Tnpb-related endonucleases

Abstract

Described herein is discovery of compositions and methods of use for TnpB-related nucleases.

Description

TNPB-RELATED ENDONUCLEASESCROSS-REFERENCE

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 510,329 filed on June 26, 2023, which is incorporated by reference herein in its entirety.BACKGROUND

[0002] CRISPR / Cas systems that comprise Cas endonucleases (such as the class 2, type II Cas9 or the class 2, type V Cpfl) are RNA-directed nuclease complexes that have been described to function as an adaptive immune system in microbes. Bioinformatic work has established that TnpB elements from IS200 / IS605 andIS607 transposon systems can share similarity in function to Cas endonucleases.SUMMARY

[0003] In some aspects, the present disclosure provides for a system comprising: (a) an RNA- guided endonuclease comprising a polypeptide sequence having at least 80% sequence identity to any one of SEQ ID NOs: 1, 3, 5, or a variant thereof, or a nucleic acid sequence encoding said RNA-guided endonuclease; and (b) an engineered or non-natural guide nucleic acid, or a template polynucleotide encoding said engineered or non-natural guide nucleic acid, wherein said engineered or non-natural guide nucleic acid is configured to form a complex with said RNA- guided endonuclease, comprising: (i) an anti-target nucleic acid sequence configured to hybridize to a target nucleic acid sequence; and (ii) a scaffold nucleic acid sequence configured to bind to said RNA-guided endonuclease. In some embodiments: (i) said engineered or non-natural guide nucleic acid comprises a sequence having at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, atleast about 88%, at least about 89%, at least about 90%, atleast about 91%, atleast about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, atleast about 98%, at least about 99%, or 100% sequence identity to any one of SEQ ID NOs: 2, 4, or 6 or an RNA cognate thereof; or (ii) said template polynucleotide encoding said engineered or non-natural guide nucleic acid encodes a sequence having at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequenceidentity to any one of SEQ ID NOs: 2, 4, or 6. In some embodiments, said sequence identity is determined over the whole length of said sequence. In some embodiments, said engineered or non-natural guide nucleic acid comprises (ii) said scaffold nucleic acid sequence and (i) said antitarget nucleic acid sequence in order from 5' to 3 '. In some embodiments, said engineered or non-natural guide nucleic acid comprises (ii) said scaffold nucleic acid sequence and (i) said antitarget nucleic acid sequence in order from 3 ' to 5 In some embodiments, said anti-target nucleic acid sequence comprises about 16-22 nucleotides. In some embodiments, said anti -target nucleic acid sequence comprises or is at least 16, at least 17, at least 18, at least 19, at least 20, or at least 21 nucleotides in length. In some embodiments, said anti-target nucleic acid sequence comprises or is at most 17, at most 18, at most 19, at most 20, at most 21, or at most 22 nucleotides in length. In some embodiments, said RNA-guided endonuclease is a class 2 Cas-like endonuclease. In some embodiments, said RNA-guided endonuclease is a class 2, type II Cas- like endonuclease. In some embodiments, said RNA-guided endonuclease is a class 2, type V Cas-like endonuclease. In some embodiments, said system further comprises a deoxyribonucleic acid (DNA) molecule comprising said target nucleic acid sequence, wherein said DNA molecule comprises a target adjacent motif (TAM) sequence 5' to said target nucleic acid sequence. In some embodiments, said RNA-guided endonuclease is configured to be selective for any of the TAM sequences described in Table 3, or 5'-NTBNHSB-3 '. In some embodiments, said target nucleic acid sequence is a eukaryotic, a mammalian, or a primate nucleic acid sequence. In some embodiments, said target nucleic acid sequence is a prokaryotic or an archaean nucleic acid sequence.

[0004] In some aspects, the present disclosure provides for a polynucleotide comprising an open reading frame (ORF) encoding a sequence having at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identity to any one of SEQ ID NOs: 1, 3, 5, or a variant thereof . In some embodiments, said ORF is codon-optimized for expression in a eukaryotic, a mammalian, or a primate organism. In some embodiments, said ORF is codon-optimized for expression in a prokaryotic or archaean organism. In some embodiments, said polynucleotide is an in vitro transcribed (IVT) RNA. In some embodiments, said polynucleotide is an in vitro transcribed (IVT) messenger ribonucleic acid (mRNA). In some embodiments, said polynucleotide is a synthetic RNA.

