Modified guide RNA

Abstract

This disclosure relates to guide RNA modified to improve performance and stability. In certain embodiments, the guide RNA of this disclosure includes one or more modifications at its 5' end and / or one or more modifications at its 3' end.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims priority to U.S. Provisional Application No. 63 / 458,017, filed on April 7, 2023, the entirety of which is incorporated herein by reference.

[0002] field This disclosure relates to guide RNA modified to improve stability and performance. [Background technology]

[0003] background Clustered, regularly spaced, short palindromic repeat sequences (CRISPR) / CRISPR-related protein (Cas) systems are widely used to edit genomes of various cell types. CRISPR / Cas systems can be classified into two classes (Class I and Class II), and further subdivided into at least six different types: Type I, Type II, Type III, Type IV, Type V, and Type VI. In Type II CRISPR / Cas systems, a Cas protein (such as Cas9) forms a complex with a guide RNA (gRNA) molecule and binds to a target nucleic acid containing a protospacer-adjacent motif (PAM) and a spacer. The gRNA molecule contains a sequence complementary to the spacer in the target nucleic acid and functions to guide the Cas protein to the target nucleic acid. Recognition and binding of the target nucleotide by the Cas protein:gRNA complex induces cleavage of the target nucleic acid.

[0004] It has been found that gRNA molecules can be degraded within cells by nuclease cleavage, such as endonuclease or exonuclease cleavage, which may affect the effectiveness of the CRISPR-Cas system. Therefore, gRNA molecules with improved stability and gene editing efficiency are needed in this field. [Overview of the Initiative]

[0005] overview This disclosure provides modified guide RNA molecules. In certain embodiments, the guide RNA molecule comprises (i) a phosphorodithioate bond between the first and second nucleotides at the 5' end of the guide RNA molecule, (ii) a phosphorodithioate bond between the nucleotide at the 3' end ("n") and the n-1 nucleotide of the guide RNA molecule, or (iii) a phosphorodithioate bond between the first and second nucleotides at the 5' end of the guide RNA molecule, and a phosphorodithioate bond between the n nucleotide and the n-1 nucleotide of the guide RNA molecule.

[0006] In certain embodiments, the guide RNA molecule further includes phosphorodithioate bonds between the second and third nucleotides at the 5' end of the guide RNA molecule, and between the third and fourth nucleotides. In certain embodiments, the guide RNA molecule includes phosphorodithioate bonds between the first and second nucleotides at the 5' end of the guide RNA molecule, between the second and third nucleotides, and between the third and fourth nucleotides. In certain embodiments, the guide RNA molecule includes phosphorodithioate bonds between the first and second nucleotides at the 5' end of the guide RNA molecule, between the second and third nucleotides, between the third and fourth nucleotides, and between the fourth and fifth nucleotides.

[0007] In certain embodiments, the guide RNA molecule includes phosphorodithioate bonds between the n nucleotide and the n-1 nucleotide of the guide RNA molecule, between the n-1 nucleotide and the n-2 nucleotide, or between the n nucleotide and the n-1 nucleotide, and between the n-2 nucleotide and the n-3 nucleotide. In certain embodiments, the guide RNA molecule includes phosphorodithioate bonds between the n nucleotide and the n-1 nucleotide, between the n-1 nucleotide and the n-2 nucleotide, and between the n-2 nucleotide and the n-3 nucleotide. In certain embodiments, the guide RNA molecule includes phosphorodithioate bonds between the n nucleotide and the n-1 nucleotide, between the n-1 nucleotide and the n-2 nucleotide, between the n-2 nucleotide and the n-3 nucleotide, and between the n-3 nucleotide and the n-4 nucleotide.

[0008] In certain embodiments, the first nucleotide at the 5' end of the guide RNA molecule includes modifications selected from the group consisting of (a) 2'-fluoro-modified nucleotide, (b) 2'-O-methyl-modified nucleotide, (c) 2'-O-(2-methoxyethyl)-modified nucleotide, (d) locked nucleic acid (LNA), (e) deoxyribose nucleotide, and two or more combinations of (a) to (e). In certain embodiments, the first nucleotide at the 5' end of the guide RNA molecule includes a 2'-fluoro-modified nucleotide. In certain embodiments, the first nucleotide at the 5' end of the guide RNA molecule includes a 2'-O-methyl-modified nucleotide. In certain embodiments, the first nucleotide at the 5' end of the guide RNA molecule includes a 2'-O-(2-methoxyethyl)-modified nucleotide. In certain embodiments, the first nucleotide at the 5' end of the guide RNA molecule includes LNA. In certain embodiments, the first nucleotide at the 5' end of the guide RNA molecule includes a deoxyribose nucleotide.

[0009] In certain embodiments, the 5' end of the guide RNA molecule may include (i) a second nucleotide, (ii) a third nucleotide, (iii) a fourth nucleotide, (iv) a second and a third nucleotide, (v) a second and a fourth nucleotide, (vi) a third and a fourth nucleotide, or (v) the second, third, and fourth nucleotides may include modifications selected from the group consisting of (a) 2'-fluoro-modified nucleotides, (b) 2'-O-methyl-modified nucleotides, (c) 2'-O-(2-methoxyethyl)-modified nucleotides, (d) locked nucleic acids (LNAs), (e) deoxyribose nucleotides, and two or more combinations of (a) to (e).

[0010] In certain embodiments, the guide RNA molecule contains three consecutive 2'-fluoromodified nucleotides in the first three nucleotides of the 5' end of the guide RNA molecule. In certain embodiments, the guide RNA molecule contains three consecutive 2'-O-methylmodified nucleotides in the first three nucleotides of the 5' end of the guide RNA molecule. In certain embodiments, the guide RNA molecule contains four consecutive 2'-O-methylmodified nucleotides in the first four nucleotides of the 5' end of the guide RNA molecule. In certain embodiments, the guide RNA molecule contains three consecutive 2'-O-(2-methoxyethyl) modified nucleotides in the first three nucleotides of the 5' end of the guide RNA molecule. In certain embodiments, the guide RNA molecule contains three consecutive LNAs in the first three nucleotides of the 5' end of the guide RNA molecule. In certain embodiments, the guide RNA molecule contains three consecutive deoxyribose nucleotides in the first three nucleotides of the 5' end of the guide RNA molecule.

[0011] In certain embodiments, the nucleotide at the 3' end ("n") of the guide RNA molecule includes modifications selected from the group consisting of (a) 2'-fluoro-modified nucleotides, (b) 2'-O-methyl-modified nucleotides, (c) 2'-O-(2-methoxyethyl)-modified nucleotides, (d) locked nucleic acid (LNA), (e) deoxyribose nucleotides, and two or more combinations of (a) to (e). In certain embodiments, the nucleotide at n of the guide RNA molecule includes a 2'-fluoro-modified nucleotide. In certain embodiments, the nucleotide at n of the guide RNA molecule includes a 2'-O-methyl-modified nucleotide. In certain embodiments, the nucleotide at n of the guide RNA molecule includes a 2'-O-(2-methoxyethyl)-modified nucleotide. In certain embodiments, the nucleotide at n of the guide RNA molecule includes LNA. In certain embodiments, the nucleotide at n of the guide RNA molecule includes a deoxyribose nucleotide.

[0012] In certain embodiments, the 3' end of the guide RNA molecule contains modifications selected from the group consisting of (i) n-1 nucleotide, (ii) n-2 nucleotide, (iii) n-3 nucleotide, (iv) n-4 nucleotide, (v) n and n-1 nucleotide, (vi) n and n-2 nucleotide, (vii) n-1 and n-2 nucleotide, (viii) n, n-1 and n-2 nucleotide, (ix) n-1, n-2 and n-3 nucleotide, (x) n, n-1, n-2 and n-3 nucleotide, or (xi) n, n-1, n-2, n-3 and n-4 nucleotide, respectively, including modifications selected from the group consisting of (a) 2'-fluoro-modified nucleotide, (b) 2'-O-methyl-modified nucleotide, (c) 2'-O-(2-methoxyethyl)-modified nucleotide, (d) locked nucleic acid (LNA), (e) deoxyribose nucleotide, and two or more combinations of (a) to (e). In certain embodiments, the n and n-1 nucleotides, n and n-2 nucleotides, n-1 and n-2 nucleotides, n, n-1 and n-2 nucleotides, n-1, n-2, n-3 and n-4 nucleotides, or n, n-1, n-2, n-3 and n-4 nucleotides of the guide RNA molecule are each 2'-fluoromodified nucleotides. In certain embodiments, the n and n-1 nucleotides, n and n-2 nucleotides, n-1 and n-2 nucleotides, n, n-1 and n-2 nucleotides, n-1, n-2, n-3 and n-4 nucleotides, or n, n-1, n-2, n-3 and n-4 nucleotides of the guide RNA molecule are each 2'-O-methylmodified nucleotides. In certain embodiments, the n and n-1 nucleotides, the n and n-2 nucleotides, the n-1 and n-2 nucleotides, the n, n-1 and n-2 nucleotides, the n-1, n-2, n-3 and n-4 nucleotides, or the n, n-1, n-2, n-3 and n-4 nucleotides of the guide RNA molecule are each 2'-O-(2-methoxyethyl) modified nucleotides.In certain embodiments, the n and n-1 nucleotides, the n and n-2 nucleotides, the n-1 and n-2 nucleotides, the n, n-1 and n-2 nucleotides, the n-1, n-2, n-3 and n-4 nucleotides, or the n, n-1, n-2, n-3 and n-4 nucleotides of the guide RNA molecule are each LNAs. In certain embodiments, the n and n-1 nucleotides, the n and n-2 nucleotides, the n-1 and n-2 nucleotides, the n, n-1 and n-2 nucleotides, the n-1, n-2, n-3 and n-4 nucleotides, or the n, n-1, n-2, n-3 and n-4 nucleotides of the guide RNA molecule are each deoxyribose nucleotides.

[0013] This disclosure further provides nucleic acids comprising polynucleotides encoding guide RNA molecules disclosed herein. In certain embodiments, the nucleic acid further comprises polynucleotides encoding RNA-inducible nucleases. In certain embodiments, the RNA-inducible nuclease is a Cas protein. In certain embodiments, the Cas protein is selected from the group consisting of Cas9, Cas12, Cas13 and combinations thereof. This disclosure provides vectors comprising nucleic acids disclosed herein.

[0014] This disclosure provides compositions comprising guide RNA molecules disclosed herein. In certain embodiments, the compositions of this disclosure comprise vectors disclosed herein. In certain embodiments, the compositions further comprise RNA-inducible nucleases. Alternatively or additionally, the compositions further comprise nucleic acids encoding RNA-inducible nucleases. In certain embodiments, the RNA-inducible nucleases are Cas proteins, e.g., Cas9, Cas12, and / or Cas13. In certain embodiments, the compositions comprise nucleic acids disclosed herein, e.g., polynucleotides encoding guide RNA molecules.

[0015] This disclosure further comprises a ribonucleoprotein (RNP) complex comprising the modification guide RNA molecule and the RNA-inducible nuclease of this disclosure. In certain embodiments, the RNA-inducible nuclease is a Cas protein. In certain embodiments, the Cas protein is selected from the group consisting of Cas9, Cas12, Cas13 and combinations thereof.

[0016] This disclosure further includes cells containing the compositions or RNP complexes disclosed herein.

[0017] This disclosure further provides methods for modifying cells. In certain embodiments, the methods include contacting cells with a composition or RNP complex disclosed herein. In certain embodiments, the contact includes introducing the composition into cells by electroporation.

[0018] This disclosure provides a method for treating subjects requiring treatment. In certain embodiments, the method includes ex vivo modification of cells of a subject by contacting the cells with a composition or RNP complex disclosed herein and returning the modified cells to the subject.

[0019] This disclosure further provides a gene editing system comprising one or more modified guide RNAs, one or more nucleic acids, one or more vectors, one or more compositions, and / or one or more RNP complexes disclosed herein. [Brief explanation of the drawing]

[0020] [Figure 1] Figure 1 shows an exemplary structure of the gRNA of this disclosure. [Figure 2] Figure 2 shows the elution profile of the exemplary modified gRNA of this disclosure. [Figure 3] Figure 3 shows the elution profile of the exemplary modified gRNA described herein. [Figure 4] Figure 4 shows the elution profile of the exemplary modified gRNA of this disclosure. [Figure 5]Figure 5 shows the elution profile of an exemplary modified gRNA of the present disclosure. [Figure 6] Figure 6 shows the editing efficiency of an exemplary modified gRNA of the present disclosure at various concentrations compared to a reference gRNA. [Figure 7] Figure 7 shows the editing efficiency of an exemplary modified gRNA of the present disclosure at various concentrations. [Figure 8] Figure 8 shows the editing efficiency of an exemplary modified gRNA of the present disclosure compared to a reference gRNA using cells from different donors. [Figure 9] Figure 9 shows the editing efficiency of an exemplary modified gRNA of the present disclosure at various concentrations compared to a reference gRNA. [Figure 10] Figure 10 shows the editing efficiency of an exemplary modified gRNA of the present disclosure at various concentrations compared to a reference gRNA. [Figure 11] Figure 11 shows the editing efficiency of an exemplary modified gRNA of the present disclosure compared to a reference gRNA. [Figure 12] Figure 12 shows the editing efficiency of an exemplary modified gRNA of the present disclosure compared to a reference gRNA. [Figure 13] Figure 13 shows the % cell viability and total number of cells in the presence of an exemplary modified gRNA of the present disclosure under knockout conditions compared to a reference gRNA. [Figure 14] Figure 14 shows the % cell viability and total number of cells in the presence of an exemplary modified gRNA of the present disclosure under knockout conditions compared to a reference gRNA. [Figure 15] Figure 15 shows the % cell viability and total number of cells in the presence of an exemplary modified gRNA of the present disclosure under knock-in conditions compared to a reference gRNA. [Figure 16] Figure 16 shows the % cell viability and total number of cells in the presence of an exemplary modified gRNA of the present disclosure under knockout conditions compared to a reference gRNA. [Figure 17] Figure 17 shows the elution profile of an exemplary modified gRNA of the present disclosure. [Figure 18]Figure 18 shows the elution profiles of reference gRNA3 under basic stress (pH 11), acidic stress (pH 5), and oxidative stress (0.3% H2O2). [Figure 19] Figure 19 shows the elution profiles of reference gRNA 3 under basic stress (pH 11), acidic stress (pH 5), and oxidative stress (0.3% H2O2) conditions over specific periods. [Figure 20] Figure 20 shows the elution profile of the exemplary modified gRNA of this disclosure under acidic stress (pH 5) conditions. [Figure 21] Figure 21 shows the elution profile of the exemplary modified gRNA of this disclosure under acidic stress (pH 5) conditions. [Figure 22] Figure 22 shows the elution profile of the exemplary modified gRNAs of this disclosure under acidic stress (pH 5) conditions. [Figure 23] Figure 23 shows the elution profile of the exemplary modified gRNA of this disclosure under acidic stress (pH 5) conditions. [Figure 24] Figure 24 shows the elution profile of the exemplary modified gRNA of this disclosure under basic stress (pH 11) conditions. [Figure 25] Figure 25 shows the elution profile of the exemplary modified gRNA of this disclosure under basic stress (pH 11) conditions. [Figure 26] Figure 26 shows the elution profile of the exemplary modified gRNA of this disclosure under basic stress (pH 11) conditions. [Figure 27] Figure 27 shows the elution profile of the exemplary modified gRNA of this disclosure under basic stress (pH 11) conditions. [Figure 28] Figure 28 shows the elution profile of the exemplary modified gRNAs of this disclosure under thermal stress. [Figure 29] Figure 29 shows the elution profile of the exemplary modified gRNAs of this disclosure under thermal stress. [Figure 30] Figure 30 shows the elution profile of the exemplary modified gRNA of this disclosure under thermal stress. [Figure 31] Figure 31 shows the elution profile of the exemplary modified gRNA of this disclosure under thermal stress. [Figure 32] Figure 32 shows the elution profile of the exemplary modified gRNA of this disclosure under oxidative stress (0.3% H2O2). [Figure 33] Figure 33 shows the elution profile of the exemplary modified gRNA of this disclosure under oxidative stress (0.3% H2O2). [Figure 34] Figure 34 shows the elution profile of the exemplary modified gRNA of this disclosure under oxidative stress (0.3% H2O2). [Figure 35] Figure 35 shows the elution profile of the exemplary modified gRNA of this disclosure under oxidative stress (0.3% H2O2). [Figure 36] Figure 36 shows the elution profile of the exemplary modified gRNA of this disclosure under oxidative stress (0.3% H2O2) for 5 days. [Figure 37] Figure 37 shows the change in purity of the exemplary modified gRNA of this disclosure over 3 days under acidic stress (pH 5) conditions. [Figure 38] Figure 38 shows the change in purity of the exemplary modified gRNA of this disclosure over 24 hours under basic stress (pH 11). [Figure 39] Figure 39 shows the change in purity of the exemplary modified gRNA of this disclosure over 7 days under heat stress. [Figure 40] Figure 40 shows the change in purity of the exemplary modified gRNA of this disclosure over 5 days under oxidative stress (0.3% H2O2). [Figure 41] Figure 41 shows the elution profile of the exemplary modified gRNA of this disclosure. [Figure 42] Figure 42 shows the elution profile of the exemplary modified gRNAs of this disclosure under acidic stress (pH 5) conditions. [Figure 43] Figure 43 shows the elution profile of the exemplary modified gRNA of this disclosure under acidic stress (pH 5) conditions. [Figure 44]Figure 44 shows the elution profile of the exemplary modified gRNA of this disclosure under acidic stress (pH 5) conditions. [Figure 45] Figure 45 shows the elution profile of the exemplary modified gRNA of this disclosure under acidic stress (pH 5) conditions. [Figure 46] Figure 46 shows the elution profile of the exemplary modified gRNA of this disclosure under basic stress (pH 11) conditions. [Figure 47] Figure 47 shows the elution profile of the exemplary modified gRNA of this disclosure under basic stress (pH 11) conditions. [Figure 48] Figure 48 shows the elution profile of the exemplary modified gRNA of this disclosure under basic stress (pH 11) conditions. [Figure 49] Figure 49 shows the elution profile of the exemplary modified gRNA of this disclosure under basic stress (pH 11) conditions. [Figure 50] Figure 50 shows the elution profile of the exemplary modified gRNA of this disclosure under thermal stress. [Figure 51] Figure 51 shows the elution profile of the exemplary modified gRNA of this disclosure under thermal stress. [Figure 52] Figure 52 shows the elution profile of the exemplary modified gRNA of this disclosure under thermal stress. [Figure 53] Figure 53 shows the elution profile of the exemplary modified gRNA of this disclosure under thermal stress. [Figure 54] Figure 54 shows the elution profile of the exemplary modified gRNA of this disclosure under oxidative stress (0.3% H2O2). [Figure 55] Figure 55 shows the elution profile of the exemplary modified gRNA of this disclosure under oxidative stress (0.3% H2O2). [Figure 56] Figure 56 shows the change in purity of the exemplary modified gRNA of this disclosure over 7 days under heat stress. [Figure 57] Figure 57 shows the change in purity of the exemplary modified gRNAs of this disclosure under acidic stress (pH 5) conditions. [Figure 58] Figure 58 shows the change in purity of the exemplary modified gRNAs of this disclosure under basic stress (pH 11) conditions. [Figure 59] Figure 59 shows the change in purity of the exemplary modified gRNAs of this disclosure under oxidative stress (0.3% H2O2). [Figure 60] Figure 60 shows the editing efficiency of the exemplary modified gRNA of this disclosure at an intermediate point compared to the reference gRNA. [Figure 61] Figure 61 shows the editing efficiency of the exemplary modified gRNA of this disclosure at the end of the process, compared to the reference gRNA. [Figure 62] Figure 62 shows cell proliferation of donor 1 cells 5 days and from day 5 to day 12 after transfection (Tfx) with the exemplary modified gRNA of this disclosure. [Figure 63] Figure 63 shows the phenotype of the exemplary modified gRNA of this disclosure at the end of the process, compared to the reference gRNA. [Figure 64] Figure 64 shows the editing efficiency of the exemplary modified gRNA of this disclosure at an intermediate point compared to the reference gRNA. [Figure 65] Figure 65 shows the editing efficiency of the exemplary modified gRNA of this disclosure at the end of the process, compared to the reference gRNA. [Figure 66] Figure 66 shows cell proliferation of donor 2 cells 5 days and from day 5 to day 12 after transfection (Tfx) with the exemplary modified gRNA of this disclosure. [Figure 67] Figure 67 shows the phenotype of the exemplary modified gRNA of this disclosure at the end of the process, compared to the reference gRNA. [Figure 68] Figure 68 shows the editing efficiency of the exemplary modified gRNA of this disclosure at an intermediate point compared to the reference gRNA. [Figure 69] Figure 69 shows the editing efficiency of the exemplary modified gRNA of this disclosure at the end of the process, compared to the reference gRNA. [Figure 70] Figure 70 shows cell proliferation of donor 3 cells 5 days and from day 5 to day 12 after transfection (Tfx) with the exemplary modified gRNA of this disclosure. [Modes for carrying out the invention]

[0021] Detailed explanation This disclosure relates to modified guide RNA molecules, pharmaceutical compositions containing such modified guide RNA, and methods for modifying cells by administering modified guide RNA molecules. This disclosure is based in part on the finding that introducing phosphorodithioate bonds into gRNA molecules eliminates the chiral centers generated by the phosphorodithioate bonds, resulting in stable gRNA molecules, gRNA compositions that do not contain diastereomers at these modified phosphate positions, and a more clearly defined gRNA population. As shown in Figures 37, 38, 57, and 58, gRNA molecules containing phosphorodithioate bonds at the 5' and / or 3' ends exhibit improved stability under forced degradation conditions, such as basic stress and acidic stress conditions, compared to gRNA molecules without phosphorodithioate bonds. Furthermore, as shown in Figures 61, 65, and 69, gRNA molecules containing phosphorodithioate bonds at the 5' and / or 3' ends have equivalent or increased editing efficiency compared to gRNA molecules without phosphorodithioate bonds.

