Methods and compositions relating to phage RPA protein production

Abstract

The technology described herein relates to methods for robust and cost-efficient nucleic acid detection using recombinase polymerase amplification (RPA). Disclosed herein are GP32, UvsX, and UvsY polypeptides that provide highly efficient RPA at significantly lower temperatures than current methods, and which can be readily produced using the technology described herein.

Description

METHODS AND COMPOSITIONS RELATING TO PHAGE RPA PROTEIN PRODUCTIONCROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims benefit under 35 U.S.C. §119 of U.S. Provisional Application No. 63 / 564,105, filed March 12, 2024, the contents of which are incorporated herein by reference in its entirety.GOVERNMENT SUPPORT

[0002] This invention was made with government support under DI 8AC00006 awarded by U.S. Department of Defense / Defense Advanced Research Projects Agency (DOD / DARPA). The government has certain rights in the invention.SEQUENCE LISTING

[0003] The instant application contains a Sequence Listing which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. Said XML copy, created on March 4, 2025, is named 002806-00013 lWOPT_SL.xml and is 86,016 bytes in size.TECHNICAL FIELD

[0004] The technology described herein relates to methods and compositions related to DNA amplification, e.g., recombinase polymerase amplification (RPA).BACKGROUND

[0005] Nucleic acid detection is most commonly performed by PCR (polymerase chain reaction). However, PCR requires careful and variable temperature control, as well-trained personnel which makes it unsuitable for most point of need applications.

[0006] Nucleic acid detection methods that avoid the precisely controlled temperature changes necessary for PCR are known. An example is recombinase polymerase amplification (RPA). RPA is a constant temperature approach to amplify a double stranded target DNA (Fig. 1). In RPA, UvsX targets primers to a DNA template. UvsY, facilitates formation of UvsX complexes on ssDNA. Once the primer is present, Gp32 helps to stabilizes the resulting structures. Finally, DNA synthesis is catalyzed by a DNA polymerase. Current commercial applications of RPA have relied on UvsX, UvsY and Gp32 from bacteriophage T4 along with Bsu Pol I.

[0007] Several current issues hamper implementation of RPA. First, these T4 enzymes function best at elevated temperatures (37-42 degrees C) which means that commercial applications of RPA have required a heating element. Second, RPA reactions require a relatively large amount of protein and T4 UvsY is difficult to produce and use in such concentrations. Third, current RPA technology requires the use of crowding agents.SUMMARY

[0008] The technology disclosed herein relates to alternatives to T4 UvsX, UvxY, and Gp32. These new alternatives are functional at lower temperatures, permitting more robust and cost-effectiveRPA. Additionally, these new alternatives are easier to produce and provide robust performance without the use of crowding agents.

[0009] In one aspect of any of the embodiments, described herein is a composition or combination comprising one or more of: a) at least one isolated Gp32 polypeptide; b) at least one isolated UvsY polypeptide; and c) at least one isolated UvsX polypeptide.

[0010] In some embodiments of any of the aspects, the at least one isolated Gp32 polypeptide, the at least one isolated UvsY polypeptide, and / or the at least one isolated UvsX polypeptide comprises an affinity tag.

[0011] In some embodiments of any of the aspects, the composition or combination comprises two or more polypeptides selected from: a) at least one isolated Gp32 polypeptide, b) at least one isolated UvsY polypeptide, and c) at least one isolated UvsX polypeptide; wherein the two or more polypeptides do not naturally occur in the same phage and / or are not naturally encoded by the same phage genome.In some embodiments of any of the aspects, the one or more polypeptides are Shigella phage Shfl2 polypeptides, Citrobacter phage CF1 ERZ-2017 polypeptides, Serratia phage CHU4 polypeptides, Edwardsiella phage PEi20 polypeptides, Escherichia phage ECD7 polypeptides, Aeromonas phage PX29 polypeptides, or Pseudomonas phage pfl6 polypeptides. In some embodiments of any of the aspects, the one or more polypeptides comprise a sequence selected from SEQ ID NOs: 1-21.

[0012] In one aspect of any of the embodiments, described herein is a polynucleotide or combination of polynucleotides comprising a sequence encoding one or more of: a) at least one Gp32 polypeptide; b) at least one UvsY polypeptide; and c) at least one UvsX polypeptide.In some embodiments of any of the aspects, the sequence encoding a polypeptide is operably connected to a heterologous promoter. In some embodiments of any of the aspects, the sequence encoding a polypeptide further encodes an affinity tag. In some embodiments of any of the aspects, the one or more polypeptides are Shigella phage Shfl2 polypeptides, Citrobacter phage CF1 ERZ- 2017 polypeptides, Serratia phage CHI14 polypeptides, Edwardsiella phage PEi20 polypeptides, Escherichia phage ECD7 polypeptides, Aeromonas phage PX29 polypeptides, or Pseudomonas phage pfl6 polypeptides. In some embodiments of any of the aspects, the one or more polypeptides comprise a sequence selected from SEQ ID NOs: 1-21.

[0013] In one aspect of any of the embodiments, described herein is a bacterium comprising a polynucleotide or combination of polynucleotides as described herein, or comprising a composition or combination as described herein. In some embodiments of any of the aspects, the bacterium is Escherichia coli.

[0014] In one aspect of any of the embodiments, described herein is a composition, combination, or kit, comprising: a) one or more of: i) at least one Gp32 polypeptide, ii) at least one UvsY polypeptide, and iii) at least one UvsX polypeptide; b) at least one of: iv) a polymerase I polypeptide; v) at least one polynucleotide comprising a sequence not naturally occurring in Shigella phage Shfl2, Citrohacter phage CF1 ERZ-2017, Serratia phage CHI 14, Edwardsiella phage PEi20, Escherichia phage ECD7, Aeromonas phage PX29, and Pseudomonas phage pfl6.In some embodiments of any of the aspects, the polymerase I polypeptide is Bsu Pol I. In some embodiments of any of the aspects, the at least one polynucleotide comprises a sequence not naturally occurring in a phage. In some embodiments of any of the aspects, the at least one polynucleotide comprises one or more primer molecules and / or one or more double -stranded target polynucleotides. In some embodiments of any of the aspects, the at least one polynucleotide comprises one or more primer molecules. In some embodiments of any of the aspects, the one or more primer molecules comprise at least one detectable label. In some embodiments of any of the aspects, the one or more double -stranded target polynucleotides comprise a coronavirus genome or fragment thereof. In some embodiments of any of the aspects, the one or more primer molecules are complementary to sequences naturally occurring in a coronavirus genome. In some embodiments of any of the aspects, the composition, combination, or kit does not comprise a crowding agent.

[0015] In some embodiments of any of the aspects, the combination, composition, or kit further comprises a buffer comprising at least one of: a) 5 mM or greater NaCl; b) 1 mM or greater Tris-HCl; c) 1 mM or greater MgCE; d) 0.1 mM or greater DTT; and e) pH 7.9 at 25°C.

[0016] In some embodiments of any of the aspects, the combination, composition, or kit further comprises a buffer comprising at least one of:a) 50 mM NaCl; b) 10 mM Tris-HCl; c) 10 mM MgCl2; d) l mM DTT; and e) pH 7.9 at 25°C.

[0017] In some embodiments of any of the aspects, the combination, composition, or kit further comprises a buffer comprising at least one of: a) 5 mM or greater Potassium acetate; b) 2 mM or greater Tris-acetate; c) 1 mM or greater Magnesium acetate; d) 0.1 mM or greater DTT; and e) pH 7.9 at 25°C.

[0018] In some embodiments of any of the aspects, the combination, composition, or kit further comprises a buffer comprising at least one of: a) 50 mM Potassium acetate; b) 20 mM Tris-acetate; c) 10 mM Magnesium acetate; d) l mM DTT; and e) pH 7.9 at 25°C.

[0019] In some embodiments of any of the aspects, the composition, combination, or kit can amplify a polynucleotide sequence at a temperature of less than 37 °C. In some embodiments of any of the aspects, the composition, combination, or kit can amplify a polynucleotide sequence with a higher fidelity than a composition, combination, or kit comprising T4 Gp32 polypeptide, T4 UvsY polypeptide, and T4 UvsX polypeptide.

[0020] In one aspect of any of the embodiments, described herein is a method of amplifying a polynucleotide sequence, the method comprising contacting a target polynucleotide with a composition, combination, or kit as described herein. In some embodiments of any of the aspects, the contacting occurs at a temperature of less than 37 °C. In some embodiments of any of the aspects, the target polynucleotide comprises a coronavirus genome or fragment thereof.

[0021] In one aspect of any of the embodiments, described herein is a method of detecting an organism or virus in a sample, the method comprising contacting a sample with a composition, combination, or kit as described herein; wherein amplification of a target polynucleotide found in the organism’s or virus’ genome indicates the organism or virus is present in the sample.

[0022] In one aspect of any of the embodiments, described herein is an assay comprising contacting a sample with a composition, combination, or kit as described herein.

[0023] In one aspect of any of the embodiments, described herein is a method of producing a composition comprising one or more polypeptides selected from: a) at least one isolated Gp32 polypeptide; b) at least one isolated UvsY polypeptide; and c) at least one isolated UvsX polypeptide; the method comprising: contacting a bacterium, a supernatant from a culture of a bacterium, or a lysate of a bacterium with at least one affinity reagent specific for an affinity tag; wherein the bacterium comprises a polynucleotide or combination of polynucleotides encoding one or more polypeptides selected from at least one Gp32 polypeptide further comprising the affinity tag, at least one isolated UvsY polypeptide further comprising the affinity tag, and at least one isolated UvsX polypeptide further comprising the affinity tag.BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figs. 1 depicts the RPA reaction, a) Formation of primer-recombinase complexes, b) Recombinases mediated primer annealing, c) Uoading of DNA polymerases, d) and e) Primer extension by strand displacement synthesis.

[0025] Fig. 2 depicts a list of novel RPA protein components that have been purified. Percent identity indicates sequence conservation with the corresponding T4 protein.

[0026] Fig. 3 depicts an exemplary protein purification protocol.

[0027] Fig. 4 depicts current testing modalities.

[0028] Fig. 5 depicts assay workflow.

[0029] Fig. 6 demonstrates that RT-RPA has 97.5% concordance with qPCR data from NP swab from patients. The two disconcordant samples have low viral titer.

[0030] Fig. 7 depicts expression and solubility of selected UvsY homologs with a C-terminal His6 tag (SEQ ID NO: 100).

[0031] Fig. 8 depicts Ni-NTA purified recombinant proteins; His6-UvsY, UvsX-His6 and His6- Gp32.

[0032] Figs. 9A-9B depict Citrobacter phage RPA protein results. Fig. 9A) Citro UvsY titration. 0.25X citro UvsY shows the greatest efficiency and there is similar activity to T4 UvsY from 0.5X to 0.05X. Fig. 9B) Citro RPA proteins show high efficiency than T4 RPA proteins at all concentrations tested.DETAILED DESCRIPTION

[0033] RPA offers a streamlined assay approach to detecting nucleic acids, which can be utilized for detecting pathogens (e.g., fungi, bacteria, and / or viruses) or environmental organisms. However, current RPA technology relies upon Gp32, UvsX, and UvsY polypeptides from T4 phage. These T4 polypeptides require high minimum temperatures that result in RPA assays that will not function at room temperature and necessarily require expensive temperature control methods. This renders RPAtechnology impractical for field or point-of-care use. Additionally, the T4 polypeptides are difficult to produce and solubilize at the necessary concentrations for effective RPA. The technology disclosed herein relates to GP32, UvsX, and UvsY polypeptides that, surprisingly, provide highly efficient RPA at significantly lower temperatures than T4, and which can be readily produced using the methods described herein. The polypeptides described herein can even provide RPA at ambient temperatures. This advance provides greatly expanded utility for RPA, lowering costs and permitting field and point-of-care use in a much greater breadth of contexts. This improves the ability and speed of diagnostic tests, improving healthcare outcomes.

[0034] In one aspect of any of the embodiments, described herein is a composition comprising one or more of: at least one Gp32 polypeptide and at least one UvsX polypeptide. In one aspect of any of the embodiments, described herein is a composition comprising one or more of: at least one isolated Gp32 polypeptide and at least one isolated UvsX polypeptide. The UvsX polypeptides described herein provide sufficient activity in the absence of UvsY, rendering UvsY dispensible for RPA when the UvsY polypeptides described herein are utilized.

[0035] In one aspect of any of the embodiments, described herein is a composition comprising one or more of: at least one Gp32 polypeptide; at least one UvsY polypeptide; and at least one UvsX polypeptide. In one aspect of any of the embodiments, described herein is a composition comprising one or more of: at least one isolated Gp32 polypeptide; at least one isolated UvsY polypeptide; and at least one isolated UvsX polypeptide.

[0036] In one aspect of any of the embodiments, described herein is a composition comprising one or more of: at least one Gp32 polypeptide and at least one UvsX polypeptide. In one aspect of any of the embodiments, described herein is a composition comprising one or more of: at least one isolated Gp32 polypeptide; and at least one isolated UvsX polypeptide.

[0037] In one aspect of any of the embodiments, described herein is a composition comprising one or more of: at least one Gp32 polypeptide that is not a T4 Gp32 polypeptide; at least one UvsY polypeptide that is not a T4 UvsY polypeptide; and at least one UvsX polypeptide that is not a T4 UvsX polypeptide. In one aspect of any of the embodiments, described herein is a composition comprising one or more of: at least one isolated Gp32 polypeptide that is not a T4 Gp32 polypeptide; at least one isolated UvsY polypeptide that is not a T4 UvsY polypeptide; and at least one isolated UvsX polypeptide that is not a T4 UvsX polypeptide.

[0038] In one aspect of any of the embodiments, described herein is a composition comprising one or more of: at least one Gp32 polypeptide that is not a T4 Gp32 polypeptide and at least one UvsX polypeptide that is not a T4 UvsX polypeptide. In one aspect of any of the embodiments, described herein is a composition comprising one or more of: at least one isolated Gp32 polypeptide that is not a T4 Gp32 polypeptide; and at least one isolated UvsX polypeptide that is not a T4 UvsX polypeptide.

[0039] In some embodiments of any of the aspects, the at least one Gp32 polypeptide is not a T4 Gp32 polypeptide. In some embodiments of any of the aspects, the at least one UvsX polypeptide is not a T4 UvsX polypeptide. In some embodiments of any of the aspects, the at least one UvsY polypeptide is not a T4 UvsY polypeptide. In some embodiments of any of the aspects, the at least one Gp32 polypeptide is not a T4 Gp32 polypeptide and the at least one UvsX polypeptide is not a T4 UvsX polypeptide. In some embodiments of any of the aspects, the at least one Gp32 polypeptide is not a T4 Gp32 polypeptide and the at least one UvsY polypeptide is not a T4 UvsY polypeptide. In some embodiments of any of the aspects, the at least one UvsX polypeptide is not a T4 UvsX polypeptide and the at least one UvsY polypeptide is not a T4 UvsY polypeptide. In some embodiments of any of the aspects, the at least one Gp32 polypeptide is not a T4 Gp32 polypeptide, the at least one UvsX polypeptide is not a T4 UvsX polypeptide, and the at least one UvsY polypeptide is not a T4 UvsY polypeptide.

[0040] In one aspect of any of the embodiments, described herein is a composition comprising: at least one Gp32 polypeptide; at least one UvsY polypeptide; and at least one UvsX polypeptide. In one aspect of any of the embodiments, described herein is a composition comprising: at least one isolated Gp32 polypeptide; at least one isolated UvsY polypeptide; and at least one isolated UvsX polypeptide.

[0041] In one aspect of any of the embodiments, described herein is a composition comprising: at least one Gp32 polypeptide and at least one UvsX polypeptide. In one aspect of any of the embodiments, described herein is a composition comprising: at least one isolated Gp32 polypeptide and at least one isolated UvsX polypeptide.

[0042] In one aspect of any of the embodiments, described herein is a composition consisting of: at least one Gp32 polypeptide; at least one UvsY polypeptide; and at least one UvsX polypeptide. In one aspect of any of the embodiments, described herein is a composition consisting of: at least one isolated Gp32 polypeptide; at least one isolated UvsY polypeptide; and at least one isolated UvsX polypeptide.

[0043] In one aspect of any of the embodiments, described herein is a composition consisting of: at least one Gp32 polypeptide and at least one UvsX polypeptide. In one aspect of any of the embodiments, described herein is a composition consisting of: at least one isolated Gp32 polypeptide and at least one isolated UvsX polypeptide.

[0044] In one aspect of any of the embodiments, described herein is a composition consisting essentially of: at least one Gp32 polypeptide; at least one UvsY polypeptide; and at least one UvsX polypeptide. In one aspect of any of the embodiments, described herein is a composition consisting essentially of: at least one isolated Gp32 polypeptide; at least one isolated UvsY polypeptide; and at least one isolated UvsX polypeptide.

[0045] In one aspect of any of the embodiments, described herein is a composition consisting essentially of: at least one Gp32 polypeptide and at least one UvsX polypeptide. In one aspect of any of the embodiments, described herein is a composition consisting essentially of: at least one isolated Gp32 polypeptide and at least one isolated UvsX polypeptide.

[0046] As used herein “Gene 32 Protein” or “Gp32” refers to a phage protein which binds to ssDNA to form protein filaments. Gp32 functions during DNA replication to promote interaction of ssDNA with polymerase and other replication proteins, while also protecting the ssDNA from nucleases. Gp32 comprises a central core that contains the ssDNA binding site, a positively-charged N-terminal domain, responsible for homotypic protein interactions, and a negatively-charged C- terminal domain. Sequences for Gp32 from different phage are known in the art and provided elsewhere herein.

[0047] As used herein “Ultra Violet Sensitive X” or “UvsX” refers to a phage protein which catalyzes ATP hydrolysis and DNA strand exchange during DNA replication. The ATP hydrolysis is stimulated by ssDNA and produces both ADP and AMP. Sequences for UvsX from different phage are known in the art and provided elsewhere herein.

[0048] As used herein “Ultra Violet Sensitive Y” or “UvsY” refers to a phage protein which stimulates UvsX activity and promotes the displacement of GP32. binds to ssDNA to form protein filaments. Gp32 functions during DNA replication to promote interaction of ssDNA with polymerase and other replication proteins, while also protecting the ssDNA from nucleases. Gp32 comprises a central core that contains the ssDNA binding site, a positively-charged N-terminal domain, responsible for homotypic protein interactions, and a negatively-charged C-terminal domain. Sequences for Gp32 from different phage are known in the art and provided elsewhere herein.

[0049] In some embodiments of any of the aspects, each of the one or more polypeptides (e.g., at least one Gp32 polypeptide, at least one UvsY polypeptide, and / or at least one UvsX polypeptide) are Shigella phage Shfl2 polypeptides, Citrohacter phage CF1 ERZ-2017 polypeptides, Serratia phage CHI14 polypeptides, Edwardsiella phage PEi20 polypeptides, Escherichia phage ECD7 polypeptides, Aeromonas phage PX29 polypeptides, or Pseudomonas phage pfl6 polypeptides. In some embodiments of any of the aspects, the one or more polypeptides (e.g., at least one Gp32 polypeptide, at least one UvsY polypeptide, and / or at least one UvsX polypeptide) are Shigella phage Shfl2 polypeptides. In some embodiments of any of the aspects, the one or more polypeptides (e.g., at least one Gp32 polypeptide, at least one UvsY polypeptide, and / or at least one UvsX polypeptide) are Citrobacter phage CF1 ERZ-2017 polypeptides. In some embodiments of any of the aspects, the one or more polypeptides (e.g., at least one Gp32 polypeptide, at least one UvsY polypeptide, and / or at least one UvsX polypeptide) are Serratia phage CHI 14 polypeptides. In some embodiments of any of the aspects, the one or more polypeptides (e.g., at least one Gp32 polypeptide, at least one UvsY polypeptide, and / or at least one UvsX polypeptide) are Edwardsiella phage PEi20 polypeptides. Insome embodiments of any of the aspects, the one or more polypeptides (e.g., at least one Gp32 polypeptide, at least one UvsY polypeptide, and / or at least one UvsX polypeptide) are Escherichia phage ECD7 polypeptides. In some embodiments of any of the aspects, the one or more polypeptides (e.g., at least one Gp32 polypeptide, at least one UvsY polypeptide, and / or at least one UvsX polypeptide) are Aeromonas phage PX29 polypeptides. In some embodiments of any of the aspects, the one or more polypeptides (e.g., at least one Gp32 polypeptide, at least one UvsY polypeptide, and / or at least one UvsX polypeptide) are Pseudomonas phage pfl6 polypeptides.

[0050] In some embodiments of any of the aspects, the at least one Gp32 polypeptide comprises a sequence with at least 80% sequence identity to the sequence of one of SEQ ID NOs: 1-7. In some embodiments of any of the aspects, the at least one Gp32 polypeptide consists of a sequence with at least 80% sequence identity to the sequence of one of SEQ ID NOs: 1-7. In some embodiments of any of the aspects, the at least one Gp32 polypeptide consists essentially of a sequence with at least 80% sequence identity to the sequence of one of SEQ ID NOs: 1-7. In some embodiments of any of the aspects, the at least one Gp32 polypeptide comprises a sequence with at least 85% sequence identity to the sequence of one of SEQ ID NOs: 1-7. In some embodiments of any of the aspects, the at least one Gp32 polypeptide consists of a sequence with at least 85% sequence identity to the sequence of one of SEQ ID NOs: 1-7. In some embodiments of any of the aspects, the at least one Gp32 polypeptide consists essentially of a sequence with at least 85% sequence identity to the sequence of one of SEQ ID NOs: 1-7. In some embodiments of any of the aspects, the at least one Gp32 polypeptide comprises a sequence with at least 90% sequence identity to the sequence of one of SEQ ID NOs: 1-7. In some embodiments of any of the aspects, the at least one Gp32 polypeptide consists of a sequence with at least 90% sequence identity to the sequence of one of SEQ ID NOs: 1-7. In some embodiments of any of the aspects, the at least one Gp32 polypeptide consists essentially of a sequence with at least 90% sequence identity to the sequence of one of SEQ ID NOs: 1-7. In some embodiments of any of the aspects, the at least one Gp32 polypeptide comprises a sequence with at least 95% sequence identity to the sequence of one of SEQ ID NOs: 1-7. In some embodiments of any of the aspects, the at least one Gp32 polypeptide consists of a sequence with at least 95% sequence identity to the sequence of one of SEQ ID NOs: 1-7. In some embodiments of any of the aspects, the at least one Gp32 polypeptide consists essentially of a sequence with at least 95% sequence identity to the sequence of one of SEQ ID NOs: 1-7. In some embodiments of any of the aspects, the at least one Gp32 polypeptide comprises a sequence with at least 99% sequence identity to the sequence of one of SEQ ID NOs: 1-7. In some embodiments of any of the aspects, the at least one Gp32 polypeptide consists of a sequence with at least 99% sequence identity to the sequence of one of SEQ ID NOs: 1- 7. In some embodiments of any of the aspects, the at least one Gp32 polypeptide consists essentially of a sequence with at least 99% sequence identity to the sequence of one of SEQ ID NOs: 1-7. In some embodiments of any of the aspects, the at least one Gp32 polypeptide comprises the sequence ofone of SEQ ID NOs: 1-7. In some embodiments of any of the aspects, the at least one Gp32 polypeptide consists of the sequence of one of SEQ ID NOs: 1-7. In some embodiments of any of the aspects, the at least one Gp32 polypeptide consists essentially of the sequence of one of SEQ ID NOs: 1-7.