[0005] In some aspects, the present disclosure provides for a polynucleotide comprising or encoding: (ii) a scaffold nucleic acid sequence configured to bind to an RNA-guided endonuclease, wherein said scaffold nucleic acid sequence comprises a sequence having at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identity to any one of SEQ ID NOs: 2, 4, or 6, or an RNA cognate thereof. In some embodiments, the polynucleotide comprising or encoding (ii) further comprises (i) an antitarget nucleic acid sequence configured to hybridize to a target eukaryotic nucleic acid sequence. In some embodiments, said anti-target nucleic acid sequence is oriented 3' to said scaffold nucleic acid sequence. In some embodiments, said anti-target nucleic acid sequence comprises about 16-22 nucleotides. In some embodiments, said anti-target nucleic acid sequence comprises or is at least 16, atleast 17, atleast 18, at least 19, at least 20, or atleast 21 nucleotides in length. In some embodiments, said anti -target nucleic acid sequence comprises or is at most 17, at most 18, at most 19, at most 20, at most 21, or at most 22 nucleotides in length. In some embodiments, said polynucleotide comprises (i) said anti-target nucleic acid sequence and (ii) said scaffold nucleic acid sequence and is a synthetic or in-vitro transcribed (IVT) RNA. In some embodiments, said polynucleotide comprises said scaffold nucleic acid sequence and is a synthetic or in-vitro transcribed (IVT) RNA. In some embodiments, said polynucleotide encodes(i) said anti -target nucleic acid sequence and (ii) said scaffold nucleic acid sequence, and said polynucleotide further comprises a vector. In some embodiments, said polynucleotide encodes(ii) said scaffold nucleic acid sequence, and said polynucleotide further comprises a vector. In some embodiments, said vector comprises a viral vector. In some embodiments, vector comprises a DNA vector. In some embodiments, said polynucleotide further comprises an open reading frame (ORF) encoding a sequence having atleast about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identity to any one of SEQ ID NOs: 1, 3, 5, or a variant thereof. In some embodiments, said RNA-guided endonuclease comprises a sequence having at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at leastabout 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identity to any one of SEQ ID NOs: 1 , 3, 5, or a variant thereof.

[0006] In some aspects, the present disclosure provides for a method of binding a target nucleic acid sequence in a deoxyribonucleic acid (DNA) molecule, comprising contacting to said DNA with any of the systems, polynucleotides, polypeptides, or enzymes described herein. In some embodiments, said binding occurs within a cell. In some embodiments, said method comprises contacting to said cell any of the systems, polynucleotides, polypeptides, or enzymes described herein. In some embodiments, said DNA molecule further comprises a target adjacent motif (TAM) sequence and said target nucleic acid sequence, wherein said TAM sequence is 5 ' to said target nucleic acid sequence. In some embodiments, said cell is a eukaryotic, a mammalian, or a primate cell. In some embodiments, said cell is a prokaryotic or an archaean cell. In some embodiments, the method further comprises introducing a double-stranded break in said DNA molecule proximal to said target nucleic acid sequence. In some embodiments, the method is a method for introducing a double-stranded break in said DNA molecule proximal to said target nucleic acid sequence. In some embodiments, the method further comprises introducing a singlestranded nick in said DNA molecule proximal to said target nucleic acid sequence. In some embodiments, the method is a method for introducing a single-stranded nick in said DNA molecule proximal to said target nucleic acid sequence. In some embodiments, said doublestranded break or said single-stranded nick is introduced about 20-40 nucleotides 3 ' to said target nucleic acid sequence. In some embodiments, said double-stranded break or said singlestranded nick is introduced at least about 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, or 40 nucleotides 3 ' to said target nucleic acid sequence. In some embodiments, said double-stranded break or said single-stranded nick is introduced at most about 22, 24, 26, 28, 30, 32, 34, 36, 38, or 40 nucleotides 3 ' to said target nucleic acid sequence. In some embodiments, said double-stranded break or said single-stranded nick is introduced about 30-40 nucleotides 3 ' to said target nucleic acid sequence. In some embodiments, said double-stranded break is introduced about 35-40 nucleotides 3 ' to said target nucleic acid sequence.

[0007] In some aspects, the present disclosure provides for a cell comprising any of the systems, polypeptides, enzymes, or polynucleotides described herein. In some embodiments, said cell is a eukaryotic, a mammalian, or a primate cell. In some embodiments, said cell is a prokaryotic or an archaean cell.

[0008] In some aspects, the present disclosure provides for a pharmaceutically acceptable composition, comprising: (i) any of the systems, polypeptides, enzymes, or polynucleotides described herein; and (ii) a pharmaceutically acceptable carrier.

[0009] In some aspects, the present disclosure provides for a system comprising: (a) an RNA- guided endonuclease comprising a sequence having at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identity to any one of SEQ ID NOs: 1, 3, 5, or a variant thereof, or a nucleic acid sequence encoding said RNA-guided endonuclease. In some embodiments, the system further comprises: (b) an engineered guide nucleic acid or a template polynucleotide encoding said engineered guide nucleic acid, wherein said engineered guide nucleic acid is configured to form a complex with said RNA-guided endonuclease. In some cases, the engineered guide nucleic acid comprises: (i) an anti-target nucleic acid sequence configured to hybridize to a target nucleic acid sequence. In some cases, the engineered guide nucleic acid further comprises: (ii) a scaffold nucleic acid sequence configured to bind to said RNA-guided endonuclease. In some embodiments, the target nucleic acid sequence comprises or is a eukaryotic, mammalian, primate, or plant nucleic acid sequence. In some embodiments, said engineered guide nucleic acid comprises a sequence having at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identity to any one of SEQ ID NOs: 2, 4, or 6. In some embodiments, said template polynucleotide encoding said engineered guide nucleic acid encodes a sequence having at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identity to any one of SEQ ID NOs: 2, 4, or 6. In some embodiments, the sequence identity is determined over the whole length of said sequence. In some embodiments, said engineered guide nucleic acid comprises (ii) and (i) in order from 5' to 3 '. In some embodiments, said engineered guide nucleic acid comprises (ii) and (i) in order from 3 ' to 5 '. In some embodiments, said anti-target sequence comprises at least 16-22 nucleotides. In someembodiments, saidRNA-guided endonuclease comprises a class 2 Cas-like endonuclease, or an endonuclease that functions or is composed similarly to a class 2 Cas-like endonuclease. In some embodiments, saidRNA-guided endonuclease comprises a class 2, type II Cas-like endonuclease, or an endonuclease that functions or is composed similarly to a class 2, type II Cas endonuclease. In some embodiments, said RNA-guided endonuclease is a class 2, type V Cas-like endonuclease, or an endonuclease that functions or is composed similarly to a class 2, type V Cas endonuclease. In some embodiments, the system further comprises a DNA molecule comprising said target nucleic acid sequence, wherein said DNA molecule comprises a target adjacent motif (TAM) sequence 5' to said target nucleic acid sequence. In some embodiments, the system further comprises a DNA molecule comprising said target sequence, wherein said DNA molecule comprises a target adjacent motif (TAM) sequence 3 ' to said target nucleic acid sequence.

