RELATED CASES This application is a continuation of U.S. Ser. No. 17/463,498, filed 31 Aug. 2021, now allowed; which claims priority to U.S. Ser. No. 63/133,502, filed 4 Jan. 2021, entitled “MAD NUCLEASES”, which is incorporated herein in its entirety.
INCORPORATION BY REFERENCE Submitted with the present application is an electronically filed sequence listing via EFS-Web as an ASCII formatted sequence listing, entitled “INSC083US2_SEQLIST_20220309”, created Mar. 9, 2022, and 359,000 bytes in size. The sequence listing is part of the specification filed Mar. 9, 2022 and is incorporated by reference in its entirety.
FIELD OF THE INVENTION The present disclosure provides new RNA-guided nuclease systems and engineered nickases for making rational, direct edits to nucleic acids in live cells.
BACKGROUND OF THE INVENTION In the following discussion certain articles and methods will be described for background and introductory purposes. Nothing contained herein is to be construed as an “admission” of prior art. Applicant expressly reserves the right to demonstrate, where appropriate, that the methods referenced herein do not constitute prior art under the applicable statutory provisions.
The ability to make precise, targeted changes to the genome of living cells has been a long-standing goal in biomedical research and development. Recently, various nucleases have been identified that allow manipulation of gene sequence: hence, gene function. These nucleases include nucleic acid-guided nucleases. The range of target sequences that nucleic acid-guided nucleases can recognize, however, is constrained by the need for a specific PAM to be located near the desired target sequence. PAMs are short nucleotide sequences recognized by a gRNA/nuclease complex where this complex directs editing of the target sequence. The precise PAM sequence and PAM length requirements for different nucleic acid-guided nucleases vary; however, PAMs typically are 2-7 base-pair sequences adjacent or in proximity to the target sequence and, depending on the nuclease, can be 5′ or 3′ to the target sequence. Engineering nucleic acid-guided nucleases or mining for new nucleic acid-guided nucleases may provide nucleases with altered PAM preferences and/or altered activity or fidelity; all changes that may increase the versatility of a nucleic acid-guided nuclease for certain editing tasks.
There is thus a need in the art of nucleic acid-guided nuclease gene editing for novel nucleases with varied PAM preferences, varied activity in cells from different organisms such as mammals and/or altered enzyme fidelity. The novel MAD nucleases described herein satisfy this need.
SUMMARY OF THE INVENTION This Summary is provided to introduce a selection of concepts in a simplified form that are further described below in the Detailed Description. This Summary is not intended to identify key or essential features of the claimed subject matter, nor is it intended to be used to limit the scope of the claimed subject matter. Other features, details, utilities, and advantages of the claimed subject matter will be apparent from the following written Detailed Description including those aspects illustrated in the accompanying drawings and defined in the appended claims.
The present disclosure provides Type II MAD nucleases (e.g., RNA-guided nucleases or RGNs) with varied PAM preferences, and/or varied activity in mammalian cells.
Thus, in one embodiment there are provided MAD nuclease systems that perform nucleic acid-guided nuclease editing including a MAD2015 system comprising SEQ ID Nos. 1 (MAD2015 nuclease), 2 (CRISPR RNA) and 3 (trans-activating crispr RNA); a MAD2016 system comprising SEQ ID Nos. 4 (MAD2016 nuclease), 5 (CRISPR RNA) and 6 (trans-activating crispr RNA); a MAD2017 system comprising SEQ ID Nos. 7 (MAD2017 nuclease), 8 (CRISPR RNA) and 9 (trans-activating crispr RNA); a MAD2019 system comprising SEQ ID Nos. 10 (MAD2019 nuclease), 11 (CRISPR RNA) and 12 (trans-activating crispr RNA); a MAD2020 system comprising SEQ ID Nos. 13 (MAD2020 nuclease), 14 (CRISPR RNA) and 15 (trans-activating crispr RNA); a MAD2021 system comprising SEQ ID Nos. 16 (MAD2021 nuclease), 17 (CRISPR RNA) and 18 (trans-activating crispr RNA); a MAD2022 system comprising SEQ ID Nos. 19 (MAD2022 nuclease), 20 (CRISPR RNA) and 21 (trans-activating crispr RNA); a MAD2023 system comprising SEQ ID Nos. 22 (MAD2023 nuclease), 23 (CRISPR RNA) and 24 (trans-activating crispr RNA); a MAD2024 system comprising SEQ ID Nos. 25 (MAD2024 nuclease), 26 (CRISPR RNA) and 27 (trans-activating crispr RNA); a MAD2025 system comprising SEQ ID Nos. 28 (MAD2025 nuclease), 29 (CRISPR RNA) and 30 (trans-activating crispr RNA); a MAD2026 system comprising SEQ ID Nos. 31 (MAD2026 nuclease), 32 (CRISPR RNA) and 33 (trans-activating crispr RNA); a MAD2027 system comprising SEQ ID Nos. 34 (MAD2034 nuclease), 35 (CRISPR RNA) and 36 (trans-activating crispr RNA); a MAD2028 system comprising SEQ ID Nos. 37 (MAD2028 nuclease), 38 (CRISPR RNA) and 39 (trans-activating crispr RNA); a MAD2029 system comprising SEQ ID Nos. 40 (MAD2029 nuclease), 41 (CRISPR RNA) and 42 (trans-activating crispr RNA); a MAD2030 system comprising SEQ ID Nos. 43 (MAD2030 nuclease), 44 (CRISPR RNA) and 45 (trans-activating crispr RNA); a MAD2031 system comprising SEQ ID Nos. 46 (MAD2031 nuclease), 47 (CRISPR RNA) and 48 (trans-activating crispr RNA); a MAD2032 system comprising SEQ ID Nos. 49 (MAD2032 nuclease), 50 (CRISPR RNA) and 51 (trans-activating crispr RNA); a MAD2033 system comprising SEQ ID Nos. 52 (MAD2033 nuclease), 53 (CRISPR RNA) and 54 (trans-activating crispr RNA); a MAD2034 system comprising SEQ ID Nos. 55 (MAD2034 nuclease), 56 (CRISPR RNA) and 57 (trans-activating crispr RNA); a MAD2035 system comprising SEQ ID Nos. 58 (MAD2035 nuclease), 59 (CRISPR RNA) and 60 (trans-activating crispr RNA); a MAD2036 system comprising SEQ ID Nos. 61 (MAD2036 nuclease), 62 (CRISPR RNA) and 63 (trans-activating crispr RNA); a MAD2037 system comprising SEQ ID Nos. 64 (MAD2031 nuclease), 65 (CRISPR RNA) and 66 (trans-activating crispr RNA); a MAD2038 system comprising SEQ ID Nos. 67 (MAD2038 nuclease), 68 (CRISPR RNA) and 69 (trans-activating crispr RNA); a MAD2039 system comprising SEQ ID Nos. 70 (MAD2039 nuclease), 71 (CRISPR RNA) and 72 (trans-activating crispr RNA); and a MAD2040 system comprising SEQ ID Nos. 73 (MAD2040 nuclease), 74 (CRISPR RNA) and 75 (trans-activating crispr RNA). In some aspects, the MAD system components are delivered as sequences to be transcribed (in the case of the gRNA components) and transcribed and translated (in the case of the MAD nuclease), and in some aspects, the coding sequence for the MAD nuclease and the gRNA component sequences are on the same vector. In other aspects, the coding sequence for the MAD nuclease and the gRNA component sequences are on a different vector and in some aspects, the gRNA component sequences are located in an editing cassette which also comprises a donor DNA (e.g., homology arm). In other aspects, the MAD nuclease is delivered to the cells as a peptide or the MAD nuclease and gRNA components are delivered to the cells as a ribonuclease complex.
Additionally there is provided engineered nickases derived from the nucleases from the above-referenced systems, including MAD2016-H851A (SEQ ID NO: 178); MAD2016-N874A (SEQ ID NO: 179); MAD2032-H590A (SEQ ID NO: 180); MAD2039-H587A (SEQ ID NO: 181); MAD2039-N610A (SEQ ID NO: 182).
These aspects and other features and advantages of the invention are described below in more detail.
BRIEF DESCRIPTION OF THE FIGURES FIG. 1 is an exemplary workflow for creating and screening mined MAD nucleases or RGNs.
FIG. 2 is a simplified depiction of an in vitro test conducted on candidate enzymes.
FIG. 3 is a list of novel Type II MADzymes that have been identified.
FIG. 4 is a map of Type II MADzymes in cluster 59.
FIG. 5 is a map of Type II MADzymes in cluster 55, 56, 57 and 58.
FIG. 6 is a map of Type II MADzymes in cluster 141.
FIG. 7 is a reproduction of a gel showing nicked plasmid formation with different MADzyme nickases compared to corresponding MADzyme nucleases.
It should be understood that the drawings are not necessarily to scale.
DETAILED DESCRIPTION The description set forth below in connection with the appended drawings is intended to be a description of various, illustrative embodiments of the disclosed subject matter. Specific features and functionalities are described in connection with each illustrative embodiment; however, it will be apparent to those skilled in the art that the disclosed embodiments may be practiced without each of those specific features and functionalities. Moreover, all of the functionalities described in connection with one embodiment are intended to be applicable to the additional embodiments described herein except where expressly stated or where the feature or function is incompatible with the additional embodiments. For example, where a given feature or function is expressly described in connection with one embodiment but not expressly mentioned in connection with an alternative embodiment, it should be understood that the feature or function may be deployed, utilized, or implemented in connection with the alternative embodiment unless the feature or function is incompatible with the alternative embodiment.
The practice of the techniques described herein may employ, unless otherwise indicated, conventional techniques and descriptions of organic chemistry, polymer technology, molecular biology (including recombinant techniques), cell biology, biochemistry, biological emulsion generation, and sequencing technology, which are within the skill of those who practice in the art. Such conventional techniques include polymer array synthesis, hybridization and ligation of polynucleotides, and detection of hybridization using a label. Specific illustrations of suitable techniques can be had by reference to the examples herein. However, other equivalent conventional procedures can, of course, also be used. Such conventional techniques and descriptions can be found in standard laboratory manuals such as Green, et al., Eds. (1999), Genome Analysis: A Laboratory Manual Series (Vols. I-IV); Weiner, Gabriel, Stephens, Eds. (2007), Genetic Variation: A Laboratory Manual; Dieffenbach, Dveksler, Eds. (2003), PCR Primer: A Laboratory Manual; Bowtell and Sambrook (2003), DNA Microarrays: A Molecular Cloning Manual; Mount (2004), Bioinformatics: Sequence and Genome Analysis; Sambrook and Russell (2006), Condensed Protocols from Molecular Cloning: A Laboratory Manual; and Sambrook and Russell (2002), Molecular Cloning: A Laboratory Manual (all from Cold Spring Harbor Laboratory Press); Stryer, L. (1995) Biochemistry (4th Ed.) W.H. Freeman, New York N.Y.; Gait, “Oligonucleotide Synthesis: A Practical Approach” 1984, IRL Press, London; Nelson and Cox (2000), Lehninger, Principles of Biochemistry 3rd Ed., W. H. Freeman Pub., New York, N.Y.; Berg et al. (2002) Biochemistry, 5th Ed., W.H. Freeman Pub., New York, N.Y.; Cell and Tissue Culture: Laboratory Procedures in Biotechnology (Doyle & Griffiths, eds., John Wiley & Sons 1998), all of which are herein incorporated in their entirety by reference for all purposes. Nuclease-specific techniques can be found in, e.g., Genome Editing and Engineering From TALENs and CRISPRs to Molecular Surgery, Appasani and Church, 2018; and CRISPR: Methods and Protocols, Lindgren and Charpentier, 2015; both of which are herein incorporated in their entirety by reference for all purposes. Basic methods for enzyme engineering may be found in, Enzyme Engineering Methods and Protocols, Samuelson, ed., 2013; Protein Engineering, Kaumaya, ed., (2012); and Kaur and Sharma, “Directed Evolution: An Approach to Engineer Enzymes”, Crit. Rev. Biotechnology, 26:165-69 (2006).
Note that as used herein and in the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “an oligonucleotide” refers to one or more oligonucleotides. Terms such as “first,” “second,” “third,” etc., merely identify one of a number of portions, components, steps, operations, functions, and/or points of reference as disclosed herein, and likewise do not necessarily limit embodiments of the present disclosure to any particular configuration or orientation.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. All publications mentioned herein are incorporated by reference for the purpose of describing and disclosing devices, methods and cell populations that may be used in connection with the presently described invention.
Where a range of values is provided, it is understood that each intervening value, between the upper and lower limit of that range and any other stated or intervening value in that stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either both of those included limits are also included in the invention.
In the following description, numerous specific details are set forth to provide a more thorough understanding of the present invention. However, it will be apparent to one of ordinary skill in the art that the present invention may be practiced without one or more of these specific details. In other instances, well-known features and procedures well known to those skilled in the art have not been described in order to avoid obscuring the invention.
The term “complementary” as used herein refers to Watson-Crick base pairing between nucleotides and specifically refers to nucleotides hydrogen bonded to one another with thymine or uracil residues linked to adenine residues by two hydrogen bonds and cytosine and guanine residues linked by three hydrogen bonds. In general, a nucleic acid includes a nucleotide sequence described as having a “percent complementarity” or “percent homology” to a specified second nucleotide sequence. For example, a nucleotide sequence may have 80%, 90%, or 100% complementarity to a specified second nucleotide sequence, indicating that 8 of 10, 9 of 10 or 10 of 10 nucleotides of a sequence are complementary to the specified second nucleotide sequence. For instance, the nucleotide sequence 3′-TCGA-5′ is 100% complementary to the nucleotide sequence 5′-AGCT-3′; and the nucleotide sequence 3′-TCGA-5′ is 100% complementary to a region of the nucleotide sequence 5′-TAGCTG-3′.
The term DNA “control sequences” refers collectively to promoter sequences, polyadenylation signals, transcription termination sequences, upstream regulatory domains, origins of replication, internal ribosome entry sites, nuclear localization sequences, enhancers, and the like, which collectively provide for the replication, transcription and translation of a coding sequence in a recipient cell. Not all of these types of control sequences need to be present so long as a selected coding sequence is capable of being replicated, transcribed and—for some components—translated in an appropriate host cell.
As used herein the term “donor DNA” or “donor nucleic acid” refers to nucleic acid that is designed to introduce a DNA sequence modification (insertion, deletion, substitution) into a locus by homologous recombination using nucleic acid-guided nucleases. For homology-directed repair, the donor DNA must have sufficient homology to the regions flanking the “cut site” or site to be edited in the genomic target sequence. The length of the homology arm(s) will depend on, e.g., the type and size of the modification being made. In many instances and preferably, the donor DNA will have two regions of sequence homology (e.g., two homology arms) to the genomic target locus. Preferably, an “insert” region or “DNA sequence modification” region—the nucleic acid modification that one desires to be introduced into a genome target locus in a cell—will be located between two regions of homology. The DNA sequence modification may change one or more bases of the target genomic DNA sequence at one specific site or multiple specific sites. A change may include changing 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 50, 75, 100, 150, 200, 300, 400, or 500 or more base pairs of the target sequence. A deletion or insertion may be a deletion or insertion of 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 40, 50, 75, 100, 150, 200, 300, 400, or 500 or more base pairs of the target sequence.
The terms “guide nucleic acid” or “guide RNA” or “gRNA” refer to a polynucleotide comprising 1) a guide sequence capable of hybridizing to a genomic target locus, and 2) a scaffold sequence capable of interacting or complexing with a nucleic acid-guided nuclease.
“Homology” or “identity” or “similarity” refers to sequence similarity between two peptides or, more often in the context of the present disclosure, between two nucleic acid molecules. The term “homologous region” or “homology arm” refers to a region on the donor DNA with a certain degree of homology with the target genomic DNA sequence. Homology can be determined by comparing a position in each sequence which may be aligned for purposes of comparison. When a position in the compared sequence is occupied by the same base or amino acid, then the molecules are homologous at that position. A degree of homology between sequences is a function of the number of matching or homologous positions shared by the sequences.
“Operably linked” refers to an arrangement of elements where the components so described are configured so as to perform their usual function. Thus, control sequences operably linked to a coding sequence are capable of effecting the transcription, and in some cases, the translation, of a coding sequence. The control sequences need not be contiguous with the coding sequence so long as they function to direct the expression of the coding sequence. Thus, for example, intervening untranslated yet transcribed sequences can be present between a promoter sequence and the coding sequence and the promoter sequence can still be considered “operably linked” to the coding sequence. In fact, such sequences need not reside on the same contiguous DNA molecule (i.e. chromosome) and may still have interactions resulting in altered regulation.
A “promoter” or “promoter sequence” is a DNA regulatory region capable of binding RNA polymerase and initiating transcription of a polynucleotide or polypeptide coding sequence such as messenger RNA, ribosomal RNA, small nuclear or nucleolar RNA, guide RNA, or any kind of RNA transcribed by any class of any RNA polymerase I, II or III. Promoters may be constitutive or inducible and, in some embodiments—particularly many embodiments in which selection is employed—the transcription of at least one component of the nucleic acid-guided nuclease editing system is under the control of an inducible promoter.
As used herein the term “selectable marker” refers to a gene introduced into a cell, which confers a trait suitable for artificial selection. General use selectable markers are well-known to those of ordinary skill in the art. Drug selectable markers such as ampicillin/carbenicillin, kanamycin, chloramphenicol, erythromycin, tetracycline, gentamicin, bleomycin, streptomycin, rhamnose, puromycin, hygromycin, blasticidin, and G418 may be employed. In other embodiments, selectable markers include, but are not limited to human nerve growth factor receptor (detected with a MAb, such as described in U.S. Pat. No. 6,365,373); truncated human growth factor receptor (detected with MAb); mutant human dihydrofolate reductase (DHFR; fluorescent MTX substrate available); secreted alkaline phosphatase (SEAP; fluorescent substrate available); human thymidylate synthase (TS; confers resistance to anti-cancer agent fluorodeoxyuridine); human glutathione S-transferase alpha (GSTA1; conjugates glutathione to the stem cell selective alkylator busulfan; chemoprotective selectable marker in CD34+cells); CD24 cell surface antigen in hematopoietic stem cells; human CAD gene to confer resistance to N-phosphonacetyl-L-aspartate (PALA); human multi-drug resistance-1 (MDR-1; P-glycoprotein surface protein selectable by increased drug resistance or enriched by FACS); human CD25 (IL-2α; detectable by Mab-FITC); Methylguanine-DNA methyltransferase (MGMT; selectable by carmustine); and Cytidine deaminase (CD; selectable by Ara-C). “Selective medium” as used herein refers to cell growth medium to which has been added a chemical compound or biological moiety that selects for or against selectable markers.
The terms “target genomic DNA sequence”, “target sequence”, or “genomic target locus” refer to any locus in vitro or in vivo, or in a nucleic acid (e.g., genome) of a cell or population of cells, in which a change of at least one nucleotide is desired using a nucleic acid-guided nuclease editing system. The target sequence can be a genomic locus or extrachromosomal locus.
A “vector” is any of a variety of nucleic acids that comprise a desired sequence or sequences to be delivered to and/or expressed in a cell. Vectors are typically composed of DNA, although RNA vectors are also available. Vectors include, but are not limited to, plasmids, fosmids, phagemids, virus genomes, synthetic chromosomes, and the like. As used herein, the phrase “engine vector” comprises a coding sequence for a nuclease to be used in the nucleic acid-guided nuclease systems and methods of the present disclosure. The engine vector may also comprise, in a bacterial system, the λ Red recombineering system or an equivalent thereto. Engine vectors also typically comprise a selectable marker. As used herein the phrase “editing vector” comprises a donor nucleic acid, optionally including an alteration to the target sequence that prevents nuclease binding at a PAM or spacer in the target sequence after editing has taken place, and a coding sequence for a gRNA. The editing vector may also comprise a selectable marker and/or a barcode. In some embodiments, the engine vector and editing vector may be combined; that is, the contents of the engine vector may be found on the editing vector. Further, the engine and editing vectors comprise control sequences operably linked to, e.g., the nuclease coding sequence, recombineering system coding sequences (if present), donor nucleic acid, guide nucleic acid, and selectable marker(s).
Editing in Nucleic Acid-Guided Nuclease Genome Systems RNA-guided nucleases (RGNs) have rapidly become the foundational tools for genome engineering of prokaryotes and eukaryotes. Clustered Rapidly Interspaced Short Palindromic Repeats (CRISPR) systems are an adaptive immunity system which protect prokaryotes against mobile genetic elements (MGEs). RGNs are a major part of this defense system because they identify and destroy MGEs. RGNs can be repurposed for genome editing in various organisms by reprogramming the CRISPR RNA (crRNA) that guides the RGN to a specific target DNA. A number of different RGNs have been identified to date for various applications; however, there are various properties that make some RGNs more desirable than others for specific applications. RGNs can be used for creating specific double strand breaks (DSBs), specific nicks of one strand of DNA, or guide another moiety to a specific DNA sequence.
The ability of an RGN to specifically target any genomic sequence is perhaps the most desirable feature of RGNs; however, RGNs can only access their desired target if the target DNA also contains a short motif called PAM (protospacer adjacent motif) that is specific for every RGN. Type V RGNs such as MAD7, AsCas12a and LbCas12a tend to access DNA targets that contain YTTN/TTTN on the 5′ end whereas type II RGNs—such as the MADzymes disclosed herein—target DNA sequences containing a specific short motif on the 3′ end. An example well known in the art for a type II RGN is SpCas9 which requires an NGG on the 3′ end of the target DNA. Type II RGNs, unlike type V RGNS, require a transactivating RNA (tracrRNA) in addition to a crRNA for optimal function. Compared to type V RGNs, the type II RGNs create a double-strand break closer to the PAM sequence, which is highly desirable for precise genome editing applications.
A number of type II RGNs have been discovered so far; however, their use in widespread applications is limited by restrictive PAMs. For example, the PAM of SpCas9 occurs less frequently in AT-rich regions of the genome. New type II RGNs with new and less restrictive PAMs are beneficial for the field. Further, not all type II nucleases are active in multiple organisms. For example, a number of RGNs have been discussed in the scientific literature but only a few have been demonstrated to be active in vitro and fewer still are active in cells, particularly in mammalian cells. The present disclosure identifies multiple type II RGNs that have novel PAMs and are active in mammalian cells.
