Variants of cas12a nucleases and methods of making and using the same

Abstract

The present invention relates to variants of Cas12a nucleases having altered protospacer adjacent motif recognition specificity. The present invention also relates to methods of making CRISPR-CAS nuclease variants and methods of using the variants to modify nucleic acids.

Description

[0001] Declaration regarding the electronic submission of the sequence list

[0002] A sequence list in ASCII text format is provided in lieu of a paper copy. This sequence list, filed in accordance with 37 CFR §1.821, is named 1499.7WO_ST25.txt, is 257,774 bytes in size, and was generated and submitted via EFS-Web on October 13, 2020. This sequence list is hereby incorporated by reference in this specification to disclose its contents.

[0003] Priority Statement

[0004] This application claims the benefit of U.S. Provisional Application No. 62 / 916,392, filed October 17, 2019, pursuant to 35 USC § 119(e), the entire contents of which are incorporated herein by reference. Technical Field

[0005] This invention relates to variants of the Cas12a CRISPR-Cas nuclease with altered prespacer neighbor motif recognition specificity. The invention also relates to methods for preparing CRISPR-Cas nuclease variants and methods for modifying nucleic acids using said variants. Background Technology

[0006] Genome editing / modification is the process of introducing mutations at target genomic locations using site-directed nucleases, such as CRISPR-Cas nucleases. Cas9, the most widely used nuclease for genome modification, can introduce mutations in genomic regions upstream of NGG motifs (e.g., a preseptal neighbor motif (PAM)). Other Cas nucleases have different PAM recognition specificities. When the PAM specificity of these nucleases is particularly stringent, they can reduce the usefulness of the nuclease for genome modification by limiting the number of genomic target sites available for modification by that nuclease.

[0007] To address the deficiencies in this field, the present invention provides modified CRISPR-Cas nucleases with improved PAM specificity, as well as methods for designing, identifying, and selecting such CRISPR-Cas nucleases. Summary of the Invention

[0008] One aspect of the present invention provides a modified Trichophyceae family ( LachnospiraceaeBacterial CRISPR (clustered regular interspaced short palindromic repeats) Cas12a (LbCas12a) polypeptide, wherein the modified LbCas12a polypeptide comprises, substantially consists of, or is composed of, the following amino acid sequence, which has at least 80% identity with the amino acid sequence SEQ ID NO: 1 (LbCas12a), and refers to SEQ ID NO: Position number 1, having mutations in any combination at one or more of the following positions (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or more): K116, K120, K121, D122, E125, T148, T149, T152, D156, E159, Q529, G532, D535, K538, D541, Y542, L585, K591, M592, K595, V596, S599, K600, K601, Y616, Y646 and / or W649, optionally, refer to SEQ ID NO: The position number 1 has mutations located at one or more of the following positions in any combination: K116, K120, K121, D122, E125, T152, D156, E159, G532, D535, K538, D541 and / or K595.

[0009] A second aspect of the invention provides a CRISPR-Cas (CRISPR-Cas) system comprising: (a) a fusion protein comprising (i) the modified LbCas12a polypeptide of the present invention, or a nucleic acid encoding the modified LbCas12a polypeptide of the present invention, and (ii) a polypeptide of interest or a nucleic acid encoding the polypeptide of interest; and (b) a guide nucleic acid (CRISPR RNA, CRISPR DNA, crRNA, crDNA) comprising a spacer sequence and a repeat sequence, wherein the guide nucleic acid is capable of forming a complex with the modified LbCas12a polypeptide or the fusion protein, and the spacer sequence is capable of hybridizing with a target nucleic acid to guide the modified LbCas12a polypeptide and the polypeptide of interest to the target nucleic acid, thereby modifying or regulating the target nucleic acid.

[0010] A third aspect of the invention provides a method for modifying a target nucleic acid, comprising: contacting the target nucleic acid with: (a) (i) a modified LbCas12a polypeptide of the present invention, or a fusion protein comprising the modified LbCas12a polypeptide of the present invention, and (ii) a guide nucleic acid (e.g., CRISPR RNA, CRISPR DNA, crRNA, crDNA); (b) a complex comprising the modified LbCas12a polypeptide of the present invention and the guide nucleic acid; (c) a composition comprising (i) the modified LbCas12a polypeptide of the present invention or the fusion protein of the present invention, and (ii) the guide nucleic acid; and / or (d) the system of the present invention, thereby modifying the target nucleic acid.

[0011] A fourth aspect of the invention provides a method for modifying a target nucleic acid, comprising contacting a cell or cell-free system containing the target nucleic acid with: (a)(i) a polynucleotide encoding the modified LbCas12a polypeptide of the present invention, or an expression cassette or vector containing thereof, and (ii) a guide nucleic acid, or an expression cassette or vector containing thereof; and / or (b) a nucleic acid construct encoding (i) a complex containing the modified LbCas12a polypeptide or fusion protein of the present invention and (ii) a guide nucleic acid, or an expression cassette or vector containing thereof, thereby modifying the target nucleic acid.

[0012] A fifth aspect of the invention provides a method for editing a target nucleic acid, comprising: contacting the target nucleic acid with: (a)(i) a fusion protein comprising the modified LbCas12a polypeptide of the present invention and (a)(ii) a guide nucleic acid; (b) a complex comprising the fusion protein and the guide nucleic acid of the present invention; (c) a composition comprising the fusion protein and the guide nucleic acid of the present invention; and / or (d) the system of the present invention, thereby editing the target nucleic acid.

[0013] A sixth aspect of the invention provides a method for editing a target nucleic acid, comprising contacting a cell or cell-free system containing the target nucleic acid with: (a)(i) a polynucleotide encoding a fusion protein comprising the modified LbCas12a polypeptide of the present invention, or an expression cassette or vector comprising thereto; and (a)(ii) a guide nucleic acid, or an expression cassette or vector comprising thereto; and / or (b) a nucleic acid construct encoding a complex or an expression cassette or vector comprising thereto, the complex comprising the fusion protein and the guide nucleic acid, the fusion protein comprising the modified LbCas12a polypeptide of the present invention; and / or (c) the system of the present invention, thereby editing the target nucleic acid.

[0014] A seventh aspect of the present invention provides a method for constructing a randomized DNA library comprising double-stranded nucleic acid molecules for determining the preseptal neighbor motif (PAM) requirement / specificity of a CRISPR-Cas nuclease having a PAM recognition site at the 5' end of the preseptal, the method comprising: preparing two or more double-stranded nucleic acid molecules, including the steps of: (a) synthesizing a non-target oligonucleotide (first) chain and a target oligonucleotide (second) chain for each of the two or more double-stranded nucleic acid molecules, wherein the non-target oligonucleotide chain comprises from 5' to 3': (i) a first sequence having about 5 to about 15 nucleotides, (ii) a second sequence having at least four randomized nucleotides, (iii) a preseptal sequence comprising about 16 to about 25 nucleotides, and (iv) having about 5 to about 20 nucleotides. The third sequence of the acid, wherein (i) the first sequence having about 5 to 15 nucleotides is adjacent to the 5' end of the second sequence of (ii), the second sequence of (ii) is adjacent to the 5' end of the prespacer sequence of (iii), and the prespacer sequence is adjacent to the 5' end of the third sequence of (iv); and the target oligonucleotide (second) strand is complementary to the non-target oligonucleotide strand; and (b) annealing the non-target oligonucleotide strand with the complementary target oligonucleotide strand to produce a double-stranded nucleic acid molecule, wherein the first sequence contains a restriction site (at its 5' end), and the third sequence contains a restriction site (at its 3' end), wherein the first sequence (i), the prespacer sequence (iii), and the third sequence (iv) of each of two or more double-stranded nucleic acid molecules are identical, thereby constructing a randomized DNA library containing double-stranded nucleic acid molecules.

[0015] An eighth aspect of the present invention provides a method for constructing a randomized DNA library comprising double-stranded nucleic acid molecules for determining the preseptal neighbor motif (PAM) requirement / specificity of a CRISPR-Cas nuclease having a PAM recognition site at the 3' end of the preseptal neighbor motif, the method comprising: preparing two or more double-stranded nucleic acid molecules, including the steps of: (a) synthesizing a non-target oligonucleotide (first) strand and a target oligonucleotide (second) strand for each of the two or more double-stranded nucleic acid molecules, wherein the non-target oligonucleotide strand comprises from 5' to 3': (i) a first sequence having about 5 to about 20 nucleotides, (ii) a preseptal sequence having about 16 to about 25 nucleotides, (iii) a second sequence having at least four randomized nucleotides, and (iv) a third sequence having about 5 to about 15 nucleotides. In (i), a first sequence of about 5 to 20 nucleotides is adjacent to the 5' end of the prespacer sequence of (ii), a second sequence of (iii) is adjacent to the 3' end of the prespacer sequence of (ii), and a third sequence of (iv) is adjacent to the 3' end of the second sequence of (iii); and the target oligonucleotide (second) strand is complementary to the non-target oligonucleotide strand; and (b) the non-target oligonucleotide strand is annealed to the complementary target oligonucleotide strand to produce a double-stranded nucleic acid molecule, wherein the first sequence (i) contains a restriction site (at its 5' end), and the third sequence (iv) contains a restriction site (at its 3' end), wherein the first sequence (i), the prespacer sequence (ii), and the third sequence (iv) of each of two or more double-stranded nucleic acid molecules are identical, thereby constructing a randomized DNA library containing double-stranded nucleic acid molecules.

[0016] A ninth aspect of the invention provides a randomized DNA library for determining the preseptal neighbor motif (PAM) requirement / specificity of a CRISPR-Cas nuclease having a PAM recognition site at the 5' end of the preseptal, the randomized DNA library comprising two or more double-stranded nucleic acid molecules, wherein each double-stranded nucleic acid molecule comprises: (a) a non-target oligonucleotide (first) strand and a target oligonucleotide (second) strand, wherein the non-target oligonucleotide strand comprises from 5' to 3': (i) having about 5 to about 1 (i) a first sequence of 5 nucleotides (e.g., about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 nucleotides, and any range or value thereof), (ii) a second sequence having at least four randomized nucleotides (e.g., at least 4, 5, 6, 7, 8, 9, 10, or more, and any range or value thereof), and (iii) a sequence comprising about 16 to about 25 nucleotides (e.g., about 16, 17, 18, 19, 20, 21, 22, 23, 24, or 2... (i) a prespacer sequence of about 5 nucleotides, and (iv) a third sequence of about 5 to about 20 nucleotides (e.g., about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 nucleotides, and any range or value thereof), wherein the first sequence of (i) having about 5 to 15 nucleotides is immediately adjacent to the 5' end of the second sequence of (ii), the second sequence of (ii) is immediately adjacent to the 5' end of the prespacer sequence of (iii), and the prespacer... The spacer sequence is adjacent to the 5' end of the third sequence in (iv); and the target oligonucleotide (second) chain is complementary to the non-target oligonucleotide chain; and (b) the non-target oligonucleotide chain is annealed to the complementary target oligonucleotide chain to produce a double-stranded nucleic acid molecule, wherein the first sequence contains a restriction site (at its 5' end) and the third sequence contains a restriction site (at its 3' end), wherein the first sequence (i), the prespacer sequence (iii), and the third sequence (iv) are identical in each of two or more double-stranded nucleic acid molecules.

[0017] A tenth aspect of the invention provides a randomized DNA library for determining the preseptal neighbor motif (PAM) requirement / specificity of a CRISPR-Cas nuclease having a PAM recognition site at the 3' end of the preseptal, the randomized DNA library comprising two or more double-stranded nucleic acid molecules, wherein each double-stranded nucleic acid molecule comprises: (a) a non-target oligonucleotide (first) strand and a target oligonucleotide (second) strand, wherein the non-target oligonucleotide strand comprises from 5' to 3': (i) having about 5 to about 20 (ii) a first sequence comprising about 16 to about 25 nucleotides (e.g., about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 nucleotides, and any range or value thereof), and (iii) having at least four randomized nucleotides (e.g., at least 4, 5, 6, 7, 8, 9, ...). (i) a second sequence having 10 or more, and any range or value thereof, and (iv) a third sequence having about 5 to about 15 nucleotides (e.g., about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 nucleotides, and any range or value thereof), wherein the first sequence of (i) having about 5 to 20 nucleotides is adjacent to the 5' end of the prespacer sequence of (ii), the second sequence of (iii) is adjacent to the 3' end of the prespacer sequence of (ii), and the third sequence of (iv) is adjacent to the 3' end of the second sequence of (iii); and the target oligonucleotide (second) chain is complementary to the non-target oligonucleotide chain; and (b) annealing the non-target oligonucleotide chain to the complementary target oligonucleotide chain to produce a double-stranded nucleic acid molecule, wherein the first sequence contains a restriction site (at its 5' end), and the third sequence contains a restriction site (at its 3' end), wherein the first sequence (i), the prespacer sequence (ii), and the third sequence (iv) of each of the two or more double-stranded nucleic acid molecules are identical.

[0018] The present invention further provides expression cassettes and / or vectors comprising polynucleotides encoding the CRISPR-Cas nuclease and / or fusion protein of the present invention, and / or cells comprising the polynucleotides, peptides and / or fusion proteins of the present invention, and / or kits comprising them.

[0019] These and other aspects of the invention will be set forth in more detail in the following description of the invention.

[0020] Sequence Description

[0021] SEQ ID NO: 1-17, 49, 50 and 51 are exemplary nucleotide sequences encoding the Cas12a nuclease.

[0022] SEQ ID NO: 18-22 are exemplary adenosine deaminases.

[0023] SEQ ID NO: 23-25 ​​and SEQ ID NO: 42-48 are exemplary cytosine deaminases.

[0024] SEQ ID NO: 26 is an exemplary nucleotide sequence encoding a uracil-DNA glycosylation inhibitor (UGI).

[0025] SEQ ID NO: 27-29 provides examples of motif positions adjacent to the prespacer for type V CRISPR-Cas12a nucleases.

[0026] SEQ ID NO: 30-39 illustrates exemplary nucleotide sequences that can be used to generate randomized libraries of the present invention for, for example, in vitro cleavage assays.

[0027] SEQ ID NO: 40-41 are exemplary regulatory sequences encoding promoters and introns.

[0028] SEQ ID NO: 52 provides the nucleotide sequence of an exemplary expression cassette.

[0029] SEQ ID NO: 53 provides the nucleotide sequence of an exemplary vector.

[0030] SEQ ID NO: 54-61 provides exemplary spacer subsequences.

[0031] SEQ ID NO: 62 provides an exemplary CRISPR RNA. Attached Figure Description

[0032] Figure 1 A schematic diagram illustrating an exemplary PAM library preparation of the present invention is shown. In this method, 5' phosphorylated oligonucleotides are annealed and cloned into a pUC19 vector digested with EcoRI and SphI. ScaI is used to linearize the vector (Lbcpf1 does not recognize the AGTACT sequence). Upper strand (SEQ ID NO: 32); Lower strand (SEQ ID NO: 33).

[0033] Figure 2 The diagram shows the average prespacer neighbor motif (PAM) for each gene within the coding sequences of maize (top) and soybean (bottom). The LbCpf1 gene has significantly fewer accessed gene sequences compared to the Cas9 variant.

[0034] Figure 3The average accessible cytosine (insets A and B) and adenine (insets C and D) restricted by PAM are shown in maize (insets A and C) and soybean (insets B and D). As shown, LbCpf1 has significantly fewer accessible cytosine and adenine compared to the Cas9 variant.

[0035] Figure 4 Provided display of Gao et al. ( Nat Biotechnol A schematic diagram of the simplified PAM assay for determining the sequence (35(8):789-792 (2017)). The amplified fragment represents the sequence that was not cleaved by the CRISPR-Cas nuclease, while the unamplified fragment was cleaved by the CRISPR-Cas nuclease. Sequencing and comparison with the enzyme-free control identified the unamplified nucleic acid sequence (i.e., present in the control population but not in the edited population), thereby determining the sequence that was recognized and cleaved by the nuclease. Top: Upper sequence (SEQ ID NO: 36), Middle sequence (SEQ ID NO: 37), Lower sequence (SEQ ID NO: 38); Middle: Upper sequence (SEQ ID NO: 39), Lower sequence (SEQ ID NO: 37); Bottom: Upper sequence (SEQ ID NO: 36), Lower sequence (SEQ ID NO: 37).

[0036] Figure 5 The average of three Illumina MiSeq NGS reads from the PAMDA library is plotted from highest to lowest count. The 1024 library members containing NNNNN follow a normal distribution with an average of 39 reads.

[0037] Figure 6 Cell sorting results of negative controls containing wtLbCas12a and non-targeting plasmid spacers.

[0038] Figure 7 Cell sorting results of wtLbCas12a and crRNA targeting plasmid spacers.

[0039] Figure 8 Cell sorting results of crRNA from LbCas12a-K595Y and target plasmid spacers.

[0040] Figure 9 Cell sorting results of crRNA from LbCas12a-G532R-K595R double mutant control and target plasmid spacers.

[0041] Figure 10 Cell sorting results of LbCas12a-T152R-K595Y double mutation (combining two point mutations in this study) and crRNA targeting plasmid spacers.

[0042] Figure 11 Cell sorting results of LbCas12a-T152R-K538W-K595Y triple mutation (combination of three point mutations) and crRNA targeting plasmid spacers.

[0043] Figure 12 Total normalized NGS counts of two separate crRNA-free controls and wild-type dLbCas12a, as well as the reporter library.

[0044] Figure 13 Single-point mutation-normalized PAM-SCANR scores for each of the 256 tetranucleotide PAMs. The line across the graph shows the highest observed score of either of the two negative controls, 1.67.

[0045] Figure 14 The PAM-SCANR score is normalized for the combined mutations of each of the 256 tetranucleotide PAMs.

[0046] Figure 15 The combination of mutants K538W and K595Y produces the enzyme LbCas12a-K538W-K595Y with a unique PAM recognition sequence. In some cases, the shared PAM recognition motif from K538W (vertical shading) or K595Y (horizontal shading) is recognized by the combined mutant, but the combination often produces a completely new PAM recognition sequence (thatched).

[0047] Figure 16 This demonstrates that combined multiple extended PAM mutations can sometimes produce additive but usually unique PAM recognition sequences.

[0048] Figure 17 All non-TTTV PAMs with a PAM-SCANR score higher than 1.67 were compared for K595Y (left) and T152R (right). With the exception of one, all other PAM-SCANR positive PAMs with a cutoff value higher than 1.67 had PAM depletion scores higher than the in vitro cutoff value of 9.2.

[0049] Figure 18 The percentage of indels formed for each type of spacer containing TTTV is shown in HEK293T cells. The individual indel percentages for TTTC, TTTA, and TTTG are shown as circles, squares, or triangles, respectively. The mean line for each spacer and the values ​​rounded to the nearest integer are also shown.

[0050] Figure 19The percentage of the maximum observed INDEL for LbCas12a_K595Y HEK293T is shown for each test PAM. Values ​​above 0.1% are outside of sequencing noise and represent true INDEL.

[0051] Figure 20 The percentage of maximum observed INDELs for LbCas12a_T152R HEK293T is shown for each test PAM. Values ​​above 0.1% are outside of sequencing noise and represent true INDELs.

[0052] Figure 21 The percentage of maximum observed INDELs for LbCas12a_K538W HEK293T is shown for each test PAM. Values ​​above 0.1% are outside of sequencing noise and represent true INDELs.

[0053] Figure 22A - 22B LbCas12a-T152R ( Figure 22A ) and LbCas12a-K595Y ( Figure 22B The linear correlation between % INDEL (maximum value) and standardized bacterial PAM-SCANR score. Invention Details

[0055] The invention will now be described with reference to the accompanying drawings and examples, which illustrate embodiments of the invention. This description is not intended to be a detailed list of all different ways in which the invention may be practiced or of all features that may be added to the invention. For example, features shown with respect to one embodiment may be incorporated into other embodiments, and features shown with respect to a particular embodiment may be removed from that embodiment. Therefore, the invention contemplates that in some embodiments of the invention, any features or combinations of features set forth herein may be excluded or omitted. Furthermore, numerous changes and additions to the various embodiments presented herein will be apparent to those skilled in the art, without departing from the invention. Therefore, the following description is intended to illustrate some specific embodiments of the invention, and not to exhaustively list all permutations, combinations, and variations thereof.

[0056] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the invention.

[0057] All publications, patent applications, patents and other references cited in this article are incorporated in their entirety by reference to obtain the teachings relevant to the sentences and / or paragraphs in which they appear.

[0058] Unless the context otherwise requires, it is specifically intended that the various features of the invention described herein can be used in any combination. Furthermore, the invention also contemplates that in some embodiments, any feature or combination of features set forth herein may be excluded or omitted. For illustrative purposes, if the specification specifies that a composition comprises components A, B, and C, it is specifically intended that any one or any combination of A, B, or C may be omitted or discarded, individually or in any combination.

[0059] As used in the description of this invention and the appended claims, the singular forms “a,” “an,” and “the” are also intended to include the plural forms, unless the context clearly specifies otherwise.

[0060] As used herein, “and / or” refers to and covers any and all possible combinations of one or more of the related listed items, as well as combinations that are missing when interpreted as alternatives (“or”).

[0061] As used herein, the term "about" when referring to a measurable value such as amount or concentration means including a variation of ±10%, ±5%, ±1%, ±0.5%, or even ±0.1% of the specified value, as well as the specified value itself. For example, "about X," where X is a measurable value, means including X and variations of X of ±10%, ±5%, ±1%, ±0.5%, or even ±0.1%. The ranges of measurable values ​​provided herein may include any other ranges and / or individual values ​​therein.

[0062] As used herein, phrases such as “between X and Y” and “between about X and Y” should be interpreted as including both X and Y. As used herein, phrases such as “between about X and Y” mean “between about X and about Y”, while phrases such as “from about X to Y” mean “from about X to about Y”.

[0063] Unless otherwise stated herein, the ranges of values ​​described herein are intended only as a convenient way to individually refer to each individual value falling within that range, and each individual value is incorporated into this specification as if it were listed separately herein. For example, if the range 10 to 15 is disclosed, then 11, 12, 13, and 14 are also disclosed.

[0064] As used herein, the terms “comprise,” “comprises,” and “comprising” specify the presence of the said feature, integer, step, operation, element, and / or component, but do not exclude the presence or addition of one or more other features, integers, steps, operations, elements, components, and / or groups thereof.

[0065] As used herein, the transitional phrase “consistently of…” means that the scope of the claim should be interpreted to include the specified materials or steps listed in the claim, as well as those materials or steps that do not substantially affect the essential and novel features of the claimed invention. Therefore, when used in the claims of this invention, the term “consistently of…” is not intended to be equivalent to “comprising or including”.

[0066] As used herein, the terms “increase,” “increasing,” “enhance,” “enhancing,” “improve,” and “improving” (and their grammatical variations) describe an increase of at least about 25%, 50%, 75%, 100%, 150%, 200%, 300%, 400%, 500%, or more compared to a control.

[0067] As used herein, the terms “reduce,” “reduced,” “reducing,” “reduction,” “diminish,” and “decrease” (and their grammatical variations) describe a reduction, for example, of at least about 5%, 10%, 15%, 20%, 25%, 35%, 50%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% compared to a control. In certain embodiments, this reduction may result in no or substantially no (i.e., negligible amounts, such as less than about 10% or even 5%) of detectable activity or amount.

[0068] "Heterologous" or "recombinant" nucleotide sequences are nucleotide sequences that are not naturally associated with the host cell to which they are introduced, including multiple non-natural copies of naturally occurring nucleotide sequences.

[0069] "Natural" or "wild-type" nucleic acid, nucleotide, polypeptide, or amino acid sequence refers to a naturally occurring or endogenous nucleic acid, nucleotide, polypeptide, or amino acid sequence. Therefore, for example, "wild-type mRNA" is mRNA that is naturally present in an organism or is endogenous to that organism. "Homologous" nucleic acid sequences are nucleotide sequences that are naturally associated with the host cell to which they are introduced.

[0070] As used herein, the terms “nucleic acid,” “nucleic acid molecule,” “nucleotide sequence,” and “polynucleotide” refer to linear or branched, single-stranded or double-stranded RNA or DNA, or hybrids thereof. The term also includes RNA / DNA hybrids. When dsRNA is produced synthetically, less common bases such as inosine, 5-methylcytosine, 6-methyladenine, and hypoxanthine can also be used for antisense, dsRNA, and ribozyme pairing. For example, polynucleotides containing C-5 propyne analogs of uridine and cytidine have been shown to bind RNA with high affinity and are potent antisense repressors of gene expression. Other modifications can also be made, such as modifications to the phosphodiester backbone or the 2'-hydroxyl group in the RNA ribose group.

[0071] As used herein, the term "nucleotide sequence" refers to a hybrid of nucleotides or the sequence of these nucleotides from the 5' to 3' ends of a nucleic acid molecule, and includes DNA or RNA molecules, including cDNA, DNA fragments or portions, genomic DNA, synthetic (e.g., chemically synthesized) DNA, plasmid DNA, mRNA, and antisense RNA, any of which may be single-stranded or double-stranded. The terms "nucleotide sequence," "nucleic acid," "nucleic acid molecule," "nucleic acid construct," "oligonucleotide," and "polynucleotide" are used interchangeably herein to refer to a hybrid of nucleotides. The nucleic acid molecules and / or nucleotide sequences provided herein are shown in a left-to-right 5' to 3' orientation and are represented using standard codes for representing nucleotide symbols as specified in U.S. Sequence Rules 37 CFR §§1.821-1.825 and the World Intellectual Property Organization (WIPO) ST.25 standard. As used herein, "5' region" can refer to the region of a polynucleotide closest to its 5' end. Thus, for example, elements of a polynucleotide's 5' region can be located anywhere from the first nucleotide at the 5' end of the polynucleotide to the nucleotide in the middle of the polynucleotide. As used herein, the term "3' region" can refer to the region of a polynucleotide closest to its 3' end. Therefore, for example, elements of the 3' region of a polynucleotide can be located anywhere from the first nucleotide at the 3' end to the nucleotide in the middle of the polynucleotide.

[0072] As used herein, the term "gene" refers to a nucleic acid molecule capable of producing mRNA, antisense RNA, miRNA, antimicroRNA antisense oligodeoxyribonucleotides (AMOs), etc. Genes may or may not be used to produce functional proteins or gene products. Genes may include coding and non-coding regions (e.g., introns, regulatory elements, promoters, enhancers, termination sequences, and / or 5' and 3' untranslated regions). Genes may be "isolated," meaning that the nucleic acid substantially or substantially lacks components typically associated with that nucleic acid in its native state. Such components include other cellular material, culture media from recombinant production, and / or various chemicals used for the chemical synthesis of nucleic acids.

[0073] The term "mutation" refers to point mutations (e.g., missense or nonsense, or insertions or deletions of single base pairs that result in frameshifts), insertions, deletions, and / or truncations. When a mutation is the substitution of one residue in an amino acid sequence for another, or the deletion or insertion of one or more residues in the sequence, the mutation is typically described by identifying the original residue, then determining the position of the residue in the sequence, and finally identifying the newly substituted residue.

[0074] As used herein, the term "complementary" or "complementarity" refers to the natural binding of polynucleotides through base pairing under permissible salt and temperature conditions. For example, the sequence "AGT" (5' to 3') binds to the complementary sequence "TCA" (3' to 5'). Complementarity between two single-stranded molecules can be "partial," where only some nucleotides bind, or it can be complete, in which case there is perfect complementarity between the single-stranded molecules. The degree of complementarity between nucleic acid strands has a significant impact on the efficiency and strength of hybridization between nucleic acid strands.

[0075] As used herein, “complementary” can mean 100% complementarity with the reference nucleotide sequence, or it can mean less than 100% complementarity (e.g., complementarity of approximately 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, etc.).

[0076] The term "part" or "fragment" of the nucleotide sequence of this invention will be understood to refer to a nucleotide sequence shortened relative to the length of a reference nucleic acid or nucleotide sequence (e.g., shortened by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more nucleotides), and comprising, substantially composed of and / or consisting of, the following nucleotide sequences: nucleotide sequences that are identical or nearly identical to the reference nucleic acid or nucleotide sequence (e.g., 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity) of consecutive nucleotides. Where appropriate, such a nucleic acid fragment or part according to the invention may be contained within a larger polynucleotide, which is a component thereof. For example, the repeat sequence of the nucleic acid guided by the present invention may include a portion of a wild-type CRISPR-Cas repeat sequence (e.g., wild-type Cas9 repeat sequence, wild-type Cas12a repeat sequence, etc.).

[0077] Different nucleic acids or proteins that are homologous are referred to herein as “homologs.” The term “homologous” includes homologous sequences from the same and other species, as well as orthologous sequences from the same and other species. “Homology” refers to the level of similarity between two or more nucleic acid and / or amino acid sequences in terms of the percentage of positional identity (i.e., sequence similarity or identity). Homology also refers to the concept of similar functional properties between different nucleic acids or proteins. Therefore, the compositions and methods of the present invention also comprise homologs of the nucleotide and polypeptide sequences of the present invention. As used herein, “orthologous” refers to homologous nucleotide and / or amino acid sequences from different species that originated from a common ancestral gene during speciation. Homologous products of the nucleotide sequence of the present invention have substantial sequence identity with the nucleotide sequence of the present invention (e.g., at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100%).

[0078] As used herein, “sequence identity” refers to the degree to which two optimally matched polynucleotide or polypeptide sequences remain unchanged within a window of the matching components (e.g., nucleotides or amino acids). “Identity” can be readily calculated using known methods, including but not limited to those described in the following literature:Computational Molecular Biology (Lesk, AM, ed.) Oxford University Press, New York (1988); Biocomputing: Informatics and Genome Projects (Smith, DW, ed.) Academic Press, New York (1993); Computer Analysis of Sequence Data, Part I (Edited by Griffin, AM and Griffin, HG) Humana Press, New Jersey (1994); Sequence Analysis in Molecular Biology (Edited by von Heinje, G.) Academic Press (1987); and Sequence Analysis Primer (Edited by Gribskov, M. and Devereux, J.) Stockton Press, New York (1991).

[0079] As used herein, the term "sequence identity percentage" or "identity percentage" refers to the percentage of identical nucleotides in the linear polynucleotide sequence of a reference ("query") polynucleotide molecule (or its complementary strand) compared to the test ("subject") polynucleotide molecule (or its complementary strand) when two sequences are optimally conjugated. In some embodiments, "identity percentage" may refer to the percentage of identical amino acids in the amino acid sequence compared to a reference polypeptide.

[0080] As used herein, in the context of two nucleic acid molecules, nucleotide sequences, or protein sequences, the phrase “substantially identical” or “substantially identical” means that two or more sequences or subsequences have at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identity of nucleotide or amino acid residues when compared and aligned to obtain maximum correspondence using one of the following sequence comparison algorithms or by visual inspection. In some embodiments of the invention, substantial identity exists in continuous nucleotide regions of the nucleotide sequence of the invention, the length of which is about 10 to about 20 nucleotides, about 10 to about 25 nucleotides, about 10 to about 30 nucleotides, about 15 to about 25 nucleotides, about 30 to about 40 nucleotides, about 50 to about 60 nucleotides, about 70 to about 80 nucleotides, about 90 to about 100 nucleotides or more, and any range therewith, up to the full length of the sequence. In some embodiments, the nucleotide sequences may be substantially identical in at least about 20 nucleotides (e.g., about 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40 nucleotides). In some embodiments, substantially identical nucleotide or protein sequences perform substantially the same function as substantially identical nucleotides (or encoded protein sequences).

[0081] For sequence comparisons, a reference sequence is typically used as the comparison target. When using a sequence comparison algorithm, the test and reference sequences are input into the computer, with subsequence coordinates specified if necessary, and the sequence comparison algorithm program parameters are also specified. The algorithm then calculates the percentage of sequence identity between the test sequence (or one or more) and the reference sequence based on the specified program parameters.

[0082] Optimal alignment of sequences for the alignment comparison window is well known to those skilled in the art and can be performed using tools such as Smith and Waterman's local homology algorithm, Needleman and Wunsch's homology alignment algorithm, Pearson and Lipman's similarity search method, and optionally through computerized execution of these algorithms, such as GAP, BESTFIT, FASTA, and TFASTA, which are provided as part of the GCG® Wisconsin Package® (Accelrys Inc., San Diego, CA). The "identity score" of the aligned segments of the test and reference sequences is the number of common components shared by the two aligned sequences divided by the total number of components in the reference sequence segment, i.e., the entire reference sequence or a smaller defined portion thereof. The sequence identity percentage is expressed as the identity score multiplied by 100. The comparison of one or more polynucleotide sequences can be a comparison of a full-length polynucleotide sequence or a portion thereof, or a comparison with a longer polynucleotide sequence. For the purposes of this invention, the “percentage of identity” can also be determined using BLASTX version 2.0 (for translated nucleotide sequences) and BLASTN version 2.0 (for polynucleotide sequences).

[0083] Two nucleotide sequences can be considered substantially complementary when they hybridize under stringent conditions. In some representative implementations, two nucleotide sequences considered substantially complementary hybridize under highly stringent conditions.

[0084] In nucleic acid hybridization experiments such as Southern and Northern hybridization, "strict hybridization conditions" and "strict hybridization washing conditions" are sequence-dependent and vary under different environmental parameters. Detailed guidelines on nucleic acid hybridization can be found in Tijssen. Laboratory Techniques in Biochemistry and Molecular Biology - Hybridization with Nucleic Acid Probes Part I, Chapter 2, “Overview of principles of hybridization and the strategy of nucleic acid probe assays,” Elsevier, New York (1993). Generally, highly stringent hybridization and washing conditions are selected at specified ionic strengths and pH values, relative to the thermal decomposition temperature (T0) of the specific sequence. m The temperature is about 5°C lower.

[0085] T m This is the temperature at which 50% of the target sequence hybridizes with a perfectly matched probe (at specified ionic strength and pH). Very stringent conditions are chosen to be equal to the Ta of the specific probe. mAn example of stringent hybridization conditions for hybridization of complementary nucleotide sequences with more than 100 complementary residues on a filter membrane in Southern or Northern blots is overnight in 50% formamide containing 1 mg heparin at 42°C. An example of highly stringent washing conditions is washing with 0.15 M NaCl for approximately 15 minutes at 72°C. An example of stringent washing conditions is washing with 0.2x SSC at 65°C for 15 minutes (see Sambrook's description of SSC buffer below). Low-stringent washes are often performed before highly stringent washes to remove background probe signals. An example of a moderately stringent wash for duplexes, for example, exceeding 100 nucleotides, is washing with 1x SSC at 45°C for 15 minutes. An example of a low-stringent wash for duplexes, for example, exceeding 100 nucleotides, is washing with 4–6x SSC at 40°C for 15 minutes. For short probes (e.g., about 10 to 50 nucleotides), stringent conditions typically involve a concentration of less than about 1.0 M Na ions, typically a salt concentration of about 0.01 to 1.0 M Na ions (or other salts) (at pH 7.0 to 8.3), and a temperature typically of at least about 30°C. Adding a destabilizing agent such as formamide can also achieve stringent conditions. Generally, a signal-to-noise ratio (SNR) of 2-fold (or higher) compared to the observed unrelated probes in a specific hybridization assay indicates that specific hybridization has been detected. Nucleotide sequences that do not hybridize under stringent conditions are still substantially identical if they encode substantially the same protein. This can occur, for example, when copies of nucleotide sequences are generated using the maximum codon degeneracy allowed by the genetic code.

[0086] Any nucleotide sequence, polynucleotide, and / or recombinant nucleic acid construct of the present invention can be codon-optimized for expression in any organism of interest. Codon optimization is well known in the art and involves modifying nucleotide sequences for codon usage preferences using species-specific codon usage tables. Codon usage tables are generated based on sequence analysis of the most expressed genes in the organism / species of interest. When the nucleotide sequence is to be expressed in the cell nucleus, the codon usage table is generated based on sequence analysis of highly expressed nuclear genes in the species of interest. Modifications to the nucleotide sequence are determined by comparing the species-specific codon usage table with codons present in the natural polynucleotide sequence. As understood in the art, codon optimization of a nucleotide sequence results in a nucleotide sequence having less than 100% identity with the natural nucleotide sequence (e.g., about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9%, and any range or value therein), but still encoding a polypeptide with the same function as the polypeptide encoded by the original natural nucleotide sequence. Therefore, in some embodiments of the invention, the polynucleotides, nucleic acid constructs, expression cassettes, and / or vectors of the invention (e.g., containing / encoding polypeptides, fusion proteins, complexes, such as modified CRISPR-Cas nucleases) are codon-optimized for expression in specific species of interest, such as specific plant species, specific bacterial species, specific animal species, etc. In some embodiments, the codon-optimized nucleic acid constructs, polynucleotides, expression cassettes, and / or vectors of the present invention have about 70% to about 99.9% (e.g., about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100%) or higher identity with the uncodon-optimized polynucleotides, nucleic acid constructs, expression cassettes, and / or vectors of the present invention.

[0087] In any of the embodiments described herein, the polynucleotide or nucleic acid constructs of the present invention can be operatively associated with a variety of promoters and / or other regulatory elements for expression in plants and / or plant cells. Therefore, in some embodiments, the polynucleotide or nucleic acid constructs of the present invention may further comprise one or more promoters, introns, enhancers, and / or terminators operatively linked to one or more nucleotide sequences. In some embodiments, the promoter may be operatively associated with an intron (e.g., the Ubi1 promoter and introns). In some embodiments, the promoter associated with an intron may be referred to as a “promoter region” (e.g., the Ubi1 promoter and introns).

[0088] The term "operably linked" or "operably associated" as used in reference to polynucleotides herein means that the specified elements are functionally related to each other and are generally also related in physical form. Therefore, the terms "operably linked" or "operably associated" as used herein refer to functionally related nucleotide sequences on a single nucleic acid molecule. Thus, a first nucleotide sequence operably linked to a second nucleotide sequence refers to the case where the first nucleotide sequence and the second nucleotide sequence are functionally related. For example, if a promoter affects the transcription or expression of a nucleotide sequence, then the promoter is operably associated with said nucleotide sequence. Those skilled in the art will understand that a regulatory sequence (e.g., a promoter) does not need to be adjacent to its operably associated nucleotide sequence, as long as the function of the regulatory sequence is to direct its expression. Therefore, for example, an intercalated untranslated but transcribed nucleic acid sequence may exist between the promoter and the nucleotide sequence, and the promoter and nucleotide sequence can still be considered "operably linked."

[0089] As used herein, the term "link" when referring to peptides refers to the attachment of one peptide to another. A peptide can be linked to another peptide directly (e.g., via peptide bonds) or through a linker (at the N-terminus or C-terminus).

[0090] The term "connector" is recognized in the art and refers to a chemical group or molecule that connects two molecules or parts, such as two domains of a fusion protein, such as the LbCas12a CRISPR-Cas nuclease domain, and a polypeptide of interest (e.g., a nucleic acid editing domain, a deaminase domain, adenosine deaminase, cytosine deaminase). A connector may consist of a single linker molecule or may contain more than one linker molecule. In some embodiments, the connector may be an organic molecule, group, polymer, or chemical moiety, such as a divalent organic moiety. In some embodiments, the connector may be an amino acid or a peptide. In some embodiments, the length of the peptide linker can be from about 4 to about 100 or more amino acids, for example, lengths of about 4, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, ... 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 or more amino acids (e.g., lengths of about 4 to about 40, about 4 to about 50, about 4 to about 60, about 5 to about 40). Approximately 5 to 50, approximately 5 to 60, approximately 9 to 40, approximately 9 to 50, approximately 9 to 60, approximately 10 to 40, approximately 10 to 50, approximately 10 to 60 amino acids, or approximately 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 amino acids to approximately 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41. 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 or more amino acids). In some embodiments, the peptide linker may be a GS linker.

[0091] A promoter is a nucleotide sequence that controls or regulates transcription of a nucleotide sequence (e.g., a coding sequence) that is operatively associated with that promoter. The coding sequence controlled or regulated by a promoter can encode polypeptides and / or functional RNA. Generally, a promoter refers to a nucleotide sequence containing an RNA polymerase II binding site that directs the initiation of transcription. Typically, a promoter is located 5' or upstream of the start of the coding region relative to the corresponding coding sequence. Promoters may contain other elements that act as regulators of gene expression; for example, promoter regions. These include TATA box concordant sequences, and often also CAAT box concordant sequences (Breathnach and Chambon, (1981)). Annu. Rev. Biochem 50:349). In plants, the CAAT box can be replaced by the AGGA box (Messing et al., (1983) in Genetic Engineering of Plants, T. Kosuge, C. Meredith and A. Hollaender (eds.), Plenum Press, pp. 211-227). In some embodiments, the promoter region may contain at least one intron (e.g., SEQ ID NO: 40 or SEQ ID NO: 41).

[0092] Promoters that can be used in this invention may include, for example, constitutive, inducible, time-regulated, developmentally regulated, chemically regulated, tissue-biased, and / or tissue-specific promoters for the preparation of recombinant nucleic acid molecules, such as “synthetic nucleic acid constructs” or “protein-RNA complexes.” These different types of promoters are known in the art.