[0022] For clarity, and without limitation, a detailed description of the subject matter disclosed herein is divided into the following subsections: I. Definition, II. Modified guide RNA, III. RNA-inducible nucleases, IV. Composition, V. How to use, VI. Gene editing systems, and VII. Exemplary Embodiments.

[0023] I. Definition Unless otherwise defined, all technical and scientific terms used herein have the meanings generally understood by those skilled in the art in the field of the subject matter of this disclosure. The following references provide general definitions of many of the terms used herein: Singleton et al., Dictionary of Microbiology and Molecular Biology (2nd ed. 1994); The Cambridge Dictionary of Science and Technology (Walker ed., 1988); The Glossary of Genetics, 5th Ed., R. Rieger et al. (eds.), Springer Verlag (1991); and Hale & Marham, The Harper Collins Dictionary of Biology (1991). Where used herein, the following terms have the meanings set forth below unless otherwise specified.

[0024] As used herein, in the claims and / or specification, the use of the phrase "a" or "an" with the term "including" may mean "one," which also coincides with the meanings of "one or more," "at least one," and "one or more."

[0025] The terms “about” or “approximately” mean that a particular value is within an acceptable margin of error, as determined by those skilled in the art, and this depends to some extent on the method by which the value is measured or determined, i.e., on the limitations of the measuring system. For example, “about” may mean a standard deviation of 3 or more than 3, according to convention in the art. Alternatively, “about” may mean a range of up to 20%, preferably up to 10%, more preferably up to 5%, and more preferably up to 1% of a given value. Or, particularly with respect to biological systems or processes, the term may mean within one order of magnitude of the value, preferably up to five times, and more preferably up to two times.

[0026] As used herein, the terms “comprise(s) / include(s),” “having / has,” “can,” and “contain(s),” and their variations, are intended to be open-ended transitional phrases, terms, or words that do not preclude any additional acts or structures. This disclosure also considers other embodiments that “comprising,” “consisting of,” and “consisting essentially of” the embodiments or elements presented herein, whether expressly provided for.

[0027] The term “coupled” can refer to two or more components connected or joined by interaction, bonding, linking, force, or binding in order to keep them together. In certain embodiments, the term “coupled” includes, for example, direct bonding of a first component to a second component, or direct or indirect bonding in which one or more intermediate molecules are placed between the first and second components. Exemplary bonding includes covalent bonds, ionic bonds, van der Waals interactions, and other bonding that can be identified by those skilled in the art.

[0028] As used herein, the term “detect” or “detect” indicates the determination of the presence and / or detection of a target, such as a nucleic acid target, in a limited portion of a space including but not limited to a sample. As used herein, the term “detect” or “detect” may include, but is not limited to, the determination of the chemical and / or biological properties of a target, including, but not limited to, its ability to interact with other compounds, particularly its ability to bind, its ability to activate other compounds, and additional properties that can be identified by those skilled in the art upon reading this disclosure. Detection can be quantitative or qualitative. Detection is “quantitative” if it refers to, relates to, or includes the measurement (also called quantification) of a quantity of a target or signal. This includes, but is not limited to, analyses designed to determine a quantity or proportion of a target or signal. Detection is “qualitative” if it refers to, relates to, or includes the identification of a quality or type of a target or signal in relation to its relative abundance to another target or signal that has not been quantified.

[0029] As used herein, the term “domain” refers to a segment of a protein or nucleic acid, such as a gRNA molecule. Unless otherwise indicated, a domain does not need to have specific functional properties.

[0030] As used herein, the term “editing efficiency” refers to the total number of sequence reads having nucleotide insertions or deletions in the target region of interest, relative to the total number of sequence reads after cleavage by RNA-induced nucleases.

[0031] The terms “guide RNA,” “gRNA,” or “gRNA molecule,” as used interchangeably herein, refer to nucleic acids that facilitate the specific targeting or homing of RNA-induced nucleases to target nucleic acids.

[0032] As used herein, the term "hybridization" refers to the process by which two single-stranded polynucleotides are non-covalently linked to form a stable double-stranded polynucleotide.

[0033] As used herein, the terms “individual” or “subject” refer to vertebrates or invertebrates, such as humans or non-human animals, such as mammals. Mammals include, but are not limited to, humans, non-human primates, livestock, sports animals, rodents, pets, etc. Non-exclusive examples of non-human animal subjects include rodents, such as mice, rats, hamsters, guinea pigs, rabbits, dogs, cats, sheep, pigs, goats, cattle, horses, apes, and monkeys. In certain embodiments, the individual or subject is a human.

[0034] As used herein, the term "in vitro" refers to an artificial environment and any process or reaction occurring within it. Examples of in vitro environments include, but are not limited to, test tubes and cell cultures.

[0035] As used herein, the term “in vivo” means a natural environment (e.g., an animal or a cell) and processes or reactions that occur within that natural environment, such as embryonic development, cell differentiation, and neural tube formation.

[0036] As used herein, “label” refers to a substance that enables direct or indirect detection. Examples of labels include, but are not limited to, fluorescent labels, colorimetric labels, electron density labels, chemiluminescent labels, and radioactive labels. Non-exclusive examples of labels include green fluorescent protein ("GFP"), mCherry, dtTomato, or other fluorescent proteins known in the art (e.g., Shaner et al., A Guide to Choosing Fluorescent Proteins, Nature Methods 2(12):905-909(2005)), which are incorporated herein by reference. 32 P, 14 C, 125 I, 3 H and 131I. Fluorescent sources (such as rare earth chelates or Lucifer Yellow and its derivatives), Rhodamine and its derivatives, Dansyl, Umbelliferone, Luciferases (such as firefly luciferase and bacterial fluorescent plain enzyme) (U.S. Patent No. 4,737,456), Fluorescein, 2,3-dihydrophthalazine diketone, and enzymes that produce detectable signals, such as horseradish peroxidase (HRP), alkaline phosphorus sour enzyme, β-galactosidase, glucoamylase, lysozyme, carbohydrate oxidases (such as glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase (G6PD)), and heterocyclic oxidases (such as uricase and xanthine oxidase).

[0037] The terms “nucleic acid” or “polynucleotide” encompass all compounds and / or substances containing polymers of nucleotides. Each nucleotide consists of a base, specifically a purine or pyrimidine base (i.e., cytosine (C), guanine (G), adenine (A), thymine (T), or uracil (U)), a sugar (i.e., deoxyribose or ribose), and a phosphate group. Often, nucleic acid molecules are described by their base sequence, which represents the primary structure (linear structure) of the nucleic acid molecule. The base sequence is typically represented 5' to 3'. The term nucleic acid encompasses, for example, deoxyribonucleic acid (DNA), such as complementary DNA (cDNA) and genomic DNA; ribonucleic acid (RNA), such as messenger RNA (mRNA); synthetic forms of DNA or RNA; and mixed polymers containing two or more of these molecules. Nucleic acid molecules can be linear or cyclic. In addition, the term nucleic acid includes both sense and antisense strands, as well as single-stranded and double-stranded forms. Furthermore, the nucleic acids described herein may contain naturally occurring or non-naturally occurring nucleotides. Examples of non-naturally occurring nucleotides include modified nucleotide bases containing derivatized sugars or phosphate backbone links or chemically modified residues.

[0038] The term "nucleoside" refers to a compound containing a purine or pyrimidine base (i.e., cytosine (C), guanine (G), adenine (A), thymine (T), or uracil (U)) and a sugar (i.e., deoxyribose or ribose).

[0039] "Isolated" nucleic acids refer to nucleic acid molecules that have been separated from their natural environment. Isolated nucleic acids include nucleic acid molecules that are normally contained within cells that contain nucleic acid molecules, but these nucleic acid molecules are located outside of chromosomes or in chromosomal locations different from their natural chromosomal locations.

[0040] The term "multiple" refers to a number greater than one. In certain embodiments, the term "multiple guide RNAs" refers to a number of guide RNAs greater than one. For example, multiple guide RNAs include, but are not limited to, at least two guide RNAs. In certain embodiments, the term "multiple nucleic acids" refers to a number of nucleic acids greater than one. For example,, but are not limited to, multiple nucleic acids include, but are not limited to, at least two nucleic acids.

[0041] As used herein, the terms “reference molecule” or “control molecule,” for example, a reference or control gRNA molecule, refer to a molecule compared to a target molecule, such as a target gRNA molecule. For example, but not limited to, a target gRNA molecule may have one or more modifications, such as nucleotide modifications, compared to a reference molecule.

[0042] As used herein, the term "specifically binds" refers to preferential binding to a target molecule, such as a protein or nucleic acid, compared to other molecules in the sample, such as proteins or nucleic acids.

[0043] As used herein, “treatment” (and its grammatical variations, e.g., “to treat” or “to treat”) refers to a clinical intervention that seeks to alter the natural course of a disease in the treated individual, and may be carried out for preventive purposes or in the course of clinicopathology. Desired effects of treatment include, but are not limited to, prevention of disease onset or recurrence, relief of symptoms, reduction of direct or indirect pathological consequences of the disease, prevention of metastasis, slowing of the rate of disease progression, improvement or relief of symptoms, and remission or improved prognosis. Reduction may be a reduction of at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 99% in the severity of complications, signs or symptoms, or the likelihood of progression to another grade. "Treatment" may also mean inhibiting cancer growth or progression to higher grades by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 99%. In certain embodiments, the gRNAs of this disclosure are used to delay the onset of disease or to slow the progression of disease.

[0044] As used herein, the term “therapeutic effect” refers to a local or systemic effect on a subject caused by a pharmacologically active substance.

[0045] As used herein, the terms “therapeutic effective dose” and “effective dose” refer to the amount of the composition of this disclosure that is effective in producing any desired therapeutic effect in at least a subpopulation of cells of interest, in terms of a benefit-to-risk ratio applicable to any medical treatment.

[0046] The term "mutation" refers to any modification at a given polypeptide residue position. For example, though not limited to, the term "mutation" can refer to replacing an amino acid at a given position with a different amino acid.

[0047] As used herein, the term “vector” refers to a nucleic acid molecule capable of replicating another nucleic acid to which it is ligated. This term includes vectors as self-replicating nucleic acid structures, as well as vectors integrated into the genome of a host cell to which they are introduced. Certain vectors can direct the expression of the nucleic acid to which they are operably ligated. Such vectors are referred herein to as “expression vectors.”

[0048] II. Modified Guide RNA Molecules This disclosure provides gRNAs containing one or more modifications. In certain embodiments, one or more modifications increase the stability of the gRNA compared to gRNAs without one or more modifications, such as control or reference gRNAs. For example, but not limited to, modifications improve the stability of the disclosed gRNA molecules by preventing degradation of the gRNA molecule by nucleases, such as endonucleases and / or exonucleases. Such improved stability can enhance the therapeutic effect of the modified gRNA molecule. Furthermore, this modification improves shelf life stability, allowing for the production of higher-quality gRNA compositions (since such compositions do not contain diastereomers). This modification can also improve editing efficiency and reduce potential off-target effects.

[0049] In certain embodiments, the modified gRNAs of this disclosure exhibit an increase in stability of approximately 1% or more, for example, approximately 2% or more, approximately 3% or more, approximately 4% or more, approximately 5% or more, approximately 6% or more, approximately 7% or more, approximately 8% or more, approximately 9% or more, approximately 10% or more, approximately 15% or more, approximately 20% or more, approximately 25% or more, approximately 30% or more, approximately 35% or more, approximately 40% or more, approximately 45% or more, approximately 50% or more, or approximately 55% or more, compared to the reference gRNA under degradation conditions, for example, under basic stress conditions and / or acidic stress conditions. In certain embodiments, the modified gRNAs of this disclosure exhibit an increase in stability of approximately 1% to approximately 60% compared to the reference gRNA under degradation conditions, for example, under basic stress conditions and / or acidic stress conditions. In certain embodiments, the modified gRNAs of this disclosure exhibit an increase in stability of approximately 1% or more, approximately 2% or more, approximately 3% or more, approximately 4% or more, or approximately 5% or more, under acidic stress conditions. In certain embodiments, the modified gRNAs of this disclosure exhibit a stability increase of approximately 1% to approximately 5% compared to the reference gRNA under acidic stress conditions. In certain embodiments, the modified gRNAs of this disclosure exhibit a stability increase of approximately 5% or more, approximately 10% or more, approximately 15% or more, approximately 20% or more, approximately 25% or more, approximately 30% or more, approximately 35% or more, approximately 40% or more, approximately 45% or more, approximately 50% or more, or approximately 55% or more under basic stress conditions. In certain embodiments, the modified gRNAs of this disclosure exhibit a stability increase of approximately 1% to approximately 60% compared to the reference gRNA under basic stress conditions. In certain embodiments, the modified gRNAs of this disclosure exhibit a reduction in degradation of approximately 1% or more, for example, approximately 2% or more, approximately 3% or more, approximately 4% or more, approximately 5% or more, approximately 6% or more, approximately 7% or more, approximately 8% or more, approximately 9% or more, approximately 10% or more, approximately 15% or more, approximately 20% or more, approximately 25% or more, approximately 30% or more, approximately 35% or more, approximately 40% or more, approximately 45% or more, approximately 50% or more, or approximately 55% or more, compared to the reference gRNA under degradation conditions, for example, under basic stress conditions and / or acidic stress conditions. In certain embodiments, the modified gRNAs of this disclosure exhibit a reduction in degradation of approximately 1% to approximately 60% compared to the reference gRNA under degradation conditions, for example, under basic stress conditions and / or acidic stress conditions. In certain embodiments, the modified gRNAs of this disclosure exhibit a reduction in degradation of approximately 1% or more, approximately 2% or more, approximately 3% or more, approximately 4% or more, or approximately 5% or more, under acidic stress conditions.In certain embodiments, the modified gRNAs of this disclosure show a degradation reduction of about 1% to about 5% compared to the reference gRNA under acidic stress conditions. In certain embodiments, the modified gRNAs of this disclosure show a degradation reduction of about 5% or more, about 10% or more, about 15% or more, about 20% or more, about 25% or more, about 30% or more, about 35% or more, about 40% or more, about 45% or more, about 50% or more, or about 55% or more under basic stress conditions. In certain embodiments, the modified gRNAs of this disclosure show a degradation reduction of about 1% to about 60% compared to the reference gRNA under basic stress conditions. In certain embodiments, increased stability and / or reduced degradation are observed about 24 hours, 48 ​​hours, 3 days, 4 days, 5 days, 6 days, or 7 days after exposure to degradation conditions. In certain embodiments, heat stress conditions involve exposing the gRNA to a high temperature, for example, about 37°C. ° This includes exposure to temperatures higher than C. In certain embodiments, oxidative stress conditions include exposure of the gRNA to free radicals and / or compounds (e.g., H2O2). In certain embodiments, basic stress conditions include exposure of the gRNA to a basic pH, e.g., greater than about 8, e.g., about 11. In certain embodiments, acidic stress conditions include exposure of the gRNA to an acidic pH, e.g., less than about 6, e.g., about 5. In certain embodiments, the reference gRNA may be a gRNA having the same nucleotide sequence as the modified gRNA (e.g., an unmodified gRNA with the same sequence as the modified gRNA). In certain embodiments, the reference gRNA may be a gRNA having a targeting domain with the same sequence as the modified gRNA (e.g., an unmodified gRNA with the same sequence as the modified gRNA). In certain embodiments, the reference gRNA may be a gRNA having the same number of nucleotides as the modified gRNA (e.g., an unmodified gRNA with the same number of nucleotides as the modified gRNA).

[0050] In certain embodiments, the gRNA of the Disclosure includes one or more, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, eleven or more, twelve or more, thirteen or more, fourteen or more, or fifteen or more modifications. In certain embodiments, the gRNA of the Disclosure includes at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten nucleotide modifications, at least eleven nucleotide modifications, at least twelve nucleotide modifications, at least thirteen nucleotide modifications, at least fourteen nucleotide modifications, or at least fifteen nucleotide modifications. In certain embodiments, the gRNA of the Disclosure includes at least about eleven modifications.

[0051] In certain embodiments, the gRNAs of the Disclosure have a length of about 20 to about 200 nucleotides, for example, about 20 to about 190, about 20 to about 180, about 20 to about 170, about 20 to about 160, about 20 to about 150, about 20 to about 140, about 20 to about 130, about 20 to about 120, about 20 to about 110, about 20 to about 100, about 30 to about 200, about 40 to about 200, about 50 to about 200, about 60 to about 200, about 70 to about 200, about 80 to about 200, about 90 to about 200, about 50 to about 150, about 80 to about 120, or about 90 to about 100 nucleotides. In certain embodiments, the gRNAs of the Disclosure have a length of about 80 to about 120 nucleotides. In certain embodiments, the gRNAs of this disclosure have nucleotide lengths of approximately 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, and 200 or more.

[0052] In a particular embodiment, approximately 1% to 20% of the nucleotides present in the gRNA of this disclosure are modified, for example, approximately 1% to 19%, 1% to 18%, 1% to 17%, 1% to 16%, 1% to 15%, 1% to 14%, 1% to 13%, 1% to 12%, 1% to 11%, 1% to 10%, 1% to 9%, 1% to 8%, 1% to 7%, 1% to 6%, 1% to 5%, 1% to 4%, and 1% of the nucleotides present in the gRNA of this disclosure. Approximately 3%, 1% to 2%, 2% to 20%, 3% to 20%, 4% to 20%, 5% to 20%, 6% to 20%, 7% to 20%, 8% to 20%, 9% to 20%, 10% to 20%, 11% to 20%, 12% to 20%, 13% to 20%, 14% to 20%, 15% to 20%, 16% to 20%, 17% to 20%, 18% to 20%, 19% to 20%, 5% to 15%, or 10% to 15% are modified. In certain embodiments, approximately 1% to 15% of the nucleotides present in the gRNA of this disclosure are modified. In certain embodiments, approximately 1% to approximately 10% of the nucleotides present in the gRNA of this disclosure are modified. In certain embodiments, approximately 1% to approximately 6% of the nucleotides present in the gRNA of this disclosure are modified. In certain embodiments, approximately 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20% or more of the nucleotides present in the gRNA are modified.

[0053] In certain embodiments, the gRNA molecules of this disclosure have the structure shown in Figure 1. For example, but not limited to, the gRNA molecules of this disclosure include a spacer region (blue region in Figure 1) at its 5' end. Further exemplary structures of the gRNA molecules of this disclosure are provided in Figure 1A of Daniel et al., Frontiers in Genome Editing 2:617910 (2021), the contents of which are incorporated herein by reference in their entirety (the spacer region is shown in blue and red in Figure 1A of Daniel et al.).

[0054] In certain embodiments, the gRNA molecules of this disclosure include one or more modifications in the spacer region of the gRNA molecule. In certain embodiments, the spacer region of the gRNA molecules disclosed herein is complementary to the sequence of the target nucleic acid, for example, at least about 80%, about 85%, about 90%, about 95%, about 98%, about 99%, or about 100% complementary. In certain embodiments, the spacer region includes the first 30 nucleotides located at the 5' end of the gRNA molecule, for example, the first 29, first 28, first 27, first 26, first 25, first 24, first 23, first 22, first 21, first 20, first 19, first 18, first 17, first 16, or first 15 nucleotides. In certain embodiments, the spacer region includes the first 20 nucleotides located at the 5' end of the gRNA molecule. In certain embodiments, the modifications to the spacer region do not impede editing efficiency, which can be evaluated using techniques known in the art or techniques described herein. In certain embodiments, modification of the spacer region can improve editing efficiency and reduce off-target effects. In certain embodiments, the spacer region contains 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 modifications. In certain embodiments, the spacer region contains 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 modifications within the first 5 nucleotides of its 5' end. In certain embodiments, the spacer region contains 1, 2, 3, 4, 5, or 6 modifications within the first 5 nucleotides of its 5' end. In certain embodiments, the spacer region contains 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 modifications within the first 3 nucleotides of its 5' end. In certain embodiments, the spacer region contains 1, 2, 3, 4, 5, or 6 modifications within the first 3 nucleotides of its 5' end. In certain embodiments, the spacer region contains at least one chemically modified nucleotide within the first 5 nucleotides of its 5' end. In certain embodiments, the spacer region contains 1, 2, 3, 4, or 5 chemically modified nucleotides within the first 5 nucleotides of its 5' end.In certain embodiments, the spacer region contains one chemically modified nucleotide within the first five nucleotides of its 5' end. In certain embodiments, the spacer region contains two chemically modified nucleotides within the first five nucleotides of its 5' end. In certain embodiments, the spacer region contains three chemically modified nucleotides within the first five nucleotides of its 5' end. In certain embodiments, the spacer region contains four chemically modified nucleotides within the first five nucleotides of its 5' end. In certain embodiments, the spacer region contains five chemically modified nucleotides within the first five nucleotides of its 5' end. In certain embodiments, the spacer region contains at least one chemically modified nucleotide within the first three nucleotides of its 5' end. In certain embodiments, the spacer region contains one, two, or three chemically modified nucleotides within the first three nucleotides of its 5' end. In certain embodiments, the spacer region contains one chemically modified nucleotide within the first three nucleotides of its 5' end. In certain embodiments, the spacer region contains two chemically modified nucleotides within the first three nucleotides of its 5' end. In certain embodiments, the spacer region contains three chemically modified nucleotides within the first three nucleotides of its 5' end. In certain embodiments, a chemically modified nucleotide may include one or more modifications, two or more modifications, or three or more modifications. In certain embodiments, a chemically modified nucleotide may include one modification, for example, a sugar modification or a phosphate backbone modification as described below. In certain embodiments, a chemically modified nucleotide may include two modifications, for example, a sugar modification and a phosphate backbone modification as described below. In certain embodiments, a chemically modified nucleotide may include three modifications, for example, two sugar modifications and a phosphate backbone modification as described below.