In performing nucleic acid-guided nuclease editing, the type II RGNs or MADzymes may be delivered to cells to be edited as a polypeptide; alternatively, a polynucleotide sequence encoding the MADzyme are transformed or transfected into the cells to be edited. The polynucleotide sequence encoding the MADzyme may be codon optimized for expression in particular cells, such as archaeal, prokaryotic or eukaryotic cells. Eukaryotic cells can be yeast, fungi, algae, plant, animal, or human cells. Eukaryotic cells may be those of or derived from a particular organism, such as a mammal, including but not limited to human, mouse, rat, rabbit, dog, or non-human mammals including non-human primates. The choice of the MADzyme to be employed depends on many factors, such as what type of edit is to be made in the target sequence and whether an appropriate PAM is located close to the desired target sequence. The MADzyme may be encoded by a DNA sequence on a vector (e.g., the engine vector) and be under the control of a constitutive or inducible promoter. In some embodiments, the sequence encoding the nuclease is under the control of an inducible promoter, and the inducible promoter may be separate from but the same as an inducible promoter controlling transcription of the guide nucleic acid; that is, a separate inducible promoter may drive the transcription of the nuclease and guide nucleic acid sequences but the two inducible promoters may be the same type of inducible promoter (e.g., both are pL promoters). Alternatively, the inducible promoter controlling expression of the nuclease may be different from the inducible promoter controlling transcription of the guide nucleic acid; that is, e.g., the nuclease may be under the control of the pBAD inducible promoter, and the guide nucleic acid may be under the control of the pL inducible promoter.
In general, a guide nucleic acid (e.g., gRNA) complexes with a compatible nucleic acid-guided nuclease and can then hybridize with a target sequence, thereby directing the nuclease to the target sequence. With the type II MADzymes described herein, the nucleic acid-guided nuclease editing system uses two separate guide nucleic acid components that combine and function as a guide nucleic acid; that is, a CRISPR RNA (crRNA) and a transactivating CRISPR RNA (tracrRNA). The gRNA may be encoded by a DNA sequence on a polynucleotide molecule such as a plasmid, linear construct, or the coding sequence may reside within an editing cassette and is under the control of a constitutive promoter, or, in some embodiments, an inducible promoter as described below.
A guide nucleic acid comprises a guide polynucleotide sequence having sufficient complementarity with a target sequence to hybridize with the target sequence and direct sequence-specific binding of a complexed nucleic acid-guided nuclease to the target sequence. The degree of complementarity between a guide sequence and the corresponding target sequence, when optimally aligned using a suitable alignment algorithm, is about or more than about 50%, 60%, 75%, 80%, 85%, 90%, 95%, 97.5%, 99%, or more. Optimal alignment may be determined with the use of any suitable algorithm for aligning sequences. In some embodiments, a guide sequence is about or more than about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 75, or more nucleotides in length. In some embodiments, a guide sequence is less than about 75, 50, 45, 40, 35, 30, 25, 20 nucleotides in length. Preferably the guide sequence is 10-30 or 15-20 nucleotides long, or 15, 16, 17, 18, 19, or 20 nucleotides in length.
In the present methods and compositions, the components of the guide nucleic acid is provided as a sequence to be expressed from a plasmid or vector and comprises both the guide sequence and the scaffold sequence as a single transcript under the control of a promoter, and in some embodiments, an inducible promoter. In general, to generate an edit in a target sequence, the gRNA/nuclease complex binds to a target sequence as determined by the guide RNA, and the nuclease recognizes a protospacer adjacent motif PAM) sequence adjacent to the target sequence. The target sequence can be any polynucleotide endogenous or exogenous to a prokaryotic or eukaryotic cell, or in vitro. For example, the target sequence can be a polynucleotide residing in the nucleus of a eukaryotic cell. A target sequence can be a sequence encoding a gene product (e.g., a protein) or a non-coding sequence (e.g., a regulatory polynucleotide, an intron, a PAM, or “junk” DNA).
The guide nucleic acid may be part of an editing cassette that encodes the donor nucleic acid. Alternatively, the guide nucleic acid may not be part of the editing cassette and instead may be encoded on the engine or editing vector backbone. For example, a sequence coding for a guide nucleic acid can be assembled or inserted into a vector backbone first, followed by insertion of the donor nucleic acid in, e.g., the editing cassette. In other cases, the donor nucleic acid in, e.g., an editing cassette can be inserted or assembled into a vector backbone first, followed by insertion of the sequence coding for the guide nucleic acid. In yet other cases, the sequence encoding the guide nucleic acid and the donor nucleic acid (inserted, for example, in an editing cassette) are simultaneously but separately inserted or assembled into a vector. In yet other embodiments, the sequence encoding the guide nucleic acid and the sequence encoding the donor nucleic acid are both included in the editing cassette.
The target sequence is associated with a PAM, which is a short nucleotide sequence recognized by the gRNA/nuclease complex. The precise PAM sequence and length requirements for different nucleic acid-guided nucleases vary; however, PAMs typically are 2-7 base-pair sequences adjacent or in proximity to the target sequence and, depending on the nuclease, can be 5′ or 3′ to the target sequence. Engineering of the PAM-interacting domain of a nucleic acid-guided nuclease may allow for alteration of PAM specificity, improve fidelity, or decrease fidelity. In certain embodiments, the genome editing of a target sequence both introduces a desired DNA change to a target sequence, e.g., the genomic DNA of a cell, and removes, mutates, or renders inactive a proto-spacer mutation (PAM) region in the target sequence. Rendering the PAM at the target sequence inactive precludes additional editing of the cell genome at that target sequence, e.g., upon subsequent exposure to a nucleic acid-guided nuclease complexed with a synthetic guide nucleic acid in later rounds of editing. Thus, cells having the desired target sequence edit and an altered PAM can be selected using a nucleic acid-guided nuclease complexed with a synthetic guide nucleic acid complementary to the target sequence. Cells that did not undergo the first editing event will be cut rendering a double-stranded DNA break, and thus will not continue to be viable. The cells containing the desired target sequence edit and PAM alteration will not be cut, as these edited cells no longer contain the necessary PAM site and will continue to grow and propagate.
As mentioned previously, the range of target sequences that nucleic acid-guided nucleases can recognize is constrained by the need for a specific PAM to be located near the desired target sequence. As a result, it often can be difficult to target edits with the precision that is necessary for genome editing. It has been found that nucleases can recognize some PAMs very well (e.g., canonical PAMs), and other PAMs less well or poorly (e.g., non-canonical PAMs). Because the mined MAD nucleases disclosed herein may recognize different PAMs, the mined MAD nucleases increase the number of target sequences that can be targeted for editing; that is, mined MAD nucleases decrease the regions of “PAM deserts” in the genome. Thus, the mined MAD nucleases expand the scope of target sequences that may be edited by increasing the number (variety) of PAM sequences recognized. Moreover, cocktails of mined MAD nucleases may be delivered to cells such that target sequences adjacent to several different PAMs may be edited in a single editing run.
Another component of the nucleic acid-guided nuclease system is the donor nucleic acid. In some embodiments, the donor nucleic acid is on the same polynucleotide (e.g., editing vector or editing cassette) as the guide nucleic acid and may be (but not necessarily) under the control of the same promoter as the guide nucleic acid (e.g., a single promoter driving the transcription of both the guide nucleic acid and the donor nucleic acid). For cassettes of this type, see U.S. Pat. Nos. 10,240,167; 10,266,849; 9,982,278; 10,351,877; 10,364,442; 10,435,715; and 10,465,207. The donor nucleic acid is designed to serve as a template for homologous recombination with a target sequence nicked or cleaved by the nucleic acid-guided nuclease as a part of the gRNA/nuclease complex. A donor nucleic acid polynucleotide may be of any suitable length, such as about or more than about 20, 25, 50, 75, 100, 150, 200, 500, or 1000 nucleotides in length. In certain preferred aspects, the donor nucleic acid can be provided as an oligonucleotide of between 20-300 nucleotides, more preferably between 50-250 nucleotides. The donor nucleic acid comprises a region that is complementary to a portion of the target sequence (e.g., a homology arm). When optimally aligned, the donor nucleic acid overlaps with (is complementary to) the target sequence by, e.g., about 20, 25, 30, 35, 40, 50, 60, 70, 80, 90 or more nucleotides. In many embodiments, the donor nucleic acid comprises two homology arms (regions complementary to the target sequence) flanking the mutation or difference between the donor nucleic acid and the target template. The donor nucleic acid comprises at least one mutation or alteration compared to the target sequence, such as an insertion, deletion, modification, or any combination thereof compared to the target sequence.
Often the donor nucleic acid is provided as an editing cassette, which is inserted into a vector backbone where the vector backbone may comprise a promoter driving transcription of the gRNA and the coding sequence of the gRNA, or the vector backbone may comprise a promoter driving the transcription of the gRNA but not the gRNA itself. Moreover, there may be more than one, e.g., two, three, four, or more guide nucleic acid/donor nucleic acid cassettes inserted into an engine vector, where each guide nucleic acid is under the control of separate different promoters, separate like promoters, or where all guide nucleic acid/donor nucleic acid pairs are under the control of a single promoter. In some embodiments the promoter driving transcription of the gRNA and the donor nucleic acid (or driving more than one gRNA/donor nucleic acid pair) is an inducible promoter. Inducible editing is advantageous in that isolated cells can be grown for several to many cell doublings to establish colonies before editing is initiated, which increases the likelihood that cells with edits will survive, as the double-strand cuts caused by active editing are largely toxic to the cells. This toxicity results both in cell death in the edited colonies, as well as a lag in growth for the edited cells that do survive but must repair and recover following editing. However, once the edited cells have a chance to recover, the size of the colonies of the edited cells will eventually catch up to the size of the colonies of unedited cells. See, e.g., U.S. Pat. Nos. 10,533,152; 10,550,363; 10,532,324; 10,550,363; 10,633,626; 10,633,627; 10,647,958; 10,760,043; 10,723,995; 10,801,008; and 10,851,339. Further, a guide nucleic acid may be efficacious directing the edit of more than one donor nucleic acid in an editing cassette; e.g., if the desired edits are close to one another in a target sequence.
In addition to the donor nucleic acid, an editing cassette may comprise one or more primer sites. The primer sites can be used to amplify the editing cassette by using oligonucleotide primers; for example, if the primer sites flank one or more of the other components of the editing cassette.
In addition, the editing cassette may comprise a barcode. A barcode is a unique DNA sequence that corresponds to the donor DNA sequence such that the barcode can identify the edit made to the corresponding target sequence. The barcode typically comprises four or more nucleotides. In some embodiments, the editing cassettes comprise a collection of donor nucleic acids representing, e.g., gene-wide or genome-wide libraries of donor nucleic acids. The library of editing cassettes is cloned into vector backbones where, e.g., each different donor nucleic acid is associated with a different barcode.
Additionally, in some embodiments, an expression vector or cassette encoding components of the nucleic acid-guided nuclease system further encodes one or more nuclear localization sequences (NLSs), such as about or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more NLSs. In some embodiments, the nuclease comprises NLSs at or near the amino-terminus of the MADzyme, NLSs at or near the carboxy-terminus of the MADzyme, or a combination.
The engine and editing vectors comprise control sequences operably linked to the component sequences to be transcribed. As stated above, the promoters driving transcription of one or more components of the mined MAD nuclease editing system may be inducible, and an inducible system is likely employed if selection is to be performed. A number of gene regulation control systems have been developed for the controlled expression of genes in plant, microbe, and animal cells, including mammalian cells, including the pL promoter (induced by heat inactivation of the CI857 repressor), the pBAD promoter (induced by the addition of arabinose to the cell growth medium), and the rhamnose inducible promoter (induced by the addition of rhamnose to the cell growth medium). Other systems include the tetracycline-controlled transcriptional activation system (Tet-On/Tet-Off, Clontech, Inc. (Palo Alto, Calif.); Bujard and Gossen, PNAS, 89(12):5547-5551 (1992)), the Lac Switch Inducible system (Wyborski et al., Environ Mol Mutagen, 28(4):447-58 (1996); DuCoeur et al., Strategies 5(3):70-72 (1992); U.S. Pat. No. 4,833,080), the ecdysone-inducible gene expression system (No et al., PNAS, 93(8):3346-3351 (1996)), the cumate gene-switch system (Mullick et al., BMC Biotechnology, 6:43 (2006)), and the tamoxifen-inducible gene expression (Zhang et al., Nucleic Acids Research, 24:543-548 (1996)) as well as others.
Typically, performing genome editing in live cells entails transforming cells with the components necessary to perform nucleic acid-guided nuclease editing. For example, the cells may be transformed simultaneously with separate engine and editing vectors; the cells may already be expressing the mined MAD nuclease (e.g., the cells may have already been transformed with an engine vector or the coding sequence for the mined MAD nuclease may be stably integrated into the cellular genome) such that only the editing vector needs to be transformed into the cells; or the cells may be transformed with a single vector comprising all components required to perform nucleic acid-guided nuclease genome editing.
A variety of delivery systems can be used to introduce (e.g., transform or transfect) nucleic acid-guided nuclease editing system components into a host cell. These delivery systems include the use of yeast systems, lipofection systems, microinjection systems, biolistic systems, virosomes, liposomes, immunoliposomes, polycations, lipid:nucleic acid conjugates, virions, artificial virions, viral vectors, electroporation, cell permeable peptides, nanoparticles, nanowires, exosomes. Alternatively, molecular trojan horse liposomes may be used to deliver nucleic acid-guided nuclease components across the blood brain barrier. Of particular interest is the use of electroporation, particularly flow-through electroporation (either as a stand-alone instrument or as a module in an automated multi-module system) as described in, e.g., U.S. Pat. Nos. 10,435,713; 10,443,074; 10,323,258; and 10,415,058.
After the cells are transformed with the components necessary to perform nucleic acid-guided nuclease editing, the cells are cultured under conditions that promote editing. For example, if constitutive promoters are used to drive transcription of the mined MAD nucleases and/or gRNA, the transformed cells need only be cultured in a typical culture medium under typical conditions (e.g., temperature, CO2 atmosphere, etc.) Alternatively, if editing is inducible—by, e.g., activating inducible promoters that control transcription of one or more of the components needed for nucleic acid-guided nuclease editing, such as, e.g., transcription of the gRNA, donor DNA, nuclease, or, in the case of bacteria, a recombineering system—the cells are subjected to inducing conditions.
EXAMPLES The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention, and are not intended to limit the scope of what the inventors regard as their invention, nor are they intended to represent or imply that the experiments below are all of or the only experiments performed. It will be appreciated by persons skilled in the art that numerous variations and/or modifications may be made to the invention as shown in the specific aspects without departing from the spirit or scope of the invention as broadly described. The present aspects are, therefore, to be considered in all respects as illustrative and not restrictive.
Example 1 Exemplary Workflow Overview The disclosed MADzyme Type II CRISPR enzymes were identified by the method depicted in FIG. 1. FIG. 1 shows an exemplary workflow for creating and for in vitro screening of MADzymes, including those in untapped clusters. In a first step, metagenome mining was performed to identify putative RGNs of interest based on, e.g., sequence (HMMER profile) and a search for CRISPR arrays. Once putative RGNs of interest were identified in silico, candidate pools were created and each MADzyme was identified by cluster, the tracrRNA was identified, and the sgRNA structure was predicted. Final candidates were identified, then the genes were synthesized. An in vitro depletion test was performed (see FIG. 2), where a synthetic target library was constructed in which to test target depletion for each of the candidate MADzymes. After target depletion, amplicons were produced for analysis for in vivo analysis. FIG. 2 depicts the in vitro depletion test in more detail.
Example 2 Metagenome Mining The NCBI Metagenome database was used to search for novel, putative CRISPR nucleases using HMMER hidden Markov model searches. Hundreds of potential nucleases were identified. For each potential nuclease candidate, putative CRISPR arrays were identified and CRISPR repeat and anti-repeats were identified. Thirteen nucleases (FIG. 3) were chosen for in vitro validation and 11 active MADzymes were identified and assigned to clusters. There was less than 40% sequence identity between clusters. Cluster 59 shown in FIG. 4 presents two unique subclusters with distinct sgRNA architecture. Clusters 55-57 are shown in FIG. 5. These new MADzymes have diverse PAM preferences and distinct sgRNA structure. Cluster 141 (FIG. 6) is a distant cluster from 55, 56, 57 and 59 and shows diverse Cas protein structure and smaller-sized enzymes (e.g., approximately 200 amino acids shorter than the counterparts from the 55, 56, 57 and 59 clusters). Table 1 lists the identified MADzymes, including amino acid sequences, origin, and nucleic acid sequences of the CRISPR RNA and the trans-activating crispr RNA.
