Engineered terminal deoxynucleotidyl transferase variants

Abstract

The present invention provides engineered terminal deoxynucleotidyl transferase (TdT) polypeptides useful in template-independent polynucleotide synthesis, as well as compositions, methods, and polynucleotides encoding the engineered terminal deoxynucleotidyl transferases. In some additional embodiments, the engineered polypeptides comprise an amino acid sequence having at least 60% sequence identity to any of the even-numbered sequences set forth in SEQ ID NOS: 4-1960, 2004-3920, 4048-5466, and 5476.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 499,770, filed May 3, 2023, and U.S. Provisional Patent Application No. 63 / 379,439, filed October 13, 2022, each of which is incorporated by reference in its entirety.

[0002] The present invention provides engineered terminal deoxynucleotidyl transferase (TdT) polypeptides useful in template-independent polynucleotide synthesis, as well as compositions and methods utilizing such engineered polypeptides.

[0003] Reference to a sequence listing, table, or computer program An official copy of the Sequence Listing has been submitted contemporaneously with the specification as an XML file having the filename "CX10-238WO3_ST26.xml," a creation date of October 11, 2023, and a size of 13,670,249 bytes. The submitted Sequence Listing is a part of the present specification and is incorporated herein by reference in its entirety. [Background technology]

[0004] Synthetic biology is becoming established in a variety of high-value, high-growth markets. From food and agriculture to therapeutics, diagnostics, and vaccines, tools such as gene editing, DNA sequencing, and gene synthesis are being used to construct highly functional, value-added products (e.g., cell bioreactors) and desired end products (e.g., drugs, chemicals, etc.). A major obstacle to the widespread realization of these technologies is the ability to efficiently synthesize RNA, DNA, and other polynucleotides.

[0005] Specifically, silencing RNA (siRNA) therapeutics are a promising class of drugs that have the potential to treat numerous difficult-to-treat conditions in a highly targeted manner by binding to known mRNA targets (Hu et al. (2020). Sig Transduct Target Ther 5, 101; Zhang et al. (2021). Bioch. Pharmac., 189, 114432). As these treatments become more commonplace and target larger patient populations, the ability to produce large quantities of oligonucleotide active pharmaceutical ingredients (APIs) will be crucial.

[0006] To date, short RNA oligonucleotides have been synthesized almost exclusively by the repetitive addition of nucleotides in the form of activated phosphoramidites to a growing immobilized nucleotide chain, followed by an additional processing step (Brown, T. Nucleic Acids Book, see www.atdbio.com / nucleic-acids-book (accessed 2022-10-10)).

[0007] Phosphoramidite chemistry has been widely developed over the years for the synthesis of small amounts of DNA and even more complex therapeutic RNAs. However, as API demand grows to three- to four-digit kilograms per year, several cost and sustainability issues pose potential limitations (Andrews et al. (2021). J. Org. Chem. 86, 49-61). Furthermore, RNA synthesis using phosphoramidite synthesis chemistry is limited to producing short oligonucleotides of approximately 200 base pairs (Beaucage & Caruthers. (1981). Tetrahedron Lett. 22(20):1859).

[0008] The phosphoramidite iteration methodology is multistep and based on phosphorus(III) coupling chemistry, requiring (i) coupling, (ii) capping, (iii) oxidation to P(V) to form a phosphodiester or phosphorothioate diester, and (iv) unblocking of the 5'O group. After chain synthesis is complete, the final oligo is cleaved from the support, which may also involve unblocking of the cyanoethyl phosphate group and nucleobases (see Brown, T. Nucleic Acids Book, www.atdbio.com / nucleic-acids-book (accessed 2022-10-10)). Organic solvent washes are also required at each step. The cyanoethyl phosphate blocking group and nucleobase protecting groups can be removed in parallel with oligonucleotide cleavage from the solid support to generate the oligonucleotide product, or, if desired, the cyanoethyl group can be removed under milder conditions prior to chain cleavage.

[0009] Current phosphoramidite methodologies and potential advances have been reviewed, focusing on many aspects of their environmental impact (Andrews et al. (2021). J. Org. Chem. 86, 49-61). High oligonucleotide loadings of 20% of the final oligonucleotide mass relative to the mass of the solid support are highly desirable, but even this requires at least a 5-fold increase in the mass of the support relative to the final product mass.

[0010] In addition to the high cost of the support mass required for oligonucleotide immobilization, the use of organic solvents and 5'-O-blocking groups entails additional waste and process inefficiencies. Organic solvents such as acetonitrile are required to solubilize the phosphoramidite coupling partner, or dichloromethane or toluene are required for the deprotection step. These solvents must be anhydrous to reduce undesired hydrolysis of the phosphoramidite partner and may be derived from non-sustainable sources, adding cost, sustainability issues, and potential supply problems to the process.

[0011] The phosphoramidite coupling partner itself possesses the necessary blocking group at the 5'O position, the nucleobase nitrogen atom (as in A, C, and G), and the nascent phosphate. The most common 5'O blocking group, dimethoxytrityl, has a molecular weight of approximately 303 Da, which is close to the molecular weight of Gp, the heaviest naturally occurring ribonucleotide fragment, which has a mass of approximately 345 Da. This protecting group requires energy, resources, and labor to generate and attach, and therefore must be disposed of upon separation from the desired material.

[0012] In conclusion, a paradigm shift in oligonucleotide synthesis is necessary to realize siRNA therapeutics by reducing environmental impact, improving economic efficiency, and increasing scalability. Therefore, new oligonucleotide synthesis methods are of great interest to the pharmaceutical industry.

[0013] Template-free enzymatic synthesis

[0014] Enzymatic synthesis can facilitate the production of large quantities of complex or long polynucleotides (>200 base pairs) while minimizing toxic waste. A variety of prokaryotic and eukaryotic DNA and RNA polymerases are known to naturally synthesize polynucleotides of several thousand base pairs or more. Most of these polymerases function during DNA replication, associated with cell division, or during transcription of RNA from DNA, associated with gene or protein expression. Both of these processes involve template-dependent polynucleotide synthesis, in which a polymerase uses an existing template polynucleotide strand to synthesize a complementary polynucleotide strand.

[0015] The potential of nontemplated enzymatic polynucleotide synthesis in generating defined sequences has long been recognized. One early report suggested the use of 3'-blocked NTPs to allow the stepwise addition of specific nucleotide residues (Bollum, 1962). JBC, 237, 1945-1949).

[0016] However, few polymerases are known to catalyze template-independent polynucleotide synthesis. These include polymerase lambda, polymerase mu, and terminal deoxynucleotidyl transferase (TdT), all of which are members of the X family of DNA polymerases, many of which are involved in DNA repair processes (Dominguez et al. (2000). EMBO, 19(7), 1731-1742). Among these, TdT is known to confer diversity to antigen receptors by indiscriminately adding nucleosides to the 3' termini of single-stranded polynucleotides in a template-independent process (Bentolila et al. (1995). EMBO, 14(17), 4221-4229).

[0017] Others have also published methods for polynucleotide synthesis using nucleoside 5'-triphosphates with the 3'-OH position protected by a removable blocking moiety, and specifically template-free polynucleotide polymerases (including terminal deoxynucleotidyl transferase) (U.S. Pat. No. 5,763,594). Blocking groups, also known to those skilled in the art as inhibitors or reversible terminators, can include a variety of groups that prevent TdT from adding additional NTPs to the nascent polynucleotide chain. These can include charged molecules, large molecules and moieties, or other blocking groups known to those skilled in the art. Suitable removable blocking groups can include carbonitriles, phosphates, carbonates, carbamates, esters, ethers, borates, nitrates, sugars, phosphoramidates, phenylsulfenates, and sulfates. Other 3' blocking groups are also known in the art, including 3'-O-amines and methylamines (US Pat. No. 7,544,794) and 3'-O-azides (US Pat. No. 10,407,721).

[0018] Although initially promising, the use of 3'-blocked NTPs in template-independent synthesis catalyzed by TdT has proven difficult in practice due to TdT's poor tolerance of 3'-O-blocked NTPs as substrates. Furthermore, wild-type TdT has poor tolerance for oligo-acceptor substrates containing one or more modified nucleotides (e.g., 2' modifications).

[0019] Additionally, synthesis of RNA chains presents unique challenges due to the presence of an additional reactive 2'-OH on ribose. While protecting the 2' position facilitates RNA synthesis, this approach is less efficient due to steric hindrance from the 2'-protecting group, necessitating its maintenance and removal (CB Reese. (2005). Org Biomol Chem 3, 3851-3868).

[0020] Recently, several reports have described template-free synthesis methods using modified NTPs with a blocking group attached to the purine or pyrimidine base, leaving the 3'-OH unmodified and available for additional synthesis rounds. These base-blocking groups can include cleavable linkers, which allow for removal of the blocking group after each NTP addition step. Cleavable linkers can also be attached to detectable labels (see, inter alia, U.S. Patent 7,057,026). A variety of cleavable linkers are known to those skilled in the art. These include linkers attached via a reducible disulfide bond, photocleavable linkers, electrophilic or nucleophilic linkers, pH-sensitive linkers, temperature-sensitive linkers, and enzymatically cleavable linkers. One drawback of using a cleavable linker is that, after cleavage, some atoms of the linker moiety typically remain attached to the NTP, leaving a "scar" that can interfere with the synthesis of the complementary strand following the initial template-free synthesis of the main polynucleotide strand.

[0021] Recently, modified NTPs have been developed in which the base is attached to the blocking group using a cleavable linker that is "scarless" and prepares the nascent DNA for the next round of synthesis. In one example, the blocking group and cleavable linker are attached to the base via a disulfide bond. Upon addition of a reducing agent, the blocking group is removed and the remaining atoms of the linker self-cyclize, leaving the nascent DNA free of any linker atoms (U.S. Patent 8,808,989, U.S. Patent 9,695,470, U.S. Patent 10,041,110). Methods for synthesizing polynucleotides using NTPs attached to cleavable blocking groups are known, including using microfluidic devices or inkjet printing technology (U.S. Patent 9,279,149). Exonucleases can also be used in methods to synthesize polynucleotides and shorten or completely degrade polynucleotide chains that do not successfully add NTPs after the polynucleotide elongation step and before removal of the blocking group (U.S. Patent 9,771,613).

[0022] However, NTP bases with bulky blocking groups attached via cleavable linkers are not optimal for the efficient synthesis of complex or long oligonucleotides. Large labels can negatively affect enzyme kinetics, and linker scars can cause unacceptable rates of misincorporation during the synthesis of oligonucleotide chains. Furthermore, the larger linkers and the required deblocking steps can increase the cost, time, and inefficiency of the overall process, making such methods economically unfeasible. Recently, several groups have explored modifying the structure or amino acid sequence of TdT or other polymerases to enable template-independent synthesis using 3'-O blocking groups. Efcavitch et al. described the incorporation of 3'-modified dNTPs by TdT in template-independent synthesis using murine or bacterial TdT with substituted amino acid residues (U.S. Patent 10,059,929). Other reports have described engineered bovine and gar (Lepisosteus oculatus) TdTs that exhibited improved activity over wild-type TdT (U.S. Patent 10,745,727, PCT / GB2020 / 050247). Similarly, various mutations to improve the activity of Pol X family enzymes have been described (WO2017216472A2). Finally, it has been demonstrated that N-terminal truncation of the BRCT domain of TdT (or alternatively, mutation of the BRCT domain) also enhances its ability to reversibly add blocked NTPs to the 3'-OH of nucleic acids (US20210164008A1). However, despite the recognized value of this technology and intensive research efforts aimed at solving problems in this field, no feasible template-independent enzymatic synthesis method for complex or long polynucleotides is currently known or commercially available. Improved engineered TdT enzymes are needed to enable template-independent enzymatic synthesis of complex or long polynucleotides or oligonucleotides of defined sequences using nucleoside triphosphates bearing 3'-O-removable blocking groups, 2' modifications, and / or other modifications. [Prior art documents] [Patent documents]

[0023] [Patent Document 1] U.S. Patent No. 5,763,594 [Patent Document 2] U.S. Patent No. 7,544,794 [Patent Document 3] U.S. Patent No. 10,407,721 [Patent Document 4] U.S. Patent No. 7,057,026 [Patent Document 5] U.S. Patent No. 8,808,989 [Patent Document 6] U.S. Patent No. 9,695,470 [Patent Document 7] U.S. Patent No. 10,041,110 [Patent Document 8] U.S. Patent No. 9,279,149 [Patent Document 9] U.S. Patent No. 9,771,613 [Patent Document 10] U.S. Patent No. 10,059,929 [Patent Document 11] U.S. Patent No. 10,745,727 [Patent Document 12] International Publication No. 2020 / 161480 [Patent Document 13] International Publication No. 2017 / 216472 [Patent Document 14] U.S. Patent Application Publication No. 2021 / 0164008

Non-licensed literature

[0024]

Non-patent document 1

Non-patent document 2

Non-patent document 3

Non-patent document 4

Non-patented document 5

[0025] The present invention provides engineered terminal deoxynucleotidyl transferase (TdT) polypeptides useful in template-independent polynucleotide synthesis, as well as compositions and methods utilizing such engineered polypeptides. The TdT of the present invention is a variant of the predicted splice variant (SEQ ID NO: 2) of the wild-type gene from Monodelphis domestica. These engineered TdTs are capable of adding nucleoside triphosphates bearing 3'-O-removable blocking groups, as well as other natural or modified NTPs, to the 3'-OH terminus of a growing oligonucleotide or polynucleotide chain in a template-independent manner. After removal of the blocking group, multiple additional rounds of NTP addition can be used to synthesize polynucleotides having defined base sequences without using a complementary template strand as a guide for NTP incorporation (template-independent synthesis).

[0026] In some embodiments, the present invention provides SEQ ID NOs: 2, 8, 16, 24, 36, 268, 648, 660, 882, 1100, 1336, 1348, 1596, 1654, 1830, 1950, 2008, 2254, 2514, 2524, 2638, 2804, 2812, 2956, 3174, 3222, 3670, 3674, 3796, 3870, 3906, 4006, 4106, 4206, 4306, 4406, 4506, 4606, 4706, 4806, 4906, 5006, 5106, 5206, 5306, 5406, 5506, 5606, 5706, 5806, 5906, 6006, 6106, 6206, 6306, 6406, 6506, 6506, 6606, 6706, 6806, 6806, 6906, 7006, 7106, 7206, 7306, 7406, 7506, 7606, 7706, 7806, 7906, 8006, 8106, 8206, 8306, 8406, 8506, 8606, 8706, 8806, 8906, 9006, 9106, 92 and / or 5246, wherein the positions are selected from the group consisting of: 3918, 4266, 4442, 4654, 4850, 4856, 4904, 5002, 5028, 5192, and / or 5246. The present invention provides engineered TdT polypeptides comprising an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to the reference sequences of: 3918, 4266, 4442, 4654, 4850, 4856, 4904, 5002, 5028, 5192, and / or 5246, and wherein the positions are selected from the group consisting of: 2, 8, 16, 24, 36, 268, 648, 660, 670, 672, 674, 676, 678, 679, 680, 681, 682, 683, 684, 685, 686, 687, 688, 689, 690, 700, 701, 702, 703, 704, 705, 706, 707, 708, 710, 711, 712, 713, 714, 715, 716, 717, 718, 719, 720, 721, 722, 723, 724, 725, 726, 727, 728, 729, 7 , 882, 1100, 1336, 1348, 1596, 1654, 1830, 1950, 2008, 2254, 2514, 2524, 2638, 2804, 2812, 2956, 3174, 3222, 3670, 3674, 3796, 3870, 3918, 4266, 4442, 4654, 4850, 4856, 4904, 5002, 5028, 5192, and / or 5246 and are numbered with reference to The engineered TdT polypeptides have improved thermostability, increased activity at high temperatures, increased soluble expression or isolated protein yield, reduced by-product formation, increased specific activity toward NTP-3'-O-RBG and other natural or modified NTP substrates, and / or increased activity toward various oligo acceptor substrates, compared to wild-type TdT or other TdTs or template-independent polymerases known to those skilled in the art. These engineered TdT polypeptides with one or more amino acid substitutions or sets of substitutions are described below in the detailed description of the invention.

[0027] In some additional embodiments, the engineered polypeptide comprises an amino acid sequence having at least 60% sequence identity to any even-numbered sequence set forth in SEQ ID NOs: 4-1960, 2004-3920, 4048-5466, and 5476.

[0028] In additional embodiments, the engineered polypeptides of the invention further comprise an N-terminal truncation of 1 to 156 amino acids of the polypeptide sequence compared to any even-numbered sequence set forth in SEQ ID NOs: 4-1960, 2004-3920, 4048-5466, and 5476.

[0029] In additional embodiments, an engineered polypeptide of the invention is fused to a second polypeptide, optionally with inorganic pyrophosphatase (IPP) activity (e.g., an IPP having an amino acid sequence selected from SEQ ID NOs: 3942 and 3944). In one embodiment, an engineered polypeptide of the invention fused to a second polypeptide with IPP activity comprises a sequence selected from SEQ ID NOs: 5468, 5470, 5472, and 5474.

[0030] The present invention also provides engineered polynucleotides encoding at least one engineered polypeptide described in the preceding paragraph. In some embodiments, the engineered polynucleotide comprises an odd-numbered sequence set forth in SEQ ID NOs: 3-1959, 2003-3919, 4047-5465, and 5475.

[0031] The present invention further provides vectors comprising at least one engineered polynucleotide described above. In some embodiments, the vector further comprises at least one regulatory sequence.

[0032] The present invention also provides host cells comprising the vectors provided herein. In some embodiments, the host cells produce at least one engineered polypeptide provided herein.

[0033] The present invention further provides methods for producing an engineered TdT polypeptide, the methods comprising culturing a host cell provided herein under conditions such that the engineered polynucleotide is expressed and the engineered polypeptide is produced. In some embodiments, the methods further comprise recovering the engineered polypeptide.

[0034] The present invention further provides template-independent synthesis methods comprising TdT or template-independent polymerases having activity toward a variety of oligo acceptor substrates and NTP-3'-O-RBG and other natural or modified NTP substrates, which methods may involve immobilized TdT or immobilized oligo acceptor substrates, or may involve neither immobilized TdT nor immobilized oligo acceptor substrates. DETAILED DESCRIPTION OF THE INVENTION

[0035] Unless otherwise defined, all technical and scientific terms used herein generally have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Generally, the nomenclature used herein and the laboratory procedures of cell culture, molecular genetics, microbiology, organic chemistry, analytical chemistry, and nucleic acid chemistry described below are well-known and widely used in the art. Such techniques are well-known and described in numerous texts and references familiar to those of skill in the art. Standard techniques, or variations thereof, are used for chemical syntheses and chemical analyses. All patents, patent applications, articles, and publications mentioned herein, both supra and infra, are hereby expressly incorporated by reference.

[0036] Although any methods and materials similar or equivalent to those described herein can be used in the practice of the present invention, several methods and materials are described herein. It is understood that the present invention is not limited to the particular methodology, protocols, and reagents described, as these may vary depending on the context in which they are used by those of skill in the art. Accordingly, the terms defined below are more fully described with reference to the invention as a whole.

[0037] It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention. The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described. Numerical ranges are inclusive of the numbers defining the range. Accordingly, every numerical range disclosed herein is intended to include every narrower numerical range that falls within such broader numerical range, as if such narrower numerical ranges were all expressly written herein. Every maximum (or minimum) numerical limitation disclosed herein is also intended to include every lower (or higher) numerical limitation, as if such lower (or higher) numerical limitations were expressly written herein.

[0038] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a polypeptide" includes a plurality of polypeptides. Similarly, "comprise," "comprises," "comprising," "include," "includes," and "including" are interchangeable and are not intended to be limiting.

[0039] When the term "comprising" is used in describing various embodiments, it is understood that one of ordinary skill in the art would understand that in some specific instances, the embodiments could alternatively be described using the words "consisting essentially of" or "consisting of." When the term "optional" or "optionally" is used in describing various embodiments, it is further understood that the subsequently described event or circumstance may or may not occur, and that the description includes both the occurrence of the event or circumstance and its non-occurrence. It is understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not limiting of the present disclosure. The section headings used herein are for organizational purposes only and should not be construed as limiting the subject matter described.

[0040] Abbreviation

[0041] The abbreviations used for the genetically encoded amino acids are conventional and are as follows: [Table 93]

[0042] When a three-letter abbreviation is used, amino acids are referred to as having an alpha carbon (C) unless specifically preceded by "L" or "D" or unless otherwise clear from the context in which the abbreviation is used. α ) can be in either the L- or D-configuration. For example, "Ala" designates alanine without specifying the configuration about the α-carbon, while "D-Ala" and "L-Ala" designate D-alanine and L-alanine, respectively.

[0043] When single-letter abbreviations are used, an uppercase letter designates an amino acid having the L-configuration about the alpha carbon, and a lowercase letter designates an amino acid having the D-configuration about the alpha carbon. For example, "A" designates L-alanine and "a" designates D-alanine. When a polypeptide sequence is presented as a series of single-letter or three-letter abbreviations (or a mixture thereof), the sequence is presented in the amino (N) to carboxy (C) orientation, according to common convention.

[0044] The abbreviations used for genetically coded nucleosides are conventional and are as follows: adenosine (A), guanosine (G), cytidine (C), thymidine (T), and uridine (U). These abbreviations are also used interchangeably for nucleosides and nucleotides (nucleosides having one or more phosphate groups). Unless otherwise specified, an abbreviated nucleoside or nucleotide may be either a ribonucleoside (or ribonucleotide) or a 2'-deoxyribonucleoside (or 2'-deoxyribonucleotide). A nucleoside or nucleotide may also be modified at the 3' position. A nucleoside or nucleotide, individually or collectively, may be identified as being either a ribonucleoside (or ribonucleotide) or a 2'-deoxyribonucleoside (or 2'-deoxyribonucleotide). When a nucleic acid sequence is presented as a series of single-letter abbreviations, the sequence is presented in the 5' to 3' direction, according to common convention, and no phosphate is indicated.

[0045] definition

[0046] In referring to the present invention, unless specifically defined otherwise, the technical and scientific terms used in the description herein have the meanings that are commonly understood by those of ordinary skill in the art. Accordingly, the following terms are intended to have the following meanings:

[0047] The "EC" number refers to the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology (NC-IUBMB) enzyme nomenclature. The IUBMB biochemical classification is a numerical classification system for enzymes based on the chemical reaction they catalyze.

[0048] "ATCC" refers to the American Type Culture Collection, whose biorepository collection includes genes and strains.

[0049] "NCBI" refers to the National Center for Biological Information and the sequence databases it hosts.

[0050] "Protein," "polypeptide," and "peptide" are used interchangeably herein to refer to a polymer of at least two amino acids covalently linked by an amide bond, regardless of length or post-translational modification (e.g., glycosylation, phosphorylation, lipidation, myristilation, ubiquitination, etc.). Included within this definition are D- and L-amino acids, as well as mixtures of D- and L-amino acids, and polymers comprising D- and L-amino acids and polymers comprising mixtures of D- and L-amino acids.

[0051] "Amino acids" are referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Similarly, nucleotides may be referred to by their commonly accepted single-letter codes.

[0052] As used herein, "polynucleotide," "oligonucleotide," and "nucleic acid" are used interchangeably herein and refer to two or more nucleosides or nucleotides covalently linked together. A polynucleotide can be composed entirely of ribonucleotides (i.e., RNA), entirely of 2'-deoxyribonucleotides (i.e., DNA), entirely of other synthetic nucleotides, or a mixture of synthetic ribonucleotides and / or 2'-deoxyribonucleotides. A polynucleotide can also contain modified nucleotides with 2'-substitutions (e.g., 2'-fluoro, 2'-O-methyl, 2'-O-methoxyethyl, locked or constrained ethyl modifications, and others known to those of skill in the art). Nucleosides are linked together via standard phosphodiester bonds or one or more non-standard linkages (including, but not limited to, phosphorothioate linkages). A polynucleotide can be single-stranded or double-stranded and can contain both single-stranded and double-stranded regions. Furthermore, polynucleotides are typically composed of naturally occurring coding nucleobases (i.e., adenine, guanine, uracil, thymine, and cytosine), but may contain one or more modified and / or synthetic nucleobases (e.g., inosine, xanthine, hypoxanthine, etc.). In some embodiments, such modified or synthetic nucleobases are nucleobases that encode an amino acid sequence. Modified or synthetic nucleobases can include any known, hypothesized, or future-discovered modification or structure that would be recognized by those of skill in the art as a modified or synthetic nucleobase. Similarly, the terms "polynucleotide," "oligonucleotide," and "nucleic acid" are intended to include any currently unknown or future-discovered modified or synthetic structure that would be recognized by those of skill in the art as, or having the functionality of, a "polynucleotide," "oligonucleotide," or "nucleic acid." One example of a modified or synthetic structure having the functionality of a "polynucleotide," "oligonucleotide," or "nucleic acid" is PNA, or peptide nucleic acid.

[0053] As used herein, "oligo acceptor substrate" and "acceptor substrate," as well as "growing oligo acceptor substrate strand," "growing oligonucleotide strand," and "growing polynucleotide strand," are used interchangeably herein and refer to any oligo or nucleotide strand or similar moiety having an exposed 3'-OH or equivalent that can be recognized by wild-type TdT or polymerase or an engineered TdT or template-free polymerase of the disclosure as a substrate for nucleoside addition or synthesis. In some embodiments, the acceptor substrate can be single-stranded. In yet other embodiments, the acceptor substrate can be double-stranded or partially double-stranded. In some embodiments, the acceptor substrate can comprise a nucleotide strand of 1-10 nucleotides, 5-20 nucleotides, 15-50 nucleotides, 30-100 nucleotides, or more than 100 nucleotides. In some embodiments, the acceptor substrate is not a nucleotide chain, but may comprise a chemical moiety containing a free -OH (referred to herein as a "3'-OH equivalent") that can be recognized as a substrate by wild-type or engineered TdT. Exemplary oligo acceptor substrates are provided in the Examples.

[0054] As used herein, "nucleoside triphosphate-3'-O-removable blocking group" and "nucleotide triphosphate-3'-O-removable blocking group," as well as "reversible terminator" and "NTP-3'-O-RBG," are used interchangeably herein and refer to a ribonucleoside triphosphate or deoxyribonucleoside triphosphate or synthetic or nucleoside triphosphate composed of an alternative or modified sugar with a removable blocking group attached to the 3' position of the sugar moiety. NTP-3'-O-RBG can also contain other modifications described herein, including, but not limited to, modifications at the 2' position, modifications to the nucleobase, and modifications to the phosphate. Nucleotides can also have a 3'-O-RBG, as expected after reaction of NTP-3'-O-RBG with the engineered TdT and oligoacceptor substrates of the present disclosure.

[0055] As used herein, "oligo acceptor product" and "growing oligonucleotide chain" and "oligo acceptor extension product" are used interchangeably herein and refer to the product of NTP-3'-O-RBG or other natural or modified NTP substrates with an oligo acceptor substrate, wherein TdT or a related polymerase has catalyzed the extension or addition of nucleotide-3'-O-RBG or other natural or modified nucleotide substrates to the oligo acceptor substrate via reaction with one or more NTP-3'-O-RBG or other natural or modified NTP substrates.

[0056] As used herein, "removable blocking group" and "blocking group," as well as "terminator group" and "reversible termination group" and "inhibitor group," and related variations of these terms, are used interchangeably herein and refer to a chemical group that will prevent the addition of a second NTP-3'-O-RBG or other natural or modified NTP substrate to the 3'-end of a growing oligo acceptor substrate chain prior to removal of the removable blocker from the first addition. In some embodiments, the NTP-3'-O-RBG or other natural or modified NTP substrate may comprise a removable blocking group selected from the group consisting of NTP-3'-O-NH or NTP-3'-O-PO. In some embodiments, the NTP-3'-O-RBG or other natural or modified NTP substrate may have a natural purine or pyrimidine base, such as adenine, guanine, cytosine, thymine, or uridine. In some embodiments, NTP-3'-O-RBG or other natural or modified NTP substrates may have a non-natural base analog, such as inosine, xanthine, hypoxanthine, or another base analog, as known in the art. In some embodiments, the blocking group may include a modification at the 2' position, or may additionally include a modification at the 2' position.

[0057] As used herein, "template-independent synthesis" refers to the synthesis of oligonucleotides or polynucleotides without using a template strand as a guide for the synthesis of complementary oligonucleotide or polynucleotide strands. Thus, template-independent synthesis refers to the iterative process in which nucleotides are successively added to a growing oligonucleotide or nucleotide strand or an acceptor substrate. Template-independent synthesis can be in a sequence-defined manner, or can be random, as is the case with wild-type TdT in generating antigen receptor diversity. The process for template-independent synthesis is further described herein.

[0058] "Coding sequence" refers to a portion of a nucleic acid (eg, a gene) that codes for the amino acid sequence of a protein.

[0059] "Naturally occurring" or "wild-type" refers to a form found in nature. For example, a naturally occurring or wild-type polypeptide or polynucleotide sequence is a sequence present in an organism that can be isolated from a natural source and has not been intentionally modified by human manipulation.

[0060] As used herein, "recombinant," "engineered," and "non-naturally occurring," when used in reference to a cell, nucleic acid, or polypeptide, refer to a material, or a material corresponding to the native or natural form of that material, that has been modified in some way and is not otherwise found in nature. In some embodiments, the cell, nucleic acid, or polypeptide is identical to a naturally occurring cell, nucleic acid, or polypeptide, but is produced or obtained from synthetic materials and / or by manipulation using recombinant techniques. Non-limiting examples include, among others, recombinant cells expressing genes not found in the native (non-recombinant) form of the cell, or expressing native genes that are expressed at different levels or in other ways.

[0061] "Percentage of sequence identity" and "percentage of homology" are used interchangeably herein to refer to a comparison between polynucleotides or polypeptides and are determined by comparing two optimally aligned sequences over a comparison window, where a portion of the polynucleotide or polypeptide sequence in the comparison window may contain additions or deletions (i.e., gaps) when compared to a reference sequence so that the two sequences are optimally aligned. The percentage can be calculated by determining the number of positions in both sequences where the same nucleic acid base or amino acid residue occurs to obtain the number of matching positions, dividing the number of matching positions by the total number of positions in the comparison window, and multiplying the result by 100 to obtain the percentage of sequence identity. 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 occurs, or where the nucleic acid base or amino acid residue is aligned with a gap, to obtain the number of matching positions, dividing the number of matching positions by the total number of positions in the comparison window, and multiplying the result by 100 to obtain the percentage of sequence identity. Those skilled in the art understand that there are many established algorithms available for aligning two sequences. Optimal alignment of sequences for comparison can be performed, for example, by the Smith and Waterman local homology algorithm (Smith and Waterman, Adv. Appl. Math., 2:482

[1981] ), the Needleman and Wunsch homology alignment algorithm (Needleman and Wunsch, J. Mol. Biol., 48:443

[1970] ), the Pearson and Lipman similarity search method (Pearson and Lipman, Proc. Natl. Acad. Sci. USA 85:2444

[1988] ), computerized implementations of these algorithms (e.g., GAP, BESTFIT, FASTA, and TFASTA in the GCG Wisconsin Software package), or by visual inspection as known in the art.Examples of algorithms suitable for determining percent sequence identity and percent sequence similarity include, but are not limited to, the BLAST and BLAST 2.0 algorithms described by Altschul et al. (See Altschul et al., J. Mol. Biol., 215:403-410

[1990] and Altschul et al., Nucl. Acids Res., 3389-3402

[1977] , respectively). Software for performing BLAST analyses is publicly available via the website of the National Center for Biotechnology Information. This algorithm involves first identifying high-scoring sequence pairs (HSPs) by identifying short strings of length W in a query sequence that match or meet some positive threshold score T when aligned with strings of the same length in a database sequence. T is referred to as the neighbor string score threshold (Altschul et al., supra). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are then extended in both directions along each sequence for as far as the cumulative alignment score can be increased. For nucleotide sequences, the cumulative score is calculated using the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatching residues; always <0). For amino acid sequences, a scoring matrix is ​​used to calculate the cumulative score. Extension of the word hits in each direction is halted when the cumulative alignment score drops by an amount X from its maximum achieved value, when the accumulation of one or more negatively scoring residue alignments causes the cumulative score to fall below 0, or when 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) uses as defaults a string length (W) of 11, an expectation (E) of 10, M=5, N=-4, and a comparison of both strands.For amino acid sequences, the BLASTP program uses as default a word length (W) of 3, an expectation (E) of 10, and the BLOSum62 scoring matrix (see Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89:10915

[1989] ). For exemplary sequence alignment and determination of percent sequence identity, the BESTFIT or GAP programs in the GCG Wisconsin Software package (Accelrys, Madison WI) can be used with the default parameters provided.

[0062] A "reference sequence" refers to a defined sequence used as a basis for sequence comparison. A reference sequence can be a subset of a larger sequence, for example, a segment of a full-length gene or polypeptide sequence. Generally, a reference sequence is at least 20 nucleotides or amino acid residues in length, at least 25 residues in length, at least 50 residues in length, or the entire length of the nucleic acid or polypeptide. Because two polynucleotide or amino acid sequences can each contain (1) similar sequences (i.e., portions of the complete sequence) between the two sequences and (2) additional sequences that differ between the two sequences, sequence comparison between two (or more) polynucleotides or polypeptides is typically performed by comparing the sequences of the two polynucleotides or polypeptides over 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, which may have one or more changes in the primary sequence. For example, "a reference sequence based on SEQ ID NO: 4 with a valine at the residue corresponding to X14," or X14V, refers to a reference sequence in which the corresponding residue at X14 in SEQ ID NO: 4, which is a tyrosine, has been changed to a valine.

[0063] A "comparison window" refers to a conceptual segment of at least about 20 contiguous nucleotide positions or amino acid residues, wherein a sequence can be compared to a reference sequence of at least 20 contiguous nucleotides or amino acids, and the portion of the sequence in the comparison window can contain no more than 20 percent additions or deletions (i.e., gaps) when compared to the reference sequence such that the two sequences are optimally aligned (no additions or deletions). The comparison window can be longer than 20 contiguous residues, and optionally includes windows of 30, 40, 50, 100, or longer.

[0064] As used herein, "substantial identity" refers to a polynucleotide or amino acid sequence having at least 80 percent sequence identity, at least 85 percent identity, at least 89-95 percent sequence identity, or more usually at least 99 percent sequence identity when compared to a reference sequence over a comparison window of at least 20 residue positions, often over a window of at least 30-50 residues, where the percentage of sequence identity is calculated by comparing the reference sequence to a sequence that contains deletions or additions totaling no more than 20 percent of the reference sequence over the comparison window. In certain embodiments, as applied to polypeptides, the term "substantial identity" means that two polypeptide sequences share at least 80 percent sequence identity, preferably at least 89 percent sequence identity, at least 95 percent sequence identity, or more (e.g., at least 99 percent sequence identity) when optimally aligned, such as by programs like GAP or BESTFIT using default gap weights. In some embodiments, non-identical residue positions in the compared sequences differ by conservative amino acid substitutions.

[0065] "Corresponding to," "with reference to," and "compared to," when used in the context of numbering a given amino acid sequence or polynucleotide sequence, refer to the numbering of residues in a particular reference sequence when the given amino acid sequence or polynucleotide sequence is compared to that reference sequence. In other words, the residue numbers or residue positions of a given polymer are specified with respect to the reference sequence, rather than the actual numerical positions of the residues in the given amino acid sequence or polynucleotide sequence. For example, a given amino acid sequence (such as that of an engineered TdT) can be aligned with a reference sequence by introducing gaps to optimize residue matches between the two sequences. In such cases, despite the presence of gaps, the numbering of residues in the given amino acid sequence or polynucleotide sequence is done with respect to the reference sequence to which it is being aligned.

[0066] An "amino acid difference" or "residue difference" refers to a change in an amino acid residue at a position in a polypeptide sequence relative to the amino acid residue at the corresponding position in a reference sequence. The position of an amino acid difference is generally referred to herein as "Xn," where n refers to the corresponding position in the reference sequence that is the basis for the residue difference. For example, a "residue difference at position X25 as compared to SEQ ID NO:2" refers to a change in the amino acid residue at the polypeptide position corresponding to position 25 of SEQ ID NO:2. Thus, if a reference polypeptide of SEQ ID NO:2 has a valine at position 25, then a "residue difference at position X25 as compared to SEQ ID NO:2" is an amino acid substitution of any residue other than valine at the polypeptide position corresponding to position 25 of SEQ ID NO:2. In most cases herein, a specific amino acid residue difference at a position will be designated as "XnY," where "Xn" identifies the corresponding position as described above and "Y" is a single-letter identifier for the amino acid found in the engineered polypeptide (i.e., the residue that differs from that in the reference polypeptide). In some embodiments, multiple amino acids may be found at a particular residue position (i.e., alternative amino acids may be written in the format XnY / Z, where Y and Z represent alternative amino acid residues).In some cases (e.g., Tables 5.1, 6.2, 7.2, 8.2, 9.2, 10.2, 11.2, 12.2, 13.2, 14.2, 15.2, 16.2, 17.2, 18.2, 19.2, 20.2, 21.2, 22.2, 23.2, 24.2, 25.2, 26.2, 26.3, 26.4, 27.2, 27.3, 27.4, 27.5, 28.1, 28.2, 28.3, 29.2, 30.2, 31.2, 32.2, 33.2, 34.2, 35.2, 36.2, 37.2, 38.2, 39.2, 40.2, 41.2, 42.2, 43.2, 44.2, 45.2, 46.2, 47.2, 48.2, 49. 2, 50.2, 51.2, 52.2, 53.2, 54.2, 55.2, 56.2, 56.3, 56.4, 61.2, 63.2, 64.2, 65.2, 66.2, 67.2, 68.2, 69.2, 70.2, 71.2, 72.2, 73.2, 74.2, 75.2, 76.2, 77.2, 78.2, 79.2, and 80.1), the present invention also provides specific amino acid differences, indicated by the conventional designation "AnB," where A is the single-letter identifier of the residue in the reference sequence, "n" is the number of the residue position in the reference sequence, and B is the single-letter identifier of the residue substitution in the sequence of the engineered polypeptide. Additionally, in some cases, the polypeptides of the present invention may contain one or more amino acid residue differences compared to the reference sequence, indicated by a list of the specific positions that are altered compared to the reference sequence. In some additional embodiments, the present invention provides engineered polypeptide sequences that include both conservative and non-conservative amino acid substitutions.

[0067] As used herein, "conservative amino acid substitution" refers to the replacement of a residue with a different residue having a similar side chain, and thus typically involves the replacement of an amino acid in a polypeptide with an amino acid within the same or similar defined class of amino acids. Non-limiting examples include an amino acid with an aliphatic side chain being replaced with another aliphatic amino acid (e.g., alanine, valine, leucine, and isoleucine), an amino acid with a hydroxyl side chain being replaced with another amino acid with a hydroxyl side chain (e.g., serine and threonine), an amino acid with an aromatic side chain being replaced with another amino acid with an aromatic side chain (e.g., phenylalanine, tyrosine, tryptophan, and histidine), an amino acid with a basic side chain being replaced with another amino acid with a basic side chain (e.g., lysine and arginine), an amino acid with an acidic side chain being replaced with another amino acid with an acidic side chain (e.g., aspartic acid or glutamic acid), and / or a hydrophobic or hydrophilic amino acid being replaced with another hydrophobic or hydrophilic amino acid, respectively. Table 1 below shows exemplary conservative substitutions. [Table 1]

[0068] "Non-conservative substitution" refers to the substitution of an amino acid in a polypeptide with an amino acid that has significantly different side chain properties. Non-conservative substitutions may use amino acids between, rather than within, a given group, and affect (a) the structure of the peptide backbone in the area of ​​the substitution (e.g., proline for glycine), (b) the charge or hydrophobicity, or (c) the bulk of the side chain. By way of non-limiting example, exemplary non-conservative substitutions may be the substitution of an acidic amino acid with a basic or aliphatic amino acid, the substitution of an aromatic amino acid with a small amino acid, and the substitution of a hydrophilic amino acid with a hydrophobic amino acid.

[0069] "Deletion" refers to the modification of a polypeptide by the removal of one or more amino acids from a reference polypeptide. Deletions can include removing 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, up to 10% or up to 20% of the total number of amino acids comprising the reference enzyme, while retaining enzymatic activity and / or improving the properties of the engineered TdT enzyme. Deletions can be directed to internal and / or terminal portions of the polypeptide. In various embodiments, deletions can include a continuous segment or can be discontinuous.

[0070] "Insertion" refers to the modification of a polypeptide by the addition of one or more amino acids from a reference polypeptide. In some embodiments, improved engineered TdT enzymes include the insertion of one or more amino acids into naturally occurring polypeptides and the insertion of one or more amino acids into other improved TdT polypeptides. Insertions can be internal to the polypeptide or can be to the carboxy- or amino-terminus. As used herein, insertions include fusion proteins known in the art. Insertions can be a contiguous segment of amino acids or can be separated by one or more of the amino acids in the naturally occurring polypeptide.

[0071] "Fragment," as used herein, refers to a polypeptide having an amino- and / or carboxy-terminal deletion, but the remaining amino acid sequence is identical to the corresponding positions in the sequence. Fragments can be at least 14 amino acids long, at least 20 amino acids long, at least 50 amino acids long, or more, and can be up to 70%, 80%, 90%, 95%, 98%, and 99% of the full-length TdT polypeptide (e.g., the polypeptide of SEQ ID NO: 2, or the TdT provided in the even-numbered sequences of SEQ ID NOs: 4-1960, 2004-3920, 4048-5466, and 5476).

[0072] An "isolated polypeptide" refers to a polypeptide that has been substantially separated from other contaminants that naturally accompany it (e.g., proteins, lipids, and polynucleotides). The term encompasses polypeptides that have been removed or purified from their natural environment or expression system (e.g., a host cell or in vitro synthesis). Engineered TdT enzymes can be present intracellularly, in cell culture medium, or prepared in various forms, such as a lysate or isolated preparation. Thus, in some embodiments, an engineered TdT enzyme can be an isolated polypeptide.

[0073] A "substantially pure polypeptide" refers to a composition in which the polypeptide species is the predominant species present (i.e., it is more abundant on a molar or weight basis than any other individual macromolecular species in the composition); generally, a composition is substantially purified when the target species constitutes at least about 50 percent of the macromolecular species present, by molar or weight percent. Generally, a substantially pure TdT composition comprises 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 macromolecular species present in the composition, by molar or weight percent. In some embodiments, the target species is purified to essential homogeneity (i.e., contaminants cannot be detected in the composition by conventional detection methods), and the composition consists essentially of a single macromolecular species. Solvent species, small molecules (<500 Daltons), and elemental ion species are not considered macromolecular species. In some embodiments, an isolated engineered TdT polypeptide is a substantially pure polypeptide composition.

[0074] As used herein, "improved enzymatic properties" refers to an improvement in at least one property of an enzyme. In some embodiments, the present invention provides engineered TdT polypeptides that exhibit an improved enzymatic property compared to a reference TdT polypeptide and / or a wild-type TdT polypeptide, and / or another engineered TdT polypeptide. For the engineered TdT polypeptides described herein, the comparison is generally made to the wild-type enzyme from which the TdT was derived, although in some embodiments, the reference enzyme can be another improved engineered TdT. Thus, the level of "improvement" can be determined and compared among various TdT polypeptides, including wild-type and engineered TdT. Improved properties include, but are not limited to, properties such as enzyme activity (which may be expressed in terms of percent conversion of substrate), thermostability, solvent stability, pH activity profile, cofactor requirements, resistance to inhibitors (e.g., substrate or product inhibition), activity at elevated temperatures, increased soluble expression, reduced by-product formation, increased specific activity towards NTP-3'-O-RBG substrate, increased incorporation efficiency in the extension of oligo acceptor substrates, and / or increased activity (including enantioselectivity) towards various oligo acceptor substrates.

[0075] "Increased enzyme activity" refers to an improvement in a property of a TdT polypeptide, which may be expressed by an increase in specific activity (e.g., product produced / time / weight of protein) or an increase in percent conversion of substrate to product (e.g., percent conversion of a starting amount of substrate to product in a specific time using a specific amount of TdT) compared to a reference TdT enzyme. Exemplary methods for determining enzyme activity are provided in the Examples. Any property associated with enzyme activity (such as the classical enzyme property K) whose change may lead to increased enzyme activity. m , V max , or k cat(including enzyme activity) may be affected. Improvements in enzyme activity may range from about 1.2-fold over the enzyme activity of the corresponding wild-type enzyme to 2-fold, 5-fold, 10-fold, 20-fold, 25-fold, 50-fold, or more enzyme activity compared to the naturally occurring TdT from which the TdT polypeptide is derived or another engineered TdT. TdT activity may be measured by any one of standard assays (such as monitoring changes in substrate, cofactor, or product properties). In some embodiments, the amount of product produced may be measured by liquid chromatography-mass spectrometry (LC-MS), HPLC, or other methods known in the art. Comparisons of enzyme activity are made using defined preparations of enzyme, defined assays under set conditions, and one or more defined substrates, as described in further detail herein. Generally, when lysates are compared, the same expression system and the same host cells are used to minimize variations in the abundance of host cell-produced enzyme in the lysates, in addition to determining the number of cells and amount of protein assayed.

[0076] "Conversion" refers to the enzymatic conversion of a substrate(s) to the corresponding product(s). "Percent conversion" refers to the percent of a substrate that is converted to a product under specified conditions and in a time period. Thus, the "enzyme activity" or "activity" of a TdT polypeptide can be expressed as the "percent conversion" of substrate to product.

[0077] "Thermostable" refers to the polypeptide maintaining similar activity (e.g., greater than 60% to 80%) after exposure to elevated temperatures (e.g., 40-80°C) for a period of time (e.g., 0.5 to 24 hours) as compared to the wild-type enzyme exposed to the same elevated temperature.

[0078] "Solvent-stable" refers to the polypeptide maintaining similar activity (e.g., greater than 60% to 80%) after exposure to various concentrations (e.g., 5% to 99%) of a solvent (e.g., ethanol, isopropyl alcohol, dimethyl sulfoxide (DMSO), tetrahydrofuran, 2-methyltetrahydrofuran, acetone, toluene, butyl acetate, methyl tert-butyl ether, etc.) for a period of time (e.g., 0.5 to 24 hours) as compared to the wild-type enzyme exposed to the same solvent at the same concentration.

[0079] "Heat and solvent stable" refers to a polypeptide that has both heat stability and solvent stability.

[0080] The term "stringent hybridization conditions" is used herein to refer to conditions under which nucleic acid hybrids are stable. As known to those skilled in the art, hybrid stability is determined by the melting temperature (T m ) Generally, hybrid stability is a function of ionic strength, temperature, G / C content, and the presence of chaotropic agents. mThe value can be calculated using known methods for predicting melting temperatures (e.g., Baldino et al., Meth. Enzymol., 168:761-777

[1989] ; Bolton et al., Proc. Natl. Acad. Sci. USA 48:1390

[1962] ; Bresslauer et al., Proc. Natl. Acad. Sci. USA 83:8893-8897

[1986] ; Freier et al., Proc. Natl. Acad. Sci. USA 83:9373-9377

[1986] ; Kierzek et al., Biochem., 25:7840-7846

[1986] ; Rychlik et al., 1990, Nucl. Acids Res., 18:6409-6412

[1990] (erratum, Nucl. Acids Res., 19:698

[1991] ; Sambrook et al. (ibid.); Suggs et al., 1981, in Developmental Biology Using Purified Genes, Brown et al. [eds.], pp. 683-693, Academic Press, Cambridge, MA

[1981] ; and Wetmur, Crit Rev. Biochem. Mol. Biol., 26:227-259

[1991] ). In some embodiments, the polynucleotide encodes a polypeptide disclosed herein and hybridizes under defined conditions (e.g., moderately stringent conditions or highly stringent conditions) to the complement of a sequence encoding an engineered TdT enzyme of the invention.

[0081] "Hybridization stringency" refers to hybridization conditions (such as washing conditions) in nucleic acid hybridization. Generally, hybridization reactions are performed under conditions of reduced stringency, followed by washing with varying degrees of stringency. The term "moderately stringent hybridization" refers to conditions that allow target DNA to bind to complementary nucleic acids that have about 60% identity with the target DNA, preferably about 75% identity, about 85% identity, or even more than about 90% identity with the target polynucleotide. Exemplary moderately stringent conditions are conditions equivalent to hybridization in 50% formamide, 5x Denhardt's solution, 5x SSPE, 0.2% SDS at 42°C, followed by washing in 0.2x SSPE, 0.2% SDS at 42°C. "High stringency hybridization" generally refers to hybridization that achieves a thermal melting temperature T determined under solution conditions for a specified polynucleotide sequence. m to about 10°C or less. In some embodiments, high stringency conditions refer to conditions that allow hybridization of only nucleic acid sequences that form stable hybrids in 0.018 M NaCl at 65°C (i.e., if a hybrid is not stable in 0.018 M NaCl at 65°C, it will not be stable under the high stringency conditions contemplated herein). High stringency conditions can be provided, for example, by hybridization under conditions equivalent to 50% formamide, 5x Denhardt's solution, 5x SSPE, and 0.2% SDS at 42°C, followed by a wash in 0.1x SSPE and 0.1% SDS at 65°C. Another high stringency condition is hybridization under conditions equivalent to hybridization in 5x SSC containing 0.1% (w:v) SDS at 65°C and a wash in 0.1x SSC containing 0.1% SDS at 65°C. Other high stringency and moderate stringency hybridization conditions are described in the references cited above.

[0082] A "heterologous" polynucleotide refers to any polynucleotide that is introduced into a host cell by laboratory techniques, and includes polynucleotides that are removed from a host cell, subjected to laboratory manipulation, and then reintroduced into the host cell.

[0083] "Codon optimization" refers to changing the codons of a polynucleotide encoding a protein to those preferentially used in a particular organism so that the encoded protein is efficiently expressed in the organism of interest. Although the genetic code is degenerate in that most amino acids are represented by a few codons, called "synonyms" or "synonymous" codons, it is well known that codon usage by a particular organism is not random but is biased toward certain codon triplets. This codon usage bias may be higher for a given gene, for genes of common function or ancestral origin, for highly expressed proteins compared to low-copy-number proteins, and for the collective protein-coding region of an organism's genome. In some embodiments, a polynucleotide encoding a TdT enzyme may be codon-optimized for optimal production from the host organism selected for expression.

[0084] As used herein, "preferred and optimal high codon usage biased codons" refer interchangeably to codons that are used more frequently in protein-coding regions than other codons that encode the same amino acid. Preferred codons can be determined in relation to codon usage in a single gene, codon usage in a set of genes of common function or origin, codon usage in highly expressed genes, codon frequency in the collective protein-coding regions of an entire organism, codon frequency in the collective protein-coding regions of related organisms, or a combination thereof. Codons whose frequency increases with the level of gene expression are typically optimal codons for expression. A variety of methods are known for determining codon frequencies (e.g., codon usage, relative synonymous codon usage) and codon preferences in a particular organism, including multivariate analyses (e.g., using cluster analysis or correspondence analysis), as well as the number of effective codons used in a gene (see, e.g., GCG CodonPreference, Genetics Computer Group Wisconsin Package; CodonW, Peden, University of Nottingham; McInerney, Bioinform., 14:372-73

[1998] ; Stenico et al., Nucl. Acids Res., 222437-46

[1994] ; Wright, Gene 87:23-29

[1990] ). Codon usage tables are available for many different organisms (see, e.g., Wada et al., Nucl. Acids Res., 20:2111-2118

[1992] ; Nakamura et al., Nucl. Acids Res., 28:292

[2000] ; Duret, et al. (ibid.); Henaut and Danchin, in Escherichia coli and Salmonella, Neidhardt, et al. (eds.), ASM Press, Washington DC, pp. 2047-2066

[1996] ). The data source for obtaining codon usage can be any available nucleotide sequence capable of encoding a protein.Such datasets include nucleic acid sequences that are actually known to encode expressed proteins (e.g., complete protein-coding sequences - CDS), expressed sequence tags (ESTS), or predicted coding regions of genomic sequences (see, e.g., Mount, Bioinformatics: Sequence and Genome Analysis, Chapter 8, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY

[2001] ; Uberbacher, Meth. Enzymol., 266:259-281

[1996] ; and Tiwari et al., Comput. Appl. Biosci., 13:263-270

[1997] ).