[0055] In certain embodiments, the gRNA molecules of this disclosure include one or more modifications at their 5' end. In certain embodiments, the term “5' end” as used herein with respect to modifications refers to the first five nucleotides at the 5' end of the gRNA molecule. For example, but not limited to, one or more nucleotides present at the 5' end of the gRNA molecules of this disclosure are modified. In certain embodiments, the first nucleotide present at the 5' end of the gRNA molecules of this disclosure is modified. In certain embodiments, the second nucleotide present at the 5' end of the gRNA molecules of this disclosure is modified. In certain embodiments, the third nucleotide present at the 5' end of the gRNA molecules of this disclosure is modified. In certain embodiments, the fourth nucleotide present at the 5' end of the gRNA molecules of this disclosure is modified. In certain embodiments, the fifth nucleotide present at the 5' end of the gRNA molecules of this disclosure is modified. In certain embodiments, the gRNA molecules of this disclosure include 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 modifications within the first five nucleotides at their 5' end.

[0056] In certain embodiments, the gRNA molecule of this disclosure includes modifications to two consecutive nucleotides, for example, two consecutive nucleotides within the first five nucleotides at the 5' end of the gRNA molecule. For example, but not limited to, the first and second nucleotides of the gRNA molecule are modified. In certain embodiments, the first and third nucleotides of the gRNA molecule are modified. In certain embodiments, the second and third nucleotides at the 5' end of the gRNA molecule are modified. In certain embodiments, the third and fourth nucleotides at the 5' end of the gRNA molecule are modified. In certain embodiments, the fourth and fifth nucleotides at the 5' end of the gRNA molecule are modified.

[0057] In certain embodiments, the gRNA molecule of this disclosure includes modifications to three consecutive nucleotides, for example, three consecutive nucleotides within the first five nucleotides at the 5' end of the gRNA molecule. For example, but not limited to, the first, second, and third nucleotides at the 5' end of the gRNA molecule are modified. In certain embodiments, the second, third, and fourth nucleotides at the 5' end of the gRNA molecule are modified. In certain embodiments, the third, fourth, and fifth nucleotides at the 5' end of the gRNA molecule are modified.

[0058] In certain embodiments, the gRNA molecule of this disclosure includes modifications to four consecutive nucleotides, for example, four consecutive nucleotides within the first five nucleotides at the 5' end of the gRNA molecule. For example, but not limited to, the first, second, third, and fourth nucleotides at the 5' end of the gRNA molecule are modified. In certain embodiments, the second, third, fourth, and fifth nucleotides at the 5' end of the gRNA molecule are modified.

[0059] In certain embodiments, the gRNA molecule of this disclosure includes modifications to five consecutive nucleotides, for example, five consecutive nucleotides within the first five nucleotides at the 5' end of the gRNA molecule. For example, but not limited to, the first, second, third, fourth, and fifth nucleotides at the 5' end of the gRNA molecule are modified.

[0060] In certain embodiments, the gRNA molecules of this disclosure include one or more modifications at their 3' end. In certain embodiments, the term “3' end” as used herein with respect to modifications refers to the last five nucleotides (n, n-1, n-2, n-3, and n-4) at the 3' end of the gRNA molecule. For example, but not limited to, one or more nucleotides present at the 3' end of the gRNA molecules of this disclosure are modified. In certain embodiments, the last nucleotide (n) present at the 3' end of the gRNA molecules of this disclosure is modified. In certain embodiments, the second-to-last nucleotide (n-1) present at the 3' end of the gRNA molecules of this disclosure is modified. In certain embodiments, the third-to-last nucleotide (n-2) present at the 3' end of the gRNA molecules of this disclosure is modified. In certain embodiments, the fourth-to-last nucleotide (n-3) present at the 3' end of the gRNA molecules of this disclosure is modified. In certain embodiments, the fifth-to-last nucleotide (n-4) present at the 3' end of the gRNA molecules of this disclosure is modified. In certain embodiments, the gRNA molecule of this disclosure includes 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 modifications within the last 5 nucleotides of its 3' end.

[0061] In certain embodiments, the gRNA molecule of this disclosure includes modifications to two consecutive nucleotides, for example, two consecutive nucleotides within five nucleotides of the 3' end of the gRNA molecule. For example, but not limited to, the n and n-1 nucleotides at the 3' end of the gRNA molecule are modified. In certain embodiments, the n and n-2 nucleotides at the 3' end of the gRNA molecule are modified. In certain embodiments, the n-1 and n-2 nucleotides at the 3' end of the gRNA molecule are modified. In certain embodiments, the n-2 and n-3 nucleotides at the 3' end of the gRNA molecule are modified. In certain embodiments, the n-3 and n-4 nucleotides at the 3' end of the gRNA molecule are modified. In certain embodiments, the n-4 and n-5 nucleotides at the 3' end of the gRNA molecule are modified.

[0062] In certain embodiments, the gRNA molecule of this disclosure includes modifications to three consecutive nucleotides, for example, three consecutive nucleotides within the last five nucleotides at the 3' end of the gRNA molecule. For example, but not limited to, the n, n-1, and n-2 nucleotides of the gRNA molecule are modified. In certain embodiments, the n-1, n-2, and n-3 nucleotides at the 3' end of the gRNA molecule are modified. In certain embodiments, the n-2, n-3, and n-4 nucleotides at the 3' end of the gRNA molecule are modified.

[0063] In certain embodiments, the gRNA molecule of this disclosure includes modifications to four consecutive nucleotides, for example, four consecutive nucleotides within five nucleotides from the 3' end of the gRNA molecule. For example, but not limited to, the n, n-1, n-2, and n-3 nucleotides of the gRNA molecule are modified. In certain embodiments, the n-1, n-2, n-3, and n-4 nucleotides of the gRNA molecule are modified.

[0064] In certain embodiments, the gRNA molecule of this disclosure includes modifications to five consecutive nucleotides, for example, five consecutive nucleotides within five nucleotides from the 3' end of the gRNA molecule. For example, but not limited to, the n, n-1, n-2, n-3, and n-4 nucleotides of the gRNA molecule may be modified.

[0065] In certain embodiments, the gRNA molecule of the Disclosure includes one or more modifications at its 5' end and one or more modifications at its 3' end. In certain embodiments, the gRNA molecule of the Disclosure includes two or more modifications at its 5' end and two or more modifications at its 3' end. In certain embodiments, the gRNA molecule of the Disclosure includes three or more modifications at its 5' end and three or more modifications at its 3' end. In certain embodiments, the gRNA molecule of the Disclosure includes four or more modifications at its 5' end and four or more modifications at its 3' end. In certain embodiments, the gRNA molecule of the Disclosure includes five or more modifications at its 5' end and five or more modifications at its 3' end.

[0066] Non-limiting examples of modifications that may be included in the gRNA molecules of this disclosure are disclosed herein. In certain embodiments, the gRNAs of this disclosure may include one or more of the following modifications: (i) phosphate backbone modifications, and (ii) sugar modifications.

[0067] Phosphate skeleton modification In certain embodiments, the phosphate groups of nucleotides present in the gRNA molecule can be modified. For example, but not limited to, the phosphate groups of nucleotides can be modified by substituting one or more oxygen atoms in the phosphodiester bond, such as cross-linked or uncross-linked oxygen atoms, with different substituents. Non-limited examples of substituents include sulfur (S), nitrogen (N), hydrogen (H), and carbon (C). In certain embodiments, one or more oxygen atoms in the phosphodiester bond are substituted with S.

[0068] In certain embodiments, a gRNA molecule can be modified with one or more phosphorothioate (PS) links. In certain embodiments, a PS linkage or bond refers to a linkage in which one non-crosslinked phosphate oxygen of an internucleotide phosphodiester bond is substituted with sulfur. In certain embodiments, "*" is used herein to indicate a nucleotide linked to an adjacent 3' nucleotide by a PS linkage. In certain embodiments, the phosphorus in an unmodified phosphodiester bond is achiral, and when one non-crosslinked phosphate oxygen is substituted with sulfur, the phosphorus becomes chiral.

[0069] In certain embodiments, gRNA molecules can be modified with one or more phosphorodithioate (PS2) links. In certain embodiments, PS2 linkage or bond refers to a linkage in which both non-crosslinking oxygen atoms of an internucleotide phosphodiester bond are replaced with sulfur. In certain embodiments, "**" is used herein to indicate a nucleotide linked to an adjacent 3' nucleotide by a PS2 linkage. Similar to naturally occurring phosphodiester backbone links, PS2 links are achiral at phosphorus, resulting in gRNA molecules that are not diastereomers at the PS2 linkage. In certain embodiments, phosphorodithioate links are resistant to nuclease degradation, and the presence of one or more phosphorodithioate links in the gRNA of this disclosure can increase the stability of the gRNA compared to, for example, unmodified gRNA.

[0070] sugar modification In certain embodiments, the sugar groups of nucleotides present in the gRNA of the Disclosure can be modified. For example, but not limited to, the nucleotides of the gRNA of the Disclosure may include one or more modifications to their sugar groups, such as ribose.

[0071] In certain embodiments, the sugar group can be modified with a 2'-hydroxyl group (OH). In certain embodiments, the 2'-hydroxyl group can be replaced with a different substituent. Non-limiting examples of substituents include hydrogen (H), halogens, alkyl or alkoxy (OR, where R may be alkyl, cycloalkyl or alkoxy).

[0072] In certain embodiments, the 2'-hydroxyl group is substituted with an alkoxy group. In certain embodiments, the 2'-hydroxyl group is substituted with a methoxy group. In certain embodiments, "m" is used herein to indicate a 2'-O-methyl modified nucleotide (i.e., a 2'-O-methyl modified nucleotide).

[0073] In certain embodiments, the hydrogen (H) of the 2'-hydroxyl group is substituted with a methoxyethyl group. In certain embodiments, "M" is used herein to indicate a 2'-O-(2-methoxyethyl) modified nucleotide (i.e., a 2'-O-(2-methoxyethyl) modified nucleotide).

[0074] In certain embodiments, the 2'-hydroxyl group can be substituted with a halogen. Non-limiting examples of halogens include fluorine (F), chlorine (Cl), bromide (Br), and iodine (I). In certain embodiments, the 2'-hydroxyl group is replaced with fluorine. In certain embodiments, "f" is used herein to indicate a 2'-fluoro-modified nucleotide (i.e., a 2'-fluoro-modified nucleotide).

[0075] In certain embodiments, the 2' hydroxyl group can be substituted with hydrogen (H) to produce a deoxyribose sugar. For example, but not limited to, nucleotides present in the gRNA of this disclosure may have a deoxyribose sugar. In certain embodiments, "d" is used herein to indicate a nucleotide having a deoxyribose sugar.

[0076] In certain embodiments, the modification of the 2' hydroxyl group may include a “locked nucleic acid” (LNA) in which the 2' hydroxyl group is bonded to the 4' carbon of the same ribose sugar. In certain embodiments, the 2' hydroxyl group is bonded to the 4' carbon by a crosslink, such as an alkylene (e.g., methylene), ether, or amino crosslink. In certain embodiments, “LNA” is used herein to refer to a nucleotide that is an LNA.

[0077] Existentially modified gRNA In certain embodiments, the gRNA of this disclosure may have at least one phosphate backbone modification, for example, a phosphorodithioate bond. In certain embodiments, the gRNA may have at least two phosphate backbone modifications, for example, a phosphorodithioate bond. In certain embodiments, the gRNA may have at least three phosphate backbone modifications, for example, a phosphorodithioate bond. In certain embodiments, the gRNA may have at least four phosphate backbone modifications, for example, a phosphorodithioate bond. In certain embodiments, the gRNA may have at least five phosphate backbone modifications, for example, a phosphorodithioate bond. In certain embodiments, the gRNA may have at least six phosphate backbone modifications, for example, a phosphorodithioate bond. In certain embodiments, the gRNA may have at least seven phosphate backbone modifications, for example, a phosphorodithioate bond. In certain embodiments, the gRNA may have at least eight phosphate backbone modifications, for example, a phosphorodithioate bond. In certain embodiments, the gRNA may have at least nine phosphate backbone modifications, for example, a phosphorodithioate bond. In certain embodiments, the gRNA may have at least 10 phosphate backbone modifications, such as phosphorodithioate bonds. In certain embodiments, the gRNA may have about 1 to about 10 phosphate backbone modifications, for example, about 1 to about 9, about 1 to about 8, about 1 to about 7, about 1 to about 6, about 1 to about 5, about 1 to about 4, about 1 to about 3, about 1 to about 2, about 2 to about 10, about 3 to about 10, about 4 to about 10, about 5 to about 10, about 6 to about 10, about 7 to about 10, about 8 to about 10, about 9 to about 10, about 2 to about 8, about 2 to about 6, about 3 to about 8, about 3 to about 6, or 3 to 5 phosphate backbone modifications, such as phosphorodithioate bonds. In certain embodiments, the gRNA may have about 2 to about 10 phosphate backbone modifications, for example, phosphorodithioate bonds. In certain embodiments, the gRNA may have about 2 to about 9 phosphate backbone modifications, such as phosphorodithioate bonds. In certain embodiments, the gRNA may have about 3 to about 8 phosphate backbone modifications, such as phosphorodithioate bonds. In certain embodiments, the gRNA may have about 3 to about 5 phosphate backbone modifications, such as phosphorodithioate bonds.

[0078] In certain embodiments, the gRNA of the Disclosure may comprise at least one phosphorodithioate bond and at least one phosphorothioate bond, as shown, for example, in Table 10. For example, but not limited to, the gRNA of the Disclosure may comprise at least two phosphorodithioate bonds and at least one phosphorothioate bond. In certain embodiments, the gRNA of the Disclosure may comprise at least three phosphorodithioate bonds and at least one phosphorothioate bond. In certain embodiments, the gRNA of the Disclosure may comprise at least four phosphorodithioate bonds and at least one phosphorothioate bond. In certain embodiments, the gRNA of the Disclosure may comprise at least two phosphorodithioate bonds and at least two phosphorothioate bonds. In certain embodiments, the gRNA of the Disclosure may comprise at least two phosphorodithioate bonds and at least three phosphorothioate bonds.

[0079] In certain embodiments, the gRNA may have at least one sugar modification. In certain embodiments, the gRNA may have at least two sugar modifications. In certain embodiments, the gRNA may have at least three sugar modifications. In certain embodiments, the gRNA may have at least four sugar modifications. In certain embodiments, the gRNA may have at least five sugar modifications. In certain embodiments, the gRNA may have at least six sugar modifications. In certain embodiments, the gRNA may have at least seven sugar modifications. In certain embodiments, the gRNA may have at least eight sugar modifications. In certain embodiments, the gRNA may have at least nine sugar modifications. In certain embodiments, the gRNA may have at least ten sugar modifications. In certain embodiments, the gRNA may have about 1 to about 10 sugar modifications, for example, about 1 to about 9, about 1 to about 8, about 1 to about 7, about 1 to about 6, about 1 to about 5, about 1 to about 4, about 1 to about 3, about 1 to about 2, about 2 to about 10, about 3 to about 10, about 4 to about 10, about 5 to about 10, about 6 to about 10, about 7 to about 10, about 8 to about 10, about 9 to about 10, about 2 to about 8, about 2 to about 6, about 3 to about 8, or about 3 to about 6 sugar modifications. In certain embodiments, the gRNA may have about 2 to about 10 sugar modifications. In certain embodiments, the gRNA may have about 3 to about 8 sugar modifications. In certain embodiments, the gRNA molecule of this disclosure may have about 3 to about 6 sugar modifications.

[0080] In a particular embodiment, the gRNA may have at least one phosphate backbone modification (e.g., at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten phosphate backbone modifications) and at least one glycosylation modification (e.g., at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten glycosylation modifications).

[0081] In certain embodiments, the gRNA may have at least two phosphate backbone modifications and at least two sugar modifications. In certain embodiments, the gRNA may have at least three phosphate backbone modifications and at least three sugar modifications. In certain embodiments, the gRNA may have at least four phosphate backbone modifications and at least four sugar modifications. In certain embodiments, the gRNA may have at least five phosphate backbone modifications and at least five sugar modifications. In certain embodiments, the gRNA may have at least five phosphate backbone modifications and at least six sugar modifications. In certain embodiments, the gRNA may have at least six phosphate backbone modifications and at least six sugar modifications. In certain embodiments, the gRNA may have at least seven phosphate backbone modifications and at least seven sugar modifications. In certain embodiments, the gRNA may have at least eight phosphate backbone modifications and at least eight sugar modifications. In certain embodiments, the gRNA may have at least eight phosphate backbone modifications and at least nine sugar modifications. In certain embodiments, the gRNA may have at least nine phosphate backbone modifications and at least nine sugar modifications. In certain embodiments, the gRNA may have at least 10 phosphate backbone modifications and at least 10 glycosylation modifications.

[0082] In certain embodiments, the gRNA molecule of the Disclosure includes at least one phosphorodithioate bond at the 5' end of the gRNA molecule. In certain embodiments, the gRNA molecule of the Disclosure includes a phosphorodithioate bond between the first and second nucleotides at the 5' end of the gRNA molecule. In certain embodiments, the gRNA molecule of the Disclosure includes a phosphorodithioate bond between the second and third nucleotides at the 5' end of the gRNA molecule. In certain embodiments, the gRNA molecule of the Disclosure includes a phosphorodithioate bond between the third and fourth nucleotides at the 5' end of the gRNA molecule. In certain embodiments, the gRNA molecule of the Disclosure includes a phosphorodithioate bond between the fourth and fifth nucleotides at the 5' end of the gRNA molecule.

[0083] In certain embodiments, the gRNA molecule of the Disclosure includes at least one phosphorodithioate bond at the 3' end of the gRNA molecule. In certain embodiments, the gRNA molecule of the Disclosure includes a phosphorodithioate bond between the nucleotide at the 3' end ("n") of the gRNA molecule and the n-1 nucleotide. In certain embodiments, the gRNA molecule of the Disclosure includes a phosphorodithioate bond between the n-1 nucleotide and the n-2 nucleotide at the 3' end of the gRNA molecule. In certain embodiments, the gRNA molecule of the Disclosure includes a phosphorodithioate bond between the n-2 nucleotide and the n-3 nucleotide at the 3' end of the gRNA molecule. In certain embodiments, the gRNA molecule of the Disclosure includes a phosphorodithioate bond between the n-3 nucleotide and the n-4 nucleotide at the 3' end of the gRNA molecule.

[0084] In certain embodiments, the gRNA molecule of the present disclosure includes a phosphorodithioate bond between the first and second nucleotides at the 5' end of the gRNA molecule, and a phosphorodithioate bond between the n nucleotide and the n-1 nucleotide of the gRNA molecule.

[0085] In certain embodiments, the gRNA molecule of the present disclosure includes phosphorodithioate bonds between the first and second nucleotides at the 5' end of the gRNA molecule, and between the second and third nucleotides.

[0086] In certain embodiments, the gRNA molecule of the present disclosure includes phosphorodithioate bonds between the first and second nucleotides at the 5' end of the gRNA molecule, and between the third and fourth nucleotides.

[0087] In certain embodiments, the gRNA molecule of the present disclosure includes phosphorodithioate bonds between the second and third nucleotides at the 5' end of the gRNA molecule, and between the third and fourth nucleotides.

[0088] In certain embodiments, the gRNA molecule of the present disclosure includes phosphorodithioate bonds between the first nucleotide and the second nucleotide at the 5' end of the gRNA molecule, between the second nucleotide and the third nucleotide, and between the third nucleotide and the fourth nucleotide.

[0089] In certain embodiments, the gRNA molecule of the present disclosure includes phosphorodithioate bonds between the first nucleotide and the second nucleotide at the 5' end of the gRNA molecule, between the second nucleotide and the third nucleotide, between the third nucleotide and the fourth nucleotide, and between the fourth nucleotide and the fifth nucleotide.

[0090] In certain embodiments, the gRNA molecule of this disclosure includes a phosphorothioate bond between a first nucleotide and a second nucleotide.

