Engineered CRISPR-Cas13f systems and their uses

Abstract

The present disclosure provides engineered Cas13f polypeptides, systems or compositions comprising the same, and methods of using the same.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of and priority to International Patent Application No. PCT / CN2022 / 083461, filed on March 28, 2022, entitled “ENGINEERED CRISPR / CAS13 SYSTEM AND USES THEREOF,” and International Patent Application No. PCT / CN2022 / 122833, filed on September 29, 2022, entitled “ENGINEERED CRISPR-CAS13F SYSTEM AND USES THEREOF,” which are incorporated herein by reference in their entirety (including any sequence listing and drawings).

[0003] References to Electronic Sequence Listings

[0004] The present disclosure contains an electronic sequence listing ("HGP020PCT2.xml", created on March 26, 2023 by the software "WIPOSequence" according to WIPO Standard ST.26), which is incorporated herein by reference in its entirety. In the case where the sequence is an RNA sequence, T in the sequence should be considered as U. Background Art

[0005] Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) genes (collectively referred to as CRISPR-Cas or CRISPR / Cas systems) are adaptive immune systems in archaea and bacteria that protect specific species from foreign genetic elements.

[0006] Citation or identification of any document in this disclosure is not an admission that such document is available as prior art to the present disclosure. Summary of the invention

[0007] Against the above background, the present disclosure provides certain advantages over the prior art. Although the disclosure herein is not limited to specific advantages, in one aspect, the present disclosure provides an engineered Cas13f polypeptide, wherein the engineered Cas13f polypeptide:

[0008] (1) having at least about 80% (e.g., at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.6%, 99.7%, or 99.8%) and less than 100% sequence identity to the amino acid sequence of SEQ ID NO:3;

[0009] (2) comprising the double mutation Y666A and Y677A corresponding to the amino acid sequence of SEQ ID NO: 3; and

[0010] (3) having increased spacer-specific cleavage activity and / or decreased spacer-independent cleavage activity compared to the amino acid sequence of SEQ ID NO: 3.

[0011] In another aspect, the present disclosure provides polynucleotides encoding the engineered Cas13f polypeptides of the present disclosure.

[0012] In yet another aspect, the present disclosure provides a CRISPR-Cas13f system, comprising:

[0013] a) an engineered Cas13f polypeptide of the present disclosure or a polynucleotide (e.g., DNA, RNA) encoding the engineered Cas13f polypeptide; and

[0014] b) a guide nucleic acid or a polynucleotide encoding the guide nucleic acid (e.g., DNA or RNA), the guide nucleic acid comprising:

[0015] i. a direct repeat (DR) sequence capable of forming a complex with the engineered Cas13f polypeptide; and

[0016] ii. A spacer sequence capable of hybridizing to a target RNA, thereby directing the complex to the target RNA.

[0017] In yet another aspect, the present disclosure provides a vector comprising a polynucleotide of the present disclosure.

[0018] In yet another aspect, the present disclosure provides a delivery system comprising (1) a delivery vehicle, and (2) an engineered Cas13f polypeptide of the present disclosure, a polynucleotide of the present disclosure, a CRISPR-Cas13f system of the present disclosure, or a vector of the present disclosure.

[0019] In yet another aspect, the present disclosure provides a method for modifying a target RNA, the method comprising contacting the target RNA with the CRISPR-Cas13f system of the present disclosure, the vector of the present disclosure, or the delivery system of the present disclosure, thereby modifying the target RNA.

[0020] In yet another aspect, the present disclosure provides a method of treating a disease in a subject in need thereof, the method comprising administering a CRISPR-Cas13f system of the present disclosure to the subject, wherein the disease is associated with a target RNA, wherein the CRISPR-Cas13f system modifies the target RNA, and wherein modification of the target RNA treats the disease.

[0021] The above generally describes the present disclosure, and a more detailed description of various aspects of the present disclosure is provided in separate sections below. However, it should be understood that, for the sake of brevity and reducing redundancy, certain embodiments of the present disclosure are described only under one section or only in the claims or examples. Therefore, it should also be understood that any one embodiment of the present disclosure, including those described only in one aspect, section, or only in the claims or examples, can be combined with any other embodiment of the present disclosure, unless specifically denied or improperly combined.

[0022] The details of one or more embodiments of the present disclosure are described in the following specification. Other features and advantages of the present disclosure will become apparent from the following drawings and detailed description of several embodiments and also from the appended claims.

[0023] definition

[0024] The present disclosure will describe specific embodiments, but the present disclosure is not limited thereto, but only by the claims. Unless otherwise defined, all technical terms and scientific terms used herein have the same meanings as those commonly understood by ordinary technicians in the field to which the present disclosure belongs. Unless otherwise specified, the terms described below are generally understood in their ordinary meanings.

[0025] Overview

[0026] Class II, type VII CRISPR-associated (Cas) proteins (Cas13), as nucleic acid programmable RNA nucleases (napRNAn), include Cas13a (C2c2), Cas13b (such as Cas13b1, Cas13b2), Cas13c, Cas13d, Cas13e and Cas13f polypeptides, capable of cutting the target RNA under the guidance of a guide nucleic acid (e.g., guide RNA) containing a guide sequence targeting the target RNA. In some embodiments, the target RNA is eukaryotic.

[0027] Without wishing to be bound by theory, in some embodiments, the guide nucleic acid comprises a scaffold sequence responsible for forming a complex with Cas13, and a guide sequence intentionally designed to be responsible for hybridizing with a target sequence of a target RNA, thereby directing the complex comprising Cas13 and the guide nucleic acid to the target RNA.

[0028] refer to Figure 7, exemplary dsRNA is described as comprising a 5' to 3' single DNA strand and a 3' to 5' single DNA strand. According to conventional transcription processes, exemplary RNA transcripts can be transcribed using the 3' to 5' single DNA strand as a synthetic template, so the 3' to 5' single DNA strand is referred to as the "template strand" or "antisense strand". The RNA transcript so transcribed has the same primary sequence as the 5' to 3' single DNA strand (except that T is replaced by U), so the 5' to 3' single DNA strand is referred to as the "coding strand" or "sense strand".

[0029] Exemplary guide nucleic acids are described as comprising a guide sequence and a scaffold sequence. The guide sequence is designed to hybridize with a portion of the RNA transcript (target RNA), so the guide sequence "targets" the portion. Therefore, the portion of the target RNA on which the guide sequence is designed and to which the guide sequence can hybridize is called the "target sequence." In some embodiments, the guide sequence is 100% (completely) reverse complementary to the target sequence. In some other embodiments, the guide sequence is reverse complementary to the target sequence and contains mismatches (such as Figure 8 exemplified in ).

[0030] Typically, the double-stranded sequence of dsDNA can be represented by the sequence of its 5' to 3' single DNA strand, and conventionally, written in a 5' to 3' direction / orientation. Typically, nucleic acid sequences (e.g., DNA sequences, RNA sequences) are written in a 5' to 3' direction / orientation.

[0031] For example, for a dsDNA having a 5' to 3' single DNA strand of 5'-ATGC-3' and a 3' to 5' single DNA strand of 3'-TACG-5', the dsDNA can be simply represented as 5'-ATGC-3'. Normally, the RNA transcript (target RNA) transcribed from the dsDNA then has a sequence of 5'-AUGC-3'.

[0032] In order to hybridize with the target RNA, in one embodiment, the guide sequence of the guide nucleic acid is designed to have a 5'-GCAU-3' sequence that is completely reverse complementary to the target RNA. According to WIPO's electronic sequence table standard ST.26, the symbol "t" is used to represent both T in DNA and U in RNA (see "Table 1: List of nucleotide symbols", the definition of the symbol "t" is "thymine in DNA / uracil in RNA (t / u)"). Therefore, in the sequence table according to ST.26, such a guide sequence will be listed in GCAT, but marked as an RNA sequence.

[0033] the term

[0034] As used herein, if a DNA sequence, e.g., 5'-ATGC-3', is transcribed into an RNA sequence (e.g., 5'-AUGC-3') in which each dT (deoxythymidine, or "T" for short) in the primary sequence is replaced with U (uridine), and the other dA (deoxyadenosine, or "A" for short), dG (deoxyguanosine, or "G" for short), and dC (deoxycytidine, or "C" for short) are replaced with A (adenosine), G (guanosine), and C (cytidine), respectively, it is said in the present disclosure that the DNA sequence "encodes" the RNA sequence.

[0035] As used herein, the term "activity" refers to biological activity. In some embodiments, the activity includes enzymatic activity, for example, the catalytic ability of the effector. For example, the activity can include nuclease activity, such as RNA nuclease activity, RNA endonuclease activity.

[0036] As used herein, the term "complex" refers to a grouping of two or more molecules. In some embodiments, the complex comprises a polypeptide and a nucleic acid, and the polypeptide and nucleic acid interact with each other (e.g., bind, contact, adhere). As used herein, the term "complex" may refer to a grouping of guide nucleic acids and polypeptides (e.g., napRNAn, such as Cas13 polypeptides). As used herein, the term "complex" may refer to a grouping of guide nucleic acids, polypeptides, and target sequences. As used herein, the term "complex" may refer to a grouping of guide nucleic acids, napRNAn, and optionally target RNA targeting target RNAs.

[0037] As used herein, the term "guide nucleic acid" refers to any nucleic acid that helps to target napRNAn (e.g., Cas13 polypeptide) to a target sequence (e.g., a sequence of a target RNA). The guide nucleic acid can be designed to include a sequence complementary to a specific nucleic acid sequence (e.g., a sequence of a target RNA). The guide nucleic acid can include a scaffold sequence that helps to guide napRNAn to the target RNA. In some embodiments, the guide nucleic acid is a guide RNA.

[0038] As used herein, the terms "nucleic acid," "polynucleotide," and "nucleotide sequence" are used interchangeably to refer to a polymeric form of nucleotides of any length, including deoxyribonucleotides, ribonucleotides, combinations thereof, and analogs or modifications thereof.

[0039] As used in the context of CRISPR-Cas technology (e.g., CRISPR-Cas13 technology), the term "guide RNA" is used interchangeably with the term "CRISPR RNA (crRNA)", "single guide RNA (sgRNA)" or "RNA guide", the term "guide sequence" is used interchangeably with the term "spacer sequence", and the term "scaffold sequence" is used interchangeably with the term "direct repeat sequence".

[0040] As described herein, the guide sequence is designed to be able to hybridize with the target sequence. As used herein, the term "hybridization (hybridize, hybridizing or hybridization)" refers to a reaction in which one or more polynucleotide sequences react to form a complex, and the complex is stabilized via hydrogen bonds between the bases of the polynucleotide sequence. Hydrogen bonds can occur by Watson Crick base pairing, Hoogstein binding or in any other sequence-specific manner. The polynucleotide sequence that can hybridize with a given polynucleotide sequence is called the "complementary sequence" of a given polynucleotide sequence. As used herein, the hybridization of a guide sequence and a target sequence is so stable that the effector polypeptide (e.g., napRNAn) or the functional domain associated with (e.g., fusion) with the nucleic acid containing the guide sequence is allowed to act (e.g., cutting, deamination) on the target sequence or its complementary sequence (e.g., the sequence of the target RNA or its complementary sequence).

[0041] For the purpose of hybridization, in some embodiments, the guide sequence is complementary or reverse complementary to the target sequence. As used herein, the term "complementary" refers to the ability of the nucleobases of a first polynucleotide sequence (such as a guide sequence) to pair with the nucleobases of a second polynucleotide sequence (such as a target sequence) by traditional Watson-Crick base pairing. Two complementary polynucleotide sequences can be non-covalently bound under appropriate temperature and solution ionic strength conditions. In some embodiments, the first polynucleotide sequence (e.g., a guide sequence) comprises 100% (complete) complementarity with the second nucleic acid (e.g., a target sequence). In some embodiments, a first polynucleotide sequence (e.g., a guide sequence) is complementary to a second polynucleotide sequence (e.g., a target sequence) if the first polynucleotide sequence comprises at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% complementarity with the second nucleic acid. As used herein, the term "substantially complementary" refers to a polynucleotide sequence (e.g., a guide sequence) having a certain degree of complementarity with a second polynucleotide sequence (e.g., a target sequence). In some embodiments, the level of complementarity enables a first polynucleotide sequence (e.g., a guide sequence) to hybridize with a second polynucleotide sequence (e.g., a target sequence) with sufficient affinity to allow an effector polypeptide (e.g., napRNAn) complexed with the first polynucleotide sequence or a nucleic acid comprising the first polynucleotide sequence or a functional domain associated with (e.g., fused to) the effector polypeptide to act (e.g., cleavage, deamination) on the target sequence or its complementary sequence (e.g., the sequence of a target RNA or its complementary sequence). In some embodiments, a guide sequence that is substantially complementary to a target sequence has less than 100% complementarity with the target sequence. In some embodiments, a guide sequence that is substantially complementary to a target sequence has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% complementarity with the target sequence.

[0042] As used herein, the term "sequence identity" is related to sequence homology. Homology comparisons can be performed by eye, or more usually with the aid of readily available sequence comparison programs. These commercially available computer programs can calculate the homology percentage (%) between two or more sequences, and can also calculate the sequence identity shared by two or more amino acid or nucleic acid sequences.

[0043] Sequence homology can be generated by any of a variety of computer programs known in the art, such as BLAST or FASTA, etc. A suitable computer program for performing such comparison is the GCG Wisconsin Bestfit package (University of Wisconsin, USA; Devereux et al., 1984, Nucleic Acids Research [nucleic acid research] 12: 387). Embodiments of other software that can be used for sequence comparison include, but are not limited to, BLAST package (see Ausubel et al., 1999 ibid.-Chapter 18), FASTA (Atschul et al., 1990, J.Mol.Biol. [Molecular Biology Magazine], 403-410) and GENEWORKS comparison tool suite. Both BLAST and FASTA can be used for offline and online searches (see Ausubel et al., 1999 ibid., pp. 7-58 to pp. 7-60). The percentage (%) of sequence homology can be calculated on a continuous sequence, that is, one sequence is aligned with other sequences, and each amino acid or nucleotide in one sequence is directly compared with the corresponding amino acid or nucleotide in the other sequence, one residue at a time. This is called an "ungapped" alignment. Typically, such ungapped alignments are performed only on multiple residues of a relatively short number. Although this is a very simple and reliable method, it does not take into account that, for example, in a pair of sequences that are originally identical, an insertion or deletion can cause the subsequent amino acid residues to no longer align, which may cause the % homology to be greatly reduced when a global alignment is performed. Therefore, most sequence comparison methods are designed to produce an optimal alignment that takes into account possible insertions and deletions, so as not to unduly impair the overall homology or identity score. This is achieved by inserting "gaps" in the sequence alignment in an attempt to maximize local homology or identity. However, these more complex methods assign a "gap penalty" to each gap that occurs in the alignment so that, for the same number of identical amino acids, a sequence alignment with as few gaps as possible - reflecting a higher relatedness between the two compared sequences - can achieve a higher score than a sequence alignment with many gaps. Typically an "affine gap cost" is used, which charges a relatively high cost for the presence of a gap, and a smaller penalty for each subsequent residue in the gap. This is the most commonly used gap scoring system. Of course, high gap penalties can produce optimal alignments with fewer gaps. Most alignment programs allow gap penalties to be modified. However, it is preferred to use the default values ​​when using such software for sequence comparisons. For example, when using the GCG Wisconsin Bestfit package, the default gap penalty for amino acid sequences is: -12 for a gap, -4 for each gap extension. Therefore, the calculation of the maximum % homology first requires the generation of an optimal alignment taking into account the gap penalties.A new tool called BLAST 2 sequences can also be used to compare protein and nucleotide sequences (see FEMS Microbiol Lett. 1999 174(2):247-50; FEMS Microbiol Lett. 1999 177(1):187-8 and the National Center for Biotechnology Information website on the National Institutes of Health website). Although the final % homology can be measured in terms of identity, the alignment process itself is typically not based on an all-or-nothing pairwise comparison. Instead, a scaled similarity score matrix is ​​usually used, which assigns a score to each pairwise comparison based on chemical similarity or evolutionary distance. An example of such a matrix that is commonly used is the BLOSUM62 matrix - the default matrix for the BLAST program package. The GCG Wisconsin program typically uses the public defaults or a custom symbol comparison table (if provided) (see the user manual for further details). For some applications, it is preferred to use the public default values ​​of the GCG software package, or in the case of other software, the default matrix, such as BLOSUM62. Alternatively, an algorithm based on a CLUSTAL-like algorithm is used in DNASIS. TMThe multiple alignment feature in the Hitachi software is used to calculate the homology percentage (Higgins DG and Sharp PM (1988), Gene [Gene] 73 (1), 237-244). Once the software has produced the best alignment, the % homology, preferably the % sequence identity, can be calculated. Typically, as part of the sequence comparison, the software performs this calculation and generates a numerical result. The sequence can also have a deletion, insertion or substitution of amino acid residues that produces a silent change and produces a functionally equivalent substance. Intentional amino acid substitutions can be made based on the similarity of amino acid properties (such as the polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphipathic nature of the residue), and it is therefore useful to group amino acids together in functional groups. Amino acids can be grouped together based solely on their side chain properties. However, it would also be more useful to include mutation data. For structural reasons, the groups of amino acids derived may therefore be conservative. These groups can be described in the form of a Venn diagram (Livingstone CD and Barton GJ (1993) "Protein sequence alignments: a strategy for the hierarchical analysis of residue conservation" Comput. Appl. Biosci. 9:745-756) (Taylor WR (1986) "The classification of amino acid conservation" J. Theor. Biol. 119; 205-218). Conservative substitutions can be made, for example, according to the following table, which describes the generally accepted Venn diagram groupings of amino acids.

