acid position is relative to the reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 44 or relative to the reference sequence corresponding to SEQ ID NO:

44.

30. The engineered RNA polymerase of claim 1 or 15, comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with respect to the reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 156 or with respect to the reference sequence corresponding to SEQ ID NO: 156, wherein the amino acid sequence comprises one or more substitutions.

31. The engineered RNA polymerase of claim 16 or 30, wherein the amino acid sequence of the engineered RNA polymerase contains at least one substitution or set of substitutions at the following amino acid positions: 207, 419, 207 / 336 / 340, 166 / 169, 207 / 670, 166 / 336 / 340, 207 / 419 / 670, 207 / 336 / 340 / 382 / 419 / 670, 2 07 / 382, 670, 336 / 670, 340 / 670, 382, ​​127 / 141 / 207 / 336 / 382 / 670, 207 / 336 / 382 / 419 / 670, 419 / 670, 166 / 168 / 207 / 336 / 340 / 419 / 670, 166 / 340 or 207 / 336 / 340 / 670, wherein the amino acid position is relative to the reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 156 or relative to the reference sequence corresponding to SEQ ID NO:

156.

32. The engineered RNA polymerase of claim 30 or 31, wherein the amino acid sequence of the engineered RNA polymerase comprises at least one substitution or set of substitutions or amino acid residues 207S, 419R, 207S / 336K / 340K, 166R / 169L, 207S / 670R, 166R / 336K / 340K, 207S / 419R / 670R, 207S / 336K / 340K / 382G / 419R / 670R, 207S / 382G, 670R, 336K / 670R, 340K / 670R, 382G, 127G / 141A / 207S / 336K / 382G / 670R, 207S / 336K / 382G / 419R / 670R, 419R / 670R, 166R / 168V / 207S / 336K / 340K / 419R / 670R, 166V / 340K or 207S / 336K / 340K / 670R, wherein the amino acid position is relative to the reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 156 or relative to the reference sequence corresponding to SEQ ID NO:

156.

33. The engineered RNA polymerase of claim 16 or 30, wherein the amino acid sequence of the engineered RNA polymerase comprises at least one substitution or set of substitutions at the following amino acid positions: 38 / 437 / 779, 38 / 437, 38, 38 / 207 / 437 / 779, 517 / 779, 38 / 437 / 527 / 664, 38 / 207 / 379 / 437, 664 / 670 / 779, 38 / 779, 38 / 437 / 517, 437 / 779, 38 / 437 / 607, 38 / 379 / 779, or 517 / 670 / 779, wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO:

156.

34. The engineered RNA polymerase of claim 30 or 33, wherein the amino acid sequence of the engineered RNA polymerase comprises at least one substitution or set of substitutions or amino acid residues 38G / 437P / 779R, 38G / 437P, 38G, 38G / 207S / 437P / 779R, 517R / 779R, 38G / 437P / 527E / 664K, 38G / 207S / 379S / 437P, 664K / 670R / 779R, 38G / 779R, 38G / 437P / 517R, 437P / 779R, 38G / 437P / 607Y, 38G / 379S / 779R, or 517R / 670R / 779R, wherein the amino acid position is relative to the position corresponding to SEQ ID NO: The reference sequence of residues 8 to 890 of SEQ ID NO: 156 or the reference sequence corresponding to SEQ ID NO:

156.

35. The engineered RNA polymerase of claim 16 or 30, wherein the amino acid sequence of the engineered RNA polymerase comprises at least one set of substitutions at amino acid positions 357 / 394 / 664 or 138 / 311 / 336 / 340 / 357 / 388 / 394 / 404 / 664, wherein the amino acid positions are relative to the reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 156 or relative to the reference sequence corresponding to SEQ ID NO:

156.

36. The engineered RNA polymerase of claim 30 or 35, wherein the amino acid sequence of the engineered RNA polymerase comprises at least one set of substitutions or amino acid residues 357E / 394K / 664P or 138N / 311A / 336K / 340K / 357E / 388A / 394K / 404S / 664P, wherein the amino acid position is relative to the reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 156 or relative to the reference sequence corresponding to SEQ ID NO:

156.

37. The engineered RNA polymerase of claim 1 or 15, comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with respect to the reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 234 or with respect to the reference sequence corresponding to SEQ ID NO: 234, wherein the amino acid sequence comprises one or more substitutions.

