Compositions and methods related to crispr-associated enzymes

Abstract

The present invention provides compositions and methods related to Clustered regularly interspaced short palindromic repeats (CRISPR)-associated (Cas) nucleases. In particular, the present disclosure provides artificial Cas-12 enzymes and corresponding methods and systems for targeted nucleic acid modification.

Description

BIOTN-41612.601COMPOSITIONS AND METHODS RELATED TO CRISPR-ASSOCIATED ENZYMESCROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to and the benefit of U.S. Provisional Patent Application No. 63 / 519,750 filed August 15, 2023, which is incorporated herein by reference in its entirety for all purposes.INCORPORATION-BY-REFERENCE OF MATERIAL SUBMITTED ELECTRONICALLY

[0002] Incorporation by reference in its entirety herein is a computer-readable nucleotide / amino acid sequence listing submitted concurrently herewith and identified as follows: One 219,068 bytes Byte ASCII (Text) file named “BIOTN_41612_601_SequenceListing” created on August 14, 2024.FIELD

[0003] The present invention provides compositions and methods related to Clustered regularly interspaced short palindromic repeats (CRISPR)-associated (Cas) nucleases. In particular, the present disclosure provides artificial Cas- 12 enzymes and corresponding methods and systems for targeted nucleic acid modification.BACKGROUND

[0004] Clustered regularly interspaced short palindromic repeats (CRISPR)-associated (Cas) nucleases dominate the nucleic acid-editing landscape because they are versatile, rapid, and easy- to-use editing tools. CRISPR-Cas systems employ diverse RNA-guided nucleases to help microbes fend off bacteriophages and other mobile genetic elements. Current genome editing technologies primarily use single effector enzymes such as Cas9 or Cas 12 from Class II CRISPR systems, for programmable DNA sequence alterations. Cas9 or Cas 12 is guided by its CRISPR RNA (crRNA) to find the complementary target site flanked by a short protospacer-adjacent motif (PAM), and then cleaves the DNA at precise locations. However, presently CRISPR / Cas systems have some limitations for use in eukaryotic organisms including inefficient delivery to mature cells in large numbers, low efficiency of editing, off-target events, target sequence preferences, and optimal temperatures and conditions for enzymatic activity.BIOTN-41612.601SUMMARY

[0005] Embodiments of the present disclosure include an engineered nuclease comprising an amino acid sequence having at least 50% identity but less than 100% identity to any of SEQ ID NOs: 10-82.

[0006] In some embodiments, the amino acid sequence has at least 60% identity to any of SEQ ID NOs: 10-82. In some embodiments, the amino acid sequence has at least 70% identity to any of SEQ ID NOs: 10-82. In some embodiments, the amino acid sequence has at least 80% identity to any of SEQ ID NOs: 10-82. In some embodiments, the amino acid sequence has at least 90% identity to any of SEQ ID NOs: 10-82.

[0007] In some embodiments, the amino acid sequence has at least 70% identity to any of SEQ ID NOs: 1-9. In some embodiments, the amino acid sequence has at least 80% identity to any of SEQ ID NOs: 1-9. In some embodiments, the amino acid sequence has at least 90% identity to any of SEQ ID NOs: 1-9. In some embodiments, the amino acid sequence has at least 95% identity to any of SEQ ID NOs: 1-9. In some embodiments, the amino acid sequence is selected from the group consisting of SEQ ID NOs: 1-9.

[0008] In some embodiments, the amino acid sequence comprises any one of SEQ ID NOs: 83- 119. In some embodiments, the amino acid sequence comprises any one of SEQ ID NOs: 83-119 in place of at least one amino acid in SEQ ID NO: 1 or SEQ ID NO: 2. In some embodiments, the amino acid sequence comprises any one of SEQ ID NOs: 83-119 in place of the corresponding amino acid sequence in SEQ ID NO: 1 or SEQ ID NO: 2, as provided in Table 1.

[0009] In some embodiments, the amino acid sequence comprises any of the mutant amino acid substitutions in Table 3. In some embodiments, the amino acid sequence has at least 70% identity to SEQ ID NO: 1 , and comprises at least one of the mutant amino acid substitutions in Table 3. In some embodiments, the amino acid sequence has at least 80% identity to SEQ ID NO: 1, and comprises at least one of the mutant amino acid substitutions in Table 3. In some embodiments, the amino acid sequence has at least 90% identity to SEQ ID NO: 1, and comprises at least one of the mutant amino acid substitutions in Table 3.

[0010] In some embodiments, the amino acid sequence comprises any of the mutant amino acid substitutions in Table 4. In some embodiments, the amino acid sequence has at least 70% identity to SEQ ID NO: 2, and comprises at least one of the mutant amino acid substitutions in Table 4. In some embodiments, the amino acid sequence has at least 80% identity to SEQ ID NO: 2, andBIOTN-41612.601 comprises at least one of the mutant amino acid substitutions in Table 4. In some embodiments, the amino acid sequence has at least 90% identity to SEQ ID NO: 2, and comprises at least one of the mutant amino acid substitutions in Table 4.

[0011] Embodiments of the present disclosure also include a nucleic acid molecule comprising a sequence encoding any of the engineered nucleases described herein.

[0012] Embodiments of the present disclosure also include a vector comprising a nucleic acid molecule comprising a sequence encoding any of the engineered nucleases described herein. In some embodiments, the vector further comprises a promoter operatively linked to the nucleic acid. In some embodiments, the vector further comprises a nucleic acid encoding a guide RNA (gRNA). In some embodiments, the vector further comprises a promoter operatively linked to the nucleic acid encoding a guide RNA (gRNA).

[0013] Embodiments of the present disclosure also include a composition comprising an engineered nuclease, wherein the engineered nuclease comprises a sequence with at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to any one of SEQ ID NOs: 1-9, or a nucleic acid comprising a sequence encoding the engineered nuclease. In some embodiments, the amino acid sequence of the engineered nuclease comprises any one of SEQ ID NOs: 1-9. In some embodiments, the engineered nuclease is comprised in a fusion protein of a peptide or protein that is fused to the nuclease. In some embodiments, the composition further comprises at least one guide RNA (gRNA) or a nucleic acid comprising a sequence encoding the at least one gRNA.

[0014] Embodiments of the present disclosure also include a system for modifying a target nucleic acid comprising: a) an engineered nuclease comprising an amino acid sequence having at least 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to any of SEQ ID NOs: 1-9, or a nucleic acid sequence encoding the engineered nuclease; and b) an engineered guide RNA (gRNA) comprising a first region configured to interact with the engineered nuclease and a second region configured to hybridize with a portion of the target nucleic acid.

[0015] In some embodiments, the engineered nuclease in the presence of the gRNA is capable of modifying the target nucleic acid. In some embodiments, modifying comprises nucleic acid cleavage. In some embodiments, modifying comprises one or more of nucleic acid binding, base editing, transcription modulation, nucleic acid modification, protein modification, and histone modification. In some embodiments, the system further comprises at least one target nucleic acid. In some embodiments, the system is a cell-free system.BIOTN-41612.601

[0016] Embodiments of the present disclosure also include a cell comprising any of the systems described herein. In some embodiments, the cell is a mammalian cell. In some embodiments, the cell is a human cell. In some embodiments, the cell is a prokaryotic or eukaryotic cell.

[0017] Embodiments of the present disclosure also include a method of modifying a target nucleic acid sequence. In accordance with these embodiments, the method comprises contacting the target nucleic acid with a targeting composition comprising any of the engineered nucleases described herein, any of the compositions described herein, any of the nucleic acids described herein, any of the vectors described herein, or any of the systems described herein.

[0018] In some embodiments of the method, the target nucleic acid sequence is in a cell. In some embodiments of the method, the cell is a prokaryotic or eukaryotic cell. In some embodiments of the method, the cell is a human cell. In some embodiments of the method, contacting a target nucleic acid sequence comprises introducing the targeting composition into the cell. In some embodiments of the method, contacting a target nucleic acid sequence comprises administering the targeting composition to a subject. In some embodiments of the method, the target nucleic acid sequence encodes a gene product.

[0019] Embodiments of the present disclosure also include a method modifying a target nucleic acid sequence in a plant. In accordance with these embodiments, the method comprises providing to the plant, or a plant cell, seed, fruit, plant part, or propagation material of the plant: a) an engineered nuclease comprising an amino acid sequence having at least 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to any of SEQ ID NOs: 1-9, or a nucleic acid sequence encoding the engineered nuclease; and b) an engineered guide RNA (gRNA) comprising a first region configured to interact with the engineered nuclease and a second region configured to hybridize with a portion of the target nucleic acid.

[0020] In some embodiments, the method further comprises providing to the plant a donor polynucleotide. In some embodiments of the method, the target nucleic acid sequence encodes a gene product. In some embodiments of the method, the plant is a monocot or a dicot. In some embodiments of the method, the plant is a grain crop, a fruit crop, a forage crop, a root vegetable crop, a leafy vegetable crop, a flowering plant, a conifer, an oil crop, a plant used in phytoremediation, an industrial crop, a medicinal crop, or a laboratory model plant.

[0021] Embodiments of the present disclosure also include a method for treating a disease or disorder in a subject. In accordance with these embodiments, the method comprises administeringBIOTN-41612.601 to the subject in need thereof: a) an engineered nuclease comprising an amino acid sequence having at least 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to any of SEQ ID NOs: 1- 9, or a nucleic acid sequence encoding the engineered nuclease; and b) an engineered guide RNA (gRNA) comprising a first region configured to interact with the engineered nuclease and a second region configured to hybridize with a portion of a target nucleic acid.

[0022] In some embodiments of the method, the engineered nuclease and the gRNA are encoded on a single nucleic acid. In some embodiments of the method, the target nucleic acid is a disease-associated gene. In some embodiments, the method further comprises administering a donor polynucleotide. In some embodiments of the method, the donor polynucleotide comprises a therapeutic protein, functional gene product, or a combination thereof. In some embodiments of the method, the subject is a human. In some embodiments, the method further comprises administering a therapeutic agent.

[0023] Other aspects and embodiments of the disclosure will be apparent in light of the following detailed description.BRIEF DESCRIPTION OF THE DRAWINGS

[0024] FIGS. 1A-1B: Representative phylogenetic trees were construction from homologous Casl2A family members using (FireProtASR vl.l (loschmidt.chemi.muni.cz / fireprotasr / )). Ancestral sequences at indicated named nodes were reconstructed and synthesized. Nodes indicate the last common ancestor of all descendant gene sequences. Ancestral reconstruction was performed using FireProt ASR. The tree and reconstruction in FIG. 1A used homologs without limitation of any catalytic residues that must be conserved, while the tree in FIG. IB used only homologs identified that included E962 and R1173 as catalytic residues.

[0025] FIG. 2: Representative sequence alignments of the artificial CRISPR-associated enzymes identified in the present disclosure (SEQ ID NOs: 1-9) against the 73 homologous natural enzymes that were used to design the artificial sequences (SEQ ID NOs: 10-82). Enzyme names and species is indicated for each natural enzyme. The alignment illustrates highly conserved positions as well as variable regions.

[0026] FIGS. 3A-3B: Representative data from an E. coli screen of the artificial CRISPR- associated enzymes identified in the present disclosure. CRISPR-associated enzymes were transformed into an E. coli strain stable for a selection plasmid. The selection plasmid contains an ampicillin resistance gene split by nonsense nucleotides and flanked by sgRNA guide cut sites,BIOTN-41612.601 where successful cutting recovers ampicillin resistance. Activity of CRISPR-associated enzymes is relative to the amount of ampicillin resistance clones due to cutting.

[0027] FIGS. 4A-4C: Representative data of CRISPR-associated enzyme activity. Artificial CRISPR-associated enzymes (e.g., artificial Casl2a.l and artificial Casl2a.2) were transfected into K562 cells and measured for cutting activity against known CRISPR enzymes Cas9, CPF1 Ultra, and MAD7. Cutting efficiency was measured via gene knockout of either CD45 (FIG. 4A) or PDL-1 (FIG. 4B) with either one or two different sgRNAs respectively. Artificial CRISPR- associated enzymes (e.g., artificial Casl2a.l and artificial Casl2a.2) were transfected into K562 cells and measured for cutting activity against known CRISPR enzymes Cas9 and CPF1 Ultra (FIG. 4C). Cutting efficiency was measured via gene knockout of B2M.

[0028] FIGS. 5A-5B: Representative data of CRISPR-associated enzyme activity. Relative activity in cellular assays of artificial CRISPR-associated enzyme mutants containing various swapped domains covering the indicated amino acid ranges is provided in FIG. 5A. Data in FIG. 5B are normalized to 100% of the parental enzyme (e.g., artificial Casl2a.2). These data indicate that a number of domain-swap mutants show higher activity than the parental artificial CRISPR enzyme.DETAILED DESCRIPTION

[0029] The disclosed compositions, systems, kits, and methods comprise nucleases useful for nucleic acid modification. The disclosed nucleases allow for high precision gene editing with improved efficacy and safety for use in in vivo and ex vivo applications of eukaryotic (e.g., mammalian (e.g., human)) therapeutics, diagnostics, and research.

[0030] Section headings as used in this section and the entire disclosure herein are merely for organizational purposes and are not intended to be limiting.Definitions

[0031] Unless otherwise defined herein, scientific, and technical terms used in connection with the present disclosure shall have the meanings that are commonly understood by those of ordinary skill in the art. For example, any nomenclature used in connection with, and techniques of cell and tissue culture, molecular biology, microbiology, genetics and protein and nucleic acid chemistry and hybridization described herein are those that are well known and commonly used in the art. The meaning and scope of the terms should be clear; in the event, however of any latentBIOTN-41612.601 ambiguity, definitions provided herein take precedent over any dictionary or extrinsic definition. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular.

[0032] The terms “comprise(s),” “include(s),” “having,” “has,” “can,” “contain(s),” and variants thereof, as used herein, are intended to be open-ended transitional phrases, terms, or words that do not preclude the possibility of additional acts or structures. As used herein, comprising a certain sequence or a certain SEQ ID NO usually implies that at least one copy of said sequence is present in recited peptide or polynucleotide. However, two or more copies are also contemplated. The singular forms “a,” “and,” and “the” include plural references unless the context clearly dictates otherwise. The present disclosure also contemplates other embodiments “comprising,” “consisting of,” and “consisting essentially of,” the embodiments or elements presented herein, whether explicitly set forth or not.

[0033] For the recitation of numeric ranges herein, each intervening number there between with the same degree of precision is explicitly contemplated. For example, for the range of 6-9, the numbers 7 and 8 are contemplated in addition to 6 and 9, and for the range 6.0-7.0, the number 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are explicitly contemplated.

[0034] As used herein, “nucleic acid” or “nucleic acid sequence” refers to a polymer or oligomer of pyrimidine and / or purine bases, preferably cytosine, thymine, and uracil, and adenine and guanine, respectively (See Albert L. Lehninger, Principles of Biochemistry, at 793-800 (Worth Pub. 1982)). The present technology contemplates any deoxyribonucleotide, ribonucleotide, or peptide nucleic acid component, and any chemical variants thereof, such as methylated, hydroxymethylated, or glycosylated forms of these bases, and the like. The polymers or oligomers may be heterogenous or homogenous in composition and may be isolated from naturally occurring sources or may be artificially or synthetically produced. In addition, the nucleic acids may be DNA or RNA, or a mixture thereof, and may exist permanently or transitionally in single-stranded or double-stranded form, including homoduplex, heteroduplex, and hybrid states. In some embodiments, a nucleic acid or nucleic acid sequence comprises other kinds of nucleic acid structures such as, for instance, a DNA / RNA helix, peptide nucleic acid (PNA), morpholino nucleic acid (see, e.g., Braasch and Corey, Biochemistry, 41(14): 4503-4510 (2002)) and U.S. Pat. No. 5,034,506), locked nucleic acid (LNA; see Wahlestedt et al., Proc. Natl. Acad. Sci. U.S.A., 97: 5633-5638 (2000)), cyclohexenyl nucleic acids (see Wang, J. Am. Chem. Soc., 122: 8595-BIOTN-41612.6018602 (2000)), and / or a ribozyme. Hence, the term “nucleic acid” or “nucleic acid sequence” may also encompass a chain comprising non-natural nucleotides, modified nucleotides, and / or nonnucleotide building blocks that can exhibit the same function as natural nucleotides (e.g., “nucleotide analogs”); further, the term “nucleic acid sequence” as used herein refers to an oligonucleotide, nucleotide or polynucleotide, and fragments or portions thereof, and to DNA or RNA of genomic or synthetic origin, which may be single or double-stranded, and represent the sense or antisense strand. The terms “nucleic acid,” “polynucleotide,” “nucleotide sequence,” and “oligonucleotide” are used interchangeably. They refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof.

[0035] Nucleic acid or amino acid sequence “identity,” as described herein, can be determined by comparing a nucleic acid or amino acid sequence of interest to a reference nucleic acid or amino acid sequence. The percent identity is the number of nucleotides or amino acid residues that are the same (e.g., that are identical) as between the sequence of interest and the reference sequence divided by the length of the longest sequence (e.g., the length of either the sequence of interest or the reference sequence, whichever is longer). A number of mathematical algorithms for obtaining the optimal alignment and calculating identity between two or more sequences are known and incorporated into a number of available software programs. Examples of such programs include CLUSTAL-W, T-Coffee, and ALIGN (for alignment of nucleic acid and amino acid sequences), BLAST programs (e.g., BLAST 2.1, BL2SEQ, and later versions thereof) and FASTA programs (e.g., FASTA3x, FAS™, and SSEARCH) (for sequence alignment and sequence similarity searches). Sequence alignment algorithms also are disclosed in, for example, Altschul et al., J. Molecular Biol., 215(3): 403-410 (1990), Beigert et al., Proc. Natl. Acad. Sci. USA, 106(10): 3770- 3775 (2009), Durbin et al., eds., Biological Sequence Analysis : Probabilistic Models of Proteins and Nucleic Acids , Cambridge University Press, Cambridge, UK (2009), Soding, Bioinformatics, 21(1): 951-960 (2005), Altschul et al., Nucleic Acids Res., 25(11): 3389-3402 (1997), and Gusfield, Algorithms on Strings, Trees and Sequences, Cambridge University Press, Cambridge UK (1997)).

[0036] The terms “non-naturally occurring,” “engineered,” and “synthetic” are used interchangeably and indicate the involvement of the hand of man. The terms, when referring to nucleic acid molecules or polypeptides mean that the nucleic acid molecule or the polypeptide is at least substantially free from at least one other component with which it is naturally associatedBIOTN-41612.601 in nature and as found in nature, and / or the nucleic acid molecule or the polypeptide is associated with at least one other component with which it is not naturally associated in nature and / or that there is one or more changes in nucleic acid or amino acid sequence as compared with such sequence as it is found in nature.

[0037] A “vector” or “expression vector” is a replicon, such as plasmid, phage, virus, or cosmid, to which another DNA segment, e.g., an “insert,” may be attached or incorporated so as to bring about the replication of the attached segment in a cell.

[0038] A cell has been “genetically modified,” “transformed,” or “transfected” by exogenous DNA, e.g., a recombinant expression vector, when such DNA has been introduced inside the cell. The presence of the exogenous DNA results in permanent or transient genetic change. The transforming DNA may or may not be integrated (covalently linked) into the genome of the cell. For example, the transforming DNA may be maintained on an episomal element such as a plasmid. With respect to eukaryotic cells, a stably transformed cell is one in which the transforming DNA has become integrated into a chromosome so that it is inherited by daughter cells through chromosome replication. This stability is demonstrated by the ability of the eukaryotic cell to establish cell lines or clones that comprise a population of daughter cells containing the transforming DNA. A “clone” is a population of cells derived from a single cell or common ancestor by mitosis. A “cell line” is a clone of a primary cell that is capable of stable growth in vitro for many generations.

[0039] The term “contacting” as used herein refers to bring or put in contact, to be in or come into contact. The term “contact” as used herein refers to a state or condition of touching or of immediate or local proximity. Contacting a composition to a target destination, such as, but not limited to, an organ, tissue, cell, or tumor, may occur by any means of administration known to the skilled artisan.

[0040] As used herein, the terms “providing,” “administering,” and “introducing,” are used interchangeably herein and refer to the placement of the composition or systems of the disclosure into a cell, organism, or subject by a method or route which results in at least partial localization to a desired site. The composition or systems can be administered by any appropriate route which results in delivery to a desired location in the cell, organism, or subject.

[0041] Preferred methods and materials are described below, although methods and materials similar or equivalent to those described herein can be used in practice or testing of the presentBIOTN-41612.601 disclosure. All publications, patent applications, patents and other references mentioned herein are incorporated by reference in their entirety. The materials, methods, and examples disclosed herein are illustrative only and not intended to be limiting.Nucleases

[0042] Advances and developments in CRISPR-Cas genome editing tools including nucleases and other Cas protein drive major advances in nucleic acid editing. Nucleic acid editing has many uses including in the diagnostics and therapeutics field. Such breadth is accompanied by a diversity of nucleic acid targets and environments in which to engineer editing activity. As such, there is a need for diverse and additional nucleases and associated methods that provide a toolbox for nucleic acid editing.

[0043] Disclosed herein are compositions that include nucleases that have Cas-like activity. Unless otherwise noted herein, “nucleases” as used herein includes and encompasses the described “engineered nucleases.” The disclosed nucleases comprise a sequence having at least 50% identity (e.g., at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 98%, or at least 99%) to an amino acid sequence of SEQ ID NOs: 10-82. In accordance with these embodiments, the engineered nucleases disclosed herein also have less than 100% identity to any of SEQ ID NOs: 10-82. In some embodiments, the engineered nucleases of the present disclosure comprise a sequence having at least 70% identity (e.g., at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 98%, or at least 99%) to an amino acid sequence of SEQ ID NOs: 1-9. In some embodiments, the nuclease comprises a sequence having at least 90% identity an amino acid sequence of SEQ ID NOs: 1 -9. In some embodiments, the nuclease comprises an amino acid sequence of SEQ ID NOs: 1-9. In some embodiments, the amino acid sequence of the nucleases of the present disclosure lacks identity or significant amino acid homology with certain known Cas nucleases. In some embodiments, the amino acid sequence of the nuclease lacks identity or significant amino acid homology with a Cas 12a (Cpfl) protein.

[0044] Any of the nucleases described herein may comprise one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 50, 100, 150, 200, etc.) amino acid substitutions. An amino acid “replacement” or “substitution” refers to the replacement of one amino acid at a given position or residue by another amino acid at the same position or residue within a polypeptide sequence. Amino acids are broadly grouped as “aromatic” or “aliphatic.” An aromatic amino acid includesBIOTN-41612.601 an aromatic ring. Examples of “aromatic” amino acids include histidine (H or His), phenylalanine (F or Phe), tyrosine (Y or Tyr), and tryptophan (W or Trp). Non- aromatic amino acids are broadly grouped as “aliphatic.” Examples of “aliphatic” amino acids include glycine (G or Gly), alanine (A or Ala), valine (V or Vai), leucine (L or Leu), isoleucine (I or He ), methionine (M or Met), serine (S or Ser), threonine (T or Thr), cysteine (C or Cys), proline (P or Pro), glutamic acid (E or Glu), aspartic acid (A or Asp), asparagine (N or Asn), glutamine (Q or Gin), lysine (K or Lys), and arginine (R or Arg).

[0045] The amino acid replacement or substitution can be conservative, semi-conservative, or non-conservative. The phrase “conservative amino acid substitution” or “conservative mutation” refers to the replacement of one amino acid by another amino acid with a common property. A functional way to define common properties between individual amino acids is to analyze the normalized frequencies of amino acid changes between corresponding proteins of homologous organisms (Schulz and Schirmer, Principles of Protein Structure, Springer- Verlag, New York (1979)). According to such analyses, groups of amino acids may be defined where amino acids within a group exchange preferentially with each other, and therefore resemble each other most in their impact on the overall protein structure (Schulz and Schirmer, supra). Examples of conservative amino acid substitutions include substitutions of amino acids within the sub-groups described above, for example, lysine for arginine and vice versa such that a positive charge may be maintained, glutamic acid for aspartic acid and vice versa such that a negative charge may be maintained, serine for threonine such that a free -OH can be maintained, and glutamine for asparagine such that a free -NH2 can be maintained. “Semi-conservative mutations” include amino acid substitutions of amino acids within the same groups listed above, but not within the same subgroup. For example, the substitution of aspartic acid for asparagine, or asparagine for lysine, involves amino acids within the same group, but different sub-groups. “Non-conservative mutations” involve amino acid substitutions between different groups, for example, lysine for tryptophan, or phenylalanine for serine, etc.

[0046] For example, in some embodiments, the engineered nucleases of the present disclosure comprise an amino acid substitution (e.g., mutation) described in Table 3. In some embodiments, the engineered nucleases of SEQ ID NOs: 1-9 comprise an amino acid substitution described in Table 3. In some embodiments, the engineered nucleases of the present disclosure comprise at least one amino acid substitution (e.g., mutation) described in Table 3. In some embodiments, theBIOTN-41612.601 engineered nucleases of the present disclosure comprise at least two amino acid substitution (e.g., mutation) described in Table 3. In some embodiments, the engineered nucleases of the present disclosure comprise at least three amino acid substitution (e.g., mutation) described in Table 3. In some embodiments, the engineered nucleases of the present disclosure comprise at least four amino acid substitution (e.g., mutation) described in Table 3. In some embodiments, the engineered nucleases of the present disclosure comprise at least five amino acid substitution (e.g., mutation) described in Table 3. In some embodiments, the engineered nucleases of the present disclosure comprise at least six amino acid substitution (e.g., mutation) described in Table 3. In some embodiments, the engineered nucleases of the present disclosure comprise at least seven amino acid substitution (e.g., mutation) described in Table 3. In some embodiments, the engineered nucleases of the present disclosure comprise at least eight amino acid substitution (e.g., mutation) described in Table 3. In some embodiments, the engineered nucleases of the present disclosure comprise at least nine amino acid substitution (e.g., mutation) described in Table 3. In some embodiments, the engineered nucleases of the present disclosure comprise at least ten amino acid substitution (e.g., mutation) described in Table 3.

[0047] In some embodiments, the engineered nucleases of the present disclosure comprise an amino acid substitution (e.g., mutation) described in Table 4. In some embodiments, the engineered nucleases of SEQ ID NOs: 1-9 comprise an amino acid substitution described in Table 3. In some embodiments, the engineered nucleases of the present disclosure comprise at least one amino acid substitution (e.g., mutation) described in Table 4. In some embodiments, the engineered nucleases of the present disclosure comprise at least two amino acid substitution (e.g., mutation) described in Table 4. In some embodiments, the engineered nucleases of the present disclosure comprise at least three amino acid substitution (e.g., mutation) described in Table 4. In some embodiments, the engineered nucleases of the present disclosure comprise at least four amino acid substitution (e.g., mutation) described in Table 4. In some embodiments, the engineered nucleases of the present disclosure comprise at least five amino acid substitution (e.g., mutation) described in Table 4. In some embodiments, the engineered nucleases of the present disclosure comprise at least six amino acid substitution (e.g., mutation) described in Table 4. In some embodiments, the engineered nucleases of the present disclosure comprise at least seven amino acid substitution (e.g., mutation) described in Table 4. In some embodiments, the engineered nucleases of the present disclosure comprise at least eight amino acid substitution (e.g.,BIOTN-41612.601 mutation) described in Table 4. In some embodiments, the engineered nucleases of the present disclosure comprise at least nine amino acid substitution (e.g., mutation) described in Table 4. In some embodiments, the engineered nucleases of the present disclosure comprise at least ten amino acid substitution (e.g., mutation) described in Table 4.

[0048] In some embodiments, one or more amino acid substitutions in the amino acid sequence of a nuclease results in an increase in editing efficiency (e.g., an increase in cleavage, modification of a target nucleic acid) relative to the nuclease without the one or more amino acid substitutions. In some cases, one or more amino acid substitutions in the amino acid sequence of a nuclease results in detectable editing efficiency where the nuclease without the one or more amino acid substitutions has no detectable or minimally detectable editing ability. In some embodiments, one or more amino acid substitutions in the amino acid sequence of a nuclease results in an alteration of preferred PAM sequences, such that the where the nuclease without the one or more amino acid substitutions can utilize an expanded set of protospacer adjacent motif (PAM) sequences as compared to the nuclease without the one or more amino acid substitutions.

[0049] In some embodiments, a nuclease comprises one or more amino acid substitutions and has an amino acid sequence having at least 70% identity (e.g., at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 98%, or at least 99% identity) to an amino acid sequence of SEQ ID NOs: 1-9. In some embodiments, a nuclease comprises one or more amino acid substitutions as compared to SEQ ID NOs: 1-9, wherein the one or more substitutions improves the editing efficiency of the nuclease.

[0050] In some embodiments, the engineered nuclease may comprise an amino acid sequence having at least 70% identity (e.g., at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 98%, or at least 99% identity) SEQ ID NO: 1. In some embodiments, the engineered nuclease may comprise an amino acid sequence having at least 70% identity (e.g., at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 98%, or at least 99% identity) SEQ ID NO: 2. In some embodiments, the engineered nuclease may comprise an amino acid sequence having at least 70% identity (e.g., at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 98%, or at least 99% identity) SEQ ID NO: 3. In some embodiments, the engineered nuclease may comprise an amino acid sequence having at least 70% identity (e.g., at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 98%, or at least 99%BIOTN-41612.601 identity) SEQ ID NO: 4. In some embodiments, the engineered nuclease may comprise an amino acid sequence having at least 70% identity (e.g., at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 98%, or at least 99% identity) SEQ ID NO: 5. In some embodiments, the engineered nuclease may comprise an amino acid sequence having at least 70% identity (e.g., at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 98%, or at least 99% identity) SEQ ID NO: 6. In some embodiments, the engineered nuclease may comprise an amino acid sequence having at least 70% identity (e.g., at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 98%, or at least 99% identity) SEQ ID NO: 7. In some embodiments, the engineered nuclease may comprise an amino acid sequence having at least 70% identity (e.g., at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 98%, or at least 99% identity) SEQ ID NO: 8. In some embodiments, the engineered nuclease may comprise an amino acid sequence having at least 70% identity (e.g., at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 98%, or at least 99% identity) SEQ ID NO: 9.

[0051] In some embodiments, the engineered nuclease may comprise an amino acid sequence having at least 50% identity (e.g., at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 98%, or at least 99%), but less than 100% identity, to SEQ ID NO: 10. In some embodiments, the engineered nuclease may comprise an amino acid sequence having at least 50% identity (e.g., at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 98%, or at least 99%), but less than 100% identity, to SEQ ID NO: 1 1. In some embodiments, the engineered nuclease may comprise an amino acid sequence having at least 50% identity (e.g., at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 98%, or at least 99%), but less than 100% identity, to SEQ ID NO: 12. In some embodiments, the engineered nuclease may comprise an amino acid sequence having at least 50% identity (e.g., at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 98%, or at least 99%), but less than 100% identity, to SEQ ID NO: 13. In some embodiments, the engineered nuclease may comprise an amino acid sequence having at least 50% identity (e.g., at least 50%, at least 55%, atBIOTN-41612.601 least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 98%, or at least 99%), but less than 100% identity, to SEQ ID NO: 14. In some embodiments, the engineered nuclease may comprise an amino acid sequence having at least 50% identity (e.g., at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 98%, or at least 99%), but less than 100% identity, to SEQ ID NO: 15. In some embodiments, the engineered nuclease may comprise an amino acid sequence having at least 50% identity (e.g., at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 98%, or at least 99%), but less than 100% identity, to SEQ ID NO: 16. In some embodiments, the engineered nuclease may comprise an amino acid sequence having at least 50% identity (e.g., at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 98%, or at least 99%), but less than 100% identity, to SEQ ID NO: 17. In some embodiments, the engineered nuclease may comprise an amino acid sequence having at least 50% identity (e.g., at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 98%, or at least 99%), but less than 100% identity, to SEQ ID NO: 18. In some embodiments, the engineered nuclease may comprise an amino acid sequence having at least 50% identity (e.g., at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 98%, or at least 99%), but less than 100% identity, to SEQ ID NO: 19. In some embodiments, the engineered nuclease may comprise an amino acid sequence having at least 50% identity (e.g., at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 98%, or at least 99%), but less than 100% identity, to SEQ ID NO: 20. In some embodiments, the engineered nuclease may comprise an amino acid sequence having at least 50% identity (e.g., at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 98%, or at least 99%), but less than 100% identity, to SEQ ID NO: 21. In some embodiments, the engineered nuclease may comprise an amino acid sequence having at least 50% identity (e.g., at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 98%, or at least 99%), but less than 100% identity, to SEQ ID NO: 22. In some embodiments, the engineeredBIOTN-41612.601 nuclease may comprise an amino acid sequence having at least 50% identity (e.g., at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 98%, or at least 99%), but less than 100% identity, to SEQ ID NO: 23. In some embodiments, the engineered nuclease may comprise an amino acid sequence having at least 50% identity (e.g., at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 98%, or at least 99%), but less than 100% identity, to SEQ ID NO: 24. In some embodiments, the engineered nuclease may comprise an amino acid sequence having at least 50% identity (e.g., at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 98%, or at least 99%), but less than 100% identity, to SEQ ID NO: 25. In some embodiments, the engineered nuclease may comprise an amino acid sequence having at least 50% identity (e.g., at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 98%, or at least 99%), but less than 100% identity, to SEQ ID NO: 26. In some embodiments, the engineered nuclease may comprise an amino acid sequence having at least 50% identity (e.g., at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 98%, or at least 99%), but less than 100% identity, to SEQ ID NO: 27. In some embodiments, the engineered nuclease may comprise an amino acid sequence having at least 50% identity (e.g., at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 98%, or at least 99%), but less than 100% identity, to SEQ ID NO: 28. In some embodiments, the engineered nuclease may comprise an amino acid sequence having at least 50% identity (e.g., at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 98%, or at least 99%), but less than 100% identity, to SEQ ID NO: 29. In some embodiments, the engineered nuclease may comprise an amino acid sequence having at least 50% identity (e.g., at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 98%, or at least 99%), but less than 100% identity, to SEQ ID NO: 30. In some embodiments, the engineered nuclease may comprise an amino acid sequence having at least 50% identity (e.g., at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 98%,BIOTN-41612.601 or at least 99%), but less than 100% identity, to SEQ ID NO: 31. In some embodiments, the engineered nuclease may comprise an amino acid sequence having at least 50% identity (e.g., at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 98%, or at least 99%), but less than 100% identity, to SEQ ID NO: 32. In some embodiments, the engineered nuclease may comprise an amino acid sequence having at least 50% identity (e.g., at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 98%, or at least 99%), but less than 100% identity, to SEQ ID NO: 33. In some embodiments, the engineered nuclease may comprise an amino acid sequence having at least 50% identity (e.g., at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 98%, or at least 99%), but less than 100% identity, to SEQ ID NO: 34. In some embodiments, the engineered nuclease may comprise an amino acid sequence having at least 50% identity (e.g., at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 98%, or at least 99%), but less than 100% identity, to SEQ ID NO: 35. In some embodiments, the engineered nuclease may comprise an amino acid sequence having at least 50% identity (e.g., at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 98%, or at least 99%), but less than 100% identity, to SEQ ID NO: 36. In some embodiments, the engineered nuclease may comprise an amino acid sequence having at least 50% identity (e.g., at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 98%, or at least 99%), but less than 100% identity, to SEQ ID NO: 37. In some embodiments, the engineered nuclease may comprise an amino acid sequence having at least 50% identity (e.g., at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 98%, or at least 99%), but less than 100% identity, to SEQ ID NO: 38. In some embodiments, the engineered nuclease may comprise an amino acid sequence having at least 50% identity (e.g., at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 98%, or at least 99%), but less than 100% identity, to SEQ ID NO: 39. In some embodiments, the engineered nuclease may comprise an amino acid sequence having at least 50% identity (e.g., at least 50%, at least 55%, at least 60%, atBIOTN-41612.601 least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 98%, or at least 99%), but less than 100% identity, to SEQ ID NO: 40. In some embodiments, the engineered nuclease may comprise an amino acid sequence having at least 50% identity (e.g., at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 98%, or at least 99%), but less than 100% identity, to SEQ ID NO: 41. In some embodiments, the engineered nuclease may comprise an amino acid sequence having at least 50% identity (e.g., at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 98%, or at least 99%), but less than 100% identity, to SEQ ID NO: 42. In some embodiments, the engineered nuclease may comprise an amino acid sequence having at least 50% identity (e.g., at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 98%, or at least 99%), but less than 100% identity, to SEQ ID NO: 43. In some embodiments, the engineered nuclease may comprise an amino acid sequence having at least 50% identity (e.g., at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 98%, or at least 99%), but less than 100% identity, to SEQ ID NO: 44. In some embodiments, the engineered nuclease may comprise an amino acid sequence having at least 50% identity (e.g., at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 98%, or at least 99%), but less than 100% identity, to SEQ ID NO: 45. In some embodiments, the engineered nuclease may comprise an amino acid sequence having at least 50% identity (e.g., at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 98%, or at least 99%), but less than 100% identity, to SEQ ID NO: 46. In some embodiments, the engineered nuclease may comprise an amino acid sequence having at least 50% identity (e.g., at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 98%, or at least 99%), but less than 100% identity, to SEQ ID NO: 47. In some embodiments, the engineered nuclease may comprise an amino acid sequence having at least 50% identity (e.g., at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 98%, or at least 99%), but less than 100% identity, to SEQ ID NO: 48. In some embodiments, the engineered nuclease mayBIOTN-41612.601 comprise an amino acid sequence having at least 50% identity (e.g., at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 98%, or at least 99%), but less than 100% identity, to SEQ ID NO: 49. In some embodiments, the engineered nuclease may comprise an amino acid sequence having at least 50% identity (e.g., at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 98%, or at least 99%), but less than 100% identity, to SEQ ID NO: 50. In some embodiments, the engineered nuclease may comprise an amino acid sequence having at least 50% identity (e.g., at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 98%, or at least 99%), but less than 100% identity, to SEQ ID NO: 51. In some embodiments, the engineered nuclease may comprise an amino acid sequence having at least 50% identity (e.g., at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 98%, or at least 99%), but less than 100% identity, to SEQ ID NO: 52. In some embodiments, the engineered nuclease may comprise an amino acid sequence having at least 50% identity (e.g., at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 98%, or at least 99%), but less than 100% identity, to SEQ ID NO: 53. In some embodiments, the engineered nuclease may comprise an amino acid sequence having at least 50% identity (e.g., at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 98%, or at least 99%), but less than 100% identity, to SEQ ID NO: 54. In some embodiments, the engineered nuclease may comprise an amino acid sequence having at least 50% identity (e.g., at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 98%, or at least 99%), but less than 100% identity, to SEQ ID NO: 55. In some embodiments, the engineered nuclease may comprise an amino acid sequence having at least 50% identity (e.g., at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 98%, or at least 99%), but less than 100% identity, to SEQ ID NO: 56. In some embodiments, the engineered nuclease may comprise an amino acid sequence having at least 50% identity (e.g., at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 98%, or at leastBIOTN-41612.60199%), but less than 100% identity, to SEQ ID NO: 57. In some embodiments, the engineered nuclease may comprise an amino acid sequence having at least 50% identity (e.g., at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 98%, or at least 99%), but less than 100% identity, to SEQ ID NO: 58. In some embodiments, the engineered nuclease may comprise an amino acid sequence having at least 50% identity (e.g., at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 98%, or at least 99%), but less than 100% identity, to SEQ ID NO: 59. In some embodiments, the engineered nuclease may comprise an amino acid sequence having at least 50% identity (e.g., at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 98%, or at least 99%), but less than 100% identity, to SEQ ID NO: 60. In some embodiments, the engineered nuclease may comprise an amino acid sequence having at least 50% identity (e.g., at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 98%, or at least 99%), but less than 100% identity, to SEQ ID NO: 61. In some embodiments, the engineered nuclease may comprise an amino acid sequence having at least 50% identity (e.g., at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 98%, or at least 99%), but less than 100% identity, to SEQ ID NO: 62. In some embodiments, the engineered nuclease may comprise an amino acid sequence having at least 50% identity (e.g., at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 98%, or at least 99%), but less than 100% identity, to SEQ ID NO: 63. In some embodiments, the engineered nuclease may comprise an amino acid sequence having at least 50% identity (e.g., at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 98%, or at least 99%), but less than 100% identity, to SEQ ID NO: 64. In some embodiments, the engineered nuclease may comprise an amino acid sequence having at least 50% identity (e.g., at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 98%, or at least 99%), but less than 100% identity, to SEQ ID NO: 65. In some embodiments, the engineered nuclease may comprise an amino acid sequence having at least 50% identity (e.g., at least 50%, at least 55%, at least 60%, at least 65%, at leastBIOTN-41612.60170%, at least 75%, at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 98%, or at least 99%), but less than 100% identity, to SEQ ID NO: 66. In some embodiments, the engineered nuclease may comprise an amino acid sequence having at least 50% identity (e.g., at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 98%, or at least 99%), but less than 100% identity, to SEQ ID NO: 67. In some embodiments, the engineered nuclease may comprise an amino acid sequence having at least 50% identity (e.g., at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 98%, or at least 99%), but less than 100% identity, to SEQ ID NO: 68. In some embodiments, the engineered nuclease may comprise an amino acid sequence having at least 50% identity (e.g., at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 98%, or at least 99%), but less than 100% identity, to SEQ ID NO: 69. In some embodiments, the engineered nuclease may comprise an amino acid sequence having at least 50% identity (e.g., at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 98%, or at least 99%), but less than 100% identity, to SEQ ID NO: 70. In some embodiments, the engineered nuclease may comprise an amino acid sequence having at least 50% identity (e.g., at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 98%, or at least 99%), but less than 100% identity, to SEQ ID NO: 71. In some embodiments, the engineered nuclease may comprise an amino acid sequence having at least 50% identity (e.g., at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 98%, or at least 99%), but less than 100% identity, to SEQ ID NO: 72. In some embodiments, the engineered nuclease may comprise an amino acid sequence having at least 50% identity (e.g., at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 98%, or at least 99%), but less than 100% identity, to SEQ ID NO: 73. In some embodiments, the engineered nuclease may comprise an amino acid sequence having at least 50% identity (e.g., at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 98%, or at least 99%), but less than 100% identity, to SEQ ID NO: 74. In some embodiments, the engineered nuclease may comprise anBIOTN-41612.601 amino acid sequence having at least 50% identity (e.g., at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 98%, or at least 99%), but less than 100% identity, to SEQ ID NO: 75. In some embodiments, the engineered nuclease may comprise an amino acid sequence having at least 50% identity (e.g., at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 98%, or at least 99%), but less than 100% identity, to SEQ ID NO: 76. In some embodiments, the engineered nuclease may comprise an amino acid sequence having at least 50% identity (e.g., at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 98%, or at least 99%), but less than 100% identity, to SEQ ID NO: 77. In some embodiments, the engineered nuclease may comprise an amino acid sequence having at least 50% identity (e.g., at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 98%, or at least 99%), but less than 100% identity, to SEQ ID NO: 78. In some embodiments, the engineered nuclease may comprise an amino acid sequence having at least 50% identity (e.g., at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 98%, or at least 99%), but less than 100% identity, to SEQ ID NO: 79. In some embodiments, the engineered nuclease may comprise an amino acid sequence having at least 50% identity (e.g., at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 98%, or at least 99%), but less than 100% identity, to SEQ ID NO: 80. In some embodiments, the engineered nuclease may comprise an amino acid sequence having at least 50% identity (e.g., at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 98%, or at least 99%), but less than 100% identity, to SEQ ID NO: 81. In some embodiments, the engineered nuclease may comprise an amino acid sequence having at least 50% identity (e.g., at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 98%, or at least 99%), but less than 100% identity, to SEQ ID NO: 82.

[0052] In some embodiments, the engineered nucleases of the present disclosure comprise amino acid sequences in which regions of amino acids (e.g., domains) have been removed, added, and / or substituted. In some embodiments, the engineered nucleases of the present disclosureBIOTN-41612.601 comprise amino acid sequences that include one more domains from homologous nucleases in place of, or in addition to, their corresponding endogenous domains. For example, in some embodiments, an engineered nuclease of the present disclosure comprises an amino acid sequence of any one of SEQ ID NOs: 83-1 19. In some embodiments, the amino acid sequence comprises any one of SEQ ID NOs: 83-1 19 in place of at least one amino acid in SEQ ID NO: 1 or SEQ ID NO: 2. In some embodiments, the amino acid sequence comprises any one of SEQ ID NOs: 83- 119 in place of the corresponding amino acid sequence in SEQ ID NO: 1 or SEQ ID NO: 2, as provided in Table 1.