[0044] As used herein, the terms "polypeptide" and "peptide" are used interchangeably herein and refer to polymers of amino acids of any length. The polymer may be linear or branched, it may contain modified amino acids, and it may be interrupted by non-amino acids. A protein may have one or more polypeptides. The term also encompasses amino acid polymers that have been modified; for example, by disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation, such as conjugation with a labeling component.

[0045] As used herein, "variant" is interpreted to mean a polynucleotide or polypeptide that is different from a reference polynucleotide or polypeptide, but retains basic properties. Typical variants of polynucleotides are different from another reference polynucleotide in nucleic acid sequence. Changes in the nucleic acid sequence of the variant may or may not change the amino acid sequence of the polypeptide encoded by the reference polynucleotide. As discussed below, nucleotide changes may result in amino acid substitutions, additions, deletions, fusions, and truncations in the polypeptide encoded by the reference sequence. Typical variants of polypeptides are different from another reference polypeptide in amino acid sequence. Usually, the differences are limited, so the sequences of the reference polypeptide and the variant are generally very similar and identical in many regions. Variants and reference polypeptides may differ in amino acid sequence by one or more substitutions, additions, deletions in any combination. The substituted or inserted amino acid residues may or may not be amino acid residues encoded by the genetic code. The variant of a polynucleotide or polypeptide may be naturally occurring, such as an allelic variant, or it may be a variant known to be not naturally occurring. Non-naturally occurring variants of polynucleotides and polypeptides may be prepared by mutagenesis techniques, by direct synthesis, and by other recombinant methods known to those skilled in the art.

[0046] As used herein, the terms "upstream" and "downstream" refer to the relative position within a sequence of individual nucleotides in a nucleic acid (e.g., DNA). Typically, a first sequence "upstream" of a second sequence means that the first sequence is 5' of the second sequence, while a first sequence "downstream" of a second sequence means that the first sequence is 3' of the second sequence.

[0047] As used herein, the term "wild type" has the meaning generally understood by those skilled in the art, and refers to an organism, strain, gene or characteristic in a typical form that distinguishes it from a mutant or variant when it occurs in nature. It can be isolated from a source in nature without being intentionally modified.

[0048] As used herein, the terms "non-naturally occurring" and "engineered" are used interchangeably and refer to human involvement. When these terms are used to describe a nucleic acid or polypeptide, it means that the nucleic acid or polypeptide is at least substantially free of at least one other component with which it is associated in nature or found in nature.

[0049] As used herein, "cell" is understood to refer not only to a particular individual cell, but also to the progeny or potential progeny of the cell. Because certain modifications may occur in subsequent generations due to mutations or environmental influences, such progeny may not in fact be different from the parent cell, but are still included within the scope of the term.

[0050] As used herein, the term "in vivo" refers to the body of an organism, while the term "ex vivo" or "in vitro" refers to outside of an organism.

[0051] As used herein, the term "treatment" is a method for obtaining a beneficial or desired result (including clinical results). For the purposes of this disclosure, a beneficial or desired clinical result includes, but is not limited to, one or more of the following: alleviating one or more symptoms caused by the disease, reducing the extent of the disease, stabilizing the disease (e.g., preventing or delaying the deterioration of the disease), preventing or delaying the spread of the disease (e.g., metastasis), preventing or delaying the recurrence of the disease, reducing the recurrence rate of the disease, delaying or slowing the progression of the disease, improving the disease state, providing relief (partial or complete) of the disease, reducing the dosage of one or more other drugs required for treating the disease, delaying disease progression, increasing the quality of life and / or prolonging survival. "Treatment" also encompasses the pathological results of reducing the disease (e.g., cancer). The method disclosed herein encompasses any one or more of these therapeutic aspects of treatment.

[0052] As used herein, the term "disease" includes the terms "disorder" and "condition", and is not limited to medically well-defined terms.

[0053] As used herein, reference to a value or parameter that is "not" generally means and describes a value or parameter that is "different from." For example, a method is not for treating cancer type X, meaning that the method can be used to treat cancers other than type X.

[0054] As used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0055] As used herein, the term "and / or" in phrases such as "A and / or B" is intended to include both A and B; A or B; A (alone); and B (alone). Similarly, the term "and / or" in phrases such as "A, B, and / or C" is intended to cover each of the following embodiments: 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).

[0056] As used herein, when the term "about" is placed before a series of numbers (e.g., about 1, 2, 3), it should be understood that each of the series of numbers is modified by the term "about" (i.e., about 1, about 2, about 3). The term "about XY" used herein has the same meaning as "about X to about Y".

[0057] It should be understood that the embodiments of the present disclosure described herein include "consisting of" and / or "consisting essentially of" the embodiments.

[0058] It is also noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as an antecedent basis for use of exclusive terminology such as "solely," "only" and the like in connection with the recitation of claim elements, or use of a "negative" limitation. BRIEF DESCRIPTION OF THE DRAWINGS

[0059] An understanding of certain features and advantages of the present disclosure will be gained by referring to the following detailed description, which sets forth illustrative embodiments in which the principles of the present disclosure may be utilized, and to the accompanying drawings, in which:

[0060] Figure 1 A view of the predicted 3D structure (by I-TASSER) of the reference Cas13f polypeptide of SEQ ID NO: 1 is shown as a ribbon. The RXXXXH motif of the two HEPN domains is the catalytic site.

[0061] Figure 2 Schematic diagram of an exemplary single-plasmid mammalian dual fluorescent reporter system for detecting the cleavage and para-cleavage activities of Cas13f mutants.

[0062] Figure 3 Twenty segments of the HEPN1, HEPN2, IDL, and Hell-3 domains of the reference Cas13f polypeptide of SEQ ID NO: 1 selected for mutagenesis are shown, wherein each segment spans 9 or 17 amino acids.

[0063] Figure 4 Schematic diagram of an exemplary two-plasmid mammalian dual fluorescent reporter system for detecting the cleavage and para-cleavage activities of Cas13f mutants.

[0064] Figure 5 The functional domain structure of hfCas13f is shown. Compared with the reference Cas13f polypeptide of SEQ ID NO: 1, the four amino acid mutations marked in red are mutations of hfCas13f.

[0065] Figure 6 Schematic diagram of an exemplary two-plasmid mammalian dual fluorescent reporter system for detecting the cleavage activity of Cas13f mutants.

[0066] Figure 7 is a schematic diagram showing an exemplary dsDNA, an exemplary RNA transcript transcribed from the dsDNA, an exemplary guide nucleic acid, and an exemplary Cas13, wherein the guide sequence is reverse complementary to the target sequence.

[0067] Figure 8is a schematic diagram showing an exemplary dsDNA, an exemplary RNA transcript transcribed from the dsDNA, an exemplary guide nucleic acid, and an exemplary Cas13, wherein the guide sequence is reverse complementary to a target sequence and contains mismatches with the target sequence.

[0068] The drawings herein are for illustration purposes only and are not necessarily drawn to scale. DETAILED DESCRIPTION

[0069] Overview

[0070] The present disclosure provides desirable engineered Cas13f polypeptides with high cleavage activity and / or low by-cleavage activity and uses thereof.

[0071] There are several subtypes of Class 2 Type VI CRISPR-associated (Cas) proteins, including at least VI-A (Cas13a / C2c2), VI-B (Cas13b1 and Cas13b2), VI-C (Cas13c), VI-D (Cas13d, CasRx), VI-E (Cas13e), and VI-F (Cas13f) subtypes. Cas13 subtypes generally share very low sequence identity / similarity, but can all be classified as Class 2 Type VI Cas proteins (e.g., generally referred to herein as "Cas13") based on the presence of two conserved HEPN-like RNase domains. Cas13 offers great opportunities for knocking down target gene products (e.g., mRNA) for gene therapy, but on the other hand, such use may be limited by its cleavage activity and / or so-called para-cleavage activity, which can pose a significant risk of cytotoxicity.

[0072] For the latter, in the Class 2 Type VI system, after target RNA binding, the higher eukaryotic and prokaryotic nucleotide binding (HEPN) domain in Cas13 confers non-specific (independent) RNA cleavage of the guide sequence, known as "bypass activity". Binding of a homologous target RNA complementary to the bound gRNA causes a substantial conformational change in Cas13, resulting in the formation of a single composite catalytic site for guide sequence non-specific "bypass" RNA cleavage, thereby converting Cas13 into a guide sequence non-specific RNA nuclease. This newly formed highly accessible active site will not only degrade the target RNA in cis (if the target RNA is long enough to reach this new active site), but will also degrade non-target RNA in trans based on this promiscuous RNase activity. Most RNAs appear to be susceptible to this promiscuous RNase activity of Cas13, and most (if not all) Cas13s have this bypass activity. It has recently been demonstrated that the side-cutting effect caused by Cas13-mediated knockdown exists in mammalian cells and animals, indicating that the clinical application of Cas13-mediated knockdown of target RNA will face major challenges in the presence of such side-cutting effects.

[0073] Cas13f has been identified as a Cas13 subtype with a very small molecular weight, making it particularly suitable for delivery, for example, by rAAV particles. In order to take advantage of its delivery in gene therapy, it is desirable to substantially maintain or improve its cleavage activity and / or substantially reduce or eliminate its by-cutting activity to prevent unnecessary spontaneous cytotoxicity. Using the reporter system disclosed herein, it was found that Cas13f mutants have been developed by mutagenesis to achieve at least one aspect of cleavage activity and by-cutting activity, or even improvements in both aspects.

[0074] In some embodiments, the wild-type Cas13f polypeptide of the present disclosure can be: (i) SEQ ID NO: 1 (Cas13f.1) of the present disclosure, any one of SEQ ID NO: 2-7 (Cas13f.2, Cas13f.3, Cas13f.4 and Cas13f.5, respectively) of PCT / CN2020 / 077211, or any one of SEQ ID NO: 9-10 (Cas13f.6 and Cas13f.7, respectively) of PCT / CN2022 / 101884, such as SEQ ID NO: 1 of the present disclosure; (ii) a naturally occurring ortholog, paralog, or homolog of SEQ ID NO: 1 (Cas13f.1) of the present disclosure, SEQ ID NO: 2-7 (Cas13f.2, Cas13f.3, Cas13f.4 and Cas13f.5, respectively) of PCT / CN2022 / 101884, such as SEQ ID NO: 1 of the present disclosure. NO: 2-7 (Cas13f.2, Cas13f.3, Cas13f.4 and Cas13f.5, respectively), or any one of SEQ ID NO: 9-10 (Cas13f.6 and Cas13f.7, respectively) of PCT / CN2022 / 101884. A naturally occurring ortholog, paralog, or homolog; or (iii) any one of SEQ ID NO: 1 (Cas13f.1) of the present disclosure, any one of SEQ ID NO: 2-7 (Cas13f.2, Cas13f.3, Cas13f.4 and Cas13f.5, respectively) of PCT / CN2020 / 077211, SEQ ID NO: 9-10 (Cas13f.6 and Cas13f.7, respectively) of PCT / CN2022 / 101884. A naturally occurring ortholog, paralog, or homolog; IDNO: 9-10 (Cas13f.6 and Cas13f.7, respectively), or any of the above naturally occurring orthologs, paralogs or homologs, compared to a wild-type Cas13 polypeptide having at least about 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity.

[0075] Representative engineered Cas13f polypeptides

[0076] In one aspect, the present disclosure provides an engineered Cas13f polypeptide, wherein the engineered Cas13f polypeptide:

[0077] (1) having at least about 80% (e.g., at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.6%, 99.7%, or 99.8%) and less than 100% sequence identity to the amino acid sequence of SEQ ID NO:3;

[0078] (2) comprising the double mutation Y666A and Y677A corresponding to the amino acid sequence of SEQ ID NO: 3; and

[0079] (3) having increased spacer-specific cleavage activity and / or decreased spacer-independent cleavage activity compared to the amino acid sequence of SEQ ID NO: 3.

[0080] In some embodiments, the engineered Cas13f polypeptide has at least about 70% (e.g., at least about 70%, 75%, 80%, 85%, 90%, 95%, 100%, 105%, 110%, 115%, 120%, 125%, 130%, 135%, 140%, 145%, or 150%) of the spacer sequence-specific cleavage activity compared to the amino acid sequence of SEQ ID NO: 3.

[0081] In some embodiments, the engineered Cas13f polypeptide has up to about 120% (e.g., up to about 120%, 115%, 110%, 105%, 100%, 95%, 90%, 85%, 80%, 75% or 70%) of spacer-independent lateral cutting activity compared to the amino acid sequence of SEQ ID NO: 3.

[0082] In some embodiments, the engineered Cas13f polypeptide (1) has at least about 75% of the spacer-specific cleavage activity compared to the amino acid sequence of SEQ ID NO: 3, and (2) has up to about 90% of the spacer-independent cleavage activity compared to the amino acid sequence of SEQ ID NO: 3.

[0083] In some embodiments, the engineered Cas13f polypeptide (1) has at least about 100% of the spacer-specific cleavage activity compared to the amino acid sequence of SEQ ID NO: 3, and (2) has up to about 90% of the spacer-independent cleavage activity compared to the amino acid sequence of SEQ ID NO: 3.

[0084] In some embodiments, the engineered Cas13f polypeptide (1) has at least about 130% of the spacer-specific cleavage activity compared to the amino acid sequence of SEQ ID NO: 3, and (2) has up to about 110% of the spacer-independent cleavage activity compared to the amino acid sequence of SEQ ID NO: 3.

[0085] In some embodiments, the engineered Cas13f polypeptide (1) has at least about 130% of the spacer-specific cleavage activity compared to the amino acid sequence of SEQ ID NO: 3, and (2) has up to about 100% of the spacer-independent cleavage activity compared to the amino acid sequence of SEQ ID NO: 3.

[0086] In some embodiments, the engineered Cas13f polypeptide (1) has at least about 130% of the spacer-specific cleavage activity compared to the amino acid sequence of SEQ ID NO: 3, and (2) has up to about 90% of the spacer-independent cleavage activity compared to the amino acid sequence of SEQ ID NO: 3.

[0087] In some embodiments, the engineered Cas13f polypeptide comprises an amino acid substitution at one or more positions corresponding to the following positions of the amino acid sequence selected from SEQ ID NO: 3: 160, 161, 183, 189, 200, 202, 204, 205, 213, 214, 222, 233, 239, 240, 241, 258, 259, 276, 282, 283, 298, 299, 300, 314, 320 , 329, 338, 339, 345, 353, 361, 383, 410, 433, 451, 455, 497, 508, 509, 518, 520, 526, 574, 595, 598, 599, 601, 631, 634, 638, 641, 642, 647, 667, 670, 762, 763 and combinations thereof.

[0088] In some embodiments, the amino acid substitution is a substitution with a non-polar amino acid residue (such as glycine (Gly / G), alanine (Ala / A), valine (Val / V), cysteine ​​(Cys / C), proline (Pro / P), leucine (Leu / L), isoleucine (Ile / I), methionine (Met / M), tryptophan (Trp / W), phenylalanine (Phe / F), or a positively charged amino acid residue (such as lysine (Lys / K), arginine (Arg / R), histidine (His / H)).

[0089] In some embodiments, the amino acid substitution is a substitution of a non-arginine (Arg / R) residue with an arginine (Arg / R) residue.

[0090] In some embodiments, the amino acid substitution is a substitution of a non-alanine (Ala / A) residue with an alanine (Ala / A) residue.

[0091] In some embodiments, the amino acid substitution is a substitution of an alanine (Ala / A) residue with a valine (Val / V) residue.

[0092] In some embodiments, the engineered Cas13f polypeptide comprises an amino acid substitution at one or more positions corresponding to the following positions of the amino acid sequence selected from SEQ ID NO: 3: 160, 161, 631, 634, 638, 641, 642, 647, 667, 670, 762, 763 and combinations thereof.

[0093] In some embodiments, the amino acid substitution is a substitution of a non-alanine (Ala / A) residue with an alanine (Ala / A) residue or a substitution of an alanine (Ala / A) residue with a valine (Val / V) residue.

[0094] In some embodiments, the engineered Cas13f polypeptide comprises an amino acid substitution with an alanine (Ala / A) residue at one or more positions corresponding to positions D160, H638, D642, and combinations thereof of an amino acid sequence selected from SEQ ID NO: 3.

[0095] In some embodiments, the engineered Cas13f polypeptide comprises an amino acid substitution with an alanine (Ala / A) residue at one or more of the following positions corresponding to the amino acid sequence of SEQ ID NO: 3:

[0096] 1) Position D160,

[0097] 2) Location H638,

[0098] 3) Position D642,

[0099] 4) Position D160 and H638,

[0100] 5) Position D160 and D642,

[0101] 6) Positions H638 and D642, or

[0102] 7) Positions D160 and L631.

[0103] In some embodiments, the engineered Cas13f polypeptide comprises a quadruple amino acid substitution with alanine (Ala / A) residues at positions corresponding to positions D160, D642, Y666, and Y677 of an amino acid sequence selected from SEQ ID NO: 1.

[0104] In some embodiments, the engineered Cas13f polypeptide comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO:4.

[0105] In some embodiments, the engineered Cas13f polypeptide comprises an amino acid substitution at one or more positions corresponding to the following positions of the amino acid sequence selected from SEQ ID NO: 3: 183, 189, 200, 202, 204, 205, 213, 214, 222, 233, 239, 240, 241, 258, 259, 276, 282, 283, 298, 299, 300, 314, 320, 329, 338, 339, 345, 353, 361, 383, 410, 433, 451, 455, 497, 508, 509, 518, 520, 526, 574, 595, 598, 599, 601, and combinations thereof.

