Modified b-family polymerases

Abstract

Provided herein are archaeal B -family polymerase variants with improved replication fidelity compared to wild-type archaeal B-family polymerases and methods of use.

Description

[0001] MODIFIED B-FAMILY POLYMERASES

[0002] RELATED APPLICATIONS

[0003] This application claims benefit under 35 U.S.C. § 119(e) to U.S. Provisional Application No. 63 / 709,385, filed October 18, 2024, entitled “Modified B-Family Polymerases”, the entire contents of which are incorporated herein by reference.

[0004] REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

[0005] The contents of the electronic sequence listing (W109470027WO00-SEQ-ARM.xml; Size: 55,542 bytes; and Date of Creation: October 16, 2025) are herein incorporated by reference in its entirety.

[0006] BACKGROUND

[0007] DNA polymerases play a central role in modern molecular biology. DNA polymerases make new copies of DNA strands using an already existing DNA strand as a template. Additionally, some DNA polymerases have proofreading / repair domains that can repair errors that occur when new copies of the DNA strand are made. DNA polymerases are important in many applications including the polymerase chain reaction (PCR), DNA sequencing, DNA cloning, synthetic biology, and molecular diagnostics. In a natural system, DNA polymerases operate in cellular physiological conditions and amplify naturally occurring DNA (typically as part of cell replication). However, in molecular biology applications, DNA polymerases may need to operate in conditions that are not physiological or be able to amplify a polynucleotide having nucleotides that do not naturally occur in DNA.

[0008] Thermococcus kodakarensis (KOD) polymerase and Pyrococcus furiosus (Pfu) polymerase are archaeal B-family thermostable DNA polymerases used in some molecular biology applications, like PCR which requires drastically changing temperatures (e.g., frequent changes between 60 °C to 95 °C).

[0009] SUMMARY

[0010] This disclosure provides archaeal B-family polymerase variants (e.g., KOD and Pfu polymerase variants) that have increased replication fidelity (i.e., reduced error rates) relative to wildtype archaeal B-family polymerase variants (e.g., wildtype KOD or Pfu polymerase). Improved archaeal B-family polymerase variants (e.g., improved KOD or Pfu polymerase variants) are useful in molecular biology applications, such as amplification of polynucleotides

[0011] #14495181v1 via polymerase chain reaction (PCR) for sequencing. Archaeal B-family polymerases with reduced error rates can be used to produce amplified polynucleotides that have fewer mutations (e.g., insertions, deletions, or substitutions) compared to target polynucleotides.

[0012] Accordingly, in some aspects, this disclosure provides a Thermococcus kodakarensis (KOD) polymerase variant comprising a threonine at a position corresponding to S407 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant comprises at least 95% identity to SEQ ID NO: 1. In some embodiments, the KOD polymerase variant further comprises a lysine at a position corresponding to E664 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant further comprises an arginine at a position corresponding to A675 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant further comprises an arginine at a position corresponding to H147 of SEQ ID NO: 1. In some embodiments, the KOD polymerase variant further comprises an isoleucine at a position corresponding to L408 of SEQ ID NO: 1.

[0013] In some embodiments, the KOD polymerase variant comprises an amino acid sequence having at least 95% identity to SEQ ID NO: 2. In some embodiments, the KOD polymerase variant comprises an amino acid sequence of SEQ ID NO: 2.

[0014] In some embodiments, the KOD polymerase variant comprises an amino acid sequence having at least 95% identity to SEQ ID NO: 3. In some embodiments, the KOD polymerase variant comprises an amino acid sequence of SEQ ID NO: 3.

[0015] In some aspects, this disclosure provides a Pyrococcus furiosus (Pfu) polymerase variant comprising a threonine at a position corresponding to A408 of SEQ ID NO: 4. In some embodiments, the Pfu polymerase variant comprises at least 95% identity to SEQ ID NO: 4. In some embodiments, the Pfu polymerase variant further comprises a lysine at a position corresponding to E665 of SEQ ID NO: 4.

[0016] In some embodiments, the Pfu polymerase variant comprises an amino acid sequence having at least 95% identity to SEQ ID NO: 5. In some embodiments, the Pfu polymerase variant comprises an amino acid sequence of SEQ ID NO: 5.

[0017] In some embodiments, the Pfu polymerase variant comprises an amino acid sequence having at least 95% identity to SEQ ID NO: 6. In some embodiments, the Pfu polymerase variant comprise an amino acid sequence of SEQ ID NO: 6.

[0018] In some embodiments, the Pfu polymerase variant further comprises an Sso7d domain. In some embodiments, the Sso7d domain comprises an amino acid sequence of SEQ ID NO: 7.

[0019] In some embodiments, the Pfu polymerase variant comprises an amino acid sequence of SEQ ID NO: 8. In some embodiments, the Pfu polymerase variant comprises an amino acid sequence of SEQ ID NO: 9.

[0020] #14495181v1 In some embodiments, this disclosure provides a method of amplifying a target polynucleotide, the method comprising the target polynucleotide with a KOD polymerase variant or a Pfu polymerase variant provided herein. In some embodiments, amplifying the target polynucleotide comprises amplifying with an error rate that is less than 0.0020.

[0021] BRIEF DESCRIPTION OF DRAWINGS

[0022] FIG. 1 shows Total Error Rate and Median Family Size (y-axes) resulting from amplification of target polynucleotides with KOD and Pfu polymerase variants (x-axis). The mutations of each variant are described by name.

[0023] FIG. 2 shows average error rates (y-axis) resulting from amplification of target polynucleotides using KOD polymerase variants at varying concentrations (x-axis) compared to wild- type KOD polymerase (horizontal cutoff line). Data for the industry standard high fidelity polymerase Q5® is also provided.

[0024] FIG. 3 provides average error rates (y-axis) resulting from amplification of target polynucleotides using KOD polymerase variants at varying concentrations (x-axis) compared to wild-type KOD polymerase (horizontal cutoff line). Reactions were performed in excess salt (lOOrnM) conditions.

[0025] DETAILED DESCRIPTION

[0026] In some aspects, this disclosure provides archaeal B-family polymerase variants (e.g., KOD and Pfu polymerase variants) that have increased replication fidelity relative to wildtype archaeal B-family polymerases. Archaeal B-family polymerases are of the domain archaea that are involved in DNA replication and repair. Such polymerases are frequently used in molecular biology, such as polymerase chain reaction (PCR), in which polynucleotides (polymers of nucleotides, also referred to herein as nucleic acids) are amplified to generate an amount of DNA material suitable for use in a variety of downstream applications for which accurate amplification of DNA is important, such as DNA cloning, sequencing (e.g., next-generation sequencing, NGS), gene analysis, diagnostics, etc. Many archaeal B-family polymerases are derived from extremophiles and, as such, exhibit high thermostability, making them useful for applications that require high temperatures or large temperature changes (such as PCR, which can include steps ranging from around 50 °C to 95 °C). While archaeal B-family polymerases have proofreading capabilities and can therefore replicate polynucleotides with slightly greater fidelity (i.e., fewer mutations) compared to some other polymerases (e.g., Taq polymerase, derived from the bacterial species Thermits aquaticus), they typically possess lower fidelity than

[0027] #14495181v1 other polymerases (e.g., Q5®) and are therefore more prone to introducing errors in replicated polynucleotides. Archaeal B-family polymerase variants with enhanced replication fidelity represent are a very useful contribution to the field of molecular biology, as such variants can accurately amplify polynucleotides, even long and complex polynucleotides, across a range of temperatures. Provided herein are archaeal B-family polymerase variants that have enhanced replication fidelity compared not only to wild-type archaeal B-family polymerases, but also to the high-fidelity DNA polymerase Q5®.

[0028] Archaeal B-family DNA polymerases used in molecule biology include, but are not limited to, Thermococcus kodakaraensis (KOD) polymerase, Thermococcus sp. 9 degree north (9dN) polymerase, Pyrococcus furiosus (Pfu) polymerase, Pyrococcus sp. GB-D (GB-D) polymerase, Thermococcus litoralis (Tli) polymerase, and Thermococcus gorgonarius (Tgo) polymerase. Other archaeal B-family polymerases are known in the art (see, e.g., Kazlauskas et al., Nucleic Acids Res, 2020. 48(18): 10142-10156).

[0029] “Replication fidelity” is related to a DNA polymerase’s error rate and refers to the accuracy (i.e., fidelity) with which the DNA polymerase replicates a target polynucleotide. The error rate of polymerases can be determined by identifying nucleotide mutations (e.g., substitutions, insertions, or deletions) in replicated nucleic acids compared to the target polynucleotide sequence, i.e., the nucleic acid sequence that was replicated by the polymerase. For example, error rates can be calculated as:

[0030] / = n / 5 where f= error rate; n = number of mutations observed; and S = the target polynucleotide size multiplied by the average target polynucleotide doublings per amplification cycle. Any suitable formula can be used to calculate error rate (e.g., as described in McInerney et al, Molecular biology international 2014.1 (2014): 287430).

[0031] The higher the average error rate of a DNA polymerase, the lower replication fidelity the DNA polymerase is considered to have. While some DNA polymerases possess inherent proofreading / repair domains that can repair replication errors, replication errors can still occur and remain uncorrected.

[0032] In some embodiments, this disclosure provides an archaeal B-family DNA polymerase variant comprising a mutation (e.g., a substitution, an insertion, or a deletion) at a position corresponding to a reference archaeal B-family DNA polymerase. An archaeal B-family DNA polymerase variant (e.g., a KOD polymerase variant or Pfu variant) having a “position corresponding to” a reference amino acid sequence (e.g., a reference KOD polymerase amino acid sequence like SEQ ID NO: 1 or a reference Pfu polymerase amino acid sequence like SEQ

[0033] #14495181v1 ID NO: 4) refers to a particular amino acid in the archaeal B-family DNA polymerase variant relative to the reference archaeal B-family polymerase.

[0034] An archaeal B-family polymerase variant (e.g., a KOD polymerase variant or Pfu polymerase variant) may be identified relative to a reference sequence. In other words, the reference sequence can be used as a standard for mapping mutations between different archaeal B-family DNA polymerase variants (e.g., different KOD polymerase variants or different Pfu polymerase variants). This mapping may be used to compare archaeal B-family DNA polymerase variants comprising insertions or deletions that shift the location of a mutation relative to the reference sequence). In some embodiments, determining a position of archaeal B- family DNA polymerase variant corresponding to a reference sequence comprises aligning the archaeal B-family DNA polymerase sequence (e.g., wild type KOD polymerase) and the archaeal B-family DNA polymerase variant sequence (e.g., a KOD polymerase variant) (e.g., using Emboss Needle as described in Madeira F et al. Nucleic Acids Research. 2024 Jul;52(Wl):W521-W525). In some embodiments, this is determined using a structural alignment (e.g., as described in Bittrich et al., Bioinformatics, Volume 40, Issue 6, June 2024, doi.org / 10.1093 / bioinformatics / btae370). Directly below are four examples all relating to a KOD polymerase variant comprising a methionine at a position corresponding to Y38 of a reference sequence. These examples apply similarly to other proteins including other archaeal B-family DNA polymerase variants. All the below KOD polymerase variants in these four examples would fall within the scope of, for example, a KOD polymerase variant comprising a methionine at a position corresponding to Y38 of a reference sequence (Ref).