[0085] "Control sequences" is defined herein to include all components necessary or advantageous for the expression of polynucleotides and / or polypeptides of the present invention. Each control sequence may be native or foreign to the nucleic acid sequence encoding the polypeptide. Such control sequences include, but are not limited to, a leader, polyadenylation sequence, propeptide sequence, promoter, signal peptide sequence, and transcription terminator. At a minimum, control sequences include a promoter, and transcriptional and translational stop signals. Control sequences may be provided with linkers for the purpose of introducing specific restriction sites facilitating ligation of the control sequences with the coding region of the nucleic acid sequence encoding the polypeptide.

[0086] "Operably linked" is defined herein as a configuration in which the control sequences are positioned in an appropriate position (i.e., in functional relationship) with a polynucleotide of interest such that expression of the polynucleotide and / or polypeptide of interest is directed or regulated by the control sequences.

[0087] A "promoter sequence" refers to a nucleic acid sequence recognized by a host cell to direct expression of a polynucleotide of interest, such as a coding sequence. The promoter sequence comprises transcriptional control sequences that mediate expression of the polynucleotide of interest. The promoter may be any nucleic acid sequence that exhibits transcriptional activity in the host cell of choice, including mutant promoters, truncated promoters, and hybrid promoters, and may be derived from a gene encoding an extracellular or intracellular polypeptide that is either homologous or heterologous to the host cell.

[0088] "Suitable reaction conditions" refer to conditions in a biocatalytic reaction solution (e.g., ranges for enzyme loading, substrate loading, cofactor loading, temperature, pH, buffers, cosolvents, etc.) that allow a TdT polypeptide of the invention to convert one or more substrate compounds into product compounds (e.g., addition of nucleotide-3'-O-RBG or other natural or modified nucleotide substrates to an oligo acceptor substrate via reaction with NTP-3'-O-RBG or other natural or modified NTP substrates). Exemplary "suitable reaction conditions" are provided herein and illustrated by the Examples.

[0089] "Composition" refers to a mixture or combination of one or more substances, where each substance or component of the composition retains its individual properties. As used herein, a biocatalyst composition refers to a combination of one or more substances useful in biocatalysis.

[0090] "Loading," as in "compound loading" or "enzyme loading" or "cofactor loading," refers to the concentration or amount of a component in the reaction mixture at the start of the reaction.

[0091] "Substrate," in the context of a biocatalyst-mediated process, refers to the compound or molecule on which the biocatalyst acts. For example, the TdT biocatalyst used in the synthetic processes disclosed herein acts on an NTP-3'-O-RBG substrate or other natural or modified NTP substrates, and an oligo acceptor substrate.

[0092] "Product" in the context of a biocatalyst-mediated process refers to a compound or molecule that results from the action of the biocatalyst. For example, exemplary products of the TdT biocatalyst used in the processes disclosed herein are the oligo acceptor extension products shown in Schemes 1 and 2.

[0093] "Alkyl" refers to a saturated hydrocarbon group of 1 to 18 carbon atoms (inclusive), more preferably 1 to 8 carbon atoms (inclusive), and most preferably 1 to 6 carbon atoms (inclusive), which is either straight-chained or branched. A specific number of carbon atoms for an alkyl is indicated in parentheses (e.g., (C1-C6) alkyl refers to an alkyl of 1 to 6 carbon atoms).

[0094] "Alkenyl" refers to a hydrocarbon group of 2 to 12 carbon atoms (inclusive) that is either straight or branched and contains at least one double bond, but optionally contains multiple double bonds.

[0095] "Alkynyl" refers to a hydrocarbon group of 2 to 12 carbon atoms (inclusive), either straight or branched, containing at least one triple bond, but optionally containing multiple triple bonds, and optionally further containing one or more double bond moieties.

[0096] "Heteroalkyl," "heteroalkenyl," and "heteroalkynyl" refer to alkyl, alkenyl, and alkynyl, respectively, as defined herein, in which one or more of the carbon atoms are independently replaced with the same or different heteroatoms or heteroatomic groups. The heteroatoms and / or heteroatomic groups that may replace the carbon atoms include, but are not limited to, -O-, -S-, -SO-, -NR γ -, -PH-, -S(O)-, -S(O)2-, -S(O)NR γ -, -S(O)NR γ and the like (including combinations thereof), wherein each R γare independently selected from hydrogen, alkyl, cycloalkyl, heterocycloalkyl, aryl, and heteroaryl.

[0097] "Amino" refers to the group -NH2. Substituted amino is -NHR η , N.R. η R η , and NR η R η R η In the formula, each R η are independently selected from substituted or unsubstituted alkyl, cycloalkyl, cycloheteroalkyl, alkoxy, aryl, heteroaryl, heteroarylalkyl, acyl, alkoxycarbonyl, sulfanyl, sulfinyl, sulfonyl, and the like. Exemplary amino groups include, but are not limited to, dimethylamino, diethylamino, trimethylammonium, triethylammonium, methylsulfonylamino, furanyl-oxy-sulfamino, and the like.

[0098] "Aminoalkyl" refers to an alkyl group in which one or more of the hydrogen atoms has been replaced with one or more amino groups (including substituted amino groups).

[0099] "Aminocarbonyl" refers to -C(O)NH2. Substituted aminocarbonyl is -C(O)NR η R η In the formula, NR η R η The amino group is as defined herein.

[0100] "Oxy" refers to the divalent group --O--, which can have a variety of substituents to form different oxy groups, including ethers and esters.

[0101] "Alkoxy" or "alkyloxy" are used interchangeably herein and refer to -OR ζ In the formula, R ζ is an alkyl group (including optionally substituted alkyl groups).

[0102] "Carboxy" refers to --COOH.

[0103] "Carbonyl" refers to -C(O)-, which can have a variety of substituents to form different carbonyl groups including acids, acid halides, aldehydes, amides, esters, and ketones.

[0104] "Carboxyalkyl" refers to an alkyl in which one or more of the hydrogen atoms has been replaced with one or more carboxy groups.

[0105] "Aminocarbonylalkyl" refers to an alkyl substituted with an aminocarbonyl group, as defined herein.

[0106] "Halogen" or "halo" refers to fluoro, chloro, bromo, and iodo.

[0107] "Haloalkyl" refers to an alkyl group in which one or more of the hydrogen atoms has been replaced with a halogen. Thus, the term "haloalkyl" is intended to include monohaloalkyl, dihaloalkyl, trihaloalkyl, etc., up to perhaloalkyl. For example, the expression "(C1-C2)haloalkyl" includes 1-fluoromethyl, difluoromethyl, trifluoromethyl, 1-fluoroethyl, 1,1-difluoroethyl, 1,2-difluoroethyl, 1,1,1 trifluoroethyl, perfluoroethyl, etc.

[0108] "Hydroxy" refers to --OH.

[0109] "Hydroxyalkyl" refers to an alkyl group in which one or more of the hydrogen atoms has been replaced with one or more hydroxy groups.

[0110] A "thiol" or "sulfanyl" refers to -SH. A substituted thiol or substituted sulfanyl is -SR. η In the formula, R η is an alkyl, aryl, or other suitable substituent.

[0111] "Sulfonyl" refers to -SO2-. Substituted sulfonyl is -SO2-R η In the formula, R η is an alkyl, aryl, or other suitable substituent.

[0112] "Alkylsulfonyl" is -SO2-R ζ In the formula, R ζ is alkyl, which may be optionally substituted. Typical alkylsulfonyl groups include, but are not limited to, methylsulfonyl, ethylsulfonyl, n-propylsulfonyl, and the like.

[0113] "Phosphate," as used herein, refers to a functional group composed of an orthophosphate ion (a phosphorus atom covalently linked to four oxygen atoms), which is commonly found with one or more hydrogen atoms or organic groups.

[0114] "Phosphorylation," as used herein, refers to the addition or presence of one or more phosphoryl groups (a phosphorus atom covalently linked to three oxygen atoms).

[0115] "Optionally substituted," as used herein with respect to the aforementioned chemical groups, means that a position in the chemical group occupied by a hydrogen is replaced with another atom (exemplified, but not limited to, by carbon, oxygen, nitrogen, or sulfur (unless otherwise specified)) or a chemical group (including, but not limited to, hydroxy, oxo, nitro, methoxy, ethoxy, alkoxy, substituted alkoxy, trifluoromethoxy, haloalkoxy, fluoro, chloro, bromo, iodo, halo, methyl, ethyl, propyl, butyl, alkyl, alkenyl, alkynyl, substituted alkyl, trifluoromethyl, haloalkyl, hydroxyalkyl, alkoxyalkyl, thio, alkylthio, acyl, carboxy, alkoxycarbonyl, carboxamido, substituted carboxamido, alkylsulfonyl, alkylsulfinyl, alkylsulfonylamino, sulfonamido, substituted sulfonamido, "Cyano" means optionally substituted with an alkyl group (exemplified by cyano, amino, substituted amino, alkylamino, dialkylamino, aminoalkyl, acylamino, amidino, amidoximo, hydroxamoyl, phenyl, aryl, substituted aryl, aryloxy, arylalkyl, arylalkenyl, arylalkynyl, pyridyl, imidazolyl, heteroaryl, substituted heteroaryl, heteroaryloxy, heteroarylalkyl, heteroarylalkenyl, heteroarylalkynyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloalkyl, cycloalkenyl, cycloalkylalkyl, substituted cycloalkyl, cycloalkyloxy, pyrrolidinyl, piperidinyl, morpholino, heterocyclo, (heterocyclo)oxy, and (heterocyclo)alkyl), where preferred heteroatoms are oxygen, nitrogen, and sulfur.Furthermore, if open valences exist on these substituted chemical groups, they may be further substituted with alkyl, cycloalkyl, aryl, heteroaryl, and / or heterocyclic groups; if these open valences exist on carbon, they may be further substituted with halogens, and with oxygen-, nitrogen-, or sulfur-linked substituents; if multiple such open valences exist, these groups may join together to form a ring, either by direct bond formation or by the formation of a bond to a new heteroatom, preferably oxygen, nitrogen, or sulfur. It is further contemplated that the above substitutions may be made provided that the replacement of hydrogen with a substituent does not introduce unacceptable instability into the molecules of the invention and is chemically reasonable in a manner that does not. Those skilled in the art will understand that with respect to any chemical group described as optionally substituted, only sterically practical and / or synthetically feasible chemical groups are intended to be included. "Optionally substituted," as used herein, refers to all subsequent phrases in a term or series of chemical groups. For example, in the term "optionally substituted arylalkyl," the "alkyl" and "aryl" portions of the molecule can be substituted or unsubstituted, and in the series "optionally substituted alkyl, cycloalkyl, aryl, and heteroaryl," the alkyl, cycloalkyl, aryl, and heteroaryl groups can be substituted or unsubstituted, independently of the others.

[0116] "Reaction," as used herein, refers to a process in which one or more substances or compounds or substrates are converted into one or more different substances, compounds, or processes.

[0117] Template-independent synthesis with engineered TdT

[0118] New methods for efficiently synthesizing highly pure strands of DNA, RNA, and other polynucleotides are needed to overcome the limitations of existing phosphoramidite chemical synthesis methods and enable a wide range of new and existing synthetic biology applications.

[0119] The present invention provides novel terminal deoxynucleotidyl transferases with improved activity for the template-independent synthesis of polynucleotides using 5'-nucleoside triphosphates ("NTPs") modified with a 3'-O-removable blocking group (NTP-3'-O-RBG) or other natural or modified NTP substrates. The TdTs of the present disclosure have improved thermostability, improved activity at high temperatures, increased soluble expression or isolated protein yield, reduced by-product formation, increased affinity for NTP-3'-O-RBG and other natural or modified NTP substrates, increased affinity for oligoacceptor substrates, increased activity or specific activity for NTP-3'-O-RBG and other natural or modified NTP substrates, and / or increased activity or specific activity for various oligoacceptor substrates, compared to wild-type TdT or other TdTs or template-independent polymerases known to those skilled in the art. The engineered polypeptides of the present disclosure are variants of SEQ ID NO: 2 (a predicted splice variant encoded by the genome of Monodelphis domestica). These engineered TdTs are capable of template-independent synthesis of oligonucleotides and polynucleotides.

[0120] Template-independent synthesis of defined polynucleotide sequences using engineered TdT is a multi-step process. In one embodiment, an oligo acceptor substrate bearing a 3'-OH allows for the addition of a defined modified NTP substrate (in this example, NTP-3'-O-RBG) by the engineered TdT, as shown in Scheme 1 below.

[0121] [ka] After reaction of NTP-3'-O-RBG with the 3'-OH of the oligoacceptor substrate or growing polynucleotide chain, TdT is blocked from further reaction by the 3'-O-RBG. The RBG is then removed, exposing the 3'-OH and allowing another round of addition. After each round of addition, the blocking group of the nucleotide-3'-O-RBG or natural or modified nucleotide from the previous round is removed, and a new NTP-3'-O-RBG or natural or modified NTP substrate is added, thereby continuously and efficiently creating a defined polynucleotide sequence by addition at the 3'-OH terminus of the polynucleotide or oligoacceptor substrate without a complementary strand or template primer sequence. After synthesis of the defined polynucleotide is complete, the oligonucleotide chain can be cleaved or released from the oligoacceptor substrate.

[0122] As can be envisioned by those skilled in the art, various oligo acceptor substrates and NTP-3'-O-RBG or natural or modified NTP substrates can be used in this process. Scheme 2 below details one example of a reaction. Scheme 2 illustrates the reaction of 5'-6-FAM-[N] as described in Example 27. 15 Although the TdT-catalyzed reaction of AT*mC with 3'-phos-mATP is shown, other examples include other suitable oligo acceptor substrates paired with NTP-3'-O-RBG or natural or modified NTPs. These examples are non-limiting.

[0123] [ka] Occasionally, unwanted synthesis products are created by TdT during the addition step, including incorporation of an NTP missing a blocking group, addition of multiple NTPs, or excision or pyrophosphorolysis of the growing polynucleotide chain by TdT.

[0124] In some embodiments, one or more additional quality control steps (such as adding an exonuclease) are performed before removing the blocking group and initiating a new round of synthesis. In some embodiments, a phosphatase (such as pyrophosphatase) is used to degrade inorganic phosphate and drive the reversible TdT reaction toward synthesis.

[0125] As further described herein, the engineered TdT polypeptides of the present disclosure exhibit, among other things, improved properties in the template-independent polynucleotide synthesis process shown in Schemes 1 and 2.

[0126] In some embodiments, the invention provides engineered TdT polypeptides comprising an amino acid sequence having at least 60% sequence identity to the amino acid reference sequence of SEQ ID NO:2 and further comprising one or more amino acid residue differences compared to the reference amino acid sequence, wherein the engineered TdT polypeptides have improved thermostability, increased activity at high temperatures, increased soluble expression or isolated protein yield, reduced by-product formation, increased specific activity toward NTP-3'-O-RBG or natural or modified NTP substrates, and / or increased activity toward various oligo acceptor substrates compared to wild-type TdT or other TdTs or template-independent polymerases known to those of skill in the art.

[0127] Specifically, the engineered TdT polypeptides of the present disclosure are engineered to efficiently synthesize polynucleotides having defined sequences using NTP-3'-O-RBG or natural or modified NTP substrates in the above process.

[0128] A variety of suitable reaction conditions will be known to those skilled in the art, as detailed below and in the Examples.

[0129] Engineered terminal deoxynucleotidyl transferase (TdT) polypeptides

[0130] The present invention provides engineered terminal deoxynucleotidyl transferase (TDT) polypeptides useful in template-independent polynucleotide synthesis using NTP-3'-O-RBG or natural or modified NTP substrates, as well as compositions and methods utilizing such engineered polypeptides in template-independent oligonucleotide synthesis.

[0131] The present invention provides TdT polypeptides, polynucleotides encoding the polypeptides, methods of preparing the polypeptides, and methods for using the polypeptides. Where a description is made of a polypeptide, it is understood that it may also be made of a polynucleotide encoding the polypeptide.

[0132] Suitable reaction conditions for achieving the desired reaction with the improved properties of the engineered polypeptide described above can be determined with respect to conditions including the concentration or amount of polypeptide, substrate, co-substrate, buffer, solvent, pH, temperature and reaction time, and / or conditions under which the polypeptide is immobilized on a solid support, as further described below and in the Examples.

[0133] In some embodiments, exemplary engineered TdTs are listed in Table 5.1, 6.2, 7.2, 8.2, 9.2, 10.2, 11.2, 12.2, 13.2, 14.2, 15.2, 16.2, 17.2, 18.2, 19.2, 20.2, 21.2, 22.2, 23.2, 24.2, 25.2, 26.2, 26.3, 26.4, 27.2, 27.3, 27.4, 27.5, 28.1, 28.2, 28.3, 29.2, 30.2, 31.2, 32.2, 33.2, 34.2, 35.2, 36.2, 37.2, 38.2, 39.2, 40.2 , 41.2, 42.2, 43.2, 44.2, 45.2, 46.2, 47.2, 48.2, 49.2, 50.2, 51.2, 52.2, 53.2, 54.2, 55.2, 56.2, 56.3, 56.4, 61.2, 63.2, 64.2, 65.2, 66.2, 67.2, 68.2, 69.2, 70.2, 71.2, 72.2, 73.2, 74.2, 75.2, 76.2, 77.2, 78.2, 79.2, and 80.1, and the amino acid sequences include those having one or more residue differences as compared to SEQ ID NO:2 at the residue positions set forth in SEQ ID NO:2.

[0134] Structural and functional information for exemplary engineered polypeptides of the invention is based on the conversion of oligo acceptor substrates and NTP-3'-O-RBG or dideoxy NTPs (e.g., 2',3'-dideoxy NTPs), and the results are shown in Tables 5.1, 6.2, 7.2, 8.2, 9.2, 10.2, 11.2, 12.2, 13.2, 14.2, 15.2, 16.2, 17.2, 18.2, 19.2, 20.2, 21.2, 22.2, 23.2, 24.2, 25.2, 26.2, 26.3, 26.4, 27.2, 27.3, 27.4, 27.5, 28.1, 28.2, 28.3, 29.2, 30.2, 31.3, 32.4, 33.5, 34.6, 35.7, 36.8, 37.9, 38.1, 38.2, 39.3, 40.4, 41.5, 42.6, 43.7, 44.8, 45.9, 46.1, 47.1, 48.1, 49.2, 50.3, 51.4, 52.1, 53.1, 54.1, 55.1, 56.1, 57.1, 58.1, 59.2, 60.2, 61.3, 62.1, 63.1, 64.1, 65.1, 66.1, 67.1, 68.1, 69.2, 70.2 31.2, 32.2, 33.2, 34.2, 35.2, 36.2, 37.2, 38.2, 39.2, 40.2, 41.2, 42.2, 43.2, 44.2, 45.2, 46.2, 47.2, 48.2, 49.2, 50.2, 51.2, 52.2, 53.2, 54.2, 55.2, 56.2, 56. The amino acid sequences of the nucleotide ... Amino acid residue differences are based on comparison to the reference sequence of SEQ ID NOs: 2, 8, 16, 24, 36, 268, 648, 660, 882, 1100, 1336, 1348, 1596, 1654, 1830, 1950, 2008, 2254, 2514, 2524, 2638, 2804, 2812, 2956, 3174, 3222, 3670, 3674, 3796, 3870, 3918, 4266, 4442, 4654, 4850, 4856, 4904, 5002, 5028, 5192, and / or 5246, as indicated.

[0135] Terminal deoxynucleotidyl transferases, members of the Pol X family, have been identified in many species. Diverse Pol X family members are known to share certain residues, which are conserved among family members. TdT also has a high level of conservation among species for residues thought to be involved in divalent metal ion binding, ternary complex formation, and dNTP and DNA ligand binding (Dominguez et al. (2000). EMBO, 19(7), 1731-1742). Furthermore, TdT is known to have splice variants that are N-terminally truncated and lack the BRCT domain. Other template-independent polymerases, including but not limited to polyA polymerase, polyU polymerase, and terminal uridyl transferase, are known in the art and can be used in the practice of the present invention. Similarly, other polymerases capable of template-independent synthesis, including but not limited to reverse transcriptase, are known and can be used in the practice of the present invention.

[0136] Wild-type TdT from Monodelphis domestica (SEQ ID NO: 2) was selected for evolution. The TdT polypeptide of the present disclosure is an engineered variant of SEQ ID NO: 2 with a 6-histidine tag at the N-terminus.

[0137] The polypeptides of the present disclosure have residue differences that provide improved properties necessary for the development of efficient TdT enzymes capable of template-independent synthesis of polynucleotides having defined sequences. The various residue differences, both at conserved and non-conserved positions, have been discovered to be associated with improved properties of the enzyme, including improved thermostability, increased activity at high temperatures, increased soluble expression or isolated protein yield, reduced by-product formation, increased specific activity toward NTP-3'-O-RBG or natural or modified NTP substrates, increased incorporation efficiency in the extension of oligo acceptor substrates, and / or increased activity toward various oligo acceptor substrates, compared to wild-type TdT or other TdTs or template-independent polymerases known to those of skill in the art. In some embodiments, the engineered TdT polypeptides exhibit an increase in incorporation efficiency of greater than 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% upon extension of an oligo acceptor substrate by addition of NTPs or NQPs. Exemplary incorporation efficiencies of engineered TdTs are provided in the Examples (e.g., Example 92).

[0138] The activity of each engineered TdT compared to the reference polypeptides of SEQ ID NOs: 2, 8, 16, 24, 36, 268, 648, 660, 882, 1100, 1336, 1348, 1596, 1654, 1830, 1950, 2008, 2254, 2514, 2524, 2638, 2804, 2812, 2956, 3174, 3222, 3670, 3674, 3796, 3870, 3918, 4266, 4442, 4654, 4850, 4856, 4904, 5002, 5028, 5192, and / or 5246 was determined as percent conversion of the substrate as described in the Examples herein. In some embodiments, shake flask-derived purified enzyme (SFP) was used to evaluate the properties of engineered TdT, the results of which are provided in the Examples.

[0139] In some embodiments, particular enzymatic properties are associated with residue differences at residue positions set forth herein compared to SEQ ID NOs: 2, 8, 16, 24, 36, 268, 648, 660, 882, 1100, 1336, 1348, 1596, 1654, 1830, 1950, 2008, 2254, 2514, 2524, 2638, 2804, 2812, 2956, 3174, 3222, 3670, 3674, 3796, 3870, 3918, 4266, 4442, 4654, 4850, 4856, 4904, 5002, 5028, 5192, and / or 5246. In some embodiments, residue differences that affect polypeptide expression can be used to increase expression of the engineered TdT.

[0140] Given the guidance provided herein, SEQ ID NOS: 2, 8, 16, 24, 36, 268, 648, 660, 882, 1100, 1336, 1348, 1596, 1654, 1830, 1950, 2008, 2254, 2514, 2524, 2638, 2804, 2812, 2956, 3174, 3222, 3670, 3674, 3796, 3870, 3918, 4266, 4442, 4654, 4850, 4856, 49 Any of the exemplary engineered polypeptides comprising the sequences of Tables 5.1, 6.2, 7.2, 8.2, 9.2, 10.2, 11.2, 12.2, 13.2, 14.2, 15.2, 16.2, 17.2, 18.2, 19.2, 20.2, 21.2, 22.2, 23.2, 24.2, 25.2, 26.2, 26.3, 26.4, 27.2, 27.3, 27.4, 28.5, 29.6, 30.7, 30.8, 31.9, 32.10, 32.11, 32.12, 33.13, 33.14, 33.15, 33.16, 33.17, 33.18, 33.19, 34.2, 34.2, 35.2, 35.2, 36.2, 36.3, 36.4, 37.2, 37.3, 37.4, 37.5, 37.6, 37.7, 37.8, 37.9, 38.10, 38.11, 38.12, 38.13, 38.14, 38.15, 38.16, 38.17, 38.18, 38.19, 39.20, 39.21, 39.22, 39.23, 39.24, 39.25, 39.26, 39.27, 39.28, 39.29, 40.20, 40 , 27.5, 28.1, 28.2, 28.3, 29.2, 30.2, 31.2, 32.2, 33.2, 34.2, 35.2, 36.2, 37.2, 38.2, 39.2, 40.2, 41.2, 42.2, 43.2, 44.2, 45.2, 46.2, 47.2, 48.2, 49.2, 50.2, 51.2, 52.2, 53.2, 54.2, 55.2, 56.2, 56.3, 56.4, 61.2, 63.2, 64.2, 65.2 , 66.2, 67.2, 68.2, 69.2, 70.2, 71.2, 72.2, 73.2, 74.2, 75.2, 76.2, 77.2, 78.2, 79.2, and 80.1, as well as other polypeptides at other residue positions described herein. Further improvements may occur by including amino acid differences at residue positions that were maintained as invariant through previous evolutionary rounds.

[0141] In some embodiments, the engineered TdT polypeptide has increased soluble protein expression and has at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reference sequence SEQ ID NO:2 and is selected from the group consisting of 80 / 106 / 121 / 185 / 190 / 205 / 289 / 290 / 293 / 313 / 315 / 336 / 342 / 359 / 391 / 470 / 474 / 499 / 522 / 523, 80 / 106 / 121 / 185 / 190 / 205 / 289 / 290 / 293 / 313 / 315 / 336 / 342 / 359 / 391 / 470 / 474 / 499 / 522 / 523, 80 / 106 / 121 / 185 / / 190 / 205 / 289 / 290 / 293 / 313 / 342 / 470 / 474 / 499 / 523, 80 / 106 / 121 / 185 / 190 / 244 / 289 / 290 / 293 / 307 / 342 / 359 / 470 / 474 / 499, 80 / 121 / 131 / 185 / 190 / 205 / 244 / 289 / 290 / 293 / 313 / 315 / 336 / 342 / 359 / 391 / 414 / 470 / 474 / 499 / 522 / 523, 80 / 121 / 131 / 185 / 190 / 205 / 244 / 289 / 290 / 293 / 313 / 336 / 342 / 359 / 391 / 414 / 470 / 474 / 499 / 522 / 523, 80 / 121 / 131 / 185 / 190 / 289 / 290 / 293 / 313 / 342 / 470 / 474 / 499 / 522 / 523, 80 / 121 / 174 / 179 / 185 / 190 / 236 / 244 / 288 / 2 89 / 290 / 293 / 313 / 315 / 317 / 336 / 342 / 359 / 363 / 391 / 394 / 408 / 426 / 462 / 470 / 474 / 499 / 522 / 523, 80 / 121 / 174 / 185 / 186 / 190 / 236 / 244 / 273 / 284 / 288 / 2 89 / 290 / 293 / 313 / 315 / 317 / 336 / 342 / 352 / 359 / 391 / 394 / 395 / 419 / 428 / 431 / 462 / 470 / 474 / 499 / 522 / 523, 80 / 121 / 174 / 185 / 190 / 193 / 196 / 244 / 273 / 2 84 / 288 / 289 / 290 / 293 / 297 / 313 / 315 / 317 / 324 / 336 / 342 / 352 / 359 / 376 / 380 / 391 / 394 / 401 / 415 / 419 / 428 / 431 / 435 / 441 / 462 / 470 / 474 / 499 / 522 / 523,80 / 121 / 174 / 185 / 190 / 193 / 244 / 273 / 284 / 288 / 289 / 290 / 293 / 297 / 313 / 315 / 317 / 336 / 342 / 352 / 359 / 391 / 394 / 415 / 419 / 428 / 431 / 462 / 470 / 474 / 499 / 522 / 523、80 / 121 / 174 / 185 / 190 / 196 / 244 / 266 / 273 / 284 / 288 / 289 / 290 / 293 / 313 / 315 / 317 / 324 / 336 / 342 / 352 / 359 / 391 / 394 / 397 / 401 / 419 / 428 / 431 / 462 / 470 / 474 / 499 / 522 / 523、80 / 121 / 174 / 185 / 190 / 236 / 244 / 273 / 282 / 284 / 288 / 289 / 290 / 293 / 313 / 315 / 317 / 336 / 342 / 352 / 359 / 391 / 394 / 395 / 419 / 428 / 431 / 462 / 470 / 474 / 499 / 522 / 523、80 / 121 / 174 / 185 / 190 / 244 / 273 / 284 / 288 / 289 / 290 / 293 / 313 / 315 / 317 / 336 / 342 / 352 / 359 / 391 / 394 / 419 / 428 / 431 / 462 / 470 / 474 / 499 / 522 / 523、80 / 121 / 174 / 185 / 190 / 244 / 273 / 284 / 288 / 289 / 290 / 293 / 313 / 315 / 317 / 336 / 342 / 359 / 391 / 394 / 428 / 431 / 462 / 470 / 474 / 499 / 522 / 523、80 / 121 / 174 / 185 / 190 / 244 / 284 / 288 / 289 / 290 / 293 / 313 / 315 / 317 / 336 / 342 / 352 / 359 / 391 / 394 / 419 / 428 / 431 / 462 / 470 / 474 / 499 / 522 / 523、80 / 121 / 174 / 185 / 190 / 244 / 284 / 288 / 289 / 290 / 293 / 313 / 315 / 317 / 336 / 342 / 352 / 359 / 391 / 394 / 428 / 431 / 462 / 470 / 474 / 499 / 522 / 523、80 / 121 / 174 / 185 / 190 / 244 / 284 / 288 / 289 / 290 / 293 / 313 / 315 / 317 / 336 / 342 / 359 / 391 / 394 / 428 / 431 / 462 / 470 / 474 / 499 / 522 / 523、80 / 121 / 185 / 190 / 196 / 244 / 289 / 290 / 293 / 313 / 315 / 317 / 336 / 342 / 359 / 391 / 470 / 474 / 499 / 522 / 523、80 / 121 / 185 / 190 / 201 / 289 / 290 / 293 / 313 / 342 / 470 / 474 / 499 / 522、80 / 121 / 185 / 190 / 244 / 273 / 289 / 290 / 293 / 313 / 315 / 317 / 336 / 342 / 352 / 359 / 391 / 419 / 435 / 470 / 474 / 499 / 522 / 523、80 / 121 / 185 / 190 / 244 / 289 / 290 / 293 / 300 / 313 / 315 / 317 / 336 / 342 / 359 / 391 / 470 / 474 / 499 / 522 / 523、80 / 121 / 185 / 190 / 244 / 289 / 290 / 293 / 313 / 315 / 317 / 336 / 342 / 359 / 380 / 391 / 401 / 419 / 470 / 474 / 499 / 522 / 523、80 / 121 / 185 / 190 / 244 / 289 / 290 / 293 / 313 / 315 / 317 / 336 / 342 / 359 / 391 / 392 / 470 / 474 / 499 / 522 / 523、80 / 121 / 185 / 190 / 244 / 289 / 290 / 293 / 313 / 315 / 317 / 336 / 342 / 359 / 391 / 395 / 470 / 474 / 499 / 522 / 523、80 / 121 / 185 / 190 / 244 / 289 / 290 / 293 / 313 / 315 / 317 / 336 / 342 / 359 / 391 / 470 / 474 / 499 / 522 / 523、80 / 121 / 185 / 190 / 244 / 289 / 290 / 293 / 313 / 336 / 342 / 359 / 391 / 414 / 470 / 474 / 499 / 522 / 523、80 / 121 / 185 / 190 / 289 / 290 / 293 / 313 / 315 / 336 / 342 / 359 / 391 / 414 / 470 / 474 / 499 / 522 / 523、80 / 121 / 185 / 190 / 289 / 290 / 293 / 313 / 336 / 342 / 359 / 391 / 470 / 474 / 499 / 522 / 523、80 / 121 / 185 / 190 / 289 / 290 / 293 / 313 / 342 / 499、80 / 121 / 185 / 315、80 / 121 / 190 / 289 / 290、80 / 185 / 236 / 289 / 293、121 / 185 / 190 / 213 / 289 / 290 / 293、and 185 / 289 / 290 / 293. In some embodiments, the engineered TdT polypeptide has increased soluble protein expression and has at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reference sequence SEQ ID NO:2, and is selected from the following amino acid sequences: 80D / 106D / 121S / 185L / 190E / 205R / 289D / 290V / 293S / 313A / 315G / 336D / 342E / 359L / 391G / 470T / 474M / 499L / 522L / 523E, 80D / 106D / 121S / 185L / 190E / 205R / 289D / 290V / 293S / 313A / 342E / 470T / 474M / 499L / 523E, 80D / 106D / 121S / 185L / 190E / 244V / 289D / 290V / 293S / 307K / 342E / 359L / 470T / 474M / 499L, 80D / 121S / 131E / 1 85L / 190E / 205R / 244V / 289D / 290V / 293S / 313A / 315G / 336D / 342E / 359L / 391G / 414H / 470T / 474M / 499L / 522L / 523E, 80D / 121S / 1 31E / 185L / 190E / 205R / 244V / 289D / 290V / 293S / 313A / 336D / 342E / 359L / 391G / 414H / 470T / 474M / 499L / 522L / 523E, 80D / 121S / 1 31E / 185L / 190E / 289D / 290V / 293S / 313A / 342E / 470T / 474M / 499L / 522L / 523E, 80D / 121S / 174L / 179K / 185L / 190E / 236P / 244V / 2 88E / 289D / 290V / 293S / 313A / 315G / 317T / 336D / 342E / 359L / 363I / 391G / 394R / 408P / 426P / 462F / 470T / 474M / 499L / 522L / 523E,80D / 121S / 174L / 185L / 186G / 190E / 236P / 244V / 273V / 284L / 288E / 289D / 290V / 293S / 313A / 315G / 317T / 336D / 342E / 352P / 359L / 391G / 394R / 395R / 419A / 428V / 431S / 462F / 470T / 474M / 499L / 522L / 523E、80D / 121S / 174L / 185L / 190E / 193G / 196Y / 244V / 273V / 284L / 288 E / 289D / 290V / 293S / 297R / 313A / 315G / 317T / 324I / 336D / 342E / 352P / 359L / 376H / 380D / 391G / 394R / 401G / 415S / 419A / 428V / 431S / 435T / 441M / 462F / 470T / 474M / 499L / 522L / 523E、80D / 121S / 174L / 185L / 190E / 193G / 244V / 273V / 284L / 288E / 289D / 290V / 293S / 297R / 313A / 315G / 317T / 336D / 342E / 352P / 359L / 391G / 394R / 415S / 419A / 428V / 431S / 462F / 470T / 474M / 499L / 522L / 523E、80D / 121S / 174L / 185L / 190E / 196R / 244V / 266K / 273V / 284L / 288E / 289D / 290V / 293S / 313A / 315G / 317T / 324I / 336D / 342E / 352P / 359L / 391G / 394R / 39 7R / 401G / 419A / 428V / 431S / 462F / 470T / 474M / 499L / 522L / 523E、80D / 121S / 174L / 185L / 190E / 236P / 244V / 273V / 282R / 284L / 288E / 289D / 290V / 293S / 313A / 315G / 317T / 336D / 342E / 352P / 359L / 391G / 394R / 395R / 419A / 428V / 431S / 462F / 470T / 474M / 499L / 522L / 523E、80D / 121S / 174L / 185L / 190E / 244V / 273V / 284L / 288E / 289D / 290V / 293S / 313A / 315G / 317T / 336D / 342E / 352P / 359L / 391G / 394R / 419A / 428V / 431S / 462F / 470T / 474M / 499L / 522L / 523E、80D / 121S / 174L / 185L / 190E / 244V / 273V / 284L / 288E / 289D / 290V / 293S / 313A / 315G / 317T / 336D / 342E / 359L / 391G / 394R / 428V / 431S / 462F / 470T / 474M / 499L / 522L / 523E、80D / 121S / 174L / 185L / 190E / 244V / 284L / 288E / 289D / 290V / 293S / 313A / 315G / 317T / 336D / 342E / 352P / 359L / 391G / 394R / 419A / 428V / 431S / 462F / 470T / 474M / 499L / 522L、 / 523E, 80D, 121S, 174L, 185L, 190E, 244V, 284L, 288E, 289D, 290V, 293S, 313A, 315G, 317T, 336D, 342E, 352P, 359L, 391G, 394R, 428V, 431S, 462F, 470T / 474M / 499L / 522L / 523E、80D / 121S / 174L / 185L / 190E / 244V / 284L / 288E / 289D / 290V / 293S / 313A / 315G / 317T / 336D / 342E / 359L / 391G / 394R / 428V / 431S / 462F / 470T / 474M / 499L / 522L / 523E、80D / 121S / 185L / 190E / 196R / 244V / 289D / 290V / 293S / 313A / 315G / 317T / 336D / 342E / 359L / 391G / 470T / 474M / 499L / 522L / 523E、80D / 121S / 185L / 190E / 201R / 289D / 290V / 293S / 313A / 342E / 470 T / 474M / 499L / 522L、80D / 121S / 185L / 190E / 244V / 273V / 289D / 290V / 293S / 31 3A / 315G / 317T / 336D / 342E / 352P / 359L / 391G / 419A / 435T / 470T / 474M / 499L / 522L / 523E、80D / 121S / 185L / 190E / 244V / 289D / 290V / 293S / 300R / 313A / 31 5G / 317T / 336D / 342E / 359L / 391G / 470T / 474M / 499L / 522L / 523E、80D / 121S / 185L / 190E / 244V / 289D / 290V / 293S / 313A / 315G / 317T / 336D / 342E / 359L / 380 D / 391G / 401G / 419A / 470T / 474M / 499L / 522L / 523E、80D / 121S / 185L / 190E / 244V / 289D / 290V / 293S / 313A / 315G / 317T / 336D / 342E / 359L / 391G / 392R / 470T / 474M / 499L / 522L / 523E、80D / 121S / 185L / 190E / 244V / 289D / 290V / 293S / 313A / 315G / 317T / 336D / 342E / 359L / 391G / 395W / 470T / 474M / 499L / 522L / 523E、80D / 121S / 185L / 190E / 244V / 289D / 290V / 293S / 313A / 315G / 317T / 336D / 342E / 359L / 391G / 470T / 474M / 499L / 522L / 523E, 80D / 121S / 185L / 190E / 244V / 289D / 290V / 293S / 313A / 336 D / 342E / 359L / 391G / 414H / 470T / 474M / 499L / 522L / 523E, 80D / 121S / 185L / 190E / 289D / 29 0V / 293S / 313A / 315G / 336D / 342E / 359L / 391G / 414H / 470T / 474M / 499L / 522L / 523E, 80D / 12 1S / 185L / 190E / 289D / 290V / 293S / 313A / 336D / 342E / 359L / 391G / 470T / 474M / 499L / 522L / 523E, 80D / 121S / 185L / 190E / 289D / 290V / 293S / 313A / 342E / 499L, 80D / 121S / 185L / 315G, 8 The amino acid sequence has one or more residue differences compared to SEQ ID NO: 2 selected from 0D / 121S / 190E / 289D / 290V, 80D / 185L / 236P / 289D / 293S, 121S / 185L / 190E / 213S / 289D / 290V / 293S, and 185L / 289D / 290V / 293S. In some embodiments, the engineered TdT polypeptide has increased soluble protein expression and has at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reference sequence SEQ ID NO:2, and has the amino acid sequence N80D / K106D / C121S / F185L / K190. E / M205R / K289D / I290V / D293S / I313A / C315G / L336D / T342E / F359L / C391G / S470T / Q474M / K499L / I522L / Q523 E, N80D / K106D / C121S / F185L / K190E / M205R / K289D / I290V / D293S / I313A / T342E / S470T / Q474M / K499L / Q523E,N80D / K106D / C121S / F185L / K190E / D244V / K289D / I290V / D293S / C307K / T342E / F359L / S470T / Q474M / K499L、N80D / C121S / K131E / F185L / K190E / M205R / D244V / K289D / I290V / D293S / I313A / C315G / L336D / T342E / F359L / C391G / F414H / S470T / Q474M / K499L / I522L / Q523E、N80D / C121S / K131E / F185L / K190E / M205R / D244V / K289D / I290V / D293S / I313A / L336D / T342E / F359L / C391G / F414H / S470T / Q474M / K499L / I522L / Q523E、N80D / C121S / K131E / F185L / K190E / K289D / I290V / D293S / I313A / T342 E / S470T / Q474M / K499L / I522L / Q523E、N80D / C121S / I174L / Q179K / F185L / K190E / G236P / D244V / N288E / K289D / I290V / D29 3S / I313A / C315G / S317T / L336D / T342E / F359L / S363I / C391G / I394R / I408P / H426P / Y462F / S470T / Q474M / K499L / I522L / Q5 23E、N80D / C121S / I174L / F185L / E186G / K190E / G236P / D244V / L273V / F284L / N288E / K289D / I290V / D293S / I313A / C315G / S317T / L336D / T342E / E352P / F359L / C391G / I394R / E395R / L419A / E428V / R431S / Y462F / S470T / Q474M / K499L / I522L / Q523E、N80D / C121S / I174L / F185L / K190E / E193G / E196Y / D244V / L273V / F284L / N288E / K289D / I290V / D293S / K297R / I313A / C315G / S317 T / V324I / L336D / T342E / E352P / F359L / Q376H / N380D / C391G / I394R / L401G / Q415S / L419A / E428V / R431S / M435T / E441M / Y462F / S 470T / Q474M / K499L / I522L / Q523E、N80D / C121S / I174L / F185L / K190E / E193G / D244V / L273V / F284L / N288E / K289D / I290V / D293S / K297R / I313A / C315G / S317T / L336D / T342E / E352P / F359L / C391G / I394R / Q415S / L419A / E428V / R431S / Y462F / S470T / Q474M / K49 9L / I522L / Q523E、N80D / C121S / I174L / F185L / K190E / E196R / D244V / T266K / L273V / F284L / N288E / K289D / I290V / D293S / I313A / C315G / S317T / V324I / L336D / T342E / E352P / F359L / C391G / I394R / T397R / L401G / L419A / E428V / R431S / Y462F / S470T / Q474M / K499 L / I522L / Q523E、N80D / C121S / I174L / F185L / K190E / G236P / D244V / L273V / M282R / F284L / N288E / K289D / I290V / D293S / I313A / C315G / S317T / L336D / T342E / E352P / F359L / C391G / I394R / E395R / L419A / E428V / R431S / Y462F / S470T / Q474M / K499L / I522L / Q523E、N80D / C121S / I174L / F185L / K190E / D244V / L273V / F284L / N288E / K289D / I 290V / D293S / I313A / C315G / S317T / L336D / T342E / E352P / F359L / C391G / I3 94R / L419A / E428V / R431S / Y462F / S470T / Q474M / K499L / I522L / Q523E、N80 D / C121S / I174L / F185L / K190E / D244V / L273V / F284L / N288E / K289D / I290V / D293S / I313A / C315G / S317T / L336D / T342E / F359L / C391G / I394R / E428V / R431S / Y462F / S470T / Q474M / K499L / I522L / Q523E、N80D / C121S / I174L / F185L / K190E / D244V / F284L / N288E / K289D / I290V / D293S / I313A / C315G / S317T / L336D / T342E / E352P / F359L / C391G / I394R / L419A / E428V / R431S / Y46 2F / S470T / Q474M / K499L / I522L / Q523E、N80D / C121S / I174L / F185L / K190E / D244V / F284L / N288E / K289D / I290V / D293S / I313A / C315G / S317T / L336D / T342E / E352P / F359L / C391G / I394R / E428V / R431S / Y462F / S470T / Q474M / K499L / I522L / Q523E、N80D / C121S / I174L / F185L / K190E / D244V / F284L / N2 88E / K289D / I290V / D293S / I313A / C315G / S317T / L336D / T342E / F359L / C391G / I394R / E428V / R431S / Y462F / S470T / Q474M / K499L / I522L / Q523E、N80 D / C121S / F185L / K190E / E196R / D244V / K289D / I290V / D293S / I313A / C315G / S317T / L336D / T342E / F359L / C391G / S470T / Q474M / K499L / I522L / Q523EN80D / C121S / F185L / K190E / C201R / K289D / I290V / D293S / I313A / T342E / S470T / Q474 M / K499L / I522L、N80D / C121S / F185L / K190E / D244V / L273V / K289D / I290V / D293S / I31 3A / C315G / S317T / L336D / T342E / E352P / F359L / C391G / L419A / M435T / S470T / Q474M / K499L / I522L / Q523E、N80D / C121S / F185L / K190E / D244V / K289D / I290V / D293S / K300R / I313A / C315G / S317T / L336D / T342E / F359L / C391G / S470T / Q474M / K499L / I522L / Q523E、N80D / C121S / F185L / K190E / D244V / K289D / I290V / D293S / I313A / C315G / S317T / L3 36D / T342E / F359L / N380D / C391G / L401G / L419A / S470T / Q474M / K499L / I522L / Q523E、N80D / C121S / F185L / K190E / D244V / K289D / I290V / D293S / I313A / C315G / S317T / L336、 D / T342E / F359L / C391G / D392R / S470T / Q474M / K499L / I522L / Q523E, N80D / C121S / F185L / K190E / D244V / K289D / I290V / D293S / I313A / C315G / S317T / L336D / T342E / F359L / C391G / E395W / S470T / Q474M / K499L / I522L / Q523E, N80D / C121S / F185L / K190E / D244V / K289D / I29 0V / D293S / I313A / C315G / S317T / L336D / T342E / F359L / C391G / S470T / Q474M / K499L / I522L / Q523E, N80D / C121S / F185L / K190E / D244V / K289D / I290V / D293S / I313A / L336D / T342E / F359L / C391G / F414H / S470T / Q474M / K499L / I522L / Q523E, N80D / C121S / F185L / K190E / K28 9D / I290V / D293S / I313A / C315G / L336D / T342E / F359L / C391G / F414H / S470T / Q474M / K499L / I522L / Q523E, N80D / C121S / F185L / K190E / K289D / I290V / D293S / I313A / L336D / T342E / F359L / C391G / S470T / Q474M / K499L / I522L / Q523E, N80D / C121S / F185L / K190E / K289D / I2 and F185L / K289D / I290V / D293S.

[0142] In some embodiments, the engineered TdT polypeptide has increased thermal stability and has at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reference sequence SEQ ID NO: 36 and is selected from the group consisting of 174, 244, 273, 284, 288, 315, 317, 336, 352, 359, 391, 394, 419, 428, 430, 440, 442, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 486, 487, 488, 489, 490, 500, 501, 502, 503, 504, 505, 506, 507, 508, 509, 510, 511, 512, 513, 514, and 336 / 359 / 391 / 470 / 474 / 522 / 523. In some embodiments, the engineered TdT polypeptide has increased thermal stability and has at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reference sequence SEQ ID NO: 36 and is selected from the group consisting of 174L / 244V / 273V / 284L / 288E / 315G / 317T / 336D / 352P / 359L / 391G / 394R / 419A / 428V / 431S / 462F / 470T / 474M / 522L / 523E, 174L / 244V / 284L / 288E / 315G / 317T / 336D / 359L / 391G / 394R / 428V / 431S / 462F / 470T / 474M / 522L / 523E, 244V / 315G / 317T / 336D / 359L / 391G / 470T / 474M / 522L / 523E, and 336D / 359L / 391G / 470T / 474M / 522L / 523E.In some embodiments, the engineered TdT polypeptide has increased thermal stability and has at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reference sequence SEQ ID NO: 36 and is selected from the group consisting of I174L / D244V / L273V / F284L / N288E / C315G / S317T / L336D / E352P / F359L / C391G / I394R / L419A / E428V / R431S / Y462F / S470T / Q474M / I52 2L / Q523E, I174L / D244V / F284L / N288E / C315G / S317T / L336D / F359L / C391G / I394R / E428V / R431S / Y462F / S470T / Q474M / I522L / Q523E, D244V / C315G / S317T / L336D / F359L / C391G / S470T / Q474M / I522L / Q523E, and L336D / F359L / C391G / S470T / Q474M / I522L / Q523E.

[0143] In some embodiments, the engineered TdT polypeptide has at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reference sequence SEQ ID NO: 8, and has 129 / 196, 173, 183, 186, 196, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 27 3, 195, 196, 263, 266, 268, 281, 282, 297, 300, 303, 316, 318, 320, 324, 343, 360, 392, 395, 397, 411, 415, 417, 421, 454, 456, 477, 481, and 492. In some embodiments, the engineered TdT polypeptide has at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reference sequence SEQ ID NO:8 and is selected from the group consisting of 129G / 196G, 173L, 183R, 186A, 186G, 186L, 186T, 193C, 193G, 193N, 193V, 195R, 195W, 196A, 196G, 196R, 196W, 196Y, 263R, 266K, 268C, 281A, 282Q, 282R, 297F, 297Q, 29 7R, 297T, 300P, 300R, 303A, 303E, 303M, 316C, 316I, 316T, 318E, 318S, 318T, 318V, 320N, 324I, 343V, 360C, 360V, 392A, 392C, 392R, 395A, 395L, 395R, 395S, 395T, 395 The amino acid sequence has one or more residue differences compared to SEQ ID NO: 8 selected from W, 395Y, 397R, 411A, 411G, 411R, 415A, 415S, 417G, 417V, 421I, 421M, 454V, 456K, 456R, 477T, 481E, 481V, and 492T.In some embodiments, the engineered TdT polypeptide has at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reference sequence SEQ ID NO: 8, and has the following amino acids: D129G / E196G, T173L, D183R, E186A, E186G, E186L, E186T, E193C, E193G, E193N, E193V, K195R, K195W, E196A, E196G , E196R, E196W, E196Y, K263R, T266K, V268C, R281A, M282Q, M282R, K297F, K297Q, K297R, K297T , K300P, K300R, K303A, K303E, K303M, V316C, V316I, V316T, K318E, K318S, K318T, K318V, E320 N, V324I, I343V, L360C, L360V, D392A, D392C, D392R, E395A, E395L, E395R, E395S, E395T, E395 The amino acid sequence has one or more residue differences compared to SEQ ID NO: 8 selected from W, E395Y, T397R, L411A, L411G, L411R, Q415A, Q415S, C417G, C417V, L421I, L421M, L454V, V456K, V456R, R477T, R481E, R481V, and D492T.

