Type ii cas proteins and uses thereof

Abstract

The present disclosure relates to Type II Cas proteins and their uses. The present disclosure provides engineered, non-naturally occurring novel Type II Cas proteins comprising an amino acid sequence having at least 70% sequence identity to SEQ ID NO. 32 or variants thereof. The Type II Cas proteins should enable the CRISPR Cas system to be applied more broadly to gene targeting or gene editing.

Description

Technical Field

[0001] This application claims priority to provisional patent application PCT / CN2024 / 080919, filed on March 11, 2024. The entire contents of that provisional application are incorporated herein by reference. Background Technology

[0002] Among the various CRISPR-Cas systems explored, the CRISPR-Cas9 system belongs to the second category of CRISPR-Cas systems, which has a particularly important impact on genome editing and holds great potential in biomedical research. Despite its versatility, the CRISPR-Cas9 system still faces several challenges, such as a limited number of known orthologs, a limited genome targeting range, and relatively limited editing efficiency. Given the vast diversity of microbial genomes, it is likely that many CRISPR-Cas variants remain undiscovered, many of which may possess superior target recognition capabilities or higher editing efficiency compared to currently available commercial versions. Summary of the Invention

[0003] This disclosure provides an engineered, non-naturally occurring type II Cas protein comprising an amino acid sequence selected from: (i) a sequence identical to SEQ ID NO. 32; or (ii) a sequence having at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO. 32.

[0004] In some embodiments, the Cas protein is capable of recognizing a prototype spacer adjacent motif (PAM) having the following sequence: NRWAAW; preferably, the Cas protein is capable of recognizing a prototype spacer adjacent motif (PAM) having the following sequence: NAAAAA, NGAAAA, NATAAA, NAAAAT, NGAAAA, NGTAAA, NGTAAT; more preferably, the Cas protein is capable of recognizing a prototype spacer adjacent motif (PAM) having the following sequence: NAAAAA.

[0005] In some embodiments, the Cas protein is able to recognize prototype spacer adjacent motifs (PAMs) having the following sequences: AAAAA, GAAAA, ATAAA, AAAAT, GAAAA, GTAAA, GTAAT.

[0006] In some implementations, the Cas protein described herein is a nicking enzyme or an inactivated Cas protein.

[0007] In some aspects, this disclosure also provides an engineered, non-naturally occurring type II Cas protein comprising an amino acid sequence selected from: (i) a sequence identical to SEQ ID NO. 32; or (ii) a sequence having at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO. 32, except for the “M” at the first position of the sequence.

[0008] In some embodiments, the Cas protein is capable of recognizing a prototype spacer adjacent motif (PAM) having the following sequence: NRWAAW; preferably, the Cas protein is capable of recognizing a prototype spacer adjacent motif (PAM) having the following sequence: NAAAAA, NGAAAA, NATAAA, NAAAAT, NGAAAA, NGTAAA, NGTAAT; more preferably, the Cas protein is capable of recognizing a prototype spacer adjacent motif (PAM) having the following sequence: NAAAAA.

[0009] In some embodiments, the Cas protein is able to recognize prototype spacer adjacent motifs (PAMs) having the following sequences: AAAAA, GAAAA, ATAAA, AAAAT, GAAAA, GTAAA, GTAAT.

[0010] In some implementations, the Cas protein described herein is a nicking enzyme or an inactivated Cas protein.

[0011] In other aspects, the present invention also provides a fusion protein comprising the engineered, non-naturally occurring type II Cas protein disclosed herein, wherein the fusion protein further comprises a fusion chaperone sequence selected from the following: nuclear localization signal sequence, nuclear export signal sequence, cell penetration peptide sequence, affinity tag sequence, deaminase sequence, reverse transcriptase sequence, recombinase sequence, methyltransferase sequence, methyltransferase sequence, acetyltransferase sequence, acetyltransferase sequence, transcription activator sequence, transcription repressor domain sequence, cryptochrome sequence, photoinducible / controllable domain sequence, and chemically inducible / controllable domain sequence.

[0012] In some embodiments, the fusion protein comprises an amino acid sequence selected from: (i) a sequence identical to SEQ ID NO. 463; or (ii) a sequence having at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO. 463; optionally, the fusion protein comprises an amino acid sequence selected from: (i) a sequence identical to SEQ ID NO. 463; or (ii) a sequence identical to SEQ ID NO. 463. NO.463 is a sequence having at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity, except for the first position "M" in the sequence.

[0013] In other aspects, the present invention also provides a polynucleotide encoding the type II Cas protein or fusion protein described herein.

[0014] In other aspects, the present invention also provides an engineered, non-naturally occurring CRISPR-Cas system comprising: (i) the Cas protein or fusion protein described herein, or a polynucleotide encoding the Cas protein or the fusion protein; and (ii) a guide RNA (gRNA), or a polynucleotide sequence encoding the guide RNA; wherein the guide RNA comprises a spacer sequence complementary to a target nucleic acid and a Cas protein-binding region that interacts with the Cas protein, wherein the Cas protein-binding region comprises a tracrRNA sequence and a direct repeat (DR) sequence, the tracrRNA sequence and the direct repeat sequence hybridizing to form a double-stranded RNA (dsRNA) duplex.

[0015] In some implementations, the guide RNA further includes a connector sequence linking the tracrRNA sequence and the DR sequence to form an sgRNA scaffold.

[0016] In some embodiments, the tracrRNA comprises a sequence having at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with any of SEQ ID NO. 190.

[0017] In some embodiments, the direct repeat sequence comprises a sequence having at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with any of SEQ ID NO. 279.

[0018] In other aspects, this disclosure provides a pharmaceutical composition comprising the Cas protein, the fusion protein, the polynucleotide, and / or the CRISPR-Cas system described herein.

[0019] The Cas protein, fusion protein, or system described herein are used to identify a prototype spacer adjacent motif (PAM) having the following sequence: NRWAAW; preferably, the PAM has the following sequence: NAAAAA, NGAAAA, NATAAA, NGTAAA, NAAAGA, NAAAGT, NGAAGA, NGTAGA, NGTAGT; more preferably, the PAM has the following sequence: NAAAAA.

[0020] In some embodiments, the Cas protein is able to recognize prototype spacer adjacent motifs (PAMs) having the following sequences: AAAAA, GAAAA, ATAAA, AAAAT, GAAAA, GTAAA, GTAAT.

[0021] In other aspects, this disclosure provides the Cas protein, the fusion protein, the polynucleotide, the CRISPR-Cas system, or the pharmaceutical composition described herein for use in methods of treatment, therapy, prevention, diagnosis, or disease detection.

[0022] In other aspects, this disclosure provides a method for modifying or targeting a target DNA locus, comprising delivering the Cas protein, the fusion protein, the polynucleotide, the CRISPR-Cas system, or the pharmaceutical composition described herein to the locus.

[0023] In other aspects, this disclosure provides an isolated eukaryotic cell containing a modified target locus of interest. In some specific embodiments, the target locus of interest has been modified according to the methods described herein. Attached Figure Description

[0024] Figures 1A to 1D A structural model of the Cas protein is shown.

[0025] Figure 2 The sequence alignment of the BH region is shown.

[0026] Figure 3 The PAM bias of Cas protein in the HEK293 cell line was shown. Detailed Implementation

[0027] The following embodiments further illustrate this disclosure, but this disclosure is not limited thereto.

[0028] It should be noted that, as used herein and in the appended claims, the singular forms or terms “a,” “an,” “one,” and “the,” as well as similar terms used in this disclosure (especially in the context of the claims), should be construed as encompassing both the singular and plural forms, unless otherwise stated herein or the context clearly contradicts it. In some embodiments, the foregoing terms may reasonably be understood as “a” or “one or more.” Furthermore, unless the context requires otherwise, singular terms should include the plural, and plural terms should include the singular.

[0029] Unless otherwise stated, all singular / plural terms are intended to cover both active and past tense forms of the term and should be understood in the context of the text.

[0030] It should be noted that in this disclosure, particularly in the claims and / or paragraphs, terms such as “comprising,” “including,” and “including” may have the meanings given by U.S. patent law; for example, they may mean “comprising,” “including,” “including,” etc.; the term “group of” refers to a specific collection or group of elements, components, or features. It may include one or more specified elements, components, or features. For example, a group of A, B, or C may refer to a collection that includes any one or more of the specified elements A, B, or C. The claims cover the possibility of having any single element (A, B, or C) alone, any combination of two elements (A and B, A and C, or B and C), or all three elements together (A, B, and C). This phrase defines the invention by the variability within the specified options, allowing different combinations of the listed elements while still remaining within the scope of the claims.

[0031] When the nucleotide sequence in this invention is an RNA sequence and the letter “t” or “T” appears, it should be interpreted as “u” or “U”.

[0032] In the context of two or more nucleic acid or polypeptide sequences, the term “identity” means that two or more sequences or subsequences are identical, or have a specified percentage of identical amino acid residues or nucleotides when measured using a sequence alignment algorithm such as BLAST or BLAST 2.0 or FASTA (with the default parameters described below).

[0033] The term “exemplary” is used herein to mean that it is used as an example, instance, or illustration. Any aspect or design described herein as “exemplary” is not necessarily to be construed as being more preferred or advantageous than other aspects, implementations, or designs.