[0035] In a first example, the reference sequence (Ref) and the target sequence (Tar) align at every amino acid between positions 4 and 37 of the reference sequence. Position 38 of the target sequence corresponds to position 38 of the reference sequence. Position 38 of the target sequence is methionine. Position 38 of the reference sequence is tyrosine (SEQ ID NOs: 30-31).

[0036] Ref 4 DTDYITEDGKPVIRIFKKENGEFKIEYDRTFEPY 38 Tar 4 DTDYITEDGKPVIRIFKKENGEFKIEYDRTFEPM 38

[0037] In a second example, the target sequence has a three amino acid deletion and a tyrosine to methionine substitution relative to the reference sequence. Because of the deletion, position 35 of the target sequence corresponds to position 38 of the reference sequence. Position 35 of

[0038] #14495181v1 the target sequence is methionine. Position 38 of the reference sequence is tyrosine (SEQ ID NOs: 30 and 32).

[0039] Re f 4 -DTDYITEDGKPVIRIFKKENGEFKIEYDRTFEPY 38

[0040] Tar 4-DT - TEDGKPVIRIFKKENGEFKIEYDRTFEPM 35

[0041] In a third example, the target sequence has a four amino acid insertion and a tyrosine to methionine substitution relative to the reference sequence. Because of the insertion, position 42 of the target sequence corresponds to position 38 of the reference sequence. Position 42 of the target sequence is methionine. Position 38 of the reference sequence is tyrosine (SEQ ID NOs: 30 and 33).

[0042] Re f 4 DTDYITED - GKPVIRIFKKENGEFKIEYDRTFEPY 38

[0043] Tar 4 DTDYITEDQRQRGKPVIRIFKKENGEFKIEYDRTFEPM 42

[0044] In a fourth example, the target sequence differs in sequence at amino position 38, 42, 43, and 44. Position 38 of the target sequence corresponds to position 38 of the reference sequence. Position 38 of the target sequence is methionine. Position 38 of the reference sequence is tyrosine (SEQ ID NOs: 34-35).

[0045] Re f 4 DTDYITEDGKPVIRIFKKENGEFKIEYDRTFEPYGGYSTV 44 Tar 4 DTDYITEDGKPVIRIFKKENGEFKIEYDRTFEPMGGYRSE 44

[0046] In some instances, two different protein sequences may not correspond with one another. For example, this may occur when the sequence identity or structural identity between two protein sequences is so low that an alignment cannot be made.

[0047] In some embodiments, this disclosure provides an archaeal B-family polymerase variant comprising a mutation at a position corresponding to S407 of SEQ ID NO: 1. In some embodiments, this disclosure provides a 9dN polymerase variant comprising a mutation at a position corresponding to S407 of SEQ ID NO: 1. In some embodiments, this disclosure provides a GB-D polymerase variant comprising a mutation at a position corresponding to S407 of SEQ ID NO: 1. In some embodiments, this disclosure provides a Tli polymerase variant comprising a mutation at a position corresponding to S407 of SEQ ID NO: 1. In some

[0048] #14495181v1 embodiments, this disclosure provides a Tgo polymerase variant comprising a mutation at a position corresponding to S407 of SEQ ID NO: 1.

[0049] In some aspects, this disclosure provides an archaeal B-family Polymerase variant (e.g., Thermococcus kodakaraensis (KOD) polymerase variant, a Thermococcus sp. 9 degree north (9dN) polymerase variant, a Pyrococcus furiosus (Pfu) polymerase, a Pyrococcus sp. GB-D (GB-D) polymerase variant, a Thermococcus litoralis (Tli) polymerase variant, or a Thermococcus gorgonarius (Tgo) polymerase) comprising a mutation at a position corresponding to S407 of SEQ ID NO: 1. In some aspects, this disclosure provide an archaeal B-family polymerase variant comprising a mutation at a position corresponding to S407 of SEQ ID NO: 1 and a mutation at a position corresponding to E664 of SEQ ID NO: 1.

[0050] In some embodiments, an archaeal B-family polymerase variant comprising a threonine at a position corresponding to S407 of SEQ ID NO: 1. In some embodiments, an archaeal B- family polymerase variant comprising a threonine at position S407 of SEQ ID NO: 1 and one, two, three or four additional mutations (e.g., amino acid substitutions). In some embodiments, an archaeal B-family Polymerase variant comprises: an amino acid sequence having at least 70% (e.g., at least 75%, at least 78%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and a threonine at a position corresponding to S407 of SEQ ID NO: 1.

[0051] In some embodiments, an archaeal B-family polymerase variant comprises a threonine at a position corresponding to S407 of SEQ ID NO: 1 and a lysine at a position corresponding to E664 of SEQ ID NO: 1. In some embodiments, an archaeal B-family polymerase variant comprises a threonine at position S407 of SEQ ID NO: 1, a lysine at a position corresponding to E664 of SEQ ID NO: 1, and one, two, three or four additional mutations (e.g., amino acid substitutions). In some embodiments, an archaeal B-family polymerase variant comprises: an amino acid sequence having at least 70% (e.g., at least 75%, at least 78%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1, a threonine at a position corresponding to S407 of SEQ ID NO: 1, and a lysine at a position corresponding to E664 of SEQ ID NO: 1.

[0052] In some embodiments, the archaeal B-family polymerase variant is a Thermococcus kodakaraensis (KOD) polymerase variant, a Thermococcus sp. 9 degree north (9dN) polymerase variant, a Pyrococcus furiosus (Pfu) polymerase, a Pyrococcus sp. GB-D (GB-D) polymerase variant, a Thermococcus litoralis (Tli) polymerase variant, or a Thermococcus gorgonarius (Tgo) polymerase variant comprising a mutation a position corresponding to S407 of SEQ ID NO: 1. In some embodiments, the archaeal B-family polymerase variant is a Thermococcus

[0053] #14495181v1 kodakaraensis (KOD) polymerase variant, a Thermococcus sp. 9 degree north (9dN) polymerase variant, a Pyrococcus furiosus (Pfu) polymerase, a Pyrococcus sp. GB-D (GB-D) polymerase variant, a Thermococcus litoralis (Tli) polymerase variant, or a Thermococcus gorgonarius (Tgo) polymerase variant comprising a mutation a position corresponding to S407 of SEQ ID NO: 1 and one, two, three or four additional mutations (e.g., amino acid substitutions.

[0054] In some embodiments, an archaeal B-family polymerase variant comprises an amino acid sequence having at least 90% identity to any one of SEQ ID NOs: 2, 13, 15, 17, or 19. In some embodiments, the archaeal B-family polymerase variant comprises an amino acid sequence having at least 95% identity to any one of SEQ ID NOs: 2, 13, 15, 17, or 19. In some embodiments, the archaeal B-family polymerase variant comprises an amino acid sequence having at least 98% identity to any one of SEQ ID NOs: 2, 13, 15, 17, or 19. In some embodiments, the archaeal B-family polymerase variant comprises an amino acid sequence having at least 99% identity to any one of SEQ ID NOs: 2, 13, 15, 17, or 19. In some embodiments, the archaeal B-family polymerase variant comprises an amino acid sequence of any one of SEQ ID NOs: 2, 13, 15, 17, or 19.

[0055] Some embodiments relate to amino acid sequences or nucleotide sequences having some percentage identity to a reference amino acid sequence or reference nucleotide sequence, respectively. As used herein, the term “identity” refers to the degree to which two or more sequences (e.g., amino acid sequences, nucleotide sequences) are related, as determined by the number of matches between each sequence. “Percent (%) identity” refers to the percentage of residues or nucleotides in a first amino acid or first nucleic acid sequence that are identical to the residues in a second amino acid sequence or second nucleic acid sequence. The skilled artisan will understand that determining percent identity may require a step of aligning a first and second sequence and / or introducing gaps. Several algorithms for aligning sequences are known in the art, for example, on the worldwide web at ebi.ac.uk / jdispatcher / psa / emboss_needle, e.g., as described in Madeira F. et al., Nucleic Acids Research. 2024 Jul;52(Wl):W521-W525.

[0056] KOD Polymerase Variants

[0057] In some aspects, this disclosure provides a KOD polymerase variant. In some embodiments, the KOD polymerase variant comprises improved replication fidelity compared to wildtype KOD polymerase. In some embodiments, this disclosure provides a KOD polymerase variant comprising a mutation at a position corresponding to position S407 of SEQ ID NO: 1.

[0058] A “Thermococcus kodakarensis (KOD) polymerase” refers to a B-family DNA polymerase of Thermococcus kodakarensis (e.g., a wildtype B-family KOD polymerase). In

[0059] #14495181v1 some embodiments, a KOD polymerase comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99% identity) to SEQ ID NO: 1. In some embodiments, a KOD polymerase comprises an amino acid sequence of SEQ ID NO: 1.

[0060] A “Thermococcus kodakarensis (KOD) polymerase variant” refers to a KOD polymerase that comprises at least one amino acid mutation relative to a KOD polymerase (e.g., relative to a wildtype KOD polymerase (SEQ ID NO: 1)). In some embodiments, a KOD polymerase variant comprises a substitution mutation relative to a KOD polymerase. In some embodiments, a KOD polymerase variant comprises one or more substitution mutations relative to a KOD polymerase. In some embodiments, a KOD polymerase variant comprises an insertion and / or deletion mutation relative to a KOD polymerase. In some embodiments, a KOD polymerase variant comprises a substitution mutation that increases salt tolerance of the KOD polymerase variant relative to a KOD polymerase (e.g., increased activity of the KOD polymerase variant in the presence of salt and / or salt concentration relative to a KOD polymerase).

[0061] In some embodiments, a KOD polymerase variant further comprises a protein tag. In some embodiments, the protein tag is a purification tag (e.g., a His tag, a Myc tag, or a Flag tag). In some embodiments, the protein tag is a secretion tag.

[0062] Sso7d KOD Polymerase Variants

[0063] In some embodiments, this disclosure provides a fusion protein comprising a KOD polymerase variant and an Sso7d domain. Sso7d is a small protein domain derived from the Sulfolobus solfataricus species of thermophilic archaeon that, when fused (e.g., directly or via a linker) to a polymerase confers or improves thermostability of the polymerase. In some embodiments, a fusion protein comprises, in N-terminal to C-terminal order, a KOD polymerase variant and an Sso7d domain. In some embodiments, a fusion protein comprises, in C-terminal to N-terminal order, an Sso7d domain and a KOD polymerase variant. In some embodiments, a fusion protein comprises, in N-terminal to C-terminal order, a KOD polymerase variant, a linker, and an Sso7d domain. In some embodiments, a fusion protein comprises, in N-terminal to C- terminal order, an Sso7d domain, a linker, and a KOD polymerase variant. In some embodiments, a linker is an amino acid linker. In some embodiments, an Sso7d domain comprises an amino acid sequence having at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 7. In some embodiments, an Sso7d domain comprises an amino acid sequence of SEQ ID NO: 7.

[0064] In some embodiments, a mutation in a KOD polymerase variant at position S407 and / or position E664 corresponding to SEQ ID NO: 1 alters the replication fidelity of the KOD

[0065] #14495181v1 polymerase. In some embodiments, a mutation in a KOD polymerase variant at position S407 and / or position E664 corresponding to SEQ ID NO: 1 alters the salt tolerance of the KOD polymerase. In some embodiments, a mutation in a KOD polymerase variant at position S407 and / or position E664 corresponding to SEQ ID NO: 1 alters the replication fidelity and salt tolerance of the KOD polymerase.