TABLE 1
Organism
MAD Clus- (meta- CRISPR
name ter Contig_id genome) Source aa_seq repeat tracrRNA
MAD2015 59 DPZI01000013.1 Vagococcus MGKNYTIGLDIGTNSVGWSVVTENQQLVKKRMKIRGDS GTTTT TGTTGGT
sp. EKKQVKKNFWGVRLFDEGETAEATRLKRTTRRRYTRRR AGAGC AGCATTC
NRVVDLQNIFKDEINQKDSNFFNRLNESFLVVEDKKQP TATGC AAAACAA
KQMIFGTVEEEASYHESFPTIYHLRKELVDNKDQADIR TGTTT CATAGCA
LVYLAMAHMIKYRGHFLIEGQLSTENTSVEEKFHLFLK TGAAT AGTTAAA
EYNSTFCKQEDGSLVNPVNEDINGEEILMGTLSRSKKA GCTTC ATAAGGC
EQIMKSFEGEKSNGVFSQFLKMIVGNQGNFKKAFNLEE CAAAA TTTGTCC
DAKIQFAKEEYDEDLTTLLSNIGDEYANVFSLAKETYE C GTTCTCA
AIELSGILSTKDKETYAKLSSSMTERYEDHEKDLASLK [SEQ ACTTTTA
SFFREHLPEKYAVMFKDVSKNGYAGYIENSNKISQEEF ID NO. GTGACGC
YKYTKKLIGQIEGADYFIKKMEQEAFLRKQRTYDNGVI 2] TGTTTCG
PYQVHLSELTHIINNQKKYYPFLLEKEEEIKSILTFKI GCG
PYYIGPLAKGNSDFAWLIRNSNDKITPSNFNEVLDIEN [SEQ ID
SASQFIERMTNNDVYLPEEKVLPKNSMLYQKYIVFNEL NO. 3]
TKVRYINDRGTECNFSGEEKLQIFERFFKDSSTKVKKV
SLENYLNKEYMIESPTIKGIEDDFNASFRTYHDFIKLG
VSREMLDDIDNEEMFEDIVKILTIFEDRQMIKKQLEKY
KDVFDSDILKKMVRRHYTGWGRLSKKLLHEMKDDNSGK
TILDYLIEDDRLPKHINRNFMQLINDSNLSFKEKIEKA
QLTDGTEDIDSVVKNLIGSPAIKKGISQSLKIVEELVS
IMGYQPTSIVVEMARENQTTSKGKRQSIQRYKRLEAAI
NELGSDLLKVCPTDNHALKDDRLYLYYLQNGRDMYTGL
ELDIHNLSQYDIDHIVPRSFITDNSIDNRVLVSSKKNR
GKLDNVPSKEIVQKNKLLWMNLKKSKLMSEKKYANLIK
GETGGLTEDDKAKFLNRQLVETRQITKNVAQILDQRFN
TQKDEKGNIIREVKVITLKSALVSQFRQNFEFYKVREV
NDFHHANDAYLNAVVANTLLKVYPKLTPDFVYGEYRKG
NPFKNTKATAKKHYYSNIMENLCHETTIIDDETGEILW
DKKCIGTIKQVLNYHQVNVVKKVETQTGRFSEETLVPR
GSTKNPIALKSHLDPQKYGGFKSPTIAYTIVIEYKKGK
KDILIKELLGISIMNRGAFEKNNKEYLEKLNYKEPRVL
MVLPKYSLFELENGRRRLLASDKESQKGNQMAVPSYLN
NLLYHTNKSLSKNAKSLEYVNEHRQQFEELLEEIIDFA
NQFTLAEKNTLLIADLYESNKEADIELLASSFINLLRF
NQMGAPAEFSFFEKPIPRKRYSSTFELLKGKVIHQSIT
GLYETHQKV [SEQ ID NO. 1]
MAD2016 59 DGLK01000042.1 Entero- New MKKDYVIGLDIGTNSVGWAVMTEDYQLVKKKMPIYGNT GTTTT TCTTTTG
coccus York EKKKIKKNFWGVRLFEEGHTAEDRRLKRTARRIISRRR AGAGT GGACTAT
faecalis City NRLRYLQAFFEEAMTDLDENFFARLQESFLVPEDKKWH CATGT TCTAAAC
MTA RHPIFAKLEDEVAYHETYPTIYHLRKKLADSSEQADLR TGTTT AACATAG
subway LIYLALAHIVKYRGHFLIEGKLSTENISVKEQFQQFMI AGAAT CAAGTTA
IYNQTFVNGESRLVSAPLPESVLIEEELTEKASRTKKS GGTAC AAATAAG
EKVLQQFPQEKANGLFGQFLKLMVGNKADFKKVFGLEE CAAAA GTTTTAA
EAKITYASESYEEDLEGILAKVGDEYSDVFLAAKNVYD C CCGTAAT
AVELSTILADSDKKSHAKLSSSMIVRFTEHQEDLKKFK [SEQ CAACTGT
RFIRENCPDEYDNLFKNEQKDGYAGYIAHAGKVSQLKF ID NO. AAAGTGG
YQYVKKIIQDIAGAEYFLEKIAQENFLRKQRTFDNGVI 5] CGCTGTT
PHQIHLAELQAIIHRQAAYYPFLKENQEKIEQLVTFRI TCGGCGC
PYYVGPLSKGDASTFAWLKRQSEEPIRPWNLQETVDLD [SEQ ID
QSATAFIERMTNFDTYLPSEKVLPKHSLLYEKFMVFNE NO. 6]
LTKISYTDDRGIKANFSGKEKEKIFDYLFKTRRKVKKK
DIIQFYRNEYNTEIVTLSGLEEDQFNASFSTYQDLLKC
GLTRAELDHPDNAEKLEDIIKILTIFEDRQRIRTQLST
FKGQFSAEVLKKLERKHYTGWGRLSKKLINGIYDKESG
KTILGYLIKDDGVSKHYNRNFMQLINDSQLSFKNAIQK
AQSSEHEETLSETVNELAGSPAIKKGIYQSLKIVDELV
AIMGYAPKRIVVEMARENQTTSTGKRRSIQRLKIVEKA
MAEIGSNLLKEQPTTNEQLRDTRLFLYYMQNGKDMYTG
DELSLHRLSHYDIDHIIPQSFMKDDSLDNLVLVGSTEN
RGKSDDVPSKEVVKDMKAYWEKLYAAGLISQRKFQRLT
KGEQGGLTLEDKAHFIQRQLVETRQITKNVAGILDQRY
NANSKEKKVQIITLKASLTSQFRSIFGLYKVREVNDYH
HGQDAYLNCVVATTLLKVYPNLAPEFVYGEYPKFQTFK
ENKATAKAIIYTNLLRFFTEDEPRFTKDGEILWSNSYL
KTIKKELNYHQMNIVKKVEVQKGGFSKESIKPKGPSNK
LIPVKNGLDPQKYGGFDSPIVAYTVLFTHEKGKKPLIK
QEILGITIMEKTRFEQNPILFLEEKGFLRPRVLMKLPK
YTLYEFPEGRRRLLASAKEAQKGNQMVLPEHLLTLLYH
AKQCLLPNQSESLTYVEQHQPEFQEILERVVDFAEVHT
LAKSKVQQIVKLFEANQTADVKEIAASFIQLMQFNAMG
APSTFKFFQKDIERARYTSIKEIFDATIIYQSTTGLYE
TRRKVVD [SEQ ID NO. 4]
MAD2017 59 DMKA01000006.1 Strepto- MKKPYSIGLDIGTNSVGWAVITDDYKVPAKKMKVLGNT GTTTT TGTTGGA
coccus DKKYIKKNLLGALLFDSGETAEVTRLKRTARRRYTRRK AGAGC ACTATTC
sp. NRLRYLQEIFAKEMTKVDESFFQRLEESFLTDDDKTFD TGTGC GAAACAA
(firmi- SHPIFGNKAEEDAYHQKFPTIYHLRKYLADSQEKADLR TGTTT CACAGCG
cutes) LVYLALAHMIKYRGHFLIEGELNAENTDVQKLFNVFVE CGAAT AGTTAAA
TYDKIVDESHLSEIEVDASSILTEKVSKSRRLENLIKQ GGTTC ATAAGGC
YPTEKKNTLFGNLIALALGLQPNFKTNFKLSEDAKLQF CAAAA TTTGTCC
SKDTYEEDLEELLGKVGDDYADLFISAKNLYDAILLSG C GTACACA
ILTVDDNSTKAPLSASMIKRYVEHHEDLEKLKEFIKIN [SEQ ACTTGTA
KLKLYHDIFKDKTKNGYAGYIDNGVKQDEFYKYLKTIL ID NO. AAAGGGG
TKIDDSDYFLDKIERDDFLRKQRTFDNGSIPHQIHLQE 8] CACCCGA
MHSILRRQGEYYPFLKENQAKIEKILTFRIPYYVGPLA TTCGGGT
RKDSRFAWANYHSDEPITPWNFDEVVDKEKSAEKFITR GCA
MTLNDLYLPEEKVLPKHSHVYETFTVYNELTKIKYVNE [SEQ ID
QGESFFFDANMKQEIFDHVFKENRKVTKAKLLSYLNNE NO. 9]
FEEFRINDLIGLDKDSKSFNASLGTYHDLKKILDKSFL
DDKTNEQIIEDIVLTLTLFEDRDMIHERLQKYSDFFTS
QQLKKLERRHYTGWGRLSYKLINGIRNKENNKTILDFL
IDDGHANRNFMQLINDESLSFKTIIQEAQVVGDVDDIE
AVVHDLPGSPAIKKGILQSVKIVDELVKVMGDNPDNIV
IEMARENQTTGYGRNKSNQRLKRLQDSLKEFGSDILSK
KKPSYVDSKVENSHLQNDRLFLYYIQNGKDMYTGEELD
IDRLSDYDIDHIIPQAFIKDNSIDNKVLTSSAKNRGKS
DDVPSIEIVRNRRSYWYKLYKSGLISKRKFDNLTKAER
GGLTEADKAGFIKRQLVETRQITKHVAQILDARFNTKR
DENDKVIRDVKVITLKSNLVSQFRKEFKFYKVREINDY
HHANDAYLNAVVGTALLKKYPKLTPEFVYGEYKKYDVR
KLIAKSSDDYSEMGKATAKYFFYSNLMNFFKTEVKYAD
GRVFERPDIETNADGEVVWNKQKDFDIVRKVLSYPQVN
IVKKVEAQTGGFSKESILSKGDSDKLIPRKTKKVYWNT
KKYGGFDSPTVAYSVLVVADIEKGKAKKLKTVKELVGI
SIMERSFFEENPVSFLEKKGYHNVQEDKLIKLPKYSLF
EFEGGRRRLLASATELQKGNEVMLPAHLVELLYHAHRI
DSFNSTEHLKYVSEHKKEFEKVLSCVENFSNLYVDVEK
NLSKVRAAAESMTNFSLEEISASFINLLTLTALGAPAD
FNFLGEKIPRKRYTSTKECLSATLIHQSVTGLYETRID
LSKLGEE [SEQ ID NO. 7]
MAD2019 59 DOTL01000042.1 Strepto- MTKPYSIGLDIGTNSVGWAVITDDYKVPSKKMKVLGNT GTTTT GGTTTGA
coccus SKKYIKKNLLGALLFDSGITAEGRRLKRTARRRYTRRR AGAGC AACCATT
sp. NRILYLQEIFSTEMATLDDAFFQRLDDSFLVPDDKRDS TGTGT CGAAACA
(firmi- KYPIFGNLVEEKAYHDEFPTIYHLRKYLADSTKKADLR TGTTT ATACAGC
cutes) LVYLALAHMIKYRGHFLIEGEFNSKNNDIQKNFQDFLD CGAAT AAAGTTA
TYNAIFESDLSLENSKQLEEIVKDKISKLEKKDRILKL GGTTC AAATAAG
FPGEKNSGIFSEFLKLIVGNQADFKKYFNLDEKASLHF CAAAA GCTAGTC
SKESYDEDLETLLGYIGDDYSDVFLKAKKLYDAILLSG C CGTATAC
ILTVTDNGTETPLSSAMIMRYKEHEEDLGLLKAYIRNI [SEQ AACGTGA
SLKTYNEVFNDDTKNGYAGYIDGKTNQEDFYVYLKKLL ID NO. AAACACG
AKFEGADYFLEKIDREDFLRKQRTFDNGSIPYQIHLQE 11] TGGCACC
MRAILDKQAKFYPFLAKNKERIEKILTFRIPYYVGPLA GATTCGG
RGNSDFAWSIRKRNEKITPWNFEDVIDKESSAEAFINR TGC
MTSFDLYLPEEKVLPKHSLLYETFTVYNELTKVRFIAE [SEQ ID
GMSDYQFLDSKQKKDIVRLYFKGKRKVKVTDKDIIEYL NO. 12]
HAIDGYDGIELKGIEKQFNSSLSTYHDLLNIINDKEFL
DDSSNEAIIEEIIHTLTIFEDREMIKQRLSKFENIFDK
SVLKKLSRRHYTGWGKLSAKLINGIRDEKSGNTILDYL
IDDGISNRNFMQLIHDDALSFKKKIQKAQIIGDKDKDN
IKEVVKSLPGSPAIKKGILQSIKIVDELVKVMGRKPES
IVVEMARENQYTNQGKSNSQQRLKRLEESLEELGSKIL
KENIPAKLSKIDNNSLQNDRLYLYYLQNGKDMYTGDDL
DIDRLSNYDIDHIIPQAFLKDNSIDNKVLVSSASNRGK
SDDVPSLEVVKKRKTLWYQLLKSKLISQRKFDNLTKAE
RGGLSPEDKAGFIQRQLVETRQITKHVARLLDEKFNNK
KDENNRAVRTVKIITLKSTLVSQFRKDFELYKVREIND
FHHAHDAYLNAVVASALLKKYPKLEPEFVYGDYPKYNS
FRERKSATEKVYFYSNIMNIFKKSISLADGRVIERPLI
EVNEETGESVWNKESDLATVRRVLSYPQVNVVKKVEVQ
SGGFSKELVQPHGNSDKLIPRKTKKMIWDTKKYGGFDS
PIVAYSVLVMAEREKGKSKKLKPVKELVRITIMEKESF
KENTIDFLERRGLRNIQDENIILLPKFSLFELENGRRR
LLASAKELQKGNEFILPNKLVKLLYHAKNIHNTLEPEH
LEYVESHRADFGKILDVVSVFSEKYILAEAKLEKIKEI
YRKNMNTEIHEMATAFINLLTFTSIGAPATFKFFGHNI
ERKRYSSVAEILNATLIHQSVTGLYETRIDLGKLGED
[SEQ ID NO. 10]
MAD2020 55 DQFW01000027.1 Achole- human MKNNEETLKKLRLGLDIGTNSVGYALLDENNKLIKKNG GTTTG TGTAAAT
plasmatales gut HTFWGVRMFDEAETAKDRGSYRKSRRRLLRRKERMEIL CTAGT AACATAA
bacterium RSFFTKEICDIDPTFFERLDDSFYYKEDKKNKNTYNLF TATGT CGAGTGC
TSEYTDKDFYLEYPTIYHLRKAMQEEDKKFDIRMVYLA TATTT AAATAAG
IAHIIKYRGNFLYPGEEFSTSEYTSIKQFFLDFNDILD ATAGT CGTTTCG
ELSNELEDNEDYSAEYFDKIENINDDFLEKLKVILMEI ATTAA CGAAAAT
KGISNKKKELLDLFNVNKKSIYNELVIPFISGSAKVNI GCAAA TTACAGT
SSLSVIKNSKYPKTEISLGSEELEGQVEEAISVAPEIK C GGCCCTG
SVLEMIIKIKEISDFYFINKILSDSKTISESMVKMYDE [SEQ CTGTGGG
HNEDLKKLKGFFKKYAEDQYNEIFKIRDEKLANYVAYV ID NO. GCCTTTT
GFNKLRKNKVERFKHASREEFYGYLKQKLNNIKYAEAQ 14] TTATTTA
EEIKYFIDKIDNNEFLLKQNSNQNGAFPMQLHLKELKT TCAAA
ILNNQEKYYPFLSEGNDGYSIKEKIILTFKYKIPYYVG [SEQ ID
PLNKESKYSWVVREDEKIYPWNFDKVVKLDETAEKFIL NO. 15]
RMQNKCTYLKGDNDYCLPKNSLIFSEYSCLSYLNKLSI
NGKPIDPIMKSKIFNEVFLIKKQPTKKDIIEFIKTNYN
ADALTTTEKELPEATCNMASYIKMKEIFGKDFNDNKEM
IENIIKDITIFEDKSILGNRLKELYKLNNDRIKQIKGL
NYKGYSRLSKNLLVGLQIVDNQTGEIKGNVIEVMRKTN
LNLQEILYLDGYRLIDAIDEYNRKNSLNDSYLCARDYI
AENLVISPSFKRALIQTCSIIQEIERIFHKKIDEFYVE
VTRTNKDKNKGKTTSSRYDKIKKIYSSCQELAMAYNFD
MKRLKNELESNKDNLKSDILYFYFTQLGKCMYSLEDID
ISDLTNNYHYDIDHIYPQSIIKDDSLSNRVLVDKKKNA
AKTDKFLFEAKVLNPKAQQFYKKLLSLELISKEKYRRL
TQKEISKDELEGFVNRQLVSTNQSVMGLIKLLKEYYKV
DEKNIIYSKGENVSDFRHTFDLVKSRTANNFHHANDAY
LNVVVGGILNKYYTSRRFYQFSDIARIENEGESLNPSR
IFTKRDILKANGKVIWDKKEDIKRIEKDLYHRFDITET
IRTYNPNKMYSKVTILPKGEGESAVPFQTTTPRVDVEK
YGGITSNKFSRYVIIEAHGKKGLDTILEAIPKTACGDN
NKIEKDIDNYIASLDEYQKYTSYKVVNYNIKANVVIQE
GSFKYIITGKSGNQYVLQNVQDRFFSKKAMITIKNIDK
YLNNKKLGIIMAKDNEKIIVSPARGKNNEEIFFEKTEL
VNLLKEIKTMYSKDIYSFSAIQNIVNNIDCSIDYSIDD
FIIICNNLLQILKTNERKNADLRLIHLSGNSGTLYLGK
KLKSGMKFIWQSITGYYEEILYEVK [SEQ ID NO.
13]
MAD2021 57 DEED01000018.1 Lachno- MSEKYFVGLDMGTSSVGWAVTDEHYHLLRRKGKDLWGA GTTTG GATAATG
spiraceae RLFDEAETAAGRRTNRVSRRRLARQRARIGWLKELFRP AGAGC TTTTACA
bacterium YLEEKDAGFLQRLEESRFFLEDKTVKQPYALFSDKEFT CTTGT AGGCGAG
DKDYYQKYPTIFHLRKELLESKAPHDVRLVFLAVLNMY AAAAC TTCAAAT
AHRGHFLNPELQEGTLGDIHDLLSRLDAYIQDLFEDQG CGTAT AAGGATT
WSILENVEEQQKVLAEKNISNTVRLEKILSAIGTSPKD ATCTC TATCCGA
KEKKPLIEIYKLICGLKGSLSLAFSGVEMNETDAQMKF TCAAG AATCGCT
SFSDSNLEENEPEIERILGERYFEMYSILKEIHAWGLL C TGCGTGC
SEIMSDDSGKTYPYISYAKVDLYQKHHEQLRMLKKIIR [SEQ ATTGGCA
TYAPDEYHRMFRSMEDNTYSAYVGSVNSKNKKQRRGAK ID NO. CCATCTA
STDFFKEVKRIIEKIEKEHGELPECEEILDLIARDSFL 17] TCTTTTA
PKQLTTANGVIPNQVYATELRQIVTNAAAYLPFLNDKD
DTGLTNAEKIVEMFKFHIPYYIGPLKNDGNGTAWVVRK AGACTTT
QQGTVYPWNIDEKVDMAKTRDQFILNLVRKCSYLNDET CTTTGAA
VLPASSLLYEKFKVLNELNNLTINGQKISVELKQDIFR AGTCTT
DLFRATGKRVTTRKLMGYLRRKAVIDADADETCLEGFD [SEQ ID
KTQGGFVSTLSSYHKFMEIFSTDVLTDRQREIAEGAIY NO. 18]
FATVYGEDKSFLKKVLRDKFSPAELSQAQIDRLSGIRF
KDWSHLSREFLLLEEADHSTGEIMTIIDRLWNTNENLM
QIIHSDEYTYKQAIEERTARLEKSLSEVSFEDIEDSYM
SAPVRRMVWQTIRILQEIEEVMGSEPARVFVEMTRSEG
EKGDKGRKDSRKKKLKELYKKCKDDDQGLLSDIEGRDE
RDFRIRKLYLYYMQKGLCMYSGHPIDFGKLFDDSYYDI
DHIYPRHYVKDDSIENNLVLVESKLNRDKKDTLLCPDI
QERMHPVWEMLHRQGFMNDEKFKRLMRKEPFSEEEFAH
FIERQLVETGQGTKEIARILNDVLGNKDENNKVIYVKA
GNVSSFRNDNKKNPEFVKCRVINDHHHAKDAYLNIVVG
NTYYTKFTLHPANFIRELRNKSHPTLEDQYNMDKLFAR
RVERNGYTAWNPDTDFQTVKQVLRKNSVLISRRSFIEH
GQIADLQLVSGRKISEVNGKGYLPIKASDIRLSGPSGT
MKYGGYNKASGAYFFLVEHELKGKLVRTIEPVYVYMMA
SIHGKEDLEKYCQEELGYIHPRICLKKIPMYSHIRING
FDYYLTGRSNDRLFICNAVQLTLSSEWSAYIKALSKAV
DEKWDAAYIEQQASRIQDSLKSEEVFISKERNDQLYKV
LLQKHLEGFFNNRINSIGTIMKEGYDSFRALPVNEQAE
TLMEILKISQLVNIGANLVSIGGKSRSGVATVSKKISD
SKSFQLISDSVTGIFQRATDLLTI [SEQ ID NO.