[0053] In some embodiments, an engineered nuclease of the present disclosure comprises an amino acid sequence that is at least 70% identical to any of SEQ ID NOs: 1-9, and comprises an amino acid sequence that is at least 70% identical to any one of SEQ ID NOs: 83-1 19. In accordance with these embodiments, the engineered nuclease comprises an amino acid sequence that is at least 70% identical (e.g., at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 98%, at least 99%, or 100% identical) to any of SEQ ID NOs: 1-9 and an amino acid sequence that is at least 70% identical (e.g., at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 98%, at least 99%, or 100% identical) to SEQ ID NO: 83. In some embodiments, the engineered nuclease comprises an amino acid sequence that is at least 70% identical (e.g., at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 98%, at least 99%, or 100% identical) to any of SEQ ID NOs: 1-9 and an amino acid sequence that is at least 70% identical (e.g., at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 98%, at least 99%, or 100% identical) to SEQ ID NO: 84. In some embodiments, the engineered nuclease comprises an amino acid sequence that is at least 70% identical (e.g., at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 98%, at least 99%, or 100% identical) to any of SEQ ID NOs: 1-9 and an amino acid sequence that is at least 70% identical (e.g., at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 98%, at least 99%, or 100% identical) to SEQ ID NO: 85. In some embodiments, the engineered nuclease comprises an amino acid sequence that is at least 70% identical (e.g., at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 98%, at least 99%, or 100% identical) to any of SEQ ID NOs: 1-9 and an amino acid sequence that is at least 70% identical (e.g., at least 70%, at least 75%, at least 80%, at least 85%, at least 90%,BIOTN-41612.601 at least 93%, at least 95%, at least 98%, at least 99%, or 100% identical) to SEQ ID NO: 86. In some embodiments, the engineered nuclease comprises an amino acid sequence that is at least 70% identical (e.g., at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 98%, at least 99%, or 100% identical) to any of SEQ ID NOs: 1-9 and an amino acid sequence that is at least 70% identical (e.g., at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 98%, at least 99%, or 100% identical) to SEQ ID NO: 87. In some embodiments, the engineered nuclease comprises an amino acid sequence that is at least 70% identical (e.g., at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 98%, at least 99%, or 100% identical) to any of SEQ ID NOs: 1-9 and an amino acid sequence that is at least 70% identical (e.g., at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 98%, at least 99%, or 100% identical) to SEQ ID NO: 88. In some embodiments, the engineered nuclease comprises an amino acid sequence that is at least 70% identical (e.g., at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 98%, at least 99%, or 100% identical) to any of SEQ ID NOs: 1-9 and an amino acid sequence that is at least 70% identical (e.g., at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 98%, at least 99%, or 100% identical) to SEQ ID NO: 89. In some embodiments, the engineered nuclease comprises an amino acid sequence that is at least 70% identical (e.g., at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 98%, at least 99%, or 100% identical) to any of SEQ ID NOs: 1-9 and an amino acid sequence that is at least 70% identical (e.g., at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 98%, at least 99%, or 100% identical) to SEQ ID NO: 90. In some embodiments, the engineered nuclease comprises an amino acid sequence that is at least 70% identical (e.g., at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 98%, at least 99%, or 100% identical) to any of SEQ ID NOs: 1-9 and an amino acid sequence that is at least 70% identical (e.g., at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 98%, at least 99%, or 100% identical) to SEQ ID NO: 91. In some embodiments, the engineered nuclease comprises an amino acid sequence that is at least 70% identical (e.g., at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 98%, at least 99%, or 100% identical) to any of SEQ ID NOs: 1-9 and an amino acid sequence that is at least 70% identical (e.g., at least 70%, atBIOTN-41612.601 least 75%, at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 98%, at least 99%, or 100% identical) to SEQ ID NO: 92. In some embodiments, the engineered nuclease comprises an amino acid sequence that is at least 70% identical (e.g., at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 98%, at least 99%, or 100% identical) to any of SEQ ID NOs: 1-9 and an amino acid sequence that is at least 70% identical (e.g., at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 98%, at least 99%, or 100% identical) to SEQ ID NO: 93. In some embodiments, the engineered nuclease comprises an amino acid sequence that is at least 70% identical (e.g., at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 98%, at least 99%, or 100% identical) to any of SEQ ID NOs: 1-9 and an amino acid sequence that is at least 70% identical (e.g., at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 98%, at least 99%, or 100% identical) to SEQ ID NO: 94. In some embodiments, the engineered nuclease comprises an amino acid sequence that is at least 70% identical (e.g., at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 98%, at least 99%, or 100% identical) to any of SEQ ID NOs: 1-9 and an amino acid sequence that is at least 70% identical (e.g., at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 98%, at least 99%, or 100% identical) to SEQ ID NO: 95. In some embodiments, the engineered nuclease comprises an amino acid sequence that is at least 70% identical (e.g., at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 98%, at least 99%, or 100% identical) to any of SEQ ID NOs: 1-9 and an amino acid sequence that is at least 70% identical (e.g., at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 98%, at least 99%, or 100% identical) to SEQ ID NO: 96. In some embodiments, the engineered nuclease comprises an amino acid sequence that is at least 70% identical (e.g., at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 98%, at least 99%, or 100% identical) to any of SEQ ID NOs: 1-9 and an amino acid sequence that is at least 70% identical (e.g., at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 98%, at least 99%, or 100% identical) to SEQ ID NO: 97. In some embodiments, the engineered nuclease comprises an amino acid sequence that is at least 70% identical (e.g., at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 98%, at least 99%, or 100% identical) to any of SEQ ID NOs: 1-9 and an amino acid sequence thatBIOTN-41612.601 is at least 70% identical (e.g., at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 98%, at least 99%, or 100% identical) to SEQ ID NO: 98. In some embodiments, the engineered nuclease comprises an amino acid sequence that is at least 70% identical (e.g., at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 98%, at least 99%, or 100% identical) to any of SEQ ID NOs: 1-9 and an amino acid sequence that is at least 70% identical (e.g., at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 98%, at least 99%, or 100% identical) to SEQ ID NO: 99. In some embodiments, the engineered nuclease comprises an amino acid sequence that is at least 70% identical (e.g., at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 98%, at least 99%, or 100% identical) to any of SEQ ID NOs: 1-9 and an amino acid sequence that is at least 70% identical (e.g., at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 98%, at least 99%, or 100% identical) to SEQ ID NO: 100. In some embodiments, the engineered nuclease comprises an amino acid sequence that is at least 70% identical (e.g., at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 98%, at least 99%, or 100% identical) to any of SEQ ID NOs: 1-9 and an amino acid sequence that is at least 70% identical (e.g., at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 98%, at least 99%, or 100% identical) to SEQ ID NO: 101. In some embodiments, the engineered nuclease comprises an amino acid sequence that is at least 70% identical (e.g., at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 98%, at least 99%, or 100% identical) to any of SEQ ID NOs: 1-9 and an amino acid sequence that is at least 70% identical (e.g., at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 98%, at least 99%, or 100% identical) to SEQ ID NO: 102. In some embodiments, the engineered nuclease comprises an amino acid sequence that is at least 70% identical (e.g., at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 98%, at least 99%, or 100% identical) to any of SEQ ID NOs: 1-9 and an amino acid sequence that is at least 70% identical (e.g., at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 98%, at least 99%, or 100% identical) to SEQ ID NO: 103. In some embodiments, the engineered nuclease comprises an amino acid sequence that is at least 70% identical (e.g., at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 98%, at least 99%, orBIOTN-41612.601100% identical) to any of SEQ ID NOs: 1-9 and an amino acid sequence that is at least 70% identical (e.g., at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 98%, at least 99%, or 100% identical) to SEQ ID NO: 104. In some embodiments, the engineered nuclease comprises an amino acid sequence that is at least 70% identical (e.g., at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 98%, at least 99%, or 100% identical) to any of SEQ ID NOs: 1-9 and an amino acid sequence that is at least 70% identical (e.g., at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 98%, at least 99%, or 100% identical) to SEQ ID NO: 105. In some embodiments, the engineered nuclease comprises an amino acid sequence that is at least 70% identical (e.g., at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 98%, at least 99%, or 100% identical) to any of SEQ ID NOs: 1-9 and an amino acid sequence that is at least 70% identical (e.g., at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 98%, at least 99%, or 100% identical) to SEQ ID NO: 106. In some embodiments, the engineered nuclease comprises an amino acid sequence that is at least 70% identical (e.g., at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 98%, at least 99%, or 100% identical) to any of SEQ ID NOs: 1-9 and an amino acid sequence that is at least 70% identical (e.g., at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 98%, at least 99%, or 100% identical) to SEQ ID NO: 107. In some embodiments, the engineered nuclease comprises an amino acid sequence that is at least 70% identical (e.g., at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 98%, at least 99%, or 100% identical) to any of SEQ ID NOs: 1-9 and an amino acid sequence that is at least 70% identical (e.g., at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 98%, at least 99%, or 100% identical) to SEQ ID NO: 108. In some embodiments, the engineered nuclease comprises an amino acid sequence that is at least 70% identical (e.g., at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 98%, at least 99%, or 100% identical) to any of SEQ ID NOs: 1-9 and an amino acid sequence that is at least 70% identical (e.g., at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 98%, at least 99%, or 100% identical) to SEQ ID NO: 109. In some embodiments, the engineered nuclease comprises an amino acid sequence that is at least 70% identical (e.g., at least 70%, at least 75%,BIOTN-41612.601 at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 98%, at least 99%, or 100% identical) to any of SEQ ID NOs: 1-9 and an amino acid sequence that is at least 70% identical (e.g., at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 98%, at least 99%, or 100% identical) to SEQ ID NO: 110. In some embodiments, the engineered nuclease comprises an amino acid sequence that is at least 70% identical (e.g., at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 98%, at least 99%, or 100% identical) to any of SEQ ID NOs: 1-9 and an amino acid sequence that is at least 70% identical (e.g., at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 98%, at least 99%, or 100% identical) to SEQ ID NO: 111. In some embodiments, the engineered nuclease comprises an amino acid sequence that is at least 70% identical (e.g., at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 98%, at least 99%, or 100% identical) to any of SEQ ID NOs: 1-9 and an amino acid sequence that is at least 70% identical (e.g., at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 98%, at least 99%, or 100% identical) to SEQ ID NO: 112. In some embodiments, the engineered nuclease comprises an amino acid sequence that is at least 70% identical (e.g., at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 98%, at least 99%, or 100% identical) to any of SEQ ID NOs: 1-9 and an amino acid sequence that is at least 70% identical (e.g., at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 98%, at least 99%, or 100% identical) to SEQ ID NO: 113. In some embodiments, the engineered nuclease comprises an amino acid sequence that is at least 70% identical (e.g., at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 98%, at least 99%, or 100% identical) to any of SEQ ID NOs: 1-9 and an amino acid sequence that is at least 70% identical (e.g., at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 98%, at least 99%, or 100% identical) to SEQ ID NO: 114. In some embodiments, the engineered nuclease comprises an amino acid sequence that is at least 70% identical (e.g., at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 98%, at least 99%, or 100% identical) to any of SEQ ID NOs: 1-9 and an amino acid sequence that is at least 70% identical (e.g., at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 98%, at least 99%, or 100% identical) to SEQ ID NO: 115. In some embodiments, the engineered nucleaseBIOTN-41612.601 comprises an amino acid sequence that is at least 70% identical (e.g., at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 98%, at least 99%, or 100% identical) to any of SEQ ID NOs: 1-9 and an amino acid sequence that is at least 70% identical (e.g., at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 98%, at least 99%, or 100% identical) to SEQ ID NO: 116. In some embodiments, the engineered nuclease comprises an amino acid sequence that is at least 70% identical (e.g., at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 98%, at least 99%, or 100% identical) to any of SEQ ID NOs: 1-9 and an amino acid sequence that is at least 70% identical (e.g., at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 98%, at least 99%, or 100% identical) to SEQ ID NO: 117. In some embodiments, the engineered nuclease comprises an amino acid sequence that is at least 70% identical (e.g., at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 98%, at least 99%, or 100% identical) to any of SEQ ID NOs: 1-9 and an amino acid sequence that is at least 70% identical (e.g., at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 98%, at least 99%, or 100% identical) to SEQ ID NO: 118. In some embodiments, the engineered nuclease comprises an amino acid sequence that is at least 70% identical (e.g., at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 98%, at least 99%, or 100% identical) to any of SEQ ID NOs: 1-9 and an amino acid sequence that is at least 70% identical (e.g., at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 98%, at least 99%, or 100% identical) to SEQ ID NO: 1 19.

[0054] In some embodiments, the nuclease or engineered nuclease is covalently attached to a peptide or protein in a fusion protein. In some embodiments, the peptide comprises a nuclear localization sequence (NLS). The nuclear localization sequence may be appended, for example, to the N-terminus, a C-terminus, or a combination thereof. In some embodiments, the nuclease comprises two or more NLSs. The two or more NLSs may be in tandem, separated by a linker, at either end terminus of the protein, or one or more may be embedded in the protein.

[0055] The NLS may be appended to the nuclease by a linker. The linker may be a polypeptide of any amino acid sequence and length. The linker may act as a spacer peptide. In some embodiments, the linker is flexible. In some embodiments, the linker comprises at least oneBIOTN-41612.601 glycine and at least one serine. In some embodiments, the linker comprises an amino acid sequence consisting of (Gly2Ser)n, where n is the number of repeats comprising an integer from 2-20.

[0056] The peptide may comprise an epitope tag (e.g., 3xFLAG tag, an HA tag, a Myc tag, and the like). In some embodiments, the epitope tag may be adjacent, either upstream or downstream, to a nuclear localization sequence. The epitope tags may be at the N-terminus, a C-terminus, or a combination thereof of the corresponding protein.

[0057] The nuclease or engineered nuclease may be part of a fusion protein comprising another protein or protein domain. For example, the nuclease or engineered nuclease may be fused to another protein or protein domain that provides for tagging or visualization (e.g., GFP). The nuclease or engineered nuclease may be fused to a protein or protein domain that has another functionality or activity useful to target to certain DNA sequences (e.g., nuclease activity such as that provide by FokI nuclease, protein modification activity such as histone modification activity including acetylation or deacetylation or demethylation or methyltransferase activity, transcription modulation activity such as activity of a transcriptional activator or repressor, base editing activity such as deaminase activity, DNA modifying activity such as DNA methylation activity, and the like).

[0058] In some embodiments, the nuclease or engineered nuclease may be fused with one or more (e.g., two, three, four, or more) protein transduction domains or PTDs, also known as a CPP - cell penetrating peptide. A protein transduction domains is a polypeptide, polynucleotide, carbohydrate, or organic or inorganic compound that facilitates traversing a lipid bilayer, micelle, cell membrane, organelle membrane, or vesicle membrane. A PTD attached to another molecule, facilitates the molecule traversing a membrane, for example going from extracellular space to intracellular space, or cytosol to within an organelle. In some embodiments, a PTD is covalently linked to a terminus of the nuclease or engineered nuclease (e.g., N-terminus, C-terminus, or both). In some embodiments, the PTD is inserted internally at a suitable insertion site. Examples of PTDs include but are not limited to a minimal undecapeptide protein transduction domain (corresponding to residues 47-57 of HIV-1 TAT comprising); a polyarginine sequence comprising a number of arginines sufficient to direct entry into a cell (e.g., 3, 4, 5, 6, 7, 8, 9, 10, or 10-50 arginines); a VP22 domain (Zender et al. (2002) Cancer Gene Ther. 9(6):489-96); a Drosophila Antennapedia protein transduction domain (Noguchi et al. (2003) Diabetes 52 iy 132- 13iy a truncated humanBIOTN-41612.601 calcitonin peptide (Trehin et al. (2004) Pharm. Research 21: 1248-1256); polylysine (Wender et al. (2000) Proc. Natl. Acad. Sci. USA 97:13003-13008); Transportan, and the like.

[0059] The nuclease or engineered nuclease may be fused via a linker polypeptide. The linker polypeptide may have any of a variety of amino acid sequences. Proteins can be joined by a spacer peptide, generally of a flexible nature, although other chemical linkages are not excluded. Suitable linkers include polypeptides of between 4 amino acids and 40 amino acids in length, or between 4 amino acids and 25 amino acids in length. These linkers can be produced by using synthetic, linkerencoding oligonucleotides to couple the proteins, or can be encoded by a nucleic acid sequence encoding the fusion protein. Peptide linkers with a degree of flexibility can be used. The linking peptides may have virtually any amino acid sequence, bearing in mind that the preferred linkers will have a sequence that results in a generally flexible peptide. The use of small amino acids, such as glycine and alanine, are of use in creating a flexible peptide. The creation of such sequences is routine to those of skill in the art. A variety of different linkers are commercially available and are considered suitable for use, including but not limited to, glycine-serine polymers, glycine-alanine polymers, and alanine-serine polymers.Compositions and Systems

[0060] Also disclosed herein are compositions comprising a nuclease or engineered nuclease as described herein or a nucleic acid molecule comprising a sequence encoding the nuclease or engineered nuclease. In some embodiments, the compositions further comprise at least one guide RNA (gRNA) or one or more nucleic acids comprising a sequence encoding the least one gRNA.

[0061] Further disclosed herein are systems for modifying a target nucleic acid comprising a nuclease as described herein (e.g., a nuclease comprising an amino acid sequence having at least 70% identity (e.g., at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 98%, at least 99% or 100% identity) to an amino acid sequence of SEQ ID NOs: 1-9 or a nucleic acid molecule comprising a sequence encoding the nuclease.

[0062] In some embodiments, the components of the system may be in the form of a composition. In some embodiments, the components of the present compositions or systems may be mixed, individually or in any combination, with a carrier which are also within the scope of the present disclosure. Exemplary carriers include buffers, antioxidants, preservatives, carbohydrates, surfactants, and the like.BIOTN-41612.601

[0063] Also disclosed is a cell comprising the compositions or systems described herein. In some embodiments, the cell is a prokaryotic cell. In some embodiments, the cell is a eukaryotic cell. In some embodiments, the cell is a mammalian cell. In some embodiments, the cell is a human cell.

[0064] The compositions or systems disclosed herein may further comprise at least one gRNA complementary to at least a portion of a target nucleic acid sequence, or a nucleic acid encoding the at least one gRNA. In instances when the composition or system comprises more than one gRNA, each may be encoded on the same or different nucleic acid as the other gRNA.

[0065] The gRNA may be a crRNA, crRNA / tracrRNA (or single guide RNA, sgRNA). The terms “gRNA,” “guide RNA,” and “CRISPR guide sequence” may be used interchangeably throughout and refer to a nucleic acid comprising a sequence that determines the sequence specificity of the nuclease. A gRNA hybridizes to (complementary to, partially or completely) a target nucleic acid sequence (e.g., the genome in a host cell).

[0066] In some embodiments, the at least one gRNA is encoded in a CRISPR RNA (crRNA) array. CRISPR arrays contain a series of direct repeats separated by short sequences called spacers. The nucleases described herein may have a preference for direct repeat sequences. These can be determined by methods known in the art including those described in the Examples herein. For example, the CRISPR RNA (crRNA) may contain multiple gRNAs or may contain more than one different sequence each configured to hybridize a distinct target nucleic acid sequence.

[0067] The gRNA or portion thereof that hybridizes to the target nucleic acid (a target site) may be between 15-40 nucleotides in length. In some embodiments, the gRNA sequence that hybridizes to the target nucleic acid is 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 nucleotides in length. gRNAs or sgRNA(s) used in the present disclosure can be between about 5 and 100 nucleotides long, or longer (e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59 60, 61, 62, 63, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 92, 93, 94, 95, 96, 97, 98, 99, or 100 nucleotides in length, or longer).

[0068] To facilitate gRNA design, many computational tools have been developed (See Prykhozhij et al. (PLoS ONE, 10(3): (2015)); Zhu et al. (PLoS ONE, 9(9) (2014)); Xiao et al. (Bioinformatics. Jan 21 (2014)); Heigwer et al. (Nat Methods, 11(2): 122-123 (2014)). MethodsBIOTN-41612.601 and tools for guide RNA design are discussed by Zhu (Frontiers in Biology, 10 (4) pp 289-296 (2015)), which is incorporated by reference herein. Additionally, there are many publicly available software tools that can be used to facilitate the design of sgRNA(s); including but not limited to, Genscript Interactive CRISPR gRNA Design Tool, WU-CRISPR, and Broad Institute GPP sgRNA Designer. There are also publicly available pre-designed gRNA sequences to target many genes and locations within the genomes of many species (human, mouse, rat, zebrafish, C. elegcms), including but not limited to, IDT DNA Predesigned Alt-R CRISPR-Cas9 guide RNAs, Addgene Validated gRNA Target Sequences, and GenScript Genome-wide gRNA databases.

[0069] In addition to a sequence that binds to a target nucleic acid, in some embodiments, the gRNA may also comprise a scaffold sequence (e.g., tracrRNA). In some embodiments, such a chimeric gRNA may be referred to as a single guide RNA (sgRNA). Exemplary scaffold sequences will be evident to one of skill in the art and can be found, for example, in Jinek, et al. Science (2012) 337(6096):816-821 , and Ran, et al. Nature Protocols (2013) 8:2281-2308, incorporated herein by reference in their entireties.

[0070] In some embodiments, the gRNA sequence does not comprise a scaffold sequence and a scaffold sequence is expressed as a separate transcript. In such embodiments, the gRNA sequence further comprises an additional sequence that is complementary to a portion of the scaffold sequence and functions to bind (hybridize) the scaffold sequence.

[0071] In some embodiments, the gRNA sequence is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or at least 100% complementary to a target nucleic acid. In some embodiments, the gRNA sequence is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or at least 100% complementary to the 3 ’ end of the target nucleic acid (e.g., the last 5, 6, 7, 8, 9, or 10 nucleotides of the 3’ end of the target nucleic acid).

[0072] The gRNA may be a non-naturally occurring gRNA.

[0073] The target sequence may or may not be flanked by a protospacer adjacent motif (PAM) sequence. In certain embodiments, a nucleic acid-guided nuclease can only cleave a target sequence if an appropriate PAM is present, see, for example Doudna et al., Science, 2014, 346(6213): 1258096, incorporated herein by reference. A PAM can be 5' or 3' of a target sequence. A PAM can be upstream or downstream of a target sequence. In one embodiment, the target sequence is immediately flanked on the 3' end by a PAM sequence. A PAM can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more nucleotides in length. In certain embodiments, a PAM is between 2-6BIOTN-41612.601 nucleotides in length. Sequence requirements for PAMs for any given nuclease can be determined using known methods, for example, the protocol of Walton et al. (Walton RT, et al., Science. 2020 Apr 17;368(6488):290-296, incorporated herein by reference in its entirety). In some embodiments, the engineered nucleases herein have an enlarged PAM preference, e.g., the engineered nuclease utilizes additional PAM sequences that are not used or are poorly used by a wildtype (non-engineered nuclease) or are not used or are poorly used by other nucleases such as a Casl2a (Cpfl) nuclease. In some embodiments, the engineered nucleases herein have a reduced PAM preference, e.g., the engineered nuclease utilizes fewer PAM sequences than are used by a wildtype (non-engineered nuclease) or by other nucleases such as a Casl2a (Cpfl) nuclease.

[0074] “Complementarity” refers to the ability of a nucleic acid to form hydrogen bond(s) with another nucleic acid sequence by either traditional Watson-Crick or other non-traditional types. A percent complementarity indicates the percentage of residues in a nucleic acid molecule, which can form hydrogen bonds (e.g., Watson-Crick base pairing) with a second nucleic acid sequence. Full complementarity is not necessarily required, provided there is sufficient complementarity to cause hybridization. There may be mismatches distal from the PAM.

[0075] In some cases, the compositions or systems disclosed herein may further comprise a donor polynucleotide. For example, in applications in which it is desirable to insert a polynucleotide sequence into the genome where a target sequence is cleaved, a donor polynucleotide (a nucleic acid comprising a donor sequence) can also be provided to the cell. By a “donor sequence” or “donor polynucleotide” or “donor template” it is meant a nucleic acid sequence to be inserted at the site targeted by the nuclease (e.g., after dsDNA cleavage, after nicking a target DNA, after dual nicking a target DNA, and the like). In some cases, the donor sequence is provided to the cell as single-stranded DNA. In some cases, the donor template is provided to the cell as double-stranded DNA. It may be introduced into a cell in linear or circular form. If introduced in linear form, the ends of the donor sequence may be protected (e.g., from exonucleolytic degradation) by any convenient method and such methods are known to those of skill in the art. For example, one or more dideoxynucleotide residues can be added to the 3' terminus of a linear molecule and / or self-complementary oligonucleotides can be ligated to one or both ends. A donor template can be introduced into a cell as part of a vector molecule having additional sequences such as, for example, replication origins, promoters and genes encoding antibiotic resistance. Moreover, donor template can be introduced as naked nucleic acid, as nucleicBIOTN-41612.601 acid complexed with an agent such as a liposome or poloxamer, or can be delivered by viruses (e.g., adenovirus, AAV).

[0076] Also disclosed herein are one or more nucleic acids encoding the nucleases, the gRNA(s), and systems thereof. In some embodiments, the one or more nucleic acids comprise one or more messenger RNAs, one or more vectors, or any combination thereof. A single nucleic acid may encode the nuclease and the at least one gRNA, or the nuclease may be encoded on a separate nucleic acid from the at least one gRNA.

[0077] In some embodiments, the nuclease is provided as a split-nuclease (e.g., a nuclease can in some cases be delivered as a split- nuclease, or a nucleic acid(s) encoding a split- nuclease) such that two separate proteins together form a functional nuclease. In some such cases the sequences that encode the two parts of the split-nuclease protein are present on the same vector. In some cases, they are present on separate vectors, e.g., as part of a vector system that encodes the nucleases, the gRNA(s), and systems thereof.

[0078] In certain embodiments, engineering the nucleases for use in eukaryotic cells may involve codon-optimization. It will be appreciated that changing native codons to those most frequently used in mammals allows for maximum expression of the system proteins in mammalian cells (e.g., human cells). Such modified nucleic acid sequences are commonly described in the art as “codon-optimized,” or as utilizing “mammalian-preferred” or “human-preferred” codons. In some embodiments, the nucleic acid sequence is considered codon-optimized if at least about 60% (e.g., 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 98%) of the codons encoded therein are mammalian preferred codons.

[0079] The present disclosure also provides for DNA segments encoding the nucleases and nucleic acids (e.g., gRNA) disclosed herein, vectors containing these segments and cells containing the vectors. The vectors may be used to propagate the segment in an appropriate cell and / or to allow expression from the segment (e.g., an expression vector). The person of ordinary skill in the art would be aware of the various vectors available for propagation and expression of a nucleic acid sequence.

[0080] The present disclosure further provides engineered, non-naturally occurring vectors and vector systems, which can encode one or more or all of the components of the present system. The vector(s) can be introduced into a cell that is capable of expressing the polypeptide encoded thereby, including any suitable prokaryotic or eukaryotic cell.BIOTN-41612.601

[0081] The vectors of the present disclosure may be delivered to a eukaryotic cell in a subject. Modification of the eukaryotic cells via the present system can take place in a cell culture, where the method comprises isolating the eukaryotic cell from a subject prior to the modification. In some embodiments, the method further comprises returning said eukaryotic cell and / or cells derived therefrom to the subject.

[0082] Viral and non-viral based gene transfer methods can be used to introduce nucleic acids encoding components of the present system into cells, tissues, or a subject. Such methods can be used to administer nucleic acids encoding components of the present system to cells in culture, or in a host organism. Non-viral vector delivery systems include DNA plasmids, cosmids, RNA (e.g., a transcript of a vector described herein), a nucleic acid, and a nucleic acid complexed with a delivery vehicle. Viral vector delivery systems include DNA and RNA viruses, which have either episomal or integrated genomes after delivery to the cell. Viral vectors include, for example, retroviral, lentiviral, adenoviral, adeno-associated and herpes simplex viral vectors.

[0083] In certain embodiments, plasmids that are non-replicative, or plasmids that can be cured by high temperature may be used, such that any or all of the necessary components of the composition or system may be removed from the cells under certain conditions. For example, this may allow for DNA integration by transforming bacteria of interest, but then being left with engineered strains that have no memory of the plasmids or vectors used for the integration.

[0084] A variety of viral constructs may be used to deliver the present composition or system (such as a nuclease and one or more gRNA(s)) to the targeted cells and / or a subject. Nonlimiting examples of such recombinant viruses include recombinant adeno-associated virus (AAV), recombinant adenoviruses, recombinant lentiviruses, recombinant retroviruses, recombinant herpes simplex viruses, recombinant poxviruses, phages, etc. The present disclosure provides vectors capable of integration in the host genome, such as retrovirus or lentivirus. See, e.g., Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, New York, 1989; Kay, M. A., et al., 2001 Nat. Medic. 7( 1):33-40; and Walther W. and Stein U., 2000 Drugs, 60(2): 249-71, incorporated herein by reference.

[0085] In one embodiment, a DNA segment encoding the nuclease is contained in a plasmid vector that allows expression of the protein and subsequent isolation and purification of the protein produced by the recombinant vector. Accordingly, the nucleases disclosed herein can be purified following expression, obtained by chemical synthesis, or obtained by recombinant methods.BIOTN-41612.601

[0086] To construct cells that express the present system, expression vectors for stable or transient expression of the system, or any of its components, may be constructed via methods as described herein or known in the art and introduced into cells. For example, nucleic acids encoding the components of the present system may be cloned into a suitable expression vector, such as a plasmid or a viral vector in operable linkage to a suitable promoter. The selection of expression vectors / plasmids / viral vectors should be suitable for integration and replication in eukaryotic cells.

[0087] In certain embodiments, vectors of the present disclosure can drive the expression of one or more sequences in prokaryotic cells. Promoters that may be used include T7 RNA polymerase promoters, constitutive E. coli promoters, and promoters that could be broadly recognized by transcriptional machinery in a wide range of bacterial organisms. The composition or system may be used with various bacterial hosts.

[0088] In certain embodiments, vectors of the present disclosure can drive the expression of one or more sequences in mammalian cells using a mammalian expression vector. Examples of mammalian expression vectors include pCDM8 (Seed, Nature (1987) 329:840, incorporated herein by reference) and pMT2PC (Kaufman, et al., EMBO J. (1987) 6: 187, incorporated herein by reference). When used in mammalian cells, the expression vector's control functions are typically provided by one or more regulatory elements. For example, commonly used promoters are derived from polyoma, adenovirus 2, cytomegalovirus, simian virus 40, and others disclosed herein and known in the art. For other suitable expression systems for both prokaryotic and eukaryotic cells see, e.g., Chapters 16 and 17 of Sambrook, et al., MOLECULAR CLONING: A LABORATORY MANUAL. 2nd eds., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989, incorporated herein by reference.

[0089] Vectors of the present disclosure can comprise any of a number of promoters known to the art, wherein the promoter is constitutive, regulatable or inducible, cell type specific, tissuespecific, or species specific. In addition to the sequence sufficient to direct transcription, a promoter sequence of the invention can also include sequences of other regulatory elements that are involved in modulating transcription (e.g., enhancers, Kozak sequences and introns). Many promoter / regulatory sequences useful for driving constitutive expression of a gene are available in the art and include, but are not limited to, for example, CMV (cytomegalovirus promoter), EFla (human elongation factor 1 alpha promoter), SV40 (simian vacuolating virus 40 promoter), PGK (mammalian phosphoglycerate kinase promoter), Ubc (human ubiquitin C promoter), human beta-BIOTN-41612.601 actin promoter, rodent beta-actin promoter, CBh (chicken beta-actin promoter), CAG (hybrid promoter contains CMV enhancer, chicken beta actin promoter, and rabbit beta-globin splice acceptor), TRE (Tetracycline response element promoter), Hl (human polymerase III RNA promoter), U6 (human U6 small nuclear promoter), and the like. Additional promoters that can be used for expression of the components of the present system, include, without limitation, cytomegalovirus (CMV) intermediate early promoter, a viral LTR such as the Rous sarcoma virus LTR, HIV-LTR, HTLV-1 LTR, Maloney murine leukemia virus (MMLV) LTR, myeoloproliferative sarcoma virus (MPSV) LTR, spleen focus-forming virus (SFFV) LTR, the simian virus 40 (SV40) early promoter, herpes simplex tk virus promoter, elongation factor 1- alpha (EFl -a) promoter with or without the EFl -a intron. Additional promoters include any constitutively active promoter. Alternatively, any regulatable promoter may be used, such that its expression can be modulated within a cell. In embodiments, a polymerase II promoter is used to drive expression of the nuclease (e.g., a CMV promoter) and a polymerase III promoter (e.g., U6 promoter) is used to drive expression of the gRNA.

[0090] Different promoters and regulatory elements may be used to achieve proper balance (expression level ratio) between the components of the systems (e.g., the nuclease, the at least one gRNA). For example, in some cases a nucleic acid includes a promoters and regulatory elements that is operably linked to (and therefore regulates / modulates translation of) a sequence encoding the nuclease. In some cases, a subject nucleic acid includes a promoters and regulatory elements that is operably linked to a sequence encoding the gRNA. In some cases, the sequence encoding the nuclease and the sequence encoding the gRNA are both operably linked to the same promoters and regulatory elements.

[0091] A variety of promoter types are suitable for use. A promoter can be a constitutively active promoter (e.g., a promoter that is constitutively in an active / ”ON” state), it may be an inducible promoter (e.g., a promoter whose state, active / ”ON” or inactive / ”OFF”, is controlled by an external stimulus, e.g., the presence of a particular temperature, compound, or protein.), it may be a spatially restricted promoter (e.g., tissue specific promoter, cell type specific promoter, etc.), and it may be a temporally restricted promoter (e.g., the promoter is in the “ON” state or “OFF” state during specific stages of embryonic development or during specific stages of a biological process, e.g., hair follicle cycle in mice).BIOTN-41612.601

[0092] Inducible and tissue specific expression of RNA or proteins can be accomplished by placing the nucleic acid encoding such a molecule under the control of an inducible or tissue specific promoter / regulatory sequence. The vectors of the present disclosure may direct expression of the nucleic acid in a particular cell type (e.g., tissue-specific regulatory elements are used to express the nucleic acid). Such regulatory elements include promoters that may be tissue specific or cell specific. The term “tissue specific” as it applies to a promoter refers to a promoter that is capable of directing selective expression of a nucleotide sequence of interest to a specific type of tissue (e.g., seeds) in the relative absence of expression of the same nucleotide sequence of interest in a different type of tissue. The term “cell type specific” as applied to a promoter refers to a promoter that is capable of directing selective expression of a nucleotide sequence of interest in a specific type of cell in the relative absence of expression of the same nucleotide sequence of interest in a different type of cell within the same tissue. The term “cell type specific” when applied to a promoter also means a promoter capable of promoting selective expression of a nucleotide sequence of interest in a region within a single tissue. Cell type specificity of a promoter may be assessed using methods well known in the art, e.g., immunohistochemical staining.

[0093] Examples of tissue specific or inducible promoter / regulatory sequences which are useful for this purpose include, but are not limited to, the rhodopsin promoter, the MMTV LTR inducible promoter, the SV40 late enhancer / promoter, synapsin 1 promoter, ET hepatocyte promoter, GS glutamine synthase promoter and many others. Various commercially available ubiquitous as well as tissue-specific promoters and tumor-specific are available, for example from InvivoGen. In addition, promoters that are well known in the art can be induced in response to inducing agents such as metals, glucocorticoids, tetracycline, hormones, and the like, are also contemplated for use with the invention. Thus, it will be appreciated that the present disclosure includes the use of any promoter / regulatory sequence known in the art that is capable of driving expression of the desired nuclease or RNA operably linked thereto.

[0094] Examples of spatially restricted promoters include, but are not limited to, neuronspecific promoters, adipocyte-specific promoters, cardiomyocyte-specific promoters, smooth muscle-specific promoters, photoreceptor-specific promoters, etc. Neuron-specific spatially restricted promoters include, but are not limited to, a neuron-specific enolase (NSE) promoter (see, e.g., EMBL HSENO2, X51956); an aromatic amino acid decarboxylase (AADC) promoter; a neurofilament promoter (see, e.g., GenBank HUMNFL, L04147); a synapsin promoter (see, e.g.,BIOTN-41612.601GenBank HUMSYNIB, M55301); a thy-1 promoter; a serotonin receptor promoter (see, e.g., GenBank S62283); a tyrosine hydroxylase promoter (TH); a GnRH promoter; an L7 promoter; a DNMT promoter; an enkephalin; a myelin basic protein (MBP) promoter; a Ca2+-calmodulin- dependent protein kinase Il-alpha (CamKIIa) promoter; a CMV enhancer / platelet-derived growth factor-p promoter; and the like. Suitable liver-specific promoters can in some cases include, but are not limited to: TTR, Albumin, and AAT promoters. Suitable CNS-specific promoters can in some cases include, but are not limited to: Synapsin 1, BM88, CHNRB2, GFAP, and CAMK2a promoters. Suitable muscle-specific promoters can in some cases include, but are not limited to: MYODI, MYLK2, SPc5-12 (synthetic), a-MHC, MLC-2, MCK, MHCK7, human cardiac troponin C (cTnC) and desmin promoters. Adipocyte-specific spatially restricted promoters include, but are not limited to, aP2 gene promoter / enhancer, e.g., a region from -5.4 kb to +21 bp of a human aP2; a glucose transporter-4 (GLUT4); a fatty acid translocase (FAT / CD36) promoter; a stearoyl-CoA desaturase- 1 (SCD1) promoter; a leptin promoter; an adiponectin promoter; an adipsin promoter; a resistin promoter; and the like. Cardiomyocyte-specific spatially restricted promoters include, but are not limited to control sequences derived from the following genes: myosin light chain-2, a-myosin heavy chain, AE3, cardiac troponin C, cardiac actin, and the like. Smooth muscle-specific spatially restricted promoters include, but are not limited to, an SM22a promoter; a smoothelin promoter; an a-smooth muscle actin promoter; and the like. For example, a 0.4 kb region of the SM22a promoter, within which lie two CArG elements, has been shown to mediate vascular smooth muscle cell-specific. Photoreceptor-specific spatially restricted promoters include, but are not limited to, a rhodopsin promoter; a rhodopsin kinase promoter; a beta phosphodiesterase gene; a retinitis pigmentosa gene promoter; an interphotoreceptor retinoid- binding protein (IRBP) gene enhancer; an IRBP gene promoter; and the like.

[0095] Examples of inducible promoters include, but are not limited to, heat shock promoter, tetracycline -regulated promoter, steroid-regulated promoter, metal-regulated promoter, estrogen receptor-regulated promoter, etc. Inducible promoters can therefore be regulated by molecules including, but not limited to, doxycycline; an estrogen receptor; an estrogen receptor fusion; an estrogen analog; IPTG; and the like. Inducible promoters suitable for use include any inducible promoter described herein or known to one of ordinary skill in the art. Examples of inducible promoters include, without limitation, chemically / biochemically-regulated and physically- regulated promoters such as alcohol-regulated promoters, tetracycline-regulated promoters (e.g.,BIOTN-41612.601 anhydrotetracycline (aTc)-responsive promoters and other tetracycline -responsive promoter systems, which include a tetracycline repressor protein (tetR), a tetracycline operator sequence (tetO) and a tetracycline transactivator fusion protein (tTA)), steroid-regulated promoters (e.g., promoters based on the rat glucocorticoid receptor, human estrogen receptor, moth ecdysone receptors, and promoters from the steroid / retinoid / thyroid receptor superfamily), metal-regulated promoters (e.g., promoters derived from metallothionein (proteins that bind and sequester metal ions) genes from yeast, mouse and human), pathogenesis-regulated promoters (e.g., induced by salicylic acid, ethylene or benzothiadiazole (BTH)), temperature / heat-inducible promoters (e.g., heat shock promoters), and light-regulated promoters (e.g., light responsive promoters from plant cells).

[0096] Inducible promoters include sugar-inducible promoters (e.g., lactose-inducible promoters; arabinose-inducible promoters); amino acid-inducible promoters; alcohol-inducible promoters; and the like. Suitable promoters include, e.g., lactose-regulated systems (e.g., lactose operon systems, sugar-regulated systems, isopropyl-beta-D-thiogalactopyranoside (IPTG) inducible systems, arabinose regulated systems (e.g., arabinose operon systems, e.g., an ARA operon promoter, pBAD, pARA, portions thereof, combinations thereof and the like), synthetic amino acid regulated systems, fructose repressors, a tac promoter / operator (pTac), tryptophan promoters, PhoA promoters, recA promoters, proU promoters, cst-1 promoters, tetA promoters, cadA promoters, nar promoters, PL promoters, cspA promoters, and the like, or combinations thereof. In certain cases, a promoter comprises a Lac-Z, or portions thereof. In some cases, a promoter comprises a Lac operon, or portions thereof. In some cases, an inducible promoter comprises an ARA operon promoter, or portions thereof. In certain embodiments an inducible promoter comprises an arabinose promoter or portions thereof. An arabinose promoter can be obtained from any suitable bacteria. In some cases, an inducible promoter comprises an arabinose operon of E. coli or B. subtilis. In some cases, an inducible promoter is activated by the presence of a sugar or an analog thereof. Non-limiting examples of sugars and sugar analogs include lactose, arabinose (e.g., L-arabinose), glucose, sucrose, fructose, IPTG, and the like. Suitable promoters include a T7 promoter; a pBAD promoter; a lacIQ promoter; and the like. In some cases, the promoter is a J23119 promoter. Many bacterial promoters are known in the art; bacterial promoters can be found on the internet at parts(dot)igem(dot)org / promoters.BIOTN-41612.601

[0097] In some cases, the promoter is a reversible promoter. Suitable reversible promoters, including reversible inducible promoters are known in the art. Such reversible promoters may be isolated and derived from many organisms. Such reversible promoters may be isolated and derived from many organisms, e.g., eukaryotes and prokaryotes. Modification of reversible promoters derived from a first organism for use in a second organism is well known in the art. Modification of reversible promoters derived from a first organism for use in a second organism, e.g., a first prokaryote and a second a eukaryote, a first eukaryote and a second a prokaryote, etc., is well known in the art. Such reversible promoters, and systems based on such reversible promoters but also comprising additional control proteins, include, but are not limited to, alcohol regulated promoters (e.g., alcohol dehydrogenase I (alcA) gene promoter, promoters responsive to alcohol transactivator proteins (AlcR)), tetracycline regulated promoters, (e.g., promoter systems including Tet Activators, TetON, TetOFF), steroid regulated promoters (e.g., rat glucocorticoid receptor promoter systems, human estrogen receptor promoter systems, retinoid promoter systems, thyroid promoter systems, ecdysone promoter systems, mifepristone promoter systems), metal regulated promoters (e.g., metallothionein promoter systems), pathogenesis-related regulated promoters (e.g., salicylic acid regulated promoters, ethylene regulated promoters, benzothiadiazole regulated promoters), temperature regulated promoters (e.g., heat shock inducible promoters (e.g., HSP-70, HSP-90, soybean heat shock promoter), light regulated promoters, synthetic inducible promoters, and the like.

[0098] The present composition and system (e.g., proteins, polynucleotides encoding these proteins, or compositions comprising the proteins and / or polynucleotides described herein) may be delivered by any suitable means. In certain embodiments, the composition or system is delivered in vivo. In other embodiments, the composition or system is delivered to isolated / cultured cells (e.g., autologous iPS cells) in vitro to provide modified cells useful for in vivo delivery to patients afflicted with a disease or condition.

[0099] Vectors according to the present disclosure can be transformed, transfected, or otherwise introduced into a wide variety of host cells. Transfection refers to the taking up of a vector by a host cell whether or not any coding sequences are in fact expressed. Numerous methods of transfection are known to the ordinarily skilled artisan, for example, lipofectamine, calcium phosphate co-precipitation, electroporation, DEAE-dextran treatment, microinjection, viral infection, and other methods known in the art. Transduction refers to entry of a virus into the cellBIOTN-41612.601 and expression (e.g., transcription and / or translation) of sequences delivered by the viral vector genome. In the case of a recombinant vector, “transduction” generally refers to entry of the recombinant viral vector into the cell and expression of a nucleic acid of interest delivered by the vector genome.

[0100] Any of the vectors comprising a nucleic acid sequence that encodes the components of the present compositions and system is also within the scope of the present disclosure. Such a vector may be delivered into host cells by a suitable method. Methods of delivering vectors to cells are well known in the art and may include DNA or RNA electroporation, transfection reagents such as liposomes or nanoparticles to delivery DNA or RNA; delivery of DNA, RNA, or protein by mechanical deformation, or viral transduction. In some embodiments, the vectors are delivered to host cells by viral transduction. Nucleic acids can be delivered as part of a larger construct, such as a plasmid or viral vector, or directly, e.g., by electroporation, lipid vesicles, viral transporters, microinjection, and biolistics (high-speed particle bombardment). Similarly, the construct containing the one or more transgenes can be delivered by any method appropriate for introducing nucleic acids into a cell. Additionally, delivery vehicles such as nanoparticle- and lipid-based mRNA or protein delivery systems can be used. Further examples of delivery vehicles include lentiviral vectors, ribonucleoprotein (RNP) complexes, lipid-based delivery system, gene gun, hydrodynamic, electroporation or nucleofection microinjection, biolistics, and the like.

[0101] In some embodiments, the vector is a viral construct, e.g., a recombinant adeno- associated virus construct, a recombinant adenoviral construct, a recombinant lentiviral construct, a recombinant retroviral construct, etc. Suitable viral vectors include, but are not limited to, viral vectors based on vaccinia virus; poliovirus; adenovirus; adeno-associated virus; SV40; herpes simplex virus; human immunodeficiency virus; a retroviral vector (e.g., Murine Leukemia Virus, spleen necrosis virus, and vectors derived from retroviruses such as Rous Sarcoma Virus, Harvey Sarcoma Virus, avian leukosis virus, a lentivirus, human immunodeficiency virus, myeloproliferative sarcoma virus, and mammary tumor virus); and the like.

[0102] In some embodiments, the vector is an AAV vector. By adeno-associated virus, or “AAV” it is meant the virus itself or derivatives thereof. The term covers all subtypes and both naturally occurring and recombinant forms, except where required otherwise, for example, AAV type 1 (AAV-1), AAV type 2 (AAV-2), AAV type 3 (AAV-3), AAV type 4 (AAV-4), AAV type 5 (AAV-5), AAV type 6 (AAV-6), AAV type 7 (AAV-7), AAV type 8 (AAV-8), AAV type 9BIOTN-41612.601(AAV-9), AAV type 10 (AAV-10), AAV type 11 (AAV-11), avian AAV, bovine AAV, canine AAV, equine AAV, primate AAV, non-primate AAV, ovine AAV, a hybrid AAV (i.e., an AAV comprising a capsid protein of one AAV subtype and genomic material of another subtype), an AAV comprising a mutant AAV capsid protein or a chimeric AAV capsid (i.e. a capsid protein with regions or domains or individual amino acids that are derived from two or more different serotypes of AAV, e.g. AAV-DJ, AAV-LK3, AAV-LK19). “Primate AAV” refers to AAV that infect primates, “non-primate AAV” refers to AAV that infect non-primate mammals, “bovine AAV” refers to AAV that infect bovine mammals, etc.

[0103] By a “recombinant AAV vector” or “rAAV vector” it is meant an AAV virus or AAV viral chromosomal material comprising a polynucleotide sequence not of AAV origin (e.g., a polynucleotide heterologous to AAV), typically a nucleic acid sequence of interest to be integrated into the cell following the subject methods. In general, the heterologous polynucleotide is flanked by at least one, and generally by two AAV inverted terminal repeat sequences (ITRs). In some instances, the recombinant viral vector also comprises viral genes important for the packaging of the recombinant viral vector material. Packaging refers to the series of intracellular events that result in the assembly and encapsulation of a viral particle, e.g., an AAV viral particle. Examples of nucleic acid sequences important for AAV packaging include the AAV “rep” and “cap” genes, which encode for replication and encapsulation proteins of adeno-associated virus, respectively. The term rAAV vector encompasses both rAAV vector particles and rAAV vector plasmids.

[0104] A “viral particle” refers to a single unit of virus comprising a capsid encapsulating a virus-based polynucleotide, e.g., the viral genome (as in a wild-type virus), or, e.g., the subject targeting vector (as in a recombinant virus). An AAV viral particle refers to a viral particle composed of at least one AAV capsid protein (typically by all of the capsid proteins of a wild-type AAV) and an encapsulated polynucleotide AAV vector. If the particle comprises a heterologous polynucleotide (e.g., a polynucleotide other than a wild-type AAV genome, such as a transgene to be delivered to a mammalian cell), it is typically referred to as an “rAAV vector particle” or simply an “rAAV vector.” Thus, production of rAAV particle necessarily includes production of rAAV vector, as such a vector is contained within an rAAV particle.

[0105] An rAAV virion can be constructed by a variety of methods. For example, the heterologous sequence(s) can be directly inserted into an AAV genome which has had the major AAV open reading frames (“ORFs”) excised therefrom. Other portions of the AAV genome canBIOTN-41612.601 also be deleted, so long as a sufficient portion of the ITRs remain to allow for replication and packaging functions. In order to produce rAAV virions, an AAV expression vector can be introduced into a suitable host cell using known techniques, such as by transfection. Particularly suitable transfection methods include calcium phosphate co-, direct micro-injection into cultured cells, electroporation, liposome mediated gene transfer, lipid-mediated transduction, and nucleic acid delivery using high-velocity microprojectiles. Suitable cells for producing rAAV virions include microorganisms, yeast cells, insect cells, and mammalian cells, that can be, or have been, used as recipients of a heterologous DNA molecule.

[0106] An AAV virus that is produced may be replication competent or replicationincompetent. A “replication-competent” virus (e.g., a replication-competent AAV) refers to a phenotypically wild-type virus that is infectious and is also capable of being replicated in an infected cell (e.g., in the presence of a helper virus or helper virus functions). In the case of AAV, replication competence generally requires the presence of functional AAV packaging genes. In general, rAAV vectors as described herein are replication-incompetent in mammalian cells (especially in human cells) by virtue of the lack of one or more AAV packaging genes. Typically, such rAAV vectors lack any AAV packaging gene sequences in order to minimize the possibility that replication competent AAV are generated by recombination between AAV packaging genes and an incoming rAAV vector.

[0107] Retroviruses, for example, lentiviruses, are suitable for use in methods of the present disclosure. Commonly used retroviral vectors are unable to produce viral proteins required for productive infection. Rather, replication of the vector requires growth in a packaging cell line. To generate viral particles comprising nucleic acids of interest, the retroviral nucleic acids comprising the nucleic acid are packaged into viral capsids by a packaging cell line. Different packaging cell lines provide a different envelope protein (ecotropic, amphotropic or xenotropic) to be incorporated into the capsid, this envelope protein determining the specificity of the viral particle for the cells (ecotropic for murine and rat; amphotropic for most mammalian cell types including human, dog, and mouse; and xenotropic for most mammalian cell types except murine cells). The appropriate packaging cell line may be used to ensure that the cells are targeted by the packaged viral particles. Methods of introducing subject vector expression vectors into packaging cell lines and of collecting the viral particles that are generated by the packaging lines are well known in the art. Nucleic acids can also introduced by direct micro-injection (e.g., injection of RNA).BIOTN-41612.601

[0108] As noted elsewhere herein, proteins may instead be provided to cells as RNA (e.g., an RNA comprising the translational control element as discussed elsewhere herein). Methods of introducing RNA into cells may include, for example, direct injection, transfection, or any other method used for the introduction of DNA. The nuclease may also be introduced into a host cell directly as protein. In such instances, the nuclease may be delivered as an RNP (ribonucleoprotein complex) in which it is already complexed with an appropriate guide RNA.

[0109] The disclosed nucleic acids (e.g., vectors) and proteins can be delivered to cells using any convenient method. Suitable methods include, e.g., viral infection (e.g., AAV, adenovirus, lentiviral), transfection, conjugation, protoplast fusion, lipofection, electroporation, calcium phosphate precipitation, polyethyleneimine (PEI)-mediated transfection, DEAE-dextran mediated transfection, liposome-mediated transfection, particle gun technology, calcium phosphate precipitation, direct micro injection, nanoparticle-mediated nucleic acid delivery, and the like.

[0110] In some cases, a nuclease is delivered to a cell in a particle, or associated with a particle. In some cases, a nuclease is delivered with a cationic lipid and a hydrophilic polymer, for instance wherein the cationic lipid comprises l,2-dioleoyl-3-trimethylammonium-propane (DOTAP) or l,2-ditetradecanoyl-sn-glycero-3 -phosphocholine (DMPC) and / or wherein the hydrophilic polymer comprises ethylene glycol or polyethylene glycol (PEG); and / or wherein the particle further comprises cholesterol.

[0111] A nuclease may be delivered using particles or lipid envelopes. For example, a biodegradable core-shell structured nanoparticle with a poly (p-amino ester) (PBAE) core enveloped by a phospholipid bilayer shell can be used. In some cases, particles / nanoparticles based on self-assembling bioadhesive polymers are used; such particles / nanoparticles may be applied to oral delivery of peptides, intravenous delivery of peptides and nasal delivery of peptides, e.g., to the brain. Other embodiments, such as oral absorption and ocular delivery of hydrophobic drugs are also contemplated. A molecular envelope technology, which involves an engineered polymer envelope which is protected and delivered to the desired cell, can be used.

[0112] Lipidoid compounds (e.g., as described in U.S. Patent Application Publication No. 2011 / 0293703) are also useful in the delivery of polynucleotides, and can be used to deliver the disclosed nucleases (or RNA or DNA encoding thereof). In one aspect, the aminoalcohol lipidoid compounds are combined with an agent to be delivered to a cell to form microparticles, nanoparticles, liposomes, or micelles. The aminoalcohol lipidoid compounds may be combinedBIOTN-41612.601 with other aminoalcohol lipidoid compounds, polymers (synthetic or natural), surfactants, cholesterol, carbohydrates, proteins, lipids, etc. to form the particles. These particles may then optionally be combined with a pharmaceutical excipient to form a pharmaceutical composition.

[0113] A poly(beta-amino alcohol) (PBAA) can be used to deliver a nuclease, or a nucleic acid encoding thereof, and gRNA, or a nucleic acid encoding thereof, to a target cell. U.S. Patent Application Publication No. 2013 / 0302401 relates to a class of poly(beta-amino alcohols) (PBAAs) that has been prepared using combinatorial polymerization.

[0114] Sugar-based particles, for example GalNAc, as described in International Patent Publication No. WO2014118272 (incorporated herein by reference in its entirety and Nair, J K et al., 2014, Journal of the American Chemical Society 136 (49), 16958-16961) can be used to deliver a nuclease, or a nucleic acid encoding thereof, and gRNA, or a nucleic acid encoding thereof, to a target cell.

[0115] In some cases, lipid nanoparticles (LNPs) are used to deliver a nuclease, or a nucleic acid encoding thereof, and gRNA, or a nucleic acid encoding thereof, to a target cell. Negatively charged polymers such as RNA may be loaded into LNPs at low pH values (e.g., pH 4) where the ionizable lipids display a positive charge. However, at physiological pH values, the LNPs exhibit a low surface charge compatible with longer circulation times. Four species of ionizable cationic lipids have been focused upon, namely l,2-dilineoyl-3-dimethylammonium-propane (DLinDAP), 1 ,2-dilinoleyloxy-3-N,N-dimethylaminopropane (DLinDMA), 1 ,2-dilinoleyloxy-keto-N,N- dimethyl-3 -aminopropane (DLinKDMA), and l,2-dilinoleyl-4-(2-dimethylaminoethyl)-[l,3]- dioxolane (DLinKC2-DMA). Preparation of LNPs and is described in, e.g., Rosin et al. (2011) Molecular Therapy 19: 1286-2200). The cationic lipids l,2-dilineoyl-3-dimethylammonium- propane (DLinDAP), l,2-dilinoleyloxy-3-N,N-dimethylaminopropane (DLinDMA), 1,2- dilinoleyloxyketo-N,N-dimethyl-3-aminopropane (DLinK-DMA), 1 ,2-dilinoleyl-4-(2- dimethylaminoethyl)-[ 1,3] -di oxolane (DLinKC2-DMA), (3-o-[2"-(methoxypolyethyleneglycol 2000) succinoyl]-l,2-dimyristoyl-sn-glycol (PEG-S-DMG), and R-3-[(.omega. -methoxy- polyethylene glycol)2000) carbamoyl]- l,2-dimyristyloxlpropyl-3-amine (PEG-C-DOMG) may be used. A nucleic acid may be encapsulated in LNPs containing DLinDAP, DLinDMA, DLinKDMA, and DLinKC2-DMA (cationic lipid:DSPC:CHOL: PEGS-DMG or PEG-C-DOMG at 40: 10:40:10 molar ratios). In some cases, 0.2% SP-DiOC18 is incorporated.BIOTN-41612.601

[0116] Spherical Nucleic Acid (SNA™) constructs and other nanoparticles (particularly gold nanoparticles) can be used to deliver a nuclease, or a nucleic acid encoding thereof, and gRNA, or a nucleic acid encoding thereof, to a target cell.