[0066] S407 KOD Polymerase Variants

[0067] In some embodiments, a KOD polymerase variant comprises a mutation at a position corresponding to position S407 of SEQ ID NO: 1.

[0068] In some embodiments, this disclosure provides a KOD polymerase variant comprising a threonine at a position corresponding to S407 of SEQ ID NO: 1 (e.g., S407T in a KOD polymerase corresponding to SEQ ID NO: 1). In some embodiments, this disclosure provides a KOD polymerase variant comprising a threonine at position S407 of SEQ ID NO: 1 and one, two, three or four additional mutations (e.g., amino acid substitutions) in SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises: an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and a threonine at a position corresponding to S407 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises a threonine at position S407 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises a threonine at a position corresponding to S407 of SEQ ID NO: 1.

[0069] In some embodiments, a KOD polymerase variant comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 2. In some embodiments, a KOD polymerase variant comprises an amino acid sequence having at least 95% identity to SEQ ID NO: 2. In some embodiments, a KOD polymerase variant comprises an amino acid sequence having at least 98% identity to SEQ ID NO: 2. In some embodiments, a KOD polymerase variant comprises an amino acid sequence having at least 99% identity to SEQ ID NO: 2. In some embodiments, a KOD polymerase variant comprises an amino acid sequence having at least 99.5% identity to SEQ ID NO: 2. In some embodiments, a KOD polymerase variant comprises an amino acid sequence of SEQ ID NO: 2. In some embodiments, a KOD polymerase variant consists of an amino acid sequence of SEQ ID NO: 2.

[0070] S407 and Sso7d KOD Polymerase Variants

[0071] In some embodiments, this disclosure provides a fusion protein comprising a KOD polymerase variant and an Sso7d domain.

[0072] #14495181v1 In some embodiments, a fusion protein comprises an Sso7d domain and a KOD polymerase variant comprising a threonine at a position corresponding to S407 of SEQ ID NO: 4. In some embodiments, a fusion protein comprises: an Sso7d domain; and a KOD polymerase variant comprising a threonine at position S407 of SEQ ID NO: 1 and one, two, three or four additional mutations (e.g., amino acid substitutions) in SEQ ID NO: 1.

[0073] In some embodiments, a fusion protein comprises: an Sso7d domain; and a KOD polymerase variant comprising a threonine at position S407 of SEQ ID NO: 4 and having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 4.

[0074] In some embodiments, a fusion protein comprises: an Sso7d domain comprising an amino acid sequence having at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 7; and a KOD polymerase variant comprising a threonine at position S407 of SEQ ID NO: 1 and having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1.

[0075] In some embodiments, a fusion protein comprises: an Sso7d domain comprising an amino acid sequence of SEQ ID NO: 7; and a KOD polymerase variant comprising a threonine at position S407 of SEQ ID NO: 1 and having an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1.

[0076] In some embodiments, a fusion protein comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 10. In some embodiments, a fusion protein comprises an amino acid sequence having at least 95% identity to SEQ ID NO: 10. In some embodiments, a fusion protein comprises an amino acid sequence having at least 98% identity to SEQ ID NO: 10. In some embodiments, a fusion protein comprises an amino acid sequence having at least 99% identity to SEQ ID NO: 10. In some embodiments, a fusion protein comprises an amino acid sequence having at least 99.5% identity to SEQ ID NO: 10. In some embodiments, a fusion protein comprises an amino acid sequence of SEQ ID NO: 10. In some embodiments, a fusion protein consists of an amino acid sequence of SEQ ID NO: 10.

[0077] S407 and E664 KOD Polymerase Variants

[0078] In some embodiments, a KOD polymerase variant comprises a mutation at a position corresponding to position E664 and a mutation at a position corresponding to S407 of SEQ ID NO: 1.

[0079] In some embodiments, a KOD polymerase variant comprises a lysine at a position corresponding to E664 of SEQ ID NO: 1 and a threonine at a position corresponding to S407 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises a lysine at

[0080] #14495181v1 position E664 of SEQ ID NO: 1; a threonine at position S407 of SEQ ID NO: 1; and one, two, three or four additional mutations (e.g., amino acid substitutions) in SEQ ID NO: 1.

[0081] In some embodiments, a KOD polymerase variant comprises: an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1; a lysine at position E664 of SEQ ID NO: 1; and a threonine at position S407 of SEQ ID NO: 1.

[0082] In some embodiments, a KOD polymerase variant comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 3. In some embodiments, a KOD polymerase variant comprises an amino acid sequence having at least 95% identity to SEQ ID NO: 3. In some embodiments, a KOD polymerase variant comprises an amino acid sequence having at least 98% identity to SEQ ID NO: 3. In some embodiments, a KOD polymerase variant comprises an amino acid sequence having at least 99% identity to SEQ ID NO: 3. In some embodiments, a KOD polymerase variant comprises an amino acid sequence having at least 99.5% identity to SEQ ID NO: 3. In some embodiments, a KOD polymerase variant comprises an amino acid sequence of SEQ ID NO: 3. In some embodiments, a KOD polymerase variant consists of an amino acid sequence of SEQ ID NO: 3.

[0083] S407, E664, and Sso7d KOD Polymerase Variants

[0084] In some embodiments, this disclosure provides a fusion protein comprising an Sso7d domain and a KOD polymerase variant comprising a mutation at a position corresponding to S407 and a mutation at a position corresponding to E664 of SEQ ID NO: 1. In some embodiments, a fusion protein comprises an Sso7d domain and a KOD polymerase variant comprising a threonine at a position corresponding to S407 and a lysine at a position corresponding to E664 of SEQ ID NO: 1. In some embodiments, a fusion protein comprises: an Sso7d domain; and a KOD polymerase variant comprising a threonine at position S407 of SEQ ID NO: 1, a lysine at a position corresponding to E664 of SEQ ID NO: 1, and one, two, three or four additional mutations e.g., amino acid substitutions) in SEQ ID NO: 1. In some embodiments, a fusion protein comprises: an Sso7d domain; and a KOD polymerase variant comprising a threonine at position S407 of SEQ ID NO: 1, a lysine at position E664 of SEQ ID NO: 1, and having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1. In some embodiments, a fusion protein comprises: an Sso7d domain comprising an amino acid sequence having at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 7; and a KOD polymerase variant comprising a threonine at position S407 of SEQ ID NO: 1, a lysine at position E664 of SEQ ID

[0085] #14495181v1 NO: 1, and having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1. In some embodiments, a fusion protein comprises: an Sso7d domain comprising an amino acid sequence of SEQ ID NO: 7; and a KOD polymerase variant comprising a threonine at position S407 of SEQ ID NO: 1, a lysine at position E664 of SEQ ID NO: 1, and having at least 90% e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1.

[0086] In some embodiments, a fusion protein comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 11. In some embodiments, a fusion protein comprises an amino acid sequence having at least 95% identity to SEQ ID NO: 11. In some embodiments, a fusion protein comprises an amino acid sequence having at least 98% identity to SEQ ID NO: 11. In some embodiments, a fusion protein comprises an amino acid sequence having at least 99% identity to SEQ ID NO: 11. In some embodiments, a fusion protein comprises an amino acid sequence having at least 99.5% identity to SEQ ID NO: 11. In some embodiments, a fusion protein comprises an amino acid sequence of SEQ ID NO: 11. In some embodiments, a fusion protein consists of an amino acid sequence of SEQ ID NO: 11.

[0087] S407 and A675 KOD Polymerase Variants

[0088] In some embodiments, a KOD polymerase variant comprises a mutation at a position corresponding to position A675 and a mutation at a position corresponding to S407 of SEQ ID NO: 1.

[0089] In some embodiments, a KOD polymerase variant comprises an arginine at a position corresponding to A675 of SEQ ID NO: 1 and a threonine at a position corresponding to S407 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises an arginine at position A675 of SEQ ID NO: 1; a threonine at position S407 of SEQ ID NO: 1; and one, two, three or four additional mutations e.g., amino acid substitutions) in SEQ ID NO: 1.

[0090] In some embodiments, a KOD polymerase variant comprises: an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1; an arginine at position A675 of SEQ ID NO: 1; and a threonine at position S407 of SEQ ID NO: 1.

[0091] In some embodiments, a KOD polymerase variant comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 20. In some embodiments, a KOD polymerase variant comprises an amino acid sequence having at least 95% identity to SEQ ID NO: 20. In some embodiments, a KOD polymerase variant comprises an amino acid sequence having at least 98% identity to SEQ ID NO: 20. In some embodiments, a KOD polymerase variant

[0092] #14495181v1 comprises an amino acid sequence having at least 99% identity to SEQ ID NO: 20. In some embodiments, a KOD polymerase variant comprises an amino acid sequence having at least 99.5% identity to SEQ ID NO: 20. In some embodiments, a KOD polymerase variant comprises an amino acid sequence of SEQ ID NO: 20. In some embodiments, a KOD polymerase variant consists of an amino acid sequence of SEQ ID NO: 20.

[0093] S407, A675, and Sso7d KOD Polymerase Variants

[0094] In some embodiments, this disclosure provides a fusion protein comprising an Sso7d domain and a KOD polymerase variant comprising a mutation at a position corresponding to S407 and a mutation at a position corresponding to A675 of SEQ ID NO: 1. In some embodiments, a fusion protein comprises an Sso7d domain and a KOD polymerase variant comprising a threonine at a position corresponding to S407 and an arginine at a position corresponding to A675 of SEQ ID NO: 1. In some embodiments, a fusion protein comprises: an Sso7d domain; and a KOD polymerase variant comprising a threonine at position S407 of SEQ ID NO: 1, an arginine at a position corresponding to A675 of SEQ ID NO: 1, and one, two, three or four additional mutations (e.g., amino acid substitutions) in SEQ ID NO: 1. In some embodiments, a fusion protein comprises: an Sso7d domain; and a KOD polymerase variant comprising a threonine at position S407 of SEQ ID NO: 1, an arginine at position A675 of SEQ ID NO: 1, and having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1. In some embodiments, a fusion protein comprises: an Sso7d domain comprising an amino acid sequence having at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 7; and a KOD polymerase variant comprising a threonine at position S407 of SEQ ID NO: 1, an arginine at position A675 of SEQ ID NO: 1, and having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1. In some embodiments, a fusion protein comprises: an Sso7d domain comprising an amino acid sequence of SEQ ID NO: 7; and a KOD polymerase variant comprising a threonine at position S407 of SEQ ID NO: 1, an arginine at position A675 of SEQ ID NO: 1, and having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1.

[0095] In some embodiments, a fusion protein comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 21. In some embodiments, a fusion protein comprises an amino acid sequence having at least 95% identity to SEQ ID NO: 21. In some embodiments, a fusion protein comprises an amino acid sequence having at least 98% identity to SEQ ID NO: 21. In some embodiments, a fusion protein comprises an amino acid sequence having at least 99%

[0096] #14495181v1 identity to SEQ ID NO: 21. In some embodiments, a fusion protein comprises an amino acid sequence having at least 99.5% identity to SEQ ID NO: 21. In some embodiments, a fusion protein comprises an amino acid sequence of SEQ ID NO: 21. In some embodiments, a fusion protein consists of an amino acid sequence of SEQ ID NO: 21.

[0097] L408 KOD Polymerase Variants

[0098] In some embodiments, a KOD polymerase variant comprises a mutation at a position corresponding to position L408 of SEQ ID NO: 1.