16]
MAD2022 57 CACYWR010000004.1 uncultured Cattle MEKEYYLGLDMGTSSVGWAVTDKEYRLLRAKGKDMWGI GTTTG GAGAATT
Lachno- rumen REFEEAQTAVERRTHRLSKRRRARQLVRIGLLKDYFHD AGAGT AACAAGA
spiraceae EIMKIDPNFYIRLENSKYYLEDKDVRLASSNGIFDDKN CTTGT CGAGTGC
bacterium YTDKDYYEQYKTIFHLRSELIHNSQKHDVRLVYLALLN TAATT AAATAAG
MFKHRGHFLFEGDAYVQGNIGDIYKEFIQLLKNEYYED CTTAA GTTTATC
ENVKLTDQIDYFKLKEILSNSEFSRTAKAEKINSLVHI AGGTG CGGAATC
DKKNKLENTYIRLLCGLEIELKILFPEIDEKIKICFAK TAAAA GTCAATA
GYDEKLVEITEILTDNQLQILENLKKIHDIAALDKIRK C TGACCTG
GKEYLSDARVAEYEKHREDLALLKKIYREYMTKQDYDR [SEQ CATTGTG
MFREGEDGSYSAYVNSYNTSKKQRRNMKHRKIDEFYGT ID NO. CAGAATC
IRKDLKLLLKQGIQDDNIERILEEIDGNNDNKFMPKQL 20] TTTAAAA
SFANGVIPNSLHKAEMKAILRNAETYLPFLLETDESGL TCATATG
TVSERILQLFSFHIPYYIGPVSVNSEKNNGNGWVVRRE ATTTCAT
DGEVLPWNIEQKIDYGETSKRFIEKMVRRCTYISGEQV ATGGTTT
LPKNSFIYEKYCVLNEINNIKIDGERITVELKQNIYND TA
LYLHGKRVTKKQLINYLNNRGMIEDENQVSGIDINLNN [SEQ ID
YLGSYGKFLPIFEEKLKEDNYIKIAEDIIYLASIYGDS NO. 21]
KKMLKSQIKSKYGDILDDKQIKRILGLKFKDWGRISRR
FLELEGLDKETGEITTIIKAMWDYNLNFMEIIHSDAFD
FKDKIEELHANSIKPLAEIEVEDLDDMYFSAPVKRMIW
QTFKVIKEIEKVMGCPPKKVFIEMTRINDKKSKGKRTN
SRKEKFLSLYKNIHDELVDWKQLIISSDESGKLNSKKM
YLYLTQQGICMYTGRRINLEELFDDNKYDIDHIYPRHF
VKDDNLENNLVLVEKQSNSRKSDTYPIDKSIRNNSQVY
KHWKSLREGNFISKEKYDRLTGKNEFTDEQKAGFIARQ
MVETSQGTKGVADIIKQALPQSRIIYSKASNVSEFRRK
YDILKSRTVNEFHHANDAYLNIVVGNVYDTKFTSNPLN
FIKKQYNVDRKANNYNLDKMFVYDVKRGNEIAWIGWNP
KKSEDSSEMSKRGTIVTVKKMLSKNTPLMTRMSFVGHG
GIAEDNLSSHFVAKNKGYMPNGKESDVTKYGGYKKAKT
AYFFVVEHGQTNNRIRTIETLPIYRRREVEKYEDGLIK
YCEQSLSLLNPIIIYKKIKIQSLMKINGYYAYISGKSN
EVYTFRNGVNMCLSQEWINYVKKLENYIEKDRQDRMIT
YEKNIELYEIILRKYSTTILNKRLSKMDKKLINAKDRF
CILNVKEQSQVLINVFVLSRIGDNQTDLSKIGIGKQSG
QITQNKKITGCKEFKLVNQSVTGLYENEIDLLTV
[SEQ ID NO. 19]
MAD2023 56 DCGJ01000048.1 Lachno- Feces MEKNNYLLGLDIGTDSVGYAVTNDKYDILKFHGEPAWG GTTTG AGACCCC
clostridium of six- VTIFDEASLSTEKRSFRVSRRRLDRRQQRVLLVQELFA AGAGT TATGGAT
sp. years SEVAKVDKDFFKRIQESNLYRSDAENQAGLFIGEDYCD AGTGT TTACATT
old REYYGQYPTIHHLISDLMNGTSPHDVRLVYLACAWLVA AAATC GCGAGTT
elephant HRGHFLSNIDKDNLSGLKDFSSVYEGLMQYFSDNGYER CATAG CAAATAA
PWNANVDVKALGDALKKKQGVTAKTKELLALLLDSAKA GGGTC AAGTTTA
EKLPREEFPFSQDGIIKLLAGGTYKLSELFGNEEYKDF TCAAA CTCAAAT
GSVKLSMDDEKLGEIMSNIGEDYELIASLRIVSDWAVL C CGTTGGC
VDVLGESATISEAKVGIYNQHKADLEVLKKIIRKYTGK [SEQ TTGACCA
EGYKKVFRQVDSKENYVAYSQHESDGKAPKEKGIDIAT ID NO. ACCGCAC
FSKFILNIVRLLDVEPEDKEVYEDMVARLELNSFLPKQ 23] AGCGTGT
VNTDNRVIPYQLYWFELHKILENASIYLPMLTEKDSNG
ISVMEKLESVFMFRIPYFVGPLNKHSKYAWLERKEGKI GCTTAAA
YPWNFENMVDLDASEANFIKRMTNTCTYLPGQNVLPKD GATCTCT
SLRYHRFMVLNEINNLRINNERISVELKQKIYSELFLN TCAGTGA
VKKVTRKRLVDFLISNGELRKGEESSLTGIDVEIKANL GGTC
APQIAFKKLMESGQLTEEDVESIIERASYAEDKARLAH [SEQ ID
WLEAKYSKLSEIDRKYICGIKIKDFGRLSKMFLSELEG NO. 24]
VDKTTGEMTTILGAMWNSQLNLMELINSELYSFREAIC
AYQTDYYSTHSSSLEERMNEMYLSNAVKRPVYRTLDIV
KDVKKAFGEPKKIFVEMTRGASEEQKGKRTKSRKEQIL
ELYKQCKDEDVRILQQQLEEMGDLADNKLQGDKLFLYY
MQKGKCMYTGTPIVLEQLGSKAYDIDHIYPQAYVKDDS
ILNNRVLVLSEANGKKKDIYPIEKETRDKMHGFWTYLN
DKGMITEEKYKRLTRTTGFTEEEKWSFINRQLTETSQA
TKAVATLLGELFPNAEIVYSKARLTSEFRQEFNLLKCR
SYNDLHHAVDAYLNIVCGNVYNMKFTKRWFNINKDYSI
KTKTVFTHPVVCGGQVVWDGQEMLNKVIRNAKKNTAHF
TKYAYIRKGGFFDQMPVKAAEGLTPLKKDMPTAVYGGY
NKPSVAFLIPTRYKAGKKTEIIILSVEHLFGERFLRDE
AYAKEYAAERLKKILGKQVDEVSFPMGMRPWKINTVLS
LDGFLICISGIGSGGKCLRAQSIMQFSSDYRWTIYLKR
LERLVEKITVNAKYVYSEEFDKVSTIENIELYDLYIEK
YKATIFSKRVNSPEEIIESGRDKFVKLDVLSQARALLC
IHQTFGRIVGGCDLGLIGGKKNSAATGNFSSTISNWAK
YYKDVRIIDQSTSGLWVRKSENLLELV
[SEQ ID NO. 22]
MAD2024 56 CADAKQ010000027.1 uncultured Cattle MNFDGEYFLGLDIGTDSVGYAVTDQRYNLVKFKGEPMW GTTTG GAGCCCT
Lachno- rumen GSHLFDAANQCAERRGFRTARRRLDRRQQRVKLVDEIF AGAGT CTGGATT
spiraceae APEVAKVDPNFYIRKMESALYPEDKSNKGDLYLYFNKQ AGTGT TACACTA
bacterium EYDEKHYYKDYPTIHHLICALMNDEKTKFDIRLINIAI AAATC CGAGTTC
DWLVAHRGHFLSEVGTDSVDKVLDFRKIYDEFMALFSD CAGAG AAATAAA
EDDAVSSKPWENINPDELGKVLKIHGKNAKRNELKKLL GGCTC AATTATT
YGGKIPTDEDSFIDRKLLIDFIAGTSVQCNKLFRNSEY CAAAA TCAAATC
EDDLKITISNSDEREVVLPQLEDFHADIIAKLSSMYDW C GCCGCTA
SVLSDILSGSTYISESKVKVYEQHKKDLKELKEFVRKY [SEQ TGTCGGC
APEKYNDIFRLASKETYNYTAYSYNLKSVKDEKDLPKG ID NO. CGCACAG
KASKEDFYSYLKKTLKLDKAENYNFVNDADTRFFDDMV 26] TGTGTGC
ERISSGTFLPKQVNSDNRVIPYQVYYIELKKILENAKK ATTAAGA
HYAFFEEKDEDGYSNVEKIMSVFTFRIPYYVGPLRNDD AAAGTCC
KSPYAWIRRKADGKIYPWDFEEKVDLDASENAFIDRMT GAAAGGG
NSCTYIPGADVLPKWSLLYTKYMVLNEINNIKVNNIGI C
SVEAKQGIYNELFCKKAKVSLKAIREYLISNGFMQKDD [SEQ ID
EMSGIDITVKSSLKSRYDFRHLLEKNELTTDDVEAIIS NO. 27]
RSTYAEDKARFKKWLKKEFPQLSDEDYKYVSKLKYKDF
GRLSRSLLNGLEGASKETGEIGTIMHFLWETNDNLMQL
LSDRYTFMEEINKKRQDYYIEHKLTLNEQMEELGISNA
VKRPVTRTLAVVKDVVSAIGYAPQKIFVEMARQEDEKK
KRSVTRKEQILELYKNVEEDTKELERQLKKMGDTANNE
LQSDALFLYYLQLGKCMYSGKPIDLTQIKTTKKYDIDH
IWPQSMVKDDSLLNNRVLVLSEINGDKKDVYPIDESIR
SKMHSYWKMLLDKNLITKEKYSRLTRPTPFTESEKLGF
INRQLVETRQSMKAVTQLLNNMYPDSEIVYVKAKLAAD
FKQDFKLAPKSRIINDLHHAKDTYLNVVAGNVYNERFT
KKWFNVNEKYSMKTKVLFGHDVKIGDRLIWDSKKDLQT
VKNTYEKNNIHLTRYAYCQKGGLFDQMPVKKGQGQIQL
KKGMDIDRYGGYNKATASFFIIARYLRGGKKEVSFVPV
ELMVSEKFLNDDNFAIEYITNVLTGMNTKKIENVELPL
GKRVIKIKTVLLLDGYKVWVNGKASGGTRVMLTSAESL
RMPKEYVEYLKKMENYSEKKKSNRNFMHDSENDGLSEE
KNILLYDKLLEKLDENHFKKMPGNQCETMKSGRVKFIE
LDFDVQISTLLNCIDLLKSGRTGGCDLKNIGGKSASGV
VYISANLSACKYNDVHIIDISPAGLHENISCNLMELFE
[SEQ ID NO. 25]
MAD2025 56 DOQG01000053.1 Rumino- human MSFKENSKFYFGLDIGTDSVGWAVTDNLYKLYKYKNNL GTTTG TTTTACT
coccaceae gut MWGVSLFEAASPAEDRRNHRTARRRLDRRQQRVALLRE AGAGT ACCCTAT
bacterium LFAKEILKTDPDFFLRLKESSLYPEDRTNKNVNTYFDD AGTGT AAATTTA
ADFKDSDYFKMYPTVHHLIKELSESDKPHDVRLVYLAC AAATT CACTACG
AFIVAHRGHFLNGADENNVQEVLDFNSSYCEFTDWFKS TATAG AGTTCAA
NDIEDNPFSESTENEFSVILRKKIGITAKEKEIKNLLF GGTAG ATAAAAA
GTTKTPDCYKDEEYPIDIDVLIKFISGGKTNLAKLFRN TAAAA TTATTTC
PAYDELDIQTVEVGKADFADTIDLLASSMEDTDVPLLS C AAATCGT
AVKAMYDWSLLIDVLKGQKTISDAKVCEYEQHKSDLKA [SEQ ACTTTTT
LKHIVRKYLDKAQYDEIFRTAGEKPNYVSYSYNVTDVK ID NO. AGTACCT
LKQLPSNFKKKYSEEFCKYINSKLEKIKPEPDDEAVYN 29] TCACAAG
ELIEKCNSKTLCPKQVTDENRVIPYQLYYHELSMILDK TGTTGTG
ASAYLDFLNETEDGISVKQKILTLMKFRIPYFVGPSVK AATATTA
RNETDNVWIVRKAEGRIYPWNFENMVDYDKSEDGFIRR ACTCACC
MTCKCTYLAGEDVLPKYSLLYSRYTVLNEINNIKVKDV TTCGGGT
KISPELKQDIFNELFMKTSRVTVKKITELLKRKGAFSE GAG
ENGDSLSGVDINIKSSLKSYLDFRRLLENGSLSESDVE [SEQ ID
RIIERITVTTDKPRLISWLKTEYPALPAEDIRYISRLS NO. 30]
YKDYGRLSAKMLTGCYELDMDTGEIGGRSIIDLMWAEN
INLMQIMSDSYGYKSFIEEENKKYYAINPTGSIAQTLR
EMYVSPSASRAIIRTMDIVKELRKIIKRDPDKIFVEMA
RGSKPEDKGKRTSSRREQIEKLFASAKEFVSDEEISHL
RSQLGSLSDEQLRSEKYYLYFTQFGKCVYSGEAIDFSR
LGDNHCYDIDHIYPQSKVKDDSLHNKVLVKSQLNGEKS
DDYPIKEQIRNKMHPIWKNLFYRDPKNPTDKIKYERLT
RSTPFTEDELAGFIERQLVETRQSTKAVATLLKEMFPD
SKIVYVKAGQVSKFRHDFDMLKCREINDLHHAKDAYLN
VVVGNVHDVKFTSNPLNFVKNADKHYTIKIKETLKHKV
ARNGETAWNPETDFDTVKRMMSKNSVRYVRYCYKRKGE
LFKQQPKKAGNPDLAWLKKNLDPVKYGGYNSKSISCFS
LIKCTGVGVVIIPVELLCEKRYFSDDSFASEYAYSVLK
NALPAKNIAKISIDDISFPLKRRPIKINTLFEFDGYRV
NIRSKDSYSVFRISSAMAAIYSKDTSDYIKAISSYIDK
SDKGSKFKPGEAFDVLSNLKAYDEIAKKCISEPFCKIS
KLAEAGKKMEEGRNKFAELSIIEQMKTLLLLVDVLKTG
RVDKCNLKPVGGVDNFHTERMSAILKNTKYSDIRIIDQ
SPTGLYENKSDNLLEL [SEQ ID NO. 28]
MAD2026 65 CADBQN010000053.1 uncultured Cattle MEQKDYYIGLDIGTNSVGWAVVDEGYQLCRFKKYDMWG GTTTG GACTACC
Firmicutes rumen VRLFDSAETAAERRMNRVNRRRNRRKKQRIDLLQGLFA AGAGT ATATGAG
bacterium EEIAKIDRTFFVRLNESRLHPEDKSTAFRHPLFNDPNY AGTGT ATTACAC
TDVDYYKEYPTIYHLRKELMDSAEPHDIRLVYLALHHI AATTT TACACGG
LKNRGHFLIEGGFEDSKKFEPTFRQLLEVLTEELGLKM CATAT TTCAAAT
DGADAALAESVLKDRGMKKTEKVKRLKNVFTLNTTDMD GGTAG AAAGAAT
QESQKKQKAQIDAVCKFLAGSKGDFKKLVADEALNELK TCAAA GTTCGAA
LDTFALGTSKAEDIGLEIEKSAPQYCVVFESVKSVFDW C ACCGCCC
KIMTQILGDESTFSSAKVKEYEKHHENLIILRELIRKY [SEQ TTTGGGG
CDKETYRHFFNNVNGGYSRYIGSLKKNGKKYYVAGCTQ ID NO. CCCGCTT
EEFYKELKGLLKSIDQRVDPEDRPVYQRVLAETEDETF 32] GTTGCGG
LPLLRSKANSAIPRQIHQKELDDILQNASVYLPFLNDV ATTTACA
DEDGLSAAEKIRSIFTFRIPYYVGPLSLRHKDKGAHVW GACTTGA
IKRKEEGYIYPWNYEKKIDREKSNEEFIRRLINQCTYL TATCAAG
KDEKVLPKKSLLYSEFMVLNELNNLRIRGKRLSEEQVE TCTG
LKQRIYRDLFMTKTRVTKKTLLNYLRKEDSDLTEEDLS [SEQ ID
GFDNDFKASLSSCLELKNKVFGDRIEEDRVRKIAEDLI NO. 33]
RWLTIYDDDKKMIKEVIRAEYPNEFTNEQLDVICRLKF
SGWGNLSEAFLCGVEGADKDTGEVFTIIEALRNTNHNL
MELLSGNYTFTEKIREHNAALSSEIKAKDYESLVRDLY
VSPACKRGIWQTIRITEEIKKIMGHEPKKIFVEMTREH
RDSGRTTSRKDQLLALYQKCEEDARDWVKEIEDREERD
FSSIKLFLYYLQQGKCMYSGEAIDLDELMSKNSRWDRD
HIYPQSKIKDDSLDNLVLVKKELNAVKDNGEIAPDIQK
RMKGFWLSLLRQGFLSKKKFDRLTRTGPFTSEELAGFI
SRQLVETSQMSKAVAELLNQLYEDSRVVYVKAGLVSQF
RQKDLGVLKSRSVNDYHHAKDAYLNVVVGDMFDRKFTS
DPARWFKKNKKVNYSINQVFRRDYEENGKLIWKGIDRG
EDGKPLFRDGLIHGGTIDLVRAIAKRNTNIRYTEYTYC
ETGQLYNLTLLPKTDTAITIPLKKELPAAKYGGFKGAG
TSYFSLIEFDDKKGHHHKQIVGVPIYVANMLEHNENAF
IEYLETVCSFRNITVLCEKIKKNALISVNGYPMRIRGE
NEILNMLKNNLQLVLSQEGEETLRHIEKYFNKKPGFEP
DKEHDGIDRDAMAALYDEMTEKLCTVYKKRPTNQGELL
KNNRGLFLNLEKRSEMAKVLSETAKMFGTTAQTTADLS
LIKGSKYAGKIVINKNTLGAAKLILIHQSVTGLFETRV
EL [SEQ ID NO. 31]
MAD2027 65 CACWRN010000001.1 uncultured Cattle MSKKFAGEYYLGLDIGTDSVGWAVTDNQYNVLKFNGKS GTTTG TTTACCA
Succini- rumen MWGIRLFDAAQTAAERRMFRTARRRVERRRWRLELLQE AGAGT TCCAGTG
clasticum LFQNEIEKKDPDFFQRMKDSALYPEDSKTGKPFALFCD AATGT AGTTTAC
sp. KDLNDKLYYKQYPTIYHLRKALLTENSKFDIRLVYLAI AAATT ATTACAA
HHILKHRGHFLFNGDFSNVTRFSFAFEQLQTCLCNELD CATAG GTTCAAA
MDFECNNVQKLSEILKDTHMSKNDKVKASVALFENSGD GATGG TAAAAAT
KKQLQAVIGLFCGAKKKLADVFLDETLNDTEMPSISIA TAAAA TTATTCA
DKPYEELRPELESILAEKCCVIDYIKAVYDWAILADML C ACCCGTT
DGGEYGNRTYISVARVRQYEKHHDDLKKLKKLVRRYCK [SEQ CTTCGGA
SEYKSFFSVAGTDNYCAYIGDDIETDDRKSVKKCKQED ID NO. ACCTCCA
FYKRIKGLLKKAIENGCPKDEVVEIIKDIDAQVFLPLQ 35] CCGTGTG
VTKDNGVIPHQVHEMELKQILKNAEKYYPFLCKKDEEG GAACATT
IVTSNKILQLFKFRIPYYVGPLNSRIGKNSWIVRRAEG AAGGTCT
KIYPWNFEEKVDFDKSEEGFIRRMTNPCTYMAGADVLP GCTTTGC
KYSLLYSEFMVLNELNNVRICGDKLSVEIKQTIIKDLF AGGCC
QRTRRVTVRKLCDKLKAEGVISRNSNQKDIDIKGIDQD [SEQ ID
LKSSMVSYVDFKNIFGKEIEKYSVQQMCERIIFLLTIH NO. 36]
HDDKRRLQKRIRAEFTEAQITDDQLQKVLRLNYQGWGR
FSAEFLKELKGVDTETGEVFSIINALRETDDNLMQLLS
NRYTFAEELEKYNSNKRKKIEALTYDNIMEGIVASPAI
KRSAWQAISIVMELSKIMGREPKRIFVEMARGPEEKKH
TISRKNQLLELYKSVKDESRDWKTELETKTESDFRSIK
LFLYYTQMGRCMYTGEPIDLDQLANTTIYDRDHIYPQS
LTKDDSLNNLVLVKKVENANKGNGLISADIQKKMRGFW
AELKKKGLISDEKFSRLTRTTPLSDDELAGFINRQLVE
TRQSSKIVADLFHQLYPTTQVVYVKAKIVSDFRHETLD
MVKVRSLNDLHHAKDAYLNIVTGNVYYEKFSGNPLTWL
RKNPDRNYSLNQMFNYDIVKKTKEGTSYVWKKGKDGSI
AVVRRTMERNDILYTRQATENKNGGLFDQNIVSSKNKP
FIPVKKGLDVNKYGGYKGITPAYFALIEFTDKKGSRQR
LLEAVPLYLRADIDNDSNVLRDFYKNVLGLENPVVILN
RIKKNSLLKINGFLIHLRGTTGFSASQLKVQNAVEFSL
PHHMEDYVKKLENYEKHIIAERGSTKNSQIKITEWDGI
SKEKNLQLYDMFINKMENTIYKFRPANQVSNLKENREV
FNSLAVEDQCSVLNQVLMLFVCKPVTANLSLIKGSKNA
GNMALSKIISNMRSAYLIHQSVTGLFEQKIDLLKVSSQ
KD [SEQ ID NO. 34]
MAD2028 66 DHKP01000031.1 Bacillales gut MANKLFIGLDVGSDSVGWAATDENFHLYRLKGKTAWGA GTTTG GCATTGT
bacterium meta- RIFSEASDAKGRRGFRVAGRRLARRKERIRLLNTLFDP AGAGC AAGACAA
genome LLKEKDPTFLLRLENSAIQNDDPNKPAQAVTDCLLFAN AGTGT CACTGCT
KQEEKGFYKRYPTIWHLRKALMDNEDCAFSDIRFLYLA TGTCT ACGTTCA
IHHIIKYRGNFLRDGEIKIGQFDYSVFDKLNETLSVLF TATAT AATAAGC
DLQSEDEDSQEGHFVGLPKSQYEAFITTANDRNLPKQT AGCTC ATATTGC
KKTKLLSMFEKDEESKSFLEMFCTLCAGGEFSTKKLNK GAAAA TACAAGG
KGEETFDDTKISFNASYDQNEPNYQEILGDAFDLVDIA C TTCTCCC
KAVFDYCDLSDILNGNDNLSNAFVELYDSHKSQLSALK [SEQ TCGGAGA
AICKQIDNQSNLKGDASVYVKLFNDPNDKSNYPAFTHN ID NO. ATGACCA
KTLVDKRCDIHTFDKYVIDTVLPYEPLLMGQDATNWQM 38] TTAGGTC
LKSLAEQDRLLQTIALRSTSVIPMQLHQKELKIILKNA ACTTAGA
ISRNVKGIAEIEEKILKLFQYKIPYYCGPLTTKSAYSN TAGCCGG
VVFKNNEYRPLKPWDYEEAIDWDETKKKFMEGLTNKCT TTCTTCT
YLKDKNVLPKQSILYQDFDAWNKLNNLKVNGSKPSLKE GGCTA
LKDLFSFVSQRPKTTMKDIQRHFKSDTNSKDKDVVVSG [SEQ ID
WNPEDYICCSSRASFGKNGVFDLNNPDSSDPKDLSKCE NO. 39]
RMIFLKTIYADSPKDADVAILKEFPDLTNDQKSLLKTI
KCKEWSPLSKEFLELRYADKYGEIRESIINLLRSGEGN
LMQILAKYDYQERIDAYNADSFQTKSKSQIVSDLIEEM
PPKMRRPVIQAVRIVHEVVKVAKKEPDQISIEVTRENN
NKEKKQQLTKKAKSRSAQIQTFLKNLVKIDTFEEKRVD
EVLEELKKYSDRSINGKHLYLYFLQNGKDAYTGKPINI
DDVLSGNKYDTDHVIPQSKMKDDSIDNLVLVERSINQH
RSNEYPLPESIRKNPANVAFWSKLKKAGMMSEKKFNNL
TRANPLTEEELSAFVAAQINVVNRSNIVIRDVLKVLYP
NAKLIFSKAQYPSQIRKELNIPKLRDLNDTHHAVDAYL
NIVSGVSLTERYGNLSFIKAAQKNENQTDYSLNMERYI
SSLIQTKEGEKTSLGKLIDQTSRRHDFLLTYRFSYQDS
AFYNQTIYKKNAGLIPVHEKLPPERYGGYNSMSTEVNC
VVTIKGKKERRYLVGVPHLLLEKGNKVADINKEIANSV
PHKENETIAVSLKDIIQLDSMVKKDGLVYLCTTQNKDL
VKLKPFGPIFLSRESEVYLSNLNKFVEKYPNIADGNEN
YSLKTNRYGEKSIDFLQEKTGNVLKELVDLSNQKRFDY
CPMICKLRTIDYRKGVEGKTLTEQLILIRSFVGVFTRK
SEALSNGSNFRKARGLVLQDGLVLCSDSITGLYHTERK