[0091] In certain embodiments, the gRNA molecule of this disclosure includes a phosphorothioate bond between the third nucleotide and the fourth nucleotide.

[0092] In certain embodiments, the gRNA molecule of the present disclosure includes phosphorothioate bonds between the first nucleotide and the second nucleotide, between the second nucleotide and the third nucleotide, and between the third nucleotide and the fourth nucleotide.

[0093] In certain embodiments, the gRNA molecule of this disclosure includes phosphorodithioate bonds between the n nucleotide and the n-1 nucleotide, and between the n-1 nucleotide and the n-2 nucleotide.

[0094] In certain embodiments, the gRNA molecule of the present disclosure includes a phosphorodithioate bond between the n nucleotide and the n-1 nucleotide of the gRNA molecule, and between the n-2 nucleotide and the n-3 nucleotide.

[0095] In certain embodiments, the gRNA molecule of the present disclosure includes a phosphorodithioate bond between the n-1 nucleotide and the n-2 nucleotide of the gRNA molecule, and between the n-2 nucleotide and the n-3 nucleotide.

[0096] In certain embodiments, the gRNA molecule of the present disclosure includes phosphorodithioate bonds between the n nucleotide and the n-1 nucleotide, between the n-1 nucleotide and the n-2 nucleotide, between the n-2 nucleotide and the n-3 nucleotide, and between the n-3 nucleotide and the n-4 nucleotide of the gRNA molecule.

[0097] In certain embodiments, the gRNA molecule of the present disclosure includes phosphorodithioate bonds between the n-1 nucleotide and the n-2 nucleotide, between the n-2 nucleotide and the n-3 nucleotide, between the n-3 nucleotide and the n-4 nucleotide, and between the n-4 nucleotide and the n-5 nucleotide of the gRNA molecule.

[0098] In certain embodiments, the gRNA molecule of this disclosure includes a phosphorothioate bond between the n nucleotide and the n-1 nucleotide.

[0099] In certain embodiments, the gRNA molecule of this disclosure includes a phosphorothioate bond between the n-1 nucleotide and the n-2 nucleotide.

[0100] In certain embodiments, the gRNA molecule of the present disclosure includes a phosphorothioate bond between the n-1 nucleotide and the n-2 nucleotide, and a phosphorothioate bond between the n nucleotide and the n-1 nucleotide. In certain embodiments, the gRNA molecule further includes phosphorothioate bonds between the first nucleotide and the second nucleotide, between the second nucleotide and the third nucleotide, and between the third nucleotide and the fourth nucleotide.

[0101] In certain embodiments, the gRNA molecule of the present disclosure includes a phosphorothioate bond between a third nucleotide and a fourth nucleotide, as well as phosphorothioate bonds between a first nucleotide and a second nucleotide, and between a second nucleotide and a third nucleotide. In certain embodiments, the gRNA further includes phosphorothioate bonds between an n nucleotide and an n-1 nucleotide, and between an n-1 nucleotide and an n-2 nucleotide.

[0102] In certain embodiments, the gRNA molecule of the present disclosure includes phosphorothioate bonds between the first nucleotide and the second nucleotide, between the second nucleotide and the third nucleotide, and between the third nucleotide and the fourth nucleotide. In certain embodiments, the gRNA further includes phosphorothioate bonds between the n nucleotide and the n-1 nucleotide, and between the n-1 nucleotide and the n-2 nucleotide.

[0103] In certain embodiments, the gRNA molecules of this disclosure contain one or more 2'-fluoro-modified nucleotides, 2'-O-methyl-modified nucleotides, 2'-O-(2-methoxyethyl)-modified nucleotides, deoxyribose nucleotides, and locked nucleic acids (LNAs) at their 5' end, for example, within the first five nucleotides of the 5' end. For example, but not limited to, the first nucleotide at the 5' end of a gRNA molecule is a 2'-fluoro-modified nucleotide, a 2'-O-methyl-modified nucleotide, a 2'-O-(2-methoxyethyl)-modified nucleotide, a deoxyribose nucleotide, or locked nucleic acid (LNA). In certain embodiments, the first nucleotide at the 5' end of a gRNA molecule is a 2'-fluoro-modified nucleotide. In certain embodiments, the first nucleotide at the 5' end of a guide RNA molecule is a 2'-O-methyl-modified nucleotide. In certain embodiments, the first nucleotide at the 5' end of a gRNA molecule is a 2'-O-(2-methoxyethyl)-modified nucleotide. In certain embodiments, the first nucleotide at the 5' end of the gRNA molecule is LNA. In certain embodiments, the first nucleotide at the 5' end of the gRNA molecule has a deoxyribose sugar.

[0104] In certain embodiments, the gRNA of this disclosure contains two or more 2'-fluoro-modified nucleotides, 2'-O-methyl-modified nucleotides, 2'-O-(2-methoxyethyl)-modified nucleotides, deoxyribose nucleotides and / or LNAs at its 5' end, for example, within the first five nucleotides of its 5' end. For example, but not limited to, the first and second nucleotides at the 5' end of a gRNA molecule are 2'-fluoro-modified nucleotides, 2'-O-methyl-modified nucleotides, 2'-O-(2-methoxyethyl)-modified nucleotides, deoxyribose nucleotides, LNAs, or a combination thereof. In certain embodiments, the first and second nucleotides at the 5' end of a gRNA molecule are 2'-fluoro-modified nucleotides. In certain embodiments, the first and second nucleotides at the 5' end of a guide RNA molecule are 2'-O-methyl-modified nucleotides. In certain embodiments, the first and second nucleotides at the terminal end of a gRNA molecule are 2'-O-(2-methoxyethyl)-modified nucleotides. In certain embodiments, the first and second nucleotides at the 5' end of the gRNA molecule are LNA. In certain embodiments, the first and second nucleotides at the 5' end of the gRNA molecule have a deoxyribose sugar.

[0105] In certain embodiments, the gRNA of this disclosure contains three or more 2'-fluoro-modified nucleotides, 2'-O-methyl-modified nucleotides, 2'-O-(2-methoxyethyl)-modified nucleotides, deoxyribose nucleotides and / or LNAs at its 5' end, for example, within the first five nucleotides of the 5' end. For example, but not limited to, the first, second and third nucleotides of the 5' end of a gRNA molecule are 2'-fluoro-modified nucleotides, 2'-O-methyl-modified nucleotides, 2'-O-(2-methoxyethyl)-modified nucleotides, deoxyribose nucleotides, LNAs, or a combination thereof. In certain embodiments, the first, second and third nucleotides of the 5' end of a gRNA molecule are 2'-fluoro-modified nucleotides. In certain embodiments, the first, second and third nucleotides of the 5' end of a gRNA molecule are 2'-O-methyl-modified nucleotides. In certain embodiments, the first, second and third nucleotides of the terminal of a gRNA molecule are 2'-O-(2-methoxyethyl)-modified nucleotides. In certain embodiments, the first, second, and third nucleotides at the 5' end of the gRNA molecule are LNA. In certain embodiments, the first, second, and third nucleotides at the 5' end of the gRNA molecule have a deoxyribose sugar.

[0106] In certain embodiments, the gRNA of this disclosure contains four or more 2'-fluoro-modified nucleotides, 2'-O-methyl-modified nucleotides, 2'-O-(2-methoxyethyl)-modified nucleotides, deoxyribose nucleotides and / or LNAs at its 5' end, for example, within the first five nucleotides of the 5' end. For example, but not limited to, the first, second, third, and fourth nucleotides of the 5' end of a gRNA molecule are 2'-fluoro-modified nucleotides, 2'-O-methyl-modified nucleotides, 2'-O-(2-methoxyethyl)-modified nucleotides, deoxyribose nucleotides, LNAs, or a combination thereof. In certain embodiments, the first, second, third, and fourth nucleotides of the 5' end of a gRNA molecule are 2'-fluoro-modified nucleotides. In certain embodiments, the first, second, third, and fourth nucleotides of the 5' end of a gRNA molecule are 2'-O-methyl-modified nucleotides. In certain embodiments, the first, second, third, and fourth nucleotides at the terminal end of the gRNA molecule are 2'-O-(2-methoxyethyl) modified nucleotides. In certain embodiments, the first, second, third, and fourth nucleotides at the 5' end of the gRNA molecule are LNAs. In certain embodiments, the first, second, third, and fourth nucleotides at the 5' end of the gRNA molecule have a deoxyribose sugar.

[0107] In certain embodiments, the gRNA of this disclosure contains five or more 2'-fluoro-modified nucleotides, 2'-O-methyl-modified nucleotides, 2'-O-(2-methoxyethyl)-modified nucleotides, deoxyribose nucleotides and / or LNAs at its 5' end, for example, within the first five nucleotides of its 5' end. For example, but not limited to, the first, second, third, fourth and fifth nucleotides of the 5' end of a gRNA molecule are 2'-fluoro-modified nucleotides, 2'-O-methyl-modified nucleotides, 2'-O-(2-methoxyethyl)-modified nucleotides, deoxyribose nucleotides, LNAs, or a combination thereof. In certain embodiments, the first, second, third, fourth and fifth nucleotides of the 5' end of a gRNA molecule are 2'-fluoro-modified nucleotides. In certain embodiments, the first, second, third, fourth and fifth nucleotides of the 5' end of a gRNA molecule are 2'-O-methyl-modified nucleotides. In certain embodiments, the first, second, third, fourth, and fifth nucleotides at the terminal end of the gRNA molecule are 2'-O-(2-methoxyethyl) modified nucleotides. In certain embodiments, the first, second, third, fourth, and fifth nucleotides at the 5' end of the gRNA molecule are LNAs. In certain embodiments, the first, second, third, fourth, and fifth nucleotides at the 5' end of the gRNA molecule have deoxyribose sugars.

[0108] In certain embodiments, the gRNAs of this disclosure contain one or more 2'-fluoro-modified nucleotides, 2'-O-methyl-modified nucleotides, 2'-O-(2-methoxyethyl)-modified nucleotides, deoxyribose nucleotides, and locked nucleic acids (LNAs) at their 3' end, for example, within the first five nucleotides of the 3' end. For example, but not limited to, the nucleotides at the 3' end ("n") of a gRNA molecule are 2'-fluoro-modified nucleotides, 2'-O-methyl-modified nucleotides, 2'-O-(2-methoxyethyl)-modified nucleotides, deoxyribose nucleotides, or locked nucleic acids (LNAs). In certain embodiments, the nucleotides at n of a gRNA molecule are 2'-fluoro-modified nucleotides. In certain embodiments, the nucleotides at n of a gRNA molecule are 2'-O-methyl-modified nucleotides. In certain embodiments, the nucleotides at n of a gRNA molecule include 2'-O-(2-methoxyethyl)-modified nucleotides. In certain embodiments, the nucleotides at n of a gRNA molecule are LNAs. In certain embodiments, the n nucleotide of the gRNA molecule has a deoxyribose sugar.

[0109] In certain embodiments, the gRNA of this disclosure contains two or more 2'-fluoro-modified nucleotides, 2'-O-methyl-modified nucleotides, 2'-O-(2-methoxyethyl)-modified nucleotides, deoxyribose nucleotides and / or LNAs at its 3' end, for example, within the last 5 nucleotides of the 3' end. For example, but not limited to, the n and n-1 nucleotides of a gRNA molecule are 2'-fluoro-modified nucleotides, 2'-O-methyl-modified nucleotides, 2'-O-(2-methoxyethyl)-modified nucleotides, deoxyribose nucleotides, LNAs, or a combination thereof. In certain embodiments, the n and n-1 nucleotides of a gRNA molecule are 2'-fluoro-modified nucleotides. In certain embodiments, the n and n-1 nucleotides of a guide RNA molecule are 2'-O-methyl-modified nucleotides. In certain embodiments, the n and n-1 nucleotides at the terminal end of a gRNA molecule are 2'-O-(2-methoxyethyl)-modified nucleotides. In certain embodiments, the n and n-1 nucleotides of a gRNA molecule are LNAs. In certain embodiments, the n and n-1 nucleotides of the gRNA molecule contain a deoxyribose sugar.

[0110] In certain embodiments, the gRNA of this disclosure contains three or more 2'-fluoro-modified nucleotides, 2'-O-methyl-modified nucleotides, 2'-O-(2-methoxyethyl)-modified nucleotides, deoxyribose nucleotides and / or LNAs at its 3' end, for example, within the last 5 nucleotides of the 3' end. For example, but not limited to, the n, n-1 and n-2 nucleotides of a gRNA molecule are 2'-fluoro-modified nucleotides, 2'-O-methyl-modified nucleotides, 2'-O-(2-methoxyethyl)-modified nucleotides, deoxyribose nucleotides, LNAs, or a combination thereof. In certain embodiments, the n, n-1 and n-2 nucleotides of a gRNA molecule are 2'-fluoro-modified nucleotides. In certain embodiments, the n, n-1 and n-2 nucleotides of a guide RNA molecule are 2'-O-methyl-modified nucleotides. In certain embodiments, the terminal n, n-1 and n-2 nucleotides of a gRNA molecule are 2'-O-(2-methoxyethyl)-modified nucleotides. In certain embodiments, the n, n-1, and n-2 nucleotides of the gRNA molecule are LNA. In certain embodiments, the n, n-1, and n-2 nucleotides of the gRNA molecule have a deoxyribose sugar.

[0111] In certain embodiments, the gRNA of this disclosure contains four or more 2'-fluoro-modified nucleotides, 2'-O-methyl-modified nucleotides, 2'-O-(2-methoxyethyl)-modified nucleotides, deoxyribose nucleotides and / or LNAs at its 3' end, for example, within the last five nucleotides of the 3' end. For example, but not limited to, the n, n-1, n-2, and n-3 nucleotides of a gRNA molecule are 2'-fluoro-modified nucleotides, 2'-O-methyl-modified nucleotides, 2'-O-(2-methoxyethyl)-modified nucleotides, deoxyribose nucleotides, LNA, or a combination thereof. In certain embodiments, the n-1, n-2, n-3, and n-4 nucleotides of a gRNA molecule are 2'-fluoro-modified nucleotides, 2'-O-methyl-modified nucleotides, 2'-O-(2-methoxyethyl)-modified nucleotides, LNA, or a combination thereof. In certain embodiments, the n, n-1, n-2, and n-3 nucleotides of a gRNA molecule are 2'-fluoro-modified nucleotides. In certain embodiments, the n, n-1, n-2, and n-3 nucleotides of the guide RNA molecule are 2'-O-methyl modified nucleotides. In certain embodiments, the terminal n, n-1, n-2, and n-3 nucleotides of the gRNA molecule are 2'-O-(2-methoxyethyl) modified nucleotides. In certain embodiments, the n, n-1, n-2, and n-3 nucleotides of the gRNA molecule are LNA. In certain embodiments, the n, n-1, n-2, and n-3 nucleotides of the gRNA molecule have a deoxyribose sugar. In certain embodiments, the n-1, n-2, n-3, and n-4 nucleotides of the gRNA molecule are 2'-O-methyl modified nucleotides.

[0112] In certain embodiments, the gRNA of this disclosure contains five or more 2'-fluoro-modified nucleotides, 2'-O-methyl-modified nucleotides, 2'-O-(2-methoxyethyl)-modified nucleotides, deoxyribose nucleotides and / or LNAs at its 3' end, for example, within the last five nucleotides of its 3' end. For example, but not limited to, the n, n-1, n-2, n-3 and n-4 nucleotides of a gRNA molecule are 2'-fluoro-modified nucleotides, 2'-O-methyl-modified nucleotides, 2'-O-(2-methoxyethyl)-modified nucleotides, deoxyribose nucleotides, LNA or a combination thereof. In certain embodiments, the n, n-1, n-2, n-3 and n-4 nucleotides of a gRNA molecule are 2'-fluoro-modified nucleotides. In certain embodiments, the n, n-1, n-2, n-3 and n-4 nucleotides of a guide RNA molecule are 2'-O-methyl-modified nucleotides. In certain embodiments, the n, n-1, n-2, n-3, and n-4 nucleotides at the ends of the gRNA molecule are 2'-O-(2-methoxyethyl) modified nucleotides. In certain embodiments, the n, n-1, n-2, n-3, and n-4 nucleotides of the gRNA molecule are LNAs. In certain embodiments, the n, n-1, n-2, n-3, and n-4 nucleotides of the gRNA molecule have a deoxyribose sugar.

[0113] In certain embodiments, the gRNA of the Disclosure comprises (i) one or more 2'-fluoro-modified nucleotides, 2'-O-methyl-modified nucleotides, 2'-O-(2-methoxyethyl)-modified nucleotides, deoxyribose nucleotides and / or LNAs at its 5' end, and (ii) one or more phosphorodithioate bonds at the 5' end of the gRNA molecule. In certain embodiments, the first nucleotide at the 5' end of the gRNA molecule is a 2'-fluoro-modified nucleotide, a 2'-O-methyl-modified nucleotide, a 2'-O-(2-methoxyethyl)-modified nucleotide, a deoxyribose nucleotide, or locked nucleic acid (LNA), and the gRNA comprises a phosphorodithioate bond between the first and second nucleotides at the 5' end of the gRNA molecule. In certain embodiments, the first nucleotide at the 5' end of the gRNA molecule is a 2'-O-methyl-modified nucleotide, and the gRNA comprises a phosphorodithioate bond between the first and second nucleotides at the 5' end of the gRNA molecule.

[0114] In certain embodiments, the gRNA of the Disclosure comprises (i) two or more 2'-fluoro-modified nucleotides, 2'-O-methyl-modified nucleotides, 2'-O-(2-methoxyethyl)-modified nucleotides, deoxyribose nucleotides and / or LNAs at its 5' end, and (ii) two or more phosphorodithioate bonds at the 5' end of the gRNA molecule. In certain embodiments, the first and second nucleotides at the 5' end of the gRNA molecule are 2'-fluoro-modified nucleotides, 2'-O-methyl-modified nucleotides, 2'-O-(2-methoxyethyl)-modified nucleotides, deoxyribose nucleotides or LNAs, and the gRNA molecule comprises phosphorodithioate bonds between the first and second nucleotides at the 5' end of the gRNA molecule and the second and third nucleotides. In certain embodiments, the first and second nucleotides at the 5' end of the gRNA molecule are 2'-O-methyl-modified nucleotides, and the gRNA molecule contains a phosphorodithioate bond between the first and second nucleotides at the 5' end of the gRNA molecule and the second and third nucleotides.

[0115] In certain embodiments, the gRNA of the Disclosure comprises (i) three or more 2'-fluoro-modified nucleotides, 2'-O-methyl-modified nucleotides, 2'-O-(2-methoxyethyl)-modified nucleotides, deoxyribose nucleotides and / or LNAs at its 5' end, and (ii) three or more phosphorodithioate bonds at the 5' end of the gRNA molecule. In certain embodiments, the first, second and third nucleotides at the 5' end of the gRNA molecule are 2'-fluoro-modified nucleotides, 2'-O-methyl-modified nucleotides, 2'-O-(2-methoxyethyl)-modified nucleotides, deoxyribose nucleotides or LNAs, and the gRNA comprises phosphorodithioate bonds between the first and second nucleotides, between the second and third nucleotides, and between the third and fourth nucleotides at the 5' end of the gRNA molecule. In certain embodiments, the first, second, and third nucleotides at the 5' end of the gRNA molecule are 2'-O-methyl-modified nucleotides, and the gRNA contains phosphorodithioate bonds between the first and second nucleotides, between the second and third nucleotides, and between the third and fourth nucleotides at the 5' end of the gRNA molecule.

[0116] In certain embodiments, the gRNA of the Disclosure comprises (i) four or more 2'-fluoro-modified nucleotides, 2'-O-methyl-modified nucleotides, 2'-O-(2-methoxyethyl)-modified nucleotides, deoxyribose nucleotides and / or LNAs at its 5' end, and (ii) four or more phosphorodithioate bonds at the 5' end of the gRNA molecule. In certain embodiments, the first, second, third and fourth nucleotides at the 5' end of the gRNA molecule are 2'-fluoro-modified nucleotides, 2'-O-methyl-modified nucleotides, 2'-O-(2-methoxyethyl)-modified nucleotides, deoxyribose nucleotides or LNAs, and the gRNA comprises phosphorodithioate bonds between the first and second nucleotides, between the second and third nucleotides, between the third and fourth nucleotides, and between the fourth and fifth nucleotides at the 5' end of the gRNA molecule. In certain embodiments, the first, second, third, and fourth nucleotides at the 5' end of the gRNA molecule are 2'-O-methyl-modified nucleotides, and the gRNA contains phosphorodithioate bonds between the first and second nucleotides, the second and third nucleotides, the third and fourth nucleotides, and the fourth and fifth nucleotides at the 5' end of the gRNA molecule.

[0117] In certain embodiments, the gRNA of the Disclosure comprises (i) one or more 2'-fluoromodified nucleotides, 2'-O-methyl-modified nucleotides, 2'-O-(2-methoxyethyl)-modified nucleotides, deoxyribose nucleotides and / or LNAs at its 3' end, and (ii) one or more phosphorodithioate bonds at the 3' end of the gRNA molecule. In certain embodiments, the n nucleotide of the gRNA molecule is a 2'-fluoromodified nucleotide, a 2'-O-methyl-modified nucleotide, a 2'-O-(2-methoxyethyl)-modified nucleotide, a deoxyribose nucleotide, or locked nucleic acid (LNA), and the gRNA comprises a phosphorodithioate bond between the n nucleotide and the n-1 nucleotide of the gRNA molecule. In certain embodiments, the n nucleotide of the gRNA molecule is a 2'-O-methyl-modified nucleotide, and the gRNA comprises a phosphorodithioate bond between the n nucleotide and the n-1 nucleotide of the gRNA molecule.