[0051] In some embodiments of any of the aspects, the at least one Gp32 polypeptide comprises a sequence with at least 80% sequence identity to the sequence of SEQ ID NO: 1. In some embodiments of any of the aspects, the at least one Gp32 polypeptide consists of a sequence with at least 80% sequence identity to the sequence of SEQ ID NO: 1. In some embodiments of any of the aspects, the at least one Gp32 polypeptide consists essentially of a sequence with at least 80% sequence identity to the sequence of SEQ ID NO: 1. In some embodiments of any of the aspects, the at least one Gp32 polypeptide comprises a sequence with at least 85% sequence identity to the sequence of SEQ ID NO: 1. In some embodiments of any of the aspects, the at least one Gp32 polypeptide consists of a sequence with at least 85% sequence identity to the sequence of SEQ ID NO: 1. In some embodiments of any of the aspects, the at least one Gp32 polypeptide consists essentially of a sequence with at least 85% sequence identity to the sequence of SEQ ID NO: 1. In some embodiments of any of the aspects, the at least one Gp32 polypeptide comprises a sequence with at least 90% sequence identity to the sequence of SEQ ID NO: 1. In some embodiments of any of the aspects, the at least one Gp32 polypeptide consists of a sequence with at least 90% sequence identity to the sequence of SEQ ID NO: 1. In some embodiments of any of the aspects, the at least one Gp32 polypeptide consists essentially of a sequence with at least 90% sequence identity to the sequence of SEQ ID NO: 1. In some embodiments of any of the aspects, the at least one Gp32 polypeptide comprises a sequence with at least 95% sequence identity to the sequence of SEQ ID NO: 1. In some embodiments of any of the aspects, the at least one Gp32 polypeptide consists of a sequence with at least 95% sequence identity to the sequence of SEQ ID NO: 1. In some embodiments of any of the aspects, the at least one Gp32 polypeptide consists essentially of a sequence with at least 95% sequence identity to the sequence of SEQ ID NO: 1. In some embodiments of any of the aspects, the at least one Gp32 polypeptide comprises a sequence with at least 99% sequence identity to the sequence of SEQ ID NO: 1. In some embodiments of any of the aspects, the at least one Gp32 polypeptide consists of a sequence with at least 99% sequence identity to the sequence of SEQ ID NO: 1. In some embodiments of any of the aspects, the at least one Gp32 polypeptide consists essentially of a sequence with at least 99% sequence identity to the sequence of SEQ ID NO: 1. In some embodiments of any of the aspects, the at least one Gp32 polypeptide comprises the sequence of SEQ ID NO: 1. In some embodiments of any of the aspects, the at least one Gp32 polypeptide consists of the sequence of SEQ ID NO: 1. In some embodiments of any of the aspects, the at least one Gp32 polypeptide consists essentially of the sequence of SEQ ID NO: 1.

[0052] In some embodiments of any of the aspects, the at least one Gp32 polypeptide comprises a sequence with at least 80% sequence identity to the sequence of SEQ ID NO: 2. In some embodiments of any of the aspects, the at least one Gp32 polypeptide consists of a sequence with at least 80% sequence identity to the sequence of SEQ ID NO: 2. In some embodiments of any of the aspects, the at least one Gp32 polypeptide consists essentially of a sequence with at least 80% sequence identity to the sequence of SEQ ID NO: 2. In some embodiments of any of the aspects, the at least one Gp32 polypeptide comprises a sequence with at least 85% sequence identity to the sequence of SEQ ID NO: 2. In some embodiments of any of the aspects, the at least one Gp32 polypeptide consists of a sequence with at least 85% sequence identity to the sequence of SEQ ID NO: 2. In some embodiments of any of the aspects, the at least one Gp32 polypeptide consists essentially of a sequence with at least 85% sequence identity to the sequence of SEQ ID NO: 2. In some embodiments of any of the aspects, the at least one Gp32 polypeptide comprises a sequence with at least 90% sequence identity to the sequence of SEQ ID NO: 2. In some embodiments of any of the aspects, the at least one Gp32 polypeptide consists of a sequence with at least 90% sequence identity to the sequence of SEQ ID NO: 2. In some embodiments of any of the aspects, the at least one Gp32 polypeptide consists essentially of a sequence with at least 90% sequence identity to the sequence of SEQ ID NO: 2. In some embodiments of any of the aspects, the at least one Gp32 polypeptide comprises a sequence with at least 95% sequence identity to the sequence of SEQ ID NO: 2. In some embodiments of any of the aspects, the at least one Gp32 polypeptide consists of a sequence with at least 95% sequence identity to the sequence of SEQ ID NO: 2. In some embodiments of any of the aspects, the at least one Gp32 polypeptide consists essentially of a sequence with at least 95% sequence identity to the sequence of SEQ ID NO: 2. In some embodiments of any of the aspects, the at least one Gp32 polypeptide comprises a sequence with at least 99% sequence identity to the sequence of SEQ ID NO: 2. In some embodiments of any of the aspects, the at least one Gp32 polypeptide consists of a sequence with at least 99% sequence identity to the sequence of SEQ ID NO: 2. In some embodiments of any of the aspects, the at least one Gp32 polypeptide consists essentially of a sequence with at least 99% sequence identity to the sequence of SEQ ID NO: 2. In some embodiments of any of the aspects, the at least one Gp32 polypeptide comprises the sequence of SEQ ID NO: 2. In some embodiments of any of the aspects, the at least one Gp32 polypeptide consists of the sequence of SEQ ID NO: 2. In some embodiments of any of the aspects, the at least one Gp32 polypeptide consists essentially of the sequence of SEQ ID NO: 2.

[0053] In some embodiments of any of the aspects, the at least one Gp32 polypeptide comprises a sequence with at least 80% sequence identity to the sequence of SEQ ID NO: 3. In some embodiments of any of the aspects, the at least one Gp32 polypeptide consists of a sequence with at least 80% sequence identity to the sequence of SEQ ID NO: 3. In some embodiments of any of the aspects, the at least one Gp32 polypeptide consists essentially of a sequence with at least 80%sequence identity to the sequence of SEQ ID NO: 3. In some embodiments of any of the aspects, the at least one Gp32 polypeptide comprises a sequence with at least 85% sequence identity to the sequence of SEQ ID NO: 3. In some embodiments of any of the aspects, the at least one Gp32 polypeptide consists of a sequence with at least 85% sequence identity to the sequence of SEQ ID NO: 3. In some embodiments of any of the aspects, the at least one Gp32 polypeptide consists essentially of a sequence with at least 85% sequence identity to the sequence of SEQ ID NO: 3. In some embodiments of any of the aspects, the at least one Gp32 polypeptide comprises a sequence with at least 90% sequence identity to the sequence of SEQ ID NO: 3. In some embodiments of any of the aspects, the at least one Gp32 polypeptide consists of a sequence with at least 90% sequence identity to the sequence of SEQ ID NO: 3. In some embodiments of any of the aspects, the at least one Gp32 polypeptide consists essentially of a sequence with at least 90% sequence identity to the sequence of SEQ ID NO: 3. In some embodiments of any of the aspects, the at least one Gp32 polypeptide comprises a sequence with at least 95% sequence identity to the sequence of SEQ ID NO: 3. In some embodiments of any of the aspects, the at least one Gp32 polypeptide consists of a sequence with at least 95% sequence identity to the sequence of SEQ ID NO: 3. In some embodiments of any of the aspects, the at least one Gp32 polypeptide consists essentially of a sequence with at least 95% sequence identity to the sequence of SEQ ID NO: 3. In some embodiments of any of the aspects, the at least one Gp32 polypeptide comprises a sequence with at least 99% sequence identity to the sequence of SEQ ID NO: 3. In some embodiments of any of the aspects, the at least one Gp32 polypeptide consists of a sequence with at least 99% sequence identity to the sequence of SEQ ID NO: 3. In some embodiments of any of the aspects, the at least one Gp32 polypeptide consists essentially of a sequence with at least 99% sequence identity to the sequence of SEQ ID NO: 3. In some embodiments of any of the aspects, the at least one Gp32 polypeptide comprises the sequence of SEQ ID NO: 3. In some embodiments of any of the aspects, the at least one Gp32 polypeptide consists of the sequence of SEQ ID NO: 3. In some embodiments of any of the aspects, the at least one Gp32 polypeptide consists essentially of the sequence of SEQ ID NO: 3.

[0054] In some embodiments of any of the aspects, the at least one Gp32 polypeptide comprises a sequence with at least 80% sequence identity to the sequence of SEQ ID NO: 4. In some embodiments of any of the aspects, the at least one Gp32 polypeptide consists of a sequence with at least 80% sequence identity to the sequence of SEQ ID NO: 4. In some embodiments of any of the aspects, the at least one Gp32 polypeptide consists essentially of a sequence with at least 80% sequence identity to the sequence of SEQ ID NO: 4. In some embodiments of any of the aspects, the at least one Gp32 polypeptide comprises a sequence with at least 85% sequence identity to the sequence of SEQ ID NO: 4. In some embodiments of any of the aspects, the at least one Gp32 polypeptide consists of a sequence with at least 85% sequence identity to the sequence of SEQ ID NO: 4. In some embodiments of any of the aspects, the at least one Gp32 polypeptide consistsessentially of a sequence with at least 85% sequence identity to the sequence of SEQ ID NO: 4. In some embodiments of any of the aspects, the at least one Gp32 polypeptide comprises a sequence with at least 90% sequence identity to the sequence of SEQ ID NO: 4. In some embodiments of any of the aspects, the at least one Gp32 polypeptide consists of a sequence with at least 90% sequence identity to the sequence of SEQ ID NO: 4. In some embodiments of any of the aspects, the at least one Gp32 polypeptide consists essentially of a sequence with at least 90% sequence identity to the sequence of SEQ ID NO: 4. In some embodiments of any of the aspects, the at least one Gp32 polypeptide comprises a sequence with at least 95% sequence identity to the sequence of SEQ ID NO: 4. In some embodiments of any of the aspects, the at least one Gp32 polypeptide consists of a sequence with at least 95% sequence identity to the sequence of SEQ ID NO: 4. In some embodiments of any of the aspects, the at least one Gp32 polypeptide consists essentially of a sequence with at least 95% sequence identity to the sequence of SEQ ID NO: 4. In some embodiments of any of the aspects, the at least one Gp32 polypeptide comprises a sequence with at least 99% sequence identity to the sequence of SEQ ID NO: 4. In some embodiments of any of the aspects, the at least one Gp32 polypeptide consists of a sequence with at least 99% sequence identity to the sequence of SEQ ID NO: 4. In some embodiments of any of the aspects, the at least one Gp32 polypeptide consists essentially of a sequence with at least 99% sequence identity to the sequence of SEQ ID NO: 4. In some embodiments of any of the aspects, the at least one Gp32 polypeptide comprises the sequence of SEQ ID NO: 4. In some embodiments of any of the aspects, the at least one Gp32 polypeptide consists of the sequence of SEQ ID NO: 4. In some embodiments of any of the aspects, the at least one Gp32 polypeptide consists essentially of the sequence of SEQ ID NO: 4.

[0055] In some embodiments of any of the aspects, the at least one Gp32 polypeptide comprises a sequence with at least 80% sequence identity to the sequence of SEQ ID NO: 5. In some embodiments of any of the aspects, the at least one Gp32 polypeptide consists of a sequence with at least 80% sequence identity to the sequence of SEQ ID NO: 5. In some embodiments of any of the aspects, the at least one Gp32 polypeptide consists essentially of a sequence with at least 80% sequence identity to the sequence of SEQ ID NO: 5. In some embodiments of any of the aspects, the at least one Gp32 polypeptide comprises a sequence with at least 85% sequence identity to the sequence of SEQ ID NO: 5. In some embodiments of any of the aspects, the at least one Gp32 polypeptide consists of a sequence with at least 85% sequence identity to the sequence of SEQ ID NO: 5. In some embodiments of any of the aspects, the at least one Gp32 polypeptide consists essentially of a sequence with at least 85% sequence identity to the sequence of SEQ ID NO: 5. In some embodiments of any of the aspects, the at least one Gp32 polypeptide comprises a sequence with at least 90% sequence identity to the sequence of SEQ ID NO: 5. In some embodiments of any of the aspects, the at least one Gp32 polypeptide consists of a sequence with at least 90% sequence identity to the sequence of SEQ ID NO: 5. In some embodiments of any of the aspects, the at least oneGp32 polypeptide consists essentially of a sequence with at least 90% sequence identity to the sequence of SEQ ID NO: 5. In some embodiments of any of the aspects, the at least one Gp32 polypeptide comprises a sequence with at least 95% sequence identity to the sequence of SEQ ID NO:5. In some embodiments of any of the aspects, the at least one Gp32 polypeptide consists of a sequence with at least 95% sequence identity to the sequence of SEQ ID NO: 5. In some embodiments of any of the aspects, the at least one Gp32 polypeptide consists essentially of a sequence with at least 95% sequence identity to the sequence of SEQ ID NO: 5. In some embodiments of any of the aspects, the at least one Gp32 polypeptide comprises a sequence with at least 99% sequence identity to the sequence of SEQ ID NO: 5. In some embodiments of any of the aspects, the at least one Gp32 polypeptide consists of a sequence with at least 99% sequence identity to the sequence of SEQ ID NO: 5. In some embodiments of any of the aspects, the at least one Gp32 polypeptide consists essentially of a sequence with at least 99% sequence identity to the sequence of SEQ ID NO: 5. In some embodiments of any of the aspects, the at least one Gp32 polypeptide comprises the sequence of SEQ ID NO: 5. In some embodiments of any of the aspects, the at least one Gp32 polypeptide consists of the sequence of SEQ ID NO: 5. In some embodiments of any of the aspects, the at least one Gp32 polypeptide consists essentially of the sequence of SEQ ID NO: 5.

[0056] In some embodiments of any of the aspects, the at least one Gp32 polypeptide comprises a sequence with at least 80% sequence identity to the sequence of SEQ ID NO: 6. In some embodiments of any of the aspects, the at least one Gp32 polypeptide consists of a sequence with at least 80% sequence identity to the sequence of SEQ ID NO: 6. In some embodiments of any of the aspects, the at least one Gp32 polypeptide consists essentially of a sequence with at least 80% sequence identity to the sequence of SEQ ID NO: 6. In some embodiments of any of the aspects, the at least one Gp32 polypeptide comprises a sequence with at least 85% sequence identity to the sequence of SEQ ID NO: 6. In some embodiments of any of the aspects, the at least one Gp32 polypeptide consists of a sequence with at least 85% sequence identity to the sequence of SEQ ID NO: 6. In some embodiments of any of the aspects, the at least one Gp32 polypeptide consists essentially of a sequence with at least 85% sequence identity to the sequence of SEQ ID NO: 6. In some embodiments of any of the aspects, the at least one Gp32 polypeptide comprises a sequence with at least 90% sequence identity to the sequence of SEQ ID NO: 6. In some embodiments of any of the aspects, the at least one Gp32 polypeptide consists of a sequence with at least 90% sequence identity to the sequence of SEQ ID NO: 6. In some embodiments of any of the aspects, the at least one Gp32 polypeptide consists essentially of a sequence with at least 90% sequence identity to the sequence of SEQ ID NO: 6. In some embodiments of any of the aspects, the at least one Gp32 polypeptide comprises a sequence with at least 95% sequence identity to the sequence of SEQ ID NO:6. In some embodiments of any of the aspects, the at least one Gp32 polypeptide consists of a sequence with at least 95% sequence identity to the sequence of SEQ ID NO: 6. In someembodiments of any of the aspects, the at least one Gp32 polypeptide consists essentially of a sequence with at least 95% sequence identity to the sequence of SEQ ID NO: 6. In some embodiments of any of the aspects, the at least one Gp32 polypeptide comprises a sequence with at least 99% sequence identity to the sequence of SEQ ID NO: 6. In some embodiments of any of the aspects, the at least one Gp32 polypeptide consists of a sequence with at least 99% sequence identity to the sequence of SEQ ID NO: 6. In some embodiments of any of the aspects, the at least one Gp32 polypeptide consists essentially of a sequence with at least 99% sequence identity to the sequence of SEQ ID NO: 6. In some embodiments of any of the aspects, the at least one Gp32 polypeptide comprises the sequence of SEQ ID NO: 6. In some embodiments of any of the aspects, the at least one Gp32 polypeptide consists of the sequence of SEQ ID NO: 6. In some embodiments of any of the aspects, the at least one Gp32 polypeptide consists essentially of the sequence of SEQ ID NO: 6.

[0057] In some embodiments of any of the aspects, the at least one Gp32 polypeptide comprises a sequence with at least 80% sequence identity to the sequence of SEQ ID NO: 7. In some embodiments of any of the aspects, the at least one Gp32 polypeptide consists of a sequence with at least 80% sequence identity to the sequence of SEQ ID NO: 7. In some embodiments of any of the aspects, the at least one Gp32 polypeptide consists essentially of a sequence with at least 80% sequence identity to the sequence of SEQ ID NO: 7. In some embodiments of any of the aspects, the at least one Gp32 polypeptide comprises a sequence with at least 85% sequence identity to the sequence of SEQ ID NO: 7. In some embodiments of any of the aspects, the at least one Gp32 polypeptide consists of a sequence with at least 85% sequence identity to the sequence of SEQ ID NO: 7. In some embodiments of any of the aspects, the at least one Gp32 polypeptide consists essentially of a sequence with at least 85% sequence identity to the sequence of SEQ ID NO: 7. In some embodiments of any of the aspects, the at least one Gp32 polypeptide comprises a sequence with at least 90% sequence identity to the sequence of SEQ ID NO: 7. In some embodiments of any of the aspects, the at least one Gp32 polypeptide consists of a sequence with at least 90% sequence identity to the sequence of SEQ ID NO: 7. In some embodiments of any of the aspects, the at least one Gp32 polypeptide consists essentially of a sequence with at least 90% sequence identity to the sequence of SEQ ID NO: 7. In some embodiments of any of the aspects, the at least one Gp32 polypeptide comprises a sequence with at least 95% sequence identity to the sequence of SEQ ID NO: 7. In some embodiments of any of the aspects, the at least one Gp32 polypeptide consists of a sequence with at least 95% sequence identity to the sequence of SEQ ID NO: 7. In some embodiments of any of the aspects, the at least one Gp32 polypeptide consists essentially of a sequence with at least 95% sequence identity to the sequence of SEQ ID NO: 7. In some embodiments of any of the aspects, the at least one Gp32 polypeptide comprises a sequence with at least 99% sequence identity to the sequence of SEQ ID NO: 7. In some embodiments of any of the aspects, the at least one Gp32 polypeptide consists of a sequence with at least 99% sequence identityto the sequence of SEQ ID NO: 7. In some embodiments of any of the aspects, the at least one Gp32 polypeptide consists essentially of a sequence with at least 99% sequence identity to the sequence of SEQ ID NO: 7. In some embodiments of any of the aspects, the at least one Gp32 polypeptide comprises the sequence of SEQ ID NO: 7. In some embodiments of any of the aspects, the at least one Gp32 polypeptide consists of the sequence of SEQ ID NO: 7. In some embodiments of any of the aspects, the at least one Gp32 polypeptide consists essentially of the sequence of SEQ ID NO: 7.

[0058] In some embodiments of any of the aspects, the at least one UvsX polypeptide comprises a sequence with at least 80% sequence identity to the sequence of one of SEQ ID NOs: 8-14. In some embodiments of any of the aspects, the at least one UvsX polypeptide consists of a sequence with at least 80% sequence identity to the sequence of one of SEQ ID NOs: 8-14. In some embodiments of any of the aspects, the at least one UvsX polypeptide consists essentially of a sequence with at least 80% sequence identity to the sequence of one of SEQ ID NOs: 8-14. In some embodiments of any of the aspects, the at least one UvsX polypeptide comprises a sequence with at least 85% sequence identity to the sequence of one of SEQ ID NOs: 8-14. In some embodiments of any of the aspects, the at least one UvsX polypeptide consists of a sequence with at least 85% sequence identity to the sequence of one of SEQ ID NOs: 8-14. In some embodiments of any of the aspects, the at least one UvsX polypeptide consists essentially of a sequence with at least 85% sequence identity to the sequence of one of SEQ ID NOs: 8-14. In some embodiments of any of the aspects, the at least one UvsX polypeptide comprises a sequence with at least 90% sequence identity to the sequence of one of SEQ ID NOs: 8-14. In some embodiments of any of the aspects, the at least one UvsX polypeptide consists of a sequence with at least 90% sequence identity to the sequence of one of SEQ ID NOs: 8- 14. In some embodiments of any of the aspects, the at least one UvsX polypeptide consists essentially of a sequence with at least 90% sequence identity to the sequence of one of SEQ ID NOs: 8-14. In some embodiments of any of the aspects, the at least one UvsX polypeptide comprises a sequence with at least 95% sequence identity to the sequence of one of SEQ ID NOs: 8-14. In some embodiments of any of the aspects, the at least one UvsX polypeptide consists of a sequence with at least 95% sequence identity to the sequence of one of SEQ ID NOs: 8-14. In some embodiments of any of the aspects, the at least one UvsX polypeptide consists essentially of a sequence with at least 95% sequence identity to the sequence of one of SEQ ID NOs: 8-14. In some embodiments of any of the aspects, the at least one UvsX polypeptide comprises a sequence with at least 99% sequence identity to the sequence of one of SEQ ID NOs: 8-14. In some embodiments of any of the aspects, the at least one UvsX polypeptide consists of a sequence with at least 99% sequence identity to the sequence of one of SEQ ID NOs: 8-14. In some embodiments of any of the aspects, the at least one UvsX polypeptide consists essentially of a sequence with at least 99% sequence identity to the sequence of one of SEQ ID NOs: 8-14. In some embodiments of any of the aspects, the at least one UvsX polypeptide comprises the sequence of one of SEQ ID NOs: 8-14. In some embodiments of anyof the aspects, the at least one UvsX polypeptide consists of the sequence of one of SEQ ID NOs: 8- 14. In some embodiments of any of the aspects, the at least one UvsX polypeptide consists essentially of the sequence of one of SEQ ID NOs: 8-14.

[0059] In some embodiments of any of the aspects, the at least one UvsX polypeptide comprises a sequence with at least 80% sequence identity to the sequence of SEQ ID NO: 8. In some embodiments of any of the aspects, the at least one UvsX polypeptide consists of a sequence with at least 80% sequence identity to the sequence of SEQ ID NO: 8. In some embodiments of any of the aspects, the at least one UvsX polypeptide consists essentially of a sequence with at least 80% sequence identity to the sequence of SEQ ID NO: 8. In some embodiments of any of the aspects, the at least one UvsX polypeptide comprises a sequence with at least 85% sequence identity to the sequence of SEQ ID NO: 8. In some embodiments of any of the aspects, the at least one UvsX polypeptide consists of a sequence with at least 85% sequence identity to the sequence of SEQ ID NO: 8. In some embodiments of any of the aspects, the at least one UvsX polypeptide consists essentially of a sequence with at least 85% sequence identity to the sequence of SEQ ID NO: 8. In some embodiments of any of the aspects, the at least one UvsX polypeptide comprises a sequence with at least 90% sequence identity to the sequence of SEQ ID NO: 8. In some embodiments of any of the aspects, the at least one UvsX polypeptide consists of a sequence with at least 90% sequence identity to the sequence of SEQ ID NO: 8. In some embodiments of any of the aspects, the at least one UvsX polypeptide consists essentially of a sequence with at least 90% sequence identity to the sequence of SEQ ID NO: 8. In some embodiments of any of the aspects, the at least one UvsX polypeptide comprises a sequence with at least 95% sequence identity to the sequence of SEQ ID NO: 8. In some embodiments of any of the aspects, the at least one UvsX polypeptide consists of a sequence with at least 95% sequence identity to the sequence of SEQ ID NO: 8. In some embodiments of any of the aspects, the at least one UvsX polypeptide consists essentially of a sequence with at least 95% sequence identity to the sequence of SEQ ID NO: 8. In some embodiments of any of the aspects, the at least one UvsX polypeptide comprises a sequence with at least 99% sequence identity to the sequence of SEQ ID NO: 8. In some embodiments of any of the aspects, the at least one UvsX polypeptide consists of a sequence with at least 99% sequence identity to the sequence of SEQ ID NO: 8. In some embodiments of any of the aspects, the at least one UvsX polypeptide consists essentially of a sequence with at least 99% sequence identity to the sequence of SEQ ID NO: 8. In some embodiments of any of the aspects, the at least one UvsX polypeptide comprises the sequence of SEQ ID NO: 8. In some embodiments of any of the aspects, the at least one UvsX polypeptide consists of the sequence of SEQ ID NO: 8. In some embodiments of any of the aspects, the at least one UvsX polypeptide consists essentially of the sequence of SEQ ID NO: 8.