[0034] As used herein, the terms “optional” or “optionally” mean that the event, situation, or substituent described below may or may not occur, and the description includes both the circumstances under which the event or situation occurs and the circumstances under which it does not occur.

[0035] The use of “or” or “ / ” is inclusive, meaning “and / or” unless otherwise stated; or may be interpreted differently depending on the context. As used herein, the term “and / or”, such as phrases like “A and / or B”, is intended to include both A and B; A or B; A (alone); and B (alone). Similarly, as used herein, the term “and / or”, such as phrases like “A, B, and / or C”, is intended to cover each of the following implementations: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0036] The term “~” used in this document when referring to measurable values ​​(such as parameters, quantities, durations, etc.) is intended to cover variations and deviations relative to a specified value. It should be understood that the value referred to by the modifier “~” is itself specifically and preferably disclosed.

[0037] The terms “subject” and “patient” are used interchangeably herein and refer to vertebrates, preferably mammals, and more preferably humans. Mammals include, but are not limited to, rodents, apes, humans, farm animals, sporting animals, and pets. This also includes tissues, cells, and their progeny from biological entities obtained in vivo or cultured in vitro.

[0038] As presented in this disclosure, the term "encoding" refers to the property of a particular nucleotide sequence (such as cDNA or mRNA) to serve as a template for the synthesis of other macromolecules (such as defined amino acid sequences). Therefore, if transcription and translation of mRNA corresponding to a gene produces that protein in a cell or other biological system, then that sequence encodes that protein. Polynucleotides encoding proteins comprise all nucleotide sequences that are degenerate variants of each other and encode the same amino acid sequence or amino acid sequences with substantially similar form and function.

[0039] The terms “non-natural” and “engineered” are used interchangeably to indicate artificial involvement. When referring to nucleic acid molecules or peptides, these terms mean that the nucleic acid molecule or peptide is at least substantially free of at least one other component that is naturally associated with and present in nature. In all aspects and embodiments, whether or not these terms are included, it should be understood that, preferably, they may be optional, and therefore preferably included or not preferably included. Furthermore, the terms “non-natural” and “engineered” are used interchangeably and therefore may be used alone or in combination, and one may replace both when mentioned together. In particular, “engineered” is preferred to be used instead of “non-natural” or “non-natural and / or engineered” or “engineered, non-natural”.

[0040] As presented herein, the term "cleavage event" refers to a DNA break in the target nucleic acid produced by the type II Cas nuclease of the CRISPR system described herein. In some embodiments, the cleavage event is a double-stranded DNA break. In some embodiments, the cleavage event is a single-stranded DNA break.

[0041] As presented in this disclosure, the term "GEBx" followed by a numerical suffix and / or some letter notation is used as a general code to represent nucleic acids or proteins. It is important to note that the use of the same code for nucleic acids and proteins or their derivatives does not imply that the substances represented by these codes are the same. In other words, GEBx0417 may refer to a specific nucleic acid sequence in one context, and a different protein in another. Some embodiments can illustrate a direct correspondence between nucleic acids and proteins represented by the same or derived codes. Similarly, for example, "GEBx0417-HPT-V1" may also represent different nucleic acid sequences (e.g., DR, tracr, and / or scaffold). The code "GEBx" is used as an indexing system to organize and reference the different biomolecules described in this disclosure, and the meaning of the code will be understood according to the provided context.

[0042] Various embodiments are described below. It should be noted that the specific embodiments are not intended as an exhaustive description or a limitation on the broader aspects discussed herein. An aspect described in connection with a particular embodiment is not necessarily limited to that embodiment and may be implemented in conjunction with any other embodiment. Throughout this specification, references to “in some embodiments,” “in some embodiments,” “in some preferred embodiments,” “in some typical embodiments,” “in some specific embodiments,” or similar expressions mean that a particular feature, structure, or characteristic described in connection with that embodiment is included in at least one embodiment of this disclosure. Furthermore, particular features, structures, or characteristics may be combined in any suitable manner, as will be apparent to those skilled in the art based on this disclosure, in one or more embodiments. Moreover, although some aspects or embodiments described herein include other features not included in other aspects or embodiments, combinations of features from different embodiments are intended to fall within the scope of this disclosure. For example, in the appended claims, any claimed embodiment may be used in any combination.

[0043] All publications, published patent documents, and patent applications cited herein are incorporated by reference to the same extent that each individual publication, published patent document, or patent application is specifically and individually indicated to be incorporated by reference.

[0044] The present invention provides an engineered, non-naturally occurring type II Cas protein comprising an amino acid sequence selected from the following: (i) a sequence identical to any one of SEQ ID NO. 1-75; or (ii) a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 98%, or at least 99% sequence identity with SEQ ID NO. 1-75.

[0045] As presented in this disclosure, the term "at least n%" means a value, proportion, or degree that is equal to or greater than a specified percentage (n%). For example, "at least 70%" in this disclosure may include at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, and so on.

[0046] The present invention also provides an engineered, non-naturally occurring type II Cas protein comprising an amino acid sequence selected from the following: (i) a sequence identical to any one of SEQ ID NO. 1-75; or (ii) a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 98%, or at least 99% sequence identity with any one of SEQ ID NO. 1-75, except for the “M” at the first position of the sequence.

[0047] As presented in this invention, “M” represents the starting amino acid methionine, which is commonly the starting point for protein synthesis in many proteins. Naturally occurring Cas proteins typically begin with methionine as the first amino acid in their sequence. However, when scientists engineer these proteins (e.g., by fusing them with nuclear localization signals (NLS) or other domains), this starting amino acid “M” may be replaced or altered to introduce new functions or properties.

[0048] This disclosure focuses particularly on the flexibility and functionality of engineered Cas proteins. Aside from intentional modification of the first starting amino acid, methionine (M), the engineered Cas proteins exhibit significant sequence identity—ranging from 70% to 100%—when compared to a reference sequence. This strategic alteration not only aligns with our goal of customizing protein properties but also ensures that the inherent core or intended function of the Cas protein is preserved. By manipulating the starting amino acid without affecting overall sequence similarity, specific properties are enhanced, such as improved cellular localization or the introduction of other advantageous features, while maintaining the essential characteristics that make Cas proteins indispensable tools in genome manipulation.

[0049] As presented in this disclosure, the terms "Cas protein," "CRISPR-related protein," or other similar terms refer to a class of CRISPR-related proteins that are components of the CRISPR-Cas system. These proteins may possess intrinsic nuclease activity, enabling them to cleave double-stranded DNA or RNA molecules in a sequence-specific manner, guided by complementary RNA molecules, such as Cas9 and Cas12, which have been widely used in genome editing applications. Furthermore, some Cas proteins can be engineered to retain only one of the two active sites required for double-strand cleavage, thereby generating nickase activity that allows them to introduce single-strand breaks into target nucleic acid sequences for controlled cleavage events. Additionally, some Cas proteins can be modified to lack any intrinsic nuclease activity, referred to as inactivated Cas or nuclease-free Cas proteins. Despite lacking enzymatic function, these inactivated Cas proteins retain the ability to specifically bind to target nucleic acid sequences and are often used in conjunction with chaperones (such as other effector or functional domains) for applications such as gene regulation, epigenome editing, and as components of advanced imaging systems. In some embodiments, the Cas protein may be a fissile form. In some embodiments, the Cas protein may also be an inducible Cas protein. In some embodiments, the type II Cas protein may be part of a self-inactivation system (SIN). In some embodiments, the type II Cas nuclease may also be part of a co-activation system (SAM).

[0050] One of the key elements of the CRISPR-Cas system's modification process is the prototype spacer adjacent motif (PAM), a short DNA sequence immediately adjacent to the target DNA sequence. PAM sequences are crucial for the binding and cleavage activity of Cas proteins, ensuring that only the intended DNA sequence is targeted for editing. The ability of Cas proteins to recognize these specific PAM sequences is attributed to their unique structural features and binding interactions with DNA. Each PAM sequence provides a unique motif that Cas proteins can recognize, ensuring accurate and efficient targeting. For example, the AAAAA sequence provides a specific nucleotide sequence that Cas proteins can bind with high affinity. These diverse PAM sequences expand the potential applications of the CRISPR-Cas system. It enables researchers and scientists to target a wider range of DNA sequences for editing, enhancing the versatility and effectiveness of this powerful gene-editing tool. Furthermore, understanding these PAM sequences helps in developing more advanced Cas proteins with stronger targeting capabilities, further advancing the field of gene editing and its potential benefits for research, medicine, and biotechnology. Cas proteins, as disclosed in this invention, exhibit a unique ability to recognize a variety of PAM sequences. These sequences include NRWAAA, NRDAAAY, NRRAVW, NRWAAW, NRWAAA, NRAARW, or NNNAAAG. The specific recognition of these Cas proteins allows for greater flexibility in selecting target DNA sequences for editing.