[0106] In some embodiments, the amino acid substitution is a substitution of a non-arginine (Arg / R) residue with an arginine (Arg / R) residue.

[0107] In some embodiments, the engineered Cas13f polypeptide has increased spacer sequence-specific cleavage activity compared to the amino acid sequence of SEQ ID NO:4.

[0108] In some embodiments, the engineered Cas13f polypeptide has at least about 105%, 110%, 115%, 120%, 125%, 130%, 135%, 140%, 145% or 150% of the spacer sequence-specific cleavage activity compared to the amino acid sequence of SEQ ID NO:4.

[0109] In some embodiments, the engineered Cas13f polypeptide comprises an amino acid substitution with an arginine (Arg / R) residue at one or more positions corresponding to positions G282, F314, Y338, E410, Q520, L526, F598, and combinations thereof of an amino acid sequence selected from SEQ ID NO: 3.

[0110] Guide nucleic acid

[0111] The engineered Cas13f polypeptides disclosed herein can be used in combination with a guide nucleic acid and guided to a target RNA by the guide nucleic acid to exert an effect on the target RNA.

[0112] In another aspect, the disclosure provides a guide nucleic acid comprising:

[0113] (1) a DR sequence capable of forming a complex with the engineered Cas13f polypeptide of the present disclosure, and

[0114] (2) A spacer sequence capable of hybridizing to a target RNA, thereby directing the complex to the target RNA.

[0115] In some embodiments, the spacer sequence is capable of hybridizing to the target sequence of the target RNA.

[0116] In some embodiments, the guide nucleic acid is RNA. In some embodiments, the guide nucleic acid comprises crRNA. In some embodiments, the guide nucleic acid does not comprise tracrRNA.

[0117] Guide nucleic acid structure

[0118] In some embodiments, the guide nucleic acid comprises a DR sequence 5' or 3' to the spacer sequence. In some embodiments, the guide nucleic acid comprises a DR sequence 3' to the spacer sequence. In some embodiments, the DR sequence is fused to the spacer sequence without a linker.

[0119] In some embodiments, the guide nucleic acid comprises a spacer sequence and a DR sequence from 5' to 3'.

[0120] In some embodiments, the guide nucleic acid comprises, from 5' to 3', a DR sequence, a spacer sequence, and a DR sequence, wherein the DR sequences are the same or different.

[0121] In some embodiments, the guide nucleic acid comprises from 5' to 3' a DR sequence, a spacer sequence, a DR sequence and a spacer sequence, wherein the DR sequences are the same or different, and wherein the spacer sequences are the same or different.

[0122] In some embodiments, the guide nucleic acid comprises from 5' to 3' a DR sequence, a spacer sequence, a DR sequence, a spacer sequence, and a DR sequence, wherein the DR sequences are the same or different, and wherein the spacer sequences are the same or different.

[0123] In some embodiments, the guide nucleic acid comprises from 5' to 3' a DR sequence, a spacer sequence, a DR sequence, a spacer sequence, a DR sequence and a spacer sequence, wherein the DR sequences are the same or different, and wherein the spacer sequences are the same or different.

[0124] Target RNA

[0125] The target RNA can be any RNA molecule of interest, including naturally occurring and engineered RNA molecules. The target RNA can be mRNA, tRNA, ribosomal RNA (rRNA), microRNA (miRNA), non-coding RNA, long non-coding (lnc) RNA, nuclear RNA, interfering RNA (iRNA), small interfering RNA (siRNA), ribozyme, riboswitch, satellite RNA, microswitch, microenzyme (microzyme) or viral RNA. In some embodiments, the target RNA is eukaryotic RNA. In some embodiments, the target RNA is encoded by eukaryotic DNA. In some embodiments, the eukaryotic DNA is mammalian DNA, such as non-human mammalian DNA, non-human primate DNA, human DNA, plant DNA, insect DNA, bird DNA, reptile DNA, rodent (e.g., mouse, rat) DNA, fish DNA, nematode DNA or yeast DNA.

[0126] In some embodiments, the target RNA is associated with a disease (e.g., an infectious disease, a genetic disease, or a cancer). Therefore, in some embodiments, the system disclosed herein can be used to treat a disease by targeting the target RNA. For example, a target RNA associated with a disease can be an RNA overexpressed in a diseased cell (e.g., a cancer cell or a tumor cell). The target RNA can also be a toxic RNA and / or a mutated RNA (e.g., an mRNA molecule with a splicing defect or a mutation). The target RNA can also be an RNA specific to a specific microorganism (e.g., a pathogenic bacterium).

[0127] Target sequence

[0128] In some embodiments, the target sequence is at least about 14 nucleotides in length, such as about 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70 or more nucleotides in length, or a range of values ​​between any two of the foregoing values ​​in length, such as about 16 to about 50 nucleotides in length. In some embodiments, the target sequence is about 30 nucleotides in length.

[0129] In some embodiments, the target sequence comprises, consists essentially of, or consists of at least about 14 consecutive nucleotides of a target RNA (e.g., about 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, or more consecutive nucleotides of a target RNA, or a numerical range between any two of the foregoing values, for example, about 14 to about 50 consecutive nucleotides of a target RNA). In some embodiments, the target sequence comprises, consists essentially of, or consists of about 30 contiguous nucleotides of a target RNA.

[0130] Spacer sequence

[0131] The spacer sequence is designed to be able to hybridize with the target RNA, more particularly, to hybridize with the target sequence of the target RNA. For this purpose, the primary sequence of the spacer sequence is designed to be complementary to the primary sequence of the target sequence. If the complementarity between the spacer sequence and the target sequence is sufficient to hybridize the spacer sequence with the target sequence, and the hybridization is stable enough to guide the engineered Cas13f polypeptide to the guide RNA by hybridization, 100% complementarity may not be required.

[0132] In some embodiments, the spacer sequence is at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% (completely), optionally about 100% (completely) complementary to the target sequence; or wherein the spacer sequence is at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 at the 5' end of the spacer sequence. , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69 or 70 nucleotides do not contain mismatches with the target sequence. It is generally believed that for the napRNAn disclosed herein (e.g., Cas13), at least 2 mismatches between the spacer sequence and the target sequence can be tolerated without significantly reducing the cleavage activity. In some embodiments, the spacer sequence is 100% (completely) complementary to the target sequence.

[0133] Typically, the spacer sequence has the same length as the target sequence. In some embodiments, the spacer sequence has a length of at least about 14 nucleotides, such as a length of about 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70 or more nucleotides, or a length range between any two of the aforementioned values, such as a length of about 16 to about 50 nucleotides. In some embodiments, the spacer sequence is about 30 nucleotides in length.

[0134] DR sequence

[0135] For the purpose of the present disclosure, the DR sequence is compatible with the engineered Cas13f polypeptide of the present disclosure, and can be compounded with the engineered Cas13f polypeptide. The DR sequence can be a naturally occurring DR sequence identified together with the engineered Cas13f polypeptide, or a variant thereof that maintains the ability to compound with the engineered Cas13f polypeptide. Generally, as long as the secondary structure of the variant is substantially the same as the secondary structure of the naturally occurring DR sequence, the ability to compound with the engineered Cas13f polypeptide is maintained. The nucleotide deletion, insertion or substitution in the primary sequence of the DR sequence may not necessarily change the secondary structure of the DR sequence (for example, the relative position and / or size of the stem, protrusion and ring of the DR sequence does not significantly deviate from the relative position and / or size of the original stem, protrusion or ring). For example, the nucleotide deletion, insertion or substitution can be in the protrusion or loop region of the DR sequence, so that the overall symmetry of the protrusion and the secondary structure thereof remain roughly the same. The nucleotide deletion, insertion or substitution may also be in the stem of the DR sequence such that the length of the stem does not deviate significantly from the length of the original stem (eg, adding or deleting one base pair in each of the two stems corresponds to a total of 4 base changes).

[0136] In some embodiments, the DR sequence has a secondary structure substantially identical to the secondary structure of the DR sequence of SEQ ID NO:2.

[0137] In some embodiments, the DR sequence comprises, consists essentially of, or consists of a sequence having at least about 80% (e.g., at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to the sequence of SEQ ID NO:2, or a sequence having at most 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotide differences (whether consecutive or not) compared to the sequence of SEQ ID NO:2.

[0138] In some embodiments, the DR sequence comprises the sequence of SEQ ID NO:2.

[0139] Guide nucleic acid modification

[0140] In some embodiments, the guide nucleic acid comprises a modification. In some embodiments, the guide nucleic acid is an unmodified RNA or a modified RNA. In some embodiments, the guide nucleic acid is a modified RNA containing modified ribonucleotides. In some embodiments, the guide nucleic acid is a modified RNA containing deoxyribonucleotides. In some embodiments, the guide nucleic acid is a modified RNA containing modified deoxyribonucleotides. In some embodiments, the guide nucleic acid comprises modified or unmodified deoxyribonucleotides and modified or unmodified ribonucleotides.

[0141] Chemical modifications can be applied to the phosphate backbone, sugars and / or bases of the guide nucleic acids. Backbone modifications such as phosphorothioates modify the charge on the phosphate backbone and aid in delivery and nuclease resistance of the oligonucleotide (see, e.g., Eckstein, “Phosphorothioates, essential components of therapeutic oligonucleotides,” Nucl. Acid Ther., 24, pp. 374-387, 2014); sugar modifications such as 2'-O-methyl (2'-OMe), 2'-F, and locked nucleic acids (LNA) enhance both base pairing and nuclease resistance (see, e.g., Allerson et al. “Fully 2'-modified oligonucleotide duplexes with improved in vitro potency and stability compared to unmodified small interfering RNA,” J. Med. Chem. 48.4:901-904, 2005). Chemically modified bases (such as 2-thiouridine or N6-methyladenosine, etc.) can allow stronger or weaker base pairing (see, e.g., Bramsen et al., "Development of therapeutic-grade small interfering RNAs by chemical engineering", Front. Genet., 2012 Aug 20; 3:154). In addition, the guide nucleic acid is suitable for conjugation to a variety of functional moieties (including fluorescent dyes, polyethylene glycol, or proteins) at both the 5' and 3' ends.

[0142] A variety of modifications can be applied to chemically synthesized guide nucleic acids. For example, modifying guide nucleic acids with 2'-OMe to improve nuclease resistance can change the binding energy of Watson-Crick base pairing. In addition, 2'-OMe modifications can affect the way guide nucleic acids interact with transfection reagents, proteins, or any other molecules in cells. The effects of these modifications can be determined by empirical testing.

[0143] Examples of chemical modifications of guide nucleic acids include, but are not limited to, incorporation of 2'-O-methyl (M), 2'-O-methyl 3'-phosphorothioate (MS), or 2'-O-methyl 3'-sulfenyl PACE (MSP) at one or more terminal nucleotides. Such chemically modified guide nucleic acids can have increased stability and / or increased activity compared to unmodified guide nucleic acids, although the on-target specificity is unpredictable relative to the off-target specificity. See Hendel, Nat Biotechnol. [Nature Biotechnology] 33 (9): 985-9, 2015, which is incorporated by reference. The chemically modified guide nucleic acid may further include, but is not limited to, a nucleic acid having a phosphorothioate bond and a locked nucleic acid (LNA) nucleotide, the locked nucleic acid (LNA) nucleotide comprising a methylene bridge between the 2' and 4' carbons of the ribose ring.

[0144] In some embodiments, the guide nucleic acid comprises one or more phosphorothioate modifications. In some embodiments, the guide nucleic acid comprises one or more locked nucleic acid nucleotides for the purpose of enhancing base pairing and / or increasing nuclease resistance.

[0145] A summary of these chemical modifications can be found, for example, in Kelley et al., “Versatility of chemicallysynthesized guide RNAs for CRISPR-Cas9 genome editing,” J. Biotechnol. 233:74-83, 2016; WO 2016205764; and U.S. Pat. No. 8,795,965 B2; each of which is incorporated by reference in its entirety.

[0146] Polynucleotide

[0147] In yet another aspect, the present disclosure provides polynucleotides encoding the engineered Cas13f polypeptides of the present disclosure.

[0148] In some embodiments, the polynucleotide is codon optimized for expression in a eukaryote, a mammal such as a non-human mammal, a non-human primate, a human, a plant, an insect, a bird, a reptile, a rodent (e.g., a mouse, a rat), a fish, a nematode, or a yeast.

[0149] In some embodiments, the polynucleotide is a synthetic polynucleotide. In some embodiments, the polynucleotide is DNA. In some embodiments, the polynucleotide is RNA (e.g., mRNA encoding the engineered Cas13f polypeptide). In some embodiments, the mRNA is capped, polyadenylated, substituted with 5-methylcytidine, substituted with pseudouridine, or a combination thereof.

[0150] CRISPR-Cas13f system

[0151] In yet another aspect, the present disclosure provides a CRISPR-Cas13f system, comprising:

[0152] a) an engineered Cas13f polypeptide of the present disclosure or a polynucleotide (e.g., DNA, RNA) encoding the engineered Cas13f polypeptide; and

[0153] b) a guide nucleic acid or a polynucleotide encoding the guide nucleic acid (e.g., DNA or RNA), the guide nucleic acid comprising:

[0154] i. a direct repeat (DR) sequence capable of forming a complex with the engineered Cas13f polypeptide; and

[0155] ii. A spacer sequence capable of hybridizing to a target RNA, thereby directing the complex to the target RNA.

[0156] In some embodiments, the system is a complex comprising an engineered Cas13f polypeptide complexed with the guide nucleic acid. In some embodiments, the complex further comprises a target RNA hybridized with the target sequence.

[0157] In some embodiments, the spacer sequence is capable of hybridizing to the target sequence of the target RNA.

[0158] The systems of the present disclosure can comprise one guide nucleic acid or more than one nucleic acid, e.g., for the purpose of increasing the efficiency of cleavage of the target RNA.

[0159] In some embodiments, the system further comprises a second guide nucleic acid comprising:

[0160] (1) a DR sequence capable of forming a complex with napRNAn or said DR sequence, and

[0161] (2) capable of hybridizing to a second target sequence of the target RNA, thereby directing the complex to a second spacer sequence of the target RNA.

[0162] In some embodiments, the system further comprises a third guide nucleic acid comprising:

[0163] (1) a DR sequence capable of forming a complex with napRNAn or said DR sequence, and

[0164] (2) capable of hybridizing to a third target sequence of the target RNA, thereby guiding the complex to a third spacer sequence of the target RNA.

[0165] In some embodiments, the system further comprises a fourth guide nucleic acid comprising:

[0166] (1) a DR sequence capable of forming a complex with napRNAn or said DR sequence, and

[0167] (2) capable of hybridizing to a fourth target sequence of the target RNA, thereby guiding the complex to a fourth spacer sequence of the target RNA.

[0168] In some embodiments, the system further comprises a fifth guide nucleic acid, a sixth guide nucleic acid, a seventh guide nucleic acid, etc.

[0169] In some embodiments, the DR sequences of the more than one guide nucleic acids can be identical or slightly different (e.g., differing by no more than 5, 4, 3, 2, or 1 nucleotides) to be compatible with the engineered Cas13f polypeptide.

[0170] The guide sequences of the multiple guide nucleic acids can be the same to improve cleavage activity on the same target RNA, or different to target different target RNAs at one time.

[0171] Regulation of engineered Cas13f peptides

[0172] In some embodiments, a polynucleotide (e.g., DNA) encoding an engineered Cas13f polypeptide of the present disclosure is operably linked to a regulatory element (e.g., a promoter) to control expression of the polynucleotide.

[0173] In some embodiments, the promoter is a broad spectrum promoter, a tissue-specific promoter, a cell type-specific promoter, a constitutive promoter, or an inducible promoter.

[0174] Suitable promoters are known in the art and include, for example, the Cbh promoter, the Cba promoter, the pol I promoter, the pol II promoter, the pol III promoter, T7 promoter, U6 promoter, H1 promoter, retrovirus Rous sarcoma virus LTR promoter, cytomegalovirus (CMV) promoter, SV40 promoter, dihydrofolate reductase promoter, β-actin promoter, elongation factor 1α short (EFS) promoter, β-glucuronidase (GUSB) promoter, cytomegalovirus (CMV) immediate early (Ie) enhancer and / or promoter, chicken β-actin (CBA) promoter or its derivatives such as CAG promoter, CB promoter, (human) elongation factor 1α-subunit (EF1α) promoter, ubiquitin C (UBC) promoter, prion promoter, neuron-specific enolase (NSE), neurofilament light chain (NFL) promoter, neurofilament heavy chain (NFH) promoter, platelet-derived growth factor (PDGF) promoter, blood Platelet-derived growth factor B chain (PDGF-β) promoter, synapsin (Syn) promoter, synapsin 1 (Syn1) promoter, methyl-CpG binding protein 2 (MeCP2) promoter, Ca2+ / calmodulin-dependent protein kinase II (CaMKII) promoter, metabotropic glutamate receptor 2 (mGluR2) promoter, neurofilament light chain (NFL) promoter, neurofilament heavy chain (NFH) promoter, β-globin minigene nβ2 promoter, preproenkephalin (PPE) promoter, enkephalin (Enk) promoter, excitatory amino acid transporter 2 (EAAT2) promoter, glial fibrillary acidic protein (GFAP) promoter, myelin basic protein (MBP) promoter, HTT promoter, GRK1 promoter, CRX promoter, NRL promoter, and RCVRN promoter.