[0117] Self-assembling nanoparticles with RNA may be constructed with polyethyleneimine (PEI) that is PEGylated with an Arg-Gly-Asp (RGD) peptide ligand attached at the distal end of the polyethylene glycol (PEG).

[0118] Nanoparticles suitable for use in delivering a nuclease, or a nucleic acid encoding thereof, and gRNA, or a nucleic acid encoding thereof, to a target cell may be provided in different forms, e.g., as solid nanoparticles (e.g., metal such as silver, gold, iron, titanium), non-metal, lipid- based solids, polymers), suspensions of nanoparticles, or combinations thereof. Metal, dielectric, and semiconductor nanoparticles may be prepared, as well as hybrid structures (e.g., core-shell nanoparticles). Nanoparticles made of semiconducting material may also be labeled quantum dots if they are small enough (typically below 10 nm) that quantization of electronic energy levels occurs. Such nanoscale particles are used in biomedical applications as drug carriers or imaging agents and may be adapted for similar purposes in the present disclosure. In general, a “nanoparticle” refers to any particle having a diameter of less than 1000 nm. In some cases, nanoparticles suitable for use in delivering a nuclease or nucleic acid to a target cell have a diameter of 500 nm or less, e.g., from 25 nm to 35 nm, from 35 nm to 50 nm, from 50 nm to 75 nm, from 75 nm to 100 nm, from 100 nm to 150 nm, from 150 nm to 200 nm, from 200 nm to 300 nm, from 300 nm to 400 nm, or from 400 nm to 500 nm. In some cases, nanoparticles suitable for use in delivering a nuclease or nucleic acid to a target cell have a diameter of from 25 nm to 200 nm.

[0119] In some cases, an exosome is used to deliver a nuclease, or a nucleic acid encoding thereof, and gRNA, or a nucleic acid encoding thereof, to a target cell. Exosomes are endogenous nano-vesicles that transport RNAs and proteins, and which can deliver RNA to the brain and other target organs.

[0120] In some cases, a liposome is used to deliver a nuclease, or a nucleic acid encoding thereof, and gRNA, or a nucleic acid encoding thereof, to a target cell. Liposomes are spherical vesicle structures composed of a uni- or multi-lamellar lipid bilayer surrounding internal aqueous compartments and a relatively impermeable outer lipophilic phospholipid bilayer. Liposomes can be made from several different types of lipids; however, phospholipids are most commonly usedBIOTN-41612.601 to generate liposomes. Although liposome formation is spontaneous when a lipid film is mixed with an aqueous solution, it can also be expedited by applying force in the form of shaking by using a homogenizer, sonicator, or an extrusion apparatus. Several other additives may be added to liposomes in order to modify their structure and properties. For instance, either cholesterol or sphingomyelin may be added to the liposomal mixture in order to help stabilize the liposomal structure and to prevent the leakage of the liposomal inner cargo. A liposome formulation may be mainly comprised of natural phospholipids and lipids such as l,2-distearoryl-sn-glycero-3- phosphatidyl choline (DSPC), sphingomyelin, egg phosphatidylcholines and monosialoganglioside.

[0121] A stable nucleic-acid-lipid particle (SNALP) can be used to deliver a nuclease, or a nucleic acid encoding thereof, and gRNA, or a nucleic acid encoding thereof, to a target cell. The SNALP formulation may contain the lipids 3-N-[(methoxypoly(ethylene glycol) 2000) carbamoyl] -1,2-dimyristyloxy-propylamine (PEG-C-DMA), l,2-dilinoleyloxy-N,N-dimethyl-3- aminopropane (DLinDMA), 1 ,2-distearoyl-sn-glycero-3 -phosphocholine (DSPC) and cholesterol, in a 2:40:10:48 molar percent ratio. The SNALP liposomes may be prepared by formulating D- Lin-DMA and PEG-C-DMA with distearoylphosphatidylcholine (DSPC), Cholesterol and siRNA using a 25:1 lipid / siRNA ratio and a 48 / 40 / 10 / 2 molar ratio of Cholesterol / D-Lin- DMA / DSPC / PEG-C-DMA. The resulting SNALP liposomes can be about 80-100 nm in size. A SNALP may comprise synthetic cholesterol (Sigma-Aldrich, St Louis, Mo., USA), dipalmitoylphosphatidylcholine (Avanti Polar Lipids, Alabaster, Ala., USA), 3-N-[(w-methoxy poly(ethylene glycol)2000)carbamoyl]-l,2-dimyrestyloxypropylamine, and cationic 1,2- dilinoleyloxy-3-N,Ndimethylaminopropane. A SNALP may comprise synthetic cholesterol (Sigma-Aldrich), 1 ,2-distearoyl-sn-glycero-3 -phosphocholine (DSPC; Avanti Polar Lipids Inc.), PEG-cDMA, and l,2-dilinoleyloxy-3-(N;N-dimethyl)aminopropane (DLinDMA).

[0122] Other cationic lipids, such as amino lipid 2,2-dilinoleyl-4-dimethylaminoethyl-[l,3]- dioxolane (DLin-KC2-DMA) can be used to deliver a nuclease or nucleic acid to a target cell. A preformed vesicle with the following lipid composition may be contemplated: amino lipid, distearoylphosphatidylcholine (DSPC), cholesterol and (R)-2,3-bis(octadecyloxy) propyl-1- (methoxy poly( ethylene glycol)2000)propylcarbamate (PEG-lipid) in the molar ratio 40 / 10 / 40 / 10, respectively, and a FV11 siRNA / total lipid ratio of approximately 0.05 (w / w). To ensure a narrow particle size distribution in the range of 70-90 nm and a low polydispersity index of 0.11.+-.0.04BIOTN-41612.601(n=56), the particles may be extruded up to three times through 80 nm membranes prior to adding the guide RNA. Particles containing the highly potent amino lipid 16 may be used, in which the molar ratio of the four lipid components 16, DSPC, cholesterol and PEG-lipid (50 / 10 / 38.5 / 1.5), may be further optimized to enhance in vivo activity.

[0123] Lipids may be formulated with a nuclease, or a nucleic acid encoding thereof, and gRNA, or a nucleic acid encoding thereof, to form lipid nanoparticles (LNPs). Suitable lipids include, but are not limited to, DLin-KC2-DMA4, Cl 2-200 and colipids disteroylphosphatidyl choline, cholesterol, and PEG-DMG may be formulated with a nuclease or nucleic acid using a spontaneous vesicle formation procedure.

[0124] A nuclease, or a nucleic acid encoding thereof, and gRNA, or a nucleic acid encoding thereof, may be delivered encapsulated in PLGA microspheres such as those further described in US published applications 20130252281, 20130245107, and 20130244279.

[0125] Supercharged proteins can be used to deliver a nuclease, or a nucleic acid encoding thereof, and gRNA, or a nucleic acid encoding thereof, to a target cell. Supercharged proteins are a class of engineered or naturally occurring proteins with unusually high positive or negative net theoretical charge. Both supemegatively and superpositively charged proteins exhibit the ability to withstand thermally or chemically induced aggregation. Superpositively charged proteins are also able to penetrate mammalian cells. Associating cargo with these proteins, such as plasmid DNA, RNA, or other proteins, can facilitate the functional delivery of these macromolecules into mammalian cells both in vitro and in vivo.

[0126] Cell Penetrating Peptides (CPPs) can be used to deliver a nuclease, or a nucleic acid encoding thereof, and gRNA, or a nucleic acid encoding thereof, to a target cell. CPPs typically have an amino acid composition that either contains a high relative abundance of positively charged amino acids such as lysine or arginine or has sequences that contain an alternating pattern of polar / charged amino acids and non-polar, hydrophobic amino acids.Methods

[0127] The disclosure also provides methods of modifying a target nucleic acid sequence (e.g., DNA or RNA). The phrase “modifying a nucleic acid sequence,” as used herein, refers to modifying at least one physical feature of a nucleic acid sequence of interest. Nucleic acid modifications include, for example, single or double strand breaks, deletion, or insertion of one or more nucleotides, and other modifications that affect the structural integrity or nucleotide sequenceBIOTN-41612.601 of the nucleic acid sequence. The modifications may comprise one or more of nucleic acid binding, base editing, transcription modulation, nucleic acid modification, protein modification, and histone modification.

[0128] The methods comprise contacting a target nucleic acid sequence with a composition as disclosed herein, a system disclosed herein or a composition comprising the system.

[0129] In one embodiment, the method introduces a single strand or double strand break in the target nucleic acid sequence. In this respect, the disclosed systems may direct cleavage of one or both strands of a target DNA sequence, such as within the target genomic DNA sequence and / or within the complement of the target sequence.

[0130] In some embodiments, modifying a DNA sequence comprises a deletion. The deletion may be upstream or downstream of the PAM binding side, so called unidirectional deletions. The deletion may encompass sequences on either side of the PAM binding site, a bidirectional deletion. The deletion of the DNA sequence may be of any size.

[0131] In some embodiments, contacting a target nucleic acid sequence comprises introducing the composition or system into the cell. As described above the composition or system may be introduced into eukaryotic or prokaryotic cells by methods known in the art.

[0132] The cell may be a prokaryotic cell, a plant cell, an insect cell, a vertebrate cell, an invertebrate cell, an animal cell, a mammalian cell, or a human cell. In some embodiments, the cell is a plant cell. In some embodiments, the cell is an insect cell. In some embodiments, the cell is a vertebrate cell. In some embodiments, the cell is an invertebrate cell. In some embodiments, the cell is a mammalian cell. In some embodiments, the cell is a human cell. In some cases, the cell is ex vivo (e.g., fresh isolate - early passage). In some cases, the cell is in vivo. In some cases, the cell is in culture in vitro (e.g., immortalized cell line).

[0133] Cells may be from established cell lines or they may be primary cells, where “primary cells,” “primary cell lines,” and “primary cultures” are used interchangeably herein to refer to cells and cells cultures that have been derived from a subject and allowed to grow in vitro for a limited number of passages of the culture. For example, primary cultures are cultures that may have been passaged 0 times, 1 time, 2 times, 4 times, 5 times, 10 times, or 15 times, but not enough times go through the crisis stage. Typically, the primary cell lines are maintained for fewer than 10 passages in culture.BIOTN-41612.601

[0134] Suitable cells include, but are not limited to: bacterial cell; an archaeal cell; a eukaryotic cell; a cell of a single-cell eukaryotic organism; a plant cell; a protozoa cell; an algal cell, e.g., Botryococcus braunii, Chlamydomonas reinhardtii, Nannochloropsis gaditana, Chlorella pyrenoidosa, Sargassum patens, C. agardh, and the like; a fungal cell (e.g., a yeast cell); an animal cell; a cell from an invertebrate animal (e.g. fruit fly, a cnidarian, an echinoderm, a nematode, etc.); a cell of an insect (e.g., a mosquito; a bee; an agricultural pest; etc.); a cell of an arachnid (e.g., a spider; a tick; etc.); a cell of a vertebrate animal (e.g., a fish, an amphibian, a reptile, a bird, a mammal); a cell of a mammal (e.g., a cell of a rodent; a cell of a human; a cell of a non-human mammal; a cell of a rodent (e.g., a mouse, a rat); a cell of a lagomorph (e.g., a rabbit); a cell of an ungulate (e.g., a cow, a horse, a camel, a llama, a vicuna, a sheep, a goat, etc.); a cell of a marine mammal (e.g., a whale, a seal, an elephant seal, a dolphin, a sea lion; etc.) and the like. Any type of cell may be of interest (e.g. a stem cell, e.g. an embryonic stem (ES) cell, an induced pluripotent stem (iPS) cell, a germ cell (e.g., an oocyte, a sperm, an oogonia, a spermatogonia, etc.), an adult stem cell, a somatic cell, e.g. a fibroblast, a hematopoietic cell, a neuron, a muscle cell, a bone cell, a hepatocyte, a pancreatic cell; an in vitro or in vivo embryonic cell of an embryo at any stage, e.g., a 1-cell, 2-cell, 4-cell, 8-cell, etc. stage zebrafish embryo; etc.). In some cases, the cell is a cell that does not originate from a natural organism (e.g., the cell can be a synthetically made cell; also referred to as an artificial cell).

[0135] Non-limiting examples of plant cell include cells from: plant crops, fruits, vegetables, grains, soybean, com, maize, wheat, seeds, tomatoes, rice, cassava, sugarcane, pumpkin, hay, potatoes, cotton, cannabis, tobacco, flowering plants, conifers, gymnosperms, angiosperms, ferns, clubmosses, homworts, liverworts, mosses, dicotyledons, monocotyledons, seaweeds (e.g., kelp), and the like.

[0136] Suitable cells include a stem cell (e.g., an embryonic stem (ES) cell, an induced pluripotent stem (iPS) cell; a germ cell (e.g., an oocyte, a sperm, an oogonia, a spermatogonia, etc.); a somatic cell, e.g., a fibroblast, an oligodendrocyte, a glial cell, a hematopoietic cell, a neuron, a muscle cell, a bone cell, a hepatocyte, a pancreatic cell, etc.

[0137] Suitable cells include human embryonic stem cells, fetal cardiomyocytes, myofibroblasts, mesenchymal stem cells, autotransplated expanded cardiomyocytes, adipocytes, totipotent cells, pluripotent cells, blood stem cells, myoblasts, adult stem cells, bone marrow cells, mesenchymal cells, embryonic stem cells, parenchymal cells, epithelial cells, endothelial cells,BIOTN-41612.601 mesothelial cells, fibroblasts, osteoblasts, chondrocytes, exogenous cells, endogenous cells, stem cells, hematopoietic stem cells, bone-marrow derived progenitor cells, myocardial cells, skeletal cells, fetal cells, undifferentiated cells, multi-potent progenitor cells, unipotent progenitor cells, monocytes, cardiac myoblasts, skeletal myoblasts, macrophages, capillary endothelial cells, xenogenic cells, allogenic cells, and post-natal stem cells.

[0138] In some cases, the cell is an immune cell, a neuron, an epithelial cell, and endothelial cell, or a stem cell. In some cases, the immune cell is a T cell, a B cell, a monocyte, a natural killer cell, a dendritic cell, or a macrophage. In some cases, the immune cell is a cytotoxic T cell. In some cases, the immune cell is a helper T cell. In some cases, the immune cell is a regulatory T cell (Treg).

[0139] In some cases, the cell is a stem cell. Stem cells include adult stem cells. Adult stem cells are also referred to as somatic stem cells.

[0140] Adult stem cells are resident in differentiated tissue but retain the properties of selfrenewal and ability to give rise to multiple cell types, usually cell types typical of the tissue in which the stem cells are found. Numerous examples of somatic stem cells are known to those of skill in the art, including muscle stem cells; hematopoietic stem cells; epithelial stem cells; neural stem cells; mesenchymal stem cells; mammary stem cells; intestinal stem cells; mesodermal stem cells; endothelial stem cells; olfactory stem cells; neural crest stem cells; and the like.

[0141] Stem cells of interest include mammalian stem cells, where the term “mammalian” refers to any animal classified as a mammal, including humans; non-human primates; domestic and farm animals; and zoo, laboratory, sports, or pet animals, such as dogs, horses, cats, cows, mice, rats, rabbits, etc. In some cases, the stem cell is a human stem cell. In some cases, the stem cell is a rodent (e.g., a mouse; a rat) stem cell. In some cases, the stem cell is a non-human primate stem cell.

[0142] In some embodiments, the stem cell is a hematopoietic stem cell (HSC). HSCs are mesoderm-derived cells that can be isolated from bone marrow, blood, cord blood, fetal liver, and yolk sac. HSCs are characterized as CD34+and CD3“. HSCs can repopulate the erythroid, neutrophil-macrophage, megakaryocyte, and lymphoid hematopoietic cell lineages in vivo. In vitro, HSCs can be induced to undergo at least some self-renewing cell divisions and can be induced to differentiate to the same lineages as is seen in vivo. As such, HSCs can be induced toBIOTN-41612.601 differentiate into one or more of erythroid cells, megakaryocytes, neutrophils, macrophages, and lymphoid cells.

[0143] In other embodiments, the stem cell is a neural stem cell (NSC). Neural stem cells (NSCs) are capable of differentiating into neurons, and glia (including oligodendrocytes, and astrocytes). A neural stem cell is a multipotent stem cell which is capable of multiple divisions, and under specific conditions can produce daughter cells which are neural stem cells, or neural progenitor cells that can be neuroblasts or glioblasts, e.g., cells committed to become one or more types of neurons and glial cells, respectively. Methods of obtaining NSCs are known in the art.

[0144] In other embodiments, the stem cell is a mesenchymal stem cell (MSC). MSCs originally derived from the embryonal mesoderm and isolated from adult bone marrow, can differentiate to form muscle, bone, cartilage, fat, marrow stroma, and tendon. Methods of isolating MSC are known in the art; and any known method can be used to obtain MSC. See, e.g., U.S. Pat. No. 5,736,396, which describes isolation of human MSC.

[0145] In some embodiments, the cell is a T cell. The invention is not limited by the type of T cell. The T cells may be selected from, for example, CD3+ T cells, CD8+ T cells, CD4+ T cells, natural killer (NK) T cells, alpha beta T cells, gamma delta T cells, or any combination thereof (e.g., a combination of CD4+ and CD8+ T cells).

[0146] In some embodiments, the T cells are naturally occurring T cells. For example, the T cells may be isolated from a subject sample. In some embodiments, the T cell is an anti-tumor T cell (e.g., a T cell with activity against a tumor (e.g., an autologous tumor) that becomes activated and expands in response to antigen). Anti-tumor T cells include, but are not limited to, T cells obtained from resected tumors or tumor biopsies (e.g., tumor infiltrating lymphocytes (TILs)) and a polyclonal or monoclonal tumor-reactive T cell (e.g., obtained by apheresis, expanded ex vivo against tumor antigens presented by autologous or artificial antigen-presenting cells). In some embodiments, the T cells are expanded ex vivo.

[0147] A cell is in some cases a plant cell. A plant cell can be a cell of a monocotyledon. A plant cell can be a cell of a dicotyledon. The cells can be root cells, leaf cells, cells of the xylem, cells of the phloem, cells of the cambium, apical meristem cells, parenchyma cells, collenchyma cells, sclerenchyma cells, and the like. Plant cells include cells of agricultural crops such as wheat, com, rice, sorghum, millet, soybean, etc. Plant cells include cells of agricultural fruit and nut plants, e.g., plants that produce apricots, oranges, lemons, apples, plums, pears, almonds, etc.BIOTN-41612.601

[0148] A plant cell can be a cell of a major agricultural plant, e.g., Barley, Beans (Dry Edible), Canola, Com, Cotton (Pima), Cotton (Upland), Flaxseed, Hay (Alfalfa), Hay (Non-Alfalfa), Oats, Peanuts, Rice, Sorghum, Soybeans, Sugarbeets, Sugarcane, Sunflowers (Oil), Sunflowers (NonOil), Sweet Potatoes , Tobacco (Burley), Tobacco (Flue-cured), Tomatoes, Wheat (Durum), Wheat (Spring), Wheat (Winter), and the like. As another example, the cell is a cell of a vegetable crops which include but are not limited to, e.g., alfalfa sprouts, aloe leaves, arrow root, arrowhead, artichokes, asparagus, bamboo shoots, banana flowers, bean sprouts, beans, beet tops, beets, bittermelon, bok choy, broccoli, broccoli rabe (rappini), brussels sprouts, cabbage, cabbage sprouts, cactus leaf (nopales), calabaza, cardoon, carrots, cauliflower, celery, chayote, Chinese artichoke (crosnes), Chinese cabbage, Chinese celery, Chinese chives, choy sum, chrysanthemum leaves (tung ho), collard greens, com stalks, com-sweet, cucumbers, daikon, dandelion greens, dasheen, dau mue (pea tips), donqua (winter melon), eggplant, endive, escarole, fiddle head fems, field cress, frisee, gai choy (Chinese mustard), gailon, galanga (siam, thai ginger), garlic, ginger root, gobo, greens, hanover salad greens, huauzontle, jemsalem artichokes, jicama, kale greens, kohlrabi, lamb's quarters (quilete), lettuce (bibb), lettuce (boston), lettuce (boston red), lettuce (green leaf), lettuce (iceberg), lettuce (lolla rossa), lettuce (oak leaf - green), lettuce (oak leaf - red), lettuce (processed), lettuce (red leaf), lettuce (romaine), lettuce (ruby romaine), lettuce (mssian red mustard), linkok, lo bok, long beans, lotus root, mache, maguey (agave) leaves, malanga, mesculin mix, mizuna, moap (smooth luffa), moo, moqua (fuzzy squash), mushrooms, mustard, nagaimo, okra, ong choy, onions green, opo (long squash), ornamental com, ornamental gourds, parsley, parsnips, peas, peppers (bell type), peppers, pumpkins, radicchio, radish sprouts, radishes, rape greens, rape greens, rhubarb, romaine (baby red), mtabagas, salicomia (sea bean), sinqua (angled / ridged luffa), spinach, squash, straw bales, sugarcane, sweet potatoes, swiss chard, tamarindo, taro, taro leaf, taro shoots, tatsoi, tepeguaje (guaje), tindora, tomatillos, tomatoes, tomatoes (cherry), tomatoes (grape type), tomatoes (plum type), tumeric, turnip tops greens, turnips, water chestnuts, yampi, yams (names), yu choy, yuca (cassava), and the like.

[0149] A cell is in some cases an arthropod cell. For example, the cell can be a cell of a suborder, a family, a sub-family, a group, a sub-group, or a species of, e.g., Chelicerata, Myriapodia, Hexipodia, Arachnida, Insecta, Archaeognatha, Thysanura, Palaeoptera, Ephemeroptera, Odonata, Anisoptera, Zygoptera, Neoptera, Exopterygota, Plecoptera, Embioptera, Orthoptera, Zoraptera, Dermaptera, Dictyoptera, Notoptera, Grylloblattidae, Mantophasmatidae,BIOTN-41612.601Phasmatodea, Blattaria, Isoptera, Mantodea, Parapneuroptera, Psocoptera, Thysanoptera, Phthiraptera, Hemiptera, Endopterygota or Holometabola, Hymenoptera, Coleoptera, Strepsiptera, Raphidioptera, Megaloptera, Neuroptera, Mecoptera, Siphonaptera, Diptera, Trichoptera, or Lepidoptera.

[0150] A cell is in some cases an insect cell. For example, in some cases, the cell is a cell of a mosquito, a grasshopper, a true bug, a fly, a flea, a bee, a wasp, an ant, a louse, a moth, or a beetle.

[0151] In some embodiments, introducing the system into a cell comprises administering the system to a subject. In some embodiments, the subject is human. The administering may comprise in vivo administration. In alternative embodiments, a vector is contacted with a cell in vitro or ex vivo and the treated cell, containing the system, is transplanted into a subject.

[0152] In some embodiments, the target nucleic acid is a nucleic acid endogenous to a target cell. In some embodiments, the target nucleic acid is a genomic DNA sequence. The term “genomic,” as used herein, refers to a nucleic acid sequence (e.g., a gene or locus) that is located on a chromosome in a cell.

[0153] In some embodiments, the target nucleic acid encodes a gene or gene product. The term “gene product,” as used herein, refers to any biochemical product resulting from expression of a gene. Gene products may be RNA or protein. RNA gene products include non-coding RNA, such as tRNA, rRNA, micro RNA (miRNA), and small interfering RNA (siRNA), and coding RNA, such as messenger RNA (mRNA). In some embodiments, the target nucleic acid sequence encodes a protein or polypeptide.

[0154] The disclosed method may modify a target DNA sequence in a cell so as to modulate expression of the target DNA sequence, e.g., expression of the target DNA sequence is increased, decreased, or completely eliminated (e.g., via deletion of a gene). In one embodiment, the disclosed system cleaves a target DNA sequence of the host cell to produce double strand DNA breaks. The double strand breaks can be repaired by the host cell by either non-homologous end joining (NHEJ) or homologous recombination. In NHEJ, the double-strand breaks are repaired by direct ligation of the break ends to one another. In homologous recombination repair, a donor nucleic acid molecule comprising a second DNA sequence with homology to the cleaved target DNA sequence is used as a template for repair of the cleaved target DNA sequence, resulting in the transfer of genetic information from the donor nucleic acid molecule to the target DNA. As a result, new nucleic acid material is inserted / copied into the DNA break site. The modifications of the targetBIOTN-41612.601 sequence due to NHEJ and / or homologous recombination repair may lead to, for example, gene correction, gene replacement, gene tagging, transgene insertion, nucleotide deletion, gene disruption, gene mutation, gene knock-down, etc.

[0155] In some embodiments, the systems and methods described herein may be used to correct one or more defects or mutations in a gene (referred to as “gene correction”). In such cases, the target sequence encodes a defective version of a gene, and the disclosed compositions and systems further comprise a donor nucleic acid molecule which encodes a wild-type or corrected version of the gene.

[0156] In another embodiment, the method of modifying a target sequence can be used to delete nucleic acids from a target sequence in a host cell by cleaving the target sequence and allowing the host cell to repair the cleaved sequence in the absence of an exogenously provided donor nucleic acid molecule. Deletion of a nucleic acid sequence in this manner can be used in a variety of applications, such as, for example, to remove disease-causing trinucleotide repeat sequences in neurons, to create gene knock-outs or knock-downs, and to generate mutations for disease models in research.

[0157] In some embodiments, the systems and methods described herein can be used to generate cell lines used for protein production (e.g., CHO cells, HEK293 cells, BHK21 cells, NSO cells, Sp2 / O cells, and the like). For example, the systems and methods of the present disclosure can be used to generate cell lines in which one or more genes that negatively impact protein production, expression, and / or purification have been knocked out. In some embodiments, the systems and methods of the present disclosure can be used to knockout genes that encode contaminating protein(s), including but not limited to, legumain or transforming growth factor (TGF-p; see, e.g., U.S. Patent Appln. Pub. No. US2023 / 0143582, which is incorporated herein by reference in its entirety and for all purposes). In some embodiments, the systems and methods of the present disclosure can be used to knockout genes than inhibit recombinant protein expression, including but not limited to, activating transcription factor 6 beta (ATF6B) or heparan sulfate proteoglycan (HSPG).

[0158] In accordance with these embodiments, the systems and methods of the present disclosure include producing recombinant proteins using a mammalian expression system. Exemplary methods can include identifying genes that encode for proteins that negatively impact protein production, expression, and / or purification; designing and synthesizing specific guideBIOTN-41612.601RNAs for each target gene-of-interest; introducing the engineered nuclease enzymes of the present disclosure that recognize the specific guide RNAs to the expression system; inducing gene knockout using the engineered nuclease enzymes; and purifying the recombinant protein products. These methods can be used to enhance recombinant protein production, yield, and / or purity for use in research, diagnostics, and therapeutics.

[0159] In some embodiments, the system and methods described herein may be used to genetically modify a plant or plant cell. As used herein, genetically modified plants include a plant into which has been introduced an exogenous polynucleotide. Genetically modified plants also include a plant that has been genetically manipulated such that endogenous nucleotides have been altered to include a mutation, such as a deletion, an insertion, a transition, a transversion, or a combination thereof. For instance, an endogenous coding region could be deleted. Such mutations may result in a polypeptide having a different amino acid sequence than was encoded by the endogenous polynucleotide. Another example of a genetically modified plant is one having an altered regulatory sequence, such as a promoter, to result in increased or decreased expression of an operably linked endogenous coding region. The genetically modified plant may promote a desired phenotypic or genotypic plant trait.

[0160] Genetically modified plants can potentially have improved crop yields, enhanced nutritional value, and increased shelf life. They can also be resistant to unfavorable environmental conditions, insects, and pesticides. The present systems and methods have broad applications in gene discovery and validation, mutational and cisgenic breeding, and hybrid breeding. The present systems and methods may facilitate the production of a new generation of genetically modified crops with various improved agronomic traits such as herbicide resistance, herbicide tolerance, drought tolerance, male sterility, insect resistance, abiotic stress tolerance, modified fatty acid metabolism, modified carbohydrate metabolism, modified seed yield, modified oil percent, modified protein percent, resistance to bacterial disease, disease (e.g. bacterial, fungal, and viral) resistance, high yield, and superior quality. The present systems and methods may also facilitate the production of a new generation of genetically modified crops with optimized fragrance, nutritional value, shelf-life, pigmentations (e.g., lycopene content), starch content (e.g., low-gluten wheat), toxin levels, propagation and / or breeding and growth time. See, for example, CRISPR / Cas Genome Editing and Precision Plant Breeding in Agriculture (Chen et aL, Annu Rev Plant Biol. 2019 Apr 29;70:667-69), incorporated herein by reference.BIOTN-41612.601

[0161] The present system and method may confer one or more of the following traits to the plant cell: herbicide tolerance, drought tolerance, male sterility, insect resistance, abiotic stress tolerance, modified fatty acid metabolism, modified carbohydrate metabolism, modified seed yield, modified oil percent, modified protein percent, resistance to bacterial disease, resistance to fungal disease, and resistance to viral disease.

[0162] The present disclosure provides for a modified plant cell produced by the present system and method, a plant comprising the plant cell, and a seed, fruit, plant part, or propagation material of the plant. Transformed or genetically modified plant cells of the present disclosure may be as populations of cells, or as a tissue, seed, whole plant, stem, fruit, leaf, root, flower, stem, tuber, grain, animal feed, a field of plants, and the like. The present disclosure provides a transgenic plant. The transgenic plant may be homozygous or heterozygous for the genetic modification. Also provided by the present disclosure are transformed or genetically modified plant cells, tissues, plants, and products that contain the transformed or genetically modified plant cells. The present disclosure further encompasses the progeny, clones, cell lines or cells of the transgenic plants.

[0163] The present system and method may be used to modify a plant stem cell. The present disclosure further provides progeny of a genetically modified cell, where the progeny can comprise the same genetic modification as the genetically modified cell from which it was derived. The present disclosure further provides a composition comprising a genetically modified cell.

[0164] In one embodiment, the transformed or genetically modified cells, and tissues and products comprise a nucleic acid integrated into the genome, and production by plant cells of a gene product due to the transformation or genetic modification.

[0165] Methods of introducing exogenous nucleic acids into plant cells are well known in the art. Such plant cells are considered “transformed.” DNA constructs can be introduced into plant cells by various methods, including, but not limited to PEG- or electroporation-mediated protoplast transformation, tissue culture or plant tissue transformation by biolistic bombardment, or the Agrobacterium-mediated transient and stable transformation. The transformation can be transient or stable transformation. Suitable methods also include viral infection (such as double stranded DNA viruses), transfection, conjugation, protoplast fusion, electroporation, particle gun technology, calcium phosphate precipitation, direct microinjection, silicon carbide whiskers technology, Agrobacterium-mediated transformation, and the like. The choice of method is generally dependent on the type of cell being transformed and the circumstances under which theBIOTN-41612.601 transformation is taking place (i.e., in vitro, ex vivo, or in vivo). Transformation methods based upon the soil bacterium Agrobacterium tumefaciens are useful for introducing an exogenous nucleic acid molecule into a vascular plant. The wild-type form of Agrobacterium contains a Ti (tumor-inducing) plasmid that directs production of tumorigenic crown gall growth on host plants. Transfer of the tumor-inducing T-DNA region of the Ti plasmid to a plant genome requires the Ti plasmid-encoded virulence genes as well as T-DNA borders, which are a set of direct DNA repeats that delineate the region to be transferred. An Agrobacterium-based vector is a modified form of a Ti plasmid, in which the tumor inducing functions are replaced by the nucleic acid sequence of interest to be introduced into the plant host.

[0166] Agrobacterium-mediated transformation generally employs cointegrate vectors or binary vector systems, in which the components of the Ti plasmid are divided between a helper vector, which resides permanently in the Agrobacterium host and carries the virulence genes, and a shuttle vector, which contains the gene of interest bounded by T-DNA sequences. A variety of binary vectors are well known in the art and are commercially available, for example, from Clontech (Palo Alto, Calif.). Methods of coculturing Agrobacterium with cultured plant cells or wounded tissue such as leaf tissue, root explants, hypocotyledons, stem pieces or tubers, for example, also are well known in the art. See., e.g., Glick and Thompson, (eds.), Methods in Plant Molecular Biology and Biotechnology, Boca Raton, Fla.: CRC Press (1993), incorporated herein by reference.

[0167] Microprojectile-mediated transformation also can be used to produce a transgenic plant. This method, first described by Klein et al. (Nature 327:70-73 (1987), incorporated herein by reference), relies on microprojectiles such as gold or tungsten that are coated with the desired nucleic acid molecule by precipitation with calcium chloride, spermidine, or polyethylene glycol. The microprojectile particles are accelerated at high speed into an angiosperm tissue using a device such as the BIOLISTIC PD-1000 (Biorad; Hercules Calif.).

[0168] In one embodiment, the present systems and methods may be adapted to use in plants. In one embodiment, a series of plant-specific RNA-guided Genome Editing vectors (pRGE plasmids) are provided for expression of the present system in plants. The vectors may be optimized for transient expression of the present system in plant protoplasts, or for stable integration and expression in intact plants via the Agrobacterium-mediated transformation. In one aspect, the vector constructs include a nucleotide sequence comprising a DNA-dependent RNABIOTN-41612.601 polymerase III promoter, wherein the promoter is operably linked to a gRNA molecule and a Pol III terminator sequence, and a nucleotide sequence comprising a DNA-dependent RNA polymerase II promoter operably linked to a nucleic acid sequence encoding the nuclease.

[0169] In certain embodiments, the present systems and methods use a monocot promoter to drive the expression of one or more components of the present systems (e.g., gRNA) in a monocot plant. In certain embodiments, the present systems and methods use a dicot promoter to drive the expression of one or more components of the present systems (e.g., gRNA) in a dicot plant. In some embodiments, the present system is transiently expressed in plant protoplasts. Vectors for transient transformation of plants include, but are not limited to, pRGE3, pRGE6, pRGE31, and pRGE32. In some embodiment, the vector may be optimized for use in a particular plant type or species, such as pStGE3.

[0170] In one embodiment, the present system may be stably integrated into the plant genome, for example via Agrobacterium-mediated transformation. Thereafter, one or more components of the present system (e.g., the transgene) may be removed by genetic cross and segregation, which may lead to the production of non-transgenic, but genetically modified plants or crops. In one embodiment, the vector is optimized for Agrobacterium-mediated transformation. In one embodiment, the vector for stable integration is pRGEB3, pRGEB6, pRGEB31, pRGEB32, or pStGEB3.

[0171] The present system may be used in various bacterial hosts, including human pathogens that are medically important, and bacterial pests that are key targets within the agricultural industry, as well as antibiotic resistant versions thereof.

[0172] The system and method may be designed to target any gene or any set of genes, such as virulence or metabolic genes, for clinical and industrial applications in other embodiments. For example, the present systems and methods may be used to target and eliminate virulence genes from the population, to perform in situ gene knockouts, or to stably introduce new genetic elements to the metagenomic pool of a microbiome. The present systems and methods may be used to treat a multi-drug resistance bacterial infection in a subject. The present systems and methods may be used for genomic engineering within complex bacterial consortia.

[0173] The present systems and methods may be used to inactivate microbial genes. In some embodiments, the gene is an antibiotic resistance gene. For example, the coding sequence ofBIOTN-41612.601 bacterial resistance genes may be disrupted in vivo by insertion of a DNA sequence, leading to non-selective re-sensitization to drug treatment.

[0174] In some embodiments, introducing the system into a cell comprises administering the system to a subject. In some embodiments, the subject is human. The administering may comprise in vivo administration. In alternative embodiments, a vector is contacted with a cell in vitro or ex vivo and the treated cell, containing the system, is transplanted into a subject.

[0175] The components of the composition, system or ex vivo treated cells may be administered to a cell or subject with a pharmaceutically acceptable carrier or excipient as a pharmaceutical composition. In some embodiments, the components of the present system may be mixed, individually or in any combination, with a pharmaceutically acceptable carrier to form pharmaceutical compositions, which are also within the scope of the present disclosure.

[0176] The methods described here also provide for treating a disease or condition in a subject. The method may comprise administering to the subject, in vivo, or by transplantation of ex vivo treated cells (e.g., disclosed T cells), a therapeutically effective amount of the present system, or components thereof. A “subject” or “patient” may be human or non-human and may include, for example, animal strains or species used as “model systems” for research purposes, such a mouse model as described herein.

[0177] In some embodiments, the systems and methods are used to treat a pathogen or parasite on or in a subject by altering the pathogen or parasite. In some embodiments, the systems and methods target a “disease-associated” gene. The term “disease-associated gene,” refers to any gene or polynucleotide whose gene products are expressed at an abnormal level or in an abnormal form in cells obtained from a disease-affected individual as compared with tissues or cells obtained from an individual not affected by the disease. A disease-associated gene may be expressed at an abnormally high level or at an abnormally low level, where the altered expression correlates with the occurrence and / or progression of the disease. A disease-associated gene also refers to a gene, the mutation or genetic variation of which is directly responsible or is in linkage disequilibrium with a gene(s) that is responsible for the etiology of a disease. Examples of genes responsible for such “single gene” or “monogenic” diseases include, but are not limited to, adenosine deaminase, a-1 antitrypsin, cystic fibrosis transmembrane conductance regulator (CFTR), p-hemoglobin (HBB), oculocutaneous albinism II (OCA2), Huntingtin (HTT), dystrophia myotonica-protein kinase (DMPK), low-density lipoprotein receptor (LDLR), apolipoprotein B (APOB),BIOTN-41612.601 neurofibromin 1 (NF1), polycystic kidney disease 1 (PKD1), polycystic kidney disease 2 (PKD2), coagulation factor VIII (F8), dystrophin (DMD), phosphate -regulating endopeptidase homologue, X-linked (PHEX), methyl-CpG-binding protein 2 (MECP2), and ubiquitin-specific peptidase 9Y, Y-linked (USP9Y). Other single gene or monogenic diseases are known in the art and described in, e.g., Chial, H. Rare Genetic Disorders: Learning About Genetic Disease Through Gene Mapping, SNPs, and Microarray Data, Nature Education 1(1): 192 (2008); Online Mendelian Inheritance in Man (OMIM); and the Human Gene Mutation Database (HGMD). In another embodiment, the target genomic DNA sequence can comprise a gene, the mutation of which contributes to a particular disease in combination with mutations in other genes. Diseases caused by the contribution of multiple genes which lack simple (i.e., Mendelian) inheritance patterns are referred to in the art as a “multifactorial” or “polygenic” disease. Examples of multifactorial or polygenic diseases include, but are not limited to, asthma, diabetes, epilepsy, hypertension, bipolar disorder, and schizophrenia. Certain developmental abnormalities also can be inherited in a multifactorial or polygenic pattern and include, for example, cleft lip / palate, congenital heart defects, and neural tube defects. In another embodiment, the target DNA sequence can comprise a cancer oncogene.

[0178] The present disclosure provides for gene editing methods that can ablate a disease- associated gene (e.g., a cancer oncogene), which in turn can be used for in vivo gene therapy for patients. In some embodiments, the gene editing methods include donor nucleic acids comprising therapeutic genes.

[0179] In some embodiments, an effective amount of the components of the present system or compositions as described herein can be administered. Within the context of the present disclosure, the term “effective amount” refers to that quantity of the components of the system such that modification of the target nucleic acid is achieved.

[0180] When utilized as a method of treatment, the effective amount may depend on the particular condition being treated, the severity of the condition, the individual patient parameters including age, physical condition, size, gender and weight, the duration of the treatment, the nature of concurrent therapy (if any), the specific route of administration and like factors within the knowledge and expertise of the health practitioner. In some embodiments, the effective amount alleviates, relieves, ameliorates, improves, reduces the symptoms, or delays the progression of any disease or disorder in the subject. In some embodiments, the subject is a human.BIOTN-41612.601

[0181] A wide range of additional therapies may be used in conjunction with the methods of the present disclosure. The additional therapy may be administration of an additional therapeutic agent or may be an additional therapy not connected to administration of another agent. Such additional therapies include, but are not limited to, surgery, immunotherapy, radiotherapy. The additional therapy may be administered at the same time as the above methods. In some embodiments, the additional therapy may precede or follow the treatment of the disclosed methods by time intervals ranging from hours to months.

[0182] In some embodiments, a therapeutically effective amount of a system (e.g., nuclease and / or gRNA) or compositions described herein, is administered alone or in combination with a therapeutically effective amount of at least one additional therapeutic agent. In some embodiments, effective combination therapy is achieved with a single composition or pharmacological formulation or with two distinct compositions or formulations, administered at the same time or separated by a time interval. The at least one additional therapeutic agent may comprise any manner of therapeutic, including protein, small molecule, nucleic acids, and the like. For example, exemplary additional therapeutic agents include, but are not limited to, immune modulators, chemotherapeutic agents, a nucleic acid (e.g., mRNA, aptamers, antisense oligonucleotides, ribozyme nucleic acids, interfering RNAs, antigene nucleic acids), decongestants, steroids, analgesics, antimicrobial agents, immunotherapies, or any combination thereof.

[0183] In the context of the present disclosure insofar as it relates to any of the disease conditions recited herein, the terms “treat,” “treatment,” and the like mean to relieve or alleviate at least one symptom associated with such condition, or to slow or reverse the progression of such condition. Within the meaning of the present disclosure, the term “treat” also denotes to arrest, delay the onset (e.g., the period prior to clinical manifestation of a disease) and / or reduce the risk of developing or worsening a disease. For example, in connection with cancer, the term “treat” may mean eliminating or reducing a patient's tumor burden, or prevent, delay, or inhibit metastasis, etc.

[0184] The phrase “pharmaceutically acceptable,” as used in connection with compositions and / or cells of the present disclosure, refers to molecular entities and other ingredients of such compositions that are physiologically tolerable and do not typically produce untoward reactions when administered to a subject (e.g., a mammal, a human). Preferably, as used herein, the term “pharmaceutically acceptable” means approved by a regulatory agency of the Federal or a stateBIOTN-41612.601 government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in mammals, and more particularly in humans. “Acceptable” means that the carrier is compatible with the active ingredient of the composition (e.g., the nucleic acids, vectors, cells, or therapeutic antibodies) and does not negatively affect the subject to which the composition(s) are administered. Any of the pharmaceutical compositions and / or cells to be used in the present methods can comprise pharmaceutically acceptable carriers, excipients, or stabilizers in the form of lyophilized formations or aqueous solutions.

[0185] Pharmaceutically acceptable carriers, including buffers, are well known in the art, and may comprise phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives; low molecular weight polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; amino acids; hydrophobic polymers; monosaccharides; disaccharides; and other carbohydrates; metal complexes; and / or non-ionic surfactants. See, e.g., Remington: The Science and Practice of Pharmacy 20th Ed. (2000) Lippincott Williams and Wilkins, Ed. K. E. Hoover.

[0186] In some cases, desirable delivery systems provide for roughly uniform distribution and have controllable rates of release of their components (e.g., vectors, proteins, nucleic acids) in vivo. A variety of different media are described below that are useful in creating composition delivery systems. It is not intended that any one medium is limiting to the present invention. Note that any medium may be combined with another medium or carrier; for example, in one embodiment a polymer microparticle attached to a compound may be combined with a gel medium. An implantable device can be used to deliver a nuclease, or a nucleic acid encoding thereof, and gRNA, or a nucleic acid encoding thereof, to, for example, a target cell in vivo.

[0187] Carriers or mediums contemplated include materials such as gelatin, collagen, cellulose esters, dextran sulfate, pentosan polysulfate, chitin, saccharides, albumin, fibrin sealants, synthetic polyvinyl pyrrolidone, polyethylene oxide, polypropylene oxide, block polymers of polyethylene oxide and polypropylene oxide, polyethylene glycol, acrylates, acrylamides, methacrylates including, but not limited to, 2 -hydroxy ethyl methacrylate, poly( ortho esters), cyanoacrylates, gelatin-resorcin-aldehyde type bioadhesives, polyacrylic acid and copolymers and block copolymers thereof.

[0188] In some cases, a carrier / medium can include a microparticle. Microparticles can include, but are not limited to, liposomes, nanoparticles, microspheres, nanospheres, microcapsules, andBIOTN-41612.601 nanocapsules. In some cases, microparticle can include one or more of the following: a poly(lactide-co-glycolide), aliphatic polyesters including, but not limited to, poly-glycolic acid and poly-lactic acid, hyaluronic acid, modified polysaccharides, chitosan, cellulose, dextran, polyurethanes, polyacrylic acids, pseudo-poly(amino acids), polyhydroxybutyrate-related copolymers, poly anhydrides, polymethylmethacrylate, poly(ethylene oxide), lecithin and phospholipids - in any combination thereof.

[0189] In some cases, a carrier / medium can include a liposome that is capable of attaching and releasing therapeutic agents (e.g., the subject nucleic acids and / or proteins). Liposomes are microscopic spherical lipid bilayers surrounding an aqueous core that are made from amphiphilic molecules such as phospholipids. For example, a liposome may trap a therapeutic agent between the hydrophobic tails of the phospholipid micelle. Water soluble agents can be entrapped in the core and lipid-soluble agents can be dissolved in the shell-like bilayer. Liposomes have a special characteristic in that they enable water soluble and water insoluble chemicals to be used together in a medium without the use of surfactants or other emulsifiers. Liposomes can form spontaneously by forcefully mixing phospholipids in aqueous media. Water soluble compounds are dissolved in an aqueous solution capable of hydrating phospholipids. Upon formation of the liposomes, therefore, these compounds are trapped within the aqueous liposomal center. The liposome wall, being a phospholipid membrane, holds fat soluble materials such as oils. Liposomes provide controlled release of incorporated compounds. In addition, liposomes can be coated with water soluble polymers, such as polyethylene glycol to increase the pharmacokinetic half-life.

[0190] In some embodiments, a cationic or anionic liposome is used as part of a subject composition or method, or liposomes having neutral lipids can also be used. Cationic liposomes can include negatively-charged materials by mixing the materials and fatty acid liposomal components and allowing them to charge-associate. The choice of a cationic or anionic liposome depends upon the desired pH of the final liposome mixture.

[0191] Any element of any suitable CRISPR / Cas gene editing system known in the art can be employed in the systems and methods described herein, as appropriate. CRISPR / Cas gene editing technology is described in detail in, for example, U.S. Patent Nos. 8,546,553, 8,697,359; 8,771,945; 8,795,965; 8,865,406; 8,871,445; 8,889,356; 8,889,418; 8,895,308; 8,9066,616; 8,932,814; 8,945,839; 8,993,233; 8,999,641; 9,115,348; 9,149,049; 9,493,844; 9,567,603; 9,637,739; 9,663,782; 9,404,098; 9,885,026; 9,951,342; 10,087,431; 10,227,610; 10,266,850;BIOTN-41612.60110,601,748; 10,604,771; and 10,760,064; and U.S. Patent Application Publication Nos. US2010 / 0076057; US2014 / 0113376; US2015 / 0050699; US2015 / 0031134; US2014 / 0357530; US2014 / 0349400; US2014 / 0315985; US2014 / 0310830; US2014 / 0310828; US2014 / 0309487; US2014 / 0294773; US2014 / 0287938; US2014 / 0273230; US2014 / 0242699; US2014 / 0242664; US2014 / 0212869; US2014 / 0201857; US2014 / 0199767; US2014 / 0189896; US2014 / 0186919; US2014 / 0186843; and US2014 / 0179770, each incorporated herein by reference.Kits

[0192] Also within the scope of the present disclosure are kits that include the compositions, systems, or components thereof as disclosed herein.

[0193] For example the kits may contain one or more reagents or other components useful, necessary, or sufficient for practicing any of the methods described herein, such as, editing reagents (nuclease, guide RNAs, vectors, compositions, etc.), transfection or administration reagents, negative and positive control samples (e.g., cells, template DNA), cells, containers housing one or more components (e.g., microcentrifuge tubes, boxes), detectable labels, detection and analysis instruments, software, instructions, and the like.

[0194] The kit may include instructions for use in any of the methods described herein. The instructions can comprise a description of administration of the present system or composition to a subject to achieve the intended effect. The instructions generally include information as to dosage, dosing schedule, and route of administration for the intended treatment. The kit may further comprise a description of selecting a subject suitable for treatment based on identifying whether the subject is in need of the treatment.

[0195] The kits provided herein are in suitable packaging. Suitable packaging includes, but is not limited to, vials, bottles, jars, flexible packaging, and the like. A kit may have a sterile access port (for example, the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). The container may also have a sterile access port.

[0196] The packaging may be unit doses, bulk packages (e.g., multi-dose packages) or sub-unit doses. Instructions supplied in the kits of the disclosure are typically written instructions on a label or package insert. The label or package insert indicates that the pharmaceutical compositions are used for treating, delaying the onset, and / or alleviating a disease or disorder in a subject.

[0197] Kits optionally may provide additional components such as buffers and interpretive information. Normally, the kit comprises a container and a label or package insert(s) on orBIOTN-41612.601 associated with the container. In some embodiment, the disclosure provides articles of manufacture comprising contents of the kits described above.

[0198] The kit may further comprise a device for holding or administering the present system or composition. The device may include an infusion device, an intravenous solution bag, a hypodermic needle, a vial, and / or a syringe.

[0199] The kit will typically be provided with its various components in one or more packages, e.g., a fiber-based, a cardboard, polymeric, or a Styrofoam box. The enclosure(s) can be configured so as to maintain a temperature differential between the interior and the exterior, for example, to provide insulating properties to keep the reagents at a preselected temperature for a preselected time. The packaging can be air-tight, waterproof (e.g., impermeable to changes in moisture or evaporation), and / or light-tight.Sequences

[0200] The various embodiments of the nucleases referenced in the present disclosure include the following.