[0093] The choice of promoter can vary depending on the temporal and spatial requirements of expression, as well as the host cell to be transformed. Promoters used for many different organisms are well-known in the art. Based on the extensive knowledge available in the field, a suitable promoter can be selected for a specific host organism of interest. Thus, for example, much is known about promoters upstream of highly constitutively expressed genes in model organisms, and this knowledge can be readily acquired and implemented in other systems where appropriate.

[0094] In some embodiments, promoters functional in plants can be used in the constructs of this invention. Non-limiting examples of promoters that can be used to drive expression in plants include the promoter of the RubisCo small subunit gene 1 (PrbcS1), the promoter of the actin gene (Pactin), the promoter of the nitrate reductase gene (Pnr), and the promoter of the double-copy carbonic anhydrase gene 1 (Pdca1) (see Walker et al.). Plant Cell Rep. 23:727-735 (2005); Li et al. Gene403:132-142 (2007); Li et al. Mol Biol. Rep. 37:1143-1154 (2010)). PrbcS1 and Pactin are constitutive promoters, while Pnr and Pdca1 are inductive promoters. Pnr is nitrate-induced and ammonium-repressed (Li et al.). Gene 403:132-142 (2007), while Pdca1 is salt-induced (Li et al.) Mol Biol. Rep. 37:1143-1154(2010)).

[0095] Examples of constitutive promoters that can be used in plants include, but are not limited to, the Cestrum nocturnum virus promoter (CMP) (US Patent No. 7,166,770) and the rice actin 1 promoter (Wang et al. (1992)). Mol. Cell. Biol. 12:3399-3406; and U.S. Patent No. 5,641,876), CaMV 35S promoter (Odell et al. (1985)). Nature 313:810-812), CaMV 19S promoter (Lawton et al. (1987)) Plant Mol. Biol. 9:315-324), nos promoter (Ebert et al. (1987) Proc. Natl. Acad. Sci USA 84:5745-5749), Adh promoter (Walker et al. (1987) Proc. Natl. Acad. Sci. USA 84:6624-6629), sucrose synthase promoter (Yang and Russell (1990) Proc. Natl. Acad. Sci. USA (87:4144-4148) and ubiquitin promoters. Constitutive promoters derived from ubiquitin accumulate in many cell types. Ubiquitin promoters have been cloned from several plant species for use in transgenic plants, such as sunflower (Binet et al., 1991). Plant Science 79: 87-94), corn (Christensen et al., 1989). Plant Molec. Biol. 12:619-632) and Arabidopsis thaliana (Norris et al. 1993). Plant Molec. Biol. 21:895-906). The maize ubiquitin promoter has been developed in transgenic monocotyledonous plant systems. UbiP The sequence of the ubiquitin promoter and its construction of vectors for monocotyledonous plant transformation are disclosed in patent publication EP 0 342 926. The ubiquitin promoter is suitable for expressing the nucleotide sequence of the present invention in transgenic plants, especially monocotyledonous plants. Furthermore, by McElroy et al. (… Mol. Gen. Genet.The promoter expression cassette described in 231: 150-160 (1991) can be readily modified to express the nucleotide sequence of the present invention and is particularly suitable for monocotyledonous plant hosts.

[0096] In some embodiments, tissue-specific / tissue-preferred promoters can be used to express heterologous polynucleotides in plant cells. Tissue-specific or preferred expression patterns include, but are not limited to, green tissue-specific or preferred, root-specific or preferred, stem-specific or preferred, flower-specific or preferred, or pollen-specific or preferred. Promoters suitable for expression in green tissues include many promoters regulating genes involved in photosynthesis, and many of these have been cloned from monocotyledonous and dicotyledonous plants. In one embodiment, the promoter used in this invention is the corn PEPC promoter (Hudspeth and Grula, from the phosphoenol carboxylase gene). Plant Molec. Biol. 12:579-589 (1989)). Non-restrictive examples of tissue-specific promoters include genes encoding seed storage proteins (such as β-conglycinin, cruciferous proteins, rapeseed storage protein (napin), and bean globulin), zein or oleosome proteins (such as olein), or proteins involved in fatty acid biosynthesis (including acyl carrier proteins, stearoyl-ACP desaturases, and fatty acid desaturases (fad 2-1)), as well as other nucleic acids expressed during embryonic development (such as Bce4, see, for example, Kridl et al. (1991)). Seed Sci. Res.Promoters related to 1:209-219; and European Patent No. 255378. Tissue-specific or tissue-preferred promoters that can be used to express the nucleotide sequences of the present invention in plants, particularly corn, include, but are not limited to, promoters that direct expression in roots, pith, leaves or pollen. For example, such a promoter is disclosed in WO 93 / 07278, the entire text of which is incorporated herein by reference. Other non-limiting examples of tissue-specific or tissue-preferred promoters that can be used in this invention include the cotton ribulose-1,5-bisphosphate carboxylase / oxygenase (rubisco) promoter disclosed in U.S. Patent No. 6,040,504; the rice sucrose synthase promoter disclosed in U.S. Patent No. 5,604,121; the root-specific promoter described by de Framond (FEBS 290:103-106 (1991); EP 0 452269 belonging to Ciba-Geigy); the stem-specific promoter described in U.S. Patent No. 5,625,136 (belonging to Ciba-Geigy), which drives the expression of the corn trpA gene; the night-blooming jasmine leaf curl virus promoter disclosed in WO 01 / 73087; and pollen-specific or tissue-preferred promoters, including but not limited to ProOsLPS10 and ProOsLPS11 from rice (Nguyen et al.). Plant Biotechnol. Reports 9(5):297-306 (2015)), ZmSTK2_USP from corn (Wang et al.) Genome 60(6):485-495 (2017)), LAT52 and LAT59 from tomatoes (Twell et al.) Development 109(3):705-713 (1990)), Zm13 (US Patent No. 10,421,972), PLA2-δ promoter from Arabidopsis thaliana (US Patent No. 7,141,424) and / or ZmC5 promoter from maize (International PCT document No. WO1999 / 042587).

[0097] Other examples of plant tissue-specific / tissue-biased promoters include, but are not limited to, root hair-specific cis-elements (RHE) (Kim et al.). The Plant Cell 18:2958-2970 (2006)), root-specific promoters RCc3 (Jeong et al.) Plant Physiol. 153:185-197 (2010) and RB7 (US Patent No. 5,459,252), lectin promoter (Lindstrom et al. (1990)). Der. Genet. 11:160-167; and Vodkin (1983) Prog. Clin. Biol. Res.138:87-98), Zeaxyl alcohol dehydrogenase 1 promoter (Dennis et al. (1984)) Nucleic Acids Res. 12:3983-4000), S-adenosine-L-methionine synthase (SAMS) (Vander Mijnsbrugge et al. (1996)). Plant and Cell Physiology , 37(8):1108-1115), maize light-collecting complex promoter (Bansal et al. (1992) Proc. Natl. Acad. Sci. USA 89:3654-3658), maize heat shock protein promoter (O'Dell et al. (1985) EMBO J. 5:451-458; and Rochester et al. (1986) EMBO J. 5:451-458), Pea small subunit RuBP carboxylase promoter (Cashmore, "Nuclear genes encoding the small subunit of ribulose-1,5-bisphosphate carboxylase" pp. 29-39, in Genetic Engineering of Plants Hollaender (ed., Plenum Press, 1983); and Poulsen et al. (1986). Mol. Gen. Genet 205:193-200), Ti plasmid mannitol synthase promoter (Langridge et al. (1989)). Proc. Natl. Acad. Sci. USA 86:3219-3223), Ti plasmid carmine synthase promoter (Langridge et al. (1989), see above), petunia chalcone isomerase promoter (van Tunen et al. (1988)). EMBO J. 7:1257-1263), Leguminosae are rich in the glycine protein 1 promoter (Keller et al. (1989)). Genes Dev. 3:1639-1646), truncated CaMV35S promoter (O'Dell et al. (1985)) Nature 313:810-812), potato tuber storage protein (patatin) promoter (Wenzler et al. (1989)). Plant Mol. Biol. 13:347-354), root cell promoter (Yamamoto et al. (1990) Nucleic Acids Res. 18:7449), corn gliadin promoter (Kriz et al. (1987) Mol. Gen. Genet. 207:90-98; Langridge et al. (1983) Cell34:1015-1022; Reina et al. (1990) Nucleic Acids Res. 18:6425; Reina et al. (1990) Nucleic Acids Res. 18:7449; and Wandelt et al. (1989) Nucleic Acids Res. 17:2354), globulin-1 promoter (Belanger et al. (1991) Genetics 129:863-872), α-tubulin cab promoter (Sullivan et al. (1989) Mol. Gen. Genet. 215:431-440), PEPCase promoter (Hudspeth and Grula (1989) Plant Mol. Biol. 12:579-589), R gene complex-related promoters (Chandler et al. (1989) Plant Cell 1:1175-1183) and chalcone synthase promoter (Franken et al. (1991) EMBO J. 10:2605-2612).

[0098] The pea globulin promoter (Czako et al. (1992)) is useful for seed-specific expression. Mol. Gen. Genet. 235:33-40); and the seed-specific promoter disclosed in U.S. Patent No. 5,625,136. Promoters that can be used for expression in mature leaves are those that switch at the onset of senescence, such as the SAG promoter from Arabidopsis thaliana (Gan et al. (1995)). Science 270:1986-1988).

[0099] In addition, promoters that function in chloroplasts can be used. Non-limiting examples of such promoters include the phage T3 gene 9 5' UTR and other promoters disclosed in U.S. Patent No. 7,579,516. Other promoters that can be used in this invention include, but are not limited to, the S-E9 small subunit RuBP carboxylase promoter and the Kunitz-type trypsin inhibitor gene promoter (Kti3).

[0100] Other control elements that can be used in this invention include, but are not limited to, introns, enhancers, termination sequences and / or 5' and 3' untranslated regions.

[0101] Introns that can be used in this invention can be introns identified and isolated from plants, and then inserted into expression cassettes for plant transformation. As those skilled in the art will understand, introns can contain sequences required for self-splicing and are integrated into nucleic acid constructs / expression cassettes in a frame-compliant manner. Introns can be used as spacers to separate multiple protein-coding sequences within a nucleic acid construct, or introns can be used within a protein-coding sequence to, for example, stabilize mRNA. If they are used within a protein-coding sequence, they are inserted “frame-compliantly” with the included splicing site. Introns may also be associated with promoters to improve or modify expression. For example, promoter / intron combinations that can be used in this invention include, but are not limited to, combinations of the corn Ubi1 promoter and introns.

[0102] Non-limiting examples of introns that can be used in this invention include introns from the following genes: ADHI gene (e.g., Adh1-S Introns 1, 2, and 6), ubiquitin gene (Ubi1), RuBisCO small subunit (rbcS) gene, RuBisCO large subunit (rbcL) gene, actin gene (e.g., actin-1 The nucleic acid constructs of the present invention may encode a base editor comprising an optimized CRISPR-Cas nuclease (e.g., SEQ ID NO: 1-11 or 23-25) and a deaminase, wherein the nucleic acid construct further comprises a promoter containing an intron or associated with an intron. As another non-limiting example, the nucleic acid constructs of the present invention may encode a base editor comprising an optimized CRISPR-Cas nuclease (e.g., SEQ ID NO: 1-11 or 23-25) and a deaminase, wherein the nuclease and / or deaminase contains one or more introns, and optionally, the nucleic acid construct further comprises a promoter containing an intron or associated with an intron.

[0103] In some embodiments, the polynucleotide and / or nucleic acid constructs of the present invention may be "expression cassettes" or may be contained within an expression cassette. As used herein, "expression cassette" means a recombinant nucleic acid molecule containing, for example, a nucleic acid construct of the present invention (e.g., encoding the modified LbCas12a of the present invention), wherein the nucleic acid construct is operatively associated with at least one regulatory sequence (e.g., a promoter). Therefore, some embodiments of the present invention provide expression cassettes designed for expressing, for example, nucleic acid constructs of the present invention (e.g., nucleic acid constructs of the present invention encoding the modified LbCas12a of the present invention).

[0104] The expression cassette containing the nucleic acid construct of the present invention can be chimeric, meaning that at least one of its components is heterologous relative to at least one of its other components (e.g., a promoter from a host organism is operatively linked to a polynucleotide of interest to be expressed in the host organism, wherein the polynucleotide of interest originates from an organism different from the host, or is normally found not to be associated with the promoter). The expression cassette can also be naturally occurring, but has been obtained in a recombinant form that can be used for heterologous expression.

[0105] The expression cassette may optionally include functional transcription and / or translation termination regions (i.e., termination regions) and / or enhancer regions in selected host cells. Various transcription terminators and enhancers are known in the art and can be used in the expression cassette. Transcription terminators are responsible for the termination of transcription and proper mRNA polyadenylation. The termination and / or enhancer regions may be natural with respect to the transcription initiation region, natural with respect to the gene encoding, for example, the LbCas12a nuclease encoded by the nucleic acid construct of the present invention, natural with respect to the host cell, or natural with respect to other sources (e.g., exogenous or heterologous with respect to the promoter, the gene encoding the LbCas12a nuclease encoded by the nucleic acid construct of the present invention, the host cell, or any combination thereof). The enhancer region may be natural with respect to the gene encoding the LbCas12a nuclease encoded by the nucleic acid construct of the present invention, natural with respect to the host cell, or may be derived from other sources (e.g., exogenous or heterogeneous with respect to the promoter, the gene encoding the LbCas12a nuclease encoded by the nucleic acid construct of the present invention, the host cell, or any combination thereof).

[0106] The expression cassette of the present invention may also include a nucleotide sequence encoding a selectable marker that can be used to select transformed host cells. As used herein, "selectable marker" means a nucleotide sequence that, when expressed, confers a unique phenotype on host cells expressing the marker, thereby allowing differentiation of such transformed cells from those without the marker. This nucleotide sequence may encode a selectable or screenable marker, depending on whether the marker confers a trait that can be selected by chemical means, such as by using a selector (e.g., antibiotics), or whether the marker is simply a trait that can be identified by observation or testing, such as by screening (e.g., fluorescence). Many examples of suitable selectable markers are known in the art and can be used in the expression cassette described herein.

[0107] In addition to expression cassettes, the nucleic acid molecules / constructs and polynucleotide sequences described herein can be used in conjunction with vectors. The term "vector" refers to a composition used to transfer, deliver, or introduce nucleic acids (or multiple nucleic acids) into cells. A vector contains a nucleic acid construct that contains one or more nucleotide sequences to be transferred, delivered, or introduced. Vectors for transforming host organisms are well known in the art. Non-limiting examples of general vector classes include viral vectors, plasmid vectors, bacteriophage vectors, phage particle vectors, fosmid vectors, bacteriophages, artificial chromosomes, minicircles, or Agrobacterium binary vectors, in double-stranded or single-stranded linear or circular form, which may or may not be self-transmitting or mobile. In some embodiments, viral vectors may include, but are not limited to, retroviruses, lentiviruses, adenoviruses, adeno-associated viruses, or herpes simplex virus vectors. Vectors as defined herein can transform prokaryotic or eukaryotic hosts by integration into the cellular genome or by presence outside the chromosome (e.g., autonomously replicating plasmids with origins of replication). Furthermore, shuttle vectors are also included, which are DNA media capable of replicating naturally or by design in two different host organisms, selectable from actinomycetes and related species, bacteria, and eukaryotes (e.g., higher plants, mammals, yeast, or fungal cells). In some embodiments, the nucleic acids in the vector are controlled and operatively linked to a suitable promoter or other regulatory element for transcription in a host cell. The vector can be a bifunctional expression vector that functions in multiple hosts. In the case of genomic DNA, this may contain its own promoter and / or other regulatory elements, while in the case of cDNA, this may be controlled by a suitable promoter and / or other regulatory elements for expression in a host cell. Accordingly, the nucleic acid constructs of the present invention and / or expression cassettes containing them can be contained in vectors as described herein and known in the art. In some embodiments, the vector can be a high copy number vector (e.g., a high copy number E. coli vector; e.g., pUC, pBluescript, pGEM, etc.). Thus, for example, high copy number vectors can be used to construct the libraries of the present invention.

[0108] As used herein, “contact,” “contacting,” “contacted,” and their grammatical variations refer to bringing together components of a desired reaction under conditions suitable for carrying out the desired reaction (e.g., transformation, transcriptional regulation, genome editing, nicking, and / or cleavage). Thus, for example, a target nucleic acid can be contacted with (a) a polynucleotide and / or nucleic acid construct of the present invention encoding the modified LbCas12a nuclease and (b) a guide nucleic acid under conditions of expressing the polynucleotide / nucleic acid construct and producing the modified LbCas12a nuclease, wherein the nuclease forms a complex with the guide nucleic acid, and the complex hybridizes with the target nucleic acid, thereby modifying the target nucleic acid. In some embodiments, the target nucleic acid may be contacted with (a) the modified LbCas12a nuclease of the present invention and / or a fusion protein comprising therein (e.g., the modified LbCas12a nuclease of the present invention and a polypeptide of interest (e.g., a deaminase)) and (b) a guide nucleic acid, wherein the modified LbCas12a nuclease forms a complex with the guide nucleic acid, and the complex hybridizes with the target nucleic acid, thereby modifying the target nucleic acid. As described herein, the target nucleic acid may be contacted with the polynucleotide / nucleic acid construct / peptide of the present invention before, simultaneously with, or after contact with the guide nucleic acid.

[0109] As used herein, “modifying” or “modification” when referring to a target nucleic acid includes editing the target nucleic acid (e.g., mutation), covalent modification, exchange / substitution of nucleic acid / nucleotide bases, deletion, cleavage, nicking, and / or transcriptional regulation.

[0110] In the context of the polynucleotide of interest, “introducing,” “introduce,” “introduced” (and its grammatical variations) means presenting a nucleotide sequence of interest (e.g., a polynucleotide, a nucleic acid construct, and / or a guide nucleic acid) to a host organism or the cells of said organism (e.g., host cells; such as plant cells) in a manner that allows the nucleotide sequence to enter the cell. Thus, for example, the polynucleotide of the present invention encoding a modified LbCas12a nuclease as described herein can be introduced into the cells of an organism, thereby transforming the cells with the modified LbCas12a nuclease and the guide nucleic acid.

[0111] As used herein, the term "transformation" refers to the introduction of a heterologous nucleic acid into a cell. Cellular transformation can be stable or transient. Therefore, in some embodiments, the polynucleotide / nucleic acid molecules of the present invention can be used to stably transform host cells or host organisms. In some embodiments, the polynucleotide / nucleic acid constructs of the present invention can be used to transiently transform host cells or host organisms.

[0112] In the context of polynucleotides, "transient conversion" refers to the introduction of polynucleotides into cells without their integration into the cell's genome.

[0113] In the context of introducing polynucleotides into cells, "stably introducing" or "stably introduced" means that the introduced polynucleotide is stably integrated into the cell's genome, thereby stably transforming the cell with that polynucleotide.

[0114] As used herein, "stable transformation" or "stably transformed" refers to the introduction of nucleic acid molecules into cells and their integration into the cell's genome. Therefore, the integrated nucleic acid molecules can be inherited by their offspring, more specifically, by multiple successive generations. As used herein, "genome" includes both the nuclear genome and the plastid genome, and thus includes the integration of nucleic acids into, for example, the chloroplast or mitochondrial genome. Stable transformation, as used herein, can also refer to transgenes maintained outside of chromosomes, for example, as microchromosomes or plasmids.

[0115] Transient transformation can be detected, for example, by enzyme-linked immunosorbent assay (ELISA) or Western blotting, which can detect the presence of peptides or polypeptides encoded by one or more transgenes introduced into the organism. Stable transformation of cells can be detected, for example, by Southern blotting of cellular genomic DNA with a nucleic acid sequence that specifically hybridizes to the nucleotide sequence of the transgene introduced into the organism (e.g., a plant). Stable transformation of cells can also be detected, for example, by Northern blotting of cellular RNA with a nucleic acid sequence that specifically hybridizes to the nucleotide sequence of the transgene introduced into the host organism. Stable transformation of cells can also be detected, for example, by polymerase chain reaction (PCR) or other amplification reactions well known in the art, using specific primer sequences that hybridize to the target sequence of the transgene, which amplifies the transgene sequence, which can be detected according to standard methods. Transformation can also be detected by direct sequencing and / or hybridization protocols well known in the art.

[0116] Accordingly, in some embodiments, the nucleotide sequences, polynucleotides and / or nucleic acid constructs of the present invention and / or expression cassettes and / or vectors containing them can be transiently expressed and / or stably integrated into the genome of a host organism. Thus, in some embodiments, the nucleic acid constructs of the present invention (e.g., encoding the modified LbCas12a nuclease of the present invention or a fusion protein thereof; e.g., a fusion protein containing a modified LbCas12a nuclease linked to, for example, a polynucleotide of interest (e.g., a deaminase domain)) can be transiently introduced into the cells of an organism along with a guide nucleic acid such that the DNA is not maintained in the cell, wherein the nucleic acid construct encoding the modified LbCas12a nuclease is codon-optimized for expression in an organism (e.g., a plant, mammal, fungus, bacteria, etc.).

[0117] The polynucleotide / nucleic acid constructs of the present invention can be introduced into cells using any method known to those skilled in the art. In some embodiments of the invention, cell transformation includes nuclear transformation. In other embodiments, cell transformation includes plastid transformation (e.g., chloroplast transformation). In still other embodiments, the polynucleotide / nucleic acid constructs of the present invention can be introduced into cells using conventional breeding techniques.

[0118] Procedures for transforming eukaryotes and prokaryotes are well-known and routine in the field, and are described throughout the literature (see, for example, Jiang et al., 2013). Nat. Biotechnol 31:233-239; Ran et al. Nature Protocols 8:2281–2308 (2013)).

[0119] Therefore, nucleotide sequences can be introduced into a host organism or its cells in a variety of ways well known in the art. The methods of the present invention do not depend on a specific method of introducing one or more nucleotide sequences into an organism, but only on their ability to enter the interior of at least one cell of the organism. In the case of introducing more than one nucleotide sequence, they can be assembled as part of a single nucleic acid construct, or assembled as separate nucleic acid constructs, and can be located on the same or different nucleic acid constructs. Accordingly, nucleotide sequences can be introduced into cells of interest in a single transformation event and / or in separate transformation events, or alternatively, in relevant cases, such as as part of a breeding program, nucleotide sequences can be incorporated into plants.

[0120] This invention relates to Cas12a nucleases modified to include non-natural PAM recognition sites / sequences (e.g., a Cas12a nuclease that includes non-natural PAM recognition specificity in addition to or in lieu of the natural PAM recognition specificity of that particular Cas12a nuclease). Furthermore, this invention relates to methods for designing, identifying, and selecting Cas12a nucleases having desired properties, including improved PAM recognition specificity.

[0121] As used herein, when referring to modified Cas12a peptides, "altered PAM specificity" means that the PAM specificity of the nuclease has changed from that of the wild-type nuclease (e.g., recognizing non-natural PAM sequences in addition to and / or replacing the natural PAM sequence). For example, if a modified Cas12a nuclease recognizes a PAM sequence different from the natural Cas12a PAM sequence TTTV (where V is A, C, or G) and / or also recognizes PAM sequences other than the natural Cas12a PAM sequence TTTV, then its PAM specificity is altered.

[0122] This invention relates to an LbCas12a nuclease having modified PAM recognition specificity. In some embodiments, the invention provides a modified CRISPR (clustered regular spaced short palindromic repeat) Cas12a (LbCas12a) polypeptide from the family Trichophyceae, wherein the modified LbCas12a polypeptide comprises the following amino acid sequence having at least 80% identity with the amino acid sequence SEQ ID NO: 1 (LbCas12a) (e.g., about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identity; e.g., about 80% to about 100%, about 85% to about 100%, about 90% to about 100%, about 95% to about 100%), and refers to SEQ ID NO: Position number 1, having a mutation at one or more of the following positions: K116, K120, K121, D122, E125, T148, T149, T152, D156, E159, Q529, G532, D535, K538, D541, Y542, L585, K591, M592, K595, V596, S599, K600, K601, Y616, Y646 and / or W649 (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or more), optionally, refer to SEQ ID NO: Position number 1, having a mutation at one or more of the following positions: K116, K120, K121, D122, E125, T152, D156, E159, G532, D535, K538, D541, and / or K595. In some embodiments, the mutation of the Cas12a (LbCas12a) polypeptide comprises, is substantially composed of, or consists of the following mutations, i.e., referring to position number SEQ ID NO: 1, a mutation at one or more of the following positions: K116, K120, K121, D122, E125, T152, D156, E159, G532, D535, K538, D541, and / or K595 in any combination.Therefore, referring to the position number in SEQ ID NO: 1, the modified LbCas12a polypeptide of the present invention may contain a single mutation at any one of the following positions: K116, K120, K121, D122, E125, T148, T149, T152, D156, E159, Q529, G532, D535, K538, D541, Y542, L585, K591, M592, K595, V596, S599, K600, K601, Y616, Y646 and / or W649, or refer to SEQ ID NO: The position number 1 can contain a combination of mutations at any two or more of the following positions: K116, K120, K121, D122, E125, T148, T149, T152, D156, E159, Q529, G532, D535, K538, D541, Y542, L585, K591, M592, K595, V596, S599, K600, K601, Y616, Y646, and / or W649.

[0123] In some embodiments, referring to the position number of SEQ ID NO: 1, the mutation of the Cas12a (LbCas12a) polypeptide comprises one or more of the following mutations, and is substantially composed of one or more of the following mutations: K116N, K116R, K120H, K120N, K120Q, K120R, K120T, K121D, K121G, K121H, K121Q, K121R, K121S, K121T, D122H, D122K, D122N, D122R, E125K, E125Q, E125R, E125Y, T148A, T148C, T148H, T148S, T149C, T149F, T149G, T149H, T149N, T1 49P, T149S, T149V, T152E, T152F, T152H, T152K, T152L, T152Q, T152R, T1 52W, T152Y, D156E, D156H, D156I, D156K, D156L, D156Q, D156R, D156W, D1 56Y, E159K, E159Q, E159R, E159Y, Q529A, Q529D, Q529F, Q529G, Q529H, Q5 29N, Q529P, Q529S, Q529T, Q529W, G532A, G532C, G532D, G532F, G532H, G53 2K, G532L, G532N, G532Q, G532S, D535A, D535H, D535K, D535N, D535S, D53 5T, D535V, K538C, K538F, K538G, K538H, K538L, K538M, K538Q, K538R, K53 8V, K538W, K538Y, D541A, D541E, D541H, D541I, D541N, D541R, D541Y, Y54 2F, Y542H, Y542K, Y542L, Y542M, Y542N, Y542R, Y542T, Y542V, L585F, L585 G, L585H, K591A, K591F, K591G, K591H, K591R, K591S, K591W, K591Y, M592 A. M592E, M592Q, K595H, K595L, K595M, K595Q, K595R, K595S, K595W, K595Y , V596H, V596T, S599G, S599H, S599N, K600G, K600H, K600R, K601H, K601Q , K601R, K601T, Y616E, Y616F, Y616H, Y616K, Y616R, Y646E, Y646H, Y646K,Y646N, Y646Q, Y646R, Y646W, W649H, W649K, W649R, W649S, and / or W649Y. It is understood that any single Cas12a polypeptide having two or more mutations will contain only a single mutation at any given position. Therefore, for example, a polypeptide may have a mutation at position D535 of any one of D535A, D535H, D535K, D535N, D535S, D535T, or D535V, but the same polypeptide may also contain mutations at one or more of any other positions as described herein. In some embodiments, referring to the residue position number of SEQ ID NO: 1, Cas12a Mutations in the (LbCas12a) polypeptide include one or more of any combination of the following mutations, or are substantially composed of one or more of any combination of the following mutations: K116N, K116R, K120H, K120N, K120Q, K120R, K120T, K121D, K121G, K121H, K121Q, K121R, K121S, K121T. D122H, D122K, D122N, D122R, E125K, E125Q, E125R, E125Y, T152E, T152F, T152H, T152K, T 152L, T152Q, T152R, T152W, T152Y, D156E, D156H, D156I, D156K, D156L, D156Q, D156R, D15 6W, D156Y, E159K, E159Q, E159R, E159Y, G532A, G532C, G532D, G532F, G532H, G532K, G532 L, G532N, G532Q, G532S, D535A, D535H, D535K, D535N, D535S, D535T, D535V, K538C, K538F K538G, K538H, K538L, K538M, K538Q, K538R, K538V, K538W, K538Y, D541A, D541E, D541H, D541I, D541N, D541R, D541Y, K595H, K595L, K595M, K595Q, K595R, K595S, K595W and / or K595Y. In some embodiments, referring to the position number of SEQ ID NO: 1, the mutation of the Cas12a (LbCas12a) polypeptide includes one or more of the following mutations, and is substantially composed of one or more of the following mutations: K116R, K116N, K120Y, K121S, K121R, D122H, D122N, E125K, T152R, T152K, T152Y, T152Q, T152E.T152F, D156R, D156W, D156Q, D156H, D156I, D156V, D156L, D156E, E159K, E159R , G532N, G532S, G532H, G532K, G532R, G532L, D535N, D535H, D535T, D535S, D535 A, D535W, K538R, K538V, K538Q, K538W, K538Y, K538F, K538H, K538L, K538M, K538C, K538G, K538A, D541E, K595R, K595Q, K595Y, K595W, K595H, K595S, and / or K595M. It is understood that any single Cas12a polypeptide with two or more mutations will contain a single mutation at any given position. Therefore, for example, a polypeptide may have a mutation at position D535 of any of the following: D535A, D535H, D535K, D535N, D535S, D535T, or D535V, and may also contain mutations at one or more of any other positions as described herein.

[0124] In some embodiments, referring to the position number of SEQ ID NO: 1, the mutation does not include, is not substantially composed of, or is not composed of, the mutations of D156R, G532R, K538R, K538V, Y542R, or K595R. In some embodiments, referring to the position number of SEQ ID NO: 1, the mutation of the Cas12a (LbCas12a) polypeptide does not include the mutation combination of G532R and K595R, the mutation combination of G532R, K538V, and Y542R, or the mutation combination of D156R, G532R, and K532R, and is not substantially composed of or is not composed of the mutation combination.

[0125] In some embodiments, the modified LbCas12a polypeptide may contain one or more amino acid mutations of SEQ ID NO: 1 as listed in Table 2 (in Example 2).

[0126] In some embodiments, the modified LbCas12a peptide may contain altered prespacer neighbor motif (PAM) specificity compared to wild-type LbCas12a (e.g., SEQ ID NO: 1). The modified LbCas12a peptide of the present invention may contain altered PAM specificity, wherein the altered PAM specificity includes, but is not limited to, NNNG, NNNT, NNNA, NNNC, NNG, NNT, NNC, NNA, NG, NT, NC, NA, NN, NNN, NNNN, wherein each N in each sequence is independently selected from any one of T, C, G, or A. In some embodiments, the altered PAM specificity may include, but is not limited to, TTTA, TTTC, TTTG, TTTT, TTCA, TTCC, TTCG, TTCT, ATTC, CTTA, CTTC, CTTG, GTTC, TATA, TATC, CTCC, TCCG, TACA, TCCG, TACA, TCCG, TCCC, TCCA, and / or TATG. In some implementations, the modified PAM specificity can be NNNN, where each N of each sequence is independently selected from any one of T, C, G, or A.

[0127] In addition to altered PAM recognition specificity, modified LbCas12a nucleases may also contain mutations (e.g., inactivation of LbCas12a, dLbCas12a) in the nuclease active site (e.g., RuvC domain). This modification may result in reduced or absent nuclease activity (e.g., nickase activity) of the LbCas12a peptide.

[0128] In some embodiments, a CRISPR-Cas (CRISPR-Cas) system is provided, comprising: (a) a fusion protein comprising (i) the modified LbCas12a nuclease of the present invention, or a nucleic acid encoding the modified LbCas12a nuclease of the present invention, and (ii) a polypeptide of interest or a nucleic acid encoding the polypeptide of interest; and (b) a guide nucleic acid (CRISPR RNA, CRISPR DNA, crRNA, crDNA) comprising a spacer sequence and a repeat sequence, wherein the guide nucleic acid is capable of forming a complex with the modified LbCas12a nuclease or the fusion protein, and the spacer sequence is capable of hybridizing with a target nucleic acid to guide the modified LbCas12a nuclease and the polypeptide of interest to the target nucleic acid, thereby enabling the system to modify (e.g., cleave or edit) or regulate (e.g., regulate transcription) the target nucleic acid. In some embodiments, the system comprises a polypeptide of interest linked to the C-terminus and / or N-terminus of a modified LbCas12a nuclease (e.g., a fusion protein), optionally linked via a peptide linker.

[0129] Furthermore, this document provides fusion proteins comprising the modified Cas12a nuclease of the present invention. In some embodiments, the fusion protein may comprise a polypeptide of interest linked to the C-terminus and / or N-terminus of the modified LbCas12a. In some embodiments, the present invention provides a fusion protein comprising a modified LbCas12a and optionally an intercalator linking the polypeptide of interest.

[0130] Any linker known in the art or subsequently identified that does not interfere with the activity of the fusion protein can be used. A linker that does not “interfere” with the activity of the fusion protein is one that does not reduce or eliminate the activity of the peptide (e.g., a nuclease and / or peptide of interest) of the fusion protein; that is, the nuclease activity, nucleic acid binding activity, editing activity, and / or any other activity of the nuclease or peptide of interest is maintained in the fusion protein, wherein the nuclease and the peptide of interest are tethered to each other via the linker. In some embodiments, the peptide linker may be attached (e.g., at its N-terminus) to the C-terminus of the modified LbCas12a, optionally wherein the fusion protein may also contain the peptide of interest attached to the C-terminus of the linker. In some embodiments, the peptide linker may be attached (e.g., at its C-terminus) to the N-terminus of the modified LbCas12a, optionally wherein the fusion protein may also contain the peptide of interest attached to the N-terminus of the linker. In some embodiments, the modified LbCas12a of the present invention may be attached to both its C-terminus and N-terminus to the linker and / or the peptide of interest (directly or via the linker).

[0131] In some embodiments, the linker used in this invention can be an amino acid or a peptide. In some embodiments, the length of the peptide linker used in this invention can be from about 4 to about 100 or more amino acids, for example, lengths of about 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52. 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 or more amino acids (e.g., lengths of about 4 to about 40, about 4 to about 50, about 4 to about 60, about 5 to about 40, about 5 to about 50, about 5 to about 60, about 9 to about 40, about 9 to about 50, about 9 to about 60, about 10 to about 40, about 10 to about 50, about 10 to about 60 amino acids, or about 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 amino acids to about 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 4 1, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 or more amino acids). In some embodiments, the peptide linker may be a GS linker.

[0132] The polypeptides of interest that may be used in this invention may include, but are not limited to, polypeptide or protein domains having the following activities: deaminase (deamination) activity, nickase activity, recombinase activity, transposase activity, methyltransferase activity, glycosylationase (DNA glycosylationase) activity, glycosylationase inhibitor activity (e.g., uracil-DNA glycosylation inhibitor (UGI)), demethylase activity, transcriptional activation activity, transcriptional repression activity, transcription release factor activity, histone modification activity, nuclease activity, single-stranded RNA cleavage activity, double-stranded RNA cleavage activity, restriction endonuclease activity (e.g., Fok1), nucleic acid binding activity, methyltransferase activity, DNA repair activity, DNA damage activity, dismutase activity, alkylation activity, depurination activity, oxidation activity, pyrimidine dimer formation activity, integrase activity, transposase activity, polymerase activity, ligase activity, helicase activity, and / or photolyase activity.

[0133] In some embodiments, the polypeptide of interest may comprise at least one polypeptide or protein domain having deaminase activity. In some embodiments, said at least one polypeptide or protein domain may be an adenine deaminase domain. The adenine deaminase (or adenosine deaminase) that can be used in this invention may be any known or subsequently identified adenine deaminase from any organism (see, for example, U.S. Patent No. 10,113,163, which discloses adenine deaminases incorporated herein by reference). Adenine deaminases can catalyze the hydrolytic deamination of adenine or adenosine. In some embodiments, adenine deaminases can catalyze the hydrolytic deamination of adenosine or deoxyadenosine to inosine or deoxyinosine, respectively. In some embodiments, adenosine deaminases can catalyze the hydrolytic deamination of adenine or adenosine in DNA. In some embodiments, the adenine deaminase encoded by the nucleic acid construct of the present invention can generate an A→G transition in the sense (e.g., "+", template) strand of the target nucleic acid, or a T→C transition in the antisense (e.g., "-", complementary) strand of the target nucleic acid.

[0134] In some embodiments, the adenosine deaminase may be a variant of a naturally occurring adenine deaminase. Therefore, in some embodiments, the adenosine deaminase used in the present invention may have about 70% to 100% identity with wild-type adenine deaminase (e.g., about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, and any range or value thereof, with naturally occurring adenine deaminase). In some embodiments, one or more deaminases are not found in nature and may be referred to as engineered, mutant, or evolved adenosine deaminases. Therefore, for example, engineered, mutated, or evolved adenine deaminase peptides or adenine deaminase domains may share approximately 70% to 99.9% identity with naturally occurring adenine deaminase peptides / domains (e.g., approximately 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9%, and any range or value thereof). In some implementations, adenosine deaminase can be derived from bacteria (e.g., Escherichia coli, Staphylococcus aureus). Staphylococcus aureus Haemophilus influenzae () Haemophilus influenzae ), Crested Bacillus ( Caulobacter crescentus (etc.). In some implementations, the polynucleotide encoding the adenine deaminase polypeptide / domain can be codon-optimized for expression in an organism.

[0135] In some implementations, the adenine deaminase domain can be a wild-type tRNA-specific adenine deaminase domain, such as tRNA-specific adenine deaminase (TadA), and / or a mutated / evolved adenine deaminase domain, such as a mutated / evolved tRNA-specific adenine deaminase domain (TadA). In some embodiments, the TadA domain may be derived from *E. coli*. In some embodiments, TadA may be modified, for example, by truncating or deleting one or more N-terminal and / or C-terminal amino acids relative to full-length TadA (e.g., possibly deleting 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 N-terminal and / or C-terminal amino acid residues relative to full-length TadA). In some embodiments, the TadA polypeptide or TadA domain does not contain an N-terminal methionine. In some embodiments, wild-type *E. coli* TadA contains the amino acid sequence SEQ ID NO: 18. In some embodiments, mutant / evolved *E. coli* TadA... It contains the amino acid sequence SEQ ID NO: 19-22. In some embodiments, it can encode TadA / TadA. The polynucleotides are codon-optimized for expression in organisms.

[0136] The cytosine deaminase (or cytidine deaminase) that can be used in this invention can be any known or subsequently identified cytosine deaminase from any organism (see, for example, U.S. Patent No. 10,167,457, which discloses cytosine deaminases incorporated herein by reference). In some embodiments, the at least one polypeptide or protein domain can be a cytosine deaminase polypeptide or domain. In some embodiments, the cytosine deaminase polypeptide / domain can be an apolipoprotein B mRNA editing catalytic polypeptide-like (APOBEC) domain. In some embodiments, the polypeptide of interest may comprise at least one polypeptide or protein domain having glycosylation inhibitor activity. In some embodiments, the polypeptide of interest can be a uracil-DNA glycosylation inhibitor (UGI) polypeptide / domain. In some embodiments, the nucleic acid construct encoding the modified LbCas12a nuclease and cytosine deaminase domain of the present invention (e.g., encoding a fusion protein comprising the modified LbCas12a nuclease and cytosine deaminase domain) may also encode a uracil-DNA glycosylase inhibitor (UGI), wherein the UGI is codon-optimized for expression in a organism. In some embodiments, the present invention provides a fusion protein comprising a modified LbCas12a nuclease, a cytosine deaminase domain, and a UGI, and / or one or more polynucleotides encoding them, optionally wherein the one or more polynucleotides are codon-optimized for expression in a organism.

[0137] Cytosine deaminases catalyze the hydrolytic deamination of cytidine or deoxycytidine to uridine or deoxyuridine, respectively. In some embodiments, the deaminase or deaminase domain may be a cytidine deaminase domain, catalyzing the hydrolytic deamination of cytosine to uracil. In some embodiments, the cytosine deaminase may be a variant of a naturally occurring cytosine deaminase (including, but not limited to, primate (e.g., human, monkey, chimpanzee, gorilla), dog, cow, rat, or mouse). Therefore, in some embodiments, the cytosine deaminase used in this invention may have about 70% to 100% identity with wild-type cytosine deaminase (e.g., about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, and any range or value thereof, with naturally occurring cytosine deaminases). In some embodiments, the polynucleotide encoding the cytosine deaminase polypeptide / domain may be codon-optimized for expression in organisms.

