Engineered terminal deoxynucleotide transferase variants

Abstract

The present invention provides engineered terminal deoxynucleotidyl transferase (TdT) polypeptides useful for template-independent polynucleotide synthesis, as well as compositions, methods of utilizing these engineered polypeptides, and polynucleotides encoding engineered terminal deoxynucleotidyl transferases.

Description

[0001] Cross - reference to related applications

[0002] This application claims the priority benefit of U.S. Provisional Patent Application No. 63 / 499,770, filed on May 3, 2023, and U.S. Provisional Patent Application No. 63 / 379,439, filed on October 13, 2022, the entire contents of each of which are incorporated herein by reference. Technical field

[0003] The present invention provides engineered terminal deoxynucleotidyl transferase (TdT) polypeptides that can be used for template - independent polynucleotide synthesis, as well as compositions and methods of using these engineered polypeptides.

[0004] Reference to a sequence listing, table, or computer program

[0005] A formal copy of the sequence listing is submitted as an XML file simultaneously with the specification, with the file name "CX10 - 238WO3_ST26.xml", a creation date of October 11, 2023, and a size of 13,670,249 bytes. The submitted sequence listing is part of the specification and is incorporated herein by reference in its entirety.

[0006] Background

[0007] Synthetic biology is being established in various 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 build value - added products with advanced functions (e.g., cell bioreactors, etc.) and desired end - products (e.g., drugs, chemicals, etc.). An obstacle to the widespread implementation of these technologies is the ability to effectively synthesize RNA, DNA, and other polynucleotides.

[0008] In particular, small interfering RNA (siRNA) therapeutics are a promising class of drugs with the potential to treat many 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 therapies become more common and target larger patient populations, the ability to produce large amounts of oligonucleotide active pharmaceutical ingredients (APIs) becomes crucial.

[0009] To date, short RNA oligonucleotides have been almost exclusively synthesized by the iterative addition of activated phosphoramidite forms of nucleotides to a growing, immobilized nucleotide chain, plus additional processing steps (Brown, T. Nucleic Acids Book. See: www.atdbio.com / nucleic-acids-book (accessed 2022-10-10)).

[0010] For many years, phosphoramidite chemistry has been extensively developed for the synthesis of small amounts of DNA and for more complex therapeutic RNA synthesis, but there are some cost and sustainability issues that may be limiting as API demand grows into the three- to four-digit kilogram per year range (Andrews et al. (2021). J. Org. Chem. 86, 49-61). Additionally, RNA synthesis using phosphoramidite synthetic chemistry is limited to producing short oligonucleotides of approximately 200 base pairs (Beaucage & Caruthers. (1981). Tetrahedron Lett. 22(20):1859).

[0011] The phosphoramidite iterative method is multi-step and based on phosphorus(III) coupling chemistry. This phosphoramidite iterative method requires (i) coupling (ii) capping (iii) oxidation to P(V) to form phosphodiester or phosphorothioate diesters (iv) deblocking of the 5’O group. After chain synthesis is complete, the final oligonucleotide is cleaved from the support, and deblocking of the cyanoethyl phosphate group and nucleobases can also occur here (Brown, T. Nucleic Acids Book. See: www.atdbio.com / nucleic-acids-book (accessed 2022-10-10)). Washing with organic solvents is also required at each step. The cyanoethyl phosphate blocking group and nucleobase protecting groups can be removed from the solid support in parallel with oligonucleotide cleavage to produce the oligonucleotide product, or, if desired, the cyanoethyl group can be removed under milder conditions prior to chain cleavage.

[0012] Many aspects of the environmental impact and potential progress of current phosphoramidite methods have been reviewed (Andrews et al. (2021). J. Org. Chem. 86, 49-61). Even at a desired high oligonucleotide loading of 20% mass of the final oligonucleotide relative to the mass of the solid support, at least 5 times the mass of the support is required relative to the mass of the final product.

[0013] In addition to the high cost of the large amount of support required to immobilize the oligonucleotides, the use of organic solvents and 5’-O-blocking groups involves additional waste and process inefficiencies. The solubilization of phosphoramidite coupling partners requires organic solvents such as acetonitrile, or the deprotection step requires dichloromethane or toluene. These solvents need to be anhydrous to reduce the unwanted hydrolysis of the phosphoramidite partners and may be sourced unsustainably, adding to the cost, sustainability issues, and potential supply problems of the process.

[0014] The phosphoramidite coupling partners themselves carry the required blocking group at the 5’O-position, the nucleobase nitrogen atoms (in A, C, and G), and the nascent phosphate ester. The most common 5’O-blocking group, dimethoxytrityl, has a molecular weight of ~303 Da, close to the molecular weight of the heaviest natural ribonucleotide fragment, Gp, of ~345 Da. This protecting group requires energy, resources, and effort to generate and attach and then needs to be disposed of when separated from the desired material.

[0015] In summary, a paradigm shift in oligonucleotide synthesis is necessary to enable siRNA therapeutics by reducing environmental impact, improving economic efficiency, and increasing scalability. Thus, new methods of oligonucleotide synthesis are of great interest to the pharmaceutical industry.

[0016] Template-independent enzymatic synthesis

[0017] Enzymatic synthesis can facilitate the production of large volumes 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 thousands of base pairs or more. Most of these polymerases function during DNA replication associated with cell division or transcription of RNA from DNA associated with gene or protein expression. Both of these processes involve template-dependent polynucleotide synthesis, where the polymerase uses an existing template polynucleotide strand to synthesize a complementary polynucleotide strand.

[0018] The potential for non-template-dependent enzymatic polynucleotide synthesis to generate defined sequences has long been recognized. An early report proposed the use of NTPs with blocked 3’ groups to allow stepwise addition of specific nucleotide residues (Bollum. (1962). JBC, 237, 1945-1949).

[0019] However, few polymerases are known to catalyze non-template-dependent polynucleotide synthesis. These include polymerase λ, polymerase μ, and terminal deoxynucleotidyl transferase (TdT), all of which are members of the DNA polymerase X family, many of which are involved in DNA repair processes (Domínguez et al. (2000). EMBO, 19(7), 1731-1742). Among them, TdT is known to generate diversity in antigen receptors by adding nucleotides to the 3'-end of single-stranded polynucleotides without selectivity in a non-template-dependent process (Bentolila et al. (1995). EMBO, 14(17), 4221-4229).

[0020] Others have published a method of polynucleotide synthesis that uses nucleoside 5'-triphosphates whose 3'-OH position is protected by a removable blocking moiety and, in particular, non-template-dependent polynucleotide polymerases, including terminal deoxynucleotidyl transferase (U.S. Patent 5,763,594). Blocking groups, also referred to by those skilled in the art as inhibitor groups or reversible terminating groups, can include a variety of groups that prevent TdT from adding additional NTPs to the nascent polynucleotide chain. This can include charged molecules, macromolecules and moieties, or other blocking groups known to those skilled in the art. Suitable removable blocking groups can include nitriles, 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 (U.S. Patent 7,544,794) and 3'-O-azides (U.S. Patent 10,407,721).

[0021] Although initially promising, the use of 3'-blocked NTPs in TdT-catalyzed non-template-dependent synthesis has proven difficult in practice because TdT has difficulty accepting 3'-O-blocked NTPs as substrates. In addition, wild-type TdT has low tolerance for oligonucleotide acceptor substrates containing one or more modified nucleotides (e.g., 2'-modifications).

[0022] Additionally, the synthesis of RNA strands presents unique challenges due to the additional reactive 2'-OH on the ribose. While protection of the 2'-position is beneficial for RNA synthesis, this approach reduces efficiency due to the steric hindrance of the 2'-protecting group and requires the maintenance and removal of the protecting group (CB Reese. (2005). Org Biomol Chem 3, 3851-3868).

[0023] Recently, several reports have described non-template-dependent synthesis methods that use modified NTPs, where the blocking group is attached to the purine or pyrimidine base and the 3'-OH is unmodified and available for additional synthesis rounds. These base blocking groups can include cleavable linkers that allow removal of the blocking group after each NTP addition step. The cleavable linker can also be attached to a detectable label (U.S. Patent 7,057,026, etc.). A variety of cleavable linkers are known to those skilled in the art. These include linkers attached via reducible disulfide bonds, photocleavable, electrophilic or nucleophilic, pH-sensitive, temperature-sensitive linkers, and enzymatically cleaved linkers. One disadvantage of using cleavable linkers is that some atoms of the linker moiety typically remain attached to the NTP after cleavage, leaving a "scar" that may interfere with complementary strand synthesis after the initial non-template-dependent synthesis of the primary polynucleotide chain.

[0024] Recently, modified NTPs have been developed in which the base is attached to a blocking group with a "scarless" cleavable linker and the nascent DNA is prepared for the next round of synthesis. In one example, the blocking group and the cleavable linker are attached to the base via a disulfide bond. When a reducing agent is added, 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 those using microfluidic devices or inkjet printing techniques (U.S. Patent 9,279,149). Exonucleases can also be used in methods for synthesizing polynucleotides to shorten or completely degrade polynucleotide chains that have not successfully added an NTP after a polynucleotide extension step and before removal of the blocking group (U.S. Patent 9,771,613).

[0025] 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 have a negative impact on enzyme kinetics, and when synthesizing oligonucleotide chains, linker scars can lead to unacceptable error incorporation rates. Additionally, larger linkers and the necessary deblocking steps can increase the cost, time, and inefficiency of the overall process, making these methods economically unfeasible.

[0026] Recently, several groups have explored modifying the structure or amino acid sequence of TdT or other polymerases to allow non-template-dependent synthesis using 3'-O-blocked groups. Efcavitch et al. described the use of murine or bacterial TdT with substituted amino acid residues in non-template-dependent synthesis to incorporate 3'-modified dNTPs via TdT (U.S. Patent 10,059,929). Other reports have described engineered bovine and gar (Lepisosteus oculatus) TdT that exhibit superior activity to wild-type TdT (U.S. Patent 10,745,727, PCT / GB2020 / 050247). Similarly, a variety of mutations have been described to enhance the activity of Pol X family enzymes (WO 2017216472 A2). Finally, N-terminal truncation of the BRCT domain of TdT (or alternatively, mutation of the BRCT domain) has also been described as enhancing activity when adding reversibly blocked NTPs to the 3'-OH of nucleic acids (US20210164008A1).

[0027] However, despite the recognized value of this technology and intensive research efforts to address the challenges in this area, there is currently no known or commercially available viable method for non-template-dependent enzymatic synthesis of complex or long polynucleotides. Improved engineered TdT enzymes are necessary for non-template-dependent enzymatic synthesis of complex or long polynucleotides or oligonucleotides with defined sequences using nucleoside triphosphates having 3'-O-removable blocking groups, having 2'-modifications, and / or having other modifications.

[0028] Overview

[0029] The present invention provides engineered terminal deoxynucleotidyl transferase (TdT) polypeptides that can be used for non-template-dependent polynucleotide synthesis, as well as compositions and methods of using these engineered polypeptides. The TdT of the present invention is a variant of a predicted splice variant (SEQ ID NO:2) of the wild-type gene from the gray short-tailed opossum (Monodelphis domestica). These engineered TdT are capable of adding nucleoside triphosphates having 3'-O-removable blocking groups and other natural or modified NTPs to the 3'-OH terminus of a growing oligonucleotide or polynucleotide chain in a non-template-dependent manner. After removal of the blocking group, additional rounds of NTP addition can be used to synthesize polynucleotides with defined base sequences without using a complementary template strand as a guide for NTP incorporation (non-template-dependent synthesis).

[0030] In some embodiments, the present invention provides an engineered TdT polypeptide comprising an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity with 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, comprising at least one substitution or a set of substitutions at one or more positions, where the positions are numbered with reference to 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, and wherein the engineered TdT polypeptide has improved thermal stability, increased activity at high temperature, increased soluble expression or isolated protein yield, reduced byproduct formation, increased specific activity towards NTP-3'-O-RBG or other native or modified NTP substrates, and / or increased activity towards a variety of oligonucleotide acceptor substrates as compared to wild-type TdT or other TdT or non-template-dependent polymerases known to those skilled in the art. These engineered TdT polypeptides having one or more amino acid substitutions or sets of substitutions are described in the detailed description of the present invention below.

[0031] In some additional embodiments, the engineered polypeptide comprises an amino acid sequence having at least 60% sequence identity with any even-numbered sequence listed in SEQ ID NO: 4-1960, 2004-3920, 4048-5466 and 5476.

[0032] In further embodiments, the engineered polypeptide of the present invention further comprises an N-terminal truncation of 1-156 amino acids relative to the polypeptide sequence of any even-numbered sequence listed in SEQ ID NO: 4-1960, 2004-3920, 4048-5466 and 5476.

[0033] In additional embodiments, the engineered polypeptides of the invention are fused to a second polypeptide; optionally, wherein the second polypeptide has inorganic pyrophosphatase (IPP) activity (e.g., IPP having an amino acid sequence selected from SEQ ID NOs: 3942 and 3944). In one embodiment, the engineered polypeptides of the invention fused to a second polypeptide having IPP activity comprise a sequence selected from SEQ ID NOs: 5468, 5470, 5472, and 5474.

[0034] The invention also provides engineered polynucleotides encoding at least one of the engineered polypeptides described in the paragraphs above. In some embodiments, the engineered polynucleotides comprise the odd-numbered sequences listed in SEQ ID NOs: 3-1959, 2003-3919, 4047-5465, and 5475.

[0035] The invention also provides vectors comprising at least one of the engineered polynucleotides described above. In some embodiments, the vectors further comprise at least one control sequence.

[0036] The invention also provides host cells comprising the vectors provided herein. In some embodiments, the host cells produce at least one of the engineered polypeptides provided herein.

[0037] The invention also provides a method for producing an engineered TdT polypeptide, the method comprising culturing the host cells provided herein under conditions such that the engineered polynucleotide is expressed and the engineered polypeptide is produced. In some embodiments, the method further comprises the step of recovering the engineered polypeptide.

[0038] The invention also provides a non-template-dependent synthesis method, comprising TdT or a non-template-dependent polymerase that is active against various oligonucleotide acceptor substrates and NTP-3'-O-RBG and other natural or modified NTP substrates, wherein the method may comprise immobilized TdT or an immobilized oligonucleotide acceptor substrate or neither immobilized TdT nor an immobilized oligonucleotide acceptor substrate.

[0039] Description of the Invention

[0040] 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. In general, the nomenclature used herein and the experimental procedures in cell culture, molecular genetics, microbiology, organic chemistry, analytical chemistry, and nucleic acid chemistry described below are those well known and commonly employed in the art. Such techniques are well known and described in many textbooks and reference works well known to those of skill in the art. Standard techniques or modifications thereof are used for chemical synthesis and chemical analysis. All patents, patent applications, articles, and publications mentioned both above and below herein are hereby expressly incorporated herein by reference.

[0041] Although any suitable methods and materials similar or equivalent to those described herein can be used in the practice of the present invention, some methods and materials are described herein. It should be understood that the present invention is not limited to the specific methods, protocols, and reagents described, as these may vary depending on the circumstances of use by those of skill in the art. Thus, the terms defined below are more fully described by reference to the entire present invention.

[0042] It should be understood that the above general description and the following detailed description are merely exemplary and illustrative, and not limiting of the present invention. The section headings used herein are for organizational purposes only and should not be construed as limiting the subject matter described. Numerical ranges include the numbers defining the range. Thus, each numerical range disclosed herein is intended to include every narrower numerical range falling within such broader numerical range, as if such narrower numerical ranges were all expressly written out herein. It is also intended that each maximum (or minimum) numerical limitation disclosed herein include every lower (or higher) numerical limitation, as if such lower (or higher) numerical limitations were all expressly written out herein.

[0043] Unless the context clearly indicates otherwise, as used in this specification and the appended claims, the singular forms "a", "an", and "the" include plural referents. Thus, for example, reference to "a polypeptide" includes more than one polypeptide. Similarly, "comprise", "comprises", "comprising", "include", "includes", and "including" are interchangeable and not intended to be limiting.

[0044] It should be understood that in cases where the term "comprising" is used in the description of various embodiments, those skilled in the art will understand that in some specific cases, the language of "consisting essentially of" or "consisting of" can optionally be used to describe the embodiments. It should also be understood that in cases where the term "optional" or "optionally" is used in the description of various embodiments, the subsequently described event or situation may or may not occur, and the description includes the case where the event or situation occurs and the case where it does not occur. It should be understood that the foregoing general description and the following detailed description are merely exemplary and illustrative, and do not limit the present disclosure. The section headings used herein are for organizational purposes only and should not be construed as limiting the subject matter described.

[0045] Abbreviations

[0046] The abbreviations for the amino acids used in genetic coding are conventional and are as follows:

[0047]

[0048]

[0049] When using three-letter abbreviations, unless specifically preceded by "L" or "D", or it is clear from the context in which the abbreviation is used, the amino acid can be in the L-configuration or D-configuration with respect to the α-carbon (C α ). For example, "Ala" represents alanine without specifying the configuration with respect to the α-carbon, while "D-Ala" and "L-Ala" represent D-alanine and L-alanine, respectively.

[0050] When using single-letter abbreviations, capital letters represent amino acids in the L-configuration with respect to the α-carbon, and lowercase letters represent amino acids in the D-configuration with respect to the α-carbon. For example, "A" represents L-alanine and "a" represents D-alanine. When a polypeptide sequence is presented as a string of single-letter or three-letter abbreviations (or a mixture thereof), the sequence is presented in the amino (N) to carboxyl (C) direction according to conventional convention.

[0051] The abbreviations used for genetically encoded nucleosides are conventional and are as follows: adenosine (A); guanosine (G); cytidine (C); thymidine (T); and uridine (U). These abbreviations may also be used interchangeably for nucleosides and nucleotides (nucleosides with one or more phosphate groups). Unless specifically described, an abbreviated nucleoside or nucleotide may be a ribonucleoside (or ribonucleotide) or a 2'-deoxyribonucleoside (or 2'-deoxyribonucleotide). The nucleoside or nucleotide may also be modified at the 3' position. The nucleoside or nucleotide may be designated as a ribonucleoside (or ribonucleotide) or a 2'-deoxyribonucleoside (or 2'-deoxyribonucleotide) either individually or collectively. When a nucleic acid sequence is presented as a string of single-letter abbreviations, the sequence is presented in the 5' to 3' direction according to conventional convention, and the phosphates are not shown.

[0052] Definitions

[0053] In reference to the present invention, the technical and scientific terms used in the description herein will have the meanings commonly understood by one of ordinary skill in the art, unless otherwise specifically defined. Accordingly, the following terms are intended to have the following meanings.

[0054] The "EC" number refers to the enzyme nomenclature of the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology (NC-IUBMB). This IUBMB biochemical classification is an enzyme number classification system based on the chemical reactions catalyzed by enzymes.

[0055] "ATCC" refers to the American Type Culture Collection, whose biological deposit collection includes genes and strains.

[0056] "NCBI" refers to the National Center for Biological Information and the sequence databases provided therein.

[0057] "Protein", "polypeptide", and "peptide" are used interchangeably herein to denote a polymer of at least two amino acids covalently linked by amide bonds, regardless of length or post-translational modifications (such as glycosylation, phosphorylation, lipidation, myristoylation, ubiquitination, etc.). This definition includes D-amino acids and L-amino acids, as well as mixtures of D-amino acids and L-amino acids, and polymers containing D-amino acids and L-amino acids and mixtures of D-amino acids and L-amino acids.

[0058] "Amino acids" are referred to herein by their commonly known three-letter symbols or by the single-letter symbols recommended by the IUPAC-IUB Commission on Biochemical Nomenclature. Similarly, nucleotides may be referred to by their commonly accepted single-letter codes.

[0059] As used herein, the terms "polynucleotide", "oligonucleotide", and "nucleic acid" are used interchangeably herein and refer to two or more nucleosides or nucleotides covalently linked together. A polynucleotide may consist entirely of ribonucleotides (i.e., RNA), consist entirely of 2'-deoxyribonucleotides (i.e., DNA), consist entirely of other synthetic nucleotides, or contain a mixture of synthetic ribonucleotides and / or 2'-deoxyribonucleotides. A polynucleotide may also contain modified nucleotides with substitutions, including 2'-substitutions (e.g., 2'-fluoro, 2'-O-methyl, 2'-O-methoxyethyl, locked nucleic acid or constrained ethyl modifications, and others known to those of skill in the art). Nucleosides will be linked together via standard phosphodiester bonds or via one or more non-standard bonds, including but not limited to phosphorothioate bonds. A polynucleotide may be single-stranded or double-stranded, or may include both single-stranded and double-stranded regions. Additionally, while polynucleotides typically contain the naturally occurring coding nucleobases (i.e., adenine, guanine, uracil, thymine, and cytosine), it may contain one or more modified and / or synthetic nucleobases, such as, for example, inosine, xanthine, hypoxanthine, etc. In some embodiments, such modified or synthetic nucleobases are the nucleobases encoding an amino acid sequence. Modified or synthetic nucleobases may contain any known or 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 modified or synthetic structure that would be recognized by those of skill in the art as being a "polynucleotide", "oligonucleotide", or "nucleic acid", or having the function of a "polynucleotide", "oligonucleotide", or "nucleic acid". Examples of modified or synthetic structures having the function of a "polynucleotide", "oligonucleotide", or "nucleic acid" are PNA or peptide nucleic acid.