[0010] In some aspects, the present disclosure provides for a polynucleotide comprising an open reading frame (ORF) encoding a sequence having at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identity to any one of SEQ ID NOs: 1, 3, 5, or a variant thereof, wherein said open reading frame is codon-optimized for expression in a eukaryotic, mammalian, primate, or plant organism. In some embodiments, said polynucleotide is an in vitro transcribed (IVT) mRNA. In some embodiments, said polynucleotide is an RNA molecule comprising a 7-methyl-guanosine (m7G) cap. In some embodiments, said m7G cap further comprises m7G followed by three phosphates.

[0011] In some aspects, the present disclosure provides for a polynucleotide comprising or encoding: (i) a non-natural or engineered anti-target nucleic acid sequence configured to hybridize to a target nucleic acid sequence; and (ii) a scaffold nucleic acid sequence configured to bind to said RNA-guided endonuclease, wherein said scaffold nucleic acid sequence comprises a sequence having at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identity to any one of SEQ ID NOs: 2, 4, or 6, or an RNA cognate thereof. In some embodiments, the target nucleic acid sequence comprises or is a eukaryotic, mammalian, primate, or plant sequence. In some embodiments, said anti-target sequence is oriented 3 ' to said scaffold nucleic acid sequence. In some embodiments, said anti-target sequence is oriented 5 ' to said scaffold nucleic acid sequence. In some embodiments, said anti-target nucleic acid sequence comprises at least 16-22 nucleotides. In some embodiments, said polynucleotide comprises (i) and (ii), wherein said polynucleotide is a synthetic or in-vitro transcribed (IVT) RNA. In some embodiments, said polynucleotide encodes (i) and (ii), wherein said polynucleotide further comprises a vector. In some embodiments, said vector comprises a viral vector. In some embodiments, said vector comprises a DNA vector. In some embodiments, said polynucleotide further comprises an open reading frame (ORF) encoding a sequence having at least about 80%, at least about 81 %, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, atleast about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identity to any one of SEQ ID NOs: 1, 3, 5, or a variant thereof.

[0012] In some aspects, the present disclosure provides for a method of binding a target nucleic acid sequence in a deoxyribonucleic acid (DNA) molecule in a eukaryotic, mammalian, primate, or plant cell, comprising contacting to said cell any of the systems, polynucleotides, or polypeptides described herein. In some embodiments, the method further comprises introducing a double-stranded break or single-stranded nick in said DNA molecule proximal to said target nucleic acid sequence. In some embodiments, said double-stranded break or said single-stranded nick is introduced about 20-40 nucleotides 3 ' to said target sequence. In some embodiments, said double-stranded break or said single-stranded nick is introduced about 30-40 nucleotides 3' to said target sequence. In some embodiments, said double-stranded break or said single-stranded nick is introduced about 35-40 nucleotides 3 ' to said target sequence. In some embodiments, said cell comprises a genomic region comprising a TAM sequence and said target nucleic acid sequence, wherein said TAM sequence is 5' to said target nucleic acid sequence. In some embodiments, said cell comprises a genomic region comprising a TAM sequence and said target nucleic acid sequence, wherein said TAM sequence is 3 ' to said target nucleic acid sequence.

[0013] In some aspects, the present disclosure provides for a cell comprising any of the systems, polypeptides, or polynucleotides described herein. In some embodiments, the cells is a eukaryotic, mammalian, or primate cell. In some embodiments, the cell is a prokaryotic or archaean cell.

[0014] In some aspects, the present disclosure provides for a pharmaceutically acceptable composition, comprising: (i) any of the systems, polynucleotides, or polypeptides described herein; and (ii) a pharmaceutically acceptable carrier.