[0138] In some embodiments, the cytosine deaminase used in this invention may be an apolipoprotein B mRNA editing complex (APOBEC) family deaminase. In some embodiments, the cytosine deaminase may be APOBEC1 deaminase, APOBEC2 deaminase, APOBEC3A deaminase, APOBEC3B deaminase, APOBEC3C deaminase, APOBEC3D deaminase, APOBEC3F deaminase, APOBEC3G deaminase, APOBEC3H deaminase, APOBEC4 deaminase, human activation-inducible deaminase (hAID), rAPOBEC1, FERNY and / or CDA1, optionally pmCDA1, atCDA1 (e.g., At2g19570), and their evolved forms. In some embodiments, the cytosine deaminase may be an APOBEC1 deaminase having the amino acid sequence SEQ ID NO: 23, SEQ ID NO: 44, or SEQ ID NO: 46. In some embodiments, the cytosine deaminase may be APOBEC3A deaminase having the amino acid sequence SEQ ID NO: 24. In some embodiments, the cytosine deaminase may be CDA1 deaminase, optionally CDA1 having the amino acid sequence SEQ ID NO: 25 or SEQ ID NO: 43. In some embodiments, the cytosine deaminase may be FERNY deaminase, optionally FERNY having the amino acid sequence SEQ ID NO: 42 or SEQ ID NO: 45. In some embodiments, the cytosine deaminase may be a human activation-inducible deaminase (hAID) having the amino acid sequence SEQ ID NO: 47 or SEQ ID NO: 48. In some embodiments, the cytosine deaminases used in this invention may have about 70% to about 100% identity with the amino acid sequence of naturally occurring cytosine deaminases (e.g., evolved deaminases) (e.g., 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100%).In some embodiments, the cytosine deaminase used in this invention may share about 70% to about 99.5% identity with the amino acid sequences SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25 or SEQ ID NO: 42-48 (e.g., about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.5% identity) (e.g., with the amino acid sequences SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25 or SEQ ID NO: 42-48). 42-48 exhibit at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identity. In some embodiments, the polynucleotide encoding cytosine deaminase may be codon-optimized for expression in an organism, and the codon-optimized polypeptide may be approximately 70% to 99.5% identical to the reference polynucleotide.

[0139] The "uracil glycosylation enzyme inhibitor" (UGI) used in this invention can be any protein capable of inhibiting uracil-DNA glycosylation enzyme base excision repair enzyme. In some embodiments, the UGI domain comprises wild-type UGI or a fragment thereof. In some embodiments, the UGI domain used in this invention can have about 70% to about 100% identity with the amino acid sequence of a naturally occurring UGI domain (e.g., about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100%, and any range or value thereof). In some embodiments, the UGI domain may contain the amino acid sequence SEQ ID NO: 26 or a polypeptide having about 70% to about 99.5% identity with the amino acid sequence SEQ ID NO: 26 (e.g., having at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identity with the amino acid sequence SEQ ID NO: 26). For example, in some embodiments, the UGI domain may contain a fragment of the amino acid sequence SEQ ID NO: 26 that is 100% identical to a portion of the amino acid sequence SEQ ID NO: 26 consisting of consecutive nucleotides (e.g., about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80 consecutive nucleotides; e.g., about 10, 15, 20, 25, 30, 35, 40, 45 to about 50, 55, 60, 65, 70, 75, 80 consecutive nucleotides). In some embodiments, the UGI domain may be a variant of a known UGI (e.g., SEQ ID NO: 26) having 70% to 99.5% identity with a known UGI (e.g., about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, and any range or value thereof). In some embodiments, the polynucleotide encoding UGA may be codon-optimized for expression in an organism, and the codon-optimized polypeptide may have about 70% to 99.5% identity with a reference polynucleotide.

[0140] In some embodiments, the modified LbCas12a nuclease may contain a mutation in its nuclease active site (e.g., RuvC). A modified LbCas12a nuclease that has a mutation in its nuclease active site and no longer contains nuclease activity is generally referred to as "inactive," such as dLbCas12a. In some embodiments, the activity (e.g., nickase activity) of the modified LbCas12a domain or polypeptide with a mutation in its nuclease active site may be impaired or reduced compared to the same LbCas12a nuclease without the mutation.

[0141] The modified LbCas12a nuclease of the present invention can be used in combination with a guide RNA (gRNA, CRISPR array, CRISPRRNA, crRNA) designed to work in conjunction with the modified LbCas12a nuclease to modify target nucleic acids. The guide nucleic acid used in the present invention comprises at least a spacer sequence and a repeat sequence. The guide nucleic acid is capable of forming a complex with the LbCas12a nuclease domain encoded and expressed by the polynucleotide / nucleic acid construct of the present invention (encoding the modified LbCas12a nuclease), and the spacer sequence is capable of hybridizing with the target nucleic acid, thereby guiding the nucleic acid construct (e.g., the modified LbCas12a nuclease (and / or the polypeptide of interest)) to the target nucleic acid, wherein the modified LbCas12a nuclease (and / or the encoded deaminase domain and / or the polypeptide of interest) can modify (e.g., cleave or edit) or regulate (e.g., regulate transcription) the target nucleic acid. For example, a nucleic acid construct encoding an LbCas12a domain (e.g., a fusion protein) linked to a cytosine deaminase domain can be combined with an LbCas12a guide nucleic acid to modify a target nucleic acid, wherein the cytosine deaminase domain of the fusion protein deaminates a cytosine base in the target nucleic acid, thereby editing the target nucleic acid. In another example, a nucleic acid construct encoding an LbCas12a domain (e.g., a fusion protein) linked to an adenine deaminase domain can be combined with an LbCas12a guide nucleic acid to modify a target nucleic acid, wherein the adenine deaminase domain of the fusion protein deaminates an adenosine base in the target nucleic acid, thereby editing the target nucleic acid.

[0142] As used herein, “guide nucleic acid,” “guide RNA,” “gRNA,” “CRISPR RNA / DNA,” “crRNA,” or “crDNA” refers to a nucleic acid comprising at least one spacer sequence complementary to (and hybridizing with) a target nucleic acid (e.g., an anterior spacer sequence) and at least one repeat sequence (e.g., a repeat sequence of the V-type Cas12a CRISPR-Cas system, or a fragment or portion thereof), wherein the repeat sequence may be linked to the 5' end and / or the 3' end of the spacer sequence. The gRNA of this invention can be designed based on the V-type Cas12a system.

[0143] In some implementations, from 5' to 3', the Cas12a gRNA may contain a repetitive sequence (full length or a portion thereof (“stalk-like structure”; e.g., a pseudoknot-like structure) and a spacer subsequence.

[0144] In some embodiments, the guide nucleic acid may contain more than one "repetitive sequence-spacer" sequence (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 or more repeat-spacer sequences) (e.g., repeat-spacer-repetition, such as repeat-spacer-repetition-spacer-repetition-spacer-repetition-spacer-repetition-spacer-repetition-spacer, etc.). The guide nucleic acid of the present invention is synthetic, artificial, and does not exist in nature. The gRNA can be long and can be used as an aptamer (e.g., in the MS2 recruitment strategy) or other RNA structures suspended on the spacer.

[0145] As used herein, “repetitive sequence” refers to, for example, any repetitive sequence from the wild-type Cas12a locus (e.g., the LbCas12a locus) or a repetitive sequence of a synthetic crRNA that functions in conjunction with the LbCas12a nuclease encoded by the nucleic acid construct of the present invention. Repetitive sequences that can be used in the present invention can be any known or subsequently identified repetitive sequence from the Cas12a locus, or they can be synthetic repetitive sequences designed to function in the Cas12a type V CRISPR-Cas system. Repetitive sequences may contain hairpin structures and / or stem-loop structures. In some embodiments, the repetitive sequence may form a pseudo-knot-like structure (i.e., a “stalk”) at its 5' end. Thus, in some embodiments, the repetitive sequence may be identical or substantially identical to a repetitive sequence from the wild-type V CRISPR-Cas locus (e.g., the wild-type Cas12a locus). Repetitive sequences from the wild-type Cas12a locus can be determined by established algorithms, for example using a CRISPRfinder provided via CRISPRdb (see Grissa et al.). Nucleic Acids Res.35 (Network Server Album): W52-7). In some implementations, the repeat sequence or a portion thereof is attached at its 3' end to the 5' end of the spacer sequence to form a repeat-spacer sequence (e.g., guide RNA, crRNA).

[0146] In some embodiments, the repeat sequence comprises at least 10 nucleotides, consists substantially of at least 10 nucleotides, or consists of at least 10 nucleotides, depending on the specific repeat and whether the guide RNA containing the repeat is processed or unprocessed (e.g., about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 to 100 or more nucleotides or any range or value thereof; for example, about). In some embodiments, the repetitive sequence comprises about 10 to about 20, about 10 to about 30, about 10 to about 45, about 10 to about 50, about 15 to about 30, about 15 to about 40, about 15 to about 45, about 15 to about 50, about 20 to about 30, about 20 to about 40, about 20 to about 50, about 30 to about 40, about 40 to about 80, about 50 to about 100, or more nucleotides, substantially consisting of, or consisting of.

[0147] The repeat sequence attached to the 5' end of the spacer sequence may comprise a portion of the repeat sequence (e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or more consecutive nucleotides of the wild-type repeat sequence). In some embodiments, the length of the portion of the repeat sequence attached to the 5' end of the spacer sequence may be about 5 to about 10 consecutive nucleotides (e.g., about 5, 6, 7, 8, 9, 10 nucleotides), and it has at least 90% identity with the same region (e.g., the 5' end) of the wild-type CRISPR Cas repeat nucleotide sequence (e.g., at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher). In some implementations, a portion of the repeating sequence may include a pseudoknot-like structure (e.g., a "stalk-like structure") at its 5' end.

[0148] As used herein, a “spacer sequence” is a nucleotide sequence complementary to a target nucleic acid (e.g., target DNA) (e.g., a prespacer). The spacer sequence may be fully or substantially complementary to the target nucleic acid (e.g., at least about 70% complementary (e.g., about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher complementarity, and any range or value thereof)). Therefore, in some embodiments, the spacer sequence may have one, two, three, four, or five mismatches compared to the target nucleic acid, and these mismatches may be sequential or discontinuous. In some embodiments, the spacer sequence may have about 70% complementarity to the target nucleic acid. In other embodiments, the spacer nucleotide sequence may have about 80% complementarity to the target nucleic acid. In other embodiments, the spacer nucleotide sequence may have complementarity with the target nucleic acid (pre-spacer sequence) of about 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5%. In some embodiments, the spacer sequence is 100% complementary to the target nucleic acid. The length of the spacer sequence may be about 15 nucleotides to about 30 nucleotides (e.g., 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides, or any range or value therein). Thus, in some embodiments, the spacer sequence may be completely or substantially complementary to a target nucleic acid region (e.g., the pre-spacer sequence) that may be at least about 15 nucleotides to about 30 nucleotides in length. In some embodiments, the length of the spacer may be about 20, 21, 22, 23, 24, or 25 nucleotides. In some embodiments, the length of the spacer region may be 23 nucleotides.

[0149] In some implementations, the 5' region of the spacer sequence of the guide RNA may be identical to the target nucleic acid, while the 3' region of the spacer may be substantially complementary to the target nucleic acid (e.g., type V CRISPR-Cas). Alternatively, the 3' region of the spacer sequence of the guide RNA may be identical to the target nucleic acid, while the 5' region of the spacer may be substantially complementary to the target nucleic acid (e.g., type II CRISPR-Cas). Thus, the overall complementarity of the spacer sequence to the target nucleic acid may be less than 100%. Therefore, for example, in the guidance of a type V CRISPR-Cas system, the first 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10 nucleotides in the 5' region (i.e., the seed region) of a 20-nucleotide spacer sequence may be 100% complementary to the target nucleic acid, while the remaining nucleotides in the 3' region of the spacer sequence may be substantially complementary to the target nucleic acid (e.g., at least about 70% complementary). In some implementations, the first 1 to 8 nucleotides (e.g., the first 1, 2, 3, 4, 5, 6, 7, 8 nucleotides, and any range thereof) of the 5' end of the spacer sequence can be 100% complementary to the target nucleic acid, while the remaining nucleotides in the 3' region of the spacer sequence can be substantially complementary to the target nucleic acid (e.g., at least about 50% complementary (e.g., about 50%, 55%, 60%, 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher)).

[0150] In some implementations, the length of the seed region of the spacer sequence can be about 8 to about 10 nucleotides, about 5 to about 6 nucleotides, or about 6 nucleotides.

[0151] As used herein, “target nucleic acid,” “target DNA,” “target nucleotide sequence,” “target region,” or “target region in the genome” refers to a region in an organism’s genome that is completely complementary (100% complementary) or substantially complementary (e.g., at least 70% complementary (e.g., about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher, and any range or value thereof)) to the spacer sequence in the guide RNA of this invention. In some embodiments, the target region that can be used in a type V CRISPR-Cas system (e.g., LbCas12a) is located at the 3' position immediately adjacent to the PAM sequence in the organism’s genome (e.g., plant genome, animal genome, bacterial genome). In some implementations, the target region may be selected from any at least 15 consecutive nucleotides located immediately adjacent to the PAM sequence (e.g., 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more nucleotides, and any range or value thereof; e.g., about 19 to about 25 nucleotides, about 20 to about 24 nucleotides, etc.).

[0152] "Pre-spacer sequence" refers to a portion of a target nucleic acid (e.g., or a target region in the genome) that is fully or substantially complementary (and hybridizes) to the spacer sequence of a CRISPR repeat-spacer sequence (e.g., guide RNA, CRISPR array, crRNA).

[0153] In the case of the V-type CRISPR-Cas Cas12a system, the prespacer sequence is located flanking (immediately adjacent) to the prespacer neighbor motif (PAM). The PAM is located at the 5' end of the non-target strand and the 3' end of the target strand (e.g., see below).

[0154]

[0155] Classic Cas12a PAM is rich in T. In some implementations, the classic Cas12a PAM sequence can be 5'-TTN, 5'-TTTN, or 5'-TTTV.

[0156] The peptides, fusion proteins, and / or systems of the present invention can be encoded by polynucleotide or nucleic acid constructs. In some embodiments, the polynucleotide / nucleic acid constructs encoding the peptides, fusion proteins, and / or systems of the present invention can be operatively associated with regulatory elements (e.g., promoters, terminators, etc.) for expression in organisms of interest and / or cells of organisms of interest as described herein. In some embodiments, the polynucleotide / nucleic acid constructs encoding the peptides, fusion proteins, and / or systems of the present invention can be codon-optimized for expression in organisms.

[0157] In some embodiments, the present invention provides a complex comprising (a) the modified LbCas12a polypeptide of the present invention or the fusion protein of the present invention and (b) a guide nucleic acid (e.g., CRISPR RNA, CRISPR DNA, crRNA, crDNA).

[0158] In some embodiments, the present invention provides a composition comprising (a) the modified LbCas12a polypeptide of the present invention or the fusion protein of the present invention and (b) a guide nucleic acid.

[0159] In some embodiments, the present invention provides expression cassettes and / or vectors comprising the polynucleotide / nucleic acid constructs of the present invention. In some embodiments, expression cassettes and / or vectors comprising the polynucleotide / nucleic acid constructs of the present invention and / or one or more guide nucleic acids may be provided. In some embodiments, nucleic acid constructs encoding modified CRISPR-Cas nucleases and / or fusion proteins comprising the modified CRISPR-Cas nucleases of the present invention may be contained in expression cassettes or vectors that are the same as or separate from those containing guide nucleic acids. When the nucleic acid construct is contained in an expression cassette or vector separate from those containing guide nucleic acids, the target nucleic acid may be contacted with the expression cassette or vector containing the nucleic acid constructs of the present invention (e.g., provided together) before, simultaneously with, or after the expression cassette containing the guide nucleic acid (e.g., contacted with the target nucleic acid).

[0160] In some embodiments, the present invention provides expression cassettes and / or carriers that encode the compositions and / or complexes of the present invention or that contain the systems of the present invention.

[0161] In some embodiments, the polynucleotides, nucleic acid constructs, expression cassettes, and / or vectors of the present invention optimized for expression in organisms may share approximately 70% to approximately 100% identity with polynucleotides, nucleic acid constructs, expression cassettes, and / or vectors encoding the same modified CRISPR-Cas nucleases or fusion proteins of the present invention but which have not been codon-optimized for expression in organisms (e.g., approximately 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100%, and any values ​​or ranges thereof). Organisms for which polynucleotides or nucleic acid constructs may be optimized may include, but are not limited to, animals, plants, fungi, archaea, or bacteria. In some embodiments, the polynucleotide or nucleic acid constructs of the present invention are codon-optimized for expression in plants.

[0162] In some embodiments, the present invention provides cells comprising one or more polynucleotides, guide nucleic acids, nucleic acid constructs, systems, expression cassettes, and / or vectors of the present invention.

[0163] The nucleic acid constructs of the present invention (e.g., encoding the modified CRISPR-Cas nuclease of the present invention and / or fusion proteins containing the modified CRISPR-Cas nuclease of the present invention) and expression cassettes / vectors containing them can be used to modify target nucleic acids and / or express them in vivo (e.g., in an organism or in the cells of an organism; such as a plant) and in vitro (e.g., in cells or cell-free systems).

[0164] This invention further provides a method for altering the PAM specificity of the Cas12a peptide. In some embodiments, a method for altering PAM specificity is provided, comprising introducing a mutation into the Cas12a peptide, wherein, referring to the position numbers of SEQ ID NO: 1, the mutation is located at amino acid residues K116, K120, K121, D122, E125, T148, T149, T152, D156, E159, Q529, D535, K538, D541, Y542, L585, K591, M592, K595, V596, S599, K600, K601, Y616, Y646, W649. In some embodiments, referring to SEQ ID NO: The mutations that introduce the Cas12a peptide, numbered 1, are K116R, K116N, K120R, K120H, K120N, K120T, K120Y, K120Q, K121S, K121T, K121H, K121R, K121G, K121D, K121Q, D122R, D122K, D122H, D122E, D122N, E125R, E125K, E125Q, E125Y, T148H, T148S, T148A, and T1 48C, T149A, T149C, T149S, T149G, T149H, T149P, T149F, T149N, T149D, T149V, T152R, T152K, T152W, T152Y, T152 H, T152Q, T152E, T152L, T152F, D156R, D156K, D156Y, D156W, D156Q, D156H, D156I, D156V, D156L, D156E, E159K, E 159R, E159H, E159Y, E159Q, Q529N, Q529T, Q529H, Q529A, Q529F, Q529G, Q529G, Q529S, Q529P, Q529W, Q529D, G53 2D, G532N, G532S, G532H, G532F, G532K, G532R, G532Q, G532A, G532L, G532C, D535N, D535H, D535V, D535T, D535S, D535A, D535W, D535K, K538R, K538V, K538Q, K538W, K538Y, K538F, K538H, K538L, K538M, K538C, K538G, K538A, K53 8P, D541N, D541H, D541R, D541K, D541Y, D541I, D541A, D541S, D541E, Y542R, Y542K, Y542H, Y542Q, Y542F, Y542L,Y542M, Y542P, Y542V, Y542N, Y542T, L585G, L585H, L585F, K591W, K591F, K591Y, K591H, K591R, K591S, K591A, K591G, K591P , M592R, M592K, M592Q, M592E, M592A, K595R, K595Q, K595Y, K595L, K595W, K595H, K595E, K595S, K595D, K595M, V596T, V596 H, V596G, V596A, S599G, S599H, S599N, S599D, K600R, K600H, K600G, K601R, K601H, K601Q, K601T, Y616K, Y616R, Y616E, Y61 6F, Y616H, Y646R, Y646E, Y646K, Y646H, Y646Q, Y646W, Y646N, W649H, W649K, W649Y, W649R, W649E, W649S, W649V and / or W649T. In some embodiments, referring to the position number of SEQ ID NO: 1, the mutation introducing the Cas12a peptide is located at amino acid residue positions K116, K120, K121, D122, E125, T152, D156, E159, G532, D535, K538, D541 and / or K595, optionally, wherein referring to SEQ ID NO: 1 The position number of NO:1, the mutations are K116R, K116N, K120Y, K121S, K121R, D122H, D122N, E125K, T152R, T152K, T152Y, T152Q, T152E, T152F, D156R, D156W, D156Q, D156H, D156I, D156V, D156L, D156E, E159K, E159R, G532N, G532S , G532H, G532K, G532R, G532L, D535N, D535H, D535T, D535S, D535A, D535W, K538R, K538V, K538Q, K538W , K538Y, K538F, K538H, K538L, K538M, K538C, K538G, K538A, D541E, K595R, K595Q, K595Y, K595W, K595H,K595S and / or K595M. The introduced mutation can be a single mutation or a combination of two or more mutations. It is understood that any single Cas12a polypeptide with two or more mutations contains only a single mutation at any given position. In some embodiments, the PAM-specific Cas12a polypeptide modified by the method of the present invention is the LbCas12a polypeptide (from Trichophytonceae bacteria).

[0165] The modified Cas12a polypeptide or nuclease (e.g., LbCas12a nuclease) of the present invention can be used to modify target nucleic acids in cells or cell-free systems (e.g., altering the target nucleic acid, altering the genome of a cell / organism). Accordingly, in some embodiments, a method for modifying a target nucleic acid is provided, the method comprising: contacting the target nucleic acid with: (a) (i) the modified LbCas12a polypeptide of the present invention, or the fusion protein of the present invention (e.g., the modified LbCas12a polypeptide of the present invention and a polypeptide of interest (e.g., a deaminase)), and (ii) a guiding nucleic acid; (b) a complex of the present invention comprising (i) the modified LbCas12a polypeptide or fusion protein of the present invention, and (ii) the guiding nucleic acid; (c) a composition comprising (i) the modified LbCas12a polypeptide of the present invention or the fusion protein of the present invention, and (ii) the guiding nucleic acid; and / or (d) the system of the present invention, thereby modifying the target nucleic acid. In some embodiments, a method for modifying / altering the genome of a cell or organism is provided, the method comprising: contacting a target nucleic acid in the genome of the cell / organism with: (a) (i) a modified LbCas12a polypeptide of the present invention, or a fusion protein of the present invention (e.g., a modified LbCas12a polypeptide of the present invention and a polypeptide of interest (e.g., a deaminase)), and (ii) a guiding nucleic acid; (b) a complex of the present invention comprising (i) a modified LbCas12a polypeptide or fusion protein of the present invention, and (ii) a guiding nucleic acid; (c) a composition comprising (i) a modified CRISPR-Cas nuclease of the present invention (e.g., a modified LbCas12a polypeptide) or a fusion protein of the present invention, and (ii) a guiding nucleic acid; and / or (d) a system of the present invention, thereby modifying / altering the genome of the cell or organism. In some embodiments, the cell or organism is a plant cell or a plant.

[0166] In some embodiments, a method for modifying a target nucleic acid is provided, the method comprising: contacting a cell or cell-free system containing the target nucleic acid with: (a)(i) a polynucleotide of the present invention (e.g., encoding a modified LbCas12a polypeptide of the present invention, or a fusion protein encoding a modified LbCas12a polypeptide of the present invention and a polypeptide of interest (e.g., a deaminase), or an expression cassette or vector containing thereof, and (ii) a guide nucleic acid, or an expression cassette and / or vector containing thereof; and / or (b) a nucleic acid construct encoding a complex of the present invention or an expression cassette and / or vector containing thereof, the complex comprising a modified LbCas12a polypeptide of the present invention, or a fusion protein comprising a modified LbCas12a polypeptide of the present invention and a polypeptide of interest (e.g., a deaminase), wherein the contact is performed under conditions of expressing the polynucleotide and / or nucleic acid construct and producing a modified LbCas12a polypeptide and / or fusion protein, the modified LbCas12a polypeptide and / or fusion protein forming a complex with the guide nucleic acid thereby modifying the target nucleic acid. In some embodiments, a method for modifying / altering the genome of a cell and / or organism is provided, the method comprising: contacting a cell and / or organism containing a target nucleic acid with: (a)(i) a polynucleotide of the present invention (e.g., encoding a modified LbCas12a polypeptide of the present invention, or a fusion protein encoding a modified LbCas12a polypeptide of the present invention and a polypeptide of interest (e.g., a deaminase), or an expression cassette or vector containing thereof; and (ii) a guide nucleic acid or an expression cassette and / or vector containing thereof; and / or (b) a nucleic acid construct encoding a complex of the present invention or an expression cassette and / or vector containing thereof, the complex containing a modified LbCas12a polypeptide of the present invention, or a fusion protein containing a modified LbCas12a polypeptide of the present invention and a polypeptide of interest (e.g., a deaminase), wherein the contact is performed under conditions of expressing the polynucleotide and / or nucleic acid construct and producing a modified LbCas12a polypeptide and / or fusion protein, the modified LbCas12a polypeptide and / or fusion protein forming a complex with the guide nucleic acid thereby modifying the target nucleic acid.

[0167] In some embodiments, the present invention provides a method for editing a target nucleic acid, the method comprising: contacting the target nucleic acid with: (a)(i) a fusion protein of the present invention (comprising the modified LbCas12a polypeptide of the present invention and a polypeptide of interest (e.g., a deaminase)); and (a)(ii) a guide nucleic acid; (b) a complex comprising the fusion protein of the present invention and the guide nucleic acid; (c) a composition comprising the fusion protein of the present invention and the guide nucleic acid; and / or (d) a system of the present invention, thereby editing the target nucleic acid.

[0168] In some embodiments, the present invention provides a method for editing a target nucleic acid, the method comprising: contacting a cell or cell-free system containing the target nucleic acid with: (a)(i) a polynucleotide encoding a fusion protein of the present invention (e.g., the modified LbCas12a polypeptide of the present invention and a polypeptide of interest (e.g., a deaminase)) or an expression cassette and / or vector containing thereof; and (a)(ii) a guide nucleic acid, or an expression cassette and / or vector containing thereof; (b) a nucleic acid construct encoding a complex or an expression cassette and / or vector containing the nucleic acid construct, the complex containing the fusion protein of the present invention and the guide nucleic acid; and / or (c) the system of the present invention, wherein the contact is performed under conditions of expressing a polynucleotide and / or nucleic acid construct and generating a modified CRISPR-Cas nuclease and / or fusion protein, the modified CRISPR-Cas nuclease and / or fusion protein forming a complex with the guide nucleic acid thereby modifying the target nucleic acid.

[0169] CRISPR-Cas nucleases with modified PAM recognition specificity can be utilized in many ways, including but not limited to: generating insertion / deletion (NHEJ), in homology-directed repair, as genome recognition elements without nuclease function (inactivated Cpf1), as genome recognition elements with partially functional nucleases (nickase Cpf1), for catalytic editing of genomic DNA in fusion proteins (DNA base editor), for catalytic editing of RNA in fusion proteins (RNA base editor), for targeting other macromolecules to specific genomic regions; for targeting small chemicals to specific genomic regions, for labeling specific genomic regions, and / or for CRISPR-directed genome recombination strategies.

[0170] When provided on different nucleic acid constructs, expression vectors and / or vectors, the nucleic acid constructs of the present invention can contact the target nucleic acid before, simultaneously with or after contacting the guide nucleic acid.

[0171] The modified CRISPR-Cas nuclease of the present invention, along with the polynucleotide and nucleic acid constructs encoding it, can be used to modify target nucleic acids in any organism, including but not limited to animals, plants, fungi, archaea, or bacteria. Animals can include, but are not limited to, mammals, insects, fish, birds, etc. Exemplary mammals to which the present invention can be used include, but are not limited to, primates (humans and non-humans (e.g., chimpanzees, baboons, monkeys, gorillas, etc.)), cats, dogs, mice, rats, ferrets, gerbils, hamsters, cattle, pigs, horses, goats, donkeys, or sheep.

[0172] The nucleic acid constructs of this invention can be used to modify and / or edit (e.g., mutate, such as base editing, cutting, notching, etc.) any target nucleic acid of a plant or plant part. The nucleic acid constructs of this invention can be used to modify any plant (or plant classification, e.g., genus or higher classification), including angiosperms, gymnosperms, monocots, dicots, C3, C4, CAM plants, bryophytes, ferns and / or pseudoferns, microalgae and / or macroalgae. The plants and / or plant parts that can be used in this invention can be plants and / or plant parts of any plant species / variety / cultivar. As used herein, the term "plant part" includes, but is not limited to, embryo, pollen, ovule, seed, leaf, stem, shoot, flower, branch, fruit, kernel, spike, stalk, shell, stalk, root, root tip, anther, plant cell (including intact plant cells in the plant and / or plant part), plant protoplast, plant tissue, plant cell tissue culture, plant callus, plant mass, etc. As used herein, “twig” refers to the above-ground parts, including leaves and stems. Furthermore, as used herein, “plant cell” refers to the structural and physiological unit of a plant, including the cell wall, and may also refer to the protoplast. A plant cell can be in the form of an isolated single cell, a cultured cell, or part of a higher unit of organization (e.g., plant tissue or plant organ).

[0173] Non-limiting examples of plants that can be used in this invention include turfgrasses (e.g., Kentucky bluegrass, bentgrass, ryegrass, fescue), feather reeds, hairgrass, miscanthus, arundo, switchgrass, vegetable crops including artichokes, kohlrabi, arugula, leeks, asparagus, lettuce (e.g., head lettuce, loose-leaf lettuce, long-leaf lettuce), taro, melons (e.g., cantaloupe, watermelon, Crenshoe melon, cantaloupe, honeydew melon), rapeseed crops (e.g., Brussels sprouts, cabbage, cauliflower, broccoli, loose-leaf cabbage, collard greens, Chinese cabbage, bok choy), spiny artichokes, carrots, etc. Napa cabbage, okra, onion, celery, parsley, chickpeas, parsley, chicory, pepper, potato, cucurbitaceae (e.g., marrow, cucumber, dense zucchini, squash, pumpkin, honeydew melon, watermelon, cantaloupe), radish, dried bulb onion, rutabaga, eggplant, ginseng, broadleaf endive, leaf onion, chicory, garlic, spinach, leeks, squash, leafy green vegetables, beets (e.g., sugar beets and feed beets), sweet potato, leaf beet, horseradish, tomato, turnip, and spices; fruit crops such as apple, apricot, cherry, nectarine, peach, pear, plum, prune, cherry, quince, fig, Nuts (e.g., chestnuts, pecans, pistachios, hazelnuts, peanuts, walnuts, macadamia nuts, almonds, etc.), citrus fruits (e.g., mandarins, kumquats, oranges, grapefruits, tangerines, lemons, limes, etc.), blueberries, black raspberries, boysonberries, cranberries, blackcurrants, currants, raspberries, strawberries, blackberries, grapes (e.g., wine grapes and table grapes), avocados, bananas, kiwifruit, persimmons, pomegranates, pineapples, tropical fruits, pears, melons, mangoes, papayas, and lychees; field crops such as clover, alfalfa, timothy grass, evening primrose, reeds, and corn / maize (e.g., forage crops). Corn, sweet corn, popcorn corn), hops, jojoba, buckwheat, safflower, quinoa, wheat, rice, barley, rye, millet / sorghum, oats, triticale, tobacco, kapok, legumes (e.g., green beans and dried beans), lentils, peas, soybeans), oilseed plants (e.g., rapeseed, rapeseed, mustard greens, poppy, olive, sunflower, coconut, castor oil plants, cocoa beans, peanuts, oil coconut, soybeans, flaxseed, etc.), duckweed, Arabidopsis thaliana, fiber plants (cotton, flax, hemp, jute), Cannabis (e.g., Cannabis sativa , Cannabis indica and Cannabis ruderalisPlants of the Lauraceae family (e.g., cinnamon, camphor tree) or some plants such as coffee trees, sugarcane, tea trees, and natural rubber plants; and / or bedding plants such as flowering plants, cacti, succulents, and / or ornamental plants (e.g., roses, tulips, violets), and some trees such as forest trees (broadleaf trees and evergreen trees, such as conifers; e.g., elm, ash, oak, maple, fir, spruce, cedar, pine, birch, cypress, eucalyptus, willow), as well as shrubs and other seedlings. In some embodiments, the nucleic acid constructs of the present invention and / or expression cassettes and / or vectors encoding them can be used to modify corn, soybean, wheat, rapeseed, rice, tomato, pepper, sunflower, raspberry, blackberry, black raspberry, and / or cherry.

[0174] The present invention further includes a kit for carrying out the method of the present invention. The kit of the present invention may contain reagents, buffers and / or devices for mixing, measuring, sorting, labeling, etc., as well as instructions, etc., as applicable to modifying target nucleic acids.

[0175] In some embodiments, the present invention provides a kit comprising one or more polynucleotide and / or nucleic acid constructs of the present invention, and / or an expression cassette and / or vector comprising thereto, and optionally, instructions for use thereof. In some embodiments, the kit may further comprise a polypeptide of interest and / or a polynucleotide encoding that polypeptide, and an expression cassette and / or vector comprising that polynucleotide. In some embodiments, a guide nucleic acid may be provided on the same expression cassette and / or vector as the nucleic acid construct of the present invention. In some embodiments, a guide nucleic acid may be provided on an expression cassette or vector separate from the expression cassette or vector comprising the nucleic acid construct of the present invention.

[0176] Accordingly, in some embodiments, a kit is provided comprising a nucleic acid construct containing (a) a polynucleotide encoding a modified CRISPR-Cas nuclease as provided herein and (b) a promoter driving the expression of the polynucleotide of (a). In some embodiments, the kit may also comprise a nucleic acid construct encoding a guide nucleic acid, wherein the construct contains a cloning site for cloning a nucleic acid sequence identical or complementary to the target nucleic acid sequence into the backbone of the guide nucleic acid.

[0177] In some embodiments, the kit may comprise a nucleic acid construct containing / encoding one or more nuclear localization signals, wherein the nuclear localization signals are fused to a CRISPR-Cas nuclease. In some embodiments, a kit is provided comprising the nucleic acid construct of the present invention encoding the modified CRISPR-Cas nuclease, or, and / or an expression cassette and / or vector comprising the nucleic acid construct, wherein the nucleic acid construct, expression cassette, and / or vector may further encode one or more optional markers (e.g., nucleic acids encoding antibiotic resistance genes, herbicide resistance genes, etc.) that can be used to identify transformants. In some embodiments, the nucleic acid construct may be an mRNA encoding one or more introns within the encoded CRISPR-Cas nuclease. In some embodiments, the kit may comprise a promoter and an intron-containing promoter for expressing the polypeptide and nucleic acid construct of the present invention.

[0178] PAM-specific methods for modifying CRISPR-Cas nucleases and related compositions

[0179] The CRISPR-Cas system utilizes two main criteria to target nucleic acids: homology between the guide RNA and the target DNA sequence, and the presence of a prespacer neighbor motif (PAM) within a specific sequence. Different CRISPR-Cas nucleases have different PAM sequence requirements; for example, NGG for SpCas9 or TTTV for LbCas12a (Cpf1) (where V is any non-thymidine nucleotide). Screening for new CRISPR nucleases or their mutants to meet their PAM requirements can be complex and unpredictable, involving numerous iterations. In vitro assays, particularly PAM determination assays (PAMDA), can be used to screen for PAM specificity in any particular CRISPR nuclease or its mutants. These assays rely on randomized DNA portions adjacent to a defined / known prespacer sequence. The guide RNA can be programmed to target a known prespacer sequence, and if the randomized PAM region contains a suitable DNA sequence (e.g., recognized by the CRISPR nuclease or its mutants), the CRISPR nuclease can bind to and cleave the target.

[0180] The PAM recognition site of CRISPR-Cas nucleases can be evaluated using PAM site consumption assays (e.g., PAM consumption assays) or PAM determination assays (PAMDA) (Kleinstiver et al.). Nat Biotechnol37: 276–282 (2019)). For the PAM consumption assay, a plasmid library carrying randomized nucleotides (base pairs) adjacent to the anterior spacer is tested in bacteria (e.g., *E. coli*) to determine whether it is cleaved by a CRISPR nuclease. The plasmid may contain, for example, antibiotic-conferring polynucleotides adjacent to the randomized PAM sequence. Due to the presence of antibiotic resistance genes, those sequences that are not cleaved upon exposure to the CRISPR-Cas nuclease allow cells to survive in the presence of antibiotics, while plasmids carrying PAM-targeting sequences are cleaved and consumed from the library due to cell death. Sequencing of the surviving (uncleaved) plasmid population allows for the calculation of the selected PAM consumption value, which is compared to a library not exposed to the CRISPR-Cas nuclease. Those sequences consumed from the sequence pool in the experimental library contain PAM sequences recognized by the CRISPR-Cas nuclease.

[0181] Another method that can be used to identify PAM sequences is the PAM determination assay (PAMDA) (Kleinstiver et al.). Nat Biotechnol 37: 276–282 (2019)). In this case, cleavage is performed outside of living cells. In PAMDA, a single DNA strand is synthesized, with randomized nucleotide motifs immediately adjacent to a defined prespacer sequence. Oligonucleotides are annealed to the 3' end of the synthesized DNA strand and extended using a negative (-exo) Klenow fragment of an exonuclease, polymerizing on a defined and randomized sequence. This generates a double-stranded library, which is then cut with a restriction endonuclease and cloned into bacteria to amplify total DNA. Plasmids are extracted and linearized with another restriction endonuclease to prepare a linear template. The template is contacted with a CRISPR-Cas nuclease-guide RNA complex. Only sequences containing PAM recognized by the CRISPR-Cas nuclease are cleaved. The experimental and control libraries (not exposed to the CRISPR-Cas nuclease) are then amplified by PCR. Only sequences not cleaved by the CRISPR-Cas nuclease are amplified. PCR amplified sequences from control and experimental libraries (treated with CRISPR-Cas nuclease) were sequenced and compared. PAM sequences present in the control (not exposed to CRISPR-Cas nuclease) library but absent in the experimental library were PAM sequences recognized by CRISPR-Cas nuclease (thus allowing the anterior spacer to be cleaved).

[0182] To assess nuclease requirements in vitro, randomized PAM libraries were prepared. The steps described in this method involve preparing an unbiased randomized DNA library containing all PAM sequences to be evaluated, cloning it into a plasmid, introducing the library into bacteria to increase the total amount of starting DNA, extracting the plasmid, linearizing the plasmid with a restriction enzyme to remove supercoiling, exposing the linearized molecule to a CRIPSR-Cas nuclease, amplifying the fragment (e.g., PCR), and finally performing sequencing analysis (e.g., next-generation sequencing, NGS). The initial steps of generating an unbiased library and restriction digestion require at least two restriction enzymes, Klenow extension, and cleaning of the product before ligation into a vector. Using two or three restriction enzymes typically removes some PAM sequences from the library, introducing bias. Furthermore, subsequent Klenow extension and cleaning steps may also remove PAM sequences, introducing further bias into the library. To avoid the loss of PAM sequences and generate a more complete unbiased library, this invention provides a novel method for generating randomized PAM libraries using a method with dangling (see, e.g.) Figure 1 Oligonucleotides synthesized in an overlapping solid-state manner (e.g., annealed oligonucleotides) are used instead of restriction endonucleases and Klenow extensions. The randomized PAM libraries generated using the method of this invention can then be used to test the PAM specificity of CRISPR-Cas nucleases with higher accuracy than libraries previously generated using prior art methods.

[0183] Accordingly, in some embodiments, the present invention provides a method for constructing a randomized DNA library comprising double-stranded nucleic acid molecules for determining the preseptal neighbor motif (PAM) requirement / specificity of a CRISPR-Cas nuclease having a PAM recognition site at the 5' end of the preseptal, the method comprising: preparing two or more double-stranded nucleic acid molecules, including the steps of: (a) synthesizing a non-target oligonucleotide (first) chain and a target oligonucleotide (second) chain for each of the two or more double-stranded nucleic acid molecules, wherein the non-target oligonucleotide chain extends from the 5' end of the preseptal. 3' comprises: (i) a first sequence having about 5 to about 15 nucleotides (e.g., about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 nucleotides, and any range thereof); (ii) a second sequence having at least four randomized nucleotides (e.g., at least 4, 5, 6, 7, 8, 9, 10, or more, and any range thereof); and (iii) a second sequence comprising about 16 to about 25 nucleotides (e.g., about 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides). (i) a first sequence having about 5 to about 20 nucleotides (e.g., about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 nucleotides, and any range thereof), wherein (i) the first sequence having about 5 to about 15 nucleotides is immediately adjacent to the 5' end of the second sequence of (ii), the second sequence of (ii) is immediately adjacent to the 5' end of the first sequence of (iii), and the first sequence of (iii) is immediately adjacent to the 5' end of the first sequence of (ii). (iv) the 5' end of the third sequence; and the target oligonucleotide (second) strand is complementary to the non-target oligonucleotide strand; and (b) the non-target oligonucleotide strand is annealed to the complementary target oligonucleotide strand to produce a double-stranded nucleic acid molecule, wherein the first sequence contains a restriction site (at its 5' end) and the third sequence contains a restriction site (at its 3' end), wherein the first sequence (i), the prespacer sequence (iii), and the third sequence (iv) are identical for each of two or more double-stranded nucleic acid molecules, thereby constructing a randomized DNA library containing double-stranded nucleic acid molecules. In some embodiments, the target strand and / or the non-target strand may be 5' phosphorylated.