[0175] In some embodiments, the polynucleotide further comprises a first nuclear localization sequence (e.g., SV40 NLS, bpSV40 NLS, npNLS) or a first coding sequence of a nuclear export signal (NES) 5' of the sequence encoding the engineered Cas13 polypeptide, and / or a second NLS (e.g., SV40 NLS, bpSV40 NLS, npNLS) or a second coding sequence of a NES 3' of the sequence encoding the engineered Cas13 polypeptide.

[0176] Directing the regulation of nucleic acids

[0177] In some embodiments, a polynucleotide (e.g., DNA) encoding the guide nucleic acid is operably linked to a regulatory element (e.g., a promoter) to control expression of the polynucleotide.

[0178] In some embodiments, the promoter is a broad spectrum promoter, a tissue-specific promoter, a cell type-specific promoter, a constitutive promoter, or an inducible promoter.

[0179] Suitable promoters are known in the art and include, for example, Cbh promoter, Cba promoter, pol I promoter, pol II promoter, pol III promoter, T7 promoter, U6 promoter, H1 promoter, retroviral Rous sarcoma virus LTR promoter, cytomegalovirus (CMV) promoter, SV40 promoter, dihydrofolate reductase promoter, β-actin promoter, elongation factor 1 alpha short (EFS) promoter, β-glucuronidase (GUSB) promoter, cytomegalovirus (CMV) immediate early (le) enhancer and / or promoter, chicken β-actin (CBA) promoter or derivatives thereof such as CAG promoter, CB promoter, (human) elongation factor 1 alpha-subunit (EF1α) promoter, ubiquitin C (UBC) promoter, prion promoter, neuron-specific enolase (NSE), neurofilament light chain (NFL) promoter, neurofilament heavy chain (NFH) promoter, plasma leukocyte antigen (PEE) promoter, leukocyte antigen (VEA) promoter, leukocyte antigen (VEA) promoter, leukocyte antigen (VEA) promoter, leukocyte antigen (VEA) promoter, leukocyte antigen (VEA) promoter, leukocyte antigen (VEA) promoter, leukocyte antigen (VEA) promoter, In some embodiments, the promoter is a U6 promoter.

[0180] How to use

[0181] The CRISPR-Cas13f system of the present disclosure comprising the engineered Cas13f polypeptide of the present disclosure has a variety of utilities similar to those of wild-type CRISPR-Cas13 systems, including modification (e.g., cutting, deletion, insertion, translocation, inactivation or activation) of target RNA in a variety of cell types. The CRISPR-Cas13f system has a wide range of applications, such as drug screening, disease diagnosis and prognosis, and treatment of various genetic disorders, which require high cleavage activity and low para-cleavage activity.

[0182] In yet another aspect, the present disclosure provides a method for modifying a target RNA, the method comprising contacting the target RNA with the CRISPR-Cas13f system of the present disclosure, the vector of the present disclosure, or the delivery system of the present disclosure, thereby modifying the target RNA.

[0183] In yet another aspect, the present disclosure provides a method of treating a disease in a subject in need thereof, the method comprising administering to the subject a CRISPR-Cas13f system of the present disclosure or an rAAV particle of the present disclosure, wherein the disease is associated with a target RNA, wherein the CRISPR-Cas13f system modifies the target RNA, and wherein modification of the target RNA treats the disease.

[0184] In some embodiments, the target RNA is mRNA, tRNA, ribosomal RNA (rRNA), microRNA (miRNA), non-coding RNA, long non-coding (lnc) RNA, nuclear RNA, interfering RNA (iRNA), small interfering RNA (siRNA), ribozyme, riboswitch, satellite RNA, microswitch, microenzyme or viral RNA.

[0185] In some embodiments, the target RNA is encoded by eukaryotic DNA.

[0186] In some embodiments, the eukaryotic DNA is mammalian DNA, such as non-human mammal DNA, non-human primate DNA, human DNA, plant DNA, insect DNA, bird DNA, reptile DNA, rodent (e.g., mouse, rat) DNA, fish DNA, nematode DNA, or yeast DNA.

[0187] The CRISPR-Cas13f system disclosed herein can have various therapeutic applications. Such applications can be based on one or more of the following in vitro and in vivo capabilities attributed to the high cleavage activity of the engineered Cas13f polypeptide: cutting or degrading target RNA, reducing or increasing transcription, reducing or increasing translation, inhibiting or activating expression, inducing or inhibiting cell senescence, inducing or inhibiting cell cycle arrest, inducing or inhibiting cell growth and / or proliferation, inducing or inhibiting apoptosis, inducing or inhibiting necrosis, etc.

[0188] In some embodiments, the CRISPR-Cas13f system can be used to treat various diseases, such as genetic disorders (e.g., monogenic diseases), diseases treatable by RNA nuclease activity (e.g., Pcsk9 targeting, Duchenne muscular dystrophy (DMD) targeting, BCL11a targeting), and various cancers.

[0189] In one aspect, the CRISPR-Cas13f system can be used to treat diseases caused by overexpression of RNA, toxic RNA and / or mutant RNA (e.g., splicing defects or truncations). For example, the expression of toxic RNA can be associated with the formation of nuclear inclusions and delayed degenerative changes in brain, heart or skeletal muscle. For example, in some embodiments, the disease is myotonic dystrophy. In myotonic dystrophy, the main pathogenic effect of the toxic RNA is to isolate (sequester) binding proteins and impair the regulation of alternative splicing (see, for example, Osborne et al., "RNA-dominant diseases [RNA dominant diseases]," Hum. Mol. Genet. [Human Molecular Genetics], April 15, 2009; 18 (8): 1471-81). Geneticists are particularly interested in myotonic dystrophy (dystrophic myotonia (DM)) because it produces extremely wide clinical features. The classic form of DM, now called type 1 DM (DM1), is caused by expansion of CTG repeats in the 3'-untranslated region (UTR) of the gene DMPK encoding a cytosolic protein kinase. The CRISPR system as described herein can target overexpressed RNA or toxic RNA, such as any misregulated alternative splicing of the DMPK gene or DM1 in skeletal muscle, heart, or brain.

[0190] The CRISPR-Cas13f system can also target trans-acting mutations that affect RNA-dependent functions, which cause a variety of diseases, such as Prader Willi syndrome, spinal muscular atrophy (SMA), and dyskeratosis congenita. The list of diseases that can be treated using the CRISPR-Cas13f system is summarized in Cooper et al., "RNA and disease," Cell, 136.4 (2009): 777-793 and WO 2016 / 205764A1, which are incorporated herein by reference in their entirety. Those skilled in the art will understand how to use the CRISPR-Cas13f system to treat these diseases.

[0191] The CRISPR-Cas13f system can also be used to treat various tau diseases, including, for example, primary and secondary tau diseases, such as primary age-related tau disease (PART) / neurofibrillary tangles (NFT) dominant senile dementia (wherein NFT is similar to those seen in Alzheimer's disease (AD), but without plaques), pugilistic dementia (chronic traumatic encephalopathy) and progressive supranuclear palsy. Available lists of tau diseases and methods for treating these diseases are described in, for example, WO2016205764, which is incorporated herein by reference in its entirety.

[0192] The CRISPR-Cas13f system can also be used to target mutations that disrupt the cis-acting splicing code, which can lead to splicing defects and diseases. These diseases include, for example, motor neuron degenerative diseases (e.g., spinal muscular atrophy), Duchenne muscular dystrophy (DMD), chromosome 17-related frontotemporal dementia with Parkinson's syndrome (FTDP-17), and cystic fibrosis caused by the loss of the SMN1 gene.

[0193] The CRISPR-Cas13f system can also be used for antiviral activity, particularly against RNA viruses. For example, the CRISPR-Cas13f system can be programmed with a guide nucleic acid targeting an RNA molecule associated with the RNA virus to prevent the reproduction of the RNA virus and / or inactivate the activity of the RNA virus.

[0194] The CRISPR-Cas13f system can also be used to treat cancer in a subject (e.g., a human subject). For example, the CRISPR-Cas13f system can be programmed with a guide nucleic acid targeting an RNA molecule that is abnormal (e.g., comprising a point mutation or via alternative splicing) and found in cancer cells to induce cell death (e.g., via apoptosis) of cancer cells.

[0195] The CRISPR-Cas13f system can also be used to treat autoimmune diseases or disorders in subjects (e.g., human subjects). For example, the CRISPR-Cas13f system can be programmed with a guide nucleic acid targeting an RNA molecule that is abnormal (e.g., comprising a point mutation or via alternative splicing) and found in cells that cause autoimmune diseases or disorders.

[0196] The CRISPR-Cas13f system can also be used to treat infectious diseases in subjects. For example, the CRISPR-Cas13f system can be programmed with a guide nucleic acid targeting an RNA molecule, which is expressed by an infectious agent (e.g., bacteria, viruses, parasites, or protozoa) to target and induce cell death in cells containing infectious agents. The CRISPR-Cas13f system can also be used to treat diseases in which intracellular infectious agents infect host subject cells. By programming the CRISPR-Cas13f system to target RNA molecules encoded by infectious agent genes, cells infected by infectious agents can be targeted and cell death can be induced.

[0197] Detailed descriptions of therapeutic applications of the CRISPR-Cas13 system described herein can be found, for example, in U.S. Pat. No. 8,795,965, EP 3009511, WO 2016205764, and WO 2017070605; each of which is incorporated herein by reference in its entirety.

[0198] In some embodiments, the target RNA is a transcript (eg, mRNA) of a target gene associated with an eye disease or disorder.

[0199] In some embodiments, the eye disease or disorder is amebic keratitis, fungal keratitis, bacterial keratitis, viral keratitis, onchocercal keratitis, keratoconjunctivitis, bacterial keratoconjunctivitis, viral keratoconjunctivitis, vernal keratoconjunctivitis, atopic keratoconjunctivitis, corneal dystrophic diseases, Fuchs' endothelial dystrophy, Sjögren's syndrome, Stevens-Johnson syndrome, autoimmune dry eye, environmental dry eye, corneal neovascularization disease, prevention and treatment of corneal transplant rejection, autoimmune uveitis, infectious uveitis, non-infectious uveitis, anterior uveitis, posterior uveitis (including toxoplasmosis), pan Uveitis, inflammatory diseases of the vitreous or retina, prevention and treatment of endophthalmitis, macular edema, macular degeneration, wet age-related macular degeneration (wet AMD), dry age-related macular degeneration (dry AMD), diabetic macular edema (DME), allergic conjunctivitis, proliferative and non-proliferative diabetic retinopathy, hypertensive retinopathy, autoimmune diseases of the retina, primary and metastatic intraocular melanoma, other metastatic intraocular tumors, open-angle glaucoma, Stargardt's disease, fundus yellow spots, angle-closure glaucoma, pigmentary glaucoma, retinitis pigmentosa (RP), Leber congenital amaurosis (LCA), Erb Sjögren's syndrome, choroideremia, rod-cone or cone-rod dystrophy, ciliopathy, mitochondrial disorders, progressive retinal atrophy, degenerative retinal disease, geographic atrophy, familial or acquired maculopathy, retinal photoreceptor disease, retinal pigment epithelium-based disease, cystoid macular edema, retinal detachment, traumatic retinal injury, iatrogenic retinal injury, macular hole, macular telangiectasia, ganglion cell disease, optic nerve cell disease, optic neuropathy, ischemic retinal disease, retinopathy of prematurity, retinal vascular occlusion, familial aneurysm, retinal vascular disease, ocular vascular disease, vascular disease, ischemia optic neuropathy, diabetic retinal edema, age-related macular degeneration due to subretinal neovascularization, myopic retinopathy, retinal ischemia, choroidal vascular insufficiency, choroidal thrombosis and neovascular retinopathy due to carotid artery ischemia, corneal neovascularization, corneal disease or opacity with exudative or inflammatory components, diffuse lamellar keratitis, neovascularization due to penetrating or contusive eye injury, erythematous iritis, Fuchs' heterochromic iridocyclitis, chronic uveitis, anterior uveitis, inflammatory conditions caused by surgery such as LASIK, LASEK, refractive surgery, IOL implantation;Irreversible corneal edema as a complication of cataract surgery, edema caused by injury or trauma, inflammation, infectious and noninfectious conjunctivitis, iridocyclitis, iritis, scleritis, episcleritis, superficial punctate keratitis, keratoconus, posterior polymorphic dystrophy, Fuchs' dystrophy, aphakic and pseudophakic bullous keratopathy, corneal edema, scleral disease, ocular cicatricial pemphigoid, pars planitis, glaucomatocyclitis syndrome, Behcet's disease, Voigt-Koyanagi-Harada syndrome, hypersensitivity reactions, ocular surface disorders, conjunctival edema, toxoplasmosis chorioretinitis, orbital inflammatory pseudotumor, chemosis, conjunctival venous congestion, periorbital cellulitis, acute dacryocystitis, nonspecific vasculitis, sarcoidosis, cytomegalovirus infection, and combinations thereof.;

[0200] In some embodiments, the target gene is selected from vascular endothelial growth factor A (VEGFA), complement factor H (CFH), age-related maculopathy susceptibility factor 2 (ARMS2), HtrA serine peptidase 1 (HTRA1), ATP binding cassette subfamily A member 4 (ABCA4), peripherin 2 (PRPH2), fibulin-5 (FBLN5), ERCC excision repair 6 chromatin remodeling factor (ERCC6), retinal and anterior neural fold homeobox 2 (RAX2), complement C3 (C3), Toll-like receptor 4 (TLR4), cystatin C (CST3), CX 3C chemokine receptor 1 (CX3CR1), complement factor I (CFI), complement C2 (C2), complement factor B (CFB), complement C9 (C9), mitochondrial encoded tRNA leucine 1 (UUA / G) (MT-TL-1), complement factor H related protein 1 (CFHR1), complement factor H related protein 3 (CFHR3), ciliary neurotrophic factor (CNTF), pigment epithelium derived factor (PEDF), rod-derived cone viability factor (RdCVF), glial cell line-derived neurotrophic factor (GDNF), myosin VIIA (MYO7A);Centrosomal protein 290 (CEP290), cadherin-related protein 23 (CDH23), eyes closed homolog (EYS), Usherin protein (USH2A), adhesion G protein-coupled receptor V1 (ADGRV1), ALMS1 centrosomal and basal body-associated protein (ALMS1), retinoid isomerohydrolase 65 kDa (RPE65), aryl-hydrocarbon interacting protein-like 1 (AIPL1), guanylate cyclase 2D, retinal (GUCY2D), Leber's congenital amaurosis 5 protein (LCA5), cone-rod homeobox (CRX), Clarin protein (CLRN1), ATP-binding cassette subfamily A member 4 (ABCA4), retinol dehydrogenase 12 (RDH12), inosine monophosphate dehydrogenase 1 (IMPDH1), debris cell polarity complex component 1 (CRB1), lecithin retinol acyltransferase (LRAT), nicotinamide nucleotide adenylyltransferase 1 (NMNAT1), TUB-like protein 1 (TULP1), MER proto-oncogene, tyrosine kinase (MERTK), retinitis pigmentosa GTPase regulator (RPGR), ARL3 RP2 activator of GTPase (RP2), X-linked retinitis pigmentosa GTPase regulator interacting protein 1 (RPGRIP), cyclic nucleotide-gated channel subunit alpha 3 (CNGA3), cyclic nucleotide-gated channel subunit beta 3 (CNGB3), G protein subunit alpha transducin 2 (GNAT2), fibroblast growth factor 2 (FGF2), erythropoietin (EPO), BCL2 apoptosis regulator (BCL2), BCL2-like 1 (BCL2L1), nuclear factor kappa B (NFκB), endostatin, angiostatin, fms-like tyrosine kinase receptor (sFlt), pigment dispersing factor receptor (Pdfr), interleukin 10 (IL10), soluble interleukin 17 (sIL17R), interleukin 1 receptor antagonist (IL1-ra), TNF receptor superfamily member 1A (TNFRSF1A), TNF receptor superfamily member 1B (TNFRSF1B), and interleukin 4 (IL4).

[0201] In some embodiments, the target RNA is a transcript (eg, mRNA) of a target gene associated with a neurodegenerative disease or disorder.