38. The engineered RNA polymerase of claim 16 or 37, wherein the amino acid sequence of the engineered RNA polymerase contains at least one substitution or set of substitutions at the following amino acid positions: 720, 196, 609, 246, 185, 594, 212, 494, 375, 721, 72, 867, 160, 718, 187, 170 / 173, 615, 137 / 138, 854, 131, 38 / 185 / 187 / 437, 185 / 187, 437 / 609, 38 / 609, 38 / 185 / 187 / 437 / 609, 437 / 609 / 720, 185 / 187 / 609, 185 / 187 / 437, 437 / 721, 609 / 720, 185 / 187 / 246 or 185 / 187 / 437 / 609 / 720, wherein the amino acid position is relative to the reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 234 or relative to the reference sequence corresponding to SEQ ID NO:

234.

39. The engineered RNA polymerase of claim 37 or 38, wherein the amino acid sequence of the engineered RNA polymerase comprises at least one substitution or set of substitutions or amino acid residues 720A, 196C, 609W, 196V, 246V, 185L, 594G, 212Y, 494G, 196A, 212R, 375F, 721T, 721A, 72R, 185I, 721S, 867Q, 721Y, 160F, 718F, 609F, 720S, 187L, 718V, 170L / 173V, 609E, 718S, 609C, 609P, 721M, 615A, 187H, 196S, 721E, 718Y, 137G / 138N, 187G, 720G, 7 20P, 854T, 720I, 160L, 131T, 38R / 185L / 187H / 437S, 185L / 187H, 185L / 187L, 437S / 609C, 38R / 609P, 38R / 185L / 187H / 437S / 609C, 437S / 609P / 720A, 185L / 187L / 609P, 185L / 187L / 437S, 185L / 187H / 437S, 437S / 609P, 437S / 721S, 185L / 187H / 609E, 609P / 720A, 185L / 187L / 246V or 185L / 187H / 437S / 609P / 720A, wherein the amino acid position is relative to the reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 234 or relative to the reference sequence corresponding to SEQ ID NO:

234.

40. The engineered RNA polymerase of claim 1 or 15, comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with respect to the reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 386 or the reference sequence corresponding to SEQ ID NO: 386, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 386 or relative to the reference sequence corresponding to SEQ ID NO:

386.

41. The engineered RNA polymerase of claim 16 or 40, wherein the amino acid sequence of the engineered RNA polymerase contains at least one substitution or set of substitutions at the following amino acid positions: 170 / 609, 38 / 185 / 187 / 609, 38 / 185 / 187, 38 / 170 / 173, 38 / 173 / 185 / 187 / 720, 170 / 185 / 187 / 609, 609, 170 / 185 / 187 / 609 / 721, 38 / 609, 38 / 170 / 246 / 609, 185 / 187 / 609 / 718, 38 / 185 / 187 / 609 / 718 / 720, 38 / 170 / 246 / 609 / 718, 720, 246 / 609, 38 / 185 / 187 / 246 / 720, 38 / 609 / 721, 38 / 72 1, 38 / 720 / 721, 38 / 170 / 609 / 718 / 720, 38 / 718, 38 / 185 / 187 / 609 / 718 / 721, 529 / 609, 38 / 609 / 718, 185 / 187 / 609 / 718 / 720, 185 / 187 / 609 / 718 / 721, 38 / 170 / 718 / 720 / 721, 170 / 185 / 18 7 / 718 / 720, 38 / 173 / 185 / 187 / 246 / 609 / 720, 609 / 718 / 720, 170 / 185 / 187 / 718 / 720 / 721, 38 / 170 / 173 / 246 / 609 / 718, 246 / 721, 38 / 718 / 720 or 38 / 609 / 718 / 720, wherein the amino acid position is relative to the reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 386 or relative to the reference sequence corresponding to SEQ ID NO:

386.