[0099] In some embodiments, this disclosure provides a KOD polymerase variant comprising an isoleucine at a position corresponding to L408 of SEQ ID NO: 1 (e.g., L408I in a KOD polymerase corresponding to SEQ ID NO: 1). In some embodiments, this disclosure provides a KOD polymerase variant comprising a isoleucine at position L408 of SEQ ID NO: 1 and one, two, three or four additional mutations (e.g., amino acid substitutions) in SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises: an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and a isoleucine at a position corresponding to L408 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises a isoleucine at position L408 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises a isoleucine at a position corresponding to L408 of SEQ ID NO: 1.

[0100] In some embodiments, a KOD polymerase variant comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 22. In some embodiments, a KOD polymerase variant comprises an amino acid sequence having at least 95% identity to SEQ ID NO: 22. In some embodiments, a KOD polymerase variant comprises an amino acid sequence having at least 98% identity to SEQ ID NO: 22. In some embodiments, a KOD polymerase variant comprises an amino acid sequence having at least 99% identity to SEQ ID NO: 22. In some embodiments, a KOD polymerase variant comprises an amino acid sequence having at least 99.5% identity to SEQ ID NO: 22. In some embodiments, a KOD polymerase variant comprises an amino acid sequence of SEQ ID NO: 22. In some embodiments, a KOD polymerase variant consists of an amino acid sequence of SEQ ID NO: 22.

[0101] L408 and Sso7d KOD Polymerase Variants

[0102] In some embodiments, this disclosure provides a fusion protein comprising a KOD polymerase variant and an Sso7d domain.

[0103] #14495181v1 In some embodiments, a fusion protein comprises an Sso7d domain and a KOD polymerase variant comprising an isoleucine at a position corresponding to L408 of SEQ ID NO: 4. In some embodiments, a fusion protein comprises: an Sso7d domain; and a KOD polymerase variant comprising an isoleucine at position L408 of SEQ ID NO: 1 and one, two, three or four additional mutations (e.g., amino acid substitutions) in SEQ ID NO: 1.

[0104] In some embodiments, a fusion protein comprises: an Sso7d domain; and a KOD polymerase variant comprising an isoleucine at position L408 of SEQ ID NO: 4 and having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 4.

[0105] In some embodiments, a fusion protein comprises: an Sso7d domain comprising an amino acid sequence having at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 7; and a KOD polymerase variant comprising a isoleucine at position L408 of SEQ ID NO: 1 and having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1.

[0106] In some embodiments, a fusion protein comprises: an Sso7d domain comprising an amino acid sequence of SEQ ID NO: 7; and a KOD polymerase variant comprising an isoleucine at position L408 of SEQ ID NO: 1 and having an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1.

[0107] In some embodiments, a fusion protein comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 23. In some embodiments, a fusion protein comprises an amino acid sequence having at least 95% identity to SEQ ID NO: 23. In some embodiments, a fusion protein comprises an amino acid sequence having at least 98% identity to SEQ ID NO: 23. In some embodiments, a fusion protein comprises an amino acid sequence having at least 99% identity to SEQ ID NO: 23. In some embodiments, a fusion protein comprises an amino acid sequence having at least 99.5% identity to SEQ ID NO: 23. In some embodiments, a fusion protein comprises an amino acid sequence of SEQ ID NO: 23. In some embodiments, a fusion protein consists of an amino acid sequence of SEQ ID NO: 23.

[0108] S407 and H147 KOD Polymerase Variants

[0109] In some embodiments, a KOD polymerase variant comprises a mutation at a position corresponding to position H147 and a mutation at a position corresponding to S407 of SEQ ID NO: 1.

[0110] In some embodiments, a KOD polymerase variant comprises an arginine at a position corresponding to H147 of SEQ ID NO: 1 and a threonine at a position corresponding to S407 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises an arginine at

[0111] #14495181v1 position H147 of SEQ ID NO: 1; a threonine at position S407 of SEQ ID NO: 1; and one, two, three or four additional mutations (e.g., amino acid substitutions) in SEQ ID NO: 1.

[0112] In some embodiments, a KOD polymerase variant comprises: an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1; an arginine at position H147 of SEQ ID NO: 1; and a threonine at position S407 of SEQ ID NO: 1.

[0113] In some embodiments, a KOD polymerase variant comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 28. In some embodiments, a KOD polymerase variant comprises an amino acid sequence having at least 95% identity to SEQ ID NO: 28. In some embodiments, a KOD polymerase variant comprises an amino acid sequence having at least 98% identity to SEQ ID NO: 28. In some embodiments, a KOD polymerase variant comprises an amino acid sequence having at least 99% identity to SEQ ID NO: 28. In some embodiments, a KOD polymerase variant comprises an amino acid sequence having at least 99.5% identity to SEQ ID NO: 28. In some embodiments, a KOD polymerase variant comprises an amino acid sequence of SEQ ID NO: 28. In some embodiments, a KOD polymerase variant consists of an amino acid sequence of SEQ ID NO: 28.

[0114] S407, H147, and Sso7d KOD Polymerase Variants

[0115] In some embodiments, this disclosure provides a fusion protein comprising an Sso7d domain and a KOD polymerase variant comprising a mutation at a position corresponding to S407 and a mutation at a position corresponding to H147 of SEQ ID NO: 1. In some embodiments, a fusion protein comprises an Sso7d domain and a KOD polymerase variant comprising a threonine at a position corresponding to S407 and an arginine at a position corresponding to H147 of SEQ ID NO: 1. In some embodiments, a fusion protein comprises: an Sso7d domain; and a KOD polymerase variant comprising a threonine at position S407 of SEQ ID NO: 1, an arginine at a position corresponding to H147 of SEQ ID NO: 1, and one, two, three or four additional mutations e.g., amino acid substitutions) in SEQ ID NO: 1. In some embodiments, a fusion protein comprises: an Sso7d domain; and a KOD polymerase variant comprising a threonine at position S407 of SEQ ID NO: 1, an arginine at position H147 of SEQ ID NO: 1, and having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1. In some embodiments, a fusion protein comprises: an Sso7d domain comprising an amino acid sequence having at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 7; and a KOD polymerase variant comprising a threonine at position S407 of SEQ ID NO: 1, an arginine at position H147 of SEQ

[0116] #14495181v1 ID NO: 1, and having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1. In some embodiments, a fusion protein comprises: an Sso7d domain comprising an amino acid sequence of SEQ ID NO: 7; and a KOD polymerase variant comprising a threonine at position S407 of SEQ ID NO: 1, an arginine at position H147 of SEQ ID NO: 1, and having at least 90% e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1.

[0117] In some embodiments, a fusion protein comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 24. In some embodiments, a fusion protein comprises an amino acid sequence having at least 95% identity to SEQ ID NO: 24. In some embodiments, a fusion protein comprises an amino acid sequence having at least 98% identity to SEQ ID NO: 24. In some embodiments, a fusion protein comprises an amino acid sequence having at least 99% identity to SEQ ID NO: 24. In some embodiments, a fusion protein comprises an amino acid sequence having at least 99.5% identity to SEQ ID NO: 24. In some embodiments, a fusion protein comprises an amino acid sequence of SEQ ID NO: 24. In some embodiments, a fusion protein consists of an amino acid sequence of SEQ ID NO: 24.

[0118] S407, H147, andA675 KOD Polymerase Variants

[0119] In some embodiments, a KOD polymerase variant comprises a mutation at a position corresponding to position S407, a mutation at a position corresponding to H147, and a mutation at a position corresponding to A675 of SEQ ID NO: 1.

[0120] In some embodiments, a KOD polymerase variant comprises a threonine at a position corresponding to S407 of SEQ ID NO: 1, an arginine at a position corresponding to H147 of SEQ ID NO: 1, and an arginine at a position corresponding to A675 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises a threonine at a position corresponding to S407 of SEQ ID NO: 1; an arginine at a position corresponding to H147 of SEQ ID NO: 1; an arginine at a position corresponding to A675 of SEQ ID NO: 1; and one, two, three, or four additional mutations e.g., amino acid substitutions) in SEQ ID NO: 1.

[0121] In some embodiments, a KOD polymerase variant comprises: an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1; a threonine at a position corresponding to S407 of SEQ ID NO: 1; an arginine at a position corresponding to H147 of SEQ ID NO: 1; and an arginine at a position corresponding to A675 of SEQ ID NO: 1.

[0122] In some embodiments, a KOD polymerase variant comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 25. In some embodiments, a KOD polymerase

[0123] #14495181v1 variant comprises an amino acid sequence having at least 95% identity to SEQ ID NO: 25. In some embodiments, a KOD polymerase variant comprises an amino acid sequence having at least 98% identity to SEQ ID NO: 25. In some embodiments, a KOD polymerase variant comprises an amino acid sequence having at least 99% identity to SEQ ID NO: 25. In some embodiments, a KOD polymerase variant comprises an amino acid sequence having at least 99.5% identity to SEQ ID NO: 25. In some embodiments, a KOD polymerase variant comprises an amino acid sequence of SEQ ID NO: 25. In some embodiments, a KOD polymerase variant consists of an amino acid sequence of SEQ ID NO: 25.

[0124] S407, H147, A675, and Sso7d KOD Polymerase Variants

[0125] In some embodiments, this disclosure provides a fusion protein comprising an Sso7d domain and a KOD polymerase variant comprising a mutation at a position corresponding to S407, a mutation at a position corresponding to H147 of SEQ ID NO: 1, and a mutation at a position corresponding to A675 of SEQ ID NO: 1.

[0126] In some embodiments, a fusion protein comprises an Sso7d domain and a KOD polymerase variant comprising a threonine at a position corresponding to S407, an arginine at a position corresponding to H147 of SEQ ID NO: 1, and an arginine at a position corresponding to A675 of SEQ ID NO: 1. In some embodiments, a fusion protein comprises: an Sso7d domain; and a KOD polymerase variant comprising a threonine at position S407 of SEQ ID NO: 1; an arginine at a position corresponding to H147 of SEQ ID NO: 1; an arginine at a position corresponding to A675 of SEQ ID NO: 1; and one, two, three or four additional mutations (e.g., amino acid substitutions) in SEQ ID NO: 1. In some embodiments, a fusion protein comprises: an Sso7d domain; and a KOD polymerase variant comprising a threonine at position S407 of SEQ ID NO: 1, an arginine at position H147 of SEQ ID NO: 1, an arginine at a position A675 of SEQ ID NO: 1, and having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1. In some embodiments, a fusion protein comprises: an Sso7d domain comprising an amino acid sequence having at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 7; and a KOD polymerase variant comprising a threonine at position S407 of SEQ ID NO: 1, an arginine at position H147 of SEQ ID NO: 1, an arginine at position A675 of SEQ ID NO: 1, and having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1. In some embodiments, a fusion protein comprises: an Sso7d domain comprising an amino acid sequence of SEQ ID NO: 7; and a KOD polymerase variant comprising a threonine at position S407 of SEQ ID NO:

[0127] #14495181v1 1, an arginine at position H147 of SEQ ID NO: 1, an arginine at position A675, and having at least 90% e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1.