[0051] As presented in this invention, in the provided PAM sequence: N: represents any of the four standard DNA nucleotides: adenine (A), thymine (T), cytosine (C), or guanine (G). This code facilitates the inclusion of any nucleotide at a given position without individual specification; R: specifically refers to a purine nucleotide, namely adenine (A) or guanine (G). Purines are essential in DNA structure and function, and this code simplifies the process of incorporating these larger nucleotides into the PAM sequence; H: represents any nucleotide other than guanine (G). Therefore, it can represent adenine (A), thymine (T), or cytosine (C). This code can be used to exclude guanine (G) at a specific position because its larger size may affect structural or functional aspects of the sequence. Y: represents a pyrimidine nucleotide, namely thymine (T) or cytosine (C). Pyrimidines are commonly found in specific regions of DNA and RNA, and this code facilitates their inclusion without specifying the exact nucleotide. W: represents a weak base, which can be adenine (A) or thymine (T). This distinction is biochemically relevant because adenine and thymine have similar properties in certain situations (e.g., hydrogen bonding). V: Reflects any nucleotide other than thymine (T), thus including adenine (A), cytosine (C), or guanine (G). This code is helpful when thymine is not preferred or is not needed due to its unique chemical properties among pyrimidines.

[0052] In some embodiments, (1) the Cas protein has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO. 28, and is capable of recognizing a prototype spacer adjacent motif (PAM) having the following sequence: NRWAAA; (2) the Cas protein has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO. 29, and is capable of recognizing a prototype spacer adjacent motif (PAM) having the following sequence: NRDAAAY; (3) the Cas protein has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO. 29, and is capable of recognizing a prototype spacer adjacent motif (PAM) having the following sequence: NRDAAAY; The amino acid sequence of NO.30 has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 98%, at least 99%, or 100% sequence identity and is able to identify a prototype spacer adjacent motif (PAM) having the following sequence: NRRAVW; (4) The Cas protein has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO.32 and is able to identify a prototype spacer adjacent motif (PAM) having the following sequence: NRWAAW; (5) The Cas protein has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO.32 and is able to identify a prototype spacer adjacent motif (PAM) having the following sequence: NRWAAW; The amino acid sequence of NO.35 has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 98%, at least 99% or 100% sequence identity and is able to identify a prototype spacer adjacent motif (PAM) with the following sequence: NRWAAA; (6) The Cas protein has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 98%, at least 99% or 100% sequence identity with the amino acid sequence of SEQ ID NO.37 and is able to identify a prototype spacer adjacent motif (PAM) with the following sequence: NRAARW; or (7) The Cas protein has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 98%, at least 99% or 100% sequence identity with the amino acid sequence of SEQ ID NO.68 and is able to identify a prototype spacer adjacent motif (PAM) with the following sequence: NNAAAG.

[0053] As presented in this invention, in this context, the term "recognition" or "identification" refers to the ability of a Cas protein to form a functional complex with sgRNA at a DNA target site, the sgRNA hybridizing to the DNA target site (i.e., the spacer sequence of the sgRNA hybridizing to it) and flanked by a PAM sequence, and the Cas protein being able to perform its native function, i.e., DNA cleavage or DNA binding. It should be noted in this context that such DNA cleavage excludes the type II Cas protein being a catalytically inactivated type II Cas nuclease. For example, in the case of an inactivated type II Cas nuclease (e.g., a dead type II Cas nuclease), a complex can still form between the type II Cas nuclease, sgRNA, and homologous target if the desired PAM sequence is present, but such a complex will not lead to DNA cleavage.

[0054] In another embodiment, the present invention also provides a fusion protein comprising the engineered, non-naturally occurring type II Cas protein described herein, and a fusion chaperone. In such cases, an active Cas nuclease, a nicking enzyme, or an inactivated Cas may also be incorporated as part of the fusion protein.

[0055] As presented in this invention, the term "fusion chaperone" refers to a protein, peptide, or polypeptide that is genetically or chemically linked to a target protein (such as the engineered type II Cas protein described herein). Fusion chaperones can confer additional functions, properties, or regulatory capabilities to the target protein, such as enhancing stability, altering subcellular localization, improving solubility, providing detection tags (e.g., fluorescent proteins), or introducing enzymatic activity. In some embodiments, the fusion chaperone sequence is selected from: nuclear localization signal sequences, nuclear export signal sequences, cell-penetrating peptide sequences, affinity tag sequences, deaminase sequences, reverse transcriptase sequences, recombinase sequences, methyltransferase sequences, methylylase sequences, acetyltransferase sequences, transcription activator sequences, transcription repressor domain sequences, cryptochrome sequences, photoinducible / controllable domain sequences, and chemically inducible / controllable domain sequences.

[0056] In some embodiments, the fusion protein includes one or more nuclear localization signals (NLS). The NLS may be located at the ends or other parts of the peptide. The NLS located at the ends or other parts of the Cas sequence may be the same or different. In some embodiments, the N-terminal NLS and the C-terminal NLS are the same. In some embodiments, the N-terminal NLS and the C-terminal NLS are different. In some embodiments, the N-terminus of the Cas sequence contains one NLS, and the C-terminus of the Cas sequence contains one NLS. The amino acid sequences of the NLS are fused to the N-terminus and / or C-terminus of the Cas9 amino acid sequence, respectively. The NLS may be an SV40 (monkey virus 40) NLS, a c-Myc NLS, or other suitable monotypic NLS. The NLS may be fused to the N-terminus and / or C-terminus of the Cas protein. In some embodiments, affinity tags (such as GST, FLAG, or hexahistine sequences) are used to purify the Cas protein by affinity chromatography.

[0057] In some embodiments, the fusion protein comprises an amino acid sequence selected from: (i) a sequence identical to any one of SEQ ID NO. 432-506; or (ii) a sequence having at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with any one of SEQ ID NO. 432-506. Optionally, the fusion protein comprises an amino acid sequence selected from: (i) a sequence identical to any one of SEQ ID NO. 432-506; or (ii) a sequence having at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with any one of SEQ ID NO. 432-506, except for the “M” at the first position of the sequence.

[0058] In another aspect, the present invention also provides a polynucleotide encoding the type II Cas protein or fusion protein disclosed herein.

[0059] As presented in this invention, the term "polynucleotide" refers to a polymeric form of nucleotides of any length, whether deoxyribonucleotides or ribonucleotides, or the like. Polynucleotides can have any three-dimensional structure and can perform any known or unknown function. Therefore, the term includes, but is not limited to, single-stranded, double-stranded, or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or polymers containing purine and pyrimidine bases or other natural, chemically or biochemically modified, non-natural, or derivatized nucleotide bases.

[0060] In some embodiments, multiple nucleotides encoding more than one portion of the type II Cas protein or fusion protein expressed herein may be operatively linked to each other and to associated regulatory sequences (such as promoters, enhancers, and termination regions). For example, a functional link may exist between the regulatory sequence and a foreign nucleic acid sequence, resulting in the expression of the latter. In another embodiment, the first nucleic acid sequence may be operatively linked to the second nucleic acid sequence when the first nucleic acid sequence is functionally related to the second nucleic acid sequence. For example, if the promoter affects the transcription or expression of the coding sequence, the promoter is operatively linked to the coding sequence. Typically, the operatively linked DNA sequences are contiguous and, where necessary or advantageous, the coding regions are linked into the same reading frame. In some embodiments, the promoter is a constitutive promoter, a tissue-specific promoter, or an inducible promoter. In some embodiments, the term may also include all introns and other DNA sequences spliced ​​from mRNA transcripts, as well as variants resulting from alternative splicing sites. These nucleic acid sequences may be DNA strand sequences transcribed into RNA or RNA sequences translated into proteins. The nucleic acid sequences include full-length nucleic acid sequences and non-full-length sequences derived from full-length proteins. The sequence may also include degenerate codons of the natural sequence or sequences that may be introduced to provide codon preference in a particular cell type.

[0061] In some embodiments, the polynucleotide is operatively linked to a promoter and is present in a vector; optionally, the vector is selected from: retroviral vectors, lentiviral vectors, bacteriophage vectors, adenovirus vectors, adeno-associated virus vectors, herpes simplex virus vectors, and plasmid vectors.

[0062] In some embodiments, the polynucleotide is a ribonucleotide sequence or a deoxyribonucleotide sequence, or an analogue thereof; optionally, the polynucleotide is codon-optimized for expression in cells of interest. In some embodiments, the polynucleotide is codon-optimized for expression in eukaryotic cells. In some embodiments, the eukaryotic cells are selected from: plant cells, fungal cells, unicellular eukaryotes, mammalian cells, reptile cells, insect cells, avian cells, fish cells, parasitic cells, arthropod cells, invertebrate cells, vertebrate cells, rodent cells, mouse cells, rat cells, primate cells, non-human primate cells, and human cells. In some embodiments, the cells are mammalian cells, preferably human cells. In some embodiments, the cells are mammalian cells, preferably human cells.

[0063] In some embodiments, the polynucleotide is mRNA and also includes a 5' cap sequence and / or a poly-A tail sequence. In some embodiments of the invention, the mRNA used may be modified to enhance its functional properties and stability. Specifically, in some embodiments, the modification process involves replacing uridine (denoted by the letter "U") with N1-methylpseudouridine or pseudouridine. This substitution aims to improve the mRNA's resistance to ribonuclease degradation, potentially increasing its intracellular half-life and translation efficiency. Incorporating N1-methylpseudouridine or pseudouridine into the mRNA structure can also have a positive impact on the immune response profile, as these modifications have been shown to reduce the immunogenicity of the mRNA molecule compared to its unmodified counterpart. This is particularly important for the development of mRNA-based therapeutics and vaccines, where minimizing adverse immune responses is crucial.