[0015] Additional aspects and advantages of the present disclosure will become readily apparent to those skilled in this art from the following detailed description, wherein only illustrative embodiments of the present disclosure are shown and described. As will be realized, the present disclosure is capable of other and different embodiments, and its several details are capable of modifications in various obvious respects, all without departing from the disclosure. Accordingly, the drawings and description are to be regarded as illustrative in nature, and not as restrictive.INCORPORATION BY REFERENCE

[0016] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.BRIEF DESCRIPTION OF THE DRAWINGS

[0017] The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings of which:

[0018] Figure 1 (FIG. 1) depicts discovery of uncharacterized TnpB-related nucleases. Panel A shows a flow chart illustrating an example of a bioinformatic analysis pipeline to identify uncharacterized TnpB nucleases. Briefly, a query TnpB sequence was used to search a database of predicted enzymes contained within a glacial metagenomic database (Liu et al., Nature Biotechnology, 2022). A Hidden Markov Model (HMM) search approach was used to rank protein candidates based on their homology scores. Candidates that contained non-coding scaffolding RNAs overlapping with the 3 ' ends of their ORFs were prioritized. Panel B shows an example of a bar chart illustrating distribution of protein lengths across CRISPR / Cas type II and type V family proteins that have evolved from the TnpB ancestor family. Panel C depicts an example of a phylogenetic tree of CRISPR / Cas (purple) and predicted TnpB enzymes. The enzymes with guide RNAs (e.g., reRNAs) predicted to have RNA secondary structure for scaffolding to TnpB nucleases are highlighted (blue). Panel D depicts an example of a multisequence alignment of 3 ' terminal regions of the predicted scaffold RNA indicating conservation specifically in regions required to form scaffolding stem loops (bottom RNAFold predictions); shown are SEQ ID NOs: 2, 4, 6, and an ISDRa2 scaffold query from top to bottom. Panel E depicts examples of predicted RNA structure of selected scaffolds from Panel D, shown are SEQ ID NOs: 4, 2, and 6 from left to right.

[0019] Figure 2 (FIG. 2) depicts editing signal observed in HEK cells expressing a scaffold RNA (SEQ ID NO: 2) configured to target a predetermined sequence, a compatible putative TnpB-related endonuclease (SEQ IDNO:1), and a reporter nucleotide sequence comprising a 5' 7-nucleotide degenerate target adjacent motif (TAM) sequence linked 3' to the predetermined sequence targeted by the scaffold RNA (“TAM reporter”). Cells were collected 3 days after transient transfection with the scaffold RNA and enzyme, after which next generation sequencing (NGS) was performed on the reporter nucleotide sequence to detect the edits made by the TnpB related enzyme. “NTC” denotes non-transfection control. The data indicates that the subject endonuclease (SEQ ID NO: 1) preferentially targets a region -20-40 nucleotides downstream of the predetermined sequence targeted by the scaffold RNA. The highlighted regions (from left to right on the x-axis) indicate the TAM region, target region bound by the reRNA (e.g., the guide RNA), and the target distal region, respectively. Peaks within the target region and target distal region indicate editing activity as measured within the TAM reporter assay by NGS.

[0020] Figure 3 (FIG. 3) depicts an example of a computational workflow for processing TAM sequences identified for TnpB-related endonucleases as described herein by a method described in Example 2.DETAILED DESCRIPTION

[0021] Definitions

[0022] While various embodiments of the invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions may occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed.

[0023] The practice of some methods disclosed herein employ, unless otherwise indicated, techniques of immunology, biochemistry, chemistry, molecular biology, microbiology, cell biology, genomics, and recombinant DNA. See for example Sambrook and Green, Molecular Cloning: A Laboratory Manual, 4thEdition (2012); the series Current Protocols in Molecular Biology (F. M. Ausubel, et al. eds.); the series Methods In Enzymology (Academic Press, Inc.), PCR 2: A Practical Approach (M.J. MacPherson, B.D. Hames and G.R. Taylor eds. (1995)), Harlow and Lane, eds. (1988) Antibodies, A Laboratory Manual, and Culture of Animal Cells: A Manual of Basic Technique and Specialized Applications, 6thEdition (R.I. Freshney, ed. (2010)) (which is entirely incorporated by reference herein).

[0024] As used herein, the singular forms “a”, “an” and “the” are generally intended to include the plural forms as well, unless the context clearly indicates otherwise. Furthermore, to the extent that the terms “including”, “includes”, “having”, “has”, “with”, or variants thereof are used in either the detailed description or the claims, such terms are intended to be inclusive in a manner similar to the term “comprising”.

[0025] The term “or”, as used herein, is generally intended to be an inclusive “or”. Accordingly, the statement “A or B” can encompass both “A and B” and “A but not B”.

[0026] The term “nucleotide,” as used herein, generally refers to a base-sugar-phosphate combination. A nucleotide may comprise a synthetic nucleotide. A nucleotide may comprise a synthetic nucleotide analog. Nucleotides may be monomeric units of a nucleic acid sequence (e.g., deoxyribonucleic acid (DNA) and ribonucleic acid (RNA)). The term nucleotide may include ribonucleoside triphosphates adenosine triphosphate (ATP), uridine triphosphate (UTP), cytosine triphosphate (CTP), guanosine triphosphate (GTP) and deoxyribonucleoside triphosphates such as dATP, dCTP, diTP, dUTP, dGTP, dTTP, or derivatives thereof. Such derivatives may include, for example, [aS]dATP, 7-deaza-dGTP and 7-deaza-dATP, and nucleotide derivatives that confer nuclease resistance on the nucleic acid molecule containing them. Nucleotides can also be labeled or marked by chemical modification. A chemically- modified single nucleotide can be biotin-dNTP. Some non-limiting examples of biotinylated dNTPs can include, biotin-dATP (e.g., bio-N6-ddATP, biotin- 14-d ATP), biotin-dCTP (e.g., biotin- 11-dCTP, biotin-14-dCTP), and biotin-dUTP (e.g., biotin- 11-dUTP, biotin- 16-dUTP, biotin-20-dUTP). In some cases, nucleotides can encompass modifications at the base moiety or modifications at the phosphate moiety (e.g., modifications intended to influence stability of nucleotide polymers).