[0144] In some embodiments, the engineered TdT polypeptide has at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reference sequence SEQ ID NO: 16, and 6 / 318, 186 / 236 / 395, 186 / 282 / 318, 193 / 196, 193 / 196 / 266 / 324 / 376 / 380, 193 / 196 / 297, 193 / 196 / 297 / 324 / 376 / 380 / 401 / 415 / 435 / 441, 193 / 196 / 324, 193 / 196 / 324 / 397 / 401 / 44 1, 193 / 196 / 376 / 380, 193 / 297 / 324 / 376 / 435, 193 / 297 / 324 / 380, 193 / 297 / 415, 193 / 435, 196, 196 / 266, 196 / 266 / 324 / 397 / 401, 196 / 297 / 324 / 435, 236 / 282, 236 / 282 / 395, 236 / 31 and 435 / 441.In some embodiments, the engineered TdT polypeptide has at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reference sequence SEQ ID NO: 16 and is selected from the group consisting of 186G, 186G / 236P / 318S, 186G / 236P / 395R ... 86G / 282R / 318S, 193G / 196R / 297R, 193G / 196Y, 193G / 196Y / 266K / 324I / 376H / 380D, 193G / 196Y / 297R / 32 4I / 376H / 380D / 401G / 415S / 435T / 441M, 193G / 196Y / 324I / 397R / 401G / 441M, 193G / 196Y / 376H / 380D, 193G / 297R / 324I / 376H / 435T, 193G / 297R / 415S, 193G / 435T, 193N / 196Y / 324I, 193N / 297R / 324I / 380D, 196R, 196R / 266K, 196R / 266K / 324I / 397R / 401G, 196Y / 297R / 324I / 435T, 236P / 282R, 236P / 282R / 395R, 236P / 31 The amino acid sequence has one or more residue differences compared to SEQ ID NO: 16 selected from 8S / 481E, 266K / 297R / 380D / 397R / 401G, 282R, 282R / 318S, 282R / 481E, 297R / 380D / 401G / 441M, 297R / 435T, 318S / 395R, 376H / 401G / 441M, 415S, and 435T / 441M.In some embodiments, the engineered TdT polypeptide has at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reference sequence SEQ ID NO: 16 and is selected from the group consisting of E186G, E186G / G236P / K318S, E186G / G236P / E395R, E186G / M282R / K318S ... 193G / E196R / K297R, E193G / E196Y, E193G / E196Y / T266K / V324I / Q376H / N380D, E193G / E196Y / K297R / V324I / Q376H / N380 D / L401G / Q415S / M435T / E441M, E193G / E196Y / V324I / T397R / L401G / E441M, E193G / E196Y / Q376H / N380D, E193G / K297R / V3 24I / Q376H / M435T, E193G / K297R / Q415S, E193G / M435T, E193N / E196Y / V324I, E193N / K297R / V324I / N380D, E196R, E196R / T266K, E196R / T266K / V324I / T397R / L401G, E196Y / K297R / V324I / M435T, G236P / M282R, G236P / M282R / E395R, G236P / K31 The amino acid sequence has one or more residue differences compared to SEQ ID NO: 16 selected from: 8S / R481E, T266K / K297R / N380D / T397R / L401G, M282R, M282R / K318S, M282R / R481E, K297R / N380D / L401G / E441M, K297R / M435T, K318S / E395R, Q376H / L401G / E441M, Q415S, and M435T / E441M.

[0145] In some embodiments, the engineered TdT polypeptide has at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reference sequence SEQ ID NO: 24, and 12, 13, 14, 17, 18, 20, 21, 22, 23, 24, 26, 27, 29, 30, 31, 33, 34, 35, 37, 41, 53, 57 , 58, 61, 92, 94, 97, 101, 102, 103, 104, 105, 106, 107, 108, 124, 126, 133, 135, 137, 138, 139, 140, 141, 142, 144, 145, 147, 149, 150, 152, 153, 154, 155, 156, 156 / 294, 159, 160, 161, 162, and 163, when compared to SEQ ID NO: 24.In some embodiments, the engineered TdT polypeptide has at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reference sequence SEQ ID NO:24 and is selected from the group consisting of 12S, 13G, 13K, 13R, 13S, 14G, 14Q, 17A, 17G, 18D, 18R , 20H, 20T, 21E, 22G, 22L, 23E, 23P, 24G, 26G, 27D, 29P, 29R, 30E, 30G, 30V, 31S, 33G, 33K, 33P, 34D, 34K, 34R, 3 4S, 35E, 35G, 37A, 37F, 37G, 37S, 37T, 37V, 41V, 53E, 57H, 58A, 58S, 61H, 61L, 92M, 92R, 92S, 92V, 92Y, 94E, 94R , 97D, 101E, 102L, 103M, 104G, 104I, 104P, 105N, 106G, 106H, 106S, 107R, 107W, 108D, 108K, 124E, 124I, 126V, 133G, 135R, 137A, 138Q, 139A, 140E, 140G, 141E, 141M, 141R, 142M, 142S, 144C, 145E, 147L, 149R, 150E, 150G, The amino acid sequence has one or more residue differences compared to SEQ ID NO: 24 selected from 152G, 152R, 153E, 153G, 153K, 153M, 153P, 153Q, 153V, 154G, 155E, 155T, 156D, 156E / 294T, 156M, 156Q, 159D, 160G, 161D, 161E, 161G, 161R, 161S, 162E, 163L, and 163V.In some embodiments, the engineered TdT polypeptide has at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reference sequence SEQ ID NO:24 and is selected from the group consisting of M12S, H13G, H13K, H13R, H13S, R14G, R14Q, T17A, T17G, I18D, I18R, S20H, S20T, D21E, F22 G, F22L, G23E, G23P, K24G, R26G, Q27D, K29P, K29R, M30E, M30G, M30V, D31S, H33G, H33K, H33P, I34D, I34K, I34R, I34S, S35E, S35 G, M37A, M37F, M37G, M37S, M37T, M37V, I41V, K53E, A57H, T58A, T58S, T61H, T61L, G92M, G92R, G92S, G92V, G92Y, D94E, D94R, A97 D, T101E, H102L, K103M, M104G, M104I, M104P, E105N, K106G, K106H, K106S, T107R, T107W, T108D, T108K, V124E, V124I, K126V, K 133G, Q135R, M137A, E138Q, S139A, R140E, R140G, V141E, V141M, V141R, D142M, D142S, A144C, N145E, D147L, T149R, A150E, A150 and I163V.

[0146] In some embodiments, the engineered TdT polypeptide has at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reference sequence SEQ ID NO:24, and 1, 32, 33, 34, 35, 44, 45, 46, 57, 65, 77, 85, 89, 93, 94, 97, 101, 102, 103, 105, 106, 108, 109, 110, 119, 123, 124, 126, 130, 131, 132, 133, 134, 135, 137, 138, 139, 149, 150, 153, and 156, when compared to SEQ ID NO: 24.In some embodiments, the engineered TdT polypeptide has at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reference sequence SEQ ID NO: 24, and is selected from the group consisting of 7Y / 135C, 12F, 13E, 1 4G, 14N, 14Y, 15L, 16V, 17A / 131R, 18Y, 20P, 23C, 23E, 23T, 24A, 24M, 24P, 25N, 26G, 27D, 27F, 2 8R, 29G, 29I, 31V, 32E, 33A, 33C, 34S, 35H, 35W, 44S, 45R, 46M, 57T, 65S, 77C, 77S, 85V, 89A, 93V , 94N, 97T, 101E, 101G, 101V, 102L, 103M, 105N, 105W, 106V, 108G, 108M, 109M, 109N, 109T, 110 M, 110V, 119F, 119Q, 123M, 123Q, 124E, 124G, 124I, 124M, 124S, 126C, 130A, 130M, 130Q, 130S, 1 The amino acid sequence has one or more residue differences compared to SEQ ID NO: 24 selected from 31G, 131W, 132S, 133G, 133M, 133Q, 134M, 134W, 135E, 135H, 135K, 137A, 137E, 138A, 139G, 149R, 150E, 153E, 153G, 153P, 153Q, and 156D.In some embodiments, the engineered TdT polypeptide has at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reference sequence SEQ ID NO:24 and is selected from the group consisting of H7Y / Q135C, M12F, H13E, R14G, R14N, R14Y, I15L, R 16V, T17A / K131R, I18Y, S20P, G23C, G23E, G23T, K24A, K24M, K24P, K25N, R26G, Q27D, Q27F, K28R, K29G, K29I , D31V, N32E, H33A, H33C, I34S, S35H, S35W, H44S, E45R, F46M, A57T, D65S, E77C, E77S, I85V, N89A, S93V, D94N , A97T, T101E, T101G, T101V, H102L, K103M, E105N, E105W, K106V, T108G, T108M, Q109M, Q109N, Q109T, F110M , F110V, I119F, I119Q, K123M, K123Q, V124E, V124G, V124I, V124M, V124S, K126C, T130A, T130M, T130Q, T130S , K131G, K131W, G132S, K133G, K133M, K133Q, Y134M, Y134W, Q135E, Q135H, Q135K, M137A, M137E, E138A, S139G, T149R, A150E, L153E, L153G, L153P, L153Q, and L156D.

[0147] In some embodiments, the engineered TdT polypeptide comprises an amino acid sequence having at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reference sequence SEQ ID NO: 268 and having one or more residue differences compared to SEQ ID NO: 268 selected from 14 / 53 / 300, 14 / 53 / 419, 106 / 300 / 415 / 419 / 456, 140, and 300 / 395 / 419. In some embodiments, an engineered TdT polypeptide comprises an amino acid sequence having at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reference sequence SEQ ID NO: 268 and having one or more residue differences compared to SEQ ID NO: 268 selected from 14G / 53K / 300P, 14G / 53K / 419L, 106V / 300P / 415A / 419L / 456R, 140G, and 300P / 395Y / 419L. In some embodiments, the engineered TdT polypeptide comprises an amino acid sequence having at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reference sequence SEQ ID NO: 268 and having one or more residue differences compared to SEQ ID NO: 268 selected from R14G / E53K / K300P, R14G / E53K / A419L, K106V / K300P / Q415A / A419L / V456R, R140G, and K300P / E395Y / A419L.

[0148] In some embodiments, the engineered TdT polypeptide has at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reference sequence SEQ ID NO: 648 and is selected from the group consisting of 12 / 14 / 34, 12 / 14 / 34 / 37 / 94 / 140 / 141 / 145, 12 / 14 / 34 / 37 / 106 / 140 / 142 / 150 / 152 / 153, 12 / 14 / 34 / 37 / 141 / 142, 12 / 14 / 34 / 37 / 142 / 145, 12 / 14 / 34 / 37 / 142 / 161, 12 / 14 / 34 / 37 / 150, 12 / 14 / 34 / 37 / 150 / 153, 12 / 14 / 34 / 140 / 142, 12 / 14 / 34 / 140 / 150, 12 / 14 / 34 / 142 / 150 / 153, 12 / 14 / 92 / 94, 12 / 14 / 94 / 150 / 152, 12 / 14 / 106 / 107 / 141 / 142, 12 / 14 / 106 / 108 / 140 / 141 / 145 / 150, 12 / 14 / 106 / 108 / 152, 12 / 14 / 141 / 142, 12 / 14 / 150 / 152 / 15 3, 12 / 14 / 153, 12 / 34 / 92 / 140, 12 / 34 / 150 / 152, 12 / 37 / 94 / 141 / 150 / 152 / 153, 12 / 37 / 140 / 141 / 150 / 162, 12 / 161, 14, 14 / 31 / 34 / 37 / 140 / 141 / 145 / 16 1 / 162, 14 / 34 / 37, 14 / 34 / 37 / 145, 14 / 34 / 37 / 152, 14 / 34 / 94 / 106 / 108 / 141, 14 / 34 / 150 / 153, 14 / 106, 14 / 140, 14 / 141 / 161, 14 / 142, 14 / 142 / 161 / 162, 14 / 153, 14 / 161, 20 / 21 / 24 / 33 / 58 / 104 / 106 / 124 / 155 / 156, 20 / 21 / 33 / 58 / 101 / 104 / 106 / 124 / 155, 20 / 21 / 58 / 104 / 106 / 155 / 156, 20 / 33 / 104 / 106 / 12 4 / 156, 20 / 58 / 101 / 104 / 106 / 156, 20 / 58 / 101 / 106, 20 / 101 / 106 / 156, 21 / 33 / 58 / 101 / 106, 21 / 33 / 58 / 106 / 155 / 156, 21 / 33 / 101 / 104 / 106, 21 / 33 / 106,21 / 58 / 101 / 104 / 106 / 155, 21 / 58 / 106 / 155 / 156, 21 / 101 / 104 / 106 / 156, 21 / 104 / 106, 21 / 104 / 106 / 124, 21 / 104 / 106 / 156, 30 / 33 / 58 / 104 / 106 / 155 / 156, 30 / 33 / 101 / 106 / 156, 30 / 33 / 104 / 106 / 155 / 156, 30 / 101 / 104 / 106 / 155 / 156, 30 / 104 / 106 / 1 55, 30 / 104 / 106 / 155 / 156, 30 / 106 / 155, 33 / 58 / 104 / 106, 33 / 101 / 104 / 106 / 155, 34 / 37, 34 / 37 / 92, 34 / 37 / 140 / 141 / 142 / 145, 34 / 37 / 141 / 142, 34 / 37 / 150 / 153, 34 / 92 / 94 / 141 / 142, 34 / 141 / 142 / 145, 34 / 150 / 152 / 153, 37 / 92 / 142, 37 / 141 / 142, 3 7 / 153, 58 / 101 / 104 / 106 / 156, 58 / 101 / 106 / 155, 58 / 104 / 106 / 155 / 156, 92 / 94 / 106 / 142 / 145, 101 / 104 / 106, 101 / 104 / 106 / 155 / 156, 101 / 104 / 106 / 156, 101 / 106, 101 / 106 / 124 / 155, 101 / 106 / 155 / 156, 104 / 106, 104 / 106 / 124, 104 / 106 / 155, 104 / 106 / 155 / 156, 104 / 106 / 156, 106 / 107 / 108 / 142 / 220, 106 / 108 / 140 / 141 / 152 / 153, 106 / 108 / 140 / 142 / 150 / 153, 106 / 156, 140 / 141 / 142, 140 / 145, 140 / 145 / 150 / 152, 141, 141 / 152, and 161. In some embodiments, the engineered TdT polypeptide has at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reference sequence SEQ ID NO: 648 and is selected from the group consisting of 12S / 14G / 34D, 12S / 14G / 34D / 37A / 106K / 140G / 142M / 150E / 152R / 153E,12S / 14G / 34D / 37A / 141E / 142S、12S / 14G / 34D / 37A / 150E、12S / 14G / 34D / 37F / 94E / 140G / 141E / 145E、12S / 14G / 34D / 37F / 142S / 145E、12S / 14G / 34D / 37F / 150E / 153E、12S / 14G / 34D / 140G / 142S、12S / 14G / 34D / 140G / 150E、12S / 14G / 34S / 37A / 142M / 161E、12S / 14G / 34S / 142S / 150E / 153E、12S / 14G / 92R / 94E、12S / 14G / 94E / 150E / 152R、12S / 14G / 106K / 107R / 141E / 142M、12S / 14G / 106K / 108K / 140G / 141E / 145E / 150E、12S / 14G / 106K / 108K / 152R、12S / 14G / 141E / 142S、12S / 14G / 150E / 152R / 153E、12S / 14G / 153E、12S / 34D / 92R / 140G、12S / 34D / 150E / 152R、12S / 37A / 94E / 141E / 150E / 152R / 153E、12S / 37F / 140G / 141E / 150E / 162E、12S / 161E、14G、14G / 31G / 34D / 37A / 140G / 141E / 145E / 161E / 162E、14G / 34D / 94E / 106K / 108K / 141E、14G / 34D / 150E / 153E、14G / 34S / 37A、14G / 34S / 37A / 152R、14G / 34S / 37F / 145E、14G / 34S / 150E / 153E、14G / 106K、14G / 140G、14G / 141E / 161E、14G / 142M、14G / 142S / 161E / 162E、14G / 153E、14G / 161E、20T / 21E / 24G / 33K / 58A / 104P / 106S / 124E / 155E / 156E、20T / 21E / 33K / 58A / 101E / 104P / 106K / 124E / 155E、20T / 21E / 58A / 104I / 106S / 155E / 156E、20T / 33K / 104P / 106K / 124E / 156E、20T / 58A / 101E / 106K、20T / 58S / 101E / 104P / 106S / 156E、20T / 101E / 106K / 156E、21E / 33K / 58A / 101E / 106K、21E / 33K / 58S / 106S / 155E / 156E、21E / 33K / 101E / 104P / 106S、21E / 33K / 106S、21E / 58A / 106K / 155E / 156E、21E / 58S / 101E / 104I / 106S / 155E、21E / 101E / 104P / 106S / 156E、21E / 104I / 106S / 124E、21E / 104P / 106S、21E / 104P / 106S / 156E、30G / 33K / 58S / 104P / 106S / 155E / 156E、30G / 33K / 101E / 106S / 156E、30G / 33K / 104I / 106S / 155E / 156E、30G / 101E / 104P / 106S / 155E / 156E、30G / 104P / 106S / 155E、30G / 104P / 106S / 155E / 156E、30G / 106K / 155E、33K / 58A / 104I / 106S、33K / 101E / 104P / 106K / 155E、34D / 37A、34D / 37A / 92R、34D / 37F / 141E / 142S、34D / 92R / 94E / 141E / 142M、34D / 141E / 142S / 145E、34D / 150E / 152R / 153E、34S / 37A / 150E / 153E、34S / 37F / 140G / 141E / 142M / 145E、37A / 141E / 142S、37F / 92R / 142M、37F / 153E、58A / 101E / 106K / 155E、58S / 101E / 104P / 106S / 156E、58S / 104I / 106K / 155E / 156E、92R / 94E / 106K / 142S / 145E、101E / 104I / 106K、101E / 104I / 106K / 156E、101E / 104I / 106S / 156E、101E / 104P / 106K / 155E / 156E、101E / 106K、101E / 106K / 155E / 156E、101E / 106S / 124E / 155E、104I / 106K、104I / 106K / 124E、104I / 106S、104I / 106S / 155E / 156E、104P / 106K、104P / 106K / 155E、104P / 106K / 156E、104P / 106S、104P / 106S / 155E、106K / 107R / 108K / 142S / 220R、106K / 108K / 140G / 141E / 152R / 153E、The amino acid sequence has one or more residue differences compared to SEQ ID NO: 648 selected from 106K / 108K / 140G / 142S / 150E / 153E, 106K / 156E, 140G / 141E / 142M, 140G / 145E, 140G / 145E / 150E / 152R, 141E, 141E / 152R, and 161E. In some embodiments, the engineered TdT polypeptide has at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reference sequence SEQ ID NO: 648 and is selected from the group consisting of M12S / R14G / I34D, M12S / R14G / I34D / M37A / V106K / R140G / D142M / A150E / T152R / L153E ... 2S / R14G / I34D / M37A / V141E / D142S, M12S / R14G / I34D / M37A / A150E, M12S / R14G / I34D / M37F / D94E / R140G / V141E / N145E, M12S / R1 4G / I34D / M37F / D142S / N145E, M12S / R14G / I34D / M37F / A150E / L153E, M12S / R14G / I34D / R140G / D142S, M12S / R14G / I34D / R140G / A 150E, M12S / R14G / I34S / M37A / D142M / K161E, M12S / R14G / I34S / D142S / A150E / L153E, M12S / R14G / G92R / D94E, M12S / R14G / D94E / A150E / T152R, M12S / R14G / V106K / T107R / V141E / D142M, M12S / R14G / V106K / T108K / R140G / V141E / N145E / A150E, M12S / R14G / V106 K / T108K / T152R, M12S / R14G / V141E / D142S, M12S / R14G / A150E / T152R / L153E, M12S / R14G / L153E, M12S / I34D / G92R / R140G, M12S / I34D / A150E / T152R, M12S / M37A / D94E / V141E / A150E / T152R / L153E, M12S / M37F / R140G / V141E / A150E / T162E, M12S / K161E, R14G,R14G / D31G / I34D / M37A / R140G / V141E / N145E / K161E / T162E、R14G / I34D / D94E / V106K / T108K / V141E、R14G / I34D / A150E / L153E、R14G / I34S / M37A、R14G / I34S / M37A / T152R、R14G / I34S / M37F / N145E、R14G / I34S / A150E / L153E、R14G / 、 V106K、R14G / R140G、R14G / V141E / K161E、R14G / D142M、R14G / D142S / K161E / T162E、R14G / L153E、R14G / K161E、S20T / D21E / K24G / H33K / T58A / M104P / V106S / V124E / I155E / L156E、S20T / D21E / H33K / T58A / T101E / M104P / V106K / V124E / I155E、S20T / D21E / T58A / M104I / V106S / I155E / L156E、S20T / H33K / M104P / V106K / V124E / L156E、S20T / T58A / T101E / V106K、S20T / T58S / T101E / M104P / V106S / L156E、S20T / T101E / V106K / L156E、D21E / H33K / T58A / T101E / V106K、D21E / H33K / T58S / V106S / I155E / L156E、D21E / H33K / T101E / M104P / V106S、D21E / H33K / V106S、D21E / T58A / V106K / I155E / L156E、D21E / T58S / T101E / M104I / V106S / I155E、D21E / T101E / M104P / V106S / L156E、D21E / M104I / V106S / V124E、D21E / M104P / V106S、D21E / M104P / V106S / L156E、M30G / H33K / T58S / M104P / V106S / I155E / L156E、M30G / H33K / T101E / V106S / L156E、M30G / H33K / M104I / V106S / I155E / L156E、M30G / T101E / M104P / V106S / I155E / L156E、M30G / M104P / V106S / I155E、M30G / M104P / V106S / I155E / L156E、M30G / V106K / I155E、H33K / T58A / M104I / V106S、H33K / T101E / M104P / V106K / I155E、I34D / M37A、I34D / M37A / G92R、I34D / M37F / V141E / D142S、I34D / G92R / D94E / V141E / D142M、I34D / V141E / D142S / N145E、I34D / A150E / T152R / L153E、I34S / M37A / A150E / L153E, I34S / M37F / R140G / V141E / D142M / N145E, M37A / V141E / D142S, M37F / G92R / D142M, M37F / L153E, T58A / T101E / V106K / I155E, T58S / T101E / M104P / V106S / L156E, T58S / M104I / V106K / I155E / L156E, G92R / D 94E / V106K / D142S / N145E, T101E / M104I / V106K, T101E / M104I / V106K / L156E, T101E / M104I / V106S / L156E, T101E / M104P / V106K / I155E / L156E, T101E / V106K, T101E / V106K / I155E / L156E, T101E / V106S / V124E / I155E, M104I / V10 6K, M104I / V106K / V124E, M104I / V106S, M104I / V106S / I155E / L156E, M104P / V106K, M104P / V106K / I155E, M104P / V106K / L156E, M104P / V106S, M104P / V106S / I155E, V106K / T107R / T108K / D142S / S220R, V106K / T108K / R140G / V141 and K161E.

[0149] In some embodiments, the engineered TdT polypeptide has at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reference sequence SEQ ID NO: 660, and is selected from the group consisting of 16 / 29 / 30, 16 / 29 / 30 / 33 / 153, 16 / 29 / 30 / 101 / 104, 16 / 30 / 104, 16 / 33, 29 / 30, 58, 92 / 94 / 108 / 141 / 155 / 392, 92 / 101 / 137 / 155 / 476, 94 / 101 / 156 / 476, 101, 101 / 104, 101 / 137 / 155, 101 / 141 / 155 / 156, and 108. In some embodiments, the engineered TdT polypeptide has at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reference sequence SEQ ID NO: 660 and is selected from the group consisting of 16V / 29I / 30E, 16V / 29I / 30E / 33K / 153P, 16V / 29I / 30E / 101E / 104V, 16V / 30 The amino acid sequence has one or more residue differences compared to SEQ ID NO: 660 selected from L / 104V, 16V / 33K, 29I / 30E, 58A, 92R / 94E / 108K / 141E / 155E / 392R, 92R / 101E / 137A / 155E / 476R, 94E / 101E / 156E / 476R, 101E, 101E / 104V, 101E / 137E / 155E, 101E / 141E / 155E / 156E, and 108K.In some embodiments, the engineered TdT polypeptide has at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reference sequence SEQ ID NO: 660 and is selected from the group consisting of R16V / K29I / M30E, R16V / K29I / M30E / H33K / E153P, R16V / K29I / M30E / T101E / M104V, R16V / M30L / M104V ... The amino acid sequence has one or more residue differences compared to SEQ ID NO: 660 selected from 16V / H33K, K29I / M30E, T58A, G92R / D94E / T108K / V141E / I155E / D392R, G92R / T101E / M137A / I155E / E476R, D94E / T101E / L156E / E476R, T101E, T101E / M104V, T101E / M137E / I155E, T101E / V141E / I155E / L156E, and T108K.

[0150] In some embodiments, the engineered TdT polypeptide has at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reference sequence SEQ ID NO: 660, and is selected from the group consisting of 195, 197, 204 / 342, 205, 236 / 297, 258, 261, 262, 264, 268, 269, 276, 278, 280, 281, 282, 290, 291, 297, 300, 303, 306, 308, 309, 310, 312, 315, 316, 342, 344, 353, 360, 385, 391, 410, 413, 419, 421, 448, 454, 456, 473, 476, 515, and 525, when compared to SEQ ID NO: 660.In some embodiments, the engineered TdT polypeptide has at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reference sequence SEQ ID NO: 660 and is selected from the group consisting of 195E, 197M, 204L / 342W, 205E, 205L, 236E / 297L, 258A, 258C, 258G, 258L, 258M, 258S, 258W, 261G, 261R, 261V, 262I, 264A, 264E, 264R, 264S , 268L, 269W, 276S, 278C, 278E, 278I, 278R, 278T, 278V, 280F, 281A, 281C, 281G, 281L, 281S, 281T, 281V , 282C, 282G, 282H, 282W, 290A, 290L, 291S, 297C, 297D, 297S, 297V, 300R, 303A, 303N, 303Q, 303S, 303 V, 306F, 308F, 308W, 309F, 310A, 310G, 310H, 310R, 310S, 312V, 315A, 315S, 316A, 316L, 342R, 342V, 344 V, 353A, 353K, 353M, 353R, 353S, 360I, 385R, 391L, 391R, 410E, 413C, 413F, 413V, 419G, 419H, 421F, 448R, 454M, 456S, 473V, 476V, 515V, 525F, and 525H.In some embodiments, the engineered TdT polypeptide has at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reference sequence SEQ ID NO: 660 and is selected from the group consisting of K195E, N197M, F204L / E342W, M205E, M205L, G236E / K297L, R258A, R 258C, R258G, R258L, R258M, R258S, R258W, S261G, S261R, S261V, F262I, L264A, L264E, L264R, L264S, V268L, F269W, A 276S, K278C, K278E, K278I, K278R, K278T, K278V, Y280F, R281A, R281C, R281G, R281L, R281S, R281T, R281V, M282C, M2 82G, M282H, M282W, V290A, V290L, R291S, K297C, K297D, K297S, K297V, P300R, K303A, K303N, K303Q, K303S, K303V, L3 06F, Y308F, Y308W, Y309F, E310A, E310G, E310H, E310R, E310S, L312V, G315A, G315S, V316A, V316L, E342R, E342V, T34 4V, F353A, F353K, F353M, F353R, F353S, L360I, Q385R, G391L, G391R, A410E, H413C, H413F, H413V, L419G, L419H, L421F, K448R, L454M, R456S, R473V, E476V, A515V, S525F, and S525H.

[0151] In some embodiments, the engineered TdT polypeptide comprises an amino acid sequence having at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reference sequence SEQ ID NO: 882 and having one or more residue differences compared to SEQ ID NO: 882 selected from 175, 196, 199, 203, 208, 275, 313, 314, 317, 321, 322, 325, 329 / 462, 379, 394, 397, 403 / 462, 406, 408, 457, 461, 462, 469, 477, 481, 484, and 495. In some embodiments, the engineered TdT polypeptide has at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reference sequence SEQ ID NO: 882 and is selected from the group consisting of 175L, 196G, 196Y, 199G, 199M, 199Q, 199R, 199S, 199V, 203A, 203G, 203L, 203R, 203S, 208V, 275V, 313I, 314G, 314K, 314L, 314R , 314V, 314Y, 317G, 321C, 321K, 321S, 322A, 325F, 325T, 325V, 325W, 329R / 462E, 379C, 394E, 394T, 397D, 397T, 403F / 462H, 406G, 406V, 408A, 408T, 457S, 457V, 461G, 461V, 462I, 462R, 462W, 469Q, 477T, 481D, 481M, 481T, 484M, 484R, and 495S.In some embodiments, the engineered TdT polypeptide has at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reference sequence SEQ ID NO: 882 and is selected from the group consisting of N175L, R196G, R196Y, D199G, D199M, D199Q, D199R, D199S, D199V, T203A, T203G, T203L, T203R, T203S, I208V, T275V, A313I, D314G, D314K, D314L, D314R, D314V ... 14Y, T317G, A321C, A321K, A321S, D322A, S325F, S325T, S325V, S325W, Q329R / F462 E, T379C, R394E, R394T, R397D, R397T, L403F / F462H, R406G, R406V, I408A, I408T, The amino acid sequence has one or more residue differences compared to SEQ ID NO: 882 selected from C457S, C457V, R461G, R461V, F462I, F462R, F462W, W469Q, R477T, R481D, R481M, R481T, T484M, T484R, and A495S.

[0152] In some embodiments, the engineered TdT polypeptide has at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reference sequence SEQ ID NO: 882, and 175, 179, 196, 199, 20 1, 203, 272, 273, 275, 307, 313, 314, 319, 321, 322, 324, 325, 350, 376, 394, 404, 406, 408, 461, 462, 477, 481, 484, 491, 492, 495, and 523, when compared to SEQ ID NO: 882. In some embodiments, the engineered TdT polypeptide has at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reference sequence SEQ ID NO: 882 and is selected from the group consisting of 175D, 175I, 175V, 179R, 196G, 199A, 199E, 199G, 199H, 199I, 199Q, 199R, 199V, 201A, 201W, 203M, 203R, 272T, 273M, 273W, 275D, 307M, 307V, 31 The amino acid sequence has one or more residue differences compared to SEQ ID NO: 882 selected from: 3M, 313Q, 313R, 313S, 314G, 314I, 319G, 319R, 321K, 322K, 322Q, 324V, 325A, 325V, 350S, 376V, 394L, 394M, 394S, 394T, 404F, 404W, 406V, 408G, 408L, 408R, 461A, 461G, 461Q, 461S, 462L, 462Q, 477Q, 481W, 484M, 484R, 491I, 492S, 492T, 495G, 495S, and 523H.In some embodiments, the engineered TdT polypeptide has at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reference sequence SEQ ID NO: 882, and , N175I, N175V, Q179R, R196G, D199A, D199E, D199G, D199H, D199I, D199Q, D199R, D199V , C201A, C201W, T203M, T203R, G272T, V273M, V273W, T275D, C307M, C307V, A313M, A313Q , A313R, A313S, D314G, D314I, A319G, A319R, A321K, D322K, D322Q, I324V, S325A, S325V , G350S, Q376V, R394L, R394M, R394S, R394T, P404F, P404W, R406V, I408G, I408L, I408R , R461A, R461G, R461Q, R461S, F462L, F462Q, R477Q, R481W, T484M, T484R, L491I, D492S, D492T, A495G, A495S, and E523H.

[0153] In some embodiments, the engineered TdT polypeptide has at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reference sequence SEQ ID NO: 1336, and 15 / 1 99 / 203 / 394, 15 / 199 / 394, 28 / 344 / 353 / 395, 195 / 199 / 203 / 278 / 297 / 394, 195 / 199 / 203 / 278 / 314 / 353 / 394, 195 / 203 / 278 / 394 / 395, 195 / 203 / 297 / 314 / 394, 195 / 203 / 297 / 39 4 / 419, 195 / 203 / 394, 195 / 278 / 297 / 394, 195 / 278 / 297 / 394 / 395, 195 / 314 / 344, 195 / 394 / 395, 199 / 203 / 297 / 394 / 395, 203 / 278 / 297 / 394, 203 / 297 / 314 / 394 / 395, 203 / 310 / 3 The amino acid sequence has one or more residue differences when compared to SEQ ID NO: 1336 selected from 14 / 394 / 395 / 419, 203 / 344 / 394, 203 / 353, 203 / 394, 203 / 394 / 395, 297 / 394, 314 / 394 / 395, 344 / 394 / 395, 353, and 394.In some embodiments, the engineered TdT polypeptide has at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reference sequence SEQ ID NO: 1336, and has at least 15F / 199R / 203A / 394E, ... 394E, 28N / 344V / 353K / 395W, 195E / 199R / 203A / 278E / 297D / 394E, 195E / 199R / 203A / 278E / 314R / 353K / 394E, 195E / 203A / 278E / 394E / 395W, 195E / 203A / 297D / 314R / 394E, 195E / 203A / 297D / 394E / 419M, 195 E / 203A / 394E, 195E / 278E / 297D / 394E, 195E / 278E / 297D / 394E / 395W, 195E / 314R / 344V, 195E / 394E / 3 95W, 199R / 203A / 297D / 394E / 395W, 203A / 278E / 297D / 394E, 203A / 297D / 314R / 394E / 395W, 203A / 310R / The amino acid sequence has one or more residue differences compared to SEQ ID NO: 1336 selected from 314R / 394E / 395W / 419M, 203A / 344V / 394E, 203A / 353K, 203A / 394E, 203A / 394E / 395W, 297D / 394E, 314R / 394E / 395W, 344V / 394E / 395W, 353K, and 394E.In some embodiments, the engineered TdT polypeptide has at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reference sequence SEQ ID NO: 1336 and is selected from the group consisting of I15F / D199R / T203A / R394E, I15F / D199R / R394E, K28N / T344V / F 353K / E395W, K195E / D199R / T203A / K278E / K297D / R394E, K195E / D199R / T203A / K278E / D314R / F353K / R394E, K195E / T 203A / K278E / R394E / E395W, K195E / T203A / K297D / D314R / R394E, K195E / T203A / K297D / R394E / L419M, K195E / T203A / R 394E, K195E / K278E / K297D / R394E, K195E / K278E / K297D / R394E / E395W, K195E / D314R / T344V, K195E / R394E / E395W, D199R / T203A / K297D / R394E / E395W, T203A / K278E / K297D / R394E, T203A / K297D / D314R / R394E / E395W, T203A / E310R / The amino acid sequence has one or more residue differences compared to SEQ ID NO: 1336 selected from D314R / R394E / E395W / L419M, T203A / T344V / R394E, T203A / F353K, T203A / R394E, T203A / R394E / E395W, K297D / R394E, D314R / R394E / E395W, T344V / R394E / E395W, F353K, and R394E.

[0154] In some embodiments, the engineered TdT polypeptide has at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reference sequence SEQ ID NO: 1336, and is 61, 152 / 503, 160, 162, 165, 177, 200, 200 / 425, 213, 217, 219, 223, 236, 246, 248, 292, 292 400 / 401 / 402, 425, 427, 435, 435 / 503, 437, 440, 441, 442, 443, 444, 446, 459, 460, 488, 490, 501, 502, 503, 504, and 506.In some embodiments, the engineered TdT polypeptide has at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reference sequence SEQ ID NO: 1336 and is selected from the group consisting of 61A, 152H / 503S, 160M, 160N, 160S, 160V, 162A, 165K ... 77L, 200C, 200C / 425K, 200K, 200V, 213S, 217Q, 219L, 219R, 223Q, 236I, 236P, 236V, 246K, 248C, 248S, 292L / 411 P, 292T, 295S, 326C, 326M, 326N, 326S, 326T, 329K, 329R, 330E, 333A, 333D, 333G, 333H, 333R, 334E, 334R, 334S, 338T, 340A, 340G, 340M, 340M / 438V, 340R, 340S, 363C, 369G, 369M, 369N, 370G, 372G, 373H, 373N, 383R, 400A / 40 1E / 402F, 425D, 425R, 425T, 427E, 427Q, 435A, 435C, 435E, 435G, 435K, 435Q, 435S, 435S / 503M, 435T, 437Q, 437S , 440E, 440V, 441N, 442G, 443T, 444R, 446E, 446P, 459Q, 460G, 488S, 490E, 490H, 490R, 490V, 490W, 501A, 502G, 502R, 503E, 503Q, 503R, 503V, 504N, 504R, 504W, and 506E.In some embodiments, the engineered TdT polypeptide has at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reference sequence SEQ ID NO: 1336 and is selected from the group consisting of T61A, R152H / I503S, T160M, T160N, T160S, T160V, T162A, Q165K, H177L, T200C, T200C / H 425K, T200K, T200V, C213S, E217Q, V219L, V219R, D223Q, G236I, G236P, G236V, E246K, L248C, L248S, S292L / L411P, S292T, T295S , L326C, L326M, L326N, L326S, L326T, Q329K, Q329R, D330E, W333A, W333D, W333G, W333H, W333R, T334E, T334R, T334S, D338T, L34 0A, L340G, L340M, L340M / G438V, L340R, L340S, S363C, E369G, E369M, E369N, Q370G, D372G, Q373H, Q373N, K383R, D400A / G401E / K402F, H425D, H425R, H425T, K427E, K427Q, M435A, M435C, M435E, M435G, M435K, M435Q, M435S, M435S / I503M, M435T, T437Q, T437 S, N440E, N440V, E441N, S442G, E443T, A444R, S446E, S446P, Y459Q, D460G, K488S, M490E, M490H, M490R, M490V, M490W, K501A, K502G, K502R, I503E, I503Q, I503R, I503V, F504N, F504R, F504W, and K506E.

[0155] In some embodiments, the engineered TdT polypeptide has at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reference sequence SEQ ID NO: 1348, and 7 / 481 / 485, 3 / 307 / 321 / 340 / 353 / 406 / 408 / 445, 148 / 175 / 201 / 457 / 485, 175 / 201 / 333 / 412 / 425 / 457 / 485, 175 / 325 / 397, 175 / 333, 175 / 333 / 369 / 481, 175 / 333 / 485, 175 / 485, 199 / 307 / 321 / 340 / 406 / 408 / 445 / 484, 201 / 213 / 333 / 344 / 397 / 425 / 481 / 485, 201 / 333 / 344 / 457 / 481, 201 / 333 / 481, 201 / 406 / 408 / 462 / 484 / 502, 213, 213 / 333 / 397, 307 / 333 / 340 / 408 / 445 / 462 / 502, 307 / 373 / 406 / 408 / 484, The amino acid sequence has one or more residue differences when compared to SEQ ID NO: 1348 selected from: 321 / 333 / 340, 325 / 333 / 369 / 425, 325 / 425 / 457 / 481, 333, 333 / 344 / 369 / 397, 333 / 344 / 369 / 485, 340 / 484, 344 / 485, 353 / 406, 373, and 406 / 408.In some embodiments, the engineered TdT polypeptide has at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reference sequence SEQ ID NO: 1348 and is selected from the group consisting of 3Q / 175D / 213S / 313R / 325T / 340R / 457V / 481W / 485D, 3Q / 307M / 321 ... K / 340R / 353K / 406G / 408A / 445N, 148T / 175D / 201A / 457V / 485D, 175D / 201A / 333A / 412N / 425D / 457V / 485D, 175D / 325T / 397Q, 175D / 333A, 175D / 333A / 369N / 481W, 175D / 333A / 485D, 175D / 485D, 199H / 307M / 321K / 340R / 406G / 408A / 445N / 4 84M, 201A / 213S / 333A / 344V / 397Q / 425D / 481W / 485D, 201A / 333A / 344V / 457V / 481W, 201A / 333A / 481W, 201R / 406G / 40 8A / 462L / 484M / 502G, 213S, 213S / 333A / 397Q, 307M / 333G / 340R / 408A / 445N / 462L / 502G, 307M / 373H / 406G / 408A / 484M , 321K / 333G / 340R, 325T / 333A / 369N / 425D, 325T / 425D / 457V / 481W, 333A / 344V / 369N / 397Q, 333A / 344V / 369N / 485D, 333G, 340R / 484M, 344V / 485D, 353K / 406G, 373H, and 406G / 408A.In some embodiments, the engineered TdT polypeptide has at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reference sequence SEQ ID NO: 1348 and is selected from the group consisting of H3Q / N175D / C213S / A313R / S325T / L340R / C457V / R481W / H485D, H3Q / C307M / A321K / L340R / F353K / R40 6G / I408A / K445N, P148T / N175D / C201A / C457V / H485D, N175D / C201A / W333A / D412N / H425D / C457V / H485D, N175D / S325T / R397Q, N175D / W333A, N175D / W333A / E369N / R481W, N175D / W333A / H485D, N175D / H485D, D199H / C307M / A321K / L340R / R406G / I408A / K445N / T484M, C201 A / C213S / W333A / T344V / R397Q / H425D / R481W / H485D, C201A / W333A / T344V / C457V / R481W, C201A / W333A / R481W, C201R / R406G / I408A / F 462L / T484M / K502G, C213S, C213S / W333A / R397Q, C307M / W333G / L340R / I408A / K445N / F462L / K502G, C307M / Q373H / R406G / I408A / T484M , A321K / W333G / L340R, S325T / W333A / E369N / H425D, S325T / H425D / C457V / R481W, W333A / T344V / E369N / R397Q, W333A / T344V / E369N / H485D, W333G, L340R / T484M, T344V / H485D, F353K / R406G, Q373H, and R406G / I408A.

[0156] In some embodiments, the engineered TdT polypeptide comprises an amino acid sequence having at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reference sequence SEQ ID NO: 1596 and having one or more residue differences compared to SEQ ID NO: 1596 selected from 160 / 219 / 460 / 503 / 506, 219 / 307 / 326, 252, 252 / 333, 406 / 408, 406 / 408 / 442 / 446, 406 / 408 / 490, 408, and 446. In some embodiments, an engineered TdT polypeptide comprises an amino acid sequence having at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reference sequence SEQ ID NO: 1596 and having one or more residue differences compared to SEQ ID NO: 1596 selected from 160M / 219R / 460G / 503E / 506E, 219R / 307M / 326T, 252K, 252K / 333H, 406G / 408A, 406G / 408A / 442G / 446P, 406G / 408A / 490R, 408A, and 446P. In some embodiments, the engineered TdT polypeptide has at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reference sequence SEQ ID NO: 1596 and is selected from the group consisting of T160M / V219R / D460G / I5 1596, comprising an amino acid sequence having one or more residue differences when compared to SEQ ID NO: 1596 selected from: 03E / K506E, V219R / C307M / L326T, A252K, A252K / A333H, R406G / I408A, R406G / I408A / S442G / S446P, R406G / I408A / M490R, I408A, and S446P.

[0157] In some embodiments, the engineered TdT polypeptide has at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reference sequence SEQ ID NO: 1596, and is selected from the group consisting of 162, 163, 165, 171, 177, 179, 188, 200, 205, 208, 23 3, 252, 253, 260, 261, 277, 307, 325, 329, 330, 353, 371, 376, 382, ​​393, 400, 402, 405, 406, 407, 410, 413, 419, 441, 442, 460, 464, 484, 488, 490, 495, 506, 508, and 520, when compared to SEQ ID NO: 1596. In some embodiments, the engineered TdT polypeptide has at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reference sequence SEQ ID NO: 1596 and is selected from the group consisting of 162H, 163V, 165P, 171K, 177Y, 179K, 188M, 200A, 205R, 208A, 233D, 252E, 253I, 260A, 261A, 261B, 261C, 261D, 261E, 261F, 261G, 261H, 261I ... , 260K, 261A, 277E, 307L, 325L, 329K, 330E, 353Q, 371D, 376H, 382L, 393I, 400E, 402Q, 405N, 406P, 407A, 410Q, 413G, 419A, 441M, 442A, 460E, 464Y, 484E, 488N, 490L, 495G, 506S, 508D, and 520D.In some embodiments, the engineered TdT polypeptide has at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reference sequence SEQ ID NO: 1596 and is selected from the group consisting of T162H, I163V, Q165P, R171K, H177Y, Q179K, L188M, T200A, M205R, I208A, E233D, A252E, V253I, Q260K, S2 61A, D277E, C307L, S325L, Q329K, D330E, F353Q, E371D, Q376H, W382L, L393I, D400E, K402Q, S405N, R406P, K407A, A410Q, H413G, L419A, E441M, S442A, D460E, F464Y, T484E, K488N, M490L, A495G, K506S, K508D, and E520D.

[0158] In some embodiments, the engineered TdT polypeptide has at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reference sequence SEQ ID NO: 1596, and is selected from the group consisting of 161, 162, 163, 165, 179, 188, 205, 208, 231, 233, 251, 252, 253, 261, 277, 306, 307, 321, 325, 327, 329, 353, 368, 370, 371, 376, 380, 393, 400, 402, 406, 410, 413, 414, 419, 426, 441, 442, 460, 464, 484, 488, 490, 495, 506, 508, and 518, when compared to SEQ ID NO: 1596. In some embodiments, the engineered TdT polypeptide has at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reference sequence SEQ ID NO: 1596 and is selected from the group consisting of 161R, 162H, 163V, 165P, 179K, 188M, 205R, 208A, 231T, 233D, 251K, 252E, 253I, 261A, 27 7E, 306F, 307L, 321K, 325L, 327I, 329K, 353Q, 368R, 370T, 371D, 376H, 380D, 393I, 400E, 402Q, 406P, 410Q, 413G, 414H, 419A, 426P, 441M, 442A, 460E, 464Y, 484E, 488N, 490L, 495G, 506S, 508D, and 518D.In some embodiments, the engineered TdT polypeptide has at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reference sequence SEQ ID NO: 1596 and is selected from the group consisting of K161R, T162H, I163V, Q165P, Q179K, L188M, M205R, I208A, G231T, E233D, Q251K, A252E, V253I, S261A, D277E, L306F , C307L, A321K, S325L, L327I, Q329K, F353Q, K368R, Q370T, E371D, Q376H, N380D, L393I, D400E, K402Q, R406P, A410Q, H413G, F414H, L419A, H426P, E441M, S442A, D460E, F464Y, T484E, K488N, M490L, A495G, K506S, K508D, and G518D.

[0159] In some embodiments, the engineered TdT polypeptide comprises an amino acid sequence having at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reference sequence SEQ ID NO: 1654 and having one or more residue differences compared to SEQ ID NO: 1654 selected from 160 / 165 / 203 / 205 / 219 / 353 / 460 / 488, 160 / 165 / 205 / 219 / 441, 160 / 203 / 205 / 441, 160 / 219 / 330 / 484, 179 / 353, 205 / 307 / 441 / 460 / 488, and 441. In some embodiments, the engineered TdT polypeptide has at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reference sequence SEQ ID NO: 1654 and is selected from the group consisting of 160M / 165P / 203E / 205R / 219R / 35 The amino acid sequence has one or more residue differences compared to SEQ ID NO: 1654 selected from: 3Q / 460G / 488N, 160M / 165P / 205R / 219R / 441M, 160M / 203E / 205R / 441M, 160M / 219R / 330K / 484E, 179K / 353Q, 205R / 307L / 441M / 460G / 488N, and 441M. In some embodiments, the engineered TdT polypeptide has at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reference sequence SEQ ID NO: 1654, and is selected from the group consisting of T160M / Q165P / T203E / M205R / V219R / F353Q / D460G / K488N, T160M / Q165P / M205R / V219R / E441M, T160M / T203E / M205R / E441M, T160M / V219R / D330K / T484E, Q179K / F353Q, M205R / C307L / E441M / D460G / K488N, and E441M.