[0060] In some embodiments of any of the aspects, the at least one UvsX polypeptide comprises a sequence with at least 80% sequence identity to the sequence of SEQ ID NO: 9. In someembodiments of any of the aspects, the at least one UvsX polypeptide consists of a sequence with at least 80% sequence identity to the sequence of SEQ ID NO: 9. In some embodiments of any of the aspects, the at least one UvsX polypeptide consists essentially of a sequence with at least 80% sequence identity to the sequence of SEQ ID NO: 9. In some embodiments of any of the aspects, the at least one UvsX polypeptide comprises a sequence with at least 85% sequence identity to the sequence of SEQ ID NO: 9. In some embodiments of any of the aspects, the at least one UvsX polypeptide consists of a sequence with at least 85% sequence identity to the sequence of SEQ ID NO: 9. In some embodiments of any of the aspects, the at least one UvsX polypeptide consists essentially of a sequence with at least 85% sequence identity to the sequence of SEQ ID NO: 9. In some embodiments of any of the aspects, the at least one UvsX polypeptide comprises a sequence with at least 90% sequence identity to the sequence of SEQ ID NO: 9. In some embodiments of any of the aspects, the at least one UvsX polypeptide consists of a sequence with at least 90% sequence identity to the sequence of SEQ ID NO: 9. In some embodiments of any of the aspects, the at least one UvsX polypeptide consists essentially of a sequence with at least 90% sequence identity to the sequence of SEQ ID NO: 9. In some embodiments of any of the aspects, the at least one UvsX polypeptide comprises a sequence with at least 95% sequence identity to the sequence of SEQ ID NO: 9. In some embodiments of any of the aspects, the at least one UvsX polypeptide consists of a sequence with at least 95% sequence identity to the sequence of SEQ ID NO: 9. In some embodiments of any of the aspects, the at least one UvsX polypeptide consists essentially of a sequence with at least 95% sequence identity to the sequence of SEQ ID NO: 9. In some embodiments of any of the aspects, the at least one UvsX polypeptide comprises a sequence with at least 99% sequence identity to the sequence of SEQ ID NO: 9. In some embodiments of any of the aspects, the at least one UvsX polypeptide consists of a sequence with at least 99% sequence identity to the sequence of SEQ ID NO: 9. In some embodiments of any of the aspects, the at least one UvsX polypeptide consists essentially of a sequence with at least 99% sequence identity to the sequence of SEQ ID NO: 9. In some embodiments of any of the aspects, the at least one UvsX polypeptide comprises the sequence of SEQ ID NO: 9. In some embodiments of any of the aspects, the at least one UvsX polypeptide consists of the sequence of SEQ ID NO: 9. In some embodiments of any of the aspects, the at least one UvsX polypeptide consists essentially of the sequence of SEQ ID NO: 9.

[0061] In some embodiments of any of the aspects, the at least one UvsX polypeptide comprises a sequence with at least 80% sequence identity to the sequence of SEQ ID NO: 10. In some embodiments of any of the aspects, the at least one UvsX polypeptide consists of a sequence with at least 80% sequence identity to the sequence of SEQ ID NO: 10. In some embodiments of any of the aspects, the at least one UvsX polypeptide consists essentially of a sequence with at least 80% sequence identity to the sequence of SEQ ID NO: 10. In some embodiments of any of the aspects, the at least one UvsX polypeptide comprises a sequence with at least 85% sequence identity to thesequence of SEQ ID NO: 10. In some embodiments of any of the aspects, the at least one UvsX polypeptide consists of a sequence with at least 85% sequence identity to the sequence of SEQ ID NO: 10. In some embodiments of any of the aspects, the at least one UvsX polypeptide consists essentially of a sequence with at least 85% sequence identity to the sequence of SEQ ID NO: 10. In some embodiments of any of the aspects, the at least one UvsX polypeptide comprises a sequence with at least 90% sequence identity to the sequence of SEQ ID NO: 10. In some embodiments of any of the aspects, the at least one UvsX polypeptide consists of a sequence with at least 90% sequence identity to the sequence of SEQ ID NO: 10. In some embodiments of any of the aspects, the at least one UvsX polypeptide consists essentially of a sequence with at least 90% sequence identity to the sequence of SEQ ID NO: 10. In some embodiments of any of the aspects, the at least one UvsX polypeptide comprises a sequence with at least 95% sequence identity to the sequence of SEQ ID NO: 10. In some embodiments of any of the aspects, the at least one UvsX polypeptide consists of a sequence with at least 95% sequence identity to the sequence of SEQ ID NO: 10. In some embodiments of any of the aspects, the at least one UvsX polypeptide consists essentially of a sequence with at least 95% sequence identity to the sequence of SEQ ID NO: 10. In some embodiments of any of the aspects, the at least one UvsX polypeptide comprises a sequence with at least 99% sequence identity to the sequence of SEQ ID NO: 10. In some embodiments of any of the aspects, the at least one UvsX polypeptide consists of a sequence with at least 99% sequence identity to the sequence of SEQ ID NO: 10. In some embodiments of any of the aspects, the at least one UvsX polypeptide consists essentially of a sequence with at least 99% sequence identity to the sequence of SEQ ID NO: 10. In some embodiments of any of the aspects, the at least one UvsX polypeptide comprises the sequence of SEQ ID NO: 10. In some embodiments of any of the aspects, the at least one UvsX polypeptide consists of the sequence of SEQ ID NO: 10. In some embodiments of any of the aspects, the at least one UvsX polypeptide consists essentially of the sequence of SEQ ID NO: 10.

[0062] In some embodiments of any of the aspects, the at least one UvsX polypeptide comprises a sequence with at least 80% sequence identity to the sequence of SEQ ID NO: 11. In some embodiments of any of the aspects, the at least one UvsX polypeptide consists of a sequence with at least 80% sequence identity to the sequence of SEQ ID NO: 11. In some embodiments of any of the aspects, the at least one UvsX polypeptide consists essentially of a sequence with at least 80% sequence identity to the sequence of SEQ ID NO: 11. In some embodiments of any of the aspects, the at least one UvsX polypeptide comprises a sequence with at least 85% sequence identity to the sequence of SEQ ID NO: 11. In some embodiments of any of the aspects, the at least one UvsX polypeptide consists of a sequence with at least 85% sequence identity to the sequence of SEQ ID NO: 11. In some embodiments of any of the aspects, the at least one UvsX polypeptide consists essentially of a sequence with at least 85% sequence identity to the sequence of SEQ ID NO: 11. In some embodiments of any of the aspects, the at least one UvsX polypeptide comprises a sequencewith at least 90% sequence identity to the sequence of SEQ ID NO: 11. In some embodiments of any of the aspects, the at least one UvsX polypeptide consists of a sequence with at least 90% sequence identity to the sequence of SEQ ID NO: 11. In some embodiments of any of the aspects, the at least one UvsX polypeptide consists essentially of a sequence with at least 90% sequence identity to the sequence of SEQ ID NO: 11. In some embodiments of any of the aspects, the at least one UvsX polypeptide comprises a sequence with at least 95% sequence identity to the sequence of SEQ ID NO: 11. In some embodiments of any of the aspects, the at least one UvsX polypeptide consists of a sequence with at least 95% sequence identity to the sequence of SEQ ID NO: 11. In some embodiments of any of the aspects, the at least one UvsX polypeptide consists essentially of a sequence with at least 95% sequence identity to the sequence of SEQ ID NO: 11. In some embodiments of any of the aspects, the at least one UvsX polypeptide comprises a sequence with at least 99% sequence identity to the sequence of SEQ ID NO: 11. In some embodiments of any of the aspects, the at least one UvsX polypeptide consists of a sequence with at least 99% sequence identity to the sequence of SEQ ID NO: 11. In some embodiments of any of the aspects, the at least one UvsX polypeptide consists essentially of a sequence with at least 99% sequence identity to the sequence of SEQ ID NO: 11. In some embodiments of any of the aspects, the at least one UvsX polypeptide comprises the sequence of SEQ ID NO: 11. In some embodiments of any of the aspects, the at least one UvsX polypeptide consists of the sequence of SEQ ID NO: 11. In some embodiments of any of the aspects, the at least one UvsX polypeptide consists essentially of the sequence of SEQ ID NO: 11.

[0063] In some embodiments of any of the aspects, the at least one UvsX polypeptide comprises a sequence with at least 80% sequence identity to the sequence of SEQ ID NO: 12. In some embodiments of any of the aspects, the at least one UvsX polypeptide consists of a sequence with at least 80% sequence identity to the sequence of SEQ ID NO: 12. In some embodiments of any of the aspects, the at least one UvsX polypeptide consists essentially of a sequence with at least 80% sequence identity to the sequence of SEQ ID NO: 12. In some embodiments of any of the aspects, the at least one UvsX polypeptide comprises a sequence with at least 85% sequence identity to the sequence of SEQ ID NO: 12. In some embodiments of any of the aspects, the at least one UvsX polypeptide consists of a sequence with at least 85% sequence identity to the sequence of SEQ ID NO: 12. In some embodiments of any of the aspects, the at least one UvsX polypeptide consists essentially of a sequence with at least 85% sequence identity to the sequence of SEQ ID NO: 12. In some embodiments of any of the aspects, the at least one UvsX polypeptide comprises a sequence with at least 90% sequence identity to the sequence of SEQ ID NO: 12. In some embodiments of any of the aspects, the at least one UvsX polypeptide consists of a sequence with at least 90% sequence identity to the sequence of SEQ ID NO: 12. In some embodiments of any of the aspects, the at least one UvsX polypeptide consists essentially of a sequence with at least 90% sequence identity to the sequence of SEQ ID NO: 12. In some embodiments of any of the aspects, the at least one UvsXpolypeptide comprises a sequence with at least 95% sequence identity to the sequence of SEQ ID NO:12. In some embodiments of any of the aspects, the at least one UvsX polypeptide consists of a sequence with at least 95% sequence identity to the sequence of SEQ ID NO: 12. In some embodiments of any of the aspects, the at least one UvsX polypeptide consists essentially of a sequence with at least 95% sequence identity to the sequence of SEQ ID NO: 12. In some embodiments of any of the aspects, the at least one UvsX polypeptide comprises a sequence with at least 99% sequence identity to the sequence of SEQ ID NO: 12. In some embodiments of any of the aspects, the at least one UvsX polypeptide consists of a sequence with at least 99% sequence identity to the sequence of SEQ ID NO: 12. In some embodiments of any of the aspects, the at least one UvsX polypeptide consists essentially of a sequence with at least 99% sequence identity to the sequence of SEQ ID NO: 12. In some embodiments of any of the aspects, the at least one UvsX polypeptide comprises the sequence of SEQ ID NO: 12. In some embodiments of any of the aspects, the at least one UvsX polypeptide consists of the sequence of SEQ ID NO: 12. In some embodiments of any of the aspects, the at least one UvsX polypeptide consists essentially of the sequence of SEQ ID NO: 12.

[0064] In some embodiments of any of the aspects, the at least one UvsX polypeptide comprises a sequence with at least 80% sequence identity to the sequence of SEQ ID NO: 13. In some embodiments of any of the aspects, the at least one UvsX polypeptide consists of a sequence with at least 80% sequence identity to the sequence of SEQ ID NO: 13. In some embodiments of any of the aspects, the at least one UvsX polypeptide consists essentially of a sequence with at least 80% sequence identity to the sequence of SEQ ID NO: 13. In some embodiments of any of the aspects, the at least one UvsX polypeptide comprises a sequence with at least 85% sequence identity to the sequence of SEQ ID NO: 13. In some embodiments of any of the aspects, the at least one UvsX polypeptide consists of a sequence with at least 85% sequence identity to the sequence of SEQ ID NO: 13. In some embodiments of any of the aspects, the at least one UvsX polypeptide consists essentially of a sequence with at least 85% sequence identity to the sequence of SEQ ID NO: 13. In some embodiments of any of the aspects, the at least one UvsX polypeptide comprises a sequence with at least 90% sequence identity to the sequence of SEQ ID NO: 13. In some embodiments of any of the aspects, the at least one UvsX polypeptide consists of a sequence with at least 90% sequence identity to the sequence of SEQ ID NO: 13. In some embodiments of any of the aspects, the at least one UvsX polypeptide consists essentially of a sequence with at least 90% sequence identity to the sequence of SEQ ID NO: 13. In some embodiments of any of the aspects, the at least one UvsX polypeptide comprises a sequence with at least 95% sequence identity to the sequence of SEQ ID NO:13. In some embodiments of any of the aspects, the at least one UvsX polypeptide consists of a sequence with at least 95% sequence identity to the sequence of SEQ ID NO: 13. In some embodiments of any of the aspects, the at least one UvsX polypeptide consists essentially of a sequence with at least 95% sequence identity to the sequence of SEQ ID NO: 13. In someembodiments of any of the aspects, the at least one UvsX polypeptide comprises a sequence with at least 99% sequence identity to the sequence of SEQ ID NO: 13. In some embodiments of any of the aspects, the at least one UvsX polypeptide consists of a sequence with at least 99% sequence identity to the sequence of SEQ ID NO: 13. In some embodiments of any of the aspects, the at least one UvsX polypeptide consists essentially of a sequence with at least 99% sequence identity to the sequence of SEQ ID NO: 13. In some embodiments of any of the aspects, the at least one UvsX polypeptide comprises the sequence of SEQ ID NO: 13. In some embodiments of any of the aspects, the at least one UvsX polypeptide consists of the sequence of SEQ ID NO: 13. In some embodiments of any of the aspects, the at least one UvsX polypeptide consists essentially of the sequence of SEQ ID NO: 13.

[0065] In some embodiments of any of the aspects, the at least one UvsX polypeptide comprises a sequence with at least 80% sequence identity to the sequence of SEQ ID NO: 14. In some embodiments of any of the aspects, the at least one UvsX polypeptide consists of a sequence with at least 80% sequence identity to the sequence of SEQ ID NO: 14. In some embodiments of any of the aspects, the at least one UvsX polypeptide consists essentially of a sequence with at least 80% sequence identity to the sequence of SEQ ID NO: 14. In some embodiments of any of the aspects, the at least one UvsX polypeptide comprises a sequence with at least 85% sequence identity to the sequence of SEQ ID NO: 14. In some embodiments of any of the aspects, the at least one UvsX polypeptide consists of a sequence with at least 85% sequence identity to the sequence of SEQ ID NO: 14. In some embodiments of any of the aspects, the at least one UvsX polypeptide consists essentially of a sequence with at least 85% sequence identity to the sequence of SEQ ID NO: 14. In some embodiments of any of the aspects, the at least one UvsX polypeptide comprises a sequence with at least 90% sequence identity to the sequence of SEQ ID NO: 14. In some embodiments of any of the aspects, the at least one UvsX polypeptide consists of a sequence with at least 90% sequence identity to the sequence of SEQ ID NO: 14. In some embodiments of any of the aspects, the at least one UvsX polypeptide consists essentially of a sequence with at least 90% sequence identity to the sequence of SEQ ID NO: 14. In some embodiments of any of the aspects, the at least one UvsX polypeptide comprises a sequence with at least 95% sequence identity to the sequence of SEQ ID NO: 14. In some embodiments of any of the aspects, the at least one UvsX polypeptide consists of a sequence with at least 95% sequence identity to the sequence of SEQ ID NO: 14. In some embodiments of any of the aspects, the at least one UvsX polypeptide consists essentially of a sequence with at least 95% sequence identity to the sequence of SEQ ID NO: 14. In some embodiments of any of the aspects, the at least one UvsX polypeptide comprises a sequence with at least 99% sequence identity to the sequence of SEQ ID NO: 14. In some embodiments of any of the aspects, the at least one UvsX polypeptide consists of a sequence with at least 99% sequence identity to the sequence of SEQ ID NO: 14. In some embodiments of any of the aspects, the at least one UvsX polypeptide consists essentially of a sequence with at least 99% sequence identity to the sequence ofSEQ ID NO: 14. In some embodiments of any of the aspects, the at least one UvsX polypeptide comprises the sequence of SEQ ID NO: 14. In some embodiments of any of the aspects, the at least one UvsX polypeptide consists of the sequence of SEQ ID NO: 14. In some embodiments of any of the aspects, the at least one UvsX polypeptide consists essentially of the sequence of SEQ ID NO: 14.

[0066] In some embodiments of any of the aspects, the at least one UvsY polypeptide comprises a sequence with at least 80% sequence identity to the sequence of one of SEQ ID NOs: 15-21. In some embodiments of any of the aspects, the at least one UvsY polypeptide consists of a sequence with at least 80% sequence identity to the sequence of one of SEQ ID NOs: 15-21. In some embodiments of any of the aspects, the at least one UvsY polypeptide consists essentially of a sequence with at least 80% sequence identity to the sequence of one of SEQ ID NOs: 15-21. In some embodiments of any of the aspects, the at least one UvsY polypeptide comprises a sequence with at least 85% sequence identity to the sequence of one of SEQ ID NOs: 15-21. In some embodiments of any of the aspects, the at least one UvsY polypeptide consists of a sequence with at least 85% sequence identity to the sequence of one of SEQ ID NOs: 15-21. In some embodiments of any of the aspects, the at least one UvsY polypeptide consists essentially of a sequence with at least 85% sequence identity to the sequence of one of SEQ ID NOs: 15-21. In some embodiments of any of the aspects, the at least one UvsY polypeptide comprises a sequence with at least 90% sequence identity to the sequence of one of SEQ ID NOs: 15-21. In some embodiments of any of the aspects, the at least one UvsY polypeptide consists of a sequence with at least 90% sequence identity to the sequence of one of SEQ ID NOs: 15-21. In some embodiments of any of the aspects, the at least one UvsY polypeptide consists essentially of a sequence with at least 90% sequence identity to the sequence of one of SEQ ID NOs: 15-21. In some embodiments of any of the aspects, the at least one UvsY polypeptide comprises a sequence with at least 95% sequence identity to the sequence of one of SEQ ID NOs: 15-21. In some embodiments of any of the aspects, the at least one UvsY polypeptide consists of a sequence with at least 95% sequence identity to the sequence of one of SEQ ID NOs: 15- 21. In some embodiments of any of the aspects, the at least one UvsY polypeptide consists essentially of a sequence with at least 95% sequence identity to the sequence of one of SEQ ID NOs: 15-21. In some embodiments of any of the aspects, the at least one UvsY polypeptide comprises a sequence with at least 99% sequence identity to the sequence of one of SEQ ID NOs: 15-21. In some embodiments of any of the aspects, the at least one UvsY polypeptide consists of a sequence with at least 99% sequence identity to the sequence of one of SEQ ID NOs: 15-21. In some embodiments of any of the aspects, the at least one UvsY polypeptide consists essentially of a sequence with at least 99% sequence identity to the sequence of one of SEQ ID NOs: 15-21. In some embodiments of any of the aspects, the at least one UvsY polypeptide comprises the sequence of one of SEQ ID NOs: 15-21. In some embodiments of any of the aspects, the at least one UvsY polypeptide consists of thesequence of one of SEQ ID NOs: 15-21. In some embodiments of any of the aspects, the at least one UvsY polypeptide consists essentially of the sequence of one of SEQ ID NOs: 15-21.

[0067] In some embodiments of any of the aspects, the at least one UvsY polypeptide comprises a sequence with at least 80% sequence identity to the sequence of SEQ ID NO: 15. In some embodiments of any of the aspects, the at least one UvsY polypeptide consists of a sequence with at least 80% sequence identity to the sequence of SEQ ID NO: 15. In some embodiments of any of the aspects, the at least one UvsY polypeptide consists essentially of a sequence with at least 80% sequence identity to the sequence of SEQ ID NO: 15. In some embodiments of any of the aspects, the at least one UvsY polypeptide comprises a sequence with at least 85% sequence identity to the sequence of SEQ ID NO: 15. In some embodiments of any of the aspects, the at least one UvsY polypeptide consists of a sequence with at least 85% sequence identity to the sequence of SEQ ID NO: 15. In some embodiments of any of the aspects, the at least one UvsY polypeptide consists essentially of a sequence with at least 85% sequence identity to the sequence of SEQ ID NO: 15. In some embodiments of any of the aspects, the at least one UvsY polypeptide comprises a sequence with at least 90% sequence identity to the sequence of SEQ ID NO: 15. In some embodiments of any of the aspects, the at least one UvsY polypeptide consists of a sequence with at least 90% sequence identity to the sequence of SEQ ID NO: 15. In some embodiments of any of the aspects, the at least one UvsY polypeptide consists essentially of a sequence with at least 90% sequence identity to the sequence of SEQ ID NO: 15. In some embodiments of any of the aspects, the at least one UvsY polypeptide comprises a sequence with at least 95% sequence identity to the sequence of SEQ ID NO: 15. In some embodiments of any of the aspects, the at least one UvsY polypeptide consists of a sequence with at least 95% sequence identity to the sequence of SEQ ID NO: 15. In some embodiments of any of the aspects, the at least one UvsY polypeptide consists essentially of a sequence with at least 95% sequence identity to the sequence of SEQ ID NO: 15. In some embodiments of any of the aspects, the at least one UvsY polypeptide comprises a sequence with at least 99% sequence identity to the sequence of SEQ ID NO: 15. In some embodiments of any of the aspects, the at least one UvsY polypeptide consists of a sequence with at least 99% sequence identity to the sequence of SEQ ID NO: 15. In some embodiments of any of the aspects, the at least one UvsY polypeptide consists essentially of a sequence with at least 99% sequence identity to the sequence of SEQ ID NO: 15. In some embodiments of any of the aspects, the at least one UvsY polypeptide comprises the sequence of SEQ ID NO: 15. In some embodiments of any of the aspects, the at least one UvsY polypeptide consists of the sequence of SEQ ID NO: 15. In some embodiments of any of the aspects, the at least one UvsY polypeptide consists essentially of the sequence of SEQ ID NO: 15.

[0068] In some embodiments of any of the aspects, the at least one UvsY polypeptide comprises a sequence with at least 80% sequence identity to the sequence of SEQ ID NO: 16. In some embodiments of any of the aspects, the at least one UvsY polypeptide consists of a sequence with atleast 80% sequence identity to the sequence of SEQ ID NO: 16. In some embodiments of any of the aspects, the at least one UvsY polypeptide consists essentially of a sequence with at least 80% sequence identity to the sequence of SEQ ID NO: 16. In some embodiments of any of the aspects, the at least one UvsY polypeptide comprises a sequence with at least 85% sequence identity to the sequence of SEQ ID NO: 16. In some embodiments of any of the aspects, the at least one UvsY polypeptide consists of a sequence with at least 85% sequence identity to the sequence of SEQ ID NO: 16. In some embodiments of any of the aspects, the at least one UvsY polypeptide consists essentially of a sequence with at least 85% sequence identity to the sequence of SEQ ID NO: 16. In some embodiments of any of the aspects, the at least one UvsY polypeptide comprises a sequence with at least 90% sequence identity to the sequence of SEQ ID NO: 16. In some embodiments of any of the aspects, the at least one UvsY polypeptide consists of a sequence with at least 90% sequence identity to the sequence of SEQ ID NO: 16. In some embodiments of any of the aspects, the at least one UvsY polypeptide consists essentially of a sequence with at least 90% sequence identity to the sequence of SEQ ID NO: 16. In some embodiments of any of the aspects, the at least one UvsY polypeptide comprises a sequence with at least 95% sequence identity to the sequence of SEQ ID NO: 16. In some embodiments of any of the aspects, the at least one UvsY polypeptide consists of a sequence with at least 95% sequence identity to the sequence of SEQ ID NO: 16. In some embodiments of any of the aspects, the at least one UvsY polypeptide consists essentially of a sequence with at least 95% sequence identity to the sequence of SEQ ID NO: 16. In some embodiments of any of the aspects, the at least one UvsY polypeptide comprises a sequence with at least 99% sequence identity to the sequence of SEQ ID NO: 16. In some embodiments of any of the aspects, the at least one UvsY polypeptide consists of a sequence with at least 99% sequence identity to the sequence of SEQ ID NO: 16. In some embodiments of any of the aspects, the at least one UvsY polypeptide consists essentially of a sequence with at least 99% sequence identity to the sequence of SEQ ID NO: 16. In some embodiments of any of the aspects, the at least one UvsY polypeptide comprises the sequence of SEQ ID NO: 16. In some embodiments of any of the aspects, the at least one UvsY polypeptide consists of the sequence of SEQ ID NO: 16. In some embodiments of any of the aspects, the at least one UvsY polypeptide consists essentially of the sequence of SEQ ID NO: 16.

[0069] In some embodiments of any of the aspects, the at least one UvsY polypeptide comprises a sequence with at least 80% sequence identity to the sequence of SEQ ID NO: 17. In some embodiments of any of the aspects, the at least one UvsY polypeptide consists of a sequence with at least 80% sequence identity to the sequence of SEQ ID NO: 17. In some embodiments of any of the aspects, the at least one UvsY polypeptide consists essentially of a sequence with at least 80% sequence identity to the sequence of SEQ ID NO: 17. In some embodiments of any of the aspects, the at least one UvsY polypeptide comprises a sequence with at least 85% sequence identity to the sequence of SEQ ID NO: 17. In some embodiments of any of the aspects, the at least one UvsYpolypeptide consists of a sequence with at least 85% sequence identity to the sequence of SEQ ID NO: 17. In some embodiments of any of the aspects, the at least one UvsY polypeptide consists essentially of a sequence with at least 85% sequence identity to the sequence of SEQ ID NO: 17. In some embodiments of any of the aspects, the at least one UvsY polypeptide comprises a sequence with at least 90% sequence identity to the sequence of SEQ ID NO: 17. In some embodiments of any of the aspects, the at least one UvsY polypeptide consists of a sequence with at least 90% sequence identity to the sequence of SEQ ID NO: 17. In some embodiments of any of the aspects, the at least one UvsY polypeptide consists essentially of a sequence with at least 90% sequence identity to the sequence of SEQ ID NO: 17. In some embodiments of any of the aspects, the at least one UvsY polypeptide comprises a sequence with at least 95% sequence identity to the sequence of SEQ ID NO: 17. In some embodiments of any of the aspects, the at least one UvsY polypeptide consists of a sequence with at least 95% sequence identity to the sequence of SEQ ID NO: 17. In some embodiments of any of the aspects, the at least one UvsY polypeptide consists essentially of a sequence with at least 95% sequence identity to the sequence of SEQ ID NO: 17. In some embodiments of any of the aspects, the at least one UvsY polypeptide comprises a sequence with at least 99% sequence identity to the sequence of SEQ ID NO: 17. In some embodiments of any of the aspects, the at least one UvsY polypeptide consists of a sequence with at least 99% sequence identity to the sequence of SEQ ID NO: 17. In some embodiments of any of the aspects, the at least one UvsY polypeptide consists essentially of a sequence with at least 99% sequence identity to the sequence of SEQ ID NO: 17. In some embodiments of any of the aspects, the at least one UvsY polypeptide comprises the sequence of SEQ ID NO: 17. In some embodiments of any of the aspects, the at least one UvsY polypeptide consists of the sequence of SEQ ID NO: 17. In some embodiments of any of the aspects, the at least one UvsY polypeptide consists essentially of the sequence of SEQ ID NO: 17.