[0060] As used herein, "oligonucleotide acceptor substrate", "acceptor substrate", "growing oligonucleotide acceptor substrate chain", "growing oligonucleotide chain", and "growing polynucleotide chain" are used interchangeably herein and refer to any oligonucleotide or nucleotide chain or similar moiety having an exposed 3'-OH or its equivalent that can be recognized by wild-type TdT or polymerase or engineered TdT or non-template-dependent polymerase of the present disclosure as a substrate for nucleoside addition or synthesis. In some embodiments, the acceptor substrate can be single-stranded. In still other embodiments, the acceptor substrate can be double-stranded or partially double-stranded. In some embodiments, the acceptor substrate can comprise a nucleotide chain consisting of 1-10 nucleotides, 5-20 nucleotides, 15-50 nucleotides, 30-100 nucleotides, or greater than 100 nucleotides. In some embodiments, the acceptor substrate can comprise a chemical moiety that is not a nucleotide chain but contains a free -OH that can be recognized as a substrate by wild-type or engineered TdT, referred to herein as a "3'-OH equivalent". Exemplary oligonucleotide acceptor substrates are provided in the Examples.

[0061] As used herein, "nucleoside triphosphate-3'-O-removable blocking group", "nucleotide triphosphate-3'-O-removable blocking group", "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 comprising an alternative or modified sugar and a removable blocking group attached at the 3' position of the sugar moiety. The NTP-3'-O-RBG can also comprise other modifications as described herein, including but not limited to modifications at the 2' position, modifications to the nucleobase, and modifications to the phosphate. The nucleotide can also have a 3'-O-RBG, as expected after the reaction of NTP-3'-O-RBG with the engineered TdT and oligonucleotide acceptor substrate of the present disclosure.

[0062] As used herein, "oligonucleotide acceptor product", "growing oligonucleotide chain", and "oligonucleotide acceptor extension product" are used interchangeably herein and refer to the product of an NTP-3'-O-RBG or other native or modified NTP substrate with an oligonucleotide acceptor substrate, wherein TdT or a related polymerase catalyzes the extension or addition of a nucleotide-3'-O-RBG or other native or modified nucleotide substrate to the oligonucleotide acceptor substrate via the reaction of the oligonucleotide acceptor substrate with one or more NTP-3'-O-RBG or other native or modified NTP substrates.

[0063] As used herein, "removable blocking group" and "blocking group" and "terminator group" and "reversible terminating group" and "inhibitor group" and related variants of these terms are used interchangeably herein and refer to a chemical group that impedes the addition of a second NTP-3'-O-RBG or other native or modified NTP substrate to the 3'-end of a growing oligonucleotide acceptor substrate chain prior to removal of the removable blocking group from the first round of addition. In some embodiments, the NTP-3'-O-RBG or other native or modified NTP substrate can comprise a removable blocking group selected from the group consisting of NTP-3'-O-NH 2 or NTP-3'-O-PO 3 . In some embodiments, the NTP-3'-O-RBG or other native or modified NTP substrate can have a native purine or pyrimidine base, such as adenine, guanine, cytosine, thymine, or uridine. In some embodiments, the NTP-3'-O-RBG or other native or modified NTP substrate can have a non-natural base analogue, such as inosine, xanthine, hypoxanthine, or another base analogue, as known in the art. In some embodiments, the blocking group can comprise or additionally comprise a modification at the 2'-position.

[0064] As used herein, "non-template-dependent synthesis" refers to the synthesis of an oligonucleotide or polynucleotide without using a template strand as a guide for the synthesis of a complementary oligonucleotide or polynucleotide chain. Thus, non-template-dependent synthesis refers to the iterative process by which successive nucleotides are added to a growing oligonucleotide chain or nucleotide chain or acceptor substrate. Non-template-dependent synthesis can be sequence-determined or random, as in the case of wild-type TdT generating antigen receptor diversity. The process of non-template-dependent synthesis is further described herein.

[0065] "Coding sequence" refers to the portion of a nucleic acid (e.g., a gene) that encodes the amino acid sequence of a protein.

[0066] "Naturally occurring" or "wild-type" refers to the form found in nature. For example, a naturally occurring or wild-type polypeptide or polynucleotide sequence is a sequence that exists in an organism, can be isolated from a natural source, and has not been deliberately modified by human manipulation.

[0067] As used herein, when used in reference to a cell, nucleic acid, or polypeptide, "recombinant," "engineered," and "non-naturally occurring" refer to a material that has been modified in a manner that does not occur naturally or a material corresponding to the native or original form of the material. In some embodiments, a cell, nucleic acid, or polypeptide is identical to a naturally occurring cell, nucleic acid, or polypeptide but is produced or derived from synthetic materials and / or by using recombinant techniques. Non-limiting examples include, among others, recombinant cells that express genes not found in cells in their natural (non-recombinant) form or that express natural genes that are otherwise expressed at different levels.

[0068] "Percent sequence identity" and "percent 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, wherein for optimal alignment of the two sequences, the portion of the polynucleotide or polypeptide sequence in the comparison window may include additions or deletions (i.e., gaps) as compared to the reference sequence. The percent may be calculated as follows: determine the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, divide the number of matched positions by the total number of positions in the comparison window, and multiply the result by 100 to yield the percent sequence identity. Alternatively, the percent may be calculated as follows: determine the number of positions at which the identical nucleic acid base or amino acid residue or a nucleic acid base or amino acid residue aligned with a gap occurs in both sequences to yield the number of matched positions, divide the number of matched positions by the total number of positions in the comparison window, and multiply the result by 100 to yield the percent sequence identity. Those skilled in the art understand that there are many established algorithms available for aligning two sequences. The optimal alignment of the sequences to be compared can be, for example, by the local homology algorithm of Smith and Waterman (Smith and Waterman, Adv. Appl. Math., 2:482

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

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

[1988] ), by the computerized execution 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 analysis is publicly available through the website of the National Center for Biotechnology Information. The algorithm involves first identifying high-scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence that match or satisfy a positive-valued threshold score T when aligned with words of the same length in the database sequence.T is referred to as the neighbor word score threshold (see, Altschul et al., supra). These initial neighbor word hits are used as seeds to initiate the search to find longer HSPs that contain them. The word hits are then extended in both directions along each sequence until the cumulative alignment score can no longer increase. For nucleotide sequences, the cumulative score is calculated using parameters M (reward score for matching residue pairs; always >0) and N (penalty score for mismatched residues; always <0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction is stopped when: the cumulative alignment score has decreased by amount X from its maximum achieved value; the cumulative score reaches 0 or less due to the accumulation of one or more negative score residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses the following as defaults: word length (W) of 11, expectation value (E) of 10, M = 5, N = -4, and comparison of both strands. For amino acid sequences, the BLASTP program uses the following as defaults: word length (W) of 3, expectation value (E) of 10, and BLOSum62 scoring matrix (see Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89:10915

[1989] ). Exemplary determination of sequence alignment and percent sequence identity can be made using the provided default parameters, using the BESTFIT or GAP programs in the GCG Wisconsin software package (Accelrys, Madison WI).

[0069] "Reference sequence" means a designated sequence that serves as a basis for sequence comparison. A reference sequence can be a subset of a larger sequence, e.g., a segment of a full-length gene or polypeptide sequence. Typically, 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 full length of a nucleic acid or polypeptide. Since two polynucleotides or polypeptides can each (1) contain sequences that are similar between the two sequences (i.e., a portion of the complete sequence), and (2) can also contain sequences that are different between the two sequences, sequence comparison between two (or more) polynucleotides or polypeptides is typically carried out 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, where the reference sequence is a sequence that can have one or more variations in the primary sequence. For example, a "reference sequence based on SEQ ID NO:4 having valine at the residue corresponding to X14" or X14V refers to a reference sequence where the corresponding residue (tyrosine) at X14 in SEQ ID NO:4 has been changed to valine.

[0070] A "comparison window" is a conceptual segment of at least about 20 contiguous nucleotide positions or amino acid residues where a sequence can be compared to a reference sequence of at least 20 contiguous nucleotides or amino acids, and where the portion of the sequence in the comparison window can contain 20% or fewer additions or deletions (i.e., gaps) compared to the reference sequence (which does not contain additions or deletions) to obtain the best alignment of the two sequences. The comparison window can be longer than 20 contiguous residues and optionally includes windows of 30, 40, 50, 100 or longer.

[0071] As used herein, "substantial identity" means a polynucleotide or polypeptide sequence having at least 80% sequence identity, at least 85% identity, at least 89% to 95% sequence identity, or more typically at least 99% sequence identity, compared to a reference sequence in a comparison window of at least 20 residue positions, typically in a window of at least 30 - 50 residues, where the percentage of sequence identity is calculated by comparing the reference sequence and the sequence containing a total of 20% or fewer deletions or additions over the comparison window. In some specific embodiments applied to polypeptides, the term "substantial identity" means that when optimally aligned using default gap weights such as by the programs GAP or BESTFIT, two polypeptide sequences share at least 80% sequence identity, preferably at least 89% sequence identity, at least 95% sequence identity or more (e.g., 99% sequence identity). In some embodiments, the residue positions that are not identical in the sequences being compared differ by conservative amino acid substitutions.

[0072] When used in the context of numbering a given amino acid or polynucleotide sequence, "corresponding to", "reference to", and "relative to" refer to the numbering of the residues of a specified reference sequence when the given amino acid or polynucleotide sequence is compared to that reference sequence. In other words, the residue numbering or residue position of a given polymer is assigned with respect to the reference sequence, rather than by the actual numerical position of the residues within the given amino acid or polynucleotide sequence. For example, given an amino acid sequence, such as the amino acid sequence of engineered TdT, the residue matching between the two sequences can be optimized by introducing gaps to align with the reference sequence. In these cases, despite the presence of gaps, the residues in the given amino acid or polynucleotide sequence are numbered with respect to the reference sequence to which it is aligned.

[0073] "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 in this document is typically referred to as "Xn", where n refers to the corresponding position in the reference sequence on which the residue difference is based. For example, "a residue difference at position X25 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 the reference polypeptide SEQ ID NO:2 has valine at position 25, then "a residue difference at position X25 compared to SEQ ID NO:2" refers to 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 instances in this document, a specific amino acid residue difference at a position is indicated as "XnY", where "Xn" designates the corresponding position as described above, and "Y" is the single-letter identifier of the amino acid present in the engineered polypeptide (i.e., the residue different from that in the reference polypeptide). In some embodiments, more than one amino acid may be present at the designated residue position (i.e., alternative amino acids may be listed in the form of XnY / Z, where Y and Z represent the alternative amino acid residues).In some instances (e.g., 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, 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 represented by the conventional symbol “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 include one or more amino acid residue differences relative to the reference sequence, as indicated by a list of specified positions that are altered relative to the reference sequence. In some additional embodiments, the present invention provides engineered polypeptide sequences that include both conservative amino acid substitutions and non-conservative amino acid substitutions.

[0074] 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 generally includes the replacement of an amino acid in a polypeptide with an amino acid within the same or a similar amino acid-defined class. By way of example and not limitation, an amino acid having an aliphatic side chain is replaced with another aliphatic amino acid (e.g., alanine, valine, leucine, and isoleucine); an amino acid having a hydroxyl side chain is replaced with another amino acid having a hydroxyl side chain (e.g., serine and threonine); an amino acid having an aromatic side chain is replaced with another amino acid having an aromatic side chain (e.g., phenylalanine, tyrosine, tryptophan, and histidine); an amino acid having a basic side chain is replaced with another amino acid having a basic side chain (e.g., lysine and arginine); an amino acid having an acidic side chain is replaced with another amino acid having an acidic side chain (e.g., aspartic acid or glutamic acid); and / or a hydrophobic amino acid or a hydrophilic amino acid is replaced with another hydrophobic amino acid or hydrophilic amino acid, respectively. Exemplary conservative substitutions are provided in Table 1 below.

[0075]

[0076] "Non-conservative substitution" refers to the replacement of an amino acid in a polypeptide with an amino acid having significantly different side chain characteristics. Non-conservative substitutions can use amino acids between rather than within defined groups and affect (a) the structure of the peptide backbone in the region of substitution (e.g., proline replacing glycine), (b) charge or hydrophobicity, or (c) side chain volume. By way of example and not limitation, exemplary non-conservative substitutions can be the replacement of an acidic amino acid with a basic or aliphatic amino acid; the replacement of an aromatic amino acid with a small amino acid; and the replacement of a hydrophilic amino acid with a hydrophobic amino acid.

[0077] "Deletion" refers to a polypeptide modification by removing one or more amino acids from a reference polypeptide. Deletions can include the removal of 1 or more amino acids, 2 or more amino acids, 5 or more amino acids, 10 or more amino acids, 15 or more amino acids, or 20 or more amino acids, up to 10% or up to 20% of the total number of amino acids that make up the reference enzyme, while retaining enzyme activity and / or retaining the improved characteristics of the engineered TdT enzyme. Deletions can involve internal portions and / or terminal portions of the polypeptide. In various embodiments, deletions can include contiguous segments or can be discontinuous.

[0078] "Insertion" refers to a polypeptide modification by adding one or more amino acids to a reference polypeptide. In some embodiments, the improved engineered TdT enzyme includes one or more amino acid insertions into a naturally occurring polypeptide, and one or more amino acid insertions into other improved TdT enzyme polypeptides. The insertion can be in the internal portion of the polypeptide or at the carboxyl or amino terminus. As used herein, an insertion includes a fusion protein as known in the art. The insertion can be a continuous segment of amino acids, or separated by one or more amino acids in the naturally occurring polypeptide.

[0079] As used herein, "fragment" refers to a polypeptide having an amino-terminal and / or carboxyl-terminal deletion but the remaining amino acid sequence being the same as at the corresponding position in the sequence. The fragment can be at least 14 amino acids long, at least 20 amino acids long, at least 50 amino acids long or longer, and 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).

[0080] "Isolated polypeptide" refers to a polypeptide that is substantially separated from other contaminants (such as proteins, lipids, and polynucleotides) that naturally accompany it. The term includes polypeptides that have been removed or purified from their natural environment or expression system (e.g., host cell or in vitro synthesis). The engineered TdT enzyme can be present intracellularly, in cell culture medium, or prepared in various forms, such as a lysate or an isolated preparation. As such, in some embodiments, the engineered TdT enzyme can be an isolated polypeptide.

[0081] "Substantially pure polypeptide" refers to a composition in which the polypeptide substance is the major substance present (i.e., on a molar or weight basis, it is more abundant than any other individual macromolecular substance in the composition), and the composition is generally substantially purified when the target substance constitutes at least about 50% of the macromolecular substances present on a molar or weight % basis. Generally, a substantially pure TdT composition will constitute 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 the macromolecular substances present in the composition on a molar or weight % basis. In some embodiments, the target class is purified to substantial homogeneity (i.e., contaminants cannot be detected in the composition by conventional detection methods), where the composition consists essentially of a single macromolecular class. Solvent substances, small molecules (<500 daltons), and elemental ionic substances are not considered macromolecular substances. In some embodiments, the isolated engineered TdT polypeptide is a substantially pure polypeptide composition.

[0082] As used herein, "improved enzyme properties" refers to at least one improved property of an enzyme. In some embodiments, the invention provides engineered TdT polypeptides that exhibit improvements in any enzyme 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, comparison is typically made with the wild-type enzyme from which the TdT was derived, although in some embodiments, the reference enzyme may be another improved engineered TdT. Therefore, the level of "improvement" between various TdT polypeptides, including wild-type and engineered TdT, can be determined and compared. Improved properties include, but are not limited to, properties such as: enzyme activity (which can be expressed as a percentage of substrate conversion), thermal stability, solvent stability, pH activity spectrum, cofactor requirements, refractoriness to inhibitors (e.g., substrate or product inhibition), activity at elevated temperatures, increased soluble expression, reduced byproduct formation, increased specific activity towards NTP-3'-O-RBG substrates, increased incorporation efficiency in oligomeric acceptor substrate extension, and / or increased activity (including enantioselectivity) towards various oligomeric acceptor substrates.

[0083] "Increased enzyme activity" refers to an improved property of a TdT polypeptide, which can be expressed as an increase in specific activity (e.g., product produced / time / weight protein) or an increase in the percentage conversion of substrate to product (e.g., the percentage conversion of a starting amount of substrate to product within a specified time period using a specified 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 can be affected, including typical enzyme properties. m 、V max or k cat , their variation can result in increased enzymatic activity.The improvement of enzymatic activity can be up to 2 times, 5 times, 10 times, 20 times, 25 times, 50 times or more of the enzymatic activity of about 1.2 times of the corresponding wild-type enzyme to the naturally occurring or another engineered TdT activity of the derived TdT polypeptide.TdT activity can be measured by any standard assay, such as by monitoring the change of the property of substrate, cofactor or product.In some embodiments, the amount of the product produced can be measured by liquid chromatography-mass spectrometry (LC-MS), HPLC or other methods known in the art.The comparison of enzyme activity uses defined enzyme products, the mensuration of the definition under the set conditions and one or more defined substrates to carry out, as described in further detail herein.Usually, when comparing lysates, determine the amount of the protein of the number of cells and the assay, and use the same expression system and the same host cell to minimize the variation of the amount of the enzyme produced by the host cell and present in the lysate.

[0084] "Conversion" refers to the enzymatic conversion of a substrate into the corresponding product. "Percentage of conversion" refers to the percentage of substrate that is converted into product over a certain period of time under specified conditions. Thus, the "enzyme activity" or "activity" of a TdT polypeptide can be expressed as the "percentage of conversion" of substrate to product.

[0085] "Thermostable" refers to a polypeptide that maintains a similar activity (e.g., more than 60% to 80%) after exposure to the same high temperature for a certain period of time (e.g., 0.5 hour - 24 hours) compared to the wild-type enzyme exposed to a high temperature (e.g., 40°C - 80°C).

[0086] "Solvent-stable" refers to a polypeptide that maintains a similar activity (more than e.g., 60% to 80%) after exposure to the same concentration of the same solvent for a certain period of time (e.g., 0.5 hour - 24 hours) compared to the wild-type enzyme exposed to solvents (ethanol, isopropanol, dimethyl sulfoxide (DMSO), tetrahydrofuran, 2-methyltetrahydrofuran, acetone, toluene, butyl acetate, methyl tert-butyl ether, etc.) at different concentrations (e.g., 5% - 99%).

[0087] "Thermostable and solvent-stable" refers to a polypeptide that is both thermostable and solvent-stable.

[0088] The term "stringent hybridization conditions" is used herein to refer to conditions under which nucleic acid hybrids are stable. As is known to those skilled in the art, the stability of a hybrid is reflected in the melting temperature (T m ) of the hybrid. Generally, the stability of a hybrid varies with ionic strength, temperature, G / C content, and the presence of chaotropes. The T m value of a polynucleotide can be calculated using known methods for predicting melting temperature (see, for example, 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., supra); Suggs et al., 1981, in Developmental Biology Using Purified GenesIn some embodiments, the polynucleotide encodes a polypeptide disclosed herein and hybridizes to the complement of a sequence encoding an engineered TdT enzyme of the invention under defined conditions, such as moderately stringent or highly stringent conditions.

[0089] "Hybridization stringency" refers to the hybridization conditions, such as washing conditions, in nucleic acid hybridization. Typically, hybridization reactions are performed under conditions of lower stringency, followed by washes of different but higher stringency. The term "moderately stringent hybridization" refers to conditions that allow target DNA to bind to complementary nucleic acids that have about 60% identity, preferably about 75% identity, about 85% identity, and greater than about 90% identity with the target polynucleotide. Exemplary moderately stringent conditions are conditions equivalent to hybridization at 42°C in 50% formamide, 5×Denhart solution, 5×SSPE, 0.2% SDS, followed by washing at 42°C in 0.2×SSPE, 0.2% SDS. "Highly stringent hybridization" generally refers to conditions that are consistent with the thermal melting temperature T as determined for a defined polynucleotide sequence under solution conditions. m Conditions that differ by about 10°C or less. In some embodiments, high stringency conditions refer to conditions that allow only hybridization of those nucleic acid sequences that form stable hybrids in 0.018M NaCl at 65°C (i.e., if the hybrid is unstable in 0.018M NaCl at 65°C, it will be unstable under high stringency conditions as contemplated herein). High stringency conditions can be provided, for example, by hybridizing under conditions equivalent to 50% formamide, 5×Denhart solution, 5×SSPE, 0.2% SDS at 42°C, followed by washing in 0.1×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 washing in 0.1×SSC containing 0.1% SDS at 65°C. Other high stringency hybridization conditions as well as moderate stringency conditions are described in the references cited above.

[0090] A "heterologous" polynucleotide refers to any polynucleotide that is introduced into a host cell by experimental techniques and includes polynucleotides that are removed from a host cell, subjected to laboratory manipulations, and then reintroduced into the host cell.

[0091] "Codon-optimized" means that the codons of a polynucleotide encoding a protein are changed to those codons that are preferentially used in a particular organism, such that the encoded protein is efficiently expressed in the organism of interest. Although the genetic code is degenerate, i.e., most amino acids are represented by several codons referred to as "synonyms" or "synonymous" codons, it is well known that codon usage in a particular organism is non-random and biased for specific codon triplets. Such codon usage bias may be higher for a given gene, genes of common function or ancestral origin, highly expressed proteins versus low-copy number proteins, and the aggregated protein-coding regions of the genome of an organism. In some embodiments, the polynucleotide encoding the TdT enzyme can be codon-optimized for optimal production from a selected host organism for expression.

[0092] As used herein, "preferred, optimal, high codon usage preference codons" are interchangeably meant to refer to codons that have a higher frequency of use in a protein-coding region than other codons encoding the same amino acid. Preferred codons can be determined based on the codon usage in a single gene, a group of genes of common function or origin, highly expressed genes, the codon frequencies in the aggregated protein-coding regions of an entire organism, the codon frequencies in the aggregated protein-coding regions of related organisms, or a combination thereof. Codons whose frequency increases with the level of gene expression are generally the optimal codons for expression. Various methods for determining codon frequencies (e.g., codon usage, relative synonymous codon usage) and codon preferences in a particular organism, as well as the effective number of codons used in a gene, are known, including multivariate analyses such as using cluster analysis or correlation analysis (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., 22:2437-46

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

[1990] ). Codon usage tables for many different organisms are available (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 salmonellaIn Neidhardt et al. (eds.), ASM Press, Washington D.C., p. 2047-2066

[1996] ). Data sources for obtaining codon usage can depend on any available nucleotide sequence capable of encoding a protein. These data sets include nucleic acid sequences actually known to encode expressed proteins (e.g., complete protein-coding sequences - CDSs), 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, N.Y.