[0160] In some embodiments, the engineered TdT polypeptide has at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reference sequence SEQ ID NO: 1654, and is 488, 160 / 165 / 179 / 203 / 307 / 376 / 413 / 495, 160 / 165 / 203 / 205 / 219 / 353 / 460 / 488, 160 / 165 / 203 / 205 / 307 / 371 / 376 / 413 / 414, 160 / 165 / 203 / 208 / 371 / 376 / 413 / 414, 160 / 165 / 205, 160 / 165 / 205 / 208, 160 / 165 / 205 / 219 / 441, 160 / 165 / 205 / 414 / 441 / 495, 160 / 165 / 307 / 353 / 413 / 414 / 441 / 488 / 4 95, 160 / 179 / 203 / 205 / 208 / 219 / 371 / 414 / 484 / 506, 160 / 179 / 208 / 219 / 307 / 413 / 414 / 503 / 506, 160 / 179 / 208 / 307 / 371 / 376 / 414, 160 / 179 / 307 / 376 / 441 / 488 / 503, 160 / 203 / 205 / 208 / 219 / 414 / 460 / 506, 160 / 203 / 205 / 208 / 413 / 460 / 484 / 488, 160 / 203 / 205 / 441, 160 / 203 / 208 / 307 / 353 / 495, 16 0 / 203 / 208 / 371 / 413 / 414, 160 / 203 / 208 / 413 / 414 / 441 / 484, 160 / 203 / 326 / 353 / 413 / 414 / 484 / 495, 160 / 205 / 208 / 219 / 326 / 441 / 484 / 488 / 503, 160 / 208 / 326 / 376 / 414 / 441 / 484 / 488, 160 / 208 / 371 / 441 / 484 / 506, 160 / 208 / 414 / 441 / 452 / 480 / 488 / 495, 160 / 219 / 307 / 371 / 506, 160 / 219 / 330 / 484,165 / 179 / 203 / 205 / 219 / 414 / 418 / 441 / 488 / 503, 165 / 179 / 203 / 205 / 484 / 503, 165 / 179 / 205 / 413 / 441, 165 / 179 / 208 / 353 / 413 / 414 / 503, 165 / 203 / 205, 165 / 203 / 205 / 307 / 414 / 441 / 484 / 495 / 503 / 506, 165 / 203 / 205 / 484 / 488, 165 / 203 / 208 / 326 / 376 / 503, 165 / 205, 165 / 208 / 326 / 413 / 414 / 484 / 495 / 506, 179, 179 / 203 / 205, 179 / 203 / 208 / 326 / 353 / 376 / 484, 179 / 205 / 208 / 353 / 414 / 441 / 460 / 484 / 488, 179 / 205 / 353, 179 / 208 / 353 / 460, 17 9 / 353, 203 / 205 / 208 / 307 / 330 / 353 / 441 / 460 / 503 / 506, 203 / 205 / 208 / 307 / 441, 203 / 205 / 208 / 353, 203 / 208 / 219 / 376 / 441, 203 / 208 / 219 / 441, 203 / 208 / 326 / 353, 203 / 413 / 503 / 506, 205 / 208 / 307 / 353 / 376 / 413, 205 / 2 205 / 307 / 441 / 460 / 488, 205 / 326 / 488 / 503 / 506, 208 / 488 / 506, 326 / 353 / 371 / 376 / 414, and 441. In some embodiments, the engineered TdT polypeptide has at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reference sequence SEQ ID NO: 1654, and is selected from the group consisting of 3Q / 160M / 205R / 208A / 219R / 307L / 353Q / 371D / 376H / 413G / 414H / 441M / 488N, 160M, 160M / 165P / 179K / 203E / 205R / 208A / 219R / 353Q / 376H / 413G / 414H / 441M / 488N,160M / 165P / 179K / 203E / 307L / 376H / 413G / 495G、160M / 165P / 203E / 205R / 219R / 353Q / 460G / 488N、160M / 165P / 203E / 205R / 307L / 371D / 376H / 413G / 414H、160M / 165P / 203E / 208A / 371D / 376H / 413G / 414H、160M / 165P / 205R、160M / 165P / 205R / 208A、160M / 165P / 205R / 219R / 441M、160M / 165P / 205R / 414H / 441M / 495G、160M / 165P / 307L / 353Q / 413G / 414H / 441M / 488N / 495G、160M / 179K / 203E / 205R / 208A / 219R / 371D / 414H / 484E / 506E、160M / 179K / 208A / 219R / 307L / 413G / 414H / 503E / 506E、160M / 179K / 208A / 307L / 371D / 376H / 414H、160M / 179K / 307L / 376H / 441M / 488N / 503E、160M / 203E / 205R / 208A / 219R / 414H / 460G / 506E、160M / 203E / 205R / 208A / 413G / 460G / 484E / 488N、160M / 203E / 205R / 441M、160M / 203E / 208A / 307L / 353Q / 495G、160M / 203E / 208A / 371D / 413G / 414H、160M / 203E / 208A / 413G / 414H / 441M / 484E、160M / 203E / 326T / 353Q / 413G / 414H / 484E / 495G、160M / 205R / 208A / 219R / 326T / 441M / 484E / 488N / 503E、160M / 208A / 326T / 376H / 414H / 441M / 484E / 488N、160M / 208A / 371D / 441M / 484E / 506E、160M / 208A / 414H / 441M / 452I / 480H / 488N / 495G、160M / 219R / 307L / 371D / 506E、160M / 219R / 330K / 484E、165P / 179K / 203E / 205R / 219R / 414H / 418I / 441M / 488N / 503E、165P / 179K / 203E / 205R / 484E / 503E、165P / 179K / 205R / 413G / 441M, 165P / 179K / 208A / 353Q / 413G / 414H / 503E, 165P / 203E / 205R, 165P / 203E / 205R / 307L / 414Y / 441M / 484E / 495G / 503E / 506E, 165P / 203E / 205R / 484E / 488N, 165P / 203E / 208A / 326T / 376H / 503E, 165P / 205R, 165P / 208A / 326T / 413G / 414H / 484E / 495G / 506E, 179K, 179K / 203E / 205R, 179K / 203E / 208A / 326T / 353Q / 376H / 484E, 179K / 205R / 208A / 353Q / 414H / 441M / 460G / 484E / 488N, 179K / 205R / 353Q, 179K / 208A / 353Q / 460G, 179K / 353Q, 203E / 205R / 208A / 307L / 330N / 3 53Q / 441M / 460G / 503E / 506E, 203E / 205R / 208A / 307L / 441M, 203E / 205R / 208A / 353Q, 203E / 208A / 219R / 376H / 441M, 203E / 2 08A / 219R / 441M, 203E / 208A / 326T / 353Q, 203E / 413G / 503E / 506E, 205R / 208A / 307L / 353Q / 376H / 413G, 205R / 208A / 414H, 20 The amino acid sequence has one or more residue differences compared to SEQ ID NO: 1654 selected from 5R / 219R / 307L / 353Q, 205R / 307L, 205R / 307L / 376H / 414H / 441M / 495G, 205R / 307L / 441M / 460G / 488N, 205R / 326T / 488N / 503E / 506E, 208A / 488N / 506E, 326T / 353Q / 371D / 376H / 414H, and 441M. In some embodiments, the engineered TdT polypeptide has at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reference sequence SEQ ID NO: 1654;And H3Q / T160M / M205R / I208A / V219R / C307L / F353Q / E371D / Q376H / H413G / F414H / E441M / K488N, T160M, T160M / Q165P / Q179K / T203E / M205R / I208A / V219R / F353Q / Q376H / H413G / F414H / E441M / K488N, T160M / Q165P / Q179K / T203E / C307L / Q376H / H413G / A495G, T160M / Q165P / T203E / M205R / V219 R / F353Q / D460G / K488N, T160M / Q165P / T203E / M205R / C307L / E371D / Q37 6H / H413G / F414H, T160M / Q165P / T203E / I208A / E371D / Q376H / H413G / F41 4H, T160M / Q165P / M205R, T160M / Q165P / M205R / I208A, T160M / Q165P / M20 5R / V219R / E441M, T160M / Q165P / M205R / F414H / E441M / A495G, T160M / Q16 5P / C307L / F353Q / H413G / F414H / E441M / K488N / A495G, T160M / Q179K / T2 03E / M205R / I208A / V219R / E371D / F414H / T484E / K506E, T160M / Q179K / I2 08A / V219R / C307L / H413G / F414H / I503E / K506E, T160M / Q179K / I208A / C 307L / E371D / Q376H / F414H, T160M / Q179K / C307L / Q376H / E441M / K488N / I 503E, T160M / T203E / M205R / I208A / V219R / F414H / D460G / K506E, T160M / T203E / M205R / I208A / H413G / D460G / T484E / K488N, T160M / T203E / M205R / E441M, T160M / T203E / I208A / C307L / F353Q / A495G, T160M / T203E / I208A / E371D / H413G / F414H, T160M / T203E / I208A / H413G / F414H / E441M / T484E,T160M / T203E / L326T / F353Q / H413G / F414H / T484E / A495G、T160M / M205R / I208A / V219R / L326T / E441M / T484E / K488N / I503E、T160M / I208A / L326T / Q376H / F414H / E441M / T484E / K488N、T160M / I208A / E371D / E441M / T484E / K506E、T160M / I208A / F414H / E441M / V452I / R480H / K488N / A495G、T160M / V219R / C307L / E371D / K506E、T160M / V219R / D330K / T484E、Q165P、 / Q179K / T203E / M205R / V219R / F414H / F418I / E441M / K488N / I503E、Q165P / Q179K / T203E / M205R / T484E / I503E、Q165P / Q179K / M205R / H413G / E441M、Q165P / Q179K / I208A / F353Q / H413G / F414H / I503E、Q165P / T203E / M205R、Q165P / T203E / M205R / C307L / F414Y / E441M / T484E / A495G / I503E / K506E、Q165P / T203E / M205R / T484E / K488N、Q165P / T203E / I208A / L326T / Q376H / I503E、Q165P / M205R、Q165P / I208A / L326T / H413G / F414H / T484E / A495G / K506E、Q179K、Q179K / T203E / M205R、Q179K / T203E / I208A / L326T / F353Q / Q376H / T484E、Q179K / M205R / I208A / F353Q / F414H / E441M / D460G / T484E / K488N、Q179K / M205R / F353Q、Q179K / I208A / F353Q / D460G、Q179K / F353Q、T203E / M205R / I208A / C307L / D330N / F353Q / E441M / D460G / I503E / K506E、T203E / M205R / I208A / C307L / E441M、T203E / M205R / I208A / F353Q、T203E / I208A / V219R / Q376H / E441M、T203E / I208A / V219R / E441M、T203E / I208A / L326T / F353Q、T203E / H413G / I503E / K506E、M205R / I208A / C307L / F353Q / Q376H / H413G、M205R / I208A / F414H、M205R / V219R / C307L / F353Q、M205R / C307L、M205R / C307L / Q376H / F414H / E441M / A495G、M205R / C307L / E441M / D460G / K488N、M205R / L326T / K488N / I503E / K506E、I208A / K488N / K506E、L326T / F353Q / E371D / Q376H / F414H、and E441M, and an amino acid sequence having one or more residue differences when compared to SEQ ID NO: 1654.

[0161] In some embodiments, the engineered TdT polypeptide comprises an amino acid sequence having at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reference sequence SEQ ID NO: 1830 and having one or more residue differences compared to SEQ ID NO: 1830 selected from 163 / 179 / 277 / 338 / 340, 163 / 414 / 441, 171 / 200 / 334 / 406 / 490, 177, and 292 / 406. In some embodiments, the engineered TdT polypeptide comprises an amino acid sequence having at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reference sequence SEQ ID NO: 1830 and having one or more residue differences compared to SEQ ID NO: 1830 selected from 163V / 179K / 277E / 338T / 340A, 163V / 414H / 441M, 171K / 200V / 334R / 406P / 490L, 177L, and 292T / 406P. In some embodiments, an engineered TdT polypeptide comprises an amino acid sequence having at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reference sequence SEQ ID NO: 1830 and having one or more residue differences compared to SEQ ID NO: 1830 selected from I163V / Q179K / D277E / D338T / L340A, I163V / F414H / E441M, R171K / T200V / T334R / G406P / M490L, H177L, and S292T / G406P.

[0162] In some embodiments, the engineered TdT polypeptide comprises an amino acid sequence having at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reference sequence SEQ ID NO: 1100 and having one or more residue differences compared to SEQ ID NO: 1100 selected from 101 / 137 / 264 / 476 / 525 and 264. In some embodiments, the engineered TdT polypeptide comprises an amino acid sequence having at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reference sequence SEQ ID NO: 1100 and having one or more residue differences compared to SEQ ID NO: 1100 selected from 101E / 137A / 264R / 476R / 525F and 264R. In some embodiments, an engineered TdT polypeptide comprises an amino acid sequence having at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reference sequence SEQ ID NO: 1100 and having one or more residue differences compared to SEQ ID NO: 1100 selected from T101E / M137A / L264R / E476R / S525F and L264R.

[0163] In some embodiments, the engineered TdT polypeptide comprises an amino acid sequence having at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reference sequence SEQ ID NO: 1654 and having one or more residue differences compared to SEQ ID NO: 1654 selected from 160 / 163 / 165 / 203 / 205 / 219 / 353 / 414 / 441 / 460 / 488 and 160 / 165 / 203 / 205 / 219 / 353 / 460 / 488. In some embodiments, an engineered TdT polypeptide comprises an amino acid sequence having at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reference sequence SEQ ID NO: 1654 and having one or more residue differences compared to SEQ ID NO: 1654 selected from 160M / 163V / 165P / 203E / 205R / 219R / 353Q / 414H / 441M / 460G / 488N and 160M / 165P / 203E / 205R / 219R / 353Q / 460G / 488N. In some embodiments, an engineered TdT polypeptide comprises an amino acid sequence having at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reference sequence SEQ ID NO: 1654 and having one or more residue differences compared to SEQ ID NO: 1654 selected from T160M / I163V / Q165P / T203E / M205R / V219R / F353Q / F414H / E441M / D460G / K488N and T160M / Q165P / T203E / M205R / V219R / F353Q / D460G / K488N.

[0164] In some embodiments, the engineered TdT polypeptide has at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reference sequence SEQ ID NO: 1596, and is selected from the group consisting of 160 / 165 / 203 / 205 / 219 / 353 / 406 / 408 / 442 / 446 / 460 / 488, ... and 406 / 408 / 442 / 446. In some embodiments, the engineered TdT polypeptide has at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reference sequence SEQ ID NO: 1596 and is selected from the group consisting of 160M / 165P / 203E / 205R / 219R / 353Q / 406G / 408A / 442G / 446P / 460G / 488N, 160M / 165P / 203E / 205R / 219R ... and 406G / 408A / 441M / 442G / 446P.In some embodiments, the engineered TdT polypeptide has at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reference sequence SEQ ID NO: 1596, and is selected from the group consisting of T160M / Q165P / T203E / M205R / V219R / F353Q / R406G / I408A / S442G / S446P / D460G / K488N, T160M / Q165P / M205R / V219R / R406G / I408A / E441M / S442G / S446P, T160M / T203E / M205R / R406G / I408A / E441M / S442G / S446P, T160M / V219R / D330K / R406G / I408A / S442G / S446P / T484E, M205R / C307L / R406G / I408A / E441M / S442G / S446P / D460G / K488N, R406G / I408A / E441M / S442G / S446P, and R406G / I408A / S442G / S446P.

[0165] In some embodiments, the engineered TdT polypeptide has at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reference sequence SEQ ID NO: 1950, and is selected from the group consisting of 163 / 169, 185, 186, 192, 193, 194, 197, 198, 245, 251, 258, 259, 261, 263, 271, 274, 278, 280, 284, 286, 290, 291, 297, 304, 306, 308, 316, 347, 352, 359, 362, 378, 393, 396, 398, 399, 405, 407, 409 / 414, 410 / 414, 411 / 414, 413 / 414, 414, 414 / 415, 414 / 417, 415, 455, 465, 466, 468, 494, and 509. In some embodiments, the engineered TdT polypeptide has at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reference sequence SEQ ID NO: 1950, and / 169E, 163I / 169R, 185C, 185F, 186C, 192C, 192G, 192I, 192L, 192R, 193R, 193S, 194A, 1 94C, 194D, 194G, 194M, 194W, 197G, 197L, 197R, 198A, 198L, 245A, 251R, 258E, 258G, 258 K, 258L, 258Q, 258W, 259N, 259V, 261A, 261G, 263I, 263S, 271C, 274M, 274N, 274P, 274Q , 274T, 274V, 278C, 278L, 278N, 280L, 280S, 284I, 284M, 286N, 286S, 290A, 291M, 291W, 2 97L, 297P, 304L, 306I, 306M, 308N, 316A, 316C, 347Q, 352G, 352R, 359V, 362S, 362Y, 378 L, 393R, 396T, 398W, 399C, 399D, 399F, 399G, 399T, 405G, 405L, 405Y, 407F, 407N, 407S,409K / 414F, 409Q / 414F, 410F / 414F, 410I / 414F, 410S / 414F, 410V / 414F, 410Y / 414F, 411A / 414F, 411 F / 414F, 411G / 414F, 411I / 414F, 411Q / 414F, 411R / 414F, 411T / 414F, 413A / 414F, 413F / 414F, 413G / 4 1950, 414F, 413I / 414F, 414F / 415W, 414F / 417W, 414G, 415F, 455I, 455L, 465E, 466M, 468M, 468Q, 468S, 468W, 494A, 494C, 494G, 494L, 494W, 509G, and 509K. In some embodiments, the engineered TdT polypeptide has at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reference sequence SEQ ID NO: 1950, and is selected from the group consisting of V163I / Q169E, V163I / Q169R, L1 85C, L185F, E186C, Y192C, Y192G, Y192I, Y192L, Y192R, E193R, E193S, F194A, F194C, F194D, F194G, F194M, F194W, N197G, N197L, N197R, D198A, D198L, G245A, Q251R, R258E, R258G, R258K, R258L, R258Q , R258W, Y259N, Y259V, S261A, S261G, K263I, K263S, V271C, K274M, K274N, K274P, K274Q, K274T, K27 4V, K278C, K278L, K278N, Y280L, Y280S, L284I, L284M, T286N, T286S, V290A, R291M, R291W, K297L, K2 97P, A304L, L306I, L306M, Y308N, V316A, V316C, F347Q, P352G, P352R, L359V, T362S, T362Y, V378L, L 393R, S396T, F398W, E399C, E399D, E399F, E399G, E399T, S405G, S405L, S405Y, K407F, K407N, K407S,D409K / H414F, D409Q / H414F, A410F / H414F, A410I / H414F, A410S / H414F, A410V / H414F, A410Y / H414F, L411A / H414F, L 411F / H414F, L411G / H414F, L411I / H414F, L411Q / H414F, L411R / H414F, L411T / H414F, H413A / H414F, H413F / H414F, H41 3G / H414F, H413I / H414F, H414F / A415W, H414F / C417W, H414G, A415F, V455I, V455L, A465E, L466M, G468M, G468Q, G468S, G468W, H494A, H494C, H494G, H494L, H494W, S509G, and S509K.

[0166] In some embodiments, the engineered TdT polypeptide has at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reference sequence SEQ ID NO: 2008 and is selected from the group consisting of 158 / 274 / 411 / 413 / 414, 185 / 274 / 413 / 414, 185 / 411 / 413 / 414, 263 / 411 / 413 / 414 / 468, 263 / 413 / 414, 274 / 286 / 41 and 468.In some embodiments, the engineered TdT polypeptide has at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reference sequence SEQ ID NO: 2008 and is selected from the group consisting of 158H / 274V / 411G / 413A / 414G, 185F / 274V / 413A / 414G, 185F / 411G / 413A / 414G, 263H / 411G / 413A / 414G / 468Q, 263H / 413A / 414G, 274V / 286N / 411G / 413A / 417W / 468Q, 274V / 411G / 417W / 468Q, 274V / 411G / 468Q, 274V / 413A / 414G / 417W / 468Q, 274V / 468Q, 278N / 411G / 413A / 468Q, 411G / 413A / 417W, 411G / 413A / 417W / 468Q, 411G / 413A / 468Q, 411G / 414G, 413A, 413A / 414G, 413A / 414G / 468Q, 413A / 417W / 468Q, 413A / 468Q, 414G, and 468Q.In some embodiments, the engineered TdT polypeptide has at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reference sequence SEQ ID NO: 2008, and is P158H / K274V / L411G / H413A / H414G, L185F / K274V / H413A / H414G, L185F / L411G / H413A / H414G, K263H / L411 G / H413A / H414G / G468Q, K263H / H413A / H414G, K274V / T286N / L411G / H413A / C417W / G468Q, K2 74V / L411G / C417W / G468Q, K274V / L411G / G468Q, K274V / H413A / H414G / C417W / G468Q, K274V / G468Q, K278N / L411G / H413A / G468Q, L411G / H413A / C417W, L411G / H413A / C417W / G468Q, L411 G / H413A / G468Q, L411G / H414G, H413A, H413A / H414G, H413A / H414G / G468Q, H413A / C417W / G468Q, H413A / G468Q, H414G, and G468Q.

[0167] In some embodiments, the engineered TdT polypeptide comprises an amino acid sequence having at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reference sequence SEQ ID NO: 2008 and having one or more residue differences compared to SEQ ID NO: 2008 selected from 3, 8, 11, 12, 15, 26, 26 / 27, 36, 39, 50, 58, 62, 76, 90, 116, 147, 151, 157, 246, 248, 249, 253, and 255. In some embodiments, the engineered TdT polypeptide has at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reference sequence SEQ ID NO: 2008, and is selected from the group consisting of 3A, 3G, 8P, 11C, 12V, 15A, 26A / 27R, 28A / 29R, 39A / 30R, 39B / 31R, 39C / 32R, 39D / 31D, 39E / 32R, 39F / 33R, 39G / 34R, 39H / 35R, 39H / 36R, 39H / 37R, 39H / 38R, 39H / 39R, 40H / 40R, 40H / 41R, 40H / 42R, 40H / 43R, 40H / 44R, 40H / 45R, 40H / 46R, 40H / 47R, 40H / 48R, 40H / 49R, 41H / 42R, 41H / 43R, 41H / 44R, 41H / 45R, 41H / 46R, 41H / 47R, 41H / 48R, 41H / 49R, 42 ...3H / 44R, 43H / 45R, 43H / 46R, 4 The amino acid sequence has one or more residue differences compared to SEQ ID NO: 2008 selected from: 26G, 26I, 26Q, 26T, 36G, 36K, 39A, 50L, 58M, 58N, 58S, 62W, 76P, 90L, 90S, 116A, 147G, 151S, 157V, 246N, 248R, 248V, 249A, 249G, 249R, 253L, and 255G. In some embodiments, the engineered TdT polypeptide has at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reference sequence SEQ ID NO: 2008 and is selected from the group consisting of H3A, H3G, G8P, G11C, S12V, I15A, R26A / Q27R, R26G, R26I , R26Q, R26T, S36G, S36K, Y39A, I50L, T58M, T58N, T58S, F62W, N76P, N90L, N90S, S116A, D147G, G151S, P157V, E246N, L248R, L248V, E249A, E249G, E249R, V253L, and N255G.

[0168] In some embodiments, the engineered TdT polypeptide comprises an amino acid sequence having at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reference sequence SEQ ID NO: 2008 and having one or more residue differences compared to SEQ ID NO: 2008 selected from 9, 11, 12, 15, 16, 26, 38, 39, 58, 70, 79, 81, 90, 151, 157, 158, and 249. In some embodiments, an engineered TdT polypeptide comprises an amino acid sequence having at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reference sequence SEQ ID NO: 2008 and having one or more residue differences compared to SEQ ID NO: 2008 selected from 9A, 9T, 11S, 12A, 12N, 12Q, 15L, 15Q, 16S, 26S, 26W, 38L, 38T, 39G, 58A, 70A, 79T, 81E, 90M, 151I, 157L, 158A, and 249T. In some embodiments, an engineered TdT polypeptide comprises an amino acid sequence having at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reference sequence SEQ ID NO: 2008 and having one or more residue differences compared to SEQ ID NO: 2008 selected from G9A, G9T, G11S, S12A, S12N, S12Q, I15L, I15Q, R16S, R26S, R26W, I38L, I38T, Y39G, T58A, K70A, S79T, S81E, N90M, G151I, P157L, P158A, and E249T.

[0169] In some embodiments, the engineered TdT polypeptide comprises an amino acid sequence having at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reference sequence SEQ ID NO: 2008 and having one or more residue differences compared to SEQ ID NO: 2008 selected from 189, 196, 205, 206, 262, 307, 314, 318, 324, 353, 397, 408, 410, 413, 469, 473, 480, 481, 491, and 493. In some embodiments, the engineered TdT polypeptide has at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reference sequence SEQ ID NO: 2008 and is selected from the group consisting of 189E, 189Q, 189R, 196A, 196S, 196T, 196Y, 205E, 206N, 262V, 307E, 307H, 307L, 307S, 314A, 314M, 314N, 314V ... 4Q, 318R, 324V, 353R, 397A, 408E, 408L, 408W, 410E, 413E, 413G, 413L, 413M, 413S, 469F, 469Y, 473A, 473D, 473G, 473P, 473Q, 473S, 480A, 480E, 480G, 480L, 480S, 480W, 481A, 481L, 481S, 481V, 491M, 493E, and 493Q.In some embodiments, the engineered TdT polypeptide has at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reference sequence SEQ ID NO: 2008 and is selected from the group consisting of A189E, A189Q, A189R, R196A, R196S, R196T, R196Y, R205E, R206N, F262V, C307E, C307H, C307L, C307S, R314A, R314M, R314Q, K318R , I324V, Q353R, Q397A, A408E, A408L, A408W, A410E, H413E, H413G, H413L, H413M, H413S, W469F, W469Y, R473A, R473D, R473G, R473P, R473Q, R473S, R480A, R480E, R480G, R480L, R480S, R480W, W481A, W481L, W481S, W481V, L491M, N493E, and N493Q.

[0170] In some embodiments, the engineered TdT polypeptide comprises an amino acid sequence having at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reference sequence SEQ ID NO: 2254 and having one or more residue differences compared to SEQ ID NO: 2254 selected from 197 / 407 / 455, 197 / 455, 284 / 398 / 466, 362 / 407 / 455, 396 / 398 / 410 / 466, 398 / 466, 399 / 411 / 416, and 466. In some embodiments, an engineered TdT polypeptide comprises an amino acid sequence having at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reference sequence SEQ ID NO: 2254 and having one or more residue differences compared to SEQ ID NO: 2254 selected from 197R / 407S / 455L, 197R / 455L, 284M / 398W / 466M, 362S / 407S / 455L, 396T / 398W / 410V / 466M, 398W / 466M, 399D / 411Q / 416Q, and 466M. In some embodiments, the engineered TdT polypeptide has at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reference sequence SEQ ID NO: 2254, and N197R / K407S / V455L , N197R / V455L, L284M / F398W / L466M, T362S / K407S / V455L, S396T / F398W / A410V / L466M, F398W / L466M, E399D / G411Q / K416Q, and L466M, when compared to SEQ ID NO: 2254.

[0171] In some embodiments, the engineered TdT polypeptide has at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reference sequence SEQ ID NO: 2514 and is selected from the group consisting of 26 / 90 / 94 / 248 / 261 / 266 / 362 / 455, 26 / 90 / 246, 26 / 90 / 246 / 2 48, 26 / 90 / 246 / 248 / 261, 26 / 90 / 246 / 248 / 362 / 455, 26 / 90 / 246 / 248 / 455, 26 / 90 / 246 / 266 / 362, 26 / 90 / 246 / 362, 26 / 90 / 246 / 455, 26 / 90 / 248, 26 / 90 / 248 / 266 / 455, 26 / 90 / 248 / 455, 26 / 90 / 248 / 455 / 459, 26 / 90 / 266, 26 / 90 / 362 / 455 , 26 / 173 / 248, 26 / 246, 26 / 246 / 248 / 362, 26 / 246 / 248 / 362 / 455, 26 / 246 / 248 / 455, 26 / 248, 26 / 248 / 261 / 266, 26 / 248 / 261 / 266 / 362 / 455, 26 / 248 / 266 / 362, 26 / 248 / 362 / 455, 26 / 248 / 455, 26 / 362, 26 / 362 / 455, 58 / 197 / 249 / 407 / 41 and 362. In some embodiments, the engineered TdT polypeptide has at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reference sequence SEQ ID NO: 2514 and is selected from the group consisting of 26G / 90L / 94K / 248R / 261A / 266V / 362S / 455L, 26G / 90L / 246N, 26G / 90L / 246N / 248R, 26G / 90L / 246N / 266V / 362S,26G / 90L / 248V / 455L, 26G / 90L / 248V / 455L / 459H, 26G / 90L / 266V, 26G / 90L / 362S / 455L, 26G / 246N, 26G / 246N / 248R / 362S, 26 G / 246N / 248V / 455L, 26G / 248R, 26G / 248V / 261A / 266V, 26I / 90L / 246N / 248R, 26I / 90L / 248R, 26I / 246N / 248R / 362S / 455L, 26 I / 248R / 266V / 362S, 26I / 248R / 455L, 26Q / 90L / 246N / 248R / 261A, 26Q / 90L / 246N / 248R / 362S / 455L, 26Q / 90L / 246N / 248R / 45 5L, 26Q / 90L / 246N / 248V / 362S / 455L, 26Q / 90L / 246N / 362S, 26Q / 90L / 246N / 455L, 26Q / 90L / 248R / 266V / 455L, 26Q / 173I / 248R , 26Q / 246N / 248V / 362S, 26Q / 248R, 26Q / 248R / 261A / 266V / 362S / 455L, 26Q / 248R / 362S / 455L, 26Q / 248R / 455L, 26Q / 248V, 26 Q / 248V / 266V / 362S, 26Q / 248V / 362S / 455L, 26Q / 362S, 26Q / 362S / 455L, 58N / 197L / 249R / 407N / 410S, 62W / 249R, 90L / 246N / 2 246N / 248R / 362S, 246N / 266V / 455L, 248R, 248R / 455L, 248V / 266V / 362S, 248V / 362S / 455L, and 362S. In some embodiments, the engineered TdT polypeptide has at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reference sequence SEQ ID NO: 2514;And R26G / N90L / E94K / L248R / S261A / K266V / T362S / V455L, R26G / N90L / E246N , R26G / N90L / E246N / L248R, R26G / N90L / E246N / K266V / T362S, R26G / N90L / L 248V / V455L, R26G / N90L / L248V / V455L / Y459H, R26G / N90L / K266V, R26G / N9 0L / T362S / V455L, R26G / E246N, R26G / E246N / L248R / T362S, R26G / E246N / L2 48V / V455L, R26G / L248R, R26G / L248V / S261A / K266V, R26I / N90L / E246N / L2 48R, R26I / N90L / L248R, R26I / E246N / L248R / T362S / V455L, R26I / L248R / K2 66V / T362S, R26I / L248R / V455L, R26Q / N90L / E246N / L248R / S261A, R26Q / N9 0L / E246N / L248R / T362S / V455L, R26Q / N90L / E246N / L248R / V455L, R26Q / N9 0L / E246N / L248V / T362S / V455L, R26Q / N90L / E246N / T362S, R26Q / N90L / E24 6N / V455L, R26Q / N90L / L248R / K266V / V455L, R26Q / T173I / L248R, R26Q / E24 6N / L248V / T362S, R26Q / L248R, R26Q / L248R / S261A / K266V / T362S / V455L, R 26Q / L248R / T362S / V455L, R26Q / L248R / V455L, R26Q / L248V, R26Q / L248V / K 266V / T362S, R26Q / L248V / T362S / V455L, R26Q / T362S, R26Q / T362S / V455L, T58N / N197L / E249R / K407N / V410S, F62W / E249R, N90L / E246N / L248R, N90L / E246N / L248R / S261A / K266V / T362S / V455L, N90L / E246N / L248R / K266V / T36 2S / V455L, E246N / L248R, E246N / L248R / T362S, E246N / K266V / V455L, L248R,The amino acid sequence has one or more residue differences compared to SEQ ID NO: 2514 selected from L248R / V455L, L248V / K266V / T362S, L248V / T362S / V455L, and T362S.

[0172] In some embodiments, the engineered TdT polypeptide has at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reference sequence SEQ ID NO: 2524 and is 9 / 16 / 62 / 157 / 246 / 249 / 362, 11 / 58 / 227 / 246, 12 / 16 / 158 / 246 / 248 / 249, 30 / 189 / 261 / 266 / 353 / 465 / 468, 30 / 189 266 / 307 / 353 / 468, 26 ...In some embodiments, the engineered TdT polypeptide has at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reference sequence SEQ ID NO: 2524 and is selected from the group consisting of 9T / 16S / 62W / 157L / 246E / 249R / 362T, 11S / 58N / 227M / 246E, 12A / 16S / 158A / 246E / 248L / 249R, 30E / 189R / 266V, 30G / 261A / 266V / 353R / 468S, 30G / 266V, 30G / 266 V / 353R, 30T / 189R / 261A / 266V / 353R / 465E / 468S, 30T / 266V / 303E, 38T, 38T / 81E / 318R, 38T / 197L, 39G, 39G / 79T, 58N / 157L / 158A / 362T, 70A / 353R, 79T / 81E, 81E, 189R, 189R / 261A, 189R / 353R, 246E / 249R, 261A / 353R, 266V / 307S / 353R / 468S, 266V / 353R / 468S, and 266V / 468S.In some embodiments, the engineered TdT polypeptide has at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reference sequence SEQ ID NO: 2524, and is selected from the group consisting of G9T / R16S / F62W / P 157L / N246E / E249R / S362T, G11S / T58N / L227M / N246E, S12A / R16S / P158A / N246E / R248L / E249 R, M30E / A189R / K266V, M30G / S261A / K266V / Q353R / Q468S, M30G / K266V, M30G / K266V / Q353R, M 30T / A189R / S261A / K266V / Q353R / A465E / Q468S, M30T / K266V / K303E, I38T, I38T / S81E / K318R , I38T / N197L, Y39G, Y39G / S79T, T58N / P157L / P158A / S362T, K70A / Q353R, S79T / S81E, S81E, A The amino acid sequence has one or more residue differences compared to SEQ ID NO: 2524 selected from: 189R, A189R / S261A, A189R / Q353R, N246E / E249R, S261A / Q353R, K266V / C307S / Q353R / Q468S, K266V / Q353R / Q468S, and K266V / Q468S.

[0173] In some embodiments, the engineered TdT polypeptide comprises an amino acid sequence having at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reference sequence SEQ ID NO: 2524 and having one or more residue differences compared to SEQ ID NO: 2524 selected from: 47 / 326, 169, 191 / 413, 200, 292, 304 / 329, 327 / 406, 329, 340, 353 / 459, 373, 379, 382, ​​402, 403, 404, 427, 429, 459, 461, 484, 490, 495, 504, and 506. In some embodiments, the engineered TdT polypeptide has at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reference sequence SEQ ID NO: 2524 and is selected from the group consisting of 47A / 326R, 169M, 191V / 413V, 200V, 292N, 304V / 329R, 327M / 406T, 329R, 340I, 340V, 35 2524, comprising an amino acid sequence having one or more residue differences compared to SEQ ID NO: 2524 selected from: 3H / 459V, 373G, 379I, 379L, 379M, 379V, 382F, 382L, 402G, 402S, 402V, 403A, 403E, 403P, 403R, 403S, 404D, 404S, 427L, 427M, 427R, 427W, 429R, 459I, 461S, 484A, 490R, 495C, 495G, 495S, 504K, and 506P.In some embodiments, the engineered TdT polypeptide has at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reference sequence SEQ ID NO: 2524 and is selected from the group consisting of V47A / L326R, Q169M, N191V / A413V, T200V, S292N, A304V / Q329R, L327M / G406T, Q329R, L340I, L340V, Q353H / Y459V, The amino acid sequence has one or more residue differences compared to SEQ ID NO: 2524 selected from Q373G, T379I, T379L, T379M, T379V, W382F, W382L, K402G, K402S, K402V, L403A, L403E, L403P, L403R, L403S, P404D, P404S, K427L, K427M, K427R, K427W, D429R, Y459I, G461S, T484A, M490R, A495C, A495G, A495S, F504K, and K506P.

[0174] In some embodiments, the engineered TdT polypeptide comprises an amino acid sequence having at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reference sequence SEQ ID NO: 2524 and having one or more residue differences compared to SEQ ID NO: 2524 selected from 175, 179, 189, 200, 203, 292, 293, 325, 340, 373, 379, 402, 403, 404, 406, 427, 459, 461, 484, 495, 506, and 508. In some embodiments, the engineered TdT polypeptide has at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reference sequence SEQ ID NO: 2524 and is selected from the group consisting of 175L, 179L, 189V, 200N, 203D, 203M, 292N, 293Q, 325W, 340V, 373C, 373G, 379L, , 379M, 402E, 402G, 402S, 403A, 403E, 403G, 403P, 404D, 404E, 404S, 406N, 427C, 427F, 427L, 427M, 427N, 427W, 427Y, 459I, 461S, 484A, 484H, 484M, 495G, 495S, 506P, 506S, 506T, 508S, and 508T.In some embodiments, the engineered TdT polypeptide has at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reference sequence SEQ ID NO: 2524 and is selected from the group consisting of N175L, Q179L, A189V, T200N, E203D, E203M, S292N, S293Q, S325W, L340V, Q373C, Q373G, T379L, T379M, K4 2524, comprising an amino acid sequence having one or more residue differences when compared to SEQ ID NO: 2524 selected from: 02E, K402G, K402S, L403A, L403E, L403G, L403P, P404D, P404E, P404S, G406N, K427C, K427F, K427L, K427M, K427N, K427W, K427Y, Y459I, G461S, T484A, T484H, T484M, A495G, A495S, K506P, K506S, K506T, K508S, and K508T.

[0175] In some embodiments, the engineered TdT polypeptide comprises an amino acid sequence having at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reference sequence SEQ ID NO: 2638 and having one or more residue differences compared to SEQ ID NO: 2638 selected from 11 / 30 / 79 / 189 / 480, 30 / 58 / 79 / 189 / 307 / 480, 79 / 189 / 307 / 410, and 79 / 307. In some embodiments, an engineered TdT polypeptide comprises an amino acid sequence having at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reference sequence SEQ ID NO: 2638 and having one or more residue differences compared to SEQ ID NO: 2638 selected from 11S / 30G / 79T / 189R / 480E, 30G / 58N / 79T / 189R / 307L / 480E, 79T / 189R / 307L / 410E, and 79T / 307L. In some embodiments, an engineered TdT polypeptide comprises an amino acid sequence having at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reference sequence SEQ ID NO: 2638 and having one or more residue differences compared to SEQ ID NO: 2638 selected from G11S / M30G / S79T / A189R / R480E, M30G / T58N / S79T / A189R / C307L / R480E, S79T / A189R / C307L / V410E, and S79T / C307L.

[0176] In some embodiments, the engineered TdT polypeptide has at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reference sequence SEQ ID NO: 2804 and is selected from the group consisting of 169 / 304 / 340 / 402 / 427, 169 / 304 / 340 / 427 / 429 / 504, 169 / 304 / 340 / 429 / 506, 169 / 304 / 340 / 504, 169 / 304 / 402 / 403 / 427 ...340 / 504, 169 / 304 / 340 / 504, 169 / 304 / 340 / 504, 169 / 304 / 340 / 504, 169 / 304 / 340 / 504, 169 / 304 / 34 04 / 403 / 427 / 504, 169 / 304 / 427 / 504 / 506, 169 / 304 / 504, 169 / 340 / 402 / 403 / 427 / 429 / 504, 169 / 340 / 402 / 403 / 427 / 504 / 506, 169 / 340 / 402 / 504, 169 / 3 40 / 427, 169 / 340 / 506, 169 / 402 / 403 / 504 / 506, 169 / 402 / 427 / 429 / 504, 169 / 402 / 504, 169 / 402 / 504 / 506, 169 / 403 / 427 / 506, 266 / 327 / 329 / 404 / 410, 29 2 / 327 / 329 / 468, 304, 304 / 340 / 402, 304 / 340 / 402 / 403 / 427 / 429 / 504 / 506, 304 / 340 / 402 / 403 / 504 / 506, 304 / 340 / 402 / 403 / 506, 304 / 340 / 402 / 427 / 50 4 / 506, 304 / 340 / 402 / 506, 304 / 340 / 403 / 427 / 429 / 504 / 506, 304 / 340 / 427, 304 / 402 / 403, 304 / 402 / 403 / 427 / 504, 304 / 402 / 403 / 429, 304 / 402 / 403 / 504 / 506, 304 / 402 / 403 / 506, 304 / 403 / 504, 304 / 504, 304 / 504 / 506, 327, 327 / 329, 327 / 329 / 379 / 404 / 406 / 410, 327 / 329 / 404 / 410, 327 / 329 / 465 / 484, 327 / 382 / 406 / 410 / 484, 327 / 410 / 484, 340, 340 / 402 / 403, 340 / 402 / 427 / 429 / 506, 340 / 402 / 429 / 504 / 506, 340 / 402 / 504, 340 / 403 / 504, 340 / 504, 340 / 506,The amino acid sequence has one or more residue differences when compared to SEQ ID NO: 2804 selected from: 379 / 382 / 468, 379 / 404 / 410, 379 / 410, 379 / 465 / 468 / 484, 379 / 468, 402 / 403 / 427 / 504 / 506, 402 / 403 / 429 / 504, 402 / 427 / 504, 402 / 504, 403 / 427, 427, 484, 504, 504 / 506, and 506. In some embodiments, the engineered TdT polypeptide has at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reference sequence SEQ ID NO: 2804 and is selected from the group consisting of 169M / 304V / 340I / 429R / 506P, 169M / 304V / 340I / 504K, ... V / 402G / 427L, 169M / 304V / 340V / 427L / 429R / 504K, 169M / 304V / 402G / 403S / 427L / 506P, 169M / 304V / 403S / 427L / 504K, 1 69M / 304V / 427M / 504K / 506P, 169M / 304V / 504K, 169M / 340I / 402G / 403A / 427M / 429R / 504K, 169M / 340I / 427L, 169M / 340I / 506P, 169M / 340V / 402G / 403P / 427M / 504K / 506P, 169M / 340V / 402G / 504K, 169M / 402G / 403A / 504K / 506P, 169M / 402G / 427 L / 429R / 504K, 169M / 402G / 504K, 169M / 402G / 504K / 506P, 169M / 403A / 427L / 506P, 266V / 327M / 329R / 404S / 410V, 292N / 32 7M / 329R / 468S, 304V, 304V / 340I / 402G, 304V / 340I / 402G / 403A / 427L / 429R / 504K / 506P, 304V / 340I / 402G / 403S / 506P, 3 04V / 340I / 402G / 506P, 304V / 340I / 403A / 427L / 429R / 504K / 506P, 304V / 340I / 427M, 304V / 340V / 402G / 403S / 504K / 506P,304V / 340V / 402G / 427L / 504K / 506P, 304V / 402G / 403A / 429R, 304V / 402G / 403A / 504K / 506P, 304V / 402G / 4 03P / 427M / 504K, 304V / 402G / 403P / 506P, 304V / 402G / 403S, 304V / 403S / 504K, 304V / 504K, 304V / 504K / 50 6P, 327M, 327M / 329R, 327M / 329R / 379M / 404D / 406T / 410V, 327M / 329R / 404D / 410V, 327M / 329R / 465E / 484 A, 327M / 382L / 406T / 410V / 484A, 327M / 410V / 484A, 340I / 402G / 429R / 504K / 506P, 340I / 402G / 504K, 340I / 403S / 504K, 340I / 506P, 340V, 340V / 402G / 403A, 340V / 402G / 427L / 429R / 506P, 340V / 504K, 379L / 465E / 468S / 484A, 379L / 468S, 379M / 382L / 468S, 379M / 404D / 410V, 379M / 410V, 379M / 468S, 402G / 403A / 427M / 5 402G / 403S / 427R / 504K, 402G / 427L / 504K, 402G / 504K, 403S / 427L, 427L, 427M, 484A, 504K, 504K / 506P, and 506P. In some embodiments, the engineered TdT polypeptide has at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reference sequence SEQ ID NO: 2804, and 04V / L340I / D429R / K506P, Q169M / A304V / L340I / F504K, Q169M / A304V / L340V / K402G / K427 L, Q169M / A304V / L340V / K427L / D429R / F504K, Q169M / A304V / K402G / L403S / K427L / K506P,Q169M / A304V / L403S / K427L / F504K、Q169M / A304V / K427M / F504K / K506P、Q169M / A304V / F504K、Q169M / L340I / K402G / L403A / K427M / D429R / F504K、Q169M / L340I / K427L、Q169M / L340I / K506P、Q169M / L340V / K402G / L403P / K427M / F504K / K506P、Q169M / L340V / K402G / F504K、Q169M / K402G / L403A / F504K / K506P、Q169M / K402G / K427L / D429R / F504K、Q169M / K402G / F504K、Q169M / K402G / F504K / K506P、Q169M / L403A / K427L / K506P、K266V / L327M / Q329R / P404S / E410V、S292N / L327M / Q329R / Q468S、A304V、A304V / L340I / K402G、A304V / L340I / K402G / L403A / K427L / D429R / F504K / K506P、A304V / L340I / K402G / L403S / K506P、A304V / L340I / K402G / K506P、A304V / L340I / L403A / K427L / D429R / F504K / K506P、A304V / L340I / K427M、A304V / L340V / K402G / L403S / F504K / K506P、A304V / L340V / K402G / K427L / F504K / K506P、A304V / K402G / L403A / D429R、A304V / K402G / L403A / F504K / K506P、A304V / K402G / L403P / K427M / F504K、A304V / K402G / L403P / K506P、A304V / K402G / L403S、A304V / L403S / F504K、A304V / F504K、A304V / F504K / K506P、L327M、L327M / Q329R、L327M / Q329R / T379M / P404D / G406T / E410V、L327M / Q329R / P404D / E410V、L327M / Q329R / A465E / T484A、L327M / W382L / G406T / E410V / T484A、L327M / E410V / T484A、L340I / K402G / D429R / F504K / K506P, L340I / K402G / F504K, L340I / L403S / F504K, L340I / K506P, L340V, L340V / K402G / L403A, L340V / K402G / K427L / D429R / K506P, L340V / F504K, T379L / A465E / Q468S / T484A, T379L / Q468S, T379M / W382L / Q468S, T379M / P404D / E410V, T379M / E410V, The amino acid sequence has one or more residue differences compared to SEQ ID NO: 2804 selected from T379M / Q468S, K402G / L403A / K427M / F504K / K506P, K402G / L403S / K427M / F504K / K506P, K402G / L403S / D429R / F504K, K402G / K427L / F504K, K402G / F504K, L403S / K427L, K427L, K427M, T484A, F504K, F504K / K506P, and K506P.

[0177] In some embodiments, the engineered TdT polypeptide has at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reference sequence SEQ ID NO: 2812, and is 30, 30 / 179 / 200 / 373 / 403, 30 / 179 / 373 / 37 9, 30 / 184 / 246 / 325 / 379 / 429 / 495, 30 / 184 / 246 / 327 / 329 / 459, 30 / 184 / 246 / 379 / 404, 30 / 184 / 246 / 459 / 461 / 495 / 500 / 504, 30 / 200 / 373, 30 / 200 / 373 / 379, 30 / 246 / 325 / 327 / 329 / 404 / 461, 30 / 246 / 325 / 379 / 404 / 4 27 / 429 / 461, 30 / 246 / 427 / 459 / 461, 30 / 325 / 327, 30 / 325 / 327 / 379 / 404 / 429 / 495, 30 / 327 / 404 / 504, 30 / 329 / 379, 30 / 373, 30 / 373 / 403, 30 / 379 / 429 / 459 / 461, 30 / 379 / 459 / 461 / 504, 30 / 403 / 441 / 460, 200 / 373 / 3 79, 246 / 325 / 329 / 379 / 461, 246 / 327 / 404 / 461 / 495, 327 / 329 / 379 / 504, 327 / 459 / 461 / 495, 353 / 403, 373 / 379, 373 / 379 / 403, 379 / 403 / 406 / 468, and 403 / 441.In some embodiments, the engineered TdT polypeptide has at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reference sequence SEQ ID NO: 2812 and is selected from the group consisting of 30G, 30G / 179A / 200N / 373G / 403E, 30G / 179A / 373G / 379M, 30G / 184S / 246E / 325W / 37 9M / 429R / 495S, 30G / 184S / 246E / 327M / 329R / 459I, 30G / 184S / 246E / 379M / 404D, 30G / 184S / 246E / 459I / 461S / 495S / 500N / 504Q , 30G / 200N / 373G, 30G / 200N / 373G / 379M, 30G / 246E / 325W / 327M / 329R / 404D / 461S, 30G / 246E / 325W / 379M / 404D / 427L / 429R / 46 1S, 30G / 246E / 427L / 459I / 461S, 30G / 325W / 327M, 30G / 325W / 327M / 379M / 404D / 429R / 495S, 30G / 327M / 404D / 504S, 30G / 329R / 3 79M, 30G / 373G, 30G / 373G / 403L, 30G / 379M / 429R / 459I / 461S, 30G / 379M / 459I / 461S / 504Q, 30G / 403E / 441K / 460P, 200N / 373G / The amino acid sequence has one or more residue differences compared to SEQ ID NO: 2812 selected from: 379M, 246E / 325W / 329R / 379M / 461S, 246E / 327M / 404D / 461S / 495S, 327M / 329R / 379M / 504S, 327M / 459I / 461S / 495S, 353R / 403E, 373G / 379M, 373G / 379M / 403L, 379M / 403L / 406N / 468S, and 403L / 441K.In some embodiments, the engineered TdT polypeptide has at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reference sequence SEQ ID NO: 2812 and is selected from the group consisting of M30G, M30G / Q179A / T200N / Q373G / S403E, M30G / Q179A / Q373G / T379M, M30G / A184S / N246E / S325W / T379M / D429R / A495S, M30G / A184S / N246E / L327M / Q329R / Y459I, M30G / A184S / N246E / T379M / P404D, M30G / A184S / N246E / Y459I / G461S / A495S / T500N / F504Q, M30G / T200N / Q3 73G, M30G / T200N / Q373G / T379M, M30G / N246E / S325W / L327M / Q329R / P404 D / G461S, M30G / N246E / S325W / T379M / P404D / K427L / D429R / G461S, M30G / N 246E / K427L / Y459I / G461S, M30G / S325W / L327M, M30G / S325W / L327M / T37 9M / P404D / D429R / A495S, M30G / L327M / P404D / F504S, M30G / Q329R / T379M , M30G / Q373G, M30G / Q373G / S403L, M30G / T379M / D429R / Y459I / G461S, M3 0G / T379M / Y459I / G461S / F504Q, M30G / S403E / M441K / G460P, T200N / Q373G / T379M, N246E / S325W / Q329R / T379M / G461S, N246E / L327M / P404D / G461S / A495S, L327M / Q329R / T379M / F504S, L327M / Y459I / G461S / A495S, Q353R / S403E, Q373G / T379M, Q373G / T379M / S403L, T379M / S403L / G406N / Q468S, and S403L / M441K.

[0178] In some embodiments, the engineered TdT polypeptide comprises an amino acid sequence having at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reference sequence SEQ ID NO: 2812 and having one or more residue differences compared to SEQ ID NO: 2812 selected from 198, 231, 233, 248, 253, 264, 266, 278, 326, 367, 370, 396, 414, 433, 435, 437, 444, 446, 485, 499, 503, 520, and 525. In some embodiments, the engineered TdT polypeptide has at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reference sequence SEQ ID NO: 2812 and is selected from the group consisting of 198E, 231S, 233R, 248K, 248T, 253I, 264E, 264L, 264T, 264V, 266R, 278M, 326R, 367D, 37 2812, comprising an amino acid sequence having one or more residue differences when compared to SEQ ID NO: 2812 selected from: 0D, 370G, 370M, 370S, 396R, 414E, 433A, 433E, 433G, 433M, 433P, 433R, 433S, 435A, 435E, 435G, 435S, 437G, 437R, 444G, 444R, 446G, 485E, 499M, 499R, 503A, 503E, 503T, 503V, 520P, 525Q, 525R, and 525S.In some embodiments, the engineered TdT polypeptide has at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reference sequence SEQ ID NO: 2812 and is selected from the group consisting of D198E, G231S, E233R, R248K, R248T, V253I, R264E, R264L, R264T, R264V, K266R, K278M, L326R, E367D, Q370D, Q370G, , Q370M, Q370S, T396R, G414E, W433A, W433E, W433G, W433M, W433P, W433R, W433S, M435A, M435E, M435G, M435S, T437G, T437R, A444G, A444R, P446G, D485E, L499M, L499R, I503A, I503E, I503T, I503V, E520P, F525Q, F525R, and F525S.

[0179] In some embodiments, the engineered TdT polypeptide comprises an amino acid sequence having at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reference sequence SEQ ID NO: 2812 and having one or more residue differences compared to SEQ ID NO: 2812 selected from 186, 188, 198, 231, 233, 235, 243, 248, 253, 264, 266, 287, 297 / 440, 366, 367, 368, 370, 414, 433, 435, 437, 439, 442, 444, 485, 501, and 515. In some embodiments, the engineered TdT polypeptide has at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reference sequence SEQ ID NO: 2812 and is selected from the group consisting of 186D, 188I, 198E, 231S, 233A, 235R, 243L, 243R, 243S, 243T, 248S, 253I, 264A, 265R, 266R, 267R, 268R, 269R, 270R, 271R, 272R, 273R, 274R, 275R, 276R, 277R, 278R, 279R, 280R, 281R, 282R, 283R, 284R, 285R, 286R, 287R, 288R, 289R, 289R, 290R, 300R, 301R, 302R, 303R, 304R, 305R, 306R, 307R, 308R, 309R, 310R, 311R, 312R, 313R, 314R, 315R, 316R, 317R, 318R, 319R, 320R, 321R, 322R, 323R, 324R, 325R, 32 The amino acid sequence has one or more residue differences compared to SEQ ID NO: 2812 selected from: 4T, 266R, 266T, 287I, 297S / 440K, 366V, 367A, 368R, 370M, 370N, 370S, 414E, 433M, 433P, 433V, 435E, 435I, 435P, 437A, 437G, 437K, 437P, 439G, 439P, 442A, 444G, 444H, 485E, 485S, 501R, and 515E.In some embodiments, the engineered TdT polypeptide has at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reference sequence SEQ ID NO: 2812 and is selected from the group consisting of E186D, L188I, D198E, G231S, E233A, K235R, E243L, E243R, E243S, E243T, R248S, V253I, R264A, R264T, K266R , K266T, L287I, K297S / N440K, A366V, E367A, K368R, Q370M, Q370N, Q370S, G414E, W433M, W433P, W433V, M435E, M435I, M435P, T437A, T437G, T437K, T437P, S439G, S439P, G442A, A444G, A444H, D485E, D485S, K501R, and A515E.

[0180] In some embodiments, the engineered TdT polypeptide has at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reference sequence SEQ ID NO: 2956 and is 140 / 142 / 153 / 177 / 427 / 4 34 / 441 / 444 / 461 / 502, 140 / 142 / 153 / 427 / 484, 140 / 142 / 177, 140 / 142 / 177 / 441, 140 / 142 / 365 / 373 / 404 / 427 / 484, 140 / 142 / 373 / 427 / 484, 140 / 148 / 161 / 177 / 404, 140 / 150 / 153 / 365 / 373 / 427 / 48 4, 140 / 150 / 177 / 404 / 436 / 441 / 484 / 502, 140 / 161 / 177 / 404 / 427 / 484, 140 / 177 / 404, 140 / 177 / 404 / 484, 142 / 150 / 158 / 177 / 427 / 445, 142 / 150 / 177 / 404, 142 / 153 / 177 / 441 / 444, 142 / 177 / 373, 142 / 177 / 373 / 441, 142 / 461 / 484 / 502, 150 / 177, 153, 153 / 161 / 325 / 404 / 427 / 441 / 484, 153 / 484, 177, 177 / 365 / 427 / 434, 325 / 427, 427, and 484.In some embodiments, the engineered TdT polypeptide has at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reference sequence SEQ ID NO: 2956 and is selected from the group consisting of 140V / 142V / 153V / 177R / 427L / 434N / 441V / 444S / 461S / 502G, ... 140V / 142V / 153V / 427L / 484L, 140V / 142V / 177R, 140V / 142V / 177R / 441V, 140V / 142V / 365A / 373R / 404D / 427L / 484L , 140V / 142V / 373R / 427L / 484L, 140V / 148T / 161H / 177R / 404D, 140V / 150P / 153V / 365A / 373R / 427L / 484L, 140V / 150P / 177R / 404D / 436S / 441V / 484L / 502G, 140V / 161H / 177R / 404D / 427L / 484L, 140V / 177R / 404D, 140V / 177R / 404D / 484 L, 142V / 150P / 158S / 177R / 427L / 445N, 142V / 150P / 177R / 404D, 142V / 153V / 177R / 441V / 444S, 142V / 177R / 373R, 142 The amino acid sequence has one or more residue differences compared to SEQ ID NO: 2956 selected from V / 177R / 373R / 441V, 142V / 461S / 484L / 502G, 150P / 177R, 153V, 153V / 161H / 325L / 404D / 427L / 441V / 484L, 153V / 484L, 177R, 177R / 365A / 427L / 434N, 325L / 427L, 427L, and 484L.In some embodiments, the engineered TdT polypeptide has at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reference sequence SEQ ID NO: 2956, and is selected from the group consisting of G140V / M142V / E153V / H177R / K427L / E434N / M441V / A444S / G461S / K502G, ... K427L / T484L, G140V / M142V / H177R, G140V / M142V / H177R / M441V, G140V / M142V / G365A / Q373R / P404D / K427L / T484L, G140V / M142V / Q 373R / K427L / T484L, G140V / P148T / K161H / H177R / P404D, G140V / E150P / E153V / G365A / Q373R / K427L / T484L, G140V / E150P / H177R / P4 04D / P436S / M441V / T484L / K502G, G140V / K161H / H177R / P404D / K427L / T484L, G140V / H177R / P404D, G140V / H177R / P404D / T484L, M14 2V / E150P / P158S / H177R / K427L / K445N, M142V / E150P / H177R / P404D, M142V / E153V / H177R / M441V / A444S, M142V / H177R / Q373R, M142 The amino acid sequence has one or more residue differences compared to SEQ ID NO: 2956 selected from V / H177R / Q373R / M441V, M142V / G461S / T484L / K502G, E150P / H177R, E153V, E153V / K161H / W325L / P404D / K427L / M441V / T484L, E153V / T484L, H177R, H177R / G365A / K427L / E434N, W325L / K427L, K427L, and T484L.