L [SEQ ID NO. 37]
MAD2029 66 DBKT01000013.1 Bacillales gut MADKLFIGLDVGSESVGWAATDENFHLYRLKGKTAWGA GTTTG GCATTGT
bacterium meta- RIFSEANDAKTRRGFRVAGRRLARRKERIRLLNTLFDP AGAGC AAGACAA
genome LLKKDPAFLLRLENSAIQNDDPNKPIQAIADCPLLVNK AGTGT CACTGCT
QEEKDYYKRYPTIWHLRKALMENDDHAFSDIRFLYLAI TGTCT ACGTTCA
HHIIKYRGNFLREGDIKIGQFDYSIFDKLNETLAVLFD TATAT AATAAGC
LQNEDGENEEGRFIGLPKSQYEAFITCANDRNLPKQPK AGCTC ATATTGC
KAKLLSMFEKTEESKAFLEMFCTLCSGGEFSTKKLNAK GAAAA TACAAGG
GEETYQDAKISFNSSYDENEGAYQEILGDFFDLVDIAK C TTCTCCA
AVFDYCDLSDILNGNDNLSSAFVELYDSHKSQLSALKS [SEQ TTGGAGA
ICKRIDNQNGFIGEKSIYVKLFNDPNDKSNYPAFTNNK ID NO. ATGACCA
TLVDKRCDIHTFDKYVKETILPYESSLTGRDAVNWQML 41] TTAGGTC
KSLAEQDRLLQTIALRSTSVIPMQLHQKELKIILKNAV GCTTAGA
SRNIKGVAEIEEKILKLFQYKIPYYCGPLTTKSDYSNV TAGCCAG
VFKNNEYRPLKPWDYEEAIDWDGTKQKFMEGLTNKCTY TTCTTCT
LKDKNVLPKQSVLYQDFDTWNKLNNLKVNGNKPSLEDL GGCTA
NDLFSFVSQRSKTTMRDIQRYLKSKTNSKENDVVVSGW [SEQ ID
NSEDYICCSSRASFNKNGIFNLNNSEVLKECERIIFLK NO. 42]
TIYTDSPKDADAAVLKEFPDLTNNQKTLLKTIKCKEWS
PLSKEFLELRYSDKYGElRQSIIDLLRNGEGNLMQILA
KYDYQEVIDACNAASFQTKSKSQIVSDLIEEMPPKMRR
PVIQAVRIVQEVAKVAKKEPDEISIEVTRENNDKEKKQ
QLTKKAKSRSTQIQNFLKNLVKIDASEKKQANEVLEEL
KKYSDQSINGKHLYLYFLQNGKDAYTGKPINIDDVLSG
NKYDTDHIIPQSKMKDDSIDNLVLVEREINQHRSNEYP
LPESIRKNPANVAFWRKLKKAGMMSEKKFNNLTRSNPL
TEEELGAFVAAQINVVNRSNVVIRDVLKILYPNAKLIF
SKAQYPSQIRKELNIPKLRDLNDTHHAVDAYLNIVSGV
TLTDRYGNMRFIKASQDEEKHSLNMERYISSLIQTKEG
QRTELGELIDQTSRRHDFLLTYRFSYQDSAFYKQTIYK
KNAGLIPAHDNLPPERYGGYDSMSTEVNCVATIIGKKT
TRYLVGVPHLLIKKAKDGIDVNDELIKLVPHKENEVVK
VDLNTTLQLDCTVKKDGFMYLCTSNNIALVKLKPFSPI
FLSRESEIYLSNLMKYVEKYPNISDENSEYEFKINREN
VDPIKFTEKQSIEVVQDLIIKAKQDRFSYCSMISKLRD
INAEEMIHSKSLTEQLKIIKSLIGVFTRKSEILSDKNN
FRKSRGAILQEDLFLCSDSITGLYHTERKL [SEQ ID
NO. 40]
MAD2030 66 DBLD01000015.1 Bacillales gut MEQNTKKLFIGLDVGTDSVGWAATDEYFNLYRLKGKTA GTTTG GCATTGT
bacterium meta- WGARLFLDAANAKDRRQHRVSGRRLARRKERIRLLNAL AGAGC AAGACAA
genome FDPLLKKVDPTFLLRLESSTLQNDDPNKDQRAVSDALL AGTGT CACTGCA
FGNKKHEKAYYAAFPTIWHLRKALIENDDKAFSDIRYL TGTCT CGTTCAA
YLAIHHIIKYRGNFLRQGEIKIGEFDFSCFDKLNQFFD TAAAT ATAAGCA
IYFSKEDEEEVEFIGLPNENYQRFIDCAADKNLGKGKK AGCTC GATTGCT
KGDLLKLMSFSEDEKPFCEMFCSLCAGLAFSTKKLNKK GAAAA ACAAGGT
DETVFEDIKVEFNGKFDDKQEEIKSVLGDAYDLVELAK C TCCCGTA
FIFDYCDLKDILGASTNRLSEAFAGIYDSHKEELKALK [SEQ AGGGAAT
GICREIDRSLGNESKNSLYREVFNDKGIPNNYAAFIHH ID NO. GACCATC
ETNSSRCGIADFNNYVLQKIEPLENLLSKQNYKNWIQL 44] TGGTCAC
KQLASQGRLLQTIAIRSTSIIPMQLHLKDLKLILANAE ATGAATA
KRDIPGIKDIKEKILLLFQFKVPYYCGPLTDRSQYSNV GCCCCCG
VLKAGTREKITPWNFADQVDLEETKKKFMEGLTNKCTY GCAACGG
LKDCNVLPRQSLMFQEYDAWNKLNNLSINGNKPSPEEM TGGCTG
NALFDFASKRRKTTMSDIKKFEKRATMSKENDVTVSGW [SEQ ID
NENDFIDLSSFVSLSGFFDLGEIHSADYMACEEAILLK NO. 45]
TIFTDAPQDADPIIAEKFPNLKPNQLAALKKMSCKGWA
TLSREFLTLKAVDADGEVMNETLLGLMKEGKGNLMQLL
HSSLYNFQDVIDSHNRAVFGDKSPKQIANDLIEEMPPQ
MRRPVIQALRIVREVSKVAKKQPDVISIEVTRESNDKK
KKEEWSKKATDRKKQIDLFLKNLKKTEDVKQTESELDG
QAINDIDSIRGKHLYLYFLQNGKDAYTGLPIDINDVLN
GTKYDTDHIIPQSLMKDDSIDNLVLVNREKNQHKSNEF
PLPRDIQTKANIERWRALKKAGGMSEKKFNNLTRTTPL
TEEELSAFVAAQINVVNRSNVVIRDVLKILYPNAKLIF
SKAQYPSQIRRDLEIPKLRDLNDTHHAVDAFLNIVSGV
ELTKQFGRMDVIKAAAKGDKDHSLNMTRYLERLLKKVD
ENKNETMTELGNHVFVTSQRHDFLLTYRFDYQDSAFYN
ATIYSPDKNLIPMHDGMDPERYGGYSSLNIEYNCIATI
KGKKKTTRYLLGVPHLLALKFKNDGIDITSDLIKLVPH
KGDEEVSIDWKNPIPLRITVKKDGVEYLLAPFNAQVME
LKPVSPVFLPREAAEYLARLKKAVDQKKQFIYQNSAEI
FQSKDKNNALQFGPEQSKNVALKIYALADAKKYDYCAM
ISKLRDAALRAEMLDSLSSEALFKQYNDLISLLSQLTR
RSKKISSKYFSKSRGALLQDGLKIVSKSITGLYETERN
L [SEQ ID NO. 43]
MAD2031 141 CACVOG010000001.1 uncultured Cattle MNYILGLDIGIASVGWAAVALDANDEPCKILDLNARIF ATTGT TTGTAAT
Seleno- rumen EAAEQPKTGASLAAPRREARGSRRRTRRRRHRMERLRH ACCAT AACCTAT
monadaceae LFAREELISAENIAALFEAPADVYRLRAEGLSRRLDEG AGCGA TTTACCT
bacterium EWARVLYHIAKRRGFKSNRKGAASDADEGKVLEAVKEN GTTAA CGCTATG
EALLKNYKTVGEMMFRDEKFQTAKRNKGGSYTFCVSRG ATTAG GCACAAT
MLAEEIGELFAAQREQGNPHASETFETAYSKIFADQRS GGAAT TTGTTAT
FDDGPDANSRSPYAGNQIEKMIGTCSLETDPPEKRAAK TACAA TACATGG
ASYSFMRFSLLQKINHLRLKDAKGEERPLTDEERAAVE C ACATTAT
ALAWKSPSLTYGAIRKALPLPDELRFTDLYYRWDKKPE [SEQ ACTAAAC
EIEKKKLPFAAPYHEIRKALDKREKGRIQSLTPDALDA ID NO. ATTTCCT
VGYAFTVFKNDAKIEAALSAAGIDGEDAVALMAAGLTF 47] AAAAAAG
RGFGHISVKACRKLIPHLEKGMTYDKACKEAGYDLQKT CAACGAA
GGEKTKLLSGNLDEIREIPNPVVRRAIAQTVKVVNAVI AAACGTG
RRYGSPVAVNVELAREMGRTFQERRDMMKSMEDNNAEN CTGGCAG
EKRKEELKGYGVVHPSGLDIVKLKLYKEQGGVCAYSLA CAA
AMPIEKVLKDHDYAEVDHILPYSRSFDDSYANKVLVLS [SEQ ID
KENRDKGNRTPMEYMANMPGRRHDFITWVKSAVRNPRK NO. 48]
RDNLLLEKFGEDKEAAWKERHLTDTKYIGSFIANLLRD
HLEFAPWLNGKKKQHVLAVNGAVTDYTRKRLGIRKIRE
DGDLHHAVDAAVIATVTQGNIQKLTDYSKQIERAFVKN
RDGRYVNPDTGEVLKKDEWIVQRSRHFPEPWPGFRHEL
EARVSDHPKEMIESLRLPTYTPEEIDGLKPPFVSRMPT
RKVRGAAHLETVVSPRLKDEGMIVKKVSLDALKLTKDK
DAIENYYAPESDHLLYEALLHRLQAFGGDGEKAFAESF
HKPKADGTPGPVVKKVKIAEKSTLSVPVHHGRGLAANG
GMVRVDVFFIPEGKDRGYYLVPVYTSDVVRGELPMRAV
VQGKSYAEWKLMREEDFIFSLYPNDLVYIEHEKGVKVK
IQKKLREISTLPREKTMTSGLFYYRTMGIAVASIHIYA
PDGVYVQESLGVKTLKEFKKWTIDILGGEPHPVQKEKR
QDFASVKRDPHAAKSTSSG [SEQ ID NO. 46]
MAD2032 141 CACVWE010000020.1 uncultured Cattle MKYIIGLDMGITSVGFATMMLDDKDEPCRIIRMGSRIF GTTGT ATTGTAT
Rumino- rumen EAAEHPKDGSSLAAPRRINRGMRRRLRRKSHRKERIKD AGTTC CATACCA
coccus LIIKNELMTADEISAIYSTGKQLSDIYQIRAEALDRKL CCTAA AGAACAA
sp. NTEEFVRLLIHLSQRRGFKSNRKVDAKEKGSDAGKLLS TTATT TTAGGTT
AVNSNKELMIEKNYRTIGEMLYKDEKFSEYKRNKADDY CTTGG ACTATGA
SNTFARSEYEDEIRQIFSAQQEHGNPYATDELKESYLD TATGG TAAGGTA
IYLSQRSFDEGPGGSSPYGGNQIEKMIGNCTLEPEEKR TATAA GTATACC
AAKATFSFEYFNLLSKVNSIKIVSSSGKRALNNDERQS T GCAAAGC
VIRLAFAKNAISYTSLRKELNMEYSERFNISYSQSDKS [SEQ TCTAACA
IEEIEKKTKFTYLTAYHTFKKAYGSVFVEWSADKKNSL ID NO. CCTCATC
AYALTAYKNDTKIIEYLTQKGFDAAETDIALTLPSFSK 50] TTCGGAT
WGNLSEKALNNIIPYLEQGMLYHDACTAAGYNFKADDT GAGGTGT
DKRMYLPAHEKEAPELDDITNPVVRRAISQTIKVINAL TATCT
IREMGESPCFVNIELARELSKNKAERSKIEKGQKENQV [SEQ ID
RNDRIMERLRNEFGLLSPTGQDLIKLKLWEEQDGICPY NO. 51]
SLKPIKIEKLFDVGYTDIDHIIPYSLSFDDTYNNKVLV
MSSENRQKGNRIPMQYLEGKRQDDFWLWVDNSNLSRRK
KQNLTKETLSEDDLSGFKKRNLQDTQYLSRFMMNYLKK
YLALAPNTTGRKNTIQAVNGAVTSYLRKRWGIQKVREN
GDTHHAVDAVVISCVTAGMTKRVSEYAKYKETEFQNPQ
TGEFFDVDIRTGEVINRFPLPYARFRNELLMRCSENPS
RILHEMPLPTYAADEKVAPIFVSRMPKHKVKGSAHKET
IRRAFEEDGKKYTVSKVPLTDLKLKNGEIENYYNPESD
GLLYNALKEQUAFGGDAAKAFEQPFYKPKSDGSEGPLV
KKVKLINKATLTVPVLNNTAVADNGSMVRVDVFFVEGE
GYYLVPIYVADTVKKELPNKAIIANKPYEEWKEMREEN
FVFSLYPNDLIKISSRKDMKFNLVNKESTLAPNCQSKE
ALVYYKGSDISTAAVTAINHDNTYKLRGLGVKTLLKIE
KYQVDVLGNVFKVGKEKRVRFK [SEQ ID NO. 49]
MAD2033 141 DCJP01000021.1 un- Feces MKNTLYGIGLDIGVASVGWAVVGLNGTGEPVGLHRLGV GTTGT TTATACC
cultivated of RIFDKAEQPKTGESLAAPRRMARGMRRRLRRKALRRAD AGTTC ATACCAA
Faecali three- VYALLERSGLSTREALAQMFEAGGLEDIYALRTRALDE CCTAA GAACTGT
bacterium weeks PVGKAEFSRILLHLAQRRGFKSNRRTASDGEDGRLLAA CAGTT TATGGTT
sp. old VNENRRRMAQGGWRTVGEMLYRHEAFALRKRNKADEYL CTTGG GCTATGA
elephant STVGRDMVAEEASLLFQRQRELGCAWATPELQAEYLSI TATGG TAAGGTC
LLRQRSFDEGPGGNSPYGGNQVEKMVGRCTFEPDEPRA TATAA TTAGCAC
AKAAYSFEYFSLLQKLNHIRLAENGETRPLTQPQRQQL T CGTAAAG
LSLAHKTPDVSLARIRKELALPETVQFNGVRCRANETL [SEQ CTCTGAC
EESEKKEKFACLPAYHKMRKALDGVVKGRISSLSISQR ID NO. GCCTCGC
DAAATALSLYKNEDTLRAKLTEAGFQAPEIDALAGLTG 53] TTTCAGC
FSKFGHLSLKACRKLIPHLEQGLTYDQACSAAGYDFKG GGGGCGT
HGAGERAFTLPAAAPEMEQITSPVVRRAVAQTIKVVNG CATCTTT
IIREMDASPAWVRIELARELSKTFGERQEMDRSMRENA TTTGCCC
AQNERLMQELRDTFHLLSPTGQDLVKYRLWKEQDGVCA AAAAGAC
YSLRRLDVERLFEPGYVDVDHIVPYSLSFDDRRSNKVL ACGGATA
VLSSENRQKGNRLPLQYLQGKRREDFIVWTNSSVRDYR TTTTT
KRQNLLREKFSGDEAEGFRQRNLQDTQHMARFLYNYIS [SEQ ID
DHLAFAQSEALGKKRVFAVSGAVTSHLRKRWGLSKVRA NO. 54]
DGDLHHALDAAVIACTTDGMIRRISGYYGHIEGEYLQD
ADGAGSQHARTKERFPAPWPRFRDELIVRLSEQPGEHL
LDINPAFYCEYGTEHICPVFVSRMPRRKVTGPGHKETI
KGAAAADEGLLTVRKALTELKLDKDGEIKDYYMPSSDT
LLYEALKAQLRRFGGDGKKAFAEPFYKPKADGTPGPLV
RKVKTIEKATLTVPVHGGAASNDTMVRVDVFLVPGDGY
YWVPVYVADTLKPELPNRAVVAFKPYSEWKEMREEDFI
FSLYPNDLVYVEHKSGLKFTLQNADSTLEKTWVPKASF
AYFVGGDISTAAISLRTHDNAYGLRGLGIKTLKVLKKY
QVDVLGNISPVHRETRQRFR [SEQ ID NO. 52]
MAD2034 141 CACXAV010000001.1 uncultured Cattle MAYGIGLDIGIASVGFATVALNEQDEPCGILRMGSRIF GTTGT TTATACC
Clostri- rumen DAAEHPKNGASLAAPRREARSARRRLRRHRHRLERIRN AGTTC ATACCAA
diales LLVESCLISQDGLGSLFEGRLEDIYALRTRALDERLTD CCTAA GAACTGT
bacterium AELCRVLIHLAQRRGFRSNRKADAADKEAGKLLKAVSE CGGTT TGGGTTA
NDRRMEENGYRTVGEMLYKDPLFAEHRRNKGEAYLSTV CTTGG CTACAAT
TRTAVEQEARLVLSTQREKGNAAITEDFVEKYLDILLS TATGG AAGGTAG
QRPFDVGPGGNSPYGGNMIEKMIGRCTFEPDELRAPKA TATAA TAAACCG
SYSFEYFQLLQKVNHIRLLRDGRSEPLSEEQRRAIIDL T AAAAGCT
ALASADVTFAKIRKALSLPDSVRFNDVYYRESAEEAEK [SEQ CTGACGT
KKKLGCMDAYHEMRKALDKVAKGRICAIPVEQRNAIAY ID NO. CTTGTTT
VLTVHKTDERILTELQNINLERSDIDQLMQMKGFSKFG 56] GCGCAGG
HLSIKACDRIIPYLEQGMTYSDACTAAGYAFRGHEGGE ACGTCAT
HSLYLPAQTPEMDEITSPVVRRAVSQTIKVVNALIREQ CTTTATA
GESPTFVNIELAREMSKDFAERNDIRRENEKNAKANEA TCAGACG
VMNELRRTFGLVNPSGQDLVKYKLFLEQGGVCPYTQRP GATG
MEPGRLFEAGYADVDHIVPYSISFDDRYCNKVLTFASV [SEQ ID
NRKEKGNRLPLQFLKGERRESFIVYVKANVRDYRKQRL NO. 57]
LLKETVTEEDRKGFRDRNLQDTKHMAAFLHSYINDHLQ
FAPFQTDRKRHVTAVNGAVTAYLRKRWGIRKVRAEGDL
HHASDALVIACTTPGMIQRLSRYAELREAEYMQTEDGA
VRFDPATGEVLEKFPYPWPCFRQEWTARVSDDPQAMLQ
DMKLTDYRGLPLEQVKPVFVSRMPKHKVTGAAHKDTVK
SAKALDRGVVLVKRALTDLKLKDGEIENYYDPASDRLL
YEALKERLIAFGGDAQKAFAEPFHKPKRDGTPGPLVKK
VKLMEKSSLTVPVHDGKGVADNDSMVRIDVFFVAGEGY
YFVPIYVADTVKPELPNRAVVANKPYAEWKEMKDEDFL
FSLYPSDLMRVTQKKGIKLSLINKESTLKKEEMAQSIL
LYYVKGSISTGSITAENHDRTYAINSLGIKTLEKLEKY
QVDVLGNVSPVGKEKRLTFC [SEQ ID NO. 55]
MAD2035 141 CADATZ010000012.1 uncultured Cattle MLPYAIGLDIGIASVGWAVVGLDTNERPFCILGMGSRI GTTGT TTATACC
Chloroflexi rumen FDKAEQPKTGASLALPRREARSLRRRLRRHRHRNERIR AGTCC ATTCCAG
bacterium NLLLREKIISESELQDLFSGTLSDIYQLRVEALDRKLD CCTGA AAACTAT
DKEFSRVLIHIAQRRGFKSNRKNAAASQEDGKLLSAVT TGGTT TATGGTC
ENQQRMNDKGYRTVSEMLLRDDKFKDHKRNKGGEYLTT TCTGG ACTACAA
VTRTMVEDEVHKIFSAQRTHGNLKADNQLESEYLEILL AATGG TAAGGTA
SQRSFDEGPGGDSPYGGSQIEKMIGKCTFFPEEKRAAK TATAA TTAGACC
ATYTFEYFNLLEKINHIRLVSKDNLPEPLSDFQRRSLI T GTAGAGC
ELAYKVENLTYDRIRKELHISPELKFNTIRYESDDLPE [SEQ ACTAACA
NEKKQKLNCLKAYHElRKALDKLGKGTINTLSKEQLNT ID NO. CCCCATT
IGTVLSMYKTSEIIKNKMEQIPAEIVDKLDEEGINFSK 59] TGGGGTG
FGHLSIKACELIIPGLEKGLNYNDACEEAGLNFKAHNN TTATCTC
EEKSFLLHPTEDDYADITSPVVKRAASQTIKVINAIIR TTTAAAC
KQGCSPTYINIEVARELSKDFYERDKINKRNEANRAEN TGTCCAA
ERSLEQIRKEYGKSNASGLDLVKFKLYQKQDGVCAYSQ AATTTAG
KQISFERLFEPNYVEVDHIIPYSKCFDDRESNKVLVFA TATTGCA
KENREKGNRLPLEYLDGKKRESFIVWVNSKVKDYRKKQ ATTATTG
NLLKESLSEEEEKQFKERNLQDTKTVSKFLMNYINDNL A
IFSSSNKRKKHVTAVSGGVTSYMRKRWGISKVREDGDQ [SEQ ID
HHAVDALVIVCTTDGMIQQVSKYVEYKECQYIQTDAGS NO. 60]
LAVDPYTGEVLRSFPYPWARFHEDAVTWTEKIFVSRMP
MRKVTGPAHKETIKSPKALGEGLLIVRKPLTELKLKNG
EIENYYKPEADLLLYNGLKERLMEFGGDAKKAFAEPFP
KPGNPQKIVKKVRLTEKSTLNVPVLKGEGRADNDSMVR
VDVFLKDGKYYLVPIYVADTLKPELPNKACIAHKPYDE
WATMDDGDFLFSLYPNDLIYIKHKKGIKLTKINKNSTL
ADSIEGKEFFLFYKTMGISSAVLTCTNHDNTYYIESLG
VKTLESLEKCVVGVLGEIHKVRKEKRTGFSGN [SEQ
ID NO. 58]
MAD2036 141 CADAWQ010000026.1 Ruminoe- Cattle MLPYAIGLDIGISSVGWASVALDEEDKPCGIIGMGSRI GTTAT TTATACC
coccacea rumen FDAAEQPKTGDSLAAPRRAARSARRRLRRRRHRNERIR AGTTC ATACCAA
bacterium ALMLREGLLSEAELAALFDGRLEDICALRVRALDEAVT CCTGT GAACGAA
NDELARILLHLSQRRGFRSNRKTAATQEDGELLAAVSA TCGTT GCAGGTT
NRALMQERGYRTVAEMLLRDERYRDHRRNKGGAYIATV CTTGG ACTATGA
GRDMVEDEVRQIFAAQRALGSTAASETLETAYLEILLS TATGG TAAGGTA
QRSFDAGPGEPSPYAGGQIERMIGRCTFEPDEPRAARA TATAA GTATACC
TYSFEYFSLLEAVNHIRLTEAGESVPLTKEQREKLIAL T GCAGAGC
AHRTADLSYAKIRKELGVPESQRFNMVTYGKTDSADEA [SEQ TCCAACG
EKKTKLKQLRAYHQMRAAFEKAAKGSFVLLTKEQRNAV ID NO. CCTCGCT
GQTLSIYKTSDNIRPRLREAGLTEAEIDVAEGLSFSKF 62] TTTGCGG
GHLSVKACDKIIPFLEQGMKYSEACVAAGYAFRGHEGQ GGCGTTG
DKQRLLPPLDNDAKDTITSPVVLRAVSQTIKVVNAIIR TCTCT
ERGGSPTFINIELAREMAKDFSERSQIKREQDSNRARN [SEQ ID
ERMMERIKTEYGKSSPTGLDLVKLKLYEEQAGVCAYSL NO. 63]
KQMSLEHLFDPNYAEIDHIIPYSISFDDGYKNKVLVLA
KENRDKGNRLPLEYLNGKRREDFIVWVNSSVRDWRKKQ
NLLKEHVTPEDEAKFKERNLQDTKTASRFLLNYIADNL
AFAPFQTERKKRVTAVNGSVTAYLRKRWGIAKVRANGD
LHHAVDALVIACTTDGLIQKVSRYACYQENRYSEAGGV
IVDSATGEVVAQFPEPWPRFRHELEARLSDDPARAVLG
LGLAHYMTGEIRPRPLFVSRMPRRKVTGAAHKETVKSP
RALDEGQLVTKTPLSALKLGKDGEIPGYYKPESDRLLY
EALKARLRQFGGDGKKAFAEPFHKPKHDGTPGPVVTKV
KLCEPATLSVPVHGGLGAANNDSMVRIDVFHVEGDGYY
FVPIYIADTLKLELPNKACVKIKKISEWKHMKPQDFMF
SLYPNDLFRIVSKKGITLNLVSKESTLPTSVNVSDTLL
YFVSAGIASACLTCRNHDNTYQIESLGIKTLEKLEKYT
VDVLGNVHRVEKEPRMSFSQKGD [SEQ ID NO.