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

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

[1997] ).

[0093] "Control sequence" is defined herein to include all components that are necessary or advantageous for the expression of the 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, leader sequences, polyadenylation sequences, propeptide sequences, promoters, signal peptide sequences, and transcription terminators. At a minimum, the control sequences include a promoter and transcription and translation termination signals. For the purpose of introducing specific restriction sites, the control sequences may be provided with linkers that facilitate the ligation of the control sequences to the coding region of the nucleic acid sequence encoding the polypeptide.

[0094] "Operably linked" is defined herein as a configuration in which a control sequence is placed appropriately (i.e., in a functional relationship) at a position relative to a polynucleotide of interest such that the control sequence directs or regulates the expression of the polynucleotide and / or polypeptide of interest.

[0095] "Promoter sequence" refers to a nucleic acid sequence that is recognized by a host cell for the expression of a polynucleotide of interest, such as a coding sequence. The promoter sequence contains transcriptional control sequences that mediate the expression of the polynucleotide of interest. The promoter may be any nucleic acid sequence that shows transcriptional activity in the selected host cell, including mutant, truncated, and hybrid promoters, and may be obtained from genes encoding extracellular or intracellular polypeptides that are homologous or heterologous to the host cell.

[0096] "Suitable reaction conditions" refers to those conditions in a biocatalytic reaction solution (e.g., ranges of enzyme loading, substrate loading, cofactor loading, temperature, pH, buffer, cosolvent, etc.) under which the TdT polypeptide of the present invention is capable of converting one or more substrate compounds into product compounds (e.g., adding nucleotide-3'-O-RBG or other natural or modified nucleotide substrates to an oligonucleotide acceptor substrate via reaction with NTP-3'-O-RBG or other natural or modified NTP substrates). Exemplary "suitable reaction conditions" are provided in the present invention and illustrated by the examples.

[0097] "Composition" refers to a mixture or combination of one or more substances, where each substance or component of the composition retains its respective properties. As used herein, a biocatalytic composition refers to a combination of one or more substances that can be used for biocatalysis.

[0098] "Loading" in terms such as "compound loading" or "enzyme loading" or "cofactor loading" refers to the concentration or amount of a component in a reaction mixture at the start of the reaction.

[0099] In the context of a biocatalyst-mediated process, "substrate" refers to a compound or molecule on which a biocatalyst acts. For example, the TdT biocatalyst used in the synthetic processes disclosed herein acts on NTP-3'-O-RBG substrates or other natural or modified NTP substrates and oligonucleotide acceptor substrates.

[0100] In the context of a biocatalyst-mediated process, "product" refers to a compound or molecule produced by the action of a biocatalyst. For example, an exemplary product of the TdT biocatalyst used in the processes disclosed herein is an oligonucleotide acceptor extension product, as depicted in Schemes 1 and 2.

[0101] "Alkyl" refers to a straight-chain or branched-chain saturated hydrocarbon group having from 1 to 18 carbon atoms (including the termini), more preferably from 1 to 8 carbon atoms (including the termini), and most preferably 1 to 6 carbon atoms (including the termini). An alkyl having a specified number of carbon atoms is indicated in parentheses (e.g., (C 1 -C 6 ) alkyl refers to an alkyl having 1 to 6 carbon atoms).

[0102] "Alkenyl" refers to a straight-chain or branched-chain hydrocarbon group having from 2 to 12 carbon atoms (including the termini) and containing at least one double bond, but optionally containing more than one double bond.

[0103] "Alkynyl" means a hydrocarbon group having from 2 to 12 carbon atoms (including the termini), straight-chain or branched-chain, containing at least one triple bond but optionally containing more than one triple bond, and further optionally containing one or more double-bonded moieties.

[0104] "Heteroalkyl", "heteroalkenyl", and "heteroalkynyl" respectively mean alkyl, alkenyl, and alkynyl as defined herein in which one or more carbon atoms are each independently replaced by the same or different heteroatoms or heteroatom groups. Heteroatoms and / or heteroatom groups that can replace carbon atoms include, but are not limited to, -O-, -S-, -S-O-, -NR γ -, -PH-, -S(O)-, -S(O)2-, -S(O)NR γ -, -S(O) 2 NR γ -, etc., including combinations thereof, where each R γ is independently selected from hydrogen, alkyl, cycloalkyl, heterocycloalkyl, aryl, and heteroaryl.

[0105] "Amino" means the group -NH 2 . Substituted amino means -NHR η , NR η R η and NR η R η R η , where each R η is independently selected from substituted or unsubstituted alkyl, cycloalkyl, cycloheteroalkyl, alkoxy, aryl, heteroaryl, heteroarylalkyl, acyl, alkoxycarbonyl, thioalkyl, sulfinyl, sulfonyl, etc. Representative amino groups include, but are not limited to, dimethylamino, diethylamino, trimethylammonium, triethylammonium, methylsulfonylamino, furanyl-oxy-sulfamino, etc.

[0106] "Aminoalkyl" means an alkyl group in which one or more hydrogen atoms are replaced by one or more amino groups (including substituted amino groups).

[0107] "Aminocarbonyl" means -C(O)NH 2 . Substituted aminocarbonyl means -C(O)NR η R η , where the amino group NR η R η is as defined herein.

[0108] "Oxy" means the divalent group "-O-", which can have various substituents to form different oxy groups, including ethers and esters.

[0109] "Alkoxy" or "alkyloxy" are used interchangeably herein to refer to the group "-ORζ ”, wherein R ζ is an alkyl group, including an optionally substituted alkyl group.

[0110] "Carboxyl" means -COOH.

[0111] "Carbonyl" means -C(O)-, which may have various substituents to form different carbonyl groups, including acids, acid halides, aldehydes, amides, esters, and ketones.

[0112] "Carboxyalkyl" means an alkyl group in which one or more hydrogen atoms are replaced by one or more carboxyl groups.

[0113] "Aminocarbonylalkyl" means an alkyl group substituted by an aminocarbonyl group as defined herein.

[0114] "Halogen" or "halo" means fluorine, chlorine, bromine, and iodine.

[0115] "Haloalkyl" means an alkyl group in which one or more hydrogen atoms are replaced by halogen. Thus, the term "haloalkyl" is intended to include monohaloalkyl, dihaloalkyl, trihaloalkyl, etc. up to perhaloalkyl. For example, the expression "(C 1 -C 2 ) haloalkyl" includes 1-fluoromethyl, difluoromethyl, trifluoromethyl, 1-fluoroethyl, 1,1-difluoroethyl, 1,2-difluoroethyl, 1,1,1-trifluoroethyl, perfluoroethyl, etc.

[0116] "Hydroxy" means -OH.

[0117] "Hydroxyalkyl" means an alkyl group in which one or more hydrogen atoms are replaced by one or more hydroxy groups.

[0118] "Thiol" or "sulfanyl" means -SH. Substituted thiol or sulfanyl means -S-R η , wherein R η is an alkyl, aryl, or other suitable substituent.

[0119] "Sulfonyl" means -SO 2 -. Substituted sulfonyl means -SO 2 -R η , wherein R η is an alkyl, aryl, or other suitable substituent.

[0120] "Alkylsulfonyl" means -SO 2 -R ζ , wherein R ζis an optionally substituted alkyl group. Typical alkylsulfonyl groups include, but are not limited to, methylsulfonyl, ethylsulfonyl, n-propylsulfonyl, and the like.

[0121] As used herein, "phosphate ester" refers to a functional group that includes an orthophosphate ion (a phosphorus atom covalently linked to four oxygen atoms). The orthophosphate ion typically has one or more hydrogen atoms or organic groups.

[0122] As used herein, "phosphorylation" refers to the addition or presence of one or more phosphoryl groups (a phosphorus atom covalently linked to three oxygen atoms).

[0123] As used herein, "optionally substituted" with respect to the above chemical groups means that the position of the chemical group occupied by hydrogen can be substituted by another atom (unless otherwise specified) or chemical group, said another atom such as but not limited to carbon, oxygen, nitrogen or sulfur, said chemical group such as but not limited to hydroxy, oxo, nitro, methoxy, ethoxy, alkoxy, substituted alkoxy, trifluoromethoxy, haloalkoxy, fluorine, chlorine, bromine, iodine, halogen, methyl, ethyl, propyl, butyl, alkyl, alkenyl, alkynyl, substituted alkyl, trifluoromethyl, haloalkyl, hydroxyalkyl, alkoxyalkyl, thio, alkylthio, acyl, carboxy, alkoxycarbonyl, carboxamido, substituted carboxamido, alkylsulfonyl, alkylsulfinyl, alkylsulfonylamino, sulfonylamino, substituted sulfonylamino, 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, heterocyclic group, (heterocyclic)oxy and (heterocyclic)alkyl; wherein the preferred heteroatoms are oxygen, nitrogen and sulfur. Additionally, in the case where there are open valences on these substituted chemical groups, they can be further substituted by alkyl, cycloalkyl, aryl, heteroaryl and / or heterocyclic groups, and in the case where these open valences are on carbon, they can be further substituted by halogen and substituents bonded by oxygen-, nitrogen- or sulfur-bonds, and in the case where there are more than one such open valences, these groups can be linked by directly forming a bond or by forming a bond with a new heteroatom (preferably oxygen, nitrogen or sulfur) to form a ring. It is also contemplated that the above substitutions can be made provided that replacing hydrogen with a substituent does not impart unacceptable instability to the molecules of the present invention and is otherwise chemically reasonable. One of ordinary skill in the art will understand that for any chemical group described as optionally substituted, only those chemical groups that are spatially achievable and / or synthetically feasible are intended to be included. As used herein, "optionally substituted" refers to all subsequent modifiers in a term or series of chemical groups. For example, in the term "optionally substituted arylalkyl", the "alkyl" portion and the "aryl" portion of the molecule can be substituted or can be unsubstituted, and for the series "optionally substituted alkyl, cycloalkyl, aryl and heteroaryl", the alkyl, cycloalkyl, aryl and heteroaryl groups can be substituted or can be unsubstituted independently of one another.

[0124] As used herein, "reaction" refers to one or more processes in which one or more substances, compounds, or substrates are converted into one or more different substances or compounds.

[0125] Non-template-dependent synthesis of engineered TdT

[0126] New methods for the efficient synthesis of high-purity strands of DNA, RNA, and other polynucleotides are necessary to overcome the limitations of existing phosphoramidite chemical synthesis methods in order to enable a range of emerging and existing synthetic biology applications.

[0127] The present invention provides new terminal deoxynucleotidyl transferases that have improved activity in the non-template-dependent synthesis of polynucleotides using 5'-nucleoside triphosphates ("NTPs") modified with 3'-O-removable blocking groups (NTP-3'-O-RBG) or other natural or modified NTP substrates. Compared to wild-type TdT or other TdT or non-template-dependent polymerases known to those skilled in the art, the TdT of the present disclosure has improved thermal stability, activity at high temperatures, increased soluble expression or isolated protein yield, reduced byproduct formation, increased affinity for NTP-3'-O-RBG and other natural or modified NTP substrates, increased activity or specific activity towards NTP-3'-O-RBG and other natural or modified NTP substrates, and / or increased activity or specific activity towards various oligonucleotide acceptor substrates. The engineered polypeptides of the present disclosure are variants of SEQ ID NO:2, which is a predicted spliced variant encoded by the genome of the species Monodelphis domestica. These engineered TdTs are capable of non-template-dependent synthesis of oligonucleotides and polynucleotides.

[0128] Non-template-dependent synthesis of defined polynucleotide sequences using engineered TdT is a multi-step process. In one embodiment, an oligonucleotide acceptor substrate having a 3'-OH allows the addition of a defined modified NTP substrate (in this instance, NTP-3'-O-RBG) by engineered TdT, as depicted in Scheme 1 below.

[0129] Solution 1

[0130]

[0131] in

[0132]

[0133] and

[0134] B = adenine, guanine, cytosine, thymine, uracil, pseudouracil, 1-methylpseudouracil

[0135] R 1 = benzyl, O-nitrobenzyl, benzoyl, acetyl, cyanoethyl, NO 2 、PO 3 2 -、SO 3 -

[0136] R 2 = H, OH, OMe, F, Me, methoxyethyl (MOE)

[0137] R 3 = H, OH, OMe, F, Me

[0138] X = O, S

[0139] Y = OS

[0140] After the reaction of NTP-3'-O-RBG with the 3'-OH of the oligonucleotide acceptor substrate or the growing polynucleotide chain, TdT is blocked by 3'-O-RBG and cannot react further. Then, RBG is 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 the 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 to sequentially and efficiently generate a defined polynucleotide sequence by addition at the 3'-OH terminus of the polynucleotide or oligonucleotide acceptor substrate, without the need for a complementary strand or a template primer sequence. After the synthesis of the defined polynucleotide is completed, the oligonucleotide chain can be cleaved or released from the oligonucleotide acceptor substrate.

[0141] As can be envisioned by those skilled in the art, a variety of oligomeric acceptor substrates and NTP-3'-O-RBG or natural or modified NTP substrates can be used in this process. An example of a reaction is detailed in Scheme 2 below. Scheme 2 depicts the TdT-catalyzed reaction of 5'-6-FAM-[N] 15 AT*mC and 3'-phos-mATP, and other examples of suitable oligomeric acceptor substrates and NTP-3'-O-RBG or natural or modified NTP pairs are described in other examples. These examples are non-limiting.

[0142] Solution 2

[0143]

[0144] Occasionally, during the addition step, TdT can produce undesired synthetic products. This includes incorporation of NTPs that have lost their blocking groups, addition of more than one NTP, or excision or pyrophosphorylation of NTPs on the growing polynucleotide chain.

[0145] In some embodiments, one or more additional quality control steps are used, such as adding exonuclease and starting a new round of synthesis before removing the blocking groups. In some embodiments, phosphatases (such as pyrophosphatase) are used to break down inorganic phosphate esters and drive the reversible TdT reaction towards synthesis.

[0146] As further described herein, the engineered TdT polypeptides of the present disclosure exhibit one or more improved properties in the non-template-dependent polynucleotide synthesis processes depicted in Schemes 1 and 2.

[0147] In some embodiments, the present invention provides engineered TdT polypeptides that comprise an amino acid sequence having at least 60% sequence identity to the amino acid reference sequence of SEQ ID NO:2 and further comprise one or more amino acid residue differences compared to the reference amino acid sequence, wherein the engineered TdT polypeptides have improved thermal stability, increased activity at high temperature, increased soluble expression or isolated protein yield, reduced byproduct formation, increased specific activity towards NTP-3'-O-RBG or native or modified NTP substrates, and / or increased activity towards various oligonucleotide acceptor substrates compared to wild-type TdT or other TdT or non-template-dependent polymerases known to those skilled in the art.

[0148] In particular, the engineered TdT polypeptides of the present disclosure have been engineered for the efficient synthesis of polynucleotides having a defined sequence using NTP-3'-O-RBG or native or modified NTP substrates in the methods described above.

[0149] Various suitable reaction conditions are known to those skilled in the art and are detailed below and in the examples.

[0150] Engineered terminal deoxynucleotidyl transferase (TdT) polypeptides

[0151] The present invention provides engineered terminal deoxynucleotidyl transferase (TDT) polypeptides that can be used for non-template-dependent polynucleotide synthesis using NTP-3'-O-RBG or native or modified NTP substrates, as well as compositions and methods for utilizing these engineered polypeptides in non-template-dependent oligonucleotide synthesis.

[0152] The present invention provides TdT polypeptides, polynucleotides encoding the polypeptides, methods for preparing the polypeptides, and methods for using the polypeptides. In cases where a polypeptide is described, it should be understood that it can describe the polynucleotide encoding the polypeptide.

[0153] Suitable reaction conditions for carrying out the desired reactions with the above-described improved properties of the engineered polypeptides can be determined by considering the following: the concentration or amount of the polypeptide, substrate, cosubstrate, buffer, solvent, pH, conditions including temperature and reaction time, and / or conditions for immobilizing the polypeptide on a solid support, as further described below and in the examples.

[0154] In some embodiments, an exemplary engineered TdT comprises an amino acid sequence having one or more residue differences at residue positions indicated 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, 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 compared to SEQ ID NO:2.

[0155] The structural and functional information of exemplary engineered polypeptides of the present invention is based on the conversion of oligomeric receptor substrates and NTP-3'-O-RBG or dideoxynucleotides (e.g., 2',3'-dideoxynucleotides), the results of which 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, 31.2, 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 further described in the Examples. The odd-numbered sequence identifiers (i.e., SEQ ID NO) in these tables refer to the nucleotide sequences encoding the amino acid sequences provided by the even-numbered SEQ ID NO in these tables. Exemplary sequences are provided in the electronic sequence listing file accompanying the present invention, which is hereby incorporated by reference herein. Amino acid residue differences are based on comparison with reference sequences 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, as indicated.

[0156] Terminal deoxynucleotidyl transferase (a member of the Pol X family) has been identified in many species. Members of different PolX families are known to share certain residues that are conserved among family members. TdT also has a high level of conservation across species for residues that are thought to be involved in binding divalent metal ions, ternary complex formation, and binding dNTP and DNA ligands (Dominnguez et al. (2000). EMBO, 19(7), 1731-1742). Additionally, TDT is known to have a spliced variant that is N-terminally truncated and lacks the BRCT domain. Other non-template-dependent polymerases (including but not limited to polyA polymerase, polyU polymerase, and terminal uridylyl transferase) are also known in the art and can be used to practice the present invention. Similarly, other polymerases are known to be capable of non-template-dependent synthesis (including but not limited to reverse transcriptase) and can be used to practice the present invention.

[0157] Wild-type TdT from the short-tailed opossum (SEQ ID NO:2) was selected for evolution. The TdT polypeptides of the present disclosure are engineered variants of SEQ ID NO:2 with an N-terminal 6-histidine tag.

[0158] The polypeptides of the present disclosure have residue differences that result in improved properties required for an effective TdT enzyme capable of non-template-dependent synthesis of polynucleotides with defined sequences. Various residue differences at both conserved and non-conserved positions have been found to be associated with improvements in various enzyme properties, including improved thermal stability, increased activity at high temperatures, increased soluble expression or isolated protein yield, reduced byproduct formation, increased specific activity towards NTP-3'-O-RBG or natural or modified NTP substrates, increased incorporation efficiency in the extension of oligonucleotide acceptor substrates, and / or increased activity towards various oligonucleotide acceptor substrates compared to wild-type TdT or other TdT or non-template-dependent polymerases known to those skilled in the art. In some embodiments, the engineered TdT polypeptides exhibit increased incorporation efficiency in the extension of oligonucleotide acceptor substrates by adding greater than 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% NTP or NQP. Exemplary incorporation efficiencies of engineered TdT are provided in the Examples (e.g., Example 92).

[0159] The activity of each engineered TdT relative to the reference polypeptides 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 was determined as the conversion of the substrates described in the examples herein. In some embodiments, shake flask purified enzyme (SFP) was used to evaluate the properties of the engineered TdT, and the results are provided in the examples.

[0160] In some embodiments, specific enzyme properties are correlated with residue differences at residue positions indicated herein relative to 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, residue differences that affect polypeptide expression can be used to increase the expression of the engineered TdT.

[0161] According to the guidance provided herein, any of the exemplary engineered polypeptides comprising the sequences of 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 can be used as, for example, the starting amino acid sequence for the synthesis of other TdT polypeptides by subsequent rounds of evolution that incorporate multiple amino acid differences from other polypeptides in the following tables and new combinations of other residue positions described herein: Table 5.1, Table 6.2, Table 7.2, Table 8.2, Table 9.2, Table 10.2, Table 11.2, Table 12.2, Table 13.2, Table 14.2, Table 15.2, Table 16.2, Table 17.2, Table 18.2, Table 19.2, Table 20.2, Table 21.2, Table 22.2, Table 23.2, Table 24.2, Table 25.2, Table 26.2, Table 26.3, Table 26.4, Table 27.2, Table 27.3, Table 27.4, Table 27.5, Table 28.1, Table 28.2, Table 28.3, Table 29.2, Table 30.2, Table 31.2, Table 32.2, Table 33.2, Table 34.2, Table 35.2, Table 36.2, Table 37.2, Table 38.2, Table 39.2, Table 40.2, Table 41.2, Table 42.2, Table 43.2, Table 44.2, Table 45.2, Table 46.2, Table 47.2, Table 48.2, Table 49.2, Table 50.2, Table 51.2, Table 52.2, Table 53.2, Table 54.2, Table 55.2, Table 56.2, Table 56.3, Table 56.4, Table 61.2, Table 63.2, Table 64.2, Table 65.2, Table 66.2, Table 67.2, Table 68.2, Table 69.2, Table 70.2, Table 71.2, Table 72.2, Table 73.2, Table 74.2, Table 75.2, Table 76.2, Table 77.2, Table 78.2, Table 79.2 and Table 80.1. Additional improvements can be generated by incorporating amino acid differences at residue positions that are maintained unchanged throughout earlier rounds of evolution.