[0064] In another aspect, the present invention also provides an engineered, non-naturally occurring CRISPR-Cas system comprising: (i) the Cas protein or fusion protein described in this disclosure, or a polynucleotide encoding the Cas protein or the fusion protein; and (ii) a guide RNA (gRNA), or a polynucleotide sequence encoding the guide RNA; wherein the guide RNA comprises a spacer sequence complementary to a target nucleic acid and a Cas protein binding segment that interacts with the Cas protein, wherein the Cas protein binding segment comprises a tracrRNA sequence and a direct repeat (DR) sequence, the tracrRNA sequence and the direct repeat sequence hybridizing to form a double-stranded RNA (dsRNA) duplex.

[0065] As presented in this invention, the term "complementarity" describes the ability of two nucleic acid strands to pair with each other through their bases. This complementarity can occur through perfect base pairing, where each base on one strand forms a specific hydrogen bond pairing with a complementary base on its complementary strand according to the standard Watson-Crick base pairing rules: adenine (A) pairs with thymine (T) or uracil (U), and cytosine (C) pairs with guanine (G). This perfect base pairing allows for precise recognition and binding between the two strands. Furthermore, complementarity can also involve imperfect base pairing, including mismatches, insertions, or deletions that may result in non-standard base pairings or reduced affinity between the strands. Despite these imperfections, the strands maintain sufficient complementarity to interact, although the specificity or stability in the duplex may be reduced.

[0066] As presented in this invention, direct repeat (DR) sequences are derived from short repetitive DNA sequence elements within a CRISPR array. These sequences are typically distributed among spacer sequences derived from foreign genetic material (such as bacteriophage or plasmid DNA). Posttranscriptionally, the DR sequence is part of the pre-crRNA transcript and is processed into mature CRISPR RNA (crRNA). The DR sequence can be wild-type or non-wild-type, such as artificially modified or mutated sequences. The DR sequence in the crRNA, as a key component, pairs with a complementary sequence in the trans-activating CRISPR RNA (tracrRNA) to form a double-stranded RNA (dsRNA) duplex. This duplex facilitates Cas protein binding and is essential for the function of the guide RNA (gRNA) complex in the CRISPR / Cas system. In the context of this invention, the DR sequence comprises the portion of the gRNA that hybridizes with the tracrRNA sequence to form the dsRNA duplex, thereby enabling the formation of the Cas protein-binding segment. In this invention, "hybridization to form a double-stranded RNA (dsRNA) duplex" or similar expressions refer to the process by which a direct repeat (DR) sequence of CRISPR RNA (crRNA) pairs with a complementary sequence in trans-activated CRISPR RNA (tracrRNA) to form a stable double-stranded RNA structure. This dsRNA duplex is a key component of the guide RNA (gRNA) complex, which facilitates the binding of the Cas protein. Specifically, the DR sequence in the crRNA and the complementary sequence in the tracrRNA undergo base pairing, forming hydrogen bonds between their complementary bases, resulting in the formation of the dsRNA duplex. This duplex is crucial for the function of the Cas protein binding region, which contains the tracrRNA sequence and the DR sequence that hybridizes to form the dsRNA duplex.

[0067] As presented in this invention, the term "target nucleic acid" refers to a specific nucleic acid substrate that comprises a nucleic acid sequence that is wholly or partially complementary to the spacer sequence in an RNA guide. In some embodiments, the target nucleic acid comprises a gene or a sequence within a gene. In some embodiments, the target nucleic acid comprises a non-coding region (e.g., a promoter). In one specific embodiment, the target nucleic acid is single-stranded. In one specific embodiment, the target nucleic acid is double-stranded. The terms "target nucleic acid" or "target sequence" are to be understood in the context of this disclosure.

[0068] In some embodiments, the guide RNA is a dual guide RNA. In some embodiments, the guide RNA is a single guide RNA. In such embodiments, the guide RNA further comprises an adapter sequence linking the tracrRNA sequence and the DR sequence. In some typical embodiments, the adapter comprises a short GAAA sequence. In some embodiments, the adapter serves as an artificial loop. In some embodiments, the sgRNA comprises the following arrangement: a) a spacer sequence capable of hybridizing to the sequence of the target nucleic acid to be manipulated; b) a DR sequence; c) an adapter sequence; and d) a tracrRNA sequence. The spacer sequence, DR sequence, adapter sequence, and tracrRNA sequence are tandemly arranged in a 5' to 3' or 3' to 5' orientation. In some embodiments, the sgRNA scaffold comprises a sequence encoded by a sequence having at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with any one of SEQ ID NO. 325-416.

[0069] In some embodiments, the spacer sequence hybridizes with one or more nucleic acids in prokaryotic or eukaryotic cells. In some embodiments, the eukaryotic cells are selected from: plant cells, fungal cells, unicellular eukaryotes, mammalian cells, reptile cells, insect cells, avian cells, fish cells, parasitic cells, arthropod cells, invertebrate cells, vertebrate cells, rodent cells, mouse cells, rat cells, primate cells, non-human primate cells, and human cells. In some embodiments, the eukaryotic cells comprise mammalian cells. In some embodiments, the mammalian cells comprise human cells. In some embodiments, the eukaryotic cells comprise plant cells.

[0070] In some embodiments, the system further comprises a donor template nucleic acid. As presented herein, the term "donor template nucleic acid" refers to a nucleic acid molecule that can be used by one or more cellular proteins to alter the structure of the target nucleic acid after the Cas protein described herein has altered it. In some embodiments, the donor template nucleic acid is a double-stranded nucleic acid. In some embodiments, the donor template nucleic acid is a single-stranded nucleic acid. In some embodiments, the donor template nucleic acid is linear. In some embodiments, the donor template nucleic acid is circular (e.g., a plasmid). In some embodiments, the donor template nucleic acid is a foreign nucleic acid molecule. In some embodiments, the donor template nucleic acid is an endogenous nucleic acid molecule (e.g., a chromosome). In some embodiments, the donor template nucleic acid is DNA or RNA or a DNA-RNA hybrid.

[0071] In another aspect, the present invention also provides an engineered carrier comprising the polynucleotides described herein.

[0072] As presented in this disclosure, a "vector" is a tool that allows or facilitates the transfer of an entity from one environment to another. It is a replicon, such as a plasmid, bacteriophage, or granule, that inserts another DNA segment into it to cause replication of the inserted segment. Typically, a vector is capable of replication when associated with appropriate control elements. Generally, the term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it is attached. Vectors include, but are not limited to, single-stranded, double-stranded, or partially double-stranded nucleic acid molecules; nucleic acid molecules containing one or more free ends, or without free ends (e.g., circular); nucleic acid molecules containing DNA, RNA, or both; and other polynucleotide variants known in the art. One type of vector is a "plasmid," which refers to a circular double-stranded DNA loop into which an additional DNA segment can be inserted using standard molecular cloning techniques. Another type of vector is a viral vector in which a virally derived DNA or RNA sequence is present in the vector to package a virus (e.g., retrovirus, replication-defective retrovirus, adenovirus, replication-defective adenovirus, and adeno-associated virus (AAV)). Viral vectors also include polynucleotides carried by the virus for transfecting host cells. Some vectors are capable of autonomous replication in the host cells to which they are introduced (e.g., bacterial vectors with bacterial origins of replication and free-living mammalian vectors). Other vectors (e.g., non-free-living mammalian vectors) integrate into the host cell's genome upon introduction and thus replicate along with the host genome. Furthermore, some vectors are capable of directing the expression of genes operatively linked to them. Such vectors are referred to herein as "expression vectors." Common expression vectors useful in recombinant DNA technology are typically in plasmid form. Recombinant expression vectors may contain the nucleic acids of the present invention suitable for expression in host cells, meaning that the recombinant expression vector contains one or more regulatory elements that are selected depending on the host cell used for expression and are operatively linked to the nucleic acid sequence to be expressed. Within a recombinant expression vector, "operatively linked" is intended to mean that the nucleotide sequence of interest is linked to a regulatory element in a manner that allows the expression of that nucleotide sequence (e.g., in an in vitro transcription / translation system or in the host cell when the vector is introduced). In some embodiments, the vector is an expression vector. In some embodiments, the vector is an inducible, conditional, or constitutive expression vector. In some embodiments, the polynucleotide encoding a Cas protein and the polynucleotide encoding a guide RNA are on the same vector or on different vectors.

[0073] In another aspect, the present invention also provides a vector system comprising one or more polynucleotides described herein and one or more polynucleotides encoding a guide RNA; wherein the guide RNA comprises a spacer sequence complementary to a target nucleic acid and a Cas protein-binding region that interacts with the Cas protein, wherein the Cas protein-binding region comprises a tracrRNA sequence and a direct repeat (DR) sequence, the tracrRNA sequence and the DR sequence hybridizing to form a double-stranded RNA (dsRNA) duplex. In some embodiments, the polynucleotide encoding the Cas protein and the polynucleotide encoding the guide RNA are on the same vector or on different vectors.