[0027] As used herein, a “cell” generally refers to a biological cell. A cell may be the basic structural, functional, or biological unit of a living organism. A cell may originate from any organism having one or more cells. Some non-limiting examples include: a prokaryotic cell, eukaryotic cell, a bacterial cell, an archaeal cell, a cell of a single-cell eukaryotic organism, a protozoa cell, a cell from a plant, an algal cell, an animal cell, a cell from an invertebrate animal, a cell from a vertebrate animal (e.g., fish, amphibian, reptile, bird, mammal), or a cell from a mammal (e.g., a pig, a cow, a goat, a sheep, a rodent, a rat, a mouse, a non-human primate, a human, etc.).

[0028] The term “nucleotide,” as used herein, generally refers to a base-sugar-phosphate combination. A nucleotide may comprise a synthetic nucleotide. A nucleotide may comprise a synthetic nucleotide analog. Nucleotides may be monomeric units of a nucleic acid sequence(e.g., deoxyribonucleic acid (DNA) and ribonucleic acid (RNA)). The term nucleotide may include ribonucleoside triphosphates adenosine triphosphate (ATP), uridine triphosphate (UTP), cytosine triphosphate (CTP), guanosine triphosphate (GTP) and deoxyribonucleoside triphosphates such as dATP, dCTP, diTP, dUTP, dGTP, dTTP, or derivatives thereof. Such derivatives may include, for example, [aS]dATP, 7-deaza-dGTP and 7-deaza-dATP, and nucleotide derivatives that confer nuclease resistance on the nucleic acid molecule containing them. Nucleotides can also be labeled or marked by chemical modification. A chemically- modified single nucleotide can be biotin-dNTP. Some non-limiting examples of biotinylated dNTPs can include, biotin-dATP (e.g., bio-N6-ddATP, biotin- 14-d ATP), biotin-dCTP (e.g., biotin- 11-dCTP, biotin- 14-dCTP), and biotin-dUTP (e.g., biotin-11-dUTP, biotin- 16 -dUTP, biotin-20-dUTP). In some cases, nucleotides can encompass modifications at the base moiety or modifications at the phosphate moiety (e.g., modifications intended to influence stability of nucleotide polymers).

[0029] The terms “polynucleotide,” “oligonucleotide,” and “nucleic acid” are generally used interchangeably to generally refer to a polymeric form of nucleotides of any length, either deoxy ribonucleotides or ribonucleotides, or analogs thereof, either in single-, double-, or multistranded form. A polynucleotide maybe exogenous or endogenous to a cell. A polynucleotide may exist in a cell-free environment. A polynucleotide may be a gene or fragment thereof. A polynucleotide may be DNA. A polynucleotide may be RNA. A polynucleotide may comprise one or more analogs (e.g., altered backbone, sugar, or nucleobase). If present, modifications to the nucleotide structure may be imparted before or after assembly of the polymer. Some nonlimiting examples of analogs include: 5 -bromouracil, peptide nucleic acid, xeno nucleic acid, morpholinos, locked nucleic acids, glycol nucleic acids, threose nucleic acids, dideoxynucleotides, cordycepin, 7-deaza-GTP, fluorophores (e.g., rhodamine or fluorescein linked to the sugar), thiol containing nucleotides, biotin linked nucleotides, fluorescent base analogs, CpG islands, methyl-7-guanosine, methylated nucleotides, inosine, thiouridine, pseudourdine, dihydrouridine, queuosine, and wyosine. Non-limiting examples of polynucleotides include coding or non-coding regions of a gene or gene fragment, loci (locus) defined from linkage analysis, exons, introns, messenger RNA (mRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), short interfering RNA (siRNA), short-hairpin RNA (shRNA), micro- RNA (miRNA), ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, cell-free polynucleotides including cell-free DNA (cfDNA) and cell-free RNA (cfRNA), nucleic acidprobes, and primers. The sequence of nucleotides may be interrupted by non-nucleotide components.

[0030] A “vector” as used herein, generally refers to a macromolecule or association of macromolecules that comprises or associates with a polynucleotide and which may be used to mediate delivery of the polynucleotide to a cell. Examples of vectors include plasmids, viral vectors, liposomes, lipid nanoparticles, and other gene delivery vehicles. The vector generally comprises genetic elements, e.g., regulatory elements, operatively linked to a gene to facilitate expression of the gene in a target.