[0202] In some embodiments, the neurodegenerative disease or disorder is alcoholism, Alexander disease, Alpers disease, Alzheimer's disease, amyotrophic lateral sclerosis (ALS), ataxia telangiectasia, neuronal ceroid lipofuscinosis, Batten disease, bovine spongiform encephalopathy (BSE), spongiform leukoencephalopathy, cerebral palsy, Cockayne syndrome, corticobasal degeneration, Creutzfeldt-Jakob disease, frontotemporal lobar degeneration, Huntington disease, HIV-associated dementia, Kennedy disease, Lewy body dementia, neuroborreliosis, primary Age-related tauopathy (PART) / neurofibrillary tangle-dominant Alzheimer's disease, Machado-Joseph disease, multiple system atrophy, multiple sclerosis, multiple sulfatase deficiency, mucolipidosis, narcolepsy, Niemann-Pick disease, Parkinson's disease, Pick's disease, Pompe disease, primary lateral sclerosis, prion disease, neuronal loss, cognitive deficits, motor neuron disease, Duchenne muscular dystrophy (DMD), frontotemporal dementia, chromosome 17-associated frontotemporal dementia with parkinsonism, Lytico-Bod Ig disease (Guam Parkinson-dementia complex), neuroaxonal dystrophy, Refsum disease, Sidley's disease, subacute combined degeneration of the spinal cord secondary to pernicious anemia, Spearmeyer-Vogt-Sjogren-Batten disease, chromosome 17-related parkinsonism (FTDP-17), Prader-Willi syndrome, myotonic dystrophy, chronic traumatic encephalopathy including dementia pugilistica, spinocerebellar ataxia, spinal muscular atrophy, Still-Richardson-Olszewski disease, tabes dorsalis, Niemann-Pick disease type C (NPC1 and / or NPC2 deficiency), S-L-O syndrome (SLOS), inborn errors of cholesterol synthesis, Tangier disease, Pelletzois-Merzbacher disease, neuronal ceroid lipofuscinosis, primary glycosphingolipidosis, Farber disease or multiple sulfatase deficiency, Gaucher disease, Fabry disease, GM1 gangliosidosis, GM2 gangliosidosis, Krabbe disease, metachromatic leukodystrophy (MLD), NPC, GM1 gangliosidosis, Fabry disease, neurodegenerative mucopolysaccharidoses, MPS I, MPS IH, MPS IS, MPS II, MPS III, MPS IIIA, MPS IIIB, MPS IIIC, MPS HID, MPS, IV, MPS IV A, MPS IV B, MPS VI, MPS VII, MPSIX, secondary lysosomal involvement, SLOS, Tangier disease, ganglioglioma, ganglioneuroma, meningioangiomatosis, postencephalitic parkinsonism, subacute sclerosing panencephalitis, lead encephalopathy, tuberous sclerosis, Hallevorden-Spatz disease, lipofuscinosis, cerebellar ataxia, parkinsonism, Lewy-Barre syndrome, multiple system atrophy, frontotemporal dementia or lower limb parkinsonism, Niemann-Pick disease, Niemann-Pick disease type C, Niemann-Pick disease type A, Tay-Sachs disease, cerebellar multisystem atrophy (MSA-C), frontotemporal dementia with parkinsonism, progressive supranuclear palsy, cerebellar downbeat nystagmus, Sandhoff disease, or mucolipidosis type II, or a combination thereof.

[0203] In some embodiments, the target RNA is a transcript (eg, mRNA) of a target gene associated with cancer.

[0204] In some embodiments, the cancer is a carcinoma, a sarcoma, a myeloma, a leukemia, a lymphoma, and a mixed tumor. Non-limiting examples of cancers that can be treated by the methods and compositions described herein include cancer cells from the bladder, blood, bone, bone marrow, brain, breast, colon, esophagus, gastrointestinal tract, gums, head, kidney, liver, lung, nasopharynx, neck, ovary, prostate, skin, stomach, testicles, tongue, or uterus. Furthermore, the cancer may in particular belong to the following histological types, but is not limited to these: neoplasm, malignant; carcinoma; carcinoma, undifferentiated; giant and spindle cell carcinoma; small cell carcinoma; papillary carcinoma; squamous cell carcinoma; lymphoepithelial carcinoma; basal cell carcinoma; pilomatrix carcinoma; transitional cell carcinoma; papillary transitional cell carcinoma; adenocarcinoma; gastrinoma, malignant; cholangiocarcinoma; hepatocellular carcinoma; hepatocellular carcinoma and cholangiocarcinoma; trabecular adenocarcinoma; adenoid cystic carcinoma; adenocarcinoma with adenomatous polyps; adenocarcinoma, familial colon polyposis; solid carcinoma; carcinoid tumor, malignant; bronchiolo-alveolar adenocarcinoma; papillary adenocarcinoma; chromophobe cell carcinoma; oncocytic cell carcinoma; oncocytic adenocarcinoma; basophilic granulocytic carcinoma; clear cell adenocarcinoma; granulocytic Granulocyte carcinoma; follicular adenocarcinoma; papillary and follicular adenocarcinoma; nonencapsulated sclerosing carcinoma; adrenocortical carcinoma; endometrioid carcinoma; carcinoma of skin appendages; apocrine adenocarcinoma; sebaceous carcinoma; ceruminous adenocarcinoma; mucoepidermoid carcinoma; cystadenocarcinoma; papillary cystadenocarcinoma; papillary serous cystadenocarcinoma; mucinous cystadenocarcinoma; mucinous adenocarcinoma; signet ring cell carcinoma; infiltrating ductal carcinoma; medullary carcinoma; lobular carcinoma; inflammatory carcinoma; Paget's disease, breast; acinar cell carcinoma; adenosquamous carcinoma; adenocarcinoma with squamous metaplasia; thymoma, malignant; stromal tumor of ovary, malignant; thecoma, malignant; granulosa cell tumor, malignant; and fibroblastoma, malignant; Sertoli cell carcinoma; Leydig cell carcinoma cell tumor, malignant; lipid cell tumor, malignant; paraganglioma, malignant; extramammary paraganglioma, malignant; pheochromocytoma; glomangiosarcoma; malignant melanoma; amelanotic melanoma; superficial spreading melanoma; malignant melanoma in giant nevus; epithelioid cell melanoma; malignant blue nevus; sarcoma; fibrosarcoma; malignant fibrous histiocytoma; myxosarcoma; liposarcoma; leiomyosarcoma; rhabdomyosarcoma; embryonal rhabdomyosarcoma; alveolar rhabdomyosarcoma; stromal sarcoma; mixed tumor, malignant; mullerian mixed tumor tumor); Wilms' tumor; Hepatoblastoma; Carcinosarcoma; Mesenchymal tumor, malignant; Brenner's tumor, malignant; Phyllodes tumor, malignant; Synovial sarcoma; Mesothelioma, malignant; Dysgerminoma; Embryonic carcinoma; Teratoma, malignant; Ovarian goiter, malignant; Choriocarcinoma; Mesonephroma, malignant; Angiosarcoma; Hemangioendothelioma, malignant; Kaposi's sarcoma; Hemangiopericytoma, malignant; Lymphangiosarcoma; Osteosarcoma;Parosteal osteosarcoma; Chondrosarcoma; Chondroblastoma, malignant; Mesenchymal chondrosarcoma; Giant cell tumor of bone; Ewing's sarcoma; Odontogenic tumor, malignant; Ameloblastic odontosarcoma; Ameloblastoma, malignant; Ameloblastic fibrosarcoma; Pineal tumor, malignant; Chordoma; Glioma, malignant; Ependymoma; Astrocytoma; Protoplasmic astrocytoma; Fibrous astrocytoma; Astroblastoma; Glioblastoma; Oligodendroglioma; Oligodendroglioma; Primitive neuroectodermal tumor; Cerebellar sarcoma; Ganglioneuroblastoma; Neuroblastoma; Retinoblastoma; Olfactory neurogenic tumor; Meningioma, malignant; Neurofibrosarcoma; Neurothecoma, malignant; Granuloma Granulocytic neoplasms, malignant; Lymphoma, malignant; Hodgkin's disease; Hodgkin's lymphoma; Paragranuloma; Small lymphocytic malignant lymphoma; Diffuse large cell malignant lymphoma; Follicular malignant lymphoma; Mycosis fungoides; Other specified non-Hodgkin's lymphomas; Malignant histiocytosis; Multiple myeloma; Mast cell sarcoma; Immunoproliferative enteropathy; Leukemia; Lymphoid leukemia; Plasma cell leukemia; Erythroleukemia; Lymphosarcoma cell leukemia; Myeloid leukemia; Basophilic leukemia; Eosinophilic leukemia; Monocytic leukemia; Mast cell leukemia; Megakaryocytic leukemia; Myeloid sarcoma; Plasmacytoma, colorectal cancer, rectal cancer, and hairy cell leukemia.

[0205] In some embodiments, the target RNA is a transcript (e.g., mRNA) associated with a disease selected from the group consisting of: (shown in the format of "Disease or Disorder - Causal Gene or Transcript")

[0206] Neuronal:

[0207] Rett syndrome-MECP2,

[0208] MDS-MECP2,

[0209] Angles syndrome-UBE3A-ATS,

[0210] AADC deficiency-AADC,

[0211] Spongiform leukoencephalopathy-ASPA,

[0212] Late infantile neuronal ceroidlipofuscinosis - CLN2 (also known as TPP1),

[0213] Friedreich's Ataxia - FRDA (also called FXN),

[0214] Giant axonal neuropathy-GAN,

[0215] Leber hereditary optic neuropathy-ND1 / ND4;

[0216] Eye:

[0217] Achromatopsia - CNGA3,

[0218] Leber congenital amaurosis 10 protein-CEP290,

[0219] Retinitis pigmentosa-RHO;

[0220] Muscular:

[0221] Dysferlin myopathy-DYSF,

[0222] Danon Disease-LAMP2,

[0223] Myotonic dystrophy type 1 (DM1) - DMPK;

[0224] Ear:

[0225] Pendley syndrome - SLC26A4,

[0226] Wolfram syndrome-WFS1,

[0227] Stickler syndrome-COL11A2,

[0228] Nonsyndromic deafness-GJB2 / OTOF / Myo6 / STRC / KCNQ4 / TECTA;

[0229] Hepatic:

[0230] Homozygous familial hypercholesterolemia-LDLR / PCSK9,

[0231] Alpha-1 antitrypsin deficiency - SERPINA1;

[0232] other:

[0233] Phenylketonuria-phenylalanine hydroxylase (PAH),

[0234] Crigler-Najjar Syndrome-UGT1A1,

[0235] Ornithine transcarbamylase (OTC) deficiency - OTC,

[0236] Glycogen storage disease type IA - G6Pase.

[0237] In some embodiments, the disease is selected from glaucoma, age-related RGC loss, optic nerve damage, retinal ischemia, Leber's hereditary optic neuropathy, a neurological disorder associated with degeneration of RGC neurons, a neurological disorder associated with degeneration of functional neurons in the striatum of a subject in need thereof, Parkinson's disease, Alzheimer's disease, Huntington's disease, schizophrenia, depression, drug addiction, movement disorders such as chorea, choreoathetosis and dyskinesia, bipolar disorder, autism spectrum disorder (ASD), dysfunction, MECP2 duplication syndrome (MDS), Angelman syndrome, age-related macular degeneration (AMD) and amyotrophic lateral sclerosis (ALS).

[0238] In some embodiments, the administering comprises local administration or systemic administration.

[0239] In some embodiments, the administration comprises intrathecal administration, intramuscular administration, intravenous administration, transdermal administration, intranasal administration, oral administration, mucosal administration, intraperitoneal administration, intracranial administration, intracerebroventricular administration, or stereotactic administration.

[0240] In some embodiments, the administering is performed by injection.

[0241] In some embodiments, the subject is a human.

[0242] The dosage of rAAV particles used to treat the disease can be a single dose or multiple doses. It should be understood by those skilled in the art that the actual dosage may vary greatly depending on a variety of factors, such as vector selection, target cells, organisms, tissues, the general condition of the subject to be treated, the degree of transformation / modification sought, the route of administration, the mode of administration, the type of transformation / modification sought, etc.

[0243] In some embodiments, the rAAV particles are administered at a therapeutically effective dose. For example, the therapeutically effective dose of the rAAV particles can be about 1.0E+8, 2.0E+8, 3.0E+8, 4.0E+8, 6.0E+8, 8.0E+8, 1.0E+9, 2.0E+9, 3.0E+9, 4.0E+9, 6.0E+9, 8.0E+9, 1.0E+10, 2.0E+10, 3.0E+10, 4.0E+10, 6.0E+10, 8.0E+10, 1.0E+11, 2.0E+11, 3.0E+11, 4.0E+11, 6.0E+11, 8.0E+11, 1.0E+12, 2.0E+12, 3.0E+12, 4.0E+12, + 15, 2.0E+15, 3.0E+15, 4.0E+15, 6.0E+15, 8.0E+15, 1.0E+16, 2.0E+16, 3.0E+16, 4.0E+16, 6.0E+16, 8.0E+16, or 1.0E+17 vg, or within any two of those point values. vg represents the vector genome of the rAAV particle used for administration.

[0244] In yet another aspect, the present disclosure provides a pharmaceutical composition comprising the system of the present disclosure or the rAAV particle of the present disclosure, and a pharmaceutically acceptable excipient.

[0245] In some embodiments, the pharmaceutical composition comprises rAAV particles at a concentration selected from about 1×10 10 vg / mL, 2×10 10 vg / mL, 3×10 10 vg / mL, 4×10 10 vg / mL, 5×10 10 vg / mL, 6×10 10 vg / mL, 7×10 10 vg / mL, 8×10 10 vg / mL, 9×10 10 vg / mL, 1×10 11 vg / mL, 2×10 11 vg / mL, 3×10 11 vg / mL, 4×10 11 vg / mL, 5×10 11 vg / mL, 6×10 11vg / mL, 7×10 11 vg / mL, 8×10 11 vg / mL, 9×10 11 vg / mL, 1×10 12 vg / mL, 2×10 12 vg / mL, 3×10 12 vg / mL, 4×10 12 vg / mL, 5×10 12 vg / mL, 6×10 12 vg / mL, 7×10 12 vg / mL, 8×10 12 vg / mL, 9×10 12 vg / mL, 1×10 13 vg / mL, or a concentration range between any two of the foregoing values, for example, a concentration from about 9×10 10 vg / mL to about 8×10 11 vg / mL.

[0246] In some embodiments, the pharmaceutical composition is an injection.

[0247] In some embodiments, the volume of the injection is selected from about 1 μl, 10 μl, 50 μl, 100 μl, 150 μl, 200 μl, 250 μl, 300 μl, 350 μl, 400 μl, 450 μl, 500 μl, 550 μl, 600 μl, 650 μl, 700 μl, 750 μl, 800 μl, 850 μl, 900 μl, 950 μl, 1000 μl, and a volume range between any two of the foregoing values, for example, a concentration of about 10 μl to about 750 μl.

[0248] deliver

[0249] Through the disclosure and the knowledge in the art, the CRISPR-Cas13f system of the present disclosure can be delivered by various delivery systems (such as vectors, such as plasmids, viral vectors, lipid nanoparticles (LNP)) using any suitable means in the art. Such methods include but are not limited to electroporation, lipofection, microinjection, transfection, ultrasound, gene guns, etc.

[0250] One or more components of the CRISPR-Cas13f system disclosed herein, for example, the engineered Cas13f polypeptide or a polynucleotide (e.g., DNA, mRNA) encoding the engineered Cas13f polypeptide, the guide nucleic acid (e.g., gRNA) or a polynucleotide encoding the guide nucleic acid, can be delivered using one or more suitable vectors, such as plasmids, viral vectors, LNPs, such as adeno-associated viruses (AAV), lentiviruses, adenoviruses, retroviral vectors, and other viral vectors, or a combination thereof. One or more components can be packaged or encoded into one or more vectors (e.g., plasmids, viral vectors, LNPs).

[0251] The vector can be a cloning vector or an expression vector. The vector can be a plasmid, a phagemid, a cosmid, etc. The vector can include one or more regulatory elements that allow the vector to propagate in a cell of interest (e.g., a bacterial cell or a mammalian cell). In some embodiments, the vector comprises a polynucleotide encoding a single component of a system described herein. In some embodiments, the vector comprises a plurality of polynucleotides, each of which encodes a single component of a system described herein.

[0252] In yet another aspect, the present disclosure provides a vector comprising a polynucleotide of the present disclosure.

[0253] In some embodiments, the polynucleotide is operably linked to a promoter. In some embodiments, the polynucleotide is operably linked to an enhancer. In some embodiments, the promoter is a constitutive promoter, an inducible promoter, a ubiquitous promoter, or a cell, tissue or organ specific promoter, as described herein.

[0254] In some embodiments, the vector comprises a first polynucleotide encoding an engineered Cas13f polypeptide of the present disclosure and a second polynucleotide encoding a guide nucleic acid of the present disclosure. In some embodiments, the first polynucleotide and the second polynucleotide are operably connected to the same promoter or separate promoters.

[0255] In some embodiments, the vector is a plasmid. In some embodiments, the delivery is carried out via a plasmid, for example for use in in vitro cell transfection. The dosage can be a sufficient number of plasmids to elicit a response. In some cases, the appropriate amount of plasmid DNA in a plasmid composition can be from about 0.1 to about 2 mg. Plasmids typically include (i) a promoter; (ii) a sequence encoding an engineered Cas13 polypeptide operably connected to (i); (iii) a selectable marker; (iv) an origin of replication; and (v) a transcription terminator located downstream of (ii) and operably connected thereto. The plasmid can further encode a guide nucleic acid of the CRISPR-Cas13f system, which can be operably connected to another promoter to produce an all-in-one plasmid.

[0256] In some embodiments, the vector is a retroviral vector, a phage vector, an adenoviral vector, a herpes simplex virus (HSV) vector, an AAV vector, or a lentiviral vector.

[0257] In some embodiments, the AAV vector is a recombinant AAV particle comprising a capsid, and the serotype of the capsid is a member of the evolutionary branch to which any one of AAV1, AAV2, AAV3A, AAV3B, AAV4, AAV5, AAV6, AAV7, AAVrh74, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV-DJ, AAV.PHP.eB, AAV1-AAV13 belongs, or a functional variant thereof (e.g., a functional truncation).

[0258] In some embodiments, the AAV vector is an AAV particle that encapsulates RNA.

[0259] In yet another aspect, the present disclosure provides a recombinant adeno-associated virus (rAAV) vector genome, the rAAV vector genome comprising:

[0260] (a) a first polynucleotide sequence comprising a sequence encoding a guide nucleic acid, the guide nucleic acid comprising:

[0261] (1) a DR sequence capable of forming a complex with the engineered Cas13f polypeptide of the present disclosure, and

[0262] (2) capable of hybridizing to a target sequence of a target RNA, thereby directing the complex to a spacer sequence of the target RNA; and

[0263] (b) a second polynucleotide sequence comprising a sequence encoding the engineered Cas13f polypeptide,

[0264] The rAAV vector genome is suitable for encapsulation into recombinant AAV particles.