[0070] In some embodiments of any of the aspects, the at least one UvsY polypeptide comprises a sequence with at least 80% sequence identity to the sequence of SEQ ID NO: 18. In some embodiments of any of the aspects, the at least one UvsY polypeptide consists of a sequence with at least 80% sequence identity to the sequence of SEQ ID NO: 18. In some embodiments of any of the aspects, the at least one UvsY polypeptide consists essentially of a sequence with at least 80% sequence identity to the sequence of SEQ ID NO: 18. In some embodiments of any of the aspects, the at least one UvsY polypeptide comprises a sequence with at least 85% sequence identity to the sequence of SEQ ID NO: 18. In some embodiments of any of the aspects, the at least one UvsY polypeptide consists of a sequence with at least 85% sequence identity to the sequence of SEQ ID NO: 18. In some embodiments of any of the aspects, the at least one UvsY polypeptide consists essentially of a sequence with at least 85% sequence identity to the sequence of SEQ ID NO: 18. In some embodiments of any of the aspects, the at least one UvsY polypeptide comprises a sequence with at least 90% sequence identity to the sequence of SEQ ID NO: 18. In some embodiments of anyof the aspects, the at least one UvsY polypeptide consists of a sequence with at least 90% sequence identity to the sequence of SEQ ID NO: 18. In some embodiments of any of the aspects, the at least one UvsY polypeptide consists essentially of a sequence with at least 90% sequence identity to the sequence of SEQ ID NO: 18. In some embodiments of any of the aspects, the at least one UvsY polypeptide comprises a sequence with at least 95% sequence identity to the sequence of SEQ ID NO: 18. In some embodiments of any of the aspects, the at least one UvsY polypeptide consists of a sequence with at least 95% sequence identity to the sequence of SEQ ID NO: 18. In some embodiments of any of the aspects, the at least one UvsY polypeptide consists essentially of a sequence with at least 95% sequence identity to the sequence of SEQ ID NO: 18. In some embodiments of any of the aspects, the at least one UvsY polypeptide comprises a sequence with at least 99% sequence identity to the sequence of SEQ ID NO: 18. In some embodiments of any of the aspects, the at least one UvsY polypeptide consists of a sequence with at least 99% sequence identity to the sequence of SEQ ID NO: 18. In some embodiments of any of the aspects, the at least one UvsY polypeptide consists essentially of a sequence with at least 99% sequence identity to the sequence of SEQ ID NO: 18. In some embodiments of any of the aspects, the at least one UvsY polypeptide comprises the sequence of SEQ ID NO: 18. In some embodiments of any of the aspects, the at least one UvsY polypeptide consists of the sequence of SEQ ID NO: 18. In some embodiments of any of the aspects, the at least one UvsY polypeptide consists essentially of the sequence of SEQ ID NO: 18.

[0071] In some embodiments of any of the aspects, the at least one UvsY polypeptide comprises a sequence with at least 80% sequence identity to the sequence of SEQ ID NO: 19. In some embodiments of any of the aspects, the at least one UvsY polypeptide consists of a sequence with at least 80% sequence identity to the sequence of SEQ ID NO: 19. In some embodiments of any of the aspects, the at least one UvsY polypeptide consists essentially of a sequence with at least 80% sequence identity to the sequence of SEQ ID NO: 19. In some embodiments of any of the aspects, the at least one UvsY polypeptide comprises a sequence with at least 85% sequence identity to the sequence of SEQ ID NO: 19. In some embodiments of any of the aspects, the at least one UvsY polypeptide consists of a sequence with at least 85% sequence identity to the sequence of SEQ ID NO: 19. In some embodiments of any of the aspects, the at least one UvsY polypeptide consists essentially of a sequence with at least 85% sequence identity to the sequence of SEQ ID NO: 19. In some embodiments of any of the aspects, the at least one UvsY polypeptide comprises a sequence with at least 90% sequence identity to the sequence of SEQ ID NO: 19. In some embodiments of any of the aspects, the at least one UvsY polypeptide consists of a sequence with at least 90% sequence identity to the sequence of SEQ ID NO: 19. In some embodiments of any of the aspects, the at least one UvsY polypeptide consists essentially of a sequence with at least 90% sequence identity to the sequence of SEQ ID NO: 19. In some embodiments of any of the aspects, the at least one UvsY polypeptide comprises a sequence with at least 95% sequence identity to the sequence of SEQ ID NO:19. In some embodiments of any of the aspects, the at least one UvsY polypeptide consists of a sequence with at least 95% sequence identity to the sequence of SEQ ID NO: 19. In some embodiments of any of the aspects, the at least one UvsY polypeptide consists essentially of a sequence with at least 95% sequence identity to the sequence of SEQ ID NO: 19. In some embodiments of any of the aspects, the at least one UvsY polypeptide comprises a sequence with at least 99% sequence identity to the sequence of SEQ ID NO: 19. In some embodiments of any of the aspects, the at least one UvsY polypeptide consists of a sequence with at least 99% sequence identity to the sequence of SEQ ID NO: 19. In some embodiments of any of the aspects, the at least one UvsY polypeptide consists essentially of a sequence with at least 99% sequence identity to the sequence of SEQ ID NO: 19. In some embodiments of any of the aspects, the at least one UvsY polypeptide comprises the sequence of SEQ ID NO: 19. In some embodiments of any of the aspects, the at least one UvsY polypeptide consists of the sequence of SEQ ID NO: 19. In some embodiments of any of the aspects, the at least one UvsY polypeptide consists essentially of the sequence of SEQ ID NO: 19.

[0072] In some embodiments of any of the aspects, the at least one UvsY polypeptide comprises a sequence with at least 80% sequence identity to the sequence of SEQ ID NO: 20. In some embodiments of any of the aspects, the at least one UvsY polypeptide consists of a sequence with at least 80% sequence identity to the sequence of SEQ ID NO: 20. In some embodiments of any of the aspects, the at least one UvsY polypeptide consists essentially of a sequence with at least 80% sequence identity to the sequence of SEQ ID NO: 20. In some embodiments of any of the aspects, the at least one UvsY polypeptide comprises a sequence with at least 85% sequence identity to the sequence of SEQ ID NO: 20. In some embodiments of any of the aspects, the at least one UvsY polypeptide consists of a sequence with at least 85% sequence identity to the sequence of SEQ ID NO: 20. In some embodiments of any of the aspects, the at least one UvsY polypeptide consists essentially of a sequence with at least 85% sequence identity to the sequence of SEQ ID NO: 20. In some embodiments of any of the aspects, the at least one UvsY polypeptide comprises a sequence with at least 90% sequence identity to the sequence of SEQ ID NO: 20. In some embodiments of any of the aspects, the at least one UvsY polypeptide consists of a sequence with at least 90% sequence identity to the sequence of SEQ ID NO: 20. In some embodiments of any of the aspects, the at least one UvsY polypeptide consists essentially of a sequence with at least 90% sequence identity to the sequence of SEQ ID NO: 20. In some embodiments of any of the aspects, the at least one UvsY polypeptide comprises a sequence with at least 95% sequence identity to the sequence of SEQ ID NO:20. In some embodiments of any of the aspects, the at least one UvsY polypeptide consists of a sequence with at least 95% sequence identity to the sequence of SEQ ID NO: 20. In some embodiments of any of the aspects, the at least one UvsY polypeptide consists essentially of a sequence with at least 95% sequence identity to the sequence of SEQ ID NO: 20. In some embodiments of any of the aspects, the at least one UvsY polypeptide comprises a sequence with atleast 99% sequence identity to the sequence of SEQ ID NO: 20. In some embodiments of any of the aspects, the at least one UvsY polypeptide consists of a sequence with at least 99% sequence identity to the sequence of SEQ ID NO: 20. In some embodiments of any of the aspects, the at least one UvsY polypeptide consists essentially of a sequence with at least 99% sequence identity to the sequence of SEQ ID NO: 20. In some embodiments of any of the aspects, the at least one UvsY polypeptide comprises the sequence of SEQ ID NO: 20. In some embodiments of any of the aspects, the at least one UvsY polypeptide consists of the sequence of SEQ ID NO: 20. In some embodiments of any of the aspects, the at least one UvsY polypeptide consists essentially of the sequence of SEQ ID NO: 20.

[0073] In some embodiments of any of the aspects, the at least one UvsY polypeptide comprises a sequence with at least 80% sequence identity to the sequence of SEQ ID NO: 21. In some embodiments of any of the aspects, the at least one UvsY polypeptide consists of a sequence with at least 80% sequence identity to the sequence of SEQ ID NO: 21. In some embodiments of any of the aspects, the at least one UvsY polypeptide consists essentially of a sequence with at least 80% sequence identity to the sequence of SEQ ID NO: 21. In some embodiments of any of the aspects, the at least one UvsY polypeptide comprises a sequence with at least 85% sequence identity to the sequence of SEQ ID NO: 21. In some embodiments of any of the aspects, the at least one UvsY polypeptide consists of a sequence with at least 85% sequence identity to the sequence of SEQ ID NO: 21. In some embodiments of any of the aspects, the at least one UvsY polypeptide consists essentially of a sequence with at least 85% sequence identity to the sequence of SEQ ID NO: 21. In some embodiments of any of the aspects, the at least one UvsY polypeptide comprises a sequence with at least 90% sequence identity to the sequence of SEQ ID NO: 21. In some embodiments of any of the aspects, the at least one UvsY polypeptide consists of a sequence with at least 90% sequence identity to the sequence of SEQ ID NO: 21. In some embodiments of any of the aspects, the at least one UvsY polypeptide consists essentially of a sequence with at least 90% sequence identity to the sequence of SEQ ID NO: 21. In some embodiments of any of the aspects, the at least one UvsY polypeptide comprises a sequence with at least 95% sequence identity to the sequence of SEQ ID NO: 21. In some embodiments of any of the aspects, the at least one UvsY polypeptide consists of a sequence with at least 95% sequence identity to the sequence of SEQ ID NO: 21. In some embodiments of any of the aspects, the at least one UvsY polypeptide consists essentially of a sequence with at least 95% sequence identity to the sequence of SEQ ID NO: 21. In some embodiments of any of the aspects, the at least one UvsY polypeptide comprises a sequence with at least 99% sequence identity to the sequence of SEQ ID NO: 21. In some embodiments of any of the aspects, the at least one UvsY polypeptide consists of a sequence with at least 99% sequence identity to the sequence of SEQ ID NO: 21. In some embodiments of any of the aspects, the at least one UvsY polypeptide consists essentially of a sequence with at least 99% sequence identity to the sequence of SEQ ID NO: 21. In some embodiments of any of the aspects, the at least one UvsY polypeptidecomprises the sequence of SEQ ID NO: 21. In some embodiments of any of the aspects, the at least one UvsY polypeptide consists of the sequence of SEQ ID NO: 21. In some embodiments of any of the aspects, the at least one UvsY polypeptide consists essentially of the sequence of SEQ ID NO: 21.

[0074] In some embodiments of any of the aspects, the at least one Gp32, UvsX, and / or UvsY polypeptide comprises a sequence with at least 80% sequence identity to the sequence of at least one of SEQ ID NOs: 1-21. In some embodiments of any of the aspects, the at least one Gp32, UvsX, and / or UvsY polypeptide consists of a sequence with at least 80% sequence identity to the sequence of at least one of SEQ ID NOs: 1 -21. In some embodiments of any of the aspects, the at least one Gp32, UvsX, and / or UvsY polypeptide consists essentially of a sequence with at least 80% sequence identity to the sequence of at least one of SEQ ID NOs: 1 -21. In some embodiments of any of the aspects, the at least one Gp32, UvsX, and / or UvsY polypeptide comprises a sequence with at least 85% sequence identity to the sequence of at least one of SEQ ID NOs: 1 -21. In some embodiments of any of the aspects, the at least one Gp32, UvsX, and / or UvsY polypeptide consists of a sequence with at least 85% sequence identity to the sequence of at least one of SEQ ID NOs: 1-21. In some embodiments of any of the aspects, the at least one Gp32, UvsX, and / or UvsY polypeptide consists essentially of a sequence with at least 85% sequence identity to the sequence of at least one of SEQ ID NOs: 1-21. In some embodiments of any of the aspects, the at least one Gp32, UvsX, and / or UvsY polypeptide comprises a sequence with at least 90% sequence identity to the sequence of at least one of SEQ ID NOs: 1-21. In some embodiments of any of the aspects, the at least one Gp32, UvsX, and / or UvsY polypeptide consists of a sequence with at least 90% sequence identity to the sequence of at least one of SEQ ID NOs: 1-21. In some embodiments of any of the aspects, the at least one Gp32, UvsX, and / or UvsY polypeptide consists essentially of a sequence with at least 90% sequence identity to the sequence of at least one of SEQ ID NOs: 1 -21. In some embodiments of any of the aspects, the at least one Gp32, UvsX, and / or UvsY polypeptide comprises a sequence with at least 95% sequence identity to the sequence of at least one of SEQ ID NOs: 1-21. In some embodiments of any of the aspects, the at least one Gp32, UvsX, and / or UvsY polypeptide consists of a sequence with at least 95% sequence identity to the sequence of at least one of SEQ ID NOs: 1-21. In some embodiments of any of the aspects, the at least one Gp32, UvsX, and / or UvsY polypeptide consists essentially of a sequence with at least 95% sequence identity to the sequence of at least one of SEQ ID NOs: 1-21. In some embodiments of any of the aspects, the at least one Gp32, UvsX, and / or UvsY polypeptide comprises a sequence with at least 99% sequence identity to the sequence of at least one of SEQ ID NOs: 1-21. In some embodiments of any of the aspects, the at least one Gp32, UvsX, and / or UvsY polypeptide consists of a sequence with at least 99% sequence identity to the sequence of at least one of SEQ ID NOs: 1-21. In some embodiments of any of the aspects, the at least one Gp32, UvsX, and / or UvsY polypeptide consists essentially of a sequence with at least 99% sequence identity to the sequence of at least one of SEQ ID NOs: 1 -21. In some embodiments of any of theaspects, the at least one Gp32, UvsX, and / or UvsY polypeptide comprises the sequence of at least one of SEQ ID NOs: 1-21. In some embodiments of any of the aspects, the at least one Gp32, UvsX, and / or UvsY polypeptide consists of the sequence of at least one of SEQ ID NOs: 1-21. In some embodiments of any of the aspects, the at least one Gp32, UvsX, and / or UvsY polypeptide consists essentially of the sequence of at least one of SEQ ID NOs: 1-21.

[0075] In some embodiments of any of the aspects, the at least one Gp32, UvsX, and / or UvsY polypeptide comprises the sequence of at least one of SEQ ID NOs: 29-49. In some embodiments of any of the aspects, the at least one Gp32, UvsX, and / or UvsY polypeptide consists of the sequence of at least one of SEQ ID NOs: 29-49. In some embodiments of any of the aspects, the at least one Gp32, UvsX, and / or UvsY polypeptide consists essentially of the sequence of at least one of SEQ ID NOs: 29-49.

[0076]

[0077] In some embodiments of any of the aspects, the at least one Gp32, UvsX, and / or UvsY polypeptide does not comprise the sequence of any of SEQ ID NOs: 22-28. In some embodiments of any of the aspects, the at least one Gp32, UvsX, and / or UvsY polypeptide does not comprise a T4 polypeptide.

[0078] In some embodiments of any of the aspects, the at least one Gp32 polypeptide does not comprise the sequence of SEQ ID NO: 22. In some embodiments of any of the aspects, the at least one Gp32 polypeptide does not comprise a T4 polypeptide.

[0079] In some embodiments of any of the aspects, the at least one UvsX polypeptide does not comprise the sequence of SEQ ID NO: 23. In some embodiments of any of the aspects, the at least one UvsX polypeptide does not comprise a T4 polypeptide.

[0080] In some embodiments of any of the aspects, the at least one UvsY polypeptide does not comprise the sequence of any of SEQ ID NOs: 24-28. In some embodiments of any of the aspects, the at least one UvsY polypeptide does not comprise a T4 polypeptide.

[0081] As used herein, “isolated” means artificially produced and / or removed from a naturally- occurring environment. As used herein with respect to proteins or peptides, the term "isolated" refers to a protein or peptide that has been isolated from its natural environment or artificially produced (e.g., by chemical synthesis, by recombinant DNA technology, etc.). As used herein with respect to nucleic acids, the term "isolated" means: (i) amplified in vitro by, for example, polymerase chain reaction (PCR); (ii) recombinantly produced by cloning; (iii) purified, as by cleavage and gel separation; or (iv) synthesized by, for example, chemical synthesis. An isolated nucleic acid is one which is readily manipulable by recombinant DNA techniques well known in the art. Thus, a nucleotide sequence contained in a vector in which 5' and 3' restriction sites are known or for which polymerase chain reaction (PCR) primer sequences have been disclosed is considered isolated but a nucleic acid sequence existing in its native state in its natural host is not. An isolated nucleic acid may be substantially purified, but need not be. For example, a nucleic acid that is isolated within a cloning or expression vector is not pure in that it may comprise only a tiny percentage of the material in thecell in which it resides. Such a nucleic acid is isolated, however, as the term is used herein because it is readily manipulable by standard techniques known to those of ordinary skill in the art.

[0082] In some embodiments of any of the aspects, the Gp32 polypeptide, UvsY polypeptide, and / or UvsX polypeptide present in a composition, or combination, of the disclosure exhibit an increased utility that is not exhibited when said Gp32 polypeptide, UvsY polypeptide, and / or UvsX polypeptide occur alone or when said Gp32 polypeptide, UvsY polypeptide, and / or UvsX polypeptide are present at a naturally occurring concentration. In some embodiments of any of the aspects, compositions of the disclosure, comprising a Gp32 polypeptide, UvsY polypeptide, and / or UvsX polypeptide as taught herein, exhibit a synergistic effect on imparting at least one improved trait in DNA amplification. In some embodiments of any of the aspects, the compositions of the disclosurecomprising a Gp32 polypeptide, UvsY polypeptide, and / or UvsX polypeptide as taught herein— exhibit markedly different characteristics / properties compared to their closest naturally occurring counterpart. That is, the compositions of the disclosure exhibit markedly different functional and / or structural characteristics / properties, as compared to their closest naturally occurring counterpart. For instance, a Gp32 polypeptide, UvsY polypeptide, and / or UvsX polypeptide of the disclosure is structurally different from a Gp32 polypeptide, UvsY polypeptide, and / or UvsX polypeptide as it naturally exists in a phage, for at least the following reasons: said Gp32 polypeptide, UvsY polypeptide, and / or UvsX polypeptide can be isolated and purified, such that it is not found in the milieu of the phage or host cell, said Gp32 polypeptide, UvsY polypeptide, and / or UvsX polypeptide can be present at concentrations that do not occur in the phage or host cell, said Gp32 polypeptide, UvsY polypeptide, and / or UvsX polypeptide can be associated with acceptable carriers that do not occur in the phage or host cell, said Gp32 polypeptide, UvsY polypeptide, and / or UvsX polypeptide can be formulated to be shelf-stable and exist outside the phage or host cell environment, and said Gp32 polypeptide, UvsY polypeptide, and / or UvsX polypeptide can be combined with other reagents / polypeptides at concentrations that do not exist in the phage or host cell. Further, the Gp32 polypeptide, UvsY polypeptide, and / or UvsX polypeptide of the disclosure are functionally different from a Gp32 polypeptide, UvsY polypeptide, and / or UvsX polypeptide as it naturally exists in a phage or host cell, for at least the following reasons: said Gp32 polypeptide, UvsY polypeptide, and / or UvsX polypeptide when applied in an isolated and purified form can perform RPA, said Gp32 polypeptide, UvsY polypeptide, and / or UvsX polypeptide can be formulated to be shelf-stable and able to exist outside the phage or host cell environment, such that the Gp32 polypeptide, UvsY polypeptide, and / or UvsX polypeptide now has a new utility in RPA, wherein the Gp32 polypeptide, UvsY polypeptide, and / or UvsX polypeptide could not have such a utility in its natural state in the phage or host cell, as the Gp32 polypeptide, UvsY polypeptide, and / or UvsX polypeptide would be unable to survive outside the phage or host cell without the intervention of the hand of man to formulate the Gp32 polypeptide, UvsY polypeptide, and / or UvsX polypeptide into a shelf-stable stateand impart this new utility that has the aforementioned functional characteristics not possessed by the Gp32 polypeptide, UvsY polypeptide, and / or UvsX polypeptide in its natural state of existence in the phage or host cell.

[0083] In some embodiments of any of the aspects, the at least one Gp32 polypeptide (e.g. isolated Gp32 polypeptide), the at least one UvsY polypeptide (e.g. isolated UvsY polypeptide), and / or the at least one UvsX polypeptide (e.g. isolated UvsX polypeptide) comprises an affinity tag. In some embodiments of any of the aspects, the at least one Gp32 polypeptide (e.g. isolated Gp32 polypeptide), the at least one UvsY polypeptide (e.g. isolated UvsY polypeptide), and the at least one UvsX polypeptide (e.g. isolated UvsX polypeptide) comprises an affinity tag. In some embodiments of any of the aspects, the affinity tag comprised by each polypeptide (Gp32, UvsY, and UvsX) is identical. In some embodiments of any of the aspects, the affinity tag on each polypeptide (Gp32, UvsY, and UvsX) is different or distinct from the affinity tag comprised by the other polypeptides, e.g., the affinity tag comprised by Gp32 is different or distinct from the affinity tag comprised by UvsY.

[0084] As used herein, “affinity tag” refers to a moiety, molecule, polypeptide sequence, or nucleic acid sequence that binds with or is bound by an affinity purification reagent. In some embodiments of any of the aspects, an affinity tag is an epitope. An affinity tag can provide a convenient means for isolating or purifying a polypeptide. When present, the affinity tag can be located anywhere in the polypeptide. For example, the affinity tag can be at the N-terminal, C- terminal or at an internal position of a polypeptide. In some embodiments, an affinity tag is at a position N-terminal of the Gp32 polypeptide, UvsX polypeptide, and / or UvsY polypeptide. In some embodiments, an epitope or affinity tag is at a position C-terminal of a Gp32 polypeptide, UvsX polypeptide, and / or UvsY polypeptide.

[0085] A number of affinity tags are known in the art. These are usually divided into 3 classes according to their size: small tags have a maximum of 12 amino acids, medium-sized ones have a maximum of 60 and large ones have more than 60. The small tags include the Arg -tag, the His-tag, the avidin biotin, or streptavidin (Strep)-tag, the Flag -tag, the T7-tag, the V5-peptide-tag and the c- Myc-tag, the medium-sized ones include the S-tag, the HAT-tag, the calmodulin-binding peptide, the chitin-binding domain (CBD) and some cellulose-binding domains. The latter can contain up to 189 amino acids and are then regarded, like the glutathione-S-transferase (GST)-and maltose binding protein (MBP)-tag, as large affinity tags.