[0074] The present invention also provides a pharmaceutical composition comprising: the Cas protein described in this disclosure, the polynucleotide described in this disclosure, the CRISPR-Cas system described in this disclosure, the vector described in this disclosure, the vector system described in this disclosure, or the cell described in this disclosure.

[0075] As presented in this disclosure, the term "pharmaceutical composition" refers to a formulation intended for pharmaceutical use. In some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable excipient. In some embodiments, the pharmaceutical composition may comprise additional therapeutic agents. In some embodiments, the pharmaceutical composition is prepared according to standard procedures for administration to a subject, such as a human patient, via intravenous, intramuscular, intradermal, intra-articular, intralesional, intraperitoneal, intracardiac, intrathecal, intraventricular, epidural, local, subconjunctival, intrastromal, periglottic, intravitreal, retroscleral, transscleral, suprachoroidal, retroglottic, subretinal, subfascial, nasal, pressurized inhalation, oral, subcutaneous, or local routes. For example, injectable compositions may be provided as a sterile isotonic aqueous solution. If desired, the pharmaceutical composition may also comprise a solubilizer and a local anesthetic, such as lidocaine, to minimize injection site discomfort. Typically, the components are provided individually or in combination in unit doses in sealed containers, for example, as lyophilized powders or anhydrous concentrated solutions, with the amount of active agent indicated on the container. When the pharmaceutical composition is used for infusion, it can be combined with an infusion bottle containing sterile pharmaceutical-grade water or saline. When the pharmaceutical composition is used for injection, it may include sterile water or saline for injection to allow mixing of the components prior to administration. Furthermore, humectants, colorants, release agents, coating agents, sweeteners, flavoring agents, aromatizers, preservatives, and antioxidants may be added to the formulation as needed.

[0076] In some embodiments, the pharmaceutical composition further comprises a delivery system selected from: AAV (adeno-associated virus), adenovirus, retrovirus, HSV (herpes simplex virus), gamma retrovirus, LV (lentivirus), eCIS (extracellular contraction injection system), eVLPs (engineered virus-like particles), VLPs (virus-like particles), liposomes, plasmids, LNPs (lipid nanoparticles), exosomes, microvesicles, nucleic acid nanoassemblies, gene guns, and / or implantable devices.

[0077] The present invention also provides an engineered, non-naturally occurring cell comprising: the Cas protein or fusion protein described in this disclosure, the polynucleotide described in this disclosure, the CRISPR-Cas system described in this disclosure, the vector described in this disclosure, or the vector system described in this disclosure.

[0078] This invention also provides a cell modified using the Cas protein or fusion protein described in this disclosure, the polynucleotide described in this disclosure, the CRISPR-Cas system described in this disclosure, the vector described in this disclosure, or the vector system described in this disclosure. In some embodiments, the cell is a eukaryotic cell or a prokaryotic cell. In some embodiments, the eukaryotic cell is selected from: plant cells, fungal cells, unicellular eukaryotes, mammalian cells, reptile cells, insect cells, bird cells, fish cells, parasite cells, arthropod cells, invertebrate cells, vertebrate cells, rodent cells, mouse cells, rat cells, primate cells, non-human primate cells, and human cells. In some embodiments, the cell is a mammalian cell, a human cell, or a plant cell.

[0079] In some embodiments, this disclosure provides an isolated eukaryotic cell containing a modified target locus of interest, wherein the target locus of interest has been modified according to the method of the present invention or using the composition of the present invention or the system of the present invention.

[0080] The present invention also provides a kit comprising: the Cas protein or fusion protein described in this disclosure, the polynucleotide described in this disclosure, the CRISPR-Cas system described in this disclosure, the vector described in this disclosure, the vector system described in this disclosure, or the cell described in this disclosure.

[0081] The kits described in this disclosure may comprise one or more containers containing components necessary for carrying out the methods of this disclosure, and may optionally include instructions for use. Any of the kits may additionally contain auxiliary components required for performing the editing methods. Each component in the kit may be provided, where applicable, in liquid form (e.g., dissolved in solution) or solid form (e.g., lyophilized powder). In specific embodiments, some components may be reconstituted or otherwise processed (e.g., to an active state) upon addition of a suitable solvent or other substance (such as water or buffer), which may or may not be provided with the kit. In some embodiments, the kit may also contain other suitable excipients, such as buffers or reagents for facilitating the application of the kit. Preferably, the kit is applicable to a variety of applications, such as medical applications including therapeutics and diagnostics, research, etc. Therefore, the type II Cas nuclease and kit of the present invention can be used to prepare therapeutic drugs and / or reagents for research purposes.

[0082] The present invention also provides the use of the Cas protein, fusion protein, polynucleotide, CRISPR-Cas system, vector, vector system, cell, kit or pharmaceutical composition described herein in the treatment, prevention, diagnosis or detection of diseases.

[0083] The present invention also provides a method for modifying or targeting a target DNA locus, wherein the method includes delivering to the locus: the Cas protein or fusion protein described in this disclosure, the polynucleotide described in this disclosure, the CRISPR-Cas system described in this disclosure, the vector described in this disclosure, the vector system described in this disclosure, the kit described in this disclosure, or the pharmaceutical composition described in this disclosure.

[0084] In some embodiments, this disclosure also provides a method for targeting and cleaving target DNA, the method comprising: contacting the target DNA with the Cas protein or fusion protein described in this disclosure, the polynucleotide described in this disclosure, the CRISPR-Cas system described in this disclosure, the vector described in this disclosure, the vector system described in this disclosure, the kit described in this disclosure, or the pharmaceutical composition described in this disclosure.

[0085] In some embodiments, the modification or targeting of the target locus includes inducing DNA strand breaks. In some embodiments, the modification or targeting of the target locus includes inducing DNA double-strand breaks or DNA single-strand breaks. In some embodiments, the modification or targeting of the target locus includes altering the gene expression of one or more genes. In some embodiments, the modification or targeting of the target locus includes epigenetic modification of the target DNA locus. In some embodiments, the method is a method of modifying a cell, cell line, or organism by manipulating one or more target sequences at a locus of interest in the genome.

[0086] In some embodiments, cleaving the target DNA or target sequence results in the formation of an insertion, deletion, or inserted nucleotide sequence. In some embodiments, cleaving the target DNA or target nucleotide includes cleaving the target DNA or target sequence at two sites and resulting in a sequence deletion or inversion between the two sites. In some embodiments, the target DNA is double-stranded DNA or single-stranded DNA, or a DNA-RNA hybrid.

[0087] In some embodiments, the modification or targeting of the target locus includes inducing DNA strand breaks, altering the gene expression of one or more genes, or epigenetic modification of the target DNA locus; optionally, the DNA strand breaks include DNA double-strand breaks or DNA single-strand breaks.

[0088] In some implementations, the method is performed in vitro or in vivo.

[0089] This disclosure also provides an isolated eukaryotic cell containing a modified target locus of interest, wherein the target locus of interest has been modified according to the methods described in this disclosure, or by using the systems described in this disclosure, or by using the Cas protein or fusion protein described in this disclosure, or by using the polynucleotide described in this disclosure, or by using the CRISPR-Cas system described in this disclosure, or by using the vector described in this disclosure, or by using the vector system described in this disclosure, or by using the kit described in this disclosure, or by using the pharmaceutical composition described in this disclosure.

[0090] The present invention also provides a system for detecting the presence of a nucleic acid target sequence in an in vitro sample, comprising: a) the Cas protein described in this disclosure; b) at least one guide polynucleotide comprising a guide sequence capable of binding to the target sequence and designed to form a complex with the Cas protein; and c) a nucleic acid-based masking construct comprising a non-target sequence; wherein the Cas protein exhibits side-branching activity of RNA and / or ssDNA and cleaves the non-target sequence of the nucleic acid-based masking construct activated by the target sequence.

[0091] The present invention also provides a method for detecting target nucleic acids in a sample, comprising: reacting one or more samples with a) the Cas protein described herein; b) at least one guide polynucleotide comprising a guide sequence designed to have a degree of complementarity with a target sequence and designed to form a complex with the Cas protein; and c) a nucleic acid-based masking construct comprising non-target sequence contact; wherein the Cas protein exhibits side-branching activity of RNA and / or ssDNA and cleaves the non-target sequence of the nucleic acid-based masking construct activated by the target sequence; and detecting a signal from the non-target sequence cleavage, thereby detecting the one or more target sequences in the sample.

[0092] Table 1. Exemplary amino acid sequences of the Cas protein in this disclosure.

[0093]

[0094] Table 2. Exemplary nucleic acid sequences encoding Cas proteins in this disclosure.

[0095]

[0096] Table 3. Exemplary tracrRNA sequences of the Cas protein in this disclosure.

[0097]

[0098]

[0099] Table 4. Exemplary sequences of DRs described in this disclosure

[0100]

[0101] Table 5. Exemplary sequences encoding Cas protein sgRNA scaffolds in this disclosure

[0102]

[0103]

[0104] Further numbering of implementation plans

[0105] 1. An engineered, non-naturally occurring type II Cas protein comprising an amino acid sequence selected from: (i) a sequence identical to any one of SEQ ID NO. 1-75 in Table 1; or (ii) a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 98%, or at least 99% sequence identity to any one of SEQ ID NO. 1-75 in Table 1.