[0265] Adeno-associated virus (AAV), when engineered to deliver, for example, a protein coding sequence of interest, may be referred to as an (r)AAV vector, (r)AAV vector particle, or (r)AAV particle, where "r" stands for "recombinant". The genome packaged in an AAV vector for delivery may be referred to as an (r)AAV vector genome, vector genome, or simply vg, and the viral genome may refer to the original viral genome of a native AAV.

[0266] The serotype of the rAAV particle capsid can be matched to the type of target cell. For example, Table 2 of WO 2018002719 A1 lists exemplary cell types that can be transduced by a specified AAV serotype (incorporated herein by reference).

[0267] In some embodiments, the rAAV particles include a capsid with a serotype suitable for delivery to neural cells (e.g., neurons). In some embodiments, the rAAV particles include a capsid, and the serotype of the capsid is a member of a clade to which any one of AAV1, AAV2, AAV3A, AAV3B, AAV4, AAV5, AAV6, AAV7, AAVrh74, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV-DJ, or AAV.PHP.eB, AAV1-AAV13 belongs, or a functional variant thereof (e.g., a functional truncation), encapsulating the rAAV vector genome. In some embodiments, the serotype of the capsid is AAV9 or AAV.PHP.eB or a mutant thereof.

[0268] The general principles of rAAV particle production are known in the art. In some embodiments, rAAV particles can be produced using the triple transfection method (described in detail in US Pat. No. 6,001,650).

[0269] Vector titer is usually expressed as vector genome / ml (vg / ml). In some embodiments, the vector titer is greater than 1×10 9 , higher than 5×10 10 , higher than 1×10 11 , higher than 5×10 11 , higher than 1×10 12 , higher than 5×10 12 , or higher than 1×10 13 vg / ml.

[0270] As an alternative to packaging single-stranded (ss) DNA sequences as vector genomes of rAAV particles, systems and methods for packaging RNA sequences as vector genomes into rAAV particles have recently been developed and can be applied herein. See PCT / CN2022 / 075366, which is incorporated herein by reference in its entirety.

[0271] When the vector genome is RNA, such as in PCT / CN2022 / 075366, for simplicity of description and declaration, the sequence elements described herein for the DNA vector genome, when present in the RNA vector genome, should generally be considered to be applicable to the RNA vector genome, except that the deoxyribonucleotides in the DNA sequence are the corresponding ribonucleotides in the RNA sequence (e.g., dT is equivalent to U, and dA is equivalent to A) and / or the elements in the DNA sequence are replaced with corresponding elements having corresponding functions in the RNA sequence, or are omitted because their functions are not required in the RNA sequence, and / or additional elements necessary for the RNA vector genome are introduced.

[0272] As used herein, a coding sequence (e.g., as a sequence element of an rAAV vector genome herein) is interpreted, understood, and considered to cover and encompass both DNA coding sequences and RNA coding sequences. When it is a DNA coding sequence, an RNA sequence can be transcribed from the DNA coding sequence, and optionally a protein can be further translated from the transcribed RNA sequence as needed. When it is an RNA coding sequence, the RNA coding sequence itself can be a functional RNA sequence for use, or the RNA sequence can be produced by the RNA coding sequence, for example, by RNA processing, or a protein can be translated from the RNA coding sequence.

[0273] For example, a Cas13 coding sequence encoding a Cas13 polypeptide covers a Cas13 DNA coding sequence from which the Cas13 polypeptide is expressed (indirectly via transcription and translation) or a Cas13 RNA coding sequence from which the Cas13 polypeptide is translated (directly).

[0274] For example, a gRNA coding sequence encoding a gRNA covers a gRNA DNA coding sequence from which the gRNA is transcribed, or a gRNA RNA coding sequence (which (1) is itself a functional gRNA for use or (2) from which a gRNA is generated by, for example, RNA processing).

[0275] In some embodiments of the rAAV RNA vector genome, the 5'-ITR and / or 3'-ITR may not be necessary as DNA packaging signals and may be at least partially omitted, but RNA packaging signals may be introduced.

[0276] In some embodiments of the rAAV RNA vector genome, a promoter driving transcription of a DNA sequence may not be necessary and may be at least partially omitted.

[0277] In some embodiments of the rAAV RNA vector genome, the sequence encoding the polyA signal may be unnecessary and may be at least partially omitted, but a polyA tail may be introduced.

[0278] Similarly, other DNA elements of the rAAV DNA vector genome can be omitted or replaced with corresponding RNA elements and / or additional RNA elements can be introduced to accommodate strategies for delivering the RNA vector genome by rAAV particles.

[0279] In some embodiments, the vector (e.g., plasmid or viral vector) is delivered to the tissue of interest by, for example, intramuscular injection, intravenous administration, transdermal administration, intranasal administration, oral administration, or mucosal administration. Such delivery can be carried out via a single dose or multiple doses. It will be appreciated by those skilled in the art that the actual dose to be delivered herein may vary greatly depending on a variety of factors, such as vector selection, target cells, organisms, tissues, the general condition of the subject to be treated, the degree of conversion / modification sought, route of administration, mode of administration, the type of conversion / modification sought, etc.

[0280] In some embodiments, the delivery is via an adenovirus, which may contain at least 1×10 5 In some embodiments, the dose is preferably at least about 1×10 6 particles, at least about 1×10 7 particles, at least about 1×10 8 particles, and at least about 1×10 9 The delivery method and the dosage are described in, for example, WO 2016205764 A1 and US Pat. No. 8,454,972 B2, both of which are incorporated herein by reference in their entirety.

[0281] In another embodiment, the delivery is carried out via liposomes or lipid transfection formulations, etc., and can be prepared by methods known to those skilled in the art. Such methods are described in, for example, WO 2016205764 and U.S. Patent Nos. 5,593,972, 5,589,466, and 5,580,859, each of which is incorporated herein by reference in its entirety.

[0282] In some embodiments, the delivery is via nanoparticles or exosomes. For example, exosomes have been shown to be particularly useful in delivering RNA.

[0283] Various delivery methods for the CRISPR-Cas13 system are also described in, for example, U.S. Pat. No. 8,795,965, EP3009511, WO 2016205764, and WO 2017070605; each of which is incorporated herein by reference in its entirety.

[0284] In yet another aspect, the present disclosure provides a delivery system comprising (1) a delivery vehicle, and (2) an engineered Cas13f polypeptide of the present disclosure, a polynucleotide of the present disclosure, a CRISPR-Cas13f system of the present disclosure, or a vector of the present disclosure.

[0285] In some embodiments, the delivery vehicle is a nanoparticle (eg, LNP), a liposome, an exosome, a microbubble, or a gene gun.

[0286] cell

[0287] The disclosed methods and / or systems can be used to modify the translation and / or transcription of one or more RNA products of a cell. For example, the modification may result in an increase in the transcription / translation / expression of the RNA product. In other embodiments, the modification may result in a decrease in the transcription / translation / expression of the RNA product.

[0288] The methods of the present disclosure can be used to introduce the systems described herein into cells and cause the cells and / or their progeny to alter the production of one or more cell products (such as antibodies, starch, ethanol, or any other desired product). Such cells and their progeny are within the scope of the present disclosure.

[0289] In yet another aspect, the disclosure provides a cell or its progeny, the cell or its progeny comprising an engineered Cas13f polypeptide of the disclosure or a system of the disclosure. In some embodiments, the cell is a eukaryotic organism. In some embodiments, the cell is a human cell.

[0290] In yet another aspect, the disclosure provides a cell or its progeny, wherein the cell or its progeny is modified by a system of the disclosure or a method of the disclosure. In some embodiments, the cell is a eukaryotic organism. In some embodiments, the cell is a human cell. In some embodiments, the cell is modified in vitro, in vivo, or ex vivo.

[0291] In some embodiments, the cell is a stem cell. In some embodiments, the cell is not a human embryonic stem cell. In some embodiments, the cell is not a human germ cell.

[0292] In some embodiments, the cell is a prokaryotic cell.

[0293] In some embodiments, the cell is a eukaryotic cell, such as a mammalian cell, including a human cell (primary human cell or an established human cell line). In some embodiments, the cell is a non-human mammalian cell, such as a cell from a non-human primate (e.g., monkey), cow / bull / cow, sheep, goat, pig, horse, dog, cat, rodent (e.g., rabbit, mouse, rat, hamster, etc.). In some embodiments, the cell is from fish (e.g., salmon), bird (e.g., poultry, including chicken, duck, goose), reptile, shellfish (e.g., oyster, clam, lobster, prawn), insect, worm, yeast, etc. In some embodiments, the cell is from a plant, such as a monocot or dicot. In certain embodiments, the plant is a food crop, such as barley, cassava, cotton, groundnut or peanut, maize, millet, oil palm fruit, potato, dry bean, rapeseed or canola, rice, rye, sorghum, soybean, sugarcane, beet, sunflower and wheat. In some embodiments, the plant is a cereal (barley, maize, millet, rice, rye, sorghum, and wheat). In some embodiments, the plant is a tuber (cassava and potato). In some embodiments, the plant is a sugar crop (sugar beet and sugar cane). In some embodiments, the plant is an oil crop (soybean, groundnut or peanut, rapeseed or canola, sunflower, and oil palm fruit). In some embodiments, the plant is a fiber crop (cotton). In some embodiments, the plant is a tree (such as a peach or nectarine tree, an apple or pear tree, a nut tree (such as an almond or walnut or pistachio tree), or a citrus tree (e.g., an orange, grapefruit, or lemon tree)), a grass, a vegetable, a fruit, or an algae. In certain embodiments, the plant is a Solanum; a Brassica; a Lactuca; a Spinacia; a Capsicum; cotton, tobacco, asparagus, carrot, cabbage, broccoli, cauliflower, tomato, eggplant, pepper, lettuce, spinach, strawberry, blueberry, raspberry, blackberry, grape, coffee, cocoa, etc.

[0294] Reagent test kit

[0295] In yet another aspect, the present disclosure provides a kit comprising an engineered Cas13f polypeptide of the present disclosure, a CRISPR-Cas13f system of the present disclosure, a vector of the present disclosure, or a delivery system of the present disclosure.

[0296] In some embodiments, the kit further comprises instructions for using the components contained therein, and / or for combining with additional components that may be obtained or required elsewhere.

[0297] In some embodiments, the kit further comprises one or more buffers, which can be used to dissolve any component contained therein and / or provide suitable reaction conditions for one or more of the components. Such buffers can include one or more of the following: PBS, HEPES, Tris, MOPS, Na2CO3, NaHCO3, NaB or a combination thereof. In some embodiments, the reaction conditions include suitable pH, such as alkaline pH. In some embodiments, the pH is between 7-10.

[0298] In some embodiments, any one or more of the kit components can be stored in a suitable container or at a suitable temperature, such as 4 degrees Celsius.

[0299] Further embodiments are described in the following examples, which are provided for illustrative purposes only and are not intended to limit the scope of the present disclosure.

[0300] The present disclosure provides the following additional embodiments.

[0301] One aspect of the present disclosure provides an engineered Cas13f polypeptide, wherein the engineered Cas13f polypeptide:

[0302] (1) comprising mutations in a region that is spatially proximal to: a) an N-terminal endonuclease catalytic RXXXXH motif (e.g., an N-terminal endonuclease catalytic RNFYSH motif) of a reference Cas13f polypeptide (e.g., SEQ ID NO: 1), and / or b) a C-terminal endonuclease catalytic RXXXXH motif (e.g., a C-terminal endonuclease catalytic RNKALH motif) of the reference Cas13f polypeptide (e.g., SEQ ID NO: 1);

[0303] (2) substantially retains (e.g., has at least about 50%, 60%, 70%, 72.5%, 75%, 77.5%, 80%, 82.5%, 85%, 87.5%, 90%, 92.5%, 95%, 96%, 97%, 98%, 99% or more) the spacer-specific cleavage activity of the reference Cas13f polypeptide (e.g., of SEQ ID NO: 1) against a target RNA complementary to the spacer sequence; and

[0304] (3) substantially lacking (e.g., having no more than about 50%, 45%, 40%, 35%, 30%, 27.5%, 25%, 22.5%, 20%, 17.5%, 15%, 12.5%, 10%, 7.5%, 5%, 4.5%, 4%, 3.5%, 3%, 2.5%, 2%, 1.5%, 1% or less) the reference Cas13f polypeptide (e.g., of SEQ ID NO: 1) spacer-independent cleavage activity against a non-target RNA that does not bind to the spacer sequence.

[0305] In some embodiments, the region includes residues within 140, 130, 120, 110, 100, 90, 80, 70, 60, 50, 40, 30, 20 or 10 amino acids of any residue of the N-terminal endonuclease catalytic RXXXXH motif or the C-terminal endonuclease catalytic RXXXXH motif.

[0306] In some embodiments, the region includes residues that are more than 100, 110, 120, or 130 residues away from any residue of the N-terminal endonuclease catalytic RXXXXH motif or the C-terminal endonuclease catalytic RXXXXH motif, but spatially within about 1 angstrom to about 10 angstroms or about 5 angstroms of any residue of the N-terminal endonuclease catalytic RXXXXH motif or the C-terminal endonuclease catalytic RXXXXH motif.

[0307] In some embodiments, the region comprises, consists essentially of, or consists of residues corresponding to the following domains of a reference Cas13f polypeptide of SEQ ID NO: 1: a HEPN1 domain (e.g., residues 1-168), an IDL domain (e.g., residues 168-185), a Helical1 domain (e.g., a Helical1-1 (Hel1-1) domain (e.g., residues 185-234), a Helical1-2 (Hel1-2) domain (e.g., residues 281-346), a Helical1-3 (Hel1-3) domain (e.g., residues 477-644)), a Helical2 domain (e.g., residues 346-477), or a HEPN2 domain (e.g., residues 644-790).

[0308] In some embodiments, the mutation comprises, consists essentially of, or consists of the following within a stretch of about 8 to about 20 (e.g., about 9 or about 17) consecutive amino acids within the region:

[0309] (a) substitution of one or more (e.g., 1, 2, 3, 4, 5 or more) non-Ala(A) residues to Ala(A) residues;

[0310] (b) substitution of one or more (e.g., 1, 2, 3, 4, 5, or more) charged residues, residues containing nitrogen side chain groups, bulky (e.g., F or Y), aliphatic residues, and / or polar residues with charge-neutral short-chain aliphatic residues (e.g., A, V, or I);

[0311] (c) substitution of one or more (e.g., 1, 2, 3, 4, 5 or more) Ile (I) and / or Leu (L) residues to Ala (A) residues; and / or

[0312] (d) substitution of one or more (e.g., 1, 2, 3, 4, 5 or more) Ala (A) residues to Val (V) residues.

[0313] In some embodiments, the one or more non-Ala residues and / or the one or more charged or polar residues comprise N, Q, R, K, H, D, E, Y, S, T, L residues or combinations thereof.

[0314] In some embodiments, the one or more non-Ala residues and / or the one or more charged or polar residues comprise N, Q, R, K, H, D, Y, L residues or combinations thereof.

[0315] In some embodiments, one or more Y residues within the stretch are substituted.

[0316] In some embodiments, the one or more Y residues correspond to Y666 and / or Y677 of the reference Cas13f polypeptide of SEQ ID NO: 1.

[0317] In some embodiments, one or more D residues within the stretch are substituted.

[0318] In some embodiments, the one or more D residues correspond to D160 and / or D642 of the reference Cas13f polypeptide of SEQ ID NO: 1.

[0319] In some embodiments, the charge-neutral short-chain aliphatic residue is Ala (A).

[0320] In some embodiments, the mutation comprises, consists essentially of, or consists of:

[0321] (a) substitution within a stretch of about 8 to about 20 (e.g., about 9 or about 17) consecutive amino acids within the region;

[0322] (b) a mutation corresponding to the following mutations (e.g., any one of Tables 1-4) or a combination thereof, which results in an engineered Cas13f polypeptide having at least about 75% spacer-specific cleavage activity and no more than about 25% spacer-independent cleavage activity; and / or

[0323] (c) mutations corresponding to the F7V2, F10V1, F10V4, F40V4, F40S22, F40S26, F40S36, F10S21, F10S24, F10S26, F10S27, F10S33, F10S34, F10S35, F10S36, F10S45, F10S46, F10S48, F10S49, F40S23, or F40S27 mutations in Tables 1-4, or a combination thereof.

[0324] In some embodiments, the engineered Cas13f polypeptide retains at least about 50%, 60%, 70%, 72.5%, 75%, 77.5%, 80%, 82.5%, 85%, 87.5%, 90%, 92.5%, 95%, 96%, 97%, 98%, 99% or more of the spacer-specific cleavage activity of the reference Cas13f polypeptide of SEQ ID NO: 1 against the target RNA.

[0325] In some embodiments, the engineered Cas13f polypeptide has no more than 50%, 45%, 40%, 35%, 30%, 27.5%, 25%, 22.5%, 20%, 17.5%, 15%, 12.5%, 10%, 7.5%, 5%, 4.5%, 4%, 3.5%, 3%, 2.5%, 2%, 1.5%, 1% or less of the spacer-independent cleavage activity of the reference Cas13f polypeptide of SEQ ID NO: 1 on the non-target RNA.