42. The engineered RNA polymerase of claim 40 or 41, wherein the amino acid sequence of said engineered RNA polymerase comprises at least one substitution or set of substitutions: 170L / 609P, 38G / 185L / 187L / 609C, 38G / 185L / 187H, 38G / 170L / 173V, 38G / 173V / 185L / 187L / 720A, 170L / 185L / 187H / 609C, 609E, 170L / 185L / 187H / 609C / 721S, 3 8G / 609E, 170L / 185L / 187L / 609E, 38G / 170L / 246V / 609P, 185L / 187H / 609E / 718S, 38G / 185L / 187L / 609W / 718N / 720A, 38G / 170L / 246V / 609C / 718S, 720A, 246V / 609E, 38G / 185L / 187L / 246V / 720A, 38G / 609C / 721S, 38G / 721S, 38G / 720A / 7 21S, 38G / 170L / 609C / 718S / 720A, 38G / 718S, 38G / 170L / 246V / 609P / 718S, 38G / 185L / 187L / 609P / 718S / 721S, 529R / 6 09E, 38G / 609P / 718S, 185L / 187L / 609C / 718S / 720A, 185L / 187H / 609C / 718S / 721S, 38G / 170L / 718S / 720A / 721S, 170L / 185L / 187L / 718S / 720A, 38G / 173V / 185L / 187L / 246V / 609E / 720A, 609E / 718S / 720A, 170L / 185L / 187H / 718S / 720A / 721S, 38G / 170L / 173V / 246V / 609E / 718S, 246V / 721S, 38G / 718S / 720A or 38G / 609E / 718S / 720A, wherein the amino acid position is relative to the reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 386 or relative to the reference sequence corresponding to SEQ ID NO:

386.

43. The engineered RNA polymerase of claim 1 or 15, comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with respect to the reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 238 or the reference sequence corresponding to SEQ ID NO: 238, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 238 or relative to the reference sequence corresponding to SEQ ID NO:

238.

44. The engineered RNA polymerase of claim 16 or 43, wherein the amino acid sequence of the engineered RNA polymerase comprises at least one substitution or set of substitutions at the following amino acid positions: 143, 105, 775 / 779, 135, 100, 246, 779 / 782, 749, 237, 162, 83, 218, 227, 115, 274, 81, 122, 61, 32, 139, 75, 239, 257, 240, 69 / 161, 253, 14, 221, 84, 133 / 150, 60, 275, 89, 15, 254, 625, 14 / 67, 21, 93, 250, 217, 19, 95, 759, 62, or 140, wherein the amino acid positions are relative to the amino acid corresponding to SEQ ID NO: The reference sequence of residues 8 to 890 of SEQ ID NO: 238 or the reference sequence corresponding to SEQ ID NO:

238.

45. The engineered RNA polymerase of claim 43 or 44, wherein the amino acid sequence of the engineered RNA polymerase comprises at least one substitution or set of substitutions or amino acid residues 143N, 143Q, 143R, 105R, 775R / 779R, 135K, 100R, 775K / 779R, 246K, 779R / 782R, 749R, 237L, 162V, 83K, 218T, 227V, 115L, 274G, 81C, 122I, 61Y, 32S, 139R, 75Y, 239R 257L, 240M, 69S / 161R, 253Y, 14Y, 221C, 84A, 115V, 133C / 150Q, 60L, 60G, 32V, 275A, 89Q, 257V, 274L, 15T, 254S, 61W, 625C, 14Q / 67R, 21G, 254G, 93Y, 250R, 217V, 60M, 239L, 75S, 32T, 19E, 95Y, 81L, 60R, 759M, 62T, or 140R, wherein the amino acid position is relative to the reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 238 or relative to the reference sequence corresponding to SEQ ID NO:

238.

46. ​​The engineered RNA polymerase of claim 1 or 15, comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with respect to the reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 394 or with respect to the reference sequence corresponding to SEQ ID NO: 394, wherein the amino acid sequence comprises one or more substitutions.

47. The engineered RNA polymerase of claim 16 or 46, wherein the amino acid sequence of the engineered RNA polymerase comprises at least one substitution or set of substitutions at the following amino acid positions: 609 / 779, 246 / 609, 246, 609 / 775 / 779, 105, 143 / 609 / 775, 105 / 135 / 609 / 775 or 111 / 609, 609, wherein the amino acid position is relative to the reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 394 or relative to the reference sequence corresponding to SEQ ID NO:

394.

48. The engineered RNA polymerase of claim 46 or 47, wherein the amino acid sequence of the engineered RNA polymerase comprises at least one substitution or set of substitutions or amino acid residues 609E / 779R, 246K / 609E, 246K, 609E / 775K / 779R, 105R, 143N / 609E / 775K, 105R / 135K / 609E / 775K, 111K / 609E or 609E, wherein the amino acid position is relative to the reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 394 or relative to the reference sequence corresponding to SEQ ID NO:

394.