[0128] In some embodiments, a fusion protein comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 28. In some embodiments, a fusion protein comprises an amino acid sequence having at least 95% identity to SEQ ID NO: 28. In some embodiments, a fusion protein comprises an amino acid sequence having at least 98% identity to SEQ ID NO: 28. In some embodiments, a fusion protein comprises an amino acid sequence having at least 99% identity to SEQ ID NO: 28. In some embodiments, a fusion protein comprises an amino acid sequence having at least 99.5% identity to SEQ ID NO: 28. In some embodiments, a fusion protein comprises an amino acid sequence of SEQ ID NO: 28. In some embodiments, a fusion protein consists of an amino acid sequence of SEQ ID NO: 28.

[0129] S407, H147, and E664 KOD Polymerase Variants

[0130] In some embodiments, a KOD polymerase variant comprises a mutation at a position corresponding to position S407, a mutation at a position corresponding to H147, and a mutation at a position corresponding to E664 of SEQ ID NO: 1.

[0131] In some embodiments, a KOD polymerase variant comprises a threonine at a position corresponding to S407 of SEQ ID NO: 1, an arginine at a position corresponding to H147 of SEQ ID NO: 1, and an arginine at a position corresponding to E664 of SEQ ID NO: 1. In some embodiments, a KOD polymerase variant comprises a threonine at a position corresponding to S407 of SEQ ID NO: 1; an arginine at a position corresponding to H147 of SEQ ID NO: 1; an arginine at a position corresponding to E664 of SEQ ID NO: 1; and one, two, three, or four additional mutations (e.g., amino acid substitutions) in SEQ ID NO: 1.

[0132] In some embodiments, a KOD polymerase variant comprises: an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1; a threonine at a position corresponding to S407 of SEQ ID NO: 1; an arginine at a position corresponding to H147 of SEQ ID NO: 1; and an arginine at a position corresponding to E664 of SEQ ID NO: 1.

[0133] In some embodiments, a KOD polymerase variant comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 26. In some embodiments, a KOD polymerase variant comprises an amino acid sequence having at least 95% identity to SEQ ID NO: 26. In some embodiments, a KOD polymerase variant comprises an amino acid sequence having at least 98% identity to SEQ ID NO: 26. In some embodiments, a KOD polymerase variant comprises an amino acid sequence having at least 99% identity to SEQ ID NO: 26. In some

[0134] #14495181v1 embodiments, a KOD polymerase variant comprises an amino acid sequence having at least 99.5% identity to SEQ ID NO: 26. In some embodiments, a KOD polymerase variant comprises an amino acid sequence of SEQ ID NO: 26. In some embodiments, a KOD polymerase variant consists of an amino acid sequence of SEQ ID NO: 26.

[0135] S407, H147, E664, and Sso7d KOD Polymerase Variants

[0136] In some embodiments, this disclosure provides a fusion protein comprising an Sso7d domain and a KOD polymerase variant comprising a mutation at a position corresponding to S407, a mutation at a position corresponding to H147 of SEQ ID NO: 1, and a mutation at a position corresponding to E664 of SEQ ID NO: 1.

[0137] In some embodiments, a fusion protein comprises an Sso7d domain and a KOD polymerase variant comprising a threonine at a position corresponding to S407, an arginine at a position corresponding to H147 of SEQ ID NO: 1, and a lysine at a position corresponding to E664 of SEQ ID NO: 1. In some embodiments, a fusion protein comprises: an Sso7d domain; and a KOD polymerase variant comprising a threonine at position S407 of SEQ ID NO: 1; an arginine at a position corresponding to H147 of SEQ ID NO: 1; a lysine at a position corresponding to E664 of SEQ ID NO: 1; and one, two, three or four additional mutations (e.g., amino acid substitutions) in SEQ ID NO: 1. In some embodiments, a fusion protein comprises: an Sso7d domain; and a KOD polymerase variant comprising a threonine at position S407 of SEQ ID NO: 1, an arginine at position H147 of SEQ ID NO: 1, a lysine at a position E664 of SEQ ID NO: 1, and having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1. In some embodiments, a fusion protein comprises: an Sso7d domain comprising an amino acid sequence having at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 7; and a KOD polymerase variant comprising a threonine at position S407 of SEQ ID NO: 1, an arginine at position H147 of SEQ ID NO: 1, a lysine at position E664 of SEQ ID NO: 1, and having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1. In some embodiments, a fusion protein comprises: an Sso7d domain comprising an amino acid sequence of SEQ ID NO: 7; and a KOD polymerase variant comprising a threonine at position S407 of SEQ ID NO: 1, an arginine at position H147 of SEQ ID NO: 1, a lysine at position E664, and having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1.

[0138] In some embodiments, a fusion protein comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 27. In some embodiments, a fusion protein comprises an amino acid sequence having at least 95% identity to SEQ ID NO: 27. In some embodiments, a fusion

[0139] #14495181v1 protein comprises an amino acid sequence having at least 98% identity to SEQ ID NO: 27. In some embodiments, a fusion protein comprises an amino acid sequence having at least 99% identity to SEQ ID NO: 27. In some embodiments, a fusion protein comprises an amino acid sequence having at least 99.5% identity to SEQ ID NO: 27. In some embodiments, a fusion protein comprises an amino acid sequence of SEQ ID NO: 27. In some embodiments, a fusion protein consists of an amino acid sequence of SEQ ID NO: 27.

[0140] Pfu Polymerase Variants

[0141] In some aspects, this disclosure provides a Pfu polymerase variant. In some embodiments, the Pfu polymerase variant comprises improved replication fidelity compared to wildtype Pfu polymerase. A “Pyrococcus furiosus (Pfu) polymerase” refers to a B-family DNA polymerase of Pyrococcus furiosus (e.g., a wildtype B-family Pfu polymerase). In some embodiments, a Pfu polymerase comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99% identity) to SEQ ID NO: 4. In some embodiments, a Pfu polymerase comprises an amino acid sequence of SEQ ID NO: 4.

[0142] A “Pyrococcus furiosus (Pfu) polymerase variant” refers to a Pfu polymerase that comprises at least one amino acid mutation relative to a Pfu polymerase (e.g., relative to a wildtype Pfu polymerase (SEQ ID NO: 4)). In some embodiments, a Pfu polymerase variant comprises a substitution mutation relative to a Pfu polymerase. In some embodiments, a Pfu polymerase variant comprises one or more substitution mutations relative to a Pfu polymerase. In some embodiments, a Pfu polymerase variant comprises an insertion and / or deletion mutation relative to a Pfu polymerase. In some embodiments, a Pfu polymerase variant comprises a substitution mutation that increases salt tolerance of the Pfu polymerase variant relative to a Pfu polymerase (e.g., increased activity of the Pfu polymerase variant in the presence of salt and / or salt concentration relative to a Pfu polymerase).

[0143] In some embodiments, a Pfu polymerase variant further comprises a protein tag. In some embodiments, the protein tag is a purification tag (e.g., a His tag, a Myc tag, or a Flag tag). In some embodiments, the protein tag is a secretion tag.

[0144] In some embodiments, this disclosure provides a fusion protein comprising a Pfu polymerase variant and an Sso7d domain. In some embodiments, a fusion protein comprising a Pfu polymerase variant and an Sso7d domain that exhibits improved thermostability compared to a fusion protein that does not comprise an Sso7d domain. In some embodiments, a fusion protein comprises, in N-terminal to C-terminal order, a Pfu polymerase variant and an Sso7d domain. In some embodiments, a fusion protein comprises, in C-terminal to N-terminal order,

[0145] #14495181v1 an Sso7d domain and a Pfu polymerase. In some embodiments, a fusion protein comprises, in N- terminal to C-terminal order, a Pfu polymerase variant, a linker, and an Sso7d domain. In some embodiments, a fusion protein comprises, in N-terminal to C-terminal order, an Sso7d domain, a linker, and a Pfu polymerase variant. In some embodiments, a linker is an amino acid linker. In some embodiments, an Sso7d domain comprises an amino acid sequence having at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 7. In some embodiments, an Sso7d domain comprises an amino acid sequence of SEQ ID NO: 7. In some embodiments, an Sso7d domain consists of an amino acid sequence of SEQ ID NO: 7.

[0146] In some embodiments, a mutation in a Pfu polymerase variant at position A408 and / or position E665 corresponding to SEQ ID NO: 4 alters the replication fidelity of the Pfu polymerase. In some embodiments, a mutation in a Pfu polymerase variant at position A408 and / or position E665 corresponding to SEQ ID NO: 4 alters the salt tolerance of the Pfu polymerase. In some embodiments, a mutation in a Pfu polymerase variant at position A408 and / or position E665 corresponding to SEQ ID NO: 4 alters the replication fidelity and salt tolerance of the Pfu polymerase.

[0147] A408 Pfu Polymerase Variants

[0148] In some embodiments, a Pfu polymerase variant comprises a mutation at a position corresponding to position A408 of SEQ ID NO: 4.

[0149] In some embodiments, this disclosure provides a Pfu polymerase variant comprising a threonine at a position corresponding to A408 of SEQ ID NO: 4 (e.g., A408T in a Pfu polymerase corresponding to SEQ ID NO: 4). In some embodiments, this disclosure provides a Pfu polymerase variant comprising a threonine at position A408 of SEQ ID NO: 4 and one, two, three or four additional mutations (e.g., amino acid substitutions) in SEQ ID NO: 4. In some embodiments, a Pfu polymerase variant comprises: an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 4 and a threonine at a position corresponding to A408 of SEQ ID NO: 4. In some embodiments, a Pfu polymerase variant comprises a threonine at position A408 of SEQ ID NO: 4. In some embodiments, a Pfu polymerase variant comprises a threonine at a position corresponding to A408 of SEQ ID NO: 4.

[0150] In some embodiments, a Pfu polymerase variant comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 5. In some embodiments, a Pfu polymerase variant comprises an amino acid sequence having at least 95% identity to SEQ ID NO: 5. In some embodiments, a Pfu polymerase variant comprises an amino acid sequence having at least 98%

[0151] #14495181v1 identity to SEQ ID NO: 5. In some embodiments, a Pfu polymerase variant comprises an amino acid sequence having at least 99% identity to SEQ ID NO: 5. In some embodiments, a Pfu polymerase variant comprises an amino acid sequence having at least 99.5% identity to SEQ ID NO: 5. In some embodiments, a Pfu polymerase variant comprises an amino acid sequence of SEQ ID NO: 5. In some embodiments, a Pfu polymerase variant consists of an amino acid sequence of SEQ ID NO: 5.

[0152] A408 and E665 Pfu Polymerase Variants

[0153] In some embodiments, a Pfu polymerase variant comprises a mutation at a position corresponding to position E665 and a mutation at a position corresponding to A408 of SEQ ID NO: 4.

[0154] In some embodiments, a Pfu polymerase variant comprises a lysine at a position corresponding to E665 of SEQ ID NO: 4 and a threonine at a position corresponding to A408 of SEQ ID NO: 4. In some embodiments, a Pfu polymerase variant comprises a lysine at position E665 of SEQ ID NO: 4; a threonine at position A408 of SEQ ID NO: 4; and one, two, three or four additional mutations (e.g., amino acid substitutions) in SEQ ID NO: 4.

[0155] In some embodiments, a Pfu polymerase variant comprises: an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 4; a lysine at position E665 of SEQ ID NO: 4; and a threonine at position A408 of SEQ ID NO: 4.