[0326] In some embodiments, the engineered Cas13f polypeptide has at least about 80% of the spacer-specific cleavage activity of the reference Cas13f polypeptide of SEQ ID NO: 1 on the target RNA, and no more than about 40% of the spacer-independent cleavage activity of the reference Cas13f polypeptide of SEQ ID NO: 1 on the non-target RNA.

[0327] In some embodiments, the mutation is F40S23 (ie, a Y666A / Y677A double mutation).

[0328] In some embodiments, the engineered Cas13f polypeptide comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 3.

[0329] In some embodiments, the engineered Cas13f polypeptide further comprises a mutation corresponding to any one, a combination of two or more (e.g., 3, 4, or 5 or more) mutations (e.g., D160A, D642A, and / or L641A) in Table 5.

[0330] In some embodiments, the mutation is a combination of any one, two or more (e.g., 3, 4 or 5 or more) single mutations (e.g., D160A, D642A and / or L641A) in Table 5 and F40S23 (i.e., Y666A / Y677A double mutation).

[0331] In some embodiments, the mutation is a combination of a Y666A / Y677A double mutation and 1, 2 or 3 mutations selected from D160A, L641A and D642A.

[0332] In some embodiments, the mutations are any combination of mutations in Tables 6-11.

[0333] In some embodiments, the mutation is a D160A / D642A / Y666A / Y677A quadruple mutation.

[0334] In some embodiments, the engineered Cas13f polypeptide has increased spacer sequence-specific cleavage activity compared to the engineered Cas13f polypeptide of SEQ ID NO: 3.

[0335] In some embodiments, the mutation is a mutation corresponding to a combination of a mutation in Tables 12-15 and a D160A / D642A / Y666A / Y677A mutation.

[0336] In some embodiments, the engineered Cas13f polypeptide comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO:4.

[0337] In some embodiments, the engineered Cas13f polypeptide further comprises an amino acid substitution of a non-basic amino acid residue to an Arg (R) residue.

[0338] In some embodiments, the engineered Cas13f polypeptide further comprises a mutation corresponding to any one, a combination of two or more (e.g., 3, 4 or 5 or more) single mutations in Tables 12-15.

[0339] In some embodiments, the engineered Cas13f polypeptide has increased spacer sequence-specific cleavage activity compared to the engineered Cas13f polypeptide of SEQ ID NO:4.

[0340] In some embodiments, the engineered Cas13f polypeptide has at least 80%, at least 85%, 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%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, and less than 100% sequence identity to the reference Cas13f polypeptide of SEQ ID NO: 1.

[0341] In some embodiments, the engineered Cas13f polypeptide further comprises a nuclear localization signal (NLS) sequence or a nuclear export signal (NES).

[0342] In some embodiments, the engineered Cas13f polypeptide further comprises an N-terminal and / or C-terminal NLS.

[0343] Another aspect of the present disclosure provides polynucleotides encoding the engineered Cas13f polypeptides of the present disclosure.

[0344] In some embodiments, the polynucleotide is codon optimized for expression in a eukaryote, a mammal such as a human or non-human mammal, a plant, an insect, a bird, a reptile, a rodent (e.g., a mouse, a rat), a fish, a worm / nematode, or a yeast.

[0345] Another aspect of the present disclosure provides a CRISPR-Cas13f system, comprising:

[0346] a) an engineered Cas13f polypeptide of the present disclosure or a polynucleotide encoding sequence thereof (e.g., a DNA encoding sequence or an RNA encoding sequence); and

[0347] b) a guide RNA (gRNA) or a polynucleotide encoding sequence thereof (e.g., a DNA encoding sequence or an RNA encoding sequence), the gRNA comprising:

[0348] i. a direct repeat (DR) sequence capable of forming a complex with the engineered Cas13f polypeptide; and

[0349] ii. A spacer sequence capable of hybridizing to a target RNA and directing or recruiting the complex to the target RNA.

[0350] In some embodiments, the DR sequence has a secondary structure substantially identical to the secondary structure of SEQ ID NO:2.

[0351] In some embodiments, the length of the spacer sequence is at least 15 nucleotides. In some embodiments, the length of the spacer sequence is 30 nucleotides.

[0352] Another aspect of the present disclosure provides a vector comprising a polynucleotide of the present disclosure.

[0353] In some embodiments, the polynucleotide is operably linked to a promoter. In some embodiments, the polynucleotide is operably linked to an enhancer.

[0354] In some embodiments, the promoter is a constitutive promoter, an inducible promoter, a broad spectrum promoter, or a cell, tissue or organ specific promoter.

[0355] In some embodiments, the vector is a plasmid.

[0356] In some embodiments, the vector is a retroviral vector, a phage vector, an adenoviral vector, a herpes simplex virus (HSV) vector, an AAV vector, or a lentiviral vector.

[0357] In some embodiments, the AAV vector is a recombinant AAV vector of serotype AAV1, AAV2, AAV4, AAV5, AAV6, AAV7, AAVrh74, AAV8, AAV9, AAV10, AAV 11, AAV 12, AAV 13, AAV.PHP.eB or AAV-DJ.

[0358] In some embodiments, the AAV vector is an RNA-encapsulated AAV vector.

[0359] Another aspect of the present disclosure provides a delivery system comprising (1) a delivery vehicle, and (2) an engineered Cas13f polypeptide of the present disclosure, a polynucleotide of the present disclosure, a CRISPR-Cas13f system of the present disclosure, or a vector of the present disclosure.

[0360] In some embodiments, the delivery vehicle is a nanoparticle (eg, LNP), a liposome, an exosome, a microbubble, or a gene gun.

[0361] Another aspect of the present disclosure provides a cell or its progeny, which comprises an engineered Cas13f polypeptide of the present disclosure, a polynucleotide of the present disclosure, a CRISPR-Cas13f system of the present disclosure, a vector of the present disclosure, or a delivery system of the present disclosure.

[0362] In some embodiments, the cell is a eukaryotic cell (eg, a non-human mammalian cell, a human cell, or a plant cell) or a prokaryotic cell (eg, a bacterial cell).

[0363] Another aspect of the present disclosure provides a non-human multicellular eukaryotic organism comprising a cell or progeny of the present disclosure.

[0364] In some embodiments, the non-human multicellular eukaryotic organism is an animal (eg, rodent or primate) model for a human genetic disorder.

[0365] Another aspect of the present disclosure provides a method of modifying a target RNA, the method comprising contacting the target RNA with the CRISPR-Cas13f system of the present disclosure, the vector of the present disclosure, the delivery system of the present disclosure, or the cell or progeny of the present disclosure.

[0366] In some embodiments, the target RNA is modified by cleavage by the engineered Cas13f polypeptide.

[0367] In some embodiments, the target RNA is mRNA, tRNA, rRNA, non-coding RNA, lncRNA, or nuclear RNA.

[0368] In some embodiments, after the complex of the engineered Cas13f polypeptide and the guide RNA binds to the target RNA, the engineered Cas13f polypeptide does not exhibit substantial (or detectable) spacer-independent side-cutting activity.

[0369] In some embodiments, the target RNA is intracellular.

[0370] In some embodiments, the cell is a cancer cell.

[0371] In some embodiments, the cell is infected with an infectious agent.

[0372] In some embodiments, the infectious agent is a virus, a prion, a protozoan, a fungus, or a parasite.

[0373] In some embodiments, the cell is a neuronal cell (eg, an astrocyte, a glial cell (eg, a Muller glia cell, an oligodendrocyte, an ependymal cell, a Schwan cell, an NG2 cell, or a satellite cell)).

[0374] In some embodiments, the CRISPR-Cas13f system is encoded by: a first polynucleotide encoding the engineered Cas13f polypeptide, and a second polynucleotide comprising or encoding the guide RNA, wherein the first polynucleotide and the second polynucleotide are introduced into the cell.

[0375] In some embodiments, the first polynucleotide and the second polynucleotide are introduced into the cell via the same vector.

[0376] In some embodiments, the contacting results in one or more of: (i) induction of cellular senescence in vitro or in vivo; (ii) cell cycle arrest in vitro or in vivo; (iii) cell growth inhibition in vitro or in vivo; (iv) induction of anergy in vitro or in vivo; (v) induction of apoptosis in vitro or in vivo; and (vi) induction of necrosis in vitro or in vivo.

[0377] Another aspect of the present disclosure provides a method for treating a disease in a subject in need thereof, the method comprising administering to the subject a composition comprising: a CRISPR-Cas13f system of the present disclosure, a vector of the present disclosure, a delivery system of the present disclosure, or a cell or progeny of the present disclosure; wherein, after administration, the engineered Cas13f polypeptide cleaves the target RNA, thereby treating the disease in the subject.

[0378] In some embodiments, the disease is a neurological disorder, cancer, an infectious disease, or a genetic disorder.

[0379] In some embodiments, the cancer is Wilms tumor, Ewing sarcoma, a neuroendocrine tumor, a glioblastoma, a neuroblastoma, a melanoma, a skin cancer, a breast cancer, a colon cancer, a rectal cancer, a prostate cancer, a liver cancer, a kidney cancer, a pancreatic cancer, a lung cancer, a biliary tract cancer, a cervical cancer, an endometrial cancer, an esophageal cancer, a gastric cancer, a head and neck cancer, a medullary thyroid cancer, an ovarian cancer, a glioma, a lymphoma, a leukemia, a myeloma, an acute lymphocytic leukemia, an acute myeloid leukemia, a chronic lymphocytic leukemia, a chronic myeloid leukemia, a Hodgkin lymphoma, a non-Hodgkin lymphoma, or a bladder cancer.

[0380] In some embodiments, the neurological disorder is glaucoma, age-related RGC loss, optic nerve damage, retinal ischemia, Leber's hereditary optic neuropathy, a neurological disorder associated with degeneration of RGC neurons, a neurological disorder associated with degeneration of functional neurons in the striatum of a subject in need thereof, Parkinson's disease, Alzheimer's disease, Huntington's disease, schizophrenia, depression, drug addiction, movement disorders such as chorea, choreoathetosis and dyskinesia, bipolar disorder, autism spectrum disorder (ASD) or a functional disorder.

[0381] In some embodiments, the method is an in vitro method, an in vivo method, or an ex vivo method.

[0382] Another aspect of the present disclosure provides a CRISPR-Cas13f complex, comprising an engineered Cas13f polypeptide of the present disclosure and a guide RNA comprising a DR sequence and a spacer sequence, wherein the DR sequence is bound to the engineered Cas13f polypeptide, and the spacer sequence is capable of hybridizing with a target RNA and guiding or recruiting the complex to the target RNA.

[0383] In some embodiments, the target RNA is encoded by eukaryotic DNA.

[0384] In some embodiments, the eukaryotic DNA is non-human mammal DNA, non-human primate DNA, human DNA, plant DNA, insect DNA, bird DNA, reptile DNA, rodent DNA, fish DNA, worm / nematode DNA, or yeast DNA.

[0385] In some embodiments, the target RNA is mRNA.

[0386] In some embodiments, the CRISPR-Cas13f complex further comprises a target RNA, wherein the target RNA comprises a sequence capable of hybridizing to the spacer sequence.

[0387] Example

[0388] The following examples are provided to further illustrate some embodiments of the present disclosure but are not intended to limit the scope of the invention; it will be understood by their exemplary nature that other procedures, methods or techniques known to those skilled in the art may alternatively be used.

[0389] Example 1 Engineering of reference Cas13f polypeptide for para-cleavage activity

[0390] This example demonstrates that the spacer-independent cleavage activity ("cleavage activity", "off-target cleavage activity") of a reference Cas13f polypeptide (wild type, "WT", SEQ ID NO: 1) can be substantially reduced by introducing one or more amino acid substitutions, and the reference Cas13f polypeptide ("cleavage activity", "on-target cleavage activity") can be substantially reduced.

[0391] Design and Build:

[0392] The publicly available online tool TASSER was used to predict the 3D structure of the reference Cas13f polypeptide (SEQ ID NO: 1), and as Figure 1 As shown in , the predicted structure was visualized using PyMOL to predict the positions of the various structural domains in 3D.

[0393] Constructed as Figure 2The single-plasmid mammalian dual fluorescent reporter system shown in was used to detect the para-cleavage activity of Cas13f mutants designed based on the reference Cas13f polypeptide.

[0394] The plasmid comprises (1) a Cas13f mutant coding sequence flanked by 5' and 3' SV40 NLS (SEQ ID NO: 5) coding sequences under the regulation of a CAG promoter and a poly A sequence, (2) an EGFP green fluorescent reporter gene under the regulation of an SV40 promoter and a poly A sequence (whose RNA transcript serves as an RNA target for the cleavage activity of the Cas13f mutant), (3) an mCherry red fluorescent reporter gene under the regulation of an SV40 promoter and a poly A sequence (whose RNA transcript serves as an RNA target for the flanking activity of the Cas13f mutant), and (4) a sequence encoding an EGFP targeting guide RNA (SEQ ID NO: 15) consisting of a 5'-DR sequence (SEQ ID NO: 2)-EGFP targeting spacer sequence (SEQ ID NO: 6)-DR sequence (SEQ ID NO: 2)-3' under the regulation of a U6 promoter.

[0395] The HEPN1, HEPN2, IDL, and Hel1-3 domains of the reference Cas13f polypeptide were selected to generate the Cas13f mutagenesis library. Figure 3 ) were selected, and except for F45V1 and F45V2 (each with 9 residues), the remaining small segments each had 17 residues.

[0396] In order to design Cas13f mutants, in several versions, the non-Ala (A) residues (if present) of each segment are replaced with Ala (A) residues, and in several versions, the Ala (A) residues (if present) of each segment are replaced with Val (V) residues. For example, for the F1 segment, F1V1-F1V4 mutants were designed. In each version, about 4-5 total mutations were introduced into each segment to generate Cas13f mutants. The amino acid sequences of the Cas13f mutants and mutant segments thus produced are provided in Table 1 below, and the other parts of each Cas13f mutant are identical to the reference Cas13f polypeptide of SEQ ID NO:1.

[0397] Table 1. Design of Cas13f mutants

[0398]

[0399]

[0400]

[0401] Transfection and detection:

[0402] HEK293T cells were cultured in 24-well tissue culture plates for 12 hours according to standard methods, and then the plasmids were transfected into the cells using standard polyethyleneimine (PEI) transfection. The transfected cells were then cultured at 37°C under 5% CO2 for about 48 hours. The cultured cells were then analyzed by flow cytometry.

[0403] A dead Cas13f ("dCas13f," "dead") without cleavage and para-cleavage activity (a Cas13f mutant having R77A, H82A, R764A and H769A mutations in the HEPN domain of the reference Cas13f polypeptide of SEQ ID NO: 1) was used as a negative control ("NT").

[0404] The cleavage activity of each tested Cas13f mutant and the percentage of EGFP-positive cells (% EGFP + ) was negatively correlated. %EGFP + The lower the cleavage activity, the higher the cleavage activity. Therefore, the cleavage activity of the Cas13f mutant relative to the reference Cas13f polypeptide of SEQ ID NO: 1 ("WT") is shown as the % EGFP of the Cas13f mutant + % EGFP relative to WT + The reciprocal of .

[0405] Cleavage activity relative to WT = 1 / (% EGFP of Cas13f mutant) + % EGFP / WT + ).

[0406] The ratio of para-cleavage activity of each tested Cas13f mutant to the percentage of mCherry-positive cells (% mCherry + ) is negatively correlated. %mCherry + The higher the value, the lower the para-cleavage activity. Therefore, the para-cleavage activity of the Cas13f mutant relative to the reference Cas13f polypeptide of SEQ ID NO: 1 ("WT") is shown as the % mCherry of the Cas13f mutant + % mCherry relative to WT + The reciprocal of .

[0407] Para-cleavage activity relative to WT = 1 / (% mCherry of Cas13f mutant) + %mCherry / WT + ).

[0408] result:

[0409] Flow cytometry results (Table 2) showed the cleavage and para-cleavage activities of Cas13f mutants.

[0410] Table 2. Average cleavage and para-cleavage activities of Cas13f mutants in Table 1 (n=3)

[0411]

[0412]

[0413] Cas13f mutants are arranged in order of low to high cleavage activity relative to WT in Table 2. Note that among those Cas13f mutants with less than 70% cleavage activity relative to WT (highlighted in gray), Cas13f mutants F7V2, F10V1, F10V4, F40V2, and F40V4 have the highest five high cleavage activities relative to WT (highlighted in gray).

[0414] By generating a variety of other Cas13f mutants with single or multiple (e.g., double, triple or quadruple) mutations, a second round of mutagenesis studies was performed in or near the selected regions of these Cas13f mutants. The amino acid sequences of the Cas13f mutants and mutant segments thus produced are provided in Table 3 below, and the other parts of each Cas13f mutant are identical to the reference Cas13f polypeptide of SEQ ID NO:1. Their cutting and flank cutting activities are listed in Table 4 below.

[0415] Table 3. Design of Cas13f mutants

[0416]

[0417]

[0418]

[0419] Table 4. Average cleavage and para-cleavage activities of Cas13f mutants in Table 3 (n=3)

[0420]

[0421]

[0422]

[0423] Cas13f mutants are arranged in order of low to high lateral cleavage activity relative to WT in Table 4. Note that among those Cas13f mutants with less than 40% lateral cleavage activity relative to WT (highlighted in gray), Cas13f mutants F10S48, F10S49, F40S23, F10S33, and F40S26 have the highest five high cleavage activities relative to WT (highlighted in gray).

[0424] Cas13f mutant F40S23 (Cas13f-Y666A, Y677A, SEQ ID NO: 3) was selected for further engineering.