49. The engineered RNA polymerase of claim 1 or 15, comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with respect to the reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 588 or the reference sequence corresponding to SEQ ID NO: 588, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 588 or relative to the reference sequence corresponding to SEQ ID NO:

588.

50. The engineered RNA polymerase of claim 16 or 49, wherein the amino acid sequence of the engineered RNA polymerase comprises at least one substitution or set of substitutions at the following amino acid positions: 32, 531 / 533, 115, 83, 32 / 60 / 61, 55, 589, 357, 617, 172, 598, 56 / 193, 56, 536, 52, 17, 261, 78, 629, 63, 82, 207, 666, 150, 45, 135, 13, 33, or 245, wherein the amino acid position is relative to the reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 588 or relative to the reference sequence corresponding to SEQ ID NO:

588.

51. The engineered RNA polymerase of claim 49 or 50, wherein the amino acid sequence of the engineered RNA polymerase comprises at least one substitution or set of substitutions 32V, 531Q / 533S, 115L, 83K, 32V / 60M / 61W, 55D, 589C, 357P, 617R, 172G, 598A, 56G / 193G, 56T, 536V, 52T, 17N, 56N, 598K, 261M, 56E, 55S, 536E, 598L, 78S, 56V, 629R, 598R, 63S, 82S, 207S, 598V, 666M, 150Y, 45K, 55W, 135G, 13N, 33E, or 245C, wherein the amino acid position is relative to the position corresponding to SEQ ID NO: The reference sequence of residues 8 to 890 of 588 or the reference sequence corresponding to SEQ ID NO:

588.

52. The engineered RNA polymerase of claim 1 or 15, comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with respect to the reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 604 or the reference sequence corresponding to SEQ ID NO: 604, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 604 or relative to the reference sequence corresponding to SEQ ID NO:

604.

53. The engineered RNA polymerase of claim 16 or 52, wherein the amino acid sequence of the engineered RNA polymerase comprises at least one substitution or set of substitutions at the following amino acid positions: 207 / 245 / 629, 17 / 56 / 150 / 598, 56 / 629, 536, 185, 56 / 150, 17 / 56 / 150 / 207 / 245 / 598 / 617 / 629, 56 / 150 / 536 / 598, 56 / 536, 185 / 666, 150, or 143 / 150 / 246 / 782, wherein the amino acid position is relative to the reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 604 or relative to the reference sequence corresponding to SEQ ID NO:

604.

54. The engineered RNA polymerase of claim 52 or 53, wherein the amino acid sequence of the engineered RNA polymerase comprises at least one substitution or set of substitutions or amino acid residues 207S / 245C / 629R, 17N / 56E / 150Y / 598A, 56E / 629R, 536V, 185P, 56T / 150Y, 17N / 56E / 150Y / 207S / 245C / 598A / 617R / 629R, 56E / 150Y / 536V / 598K, 56V / 536V, 185P / 666M, 150Y, or 143N / 150Y / 246V / 782V, wherein the amino acid position is relative to the reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 604 or relative to the reference sequence corresponding to SEQ ID NO:

604.

55. The engineered RNA polymerase of claim 1 or 15, comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with respect to the reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 686 or the reference sequence corresponding to SEQ ID NO: 686, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 686 or relative to the reference sequence corresponding to SEQ ID NO:

686.

56. The engineered RNA polymerase of claim 16 or 55, wherein the amino acid sequence of the engineered RNA polymerase comprises at least one substitution or set of substitutions at the following amino acid positions: 143, 246 / 424 / 648 / 782, 246 / 659 / 782, 56 / 150 / 629 / 660 / 782, 721 / 782, 782, 196 / 659 / 782, or 720 / 721 / 782, wherein the amino acid position is relative to the reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 686 or relative to the reference sequence corresponding to SEQ ID NO:

686.

57. The engineered RNA polymerase of claim 55 or 56, wherein the amino acid sequence of the engineered RNA polymerase comprises at least one substitution or set of substitutions or amino acid residues 143N, 246R / 424A / 648F / 782V, 246R / 659E / 782V, 56T / 150Y / 629A / 660W / 782V, 721T / 782V, 782V, 196G / 659E / 782V, or 720S / 721A / 782V, wherein the amino acid position is relative to the reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 686 or relative to the reference sequence corresponding to SEQ ID NO:

686.