[0201] Artificial Casl2a.l (anCPFl.l; also referred to as anCPFl):MEKEKNGTNNFQEFIGISPLQKTLRNELIPTETTQKHIAKYGILEEDELRAQNREKLKEIM DDYYRNFIDETLRSVHDIDWNSLFEAMKNHLKNNSDDSKKTLEKEQEKMRKEIHQKFA DDAKFKDMFGAKLISEILPEFIKHNNKYSEKEKEEKIKVIKLFNRFTTSFTDYFENRKNVF SKEDISSAVGYRIVHENAEIFLENMLAYQRICKSASDDINKIEGEMKGYLKGMTLEEIFSA EYYAKLITQEGIDYYNEICGAVNQYMNLYCQKNKENKNLYKMKRLHKQILCNATTSFEI PEKFENDEEVYESVNGFLENVTKKNVLERLLKITNNYNNYDLDKIYIVAKYYEKISQYIY GDWNTIEDCLTDYYSNQLPGKGKAKENKVKKAVKEDKYKSISEINELIKKYALAKDSN RKAESYISEIANIINLKEAHELEYNPEINLIENEEKASEIKDVLDMYMDVFHWIKVFRVEE VVDKDENFYAELDEIYEEIREIVPLYNKVRNYVTQKPYSTEKIKLNFGTPTLANGWSKSK EYDNNAIILMRDGKYYLGIFNAKKKPDKEIIEGNSSPSENEYKKMIYYLLPGPNKMLPKV FLSKKGIETYKPSAYIIEGYKEKKHIKGSKNFDIKFCHDLIDYFKDCIEKHPDWKKFGFDF SDTETYEDISGFYREVEKQGYKIDWTYISEEDIDKLQEEGQLYLFQIYNKDFAEGSTGKP NLHTMYLKNLFSEENLRDIVLKLNGEAEIFFRKSSVKKPIIHKKGSILVNRTYEAEEKGQTVKKNIPENEYQELYKYFNNKKKKELSDEAKKYLDKVACHEAKTDIVKDYRYTIDKFFIH LPITINFKANKKGNINSIAQQYIAEQKDLHVIGIDRGERNLIYVSVIDMCGKIIEQKSFNIV NGYDYKEKLKQREEERDEARKSWKEIGKIKELKEGYLSSVIHEIAKMVIKYNAIIAMEDLBIOTN-41612.601NYGFKRGRFKVERQVYQKFETMLINKLNYLVFKDRSVDEPGGILRGYQLTYIPDSIKNVGRQCGIIFYVPAAYTSKIDPTTGFVNIFKYKAVTTDDAKREFIMKFDSIRYDVEKGLFRFTFDYKNFATHNTTLAKSNWTVYTNGTRIQKKFVNGRFSNMSETIDLTKKMKKLLEMSGIEYEDGQNLLPDLKEIKDYEIVHEIFEIFRLTVQMRNSKSELDDPDYDRIISPVLNEDGKFFDSSKYNNKEDAQKSALPIDADANGAYCIALKGLYTMKQIKENWKEDGKLSADKLKISNKDWFDFMQNKRYL (SEQ ID NO: 1).

[0202] Artificial Casl2a,2 (anCPF1.2; also referred to as anCPF2):MEEEKNFMENFKEFIGISPLSKTLRNELIPVGTTQKHIAKYGILEEDELRAQNREKLKEIMDDYYRNFIDETLRDVHDIDWNSLFEAMKEHLKNNSDDSKKSLEKEQEKMRKEIHQKFADDAKFKDMFSAKLISEILPEFIKHNNKYSEKEKEEKIKVIKLFNRFTTSFTDFFENRKNVFSKEDISTAIGYRIVHENAEIFLENMVAYQRIQKSASEDINKIEGEMKGYLKGMTLEEIFSAEYYAKLITQEGIDYYNEICGAVNQYMNLYCQKNKENKNLYKMKRLHKQILSNATTSFEIPEKFENDEEVYESVNGFLENVTKKNVLERILKLTNNYNNYDLDKIYITAPYYEKISQYLYGDWNTIEDCLTDYYSNQLPGKGAAKENKVKKAVKEDKYKSLSEINELIKKYALAEEVNRKAESYISEIANIINLKEAHELEYNPEVNLIENEKKASEIKDVLDTYMDLFHWMKVFRVEEAVDKDENFYAELDEIYEEIRDIVPLYNKVRNYVTQKPYSTEKIKLNFGTPTLANGWSKSKEYDNNAIILMRDGKYYLGIFNAKKKPDKEIIEGNSSPSEGNEYKKMVYYLLPGPNKMLPKVFLSKKGIETYKPSEYIIEGYKEKKHIKGSKNFDIKFCHDLIDYFKDCIEKHPDWKKFGFDFSDTETYEDISGFYREVEKQGYKIDWTYISEEDIDKLQEEGQLYLFQIYNKDFAEGSTGKPNLHTMYLKNLFSEENLRDIVLKLNGEAEIFFRKSSVKKPIIHKKGSILVNRTYEAEEKGETVKKNIPENEYQELYKYFNNKKKKELSDEAKKYLDKVACHEAKTDIVKDYRYTIDKFFIHLPITINFKANGKGNINSVAQQYIAEQKDLHVIGIDRGERNLIYVSVIDMQGKIIEQKSFNVVNGYDYKEKLKQREQERDEARKSWKEIGKIKELKEGYLSSVIHEIAKMMIKYNAIVAMEDLNYGFKRGRFKVERQVYQKFETMLINKLNYLVFKDRSVDEPGGVLRGYQLTYIPDSIKKLGRQCGIIFYVPAAYTSKIDPTTGFVNIFNYKAVTTDDAKREFIMKFDSIRYDVEKGLFRFTFDYKNFATHNTTLARNNWTVYTNGTRIQKKFVNGRFRNMKETFDPTKKMKKLLEMSGIEYEDGQNLLPDLKEIKDPNLVHEIFEIFRLTVQMRNSKSELDDTDYDHIISPVLNEDGKFFDSSKYNNKEDAQESALPIDADANGAYCIALKGLYIMKAIKENWKEDGKLSPDKLKISNKDWFDFMQNKRYL (SEQ ID NO: 2).

[0203] Artificial Casl2a,3 (anCPF1.3; also referred to as anCPF3):EEEKNLMNNFKEFTGIYPLSKTLRFELKPVGKTQEHIEKYGILEEDEKRAENYKKLKELIBIOTN-41612.601DDYHRKFIDETLKNVHDIDWNPLAEALEKYHKNKSDKNKKALEAEQKKMRKEIAAYF KDDAKFKELFGKKLFSELLPEFIKSNLKVSEKEKEEEIDALKSFNKFSTYFTGFHENRKNI YSKEDISTSIAYRIVHENFPKFLSNLKAYQRIKEKCPEVINKAEGELTGFLGGIKLDEIFSIE YYNNLLTQDGIDYYNQIIGGVSGKDGEKKLQGVNEFLNLYHQKNKEIAKKKGRIKMKP LYKQILSDRTTLSYIPEAFENDEEVIDSVNGFLSNIEKNNILDRILKLLGNLNSYDLDKIYIT ASNLSAISQKLFGDWSTIEDALKEYKANEFGGAGVAKANKKVEAWLKSKAFSLSDINA AVKKYSEAEAADINRKFSSYISELSNIIETIEAHELEFNPSKKLLGDEEKTSVIKSLLDSLM ELFHWLKVFKVEEEIDIDSSFYSEFDEIYEEIKDIIPLYNKVRNYVTQKPYSTEKIKLNFEN PTLANGWDQNKEYDNNAIILMKDGKYYLGILNAKKKPNIEIIEGDGSPSEGSSYKKMVYKLLPGPNKMLPKVFLSSKKGKEKYKPSKEILEGYEEKKHIKGSKNFDLKFCHKLIDYFKD CIEKHPDWKKFGFDFSPTESYEDISDFYREVEKQGYRINFTNISEEEIDKWVEEGQLFLFQ IYNKDFAEGATGKPNLHTMYWKALFSPENLKDVVLKLNGEAELFFRKKSVKKPWHKK GSMLVNRTYKDEEKGETVGTPIPESIYKELYKYYNGKKKKAGLSDEAKEYLDKVRVHE AKHDIVKDRRYTIDKFFFHVPLTINFKANGKGNINSKVQEYLKDNPDVHIIGIDRGERNLI YVSVIDRKGKIIEQKSFNVINGYDYREKLNQREKERDEARKSWKTIGKIKELKEGYLSAV VHEITKMMIEYNAIVVMEDLNYGFKRGRFKVEKQVYQKFEKMLIDKLNYLVFKDASAD EPGGVLRGYQLTNKFESFAKLGKQCGIIFYIPAAYTSKIDPTTGFVNLFNFSDITTAEERK EFIAKFDSIKYDAKKGLFAFTFDYKKFKTYQTSLYRNKWTVYTNGKRIQYKKENKRYK RMNEDYEPTKEMKEVLKKSGIAYEGGQDILPDIIEIKNSDNSGLIHEIFYVFRLTLQMRNS KSELDDTDEDYIISPVLNDKGKFFDSKKANKGKDAQKSALPIDADANGAYHIALKGLLIL RAIEEKFKEDGKLSLKKLKISNKDWFEFMQTRRYL (SEQ ID NO: 3).

[0204] Artificial Casl2a,4 (anCPF1.4; also referred to as anCPF4):MSYNREKMEEEEKNLMNNFKEFTGIYPLSKTLRFELKPVGKTQEHIEKYGILEEDEKRAE NYKKLKELIDDYHRKFIDETLKNVHDIDWNPLAEALEKYHKNKSDKNKAKALEAEQKK MRKEIAAYFKDKVPDKGSKDLKEFNKTQDAKFKELFGKKLFSELLPELIKSNLKVSEKE KEEEIDALKSFDKFSTYFTGFHENRKNIYSKEDISTSIAYRIVHENFPKFLSNLKAYQRIKE KCPNEVINKAEGTAELTGILGGIKLDDIFSIEYYNNLLTQDGIDYYNQIIGGVSGKDGEKK LQGVNEFLNLYHQKNKEIAKKLKKGRIKMTPLYKQILSDRTTLSYIPEAFENDEEVIDSV NGFLSNIEKFEHDGKDNNILDRILKLLGNLNSYDLDKIYITASNLSAISQKLFGDWSTIED ALKEYKANEFGGAGVAKANKKVEAWLKSKAFSLSDINAAVKKYSEAAAADISNRKED FSSYISELSNIIETIEAHGLEFSYFCKANPSKKLLGDEEKTEVIKSLLDSLMELFHWLKVFKBIOTN-41612.601VEEEIDIDSSFYSEFDEIYEEIKDIIPLYNKVRNYVTQKPYSTEKIKLNFENPTLANGWDQNKEYDNNAIILMKDGKYYLGILNAKKKPNIKIIKGDGSPEEGSSYKKMVYKLLPGPNKMLPKVFLSSKKGKEKYKPSKEILVLYNKKFIKPLTITKEGYEAKNKHIKGSKNFDLKFCHKLIDFFKDCIEKHPDWKKFGFDFVASYESTSIYDTSTVSPTESYEDISDFYREVEKQGYRINFTNISEEEIDKWVEEGQLFLFQIYNKDFAEGATGKPNLHTMYWKALFSPENLKDVVLKLNGEAELFFRKKSVKKDAPVVHKKGSMLVNRTYKDEEKGQFGNIETVGTPIPESIYKELYKYYNGKKKEAGLSDEAKGLEYLDKVRVHEAKHDIVKDRRYTIDKFFFHVPLTINFKANGKGVNINSKVQEYLKDNPDVHIIGIDRGERNLIYVSIIDRKGKIIEQKSFNVINQVSSQGTKRGYDYREKLNQREKERDEARKSWKTIGKIKELKEGYLSAVVHEITKMMIEYNAIVVMEDLNYGFKRGRFKVAEKQVYQKFEKMLIDKLNYLVFKDASADEPGGVLRGYQLTNKFESFAKLGKQCGIIFYIPAAYTSKIDPTTGFVNLFNFSDITTAEERKEFIAKFDSIKYDAKDKGMFAFTFDYKKFKTYQTSLEGYRNKWTVVVEANKDRRDAEGKTYTNGKRIQYKKENKRYKRMNEDYEPTKEMKEVLKKSGIAYEGGQDILPDIIEIEANSDNSGLIHEIFYVFRLTLQMRNSKAELDDTDEDYIISPVLNDKGKFFDSKKANKGKDAQGNWKSALPIDADANGAYHIALKGLLILRAIEEKFKEDGKLSLKKLKISNKDWFEFMQTRRYLFKN (SEQ ID NO: 4).

[0205] Artificial Casl2a,5 (anCPF1.5; also referred to as anCPF5):MMNNMNTNFEEFIGISSVSKTLRNELIPVGSTLEHIEQNGIITEDELRAEKRQELKKIMDDYYRDYIDKTLSNVHDLDWNPLFEAMEEVQKNKSDDNKKELEKVQDEKRKEICKLLSSDDKFKNMFNAKLITEILPEFIKQNNNYSEEEKEEKLETVALFNRFTTYFTDFFENRKNVFSKEAISTSICYRIVHENAEIFLDNMTAFQRIKKKAEDEIEVIEKEMKDYLDGWSLEQIFSADFYNRLLTQSGIDFYNEICGAVNQHMNLYCQQNKKNKNLFKMKRLHKQILSNSETSFEIPEKFENDEEVYDAVNGFIDNIEEKNIIERLKNILQNCDDYDLNKIYISSKYYENISQYMCGNWNVIKDCLENYYDETIAGKGKSKEEKVKKAVKEDKYRSIAEINELVEKYSDEKDRNRKAEEYISEISDIISDMEVHHLEYDDDINLIENEEKAAEIKNVLDSYMDIFHWMKTFIVDEVIDKDTAFYSELEDIYDELEDIVPLYNRVRNYVTQKPYSTEKIKLNFGTPTLANGWSKSKEYDNNAIILMRDNKYYLGIFNAKNKPDKKIIEGHSNKKNDNDYKKMVYNLLPGPNKMLPKVFLSKKGIETYKPSEYILEGYEQNKHIKSSENFDINFCHDLIDYFKECINKHPDWKNFGFKFSDTDTYEDISEFYREVEKQGYKIDWTYISEADIDKLDEEGKLYLFQIYNKDFSENSTGKDNLHTMYLKNLFSEENLKDIVLKLNGEAELFYRKSSIKKPIVHKKGSILVNKTYKEEVNNNAVRKPIPDEVYMELYKYYNGKLKAELSDEAKKYMDEKVEHREAKKDIVKDYRYTVDKFFIHLPITINYKADGKSNVNDMVLKYIAQQDDMHVIGIDRGERNLIYVSVIDMHGNIVEBIOTN-41612.601QKSFNIVNNYDYKEKLKQREKSRDNARKNWKEIGKIKELKEGYLSGVIHEIAKMMVEY NAIIAMEDLNYGFKRGRFKVERQVYQKFENMLINKLNYLVFKDRSVDEPGGVLRGYQL TYIPDSLKKLGKQCGFIFYVPAAYTSKIDPTTGFVNIFNFKSITTSEARKEFLMKFDSIRYD AERDMFAFSFDYNNFATHNTTMARTKWTVYTNGERIKKEFKNGRWTGTEEIDLTEEMK ELLEDSGIEYKDGHDLLPDIVEMDEKKKSNFFKELLEIFRLTVQMRNSKSESEDTDYDRII SPVLNEEGEFFDSSKYKENEKKKEATLPIDADANGAYCIALKGLYEVQQIKENWKEDEK FSRNTLKISNADWFDFIQNRRYE (SEQ ID NO: 5).

[0206] Artificial Casl2a,6 (anCPF1.6; also referred to as anCPF6):MNNMNTNFEEFIGISSVSKTLRNELIPTESTKKHIEENGIITEDELRAEKRQELKEIMDDYY RDFIDKTLSNVHDIDWKPLFEAMEETKKNKSDETKKELEKVQDEKRKEIHKIFSSDDKFK DMFNAKMITEILPEFIKQNNNYSEEEKEEKLETVKLFNRFTTSFTDFFENRKNVFSKEAIS TSICYRIVNENAEIFLDNMTAFQRIKKTAEEEIEKIEKEMKDYLDGWSLEEIFSADFYGRL MTQSGIDFYNDICGAVNQHMNLYCQKNKVNKNKFKMKRLHKQILCNSETSFEIPKKFE NDEEVYDAVNGFLDNIKEKNIIERLRNIGQNCDDYDLNKIYISSKAYENISQFMCGNWN VINDCLENYYNETIPGKGKSKEEKVKKAVKEDKYRSIAEINELVEKYNSEKDTNRKAEE YISEISDIINDMEVHELEYDDDINLIENEEKASEIKNVLDSYMDIFHWVKTFIVDEVIDKDT SFYSELEDIYDELQDIVPLYNRVRNYVTQKPYSTEKIKLNFGTPTLANGWSKSKEYDNN AIILMRDNKYYLGIFNAKNKPDKKIIEGHENEKDNDYKKMVYNLLPGPNKMLPKVFLSK KGIETYKPSEYILEGYEQNKHIKSSKNFDIQFCHDLIDYFKECINRHPDWKNFNFKFSDTE TYEDISEFYREVEKQGYKIDWTYISEEDIDKLDEEGQLYLFQIYNKDFSEKSTGKDNLHT MYLKNLFSEENLKDIVLKLNGEAELFFRKSSIKKPIVHKKGSILVNKTYKEQENNKTVRK SIPEEVYMELYKYFNNKGKGELSDEAKKYMDKVEHHEATKDIVKDYRYTVDKFFIHLPI TINFKADGRSNVNDMVLKYIAQQDDMHVIGIDRGERNLIYVSVIDMHGNIVEQKSFNIV NNYDYKEKLKQREKSRDNARKNWKEIGKIKELKEGYLSLVIHEIAKMVVKYNAIIAME DLNYGFKRGRFKVERQVYQKFETMLINKLNYLVDKDRAVDEPGGVLRGYQLTYIPDSL KKLGRCGFIFYVPAAYTSKIDPTTGFVNIFNFKELTTSEARRDFITKFDSIRYDAERNMFA FSFDYNNFATHNTTMARTKWTVYTNGTRIKKKFVNGRWTSSEEVDLTEKMKELLENSD IEYKDGHDLLEDIVEMDNQSNIVKEILEIFRLTVQMRNSKSENEDTDYDRIISPVLNEDGE FFDSSKYKDNKEATLPIDADANGAYCIALKGLYEVKQIKENWKEGEKFPRDTLKISNAD WFDFIQNRRYL (SEQ ID NO: 6).BIOTN-41612.601

[0207] Artificial Casl2a,7 (anCPF1.7; also referred to as anCPF7):MTNFKEFTNLYPISKTLRFELKPVGKTLEHIEKNGLLEEDEQRAEDYKKVKKIIDDYHRH FIDEALSNVNLDWNPLAEALEKYHKNKDDKDKKELEKVQDKMRKEIVKFFSNDERFKN LFKAKKELFTEILPEWIKKEETTSEKEKEEELEAVESFKKFTTYFTGFHENRKNMYSDEDI STAIAYRIVHENFPKFLDNVKVFERIKEKCPEVIEKVEKELKGYLGGNSLDDIFSIDYFNH LLTQSGIDYYNQIIGGITAEEGDTKIQGINEFLNLYRQQHKELAKSKRLFKMTPLYKQILS DRESLSFIPEAFENDEELIDAVNGFYKNLEKGKNVLDRIKELLSNLNDYDLDKIYINNKSL TAISQKLFGDWSVIKDALKEYAEETIGGKSKKREKKKEKALKKSKYFSLAEINEAVESYS DESSAASASIAEYFAKLTSASKISDLIEQIEANYSEVKKLFATPSEDKKLAENEEEVEQIKD FLDSIMDLLHRLKPFHVDDVLDKDTAFYSEFDEIYDELEDIVPLYNKVRNYLTQKPYSTE KFKLNFENPTLANGWDQNKEQDNTAIILMKDGKYYLGIMNAKNKPKFENTAGDSAEKS DNCYEKMVYKLLPGPNKMLPKVFFSKKGIEHYNPSEEILEGYEKGKHKKGENFDLDFC HKLIDFFKDSINKHPDWKKFGFKFSPTESYEDISEFYREVEQQGYKITFTNISESTIDKLVE EGKLYLFQIYNKDFSPGSTGKPNLHTLYWKALFSEENLKDVVLKLNGEAELFYRKKSIK KPIVHKAGSVLVNKTCKDKSTPIPEEIYKELYKYYNGKLKEADLTAEAKLDEDKVKIKE AKHDIVKDRRYTEDKFFFHVPITINFKAEGNSSNINEKVNEYLKDNPDVHIIGIDRGERHL IYVTLIDQKGKILEQKSFNVINNEQNEHGFDYHEKLDQREKERDAARKSWTTIGKIKELK EGYLSQVVHEIAKMMIEYNAIVVMEDLNFGFKRGRFKVEKQVYQKFEKMLIDKLNYLV FKDKGADEPGGVLNGYQLTNPFESFQKLGKQSGFIFYVPAAYTSKIDPTTGFVNLFNLSS ITNVEKRKEFFSKFDSIRYDAEKGMFAFAFDYKNFNTKGTAARTKWTVYTHGERIVYFR KNNRWESYDVNPTEELKELLEKHGIEYKDGNDLLPEIVEIDQDDSGFFKELLHVFKLTLQ MRNSKAETETEEDYIISPVANDDGEFFDSRKAKKDATLPIDADANGAYHIALKGLLLLQ RIKENYDEDEKLKKAKLAISNADWFEFVQERRYQ (SEQ ID NO: 7).

[0208] Artificial Casl2a,8 (anCPF1.8; also referred to as anCPF8):MNNMNTNFEDFIGISSVSKTLRNALIPTESTKKHIEENGIIKEDELRAEKRQELKEIMDDY YRSFIDETLSNVHDIDWKPLFEKMEETLKNKSTKKELEKVQDEKRKEIHKIFSSDDRFKN MFNAKLITDILPEFIKHNNNYSEEEKEEKLQTVKLFNRFATSFTDFFKNRKNCFSEDDIST SICYRIVNENAEIFLQNMRAFQRIKKTAEEEIEKIEKEMKDSLDGWSLEEIFSFDFYGRLLT QSGIDFYNDICGKVNQHMNLYCQKNKVNKNKFKMKRLHKQILCISESSYEVPYKFESDE EVYDAVNGFLDNIKEKNIIERLRKIGQNCDDYDLNKIYISSKHYENISQYMYGNWNVIND CLKTYYNDTLPGKGKSKEEKVKKAVKEDKYRSIAEINELVDKYNSESDKNIKAEEYISEIBIOTN-41612.601SDIINDMEVNELEYDSDINLIENEEKASEIKNVLDSFMNIFHWCKTFIVDEVVDKDTNFYS ELEDIYDELQPIVSLYNRVRNYVTQKPYSTEKIKLNFGTPTLANGWSKSKEYDNNAIILM RDNKYYLGIFNAKNKPDKKIIEGHESEKDGDYKKMVYNLLPGPNKMLPKVFLSSKKGIE TYKPSEYILEGYKQNKHIKSSKNFDINFCHDLIDYFKECINRHPDWKNFNFKFSDTETYEDISGFYREVEKQGYKIDWTYISEEDIDKLDEKGQLYLFQIYNKDFSEKSTGKDNLHTMYL KNLFSEENLKDIVLKLNGEAELFFRKSSIKKPIVHKKGSILVNRTYNEQENNKTVRKSIPE EVYMELYKYFNNKGKGELSDEAKKYMDEGKVEHHEATKDIVKDYRYTVDKFFIHLPIT INFKAEGRSNVNDMVLKYIAKQDDMHVIGIDRGERNLIYVSVIDTHGNIVEQKSFNIVNGYDYQEKLKQREKSRDAARKNWKEIGKIKELKEGYLSLVIHEIAKMVVKYNAIIAMEDLN YGFKRGRFKVERQVYQKFETMLINKLNYLVFKDRAVDEEGGVLRGYQLTYIPESLKKV GRQCGFIFYVPAAYTSKIDPTTGFVNIFNFKDLTTNEARRDFITKFDSIRYDAEKNMFAFS FDYNNFVTHNTTMARTKWTVYTNGTRIKRKFVNGRWTSKSEEVDLTEEMKKLLEKSDIEYKDGHDLREDIVEMEGIVKEILEIFRLTVQMRNSLSESEDRDYDRLISPVLNENGEFFDS AKADKTLPIDADANGAYCIALKGLYEVKQIKENWKEGEKFPRDKLKISNKDWFDFIQNK RYL (SEQ ID NO: 8).

[0209] Artificial Casl2a,9 (anCPF1.9; also referred to as anCPF9):KNMMTNFKDFTNLYPISKTLRFELKPVGKTLEHIEKNGLLEEDEQRAEDYKKVKKIIDD YHRHFIDETLSNVNLDWNPLAEALEKYHKNKDDKDKKELEKEQDKMRKEIVKMFSND ERFKNLFAAKKELFSEILPEWIKKTTSEKEKEEELEAVKSFKKFSTYFTGFHENRKNMYS DEDISTAIAYRIVHENFPKFLDNVKVFERIKEKCPEVIEKVEKELKGYLGGNSLDDIFSIDYFNHLLTQSGIDYYNQIIGGITAKEGDKKLQGINEFLNLYRQQHKELAKSKRRIKMTPLYK QILSDRESLSFIPEAFENDEELIDAVNGFYKNLEKGKNILDRIKELLSNLNDYDLDKIYINN KNLTAISQKLFGDWSVIEDALKEYAEETIGGKSQKREKKKVDAWTSKKSKYFSLAEINE AVESYSDESSAVSASIAEYFAKLTSALSKISDLCEQIEANYKEVNKLFATPSEDKKLAENEEEVEKIKNFLDSIMDLLHRLKPFKVNDVLDKDTAFYSEFDEIYDELHEIVPLYNKVRNYL TQKPYSTEKFKLNFENPTLANGWDQNKEQDNTAIILMKDGKYYLGIMNAKNKPKFEDT AGDSSDNCYQKMVYKLLPGPNKMLPKVFFSKKGIEHYNPSEEILEGYEKGKHKKGENF DLDFCHKLIDFFKDSINKHPDWKKFGFKFSPTESYEDISEFYREVEKQGYKITFTNISESTIDKLVEEGKLYLFQIYNKDFAEGATGKPNLHTLYWKALFSEENLKDVVLKLNGEAELFY RKKSIKKPIVHKAGSVLVNKTCKDGTPIPEEIYKELYKYYNGKKKEADLTAEAKIDEDK VKIKEANHDIVKDRRYTEDKFFFHVPITINFKAEGNSSNINEKVNEYLKDNPDVHIIGIDRBIOTN-41612.601GERHLIYVTLIDQKGKILEQKSFNVINNKQNTEHGFDYHEKLDQREKERDAARKSWTTI GKIKELKEGYLSQVVHEIAKMMIEYNAIVVMEDLNFGFKRGRFKVEKQVYQKFEKMLI DKLNYLVFKDKGFDEPGGVLNGYQLTNPFESFQKLGKQSGFIFYVPAAYTSKIDPTTGF VNLFNLSDITNVEKRKEFFSKFDSIRYDAEKGMFAFAFDYKNFNTKGTTSARTKWTVYTHGERIVYFRKNKRYERMDVNPTEELKEALEKHGIEYKDGEDLLPEIVEIDANQDDSGFF KELFHVFKLTLQMRNSKAETEEDYIISPVANDDGEFFDSRKAKKKDATLPIDADANGAY HIALKGLLLLRRIKEKYDEDAKLKKAKLAISNADWFEFVQERRYQK (SEQ ID NO: 9.)

[0210] Cpfl [Eubacterium rectale]:

[0211] MNNGTNNFQNFIGIS SLQKTLRNALIPTETTQQFIVKNGIIKEDELRGENRQILK DIMDDYYRGFISETLSSIDDIDWTSLFEKMEIQLKNGDNKDTLIKEQAEKRKAIYKKFAD DDRFKNMFSAKLISDILPEFVIHNNNYSASEKEEKTQVIKLFSRFATSFKDYFKNRANCFS ADDISSSSCHRIVNDNAEIFFSNALVYRRIVKNLSNDDINKISGDIKDSLKEMSLEEIYSYEKYGEFITQEGISFYNDICGKVNSFMNLYCQKNKENKNLYKLRKLHKQILCIADTSYEVPY KFESDEEVYQSVNGFLDNISSKHIVERLRKIGDNYNGYNLDKIYIVSKFYESVSQKTYRD WETINTALEIHYNNILPGNGKSKADKVKKAVKNDLQKSITEINELVSNYKLCPDDNIKAE TYIHEISHILNNFEAQELKYNPEIHLVESELKASELKNVLDVIMNAFHWCSVFMTEELVD KDNNFYAELEEIYDEIYTVISLYNLVRNYVTQKPYSTKKIKLNFGIPTLADGWSKSKEYS NNAIILMRDNLYYLGIFNAKNKPDKKIIEGNTSENKGDYKKMIYNLLPGPNKMIPKVFLS SKTGVETYKPSAYILEGYKQNKHLKSSKDFDITFCHDLIDYFKNCIAIHPEWKNFGFDFS DTSTYEDISGFYREVELQGYKIDWTYISEKDIDLLQEKGQLYLFQIYNKDFSKKSSGNDN LHTMYLKNLFSEENLKDIVLKLNGEAEIFFRKSSIKNPIIHKKGSILVNRTYEAEEKDQFG NIQIVRKTIPENIYQELYKYFNDKSDKELSDEAAKLKNVVGHHEAATNIVKDYRYTYDK YFLHMPITINFKANKTSFINDRILQYIAKEKDLHVIGIDRGERNLIYVSVIDTCGNIVEQKS FNIVNGYDYQIKLKQQEGARQIARKEWKEIGKIKEIKEGYLSLVIHEISKMVIKYNAIIAMEDLSYGFKKGRFKVERQVYQKFETMLINKLNYLVFKDISITENGGLLKGYQLTYIPEKLK NVGHQCGCIFYVPAAYTSKIDPTTGFVNIFKFKDLTVDAKREFIKKFDSIRYDSEKNLFCF TFDYNNFITQNTVMSKSSWSVYTYGVRIKRRFVNGRFSNESDTIDITKDMEKTLEMTDIN WRDGHDLRQDIIDYEIVQHIFEIFKLTVQMRNSLSELEDRDYDRLISPVLNENNIFYDSAK AGDALPKDADANGAYCIALKGLYEIKQITENWKEDGKFSRDKLKISNKDWFDFIQNKR YL (SEQ ID NO: 10).

[0212] Hypothetical [Synergistesjonesii]:BIOTN-41612.601

[0213] MANSLKDFTNIYQLSKTLRFELKPIGKTEEHINRKLIIMHDEKRGEDYKSVTKL IDDYHRKFIHETLDPAHFDWNPLAEALIQSGSKNNKALPAEQKEMREKIISMFTSQAVYK KLFKKELFSELLPEMIKSELVSDLEKQAQLDAVKSFDKFSTYFTGFHENRKNIYSKKDTS TSIAFRIVHQNFPKFLANVRAYTLIKERAPEVIDKAQKELSGILGGKTLDDIFSIESFNNVL TQDKIDYYNQIIGGVSGKAGDKKLRGVNEFSNLYRQQHPEVASLRIKMVPLYKQILSDR TTLSFVPEALKDDEQAINAVDGLRSELERNDIFNRIKRLFGKNNLYSLDKIWIKNSSISAF SNELFKNWSFIEDALKEFKENEFNGARSAGKKAEKWLKSKYFSFADIDAAVKSYSEQVS ADISSAPSASYFAKFTNLIETAAENGRKFSYFAAESKAFRGDDGKTEIIKAYLDSLNDILH CLKPFETEDISDIDTEFYSAFAEIYDSVKDVIPVYNAVRNYTTQKPFSTEKFKLNFENPAL AKGWDKNKEQNNTAIILMKDGKYYLGVIDKNNKLRADDLADDGSAYGYMKMNYKFI PTPHMELPKVFLPKRAPKRYNPSREILLIKENKTFIKDKNFNRTDCHKLIDFFKDSINKHK DWRTFGFDFSDTDSYEDISDFYMEVQDQGYKLTFTRLSAEKIDKWVEEGRLFLFQIYNK DFADGAQGSPNLHTLYWKAIFSEENLKDVVLKLNGEAELFFRRKSIDKPAVHAKGSMK VNRRDIDGNPIDEGTYVEICGYANGKRDMASLNAGARGLIESGLVRITEVKHELVKDKR YTIDKYFFHVPFTINFKAQGQGNINSDVNLFLRNNKDVNIIGIDRGERNLVYVSLIDRDGH IKLQKDFNIIGGMDYHAKLNQKEKERDTARKSWKTIGTIKELKEGYLSQVVHEIVRLAV DNNAVIVMEDLNIGFKRGRFKVEKQVYQKFEKMLIDKLNYLVFKDAGYDAPCGILKGL QLTEKFESFTKLGKQCGIIFYIPAGYTSKIDPTTGFVNLFNINDVSSKEKQKDFIGKLDSIR FDAKRDMFTFEFDYDKFRTYQTSYRKKWAVWTNGKRIVREKDKDGKFRMNDRLLTED MKNILNKYALAYKAGEDILPDVISRDKSLASEIFYVFKNTLQMRNSKRDTGEDFIISPVL NAKGRFFDSRKTDAALPIDADANGAYHIALKGSLVLDAIDEKLKEDGRIDYKDMAVSNP KWFEFMQTRKFDF (SEQ ID NO: 11).

[0214] Hypothetical [Clostridium sp.]:

[0215] MEDKQFLERYKEFIGLNSLSKTLRNSLIPVGSTLKHIQEYGILEEDSLRAQKREE LKGIMDDYYRNYIEMHLRDVHDIDWNELFEALTEVKKNQTDDAKKCLEKIQEKKRKEI YQYLSDDAVFSEMFKEKMISGILPDFIRCNEEYSEEEKEEKLKTVALFHRFTSSFNDFFLN RKNVFTKEAIATAIGYRVVHENAEIFLENMVAFQNIQKSAESQISIIERKNEHYFMEWKL SHIFTADYYMMLMTQKAIEHYNEMCGVVNQHMKEYCQKEKKNWNLYRMKRLHKQIL SNASTSFKIPEKYENDAEVYESVNSFLQNVMEKTVMERIAVLKNNTDNFDLSKIYITAPY YEKISNYLCGSWNTIADCLTHYYEQQIAGKGARKDQKVKAAVKADKWKSLSEIEQLLK EYARAEEVKRKPEEYIAEIENIVSLKEVHLLEYHPEVNLIENEKYATEIKDVLDNYMELFBIOTN-41612.601HWMKWFYIEEAVEKEVNFYGELDDLYEEIRDIVPLYNKVRNYVTQKPYSDTKIKLNFGTPTLANGWSKSKEYDYNAILLQKDGKYYMGIFNPVQKPEKEIIEGHSHPLEGNEYKKMVYYYLPSANKMLPKVLLSKKGMEIYQPSEYIINGYKERRHIKSEEKFDLQFCHDLIDYFKSGIERNPDWKVFGFHFSDTDTYQDISGFYREVEDQGYKIDWTYIKEADIDRLNEEGKLYLFQIYNKDFSEKSTGRENLHTMYLKNLFSEENIREQVLKLNGEAEIFFRKSSVKKPIIHKKGTMLVNRTYMEEMHGESVKKNIPEKEYQEIYNYMNHRWKGELSAEAKEYLKKAVCHETKKDIVKDYRYSVDKFFIHLPITINYRASGKEALNSVAQRYIAHQNDMHVIGIDRGERNLIYVSVINMQGEIIEQKSFNVVNKYNYKEKLKEREQNRDEARKNWKEIGQIKDLKEGYLSGVIHEIAKMMIKYHAIVAMEDLNYGFKRGRFKVERQVYQKFENMLIQKLNYLVFKDRSADEDGGVLRGYQLAYIPDSVKKLGRQCGMIFYVPAAFTSKIDPATGFVDIFNHKAYTTDQAKREFILSFDEICYDVERQLFRFTFDYANFATHNVTLARNNWTIYTNGTRTQKEFVNRRVRDKKEVFDPTEKMLKLLELEGVEYQSGANLLPKLEKISDPHLFHELQRIVRFTVQLRNSKNEENDVDYDHVISPVLNEEGKFFDSSKYENKEEKKESLLPVDADANGAYCIALKGLYIMQAIQKNWSEEKALSPDVLRLNNNDWFDYIQNKRYR (SEQ ID NO: 12).

[0216] Cpfl [Candidatus Methanomethylophilus alvus]:

[0217] MDAKEFTGQYPLSKTLRFELRPIGRTWDNLEASGYLAEDRHRAECYPRAKELLDDNHRAFLNRVLPQIDMDWHPIAEAFCKVHKNPGNKELAQDYNLQLSKRRKEISAYLQDADGYKGLFAKPALDEAMKIAKENGNESDIEVLEAFNGFSVYFTGYHESRENIYSDEDMVSVAYRITEDNFPRFVSNALIFDKLNESHPDIISEVSGNLGVDDIGKYFDVSNYNNFLSQAGIDDYNHIIGGHTTEDGLIQAFNVVLNLRHQKDPGFEKIQFKQLYKQILSVRTSKSYIPKQFDNSKEMVDCICDYVSKIEKSETVERALKLVRNISSFDLRGIFVNKKNLRILSNKLIGDWDAIETALMHSSSSENDKKSVYDSAEAFTLDDIFSSVKKFSDASAEDIGNRAEDICRVISETAPFINDLRAVDLDSLNDDGYEAAVSKIRESLEPYMDLFHELEIFSVGDEFPKCAAFYSELEEVSEQLIEIIPLFNKARSFCTRKRYSTDKIKVNLKFPTLADGWDLNKERDNKAAILRKDGKYYLAILDMKKDLSSIRTSDEDESSFEKMEYKLLPSPVKMLPKIFVKSKAAKEKYGLTDRMLECYDKGMHKSGSAFDLGFCHELIDYYKRCIAEYPGWDVFDFKFRETSDYGSMKEFNEDVAGAGYYMSLRKIPCSEVYRLLDEKSIYLFQIYNKDYSENAHGNKNMHTMYWEGLFSPQNLESPVFKLSGGAELFFRKSSIPNDAKTVHPKGSVLVPRNDVNGRRIPDSIYRELTRYFNRGDCRISDEAKSYLDKVKTKKADHDIVKDRRFTVDKMMFHVPIAMNFKAISKPNLNKKVIDGIIDDQDLKIIGIDRGERNLIYVTMVDRKGNILYQDSLNILNGYDYRKALDVREYDNKEARRNWTKVEGIRKMKEGYLSLAVSKLADMIIENNAHVMEDLNHGFKAGRSKIBIOTN-41612.601EKQVYQKFESMLINKLGYMVLKDKSIDQSGGALHGYQLANHVTTLASVGKQCGVIFYI PAAFTSKIDPTTGFADLFALSNVKNVASMREFFSKMKSVIYDKAEGKFAFTFDYLDYNV KSECGRTLWTVYTVGERFTYSRVNREYVRKVPTDIIYDALQKAGISVEGDLRDRIAESD GDTLKSIFYAFKYALDMRVENREEDYIQSPVKNASGEFFCSKNAGKSLPQDSDANGAYN IALKGILQLRMLSEQYDPNAESIRLPLITNKAWLTFMQSGMKTWKN (SEQ ID NO: 13).

[0218] Cpfl [Candidatus Methanoplasma termitum]:

[0219] MNNYDEFTKLYPIQKTIRFELKPQGRTMEHLETFNFFEEDRDRAEKYKILKEAI DEYHKKFIDEHLTNMSLDWNSLKQISEKYYKSREEKDKKVFLSEQKRMRQEIVSEFKKD DRFKDLFSKKLFSELLKEEIYKKGNHQEIDALKSFDKFSGYFIGLHENRKNMYSDGDEIT AISNRIVNENFPKFLDNLQKYQEARKKYPEWIIKAESALVAHNIKMDEVFSLEYFNKVLNQEGIQRYNLALGGYVTKSGEKMMGLNDALNLAHQSEKSSKGRIHMTPLFKQILSEKESF SYIPDVFTEDSQLLPSIGGFFAQIENDKDGNIFDRALELISSYAEYDTERIYIRQADINRVSN VIFGEWGTLGGLMREYKADSINDINLERTCKKVDKWLDSKEFALSDVLEAIKRTGNND AFNEYISKMRTAREKIDAARKEMKFISEKISGDEESIHIIKTLLDSVQQFLHFFNLFKARQ DIPLDGAFYAEFDEVHSKLFAIVPLYNKVRNYLTKNNLNTKKIKLNFKNPTLANGWDQN KVYDYASLIFLRDGNYYLGIINPKRKKNIKFEQGSGNGPFYRKMVYKQIPGPNKNLPRVF LTSTKGKKEYKPSKEIIEGYEADKHIRGDKFDLDFCHKLIDFFKESIEKHKDWSKFNFYFS PTESYGDISEFYLDVEKQGYRMHFENISAETIDEYVEKGDLFLFQIYNKDFVKAATGKKD MHTIYWNAAFSPENLQDVVVKLNGEAELFYRDKSDIKEIVHREGEILVNRTYNGRTPVP DKIHKKLTDYHNGRTKDLGEAKEYLDKVRYFKAHYDITKDRRYLNDKIYFHVPLTLNF KANGKKNLNKMVIEKFLSDEKAHIIGIDRGERNLLYYSIIDRSGKIIDQQSLNVIDGFDYR EKLNQREIEMKDARQSWNAIGKIKDLKEGYLSKAVHEITKMAIQYNAIVVMEELNYGF KRGRFKVEKQIYQKFENMLIDKMNYLVFKDAPDESPGGVLNAYQLTNPLESFAKLGKQ TGILFYVPAAYTSKIDPTTGFVNLFNTSSKTNAQERKEFLQKFESISYSAKDGGIFAFAFD YRKFGTSKTDHKNVWTAYTNGERMRYIKEKKRNELFDPSKEIKEALTSSGIKYDGGQNI LPDILRSNNNGLIYTMYSSFIAAIQMRVYDGKEDYIISPIKNSKGEFFRTDPKRRELPIDAD ANGAYNIALRGELTMRAIAEKFDPDSEKMAKLELKHKDWFEFMQTRGD (SEQ ID NO: 14).

[0220] Cpfl [Succiniclasticum ruminis]:

[0221] MGNFGEFTHKYQVSKTLRFELIPQGKTLENVAKYGIVDDDKRRSENYKKLKP VIDRIYKYFIDESLKNVSIDWQPLYEAIIAYRKEQTTANVVRLKEEQEACRKAIAAWFEGBIOTN-41612.601KVPDKGSKDLKEFNKTQSKLFKELFGKELFTESVTQLLPGLSLTEEEKELLASFNKFTSY FKGFYVNRKNVFSADDISTSIPHRLVQENFPKFMDNCEAYRRIVEEYPELKAKLEGTAQ ATGIFIGFKLDNIFKVSFYNHLLQQSQIDLYNQFLCGIAGEEGTMRVQGLNVTLNLAMKQ DKVLGQKLKSMPHRFIPLYKQILSDRTTLSFIPEAFQNDEEVLLTVEEYRKSLEAERTTGA VSDIFNSLQAADLRHVYVNPAKLTAFSQMLFEDWSLCRESLRNWKLRSYGKAATKKVR EEIESWLKESAISLDELQAALADGTLSVIINQKVQSVITTLEQELAKPLPKKLKTAEEKES LKSLLDSVQEACHSLEMFAVGENMDTDPCFYVPLREAMEAIQPIIPLYNKVRNFATQKP YSIEKFKLNFSNPILASGWDENRERQTCAILFRKGEKYYLGIYNAKVKPDFSIIKAVKGG NCFEKWYRQFPDFSKMMPKCTTQLKEVQQHFAS S SEDYVLYNKKFIKPLTITKEIYDL NNVLFDGKKKFQIDYLRKTKDEDGYYHALHTWINFAKEFVASYESTSIYDTSTVLSTEQ YVKLNDFYGDLDNLFYRIKFESVSEETISEFVDEGKLFLFQIYNKDFAEGATGAPNLHTIY WKAVFDPENMKNVVVKLNGQAELFYRPKSAMDIVRHKVGEKLVNRRLKDGTSLTEEL HEELYLYANGKLKKKLSEAAAAVLPQAVIYDVHHEIVKDRRFTEDKFFFHVPLTLNYKC DKNAVQFNASVQEYLKENPDTYIIGIDRGERNLIYAVVIDPQGNIVEQKSFNVINGFDYH NKLEQREKERNKARQDWTTVGKIKELKQGYLSLVVHEITSMMVKYNAIVVLENLNVGF KRIRSGIAEKAVYQQFEKMLINKLNYLMFKDVEGAKPGSVLNAYQLTDRFESFASMRN QTGFLFYIPAAFTSKIDPATGFVDPFCWSAIKTLDDKKTFISGFDTLKYDNVTGNFILHFE MKKNKDFQKKLEGFMPEWDIVVEANKDRRDAEGKTFISGKRIEFVRENNGHGHYEDYL PCKKLVEILRQYDILFEDGKDVLPLIMKNGDSKLIHEVFKVIRLSLQMRNSNAESGEDFIS SPVENNEGICFDSRLGVETLPKDADANGAYHIALKGLLLLEKIRHDERKLGISNSEWLNH IQSLRG (SEQ ID NO: 15).

[0222] Cpfl [Prevotella ruminicola] :

[0223] MIDLKQFIGIYPVSKTLRFELRPVGKTQEWIEKNRVLEGDEQKAADYPVVKKL IDDYHKVCIHDSLNHVHFDWEPLKDAIEIFQKTKSDEAKKRLEAEQAMMRKKIAAAIKD FKHFKELTAATPSDLITSVLPEFSDDGSLKSFRGFATYFSGFQENRNNIYSQEAISTGVPY RLVHDNFPKFLSDLEVFERIKSTCPEVINQASAELQPFLEGVMIDDIFSLDFYNSLLTQNGI DFFNQVIGGVSEKDKQKYRGINEFSNLYRQQHKEIAASKKAMTMIPLFKQILSDRDTLSY IPAQIRTEDELVSSITQFYDHITHFEHDGKTINVLSEIVALLGKLDTYDPNGICITARKLTDI SQKVYGKWSVIEEKMKEKAIQQYGDISVAKNKKKVDAFLSRKAYSLSDLCFDEEISFSR YYSELPQTLNAISGYWLQFNEWCKSDEKQKFLNNQTGTEVVKSLLDAMMELFHKCSVL VMPEEYEVDKSFYNEFLPLYEELDTLFLLYNKVRNYLTQKPSDVKKFKLNFESPSLASGBIOTN-41612.601WDQNKEMKNNAILLFKDGKSYLGVLNAKNKAKIKDAKGDVSSSSYKKMIYKLLSDPS KDLPHKIFAKGNLDFYKPSEYILEGRELGKYKKGPNFDKKFLHDFIDFYKAAISIDPDWSKFNFQYSPTESYDDIGMFFSEIKKQAYKIRFTDISEAQVNEWVDNGQLYLFQLYNKDYA EGAHGRKNLHTLYWENLFTDENLSNLVLKLNGQAELFCRPQSIKKPVSHKIGSKMLNRR DKSGMPIPESIYRSLYQYYNGKKKESELTVAEKQYIDQVIVKDVTHEIIKDRRYTRQEYF FHVPLTFNANADGNEYINEHVLNYLKDNPDVNIIGIDRGERHLIYLTLINQRGEILKQKTF NVVNSYNYQAKLEQREKERDEARKSWDSVGKIKDLKEGFLSAVIHEITNMMIENNAIVV LEDLNFGFKRGRFKVERQVYQKFEKMLIDKLNYLSFKDREAGEEGGILRGYQMAQKFIS FQRLGKQSGFLFYIPAAYTSKIDPVSGFVNHFNFSDITNAEKRKDFLMKMDRIEMKNGNI EFTFDYRKFKTFQTDYQNVWTVSTFGKRIVMRIDEKGYKKMVDYEPTNDIIKAFKNKGI LLSEGSDLKALIAEIEANATNAGFYSTLLYAFQKTLQMRNSNAVTEEDYILSPVAKDGH QFCSTDEANKGKDAQGNWVSKLPVDADANGAYHIALKGLYLLRNPETKKIENEKWLQ FMVEKPYLE (SEQ ID NO: 16).

[0224] Cpfl [Francisella tularensi]:

[0225] MSIYQEFVNKYSLSKTLRFELIPQGKTLENIKARGLILDDEKRAKDYKKAKQII DKYHQFFIEEILSSVCISEDLLQNYSDVYFKLKKSDDDNLQKDFKSAKDTIKKQISEYIKD SEKFKNLFNQNLIDAKKGQESDLILWLKQSKDNGIELFKANSDITDIDEALEIIKSFKGWT TYFKGFHENRKNVYSSNDIPTSIIYRIVDDNLPKFLENKAKYESLKDKAPEAINYEQIKKD LAEELTFDIDYKTSEVNQRVFSLDEVFEIANFNNYLNQSGITKFNTIIGGKFVNGENTKRK GINEYINLYSQQINDKTLKKYKMSVLFKQILSDTESKSFVIDKLEDDSDVVTTMQSFYEQI AAFKTVEEKSIKETLSLLFDDLKAQKLDLSKIYFKNDKSLTDLSQQVFDDYSVIGTAVLE YITQQIAPKNLDNPSKKEQELIAKKTEKAKYLSLETIKLALEEFNKHRDIDKQCRFEEILA NFAAIPMIFDEIAQNKDNLAQISIKYQNQGKKDLLQASAEDDVKAIKDLLDQTNNLLHKLKIFHISQSEDKANILDKDEHFYLVFEECYFELANIVPLYNKIRNYITQKPYSDEKFKLNFE NSTLANGWDKNKEPDNTAILFIKDDKYYLGVMNKKNNKIFDDKAIKENKGEGYKKIVY KLLPGANKMLPKVFFSAKSIKFYNPSEDILRIRNHSTHTKNGSPQKGYEKFEFNIEDCRKF IDFYKQSISKHPEWKDFGFRFSDTQRYNSIDEFYREVENQGYKLTFENISESYIDSVVNQG KLYLFQIYNKDFSAYSKGRPNLHTLYWKALFDERNLQDVVYKLNGEAELFYRKQSIPK KITHPAKEAIANKNKDNPKKESVFEYDLIKDKRFTEDKFFFHCPITINFKSSGANKFNDEINLLLKEKANDVHILSIDRGERHLAYYTLVDGKGNIIKQDTFNIIGNDRMKTNYHDKLAAI EKDRDSARKDWKKINNIKEMKEGYLSQVVHEIAKLVIEYNAIVVFEDLNFGFKRGRFKVBIOTN-41612.601EKQVYQKLEKMLIEKLNYLVFKDNEFDKTGGVLRAYQLTAPFETFKKMGKQTGIIYYV PAGFTSKICPVTGFVNQLYPKYESVSKSQEFFSKFDKICYNLDKGYFEFSFDYKNFGDKA AKGKWTIASFGSRLINFRNSDKNHNWDTREVYPTKELEKLLKDYSIEYGHGECIKAAIC GESDKKFFAKLTSVLNTILQMRNSKTGTELDYLISPVADVNGNFFDSRQAPKNMPQDAD ANGAYHIGLKGLMLLGRIKNNQEGKKLNLVIKNEEYFEFVQNRNN (SEQ ID NO: 17).