[0118] In certain embodiments, the gRNA of the Disclosure comprises (i) two or more 2'-fluoromodified nucleotides, 2'-O-methyl-modified nucleotides, 2'-O-(2-methoxyethyl)-modified nucleotides, deoxyribose nucleotides and / or LNAs at its 3' end, and (ii) two or more phosphorodithioate bonds at the 3' end of the gRNA molecule. In certain embodiments, the n and n-1 or n and n-2 nucleotides of the gRNA molecule are 2'-fluoromodified nucleotides, 2'-O-methyl-modified nucleotides, 2'-O-(2-methoxyethyl)-modified nucleotides or LNAs, and the gRNA molecule comprises phosphorodithioate bonds between the n nucleotide and the n-1 nucleotide, the n-1 nucleotide and the n-2 nucleotide, or between the n nucleotide and the n-1 nucleotide and the n-2 nucleotide and the n-3 nucleotide. In certain embodiments, the n and n-1 nucleotides or the n and n-2 nucleotides of the gRNA molecule are 2'-O-methyl-modified nucleotides, and the gRNA molecule contains phosphorodithioate bonds between the n nucleotide and the n-1 nucleotide, between the n-1 nucleotide and the n-2 nucleotide, or between the n nucleotide and the n-1 nucleotide and between the n-2 nucleotide and the n-3 nucleotide.

[0119] In certain embodiments, the gRNA of the Disclosure comprises (i) three or more 2'-fluoromodified nucleotides, 2'-O-methyl-modified nucleotides, 2'-O-(2-methoxyethyl)-modified nucleotides, deoxyribose nucleotides and / or LNAs at its 3' end, and (ii) two or more phosphorodithioate bonds at the 3' end of the gRNA molecule. In certain embodiments, the n, n-1 and n-2 nucleotides of the gRNA molecule are 2'-fluoromodified nucleotides, 2'-O-methyl-modified nucleotides, 2'-O-(2-methoxyethyl)-modified nucleotides, deoxyribose nucleotides or LNAs, and the gRNA molecule comprises phosphorodithioate bonds between the n nucleotide and the n-1 nucleotide and between the n-1 nucleotide and the n-2 nucleotide, or between the n nucleotide and the n-1 nucleotide and between the n-2 nucleotide and the n-3 nucleotide. In certain embodiments, the n, n-1, and n-2 nucleotides of the gRNA molecule are 2'-O-methyl-modified nucleotides, and the gRNA molecule contains phosphorodithioate bonds between the n nucleotide and the n-1 nucleotide, between the n-1 nucleotide and the n-2 nucleotide, or between the n nucleotide and the n-1 nucleotide, and between the n-2 nucleotide and the n-3 nucleotide.

[0120] In certain embodiments, the gRNA of the Disclosure comprises (i) four or more 2'-fluoro-modified nucleotides, 2'-O-methyl-modified nucleotides, 2'-O-(2-methoxyethyl)-modified nucleotides, deoxyribose nucleotides and / or LNAs at its 3' end, and (ii) four or more phosphorodithioate bonds at the 3' end of the gRNA molecule. In certain embodiments, the n, n-1, n-2 and n-3 nucleotides of the gRNA molecule are 2'-fluoro-modified nucleotides, 2'-O-methyl-modified nucleotides, 2'-O-(2-methoxyethyl)-modified nucleotides, deoxyribose nucleotides or LNAs, and the gRNA molecule comprises phosphorodithioate bonds between the n nucleotide and the n-1 nucleotide, between the n-1 nucleotide and the n-2 nucleotide, between the n-2 nucleotide and the n-3 nucleotide and between the n-3 nucleotide and the n-4 nucleotide. In certain embodiments, the n, n-1, n-2, and n-3 nucleotides of the gRNA molecule are 2'-O-methyl-modified nucleotides, and the gRNA molecule contains phosphorodithioate bonds between the n nucleotide and the n-1 nucleotide, between the n-1 nucleotide and the n-2 nucleotide, between the n-2 nucleotide and the n-3 nucleotide, and between the n-3 nucleotide and the n-4 nucleotide.

[0121] In certain embodiments, the gRNA of the Disclosure comprises (i) five or more 2'-fluoro-modified nucleotides, 2'-O-methyl-modified nucleotides, 2'-O-(2-methoxyethyl)-modified nucleotides, deoxyribose nucleotides and / or LNAs at its 3' end, and (ii) four or more phosphorodithioate bonds at the 3' end of the gRNA molecule. In certain embodiments, the n, n-1, n-2, n-3 and n-4 nucleotides of the gRNA molecule are 2'-fluoro-modified nucleotides, 2'-O-methyl-modified nucleotides, 2'-O-(2-methoxyethyl)-modified nucleotides, deoxyribose nucleotides or LNAs, and the gRNA molecule comprises phosphorodithioate bonds between the n nucleotide and the n-1 nucleotide, between the n-1 nucleotide and the n-2 nucleotide, between the n-2 nucleotide and the n-3 nucleotide and between the n-3 nucleotide and the n-4 nucleotide. In certain embodiments, the n, n-1, n-2, n-3, and n-4 nucleotides of the gRNA molecule are 2'-O-methyl-modified nucleotides, and the gRNA molecule contains phosphorodithioate bonds between the n nucleotide and the n-1 nucleotide, between the n-1 nucleotide and the n-2 nucleotide, between the n-2 nucleotide and the n-3 nucleotide, and between the n-3 nucleotide and the n-4 nucleotide.

[0122] In certain embodiments, the gRNA of the Disclosure comprises (i) one or more 2'-fluoro-modified nucleotides, 2'-O-methyl-modified nucleotides, 2'-O-(2-methoxyethyl)-modified nucleotides, deoxyribose nucleotides and / or LNA at the 5' end, (ii) one or more phosphorodithioate bonds at the 5' end, (iii) one or more 2'-fluoro-modified nucleotides, 2'-O-methyl-modified nucleotides, 2'-O-(2-methoxyethyl)-modified nucleotides, deoxyribose nucleotides and LNA at the 3' end, and (iv) one or more phosphorodithioate bonds at the 3' end. In certain embodiments, the gRNA of the Disclosure comprises (i) one or more 2'-O-methyl-modified nucleotides at the 5' end, (ii) one or more phosphorodithioate bonds at the 5' end, (iii) one or more 2'-O-methyl-modified nucleotides at the 3' end, and (iv) one or more phosphorodithioate bonds at the 3' end.

[0123] In certain embodiments, the gRNA of the Disclosure comprises (i) two or more 2'-fluoro-modified nucleotides, 2'-O-methyl-modified nucleotides, 2'-O-(2-methoxyethyl)-modified nucleotides, deoxyribose nucleotides and / or LNA at the 5' end, (ii) two or more phosphorodithioate bonds at the 5' end, (iii) two or more 2'-fluoro-modified nucleotides, 2'-O-methyl-modified nucleotides, 2'-O-(2-methoxyethyl)-modified nucleotides, deoxyribose nucleotides and LNA at the 3' end, and (iv) two or more phosphorodithioate bonds at the 3' end. In certain embodiments, the gRNA of the Disclosure comprises (i) two or more 2'-O-methyl-modified nucleotides at the 5' end, (ii) two or more phosphorodithioate bonds at the 5' end, (iii) two or more 2'-O-methyl-modified nucleotides at the 3' end, and (iv) two or more phosphorodithioate bonds at the 3' end.

[0124] In certain embodiments, the gRNA of the Disclosure comprises (i) three or more 2'-fluoro-modified nucleotides, 2'-O-methyl-modified nucleotides, 2'-O-(2-methoxyethyl)-modified nucleotides, deoxyribose nucleotides and / or LNA at the 5' end, (ii) two or more phosphorodithioate bonds at the 5' end, (iii) three or more 2'-fluoro-modified nucleotides, 2'-O-methyl-modified nucleotides, 2'-O-(2-methoxyethyl)-modified nucleotides, deoxyribose nucleotides and LNA at the 3' end, and (iv) two or more phosphorodithioate bonds at the 3' end. In certain embodiments, the gRNA of the Disclosure comprises (i) three or more 2'-O-methyl-modified nucleotides at the 5' end, (ii) two or more phosphorodithioate bonds at the 5' end, (iii) three or more 2'-O-methyl-modified nucleotides at the 3' end, and (iv) two or more phosphorodithioate bonds at the 3' end.

[0125] In certain embodiments, the gRNA of the Disclosure comprises (i) three or more 2'-fluoro-modified nucleotides, 2'-O-methyl-modified nucleotides, 2'-O-(2-methoxyethyl)-modified nucleotides, deoxyribose nucleotides and / or LNA at the 5' end, (ii) three or more phosphorodithioate bonds at the 5' end, (iii) three or more 2'-fluoro-modified nucleotides, 2'-O-methyl-modified nucleotides, 2'-O-(2-methoxyethyl)-modified nucleotides, deoxyribose nucleotides and LNA at the 3' end, and (iv) one or more phosphorodithioate bonds at the 3' end. In certain embodiments, the gRNA of the Disclosure comprises (i) three or more 2'-O-methyl-modified nucleotides at the 5' end, (ii) three or more phosphorodithioate bonds at the 5' end, (iii) three or more 2'-O-methyl-modified nucleotides at the 3' end, and (iv) one or more phosphorodithioate bonds at the 3' end.

[0126] In certain embodiments, the gRNA of the Disclosure comprises (i) three or more 2'-fluoro-modified nucleotides, 2'-O-methyl-modified nucleotides, 2'-O-(2-methoxyethyl)-modified nucleotides, deoxyribose nucleotides and / or LNA at the 5' end, (ii) three or more phosphorodithioate bonds at the 5' end, (iii) three or more 2'-fluoro-modified nucleotides, 2'-O-methyl-modified nucleotides, 2'-O-(2-methoxyethyl)-modified nucleotides, deoxyribose nucleotides and LNA at the 3' end, and (iv) two or more phosphorodithioate bonds at the 3' end. In certain embodiments, the gRNA of the Disclosure comprises (i) three or more 2'-O-methyl-modified nucleotides at the 5' end, (ii) three or more phosphorodithioate bonds at the 5' end, (iii) three or more 2'-O-methyl-modified nucleotides at the 3' end, and (iv) two or more phosphorodithioate bonds at the 3' end.

[0127] In certain embodiments, the gRNA of the Disclosure comprises (i) four or more 2'-fluoro-modified nucleotides, 2'-O-methyl-modified nucleotides, 2'-O-(2-methoxyethyl)-modified nucleotides, deoxyribose nucleotides and / or LNA at the 5' end, (ii) four or more phosphorodithioate bonds at the 5' end, (iii) four or more 2'-fluoro-modified nucleotides, 2'-O-methyl-modified nucleotides, 2'-O-(2-methoxyethyl)-modified nucleotides, deoxyribose nucleotides and LNA at the 3' end, and (iv) four or more phosphorodithioate bonds at the 3' end. In certain embodiments, the gRNA of the Disclosure comprises (i) four or more 2'-O-methyl-modified nucleotides at the 5' end, (ii) four or more phosphorodithioate bonds at the 5' end, (iii) four or more 2'-O-methyl-modified nucleotides at the 3' end, and (iv) four or more phosphorodithioate bonds at the 3' end.

[0128] In certain embodiments, the gRNA of the Disclosure comprises (i) four or more 2'-fluoro-modified nucleotides, 2'-O-methyl-modified nucleotides, 2'-O-(2-methoxyethyl)-modified nucleotides, deoxyribose nucleotides and LNA at the 5' end, (ii) four or more phosphorodithioate bonds at the 5' end, (iii) five or more 2'-fluoro-modified nucleotides, 2'-O-methyl-modified nucleotides, 2'-O-(2-methoxyethyl)-modified nucleotides, deoxyribose nucleotides and LNA at the 3' end, and (iv) four or more phosphorodithioate bonds at the 3' end. In certain embodiments, the gRNA of the Disclosure comprises (i) four or more 2'-O-methyl-modified nucleotides at the 5' end, (ii) four or more phosphorodithioate bonds at the 5' end, (iii) five or more 2'-O-methyl-modified nucleotides at the 3' end, and (iv) four or more phosphorodithioate bonds at the 3' end.

[0129] In certain embodiments, the gRNA of the Disclosure comprises (i) a 2'-O-methyl-modified nucleotide (e.g., the first nucleotide) at the 5' end, (ii) a phosphorodithioate bond at the 5' end (e.g., between the first and second nucleotides), (iii) a 2'-O-methyl-modified nucleotide (e.g., the n nucleotide) at the 3' end, and (iv) a phosphorodithioate bond at the 3' end (e.g., between the n nucleotide and the n-1 nucleotide).

[0130] In certain embodiments, the gRNA of the Disclosure comprises (i) two 2'-O-methyl-modified nucleotides at the 5' end (e.g., the first and second nucleotides), (ii) two phosphorodithioate bonds at the 5' end (e.g., between the first and second nucleotides and between the second and third nucleotides), (iii) two 2'-O-methyl-modified nucleotides at the 3' end (e.g., the n and n-1 nucleotides), and (iv) two phosphorodithioate bonds at the 3' end (e.g., between the n nucleotide and the n-1 nucleotide and between the n-1 nucleotide and the n-2 nucleotide).

[0131] In certain embodiments, the gRNA of the Disclosure comprises (i) three 2'-O-methyl-modified nucleotides at the 5' end (e.g., the first, second, and third nucleotides), (ii) three phosphorodithioate bonds at the 5' end (e.g., between the first and second nucleotides, between the second and third nucleotides, and between the third and fourth nucleotides), (iii) three 2'-O-methyl-modified nucleotides at the 3' end (e.g., the n, n-1, and n-2 nucleotides), and (iv) two phosphorodithioate bonds at the 3' end (e.g., between the n nucleotide and the n-1 nucleotide, and between the n-1 nucleotide and the n-2 nucleotide).

[0132] In certain embodiments, the gRNA of the Disclosure comprises (i) three 2'-O-methyl-modified nucleotides at the 5' end (e.g., the first, second, and third nucleotides), (ii) three phosphorothioate bonds at the 5' end (e.g., between the first and second nucleotides, between the second and third nucleotides, and between the third and fourth nucleotides), (iii) three 2'-O-methyl-modified nucleotides at the 3' end (e.g., the n, n-1, and n-2 nucleotides), and (iv) two phosphorothioate bonds at the 3' end (e.g., between the n nucleotide and the n-1 nucleotide, and between the n-1 nucleotide and the n-2 nucleotide).

[0133] In certain embodiments, the gRNA of the Disclosure comprises (i) three 2'-O-methyl-modified nucleotides at the 5' end (e.g., the first, second, and third nucleotides), (ii) two phosphorodithioate bonds at the 5' end (e.g., between the first and second nucleotides and between the second and third nucleotides, or between the second and third nucleotides and between the third and fourth nucleotides), (iii) three 2'-O-methyl-modified nucleotides at the 3' end (e.g., n, n-1, and n-2 nucleotides), and (iv) two phosphorodithioate bonds at the 3' end (e.g., between the n nucleotide and the n-1 nucleotide and between the n-1 nucleotide and the n-2 nucleotide, or between the n-1 nucleotide and the n-2 nucleotide and between the n-3 nucleotide).

[0134] In certain embodiments, the gRNA of the Disclosure has (i) three 2'-O-methyl-modified nucleotides at its 5' end (e.g., the first, second, and third nucleotides), (ii) two phosphorothioate bonds at its 5' end (e.g., between the first and second nucleotides and between the second and third nucleotides, or between the second and third nucleotides and between the third and fourth nucleotides), and (iii) one phosphorothioate bond at its 5' end (e.g., between the first and second nucleotides). (iv) containing a 2'-O-methyl modified nucleotide (e.g., between the first and second nucleotides, between the second and third nucleotides, or between the third and fourth nucleotides), (iv) three 2'-O-methyl modified nucleotides at the 3' end (e.g., between the n, n-1, and n-2 nucleotides), and (v) two phosphorodithioate bonds at the 3' end (e.g., between the n nucleotide and the n-1 nucleotide, and between the n-1 nucleotide and the n-2 nucleotide, or between the n-1 nucleotide and the n-2 nucleotide, and between the n-2 nucleotide and the n-3 nucleotide).

[0135] In certain embodiments, the gRNA of the Disclosure comprises (i) three 2'-O-methyl-modified nucleotides at the 5' end (e.g., the first, second, and third nucleotides), (ii) three phosphorodithioate bonds at the 5' end (e.g., between the first and second nucleotides, between the second and third nucleotides, and between the third and fourth nucleotides), (iii) three 2'-O-methyl-modified nucleotides at the 3' end (e.g., n, n-1, and n-2 nucleotides), and (iv) one phosphorodithioate bond at the 3' end (e.g., between the n nucleotide and the n-1 nucleotide, between the n-1 nucleotide and the n-2 nucleotide, or between the n-2 nucleotide and the n-3 nucleotide).

[0136] In certain embodiments, the gRNA of the Disclosure has (i) three 2'-O-methyl-modified nucleotides at the 5' end (e.g., the first, second, and third nucleotides), (ii) three phosphorodithioate bonds at the 5' end (e.g., between the first and second nucleotides, between the second and third nucleotides, and between the third and fourth nucleotides), and (iii) three 2'-O-methyl-modified nucleotides at the 3' end (e.g., n, n-1, and n-2 nucleotides). (iv) containing one phosphorothioate bond at the 3' end (e.g., between the n nucleotide and the n-1 nucleotide, between the n-1 nucleotide and the n-2 nucleotide, or between the n-2 nucleotide and the n-3 nucleotide) and (v) containing one phosphorothioate bond at the 3' end (e.g., between the n nucleotide and the n-1 nucleotide, between the n-1 nucleotide and the n-2 nucleotide, or between the n-2 nucleotide and the n-3 nucleotide).

[0137] In certain embodiments, the gRNA of the Disclosure comprises (i) four 2'-O-methyl-modified nucleotides at the 5' end (e.g., the first, second, third, and fourth nucleotides), (ii) four phosphorodithioate bonds at the 5' end (e.g., between the first and second nucleotides, between the second and third nucleotides, between the third and fourth nucleotides, and between the fourth and fifth nucleotides), (iii) four 2'-O-methyl-modified nucleotides at the 3' end (e.g., n, n-1, n-2, and n-3 nucleotides), and (iv) four phosphorodithioate bonds at the 3' end (e.g., between the n nucleotide and the n-1 nucleotide, between the n-1 nucleotide and the n-2 nucleotide, between the n-2 nucleotide and the n-3 nucleotide, and between the n-3 nucleotide and the n-4 nucleotide).

[0138] In certain embodiments, the gRNA of the Disclosure comprises (i) four 2'-O-methyl-modified nucleotides at the 5' end (e.g., the first, second, third, and fourth nucleotides), (ii) four phosphorodithioate bonds at the 5' end (e.g., between the first and second nucleotides, between the second and third nucleotides, between the third and fourth nucleotides, and between the fourth and fifth nucleotides), (iii) five 2'-O-methyl-modified nucleotides at the 3' end (e.g., n, n-1, n-2, n-3, and n-4 nucleotides), and (iv) four phosphorodithioate bonds at the 3' end (e.g., between the n nucleotide and the n-1 nucleotide, between the n-1 nucleotide and the n-2 nucleotide, between the n-2 nucleotide and the n-3 nucleotide, and between the n-3 nucleotide and the n-4 nucleotide).

[0139] In certain embodiments, the gRNA of the Disclosure comprises (i) four 2'-O-methyl-modified nucleotides at the 5' end (e.g., the first, second, third, and fourth nucleotides), (ii) four phosphorodithioate bonds at the 5' end (e.g., between the first and second nucleotides, between the second and third nucleotides, between the third and fourth nucleotides, and between the fourth and fifth nucleotides), (iii) five 2'-O-methyl-modified nucleotides at the 3' end (e.g., n, n-1, n-2, n-3, and n-4 nucleotides), and (iv) four phosphorodithioate bonds at the 3' end (e.g., between the n-1 nucleotide and the n-2 nucleotide, between the n-2 nucleotide and the n-3 nucleotide, between the n-3 nucleotide and the n-4 nucleotide, and between the n-4 nucleotide and the n-5 nucleotide).