[0106] 2. An engineered, non-naturally occurring type II Cas protein comprising an amino acid sequence selected from: (i) a sequence identical to any one of SEQ ID NO. 1-75 in Table 1; or (ii) a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 98%, or at least 99% sequence identity with any one of SEQ ID NO. 1-75 in Table 1, except for the “M” at the first position of the sequence.

[0107] 3. The Cas protein according to embodiment 1 or 2, wherein the Cas protein is capable of recognizing a prototype spacer adjacent motif (PAM) having any of the following sequences: NRWAAA, NRDAAAY, NRRAVW, NRWAAW, NRWAAA, NRAARW, or NNNAAAG.

[0108] 4. The Cas protein according to any one of embodiments 1-3, wherein (1) the Cas protein has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 98%, at least 99% or 100% sequence identity with the amino acid sequence of SEQ ID NO. 28, and is capable of recognizing a prototype spacer adjacent motif (PAM) having the following sequence: NRWAAA; (2) the Cas protein has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 98%, at least 99% or 100% sequence identity with the amino acid sequence of SEQ ID NO. 29, and is capable of recognizing a prototype spacer adjacent motif (PAM) having the following sequence: NRDAAAY; (3) the Cas protein has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 98%, at least 99% or 100% sequence identity with the amino acid sequence of SEQ ID NO. 29, and is capable of recognizing a prototype spacer adjacent motif (PAM) having the following sequence: NRDAAAY; The amino acid sequence of ID NO. 30 has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 98%, at least 99%, or 100% sequence identity and is able to identify a prototype spacer adjacent motif (PAM) having the following sequence: NRRAVW; (4) The Cas protein has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO. 32 and is able to identify a prototype spacer adjacent motif (PAM) having the following sequence: NRWAAW; (5) The Cas protein has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO. 32 and is able to identify a prototype spacer adjacent motif (PAM) having the following sequence: NRWAAW; The amino acid sequence of ID NO. 35 has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 98%, at least 99% or 100% sequence identity and is able to identify a prototype spacer adjacent motif (PAM) having the following sequence: NRWAAA; (6) The Cas protein has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 98%, at least 99% or 100% sequence identity with the amino acid sequence of SEQ ID NO. 37 and is able to identify a prototype spacer adjacent motif (PAM) having the following sequence: NRAARW; or (7) The Cas protein has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 98%, at least 99% or 100% sequence identity with the amino acid sequence of SEQ ID NO. 68 and is able to identify a prototype spacer adjacent motif (PAM) having the following sequence: NNAAAG.

[0109] 4. The Cas protein according to any one of embodiments 1-3, wherein the Cas protein is a nicking enzyme or an inactivated Cas protein.

[0110] 5. A fusion protein comprising an engineered, non-naturally occurring type II Cas protein according to any one of embodiments 1-4, wherein the fusion protein further comprises a fusion chaperone sequence selected from: nuclear localization signal sequence, nuclear export signal sequence, cell penetration peptide sequence, affinity tag sequence, deaminase sequence, reverse transcriptase sequence, recombinase sequence, methyltransferase sequence, methyltransferase sequence, acetyltransferase sequence, acetyltransferase sequence, transcription activator sequence, transcription repressor domain sequence, cryptochrome sequence, photoinducible / controllable domain sequence, and chemically inducible / controllable domain sequence.

[0111] 6. The fusion protein according to embodiment 5, wherein the fusion protein comprises an amino acid sequence selected from: (i) a sequence identical to any one of SEQ ID NO. 432-506; or (ii) a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 98%, or at least 99% sequence identity with any one of SEQ ID NO. 432-506; optionally, the fusion protein comprises an amino acid sequence selected from: (i) a sequence identical to any one of SEQ ID NO. 432-506; or (ii) a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 98%, or at least 99% sequence identity with any one of SEQ ID NO. 432-506, except for the "M" at the first position of the sequence.

[0112] 7. A polynucleotide encoding a type II Cas protein according to any one of embodiments 1-4 or a fusion protein according to any one of embodiments 5-6.

[0113] 8. The polynucleotide according to embodiment 7, wherein the polynucleotide is a ribonucleotide sequence or a deoxyribonucleotide sequence, or an analogue thereof; optionally, the polynucleotide is codon-optimized for expression in cells of interest; preferably, the polynucleotide is mRNA, wherein the mRNA further comprises a 5' cap sequence and a poly-A tail sequence.

[0114] The polynucleotide according to embodiment 7 or 8, wherein the polynucleotide is codon-optimized for expression in eukaryotic cells; optionally, the eukaryotic cells are selected from: plant cells, fungal cells, single-celled eukaryotes, mammalian cells, reptile cells, insect cells, bird cells, fish cells, parasite cells, arthropod cells, invertebrate cells, vertebrate cells, rodent cells, mouse cells, rat cells, primate cells, non-human primate cells, and human cells.

[0115] 10. The polynucleotide according to embodiment 7 or 8, wherein the polynucleotide comprises a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 98%, at least 99%, or 100% identity with any one of SEQ ID NOs 76-150, 510-584 in Tables 2 and 8.

[0116] 11. An engineered, non-naturally occurring CRISPR-Cas system comprising: (i) a Cas protein according to any one of embodiments 1-4, a fusion protein according to any one of embodiments 5-6, or a polynucleotide encoding said Cas protein or said fusion protein; and (ii) a guide RNA (gRNA), or a polynucleotide sequence encoding said guide RNA; wherein said guide RNA comprises a spacer sequence complementary to a target nucleic acid and a Cas protein binding segment that interacts with said Cas protein, wherein said Cas protein binding segment comprises a tracrRNA sequence and a direct repeat (DR) sequence, said tracrRNA sequence and direct repeat sequence hybridizing to form a double-stranded RNA (dsRNA) duplex.

[0117] 12. The system according to embodiment 11, wherein the guide RNA further comprises a connector sequence connecting the tracrRNA sequence and the DR sequence to form an sgRNA scaffold.

[0118] 13. The system according to embodiment 11 or 12, wherein the spacer sequence hybridizes with a target sequence in a prokaryotic or eukaryotic cell; optionally, the eukaryotic cell is selected from: plant cells, fungal cells, unicellular eukaryotes, mammalian cells, reptile cells, insect cells, bird cells, fish cells, parasite cells, arthropod cells, invertebrate cells, vertebrate cells, rodent cells, mouse cells, rat cells, primate cells, non-human primate cells, and human cells.

[0119] 14. The system according to any one of embodiments 11-13, wherein the target sequence is DNA; optionally, the target sequence is selected from: double-stranded DNA, single-stranded DNA, genomic DNA or extrachromosomal DNA.

[0120] 15. The system according to any one of embodiments 11-14, wherein the tracrRNA comprises a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 98%, at least 99%, or 100% identity with any one of SEQ ID NO. 151-242 in Table 3.

[0121] 16. The system according to any one of embodiments 11-15, wherein the direct repeat sequence comprises a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 98%, at least 99%, or 100% identity with any one of SEQ ID NO. 243-324 in Table 4.

[0122] 17. The system according to any one of embodiments 11-16, wherein the polynucleotide encoding the Cas protein is operatively linked to a promoter; optionally, the promoter is a constitutive promoter, a tissue-specific promoter, or an inducible promoter.

[0123] 18. The system according to any one of embodiments 11-17, wherein the polynucleotide encoding the Cas protein and operatively linked to the promoter is located in a vector; optionally, the vector is selected from: retroviral vectors, lentiviral vectors, bacteriophage vectors, adenovirus vectors, adeno-associated virus vectors, and herpes simplex virus vectors.

[0124] 19. An engineered vector comprising a polynucleotide according to any one of embodiments 7-10; optionally, said vector is an inducible, conditional, or constitutive expression vector.

[0125] 20. A vector system comprising one or more polynucleotides according to any one of embodiments 7-10 and one or more polynucleotides encoding guide RNA; optionally, the polynucleotides according to any one of embodiments 7-10 and the polynucleotides encoding said RNA are on the same vector or on different vectors.

[0126] 21. An engineered, non-naturally occurring cell comprising the Cas protein according to any one of embodiments 1-4, the fusion protein according to any one of embodiments 5-6, the polynucleotide according to any one of embodiments 7-10, the CRISPR-Cas system according to any one of embodiments 11-18, the vector according to embodiment 19, or the vector system according to embodiment 20.

[0127] 22. The cell according to embodiment 21, wherein the cell is a eukaryotic cell or a prokaryotic cell; preferably, the eukaryotic cell is selected from: plant cells, fungal cells, single-celled eukaryotes, mammalian cells, reptile cells, insect cells, bird cells, fish cells, parasite cells, arthropod cells, invertebrate cells, vertebrate cells, rodent cells, mouse cells, rat cells, primate cells, non-human primate cells, and human cells.

[0128] 23. A kit comprising the Cas protein according to any one of embodiments 1-4, the fusion protein according to any one of embodiments 5-6, the polynucleotide according to any one of embodiments 7-10, the CRISPR-Cas system according to any one of embodiments 11-18, the vector according to embodiment 19, or the vector system according to embodiment 20.