[0226] Cpfl [Flavobacterium sp]:

[0227] MKNFSNLYQVSKTVRFELKPIGNTLENIKNKSLLKNDSIRAESYQKMKKTIDEF HKYFIDLALNNKKLSYLNEYIALYTQSAEAKKEDKFKADFKKVQDNLRKEIVSSFTEGE AKAIFSVLDKKELITIELEKWKNENNLAVYLDESFKSFTTYFTGFHQNRKNMYSAEANS TAIAYRLIHENLPKFIENSKAFEKSSQIAELQPKIEKLYKEFEAYLNVNSISELFEIDYFNEV LTQKGITVYNNIIGGRTATEGKQKIQGLNEIINLYNQTKPKNERLPKLKQLYKQILSDRIS LSFLPDAFTEGKQVLKAVFEFYKINLLSYKQDGVEESQNLLELIQQVVKNLGNQDVNKI YLKNDTSLTTIAQQLFGDFSVFSAALQYRYETVVNPKYTAEYQKANEAKQEKLDKEKIK FVKQDYFSIAFLQEVVADYVKTLDENLDWKQKYTPSCIADYFTTHFIAKKENEADKTFN FIANIKAKYQCIQGILEQADDYEDELKQDQKLIDNIKFFLDAILEVVHFIKPLHLKSESITE KDNAFYDVFENYYEALNVVTPLYNMVRNYVTQKPYSTEKIKLNFENAQLLNGWDANK EKDYLTTILKRDGNYFLAIMDKKHNKTFQQFTEDDENYEKIVYKLLPGVNKMLPKVFFS NKNIAFFNPSKEILDNYKNNTHKKGATFNLKDCHALIDFFKDSLNKHEDWKYFDFQFSE TKTYQDLSGFYKEVEHQGYKINFKKVSVSQIDTLIEEGKMYLFQIYNKDFSPYAKGKPN MHTLYWKALFETQNLENVIYKLNGQAEIFFRKASIKKKNIITHKAHQPIAAKNPLTPTAK NTFAYDLIKDKRYTVDKFQFHVPITMNFKATGNSYINQDVLAYLKDNPEVNIIGLDRGE RHLVYLTLIDQKGTILLQESLNVIQDEKTHTPYHTLLDNKEIARDKARKNWGSIESIKELK EGYISQVVHKITKMMIEHNAIVVMEDLNFGFKRGRFKVEKQIYQKLEKMLIDKLNYLVL KDKQPHELGGLYNALQLTNKFESFQKMGKQSGFLFYVPAWNTSKIDPTTGFVNYFYTK YENVEKAKTFFSKFDSILYNKTKGYFEFVVKNYSDFNPKAADTRQEWTICTHGERIETK RQKEQNNNFVSTTIQLTEQFVNFFEKVGLDLSKELKTQLIAQNEKSFFEELFHLLKLTLQ MRNSESHTEIDYLISPVANEKGIFYDSRKATASLPIDADANGAYHIAKKGLWIMEQINKT NSEDDLKKVKLAISNREWLQYVQQVQKK (SEQ ID NO: 18).

[0228] Cpfl [Flavobacterium branchiophilu] :

[0229] MTNNFTNQYSLSKTLRFELIPQGKTLEFIQEKGLLSQDKQRAESYQEMKKTID KFHKYFIDLALSNAKLTHLETYLELYNKSAETKKEPKFKDDLKKVQDNLRKEIVKSFSEBIOTN-41612.601GEAKSIFAIIDKKELITVELEKWFENNEQKDIYFDEKFKTFTTYFTGFHQNRKNMYSVEP NSTAIAYRLIHENLPKFLENAKAFEKIKQVESLQVNFRELMGEFGDEGLIFVNELEEMFQI NYYNDVLSQNGITIYNSIISGFTKNDIKYKGLNEYINNYNQTKDKKDRLPKLKQLYKQIL SDRVSLSFLPDAFTDGKQVLKAIFDFYKINLLSYTIEGQEESQNLLQLIRQTIDNLSSFDIQKIYLKNDTHLTTISQQLFGDFSVFSSSLNYWYETKVNPKFETEYCKANEKKRETLDKAK AVFTKQDYFSIAFLQEVLSEYILTLDDTSDIVIKHSSNCVANYFKNHFVAKKENETDKTF DLIANITAKYQCIQGILENADQYEDELKQDQKLIDNLKFFLDAILELLHFIKPLHLKSDSIT EKDTAFYDVFENYYEALSLLTPLYNMVRNYVTQKPYSTEKIKLNFENAQLLNGWDANKEADYLTTILKKDGNYFLAIMDKKHNKAFQKFPEGKENYEKMVYKLLPGVNKMLPKVFF SNKNIAYFNPSKELLENYKKETHKKGDTFNLEHCHTLIDFFKDSLNKHEDWKYFDFQFS ETKSYQDLSGFYREVEHQGYKINFKNIDSEYIDGLVNEGKLFLFQIYSKDLSPFSKGKPN MHTLYWKALFEEQNLQNVIYKLNGQAEIFFRKASIKPKNIILHKKKIKIAKKHFIDKKTKTSEIVPVQTIKNLNMYYQGKISEKELTQDDLRYIDNFSIFNEENKTIDIIKDKRFTVDKFQF HVPITMNFKATGGSYINQTVLEYLQNNPEVKIIGLDRGERHLVYLTLIDQQGNILKQESL NTITDSKISTPYHKLLDNKEKERDLARKNWGTVENIKELKEGYISQVVHKIATLMAEEN AIVVMEELNFGFKRGRFKVEKQIYQKLEKMLIDKLNYLVLKDKQPQELGGLYNALQLTNKFESFQKMGKQSGFLFYVPAWNTSKIDPTTGFVNYFYTKYENLKNAKEFFDKFQSIRY NVKEKYFEFEVDKYSDFNPKAEGTQQAWTICTYGERIETKRQKDQNNKFVSTPINLTEKI EDFLGENKIVYGDGNCIKSQIASKDDKAFFETLLYWFKMTLQMRNSETRTDIDYLISPVM NDNGTFYNSRDYEKLENPTLPKDADANGAYHIAKKGLMLLNKIDQADLTKKVDLSISN RDWLQFVQKNK (SEQ ID NO: 19).

[0230] Cpfl [Brumimicrobium aurantiacu] :

[0231] MKNQINLFTNKFQLSKTLRFELKPQGKTLEHINSKGFIKNDEKRADSYKKMKA TIDAFHRDFIDLAMSNVKLTNLIDFEEIYNASNADKKDEKYKTKLSKIQEILRKEIAKGFK GEEVKDIFSKIDKKDLITKLLEEWIIENKIEDIHFDPEFKNFTTYFSGFHQNRKNMYTDQE QSTAIAYRLIHENLPRFIDNINIFQKINKVPDLEENLKKLYQEIEEYLGINAINEAFELEYFNETLSQKGIDIYNLILGGRTAEEGKQKIQGLNEYINLYNQKQDKKNRVPKLKVLYKQILSD RTRTSFLPDTFEDDEESSASQKVLDSINNFYLENLIDYLPNDKNSTINVLENLKLLLAELIN FELDKVYIKNDTSITNISMKIFKNYSVIREALNYFYENKIDPNFAHNFNNANTDKKREKL EKEKAKITKQTYLSISFIEEAIHLYINENSNGNQYKNTYKPNCIANYFKDFFIAENKEGSN KEFDFISKIKARYNTIKGVLNTPFPDNKRLHQEKNNIDNIKHFLDSIMEYLHFAKPLVLSGBIOTN-41612.601SFAFEKDEQFYTNFDELYNQLELIIPLYNKVRNYATQKPYSTEKFKLNFENSTLLNGWDVNKEEANTSILFIKNGFYYLGIMDKNHNKIFRNTPKSTNTDIYKKVNYKLLPGASKMLPKVFFGKKNLDYYKPSKDILRIRNHGTHTKGGKPQSGFDKLDFNLNDCHKLIDFFKDSIQKHPDWSKFKFKFSDTQIYESIDQFYRELEPQAYSITYTNIDSSFIEEQINEGKLYLFQIYNKDFSKFSNGKPNLHTLYWKALFDEQNLKDVTYKLNGEAEIFYRKKSIQHDRQIIHKRNQPIINKNPNNEKKESIFKYNIIKDKRYTIDKFQFHVPITLNFKAKGTDYINYDVLDYLKENPDVKIIGLDRGERHLIYLTLIDQKGKILEQISLNEIVNKKHNITTSYHNLLETKEIERDKARKNWGTVETIKELKEGYISQVVHKISKMMIEHNAIWMEDLNMGFKRGRFKVEKQVYQKLEKMLIDKLNYLVLKDRQPNEPAGIYNALQLTNKFESFQKLGKQSGFLFYVPAWNTSKIDPTTGFVNLFHVKYESVRKSQEFFNKFNSIKYNPKEAIFEFDFDYNEFTTRAEGTKTNWTVCTYGDRIKTFRNPEKLNQWDNKEINITTAFEDFFGRHNITYGNGSDIKSQLISREEKDFFSELIHLFRLTLQMRNSKTNSEIDYLISPVKNENGFFYDSRHADKNLPKDADANGAYHIAKKGLQWIKEIQSFEGNEWKKLKLDKTNKGWLKFVQENQ (SEQ ID NO: 20).

[0232] Cpfl [Candidatus Woesearchaeota]:

[0233] MSKLHFATLLTGAIMGVVRIDDKLKKLFWDLYSLRKTLRFELKPQGKTLEHIENKGLISQDEQRAESYKKMKKTINGFHKHFIEIAMSQVKLTKLNEFADLYNASPERKKEDGFKKAFEKIQADLRKDIAKAFNVGEAKEIFSKIDKKELVTELLEKWIRTQEEDLYFDERFKTFTTYFGGFHENRKNMYSDKEQSTAIAYRLIHENLPKFLDNIKVFERIKEISELYKNFPALYKEIKEYLDIEQIDEAFELYYYNKILTQKQIDAYNLIIGGMTAQEGKKKIQGLNEYINLYNQKQDKKDRIPTLKQLYKQILSDKESVSFLPDEFEDDAEETASQKVLNAIEEYYQANLVSFHPSGKDEAENVLEKIKELLTSLKDYDLNKIYLRNDKAITDISKTLFGDWNIIKAALEFAFLQTLETGKKGLSKKQEKEKEKYLEQSYFSIAEIEKALFAYKNEAEVLKDLKENTTPIADYFHTHFLAKKKEESDKEFDMIANIQAKYSCIKGILNTDYPKENRLHQDKKTIGDIKAFLDSLMELLHFVKQISLPSDTPLEKDEIFYSQFQPWYDQLQLLIPLYNKVRNYASQKPYSIEKFKLNFQSSTLAQGWDLNKEHNKNYSLIFKENDSFFLGIINIDDDKDVLRKDKRPEIFVDDSPLKKMVYKDLGDIKRQLPRIAFSDKAKKDVEDIGWNQNIEAIKKEFDKFQDAKKNDKDLWKNEFDRNKLSELIDYYKGVLKNHSERYEDTYKLEYKSTESYKNLGEFFDHIASQTYKVTFVGIDKNYINQLVDQGKLYLFQIYNKDFSKYSKGKPNLHTLYWKALFDPENLKNVMYKLNGGAELFYRKKSIKDLTIHKSNEPIKNKNPNNPKKESFFEYDIVKDRRYSVDKFQFHVPITLNFKATEKKYINNTVLGFLKDNPDVNIIGIDRGERHLLYLTLIDQKGKILEQRSLNTIVNNRVNIKTDYHKLLHNREKERDKARKDWGTIETIKELKDGYISIIVHEISRMMVEHNAIVBIOTN-41612.601VMEDLNFGFKRGRFKVEKQIYQKFEKKLIDKLNYLVFKDKANNETGGLYKALQLTSKF ESFTKLGKQSGFLFYVPAWNTSKIDPTTGFVNLFNTRYENVDKAKTFFNNFENIRYNTD KDNFEFEVKKYSLFNSKVDDTIQDWVICTHGERIKTFRNSEKNNQWDNKEIVLTEEFKN LFKEYNINYKRDLKNQINSQTNKDFFEQLLNLFKYTVQMRNSITNSETDYLISPVINSEGK FYDSRNAGDELPKDADANGAYHIAKKGLWVLQQINAHEGEDWKKLKLAISNKEWLQF VQARAK (SEQ ID NO: 21).

[0234] Cpfl [Bacteroidetes bacterium]:

[0235] MESPTTQLKKFTNLYQLSKTLRFELKPVGKTKEHIETKGILKKDEERAVNYKLI KKIIDGFHKHFIELAMQQVKLSKLDELAELYNASAERKKEESYKKELEQVQAALRKEIV KGFNIGEAKEIFSKIDKKELFTELLDEWVKNLEEKKLVDDFKTFTTYFTGFHENRKNMY TDKAQSTAIAYRLVHENLPKFLDNTKIFKQIETKFEASKIEEIETKLEPIIQGTSLSEIFTLD YYNHALTQAGIDFINNIIGGYTEDEGKKKIQGLNEYINLYNQKQEKKNRIPKLKILYKQIL SDRDSISFLPDAFEDSQEVLNAIQNYYQTNLIDFKPKDKEETENVLEETKKLLTELFSNEL SKIYIRNDKAITDISQALFNDWGVFKSALEYKFIQDLELGTKELSKKQENEKEKYLKQAY FSIAEIENALFAYQNETDVLNEIKENSHPIADYFTKHFKAKKKVDTSTSSVEKDFDLIANI DAKYSCIKGILNTDYPKDKKLNQEKKTIDDLKVFLDSLMELLHFVKPLALPNDSILEKDE NFYSHFESYYEQLELLIPLYNKVRNYAAKKPYSTEKFKLNFENATLLKGWDKNKEIDNT SVILRKRGLYYLAIMPQDNKNVFKKSPNLKNNESCFEKMDYKQMALPMGFGAFVRKCF GTAFQLGWNCPKSCINEEDKIIIKEDEVKNNRAEIIDCYKDFLNIYEKDGFQYKEYGFNF KESKEYESLREFFIDVEQKGYKIEFQNISENYIHQLVNEGKLYLFQIYNKDFSSYSKGKPN MHTMYWKALFDPENLKDVVYKLNGQAEVFYRKKSIEDKNIITHKANEPIENKNPKAKK TQSTFEYDLIKDKRYTVDKFHFHVPITINFKATGNNYINQQVLDHLKNNTDVNIIGLDRG ERHLIYLTLINQKGEILLQESLNTIVNKKFDIETPYHTLLQNKEDERAKARENWGVIENIKELKEGYLSQVVHKIAKLMVDYNAIVVMEDLNTGFKRGRFKVEKQVYQKLEKMLIDKL NYLVFKDKDPNEVGGLYNALQLTNKFESFSKMGKQSGFLFYVPAWNTSKIDPTTGFVN LFYAKYESIPKAQDFFTKFKSIRYNSDENYFEFAFDYNDFTTRAEGTKSDWTVCTYGDRI KTFRNPEKNNQWDNQEVNLIEQFEAFFGKHNITYGDGNCIKKQLIEQDKKEFFEELFHLF KLTLQMRNSITNSEIDYLISPVKNSKKEFYDSRKADSTLPKDADANGAYHIAKKGLMWL EKINSFKGSDWKKLDLDKTNKTWLNFVQETASEKHKKLQTV (SEQ ID NO: 22).

[0236] Cpfl [Sulfurimonas crateri]:BIOTN-41612.601

[0237] MLSNFTNQYQLSKTLRFELKPVGDTLKHIEKSGLIAQDEIRSQEYQEVKTIIDK YHKAFIDEALQNVVLSNLEEYEALFFERNRDEKAFEKLQAVLRKEIVAHFKQHPQYKTL FKKELIKADLKNWQELSDAEKELVSHFDNFTTYFTGFHENRANMYTDEAKHSSIAYRIIH ENLPIFLTNKKLFETIKQKAPHLAQETQDALLEYLSGAIVEDMFELSYFNHLLSQTHIDLY NQMIGGVKQDSLKIQGLNEKINLYRQANGLSKRELPNLKPLHKQILSDRETLSWLPESFE SDEELMQGVQAYFESEVLAFECCDGKVNLLEKLPELLHQTQDYDFSKVYFKNDLALTA ASQAIFKDYRIIKEALWEVNKPKKSKDLVADEEKFFNKKNSYFSIEQIDGALNSAQLSAN MMHYFQSESTKVIEQIQLTYNDWKRNSSNKELLKAFLDALLSYQRLLKPLNAPNDLEKD VAFYAYFDAYFTSLCGVVKLYDKVRNFMTKKPYSLEKFKLNFENSTLLDGWDVNKES DNTAILFRKEGLYYLGIMNKKYNKVFRNISSSQDEGYQKIDYKLLPGANKMLPKVFFSD KNKEYFKPNAKLLERYKAGEHKKGDNFDLDFCHELIDFFKTSIEKHQDWKHFAYQFSPT ESYEDLSGFYREVEQQGYKISYKNIAASFIDTLVAEGKLYFFQIYNKDFSPYSKGTPNMH TLYWRALFDEKNLADVIYKLNGQAEIFFRKKSIEYSQEKLQKGHHHEMLKDKFAYPIIK DRRFAFDKFQFHVPITLNFKAEGNENITPKTFEYIRSNPDNIKVIGIDRGERHLLYLSLIDA EGKIVEQFTLNQIINSYNGKDHVIDYHAKLDAKEKDRDKARKEWGTVENIKELKEGYLS HVIHKIATLIIEHGAVVAMEDLNFGFKRGRFKVEKQVYQKFEKALIDKLNYLVDKKKEP HKLGGLLNALQLTSKFQSFEKMGKQNGFLFYVPAWNTSKIDPVTGFVNLFDTRYASVE KSKAFFTKFQSICYNEAKDYFELVFDYNDFTEKAKETRSEWTLCTYGERIVSFRNAEKN HQWDSKTIHLTTEFKNLFGELHGNDVKEYILEQNSVEFFKSLIYLLKITLQMRNSITGTDI DYLVSPVADEAGNFYDSRKADTSLPKDADANGAYNIARKGLMLMHRIQNAEDLKKVN LAISNRDWLRNAQGLDK (SEQ ID NO: 23).

[0238] Cpfl [Bacteroidetes bacterium]:

[0239] MKNMESFINLYPVSKTLRFELKPIGKTLETFSRWIEELKEKEAIELKETGNLLA QDEHRAESYKKVKKILDEYHKWFITESLQNTKLNGLDVFYHNYMLPKKEDHEKKAFAS CQDNLRKQIVNAFRQETGLFNKLSGKELFKDSKEEVALLKAIVPYFDNKTLENIGVKSNE GALLLIEEFKDFTTYFGGFHENRKNMYSDEAKSTAVAFRLIHENLPRFIDNKKVFEEKIM NSELKDKFPEILKELEQILQVNEIEEMFQLDYFNDTLIQNGIDVYNHLIGGYAEEGKKKIQ GLNEHINLYNQIQKEKNKRIPRLKPLYKQILSDRETASFVIEAFENDGELLESLEKSYRLL QQEVFTPEGKEGLANLLAAIAESETHKIFLKNDLGLTEISQQIYESWSLIEEAWNKQYDN KQKKVTETETYVDNRKKAFKSIKSFSIAEVEEWVKALGNEKHKGKSVATYFKSLGKTD EKVSLIEQVENNYNIIKDLLNTPYPPSKDLAQQKDDVEKIKNYLDSLKALQRFIKPLLGSGBIOTN-41612.601EESDKDAHFYGEFTAFWDVLDKVTPLYNKVRNYMTKKPYSTEKFKLNFENSYFLNGWAQDYETKAGLIFLKDGNYFLAINNKKLDEKEKKQLKTNYEKNPAKRIILDFQKPDNKNIP RLFIRSKGDNFAPAVEKYNLPISDVIDIYDEGKFKTEYRKINEPEYLKSLHKLIDYFKLGFS KHESYKHYSFSWKKTHEYENIAQFYHDVEVSCYQVLDENINWDSLMEYVEQNKLYLFQ IYNKDFSPNSKGTPNMHTLYWKMLFNPDNLKDVVYKLNGQAEVFYRKASIKKENKIVH KANDPIDNKNELNKKKQNTFEYDIVKDKRYTVDKFQFHVPITLNFKAEGLNNLNSKVNE YIKECDDLHIIGIDRGERHLLYLSLIDMKGNIVKQFSLNEIVNEHKGNTYRTNYHNLLDK REKEREKERESWKTIETIKELKEGYISQVVHKITQLMIEYNAIVVLEDLNFGFKRGRFKVE KQVYQKFEKMLIDKLNYLVDKKKEANESGGTLKAYQLTDSYADFMKYKKKQCGFLFY VPAWNTSKIDPTTGFVNLFDTHYVNVSKAQEFFSKFKSIRYNAANNYFEFEVTDYFSFSG KAEGTKQNWIICTHGTRIINFRNPEKNSQWDNKEWITDEFKKLFEKHGIDYKNSSDLKG QIASQSEKAFFHNEKKDTKDPDGLLQLFKLALQMRNSFIKSEEDYLVSPVMNDEGEFFD SRKAQPNQPENADANGAYNIAMKGKWVVKQIRESEDLDKLKLAISNKEWLNFAQRS (SEQ ID NO: 24).

[0240] Cpfl [Prevotella brevi]:

[0241] MKQFTNLYQLSKTLRFELKPIGKTLEHINANGFIDNDAHRAESYKKVKKLIDD YHKDYIENVLNNFKLNGEYLQAYFDLYSQDTKDKQFKDIQDKLRKSIASALKGDDRYK TIDKKELIRQDMKTFLKKDTDKALLDEFYEFTTYFTGYHENRKNMYSDEAKSTAIAYRLIHDNLPKFIDNIAVFKKIANTSVADNFSTIYKNFEEYLNVNSIDEIFSLDYYNIVLTQTQIEV YNSIIGGRTLEDDTKIQGINEFVNLYNQQLANKKDRLPKLKPLFKQILSDRVQLSWLQEE FNTGADVLNAVKEYCTSYFDNVEESVKVLLTGISDYDLSKIYITNDLALTDVSQRMFGE WSIIPNAIEQRLRSDNPKKTNEKEEKYSDRISKLKKLPKSYSLGYINECISELNGIDIADYY ATLGAINTESKQEPSIPTSIQVHYNALKPILDTDYPREKNLSQDKLTVMQLKDLLDDFKA LQHFIKPLLGNGDEAEKDEKFYGELMQLWEVIDSITPLYNKVRNYCTRKPFSTEKIKVNF ENAQLLDGWDENKESTNASIILRKNGMYYLGIMKKEYRNILTKPMPSDGDCYDKVVYK FFKDITTMVPKCTTQMKSVKEHFSNSNDDYTLFEKDKFIAPVVITKEIFDLNNVLYNGVK KFQIGYLNNTGDSFGYNHAVEIWKSFCLKFLKAYKSTSIYDFSSIEKNIGCYNDLNSFYG AVNLLLYNLTYRKVSVDYIHQLVDEDKMYLFMIYNKDFSTYSKGTPNMHTLYWKMLF DESNLNDWYKLNGQAEVFYRKKSITYQHPTHPANKPIDNKNVNNPKKQSNFEYDLIK DKRYTVDKFMFHVPITLNFKGMGNGDINMQVREYIKTTDDLHFIGIDRGERHLLYICVIN GKGEIVEQYSLNEIVNNYKGTEYKTDYHTLLSERDKKRKEERSSWQTIEGIKELKSGYLSBIOTN-41612.601QVIHKITQLMIKYNAIVLLEDLNMGFKRGRQKVESSVYQQFEKALIDKLNYLVDKNKDA NEIGGLLHAYQLTNDPKLPNKNSKQSGFLFYVPAWNTSKIDPVTGFVNLLDTRYENVAK AQAFFKKFDSIRYNKEYDRFEFKFDYSNFTAKAEDTRTQWTLCTYGTRIETFRNAEKNS NWDSREIDLTTEWKTLFTQHNIPLNANLKEAILLQANKNFYTDILHLMKLTLQMRNSVT GTDIDYMVSPVANECGEFFDSRKVKEGLPVNADANGAYNIARKGLWLAQQIKNANDLS DVKLAITNKEWLQFAQKKQYLKD (SEQ ID NO: 25).

[0242] Cpfl [Prevotella albensi]:

[0243] MNIKNFTGLYPLSKTLRFELKPIGKTKENIEKNGILTKDEQRAKDYLIVKGFIDE YHKQFIKDRLWDFKLPLESEGEKNSLEEYQELYELTKRNDAQEADFTEIKDNLRSSITEQ LTKSGSAYDRIFKKEFIREDLVNFLEDEKDKNIVKQFEDFTTYFTGFYENRKNMYSSEEK STAIAYRLIHQNLPKFMDNMRSFAKIANSSVSEHFSDIYESWKEYLNVNSIEEIFQLDYFS ETLTQPHIEVYNYIIGKKVLEDGTEIKGINEYVNLYNQQQKDKSKRLPFLVPLYKQILSDR EKLSWIAEEFDSDKKMLSAITESYNHLHNVLMGNENESLRNLLLNIKDYNLEKINITNDL SLTEISQNLFGRYDVFTNGIKNKLRVLTPRKKKETDENFEDRINKIFKTQKSFSIAFLNKLP QPEMEDGKPRNIEDYFITQGAINTKSIQKEDIFAQIENAYEDAQVFLQIKDTDNKLSQNKT AVEKIKTLLDALKELQHFIKPLLGSGEENEKDELFYGSFLAIWDELDTITPLYNKVRNWL TRKPYSTEKIKLNFDNAQLLGGWDVNKEHDCAGILLRKNDSYYLGIINKKTNHIFDTDIT PSDGECYDKIDYKLLPGANKMLPKVFFSKSRIKEFEPSEAIINCYKKGTHKKGKNFNLTD CHRLINFFKTSIEKHEDWSKFGFKFSDTETYEDISGFYREVEQQGYRLTSHPVSASYIHSL VKEGKLYLFQIWNKDFSQFSKGTPNLHTLYWKMLFDKRNLSDVVYKLNGQAEVFYRK SSIEHQNRIIHPAQHPITNKNELNKKHTSTFKYDIIKDRRYTVDKFQFHVPITINFKATGQN NINPIVQEVIRQNGITHIIGIDRGERHLLYLSLIDLKGNIIKQMTLNEIINEYKGVTYKTNYH NLLEKREKERTEARHSWSSIESIKELKDGYMSQVIHKITDMMVKYNAIVVLEDLNGGFM RGRQKVEKQVYQKFEKKLIDKLNYLVDKKLDANEVGGVLNAYQLTNKFESFKKIGKQS GFLFYIPAWNTSKIDPITGFVNLFNTRYESIKETKVFWSKFDIIRYNKEKNWFEFVFDYNT FTTKAEGTRTKWTLCTHGTRIQTFRNPEKNAQWDNKEINLTESFKALFEKYKIDITSNLK ESIMQETEKKFFQELHNLLHLTLQMRNSVTGTDIDYLISPVADEDGNFYDSRINGKNFPE NADANGAYNIARKGLMLIRQIKQADPQKKFKFETITNKDWLKFAQDKPYLKD (SEQ ID NO: 26).

[0244] Cpfl [Parabacteroides distasoni]:BIOTN-41612.601

[0245] MKNILEQFVGLYPLSKTLRFELKPLGKTLEHIEKKGLIAQDEQRAEEYKLVKDI IDRYHKAFIHMCLKHFKLKMYSEQGYDSLEEYRKLASISKRNEKEEQQFDKVKENLRK QIVDAFKNGGSYDDLFKKELIQKHLPRFIEGEEEKRIVDNFNKFTTYFTGFHENRKNMYSDEKESTAIAYRLIHENLPLFLDNMKSFAKIAESEVAARFTEIETAYRTYLNVEHISELFTLD YFSTVLTQEQIEVYNNIIGGRVDDDNVKIQGLNEYVNLYNQQQKDRSKRLPLLKSLYKM ILSDRIAISWLPEEFKSDKEMIEAINNMHDDLKDILAGDNEDSLKSLLQHIGQYDLSKIYIA NNPGLTDISQQMFGCYDVFTNGIKQELRNSITPSKKEKADNEIYEERINKMFKSEKSFSIA YLNSLPHPKTDAPQKNVEDYFALLGTCNQNDEQPINLFAQIEMARLVASDILAGRHVNL NQSENDIKLIKDLLDAYKALQHFVKPLLGSGDEAEKDNEFDARLRAAWNALDIVTPLYN KVRNWLTRKPYSTEKIKLNFENAQLLGGWDQNKEPDCTSVLLRKDGMYYLAIMDKKA NHAFDCDCLPSDGACFEKIDYKLLPGANKMLPKVFFSKSRIKEFSPSESIIAAYKKGTHK KGPNFSLSDCHRLIDFFKASIDKHEDWSKFRFRFSDTKTYEDISGFYREVEQQGYMLGFR KVSEAFVNKLVDEGKLYLFHIWNKDFSKHSKGTPNLHTIYWKMLFDEKNLTDVIYKLN GQAEVFYRKKSLDLNKTTTHKAHAPITNKNTQNAKKGSVFDYDIIKNRRYTVDKFQFH VPITLNFKATGRNYINEHTQEAIRNNGIEHIIGIDRGERHLLYLSLIDLKGNIVKQMTLNDI VNEYNGRTYATNYKDLLATREGERTDARRNWQKIENIKEIKEGYLSQVVHILSKMMVD YKAIVVLEDLNTGFMRNRQKIERQVYEKFEKMLIDKLNCYVDKQKDADETGGALHPLQ LTNKFESFRKLGKQSGWLFYIPAWNTSKIDPVTGFVNMLDTRYENADKARCFFSKFDSI RYNADKDWFEFAMDYSKFTDKAKDTYTWWTLCSYGTRIKTFRNPAKNNLWDNEEVV LTDEFKKVFAAAGIDVHENLKEAICALTDKKYLEPLMRLMTLLVQMRNSATNSETDYL LSPVADESGMFYDSREGKETLPKDADANGAYNIARKGLWTIRRIQATNCEEKVNLVLSN REWLQFAQQKPYLND (SEQ ID NO: 27).

[0246] Cpfl [Bacteroidetes oral]:

[0247] MRKFNEFVGLYPISKTLRFELKPIGKTLEHIQRNKLLEHDAVRADDYVKVKKII DKYHKCLIDEALSGFTFDTEADGRSNNSLSEYYLYYNLKKRNEQEQKTFKTIQNNLRKQ IVNKLTQSEKYKRIDKKELITTDLPDFLTNESEKELVEKFKNFTTYFTEFHKNRKNMYSK EEKSTAIAFRLINENLPKFVDNIAAFEKVVSSPLAEKINALYEDFKEYLNVEEISRVFRLD YYDELLTQKQIDLYNAIVGGRTEEDNKIQIKGLNQYINEYNQQQTDRSNRLPKLKPLYK QILSDRESVSWLPPKFDSDKNLLIKIKECYDALSEKEKVFDKLESILKSLSTYDLSKIYISN DSQLSYISQKMFGRWDIISKAIREDCAKRNPQKSRESLEKFAERIDKKLKTIDSISIGDVDE CLAQLGETYVKRVEDYFVAMGESEIDDEQTDTTSFKKNIEGAYESVKELLNNADNITDNBIOTN-41612.601NLMQDKGNVEKIKTLLDAIKDLQRFIKPLLGKGDEADKDGVFYGEFTSLWTKLDQVTPL YNMVRNYLTSKPYSTKKIKLNFENSTLMDGWDLNKEPDNTTVIFCKDGLYYLGIMGKK YNRVFVDREDLPHDGECYDKMEYKLLPGANKMLPKVFFSETGIQRFLPSEELLGKYERG THKKGAGFDLGDCRALIDFFKKSIERHDDWKKFDFKFSDTSTYQDISEFYREVEQQGYK MSFRKVSVDYIKSLVEEGKLYLFQIYNKDFSAHSKGTPNMHTLYWKMLFDEENLKDVV YKLNGEAEVFFRKSSITVQSPTHPANSPIKNKNKDNQKKESKFEYDLIKDRRYTVDKFLF HVPITMNFKSVGGSNINQLVKRHIRSATDLHIIGIDRGERHLLYLTVIDSRGNIKEQFSLNE IVNEYNGNTYRTDYHELLDTREGERTEARRNWQTIQNIRELKEGYLSQVIHKISELAIKY NAVIVLEDLNFGFMRSRQKVEKQVYQKFEKMLIDKLNYLVDKKKPVAETGGLLRAYQL TGEFESFKTLGKQSGILFYVPAWNTSKIDPVTGFVNLFDTHYENIEKAKVFFDKFKSIRY NSDKDWFEFVVDDYTRFSPKAEGTRRDWTICTQGKRIQICRNHQRNNEWEGQEIDLTK AFKEHFEAYGVDISKDLREQINTQNKKEFFEELLRLLRLTLQMRNSMPSSDIDYLISPVA NDTGCFFDSRKQAELKENAVLPMNADANGAYNIARKGLLAIRKMKQEENDSAKISLAIS NKEWLKFAQTKPYLED (SEQ ID NO: 28).

[0248] Cpfl [Bamesiella sp]:

[0249] MKTLSDFTNLFPLSKTLRFKLIPIGNTLKNIEASGILDEDRHRAESYVKVKAIID EYHKAFIDRVLSDTCLQTESIGKHNSLEEFFFYYQIGAKSEQQKKTFKKIQDALRKQIADS LTKDKHFSRIDKKELIQEDLIQFVRDGEDAAEKTSLISEFQNFTVYFTGFHENRQNMYSP DEKSTAIAYRLINENLPKFVDNMKVFDRIAASELASCFDELYHNFEEYLQVERLHDIFSL DYFNLLLTQKHIDVYNALIGGKATETGEKIKGLNEYINLYNQRHKQEKLPKFKMLFKQI LTDREAISWLPRQFDDNSQLLSAIEQCYNHLSTYTLKDGSLKYLLENLHTYDTEKIFIRN DSLLTEISQRHYGSWSILPEAIKRHLERANPQKRRETYEAYQSRIEKAFKAYPGFSIAFLN GCLTETGKESPSIESYFESLGAVETETSQQENWFARIANAYTDFREMQNRLHATDVPLA QDAEAVARIKKLLDALKGLQLFIKPLLDTGEEAEKDERFYGDFTEFWNELDTITPLYNM VRNYLTRKPYSEEKIKLNFQNPTLLNGWDLNKEVDNTSVILRRNGRYYLAIMHRNHRR VFSQYPGTERGDCYEKMEYKLLPGANKMLPKVFFSKSRIDEFNPSEELLARYQQGTHKK GENFNLHDCHALIDFFKDSIEKHEEWRNFHFKFSDTSSYTDMSGFYREIETQGYKLSFVP VACEYIDELVRDGKIFLFQIYNKDFSTYSKGKPNMHTLYWEMLFDERNLMNVVYKLNG QAEIFFRKASLSARHPEHPAGLPIKKKQAPTEESCFPYDLIKNKRYTVDQFQFHVPITINFKATGTSNINPSVTDYIRTADDLHIIGIDRGERHLLYLVVIDSQGRICEQFSLNEIVTQYQGH QYRTDYHALLQKKEDERQKARQSWQSIENIKELKEGYLSQVVHKVSELMIKYKAIVVLBIOTN-41612.601EDLNAGFKRSRQKVEKQVYQKFEKMLIDKLNYLVFKTAEADQPGGLLHAYQLTNKFES FKKMGKQSGFLFYIPAWNTSKIDPTTGFVNLFDTRYENVDKSRAFFGKFDSIRYRADKG TFEWTFDYNNFHKKAEGTRSSWCLSSHGNRVRTFRNPAKNNQWDNEEIDLTQAFRDLF EAWGIEITSNLKEAICNQSEKKFFSELFELFKLMIQLRNSVTGTNIDYMVSPVENHYGTFF DSRTCDSSLPANADANGAYNIARKGLMLARRIQATPENDPISLTLSNKEWLRFAQGLDE TTTYE (SEQ ID NO: 29).

[0250] Cpfl [Phocaeicola_plebeiu]:

[0251] METFSGFTNLYPLSKTLRFRLIPVGETLKHFIDSGILEEDQHRAESYVKVKAIID DYHRAYIENSLSGFELPLESTGKFNSLEEYYLYHNIRNKTEEIQNLSSKVRTNLRKQVVA QLTKNEIFKRIDKKELIQSDLIDFVKNEPDANEKIALISEFRNFTVYFKGFHENRRNMYSD EEKSTSIAFRLIHENLPKFIDNMEVFAKIQNTSISENFDAIQKELCPELVTLCEMFKLGYFN KTLSQKQIDAYNTVIGGKTTSEGKKIKGLNEYINLYNQQHKQEKLPKMKLLFKQILSDRE SASWLPEKFENDSQVVGAIVNFWNTIHDTVLAEGGLKTIIASLGSYGLEGIFLKNDLQLT DISQKATGSWGKISSEIKQKIEVMNPQKKKESYETYQERIDKIFKSYKSFSLAFINECLRG EYKIEDYFLKLGAVNSSSLQKENHFSHILNTYTDVKEVIGLYSESTDTKLIQDNDSIQKIK QFLDAVKDLQAYVKPLLGNGDETGKDERFYGDLIEYWSLLDLITPLYNMVRNYVTQKP YSVDKIKINFQNPTLLNGWDLNKETDNTSVILRRDGKYYLAIMNNKSRKVFLKYPSGTD RNCYEKMEYKLLPGANKMLPKVFFSKSRINEFMPNERLLSNYEKGTHKKSGTCFSLDDC HTLIDFFKKSLDKHEDWKNFGFKFSDTSTYEDMSGFYKEVENQGYKLSFKPIDATYVDQ LVDEGKIFLFQIYNKDFSEHSKGTPNMHTLYWKMLFDETNLGDVVYKLNGEAEVFFRK ASINVSHPTHPANIPIKKKNLKHKDEERILKYDLIKDKRYTVDQFQFHVPITMNFKADGN GNINQKAIDYLRSASDTHIIGIDRGERNLLYLVVIDGNGKICEQFSLNEIEVEYNGEKYST NYHDLLNVKENERKQARQSWQSIANIKDLKEGYLSQVIHKISELMVKYNAIVVLEDLNA GFMRGRQKVEKQVYQKFEKKLIEKLNYLVFKKQSSDLPGGLMHAYQLANKFESFNTLG KQSGFLFYIPAWNTSKMDPVTGFVNLFDVKYESVDKAKSFFSKFDSIRYNVERDMFEW KFNYGEFTKKAEGTKTDWTVCSYGNRIITFRNPDKNSQWDNKEINLTENIKLLFERFGID LSSNLKDEIMQRTEKEFFIELISLFKLVLQMRNSWTGTDIDYLVSPVCNENGEFFDSRNV DETLPQNADANGAYNIARKGMILLDKIKKSNGEKKLALSITNREWLSFAQGCCKNG (SEQ ID NO: 30).

[0252] Cpfl [Sodaliphilus jjleomorphu]:BIOTN-41612.601

[0253] MKYTDFTGIYPVSKTLRFELIPQGSTVENMKREGILNNDMHRADSYKEMKKLI DEYHKVFIERCLSDFSLKYDDTGKHDSLEEYFFYYEQKRNDKTKKIFEDIQVALRKQISK RFTGDTAFKRLFKKELIKEDLPSFVKNDPVKTELIKEFSDFTTYFQEFHKNRKNMYTSDA KSTAIAYRIINENLPKFIDNINAFHIVAKVPEMQEHFKTIADELRSHLQVGDDIDKMFNLQ FFNKVLTQSQLAVYNAVIGGKSEGNKKIQGINEYVNLYNQQHKKARLPMLKLLYKQILS DRVAISWLQDEFDNDQDMLDTIEAFYNKLDSNETGVLGEGKLKQILMGLDGYNLDGVF LRNDLQLSEVSQRLCGGWNIIKDAMISDLKRSVQKKKKETGADFEERVSKLFSAQNSFSI AYINQCLGQAGIRCKIQDYFACLGAKEGENEAETTPDIFDQIAEAYHGAAPILNARPSSH NLAQDIEKVKAIKALLDALKRLQRFVKPLLGRGDEGDKDSFFYGDFMPIWEVLDQLTPL YNKVRNRMTRKPYSQEKIKLNFENSTLLNGWDLNKEHDNTSVILRREGLYYLGIMNKN YNKIFDANNVETIGDCYEKMIYKLLPGPNKMLPKVFFSKSRVQEFSPSKKILEIWESKSFK KGDNFNLDDCHALIDFYKDSIAKHPDWNKFNFKFSDTQSYTNISDFYRDVNQQGYSLSF TKVSVDYVNRMVDEGKLYLFQIYNKDFSPQSKGTPNMHTLYWRMLFDERNLHNVIYK LNGEAEVFYRKASLRCDRPTHPAHQPITCKNENDSKRVCVFDYDIIKNRRYTVDKFMFH VPITINYKCTGSDNINQQVCDYLRSAGDDTHIIGIDRGERNLLYLVIIDQHGTIKEQFSLNE IVNEYKGNTYCTNYHTLLEEKEAGNKKARQDWQTIESIKELKEGYLSQVIHKISMLMQR YHAIVVLEDLNGSFMRSRQKVEKQVYQKFEHMLINKLNYLVNKQYDAAEPGGLLHAL QLTSRMDSFKKLGKQSGFLFYIPAWNTSKIDPVTGFVNLFDTRYCNEAKAKEFFEKFDDI SYNDERDWFEFSFDYRHFTNKPTGTRTQWTLCTQGTRVRTFRNPEKSNHWDNEEFDLTQAFKDLFNKYGIDIASGLKARIVNGQLTKETSAVKDFYESLLKLLKLTLQMRNSVTGTDI DYLVSPVADKDGIFFDSRTCGSLLPANADANGAFNIARKGLMLLRQIQQSSIDAEKIQLA PIKNEDWLEFAQEKPYL (SEQ ID NO: 31).

[0254] Cpfl [Prevotella bryanti]:

[0255] MKFTDFTGLYSLSKTLRFELKPIGKTLENIKKAGLLEQDQHRADSYKKVKKIID EYHKAFIEKSLSNFELKYQSEDKLDSLEEYLMYYSMKRIEKTEKDKFAKIQDNLRKQIAD HLKGDESYKTIFSKDLIRKNLPDFVKSDEERTLIKEFKDFTTYFKGFYENRENMYSAEDK STAISHRIIHENLPKFVDNINAFSKIILIPELREKLNQIYQDFEEYLNVESIDEIFHLDYFSMV MTQKQIEVYNAIIGGKSTNDKKIQGLNEYINLYNQKHKDCKLPKLKLLFKQILSDRIAIS WLPDNFKDDQEALDSIDTCYKNLLNDGNVLGEGNLKLLLENIDTYNLKGIFIRNDLQLT DISQKMYASWNVIQDAVILDLKKQVSRKKKESAEDYNDRLKKLYTSQESFSIQYLNDCL RAYGKTENIQDYFAKLGAVNNEHEQTINLFAQVRNAYTSVQAILTTPYPENANLAQDKEBIOTN-41612.601TVALIKNLLDSLKRLQRFIKPLLGKGDESDKDERFYGDFTPLWETLNQITPLYNMVRNY MTRKPYSQEKIKLNFENSTLLGGWDLNKEHDNTAIILRKNGLYYLAIMKKSANKIFDKD KLDNSGDCYEKMVYKLLPGANKMLPKVFFSKSRIDEFKPSENIIENYKKGTHKKGANFN LADCHNLIDFFKSSISKHEDWSKFNFHFSDTSSYEDLSDFYREVEQQGYSISFCDVSVEYI NKMVEKGDLYLFQIYNKDFSEFSKGTPNMHTLYWNSLFSKENLNNIIYKLNGQAEIFFR KKSLNYKRPTHPAHQAIKNKNKCNEKKESIFDYDLVKDKRYTVDKFQFHVPITMNFKST GNTNINQQVIDYLRTEDDTHIIGIDRGERHLLYLVVIDSHGKIVEQFTLNEIVNEYGGNIY RTNYHDLLDTREQNREKARESWQTIENIKELKEGYISQVIHKITDLMQKYHAVVVLEDL NMGFMRGRQKVEKQVYQKFEEMLINKLNYLVNKKADQNSAGGLLHAYQLTSKFESFQ KLGKQSGFLFYIPAWNTSKIDPVTGFVNLFDTRYESIDKAKAFFGKFDSIRYNADKDWFE FAFDYNNFTTKAEGTRTNWTICTYGSRIRTFRNQAKNSQWDNEEIDLTKAYKAFFAKHG INIYDNIKEAIAMETEKSFFEDLLHLLKLTLQMRNSITGTTTDYLISPVHDSKGNFYDSRIC DNSLPANADANGAYNIARKGLMLIQQIKDSTSSNRFKFSPITNKDWLIFAQEKPYLND (SEQ ID NO: 32).

[0256] Cpfl [Prevotella copr]:

[0257] MAKNFEDFKRLYSLSKTLRFEAKPIGATLDNIVKSDLLDEDEHRAASYVKVKK LIDEYHKVFIDRVLDDGCLPLENKGNNNSLAEYYESYVSRAQDEDAKKKFKEIQQNLRS VIAKKLTEDKAYANLFGNKLIESYKDKEDKKKIIDSDLIQFINTAESTQLDSMSQDEAKE LVKEFWGFVTYFYGFFDNRKNMYTAEEKSTGIAYRLVNENLPKFIDNIEAFNRAITRPEI QENMGVLYSDFSEYLNVESIQEMFQLDYYNMLLTQKQIDVYNAIIGGKTDDEHDVKIKG INDYINLYNQKHKDDKLPKLKALFKQILSDRNAISWLPEEFNSDQEVLNAIKDCYERLSE NVLGDKVLKSMLGSLADYSLDGIFIRNDLQLTDISQKMFGNWSVIQNAIMQNIKHVAPA RKHKESEEEYENRIAGIFKKADSFSISYIDACLNETDPNNAYFVENYFATLGAVDTPTMQ RENLFALVQNAYTEITALLHSDYPTEKNLAQDKANVAKIKALLDAIKSLQHFVKPLLGK GDESDKDERFYGELASLWAELDTMTPLYNMIRNYMTRKPYSQKKIKLNFENPQLLGGW DANKEKDYATIILRRNGLYYLAIMNKDSKKLLGKAMPSDGECYEKMVYKLLPGANKM LPKVFFAKSRMEDFKPSKELVEKYYNGTHKKGKNFNIQDCHNLIDYFKQSIDKHEDWS KFGFKFSDTSTYEDLSGFYREVEQQGYKLSFARVSVSYINQLVEEGKMYLFQIYNKDFSE YSKGTPNMHTLYWKALFDERNLADVVYKLNGQAEMFYRKKSIENTHPTHPANHPILNK NKDNKKKESLFGYDLIKDRRYTVDKFLFHVPITMNFKSSGSENINQDVKAYLRHADDM HIIGIDRGERHLLYLVVIDLQGNIKEQFSLNEIVNDYNGNTYHTNYHDLLDVREDERLKABIOTN-41612.601RQSWQTIENIKELKEGYLSQVIHKITQLMVKYHAIVVLEDLNMGFMRGRQKVEKQVYQ KFEKMLIEKLNYLVDKKADASVSGGLLNAYQLTSKFDSFQKLGKQSGFLFYIPAWNTSK IDPVTGFVNLLDTRYQNVEKAKSFFSKFDAIRYNKDKEWFEFNLDYDKFGKKAEGTRT KWTLCTRGMRIDTFRNKEKNSQWDNQEVDLTAEMKSLLEHYYIDIHSNLKDAISAQTD KAFFTGLLHILKLTLQMRNSITGTETDYLVSPVVDENGIFYDSRSCGDELPENADANGAY NIARKGLMMIEQIKDAKDLDNLKFDISNKAWLNFAQQKPYKNG (SEQ ID NO: 33).