[0140] In certain embodiments, the modified gRNAs of this disclosure can be modified according to the modification schemes disclosed in Tables 1 and 10. For example, but not limited to, the modified gRNAs can be modified according to modification scheme "A" in Table 1. In certain embodiments, the modified gRNAs can be modified according to modification scheme "B" in Table 1. In certain embodiments, the modified gRNAs can be modified according to modification scheme "C" in Table 1. In certain embodiments, the modified gRNAs can be modified according to modification scheme "D" in Table 1. In certain embodiments, the modified gRNAs can be modified according to modification scheme "E" in Table 1. In certain embodiments, the modified gRNAs can be modified according to modification scheme "F" in Table 1. In certain embodiments, the modified gRNAs can be modified according to modification scheme "G" in Table 1. In certain embodiments, the modified gRNAs can be modified according to modification scheme "H" in Table 1. In certain embodiments, the modified gRNAs can be modified according to modification scheme "I" in Table 1. In certain embodiments, the modified gRNA can be modified according to modification scheme "J" in Table 1. In certain embodiments, the modified gRNA can be modified according to modification scheme "K" in Table 1. In certain embodiments, the modified gRNA can be modified according to modification scheme "L" in Table 1. In certain embodiments, the modified gRNA can be modified according to modification scheme "M" in Table 10. In certain embodiments, the modified gRNA can be modified according to modification scheme "N" in Table 10. In certain embodiments, the modified gRNA can be modified according to modification scheme "O" in Table 10. In certain embodiments, the modified gRNA can be modified according to modification scheme "P" in Table 10. In certain embodiments, the modified gRNA can be modified according to modification scheme "Q" in Table 10. In certain embodiments, the modified gRNA can be modified according to modification scheme "R" in Table 10. In certain embodiments, the modified gRNA can be modified according to modification scheme "S" in Table 10.In certain embodiments, the modified gRNA can be modified according to modification scheme "T" in Table 10. In certain embodiments, the modified gRNA can be modified according to modification scheme "U" in Table 10. In certain embodiments, the modified gRNA can be modified according to modification scheme "V" in Table 10.

[0141] In certain embodiments, the modified gRNAs of this disclosure are present in concentrations of approximately 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, and 24%. %, approximately 25%, approximately 26%, approximately 27%, approximately 28%, approximately 29%, approximately 30%, approximately 31%, approximately 32%, approximately 33%, approximately 34%, approximately 35%, approximately 36%, approximately 37%, approximately 38%, approximately 39%, approximately 40%, approximately 41%, approximately 42%, approximately 43%, approximately 44%, approximately 45%, approximately 46%, approximately 47%, approximately 48%, approximately 49%, approximately 50%, Approximately 51%, approximately 52%, approximately 53%, approximately 54%, approximately 55%, approximately 56%, approximately 57%, approximately 58%, approximately 59%, approximately 60%, approximately 61%, approximately 62%, approximately 63%, approximately 64%, approximately 65%, approximately 66%, approximately 67%, approximately 68%, approximately 69%, approximately 70%, approximately 71%, approximately 72%, approximately 73%, approximately 74%, approximately 75%, approximately 76%, approximately It may have an editing efficiency of 77%, approximately 78%, approximately 79%, approximately 80%, approximately 81%, approximately 82%, approximately 83%, approximately 84%, approximately 85%, approximately 86%, approximately 87%, approximately 88%, approximately 89%, approximately 90%, approximately 91%, approximately 92%, approximately 93%, approximately 94%, approximately 95%, approximately 96%, approximately 97%, approximately 98%, approximately 99%, or approximately 100%.

[0142] In certain embodiments, the modified gRNAs of the Disclosure may have an editing efficiency of about 5% or more, about 10% or more, about 15% or more, about 20% or more, about 25% or more, about 30% or more, about 35% or more, about 40% or more, about 45% or more, about 50% or more, about 55% or more, about 60% or more, about 65% or more, about 70% or more, about 75% or more, about 80% or more, about 85% or more, about 90% or more, or about 95% or more. In certain embodiments, the modified gRNAs of the Disclosure have an editing efficiency of about 60% or more. In certain embodiments, the modified gRNAs of the Disclosure have an editing efficiency of about 65% or more. In certain embodiments, the modified gRNAs of the Disclosure have an editing efficiency of about 70% or more. In certain embodiments, the modified gRNAs of the Disclosure have an editing efficiency of about 75% or more. In certain embodiments, the modified gRNAs of the Disclosure have an editing efficiency of about 80% or more. In certain embodiments, the modified gRNA of this disclosure has an editing efficiency of approximately 85% or more. In certain embodiments, the modified gRNA of this disclosure has an editing efficiency of approximately 90% or more. In certain embodiments, the modified gRNA of this disclosure has an editing efficiency of approximately 95% or more.

[0143] In certain embodiments, the modified gRNAs of the Disclosure may have editing efficiencies of about 5% to about 100%. For example, but not limited to, the modified gRNAs of the Disclosure may have editing efficiencies of about 5% to about 100%, about 10% to about 100%, about 20% to about 100%, about 30% to about 100%, about 40% to about 100%, about 45% to about 100%, about 50% to about 100%, about 55% to about 100%, about 60% to about 100%, about 65% to about 100%, about 70% to about 100%, about 75% to about 100%, about 80% to about 100%, about 85% to about 100%, about 90% to about 100%, or about 95% to about 100%. In certain embodiments, the modified gRNAs of this disclosure may have an editing efficiency of about 50% to about 100%. In certain embodiments, the modified gRNAs of this disclosure may have an editing efficiency of about 60% to about 100%. In certain embodiments, the modified gRNAs of this disclosure may have an editing efficiency of about 70% to about 100%. In certain embodiments, the modified gRNAs of this disclosure may have an editing efficiency of about 80% to about 100%. In certain embodiments, the modified gRNAs of this disclosure may have an editing efficiency of about 90% to about 100%.

[0144] In certain embodiments, the modified gRNAs of the Disclosure may retain the editing efficiency of a reference gRNA or have improved editing efficiency compared to the reference gRNA. For example, but not limited to, the modified gRNAs of the Disclosure may retain about 50% or more, about 55% or more, about 60% or more, about 65% or more, about 70% or more, about 75% or more, about 80% or more, about 85% or more, about 90% or more, or about 95% or more of the editing efficiency of the reference gRNA. In certain embodiments, the modified gRNAs of the Disclosure may retain about 50% or more of the editing efficiency of the reference gRNA. In certain embodiments, the modified gRNAs of the Disclosure may retain about 60% or more of the editing efficiency of the reference gRNA. In certain embodiments, the modified gRNAs of the Disclosure may retain about 70% or more of the editing efficiency of the reference gRNA. In certain embodiments, the modified gRNAs of the Disclosure may retain about 80% or more of the editing efficiency of the reference gRNA. In certain embodiments, the modified gRNA of this disclosure may retain approximately 90% or more of the editing efficiency of the reference gRNA. In certain embodiments, the modified gRNAs of this disclosure may retain about 50% to about 100% of the editing efficiency of the reference gRNA, for example, about 55% to about 100%, about 60% to about 100%, about 65% to about 100%, about 70% to about 100%, about 75% to about 100%, about 80% to about 100%, about 85% to about 100%, about 90% to about 100%, about 91% to about 100%, about 92% to about 100%, about 93% to about 100%, about 94% to about 100%, about 95% to about 100%, about 96% to about 100%, about 97% to about 100%, about 98% to about 100%, or about 99% to about 100%. In certain embodiments, the modified gRNA of the Disclosure may retain about 60% to about 100% of the editing efficiency of the reference gRNA. In certain embodiments, the modified gRNA of the Disclosure may retain about 70% to about 100% of the editing efficiency of the reference gRNA. In certain embodiments, the modified gRNA of the Disclosure may retain about 80% to about 100% of the editing efficiency of the reference gRNA. In certain embodiments, the modified gRNA of the Disclosure may retain about 90% to about 100% of the editing efficiency of the reference gRNA. In certain embodiments, the modified gRNA of the Disclosure may exhibit an increase in editing efficiency compared to the reference gRNA, for example, an increase of about 1% or more, about 2% or more, about 3% or more, about 4% or more, about 5% or more, about 6% or more, about 7% or more, about 8% or more, about 9% or more, or about 10% or more.In certain embodiments, the modified gRNAs of this disclosure may exhibit an increase in editing efficiency of approximately 1% to approximately 10%. In certain embodiments, the reference gRNA may be a gRNA having the same nucleotide sequence as the modified gRNA (e.g., an unmodified gRNA with the same sequence as the modified gRNA). In certain embodiments, the reference gRNA may be a gRNA having a targeting domain with the same sequence as the modified gRNA (e.g., an unmodified gRNA with the same sequence as the modified gRNA). In certain embodiments, the reference gRNA may be a gRNA having the same number of nucleotides as the modified gRNA (e.g., an unmodified gRNA with the same sequence as the modified gRNA).

[0145] III. RNA-inducible nucleases Various types and species of RNA-inducible nucleases can be used in this disclosure. The RNA-inducible nucleases are used in combination with one or more modified gRNAs disclosed herein, for example, in Section II.

[0146] In certain embodiments, the RNA-induced nuclease is a Cas protein. Non-limiting examples of Cas proteins are disclosed in Makarova and Koonin, Methods Mol. Biol. 1311:47-75 (2015), the contents of which are incorporated herein by reference in their entirety. In certain embodiments, Cas proteins include Cas1, Cas2, Cas3, Cas3-HD, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9, Cas10, Cas12a (Cpf1), Cas13, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csy1, Csy2, Csy3, Cse1, Cse2 These include Csc1, Csc2, Csa5, CsaX, Csm2, Csm3, Csm4, Csm5, Csm6, Csn2, Csb1, Csb2, Csb3, Csx1, Csx3, Csx10, Csx14, Csx15, Csx16, Csx17, Csf1, Csf2, Csf3, Csf4, C2c1, C2c2, and C2c3. In certain embodiments, the Cas protein is selected from the group consisting of Cas9, Cas12a, Cas13, and combinations thereof. In certain embodiments, the Cas protein is the Cas9 protein.

[0147] In certain embodiments, the Cas protein is an engineered Cas protein that differs from a reference Cas protein, e.g., wild-type Cas protein. In certain embodiments, the reference Cas protein is a naturally occurring Cas protein. In certain embodiments, the Cas protein contains one or more amino acid mutations compared to a reference Cas protein, e.g., wild-type Cas protein. In certain embodiments, the engineered Cas protein retains or substantially retains the nuclease (e.g., endonuclease) activity of the reference Cas protein. In certain embodiments, the engineered Cas protein retains at least about 70%, about 80%, about 90%, about 95%, or about 99% of the nuclease activity of the reference Cas protein. In certain embodiments, the engineered Cas protein has no cleavage activity or substantially no cleavage activity. In certain embodiments, the Cas protein may lack cleavage activity or have a substantially low amount of cleavage activity of the reference Cas protein, e.g., less than 20%, about 10%, about 5%, or about 1%.

[0148] In certain embodiments, the engineered Cas protein contains one or more deletions that reduce the size of the Cas protein while at least partially retaining the nuclease activity of the Cas protein. In certain embodiments, the reduction in size of the engineered Cas protein may allow for flexibility with respect to the method of delivering such an engineered Cas protein.

[0149] In certain embodiments, the Cas protein interacts with the gRNA molecule of this disclosure and, in cooperation with the gRNA molecule, localizes to a target nucleic acid containing a target sequence (e.g., a sequence complementary to the gRNA molecule's sequence) and a PAM sequence. In certain embodiments, the Cas protein's ability to interact with and cleave the target nucleic acid is PAM sequence-dependent. In certain embodiments, cleavage of the target nucleic acid occurs upstream of the PAM sequence. In certain embodiments, cleavage of the target nucleic acid occurs downstream of the PAM sequence. Cas molecules from different species, e.g., bacterial species, may recognize different PAM sequences.

[0150] In certain embodiments, the Cas protein for use in this disclosure may be derived from any one of the following species:Streptococcus pyogenes, Streptococcus pneumoniae, Streptococcus thermophilus, Streptococcus agalactiae, Streptococcus parasanguinis, Streptococcus oralis, Streptococcus salivarius, Streptococcus macacae, Streptococcus dysgalactiae, Streptococcus anginosus, Streptococcus constellatus, Streptococcus pseudoporcinus, Streptococcus mutans, Listeria innocua, Spiroplasma apis, Spiroplasma syrphidicola, Porphyromonas catoniae, Prevotella intermedia, Treponema socranskii, Finegoldia magna, Pasteurella bettyae, Olivibacter sitiensis, Epilithonimonas tenax, Mesonia mobilis, Lactobacillus plantarum, Coriobacteriaceae, Olsenella profusa, Haemophilus spitorum, Haemophilus pittmaniae, Pasteurella bettyae, Olivibacter sitiensis, Epilithonimonas tenax, Mesonia mobilis, Lactobacillus plantarum, Bacillus cereus, Aquimarina muellen, Chryseobacterium palustre, Bacteroides graminisolvens, Neisseria meningitidis, Francisella novicida, Flavobacterium frigidarium, Flavobacterium soli and / or Treponema denticola.

[0151] In certain embodiments, the Cas protein for use in this disclosure directs the cleavage of one or both strands at the site of the target nucleic acid. For example, but not limited to, the Cas protein for use in this disclosure directs the cleavage of one or both strands within the target nucleic acid. Alternatively, the Cas protein directs the cleavage of one or both strands within about 500 base pairs from the target nucleic acid (e.g., within about 400, 300, 200, 100, 80, 60, 40, 20, 10, or 5 base pairs).

[0152] In certain embodiments, a Cas protein, e.g., Cas9, containing functional RuvC and HNH nuclease domains, can cleave both strands of a target nucleic acid sequence. In certain embodiments, a Cas protein, e.g., Cas9, contains a single functional endonuclease domain that allows the Cas protein to cleave only one strand (i.e., a nick) of the target nucleic acid sequence. For example, but not limited to, a Cas9 nickase may contain (i) a non-functional RuvC domain (e.g., a mutant RuvC domain) and (ii) a functional HNH domain (e.g., a wild-type HNH domain). In certain embodiments, a Cas9 nickase may contain (i) a functional RuvC domain (e.g., a wild-type RuvC domain) and (ii) a non-functional HNH domain (e.g., a mutant HNH domain). In certain embodiments, a Cas9 nickase contains a functional HNH-like sequence containing a mutation in D10, e.g., D10A. In certain embodiments, Cas9 nickase includes a functional RuvC domain and is mutated to H840, for example, H840A. In certain embodiments, Cas9 nickase includes a functional RuvC domain and is mutated to N863, for example, N863A.

[0153] In certain embodiments, the nucleotide sequence encoding the Cas protein is codon-optimized. For example, but not limited to, the nucleotide sequence encoding the Cas protein may be codon-optimized (e.g., by replacing at least one non-common or less common codon with a common codon) to optimize expression in a particular cell type, such as mammalian cells.

[0154] In certain embodiments, the Cas protein is a fusion protein comprising one or more heterologous protein domains. In certain embodiments, the Cas fusion protein may include any additional protein domains, such as an epitope tag, a reporter sequence, and one or more protein domains having the following activities: methylase activity, demethylase activity, transcriptional activation activity, transcriptional repression activity, transcriptional release factor activity, histone modification activity, RNA cleavage activity, and nucleic acid binding activity.

[0155] In certain embodiments, the Cas protein includes one or more nuclear localization sequences to facilitate the accumulation of a detectable amount of Cas protein in the cell nucleus. Nuclear localization sequences are known in the art. For example, but not limited to, the Cas protein may include nuclear localization sequences (e.g., derived from SV40) at its N-terminus and / or C-terminus.

[0156] IV. Composition This disclosure further provides compositions comprising one or more modified gRNAs disclosed herein. In certain embodiments, the compositions of this disclosure may comprise two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, or ten or more modified gRNAs disclosed herein.

[0157] In certain embodiments, the compositions of this disclosure include gRNA having one or more modifications disclosed herein, for example, as described in Section II. In certain embodiments, the compositions may include nucleic acids encoding gRNA molecules disclosed herein. Alternatively or additionally, the compositions may include gRNA molecules as transcribed or synthesized RNA molecules.

[0158] In certain embodiments, the compositions of the present disclosure may further comprise an RNA-inducible nuclease. In certain embodiments, the composition comprises a nucleic acid encoding an RNA-inducible nuclease. Alternatively or additionally, the composition comprises an RNA-inducible nuclease as a protein. In certain embodiments, the RNA-inducible nuclease is a Cas protein, such as a Cas9 protein.

[0159] This disclosure further provides nucleic acid compositions comprising one or more modified gRNAs disclosed herein. In certain embodiments, the nucleic acid composition comprises a polynucleotide encoding a gRNA molecule as described herein. In certain embodiments, the nucleic acid composition may further comprise a polynucleotide encoding an RNA-inducible nuclease, such as a Cas protein. In certain embodiments, the nucleic acid composition of this disclosure may comprise a nucleic acid comprising a nucleic acid encoding a gRNA molecule and an RNA-inducible nuclease, such as a polynucleotide encoding a gRNA molecule (e.g., coupled to a first promoter) and a polynucleotide encoding an RNA-inducible nuclease (e.g., coupled to a second promoter). In certain embodiments, the nucleic acid composition of this disclosure may comprise a first nucleic acid encoding a gRNA molecule and a second nucleic acid encoding an RNA-inducible nuclease.

[0160] In certain embodiments, a nucleic acid composition encoding one or more gRNA molecules and / or one or more RNA-inducible nucleases can be administered to a subject or delivered to cells by methods known in the Art or by methods described herein. For example, but not limited to, nucleic acids encoding gRNA molecules and / or RNA-inducible nucleases can be delivered to a subject or to cells by non-vector-based methods (e.g., by the use of DNA complexes or naked DNA), vector-based methods, or a combination thereof. In certain embodiments, the vector may be a viral vector. In certain embodiments, the virus may be an RNA virus or a DNA virus. Exemplary viral vectors / viruses include, but are not limited to, retroviruses, lentiviruses, adenoviruses, adeno-associated viruses (AAVs), vaccinia viruses, poxviruses, and herpes simplex viruses. In certain embodiments, the vector of this disclosure comprises a polynucleotide sequence encoding a gRNA molecule and / or RNA-inducible nuclease. In certain embodiments, the vector further includes, for example, a sequence encoding a nuclear localization sequence fused to the RNA-inducible nuclease at the N-terminus and / or C-terminus of the RNA-inducible nuclease.

[0161] This disclosure further provides compositions comprising ribonucleoprotein (RNP) complexes. In certain embodiments, the RNP complex comprises, for example, a gRNA molecule as transcribed or synthesized RNA, and an RNA-inducible nuclease. In certain embodiments, the gRNA molecule forms an RNP complex with the RNA-inducible nuclease under appropriate conditions before delivery to target cells.

[0162] This disclosure further provides cells comprising one or more compositions of this disclosure. For example, but not limited to, cells may comprise the nucleic acid compositions of this disclosure. In certain embodiments, cells may comprise the RNP complexes of this disclosure. Various cells can be modified using the disclosed compositions. For example, but not limited to, cells may be immune cells, such as T cells. In certain embodiments, T cells may comprise CD8+ T cells and / or CD4 + They may be T cells. In certain embodiments, the cells may be stem cells. In certain embodiments, the cells may be induced pluripotent stem (iPS) cells. In certain embodiments, the cells may be stem cells or cells derived from iPS cells, such as immune cells derived from stem cells or iPS cells.

[0163] In certain embodiments, the compositions of the Disclosure, for example, the nucleic acid compositions of the Disclosure, can be delivered to target cells by methods known in the Art or by methods described herein. For example, but not limited to, the compositions of the Disclosure can be delivered to cells by microinjection, electroporation, transient cell compression or cell squeezing, lipid-mediated transfection, peptide-mediated delivery, or a combination thereof. In certain embodiments, the compositions, for example, compositions comprising an RNP complex, are delivered to target cells by electroporation.

[0164] V. How to use This disclosure further provides methods for using the gRNAs disclosed herein. In certain embodiments, the gRNA molecules provided herein can be used to modify target nucleic acids within cells. For example, but not limited to, the gRNA molecules of this disclosure can be used to modify target nucleic acids within cells, for example, ex vivo or in vivo.

[0165] In certain embodiments, the gRNA molecules of the Disclosure can be used in a method to reduce the expression of a target nucleic acid in a cell. In certain embodiments, the gRNA molecules of the Disclosure can be used in a method to increase the expression of a target nucleic acid in a cell. In certain embodiments, the gRNA molecules of the Disclosure can be used in a method to introduce an insertion or deletion of one or more nucleotides adjacent to a target nucleic acid. In certain embodiments, the gRNA molecules of the Disclosure can be used in a method to introduce a deletion adjacent to a target nucleic acid. In certain embodiments, the gRNA molecules of the Disclosure can be used in a method to introduce an insertion adjacent to a target nucleic acid. In certain embodiments, the gRNA molecules of the Disclosure can be used in a method to introduce one or more cleavage (e.g., a double-strand break or a single-strand break) adjacent to a target nucleic acid.

[0166] In certain embodiments, a method for modifying a target nucleic acid in a cell involves contacting the cell with a gRNA molecule containing a sequence-specific spacer (e.g., a targeting domain) within the target nucleic acid. In certain embodiments, this method involves contacting the cell with a composition disclosed herein, for example, a composition comprising a modified gRNA molecule and an RNA-inducible nuclease.

[0167] This disclosure further provides methods for treating a subject. For example, but not limited to, further compositions comprising the gRNA molecules of this disclosure can be used in therapeutic methods. In certain embodiments, this disclosure provides gRNA molecules for use in treating an individual requiring treatment. In certain embodiments, this disclosure provides gRNA molecules for use in treating an individual having a disease. In certain embodiments, the gRNA molecule comprises a spacer complementary to a target nucleic acid, e.g., a gene associated with the disease being treated. In certain embodiments, the method comprises ex vivo modification of cells of interest by contacting the cells with an effective amount of the gRNA molecules disclosed herein or a composition comprising the RNP complex disclosed herein, for example, by any delivery method disclosed herein or known in the art. In certain embodiments, the method may further include returning the modified cells to the subject.