[0086] Some exemplary affinity tag sequences include, but are not limited to, 6-HIS tag (HHHHHH (SEQ ID NO: 100), c-Myc epitope (EQKEISEEDE, SEQ ID NO: 101), Flag-tag (DYKDDDDK, SEQ ID NO: 102), AU1 HA tag (YPYDVPDYA, SEQ ID NO: 103), (DTYRYI, SEQ ID NO: 104), E tag (GAPVPYPDPLEPR, SEQ ID NO: 105); calmodulin tag (or CBP tag, KRRWKKNFIAVSAANRFKKISSSGAL, SEQ ID NO: 106), NE tag(TKENPRSNQEESYDDNESC, SEQ ID NO: 107); S tag (KETAAAKFERQHMDS, SEQ ID NO: 120); Streptavidin-Binding Peptide (SBP) tag (MDEKTTGWRGGHVVEGLAGELEQLRARLEHHPQGQREP, SEQ ID NO: 108); Strep tag (AWRHPQFGG, SEQ ID NO: 109) with high affinity for streptavidin; Spot tag (PDRVRAVSHWSS, SEQ ID NO: 110); pilin-C tag (TDKDMTITFTNKKDA, SEQ ID NO: 111); tetracysteine (TC) tag (CCPGCC, SEQ ID NO: 112); Ty tag (EVHTNQDPLD, SEQ ID NO: 113); V5 tag (GKPIPNPLLGLDST, SEQ ID NO: 114); and vesicular stomatitis virus (VSV) tag (YTDIEMNRLGK, SEQ ID NO: 115).

[0087] In some embodiments of any of the embodiments, the affinity tag is a His tag. In some embodiments, the tag comprises a His6-tag (HHHHHH, SEQ ID NO: 100), a His8-tag (HHHHHHHH, SEQ ID NO: 116), a HislO-tag (HHHHHHHHHH, SEQ ID NO: 117), or a Hisl4-tag (HHHHHHHHHHHHHH, SEQ ID NO: 118). In some embodiments of any of the aspects, the affinity tag is a His-Sumo tag.

[0088] In some embodiments of any of the aspects, described herein is a composition comprising two or more polypeptides selected from at least one Gp32 polypeptide (e.g., isolated Gp32 polypeptide), at least one UvsY polypeptide (e.g., isolated UvsY polypeptide), and at least one isolated UvsX polypeptide (e.g., isolated UvsX polypeptide); wherein the two or more polypeptides do not naturally occur in the same phage and / or are not naturally encoded by the same phage genome. In some embodiments of any of the aspects, described herein is a composition comprising two or more polypeptides selected from at least one Gp32 polypeptide (e.g., isolated Gp32 polypeptide), at least one UvsY polypeptide (e.g., isolated UvsY polypeptide), and at least one isolated UvsX polypeptide (e.g., isolated UvsX polypeptide); wherein the two or more polypeptides do not naturally occur in the same phage (e.g., T4). In some embodiments of any of the aspects, described herein is a composition comprising two or more polypeptides selected from at least one Gp32 polypeptide (e.g., isolated Gp32 polypeptide), at least one UvsY polypeptide (e.g., isolated UvsY polypeptide), and at least one isolated UvsX polypeptide (e.g., isolated UvsX polypeptide); wherein the two or more polypeptides are not naturally encoded by the same phage genome (e.g., T4 genome).

[0089] In one aspect of any of the embodiments, described herein is a polynucleotide or combination of polynucleotides comprising a sequence encoding one or more of: at least one Gp32 polypeptide; at least one UvsY polypeptide; and at least one UvsX polypeptide. In one aspect of any of the embodiments, described herein is a polynucleotide or combination of polynucleotides comprising a sequence encoding at least one Gp32 polypeptide and at least one UvsY polypeptide. In one aspect of any of the embodiments, described herein is a polynucleotide or combination of polynucleotides comprising a sequence encoding: at least one Gp32 polypeptide and at least one UvsX polypeptide. In one aspect of any of the embodiments, described herein is a polynucleotide or combination of polynucleotides comprising a sequence encoding: at least one UvsY polypeptide andat least one UvsX polypeptide. In one aspect of any of the embodiments, described herein is a polynucleotide or combination of polynucleotides comprising a sequence encoding: at least one Gp32 polypeptide; at least one UvsY polypeptide; and at least one UvsX polypeptide. In some embodiments of any of the aspects, the sequence encoding a polypeptide is operably connected to a heterologous promoter. In some embodiments of any of the aspects, the sequence encoding a polypeptide further encodes an affinity tag. In some embodiments of any of the aspects, the polynucleotide or combination of polynucleotides is isolated. In some embodiments of any of the aspects, the polynucleotide or combination of polynucleotides is engineered.

[0090] In some embodiments of any of the aspects, the heterologous promoter is a constitutive promoter. In some embodiments of any of the aspects, the heterologous promoter is an inducible promoter. As described herein, an “inducible promoter" is one that is characterized by initiating or enhancing transcriptional activity when in the presence of, influenced by, or contacted by an inducer or inducing agent than when not in the presence of, under the influence of, or in contact with the inducer or inducing agent. An “inducer" or “inducing agent" may be endogenous, or a normally exogenous compound or protein that is administered in such a way as to be active in inducing transcriptional activity from the inducible promoter. In some embodiments, the inducer or inducing agent, e.g., a chemical, a compound or a protein, can itself be the result of transcription or expression of a nucleic acid sequence ( e.g., an inducer can be a transcriptional repressor protein), which itself may be under the control or an inducible promoter. Non-limiting examples of inducible promoters include but are not limited to, the lac operon promoter, a nitrogen-sensitive promoter, an IPTG- inducible promoter, a salt-inducible promoter, and tetracycline, steroid-responsive promoters, rapamycin responsive promoters and the like. Inducible promoters for use in prokaryotic systems are well known in the art, see, e.g. the beta.-lactamase and lactose promoter systems (Chang et al., Nature, 275: 615 (1978, which is incorporated herein by reference); Goeddel et al., Nature, 281: 544 (1979), which is incorporated herein by reference), the arabinose promoter system, including the araBAD promoter (Guzman et al., J . Bacteriol., 174: 7716-7728 (1 992), which is incorporated herein by reference; Guzman et al., J. Bacteriol., 177: 4121-4130 (1995), which is incorporated herein by reference; Siegele and Hu, Proc. Natl. Acad. Sci. USA, 94: 8168-8172 (1997), which is incorporated herein by reference), the rhamnose promoter (Haldimann et al., J. Bacteriol., 180: 1277-1286 (1998), which is incorporated herein by reference), the alkaline phosphatase promoter, a tryptophan (trp) promoter system (Goeddel, Nucleic Acids Res., 8: 4057 (1980), which is incorporated herein by reference), the PUtetO-1 and Plac / are-1 promoters (Uutz and Bujard, Nucleic Acids Res., 25: 1203- 1210 (1997), which is incorporated herein by reference), and hybrid promoters such as the tac promoter. deBoer et al., Proc. Natl. Acad. Sci. USA, 80: 21-25 (1983), which is incorporated herein by reference. Non-limiting examples of mammalian and insect promoters can include CMV, SV40, UTR, and polyhedrin promoter. Exemplary promoters include but are not limited to trc promoter, tacpromoter, lac promoter, lambda phage promoter PL, the L-arabinose inducible araBAD promoter, the L-rhamnose inducible rhaP promoter, and the anhydrotetracycline -inducible tetA promoter / operator.

[0091] In one aspect of any of the embodiments, described herein is a cell comprising a polynucleotide or combination of polynucleotides as described herein, or comprising one or more of Gp32 polypeptide, UvsX polypeptide, and UvsY polypeptide. In one aspect of any of the embodiments, described herein is an organism comprising a polynucleotide or combination of polynucleotides as described herein, or comprising one or more of Gp32 polypeptide, UvsX polypeptide, and UvsY polypeptide. In one aspect of any of the embodiments, described herein is a bacterium comprising a polynucleotide or combination of polynucleotides as described herein, or comprising one or more of Gp32 polypeptide, UvsX polypeptide, and UvsY polypeptide. In some embodiment of any of the aspects, a genome comprises the polynucleotide or combination of polynucleotides. In some embodiment of any of the aspects, a vector comprises the polynucleotide or combination of polynucleotides. In some embodiment of any of the aspects, an expression vector comprises the polynucleotide or combination of polynucleotides. In some embodiment of any of the aspects, a plasmid comprises the polynucleotide or combination of polynucleotides. In some embodiments of any of the aspects, the cell is engineered. In some embodiments of any of the aspects, the organism is engineered. In some embodiments of any of the aspects, the bacterium is engineered.

[0092] The cell comprising the polynucleotide or combination of polynucleotides can be, e.g. a microbial cell or a mammalian cell. In some embodiments, the cell as described herein is cultured under conditions suitable for the expression of the polynucleotide or combination of polynucleotides. Such conditions can include, but are not limited to, conditions under which the cell is capable of growth and / or polypeptide synthesis. Conditions may vary depending upon the species and strain of cell selected. Conditions for the culture of cells, e.g. prokaryotic and mammalian cells, are well known in the art. If the recombinant polypeptide is operatively linked to an inducible promoter, such conditions can include the presence of the suitable inducing molecule(s).

[0093] Exemplary bacteria include Bacillus subtilis, Bacillus brevis, Bacillus megaterium, Caulobacter crescenius. and Escherichia coli BE21 and E. coli K12 and their derivatives. In some embodiments of any of the aspects, the bacterium is Escherichia coli.

[0094] In one aspect of any of the embodiments, described herein is a composition, combination, or kit, comprising: a) one or more of: i) at least one Gp32 polypeptide, ii) at least one UvsY polypeptide, and iii) at least one UvsX polypeptide; b) at least one of:i) a polymerase I polypeptide; and ii) at least one polynucleotide comprising a sequence not naturally occurring in Shigella phage Shfl2, Citrobacter phage CF1 ERZ-2017, Serratia phage CHI 14, Edwardsiella phage PEi20, Escherichia phage ECD7, Aeromonas phage PX29, and Pseudomonas phage pfl6.

[0095] As used herein “combination” refers to a group of two or more substances (e.g., reagents, molecules, biomolecules, or elements described herein) for use together, e.g., for RPA. The two or more substances can be present in the same formulation in any molecular or physical arrangement, e.g, in an admixture, in a solution, in a mixture, in a suspension, in a colloid, in an emulsion. The formulation can be a homogeneous or heterogenous mixture. In some embodiments of any of the aspects, the two or more substances can be comprised by the same or different superstructures, e.g., nanoparticles, liposomes, vectors, cells, scaffolds, or the like, and said superstructure is in solution, mixture, admixture, suspension with a solvent, carrier, or some of the two or more substances. Alternatively, the two or more substances can be present in two or more separate formulations, e.g., in a kit or package comprising multiple formulations in separate containers, to be mixed or brought into contact with each other when an assay is to be performed.

[0096] A kit is an assemblage of materials or components, including at least one substance (e.g., reagent, molecule, biomolecule, or element) described herein. The exact nature of the components configured in the kit depends on its intended purpose. In some embodiments of any of the aspects, a kit includes instructions for use. “Instructions for use” typically include a tangible expression describing the technique to be employed in using the components of the kit, e.g., to perform RPA or detect the presence of a target. Still in accordance with the present invention, “instructions for use” may include a tangible expression describing the preparation of at least one element described herein, such as dilution, mixing, or dosing instructions, and the like, typically for an intended purpose. Optionally, the kit also contains other useful components, such as, measuring tools, swabs, diluents, buffers, syringes, pharmaceutically acceptable carriers, or other useful paraphernalia as will be readily recognized by those of skill in the art.

[0097] The materials or components assembled in the kit can be provided to the practitioner stored in any convenient and suitable ways that preserve their operability and utility. For example, the components can be in dissolved, dehydrated, or lyophilized form; they can be provided at room, refrigerated or frozen temperatures. The components are typically contained in suitable packaging material(s). As employed herein, the phrase “packaging material” refers to one or more physical structures used to house the contents of the kit, such as inventive compositions and the like. The packaging material is constructed by well-known methods, preferably to provide a sterile, contaminant-free environment. The packaging may also preferably provide an environment that protects from light, humidity, and oxygen. As used herein, the term “package” refers to a suitablesolid matrix or material such as glass, plastic, paper, foil, polyester (such as polyethylene terephthalate, or Mylar) and the like, capable of holding the individual kit components. Thus, for example, a package can be a glass vial used to contain suitable quantities of a composition containing a volume of at least one reagent described herein. The packaging material generally has an external label which indicates the contents and / or purpose of the kit and / or its components.

[0098] As used herein, “polymerase” refers to an enzyme that synthesizes polymer chains, e.g., DNA or RNA molecules. As used herein “polymerase I” or “pol I” refers to a type of DNA polymerase that functions in DNA replication and comprises a thumb, finger, and palm domain that sustain polymerase activity, and two exonuclease domains. Pol I is also known as polA in the art. The sequences of Pol I from a number of species are known in the art. In some embodiments of any of the aspects, the polymerase I polypeptide is a phage Pol I. In some embodiments of any of the aspects, the polymerase I polypeptide is a T4 Pol I. In some embodiments of any of the aspects, the polymerase I polypeptide is Bacillus subtilis (Bsu) Pol I. Bsu Pol I can have the sequence of SEQ ID NO: 119. In some embodiments of any of the aspects, the polymerase I polypeptide comprises a sequence with at least 80% sequence identity to SEQ ID NO: 119. In some embodiments of any of the aspects, the polymerase I polypeptide comprises, consists of, or consists essentially of a sequence with at least 80% sequence identity to SEQ ID NO: 119. In some embodiments of any of the aspects, the polymerase I polypeptide comprises, consists of, or consists essentially of a sequence with at least 85% sequence identity to SEQ ID NO: 119. In some embodiments of any of the aspects, the polymerase I polypeptide comprises, consists of, or consists essentially of a sequence with at least 90% sequence identity to SEQ ID NO: 119. In some embodiments of any of the aspects, the polymerase I polypeptide comprises, consists of, or consists essentially of a sequence with at least 95% sequence identity to SEQ ID NO: 119.

[0099] SEQ ID NO: 1191 mterkklvlv dgnslayraf falpllsndk gvhtnavygf amilmkmled ekpthmlvaf61 dagkttfrhg tfkeykggrq ktppelseqm pfirellday qisryeleqy eaddiigtla121 ksaekdgfev kvfsgdkdlt qlatdkttva itrkgitdve fytpehvkek ygltpeqiid181 mkglmgdssd nipgvpgvge ktaikllkqf dsvekllesi devsgkklke kleefkdqal241 mskelatimt dapievsvsg leyqgfnreq viai fkdlgf ntllerlged saeaeqdqsl301 edinvktvtd vtsdilvsps afvveqigdn yheepilgfs ivnemgayfi pkdiavesev361 fkewvendeq kkwvfdskra vvalrwqgie Ikgaefdtll aayiinpgns yddvasvakd421 yglhivssde svygkgakra vpsedvlseh Igrkalaiqs Ireklvqele nndqlel fee481 lemplalilg emestgvkvd vdrlkrmgee Igaklkeyee kiheiagepf ninspkqlgv541 il fekiglpv vkktktgyst sadvleklad khdivdyilq yrqigklqst yiegllkvtr601 pdshkvhtrf nqaltqtgrl sstdpnlqni pirleegrki rqafvpsekd wli faadysq661 ielrvlahis kdenlieaft ndmdihtkta mdvfhvakde vtsamrrqak avnfgivygi721 sdyglsqnlg itrkeagafi dryles fqgv kaymedsvqe akqkgyvttl mhrrryipel781 tsrnfnirs f aertamntpi qgsaadiikk amidmaaklk ekqlkarlll qvhdeli fea841 pkeeieilek Ivpevmehal aldvplkvdf asgpswydak

[0100] In some embodiments of any of the aspects, the composition, combination, or kit described herein comprises: a) one or more of:i) at least one Gp32 polypeptide, ii) at least one UvsY polypeptide, and iii) at least one UvsX polypeptide; and b) a polymerase I polypeptide.

[0101] In some embodiments of any of the aspects, the composition, combination, or kit described herein comprises: at least one Gp32 polypeptide and a polymerase I polypeptide. In some embodiments of any of the aspects, the composition, combination, or kit described herein comprises: at least one UvsX polypeptide and a polymerase I polypeptide. In some embodiments of any of the aspects, the composition, combination, or kit described herein comprises: at least one UvsX polypeptide and a polymerase I polypeptide.

[0102] In some embodiments of any of the aspects, the composition, combination, or kit described herein comprises: at least one Gp32 polypeptide, at least one UvsY polypeptide, and a polymerase I polypeptide. In some embodiments of any of the aspects, the composition, combination, or kit described herein comprises: at least one Gp32 polypeptide, at least one UvsX polypeptide, and a polymerase I polypeptide. In some embodiments of any of the aspects, the composition, combination, or kit described herein comprises: at least one UvsX polypeptide, at least one UvsY polypeptide, and a polymerase I polypeptide. In some embodiments of any of the aspects, the composition, combination, or kit described herein comprises: at least one Gp32 polypeptide, at least one UvsY polypeptide, and at least one UvsX polypeptide; and a polymerase I polypeptide.

[0103] In some embodiments of any of the aspects, a composition, combination, or kit described herein comprises at least one polynucleotide comprising a sequence not naturally occurring in a phage. In some embodiments of any of the aspects, a composition, combination, or kit described herein comprises at least one polynucleotide comprising a sequence not naturally occurring in Shigella phage Shfl2, Citrohacter phage CF1 ERZ-2017, Serratia phage CHI 14, Edwardsiella phage PEi20, Escherichia phage ECD7, Aeromonas phage PX29, and Pseudomonas phage pfl6.

[0104] In some embodiments of any of the aspects, the at least one polynucleotide comprising a sequence not naturally occurring in a phage has a sequence which is less than 90% identical to a sequence of the same length which is naturally occurring in a phage. In some embodiments of any of the aspects, the at least one polynucleotide comprising a sequence not naturally occurring in a phage has a sequence which is less than 90% identical to a sequence of the same length which is naturally occurring in Shigella phage Shfl2, Citrohacter phage CF1 ERZ-2017, Serratia phage CHI 14, Edwardsiella phage PEi20, Escherichia phage ECD7, Aeromonas phage PX29, and Pseudomonas phage pfl6.

[0105] In some embodiments of any of the aspects, the at least one polynucleotide comprising a sequence not naturally occurring in a phage has a sequence which is less than 80% identical to a sequence of the same length which is naturally occurring in a phage. In some embodiments of any ofthe aspects, the at least one polynucleotide comprising a sequence not naturally occurring in a phage has a sequence which is less than 80% identical to a sequence of the same length which is naturally occurring in Shigella phage Shfl2, Citrohacter phage CF1 ERZ-2017, Serratia phage CHI 14, Edwardsiella phage PEi20, Escherichia phage ECD7, Aeromonas phage PX29, and Pseudomonas phage pfl6.

[0106] In some embodiments of any of the aspects, the at least one polynucleotide comprising a sequence not naturally occurring in a phage has a sequence which is less than 70% identical to a sequence of the same length which is naturally occurring in a phage. In some embodiments of any of the aspects, the at least one polynucleotide comprising a sequence not naturally occurring in a phage has a sequence which is less than 70% identical to a sequence of the same length which is naturally occurring in Shigella phage Shfl2, Citrohacter phage CF1 ERZ-2017, Serratia phage CHI 14, Edwardsiella phage PEi20, Escherichia phage ECD7, Aeromonas phage PX29, and Pseudomonas phage pfl6.

[0107] In some embodiments of any of the aspects, the at least one polynucleotide comprising a sequence not naturally occurring in a phage comprises one or more primer molecules and / or one or more double -stranded target polynucleotides.

[0108] In some embodiments of any of the aspects, the at least one polynucleotide comprising a sequence not naturally occurring in a phage comprises one or more primer molecules. In some embodiments of any of the aspects, the at least one polynucleotide comprising a sequence not naturally occurring in a phage consists of one or more primer molecules. In some embodiments of any of the aspects, the at least one polynucleotide comprising a sequence not naturally occurring in a phage consists essentially of one or more primer molecules.

[0109] As used herein, “primer" refers to a polynucleotide molecule or an analog thereof capable of sequence -specifically annealing to a polynucleotide template and providing a 3' end that serves as a substrate for a template-dependent polymerase to produce an extension product which is complementary to the polynucleotide template. The conditions for initiation and extension usually include the presence of at least one, but more preferably all four different deoxyribonucleoside triphosphates and a polymerization-inducing agent such as DNA polymerase (e.g, Pol I) or reverse transcriptase, in a suitable buffer (in this context "buffer" includes solvents (generally aqueous) plus necessary cofactors and reagents which affect pH, ionic strength, etc.) and at a suitable temperature. A primer useful in the methods described herein is generally single-stranded, and a primer and its complement can anneal to form a double -stranded polynucleotide. Primers according to the methods and compositions described herein can be less than or equal to 300 nucleotides in length, e.g., less than or equal to 300, or 250, or 200, or 150, or 100, or 90, or 80, or 70, or 60, or 50, or 40, and preferably 30 or fewer, or 20 or fewer, or 15 or fewer, but at least 10 nucleotides in length.

[0110] In some embodiments of any of the aspects, the methods described herein relate to the use of a set of primers. As used herein, the term "set of primers” refers to a group of at least two primers, including a forward primer and a reverse primer, one of which anneals to a first strand of a target polynucleotide and the other of which anneals to a complement of the first strand. In some embodiments of any of the aspects, the first primer of a primer pair subset can anneal to a first strand of the target polynucleotide and the second primer of a primer pair subset (e.g., reverse primer), can anneal to the complement of that strand. The orientation of the primers when annealed to the target and / or its complement can be such that nucleic acid synthesis proceeding from primer extension of a one primer of the primer pair subset would produce a nucleic acid sequence that is complementary to at least one region of the second primer of the primer pair subset. The “first strand" of a nucleic acid target and / or sequence can be either strand of a double-stranded nucleic acid comprising the sequence of the target nucleotide and / or target site locus, but once chosen, defines its complement as the second strand. Thus, as used herein, a “forward primer" is a primer which anneals to a first strand of a nucleic acid target, while a “reverse primer" of the same set is a primer which anneals to the complement of the first strand of the nucleic acid target. As used herein, “specific" when used in the context of a primer specific for a target polynucleotide refers to a level of complementarity between the primer and the target such that there exists an annealing temperature at which the primer will anneal to and mediate amplification of the target nucleic acid and will not anneal to or mediate amplification of non-target sequences present in a sample.

[0111] As used herein, "amplified product" refers to polynucleotides resulting from a reaction that are copies of a portion of a particular target nucleic acid sequence and / or its complementary sequence, which correspond in nucleotide sequence to the target sequence and / or its complementary sequence. An amplified product can be double or single stranded.

[0112] As used herein, “anneal" refers to permitting two complementary or substantially complementary nucleic acids strands to hybridize, and more particularly, when used in the context of RPA, to hybridize such that a primer extension substrate for a template -dependent polymerase enzyme is formed. Conditions for primer-target annealing vary with the length and sequence of the primer and are based upon the calculated Tm for the primer. In some embodiments of any of the aspects, the annealing temperature is no higher than the contacting temperature. Tm can be readily predicted by one of skill in the art using any of a number of widely available algorithms (e.g., OLIGOTM (Molecular Biology Insights Inc. Colorado) primer design software and VENTRO NTI™ (Invitrogen, Inc. California) primer design software and programs available on the internet, including Primer3 and Oligo Calculator). For example, Tm’s can be calculated using the NetPrimer software (Premier Biosoft; Palo Alto, CA; and freely available on the world wide web at premierbiosoft.com / netprimer / netprlaunch / Help / xnetprlaunch.html). The Tm of a primer can also be calculated using the following formula, which is used by NetPrimer software and is described in moredetail in Frieir et al. PNAS 1986 83:9373-9377 which is incorporated by reference herein in its entirety. Tm = AH / (AS + R * ln(C / 4)) + 16.6 log ([K+] / ( 1 + 0.7 [K+])) - 273.15 wherein, AH is enthalpy for helix formation; AS is entropy for helix formation; R is molar gas constant (1.987 cal / °C * mol); C is the nucleic acid concentration; and [K+] is salt concentration. As used herein, “substantially anneal" refers to a degree of annealing during a RPA regimen which is sufficient to produce a detectable level of a specifically amplified product.

[0113] Methods of making primers are well known in the art, and numerous commercial sources offer oligonucleotide synthesis services suitable for providing primers according to the methods and compositions described herein, e.g. INVITROGEN™ Custom DNA Oligos; Life Technologies; Grand Island, NY or custom DNA Oligos from IDT; Coralville, IA).

[0114] In some embodiments of any of the aspects, the one or more primer molecules are complementary to sequences naturally occurring in a coronavirus genome.

[0115] In some embodiments of any of the aspects, a primer can comprise at least one detectable label. Detectable labels are described elsewhere herein. Affinity tags can also be suitably used as detectable labels in conjunction with detection reagents that bind to / with the affinity tag.

[0116] In some embodiments of any of the aspects, the at least one polynucleotide comprising a sequence not naturally occurring in a phage comprises one or more double -stranded target polynucleotides. In some embodiments of any of the aspects, the at least one polynucleotide comprising a sequence not naturally occurring in a phage consists of one or more double -stranded target polynucleotides. In some embodiments of any of the aspects, the at least one polynucleotide comprising a sequence not naturally occurring in a phage consists essentially of one or more doublestranded target polynucleotides.