[0258] Cpfl [Porphyromonas crevioricani]:

[0259] MDSLKDFTNLYPVSKTLRFELKPVGKTLENIEKAGILKEDEHRAESYRRVKKII DTYHKVFIDSSLENMAKMGIENEIKAMLQSFCELYKKDHRTEGEDKALDKIRAVLRGLI VGAFTGVCGRRENTVQNEKYESLFKEKLIKEILPDFVLSTEAESLPFSVEEATRSLKEFDS FTSYFAGFYENRKNIYSTKPQSTAIAYRLIHENLPKFIDNILVFQKIKEPIAKELEHIRADFS AGGYIKKDERLEDIFSLNYYIHVLSQAGIEKYNALIGKIVTEGDGEMKGLNEHINLYNQQ RGREDRLPLFRPLYKQILSDREQLSYLPESFEKDEELLRALKEFYDHIAEDILGRTQQLMT SISEYDLSRIYVRNDSQLTDISKKMLGDWNAIYMARERAYDHEQAPKRITAKYERDRIK ALKGEESISLANLNSCIAFLDNVRDCRVDTYLSTLGQKEGPHGLSNLVENVFASYHEAE QLLSFPYPEENNLIQDKDNVVLIKNLLDNISDLQRFLKPLWGMGDEPDKDERFYGEYNYI RGALDQVIPLYNKVRNYLTRKPYSTRKVKLNFGNSQLLSGWDRNKEKDNSCVILRKGQ NFYLAIMNNRHKRSFENKVLPEYKEGEPYFEKMDYKFLPDPNKMLPKVFLSKKGIEIYK PSPKLLEQYGHGTHKKGDTFSMDDLHELIDFFKHSIEAHEDWKQFGFKFSDTATYENVS SFYREVEDQGYKLSFRKVSESYVYSLIDQGKLYLFQIYNKDFSPCSKGTPNLHTLYWRM LFDERNLADVIYKLDGKAEIFFREKSLKNDHPTHPAGKPIKKKSRQKKGEESLFEYDLVK DRHYTMDKFQFHVPITMNFKCSAGSKVNDMVNAHIREAKDMHVIGIDRGERNLLYICVI DSRGTILDQISLNTINDIDYHDLLESRDKDRQQERRNWQTIEGIKELKQGYLSQAVHRIAE LMVAYKAVVALEDLNMGFKRGRQKVESSVYQQFEKQLIDKLNYLVDKKKRPEDIGGL LRAYQFTAPFKSFKEMGKQNGFLFYIPAWNTSNIDPTTGFVNLFHAQYENVDKAKSFFQ KFDSISYNPKKDWFEFAFDYKNFTKKAEGSRSMWILCTHGSRIKNFRNSQKNGQWDSEE FALTEAFKSLFVRYEIDYTADLKTAIVDEKQKDFFVDLLKLFKLTVQMRNSWKEKDLDY LISPVAGADGRFFDTREGNKSLPKDADANGAYNIALKGLWALRQIRQTSEGGKLKLAIS NKEWLQFVQERSYEKD (SEQ ID NO: 34).

[0260] Cpfl [Bacteroidales bacterium]:BIOTN-41612.601

[0261] MKKFTNLYPVQKTLRFELIPQGNTSKHLCKIIQEDEQIAEDSQEVKKLLDRYH KEFIAIALSSFPTSPLAKEIIPKLKEFAQIRATGDAKQISTIQDELRELVVKGFKGEGEQERR YKILIGAKGNPNADELFNTELINFLKDPAEQALVKKFQKHTGYFLGFNENRKNMYSAKA QSTAIAYRLIHENLPRFLDNITTYEKVKTYLKEEIPQLEKELVRAGASLVSHVDSVFTIDFF LEVFTQSGIDQYNALIGKIVNQEQGEVKGLNERINLYNQQHKQEAKLPLFKPLYKQILSD REQLSWLAEAYNSDKDLLDSIQKYYQLLIDNQIFERIPRLMHTLEKAPLDKIWITYDTQL TSISNTLYGSWRVIGEALGRNAKSEKERKSSQKKALNYSLESINQAIAKMPSDEELPPIQK YFIALGSNPSKKDASSGTLVDKVRSSYKACQDILTNPDHTGKKLIQDKKQVDLLKQLLD DLLILQRFIKPLLYSNNENETHKDEVFYTELTDIMDLLNPIVGLYNKVRNYLTQKPYSTE KFKINFKSSSLLAGWDRNKEKDNLGVILKREDKYYLAIMDKAHNATFKNKSLPTQGEC YEKMEYKLLPGANKMLPKVYITSKKGIESFHPSEELQKKYKLGTHKKGASFNLSDMRA LIDYFKESLEKHEEHSQFGFHFSDTSTYEDISGFYREVEQQAYKITFRKVSVEYIDQLVNE GKLYLFQIYNKDFSPYSKGTPNLHTLYWKMLFDPANLQDIVYKLNGEAEVFFRKKSLQ YDRPTHPKGQPINKKSLLNEGETSLFDYDLIKDRRFTVDKFQFHVPITMNFKATQGTKV NQMVQEEVKKSKGFHLIGIDRGERNLLYIVVINERGEIIEQCSLNKIVNTYQEKEHTVDY KALLEKRSQSRLEERKSWQTIENIKELKGGYLSQVVHKIAQLMIKYNAIAVLEDLNFGFI RTRKKFEFSVYQEFEKKLIDKLGYVVDKKAPIQQEGGLLQAYQLTAPFKSFREMGKQN GFLFYVPAWNTSAIDPRTGFVNLLDTRYESIAKTKELIKKLKDIRYNSQKDWFEIDLDYN AFGNRAKGSRSKWRLCSYGERIEHTRKQDSNGQEESDSMVVLTEAFKDVFTKYQIDYR ENLKEQLLLQSDKAFFVDFLSLLRLTLQLRNSLSNSLIDYILSPVADENGEFFDSRKALSN EPQDADANGAYHIALKGLWVLDKIRKTEKVTPAKLALSNQEWLSFAQEKPFFNE (SEQ ID NO: 35).

[0262] Cpfl [Lachnospiraceae bacterium]:

[0263] MGLYDGFVNRYSVSKTLRFELIPQGRTREYIETNGILSDDEERAKDYKTIKRLI DEYHKDYISRCLKNVNISCLEEYYHLYNSSNRDKRHEELDALSDQMRGEIASFLTGNDE YKEQKSRDIIINERIINFASTDEELAAVKRFRKFTSYFTGFFTNRENMYSAEKKSTAIAHRI IDVNLPKYVDNIKAFNTAIEAGVFDIAEFESNFKAITDEHEVSDLLDITKYSRFIRNEDIIIY NTLLGGISMKDEKIQGLNELINLHNQKHPGKKVPLLKVLYKQILGDSQTHSFVDDQFED DQQVINAVKAVTDTFSETLLGSLKIIINNIGHYDLDRIYIKAGQDITTLSKRALNDWHIITE CLESEYDDKFPKNKKSDTYEEMRNRYVKSFKSFSIGRLNSLVTTYTEQACFLENYLGSF GGDTDKNCLTDFTNSLMEVEHLLNSEYPVTNRLITDYESVRILKRLLDSEMEVIHFLKPLBIOTN-41612.601LGNGNESDKDLVFYGEFEAEYEKLLPVIKVYNRVRNYLTRKPFSTEKIKLNFNSPTLLCG WSQSKEKEYMGVILRKDGQYYLGIMTPSNKKIFSEAPKPDEDCYEKMVLRYIPHPYQM LPKVFFSKSNIAFFNPSDEILRIKKQESFKKGKSFNRDDCHKFIDFYKDSINRHEEWRKFN FKFSDTDSYEDISRFYKEVENQAFSMSFTKIPTVYIDSLVDEGKLYLFKLHNKDFSEHSKGKPNLHTVYWNALFSEYNLQNTVYQLNGSAEIFFRKASIPENERVIHKKNVPITRKVAEL NGKKEVSVFPYDIIKNRRYTVDKFQFHVPLKMNFKADEKKRINDDVIEAIRSNKGIHVIG IDRGERNLLYLSLINEEGRIIEQRSLNIIDSGEGHTQNYRDLLDSREKDREKARENWQEIQ EIKDLKTGYLSQAIHTITKWMKEYNAIIVLEDLNDRFTNGRKKVEKQVYQKFEKMLIDK LNYYVDKDEEFDRMGGTHRALQLTEKFESFQKLGRQTGFIFYVPAWNTSKLDPTTGFVDLLYPKYKSVDATKDFIKKFDFIRFNSEKNYFEFGLHYSNFTERAIGCRDEWILCSYGNRI VNFRNAAKNNSWDYKEIDITKQLLDLFEKNGIDVKQENLIDSICEMKDKPFFKSLIANIK LILQIRNSASGTDIDYMISPAMNDRGEFFDTRKGLQQLPLDADANGAYNIAKKGLWIVD QIRNTTGNNVKMAMSNREWMHFAQESRLA (SEQ ID NO: 36).

[0264] Cpfl [Porphyromonas macaca] :

[0265] MVQSSISEYNRFVGGYSTEDGTKHQGINEWINIYRQRNKEMRLPGLVFLHKQILAKVDSSSFILDTLENDDQVFCVLRQFRKLFWNTVSSKEDDAASLKDLFCGLSGYDPEAI YVSDAHLATLSKNIFDRWNYISDAIRRKTEVLMPRKKESVERYAEKISKQIKKRESYSLA ELDDLLAHYSEESLPAGFSLLSYFTSLGGQKYLVSDGEVILYEEGSNIWDEVLIAFRDLQ VILDKDFTEKKLGKDEEAVSVIKKALDSALRLRKFFDLLSGTGAEIRRDSSFYALYTDRM DKLKGLLKMYDKVRNYLTKKPYSIEKFKLHFDNPSLLSGWDKNKELNNLSVIFRQNGY YYLGIMTPKGKNLFKTLPKLGAEEMFYEKMEYKQIAEPMLMLPKVFFPKKTKPAFAPD QSVVDIYNKKTFKTGQKGFNKKDLYRLFDFYKEALTVHEWKLFNFSFSPTEQYRNIGEF FDEVREQAYKVSMVNVPASYIDEAVENGKLYLFQIYNKDFSPYSKGIPNLHTLYWKALFSEQNQSRVYKLCGGGELFYRKASLHMQDTTVHPKGISIHKKNLNKKGETSLFNYDLVK DKRFTEDKFFFHVPISINYKNKKITNVNQMVRDYIAQNDDLQIIGIDRGERNLLYISRIDT RGNLLEQFSLNVIESDKGDLRTDYQKILGDREQERLRRRQEWKSIESIKDLKDGYMSQVVHKICNMVVEHKAIVVLENLNLSFMKGRKKVEKSVYEKFEKMLVDKLNYLVVDKKNL SNEPGGLYAAYQLTNPLFSFEELHRYPQSGILFFVDPWNTSLTDPSTGFVNLLGRINYTN VGDARKFFDRFNAIRYDGKGNILFDLDLSRFDVRVETQRKLWTLTTFGSRIAKSKKSGKWMVERIENLSLCFLELFEQFNIGYRVEKDLKKAILSQDRKEFYVRLIYLFNLMMQIRNSDBIOTN-41612.601SEEDYILSPALNEKNLQFDSRLIEAKDLPVDADANGAYNVARKGLMVVQRIKRGDHESI HRIGHAQWLRYVQEGIVE (SEQ ID NO: 37).

[0266] Cpfl [Lachnospiraceae bacterium]:

[0267] MENYYDSLTRQYPVTKTIRQELKPVGKTLENIKNAEIIEADKQKKEAYVKVKE LMDEFHKSIIEKSLVGIKLDGLSEFEKLYKIKTKTDEDKNRISELFYYMRKQIADALKNSR DYGYVDNKDLIEKILPERVKDENSLNALSCFKGFTTYFTDYYKNRKNIYSDEEKHSTVG YRCINENLLIFMSNIEVYQIYKKANIKNDNYDEETLDKTFMIESFNECLTQSGVEAYNSV VASIKTATNLYIQKNNKEENFVRVPKMKVLFKQILSDRTSLFDGLIIESDDELLDKLCSFS AEVDKFLPINIDRYIKTLMDSNNGTGIYVKNDSSLTTLSNYLTDSWSSIRNAFNENYDAKYTGKVNDKYEEKREKAYKSNDSFELNYIQNLLGINVIDKYIERINFDIKEICEAYKEMTK NCFEDHDKTKKLQKNIKAVASIKSYLDSLKNIERDIKLLNGTGLESRNEFFYGEQSTVLE EITKVDELYNITRNYLTKKPFSTEKMKLNFNNPQLLGGWDVNKERDCYGVILIKDNNYY LGIMDKSANKSFLNIKESKNENAYKKVNCKLLPGPNKMFPKVFFAKSNIDYYDPTHEIKKLYDKGTFKKGNSFNLEDCHKLIDFYKESIKKNDDWKNFNFNFSDTKDYEDISGFFREV EAQNYKITYTNVSCDFIESLVDEGKLYLFQIYNKDFSEYATGNLNLHTLYLKMLFDERN LKDLCIKMNGEAEVFYRPASILDEDKVVHKANQKITNKNTNSKKKESIFSYDIVKDKRY TVDKFFIHLPITLNYKEQNVSRFNDYIREILKKSKNIRVIGIDRGERNLLYVVVCDSDGSIL YQRSINEIVSGSHKTDYHKLLDNKEKERLSSRRDWKTIENIKDLKAGYMSQWNEIYNLI LKYNAIVVLEDLNIGFKNGRKKVEKQVYQNFEKALIDKLNYLCIDKTREQLSPSSPGGVL NAYQLTAKFESFEKIGKQTGCIFYVPAYLTSQIDPTTGFVNLFYQKDTSKQGLQLFFRKF KKINFDKVASNFEFVFDYNDFTNKAEGTKTNWTISTQGTRIAKYRSDDANGKWISRTVH PTDIIKEALNREKINYNDGHDLIDEIVSIEKSAVLKEIYYGFKLTLQLRNSTLANEEEQEDY IISPVKNSSGNYFDSRITSKELPCDADANGAYNIARKGLWALEQIRNSENVSKVKLAISN KEWFEYTQNNIPSL (SEQ ID NO: 38).

[0268] Cpfl [Butyrivibrio sp]:

[0269] MYYQNLTKKYPVSKTIRNELIPIGKTLENIRKNNILESDVKRKQDYEHVKGIM DEYHKQLINEALDNYMLPSLNQAAEIYLKKHVDVEDREEFKKTQDLLRREVTGRLKEH ENYTKIGKKDILDLLEKLPSISEEDYNALESFRNFYTYFTSYNKVRENLYSDEEKSSTVAY RLINENLPKFLDNIKSYAFVKAAGVLADCIEEEEQDALFMVETFNMTLTQEGIDMYNYQI GKVNSAINLYNQKNHKVEEFKKIPKMKVLYKQILSDREEVFIGEFKDDETLLSSIGAYGN VLMTYLKSEKINIFFDALRESEGKNVYVKNDLSKTTMSNIVFGSWSAFDELLNQEYDLABIOTN-41612.601NENKKKDDKYFEKRQKELKKNKSYTLEQMSNLSKEDISPIENYIERISEDIEKICIYNGEF EKIVVNEHDSSRKLSKNIKAVKVIKDYLDSIKELEHDIKLINGSGQELEKNLVVYVGQEE ALEQLRPVDSLYNLTRNYLTKKPFSTEKVKLNFNKSTLLNGWDKNKETDNLGILFFKDG KYYLGIMNTTANKAFVNPPAAKTENVFKKVDYKLLPGSNKMLPKVFFAKSNIGYYNPS TELYSNYKKGTHKKGPSFSIDDCHNLIDFFKESIKKHEDWSKFGFEFSDTADYRDISEFYR EVEKQGYKLTFTDIDESYINDLIEKNELYLFQIYNKDFSEYSKGKLNLHTLYFMMLFDQR NLDNVVYKLNGEAEVFYRPASIAENELVIHKAGEGIKNKNPNRAKVKETSTFSYDIVKD KRYSKYKFTLHIPITMNFGVDEVRRFNDVINNALRTDDNVNVIGIDRGERNLLYVVVINS EGKILEQISLNSIINKEYDIETNYHALLDEREDDRNKARKDWNTIENIKELKTGYLSQVVN VVAKLVLKYNAIICLEDLNFGFKRGRQKVEKQVYQKFEKMLIEKLNYLVIDKSREQVSP EKMGGALNALQLTSKFKSFAELGKQSGIIYYVPAYLTSKIDPTTGFVNLFYIKYENIEKA KQFFDGFDFIRFNKKDDMFEFSFDYKSFTQKACGIRSKWIVYTNGERIIKYPNPEKNNLF DEKVINVTDEIKGLFKQYRIPYENGEDIKEIIISKAEADFYKRLFRLLHQTLQMRNSTSDG TRDYIISPVKNDRGEFFCSEFSEGTMPKDADANGAYNIARKGLWVLEQIRQKDEGEKVNLSMTNAEWLKYAQLHLL (SEQ ID NO: 39).

[0270] Cpfl [Oribacterium sp]:

[0271] METEILKYDFFEREGKYMYYDGLTKQYALSKTIRNELVPIGKTLDNIKKNRILE ADIKRKSDYEHVKKLMDMYHKKIINEALDNFKLSVLEDAADIYFNKQNDERDIDAFLKI QDKLRKEIVEQLKGHTDYSKVGNKDFLGLLKAASTEEDRILIESFDNFYTYFTSYNKVRS NLYSAEDKSSTVAYRLINENLPKFFDNIKAYRTVRNAGVISGDMSIVEQDELFEVDTFNH TLTQYGIDTYNHMIGQLNSAINLYNQKMHGAGSFKKLPKMKELYKQLLTEREEEFIEEYTDDEVLITSVHNYVSYLIDYLNSDKVESFFDTLRKSDGKEVFIKNDVSKTTMSNILFDNW STIDDLINHEYDSAPENVKKTKDDKYFEKRQKDLKKNKSYSLSKIAALCRDTTILEKYIR RLVDDIEKIYTSNNVFSDIVLSKHDRSKKLSKNTNAVQAIKNMLDSIKDFEHDVMLINGSGQEIKKNLNVYSEQEALAGILRQVDHIYNLTRNYLTKKPFSTEKIKLNFNRPTFLDGWDK NKEEANLGILLIKDNRYYLGIMNTSSNKAFVNPPKAISNDIYKKVDYKLLPGPNKMLPK VFFATKNIAYYAPSEELLSKYRKGTHKKGDSFSIDDCRNLIDFFKSSINKNTDWSTFGFNF SDTNSYNDISDFYREVEKQGYKLSFTDIDACYIKDLVDNNELYLFQIYNKDFSPYSKGKL NLHTLYFKMLFDQRNLDNVVYKLNGEAEVFYRPASIESDEQIIHKSGQNIKNKNQKRSN CKKTSTFDYDIVKDRRYCKDKFMLHLPITVNFGTNESGKFNELVNNAIRADKDVNVIGI DRGERNLLYVVVVDPCGKIIEQISLNTIVDKEYDIETDYHQLLDEKEGSRDKARKDWNTIBIOTN-41612.601ENIKELKEGYLSQWNIIAKLVLKYDAIICLEDLNFGFKRGRQKVEKQVYQKFEKMLIDK MNYLVLDKSRKQESPQKPGGALNALQLTSAFKSFKELGKQTGIIYYVPAYLTSKIDPTTG FANLFYIKYESVDKARDFFSKFDFIRYNQMDNYFEFGFDYKSFTERASGCKSKWIACTN GERIVKYRNSDKNNSFDDKTVILTDEYRSLFDKYLQNYIDEDDLKDQILQIDSADFYKNL IKLFQLTLQMRNSSSDGKRDYIISPVKNYREEFFCSEFSDDTFPRDADANGAYNIARKGL WVIKQIRETKSGTKINLAMSNSEWLEYAQCNLL (SEQ ID NO: 40).

[0272] Cpfl [Pseudobutyrivibrio xylanivoran]:

[0273] MYYQGLTKLYPISKTIRNELIPVGKTLEHIRMNNILEADIQRKSDYERVKKLMDDYHKQLINESLQDVHLSYVEEAADLYLNASKDKDIVDKFSKCQDKLRKEIVNLLKSHEN FPKIGNKEIIKLLQSLSDTEKDYNALDSFSKFYTYFTSYNEVRKNLYSDEEKSSTAAYRLI NENLPKFLDNIKAYSIAKSAGVRAKELTEEEQDCLFMTETFERTLTQDGIDNYNELIGKL NFAINLYNQQNNKLKGFRKVPKMKELYKQILSEREASFVDEFVDDEALLTNVESFSAHI KEFLESDSLSRFAEVLEESGGEMVYIKNDTSKTTFSNIVFGSWNVIDERLAEEYDSANSK KKKDEKYYDKRHKELKKNKSYSVEKIVSLSTETEDVIGKYIEKLQADIIAIKETREVFEK VVLKEHDKNKSLRKNTKAIEAIKSFLDTIKDFERDIKLISGSEHEMEKNLAVYAEQENILS SIRNVDSLYNMSRNYLTQKPFSTEKFKLNFNRATLLNGWDKNKETDNLGILLVKEGKY YLGIMNTKANKSFVNPPKPKTDNVYHKVNYKLLPGPNKMLPKVFFAKSNLEYYKPSEDLLAKYQAGTHKKGENFSLEDCHSLISFFKDSLEKHPDWSEFGFKFSDTKKYDDLSGFYR EVEKQGYKITYTDIDVEYIDSLVEKDELYLFQIYNKDFSPYSKGNYNLHTLYLTMLFDER NLRNVVYKLNGEAEVFYRPASIGKDELIIHKSGEEIKNKNPKRAIDKPTSTFEYDIVKDRRYTKDKFMLHIPVTMNFGVDETRRFNEVVNDAIRGDDKVRVIGIDRGERNLLYVVVVDS DGTILEQISLNSIINNEYSIETDYHKLLDEKEGDRDRARKNWTTIENIKELKEGYLSQVVN VIAKLVLKYDAIICLEDLNFGFKRGRQKVEKQVYQKFEKMLIDKLNYLVIDKSRSQENPE EVGHVLNALQLTSKFTSFKELGKQTGIIYYVPAYLTSKIDPTTGFANLFYVKYESVEKSK DFFNRFDSICFNKVAGYFEFSFDYKNFTDRACGMRSKWKVCTNGERIIKYRNEEKNSSF DDKVIVLTEEFKKLFNEYGIAFNDCMDLTDAINAIDDASFFRKLTKLFQQTLQMRNSSA DGSRDYIISPVENDNGEFFNSEKCDKSKPKDADANGAFNIARKGLWVLEQLYNSSSGEK LNLAMTNAEWLEYAQQHTI (SEQ ID NO: 41).

[0274] Cpfl [Pseudobutyrivibrio xylanivoran]:

[0275] MIIGRDFNMYYQNLTKMYPISKTLRNELIPVGKTLENIRKNGILEADIQRKADY EHVKKLMDNYHKQLINEALQGVHLSDLSDAYDLYFNLSKEKNSVDAFSKCQDKLRKEIBIOTN-41612.601VSFLKNHENFPKIGNKEIIKLIQSLNDNDADNNALDSFSNFYTYFSSYNEVRKNLYSDEE KSSTVAYRLINENLPKSLDNIKAYAIAKKAGVRAEGLSEEEQDCLFIIETFERTLTQDGID NYNADIGKLNTAINLYNQQNKKQEGFRKVPQMKCLYKQILSDREEAFIDEFSDDEDLIT NIESFAENMNVFLNSEIITDFKNALVESDGSLVYIKNDVSKTLFSNIVFGSWNAIDEKLSD EYDLANSKKKKDEKYYEKRQKELKKNKSYDLETIIGLFDDSIDVIGKYIEKLESDITAIAE AKNDFDEIVLRKHDKNKSLRKNTNAVEAIKSYLDTVKDFERDIKLINGSGQEVEKNLVV YAEQENILAEIKNVDSLYNMSRNYLTQKPFSTEKFKLNFENPTLLNGWDRNKEKDYLGI LFEKEGMYYLGIINNNHRKIFENEKLCTGKESCFNKIVYKQISNAAKYLSSKQINPQNPPK EIAEILLKRKADSSSLSRKETELFIDYLKDDFLVNYPMIINSDGENFFNFHFKQAKDYGSL QEFFKEVEHQAYSLKTRPIDDSYIYRMIDEGKLYLFQIHNKDFSPYSKGNLNLHTIYLQM LFDQRNLNNVVYKLNGEAEVFYRPASINDEEVIIHKAGEEIKNKNSKRAVDKPTSKFGY DIIKDRRYSKDKFMLHIPVTMNFGVDETRRFNDVVNDALRNDEKVRVIGIDRGERNLLY VVVVDTDGTILEQISLNSIINNEYSIETDYHKLLDEKEGDRDRARKNWTTIENIKELKEGY LSQVVNVIAKLVLKYNAIICLEDLNFGFKRGRQKVEKQVYQKFEKMLIDKLNYLVIDKS RKQEKPEEFGGALNALQLTSKFTSFKDMGKQTGIIYYVPAYLTSKIDPTTGFANLFYVKY ENVEKAKEFFSRFDSISYNNESGYFEFAFDYKKFTDRACGARSQWTVCTYGERIIKYRN ADKNNSFDDKTIVLSEEFKELFSIYGISYEDGAELKNKIMSVDEADFFRCLTGLLQKTLQ MRNSSNDGTRDYIISPIMNDRGEFFNSEACDASKPKDADANGAFNIARKGLWVLEQIRN TPSGDKLNLAMSNAEWLEYAQRNQI (SEQ ID NO: 42).

[0276] Cpfl [Pseudobutyrivibrio rumini]:

[0277] MYYQNLTKMYPISKTLRNELIPVGKTLENIRKNGILEADIQRKADYEHVKKLM DNYHKQLINEALQGVHLSDLSDAYDLYFNLSKEKNSVDAFSKCQDKLRKEIVSLLKNHE NFPKIGNKEIIKLLQSLYDNDTDYKALDSFSNFYTYFSSYNEVRKNLYSDEEKSSTVAYR LINENLPKFLDNIKAYAIAKKAGVRAEGLSEEDQDCLFIIETFERTLTQDGIDNYNAAIGK LNTAINLFNQQNKKQEGFRKVPQMKCLYKQILSDREEAFIDEFSDDEDLITNIESFAENM NVFLNSEIITDFKIALVESDGSLVYIKNDVSKTSFSNIVFGSWNAIDEKLSDEYDLANSKK KKDEKYYEKRQKELKKNKSYDLETIIGLFDDNSDVIGKYIEKLESDITAIAEAKNDFDEIV LRKHDKNKSLRKNTNAVEAIKSYLDTVKDFERDIKLINGSGQEVEKNLVVYAEQENILA EIKNVDSLYNMSRNYLTQKPFSTEKFKLNFNRATLLNGWDKNKETDNLGILFEKDGMY YLGIMNTKANKIFVNIPKATSNDVYHKVNYKLLPGPNKMLPKVFFAQSNLDYYKPSEEL LAKYKAGTHKKGDNFSLEDCHALIDFFKASIEKHPDWSSFGFEFSETCTYEDLSGFYREVBIOTN-41612.601EKQGYKITYTDVDADYITSLVERDELYLFQIYNKDFSPYSKGNLNLHTIYLQMLFDQRNL NNVVYKLNGEAEVFYRPASINDEEVIIHKAGEEIKNKNSKRAVDKPTSKFGYDIIKDRRY SKDKFMLHIPVTMNFGVDETRRFNDVVNDALRNDEKVRVIGIDRGERNLLYVVVVDTD GTILEQISLNSIINNEYSIETDYHKLLDEKEGDRDRARKNWTTIENIKELKEGYLSQVVNV IAKLVLKYNAIICLEDLNFGFKRGRQKVEKQVYQKFEKMLIDKLNYLVIDKSRKQDKPE EFGGALNALQLTSKFTSFKDMGKQTGIIYYVPAYLTSKIDPTTGFANLFYVKYENVEKA KEFFSRFDSISYNNESGYFEFAFDYKKFTDRACGARSQWTVCTYGERIIKFRNTEKNNSF DDKTIVLSEEFKELFSIYGISYEDGAELKNKIMSVDEADFFRSLTRLFQQTMQMRNSSND VTRDYIISPIMNDRGEFFNSEACDASKPKDADANGAFNIARKGLWVLEQIRNTPSGDKLN LAMSNAEWLEYAQRNQI (SEQ ID NO: 43).

[0278] Cpfl [Butyrivibrio fibrisolven]:

[0279] MYYESLTKLYPIKKTIRNELVPIGKTLENIKKNNILEADEDRKIAYIRVKAIMDD YHKRLINEALSGFALIDLDKAANLYLSRSKSADDIESFSRFQDKLRKAIAKRLREHENFG KIGNKDIIPLLQKLSENEDDYNALESFKNFYTYFESYNDVRLNLYSDKEKSSTVAYRLIN ENLPRFLDNIRAYDAVQKAGITSEELSSEAQDGLFLVNTFNNVLIQDGINTYNEDIGKLN VAINLYNQKNASVQGFRKVPKMKVLYKQILSDREESFIDEFESDTELLDSLESHYANLAKYFGSNKVQLLFTALRESKGVNVYVKNDIAKTSFSNVVFGSWSRIDELINGEYDDNNNR KKDEKYYDKRQKELKKNKSYTIEKIITLSTEDVDVIGKYIEKLESDIDDIRFKGKNFYEAV LCGHDRSKKLSKNKGAVEAIKGYLDSVKDFERDLKLINGSGQELEKNLVVYGEQEAVL SELSGIDSLYNMTRNYLTKKPFSTEKIKLNFNKPTFLDGWDYGNEEAYLGFFMIKEGNYFLAVMDANWNKEFRNIPSVDKSDCYKKVIYKQISSPEKSIQNLMVIDGKTVKKNGRKEK EGIHSGENLILEELKNTYLPKKINDIRKRRSYLNGDTFSKKDLTEFIGYYKQRVIEYYNGY SFYFKSDDDYASFKEFQEDVGRQAYQISYVDVPVSFVDDLINSGKLYLFRVYNKDFSEY SKGRLNLHTLYFKMLFDERNLKNVVYKLNGQAE VFYRP S SIKKEELIVHRAGEEIKNKN PKRAAQKPTRRLDYDIVKDRRYSQDKFMLHTSIIMNFGAEENVSFNDIVNGVLRNEDKV NVIGIDRGERNLLYVVVIDPEGKILEQRSLNCITDSNLDIETDYHRLLDEKESDRKIARRD WTTIENIKELKAGYLSQVVHIVAELVLKYNAIICLEDLNFGFKRGRQKVEKQVYQKFEK MLIDKLNYLVMDKSREQLSPEKISGALNALQLTPDFKSFKVLGKQTGIIYYVPAYLTSKI DPMTGFANLFYVKYENVDKAKEFFSKFDSIKYNKDGKNWNTKGYFEFAFDYKKFTDR AYGRVSEWTVCTVGERIIKFKNKEKNNSYDDKVIDLTNSLKELFDSYKVTYESEVDLKDBIOTN-41612.601AILAIDDPAFYRDLTRRLQQTLQMRNSSCDGSRDYIISPVKNSKGEFFCSDNNDDTTPND ADANGAFNIARKGLWVLNEIRNSEEGSKINLAMSNAQWLEYAQDNTI (SEQ ID NO: 44).

[0280] Cpfl [Ruminococcus sp]:

[0281] MSININKFSDECRKIDFFTDLYNIQKTLRFSLIPIGATADNFEFKGRLSKEKDLL DSAKRIKEYISKYLADESDICLSQPVKLKHLDEYYELYITKDRDEQKFKSVEEKLRKELA DLLKEILKRLNKKILSDYLPEYLEDDEKALEDIANLSSFSTYFNSYYDNCKNMYTDKEQS TAIPYRCINDNLPKFIDNMKAYEKALEELKPSDLEELRNNFKGVYDTTVDDMFTLDYFN CVLSQSGIDSYNAIIGNDKVKGINEYINLHNQTAEQGHKVPNLKRLYKQIGSQKKTISFLP SKFESDNELLKAVYDFYNTGDAEKNFTALKDTITEFEKIFDNLSEYNLDGVFVRNDISLT NLSQSMFNDWSVFRNLWNDQYDKVNNPEKAKDIDKYNDKRHKVYKKSESFSINQLQE LIATTLEEDINSKKITDYFSCDFHRVTTEVENKYQLVKDLLSSDYPKNKNLKTSEEDVALI KDFLDSVKSLESFVKILTGTGKESGKDELFYGSFTKWFDQLRYIDKLYDKVRNYITEKPY SLDKIKLSFDNPQFLGGWQHSKETDYSAQLFMKDGLYYLGVMDKETKREFKTQYNTPE NDSDTMVKIEYNQIPNPGRVIQNLMLVDGKIVKKNGRKNADGVNAVLEELKNQYLPEN INRIRKTESYKTTSNNFNKDDLKAYLEYYIARTKEYYCKYNFVFKSADEYGSFNEFVDD VNNQAYQITKVKVSEKQLLSLVEQGKLYLFKIYNKDFSEYSKGKKNLHTMYFQMLFDD RNLENLVYKLQGGAEMFYRPASIKKDSEFKHDANVEIIKRTCEDKVNDKDNPTDDEKA KYYSKFDYDIVKNKRFTKDQFSLHLTLAMNCNQPDHYWLNNDVRELLKKSNKNHIIGI DRGERNLIYVTIINSDGVIVDQINFNIIENSYNGKKYKTDYQKKLNQREEDRQKARKTW KTIETIKELKDGYISQVVHQICKLIVQYDAIVVMENLNGGFKRGRTKVEKQVYQKFETM LINKLNYYVDKGTDYKECGGLLKAYQLTNKFETFERIGKQSGIIFYVDPYLTSKIDPVTGFANLLYPKYETIPKTHNFISNIDDIRYNQSEDYFEFDIDYDKFPQGSYNYRKKWTICSYGN RIKYYKDSRNKTASVVVDITEKFKETFTNAGIDFVNDNIKEKLLLVNSKELLKSFMDTLK LTVQLRNSEINSDVDYIISPIKDRNGNFYYSENYKKSNNEVPSQPQDGDANGAYNIARKG LMIINKLKKADDVTNNELLKISKKEWLEFAQKGDLGE (SEQ ID NO: 45).

[0282] Cpfl [Ruminococcus sp]:

[0283] MQEHKKISHLTHRNSVQKTIRMQLNPVGKTMDYFQAKQILENDEKLKENYQ KIKEIADRFYRNLNEDVLSKTRLDKLKDYTDIYYHCNTDADRKRLDECASELRKEIVKNFKNRDEYNKLFNKKMIEIVLPKHLKNEDEKEVVTSFKNFTTYFTGFFTNRKNMYSDGEE STAIAYRCINENLPKHLDNVKAFEKAISKLSKNAIDDLDTTYSGLCGTNLYDVFTVDYFN FLLPQSGITEYNKIIGGYTTNDGTKVKGINEYINLYNQQVSKRDKIPNLKILYKQILSESEKBIOTN-41612.601VSFIPPKFEDDNELLSAVSEFYANDETFDGMPLKKAIDETKLLFGNLDNPSLNGIYIQNDR SVTNLSNSMFGSWSVIEDLWNKNYDSVNSNSRIKDIQKREDKRKKAYKAEKKLSLSFLQ VLISNSENDEIREKSIVDYYKTSLMQLTDNLSDKYNEAAPLLNENYSNEKGLKNDDKSIS LIKNFLDAIKEIEKFIKPLSETNITGEKNDLFYSQFTPLLDNISRIDILYDKVRNYVTQKPFS TDKIKLNFGNSQLLNGWDRNKEKDCGAVWLCKDEKYYLAIIDKSNNSILENIDFQDCDE SDCYEKIIYKLLPGPNKMLPKVFFSEKCKKLLSPSDEILKIYKSGTFKTGDKFSLDDCHKLIDFYKESFKKYPNWLIYNFKFKKTNEYNDIREFYNDVALQGYNISKMKIPTSFIDKLVDE GKIYLFQLYNKDFSPHSKGTPNLHTLYFKMLFDERNLEDVVYRLNGEAEMFYRPASIKY DKPTHPKNTPIKNKNTLNDKKTSTFPYDLIKDKRYTKWQFSLHFPITMNFKAPDKAMIN DDVRNLLKSCNNNFIIGIDRGERNLLYVSVIDSNGAIIYQHSLNIIGNKFKEKTYETNYRE KLATREKERTEQRRNWKAIESIKELKEGYISQAVHVICQLWKYDAIIVMEKLTDGFKRG RTKFEKQVYQKFEKMLIDKLNYYVDKKLDPDEEGGLLHAYQLTNKLESFDKLGMQSGF IFYVRPDFTSKIDPVTGFVNLLYPQYENIDKAKDMISRFDEIRYNAGEDFFEFDIDYDEFPKTASDYRKKWTICTNGERIEAFRNPANNNEWSYRTIILAEKFKELFDNNSINYRDSDDLK AEILSQTKGKFFEDFFKLLRLTLQMRNSNPETGEDRILSPVKDKNGNFYDSSKYDEKSKL PCDADANGAYNIARKGLWFVEQFKKADNVSTVEPVIHNDQWLKFVQENDMANN (SEQ ID NO: 46).

[0284] Cpfl [Lachnospiraceae bacteriu] :

[0285] MSKLEKFTNCYSLSKTLRFKAIPVGKTQENIDNKRLLVEDEKRAEDYKGVKKLLDRYYLSFINDVLHSIKLKNLNNYISLFRKKTRTEKENKELENLEINLRKEIAKAFKGNEGYKSLFKKDIIETILPEFLDDKDEIALVNSFNGFTTAFTGFFDNRENMFSEEAKSTSIAFRC INENLTRYISNMDIFEKVDAIFDKHEVQEIKEKILNSDYDVEDFFEGEFFNFVLTQEGIDV YNAIIGGFVTESGEKIKGLNEYINLYNQKTKQKLPKFKPLYKQVLSDRESLSFYGEGYTSDEEVLEVFRNTLNKNSEIFSSIKKLEKLFKNFDEYSSAGIFVKNGPAISTISKDIFGEWNVI RDKWNAEYDDIHLKKKAVVTEKYEDDRRKSFKKIGSFSLEQLQEYADADLSVVEKLKE IIIQKVDEIYKVYGSSEKLFDADFVLEKSLKKNDAVVAIMKDLLDSVKSFENYIKAFFGE GKETNRDESFYGDFVLAYDILLKVDHIYDAIRNYVTQKPYSKDKFKLYFQNPQFMGGW DKDKETDYRATILRYGSKYYLAIMDKKYAKCLQKIDKDDVNGNYEKINYKLLPGPNKM LPKVFFSKKWMAYYNPSEDIQKIYKNGTFKKGDMFNLNDCHKLIDFFKDSISRYPKWSN AYDFNFSETEKYKDIAGFYREVEEQGYKVSFESASKKEVDKLVEEGKLYMFQIYNKDFSDKSHGTPNLHTMYFKLLFDENNHGQIRLSGGAELFMRRASLKKEELVVHPANSPIANKNBIOTN-41612.601PDNPKKTTTLSYDVYKDKRFSEDQYELHIPIAINKCPKNIFKINTEVRVLLKHDDNPYVIG IDRGERNLLYIVVVDGKGNIVEQYSLNEIINNFNGIRIKTDYHSLLDKKEKERFEARQNW TSIENIKELKAGYISQVVHKICELVEKYDAVIALEDLNSGFKNSRVKVEKQVYQKFEKML IDKLNYMVDKKSNPCATGGALKGYQITNKFESFKSMSTQNGFIFYIPAWLTSKIDPSTGF VNLLKTKYTSIADSKKFISSFDRIMYVPEEDLFEFALDYKNFSRTDADYIKKWKLYSYGN RIRIFRNPKKNNVFDWEEVCLTSAYKELFNKYGINYQQGDIRALLCEQSDKAFYSSFMA LMSLMLQMRNSITGRTDVDFLISPVKNSDGIFYDSRNYEAQENAILPKNADANGAYNIA RKVLWAIGQFKKAEDEKLDKVKIAISNKEWLEYAQTSVKH (SEQ ID NO: 47).

[0286] Cpfl [Clostridia bacteriu]:

[0287] MKEQLIGRYSLSKTLRFSLIPVGKTEETFHAHKLLEKDKKRAADYEKVKGYID RFHKAYIESVLTQVQLADVPAYAALYRKSDKTDADLKQMEAYEEKMRKQIAKALTSGE RYKRLFGKELITVDLPAFLTEEEEKEAVRPFESFTTYFVGFYDNRKNMYTAAPQPTAVA YRCIRENLPRFLDNVKSFAKIEKALPPETLRALDDDFLGLFGRNVRDIFCDGFFAFVLPQS GIDMYNGMIGGYTCSDGTKVQGVNEYVNLYNQSVAKNDKSLRLPLIKPLYKQILSDRET VSFIPEKFSSDDALLAAVKEYYTRNKAAFAALEGLFADLKEFRADGIYIRAGAAVTAVS NGVYGDWHALRAAWDAAYAAENPPKAKGDPEKYAEKREKLYKSIKSFSLAQLQALGV HADGDIAAWCTGTVAACAKTIGESYARCADLLNAPYAESHDKKLVQNSDATERIKNLL DAVKALELAVKPFLGTGKEEDKDDLFYGRFVTLYDAVSTVDKLYDKVRNYMTQKPYS QEKIKLNFDNPQLLGGWDKSKERDYRTVLLRKDGLYYLAVIDREHSSMFADAPAGNGE DCYEKMEYKLLPGPNKMLPKVVFAASNADLFKPTERILDIRRRESFKKGDAFNLDDCHE FIDFFKQSISKYDSWAGFGFTFTPTAEYKDISGFYREVADQGYTLRFQPVPAAYIDDLVE NGCLYLFKIYSKDFSPFSHGTPNLHTLYFRMLFDERNLKDVVYKLNGEAEMFYREASIN DKERIVHPANLSLKNKNPDNQKVESVFAYDLVKDKRYTKRQFSLHVPITINFKADGQEF LNYDVRRLVKNNPHPFVIGVDRGERNLIYISVIDGDGKIVEQTSLNEIIGDTGHRVDYQK LLDKKEKDRDSARKNWTTVESIKELKEGYLSQVVHKIGELVLKYDAVIALEDLNFGFKR SRFGVEKQVYQKFENMLVSKLNLLIDKNAAPDADGGLLRAYQLTNKFDGVNKSKQNGI LFYVPAWDTSKIDPATGFVDLLKPRYTDVAASQRLFAALDGMRYNTATDMFEWDIDY GKFPKCCAAFRKKWTICTYKDRIETFRNKEKNNAWDHRRIMLTEAFKETFAAFGVDYT GDLQRTVVSNKSADFHKRLTRLFALTLQMRNSVTGSTLPEDDYLISPVADKNGDFYDSR RFSGKNAAMPCDADANGAYNIARKALWAIGVLKNTDDDRLDKADLTIRNADWLAYV QK (SEQ ID NO: 48).BIOTN-41612.601

[0288] Cpfl [Eubacterium coprostanoligene]:

[0289] MGQFMDFFKNDMYFLCINGIIVISKLFAYLFLMYKRGVVMIKDNFVNVYSLSKTIRMALIPWGKTEDNFYKKFLLEEDEERAKNYIKVKGYMDEYHKNFIESALNSVVLNGVDEYCELYFKQNKSDSEVKKIESLEASMRKQISKAMKEYTVDGVKIYPLLSKKEFIRELLP EFLTQDEEIETLEQFNDFSTYFQGFWENRKNIYTDEEKSTGVPYRCINDNLPKFLDNVKS FEKVILALPQKAVDELNANFNGVYNVDVQDVFSVDYFNFVLSQSGIEKYNNIIGGYSNS DASKVQGLNEKINLYNQQIAKSDKSKKLPLLKPLYKQILSDRSSLSFIPEKFKDDNEVLNS INVLYDNIAESLEKANDLMSDIANYNTDNIFISSGVAVTDISKKVFGDWSLIRNNWNDEY ESTHKKGKNEEKFYEKEDKEFKKIKSFSVSELQRLANSDLSIVDYLVDESASLYADIKTA YNNAKDLLSNEYSHSKRLSKNDDAIELIKSFLDSIKNYEAFLKPLCGTGKEESKDNAFYG AFLECFEEIRQVDAVYNKVRNHITQKPYSNDKIKLNFQNPQFLAGWDKNKERAYRSVLL RNGEKYYLAIMEKGKSKLFEDFPEDESSPFEKIDYKLLPEPSKMLPKVFFATSNKDLFNP SDEILNIRATGSFKKGDSFNLDDCHKFIDFYKASIENHPDWSKFDFDFSETNDYEDISKFF KEVSDQGYSIGYRKISESYLEEMVDNGSLYMFQLYNKDFSENRKSKGTPNLHTLYFKML FDERNLEDVVYKLSGGAEMFYRKPSIDKNEMIVHPKNQPIDNKNPNNVKKTSTFEYDIV KDMRYTKPQFQLHLPIVLNFKANSKGYINDDVRNVLKNSEDTYVIGIDRGERNLVYACV VDGNGKLVEQVPLNVIEADNGYKTDYHKLLNDREEKRNEARKSWKTIGNIKELKEGYI SQVVHKICQLVVKYDAVIAMEDLNSGFVNSRKKVEKQVYQKFERMLTQKLNYLVDKK LDPNEMGGLLNAYQLTNEATKVRNGRQDGIIFYIPAWLTSKIDPTTGFVNLLKPKYNSVS ASKEFFSKFDEIRYNEKENYFEFSFNYDNFPKCNADFKREWTVCTYGDRIRTFRDPENNNKFNSEVWLNDEFKNLFVEFDIDYTDNLKEQILAMDEKSFYKKLMGLLSLTLQMRNSIS KNVDVDYLISPVKNSNGEFYDSRNYDITSSLPCDADSNGAYNIARKGLWAINQIKQADD ETKANISIKNSEWLQYAQNCDEV (SEQ ID NO: 49).

[0290] Cpfl [Lachnospiraceae bacterium]:

[0291] MHENNGKIADNFIGIYPVSKTLRFELKPVGKTQEYIEKHGILDEDLKRAGDYKSVKKIIDAYHKYFIDEALNGIQLDGLKNYYELYEKKRDNNEEKEFQKIQMSLRKQIVKRFSEHPQYKYLFKKELIKNVLPEFTKDNAEEQTLVKSFQEFTTYFEGFHQNRKNMYSDEEK STAIAYRVVHQNLPKYIDNMRIFSMILNTDIRSDLTELFNNLKTKMDITIVEEYFAIDGFN KVVNQKGIDVYNTILGAFSTDDNTKIKGLNEYINLYNQKNKAKLPKLKPLFKQILSDRD KISFIPEQFDSDTEVLEAVDMFYNRLLQFVIENEGQITISKLLTNFSAYDLNKIYVKNDTTI SAISNDLFDDWSYISKAVRENYDSENVDKNKRAAAYEEKKEKALSKIKMYSIEELNFFVBIOTN-41612.601KKYSCNECHIEGYFERRILEILDKMRYAYESCKILHDKGLINNISLCQDRQAISELKDFLD SIKEVQWLLKPLMIGQEQADKEEAFYTELLRIWEELEPITLLYNKVRNYVTKKPYTLEKV KLNFYKSTLLDGWDKNKEKDNLGIILLKDGQYYLGIMNRRNNKIADDAPLAKTDNVYR KMEYKLLTKVSANLPRIFLKDKYNPSEEMLEKYEKGTHLKGENFCIDDCRELIDFFKKGI KQYEDWGQFDFKFSDTESYDDISAFYKEVEHQGYKITFRDIDETYIDSLVNEGKLYLFQI YNKDFSPYSKGTKNLHTLYWEMLFSQQNLQNIVYKLNGNAEIFYRKASINQKDVVVHK ADLPIKNKDPQNSKKESMFDYDIIKDKRFTCDKYQFHVPITMNFKALGENHFNRKVNRL IHDAENMHIIGIDRGERNLIYLCMIDMKGNIVKQISLNEIISYDKNKLEHKRNYHQLLKTR EDENKSARQSWQTIHTIKELKEGYLSQVIHVITDLMVEYNAIVVLEDLNFGFKQGRQKFE RQVYQKFEKMLIDKLNYLVDKSKGMDEDGGLLHAYQLTDEFKSFKQLGKQSGFLYYIP AWNTSKLDPTTGFVNLFYTKYESVEKSKEFINNFTSILYNQEREYFEFLFDYSAFTSKAE GSRLKWTVCSKGERVETYRNPKKNNEWDTQKIDLTFELKKLFNDYSISLLDGDLREQM GKIDKADFYKKFMKLFALIVQMRNSDEREDKLISPVLNKYGAFFETGKNERMPLDADA NGAYNIARKGLWIIEKIKNTDVEQLDKVKLTISNKEWLQYAQEHIL (SEQ ID NO: 50).

[0292] Cpfl [Moraxella sp]:

[0293] MNTSLFSSFTRQYPVTKTLRFELKPMGATLGHIQQKGFLHKDEELAKIYKKIK ELLDEYHRAFIADTLGDAQLVGLDDFYADYQALKQDSKNSHLKDKLTKTQDNLRKQIT KNFEKTPQLKERYKRLFTKELFKAGKDKGDLEKWLINHDSEPNKAEKISWIHQFENFTT YFQGFYENRKNMYSDEVKHTAIAYRLIHENLPRFVDNIQVLSKIKSDYPDLYHELNHLD SRTIDFADFKFDDMLQMDFYHHLLIQSGITAYNTLLGGKVLEGGKKLQGINELINLYGQ KHKIKIAKLKPLHKQILSDGQSVSFLPKKFDNDYELCQTVNHFYREYVAIFDELVVLFQK FYDYDKDNIYINHQQLNQLSHELFADFRLLSRALDFYYCQIIDGDFNNKINNAKSQNAKE KLLKEKERYTKSNHSINELQKAINHYASHHEDTEVKVISDYFSATNIRNMIDGIHHHFSTI KGFLEKDNNQGESYLPKQKNSNDVKNLKLFLDGVLRLIHFIKPLALKSDDTLEKEEHFY GEFMPLYDKLVMFTLLYNKVRDYISQKPYNDEKIKLNFGNSTLLNGWDVNKEKDNFGV ILCKEGLYYLAILDKSHKKVFDNAPKATSSHTYQKMVYKLLPGPNKMLPKVFFAKSNIG YYQPSAQLLENYEKGTHKKGSNFSLTDCHHLIDFFKSSIAKHPEWKEFGFRFSDTHTYQ DLSDFYKEIEPQSYKVKFIDIDADYIDDLVEKGQLYLFQLYNKDFSKQSYGKPNLHTLYF KSLFSDDNLKNPIYKLNGEAEIFYRRASLSVSDTTIHQAGEILTPKNPNNTHNRTLSYDVI KNKRYTTDKFFLHIPITMNFGIENTGFKAFNHQVNTTLKNADKKDVHIIGIDRGERHLLY VSVIDGDGRIVEQRTLNDIVSISNNGMSMSTPYHQILDNREKERLAARTDWGDIKNIKELBIOTN-41612.601KAGYLSHVVHEVVQMMLKYNAMIVLEDLNFGFKHGRFKVEKQVYQNFENALIKKLNY LVLKNADNHQLGSVRKALQLTNNFTDIKSIGKQTGFIFYVPAWNTSKIDPTTGFVDLLKP RYENMAQAQSFISRFKKIAYNHQLDYFEFEFDYADFYQKTIDKKRIWTLCTYGDVRYYY DHKTKETKTVNITKELKSLLDKHDLSYQNGHNLVDELANSHDKSLLSGVMYLLKVLLA LRYSHAQKNEDFILSPVMNKDGVFFDSRFADDVLPNNADANGAYHIALKGLWVLNQIQ SADNMDKIDLSISNEQWLHFTQSR (SEQ ID NO: 51).