58. The engineered RNA polymerase of claim 1 or 15, comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with respect to the reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 692 or with respect to the reference sequence corresponding to SEQ ID NO: 692, wherein the amino acid sequence comprises one or more substitutions.

59. The engineered RNA polymerase of claim 16 or 58, wherein the amino acid sequence of the engineered RNA polymerase comprises at least one substitution or set of substitutions at the following amino acid positions: 143 / 659 / 782, 166 / 171 / 246 / 311, 143 / 721, 782, or 143 / 171 / 659 / 782, wherein the amino acid position is relative to a reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 692 or relative to a reference sequence corresponding to SEQ ID NO:

692.

60. The engineered RNA polymerase of claim 58 or 59, wherein the amino acid sequence of the engineered RNA polymerase comprises at least one substitution or set of substitutions or amino acid residues 143N / 659E / 782V, 166T / 171E / 246R / 311A, 143N / 721T, 782V or 143N / 171E / 659E / 782V, wherein the amino acid position is relative to the reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 692 or relative to the reference sequence corresponding to SEQ ID NO:

692.

61. The engineered RNA polymerase of claim 1 or 15, comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with respect to the reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 712 or with respect to the reference sequence corresponding to SEQ ID NO: 712, wherein the amino acid sequence comprises one or more substitutions.

62. The engineered RNA polymerase of claim 16 or 61, wherein the amino acid sequence of said engineered RNA polymerase contains at least one substitution or set of substitutions at the following amino acid positions: 367, 382, ​​670, 672, 357, 388, 693, 397, 375, 137 / 382 / 782, 655, 137 / 382 / 392, 391, 137 / 655 / 721 / 7 63, 696, 663, 137 / 382, 137 / 382 / 392 / 650 / 655 / 782, 137 / 382 / 629 / 660 / 672, 616, 690, 179, 137, 357 / 382 / 670, 388 / 670 / 672, 357 / 375, 357 / 672, 629, or 655 / 670, wherein the amino acid position is relative to the reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 712 or relative to the reference sequence corresponding to SEQ ID NO:

712.

63. The engineered RNA polymerase of claim 61 or 62, wherein the amino acid sequence of the engineered RNA polymerase comprises at least one substitution or set of substitutions or amino acid residues 367P, 382R, 670R, 382L, 672P, 357P, 388R, 693R, 672S, 397S, 375P, 672G, 137G / 382R / 782E, 655R, 137G / 382R / 392A, 391F, 137G / 655M / 721T / 763S, 696L, 67 2R, 663L, 367T, 367F, 137G / 382G, 137G / 382G / 392R / 650M / 655M / 782E, 137G / 382R / 629R / 660Y / 672T, 391S, 616A, 690S, 179N, 137G, 357P / 382R / 670R, 388R / 670R / 672P, 357P / 375P, 357P / 672G, 629K, or 655R / 670R, wherein the amino acid position is relative to the reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 712 or relative to the reference sequence corresponding to SEQ ID NO:

712.

64. The engineered RNA polymerase of claim 1 or 15, comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with respect to the reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 800 or the reference sequence corresponding to SEQ ID NO: 800, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 800 or relative to the reference sequence corresponding to SEQ ID NO:

800.

65. The engineered RNA polymerase of claim 16 or 64, wherein the amino acid sequence of the engineered RNA polymerase comprises at least one substitution or set of substitutions at the following amino acid positions: 137, 56 / 311 / 382, 56 / 137 / 143 / 721, 137 / 392, 56 / 137 / 392, 56 / 137 / 143 / 404 / 720, 56 / 143 / 246, 56 / 392, 392 / 404, 392, 56 / 137, or 56 / 196 / 392, wherein the amino acid position is relative to the reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 800 or relative to the reference sequence corresponding to SEQ ID NO:

800.

66. The engineered RNA polymerase of claim 64 or 65, wherein the amino acid sequence of the engineered RNA polymerase comprises at least one substitution or set of substitutions or amino acid residues 137G, 56E / 311A / 382R, 56E / 137G / 143N / 721A, 137G / 392A, 56E / 137G / 392A, 56A / 137G / 143N / 404Y / 720S, 56E / 143N / 246R, 56E / 392R, 392R / 404Y, 392R, 137G / 392R, 56E / 137G, or 56E / 196S / 392A, wherein the amino acid position is relative to the reference sequence corresponding to residues 8 to 890 of SEQ ID NO: 800 or relative to the reference sequence corresponding to SEQ ID NO:

800.