[0184] In some embodiments, the present invention provides a method for constructing a randomized DNA library comprising double-stranded nucleic acid molecules for determining the preseptal neighbor motif (PAM) requirement / specificity of a CRISPR-Cas nuclease having a PAM recognition site at the 3' end of the preseptal, the method comprising: preparing two or more double-stranded nucleic acid molecules, including the steps of: (a) synthesizing a non-target oligonucleotide (first) strand and a target oligonucleotide (second) strand for each of the two or more double-stranded nucleic acid molecules, wherein the non-target oligonucleotide strand comprises from 5' to 3': (i) having a first sequence of about 5 to about 20 nucleotides (e.g., about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 nucleotides, and any range thereof), (ii) having a prespacer sequence of about 16 to about 25 nucleotides (e.g., about 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides, and any range thereof), and (iii) having at least four randomized nucleotides (e.g., at least 4, 5, 6, 7). (i) a second sequence having about 5 to about 15 nucleotides (e.g., about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 nucleotides, and any range thereof), wherein the first sequence of (i) having about 5 to 20 nucleotides is immediately adjacent to the 5' end of the pre-spacer sequence of (ii), the second sequence of (iii) is immediately adjacent to the 3' end of the pre-spacer sequence of (ii), and the third sequence of (iv) is immediately adjacent to the first sequence of (iii). (i) The 3' end of the second sequence; and the target oligonucleotide (second) strand is complementary to the non-target oligonucleotide strand; and (b) the non-target oligonucleotide strand is annealed to the complementary target oligonucleotide strand to produce a double-stranded nucleic acid molecule, wherein the first sequence (i) contains a restriction site (at its 5' end) and the third sequence (iv) contains a restriction site (at its 3' end), wherein the first sequence (i), the prespacer sequence (ii), and the third sequence (iv) are identical for each of two or more double-stranded nucleic acid molecules, thereby constructing a randomized DNA library containing double-stranded nucleic acid molecules. In some embodiments, the target strand and / or the non-target strand may be 5' phosphorylated.

[0185] In some embodiments, a double-stranded nucleic acid molecule can be ligated into a vector to generate a vector containing a randomized DNA library. In some embodiments, the vector can be a high-copy-number vector. In some embodiments, the randomized DNA library can be amplified, for example, by introducing the vector containing the randomized DNA library into one or more bacterial cells and culturing the one or more bacterial cells. In some embodiments, the vector containing the randomized DNA library can be isolated from one or more bacterial cells after culturing. The isolated vector can then be linearized (e.g., by contacting the vector with one or more restriction enzymes such as ScaI or PfoI) for, for example, to analyze the PAM recognition specificity of CRISPR-Cas nucleases. In some embodiments, PfoI can be used to linearize the isolated vector.

[0186] In some embodiments, a randomized DNA library may be provided for determining the preseptal neighbor motif (PAM) requirement / specificity of a CRISPR-Cas nuclease with a PAM recognition site at the 5' end of the preseptal spacer. This randomized DNA library comprises two or more double-stranded nucleic acid molecules, each comprising: (a) a non-target oligonucleotide (first) strand and a target oligonucleotide (second) strand, wherein the non-target oligonucleotide strand from 5' to 3' comprises: (i) a first sequence having about 5 to about 15 nucleotides (e.g., about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 nucleotides, and any range thereof); (ii) a second sequence having at least four randomized nucleotides (e.g., at least 4, 5, 6, 7, 8, 9, 10, or more, and any range thereof); and (iii) a second sequence comprising about 16 to about 25 nucleotides (e.g., about 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides). (i) a first sequence having about 5 to about 20 nucleotides (e.g., about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 nucleotides, and any range thereof), wherein the first sequence having about 5 to 15 nucleotides in (i) is immediately adjacent to the 5' end of the second sequence in (ii), and the second sequence in (ii) is immediately adjacent to the 5' end of the first sequence having about 5 to 15 nucleotides in (iii), and The prespacer sequence is immediately adjacent to the 5' end of the third sequence in (iv); and the target oligonucleotide (second) strand is complementary to the non-target oligonucleotide strand; and (b) the non-target oligonucleotide strand is annealed to the complementary target oligonucleotide strand to produce a double-stranded nucleic acid molecule, wherein the first sequence contains a restriction site (at its 5' end) and the third sequence contains a restriction site (at its 3' end), wherein the first sequence (i), the prespacer sequence (iii), and the third sequence (iv) are identical in each of two or more double-stranded nucleic acid molecules. In some embodiments, the target strand and / or the non-target strand may be 5' phosphorylated.

[0187] In some implementations, a randomized DNA library can be provided for determining the preseptal neighbor motif (PAM) requirement / specificity of a CRISPR-Cas nuclease with a PAM recognition site at the 3' end of the preseptal, the randomized DNA library comprising two or more double-stranded nucleic acid molecules, wherein each double-stranded nucleic acid molecule comprises: (a) a non-target oligonucleotide (first) strand and a target oligonucleotide (second) strand, wherein the non-target oligonucleotide strand comprises from 5' to 3': (i) having about 5 to about 20 nuclei. (ii) a first sequence comprising about 16 to about 25 nucleotides (e.g., about 16, 17, 18, 19, 20 nucleotides, and any range thereof), and (iii) a first sequence comprising at least four randomized nucleotides (e.g., at least 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides, and any range thereof). (i) a second sequence having 7, 8, 9, 10 or more, and any range thereof, and (iv) a third sequence having about 5 to about 15 nucleotides (e.g., about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 nucleotides, and any range thereof), wherein (i) the first sequence having about 5 to 20 nucleotides is adjacent to the 5' end of the prespacer sequence of (ii), (iii) the second sequence is adjacent to the 3' end of the prespacer sequence of (ii), and (iv) the third sequence is adjacent to the 3' end of the second sequence of (iii); and the target oligonucleotide (second) chain is complementary to the non-target oligonucleotide chain; and (b) annealing the non-target oligonucleotide chain to the complementary target oligonucleotide chain to produce a double-stranded nucleic acid molecule, wherein the first sequence contains a restriction site (at its 5' end) and the third sequence contains a restriction site (at its 3' end), wherein the first sequence (i), the prespacer sequence (ii), and the third sequence (iv) of each of two or more double-stranded nucleic acid molecules are identical. In some implementations, the target chain and / or non-target chain may be 5' phosphorylated.

[0188] In some embodiments, the present invention provides a method for determining the prespacer neighbor motif (PAM) specificity of a CRISPR-Cas nuclease, the method comprising: contacting a CRISPR-Cas nuclease with a randomized DNA library of the present invention; and sequencing double-stranded nucleic acid molecules of the randomized DNA library before (e.g., as a control) and after contact with the CRISPR-Cas nuclease, wherein double-stranded nucleic acid molecules present in the randomized DNA library before contact with the CRISPR-Cas nuclease but not present in the randomized DNA library after contact with the CRISPR-Cas nuclease, identifying the PAM recognition sequence of the CRISPR-Cas nuclease, thereby determining the PAM specificity of the CRISPR-Cas nuclease.

[0189] In some embodiments, a method for determining the prespacer neighbor motif (PAM) specificity of a CRISPR-Cas nuclease is provided, comprising: contacting a CRISPR-Cas nuclease with a randomized DNA library of the present invention; sequencing double-stranded nucleic acid molecules of the randomized DNA library before (e.g., as a control) and after contact with the CRISPR-Cas nuclease, and identifying the PAM recognition sequence of the nuclease, wherein the identification comprises comparing double-stranded nucleic acid molecules present in the library before contact with the CRISPR-Cas nuclease with double-stranded nucleic acid molecules present in the library after contact with the CRISPR-Cas nuclease, and wherein double-stranded nucleic acid molecules present in the randomized DNA library before contact with the CRISPR-Cas nuclease but not present in the randomized DNA library after contact with the CRISPR-Cas nuclease are identified as having PAM specificity for the CRISPR-Cas nuclease.

[0190] Sequencing results from randomized libraries prior to contact can serve as a control for sequencing results after contact. In some implementations, determining the PAM specificity of a CRISPR-Cas nuclease may include performing nucleic acid sequencing. In some implementations, sequencing may include next-generation sequencing (NGS).

[0191] Any CRISPR-Cas nuclease can be used in the method of this invention to modify PAM recognition specificity. Accordingly, CRISPR-Cas nucleases that can be modified to have different PAM specificity compared to the wild type may include, but are not limited to, Cas9, C2c1, C2c3, Cas12a (also known as Cpf1), Cas12b, Cas12c, Cas12d, Cas12e, Cas13a, Cas13b, Cas13c, Cas13d, Cas1, Cas1B, Cas2, Cas3, Cas3', Cas3", Cas4, Cas5, Cas6, Cas7, Cas8, and Cas9. (Also known as Csnl and Csx12), Cas10, Csyl, Csy2, Csy3, Csel, Cse2, Cscl, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Csbl, Csb2, Csb3, Csxl7, Csxl4, Csx10, Csx16, CsaX, Csx3, Csxl, Csxl5, Csfl, Csf2, Csf3, Csf4 (dinG) and / or Csf5 polypeptides or domains.

[0192] Cas12a is a species originally found in the genus Prevotella ( Prevotella spp) and species of the genus Francisella ( FrancisellaCas12a is a type V clustered regular spaced short palindromic repeat (CRISPR)-Cas nuclease identified in spp. Cas12a (formerly known as Cpf1) differs from the more well-known type II CRISPR Cas9 nuclease in several ways. For example, Cas9 recognizes a G-rich prespacer neighbor motif (PAM) (3'-NGG) located at the 3' of its guide RNA (gRNA, sgRNA) binding site (prespacer, target nucleic acid, target DNA), while Cas12a recognizes a T-rich PAM (5'-TTN, 5'-TTTN) located at the 5' of its binding site (prespacer, target nucleic acid, target DNA). In fact, the orientation of the guide RNA binding of Cas9 and Cas12a is almost opposite relative to their N and C ends. Moreover, the Cas12a enzyme uses a single guide RNA (gRNA, CRISPR array, crRNA) instead of the dual guide RNA (sgRNA (e.g., crRNA and tracrRNA)) seen in the native Cas9 system, and Cas12a processes its own gRNA. Furthermore, Cas12a nuclease activity produces staggered DNA double-strand breaks, rather than blunt ends produced by Cas9 nuclease activity, and Cas12a relies on a single RuvC domain to cut both DNA strands, while Cas9 uses both HNH and RuvC domains for cutting.

[0193] The CRISPR Cas12a polypeptide or CRISPR Cas12a domain used in this invention can be any known or later identified Cas12a nuclease (see, for example, U.S. Patent No. 9,790,490, the disclosed Cpf1 (Cas12a) sequence of which is incorporated herein by reference). The terms “Cas12a,” “Cas12a polypeptide,” or “Cas12a domain” refer to an RNA-guided nuclease comprising a Cas12a polypeptide or a fragment thereof, comprising a Cas12a guide nucleic acid-binding domain and / or an active, inactive, or partially active DNA-cutting domain of Cas12a. In some embodiments, the Cas12a used in this invention may contain a mutation in the nuclease active site (e.g., the RuvC site of the Cas12a domain). A Cas12a domain or Cas12a polypeptide that is mutated at its nuclease active site and therefore no longer contains nuclease activity is commonly referred to as inactivated Cas12a (e.g., dCas12a). In some implementations, the activity (e.g., nicking enzyme activity) of a Cas12a peptide with a mutated Cas12a domain in its nuclease active site may be impaired / reduced compared to the same Cas12a peptide without the same mutation.

[0194] In some embodiments, the Cas12a domain may include, but is not limited to, an amino acid sequence or a polynucleotide encoding any one of SEQ ID NO: 1-17 (e.g., SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 and / or 17). In some embodiments, the fusion protein of the present invention may comprise a Cas12a domain from the bacteria ND2006 Cas12a (LbCas12a) (e.g., SEQ ID NO: 1).

[0195] The CRISPR Cas9 peptide or CRISPR Cas9 domain used in this invention can be any known or later identified Cas9 nuclease. In some embodiments, the Cas9 peptide used in this invention contains at least 70% identity with the amino acid sequence of any known Cas9 (e.g., about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, etc.). The CRISPR-Cas9 system is well known in the art and includes, but is not limited to, those derived from Legionella pneumophila strain Paris. Legionella pneumophila str. Paris), Streptococcus thermophilus ( Streptococcus thermophilus CNRZ1066, Streptococcus pyogenes MI or Neisseria lactis ( Neisseria lactamica Cas9 peptides such as 020-06.

[0196] Other nucleases that can be used in this invention to identify novel PAM recognition sequences include, but are not limited to, C2c1, C2c3, Cas12b, Cas12c, Cas12d, Cas12e, Cas13a, Cas13b, Cas13c, Cas13d, Cas1, Cas1B, Cas2, Cas3, Cas3', Cas3", Cas4, Cas5, Cas6, Cas7, Cas8, and Cas9. (Also known as Csnl and Csx12), Cas10, Csyl, Csy2, Csy3, Csel, Cse2, Cscl, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Csbl, Csb2, Csb3, Csxl7, Csxl4, Csx10, Csx16, CsaX, Csx3, Csxl, Csxl5, Csfl, Csf2, Csf3, Csf4 (dinG) and / or Csf5.

[0197] The invention will now be described with reference to the following embodiments. It should be understood that these embodiments are not intended to limit the scope of the claims of the invention, but are intended as examples of certain implementations. Any variations of the exemplary methods that will occur to those skilled in the art are intended to fall within the scope of the invention.

[0198] Example

[0199] Example 1

[0200] Randomized library

[0201] An example of the method of the present invention is provided for the efficient and cost-effective generation of libraries for in vitro cleavage assays (PAM determination assay (PAMDA)). Two libraries were generated for anterior spacers 1 and 2 (see Table 1). Oligonucleotides with five randomized nucleotide sequences at the 5' end (Integrated DNA Technologies) were synthesized and validated to ensure that each anterior spacer sequence occupied an equal molar ratio (Table 1). The oligonucleotides for anterior spacers 1 (PM0518, PM0519) and for anterior spacers 2 (PM0520, PM0521) were annealed by placing the mixture in a thermal cycler at 95°C for 5 minutes and cooling to 25°C / room temperature at 0.1°C / second.

[0202] Table 1

[0203]

[0204] Annealed double-stranded fragments were directly ligated into the pUC19 vector digested with SphI and EcoRI. The ligated prespacer construct was used to transform XL1-blue electroporated competent *E. coli* cells (Agilent), and the cells were incubated in 1 ml SOC medium at 37°C for 1 h. The presence of ligation products in *E. coli* cells was checked using carbenicillin plates. The transformed *E. coli* cells were grown in 200 ml LB broth supplemented with carbenicillin (50 mg / mL) for 16 h. The plasmid containing the prespacer construct was purified using the Zymo extraction kit. Deep sequencing analysis of the plasmid / vector was performed using Illumina Miseq to calculate the A / T / G / C frequencies at each PAM site.

[0205] This method can be used to generate a library for PAM determination using any selected anterior spacer oligonucleotide, wherein the annealed oligonucleotide may contain any appropriate restriction site of choice in order to preserve the full complementary sequence of the PAM sequence in the library.

[0206] Example 2

[0207] The bacteria ND2006 Cpf1 (LbCpf1) of the family Trichophyceae requires a highly specific anterior spacer neighbor motif (PAM). Compared to random nucleotides, the “TTTV” sequence appears only about once in 85 bases. This contrasts with the relative heterogeneity of SpCas9’s NGG, which appears about once in 16 bases of random DNA, and AaC2c1’s TTN, which appears about once in 16 bases of random DNA, while xCas9 / Cas9-NG’s NG PAM requires about once in 4 bases. The abundance of Cpf1 PAM in maize and soybean genes is significantly lower than that of Cas9 PAM. Figure 2 Furthermore, given LbCpf1's stringent requirement for PAM, adenine and cytosine (current targets of the base editor) are far more difficult for LbCpf1 to access. Figure 3 ).

[0208] like Figure 3 The stringency of the CRISPR-Cas nucleases shown significantly reduces the generation of potential targets and novel traits. This invention relates to the generation of CRISPR-Cas nucleases, particularly LbCpf1 (Cas12a) nucleases with improved accessible PAM sequence ratios (e.g., nucleases with PAM recognition sites appearing in a ratio of about 1:4 or better). Such engineered Cas12a PAM mutants can be used as nucleases (for NHEJ or HDR applications), or in their inactivated form as genome recognition elements in genome editing tools.

[0209] PAMDA assay

[0210] PAM Deterministic Assays (PAMDA) can be used to test the PAM requirement of CRISPR enzymes with unknown PAM recognition. These are in vitro assays that utilize the ability of CRISPR-Cas nucleases to cleave target sequences only after successful PAM binding. In short, a DNA substrate library with randomized PAM sequences is incubated with a CRISPR nuclease, and the DNA is then amplified by PCR. Only intact fragments (e.g., those not recognized by the nuclease) are amplified. Cleavage fragments (those recognized by the nuclease) are not amplified. DNA from libraries exposed to the nuclease and control libraries (not exposed to the nuclease) is sequenced. The two sequencing results are compared to determine which sequences were cleaved and therefore not present in the sequencing set after exposure to the nuclease (e.g., see [link to related documentation]). Figure 4 A modified PAMDA algorithm at multiple time points will be used to determine PAM binding and subsequent cleavage.

[0211] LbCpf1 mutagenesis

[0212] One hundred and eighty-six (186) point mutations were designed (Table 2) and tested individually in the PAMDA assays described herein. Successful engineering could alter the PAM recognition sequence to generate LbCpf1 that recognizes new PAMs, or potentially relax the PAM strictness, resulting in a more heterogeneous LbCpf1.

[0213] Table 2. Substituted residues in SEQ ID NO: 1

[0214]

[0215]

[0216] In addition to single mutations, combinations of mutations that alter PAM identification are combined and evaluated via PAMDA to provide second-generation LbCpf1 mutations.

[0217] Example 3

[0218] Three methods were used to test the 186 mutations:

[0219] (1) A method called PAMDA assay (Kleinstiver et al.) Nat BiotechnolThe in vitro method described in 37, 276–282 (2019) uses purified protein and plasmid libraries to test each point mutation throughout the library. Next-generation sequencing (NGS) is used to score the consumption of library members. Consumption is calculated against the library itself (to determine absolute activity against a specific PAM) or by cleavage with wild-type LbCas12a (to determine whether the mutation confers new PAM recognition compared to wild-type).

[0220] (2) A type called PAM-SCANR (Leenay et al.) Mol Cell The bacterial method described in 62:137–147 (2016) uses a library from *E. coli* to test the binding of the Cas12a mutation to 256 possible PAM NNNN variants. It does not test for cleavage, only for binding. Since the mutations are not near the catalytic region, binding is expected to reflect cleavage as well (this was later validated in the 293T assay). The advantage of PAM-SCANR is that it can rapidly test not only point mutations but also combinations of amino acid point mutations in a rapid and accurate manner. This assay may be more stringent than in vitro cleavage assays.

[0221] (3) INDEL assay in human HEK293T cells. This assay provides valuable eukaryotic INDEL data. For insertions and deletions to occur in eukaryotes, several criteria must be met: the CRISPR enzyme must be expressed and stable in the cell; crRNA must be expressed and properly processed; a protein:RNA complex must be formed; the complex must be stable; the complex must translocate to the nucleus in sufficient quantities; the target DNA must be accessible; the DNA must be strongly targeted via a specific guide RNA design; and double-strand breaks must occur at a sufficiently high rate to allow for accidental DNA repair errors via insertion or deletion (INDEL). This makes the eukaryotic assay the most rigorous of this study. Due to the low-throughput nature of the experiments, several dozen PAMs were tested for each of the three point mutants described below, instead of all 256. Three different targets were chosen for each PAM-mutant combination to minimize false negatives, as specific guides are often ineffective due to target accessibility.

[0222] 1. In vitro determination of PAM binding and cleavage

[0223] Constructing plasmid-based PAM libraries

[0224] A DNA library was prepared consisting of 5 random nucleotides directly located at the 5' position of a 23-nucleotide spacer sequence. LbCas12a is known to have a 4-nucleotide prespacer neighbor motif (PAM), but we chose to use 5 random nucleotides instead of 4 to allow for replication in experiments. The spacer sequence used was 5′-GGAATCCCTTCTGCAGCACCTGG (SEQ ID NO: 30). The library contained the sequence 5′-NNNNNGGAATCCCTTCTGCAGCACCTGG (SEQ ID NO: 36). The 5 random nucleotides generated 1024 possible PAMs, which were determined in this library.

[0225] We used a new method to generate this library. It is not as previously described (Kleinstiver et al.) Nat Biotechnol 37,:276–282 (2019)) Using a single randomized pool of PAM-spacer fusion bodies and polymerase to generate complementary strands, we opted for a more direct approach. Two 5′-phosphorylated sequences were synthesized:

[0226] 5′phos / CGATGTNNNNNGGAATCCCTTCTGCAGCACCTGGGCGCAGGTCACGAGG (SEQ ID NO:32) and

[0227] AATTCCTCGTGACCTGCGCCCAGGTGCTGCAGAAGGGATTCCNNNNNACATCGCATG / 5′phos (SEQ ID NO: 35).

[0228] Upon heating and annealing, the complementary sequence between the two NNNNN sequences annealed, and the resulting ends had ventral ends corresponding to the ventral ends generated by the SphI and EcoRI restriction endonucleases. The two oligonucleotides were annealed in an equimolar ratio at 95°C for 5 min in a thermal cycler and cooled to 25°C / room temperature at a rate of 0.1°C / s.

[0229] The annealed double-stranded fragment was directly ligated into the pUC19 vector digested with SphI and EcoRI. The ligated spacer construct was used to transform XL1-blue electrocompetent *E. coli* cells (Agilent) and incubated in 1 ml Super Optimal broth (SOC) containing glucose at 37°C for 1 hour. An aliquot was plated on a carbenicillin-supplemented plate to check for the presence of the ligation product. The remaining transformed cells were grown in 200 ml Luria broth (LB) supplemented with 50 mg / mL carbenicillin for 16 hours. The spacer plasmid was purified using a plasmid extraction kit (Zymo Research).

[0230] Validation of PAM Library

[0231] Using Illumina Miseq, deep sequencing analysis of the spacer vector was performed according to the manufacturer's protocol to calculate the A / T / G / C frequencies at each location of the PAM. Briefly, 10 ng of DNA was used as a template for PCR. Phasing gene-specific forward and reverse PCR primers were designed to amplify across the entire target site. A two-step PCR method was used to generate an amplicon library, where a preliminary PCR with a 5' tail allowed for the addition of Illumina i5 and i7 adaptor sequences and barcodes in the secondary PCR for multiplex sample sorting. PCR amplification was performed using the following parameters: 98°C for 30 s; PCR1 for 25 cycles, and PCR2 for 8 cycles (98°C 10 s, 55°C 20 s, 72°C 30 s); 72°C for 5 minutes; and maintained at 12°C. PCR reactions were performed using Q5 high-fidelity DNA polymerase (New England BioLabs, Beverly, MA, United States). Secondary PCR amplicon samples were purified separately using AMPure XP beads according to the manufacturer's instructions (Beckman Coulter, Brea, CA, United States); all purified samples were quantified using a plate reader, pooled in equimolar ratios, and run on an AATI fragment analyzer (Agilent Technologies, Palo Alto, CA, United States). The pooled amplicon libraries (2 × 250 paired ends) were sequenced on an Illumina MiSeq system using the MiSeq Seq kit v2 (Illumina, San Diego, CA, United States).

[0232] Three separate reads were generated for the library and averaged. The resulting mean read count for the 1024 library members was 39 reads with a standard deviation of 11.9 reads. The maximum mean read count for any PAM sequence was 74, and the minimum was 12. The PAM counts followed a normal distribution. Figure 5 ).

[0233] Cloning LbCas12a mutation

[0234] A DNA cassette (GeneWiz) consisting of the LbCas12a sequence, followed by the nucleoplasmic protein NLS and a 6x histidine tag (SEQ ID NO: 52) was synthesized and cloned into the pET28a vector between NcoI and XhoI to generate pWISE450 (SEQ ID NO: 53). For ease of cloning, an extra glycine was added to the sequence between Met-1 and Ser-2. This extra glycine is not included in the numbering throughout this document. Then, using a similar strategy, 186 different amino acid point mutations were generated (Table 2), resulting in 186 different plasmid vectors.

[0235] Expression and purification of LbCas12a mutation

[0236] For each mutant, the glycerol reserve in BL21 Star (DE3) cells (ThermoFisher Scientific) was used to seed 1 mL of medium containing 50 µg / mL kanamycin in 24-well blocks. The cultures were sealed with AirPore tape (Qiagen) and incubated overnight at 37°C with shaking. The next morning, 100 µL of the overnight culture was seeded into 4 mL of ZYP self-induction medium containing kanamycin and incubated at 37°C with shaking until the OD 600 nm range was 0.2–0.5. The temperature was lowered to 18°C, and the cultures were grown overnight to express the protein. Cells were harvested by centrifugation, and the pellet was stored at -80°C.

[0237] The following buffers are used for cell lysis and purification. Lysis buffer contains a nonionic detergent, lysis agent, reducing agent, protease inhibitor, buffer, and salts. This solution lyses bacteria, reduces viscosity, and allows downstream purification of enzymes without interfering with nucleases. Buffer A consists of 20 mM Hepes-KOH (pH 7.5), 0.5 M NaCl, 10% glycerol, 2 mM TCEP, and 10 mM imidazole (pH 7.5). Buffer B is the same as Buffer A, except it contains 20 mM imidazole. Buffer C contains 20 mM Hepes-KOH (pH 7.5), 150 mM NaCl, 10% glycerol, 0.5 mM TCEP, and 200 mM imidazole (pH 7.5).

[0238] Purification was performed using a multi-well setup. Two stainless steel 5 / 32" BB filters were added to all wells containing the cell pellet. The pellet was resuspended in 0.5 mL of cold lysis buffer and incubated at room temperature for 30 minutes with surrounding mixing. 0.5 mL of the crude lysate was added to pre-equilibrated His MultiTrap buffer. TM Incubate the plate (Cytiva LifeSciences) at room temperature for 5 minutes to allow protein binding. Follow the manufacturer's instructions for the remaining steps. In short, wash the plate twice with 0.5 mL buffer A, then once with 0.5 mL buffer B, and finally elute with 0.2 mL buffer C. Determine the protein concentration using the Pierce™ Coomassie Plus (Bradford) assay reagent. Store the protein eluent at 4°C.

[0239] Testing wtLbCas12a's ability to cut PAM libraries

[0240] Pre-tests were performed to evaluate three aspects of the experiment: ensuring the experiment was not affected by non-specific nucleases, ensuring that NTTTV PAM in the library was consumed after adding crRNA as a guide, and observing the degree of consumption of the CTTTA-spiked sample at 15 minutes.

[0241] The reaction conditions for the test were as follows: a total volume of 27 μL containing nuclease-free water, 3 μL NEB buffer 2.1 (NewEngland Biolabs), 3 μL of 300 nM crRNA stock solution (5′-AAUUUCUACUAAGUGUAGAUGGAAUCCCUUCUGCAGCACCUGG-3′ (SEQ ID NO: 62), Synthego Corporation), and 1 μL of 1 μM purified wtLbCas12a stock solution was incubated at room temperature for 10 minutes. 3 μL of 10 ng / μL stock solution was added to initiate the reaction. The library was added as is, or 1 μL of plasmid containing CTTTA was added first at 0.75 ng / μL. The total volume for both was 30 μL. The reaction was incubated at 37°C for 15 minutes.

[0242] Table 3 presents the experimental results. Library counts for the TTTV sequence ranged from 219 to 515 (column 2). Adding the purified wild-type protein as described above in the absence of crRNA did not lead to the consumption of library members (column 3). Adding crRNA and protein resulted in the consumption of all NTTTV-containing PAMs (column 4). The result for CTTTA-tagged libraries was approximately a 35-fold increase in CTTTA NGS counts (column 5). Adding wtLbCas12a and crRNA resulted in the consumption of all library members, including a reduction in CTTTA counts from 10,776 to 193. Library members containing NACGA PAMs were also shown, which did not show consumption under the test conditions, as expected, since ACGA is not a PAM recognized by LbCas12a. Therefore, as shown in Table 3, NTTTV PAM library members were effectively cleaved and consumed by wtLbCas12a, while this was not the case for PAMs not recognized by wtLbCas12a (NACGA).

[0243] Table 3. Segmentation and Consumption of PAM Libraries

[0244]

[0245] The results in Table 3 indicate that (1) efficient and nuclease-free purification of wtLbCAs12a was achieved, (2) the library could be consumed under conditions where members containing PAM substrates were tested, (3) the consumption results were based on crRNA with added target spacers, and (4) the enzyme-crRNA complex was in great excess of individual library members because the large amount of CTTTA substrate did not alter the consumption of the substrate.

[0246] LbCas12a mutant cleavage of PAM library

[0247] As shown in the test example for wtLbCas12a, the same reaction conditions were tested for each of the 186 PAM mutations. Three time points were selected for each mutation: 37°C, 75, 435, and 900 seconds. Several library-only controls were included. The products were analyzed using Illumina HiSeq (Genewiz). The data are reported in Table 4.

[0248] Absolute consumption score processing

[0249] We observed that for any 5-nucleotide PAM, there was little difference in consumption among the four possibilities. In other words, for any 4-nucleotide sequence, ANNNN, CNNNN, GNNNN, and TNNNN exhibited similar PAM consumption. This is consistent with our observations in wild-type LbCAs12a experiments, which showed that the NTTTV sequence was consumed in similar amounts regardless of whether N was A, C, G, or T (Table 3). Secondly, we observed similar consumption at the three time points of 75, 435, and 900 seconds. This indicates that the reaction was almost complete after only 75 seconds at 37°C. Therefore, we were able to average all four 4-nucleotide PAMs from the 5-nucleotide library and average over all three time points, effectively generating 12 data points for each PAM. We then took this average and divided it by the library median for each PAM. This provides us with a consumption score for each 4-nucleotide PAM across all 186 mutants. A score of 10 indicates that 90% of the four parent plasmid library members with the 4 nt PAM are consumed, while a score of 20 indicates 95% consumption.

[0250] Wild-type LbCas12a has a consumption score of 9.2 for TTTV sequences; therefore, using wild-type as a benchmark, any mutant that cuts PAM with a score of 9.2 or better is considered effective. For example, Table 4 shows that the mutant LbCas12a-K595Y consumed 45 different PAM tetramers from the library in vitro, at or above the level of wtLbCas12a cleavage of TTTV sequences. This analysis was used to score each of the 186 mutants to determine the in vitro PAM recognition and cleavage for each mutant. Data containing the recognition sequences are shown in Table 4, which shows the LbCas12a-K595Y PAMDA consumption score that reaches or exceeds the wtLbCas12a score of 9.2 for TTTV sequences.

[0251] Table 4.

[0252] PAM consumption data

[0253]

[0254]

[0255]

[0256]

[0257]

[0258]

[0259]

[0260]

[0261]

[0262] We observed 36 unique PAM sequences cleaved in vitro using two LbCas12a controls. This is consistent with the observation that wtLbCas12a can recognize and cleave more sequences than just TTTV in vitro. It has been demonstrated that TTCN, CTTN, TCTN, etc., are recognized and cleaved by LbCas12a in vitro, while AsCas12a shows only cleavage of TTTN (Zetsche et al.). Cell 163:759–771 (2015)).

[0263] Compared to wtLbCas12a, some mutants increased the total number of PAM sequences recognized and cleaved in vitro (Table 5). This does not refer to the absolute number of PAM recognition sequences, but rather to the overall heterogeneity conferred by individual mutations. Some individual point mutants are more heterogeneous than the wild type. For example, T152R recognizes 57 different PAMs, while K959Y recognizes 45.

[0264] Table 5: Some more heterogeneous LbCas12a mutants identified based on the number of PAMs identified and cleaved in vitro (nPAM).

[0265]

[0266] Comparison with wild-type LbCas12a consumption

[0267] In vitro, wtLbCas12a can recognize and cleave more sequences than just TTTV (Zetsche et al.) Cell 163:759–771 (2015)). It has been demonstrated that TTCN, CTTN, TCTN, etc., are recognized and cleaved by LbCas12a in vitro, while AsCas12a shows that it only cleaves TTTN (Zetsche et al.). Cell 163:759–771 (2015)). The aim of this study was to extend the PAM recognition of LbCas12a beyond its wild-type capabilities. To this end, we employed different analytical methods, not just library consumption scores. These scores are important for determining absolute PAM recognition and cleavage in vitro, but do not readily highlight changes in enzyme PAM recognition due to introduced point mutations.

[0268] First, as previously stated, the consumption results for 5-nucleotide PAMs were generalized to 4-nucleotide PAMs, each time point retained separately. The total NGS counts for each mutant-time point were normalized to 100 counts per PAM to account for loading differences on the NGS array. Then, the global median for each 4-nt PAM was compared to each mutant-time point. This provides consumption compared to the wild type, rather than consumption compared to the total library. This result highlights which mutations alter the PAM recognition profile. We took a conservative approach and selected consumption scores of 4 or higher as indicators of mutant recognition of novel PAMs. A consumption score of 4 indicates that the number of library members containing that particular PAM was cut four times the median for the wild type. For example, if 100 NGS counts were retained for wild-type PAMs containing GCGC, and 25 counts were retained for a particular mutant-time point, a score of 4 was calculated.

[0269] A summary of each of the 186 mutations is shown in Table 6 below. Mutations in bold indicate that, compared to the wild type, the mutation recognizes and cleaves more than 3 new PAM sequences, with a score higher than 4. Mutations in italics indicate that, compared to the wild type, the mutant acquires 1 to 3 new PAM sequences, with a score higher than 4. Mutations shown in regular font (not bold or italic) indicate that, compared to the wild type, the point mutation does not cleave new PAM sequences with a score higher than 4. Some amino acids, such as T149, did not acquire new PAM recognition, despite being close to the PAM recognition domain of the protein and 10 new amino acids were tested. Other amino acids, such as D156, appear to be hotspots for engineering new PAM recognition motifs. When aspartic acid 156 is changed to 10 different amino acids, compared to wtLbCAs12a, 7 mutations recognize multiple new PAMs, 1 shows several new PAMs, and 2 do not acquire new PAMs. Generally, any position showing a difference in PAM recognition and cleavage compared to the wild type can be combined into double, triple, or multiple mutations to further alter PAM recognition. Overall, of the 186 point mutations, 130 did not yield a new PAM (regular / non-bold or italic) relative to wtLbCas12a with a score higher than 4, 40 yielded many new PAMs (bold), and 16 yielded 1–3 new PAMs (italic). The overall success rate of 30% (56 / 186) indicates the use of an effective method for designing novel PAM recognition motifs through point mutations of LbCas12a.

[0270] Table 6. Summary of 186 LbCas12a point mutations (reference sequence SEQ ID NO: 1).

[0271]

[0272]

[0273] Many point mutations conferred novel PAM recognitions on LbCas12a, allowing it to cleave DNA prior to these sequences in vitro. Some mutations led to increased overall heterogeneity, while others designed and tested did not show alterations to wtLbCas12a recognition and cleavage. Overall, 130 of the 186 point mutations failed to yield novel PAMs exceeding wtLbCas12a with a score higher than 4 (Table 6, regular font / non-italic or bold), 40 of the 186 point mutations yielded many novel PAMs (Table 6, bold), and 16 of the 186 point mutations yielded 1–3 novel PAMs (Table 6, italic). The overall success rate of 30% (56 / 186) indicates that the method used to design novel PAM recognition motifs by point mutation of LbCas12a is effective.

[0274] 2. Determine the combination of point mutations and combinations in prokaryotes.

[0275] Combinations of single mutations can alter PAM recognition even more significantly than single mutations. However, such experiments can be rapidly scaled up to test a large number of combinations. Using only 40 mutations that induce LbCas12a to recognize three or more novel PAMs and constructing double-mutant libraries, a total of 40 combinations can be tested. 2 Or 1,600 enzymes. Creating a triple mutant library will yield 40 3 Purifying and repeatedly measuring 64,000 enzymes is impractical. Therefore, we modified a method called PAM-SCANR (Leenay et al.) Mol Cell The bacterial method described in 62, 137–147 (2016) was used to evaluate combinatorial mutations. We used a library from *E. coli* to test the binding of the Cas12a mutation to 256 possible PAM NNNN variants. This assay does not test for cleavage, but rather for in vivo binding. Since the prepared mutations are not near the catalytic region, binding was expected to reflect cleavage (this was later validated in the 293T assay). The advantage of PAM-SCANR is that it can rapidly test not only point mutations, but also combinations of amino acid point mutations in a rapid and accurate manner. It is also often more stringent than in vitro cleavage assays.

[0276] Report plasmid

[0277] The plasmid pWISE1963 was used as the base vector to generate reporter tokens for each of the 256 PAMs. This plasmid contained spectinomycin resistance, ColE1 origin of replication, LacI, and [other markers / signatures]. laceGFP under promoter control. Contains a fragment between NotI and SmaI restriction sites (from... lacI The promoter's exact 5' to lacI The 256 gene blocks (in the gene) were synthesized by Twist Bioscience. Each fragment contains a different tetramer PAM, located directly in... lacI The 5′ promoter. Each gene block was cloned into pWISE1963 by restriction and ligation. Clones of each variant were selected, and the identity of PAM was verified by Sanger sequencing.

[0278] CRISPR-Cas plasmid

[0279] Plasmid pWISE2031 was used as the base vector to generate all CRISPR-Cas plasmids. This plasmid contains chloramphenicol resistance, CloDF13 origin of replication, dLbCas12a driven by promoter BbaJ23108, and targeted plasmids driven by promoter BbaJ23119. lacI The promoter crRNA is LbCas12a. The negative control plasmid pWISE1961 contains the same components as pWISE2031, except that a non-targeting crRNA was used. Each point mutation and combinatorial mutant (pWISE2984–pWISE3007) was constructed in Genewiz by site-directed mutagenesis of pWISE2031.

[0280] cell lines

[0281] Obtain from Dharmacon Horizon Discovery lacI The *E. coli* cell line JW0336, with a gene deletion on the chromosome. Based on the described protocol (Sambrook, J. and Russell, DW (2006)). Transformation of E. coli by Electroporation Electrocompetent cells were prepared using Cold Spring Harb Protoc 2006 (pdb.prot3933). Escherichia coli JW0336 was used for all library transformations and cell sorting experiments.

[0282] Preparation of report libraries

[0283] Mix 10 ng of each reporter plasmid described in the previous section into a single tube to generate a library for transformation and amplification. Transform 1 / 20 of the mixed plasmid library (approximately 0.5 ng of each reporter) into supercompetent XL1-Blue cells according to the manufacturer's instructions. After 1 hour of recovery at 37°C and 225 rpm shaking, transfer the entire transformant to 1-L LB spectinomycin and grow overnight at 37°C and 225 rpm shaking. The next day, extract plasmid DNA from the overnight culture using the ZymoPURE Plasmid Ultra-Large Kit according to the manufacturer's instructions. Quantify the DNA using Nanodrop and use it for all subsequent library transformations.

[0284] Library transformation and cell sorting

[0285] 100 ng of reporter plasmid library and 100 ng of Crispr / Cas plasmid were co-transformed into 40 μL JW0336 via electroporation. The transformants were allowed to recover at 37°C with shaking at 225 rpm for 1 hour. At the end of recovery, 10 μL of transformants was taken and mixed with 90 μL LB agar, and plated onto LB agar plates containing chloramphenicol and spectinomycin to determine transformation efficiency. The remaining recovery volume (990 μL) was transferred to 29 mL LB agar containing spectinomycin and chloramphenicol and incubated overnight. The culture was allowed to grow overnight at 37°C with shaking at 225 rpm. The next morning, colonies were counted on the transformation plates to determine transformation efficiency; all but two showed >2,000 transformants, equivalent to 10X or higher coverage of the reporter library. Two samples that did not show 10X or higher coverage were replicated. Then, a glycerol stock of the overnight cultures was prepared and stored at -80°C, and small-volume extractions of 6 mL of each culture were performed using the Qiagen Mini Extract Kit according to the manufacturer's instructions. These mini extracts were labeled "before sorting" and stored at 4°C.

[0286] Centrifuge each overnight library culture at 1 optical density (OD) in a benchtop microcentrifuge at 8,000 rpm for 5 minutes at 4°C. Aspirate the supernatant and add 1 mL of filtered sterile 1X PBS buffer to each tube. Carefully resuspend the pellet by pipetting. Wash twice more with 1X PBS, and after the final resuspending, add cells (approximately 10 cells per mL of 1X PBS). 8(100 cells) were placed on ice. Each sample was sorted on a Beckman-Coulter MoFlo XDP cell sorter. Negative controls (WT-dLbCas12a + non-targeted crRNA + reporter library) and positive controls (WT-dLbCas12a + targeted crRNA + reporter library) were used to set the gating parameters for cell sorting. Samples were sorted in single-cell purity mode at 425V, SSC voltage at 535V, and FSC voltage (gain) at 4.0. Typical sorting rates were approximately 4000 events / second. Each sample had a minimum of 1.0 x 10⁻⁶ cells. 6 Perform cell sorting until 50,000 GFP-positive events are collected or the sample is exhausted. If the sample is exhausted, collect at least 200 GFP-positive events. Collect the GFP-positive events into tubes containing 2 mL of LB tincture containing spectinomycin and chloramphenicol. After sorting, dilute the sample to 6 mL with an additional LB tincture containing spectinomycin and chloramphenicol, and then grow overnight at 37°C with shaking at 225 rpm.