[0181] In some embodiments, the engineered TdT polypeptide has at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reference sequence SEQ ID NO: 3174 and is selected from the group consisting of 186 / 188 / 248 / 253 / 365 / 366 / 444 / 445, 186 / 231 / 248 / 253 / 484, 186 / 231 / 248 / 365 / 366 / 368 / 484, 186 / 231 / 248 / 366 / 368 / 484, 186 / 231 / 248 / 366 / 368 / 445 ... 44, 186 / 231 / 248 / 484, 186 / 231 / 368 / 416 / 441 / 442 / 444 / 484 / 485, 186 / 231 / 441 / 444 / 445, 186 / 484 / 485, 188 / 231 / 248 / 253 / 365 / 441 / 442 / 444 / 445 / 484, 198 / 243 / 264 / 431 / 441, 198 / 243 / 396 / 414 / 431 / 433 / 441 / 499, 198 / 243 / 396 / 414 / 433 / 437 / 441 / 515, 198 / 264 / 266 / 414, 198 / 264 / 396 / 41 4 / 433 / 441 / 515, 198 / 266 / 396 / 414 / 433 / 441 / 499 / 501, 198 / 266 / 414 / 433 / 441 / 515, 198 / 266 / 437 / 441 / 499, 198 / 414 / 431 / 441 / 499 / 520, 198 / 414 / 433 / 441 / 515 / 520, 231 / 248 / 441 / 484, 243 / 264 / 515 / 520, 243 / 396 / 414 / 433 / 441 / 515 / 520, 243 / 414 / 433 / 437 / 441, 243 / 414 / 437 / 441 / 515, 248 In some embodiments, the engineered TdT polypeptide comprises an amino acid sequence having one or more residue differences compared to SEQ ID NO: 3174 selected from: 264 / 442 / 444 / 445 / 484, 264 / 266 / 396 / 414 / 433 / 441 / 515, 264 / 266 / 414 / 441 / 499 / 501 / 515 / 520, 264 / 414, 264 / 414 / 441, 264 / 433 / 441, 266 / 437 / 441 / 499 / 515 / 520, 365 / 366 / 441 / 484 / 485, 396 / 414 / 441, 396 / 414 / 441 / 515, and 414 / 441 / 520.366V / 368R / 444H / 445N, 186D / 231S / 248K / 253I / 484L, 186D / 231S / 248K / 365A / 366V / 368R / 484L, 186D / 231S / 248K / 366V / 368R / 444H, 186 D / 231S / 248K / 484L, 186D / 231S / 368R / 416N / 441M / 442K / 444A / 484L / 485E , 186D / 231S / 441M / 444H / 445N, 186D / 484L / 485E, 188I / 231S / 248K / 253I / 365A / 441M / 442K / 444A / 445N / 484L, 198E / 243S / 264A / 431R / 441M, 198E / 2 43S / 396S / 414E / 431R / 433V / 441M / 499M, 198E / 243S / 396S / 414E / 433R / 43 7R / 441M / 515E, 198E / 264A / 266T / 414E, 198E / 264A / 396S / 414E / 433V / 441 M / 515E, 198E / 266T / 396S / 414E / 433V / 441M / 499M / 501R, 198E / 266T / 414E / 433S / 441M / 515E, 198E / 266T / 437R / 441M / 499M, 198E / 414E / 431R / 441M / 499M / 520D, 198E / 414E / 433V / 441M / 515E / 520D, 231S / 248K / 441M / 484L, 2 43S / 264A / 515E / 520D, 243S / 396S / 414E / 433V / 441M / 515E / 520D, 243S / 41 4E / 433S / 437R / 441M, 243S / 414E / 437R / 441M / 515E, 248K / 442K / 444A / 445 N / 484L, 264A / 266T / 396S / 414E / 433R / 441M / 515E, 264A / 266T / 414E / 441M / 499M / 501R / 515E / 520D, 264A / 414E, 264A / 414E / 441M, 264A / 433S / 441M,The amino acid sequence has one or more residue differences compared to SEQ ID NO: 3174 selected from 266T / 437R / 441M / 499M / 515E / 520D, 365A / 366V / 441M / 484L / 485E, 396S / 414E / 441M, 396S / 414E / 441M / 515E, and 414E / 441M / 520D. In some embodiments, the engineered TdT polypeptide has at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reference sequence SEQ ID NO: 3174 and is selected from the group consisting of E186D / L188I / R248K / V253I / G365A / A366V / S444H / K445N, E186D / G231S / R248K / V253I / T484L, E186D / G231S / R248K / G365A / A366V / K368R / T484L, E186D / G231S / R248K / A366V / K368R / S444H, E186D / G231S / R 248K / T484L, E186D / G231S / K368R / K416N / V441M / G442K / S444A / T484L / D485E, E186D / G231S / V441M / S444H / K445N, E186D / T4 84L / D485E, L188I / G231S / R248K / V253I / G365A / V441M / G442K / S444A / K445N / T484L, D198E / E243S / R264A / S431R / V441M, D19 8E / E243S / T396S / G414E / S431R / W433V / V441M / L499M, D198E / E243S / T396S / G414E / W433R / T437R / V441M / A515E, D198E / R264 A / K266T / G414E, D198E / R264A / T396S / G414E / W433V / V441M / A515E, D198E / K266T / T396S / G414E / W433V / V441M / L499M / K501R , D198E / K266T / G414E / W433S / V441M / A515E, D198E / K266T / T437R / V441M / L499M, D198E / G414E / S431R / V441M / L499M / E520D,D198E / G414E / W433V / V441M / A515E / E520D, G231S / R248K / V441M / T484L, E243S / R264A / A515E / E520D, E243S / T396S / G414E / W433V / V441M / A515E / E520D, E243S / G414E / W433S / T437R / V441M, E243S / G414E / T437R / V441M / A515E, R248K / G442K / S444A / K445N / T484L, R264A / K266T / T396S / G414E / W433R / V441M / A515E, R264A / and G414E / V441M / E520D, R264A / G414E, R264A / G414E / V441M, R264A / W433S / V441M, K266T / T437R / V441M / L499M / A515E / E520D, G365A / A366V / V441M / T484L / D485E, T396S / G414E / V441M, T396S / G414E / V441M / A515E, and G414E / V441M / E520D.

[0182] In some embodiments, the engineered TdT polypeptide has at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reference sequence SEQ ID NO: 3174, and is 122 / 473, 189, 190, 193, 194, 196, 263, 264, 266, 267, 268, 269, 273, 273 / 50 1, 274, 278, 279, 281, 297, 298, 299, 300, 301, 302, 304, 309, 312, 315, 347, 350, 352, 353, 359, 390, 392, 394, 407, 408, 410, 411, 413, 414, 416, 436, 454, 468, 472, 473, 477, 479, 480, and 493 when compared to SEQ ID NO: 3174. In some embodiments, the engineered TdT polypeptide has at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reference sequence SEQ ID NO: 3174, and , 189L, 190L, 190M, 190R, 193S, 193T, 194C, 194L, 194S, 194W, 196G, 196L, 196M, 196N, 196 T, 263M, 263R, 264C, 266F, 266I, 266R, 266T, 266V, 266Y, 267A, 267C, 267H, 267V, 267Y, 268 C, 268I, 268L, 268T, 269W, 273C, 273D, 273E, 273E / 501N, 273F, 273G, 273I, 273L, 273Y, 27 4A, 274G, 274I, 274V, 278F, 278H, 278M, 278R, 278V, 279Y, 281K, 281L, 297C, 297L, 297M, 2 97R, 297T, 297V, 298I, 298M, 299A, 299L, 299M, 299N, 299Y, 300H, 301S, 301T, 301V, 302N, 302S, 304D, 304M, 304T, 309F, 309G, 309I, 309L, 309M, 309V, 309W, 312M, 312T, 312V, 315Q,347I, 350L, 350R, 350W, 352V, 353A, 359G, 390C, 390I, 390L, 390V, 392V, 394A, 394F, 394G, 394M, 394V , 394Y, 407L, 407M, 407N, 407R, 407W, 408G, 408I, 408L, 408M, 408T, 408V, 410F, 410G, 410I, 411E, 411N , 413F, 413L, 413P, 413S, 413V, 414A, 416G, 436S, 454F, 454M, 454V, 468F, 468H, 468M, 468T, 472G, 473G, 477G, 479F, 479V, 480H, 480K, 493V, and 493Y. In some embodiments, the engineered TdT polypeptide has at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reference sequence SEQ ID NO: 3174 and is selected from the group consisting of M122I / R473K, R189L, E190L, E191L, M122I / R473K, R189L, E191L, E192L, M122I / R473K, R189L, E192L, E193L, M122I / R473K, R189L, E193L, E194L, M122I / R473K, R189L, E194L, E195L, M122I / R473K, R189L, E195L, E196L, M122I / R473K, R189L, E196L, E197L, M122I / R473K, R189L, E197L, E198L, M122I / R473K, R189L, E198L, E199L, M122I / R473K, R189L, E19 ... 90M, E190R, E193S, E193T, F194C, F194L, F194S, F194W, R196G, R196L, R196M, R196N, R196T, H263M, H263R, R264C, K266F, K266I, K266R, K266T, K266V, K266Y, S267A, S267C, S267H, S267V, S267Y, V268C , V268I, V268L, V268T, F269W, V273C, V273D, V273E, V273E / K501N, V273F, V273G, V273I, V273L, V27 3Y, K274A, K274G, K274I, K274V, K278F, K278H, K278M, K278R, K278V, W279Y, R281K, R281L, K297C, K2 97L, K297M, K297R, K297T, K297V, L298I, L298M, T299A, T299L, T299M, T299N, T299Y, P300H, M301S, M 301T, M301V, Q302N, Q302S, V304D, V304M, V304T, Y309F, Y309G, Y309I, Y309L, Y309M, Y309V, Y309W,L312M, L312T, L312V, G315Q, F347I, G350L, G350R, G350W, P352V, Q353A, L359G, Y390C, Y390I, Y390L, Y390V, R392V, E394A, E39 4F, E394G, E394M, E394V, E394Y, K407L, K407M, K407N, K407R, K407W, A408G, A408I, A408L, A408M, A408T, A408V, E410F, E410G, The amino acid sequence has one or more residue differences compared to SEQ ID NO: 3174 selected from E410I, G411E, G411N, A413F, A413L, A413P, A413S, A413V, G414A, K416G, P436S, L454F, L454M, L454V, Q468F, Q468H, Q468M, Q468T, S472G, R473G, R477G, L479F, L479V, R480H, R480K, N493V, and N493Y.

[0183] In some embodiments, the engineered TdT polypeptide comprises an amino acid sequence having at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reference sequence SEQ ID NO: 3174 and having one or more residue differences compared to SEQ ID NO: 3174 selected from 196, 263, 266, 268, 273, 281, 394, 416, 454, 468, 473, 477, and 493. In some embodiments, the engineered TdT polypeptide has at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reference sequence SEQ ID NO: 3174, and is selected from the group consisting of 196G, 263R, 266I, 26 The amino acid sequence has one or more residue differences compared to SEQ ID NO: 3174 selected from: 6T, 266V, 268I, 273I, 273L, 281K, 394A, 394F, 394G, 394M, 394V, 394Y, 416S, 454M, 468A, 468F, 468H, 468M, 468T, 473G, 477S, and 493V. In some embodiments, the engineered TdT polypeptide has at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reference sequence SEQ ID NO: 3174, and is selected from the group consisting of R196G, H263R, K266I, K266T, K266V , V268I, V273I, V273L, R281K, E394A, E394F, E394G, E394M, E394V, E394Y, K416S, L454M, Q468A, Q468F, Q468H, Q468M, Q468T, R473G, R477S, and N493V.

[0184] In some embodiments, the engineered TdT polypeptide has at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reference sequence SEQ ID NO: 1830, and is 194, 196, 266, 267, 268, 269, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374 9, 273, 273 / 501, 274, 277, 278, 297, 298, 299, 301, 302, 309, 312, 347, 359, 390, 392, 394, 407, 408, 413, 416, 454, 468, 473, 477, 479, and 493 when compared to SEQ ID NO: 1830. In some embodiments, the engineered TdT polypeptide has at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reference sequence SEQ ID NO: 1830, and is selected from the group consisting of 194L, 194W, 196G, 196M, 196N, 266R, 266T, 266V, 267A, 268L, 268T, 269W, 273D, 273E, 273E / 501N, 273F, 273G, 273I, 273L, 273Y, 274A, 275L, 275W, 276G, 276M, 27 ... 4G, 274I, 274V, 277E, 278Y, 297R, 298M, 299A, 299N, 299S, 301S, 301T, 301V, 302S, 309F, 309L, 309M, 309W, 312V, 347I, 359G, 390C, 390V, 392V, 394Y, 407D, 407L, 407M, 407N, 407R, 408I, 413S, 416G, 416S, 454M, 454V, 468T, 473G, 477G, 479V, and 493V.In some embodiments, the engineered TdT polypeptide has at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reference sequence SEQ ID NO: 1830, and 94L, F194W, R196G, R196M, R196N, K266R, K266T, K266V, S267A, V268L, V268T, F269W, V273D, V273E, V273E / K501N, V273F, V273G, V273I, V273L, V273Y, K274A, K274G, K274I , K274V, D277E, K278Y, K297R, L298M, T299A, T299N, T299S, M301S, M301T, M301V, Q30 2S, Y309F, Y309L, Y309M, Y309W, L312V, F347I, L359G, Y390C, Y390V, R392V, E394Y, K4 07D, K407L, K407M, K407N, K407R, A408I, A413S, K416G, K416S, L454M, L454V, Q468T, R473G, R477G, L479V, and N493V.

[0185] In some embodiments, the engineered TdT polypeptide has at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reference sequence SEQ ID NO: 3222 and is selected from the group consisting of 186 / 194 / 248 / 396, 186 / 198 / 243 / 248 / 366 / 368 / 394 / 501, 186 / 231 / 243 / 368 / 394 / 485 / 499, ... 6 / 231 / 248 / 485, 186 / 231 / 366 / 368 / 394 / 485, 186 / 243, 186 / 243 / 248 / 366 / 368 / 394, 186 / 243 / 248 / 394 / 484 / 485, 186 / 243 / 484 / 520, 186 / 248, 186 / 365, 186 / 365 / 366 / 368 / 394 / 499, 186 / 365 / 366 / 394 / 485, 186 / 366 / 368 / 394 / 396 / 484, 186 / 366 / 368 / 394 / 396 / 484 485, 186 / 394 / 396 / 485, 194 / 198 / 243 / 366 / 368 / 499, 194 / 515 / 520, 198 / 231 / 243 / 248 / 485, 198 / 243 / 248 / 365 / 394 / 501, 198 / 248 / 394 / 396 / 484 / 485 / 499, 198 / 394 / 396 / 484 / 485 / 499, 198 / 394 / 396 / 499 / 515 / 520, 198 / 394 / 499 / 501, 231 / 365 / 368 / 394 / 499 / 520, 23 1 / 368 / 394, 231 / 484 / 485 / 499 / 501, 243 / 248 / 394 / 396 / 484 / 485, 243 / 484, 243 / 484 / 485 / 499, 248 / 365 / 366 / 368 / 394 / 484 / 520, 248 / 394 / 484, 365 / 366 / 368 / 394, 365 / 368 / 394 / 396 / 520, 394 / 396, and 394 / 499. In some embodiments, the engineered TdT polypeptide has an identity that is at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 110%, 111%, 112%, 113%, 114%, 115%, 116%, 117%, 118%, 119%, 120%, 121%, 122%, 123%, 124%, 125%, 126%, 127%, 128%, 129%, 130%, 131%, 132%, 133%, 134%, 135%, 136%, 137%, 138%, 139%, 140%, 141%, 142%, 143%, 144%, 145%, 146%, 147%, 148%, 149%, 150%, 151%, 152%, 153%, 154%, 155%, 156%, 157%, 158%, 159%, 160%, 161%, 162%, 163%, 164%, 165%, 166%, 167%, 168%, 169%, 170%, 171%, 172%, 173%, 174%, 175%, 176%, 177%, 178%, 179%, 180%, 181%, 182%, 183%,or more sequence identity with 186D / 194L / 248K / 396T, 186D / 198E / 243S / 248K / 366V / 368R / 394Y / 501R, 186D / 231S / 243S / 368R / 394Y / 485E / 499M, 186D / 231S / 248K / 485E, 186D / 231S / 366V / 368R / 394Y / 485E, 186D / 243S, 186D / 243S / 248K / 366V / 368R / 394Y, 186D / 243S / 248K / 394Y / 484L / 48 5E, 186D / 243S / 484L / 520D, 186D / 248K, 186D / 365A, 186D / 365A / 366V / 368R / 394Y / 499M, 186D / 365A / 366V / 394Y / 485E, 186D / 366V / 368R / 39 4Y / 396T / 484L, 186D / 366V / 368R / 394Y / 396T / 484L / 485E, 186D / 394Y / 396T / 485E, 194L / 198E / 243S / 366V / 368R / 499M, 194L / 515A / 520D, 19 8E / 231S / 243S / 248K / 485E, 198E / 243S / 248K / 365A / 394Y / 501R, 198E / 248K / 394Y / 396T / 484L / 485E / 499M, 198E / 394Y / 396T / 484L / 485E / 49 9M, 198E / 394Y / 396T / 499M / 515A / 520D, 198E / 394Y / 499M / 501R, 231S / 365A / 368R / 394Y / 499M / 520D, 231S / 368R / 394Y, 231S / 484L / 485E / 49 394Y / 499M, 248K / 365A / 366V / 368R / 394Y / 484L / 520D, 248K / 394Y / 484L, 365A / 366V / 368R / 394Y, 365A / 368R / 394Y / 396T / 520D, 394Y / 396T, and 394Y / 499M. In some embodiments, the engineered TdT polypeptide has an identity that is at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%,having 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity and E186D / F194L / R248K / S396T, E186D / D198E / E243S / R248K / A366V / K368R / E394Y / K501R, E186D / G231S / E243S / K368R / E394Y / D485E / L499M, E186D / G231S / R248K / D485E, E186D / G231S / A366V / K368R / E394Y / D485E, E186D / E243S, E186D / E243S / R248K / A366V / K368R / E394Y, E186D / E243S / R248K / E394Y / T4 84L / D485E, E186D / E243S / T484L / E520D, E186D / R248K, E186D / G365A, E186 D / G365A / A366V / K368R / E394Y / L499M, E186D / G365A / A366V / E394Y / D485E, E186D / A366V / K368R / E394Y / S396T / T484L, E186D / A366V / K368R / E394Y / S3 96T / T484L / D485E, E186D / E394Y / S396T / D485E, F194L / D198E / E243S / A366 V / K368R / L499M, F194L / E515A / E520D, D198E / G231S / E243S / R248K / D485E, D198E / E243S / R248K / G365A / E394Y / K501R, D198E / R248K / E394Y / S396T / T4 84L / D485E / L499M, D198E / E394Y / S396T / T484L / D485E / L499M, D198E / E394 Y / S396T / L499M / E515A / E520D, D198E / E394Y / L499M / K501R, G231S / G365A / K368R / E394Y / L499M / E520D, G231S / K368R / E394Y, G231S / T484L / D485E / L4 99M / K501R, E243S / R248K / E394Y / S396T / T484L / D485E, E243S / T484L, E243 S / T484L / D485E / L499M, R248K / G365A / A366V / K368R / E394Y / T484L / E520D,The amino acid sequence has one or more residue differences compared to SEQ ID NO: 3222 selected from R248K / E394Y / T484L, G365A / A366V / K368R / E394Y, G365A / K368R / E394Y / S396T / E520D, E394Y / S396T, and E394Y / L499M.

[0186] In some embodiments, the engineered TdT polypeptide has at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reference sequence SEQ ID NO: 3670, and 499, 273 / 493 / 499, 273 / 499, 274 / 299 / 408 / 416, 274 / 408 / 416, 274 / 416, 288 / 299 / 416, 298 / 299 / 416, 309, 309 / 499, 416, and 493 / 499. In some embodiments, the engineered TdT polypeptide has at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reference sequence SEQ ID NO: 3670 and is selected from the group consisting of 273I, 273I / 309M / 493V / 499L, 273I / 49 The amino acid sequence has one or more residue differences compared to SEQ ID NO: 3670 selected from 3V / 499L, 273I / 499L, 274A / 299N / 408I / 416S, 274A / 408I / 416S, 274I / 416S, 288V / 299N / 416S, 298M / 299N / 416S, 309M, 309M / 499L, 416S, and 493V / 499L.In some embodiments, the engineered TdT polypeptide has at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reference sequence SEQ ID NO: 3670, and is selected from the group consisting of V273I, V273I / Y309M / N493V / M499L, V273I / N493V / M499L, V273I / Y309M ... , V273I / M499L, K274A / T299N / A408I / K416S, K274A / A408I / K416S, K274I / K416S, E288V / T299N / K416S, L298M / T299N / K416S, Y309M, Y309M / M499L, K416S, and N493V / M499L.

[0187] In some embodiments, the engineered TdT polypeptide comprises an amino acid sequence having at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reference sequence SEQ ID NO: 3670 and having one or more residue differences compared to SEQ ID NO: 3670 selected from 168, 198, 267, 301, 307, 308, 308 / 361, 313, 372, 392, 397, 415, 419, 451, 452, 456, 472, 473, 475, 493 / 499, and 528. In some embodiments, the engineered TdT polypeptide has at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reference sequence SEQ ID NO: 3670 and is selected from the group consisting of 168S, 198S, 267M, 267R, 301G, 301Q, 301S, 307A, 307G, 307S , 308A, 308A / 361T, 308G, 308H, 308K, 308S, 308V, 313M, 372E, 392V, 397F, 397W, 415L, 415W, 419G, 419M, 451K, 452L, 456P, 456T, 472A, 473A, 473S, 475V, 493R / 499L, and 528L.In some embodiments, the engineered TdT polypeptide has at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reference sequence SEQ ID NO: 3670 and is selected from the group consisting of C168S, D198S, S267M, S267R, M301G, M301Q, M301S, L307A, L307G, L307S, Y308A, Y308B, Y308C, Y308D, Y308E, Y308F, Y308G, Y308H ... 08A / I361T, Y308G, Y308H, Y308K, Y308S, Y308V, A313M, D372E, R392V, Q397F, Q397W, A415L, A415W, L419G, L419M, R451K, V452L, R456P, R456T, S472A, R473A, R473S, F475V, N493R / M499L, and N528L.

[0188] In some embodiments, the engineered TdT polypeptide comprises an amino acid sequence having at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reference sequence SEQ ID NO: 3670 and having one or more residue differences compared to SEQ ID NO: 3670 selected from 303 / 396, 308, 473, and 493 / 499. In some embodiments, the engineered TdT polypeptide comprises an amino acid sequence having at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reference sequence SEQ ID NO: 3670 and having one or more residue differences compared to SEQ ID NO: 3670 selected from 303H / 396A, 308L, 473A, 473M, 473S, and 493V / 499L. In some embodiments, the engineered TdT polypeptide comprises an amino acid sequence having at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reference sequence SEQ ID NO: 3670 and having one or more residue differences compared to SEQ ID NO: 3670 selected from K303H / S396A, Y308L, R473A, R473M, R473S, and N493V / M499L.

[0189] In some embodiments, the engineered TdT polypeptide has at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reference sequence SEQ ID NO: 3670 and is selected from the group consisting of 273, 273 / 309 / 413 / 499, 273 / 309 / 493 / 499, 273 / 493, 273 / 309 / 413 / 499, 273 / 309 / 493 ... The amino acid sequence has one or more residue differences when compared to SEQ ID NO: 3670 selected from: 3 / 493 / 499, 273 / 499, 274 / 299 / 408 / 416, 274 / 408 / 416, 274 / 416, 281 / 413 / 499, 288 / 299 / 416, 298 / 299 / 416, 309, 309 / 413, 413, 413 / 493 / 499, 413 / 499, 416, and 493 / 499. In some embodiments, the engineered TdT polypeptide has at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reference sequence SEQ ID NO: 3670, and is selected from the group consisting of 273E / 493V, 273I, 273I / 309M / 413S / 499L, 273I / 309M / 493V / 499L, 273I / 493V / 499L, 273I / 499L , 273S / 309M / 413S / 499L, 274A / 299N / 408I / 416S, 274A / 408I / 416S, 274I / 416S, 281K / 413S / 499L, 288V / 299N / 416S, 298M / 299N / 416S, 309M, 309M / 413S, 413S, 413S / 493V / 499L, 413S / 499L, 416S, and 493V / 499L.In some embodiments, the engineered TdT polypeptide has at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reference sequence SEQ ID NO: 3670 and is selected from the group consisting of V273E / N493V, V273I, V273I / Y309M / A413S / M499L, V273I / Y309M / A413S / M499L, V273I / N493V / M499L, V273I / Y309M / A413S ... 3670, comprising an amino acid sequence having one or more residue differences when compared to SEQ ID NO: 3670 selected from: O9M / A413S / M499L, K274A / T299N / A408I / K416S, K274A / A408I / K416S, K274I / K416S, R281K / A413S / M499L, E288V / T299N / K416S, L298M / T299N / K416S, Y309M, Y309M / A413S, A413S, A413S / N493V / M499L, A413S / M499L, K416S, and N493V / M499L.

[0190] In some embodiments, the engineered TdT polypeptide has at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reference sequence SEQ ID NO: 3674, and 194 / 196 / 390 / 394 / 460 / 480, 194 / 196 / 390 / 394 / 480, 194 / 196 / 390 / 454 / 480, 194 / 196 / 390 / 480, 194 / 196 / 394 / 454 / 480, 194 / 196 / 454, 194 / 390, 194 / 394, 194 / 394 / 454, 194 / 394 / 454 / 480, 196, 196 / 390, 196 / 390 / 394, 196 / 390 / 394 / 454, 196 / 390 / 394 / 454 / 480, 196 / 390 / 394 / 480, 196 / 390 / 454, 196 / 394, 196 / 394 / 454, 196 / 394 / 454 / 480, 196 / 394 / 480, 196 / 454, 297 / The amino acid sequence has one or more residue differences when compared to SEQ ID NO: 3674 selected from: 470 / 473, 297 / 473 / 493, 390, 390 / 394, 390 / 394 / 454, 390 / 394 / 454 / 480, 390 / 394 / 480, 390 / 454, 390 / 480, 394, 394 / 480, and 454.In some embodiments, the engineered TdT polypeptide has at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reference sequence SEQ ID NO: 3674, and is selected from the group consisting of 194L / 196G / 390C, 194L / 196G / 390C / 394F / 460V ... / 480K, 194L / 196G / 390C / 394F / 480K, 194L / 196G / 390C / 454M / 480K, 194L / 196G / 390C / 480K, 194L / 196G / 394 F / 454M / 480K, 194L / 196G / 454M, 194L / 390C, 194L / 394F, 194L / 394F / 454M, 194L / 394F / 454M / 480K, 196G, 19 6G / 390C, 196G / 390C / 394F, 196G / 390C / 394F / 454M, 196G / 390C / 394F / 454M / 480K, 196G / 390C / 394F / 480K, 1 96G / 390C / 454M, 196G / 394F, 196G / 394F / 454M, 196G / 394F / 454M / 480K, 196G / 394F / 480K, 196G / 454M, 297R / The amino acid sequence has one or more residue differences compared to SEQ ID NO: 3674 selected from 470S / 473G, 297R / 473G / 493V, 390C, 390C / 394F, 390C / 394F / 454M, 390C / 394F / 454M / 480K, 390C / 394F / 480K, 390C / 454M, 390C / 480K, 394F, 394F / 480K, and 454M.In some embodiments, the engineered TdT polypeptide has at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reference sequence SEQ ID NO: 3674 and is selected from the group consisting of F194L / R196G / Y390C, F194L / R196G / Y390C / E394F / G460V / R480K, F194L / R196G / Y390C / E394F / R480K, F194L / R196G / Y390C / L454M / R480K, F194L / R196G / Y390C / R480K, F194L / R196G / E394F / L454M / R480K , F194L / R196G / L454M, F194L / Y390C, F194L / E394F, F194L / E394F / L454M, F194L / E394F / L454M / R480K, R196G, R196G / Y390C , R196G / Y390C / E394F, R196G / Y390C / E394F / L454M, R196G / Y390C / E394F / L454M / R480K, R196G / Y390C / E394F / R480K, R196G / Y390C / L454M, R196G / E394F, R196G / E394F / L454M, R196G / E394F / L454M / R480K, R196G / E394F / R480K, R196G / L454M, K297R / T470S / R473G, K297R / R473G / N493V, Y390C, Y390C / E394F, Y390C / E394F / L454M, Y390C / E394F / L454M / R480K, Y390C / E394F / R480K, Y390C / L454M, Y390C / R480K, E394F, E394F / R480K, and L454M.

[0191] In some embodiments, the engineered TdT polypeptide has at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reference sequence SEQ ID NO: 3796 and is selected from the group consisting of 198, 198 / 267 / 313 / 451 / 475 / 494 / 499, 198 / 267 / 314 / 451, 198 / 267 / 314 / 475, 198 / 267 / 409 / 451, 198 / 267 / 475, 198 / 267 / 409 ... 8 / 451 / 493, 208 / 308, 208 / 308 / 461, 267, 267 / 314 / 328 / 451 / 494 / 499, 267 / 451, 267 / 451 / 494 / 499, 308, 314 / 328 / 451 / 499, 314 / 451, 328 / 409 / 451, 328 / 451, 409, 409 / 475 / 494, 451, 451 / 493 / 494, 451 / 493 / 499, 451 / 494, and 475.In some embodiments, the engineered TdT polypeptide has at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reference sequence SEQ ID NO: 3796 and is selected from the group consisting of 198P, 198P / 267Q / 314A / 451K, 198P / 267Q / 314A / 475V, 198P / 267Q / 409L / 451K, 198P / 267Q / 475V, 198P / 267R / 313L / 451K / 475V / 494V / 499L, 198P / 451K / 493V, 20 The amino acid sequence has one or more residue differences compared to SEQ ID NO: 3796 selected from: 8V / 308A / 461N, 208V / 308V, 267Q / 451K / 494V / 499L, 267R, 267R / 314A / 328T / 451K / 494R / 499L, 267R / 451K, 308A, 308V, 314A / 328T / 451K / 499L, 314A / 451K, 328T / 409L / 451K, 328T / 451K, 409L, 409L / 475V / 494V, 451K, 451K / 493V / 494V, 451K / 493V / 499L, 451K / 494V, and 475V.In some embodiments, the engineered TdT polypeptide has at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reference sequence SEQ ID NO: 3796, and is selected from the group consisting of D198P, D198P / S2, D198P / S3, D198P / S4, D198P / S5, D198P / S6, D198P / S7, D198P / S8, D198P / S9, D198P / S10, D198P / S11, D198P / S12, D198P / S13, D198P / S14, D198P / S15, D198 67Q / R314A / R451K, D198P / S267Q / R314A / F475V, D198P / S267Q / D409L / R451K, D198P / S267Q / F475V, D198P / S267R / A313L / R451K / F475V / H494V / M499L, D198P / R451K / N493V, I208V / Y308A / S461N, I208V / Y308V, S267Q / R451K / H494V / M499L, S267R, S267R / R314A / V328T / R451K / H49 4R / M499L, S267R / R451K, Y308A, Y308V, R314A / V328T / R451K / M499L, R314A / R451K, V328T / D4 09L / R451K, V328T / R451K, D409L, D409L / F475V / H494V, R451K, R451K / N493V / H494V, R451K / N493V / M499L, R451K / H494V, and F475V.

[0192] In some embodiments, the engineered TdT polypeptide has at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reference sequence SEQ ID NO: 3870, and is 165, 169, 171, 173, 175, 176, 179, 183, 187, 191, 192, 195, 197, 199, 200, 203, The amino acid sequence comprises an amino acid sequence having one or more residue differences compared to SEQ ID NO: 3870 at a position or set of positions selected from: 204, 210, 257, 259, 267, 291, 293, 295, 301, 319, 325, 340, 341, 342, 374, 387, 398, 399, 403, 404, 406, 429, 480, 481, 483, 484, 490, 491, 493, 494, 495, 521, 507, 508, 509, and 522.In some embodiments, the engineered TdT polypeptide has at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reference sequence SEQ ID NO: 3870, and is selected from the group consisting of P165A, P165C, P165R, P165T, Q169M, Q169T, R171G, T173S, N175L, N175M, N175V, N176V, Q17 9K, N175I, Q179M, Q179P, Q179S, Q179T, Q179V, D183C, D183W, I187R, N191F, N191M, N191Q, N191V, Y192A, Y192M, Y192T, K195R, N1 97C, N197E, N197F, N197R, N197T, D199G, D199R, D199S, D199V, D199W, T200K, T200M, E203F, E203G, E203M, E203V, F204I, F204V, V2 10M, E257P, E257S, Y259F, R267S, R291M, S293C, S293T, T295A, T295G, T295N, T295S, T295V, M301G, M301W, A319E, W325G, W325L, I 340S, V341C, E342Q, L374V, L387I, W398V, E399S, S403K, S403L, S403R, S403T, S403V, ​​S403W, P404G, P404M, G406L, G406R, G406S, G 406T, R429F, R429H, R429V, R480A, R480Q, W481Q, A483C, L484S, M490A, M490E, M490L, M490R, M490S, M490V, L491V, N493K, H494A, H494T, S495C, S495M, S495N, S495R, A507G, K508N, S509R, Y521V, and L522I.

[0193] In some embodiments, the engineered TdT polypeptide has at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reference sequence SEQ ID NO: 3918, and is 3, 7 / 400 / 459 / 504, 164, 173 / 367 / 459 / 500, 184, 203, 210, 220, 230, 240, 250, 260, 270, 280, 290, 310, 320, 330, 340, 350, 360, 370, 380, 390, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930 03 / 367 / 459, 203 / 367 / 459 / 500 / 501, 203 / 400 / 459 / 501, 203 / 459, 203 / 459 / 499 / 504, 203 / 459 / 500, 215, 218, 294, 335, 335 / 402 / 481 / 484, 335 / 402 / 512, 335 / 481 / 484, 335 / 481 / 493, 335 / 481 / 512, 336, 338, 339, 367, 367 / 45 9 / 500, 370, 373, 376, 380, 384, 390, 395, 395 / 402, 395 / 402 / 481 / 484, 395 / 481, 395 / 481 / 512, 395 / 484, 400, 400 / 459, 400 / 459 / 499 / 500 / 504, 400 / 459 / 500 / 501, 400 / 459 / 501 / 504, 400 / 501, 400 / 504, 402, 402 / 481, 402 / 48 1 / 484, 402 / 481 / 484 / 512, 402 / 481 / 493, 402 / 484 / 493, 402 / 512, 458, 459, 459 / 501, 460, 481, 481 / 512, 484, 485, 493, 499, 500, 501, 504, 512, 515, and 516, an amino acid sequence having one or more residue differences compared to SEQ ID NO: 3918. In some embodiments, the engineered TdT polypeptide has at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reference sequence SEQ ID NO: 3918 and is selected from the group consisting of 3Q, H7- / D400W / Y459R / F504Q, S164G, T173I / E367S / Y459V / T500S, S184A, E203R, E203R / E367S / Y459R, E203R / E367S / Y459V / T500S / R501P,E203R / D400W / Y459R / R501P, E203R / Y459R / M499R / F504G, E203R / Y459R / T500S, E203R / Y459V, P215Q, V218L, K294D, F335V, F335V / G402K / W481R / L484E, F335V / G402K / W481R / L484R, F335V / G402K / E512R, F335V / G402R / W481R / L484R, F335V / W481D / L484R, F335V / W481R / L484 R, F335V / W481R / N493T, F335V / W481R / E512R, D336Q, D338E, D338Q, A339R, E367R, E367S, E367S / Y459V / T500S, E367T, Q370E, Q373G, Q37 6S, N380G, K384S, C390A, W395A, W395E, W395N, W395N / G402K, W395N / W481R / E512R, W395N / L484R, W395T, W395T / G402K / W481R / L484R, W39 5T / W481R, D400W, D400W / Y459R / T500S / R501P, D400W / Y459V, D400W / Y459V / M499R / T500S / F504Q, D400W / Y459V / R501P / F504G, D400W / R5 01P, D400W / F504Q, G402K, G402K / W481D, G402K / W481D / L484R / E512R, G402K / W481D / N493T, G402K / W481R / L484R, G402K / E512R, G402R, G The amino acid sequence has one or more residue differences compared to SEQ ID NO: 3918 selected from: 402R / L484R / N493T, G402S, P458R, Y459R, Y459V, Y459V / R501P, G460M, G460R, W481D, W481R, W481R / E512R, L484E, L484R, E485Q, N493T, M499P, M499R, T500S, R501N, R501P, F504G, F504Q, E512G, E512R, E515P, and H516T.

[0194] In some embodiments, the engineered TdT polypeptide has at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reference sequence SEQ ID NO: 4266, and is 95 / 428 / 480, 163 / 190, 163 / 203 / 366, 163 / 328 / 363 / 480, 163 / 363 / 480 / 485 , 172 / 174 / 178 / 340, 178, 188, 190 / 202 / 203 / 363 / 366 / 480 / 483 / 485, 190 / 202 / 203 / 480, 190 / 480 / 485, 192, 192 / 498 / 499 / 503, 202, 202 / 203 / 328 / 362 / 363 / 366 / 428 / 480 / 485 / 498 / 499 / 503, 202 / 203 / 485, 203 / 328 / 363 / 428 / 483, 203 / 328 / 428, 203 / 328 / 480 / 485, 203 / 362 / 366, 203 / 498 / 499 / 503, 272, 280, 280 / 498 / 499 / 503, 296, 297, 299, 299 / 498 / 499 / 503, 301, 301 / 503, 308, 308 / 503, 311, 328 / 428, 328 / 480 / 483, 328 / 485, 343, 346, 349, 358, 359 / 498 / 499 / 503, 362 / 363, 362 / 363 / 3 The amino acid sequence comprises an amino acid sequence having one or more residue differences compared to SEQ ID NO: 4266 at a position or set of positions selected from 66 / 428 / 480 / 483 / 498 / 499 / 503, 392 / 498 / 499 / 503, 406, 407, 410, 411, 418 / 498 / 499 / 503, 418 / 503, 419 / 503, 465, 471, 472 / 498 / 499 / 503, 473, 480 / 483, 491, and 498 / 499 / 503. In some embodiments, the engineered TdT polypeptide has at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reference sequence SEQ ID NO: 4266 and comprises the following amino acids: L95M / R428V / W480D, S163Q / N190M, S163Q / F203I / E366S,S163Q / Q328L / P363G / W480D, S163Q / P363G / W480D / E485Q, T172S / N174M / Q1 78K / V340C, Q178K, R188K, N190M / E202R / F203I / P363G / E366S / W480D / L483 R / E485Q, N190M / E202R / F203I / W480D, N190M / W480D / E485Q, E192G, E192S / M498P / T499S / Q503G, E192T, E192V, E192Y, E192Y / M498P / T499S / Q503G, E20 2R, E202R / F203I / Q328A / S362T / P363G / E366S / R428V / W480D / E485Q / M498P / T499S / Q503G, E202R / F203I / E485Q, F203I / Q328A / P363G / R428V / L483R, F2 03I / Q328A / R428V, F203I / Q328A / W480D / E485Q, F203I / S362T / E366S, F203 I / M498P / T499S / Q503G, V272P, R280G / M498P / T499S / Q503G, R280H, K296G, K 296P, L297F, P299E, P299K / M498P / T499S / Q503G, P299S, P299T, Q301G, Q30 1S / Q503G, Y308H / Q503G, Y308K, Y308Q, Y308R, Y308T, L311R, Q328A / E485Q, Q328L / R428V, Q328L / W480D / L483R, V343C, V343L, F346Q, F346W, G349Q, L3 58G, L359I / M498P / T499S / Q503G, L392R / M498P / T499S / Q503G, S362T / P363G , S362T / P363R / E366S / R428V / W480D / L483R / M498P / T499S / Q503G, K406S, A407S, G410A, G410Q, D411E, L418G / M498P / T499S / Q503G, L418V / Q503G, I419V / Q503G, M465E, S471N, R472G / M498P / T499S / Q503G, R472S / M498P / T499S / Q503G, M473Q, W480D / L483R, D491G, D491R, and M498P / T499S / Q503G;It comprises an amino acid sequence having one or more residue differences when compared to SEQ ID NO: 4266.

[0195] In some embodiments, the engineered TdT polypeptide has at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reference sequence SEQ ID NO: 4558, and is selected from the group consisting of 94 / 365 / 367, 172 / 174 / 178 / 401 / 403, 172 / 174 / 178 / 401 / 403, 172 / 174 / 178 / 402 / 508, 172 / 174 / 401 / 403 / 507, 172 / 178, 172 / 178 / 401, 174 / 178, 178 / 401 / and 483.In some embodiments, the engineered TdT polypeptide has at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reference sequence SEQ ID NO: 4558, and 402L / S508R, T172S / N174M / Q178R / G401K / P403G, T172S / N174M / Q178R / G401K / P403G / K507R, T172S / N174M / G401K / P403G / K507R, T172S / Q178K, T172S / Q178K / G401K, N174M / Q178R, Q178K / G401K / P403G, Q178K / S40 2L / P403G, A318E / Q375G / L380V, W324L / N379T / G405L / L483R, V340C, V340C / W394T, A365F, A365F / K367R / R428C, A365H / C389A / W394E, K367R, Q375G / K376H, Q375G / N379T / L483R, Q375G / L380V, Q375G / L380V / G400 and L483R, Q375G / L380V / L483R, K376H / L483R, N379T / L483R, C389A / W394E, W394T, G401K, G401K / S402L / P403G / K507N, S402L / P403G, G405L / L483R, and L483R.

[0196] In some embodiments, the engineered TdT polypeptide has at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reference sequence SEQ ID NO: 4442 and is 174 / 296 / 299, 182, 185 / 190, 189 / 190, 190, 190 / 193, 192 / 280, 192 / 402 / 507, 257, 25 9, 260, 281, 289, 296 / 299, 305, 306, 307, 308, 312, 313, 316, 318, 327, 374, 381, 394, 395, 402, 402 / 507, 404, 405, 414, 432, 451, 455, 460, 461, 476 / 480, 480, 480 / 481, 493, 494, and 522.In some embodiments, the engineered TdT polypeptide has at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reference sequence SEQ ID NO: 4442, and is selected from the group consisting of N174M / K296P / P299E, D182A, D182C, D182N, D182R, E185G / N190M , E189A / N190M, E189H / N190M, E189R / N190M, E189V / N190M, N190M, N190M / L193F, E192T / R280H, E192T / S402L / K507R , W257K, W257R, Q259C, Q259K, Q259R, S260A, M281C, M281L, V289I, K296P / P299T, L306A, L306C, L305F, L306M, L306R , L306T, Y307N, Y308Q, A312H, A312K, A312Q, A312R, A312V, R313A, R313I, R313L, T316F, A318L, A318M, A318N, A318P , A318R, A318T, A318V, A318Y, V327A, L374M, W381F, W381M, W394L, W394M, W394Y, S395G, S402L, S402L / K507R, S404A , G405L, A414C, A414E, A414P, R432H, V451L, R455A, S460A, F461M, R476L / W480D, R476W / W480D, W480D, W480E, W480L, W480M, W480D / Y481W, H493K, S494Q, S494R, S494T, and E522T.

[0197] In some embodiments, the engineered TdT polypeptide has at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reference sequence SEQ ID NO: 4654 and is selected from the group consisting of 190, 190 / 197 / 308, 190 / 308 / 380 / 405, 190 / 375, 190 / 375 / 380, The amino acid sequence comprises an amino acid sequence having one or more residue differences compared to SEQ ID NO: 4654 at a position or set of positions selected from 190 / 380 / 405, 190 / 405 / 406, 272 / 301 / 393 / 394 / 480, 272 / 318 / 480 / 483, 301 / 394 / 480, 318, 375, 375 / 380, 375 / 405, 375 / 405 / 406, 380, 394, 394 / 480, and 480 / 483. In some embodiments, the engineered TdT polypeptide has at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reference sequence SEQ ID NO: 4654 and is selected from the group consisting of N190M, N190M / D197N / Y308Q, N190M / Y308Q / L380V / G405L, N190M / Q375G, N190M / Q375G / L380V, N190M ... 05L, N190M / G405L / K406S, V272A / Q301S / E393K / W394T / W480D, V272A / A318E / W480D / L483R, Q301S / W394T / W480D, A318E, Q375G, Q375G / L380V, Q375G / G405L, Q375G / G405L / K406S, L380V, W394T, W394T / W480D, and W480D / L483R.

[0198] In some embodiments, the engineered TdT polypeptide has at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reference sequence SEQ ID NO: 4850 and is selected from the group consisting of 189, 189 / 193 / 207 / 307 / 353, 190 / 322, 193, 193 / 307, 261 / 322 / 421, 297 / 298 / 300 / 392, 29 7 / 300, 297 / 300 / 328, 298 / 300 / 328, 298 / 300 / 328 / 395, 298 / 300 / 360, 298 / 300 / 392 / 395, 298 / 300 / 392 / 395 / 492, 298 / 300 / 395, 298 / 300 / 481, 300, 300 / 392 / 395, 319, 322, 392, 421, and 492. In some embodiments, the engineered TdT polypeptide has at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reference sequence SEQ ID NO: 4850 and is selected from the group consisting of R189K, R189K / E193V / A207V / L307T / Q353R, E190R / D322N, E193V, E193V / L307T, S261A / D322N / L421V, K297P / L298F / P300T / R392S, K297P / P300E / V328A, K297P / P300S, L297P / P300T / R392S, L297P / P300E / V328A, L297P / P300S, L297P / P300T / R392 ...T / R392S, L297P / P300T / R392S, L297P / P300T / R392S, L297P / P300T / R392S, L29 98F / P300E / V328A / W395L, L298F / P300E / R392S / W395L, L298F / P300E / W395L, L298F / P300E / D481W, L298F / P300S / V328A, L298F / P300S / R392S / W395L, L298F / P300S / R392 S / W395L / D492E, L298F / P300T / L360V, P300E, P300E / R392S / W395L, P300T, A319P, D322N, R392S, L421V, and D492E.

[0199] In some embodiments, an engineered TdT polypeptide comprises an amino acid sequence having at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reference sequence SEQ ID NO:4856 and having one or more residue differences compared to SEQ ID NO:4856 at a position or set of positions selected from 10 / 413, 260, 268, 302 / 307, 317, 353, 354, 362, 364, 392, 393, 394, 395, 397, 402, 404, 412, 413, 419, 436 / 512, 460, 477, 486, 490, 495, and 518. In some embodiments, the engineered TdT polypeptide has at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reference sequence SEQ ID NO: 4856, and is selected from the group consisting of S10N / A413S, Q260R, V268T, Q302A / T307L, Q302S / T307L, T317C, Q353R, Q353S, G354S, S362V, P364Q, R392G, R392H, R392K, L393R, L393V, The amino acid sequence has one or more residue differences compared to SEQ ID NO: 4856 selected from E394A, E394V, W395Y, Q397G, Q397S, G402K, G402L, G402P, G402T, G402V, P404L, P404Q, D412G, D412M, A413G, L419G, L419V, P436S / E512D, G460A, G460P, G460S, R477Q, R477S, E486V, M490E, M490N, M490Q, M490S, M490T, M490V, S495Q, S495R, and G518S.

[0200] In some embodiments, the engineered TdT polypeptide has at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reference sequence SEQ ID NO: 4904, and has at least 190, 190 / 287 / 300 / 302 ... The present invention also includes an amino acid sequence having one or more residue differences compared to SEQ ID NO: 4904 at a position or set of positions selected from: 0 / 300 / 477 / 490, 194 / 300 / 302 / 413, 194 / 300 / 302 / 481, 297 / 298 / 308 / 392 / 395, 298 / 392 / 525, 300, 300 / 317, 300 / 490, and 395. In some embodiments, the engineered TdT polypeptide has at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reference sequence SEQ ID NO: 4904, and is selected from the group consisting of E190R, E190R / L287V / P300T / Q302A, E190R / P300T / R477Q / M490E, L194F / P300E / Q302S / D481M, L194F / P300T / Q302A / A413G, K297P / L298F / Y308N / R392K / W395Y, L298F / R392K / F525A, P300E, P300E / T317C, P300T / M490E, and W395Y.

[0201] In some embodiments, the engineered TdT polypeptide has at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reference sequence SEQ ID NO: 5002, and is 11 / 523, 190 / 194, 194, 194 / 198, 202, 208, 264, 273, 273 / 347 / 354, 274, 281, 290, 298 / 300 / 302, 298 / 302 / 392 / 393 / 394 / 433, 298 / 302 / 392 / 394, 298 / 392 / 393 / 394, 298 / 392 / 393 / 394 / 477, 298 / 392 / 394 / 490, 298 / 393 / 394 / 395 / 433 / 477, 298 / 393 / 394 / 477 / 495, 298 / 394 / 433, 300 / 302 / 303, 308 / 402 / 460, 309, 313, 314, 324, 352, 359, 360, 361, 392 / 393 / 394 / 433, 392 / 393 / 394 / 477, 392 / 393 / 394 / 477 / 495, 392 / 393 / 394 / 490, 392 / 394, 392 / 394 / 395, 392 / 394 / 433 / 477, 392 / 394 / 433 / 495, 392 / 394 / 477 / 4 95, 392 / 394 / 495, 393 / 394, 393 / 394 / 433 / 477 / 490, 394 / 477, 394 / 490, 405, 408 / 413, 411 / 413, 413, 460 / 525, 463, 466, 467, 472, 473, 477, 492, 523, and 526. In some embodiments, the engineered TdT polypeptide has at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reference sequence SEQ ID NO: 5002 and is selected from the group consisting of G11D / E523R, E190L / F194L, E190R / F194L, E190T / F194L, F194L, F194L / D198G, F194L / D198P, F194L / D198Q, F194L / D198V, F194W, F194Y, L202T,I208G, I208S, I208V, A264S, V273M / Q347F / S354G, V273Q, V273S, K274G, K274V, K274W, R281G, R281Q, V290A, L298F / A302S / R392K / L39 3V / E394A / R433H, L298F / A302S / R392K / E394A, L298F / R392K / L393V / E394A / R477L, L298F / R392K / L393V / E394V, L298F / R392K / E394V / M490E, L298F / L393V / E394V / W395Y / R433H / R477V, L298F / L393V / E394V / R477L / S495Q, L298F / E394V / R433H, L298I / T300P / A302Q, T30 0P / A302Q / K303V, Y308N / G402V / G460A, Y309R, A313S, R314L, I324M, I324T, P352T, L359C, L359V, L360V, I361L, R392H / L393V / E394A / R477L, R392H / L393V / E394V / M490E, R392H / E394A / R433H / R477L, R392H / E394V / R433H / S495Q, R392K / L393V / E394V / R433H, R392K / L39 3V / E394V / R477V / S495Q, R392K / E394A / W395Y, R392K / E394V, R392K / E394V / R477V / S495Q, R392K / E394V / S495Q, L393V / E394A, L393V / The amino acid sequence has one or more residue differences compared to SEQ ID NO: 5002 selected from E394V / R433H / R477V / M490E, E394V / R477V, E394V / M490E, S405T, A408G / G413A, A408L / G413A, G411L / G413A, G413A, G460S / F525A, A463P, A463V, M466V, L467A, S472T, R473S, R477L, D492R, E523T, E526L, and E526Y.