67. The engineered RNA polymerase of claim 1, wherein the amino acid sequence of the engineered RNA polymerase comprises at least one substitution as described in Tables 5.1, 6.1, 7.1, 8.1, 9.1, 10.1, 11.1, 12.1, 13.1, 14.1, 15.1, 16.1, 17.1, 18.1, 19.1, 23.1, and 24.1, wherein the amino acid position is relative to the reference sequence of residues 8 to 890 corresponding to SEQ ID NO: 2, 4, 32, 44, 156, 234, 238, 386, 394, 588, 604, 686, 692, 712, or 800, or relative to the reference sequence corresponding to SEQ ID NO: Reference sequences of 2, 4, 32, 44, 156, 234, 238, 386, 394, 588, 604, 686, 692, 712 or 800.

68. The engineered RNA polymerase of claim 1, wherein the amino acid sequence of the engineered RNA polymerase comprises at least one substitution or set of substitutions of the engineered RNA polymerases described in Tables 5.1, 6.1, 7.1, 8.1, 9.1, 10.1, 11.1, 12.1, 13.1, 14.1, 15.1, 16.1, 17.1, 18.1, 19.1, 23.1, and 24.1, wherein the amino acid position is relative to the reference sequence of residues 8 to 890 corresponding to SEQ ID NO: 2, 4, 32, 44, 156, 234, 238, 386, 394, 588, 604, 686, 692, 712, or 800, or relative to the reference sequence of residues 8 to 890 corresponding to SEQ ID NO: Reference sequences of 2, 4, 32, 44, 156, 234, 238, 386, 394, 588, 604, 686, 692, 712 or 800.

69. The engineered RNA polymerase of claim 1, comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 97%, 98%, 99% or higher sequence identity with a reference sequence comprising at least one substitution or set of substitutions as described in Tables 5.1, 6.1, 7.1, 8.1, 9.1, 10.1, 11.1, 12.1, 13.1, 14.1, 15.1, 16.1, 17.1, 18.1, 19.1, 23.1, and 24.1, wherein the amino acid position is relative to the position corresponding to SEQ ID NO: Reference sequences of residues 8 to 890 of 2, 4, 32, 44, 156, 234, 238, 386, 394, 588, 604, 686, 692, 712, or 800, or reference sequences corresponding to SEQ ID NO: 2, 4, 32, 44, 156, 234, 238, 386, 394, 588, 604, 686, 692, 712, or 800.

70. The engineered RNA polymerase of claim 1, wherein the amino acid sequence of the engineered RNA polymerase comprises residues 8 to 890 of SEQ ID NO. with even numbers from SEQ ID NO: 4-828 or comprises SEQ ID NO. with even numbers from SEQ ID NO: 4-828, optionally wherein the amino acid sequence has 1, 2, 3, 4, 5, 6, 7, 8, 9 or up to 10 substitutions.

71. The engineered RNA polymerase according to any one of claims 1-70, wherein the engineered RNA polymerase has RNA polymerase activity.

72. The engineered RNA polymerase according to any one of claims 1-71, having at least one improved property compared to a reference RNA polymerase, wherein the reference RNA polymerase has a sequence of residues 8 to 890 corresponding to SEQ ID NO: 2, 4, 32, 44, 156, 234, 238, 386, 394, 588, 604, 686, 692, 712 or 800, or a sequence corresponding to SEQ ID NO: 2, 4, 32, 44, 156, 234, 238, 386, 394, 588, 604, 686, 692, 712 or 800.

73. The engineered RNA polymerase of claim 72, wherein the improved property compared to a reference RNA polymerase is selected from i) increased activity, ii) increased thermostability, iii) increased persistence, or iv) increased RNA product integrity, or any combination of i), ii), iii) and iv), wherein the reference RNA polymerase has a sequence of residues 8 to 890 corresponding to SEQ ID NO: 2, 4, 32, 44, 156, 234, 238, 386, 394, 588, 604, 686, 692, 712, or 800, or a sequence corresponding to SEQ ID NO: 2, 4, 32, 44, 156, 234, 238, 386, 394, 588, 604, 686, 692, 712, or 800.