[0287] Figure 6-11 An example sorting method is provided in the document. Figure 6 Cell sorting results for a negative control containing wtLbCas12a and non-targeting plasmid spacers are shown. Sorted cells from high-GFP samples show a single population with no cells in the sorted section (left panel) and a single-peaked GFP signal (right panel). Figure 7 Cell sorting results for wtLbCas12a and crRNA targeting plasmid spacers are shown. Sorted cells from high-GFP samples are shown in the sorted portion (left panel, GFP hi) and two populations of GFP signal indicated by two major peaks (right panel, GFP neg and GFP high). Fluorescence of high-GFP sorted cells is also shown (bottom right inset). Figure 8 Cell sorting results for LbCas12a-K595Y and crRNA targeting plasmid spacers are shown. Sorted cells from high-GFP samples are shown in the sorted portion (left panel, GFP hi) and the population indicated by two major peaks (right panel, GFP neg and GFP high). Figure 9 The LbCas12a-G532R-K595R double mutation control is shown (Gao et al.) Nat Biotechnol Cell sorting results of crRNA targeting plasmid spacers (35, nbt.3900 (2017)). Sorted cells from high GFP samples are shown in the sorted portion (left panel, GFP hi) and the population indicated by two major peaks (right panel). Figure 10The cell sorting results for the LbCas12a-T152R-K595Y double mutation (a combination of two point mutations used with crRNA targeting the plasmid spacer) are shown. Sorted cells from high-GFP samples are shown in the sorted portion (left panel, GFP hi) and the population indicated by two major peaks (right panel). Figure 11 The cell sorting results for the LbCas12a-T152R-K538W-K595Y triple mutation (a combination of three point mutations used with crRNA targeting the plasmid spacer) are shown. Sorted cells from high-GFP samples are shown in the sorting section as cells (left, GFP hi) and the indicated population (right, green line).

[0288] Next-generation sequencing

[0289] The morning after sorting, glycerol reserves of each overnight culture were prepared and stored at -80°C. The remaining 6 mL of each culture was micro-extracted using the Qiagen Prep Kit according to the manufacturer's instructions. These extracts were labeled "Post-Sorting" and stored at 4°C. Pre- and post-sorting extracts were quantified using Nanodrop, diluted 10-fold, and transferred for sequencing on Illumina Mi-Seq.

[0290] Deep sequencing analysis of the spacer vector was performed using Illumina Miseq according to the manufacturer's protocol to calculate the A / T / G / C frequencies at each PAM site. Briefly, 10 ng of DNA was used as a template for PCR. Phasing gene-specific forward and reverse PCR primers were designed for amplification across the entire target site. A two-step PCR method was used to generate an amplicon library, where a preliminary PCR with a 5' tail allowed for the addition of Illumina i5 and i7 adaptor sequences and barcodes in the secondary PCR for multiplex sample sorting. PCR amplification was performed using the following parameters: 98°C for 30 s; PCR1 for 25 cycles, and PCR2 for 8 cycles (98°C 10 s, 55°C 20 s, 72°C 30 s); 72°C for 5 minutes; and maintained at 12°C. PCR reactions were performed using Q5 high-fidelity DNA polymerase (New England BioLabs, Beverly, MA, United States). Secondary PCR amplicon samples were individually purified using AMPure XP beads according to the manufacturer's instructions (Beckman Coulter, Brea, CA, United States); all purified samples were quantified using a plate reader, pooled in equimolar ratios, and run on an AATI fragment analyzer (Agilent Technologies, Palo Alto, CA, United States). The pooled amplicon libraries (2 × 250 paired ends) were sequenced on an Illumina MiSeq using the MiSeq Reagen kit v2 (Illumina, San Diego, CA, United States).

[0291] Sequencing results of high fluorescence sorted cells

[0292] With a report library of 256 members available, two negative control samples containing wtdLbCas12a and non-targeting crRNA were run. Values ​​for each member in the library were normalized to 1.0 and plotted as a histogram. Figure 12 ). Figure 12The total normalized NGS counts for two separate crRNA-free controls and wild-type dLbCas12a, as well as the reporter library, are shown. Two separate samples (512 points total, representing 256 PAMs x 2) were analyzed and pooled. We selected a conservative value of 1.67, the highest count, as the cutoff value for these experiments; values ​​above this value were denoted as PAM binding. The standard deviation was 0.16. Instead of choosing a multiple of the standard deviation as the cutoff value, we chose the absolute maximum value of 1.67 found in one of the two negative controls. This gave a very tight cutoff value, exceeding 10 times the standard deviation of the data. In fact, only 3 PAM sequences were found to be above 1.5.

[0293] Pools were sequenced prior to sorting. The average read count for each PAM was approximately 250 to 500 NGS reads, depending on the sample. Pools sorted after high fluorescence were sequenced, with each PAM having a similar read count of approximately 250-500 reads per PAM. The two samples were then normalized relative to the control, targeting small loading differences in each NGS experiment. These two values ​​were then subtracted and normalized to 1.0. Many PAM sequences were bound to point mutation libraries with a cutoff value above 1.67. Figure 13 (Table 7).

[0294] Table 7. The ability of point mutations above the threshold of 1.67 to bind PAM, sorted by normalized score. TTTV sequences for wild type are shown in bold.

[0295]

[0296]

[0297] We combined the three point mutations T152R, K538W, and K595Y in various combinations to generate double and triple dLbCas12a mutants (T152R+K538W, K538W+K595Y, and T152R+K538W+K595Y). These were compared to a previously described control generated in AsCas12a (referred to as “RR”), whose LbCas12a mutation corresponds to the G532R+K595R control (Gao et al.). Nat Biotechnol 35(8):789-792 (2017)). It is described that “RR” can cause INDEL in TYCV+CCCC sequences in AsCas12a and subsequently LbCas12a.

[0298] The same method was applied to score combinatorial mutations. Pre- and post-sorting pools were sequenced, with each PAM library member averaging approximately 250-500 MiSeq NGS reads. The pre- and post-sorting pools were normalized and subtracted, with the difference normalized to 1.0. Many PAM sequences were bound by combinations exceeding the cutoff value of 1.67. Figure 14 (Table 8).

[0299] Table 8. The ability of combined mutations above the threshold of 1.67 to bind PAM, sorted by normalized score. Wild-type TTTV sequences are shown in bold. The TYCV+CCCC sequences of the G532R+K595R control are underlined.

[0300]

[0301] Overall Analysis of PAM-SCANR Data

[0302] Wild-type LbCas12a showed strong TTTV binding, while the LbCas12a-G532R-K595R control showed strong TYCV and CCCC binding. This mutation, termed "RR," arises in AsCas12a and shows binding to both TYCV and CCCC (Gao et al.). Nat Biotechnol 35(8):789-792 (2017)). However, in vitro, wtLbCas12a can identify and cleave TTCN, CTTN, TCTN, etc., while AsCas12a only shows cleavage of TTTN (Zetsche et al., Cell 163:759–771 (2015)). We infer that the “RR” mutation placed in an LbCas12a background is more heterogeneous than when placed in an AsCas12a background, which is what was observed for this control, namely that LbCas12a-RR recognizes 45 sequences. The results for both wild-type and LbCas12a-RR demonstrate the effectiveness of the selection and sorting parameters.

[0303] The tested mutations clearly demonstrate that a single point mutation identified in vitro recognizes a novel PAM in vivo. For example, K595Y binds to 13 PAMs at a threshold of 1.67, 11 of which are not recognized by wtLbCas12a, and none contain a TTTV sequence known to be bound by Cas12a. Similarly, T152R recognizes 15 different PAMs; however, in this case, it retains the wild-type TTTV. Overall, of the 12 point mutations tested, each possesses a novel PAM-binding sequence outside the classic TTTV motif and distinct from the dLbCas12a control.

[0304] Combination effect

[0305] We found that combining multiple point mutations did not cause a linear increase in the number of point-mutated PAM sequences. Figure 15 For example, combining the LbCas12a point mutations K538W and K595Y produces the enzyme LbCas12a-K538W-K595Y. In some cases, it shares a PAM recognition motif with either K538W (vertical shading) or K595Y (horizontal shading), but more commonly it produces a novel PAM recognition sequence (thatched). Using the same example, K538W recognizes AGCT, but K538W+K595Y does not. K595Y recognizes ACGC, but K538W+K595Y does not. CCCC is neither recognized by K538W nor K595Y, yet the double mutant binds to it with high affinity.

[0306] Overall, combinations of mutations lead to more than linear expansion of PAM recognition. For example, K538W recognizes 6 PAM sequences, K595Y recognizes 13, but together they recognize 32 sequences. Figure 15 A simple additive effect results in 19 PAMs in the double mutant, instead of the 32 we observed. Furthermore, only 11 of the 32 sequences recognized by the double mutant were recognized by either of the two single mutations. A similar pattern was observed when combining three mutations. Figure 16 The combination of T152R, K538W, and K595Y produces a triple mutation whose PAM recognition differs from that of any one of the three individual mutations alone. For example, GGCA, GGCC, GGGC, and GGGG are only recognized when all three mutations occur on LbCas12a. None of these PAMs can be bound by any single or double mutation, but they will only bind when T152R, K538W, and K595Y are all mutated together.

[0307] Comparison of PAM-SCANR and in vitro PAMDA recognition of point mutation PAM

[0308] Overall, for the 12 point mutations tested, PAM-SCANR hits above 1.67 were well reflected in the in vitro PAMDA depletion assay. Examples of K595Y and T152R are shown below. Figure 17 ). Figure 17We compared all non-TTTV PAMs from PAM-SCANR (gray box) to K595Y (left inset) and T152R (right inset) showing scores higher than 1.67. With one exception, all PAM-SCANR-positive PAMs with cutoff values ​​higher than 1.67 had in vitro PAM consumption scores higher than 9.2. However, the PAM-SCANR method and analysis are more stringent than in vitro assays and analyses. For example, sorting, ranking, and normalizing 13 different PAMs yielded values ​​higher than 1.67 in PAM-SCANR. This contrasts with the PAMDA assay, which identified 45 readily cleavable sequences in vitro. This could be due to the influence of intracellular relative concentrations compared to in vitro concentrations, but it could also be due to overly stringent cutoff values ​​for PAM-SCANR or overly lenient cutoff values ​​for the PAMDA assay.

[0309] The correlations between the datasets indicate that our engineering of residues far from the catalytic site affected PAM recognition and binding, rather than catalysis. If mutations at these residues affected nuclease activity and PAM binding, there would be many hits in the PAM-SCANR assay (which measures binding, not cleavage) that do not show cleavage in the PAMDA assay. We did not observe this pattern. We observed that mutations affecting binding changes (PAM-SCANR) also resulted in in vitro cleavage (PAMDA).

[0310] 3. Determination of binding, cleavage, and INDEL formation in eukaryotes.

[0311] We selected three mutants, T152R, K538W, and K595Y, to test their ability to induce insertions or deletions (INDELs) in eukaryotic HEK293T cells. This assay provides valuable eukaryotic INDEL data. To obtain insertions and deletions in eukaryotes, several criteria must be met simultaneously: the CRISPR enzyme needs to be expressed and stable in the cell; crRNA needs to be expressed and properly processed; the protein:RNA complex needs to form; the complex needs to be stable; the complex needs to translocate to the nucleus in sufficient quantities; the target DNA needs to be accessible; the DNA must be well-targeted via a specific guide RNA design; and double-strand breaks need to occur at a sufficiently high rate to elicit accidental DNA repair errors via insertions or deletions (INDELs). This makes the eukaryotic assay the most rigorous in this study. Due to the low-throughput nature of the experiments, dozens of PAMs were tested for each of the three point mutants described below, instead of all 256. Three different targets were selected for each PAM-mutant combination to minimize false negatives, as specific guide RNAs are often ineffective due to target accessibility.

[0312] HEK293T cell assay

[0313] Eukaryotic HEK293T (ATCC CRL-3216) cells were cultured in Durbecco's Modified Eagle's medium supplemented with 10% (v / v) FBS and GlutaMax (ThermoFisher) at 37°C and 5% CO2. Wild-type and mutant LbCas12 were synthesized using a solid-state synthesis method and subsequently cloned into a plasmid following the CMV promoter. CRISPR RNA (crRNA) was cloned following the human U6 promoter (Table 9). HEK293T cells were seeded in 48-well collagen-coated BioCoat plates (Corning). Cells were transfected at approximately 70% confluence. 750 ng of protein plasmid and 250 ng of crRNA expression plasmid were transfected per well using 1.5 μl of Lipofectamine 3000 (ThermoFisher Scientific) according to the manufacturer's protocol. Genomic DNA was obtained from transfected cells 3 days later, and indels were detected and quantified using high-throughput Illumina amplicon sequencing.

[0314] Deep sequencing analysis of the spacer vector was performed using Illumina Miseq according to the manufacturer's protocol to calculate the A / T / G / C frequencies at each PAM site. Briefly, 10 ng of DNA was used as a template for PCR. Phasing gene-specific forward and reverse PCR primers were designed for amplification across the entire target site. A two-step PCR method was used to generate an amplicon library, where a preliminary PCR with a 5' tail allowed for the addition of Illumina i5 and i7 adaptor sequences and barcodes in the secondary PCR for multiplex sample sorting. PCR amplification was performed using the following parameters: 98°C for 30 s; PCR1 for 25 cycles, and PCR2 for 8 cycles (98°C 10 s, 55°C 20 s, 72°C 30 s); 72°C for 5 minutes; and maintained at 12°C. PCR reactions were performed using Q5 high-fidelity DNA polymerase (New England BioLabs, Beverly, MA, United States). Secondary PCR amplicon samples were purified separately using AMPure XP beads according to the manufacturer's instructions (Beckman Coulter, Brea, CA, United States). All purified samples were quantified using a plate reader, pooled in equimolar ratios, and run on an AATI fragment analyzer (Agilent Technologies, Palo Alto, CA, United States). The pooled amplicon libraries (2 × 250 paired ends) were sequenced on an Illumina MiSeq system using the MiSeq Reagent kit v2 (Illumina, San Diego, CA, United States).

[0315] Wild-type control

[0316] Wild-type LbCas12a (wtLbCas12a) recognizes TTTV (TTTA, TTTC, and TTTG). We used crRNA spacers (Table 9) to test the response of wild-type proteins to 23 nucleotide spacer targets containing TTTV. Figure 18 ).

[0317] Table 9. Spacers and Targets

[0318]

[0319] Selecting proteins and targets

[0320] Numerous point mutations in the PAMDA in vitro assay demonstrate enhanced PAM accessibility. Given our large number of point mutations and 256 possible 4-nucleotide (nt) PAMs, testing all effective PAM mutants for endogenous 293T cell targets is practically impossible due to experimental complexity, cost, and time. Therefore, we selected three point mutations to test a subset of PAMs. The three point mutations tested were T152R, K538W, and K595Y.

[0321] Genomic targets in 293T cells were selected based on their PAM sequences. Three point-mutated genomic targets were randomly selected without using any specific rules, except for having a suitable 4-nt PAM and selecting the 23 nucleotides downstream of that PAM. Three different spacers were selected to determine each PAM. This is because CRISPR enzyme activity is observed to be target-specific and often unpredictable.

[0322] On average, we observed that approximately half of the 23 nucleotide wtLbCas12a spacers tested were invalid despite having the correct PAM TTTV sequence. Since there are only three data points for each PAM and approximately 50% of the targets were observed not to produce INDELs, assessing PAM identification by visualizing the maximum percentage of INDELs for each PAM, rather than the average of randomly designed spacers, yields richer information. Figure 19-21 If a larger number of spacers for each PAM are determined, then statistical tests can be used to assess their average editing efficiency.

[0323] Our overall transfection and assay results showed that wild-type LbCAs12a induced INDEL in HEK293T cells at approximately 11-26% in TTTC, 10% in TTTA, and 4-10% in TTTG. Figure 18 These predefined HEK293T target sites and guides from the literature are therefore expected to be more efficient than any randomly selected guide. Although the crRNA guide for mutants was designed randomly, many novel Cas12a PAM recognition sites produced INDEL at a rate similar to that of the wild type at the TTTV sequence. Figure 19-21 Any INDEL higher than 0.1% is higher than the noise of sequencing assays read at a depth of 10,000 NGS.

[0324] The INDEL levels that K595Y can cause are as follows: ACCG 25.5%, CCGC 10.9%, TCGC 10.1%, CCCG 9.5%, GCGC 8.3%, CTGG 7.8%, ACGG 6.3%, CCCG 6.0%, and TGGC 5.3%, etc. Figure 19 Although randomly designed, these figures are all within the range of the TTTV controls for wtLbCas12a. A key characteristic of Cas12a proteins is their recognition of T-rich PAMs (Zetsche et al.). Cell 163:759–771 (2015)). This limits their utility in genome editing technologies. K595Y clearly prefers C and G-rich PAMs, which would expand the utility of Cas2a to target the previous primary targets of Cas9 CRISPR enzymes (which utilize G-rich PAMs) (Jinek et al.). Science 337, 816–821 (2012)). For K595Y, only 31 (or 12%) of the total 256 possible tetranucleotide PAMs were tested in 293T cells. There are likely many other PAMs that can be recognized by K595Y and cause INDELs in eukaryotic cells.

[0325] T152R can cause INDEL levels of 11.5% for CCTC, 10.0% for CCTG, 9.6% for CCCA, 8.4% for GCCA, 7.2% for GCCC, and 5.1% for CTGC, etc. Figure 20 Interestingly, T152R retained TTTV recognition of wtLbCas12a, resulting in INDELs of 34.9% for TTTC, 10.2% for TTTA, and 6.2% for TTTG. It also acquired TTTT recognition, resulting in an INDEL of 8.3%. For T152R, only 22 (or 9%) of the total 256 possible tetranucleotide PAMs were tested in 293T cells. There are likely many other PAMs that can be recognized by T152R and cause INDELs in eukaryotic cells.

[0326] like Figure 21As shown, 22 out of 28 PAM targets did indeed exhibit activity above 0.1% background, indicating that 79% of the tested PAMs were recognized and cleaved by the enzyme, although this was sometimes lower than expected for some applications. For the three selected targets, none of the six tested PAMs showed any above-background editing. The three TTTV targets maintained good activity, with TTTC, TTTG, and TTTA at 15.6%, 6.2%, and 5.8%, respectively. Other PAM sequences with more than 1% INDEL formation included ATTA (3.5%), TTTT (3.2%), TGTC (1.8%), AGCG (1.8%), AGTC (1.6%), AGCA (1.4%), and GGTC (1.1%). In PAM-SCANR assays, this point mutation was used in combination with T152R and / or K595Y to generate a wide variety of PAM recognitions; however, on its own, it bound relatively few PAMs when used with this assay. Using it in a future double mutation, rather than alone, may be a superior option to generate INDEL in HEK293T cells. Similar to the other two point mutations, only 28 (11%) of the possible 256 tetranucleotide PAMs were tested, and this mutant may recognize PAMs or targets not tested here.

[0327] Correlation between HEK293T INDEL and PAM-SCANR binding

[0328] A correlation was observed between the observed maximum percentage of INDEL for T152R and K595Y and the PAM-SCANR score. Figures 22A-22B ). Figures 22A-22B LbCas12a-T152R (shown) Figure 22A ) and LbCas12a-K595Y ( Figure 22B The linear correlation between % INDEL (maximum value) and the standardized bacterial PAM-SCANR score.

[0329] Notably, any point mutation with a PAM-SCANR score higher than 1.5 tested produced INDELs at a rate greater than 5% in 293T cells. This suggests that any mutation with a normalized score greater than 1.5 (instead of our strict cutoff value of 1.67) tested in the PAM-SCANR experiment is likely to be able to produce INDELs at a rate useful for most eukaryotic applications.