[0031] As used herein, a “guide nucleic acid” can generally refer to a nucleic acid that may hybridize to another nucleic acid. A guide nucleic acid may be RNA. A guide nucleic acid may be DNA. The guide nucleic acid may be programmed to bind to a sequence of nucleic acid site- specifically. The nucleic acid to be targeted, or the target nucleic acid, may comprise nucleotides. The guide nucleic acid may comprise nucleotides. A portion of the target nucleic acid may be complementary to a portion of the guide nucleic acid. The strand of a doublestranded target polynucleotide that is complementary to and hybridizes with the guide nucleic acid may be called the complementary strand. The strand of the double-stranded target polynucleotide that is complementary to the complementary strand, and therefore may not be complementary to the guide nucleic acid may be called noncomplementary strand. A guide nucleic acid may comprise a polynucleotide chain and can be called a “single guide nucleic acid.” A guide nucleic acid may comprise two polynucleotide chains and may be called a “double guide nucleic acid.” If not otherwise specified, the term “guide nucleic acid” may be inclusive, referring to both single guide nucleic acids and double guide nucleic acids. A guide nucleic acid may comprise a segment that can be referred to as an “anti-target nucleic acid sequence” or a “nucleic acid-targeting sequence.” A guide nucleic acid may comprise a sub-segment that may be referred to as a “protein binding segment” or “protein binding sequence” or “scaffold nucleic acid sequence”.

[0032] The term “scaffold nucleic acid sequence”, when used in the context of an engineered guide nucleic acid, can generally refer to a nucleic acid with at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100% sequence identity or sequence similarity to a wild-type sequence that is configured to form a complex with a endonuclease polypeptide (e.g., a direct-repeat sequence). “Scaffold nucleic acid sequence” may refer to a modified form of a scaffold nucleic acid sequence (e.g., a direct-repeat sequence) that can comprise a nucleotide change such as a deletion, insertion, or substitution, variant, mutation, or chimera. In some cases,a scaffold nucleic acid sequence comprises all or part of a right-end element RNA sequence (reRNA, e.g., when derived from an IS200 / IS605 transposon).

[0033] The term “sequence identity” or “percent identity” in the context of two or more nucleic acids or polypeptide sequences, generally refers to two (e.g., in a pairwise alignment) or more (e.g., in a multiple sequence alignment) sequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same, when compared and aligned for maximum correspondence over a local or global comparison window, as measured using a sequence comparison algorithm. Suitable sequence comparison algorithms for polypeptide sequences include, e.g., BLASTP using parameters of a wordlength (W) of 3, an expectation I of 10, and the BLOSUM62 scoring matrix setting gap costs at existence of 11, extension of 1, and using a conditional compositional score matrix adjustment for polypeptide sequences longer than 30 residues.

[0034] The term “pharmaceutically acceptable carrier” is generally intended to denote any material, which is inert in the sense that it substantially does not have a therapeutic and / or prophylactic effect per se. Such an excipient is added with the purpose of making it possible to obtain a pharmaceutical composition having acceptable technical properties. Examples of suitable pharmaceutically acceptable carriers or diluents include, but are not limited to, ethanol, water, glycerol, propylene glycol, glycerin, diethylene glycol monoethylether, vitamin A and E oils, mineral oil, PPG2 myristyl propionate, magnesium carbonate, potassium phosphate, silicon dioxide, vegetable oils such as castor oil and derivatives thereof, plant gums, gelatin, animal oils, solketal, calcium, carbonate, dibasic calcium phosphate, tribasic calcium phosphate, calcium sulfate, microcrystalline cellulose, powdered cellulose, dextrans, dextrin, dextrose, fructose, kaolin, lactose, mannitol, sorbitol, starch, pre-gelatinized starch, sucrose, sugar etc.

[0035] Included in the current disclosure are variants of any of the polypeptides, proteins, endonucleases, or enzymes described herein, such as variants with one or more conservative or nonconservative amino acid substitutions. Conservative substitutions can be made in the amino acid sequence of a polypeptide, protein, endonuclease, or enzyme without disrupting the three- dimensional structure or function of the polypeptide, protein, endonuclease, or enzyme. Conservative substitutions can be accomplished by substituting amino acids with similar hydrophobicity, polarity, and R chain length for one another. Additionally or alternatively, by comparing aligned sequences of homologous polypeptides, proteins, endonucleases, or enzymes from different species, conservative substitutions can be identified by locating amino acid residues that have been mutated between species (e.g. non-conserved residues) without altering the basic functions of the encoded polypeptides, proteins, endonucleases, or enzymes. Suchconservative variants may include variants with at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% identity to any one of the polypeptide or protein sequences described herein.

[0036] In some embodiments, such conservatively substituted variants are functional variants. Such functional variants can encompass sequences with substitutions such that the activity of critical active site residues of the polypeptide, protein, endonuclease, or enzyme are not disrupted. In some embodiments, a functional variant of any of the polypeptide, protein, endonuclease, or enzyme described herein lacks substitution of at least one conserved or functional residue.

[0037] Conservative substitution tables providing functionally similar amino acids are available from a variety of references (see, for e.g., Creighton, Proteins: Structures and Molecular Properties (W H Freeman & Co.; 2nd edition (December 1993), which is incorporated by reference in its entirety herein). The following eight groups each contain amino acids that can be conservative substitutions for one another:

[0038] 1) Alanine (A), Glycine (G);

[0039] 2) Aspartic acid (D), Glutamic acid (E);

[0040] 3) Asparagine (N), Glutamine (Q);

[0041] 4) Arginine (R), Lysine (K);

[0042] 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V);

[0043] 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W);

[0044] 7) Serine (S), Threonine (T); and

[0045] 8) Cysteine (C), Methionine (M)

[0046] In some aspects, the present disclosure provides for an enzyme, protein, polypeptide, or nucleic acid as described in Table 1.Table 1: Sequences of Genes and Components Described Herein

[0047] In some embodiments, nucleotide sequences as described herein are depicted using the symbolic scheme shown in Table 2 below.