[0162] In some embodiments, the engineered TdT polypeptide has increased soluble protein expression and 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:2 and having one or more residue differences selected from the following compared to SEQ ID NO:2: 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 / 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 / 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 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:2 and having one or more residue differences selected from the following compared to SEQ ID NO:2: 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 / 185L / 190E / 205R / 244V / 289D / 290V / 293S / 313A / 315G / 336D / 342E / 359L / 391G / 414H / 470T / 474M / 499L / 522L / 523E, 80D / 121S / 131E / 185L / 190E / 205R / 244V / 289D / 290V / 293S / 313A / 336D / 342E / 359L / 391G / 414H / 470T / 474M / 499L / 522L / 523E, 80D / 121S / 131E / 185L / 190E / 289D / 290V / 293S / 313A / 342E / 470T / 474M / 499L / 522L / 523E,80D / 121S / 174L / 179K / 185L / 190E / 236P / 244V / 288E / 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 / 288E / 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 / 397R / 401G / 419A / 428V / 431S / 462F / 470T / 474M / 499L / 522L / 523E80D / 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 / 523E80D / 121S / 185L / 190E / 201R / 289D / 290V / 293S / 313A / 342E / 470T / 474M / 499L / 522L, 80D / 121S / 185L / 190E / 244V / 273V / 289D / 290V / 293S / 313A / 315G / 317T / 336D / 342E / 352P / 359L / 391G / 419A / 435T / 470T / 474M / 499L / 522L / 523E, 80D / 121S / 185L / 190E / 244V / 289D / 290V / 293S / 300R / 313A / 315G / 317T / 336D / 342E / 359L / 391G / 470T / 474M / 499L / 522L / 523E, 80D / 121S / 185L / 190E / 244V / 289D / 290V / 293S / 313A / 315G / 317T / 336D / 342E / 359L / 380D / 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 / 336D / 342E / 359L / 391G / 414H / 470T / 474M / 499L / 522L / 523E, 80D / 121S / 185L / 190E / 289D / 290V / 293S / 313A / 315G / 336D / 342E / 359L / 391G / 414H / 470T / 474M / 499L / 522L / 523E, 80D / 121S / 185L / 190E / 289D / 290V / 293S / 313A / 336D / 342E / 359L / 391G / 470T / 474M / 499L / 522L / 523E80D / 121S / 185L / 190E / 289D / 290V / 293S / 313A / 342E / 499L, 80D / 121S / 185L / 315G, 80D / 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 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:2 and having one or more residue differences selected from the following compared to SEQ ID NO:2: N80D / K106D / C121S / F185L / K190E / M205R / K289D / I290V / D293S / I313A / C315G / L336D / T342E / F359L / C391G / S470T / Q474M / K499L / I522L / Q523E, 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 / T342E / S470T / Q474M / K499L / I522L / Q523E,N80D / C121S / I174L / Q179K / F185L / K190E / G236P / D244V / N288E / K289D / I290V / D293S / I313A / C315G / S317T / L336D / T342E / F359L / S363I / C391G / I394R / I408P / H426P / Y462F / S470T / Q474M / K499L / I522L / Q523E, 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 / S317T / V324I / L336D / T342E / E352P / F359L / Q376H / N380D / C391G / I394R / L401G / Q415S / L419A / E428V / R431S / M435T / E441M / Y462F / S470T / 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 / K499L / 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 / K499L / 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 / I290V / D293S / I313A / C315G / S317T / L336D / T342E / E352P / F359L / C391G / I394R / L419A / E428V / R431S / Y462F / S470T / Q474M / K499L / I522L / Q523E, N80D / 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 / 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 / E428V / R431S / Y462F / S470T / Q474M / K499L / I522L / Q523E, N80D / C121S / I174L / F185L / K190E / D244V / F284L / N288E / K289D / I290V / D293S / I313A / C315G / S317T / L336D / T342E / F359L / C391G / I394R / E428V / R431S / Y462F / S470T / Q474M / K499L / I522L / Q523E, N80D / C121S / F185L / K190E / E196R / D244V / K289D / I290V / D293S / I313A / C315G / S317T / L336D / T342E / F359L / C391G / S470T / Q474M / K499L / I522L / Q523E, N80D / C121S / F185L / K190E / C201R / K289D / I290V / D293S / I313A / T342E / S470T / Q474M / K499L / I522L, N80D / C121S / F185L / K190E / D244V / L273V / K289D / I290V / D293S / I313A / 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 / L336D / T342E / F359L / N380D / C391G / L401G / L419A / S470T / Q474M / K499L / I522L / Q523EN80D / C121S / F185L / K190E / D244V / K289D / I290V / D293S / I313A / C315G / S317T / L336D / 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 / I290V / 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 / K289D / 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 / I290V / D293S / I313A / T342E / K499L, N80D / C121S / F185L / C315G, N80D / C121S / K190E / K289D / I290V, N80D / F185L / G236P / K289D / D293S, C121S / F185L / K190E / C213S / K289D / I290V / D293S, and F185L / K289D / I290V / D293S.,

[0163] In some embodiments, the engineered TdT polypeptide has increased thermal stability and 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:36 and having one or more residue differences selected from the following compared to SEQ ID NO:36: 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, 244 / 315 / 317 / 336 / 359 / 391 / 470 / 474 / 522 / 523, and 336 / 359 / 391 / 470 / 474 / 522 / 523. In some embodiments, the engineered TdT polypeptide has increased thermal stability and 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:36 and having one or more residue differences selected from the following compared to SEQ ID NO:36: 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 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:36 and having one or more residue differences selected from the following compared to SEQ ID NO:36: I174L / D244V / L273V / F284L / N288E / C315G / S317T / L336D / E352P / F359L / C391G / I394R / L419A / E428V / R431S / Y462F / S470T / Q474M / I522L / 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.

[0164] 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:8 and having one or more residue differences selected from the following compared to SEQ ID NO:8: 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. 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:8 and having one or more residue differences selected from the following compared to SEQ ID NO:8: 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, 297R, 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, 395W, 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 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:8 and having one or more residue differences selected from the following compared to SEQ ID NO:8: 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, E320N, V324I, I343V, L360C, L360V, D392A, D392C, D392R, E395A, E395L, E395R, E395S, E395T, E395W, E395Y, T397R, L411A, L411G, L411R, Q415A, Q415S, C417G, C417V, L421I, L421M, L454V, V456K, V456R, R477T, R481E, R481V, and D492T.

[0165] 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:16 and having one or more residue differences selected from the following compared to SEQ ID NO:16: 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 / 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.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:16 and having one or more residue differences selected from the following compared to SEQ ID NO:16: 186G, 186G / 236P / 318S, 186G / 236P / 395R, 186G / 282R / 318S, 193G / 196R / 297R, 193G / 196Y, 193G / 196Y / 266K / 324I / 376H / 380D, 193G / 196Y / 297R / 324I / 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 / 318S / 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 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:16 and having one or more residue differences selected from the following compared to SEQ ID NO:16: E186G, E186G / G236P / K318S, E186G / G236P / E395R, E186G / M282R / K318S, E193G / E196R / K297R, E193G / E196Y, E193G / E196Y / T266K / V324I / Q376H / N380D, E193G / E196Y / K297R / V324I / Q376H / N380D / L401G / Q415S / M435T / E441M, E193G / E196Y / V324I / T397R / L401G / E441M, E193G / E196Y / Q376H / N380D, E193G / K297R / V324I / 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 / K318S / 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.

[0166] 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:24 and having one or more residue differences selected from the following compared to SEQ ID NO:24: 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.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:24 and having one or more residue differences selected from the following compared to SEQ ID NO:24: 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, 34S, 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, 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 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:24 and having one or more residue differences selected from the following compared to SEQ ID NO:24: M12S, H13G, H13K, H13R, H13S, R14G, R14Q, T17A, T17G, I18D, I18R, S20H, S20T, D21E, F22G, F22L, G23E, G23P, K24G, R26G, Q27D, K29P, K29R, M30E, M30G, M30V, D31S, H33G, H33K, H33P, I34D, I34K, I34R, I34S, S35E, S35G, M37A, M37F, M37G, M37S, M37T, M37V, I41V, K53E, A57H, T58A, T58S, T61H, T61L, G92M, G92R, G92S, G92V, G92Y, D94E, D94R, A97D, T101E, H102L, K103M, M104G, M104I, M104P, E105N, K106G, K106H, K106S, T107R, T107W, T108D, T108K, V124E, V124I, K126V, K133G, Q135R, M137A, E138Q, S139A, R140E, R140G, V141E, V141M, V141R, D142M, D142S, A144C, N145E, D147L, T149R, A150E, A150G, T152G, T152R, L153E, L153G, L153K, L153M, L153P, L153Q, L153V, N154G, I155E, I155T, L156D, L156E / K294T, L156M, L156Q, T159D, T160G, K161D, K161E, K161G, K161R, K161S, T162E, I163L and I163V.

[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:24 and having one or more residue differences selected from the following compared to SEQ ID NO:24: 7 / 135, 12, 13, 14, 15, 16, 17 / 131, 18, 20, 23, 24, 25, 26, 27, 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. 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:24 and having one or more residue differences selected from the following compared to SEQ ID NO:24: 7Y / 135C, 12F, 13E, 14G, 14N, 14Y, 15L, 16V, 17A / 131R, 18Y, 20P, 23C, 23E, 23T, 24A, 24M, 24P, 25N, 26G, 27D, 27F, 28R, 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, 110M, 110V, 119F, 119Q, 123M, 123Q, 124E, 124G, 124I, 124M, 124S, 126C, 130A, 130M, 130Q, 130S, 131G, 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 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:24 and having one or more residue differences selected from the following compared to SEQ ID NO:24: H7Y / Q135C, M12F, H13E, R14G, R14N, R14Y, I15L, R16V, 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.

[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:268 and having one or more residue differences selected from the following compared to SEQ ID NO:268: 14 / 53 / 300, 14 / 53 / 419, 106 / 300 / 415 / 419 / 456, 140 and 300 / 395 / 419. 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 selected from the following compared to SEQ ID NO:268: 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 selected from the following compared to SEQ ID NO:268: R14G / E53K / K300P, R14G / E53K / A419L, K106V / K300P / Q415A / A419L / V456R, R140G and K300P / E395Y / A419L.

[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: 648 and having one or more residue differences selected from the following compared to SEQ ID NO: 648: 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 / 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 / 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, 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 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:648 and having one or more residue differences selected from the following compared to SEQ ID NO:648: 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 / 106K21E / 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 / 153E106K / 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 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:648 and having one or more residue differences selected from the following compared to SEQ ID NO:648: M12S / R14G / I34D, M12S / R14G / I34D / M37A / V106K / R140G / D142M / A150E / T152R / L153E, M12S / R14G / I34D / M37A / V141E / D142S, M12S / R14G / I34D / M37A / A150E, M12S / R14G / I34D / M37F / D94E / R140G / V141E / N145E, M12S / R14G / I34D / M37F / D142S / N145E, M12S / R14G / I34D / M37F / A150E / L153E, M12S / R14G / I34D / R140G / D142S, M12S / R14G / I34D / R140G / A150E, 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 / V106K / 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 / I155EH33K / 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 / D94E / 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 / V106K, 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 / V141E / T152R / L153E, V106K / T108K / R140G / D142S / A150E / L153E, V106K / L156E, R140G / V141E / D142M, R140G / N145E, R140G / N145E / A150E / T152R, V141E, V141E / T152R and K161E.

[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: 660 and having one or more residue differences selected from the following compared to SEQ ID NO: 660: 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 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: 660 and having one or more residue differences selected from the following compared to SEQ ID NO: 660: 16V / 29I / 30E, 16V / 29I / 30E / 33K / 153P, 16V / 29I / 30E / 101E / 104V, 16V / 30L / 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 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: 660 and having one or more residue differences selected from the following compared to SEQ ID NO: 660: R16V / K29I / M30E, R16V / K29I / M30E / H33K / E153P, R16V / K29I / M30E / T101E / M104V, R16V / M30L / M104V, R16V / 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.

[0171] 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: 660 and having one or more residue differences selected from the following compared to SEQ ID NO: 660: 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.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:660 and having one or more residue differences selected from the following compared to SEQ ID NO:660: 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, 303V, 306F, 308F, 308W, 309F, 310A, 310G, 310H, 310R, 310S, 312V, 315A, 315S, 316A, 316L, 342R, 342V, 344V, 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 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: 660 and having one or more residue differences selected from the following compared to SEQ ID NO: 660: K195E, N197M, F204L / E342W, M205E, M205L, G236E / K297L, R258A, R258C, R258G, R258L, R258M, R258S, R258W, S261G, S261R, S261V, F262I, L264A, L264E, L264R, L264S, V268L, F269W, A276S, K278C, K278E, K278I, K278R, K278T, K278V, Y280F, R281A, R281C, R281G, R281L, R281S, R281T, R281V, M282C, M282G, M282H, M282W, V290A, V290L, R291S, K297C, K297D, K297S, K297V, P300R, K303A, K303N, K303Q, K303S, K303V, L306F, Y308F, Y308W, Y309F, E310A, E310G, E310H, E310R, E310S, L312V, G315A, G315S, V316A, V316L, E342R, E342V, T344V, 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.

[0172] 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 selected from the following compared to SEQ ID NO:882: 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 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 selected from the following compared to SEQ ID NO:882: 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 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 selected from the following compared to SEQ ID NO:882: N175L, R196G, R196Y, D199G, D199M, D199Q, D199R, D199S, D199V, T203A, T203G, T203L, T203R, T203S, I208V, T275V, A313I, D314G, D314K, D314L, D314R, D314V, D314Y, T317G, A321C, A321K, A321S, D322A, S325F, S325T, S325V, S325W, Q329R / F462E, T379C, R394E, R394T, R397D, R397T, L403F / F462H, R406G, R406V, I408A, I408T, C457S, C457V, R461G, R461V, F462I, F462R, F462W, W469Q, R477T, R481D, R481M, R481T, T484M, T484R, and A495S.

[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:882 and having one or more residue differences selected from the following compared to SEQ ID NO:882: 175, 179, 196, 199, 201, 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. 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 selected from the following compared to SEQ ID NO:882: 175D, 175I, 175V, 179R, 196G, 199A, 199E, 199G, 199H, 199I, 199Q, 199R, 199V, 201A, 201W, 203M, 203R, 272T, 273M, 273W, 275D, 307M, 307V, 313M, 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 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 selected from the following compared to SEQ ID NO:882: N175D, 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.

[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: 1336 and having one or more residue differences selected from the following compared to SEQ ID NO: 1336: 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, 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 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: 1336 and having one or more residue differences selected from the following compared to SEQ ID NO: 1336: 15F / 199R / 203A / 394E, 15F / 199R / 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, 195E / 203A / 394E, 195E / 278E / 297D / 394E, 195E / 278E / 297D / 394E / 395W, 195E / 314R / 344V, 195E / 394E / 395W, 199R / 203A / 297D / 394E / 395W, 203A / 278E / 297D / 394E, 203A / 297D / 314R / 394E / 395W, 203A / 310R / 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 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: 1336 and having one or more residue differences selected from the following compared to SEQ ID NO: 1336: I15F / D199R / T203A / R394E, I15F / D199R / R394E, K28N / T344V / F353K / E395W, K195E / D199R / T203A / K278E / K297D / R394E, K195E / D199R / T203A / K278E / D314R / F353K / R394E, K195E / T203A / K278E / R394E / E395W, K195E / T203A / K297D / D314R / R394E, K195E / T203A / K297D / R394E / L419M, K195E / T203A / R394E, 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 / 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.

[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: 1336 and having one or more residue differences selected from the following compared to SEQ ID NO: 1336: 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.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: 1336 and having one or more residue differences selected from the following compared to SEQ ID NO: 1336: 61A, 152H / 503S, 160M, 160N, 160S, 160V, 162A, 165K, 177L, 200C, 200C / 425K, 200K, 200V, 213S, 217Q, 219L, 219R, 223Q, 236I, 236P, 236V, 246K, 248C, 248S, 292L / 411P, 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 / 401E / 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 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: 1336 and having one or more residue differences selected from the following compared to SEQ ID NO: 1336: T61A, R152H / I503S, T160M, T160N, T160S, T160V, T162A, Q165K, H177L, T200C, T200C / H425K, 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, L340A, 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, T437S, 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.

[0176] 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: 1348 and having one or more residue differences selected from the following compared to SEQ ID NO: 1348: 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, 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.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: 1348 and having one or more residue differences selected from the following compared to SEQ ID NO: 1348: 3Q / 175D / 213S / 313R / 325T / 340R / 457V / 481W / 485D, 3Q / 307M / 321K / 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 / 484M, 201A / 213S / 333A / 344V / 397Q / 425D / 481W / 485D, 201A / 333A / 344V / 457V / 481W, 201A / 333A / 481W, 201R / 406G / 408A / 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 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: 1348 and having one or more residue differences selected from the following compared to SEQ ID NO: 1348: H3Q / N175D / C213S / A313R / S325T / L340R / C457V / R481W / H485D, H3Q / C307M / A321K / L340R / F353K / R406G / 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, C201A / C213S / W333A / T344V / R397Q / H425D / R481W / H485D, C201A / W333A / T344V / C457V / R481W, C201A / W333A / R481W, C201R / R406G / I408A / F462L / 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.

[0177] 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 selected from the following compared to SEQ ID NO: 1596: 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, 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 selected from the following compared to SEQ ID NO: 1596: 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 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 selected from the following compared to SEQ ID NO: 1596: T160M / V219R / D460G / I503E / K506E, V219R / C307M / L326T, A252K, A252K / A333H, R406G / I408A, R406G / I408A / S442G / S446P, R406G / I408A / M490R, I408A and S446P.

[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: 1596 and having one or more residue differences selected from the following compared to SEQ ID NO: 1596: 162, 163, 165, 171, 177, 179, 188, 200, 205, 208, 233, 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. 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 selected from the following compared to SEQ ID NO: 1596: 162H, 163V, 165P, 171K, 177Y, 179K, 188M, 200A, 205R, 208A, 233D, 252E, 253I, 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 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 selected from the following compared to SEQ ID NO: 1596: T162H, I163V, Q165P, R171K, H177Y, Q179K, L188M, T200A, M205R, I208A, E233D, A252E, V253I, Q260K, S261A, 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.

[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: 1596 and having one or more residue differences selected from the following compared to SEQ ID NO: 1596: 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. 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 selected from the following compared to SEQ ID NO: 1596: 161R, 162H, 163V, 165P, 179K, 188M, 205R, 208A, 231T, 233D, 251K, 252E, 253I, 261A, 277E, 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 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 selected from the following compared to SEQ ID NO: 1596: 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.

[0180] 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 selected from the following compared to SEQ ID NO: 1654: 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 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 selected from the following compared to SEQ ID NO: 1654: 160M / 165P / 203E / 205R / 219R / 353Q / 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 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 selected from the following compared to SEQ ID NO: 1654: 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.

[0181] 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 selected from the following compared to SEQ ID NO: 1654: 3 / 160 / 205 / 208 / 219 / 307 / 353 / 371 / 376 / 413 / 414 / 441 / 488, 160, 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 / 219 / 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、326 / 353 / 371 / 376 / 414 and 441. 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 selected from the following compared to SEQ ID NO:1654: 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 / 503E165P / 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 / 353Q / 441M / 460G / 503E / 506E, 203E / 205R / 208A / 307L / 441M, 203E / 205R / 208A / 353Q, 203E / 208A / 219R / 376H / 441M, 203E / 208A / 219R / 441M, 203E / 208A / 326T / 353Q, 203E / 413G / 503E / 506E, 205R / 208A / 307L / 353Q / 376H / 413G, 205R / 208A / 414H, 205R / 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 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 selected from the following compared to SEQ ID NO:1654: 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 / V219R / F353Q / D460G / K488N, T160M / Q165P / T203E / M205R / C307L / E371D / Q376H / H413G / F414H, T160M / Q165P / T203E / I208A / E371D / Q376H / H413G / F414H, T160M / Q165P / M205R, T160M / Q165P / M205R / I208A, T160M / Q165P / M205R / V219R / E441M, T160M / Q165P / M205R / F414H / E441M / A495G, T160M / Q165P / C307L / F353Q / H413G / F414H / E441M / K488N / A495G, T160M / Q179K / T203E / M205R / I208A / V219R / E371D / F414H / T484E / K506E, T160M / Q179K / I208A / V219R / C307L / H413G / F414H / I503E / K506E, T160M / Q179K / I208A / C307L / E371D / Q376H / F414H, T160M / Q179K / C307L / Q376H / E441M / K488N / I503E, 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 / I503ET160M / 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 / H413GM205R / 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.

[0182] 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 selected from the following compared to SEQ ID NO: 1830: 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 selected from the following compared to SEQ ID NO: 1830: 163V / 179K / 277E / 338T / 340A, 163V / 414H / 441M, 171K / 200V / 334R / 406P / 490L, 177L, and 292T / 406P. 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 selected from the following compared to SEQ ID NO: 1830: I163V / Q179K / D277E / D338T / L340A, I163V / F414H / E441M, R171K / T200V / T334R / G406P / M490L, H177L, and S292T / G406P.

[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: 1100 and having one or more residue differences selected from the following compared to SEQ ID NO: 1100: 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 selected from the following compared to SEQ ID NO: 1100: 101E / 137A / 264R / 476R / 525F and 264R. 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 selected from the following compared to SEQ ID NO: 1100: T101E / M137A / L264R / E476R / S525F and L264R.

[0184] 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 selected from the following compared to SEQ ID NO: 1654: 160 / 163 / 165 / 203 / 205 / 219 / 353 / 414 / 441 / 460 / 488 and 160 / 165 / 203 / 205 / 219 / 353 / 460 / 488. 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 selected from the following compared to SEQ ID NO: 1654: 160M / 163V / 165P / 203E / 205R / 219R / 353Q / 414H / 441M / 460G / 488N and 160M / 165P / 203E / 205R / 219R / 353Q / 460G / 488N. 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 selected from the following compared to SEQ ID NO: 1654: T160M / I163V / Q165P / T203E / M205R / V219R / F353Q / F414H / E441M / D460G / K488N and T160M / Q165P / T203E / M205R / V219R / F353Q / D460G / K488N.