[0156] In some embodiments, the Pfu polymerase variant comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 6. In some embodiments, the Pfu polymerase variant comprises an amino acid sequence having at least 95% identity to SEQ ID NO: 6. In some embodiments, the Pfu polymerase variant comprises an amino acid sequence having at least 98% identity to SEQ ID NO: 6. In some embodiments, the Pfu polymerase variant comprises an amino acid sequence having at least 99% identity to SEQ ID NO: 6. In some embodiments, the Pfu polymerase variant comprises an amino acid sequence having at least 99.5% identity to SEQ ID NO: 6. In some embodiments, the Pfu polymerase variant comprises an amino acid sequence of SEQ ID NO: 6. In some embodiments, the Pfu polymerase variant consists of an amino acid sequence of SEQ ID NO: 6.

[0157] A408 and Sso7d Pfu Polymerase Variants

[0158] In some embodiments, this disclosure provides a fusion protein comprising a Pfu polymerase variant and an Sso7d domain.

[0159] #14495181v1 In some embodiments, a fusion protein comprises an Sso7d domain and a Pfu polymerase variant comprising a threonine at a position corresponding to A408 of SEQ ID NO: 4. In some embodiments, a fusion protein comprises: an Sso7d domain; and a Pfu polymerase variant comprising a threonine at position A408 of SEQ ID NO: 4 and one, two, three or four additional mutations (e.g., amino acid substitutions) in SEQ ID NO: 4.

[0160] In some embodiments, a fusion protein comprises: an Sso7d domain; and a Pfu polymerase variant comprising a threonine at position A408 of SEQ ID NO: 4 and having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 4.

[0161] In some embodiments, a fusion protein comprises: an Sso7d domain comprising an amino acid sequence having at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 7; and a Pfu polymerase variant comprising a threonine at position A408 of SEQ ID NO: 4 and having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 4.

[0162] In some embodiments, a fusion protein comprises: an Sso7d domain comprising an amino acid sequence of SEQ ID NO: 7; and a Pfu polymerase variant comprising a threonine at position A408 of SEQ ID NO: 4 and having an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 4.

[0163] In some embodiments, a fusion protein comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 8. In some embodiments, a fusion protein comprises an amino acid sequence having at least 95% identity to SEQ ID NO: 8. In some embodiments, a fusion protein comprises an amino acid sequence having at least 98% identity to SEQ ID NO: 8. In some embodiments, a fusion protein comprises an amino acid sequence having at least 99% identity to SEQ ID NO: 8. In some embodiments, a fusion protein comprises an amino acid sequence having at least 99.5% identity to SEQ ID NO: 8. In some embodiments, a fusion protein comprises an amino acid sequence of SEQ ID NO: 8.

[0164] A408, E665, and Sso7d Pfu Polymerase Variants

[0165] In some embodiments, this disclosure provides a fusion protein comprising an Sso7d domain and a Pfu polymerase variant comprising a mutation at a position corresponding to A408 and a mutation at a position corresponding to E665 of SEQ ID NO: 4. In some embodiments, a fusion protein comprises an Sso7d domain and a Pfu polymerase variant comprising a threonine at a position corresponding to A408 and a lysine at a position corresponding to E665 of SEQ ID NO: 4. In some embodiments, a fusion protein comprises: an Sso7d domain; and a Pfu polymerase variant comprising a threonine at position A408 of SEQ ID NO: 4, a lysine at a

[0166] #14495181v1 position corresponding to E665 of SEQ ID NO: 4, and one, two, three or four additional mutations (e.g., amino acid substitutions) in SEQ ID NO: 4. In some embodiments, a fusion protein comprises: an Sso7d domain; and a Pfu polymerase variant comprising a threonine at position A408 of SEQ ID NO: 4, a lysine at position E665 of SEQ ID NO: 4, and having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 4. In some embodiments, a fusion protein comprises: an Sso7d domain comprising an amino acid sequence having at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 7; and a Pfu polymerase variant comprising a threonine at position A408 of SEQ ID NO: 4, a lysine at position E665 of SEQ ID NO: 4, and having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 4. In some embodiments, a fusion protein comprises: an Sso7d domain comprising an amino acid sequence of SEQ ID NO: 7; and a Pfu polymerase variant comprising a threonine at position A408 of SEQ ID NO: 4, a lysine at position E665 of SEQ ID NO: 4, and having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 4.

[0167] In some embodiments, a fusion protein comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 9. In some embodiments, a fusion protein comprises an amino acid sequence having at least 95% identity to SEQ ID NO: 9. In some embodiments, a fusion protein comprises an amino acid sequence having at least 98% identity to SEQ ID NO: 9. In some embodiments, a fusion protein comprises an amino acid sequence having at least 99% identity to SEQ ID NO: 9. In some embodiments, a fusion protein comprises an amino acid sequence having at least 99.5% identity to SEQ ID NO: 9. In some embodiments, a fusion protein comprises an amino acid sequence of SEQ ID NO: 9.

[0168] Additional Pfu Polymerase Variants

[0169] In some embodiments, a Pfu variant comprises an arginine at a position corresponding to A675R of SEQ ID NO: 1. In some embodiments, a Pfu variant comprises an arginine at a position corresponding to H147 of SEQ ID NO: 1. In some embodiments, a Pfu variant comprises an isoleucine at a position corresponding to L408 of SEQ ID NO: 1. In some embodiments, a Pfu variant comprises one or more of: a threonine at a position corresponding to S407 of SEQ ID NO: 1; a lysine at a position corresponding to E664 of SEQ ID NO: 1; an arginine at a position corresponding to A675R of SEQ ID NO: 1; an arginine at a position corresponding to H147 of SEQ ID NO: 1; and an isoleucine at a position corresponding to L408 of SEQ ID NO: 1.

[0170] #14495181v1 In some embodiments, a Pfu variant comprises (i) an arginine at a position corresponding to A675R of SEQ ID NO: 1 and (ii) a sequence having at least 90% identity (e.g., at least 95, at least 98, or at least 99%) to SEQ ID NO: 4. In some embodiments, a Pfu variant comprises (i) an arginine at a position corresponding to H147 of SEQ ID NO: 1 and (ii) a sequence having at least 90% identity (e.g., at least 95, at least 98, or at least 99%) to SEQ ID NO: 4. In some embodiments, a Pfu variant comprises (i) an isoleucine at a position corresponding to L408 of SEQ ID NO: 1 and (ii) a sequence having at least 90% identity (e.g., at least 95, at least 98, or at least 99%) to SEQ ID NO: 4.

[0171] In some embodiments, a Pfu variant comprises (i) one or more of a threonine at a position corresponding to S407 of SEQ ID NO: 1; a lysine at a position corresponding to E664 of SEQ ID NO: 1; an arginine at a position corresponding to A675R of SEQ ID NO: 1; an arginine at a position corresponding to H147 of SEQ ID NO: 1; and an isoleucine at a position corresponding to L408 of SEQ ID NO: 1 and (ii) a sequence having at least 90% identity (e.g., at least 95, at least 98, or at least 99%) to SEQ ID NO: 4.

[0172] Methods of Use

[0173] Aspects of this disclosure are drawn to methods of amplifying target polynucleotides using archaeal B -family polymerase variants with improved replication fidelity. In some embodiments, this disclosure provides a method of amplifying a target polynucleotide, the method comprising contacting a target polynucleotide with an archaeal B -family DNA polymerase variant. In some embodiments, this disclosure provides a method of amplifying a target polynucleotide, the method comprising contacting the target polynucleotide with a KOD polymerase variant or a Pfu polymerase variant provided herein.

[0174] In some embodiments, an error rate of an archaeal B -family polymerase variant is calculated based on (i) identifying errors (e.g., a nucleotide at a position in an amplified polynucleotide that differs from the nucleotide in the same position in the target polynucleotide, or the “expected nucleotide”) in an amplified polynucleotide (e.g., a cytosine at a position in an amplified polynucleotide where there is a thymine at the same position in the target polynucleotide); (ii) summing the errors in the amplified polynucleotide; and (iii) dividing the sum of the errors by the number of positions in which the expected nucleotide is present in the target polynucleotide. Such a calculation provides an absolute value error rate based on the nucleotides present in the target polynucleotide and the deviations from the expected nucleotide (i.e., the nucleotide present at a particular position in the target polynucleotide) in the amplified polynucleotide.

[0175] #14495181v1 In some embodiments, amplifying the target polynucleotide comprises amplifying with an error rate that is less than 0.0020. In some embodiments, amplifying the target polynucleotide comprises amplifying with an error rate that is less than 0.010. In some embodiments, amplifying the target polynucleotide comprises amplifying with an error rate that is less than 0.0080. In some embodiments, amplifying the target polynucleotide comprises amplifying with an error rate that is less than 0.0060. In some embodiments, amplifying the target polynucleotide comprises amplifying with an error rate that is less than 0.0040. In some embodiments, amplifying the target polynucleotide comprises amplifying with an error rate that is less than 0.0020. In some embodiments, amplifying the target polynucleotide comprises amplifying with an error rate that is less than 0.0010. In some embodiments, amplifying the target polynucleotide comprises amplifying with an error rate that is less than 0.0005. In some embodiments, amplifying the target polynucleotide comprises amplifying with an error rate from 0.0005 to 0.0080. In some embodiments, amplifying the target polynucleotide comprises amplifying with an error rate from 0.0010 to 0.0070. In some embodiments, amplifying the target polynucleotide comprises amplifying with an error rate from 0.0020 to 0.0060. In some embodiments, amplifying the target polynucleotide comprises amplifying with an error rate from 0.0030 to 0.0050. In some embodiments, amplifying the target polynucleotide comprises amplifying with an error rate from 0 to 0.0020. In some embodiments, amplifying the target polynucleotide comprises amplifying with an error rate from 0.0005 to 0.0020. In some embodiments, amplifying the target polynucleotide comprises amplifying with an error rate from 0.0010 to 0.0030. In some embodiments, amplifying the target polynucleotide comprises amplifying with an error rate from 0.0020 to 0.0040. In some embodiments, amplifying the target polynucleotide comprises amplifying with an error rate from 0.0015 to 0.0025.

[0176] In some embodiments, amplifying the target polynucleotide with an archaeal B-family polymerase variant comprises amplifying with an error rate at least 0.15 times (at least 0.15 times, at least 0.25 times, at least 0.25 times, at least 0.5 times, at least 0.75 times, at least 1 times, at least 2 times, at least 5 times, or at least 10 times) lower than with a wild- type archaeal B-family polymerase.