[0202] In some embodiments, the engineered TdT polypeptide has at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reference sequence SEQ ID NO: 5028, and is selected from the group consisting of 82 / 194 / 198 / 313, 194, 194 / 198, 194 / 198 / 208 / 313, 194 / 198 / 309, 194 / 198 / 313, 194 / 198 / 411, 194 / 20 and 526. The amino acid sequence comprises an amino acid sequence having one or more residue differences compared to SEQ ID NO: 5028 at a position or set of positions selected from: 8 / 411, 194 / 309, 194 / 313, 194 / 411, 198, 198 / 208, 198 / 208 / 309 / 411, 198 / 208 / 313 / 411, 273 / 274, 274, 274 / 281 / 526, 274 / 359 / 526, 274 / 523, 309, 309 / 313 / 411, 324 / 526, 411, 466, 466 / 526, 523, and 526.In some embodiments, the engineered TdT polypeptide has at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reference sequence SEQ ID NO: 5028, and 313S, F194L, F194L / D198Q, F194L / D198Q / I208V / A313S, F194L / D198V, F194L / D198V / Y309R, F1 94L / D198V / G411L, F194L / I208V / G411L, F194L / Y309R, F194W, F194W / D198Q, F194W / D198V, F19 4W / D198V / A313S, F194W / A313S, F194W / G411L, D198G, D198Q, D198Q / I208S / A313S / G411L, D19 8Q / I208V / Y309R / G411L, D198V / I208S, V273Q / K274G, V273S / K274W, K274G / R281Q / E526Y, K274 The amino acid sequence has one or more residue differences compared to SEQ ID NO: 5028 selected from: M / E523T, K274V, K274V / L359V / E526Y, K274W, Y309R, Y309R / A313S / G411L, I324M / E526Y, G411L, M466V, M466V / E526Y, E523T, and E526Y.

[0203] In some embodiments, the engineered TdT polypeptide has at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reference sequence SEQ ID NO: 5192, and is 96 / 295, 169, 176, 177, 179, 184, 187, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 5, 197, 197 / 307, 198, 199, 200, 203, 292, 295, 300 / 394, 304, 325, 326, 326 / 380, 329, 373, 376, 377, 383, 394, 403, 409, 430, 485, 508, and 520 / 526.In some embodiments, the engineered TdT polypeptide has at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reference sequence SEQ ID NO: 5192, and is selected from the group consisting of L96S / T295N, Q169E, N176H, R177L, R177T, R177W, Q179G, Q179L, S184G, S184H, S184P, I187V, V193C, V193F, V193R, V193S, K195L, N197F, N197M / T307A, D198L, D198S, D199G, D199R, D199S, T200A, T200E, T200K, T200Q, T200R, T200S, T200V, T200Y, E203A, E203C, E203G, E203L, S292L, S292M, S292Q, S292R, T295G, T295H, T295K, T295L, T300A / V394F, V304I, W325M, L326Q, L326R, L326T, L326M / N380R, Q329K, Q329L, Q373G, Q376R, K377N, K377R, The amino acid sequence has one or more residue differences compared to SEQ ID NO: 5192 selected from: K383H, K383R, K383S, V394F, V394H, V394I, V394L, V394Q, V394S, V394W, V394Y, L403R, L403V, ​​D409S, K430R, E485L, K508A, K508S, and E520I / Y526E.

[0204] In some embodiments, the engineered TdT polypeptide has at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reference sequence SEQ ID NO: 5246 and is selected from the group consisting of 177 / 198 / 200, 177 / 200 / 203, 177 / 200 / 203 / 295, 177 / 200 / 295 / 326, 180, 184 / 198 / 200 / 203 / 295, 184 / 200 / 295 / 326, 190 / 198 / 200 / 203, 190 / 200 / 203 / 295 / 380, 190 / 200 / 295, 197, 198, 198 / 200, 198 / 200 / 203, 198 / 200 / 203 / 295, 200 / 326, 200 / 380, 203, 203 / 380, 233, 252, 295, 336, 364, 365, 367, 381, 384, 441, 459, and 485.In some embodiments, the engineered TdT polypeptide has at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reference sequence SEQ ID NO: 5246, and R177W / D198L / T200K, R177W / T200K / E203G, R177W / T200K / E203G / T295N, R177W / T200K / T295N / L326M, R180 K, S184H / D198L / T200K / E203G / T295N, S184H / T200K / T295N / L326M, E190M / D198L / T200K / E2 03G, E190M / T200K / E203G / T295N / N380R, E190M / T200K / T295N, N197Q, D198L, D198L / T200K, D198L / T200K / E203G, D198L / T200K / E203G / T295N, T200K / L326M, T200K / N380R, E203G, E203 G / N380R, E233L, E233R, A252R, T295N, D336C, P364A, G365H, G365R, E367S, L381H, L381Y, K384A, M441E, R459M, R459W, and E485L.

[0205] In some embodiments, the engineered TdT polypeptide has at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reference sequence SEQ ID NO: 2 or 660, and is 26 / 30 / 38 / 79 / 81 / 90 / 92 / 94 / 101 / 108 / 137 / 140 / 141 / 142 / 153 / 155 / 160 / 163 / 165 / 177 / 184 / 189 / 194 / 196 / 201 / 203 / 205 / 213 / 219 / 231 / 248 / 258 / 263 / 264 / 266 / 267 / 301 / 304 / 307 / 314 / 318 / 325 / 333 / 340 / 344 / 353 / 362 / 379 / 390 / 392 / 394 / 395 / 397 / 398 / 402 / 403 / 406 / 408 / 410 / 411 / 413 / 414 / 416 / 425 / 427 / 429 / 433 / 434 / 441 / 4 42 / 444 / 446 / 451 / 455 / 460 / 461 / 466 / 468 / 476 / 481 / 484 / 485 / 488 / 495 / 499 / 501 / 502 / 506 / 515 / 525, 26 / 30 / 38 / 79 / 81 / 90 / 92 / 94 / 101 / 108 / 137 / 140 / 141 / 142 / 153 / 155 / 160 / 163 / 165 / 177 / 184 / 189 / 201 / 203 / 205 / 213 / 219 / 231 / 248 / 258 / 263 / 264 / 26 6 / 304 / 307 / 314 / 318 / 325 / 333 / 340 / 344 / 353 / 362 / 379 / 392 / 394 / 395 / 397 / 398 / 402 / 403 / 406 / 408 / 410 / 411 / 413 / 414 / 416 / 425 / 427 / 429 / 433 / 434 / 441 / 442 / 444 / 446 / 455 / 460 / 461 / 466 / 468 / 476 / 481 / 484 / 485 / 488 / 495 / 499 / 501 / 502 / 506 / 515 / 525,26 / 38 / 79 / 81 / 90 / 92 / 94 / 101 / 108 / 137 / 141 / 155 / 160 / 163 / 165 / 189 / 201 / 203 / 205 / 213 / 219 / 246 / 248 / 258 / 263 / 264 / 304 / 307 / 314 / 318 / 333 / 340 / 344 / 353 / 362 / 392 / 394 / 395 / 396 / 397 / 398 / 402 / 403 / 406 / 408 / 410 / 411 / 413 / 414 / 425 / 441 / 442 / 446 / 455 / 460 / 461 / 466 / 468 / 476 / 481 / 485 / 488 / 506 / 525, 92 / 94 / 101 / 108 / 137 / 141 / 155 / 160 / 163 / 165 / 2 01 / 203 / 205 / 213 / 219 / 258 / 263 / 264 / 314 / 333 / 344 / 353 / 392 / 394 / 395 / 397 / 406 / 408 / 411 / 413 / 414 / 425 / 441 / 442 / 446 / 460 / 461 / 468 / 476 / 481 / 485 / 488 / 525, and 92 / 94 / 101 / 1 08 / 137 / 141 / 155 / 201 / 213 / 264 / 314 / 333 / 344 / 392 / 394 / 395 / 397 / 406 / 408 / 425 / 442 / 446 / 461 / 476 / 481 / 485 / 525. In some embodiments, the engineered TdT polypeptide has at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reference sequence SEQ ID NO: 2 or 660;Also 26Q / 30G / 38T / 79T / 81E / 90L / 92R / 94E / 101E / 108K / 137A / 140V / 141E / 142V / 153V / 155E / 160M / 163V / 165P / 177R / 184S / 189R / 194L / 196G / 201A / 203E / 205R / 213S / 219R / 231S / 248R / 258W / 263H / 264A / 266T / 267R / 301W / 304V / 307L / 314R / 318R / 325W / 333A / 340I / 344V / 353Q / 362S / 379M / 390C / 392R / 394E / 395W / 397Q / 398W / 402G / 403S / 406G / 408A / 410E / 411G / 413A / 414E / 416S / 425D / 427L / 42 9R / 433R / 434N / 441M / 442G / 444S / 446P / 451K / 455L / 460G / 461S / 466M / 468Q / 476R / 481W / 484L / 485E / 488N / 495S / 499M / 501R / 502G / 506P / 515E / 525F, 26Q / 30G / 38T / 79T / 81E / 90L / 92R / 94E / 101E / 108K / 137A / 140V / 141E / 142V / 153V / 155E / 160M / 163V / 165P / 177R / 184S / 189R / 201A / 203E / 205R / 213S / 219R / 231S / 248R / 258W / 263H / 264A / 266T / 304V / 307L / 314R / 318R / 325W / 333A / 340I / 344V / 3 53Q / 362S / 379M / 392R / 394E / 395W / 397Q / 398W / 402G / 403S / 406G / 408A / 410E / 411G / 413A / 414E / 416S / 425D / 427L / 429R / 433R / 434 N / 441M / 442G / 444S / 446P / 455L / 460G / 461S / 466M / 468Q / 476R / 481W / 484L / 485E / 488N / 495S / 499M / 501R / 502G / 506P / 515E / 525F,26Q / 38T / 79T / 81E / 90L / 92R / 94E / 101E / 108K / 137A / 141E / 155E / 160M / 163V / 165P / 189R / 201A / 203E / 205R / 213S / 219R / 246N / 248R / 258W / 263H / 264R / 304V / 307L / 314R / 318R / 333A / 340I / 344V / 353Q / 362S / 392R / 394E / 395W / 396T / 397Q / 398W / 402G / 403S / 406G / 408A / 410E / 411G / 413A / 414G / 425D / 441M / 442G / 446P / 455L / 460 G / 461G / 466M / 468Q / 476R / 481W / 485D / 488N / 506P / 525F, 92R / 94E / 101E / 108K / 137A / 141E / 155E / 160M / 163V / 165P / 201A / 203E / 205R / 213S / 219R / 258W / 263H / 264R / 314R / 333A / 344V / 353Q / 392R / 394E / 395W / 397Q / 406G / 408A / 411G / 413A / 414G / 425D / 441M / 442G / 446P / 460G / 461G / 468Q / 476R / 481W / 485D / 488N / 525F, and 92R / The amino acid sequence has one or more residue differences compared to SEQ ID NO: 2 or 660 selected from 94E / 101E / 108K / 137A / 141E / 155E / 201A / 213S / 264R / 314R / 333A / 344V / 392R / 394E / 395W / 397Q / 406G / 408A / 425D / 442G / 446P / 461G / 476R / 481W / 485D / 525F. In some embodiments, the engineered TdT polypeptide has at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence, SEQ ID NO: 2 or 660;And R26Q / M30G / I38T / S79T / S81E / N90L / G92R / D94E / T101E / T108K / M137A / G140V / V141E / M142V / E153V / I155E / T160M / I163V / Q165P / H177R / A184S / A189R / F194L / R 196G / C201A / T203E / M205R / C213S / V219R / G231S / L248R / R258W / K263H / L264A / K2 66T / S267R / M301W / A304V / C307L / D314R / K318R / S325W / W333A / L340I / T344V / F35 3Q / T362S / T379M / Y390C / D392R / R394E / E395W / R397Q / F398W / K402G / L403S / R40 6G / I408A / A410E / L411G / H413A / F414E / K416S / H425D / K427L / D429R / W433R / E434 N / E441M / S442G / A444S / S446P / R451K / V455L / D460G / R461S / L466M / G468Q / E476R / R481W / T484L / H485E / K488N / A495S / L499M / K501R / K502G / K506P / A515E / S525F,R26Q / M30G / I38T / S79T / S81E / N90L / G92R / D94E / T101E / T108K / M137A / G140V / V141E / M142V / E153V / I155E / T160M / I163V / Q165P / H177R / A184S / A189R / C201A / T203E / M205R / C213S / V219R / G231S / L248R / R258W / K263H / L264A / K266T / A304V / C307L / D314R / K318R / S325W / W333A / L340I / T344V / F353Q / T362S / T379M / D392R / R394E / E395W / R397Q / F398W / K402G / L403S / R406G / I408A / A410E / L411G / H413A / F414E / K416S / H425D / K427L / D429R / W433R / E434N / E441M / S442G / A444S / S446P / V455L / D460G / R461S / L466M / G468Q / E476R / R481W / T484L / H485E / K488N / A495S / L499M / K501R / K502G / K506P / A515E / S525F、R26Q / I38T / S79T / S81E / N90L / G92R / D94E / T101E / T108K / M137A / V141E / I155E / T160M / I163V / Q165P / A189R / C201A / T203E / M205R / C213S / V219R / E246N / L248R / R258W / K263H / L264R / A304V / C307L / D314R / K318R / W333A / L340I / T344V / F353Q / T362S / D392R / R394E / E395W / S396T / R397Q / F398W / K402G / L403S / R406G / I408A / A410E / L411G / H413A / F414G / H425D / E441M / S442G / S446P / V455L / D460G / R461G / L466M / G468Q / E476R / R481W / H485D / K488N / K506P / S525F、G92R / D94E / T101E / T108K / M137A / V141E / I155E / T160M / I163V / Q165P / C201A / T203E / M205R / C213S / V219R / R258W / K263H / L264R / D314R / W333 A / T344V / F353Q / D392R / R394E / E395W / R397Q / R406G / I408A / L411G / H4 13A / F414G / H425D / E441M / S442G / S446P / D460G / R461G / G468Q / E476R / R 481W / H485D / K488N / S525F, and G92R / D94E / T101E / T108K / M137A / V141E / I155E / C201A / C213S / L264R / D314R / W333A / T344V / D392R / R394E / E395W / R397Q / R406G / I408A / H425D / S442G / S446P / R461G / E476R / R481W / H485D / S525F.

[0206] As will be appreciated by those skilled in the art, in some embodiments, one or a combination of the above selected residue differences can be held constant (i.e., maintained) as core features in the engineered TdT, and additional residue differences at other residue positions can be incorporated into the sequence to yield additional engineered TdT polypeptides with improved properties. Thus, for any engineered TdT that contains one or a subset of the above residue differences, it should be understood that the present invention also contemplates other engineered TdTs that contain one or a subset of the residue differences and that additionally contain one or more residue differences at other residue positions disclosed herein.

[0207] As described above, engineered TdT polypeptides are also capable of converting a substrate (e.g., NTP-3'-O-RBG or a natural or modified NTP and an oligo acceptor substrate) into a product (e.g., an oligo acceptor substrate to which the nucleotide-3'-O-RBG has been added). In some embodiments, the engineered TdT polypeptide is selected from the group consisting of SEQ ID NOs: 2, 8, 16, 24, 36, 268, 648, 660, 882, 1100, 1336, 1348, 1596, 1654, 1830, 1950, 2008, 2254, 2514, 2524, 2638, 2804, 2812, 2956, 3174, 3222, 3670, 3674, 3796, 3870, 3918, 4266, 4306, 4406, 4506, 4606, 4706, 4806, 4906, 5006, 5106, 5206, 5306, 5406, 5506, 5606, 5706, 5806, 5906, 6006, 6106, 6206, 6306, 6406, 6506, 6606, 6706, 6806, 6906, 7006, 7106, 7206, 7306, 7406, 7506, 7606, 7706, 7806, 7906, 8006, 81 , 4442, 4654, 4850, 4856, 4904, 5002, 5028, 5192, and / or 5246, or more, as compared to the activity of the reference polypeptides.

[0208] In some embodiments, SEQ ID NOs: 2, 8, 16, 24, 36, 268, 648, 660, 882, 1100, 1336, 1348, 1596, 1654, 1830, 1950, 2008, 2254, 2514, 2524, 2638, 2804, 2812, 2956, 3174, 3222, 3670, 3674, 3796, 3870, 3918, 4266, 44 The engineered TdT capable of converting a substrate compound to a product compound with at least twice the activity as SEQ ID NOs: 42, 4654, 4850, 4856, 4904, 5002, 5028, 5192, and / or 5246 comprises an amino acid sequence selected from the even-numbered sequences in SEQ ID NOs: 4-1960, 2004-3920, 4048-5466, and 5476.

[0209] In some embodiments, the engineered TdT increases soluble expression or isolated protein yield of the engineered TdT in a bacterial host cell, specifically E. coli, compared to wild-type TdT or an engineered reference TdT. 804, 2812, 2956, 3174, 3222, 3670, 3674, 3796, 3870, 3918, 4266, 4442, 4654, 4850, 4856, 4904, 5002, 5028, 5192, and / or 5246, and includes an amino acid sequence selected from the even-numbered sequences in SEQ ID NOs: 4-1960, 2004-3920, 4048-5466, and 5476.

[0210] In some embodiments, the engineered TdT is selected from the group consisting of SEQ ID NOs: 2, 8, 16, 24, 36, 268, 648, 660, 882, 1100, 1336, 1348, 1596, 1654, 1830, 1950, 2008, 2254, 2514, 2524, 2638, 2804, 2812, 2956, 317, which increase the thermal stability of the engineered TdT compared to wild-type TdT or an engineered reference TdT. 4, 3222, 3670, 3674, 3796, 3870, 3918, 4266, 4442, 4654, 4850, 4856, 4904, 5002, 5028, 5192, and / or 5246, and includes an amino acid sequence selected from the even-numbered sequences of SEQ ID NOs: 4-1960, 2004-3920, 4048-5466, and 5476.

[0211] In some embodiments, the engineered TdT increases the activity of the engineered TdT at elevated temperatures (for example, but not limited to, 40°C, 45°C, 50°C, 55°C, 60°C, or 65°C) compared to wild-type TdT or an engineered reference TdT. 8, 2804, 2812, 2956, 3174, 3222, 3670, 3674, 3796, 3870, 3918, 4266, 4442, 4654, 4850, 4856, 4904, 5002, 5028, 5192, and / or 5246, and includes an amino acid sequence selected from the even-numbered sequences of SEQ ID NOs: 4-1960, 2004-3920, 4048-5466, and 5476.

[0212] In some embodiments, the engineered TdT is selected from the group consisting of SEQ ID NOs: 2, 8, 16, 24, 36, 268, 648, 660, 882, 1100, 1336, 1348, 1596, 1654, 1830, 1950, 2008, 2254, 2514, 2524, 2638, 2804, 2812, 2956, 31 74, 3222, 3670, 3674, 3796, 3870, 3918, 4266, 4442, 4654, 4850, 4856, 4904, 5002, 5028, 5192, and / or 5246, and includes an amino acid sequence selected from the even-numbered sequences of SEQ ID NOs: 4 to 1960, 2004 to 3920, 4048 to 5466, and 5476.

[0213] In some embodiments, the engineered TdT increases the specific activity of the engineered TdT for one or more NTP-3'-O-RBG or natural or modified NTP substrates compared to wild-type TdT or an engineered reference TdT. 8, 2804, 2812, 2956, 3174, 3222, 3670, 3674, 3796, 3870, 3918, 4266, 4442, 4654, 4850, 4856, 4904, 5002, 5028, 5192, and / or 5246, and includes an amino acid sequence selected from the even-numbered sequences of SEQ ID NOs: 4-1960, 2004-3920, 4048-5466, and 5476.

[0214] In some embodiments, the engineered TdT increases the specific activity of the engineered TdT toward one or more oligo acceptor substrates compared to wild-type TdT or an engineered reference TdT. , 2956, 3174, 3222, 3670, 3674, 3796, 3870, 3918, 4266, 4442, 4654, 4850, 4856, 4904, 5002, 5028, 5192, and / or 5246, and including an amino acid sequence selected from the even-numbered sequences of SEQ ID NOs: 4 to 1960, 2004 to 3920, 4048 to 5466, and 5476.

[0215] In some embodiments, the engineered TdT increases the incorporation efficiency of the extension of an oligo acceptor substrate by the addition of NTPs or NQPs by more than 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% compared to the incorporation efficiency of wild-type TdT or an engineered reference TdT, as defined in SEQ ID NOs: 2, 8, 16, 24, 36, 268, 648, 660, 882, 1100, 1336, 1348, 1596, 1654, 18 30, 1950, 2008, 2254, 2514, 2524, 2638, 2804, 2812, 2956, 3174, 3222, 3670, 3674, 3796, 3870, 3918, 4266, 4442, 4654, 4850, 4856, 4904, 5002, 5028, 5192, and / or 5246, and includes an amino acid sequence selected from the even-numbered sequences of SEQ ID NOs: 4-1960, 2004-3920, 4048-5466, and 5476.

[0216] In some embodiments, the engineered TdT with improved properties has at least 60%, 70%, or 80% similarity to the reference sequence of SEQ ID NOs: 2, 8, 16, 24, 36, 268, 648, 660, 882, 1100, 1336, 1348, 1596, 1654, 1830, 1950, 2008, 2254, 2514, 2524, 2638, 2804, 2812, 2956, 3174, 3222, 3670, 3674, 3796, 3870, 3918, 4266, 4442, 4654, 4850, 4856, 4904, 5002, 5028, 5192, and / or 5246. 0%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity and at least one substitution at an amino acid position selected from: 25, 333, 340, 344, 353, 362, 379, 390, 392, 394, 395, 396, 397, 398, 402, 403, 406, 408, 410, 411, 413, 414, 416, 425, 427, 429, 433, 434, 441, 442, 444, 446, 451, 455, 460, 461, 466, 468, 476, 481, 484, 485, 488, 495, 499, 501, 502, 506, 515, and 525, and / or any combination thereof; or an amino acid sequence having a substitution set, wherein the amino acid positions are numbered with reference to SEQ ID NOs: 2, 8, 16, 24, 36, 268, 648, 660, 882, 1100, 1336, 1348, 1596, 1654, 1830, 1950, 2008, 2254, 2514, 2524, 2638, 2804, 2812, 2956, 3174, 3222, 3670, 3674, 3796, 3870, 3918, 4266, 4442, 4654, 4850, 4856, 4904, 5002, 5028, 5192, and / or 5246.

[0217] In some embodiments, the engineered TdT with improved properties has at least 60%, 70%, 80%, or 90% similarity to the reference sequence of SEQ ID NOs: 2, 8, 16, 24, 36, 268, 648, 660, 882, 1100, 1336, 1348, 1596, 1654, 1830, 1950, 2008, 2254, 2514, 2524, 2638, 2804, 2812, 2956, 3174, 3222, 3670, 3674, 3796, 3870, 3918, 4266, 4442, 4654, 4850, 4856, 4904, 5002, 5028, 5192, and / or 5246. , 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity and at amino acid positions 12, 13, 14, 15, 16, 17, 18, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 41, 44, 45, 46, 47, 50, 53, 57, 58, 61, 62, 65, 70, 76, 77, 79, 80, 81, 85, 89, 90, 92, 93, 94, 97, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 116, 119, 121, 122, 123, 124, 126, 129, 130, 131, 132, 133, 134, 135, 137, 138, 139, 140, 141, 142, 144, 145, 147, 1 48, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 165, 168, 169, 171, 173, 174, 175, 177, 179, 183, 184, 185, 186, 188, 189, 19 0, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 203, 204, 205, 206, 208, 213, 217, 219, 220, 223, 227, 231, 233, 235, 236, 243, 244, 245, 246, 248 , 249, 251, 252, 253, 255, 258, 259, 260, 261, 262, 263, 264, 266, 267, 268, 269, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 284, 286, 287,288, 289, 290, 291, 292, 293, 294, 295, 297, 298, 299, 300, 301, 302, 303, 304, 306, 307, 308, 309, 310, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 324, 325, 326, 327, 328, 329, 330, 333, 334, 336, 338, 340, 342, 343, 344, 347, 350, 352, 353, 359, 360, 361, 362, 363, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 9, 370, 371, 372, 373, 376, 378, 379, 380, 382, ​​383, 385, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 421, 425, 426, 427, 428, 429, 431, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 448, 451, 452, 454, 455, 456, 457, 459, 460, 461, 462, 464, 465, 466, 468, 469, 470, 472, 473, 474, 475, 476, 477, 479, 480, 481, 484, 485, 488, 490, 491, 492, 493, 494, 495, 499, 500, 501, 502, 503, 504, 506, 508, 509, 515, 518, 520, 522, 523, 525, 526, or 528, or any combination thereof and an amino acid sequence having one or more substitutions, wherein the amino acid positions are numbered with reference to SEQ ID NOs: 2, 8, 16, 24, 36, 268, 648, 660, 882, 1100, 1336, 1348, 1596, 1654, 1830, 1950, 2008, 2254, 2514, 2524, 2638, 2804, 2812, 2956, 3174, 3222, 3670, 3674, 3796, 3870, 3918, 4266, 4442, 4654, 4850, 4856, 4904, 5002, 5028, 5192, and / or 5246.

[0218] In some embodiments, the engineered TdT with improved properties has at least 60%, 70%, 80%, or 90% similarity to the reference sequence of SEQ ID NOs: 2, 8, 16, 24, 36, 268, 648, 660, 882, 1100, 1336, 1348, 1596, 1654, 1830, 1950, 2008, 2254, 2514, 2524, 2638, 2804, 2812, 2956, 3174, 3222, 3670, 3674, 3796, 3870, 3918, 4266, 4442, 4654, 4850, 4856, 4904, 5002, 5028, 5192, and / or 5246. 8A / G / M / P, 8B / G / I / Q / S / T / W, 8C / E / P / T, 8D / F / R, 8E / G / I / Q / S / T / W, 8E / G / I / P / R, 8F / G / I / Q / S / T / W, 8F / F / R, 8F / G / I / Q / S / T / W, 8F / F / R, 8F / G / I / P / R, 8F / G / I / Q / S / T / W ... E, 33A / C / G / K / P, 34D / K / R / S, 35E / G / H / W, 36G / K, 37A / F / G / S / T / V, 38L / T, 39 A / G, 41V, 44S, 45R, 46M, 47A, 50L, 53K, 53E, 57H / T, 58A / M / N / S, 61A / H / L, 62W , 65S, 70A, 76P, 77C / S, 79T, 80D, 81E, 85V, 89A, 90L / M / S, 92M / R / S / V / Y, 93V , 94E / K / N / R, 97D / T, 101E / G / V, 102L, 103M, 104G / I / P / V, 105N / W, 106D / G / H / K / S / V, 107R / W, 108D / G / K / M, 109M / N / T, 110M / V, 116A, 119F / Q, 121S, 122I, 123M / Q, 124E / G / I / M / S, 126C / V, 129G, 130A / M / Q / S, 131E / G / R / W, 132S, 133G / M / Q, 134M / W, 135C / E / H / K / R, 137A / E, 138A / Q, 139A / G, 140E / G / V, 141E / M / R, 142M / S / V, 144C, 145E, 147G / L, 148T, 149R, 150E / G / P, 151I / S, 152G / H / R,153E / G / K / M / P / Q / V、154G、155E / T、156D / E / M / Q、157L / V、158A / H / S、159D、160G / M / N / S / V、161D / E / G / H / R / S、162A / E / H、163I / L / V、165K / P、168S、169E / M / R、171K、173I / L、174L、175D / I / L / V、177L / R / Y、179A / K / L / R、183R、184S、185C / F / L、186A / C / D / G / L / T、188I / M、189E / L / Q / R / V、190L / M / R、190E、191V、192C / G / I / L / R、193C / G / N / R / S / T / V、194A / C / D / E / F / G / L / M / R / S / W、196A / G / L / M / N / R / S / T / W / Y、197G / L / M / R、198A / E / L / P / S、199A / E / G / H / I / M / Q / R / S / V、200A / C / K / N / V、201A / R / W、203A / D / E / G / L / M / R / S、204L、205E / L / R、206N、208A / V、213S、217Q、219L / R、220R、223Q、227M、231S / T、233A / D / R、235R、236E / I / P / V、243L / R / S / T、244V、245A、246E / K / N、248C / K / L / R / S / T / V、249A / G / R / T、251K / R、252E / K、253I / L、255G、258A / C / E / G / K / L / M / Q / S / W、259N / V、260K、261A / G / R / V、262I / V、263H / I / M / R / S、264A / C / E / L / R / S / T / V、266F / I / K / R / T / V / Y、267A / C / H / M / Q / R / V / Y、268C / I / L / T、269W、271C、272T、273C / D / E / F / G / I / L / M / S / V / W / Y、274A / G / I / M / N / P / Q / T / V、275D / V、276S、277E、278C / E / F / H / I / L / M / N / R / T / V / Y、279Y、280F / L / S、281A / C / G / K / L / Q / S / T / V、282C / G / H / Q / R / W、284I / L / M、286N / S、287I、288E / V、289D、290A / L / V、291M / S / W、292L / N / T、293S / Q、294T、295S、297C / D / F / L / M / P / Q / R / S / T / V、298I / M、299A / L / M / N / S / Y、300H / P / R / T、301G / Q / S / T / V / W、302A / N / S、303A / E / H / M / N / Q / S / V、304D / L / M / T / V、306F / I / M、307A / E / G / H / K / L / M / S / V、308A / F / G / H / K / L / N / S / V / W、309F / G / I / L / M / V / W、310A / G / H / R / S、312M / T / V、313A / I / L / M / Q / R / S、314A / G / I / K / L / M / Q / R / V / Y、315A / G / Q / S、316A / C / I / L / T、317G / T、318E / R / S / T / V、319G / R、320N、321C / K / S、322A / K / Q、324I / V、325A / F / L / T / V / W、326C / M / N / R / S / T、327I / M、328T、329K / R、330E / K / N、333A / D / G / H / R、334E / R / S、336D、338T、340A / G / I / M / R / S / V、342E / R / V / W、343V、344V、347I / Q、350L / R / S / W、352G / P / R / V、353A / H / K / M / Q / R / S、354S、359G / L / V、360C / I / V、361T、362S / T / Y、363C / I、365A、366V、367A / D、368R、369G / M / N、370D / G / M / N / S / T、371D、372E / G、373C / G / H / N / R、376H / V、378L、379C / I / L / M / V、380D、382F / L、383R、385R、390C / I / L / V、391G / L / R、392A / C / K / R / V、393I / R / V、394A / E / F / G / L / M / R / S / T / V / Y、395A / L / R / S / T / W / Y、396A / R / S / T、397A / D / F / Q / R / T / W、398W、399C / D / F / G / T、400A / E / W、401E / G、402E / F / G / Q / S / V、403A / E / F / G / L / P / R / S、404D / E / F / S / W、405G / L / N / Y、406G / N / P / T / V、407A / D / F / L / M / N / R / S / W、408A / E / G / I / L / M / P / R / T / V / W、409K / L / Q、410E / F / G / I / Q / S / V / Y、411A / E / F / G / I / N / P / Q / R / T、412N、413A / C / E / F / G / I / L / M / P / S / V、414A / E / F / G / H / Y、415A / F / L / S / W, 416G / N / Q / S, 417G / V / W, 418I, 419A / G / H / L / M, 421F / I / M, 42 5D / K / R / T, 426P, 427C / E / F / L / M / N / Q / R / W / Y, 428V, 429R, 431R / S, 433A / E / G / H / M / P / R / S / V, 434N, 435A / C / E / G / I / K / P / Q / S / T, 436S, 437A / G / K / P / Q / R / S , 438V, 439G / P, 440E / K / V, 441K / M / N / V, 442A / G / K, 443T, 444A / G / H / R / S, 445 N, 446E / G / P, 448R, 451K, 452I / L, 454F / M / V, 455I / L, 456K / P / R / S / T, 457S / V, 459H / I / Q / R / V, 460E / G / P / V, 461A / G / N / Q / S / V, 462E / F / H / I / L / Q / R / W, 46 4Y, 465E, 466M, 468A / F / H / M / Q / S / T / W, 469F / Q / Y, 470S / T, 472A / G, 473A / D / G / K / M / P / Q / S / V, 474M, 475V, 476R / V, 477G / Q / S / T, 479F / V, 480A / E / G / H / K / L / S / W, 481A / D / E / L / M / S / T / V / W, 484A / E / H / L / M / R, 485D / E / S, 488N / S, 490E / H / L / R / V / W, 491I / M, 492S / T, 493E / Q / R / V / Y, 494A / C / G / L / R / V / W, 495C / G / S , 499L / M / R, 500N, 501A / N / R, 502G / R, 503A / E / M / Q / R / S / T / V, 504K / N / Q / R / S / W, 506E / P / S / T, 508D / S / T, 509G / K, 515E / V, 515A, 518D, 520D / P, 522L, 523E 525F / H / Q / R / S, 526L / Y, or 528L, or any combination thereof, and the amino acid positions are set forth in SEQ ID NOs: 2, 8, 16, 24, 36, 268, 648, 660, 882, 1100, 1336, 1348, 1596, 1654, 1830, 1950, 2008, 2254, 2514, 2524, 2638, 2804, 2812, 2956, 3174, 3222, 3670, 3674, 3796, 3870, 3918, 4266, 4442, 4446, 4448 ...4654, 4850, 4856, 4904, 5002, 5028, 5192, and / or 5246.

[0219] In some embodiments, the engineered TdT with improved properties has an amino acid sequence comprising a sequence selected from the even-numbered sequences of SEQ ID NOs: 4-1960, 2004-3920, 4048-5466, and 5476. In some embodiments, the engineered TdT with improved properties has an amino acid sequence comprising a sequence selected from SEQ ID NOs: 2, 8, 16, 24, 36, 268, 648, 660, 882, 1100, 1336, 1348, 1596, 1654, 1830, 1950, 2008, 2254, 2514, 2524, 2638, 2804, 2812, 2956, 3174, 3222, 3670, 3674, 3796, 3870, 3918, 4266, 4442, 4654, 4850, 4856, 4904, 5002, 5028, 5192, and / or 5246.

[0220] In some embodiments, the engineered TdT is selected from the group consisting of SEQ ID NOs: 2, 8, 16, 24, 36, 268, 648, 660, 882, 1100, 1336, 1348, 1596, 1654, 1830, 1950, 2008, 2254, 2514, 2524, 2638, 2804, 2812, 2956, 3174, 3222, 3670, 3674, 3675, 3676, 3677, 3678, 3679, 3680, 3681, 3682, 3683, 3684, 3685, 3686, 3687, 3688, 3689, 3690, 3691, 3692, 3693, 3694, 3695, 3700, 3701, 3702, 3703, 3704, 3705, 3706, 3707, 3708, 3709, 3710, 3711, 3712, 3713, 3714, 3715, 3716, 3717, 3718, 3719, 3720, 3721, 3722, 3723, 3724, 3725, 3726, 3727, 3728, 3729, 3730, 3731, 3732, 3733, 3734, and / or 5246.

[0221] In addition to the residue positions specified above, any of the engineered TdT polypeptides disclosed herein may further include other residue differences at other residue positions (i.e., residue positions other than those included herein) compared to SEQ ID NOs: 2, 8, 16, 24, 36, 268, 648, 660, 882, 1100, 1336, 1348, 1596, 1654, 1830, 1950, 2008, 2254, 2514, 2524, 2638, 2804, 2812, 2956, 3174, 3222, 3670, 3674, 3796, 3870, 3918, 4266, 4442, 4654, 4850, 4856, 4904, 5002, 5028, 5192, and / or 5246. Residue differences at these other residue positions can provide additional variation in the amino acid sequence without adversely affecting the ability of the polypeptide to carry out a substrate to product conversion. Thus, in some embodiments, in addition to the amino acid residue differences present in any one of the engineered TdT polypeptides selected from the even-numbered sequences within the ranges of SEQ ID NOs: 4-1960, 2004-3920, 4048-5466, and 5476, the sequence can be selected from any one of SEQ ID NOs: 2, 8, 16, 24, 36, 268, 648, 660, 882, 1100, 1336, 1348, 1596, 1654, 1830, 1950, 2008, 2254, 2514, 2524, 2638, 2804, 2812, 2956, 3174, 3222, 3324, 3426, 3428, 3429, 3529, 3626, 3638, 3728, 3736, 3748, 3756, 3764, 3776, 3784, 3782, 3796, 3804, 3812, 3822, 3824, 3826, 3828, 3830, 3848, 3856, 3864, 3874, 3876, 3882, 3896, 3900, 4000, 4000, 4000, 4000, 4000, 4000, 4000, 4000, 4 and / or 5246. The amino acid sequence of any one of the sequences of the present invention may further comprise 1 to 2, 1 to 3, 1 to 4, 1 to 5, 1 to 6, 1 to 7, 1 to 8, 1 to 9, 1 to 10, 1 to 11, 1 to 12, 1 to 14, 1 to 15, 1 to 16, 1 to 18, 1 to 20, 1 to 22, 1 to 24, 1 to 26, 1 to 30, 1 to 35, 1 to 40, 1 to 45, 1 to 50, 1 to 100, or 1 to 150 residue differences at other amino acid residue positions compared to 670, 3674, 3796, 3870, 3918, 4266, 4442, 4654, 4850, 4856, 4904, 5002, 5028, 5192, and / or 5246. In some embodiments, the number of amino acid residue differences compared to the reference sequence may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 30, 35, 40, 45, 50, 100, or 150 residue positions.In some embodiments, the number of amino acid residue differences compared to the reference sequence can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 18, 20, 21, 22, 23, 24, or 25 residue positions. Residue differences at these other positions can be conservative or non-conservative changes. In some embodiments, residue differences can include conservative and non-conservative substitutions compared to the TdT polypeptides of SEQ ID NOs: 2, 8, 16, 24, 36, 268, 648, 660, 882, 1100, 1336, 1348, 1596, 1654, 1830, 1950, 2008, 2254, 2514, 2524, 2638, 2804, 2812, 2956, 3174, 3222, 3670, 3674, 3796, 3870, 3918, 4266, 4442, 4654, 4850, 4856, 4904, 5002, 5028, 5192, and / or 5246.

[0222] In some embodiments, the present invention also provides engineered polypeptides, including fragments of any of the engineered TdT polypeptides described herein, that retain the improved functional activity and / or properties of the engineered TdT. Thus, in some embodiments, the present invention provides polypeptide fragments capable of converting a substrate to a product under appropriate reaction conditions, wherein the fragments comprise at least about 90%, 95%, 96%, 97%, 98%, or 99% of the full-length or truncated amino acid sequence of an engineered TdT of the present invention, such as exemplary TdT polypeptides selected from even-numbered sequences within the ranges of SEQ ID NOs: 4-1960, 2004-3920, 4048-5466, and 5476. In some embodiments, the engineered TdT can have an amino acid sequence that includes a deletion in any one of the TdT polypeptide sequences described herein, such as exemplary engineered polypeptides of even-numbered sequences within the ranges of SEQ ID NOs: 4-1960, 2004-3920, 4048-5466, and 5476.

[0223] Thus, for any and all embodiments of the engineered TdT polypeptides of the present invention, the amino acid sequence may include one or more amino acid deletions, two or more amino acid deletions, three or more amino acid deletions, four or more amino acid deletions, five or more amino acid deletions, six or more amino acid deletions, eight or more amino acid deletions, ten or more amino acid deletions, fifteen or more amino acid deletions, or twenty or more amino acid deletions, up to 10% of the total number of amino acids in the TdT polypeptide, up to 20% of the total number of amino acids in the TdT polypeptide, or up to 30% of the total number of amino acids in the TdT polypeptide, while maintaining the relevant improved functional activity and / or properties of the engineered TdT described herein. In some embodiments, the deletions may comprise 1 to 2, 1 to 3, 1 to 4, 1 to 5, 1 to 6, 1 to 7, 1 to 8, 1 to 9, 1 to 10, 1 to 15, 1 to 20, 1 to 21, 1 to 22, 1 to 23, 1 to 24, 1 to 25, 1 to 30, 1 to 35, 1 to 40, 1 to 45, or 1 to 50 amino acid residues. In some embodiments, the number of deletions may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 30, 35, 40, 45, or 50 amino acid residues. In some embodiments, the deletion may comprise the deletion of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 18, 20, 21, 22, 23, 24, or 25 amino acid residues.

[0224] In some embodiments, the engineered TdT herein can have an amino acid sequence that includes an insertion compared to any one of the engineered TdT polypeptides described herein, such as the exemplary engineered polypeptides of even-numbered sequences within the ranges of SEQ ID NOs: 4-1960, 2004-3920, 4048-5466, and 5476. Thus, for any and all embodiments of the TdT polypeptides of the present invention, the insertion can include one or more amino acids, two or more amino acids, three or more amino acids, four or more amino acids, five or more amino acids, six or more amino acids, eight or more amino acids, ten or more amino acids, fifteen or more amino acids, twenty or more amino acids, thirty or more amino acids, forty or more amino acids, or fifty or more amino acids, while maintaining the relevant improved functional activity and / or properties of the engineered TdT described herein. The insertion can be to the amino or carboxy terminus or an internal portion of the TdT polypeptide.

[0225] In some embodiments, the engineered TdT described herein can have an amino acid sequence selected from even-numbered sequences within the ranges of SEQ ID NOs: 4-1960, 2004-3920, 4048-5466, and 5476, and optionally including one or several (e.g., up to 3, 4, 5, or up to 10) amino acid residue deletions, insertions, and / or substitutions. In some embodiments, the amino acid sequence optionally includes 1 to 2, 1 to 3, 1 to 4, 1 to 5, 1 to 6, 1 to 7, 1 to 8, 1 to 9, 1 to 10, 1 to 15, 1 to 20, 1 to 21, 1 to 22, 1 to 23, 1 to 24, 1 to 25, 1 to 30, 1 to 35, 1 to 40, 1 to 45, 1 to 50, 1 to 75, 1 to 100, or 1 to 150 amino acid residue deletions, insertions, and / or substitutions. In some embodiments, the amino acid sequence optionally has about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 30, 35, 40, 45, 50, 55, 60, 65, 70, 80, 90, 100, 110, 120, 130, 140, or 150 amino acid residue deletions, insertions, and / or substitutions. In some embodiments, the substitutions may be conservative or non-conservative.

[0226] In the above embodiments, suitable reaction conditions for the engineered polypeptides are set forth in Tables 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, 20.1, 21.1, 22.1, 23.1, 24.1, 25.1, 26.1, 27.1, 28.1, 29.1, 30.1, 31.1, 32.1, 33.1, 34.1, 35.1, 36.1, 37.1, 38.1, 39.1, 40.1, 41.1, 42.1, 43.1, 44.1, 45.1, 46.1, 47.1, 48.1, 49.1, 50.1, 51.1, 52.1, 53.1, 54.1, 55.1, 56.1, 57.1, 58.1, 59.1, 60.1, 61.1, 62.1, 63.1, 64.1, 65.1, 66.1, 67.1, 68.1, 69.1, 70.1, 71.1, 72.1, 73.1, 74.1, 75.1, 76.1, 77.1, 78.1, 79.1, 80.1, 81.1, 82.1, 83.1, 84.1, 85.1, 86.1, 86.1, 87 .1, 39.1, 40.1, 41.1, 42.1, 43.1, 44.1, 45.1, 46.1, 47.1, 48.1, 49.1, 50.1, 51.1, 52.1, 53.1, 54.1, 55.1, 56.1, 59.1, 60.1, 61.1, 63.1, 64.1, 65.1, 66.1, 67.1, 68.1, 69.1, 70.1, 71.1, 72.1, 73.1, 74.1, 75.1, 76.1, 77.1, 78.1, 79.1, and 80.1.

[0227] In some embodiments, the polypeptides of the present invention are fusion polypeptides in which the engineered polypeptide is fused to another polypeptide, including, but not limited to, an antibody tag (e.g., a myc epitope), a purification sequence (e.g., a His tag for binding to metals), a cellular localization signal (e.g., a secretion signal), and a polypeptide with enzymatic activity. Thus, the engineered polypeptides described herein can be used with or without fusion to another polypeptide.

[0228] In one embodiment of an engineered TdT polypeptide of the invention, the polypeptide further comprises an N-terminal truncation of 1 to 156 amino acids of the polypeptide sequence compared to any of the even-numbered sequences set forth in SEQ ID NOS: 4-1960, 2004-3920, 4048-5466, and 5476. For example, a 156 amino acid truncated version of the TdT variant polypeptide SEQ ID NO: 5028 has been prepared and demonstrated to have TdT activity in Examples 81 and 82.

[0229] In some embodiments, the engineered TdT polypeptides of the present invention can be fused to another second polypeptide (e.g., a polypeptide having a different enzymatic activity). In some embodiments, the present invention provides a fusion polypeptide comprising an engineered TdT polypeptide fused to a second polypeptide having inorganic pyrophosphatase (IPP) activity. For example, a synthetic gene encoding an N- and C-terminal hexahistidine-tagged version of a wild-type (WT) inorganic pyrophosphatase (IPP) polypeptide (e.g., the polypeptide of SEQ ID NO: 3942 or 3944) can be fused to a gene encoding a TdT variant polypeptide. Typically, the polypeptides (e.g., IPP and TdT) are fused via a polypeptide linker (e.g., a GSGGTG linker) introduced into the construct between the genes encoding the polypeptides. Such fusion proteins can be constructed using well-established techniques (e.g., Gibson assembly cloning) and expressed in E. coli (e.g., strains derived from W3110). Exemplary IPP-TdT polypeptide fusion constructs (e.g., the fusion constructs of SEQ ID NOs: 5468, 5470, 5472, and 5474) are provided and demonstrated in Examples 81 and 82. While the Examples demonstrate fusions of particular engineered TdT polypeptides of the invention with a second polypeptide having IPP activity, it is contemplated that in any of the embodiments, engineered TdT polypeptides of the even-numbered sequences set forth in SEQ ID NOs: 4-1960, 2004-3920, 4048-5466, and 5476 can be used in such fusions with a second polypeptide.

[0230] It is understood that the polypeptides described herein are not limited to genetically encoded amino acids. In addition to genetically encoded amino acids, the polypeptides described herein may be composed, in whole or in part, of naturally occurring and / or synthetic non-encoded amino acids. Certain commonly found non-encoded amino acids that may comprise the polypeptides described herein include, but are not limited to, the D-stereoisomers of the genetically encoded amino acids, 2,3-diaminopropionic acid (Dpr), α-aminoisobutanoic acid (Aib), ε-aminohexanoic acid (Aha), δ-aminovaleric acid (Ava), N-methylglycine or sarcosine (MeGly or Sar), ornithine (Orn), citrulline (Cit), t-butylalanine (Bua), t-aminopropyl methyl ... Butylglycine (Bug), N-methylisoleucine (MeIle), phenylglycine (Phg), cyclohexylalanine (Cha), norleucine (Nle), naphthylalanine (Nal), 2-chlorophenylalanine (Ocf), 3-chlorophenylalanine (Mcf), 4-chlorophenylalanine (Pcf), 2-fluorophenylalanine (Off), 3-fluorophenylalanine (Mff), 4-fluorophenylalanine (Pff), 2-bromophenylalanine phenylalanine (Obf), 3-bromophenylalanine (Mbf), 4-bromophenylalanine (Pbf), 2-methylphenylalanine (Omf), 3-methylphenylalanine (Mmf), 4-methylphenylalanine (Pmf), 2-nitrophenylalanine (Onf), 3-nitrophenylalanine (Mnf), 4-nitrophenylalanine (Pnf), 2-cyanophenylalanine (Ocf), 3-cyanophenylalanine (Mcf), 4-cyanophenylalanine (Pcf), 2-trifluoromethylphenylalanine (Otf), 3-trifluoromethylphenylalanine (Mtf), 4-trifluoromethylphenylalanine (Ptf), 4-aminophenylalanine (Paf), 4-iodophenylalanine (Pif), 4-aminomethylphenylalanine (Pamf), 2,4-dichlorophenylalanine (Opef), 3,4-dichlorophenylalanine (Mpcf), 2,4-difluorophenylalanine (Opff), 3,4-difluorophenylalanine (Mpff), pyrid-2-ylalanine (2pAla), pyrid-3-ylalanine (3pAla), pyrid-4-ylalanine (4pAla), naphth-1-ylalanine (1nAla), naphth-2-ylalanine (2nAla), thiazolylalanine (taAla), benzothienylalanine (bAla), thienylalanine (tAla), furylalanine (fAla), homophenylalanine (hPhe), homotyrosine (hTyr), homotryptophan (hTrp), pentafluorophenylalanine (5ff), styrylalanine ( sAla), authrylalanine (aAla), 3,3-diphenylalanine (Dfa), 3-amino-5-phenylpentanoic acid (Afp), penicillamine (Pen), 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid (Tic), β-2-thienylalanine (Thi), methionine sulfoxide (Mso), N(w)-nitroarginine (nArg), homolysine (hLys), phosphonomethylphenylalanine (pmPhe), phosphoserine (pSer), phosphothreonine (pThr), homoaspartic acid (hAsp), homoglutanic acid (homoglutanic acid) (hGlu), 1-aminocyclopent-(2 or 3)-ene-4-carboxylic acid, pipecolic acid (PA), azetidine-3-carboxylic acid (ACA), 1-aminocyclopentane-3-carboxylic acid, allylglycine (aGly), propargylglycine (pgGly), homoalanine (hAla), norvaline (nVal), homoleucine (hLeu), homovaline (hVal), homoisoleucine (hIle), homoarginine (hArg), N-acetyllysine (AcLys), 2,4-diaminobutanoic acid (Dbu), 2,3-diaminobutanoic acid (Dab), N-methylvaline (MeVal), homocysteine ​​(hCys), homoserine (hSer), hydroxyproline (Hyp), and homoproline (hPro). Additional non-encoded amino acids that may comprise the polypeptides described herein will be apparent to those of skill in the art (see, e.g., Fasman,See the various amino acids provided in CRC Practical Handbook of Biochemistry and Molecular Biology, CRC Press, Boca Raton, FL, pp. 3-70 (1989) and the references cited therein, all of which are incorporated by reference. These amino acids can be in either the L- or D-configuration.