[0185] 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 selected from the following compared to SEQ ID NO: 1596: 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, 406 / 408 / 441 / 442 / 446, and 406 / 408 / 442 / 446. 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 selected from the following compared to SEQ ID NO: 1596: 160M / 165P / 203E / 205R / 219R / 353Q / 406G / 408A / 442G / 446P / 460G / 488N, 160M / 165P / 205R / 219R / 406G / 408A / 441M / 442G / 446P, 160M / 203E / 205R / 406G / 408A / 441M / 442G / 446P, 160M / 219R / 330K / 406G / 408A / 442G / 446P / 484E, 205R / 307L / 406G / 408A / 441M / 442G / 446P / 460G / 488N, 406G / 408A / 441M / 442G / 446P, and 406G / 408A / 442G / 446P.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 selected from the following compared to SEQ ID NO: 1596: 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.

[0186] 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: 1950 and having one or more residue differences selected from the following compared to SEQ ID NO: 1950: 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 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: 1950 and having the same residue differences as SEQ IDAn amino acid sequence having one or more residue differences selected from the following compared to NO:1950: 163I / 169E, 163I / 169R, 185C, 185F, 186C, 192C, 192G, 192I, 192L, 192R, 193R, 193S, 194A, 194C, 194D, 194G, 194M, 194W, 197G, 197L, 197R, 198A, 198L, 245A, 251R, 258E, 258G, 258K, 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, 297L, 297P, 304L, 306I, 306M, 308N, 316A, 316C, 347Q, 352G, 352R, 359V, 362S, 362Y, 378L, 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, 411F / 414F, 411G / 414F, 411I / 414F, 411Q / 414F, 411R / 414F, 411T / 414F, 413A / 414F, 413F / 414F, 413G / 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 comprises 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 identical to SEQ IDAn amino acid sequence having one or more residue differences selected from the following compared to NO:1950: V163I / Q169E, V163I / Q169R, L185C, 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, K274V, K278C, K278L, K278N, Y280L, Y280S, L284I, L284M, T286N, T286S, V290A, R291M, R291W, K297L, K297P, A304L, L306I, L306M, Y308N, V316A, V316C, F347Q, P352G, P352R, L359V, T362S, T362Y, V378L, L393R, 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, L411F / H414F, L411G / H414F, L411I / H414F, L411Q / H414F, L411R / H414F, L411T / H414F, H413A / H414F, H413F / H414F, H413G / 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.

[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:2008 and having one or more residue differences selected from the following compared to SEQ ID NO:2008: 158 / 274 / 411 / 413 / 414, 185 / 274 / 413 / 414, 185 / 411 / 413 / 414, 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 / 411 / 413 / 468, 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. 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 selected from the following compared to SEQ ID NO:2008: 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 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 selected from the following compared to SEQ ID NO: 2008: P158H / K274V / L411G / H413A / H414G, L185F / K274V / H413A / H414G, L185F / L411G / H413A / H414G, K263H / L411G / H413A / H414G / G468Q, K263H / H413A / H414G, K274V / T286N / L411G / H413A / C417W / G468Q, K274V / L411G / C417W / G468Q, K274V / L411G / G468Q, K274V / H413A / H414G / C417W / G468Q, K274V / G468Q, K278N / L411G / H413A / G468Q, L411G / H413A / C417W, L411G / H413A / C417W / G468Q, L411G / H413A / G468Q, L411G / H414G, H413A, H413A / H414G, H413A / H414G / G468Q, H413A / C417W / G468Q, H413A / G468Q, H414G, and G468Q.

[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: 2008 and having one or more residue differences selected from the following compared to SEQ ID NO: 2008: 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 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 selected from the following compared to SEQ ID NO: 2008: 3A, 3G, 8P, 11C, 12V, 15A, 26A / 27R, 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 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 selected from the following compared to SEQ ID NO: 2008: 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.

[0189] 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 selected from the following compared to SEQ ID NO:2008: 9, 11, 12, 15, 16, 26, 38, 39, 58, 70, 79, 81, 90, 151, 157, 158 and 249. 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 selected from the following compared to SEQ ID NO:2008: 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, 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 selected from the following compared to SEQ ID NO:2008: G9A, G9T, G11S, S12A, S12N, S12Q, I15L, I15Q, R16S, R26S, R26W, I38L, I38T, Y39G, T58A, K70A, S79T, S81E, N90M, G151I, P157L, P158A and E249T.

[0190] 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 selected from the following compared to SEQ ID NO: 2008: 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 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 selected from the following compared to SEQ ID NO: 2008: 189E, 189Q, 189R, 196A, 196S, 196T, 196Y, 205E, 206N, 262V, 307E, 307H, 307L, 307S, 314A, 314M, 314Q, 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 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 selected from the following compared to SEQ ID NO: 2008: 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.

[0191] 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 selected from the following compared to SEQ ID NO: 2254: 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, 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 selected from the following compared to SEQ ID NO: 2254: 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 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 selected from the following compared to SEQ ID NO: 2254: N197R / K407S / V455L, N197R / V455L, L284M / F398W / L466M, T362S / K407S / V455L, S396T / F398W / A410V / L466M, F398W / L466M, E399D / G411Q / K416Q and L466M.

[0192] 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: 2514 and having one or more residue differences selected from the following compared to SEQ ID NO: 2514: 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 / 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 / 410, 62 / 249, 90 / 246 / 248, 90 / 246 / 248 / 261 / 266 / 362 / 455, 90 / 246 / 248 / 266 / 362 / 455, 246 / 248, 246 / 248 / 362, 246 / 266 / 455, 248, 248 / 266 / 362, 248 / 362 / 455, 248 / 455 and 362. 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: 2514 and having one or more residue differences selected from the following compared to SEQ ID NO: 2514: 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, 26G / 246N / 248V / 455L, 26G / 248R, 26G / 248V / 261A / 266V, 26I / 90L / 246N / 248R, 26I / 90L / 248R, 26I / 246N / 248R / 362S / 455L, 26I / 248R / 266V / 362S, 26I / 248R / 455L, 26Q / 90L / 246N / 248R / 261A, 26Q / 90L / 246N / 248R / 362S / 455L, 26Q / 90L / 246N / 248R / 455L, 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, 26Q / 248V / 266V / 362S, 26Q / 248V / 362S / 455L, 26Q / 362S, 26Q / 362S / 455L, 58N / 197L / 249R / 407N / 410S, 62W / 249R, 90L / 246N / 248R, 90L / 246N / 248R / 261A / 266V / 362S / 455L, 90L / 246N / 248R / 266V / 362S / 455L, 246N / 248R, 246N / 248R / 362S, 246N / 266V / 455L, 248R, 248R / 455L, 248V / 266V / 362S, 248V / 362S / 455L, and 362S. 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: 2514 and having one or more residue differences selected from the following compared to SEQ ID NO: 2514: R26G / N90L / E94K / L248R / S261A / K266V / T362S / V455L, R26G / N90L / E246N,R26G / N90L / E246N / L248R, R26G / N90L / E246N / K266V / T362S, R26G / N90L / L248V / V455L, R26G / N90L / L248V / V455L / Y459H, R26G / N90L / K266V, R26G / N90L / T362S / V455L, R26G / E246N, R26G / E246N / L248R / T362S, R26G / E246N / L248V / V455L, R26G / L248R, R26G / L248V / S261A / K266V, R26I / N90L / E246N / L248R, R26I / N90L / L248R, R26I / E246N / L248R / T362S / V455L, R26I / L248R / K266V / T362S, R26I / L248R / V455L, R26Q / N90L / E246N / L248R / S261A, R26Q / N90L / E246N / L248R / T362S / V455L, R26Q / N90L / E246N / L248R / V455L, R26Q / N90L / E246N / L248V / T362S / V455L, R26Q / N90L / E246N / T362S, R26Q / N90L / E246N / V455L, R26Q / N90L / L248R / K266V / V455L, R26Q / T173I / L248R, R26Q / E246N / L248V / T362S, R26Q / L248R, R26Q / L248R / S261A / K266V / T362S / V455L, R26Q / L248R / T362S / V455L, R26Q / L248R / V455L, R26Q / L248V, R26Q / L248V / K266V / 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 / T362S / V455L, E246N / L248R, E246N / L248R / T362S, E246N / K266V / V455L, L248R, L248R / V455L, L248V / K266V / T362S, L248V / T362S / V455L, and T362S.,

[0193] 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 selected from the following compared to SEQ ID NO:2524: 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, 30 / 261 / 266 / 353 / 468, 30 / 266, 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 / 353 / 468 and 266 / 468. 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 selected from the following compared to SEQ ID NO:2524: 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 / 266V / 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 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 selected from the following compared to SEQ ID NO: 2524: G9T / R16S / F62W / P157L / N246E / E249R / S362T, G11S / T58N / L227M / N246E, S12A / R16S / P158A / N246E / R248L / E249R, M30E / A189R / K266V, M30G / S261A / K266V / Q353R / Q468S, M30G / K266V, M30G / K266V / Q353R, M30T / 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, A189R, A189R / S261A, A189R / Q353R, N246E / E249R, S261A / Q353R, K266V / C307S / Q353R / Q468S, K266V / Q353R / Q468S, and K266V / Q468S.

[0194] 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 selected from the following compared to SEQ ID NO:2524: 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 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 selected from the following compared to SEQ ID NO:2524: 47A / 326R, 169M, 191V / 413V, 200V, 292N, 304V / 329R, 327M / 406T, 329R, 340I, 340V, 353H / 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 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 selected from the following compared to SEQ ID NO: 2524: V47A / L326R, Q169M, N191V / A413V, T200V, S292N, A304V / Q329R, L327M / G406T, Q329R, L340I, L340V, Q353H / Y459V, 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.

[0195] 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 selected from the following compared to SEQ ID NO:2524: 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 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 selected from the following compared to SEQ ID NO:2524: 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 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 selected from the following compared to SEQ ID NO: 2524: N175L, Q179L, A189V, T200N, E203D, E203M, S292N, S293Q, S325W, L340V, Q373C, Q373G, T379L, T379M, K402E, 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.

[0196] 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 selected from the following compared to SEQ ID NO:2638: 11 / 30 / 79 / 189 / 480, 30 / 58 / 79 / 189 / 307 / 480, 79 / 189 / 307 / 410, and 79 / 307. 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 selected from the following compared to SEQ ID NO:2638: 11S / 30G / 79T / 189R / 480E, 30G / 58N / 79T / 189R / 307L / 480E, 79T / 189R / 307L / 410E, and 79T / 307L. 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 selected from the following compared to SEQ ID NO:2638: G11S / M30G / S79T / A189R / R480E, M30G / T58N / S79T / A189R / C307L / R480E, S79T / A189R / C307L / V410E, and S79T / C307L.

[0197] 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: 2804 and having one or more residue differences selected from the following compared to SEQ ID NO: 2804: 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 / 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, 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, 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, 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 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:2804 and having one or more residue differences selected from the following compared to SEQ ID NO:2804: 169M / 304V / 340I / 429R / 506P, 169M / 304V / 340I / 504K, 169M / 304V / 340V / 402G / 427L, 169M / 304V / 340V / 427L / 429R / 504K, 169M / 304V / 402G / 403S / 427L / 506P, 169M / 304V / 403S / 427L / 504K, 169M / 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 / 427L / 429R / 504K, 169M / 402G / 504K, 169M / 402G / 504K / 506P, 169M / 403A / 427L / 506P, 266V / 327M / 329R / 404S / 410V, 292N / 327M / 329R / 468S, 304V, 304V / 340I / 402G, 304V / 340I / 402G / 403A / 427L / 429R / 504K / 506P, 304V / 340I / 402G / 403S / 506P, 304V / 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 / 403P / 427M / 504K, 304V / 402G / 403P / 506P, 304V / 402G / 403S, 304V / 403S / 504K, 304V / 504K, 304V / 504K / 506P, 327M, 327M / 329R, 327M / 329R / 379M / 404D / 406T / 410V, 327M / 329R / 404D / 410V, 327M / 329R / 465E / 484A, 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 / 504K / 506P, 402G / 403S / 427M / 504K / 506P, 402G / 403S / 429R / 504K, 402G / 427L / 504K, 402G / 504K, 403S / 427L, 427L, 427M, 484A, 504K, 504K / 506P, and 506P. 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:2804 and having one or more residue differences selected from the following compared to SEQ ID NO:2804: Q169M / A304V / L340I / D429R / K506P, Q169M / A304V / L340I / F504K, Q169M / A304V / L340V / K402G / K427L, 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 / K506PL340I / 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, 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.

[0198] 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 selected from the following compared to SEQ ID NO: 2812: 30, 30 / 179 / 200 / 373 / 403, 30 / 179 / 373 / 379, 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 / 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 / 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 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 selected from the following compared to SEQ ID NO:2812: 30G, 30G / 179A / 200N / 373G / 403E, 30G / 179A / 373G / 379M, 30G / 184S / 246E / 325W / 379M / 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 / 461S, 30G / 246E / 427L / 459I / 461S, 30G / 325W / 327M, 30G / 325W / 327M / 379M / 404D / 429R / 495S, 30G / 327M / 404D / 504S, 30G / 329R / 379M, 30G / 373G, 30G / 373G / 403L, 30G / 379M / 429R / 459I / 461S, 30G / 379M / 459I / 461S / 504Q, 30G / 403E / 441K / 460P, 200N / 373G / 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 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 as compared to SEQ ID NO: 2812 selected from: 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 / Q373G, M30G / T200N / Q373G / T379M, M30G / N246E / S325W / L327M / Q329R / P404D / G461S, M30G / N246E / S325W / T379M / P404D / K427L / D429R / G461S, M30G / N246E / K427L / Y459I / G461S, M30G / S325W / L327M, M30G / S325W / L327M / T379M / P404D / D429R / A495S, M30G / L327M / P404D / F504S, M30G / Q329R / T379M, M30G / Q373G, M30G / Q373G / S403L, M30G / T379M / D429R / Y459I / G461S, M30G / 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.

[0199] 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 selected from the following compared to SEQ ID NO: 2812: 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 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 selected from the following compared to SEQ ID NO: 2812: 198E, 231S, 233R, 248K, 248T, 253I, 264E, 264L, 264T, 264V, 266R, 278M, 326R, 367D, 370D, 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 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 selected from the following compared to SEQ ID NO: 2812: 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.

[0200] 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 selected from the following compared to SEQ ID NO: 2812: 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 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 selected from the following compared to SEQ ID NO: 2812: 186D, 188I, 198E, 231S, 233A, 235R, 243L, 243R, 243S, 243T, 248S, 253I, 264A, 264T, 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 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 selected from the following compared to SEQ ID NO: 2812: 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.

[0201] 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:2956 and having one or more residue differences selected from the following compared to SEQ ID NO:2956: 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 / 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 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: 2956 and having one or more residue differences selected from the following compared to SEQ ID NO: 2956: 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 / 484L, 142V / 150P / 158S / 177R / 427L / 445N, 142V / 150P / 177R / 404D, 142V / 153V / 177R / 441V / 444S, 142V / 177R / 373R, 142V / 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 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: 2956 and having one or more residue differences selected from the following compared to SEQ ID NO: 2956: G140V / M142V / E153V / H177R / K427L / E434N / M441V / A444S / G461S / K502G, G140V / M142V / E153V / K427L / T484L, G140V / M142V / H177R, G140V / M142V / H177R / M441V, G140V / M142V / G365A / Q373R / P404D / K427L / T484L, G140V / M142V / Q373R / K427L / T484L, G140V / P148T / K161H / H177R / P404D, G140V / E150P / E153V / G365A / Q373R / K427L / T484L, G140V / E150P / H177R / P404D / P436S / M441V / T484L / K502G, G140V / K161H / H177R / P404D / K427L / T484L, G140V / H177R / P404D, G140V / H177R / P404D / T484L, M142V / E150P / P158S / H177R / K427L / K445N, M142V / E150P / H177R / P404D, M142V / E153V / H177R / M441V / A444S, M142V / H177R / Q373R, M142V / 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.

[0202] 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 selected from the following compared to SEQ ID NO: 3174: 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 and 414 / 441 / 520. In some embodiments, the engineered TdT polypeptide comprises an amino acid sequence having at least 60%, 70%, 80%, 85%,An amino acid sequence having 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity and having one or more residue differences selected from the following compared to SEQ ID NO: 3174: 186D / 188I / 248K / 253I / 365A / 366V / 444H / 445N, 186D / 231S / 248K / 253I / 484L, 186D / 231S / 248K / 365A / 366V / 368R / 484L, 186D / 231S / 248K / 366V / 368R / 444H, 186D / 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 / 243S / 396S / 414E / 431R / 433V / 441M / 499M, 198E / 243S / 396S / 414E / 433R / 437R / 441M / 515E, 198E / 264A / 266T / 414E, 198E / 264A / 396S / 414E / 433V / 441M / 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, 243S / 264A / 515E / 520D, 243S / 396S / 414E / 433V / 441M / 515E / 520D, 243S / 414E / 433S / 437R / 441M, 243S / 414E / 437R / 441M / 515E, 248K / 442K / 444A / 445N / 484L, 264A / 266T / 396S / 414E / 433R / 441M / 515E, 264A / 266T / 414E / 441M / 499M / 501R / 515E / 520D, 264A / 414E, 264A / 414E / 441M, 264A / 433S / 441M, 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 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 selected from the following compared to SEQ ID NO: 3174: 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 / R248K / T484L, E186D / G231S / K368R / K416N / V441M / G442K / S444A / T484L / D485E, E186D / G231S / V441M / S444H / K445N, E186D / T484L / D485E, L188I / G231S / R248K / V253I / G365A / V441M / G442K / S444A / K445N / T484L, D198E / E243S / R264A / S431R / V441M, D198E / E243S / T396S / G414E / S431R / W433V / V441M / L499M, D198E / E243S / T396S / G414E / W433R / T437R / V441M / A515E, D198E / R264A / 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 / K266T / G414E / V441M / L499M / K501R / A515E / 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.

[0203] 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 selected from the following compared to SEQ ID NO: 3174: 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 and 493. 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 the same compared to SEQ IDAn amino acid sequence having one or more residue differences selected from the following compared to NO:3174: 122I / 473K, 189L, 190L, 190M, 190R, 193S, 193T, 194C, 194L, 194S, 194W, 196G, 196L, 196M, 196N, 196T, 263M, 263R, 264C, 266F, 266I, 266R, 266T, 266V, 266Y, 267A, 267C, 267H, 267V, 267Y, 268C, 268I, 268L, 268T, 269W, 273C, 273D, 273E, 273E / 501N, 273F, 273G, 273I, 273L, 273Y, 274A, 274G, 274I, 274V, 278F, 278H, 278M, 278R, 278V, 279Y, 281K, 281L, 297C, 297L, 297M, 297R, 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 comprises 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 identical to SEQ IDAn amino acid sequence having one or more residue differences selected from the following compared to NO:3174: M122I / R473K, R189L, E190L, E190M, 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, V273Y, K274A, K274G, K274I, K274V, K278F, K278H, K278M, K278R, K278V, W279Y, R281K, R281L, K297C, K297L, K297M, K297R, K297T, K297V, L298I, L298M, T299A, T299L, T299M, T299N, T299Y, P300H, M301S, M301T, 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, E394F, E394G, E394M, E394V, E394Y, K407L, K407M, K407N, K407R, K407W, A408G, A408I, A408L, A408M, A408T, A408V, E410F, E410G, 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.

[0204] 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 selected from the following compared to SEQ ID NO: 3174: 196, 263, 266, 268, 273, 281, 394, 416, 454, 468, 473, 477, and 493. 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 selected from the following compared to SEQ ID NO: 3174: 196G, 263R, 266I, 266T, 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 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 selected from the following compared to SEQ ID NO: 3174: 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.

[0205] 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 selected from the following compared to SEQ ID NO: 1830: 194, 196, 266, 267, 268, 269, 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. 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 selected from the following compared to SEQ ID NO: 1830: 194L, 194W, 196G, 196M, 196N, 266R, 266T, 266V, 267A, 268L, 268T, 269W, 273D, 273E, 273E / 501N, 273F, 273G, 273I, 273L, 273Y, 274A, 274G, 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 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 selected from the following compared to SEQ ID NO: 1830: F194L, 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, Q302S, Y309F, Y309L, Y309M, Y309W, L312V, F347I, L359G, Y390C, Y390V, R392V, E394Y, K407D, K407L, K407M, K407N, K407R, A408I, A413S, K416G, K416S, L454M, L454V, Q468T, R473G, R477G, L479V and N493V.