[0168] In certain embodiments, the gRNA molecules of this disclosure can be used as pharmaceuticals. In certain embodiments, this disclosure provides the use of the gRNA molecules disclosed herein in the manufacture or preparation of pharmaceuticals. In certain embodiments, the pharmaceuticals are for the treatment of diseases. In certain embodiments, the pharmaceuticals are used in methods for treating diseases, which include administering an effective amount of the pharmaceuticals to an individual having a disease.

[0169] In certain embodiments, a composition comprising any of the gRNA molecules provided herein can be used in any of the therapeutic methods described above. In certain embodiments, a composition comprising any of the gRNA molecules and / or RNA-inducible nucleases provided herein can be used in therapeutic methods such as ex vivo cell modification.

[0170] In certain embodiments, one or more compositions disclosed herein, such as nucleic acid compositions, may be administered to a subject or brought into contact with cells derived from the subject, for example, ex vivo. In certain embodiments, the composition may comprise the gRNA and RNA-inducible nucleases of the Disclosure. Alternatively, the composition may comprise the gRNA but not the RNA-inducible nucleases of the Disclosure. In certain embodiments, the composition comprising gRNA, such as the nucleic acid composition, is administered simultaneously with the composition comprising the RNA-inducible nuclease. In certain embodiments, the composition comprising gRNA, such as the nucleic acid composition, is administered after the composition comprising the RNA-inducible nuclease. In certain embodiments, the composition comprising gRNA, such as the nucleic acid composition, is administered before the composition comprising the RNA-inducible nuclease. In certain embodiments, the nucleic acid composition may be delivered by any of the delivery methods described herein; for example, the nucleic acid encoding the gRNA may be delivered by a viral vector. In certain embodiments, the composition comprising the RNA-inducible nuclease may be delivered to cells by electroporation.

[0171] VI. Gene editing systems This disclosure also provides a gene editing system comprising one or more compositions disclosed herein and / or materials useful for carrying out the methods described herein.

[0172] In certain embodiments, the gene editing system of this disclosure may include one or more modified gRNA molecules disclosed herein. For example, but not limited to, the gene editing system of this disclosure may include one or more gRNA molecules disclosed in Section II.

[0173] In certain embodiments, the gRNA molecule of the gene editing system of the present disclosure includes (a) a phosphorodithioate bond between the first and second nucleotides at the 5' end of the guide RNA molecule; (b) a phosphorodithioate bond between the nucleotide at the 3' end ("n") of the guide RNA molecule and the n-1 nucleotide; or (c) a phosphorodithioate bond between the first and second nucleotides at the 5' end of the guide RNA molecule, and a phosphorodithioate bond between the n nucleotide and the n-1 nucleotide of the guide RNA molecule. In certain embodiments, the phosphorodithioate bond is present between the first and second nucleotides at the 5' end of the gRNA molecule, between the second and third nucleotides, and between the third and fourth nucleotides. In certain embodiments, phosphorodithioate bonds are present between the n nucleotide and the n-1 nucleotide of the gRNA molecule, and between the n-1 nucleotide and the n-2 nucleotide, or between the n nucleotide and the n-1 nucleotide, and between the n-2 nucleotide and the n-3 nucleotide.

[0174] In certain embodiments, the first, second, and / or third nucleotides at the 5' end of the gRNA molecule include modifications selected from the group consisting of 2'-fluoro-modified nucleotides; 2'-O-methyl-modified nucleotides; 2'-O-(2-methoxyethyl)-modified nucleotides; locked nucleic acids (LNA); deoxyribose nucleotides; and two or more combinations of the aforementioned modifications. In certain embodiments, the first, second, third, and / or fourth nucleotides at the 5' end of the gRNA molecule include modifications selected from the group consisting of 2'-fluoro-modified nucleotides; 2'-O-methyl-modified nucleotides; 2'-O-(2-methoxyethyl)-modified nucleotides; locked nucleic acids (LNA); deoxyribose nucleotides; and two or more combinations of the aforementioned modifications. In certain embodiments, the first, second, third, fourth, and / or fifth nucleotides at the 5' end of the gRNA molecule include modifications selected from the group consisting of 2'-fluoromodified nucleotides; 2'-O-methyl modified nucleotides; 2'-O-(2-methoxyethyl) modified nucleotides; locked nucleic acids (LNAs); deoxyribose nucleotides; and two or more combinations of the aforementioned modifications.

[0175] In certain embodiments, the n, n and n-1 nucleotides, n and n-2, n-1 and n-2, or n, n-1 and n-2 nucleotides of a gRNA molecule include modifications selected from the group consisting of 2'-fluoro-modified nucleotides; 2'-O-methyl-modified nucleotides; 2'-O-(2-methoxyethyl)-modified nucleotides; locked nucleic acids (LNA); deoxyribose nucleotides; and two or more combinations of the aforementioned modifications. In certain embodiments, the n, n-1, n-2, and n-3 nucleotides or n-1, n-2, n-3, and n-4 nucleotides of a gRNA molecule include modifications selected from the group consisting of 2'-fluoro-modified nucleotides; 2'-O-methyl-modified nucleotides; 2'-O-(2-methoxyethyl)-modified nucleotides; locked nucleic acids (LNA); deoxyribose nucleotides; and two or more combinations of the aforementioned modifications. In certain embodiments, the n, n-1, n-2, n-3, and n-4 nucleotides of the gRNA molecule include modifications selected from the group consisting of 2'-fluoro-modified nucleotides; 2'-O-methyl-modified nucleotides; 2'-O-(2-methoxyethyl)-modified nucleotides; locked nucleic acids (LNAs); deoxyribose nucleotides; and two or more combinations of the aforementioned modifications.

[0176] In certain embodiments, the gene editing system of this disclosure may include one or more modified gRNAs, such as those disclosed in Section II, and an RNA-inducible nuclease, such as that disclosed in Section III. In certain embodiments, the RNA-inducible nuclease is a Cas protein. In certain embodiments, the Cas protein is selected from the group consisting of Cas9, Cas12, Cas13, and combinations thereof.

[0177] In certain embodiments, the gene editing system of the present disclosure comprises one or more nucleic acids encoding a gRNA, such as those disclosed herein, for example, in Section II. In certain embodiments, the one or more nucleic acids further comprise an RNA-inducible nuclease, such as a polynucleotide encoding an RNA-inducible nuclease, for example, as disclosed in Section III. In certain embodiments, the RNA-inducible nuclease is a Cas protein. In certain embodiments, the Cas protein is selected from the group consisting of Cas9, Cas12, Cas13 and combinations thereof.

[0178] In certain embodiments, the gene editing system includes one or more vectors containing nucleic acids, such as those described herein, for example, as disclosed in Section IV.

[0179] In certain embodiments, the gene editing system comprises one or more compositions comprising gRNA, such as that disclosed herein, for example, in Section II. In certain embodiments, the composition further comprises an RNA-inducible nuclease. In certain embodiments, the composition comprises nucleic acids described herein, such as that disclosed herein, for example, in Section IV.

[0180] In certain embodiments, the gene editing system comprises one or more RNP complexes comprising gRNAs and RNA-inducible nucleases as disclosed in Section II herein. In certain embodiments, the RNA-inducible nuclease is a Cas protein. In certain embodiments, the Cas protein is selected from the group consisting of Cas9, Cas12, Cas13 and combinations thereof.

[0181] VII. Exemplary Embodiments A1. The subject matter of this disclosure is: (a) A phosphorodithioate bond between the first and second nucleotides at the 5' end of the guide RNA molecule. (b) A phosphorodithioate bond between the nucleotide at the 3' end ("n") of the guide RNA molecule and the n-1 nucleotide, or (c) A phosphorodithioate bond between the first nucleotide and the second nucleotide at the 5' end of the guide RNA molecule, and a phosphorodithioate bond between the n nucleotide and the n-1 nucleotide of the guide RNA molecule. This provides a guide RNA molecule that includes [the specified element].

[0182] A2. The guide RNA molecule of A1, further comprising phosphorodithioate bonds between the second and third nucleotides at the 5' end of the guide RNA molecule, and between the third and fourth nucleotides.

[0183] A3. A guide RNA molecule of type A1 or A2, containing phosphorodithioate bonds between the first and second nucleotides, between the second and third nucleotides, and between the third and fourth nucleotides at the 5' end of the guide RNA molecule.

[0184] A4. A guide RNA molecule according to any one of A1 to A3, wherein the guide RNA molecule contains a phosphorodithioate bond between the first nucleotide and the second nucleotide, between the second nucleotide and the third nucleotide, between the third nucleotide and the fourth nucleotide, and between the fourth nucleotide and the fifth nucleotide at the 5' end of the guide RNA molecule.

[0185] A5. A guide RNA molecule according to any one of A1 to A4, wherein the guide RNA molecule contains a phosphorodithioate bond between the n nucleotide and the n-1 nucleotide, and between the n-1 nucleotide and the n-2 nucleotide, or between the n nucleotide and the n-1 nucleotide, and between the n-2 nucleotide and the n-3 nucleotide.

[0186] A6. A guide RNA molecule according to any one of A1 to A5, wherein the guide RNA molecule contains phosphorodithioate bonds between the n nucleotide and the n-1 nucleotide, between the n-1 nucleotide and the n-2 nucleotide, and between the n-2 nucleotide and the n-3 nucleotide.

[0187] A7. A guide RNA molecule according to any one of A1 to A6, wherein the guide RNA molecule contains a phosphorodithioate bond between the n nucleotide and the n-1 nucleotide, between the n-1 nucleotide and the n-2 nucleotide, between the n-2 nucleotide and the n-3 nucleotide, and between the n-3 nucleotide and the n-4 nucleotide.

[0188] A8. The first nucleotide at the 5' end of the guide RNA molecule is (a) 2'-fluoromodified nucleotide, (b) 2'-O-methyl-modified nucleotide, (c) 2'-O-(2-methoxyethyl) modified nucleotide, (d) Locked nucleic acid (LNA), (e) Deoxyribose nucleotides, and (f)(a)~(e) Two or more combinations A guide RNA molecule described in any one of items A1 to A7, including a modification selected from the group consisting of the following.

[0189] A9. The guide RNA molecule described in A8, wherein the first nucleotide at the 5' end of the guide RNA molecule contains a 2'-fluoromodified nucleotide.

[0190] A10. The guide RNA molecule described in A8, wherein the first nucleotide at the 5' end of the guide RNA molecule contains a 2'-O-methyl modified nucleotide.

[0191] A11. The guide RNA molecule described in A8, wherein the first nucleotide at the 5' end of the guide RNA molecule contains a 2'-O-(2-methoxyethyl) modified nucleotide.

[0192] A12. The guide RNA molecule described in A8, wherein the first nucleotide at the 5' end of the guide RNA molecule contains LNA.

[0193] A13. The guide RNA molecule described in A8, wherein the first nucleotide at the 5' end of the guide RNA molecule contains a deoxyribose nucleotide.

[0194] A14. The 5' end of the guide RNA molecule contains (i) the second nucleotide, (ii) the third nucleotide, (iii) the fourth nucleotide, (iv) the second and third nucleotides, (v) the second and fourth nucleotides, (vi) the third and fourth nucleotides, or (v) the second, third and fourth nucleotides. (a) 2'-fluoromodified nucleotide, (b) 2'-O-methyl-modified nucleotide, (c) 2'-O-(2-methoxyethyl) modified nucleotide, (d) Locked nucleic acid (LNA), (e) Deoxyribose nucleotides, and (f)(a)~(e) Two or more combinations A guide RNA molecule described in any one of items A1 to A13, including a modification selected from the group consisting of the following.

[0195] A15. A guide RNA molecule as described in any one of A1-A9 and A14, wherein the guide RNA molecule contains three consecutive 2'-fluoromodified nucleotides in the first three nucleotides of the 5' end of the guide RNA molecule.

[0196] A16. A guide RNA molecule as described in any one of A1-A8, A10, and A14, wherein the guide RNA molecule contains three consecutive 2'-O-methyl-modified nucleotides in the first three nucleotides of the 5' end of the guide RNA molecule.

[0197] A17. A guide RNA molecule as described in any one of A1-A8, A10, and A14, wherein the guide RNA molecule contains four consecutive 2'-O-methyl-modified nucleotides in the first four nucleotides of the 5' end of the guide RNA molecule.

[0198] A18. A guide RNA molecule as described in any one of A1-A8, A11, and A14, wherein the guide RNA molecule contains three consecutive 2'-O-(2-methoxyethyl) modified nucleotides in the first three nucleotides of the 5' end of the guide RNA molecule.

[0199] A19. A guide RNA molecule as described in any one of A1-A8, A12, and A14, wherein the guide RNA molecule contains three consecutive LNAs in the first three nucleotides of the 5' end of the guide RNA molecule.

[0200] A20. A guide RNA molecule as described in any one of A1-A8, A13, and A14, wherein the guide RNA molecule contains three consecutive deoxyribose nucleotides in the first three nucleotides of the 5' end of the guide RNA molecule.

[0201] A21. The nucleotide at the 3' end ("n") of the guide RNA molecule is (a) 2'-fluoromodified nucleotide, (b) 2'-O-methyl-modified nucleotide, (c) 2'-O-(2-methoxyethyl) modified nucleotide, (d) Locked nucleic acid (LNA), (e) Deoxyribose nucleotides, and (f)(a)~(e) Two or more combinations A guide RNA molecule described in any one of items A1 to A20, including a modification selected from the group consisting of the following.

[0202] A22. The guide RNA molecule described in A21, wherein the n nucleotide of the guide RNA molecule contains a 2'-fluoromodified nucleotide.

[0203] A23. The guide RNA molecule described in A21, wherein the n nucleotide of the guide RNA molecule contains a 2'-O-methyl modified nucleotide.

[0204] A24. The guide RNA molecule described in A21, wherein the n nucleotide of the guide RNA molecule contains a 2'-O-(2-methoxyethyl) modified nucleotide.

[0205] A25. The guide RNA molecule described in A21, wherein the n nucleotide of the guide RNA molecule contains LNA.

[0206] A26. The guide RNA molecule described in A21, wherein the n nucleotide of the guide RNA molecule contains a deoxyribose nucleotide.

[0207] A27. The (i) n-1 nucleotide, (ii) n-2 nucleotide, (iii) n-3 nucleotide, (iv) n-4 nucleotide, (v) n and n-1 nucleotide, (vi) n and n-2 nucleotide, (vii) n-1 and n-2 nucleotide, (viii) n, n-1, and n-2 nucleotide, (ix) n-1, n-2, and n-3 nucleotide, (x) n, n-1, n-2, and n-3 nucleotide, or (xi) n, n-1, n-2, n-3, and n-4 nucleotide, respectively. (a) 2'-fluoromodified nucleotide, (b) 2'-O-methyl-modified nucleotide, (c) 2'-O-(2-methoxyethyl) modified nucleotide, (d) Locked nucleic acid (LNA), (e) Deoxyribose nucleotides, and (f)(a)~(e) Two or more combinations A guide RNA molecule described in any one of items A1 to A26, including a modification selected from the group consisting of the following.

[0208] A28. A guide RNA molecule according to any one of A1 to A22 and A27, wherein the n and n-1 nucleotides, the n and n-2 nucleotides, the n-1 and n-2 nucleotides, the n, n-1 and n-2 nucleotides, the n-1, n-2, n-3 and n-4 nucleotides, or the n, n-1, n-2, n-3 and n-4 nucleotides are each 2'-fluoromodified nucleotides.

[0209] A29. A guide RNA molecule according to any one of A1 to A21, A23, and A27, wherein the n and n-1 nucleotides, the n and n-2 nucleotides, the n-1 and n-2 nucleotides, the n, n-1 and n-2 nucleotides, the n-1, n-2, n-3, and n-4 nucleotides, or the n, n-1, n-2, n-3, and n-4 nucleotides are each 2'-O-methyl modified nucleotides.

[0210] A30. A guide RNA molecule as described in any one of A1-A21, A23, A27, and A29, wherein the n, n-1, n-2, n-3, and n-4 nucleotides of the guide RNA molecule are each 2'-O-methyl modified nucleotides.

[0211] A31. A guide RNA molecule according to any one of A1 to A21, A24, and A27, wherein the n and n-1 nucleotides, the n and n-2 nucleotides, the n-1 and n-2 nucleotides, the n, n-1, and n-2 nucleotides, the n-1, n-2, n-3, and n-4 nucleotides, or the n, n-1, n-2, n-3, and n-4 nucleotides are each 2'-O-(2-methoxyethyl) modified nucleotides.

[0212] A32. A guide RNA molecule according to any one of A1 to A21, A25, and A27, wherein the n and n-1 nucleotides, the n and n-2 nucleotides, the n-1 and n-2 nucleotides, the n, n-1 and n-2 nucleotides, the n-1, n-2, n-3, and n-4 nucleotides, or the n, n-1, n-2, n-3, and n-4 nucleotides are each LNAs.

[0213] A33. A guide RNA molecule according to any one of A1 to A21, A26, and A27, wherein the n and n-1 nucleotides, the n and n-2 nucleotides, the n-1 and n-2 nucleotides, the n, n-1 and n-2 nucleotides, the n-1, n-2, n-3, and n-4 nucleotides, or the n, n-1, n-2, n-3, and n-4 nucleotides are each deoxyribose nucleotides.

[0214] B. The subject matter of this disclosure provides nucleic acids comprising polynucleotides encoding a guide RNA molecule as described in any one of A1 to A33.

[0215] B1. The nucleic acid described in B, further comprising a polynucleotide encoding an RNA-inducible nuclease.

[0216] B2. The nucleic acid described in B1, in which the RNA-induced nuclease is a Cas protein.

[0217] B3. The nucleic acid described in B2, wherein the Cas protein is selected from the group consisting of Cas9, Cas12, Cas13 and combinations thereof.

[0218] C. The subject matter of this disclosure provides vectors comprising nucleic acids as described in any one of sections B to B3.

[0219] D. The subject matter of this disclosure provides compositions comprising a guide RNA molecule as described in any one of A1 to A33.

[0220] The composition according to D, further comprising an RNA-guided nuclease.

[0221] The composition according to D or D1, further comprising a nucleic acid encoding an RNA-guided nuclease.

[0222] The composition according to D1 or D2, wherein the RNA-guided nuclease is a Cas protein.

[0223] E. The subject matter of the present disclosure provides a composition comprising the nucleic acids of B to B3.

[0224] F. The subject matter of the present disclosure provides a composition comprising the vector of C.

[0225] G. The subject matter of the present disclosure provides a ribonucleoprotein (RNP) complex comprising a guide RNA molecule according to any one of A1 to A33 and an RNA-guided nuclease.

[0226] G1. The RNP complex of G, wherein the RNA-guided nuclease is a Cas protein.

[0227] G2. The RNP complex according to G1, wherein the Cas protein is selected from the group consisting of Cas9, Cas12, Cas13, and combinations thereof.

[0228] H. The subject matter of the present disclosure provides a cell comprising the composition according to any one of D to D3, E, or F.

[0229] I. The subject matter of the present disclosure provides a cell comprising the RNP complex according to any one of G to G2.

[0230] J. The subject matter of the present disclosure provides a method of modifying a cell, the method comprising contacting the cell with a composition according to any one of D to D3, E or F, or an RNP complex according to any one of G to G2.

[0231] J1. The method according to J, wherein said contacting comprises introducing the composition into the cells by electroporation. K. The subject matter of the present disclosure is a method of treating a subject in need of treatment, comprising: (a) modifying the cells of the subject ex vivo by contacting the cells with the composition according to any one of D to D3, E or F, or the RNP complex according to any one of G to G2; (b) returning the modified cells to the subject. A method comprising:

[0232] L. The subject matter of the present disclosure is: (a) one or more guide RNA molecules according to any one of A1 to A33; (b) one or more nucleic acids according to any one of B to B3; (c) one or more vectors according to C; (d) one or more compositions according to D to D3, E, or F, and / or (e) one or more RNP complexes according to any one of G to G2. A gene editing system is provided.

Examples

[0233] The subject matter disclosed herein will be better understood by reference to the following examples, which are provided as illustrations of the subject matter disclosed herein and not by way of limitation.

[0234] Example 1: Modification of gRNA molecules This example provides gRNA molecules chemically synthesized to contain multiple modifications at the 5' and 3' ends of the gRNA molecule. As shown in Table 1, this example provides modification strategies A to L. Table 1 shows a summary of the modifications for each of these strategies compared to a reference gRNA molecule.

[0235] Nucleotide modifications are shown in Table 1 as follows: *: phosphorothioate (PS) linkage; **: phosphorodithioate (PS2) linkage; m: 2'-OMe; ln: locked nucleic acid (LNA); f: 2'-fluoro; M: 2'-O-MOE; d: deoxyribonucleotide. For example, mA represents 2'-O-methyladenosine and dA represents adenosine deoxyribonucleotide. In the sequences in Table 1, "N" represents any nucleotide base, e.g., guanine (G), adenine (A), thymine (T), or cytosine (C). The reference gRNAs disclosed in Table 1 (referred to as reference gRNA1, reference gRNA2, and reference gRNA3) have the same nucleotide sequences as the modified gRNAs of strategies A-L. Reference gRNAs 1 and 3 have the same modification strategies as those shown in Table 1. Reference gRNA2, compared to reference gRNAs 1 and 3, has three phosphorothioate bonds at its 3' end and terminates with rU. [Table 1] TIFF2026512014000003.tif255170

[0236] The identity of the synthesized modified gRNA was confirmed by mass spectrometry, and the mass error between the experimental and theoretical masses was less than 200 ppm. As shown in Tables 2 and 3, the experimental masses were consistent with the calculated masses, confirming the correct length and nucleic acid base composition.