[0231] Those skilled in the art will recognize that amino acids or residues bearing side chain protecting groups can also comprise the polypeptides described herein. Non-limiting examples of such protected amino acids in the aromatic category in this context include, but are not limited to, the following (protecting groups listed in parentheses): Arg(tos), Cys(methylbenzyl), Cys(nitropyridine sulfenyl), Glu(δ-benzyl ester), Gln(xanthyl), Asn(N-δ-xanthyl), His(bom), His(benzyl), His(tos), Lys(fmoc), Lys(tos), Ser(O-benzyl), Thr(O-benzyl), and Tyr(O-benzyl).

[0232] Conformationally constrained non-encoded amino acids that may comprise the polypeptides described herein include, but are not limited to, N-methyl amino acids (L-configuration), 1-aminocyclopent-(2 or 3)-ene-4-carboxylic acid, pipetic acid, azetidine-3-carboxylic acid, homoproline (hPro), and 1-aminocyclopentane-3-carboxylic acid.

[0233] In some embodiments, the engineered polypeptide may be in various forms (e.g., as an isolated preparation, a substantially purified enzyme, whole cells transformed with a gene(s) encoding the enzyme, and / or as cell extracts and / or lysates of such cells, etc.). The enzyme may be lyophilized, spray-dried, precipitated, or in a crude paste form, as discussed further below.

[0234] In some embodiments, the engineered polypeptide may be in the form of a biocatalytic composition. In some embodiments, the biocatalytic composition comprises (a) a means for converting NTP-3-O-RBG or a natural or modified NTP substrate and an oligoacceptor compound into a one-nucleotide extended oligoacceptor product by contact with TdT, and (b) a suitable cofactor. The suitable cofactor may be cobalt, manganese, or any other suitable cofactor.

[0235] In some embodiments, the polypeptides described herein are provided in the form of a kit. The enzymes in the kit may be present individually or as multiple enzymes. The kit may further include reagents for carrying out the enzymatic reaction, substrates for assessing the activity of the enzyme, and reagents for detecting the product. The kit may also include a reagent dispenser and instructions for using the kit.

[0236] In some embodiments, the kits of the invention include an array comprising multiple different TdT polypeptides at different addressable locations, where the different polypeptides are different variants of a reference sequence, each having at least one different improved enzymatic property. In some embodiments, multiple polypeptides immobilized on a solid support are arranged on the array at different locations addressable for robotic delivery of reagents or by detection methods and / or instruments. The array can be used to test a variety of substrate compounds for conversion by the polypeptides. Such arrays comprising multiple engineered polypeptides and methods for their use are known in the art (see, e.g., WO2009 / 008908A2).

[0237] Polynucleotides, expression vectors, and host cells encoding engineered terminal deoxynucleotidyl transferases

[0238] In another aspect, the present invention provides polynucleotides encoding the engineered TdT polypeptides described herein. The polynucleotides can be operably linked to one or more heterologous regulatory sequences that control gene expression, to create recombinant polynucleotides capable of expressing the polypeptides. Introducing an expression construct containing a heterologous polynucleotide encoding an engineered TdT into a suitable host cell results in expression of the corresponding TdT polypeptide.

[0239] As will be apparent to those skilled in the art, the availability of protein sequences and knowledge of the codons corresponding to various amino acids accounts for all polynucleotides capable of encoding a subject polypeptide. The degeneracy of the genetic code (where the same amino acid is coded for by alternative or synonymous codons) allows for the creation of a vast number of nucleic acids, all of which will encode improved TdT enzymes. Thus, armed with knowledge of a particular amino acid sequence, one skilled in the art can create any number of different nucleic acids by simply altering the sequence of one or more codons in a manner that does not alter the amino acid sequence of the protein. In this regard, the present invention specifically contemplates any and all possible variations that can be made of polynucleotides encoding the polypeptides described herein by selecting combinations based on possible codon choices; all such variations are set forth in Tables 5.1, 6.2, 7.2, 8.2, 9.2, 10.2, 11.2, 12.2, 13.2, 14.2, 15. .2, 16.2, 17.2, 18.2, 19.2, 20.2, 21.2, 22.2, 23.2, 24.2, 25.2, 26.2, 26.3, 26.4, 27.2, 27.3, 27.4, 27.5, 28.1, 28.2, 28.3, 29.2, 30.2, 31.2, 32.2, 33.2, 34.2, 35.2, 36.2, 37.2, 38.2, 39.2, 40.2, 41.2, 42.2, 43.2, 44.2, 45.2, 46.2, 47.2, 48.2, 49.2, 50.2, 51.2, 52.2, 53.2, 54.2, 55.2, 56.2, 57.2, 58.2, 59.2, 60.2, 61.2, 62.2, 63.2, 64.2, 65.2, 66.2, 67.2, 68.2, 69.2, 70.2, 71.2, 72.2, 73.2, 74.2, 75.2, 76.2, 77.2, 78.2, 79.2, 80.2, 81.2, 82.2, 83.2, 84.2, 85.2, 86.2, 87.2, 88.2, 89.2, 90.2, 91.2, 92.2, 93 1.2, 42.2, 43.2, 44.2, 45.2, 46.2, 47.2, 48.2, 49.2, 50.2, 51.2, 52.2, 53.2, 54.2, 55.2, 56.2, 56.3, 56.4, 61.2, 63.2, 64.2, 65.2, 66.2, 67.2, 68.2, 69.2, 70.2, 71.2, 72.2, 73.2, 74.2, 75.2, 76.2 , 77.2, 78.2, 79.2, and 80.1, as well as the amino acid sequences disclosed in the Sequence Listing, which is incorporated herein by reference as even-numbered sequences within the ranges of SEQ ID NOs: 4-1960, 2004-3920, 4048-5466, and 5476, are considered to be specifically disclosed for any polypeptide described herein.

[0240] In various embodiments, codons are preferably selected to be compatible with the host cell in which the protein is being produced. For example, to express a gene in bacteria, preferred codons used in bacteria are used, to express in yeast, preferred codons used in yeast are used, and to express in mammalian cells, preferred codons used in mammals are used. In some embodiments, optimizing the codon usage of TdT does not require replacing every codon, since the native sequence may contain preferred codons and may not use preferred codons for every amino acid residue. Consequently, a codon-optimized polynucleotide encoding a TdT enzyme may contain preferred codons at more than about 40%, 50%, 60%, 70%, 80%, or 90% of the codon positions in the full-length coding region.

[0241] In some embodiments, the polynucleotide comprises a codon-optimized nucleotide sequence encoding a TdT polypeptide amino acid sequence represented by SEQ ID NO: 2, 8, 16, 24, 36, 268, 648, 660, 882, 1100, 1336, 1348, 1596, 1654, 1830, 1950, 2008, 2254, 2514, 2524, 2638, 2804, 2812, 2956, 3174, 3222, 3670, 3674, 3796, 3870, 3918, 4266, 4442, 4654, 4850, 4856, 4904, 5002, 5028, 5192, and / or 5246. In some embodiments, the polynucleotide has a nucleic acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to a codon-optimized nucleic acid sequence encoding an even-numbered sequence within the ranges of SEQ ID NOs: 4-1960, 2004-3920, 4048-5466, and 5476. In some embodiments, the polynucleotide has a nucleic acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to a codon-optimized nucleic acid sequence encoding an odd-numbered sequence within the ranges of SEQ ID NOs: 3-1959, 2003-3919, 4047-5465, and 5475. In some embodiments, codon-optimized sequences of odd-numbered sequences within the ranges of SEQ ID NOs: 3-1959, 2003-3919, 4047-5465, and 5475 enhance expression of the encoded TdT to provide preparations of the enzyme capable of converting substrates to products.

[0242] In some embodiments, the polynucleotide sequence is selected from the group consisting of SEQ ID NOs: 1, 7, 15, 23, 35, 267, 647, 659, 881, 1099, 1335, 1347, 1595, 1653, 1829, 1949, 2007, 2253, 2513, 2523, 2637, 2803, 2811, 2955, 3173, 3221, 3669, 3673, 3795, 3869, 3917, 4265, 4441, 4653, 4703, 4803, 4904, 5003, 5103, 5203, 5303, 5403, 5503, 5603, 5703, 5803, 5903, 6003, 6103, 6203, 6303, 6403, 6503, 6603, 6703, 6803, 6903, 7003, 7103, 7203, 7303, 7403, 7503, 7603, 7703, 7803, 7903, 8003, 8103, 8203, 8303, 8403, 8503, 8603, 8703, 8803, 8903, 9003, 9103, 9203, 9303, 9403, 9503, 9603, 9 and / or 5245, and / or a functional fragment thereof, wherein the polynucleotide sequence encodes an engineered polypeptide comprising at least one substitution at one or more amino acid positions.

[0243] In some embodiments, the polynucleotide sequence is selected from the group consisting of SEQ ID NOs: 2, 8, 16, 24, 36, 268, 648, 660, 882, 1100, 1336, 1348, 1596, 1654, 1830, 1950, 2008, 2254, 2514, 2524, 2638, 2804, 2812, 2956, 3174, 3222, 3670, 3674, 3796, 3870, 3918, 4266 , 4442, 4654, 4850, 4856, 4904, 5002, 5028, 5192, and / or 5246, each encoding at least one engineered terminal deoxynucleotidyl transferase comprising a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reference sequence of any of the following:

[0244] In some embodiments, the polynucleotide sequence comprises SEQ ID NO: 7, 15, 23, 35, 267, 647, 659, 881, 1099, 1335, 1347, 1595, 1653, 1829, 1949, 2007, 2253, 2513, 2523, 2637, 2803, 2811, 2955, 3173, 3221, 3669, 3673, 3795, 3869, 3917, 4265, 4441, 4653, 4849, 4855, 4903, 5001, 5027, 5191, and / or 5245.

[0245] In some embodiments, the polynucleotide is capable of hybridizing under high stringency conditions to a reference sequence selected from the odd-numbered sequences of SEQ ID NOs: 3-1959, 2003-3919, 4047-5465, and 5475, or its complement, and encodes TdT.

[0246] In some embodiments, as described above, the polynucleotide is selected from the group consisting of SEQ ID NOs: 2, 8, 16, 24, 36, 268, 648, 660, 882, 1100, 1336, 1348, 1596, 1654, 1830, 1950, 2008, 2254, 2514, 2524, 2638, 2804, 2812, 2956, 3174, 3222, 3670, 3674, 3796, 3870, 3918, 4266, 4442, 4654, 4850, 4856, 4904, 5002, 5028, 5056, 5102, 5116, 5126, 5136, 5144, 5156, 5168, 5170, 5182, 5184, 5186, 5188, 5188, 5190, 5200, 5210, 5212, 5214, 5216, 5218, 5222, 5222, 5222, 5230, 5230, 5240, 5242, 5250, 5252, 5254, 5260, 5262, 5264, 5266, 5270, 5272, 5276, 5278, 5278, 5276, 5278, 5278, 5280, 5282 192, and / or 5246, and the polypeptides are selected from the group consisting of SEQ ID NOs: 2, 8, 16, 24, 36, 268, 648, 660, 882, 1100, 1336, 1348, 1596, 1654, 1830, 1950, 2008, 2254, 2514, 2524, 2638, 2804, 2812, 2956, 3174, 3222, 3670, 3674, 3796, 3870, 3918, 4266, 4442, 4654, 4850, 4856, 4904, 5002, 5028, 5192, and / or 5246, and having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to a reference sequence selected from SEQ ID NOs: 2, 8, 16, 24, 36, 268, 648, 660, 882, 1100, 1336, 1348, 1596, 1654, 1830, 1950, 2008, 225 and / or 5246, wherein the sequence is selected from even-numbered sequences within the ranges of SEQ ID NOs: 4-1960, 2004-3920, 4048-5466, and 5476. In some embodiments, the reference amino acid sequence is selected from even-numbered sequences within the ranges of SEQ ID NOs: 4-1960, 2004-3920, 4048-5466, and 5476.In some embodiments, the reference amino acid sequence is SEQ ID NO:2, while in some other embodiments, the reference sequence is SEQ ID NO:8, while in some other embodiments, the reference sequence is SEQ ID NO:16. In some embodiments, the reference amino acid sequence is SEQ ID NO:24, while in some other embodiments, the reference sequence is SEQ ID NO:36, while in some other embodiments, the reference sequence is SEQ ID NO:268. In some embodiments, the reference amino acid sequence is SEQ ID NO:648, while in some other embodiments, the reference sequence is SEQ ID NO:660, while in some other embodiments, the reference sequence is SEQ ID NO:882. In some embodiments, the reference amino acid sequence is SEQ ID NO:1100, while in some other embodiments, the reference sequence is SEQ ID NO:1336, while in some other embodiments, the reference sequence is SEQ ID NO:1348. In some embodiments, the reference amino acid sequence is SEQ ID NO:1596, while in some other embodiments, the reference sequence is SEQ ID NO:1654, while in some other embodiments, the reference sequence is SEQ ID NO:1830. In some embodiments, the reference amino acid sequence is SEQ ID NO: 1950, while in some other embodiments, the reference sequence is SEQ ID NO: 2008, while in some other embodiments, the reference sequence is SEQ ID NO: 2254. In some embodiments, the reference amino acid sequence is SEQ ID NO: 2514, while in some other embodiments, the reference sequence is SEQ ID NO: 2524, while in some other embodiments, the reference sequence is SEQ ID NO: 2638. In some embodiments, the reference amino acid sequence is SEQ ID NO: 2804, while in some other embodiments, the reference sequence is SEQ ID NO: 2812, while in some other embodiments, the reference sequence is SEQ ID NO: 2956. In some embodiments, the reference amino acid sequence is SEQ ID NO: 3174, while in some other embodiments, the reference sequence is SEQ ID NO: 3222, while in some other embodiments, the reference sequence is SEQ ID NO: 3670.In some embodiments, the reference amino acid sequence is SEQ ID NO: 3674, while in some other embodiments, the reference sequence is SEQ ID NO: 3796, while in some other embodiments, the reference sequence is SEQ ID NO: 3870.

[0247] In some embodiments, the polynucleotide encodes a TdT polypeptide capable of converting one or more substrates to products with improved properties compared to a reference sequence, SEQ ID NO: 2, 8, 16, 24, 36, 268, 648, 660, 882, 1100, 1336, 1348, 1596, 1654, 1830, 1950, 2008, 2254, 2514, 2524, 2638, 2804, 2812, 2956, 3174, 3222, 3670, 3674, 3796, 3870, 3918, 4266, 4442, 4654, 4850, 4856, 4904, 5002, 5028, 5192, and / or 5246, No. 2, 8, 16, 24, 36, 268, 648, 660, 882, 1100, 1336, 1348, 1596, 1654, 1830, 1950, 2008, 22 54, 2514, 2524, 2638, 2804, 2812, 2956, 3174, 3222, 3670, 3674, 3796, 3870, 3918, 4266, and / or 5246.

[0248] In some embodiments, the polynucleotide encoding the engineered TdT comprises a polynucleotide sequence selected from odd-numbered sequences within the ranges of SEQ ID NOs: 3-1959, 2003-3919, 4047-5465, and 5475.

[0249] In some embodiments, a polynucleotide is capable of hybridizing under high stringency conditions to a reference polynucleotide sequence selected from the odd-numbered sequences within the ranges of SEQ ID NOs: 3-1959, 2003-3919, 4047-5465, and 5475, or its complement, and encodes a TdT polypeptide having one or more of the improved properties described herein. In some embodiments, a polynucleotide capable of hybridizing under high stringency conditions is selected from the odd-numbered sequences within the ranges of SEQ ID NOs: 2, 8, 16, 24, 36, 268, 648, 660, 882, 1100, 1336, 1348, 1596, 1654, 1830, 1950, 2008, 2254, 2514, 2524, 2638, 2804, 2812, 2824, 2836, 2848, 2852, 2864, 2872, 2882, 2892, 2904, 2912, 2924, 2936, 2948, 2950, ​​3008, 3014, 3024, 3036, 3048, 3052, 3064, 3072, 3084, 3092, 3106, 3112, 3114, 3116, 3118, 3126, 3128, 3136, 3136, 3148, 3156, 3156, 3166, 3172, 3176, 3178, 3178, 3180 , 2956, 3174, 3222, 3670, 3674, 3796, 3870, 3918, 4266, 4442, 4654, 4850, 4856, 4904, 5002, 5028, 5192, and / or 5246 and at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, TdT comprising an amino acid sequence having 96%, 97%, 98%, 99% or more identity to any of SEQ ID NOs: 2, 8, 16, 24, 36, 268, 648, 660, 882, 1100, 1336, 1348, 1596, 1654, 1830, 1950, 2008, 2254, 2514, 2516, 2520, 2530, 2540, 2550, 2560, 2570, 2580, 2590, 2600, 2610, 2611, 2612, 2613, 2614, 2615, 2616, 2617, 2618, 2619, 2620, 2621, 2622, 2623, 2624, 2625, 2626, 2627, 2628, 2629, 2630, 2631, 2632, 2633, 2634, 2635, 2636, 2637, 2638, 2639, 2640, 2641, 2642, 2643, 2644, 2645, 2646, 2647, 2648, 2649, 2650, 2651, 2652, 2653, 2654, 2655, 2656, 2657, 2658, 2659, 2700, 2701, 270 24, 2638, 2804, 2812, 2956, 3174, 3222, 3670, 3674, 3796, 3870, 3918, 4266, 4442, 4654, 4850, 4856, 4904, 5002, 5028, 5192, and / or 5246.

[0250] In some embodiments, polynucleotides capable of hybridizing under high stringency conditions are those set forth in SEQ ID NOs: 2, 8, 16, 24, 36, 268, 648, 660, 882, 1100, 1336, 1348, 1596, 1654, 1830, 1950, 2008, 2254, 2514, 2524, 2638, 2804, 2812, 2956, 3174, 3222, 3670, 3674, 3796, 3870, 3906, 4006, 4106, 4206, 4306, 4406, 4506, 4606, 4706, 4806, 4906, 5006, 5106, 5206, 5306, 5406, 5506, 5606, 5706, 5806, 5906, 6006, 6106, 6206, 6306, 6406, 6506, 6506, 6606, 6706, 6806, 6806, 6906, 7006, 7106, 7206, 7306, 7406, 7506, 7606, 7706, 7806, 7906, 8006, 8106, 8206, 8306, 8406, 8506, 8606, 8706, 8806, 8906, 9 , 3918, 4266, 4442, 4654, 4850, 4856, 4904, 5002, 5028, 5192, and / or 5246, encoding engineered TdT polypeptides with improved properties, comprising an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to 5002, 5028, 5192, and / or 5246. In some embodiments, a polynucleotide encodes a polypeptide described herein but has at least about 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more sequence identity at the nucleotide level to a reference polynucleotide encoding an engineered TdT. In some embodiments, the reference polynucleotide sequence is selected from SEQ ID NOs: 3-1959, 2003-3919, 4047-5465, and 5475.

[0251] In some embodiments, polynucleotides capable of hybridizing under high stringency conditions are those set forth in SEQ ID NOs: 2, 8, 16, 24, 36, 268, 648, 660, 882, 1100, 1336, 1348, 1596, 1654, 1830, 1950, 2008, 2254, 2514, 2524, 2638, 2804, 2812, 2956, 3174, 3222, 3670, 3674, 3796, 3870, 3906, 4006, 4106, 4206, 4306, 4406, 4506, 4606, 4706, 4806, 4906, 5006, 5106, 5206, 5306, 5406, 5506, 5606, 5706, 5806, 5906, 6006, 6106, 6206, 6306, 6406, 6506, 6506, 6606, 6706, 6806, 6806, 6906, 7006, 7106, 7206, 7306, 7406, 7506, 7606, 7706, 7806, 7906, 8006, 8106, 8206, 8306, 8406, 8506, 8606, 8706, 8806, 8906, 9 , 3918, 4266, 4442, 4654, 4850, 4856, 4904, 5002, 5028, 5192, and / or 5246, encoding engineered TdT polypeptides with improved properties, comprising an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to 5002, 5028, 5192, and / or 5246. In some embodiments, a polynucleotide encodes a polypeptide described herein but has at least about 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more sequence identity at the nucleotide level to a reference polynucleotide encoding an engineered TdT. In some embodiments, the reference polynucleotide sequence is selected from SEQ ID NOs: 3-1959, 2003-3919, 4047-5465, and 5475.

[0252] In some embodiments, an isolated polynucleotide encoding any of the engineered TdT polypeptides provided herein is engineered in various ways to allow for expression of the polypeptide. In some embodiments, the polynucleotide encoding the polypeptide is provided as an expression vector in which one or more regulatory sequences are present to regulate expression of the polynucleotide and / or polypeptide. Depending on the expression vector, it may be desirable or necessary to manipulate the isolated polynucleotide prior to its insertion into the vector. Techniques for modifying polynucleotides and nucleic acid sequences utilizing recombinant DNA methods are well known in the art.

[0253] In some embodiments, control sequences include, among other sequences, a promoter, leader sequence, polyadenylation sequence, propeptide sequence, signal peptide sequence, and transcription terminator. As is known in the art, appropriate promoters can be selected based on the host cell used. For bacterial host cells, suitable promoters for directing transcription of the nucleic acid constructs of the present application include, but are not limited to, promoters from the lac operon of E. coli, the agarase gene (dagA) of Streptomyces coelicolor, the levansucrase gene (sacB) of Bacillus subtilis, the alpha-amylase gene (amyL) of Bacillus licheniformis, the maltogenic amylase gene (amyM) of Bacillus stearothermophilus, the alpha-amylase gene (amyQ) of Bacillus amyloliquefaciens, the penicillinase gene (penP) of Bacillus licheniformis, the xylA and xylB genes of Bacillus subtilis, and prokaryotic beta-lactamase genes (see, e.g., Villa-Kamaroff et al., Proc. Natl. Acad. Sci. USA 75:3727-3731

[1978] ), as well as promoters derived from the tac promoter (see, e.g., DeBoer et al., Proc. Natl. Acad. Sci. USA 80:21-25

[1983] ).Exemplary promoters for filamentous fungal host cells include promoters obtained from the genes for Aspergillus oryzae TAKA amylase, Rhizomucor miehei aspartic proteinase, Aspergillus niger neutral alpha-amylase, Aspergillus niger acid-stable alpha-amylase, Aspergillus niger or Aspergillus awamori glucoamylase (glaA), Rhizomucor miehei lipase, Aspergillus oryzae alkaline protease, Aspergillus oryzae triosephosphate isomerase, Aspergillus nidulans acetamidase, and Fusarium oxysporum trypsin-like protease (see, e.g., WO 96 / 00787), as well as the NA2-tpi promoter (Aspergillus niger neutral alpha-amylase and Aspergillus Examples of promoters that can be used include promoters derived from the Saccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae galactokinase (GAL1), Saccharomyces cerevisiae alcohol dehydrogenase / glyceraldehyde-3-phosphate dehydrogenase (ADH2 / GAP), and Saccharomyces cerevisiae 3-phosphoglycerate kinase genes. Other promoters useful for yeast host cells are known in the art (see, e.g., Romanos et al., Yeast 8:423-488

[1992] ).

[0254] In some embodiments, the control sequence is a suitable transcription terminator sequence (a sequence recognized by a host cell to terminate transcription). The terminator sequence is operably linked to the 3' end of the nucleic acid sequence encoding the polypeptide. Any terminator that functions in the selected host cell can be used in the present invention. For example, exemplary transcription terminators for filamentous fungal host cells can be obtained from the genes for Aspergillus oryzae TAKA amylase, Aspergillus niger glucoamylase, Aspergillus nidulans anthranilate synthase, Aspergillus niger alpha-glucosidase, and Fusarium oxysporum trypsin-like protease. Exemplary terminators for yeast host cells can be obtained from the genes for Saccharomyces cerevisiae enolase, Saccharomyces cerevisiae cytochrome C (CYC1), and Saccharomyces cerevisiae glyceraldehyde-3-phosphate dehydrogenase. Other terminators useful for yeast host cells are known in the art (see, e.g., Romanos et al., supra).

[0255] In some embodiments, the control sequence is a suitable leader sequence (a nontranslated region of an mRNA important for translation by the host cell). The leader sequence is operably linked to the 5' end of the nucleic acid sequence encoding the polypeptide. Any leader sequence that is functional in the host cell of choice may be used. Exemplary leaders for filamentous fungal host cells are obtained from the Aspergillus oryzae TAKA amylase and Aspergillus nidulans triosephosphate isomerase genes. Suitable leaders for yeast host cells include, but are not limited to, those obtained from the Saccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae 3-phosphoglycerate kinase, Saccharomyces cerevisiae alpha factor, and Saccharomyces cerevisiae alcohol dehydrogenase / glyceraldehyde-3-phosphate dehydrogenase (ADH2 / GAP) genes. The control sequence may also be a polyadenylation sequence (a sequence operably linked to the 3' end of a nucleic acid sequence and recognized by a host cell during transcription as a signal for the addition of polyadenosine residues to the transcribed mRNA). Any polyadenylation sequence that functions in the host cell of choice may be used in the present invention. Exemplary polyadenylation sequences for filamentous fungal host cells include, but are not limited to, those derived from the genes for Aspergillus oryzae TAKA amylase, Aspergillus niger glucoamylase, Aspergillus nidulans anthranilate synthase, Fusarium oxysporum trypsin-like protease, and Aspergillus niger alpha-glucosidase. Useful polyadenylation sequences for yeast host cells are also known in the art (see, e.g., Guo and Sherman, Mol. Cell. Bio., 15:5983-5990

[1995] ).

[0256] In some embodiments, the control sequence is a signal peptide coding region that encodes an amino acid sequence linked to the amino terminus of a polypeptide and that directs the encoded polypeptide into the secretory pathway of a cell. The 5' end of the coding sequence of the nucleic acid sequence may inherently contain a signal peptide coding region naturally linked in translation reading frame with the segment of the coding region that encodes the secreted polypeptide. Alternatively, the 5' end of the coding sequence may contain a signal peptide coding region that is foreign to the coding sequence. Any signal peptide coding region that directs the expressed polypeptide into the secretory pathway of a host cell of choice can be used to express the engineered TdT polypeptides provided herein. Signal peptide coding regions useful in bacterial host cells include, but are not limited to, the signal peptide coding regions obtained from the genes for Bacillus NC1B 11837 maltogenic amylase, Bacillus stearothermophilus alpha-amylase, Bacillus licheniformis subtilisin, Bacillus licheniformis beta-lactamase, Bacillus stearothermophilus neutral protease (nprT, nprS, nprM), and Bacillus subtilis prsA. Additional signal peptides are known in the art (see, e.g., Simonen and Palva, Microbiol. Rev., 57:109-137

[1993] ). Signal peptide coding regions useful in filamentous fungal host cells include, but are not limited to, the signal peptide coding regions obtained from the genes for Aspergillus oryzae TAKA amylase, Aspergillus niger neutral amylase, Aspergillus niger glucoamylase, Rhizomucor miehei aspartic proteinase, Humicola insolens cellulase, and Humicola lanuginosa lipase.Useful signal peptides for yeast host cells include, but are not limited to, those derived from the genes for Saccharomyces cerevisiae alpha factor and Saccharomyces cerevisiae invertase.

[0257] In some embodiments, the control sequence is a propeptide-coding region that encodes an amino acid sequence positioned at the amino terminus of a polypeptide. The resulting polypeptide is sometimes referred to as a "proenzyme," "propolypeptide," or "zymogen." A propolypeptide can be converted to a mature, active polypeptide by catalytic or autocatalytic cleavage of the propeptide from the propolypeptide. Examples of propeptide-coding regions include, but are not limited to, the genes for alkaline protease (aprE) of Bacillus subtilis, neutral protease (nprT) of Bacillus subtilis, alpha-factor of Saccharomyces cerevisiae, aspartic proteinase of Rhizomucor miehei, and lactase of Myceliophthora thermophila (see, e.g., WO 95 / 33836). When both a signal peptide region and a propeptide region are present at the amino terminus of a polypeptide, the propeptide region is located adjacent to the amino terminus of the polypeptide, and the signal peptide region is located adjacent to the amino terminus of the propeptide region.

[0258] In some embodiments, regulatory sequences are also utilized. Such sequences facilitate regulation of polypeptide expression relative to the growth 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 a regulatory compound. In prokaryotic host cells, suitable regulatory sequences include, but are not limited to, the lac, tac, and trp operator 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 alpha-amylase promoter, the Aspergillus niger glucoamylase pro...

Claims

1. SEQ ID NOs: 2, 8, 12, 16, 24, 36, 54, 268, 648, 660, 882, 1100, 1336, 1348, 1596, 1654, 1830, 1950, 2008, 2254, 2514, 2524, 2638, 2804, 2812, 2956, 3174, 3222, 3670, 3674, 3796, 3870, 3918, 4266, 4442, 4654, 4850, 4856, 4904, 50 5028, 5192, 5246, or a functional fragment thereof, and having at least one amino acid residue difference compared to said reference sequence.

2. The polypeptide sequences are: 80 / 121 / 185 / 315; 80 / 121 / 185 / 190 / 289 / 290 / 293 / 313 / 342 / 499; 80 / 121 / 185 / 190 / 289 / 290 / 293 / 313 / 315 / 336 / 342 / 359 / 391 / 414 / 470 / 474 / 499 / 522 / 523; 80 / 121 / 185 / 190 / 244 / 289 / 290 / 293 / 313 / 315 / 317 / 336 / 342 / 359 / 391 / 470 / 474 / 499 / 522 / 523; 1 / 174 / 185 / 190 / 196 / 244 / 266 / 273 / 284 / 288 / 289 / 290 / 293 / 313 / 315 / 317 / 324 / 336 / 342 / 352 / 359 / 391 / 394 / 397 / 401 / 419 / 428 / 431 / 462 / 470 / 474 / 499 / 522 / 523; 80 / 106 / 121 / 185 / 190 / 205 / 289 / 290 / 293 / 313 / 315 / 336 / 342 / 359 / 391 / 470 / 474 / 499 / 522 / 523; 80 / 106 / 121 / 1 85 / 190 / 205 / 289 / 290 / 293 / 313 / 342 / 470 / 474 / 499 / 523; 80 / 106 / 121 / 185 / 190 / 244 / 289 / 290 / 293 / 307 / 342 / 359 / 470 / 474 / 499; 80 / 121 / 131 / 185 / 190 / 205 / 244 / 289 / 290 / 293 / 313 / 315 / 336 / 342 / 359 / 391 / 414 / 470 / 474 / 499 / 522 / 523; 80 / 121 / 131 / 185 / 190 / 205 / 244 / 289 / 2 90 / 293 / 313 / 336 / 342 / 359 / 391 / 414 / 470 / 474 / 499 / 522 / 523; 80 / 121 / 131 / 185 / 190 / 289 / 290 / 293 / 313 / 342 / 470 / 474 / 499 / 522 / 523; 80 / 121 / 174 / 179 / 185 / 190 / 236 / 244 / 288 / 289 / 290 / 293 / 313 / 315 / 317 / 336 / 342 / 359 / 363 / 391 / 394 / 408 / 426 / 462 / 470 / 474 / 499 / 522 / 523;８０／１２１／１７４／１８５／１８６／１９０／２３６／２４４／２７３／２８４／２８８／２８９／２９０／２９３／３１３／３１５／３１７／３３６／３４２／３５２／３５９／３９１／３９４／３９５／４１９／４２８／４３１／４６２／４７０／４７４／４９９／５２２／５２３；８０／１２１／１７４／１８５／１９０／１９３／１９６／２４４／２７３／２８４／２８８／２８９／２９０／２９３／２９７／３１３／３１５／３１７／３２４／３３６／３４２／３５２／３５９／３７６／３８０／３９１／３９４／４０１／４１５／４１９／４２８／４３１／４３５／４４１／４６２／４７０／４７４／４９９／５２２／５２３；８０／１２１／１７４／１８５／１９０／１９３／２４４／２７３／２８４／２８８／２８９／２９０／２９３／２９７／３１３／３１５／３１７／３３６／３４２／３５２／３５９／３９１／３９４／４１５／４１９／４２８／４３１／４６２／４７０／４７４／４９９／５２２／５２３；８０／１２１／１７４／１８５／１９０／２３６／２４４／２７３／２８２／２８４／２８８／２８９／２９０／２９３／３１３／３１５／３１７／３３６／３４２／３５２／３５９／３９１／３９４／３９５／４１９／４２８／４３１／４６２／４７０／４７４／４９９／５２２／５２３；８０／１２１／１７４／１８５／１９０／２４４／２７３／２８４／２８８／２８９／２９０／２９３／３１３／３１５／３１７／３３６／３４２／３５２／３５９／３９１／３９４／４１９／４２８／４３１／４６２／４７０／４７４／４９９／５２２／５２３；８０／１２１／１７４／１８５／１９０／２４４／２７３／２８４／２８８／２８９／２９０／２９３／３１３／３１５／３１７／３３６／３４２／３５９／３９１／３９４／４２８／４３１／４６２／４７０／４７４／４９９／５２２／５２３；８０／１２１／１７４／１８５／１９０／２４４／２８４／２８８／２８９／２９０／２９３／３１３／３１５／３１７／３３６／３４２／３５２／３５９／３９１／３９４／４１９／４２８／４３１／４６２／４７０／４７４／４９９／５２２／５２３；８０／１２１／１７４／１８５／１９０／２４４／２８４／２８８／２８９／２９０／２９３／３１３／３１５／３１７／３３６／３４２／３５２／３５９／３９１／３９４／４２８／４３１／４６２／４７０／４７４／４９９／５２２／５２３；８０／１２１／１７４／１８５／１９０／２４４／２８４／２８８／２８９／２９０／２９３／３１３／３１５／３１７／３３６／３４２／３５９／３９１／３９４／４２８／４３１／４６２／４７０／４７４／４９９／５２２／５２３；８０／１２１／１８５／１９０／１９６／２４４／２８９／２９０／２９３／３１３／３１５／３１７／３３６／３４２／３５９／３９１／４７０／４７４／４９９／５２２／５２３；８０／１２１／１８５／１９０／２０１／２８９／２９０／２９３／３１３／３４２／４７０／４７４／４９９／５２２；８０／１２１／１８５／１９０／２４４／２７３／２８９／２９０／２９３／３１３／３１５／３１７／３３６／３４２／３５２／３５９／３９１／４１９／４３５／４７０／４７４／４９９／５２２／５２３；８０／１２１／１８５／１９０／２４４／２８９／２９０／２９３／３００／３１３／３１５／３１７／３３６／３４２／３５９／３９１／４７０／４７４／４９９／５２２／５２３；８０／１２１／１８５／１９０／２４４／２８９／２９０／２９３／３１３／３１５／３１７／３３６／３４２／３５９／３８０／３９１／４０１／４１９／４７０／４７４／４９９／５２２／５２３；８０／１２１／１８５／１９０／２４４／２８９／２９０／２９３／３１３／３１５／３１７／３３６／３４２／３５９／３９１／３９２／４７０／４７４／４９９／５２２／５２３；８０／１２１／１８５／１９０／２４４／２８９／２９０／２９３／３１３／３１５／３１７／３３６／３４２／３５９／３９１／３９５／４７０／４７４／４９９／５２２／５２３；８０／１２１／１８５／１９０／２４４／２８９／２９０／２９３／３１３／３３６／３４２／３５９／３９１／４１４／４７０／４７４／４９９／５２２／５２３；８０／１２１／１８５／１９０／２８９／２９０／２９３／３１３／３３６／３４２／３５９／３９１／４７０／４７４／４９９／５２２／５２３；８０／１２１／１９０／２８９／２９０；８０／１８５／２３６／２８９／２９３；１２１／１８５／１９０／２１３／２８９／２９０／２９３；2. The engineered terminal deoxynucleotidyl transferase of claim 1, comprising residue differences compared to the reference sequence of SEQ ID NO: 2 at a set of amino acid positions selected from: 185 / 289 / 290 / 293;

3. The polypeptide sequences are: 174 / 244 / 273 / 284 / 288 / 315 / 317 / 336 / 352 / 359 / 391 / 394 / 419 / 428 / 431 / 462 / 470 / 474 / 522 / 523; 174 / 244 / 284 / 288 / 315 / 317 / 336 / 359 / 391 / 394 / 428 / 431 / 462 / 470 / 474 / 522 / 523; 2. The engineered terminal deoxynucleotidyl transferase of claim 1, comprising at least one substitution set comprising substitutions compared to the reference sequence of SEQ ID NO: 36 at amino acid positions selected from: 36 / 359 / 391 / 470 / 474 / 522 / 523; and 336 / 359 / 391 / 470 / 474 / 522 / 523; and / or any combination thereof.

4. 2. The engineered terminal deoxynucleotidyl transferase of claim 1, wherein the polypeptide sequence comprises at least one substitution or set of substitutions comprising a substitution compared to the reference sequence of SEQ ID NO: 8 at amino acid positions selected from 129 / 196, 173, 183, 186, 193, 195, 196, 263, 266, 268, 281, 282, 297, 300, 303, 316, 318, 320, 324, 343, 360, 392, 395, 397, 411, 415, 417, 421, 454, 456, 477, 481, and 492, and / or any combination thereof.

5. the polypeptide sequences are: 186, 186 / 236 / 318, 186 / 236 / 395, 186 / 282 / 318, 193 / 196, 193 / 196 / 266 / 324 / 376 / 380, 193 / 196 / 297, 193 / 196 / 297 / 324 / 376 / 380 / 401 / 415 / 435 / 441, 193 / 196 / 324, 193 / 196 / 324 / 397 / 401 / 441, 193 / 196 / 376 / 380, 193 / 297 / 324 / 376 / 435, 193 / 297 / 324 / 380, 193 / 297 / 415, 193 / 435, 196, 196 / 266, 196 / 266 / 324 / 397 / 2. The engineered terminal deoxynucleotidyl transferase of claim 1, comprising at least one substitution or set of substitutions comprising substitutions when compared to the reference sequence of SEQ ID NO: 16 at amino acid positions selected from: 401, 196 / 297 / 324 / 435, 236 / 282, 236 / 282 / 395, 236 / 318 / 481, 266 / 297 / 380 / 397 / 401, 282, 282 / 318, 282 / 481, 297 / 380 / 401 / 441, 297 / 435, 318 / 395, 376 / 401 / 441, 415, and 435 / 441, and / or any combination thereof.

6. The polypeptide sequence is 12, 13, 14, 17, 18, 20, 21, 22, 23, 24, 26, 27, 29, 30, 31, 33, 34, 35, 37, 41, 53, 57, 58, 61, 92, 94, 97, 101, 102, 103, 104, 105, 106, 107, 108, 124, 126, 133, 135 , 137, 138, 139, 140, 141, 142, 144, 145, 147, 149, 150, 152, 153, 154, 155, 156, 156 / 294, 159, 160, 161, 162, 163, 7 / 135, 12, 13, 14, 15, 16, 17 / 131, 18, 20, 23, 24, 25, 26, 2 2. The engineered terminal deoxynucleotidyl transferase of claim 1, comprising at least one substitution or set of substitutions comprising a substitution compared to the reference sequence of SEQ ID NO:24 at amino acid positions selected from: 7, 28, 29, 31, 32, 33, 34, 35, 44, 45, 46, 57, 65, 77, 85, 89, 93, 94, 97, 101, 102, 103, 105, 106, 108, 109, 110, 119, 123, 124, 126, 130, 131, 132, 133, 134, 135, 137, 138, 139, 149, 150, 153, and 156, and / or any combination thereof.

7. 2. The engineered terminal deoxynucleotidyl transferase of claim 1, wherein the polypeptide sequence comprises at least one substitution or set of substitutions comprising substitutions compared to the reference sequence of SEQ ID NO: 268 at amino acid positions selected from 14 / 53 / 300, 14 / 53 / 419, 106 / 300 / 415 / 419 / 456, 140, and 300 / 395 / 419, and / or any combination thereof.

8. The polypeptide sequences are 12 / 14 / 34, 12 / 14 / 34 / 37 / 94 / 140 / 141 / 145, 12 / 14 / 34 / 37 / 106 / 140 / 142 / 150 / 152 / 153, 12 / 14 / 34 / 37 / 141 / 142, 12 / 14 / 34 / 37 / 142 / 145, 12 / 14 / 34 / 37 / 142 / 161, 12 / 14 / 34 / 37 / 150, 12 / 14 / 34 / 37 / 150 / 153, 12 / 14 / 34 / 140 / 142, 12 / 14 / 34 / 140 / 150, 12 / 14 / 34 / 142 / 150 / 153, 12 / 14 / 92 / 94, 12 / 14 / 94 / 150 / 152, 12 / 14 / 106 / 107 / 141 / 142, 12 / 14 / 106 / 108 / 140 / 141 / 145 / 150, 12 / 14 / 106 / 108 / 152, 12 / 14 / 141 / 142, 12 / 14 / 150 / 152 / 153, 12 / 14 / 1 53, 12 / 34 / 92 / 140, 12 / 34 / 150 / 152, 12 / 37 / 94 / 141 / 150 / 152 / 153, 12 / 37 / 140 / 141 / 150 / 162, 12 / 161, 14, 14 / 31 / 34 / 37 / 140 / 141 / 145 / 161 / 162, 14 / 34 / 37, 14 / 34 / 37 / 145, 14 / 34 / 37 / 152, 14 / 34 / 94 / 106 / 108 / 141, 14 / 34 / 150 / 153, 14 / 106, 14 / 140, 14 / 141 / 161, 14 / 142, 14 / 142 / 161 / 162, 14 / 153, 14 / 161 , 20 / 21 / 24 / 33 / 58 / 104 / 106 / 124 / 155 / 156, 20 / 21 / 33 / 58 / 101 / 104 / 106 / 124 / 155, 20 / 21 / 58 / 104 / 106 / 155 / 156, 20 / 33 / 104 / 106 / 124 / 156, 20 / 58 / 101 / 104 / 106 / 156, 20 / 58 / 101 / 106, 20 / 101 / 106 / 156, 21 / 33 / 58 / 101 / 106, 21 / 33 / 58 / 106 / 155 / 156, 21 / 33 / 101 / 104 / 106, 21 / 33 / 106, 21 / 58 / 101 / 104 / 106 / 155, 21 / 58 / 106 / 155 / 156, 21 / 101 / 104 / 106 / 156, 21 / 104 / 106, 21 / 104 / 106 / 124, 21 / 104 / 106 / 156, 30 / 33 / 58 / 104 / 106 / 155 / 156, 30 / 33 / 101 / 106 / 156,30 / 33 / 104 / 106 / 155 / 156, 30 / 101 / 104 / 106 / 155 / 156, 30 / 104 / 106 / 155, 30 / 104 / 106 / 155 / 156, 30 / 106 / 155, 33 / 58 / 104 / 106, 33 / 101 / 104 / 106 / 155, 34 / 37, 34 / 37 / 92, 34 / 37 / 140 / 141 / 142 / 145, 34 / 37 / 141 / 142, 34 / 37 / 150 / 153, 34 / 92 / 94 / 141 / 142, 34 / 141 / 142 / 145, 34 / 150 / 152 / 153, 37 / 92 / 142, 37 / 141 / 142, 37 / 153, 58 / 101 / 104 / 106 / 156, 58 / 101 / 106 / 155, 58 / 104 / 106 / 155 / 156, 92 / 94 / 106 / 142 / 145, 101 / 104 / 106, 101 / 104 / 106 / 155 / 156, 1 01 / 104 / 106 / 156, 101 / 106, 101 / 106 / 124 / 155, 101 / 106 / 155 / 156, 104 / 106, 104 / 106 / 124, 104 / 106 / 155, 104 / 106 / 155 / 156, 104 / 106 / 156, 106 / 107 / 108 / 142 / 220, 106 / 108 / 140 / 141 / 152 / 153, 106 / 108 / 140 / 142 / 150 / 153, 1 2. The engineered terminal deoxynucleotidyl transferase of claim 1, comprising at least one substitution or set of substitutions comprising a substitution compared to the reference sequence of SEQ ID NO: 648 at an amino acid position selected from: 06 / 156, 140 / 141 / 142, 140 / 145, 140 / 145 / 150 / 152, 141, 141 / 152, and 161, and / or any combination thereof.

9. The polypeptide sequences are 16 / 29 / 30, 16 / 29 / 30 / 33 / 153, 16 / 29 / 30 / 101 / 104, 16 / 30 / 104, 16 / 33, 29 / 30, 58, 92 / 94 / 108 / 141 / 155 / 392, 92 / 101 / 137 / 155 / 476, 94 / 101 / 156 / 476, 101, 101 / 104, 101 / 137 / 155, 101 / 141 / 155 / 156, 108, 195, 197, 204 / 342, 205, 236 / 297, 258, 261, 262, 264, 268, 269, 276, 278, 280, 2 2. The engineered terminal deoxynucleotidyl transferase of claim 1, comprising at least one substitution or set of substitutions comprising a substitution compared to the reference sequence of SEQ ID NO: 660 at an amino acid position selected from: 81, 282, 290, 291, 297, 300, 303, 306, 308, 309, 310, 312, 315, 316, 342, 344, 353, 360, 385, 391, 410, 413, 419, 421, 448, 454, 456, 473, 476, 515, 525, and / or any combination thereof.

10. 2. The engineered terminal deoxynucleotidyl transferase of claim 1, wherein the polypeptide sequence comprises at least one substitution or set of substitutions comprising a substitution compared to the reference sequence of SEQ ID NO: 882 at amino acid positions selected from 175, 179, 196, 199, 201, 203, 208, 272, 273, 275, 313, 314, 317, 319, 321, 322, 324, 325, 329 / 462, 350, 376, 379, 394, 397, 403 / 462, 404, 406, 408, 457, 461, 462, 469, 477, 481, 484, 491, 492, and 495, and / or any combination thereof, wherein the amino acid positions are numbered with reference to SEQ ID NO:

882.

11. 2. The engineered terminal deoxynucleotidyl transferase of claim 1, wherein the polypeptide sequence comprises at least one substitution or set of substitutions comprising a substitution compared to the reference sequence of SEQ ID NO: 1100 at amino acid positions selected from 101 / 137 / 264 / 476 / 525 and 264 and / or any combination thereof.

12. The polypeptide sequences are: 61, 152 / 503, 160, 162, 165, 177, 200, 200 / 425, 213, 217, 219, 223, 236, 246, 248, 292, 292 / 411, 295, 326, 329, 330, 333, 334, 338, 340, 340 / 438, 363, 369, 370, 372, 373, 383, 400 / 401 / 402, 425, 427, 435, 435 / 503, 437, 440, 441, 442, 443, 444, 446, 459, 460, 488, 490, 501, 502, 503, 504, and 506, 15 / 199 / 203 / 394, 15 / 199 / 394, 28 / 344 / 353 / 395, 195 / 199 / 203 / 278 / 297 / 394, 195 / 199 / 203 / 278 / 314 / 353 / 394, 195 / 203 / 278 / 394 / 395, 195 / 203 / 297 / 314 / 394, 195 / 203 / 297 / 394 / 419, 195 / 203 / 394, 195 / 278 / 297 / 394, 195 / 278 / 297 / 394 / 395, 195 / 314 / 344, 195 / 394 / 395, 199 / 203 / 297 / 394 / 395, 203 / 278 / 297 / 394, 203 / 297 / 314 / 394 / 395, 203 / 310 / 314 / 394 / 395 / 419, 203 / 344 / 394, 20 2. The engineered terminal deoxynucleotidyl transferase of claim 1, comprising at least one substitution or set of substitutions comprising substitutions compared to the reference sequence of SEQ ID NO: 1336 at amino acid positions selected from: 3 / 353, 203 / 394, 203 / 394 / 395, 297 / 394, 314 / 394 / 395, 344 / 394 / 395, 353, and 394, and / or any combination thereof.

13. The polypeptide sequences are: 3 / 175 / 213 / 313 / 325 / 340 / 457 / 481 / 485, 3 / 307 / 321 / 340 / 353 / 406 / 408 / 445, 148 / 175 / 201 / 457 / 485, 175 / 201 / 333 / 412 / 425 / 457 / 485, 175 / 325 / 397, 175 / 333, 175 / 333 / 369 / 481, 175 / 333 / 485, 175 / 485, 199 / 307 / 321 / 340 / 406 / 408 / 445 / 484, 201 / 213 / 333 / 344 / 397 / 425 / 481 / 485, 201 / 333 / 344 / 457 / 481, 201 / 333 / 481, 201 / 406 / 408 / 462 / 484 / 502, 213 2. The engineered terminal deoxynucleotidyl transferase of claim 1, comprising at least one substitution or set of substitutions comprising a substitution when compared to the reference sequence of SEQ ID NO: 1348 at an amino acid position selected from: 213 / 333 / 397, 307 / 333 / 340 / 408 / 445 / 462 / 502, 307 / 373 / 406 / 408 / 484, 321 / 333 / 340, 325 / 333 / 369 / 425, 325 / 425 / 457 / 481, 333, 333 / 344 / 369 / 397, 333 / 344 / 369 / 485, 340 / 484, 344 / 485, 353 / 406, 373, and 406 / 408, and / or any combination thereof.

14. The polypeptide sequences are: 161, 162, 163, 165, 171, 177, 179, 188, 200, 205, 208, 231, 233, 251, 252, 253, 260, 261, 277, 306, 307, 321, 325, 327, 329, 330, 353, 368, 370, 371, 376, 380, 382, ​​393, 400, 402, 405, 406, 407, 408, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 440, 441, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 7, 408, 410, 413, 414, 419, 426, 441, 442, 446, 460, 464, 484, 488, 490, 495, 506, 508, 518, 520, 160 / 219 / 460 / 503 / 506, 160 / 165 / 203 / 205 / 219 / 353 / 406 / 408 / 442 / 446 / 460 / 488, 160 / 165 / 205 / 219 / 406 / 408 / 441 / 442 / 446, 160 / 203 / 205 / 406 / 408 / 441 / 442 / 446, 160 / 219 / 330 / 406 / 408 / 442 / 446 / 484, 205 / 307 / 406 / 408 / 441 / 442 / 446 / 460 / 488, 252 / 333, 406 / 408 / 441 / 442 / 446, 406 / 408 / 442 / 446219 / 307 2. The engineered terminal deoxynucleotidyl transferase of claim 1, comprising at least one substitution or set of substitutions comprising substitutions compared to the reference sequence of SEQ ID NO: 1596 at amino acid positions selected from: 406 / 408, 406 / 408 / 442 / 446, and 406 / 408 / 490, and / or any combination thereof.