[0330] The foregoing is a description of the present invention and should not be construed as limiting it. The present invention is defined by the following claims, including their equivalents. sequence list <110> Pairwise Plants Services, Inc. Watts, Joseph Matthew Jali, Sathya Sheela <120> Variants of CAS12A nuclease, their preparation methods, and applications <130> 1499.7.WO <150> US 62 / 916,392 <151> 2019-10-17 <160> 62 <170> PatentIn version 3.5 <210> 1 <211> 1228 <212> PRT <213> unknown <220> <223> Bacteria of the family Trichophyceae <400> 1 Met Ser Lys Leu Glu Lys Phe Thr Asn Cys Tyr Ser Leu Ser Lys Thr 1 5 10 15 Leu Arg Phe Lys Ala Ile Pro Val Gly Lys Thr Gln Glu Asn Ile Asp 20 25 30 Asn Lys Arg Leu Leu Val Glu Asp Glu Lys Arg Ala Glu Asp Tyr Lys 35 40 45 Gly Val Lys Lys Leu Leu Asp Arg Tyr Tyr Leu Ser Phe Ile Asn Asp 50 55 60 Val Leu His Ser Ile Lys Leu Lys Asn Leu Asn Asn Tyr Ile Ser Leu 65 70 75 80 Phe Arg Lys Lys Thr Arg Thr Glu Lys Glu Asn Lys Glu Leu Glu Asn 85 90 95 Leu Glu Ile Asn Leu Arg Lys Glu Ile Ala Lys Ala Phe Lys Gly Asn 100 105 110 Glu Gly Tyr Lys Ser Leu Phe Lys Lys Asp Ile Ile Glu Thr Ile Leu 115 120 125 Pro Glu Phe Leu Asp Asp Lys Asp Glu Ile Ala Leu Val Asn Ser Phe 130 135 140 Asn Gly Phe Thr Thr Ala Phe Thr Gly Phe Phe Asp Asn Arg Glu Asn 145 150 155 160 Met Phe Ser Glu Glu Ala Lys Ser Thr Ser Ile Ala Phe Arg Cys Ile 165 170 175 Asn Glu Asn Leu Thr Arg Tyr Ile Ser Asn Met Asp Ile Phe Glu Lys 180 185 190 Val Asp Ala Ile Phe Asp Lys His Glu Val Gln Glu Ile Lys Glu Lys 195 200 205 Ile Leu Asn Ser Asp Tyr Asp Val Glu Asp Phe Phe Glu Gly Glu Phe 210 215 220 Phe Asn Phe Val Leu Thr Gln Glu Gly Ile Asp Val Tyr Asn Ala Ile 225 230 235 240 Ile Gly Gly Phe Val Thr Glu Ser Gly Glu Lys Ile Lys Gly Leu Asn 245 250 255 Glu Tyr Ile Asn Leu Tyr Asn Gln Lys Thr Lys Gln Lys Leu Pro Lys 260 265 270 Phe Lys Pro Leu Tyr Lys Gln Val Leu Ser Asp Arg Glu Ser Leu Ser 275 280 285 Phe Tyr Gly Glu Gly Tyr Thr Ser Asp Glu Glu Val Leu Glu Val Phe 290 295 300 Arg Asn Thr Leu Asn Lys Asn Ser Glu Ile Phe Ser Ser Ile Lys Lys 305 310 315 320 Leu Glu Lys Leu Phe Lys Asn Phe Asp Glu Tyr Ser Ser Ala Gly Ile 325 330 335 Phe Val Lys Asn Gly Pro Ala Ile Ser Thr Ile Ser Lys Asp Ile Phe 340 345 350 Gly Glu Trp Asn Val Ile Arg Asp Lys Trp Asn Ala Glu Tyr Asp Asp 355 360 365 Ile His Leu Lys Lys Lys Ala Val Val Thr Glu Lys Tyr Glu Asp Asp 370 375 380 Arg Arg Lys Ser Phe Lys Lys Ile Gly Ser Phe Ser Leu Glu Gln Leu 385 390 395 400 Gln Glu Tyr Ala Asp Ala Asp Leu Ser Val Val Glu Lys Leu Lys Glu 405 410 415 Ile Ile Ile Gln Lys Val Asp Glu Ile Tyr Lys Val Tyr Gly Ser Ser 420 425 430 Glu Lys Leu Phe Asp Ala Asp Phe Val Leu Glu Lys Ser Leu Lys Lys 435 440 445 Asn Asp Ala Val Val Ala Ile Met Lys Asp Leu Leu Asp Ser Val Lys 450 455 460 Ser Phe Glu Asn Tyr Ile Lys Ala Phe Phe Gly Glu Gly Lys Glu Thr 465 470 475 480 Asn Arg Asp Glu Ser Phe Tyr Gly Asp Phe Val Leu Ala Tyr Asp Ile 485,490,495 Leu Leu Lys Val Asp His With Tyr Asp With Arg Asn Tyr Val Thr 500 505 510 Gln Lys Pro Tyr Ser Lys Asp Lys Phe Lys Leu Tyr Phe Gln Asn Pro 515,520,525 Gln Phe Met Gly Gly Trp Asp Lys Asp Lys Glu Thr Asp Tyr Arg Ala 530 535 540 Thr Ile Leu Arg Tyr Gly Ser Lys Tyr Leu Ala Ile Met Asp Lys 545 550 555 560 Lys Tyr Ala Lys Cys Leu Gln Lys Ile Asp Lys Asp Val Asn Gly 565,570,575 Asn Tyr Glu Lys And Asn Tyr Lys Leu Pro Gly Pro Asn Lys Met 580,585,590 Leu Pro Lys Val Phe Phe Ser Lys Lys Trp Met Ala Tyr Tyr Asn Pro 595,600,605 Serving Glu Asp With Gln Lys Ile Tyr Lys Asn Gly Thr Phe Lys Gly 610 615 620 Asp Met Phe Asn Leu Asn Asp Cys His Lys Leu Ile Asp Phe Phe Lys 625 630 635 640 Asp Ser Ile Ser Arg Tyr Pro Lys Trp Ser Asn Ala Tyr Asp Phe Asn 645,650,655 Phe Ser Glu Thr Glu Lys Tyr Lys Asp Ile Ala Gly Phe Tyr Arg Glu 660,665,670 Val Glu Glu Gln Gly Tyr Lys Val Ser Phe Glu Ser Ala Ser Lys Lys 675,680,685 Glu Val Asp Lys Leu Val Glu Glu Gly Lys Leu Tyr Met Phe Gln Ile 690,695,700 Tyr Asn Lys Asp Phe Ser Asp Lys Ser His Gly Thr Pro Asn Leu His 705 710 715 720 Thr Met Tyr Phe Lys Leu Leu Phe Asp Glu Asn Asn His Gly Gln Ile 725 730 735 Arg Leu Ser Gly Gly Ala Glu Leu Phe Met Arg Arg Ala Ser Leu Lys 740 745 750 Lys Glu Glu Leu Val Val His Pro Ala Asn Ser Pro Ile Ala Asn Lys 755 760 765 Asn Pro Asp Asn Pro Lys Lys Thr Thr Thr Leu Ser Tyr Asp Val Tyr 770 775 780 Lys Asp Lys Arg Phe Ser Glu Asp Gln Tyr Glu Leu His Ile Pro Ile 785 790 795 800 Ala Ile Asn Lys Cys Pro Lys Asn Ile Phe Lys Ile Asn Thr Glu Val 805 810 815 Arg Val Leu Leu Lys His Asp Asp Asn Pro Tyr Val Ile Gly Ile Asp 820 825 830 Arg Gly Glu Arg Asn Leu Leu Tyr Ile Val Val Val Asp Gly Lys Gly 835 840 845 Asn Ile Val Glu Gln Tyr Ser Leu Asn Glu Ile Ile Asn Asn Phe Asn 850 855 860 Gly Ile Arg Ile Lys Thr Asp Tyr His Ser Leu Leu Asp Lys Lys Glu 865 870 875 880 Lys Glu Arg Phe Glu Ala Arg Gln Asn Trp Thr Ser Ile Glu Asn Ile 885 890 895 Lys Glu Leu Lys Ala Gly Tyr Ile Ser Gln Val Val His Lys Ile Cys 900 905 910 Glu Leu Val Glu Lys Tyr Asp Ala Val Ile Ala Leu Glu Asp Leu Asn 915 920 925 Ser Gly Phe Lys Asn Ser Arg Val Lys Val Glu Lys Gln Val Tyr Gln 930 935 940 Lys Phe Glu Lys Met Leu Ile Asp Lys Leu Asn Tyr Met Val Asp Lys 945 950 955 960 Lys Ser Asn Pro Cys Ala Thr Gly Gly Ala Leu Lys Gly Tyr Gln Ile 965 970 975 Thr Asn Lys Phe Glu Ser Phe Lys Ser Met Ser Thr Gln Asn Gly Phe 980 985 990 Ile Phe Tyr Ile Pro Ala Trp Leu Thr Ser Lys Ile Asp Pro Ser Thr 995 1000 1005 Gly Phe Val Asn Leu Leu Lys Thr Lys Tyr Thr Ser Ile Ala Asp 1010 1015 1020 Ser Lys Lys Phe Ile Ser Phe Asp Arg Ile Met Tyr Val Pro 1025 1030 1035 Glu Glu Asp Leu Phe Glu Phe Ala Leu Asp Tyr Lys Asn Phe Ser 1040 1045 1050 Arg Thr Asp Ala Asp Tyr Ile Lys Trp Lys Leu Tyr Ser Tyr 1055 1060 1065 Gly Asn Arg Ile Arg Ile Phe Arg Asn Pro Lys Lys Asn Asn Val 1070 1075 1080 Phe Asp Trp Glu Glu Val Cys Leu Thr Ser Ala Tyr Lys Glu Leu 1085 1090 1095 Phe Asn Lys Tyr Gly And Asn Tyr Gln Gln Gly Asp With Arg Ala 1100 1105 1110 Leu Leu Cys Glu Gln Ser Asp Lys Ala Phe Tyr Ser Ser Phe Met 1115 1120 1125 Only One Met Ser Many Met Many Gln Met Arg Asn Ser Ile Thr Gly 1130 1135 1140 Arg Thr Asp Val Asp Phe Leu Ile Ser Pro Val Lys Asn Ser Asp 1145 1150 1155 Gly With Phe Tyr Asp Ser Arg Asn Tyr Glu Ala Gln Glu Asn Ala 1160 1165 1170 Ile Leu Pro Lys Asn Ala Asp Ala Asn Gly Ala Tyr Asn Ile Ala 1175 1180 1185 Arg Lys Val Leu Trp Ala Ile Gly Gln Phe Lys Lys Ala Glu Asp 1190 1195 1200 Glu Lys Leu Asp Lys Val Lys Ile Ala Ile Ser Asn Lys Glu Trp 1205 1210 1215 Leu Glu Tyr Ala Gln Thr Ser Val Lys His 1220 1225 <210> 2 <211> 1307 <212> PRT <213> Acidaminococcus sp. <400> 2 Met Thr Gln Phe Glu Gly Phe Thr Asn Leu Tyr Gln Val Ser Lys Thr 1 5 10 15 Leu Arg Phe Glu Leu Ile Pro Gln Gly Lys Thr Leu Lys His Ile Gln 20 25 30 Glu Gln Gly Phe Ile Glu Glu Asp Lys Ala Arg Asn Asp His Tyr Lys 35 40 45 Glu Leu Lys Pro Ile Ile Asp Arg Ile Tyr Lys Thr Tyr Ala Asp Gln 50 55 60 Cys Leu Gln Leu Val Gln Leu Asp Trp Glu Asn Leu Ser Ala Ala Ile 65 70 75 80 Asp Ser Tyr Arg Lys Glu Lys Thr Glu Glu Thr Arg Asn Ala Leu Ile 85 90 95 Glu Glu Gln Ala Thr Tyr Arg Asn Ala Ile His Asp Tyr Phe Ile Gly 100 105 110 Arg Thr Asp Asn Leu Thr Asp Ala Ile Asn Lys Arg His Ala Glu Ile 115 120 125 Tyr Lys Gly Leu Phe Lys Ala Glu Leu Phe Asn Gly Lys Val Leu Lys 130 135 140 Gln Leu Gly Thr Val Thr Thr Thr Glu His Glu Asn Ala Leu Leu Arg 145 150 155 160 Ser Phe Asp Lys Phe Thr Thr Tyr Phe Ser Gly Phe Tyr Glu Asn Arg 165 170 175 Lys Asn Val Phe Ser Ala Glu Asp Ile Ser Thr Ala Ile Pro His Arg 180 185 190 Ile Val Gln Asp Asn Phe Pro Lys Phe Lys Glu Asn Cys His Ile Phe 195 200 205 Thr Arg Leu Ile Thr Ala Val Pro Ser Leu Arg Glu His Phe Glu Asn 210 215 220 Val Lys Lys Ala Ile Gly Ile Phe Val Ser Thr Ser Ile Glu Glu Val 225 230 235 240 Phe Ser Phe Pro Phe Tyr Asn Gln Leu Leu Thr Gln Thr Gln Ile Asp 245 250 255 Leu Tyr Asn Gln Leu Leu Gly Gly Ile Ser Arg Glu Ala Gly Thr Glu 260 265 270 Lys Ile Lys Gly Leu Asn Glu Val Leu Asn Leu Ala Ile Gln Lys Asn 275 280 285 Asp Glu Thr Ala His Ile Ile Ala Ser Leu Pro His Arg Phe Ile Pro 290 295 300 Leu Phe Lys Gln Ile Leu Ser Asp Arg Asn Thr Leu Ser Phe Ile Leu 305 310 315 320 Glu Glu Phe Lys Ser Asp Glu Glu Val Ile Gln Ser Phe Cys Lys Tyr 325 330 335 Lys Thr Leu Leu Arg Asn Glu Asn Val Leu Glu Thr Ala Glu Ala Leu 340 345 350 Phe Asn Glu Leu Asn Ser Ile Asp Leu Thr His Ile Phe Ile Ser His 355 360 365 Lys Lys Leu Glu Thr Ile Ser Ser Ala Leu Cys Asp His Trp Asp Thr 370 375 380 Leu Arg Asn Ala Leu Tyr Glu Arg Arg Ile Ser Glu Leu Thr Gly Lys 385 390 395 400 Ile Thr Lys Ser Ala Lys Glu Lys Val Gln Arg Ser Leu Lys His Glu 405 410 415 Asp Ile Asn Leu Gln Glu Ile Ile Ser Ala Ala Gly Lys Glu Leu Ser 420 425 430 Glu Ala Phe Lys Gln Lys Thr Ser Glu Ile Leu Ser His Ala His Ala 435 440 445 Ala Leu Asp Gln Pro Leu Pro Thr Thr Leu Lys Lys Gln Glu Glu Lys 450 455 460 Glu Ile Leu Lys Ser Gln Leu Asp Ser Leu Leu Gly Leu Tyr His Leu 465 470 475 480 Leu Asp Trp Phe Ala Val Asp Glu Ser Asn Glu Val Asp Pro Glu Phe 485 490 495 Ser Ala Arg Leu Thr Gly Ile Lys Leu Glu Met Glu Pro Ser Leu Ser 500 505 510 Phe Tyr Asn Lys Ala Arg Asn Tyr Ala Thr Lys Lys Pro Tyr Ser Val 515 520 525 Glu Lys Phe Lys Leu Asn Phe Gln Met Pro Thr Leu Ala Ser Gly Trp 530 535 540 Asp Val Asn Lys Glu Lys Asn Asn Gly Ala Ile Leu Phe Val Lys Asn 545 550 555 560 Gly Leu Tyr Tyr Leu Gly Ile Met Pro Lys Gln Lys Gly Arg Tyr Lys 565 570 575 Ala Leu Ser Phe Glu Pro Thr Glu Lys Thr Ser Glu Gly Phe Asp Lys 580 585 590 Met Tyr Tyr Asp Tyr Phe Pro Asp Ala Ala Lys Met Ile Pro Lys Cys 595 600 605 Ser Thr Gln Leu Lys Ala Val Thr Ala His Phe Gln Thr His Thr Thr 610 615 620 Pro Ile Leu Leu Ser Asn Asn Phe Ile Glu Pro Leu Glu Ile Thr Lys 625 630 635 640 Glu Ile Tyr Asp Leu Asn Asn Pro Glu Lys Glu Pro Lys Lys Phe Gln 645 650 655 Thr Ala Tyr Ala Lys Lys Thr Gly Asp Gln Lys Gly Tyr Arg Glu Ala 660 665 670 Leu Cys Lys Trp Ile Asp Phe Thr Arg Asp Phe Leu Ser Lys Tyr Thr 675 680 685 Lys Thr Thr Ser Ile Asp Leu Ser Ser Leu Arg Pro Ser Ser Gln Tyr 690 695 700 Lys Asp Leu Gly Glu Tyr Tyr Ala Glu Leu Asn Pro Leu Leu Tyr His 705 710 715 720 Ile Ser Phe Gln Arg Ile Ala Glu Lys Glu Ile Met Asp Ala Val Glu 725 730 735 Thr Gly Lys Leu Tyr Leu Phe Gln Ile Tyr Asn Lys Asp Phe Ala Lys 740 745 750 Gly His His Gly Lys Pro Asn Leu His Thr Leu Tyr Trp Thr Gly Leu 755 760 765 Phe Ser Pro Glu Asn Leu Ala Lys Thr Ser Ile Lys Leu Asn Gly Gln 770 775 780 Ala Glu Leu Phe Tyr Arg Pro Lys Ser Arg Met Lys Arg Met Ala His 785 790 795 800 Arg Leu Gly Glu Lys Met Leu Asn Lys Lys Leu Lys Asp Gln Lys Thr 805 810 815 Pro Ile Pro Asp Thr Leu Tyr Gln Glu Leu Tyr Asp Tyr Val Asn His 820 825 830 Arg Leu Ser His Asp Leu Ser Asp Glu Ala Arg Ala Leu Leu Pro Asn 835 840 845 Val Ile Thr Lys Glu Val Ser His Glu Ile Ile Lys Asp Arg Arg Phe 850 855 860 Thr Ser Asp Lys Phe Phe Phe His Val Pro Ile Thr Leu Asn Tyr Gln 865 870 875 880 Ala Ala Asn Ser Pro Ser Lys Phe Asn Gln Arg Val Asn Ala Tyr Leu 885 890 895 Lys Glu His Pro Glu Thr Pro Ile Ile Gly Ile Asp Arg Gly Glu Arg 900 905 910 Asn Leu Ile Tyr Ile Thr Val Ile Asp Ser Thr Gly Lys Ile Leu Glu 915 920 925 Gln Arg Ser Leu Asn Thr Ile Gln Gln Phe Asp Tyr Gln Lys Lys Leu 930 935 940 Asp Asn Arg Glu Lys Glu Arg Val Ala Ala Arg Gln Ala Trp Ser Val 945 950 955 960 Val Gly Thr Ile Lys Asp Leu Lys Gln Gly Tyr Leu Ser Gln Val Ile 965 970 975 His Glu Ile Val Asp Leu Met Ile His Tyr Gln Ala Val Val Val Leu 980 985 990 Glu Asn Leu Asn Phe Gly Phe Lys Ser Lys Arg Thr Gly Ile Ala Glu 995 1000 1005 Lys Ala Val Tyr Gln Gln Phe Glu Lys Met Leu Ile Asp Lys Leu 1010 1015 1020 Asn Cys Leu Val Leu Lys Asp Tyr Pro Ala Glu Lys Val Gly Gly 1025 1030 1035 Val Leu Asn Pro Tyr Gln Leu Thr Asp Gln Phe Thr Ser Phe Ala 1040 1045 1050 Lys Met Gly Thr Gln Ser Gly Phe Leu Phe Tyr Val Pro Ala Pro 1055 1060 1065 Tyr Thr Ser Lys Ile Asp Pro Leu Thr Gly Phe Val Asp Pro Phe 1070 1075 1080 Val Trp Lys Thr Ile Lys Asn His Glu Ser Arg Lys His Phe Leu 1085 1090 1095 Glu Gly Phe Asp Phe Leu His Tyr Asp Val Lys Thr Gly Asp Phe 1100 1105 1110 Ile Leu His Phe Lys Met Asn Arg Asn Leu Ser Phe Gln Arg Gly 1115 1120 1125 Leu Pro Gly Phe Met Pro Ala Trp Asp Ile Val Phe Glu Lys Asn 1130 1135 1140 Glu Thr Gln Phe Asp Ala Lys Gly Thr Pro Phe Ile Ala Gly Lys 1145 1150 1155 Arg Ile Val Pro Val Ile Glu Asn His Arg Phe Thr Gly Arg Tyr 1160 1165 1170 Arg Asp Leu Tyr Pro Ala Asn Glu Leu Ile Ala Leu Leu Glu Glu 1175 1180 1185 Lys Gly Ile Val Phe Arg Asp Gly Ser Asn Ile Leu Pro Lys Leu 1190 1195 1200 Leu Glu Asn Asp Asp Ser His Ala Ile Asp Thr Met Val Ala Leu 1205 1210 1215 Ile Arg Ser Val Leu Gln Met Arg Asn Ser Asn Ala Ala Thr Gly 1220 1225 1230 Glu Asp Tyr Ile Asn Ser Pro Val Arg Asp Leu Asn Gly Val Cys 1235 1240 1245 Phe Asp Ser Arg Phe Gln Asn Pro Glu Trp Pro Met Asp Ala Asp 1250 1255 1260 Ala Asn Gly Ala Tyr His Ile Ala Leu Lys Gly Gln Leu Leu Leu 1265 1270 1275 Asn His Leu Lys Glu Ser Lys Asp Leu Lys Leu Gln Asn Gly Ile 1280 1285 1290 Ser Asn Gln Asp Trp Leu Ala Tyr Ile Gln Glu Leu Arg Asn 1295 1300 1305 <210> 3 <211> 1241 <212> PRT <213> Utyrivibrio proteoclasticus <400> 3 Met Leu Leu Tyr Glu Asn Tyr Thr Lys Arg Asn Gln Ile Thr Lys Ser 1 5 10 15 Leu Arg Leu Glu Leu Arg Pro Gln Gly Lys Thr Leu Arg Asn Ile Lys 20 25 30 Glu Leu Asn Leu Leu Glu Gln Asp Lys Path To Tyr Path Leu Leu Glu 35 40 45 Arg Leu Lys Pro Val Ile Asp Glu Gly Ile Lys Asp Ile Ala Arg Asp 50 55 60 Thr Leu Lys Asn Cys Glu Leu Ser Phe Glu Lys Leu Tyr Glu His Phe 65 70 75 80 Leu Ser Gly Asp Lys Lys Ala Tyr Ala Lys Glu Ser Glu Arg Leu Lys 85 90 95 Lys Glu Ile Val Lys Thr Leu Ile Lys Asn Leu Pro Glu Gly Ile Gly 100 105 110 Lys Ile Ser Glu Ile Asn Ser Ala Lys Tyr Leu Asn Gly Val Leu Tyr 115 120 125 Asp Phe Ile Asp Lys Thr His Lys Asp Ser Glu Glu Lys Gln Asn Ile 130 135 140 Leu Ser Asp Ile Leu Glu Thr Lys Gly Tyr Leu Ala Leu Phe Ser Lys 145 150 155 160 Phe Leu Thr Ser Arg Ile Thr Thr Leu Glu Gln Ser Met Pro Lys Arg 165 170 175 Val Ile Glu Asn Phe Glu Ile Tyr Ala Ala Asn Ile Pro Lys Met Gln 180 185 190 Asp Ala Leu Glu Arg Gly Ala Val Ser Phe Ala Ile Glu Tyr Glu Ser 195 200 205 Ile Cys Ser Val Asp Tyr Tyr Asn Gln Ile Leu Ser Gln Glu Asp Ile 210 215 220 Asp Ser Tyr Asn Arg Leu Ile Ser Gly Ile Met Asp Glu Asp Gly Ala 225 230 235 240 Lys Glu Lys Gly Ile Asn Gln Thr Ile Ser Glu Lys Asn Ile Lys Ile 245 250 255 Lys Ser Glu His Leu Glu Glu Lys Pro Phe Arg Ile Leu Lys Gln Leu 260 265 270 His Lys Gln Ile Leu Glu Glu Arg Glu Lys Ala Phe Thr Ile Asp His 275 280 285 Ile Asp Ser Asp Glu Glu Val Val Gln Val Thr Lys Glu Ala Phe Glu 290 295 300 Gln Thr Lys Glu Gln Trp Glu Asn Ile Lys Lys Ile Asn Gly Phe Tyr 305 310 315 320 Ala Lys Asp Pro Gly Asp Ile Thr Leu Phe Ile Val Val Gly Pro Asn 325 330 335 Gln Thr His Val Leu Ser Gln Leu Ile Tyr Gly Glu His Asp Arg Ile 340 345 350 Arg Leu Leu Leu Glu Glu Tyr Glu Lys Asn Thr Leu Glu Val Leu Pro 355 360 365 Arg Arg Thr Lys Ser Glu Asp Ala Arg Tyr Asp Lys Phe Val Asn Ala 370 375 380 Val Pro Lys Lys Val Ala Lys Glu Ser His Thr Phe Asp Gly Leu Gln 385 390 395 400 Lys Met Thr Gly Asp Asp Arg Leu Phe Ile Leu Tyr Arg Asp Glu Leu 405 410 415 Ala Arg Asn Tyr Met Arg Ile Lys Glu Ala Tyr Gly Thr Phe Glu Arg 420 425 430 Asp Ile Leu Lys Ser Arg Arg Gly Ile Lys Gly Asn Arg Asp Val Gln 435 440 445 Glu Ser Leu Val Ser Phe Tyr Asp Glu Leu Thr Lys Phe Arg Ser Ala 450 455 460 Leu Arg Ile Ile Asn Ser Gly Asn Asp Glu Lys Ala Asp Pro Ile Phe 465 470 475 480 Tyr Asn Thr Phe Asp Gly Ile Phe Glu Lys Ala Asn Arg Thr Tyr Lys 485 490 495 Ala Glu Asn Leu Cys Arg Asn Tyr Val Thr Lys Ser Pro Ala Asp Asp 500 505 510 Ala Arg Ile Met Ala Ser Cys Leu Gly Thr Pro Ala Arg Leu Arg Thr 515 520 525 His Trp Trp Asn Gly Glu Glu Asn Phe Ala Ile Asn Asp Val Ala Met 530 535 540 Ile Arg Arg Gly Asp Glu Tyr Tyr Tyr Phe Val Leu Thr Pro Asp Val 545 550 555 560 Lys Pro Val Asp Leu Lys Thr Lys Asp Glu Thr Asp Ala Gln Ile Phe 565 570 575 Val Gln Arg Lys Gly Ala Lys Ser Phe Leu Gly Leu Pro Lys Ala Leu 580 585 590 Phe Lys Cys Ile Leu Glu Pro Tyr Phe Glu Ser Pro Glu His Lys Asn 595 600 605 Asp Lys Asn Cys Val Ile Glu Glu Tyr Val Ser Lys Pro Leu Thr Ile 610 615 620 Asp Arg Arg Ala Tyr Asp Ile Phys Lys Thr 625 630 635 640 Asn Ile Gly Ile Asp Gly Leu Thr Glu Glu Lys Phe Lys Asp Asp Cys 645,650,655 Arg Tyr Leu Ile Asp Val Tyr Lys Glu Phe Ile Ala Val Tyr Thr Arg 660,665,670 Tyr Ser Cys Phe Asn Met Ser Gly Leu Lys Arg Ala Asp Glu Tyr Asn 675,680,685 Asp Ile Gly Glu Phe Phe Ser Asp Val Asp Thr Arg Leu Cys Thr Met 690,695,700 Glu Trp Ile Pro Val Ser Phe Glu Arg Ile Asn Asp Met Val Asp Lys 705 710 715 720 Lys Glu Gly Leu Leu Phe Leu Val Arg Served Met Phe Leu Tyr Asn Arg 725 730 735 Pro Arg Lys Pro Tyr Glu Arg Thr Phe Ile Gln Leu Phe Ser Asp Ser 740,745,750 Asn Met Glu His Thr Ser Met Leu Leu Asn Ser Arg Ala Met Ile Gln 755,760,765 Tyr Arg Ala Ala Ser Leu Pro Arg Arg Val Thr His Lys Lys Gly Ser 770 775 780 Ile Leu Val Ala Leu Arg Asp Ser Asn Gly Glu His Ile Pro Met His 785 790 795 800 Ile Arg Glu Ala Ile Tyr Lys Met Lys Asn Asn Phe Asp Ile Ser Ser 805 810 815 Glu Asp Phe Ile Met Ala Lys Ala Tyr Leu Ala Glu His Asp Val Ala 820 825 830 Ile Lys Lys Ala Asn Glu Asp Ile Ile Arg Asn Arg Arg Tyr Thr Glu 835 840 845 Asp Lys Phe Phe Leu Ser Leu Ser Tyr Thr Lys Asn Ala Asp Ile Ser 850 855 860 Ala Arg Thr Leu Asp Tyr Ile Asn Asp Lys Val Glu Glu Asp Thr Gln 865 870 875 880 Asp Ser Arg Met Ala Val Ile Val Thr Arg Asn Leu Lys Asp Leu Thr 885 890 895 Tyr Val Ala Val Val Asp Glu Lys Asn Asn Val Leu Glu Glu Lys Ser 900 905 910 Leu Asn Glu Ile Asp Gly Val Asn Tyr Arg Glu Leu Leu Lys Glu Arg 915 920 925 Thr Lys Ile Lys Tyr His Asp Lys Thr Arg Leu Trp Gln Tyr Asp Val 930 935 940 Ser Ser Lys Gly Leu Lys Glu Ala Tyr Val Glu Leu Ala Val Thr Gln 945 950 955 960 Ile Ser Lys Leu Ala Thr Lys Tyr Asn Ala Val Val Val Val Glu Ser 965 970 975 Met Ser Ser Thr Phe Lys Asp Lys Phe Ser Phe Leu Asp Glu Gln Ile 980 985 990 Phe Lys Ala Phe Glu Ala Arg Leu Cys Ala Arg Met Ser Asp Leu Ser 995 1000 1005 Phe Asn Thr Ile Lys Glu Gly Glu Ala Gly Ser Ile Ser Asn Pro 1010 1015 1020 Ile Gln Val Ser Asn Asn Asn Gly Asn Ser Tyr Gln Asp Gly Val 1025 1030 1035 Ile Tyr Phe Leu Asn Asn Ala Tyr Thr Arg Thr Leu Cys Pro Asp 1040 1045 1050 Thr Gly Phe Val Asp Val Phe Asp Lys Thr Arg Leu Ile Thr Met 1055 1060 1065 Gln Ser Lys Arg Gln Phe Phe Ala Lys Met Lys Asp Ile Arg Ile 1070 1075 1080 Asp Asp Gly Glu Met Leu Phe Thr Phe Asn Leu Glu Glu Tyr Pro 1085 1090 1095 Thr Lys Arg Leu Leu Asp Arg Lys Glu Trp Thr Val Lys Ile Ala 1100 1105 1110 Gly Asp Gly Ser Tyr Phe Asp Lys Asp Lys Gly Glu Tyr Val Tyr 1115 1120 1125 Val Asn Asp Ile Val Arg Glu Gln Ile Ile Pro Ala Leu Leu Glu 1130 1135 1140 Asp Lys Ala Val Phe Asp Gly Asn Met Ala Glu Lys Phe Leu Asp 1145 1150 1155 Lys Thr Ala Ile Ser Gly Lys Ser Val Glu Leu Ile Tyr Lys Trp 1160 1165 1170 Phe Ala Asn Ala Leu Tyr Gly Ile Ile Thr Lys Lys Asp Gly Glu 1175 1180 1185 Lys Ile Tyr Arg Ser Pro Ile Thr Gly Thr Glu Ile Asp Val Ser 1190 1195 1200 Lys Asn Thr Thr Tyr Asn Phe Gly Lys Lys Phe Met Phe Lys Gln 1205 1210 1215 Glu Tyr Arg Gly Asp Gly Asp Phe Leu Asp Ala Phe Leu Asn Tyr 1220 1225 1230 Met Gln Only Gln Asp To Only Val 1235 1240 <210> 4 <211> 1238 <212> PRT <213> Candidatus Methanoplasma termitum <400> 4 Met Asn Asn Tyr Asp Glu Phe Thr Lys Leu Tyr Pro Ile Gln Lys Thr 1 5 10 15 Ile Arg Phe Glu Leu Lys Pro Gln Gly Arg Thr Met Glu His Leu Glu 20 25 30 Thr Phe Asn Phe Phe Glu Glu Asp Arg Asp Arg Ala Glu Lys Tyr Lys 35 40 45 Ile Leu Lys Glu Ala Ile Asp Glu Tyr His Lys Lys Phe Ile Asp Glu 50 55 60 His Thr Asn Met Ser Leu Asp Trp Asn Ser Leu Lys Gln Ile Ser 65 70 75 80 Glu Glu Lys Tyr Lys Ser Arg Glu Glu Lys Asp Lys Val Phe Leu 85 90 95 Ser Glu Gln Lys Arg Met Arg Gln Glu Ile Val Ser Glu Phe Lys Lys 100 105 110 Asp Asp Arg Phe Lys Asp Leu Phe Ser Lys Lys Leu Phe Ser Glu Leu 115 120 125 Leu Lys Glu Glu Ile Tyr Lys Lys Gly Asn His Gln Glu Ile Asp Ala 130 135 140 Leu Lys Ser Phe Asp Lys Phe Ser Gly Tyr Phe Ile Gly Leu His Glu 145 150 155 160 Asn Arg Lys Asn Met Tyr Ser Asp Gly Asp Glu Ile Thr Ala Ile Ser 165 170 175 Asn Arg Ile Val Asn Glu Asn Phe Pro Lys Phe Leu Asp Asn Leu Gln 180 185 190 Lys Tyr Gln Glu Ala Arg Lys Lys Tyr Pro Glu Trp Ile Ile Lys Ala 195 200 205 Glu Ser Ala Leu Val Ala His Asn Ile Lys Met Asp Ile Val Phe Ser 210 215 220 Leu Glu Tyr Phe Asn Lys Val Leu Asn Gln Glu Gly Ile Gln Arg Tyr 225 230 235 240 Asn Leu Ala Leu Gly Gly Tyr Val Thr Lys Ser Gly Glu Lys Met Met 245 250 255 Gly Leu Asn Asp Ala Leu Asn Leu Ala His Gln Ser Glu Lys Ser Ser 260 265 270 Lys Gly Arg Ile His Met Thr Pro Leu Phe Lys Gln Ile Leu Ser Glu 275 280 285 Lys Glu Ser Phe Ser Tyr Ile Pro Asp Val Phe Thr Glu Asp Ser Gln 290 295 300 Leu Leu Pro Ser Ile Gly Gly Phe Phe Ala Gln Ile Glu Asn Asp Lys 305 310 315 320 Asp Gly Asn Ile Phe Asp Arg Ala Leu Glu Leu Ile Ser Ser Tyr Ala 325 330 335 Glu Tyr Asp Thr Glu Arg Ile Tyr Ile Arg Gln Ala Asp Ile Asn Arg 340 345 350 Val Ser Asn Val Ile Phe Gly Glu Trp Gly Thr Leu Gly Gly Leu Met 355 360 365 Arg Glu Tyr Lys Ala Asp Ser Ile Asn Asp Ile Asn Leu Glu Arg Thr 370 375 380 Cys Lys Lys Val Asp Lys Trp Leu Asp Ser Lys Glu Phe Ala Leu Ser 385 390 395 400 Asp Val Leu Glu Ala Ile Asp Arg Thr Gly Asn Asn Asp Ala Phe Asn 405 410 415 Glu Tyr Ile Ser Lys Met Arg Thr Ala Arg Glu Lys Ile Asp Ala Ala 420 425 430 Arg Lys Glu Met Lys Phe Ile Ser Glu Lys Ile Ser Gly Asp Glu Glu 435 440 445 I'm Not Afraid I'm Not Afraid I'm Not Afraid Asp I'm Val Gln Gln Phe Leu 450 455 460 His Phe Phe Asn Leu Phe Lys Ala Arg Gln Asp Ile Pro Leu Asp Gly 465 470 475 480 Ala Phe Tyr Ala Glu Phe Asp Glu Val His Ser Lys Leu Phe Ala Ile 485,490,495 Val Pro Leu Tyr Asn Lys Val Arg Asn Tyr Leu Thr Lys Asn Leu 500 505 510 Asn Thr Lys Lys Ile Lys Leu Asn Phe Lys Asn Pro Thr Leu Ala Asn 515,520,525 Gly Trp Asp Gln Asn Lys Val Tyr Asp Tyr Ala Ser Leu Ile Phe Leu 530 535 540 Arg Asp Gly Asn Tyr Tyr Leu Gly Ile Ile Asn Pro Lys Arg Lys Lys 545 550 555 560 Asn Ile Lys Phe Glu Gln Gly Ser Gly Asn Gly Pro Phe Tyr Arg Lys 565,570,575 Met Val Tyr Lys Gln Ile Pro Gly Pro Asn Lys Asn Leu Arg Pro Val 580,585,590 Phe Leu Thr Ser Thr Lys Gly Lys Glu Tyr Lys Pro Ser Lys Glu 595 600 605 Ile Ile Glu Gly Tyr Glu Ala Asp Lys His Ile Arg Gly Asp Lys Phe 610 615 620 Asp Leu Asp Phe Cys His Lys Leu Ile Asp Phe Phe Lys Glu Ser Ile 625 630 635 640 Glu Lys His Lys Asp Trp Ser Lys Phe Asn Phe Tyr Phe Ser Pro Thr 645 650 655 Glu Ser Tyr Gly Asp Ile Ser Glu Phe Tyr Leu Asp Val Glu Lys Gln 660 665 670 Gly Tyr Arg Met His Phe Glu Asn Ile Ser Ala Glu Thr Ile Asp Glu 675 680 685 Tyr Val Glu Lys Gly Asp Leu Phe Leu Phe Gln Ile Tyr Asn Lys Asp 690 695 700 Phe Val Lys Ala Ala Thr Gly Lys Lys Asp Met His Thr Ile Tyr Trp 705 710 715 720 Asn Ala Ala Phe Ser Pro Glu Asn Leu Gln Asp Val Val Val Lys Leu 725 730 735 Asn Gly Glu Ala Glu Leu Phe Tyr Arg Asp Lys Ser Asp Ile Lys Glu 740 745 750 Ile Val His Arg Glu Gly Glu Ile Leu Val Asn Arg Thr Tyr Asn Gly 755 760 765 Arg Thr Pro Val Pro Asp Lys Ile His Lys Lys Leu Thr Asp Tyr His 770 775 780 Asn Gly Arg Thr Lys Asp Leu Gly Glu Ala Lys Glu Tyr Leu Asp Lys 785 790 795 800 Val Arg Tyr Phe Lys Ala His Tyr Asp Ile Thr Lys Asp Arg Arg Tyr 805 810 815 Leu Asn Asp Lys Ile Tyr Phe His Val Pro Leu Thr Leu Asn Phe Lys 820 825 830 Ala Asn Gly Lys Lys Asn Leu Asn Lys Met Val Ile Glu Lys Phe Leu 835 840 845 Ser Asp Glu Lys Ala His Ile Ile Gly Ile Asp Arg Gly Glu Arg Asn 850 855 860 Leu Leu Tyr Tyr Ser Ile Ile Asp Arg Ser Gly Lys Ile Ile Asp Gln 865 870 875 880 Gln Ser Leu Asn Val Ile Asp Gly Phe Asp Tyr Arg Glu Lys Leu Asn 885 890 895 Gln Arg Glu Ile Glu Met Lys Asp Ala Arg Gln Ser Trp Asn Ala Ile 900 905 910 Gly Lys Ile Lys Asp Leu Lys Glu Gly Tyr Leu Ser Lys Ala Val His 915 920 925 Glu Ile Thr Lys Met Ala Ile Gln Tyr Asn Ala Ile Val Val Met Glu 930 935 940 Glu Leu Asn Tyr Gly Phe Lys Arg Gly Arg Phe Lys Val Glu Lys Gln 945 950 955 960 Ile Tyr Gln Lys Phe Glu Asn Met Leu Ile Asp Lys Met Asn Tyr Leu 965 970 975 Val Phe Lys Asp Ala Pro Asp Glu Ser Pro Gly Gly Val Leu Asn Ala 980 985 990 Tyr Gln Leu Thr Asn Pro Leu Glu Ser Phe Ala Lys Leu Gly Lys Gln 995 1000 1005 Thr Gly Ile Leu Phe Tyr Val Pro Ala Ala Tyr Thr Ser Lys Ile 1010 1015 1020 Asp Pro Thr Thr Gly Phe Val Asn Leu Phe Asn Thr Ser Ser Lys 1025 1030 1035 Thr Asn Ala Gln Glu Arg Lys Glu Phe Leu Gln Lys Phe Glu Ser 1040 1045 1050 Ile Ser Tyr Ser Ala Lys Asp Gly Gly Ile Phe Ala Phe Ala Phe 1055 1060 1065 Asp Tyr Arg Lys Phe Gly Thr Ser Lys Thr Asp His Lys Asn Val 1070 1075 1080 Trp Thr Ala Tyr Thr Asn Gly Glu Arg Met Arg Tyr Ile Lys Glu 1085 1090 1095 Lys Lys Arg Asn Glu Leu Phe Asp Pro Ser Lys Glu Ile Lys Glu 1100 1105 1110 Ala Leu Thr Ser Ser Gly Ile Lys Tyr Asp Gly Gly Gln Asn Ile 1115 1120 1125 Leu Pro Asp Ile Leu Arg Ser Asn Asn Asn Gly Leu Ile Tyr Thr 1130 1135 1140 Met Tyr Ser Ser Phe Ile Ala Ala Ile Gln Met Arg Val Tyr Asp 1145 1150 1155 Gly Lys Glu Asp Tyr Ile Ile Ser Pro Ile Lys Asn Ser Lys Gly 1160 1165 1170 Glu Phe Phe Arg Thr Asp Pro Lys Arg Arg Glu Leu Pro Ile Asp 1175 1180 1185 Ala Asp Ala Asn Gly Ala Tyr Asn Ile Ala Leu Arg Gly Glu Leu 1190 1195 1200 Thr Met Arg Ala Ile Ala Glu Lys Phe Asp Pro Asp Ser Glu Lys 1205 1210 1215 Met Ala Lys Leu Glu Leu Lys His Lys Asp Trp Phe Glu Phe Met 1220 1225 1230 Gln Thr Arg Gly Asp 1235 <210> 5 <211> 1281 <212> PRT <213> Eubacterium eligens <400> 5 Met Asn Gly Asn Arg Ser Ile Val Tyr Arg Glu Phe Val Gly Val Ile 1 5 10 15 Pro Val Ala Lys Thr Leu Arg Asn Glu Leu Arg Pro Val Gly His Thr 20 25 30 Gln Glu His Ile Ile Gln Asn Gly Leu Ile Gln Glu Asp Glu Leu Arg 35 40 45 Gln Glu Lys Ser Thr Glu Leu Lys Asn Ile Met Asp Asp Tyr Tyr Arg 50 55 60 Glu Tyr Ile Asp Lys Ser Leu Ser Gly Val Thr Asp Leu Asp Phe Thr 65 70 75 80 Leu Leu Phe Glu Leu Met Asn Leu Val Gln Ser Ser Pro Ser Lys Asp 85 90 95 Asn Lys Lys Ala Leu Glu Lys Glu Gln Ser Lys Met Arg Glu Gln Ile 100 105 110 Cys Thr His Leu Gln Ser Asp Ser Asn Tyr Lys Asn Ile Phe Asn Ala 115 120 125 Lys Leu Leu Lys Glu Ile Leu Pro Asp Phe Ile Lys Asn Tyr Asn Gln 130 135 140 Tyr Asp Val Lys Asp Lys Ala Gly Lys Leu Glu Thr Leu Ala Leu Phe 145 150 155 160 Asn Gly Phe Ser Thr Tyr Phe Thr Asp Phe Phe Glu Lys Arg Lys Asn 165 170 175 Val Phe Thr Lys Glu Ala Val Ser Thr Ser Ile Ala Tyr Arg Ile Val 180 185 190 His Glu Asn Ser Leu Ile Phe Leu Ala Asn Met Thr Ser Tyr Lys Lys 195 200 205 Ile Ser Glu Lys Ala Leu Asp Glu Ile Glu Val Ile Glu Lys Asn Asn 210 215 220 Gln Asp Lys Met Gly Asp Trp Glu Leu Asn Gln Ile Phe Asn Pro Asp 225 230 235 240 Phe Tyr Asn Met Val Leu Ile Gln Ser Gly Ile Asp Phe Tyr Asn Glu 245 250 255 Ile Cys Gly Val Val Asn Ala His Met Asn Leu Tyr Cys Gln Gln Thr 260 265 270 Lys Asn Asn Tyr Asn Leu Phe Lys Met Arg Lys Leu His Lys Gln Ile 275 280 285 Leu Ala Tyr Thr Ser Thr Ser Phe Glu Val Pro Lys Met Phe Glu Asp 290 295 300 Asp Met Ser Val Tyr Asn Ala Val Asn Ala Phe Ile Asp Glu Thr Glu 305 310 315 320 Lys Gly Asn Ile Ile Gly Lys Leu Lys Asp Ile Val Asn Lys Tyr Asp 325 330 335 Glu Leu Asp Glu Lys Arg Ile Tyr Ile Ser Lys Asp Phe Tyr Glu Thr 340 345 350 Leu Ser Cys Phe Met Ser Gly Asn Trp Asn Leu Ile Thr Gly Cys Val 355 360 365 Glu Asn Phe Tyr Asp Glu Asn Ile His Ala Lys Gly Lys Ser Lys Glu 370 375 380 Glu Lys Val Lys Lys Ala Val Lys Glu Asp Lys Tyr Lys Ser Ile Asn 385 390 395 400 Asp Val Asn Asp Leu Val Glu Lys Tyr Ile Asp Glu Lys Glu Arg Asn 405 410 415 Glu Phe Lys Asn Ser Asn Ala Lys Gln Tyr Ile Arg Glu Ile Ser Asn 420 425 430 Ile Ile Thr Asp Thr Glu Thr Ala His Leu Glu Tyr Asp Asp His Ile 435 440 445 Ser Leu Ile Glu Ser Glu Glu Lys Ala Asp Glu Met Lys Lys Arg Leu 450 455 460 Asp Met Tyr Met Asn Met Tyr His Trp Ala Lys Ala Phe Ile Val Asp 465 470 475 480 Glu Val Leu Asp Arg Asp Glu Met Phe Tyr Ser Asp Ile Asp Asp Ile 485 490 495 Tyr Asn Ile Leu Glu Asn Ile Val Pro Leu Tyr Asn Arg Val Arg Asn 500 505 510 Tyr Val Thr Gln Lys Pro Tyr Asn Ser Lys Lys Ile Lys Leu Asn Phe 515 520 525 Gln Ser Pro Thr Leu Ala Asn Gly Trp Ser Gln Ser Lys Glu Phe Asp 530 535 540 Asn Asn Ala Ile Ile Leu Ile Arg Asp Asn Lys Tyr Tyr Leu Ala Ile 545 550 555 560 Phe Asn Ala Lys Asn Lys Pro Asp Lys Lys Ile Ile Gln Gly Asn Ser 565 570 575 Asp Lys Lys Asn Asp Asn Asp Tyr Lys Lys Met Val Tyr Asn Leu Leu 580,585,590 Pro Gly Ala Asn Lys Met Leu Pro Lys Val Phe Leu Ser Lys Lys Gly 595,600,605 Glu Thr Phe Lys Pro Ser Asp Tyr I Ile Gly Tyr Asn Ala 610 615 620 His Lys His Ile Lys Thr Ser Glu Asn Phe Asp Ile Ser Phe Cys Arg 625 630 635 640 Asp Leu With Asp Tyr Phe Lys Asn Ser Ile Glu Lys His Ala Glu Trp 645,650,655 Arg Lys Tyr Glu Phe Lys Phe Ser Ala Thr Asp Ser Tyr Ser Asp Ile 660,665,670 Ser Glu Phe Tyr Arg Glu Val Glu Met Gln Gly Tyr Arg Ile Asp Trp 675,680,685 Thr Tyr Ile Ser Glu Ala Asp Ile Asn Lys Leu Asp Glu Glu Gly Lys 690,695,700 Gln and Tyre Asn Lys Asp With Glu Asn Ser Thr 705 710 715 720 Gly Lys Glu Asn Leu His Thr Met Tyr Phe Lys Asn Ile Phe Ser Glu 725 730 735 Glu Asn Leu Asp Lys Ile Ile Lys Leu Asn Gly Gln Ala Glu Leu Phe 740 745 750 Tyr Arg Arg Ala Ser Val Lys Asn Pro Val Lys His Lys Lys Asp Ser 755 760 765 Val Leu Val Asn Lys Thr Tyr Lys Asn Gln Leu Asp Asn Gly Asp Val 770 775 780 Val Arg Ile Pro Ile Pro Asp Asp Ile Tyr Asn Glu Ile Tyr Lys Met 785 790 795 800 Tyr Asn Gly Tyr Ile Lys Glu Ser Asp Leu Ser Glu Ala Ala Lys Glu 805 810 815 Tyr Leu Asp Lys Val Glu Val Arg Thr Ala Gln Lys Asp Ile Val Lys 820 825 830 Asp Tyr Arg Tyr Thr Val Asp Lys Tyr Phe Ile His Thr Pro Ile Thr 835 840 845 Ile Asn Tyr Lys Val Thr Ala Arg Asn Asn Val Asn Asp Met Val Val 850 855 860 Lys Tyr Ile Ala Gln Asn Asp Asp Ile His Val Ile Gly Ile Asp Arg 865 870 875 880 Gly Glu Arg Asn Leu Ile Tyr Ile Ser Val Ile Asp Ser His Gly Asn 885 890 895 Ile Val Lys Gln Lys Ser Tyr Donkey Ile Leu Donkey Donkey Tyr Asp Tyr Lys 900 905 910 Lys Lys Leu Val Glu Lys Glu Lys Thr Arg Glu Tyr Ala Arg Lys Asn 915 920 925 Trp Lys Ser Ile Gly Asn Ile Lys Glu Leu Lys Glu Gly Tyr Ile Ser 930 935 940 Gly Val Val His Glu Ile Ala Met Leu Ile Val Glu Tyr Asn Ala Ile 945 950 955 960 Ile Ala Met Glu Asp Leu Asn Tyr Gly Phe Lys Arg Gly Arg Phe Lys 965 970 975 Val Glu Arg Gln Val Tyr Gln Lys Phe Glu Ser Met Leu Ile Asn Lys 980 985 990 Leu Asn Tyr Phe Ala Ser Lys Glu Lys Ser Val Asp Glu Pro Gly Gly 995 1000 1005 Leu Leu Lys Gly Tyr Gln Leu Thr Tyr Val Pro Asp Asn Ile Lys 1010 1015 1020 Asn Leu Gly Lys Gln Cys Gly Val Ile Phe Tyr Val Pro Ala Ala 1025 1030 1035 Phe Thr Ser Lys Ile Asp Pro Ser Thr Gly Phe Ile Ser Ala Phe 1040 1045 1050 Asn Phe Lys Ser Ile Ser Thr Asn Ala Ser Arg Lys Gln Phe Phe 1055 1060 1065 Met Gln Phe Asp Glu Ile Arg Tyr Cys Ala Glu Lys Asp Met Phe 1070 1075 1080 Ser Phe Gly Phe Asp Tyr Asn Asn Phe Asp Thr Tyr Asn Ile Thr 1085 1090 1095 Met Gly Lys Thr Gln Trp Thr Val Tyr Thr Asn Gly Glu Arg Leu 1100 1105 1110 Gln Ser Glu Phe Asn Asn Ala Arg Arg Thr Gly Lys Thr Lys Ser 1115 1120 1125 Ile Asn Leu Thr Glu Thr Ile Lys Leu Leu Leu Glu Asp Asn Glu 1130 1135 1140 Ile Asn Tyr Ala Asp Gly His Asp Ile Arg Ile Asp Met Glu Lys 1145 1150 1155 Met Asp Glu Asp Lys Lys Ser Glu Phe Phe Ala Gln Leu Leu Ser 1160 1165 1170 Leu Tyr Lys Leu Thr Val Gln Met Arg Asn Ser Tyr Thr Glu Ala 1175 1180 1185 Glu Glu Gln Glu Asn Gly Ile Ser Tyr Asp Lys Ile Ile Ser Pro 1190 1195 1200 Val Ile Asn Asp Glu Gly Glu Phe Phe Asp Ser Asp Asn Tyr Lys 1205 1210 1215 Glu Ser Asp Asp Lys Glu Cys Lys Met Pro Lys Asp Ala Asp Ala 1220 1225 1230 Asn Gly Ala Tyr Cys Ile Ala Leu Lys Gly Leu Tyr Glu Val Leu 1235 1240 1245 Lys Ile Lys Ser Glu Trp Thr Glu Asp Gly Phe Asp Arg Asn Cys 1250 1255 1260 Leu Lys Leu Pro His Ala Glu Trp Leu Asp Phe Ile Gln Asn Lys 1265 1270 1275 Arg Tyr Glu 1280 <210> 6 <211> 1300 <212> PRT <213> Francisella novicida <400> 6 Met Ser Ile Tyr Gln Glu Phe Val Asn Lys Tyr Ser Leu Ser Lys Thr 1 5 10 15 Leu Arg Phe Glu Leu Ile Pro Gln Gly Lys Thr Leu Glu Asn Ile Lys 20 25 30 Ala Arg Gly Leu Ile Leu Asp Asp Glu Lys Arg Ala Lys Asp Tyr Lys 35 40 45 Lys Ala Lys Gln Ile Ile Asp Lys Tyr His Gln Phe Phe Ile Glu Glu 50 55 60 Ile Leu Ser Ser Val Cys Ile Ser Glu Asp Leu Leu Gln Asn Tyr Ser 65 70 75 80 Asp Val Tyr Phe Lys Leu Lys Lys Ser Asp Asp Asp Asn Leu Gln Lys 85 90 95 Asp Phe Lys Ser Ala Lys Asp Thr Ile Lys Lys Gln Ile Ser Glu Tyr 100 105 110 Ile Lys Asp Ser Glu Lys Phe Lys Asn Leu Phe Asn Gln Asn Leu Ile 115 120 125 Asp Ala Lys Lys Gly Gln Glu Ser Asp Leu Ile Leu Trp Leu Lys Gln 130 135 140 Ser Lys Asp Asn Gly Ile Glu Leu Phe Lys Ala Asn Ser Asp Ile Thr 145 150 155 160 Asp Ile Asp Glu Ala Leu Glu Ile Ile Lys Ser Phe Lys Gly Trp Thr 165 170 175 Thr Tyr Phe Lys Gly Phe His Glu Asn Arg Lys Val Asn Tyr Ser Ser 180 185 190 Asn Asp Ile Pro Thr Ser Ile Ile Tyr Arg Ile Val Asp Asp Asn Leu 195 200 205 Pro Lys Phe Leu Glu Asn Lys Ala Lys Tyr Glu Ser Leu Lys Asp Lys 210 215 220 Ala Pro Glu Ala Ile Asn Tyr Glu Gln Ile Lys Lys Asp Leu Ala Glu 225 230 235 240 Glu Leu Thr Phe Asp Ile Asp Tyr Lys Thr Ser Glu Val Asn Gln Arg 245 250 255 Val Phe Ser Leu Asp Glu Val Phe Glu Ile Ala Asn Phe Asn Asn Tyr 260 265 270 Leu Asn Gln Ser Gly Ile Thr Lys Phe Asn Thr Ile Ile Gly Gly Lys 275 280 285 Phe Val Asn Gly Glu Asn Thr Lys Arg Lys Gly Ile Asn Glu Tyr Ile 290 295 300 Asn Leu Tyr Ser Gln Gln Ile Asn Asp Lys Thr Leu Lys Lys Tyr Lys 305 310 315 320 Met Ser Val Leu Phe Lys Gln Ile Leu Ser Asp Thr Glu Ser Lys Ser 325 330 335 Phe Val Ile Asp Lys Leu Glu Asp Asp Ser Asp Val Val Thr Thr Met 340 345 350 Gln Ser Phe Tyr Glu Gln Ile Ala Ala Phe Lys Thr Val Glu Glu Lys 355 360 365 Ser Ile Lys Glu Thr Leu Ser Leu Leu Phe Asp Asp Leu Lys Ala Gln 370 375 380 Lys Leu Asp Leu Ser Lys Ile Tyr Phe Lys Asn Asp Lys Ser Leu Thr 385 390 395 400 Asp Leu Ser Gln Gln Val Phe Asp Asp Tyr Ser Val Ile Gly Thr Ala 405 410 415 Val Leu Glu Tyr Ile Thr Gln Gln Ile Ala Pro Lys Asn Leu Asp Asn 420 425 430 Pro Ser Lys Lys Glu Gln Glu Leu Ile Ala Lys Lys Thr Glu Lys Ala 435 440 445 Lys Tyr Leu Ser Leu Glu Thr Ile Lys Leu Ala Leu Glu Glu Phe Asn 450 455 460 Lys His Arg Asp Ile Asp Lys Gln Cys Arg Phe Glu Glu Ile Leu Ala 465 470 475 480 Asn Phe Ala Ala Ile Pro Met Ile Phe Asp Glu Ile Ala Gln Asn Lys 485 490 495 Asp Asn Leu Ala Gln Ile Ser Ile Lys Tyr Gln Asn Gln Gly Lys Lys 500 505 510 Asp Leu Leu Gln Ala Ser Ala Glu Asp Asp Val Lys Ala Ile Lys Asp 515,520,525 Leu Leu Asp Gln Thr Asn Asn Leu Leu His Lys Leu Lys Ile Phe His 530 535 540 Ser Gln Ser Glu Asp Lys Ala Asn Ileu Asp Lys Asp Glu His 545 550 555 560 Phe Tyr Leu Val Phe Glu Glu Cys Tyr Phe Glu Leu Ala Asn Ile Val 565,570,575 Pro Leu Tyr Asn With Arg Asn Tyr With Thr Gln Lys Pro Tyr Ser 580,585,590 Asp Glu Lys Phe Lys Leu Asn Phe Glu Asn Ser Thr Leu Ala Asn Gly 595,600,605 Trp Asp Lys Asn Lys Glu Pro Asp Asn Thr Ala Ile Leu Phe Ile Lys 610 615 620 Asp Asp Lys Tyr Leu Gly Val Met Lys Lys Asn Asn Lys Ile 625 630 635 640 Phe Asp Asp Lys Ala Ile Lys Glu Asn Gly Glu Gly Tyr Lys Lys 645,650,655 Ile Val Tyr Lys Leu Leu Pro Gly Ala Asn Lys Met Leu Pro Lys Val 660,665,670 Phe Phe Ser Ala Lys Ser Ile Lys Phe Tyr Asn Pro Ser Glu Asp Ile 675 680 685 Leu Arg Ile Arg Asn His Ser Thr His Thr Lys Asn Gly Ser Pro Gln 690 695 700 Lys Gly Tyr Glu Lys Phe Glu Phe Asn Ile Glu Asp Cys Arg Lys Phe 705 710 715 720 Ile Asp Phe Tyr Lys Gln Ser Ile Ser Lys His Pro Glu Trp Lys Asp 725 730 735 Phe Gly Phe Arg Phe Ser Asp Thr Gln Arg Tyr Asn Ser Ile Asp Glu 740 745 750 Phe Tyr Arg Glu Val Glu Asn Gln Gly Tyr Lys Leu Thr Phe Glu Asn 755 760 765 Ile Ser Glu Ser Tyr Ile Asp Ser Val Val Asn Gln Gly Lys Leu Tyr 770 775 780 Leu Phe Gln Ile Tyr Asn Lys Asp Phe Ser Ala Tyr Ser Lys Gly Arg 785 790 795 800 Pro Asn Leu His Thr Leu Tyr Trp Lys Ala Leu Phe Asp Glu Arg Asn 805 810 815 Leu Gln Asp Val Val Tyr Lys Leu Asn Gly Glu Ala Glu Leu Phe Tyr 820 825 830 Arg Lys Gln Ser Ile Pro Lys Lys Ile Thr His Pro Ala Lys Glu Ala 835 840 845 Ile Ala Asn Lys Asn Lys Asp Asn Pro Lys Lys Glu Ser Val Phe Glu 850 855 860 Tyr Asp Leu Ile Lys Asp Lys Arg Phe Thr Glu Asp Lys Phe Phe Phe 865 870 875 880 His Cys Pro Ile Thr Ile Asn Phe Lys Ser Ser Gly Ala Asn Lys Phe 885 890 895 Asn Asp Glu Ile Asn Leu Leu Leu Lys Glu Lys Ala Asn Asp Val His 900 905 910 Ile Leu Ser Ile Asp Arg Gly Glu Arg His Leu Ala Tyr Tyr Thr Leu 915 920 925 Val Asp Gly Lys Gly Asn Ile Ile Lys Gln Asp Thr Phe Asn Ile Ile 930 935 940 Gly Asn Asp Arg Met Lys Thr Asn Tyr His Asp Lys Leu Ala Ala Ile 945 950 955 960 Glu Lys Asp Arg Asp Ser Ala Arg Lys Asp Trp Lys Lys Ile Asn Asn 965 970 975 Ile Lys Glu Met Lys Glu Gly Tyr Leu Ser Gln Val Val His Glu Ile 980 985 990 Only Lys Leu Will Have Glu Tyr Asn Only Will Have Phe Glu Asp Leu 995 1000 1005 Asn Phe Gly Phe Lys Arg Gly Arg Phe Lys Val Glu Lys Gln Val 1010 1015 1020 Tyr Gln Lys Leu Glue Met Leu And Leu Glue Asn Tyr Leu 1025 1030 1035 Val Phe Lys Asp Asn Glu Phe Asp Lys Thr Gly Gly Val Leu Arg 1040 1045 1050 Ala Tyr Gln Leu Thr Ala Pro Phe Glu Thr Phe Lys Lys Met Gly 1055 1060 1065 Lys Gln Thr Gly Ile Tyr Tyr Val Pro Ala Gly Phe Thr Ser 1070 1075 1080 Lys Ile Cys Pro Val Thr Gly Phe Val Asn Gln Leu Tyr Pro Lys 1085 1090 1095 Tyr Glu Ser Val Ser Lys Ser Gln Glu Phe Phe Ser Lys Phe Asp 1100 1105 1110 Lys Ile Cys Tyr Asn Leu Asp Lys Gly Tyr Phe Glu Phe Ser Phe 1115 1120 1125 Asp Tyr Lys Asn Phe Gly Asp Lys Ala Ala Lys Gly Lys Trp Thr 1130 1135 1140 Ile Ala Ser Phe Gly Ser Arg Leu Ile Asn Phe Arg Asn Ser Asp 1145 1150 1155 Lys Asn His Asn Trp Asp Thr Arg Glu Val Tyr Pro Thr Lys Glu 1160 1165 1170 Leu Glu Lys Leu Leu Lys Asp Tyr Ser Ile Glu Tyr Gly His Gly 1175 1180 1185 Glu Cys Ile Lys Ala Ala Ile Cys Gly Glu Ser Asp Lys Lys Phe 1190 1195 1200 Phe Ala Lys Leu Thr Ser Val Leu Asn Thr Ile Leu Gln Met Arg 1205 1210 1215 Asn Ser Lys Thr Gly Thr Glu Leu Asp Tyr Leu Ile Ser Pro Val 1220 1225 1230 Ala Asp Val Asn Gly Asn Phe Phe Asp Ser Arg Gln Ala Pro Lys 1235 1240 1245 Asn Met Pro Gln Asp Ala Asp Ala Asn Gly Ala Tyr His Ile Gly 1250 1255 1260 Leu Lys Gly Leu Met Leu Leu Gly Arg Ile Lys Asn Asn Gln Glu 1265 1270 1275 Gly Lys Lys Leu Asn Leu Val Ile Lys Asn Glu Glu Tyr Phe Glu 1280 1285 1290 Phe Val Gln Asn Arg Asn Asn 1295 1300 <210> 7 <211> 1206 <212> PRT <213> Unknown <220> <223> Lachnospiraceae bacterium <400> 7 Met Tyr Tyr Glu Ser Leu Thr Lys Gln Tyr Pro Val Ser Lys Thr Ile 1 5 10 15 Arg Asn Glu Leu Ile Pro Ile Gly Lys Thr Leu Asp Asn Ile Arg Gln 20 25 30 Asn Asn Ile Leu Glu Ser Asp Val Lys Arg Lys Gln Asn Tyr Glu His 35 40 45 Val Lys Gly Ile Leu Asp Glu Tyr His Lys Gln Leu Ile Asn Glu Ala 50 55 60 Leu Asp Asn Cys Thr Leu Pro Ser Leu Lys Ile Ala Ala Glu Ile Tyr 65 70 75 80 Leu Lys Asn Gln Lys Glu Val Ser Asp Arg Glu Asp Phe Asn Lys Thr 85 90 95 Gln Asp Leu Leu Arg Lys Glu Val Val Glu Lys Leu Lys Ala His Glu 100 105 110 Asn Phe Thr Lys Ile Gly Lys Lys Asp Ile Leu Asp Leu Leu Glu Lys 115 120 125 Leu Pro Ser Ile Ser Glu Asp Asp Tyr Asn Ala Leu Glu Ser Phe Arg 130 135 140 Asn Phe Tyr Thr Tyr Phe Thr Ser Tyr Asn Lys Val Arg Glu Asn Leu 145 150 155 160 Tyr Ser Asp Lys Glu Lys Ser Ser Thr Val Ala Tyr Arg Leu Ile Asn 165 170 175 Glu Asn Phe Pro Lys Phe Leu Asp Asn Val Lys Ser Tyr Arg Phe Val 180 185 190 Lys Thr Ala Gly Ile Leu Ala Asp Gly Leu Gly Glu Glu Glu Gln Asp 195 200 205 Ser Leu Phe Ile Val Glu Thr Phe Asn Lys Thr Leu Thr Gln Asp Gly 210 215 220 Ile Asp Thr Tyr Asn Ser Gln Val Gly Lys Ile Asn Ser Ser Ile Asn 225 230 235 240 Leu Tyr Asn Gln Lys Asn Gln Lys Ala Asn Gly Phe Arg Lys Ile Pro 245 250 255 Lys Met Lys Met Leu Tyr Lys Gln Ile Leu Ser Asp Arg Glu Glu Ser 260 265 270 Phe Ile Asp Glu Phe Gln Ser Asp Glu Val Leu Ile Asp Asn Val Glu 275 280 285 Ser Tyr Gly Ser Val Leu Ile Glu Ser Leu Ser Ser Lys Val Ser 290,295,300 Ala Phe Phe Asp Ala Leu Arg Glu Ser Lys Gly Lys Asn Val Tyr Val 305 310 315 320 Lys Asn Asp Leu Ala Lys Thr Ala Met Ser Val Ile Val Phe Glu Asn 325 330 335 Trp Arg Thr Phe Asp Asp Leu Leu Asn Gln Glu Tyr Asp Leu Ala Asn 340 345 350 Glu Asn Lys Lys Asp Asp Lys Tyr Phe Glu Lys Arg Gln Lys Glu 355 360 365 Leu Lys Lys Asn Lys Ser Tyr Ser Leu Glu His Leu Cys Asn Leu Ser 370 375 380 Glue Asp Ser to Cys Asn Leu To Glue Asn Tyr To His Gln To Ser Asp 385 390 395 400 Asp With Asn Glue With Asn With Asn Glue Thr Phe Leu Arg With Val 405 410 415 Ile Asn Glu His Asp Arg Ser Arg Lys Leu Ala Lys Asn Arg Lys Ala 420 425 430 Val Lys Ala Ile Lys Asp Phe Leu Asp Ser Ile Lys Val Leu Glu Arg 435 440 445 Glu Leu Lys Leu Ile Asn Ser Ser Gly Gln Glu Leu Glu Lys Asp Leu 450 455 460 Ile Val Tyr Ser Ala His Glu Glu Leu Leu Val Glu Leu Lys Gln Val 465 470 475 480 Asp Ser Leu Tyr Asn Met Thr Arg Asn Tyr Leu Thr Lys Lys Pro Phe 485 490 495 Ser Thr Glu Lys Val Lys Leu Asn Phe Asn Arg Ser Thr Leu Leu Asn 500 505 510 Gly Trp Asp Arg Asn Lys Glu Thr Asp Asn Leu Gly Val Leu Leu Leu 515 520 525 Lys Asp Gly Lys Tyr Tyr Leu Gly Ile Met Asn Thr Ser Ala Asn Lys 530 535 540 Ala Phe Val Asn Pro Pro Val Ala Lys Thr Glu Lys Val Phe Lys Lys 545 550 555 560 Val Asp Tyr Lys Leu Leu Pro Val Pro Asn Gln Met Leu Pro Lys Val 565 570 575 Phe Phe Ala Lys Ser Asn Ile Asp Phe Tyr Asn Pro Ser Ser Glu Ile 580 585 590 Tyr Ser Asn Tyr Lys Lys Gly Thr His Lys Lys Gly Asn Met Phe Ser 595 600 605 Leu Glu Asp Cys His Asn Leu Ile Asp Phe Phe Lys Glu Ser Ile Ser 610 615 620 Lys His Glu Asp Trp Ser Lys Phe Gly Phe Lys Phe Asp Thr Gln Ala 625 630 635 640 Ser Tyr Asn Asp Ile Ser Glu Phe Tyr Arg Glu Val Glu Lys Gln Gly 645 650 655 Tyr Lys Leu Thr Tyr Thr Asp Ile Asp Glu Thr Tyr Ile Asn Asp Leu 660 665 670 Ile Glu Arg Asn Glu Leu Tyr Leu Phe Gln Ile Tyr Asn Lys Asp Phe 675 680 685 Ser Met Tyr Ser Lys Gly Lys Leu Asn Leu His Thr Leu Tyr Phe Met 690 695 700 Met Leu Phe Asp Gln Arg Asn Ile Asp Asp Val Val Tyr Lys Leu Asn 705 710 715 720 Gly Glu Ala Glu Val Phe Tyr Arg Pro Ala Ser