[0206] 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: 3222 and having one or more residue differences selected from the following compared to SEQ ID NO: 3222: 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 / 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, 231 / 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 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%,An amino acid sequence having 99% or more sequence identity and having one or more residue differences selected from the following compared to SEQ ID NO: 3222: 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 / 485E, 186D / 243S / 484L / 520D, 186D / 248K, 186D / 365A, 186D / 365A / 366V / 368R / 394Y / 499M, 186D / 365A / 366V / 394Y / 485E, 186D / 366V / 368R / 394Y / 396T / 484L, 186D / 366V / 368R / 394Y / 396T / 484L / 485E, 186D / 394Y / 396T / 485E, 194L / 198E / 243S / 366V / 368R / 499M, 194L / 515A / 520D, 198E / 231S / 243S / 248K / 485E, 198E / 243S / 248K / 365A / 394Y / 501R, 198E / 248K / 394Y / 396T / 484L / 485E / 499M, 198E / 394Y / 396T / 484L / 485E / 499M, 198E / 394Y / 396T / 499M / 515A / 520D, 198E / 394Y / 499M / 501R, 231S / 365A / 368R / 394Y / 499M / 520D, 231S / 368R / 394Y, 231S / 484L / 485E / 499M / 501R, 243S / 248K / 394Y / 396T / 484L / 485E, 243S / 484L, 243S / 484L / 485E / 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 comprises at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%An amino acid sequence having 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity and having one or more residue differences selected from the following compared to SEQ ID NO: 3222: 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 / T484L / D485E, E186D / E243S / T484L / E520D, E186D / R248K, E186D / G365A, E186D / G365A / A366V / K368R / E394Y / L499M, E186D / G365A / A366V / E394Y / D485E, E186D / A366V / K368R / E394Y / S396T / T484L, E186D / A366V / K368R / E394Y / S396T / T484L / D485E, E186D / E394Y / S396T / D485E, F194L / D198E / E243S / A366V / K368R / L499M, F194L / E515A / E520D, D198E / G231S / E243S / R248K / D485E, D198E / E243S / R248K / G365A / E394Y / K501R, D198E / R248K / E394Y / S396T / T484L / D485E / L499M, D198E / E394Y / S396T / T484L / D485E / L499M, D198E / E394Y / S396T / L499M / E515A / E520D, D198E / E394Y / L499M / K501R, G231S / G365A / K368R / E394Y / L499M / E520D, G231S / K368R / E394Y, G231S / T484L / D485E / L499M / K501R, E243S / R248K / E394Y / S396T / T484L / D485E, E243S / T484L, E243S / T484L / D485E / L499M,R248K / G365A / A366V / K368R / E394Y / T484L / E520D, R248K / E394Y / T484L, G365A / A366V / K368R / E394Y, G365A / K368R / E394Y / S396T / E520D, E394Y / S396T, and E394Y / L499M.

[0207] 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 with the reference sequence SEQ ID NO: 3670 and having one or more residue differences selected from the following compared to SEQ ID NO: 3670: 273, 273 / 309 / 493 / 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 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 with the reference sequence SEQ ID NO: 3670 and having one or more residue differences selected from the following compared to SEQ ID NO: 3670: 273I, 273I / 309M / 493V / 499L, 273I / 493V / 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 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 with the reference sequence SEQ ID NO: 3670 and having one or more residue differences selected from the following compared to SEQ ID NO: 3670: V273I, V273I / Y309M / N493V / M499L, V273I / N493V / M499L, V273I / M499L, K274A / T299N / A408I / K416S, K274A / A408I / K416S, K274I / K416S, E288V / T299N / K416S, L298M / T299N / K416S, Y309M, Y309M / M499L, K416S and N493V / M499L.

[0208] 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 selected from the following compared to SEQ ID NO: 3670: 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 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 selected from the following compared to SEQ ID NO: 3670: 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 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 selected from the following compared to SEQ ID NO: 3670: C168S, D198S, S267M, S267R, M301G, M301Q, M301S, L307A, L307G, L307S, Y308A, Y308A / 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.

[0209] 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 selected from the following compared to SEQ ID NO: 3670: 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 selected from the following compared to SEQ ID NO: 3670: 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 selected from the following compared to SEQ ID NO: 3670: K303H / S396A, Y308L, R473A, R473M, R473S, and N493V / M499L.

[0210] 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 selected from the following compared to SEQ ID NO: 3670: 273, 273 / 309 / 413 / 499, 273 / 309 / 493 / 499, 273 / 493, 273 / 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 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 selected from the following compared to SEQ ID NO: 3670: 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 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 selected from the following compared to SEQ ID NO: 3670: V273E / N493V, V273I, V273I / Y309M / A413S / M499L, V273I / Y309M / N493V / M499L, V273I / N493V / M499L, V273I / M499L, V273S / Y309M / 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.

[0211] 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: 3674 and having one or more residue differences selected from the following compared to SEQ ID NO: 3674: 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 / 394 / 454, 196 / 394 / 454 / 480, 196 / 394 / 480, 196 / 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.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: 3674 and having one or more residue differences selected from the following compared to SEQ ID NO: 3674: 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 / 394F / 454M / 480K, 194L / 196G / 454M, 194L / 390C, 194L / 394F, 194L / 394F / 454M, 194L / 394F / 454M / 480K, 196G, 196G / 390C, 196G / 390C / 394F, 196G / 390C / 394F / 454M, 196G / 390C / 394F / 454M / 480K, 196G / 390C / 394F / 480K, 196G / 390C / 454M, 196G / 394F, 196G / 394F / 454M, 196G / 394F / 454M / 480K, 196G / 394F / 480K, 196G / 454M, 297R / 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 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: 3674 and having one or more residue differences selected from the following compared to SEQ ID NO: 3674: 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.

[0212] 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: 3796 and having one or more residue differences selected from the following compared to SEQ ID NO: 3796: 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, 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 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: 3796 and having one or more residue differences selected from the following compared to SEQ ID NO: 3796: 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, 208V / 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 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: 3796 and having one or more residue differences selected from the following compared to SEQ ID NO: 3796: D198P, D198P / S267Q / 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 / H494R / M499L, S267R / R451K, Y308A, Y308V, R314A / V328T / R451K / M499L, R314A / R451K, V328T / D409L / R451K, V328T / R451K, D409L, D409L / F475V / H494V, R451K, R451K / N493V / H494V, R451K / N493V / M499L, R451K / H494V and F475V.

[0213] 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: 3870 and having one or more residue differences at a position or set of positions selected from the following compared to SEQ ID NO: 3870: 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, 483, 484, 490, 491, 493, 494, 495, 521, 507, 508, 509 and 522.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: 3870 and having one or more residue differences selected from the following compared to SEQ ID NO: 3870: P165A, P165C, P165R, P165T, Q169M, Q169T, R171G, T173S, N175L, N175M, N175V, N176V, Q179K, N175I, Q179M, Q179P, Q179S, Q179T, Q179V, D183C, D183W, I187R, N191F, N191M, N191Q, N191V, Y192A, Y192M, Y192T, K195R, N197C, N197E, N197F, N197R, N197T, D199G, D199R, D199S, D199V, D199W, T200K, T200M, E203F, E203G, E203M, E203V, F204I, F204V, V210M, E257P, E257S, Y259F, R267S, R291M, S293C, S293T, T295A, T295G, T295N, T295S, T295V, M301G, M301W, A319E, W325G, W325L, I340S, V341C, E342Q, L374V, L387I, W398V, E399S, S403K, S403L, S403R, S403T, S403V, S403W, P404G, P404M, G406L, G406R, G406S, G406T, 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.

[0214] 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: 3918 and having one or more residue differences at a position or set of positions selected from the following compared to SEQ ID NO: 3918: 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 / 484, 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 / 512, 484, 485, 493, 499, 500, 501, 504, 512, 515 and 516. 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: 3918 and having one or more residue differences selected from the following compared to SEQ ID NO: 3918: 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 / L484R, F335V / W481R / N493T, F335V / W481R / E512R, D336Q, D338E, D338Q, A339R, E367R, E367S, E367S / Y459V / T500S, E367T, Q370E, Q373G, Q376S, N380G, K384S, C390A, W395A, W395E, W395N, W395N / G402K, W395N / W481R / E512R, W395N / L484R, W395T, W395T / G402K / W481R / L484R, W395T / W481R, D400W, D400W / Y459R / T500S / R501P, D400W / Y459V, D400W / Y459V / M499R / T500S / F504Q, D400W / Y459V / R501P / F504G, D400W / R501P, D400W / F504Q, G402K, G402K / W481D, G402K / W481D / L484R / E512R, G402K / W481D / N493T, G402K / W481R / L484R, G402K / E512R, G402R, G402R / 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.

[0215] 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: 4266 and having one or more residue differences at a position or set of positions selected from the following compared to SEQ ID NO: 4266: 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 / 366 / 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 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: 4266 and having one or more residue differences selected from the following compared to SEQ ID NO: 4266: L95M / R428V / W480D, S163Q / N190M, S163Q / F203I / E366S,S163Q / Q328L / P363G / W480D, S163Q / P363G / W480D / E485Q, T172S / N174M / Q178K / V340C, Q178K, R188K, N190M / E202R / F203I / P363G / E366S / W480D / L483R / E485Q, N190M / E202R / F203I / W480D, N190M / W480D / E485Q, E192G, E192S / M498P / T499S / Q503G, E192T, E192V, E192Y, E192Y / M498P / T499S / Q503G, E202R, E202R / F203I / Q328A / S362T / P363G / E366S / R428V / W480D / E485Q / M498P / T499S / Q503G, E202R / F203I / E485Q, F203I / Q328A / P363G / R428V / L483R, F203I / Q328A / R428V, F203I / Q328A / W480D / E485Q, F203I / S362T / E366S, F203I / M498P / T499S / Q503G, V272P, R280G / M498P / T499S / Q503G, R280H, K296G, K296P, L297F, P299E, P299K / M498P / T499S / Q503G, P299S, P299T, Q301G, Q301S / Q503G, Y308H / Q503G, Y308K, Y308Q, Y308R, Y308T, L311R, Q328A / E485Q, Q328L / R428V, Q328L / W480D / L483R, V343C, V343L, F346Q, F346W, G349Q, L358G, 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.,

[0216] 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: 4558 and having one or more residue differences at a position or set of positions selected from the following compared to SEQ ID NO: 4558: 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, 375 / 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.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: 4558 and having one or more residue differences selected from the following compared to SEQ ID NO: 4558: V94I / A365F / K367R, T172S / N174M / Q178K / S402L / 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 / S402L / 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 / G400P / L483R, Q375G / L380V / L483R, K376H / L483R, N379T / L483R, C389A / W394E, W394T, G401K, G401K / S402L / P403G / K507N, S402L / P403G, G405L / L483R and L483R.

[0217] 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: 4442 and having one or more residue differences at a position or set of positions selected from the following compared to SEQ ID NO: 4442: 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, 432, 451, 455, 460, 461, 476 / 480, 480, 480 / 481, 493, 494 and 522.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: 4442 and having one or more residue differences selected from the following compared to SEQ ID NO: 4442: 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.

[0218] 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: 4654 and having one or more residue differences at a position or set of positions selected from the following compared to SEQ ID NO: 4654: 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. 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: 4654 and having one or more residue differences selected from the following compared to SEQ ID NO: 4654: N190M, N190M / D197N / Y308Q, N190M / Y308Q / L380V / G405L, N190M / Q375G, N190M / Q375G / L380V, N190M / L380V / G405L, 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.

[0219] 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: 4850 and having one or more residue differences at a position or set of positions selected from: 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 / 300 / 395, 298 / 300 / 481, 300, 300 / 392 / 395, 319, 322, 392, 421 and 492. 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: 4850 and having one or more residue differences selected from: R189K, R189K / E193V / A207V / L307T / Q353R, E190R / D322N, E193V, E193V / L307T, S261A / D322N / L421V, K297P / L298F / P300T / R392S, K297P / P300E / V328A, K297P / P300S, L298F / P300E / V328A / W395L, L298F / P300E / R392S / W395L, L298F / P300E / W395L, L298F / P300E / D481W, L298F / P300S / V328A, L298F / P300S / R392S / W395L, L298F / P300S / R392S / W395L / D492E, L298F / P300T / L360V, P300E, P300E / R392S / W395L, P300T, A319P, D322N, R392S, L421V and D492E.

[0220] 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: 4856 and having one or more residue differences at a position or set of positions selected from the following compared to SEQ ID NO: 4856: 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 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 selected from the following compared to SEQ ID NO: 4856: S10N / A413S, Q260R, V268T, Q302A / T307L, Q302S / T307L, T317C, Q353R, Q353S, G354S, S362V, P364Q, R392G, R392H, R392K, L393R, L393V, 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.

[0221] 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: 4904 and having one or more residue differences at a position or set of positions selected from the following compared to SEQ ID NO: 4904: 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. 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: 4904 and having one or more residue differences selected from the following compared to SEQ ID NO: 4904: 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.

[0222] 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:5002 and having one or more residue differences at a position or set of positions selected from the following compared to SEQ ID NO:5002: 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 / 495, 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 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:5002 and having the same compared to SEQ IDAn amino acid sequence having one or more residue differences selected from the following compared to NO:5002: 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 / L393V / 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, T300P / 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 / L393V / E394V / R477V / S495Q, R392K / E394A / W395Y, R392K / E394V, R392K / E394V / R477V / S495Q, R392K / E394V / S495Q, L393V / E394A, L393V / 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.

[0223] 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: 5028 and having one or more residue differences at a position or set of positions selected from the following compared to SEQ ID NO: 5028: 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, 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 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: 5028 and having one or more residue differences selected from the following compared to SEQ ID NO: 5028: V82I / F194L / D198G / A313S, F194L, F194L / D198Q, F194L / D198Q / I208V / A313S, F194L / D198V, F194L / D198V / Y309R, F194L / D198V / G411L, F194L / I208V / G411L, F194L / Y309R, F194W, F194W / D198Q, F194W / D198V, F194W / D198V / A313S, F194W / A313S, F194W / G411L, D198G, D198Q, D198Q / I208S / A313S / G411L, D198Q / I208V / Y309R / G411L, D198V / I208S, V273Q / K274G, V273S / K274W, K274G / R281Q / E526Y, K274M / E523T, K274V, K274V / L359V / E526Y, K274W, Y309R, Y309R / A313S / G411L, I324M / E526Y, G411L, M466V, M466V / E526Y, E523T and E526Y.

[0224] 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: 5192 and having one or more residue differences at a position or set of positions selected from the following compared to SEQ ID NO: 5192: 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. 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: 5192 and having one or more residue differences selected from the following compared to SEQ ID NO: 5192: 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, K383H, K383R, K383S, V394F, V394H, V394I, V394L, V394Q, V394S, V394W, V394Y, L403R, L403V, D409S, K430R, E485L, K508A, K508S and E520I / Y526E.

[0225] 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: 5246 and having one or more residue differences at a position or set of positions selected from the following compared to SEQ ID NO: 5246: 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 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: 5246 and having one or more residue differences selected from the following compared to SEQ ID NO: 5246: R177W / D198L / T200K, R177W / T200K / E203G, R177W / T200K / E203G / T295N, R177W / T200K / T295N / L326M, R180K, S184H / D198L / T200K / E203G / T295N, S184H / T200K / T295N / L326M, E190M / D198L / T200K / E203G, E190M / T200K / E203G / T295N / N380R, E190M / T200K / T295N, N197Q, D198L, D198L / T200K, D198L / T200K / E203G, D198L / T200K / E203G / T295N, T200K / L326M, T200K / N380R, E203G, E203G / N380R, E233L, E233R, A252R, T295N, D336C, P364A, G365H, G365R, E367S, L381H, L381Y, K384A, M441E, R459M, R459W and E485L.

[0226] 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: 2 or 660 and having one or more residue differences selected from the following compared to SEQ ID NO: 2 or 660: 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 / 266 / 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 / 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 / 485 / 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. In some embodiments, the engineered TdT polypeptide comprises at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% identity to the reference sequence SEQ ID NO:2 or 660An amino acid sequence having 99% or more sequence identity and having one or more residue differences selected from the following compared to SEQ ID NO: 2 or 660: 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 / 429R / 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 / 353Q / 362S / 379M / 392R / 394E / 395W / 397Q / 398W / 402G / 403S / 406G / 408A / 410E / 411G / 413A / 414E / 416S / 425D / 427L / 429R / 433R / 434N / 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 / 460G / 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 / 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 comprises at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% identity to the reference sequence SEQ ID NO:2 or 660An amino acid sequence having 99% or more sequence identity and having one or more residue differences selected from the following compared to SEQ ID NO: 2 or 660: R26Q / M30G / I38T / S79T / S81E / N90L / G92R / D94E / T101E / T108K / M137A / G140V / V141E / M142V / E153V / I155E / T160M / I163V / Q165P / H177R / A184S / A189R / F194L / R196G / C201A / T203E / M205R / C213S / V219R / G231S / L248R / R258W / K263H / L264A / K266T / S267R / M301W / A304V / C307L / D314R / K318R / S325W / W333A / L340I / T344V / F353Q / T362S / T379M / Y390C / D392R / R394E / E395W / R397Q / F398W / K402G / L403S / R406G / I408A / A410E / L411G / H413A / F414E / K416S / H425D / K427L / D429R / W433R / E434N / 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 / W333A / T344V / F353Q / D392R / R394E / E395W / R397Q / R406G / I408A / L411G / H413A / F414G / H425D / E441M / S442G / S446P / D460G / R461G / G468Q / E476R / R481W / 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。,

[0227] As those skilled in the art will appreciate, in some embodiments, one or a combination of the above-selected residue differences may be maintained (i.e., held constant) as a core feature in engineered TdT, and additional residue differences at other residue positions may be incorporated into the sequence to generate additional engineered TdT polypeptides with improved properties. It should thus be understood that for any engineered TdT comprising one or a subset of the residue differences described above, the present invention contemplates other engineered TdT comprising one or a subset of the residue differences, and additionally comprising one or more residue differences at other residue positions disclosed herein.

[0228] As described above, engineered TdT polypeptides are also capable of converting substrates (e.g., NTP-3'-O-RBG or natural or modified NTPs and oligonucleotide acceptor substrates) into products (e.g., oligonucleotide acceptor substrates with added nucleotide-3'-O-RBG). In some embodiments, the engineered TdT polypeptides are capable of converting substrate compounds into product compounds with an activity that is at least 1.2-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold or higher relative to the activity of the reference polypeptides 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.

[0229] In some embodiments, an engineered TdT that is capable of converting substrate compounds into product compounds with an activity that is at least 2-fold relative to 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 comprises an amino acid sequence selected from the even-numbered sequences of SEQ ID NO:4-1960, 2004-3920, 4048-5466 and 5476.

[0230] In some embodiments, the engineered TdT has an amino acid sequence comprising one or more residue differences compared to 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, which increases the soluble expression or isolated protein yield of the engineered TdT in a bacterial host cell, particularly in Escherichia coli (E. coli), compared to wild-type or engineered reference TdT, and which comprises an amino acid sequence selected from the even-numbered sequences of SEQ ID NO: 4-1960, 2004-3920, 4048-5466, and 5476.

[0231] In some embodiments, the engineered TdT has an amino acid sequence comprising one or more residue differences compared to 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, which increases the thermal stability of the engineered TdT compared to wild-type or engineered reference TdT, and which comprises an amino acid sequence selected from the even-numbered sequences of SEQ ID NO: 4-1960, 2004-3920, 4048-5466, and 5476.

[0232] In some embodiments, the engineered TdT has an amino acid sequence comprising one or more residue differences compared to 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, and increases the activity of the engineered TdT at high temperatures (such as but not limited to 40°C, 45°C, 50°C, 55°C, 60°C, or 65°C) compared to wild-type or engineered reference TdT, and which comprises an amino acid sequence selected from the even-numbered sequences of SEQ ID NO: 4 - 1960, 2004 - 3920, 4048 - 5466, and 5476.

[0233] In some embodiments, the engineered TdT has an amino acid sequence comprising one or more residue differences compared to 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, and decreases the byproduct formation of the engineered TdT compared to wild-type or engineered reference TdT, and which comprises an amino acid sequence selected from the even-numbered sequences of SEQ ID NO: 4 - 1960, 2004 - 3920, 4048 - 5466, and 5476.

[0234] In some embodiments, the engineered TdT has an amino acid sequence comprising one or more residue differences compared to 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, and that increases the specific activity of the engineered TdT for one or more NTP-3'-O-RBGs or natural or modified NTPs compared to wild-type or engineered reference TdT, and that comprises an amino acid sequence selected from the even-numbered sequences of SEQ ID NOs: 4-1960, 2004-3920, 4048-5466, and 5476.

[0235] In some embodiments, the engineered TdT has an amino acid sequence comprising one or more residue differences compared to 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, and that increases the specific activity of the engineered TdT for one or more oligonucleotide acceptor substrates compared to wild-type or engineered reference TdT, and that comprises an amino acid sequence selected from the even-numbered sequences of SEQ ID NOs: 4-1960, 2004-3920, 4048-5466, and 5476.

[0236] In some embodiments, the engineered TdT has one or more residue differences compared to 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, and when compared to the incorporation efficiency of wild-type or engineered reference TdT, the residue differences increase the incorporation efficiency in the extension of oligonucleotide acceptor substrates by more than 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% by adding NTP or NQP, and it has an amino acid sequence selected from the even-numbered sequences of SEQ ID NO: 4-1960, 2004-3920, 4048-5466, and 5476.

[0237] In some embodiments, engineered TdT with improved properties has 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 sequences 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 and having at least one substitution or set of substitutions at amino acid positions selected from: 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, 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, wherein the amino acid positions are numbered with reference to 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.

[0238] In some embodiments, engineered TdT with improved properties 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 with reference sequences 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 and has an amino acid sequence with at least one substituted amino acid at the following 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, 148, 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, 190, 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, 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, where the amino acid positions are referenced according to 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 numbering.

[0239] In some embodiments, engineered TdT with improved properties 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 with reference sequences 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 and at least one of the following substitutions or amino acid sequences of amino acid residues: 12A / F / N / Q / S / V, 13E / G / K / R / S, 14G / N / Q / Y, 15A / F / L / Q, 16S / V, 17A / G, 18D / R / Y, 20H / P / T, 21E, 22G / L, 23C / E / P / T, 24A / G / M / P, 25N, 26A / G / I / Q / S / T / W, 27D / F / R, 28N / R, 29G / I / P / R, 30E / G / L / T / V, 31G / S / V, 32E, 33A / C / G / K / P, 34D / K / R / S, 35E / G / H / W, 36G / K, 37A / F / G / S / T / V, 38L / T, 39A / 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 / Y415A / F / L / S / W, 416G / N / Q / S, 417G / V / W, 418I, 419A / G / H / L / M, 421F / I / M, 425D / 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, 445N, 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, 464Y, 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 / H, 525F / H / Q / R / S, 526L / Y or 528L, or any combination thereof, wherein the amino acid positions are numbered with reference to 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.,

[0240] In some embodiments, engineered TdT having improved properties has an amino acid sequence comprising an even-numbered sequence selected from SEQ ID NOs: 4-1960, 2004-3920, 4048-5466, and 5476. In some embodiments, engineered TdT having 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.