15. The polypeptide sequences are 14 / 53 / 300, 14 / 53 / 419, 106 / 300 / 415 / 419 / 456, 140, 160, 160 / 163 / 165 / 203 / 205 / 219 / 353 / 414 / 441 / 460 / 488, 160 / 165 / 203 / 205 / 219 / 353 / 460 / 488, 160 / 165 / 179 / 203 / 205 / 208 / 219 / 353 / 376 / 413 / 414 / 441 / 488, 160 / 165 / 179 / 203 / 307 / 376 / 413 / 495, 160 / 165 / 203 / 205 / 219 / 353 / 460 / 488, 160 / 165 / 203 / 205 / 307 / 371 / 376 / 413 / 414, 160 / 165 / 203 / 208 / 371 / 376 / 413 / 414, 160 / 165 / 205, 160 / 165 / 205 / 208, 160 / 165 / 205 / 219 / 441, 160 / 165 / 205 / 414 / 441 / 495, 160 / 165 / 307 / 353 / 413 / 414 / 441 / 488 / 495, 160 / 179 / 203 / 205 / 208 / 219 / 371 / 414 / 484 / 506, 160 / 179 / 208 / 219 / 307 / 413 / 414 / 503 / 506, 160 / 179 / 208 / 307 / 371 / 376 / 414, 160 / 179 / 307 / 376 / 441 / 488 / 503, 160 / 203 / 205 / 208 / 219 / 414 / 460 / 506, 160 / 203 / 205 / 208 / 413 / 460 / 484 / 488, 160 / 203 / 205 / 441, 160 / 203 / 208 / 307 / 353 / 495, 160 / 203 / 208 / 371 / 413 / 414, 160 / 203 / 208 / 413 / 414 / 441 / 484, 160 / 203 / 326 / 353 / 413 / 414 / 484 / 495, 160 / 205 / 208 / 219 / 326 / 441 / 484 / 488 / 503, 160 / 208 / 326 / 376 / 414 / 441 / 484 / 488, 160 / 208 / 371 / 441 / 484 / 506, 160 / 208 / 414 / 441 / 452 / 480 / 488 / 495, 160 / 219 / 307 / 371 / 506, 160 / 219 / 330 / 484, 165 / 179 / 203 / 205 / 219 / 414 / 418 / 441 / 488 / 503, 165 / 179 / 203 / 205 / 484 / 503, 165 / 179 / 205 / 413 / 441,165 / 179 / 208 / 353 / 413 / 414 / 503, 165 / 203 / 205, 165 / 203 / 205 / 307 / 414 / 441 / 484 / 495 / 503 / 506, 165 / 203 / 205 / 484 / 488, 165 / 203 / 208 / 326 / 376 / 503, 165 / 205, 165 / 208 / 326 / 413 / 414 / 484 / 495 / 506, 179, 179 / 203 / 205, 179 / 203 / 208 / 326 / 353 / 376 / 484, 179 / 205 / 208 / 353 / 414 / 441 / 460 / 484 / 488, 179 / 205 / 353, 179 / 208 / 353 / 460, 179 / 353, 203 / 205 / 208 / 307 / 330 / 353 / 441 / 460 / 503 / 506, 203 / 205 / 208 / 307 / 441, 203 / 205 / 208 / 353, 203 / 208 / 219 / 376 / 441, 203 / 208 / 2 19 / 441, 203 / 208 / 326 / 353, 203 / 413 / 503 / 506, 205 / 208 / 307 / 353 / 376 / 413, 205 / 208 / 414, 205 / 219 / 307 / 353, 205 / 307, 205 / 307 / 376 / 414 / 441 / 495, 205 / 307 / 441 / 460 / 488, 205 / 326 / 488 / 503 / 506, 208 / 488 / 506, 300 / 395 / 4193 / 160 / 2 2. The engineered terminal deoxynucleotidyl transferase of claim 1, comprising at least one substitution or set of substitutions comprising a substitution compared to the reference sequence of SEQ ID NO: 1654 at an amino acid position selected from: 05 / 208 / 219 / 307 / 353 / 371 / 376 / 413 / 414 / 441 / 488, 326 / 353 / 371 / 376 / 414, and 441, and / or any combination thereof.

16. The polypeptide sequences are: 163 / 179 / 277 / 338 / 340, 163 / 414 / 441, 171 / 200 / 334 / 406 / 490, 177, 194, 196, 266, 267, 268, 269, 273, 273 / 501, 274, 277, 278, 292 / 406, 297, 298, 299, 301, 302, 309, 312, 347, 359, 390, 392, 3 2. The engineered terminal deoxynucleotidyl transferase of claim 1, comprising at least one substitution or set of substitutions comprising a substitution compared to the reference sequence of SEQ ID NO: 1830 at amino acid positions selected from: 94, 407, 408, 413, 416, 454, 468, 473, 477, 479, and 493, and / or any combination thereof.

17. The polypeptide sequence is 163 / 169, 185, 186, 192, 193, 194, 197, 198, 245, 251, 258, 259, 261, 263, 271, 274, 278, 280, 284, 286, 290, 291, 297, 304, 306, 308, 316, 347, 352, 359, 362, 378, 393, 396, 398, 399, 405, 407, 409 / 414, 410 / 414, 411 2. The engineered terminal deoxynucleotidyl transferase of claim 1, comprising at least one substitution or set of substitutions comprising substitutions compared to the reference sequence of SEQ ID NO: 1950 at amino acid positions selected from: 414, 413 / 414, 414, 414 / 415, 414 / 417, 415, 455, 465, 466, 468, 494, and 509, and / or any combination thereof.

18. The polypeptide sequences are: 3, 8, 9, 11, 12, 15, 16, 26, 26 / 27, 36, 38, 39, 50, 58, 62, 70, 76, 79, 81, 90, 116, 147, 151, 157, 158, 158 / 274 / 411 / 413 / 414, 185 / 274 / 413 / 414, 185 / 411 / 413 / 414, 189, 1 96, 205, 206, 246, 248, 249, 253, 255, 262, 263 / 411 / 413 / 414 / 468, 263 / 413 / 414, 274 / 286 / 411 / 413 / 417 / 468, 274 / 411 / 417 / 468, 274 / 411 / 468, 274 / 413 / 414 / 417 / 468, 274 / 468, 278 2. The engineered terminal deoxynucleotidyl transferase of claim 1, wherein the engineered terminal deoxynucleotidyl transferase comprises at least one substitution or set of substitutions comprising a substitution when compared to the reference sequence of SEQ ID NO: 2008 at an amino acid position selected from: 411 / 413 / 468, 307, 314, 318, 324, 353, 397, 408, 410, 411 / 413 / 417, 411 / 413 / 417 / 468, 411 / 413 / 468, 411 / 414, 413, 413 / 414, 413 / 414 / 468, 413 / 417 / 468, 413 / 468, 414, and 468, 469, 473, 480, 481, 491, and 493, and / or any combination thereof.

19. 2. The engineered terminal deoxynucleotidyl transferase of claim 1, wherein the polypeptide sequence comprises at least one substitution or set of substitutions comprising substitutions compared to the reference sequence of SEQ ID NO: 2254 at amino acid positions selected from 197 / 407 / 455, 197 / 455, 284 / 398 / 466, 362 / 407 / 455, 396 / 398 / 410 / 466, 398 / 466, 399 / 411 / 416, and 466, and / or any combination thereof.

20. The polypeptide sequences are: 26 / 90 / 94 / 248 / 261 / 266 / 362 / 455, 26 / 90 / 246, 26 / 90 / 246 / 248, 26 / 90 / 246 / 248 / 261, 26 / 90 / 246 / 248 / 362 / 455, 26 / 90 / 246 / 248 / 455, 26 / 90 / 246 / 266 / 362, 26 / 90 / 246 / 362, 26 / 90 / 246 / 455, 26 / 90 / 24 8, 26 / 90 / 248 / 266 / 455, 26 / 90 / 248 / 455, 26 / 90 / 248 / 455 / 459, 26 / 90 / 266, 26 / 90 / 362 / 455, 26 / 173 / 248, 26 / 246, 26 / 246 / 248 / 362, 26 / 246 / 248 / 362 / 455, 26 / 246 / 248 / 455, 26 / 248, 26 / 248 / 261 / 266, 26 / 248 / 261 / 2 66 / 362 / 455, 26 / 248 / 266 / 362, 26 / 248 / 362 / 455, 26 / 248 / 455, 26 / 362, 26 / 362 / 455, 58 / 197 / 249 / 407 / 410, 62 / 249, 90 / 246 / 248, 90 / 246 / 248 / 261 / 266 / 362 / 455, 90 / 246 / 248 / 266 / 362 / 455, 246 / 248, 246 / 248 / 362, 2. The engineered terminal deoxynucleotidyl transferase of claim 1, comprising at least one substitution or set of substitutions comprising substitutions compared to the reference sequence of SEQ ID NO: 2514 at amino acid positions selected from 246 / 266 / 455, 248, 248 / 266 / 362, 248 / 362 / 455, 248 / 455, and 362, and / or any combination thereof.

21. The polypeptide sequences are: 9 / 16 / 62 / 157 / 246 / 249 / 362, 11 / 58 / 227 / 246, 12 / 16 / 158 / 246 / 248 / 249, 30 / 189 / 261 / 266 / 353 / 465 / 468, 47 / 326, 30 / 189 / 266, 30 / 261 / 266 / 353 / 468, 30 / 2 66, 30 / 266 / 303, 30 / 266 / 353, 38, 38 / 81 / 318, 38 / 197, 39, 39 / 79, 58 / 157 / 158 / 362, 70 / 353, 79 / 81, 81, 189, 189 / 261, 189 / 353, 246 / 249, 261 / 353, 266 / 307 / 353 / 468, 266 / 2. The engineered terminal deoxynucleotidyl transferase of claim 1, comprising at least one substitution or set of substitutions comprising a substitution compared to the reference sequence of SEQ ID NO: 2524 at amino acid positions selected from: 353 / 468, 266 / 468, 169, 175, 179, 191 / 413, 200, 203, 292, 293, 304 / 329, 325, 327 / 406, 329, 340, 353 / 459, 373, 379, 382, ​​402, 403, 404, 406, 427, 429, 459, 461, 484, 490, 495, 504, 506, and 508, and / or any combination thereof.

22. 2. The engineered terminal deoxynucleotidyl transferase of claim 1, wherein the polypeptide sequence comprises at least one substitution or set of substitutions comprising substitutions compared to the reference sequence of SEQ ID NO: 2638 at amino acid positions selected from 11 / 30 / 79 / 189 / 480, 30 / 58 / 79 / 189 / 307 / 480, 79 / 189 / 307 / 410, and 79 / 307, and / or any combination thereof.

23. The polypeptide sequences are: 169 / 304 / 340 / 402 / 427, 169 / 304 / 340 / 427 / 429 / 504, 169 / 304 / 340 / 429 / 506, 169 / 304 / 340 / 504, 169 / 304 / 402 / 403 / 427 / 506, 169 / 304 / 403 / 427 / 504, 169 / 304 / 427 / 504 / 506, 169 / 304 / 504, 169 / 340 / 402 / 403 / 427 / 429 / 504, 169 / 340 / 402 / 403 / 427 / 504 / 506, 169 / 340 / 402 / 504, 169 / 340 / 42 7, 169 / 340 / 506, 169 / 402 / 403 / 504 / 506, 169 / 402 / 427 / 429 / 504, 169 / 402 / 504, 169 / 402 / 504 / 506, 169 / 403 / 427 / 506, 266 / 327 / 329 / 404 / 410, 292 / 327 / 329 / 468, 304, 304 / 340 / 402, 304 / 340 / 402 / 403 / 427 / 429 / 504 / 506, 304 / 340 / 402 / 403 / 504 / 506, 304 / 340 / 402 / 403 / 506, 304 / 340 / 402 / 427 / 504 / 506, 304 / 340 / 402 / 506, 304 / 340 / 403 / 427 / 429 / 504 / 506, 304 / 340 / 427, 304 / 402 / 403, 304 / 402 / 403 / 427 / 504, 304 / 402 / 403 / 429, 304 / 402 / 403 / 504 / 506, 3 04 / 402 / 403 / 506, 304 / 403 / 504, 304 / 504, 304 / 504 / 506, 327, 327 / 329, 327 / 329 / 379 / 404 / 406 / 410, 327 / 329 / 404 / 410, 327 / 329 / 465 / 484, 327 / 382 / 40 6 / 410 / 484, 327 / 410 / 484, 340, 340 / 402 / 403, 340 / 402 / 427 / 429 / 506, 340 / 402 / 429 / 504 / 506, 340 / 402 / 504, 340 / 403 / 504, 340 / 504, 340 / 506, 379 / 382 / 468, 379 / 404 / 410, 379 / 410, 379 / 465 / 468 / 484, 379 / 468, 402 / 403 / 427 / 504 / 506, 402 / 403 / 429 / 504, 402 / 427 / 504, 402 / 504, 403 / 427, 427, 484, 504,2. The engineered terminal deoxynucleotidyl transferase of claim 1, comprising at least one substitution or set of substitutions comprising a substitution compared to the reference sequence of SEQ ID NO: 2804 at an amino acid position selected from 504 / 506, and 506, and / or any combination thereof.

24. The polypeptide sequences are: 30, 186, 188, 198, 231, 233, 235, 243, 248, 253, 264, 266, 278, 287, 297 / 440, 326, 366, 367, 368, 370, 396, 414, 433, 435, 437, 439, 442, 444, 446, 485, 499, 503, 515, 520, 525, 30 / 179 / 200 / 373 / 403, 30 / 179 / 373 / 379, 30 / 184 / 20 ... 46 / 325 / 379 / 429 / 495, 30 / 184 / 246 / 327 / 329 / 459, 30 / 184 / 246 / 379 / 404, 30 / 184 / 246 / 459 / 461 / 495 / 500 / 504, 30 / 200 / 373, 30 / 200 / 373 / 379, 30 / 246 / 325 / 327 / 329 / 404 / 461, 30 / 246 / 325 / 379 / 404 / 427 / 429 / 461, 30 / 246 / 427 / 459 / 461, 30 / 325 / 327, 30 / 325 / 327 / 379 / 404 / 429 / 495, 30 / 327 / 404 / 504, 30 / 329 / 379, 30 / 373, 30 / 373 / 403, 30 / 379 / 429 / 459 / 461, 30 / 379 / 459 / 461 / 504, 30 / 403 / 441 / 460, 200 / 373 / 379, 246 / 325 / 329 / 379 / 461, 246 / 327 / 404 / 461 / 495, 327 / 329 / 379 / 2. The engineered terminal deoxynucleotidyl transferase of claim 1, comprising at least one substitution or set of substitutions comprising substitutions compared to the reference sequence of SEQ ID NO: 2812 at amino acid positions selected from: 504, 327 / 459 / 461 / 495, 353 / 403, 373 / 379, 373 / 379 / 403, 379 / 403 / 406 / 468, and 403 / 441, and / or any combination thereof.

25. The polypeptide sequences are: 140 / 142 / 153 / 177 / 427 / 434 / 441 / 444 / 461 / 502, 140 / 142 / 153 / 427 / 484, 140 / 142 / 177, 140 / 142 / 177 / 441, 140 / 142 / 365 / 373 / 404 / 427 / 484, 140 / 142 / 373 / 427 / 484, 140 / 148 / 161 / 177 / 404, 140 / 150 / 153 / 365 / 373 / 427 / 484, 140 / 150 / 177 / 404 / 436 / 441 / 484 / 502, 140 / 161 / 177 / 404 / 427 / 484, 140 / 177 / 404, 140 / 177 / 404 / 484, 142 / 150 / 158 / 177 / 4 2. The engineered terminal deoxynucleotidyl transferase of claim 1, comprising at least one substitution or set of substitutions comprising a substitution compared to the reference sequence of SEQ ID NO: 2956 at amino acid positions selected from: 27 / 445, 142 / 150 / 177 / 404, 142 / 153 / 177 / 441 / 444, 142 / 177 / 373, 142 / 177 / 373 / 441, 142 / 461 / 484 / 502, 150 / 177, 153, 153 / 161 / 325 / 404 / 427 / 441 / 484, 153 / 484, 177, 177 / 365 / 427 / 434, 325 / 427, 427, and 484, and / or any combination thereof.

26. the polypeptide sequence is 122 / 473, 189, 190, 193, 194, 196, 263, 264, 266, 267, 268, 269, 273, 273 / 501, 274, 278, 279, 281, 297, 298, 299, 300, 301, 302, 304, 309, 312, 315, 347, 350, 352, 353, 359, 390, 392, 394, 407, 408, 410, 411, 413, 414, 416, 436, 454, 468, 472, 473, 477, 479, 480, 493, 186 / 188 / 248 / 253 / 365 / 366 / 444 / 445, 186 / 231 / 248 / 253 / 484, 186 / 231 / 248 / 365 / 366 / 368 / 484, 186 / 231 / 248 / 366 / 368 / 444, 186 / 231 / 248 / 484, 186 / 231 / 368 / 416 / 441 / 442 / 444 / 484 / 485, 186 / 231 / 441 / 444 / 445, 186 / 484 / 485, 188 / 231 / 248 / 253 / 365 / 441 / 442 / 444 / 445 / 484, 198 / 243 / 264 / 431 / 441, 198 / 243 / 396 / 414 / 431 / 433 / 441 / 499, 198 / 243 / 396 / 414 / 433 / 437 / 441 / 515, 198 / 264 / 266 / 414, 198 / 264 / 396 / 414 / 433 / 441 / 515, 198 / 266 / 396 / 414 / 433 / 441 / 499 / 501, 198 / 266 / 414 / 433 / 441 / 515, 198 / 266 / 437 / 441 / 499, 198 / 414 / 431 / 441 / 499 / 520, 198 / 414 / 433 / 441 / 515 / 520, 231 / 248 / 441 / 484, 243 / 264 / 515 / 520, 243 / 396 / 414 / 433 / 441 / 515 / 520, 243 / 414 / 433 / 437 / 441, 243 / 414 / 437 / 441 / 515, 248 / 442 / 444 / 445 / 484, 264 / 266 / 396 / 414 / 433 / 441 / 515, 264 / 266 / 414 / 441 / 499 / 501 / 515 / 520, 264 / 414, 264 / 414 / 441, 264 / 433 / 441, 266 / 437 / 441 / 499 / 515 / 520, 365 / 366 / 441 / 484 / 485, 396 / 414 / 441, 396 / 414 / 441 / 515,2. The engineered terminal deoxynucleotidyl transferase of claim 1, comprising at least one substitution or set of substitutions comprising substitutions compared to the reference sequence of SEQ ID NO: 3174 at amino acid positions selected from: 414 / 441 / 520, and 414 / 441 / 520, and / or any combination thereof.

27. The polypeptide sequences are 186 / 194 / 248 / 396, 186 / 198 / 243 / 248 / 366 / 368 / 394 / 501, 186 / 231 / 243 / 368 / 394 / 485 / 499, 186 / 231 / 248 / 485, 186 / 231 / 366 / 368 / 394 / 485, 186 / 243, 186 / 243 / 248 / 366 / 368 / 394, 186 / 243 / 248 / 394 / 484 / 485, 186 / 243 / 484 / 520, 186 / 2 48, 186 / 365, 186 / 365 / 366 / 368 / 394 / 499, 186 / 365 / 366 / 394 / 485, 186 / 366 / 368 / 394 / 396 / 484, 186 / 366 / 368 / 394 / 396 / 484 / 485, 186 / 394 / 396 / 485, 194 / 198 / 243 / 366 / 368 / 499, 194 / 515 / 520, 198 / 231 / 243 / 248 / 485, 198 / 243 / 248 / 365 / 394 / 501, 198 / 248 / 394 / 396 / 484 / 485 / 499, 198 / 394 / 396 / 484 / 485 / 499, 198 / 394 / 396 / 499 / 515 / 520, 198 / 394 / 499 / 501, 231 / 365 / 368 / 394 / 499 / 520, 231 / 368 / 394, 231 / 484 / 485 / 499 / 501, 243 / 248 / 394 / 396 / 484 / 485, 243 / 484, 243 / 484 / 485 / 499, 248 / 365 / 366 / 2. The engineered terminal deoxynucleotidyl transferase of claim 1, comprising at least one substitution or set of substitutions comprising substitutions compared to the reference sequence of SEQ ID NO: 3222 at amino acid positions selected from 368 / 394 / 484 / 520, 248 / 394 / 484, 365 / 366 / 368 / 394, 365 / 368 / 394 / 396 / 520, 394 / 396, and 394 / 499, and / or any combination thereof.

28. The polypeptide sequences are: 168, 198, 267, 273, 273 / 493, 273 / 493 / 499, 273 / 309 / 493 / 499, 273 / 309 / 413 / 499, 273 / 499, 274 / 299 / 408 / 416, 274 / 408 / 416, 274 / 416, 288 / 299 / 416, 298 / 299 / 416, 301, 303 / 396, 307, 308, 308 / 361, 309, 309 / 413, 309 / 499, 313, 372, 3 2. The engineered terminal deoxynucleotidyl transferase of claim 1, comprising at least one substitution or set of substitutions comprising a substitution compared to the reference sequence of SEQ ID NO: 3670 at amino acid positions selected from: 92, 397, 413, 413 / 493 / 499, 413 / 499, 415, 416, 419, 451, 452, 456, 472, 473, 475, 493 / 499, and 528, and / or any combination thereof.

29. The polypeptide sequences are 194 / 196 / 390, 194 / 196 / 390 / 394 / 460 / 480, 194 / 196 / 390 / 394 / 480, 194 / 196 / 390 / 454 / 480, 194 / 196 / 390 / 480, 194 / 196 / 394 / 454 / 480, 194 / 196 / 454, 194 / 390, 194 / 394, 194 / 394 / 454, 194 / 394 / 454 / 480, 196, 196 / 390, 196 / 390 / 394, 196 / 390 / 394 / 454, 196 / 390 / 394 / 454 / 480, 196 / 390 / 394 / 480, 196 / 390 / 454, 196 / 394, 196 2. The engineered terminal deoxynucleotidyl transferase of claim 1, wherein the engineered terminal deoxynucleotidyl transferase comprises at least one substitution or set of substitutions comprising a substitution when compared to the reference sequence of SEQ ID NO: 3674 at an amino acid position selected from: 196 / 394 / 454, 196 / 394 / 454 / 480, 196 / 394 / 480, 196 / 394 / 454, 297 / 470 / 473, 297 / 473 / 493, 390, 390 / 394, 390 / 394 / 454, 390 / 394 / 454 / 480, 390 / 394 / 480, 390 / 454, 390 / 480, 394, 394 / 480, and 454, and / or any combination thereof.

30. The polypeptide sequences are: 198, 198 / 267 / 313 / 451 / 475 / 494 / 499, 198 / 267 / 314 / 451, 198 / 267 / 314 / 475, 198 / 267 / 409 / 451, 198 / 267 / 475, 198 / 451 / 493, 208 / 308, 208 / 308 / 461, 267, 267 / 314 / 328 / 451 / 494 / 499, 267 / 451, 267 / 451 / 494 / 499, 308, 314 / 328 / 451 / 499, 3 2. The engineered terminal deoxynucleotidyl transferase of claim 1, comprising at least one substitution or set of substitutions comprising substitutions compared to the reference sequence of SEQ ID NO: 3796 at amino acid positions selected from 14 / 451, 328 / 409 / 451, 328 / 451, 409, 409 / 475 / 494, 451, 451 / 493 / 494, 451 / 493 / 499, 451 / 494, and 475, and / or any combination thereof.

31. The polypeptide sequence is 26, 30, 38, 79, 81, 90, 92, 94, 101, 108, 137, 140, 141, 142, 153, 155, 160, 163, 165, 177, 184, 189, 194, 196, 201, 203, 205, 213, 219, 231, 246, 248, 258, 263, 264, 266, 267, 301, 304, 307, 314, 318, 325, 333, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​383, 384, 385, 386, 387, 388, 389, 390, 400, 401, 402, 403, 404, 405, 406, 407, 40, 344, 353, 362, 379, 390, 392, 394, 395, 396, 397, 398, 402, 403, 406, 408, 410, 411, 413, 414, 416, 425, 427, 429, 433, 434, 441, 442, 444, 446, 451, 455, 460, 461, 466, 468, 476, 481, 484, 485, 488, 495, 499, 501, 502, 50 6, 515, 525, 26 / 30 / 38 / 79 / 81 / 90 / 92 / 94 / 101 / 108 / 137 / 140 / 141 / 142 / 153 / 155 / 160 / 163 / 165 / 177 / 184 / 189 / 194 / 196 / 201 / 203 / 205 / 213 / 219 / 231 / 248 / 258 / 263 / 264 / 266 / 267 / 301 / 304 / 307 / 314 / 318 / 325 / 333 / 340 / 344 / 353 / 362 / 379 / 390 / 392 / 394 / 395 / 397 / 398 / 402 / 403 / 406 / 408 / 410 / 411 / 413 / 414 / 416 / 425 / 427 / 429 / 433 / 434 / 441 / 442 / 444 / 446 / 451 / 455 / 460 / 461 / 466 / 468 / 476 / 481 / 484 / 485 / 488 / 495 / 499 / 501 / 502 / 506 / 515 / 525,26 / 30 / 38 / 79 / 81 / 90 / 92 / 94 / 101 / 108 / 137 / 140 / 141 / 142 / 153 / 155 / 160 / 163 / 165 / 177 / 184 / 189 / 201 / 203 / 205 / 213 / 219 / 231 / 248 / 258 / 263 / 264 / 26 6 / 304 / 307 / 314 / 318 / 325 / 333 / 340 / 344 / 353 / 362 / 379 / 392 / 394 / 395 / 397 / 398 / 402 / 403 / 406 / 408 / 410 / 411 / 413 / 414 / 416 / 425 / 427 / 429 / 433 / 434 / 4 41 / 442 / 444 / 446 / 455 / 460 / 461 / 466 / 468 / 476 / 481 / 484 / 485 / 488 / 495 / 499 / 501 / 502 / 506 / 515 / 525, 26 / 38 / 79 / 81 / 90 / 92 / 94 / 101 / 108 / 137 / 141 / 155 / 160 / 163 / 165 / 189 / 201 / 203 / 205 / 213 / 219 / 246 / 248 / 258 / 263 / 264 / 304 / 307 / 314 / 318 / 333 / 340 / 344 / 353 / 362 / 392 / 394 / 395 / 396 / 397 / 398 / 402 / 40 3 / 406 / 408 / 410 / 411 / 413 / 414 / 425 / 441 / 442 / 446 / 455 / 460 / 461 / 466 / 468 / 476 / 481 / 485 / 488 / 506 / 525, 92 / 94 / 101 / 108 / 137 / 141 / 155 / 160 / 163 / 16 5 / 201 / 203 / 205 / 213 / 219 / 258 / 263 / 264 / 314 / 333 / 344 / 353 / 392 / 394 / 395 / 397 / 406 / 408 / 411 / 413 / 414 / 425 / 441 / 442 / 446 / 460 / 461 / 468 / 476 / 481 / 4 2. The engineered terminal deoxynucleotidyl transferase of claim 1, comprising at least one substitution or set of substitutions comprising a substitution when compared to a reference sequence of SEQ ID NO: 2 or 660 at an amino acid position selected from: 85 / 488 / 525, and 92 / 94 / 101 / 108 / 137 / 141 / 155 / 201 / 213 / 264 / 314 / 333 / 344 / 392 / 394 / 395 / 397 / 406 / 408 / 425 / 442 / 446 / 461 / 476 / 481 / 485 / 525, and / or any combination thereof.

32. The polypeptide sequences are: 165, 169, 171, 173, 175, 176, 179, 183, 187, 191, 192, 195, 197, 199, 200, 203, 204, 210, 257, 259, 267, 291, 293, 295, 301, 319, 325, 340, 341, 342, 374, 387, 398, 399, 403, 404, 406, 429, 480, 481, 482, 483, 484, 485, 486, 487, 488, 489, 490, 500, 501, 502, 503, 504, 505, 506, 507, 508, 509, 510, 511, 512, 513, 514, 515, 516, 517, 518, 519, 520, 521, 522, 523, 524, 525, 526, 527, 528, 529, 530, 531, 532, 533, 534, 535, 536, 537, 538, 539, 540, 541, 542, 543, 544, 545, 546, 547, 548, 549, 550, 551, 2. The engineered terminal deoxynucleotidyl transferase of claim 1, comprising at least one substitution or set of substitutions comprising a substitution compared to the reference sequence of SEQ ID NO: 3870 at amino acid positions selected from: 83, 484, 490, 491, 493, 494, 495, 521, 507, 508, 509, and 522, and / or any combination thereof.

33. The polypeptide sequences are: 3, 7 / 400 / 459 / 504, 164, 173 / 367 / 459 / 500, 184, 203, 203 / 367 / 459, 203 / 367 / 459 / 500 / 501, 203 / 400 / 459 / 501, 203 / 459, 203 / 459 / 499 / 504, 203 / 459 / 500, 215, 218, 294, 335, 335 / 402 / 481 / 4 84, 335 / 402 / 512, 335 / 481 / 484, 335 / 481 / 493, 335 / 481 / 512, 336, 338, 339, 367, 367 / 459 / 500, 370, 373, 376, 380, 384, 390, 395, 395 / 402, 395 / 402 / 481 / 484, 395 / 481, 395 / 481 / 512, 395 / 484, 400, 400 / 459 , 400 / 459 / 499 / 500 / 504, 400 / 459 / 500 / 501, 400 / 459 / 501 / 504, 400 / 501, 400 / 504, 402, 402 / 481, 402 / 481 / 484, 402 / 481 / 484 / 512, 402 / 481 / 493, 402 / 484 / 493, 402 / 512, 458, 459, 459 / 501, 460, 481, 481 / 2. The engineered terminal deoxynucleotidyl transferase of claim 1, comprising at least one substitution or set of substitutions comprising a substitution compared to the reference sequence of SEQ ID NO: 3918 at an amino acid position selected from: 512, 484, 485, 493, 499, 500, 501, 504, 512, 515, and 516, and / or any combination thereof.

33. The polypeptide sequences are 95 / 428 / 480, 163 / 190, 163 / 203 / 366, 163 / 328 / 363 / 480, 163 / 363 / 480 / 485, 172 / 174 / 178 / 340, 178, 188, 190 / 202 / 203 / 363 / 366 / 480 / 483 / 485, 190 / 202 / 203 / 480, 190 / 480 / 485, 192, 192 / 498 / 499 / 503, 202, 202 / 203 / 328 / 362 / 363 / 366 / 428 / 480 / 485 / 498 / 499 / 503, 202 / 203 / 485, 203 / 328 / 363 / 428 / 483, 203 / 328 / 428, 203 / 328 / 480 / 485, 203 / 362 / 366, 203 / 498 / 499 / 503, 272, 280, 280 / 498 / 499 / 503, 296, 297, 299, 299 / 498 / 499 / 503, 3 01, 301 / 503, 308, 308 / 503, 311, 328 / 428, 328 / 480 / 483, 328 / 485, 343, 346, 349, 358, 359 / 498 / 499 / 503, 362 / 363, 362 / 363 / 366 / 428 / 480 / 483 / 498 / 499 / 503, 392 / 498 / 499 / 503, 406, 407, 410, 411, 418 / 498 / 499 / 503, 418 / 503 2. The engineered terminal deoxynucleotidyl transferase of claim 1, comprising at least one substitution or set of substitutions comprising substitutions compared to the reference sequence of SEQ ID NO: 4266 at amino acid positions selected from: 419 / 503, 465, 471, 472 / 498 / 499 / 503, 473, 480 / 483, 491, and 498 / 499 / 503, and / or any combination thereof.

34. The polypeptide sequences are 94 / 365 / 367, 172 / 174 / 178 / 401 / 403, 172 / 174 / 178 / 401 / 403 / 507, 172 / 174 / 178 / 402 / 508, 172 / 174 / 401 / 403 / 507, 172 / 178, 172 / 178 / 401, 174 / 178, 178 / 401 / 403, 178 / 402 / 403, 318 / 375 / 380, 324 / 379 / 405 / 483, 340, 340 / 394, 365, 365 / 367 / 428, 365 / 389 / 394, 367, 375 / 376, 3 2. The engineered terminal deoxynucleotidyl transferase of claim 1, comprising at least one substitution or set of substitutions comprising substitutions compared to the reference sequence of SEQ ID NO: 4558 at amino acid positions selected from: 75 / 379 / 483, 375 / 380, 375 / 380 / 400 / 483, 375 / 380 / 483, 376 / 483, 379 / 483, 389 / 394, 394, 401, 401 / 402 / 403 / 507, 402 / 403, 405 / 483, and 483, and / or any combination thereof.

35. The polypeptide sequences are: 174 / 296 / 299, 182, 185 / 190, 189 / 190, 190, 190 / 193, 192 / 280, 192 / 402 / 507, 257, 259, 260, 281, 289, 296 / 299, 305, 306, 307, 308, 312, 313, 316, 318, 327, 374, 381, 394, 395, 402, 402 / 507, 404, 405, 414 2. The engineered terminal deoxynucleotidyl transferase of claim 1, comprising at least one substitution or set of substitutions comprising a substitution compared to the reference sequence of SEQ ID NO: 4442 at amino acid positions selected from: 432, 451, 455, 460, 461, 476 / 480, 480, 480 / 481, 493, 494, and 522, and / or any combination thereof.

36. 2. The engineered terminal deoxynucleotidyl transferase of claim 1, wherein the polypeptide sequence comprises at least one substitution or set of substitutions comprising a substitution compared to the reference sequence of SEQ ID NO: 4654 at an amino acid position selected from: 190, 190 / 197 / 308, 190 / 308 / 380 / 405, 190 / 375, 190 / 375 / 380, 190 / 380 / 405, 190 / 405 / 406, 272 / 301 / 393 / 394 / 480, 272 / 318 / 480 / 483, 301 / 394 / 480, 318, 375, 375 / 380, 375 / 405, 375 / 405 / 406, 380, 394, 394 / 480, and 480 / 483, and / or any combination thereof.

37. The polypeptide sequences are: 189, 189 / 193 / 207 / 307 / 353, 190 / 322, 193, 193 / 307, 261 / 322 / 421, 297 / 298 / 300 / 392, 297 / 300, 297 / 300 / 328, 298 / 300 / 328, 298 / 300 / 328 / 395, 298 / 300 / 360, 298 / 300 / 392 / 395, 298 / 300 / 392 / 395 / 492, 298 2. The engineered terminal deoxynucleotidyl transferase of claim 1, comprising at least one substitution or set of substitutions comprising substitutions compared to the reference sequence of SEQ ID NO: 4850 at amino acid positions selected from: 1) 300 / 395, 298 / 300 / 481, 300, 300 / 392 / 395, 319, 322, 392, 421, and 492, and / or any combination thereof.

38. 2. The engineered terminal deoxynucleotidyl transferase of claim 1, wherein the polypeptide sequence comprises at least one substitution or set of substitutions comprising a substitution compared to the reference sequence of SEQ ID NO: 4856 at amino acid positions selected from 10 / 413, 260, 268, 302 / 307, 317, 353, 354, 362, 364, 392, 393, 394, 395, 397, 402, 404, 412, 413, 419, 436 / 512, 460, 477, 486, 490, 495, and 518, and / or any combination thereof.

39. 2. The engineered terminal deoxynucleotidyl transferase of claim 1, wherein the polypeptide sequence comprises at least one substitution or set of substitutions comprising a substitution compared to the reference sequence of SEQ ID NO: 4904 at an amino acid position selected from 190, 190 / 287 / 300 / 302, 190 / 300 / 477 / 490, 194 / 300 / 302 / 413, 194 / 300 / 302 / 481, 297 / 298 / 308 / 392 / 395, 298 / 392 / 525, 300, 300 / 317, 300 / 490, and 395, and / or any combination thereof.

40. The polypeptide sequences are 11 / 523, 190 / 194, 194, 194 / 198, 202, 208, 264, 273, 273 / 347 / 354, 274, 281, 290, 298 / 300 / 302, 298 / 302 / 392 / 393 / 394 / 433, 298 / 302 / 392 / 394, 298 / 392 / 393 / 394, 298 / 392 / 393 / 394 / 477, 298 / 392 / 394 / 490, 298 / 393 / 394 / 395 / 433 / 477, 298 / 393 / 394 / 477 / 495, 298 / 394 / 433, 300 / 302 / 303, 308 / 402 / 460, 309, 313, 314, 324, 352, 359, 360, 361, 392 / 393 / 394 / 433, 392 / 393 / 394 / 477, 392 / 393 / 394 / 4 77 / 495, 392 / 393 / 394 / 490, 392 / 394, 392 / 394 / 395, 392 / 394 / 433 / 477, 392 / 394 / 433 / 495, 392 / 394 / 477 / 495, 392 / 394 / 495, 393 / 394, 393 / 394 / 433 / 477 / 490, 394 / 477, 394 / 490, 405, 408 / 413, 411 / 413, 413 2. The engineered terminal deoxynucleotidyl transferase of claim 1, comprising at least one substitution or set of substitutions comprising a substitution compared to the reference sequence of SEQ ID NO: 5002 at an amino acid position selected from: 460 / 525, 463, 466, 467, 472, 473, 477, 492, 523, and 526, and / or any combination thereof.

41. The polypeptide sequences are 82 / 194 / 198 / 313, 194, 194 / 198, 194 / 198 / 208 / 313, 194 / 198 / 309, 194 / 198 / 313, 194 / 198 / 411, 194 / 208 / 411, 194 / 309, 194 / 313, 194 / 411, 198, 198 / 208, 198 / 208 / 309 / 411, 198 / 208 / 313 / 411, 273 / 274, 274, 274 / 281 / 526 2. The engineered terminal deoxynucleotidyl transferase of claim 1, comprising at least one substitution or set of substitutions comprising a substitution compared to the reference sequence of SEQ ID NO: 5028 at amino acid positions selected from: 274 / 359 / 526, 274 / 523, 309, 309 / 313 / 411, 324 / 526, 411, 466, 466 / 526, 523, and 526, and / or any combination thereof.

42. 2. The engineered terminal deoxynucleotidyl transferase of claim 1, wherein the polypeptide sequence comprises at least one substitution or set of substitutions comprising a substitution compared to the reference sequence of SEQ ID NO: 5192 at amino acid positions selected from 96 / 295, 169, 176, 177, 179, 184, 187, 193, 195, 197, 197 / 307, 198, 199, 200, 203, 292, 295, 300 / 394, 304, 325, 326, 326 / 380, 329, 373, 376, 377, 383, 394, 403, 409, 430, 485, 508, and 520 / 526, and / or any combination thereof.

43. The polypeptide sequences are: 177 / 198 / 200, 177 / 200 / 203, 177 / 200 / 203 / 295, 177 / 200 / 295 / 326, 180, 184 / 198 / 200 / 203 / 295, 184 / 200 / 295 / 326, 190 / 198 / 200 / 203, 190 / 200 / 203 / 295 / 380, 190 / 200 / 295, 197, 198, 198 / 200, 198 / 200 / 203, 198 / 200 / 203 / 295, 2 2. The engineered terminal deoxynucleotidyl transferase of claim 1, comprising at least one substitution or set of substitutions comprising a substitution compared to the reference sequence of SEQ ID NO: 5246 at amino acid positions selected from: 00 / 326, 200 / 380, 203, 203 / 380, 233, 252, 295, 336, 364, 365, 367, 381, 384, 441, 459, and 485, and / or any combination thereof.

44. 44. The engineered terminal deoxynucleotidyl transferase of any of claims 1 to 43, wherein the polypeptide sequence further comprises an N-terminal truncation of 1 to 156 amino acids.

45. 45. The engineered terminal deoxynucleotidyl transferase of any of claims 1-44, wherein the engineered terminal deoxynucleotidyl transferase polypeptide is fused to a second polypeptide, and optionally the second polypeptide has inorganic pyrophosphatase (IPP) activity.

46. 46. ​​The engineered terminal deoxynucleotidyl transferase of claim 45, wherein the second polypeptide having IPP activity comprises an amino acid sequence selected from SEQ ID NOs: 3942 and 3944.

47. 46. ​​The engineered terminal deoxynucleotidyl transferase polypeptide of claim 45, wherein the engineered terminal deoxynucleotidyl transferase polypeptide fused to the second polypeptide comprises a sequence selected from SEQ ID NOs: 5468, 5470, 5472, and 5474.

48. 48. The engineered terminal deoxynucleotidyl transferase of any one of claims 1 to 47, wherein the engineered terminal deoxynucleotidyl transferase is capable of template-independent synthesis.

49. 48. The engineered terminal deoxynucleotidyl transferase of any of claims 1 to 47, having at least one improved property compared to a wild-type or reference terminal deoxynucleotidyl transferase or template-independent polymerase.

50. 50. The engineered terminal deoxynucleotidyl transferase of claim 49, wherein the improved property is selected from increased thermostability, increased activity at high temperatures, increased soluble expression or isolated protein yield, decreased by-product formation, increased specific activity toward one or more NTP-3'-O-RBG or natural or modified NTP substrates, increased incorporation efficiency in the extension of oligo acceptor substrates, and increased activity toward one or more oligo acceptor substrates.

51. 51. The engineered terminal deoxynucleotidyl transferase of any of claims 1-50, wherein the engineered terminal deoxynucleotidyl transferase comprises increased soluble expression or isolated protein yield compared to a wild-type or reference terminal deoxynucleotidyl transferase or template-independent polymerase.

52. 52. The engineered terminal deoxynucleotidyl transferase of any of claims 1-51, wherein the engineered terminal deoxynucleotidyl transferase comprises increased thermostability compared to a wild-type or reference terminal deoxynucleotidyl transferase or template-independent polymerase.

53. 53. The engineered terminal deoxynucleotidyl transferase of any of claims 1-52, wherein the engineered terminal deoxynucleotidyl transferase comprises increased activity toward one or more NTP-3'-O-RBG or natural or modified NTP substrates compared to a wild-type or reference terminal deoxynucleotidyl transferase or template-independent polymerase.

54. 54. The engineered terminal deoxynucleotidyl transferase of any of claims 1-53, wherein the terminal deoxynucleotidyl transferase is purified.

55. 55. A polynucleotide sequence encoding at least one engineered terminal deoxynucleotidyl transferase according to any one of claims 1 to 54.

56. SEQ ID NOs: 1, 7, 11, 15, 23, 35, 53, 267, 647, 659, 881, 1099, 1335, 1347, 1595, 1653, 1829, 1949, 2007, 2253, 2513, 2523, 2637, 2803, 2811, 2955, 3173, 3221, 3669, 3673, 3795, 3869, 3917, 4265, 4441, 4653, 4849, 4855, 4903, 5001, 5002, 5003 1. A polynucleotide sequence comprising at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence of 5'- ...

57. The polynucleotide sequences are selected from the group consisting of SEQ ID NOs: 2, 8, 12, 16, 24, 36, 54, 268, 648, 660, 882, 1100, 1336, 1348, 1596, 1654, 1830, 1950, 2008, 2254, 2514, 2524, 2638, 2804, 2812, 2956, 3174, 3222, 3670, 3674, 3796, 3870, 3918, 4266, 4442, 4654 57. The polynucleotide sequence of claim 56, encoding at least one engineered terminal deoxynucleotidyl transferase comprising a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reference sequences of: 4850, 4856, 4904, 5002, 5028, 5192, and 5246.

58. 57. The polynucleotide sequence of claim 56, wherein the sequence comprises SEQ ID NOs: 7, 11, 15, 23, 35, 53, 267, 647, 659, 881, 1099, 1335, 1347, 1595, 1653, 1829, 1949, 2007, 2253, 2513, 2523, 2637, 2803, 2811, 2955, 3173, 3221, 3669, 3673, 3795, 3869, 3917, 4265, 4441, 4653, 4849, 4855, 4903, 5001, 5027, 5191, and 5245.

59. 57. The polynucleotide sequence of claim 56, wherein the polynucleotide sequence is operably linked to a control sequence.

60. 57. The polynucleotide sequence of claim 56, wherein the polynucleotide sequence is codon optimized.

61. An expression vector comprising at least one polynucleotide sequence according to any one of claims 55 to 60.

62. 62. A host cell comprising at least one expression vector of claim 61.

63. 63. A method for producing an engineered terminal deoxynucleotidyl transferase polypeptide in a host cell, the method comprising culturing the host cell of claim 62 under suitable culture conditions so that at least one engineered terminal deoxynucleotidyl transferase polypeptide is produced.

64. 64. The method of claim 63, further comprising recovering the at least one engineered terminal deoxynucleotidyl transferase from the culture and / or the host cell.

65. 65. The method of claim 63 or 64, further comprising purifying said at least one engineered terminal deoxynucleotidyl transferase.

66. 55. A composition comprising at least one engineered terminal deoxynucleotidyl transferase according to any one of claims 1 to 54.

67. 1. A method for template-independent synthesis of oligonucleotides, said method comprising: a) providing at least one terminal deoxynucleotidyl transferase or template-independent polymerase; b) providing at least one oligo acceptor substrate, wherein the oligo acceptor substrate comprises a 3'-OH or equivalent; c) contacting the oligo acceptor substrate with the terminal deoxynucleotidyl transferase or the template-free polymerase and one or more nucleotide triphosphates, modified nucleotide triphosphates, or NTP-3'-O-RBG under conditions sufficient for the addition of the nucleotide, the modified nucleotide, or the nucleotide-3'-O-RBG to the 3' end of the oligo acceptor substrate; The method comprising:

68. 68. The method of claim 67, wherein the method further comprises (d) unblocking the oligonucleotide formed in step (c) at the protected 3'-O position of the oligonucleotide product.

69. 69. The method of claim 67 or 68, wherein the method further comprises e) inactivating unreacted nucleotide triphosphate, modified nucleotide triphosphate, or NTP-3'-O-RBG.

70. 70. The method of any of claims 67-69, wherein the method further comprises the optional step (f) of removing excess nucleoside and / or excess inorganic phosphate or pyrophosphate from the reaction.

71. 71. The method of any of claims 67-70, wherein the method further comprises repeating steps (a)-(c), or (a)-(d), or (a)-(e), or (a)-(f) until the desired oligonucleotide sequence is obtained.

72. 72. The method of any of claims 67-71, wherein the method further comprises (g) cleaving or liberating the growing oligonucleotide strand or the completed oligonucleotide strand.

73. 73. The method of any of claims 68-72, wherein step (d) of unblocking the oligonucleotide formed in step (c) at the protected 3'-O position and step (e) of inactivating the unreacted nucleotide triphosphate, modified nucleotide triphosphate, or NTP-3'-O-RBG are performed simultaneously.

74. 74. The method of any of claims 67 to 73, wherein a phosphatase is used to unblock the NTP-3'-O-RBG and inactivate unreacted NTP-3'-O-RBG by removing at least one of the 5' phosphates.

75. 75. The method of claim 74, wherein the phosphatase comprises an alkaline phosphatase set forth in SEQ ID NO: 3922, 3924, 3926, 3928, 3930, 3932, or 3934.

76. 76. The method of any of claims 67-75, wherein step (c) optionally comprises contacting the oligo acceptor substrate, the terminal deoxynucleotidyl transferase or the template-independent polymerase, and either a nucleotide triphosphate or a modified nucleotide triphosphate or NTP-3'-O-RBG with a phosphatase (such as an inorganic pyrophosphatase) to convert pyrophosphate to inorganic phosphate.

77. 77. The method of claim 76, wherein the phosphatase comprises an inorganic pyrophosphatase set forth in SEQ ID NO: 3936, 3938, 3940, 3942, 3944, 3946, or 3948.

78. 78. The method of any one of claims 67 to 77, further comprising an NTP-3'-O-RBG that comprises a carbonitrile, phosphate, carbonate, carbamate, ester, ether, borate, nitrate, sugar, phosphoramidate, phenylsulfenate, or sulfate.

79. 79. The method of any of claims 67 to 78, wherein the nucleotide triphosphate, the modified nucleotide triphosphate, or the NTP-3'-O-RBG comprises a 2' modification.

80. 80. The method of claim 79, wherein the 2' modifications comprise 2'-F, 2'-O-alkyl, 2'-O-methoxyethyl, or locked or constrained ethyl.

81. 81. The method of claim 80, wherein the modification is 2'-F and the 2'-modified nucleotide triphosphate, the modified nucleotide triphosphate, or the NTP-3'-O-RBG is selected from 2'-fluoro-2'-deoxyadenosine-5'-triphosphate, 2'-fluoro-2'-deoxycytidine-5'-triphosphate, 2'-fluoro-2'-deoxyguanosine-5'-triphosphate, and 2'-fluoro-2'-deoxyuridine-5'-triphosphate.

82. 81. The method of claim 80, wherein the modification is a 2'-O-alkyl and the 2'-modified nucleotide triphosphate, the modified nucleotide triphosphate, or the NTP-3'-O-RBG is selected from 2'-O-methyl adenosine-5'-triphosphate, 2'-O-methyl cytidine-5'-triphosphate, 2'-O-methyl guanosine-5'-triphosphate, 2'-O-methyl uridine-5'-triphosphate, and 2'-O-methyl inosine-5'-triphosphate.

83. The method of any one of claims 67 to 82, wherein the oligonucleotide chain comprises one or more phosphorothioate bonds.

84. 84. The method of any of claims 67 to 83, wherein the oligonucleotide, the oligonucleotide sequence, or the oligonucleotide strand comprises RNA.

85. 85. The method of any one of claims 67 to 84, wherein the oligonucleotide, the oligonucleotide sequence, or the oligonucleotide strand comprises DNA.

86. 86. The method of any of claims 67-85, wherein the terminal deoxynucleotidyl transferase or the template-independent polymerase comprises one or more engineered terminal deoxynucleotidyl transferases.

87. 87. The method of claim 86, wherein the engineered terminal deoxynucleotidyl transferase is according to any one of claims 1 to 54.

88. The engineered terminal deoxynucleotidyl transferase is selected from the group consisting of SEQ ID NOs: 2, 8, 12, 16, 24, 36, 54, 268, 648, 660, 882, 1100, 1336, 1348, 1596, 1654, 1830, 1950, 2008, 2254, 2514, 2524, 2638, 2804, 2812, 2956, 3174, 3222, 3670, 3674, 3796, 3870, 3918, 4266, 4442, 4654, 4850, 4856, 4918, 5018, 5118, 5218, 5222, 5318, 5322, 5418, 5422, 5432, 5442, 5454, 5460, 5470, 5482, 5492, 5518, 5524, 5536, 5546, 5556, 5566, 5570, 5576, 5578, 5578, 5579, 5580, 5581, 5582, 5583, 5584, 5585, 5586, 5587, 5588, 5589, 5590, 5591, 5592, 5593, 5594, 5595, 5596, 5597, 5598, 904, 5002, 5028, 5192, and / or 5246, or a functional fragment thereof, and comprising an amino acid residue difference or set of amino acid residue differences in the polypeptide sequence compared to said reference sequence, wherein said amino acid residue difference or set of differences is selected from the group consisting of: , 7.2, 8.2, 9.2, 10.2, 11.2, 12.2, 13.2, 14.2, 15.2, 16.2, 17.2, 18.2, 19.2, 20.2, 21.2, 22.2, 23.2, 24.2, 25.2, 26.2, 26.3, 26.4, 27.2, 27.3, 27.4, 27.5, 28.1, 28.2, 28.3, 29.2, 30.2, 31.2, 32.2, 33.2, 34.2, 35.2, 36.2, 37.2, 38.2, 39.2, 40.2, 41.2, 42.4 87. The method of claim 86, wherein the amino acid difference is selected from the amino acid differences set forth in claim 86: 2.2, 43.2, 44.2, 45.2, 46.2, 47.2, 48.2, 49.2, 50.2, 51.2, 52.2, 53.2, 54.2, 55.2, 56.2, 56.3, 56.4, 61.2, 63.2, 64.2, 65.2, 66.2, 67.2, 68.2, 69.2, 70.2, 71.2, 72.2, 73.2, 74.2, 75.2, 76.2, 77.2, 78.2, 79.2, and 80.

1.

89. 87. The method of claim 86, wherein the engineered terminal deoxynucleotidyl transferase comprises a polypeptide sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to an amino acid sequence selected from the even-numbered sequences of SEQ ID NOs: 4-1960, 2004-3920, 4048-5466, and 5476.

90. 90. The method of any of claims 67 to 89, wherein the terminal deoxynucleotidyl transferase or the template-independent polymerase is immobilized.

91. 91. The method of Claim 90, wherein said method further comprises a column solid support for immobilization of said terminal deoxynucleotidyl transferase or said template-independent polymerase.

92. 91. The method of claim 90, wherein the method further comprises a batch method using a solid support for immobilization of the terminal deoxynucleotidyl transferase or the template-independent polymerase.

93. 90. The method of any of claims 67 to 89, wherein the oligo acceptor substrate and subsequent oligonucleotide product are immobilized.

94. 90. The method of any of claims 67-89, wherein neither the oligo acceptor substrate nor the terminal deoxynucleotidyl transferase or the template-independent polymerase is immobilized.

95. 95. The method of any one of claims 67 to 94, further comprising an aqueous liquid phase.

96. 55. The engineered terminal deoxynucleotidyl transferase of any one of claims 1 to 54, wherein the engineered terminal deoxynucleotidyl transferase is immobilized.

Images