[0117] As used herein, the term "target" refers to a biological molecule (e.g., a polynucleotide) to which is to be detected, e.g, amplified by the compositions, combinations, or kits described herein. The target can be, for example, a sequence found in, or found specifically in an organism or virus of interest, e.g., a pathogenic organism or virus. A target polynucleotide for a specific organism or virus of interest can be readily identified by one of skill in the art by comparing sequence information for the organism or virus of interest to other known sequences, e.g., using NCBI database information or other published sequence information.

[0118] In some embodiments of any of the aspects, the one or more double-stranded target polynucleotides comprise a microbial genome or fragment thereof. In some embodiments of any of the aspects, the one or more double-stranded target polynucleotides comprise a coronavirus genome or fragment thereof. In some embodiments of any of the aspects, the one or more double-stranded target polynucleotides comprise a coronavirus genome sequence.

[0119] A composition, combination, or kit can be provided as, e.g., an aqueous solution or a lyophilized powder. In some embodiments of any of the aspects, a composition, combination, or kitfurther comprises a buffer, e.g., the elements described herein are dissolved or suspended in a buffer, or a buffer is provided in a separate formulation or container. Exemplary buffers are provided herein.

[0120] RPA reactions and reagents utilizing T4 proteins utilize crowding agents. Crowding agents have been reported to enhance the interaction of polymerase enzymes with DNA (Zimmerman and Harrison, 1987), to improve the activity of polymerases (Chan E. W. et al., 1980), to influence the kinetics of RecA binding to DNA in the presence of SSB (Layery and Kowalczykowski, 1992). Crowding agents are reported to have marked influence on systems in which co-operative binding of monomers is known to occur such as during rod and filament formation (Rivas et al., 2003) by increasing association constants by potentially several orders of magnitude (see Minton, 2001). In the RPA system multiple components rely on co-operative binding to nucleic acids, including the formation of SSB filaments, recombinase filaments, and possibly the condensation of loading agents such as UvsY. Crowding agents are also well known to enhance the hybridization of nucleic acids (Amasino, 1986), and this is a process that is also necessary within RPA reactions. Finally, and not least, the crowding agent PEG is known to drive the condensation of DNA molecules in which they change from elongated structures to compact globular or toroidal forms, thus mimicking structures more common in many in vivo contexts (see Lerman, 1971; also see Vasilevskaya. Et. AL, 1995; also see Zinchenko and Anatoly, 2005) and also to affect the supercoiling free energy of DNA (Naimushin et al., 2001). Each of the foregoing references is incorporated by reference herein in its entirety.

[0121] In some embodiments of any of the aspects, the crowding agent may be selected from the group comprising polyethylene glycol, polyethylene oxide, polyvinyl alcohol, polystyrene, Ficoll, dextran, PVP, albumin. In some embodiments of any of the aspects, the crowding agent has a molecular weight of less than 200,000 daltons.

[0122] In some embodiments of any of the aspects, the crowding agent has a molecular weight greater than 10,000 Daltons in size. In some embodiments of any of the aspects, the crowding agent is a PEG which has a molecular weight greater than 10,000 Daltons in size. In some embodiments of any of the aspects, the crowding agent is or comprises PEG. Suitable PEGs are known in the art, e.g, Carbowax 20M molecular weight 15-20,000, or PEG molecular weight 35,000).

[0123] The compositions, combinations, and kits described herein can provide effective RPA without crowding agents, or with minimal crowding agents. In some embodiments of any of the aspects, the composition, combination, or kit described herein does not comprise a crowding agent. In some embodiments of any of the aspects, the composition, combination, or kit described herein comprises less than 0.5% (w / v) of a crowding agent.

[0124] In some embodiments of any of the aspects, the method described herein does not comprise contacting a UvsX, UvsY, and / or Gp32 polypeptide with a crowding agent. In some embodiments of any of the aspects, the method described herein comprises contacting a UvsX, UvsY, and / or Gp32 polypeptide with less than 0.5% (w / v) of a crowding agent.

[0125] In some embodiments of any of the aspects, a suitable buffer comprises at least one of: a) 5 mM or greater NaCl; b) 1 mM or greater Tris-HCl; c) 1 mM or greater MgCl2; d) 0.1 mM or greater DTT; and e) pH 7.9 at 25°C.In some embodiments of any of the aspects, a suitable buffer comprises: a) 5 mM or greater NaCl; b) 1 mM or greater Tris-HCl; c) 1 mM or greater MgCl2; and d) 0.1 mM or greater DTT.In some embodiments of any of the aspects, a suitable buffer comprises at least one of: a) 50 mM NaCl; b) 10 mM Tris-HCl; c) 10 mM MgCl2; d) l mM DTT; and e) pH 7.9 at 25°C.In some embodiments of any of the aspects, a suitable buffer comprises: a) 50 mM NaCl; b) 10 mM Tris-HCl; c) 10 mM MgCl2; and d) I mM DTT.In some embodiments of any of the aspects, a suitable buffer comprises at least one of: a) 5 mM or greater Potassium acetate; b) 2 mM or greater Tris-acetate; c) 1 mM or greater Magnesium acetate; d) 0.1 mM or greater DTT; and e) pH 7.9 at 25°C.In some embodiments of any of the aspects, a suitable buffer comprises: a) 5 mM or greater Potassium acetate; b) 2 mM or greater Tris-acetate; c) 1 mM or greater Magnesium acetate; and d) 0.1 mM or greater DTT.In some embodiments of any of the aspects, a suitable buffer comprises at least one of: a) 50 mM Potassium acetate; b) 20 mM Tris-acetate;c) 10 mM Magnesium acetate; d) l mM DTT; and e) pH 7.9 at 25°C.In some embodiments of any of the aspects, a suitable buffer comprises: a) 50 mM Potassium acetate; b) 20 mM Tris-acetate; c) 10 mM Magnesium acetate; and d) I mM DTT.

[0126] In some embodiments of any of the aspects, a composition, combination, or kit can amplify a polynucleotide sequence at a temperature of less than 37 °C. In some embodiments of any of the aspects, a composition, combination, or kit can amplify a polynucleotide sequence at a temperature of less than 36 °C. In some embodiments of any of the aspects, a composition, combination, or kit can amplify a polynucleotide sequence at a temperature of less than 35 °C. In some embodiments of any of the aspects, a composition, combination, or kit can amplify a polynucleotide sequence at a temperature of less than 34 °C. In some embodiments of any of the aspects, a composition, combination, or kit can amplify a polynucleotide sequence at a temperature of less than 33 °C. In some embodiments of any of the aspects, a composition, combination, or kit can amplify a polynucleotide sequence at a temperature of less than 32 °C. In some embodiments of any of the aspects, a composition, combination, or kit can amplify a polynucleotide sequence at a temperature of less than 31 °C. In some embodiments of any of the aspects, a composition, combination, or kit can amplify a polynucleotide sequence at a temperature of less than 30 °C. In some embodiments of any of the aspects, a composition, combination, or kit can amplify a polynucleotide sequence at a temperature of less than 29 °C. In some embodiments of any of the aspects, a composition, combination, or kit can amplify a polynucleotide sequence at a temperature of less than 28 °C. In some embodiments of any of the aspects, a composition, combination, or kit can amplify a polynucleotide sequence at a temperature of less than 27 °C. In some embodiments of any of the aspects, a composition, combination, or kit can amplify a polynucleotide sequence at a temperature of less than 26 °C. In some embodiments of any of the aspects, a composition, combination, or kit can amplify a polynucleotide sequence at a temperature of less than 25 °C.

[0127] In some embodiments of any of the aspects, a composition, combination, or kit can amplify a polynucleotide sequence without need for a constant temperature or temperature-controlled environment.

[0128] In some embodiments of any of the aspects, a composition, combination, or kit can amplify a polynucleotide sequence with a higher fidelity than a composition, combination, or kit comprising T4 Gp32 polypeptide, T4 UvsY polypeptide, and T4 UvsX polypeptide.

[0129] The compositions, combinations, and kits described herein can amplify a polynucleotide sequence, e.g., by RPA. In one aspect of any of the embodiments, described herein is a method of amplifying a polynucleotide sequence, the method comprising contacting a target polynucleotide with a composition, combination, or kit as described herein. In some embodiments of any of the aspect, the target polynucleotide is a double -stranded polynucleotide. In some embodiments of any of the aspect, the target polynucleotide is or is found in a coronavirus genome or fragment thereof.

[0130] In one aspect of any of the embodiments, described herein is a method of detecting an organism or virus in a sample, the method comprising contacting a sample with a composition, combination, or kit as described herein; wherein amplification of a target polynucleotide found in the organism’s or virus’ genome indicates the organism or virus is present in the sample. In one aspect of any of the embodiments, described herein is an assay comprising contacting a sample with a composition, combination, or kit as described herein.

[0131] In some embodiments of any of the aspect, the contacting occurs at a temperature of less than 37 °C. In some embodiments of any of the aspect, the contacting occurs at a temperature of less than 36 °C. In some embodiments of any of the aspect, the contacting occurs at a temperature of less than 35 °C. In some embodiments of any of the aspect, the contacting occurs at a temperature of less than 34 °C. In some embodiments of any of the aspect, the contacting occurs at a temperature of less than 33 °C. In some embodiments of any of the aspect, the contacting occurs at a temperature of less than 32 °C. In some embodiments of any of the aspect, the contacting occurs at a temperature of less than 31 °C. In some embodiments of any of the aspect, the contacting occurs at a temperature of less than 30 °C. In some embodiments of any of the aspect, the contacting occurs at a temperature of less than 29 °C. In some embodiments of any of the aspect, the contacting occurs at a temperature of less than 28 °C. In some embodiments of any of the aspect, the contacting occurs at a temperature of less than 27 °C. In some embodiments of any of the aspect, the contacting occurs at a temperature of less than 26 °C. In some embodiments of any of the aspect, the contacting occurs at a temperature of less than 25 °C.

[0132] Methods to measure or detect amplification products are known to a skilled artisan.

[0133] In some embodiments of any of the aspects, one or more of the reagents (e.g. a primer) described herein can comprise a detectable label and / or comprise the ability to generate a detectable signal (e.g. by catalyzing reaction converting a compound to a detectable product). Detectable labels can comprise, for example, a light-absorbing dye, a fluorescent dye, or a radioactive label. Detectable labels, methods of detecting them, and methods of incorporating them into reagents (e.g. antibodies and nucleic acid probes) are well known in the art.

[0134] In some embodiments of any of the aspects, detectable labels can include labels that can be detected by spectroscopic, photochemical, biochemical, immunochemical, electromagnetic, radiochemical, or chemical means, such as fluorescence, chemifluoresence, or chemiluminescence, orany other appropriate means. The detectable labels used in the methods described herein can be primary labels (where the label comprises a moiety that is directly detectable or that produces a directly detectable moiety) or secondary labels (where the detectable label binds to another moiety to produce a detectable signal, e.g., as is common in immunological labeling using secondary and tertiary antibodies). The detectable label can be linked by covalent or non-covalent means to the reagent. Alternatively, a detectable label can be linked such as by directly labeling a molecule that achieves binding to the reagent via a ligand-receptor binding pair arrangement or other such specific recognition molecules. Detectable labels can include, but are not limited to radioisotopes, bioluminescent compounds, chromophores, antibodies, chemiluminescent compounds, fluorescent compounds, metal chelates, and enzymes.

[0135] In other embodiments, the detection reagent is labeled with a fluorescent compound. When the fluorescently labeled reagent is exposed to light of the proper wavelength, its presence can then be detected due to fluorescence. In some embodiments of any of the aspects, a detectable label can be a fluorescent dye molecule, or fluorophore including, but not limited to fluorescein, phycoerythrin, phycocyanin, o-phthaldehyde, fluorescamine, Cy3™, Cy5™, allophy cocyanine, Texas Red, peridenin chlorophyll, cyanine, tandem conjugates such as phycoerythrin-Cy5™, green fluorescent protein, rhodamine, fluorescein isothiocyanate (FITC) and Oregon Green™, rhodamine and derivatives (e.g., Texas red and tetrarhodimine isothiocynate (TRITC)), biotin, phycoerythrin, AMCA, CyDyes™, 6-carboxyfhiorescein (commonly known by the abbreviations FAM and F), 6- carboxy-2',4',7',4,7-hexachlorofiuorescein (HEX), 6-carboxy-4',5'-dichloro-2',7'-dimethoxyfiuorescein (JOE or J), N,N,N',N'-tetramethyl-6carboxyrhodamine (TAMRA or T), 6-carboxy-X-rhodamine (ROX or R), 5-carboxyrhodamine-6G (R6G5 or G5), 6-carboxyrhodamine-6G (R6G6 or G6), and rhodamine 110; cyanine dyes, e.g. Cy3, Cy5 and Cy7 dyes; coumarins, e.g umbelliferone; benzimide dyes, e.g. Hoechst 33258; phenanthridine dyes, e.g. Texas Red; ethidium dyes; acridine dyes; carbazole dyes; phenoxazine dyes; porphyrin dyes; polymethine dyes, e.g. cyanine dyes such as Cy3, Cy5, etc; BODIPY dyes and quinoline dyes. In some embodiments of any of the aspects, a detectable label can be a radiolabel including, but not limited to3H,1251,35S,14C,32P, and33P. In some embodiments of any of the aspects, a detectable label can be an enzyme including, but not limited to horseradish peroxidase and alkaline phosphatase. An enzymatic label can produce, for example, a chemiluminescent signal, a color signal, or a fluorescent signal. Enzymes contemplated for use to detectably label an antibody reagent include, but are not limited to, malate dehydrogenase, staphylococcal nuclease, delta-V-steroid isomerase, yeast alcohol dehydrogenase, alphaglycerophosphate dehydrogenase, triose phosphate isomerase, horseradish peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, beta-galactosidase, ribonuclease, urease, catalase, glucose-VI-phosphate dehydrogenase, glucoamylase and acetylcholinesterase. In some embodiments of any of the aspects, a detectable label is a chemiluminescent label, including, but not limited tolucigenin, luminol, luciferin, isoluminol, theromatic acridinium ester, imidazole, acridinium salt and oxalate ester. In some embodiments of any of the aspects, a detectable label can be a spectral colorimetric label including, but not limited to colloidal gold or colored glass or plastic (e.g., polystyrene, polypropylene, and latex) beads.

[0136] A reagent can also be detectably labeled using fluorescence emitting metals such as152Eu, or others of the lanthanide series. These metals can be attached to the reagent using such metal chelating groups as diethylenetriaminepentaacetic acid (DTP A) or ethylenediaminetetraacetic acid (EDTA).

[0137] In some embodiments of any of the aspects, reagents can also be labeled with a detectable tag, such as c-Myc, HA, VSV-G, HSV, FLAG, V5, HIS, or biotin. A secondary reagent then binds to the detectable tag and the secondary agent generates a signal or is otherwise detected. A non-limiting example is a biotin-streptavidin system. In this system, a biotinylated agent is detected using a streptavidin-peroxidase conjugate and a chromagenic substrate. Such streptavidin peroxidase detection kits are commercially available, e. g. from DAKO; Carpinteria, CA. Assays for detecting the binding of peptide secondary reagents to a detectable tag also include Western blots, radioimmunoassay (RIA), ELISA (enzyme linked immunosorbent assay), "sandwich" immunoassays, immunoprecipitation assays, immunodiffusion assays, agglutination assays, e.g. latex agglutination, complement-fixation assays, immunoradiometric assays, fluorescent immunoassays, e.g. FIA (fluorescence-linked immunoassay), chemiluminescence immunoassays (CLIA), electrochemiluminescence immunoassay (ECLIA, counting immunoassay (CIA), lateral flow tests or immunoassay (LFIA), magnetic immunoassay (MIA), and protein A immunoassays. In some embodiments of any of the aspects, the immunoassay can be a quantitative or a semi-quantitative immunoassay. In some embodiments of any of the aspects, the methods described herein comprise a lateral flow assay.

[0138] In some embodiments of any of the aspects, the level or presence of no more than 200 target polynucleotides is determined. In some embodiments of any of the aspects, the level or presence of no more than 100 target polynucleotides is determined. In some embodiments of any of the aspects, the level or presence of no more than 50 target polynucleotides is determined. In some embodiments of any of the aspects, the level or presence of no more than 20 target polynucleotides is determined. In some embodiments of any of the aspects, the level or presence of no more than 10 target polynucleotides is determined.

[0139] The term “sample” or “test sample” as used herein denotes a sample taken or isolated from a source, e.g., an environmental sample or a sample from a biological organism, e.g., a blood or plasma sample from a subject. The term “sample” or “test sample” also includes untreated or pretreated (or pre-processed) samples. The term “biological sample” or “biological test sample” asused herein denotes a sample taken or isolated from a source, e.g., from a biological organism, e.g., a blood or plasma sample from a subject.

[0140] In some embodiments of any of the aspects, the present technology encompasses several examples of a biological sample. In some embodiments of any of the aspects, the biological sample is cells, or tissue, or peripheral blood, or bodily fluid. Exemplary biological samples include, but are not limited to, a biopsy, a tumor sample, biofluid sample; blood; serum; plasma; urine; sperm; mucus; tissue biopsy; organ biopsy; synovial fluid; bile fluid; cerebrospinal fluid; mucosal secretion; effusion; sweat; saliva; and / or tissue sample etc. The term also includes a mixture of the above-mentioned samples. In some embodiments of any of the aspects, a test sample can comprise cells from a subject.

[0141] The sample can be obtained by removing a sample from a subject or location, but can also be accomplished by using a previously isolated sample (e.g. isolated at a prior timepoint and isolated by the same or another person).

[0142] In some embodiments of any of the aspects, the test sample can be an untreated test sample. As used herein, the phrase “untreated test sample” refers to a test sample that has not had any prior sample pre -treatment except for dilution and / or suspension in a solution. Exemplary methods for treating a test sample include, but are not limited to, centrifugation, filtration, sonication, homogenization, heating, freezing and thawing, and combinations thereof. In some embodiments of any of the aspects, the test sample can be a frozen test sample, e.g., a frozen tissue. The frozen sample can be thawed before employing methods, assays and systems described herein. After thawing, a frozen sample can be centrifuged before being subjected to methods, assays and systems described herein. In some embodiments of any of the aspects, the test sample is a clarified test sample, for example, by centrifugation and collection of a supernatant comprising the clarified test sample. In some embodiments of any of the aspects, a test sample can be a pre-processed test sample, for example, supernatant or filtrate resulting from a treatment selected from the group consisting of centrifugation, filtration, thawing, purification, and any combinations thereof. In some embodiments of any of the aspects, the test sample can be treated with a chemical and / or biological reagent. Chemical and / or biological reagents can be employed to protect and / or maintain the stability of the sample, including biomolecules (e.g., nucleic acid and protein) therein, during processing. One exemplary reagent is a protease inhibitor, which is generally used to protect or maintain the stability of protein during processing. The skilled artisan is well aware of methods and processes appropriate for pre-processing of biological samples required for determination of the level of an expression product as described herein.

[0143] In some embodiments of any of the aspects, the methods, assays, and systems described herein can further comprise a step of obtaining or having obtained a test sample from a subject. In some embodiments of any of the aspects, the subject can be a human subject.

[0144] Nucleic acid molecules can be isolated from a particular biological sample using any of a number of procedures, which are well-known in the art, the particular isolation procedure chosen being appropriate for the particular biological sample. For example, freeze-thaw and alkaline lysis procedures can be useful for obtaining nucleic acid molecules from solid materials; heat and alkaline lysis procedures can be useful for obtaining nucleic acid molecules from urine; and proteinase K extraction can be used to obtain nucleic acid from blood (Roiff, A et al. PCR: Clinical Diagnostics and Research, Springer (1994)).

[0145] The polypeptides and compositions described herein can be prepared by, e.g., contacting a bacterium, a supernatant from a culture of a bacterium, or a lysate of a bacterium with at least one affinity reagent specific for an affinity tag; wherein the bacterium comprises a polynucleotide or combination of polynucleotides encoding one or more polypeptides selected from at least one Gp32 polypeptide further comprising the affinity tag, at least one isolated UvsY polypeptide further comprising the affinity tag, and at least one isolated UvsX polypeptide further comprising the affinity tag.

[0146] In one respect, the present invention relates to the herein described compositions, methods, and respective component(s) thereof, as essential to the technology, yet open to the inclusion of unspecified elements, essential or not ("comprising). In some embodiments of any of the aspects, other elements to be included in the description of the composition, method or respective component thereof are limited to those that do not materially affect the basic and novel characteristic(s) of the technology (e.g., the composition, method, or respective component thereof “consists essentially of’ the elements described herein). This applies equally to steps within a described method as well as compositions and components therein. In other embodiments of any of the aspects, the compositions, methods, and respective components thereof, described herein are intended to be exclusive of any element not deemed an essential element to the component, composition or method (e.g., the composition, method, or respective component thereof “consists of’ the elements described herein). This applies equally to steps within a described method as well as compositions and components therein.

[0147] For convenience, the meaning of some terms and phrases used in the specification, examples, and appended claims, are provided below. Unless stated otherwise, or implicit from context, the following terms and phrases include the meanings provided below. The definitions are provided to aid in describing particular embodiments, and are not intended to limit the claimed invention, because the scope of the invention is limited only by the claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. If there is an apparent discrepancy between the usage of a term in the art and its definition provided herein, the definition provided within the specification shall prevail.

[0148] For convenience, certain terms employed herein, in the specification, examples and appended claims are collected here.

[0149] The terms “increased”, “increase”, “enhance”, or “activate” are all used herein to mean an increase by a statistically significant amount. In some embodiments, the terms “increased”, “increase”, “enhance”, or “activate” can mean an increase of at least 10% as compared to a reference level, for example an increase of at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% or up to and including a 100% increase or any increase between 10-100% as compared to a reference level, or at least about a 2-fold, or at least about a 3 -fold, or at least about a 4-fold, or at least about a 5-fold or at least about a 10-fold increase, or any increase between 2-fold and 10-fold or greater as compared to a reference level.

[0150] As used herein, a "subject" means a human or animal. Usually the animal is a vertebrate such as a primate, rodent, domestic animal or game animal. Primates include chimpanzees, cynomologus monkeys, spider monkeys, and macaques, e.g., Rhesus. Rodents include mice, rats, woodchucks, ferrets, rabbits and hamsters. Domestic and game animals include cows, horses, pigs, deer, bison, buffalo, feline species, e.g., domestic cat, canine species, e.g., dog, fox, wolf, avian species, e.g., chicken, emu, ostrich, and fish, e.g., trout, catfish and salmon. In some embodiments, the subject is a mammal, e.g., a primate, e.g., a human. The terms, “individual,” “patient” and “subject” are used interchangeably herein.

[0151] Preferably, the subject is a mammal. The mammal can be a human, non-human primate, mouse, rat, dog, cat, horse, or cow, but is not limited to these examples. Mammals other than humans can be advantageously used as subjects that represent animal models of infection, e.g., microbial, bacterial, fungal, or viral infection. A subject can be male or female.

[0152] As used herein, the terms “protein" and “polypeptide" are used interchangeably herein to designate a series of amino acid residues, connected to each other by peptide bonds between the alpha-amino and carboxy groups of adjacent residues. The terms "protein", and "polypeptide" refer to a polymer of amino acids, including modified amino acids (e.g., phosphorylated, glycated, glycosylated, etc.) and amino acid analogs, regardless of its size or function. "Protein" and “polypeptide” are often used in reference to relatively large polypeptides, whereas the term "peptide" is often used in reference to small polypeptides, but usage of these terms in the art overlaps. The terms "protein" and "polypeptide" are used interchangeably herein when referring to a gene product and fragments thereof. Thus, exemplary polypeptides or proteins include gene products, naturally occurring proteins, homologs, orthologs, paralogs, fragments and other equivalents, variants, fragments, and analogs of the foregoing. The terms also refer to fragments or variants of the polypeptide that maintain at least 50% of the activity or effect, e.g. DNA amplification or binding, of the full length polypeptide, e.g., of a wild type sequence provided herein (e.g., one of SEQ ID NOs: 1-21), e.g. as measured by an RPA assay as described herein. Conservative substitution variants that maintain the activity of wildtype Gp32, UvsX, and / or UvsY will include a conservative substitution as defined herein. The identification of amino acids most likely to be tolerant of conservative substitution while maintaining at least 50% of the activity of the wildtype is guided by, for example, sequence alignment with Gp32, UvsX, and / or UvsY homologs or paralogs from other species. Amino acids that are identical between Gp32, UvsX, and / or UvsY homologs are less likely to tolerate change, while those showing conservative differences are obviously much more likely to tolerate conservative change in the context of an artificial variant. Similarly, positions with non-conservative differences are less likely to be critical to function and more likely to tolerate conservative substitution in an artificial variant. Variants, fragments, and / or fusion proteins can be tested for activity, for example, by utilizing an RPA assay as described herein. Details of the structure of Gp32, UvsX, and / or UvsY are known in the art.