61]
MAD2037 141 DGSQ01000028.1 Clostri- low MLPYGIGLDIGITSVGWATVALDENDRPYGIIGMGSRI GTTAT TTATACC
diales methane FDAAEQPKTGESLAAPRRAARSARRRLRRHRHRNERIR AGTTC ATACCAA
bacterium producing ALILRENLLSEGQLLHLYDGQLSDVYSLRVKALDERVS CCTGA GAACTAT
sheep NEEFARILIHISQRRGFKSNRKGASSKEDSELLAAISA TAGTT GAGGTTG
NQVRMQQQGYRTVAEMYLKDPIYQEHRRNKGGNYIATV CTTGG CTATAAT
SRAMVEDEVHQIFTGQRACGNPAATKELEEAYVEILLS TATGG AAGGTAG
QRSFDDGPGDGSPYAGSQIERMIGKCQLEKEAGEPRAA TATAA TAAACCG
KATYSFEYFSLLAAINNISIISNGQLSPLTKEQREMLI T CAGAGCT
ALAHKTSELNYARIRKELGLSEAQRFNTVSYGKMEIAE [SEQ CTAACGC
AEKKTKFEHLKAYHKMRREFERIAKGHFASITIEQRNA ID NO. CTCACAT
IGDVLSKYKTDAKIRPALREAGLTELDIDAAEALNFSK 65] TTGTGGG
FGHISIKACKKIIPWLEQGMKYSEACNAAGYNFKGHDG GCGTTAT
QEKSHLLPPLDEESRNVITSPVALRAISQTIKVVNAII CTCT
RERGCSPTFINIELAREMSKDFYERIEIKKEQDGNRAK
NERMMERIRTEYGKASPTGQDLVKFKLYEEQGGVCAYS [SEQ ID
LKQMSLAHLFEPDYAEVDHIVPYSISFDDGYKNKVLVL NO. 66]
AKENRDKGNRLPLQYLQGKRREDFIAWVNSCVRDYKKR
QRLLKESISEDDLRAFKERNLQDTKTASRFLLNYISDH
LEFTQFATERKKHVTAVNGSVTAYLRKRWGITKIRENG
DLHHAVDALVIACTTDGMIQQVSRFAQHRENQYSLAED
SRFIIDPETGEVIKEFPYPWPRFRQELEARLSSNPGLA
VRDRGFLLYMAESIPVHPLFVSRMPRRKVTGAAHKETI
KSGKAQKDGLLIVKKPLTDLKLDKEGEIANYYNPMSDR
LLYEALKKRLTAFNGDGKKAFADPFYKPKSDGTQGPLV
NKVKLCEPSTLNVSVIGGKGVAENDSMVRIDVFRVEGD
GYYFVPVYVADTVKPELPNKACVANKPYTDWKEMRESD
FLFSLYPNDLLKVTHKKALILTKAQKDSDLPDCKETKS
EMLYFVSASISTASLACRTHDNSYRINSLGIKTLEALE
KYTVDVLGEYHPVRRETRQTFTGRESSGHSGIS [SEQ
ID NO. 64]
MAD2038 141 CACWHR010000008.1 Rumino- Cattle MRPYGIGLDIGISSVGWAAIALDHQDSPCGILDMGARI GTTGT TTATACC
coccaceae rumen FDAAENPKDGASLAAPRREKRSQRRRLRRHRHRNERIR AGTTC ATACCAA
bacterium RMLLKEGLLTEAELTGLFDGALEDIYALRTRALDEALT CCTGA GAACGAT
KQEFARVLLHLSQRRGFRSNRRATAAQEDGKLLDAVSE TCGTT CAGGTTG
NAKRMADCGYRTVGEMLCRDATFAKHKRNKGGEYLTTV CTTGG CTACAAT
SRAMIEDEVKLVFASQRRLGSAFASEALEQGYLDILLS TATGG AAGGTAG
QRSFDEGPGGNSPYGGAQIERMIGKCTFYPEEPRAARA TATAA TAAACCG
CYSFEYFSLLQKVNHIRLQKDGESTPLTSEQRLQLIEL T AAGAGCT
ANKTENLDYARIRRALQIPDAYRFNTVSYRIESDPAAA [SEQ CTAACGC
EKKEKFQYLRAYHTMRKAIDGASKGRFALLSQEQRDQI ID NO. CCCGTTT
GTVLTLYKSQERISEKLTEAGIEPCDIAALESVSGFSK 68] CTTTACG
TGHISLRACKELIPYLEQGMNYNEACAAAGIEFHGHSG GGGCGTT
TERTVVLHPTPDDLADITSPVVRRAVAQTVKVINAVIR ATCTCT
RYGSPVFVNIELARELAKDFTERKKLEKDNKTNRAENE [SEQ ID
RLMRRIREEYGKMNPTGLDLVKLRLYEEQAGVCPYSQK NO. 69]
QMSLQRLFEPNYAEVDHIIPYSISFDDSRRNKVLVLAE
ENRNKGNRLPLQYLTGERRDNFIVWVNSSVRDYRKKQK
LLKPTVTDEDKQQFKERNLQDTKTMSRFLMNYINDHLQ
FGVSAKERKKRVTAVNGIVTSYLRKRWGITKIRGDGDL
HHAVDALVIACATDGMIRQITRYAQYRECRYMQTDTGS
AAIDEATGEVLRIFPYPWEHFRKELEARLSSDPARAVN
ALRLPFYLDSGEPLPKPLFVSRMPRRKVSGAAHKDTVK
SPKAMAEGKVIVRRALTDLKLKNGEIENYFDPGSDRLL
YDALKARLAAFGGDGAKAFREPFYKPRHDGTPGPLVKK
VKLCEPTTLNVAVHGGKGVADNDSMVRIDVFRVEGDGY
YFVPIYIADTLKPVLPNKACVAFKPYSEWRTMDDRDFI
FSLYPNDLIRVTHKSALKLSRVSKESTLPESIESKTAL
LYYVSAGISGAAVSCRNHDNSYEIKSMGIKTLEKLEKY
TVDVLGEYHKVEKERRMPFTGKRS [SEQ ID NO.
67]
MAD2039 141 CACZLL010000017.1 Rumino- Cattle MRPYAIGLDIGITSVGWATVALDADESPCGIIGLGSRI GTTAT TTATACC
coccaceae rumen FDAAEQPKTGESLAAPRRAARGSRRRLRRHRHRNERIR AGTTC ATACCAA
bacterium SLMLEERLISQDELETLFDGRLEDIYALRVKALDEIVS CCTGA GAACTAT
RTDFARILLHISQRRGFKSNRKNPTTKEDGVLLAAVNE TAGTT TTAGGTT
NKQRMSEHGYRTVGEMFLLDETFKDHKRNKGGNYITTV CTTGG ACTATGA
ARDMVADEVRAIFSAQRELGASFASEEFEERYLEILLS TATGG TAAGGTT
QRSFDEGPGGNSPYGGSQIERMVGRCTFFPDEPRAAKA TATAA TAGTACA
TYSFEYFTLLQKVNHIRIVENGVASKLTDEQRRIIIEL T CCTTAGA
AHTTKDVSYAKIRKVLKLSDKQLFNIRYSDNSPAEDSE [SEQ GCTCTGA
KKEKLGIMKAYHQMRSAIDRVSKGRFAMMPRAQRNAIG ID NO. CGCCTCG
TALSLYKTSDKIRKYLTDAGLDEIDINSADSIGSFSKF 71] CTTTTGC
GHISVKACDMLIPFLEQGMNYNEACAAAGLNFKGHDAG GAGGCGT
EKSKLLHPKEEDYEDITSPVVRRAIAQTIKVINAIIRR TATCTCT
EGCSPTFINIELAREMAKDFRERNRIKKENDDNRAKNE TTATATT
RLLERIRTEYGKNNPTGLDLVKLRLYEEQSGVCMYSLK GCCAAAA
QMSLEKLFEPNYAEVDHIVPYSISFDDSRKNKVLVLTE ATGCAAA
ENRNKGNRLPLQYLKGRRREDFIVWVNNNVKDYRKRRL TATATCG
LLKEELTAEDESGFKERNLQDTKTMSRFLLNYIADNLE TACAATG
FAESTRGRKKKVTAVNGAVTAYMRKRWGITKIREDGDC GTGGC
HHAVDAVVIACTTDAMIRQVSRYAQFRECEYMQTESGS [SEQ ID
VAVDTGTGEVLRTFPYPWPDFRKELEARLANDPAKVIN NO. 72]
DLHLPFYMSAGRPLPEPVFVSRMPRRKVTGAAHKDTIK
SARELDNGYLIVKRPLTDLKLKNGEIENYYNPQSDKCL
YDALKNALIEHGGDAKKAFAGEFRKPKRDGTPGPIVKK
VKLLEPTTMCVPVHGGKGAADNDSMVRVDVFLSGGKYY
LVPIYVADTLKPELPNKAVTRGKKYSEWLEMADEDFIF
SLYPNDLICATSKNGITLSVCRKDSTLPPTVESKSFML
YYRGTDISTGSISCITHDNAYKLRGLGVKTLEKLEKYT
VDVLGEYHKVGKEVRQPFNIKRRKACPSEML [SEQ
ID NO. 70]
MAD2040 141 DHKF01000115.1 Clostri- Feces MHRYAIGLDIGITSVGWAAIALDAEENPCGMLDFGSRI GTTGT TTATACC
diales FTGAEHPKTGASLAAPRREARGARRRLRRHRHRNERIR AGTTC ATACCAA
bacterium RLMVSGGLISQEQLESLFAGQLEDIYALRTRALDEQVA CCTGA GAACTGC
UBA4701 REELARIMLHLSQRRGFRSNRKGGADAEDGKLLEAVGD TGGTT TCAGGTT
NKRRMDEKGYRTAGEMFFKDEAFAAHKRNKGGNYIATV CTTGG ACTATGA
TRAMTEDEVHRIFAAQRGFGAEYANEKLEAAYLDILLS TATGG TAAGGTA
QRSFDEGPGGDSPYGGSQIERMIGTCAFEPDQPRAAKA TATAA GTAAACC
AYSFEYFSLLEKLNHIRLVSGGKSEPLTDAQRKKLIEL T GAAGAGC
AHKQDTLSYAKIRKELELNEAVRFNSVRYTDDATFEEQ [SEQ TCTAATG
EKKEKIVCMKAYHAMRKAVDKNAKGRFAYLTIPQRNEI ID NO. CCCCGTC
GRVLSTYKTSAKIEPALAAAGIEPCDIAALEGLSFSKF 74] TCGCACG
GHLSIKACDKLIPFLEKAMNYNDACAAAGYDFRGHSRD GGGCATT
GRQMYLPPLGGDCTEITSPVVRRAVSQTIKVINAIIRR ATCTCTA
YGTSPVYVNIELAREMSKDFAERNKIKKQNDDNRSKNE ACAGCGA
KIKEQVAEYKHGAATGLDIVKMKLFNEQGGICAYSQRQ AAAGGCA
MSLERLFDPNYAEVDHIVPYSISFDDRYKNKVLVLTEE AA
NRNKGNRLPLQYLTGERRDRFIVWVNNSVRDFQKRKLL [SEQ ID
LKEALTPEEENDWKERNLQDTKFVSSFLLNYINDNLLF NO. 75]
APSVRRKKRVTAVNGAVTDYMRKRWGISKVREDGDRHH
AVDAVVIACTNDALIQKVSRYESWHERHYMPTENGSIL
VDPATGEIKQTFPYPWAMFRKELEARLSNDPSRAVADL
KLPFYMDADAPPVKPLFVSRMPTRKVTGAAHKDTVKSA
RALADGLAIVRRPLTALKLDKDGEIAGYYNKDSDRLLY
DALKARLTEYGGNAAKAFAEPFYKPKSDGTPGPVVNKV
KLTEPTTLSVPVQDGTGIADNDSMVRIDVFRVVGDGYY
FVPVYVADTLKQELPDRAVVAFKAHSEWKVMSDGDFVF
SLYPNDLVKVTRKKDVILKRSFDNSTLPETIASNECLL
YYAGADISTGAISCVTNDNAYSIRGLGIKTLVSMEKYT
VDILGEYHPVRKEERQRFNTKR [SEQ ID NO. 73]
Example 3 Vector Cloning, MADZYME Library Construction and PCR The MADzyme coding sequences were cloned into a pUC57 vector with T7-promoter sequence attached to the 5′-end of the coding sequence and a T7-terminator sequence attached to the 3′-end of the coding sequence.
First, Q5 Hot Start 2× master mix reagent (NEB, Ipswich, MA) was used to amplify the MADzyme sequences cloned in the pUC57 vector. The forward primer 5′-TTGGGTAACGCCAGGGTTTT [SEQ ID No. 172] and reverse primer 5′-TGTGTGGAATTGTGAGCGGA [SEQ ID No. 173] amplified the sequences flanking the MADzyme in the pUC57 vector including the T7-promoter and T7-terminator components at the 5′- and 3′-end of the MADzymes, respectively. 1 μM primers were used in a 10 μL PCR reaction using 3.3 μL boiled cell samples as templates in 96 well PCR plates. The PCR conditions shown in Table 2 were used:
TABLE 2
STEP TEMPERATURE TIME
DENATURATION 98° C. 30 SEC
30 CYCLES 98° C. 10 SEC
66° C. 30 SEC
72° C. 3 MIN
FINAL EXTENSION 72° C. 2 MIN
HOLD 12° C.
Example 4 gRNA Construction Several functional gRNAs associated with each MADzyme was designed by truncating the 5′ region, the 3′ region and the repeat/anti-repeat duplex (see Table 3).
TABLE 3
gRNA
name sgRNAv1 sgRNAv2 sgRNAv3 sgRNAv4 sgRNAv5
sgM GTTTTAGAGCTATGC GTTTTAGAGCTATGC GTTTTAGAGCTATGC GTTTTAGAGCT NONE
2015 TGTTTTGAATGCTTC TGTTTTGAATGCTTC TGTTAACAACATAGC ATGCAAACATA
CAAAACGAAATGTTG GTAGCATTCAAAACA AAGTTAAAATAAGGC GCAAGTTAAAA
GTAGCATTCAAAACA ACATAGCAAGTTAAA TTTGTCCGTTCTCAA TAAGGCTTTGT
ACATAGCAAGTTAAA ATAAGGCTTTGTCCG CTTTTAGTGACGCTG CCGTTCTCAAC
ATAAGGCTTTGTCCG TTCTCAACTTTTAGT TTTCGGCG TTTTAGTGACG
TTCTCAACTTTTAGT GACGCTGTTTCGGCG [SEQ ID NO. 78] CTGTTTCGGCG
GACGCTGTTTCGGCG [SEQ ID NO. 77] [SEQ ID NO.
[SEQ ID NO. 76] 79]
sgM GTTTTAGAGTCATGT GTTTTAGAGTCATGT GTTTTAGAGTCATGT NONE NONE
2016 TGTTTAGAATGGTAC TGTAAAAACAACATA TGTAAAAACAACATA
CAAAACATCTTTTGG GCAAGTTAAAATAAG GCAAGTTAAAATAAG
GACTATTCTAAACAA GTTTTAACCGTAATC CGTAATCAACTGTAA
CATAGCAAGTTAAAA AACTGTAAAGTGGCG AGTGGCGCTGTTTCG
TAAGGTTTTAACCGT CTGTTTCGGCGC GCGC
AATCAACTGTAAAGT [SEQ ID NO. 81] [SEQ ID NO. 82]
GGCGCTGTTTCGGCG
C [SEQ ID NO.
80]
sgM GTTTTAGAGCTGTGC GTTTTAGAGCTGTGC GTTTTAGAGCTGTGC GTTTTAGAGCT NONE
2017 TGTTTCGAATGGTTC TGTTTCGAAAAATCG TGTAAAAACAACACA GTGCAAACACA
CAAAACGAAATGTTG AAACAACACAGCGAG GCGAGTTAAAATAAG GCGAGTTAAAA
GAACTATTCGAAACA TTAAAATAAGGCTTT GCTTTGTCCGTACAC TAAGGCTTTGT
ACACAGCGAGTTAAA GTCCGTACACAACTT AACTTGTAAAAGGGG CCGTACACAAC
ATAAGGCTTTGTCCG GTAAAAGGGGCACCC CACCCGATTCGGGTG TTGTAAAAGGG
TACACAACTTGTAAA GATTCGGGTGC C GCACCCGATTC
AGGGGCACCCGATTC [SEQ ID NO. 84] [SEQ ID NO. 85] GGGTGC
GGGTGCA [SEQ ID NO.
[SEQ ID NO. 83] 86]
sgM GTTTTAGAGCTGTGT GTTTTAGAGCTGTGT GTTTTAGAGCTGTGT NONE NONE
2019 TGTTTCGAATGGTTC TGTAAAAACAATACA TGTAAAAACAATACA
CAAAACGGTTTGAAA GCAAAGTTAAAATAA GCAAGTTAAAATAAG
CCATTCGAAACAATA GGCTAGTCCGTATAC GCTAGTCCGTATACA
CAGCAAAGTTAAAAT AACGTGAAAACACGT ACGTGAAAACACGTG
AAGGCTAGTCCGTAT GGCACCGATTCGGTG GCACCGATTCGGTGC
ACAACGTGAAAACAC C [SEQ ID NO. 89
GTGGCACCGATTCGG [SEQ ID NO. 88]
TGC [SEQ ID NO.
87]
sgM GTTTGCTAGTTATGT GTTTGCTAGTTATGT GTTTGCTAGTTATGT NONE NONE
2020 TATTTATAGTATTAA TATAAAAATAACATA TATAAAAATAACATA
GCAAACTGTAAATAA ACGAGTGCAAATAAG ACGAGTGCAAATAAG
CATAACGAGTGCAAA CGTTTCGCGAAAATT CGTTTCGCGAAAATT
TAAGCGTTTCGCGAA TACAGTGGCCCTGCT TACAGTGGCCCTGCT
AATTTACAGTGGCCC GTGGGGCCTTTTTTA GTGGGGCC
TGCTGTGGGGCCTTT TTTATCAAA [SEQ ID NO. 92]
TTTATTTATCAAA [SEQ ID NO. 91]
[SEQ ID NO. 90]
sgM GTTTGAGAGCCTTGT NONE NONE NONE NONE
2021 AAAACCGTATATCTC
TCAAGCGAAAGATAA
TGTTTTACAAGGCGA
GTTCAAATAAGGATT
TATCCGAAATCGCTT
GCGTGCATTGGCACC
ATCTATCTTTTAAGA
CTTTCTTTGAAAGTC
TT [SEQ ID NO.
93]
sgM GTTTGAGAGTCTTGT GTTTGAGAGTCTTGT GTTTGAGAGTCTTGT GTTTGAGAGTC NONE
2022 TAATTCTTAAAGGTG AAAAACAAGACGAGT AAAAACAAGACGAGT TTGTTAATTCA
TAAAACGAGAATTAA GCAAATAAGGTTTAT GCAAATAAGGTTTAT AAAGAATTAAC
CAAGACGAGTGCAAA CCGGAATCGTCAATA CCGGAATCGTCAATA AAGACGAGTGC
TAAGGTTTATCCGGA TGACCTGCATTGTGC TGACCTGCATTGTGC AAATAAGGTTT
ATCGTCAATATGACC AGAATCTTTAAAATC AG [SEQ ID NO. ATCCGGAATCG
TGCATTGTGCAGAAT ATATGATTTCATATG 96] TCAATATGACC
CTTTAAAATCATATG GTTTTA [SEQ ID TGCATTGTGCA
ATTTCATATGGTTTT NO. 95] GAATCTTTAAA
A [SEQ ID NO. ATCATATGATT
94] TCATATGGTTT
TA [SEQ ID
NO. 97]
sgM GTTTGAGAGTAGTGT NONE NONE NONE NONE
2023 AAATCCATAGGGGTC
TCAAACGAAAAGACC
CCTATGGATTTACAT
TGCGAGTTCAAATAA
AAGTTTACTCAAATC
GTTGGCTTGACCAAC
CGCACAGCGTGTGCT
TAAAGATCTCTTCAG
TGAGGTC [SEQ ID
NO. 98]
sgM GTTTGAGAGTAGTGT NONE NONE NONE NONE
2024 AAATCCAGAGGGCTC
CAAAACGAGCCCTCT
GGATTTACACTACGA
GTTCAAATAAAAATT
ATTTCAAATCGCCGC
TATGTCGGCCGCACA
GTGTGTGCATTAAGA
AAAGTCCGAAAGGGC
[SEQ ID NO. 99]
sgM GTTTGAGAGTAGTGT GTTTGAGAGTAGTGT GTTTGAGAGTAGTGT GTTTGAGAGTA NONE
2025 AAATTTATAGGGTAG AAAAATACACTACGA AAAAATACACTACGA GTGTAAATTTA
TAAAACAAATTTTAC GTTCAAATAAAAATT GTTCAAATAAAAATT TAGGAAAACCT
TACCCTATAAATTTA ATTTCAAATCGTACT ATTTCAAATCGTACT ATAAATTTACA
CACTACGAGTTCAAA TTTTAGTACCTTCAC TTTTAGTACCTTCAC CTACGAGTTCA
TAAAAATTATTTCAA AAGTGTTGTGAATAT AAGTGTTGTGAA AATAAAAATTA
ATCGTACTTTTTAGT TAACTCACCTTCGGG [SEQ ID NO. TTTCAAATCGT
ACCTTCACAAGTGTT TGAG [SEQ ID 102] ACTTTTTAGTA
GTGAATATTAACTCA NO. 101] CCTTCACAAGT
CCTTCGGGTGAG GTTGTGAATAT
[SEQ ID NO. TAACTCACCTT
100] CGGGTGAG
[SEQ ID NO.