[0177] In some embodiments, amplifying the target polynucleotide with an archaeal B-family polymerase variant comprises amplifying with an error rate 0.15 times (0.15x) to 0.65x lower than with a wild-type archaeal B-family polymerase. In some embodiments, amplifying the target polynucleotide with an archaeal B-family polymerase variant comprises amplifying with an error rate 0.20x to 0.65x lower than with a wild-type archaeal B-family polymerase. In some embodiments, amplifying the target polynucleotide with an archaeal B-family polymerase

[0178] #14495181v1 variant comprises amplifying with an error rate 0.25x to 0.65x lower than with a wild-type archaeal B-family polymerase. In some embodiments, amplifying the target polynucleotide with an archaeal B-family polymerase variant comprises amplifying with an error rate 0.30x to 0.65x lower than with a wild-type archaeal B-family polymerase. In some embodiments, amplifying the target polynucleotide with an archaeal B-family polymerase variant comprises amplifying with an error rate 0.35x to 0.65x lower than with a wild-type archaeal B-family polymerase. In some embodiments, amplifying the target polynucleotide with an archaeal B-family polymerase variant comprises amplifying with an error rate 0.40x to 0.65x lower than with a wild-type archaeal B-family polymerase. In some embodiments, amplifying the target polynucleotide with an archaeal B-family polymerase variant comprises amplifying with an error rate 0.45x to 0.65x lower than with a wild-type archaeal B-family polymerase. In some embodiments, amplifying the target polynucleotide with an archaeal B-family polymerase variant comprises amplifying with an error rate 0.50x to 0.65x lower than with a wild-type archaeal B-family polymerase. In some embodiments, amplifying the target polynucleotide with an archaeal B-family polymerase variant comprises amplifying with an error rate 0.55x to 0.65x lower than with a wild-type archaeal B-family polymerase. In some embodiments, amplifying the target polynucleotide with an archaeal B-family polymerase variant comprises amplifying with an error rate 0.60x to 0.65x lower than with a wild-type archaeal B-family polymerase. In some embodiments, amplifying the target polynucleotide with an archaeal B-family polymerase variant comprises amplifying with an error rate 0.15x to 0.60x lower than with a wild-type archaeal B-family polymerase. In some embodiments, amplifying the target polynucleotide with an archaeal B-family polymerase variant comprises amplifying with an error rate 0.15x to 0.55x lower than with a wild-type archaeal B-family polymerase. In some embodiments, amplifying the target polynucleotide with an archaeal B-family polymerase variant comprises amplifying with an error rate 0.15x to 0.50x lower than with a wild-type archaeal B-family polymerase. In some embodiments, amplifying the target polynucleotide with an archaeal B-family polymerase variant comprises amplifying with an error rate 0.15x to 0.45x lower than with a wild-type archaeal B-family polymerase. In some embodiments, amplifying the target polynucleotide with an archaeal B-family polymerase variant comprises amplifying with an error rate 0.15x to 0.40x lower than with a wild-type archaeal B-family polymerase. In some embodiments, amplifying the target polynucleotide with an archaeal B-family polymerase variant comprises amplifying with an error rate 0.15x to 0.35x lower than with a wild-type archaeal B-family polymerase. In some embodiments, amplifying the target polynucleotide with an archaeal B-family polymerase variant comprises amplifying with an error rate 0.15x to 0.30x lower than with a wild-type archaeal B-family polymerase. In

[0179] #14495181v1 some embodiments, amplifying the target polynucleotide with an archaeal B-family polymerase variant comprises amplifying with an error rate 0.15x to 0.25x lower than with a wild-type archaeal B-family polymerase. In some embodiments, amplifying the target polynucleotide with an archaeal B-family polymerase variant comprises amplifying with an error rate 0.15x to 0.20x lower than with a wild-type archaeal B-family polymerase. In some embodiments, amplifying the target polynucleotide with an archaeal B-family polymerase variant comprises amplifying with an error rate 0.15x to 0.50x lower than with a wild-type archaeal B-family polymerase.

[0180] In some embodiments, amplifying the target polynucleotide with an archaeal B-family polymerase variant comprises amplifying with an error rate 15% to 60% lower than with a wildtype archaeal B-family polymerase. In some embodiments, amplifying the target polynucleotide with an archaeal B-family polymerase variant comprises amplifying with an error rate 25% to 60% lower than with a wild-type archaeal B-family polymerase. In some embodiments, amplifying the target polynucleotide with an archaeal B-family polymerase variant comprises amplifying with an error rate 35% to 60% lower than with a wild-type archaeal B-family polymerase. In some embodiments, amplifying the target polynucleotide with an archaeal B- family polymerase variant comprises amplifying with an error rate 45% to 60% lower than with a wild-type archaeal B-family polymerase. In some embodiments, amplifying the target polynucleotide with an archaeal B-family polymerase variant comprises amplifying with an error rate 55% to 60% lower than with a wild-type archaeal B-family polymerase. In some embodiments, amplifying the target polynucleotide with an archaeal B-family polymerase variant comprises amplifying with an error rate 15% to 50% lower than with a wild-type archaeal B-family polymerase. In some embodiments, amplifying the target polynucleotide with an archaeal B-family polymerase variant comprises amplifying with an error rate 15% to 40% lower than with a wild-type archaeal B-family polymerase. In some embodiments, amplifying the target polynucleotide with an archaeal B-family polymerase variant comprises amplifying with an error rate 15% to 30% lower than with a wild-type archaeal B-family polymerase. In some embodiments, amplifying the target polynucleotide with an archaeal B-family polymerase variant comprises amplifying with an error rate 15% to 20% lower than with a wild-type archaeal B-family polymerase. In some embodiments, amplifying the target polynucleotide with an archaeal B-family polymerase variant comprises amplifying with an error rate 25% to 50% lower than with a wild-type archaeal B-family polymerase.

[0181] Archaeal B-family polymerases provided herein are useful for molecular biology applications, such as polymerase chain reaction (PCR). PCR amplifies DNA through repeated cycles of denaturing DNA, annealing primers complementary to the DNA, and extending DNA,

[0182] #14495181v1 which requires a wide range of temperatures (e.g., approximately 50 °C to 95 °C), to create large amounts of DNA from even small amounts of starting material. PCR reactions combine target DNA to be amplified, primers complementary to the target DNA or a portion thereof, nucleotides (dNTPs), buffer solutions, ions, and a DNA polymerase (e.g., a KOD polymerase variant or a Pfu polymerase variant provided herein) to amplify the target DNA.

[0183] A range of pH values of PCR reaction conditions can be used in the methods described herein. In some embodiments, PCR reaction conditions comprise a pH of 7.0 to 10.0. In some embodiments, PCR reaction conditions comprise a pH of 7.5 to 10.0. In some embodiments, PCR reaction conditions comprise a pH of 8.0 to 10.0. In some embodiments, PCR reaction conditions comprise a pH of 8.5 to 10.0. In some embodiments, PCR reaction conditions comprise a pH of 9.0 to 10.0. In some embodiments, PCR reaction conditions comprise a pH of 9.5 to 10.0. In some embodiments, PCR reaction conditions comprise a pH of 8.2 to 9.2. In some embodiments, PCR reaction conditions comprise a pH of 8.4 to 9.0. In some embodiments, PCR reaction conditions comprise a pH of 8.6 to 8.8. In some embodiments, PCR reaction conditions comprise a pH of 8.8 to 9.8. In some embodiments, PCR reaction conditions comprise a pH of 9.0 to 9.6. In some embodiments, PCR reaction conditions comprise a pH of 9.2 to 9.4.

[0184] In some embodiments, PCR reaction conditions comprise a magnesium salt concentration (e.g., MgCh) of 0.5 mM to 5.0 mM. In some embodiments, PCR reaction conditions comprise a magnesium salt concentration (e.g., MgCh) of 1.5 mM to 5.0 mM. In some embodiments, PCR reaction conditions comprise a magnesium salt concentration (e.g., MgCh) of 2.0 mM to 5.0 mM. In some embodiments, PCR reaction conditions comprise a magnesium salt concentration (e.g., MgCh) of 2.5 mM to 5.0 mM. In some embodiments, PCR reaction conditions comprise a magnesium salt concentration (e.g., MgCh) of 3.0 mM to 5.0 mM. In some embodiments, PCR reaction conditions comprise a magnesium salt concentration (e.g., MgCh) of 3.5 mM to 5.0 mM. In some embodiments, PCR reaction conditions comprise a magnesium salt concentration (e.g., MgCh) of 4.0 mM to 5.0 mM. In some embodiments, PCR reaction conditions comprise a magnesium salt concentration (e.g., MgCh) of 4.5 mM to 5.0 mM. In some embodiments, PCR reaction conditions comprise a magnesium salt concentration (e.g., MgCh) of 1.0 mM to 4.0 mM. In some embodiments, PCR reaction conditions comprise a magnesium salt concentration (e.g., MgCh) of 1.5 mM to 4.0 mM. In some embodiments, PCR reaction conditions comprise a magnesium salt concentration (e.g., MgCh) of 1.5 mM to 3.5 mM. In some embodiments, PCR reaction conditions comprise a magnesium salt concentration (e.g., MgCh) of 1.5 mM to 3.0 mM. In some embodiments, PCR reaction conditions comprise a

[0185] #14495181v1 magnesium salt concentration (e.g., MgCh) of 1.5 mM to 2.5 mM. In some embodiments, PCR reaction conditions comprise a magnesium salt concentration e.g., MgCh) of 1.5 mM to 2.0 mM.

[0186] In some embodiments, PCR reaction conditions comprise a potassium salt concentration e.g., KC1) of 30 mM to 400 mM. In some embodiments, PCR reaction conditions comprise a potassium salt concentration (e.g., KC1) of 30 mM to 350 mM. In some embodiments, PCR reaction conditions comprise a potassium salt concentration (e.g., KC1) of 30 mM to 300 mM. In some embodiments, PCR reaction conditions comprise a potassium salt concentration (e.g., KC1) of 30 mM to 250 mM. In some embodiments, PCR reaction conditions comprise a potassium salt concentration (e.g., KC1) of 30 mM to 200 mM. In some embodiments, PCR reaction conditions comprise a potassium salt concentration (e.g., KC1) of 30 mM to 150 mM. In some embodiments, PCR reaction conditions comprise a potassium salt concentration (e.g., KC1) of 30 mM to 100 mM. In some embodiments, PCR reaction conditions comprise a potassium salt concentration (e.g., KC1) of 30 mM to 50 mM. In some embodiments, PCR reaction conditions comprise a potassium salt concentration (e.g., KC1) of 35 mM to 45 mM. In some embodiments, PCR reaction conditions comprise a potassium salt concentration (e.g., KC1) of 50 mM to 80 mM. In some embodiments, PCR reaction conditions comprise a potassium salt concentration (e.g., KC1) of 50 mM to 70 mM. In some embodiments, PCR reaction conditions comprise a potassium salt concentration (e.g., KC1) of 50 mM to 60 mM. In some embodiments, PCR reaction conditions comprise a potassium salt concentration (e.g., KC1) of up to 500 mM. In some embodiments, PCR reaction conditions comprise a potassium salt concentration (e.g., KC1) of up to 450 mM. In some embodiments, PCR reaction conditions comprise a potassium salt concentration (e.g., KC1) of up to 400 mM. In some embodiments, PCR reaction conditions comprise a potassium salt concentration (e.g., KC1) of 100 mM to 400 mM. In some embodiments, PCR reaction conditions comprise a potassium salt concentration (e.g., KC1) of 150 mM to 400 mM. In some embodiments, PCR reaction conditions comprise a potassium salt concentration (e.g., KC1) of 200 mM to 400 mM. In some embodiments, PCR reaction conditions comprise a potassium salt concentration (e.g., KC1) of 250 mM to 400 mM. In some embodiments, PCR reaction conditions comprise a potassium salt concentration (e.g., KC1) of 300 mM to 400 mM. In some embodiments, PCR reaction conditions comprise a potassium salt concentration (e.g., KC1) of 350 mM to 400 mM.

[0187] In some embodiments, PCR reaction conditions comprise an extension temperature of 65 °C to 80 °C. In some embodiments, PCR reaction conditions comprise an extension temperature of 70 °C to 80 °C. In some embodiments, PCR reaction conditions comprise an extension

[0188] #14495181v1 temperature of 75 °C to 80 °C. In some embodiments, PCR reaction conditions comprise an extension temperature of 65 °C to 78 °C. In some embodiments, PCR reaction conditions comprise an extension temperature of 65 °C to 76 °C. In some embodiments, PCR reaction conditions comprise an extension temperature of 65 °C to 74 °C. In some embodiments, PCR reaction conditions comprise an extension temperature of 65 °C to 72 °C. In some embodiments, PCR reaction conditions comprise an extension temperature of 65 °C to 70 °C. In some embodiments, PCR reaction conditions comprise an extension temperature of 68 °C to 74 °C.