[0294] Cpfl [Moraxella bovocul]:

[0295] MLFQDFTHLYPLSKTVRFELKPIGRTLEHIHAKNFLSQDETMADMYQKVKVIL DDYHRDFIADMMGEVKLTKLAEFYDVYLKFRKNPKDDELQKQLKDLQAVLRKESVKPI GNGGKYKAGHDRLFGAKLFKDGKELGDLAKFVIAQEGKSSPKLAHLAHFEKFSTYFTG FHDNRKNMYSDEDKHTAIAYRLIHENLPRFIDNLQILTTIKQKHSALYDQIINELTASGLD VSLASHLDGYHKLLTQEGITAYNRIIGEVNGYTNKHNQICHKSERIAKLRPLHKQILSDG MGVSFLPSKFADDSEMCQAVNEFYRHYADVFAKVQSLFDGFDDHQKDGIYVEHKNLN ELSKQAFGDFALLGRVLDGYYVDVVNPEFNERFAKAKTDNAKAKLTKEKDKFIKGVHS LASLEQAIKHHTARHDDESVQAGKLGQYFKHGLAGVDNPIQKIHNNHSTIKGFLERERP AGERALPKIKSGKNPEMTQLRQLKELLDNALNVAHFAKLLMTKTTLDNQDGNFYGEFG VLYDELAKIPTLYNKVRDYLSQKPFSTEKYKLNFGNPTLLNGWDLNKEKDNFGVILQKD GCYYLALLDKAHKKVFDNAPNTGKNVYQKMIYKLLPGPNKMLPRVFFAKSNLDYYNP SAELLDKYAQGTHKKGDNFNLKDCHALIDFFKAGINKHPEWQNFGFKFSPTSSYRDLSD FYREVEPQGYQVKFVDINADYIDELVEQGQLYLFQIYNKDFSPKAHGKPNLHTLYFRAL FSEDNLANPIYKLNGEAQIFYRKASLGMNETTIHRAGEILENKNPDNPKERVFTYDIIKDR RYTQDKFMLHVPITMNFGVQGMTIKEFNKKVNQSIRQYDDVNVIGIDRGERHLLYLTVI NSKGEILEQRSLNDITTASANGTQMTTPYHKILDKREIERLNARVGWGEIETIKELKSGYL SHVVHQVSQLMLKYNAIWLEDLNFGFKRGRFKVEKQIYQNFENALIKKLNHLELKDK ADDEIGSYKNALQLTNNFTDLKNIGKQTGFLFYVPAWNTSKIDPETGFVDLLKPRYENIA QSQAFFGKFDKICYNADKDYFEFHIDYAKFTDKAKNSRQTWTICSHGDKRYVYDKTAN QNKGATKGINVNDELKSLFARYHINEKQPNLVMDICQNNDKEFHKSLMYLLKTLLALR YSNASSDEDFILSPVANDEGVFFNSALADDTQPQNADANGAYHIALKGLWLLNELKNS DDLNKVKLAIDNQTWLNFAQNR (SEQ ID NO: 52).

[0296] Cpfl [Moraxella lacunat] :BIOTN-41612.601

[0297] MLFQDFTHLYPLSKTVRFELKPIGKTLEHIHAKNFLSQDKTMADMYQKVKAIL DDYHRDFIADMMGEVKLTKLAEFCDVYLKFRKNPKDDGLQKQLKDLQAVLRKEIVKPI GNGGKYKVGYDRLFGAKLFKDGKELGDLAKFVIAQESESSPKLPQIAHFEKFSTYFTGF HDNRKNMYSSDDKHTAIAYRLIHENLPRFIDNLQILATIKQKHSALYDQIASELTASGLD VSLASHLGGYHKLLTQEGITAYNRIIGEVNSYTNKHNQICHKSERIAKLRPLHKQILSDG MGVSFLPSKFADDSEMCQAVNEFYRHYADVFAKVQSLFDRFDDYQKDGIYVEHKNLN ELSKRAFGDFGFLKRFLEEYYADVIDPEFNEKFAKTEPDSDEQKKLAGEKDKFVKGVHS LASLEQVIEYYTAGYDDESVQADKLGQYFKHRLAGVDNPIQKIHNSHSTIKGFLERERPA GERALPKIKSDKSPEMTQLRQLKELLDNALNVVHFAKLVSTETVLDTRSDKFYGEFRPL YVELAKITTLYNKVRDYLSQKPFSTEKYKLNFGNPTLLNGWDLNKEKDNFGVILQKDG CYYLALLDKAHKKVFDNAPNTGKSVYQKMVYKQIANARRDLACLLIINGKVVRKTKG LDDLREKYLPYDIYKIYQSESYKVLSPNFNHQDLVKYIDYNKILASGYFEYFDFRFKESS EYKSYKEFLDDVDNCGYKISFCNINADYIDELVEQGQLYLFQIYNKDFSPKAHGKPNLH TLYFKALFSEDNLANPIYKLNGEAQIFYRKASLDMNETTIHRAGEVLENKNPDNPKQRQ FVYDIIKDKRYTQDKFMLHVPITMNFGVQGMTIEGFNKKVNQSIQQYDDVNVIGIDRGE RHLLYLTVINSKGEILEQRSLNDIITTSANGTQMTTPYHKILNKKKEGRLQARKDWGEIETIKELKAGYLSHVVHQISQLMLKYNAIVVLEDLNFGFKRGRLKVENQVYQNFENALIKK LNHLVLKDKTDDEIGSYKNALQLTNNFTDLKSIGKQTGFLFYVPARNTSKIDPETGFVDL LKPRYENITQSQAFFGKFDKICYNTDKGYFEFHIDYAKFTDEAKNSRQTWVICSHGDKR YVYNKTANQNKGATKGINVNDELKSLFACHHINDKQPNLVMDICQNNDKEFHKSLMY LLKALLALRYSNANSDEDFILSPVANDEGVFFNSALADDTQPQNADANGAYHIALKGL WVLEQIKNSDDLDKVDLEIKDDEWRNFAQNR (SEQ ID NO: 53).

[0298] Cpfl [Moraxella bovocul]:

[0299] MLFQDFTHLYPLSKTVRFELKPIDRTLEHIHAKNFLSQDETMADMHQKVKVIL DDYHRDFIADMMGEVKLTKLAEFYDVYLKFRKNPKDDELQKQLKDLQAVLRKEIVKPI GNGGKYKAGYDRLFGAKLFKDGKELGDLAKFVIAQEGESSPKLAHLAHFEKFSTYFTG FHDNRKNMYSDEDKHTAIAYRLIHENLPRFIDNLQILTTIKQKHSALYDQIINELTASGLD VSLASHLDGYHKLLTQEGITAYNTLLGGISGEAGSPKIQGINELINSHHNQHCHKSERIAK LRPLHKQILSDGMSVSFLPSKFADDSEMCQAVNEFYRHYADVFAKVQSLFDGFDDHQK DGIYVEHKNLNELSKQAFGDFALLGRVLDGYYVDVVNPEFNERFAKAKTDNAKAKLT KEKDKFIKGVHSLASLEQAIEHYTARHDDESVQAGKLGQYFKHGLAGVDNPIQKIHNNHBIOTN-41612.601STIKGFLERERPAGERALPKIKSGKNPEMTQLRQLKELLDNALNVAHFAKLLTTKTTLDN QDGNFYGEFGVLYDELAKIPTLYNKVRDYLSQKPFSTEKYKLNFGNPTLLNGWDLNKE KDNFGVILQKDGCYYLALLDKAHKKVFDNAPNTGKSIYQKMIYKYLEVRKQFPKVFFS KEAIAINYHPSKELVEIKDKGRQRSDDERLKLYRFILECLKIHPKYDKKFEGAIGDIQLFK KDKKGREVPISEKDLFDKINGIFSSKPKLEMEDFFIGEFKRYNPSQDLVDQYNIYKKIDSN DNRKKENFYNNHPKFKKDLVRYYYESMCKHEEWEESFEFSKKLQDIGCYVDVNELFTE IETRRLNYKISFCNINADYIDELVEQGQLYLFQIYNKDFSPKAHGKPNLHTLYFKALFSED NLADPIYKLNGEAQIFYRKASLDMNETTIHRAGEVLENKNPDNPKKRQFVYDIIKDKRY TQDKFMLHVPITMNFGVQGMTIKEFNKKVNQSIQQYDEVNVIGIDRGERHLLYLTVINS KGEILEQCSLNDITTASANGTQMTTPYHKILDKREIERLNARVGWGEIETIKELKSGYLSH VVHQISQLMLKYNAIVVLEDLNFGFKRGRFKVEKQIYQNFENALIKKLNHLVLKDKAD DEIGSYKNALQLTNNFTDLKSIGKQTGFLFYVPAWNTSKIDPETGFVDLLKPRYENIAQS QAFFGKFDKICYNADKDYFEFHIDYAKFTDKAKNSRQIWTICSHGDKRYVYDKTANQN KGAAKGINVNDELKSLFARHHINEKQPNLVMDICQNNDKEFHKSLMYLLKTLLALRYS NASSDEDFILSPVANDEGVFFNSALADDTQPQNADANGAYHIALKGLWLLNELKNSDD LNKVKLAIDNQTWLNFAQNR (SEQ ID NO: 54).

[0300] Cpfl [Thiomicrospira sp]:

[0301] MTKTFDSEFFNLYSLQKTVRFELKPVGETASFVEDFKNEGLKRVVSEDERRAV DYQKVKEIIDDYHRDFIEESLNYFPEQVSKDALEQAFHLYQKLKAAKVEEREKALKEWE ALQKKLREKVVKCFSDSNKARFSRIDKKELIKEDLINWLVAQNREDDIPTVETFNNFTTY FTGFHENRKNIYSKDDHATAISFRLIHENLPKFFDNVISFNKLKEGFPELKFDKVKEDLEV DYDLKHAFEIEYFVNFVTQAGIDQYNYLLGGKTLEDGTKKQGMNEQINLFKQQQTRDK ARQIPKLIPLFKQILSERTESQSFIPKQFESDQELFDSLQKLHNNCQDKFTVLQQAILGLAE ADLKKVFIKTSDLNALSNTIFGNYSVFSDALNLYKESLKTKKAQEAFEKLPAHSIHDLIQ YLEQFNSSLDAEKQQSTDTVLNYFIKTDELYSRFIKSTSEAFTQVQPLFELEALSSKRRPP ESEDEGAKGQEGFEQIKRIKAYLDTLMEAVHFAKPLYLVKGRKMIEGLDKDQSFYEAFE MAYQELESLIIPIYNKARSYLSRKPFKADKFKINFDNNTLLSGWDANKETANASILFKKD GLYYLGIMPKGKTFLFDYFVSSEDSEKLKQRRQKTAEEALAQDGESYFEKIRYKLLPGA SKMLPKVFFSNKNIGFYNPSDDILRIRNTASHTKNGTPQKGHSKVEFNLNDCHKMIDFFKSSIQKHPEWGSFGFTFSDTSDFEDMSAFYREVENQGYVISFDKIKETYIQSQVEQGNLYLF QIYNKDFSPYSKGKPNLHTLYWKALFEEANLNNVVAKLNGEAEIFFRRHSIKASDKVVHBIOTN-41612.601PANQAIDNKNPHTEKTQSTFEYDLVKDKRYTQDKFFFHVPISLNFKAQGVSKFNDKVNG FLKGNPDVNIIGIDRGERHLLYFTVVNQKGEILVQESLNTLMSDKGHVNDYQQKLDKKE QERDAARKSWTTVENIKELKEGYLSHVVHKLAHLIIKYNAIVCLEDLNFGFKRGRFKVE KQVYQKFEKALIDKLNYLVFKEKELGEVGHYLTAYQLTAPFESFKKLGKQSGILFYVPA DYTSKIDPTTGFVNFLDLRYQSVEKAKQLLSDFNAIRFNSVQNYFEFEIDYKKLTPKRKV GTQSKWVICTYGDVRYQNRRNQKGHWETEEVNVTEKLKALFASDSKTTTVIDYANDD NLIDVILEQDKASFFKELLWLLKLTMTLRHSKIKSEDDFILSPVKNEQGEFYDSRKAGEV WPKDADANGAYHIALKGLWNLQQINQWEKGKTLNLAIKNQDWFSFIQEKPYQE (SEQ ID NO: 55).

[0302] Cpfl [Limihaloglobus sulfuriphilu]:

[0303] MSAVFKIKESTMKDFTHQYSLSKTLRFELKPVGETAERIEDFKNQGLKSIVEED RQRAEDYKKMKRILDDYHKEFIEEVLNDDIFTANEMESAFEVYRKYMASKNDDKLKKE ITEIFTDLRKKIAKAFENKSKEYCLYKGDFSKLINEKKTGKDKGPGKLWYWLKAKADAGVNEFGDGQTFEQAEEALAKFNNFSTYFTGFNQNRDNIYTDAEQQTAISYRVINENMTR YFDNCIRYSSIENKYPELVKQLEPLSGKFAPGNYKDYLSQTAIDIYNEAVGHKSDDINAK GINQFINEYRQRNSIKGRELPIMSVLYKQILSDINKDLIIDKFENAGELLDAVKTLHRELTD KKILLKIKQTLNEFLTEDNSEDIYIKSGTDLTAVSNAIWGEWSVIPKALEMYAENITDMN AKAREKWLKREAYHLKTVQEAIEAYLKDNEEFETRNISEYFTNFKSGENDLIQVVQSAY AKMESIFGIEDFHKDRRPVTESGEPGEGFRQVELVREYLDSLINVEHFIKPLHMFRSGKPI ELEDCNSNFYDPLNEAYKELDVVFGIYNKVRNYVTQKPYSKDKFKINFQNSTLLDGWD VNKESANSSVLLLKNGKYYLGVMKQGASNILNYRPEPSDSKNKINAKKQLSEIALAGAT DDYYEKMIYKLLPDPAKMLPKVFFSAKNIEFYNPSQEIIYIRENGLFKKDAGDKESLKKW IGFMKTSLLKHPEWGSYFNFEFEPAEDYQDISIFYKQVAEQGYSVTFDKIKTSYIEEKVAS GELYLFEIYNKDFSPHSKGRPNLHTMYWKSLFEKENLQNLVTKLNGEAEVFFRQHSIKR NEKVVHRANRPIQNKNPLTEKKQSIFEYDLVKDRRFTKDKFFLHCPITLNFKEAGPGRFN DKVNKYIAGNPDIRIIGIDRGERHLLYYSLIDQSGRIVEQGTLNQITSTLNSGGREIPKTTD YRGLLDTKEKERDKARKSWSMIENIKELKSGYLSHIVHKLAKLMVKNNAWVLEDLNF GFKRGRFKVEKQVYQKFEKALIEKLNYLVFKDARPAEPGHYLNAYQLTAPLESFKKLG KQSGFIYYVPAWNTSKIDPVTGFVNQFYIEKNSMQYLKNFFGKFDSIRFNPDKNYFEFGF DYKNFHNKAAKSKWTICTHGDKRSWYNRKQRKLEIHNVTENLASLLSGKGINFADGGSIKDKILSVDDASFFKSLAFNFKLTAQLRHTFEDNGEEIDCIISPVAAADGTFFCSETAKKLBIOTN-41612.601NMELPHDADANGAYNIARKGLMVLRQIRESGKPKPISNADWLDFAQQNED (SEQ ID NO: 56).

[0304] Cpfl [Anaerovibrio sp]:

[0305] MVAFIDEFVGQYPVSKTLRFEARPVPETKKWLESDQCSVLFNDQKRNEYYGV LKELLDDYYRAYIEDALTSFTLDKALLENAYDLYCNRDTNAFSSCCEKLRKDLVKAFGN LKDYLLGSDQLKDLVKLKAKVDAPAGKGKKKIEVDSRLINWLNNNAKYSAEDREKYIK AIESFEGFVTYLTNYKQARENMFSSEDKSTAIAFRVIDQNMVTYFGNIRIYEKIKAKYPEL YSALKGFEKFFSPTAYSEILSQSKIDEYNYQCIGRPIDDADFKGVNSLINEYRQKNGIKAR ELPVMSMLYKQILSDRDNSFMSEVINRNEEAIECAKNGYKVSYALFNELLQLYKKIFTED NYGNIYVKTQPLTELSQALFGDWSILRNALDNGKYDKDIINLAELEKYFSEYCKVLDAD DAAKIQDKFNLKDYFIQKNALDATLPDLDKITQYKPHLDAMLQAIRKYKLFSMYNGRK KMDVPENGIDFSNEFNAIYDKLSEFSILYDRIRNFATKKPYSDEKMKLSFNMPTMLAGW DYNNETANGCFLFIKDGKYFLGVADSKSKNIFDFKKNPHLLDKYSSKDIYYKVKYKQVS GSAKMLPKWFAGSNEKIFGHLISKRILEIREKKLYTAAAGDRKAVAEWIDFMKSAIAIH PEWNEYFKFKFKNTAEYDNANKFYEDIDKQTYSLEKVEIPTEYIDEMVSQHKLYLFQLY TKDFSDKKKKKGTDNLHTMYWHGVFSDENLKAVTEGTQPIIKLNGEAEMFMRNPSIEFQVTHEHNKPIANKNPLNTKKESVFNYDLIKDKRYTERKFYFHCPITLNFRADKPIKYNEK INRFVENNPDVCIIGIDRGERHLLYYTVINQTGDILEQGSLNKISGSYTNDKGEKVNKETD YHDLLDRKEKGKHVAQQAWETIENIKELKAGYLSQVVYKLTQLMLQYNAVIVLENLN VGFKRGRTKVEKQVYQKFEKAMIDKLNYLVFKDRGYEMNGSYAKGLQLTDKFESFDK IGKQTGCIYYVIPSYTSHIDPKTGFVNLLNAKLRYENITKAQDTIRKFDSISYNAKADYFE FAFDYRSFGVDMARNEWVVCTCGDLRWEYSAKTRETKAYSVTDRLKELFKAHGIDYV GGENLVSHITEVADKHFLSTLLFYLRLVLKMRYTVSGTENENDFILSPVEYAPGKFFDSR EATSTEPMNADANGAYHIALKGLMTIRGIEDGKLHNYGKGGENAAWFKFMQNQEYKN NG (SEQ ID NO: 57).

[0306] Cpfl [Nitrospinae bacterium]:

[0307] MENKELFNSFTKKCQLSKTLRFELVPQGETEKFIKKKGLLKQDKDRADDYRK AKKLIDEYHKDFIEHALSGKQLKKLQAYYDEFTALLGKPAKERDTRVLSNISEALRKEIA GWLKDNPIKDEKKLIKEIVPNFLKSKDRNDDAALVLKFKGFTTYFGGFNENRRNMYSAE EKSTAIAYRIVHENLPKFISNMKTFQRLTENHKIDFLEVETQMNDELDGNKLKDVFSIDY FNQCLTQSGIDRYNTILGGKTDSRGAQIQGVNIKINLFGQKNNLKSKDAPVLARLYKQILBIOTN-41612.601SDRTSASFLPEKFETAKELIASIREFYEKAINGFIKNNERKNVIDEIESLFNKKLNPTDCDL SQIYIRNIFISAVSQQIFGSYSVITSALNAFIDKTYKTKKEKEKQQKKSYFGIAEIEAALKA YLQETEIENKEKREEVLKHANPVLQYLNSLTTNIEINNKKEKANVIVRFRQQHEAVQSVL NKEYGNNSELIQDKNAVAAIKDYLDSCQHILHFVKPLFVVPPKKENAELPDKDAFYSEF DELFEQLKSVTPLYNMVRNFLTQKPYSTEKIKLNFENVSLLSGWDVNKETDNTSVIFRKDSLYYLGIMDKKHNKVFHDIPASSDNDFYEKMNYKLLPGANKMLPKVIFSDRWIEYFNP SKELIEKYKKGTHKKGEIFSLSDCHALIDFFKTSIAKHEDWKQFNFQFSPTKTYQDINEFY REVEHQGYKISFQNVSAKYIHQLVSEGKLFLFKIYNKDFSPYSKGKPNLHTLYWKAVFD KENLKDVVVKLNGEAEVFYRPQSIPKKSKIVHKKNKPIVNKNPNNPKKQSLFKEYDIVKDKRYTEDKYHFHVPITLNFKEAKVPSENDRKKAFAYTFNKGANETLKENFPNIIGIDRGE RHLAYYSLINSKGEILEQGSFNVIKNEFNGTKHETDYYASLVEKEKARDAARKSWDTIGKIKELKEGYLSQVVHKIARMMIEHNAIIVLEDLTMKFKDIRKKVERQVYQKLEKMLIDK LNYLVFKDREAYEPGGVLNAYQLAAPFTSFKDMGKQTGFIFYVPAANTSKIDYATGFM NFIYPKIEDAVEFFQKFEYIRFNPDKNYFEFAARYNNFVKDDEAKLPDEYNEPWIICTHG EQRFQYQPSYKSFKEVNVTQELQDLFSKHAIMPYAHGNDLSPQIKDKGNTSFYKGLIALLRLVLQMRYTDGKGRDFILSPVANDEGVFFNSEKAKETEPNDADANGAYHIALKGLLLL KRIKGEFKKEEKTGKKKTQKENWIDLSISNKEWYEFAQKKEYRK (SEQ ID NO: 58).

[0308] Hypothetical [Planctomycetes bacterium]:

[0309] MNSSNDAKKDFSSFTNKYQLSKTLRFELIPQGKTREHIETKGLLQRDEHRAESYKKVKKIIDEYHKVFIEKAMKDFRLEGLENFKLLYLKKEKEDKDKKELQEVMKGLRKQ IAACFSNNKDESVAAIYKNLFAKELIKKDLKDFVKEEDKELIEEFEDFTTYFTGFHENRKNIYTHEEKSTAIAYRLIHENLPSFLDNLGVYQSIKEDHPDLDFSPMEKELYEVLNGMRVK GIEEVFTLDFFNNMLTQKGIDAYNAILGGKSEKGQRKIKGVNEYIHEHRQLKGLTLRQLP NLKPLYKQILSDRESISFLPEYFEKDQELLDSIKAFYQNGLCSWEKDGKTQDVLQEIENLL LPLNEFDLEKIFIKNDISLTGISQKIFGDWSFIGDALGSYYEHKNPLPKKLTKTYEEKKEK WVTETTHFSIADIQRALDLYKTKNEEAKEKITASILCSFFGSMGEVQVAGEPKAPNLLQR IEESYGPVTDLLNTPYPENRKLAQDKAEVSKIKNFLDTVMDLLHFIKPLHPRENIQEKDV KFYNAYSTLFDQLNLITDLYNKTRNYLTKKPYSTEKIKLNFENSTLLDGWDANKETDNT SVLLRKNGFFYLAIMDKKHNRIFNQIPKLNNGEASYEKMNYKLLPLINQQLPRVFFAKSRIEYFNPSQEILKNYENDTHKKGETFNINDCHALIDFFKASLERHEDWKNFNFKFSPTTSYE DLSGFYREVEQQGYKVTFQNVPENYINRMVEEGKLYLFQIYNKDFSPFSKGKPNLHTLYBIOTN-41612.601WKMLFAEENLKNVVYKLNGQAEVFFRKKSLNYDEKILKTGHHAEKLKDRFNYPIISNR RFAFDKFQFHVPITLNFKATGNGNINQDVLTYLKNNPNVNIIGIDRGERHLLYVTLIDQQ RNIKQQFSLNEIVNRYNGKEYATNYHAKLDAKEKDRDVARKSWGVIENIRELKEGYLS QVVHQIAGMMVEHNAVVVMEDLNFGFKRGRQKVEKQVYQKFEKMLIDKLNYMAFKN KEAAETGGVLNALQLSNKFDSFQKLGKQSGFIFYVPAWHTSKIDPATGFVDFFKPKYENI EKAKEFFGNFKSICFNRGKNWFEFDFDYNKFTPKAEDTRTQWTVCTTPEVRYVWNRNK ETANGRGGYDEYLITEKMKSLFDGEDIRIRYQNGSDIKREIVGQSSGQFFKELTRLLGVT LSLRYNNGKKGDAEKDFILSPVANNKGEFFNSLKADASQPQNADANGAYHIALKGLWV LHQINEANDLKKVKLAISNKEWLQFVQRYES (SEQ ID NO: 59).

[0310] Cpfl [Bacteroidetes bacterium]:

[0311] MNHMKQFTNQFSLSKTLRFELIPQGKTKEFIEINGLIEKDNERAVSYKKVKKII DEYHKYFIEMVLCDFKLHGLETYETIFNKKEKDDTDKKEFDNIRNSLRKQIADAFAKNPNDEIKERFKNLFAKELIKQDLLNFVDDEQKELVNEFKDFTTYFTGFHQNRRNMYVADEK ATAIAYRLVNENLPKFIDNLKIYEKIKKDAPELISDLNKTLVEMEEIVQGKTLDEIFSLSFF NQTLTQTGIELYNIVIGGRTADEGKTKIKGLNEYINTDYNQKQTDKKKKQAKFKQLYKQ ILSDRHSVSFVAETFETDAQLLENIEQFYSSVLCNYEDDGHTTNIFEAIKNLIIGLKTFDLS KIYLRNDTSLTDISQKLFGDWSIISSALNDYYEKQNPISSKEKQEKYDERKAKWLKQDFN IETIQTALNECDSEIIKEKNNKNIVSEYFAKLGLDKDNKIDLLQKIHHNYVVIKDLLNEPY PENIKLGNQKEQVSQIKDFLDSILNLIHFLKPLSLKDKDKEKDELFYSLFTALFEHLSQTISI YNKVRNYLTQKAYSTEKIKLNFENSTLLNGWDVNKEPVNTSVIFRKNGLFYLGIMSKSN NRIFERNVPVCKNEETAFEKMNYKLLPGANKMLPKVFLSAKGIESFQPSAEIQSKYQKET HKKGDAFVRKDMENLIDFFKQSIAKHTDWKHFNHQFSKTETYNDLSEFYKEVEKQGYK LTFTKLDETYINQLVDEGKLYLFQIYNKDFSPFSKGKPNMHTLYWKMLFDEQNLQNVV YKLNGEAEVFFRQSSIKQTDRIIHKANQAIDNKNPLNNKKQSSFNYDLIKDKRFTLDKFQ FHVPITLNFKAEGNEYLNTKVNEYLKSNSDVKIIGLDRGERHLIYLTLINQKGELLKQQSL NVIATSQEHETDYKNLLVNKENERANARQDWKTIETIKELKEGYLSQVVHQIATMMVD ENAIWMEDLNAGFMRGRQKVERQVYQKLEKMLIEKLNYLVFKNNDVNETAGVLNAL QLTNKFESFEKMGKQSGFLFYVPAWNTSKIDPATGFVDFLKPKYESVEKAKLFFEKFESI KFNADKNYFEFEFDYKKFTEKAEGSQTKWTVCTHSDVRYRYNPQTKASDEVNVTNEL KLIFDKFKIEYKNGKNLKTELLLQDDKQLFSKLLHYLALTLMLRQSKSGTDIDFILSPVABIOTN-41612.601KNGVFYDSRNAMPNLPKDADANGAFHIALKGLWCVQQIKKADDLKKIKLAISNKEWLS FVQNLK (SEQ ID NO: 60).

[0312] Hypothetical[Smithella_sp] :

[0313] MQTLFENFTNQYPVSKTLRFELIPQGKTKDFIEQKGLLKKDEDRAEKYKKVKN IIDEYHKDFIEKSLNGLKLDGLEKYKTLYLKQEKDDKDKKAFDKEKENLRKQIANAFRN NEKFKTLFAKELIKNDLMSFACEEDKKNVKEFEAFTTYFTGFHQNRANMYVADEKRTAI ASRLIHENLPKFIDNIKIFEKMKKEAPELLSPFNQTLKDMKDVIKGTTLEEIFSLDYFNKTL TQSGIDIYNSVIGGRTPEEGKTKIKGLNEYINTDFNQKQTDKKKRQPKFKQLYKQILSDR QSLSFIAEAFKNDTEILEAIEKFYVNELLHFSNEGKSTNVLDAIKNAVSNLESFNLTKMYF RSGASLTDVSRKVFGEWSIINRALDNYYATTYPIKPREKSEKYEERKEKWLKQDFNVSLI QTAIDEYDNETVKGKNSGKVIADYFAKFCDDKETDLIQKVNEGYIAVKDLLNTPCPENE KLGSNKDQVKQIKAFMDSIMDIMHFVRPLSLKDTDKEKDETFYSLFTPLYDHLTQTIAL YNKVRNYLTQKPYSTEKIKLNFENSTLLGGWDLNKETDNTAIILRKDNLYYLGIMDKRH NRIFRNVPKADKKDFCYEKMVYKLLPGANKMLPKVFFSQSRIQEFTPSAKLLENYANET HKKGDNFNLNHCHKLIDFFKDSINKHEDWKNFDFRFSATSTYADLSGFYHEVEHQGYKI SFQSVADSFIDDLVNEGKLYLFQIYNKDFSPFSKGKPNLHTLYWKMLFDENNLKDVVYK LNGEAEVFYRKKSIAEKNTTIHKANESIINKNPDNPKATSTFNYDIVKDKRYTIDKFQFHI PITMNFKAEGIFNMNQRVNQFLKANPDINIIGIDRGERHLLYYALINQKGKILKQDTLNVI ANEKQKVDYHNLLDKKEGDRATARQEWGVIETIKELKEGYLSQVIHKLTDLMIENNAII VMEDLNFGFKRGRQKVEKQVYQKFEKMLIDKLNYLVDKNKKANELGGLLNAFQLANK FESFQKMGKQNGFIFYVPAWNTSKTDPATGFIDFLKPRYENLNQAKDFFEKFDSIRLNSK ADYFEFAFDFKNFTEKADGGRTKWTVCTTNEDRYAWNRALNNNRGSQEKYDITAELK SLFDGKVDYKSGKDLKQQIASQESADFFKALMKNLSITLSLRHNNGEKGDNEQDYILSP VADSKGRFFDSRKADDDMPKNADANGAYHIALKGLWCLEQISKTDDLKKVKLAISNKE WLEFVQTLKG (SEQ ID NO: 61).

[0314] Cpfl [Leptospira inada]:

[0315] MEDYSGFVNIYSIQKTLRFELKPVGKTLEHIEKKGFLKKDKIRAEDYKAVKKII DKYHRAYIEEVFDSVLHQKKKKDKTRFSTQFIKEIKEFSELYYKTEKNIPDKERLEALSE KLRKMLVGAFKGEFSEEVAEKYKNLFSKELIRNEIEKFCETDEERKQVSNFKSFTTYFTG FHSNRQNIYSDEKKSTAIGYRIIHQNLPKFLDNLKIIESIQRRFKDFPWSDLKKNLKKIDKN IKLTEYFSIDGFVNVLNQKGIDAYNTILGGKSEESGEKIQGLNEYINLYRQKNNIDRKNLPBIOTN-41612.601NVKILFKQILGDRETKSFIPEAFPDDQSVLNSITEFAKYLKLDKKKKSIIAELKKFLSSFNR YELDGIYLANDNSLASISTFLFDDWSFIKKSVSFKYDESVGDPKKKIKSPLKYEKEKEKW LKQKYYTISFLNDAIESYSKSQDEKRVKIRLEAYFAEFKSKDDAKKQFDLLERIEEAYAIV EPLLGAEYPRDRNLKADKKEVGKIKDFLDSIKSLQFFLKPLLSAEIFDEKDLGFYNQLEG YYEEIDSIGHLYNKVRNYLTGKIYSKEKFKLNFENSTLLKGWDENREVANLCVIFREDQ KYYLGVMDKENNTILSDIPKVKPNELFYEKMVYKLIPTPHMQLPRIIFSSDNLSIYNPSKSI LKIREAKSFKEGKNFKLKDCHKFIDFYKESISKNEDWSRFDFKFSKTSSYENISEFYREVE RQGYNLDFKKVSKFYIDSLVEDGKLYLFQIYNKDFSIFSKGKPNLHTIYFRSLFSKENLKD VCLKLNGEAEMFFRKKSINYDEKKKREGHHPELFEKLKYPILKDKRYSEDKFQFHLPISLNFKSKERLNFNLKVNEFLKRNKDINIIGIDRGERNLLYLVMINQKGEILKQTLLDSMQSG KGRPEINYKEKLQEKEIERDKARKSWGTVENIKELKEGYLSIVIHQISKLMVENNAIVVL EDLNIGFKRGRQKVERQVYQKFEKMLIDKLNFLVFKENKPTEPGGVLKAYQLTDEFQSF EKLSKQTGFLFYVPSWNTSKIDPRTGFIDFLHPAYENIEKAKQWINKFDSIRFNSKMDWF EFTADTRKFSENLMLGKNRVWVICTTNVERYFTSKTANSSIQYNSIQITEKLKELFVDIPF SNGQDLKPEILRKNDAVFFKSLLFYIKTTLSLRQNNGKKGEEEKDFILSPVVDSKGRFFNS LEASDDEPKDADANGAYHIALKGLMNLLVLNETKEENLSRPKWKIKNKDWLEFVWER NR (SEQ ID NO: 62).

[0316] Cpfl [Leptospira ryugeni]:

[0317] MKNLANFTNLYSLQKTLRFELKPIGKTLDWIIKKDLLKQDEILAEDYKIVKKII DRYHKDFIDLAFESAYLQKKSSDSFTAIMEASIQSYSELYFIKEKSDRDKKAMEEISGIMR KEIVECFTGKYSEVVKKKFGNLFKKELIKEDLLNFCEPDELPIIQKFADFTTYFTGFHENRENMYSNEEKATAIANRLIRENLPRYLDNLRIIRSIQGRYKDFGWKDLESNLKRIDKNLQY SDFLTENGFVYTFSQKGIDRYNLILGGQSVESGEKIQGLNELINLYRQKNQLDRRQLPNL KELYKQILSDRTRHSFVPEKFSSDKALLRSLLDFHKEVIQNKNLFEEKQVSLLQAIRETLT DLKSFDLDRIYLTNDTSLTQISNFVFGDWSKVKTILAIYFDENIANPKDRQRQSNSYLKA KENWLKKNYYSIHELNEAISVYGKHSDEELPNTKIEDYFSGLQTKDETKKPIDVLDAIVS KYADLESLLTKEYPEDKNLKSDKGSIEKIKNYLDSIKLLQNFLKPLKPKKVQDEKDLGFY NDLELYLESLESANSLYNKVRNYLTGKEYSDEKIKLNFKNSTLLDGWDENKETSNLSVIF RDTNNYYLGILDKQNNRIFESIPEIQSGEETIQKMVYKLLPGANNMLPKVFFSEKGLLKF NPSDEITSLYSEGRFKKGDKFSINSLHTLIDFYKKSLAVHEDWSVFNFKFDETSHYEDISQFYRQVESQGYKITFKPISKKYIDTLVEDGKLYLFQIYNKDFSQNKKGGGKPNLHTIYFKSBIOTN-41612.601LFEKENLKDVIVKLNGQAEVFFRKKSIHYDENITRYGHHSELLKGRFSYPILKDKRFTED KFQFHFPITLNFKSGEIKQFNARVNSYLKHNKDVKIIGIDRGERHLLYLSLIDQDGKILRQ ESLNLIKNDQNFKAINYQEKLHKKEIERDQARKSWGSIENIKELKEGYLSQVVHTISKLM VEHNAIVVLEDLNFGFKRGRQKVERQVYQKFEKMLIEKLNFLVFKDKEMDEPGGILKA YQLTDNFVSFEKMGKQTGFVFYVPAWNTSKIDPKTGFVNFLHLNYENVNQAKELIGKF DQIRYNQDRDWFEFQVTTDQFFTKENAPDTRTWIICSTPTKRFYSKRTVNGSVSTIEIDV NQKLKELFNDCNYQDGEDLVDRILEKDSKDFFSKLIAYLRILTSLRQNNGEQGFEERDFI LSPVVGSDGKFFNSLDASSQEPKDADANGAYHIALKGLMNLHVINETDDESLGKPSWKI SNKDWLNFVWQRPSLKA (SEQ ID NO: 63).

[0318] Cpfl [Proteocatella sphenisc]:

[0319] MIILYISTSNMNMEGVFMENFKNLYPINKTLRFELRPYGKTLENFKKSGLLEKD AFKANSRRSMQAIIDEKFKETIEERLKYTEFSECDLGNMTSKDKKITDKAATNLKKQVIL SFDDEIFNNYLKPDKNIDALFKNDPSNPVISTFKGFTTYFVNFFEIRKHIFKGESSGSMAY RIIDENLTTYLNNIEKIKKLPEELKSQLEGIDQIDKLNNYNEFITQSGITHYNEIIGGISKSEN VKIQGINEGINLYCQKNKVKLPRLTPLYKMILSDRVSNSFVLDTIENDTELIEMISDLINKT EISQDVIMSDIQNIFIKYKQLGNLPGISYSSIVNAICSDYDNNFGDGKRKKSYENDRKKHL ETNVYSINYISELLTDTDVSSNIKMRYKELEQNYQVCKENFNATNWMNIKNIKQSEKTN LIKDLLDILKSIQRFYDLFDIVDEDKNPSAEFYTWLSKNAEKLDFEFNSVYNKSRNYLTR KQYSDKKIKLNFDSPTLAKGWDANKEIDNSTIIMRKFNNDRGDYDYFLGIWNKSTPANE KIIPLEDNGLFEKMQYKLYPDPSKMLPKQFLSKIWKAKHPTTPEFDKKYKEGRHKKGPD FEKEFLHELIDCFKHGLVNHDEKYQDVFGFNLRNTEDYNSYTEFLEDVERCNYNLSFNK IADTSNLINDGKLYVFQIWSKDFSIDSKGTKNLNTIYFESLFSEENMIEKMFKLSGEAEIFY RPASLNYCEDIIKKGHHHAELKDKFDYPIIKDKRYSQDKFFFHVPMVINYKSEKLNSKSL NNRTNENLGQFTHIIGIDRGERHLIYLTVVDVSTGEIVEQKHLDEIINTDTKGVEHKTHYL NKLEEKSKTRDNERKSWEAIETIKELKEGYISHVINEIQKLQEKYNALIVMENLNYGFKN SRIKVEKQVYQKFETALIKKFNYIIDKKDPETYIHGYQLTNPITTLDKIGNQSGIVLYIPAW NTSKIDPVTGFVNLLYADDLKYKNQEQAKSFIQKIDNIYFENGEFKFDIDFSKWNNRYSI SKTKWTLTSYGTRIQTFRNPQKNNKWDSAEYDLTEEFKLILNIDGTLKSQDVETYKKFM SLFKLMLQLRNSVTGTDIDYMISPVTDKTGTHFDSRENIKNLPADADANGAYNIARKGI MAIENIMNGISDPLKISNEDYLKYIQNQQE (SEQ ID NO: 64).

[0320] Cpfl [Helcococcus ovi]:BIOTN-41612.601

[0321] MLNKIIKEKIMSFERLTNIASISKTLRFRLKPVGKTLENLEKLGKLDKDFERNNF YPILKNIADDYYRQYIRNRLTDLNLDWIKLYYAHELLNSTDKESKKNLTTIQSEYRKILL NILSGELDKNGEKFSKDIVKKNKELYGKLFKKEFILEILPKFVETTNIYNKEYFNGINLYN KFTTRLSNFWEARKNIFTDKDIATGIPFRVVNENFVYFYKNIQVFNKNIKYLEDKLDNLE KNLKSEGIMSIDKSIKDFFNPNGFNYVITQKGIDTYQAIRGGFTKENGEKVQGINEILNLT QQKLRRNPYTKNIKLGVLTKLRKQILEYSESTSFLIDQIEDDNDLVDRINKFNVSFFESTE VSPSIFVQLENLYNSLRTANSEDIYIDARNTQKFSQMLFGQWDVIRRGYSLKITEGTKEE KKKYKKYIELDETSKAKGYLTLMEIQELVSSVEGYEEIDVFNVLLEKFKINIIERLKVETPI YGSPMKLEAIKEYLEKHLEEYHKWKLLLINNDELDLDEAFYPLLNEVISDYNIIQLYNLT RNYLTRKYSDKEKIKINFDFPTLADGWSESKISDNRSIILRKDGNYYLGILEDNKLLDNNI TNFLENCYEIMKYNLFPDAAKMIPKCSISKKEVKNHFENGEDKSIYLSNQFVGRLEISKE LYELQNNLVDGKKKYQIDYLRNTDDKVGYRNALNQWITFCKKFLNKYQGTQDFDYSK LKEAKYYDKLDQFYADVDSYGYSLDFDTINEDLVNKAVEDGKLLLFQIYNKDFSPESKG KKNLHTLYWLSMFSDENLKARKLKLNGQAEIFYRKKLEKKPIIHKEGSILLNKIDKDGNT IPENIYHECYRYLNKKIGRKDLSDKAITLFNKDVLNYKEARFDIIKDRRYSESQFFFHVPIT FNWDLKSNQNVNSIVQNMIKDREIKHIIGIDRGERHLLYYSVIDLEGNIVEQGSLNTLNQ NRFDNSIVEVDYQDKLRTREEDRDRARKNWTNINKIKELKDGYLSHVVHKLSKLIIDYE AIVILENLNQGFKRGRFKVERQVYQKFELALMNKLSALSFKETYDEGKNLEPSGILNPIQ ACYPVDSYQDLQGQNGIVFYLPAAYTSVIDPVTGFTNLFRLNSINTTKYEEFIKGFKNIYF DNEDLDFKFIFDYKNFEKFNFVSFKNKKSKKWIVSTRGERISYNSKKKEYFYVKPTEILK NKLIELGINFEDKDKDIISLIDKINDSKKIKLLKVVFDAFKYSVQLRNHDNIQDYIISPVAD ENGNYYNSNDVAIKNLKLPDNGDANGAFNIARKGLLLIERISNSDDSKVDLKIKNEDWI DFIIS (SEQ ID NO: 65).

[0322] Cpfl [Helcococcus kunzi]:

[0323] MFEKLSNIVSISKTIRFKLIPVGKTLENIEKLGKLEKDFERSDFYPILKNISDDYY RQYIKEKLSDLNLDWQKLYDAHELLDSSKKESQKNLEMIQAQYRKVLFNILSGELDKSG EKNSKDLIKNNKALYGKLFKKQFILEVLPDFVNNNDSYSEEDLEGLNLYSKFTTRLKNF WETRKNVFTDKDIVTAIPFRAVNENFGFYYDNIKIFNKNIEYLENKIPNLENELKEADILD DNRSVKDYFTPNGFNYVITQDGIDVYQAIRGGFTKENGEKVQGINEILNLTQQQLRRKPE TKNVKLGVLTKLRKQILEYSESTSFLIDQIEDDNDLVDRINKFNVSFFESTEVSPSLFEQIE RLYNALKSIKKEEVYIDARNTQKFSQMLFGQWDVIRRGYTVKITEGSKEEKKKYKEYLEBIOTN-41612.601LDETSKAKRYLNIREIEELVNLVEGFEEVDVFSVLLEKFKMNNIERSEFEAPIYGSPIKLEA IKEYLEKHLEEYHKWKLLLIGNDDLDTDETFYPLLNEVISDYYIIPLYNLTRNYLTRKHS DKDKIKVNFDFPTLADGWSESKISDNRSIILRKGGYYYLGILIDNKLLINKKNKSKKIYEIL IYNQIPEFSKSIPNYPFTKKVKEHFKNNVSDFQLIDGYVSPLIITKEIYDIKKEKKYKKDFY KDNNTNKNYLYTIYKWIEFCKQFLYKYKGPNKESYKEMYDFSTLKDTSLYVNLNDFYA DVNSCAYRVLFNKIDENTIDNAVEDGKLLLFQIYNKDFSPESKGKKNLHTLYWLSMFSE ENLRTRKLKLNGQAEIFYRKKLEKKPIIHKEGSILLNKIDKEGNTIPENIYHECYRYLNKKI GREDLSDEAIALFNKDVLKYKEARFDIIKDRRYSESQFFFHVPITFNWDIKTNKNVNQIV QGMIKDGEIKHIIGIDRGERHLLYYSVIDLEGNIVEQGSLNTLEQNRFDNSTVKVDYQNK LRTREEDRDRARKNWTNINKIKELKDGYLSHVVHKLSRLIIKYEAIVIMENLNQGFKRGR FKVERQVYQKFELALMNKLSALSFKEKYDERKNLEPSGILNPIQACYPVDAYQELQGQN GIVFYLPAAYTSVIDPVTGFTNLFRLKSINSSKYEEFIKKFKNIYFDNEEEDFKFIFNYKDF AKANLVILNNIKSKDWKISTRGERISYNSKKKEYFYVQPTEFLINKLKELNIDYENIDIIPLI DNLEEKAKRKILKALFDTFKYSVQLRNYDFENDYIISPTADDNGNYYNSNEIDIDKTNLPNNGDANGAFNIARKGLLLKDRIVNSNESKVDLKIKNEDWINFIIS (SEQ ID NO: 66).

[0324] Cpfl [Clostri diales]:

[0325] MENKRDENIYGRYEEFIGLNSLSKTLRNSLIPVGSTLKHIQEYGILEEDSLRAQK REELKGIMDDYYRNYIEMHLRDVHDIDWNELFEALTEVKKNQTDDAKKRLEKIQEKKR KEIYQYLSDDAVFSEMFKEKMISGILPDFIRCNEEYSEEEKEKKLKTVALFHRFTSSFNDF FLNRKNVFTKEAIATAIGYRWHENAEIFLENMVAFQNIQKSAESQISIIERKNEHYFME WKLSHIFTADYYMMLMTQKAIEHYNEMCGVVNQQMKEYCQKEKKNWNLYRMKRLH KQILSNASTSFKIPEKYENDAEVYESVNSFLQNVMEKTVMERIAVLKNSTDNFDLSKIYI TAPYYEKISNYLCGSWNTITDCLTHYYEQQIAGKGARKDQKVKAAVKADKWKSLSEIE QLLKEYARAEEVKRKPEEYIAEIENIVSLKEVHFLEYHPEVNLIENEKYAIEIKDVLDNYM ELFHWMKWFYIEEAVEKEVNFYGELDDLYEEIKDIVPLYNKVRNYVTQKPYSDTKIKLN FGTPTLANGWSKSKEYDYNAILLQKDGKYYMGIFNPIQKPEKEIIEGHSQPLEGNEYKK MVYYYLPSANKMLPKVLLSKKGMEIYQPSEYIINGYKERRHIKSEEKFDLQFCHDLIDYF KSGIERNPDWKVFGFNFSDTDTYQDISGFYREVEDQGYKIDWTYIKEADIDRLNEEGKL YLFQIYNKDFSEKSTGRENLHTMYLKNLFSEENVKEQVLKLNGEAEIFFRKSSVKKPIIHKKGTMLVNRTYMEEVNGNSVRRNIPEKEYQEIYNYKNHRLKGELSTEAKKYLEKAVCH ETKKDIVKDYRYSVDKFFIHLPITINYRASGKEMLNSVAQRYIAHQNDMHVIGIDRGERNBIOTN-41612.601LIYVSVINMQGEIKEQKSFNIINEFNYKEKLKEREQSRGAARRNWKEIGQIKDLKEGYLS GVIHEIARMMIEYHAIIAMEDLNYGFKRGRFKVERQVYQKFENMLIQKLNYLVFKDRSA DEDGGVLRGYQLAYIPDSVKKLGRQCGMIFYVPAAFTSKIDPTTGFVDIFNHKAYTTDQ AKREFILSFDEICYDVERQLFRFTFDYANFATHNVTLARNNWTIYTNGTRTQKEFVNRR VRDKEDYNPKDKMVELLESEEIEFKSGQNLLPALKKVSNAKVFEELQKIVRFTVQLRNS KSEENDVDYDHVISPVLNEEGKFFDSSKYKNKEEKKESLLPVDADANGAYCIALKGLYI MQAIQKNWSEEKALSPDVLRLNNNDWFDYIQNKRYR (SEQ ID NO: 67).

[0326] Cpfl [Firmicutes bacterium]:

[0327] MERKDFINYDQVSKTIRNGLIPTEYTLKNIEETGVLVSDEYRAEVRNVLKKIM DDYYKEYINMCLNREIKMDWRPLFDAYELVKKGKMKPKEIENIQDEKRKEIYDILSAHD EFKKMFSAKMITELLPQFISQSTGYNEDEKKQYEEVIHIYSRFTSDFTDFFQNRKNVFSSA GIATSICNRIVNENAEIFSDNKSTFDRIKKDISYIDEIIDSDLKTYLDGWELEQIYSVDFYSR LMSQSGIDFYNMISGAVNKAVNEYCQKNGLNKNKYLLRKLRKQILSNSESTFQIPEKFL ADEEVYKAVGQFIDNIKSKNVIEILKDIGSNCENYDLRRIYIAESAYEDVSIFMGYGWNGI KGCLEKKYEKEIAPGKAKDIKIKKLISQEKERSIGEIDELFHIYGEEKDGKAAIQYIEEVVE LCNEVPTRLVYDSSEKLTENDNKAAEIKNVLDYYFKIFHWIKTFITDDILDKDIVFYSGIE FAYEEIKDIVPLYNKVRNYVTQKPYSSEKIKLKFGTPTLANGWSKSKEFDNNAILLVRDN KYYLGIFNVLNKPDKKIMAGYANKKNSSDYAKMVYNLLPGPSKMLPKVCLSQKGKDT YHPSAYIVDNYERKAHVKSSEHFSIQYCHDLIDYFKECIKRNDDWKCFDFKFSDTEQYN DISEFYKEVELQGYSIGWTYISGEDIEALDKEGKLYLFQIYNKDFSEQSHGTDNLHTMYL KNLFSPENLNNIVLKLNGEAELFYRKSSIKKPVVHKKGSILVNKTYTEKTGNGEEVRRPV PDDVYLELVNYYNGEQKAILSDEAKKYMKVVEHHEAVKDITKDYRYTVNKFFIHLPITI NFKAEHTGNLNEMVIQYIAKQENMHVIGIDRGERNLIYVSVIDMNGKIKEQKSFNIVNSY NYQEKLRGREKDRLNARKNWKQIANIKEMKEGYLSLVIHEITEMMMKYNAIIAMEDLN YGFKRGRFKVERQVYQKFETMLISKLNYLVDKHKKVDEPGGLLRGYQFAYVPASLDRL GRQCGFIFYVPAAYTSKIDPTTGFVDLFNHSELIKAGKRRDNLSKFDGIYYDEQKDMFCY AFDYKNFVTHNTDIYQNSWEIYTNKERLRKIFGNGRPTGKTEKIELTQMMKEVLTGAGV EYKDGHNLIDDILNSNDNCIKQVLDIFLYSIQLRNSKGENEDSKESDYDRIISPVLNQENE FFDSVVYADKYKKDEKLADKPIDADANGAYCIALKGLYEILQIKNNWKEGEVFSRDTL KITNADWLRFMQSRGFE (SEQ ID NO: 68).