103]
sgM GTTTGAGAGTAGTGT NONE NONE NONE NONE
2026 AATTTCATATGGTAG
TCAAACGACTACCAT
ATGAGATTACACTAC
ACGGTTCAAATAAAG
AATGTTCGAAACCGC
CCTTTGGGGCCCGCT
TGTTGCGGATTTACA
GACTTGATATCAAGT
CTG [SEQ ID NO.
104]
sgM GTTTGAGAGTAATGT GTTTGAGAGTAATGT GTTTGAGAGTAATGT GTTTGAGAGTA NONE
2027 AAATTCATAGGATGG AAAAATACATTACAA AAAAATACATTACAA ATGTAAATTCA
TAAAACGAAATTTAC GTTCAAATAAAAATT GTTCAAATAAAAATT TAAAAGTGAGT
CATCCAGTGAGTTTA TATTCAACCCGTTCT TATTCAACCCGTTCT TTACATTACAA
CATTACAAGTTCAAA TCGGAACCTCCACCG TCGGAACCTCCACCG GTTCAAATAAA
TAAAAATTTATTCAA TGTGGAACATTAAGG TGTGGA [SEQ ID AATTTATTCAA
CCCGTTCTTCGGAAC TCTGCTTTGCAGGCC NO. 107] CCCGTTCTTCG
CTCCACCGTGTGGAA [SEQ ID NO. GAACCTCCACC
C [SEQ ID NO. 106] GTGTGGAACAT
105] TAAG [SEQ
ID NO. 108]
sgM GTTTGAGAGCAGTGT NONE NONE NONE NONE
2028 TGTCTTATATAGCTC
GAAAACGCATTGTAA
GACAACACTGCTACG
TTCAAATAAGCATAT
TGCTACAAGGTTCTC
CCTCGGAGAATGACC
ATTAGGTCACTTAGA
TAGCCGGTTCTTCTG
GCTA [SEQ ID
NO. 109]
sgM GTTTGAGAGCAGTGT GTTTGAGAGCAGTGT GTTTGAGAGCAGTGT GTTTGAGAGCA NONE
2029 TGTCTTATATAGCTC AAAAACACTGCTACG AAAAACACTGCTACG GTGTTGTCAAA
GAAAACGCATTGTAA TTCAAATAAGCATAT TTCAAATAAGCATAT AGACAACACTG
GACAACACTGCTACG TGCTACAAGGTTCTC TGCTACAAGGTTCTC CTACGTTCAAA
TTCAAATAAGCATAT CATTGGAGAATGACC CATTGGAGAATGACC TAAGCATATTG
TGCTACAAGGTTCTC ATTAGGTCGCTTAGA ATTAGGTC [SEQ CTACAAGGTTC
CATTGGAGAATGACC TAGCCAGTTCTTCTG ID NO. 112] TCCATTGGAGA
ATTAGGTCGCTTAGA GCTA [SEQ ID ATGACCATTAG
TAGCCAGTTCTTCTG NO. 111] GTCGCTTAGAT
GCTA [SEQ ID AGCCAGTTCTT
NO. 110] CTGGCTA
[SEQ ID NO.
113]
sgM GTTTGAGAGCAGTGT NONE NONE NONE NONE
2030 TGTCTTAAATAGCTC
GAAAACGCATTGTAA
GACAACACTGCACGT
TCAAATAAGCAGATT
GCTACAAGGTTCCCG
TAAGGGAATGACCAT
CTGGTCACATGAATA
GCCCCCGGCAACGGT
GGCTG [SEQ ID
NO. 114]
sgM ATTGTACCATAGCGA NONE NONE NONE NONE
2031 GTTAAATTAGGGAAT
TACAACGAAATTGTA
ATAACCTATTTTACC
TCGCTATGGCACAAT
TTGTTATTACATGGA
CATTATACTAAACAT
TTCCTAAAAAAGCAA
CGAAAAACGTGCT
[SEQ ID NO.
115]
sgM GTTGTAGTTCCCTAA GTTGTAGTTCCCTAA GTTGTAGTTCCCTAA GTTGTAGTTCC NONE
2032 TTATTCTTGGTATGG TTATTCTTGGTAAAA TTATTCTTGGTAAAA CTAATTATTCT
TATAATGAAAATTGT ACCAAGAACAATTAG ACCAAGAACAATTAG TGGTATGGTAA
ATCATACCAAGAACA GTTACTATGATAAGG GTTACTATGATAAGG AAATATCATAC
ATTAGGTTACTATGA TAGTATACCGCAAAG TAGTATACCGCAAAG CAAGAACAATA
TAAGGTAGTATACCG CTCTAACACCTCATC CTCTAACACCTCATC GGTTACTATGA
CAAAGCTCTAACACC TTCGGATGAGGTGTT TTCGGATGAG [SEQ TAAGGTAGTAT
TCATCTTCGGATGAG A [SEQ ID NO. ID NO. 118] ACCGCAAAGCT
GTGTTATCT [SEQ 117] CTAACACCTCA
ID NO. 116] TCTTCGGATGA
GGTGTTATCT
[SEQ ID NO.
119]
sgM GTTGTAGTTCCCTAA GTTGTAGTTCCCTAA GTTGTAGTTCCCTAA GTTGTAGTTCC NONE
2033 CAGTTCTTGGTATGG CAGTTCTAAAAAGAA CAGTTCTAAAAAGAA CTAACAGTAAA
TATAATAAAAATTAT CTGTTATGGTTGCTA CTGTTATGGTTGCTA AACTGTTATGG
ACCATACCAAGAACT TGATAAGGTCTTAGC TGATAAGGTCTTAGC TTGCTATGATA
GTTATGGTTGCTATG ACCGTAAAGCTCTGA ACCGTAAAGCTCTGA AGGTCTTAGCA
ATAAGGTCTTAGCAC CGCCTCGCTTTCAGC CGCCTCGCTTTCAGC CCGTAAAGCTC
CGTAAAGCTCTGACG GGGGCGTCA [SEQ GGGG [SEQ ID TGACGCCTCGC
CCTCGCTTTCAGCGG ID NO. 121] NO. 122] TTTCAGCGGGG
GGCGTCATCTTTTTT CGTCA
GCCCAAAAGACACGG [SEQ ID NO.
ATATTTTT [SEQ 123]
ID NO. 120]
sgM GTTGTAGTTCCCTAA GTTGTAGTTCCCTAA GTTGTAGTTCCCTAA GTTGTAGTTCC NONE
2034 CGGTTCTTGGTATGG CGGTACTGTTGGGTT CGGTACTGTTGGGTT CTAACGGTTCT
TATAATGAATTATAC ACTACAATAAGGTAG ACTACAATAAGGTAG TGAAAACAAGA
CATACCAAGAACTGT TAAACCGAAAAGCTC TAAACCGAAAAGCTC ACTGTTGGGTT
TGGGTTACTACAATA TGACGTCTTGTTTGC TGACGTCTTGTTTGC ACTACAATAAG
AGGTAGTAAACCGAA GCAGGACGTCATCTT GCAGGACGTCATCTT GTAGTAAACCG
AAGCTCTGACGTCTT TATATCAGACGGATG T [SEQ ID NO. AAAAGCTCTGA
GTTTGCGCAGGACGT [SEQ ID NO. 126] CGTCTTGTTTG
CATCTTTATATCAGA 125] CGCAGGACGTC
CGGATG [SEQ ID ATCTTTATATC
NO. 124] AGACGGATG
[SEQ ID NO.
127]
sgM GTTGTAGTCCCCTGA NONE NONE NONE NONE
2035 TGGTTTCTGGAATGG
TATAATGAAATTATA
CCATTCCAGAAACTA
TTATGGTCACTACAA
TAAGGTATTAGACCG
TAGAGCACTAACACC
CCATTTGGGGTGTTA
TCTCTTTAAACTGTC
CAAAATTTAGTATTG
CAATTATTGA [SEQ
ID NO. 128]
sgM GTTATAGTTCCCTGT NONE NONE NONE NONE
2036 TCGTTCTTGGTATGG
TATAATGAAATTATA
CCATACCAAGAACGA
AGCAGGTTACTATGA
TAAGGTAGTATACCG
CAGAGCTCCAACGCC
TCGCTTTTGCGGGGC
GTTGTCTCT [SEQ
ID NO. 128]
sgM GTTATAGTTCCCTGA NONE NONE NONE NONE
2037 TAGTTCTTGGTATGG
TATAATGAAATTATA
CCATACCAAGAACTA
TGAGGTTGCTATAAT
AAGGTAGTAAACCGC
AGAGCTCTAACGCCT
CACATTTGTGGGGCG
TTATCTCT [SEQ
ID NO. 129]
sgM GTTGTAGTTCCCTGA NONE NONE NONE NONE
2038 TCGTTCTTGGTATGG
TATAATGAAATTATA
CCATACCAAGAACGA
TCAGGTTGCTACAAT
AAGGTAGTAAACCGA
AGAGCTCTAACGCCC
CGTTTCTTTACGGGG
CGTTATCTCT [SEQ
ID NO. 130]
sgM GTTATAGTTCCCTGA GTTATAGTTCCCTGA GTTATAGTTCCCTGA GTTATAGTTCC GTTATAGTTC
2039 TAGTTCTTGGTATGG TAGTTCTTGGTATGG TAGTTCTTAACCAAG CTGATAGTTCT CCTGATAGTT
TATAATGAATTATAC TATAATGAATTATAC AACTATTTAGGTTAC TGCAAGAACTA CTTGCAAGAA
CATACCAAGAACTAT CATACCAAGAACTAT TATGATAAGGTTTAG TTTAGGTTACT CTATTTAGGT
TTAGGTTACTATGAT TTAGGTTACTATGAT TACACCTTAGAGCTC ATGATAAGGTT TACTATGATA
AAGGTTTAGTACACC AAGGTTTAGTACACC TGACGCCTCGCTTTT TAGTACACCTT AGGTTTAGTA
TTAGAGCTCTGACGC TTAGAGCTCTGACGC GCGAGGCGTTATCTC AGAGCTCTGAC CACCTTAGAG
CTCGCTTTTGCGAGG CTCGCTTTTGCGAGG T [SEQ ID NO. GCCTCGCTTTT CTCTGACGCC
CGTTATCTCTTTATA CGTTATCTCT [SEQ 133] GCGAGGCGTTA AAAAGGCGTT
TTGCCAAAAATGCAA ID NO. 132] TCTCT ATCTCT
ATATATCGTACAATG [SEQ ID [SEQ ID
GTGGC [SEQ ID NO. 134] NO. 135]
NO. 131]
sgM GTTGTAGTTCCCTGA NONE GTTGTAGTTCCCTGA GTTGTAGTTCC NONE
2040 TGGTTCTTGGTATGG TGGTTCTTGAAAAAG CTGATGGTTCT
TATAATAAATTATAC AACTGCTCAGGTTAC TGAAAAAGAAC
CATACCAAGAACTGC TATGATAAGGTAGTA TGCTCAGGTTA
TCAGGTTACTATGAT AACCGAAGAGCTCTA CTATGATAAGG
AAGGTAGTAAACCGA ATGCCCCGTCTCGCA TAGTAAACCGA
AGAGCTCTAATGCCC CGGGGCATTATCTCT AGAGCTCTAAT
CGTCTCGCACGGGGC [SEQ ID NO. GCCAAAGGGCA
ATTATCTCT [SEQ 137] TTATCTCT
ID NO. 136] [SEQ ID NO.
138]
To find the optimal gRNA length, different lengths of spacer, repeat:anti-repeat duplex and 3′ end of the tracrRNA were included. These gRNAs were then synthesized as a single stranded DNA downstream of the T7 promoter (see Table 4). These sgRNAs were amplified using two primers (5′-AAACCCCTCCGTTTAGAGAG [SEQ ID NO. 174] and 5′-AAGCTAATACGACTCACTATAGGCCAGTC [SEQ ID NO. 175]) and 1 uL of 10 uM diluted single stranded DNA as a template in 25 uL PCR reactions for each sgRNA according to the conditions of Table 5.
TABLE 4
Name Sequence
sg M201 AAACCCCTCCGTTTAGAGAGGGGTTATGCTAGTTAGCGCCGAAACAGCGCCACTTTACAGTTGATTACGGT
6v1 TAAAACCTTATTTTAACTTGCTATGTTGTTTAGAATAGTCCCAAAAGATGTTTTGGTACCATTCTAAACAA
CATGACTCTAAAACCCAGTAACATTACTGACTGGCCTATAGTGAGTCGTATTA [SEQ ID NO. 139]
sg M201 AAACCCCTCCGTTTAGAGAGGGGTTATGCTAGTTAGCGCCGAAACAGCGCCACTTTACAGTTGATTACGGT
6v2 TAAAACCTTATTTTAACTTGCTATGTTGTTTTTACAACATGACTCTAAAACCCAGTAACATTACTGACTGG
CCTATAGTGAGTCGTATTA [SEQ ID NO. 140]
sg M201 AAACCCCTCCGTTTAGAGAGGGGTTATGCTAGTTAGCGCCGAAACAGCGCCACTTTACAGTTGATTACGCT
6v3 TATTTTAACTTGCTATGTTGTTTTTACAACATGACTCTAAAACCCAGTAACATTACTGACTGGCCTATAGT
GAGTCGTATTA [SEQ ID NO. 141]
sg M201 AAACCCCTCCGTTTAGAGAGGGGTTATGCTAGTTAGCACCGAATCGGTGCCACGTGTTTTCACGTTGTATA
9v1 CGGACTAGCCTTATTTTAACTTTGCTGTATTGTTTCGAATGGTTTCAAACCGTTTTGGAACCATTCGAAAC
AACACAGCTCTAAAACCCAGTAACATTACTGACTGGCCTATAGTGAGTCGTATTA [SEQ ID NO.
142]
sg M201 AAACCCCTCCGTTTAGAGAGGGGTTATGCTAGTTAGCACCGAATCGGTGCCACGTGTTTTCACGTTGTATA
9v2 CGGACTAGCCTTATTTTAACTTTGCTGTATTGTTTTTACAACACAGCTCTAAAACCCAGTAACATTACTGA
CTGGCCTATAGTGAGTCGTATTA [SEQ ID NO. 143]
sg M201 AAACCCCTCCGTTTAGAGAGGGGTTATGCTAGTTAGCACCGAATCGGTGCCACGTGTTTTCACGTTGTATA
9v3 CGGACTAGCCTTATTTTAACTTGCTGTATTGTTTTTACAACACAGCTCTAAAACCCAGTAACATTACTGAC
TGGCCTATAGTGAGTCGTATTA [SEQ ID NO. 144]
sg M202 AAACCCCTCCGTTTAGAGAGGGGTTATGCTAGTTATTTGATAAATAAAAAAGGCCCCACAGCAGGGCCACT
0v1 GTAAATTTTCGCGAAACGCTTATTTGCACTCGTTATGTTATTTACAGTTTGCTTAATACTATAAATAACAT
AACTAGCAAACCCAGTAACATTACTGACTGGCCTATAGTGAGTCGTATTA [SEQ ID NO. 145]
sg M202 AAACCCCTCCGTTTAGAGAGGGGTTATGCTAGTTATTTGATAAATAAAAAAGGCCCCACAGCAGGGCCACT
0v2 GTAAATTTTCGCGAAACGCTTATTTGCACTCGTTATGTTATTTTTATAACATAACTAGCAAACCCAGTAAC
ATTACTGACTGGCCTATAGTGAGTCGTATTA [SEQ ID NO. 146]
sg M202 AAACCCCTCCGTTTAGAGAGGGGTTATGCTAGTTAGGCCCCACAGCAGGGCCACTGTAAATTTTCGCGAAA
0v3 CGCTTATTTGCACTCGTTATGTTATTTTTATAACATAACTAGCAAACCCAGTAACATTACTGACTGGCCTA
TAGTGAGTCGTATTA [SEQ ID NO. 147]
sg M202 AAACCCCTCCGTTTAGAGAGGGGTTATGCTAGTTATAAAACCATATGAAATCATATGATTTTAAAGATTCT
2v1 GCACAATGCAGGTCATATTGACGATTCCGGATAAACCTTATTTGCACTCGTCTTGTTAATTCTTTTGAATT
AACAAGACTCTCAAACCCAGTAACATTACTGACTGGCCTATAGTGAGTCGTATTA [SEQ ID NO.
148]
sg M202 AAACCCCTCCGTTTAGAGAGGGGTTATGCTAGTTATAAAACCATATGAAATCATATGATTTTAAAGATTCT
2v2 GCACAATGCAGGTCATATTGACGATTCCGGATAAACCTTATTTGCACTCGTCTTGTTTTTACAAGACTCTC
AAACCCAGTAACATTACTGACTGGCCTATAGTGAGTCGTATTA [SEQ ID NO. 149]
sg M202 AAACCCCTCCGTTTAGAGAGGGGTTATGCTAGTTACTGCACAATGCAGGTCATATTGACGATTCCGGATAA
2v3 ACCTTATTTGCACTCGTCTTGTTTTTACAAGACTCTCAAACCCAGTAACATTACTGACTGGCCTATAGTGA
GTCGTATTA [SEQ ID NO. 150]
sg M202 AAACCCCTCCGTTTAGAGAGGGGTTATGCTAGCTCACCCGAAGGTGAGTTAATATTCACAACACTTGTGAA
5v1 GGTACTAAAAAGTACGATTTGAAATAATTTTTATTTGAACTCGTAGTGTAAATTTATAGGTTTTCCTATAA
ATTTACACTACTCTCAAACCCAGTAACATTACTGACTGGCCTATAGTGAGTCGTATTA [SEQ ID NO.
151]
sg M202 AAACCCCTCCGTTTAGAGAGGGGTTATGCTAGTTACTCACCCGAAGGTGAGTTAATATTCACAACACTTGT
5v2 GAAGGTACTAAAAAGTACGATTTGAAATAATTTTTATTTGAACTCGTAGTGTATTTTTACACTACTCTCAA
ACCCAGTAACATTACTGACTGGCCTATAGTGAGTCGTATTA [SEQ ID NO. 152]
sg M202 AAACCCCTCCGTTTAGAGAGGGGTTATGCTAGTTATTCACAACACTTGTGAAGGTACTAAAAAGTACGATT
5v3 TGAAATAATTTTTATTTGAACTCGTAGTGTATTTTTACACTACTCTCAAACCCAGTAACATTACTGACTGG
CCTATAGTGAGTCGTATTA [SEQ ID NO. 153]
sg M202 AAACCCCTCCGTTTAGAGAGGGGTTATGCTAGTTAGGCCTGCAAAGCAGACCTTAATGTTCCACACGGTGG
7v1 AGGTTCCGAAGAACGGGTTGAATAAATTTTTATTTGAACTTGTAATGTAAACTCACTTTTATGAATTTACA
TTACTCTCAAACCCAGTAACATTACTGACTGGCCTATAGTGAGTCGTATTA [SEQ ID NO. 154]
sg M202 AAACCCCTCCGTTTAGAGAGGGGTTATGCTAGTTAGGCCTGCAAAGCAGACCTTAATGTTCCACACGGTGG
7v2 AGGTTCCGAAGAACGGGTTGAATAAATTTTTATTTGAACTTGTAATGTATTTTTACATTACTCTCAAACCC
AGTAACATTACTGACTGGCCTATAGTGAGTCGTATTA [SEQ ID NO. 155]
sg M202 AAACCCCTCCGTTTAGAGAGGGGTTATGCTAGTTATCCACACGGTGGAGGTTCCGAAGAACGGGTTGAATA
7v3 AATTTTTATTTGAACTTGTAATGTATTTTTACATTACTCTCAAACCCAGTAACATTACTGACTGGCCTATA
GTGAGTCGTATTA [SEQ ID NO. 156]
sg M202 AAACCCCTCCGTTTAGAGAGGGGTTATGCTAGTTATAGCCAGAAGAACTGGCTATCTAAGCGACCTAATGG
9v1 TCATTCTCCAATGGAGAACCTTGTAGCAATATGCTTATTTGAACGTAGCAGTGTTGTCTTTTGACAACACT
GCTCTCAAACCCAGTAACATTACTGACTGGCCTATAGTGAGTCGTATTA [SEQ ID NO. 157]
sg M202 AAACCCCTCCGTTTAGAGAGGGGTTATGCTAGTTATAGCCAGAAGAACTGGCTATCTAAGCGACCTAATGG
9v2 TCATTCTCCAATGGAGAACCTTGTAGCAATATGCTTATTTGAACGTAGCAGTGTTTTTACACTGCTCTCAA
ACCCAGTAACATTACTGACTGGCCTATAGTGAGTCGTATTA [SEQ ID NO. 158]
sg M202 AAACCCCTCCGTTTAGAGAGGGGTTATGCTAGTTAGACCTAATGGTCATTCTCCAATGGAGAACCTTGTAG
9v3 CAATATGCTTATTTGAACGTAGCAGTGTTTTTACACTGCTCTCAAACCCAGTAACATTACTGACTGGCCTA
TAGTGAGTCGTATTA [SEQ ID NO. 159]
sg M203 AAACCCCTCCGTTTAGAGAGGGGTTATGCTAGTTAAGATAACACCTCATCCGAAGATGAGGTGTTAGAGCT
2v1 TTGCGGTATACTACCTTATCATAGTAACCTAATTGTTCTTGGTATGATATTTTTACCATACCAAGAATAAT
TAGGGAACTACAACCCAGTAACATTACTGACTGGCCTATAGTGAGTCGTATTA [SEQ ID NO. 160]
sg M203 AAACCCCTCCGTTTAGAGAGGGGTTATGCTAGTTATAACACCTCATCCGAAGATGAGGTGTTAGAGCTTTG
2v2 CGGTATACTACCTTATCATAGTAACCTAATTGTTCTTGGTTTTTACCAAGAATAATTAGGGAACTACAACC
CAGTAACATTACTGACTGGCCTATAGTGAGTCGTATTA [SEQ ID NO. 161]
sg M203 AAACCCCTCCGTTTAGAGAGGGGTTATGCTAGTTACTCATCCGAAGATGAGGTGTTAGAGCTTTGCGGTAT
2v3 ACTACCTTATCATAGTAACCTAATTGTTCTTGGTTTTTACCAAGAATAATTAGGGAACTACAACCCAGTAA
CATTACTGACTGGCCTATAGTGAGTCGTATTA [SEQ ID NO. 162]
sg M203 AAACCCCTCCGTTTAGAGAGGGGTTATGCTAGTTATGACGCCCCGCTGAAAGCGAGGCGTCAGAGCTTTAC
3v1 GGTGCTAAGACCTTATCATAGCAACCATAACAGTTTTTACTGTTAGGGAACTACAACCCAGTAACATTACT
GACTGGCCTATAGTGAGTCGTATTA [SEQ ID NO. 163]
sg M203 AAACCCCTCCGTTTAGAGAGGGGTTATGCTAGTTATGACGCCCCGCTGAAAGCGAGGCGTCAGAGCTTTAC
3v2 GGTGCTAAGACCTTATCATAGCAACCATAACAGTTCTTTTTAGAACTGTTAGGGAACTACAACCCAGTAAC
ATTACTGACTGGCCTATAGTGAGTCGTATTA [SEQ ID NO. 164]
sg M203 AAACCCCTCCGTTTAGAGAGGGGTTATGCTAGTTACCCCGCTGAAAGCGAGGCGTCAGAGCTTTACGGTGC
3v3 TAAGACCTTATCATAGCAACCATAACAGTTCTTTTTAGAACTGTTAGGGAACTACAACCCAGTAACATTAC
TGACTGGCCTATAGTGAGTCGTATTA [SEQ ID NO. 165]
sg M203 AAACCCCTCCGTTTAGAGAGGGGTTATGCTAGTTACATCCGTCTGATATAAAGATGACGTCCTGCGCAAAC
4v1 AAGACGTCAGAGCTTTTCGGTTTACTACCTTATTGTAGTAACCCAACAGTTCTTGTTTTCAAGAACCGTTA
GGGAACTACAACCCAGTAACATTACTGACTGGCCTATAGTGAGTCGTATTA [SEQ ID NO. 166]
sg M203 AAACCCCTCCGTTTAGAGAGGGGTTATGCTAGTTACATCCGTCTGATATAAAGATGACGTCCTGCGCAAAC
4v2 AAGACGTCAGAGCTTTTCGGTTTACTACCTTATTGTAGTAACCCAACAGTACCGTTAGGGAACTACAACCC
AGTAACATTACTGACTGGCCTATAGTGAGTCGTATTA [SEQ ID NO. 167]
sg M203 AAACCCCTCCGTTTAGAGAGGGGTTATGCTAGTTAAAAGATGACGTCCTGCGCAAACAAGACGTCAGAGCT
4v3 TTTCGGTTTACTACCTTATTGTAGTAACCCAACAGTACCGTTAGGGAACTACAACCCAGTAACATTACTGA
CTGGCCTATAGTGAGTCGTATTA [SEQ ID NO. 168]
sg M203 AAACCCCTCCGTTTAGAGAGGGGTTATGCTAGTTAAGAGATAACGCCTCGCAAAAGCGAGGCGTCAGAGCT
9v1 CTAAGGTGTACTAAACCTTATCATAGTAACCTAAATAGTTCTTGCAAGAACTATCAGGGAACTATAACCCA
GTAACATTACTGACTGGCCTATAGTGAGTCGTATTA [SEQ ID NO. 169]
sg M203 AAACCCCTCCGTTTAGAGAGGGGTTATGCTAGTTAAGAGATAACGCCTTTTGGCGTCAGAGCTCTAAGGTG
9v2 TACTAAACCTTATCATAGTAACCTAAATAGTTCTTGCAAGAACTATCAGGGAACTATAACCCAGTAACATT
ACTGACTGGCCTATAGTGAGTCGTATTA [SEQ ID NO. 170]
sg M203 AAACCCCTCCGTTTAGAGAGGGGTTATGCTAGTTAAGAGATAACGCCTCGCAAAAGCGAGGCGTCAGAGCT
9v3 CTAAGGTGTACTAAACCTTATCATAGTAACCTAAATAGTTCTTGGTTAAGAACTATCAGGGAACTATAACC
CAGTAACATTACTGACTGGCCTATAGTGAGTCGTATTA [SEQ ID NO. 171]
TABLE 5
STEP TEMPERATURE TIME
DENATURATION 98° C. 30 SEC
12 CYCLES 98° C. 10 SEC
66° C. 30 SEC
72° C. 2 MIN
FINAL EXTENSION 72° C. 2 MIN
HOLD 12° C.