[0048] Table 2: Nucleotide Symbols for Sequences Described hereinEXAMPLES

[0049] Example 1. - Discovery of Uncharacterized TnpB-related endonucleases

[0050] A novel bioinformatics pipeline was constructed to discover low-molecular weight TnpB- related nucleases. Figure 1 (FIG. 1) panel A shows a flow chart illustrating such a bioinformatic analysis pipeline useful to identify uncharacterized TnpB nucleases. Briefly, a query TnpB sequence was used to search a database of predicted enzymes contained within a glacial metagenomic database (see e.g., Liu et al. Nat Biotechnol. 2022;40(9):1341-1348. doi:10. 1038 / s41587-022-01367-2, which is incorporated by reference herein in its entirety). A Hidden Markov Model (HMM) search approach was used to rank protein candidates from the database based on their homology scores. Candidates that contained non-coding scaffolding RNAs overlapping with the 3 ' ends of their open reading frames (ORFs) were prioritized.

[0051] Figure 1 (FIG. 1) panel B shows a bar chart illustrating distribution of protein lengths across CRISPR / Cas type II and type V family proteins that have evolved from the TnpB ancestor family. Figure 1 (FIG. 1) panel C depicts a phylogenetic tree of CRISPR / Cas (purple) and predicted TnpB enzymes. The enzymes with guide RNAs (e.g., reRNAs) predicted to have RNA secondary structure for scaffolding to TnpB nucleases are highlighted in (blue). Figure 1 (FIG. 1) panel D depicts a multi-sequence alignment of 3 ' terminal regions of the predicted scaffold RNA indicating conservation specifically in regions required to form scaffolding stem loops. Figure 1 (FIG.l) panel E depicts RNAFold predictions of the scaffold RNA structures shown in panel D.

[0052] Example 2. - Characterization of TAM sequence for TnpB-related endonucleases

[0053] An assay in HEK cells was used to characterize TAM sequences for TnpB-related endonucleases. First, the RNA sequences encoding the TnpB-related protein (SEQ ID NO: 1) and a guide RNA comprising the scaffold RNA sequence (SEQ ID NO: 2) were provided to the HEK cells on a plasmid downstream of a human U6 promoter in an inducible manner. Second, an 7N TAM library was provided to the HEK cells using a library plasmid that included a random 7 bp DNA sequence upstream of a target sequence that the TnpB-scaffold RNA binary complex was configured to target. Finally, expression of the RNA sequences encoding the TnpB-related protein and the guide RNA were induced in the HEK cells, thereby resulting in expression of the TnpB-related endonuclease complex with guide RNA, and targeting of the 7N TAM library were to promote DNA cleavage. Cleaved DNA fragments were amplified from genomic DNA and Illumina-based adaptors were subsequently incorporated to enable sequencing to measure DNA repair outcomes within target regions that accumulate due to cleavage downstream of associated TAM sequences.

[0054] Putative TAM sequences for enzymes described herein (e.g., SEQ ID NO: 1) were then identified by the workflow shown in FIG. 3, wherein editing activity was computed across all TAMs, editing activated was normalized to a non-transfection control (“NTC”), TAMs with low read depth (e.g., <= 50 reads) were removed, and TAMs were ranked by editing activity.

[0055] Results for TAM editing activity for SEQ ID NO: 1 are shown in Table 3 below.Table 3: Editing Activity for Putative TAM Sequences According to Example 2

[0056] A seqLogo (see e.g., Huber et al. Nat Methods. 2015 Feb;12(2):l 15-21) depiction was constructed using the highly ranked TAM sequences to generate a consensus TAM sequence of 5 '-NTBNHSB-3 ' for SEQ ID NO: 1.

[0057] While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby.

Claims

CLAIMSWHAT IS CLAIMED IS:1 . A system comprising:(a) an RNA-guided endonuclease comprising a polypeptide sequence having at least 80% sequence identity to any one of SEQ ID NOs: 1, 3, 5, or a variant thereof, or a nucleic acid sequence encoding said RNA-guided endonuclease; and(b) an engineered or non-natural guide nucleic acid, or a template polynucleotide encoding said engineered or non-natural guide nucleic acid, wherein said engineered or nonnatural guide nucleic acid is configured to form a complex with said RNA-guided endonuclease, comprising:(i) an anti-target nucleic acid sequence configured to hybridize to a target nucleic acid sequence; and(ii) a scaffold nucleic acid sequence configured to bind to said RNA- guided endonuclease.

2. The system of claim 1, wherein: (i) said engineered or non-natural guide nucleic acid comprises a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 2, 4, or 6 or an RNA cognate thereof; or (ii) said template polynucleotide encoding said engineered or non-natural guide nucleic acid encodes a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 2, 4, or 6.

3. The system of claim 1 or 2, wherein said sequence identity is determined over the whole length of said sequence.

4. The system of claim 1, wherein said engineered or non-natural guide nucleic acid comprises (ii) said scaffold nucleic acid sequence and (i) said anti-target nucleic acid sequence in order from 5' to 3 '.