[0189] PCR protocols are known in the art (see, e.g., Zhu et al. PCR past, present and future. Biotechniques. 2020, 10.2144 / btn-2020-0057).

[0190] EXAMPLES

[0191] Example 1. S407T KOD polymerase variants and A408A Pfu polymerase variants improve replication fidelity.

[0192] Archaeal B-family polymerases possess proofreading capabilities, which typically results in greater replication fidelity (i.e., lower error rate) compared to other polymerases. Nevertheless, errors (e.g., mutations such as substitutions, insertions, and / or deletions) can still be introduced during replication of polynucleotides, such as during polymerase chain reaction (PCR) amplification. Errors in replicated polynucleotides can affect downstream applications such as cloning, sequencing, and gene expression studies. Additionally, in the case of proteincoding nucleic acids, errors in replication can cause functional alterations in proteins. Moreover, amplification reactions typically require buffers containing salts, which can affect polymerase function and lead to sub-optimal yield of amplified polynucleotides. Thus, molecular biology applications can benefit from archaeal B-family polymerase variants (e.g., KOD polymerase variants and / or Pfu polymerase variants) that have enhanced replication fidelity.

[0193] Previous work has identified mutations that improve the fidelity of T4 DNA polymerase (e.g., Reha-Krantz. J Mol Biol, 1988. 202:711-724; Reha-Krantz and Nonay. J Biol Chem, 1994. 269:5635-5643; Reha-Krantz. Mutat Res, 1996. 350:9-16; Li et al. JMol Biol, 2010. 400:295- 308). T4 DNA polymerase is a phage DNA polymerase that has about 30% identity to archaeal family B polymerases like KOD and Pfu. Here, mutations from T4 DNA polymerase were made in corresponding positions in KOD and Pfu polymerase to determine whether these mutations could have a beneficial effect on fidelity. Given the 30% sequence similarity between T4 DNA polymerase and archaeal family polymerases, it was difficult to predict what the effects of T4 DNA polymerase mutations would be in KOD polymerase and Pfu polymerase. Surprisingly, results showed that one mutation consistently enhanced KOD and Pfu polymerase

[0194] #14495181v1 fidelity (e.g., KOD S407T and Pfu A408T), another mutation improved fidelity in some conditions tested but not others (e.g., KOD L408I), and other mutations had a negative effect on fidelity (KOD S340F and KOD I413V).

[0195] The error rate of KOD and Pfu polymerase variants were tested in amplification reactions (polymerase chain reactions, PCR) at polymerase concentrations of 0.5, 1, 2.5, 3.5, 4, 7.5, or 15 ng / pL (x-axis, third row from bottom). For some KOD variants, an increased enzyme concentration was used in fidelity measuring experiments to account for decreases in variant activity typically observed in high fidelity polymerases. The high-fidelity DNA polymerase Q5® was used as a control. Results show that select KOD and Pfu polymerase variants had lower error rates than Q5® (FIG. 1). Additionally, some KOD polymerase variants performed better than wild-type KOD polymerase (FIGs. 2-3).

[0196] The amplification template consisted of fragmented human genomic DNA with unique molecular identifiers (UMI)-containing ligated adaptors (FIG. 1) or a panel of regions of the human genome generated as synthetic constructs (FIG. 2) by gene synthesis (IDT). UMIs were included to enable downstream discrimination of errors introduced by sequencing vs those introduced by the amplification polymerases themselves.

[0197] KOD polymerase variants with a threonine at a position S407, a lysine at a position E664, and an Sso7d domain had markedly lower error rates than Q5® (FIG. 1, indicated by error rates below the dashed line), as well as wild- type KOD polymerase (FIGs. 2-3, horizontal line), at all enzyme concentrations tested compared to KOD polymerase with only the E664K mutation and Sso7d fusion. Additionally, KOD polymerase variants with only a threonine at position S407 of SEQ ID NO: 1, or a threonine at a position corresponding to S407 of SEQ ID NO: 1 and an arginine at a position corresponding to A675 of SEQ ID NO: 1, had lower error rates than wild-type KOD polymerase (FIG. 2, below the horizontal line), on par with the error rates observed using Q5®. KOD polymerase variants having an isoleucine at a position corresponding to L408 of SEQ ID NO: 1 and an Sso7d domain had lower error rates than wildtype KOD polymerase at lower enzyme concentrations, and lower error rates than wild-type KOD polymerase at all tested enzyme concentrations when the salt content was increased (FIG. 3). KOD polymerase variants having an arginine at a position corresponding to H147 of SEQ ID NO: 1 and (i) a threonine at a position corresponding to S407 of SEQ ID NO: 1 and an arginine at a position corresponding to A675 of SEQ ID NO: 1; (ii) a threonine at a position corresponding to S407 of SEQ ID NO: 1 and a lysine at a position corresponding to E664 of SEQ ID NO: 1; (iii) a threonine at a position corresponding to S407 of SEQ ID NO: 1, a lysine at a position corresponding to E664 of SEQ ID NO: 1, and an Sso7d domain; or (iv) a threonine

[0198] #14495181v1 at a position corresponding to S407 of SEQ ID NO: 1 and an Sso7d domain had improved replication fidelity compared to wild-type KOD polymerase, largely on par with the replication fidelity of Q5® (FIG. 2).

[0199] In contrast, other mutations identified in T4 polymerase as improving replication fidelity surprisingly worsened replication fidelity in KOD polymerase. KOD polymerase variants with a valine at a position corresponding to 1413 (a mutation associated with improved replication fidelity in T4 polymerase) of SEQ ID NO: 1, a lysine at a position corresponding to E664 of SEQ ID NO: 1, and an Sso7d domain had higher error rates than Q5® (FIG. 1). KOD polymerase variants with an Sso7d domain and a phenylalanine at a position corresponding to S340 of SEQ ID NO: 1 (a mutation associated with improved replication fidelity in T4 polymerase) had markedly higher error rates than Q5® and wild-type KOD polymerase (FIGs. 2- 3).

[0200] Pfu polymerase variants having a threonine at a position corresponding to A408 and a lysine at a position corresponding to E665 of SEQ ID NO: 4 had markedly lower error rates than Q5® (FIG. 1) at all enzyme concentrations tested compared to Pfu polymerase with only the E665K mutation and Sso7d fusion. In contrast, Pfu polymerase variants comprising a valine at a position corresponding to 1414 of SEQ ID NO: 4, a lysine at a position corresponding to E665 of SEQ ID NO: 4, and an Sso7d domain had higher error rates than Q5®.

[0201] Taken together, the data demonstrate that KOD and Pfu polymerase variants with particular mutations (e.g., a KOD polymerase variant with a threonine at position S407 and Pfu polymerase variant with a threonine at position A408 of SEQ ID NO: 4 have improved replication fidelity, as determined by error rate, compared to the same variants without the corresponding S407mutation(s) and the industry standard high fidelity polymerase Q5®. Mutations identified herein as improving replication fidelity could not have been predicted based on their impact on replication fidelity in the DNA polymerase T4.

[0202] Error Rate Calculation

[0203] First, the rate for each error type was calculated individually (e.g., C-to-T errors). These were bases in reads where the reference sequence had a C, but the read had a T at that position. The rate was calculated by dividing the total number of times a T is observed at a C position by the total count of Cs or Ts observed at positions expected to be Cs according to the reference. These rates per error type were then summed together for all error types to derive the plotted error rate.

[0204] #14495181v1 Note that the absolute values for these measurements were influenced by the assay used. The data from FIG. 1 comes from low copy number whole genome input. FIG.2 was a synthetic assay with difficult to amplify templates.

[0205] Table 1. Sequences of Exemplary KOD Polymerase Variants

[0206] #14495181v1

[0207] #14495181v1

[0208] #14495181v1

[0209] Table 2. Sequences of Exemplary Pfu Polymerase Variants

[0210] #14495181v1

[0211] #14495181v1

[0212] Table 3. Sequences of Additional Exemplary Archaeal B-Family Polymerase Variants

[0213] #14495181v1

[0214] #14495181v1

[0215] #14495181v1

Claims

CLAIMSWhat is claimed is:

1. A Thermococcus kodakaraensis (KOD) polymerase variant comprising a threonine at a position corresponding to S407 of SEQ ID NO: 1.

2. The KOD polymerase variant of claim 1, wherein the KOD polymerase variant comprises at least 95% identity to SEQ ID NO: 1.

3. The KOD polymerase variant of claim 1 or claim 2, further comprising a lysine at a position corresponding to E664 of SEQ ID NO: 1.

4. The KOD polymerase variant of any one of claims 1-3, further comprising an arginine at a position corresponding to A675 of SEQ ID NO: 1.

5. The KOD polymerase variant of any one of claims 1-4, further comprising an arginine at a position corresponding to H147 of SEQ ID NO: 1.

6. The KOD polymerase variant of any one of claims 1-5, further comprising an isoleucine at a position corresponding to L408 of SEQ ID NO: 1.

7. The KOD polymerase variant of claim 1, comprising an amino acid sequence having at least 95% identity to SEQ ID NO: 2.

8. The KOD polymerase variant of claim 1, comprising an amino acid sequence of SEQ ID NO: 2.

9. The KOD polymerase variant of claim 1, comprising an amino acid sequence having at least 95% identity to SEQ ID NO: 3.

10. The KOD polymerase variant of claim 7, comprising an amino acid sequence of SEQ ID NO: 3.#14495181v111. A Pyrococcus furiosus (Pfu) polymerase variant comprising a threonine at a position corresponding to A408 of SEQ ID NO: 4.

12. The Pfu polymerase variant of claim 11, wherein the Pfu polymerase variant comprises at least 95% identity to SEQ ID NO: 4.

13. The Pfu polymerase variant of claim 11 or claim 12, further comprising a lysine at a position corresponding to E665 of SEQ ID NO: 4.

14. The Pfu polymerase variant of claim 11, comprising an amino acid sequence having at least 95% identity to SEQ ID NO: 5.

15. The Pfu polymerase variant of claim 11, comprising an amino acid sequence of SEQ ID NO: 5.

16. The Pfu polymerase variant of claim 15, comprising an amino acid sequence having at least 95% identity to SEQ ID NO: 6.

17. The Pfu polymerase variant of claim 16, comprising an amino acid sequence of SEQ ID NO: 6.

18. The Pfu polymerase variant of any one of claims 11-17, further comprising a Sso7d domain.

19. The Pfu polymerase variant of claim 18, wherein the Sso7d domain comprises an amino acid sequence of SEQ ID NO: 7.

20. The Pfu polymerase variant of claim 11, comprising an amino acid sequence of SEQ ID NO: 8.

21. The Pfu polymerase variant of claim 11, comprising an amino acid sequence of SEQ ID NO: 9.#14495181v122. A method of amplifying a target polynucleotide, the method comprising contacting the target polynucleotide with the KOD polymerase variant of any one of claims 1-10 or the Pfu polymerase variant of any one of claims 11-21.

23. The method of claim 22, wherein amplifying the target polynucleotide comprises amplifying with an error rate that is less than 0.0020.#14495181v1

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