74. The engineered RNA polymerase of claim 72 or 73, wherein the reference RNA polymerase has a sequence corresponding to residues 8 to 890 of SEQ ID NO: 2 or a sequence corresponding to SEQ ID NO:

2.

75. The engineered RNA polymerase according to any one of claims 1-74, wherein the engineered RNA polymerase is purified.

76. A recombinant polynucleotide encoding an engineered RNA polymerase according to any one of claims 1-74.

77. The recombinant polynucleotide of claim 76, comprising a reference polynucleotide sequence of nucleotide residues 21 to 2670 of SEQ ID NO. corresponding to odd-numbered SEQ ID NO: 3-827 or a polynucleotide sequence having at least 70%, 75%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with SEQ ID NO. corresponding to odd-numbered SEQ ID NO: 3-827, wherein the recombinant polynucleotide encodes an RNA polymerase.

78. The recombinant polynucleotide of claim 76, comprising a reference polynucleotide sequence having at least 70%, 75%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with nucleotide residues 21 to 2670 corresponding to SEQ ID NO: 3, 31, 43, 155, 233, 237, 381, 393, 587, 603, 685, 691, 711 or 799, or a reference polynucleotide sequence corresponding to SEQ ID NO: 3, 31, 43, 155, 233, 237, 381, 393, 587, 603, 685, 691, 711 or 799, wherein the recombinant polynucleotide encodes an RNA polymerase.

79. The recombinant polynucleotide of any one of claims 76-78, wherein the polynucleotide sequence is codon-optimized for the expression of the engineered RNA polymerase it encodes.

80. The recombinant polynucleotide of claim 76, comprising nucleotide residues 21 to 2670 of SEQ ID NO. with odd numbers of SEQ ID NO: 3-827 or a polynucleotide sequence with odd numbers of SEQ ID NO: 3-827.

81. The recombinant polynucleotide of claim 76, wherein the polynucleotide sequence comprises nucleotide residues 21 to 2670 of SEQ ID NO: 3, 31, 43, 155, 233, 237, 381, 393, 587, 603, 685, 691, 711, or 799, or comprises nucleotide residues 21 to 2670 of SEQ ID NO: 3, 31, 43, 155, 233, 237, 381, 393, 587, 603, 685, 691, 711, or 799.

82. An expression vector comprising at least one recombinant polynucleotide as described in any one of claims 76-81.

83. The expression vector of claim 82, wherein the polynucleotide is operatively linked to a control sequence.

84. The expression vector of claim 83, wherein the control sequence comprises at least a promoter.

85. A host cell comprising the expression vector according to any one of claims 82-84.

86. The host cell of claim 85, comprising a prokaryotic cell or a eukaryotic cell.

87. A method for producing an engineered RNA polymerase polypeptide in a host cell, comprising culturing the host cell of claim 85 or 86 under suitable culture conditions to produce at least one engineered RNA polymerase.

88. The method of claim 87, further comprising recovering at least one engineered RNA polymerase from the culture and / or host cells.

89. The method of claim 87 or 88, further comprising the step of purifying the at least one engineered RNA polymerase.

90. A composition comprising the RNA polymerase according to any one of claims 1-75.

91. The composition of claim 90, further comprising at least one buffer and at least one rNTP.

92. The composition of claim 90 or 91, further comprising a target DNA template.

93. The composition according to any one of claims 90-92, further comprising a cap analogue.

94. The composition according to any one of claims 90-93, further comprising pyrophosphatase and / or RNase inhibitors.

95. The composition according to any one of claims 90-94, wherein the RNA polymerase is a lyophilized product.

96. A method for producing RNA in vitro, comprising contacting a target DNA template with an engineered RNA polymerase according to any one of claims 1-74 in the presence of one or more nucleoside triphosphates under conditions suitable for DNA template transcription.

97. The method of claim 96, wherein the target DNA template comprises a promoter recognized by the engineered RNA polymerase.

98. The method of claim 94, further comprising providing a cap analogue.

99. The method according to any one of claims 96-98, further comprising pyrophosphatase and / or RNase inhibitors.

100. The method of any one of claims 96-99, wherein the suitable conditions include a temperature of about 25°C to 45°C.

101. A kit comprising the engineered RNA polymerase according to any one of claims 1-75.

102. The kit of claim 101, further comprising at least one buffer, one or more rNTPs, cap analogs, or molecular crowding agents.

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