[0153] In some embodiments, a polypeptide, e.g., a Gp32, UvsX, and / or UvsY polypeptide, can be a variant of a sequence described herein, e.g. a variant of a Gp32, UvsX, and / or UvsY polypeptide comprising the amino acid sequence of one of SEQ ID NOs: 1-21. In some embodiments, the variant is a conservative substitution variant. Variants can be obtained by mutations of native nucleotide sequences, for example. A “variant,” as referred to herein, is a polypeptide substantially homologous to a native or reference polypeptide, but which has an amino acid sequence different from that of the native or reference polypeptide because of one or a plurality of deletions, insertions or substitutions. Polypeptide -encoding DNA sequences encompass sequences that comprise one or more additions, deletions, or substitutions of nucleotides when compared to a native or reference DNA sequence, but that encode a variant protein or fragment thereof that retains the relevant biological activity relative to the reference protein, e.g., can bind DNA or promote DNA amplification at least 50% as well as wildtype Gp32, UvsX, and / or UvsY. As to amino acid sequences, one of skill will recognize that individual substitutions, deletions or additions to a nucleic acid, peptide, polypeptide, or protein sequence which alters a single amino acid or a small percentage, (i.e. 5% or fewer, e.g. 4% or fewer, or 3% or fewer, or 1% or fewer) of amino acids in the encoded sequence is a “conservatively modified variant” where the alteration results in the substitution of an amino acid with a chemically similar amino acid. It is contemplated that some changes can potentially improve the relevant activity, such that a variant, whether conservative or not, has more than 100% of the activity of wildtype Gp32, UvsX, and / or UvsY, e.g. 110%, 125%, 150%, 175%, 200%, 500%, 1000% or more.

[0154] One method of identifying amino acid residues which can be substituted is to align, for example, Gp32, UvsX, and / or UvsY to a Gp32, UvsX, and / or UvsY homolog from one or more different phage. Alignment can provide guidance regarding not only residues likely to be necessary for function but also, conversely, those residues likely to tolerate change. Where, for example, an alignment shows two identical or similar amino acids at corresponding positions, it is more likely thatthat site is important functionally. Where, conversely, alignment shows residues in corresponding positions to differ significantly in size, charge, hydrophobicity, etc., it is more likely that that site can tolerate variation in a functional polypeptide. The variant amino acid or DNA sequence can be at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more, identical to a native or reference sequence, e.g. Gp32, UvsX, and / or UvsY or a nucleic acid encoding one of those amino acid sequences. The degree of homology (percent identity) between a native and a mutant sequence can be determined, for example, by comparing the two sequences using freely available computer programs commonly employed for this purpose on the world wide web. The variant amino acid or DNA sequence can be at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more, similar to the sequence from which it is derived (referred to herein as an “original” sequence). The degree of similarity (percent similarity) between an original and a mutant sequence can be determined, for example, by using a similarity matrix. Similarity matrices are well known in the art and a number of tools for comparing two sequences using similarity matrices are freely available online, e.g. BLASTp or BLASTn (available on the world wide web at blast.ncbi.nlm.nih.gov), with default parameters set.

[0155] In the various embodiments described herein, it is further contemplated that variants (naturally occurring or otherwise), alleles, homologs, conservatively modified variants, and / or conservative substitution variants of any of the particular polypeptides described are encompassed. As to amino acid sequences, one of skill will recognize that individual substitutions, deletions or additions to a nucleic acid, peptide, polypeptide, or protein sequence which alters a single amino acid or a small percentage of amino acids in the encoded sequence is a “conservatively modified variant" where the alteration results in the substitution of an amino acid with a chemically similar amino acid and retains the desired activity of the polypeptide. Such conservatively modified variants are in addition to and do not exclude polymorphic variants, interspecies homologs, and alleles consistent with the disclosure.

[0156] A given amino acid can be replaced by a residue having similar physiochemical characteristics, e.g., substituting one aliphatic residue for another (such as He, Vai, Leu, or Ala for one another), or substitution of one polar residue for another (such as between Lys and Arg; Glu and Asp; or Gin and Asn). Other such conservative substitutions, e.g., substitutions of entire regions having similar hydrophobicity characteristics, are well known. Polypeptides comprising conservative amino acid substitutions can be tested in any one of the assays described herein to confirm that a desired activity, e.g. DNA binding or DNA amplification activity and specificity of a native or reference polypeptide is retained.

[0157] A given amino acid can be replaced by a residue having similar physiochemical characteristics, e.g., substituting one aliphatic residue for another (such as He, Vai, Leu, or Ala for one another), or substitution of one polar residue for another (such as between Lys and Arg; Glu and Asp;or Gin and Asn). Other such conservative substitutions, e.g., substitutions of entire regions having similar hydrophobicity characteristics, are well known. Polypeptides comprising conservative amino acid substitutions can be tested in any one of the assays described herein to confirm that a desired activity of a native or reference polypeptide is retained. Conservative substitution tables providing functionally similar amino acids are well known in the art. Such conservatively modified variants are in addition to and do not exclude polymorphic variants, interspecies homologs, and alleles consistent with the disclosure.

[0158] Amino acids can be grouped according to similarities in the properties of their side chains (in A. L. Lehninger, in Biochemistry, second ed., pp. 73-75, Worth Publishers, New York (1975)): (1) non-polar: Ala (A), Vai (V), Leu (L), He (I), Pro (P), Phe (F), Trp (W), Met (M); (2) uncharged polar: Gly (G), Ser (S), Thr (T), Cys (C), Tyr (Y), Asn (N), Gin (Q); (3) acidic: Asp (D), Glu (E); (4) basic: Lys (K), Arg (R), His (H). Alternatively, naturally occurring residues can be divided into groups based on common side-chain properties: (1) hydrophobic: Norleucine, Met, Ala, Vai, Leu, He; (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gin; (3) acidic: Asp, Glu; (4) basic: His, Lys, Arg; (5) residues that influence chain orientation: Gly, Pro; (6) aromatic: Trp, Tyr, Phe. Non-conservative substitutions will entail exchanging a member of one of these classes for another class. Particular conservative substitutions include, for example; Ala into Gly or into Ser; Arg into Lys; Asn into Gin or into His; Asp into Glu; Cys into Ser; Gin into Asn; Glu into Asp; Gly into Ala or into Pro; His into Asn or into Gin; He into Leu or into Vai; Leu into He or into Vai; Lys into Arg, into Gin or into Glu; Met into Leu, into Tyr or into He; Phe into Met, into Leu or into Tyr; Ser into Thr; Thr into Ser; Trp into Tyr; Tyr into Trp; and / or Phe into Vai, into He or into Leu. Typically conservative substitutions for one another also include: 1) Alanine (A), Glycine (G); 2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W); 7) Serine (S), Threonine (T); and 8) Cysteine (C), Methionine (M) (see, e.g., Creighton, Proteins (1984)).

[0159] In some embodiments, the polypeptide described herein (or a nucleic acid encoding such a polypeptide) can be a functional fragment of one of the amino acid sequences described herein. As used herein, a “functional fragment” is a fragment or segment of a peptide which retains at least 50% of the wildtype reference polypeptide’s activity according to the assays described below herein. A functional fragment can comprise conservative substitutions of the sequences disclosed herein.

[0160] In some embodiments, the polypeptide described herein can be a variant of a sequence described herein. In some embodiments, the variant is a conservatively modified variant. Conservative substitution variants can be obtained by mutations of native nucleotide sequences, for example. A “variant," as referred to herein, is a polypeptide substantially homologous to a native or reference polypeptide, but which has an amino acid sequence different from that of the native or reference polypeptide because of one or a plurality of deletions, insertions or substitutions. Variant polypeptide-encoding DNA sequences encompass sequences that comprise one or more additions, deletions, or substitutions of nucleotides when compared to a native or reference DNA sequence, but that encode a variant protein or fragment thereof that retains activity. A wide variety of PCR-based site-specific mutagenesis approaches are known in the art and can be applied by the ordinarily skilled artisan.

[0161] In some embodiments, a polypeptide, e.g., a Gp32, UvsX, and / or UvsY polypeptide can comprise one or more amino acid substitutions or modifications. In some embodiments, the substitutions and / or modifications can prevent or reduce proteolytic degradation and / or prolong halflife of the polypeptide in a subject. In some embodiments, a polypeptide can be modified by conjugating or fusing it to other polypeptide or polypeptide domains such as, by way of non-limiting example, transferrin (WO06096515A2), albumin (Yeh et al., 1992), growth hormone (US2003104578AA); cellulose (Levy and Shoseyov, 2002); and / or Fc fragments (Ashkenazi and Chamow, 1997). The references in the foregoing paragraph are incorporated by reference herein in their entireties.

[0162] In some embodiments, a polypeptide, e.g., a Gp32, UvsX, and / or UvsY polypeptide, as described herein can comprise at least one peptide bond replacement. A Gp32, UvsX, and / or UvsY polypeptide as described herein can comprise one type of peptide bond replacement or multiple types of peptide bond replacements, e.g. 2 types, 3 types, 4 types, 5 types, or more types of peptide bond replacements. Non-limiting examples of peptide bond replacements include urea, thiourea, carbamate, sulfonyl urea, trifluoroethylamine, ortho-(aminoalkyl)-phenylacetic acid, para- (aminoalkyl) -phenylacetic acid, meta-(aminoalkyl) -phenylacetic acid, thioamide, tetrazole, boronic ester, olefinic group, and derivatives thereof.

[0163] In some embodiments, a polypeptide, e.g., a Gp32, UvsX, and / or UvsY polypeptide, as described herein can comprise naturally occurring amino acids commonly found in polypeptides and / or proteins produced by living organisms, e.g. Ala (A), Vai (V), Leu (L), He (I), Pro (P), Phe (F), Trp (W), Met (M), Gly (G), Ser (S), Thr (T), Cys (C), Tyr (Y), Asn (N), Gin (Q), Asp (D), Glu (E), Lys (K), Arg (R), and His (H). In some embodiments, a Gp32, UvsX, and / or UvsY polypeptide as described herein can comprise alternative amino acids. Non-limiting examples of alternative amino acids include, D-amino acids; beta-amino acids; homocysteine, phosphoserine, phosphothreonine, phosphotyrosine, hydroxyproline, gamma-carboxyglutamate; hippuric acid, octahydroindole-2 - carboxylic acid, statine, 1,2, 3, 4, -tetrahydroisoquinoline-3 -carboxylic acid, penicillamine (3-mercapto- D-valine), ornithine, citruline, alpha-methyl-alanine, para-benzoylphenylalanine, para-amino phenylalanine, p-fluorophenylalanine, phenylglycine, propargylglycine, sarcosine, and tertbutylglycine), diaminobutyric acid, 7-hydroxy-tetrahydroisoquinoline carboxylic acid, naphthylalanine, biphenylalanine, cyclohexylalanine, amino-isobutyric acid, norvaline, norleucine, tert-leucine, tetrahydroisoquinoline carboxylic acid, pipecolic acid, phenylglycine, homophenylalanine, cyclohexylglycine, dehydroleucine, 2,2-diethylglycine, 1-amino-l-cyclopentanecarboxylic acid, 1-amino-l -cyclohexanecarboxylic acid, amino-benzoic acid, aminonaphthoic acid, gamma-aminobutyric acid, difluorophenylalanine, nipecotic acid, alpha-amino butyric acid, thienyl-alanine, t-butylglycine, trifluoro valine; hexafluoroleucine; fluorinated analogs; azide- modified amino acids; alkyne-modified amino acids; cyano-modified amino acids; and derivatives thereof.

[0164] In some embodiments, a polypeptide, e.g. a Gp32, UvsX, and / or UvsY polypeptide, can be modified, e.g. by addition of a moiety to one or more of the amino acids that together comprise the peptide. In some embodiments, a polypeptide as described herein can comprise one or more moiety molecules, e.g. 1 or more moiety molecules per polypeptide, 2 or more moiety molecules per polypeptide, 5 or more moiety molecules per polypeptide, 10 or more moiety molecules per polypeptide or more moiety molecules per polypeptide. In some embodiments, a polypeptide as described herein can comprise one more types of modifications and / or moieties, e.g. 1 type of modification, 2 types of modifications, 3 types of modifications or more types of modifications. Nonlimiting examples of modifications and / or moieties include PEGylation; glycosylation; HESylation; ELPylation; lipidation; acetylation; amidation; end-capping modifications; cyano groups; phosphorylation; albumin, and cyclization. In some embodiments, an end-capping modification can comprise acetylation at the N-terminus, N-terminal acylation, and N-terminal formylation. In some embodiments, an end-capping modification can comprise amidation at the C-terminus, introduction of C-terminal alcohol, aldehyde, ester, and thioester moieties. The half-life of a polypeptide can be increased by the addition of moieties, e.g. PEG, albumin, or other fusion partners (e.g. Fc fragment of an immunoglobin).

[0165] Any cysteine residue not involved in maintaining the proper conformation of the polypeptide also can be substituted, generally with serine, to improve the oxidative stability of the molecule and prevent aberrant crosslinking. Conversely, cysteine bond(s) can be added to the polypeptide to improve its stability or facilitate oligomerization.

[0166] Alterations of the native amino acid sequence can be accomplished by any of a number of techniques known to one of skill in the art. Mutations can be introduced, for example, at particular loci by synthesizing oligonucleotides containing a mutant sequence, flanked by restriction sites enabling ligation to fragments of the native sequence. Following ligation, the resulting reconstructed sequence encodes an analog having the desired amino acid insertion, substitution, or deletion. Alternatively, oligonucleotide-directed site-specific mutagenesis procedures can be employed to provide an altered nucleotide sequence having particular codons altered according to the substitution, deletion, or insertion required. Techniques for making such alterations are very well established. Alterations of the original amino acid sequence can be accomplished by any of a number of techniques known to one of skill in the art. Mutations can be introduced, for example, at particular loci by synthesizing oligonucleotides containing a mutant sequence, flanked by restriction sitespermitting ligation to fragments of the native sequence. Following ligation, the resulting reconstructed sequence encodes an analog having the desired amino acid insertion, substitution, or deletion. Alternatively, oligonucleotide -directed site-specific mutagenesis procedures can be employed to provide an altered nucleotide sequence having particular codons altered according to the substitution, deletion, or insertion required. Techniques for making such alterations include those disclosed by Khudyakov et al. “Artificial DNA: Methods and Applications” CRC Press, 2002; Braman “In Vitro Mutagenesis Protocols” Springer, 2004; and Rapley “The Nucleic Acid Protocols Handbook” Springer 2000; which are herein incorporated by reference in their entireties. In some embodiments, a polypeptide as described herein can be chemically synthesized and mutations can be incorporated as part of the chemical synthesis process.

[0167] As used herein, the term “nucleic acid” “polynucleotide” or “nucleic acid sequence” refers to any molecule, preferably a polymeric molecule, incorporating units of ribonucleic acid, deoxyribonucleic acid or an analog thereof. The nucleic acid can be either single-stranded or doublestranded. A single -stranded nucleic acid can be one nucleic acid strand of a denatured doublestranded DNA. Alternatively, it can be a single-stranded nucleic acid not derived from any doublestranded DNA. In one aspect, the nucleic acid can be DNA. In another aspect, the nucleic acid can be RNA. Suitable DNA can include, e.g., genomic DNA or cDNA. Suitable RNA can include, e.g., mRNA.

[0168] The term "expression" refers to the cellular processes involved in producing RNA and proteins and as appropriate, secreting proteins, including where applicable, but not limited to, for example, transcription, transcript processing, translation and protein folding, modification and processing. Expression can refer to the transcription and stable accumulation of sense (mRNA) or antisense RNA derived from a nucleic acid fragment or fragments of the invention and / or to the translation of mRNA into a polypeptide.

[0169] "Expression products" include RNA transcribed from a gene, and polypeptides obtained by translation of mRNA transcribed from a gene. The term "gene" means the nucleic acid sequence which is transcribed (DNA) to RNA in vitro or in vivo when operably linked to appropriate regulatory sequences. The gene may or may not include regions preceding and following the coding region, e.g. 5’ untranslated (5’UTR) or "leader" sequences and 3’ UTR or "trailer" sequences, as well as intervening sequences (introns) between individual coding segments (exons).

[0170] “Operably linked” refers to an arrangement of elements wherein the components so described are configured so as to perform their usual function. Thus, control elements operably linked to a coding sequence are capable of effecting the expression of the coding sequence. The control elements need not be contiguous with the coding sequence, so long as they function to direct the expression thereof. Thus, for example, intervening untranslated yet transcribed sequences can bepresent between a promoter sequence and the coding sequence and the promoter sequence can still be considered "operably linked" to the coding sequence.

[0171] In some embodiments, the methods described herein relate to measuring, detecting, or determining the level of at least one target. As used herein, the term "detecting" or “measuring” refers to observing a signal from, e.g. a probe, label, or target molecule to indicate the presence of an analyte in a sample. Any method known in the art for detecting a particular label moiety can be used for detection. Exemplary detection methods include, but are not limited to, spectroscopic, fluorescent, photochemical, biochemical, immunochemical, electrical, optical or chemical methods. In some embodiments of any of the aspects, measuring can be a quantitative observation.

[0172] In some embodiments of any of the aspects, a polypeptide, nucleic acid, or cell as described herein can be engineered. As used herein, “engineered" refers to the aspect of having been manipulated by the hand of man. For example, a polypeptide is considered to be “engineered" when at least one aspect of the polypeptide, e.g., its sequence, has been manipulated by the hand of man to differ from the aspect as it exists in nature. As is common practice and is understood by those in the art, progeny of an engineered cell are typically still referred to as “engineered" even though the actual manipulation was performed on a prior entity.

[0173] In some embodiments of any of the aspects, a polypeptide or polynucleotide described herein is exogenous. In some embodiments of any of the aspects, a polypeptide or polynucleotide described herein is ectopic. In some embodiments of any of the aspects, a polypeptide or polynucleotide described herein is not endogenous.

[0174] The term "exogenous" refers to a substance present in a cell other than its native source. The term "exogenous" when used herein can refer to a nucleic acid (e.g. a nucleic acid encoding a polypeptide) or a polypeptide that has been introduced by a process involving the hand of man into a biological system such as a cell or organism in which it is not normally found and one wishes to introduce the nucleic acid or polypeptide into such a cell or organism. Alternatively, “exogenous” can refer to a nucleic acid or a polypeptide that has been introduced by a process involving the hand of man into a biological system such as a cell or organism in which it is found in relatively low amounts and one wishes to increase the amount of the nucleic acid or polypeptide in the cell or organism, e.g., to create ectopic expression or levels. In contrast, the term "endogenous" refers to a substance that is native to the biological system or cell. As used herein, “ectopic” refers to a substance that is found in an unusual location and / or amount. An ectopic substance can be one that is normally found in a given cell, but at a much lower amount and / or at a different time. Ectopic also includes substance, such as a polypeptide or nucleic acid that is not naturally found or expressed in a given cell in its natural environment.

[0175] In some embodiments, a nucleic acid encoding a polypeptide as described herein (e.g. a Gp32, UvsX, and / or UvsY polypeptide) is comprised by a vector. In some of the aspects describedherein, a nucleic acid sequence encoding a given polypeptide as described herein, or any module thereof, is operably linked to a vector. The term "vector", as used herein, refers to a nucleic acid construct designed for delivery to a host cell or for transfer between different host cells. As used herein, a vector can be viral or non-viral. The term “vector” encompasses any genetic element that is capable of replication when associated with the proper control elements and that can transfer gene sequences to cells. A vector can include, but is not limited to, a cloning vector, an expression vector, a plasmid, phage, transposon, cosmid, chromosome, virus, virion, etc.

[0176] In some embodiments of any of the aspects, the vector is recombinant, e.g., it comprises sequences originating from at least two different sources. In some embodiments of any of the aspects, the vector comprises sequences originating from at least two different species. In some embodiments of any of the aspects, the vector comprises sequences originating from at least two different genes, e.g., it comprises a fusion protein or a nucleic acid encoding an expression product which is operably linked to at least one non-native (e.g., heterologous) genetic control element (e.g., a promoter, suppressor, activator, enhancer, response element, or the like).

[0177] In some embodiments of any of the aspects, the vector or nucleic acid described herein is codon-optimized, e.g., the native or wild-type sequence of the nucleic acid sequence has been altered or engineered to include alternative codons such that altered or engineered nucleic acid encodes the same polypeptide expression product as the native / wild-type sequence, but will be transcribed and / or translated at an improved efficiency in a desired expression system. In some embodiments of any of the aspects, the expression system is an organism other than the source of the native / wild-type sequence (or a cell obtained from such organism). In some embodiments of any of the aspects, the vector and / or nucleic acid sequence described herein is codon-optimized for expression in a mammal or mammalian cell, e.g., a mouse, a murine cell, or a human cell. In some embodiments of any of the aspects, the vector and / or nucleic acid sequence described herein is codon-optimized for expression in a human cell. In some embodiments of any of the aspects, the vector and / or nucleic acid sequence described herein is codon-optimized for expression in a yeast or yeast cell. In some embodiments of any of the aspects, the vector and / or nucleic acid sequence described herein is codon-optimized for expression in a bacterial cell. In some embodiments of any of the aspects, the vector and / or nucleic acid sequence described herein is codon-optimized for expression in an E. coli cell.

[0178] As used herein, the term "expression vector" refers to a vector that directs expression of an RNA or polypeptide from sequences linked to transcriptional regulatory sequences on the vector. The sequences expressed will often, but not necessarily, be heterologous to the cell. An expression vector may comprise additional elements, for example, the expression vector may have two replication systems, thus allowing it to be maintained in two organisms, for example in human cells for expression and in a prokaryotic host for cloning and amplification.

[0179] As used herein, the term “viral vector" refers to a nucleic acid vector construct that includes at least one element of viral origin and has the capacity to be packaged into a viral vector particle. The viral vector can contain the nucleic acid encoding a polypeptide as described herein in place of non-essential viral genes. The vector and / or particle may be utilized for the purpose of transferring any nucleic acids into cells either in vitro or in vivo. Numerous forms of viral vectors are known in the art.

[0180] It should be understood that the vectors described herein can, in some embodiments, be combined with other suitable compositions and therapies. In some embodiments, the vector is episomal. The use of a suitable episomal vector provides a means of maintaining the nucleotide of interest in the subject in high copy number extra chromosomal DNA thereby eliminating potential effects of chromosomal integration.

[0181] As used herein, “contacting" refers to any suitable means for delivering, or exposing, a first agent or element to a second agent or element. Exemplary delivery methods include, but are not limited to, direct delivery to cell culture medium, perfusion, injection, or other delivery method well known to one skilled in the art. In some embodiments, contacting comprises physical human activity, e.g., an injection; an act of dispensing, mixing, and / or decanting; and / or manipulation of a delivery device or machine.

[0182] The term “statistically significant" or “significantly" refers to statistical significance and generally means a two standard deviation (2SD) or greater difference.

[0183] Other than in the operating examples, or where otherwise indicated, all numbers expressing quantities of ingredients or reaction conditions used herein should be understood as modified in all instances by the term “about.” The term “about” when used in connection with percentages can mean ±1%.

[0184] As used herein, the term “comprising” means that other elements can also be present in addition to the defined elements presented. The use of “comprising” indicates inclusion rather than limitation.

[0185] The term "consisting of refers to compositions, methods, and respective components thereof as described herein, which are exclusive of any element not recited in that description of the embodiment.

[0186] As used herein the term "consisting essentially of refers to those elements required for a given embodiment. The term permits the presence of additional elements that do not materially affect the basic and novel or functional characteristic(s) of that embodiment of the invention.

[0187] As used herein, the term “corresponding to” refers to an amino acid or nucleotide at the enumerated position in a first polypeptide or nucleic acid, or an amino acid or nucleotide that is equivalent to an enumerated amino acid or nucleotide in a second polypeptide or nucleic acid.Equivalent enumerated amino acids or nucleotides can be determined by alignment of candidate sequences using degree of homology programs known in the art, e.g., BLAST.

[0188] As used herein, the term “specific binding” refers to a chemical interaction between two molecules, compounds, cells and / or particles wherein the first entity binds to the second entity with greater specificity and affinity than it binds to a third entity which is a non-target. In some embodiments, specific binding can refer to an affinity of the first entity for the second entity which is at least 10 times, at least 50 times, at least 100 times, at least 500 times, at least 1000 times or greater than the affinity for the third nontarget entity. A reagent specific for a given target is one that exhibits specific binding for that target under the conditions of the assay being utilized.