[0129] 24. A pharmaceutical composition comprising a Cas protein according to any one of embodiments 1-4, a fusion protein according to any one of embodiments 5-6, a polynucleotide according to any one of embodiments 7-10, a CRISPR-Cas system according to any one of embodiments 11-18, a vector according to embodiment 19, or a vector system according to embodiment 20.

[0130] 25. The pharmaceutical composition according to embodiment 24, wherein the pharmaceutical composition further comprises a delivery system selected from: AAV (adeno-associated virus), adenovirus, retrovirus, HSV (herpes simplex virus), gamma retrovirus, LV (lentivirus), eCIS (extracellular contraction injection system), eVLPs (engineered virus-like particles), VLPs (virus-like particles), liposomes, plasmids, LNPs (lipid nanoparticles), exosomes, microvesicles, nucleic acid nanoassemblies, gene guns and / or implantable devices.

[0131] 26. The Cas protein according to any one of embodiments 1-4, the fusion protein according to any one of embodiments 5-6, the polynucleotide according to any one of embodiments 7-10, the CRISPR-Cas system according to any one of embodiments 11-18, the vector according to embodiment 19, the vector system according to embodiment 20, the cell according to any one of embodiments 21-22, the kit according to embodiment 23, or the pharmaceutical composition according to any one of embodiments 24-25, for use in a method for the treatment or management or prevention or diagnosis or detection of a disease.

[0132] 27. The Cas protein according to any one of embodiments 1-4 or the fusion protein according to any one of embodiments 5-6, for recognizing a prototype spacer adjacent motif (PAM) having any of the following sequences: NRWAAA, NRDAAAY, NRRAVW, NRWAAW, NRWAAA, NRAARW, or NNNAAAG.

[0133] 28. A method for modifying or targeting a target DNA locus, the method comprising delivering to the locus a Cas protein according to any one of embodiments 1-4, a fusion protein according to any one of embodiments 5-6, a polynucleotide according to any one of embodiments 7-10, a CRISPR-Cas system according to any one of embodiments 11-18, or a pharmaceutical composition according to any one of embodiments 24-25.

[0134] According to the method of embodiment 27, the modification or targeting of the target locus includes inducing DNA strand breaks, altering the gene expression of one or more genes, or epigenetic modification of the target DNA locus; optionally, the DNA strand breaks include DNA double-strand breaks or DNA single-strand breaks.

[0135] 30. The method according to embodiment 27 or 28, wherein the modification or targeting of the target locus includes epigenetic modification of the target DNA locus.

[0136] 31. The method according to any one of embodiments 27-29, wherein the method is performed in vitro or in vivo.

[0137] 32. A method for targeting and cleaving double-stranded target DNA, the method comprising: contacting the double-stranded target DNA with a Cas protein according to any one of embodiments 1-4, a fusion protein according to any one of embodiments 5-6, a polynucleotide according to any one of embodiments 7-10, a CRISPR-Cas system according to any one of embodiments 11-18, or a pharmaceutical composition according to any one of embodiments 24-25.

[0138] 33. The method according to embodiment 31, wherein cutting the target DNA results in the formation of a deletion or insertion of a nucleotide sequence.

[0139] 34. The method according to embodiment 31 or 32, wherein cutting the target DNA or target nucleotide comprises cutting the target DNA at two sites and causing a sequence deletion or inversion between the two sites.

[0140] 35. An isolated eukaryotic cell comprising a modified target locus of interest, wherein the target locus of interest has been modified according to the method of any one of embodiments 28-34, or the target locus of interest has been modified by using a composition of any one of embodiments 24-25, or the target locus of interest has been modified by using a system of any one of embodiments 7-18.

[0141] 36. A system for detecting the presence of a nucleic acid target sequence in an in vitro sample, comprising: (a) a Cas protein according to any one of embodiments 1-4; (b) at least one guiding polynucleotide comprising a spacer sequence capable of binding the target sequence and designed to form a complex with said Cas protein; and (c) a nucleic acid-based masking construct comprising a non-target sequence; wherein said Cas protein exhibits side-branching activity of RNA and / or ssDNA and cleaves the non-target sequence of the nucleic acid-based masking construct activated by the target sequence.

[0142] 37. A method for detecting a target nucleic acid in a sample, comprising: contacting one or more samples with: (a) a Cas protein according to any one of embodiments 1-4; (b) at least one guiding polynucleotide comprising a spacer sequence designed to have a degree of complementarity to a target sequence and designed to form a complex with said Cas protein; and (c) a nucleic acid-based masking construct comprising a non-target sequence; wherein said Cas protein exhibits side-branching activity of RNA and / or ssDNA and cleaves the non-target sequence of the nucleic acid-based masking construct activated by the target sequence; and detecting a signal from the cleavage of the non-target sequence to detect said one or more target sequences in the sample.

[0143] Although this description relates to specific implementations and aspects, this disclosure should not be construed as being limited to the implementations set forth herein. Example

[0144] This document provides embodiments that describe certain implementations of the present disclosure in more detail. The embodiments provided herein are for illustrative purposes only and are not intended to limit the scope of the invention in any way. All references set forth herein and elsewhere are incorporated herein by reference.

[0145] Example 1: Metagenomic Analysis Methods for Proteins

[0146] Hidden Markov models based on known Cas protein sequences (including type II Cas effector proteins) were used to search metagenomic sequence data in public databases. Potential active sites were identified by comparing the search-identified CRISPR-Cas proteins with known proteins. From hundreds of potential sequences, this metagenomic workflow ultimately determined the type II Cas protein.

[0147] A phylogenetic tree was constructed using MUSCLE 3 (Veen et al., 2020) to visualize orthologous relationships at the amino acid level using 139 Type II II-A / B / C sequences from the National Center for Biotechnology Information (NCBI), various publications, and patents. Tree branches corresponding to the Type II Cas proteins provided in this disclosure were labeled.

[0148] Structural modeling of type II Cas proteins was achieved using AlphaFold2 (Figure 1). All GEBx Cas proteins exhibit a typical Cas9 structure, comprising a RuvC domain, a BH (bridged helix) domain, a REC domain, an HNH domain, and a CTD (C-terminal domain). The RuvC domain contains three split RuvC subdomains, and the BH domain is an arginine-rich region containing the RRxxRxxRRxxxRxxxR motif ( ). Figure 2 ).

[0149] Example 2: A scheme for predicting crRNA folding

[0150] Using the RNAfold network server developed by Lorenz et al. in 2011, predicted RNA folding of the putative guide RNA sequence of the GEBx type II Cas protein was calculated.

[0151] Example 3: Determination of PAM in mammalian cell lines

[0152] In one set of experiments, HEK293T cells were cultured in DMEM medium supplemented with 10% fetal bovine serum (Gibco™). For reverse transfection, HEK293T cells were cultured in DMEM medium supplemented with 10% fetal bovine serum (Gibco™). 450 µL of cells at a density of 100,000 cells / well were mixed with 50 µL of a mixture containing Lipofectamine™ 3000 (ThermoFisher Scientific, catalog number L3000008), Opti-Mem (to a final volume of 50 µL), 1 µL of dsODN (10 pM), 100 ng (approximately 1 µL) of psgRNA carrying an sgRNA scaffold (Table 5) and a Humanspacer3 spacer sequence (SEQ ID NO. 417, Table 6), and 400 ng (approximately 1 µL) of pCasX plasmid carrying Cas9CDS with NLS and FLAG (Table 8), according to the manufacturer's protocol. The cell mixture was then seeded into 24-well plates and cultured at 37°C and 5% CO2. 10 pM dsODN was prepared by annealing with dsODN-Top and dsODN-BoT oligonucleotides prior to transfection. The amino acid sequences of the Cas proteins tagged with NLS and Flag are shown in Table 7.

[0153] 72 hours after transfection, the supernatant was removed and the cell layer was washed with PBS. Genomic DNA was then extracted from each well of a 24-well plate using DNA extraction buffer (Denogen (Beijing) Biotechnology Co., Ltd., catalog number DNS033-48) according to the manufacturer's protocol. All DNA samples (500 ng, 260 / 280 value: 1.8–2.0) were analyzed by Guide-Seq NGS.

[0154] The basic method for preparing guide-Seq libraries was described by Nikolay et al. (Nat. Protoc. 2021). First, the extracted DNA sample was sheared using the KAPA Frag Kit (catalog number KK8602, Roche). The fragmented DNA was purified and then phosphorylated using T4 polynucleotide kinase (catalog number M0201S, NEB). The SS5 aptamer (generated by annealing 10 µM SS5TOP oligonucleotides and 10 µM SS5BTM oligonucleotides) was ligated into the fragmented DNA using the Quick Ligation™ Kit (catalog number M2200S, NEB), followed by a two-step off-target PCR to add the chemical components for sequencing.

[0155] Off-target PCR 1 was performed using Platinum™ Taq DNA polymerase (catalog number 15966005, Invitrogen) and GSP1 (a mixture of GSP1-Top and GSP1-BoT) and Y_XX oligonucleotides. Off-target PCR 2 was performed using Platinum™ Taq DNA polymerase and GSP2 (a mixture of GSP2-TopA / B / C and GSP1-BoTA / B / C), Y_XX (same as PCR 1), and i753_XX oligonucleotides. DNA products from each step were purified using SPRI Select (catalog number B23318, Beckman Coulter). The final library was quantified by qPCR and sequenced on an Illumina NextSeq 1000. After removing low-quality reads, the reads were aligned to a reference genome. The Q30 rate was greater than 0.9. Read lengths were between 130 bp and 140 bp. The resulting file containing the reads was aligned to the reference genome (BAM file), and reads overlapping with the target region of interest were selected. The sequences used in this paper are shown in Table 6.