5. The system of claim 1, wherein said engineered or non-natural guide nucleic acid comprises (ii) said scaffold nucleic acid sequence and (i) said anti-target nucleic acid sequence in order from 3' to 5'.

6. The system of any one of claims 1-5, wherein said anti-target nucleic acid sequence comprises about 16-22 nucleotides.

7. The system of any one of claims 1-6, wherein said RNA-guided endonuclease is a class 2 Cas-like endonuclease.

8. The system of claim 7, wherein said RNA-guided endonuclease is a class 2, type II Cas-like endonuclease.

9. The system of claim 7, wherein said RNA-guided endonuclease is a class 2, type V Cas-like endonuclease.

10. The system of any one of claims 1-9, wherein said system further comprises a deoxyribonucleic acid (DNA) molecule comprising said target nucleic acid sequence, wherein said DNA molecule comprises a target adjacent motif (TAM) sequence 5' to said target nucleic acid sequence.

11. The system of any one of claims 1-10, wherein said RNA-guided endonuclease is configured to be selective for any of the TAM sequences described in Table 3, or 5'-NTBNHSB-3 '.

12. The system of any one of claims 1-11, wherein said target nucleic acid sequence is a eukaryotic, a mammalian, or a primate nucleic acid sequence.

13. The system of any one of claims 1-11, wherein said target nucleic acid sequence is a prokaryotic or an archaean nucleic acid sequence.

14. A polynucleotide comprising an open reading frame (ORF) encoding a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 1, 3, 5, or a variant thereof, wherein said ORF is codon-optimized for expression in a eukaryotic, a mammalian, or a primate organism.

15. The polynucleotide of claim 14, wherein said polynucleotide is an in vitro transcribed (IVT) messenger ribonucleic acid (mRNA).

16. A polynucleotide comprising or encoding:(i) an anti-target nucleic acid sequence configured to hybridize to a target eukaryotic nucleic acid sequence; and(ii) a scaffold nucleic acid sequence configured to bind to an RNA-guided endonuclease, wherein said scaffold nucleic acid sequence comprises a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 2, 4, or 6, or an RNA cognate thereof.

17. The polynucleotide of claim 16, wherein said anti-target nucleic acid sequence is oriented 3 ' to said scaffold nucleic acid sequence.

18. The polynucleotide of claim 16 or 17, wherein said anti -target nucleic acid sequence comprises at least 16-22 nucleotides.

19. The polynucleotide of any one of claims 16-18, wherein said polynucleotide comprises (i) said anti-target nucleic acid sequence and (ii) said scaffold nucleic acid sequence and said polynucleotide is a synthetic or in-vitro transcribed (IVT) RNA.

20. The polynucleotide of any one of claims 16-19, wherein said polynucleotide encodes (i) said anti-target nucleic acid sequence and (ii) said scaffold nucleic acid sequence, wherein said polynucleotide further comprises a vector.

21. The polynucleotide of claim 20, wherein said vector comprises a viral vector.

22. The polynucleotide of claim 20, wherein said vector comprises a DNA vector.

23. The polynucleotide of any one of claims 16-22, wherein said polynucleotide further comprises an open reading frame (ORF) encoding a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 1, 3, 5, or a variant thereof.

24. The polynucleotide of any one of claims 16-23, wherein said RNA-guided endonuclease comprises a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 1, 3, 5, or a variant thereof.

25. A method of binding a target nucleic acid sequence in a deoxyribonucleic acid (DNA) molecule, comprising contacting to said DNA with said system of any one of claims 1-13.

26. The method of claim 25, wherein said binding occurs within a cell, and said method comprises contacting to said cell said system of any one of claims 1-13.

27. The method of claim 26, wherein said DNA molecule further comprises a target adjacent motif (TAM) sequence and said target nucleic acid sequence, wherein said TAM sequence is 5' to said target nucleic acid sequence.

28. The method of claim 26 or 27, wherein said cell is a eukaryotic, a mammalian, or a primate cell.

29. The method of claim 26 or 27, wherein said cell is a prokaryotic or an archaean cell.

30. The method of any one of claims 25-29, further comprising introducing a double-stranded break in said DNA molecule proximal to said target nucleic acid sequence.

31. The method of any one of claims 25-29, further comprising introducing a single-stranded nick in said DNA molecule proximal to said target nucleic acid sequence.

32. The method of claim 30 or 31, wherein said double-stranded break or said single-stranded nick is introduced about 20-40 nucleotides 3 ' to said target nucleic acid sequence.

33. The method of claim 32, wherein said double-stranded break or said single-stranded nick is introduced about 30-40 nucleotides 3 ' to said target nucleic acid sequence.

34. The method of claim 33, wherein said double-stranded break is introduced about 35-40 nucleotides 3' to said target nucleic acid sequence.

35. A cell comprisingthe system of any one of claims 1-13 or the polynucleotide of any one of claims 14-24.

36. The cell of claim 35, wherein said cell is a eukaryotic, a mammalian, or a primate cell.

37. The cell of claim 35, wherein said cell is a prokaryotic or an archaean cell.

8. A pharmaceutically acceptable composition, comprising: (i) the system of any one of claims 1-13, the polynucleotide of any oneof claims 14-24, or the cell of any one of claims 35-37; and (ii) a pharmaceutically acceptable carrier.

Images