Ile Ser Glu Asp Glu 725 730 735 Leu Ile Ile His Lys Ala Gly Glu Glu Ile Lys Asn Lys Asn Pro Asn 740 745 750 Arg Ala Arg Thr Lys Glu Thr Ser Thr Phe Ser Tyr Asp Ile Val Lys 755 760 765 Asp Lys Arg Tyr Ser Lys Asp Lys Phe Thr Leu His Ile Pro Ile Thr 770 775 780 Met Asn Phe Gly Val Asp Glu Val Lys Arg Phe Asn Asp Ala Val Asn 785 790 795 800 Ser Ala Ile Arg Ile Asp Glu Asn Val Asn Val Ile Gly Ile Asp Arg 805 810 815 Gly Glu Arg Asn Leu Leu Tyr Val Val Val Ile Asp Ser Lys Gly Asn 820 825 830 Ile Leu Glu Gln Ile Ser Leu Asn Ser Ile Ile Asn Lys Glu Tyr Asp 835 840 845 Ile Glu Thr Asp Tyr His Ala Leu Leu Asp Glu Arg Glu Gly Gly Arg 850 855 860 Asp Lys Ala Arg Lys Asp Trp Asn Thr Val Glu Asn Ile Arg Asp Leu 865 870 875 880 Lys Ala Gly Leu Tyr Leu Gln Val Val Asn Val Val Ala Lys Leu Val 885 890 895 Leu Lys Tyr Asn Ala Ile Ile Cys Leu Glu Asp Leu Asn Phe Gly Phe 900 905 910 Lys Arg Gly Arg Gln Lys Val Glu Lys Gln Val Tyr Gln Lys Phe Glu 915,920,925 Lys Met Lion With Asp Lys Lion Asn Tyr Lion Val With Asp Lys Ser Arg 930,935,940 Glu Gln Thr Pro Lys Ser Glu Leu Gly Gly Ala Leu Asn Ala Leu Gln 945 950 955 960 Leu Thr Ser Lys Phe Lys Ser Phe Lys Glu Leu Gly Lys Gln Ser Gly 965,970,975 Val Ile Tyr Val Pro Ala Tire Leu Thr Ser Lys Ile Asp Pro Thr 980,985,990 Thr Gly Phe Ala Asn Leu Phe Tyr Met Lys Cys Glu Asn Val Glu Lys 995 1000 1005 Ser Lys Arg Phe Phe Asp Gly Phe Asp Phe Ile Arg Phe Asn Ala 1010 1015 1020 Leu Glu Asn Val Phe Glu Phe Gly Phe Asp Tyr Arg Ser Phe Thr 1025 1030 1035 Gln Arg Ala Cys Gly Ile Asn Served Trp Thr Val Cys Thr Asn 1040 1045 1050 Gly Glu Arg With Tyr Arg Asn Pro Asp Lys Asn Asn Met 1055 1060 1065 Phe Asp Glu Lys Val Val Val Val Thr Asp Glu Met Lys Asn Leu 1070 1075 1080 Phe Glu Gln Tyr Lys Ile Pro Tyr Glu Asp Gly Arg Asn Val Lys 1085 1090 1095 Asp Met Ile Ile Ser Asn Glu Glu Ala Glu Phe Tyr Arg Arg Leu 1100 1105 1110 Tyr Arg Leu Leu Gln Gln Thr Leu Gln Met Arg Asn Ser Thr Ser 1115 1120 1125 Asp Gly Thr Arg Asp Tyr Ile Ile Ser Pro Val Lys Asn Lys Arg 1130 1135 1140 Glu Ala Tyr Phe Asn Ser Glu Leu Ser Asp Gly Ser Val Pro Lys 1145 1150 1155 Asp Ala Asp Ala Asn Gly Ala Tyr Asn Ile Ala Arg Lys Gly Leu 1160 1165 1170 Trp Val Leu Glu Gln Ile Arg Gln Lys Ser Glu Gly Glu Lys Ile 1175 1180 1185 Asn Leu Ala Met Thr Asn Ala Glu Trp Leu Glu Tyr Ala Gln Thr 1190 1195 1200 His Leu Leu 1205 <210> 8 <211> 1233 <212> PRT <213> Unknown <220> <223> Lachnospiraceae bacterium <400> 8 Met Asp Tyr Gly Asn Gly Gln Phe Glu Arg Arg Ala Pro Leu Thr Lys 1 5 10 15 Thr Ile Thr Leu Arg Leu Lys Pro Ile Gly Glu Thr Arg Glu Thr Ile 20 25 30 Arg Glu Gln Lys Leu Leu Glu Gln Asp Ala Ala Phe Arg Lys Leu Val 35 40 45 Glu Thr Val Thr Pro Ile Val Asp Asp Cys Ile Arg Lys Ile Ala Asp 50 55 60 Asn Ala Leu Cys His Phe Gly Thr Glu Tyr Asp Phe Ser Cys Leu Gly 65 70 75 80 Asn Ala Ile Ser Lys Asn Asp Ser Lys Ala Ile Lys Lys Glu Thr Glu 85 90 95 Lys Val Glu Lys Leu Leu Ala Lys Val Leu Thr Glu Asn Leu Pro Asp 100 105 110 Gly Leu Arg Lys Val Asn Asp Ile Asn Ser Ala Ala Phe Ile Gln Asp 115 120 125 Thr Leu Thr Ser Phe Val Gln Asp Asp Ala Asp Lys Arg Val Leu Ile 130 135 140 Gln Glu Leu Lys Gly Lys Thr Val Leu Met Gln Arg Phe Leu Thr Thr 145 150 155 160 Arg Ile Thr Ala Leu Thr Val Trp Leu Pro Asp Arg Val Phe Glu Asn 165 170 175 Phe Asn Ile Phe Ile Glu Asn Ala Glu Lys Met Arg Ile Leu Leu Asp 180 185 190 Ser Pro Leu Asn Glu Lys Ile Met Lys Phe Asp Pro Asp Ala Glu Gln 195 200 205 Tyr Ala Ser Leu Glu Phe Tyr Gly Gln Cys Leu Ser Gln Lys Asp Ile 210 215 220 Asp Ser Tyr Asn Leu Ile Ile Ser Gly Ile Tyr Ala Asp Asp Glu Val 225 230 235 240 Lys Asn Pro Gly Ile Asn Glu Ile Val Lys Glu Tyr Asn Gln Gln Ile 245 250 255 Arg Gly Asp Lys Asp Glu Ser Pro Leu Pro Lys Leu Lys Lys Leu His 260 265 270 Lys Gln Ile Leu Met Pro Val Glu Lys Ala Phe Phe Val Arg Val Leu 275 280 285 Ser Asn Asp Ser Asp Ala Arg Ser Ile Leu Glu Lys Ile Leu Lys Asp 290 295 300 Thr Glu Met Leu Pro Ser Lys Ile Ile Glu Ala Met Lys Glu Ala Asp 305 310 315 320 Ala Gly Asp Ile Ala Val Tyr Gly Ser Arg Leu His Glu Leu Ser His 325 330 335 Val Ile Tyr Gly Asp His Gly Lys Leu Ser Gln Ile Ile Tyr Asp Lys 340 345 350 Glu Ser Lys Arg Ile Ser Glu Leu Met Glu Thr Leu Ser Pro Lys Glu 355 360 365 Arg Lys Glu Ser Lys Lys Arg Leu Glu Gly Leu Glu Glu His Ile Arg 370 375 380 Lys Ser Thr Tyr Thr Phe Asp Glu Leu Asn Arg Tyr Ala Glu Lys Asn 385 390 395 400 Val Met Ala Ala Tyr Ile Ala Ala Val Glu Glu Ser Cys Ala Glu Ile 405 410 415 Met Arg Lys Glu Lys Asp Leu Arg Thr Leu Leu Ser Lys Glu Asp Val 420 425 430 Lys Ile Arg Gly Asn Arg His Asn Thr Leu Ile Val Lys Asn Tyr Phe 435 440 445 Asn Ala Trp Thr Val Phe Arg Asn Leu Ile Arg Ile Leu Arg Arg Lys 450 455 460 Ser Glu Ala Glu Ile Asp Ser Asp Phe Tyr Asp Val Leu Asp Asp Ser 465 470 475 480 Val Glu Val Leu Ser Leu Thr Tyr Lys Gly Glu Asn Leu Cys Arg Ser 485 490 495 Tyr Ile Thr Lys Lys Ile Gly Ser Asp Leu Lys Pro Glu Ile Ala Thr 500 505 510 Tyr Gly Ser Ala Leu Arg Pro Asn Ser Arg Trp Trp Ser Pro Gly Glu 515 520 525 Lys Phe Asn Val Lys Phe His Thr Ile Val Arg Arg Asp Gly Arg Leu 530 535 540 Tyr Tyr Phe Ile Leu Pro Lys Gly Ala Lys Pro Val Glu Leu Glu Asp 545 550 555 560 Met Asp Gly Asp Ile Glu Cys Leu Gln Met Arg Lys Ile Pro Asn Pro 565 570 575 Thr Ile Phe Leu Pro Lys Leu Val Phe Lys Asp Pro Glu Ala Phe Phe 580 585 590 Arg Asp Asn Pro Glu Ala Asp Glu Phe Val Phe Leu Ser Gly Met Lys 595 600 605 Ala Pro Val Thr Ile Thr Arg Glu Thr Tyr Glu Ala Tyr Arg Tyr Lys 610 615 620 Leu Tyr Thr Val Gly Lys Leu Arg Asp Gly Glu Val Ser Glu Glu Glu 625 630 635 640 Tyr Lys Arg Ala Leu Leu Gln Val Leu Thr Ala Tyr Lys Glu Phe Leu 645 650 655 Glu Asn Arg Met Ile Tyr Ala Asp Leu Asn Phe Gly Phe Lys Asp Leu 660 665 670 Glu Glu Tyr Lys Asp Ser Ser Glu Phe Ile Lys Gln Val Glu Thr His 675 680 685 Asn Thr Phe Met Cys Trp Ala Lys Val Ser Ser Ser Gln Leu Asp Asp 690 695 700 Leu Val Lys Ser Gly Asn Gly Leu Leu Phe Glu Ile Trp Ser Glu Arg 705 710 715 720 Leu Glu Ser Tyr Tyr Lys Tyr Gly Asn Glu Lys Val Leu Arg Gly Tyr 725 730 735 Glu Gly Val Leu Leu Ser Ile Leu Lys Asp Glu Asn Leu Val Ser Met 740 745 750 Arg Thr Leu Leu Asn Ser Arg Pro Met Leu Val Tyr Arg Pro Lys Glu 755 760 765 Ser Ser Lys Pro Met Val Val His Arg Asp Gly Ser Arg Val Val Asp 770 775 780 Arg Phe Asp Lys Asp Gly Lys Tyr Ile Pro Pro Glu Val His Asp Glu 785 790 795 800 Leu Tyr Arg Phe Phe Asn Asn Leu Leu Ile Lys Glu Lys Leu Gly Glu 805 810 815 Lys Ala Arg Lys Ile Leu Asp Asn Lys Lys Val Lys Val Lys Val Leu 820 825 830 Glu Ser Glu Arg Val Lys Trp Ser Lys Phe Tyr Asp Glu Gln Phe Ala 835 840 845 Val Thr Phe Ser Val Lys Lys Asn Ala Asp Cys Leu Asp Thr Thr Lys 850 855 860 Asp Leu Asn Ala Glu Val Met Glu Gln Tyr Ser Glu Ser Asn Arg Leu 865 870 875 880 Ile Leu Ile Arg Asn Thr Thr Asp Ile Leu Tyr Tyr Leu Val Leu Asp 885 890 895 Lys Asn Gly Lys Val Leu Lys Gln Arg Ser Leu Asn Ile Ile Asn Asp 900 905 910 Gly Ala Arg Asp Val Asp Trp Lys Glu Arg Phe Arg Gln Val Thr Lys 915 920 925 Asp Arg Asn Glu Gly Tyr Asn Glu Trp Asp Tyr Ser Arg Thr Ser Asn 930 935 940 Asp Leu Lys Glu Val Tyr Leu Asn Tyr Ala Leu Lys Glu Ile Ala Glu 945 950 955 960 Ala Val Ile Glu Tyr Asn Ala Ile Leu Ile Ile Glu Lys Met Ser Asn 965 970 975 Ala Phe Lys Asp Lys Tyr Ser Phe Leu Asp Asp Val Thr Phe Lys Gly 980 985 990 Phe Glu Thr Lys Lys Leu Ala Lys Leu Ser Asp Leu His Phe Arg Gly 995 1000 1005 Ile Lys Asp Gly Glu Pro Cys Ser Phe Thr Asn Pro Leu Gln Leu 1010 1015 1020 Cys Gln Asn Asp Ser Asn Lys Ile Leu Gln Asp Gly Val Ile Phe 1025 1030 1035 Met Val Pro Asn Ser Met Thr Arg Ser Leu Asp Pro Asp Thr Gly 1040 1045 1050 Phe Ile Phe Ala Ile Asn Asp His Asn Ile Arg Thr Lys Lys Ala 1055 1060 1065 Lys Leu Asn Phe Leu Ser Lys Phe Asp Gln Leu Lys Val Ser Ser 1070 1075 1080 Glu Gly Cys Leu Ile Met Lys Tyr Ser Gly Asp Ser Leu Pro Thr 1085 1090 1095 His Asn Thr Asp Asn Arg Val Trp Asn Cys Cys Cys Asn His Pro 1100 1105 1110 Ile Thr Asn Tyr Asp Arg Glu Thr Lys Lys Val Glu Phe Ile Glu 1115 1120 1125 Glu Pro Val Glu Glu Leu Ser Arg Val Leu Glu Glu Asn Gly Ile 1130 1135 1140 Glu Thr Asp Thr Glu Leu Asn Lys Leu Asn Glu Arg Glu Asn Val 1145 1150 1155 Pro Gly Lys Val Val Asp Ala Ile Tyr Ser Leu Val Leu Asn Tyr 1160 1165 1170 Leu Arg Gly Thr Val Ser Gly Val Ala Gly Gln Arg Ala Val Tyr 1175 1180 1185 Tyr Ser Pro Val Thr Gly Lys Lys Tyr Asp Ile Ser Phe Ile Gln 1190 1195 1200 Ala Met Asn Leu Asn Arg Lys Cys Asp Tyr Tyr Arg Ile Gly Ser 1205 1210 1215 Lys Glu Arg Gly Glu Trp Thr Asp Phe Val Ala Gln Leu Ile Asn 1220 1225 1230 <210> 9 <211> 1227 <212> PRT <213> Unknown <220> <223> Lachnospiraceae bacterium <400> 9 Met Ser Lys Leu Glu Lys Phe Thr Asn Cys Tyr Ser Leu Ser Lys Thr 1 5 10 15 Leu Arg Phe Lys Ala Ile Pro Val Gly Lys Thr Gln Glu Asn Ile Asp 20 25 30 Asn Lys Arg Leu Leu Val Glu Asp Glu Lys Arg Ala Glu Asp Tyr Lys 35 40 45 Gly Val Lys Lys Leu Leu Asp Arg Tyr Tyr Leu Ser Phe Ile Asn Asp 50 55 60 Val Leu His Ser Ile Lys Leu Lys Asn Leu Asn Asn Tyr Ile Ser Leu 65 70 75 80 Phe Arg Lys Lys Thr Arg Thr Glu Lys Glu Asn Lys Glu Leu Glu Asn 85 90 95 Leu Glu Ile Asn Leu Arg Lys Glu Ile Ala Lys Ala Phe Lys Gly Asn 100 105 110 Glu Gly Tyr Lys Ser Leu Phe Lys Lys Asp Ile Ile Glu Thr Ile Leu 115 120 125 Pro Glu Phe Leu Asp Asp Lys Asp Glu Ile Ala Leu Val Asn Ser Phe 130 135 140 Asn Gly Phe Thr Thr Ala Phe Thr Gly Phe Phe Asp Asn Arg Glu Asn 145 150 155 160 Met Phe Ser Glu Glu Ala Lys Ser Thr Ser Ile Ala Phe Arg Cys Ile 165 170 175 Asn Glu Asn Leu Thr Arg Tyr Ile Ser Asn Met Asp Ile Phe Glu Lys 180 185 190 Val Asp Ala Ile Phe Asp Lys His Glu Val Gln Glu Ile Lys Glu Lys 195 200 205 Ile Leu Asn Ser Asp Tyr Asp Val Glu Asp Phe Phe Glu Gly Glu Phe 210 215 220 Phe Asn Phe Val Leu Thr Gln Glu Gly Ile Asp Val Tyr Asn Ala Ile 225 230 235 240 Ile Gly Gly Phe Val Thr Glu Ser Gly Glu Lys Ile Lys Gly Leu Asn 245 250 255 Glu Tyr Ile Asn Leu Tyr Asn Gln Lys Thr Lys Gln Lys Leu Pro Lys 260 265 270 Phe Lys Pro Leu Tyr Lys Gln Val Leu Ser Asp Arg Glu Ser Leu Ser 275 280 285 Phe Tyr Gly Glu Gly Tyr Thr Ser Asp Glu Glu Val Leu Glu Val Phe 290 295 300 Arg Asn Thr Leu Asn Lys Asn Ser Glu Ile Phe Ser Ser Ile Lys Lys 305 310 315 320 Leu Glu Lys Leu Phe Lys Asn Phe Asp Glu Tyr Ser Ser Ala Gly Ile 325 330 335 Phe Val Lys Asn Gly Pro Ala Ile Ser Thr Ile Ser Lys Asp Ile Phe 340 345 350 Gly Glu Trp Asn Val Ile Arg Asp Lys Trp Asn Ala Glu Tyr Asp Asp 355 360 365 Ile His Leu Lys Lys Lys Ala Val Val Thr Glu Lys Tyr Glu Asp Asp 370 375 380 Arg Arg Lys Ser Phe Lys Lys Ile Gly Ser Phe Ser Leu Glu Gln Leu 385 390 395 400 Gln Glu Tyr Ala Asp Ala Asp Leu Ser Val Val Glu Lys Leu Lys Glu 405 410 415 Ile Ile Ile Gln Lys Val Asp Glu Ile Tyr Lys Val Tyr Gly Ser Ser 420 425 430 Glu Lys Leu Phe Asp Ala Asp Phe Val Leu Glu Lys Ser Leu Lys Lys 435 440 445 Asn Asp Ala Val Val Ala Ile Met Lys Asp Leu Leu Asp Ser Val Lys 450 455 460 Ser Phe Glu Asn Tyr Ile Lys Ala Phe Phe Gly Glu Gly Lys Glu Thr 465 470 475 480 Asn Arg Asp Glu Ser Phe Tyr Gly Asp Phe Val Leu Ala Tyr Asp Ile 485,490,495 Leu Leu Lys Val Asp His With Tyr Asp With Arg Asn Tyr Val Thr 500 505 510 Gln Lys Pro Tyr Ser Lys Asp Lys Phe Lys Leu Tyr Phe Gln Asn Pro 515,520,525 Gln Phe Met Gly Gly Trp Asp Lys Asp Lys Glu Thr Asp Tyr Arg Ala 530 535 540 Thr Ile Leu Arg Tyr Gly Ser Lys Tyr Leu Ala Ile Met Asp Lys 545 550 555 560 Lys Tyr Ala Lys Cys Leu Gln Lys Ile Asp Lys Asp Val Asn Gly 565,570,575 Asn Tyr Glu Lys And Asn Tyr Lys Leu Pro Gly Pro Asn Lys Met 580,585,590 Leu Pro Lys Val Phe Phe Ser Lys Lys Trp Met Ala Tyr Tyr Asn Pro 595 600 605 Ser Glu Asp Ile Gln Lys Ile Tyr Lys Asn Gly Thr Phe Lys Lys Gly 610 615 620 Asp Met Phe Asn Leu Asn Asp Cys His Lys Leu Ile Asp Phe Phe Lys 625 630 635 640 Asp Ser Ile Ser Arg Tyr Pro Lys Trp Ser Asn Ala Tyr Asp Phe Asn 645 650 655 Phe Ser Glu Thr Glu Lys Tyr Lys Asp Ile Ala Gly Phe Tyr Arg Glu 660 665 670 Val Glu Glu Gln Gly Tyr Lys Val Ser Phe Glu Ser Ala Ser Lys Lys 675 680 685 Glu Val Asp Lys Leu Val Glu Glu Gly Lys Leu Tyr Met Phe Gln Ile 690 695 700 Tyr Asn Lys Asp Phe Ser Asp Lys Ser His Gly Thr Pro Asn Leu His 705 710 715 720 Thr Met Tyr Phe Lys Leu Leu Phe Asp Glu Asn Asn His Gly Gln Ile 725 730 735 Arg Leu Ser Gly Gly Ala Glu Leu Phe Met Arg Arg Ala Ser Leu Lys 740 745 750 Lys Glu Glu Leu Val Val His Pro Ala Asn Ser Pro Ile Ala Asn Lys 755 760 765 Asn Pro Asp Asn Pro Lys Lys Thr Thr Thr Leu Ser Tyr Asp Val Tyr 770 775 780 Lys Asp Lys Arg Phe Ser Glu Asp Gln Tyr Glu Leu His Ile Pro Ile 785 790 795 800 Ala Asn Ile Asn Lys Cys Pro Lys Asn Ile Phe Lys Ile Asn Thr Glu 805 810 815 Val Arg Val Leu Leu Lys His Asp Asp Asn Pro Tyr Val Ile Gly Ile 820 825 830 Asp Arg Gly Glu Arg Asn Leu Leu Tyr Ile Val Val Val Asp Gly Lys 835 840 845 Gly Asn Ile Val Glu Gln Tyr Ser Leu Asn Glu Ile Ile Asn Asn Phe 850 855 860 Asn Gly Ile Arg Ile Lys Thr Asp Tyr His Ser Leu Leu Asp Lys Lys 865 870 875 880 Glu Lys Glu Arg Phe Glu Ala Arg Gln Asn Trp Thr Ser Ile Glu Asn 885 890 895 Ile Lys Glu Leu Lys Ala Gly Tyr Ile Ser Gln Val Val His Lys Ile 900 905 910 Cys Glu Leu Val Glu Lys Tyr Asp Ala Val Ile Ala Leu Glu Asp Leu 915,920,925 Asn Ser Gly Phe Lys Asn Ser Arg Val Lys Val Glu Lys Gln Val Tyr 930,935,940 Gln Lys Phe Glu Lys Met Leu Ile Asp Lys Leu Asn Tyr Met Val Asp 945 950 955 960 Lys Lys Ser Asn Pro Cys Ala Thr Gly Gly Ala Leu Lys Gly Tyr Gln 965,970,975 Thr Asn Lys Phe Glu Ser Phe Lys Ser Met Ser Met Ser Gln Asn Gly 980,985,990 Phe Ile Phe Tyr Ile Pro Ala Trp Leu Thr Ser Lys Ile Asp Pro Ser 995 1000 1005 Thr Gly Phe Val Asn Leu Leu Lys Thr Lys Tyr Thr Ser Ile Ala 1010 1015 1020 Asp Lys Lys Phe Ile Ser Ser Phe Asp Arg Ile Met Tyr Val Pro 1025 1030 1035 Glu Glu Asp Leu Phe Glu Phe Ala Leu Asp Tyr Lys Asn Phe Ser 1040 1045 1050 Arg Thr Asp Ala Asp Tyr Ile Lys Trp Lys Leu Tyr Ser Tyr 1055 1060 1065 Gly Asn Arg Ile Arg Ile Phe Arg Asn Pro Lys Lys Asn Asn Val 1070 1075 1080 Phe Asp Trp Glu Glu Val Cys Leu Thr Ser Ala Tyr Lys Glu Leu 1085 1090 1095 Phe Asn Lys Tyr Gly And Asn Tyr Gln Gln Gly Asp With Arg Ala 1100 1105 1110 Leu Leu Cys Glu Gln Ser Asp Lys Ala Phe Tyr Ser Ser Phe Met 1115 1120 1125 Only One Met Ser Many Met Many Gln Met Arg Asn Ser Ile Thr Gly 1130 1135 1140 Arg Thr Asp Val Asp Phe Leu Ile Ser Pro Val Lys Asn Ser Asp 1145 1150 1155 Gly With Phe Tyr Asp Ser Arg Asn Tyr Glu Ala Gln Glu Asn Ala 1160 1165 1170 Ile Leu Pro Lys Asn Ala Asp Ala Asn Gly Ala Tyr Asn Ile Ala 1175 1180 1185 Arg Lys Val Leu Trp Ala Ile Gly Gln Phe Lys Lys Ala Glu Asp 1190 1195 1200 Glu Lys Leu Asp Lys Val Lys Ile Ala Ser Asn Lys Glu Trp Leu 1205 1210 1215 Glu Tyr Only Gln Thr Serves Val Lys His 1220 1225 <210> 10 <211> 1264 <212> PRT <213> Leptospira inadequate <400> 10 Met Glu Asp Tyr Ser Gly Phe Val Asn Ile Tyr Ser Ile Gln Lys Thr 1 5 10 15 Leu Arg Phe Glu Leu Lys Pro Val Gly Lys Thr Leu Glu His Ile Glu 20 25 30 Lys Lys Gly Phe Leu Lys Lys Asp Lys Ile Arg Ala Glu Asp Tyr Lys 35 40 45 Ala Val Lys Ile Ile Asp Lys Tyr His Arg Ala Tyr Ile Glu Glu 50 55 60 Val Phe Asp Ser Val Leu His Gln Lys Lys Lys Asp Lys Thr Arg 65 70 75 80 Phe Ser Thr Gln Phe Ile Lys Glu Ile Lys Glu Phe Ser Glu Leu Tyr 85 90 95 Tyr Lys Thr Glu Lys Asn Ile Pro Asp Lys Glu Arg Leu Glu Ala Leu 100 105 110 Ser Glu Lys Leu Arg Lys Met Leu Val Gly Ala Phe Lys Gly Glu Phe 115 120 125 Ser Glu Glu Val Ala Glu Lys Tyr Asn Lys Asn Leu Phe Ser Lys Glu 130 135 140 Leu Ile Arg Asn Glu Ile Glu Lys Phe Cys Glu Thr Asp Glu Glu Arg 145 150 155 160 Lys Gln Val Ser Asn Phe Lys Ser Phe Thr Thr Tyr Phe Thr Gly Phe 165 170 175 His Ser Asn Arg Gln Asn Ile Tyr Ser Asp Glu Lys Lys Ser Thr Ala 180 185 190 Ile Gly Tyr Arg Ile Ile His Gln Asn Leu Pro Lys Phe Leu Asp Asn 195 200 205 Leu Lys Ile Ile Glu Ser Ile Gln Arg Arg Phe Lys Asp Phe Pro Trp 210 215 220 Ser Asp Leu Lys Lys Asn Leu Lys Lys Ile Asp Lys Asn Ile Lys Leu 225 230 235 240 Thr Glu Tyr Phe Ser Ile Asp Gly Phe Val Asn Val Leu Asn Gln Lys 245 250 255 Gly Ile Asp Ala Tyr Asn Thr Ile Leu Gly Gly Lys Ser Glu Glu Ser 260 265 270 Gly Glu Lys Ile Gln Gly Leu Asn Glu Tyr Ile Asn Leu Tyr Arg Gln 275 280 285 Lys Asn Asn Ile Asp Arg Lys Asn Pro Leu Asn Val Lys Ile Leu Phe 290 295 300 Lys Gln Ile Leu Gly Asp Arg Glu Thr Lys Ser Phe Ile Pro Glu Ala 305 310 315 320 Phe Pro Asp Asp Gln Ser Val Leu Asn Ser Ile Thr Glu Phe Ala Lys 325 330 335 Tyr Leu Lys Leu Asp Lys Lys Lys Lys Ser Ile Ile Ala Glu Leu Lys 340 345 350 Lys Phe Leu Ser Ser Phe Asn Arg Tyr Glu Leu Asp Gly Ile Tyr Leu 355 360 365 Ala Asn Asp Asn Ser Leu Ala Ser Ile Ser Thr Phe Leu Phe Asp Asp 370 375 380 Trp Ser Phe Ile Lys Lys Ser Val Ser Phe Lys Tyr Asp Glu Ser Val 385 390 395 400 Gly Asp Pro Lys Lys Lys Ile Lys Ser Pro Leu Lys Tyr Glu Lys Glu 405 410 415 Lys Glu Lys Trp Leu Lys Gln Lys Tyr Tyr Thr Ile Ser Phe Leu Asn 420 425 430 Asp Ala Ile Glu Ser Tyr Ser Lys Ser Gln Asp Glu Lys Arg Val Lys 435 440 445 Ile Arg Leu Glu Ala Tyr Phe Ala Glu Phe Lys Ser Lys Asp Asp Ala 450 455 460 Lys Lys Gln Phe Asp Leu Leu Glu Arg Ile Glu Glu Ala Tyr Ala Ile 465 470 475 480 Val Glu Pro Leu Leu Gly Ala Glu Tyr Pro Arg Asp Arg Asn Leu Lys 485,490,495 Ala Asp Lys Lys Glu Val Gly Lys Ile Lys Asp Phe Leu Asp Ser Ile 500 505 510 Lys Ser Leu Gln Phe Phe Leu Lys Pro Leu Leu Ser Ala Glu Ile Phe 515,520,525 Asp Glu Lys Asp Leu Gly Phe Tyr Asn Gln Leu Glu Gly Tyr Tyr Glu 530 535 540 Glu Ile Asp Ile Ser Gly His Leu Tyr Asn Lys Val Arg Asn Tyr Leu 545 550 555 560 Thr Gly Lys Ile Tyr Ser Lys Glu Lys Phe Lys Leu Asn Phe Glu Asn 565,570,575 Ser Thr Leu Lys Gly Trp Asp Glu Asn Arg Glu Val Ala Asn Leu 580,585,590 Cys Validation Of Glu Asp Gln Lys Tyr Tyr Leu Gly Val Met Asp 595,600,605 Lys Glu Asn Asn Thr Ile Leu Ser Asp Ile Pro Lys Val Lys Pro Asn 610 615 620 Glu Leu Phe Tyr Glu Lys Met Val Tyr Lys Leu Ile Pro Thr Pro His 625 630 635 640 Met Gln Pro Arg - I'm Not Afraid To Be Asp Asn I'm Not A Tyr 645,650,655 Asn Pro Ser Lys Ser Ile Leu Lys Ile Arg Glu Ala Lys Ser Phe Lys 660,665,670 Glu Gly Lys Asn Phe Lys Leu Lys Asp Cys His Lys Phe Ile Asp Phe 675,680,685 Tyr Lys Glu Ser Ile Ser Lys Asn Glu Asp Trp Ser Arg Phe Asp Phe 690,695,700 Lys Phe Server Lys Thr Ser Tyr Glu Asn Ile Ser Glu Phe Tyr Arg 705 710 715 720 Glu Val Glu Arg Gln Gly Tyr Asn Leu Asp Phe Lys Val Ser Lys 725 730 735 Phe Tyr Ile Asp Ser Leu Val Glu Asp Gly Lys Leu Tyr Leu Phe Gln 740,745,750 Ile Tyr Asn Lys Asp Phe Ser Ile Phe Ser Lys Gly Lys Pro Asn Leu 755,760,765 His Thr Ile Tyr Phe Arg Ser Leu Phe Ser Lys Glu Asn Leu Lys Asp 770,775,780 Val Cys Leu Lys Leu Asn Gly Glu Ala Glu Met Phe Phe Arg Lys Lys 785,790,795,800 Ser Ile Asn Tyr Asp Glu Lys Lys Arg Glu Gly His His Pro Glu 805 810 815 Leu Phe Glu Lysate Lysa Tyr Pro and Asp Lysate Lysa Arg Tyr Ser 820 825 830 Glu Asp Lys Phe Gln Phe His Leu Pro Ile Ser Leu Asn Phe Lys Ser 835 840 845 Lys Glu Arg Leu Asn Phe Asn Leu Lys Val Asn Glu Phe Leu Lys Arg 850 855 860 Asn Lys Asp Ile Asn Ile Ile Gly Ile Asp Arg Gly Glu Arg Asn Leu 865 870 875 880 Leu Tyr Val Met with Gln Lys Gly Glu and Leu Lys Gln Thr 885,890,895 Leu Leu Asp Ser Met Gln Ser Gly Gly Arg Pro Glu Ile Asn Tyr 900 905 910 Lys Glu Lys Leu Gln Glu Lys Glu Ile Glu Arg Asp Lys Ala Arg Lys 915 920 925 Ser Trp Gly Thr Val Glu Asn Ile Lys Glu Leu Lys Glu Gly Tyr Leu 930 935 940 Ser Ile Val Ile His Gln Ile Ser Lys Leu Met Val Glu Asn Asn Ala 945 950 955 960 Ile Val Val Leu Glu Asp Leu Asn Ile Gly Phe Lys Arg Gly Arg Gln 965 970 975 Lys Val Glu Arg Gln Val Tyr Gln Lys Phe Glu Lys Met Leu Ile Asp 980 985 990 Lys Leu Asn Phe Leu Val Phe Lys Glu Asn Lys Pro Thr Glu Pro Gly 995 1000 1005 Gly Val Leu Lys Ala Tyr Gln Leu Thr Asp Glu Phe Gln Ser Phe 1010 1015 1020 Glu Lys Leu Ser Lys Gln Thr Gly Phe Leu Phe Tyr Val Pro Ser 1025 1030 1035 Trp Asn Thr Ser Lys Ile Asp Pro Arg Thr Gly Phe Ile Asp Phe 1040 1045 1050 Leu His Pro Ala Tyr Glu Asn Ile Glu Lys Ala Lys Gln Trp Ile 1055 1060 1065 Asn Lys Phe Asp Ser Ile Arg Phe Asn Ser Lys Met Asp Trp Phe 1070 1075 1080 Glu Phe Thr Ala Asp Thr Arg Lys Phe Ser Glu Asn Leu Met Leu 1085 1090 1095 Gly Lys Asn Arg Will Trp Val Ile Cys Thr Thr Asn Will Glu Arg 1100 1105 1110 Tyr Phe Thr Served Lys Thr Ala Asn Ser Ser Ile Gln Tyr Asn Ser 1115 1120 1125 Gln Ile Thr Glu Lys Leu Lys Glu Leu Phe Val Asp Ile Pro 1130 1135 1140 Phe Ser Asn Gly Gln Asp Leu Lys Pro Glu Ile Leu Arg Lys Asn 1145 1150 1155 Asp Ala Val Phe Lys Ser Leu Leu Phe Tyr Ile Lys Thr Thr 1160 1165 1170 Leu Ser Leu Arg Gln Asn Asn Gly Lys Lys Gly Glu Glu Glu Lys 1175 1180 1185 Asp Phe Ile Leu Ser Pro Val Val Asp Ser Lys Gly Arg Phe Phe 1190 1195 1200 Asn Ser Leu Glu Ala Ser Asp Asp Glu Pro Lys Asp Ala Asp Ala 1205 1210 1215 Asn Gly Ala Tyr His Ile Ala Leu Lys Gly Leu Met Asn Leu Leu 1220 1225 1230 Val Leu Asn Glu Thr Lys Glu Glu Asn Leu Ser Arg Pro Lys Trp 1235 1240 1245 Lys Ile Lys Asn Lys Asp Trp Leu Glu Phe Val Trp Glu Arg Asn 1250 1255 1260 Arg <210> 11 <211> 1373 <212> PRT <213> Moraxella bovoculi <400> 11 Met Leu Phe Gln Asp Phe Thr His Leu Tyr Pro Leu Ser Lys Thr Val 1 5 10 15 Arg Phe Glu Leu Phe Ile Asp Arg Thr Leu Glu His Ile His Ala Lys 20 25 30 Asn Phe Leu Ser Gln Asp Glu Thr Met Ala Asp Met His Gln Lys Val 35 40 45 Lys Val Ile Leu Asp Asp Tyr His Arg Asp Phe Ile Ala Asp Met Met 50 55 60 Gly Glu Val Lys Leu Thr Lys Leu Ala Glu Phe Tyr Asp Val Tyr Leu 65 70 75 80 Lys Phe Arg Lys Asn Pro Lys Asp Asp Glu Leu Gln Lys Ala Gln Leu 85 90 95 Lys Asp Leu Gln Ala Val Leu Arg Lys Glu Ile Val Lys Pro Ile Gly 100 105 110 Asn Gly Gly Lys Tyr Lys Ala Gly Tyr Asp Arg Leu Phe Gly Ala Lys 115 120 125 Leu Phe Lys Asp Gly Lys Glu Leu Gly Asp Leu Ala Lys Phe Val Ile 130 135 140 Ala Gln Glu Gly Glu Ser Ser Pro Lys Leu Ala His Leu Ala His Phe 145 150 155 160 Glu Lys Phe Ser Thr Tyr Phe Thr Gly Phe His Asp Asn Arg Lys Asn 165 170 175 Met Tyr Ser Asp Glu Asp Lys His Thr Ala Ile Ala Tyr Arg Leu Ile 180 185 190 His Glu Asn Leu Pro Arg Phe Ile Asp Asn Leu Gln Ile Leu Thr Thr 195 200 205 Ile Lys Gln Lys His Ser Ala Leu Tyr Asp Gln Ile Ile Asn Glu Leu 210 215 220 Thr Ala Ser Gly Leu Asp Val Ser Leu Ala Ser His Leu Asp Gly Tyr 225 230 235 240 His Lys Leu Leu Thr Gln Glu Gly Ile Thr Ala Tyr Asn Thr Leu Leu 245 250 255 Gly Gly Ile Ser Gly Glu Ala Gly Ser Pro Lys Ile Gln Gly Ile Asn 260 265 270 Glu Leu Ile Asn Ser His His Asn Gln His Cys His Lys Ser Glu Arg 275 280 285 Ile Ala Lys Leu Arg Pro Leu His Lys Gln Ile Leu Ser Asp Gly Met 290 295 300 Ser Val Ser Phe Leu Pro Ser Lys Phe Ala Asp Asp Ser Glu Met Cys 305 310 315 320 Gln Ala Val Asn Glu Phe Tyr Arg His Tyr Ala Asp Val Phe Ala Lys 325 330 335 Val Gln Ser Leu Phe Asp Gly Phe Asp Asp His Gln Lys Asp Gly Ile 340 345 350 Tyr Val Glu His Lys Asn Leu Asn Glu Leu Ser Lys Gln Ala Phe Gly 355 360 365 Asp Phe Ala Leu Leu Gly Arg Val Leu Asp Gly Tyr Tyr Val Asp Val 370 375 380 Val Asn Pro Glu Phe Asn Glu Arg Phe Ala Lys Ala Lys Thr Asp Asn 385 390 395 400 Ala Lys Ala Lys Leu Thr Lys Glu Lys Asp Lys Phe Ile Lys Gly Val 405 410 415 His Ser Leu Ala Ser Leu Glu Gln Ala Ile Glu His Tyr Thr Ala Arg 420 425 430 His Asp Asp Glu Ser Val Gln Ala Gly Lys Leu Gly Gln Tyr Phe Lys 435 440 445 His Gly Leu Ala Gly Val Asp Asn Pro Ile Gln Lys Ile His Asn Asn 450 455 460 His Ser Thr Ile Lys Gly Phe Leu Glu Arg Glu Arg Pro Ala Gly Glu 465 470 475 480 Arg Ala Leu Pro Lys Ile Lys Ser Gly Lys Asn Pro Glu Met Thr Gln 485 490 495 Leu Arg Gln Leu Lys Glu Leu Leu Asp Asn Ala Leu Asn Val Ala His 500 505 510 Phe Ala Lys Leu Leu Thr Thr Lys Thr Thr Leu Asp Asn Gln Asp Gly 515 520 525 Asn Phe Tyr Gly Glu Phe Gly Val Leu Tyr Asp Glu Leu Ala Lys Ile 530 535 540 Pro Thr Leu Tyr Asn Lys Val Arg Asp Tyr Leu Ser Gln Lys Pro Phe 545 550 555 560 Ser Thr Glu Lys Tyr Lys Leu Asn Phe Gly Asn Pro Thr Leu Leu Asn 565,570,575 Gly Trp Asp Leu Asn Lys Glu Lys Asp Asn Phe Gly Val Ile Leu Gln 580,585,590 Lys Asp Gly Cys Tyr Tyr Leu Ala Leu Leu Asp Lys Ala His Lys Lys 595,600,605 Val Phe Asp Asn Ala Pro Asn Thr Gly Lys Ser Ile Tyr Gln Lys Met 610 615 620 Ile Tyr Lys Tyr Leu Glu Val Arg Lys Gln Phe Pro Lys Val Phe Phe 625 630 635 640 Ser Lys Glu Ala Ile Ala Ile Asn Tyr His Pro Ser Lys Glu Leu Val 645,650,655 Glu Ile Lys Asp Lys Gly Arg Gln Arg Ser Asp Asp Glu Arg Leu Lys 660,665,670 Tyr Arg Phe has Glu Cys and His Pro Tyr Asp 675,680,685 Lys Phe Glu Gly Ala Ile Gly Asp Ile Gln Leu Phe Lys Lys Asp 690,695,700 Lys Lys Gly Arg Glu Val Pro Ile Ser Glu Lys Asp Leu Phe Lys Asp 705 710 715 720 Ile Asn Gly Ile Phe Ser Ser Lys Pro Lys Leu Glu Met Glu Asp Phe 725 730 735 Phe Ile Gly Glu Phe Lys Arg Tyr Asn Pro Ser Gln Asp Leu Val Asp 740,745,750 Gln Tyr Asn With Tyr Lys Lys With Asp Ser Asn Asp Asn Arg Lys Lys 755,760,765 Glu Asn Phe Tyr Asn Asn His Pro Lys Phe Lys Lys Asp Leu Val Arg 770,775,780 Tyr Tyr Tyr Glu Ser Met Cys Lys His Glu Glu Trp Glu Glu Ser Phe 785,790,795,800 Glu Phe Is Lys Lys Leu Gln Asp And Gly Cys Tyr Val Asp Val Asn 805 810 815 Glu Leu Phe Thr Glu Ile Glu Thr Arg Arg Leu Asn Tyr Lys Ile Ser 820 825 830 Phe Cys Asn Ile Asn Ala Asp Tyr Ile Asp Glu Leu Val Glu Gln Gly 835 840 845 Gln Leu Tyr Leu Phe Gln Ile Tyr Asn Lys Asp Phe Ser Pro Lys Ala 850 855 860 His Gly Lys Pro Asn Leu His Thr Leu Tyr Phe Lys Ala Leu Phe Ser 865 870 875 880 Glu Asp Asn Leu Ala Asp Pro Ile Tyr Lys Leu Asn Gly Glu Ala Gln 885 890 895 Ile Phe Tyr Arg Lys Ala Ser Leu Asp Met Asn Glu Thr Thr Ile His 900 905 910 Arg Ala Gly Glu Val Leu Glu Asn Lys Asn Pro Asp Asn Pro Lys Lys 915 920 925 Arg Gln Phe Val Tyr Asp Ile Ile Lys Asp Lys Arg Tyr Thr Gln Lys 930 935 940 Asp Phe Met Leu His Val Pro Ile Thr Met Asn Phe Gly Val Gln Gly 945 950 955 960 Met Thr Ile Lys Glu Phe Asn Lys Lys Val Asn Gln Ser Ile Gln Gln 965 970 975 Tyr Asp Glu Val Asn Val Ile Gly Ile Asp Arg Gly Glu Arg His Leu 980 985 990 Leu Tyr Leu Thr Val Ile Asn Ser Lys Gly Glu Ile Leu Glu Gln Cys 995 1000 1005 Ser Leu Asn Asp Ile Thr Thr Ala Ser Ala Asn Gly Thr Gln Met 1010 1015 1020 Thr Thr Pro Tyr His Lys Ile Leu Asp Lys Arg Glu Ile Glu Arg 1025 1030 1035 Leu Asn Ala Arg Val Gly Trp Gly Glu Ile Glu Thr Ile Lys Glu 1040 1045 1050 Leu Lys Ser Gly Tyr Leu Ser His Val Val His Gln Ile Ser Gln 1055 1060 1065 Leu Met Leu Lys Tyr Asn Ala Ile Val Val Leu Glu Asp Leu Asn 1070 1075 1080 Phe Gly Phe Lys Arg Gly Arg Phe Lys Val Glu Lys Gln Ile Tyr 1085 1090 1095 Gln Asn Phe Glu Asn Ala Leu Ile Lys Lys Leu Asn His Leu Val 1100 1105 1110 Leu Lys Asp Lys Ala Asp Asp Glu Ile Gly Ser Tyr Lys Asn Ala 1115 1120 1125 Leu Gln Leu Thr Asn Asn Phe Thr Asp Leu Lys Ser Ile Gly Lys 1130 1135 1140 Gln Thr Gly Phe Leu Phe Tyr Val Pro Ala Trp Asn Thr Ser Lys 1145 1150 1155 Ile Asp Pro Glu Thr Gly Phe Val Asp Leu Leu Lys Pro Arg Tyr 1160 1165 1170 Glu Asn Ile Gln Ala Ser Gln Ala Phe Phe Gly Lys Phe Asp Lys 1175 1180 1185 With Cys Tyr Asn Ala Asp Lys Asp Tyr Phe Glu Phe His Ile Asp 1190 1195 1200 Tyr Ala Lys Phe Thr Asp Lys Ala Lys Asn Ser Arg Gln Ile Trp 1205 1210 1215 Thr Ile How To Serve His Gly Asp With Tyr Val Tyr Asp With Thr 1220 1225 1230 Only Asn Gln Asn Lys Gly Only Only Only Lys Gly Ile Asn Val Asn Asp 1235 1240 1245 Ile Leu Lys Ser Leu Phe Ala Arg His Ile Asn Glu Lys Gln 1250 1255 1260 Pro Asn Leu Val Met Asp With Cys Gln Asn Asp Lys Glu Phe 1265 1270 1275 His Lys Ser Leu Met Tyr Leu Lys Thr Le Leu Ala Leu Arg 1280 1285 1290 Tyr Ser Asn Ala Ser Ser Asp Glu Asp Phe Ile Leu Ser Pro Val 1295 1300 1305 Ala Asn Asp Glu Gly Val Phe Phe Asn Ser Ala Leu Ala Asp Asp 1310 1315 1320 Thr Gln Pro Gln Asn Ala Asp Ala Asn Gly Ala Tyr His Ile Ala 1325 1330 1335 Leu Lys Gly Leu Trp Leu Leu Asn Glu Leu Lys Asn Ser Asp Asp 1340 1345 1350 Leu Asn Lys Val Lys Leu Ala Ile Asp Asn Gln Thr Trp Leu Asn 1355 1360 1365 Phe Ala Gln Asn Arg 1370 <210> 12 <211> 1352 <212> PRT <213> Unknown <220> <223> Parcubacteria bacteria <400> 12 Met Glu Asn Ile Phe Asp Gln Phe Ile Gly Lys Tyr Ser Leu Ser Lys 1 5 10 15 Thr Leu Arg Phe Glu Leu Lys Pro Val Gly Lys Thr Glu Asp Phe Leu 20 25 30 Lys Ile Asn Lys Val Phe Glu Lys Asp Gln Thr Ile Asp Asp Ser Tyr 35 40 45 Asn Gln Ala Lys Phe Tyr Phe Asp Ser Leu His Gln Lys Phe Ile Asp 50 55 60 Ala Ala Leu Ala Ser Asp Lys Thr Ser Glu Leu Ser Phe Gln Asn Phe 65 70 75 80 Ala Asp Val Leu Glu Lys Gln Asn Lys Ile Ile Leu Asp Lys Lys Arg 85 90 95 Glu Met Gly Ala Leu Arg Lys Arg Asp Lys Asn Ala Val Gly Ile Asp 100 105 110 Arg Leu Gln Lys Glu Ile Asn Asp Ala Glu Asp Ile Ile Gln Lys Glu 115 120 125 Lys Glu Lys Ile Tyr Lys Asp Val Arg Thr Leu Phe Asp Asn Glu Ala 130 135 140 Glu Ser Trp Lys Thr Tyr Tyr Gln Glu Arg Glu Val Asp Gly Lys Lys 145 150 155 160 Ile Thr Glu Ser Lys Ala Asp Leu Lys Gln Lys Gly Ala Asp Phe Leu 165 170 175 Thr Ala Ala Gly Ile Leu Lys Val Leu Lys Tyr Glu Phe Pro Glu Glu 180 185 190 Lys Glu Lys Glu Phe Gln Ala Lys Asn Gln Pro Ser Leu Phe Val Glu 195 200 205 Glu Lys Glu Asn Pro Gly Gln Lys Arg Tyr Ile Phe Asp Ser Phe Asp 210 215 220 Lys Phe Ala Gly Tyr Leu Thr Lys Phe Gln Gln Thr Lys Lys Asn Leu 225 230 235 240 Tyr Ala Ala Asp Gly Thr Ser Thr Ala Val Ala Thr Arg Ile Ala Asp 245 250 255 Asn Phe Ile Ile Phe His Gln Asn Thr Lys Val Phe Arg Asp Lys Tyr 260 265 270 Lys Asn Asn His Thr Asp Leu Gly Phe Asp Glu Glu Asn Ile Phe Glu 275 280 285 Ile Glu Arg Tyr Lys Asn Cys Leu Leu Gln Arg Glu Ile Glu His Ile 290 295 300 Lys Asn Glu Asn Ser Tyr Asn Lys Ile Ile Gly Arg Ile Asn Lys Lys 305 310 315 320 Ile Lys Glu Tyr Arg Asp Gln Lys Ala Lys Asp Thr Lys Leu Thr Lys 325 330 335 Ser Asp Phe Pro Phe Phe Lys Asn Leu Asp Lys Gln Ile Leu Gly Glu 340 345 350 Val Glu Lys Glu Lys Gln Leu Ile Glu Lys Thr Arg Glu Lys Thr Glu 355 360 365 Glu Asp Val Leu Ile Glu Arg Phe Lys Glu Phe Ile Glu Asn Asn Glu 370 375 380 Glu Arg Phe Thr Ala Ala Lys Lys Leu Met Asn Ala Phe Cys Asn Gly 385 390 395 400 Glu Phe Glu Ser Glu Tyr Glu Gly Ile Tyr Leu Lys Asn Lys Ala Ile 405 410 415 Asn Thr Ile Ser Arg Arg Trp Phe Val Ser Asp Arg Asp Phe Glu Leu 420 425 430 Lys Leu Pro Gln Gln Lys Ser Lys Asn Lys Ser Glu Lys Asn Glu Pro 435 440 445 Lys Val Lys Lys Phe Ile Ser Ile Ala Glu Ile Lys Asn Ala Val Glu 450 455 460 Glu Leu Asp Gly Asp Ile Phe Lys Ala Val Phe Tyr Asp Lys Lys Ile 465 470 475 480 Only Gln Gly Gly Served By Lys Glu Gln Phe 485,490,495 Tyr Glu Phe Glu Tyr Leu Phe Arg Asp Ile Glu Arg Glu Asn Gly Glu 500 505 510 Lys Leu Gly Tyr Asp Ser Cys Leu Lys Ile Ala Lys Gln Leu Gly 515,520,525 Ile Phe Pro Gln Glu Lys Glu Ala Arg Glu Lys Ala Thr Ala Val Ile 530 535 540 Lys Tyr Ala Asp Ala Gly Leu Gly Ile Phe Gln Met Met Lys Tyr 545 550 555 560 Phe Ser Leu Asp Asp Lys Asp Arg Lys Asn Thr Pro Gly Gln Leu Ser 565,570,575 Thr Asn Phe Tyr Ala Glu Tyr Asp Gly Tyr Tyr Lys Asp Phe Glu Phe 580,585,590 Ile Lys Tyr Asn Glu Phe Arg Asn Phe Ile Thr Lys Lys Pro Phe 595,600,605 Asp Glu Asp Lys Ile Lys Leu Asn Phe Glu Asn Gly Ala Leu Leu Lys 610 615 620 Gly Trp Asp Glu Asn Lys Glu Tyr Asp Phe Met Gly Val Ile Leu Lys 625 630 635 640 Lys Glu Gly Arg Leu Tyr Leu Gly Ile Met His Lys Asn His Arg Lys 645,650,655 Leu Phe Gln Ser Met Gly Asn Ala Lys Gly Asp Asn Ala Asn Arg Tyr 660,665,670 Gln Lys Met Tyr Gln Ile Al Asp Alone Ser Lys Asp Val Pro 675,680,685 Arg Leu Leu Leu Thr Ser Lys Lys Ala Met Glu Lys Phe Lys Pro Ser 690,695,700 Gln Glu Ile Leu Arg Ile Lys Lys Glu Lys Thr Phe Lys Arg Glu Ser 705 710 715 720 Lys Asn Phe Ser Leu Arg Asp Leu His Ala Leu Ile Glu Tyr Tyr Arg 725 730 735 Asn Cys Ile Pro Gln Tyr Ser Asn Trp Ser Phe Tyr Asp Phe Gln Phe 740 745 750 Gln Asp Thr Gly Lys Tyr Gln Asn Ile Lys Glu Phe Thr Asp Asp Val 755 760 765 Gln Lys Tyr Gly Tyr Lys Ile Ser Phe Arg Asp Ile Asp Asp Glu Tyr 770 775 780 Ile Asn Gln Ala Leu Asn Glu Gly Lys Met Tyr Leu Phe Glu Val Val 785 790 795 800 Asn Lys Asp Ile Tyr Asn Thr Lys Asn Gly Ser Lys Asn Leu His Thr 805 810 815 Leu Tyr Phe Glu His Ile Leu Ser Ala Glu Asn Leu Asn Asp Pro Val 820 825 830 Phe Lys Leu Ser Gly Met Ala Glu Ile Phe Gln Arg Gln Pro Ser Val 835 840 845 Asn Glu Arg Glu Lys Ile Thr Thr Gln Lys Asn Gln Cys Ile Leu Asp 850 855 860 Lys Gly Asp Arg Ala Tyr Lys Tyr Arg Arg Tyr Thr Glu Lys Lys Ile 865 870 875 880 Met Phe His Met Ser Leu Val Leu Asn Thr Gly Lys Gly Glu Ile Lys 885 890 895 Gln Val Gln Phe Asn Lys Ile Ile Asn Gln Arg Ile Ser Ser Ser Asp 900 905 910 Asn Glu Met Arg Val Asn Val Ile Gly Ile Asp Arg Gly Glu Lys Asn 915 920 925 Leu Leu Tyr Tyr Ser Val Val Lys Gln Asn Gly Glu Ile Ile Glu Gln 930 935 940 Ala Ser Leu Asn Glu Ile Asn Gly Val Asn Tyr Arg Asp Lys Leu Ile 945 950 955 960 Glu Arg Glu Lys Glu Arg Leu Lys Asn Arg Gln Ser Trp Lys Pro Val 965 970 975 Val Lys Ile Lys Asp Leu Lys Lys Gly Tyr Ile Ser His Val Ile His 980 985 990 Lys Ile Cys Gln Leu Ile Glu Lys Tyr Ser Ala Ile Val Val Leu Glu 995 1000 1005 Asp Leu Asn Met Arg Phe Lys Gln Ile Arg Gly Gly Ile Glu Arg 1010 1015 1020 Ser Val Tyr Gln Gln Phe Glu Lys Ala Leu Ile Asp Lys Leu Gly 1025 1030 1035 Tyr Leu Val Phe Lys Asp Asn Arg Asp Leu Arg Ala Pro Gly Gly 1040 1045 1050 Val Leu Asn Gly Tyr Gln Leu Ser Ala Pro Phe Val Ser Phe Glu 1055 1060 1065 Lys Met Arg Lys Gln Thr Gly Ile Leu Phe Tyr Thr Gln Ala Glu 1070 1075 1080 Tyr Thr Ser Lys Thr Asp Pro Ile Thr Gly Phe Arg Lys Asn Val 1085 1090 1095 Tyr Ile Ser Asn Ser Ala Ser Leu Asp Lys Ile Lys Glu Ala Val 1100 1105 1110 Lys Lys Phe Asp Ala Ile Gly Trp Asp Gly Lys Glu Gln Ser Tyr 1115 1120 1125 Phe Phe Lys Tyr Asn Pro Tyr Asn Leu Ala Asp Glu Lys Tyr Lys 1130 1135 1140 Asn Ser Thr Val Ser Lys Glu Trp Ala Ile Phe Ala Ser Ala Pro 1145 1150 1155 Arg Ile Arg Arg Gln Lys Gly Glu Asp Gly Tyr Trp Lys Tyr Asp 1160 1165 1170 Arg Val Lys Val Asn Glu Glu Phe Glu Lys Leu Leu Lys Val Trp 1175 1180 1185 Asn Phe Val Asn Pro Lys Ala Thr Asp Ile Lys Gln Glu Ile Ile 1190 1195 1200 Lys Lys Ile Lys Ala Gly Asp Leu Gln Gly Glu Lys Glu Leu Asp 1205 1210 1215 Gly Arg Leu Arg Asn Phe Trp His Ser Phe Ile Tyr Leu Phe Asn 1220 1225 1230 Leu Val Leu Glu Leu Arg Asn Ser Phe Ser Leu Gln Ile Lys Ile 1235 1240 1245 Lys Ala Gly Glu Val Ile Ala Val Asp Glu Gly Val Asp Phe Ile 1250 1255 1260 Ala Ser Pro Val Lys Pro Phe Phe Thr Thr Pro Asn Pro Tyr Ile 1265 1270 1275 Pro Ser Asn Leu Cys Trp Leu Ala Val Glu Asn Ala Asp Ala Asn 1280 1285 1290 Gly Ala Tyr Asn Ile Ala Arg Lys Gly Val Met Ile Leu Lys Lys 1295 1300 1305 Ile Arg Glu His Ala Lys Lys Asp Pro Glu Phe Lys Lys Leu Pro 1310 1315 1320 Asn Leu Phe Ile Ser Asn Ala Glu Trp Asp Glu Ala Ala Arg Asp 1325 1330 1335 Trp Gly Lys Tyr Ala Gly Thr Thr Ala Leu Asn Leu Asp His 1340 1345 1350 <210> 13 <211> 1260 <212> PRT <213> Porphyromonas crevioricanis <400> 13 Met Asp Ser Leu Lys Asp Phe Thr Asn Leu Tyr Pro Val Ser Lys Thr 1 5 10 15 Leu Arg Phe Glu Leu Lys Pro Val Gly Lys Thr Leu Glu Asn Ile Glu 20 25 30 Lys Ala Gly Ile Leu Lys Glu Asp Glu His Arg Ala Glu Ser Tyr Arg 35 40 45 Arg Val Lys Lys Ile Ile Asp Thr Tyr His Lys Val Phe Ile Asp Ser 50 55 60 Ser Leu Glu Asn Met Ala Lys Met Gly Ile Glu Asn Glu Ile Lys Ala 65 70 75 80 Met Leu Gln Ser Phe Cys Glu Leu Tyr Lys Lys Asp His Arg Thr Glu 85 90 95 Gly Glu Asp Lys Ala Leu Asp Lys Ile Arg Ala Val Leu Arg Gly Leu 100 105 110 Ile Val Gly Ala Phe Thr Gly Val Cys Gly Arg Arg Glu Asn Thr Val 115 120 125 Gln Asn Glu Lys Tyr Glu Ser Leu Phe Lys Glu Lys Leu Ile Lys Glu 130 135 140 Ile Leu Pro Asp Phe Val Leu Ser Thr Glu Ala Glu Ser Leu Pro Phe 145 150 155 160 Ser Val Glu Glu Ala Thr Arg Ser Leu Lys Glu Phe Asp Ser Phe Thr 165 170 175 Ser Tyr Phe Ala Gly Phe Tyr Glu Asn Arg Lys Asn Ile Tyr Ser Thr 180 185 190 Lys Pro Gln Ser Thr Ala Ile Ala Tyr Arg Leu Ile His Glu Asn Leu 195 200 205 Pro Lys Phe Ile Asp Asn Ile Leu Val Phe Gln Lys Ile Lys Glu Pro 210 215 220 Ile Ala Lys Glu Leu Glu His Ile Arg Ala Asp Phe Ser Ala Gly Gly 225 230 235 240 Tyr Ile Lys Lys Asp Glu Arg Leu Glu Asp Ile Phe Ser Leu Asn Tyr 245 250 255 Tyr Ile His Val Leu Ser Gln Ala Gly Ile Glu Lys Tyr Asn Ala Leu 260 265 270 Ile Gly Lys Ile Val Thr Glu Gly Asp Gly Glu Met Lys Gly Leu Asn 275 280 285 Glu His Ile Asn Leu Tyr Asn Gln Gln Arg Gly Arg Glu Asp Arg Leu 290 295 300 Pro Leu Phe Arg Pro Leu Tyr Lys Gln Ile Leu Ser Asp Arg Glu Gln 305 310 315 320 Leu Ser Tyr Leu Pro Glu Ser Phe Glu Lys Asp Glu Glu Leu Leu Arg 325 330 335 Ala Leu Lys Glu Phe Tyr Asp His Ile Ala Glu Asp Ile Leu Gly Arg 340 345 350 Thr Gln Gln Leu Met Thr Ser Ile Ser Glu Tyr Asp Leu Ser Arg Ile 355 360 365 Tyr Val Arg Asn Asp Ser Gln Leu Thr Asp Ile Ser Lys Lys Met Leu 370 375 380 Gly Asp Trp Asn Ala Ile Tyr Met Ala Arg Glu Arg Ala Tyr Asp His 385 390 395 400 Glu Gln Ala Pro Lys Arg Ile Thr Ala Lys Tyr Glu Arg Asp Arg Ile 405 410 415 Lys Ala Leu Lys Gly Glu Glu Ser Ile Ser Leu Ala Asn Leu Asn Ser 420 425 430 Cys Ile Ala Phe Leu Asp Asn Val Arg Asp Cys Arg Val Asp Thr Tyr 435 440 445 Leu Ser Thr Leu Gly Gln Lys Glu Gly Pro His Gly Leu Ser Asn Leu 450 455 460 Val Glu Asn Val Phe Ala Ser Tyr His Glu Ala Glu Gln Leu Leu Ser 465 470 475 480 Phe Pro Tyr Pro Glu Glu Asn Asn Leu Ile Gln Asp Lys Asp Asn Val 485 490 495 Val Leu Ile Lys Asn Leu Leu Asp Asn Ile Ser Asp Leu Gln Arg Phe 500 505 510 Leu Lys Pro Leu Trp Gly Met Gly Asp Glu Pro Asp Lys Asp Glu Arg 515 520 525 Phe Tyr Gly Glu Tyr Asn Tyr Ile Arg Gly Ala Leu Asp Gln Val Ile 530 535 540 Pro Leu Tyr Asn Lys Val Arg Asn Tyr Leu Thr Arg Lys Pro Tyr Ser 545 550 555 560 Thr Arg Lys Val Lys Leu Asn Phe Gly Asn Ser Gln Leu Leu Ser Gly 565 570 575 Trp Asp Arg Asn Lys Glu Lys Asp Asn Ser Cys Val Ile Leu Arg Lys 580 585 590 Gly Gln Asn Phe Tyr Leu Ala Ile Met Asn Asn Arg His Lys Arg Ser 595 600 605 Phe Glu Asn Lys Met Leu Pro Glu Tyr Lys Glu Gly Glu Pro Tyr Phe 610 615 620 Glu Lys Met Asp Tyr Lys Phe Leu Pro Asp Pro Asn Lys Met Leu Pro 625 630 635 640 Lys Val Phe Leu Ser Lys Lys Gly Ile Glu Ile Tyr Lys Pro Ser Pro 645 650 655 Lys Leu Leu Glu Gln Tyr Gly His Gly Thr His Lys Lys Gly Asp Thr 660 665 670 Phe Ser Met Asp Asp Leu His Glu Leu Ile Asp Phe Phe Lys His Ser 675 680 685 Ile Glu Ala His Glu Asp Trp Lys Gln Phe Gly Phe Lys Phe Ser Asp 690 695 700 Thr Ala Thr Tyr Glu Asn Val Ser Ser Phe Tyr Arg Glu Val Glu Asp 705 710 715 720 Gln Gly Tyr Lys Leu Ser Phe Arg Lys Val Ser Glu Ser Tyr Val Tyr 725 730 735 Serving Asp with Gln Gly Lys and Tyr Phe with Gln and Tyr Asn Lys 740,745,750 Asp Phe Ser Pro Cys Ser Lys Gly Thr Pro Asn Leu His Thr Leu Tyr 755,760,765 Trp Arg Met Phe Asp Glu Arg Asn Leu Ala Asp Val Ile Tyr Lys 770,775,780 Leu Asp Gly Lys Ala Glu Ile Phe Phe Arg Glu Lys Ser Leu Lys Asn 785,790,795,800 Asp His Pro Thr His Pro Ala Gly Lys Pro Ile Lys Lys Lys Ser Arg 805 810 815 Gln Lys Gly Glu Glu Ser Leu Phe Glu Tyr Asp Leu Val Lys Asp 820 825 830 Arg Arg Tyr Thr Met Asp Lys Phe Gln Phe His Val Pro Ile Thr Met 835 840 845 Asn Phe Lys Cys Ser Ala Gly Ser Lys Val Asn Asp Met Val Asn Ala 850 855 860 His Ile Arg Glu Ala Lys Asp Met His Val Ile Gly Ile Asp Arg Gly 865 870 875 880 Glu Arg Asn Leu Leu Tyr Ile Cys Val Ile Asp Ser Arg Gly Thr Ile 885 890 895 Leu Asp Gln Ile Ser Leu Asn Thr Ile Asn Asp Ile Asp Tyr His Asp 900 905 910 Leu Leu Glu Ser Arg Asp Lys Asp Arg Gln Gln Glu His Arg Asn Trp 915 920 925 Gln Thr Ile Glu Gly Ile Lys Glu Leu Lys Gln Gly Tyr Leu Ser Gln 930 935 940 Ala Val His Arg Ile Ala Glu Leu Met Val Ala Tyr Lys Ala Val Val 945 950 955 960 Ala Leu Glu Asp Leu Asn Met Gly Phe Lys Arg Gly Arg Gln Lys Val 965 970 975 Glu Ser Ser Val Tyr Gln Gln Phe Glu Lys Gln Leu Ile Asp Lys Leu 980 985 990 Asn Tyr Leu Val Asp Lys Lys Lys Arg Pro Glu Asp Ile Gly Gly Leu 995 1000 1005 Leu Arg Ala Tyr Gln Phe Thr Ala Pro Phe Lys Ser Phe Lys Glu 1010 1015 1020 Met Gly Lys Gln Asn Gly Phe Leu Phe Tyr Ile Pro Ala Trp Asn 1025 1030 1035 Thr Ser Asn Ile Asp Pro Thr Thr Gly Phe Val Asn Leu Phe His 1040 1045 1050 Val Gln Tyr Glu Asn Val Asp Lys Ala Lys Ser Phe Phe Gln Lys 1055 1060 1065 Phe Asp Ser Ile Ser Tyr Asn Pro Lys Lys Asp Trp Phe Glu Phe 1070 1075 1080 Ala Phe Asp Tyr Lys Asn Phe Thr Lys Lys Ala Glu Gly Ser Arg 1085 1090 1095 Ser Met Trp Ile Leu Cys Thr His Gly Ser Arg Ile Lys Asn Phe 1100 1105 1110 Arg Asn Ser Gln Lys Asn Gly Gln Trp Asp Ser Glu Glu Phe Ala 1115 1120 1125 Leu Thr Glu Ala Phe Lys Ser Leu Phe Val Arg Tyr Glu Ile Asp 1130 1135 1140 Tyr Thr Ala Asp Leu Lys Thr Ala Ile Val Asp Glu Lys Gln Lys 1145 1150 1155 Asp Phe Phe Val Asp Leu Leu Lys Leu Phe Lys Leu Thr Val Gln 1160 1165 1170 Met Arg Asn Ser Trp Lys Glu Lys Asp Leu Asp Tyr Leu Ile Ser 1175 1180 1185 Pro Val Ala Gly Ala Asp Gly Arg Phe Phe Asp Thr Arg Glu Gly 1190 1195 1200 Asn Lys Ser Leu Pro Lys Asp Ala Asp Ala Asn Gly Ala Tyr Asn 1205 1210 1215 Ile Ala Leu Lys Gly Leu Trp Ala Leu Arg Gln Ile Arg Gln Thr 1220 1225 1230 Ser Glu Gly Gly Lys Leu Lys Leu Ala Ile Ser Asn Lys Glu Trp 1235 1240 1245 Leu Gln Phe Val Gln Glu Arg Ser Tyr Glu Lys Asp 1250 1255 1260 <210> 14 <211> 1324 <212> PRT <213> Prevotella disiens <400> 14 Met Glu Asn Tyr Gln Glu Phe Thr Asn Leu Phe Gln Leu Asn Lys Thr 1 5 10 15 Leu Arg Phe Glu Leu Lys Pro Ile Gly Lys Thr Cys Glu Leu Leu Glu 20 25 30 Glu Gly Lys Ile Phe Ala Ser Gly Ser Phe Leu Glu Lys Asp Lys Val 35 40 45 Arg Ala Asp Asn Val Ser Tyr Val Lys Lys Glu Ile Asp Lys Lys His 50 55 60 Lys Ile Phe Ile Glu Glu Thr Leu Ser Ser Phe Ser Ile Ser Asn Asp 65 70 75 80 Leu Leu Lys Gln Tyr Phe Asp Cys Tyr Asn Glu Leu Lys Ala Phe Lys 85 90 95 Lys Asp Cys Ser Asp Glu Glu Glu Val Lys Thr Ala Leu Arg 100 105 110 Asn Lys Cys Thr Ser With Gln Arg Is Met Arg Glu With Ser Gln 115 120 125 Gln Lys Lys Is Pro Gln Lys Lys 130 135 140 Ile Glu Asn Val Phe Lys Ala Asp Glu Asn Val Gln His Phe Ser Glu 145 150 155 160 Phe Thr Ser Tyr Phe Ser Gly PHE Glu Thr Asn Arg Glu Asn Phe Tyr 165 170 175 Ser Asp Glu Glu Lys Ser Ser Ile Ala Tyr Arg Leu Val His Asp 180 185 190 Asn Leu Pro Has Glu Lys Asn Has Tyr Has Glu Lys Leu Lys 195 200 205 Glu Gln Phe Asp Ala Lys Thr Leu Ser Glu Ile Phe Glu Asn Tyr Lys 210 215 220 Leu Tyr Val Ala Gly Ser Ser Leu Asp Glu Val Phe Ser Leu Glu Tyr...