[0241] In some embodiments, engineered TdT comprises an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to one of the sequences 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 provided in the Examples.

[0242] In addition to the residue positions specified above, any engineered TdT polypeptide disclosed herein can also contain residue differences at other residue positions (i.e., residue positions other than those included herein) relative to 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. The residue differences at these other residue positions can provide additional variations in the amino acid sequence without adversely affecting the ability of the polypeptide to effect the conversion of substrate to product. Thus, in some embodiments, the sequence can also contain 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 1-11, 1-12, 1-14, 1-15, 1-16, 1-18, 1-20, 1-22, 1-24, 1-26, 1-30, 1-35, 1-40, 1-45, 1-50, 1-100, or 1-150 residue differences at amino acid residue positions other than the amino acid residue differences present in any one of the engineered TdT polypeptides having an even-numbered sequence within the ranges selected from SEQ ID NO: 4-1960, 2004-3920, 4048-5466, and 5476 compared to the reference sequence. 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, 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.The residue differences at these other positions can be conservative or non-conservative changes. In some embodiments, the residue differences can include conservative and non-conservative substitutions compared to the TdT polypeptides of 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.

[0243] In some embodiments, the invention also provides engineered polypeptides that retain the functional activity and / or improved properties of any of the engineered TdTs described herein and that comprise fragments of the engineered TdT polypeptides. Thus, in some embodiments, the invention provides polypeptide fragments that are capable of converting a substrate into a product under suitable reaction conditions, wherein the fragment comprises 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 invention (such as exemplary TdT polypeptides selected from the 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 contains deletions in any one of the TdT polypeptide sequences described herein (such as exemplary engineered polypeptides of the even-numbered sequences within the ranges of SEQ ID NOs: 4-1960, 2004-3920, 4048-5466, and 5476).

[0244] Thus, for each and every embodiment of the engineered TdT polypeptides of the present invention, the amino acid sequence can comprise a deletion of 1 or more amino acids, 2 or more amino acids, 3 or more amino acids, 4 or more amino acids, 5 or more amino acids, 6 or more amino acids, 8 or more amino acids, 10 or more amino acids, 15 or more amino acids, or 20 or more amino acids, up to 10% of the total number of amino acids of the TdT polypeptide, up to 20% of the total number of amino acids, or up to 30% of the total number of amino acids, wherein the relevant functional activity and / or improved properties of the engineered TdT described herein are maintained. In some embodiments, the deletion can comprise a deletion of 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 1-15, 1-20, 1-21, 1-22, 1-23, 1-24, 1-25, 1-30, 1-35, 1-40, 1-45 or 1-50 amino acid residues. In some embodiments, the number of deletions can 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 can include a 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.

[0245] In some embodiments, the engineered TdT polypeptides herein can have an amino acid sequence comprising an insertion as compared to any of the engineered TdT polypeptides described herein (such as the exemplary engineered polypeptides of the even-numbered sequences within the ranges of SEQ ID NO: 4-1960, 2004-3920, 4048-5466 and 5476). Thus, for each and every embodiment of the TdT polypeptides of the present invention, the insertion can comprise 1 or more amino acids, 2 or more amino acids, 3 or more amino acids, 4 or more amino acids, 5 or more amino acids, 6 or more amino acids, 8 or more amino acids, 10 or more amino acids, 15 or more amino acids, 20 or more amino acids, 30 or more amino acids, 40 or more amino acids or 50 or more amino acids, wherein the relevant functional activity and / or improved properties of the engineered TdT described herein are maintained. The insertion can be inserted at the amino terminus or carboxyl terminus of the TdT polypeptide, or an internal portion.

[0246] In some embodiments, the engineered TdT herein can have a sequence comprising an even-numbered sequence selected from the ranges of SEQ ID NO: 4-1960, 2004-3920, 4048-5466, and 5476, and an amino acid sequence optionally having 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 has 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 1-15, 1-20, 1-21, 1-22, 1-23, 1-24, 1-25, 1-30, 1-35, 1-40, 1-45, 1-50, 1-75, 1-100, or 1-150 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 can be conservative substitutions or non-conservative substitutions.

[0247] In the above embodiments, suitable reaction conditions for the engineered polypeptides are provided 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, 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, as described in the examples herein.

[0248] In some embodiments, the polypeptides of the invention are fusion polypeptides in which the engineered polypeptide is fused to other polypeptides, such other polypeptides being, for example but not limited to, antibody tags (e.g., myc epitope), purification sequences (e.g., His tag for binding to metal), cell localization signals (e.g., secretion signal), and polypeptides having enzymatic activity. Thus, the engineered polypeptides described herein can be used with or without fusion to other polypeptides.

[0249] In one embodiment of the engineered TdT polypeptide of the invention, the polypeptide further comprises an N-terminal truncation of 1 to 156 amino acids relative to the polypeptide sequence of any of the even-numbered sequences listed in SEQ ID NOs: 4-1960, 2004-3920, 4048-5466, and 5476. For example, in Examples 81 and 82, a 156-amino acid truncated form of the TdT variant polypeptide SEQ ID NO: 5028 was prepared and shown to have TdT activity.

[0250] In some embodiments, the engineered TdT polypeptide of the invention can be fused to another second polypeptide, such as a polypeptide having a different enzymatic activity. In some embodiments, the 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 the N-terminal and C-terminal hexahistidine-tagged form of the wild-type (WT) inorganic pyrophosphatase (IPP) polypeptide (e.g., the polypeptide of SEQ ID NO: 3942 or 3944) can be fused to the gene encoding the TdT variant polypeptide. Generally, the polypeptides (e.g., IPP and TdT) are fused via a polypeptide linker (e.g., GSGGTG linker) introduced between the genes encoding the polypeptides in the construct. Such fusion proteins can be constructed using well-established techniques (e.g., Gibson assembly cloning) and expressed in Escherichia coli (e.g., a strain 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 shown in Examples 81 and 82. Although the examples show the fusion of a particular engineered TdT polypeptide of the invention to a second polypeptide having IPP activity, it is contemplated that in any embodiment, the engineered TdT polypeptides of the even-numbered sequences listed in SEQ ID NOs: 4-1960, 2004-3920, 4048-5466, and 5476 can be used in such fusions to a second polypeptide.

[0251] It should be 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 can wholly or partially contain naturally occurring and / or synthetic non-coded amino acids. Some common non-coded amino acids that the polypeptides described herein can contain include, but are not limited to: D-stereoisomers of genetically encoded amino acids; 2,3-diaminopropionic acid (Dpr); α-aminoisobutyric acid (Aib); ε-aminohexanoic acid (Aha); δ-aminovaleric acid (Ava); N-methylglycine or sarcosine (MeGly or Sar); ornithine (Orn); citrulline (Cit); tert-butylalanine (Bua); tert-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 (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); pyridin-2-ylalanine (2pAla); pyridin-3-ylalanine (3pAla); pyridin-4-ylalanine (4pAla); naphthalen-1-ylalanine (1nAla); naphthalen-2-ylalanine (2nAla); thiazolylalanine (taAla); benzothienylalanine (bAla); thienylalanine (tAla); furylalanine (fAla); homophenylalanine (hPhe); homotyrosine (hTyr); homotryptophan (hTrp); pentafluorophenylalanine (5ff); styrylkalanine (sAla); anthracenylalanine (aAla); 3,3-diphenylalanine (Dfa); 3-amino-5-phenylvaleric 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); homolysine (hAsp); homoglutamic 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); homolysine (hAla); norvaline (nVal); homolysine (hLeu), homolysine (hVal); homoisoleucine (hIle); homolysine (hArg); N-acetyllysine (AcLys); 2,4-diaminobutyric acid (Dbu); 2,3-diaminobutyric acid (Dab); N-methylvaline (MeVal); homocysteine (hCys); homoserine (hSer); hydroxyproline (Hyp) and homolysine (hPro). Additional non-coding amino acids that the polypeptides described herein may contain will be apparent to those skilled in the art (see, for example, the various amino acids provided in the following: Fasman,; CRC Practical Handbook ofBiochemistry and Molecular Biology, CRC Press, Boca Raton, FL, pp. 3-70

[1989] and references cited therein, which are incorporated herein by reference in their entirety). These amino acids can be in the L-configuration or D-configuration.

[0252] Those skilled in the art will recognize that amino acids or residues with side-chain protecting groups can also constitute the polypeptides described herein. Non-limiting examples of such protected amino acids (in this case belonging to the aromatic class) include (the protecting groups are listed in parentheses), but are not limited to: Arg(tos), Cys(methylbenzyl), Cys(nitropyridinesulfenyl), Glu(δ-benzyl ester), Gln(xanthenyl), Asn(N-δ-xanthenyl), His(bom), His(benzyl), His(tos), Lys(fmoc), Lys(tos), Ser(O-benzyl), Thr(O-benzyl) and Tyr(O-benzyl).

[0253] Conformationally constrained non-coding amino acids that can constitute 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; pipecolic acid; azetidine-3-carboxylic acid; homolysine (hPro) and 1-aminocyclopentane-3-carboxylic acid.

[0254] In some embodiments, the engineered polypeptide can be in various forms, such as an isolated article, as a substantially purified enzyme, a whole cell transformed with one or more genes encoding the enzyme, and / or a cell extract and / or lysate of such a cell. The enzyme can be freeze-dried, spray-dried, precipitated, or in the form of a crude paste, as further discussed below.

[0255] In some embodiments, the engineered polypeptide can be in the form of a biocatalytic composition. In some embodiments, the biocatalytic composition comprises (a) means for converting an NTP-3-O-RBG or a native or modified NTP substrate and an oligonucleotide acceptor compound into an oligonucleotide acceptor product extended by one nucleotide upon contact with TdT and (b) a suitable cofactor. Suitable cofactors can be cobalt, manganese, or any other suitable cofactor.

[0256] In some embodiments, the polypeptides described herein are provided in the form of a kit. The enzymes in the kit can be present individually or as more than one enzyme. The kit can also include reagents for performing the enzymatic reaction, substrates for assessing the activity of the enzyme, and reagents for detecting the product. The kit can also include a reagent dispenser and instructions for using the kit.

[0257] In some embodiments, the kit of the invention includes an array comprising more than one different TdT polypeptide at different addressable positions, wherein the different polypeptides are different variants of a reference sequence, and each of the variants has at least one different improved enzymatic property. In some embodiments, more than one polypeptide immobilized on a solid support is configured at multiple positions of the array, and the positions are addressable by automated delivery of reagents or by detection methods and / or instruments. The array can be used to test the conversion of various substrate compounds by the polypeptides. Such arrays comprising multiple engineered polypeptides and methods of using them are known in the art (see, for example, WO2009 / 008908A2).

[0258] Polynucleotides, expression vectors, and host cells encoding engineered terminal deoxynucleotidyl transferase

[0259] In another aspect, the invention provides polynucleotides encoding the engineered TdT polypeptides described herein. The polynucleotide can be operably linked to one or more heterologous regulatory sequences that control gene expression to create a recombinant polynucleotide capable of expressing the polypeptide. An expression construct containing the heterologous polynucleotide encoding engineered TdT is introduced into a suitable host cell to express the corresponding TdT polypeptide.

[0260] As will be apparent to the skilled artisan, the availability of protein sequences and knowledge of the codons corresponding to the various amino acids provide a description of all polynucleotides capable of encoding the subject polypeptides. The degeneracy of the genetic code, where the same amino acid is encoded by alternative or synonymous codons, allows for the generation of a vast number of nucleic acids, all of which encode improved TdT enzymes. Thus, given a particular amino acid sequence, one of ordinary skill in the art can prepare any number of different nucleic acids by simply altering the sequence of one or more codons in a manner that does not change the amino acid sequence of the protein. In this regard, the present invention specifically contemplates all and every possible variation of polynucleotides encoding the polypeptides described herein that can be prepared by selecting combinations based on the possible codon options, and for any polypeptide described herein, all such variations are considered to be specifically disclosed, including the amino acid sequences presented 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, 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 disclosed as the even numbered sequences within the ranges of SEQ ID NO:4 - 1960, 2004 - 3920, 4048 - 5466 and 5476 in the Sequence Listing incorporated herein by reference.

[0261] In various embodiments, codons are preferably selected to accommodate the host cell in which the protein is to be produced. For example, the preferred codons used in bacteria are used for gene expression in bacteria; the preferred codons used in yeast are used for expression in yeast; and the preferred codons used in mammals are used for expression in mammalian cells. In some embodiments, not all codons need to be replaced to optimize the codon usage of TdT because the native sequence will include the preferred codons and because the use of the preferred codons may not be required for all amino acid residues. Thus, the polynucleotide encoding the codon-optimized TdT enzyme may contain the preferred codons at about 40%, 50%, 60%, 70%, 80%, or greater than 90% of the codon positions in the full-length coding region.

[0262] In some embodiments, the polynucleotide comprises a codon-optimized nucleotide sequence encoding a TdT polypeptide amino acid sequence as set forth 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 comprising at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to a codon-optimized nucleic acid sequence encoding an even-numbered sequence in the range of SEQ ID NO: 4 - 1960, 2004 - 3920, 4048 - 5466, and 5476. In some embodiments, the polynucleotide has a nucleic acid sequence comprising at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to a codon-optimized nucleic acid sequence encoding an odd-numbered sequence in the range of SEQ ID NO: 3 - 1959, 2003 - 3919, 4047 - 5465, and 5475. In some embodiments, the codon-optimized sequences of the odd-numbered sequences in the range of SEQ ID NO: 3 - 1959, 2003 - 3919, 4047 - 5465, and 5475 enhance the expression of the encoded TdT, providing an article of manufacture of an enzyme capable of converting a substrate to a product.

[0263] In some embodiments, the polynucleotide sequence comprises at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with a reference sequence SEQ ID NO: 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, 4849, 4855, 4903, 5001, 5027, 5191 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.

[0264] In some embodiments, the polynucleotide sequence encodes 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 with 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.

[0265] 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.

[0266] In some embodiments, the polynucleotide is capable of hybridizing under highly stringent conditions to a reference sequence selected from the odd-numbered sequences of SEQ ID NO: 3-1959, 2003-3919, 4047-5465, and 5475 or its complement, and encodes TdT.

[0267] In some embodiments, as described above, the polynucleotide encodes an engineered TdT polypeptide having improved properties compared to 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, wherein the polypeptide comprises 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 a reference sequence selected from 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 and having one or more residue differences compared to 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, wherein the sequence is selected from the even-numbered sequences within the range of SEQ ID NO: 4 - 1960, 2004 - 3920, 4048 - 5466, and 5476. In some embodiments, the reference amino acid sequence is selected from the even-numbered sequences within the range of SEQ ID NO: 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, and 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, and 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, and 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, and 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, and 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, and 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, and 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, and 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, and 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, and in some other embodiments, the reference sequence is SEQ ID NO: 3870.

[0268] In some embodiments, the polynucleotide encodes a TdT polypeptide capable of converting one or more substrates into products with improved properties as compared to 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, wherein the polypeptide comprises 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 sequence identity to the reference sequences 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.

[0269] In some embodiments, the polynucleotide encoding the engineered TdT comprises a polynucleotide sequence selected from the odd-numbered sequences within the ranges of SEQ ID NO:3 - 1959, 2003 - 3919, 4047 - 5465, and 5475.

[0270] In some embodiments, the polynucleotide is capable of hybridizing under highly stringent conditions to a reference polynucleotide sequence of an odd-numbered sequence selected from within the ranges of SEQ ID NO: 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, the polynucleotide capable of hybridizing under highly stringent conditions encodes an amino acid sequence comprising at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to 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, and having a TdT with an amino acid sequence having one or more residue differences compared to 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, as described in the examples above and below.

[0271] In some embodiments, polynucleotides capable of hybridizing under highly stringent conditions encode engineered TdT polypeptides having improved properties and 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 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 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 to a reference polynucleotide encoding engineered TdT at the nucleotide level. In some embodiments, the reference polynucleotide sequence is selected from SEQ ID NO: 3-1959, 2003-3919, 4047-5465, and 5475.

[0272] In some embodiments, a polynucleotide capable of hybridizing under highly stringent conditions encodes an engineered TdT polypeptide having improved properties and 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 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 encodes a polypeptide as 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 to a reference polynucleotide encoding an engineered TdT at the nucleotide level. In some embodiments, the reference polynucleotide sequence is selected from SEQ ID NO: 3-1959, 2003-3919, 4047-5465 and 5475.

[0273] In some embodiments, an isolated polynucleotide encoding any of the engineered TdT polypeptides provided herein is manipulated in various ways to provide for the expression of the polypeptide. In some embodiments, the polynucleotide encoding the polypeptide is provided in an expression vector that contains one or more control sequences to regulate the expression of the polynucleotide and / or polypeptide. Depending on the expression vector, manipulation of the isolated polynucleotide prior to insertion of the polynucleotide into the vector may be desirable or necessary. Techniques for modifying polynucleotides and nucleic acid sequences using recombinant DNA methods are well known in the art.

[0274] In some embodiments, the control sequences include, among other sequences, a promoter, a leader sequence, a polyadenylation sequence, a propeptide sequence, a signal peptide sequence, and a transcription terminator. As is known in the art, suitable 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 obtained from the following: the Escherichia coli lac operon, the Streptomyces coelicolor agarase gene (dagA), the Bacillus subtilis levansucrase gene (sacB), the Bacillus licheniformis α-amylase gene (amyL), the Bacillus stearothermophilus maltogenic amylase gene (amyM), the Bacillus amyloliquefaciens α-amylase gene (amyQ), the Bacillus licheniformis penicillinase gene (penP), the Bacillus subtilis xylA and xylB genes, and the prokaryotic β-lactamase gene (see, e.g., Villa-Kamaroff et al., Proc. Natl. Acad. Sci. USA 75:3727-3731

[1978] ), as well as 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 following genes: Aspergillus oryzae TAKA amylase, Rhizomucor miehei aspartic proteinase, Aspergillus niger neutral α-amylase, Aspergillus niger acid-stable α-amylase, Aspergillus niger or Aspergillus awamori glucoamylase (glaA), Rhizomucor miehei lipase, Aspergillus oryzae alkaline protease, Aspergillus oryzae triose phosphate isomerase, Aspergillus nidulans acetamidase, and Fusarium oxysporum trypsin-like protease (see, e.g., WO 96 / 00787), as well as the NA2-tpi promoter (a hybrid of the promoters from the Aspergillus niger neutral α-amylase gene and the Aspergillus oryzae triose phosphate isomerase gene), and mutants, truncated, and hybrid promoters thereof.Exemplary yeast cell promoters can be from the genes: 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. Other useful promoters for yeast host cells are known in the art (see, e.g., Romanos et al., Yeast 8:423-488

[1992] ).

[0276] In some embodiments, the control sequence is a suitable transcription terminator sequence (a sequence recognized by the 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 functional 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: Aspergillus oryzae TAKA amylase, Aspergillus niger glucoamylase, Aspergillus nidulans anthranilate synthase, Aspergillus niger α-glucosidase, and Fusarium oxysporum trypsin-like protease. Exemplary terminators for yeast host cells can be obtained from the genes: Saccharomyces cerevisiae enolase, Saccharomyces cerevisiae cytochrome C (CYC1), and Saccharomyces cerevisiae glyceraldehyde-3-phosphate dehydrogenase. Other useful terminators for yeast host cells are known in the art (see, e.g., Romanos et al., supra).

[0277] In some embodiments, the control sequence is a suitable leader sequence (untranslated region of the 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 functional in the selected host cell can be used. Exemplary leader sequences for filamentous fungal host cells are obtained from the genes: Aspergillus oryzae TAKA amylase and Aspergillus nidulans triose phosphate isomerase. Suitable leader sequences for yeast host cells include, but are not limited to, those obtained from the genes: Saccharomyces...

Claims

1. An engineered terminal deoxynucleotidyl transferase comprising a polypeptide sequence having at least 85%, 90%, 91%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with a reference sequence selected from SEQ ID NO: 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, 5002, 5028, 5192, 5246 or a functional fragment thereof and at least one amino acid residue difference relative to the reference sequence.

2. The engineered terminal deoxynucleotidyl transferase according to claim 1, wherein the polypeptide sequence comprises residue differences at a set of amino acid positions selected from the following with respect to the reference sequence SEQ ID NO: 2: 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; 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 / 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 / 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；and 185 / 289 / 290 / 293.; 3. The engineered terminal deoxynucleotidyl transferase according to claim 1, wherein the polypeptide sequence comprises at least one substitution set including substitutions at amino acid positions selected from the following compared with reference sequence SEQ ID NO: 36: 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; 244 / 315 / 317 / 336 / 359 / 391 / 470 / 474 / 522 / 523; and 336 / 359 / 391 / 470 / 474 / 522 / 523; and / or any combination thereof.