[0156] Table 6. Additional sequences used in the examples

[0157]

[0158] Note: p: phosphorylation modification; *: phosphate thioester (PS) bond; "N" can be any natural or non-natural nucleotide.

[0159] The complete amino acid sequence of the Cas protein with nuclear localization signal (NLS) and FLAG tag sequence is shown in SEQ ID NO. 432-506.

[0160] Table 7. Amino acid sequences of Cas proteins with NLS and Flag tags

[0161]

[0162] Table 8. Sequences encoding Cas proteins with NLS and FLAG tags

[0163]

[0164] PAM bias of Cas protein in HEK293 cell line as follows Figure 3 As shown in Table 9, GEBx0413, GEBx0414, GEBx0415, GEBx0417, GEBx0421, GEBx0423, and GEBx0454 exhibit a preference for A-rich PAM sequences.

[0165] Table 9. PAM Preferences of Exemplary Cas Proteins

[0166]

[0167] Example 4: In vitro gene editing effect of the CRISPR-Cas system in mammalian cell lines

[0168] In one set of experiments, HEK293T cells were cultured in DMEM medium supplemented with 10% fetal bovine serum (Gibco™). For reverse transfection, HEK293T cells were cultured in DMEM medium supplemented with 10% fetal bovine serum (Gibco™). 250 µL of cells at a density of 100,000 cells / well were mixed with 1 µL of a mixture containing Lipofectamine™ 3000 (ThermoFisher Scientific, catalog number L3000008), 2 µL of P3000, Opti-Mem (to a final volume of 25 µL), and pCasX plasmid (375 ng) and a psgRNA plasmid targeting an endogenous gene (125 ng), according to the manufacturer's protocol. The cell mixture was then seeded into 48-well plates and cultured at 37°C and 5% CO2.

[0169] 72 hours after transfection, the supernatant was removed and the cell layer was washed with PBS. Genomic DNA was then extracted from each well of a 24-well plate using DNA extraction buffer (Denogen (Beijing) Biotechnology Co., Ltd., catalog number DNS033-48) according to the manufacturer's protocol. All DNA samples (500 ng, 260 / 280 value: 1.8–2.0) were subjected to amplicon NGS analysis.

[0170] To quantitatively determine the editing efficiency of target sites in the genome, NGS was used to identify the presence of insertions and deletions introduced by gene editing. Primers for NGS were designed flanking the target region within the endogenous gene. Additional PCR was performed according to the manufacturer's (Illumina) protocol to add the chemical components for sequencing. Amplicon sequencing was performed on an Illumina iSeq 100. After removing low-quality reads, the reads were aligned to a reference genome. The Q30 rate was greater than 0.9. Read lengths were between 130 bp and 140 bp. The resulting file containing the reads was aligned to the reference genome (BAM file), reads overlapping with the target region of interest were selected, and the number of wild-type reads and reads containing insertions, substitutions, or deletions were calculated. The number of reads aligned to the reference genome was greater than 1000.

[0171] Therefore, exemplary embodiments of the present invention have been fully described. Although this description relates to specific embodiments, it will be apparent to those skilled in the art that the invention may be practiced with variations of these specific details. Therefore, the invention should not be construed as limited to the embodiments set forth herein.

Claims

1. An engineered, non-naturally occurring type II Cas protein comprising an amino acid sequence selected from: (i) a sequence identical to SEQ ID NO. 32; or (ii) a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 98%, or at least 99% sequence identity with SEQ ID NO.

32.

2. An engineered, non-naturally occurring type II Cas protein comprising an amino acid sequence selected from: (i) a sequence identical to SEQ ID NO. 32; or (ii) a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 98%, or at least 99% sequence identity with SEQ ID NO. 32, except for the "M" at the first position of the sequence.

3. The Cas protein according to claim 1 or 2, wherein the Cas protein is capable of recognizing a prototype spacer adjacent motif (PAM) having the following sequence: NRWAAW; preferably, the Cas protein is capable of recognizing a prototype spacer adjacent motif (PAM) having the following sequence: AAAAA, GAAAA, ATAAA, AAAAT, GAAAA, GTAAA, GTAAT; more preferably, the Cas protein is capable of recognizing a prototype spacer adjacent motif (PAM) having the following sequence: AAAAA.

4. The Cas protein according to any one of claims 1-3, wherein the Cas protein is a nicking enzyme or an inactivated Cas protein.

5. A fusion protein comprising an engineered, non-naturally occurring type II Cas protein according to any one of claims 1-4, wherein the fusion protein further comprises a fusion chaperone sequence selected from the following: nuclear localization signal sequence, nuclear export signal sequence, cell penetration peptide sequence, affinity tag sequence, deaminase sequence, reverse transcriptase sequence, recombinase sequence, methyltransferase sequence, methyltransferase sequence, acetyltransferase sequence, transcription activator sequence, transcription repressor domain sequence, cryptochrome sequence, photoinducible / controllable domain sequence, and chemically inducible / controllable domain sequence.

6. The fusion protein according to claim 5, wherein the fusion protein comprises an amino acid sequence selected from: (i) a sequence identical to SEQ ID NO. 463; or (ii) a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 98%, or at least 99% sequence identity with SEQ ID NO. 463; optionally, the fusion protein comprises an amino acid sequence selected from: (i) a sequence identical to SEQ ID NO. 463; or (ii) a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 98%, or at least 99% sequence identity with SEQ ID NO. 463, except for the "M" at the first position of the sequence.

7. A polynucleotide encoding a type II Cas protein according to any one of claims 1-4 or a fusion protein according to claim 5 or 6.

8. An engineered, non-naturally occurring CRISPR Cas system comprising: (i) a Cas protein according to any one of claims 1-4 or a fusion protein according to claim 5 or 6, or a polynucleotide encoding said Cas protein or said fusion protein; and (ii) a guide RNA (gRNA), or a polynucleotide sequence encoding said guide RNA; wherein said guide RNA comprises a spacer sequence complementary to a target nucleic acid and a Cas protein binding segment that interacts with said Cas protein, wherein said Cas protein binding segment comprises a tracrRNA sequence and a direct repeat (DR) sequence, said tracrRNA sequence and direct repeat sequence hybridizing to form a double-stranded RNA (dsRNA) duplex.

9. The system of claim 8, wherein the guide RNA further comprises a connector sequence linking the tracrRNA sequence and the DR sequence to form an sgRNA scaffold.

10. The system according to claim 8 or 9, wherein the tracrRNA comprises a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 98%, at least 99%, or 100% identity with any one of SEQ ID NO.

190.

11. The system according to any one of claims 8-10, wherein the direct repeat sequence comprises a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 98%, at least 99%, or 100% identity with any one of SEQ ID NO.

279.

12. A pharmaceutical composition comprising the Cas protein according to any one of claims 1-4, the fusion protein according to claim 5 or 6, the polynucleotide according to claim 7, or the CRISPR Cas system according to any one of claims 8-11.

13. A Cas protein according to any one of claims 1-4, a fusion protein according to any one of claims 5-6, a polynucleotide according to claim 7, or a CRISPRCas system according to any one of claims 8-11, for recognizing a prototype spacer adjacent motif (PAM) having the following sequence: NRWAAW; preferably, the PAM has the following sequence: NAAAAA, NGAAAA, NATAAA, NGTAAA, NAAAGA, NAAAGT, NGAAGA, NGTAGA, NGTAGT; more preferably, the PAM has the following sequence: NAAAAA.

14. A Cas protein according to any one of claims 1-4, a fusion protein according to claim 5 or 6, a polynucleotide according to claim 7, a CRISPR Cas system according to any one of claims 8-11, or a pharmaceutical composition according to claim 12, for use in a method of treating or managing, preventing, diagnosing, or detecting a disease.

15. A method of treating, managing, preventing, diagnosing, or detecting a disease, comprising administering to a subject the Cas protein according to any one of claims 1-4, the fusion protein according to claim 5 or 6, the polynucleotide according to claim 7, the CRISPR Cas system according to any one of claims 8-11, or the pharmaceutical composition according to claim 12.

16. Use of the Cas protein according to any one of claims 1-4, the fusion protein according to claim 5 or 6, the polynucleotide according to claim 7, the CRISPR Cas system according to any one of claims 8-11, or the pharmaceutical composition according to claim 12 in the preparation of a medicament for treating or managing, preventing, diagnosing, or detecting a disease.

17. A method of modifying or targeting a target DNA locus, comprising delivering to the locus a Cas protein according to any one of claims 1-4, a fusion protein according to claim 5 or 6, a polynucleotide according to claim 7, a CRISPR Cas system according to any one of claims 8-11, or a pharmaceutical composition according to claim 12.

18. An isolated eukaryotic cell comprising a modified target locus of interest, wherein the modified target locus of interest... The locus has been modified according to the method of claim 17.

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