Claims

1. A modified species of the Trichophyceae family ( Lachnospiraceae Bacterial CRISPR (clustered regular interspaced short palindromic repeat) Cas12a (LbCas12a) polypeptide, wherein the modified LbCas12a polypeptide consists of the following amino acid sequence, which differs from SEQ ID NO:1 only in the presence of the following mutation at the position numbered in SEQ ID NO:1: (a) K595Y; (b) K595Y and K538W; (c) K595Y and T152R; or (d) K595Y, T152R and K538W.

2. A polynucleotide encoding the modified LbCas12a polypeptide of claim 1.

3. The polynucleotide of claim 2, wherein the polynucleotide encoding the modified LbCas12a polypeptide is operatively associated with a promoter.

4. The polynucleotide of claim 3, wherein the promoter is a promoter region containing introns.

5. The polynucleotide of claim 2 or claim 3, wherein the polynucleotide is codon-optimized for expression in an organism.

6. The polynucleotide of claim 5, wherein the organism is an animal, plant, fungus, archaea, or bacteria.

7. A complex comprising the modified LbCas12a polypeptide of claim 1, and a guide nucleic acid.

8. A nucleic acid construct encoding the complex of claim 7.

9. A composition comprising (a) the modified LbCas12a polypeptide of claim 1, and (b) a guiding nucleic acid.

10. An expression cassette or vector comprising the polynucleotide of any one of claims 2 to 6, or the nucleic acid construct of claim 8.

11. A CRISPR-Cas (CRISPR-Cas) system comprising: (a) The modified LbCas12a polypeptide of claim 1, or the nucleic acid encoding the modified LbCas12a polypeptide; and (b) A guide nucleic acid comprising a spacer sequence and a repeat sequence, wherein the guide nucleic acid is capable of forming a complex with the modified LbCas12a polypeptide, and the spacer sequence is capable of hybridizing with a target nucleic acid, thereby guiding the modified LbCas12a polypeptide to the target nucleic acid, thereby modifying or regulating the target nucleic acid.

12. The system of claim 11, wherein one or both of (a) and (b) are contained in one or more expression cassettes and / or carriers.

13. A host cell that does not develop into a host organism, comprising the polynucleotide of any one of claims 2 to 6, the nucleic acid construct of claim 8, the expression cassette or vector of claim 10, or the system of claim 11 or 12.

14. A cell comprising any one of the polynucleotides of claims 2 to 6, the nucleic acid construct of claim 8, the expression cassette or vector of claim 10, or the system of claim 11 or 12, wherein the cell is a fungal, archaea, or bacterial cell.

15. A method for generating modified plant cells, comprising introducing a polynucleotide of any one of claims 2 to 6, a nucleic acid construct of claim 8, an expression cassette or vector of claim 10, or a system of claim 11 or 12 into a plant cell.

16. A method for modifying a target nucleic acid in vitro, comprising: Contact the target nucleic acid with the following: (a)(i) the modified LbCas12a polypeptide of claim 1, and (ii) the guiding nucleic acid; (b) The complex and guiding nucleic acid as described in claim 7; (c) A composition comprising (i) the modified LbCas12a polypeptide of claim 1, and (ii) a guiding nucleic acid; and / or (d) The system according to claim 11 or 12, This modifies the target nucleic acid.

17. A method for modifying a target nucleic acid in vitro, comprising: Contact the cells or cell-free system containing the target nucleic acid with the following: (a)(i) the polynucleotide of any one of claims 2 to 6, or an expression cassette or vector containing thereof, and (ii) the guiding nucleic acid, or an expression cassette or vector containing thereof; and / or (b) The nucleic acid construct of claim 8, or an expression cassette or vector containing therein. This modifies the target nucleic acid.

18. A kit comprising: a polynucleotide as described in any one of claims 2 to 6, and / or an expression cassette or vector containing the polynucleotide.

19. The kit of claim 18, further comprising instructions for use of the kit.

20. The kit of claim 18 further comprises: a CRISPR-Cas12a guide nucleic acid and / or an expression cassette or vector containing the guide nucleic acid.

21. The kit of claim 20, wherein the guide nucleic acid includes a cloning site for cloning a nucleic acid sequence that is identical to or complementary to the target nucleic acid sequence into the backbone of the guide nucleic acid.

22. The kit of any one of claims 18 to 21, wherein the polynucleotide further encodes one or more nuclear localization signals, wherein the one or more nuclear localization signals are fused with a CRISPR-Cas12a nuclease.

23. The kit of any one of claims 18 to 21, wherein the polynucleotide, expression cassette, or vector further encodes one or more optional markers.

24. The kit of any one of claims 18 to 21, wherein the polynucleotide is mRNA and encodes one or more introns within the encoded CRISPR-Cas12a nuclease.

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