[0328] Cpfl [Lachnospira eligen] :BIOTN-41612.601

[0329] MNGNRSIVYREFVGVIPVAKTLRNELRPVGHTQEHIIQNGLIQEDELRQEKSTE LKNIMDDYYREYIDKSLSGVTDLDFTLLFELMNLVQSSPSKDNKKALEKEQSKMREQIC THLQSDSNYKNIFNAKLLKEILPDFIKNYNQYDVKDKAGKLETLALFNGFSTYFTDFFEK RKNVFTKEAVSTSIAYRFVHENSLIFLANMTSYKKISEKALDEIEVIEKNNQDKMGDWEL NQIFNPDFYNMVLIQSGIDFYNEICGVVNAHMNLYCQQTKNNYNLFKMRKLHKQILAY TSTSFEVPKMFEDDMSVYNAVNAFIDETEKGNIIGKLKDIVNKYDELDEKRIYISKDFYE TLSCFMSGNWNLITGCVENFYDENIHAKGKSKEEKVKKAVKEDKYKSINDVNDLVEKY IDEKERNEFKNSNAKQYIREISNIITDTETAHLEYDDHISLIESEEKADEMKKRLDMYMN MYHWAKAFIVDEVLDRDEMFYSDIDDIYNILENIVPLYNRVRNYVTQKPYNSKKIKLNF QSPTLANGWSQSKEFDNNAIILIRDNKYYLAIFNAKNKPDKKIIQGNSDKKNDNDYKKM VYNLLPGANKMLPKVFLSKKGIETFKPSDYIISGYNAHKHIKTSENFDISFCRDLIDYFKNSIEKHAEWRKYEFKFSATDSYSDISEFYREVEMQGYRIDWTYISEADINKLDEEGKIYLF QIYNKDFAENSTGKENLHTMYFKNIFSEENLKDIIIKLNGQAELFYRRASVKNPVKHKKD SVLVNKTYKNQLDNGDVVRIPIPDDIYNEIYKMYNGYIKESDLSEAAKEYLDKVEVRTA QKDIVKDYRYTVDKYFIHTPITINYKVTARNNVNDMVVKYIAQNDDIHVIGIDRGERNLI YISVIDSHGNIVKQKSYNILNNYDYKKKLVEKEKTREYARKNWKSIGNIKELKEGYISGV VHEIAMLIVEYNAIIAMEDLNYGFKRGRFKVERQVYQKFESMLINKLNYFASKEKSVDE PGGLLKGYQLTYVPDNIKNLGKQCGVIFYVPAAFTSKIDPSTGFISAFNFKSISTNASRKQ FFMQFDEIRYCAEKDMFSFGFDYNNFDTYNITMGKTQWTVYTNGERLQSEFNNARRTG KTKSINLTETIKLLLEDNEINYADGHDIRIDMEKMDEDKKSEFFAQLLSLYKLTVQMRNS YTEAEEQENGISYDKIISPVINDEGEFFDSDNYKESDDKECKMPKDADANGAYCIALKGL YEVLKIKSEWTEDGFDRNCLKLPHAEWLDFIQNKRYE (SEQ ID NO: 69).

[0330] Cpfl [Lachnospira_pectinoschiz]:

[0331] MIMNNVTGDFSEFVAISKVQKTLRNELRPTPLTMKHIKQKGIITEDEYKTQQSL ELKRIADGYYRDYITHKLNDTNNLDFRNLFEAIEEKYKKNDKDNRDKLDLVEKSKRGEI AKLLSADDNFKSMFEAKLITQLLPVYVEQNYIGEDKEKALETIALFKGFTTYFTDYFNIRKNMFKENGGASSICYRIVNVNASIFYDNLKTFMCIKEKAETEIALIEEELTELLDSWRLEH IFSEDYYNELLAQKGIDYYNQICGDVNKHMNLYCQQNKLKANVFKMTKLQKQIMGISE KAFEIPPMYQNDEEVYAAFNGFISRLEEVKLIDRLGNVLQNSNIYDTAKIYINARCYTNV SSYVYGGWGVIESAIERYWYNTIAGKGQSKAKKIEKAKKDNKFMSVKELDSIVSDYEPD YFNASNMDDDNSGRAFSGHGVLGYFNKMSKLLANMSLHTITYDSGDSLIENKETALNIBIOTN-41612.601KKDLDDIMSIYHWLQTFIIDEVVEKDNAFYAELEDIYYELENVVTLYDRIRNYVTRKPYS TQKFKLNFASPTLASGWSRSKEFDNNAIILLRNNKYYIAIFNVNNKPDKQIIKGSEEQRLS TDYKKMVYNLLPGPNKMLPWVFIKSNTGKRDYNPSSYILEGYEKNRHIKSSGNFDINYC HDLIDYYKACINKHPEWKNYGFKFKETTQYNDIGQFYKDVEKQGYSISWAYISEADINR LDEEGKIYLFEIYNKDLSSHSTGKDNLHTMYLKNIFSEDNLKNICIELNGNAELFYRKSS MKRNITHKKDTVLVNKTYINEAGVRVSLTDEDYIKVYNYYNNDYVIDVEKDKKLVEIL ERIGHRKNPIDIIKDKRYTEDKYFLHFPITINYGVDDENINAKMIEYIAKHNNMNVIGIDR GERNLIYISVINNKGNIIEQKSFNLVNNYDYKNKLKNMEKTRDNARKNWQEIGKIKDVK NGYLSGVISKIARMVVDYNAIIVMEDLNRGFKRGRFKVERQVYQKFENMLISKLNYLVF KEKKADENGGILKGYQLTYLPKSALQIGKQCGCIFYVPAAYTSKIDPATGFINIFDFKKYS GSAINAKVKDKKEFLMSMNSIRYVNEGSAEYEKIGHRQLFAFSFDYNNFKTYNVSIPVN EWTTYTYGERIKKLYKDGRWSGSEVLNLTEDLIELMEQYGIEYKDGHDIREDISHMDEM RNADFICNLFEKFKYTVQLRNSKSEAEGDDYDRLVSPVLNSHNGFFDSSDYKENEKSDD IIDDKQIMPKDADANGAYCIALKGLYEINKIKENWSDDKKLKESELYIGVTEWLDYIQNR RFE (SEQ ID NO: 70).

[0332] Hypothetical [Eubacterium sp] :

[0333] MNKAADNYTGGNYDEFIALSKVQKTLRNELKPTPFTAEHIKQRGIISEDEYRAQQSLELKKIADEYYRNYITHKLNDINNLDFYNLFDAIEEKYKKNDKDNRDKLDLVEKSK RGEIAKMLSADDNFKSMFEAKLITKLLPDYVERNYTGEDKEKALETLALFKGFTTYFKG YFKTRKNMFSGEGGASSICHRIVNVNASIFYDNLKTFMRIQEKAGDEIALIEEELTEKLDG WRLEHIFSRDYYNEVLAQKGIDYYNQICGDINKHMNLYCQQNKFKANIFKMMKIQKQI MGISEKAFEIPPMYQNDEEVYASFNEFISRLEEVKLTDRLINILQNINIYNTAKIYINARYY TNVSSYVYGGWGVIDSAIERYLYNTIAGKGQSKVKKIENAKKDNKFMSVKELDSIVAEY EPDYFNAPYIDDDDNAVKAFGGQGVLGYFNKMSELLADVSLYTIDYNSDDSLIENKESA LRIKKQLDDIMSLYHWLQTFIIDEVVEKDNAFYAELEDICCELENVVTLYDRIRNYVTKK PYSTQKFKLNFASPTLAAGWSRSKEFDNNAIILLRNNKYYIAIFNVNNKPDKQIIKGSEEQ RLSTDYKKMVYNLLPGPNKMLPKVFIKSDTGKRDYNPSSYILEGYEKNRHIKSSGNFDIN YCHDLIDYYKACINKHPEWKNYGFKFKETNQYNDIGQFYKDVEKQGYSISWAYISEEDI NKLDEEGKIYLFEIYNKDLSAHSTGRDNLHTMYLKNIFSEDNLKNICIELNGEAELFYRK SSMKSNITHKKDTILVNKTYINETGVRVSLSDEDYMKVYNYYNNNYVIDTENDKNLIDII EKIGHRKSKIDIVKDKRYTEDKYFLYLPITINYGIEDENVNSKIIEYIAKQDNMNVIGIDRGBIOTN-41612.601ERNLIYISVIDNKGNIIEQKSFNLVNNYDYKNKLKNMEKTRDNARKNWQEIGKIKDVKS GYLSGVISKIARMVIDYNAIIVMEDLNKGFKRGRFKVERQVYQKFENMLISKLNYLVFK ERKADENGGILRGYQLTYIPKSIKNVGKQCGCIFYVPAAYTSKIDPATGFINIFDFKKYSG SGINAKVKDKKEFLMSMNSIRYINECSEEYEKIGHRELFAFSFDYNNFKTYNVSSPVNEW TAYTYGERIKKLYKDGRWLRSEVLNLTENLIKLMEQYNIEYKDGHDIREDISHMDETRN ADFICSLFEELKYTVQLRNSKSEAEDENYDRLVSPILNSSNGFYDSSDYMENENNTTHTM PKDADANGAYCIALKGLYEINKIKQNWSDDKKFKENELYINVTEWLDYIQNRRFE (SEQ ID NO: 71).

[0334] Cpfl [Clostridium sp]:

[0335] MSYNREKMEEKELGKNQNFQEFIGVSPLQKTLRNELIPTETTKKNIAQLDLLTE DEVRAQNREKLKEMMDDYYRDVIDSTLRGELLIDWSYLFSCMRNHLSENSKESKRELE RTQDSVRSQIHDKFAERADFKDMFGASIITKLLPTYIKQNSKYSERYDESVKIMKLYGKF TTSLTDYFETRKNIFSKEKISSAVGYRIVEENAEIFLQNQNAYDRICKIAGLDLHGLDNEIT AYVDGKTLKEVCSDEGFAKVITQGGIDRYNEAIGAVNQYMNLLCQKNKALKPGQFKM KRLHKQILCKGTTSFDIPKKFENDKQVYDAVNSFTEIVTKNNDLKRLLNITQNANDYDM NKIYVVADAYSMISQFISKKWNLIEECLLDYYSDNLPGKGNAKENKVKKAVKEETYRS VSQLNEVIEKYYVEKTGQSVWKVESYISSLAEMIKLELCHEIDNDEKHNLIEDDEKISEIK ELLDMYMDVFHIIKVFRVNEVLNFDETFYSEMDEIYQDMQEIVPLYNHVRNYVTQKPY KQEKYRLYFHTPTLANGWSKSKEYDNNAIILVREDKYYLGILNAKKKPSKEIMAGKEDC SEHAYAKMNYYLLPGANKMLPKVFLSKKGIQDYHPSSYIVEGYNEKKHIKGSKNFDIRF CRDLIDYFKECIKKHPDWNKFNFEFSATETYEDISVFYREVEKQGYRVEWTYINSEDIQK LEEDGQLFLFQIYNKDFAVGSTGKPNLHTLYLKNLFSEENLRDIVLKLNGEAEIFFRKSSV QKPVIHKCGSILVNRTYEITESGTTRVQSIPESEYMELYRYFNSEKQIELSDEAKKYLDKV QCNKAKTDIVKDYRYTMDKFFIHLPITINFKVDKGNNVNAIAQQYIAEQEDLHVIGIDRG ERNLIYVSVIDMYGRILEQKSFNLVEQVSSQGTKRYYDYKEKLQNREEERDKARKSWK TIGKIKELKEGYLSSVIHEIAQMVVKYNAIIAMEDLNYGFKRGRFKVERQVYQKFETMLI SKLNYLADKSQAVDEPGGILRGYQMTYVPDNIKNVGRQCGIIFYVPAAYTSKIDPTTGFI NAFKRDVVSTNDAKENFLMKFDSIQYDIEKGLFKFSFDYKNFATHKLTLAKTKWDVYT NGTRIQNMKVEGHWLSMEVELTTKMKELLDDSHIPYEEGQNILDDLREMKDITTIVNGI LEIFWLTVQLRNSRIDNPDYDRIISPVLNNDGEFFDSDEYNSYIDAQKAPLPIDADANGAF CIALKGMYTANQIKENWVEGEKLPADCLKIEHASWLAFMQGERG (SEQ ID NO: 72).BIOTN-41612.601

[0336] Cpfl [Coprococcus sp]:

[0337] MVGNKISNSFDSFTGINALSKTLRNELIPSDYTKRHIAESDFIAADTNKNEDQY VAKEMMDDYYRDFISKVLDNLHDIEWKNLFELMHKAKIDKSDATSKELIKIQDMLRKKI GKKFSQDPEYKVMLSAGMITKILPKYILEKYETDREDRLEAIKRFYGFTVYFKEFWASR QNVFSDKAIASSISYRIIHENAKIYMDNLDAYNRIKQIACEEIEKIEEEAYDFLQGDQLDV VYTEEAYGRFISQSGIDLYNNICGVINAHMNLYCQSKKCSRSKFKMQKLHKQILCKAET GFEIPLGFQDDAQVINAINSFNALIKEKNIISRLRTIGKSISLYDVNKIYISSKAFENVSVYID HKWDVIASSLYKYFSEIVKGNKDNREEKIQKEIKKVKSCSLGDLQRLVNSYYKIDSTCLE HEVTEFVTKIIDEIDNFQITDFKFNDKISLIQNEQIVMDIKTYLDKYMSIYHWMKSFVIDEL VDKDMEFYSELDELNEDMSEIVNLYNKVRNYVTQKPYSQEKIKLNFGSPTLADGWSKS KEFDNNAIILIRDEKIYLAIFNPRNKPAKTVISGHDVCNSETDYKKMNYYLLPGASKTLPHVFIKSRLWNESHGIPDEILRGYELGKHLKSSVNFDVEFCWKLIDYYKECISCYPNYKAYN FKFADTESYNDISEFYREVECQGYKIDWTYISSEDVEQLDRDGQIYLFQIYNKDFAPNSK GMDNLHTKYLKNIFS EDNLKNIVIKLNGEAELFYRKS S VKKKVEHKKGTILVNKTYKVE DNTENSKEKRVIIESVPDDCYMELVDYWRNGGIGILSDKAVQYKDKVSHYEATMDIVK DRRYTVDKFFIHLPITINFKADGRININEKVLKYIAENDELHVIGIDRGERNLLYVSVINKK GKIVEQKSFNMIESYETVTNFVRRYNYKDKLVNKESARTDARKNWKEIGKIKEIKEGYLS QVIHEISKMVLKYNAIIVMEDLNYGFKRGRFRVERQVYQKFENMLISKLAYLVDKSRKA DEPGGVLRGYQLTYIPDSLEKLGSQCGIIFYVPAAYTSKIDPLTGFVNVFNFREYSNFETK LDFVRSLDSIRYDTEKKLFSISFDYDNFKTHNTTLAKTKWVIYLRGERIKKEHTSYGWKD DVWNVESRIKDLFDSSHMKYDDGHNLIEDILELESSVQKKLINELIEIIRLTVQLRNSKSE RYDRTEAEYDRIVSPVMDENGRFYDSENYIFNEETELPKDADANGAYCIALKGLYNVIAIKNNWKEGEKFNRKLLSLNNYNWFDFIQNRRF (SEQ ID NO: 73).

[0338] Cpfl [Coprococcus sp]:

[0339] MCYDLNNIKTKLREREVETMGNNMDNSFEPFIGGNSVSKTLRNELRVGSEYT GKHIKECAIIAEDAVKAENQYIVKEMMDDFYRDFINRKLDALQGINWEQLFDIMKKAKL DKSNKVSKELDKIQESTRKEIGKIFSSDPIYKDMLKADMISKILPEYIVDKYGDAASRIEA VKVFYGFSGYFIDFWASRKNVFSDKNIASAIPHRIVNVNARIHLDNITAFNRIAEIAGDEV AGIAEDACAYLQNMSLEDVFTGACYGEFICQKDIDRYNNICGVINQHMNQYCQNKKISR SKFKMERLHKQILCRSESGFEIPIGFQTDGEVIDAINSFSTILEEKDILDRLRTLSQEVTGYD MERIYVSSKAFESVSKYIDHKWDVIASSMYNYFSGAVRGKDDKKDVKIQTEIKKIKSCSBIOTN-41612.601LLDLKKLVDMYYKMDGMCLEHEATEYVAGITEILVDFNYKTFDMDDSVKMIQNEHMI NEIKEYLDTYMSIYHWAKDFMIDELVDRDMEFYSELDEIYYDLSDIVPLYNKVRNYVTQ KPYSQDKIKLNFGSPTLANGWSKSKEFDNNVVVLLRDEKIYLAILNVGNKPSKDIMAGE DRRRSDTDYKKMNYYLLPGASKTLPHVFISSNAWKKSHGIPDEIMYGYNQNKHLKSSP NFDLEFCRKLIDYYKECIDSYPNYQIFNFKFAATETYNDISEFYKDVERQGYKIEWSYISE DDINQMDRDGQIYLFQIYNKDFAPNSKGMQNLHTLYLKNIFSEENLSDVVIKLNGEAELF FRKSSIQHKRGHKKGSVLVNKTYKTTEKTENGQGEIEVIESVPDQCYLELVKYWSEGGV GQLSEEASKYKDKVSHYAATMDIVKDRRYTEDKFFIHMPITINFKADNRNNVNEKVLKF IAENDDLHVIGIDRGERNLLYVSVIDSRGRIVEQKSFNIVENYESSKNVIRRHDYRGKLVN KEHYRNEARKSWKEIGKIKEIKEGYLSQVIHEISKLVLKYNAIIVMEDLNYGFKRGRFKV ERQVYQKFETMLINKLAYLVDKSRAVDEPGGLLKGYQLTYVPDNLGELGSQCGIIFYVP AAYTSKIDPVTGFVDVFDFKAYSNAEARLDFINKLDCIRYDAPRNKFEIAFDYGNFRTHH TTLAKTSWTIFIHGDRIKKERGSYGWKDEIIDIEARIRKLFEDTDIEYADGHNLIGDINELESPIQKKFVGELFDIIRFTVQLRNSKSEKYDGTEKEYDKIISPVMDEEGVFFTTDSYIRADG TELPKDADANGAYCIALKGLYDVLAVKKYWKEGEKFDRKLLAITNYNWFDFIQNRRF (SEQ ID NO: 74).

[0340] Cpfl [Eubacteriaceae bacteriu]:

[0341] MNKNKTIFSETIGQTYVSKTLKNTIIPIGATQLNIERNGLITEDKIRSEKRQELKK IMDDYYRYFINNTLSGNLAKTIEWGELFRAIDNNYINNSDKTKNELKNIQKKKRTELNKL FKSNDDYKKMFNAKLLSDILPEFINKQKISNTEKEERLNTVNLFNKFTSSFTDFFINRENIF ADDKSTSICYRIVNENAWIFYQNYLIYNKIKEDAEKEIITIENNNVDGLDGFTLNSIFNFDF YGMLMNQKGIRFYNDICGIVNAHMNLYCQKYKKKAAKYRLKRMHKQILSIDESTFEVP KMLESDKEVYDLVNGFLYKLNEKKVLDRIRKISEDVTSYDLNKIYVCEKNYENLSLFMC RNWSVLRDSIKAFYQENTTGSNAGKEKKIEKLMKEDKYRSLQNINELINKYNSNSIERK AEEYIVNINEKLENKELVQLTNKEDISLIEDEESGEELKRVLDLIMSINHWIKMFIIDEEVD KDMAFYTEIEDIYYELEPLKSIYDKVRNYITQKPYSKEKIKLNFGIPTLAKGWSKTKEYD NNAIILIRENKYYIGIFNAKNKPDKTVIEGHKKKNDSDYKKMVYMFLTKPRLMLPKVFL SKKGIATYKTSEYILDGYRKGKHNKSSKNFDVNYCRDLIDYFKDCINKRDEWKGYKFKFSETSTYEDINDFYKEIEKQAYTIQWTYIDEKDIDEMNEKGQLYLFQIYNKDFSEKSTGTK NLHTMYFENLFSEENLKNIVLKLDGNAEVFFRKSSIKQPIIHKKGSILVNKGYNEQKENE ERKTIPDEVYTELYKYFNKIGKVKLSNEAKKLIDEDKVEYFTSDYDIVKDYRYTVDKYFBIOTN-41612.601LHLPIKINCNAENYSPVNDIALKNIAENNDMHIIGIDRGERNLIYVSVIDLHGNIIEQKNFNI INGVNYKEKLKQRESERDSARKNWKQIGKIKDIKEGYLSLVIHEITSLVLKYNAIIAMED LNMGFKRGRFKVERQVYQKFENMLISKLNYLVDKNKAVNEEGGVLKGYQMTYIPKSL KELGRQCGYIFYVPAAYTSKIDPTTGFVSVFNYKEMDDEKFVTSFDSIKYDKNRQKFAL KFDYTNFITHNVKLAKNEWTIYSNGTRKIRKYRNDKWNSPDNVNLTNEIIKLMEQYNIQ YDNEQDILEQVETLETKNKNNFCKEMKKLVKYIVQMRNSLPDNDSIEYDEIISPVLNESG EFYNSEEKNKKLPLDADANGAYCIALKGLYEVKQIKNNWKKGEKFPRKTLKISHNDWF DFIQNKRYL (SEQ ID NO: 75).

[0342] Cpfl [Eubacterium ventriosu]:

[0343] MESNNKIFTETIGTSSIAKTMRNSLVPTESTKRNIEKNGIIIDDQLRAEKRQQLK EIMDEYYRTYIDNKLSNVALTRTIDWKELFQAIEDNYKQNTTKTKNELEKKQKEKRTEI YKILSDDEKFKQLFNAKLLTNVLPEFIKNQNIDNEEKQEKISTVELFQRFTSSFTDFFKNR KNVFSKDEISTSICYRVVQENAWIFYQNLLAFEEIKKTAEQEIEKIEAENRDSISDYSLKEI FDFDFYGLLLNQGGIRFYNDVCGKINYHMNLYGQKHNIKSNKFKMKRMHKQILSIDEST FEVPTMFENDKEVYQVLNEFLSDLASKKILERVEKIGENVSEYEINKIYIQSKNFEKFSSF MCGNWQIINDSLKTYYNEKIKSKGKAKEEKVKKAIKAIEYKSLADINQLVERYNNDELN RKAEEYISAINEKIKDLDVNEIEYDEKINLIENETKSEEIKSKLDSIMEIMHWTKMFIIEEEI EKDVNFYNEIEEIYDELQPLVTIYNRIRNYVTQKPYSEEKIKLNFGIPTLANGWSKTKEYD NNAIIMIRDGKYYLGIFNAKNKPDKKIMEGHQSEENGDYKKMIYRLLPGPNKMLPKVF MSKTGIAEYKPSQYILECYEQNKHIKSDKNFDIKFCRDLIDFFKTSINRHPEWSKFNFKFS ETSEYEDISTFYREVEKQGYKIEWTYISEKEIKELDENGQLYLFQIYNKDFSEKSKGKENL HTMYLKNLFSEENLKNIVLKLNGEAEVFFRKSSIKKPIIHKKGSVLVNKTYNENGERKSIP EEQYTEIYKYLNSIGTNELSEKSKKLMEEGKVEYYKANYDIVKDYRYSVDKFFIHLPMTI NFKAAGFSPINNIALKNIALKDDMHIIGIDRGERNLIYVSVIDTKGNIVEQRNFNIVNGIDY KEKLKQKELDRDNARKNWKEIGKIKDLKEGYLSLVVHEIAKLVVKYNAIITMEDLNQG FKRGRFKVERQVYQKFETMLINKLNYLVDKDLAVDQEGGLLRGYQLTYIPESLKVLGR QCGYIFYVPAAYTSKIDPTTGFVAIFNYKGMTDKDFVTSFDSIKYDDERGLFAFEFDYEN FVTHKVEMARNKWTVYTYGERIKRKFKNGSWDTAEKVDLTYQMRSILEKYEIEYNKG QDILEQIEELDEKAQNGICKEIKYLVKDIVQMRNSLPDNAAEDYDAIISPVINNNGEFFDS TRGDEDKPLDADANGAYCIALKGLYEVMQIKKNWNEETEFPRKELKIRHQDWFDFIQN KRYL (SEQ ID NO: 76).BIOTN-41612.601

[0344] Cpfl [Lacrimispora saccharo lytic] :

[0345] MNYKTGLEDFIGKESLSKTLRNALIPTESTKIHMEEMGVIRDDELRAEKQQEL KEIMDDYYRTFIEEKLGQIQGIQWNSLFQKMEETMEDISVRKDLDKIQNEKRKEICCYFT SDKRFKDLFNAKLITDILPNFIKDNKEYTEEEKAEKEQTRVLFQRFATAFTNYFNQRRNN FSEDNISTAISFRIVNENSEIHLQNMRAFQRIEQQYPEEVCGMEEEYKDMLQEWQMKHIY SVDFYDRELTQPGIEYYNGICGKINEHMNQFCQKNRINKNDFRMKKLHKQILCKKSSYY EIPFRFESDQEVYDALNEFIKTMKKKEIIRRCVHLGQECDDYDLGKIYISSNKYEQISNAL YGSWDTIRKCIKEEYMDALPGKGEKKEEKAEAAAKKEEYRSIADIDKIISLYGSEMDRTI SAKKCITEICDMAGQISIDPLVCNSDIKLLQNKEKTTEIKTILDSFLHVYQWGQTFIVSDIIE KDSYFYSELEDVLEDFEGITTLYNHVRSYVTQKPYSTVKFKLHFGSPTLANGWSQSKEY DNNAILLMRDQKFYLGIFNVRNKPDKQIIKGHEKEEKGDYKKMIYNLLPGPSKMLPKVF ITSRSGQETYKPSKHILDGYNEKRHIKSSPKFDLGYCWDLIDYYKECIHKHPDWKNYDF HFSDTKDYEDISGFYREVEMQGYQIKWTYISADEIQKLDEKGQIFLFQIYNKDFSVHSTG KDNLHTMYLKNLFSEENLKDIVLKLNGEAELFFRKASIKTPIVHKKGSVLVNRSYTQTV GNKEIRVSIPEEYYTEIYNYLNHIGKGKLSSEAQRYLDEGKIKSFTATKDIVKNYRYCCD HYFLHLPITINFKAKSDVAVNERTLAYIAKKEDIHIIGIDRGERNLLYISVVDVHGNIREQR SFNIVNGYDYQQKLKDREKSRDAARKNWEEIEKIKELKEGYLSMVIHYIAQLWKYNA VVAMEDLNYGFKTGRFKVERQVYQKFETMLIEKLHYLVFKDREVCEEGGVLRGYQLT YIPESLKKVGKQCGFIFYVPAGYTSKIDPTTGFVNLFSFKNLTNRESRQDFVGKFDEIRYD RDKKMFEFSFDYNNYIKKGTILASTKWKVYTNGTRLKRIVVNGKYTSQSMEVELTDAM EKMLQRAGIEYHDGKDLKGQIVEKGIEAEIIDIFRLTVQMRNSRSESEDREYDRLISPVLN DKGEFFDTATADKTLPQDADANGAYCIALKGLYEVKQIKENWKENEQFPRNKLVQDN KTWFDFMQKKRYL (SEQ ID NO: 77).

[0346] Cpfl [Collinsella tanakae] :

[0347] MIYRENFKRKKEKIEMNTGFNDFTNLSSVTKTLCNRLIPTEITAKYIKEHGVIEA DQERNMMSQELKNILNDFYRSFLNENLVKVHELDFKPLFTEMKKYLETKDNKEALEKA QDDMRKAIHDIFESDDRYKKMFKAEITASILPEFILHNGAYSAEEKEEKMQWKMFNGF MTSFSAFFTNRENCFSKEKISSSACYRIVDDNAKIHFDNIRIYKNIANKFDYEIEMIEKIEE AAGGADIRNIFSYNFDHFAFNHFVSQDDISFYNYVVGGINKFMNLYCQATKEKLSPYKL RHLHKQILCIEESLYDVPAKFNCDEDVYAAVNDFLNNVRTKSVIERLQMLGKNADSYDL DKIYISKKHFTNISQTLYRDFSVINTALTMSYIDTLPGKGKTKEKKAASMAKNTELISLGEBIOTN-41612.601IDKLVDKYNLCPDKAASTRSLIRSISDIVADYKANPLTMNSGIPLAENETEIAVLKEAIEPF MDIFRWCAKFKTDEPVDKDTDFYTELEDINDEIHSIVSLYNRTRNYVTKKPYNTDKFGL YFGTSSFASGWSESKEFTNNAILLAKDDKFYLGVFNAKNKPAKSIIKGHDTIQDGDYKK MVYSLLTGPNKMLPHMFISSSKAVPVYGLTDELLSDYKKGRHLKTSKNFDIDYCHKLID YFKHCLALYTDWDCFNFKFSDTESYNDIGEFYKEVAEQGYYMNWTYIGSDDIDSLQEN GQLYLFQIYNKDFSEKSFGKPSKHTAILRSLFSDENVADPVIKLCGGTEVFFRPKSIKTPV VHKKGSILVSKTYNAQEMDENGNIITVRKCVPDDVYMELYGYYNNSGTPLSAEALKYK DIVDHRTAPYDIIKDRRYTEDEFFINMPVSLNYKAENRRVNVNEMALKYIAQTKDTYIIG IDRGERNLLYVSVIDTDGNIVEQKSLNIINNVDYQAKLKQVEIMRKLARQNWKQGVKIA DLKKGYLSQAVHEVAELVIKYNGIVVMEDLNSRFKEKRSKIERGVYQQFETSLIKTLNY LTFKDRKPLEAGGIANGYQLTYIPESLKNVGSQCGCILYVPAAYTSKIDPTTGFVTLFKFK DISSEKAKTDFIGRFDCIRYDAEKDLFAFEFDYDNFETYETCARTKWCAYTYGTRVKKT FRNRKFVSEVIIDITEEIKKTLAATDINWIDSHDIKQEIIDYALSSHIFEMFKLTVQMRNSLC ESKDREYDKFVSPILNASGKFFDTDAADKSLPIEADANDAYGIAMKGLYNVLQVKNNWAEGEKFKFSRLSNEDWFNFMQKR (SEQ ID NO: 78).

[0348] Cpfl [Succinivibrio dextrinosolven]:

[0349] MSSLTKFTNKYSKQLTIKNELIPVGKTLENIKENGLIDGDEQLNENYQKAKIIV DDFLRDFINKALNNTQIGNWRELADALNKEDEDNIEKLQDKIRGIIVSKFETFDLFSSYSIKKDEKIIDDDNDVEEEELDLGKKTSSFKYIFKKNLFKLVLPSYLKTTNQDKLKIISSFDNF STYFRGFFENRKNIFTKKPISTSIAYRIVHDNFPKFLDNIRCFNVWQTECPQLIVKADNYL KSKNVIAKDKSLANYFTVGAYDYFLSQNGIDFYNNIIGGLPAFAGHEKIQGLNEFINQEC QKDSELKSKLKNRHAFKMAVLFKQILSDREKSFVIDEFESDAQVIDAVKNFYAEQCKDN NVIFNLLNLIKNIAFLSDDELDGIFIEGKYLSSVSQKLYSDWSKLRNDIEDSANSKQGNKE LAKKIKTNKGDVEKAISKYEFSLSELNSIVHDNTKFSDLLSCTLHKVASEKLVKVNEGD WPKHLKNNEEKQKIKEPLDALLEIYNTLLIFNCKSFNKNGNFYVDYDRCINELSSVVYLY NKTRNYCTKKPYNTDKFKLNFNSPQLGEGFSKSKENDCLTLLFKKDDNYYVGIIRKGAK INFDDTQAIADNTDNCIFKMNYFLLKDAKKFIPKCSIQLKEVKAHFKKSEDDYILSDKEK FASPLVIKKSTFLLATAHVKGKKGNIKKFQKEYSKENPTEYRNSLNEWIAFCKEFLKTYK AATIFDITTLKKAEEYADIVEFYKDVDNLCYKLEFCPIKTSFIENLIDNGDLYLFRINNKDF SSKSTGTKNLHTLYLQAIFDERNLNNPTIMLNGGAELFYRKESIEQKNRITHKAGSILVNK VCKDGTSLDDKIRNEIYQYENKFIDTLSDEAKKVLPNVIKKEATHDITKDKRFTSDKFFFBIOTN-41612.601HCPLTINYKEGDTKQFNNEVLSFLRGNPDINIIGIDRGERNLIYVTVINQKGEILDSVSFNT VTNKSSKIEQTVDYEEKLAVREKERIEAKRSWDSISKIATLKEGYLSAIVHEICLLMIKHN AIVVLENLNAGFKRIRGGLSEKSVYQKFEKMLINKLNYFVSKKESDWNKPSGLLNGLQL SDQFESFEKLGIQSGFIFYVPAAYTSKIDPTTGFANVLNLSKVRNVDAIKSFFSNFNEISYS KKEALFKFSFDLDSLSKKGFSSFVKFSKSKWNVYTFGERIIKPKNKQGYREDKRINLTFE MKKLLNEYKVSFDLENNLIPNLTSANLKDTFWKELFFIFKTTLQLRNSVTNGKEDVLISP VKNAKGEFFVSGTHNKTLPQDCDANGAYHIALKGLMILERNNLVREEKDTKKIMAISN VDWFEYVQKRRGVL (SEQ ID NO: 79).

[0350] Cpfl [Acidaminococcus massiliensi]:

[0351] MAAFDKFIHQYQVSKTLRFALIPQGKTLENTKNNVLQEDDERQKNYEKVKPIL DRIYKVFAEESLKDCSVDWNDLNACLDAYQKNPSADKRQKVKAAQDALRDEIAGYFT GKQYANGKNKNAVKEKEQAELYKDIFSKKIFDGTVTNNKLPQVNLSAEETELLGCFDKFTTYFVGFYQNRENVFSGEDIATAIPHRIVQDNFPKFRENCRIYQDLIKNEPALKPLLQQA AAAVMAQNPKGIYQPRKSLDDIFVIPFYNHLLLQDDIDYFNQILGGISGAAGQKKIQGLN ETINLFMQQHPQEADKLKKKKIRHRFIPLYKQILSDRTSFSFIPEAFSNSQEALDGIETFKK SLKKNDTFGALERLIQNLASLDLKYVYLSNKKVNEISQALYGEWHCIQDVLKQDFSLES LIQINPQNSSNGFLATLTDEGKKRISQCRNVLGNPLPVKLADDQDKAQVKNQLDTLLAA VHYLEWFKADPDLETDPNFTVPFEKIWEELVPLLSLYSKVRNFVTKKPYSTAKFKLNFA NPTLADGWDIHKESDNGALLFEKGGLYYLGIMNPKDKPNFKSYQGAEPYYQKMVYRFF PDCSKTIPKCSTQRKDVKKYFEDHPQATSYQIHDSKKEKFRQDFFEIPREIYELNNTTYGT GKSKYKKFQTQYYQKTQDKSGYQKALRKWIDFSKKFLQTYVSTSIFDFKGLRPSKDYQ DLGEFYKDVNSRCYRVTFEKIRVQDIHEAVKNGQLYLFQLYNKDFSPKSHGLPNLHTLY WKAVFDPENLKDPIVKLNGQAELFYRPKSNMQIIQHKTGEEIVNKKLKDGTPVPDDIYR EISAYVQGKCQGNLSPEAEKWLPSVTIKKAAHDITKDRRFTEDKFFFHVPITLNYQSSGK PTAFNSQVNDFLTEHPETNIIGIDRGERNLIYAVVITPDGKILEQKSFNVIHDFDYHESLSQ REKQRVAARQAWTAIGRIKDLKEGYLSLVVHEIAQMMIKYQAVVVLENLNTGFKRVRG GISEKAVYQQFEKMLIEKLNFLVFKDRAINQEGGVLKAYQLTDSFTSFAKLGNQSGFLF YIPSAYTSKIDPGTGFVDPFIWSHVTASEENRNEFLKGFDSLKYDAQSSAFVLHFKMKSN KQFQKNNVEGFMPEWDICFEKNEEKISLQGSKYTAGKRIIFDSKKKQYMECFPQNELMK ALQDVGITWNTGNDIWQDVLKQASTDTGFRHRMINLIRSVLQMRSSNGATGEDYINSPVBIOTN-41612.601MDLDGRFFDTRAGIRDLPLDADANGAYHIALKGRMVLERIRSQKNTAIKNTDWLYAIQE ERN (SEQ ID NO: 80).

[0352] Cpfl [Acidaminococcus sp]:

[0353] MTQFEGFTNLYQVSKTLRFELIPQGKTLKHIQEQGFIEEDKARNDHYKELKPII DRIYKTYADQCLQLVQLDWENLSAAIDSYRKEKTEETRNALIEEQATYRNAIHDYFIGRT DNLTDAINKRHAEIYKGLFKAELFNGKVLKQLGTVTTTEHENALLRSFDKFTTYFSGFYE NRKNVFSAEDISTAIPHRIVQDNFPKFKENCHIFTRLITAVPSLREHFENVKKAIGIFVSTSI EEVFSFPFYNQLLTQTQIDLYNQLLGGISREAGTEKIKGLNEVLNLAIQKNDETAHIIASLP HRFIPLFKQILSDRNTLSFILEEFKSDEEVIQSFCKYKTLLRNENVLETAEALFNELNSIDLT HIFISHKKLETISSALCDHWDTLRNALYERRISELTGKITKSAKEKVQRSLKHEDINLQEII SAAGKELSEAFKQKTSEILSHAHAALDQPLPTTLKKQEEKEILKSQLDSLLGLYHLLDWF AVDESNEVDPEFSARLTGIKLEMEPSLSFYNKARNYATKKPYSVEKFKLNFQMPTLASG WDVNKEKNNGAILFVKNGLYYLGIMPKQKGRYKALSFEPTEKTSEGFDKMYYDYFPD AAKMIPKCSTQLKAVTAHFQTHTTPILLSNNFIEPLEITKEIYDLNNPEKEPKKFQTAYAK KTGDQKGYREALCKWIDFTRDFLSKYTKTTSIDLSSLRPSSQYKDLGEYYAELNPLLYHI SFQRIAEKEIMDAVETGKLYLFQIYNKDFAKGHHGKPNLHTLYWTGLFSPENLAKTSIKL NGQAELFYRPKSRMKRMAHRLGEKMLNKKLKDQKTPIPDTLYQELYDYVNHRLSHDL SDEARALLPNVITKEVSHEIIKDRRFTSDKFFFHVPITLNYQAANSPSKFNQRVNAYLKEH PETPIIGIDRGERNLIYITVIDSTGKILEQRSLNTIQQFDYQKKLDNREKERVAARQAWSV VGTIKDLKQGYLSQVIHEIVDLMIHYQAVVVLENLNFGFKSKRTGIAEKAVYQQFEKML IDKLNCLVLKDYPAEKVGGVLNPYQLTDQFTSFAKMGTQSGFLFYVPAPYTSKIDPLTG FVDPFVWKTIKNHESRKHFLEGFDFLHYDVKTGDFILHFKMNRNLSFQRGLPGFMPAW DIVFEKNETQFDAKGTPFIAGKRIVPVIENHRFTGRYRDLYPANELIALLEEKGIVFRDGS NILPKLLENDDSHAIDTMVALIRSVLQMRNSNAATGEDYINSPVRDLNGVCFDSRFQNPE WPMDADANGAYHIALKGQLLLNHLKESKDLKLQNGISNQDWLAYIQELRN (SEQ ID NO: 81).

[0354] Cpfl [Treponema jjorcinu]:

[0355] MKISEEFCGQGNGYSISKTLRFELKPKGKTLENIEKEKLLESDFKKSQDYKDVK IILDNYHKYFIDDVLQKVNLDWTKLAASITDYNKNKEDDSSVIKEQDFLRKEIVKIISKDK RFACLTASTPKDLFNSILLEWFEKSTEFSLNKKAVETFKRFSSYFKGFQENRKNMYKDEP IPTAVPYRIVNENFPKFLQNAESFKEIQKKCPEVIELVEKELSAYLGNDKLSDIFCVKNFNBIOTN-41612.601RYLCQTGAENQRGIDYYNQIIGGIVQKENDVKLRGINEFLNLFWQQHVDFAKDNRRIKF VPLYKQILSDRSSLSFKIQTLESDEELKEAVLSFAKKLDSKNKDGKNIFDLVMELTENINQ YDLSQIYINQKDMNAVSKTLTGDWAYLQKRMNIFAEETLTKSEQKRWKKELDDDTSKS KGIFSFEELNKVLEYSSENCSAVSIKIQEYFETTKRWYFEKQTGIFTKGEEIIEPSISGLCGQ IKSNFDEVNKVFGNVSSENTLRENPEEVEKIKNYLDSVQNLLHRIKPLKVNGIGDTSFYS EYDEIYSVLYEVISLYNKTRNYISKKSGIPEKFKLNFDNPTLADGWDQNKEQANTSVILIK DDEYFLGIMNANNKPKFLENYEGNTEKCYQKMIYKLLPGPNKMLPKVFFSTKGIETFNP PKEILNGYNAGKHKKGDSFDLDFCHSLIDWFKDAINRHEDWKKFDFKFSETSSYKDISEF YREISEQGYKLTFTAIPES...

Claims

BIOTN-41612.601CLAIMSWhat is claimed is:

1. An engineered nuclease comprising an amino acid sequence having at least 50% identity but less than 100% identity to any of SEQ ID NOs: 10-82.

2. The engineered nuclease of claim 1, wherein the amino acid sequence has at least 60% identity to any of SEQ ID NOs: 10-82.

3. The engineered nuclease of claim 1, wherein the amino acid sequence has at least 70% identity to any of SEQ ID NOs: 10-82.

4. The engineered nuclease of claim 1, wherein the amino acid sequence has at least 80% identity to any of SEQ ID NOs: 10-82.

5. The engineered nuclease of claim 1, wherein the amino acid sequence has at least 90% identity to any of SEQ ID NOs: 10-82.

6. The engineered nuclease of any one of claims 1 to 5, wherein the amino acid sequence has at least 70% identity to any of SEQ ID NOs: 1-9.

7. The engineered nuclease of any one of claims 1 to 5, wherein the amino acid sequence has at least 80% identity to any of SEQ ID NOs: 1-9.

8. The engineered nuclease of any one of claims 1 to 5, wherein the amino acid sequence has at least 90% identity to any of SEQ ID NOs: 1-9.

9. The engineered nuclease of any one of claims 1 to 5, wherein the amino acid sequence has at least 95% identity to any of SEQ ID NOs: 1-9.BIOTN-41612.60110. The engineered nuclease of any one of claims 1 to 9, wherein the amino acid sequence is selected from the group consisting of SEQ ID NOs: 1-9.

11. The engineered nuclease of any one of claims 1 to 10, wherein the amino acid sequence comprises any one of SEQ ID NOs: 83-119.

12. The engineered nuclease of any one of claims 1 to 10, wherein the amino acid sequence comprises any one of SEQ ID NOs: 83-119 in place of at least one amino acid in SEQ ID NO: 1 or SEQ ID NO: 2.

13. The engineered nuclease of any one of claims 1 to 12, wherein the amino acid sequence comprises any of the mutant amino acid substitutions in Table 3.

14. The engineered nuclease of any one of claims 1 to 12, wherein the amino acid sequence comprises any of the mutant amino acid substitutions in Table 4.

15. The engineered nuclease of any one of claims 1 to 12, wherein the amino acid sequence has at least 70% identity to SEQ ID NO: 1, and comprises at least one of the mutant amino acid substitutions in Table 3.

16. The engineered nuclease of any one of claims 1 to 12, wherein the amino acid sequence has at least 80% identity to SEQ ID NO: 1, and comprises at least one of the mutant amino acid substitutions in Table 3.

17. The engineered nuclease of any one of claims 1 to 12, wherein the amino acid sequence has at least 90% identity to SEQ ID NO: 1, and comprises at least one of the mutant amino acid substitutions in Table 3.

18. The engineered nuclease of any one of claims 1 to 12, wherein the amino acid sequence has at least 70% identity to SEQ ID NO: 2, and comprises at least one of the mutant amino acid substitutions in Table 4.BIOTN-41612.60119. The engineered nuclease of any one of claims 1 to 12, wherein the amino acid sequence has at least 80% identity to SEQ ID NO: 2, and comprises at least one of the mutant amino acid substitutions in Table 4.

20. The engineered nuclease of any one of claims 1 to 12, wherein the amino acid sequence has at least 90% identity to SEQ ID NO: 2, and comprises at least one of the mutant amino acid substitutions in Table 4.

21. A nucleic acid molecule comprising a sequence encoding an engineered nuclease of any one of claims 1 to 20.

22. A vector comprising the nucleic acid of claim 21.

23. The vector of claim 22, further comprising a promoter operatively linked to the nucleic acid.

24. The vector of claim 22 or 23 , further comprising a nucleic acid encoding a guide RNA (gRNA).

25. The vector of claim 24, further comprising a promoter operatively linked to the nucleic acid encoding a guide RNA (gRNA).

26. A composition comprising: an engineered nuclease, wherein the engineered nuclease comprises a sequence with at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to any one of SEQ ID NOs: 1-9, or a nucleic acid comprising a sequence encoding the engineered nuclease.

27. The composition of claim 26, wherein the amino acid sequence of the engineered nuclease comprises any one of SEQ ID NOs: 1-9.BIOTN-41612.60128. The composition of claim 26 or 27, wherein the engineered nuclease is comprised in a fusion protein of a peptide or protein that is fused to the nuclease.

29. The composition of any of claims 26 to 28, wherein the composition further comprises at least one guide RNA (gRNA) or a nucleic acid comprising a sequence encoding the at least one gRNA.

30. A system for modifying a target nucleic acid comprising: a) an engineered nuclease comprising an amino acid sequence having at least 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to any of SEQ ID NOs: 1-9, or a nucleic acid sequence encoding the engineered nuclease; and b) an engineered guide RNA (gRNA) comprising a first region configured to interact with the engineered nuclease and a second region configured to hybridize with a portion of the target nucleic acid.

31. The system of claim 30, wherein the engineered nuclease in the presence of the gRNA is capable of modifying the target nucleic acid.

32. The system of claim 30 or 31, wherein modifying comprises nucleic acid cleavage.

33. The system of any one of claims 30 to 32, wherein modifying comprises one or more of nucleic acid binding, base editing, transcription modulation, nucleic acid modification, protein modification, and histone modification.

34. The system of any one of claims 30 to 33, wherein the system further comprises at least one target nucleic acid.

35. The system of any one of claims 30 to 34, wherein the system is a cell-free system.

36. A cell comprising the system of any one of claims 30 to 35.BIOTN-41612.60137. The cell of claim 36, wherein the cell is a mammalian cell.

38. The cell of claim 37, wherein the cell is a human cell.

39. The cell of claim 37, wherein the cell is a prokaryotic or eukaryotic cell.

40. A method of modifying a target nucleic acid sequence comprising contacting the target nucleic acid with a targeting composition comprising an engineered nuclease of any one of claims 1 to 20, a composition of any one of claims 26 to 29, a nucleic acid of claim 21, a vector of any one of claims 22 to 25, or a system of any one of claims 30 to 35.

41. The method of claim 40, wherein the target nucleic acid sequence is in a cell.

42. The method of claim 41, wherein the cell is a prokaryotic or eukaryotic cell.

43. The method of claim 41, wherein the cell is a human cell.

44. The method of any of claims 40 to 43, wherein contacting a target nucleic acid sequence comprises introducing the targeting composition into the cell.

45. The method of any of claims 40 to 44, wherein contacting a target nucleic acid sequence comprises administering the targeting composition to a subject.

46. The method of any of claims 40 to 45, wherein the target nucleic acid sequence encodes a gene product.

47. A method modifying a target nucleic acid sequence in a plant, the method comprising providing to the plant, or a plant cell, seed, fruit, plant part, or propagation material of the plant: a) an engineered nuclease comprising an amino acid sequence having at least 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to any of SEQ ID NOs: 1-9, or a nucleic acid sequence encoding the engineered nuclease; andBIOTN-41612.601 b) an engineered guide RNA (gRNA) comprising a first region configured to interact with the engineered nuclease and a second region configured to hybridize with a portion of the target nucleic acid.

48. The method of claim 47, further comprising providing to the plant a donor polynucleotide.

49. The method of claim 47 or 48, wherein the target nucleic acid sequence encodes a gene product.

50. The method of any one of claims 47 to 49, wherein the plant is a monocot or a dicot.

51. The method of any one of claims 47 to 50, wherein the plant is a grain crop, a fruit crop, a forage crop, a root vegetable crop, a leafy vegetable crop, a flowering plant, a conifer, an oil crop, a plant used in phytoremediation, an industrial crop, a medicinal crop, or a laboratory model plant.

52. A method for treating a disease or disorder in a subject, the method comprising administering to the subject in need thereof: a) an engineered nuclease comprising an amino acid sequence having at least 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to any of SEQ ID NOs: 1-9, or a nucleic acid sequence encoding the engineered nuclease; and b) an engineered guide RNA (gRNA) comprising a first region configured to interact with the engineered nuclease and a second region configured to hybridize with a portion of a target nucleic acid.

53. The method of claim 52, wherein the engineered nuclease and the gRNA are encoded on a single nucleic acid.

54. The method of claim 52 or 53, wherein the target nucleic acid is a disease-associated gene.BIOTN-41612.60155. The method of any one of claims 52 to 54, further comprising administering a donor polynucleotide.

56. The method of claim 55, wherein the donor polynucleotide comprises a therapeutic protein, functional gene product, or a combination thereof.

57. The method of any one of claims 52 to 56, wherein the subject is a human.

58. The method of any one of claims 52 to 57, further comprising administering a therapeutic agent.

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