4. The engineered terminal deoxynucleotidyl transferase according to claim 1, wherein the polypeptide sequence comprises at least one substitution or substitution set including substitutions at amino acid positions selected from the following compared with reference sequence SEQ ID NO: 8: 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 engineered terminal deoxynucleotidyl transferase according to claim 1, wherein the polypeptide sequence comprises at least one substitution or set of substitutions including substitutions at amino acid positions selected from the following compared to the reference sequence SEQ ID NO: 16: 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 / 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 engineered terminal deoxynucleotidyl transferase according to claim 1, wherein the polypeptide sequence comprises at least one substitution or set of substitutions including substitutions at amino acid positions selected from the following compared to the reference sequence SEQ ID NO: 24: 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, 27, 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. The engineered terminal deoxynucleotidyl transferase according to claim 1, wherein the polypeptide sequence comprises at least one substitution or set of substitutions including substitutions at amino acid positions selected from the following compared to the reference sequence SEQ ID NO: 268: 14 / 53 / 300, 14 / 53 / 419, 106 / 300 / 415 / 419 / 456, 140 and 300 / 395 / 419, and / or any combination thereof.

8. The engineered terminal deoxynucleotidyl transferase according to claim 1, wherein the polypeptide sequence comprises at least one substitution or set of substitutions including substitutions at amino acid positions selected from the following compared to the reference sequence SEQ ID NO: 648: 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 / 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 / 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, 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, and / or any combination thereof., 9. The engineered terminal deoxynucleotidyl transferase according to claim 1, wherein the polypeptide sequence comprises at least one substitution or set of substitutions comprising substitutions at amino acid positions selected from the following compared to the reference sequence SEQ ID NO: 660: 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, 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, 525, and / or any combination thereof.

10. The engineered terminal deoxynucleotidyl transferase according to claim 1, wherein the polypeptide sequence comprises at least one substitution or set of substitutions comprising substitutions at amino acid positions selected from the following compared to the reference sequence SEQ ID NO: 882: 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. The engineered terminal deoxynucleotidyl transferase according to claim 1, wherein the polypeptide sequence comprises at least one substitution or set of substitutions comprising substitutions at amino acid positions selected from the following compared to the reference sequence SEQ ID NO: 1100: 101 / 137 / 264 / 476 / 525 and 264, and / or any combination thereof.

12. The engineered terminal deoxynucleotidyl transferase according to claim 1, wherein the polypeptide sequence comprises at least one substitution or set of substitutions including substitutions at amino acid positions selected from the following compared to the reference sequence SEQ ID NO: 1336: 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, 203 / 353, 203 / 394, 203 / 394 / 395, 297 / 394, 314 / 394 / 395, 344 / 394 / 395, 353 and 394, and / or any combination thereof.

13. The engineered terminal deoxynucleotidyl transferase according to claim 1, wherein the polypeptide sequence comprises at least one substitution or set of substitutions including substitutions at amino acid positions selected from the following compared to the reference sequence SEQ ID NO: 1348: 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, 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 engineered terminal deoxynucleotidyl transferase according to claim 1, wherein the polypeptide sequence comprises at least one substitution or set of substitutions comprising substitutions at amino acid positions selected from the following compared to the reference sequence SEQ ID NO: 1596: 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, 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 / 326, 406 / 408, 406 / 408 / 442 / 446 and 406 / 408 / 490, and / or any combination thereof.

15. The engineered terminal deoxynucleotidyl transferase according to claim 1, wherein the polypeptide sequence comprises at least one substitution or set of substitutions including substitutions at amino acid positions selected from the following compared to the reference sequence SEQ ID NO: 1654: 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 / 219 / 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 / 205 / 208 / 219 / 307 / 353 / 371 / 376 / 413 / 414 / 441 / 488、326 / 353 / 371 / 376 / 414 and 441, and / or any combination thereof.

16. The engineered terminal deoxynucleotidyl transferase according to claim 1, wherein the polypeptide sequence comprises at least one substitution or set of substitutions comprising substitutions at amino acid positions selected from the following compared to the reference sequence SEQ ID NO: 1830: 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, 394, 407, 408, 413, 416, 454, 468, 473, 477, 479 and 493, and / or any combination thereof.

17. The engineered terminal deoxynucleotidyl transferase according to claim 1, wherein the polypeptide sequence comprises at least one substitution or set of substitutions including substitutions at amino acid positions selected from the following compared to the reference sequence SEQ ID NO: 1950: 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, and / or any combination thereof.

18. The engineered terminal deoxynucleotidyl transferase according to claim 1, wherein the polypeptide sequence comprises at least one substitution or set of substitutions including substitutions at amino acid positions selected from the following compared to the reference sequence SEQ ID NO: 2008: 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, 196, 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 / 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. The engineered terminal deoxynucleotidyl transferase according to claim 1, wherein the polypeptide sequence comprises at least one substitution or set of substitutions comprising substitutions at amino acid positions selected from the following compared to the reference sequence SEQ ID NO: 2254: 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 engineered terminal deoxynucleotidyl transferase according to claim 1, wherein the polypeptide sequence comprises at least one substitution or set of substitutions comprising substitutions at amino acid positions selected from the following compared to the reference sequence SEQ ID NO: 2514: 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 / 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 / 410, 62 / 249, 90 / 246 / 248, 90 / 246 / 248 / 261 / 266 / 362 / 455, 90 / 246 / 248 / 266 / 362 / 455, 246 / 248, 246 / 248 / 362, 246 / 266 / 455, 248, 248 / 266 / 362, 248 / 362 / 455, 248 / 455 and 362, and / or any combination thereof.

21. The engineered terminal deoxynucleotidyl transferase according to claim 1, wherein the polypeptide sequence comprises at least one substitution or set of substitutions comprising substitutions at amino acid positions selected from the following compared to the reference sequence SEQ ID NO: 2524: 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 / 266, 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 / 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. The engineered terminal deoxynucleotidyl transferase according to claim 1, wherein the polypeptide sequence comprises at least one substitution or set of substitutions comprising substitutions at amino acid positions selected from the following compared to the reference sequence SEQ ID NO: 2638: 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 engineered terminal deoxynucleotidyl transferase according to claim 1, wherein the polypeptide sequence comprises at least one substitution or set of substitutions including substitutions at amino acid positions selected from the following compared to the reference sequence SEQ ID NO: 2804: 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 / 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, 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, 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, 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, and / or any combination thereof.

24. The engineered terminal deoxynucleotidyl transferase according to claim 1, wherein the polypeptide sequence comprises at least one substitution or set of substitutions including substitutions at amino acid positions selected from the following compared to the reference sequence SEQ ID NO: 2812: 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 / 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 / 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 / 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 engineered terminal deoxynucleotidyl transferase according to claim 1, wherein the polypeptide sequence comprises at least one substitution or set of substitutions including substitutions at amino acid positions selected from the following compared to the reference sequence SEQ ID NO: 2956: 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 / 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, and / or any combination thereof.

26. The engineered terminal deoxynucleotidyl transferase according to claim 1, wherein the polypeptide sequence comprises at least one substitution or set of substitutions including substitutions at amino acid positions selected from the following compared to the reference sequence SEQ ID NO: 3174: 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, and 414 / 441 / 520, and / or any combination thereof.

27. The engineered terminal deoxynucleotidyl transferase according to claim 1, wherein the polypeptide sequence comprises at least one substitution or set of substitutions including substitutions at amino acid positions selected from the following compared to the reference sequence SEQ ID NO: 3222: 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 / 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, 231 / 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, and / or any combination thereof.

28. The engineered terminal deoxynucleotidyl transferase according to claim 1, wherein the polypeptide sequence comprises at least one substitution or set of substitutions including substitutions at amino acid positions selected from the following compared to the reference sequence SEQ ID NO: 3670: 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, 392, 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 engineered terminal deoxynucleotidyl transferase according to claim 1, wherein the polypeptide sequence comprises at least one substitution or set of substitutions including substitutions at amino acid positions selected from the following compared to the reference sequence SEQ ID NO: 3674: 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 / 394 / 454, 196 / 394 / 454 / 480, 196 / 394 / 480, 196 / 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 engineered terminal deoxynucleotidyl transferase according to claim 1, wherein the polypeptide sequence comprises at least one substitution or set of substitutions comprising substitutions at amino acid positions selected from the following compared to the reference sequence SEQ ID NO: 3796: 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, 314 / 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 engineered terminal deoxynucleotidyl transferase according to claim 1, wherein the polypeptide sequence comprises at least one substitution or set of substitutions including substitutions at amino acid positions selected from the following compared to the reference sequence SEQ ID NO: 2 or 660: 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, 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, 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 / 266 / 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 / 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 / 485 / 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 engineered terminal deoxynucleotidyl transferase according to claim 1, wherein the polypeptide sequence comprises at least one substitution or set of substitutions comprising substitutions at amino acid positions selected from the following compared to the reference sequence SEQ ID NO: 3870: 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, 483, 484, 490, 491, 493, 494, 495, 521, 507, 508, 509 and 522, and / or any combination thereof.

33. The engineered terminal deoxynucleotidyl transferase according to claim 1, wherein the polypeptide sequence comprises at least one substitution or set of substitutions including substitutions at amino acid positions selected from the following compared to the reference sequence SEQ ID NO: 3918: 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 / 484, 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 / 512, 484, 485, 493, 499, 500, 501, 504, 512, 515 and 516, and / or any combination thereof.

34. The engineered terminal deoxynucleotidyl transferase according to claim 1, wherein the polypeptide sequence comprises at least one substitution or set of substitutions including substitutions at amino acid positions selected from the following compared to the reference sequence SEQ ID NO: 4266: 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 / 366 / 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, and / or any combination thereof.

35. The engineered terminal deoxynucleotidyl transferase according to claim 1, wherein the polypeptide sequence comprises at least one substitution or set of substitutions including substitutions at amino acid positions selected from the following compared to the reference sequence SEQ ID NO: 4558: 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, 375 / 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.

36. The engineered terminal deoxynucleotidyl transferase according to claim 1, wherein the polypeptide sequence comprises at least one substitution or set of substitutions including substitutions at amino acid positions selected from the following compared to the reference sequence SEQ ID NO: 4442: 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, 432, 451, 455, 460, 461, 476 / 480, 480, 480 / 481, 493, 494 and 522, and / or any combination thereof.

37. The engineered terminal deoxynucleotidyl transferase according to claim 1, wherein the polypeptide sequence comprises at least one substitution or set of substitutions including substitutions at amino acid positions selected from the following compared to the reference sequence SEQ ID NO: 4654: 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.

38. The engineered terminal deoxynucleotidyl transferase according to claim 1, wherein the polypeptide sequence comprises at least one substitution or set of substitutions including substitutions at amino acid positions selected from the following compared to the reference sequence SEQ ID NO: 4850: 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 / 300 / 395, 298 / 300 / 481, 300, 300 / 392 / 395, 319, 322, 392, 421, and 492, and / or any combination thereof.

39. The engineered terminal deoxynucleotidyl transferase according to claim 1, wherein the polypeptide sequence comprises at least one substitution or set of substitutions including substitutions at amino acid positions selected from the following compared to the reference sequence SEQ ID NO: 4856: 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.

40. The engineered terminal deoxynucleotidyl transferase according to claim 1, wherein the polypeptide sequence comprises at least one substitution or set of substitutions comprising substitutions at amino acid positions selected from the following compared to the reference sequence SEQ ID NO: 4904: 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.

41. The engineered terminal deoxynucleotidyl transferase according to claim 1, wherein the polypeptide sequence comprises at least one substitution or set of substitutions comprising substitutions at amino acid positions selected from the following compared to the reference sequence SEQ ID NO: 5002: 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 / 495, 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, and / or any combination thereof.

42. The engineered terminal deoxynucleotidyl transferase according to claim 1, wherein the polypeptide sequence comprises at least one substitution or set of substitutions comprising substitutions at amino acid positions selected from the following compared to the reference sequence SEQ ID NO: 5028: 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, 274 / 359 / 526, 274 / 523, 309, 309 / 313 / 411, 324 / 526, 411, 466, 466 / 526, 523 and 526, and / or any combination thereof.

43. The engineered terminal deoxynucleotidyl transferase according to claim 1, wherein the polypeptide sequence comprises at least one substitution or set of substitutions comprising substitutions at amino acid positions selected from the following compared to the reference sequence SEQ ID NO: 5192: 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.

44. The engineered terminal deoxynucleotidyl transferase according to claim 1, wherein the polypeptide sequence comprises at least one substitution or set of substitutions comprising substitutions at amino acid positions selected from the following compared to the reference sequence SEQ ID NO: 5246: 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, and / or any combination thereof.

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

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

47. The engineered terminal deoxynucleotidyl transferase according to claim 45, wherein the second polypeptide having IPP activity comprises an amino acid sequence selected from SEQ ID NO: 3942 and 3944.

48. The engineered terminal deoxynucleotidyl transferase according to claim 45, wherein the engineered terminal deoxynucleotidyl transferase polypeptide fused to the second polypeptide comprises a sequence selected from SEQ ID NO: 5468, 5470, 5472 and 5474.

49. The engineered terminal deoxynucleotidyl transferase according to any one of claims 1-47, wherein the engineered terminal deoxynucleotidyl transferase is capable of non-template-dependent synthesis.

50. The engineered terminal deoxynucleotidyl transferase according to any one of claims 1-47, wherein the engineered terminal deoxynucleotidyl transferase has at least one improved property compared to a wild-type or reference terminal deoxynucleotidyl transferase or a non-template-dependent polymerase.

51. The engineered terminal deoxynucleotidyl transferase according to claim 49, wherein the improved property is selected from increased thermal stability, increased activity at high temperature, increased soluble expression or isolated protein yield, reduced by-product formation, increased specific activity towards one or more NTP-3'-O-RBG or natural or modified NTP substrates, increased incorporation efficiency of oligonucleotide acceptor substrate extension, and increased activity towards one or more oligonucleotide acceptor substrates.

52. The engineered terminal deoxynucleotidyl transferase according to any one 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 a non-template-dependent polymerase.

53. The engineered terminal deoxynucleotidyl transferase according to any one of claims 1-51, wherein the engineered terminal deoxynucleotidyl transferase comprises increased thermal stability compared to a wild-type or reference terminal deoxynucleotidyl transferase or a non-template-dependent polymerase.

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

55. The engineered terminal deoxynucleotidyl transferase according to any one of claims 1-53, wherein the terminal deoxynucleotidyl transferase is purified.

56. A polynucleotide sequence that encodes at least one engineered terminal deoxynucleotidyl transferase according to any one of claims 1-54.

57. A polynucleotide sequence that comprises at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with a reference sequence SEQ ID NO: 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, 5027, 5191 and 5245 and / or a functional fragment thereof, wherein the polynucleotide sequence encodes an engineered polypeptide that comprises at least one substitution at one or more amino acid positions.

58. The polynucleotide sequence according to claim 56, wherein the polynucleotide sequence encodes at least one engineered terminal deoxynucleotidyl transferase that comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with a reference sequence SEQ ID NO: 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, 5002, 5028, 5192 and 5246.

59. The polynucleotide sequence according to claim 56, wherein the sequence comprises SEQ ID NO: 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.

60. The polynucleotide sequence according to claim 56, wherein the polynucleotide sequence is operably linked to a control sequence.

61. The polynucleotide sequence according to claim 56, wherein the polynucleotide sequence is codon-optimized.

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

63. A host cell comprising at least one expression vector according to claim 61.

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

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

66. The method according to claim 63 or 64, further comprising the step of purifying the at least one engineered terminal deoxynucleotidyl transferase.

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

68. A method for non-template-dependent synthesis of oligonucleotides, the method comprising: a) providing at least one terminal deoxynucleotidyl transferase or non-template-dependent polymerase; b) providing at least one oligonucleotide acceptor substrate, wherein the oligonucleotide acceptor substrate comprises a 3'-OH or its equivalent; c) contacting the oligonucleotide acceptor substrate, the terminal deoxynucleotidyl transferase or non-template-dependent polymerase with one or more nucleotide triphosphates, modified nucleotide triphosphates or NTP-3'-O-RBG under conditions sufficient to add the nucleotide, modified nucleotide or nucleotide-3'-O-RBG to the 3'-end of the oligonucleotide acceptor substrate.

69. The method according to claim 67, wherein the method further comprises (d) deblocking the protected 3'-O-position of the oligonucleotide product formed in step (c).

70. The method according to claim 67 or 68, wherein the method further comprises e) inactivating unreacted nucleotide triphosphates, modified nucleotide triphosphates or NTP-3'-O-RBG.

71. The method according to any one of claims 67 - 69, wherein the method further comprises an optional step (f) of removing excess nucleosides and / or excess inorganic phosphate or pyrophosphate from the reaction.

72. The method according to any one 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.

73. The method according to any one of claims 67 - 71, wherein the method further comprises (g) cleaving or releasing the growing or intact oligonucleotide chain.

74. The method according to any one of claims 68 - 72, wherein step (d) deblocks the oligonucleotide formed in step (c) at the protected 3'-O-position and step (e) inactivates unreacted nucleotide triphosphates, modified nucleotide triphosphates or NTP-3'-O-RBG simultaneously.

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

76. The method according to claim 74, wherein the phosphatase comprises an alkaline phosphatase listed in SEQ ID NO: 3922, 3924, 3926, 3928, 3930, 3932 or 3934.

77. The method according to any one of claims 67 - 75, wherein step (c) optionally comprises contacting the oligonucleotide acceptor substrate, the terminal deoxynucleotidyl transferase or non-template-dependent polymerase and nucleotide triphosphates or modified nucleotide triphosphates or NTP-3'-O-RBG with a phosphatase such as inorganic pyrophosphatase to convert pyrophosphate to inorganic phosphate.

78. The method according to claim 76, wherein the phosphatase comprises an inorganic pyrophosphatase listed in SEQ ID NO: 3936, 3938, 3940, 3942, 3944, 3946 or 3948.

79. The method according to any one of claims 67 - 77, further comprising NTP-3'-O-RBG, wherein the NTP-3'-O-RBG comprises a nitrile, phosphate ester, carbonate, carbamate, ester, ether, borate, nitrate, sugar, aminophosphate, phenylsulfinate or sulfate.

80. The method according to any one of claims 67 - 78, wherein the nucleotide triphosphates, the modified nucleotide triphosphates or the NTP-3'-O-RBG comprise a 2'-modification.

81. The method according to claim 79, wherein the 2'-modification comprises 2'-F, 2'-O-alkyl, 2'-O-methoxyethyl or locked ethyl or constrained ethyl.

82. The method according to claim 80, wherein the modification is 2'-F, and the 2'-modified nucleotide triphosphates, the modified nucleotide triphosphates or the NTP-3'-O-RBG are 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.

83. The method according to claim 80, wherein the modification is 2'-O-alkyl, and the 2'-modified nucleotide triphosphate, the modified nucleotide triphosphate, or the NTP-3'-O-RBG is selected from: 2'-O-methyladenosine-5'-triphosphate, 2'-O-methylcytidine-5'-triphosphate, 2'-O-methylguanosine-5'-triphosphate, 2'-O-methyluridine-5'-triphosphate, and 2'-O-methylinosine-5'-triphosphate.

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

85. The method according to any one of claims 67-83, wherein the oligonucleotide, oligonucleotide sequence, or oligonucleotide chain comprises RNA.

86. The method according to any one of claims 67-84, wherein the oligonucleotide, oligonucleotide sequence, or oligonucleotide chain comprises DNA.

87. The method according to any one of claims 67-85, wherein the terminal deoxynucleotidyl transferase or non-template-dependent polymerase comprises one or more engineered terminal deoxynucleotidyl transferases.

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

89. The method according to 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 with a reference sequence selected from SEQ ID NO: 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, 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 relative to the reference sequence in its polypeptide sequence, wherein the amino acid residue difference or set of amino acid residue differences is selected from the amino acid differences listed in the following: Table 5.1, Table 6.2, Table 7.2, Table 8.2, Table 9.2, Table 10.2, Table 11.2, Table 12.2, Table 13.2, Table 14.2, Table 15.2, Table 16.2, Table 17.2, Table 18.2, Table 19.2, Table 20.2, Table 21.2, Table 22.2, Table 23.2, Table 24.2, Table 25.2, Table 26.2, Table 26.3, Table 26.4, Table 27.2, Table 27.3, Table 27.4, Table 27.5, Table 28.1, Table 28.2, Table 28.3, Table 29.2, Table 30.2, Table 31.2, Table 32.2, Table 33.2, Table 34.2, Table 35.2, Table 36.2, Table 37.2, Table 38.2, Table 39.2, Table 40.2, Table 41.2, Table 42.2, Table 43.2, Table 44.2, Table 45.2, Table 46.2, Table 47.2, Table 48.2, Table 49.2, Table 50.2, Table 51.2, Table 52.2, Table 53.2, Table 54.2, Table 55.2, Table 56.2, Table 56.3, Table 56.4, Table 61.2, Table 63.2, Table 64.2, Table 65.2, Table 66.2, Table 67.2, Table 68.2, Table 69.2, Table 70.2, Table 71.2, Table 72.2, Table 73.2, Table 74.2, Table 75.2, Table 76.2, Table 77.2, Table 78.2, Table 79.2 and Table 80.

1.

90. The method according to 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 NO: 4-1960, 2004-3920, 4048-5466 and 5476.

91. The method according to any one of claims 67-89, wherein the terminal deoxynucleotidyl transferase or non-template-dependent polymerase is immobilized.

92. The method according to claim 90, wherein the method further comprises a column solid support for immobilizing the terminal deoxynucleotidyl transferase or non-template-dependent polymerase.

93. The method according to claim 90, wherein the method further comprises a batch method for immobilizing the terminal deoxynucleotidyl transferase or non-template-dependent polymerase with a solid support.

94. The method according to any one of claims 67-89, wherein the oligonucleotide acceptor substrate and the subsequent oligonucleotide product are immobilized.

95. The method according to any one of claims 67-89, wherein neither the oligonucleotide acceptor substrate nor the terminal deoxynucleotidyl transferase of the non-template-dependent polymerase is immobilized.

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

97. The engineered terminal deoxynucleotidyl transferase according to any one of claims 1-54, wherein the engineered terminal deoxynucleotidyl transferase is immobilized.

Images