[0425] Example 2 Engineering of mutant F40S23 for increased cleavage activity

[0426] In Example 1, Cas13f mutants were screened for low spacer-independent cleavage activity ("cleavage activity", "off-target cleavage activity"). In order to improve the spacer-specific cleavage activity ("cleavage activity", "on-target cleavage activity") while maintaining low cleavage activity, one or more mutations identified in Example 1 shown in Table 5 were further introduced into the Cas13f mutant F40S23.

[0427] This example demonstrates that by introducing one or more amino acid substitutions, the cleavage activity of F40S23 can be significantly increased, and the para-cleavage activity can be substantially maintained or even reduced.

[0428] Table 5. Available mutations for introduction into mutant F40S23

[0429] mutation The corresponding mutant in Example 1 D160A F10S6 T161A F10S7 L631A F38S1 L634A F38S4 H638A F38S8 L641A F38S11 D642A F38S12 T647A F38S17 P667A F40S3 V670A F40S6 D762A F46S6 A763V F46S7

[0430] Design and Build:

[0431] A two-plasmid mammalian fluorescent reporter system consisting of a reporter plasmid and an expression plasmid was constructed ( Figure 4 ), used to detect the cleavage and para-cleavage activities of Cas13f mutants further engineered based on F40S23.

[0432] The reporter plasmid contains the ATXN2 cDNA coding sequence (its RNA transcript serves as the RNA target for the cleavage activity of the Cas13f mutant), followed by p2A (self-cleaving peptide) and EGFP reporter gene (SEQ ID NO: 7) under the regulation of the SV40 promoter and poly A sequence. EGFP mRNA is transcribed together with the ATXN2 RNA transcript from the reporter plasmid to form a chimeric transcript. When the ATXN2 RNA transcript as a part of the chimeric transcript is cut by the Cas13f mutant guided by the ATXN2 targeting gRNA (SEQ ID NO: 16), the EGFR mRNA as another part of the chimeric transcript will also be gradually degraded due to, for example, total RNA instability, resulting in a decrease in the fluorescence intensity of EGFP (green).

[0433] The expression plasmid comprises (1) a Cas13f mutant coding sequence flanked by both 5' and 3' SV40 NLS (SEQ ID NO: 5) coding sequences under the regulation of the Cbh promoter and poly A sequence, (2) a sequence encoding an ATXN2 targeting gRNA (SEQ ID NO: 16) consisting of 5'-DR sequence (SEQ ID NO: 2)-AXTN2 targeting spacer sequence (SEQ ID NO: 8)-DR sequence (SEQ ID NO: 2)-3' under the regulation of the U6 promoter, and (3) an mCherry reporter gene (its RNA transcript serves as an RNA target for the lateral cutting activity of the Cas13f mutant) under the regulation of the SV40 promoter and poly A sequence. In the case where the Cas13f mutant retains basic lateral cutting activity, the mCherry RNA transcript may be cleaved, resulting in a decrease in the fluorescence intensity of mCherry (red).

[0434] A pair of similar reporter and expression plasmids were constructed in the same manner with the RHO cDNA coding sequence, followed by p2A (self-cleaving peptide) and EGFP reporter genes (SEQ ID NO: 10) and RHO targeting spacer sequence (SEQ ID NO: 11) for additional evaluation of the cleavage and framing activities of Cas13f mutants. The RHO targeting guide RNA consisting of 5'-DR sequence (SEQ ID NO: 2)-RHO targeting spacer sequence (SEQ ID NO: 11)-DR sequence (SEQ ID NO: 2)-3' is shown in SEQ ID NO: 17.

[0435] Transfection and detection:

[0436] According to standard cell culture methods, HEK293T cells were grown to a suitable density in a 24-well tissue culture plate, and then the cells were transfected with two plasmids using PEI transfection reagent. The transfected cells were cultured in an incubator at 37°C under 5% CO2 for about 72 hours, and then the EGFP and mCherry fluorescence signals in the cells were measured using FACS. Low EGFP mean fluorescence intensity (MFI) indicates the desired high cleavage activity. High mCherry MFI indicates the desired low para-cleavage activity.

[0437] As a negative control ("NT"), an expression plasmid encoding F40S23 and a gRNA containing a non-targeting spacer sequence (SEQ ID NO: 9) instead of a targeting spacer sequence were used for transfection together with the reporter plasmid. Since side cutting is only triggered by on-target cutting, in theory, side cutting or on-target cutting should not occur when a non-targeting spacer sequence is used. Therefore, all MFI results (mean ± SD) of Cas13f mutants are normalized relative to the negative control.

[0438] As a positive control ("PT" or "F40S23"), an expression plasmid encoding F40S23 and a gRNA containing a targeting spacer sequence were used for transfection together with the reporter plasmid.

[0439] In addition, RT-qPCR was performed on the endogenous genomic site SOD1 in Cos7 cells for testing to study the SOD1 mRNA knockdown indicating the cleavage activity of the Cas13f mutant. According to standard cell culture methods, Cos7 cells were grown to a suitable density in a 6-well tissue culture plate, and then transfected with each of the expression plasmids encoding the Cas13f mutant and the SOD1 targeting guide RNA (SEQ ID NO: 18) using PEI transfection reagent. After 72 hours, the mCherry positive cells of the first 30% of the amount were sorted by flow sorting, total RNA was extracted from the positive cells, and the SOD1 mRNA level was measured by RT-qPCR and normalized to the housekeeping gene GAPDH.

[0440] The cleavage activity of each tested Cas13f mutant was negatively correlated with the EGFP MFI. The lower the EGFP MFI, the higher the cleavage activity. Therefore, the cleavage activity of the Cas13f mutant relative to the reference F40S23 is shown as the reciprocal of the EGFP MFI of the Cas13f mutant relative to the EGFP MFI of F40S23.

[0441] Cleavage activity relative to F40S23 = 1 / (EGFP MFI of Cas13f mutant / EGFP MFI of F40S23).

[0442] The para-cleavage activity of each tested Cas13f mutant was negatively correlated with the mCherry MFI. The higher the mCherry MFI, the lower the para-cleavage activity. Therefore, the para-cleavage activity of the Cas13f mutant relative to the reference F40S23 is shown as the reciprocal of the mCherry MFI of the Cas13f mutant relative to the mCherry MFI of F40S23.

[0443] Para-cleavage activity relative to F40S23 = 1 / (mCherry MFI of Cas13f mutant / mCherry MFI of F40S23).

[0444] In addition, for RT-qPCR testing, the cleavage activity of each tested Cas13f mutant was negatively correlated with the SOD1 mRNA level. The lower the SOD1 mRNA level, the higher the cleavage activity. Therefore, the cleavage activity of the Cas13f mutant relative to the reference F40S23 is shown as the reciprocal of the SOD1 mRNA level of the Cas13f mutant relative to the SOD1 mRNA level of F40S23.

[0445] Cleavage activity relative to F40S23 = 1 / (SOD1 mRNA level of Cas13f mutant / SOD1 mRNA level of F40S23).

[0446] result:

[0447] Note that the Cas13f mutants listed in Table 6 not only have higher cleavage activity than F40S23, but also have lower side cutting activity than F40S23.

[0448] Table 6. Average cleavage and cleavage activities of the listed Cas13f mutants, as presented by MFI, where gRNA targets ATXN2 RNA transcript (n=3)

[0449]

[0450]

[0451] In addition, the RT-qPCR results in Table 7 below show that the SOD1 mRNA knockdown efficiency of all listed Cas13f mutants was improved compared with F40S23.

[0452] Table 7. Average SOD1 mRNA levels in Cos7 cells by RT-qPCR for the listed Cas13f mutants (n=3)

[0453]

[0454] The above results indicate that the additional introduction of the single point mutations listed in Table 5 into F40S23 enhances the cleavage activity while maintaining or even reducing the para-cleavage activity of F40S23.

[0455] Based on the above results and the same experimental procedure, single mutations were subsequently combined in pairs to be introduced into F40S23 to further evaluate the cleavage and para-cleavage activities of the resulting mutants, as shown in Table 8 below.

[0456] Table 8. Average cleavage and framing activities of the listed Cas13f mutants, as presented by MFI, where gRNA targets RHO RNA transcripts (n=3)

[0457]

[0458]

[0459] The results in Table 8 show that the listed Cas13f mutants have reduced cleavage activity compared to F40S23, and the listed Cas13f mutants have at least 75% of the cleavage activity of F40S23 (e.g., F40S23+L631A and L641A), at least 95% of the cleavage activity of F40S23 (e.g., F40S23+H638A and L641A), or the cleavage activity of the listed Cas13f mutants is increased compared to F40S23.

[0460] In particular, mutants F40S23+D160A and D642A (Cas13f-D160A+D642A+Y666A+Y677A, whose full-length amino acid sequence is shown in SEQ ID NO: 4, named "hfCas13f") achieved both the highest cleavage activity and the lowest side-cutting activity.

[0461] Additional evaluations were performed to verify the cleavage and para-cleavage activities of specific mutants in Tables 9-11.

[0462] Table 9. Average cleavage and framing activities of Cas13f mutants, as presented by MFI, wherein the gRNA targets EGFP RNA transcripts (gRNA shown in SEQ ID NO: 15) (n=3)

[0463]

[0464] The results in Table 9 show that compared to F40S23, the mutant has significantly improved cleavage activity for the EGFP target while maintaining the para-cleavage activity.

[0465] Table 10. Average cleavage and framing activities of Cas13f mutants, as presented by MFI, wherein the gRNA targets the ATXN2 RNA transcript (gRNA shown in SEQ ID NO: 16) (n=3)

[0466]

[0467] The results in Table 10 show that the mutants have significantly improved cleavage activity and significantly reduced para-cleavage activity for the ATXN2 target compared to F40S23.

[0468] Table 11. Average SOD1 mRNA levels in Cos7 cells by RT-qPCR for Cas13f mutants (gRNA shown in SEQ ID NO: 18) (n=3)

[0469]

[0470] The results in Table 11 show that the mutants have significantly improved cleavage activity for the SOD1 target compared to the mutant F40S23.

[0471] Example 3 Engineering of hfCas13f for increased cleavage activity

[0472] This example demonstrates that the cleavage activity can be further significantly increased by introducing specific amino acid mutations into hfCas13f.

[0473] Design and Build:

[0474] In order to obtain a Cas13f mutant with increased cleavage activity, one non-basic amino acid in hfCas13f except the non-basic amino acids in the HEPN1 and HEPN2 domains was mutated to arginine (R, a common positively charged basic amino acid) to construct a Cas13f mutant ( Figure 5 ).

[0475] A two-plasmid mammalian fluorescent reporter system containing a mutant encoding plasmid and a gRNA encoding plasmid was constructed ( Figure 6 ) was used to detect the cleavage activity of Cas13f mutants further engineered based on hfCas13f.

[0476] The mutant encoding plasmid contains (1) mCherry red fluorescent reporter gene under the regulation of SV40 promoter and poly A sequence (its RNA transcript serves as the RNA target of the cleavage activity of Cas13f mutant), (2) Cas13f mutant coding sequence flanked by 5' and 3' SV40 NLS (SEQ ID NO: 5) coding sequences under the regulation of Cbh promoter and poly A sequence, and (3) BFP fluorescent reporter gene under the regulation of CMV promoter and poly A sequence. Blue fluorescence from BFP will indicate successful transfection and expression of mutant encoding plasmid in host cells.

[0477] The gRNA encoding plasmid contains a sequence encoding an mCherry targeting gRNA (SEQ ID NO: 19) consisting of 5'-DR sequence (SEQ ID NO: 2)-mCherry targeting spacer sequence (SEQ ID NO: 13)-DR sequence (SEQ ID NO: 2)-3' under the regulation of U6 promoter.

[0478] Transfection and detection:

[0479] HEK293T cells were cultured in 24-well tissue culture plates for 12 hours according to standard methods, and then two plasmids were co-transfected into the cells using standard polyethyleneimine (PEI) transfection. The transfected cells were then cultured at 37°C in 5% CO2 for about 48 hours. The BFP-positive cultured cells were then analyzed by flow cytometry.

[0480] As a negative control ("NT"), a mutant encoding plasmid encoding hfCas13f was used for transfection together with a gRNA encoding plasmid encoding a non-targeting spacer sequence (SEQ ID NO: 14) instead of an mCherry targeting spacer sequence (SEQ ID NO: 13). All mCherry (RFP) MFI results (mean ± SD) of Cas13f mutants were normalized to the negative control.

[0481] As a positive control ("PT" or "hfCas13f"), a mutant encoding plasmid encoding hfCas13f and a gRNA encoding plasmid encoding an mCherry targeting spacer sequence (SEQ ID NO: 13) were used for transfection.

[0482] The cleavage activity of each tested Cas13f mutant was negatively correlated with the mCherry MFI. The lower the mCherry MFI, the higher the cleavage activity. Therefore, the cleavage activity of the Cas13f mutant relative to the reference hfCas13f is shown as the reciprocal of the mCherry MFI of the Cas13f mutant relative to the mCherry MFI of hfCas13f.

[0483] Cleavage activity relative to hfCas13f = 1 / (mCherry MFI of Cas13f mutant / mCherry MFI of hfCas13f).

[0484] result:

[0485] The Cas13f mutants were tested in four batches with hfCas13f as a positive control to exclude the effect of transfection efficiency on cleavage activity. Flow cytometry results show the mCherry MFI of each Cas13f mutant with a single amino acid substitution to R. Among them, each Cas13f mutant has a single amino acid substitution to R at position 183, 189, 200, 202, 204, 205, 213, 214, 222, 233, 239, 240, 241, 258, 259, 276, 282, 283, 298, 299, 300, 314, 320, 329, 338, 339, 345, 353, 361, 383, 410, 433, 451, 455, 497, 508, 509, 518, 520, 526, 574, 595, 598, 599 or 601 (highlighted in gray), and the mCherry MFI of the Cas13f mutant in one or more batches is weaker than that of hfCas13f, indicating increased cleavage activity (Tables 12-15).

[0486] Table 12. Average mCherry MFI of Cas13f mutants (n=2)

[0487]

[0488] Table 13. Average mCherry MFI of Cas13f mutants (n=2 or 1)

[0489]

[0490] Table 14. Average mCherry MFI of Cas13f mutants (n=2)

[0491]

[0492]

[0493] Table 15. Average mCherry MFI of Cas13f mutants (n=2 or 1)

[0494]

[0495] ***

[0496] Without departing from the scope and spirit of the present invention, various modifications and variations of the described methods, pharmaceutical compositions and kits of the present invention will be apparent to those skilled in the art. Although the present invention has been described in conjunction with specific embodiments, it should be understood that further modifications can be made, and the claimed invention should not be unduly limited to such specific embodiments. In fact, various modifications of the described modes for carrying out the present invention that are apparent to those skilled in the art are intended to fall within the scope of the present invention. This application is intended to cover any changes, uses or adaptations that generally follow the principles of the present invention, and includes such deviations from the present disclosure: it is within the known or customary operation of the field to which the present invention belongs and is applicable to the basic features shown heretofore.

Claims

1. An engineered Cas13f polypeptide, whose amino acid sequence is as described in SEQ ID NO:

4.

2. A fusion protein comprising the engineered Cas13f polypeptide according to claim 1 and a nuclear localization signal sequence as shown in SEQ ID NO: 5 at the N-terminus and / or C-terminus of the engineered Cas13f polypeptide.

3. A polynucleotide encoding the engineered Cas13f polypeptide of claim 1 or the fusion protein of claim 2.

4. A CRISPR-Cas13f system, comprising: a) the engineered Cas13f polypeptide of claim 1 or the fusion protein of claim 2 or a polynucleotide encoding the engineered Cas13f polypeptide or the fusion protein; and b) a guide nucleic acid or a polynucleotide encoding the guide nucleic acid, the guide nucleic acid comprising: i. a direct repeat sequence capable of forming a complex with the engineered Cas13f polypeptide, wherein the direct repeat sequence is as shown in SEQ ID NO: 2; and ii. A spacer sequence capable of hybridizing to a target RNA, thereby directing the complex to the target RNA.

5. The CRISPR-Cas13f system of claim 4, wherein the direct repeat sequence is at the 5' end and / or 3' end of the spacer sequence.

6. The CRISPR-Cas13f system of claim 4, wherein the spacer sequence has a length of at least 15 nucleotides.

7. The CRISPR-Cas13f system of claim 4, wherein the spacer sequence has a length of 30 nucleotides.

8. The CRISPR-Cas13f system of claim 4, wherein the guide nucleic acid is a guide RNA.

9. The CRISPR-Cas13f system of claim 4, wherein the target RNA is a eukaryotic RNA.

10. The CRISPR-Cas13f system of claim 4, wherein the target RNA is mRNA or non-coding RNA. A vector comprising the polynucleotide according to claim 3.

12. The vector of claim 11, wherein the vector is selected from the group consisting of a plasmid, an adeno-associated virus vector, and a lentiviral vector.

13. A delivery system comprising (1) a delivery vehicle, and (2) the engineered Cas13f polypeptide of claim 1, the fusion protein of claim 2, the polynucleotide of claim 3, the CRISPR-Cas13f system of any one of claims 4-10, or the vector of claim 11 or 12.

14. A method for modifying a target RNA, the method comprising contacting the target RNA with the CRISPR-Cas13f system of any one of claims 4-10, thereby modifying the target RNA.

15. Use of the engineered Cas13f polypeptide of claim 1, the fusion protein of claim 2, the polynucleotide of claim 3, the CRISPR-Cas13f system of any one of claims 4-10, the vector of claim 11 or 12, or the delivery system of claim 13 for preparing an agent for modifying a target RNA.

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