The target library was designed based on an assumption that the eight randomized NNNNNNNN [SEQ ID NO. 176] PAMs of these nucleases reside on the 3′ end of the target sequence (5′-CCAGTCAGTAATGTTACTGG [SEQ ID NO. 177]).
Example 5 In Vitro Transcription and Translation for Production of MAD Nucleases and gRNAs The MADZYMEs were tested for activity by in vitro transcription and translation (txtl). Both the gRNA plasmid and nuclease plasmid were included in each txtl reaction. A PURExpress® In Vitro Protein Synthesis Kit (NEB, Ipswich, Mass.) was used to produce MADzymes from the PCR-amplified MADZYME library and also to produce the gRNA libraries. In each well in a 96-well plate, the reagents listed in Table 6 were mixed to start the production of MADzymes and gRNAs:
TABLE 6
REAGENTS VOLUME (μl)
1 SolA (NEB kit) 10
2 SolB (NEB kit) 7.5
3 PCR amplified gRNA 0.4
4 Murine RNase inhibitor (NEB) 0.5
5 Water 3.0
6 PCR amplified T7 MADZYMEs 3.6
A master mix with all reagents was mixed on ice with the exception of the PCR-amplified T7-MADZYMEs to cover enough 96-well plates for the assay. After 21 μL of the master mix was distributed in each well in 96 well plates, 4 μL of the mixture of PCR amplified MADZYMEs and gRNA under the control of T7 promoter was added. The 96-well plates were sealed and incubated for 4 hrs at 37° C. in a thermal cycler. The plates were kept at room temperature until the target pool was added to perform the target depletion reaction.
After 4 hours incubation to allow production of the MADzymes and gRNAs, 4 μL of the target library pool (10 ng/μL) was added to the 10 μL aliquots of in vitro transcription/translation reaction mixture and allowed to deplete for 30 min, 3 hrs or overnight at 37° C. and 48° C. The target depletion reaction mixtures were diluted into PCR-grade water that contains RNAse A incubated for 5 min at room temperature. Proteinase K was then added and the mixtures were incubated for 5 min at 55° C. RNAseA/Proteinase K treated samples were purified with DNA purification kits and the purified DNA samples were then amplified and sequenced. The PCR conditions are shown in Table 7:
TABLE 7
STEP TEMPERATURE TIME
DENATURATION 98° C. 30 SEC
4 CYCLES 98° C. 10 SEC
66° C. 30 SEC
72° C. 20 SEC
12 CYCLES 98° C. 10 SEC
72° C. 20 SEC
FINAL EXTENSION 72° C. 2 MINUTES
HOLD 12° C.
Example 6 Measurement of Nicked Plasmid with Nickase RNP Complexes Proteins were produced in vitro under a PURExpress® In Vitro Protein Synthesis Kit (NEB, Ipswich, Mass.). Guide RNAs that target the target plasmid were also produced under a T7 promoter in the same mixture. The MADzyme Nickase or Nuclease and guide complexes (RNP complex) formed as they were produced in the in vitro transcription and translation reagent. Supercoiled plasmid target was diluted into the digestion buffer, then the RNP complex was added to the same digestion buffer to initiate the plasmid digestion. After incubation at 37° C. to allow digestion of the plasmid, the resulting mixtures were treated with RNAase and Proteinase K, then the target plasmid was purified with a PCR cleanup kit, and run on TAE-agarose gel to observe the formation of nicked or double stand cut plasmid. The results are shown in FIG. 7. Table 8 lists the identified MADzyme nickases, including the variations from the nuclease sequence in Table 1 and the amino acid sequence.
TABLE 8
MAD
zyme SEQ
Nickase ID
Name NO Amino Acid Sequence
MAD2016- 178 MKKDYVIGLDIGTNSVGWAVMTEDYQLVKKKMPIYGNTEKKKIKKNFWGVRLFEEGHTAEDRR
H851A LKRTARRIISRRRNRLRYLQAFFEEAMTDLDENFFARLQESFLVPEDKKWHRHPIFAKLEDEV
AYHETYPTIYHLRKKLADSSEQADLRLIYLALAHIVKYRGHFLIEGKLSTENISVKEQFQQFM
IIYNQTFVNGESRLVSAPLPESVLIEEELTEKASRTKKSEKVLQQFPQEKANGLFGQFLKLMV
GNKADFKKVFGLEEEAKITYASESYEEDLEGILAKVGDEYSDVFLAAKNVYDAVELSTILADS
DKKSHAKLSSSMIVRFTEHQEDLKKFKRFIRENCPDEYDNLFKNEQKDGYAGYIAHAGKVSQL
KFYQYVKKIIQDIAGAEYFLEKIAQENFLRKQRTFDNGVIPHQIHLAELQAIIHRQAAYYPFL
KENQEKIEQLVTFRIPYYVGPLSKGDASTFAWLKRQSEEPIRPWNLQETVDLDQSATAFIERM
TNFDTYLPSEKVLPKHSLLYEKFMVFNELTKISYTDDRGIKANFSGKEKEKIFDYLFKTRRKV
KKKDIIQFYRNEYNTEIVTLSGLEEDQFNASFSTYQDLLKCGLTRAELDHPDNAEKLEDIIKI
LTIFEDRQRIRTQLSTFKGQFSAEVLKKLERKHYTGWGRLSKKLINGIYDKESGKTILGYLIK
DDGVSKHYNRNFMQLINDSQLSFKNAIQKAQSSEHEETLSETVNELAGSPAIKKGIYQSLKIV
DELVAIMGYAPKRIVVEMARENQTTSTGKRRSIQRLKIVEKAMAEIGSNLLKEQPTTNEQLRD
TRLFLYYMQNGKDMYTGDELSLHRLSHYDIDAIIPQSFMKDDSLDNLVLVGSTENRGKSDDVP
SKEVVKDMKAYWEKLYAAGLISQRKFQRLTKGEQGGLTLEDKAHFIQRQLVETRQITKNVAGI
LDQRYNANSKEKKVQIITLKASLTSQFRSIFGLYKVREVNDYHHGQDAYLNCVVATTLLKVYP
NLAPEFVYGEYPKFQTFKENKATAKAIIYTNLLRFFTEDEPRFTKDGEILWSNSYLKTIKKEL
NYHQMNIVKKVEVQKGGFSKESIKPKGPSNKLIPVKNGLDPQKYGGFDSPIVAYTVLFTHEKG
KKPLIKQEILGITIMEKTRFEQNPILFLEEKGFLRPRVLMKLPKYTLYEFPEGRRRLLASAKE
AQKGNQMVLPEHLLTLLYHAKQCLLPNQSESLTYVEQHQPEFQEILERVVDFAEVHTLAKSKV
QQIVKLFEANQTADVKEIAASFIQLMQFNAMGAPSTFKFFQKDIERARYTSIKEIFDATIIYQ
STTGLYETRRKVVD
MAD2016- 179 MKKDYVIGLDIGTNSVGWAVMTEDYQLVKKKMPIYGNTEKKKIKKNFWGVRLFEEGHTAEDRR
N874A LKRTARRIISRRRNRLRYLQAFFEEAMTDLDENFFARLQESFLVPEDKKWHRHPIFAKLEDEV
AYHETYPTIYHLRKKLADSSEQADLRLIYLALAHIVKYRGHFLIEGKLSTENISVKEQFQQFM
IIYNQTFVNGESRLVSAPLPESVLIEEELTEKASRTKKSEKVLQQFPQEKANGLFGQFLKLMV
GNKADFKKVFGLEEEAKITYASESYEEDLEGILAKVGDEYSDVFLAAKNVYDAVELSTILADS
DKKSHAKLSSSMIVRFTEHQEDLKKFKRFIRENCPDEYDNLFKNEQKDGYAGYIAHAGKVSQL
KFYQYVKKIIQDIAGAEYFLEKIAQENFLRKQRTFDNGVIPHQIHLAELQAIIHRQAAYYPFL
KENQEKIEQLVTFRIPYYVGPLSKGDASTFAWLKRQSEEPIRPWNLQETVDLDQSATAFIERM
TNFDTYLPSEKVLPKHSLLYEKFMVFNELTKISYTDDRGIKANFSGKEKEKIFDYLFKTRRKV
KKKDIIQFYRNEYNTEIVTLSGLEEDQFNASFSTYQDLLKCGLTRAELDHPDNAEKLEDIIKI
LTIFEDRQRIRTQLSTFKGQFSAEVLKKLERKHYTGWGRLSKKLINGIYDKESGKTILGYLIK
DDGVSKHYNRNFMQLINDSQLSFKNAIQKAQSSEHEETLSETVNELAGSPAIKKGIYQSLKIV
DELVAIMGYAPKRIVVEMARENQTTSTGKRRSIQRLKIVEKAMAEIGSNLLKEQPTTNEQLRD
TRLFLYYMQNGKDMYTGDELSLHRLSHYDIDHIIPQSFMKDDSLDNLVLVGSTEARGKSDDVP
SKEVVKDMKAYWEKLYAAGLISQRKFQRLTKGEQGGLTLEDKAHFIQRQLVETRQITKNVAGI
LDQRYNANSKEKKVQIITLKASLTSQFRSIFGLYKVREVNDYHHGQDAYLNCVVATTLLKVYP
NLAPEFVYGEYPKFQTFKENKATAKAIIYTNLLRFFTEDEPRFTKDGEILWSNSYLKTIKKEL
NYHQMNIVKKVEVQKGGFSKESIKPKGPSNKLIPVKNGLDPQKYGGFDSPIVAYTVLFTHEKG
KKPLIKQEILGITIMEKTRFEQNPILFLEEKGFLRPRVLMKLPKYTLYEFPEGRRRLLASAKE
AQKGNQMVLPEHLLTLLYHAKQCLLPNQSESLTYVEQHQPEFQEILERVVDFAEVHTLAKSKV
QQIVKLFEANQTADVKEIAASFIQLMQFNAMGAPSTFKFFQKDIERARYTSIKEIFDATIIYQ
STTGLYETRRKVVD
MAD2032- 180 MKYIIGLDMGITSVGFATMMLDDKDEPCRIIRMGSRIFEAAEHPKDGSSLAAPRRINRGMRRR
H590A LRRKSHRKERIKDLIIKNELMTADEISAIYSTGKQLSDIYQIRAEALDRKLNTEEFVRLLIHL
SQRRGFKSNRKVDAKEKGSDAGKLLSAVNSNKELMIEKNYRTIGEMLYKDEKFSEYKRNKADD
YSNTFARSEYEDEIRQIFSAQQEHGNPYATDELKESYLDIYLSQRSFDEGPGGSSPYGGNQIE
KMIGNCTLEPEEKRAAKATFSFEYFNLLSKVNSIKIVSSSGKRALNNDERQSVIRLAFAKNAI
SYTSLRKELNMEYSERFNISYSQSDKSIEEIEKKTKFTYLTAYHTFKKAYGSVFVEWSADKKN
SLAYALTAYKNDTKIIEYLTQKGFDAAETDIALTLPSFSKWGNLSEKALNNIIPYLEQGMLYH
DACTAAGYNFKADDTDKRMYLPAHEKEAPELDDITNPVVRRAISQTIKVINALIREMGESPCF
VNIELARELSKNKAERSKIEKGQKENQVRNDRIMERLRNEFGLLSPTGQDLIKLKLWEEQDGI
CPYSLKPIKIEKLFDVGYTDIDAIIPYSLSFDDTYNNKVLVMSSENRQKGNRIPMQYLEGKRQ
DDFWLWVDNSNLSRRKKQNLTKETLSEDDLSGFKKRNLQDTQYLSRFMMNYLKKYLALAPNTT
GRKNTIQAVNGAVTSYLRKRWGIQKVRENGDTHHAVDAVVISCVTAGMTKRVSEYAKYKETEF
QNPQTGEFFDVDIRTGEVINRFPLPYARFRNELLMRCSENPSRILHEMPLPTYAADEKVAPIF
VSRMPKHKVKGSAHKETIRRAFEEDGKKYTVSKVPLTDLKLKNGEIENYYNPESDGLLYNALK
EQUAFGGDAAKAFEQPFYKPKSDGSEGPLVKKVKLINKATLTVPVLNNTAVADNGSMVRVDVF
FVEGEGYYLVPIYVADTVKKELPNKAIIANKPYEEWKEMREENFVFSLYPNDLIKISSRKDMK
FNLVNKESTLAPNCQSKEALVYYKGSDISTAAVTAINHDNTYKLRGLGVKTLLKIEKYQVDVL
GNVFKVGKEKRVRFK
MAD2039- 181 MRPYAIGLDIGITSVGWATVALDADESPCGIIGLGSRIFDAAEQPKTGESLAAPRRAARGSRR
H587A RLRRHRHRNERIRSLMLEERLISQDELETLFDGRLEDIYALRVKALDEIVSRTDFARILLHIS
QRRGFKSNRKNPTTKEDGVLLAAVNENKQRMSEHGYRTVGEMFLLDETFKDHKRNKGGNYITT
VARDMVADEVRAIFSAQRELGASFASEEFEERYLEILLSQRSFDEGPGGNSPYGGSQIERMVG
RCTFFPDEPRAAKATYSFEYFTLLQKVNHIRIVENGVASKLTDEQRRIIIELAHTTKDVSYAK
IRKVLKLSDKQLFNIRYSDNSPAEDSEKKEKLGIMKAYHQMRSAIDRVSKGRFAMMPRAQRNA
IGTALSLYKTSDKIRKYLTDAGLDEIDINSADSIGSFSKFGHISVKACDMLIPFLEQGMNYNE
ACAAAGLNFKGHDAGEKSKLLHPKEEDYEDITSPVVRRAIAQTIKVINAIIRREGCSPTFINI
ELAREMAKDFRERNRIKKENDDNRAKNERLLERIRTEYGKNNPTGLDLVKLRLYEEQSGVCMY
SLKQMSLEKLFEPNYAEVDAIVPYSISFDDSRKNKVLVLTEENRNKGNRLPLQYLKGRRREDF
IVWVNNNVKDYRKRRLLLKEELTAEDESGFKERNLQDTKTMSRFLLNYIADNLEFAESTRGRK
KKVTAVNGAVTAYMRKRWGITKIREDGDCHHAVDAVVIACTTDAMIRQVSRYAQFRECEYMQT
ESGSVAVDTGTGEVLRTFPYPWPDFRKELEARLANDPAKVINDLHLPFYMSAGRPLPEPVFVS
RMPRRKVTGAAHKDTIKSARELDNGYLIVKRPLTDLKLKNGEIENYYNPQSDKCLYDALKNAL
IEHGGDAKKAFAGEFRKPKRDGTPGPIVKKVKLLEPTTMCVPVHGGKGAADNDSMVRVDVFLS
GGKYYLVPIYVADTLKPELPNKAVTRGKKYSEWLEMADEDFIFSLYPNDLICATSKNGITLSV
CRKDSTLPPTVESKSFMLYYRGTDISTGSISCITHDNAYKLRGLGVKTLEKLEKYTVDVLGEY
HKVGKEVRQPFNIKRRKACPSEML
MAD2039- 182 MRPYAIGLDIGITSVGWATVALDADESPCGIIGLGSRIFDAAEQPKTGESLAAPRRAARGSRR
N610A RLRRHRHRNERIRSLMLEERLISQDELETLFDGRLEDIYALRVKALDEIVSRTDFARILLHIS
QRRGFKSNRKNPTTKEDGVLLAAVNENKQRMSEHGYRTVGEMFLLDETFKDHKRNKGGNYITT
VARDMVADEVRAIFSAQRELGASFASEEFEERYLEILLSQRSFDEGPGGNSPYGGSQIERMVG
RCTFFPDEPRAAKATYSFEYFTLLQKVNHIRIVENGVASKLTDEQRRIIIELAHTTKDVSYAK
IRKVLKLSDKQLFNIRYSDNSPAEDSEKKEKLGIMKAYHQMRSAIDRVSKGRFAMMPRAQRNA
IGTALSLYKTSDKIRKYLTDAGLDEIDINSADSIGSFSKFGHISVKACDMLIPFLEQGMNYNE
ACAAAGLNFKGHDAGEKSKLLHPKEEDYEDITSPVVRRAIAQTIKVINAIIRREGCSPTFINI
ELAREMAKDFRERNRIKKENDDNRAKNERLLERIRTEYGKNNPTGLDLVKLRLYEEQSGVCMY
SLKQMSLEKLFEPNYAEVDHIVPYSISFDDSRKNKVLVLTEENRNKGNRLPLQYLKGRRREDF
IVWVNNNVKDYRKRRLLLKEELTAEDESGFKERNLQDTKTMSRFLLNYIADNLEFAESTRGRK
KKVTAVNGAVTAYMRKRWGITKIREDGDCHHAVDAVVIACTTDAMIRQVSRYAQFRECEYMQT
ESGSVAVDTGTGEVLRTFPYPWPDFRKELEARLANDPAKVINDLHLPFYMSAGRPLPEPVFVS
RMPRRKVTGAAHKDTIKSARELDNGYLIVKRPLTDLKLKNGEIENYYNPQSDKCLYDALKNAL
IEHGGDAKKAFAGEFRKPKRDGTPGPIVKKVKLLEPTTMCVPVHGGKGAADNDSMVRVDVFLS
GGKYYLVPIYVADTLKPELPAKAVTRGKKYSEWLEMADEDFIFSLYPNDLICATSKNGITLSV
CRKDSTLPPTVESKSFMLYYRGTDISTGSISCITHDNAYKLRGLGVKTLEKLEKYTVDVLGEY
HKVGKEVRQPFNIKRRKACPSEML
While this invention is satisfied by embodiments in many different forms, as described in detail in connection with preferred embodiments of the invention, it is understood that the present disclosure is to be considered as exemplary of the principles of the invention and is not intended to limit the invention to the specific embodiments illustrated and described herein. Numerous variations may be made by persons skilled in the art without departure from the spirit of the invention. The scope of the invention will be measured by the appended claims and their equivalents. The abstract and the title are not to be construed as limiting the scope of the present invention, as their purpose is to enable the appropriate authorities, as well as the general public, to quickly determine the general nature of the invention. In the claims that follow, unless the term “means” is used, none of the features or elements recited therein should be construed as means-plus-function limitations pursuant to 35 U.S.C. § 112, ¶6.