[0189] The singular terms "a," "an," and "the" include plural referents unless context clearly indicates otherwise. Similarly, the word "or" is intended to include "and" unless the context clearly indicates otherwise. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of this disclosure, suitable methods and materials are described below. The abbreviation, "e.g." is derived from the Latin exempli gratia, and is used herein to indicate a non-limiting example. Thus, the abbreviation "e.g." is synonymous with the term "for example."

[0190] Groupings of alternative elements or embodiments of the invention disclosed herein are not to be construed as limitations. Each group member can be referred to and claimed individually or in any combination with other members of the group or other elements found herein. One or more members of a group can be included in, or deleted from, a group for reasons of convenience and / or patentability. When any such inclusion or deletion occurs, the specification is herein deemed to contain the group as modified thus fulfilling the written description of all Markush groups used in the appended claims.

[0191] Unless otherwise defined herein, scientific and technical terms used in connection with the present application shall have the meanings that are commonly understood by those of ordinary skill in the art to which this disclosure belongs. It should be understood that this invention is not limited to the particular methodology, protocols, and reagents, etc., described herein and as such can vary. The terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention, which is defined solely by the claims. Definitions of common terms in immunology and molecular biology can be found in The Merck Manual of Diagnosis and Therapy, 20th Edition, published by Merck Sharp & Dohme Corp., 2018 (ISBN 0911910190, 978-0911910421); Robert S. Porter et al. (eds.), The Encyclopedia of Molecular Cell Biology and Molecular Medicine, published by Blackwell Science Ltd., 1999-2012 (ISBN 9783527600908); and Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995 (ISBN 1-56081-569-8); Immunology by Werner Luttmann, published by Elsevier, 2006; Janeway's Immunobiology, Kenneth Murphy, Allan Mowat, Casey Weaver (eds.), W. W. Norton & Company, 2016 (ISBN 0815345054,978-0815345053); Lewin's Genes XI, published by Jones & Bartlett Publishers, 2014 (ISBN- 1449659055); Michael Richard Green and Joseph Sambrook, Molecular Cloning: A Laboratory Manual, 4th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., USA (2012) (ISBN 1936113414); Davis et al., Basic Methods in Molecular Biology, Elsevier Science Publishing, Inc., New York, USA (2012) (ISBN 044460149X); Laboratory Methods in Enzymology: DNA, Jon Lorsch (ed.) Elsevier, 2013 (ISBN 0124199542); Current Protocols in Molecular Biology (CPMB), Frederick M. Ausubel (ed.), John Wiley and Sons, 2014 (ISBN 047150338X, 9780471503385), Current Protocols in Protein Science (CPPS), John E. Coligan (ed.), John Wiley and Sons, Inc., 2005; and Current Protocols in Immunology (CPI) (John E. Coligan, ADA M Kruisbeek, David H Margulies, Ethan M Shevach, Warren Strobe, (eds.) John Wiley and Sons, Inc., 2003 (ISBN 0471142735, 9780471142737), the contents of which are all incorporated by reference herein in their entireties.

[0192] In all embodiments where a sample is obtained or has been obtained or provided, the sample can be sample taken, obtained, or provided via minimally invasive methods and / or involves only a minor intervention. In some embodiments of any of the aspects, a sample is taken, obtained, or provided by one or more of a blood draw or prick, an epidermal or mucus membrane swab, buccal sampling, saliva sample, a epidermal skin sampling technique, and / or collection of a secreted or expelled bodily fluid (e.g., mucus, urine, sweat, etc), fecal sampling, semen / seminal fluid sampling, or clippings (e.g., of hair or nails). In some embodiments of any of the aspects, the sample comprises, consists of, or consists essentially of blood (or any fraction or component thereof), serum, urine, mucus, epithelial cells, saliva, buccal cells, a secreted or expelled bodily fluid, and / or hair or nail clippings.

[0193] Other terms are defined herein within the description of the various aspects of the invention.

[0194] All patents and other publications; including literature references, issued patents, published patent applications, and co-pending patent applications; cited throughout this application are expressly incorporated herein by reference for the purpose of describing and disclosing, for example, the methodologies described in such publications that might be used in connection with the technology described herein. These publications are provided solely for their disclosure prior to the filing date of the present application. Nothing in this regard should be construed as an admission that the inventors are not entitled to antedate such disclosure by virtue of prior invention or for any other reason. All statements as to the date or representation as to the contents of these documents are based on the information available to the applicants and does not constitute any admission as to the correctness of the dates or contents of these documents.

[0195] The description of embodiments of the disclosure is not intended to be exhaustive or to limit the disclosure to the precise form disclosed. While specific embodiments of, and examples for, the disclosure are described herein for illustrative purposes, various equivalent modifications arepossible within the scope of the disclosure, as those skilled in the relevant art will recognize. For example, while method steps or functions are presented in a given order, alternative embodiments may perform functions in a different order, or functions may be performed substantially concurrently. The teachings of the disclosure provided herein can be applied to other procedures or methods as appropriate. The various embodiments described herein can be combined to provide further embodiments. Aspects of the disclosure can be modified, if necessary, to employ the compositions, functions and concepts of the above references and application to provide yet further embodiments of the disclosure. Moreover, due to biological functional equivalency considerations, some changes can be made in protein structure without affecting the biological or chemical action in kind or amount. These and other changes can be made to the disclosure in light of the detailed description. All such modifications are intended to be included within the scope of the appended claims.

[0196] Specific elements of any of the foregoing embodiments can be combined or substituted for elements in other embodiments. Furthermore, while advantages associated with certain embodiments of the disclosure have been described in the context of these embodiments, other embodiments may also exhibit such advantages, and not all embodiments need necessarily exhibit such advantages to fall within the scope of the disclosure.

[0197] In some embodiments, the present technology may be defined in any of the following numbered paragraphs:1. A composition comprising one or more of: a) at least one isolated Gp32 polypeptide; b) at least one isolated UvsY polypeptide; and c) at least one isolated UvsX polypeptide.2. The composition of paragraph 1, wherein the at least one isolated Gp32 polypeptide, the at least one isolated UvsY polypeptide, and / or the at least one isolated UvsX polypeptide comprises an affinity tag.3. The composition of any one of paragraphs 1-2, comprising two or more polypeptides selected from: a) at least one isolated Gp32 polypeptide, b) at least one isolated UvsY polypeptide, and c) at least one isolated UvsX polypeptide; wherein the two or more polypeptides do not naturally occur in the same phage and / or are not naturally encoded by the same phage genome.4. The composition of any one of paragraphs 1-3, wherein the one or more polypeptides are Shigella phage Shfl2 polypeptides, Citrohacter phage CF1 ERZ-2017 polypeptides, Serratia phage CHI14 polypeptides, Edwardsiella phage PEi20 polypeptides, Escherichia phageECD7 polypeptides, Aeromonas phage PX29 polypeptides, or Pseudomonas phage pfl6 polypeptides. The composition of any one of paragraphs 1-4, wherein the one or more polypeptides comprise a sequence selected from SEQ ID NOs: 1-21. A polynucleotide or combination of polynucleotides comprising a sequence encoding one or more of: a) at least one Gp32 polypeptide; b) at least one UvsY polypeptide; and c) at least one UvsX polypeptide. The nucleic acid or combination of nucleic acids of paragraph 6, wherein the sequence encoding a polypeptide is operably connected to a heterologous promoter. The nucleic acid or combination of nucleic acids of paragraph 6 or 7, wherein the sequence encoding a polypeptide further encodes an affinity tag. The nucleic acid or combination of nucleic acids of any one of paragraphs 6-8, wherein the one or more polypeptides are Shigella phage Shfl2 polypeptides, Citrobacter phage CF 1 ERZ-2017 polypeptides, Serratia phage CHI14 polypeptides, Edwardsiella phage PEi20 polypeptides, Escherichia phage ECD7 polypeptides, Aeromonas phage PX29 polypeptides, or Pseudomonas phage pfl6 polypeptides. The nucleic acid or combination of nucleic acids of any one of paragraphs 6-9, wherein the one or more polypeptides comprise a sequence selected from SEQ ID NOs: 1-21. A bacterium comprising a polynucleotide or combination of polynucleotides of any one of paragraphs 6-10, or comprising a composition of any one of paragraphs 1-5. The bacterium of paragraph 11, wherein the bacterium is Escherichia coli. A composition, combination, or kit, comprising: a) one or more of: i) at least one Gp32 polypeptide, ii) at least one UvsY polypeptide, and iii) at least one UvsX polypeptide; b) at least one of: iv) a polymerase I polypeptide; v) at least one polynucleotide comprising a sequence not naturally occurring in Shigella phage Shfl2, Citrobacter phage CF1 ERZ-2017, Serratia phage CHI 14, Edwardsiella phage PEi20, Escherichia phage ECD7, Aeromonas phage PX29, and Pseudomonas phage pfl6. The composition, combination, or kit of paragraph 13, wherein the polymerase I polypeptide is Bsu Pol I.The composition, combination, or kit of paragraphs 13 or 14, wherein the at least one polynucleotide comprises a sequence not naturally occurring in a phage. The composition, combination, or kit of any one of paragraphs 13-15, wherein the at least one polynucleotide comprises one or more primer molecules and / or one or more double -stranded target polynucleotides. The composition, combination, or kit of any one of paragraphs 13-15, wherein the at least one polynucleotide comprises one or more primer molecules. The composition, combination, or kit of paragraph 16 or 17, wherein the one or more primer molecules comprise at least one detectable label. The composition, combination, or kit of any one of paragraphs 13-18, wherein the one or more double-stranded target polynucleotides comprise a coronavirus genome or fragment thereof. The composition, combination, or kit of any one of paragraphs 13-19, wherein the one or more primer molecules are complementary to sequences naturally occurring in a coronavirus genome. The composition, combination, or kit of any one of paragraphs 13-20, wherein the composition, combination, or kit does not comprise a crowding agent. The composition, combination, or kit of any one of paragraphs 1-5 and 13-21, further comprising a buffer comprising at least one of: a) 5 mM or greater NaCl; b) 1 mM or greater Tris-HCl; c) 1 mM or greater MgCl2; d) 0.1 mM or greater DTT; and e) pH 7.9 at 25°C. The composition, combination, or kit of any one of paragraphs 1-5 and 13-21, further comprising a buffer comprising at least one of: a) 50 mM NaCl; b) 10 mM Tris-HCl; c) 10 mM MgCl2; d) l mM DTT; and e) pH 7.9 at 25°C. The composition, combination, or kit of any one of paragraphs 1-5 and 13-21, further comprising a buffer comprising at least one of: a) 5 mM or greater Potassium acetate; b) 2 mM or greater Tris-acetate; c) 1 mM or greater Magnesium acetate;d) 0.1 mM or greater DTT; and e) pH 7.9 at 25°C. The composition, combination, or kit of any one of paragraphs 1-5 and 13-20, further comprising a buffer comprising at least one of: a) 50 mM Potassium acetate; b) 20 mM Tris-acetate; c) 10 mM Magnesium acetate; d) l mM DTT; and e) pH 7.9 at 25°C. The composition, combination, or kit of any one of paragraphs 1-25, whereby the composition, combination, or kit can amplify a polynucleotide sequence at a temperature of less than 37 °C. The composition, combination, or kit of any one of paragraphs 1-26, whereby the composition, combination, or kit can amplify a polynucleotide sequence with a higher fidelity than a composition, combination, or kit comprising T4 Gp32 polypeptide, T4 UvsY polypeptide, and T4 UvsX polypeptide. A method of amplifying a polynucleotide sequence, the method comprising contacting a target polynucleotide with a composition, combination, or kit of any one of paragraphs 1-5 and 13-27. The method of paragraph 28, wherein the contacting occurs at a temperature of less than 37 °C. The method of paragraph 28 or 29, wherein the target polynucleotide comprises a coronavirus genome or fragment thereof. A method of detecting an organism or virus in a sample, the method comprising contacting a sample with a composition, combination, or kit of any one of paragraphs 1-5 and 13-27; wherein amplification of a target polynucleotide found in the organism’s or virus’ genome indicates the organism or virus is present in the sample. An assay comprising contacting a sample with a composition, combination, or kit of any one of paragraphs 1-5 and 13-27. A method of producing a composition comprising one or more polypeptides selected from: a) at least one isolated Gp32 polypeptide; b) at least one isolated UvsY polypeptide; and c) at least one isolated UvsX polypeptide; the method comprising: contacting a bacterium, a supernatant from a culture of a bacterium, or a lysate of a bacterium with at least one affinity reagent specific for an affinity tag;wherein the bacterium comprises a polynucleotide or combination of polynucleotides encoding one or more polypeptides selected from at least one Gp32 polypeptide further comprising the affinity tag, at least one isolated UvsY polypeptide further comprising the affinity tag, and at least one isolated UvsX polypeptide further comprising the affinity tag.

[0198] The technology described herein is further illustrated by the following examples which in no way should be construed as being further limiting.EXAMPLES

[0199] Example 1 : Protein Production of Phage Recombinases and Cofactors for Isothermal Amplification

[0200] While PCR remains the gold standard in nucleic acid detection it requires expensive equipment and well-trained personnel which makes it unsuitable for most point of need applications. Isothermal amplification approaches offer a potential solution to the problems of PCR in that they do not require expensive equipment or highly trained personnel. Recombinase polymerase amplification (RPA) is an isothermal approach to amplify a double stranded target DNA (Fig. 1). In RPA, a RecA- like enzyme (UvsX) performs recombinase-mediated primer targeting of the DNA template. A recombinase cofactor, UvsY, facilitates formation of the UvsX filament on ssDNA. Strand invasion of the primer results in formation of displaced single-stranded DNA which is stabilized by a singlestranded DNA binding protein, Gp32. Finally, strand displacement synthesis is catalyzed by a DNA polymerase.

[0201] Current commercial applications of RPA have relied on UvsX, UvsY and Gp32 from bacteriophage T4 along with Bsu Pol I. Two current issues hamper implementation of RPA. First, these enzymes function best at elevated temperatures (37-42 degrees C) which means that commercial applications of RPA have required a heating element. Second, RPA reactions consume a relatively large amount of protein and T4 UvsY does not express well and has issues with crashing out of solution.

[0202] In an effort to address the limitations of the T4 RPA proteins, a large collection of phage genomes that encode homologs of the recombinase UvsX, the recombinase loader UvsY, and the single -stranded DNA binding protein Gp32 are disclosed herein. Seven sets of these three proteins have been purified (See Fig. 2). Phage were selected to sample a broad range of sequence conservation with T4 and to include phages that infect either mesophiles or psychrophiles as these are more likely to contain homologs that function at room temperature.

[0203] To facilitate industrial scale protein purification, the inventors appended an N-terminal His6 tag (SEQ ID NO: 100) onto UvsY and Gp32 and a C-terminal His6 tag (SEQ ID NO: 100) onto UvsX. The inventors have engineered other tags as well to potentially increase solubility or to allowfor removal of the tag such as the His6-Sumo tag. Protein expression and solubility is quantified as compared to the T4 proteins. Qualitatively a number of UvsY homologs express better than T4 UvsY and are more stable in solution. The performance of these proteins is quantified in RPA assays both at elevated temperatures and at room temperature.

[0204] The protein purification protocol is depicted in Fig. 3.

[0205] Example 2

[0206] Testing paradigms implemented during the COVID pandemic include centralized labs (e.g. HUCL) and at home testing (Rapid antigen tests) (Fig. 4). The goal of the work described herein is to provide at home testing with the sensitivity and specificity of a centralized lab test with the speed of a rapid antigen test.

[0207] Methodologies utilized herein include RT-qPRA and quantitative PCR:

[0208] RT-qRPA: Recombinase Polymerase Amplification (RPA). Exemplary reaction mixture is a master mix containing salt, crowding agent, sugar additives, energy reservoirs, dNTPs, detergents, and RPA enzymes gp32, uvsX, uvsY, SSIV, and Bsu. Amplification occurs isothermally at 42°C on heat block for ~30 min. N-gene RNA input used at concentrations of 20,100, and 1000 molecules.

[0209] Quantitative PCR: cDNA product quantified by qPCR using N-gene targeting primers. Thermocycling conditions include 15 sec at 95°C, 15 sec at 60°C, 1 min at 72°C.

[0210] To optimize RPA for at home testing applications, 4 areas to improve have been identified: 1) Express and purify novel proteins, 2) Define the range of robustness of the assay, 3) Optimize for room temperature and no instrumentation, and 4) Decrease the total number of reagents.

[0211] Clinical samples were validated (Fig. 6) according to the workflow show in Fig. 5.

[0212] Selection of candidate UvsY, UvsX and Gp32 homologs. 7 sets of UvsY, UvsX andGp32 homologs were identified from 178 phages (Fig. 2). Sequence identities to T4 phage range from 29 to 98%. Some phages possibly infect psychrophiles.

[0213] Requirements for feasible purification include 1) Good expression of recombinant proteins in E. coli expression strains, 2) Reasonable solubility in expression strains, 3) Epitope tagging for easy purification -minimal functional interference. (Fig. 7)

[0214] Purification of recombinant homologs, mg quantities of reasonably pure recombinant proteins were obtained from one step Ni-NTA purification with 2-4 L cultures (Fig. 8). Storage buffers were optimized to keep recombinant proteins soluble at >100 pM.

[0215] Citrobacter phage proteins were tested (Fig. 9). Citro UvsY activity is concentration dependent and is more efficient at lower concentration than T4. Citro RPA proteins are more efficient than T4 proteins at lower concentrations.

[0216] The data presented herein demonstrate that the pipeline for recombinant protein purification is working and that citrobacter phage proteins outperform current commercial product.

[0217] More proteins can be expressed and tested. Optimal protein concentrations and mixtures can be determined. Behavior at room temperature and the robustness of the assays can be measured.

[0218]

Claims

What is claimed herein is:

1. A composition comprising one or more of: a) at least one isolated Gp32 polypeptide; b) at least one isolated UvsY polypeptide; and c) at least one isolated UvsX polypeptide.

2. The composition of claim 1, wherein the at least one isolated Gp32 polypeptide, the at least one isolated UvsY polypeptide, and / or the at least one isolated UvsX polypeptide comprises an affinity tag.

3. The composition of any one of claims 1-2, comprising two or more polypeptides selected from: a) at least one isolated Gp32 polypeptide, b) at least one isolated UvsY polypeptide, and c) at least one isolated UvsX polypeptide; wherein the two or more polypeptides do not naturally occur in the same phage and / or are not naturally encoded by the same phage genome.

4. The composition of any one of claims 1-3, wherein the one or more polypeptides are Shigella phage Shfl2 polypeptides, Citrohacter phage CF1 ERZ-2017 polypeptides, Serratia phage CHI14 polypeptides, Edwardsiella phage PEi20 polypeptides, Escherichia phage ECD7 polypeptides, Aeromonas phage PX29 polypeptides, or Pseudomonas phage pfl6 polypeptides.

5. The composition of any one of claims 1-4, wherein the one or more polypeptides comprise a sequence selected from SEQ ID NOs: 1-21.

6. A polynucleotide or combination of polynucleotides comprising a sequence encoding one or more of: a) at least one Gp32 polypeptide; b) at least one UvsY polypeptide; and c) at least one UvsX polypeptide.

7. The nucleic acid or combination of nucleic acids of claim 6, wherein the sequence encoding a polypeptide is operably connected to a heterologous promoter.

8. The nucleic acid or combination of nucleic acids of claim 6 or 7, wherein the sequence encoding a polypeptide further encodes an affinity tag.

9. The nucleic acid or combination of nucleic acids of any one of claims 6-8, wherein the one or more polypeptides are Shigella phage Shfl2 polypeptides, Citrobacter phage CF1 ERZ-2017 polypeptides, Serratia phage CHI14 polypeptides, Edwardsiella phage PEi20 polypeptides,Escherichia phage ECD7 polypeptides, Aeromonas phage PX29 polypeptides, or Pseudomonas phage pfl6 polypeptides.

10. The nucleic acid or combination of nucleic acids of any one of claims 6-9, wherein the one or more polypeptides comprise a sequence selected from SEQ ID NOs: 1-21.

11. A bacterium comprising a polynucleotide or combination of polynucleotides of any one of claims 6-10, or comprising a composition of any one of claims 1-5.

12. The bacterium of claim 11, wherein the bacterium is Escherichia coli.

13. A composition, combination, or kit, comprising: a) one or more of: i) at least one Gp32 polypeptide, ii) at least one UvsY polypeptide, and iii) at least one UvsX polypeptide; b) at least one of: iv) a polymerase I polypeptide; v) at least one polynucleotide comprising a sequence not naturally occurring in Shigella phage Shfl2, Citrohacter phage CF1 ERZ-2017, Serratia phage CHI 14, Edwardsiella phage PEi20, Escherichia phage ECD7, Aeromonas phage PX29, and Pseudomonas phage pfl6.

14. The composition, combination, or kit of claim 13, wherein the polymerase I polypeptide is Bsu Pol I.

15. The composition, combination, or kit of claim 13 or 14, wherein the at least one polynucleotide comprises a sequence not naturally occurring in a phage.

16. The composition, combination, or kit of any one of claims 13-15, wherein the at least one polynucleotide comprises one or more primer molecules and / or one or more double -stranded target polynucleotides.

17. The composition, combination, or kit of any one of claims 13-15, wherein the at least one polynucleotide comprises one or more primer molecules.

18. The composition, combination, or kit of claim 16 or 17, wherein the one or more primer molecules comprise at least one detectable label.

19. The composition, combination, or kit of any one of claims 13-18, wherein the one or more double -stranded target polynucleotides comprise a coronavirus genome or fragment thereof.

20. The composition, combination, or kit of any one of claims 13-19, wherein the one or more primer molecules are complementary to sequences naturally occurring in a coronavirus genome.

21. The composition, combination, or kit of any one of claims 13-20, wherein the composition, combination, or kit does not comprise a crowding agent.

22. The composition, combination, or kit of any one of claims 1-5 and 13-21, further comprising a buffer comprising at least one of: a) 5 mM or greater NaCl; b) 1 mM or greater Tris-HCl; c) 1 mM or greater MgCl2; d) 0.1 mM or greater DTT; and e) pH 7.9 at 25°C.

23. The composition, combination, or kit of any one of claims 1-5 and 13-21, further comprising a buffer comprising at least one of: a) 50 mM NaCl; b) 10 mM Tris-HCl; c) 10 mM MgCl2; d) l mM DTT; and e) pH 7.9 at 25°C.

24. The composition, combination, or kit of any one of claims 1-5 and 13-21, further comprising a buffer comprising at least one of: a) 5 mM or greater Potassium acetate; b) 2 mM or greater Tris-acetate; c) 1 mM or greater Magnesium acetate; d) 0.1 mM or greater DTT; and e) pH 7.9 at 25°C.

25. The composition, combination, or kit of any one of claims 1-5 and 13-20, further comprising a buffer comprising at least one of: a) 50 mM Potassium acetate; b) 20 mM Tris-acetate; c) 10 mM Magnesium acetate; d) l mM DTT; and e) pH 7.9 at 25°C.

26. The composition, combination, or kit of any one of claims 1-25, whereby the composition, combination, or kit can amplify a polynucleotide sequence at a temperature of less than 37 °C.

27. The composition, combination, or kit of any one of claims 1-26, whereby the composition, combination, or kit can amplify a polynucleotide sequence with a higher fidelity than a composition, combination, or kit comprising T4 Gp32 polypeptide, T4 UvsY polypeptide, and T4 UvsX polypeptide.

28. A method of amplifying a polynucleotide sequence, the method comprising contacting a target polynucleotide with a composition, combination, or kit of any one of claims 1-5 and 13-27.

29. The method of claim 28, wherein the contacting occurs at a temperature of less than 37 °C.

30. The method of claim 28 or 29, wherein the target polynucleotide comprises a coronavirus genome or fragment thereof.

31. A method of detecting an organism or virus in a sample, the method comprising contacting a sample with a composition, combination, or kit of any one of claims 1-5 and 13-27; wherein amplification of a target polynucleotide found in the organism’s or virus’ genome indicates the organism or virus is present in the sample.

32. An assay comprising contacting a sample with a composition, combination, or kit of any one of claims 1-5 and 13-27.

33. A method of producing a composition comprising one or more polypeptides selected from: a) at least one isolated Gp32 polypeptide; b) at least one isolated UvsY polypeptide; and c) at least one isolated UvsX polypeptide; the method comprising: contacting a bacterium, a supernatant from a culture of a bacterium, or a lysate of a bacterium with at least one affinity reagent specific for an affinity tag; wherein the bacterium comprises a polynucleotide or combination of polynucleotides encoding one or more polypeptides selected from at least one Gp32 polypeptide further comprising the affinity tag, at least one isolated UvsY polypeptide further comprising the affinity tag, and at least one isolated UvsX polypeptide further comprising the affinity tag.

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