[0237] The purity of the synthesized modified gRNAs was analyzed. As shown in Figures 2 and 3, similar purity profiles were observed for gRNAs modified according to modification schemes A-F. The purity of these modified gRNAs ranged from 80.1% to 99.5%. Figures 2 and 4 and Table 2 provide the purity profiles of gRNAs targeting reference target 1 and modified according to modification schemes A-L, while Figures 3 and 5 and Table 3 provide the purity profiles of gRNAs targeting reference target 2 and modified according to modification schemes A-L. As shown in Figures 2 and 3, the gRNA sequence had little effect on the purity and ion-paired reversed-phase liquid chromatography (IPRP) profile of modified gRNAs modified according to modification schemes A-F. Similar results were observed for gRNAs targeting these two target sequences and modified according to modification schemes G-L (Figures 4-5 and Tables 2 and 3). Furthermore, impurities were observed to be mainly shortmers.

[0238] As shown in Figure 4, the 2'-fluoro modification significantly increased the hydrophobicity of the sgRNA. While not bound by any particular theory, increased hydrophobicity may allow oligonucleotides to bind and interact with specific domains of the protein, thereby regulating its activity (see Crooke et al., Nucleic Acids Research 48(10):5235-5253 (2020), the entire content of which is incorporated herein by reference). [Table 2] [Table 3]

[0239] The efficacy of the above modified gRNA molecules, targeting reference target 1 as shown in Table 1, was analyzed as follows: Cells were seeded in Prime-XV (IL-7 (25 ng / ml), IL-15 (50 ng / ml), TransAct1:100) (= Day 0). 4 mg of each sgRNA was resuspended in 611-624 μL (depending on MW) of IDT Duplex buffer (catalog no. 1072570) to obtain a 200 μM solution. RNP was generated using a 3:1 sgRNA:Cas9 ratio. Under knockout conditions, 1.11, 3.33, 10, and 30 pmol of RNP were titrated. Under knock-in conditions, 30 pmol of RNP was used with the addition of 2 μg of reference target 1-mNeon nanoplasmid. The components were added in the following order: 1-RNP (sgRNA and Cas9 pre-incubated in RT for 15 minutes), 2-template (3 μg), and 3-cells (2 million). Cells were electroporated in P3 buffer in a Lonza 4D electroporator 46 hours after stimulation. After electroporation, the cells were left at 37°C for 15 minutes. 75 μl of plain Prime-XV was added, and the cells were transferred to 1 ml of TransAct-free complete medium in a 48-well plate. On day 5, 3 ml of complete medium was added, followed by transfer of the cells to a 12-well plate. 0.5 mL of culture was taken out on day 5 for FAC reading (KO samples only). 0.5 mL of culture was taken out and FAC reading was performed on day 6 (KI samples only). Subsequently, cells were stained on days 5 and 6 after stimulation to detect a decrease or increase in the expression of reference target 1.

[0240] The editing efficiencies of the modified gRNAs shown in Figures 6-12 are summarized in Tables 4 and 5. As shown in Figures 9 and 11 and Table 4, gRNA "B" functioned very similarly to the reference gRNA under both knockout and knock-in conditions. gRNA "D" performed better than gRNA "C", and gRNA "C" performed better than gRNA "A". gRNAs "E" and "F" showed very low activity compared to the reference gRNA under both knockout and knock-in conditions.

[0241] As shown in FIGS. 6 to 8, FIGS. 10 and 12, and Table 5, the gRNAs "G", "J", and "L" showed a moderate level of knockout activity compared to the reference gRNAs, but showed the same knock-in activity as the two reference gRNAs. The gRNAs "H" and "I" did not result in knockout or knock-in activity. Furthermore, the gRNA "K" produced a very small amount of knockout or knock-in activity compared to the reference gRNA.

[0242] The cell viability was tested under both knockout and knock-in conditions in the presence of the modified gRNAs. As shown in FIGS. 13 to 16, the cell viability was very similar for all the modified gRNAs tested under knockout (FIGS. 13 to 14) and knock-in (FIGS. 15 to 16) conditions. Similarly, the total number of cells was very similar for most of the gRNAs tested under knockout (FIGS. 13 to 14) and knock-in (FIGS. 15 to 16) conditions. However, in the case of the knock-in experiment, a larger number of cells was measured for the gRNAs "E" and "F", which may be due to the fact that these gRNAs were unable to efficiently cleave the target nucleic acid (FIG. 15). [Table 4] [Table 5]

[0243] As summarized in Tables 4 and 5, the gRNAs "B", "C", and "D" showed the highest editing effect compared to the other gRNAs tested and were more equivalent compared to the control.

[0244] Example 2: Forced degradation of modified gRNA molecules This example shows the degradation of the exemplary gRNA molecules described in Example 1 under forced conditions. The gRNA molecules of Example 1 were subjected to forced degradation conditions including acidic stress (pH 5), basic stress (pH 11), oxidative stress, and heat stress conditions.

[0245] Forced degradation conditions: Acidic stress (pH 5): Acidic buffer: 20 mM sodium acetate, pH 5.0. Temperature: 40 °C. Time points: 0, 1, 2, 3 days (gRNAs "A" to "L"); 0, 1, 2, 3, 5, and 7 days (gRNAs "M" to "V"). Basic stress (pH 11): Basic buffer: 20 mM sodium carbonate, pH 11.0. Temperature: 40 °C. Time points: 0, 8, 16, and 24 hours (gRNAs "A" to "L"); 0, 8, 16, 24, and 48 hours (gRNAs "M" to "V"). Oxidative stress: 0.3% H2O 2。 Temperature: 40 °C. Time points: 0, 1, 2, 3, and 5 days (gRNAs "A" to "V"). Heat stress: Tris-EDTA (TE) buffer, pH 8.0. Temperature: 40 °C. Time points: 0, 1, 3, 5, and 7 days (gRNAs "A" to "V").

[0246] Sample preparation method: Analysis of acid stress (pH 5): To prepare the acid stress (pH 5) solution, 164 mg of sodium acetate was added to a 100 mL volumetric flask. 80 mL of LC-grade water was added to the flask, and the pH was adjusted to 5.0 using acetic acid. The volume of the solution was increased with water. 10 μL of sgRNA was pipetted into an HPLC insert, and after adding 55 μL of the acid stress (pH 5) solution, it was gently vortexed for 5 seconds. Analysis of basic stress (pH 11): To prepare the basic stress (pH 11) solution, 212 mg of sodium carbonate was weighed and placed in a 100 mL volumetric flask. Approximately 80 mL of LC-grade water was added to the flask to dissolve the sodium carbonate, and the pH was adjusted to 11.0 using 1N HCl. The volume of the solution was increased with water. 10 μL of sgRNA was pipetted into an HPLC insert, and after adding 55 μL of the basic stress (pH 11) solution, it was gently vortexed for 5 seconds. Analysis of heat (liquid): 10 μL of sgRNA was pipetted into an HPLC insert, and after adding 55 μL of TE buffer (pH 8), it was gently vortexed for 5 seconds. Oxidation analysis: To prepare the oxidation solution, 0.59 mL of 3% H2O2 was added to a 5 mL volumetric flask, the volume was increased with TE buffer, and then mixed. 10 μL of sgRNA was pipetteed into an HPLC insert, 55 μL of 0.35% H2O2 was added, and the mixture was gently vortexed for 5 seconds. HPLC: HPLC was performed as shown in Table 6. [Table 6]

[0247] Figure 17 shows the elution profiles of modified gRNAs under normal conditions. As shown in Figure 17, gRNAs using modification strategies A-L had initial purities ranging from 47% to 92% at time point 0 (T0). The results of forced degradation studies of these modified gRNAs are shown in Tables 7-9 and Figures 18-40. [Table 7]

[0248] Figure 18 shows chromatograms of reference gRNA3 under basic stress (pH 11), acidic stress (pH 5), and oxidative stress (0.3% H2O2). Figure 19 shows overlapping chromatograms of reference gRNA3 under basic stress (pH 11), acidic stress (pH 5), and oxidative stress (0.3% H2O2) over specific periods. As shown in Figures 18-19, reference gRNA3 was more resistant to acid hydrolysis (e.g., it withstood degradation due to exposure to acid) compared to basic stress (pH 11), which leads to shorter formation from rapid degradation.

[0249] Figure 20 shows the chromatograms of gRNAs A, B, and C under acidic stress (pH 5). Figure 21 shows the chromatograms of modified gRNAs D, E, and F under acidic stress (pH 5). Figure 22 shows the chromatograms of modified gRNAs G, H, and I under acidic stress (pH 5). Figure 23 shows the chromatograms of gRNAs J, K, and L under acidic stress (pH 5). As shown in Figures 20-23, gRNAs E, F, H, I, and J were more resistant to acidic stress.

[0250] Figure 24 shows chromatograms of gRNAs A, B, and C under basic stress (pH 11). Figure 25 shows chromatograms of modified gRNAs D, E, and F under basic stress (pH 11). Figure 26 shows chromatograms of modified gRNAs G, H, and I under basic stress (pH 11). Figure 27 shows chromatograms of modified gRNAs J, K, and L under basic stress (pH 11). As shown in Figures 24-27, modified gRNAs D, E, F, H, I, J, and K were resistant to basic stress (pH 11).

[0251] Figure 28 shows chromatograms of gRNAs A, B, and C under heat stress. Figure 29 shows chromatograms of modified gRNAs D, F, and G under heat stress. Figure 30 shows chromatograms of modified gRNAs H, I, and J under heat stress. Figure 31 shows chromatograms of modified gRNAs K and L under heat stress. As shown in Figures 28 to 31, most gRNAs were resistant to thermal degradation.

[0252] Figure 32 shows the chromatograms of modified gRNAs A, B, and C under oxidative stress (0.3% H2O2). Figure 33 shows the chromatograms of modified gRNAs D, E, and F under oxidative stress (0.3% H2O2). Figure 34 shows the chromatograms of modified gRNAs G, H, and I under oxidative stress (0.3% H2O2). Figure 35 shows the chromatograms of modified gRNAs J, K, and L under oxidative stress (0.3% H2O2). Figure 36 shows the overlapping chromatograms of modified gRNAs A-L under oxidative stress (0.3% H2O2) over 5 days. As shown in Figures 32-36, gRNAs E, G, I, J, K, and L were more resistant to oxidative stress, with the exception of gRNAs C and H.

[0253] Figure 37 shows the change in purity over 3 days under acidic stress (pH 5). Figure 38 shows the change in purity over 24 hours under basic stress (pH 11). Figure 39 shows the change in purity over 7 days under heat stress. As shown in Figure 39, there was not enough gRNA E available for the heat stress test. Figure 40 shows the change in purity over 5 days under oxidative stress (0.3% H2O2).

[0254] As shown in Table 7, many of the modified gRNAs withstood degradation under forced degradation conditions. For example, gRNAs "D", "E", "F", and "J" withstood degradation conditions, including basic stress (pH 11), compared to the reference gRNA (Table 7).

[0255] Tables 8 and 9 summarize the changes in the purity of modified gRNA under acid hydrolysis (Table 8) and heat stress (Table 9). The slope indicates the decrease in purity per day, the intercept indicates the number of days until purity drops to 0, and R is the correlation coefficient of the linear curve. [Table 8] [Table 9]

[0256] Example 3: Modification of gRNA molecules This example provides additional gRNA molecules that have been chemically synthesized to include multiple modifications at the 5' and 3' ends of the gRNA molecule. As shown in Table 10, this example provides modification strategies B and M-V. Modification strategy B shown in Table 10 is the same strategy as modification strategy B shown in Example 1 and Table 1. Table 10 provides an overview of the modifications for each of these strategies, and Table 11 provides a comparison of the modifications for each of these strategies with reference gRNA3.

[0257] Nucleotide modifications are shown in Table 10 below: *: phosphorothioate (PS) linkage; **: phosphorodithioate (PS2) linkage; m: 2'-OMe; ln: locked nucleic acid (LNA); f: 2'-fluoro; M: 2'-O-MOE; and d: deoxyribonucleotide. In the sequences in Table 10, "N" represents any nucleotide base, e.g., guanine (G), adenine (A), thymine (T), or cytosine (C). [Table 10] TIFF2026512014000013.tif255170 [Table 11] TIFF2026512014000015.tif255170

[0258] Figure 41 and Table 12 show the initial purity profiles of reference gRNA3 and gRNAs modified according to modification schemes B and M-V. Similar purity profiles were observed for gRNAs modified according to modification schemes B and M-V (Table 12 and Figure 41). The results of forced degradation studies of these modified gRNAs are shown in Table 12 and Figures 41-59. Details of the experimental conditions for the forced degradation studies are described in Example 2. [Table 12]

[0259] Figures 42–45 show chromatograms of gRNA B and M–V under acidic stress (pH 5). As shown in Figure 44, gRNA modified according to scheme "T" appears to be the most resistant to acidic stress (pH 5) conditions. These data indicate that including a phosphorodithioate bond at the 3' end of gRNA improves stability under acidic stress conditions.

[0260] Figures 46–49 show chromatograms of gRNAs B and M–V under basic stress (pH 11). As shown in Figure 47, gRNAs modified according to scheme "P" appear to be the most tolerant to basic stress (pH 11) conditions. These data indicate that including a phosphorodithioate bond at the 5' end of gRNA improves stability under basic stress conditions.

[0261] Figures 50-53 show chromatograms of gRNAs B and M-V under heat stress. Most gRNAs were resistant to thermal degradation.

[0262] Figures 54-55 show chromatograms of gRNA B and M-V under oxidative stress (0.3% H2O2). Most gRNAs were susceptible to oxidative degradation.

[0263] Figure 56 shows the change in purity over 7 days under thermal stress. Figure 57 shows the change in purity over 7 days under acidic stress (pH 5). Figure 58 shows the change in purity over 48 hours under basic stress (pH 11). Figure 59 shows the change in purity over 5 days under oxidative stress (0.3% H2O2).

[0264] Further analysis of the characteristics of gRNAs modified according to modification schemes B and M-V, targeting reference target 1, was performed in three donor cell lines, Donor 1, Donor 2, and Donor 3. Using the Lonza 4D system, the editing efficiency, cell proliferation, and cellular phenotype of these gRNAs were analyzed at a 100 μL scale using approximately 3.0 × 10⁷ cells / tfx. Low editing was observed in gRNAs modified according to scheme "N" at the midpoint and endpoint in Donor 1 (Figures 60 and 61), Donor 2 (Figures 64 and 65), and Donor 3 (Figures 68 and 69). Furthermore, similar rates of knock-in (KI) and knock-out (KO) editing were observed for each modified gRNA in all three donors, except for gRNAs modified using scheme "N". As shown in Figures 61, 65, and 69, improved editing efficiency was observed in gRNAs modified according to modification schemes "S" and "T" compared to the reference gRNA. In particular, the number of wild-type cells (i.e., cells not edited by gRNA) for the reference gRNA was observed to be 22%–39%, compared to 18%–36% for the “S” and “T” modified gRNAs at the end of the study. Furthermore, at the end of the study, the KO editing rate was 51%–78% for reference gRNA3 compared to 64%–82% for “S” and “T” modified gRNAs, and the KI editing rate was 16%–23% for reference gRNA3 compared to 13%–28% for “S” and “T” modified gRNAs. These results indicate that “S” and “T” modified gRNAs showed equivalent or improved editing efficiency compared to reference gRNA3.

[0265] Similar cell proliferation rates were observed in all modified gRNAs in donor 1 (Figure 62), donor 2 (Figure 66), and donor 3 (Figure 70).

[0266] All publications, patents, and patent applications described herein are incorporated herein by reference in whole, as if each individual publication, patent, or patent application were specifically and individually indicated to be incorporated by reference. In the event of any conflict, this application, including any definitions herein, shall prevail.

Claims

1. A guide RNA molecule, (a) A phosphorodithioate bond between the first nucleotide and the second nucleotide at the 5' end of the guide RNA molecule, (b) A phosphorodithioate bond between the 3' terminal ("n") nucleotide and the n-1 nucleotide of the guide RNA molecule, or (c) A phosphorodithioate bond between the first nucleotide and the second nucleotide at the 5' end of the guide RNA molecule, and a phosphorodithioate bond between the n nucleotide and the n-1 nucleotide of the guide RNA molecule. A guide RNA molecule containing this molecule.

2. The guide RNA molecule according to claim 1, further comprising a phosphorodithioate bond between the second nucleotide and the third nucleotide at the 5' end of the guide RNA molecule, and between the third nucleotide and the fourth nucleotide.

3. The guide RNA molecule according to claim 1 or 2, wherein the guide RNA molecule includes a phosphorodithioate bond between the first nucleotide and the second nucleotide at the 5' end, between the second nucleotide and the third nucleotide, and between the third nucleotide and the fourth nucleotide.

4. The guide RNA molecule according to any one of claims 1 to 3, wherein the guide RNA molecule includes a phosphorodithioate bond between the first nucleotide and the second nucleotide at the 5' end, between the second nucleotide and the third nucleotide, between the third nucleotide and the fourth nucleotide, and between the fourth nucleotide and the fifth nucleotide.

5. The guide RNA molecule according to any one of claims 1 to 4, wherein the guide RNA molecule includes a phosphorodithioate bond between the n nucleotide and the n-1 nucleotide, and between the n-1 nucleotide and the n-2 nucleotide, or between the n nucleotide and the n-1 nucleotide, and between the n-2 nucleotide and the n-3 nucleotide.

6. The guide RNA molecule according to any one of claims 1 to 5, wherein the guide RNA molecule includes a phosphorodithioate bond between the n nucleotide and the n-1 nucleotide, between the n-1 nucleotide and the n-2 nucleotide, and between the n-2 nucleotide and the n-3 nucleotide.

7. The guide RNA molecule according to any one of claims 1 to 6, wherein the guide RNA molecule includes a phosphorodithioate bond between the n nucleotide and the n-1 nucleotide, between the n-1 nucleotide and the n-2 nucleotide, between the n-2 nucleotide and the n-3 nucleotide, and between the n-3 nucleotide and the n-4 nucleotide.

8. The first nucleotide at the 5' end of the guide RNA molecule is (a) 2'-fluoromodified nucleotides, (b) 2'-O-methyl modified nucleotide, (c) 2'-O-(2-methoxyethyl) modified nucleotide, (d) Locked nucleic acid (LNA), (e) Deoxyribose nucleotides, and (f) Two or more combinations of (a) to (e) A guide RNA molecule according to any one of claims 1 to 7, comprising a modification selected from the group consisting of the following.

9. The guide RNA molecule according to claim 8, wherein the first nucleotide at the 5' end of the guide RNA molecule comprises a 2'-fluoromodified nucleotide.

10. The guide RNA molecule according to claim 8, wherein the first nucleotide at the 5' end of the guide RNA molecule comprises a 2'-O-methyl modified nucleotide.

11. The guide RNA molecule according to claim 8, wherein the first nucleotide at the 5' end of the guide RNA molecule comprises a 2'-O-(2-methoxyethyl) modified nucleotide.

12. The guide RNA molecule according to claim 8, wherein the first nucleotide at the 5' end of the guide RNA molecule contains LNA.

13. The guide RNA molecule according to claim 8, wherein the first nucleotide at the 5' end of the guide RNA molecule comprises a deoxyribose nucleotide.

14. The 5' end of the guide RNA molecule contains (i) the second nucleotide, (ii) the third nucleotide, (iii) the fourth nucleotide, (iv) the second and third nucleotides, (v) the second and fourth nucleotides, (vi) the third and fourth nucleotides, or (v) the second, third and fourth nucleotides. (a) 2'-fluoromodified nucleotides, (b) 2'-O-methyl modified nucleotide, (c) 2'-O-(2-methoxyethyl) modified nucleotide, (d) Locked nucleic acid (LNA), (e) Deoxyribose nucleotides, and (f) Two or more combinations of (a) to (e) A guide RNA molecule according to any one of claims 1 to 13, comprising a modification selected from the group consisting of the following.

15. The guide RNA molecule according to any one of claims 1 to 9 and 14, wherein the guide RNA molecule comprises three consecutive 2'-fluoromodified nucleotides in the first three nucleotides of the 5' end of the guide RNA molecule.

16. The guide RNA molecule according to any one of claims 1 to 8, 10, and 14, wherein the guide RNA molecule comprises three consecutive 2'-O-methyl-modified nucleotides in the first three nucleotides of the 5' end of the guide RNA molecule.

17. The guide RNA molecule according to any one of claims 1 to 8, 10, and 14, wherein the guide RNA molecule comprises four consecutive 2'-O-methyl-modified nucleotides in the first four nucleotides of the 5' end of the guide RNA molecule.

18. The guide RNA molecule according to any one of claims 1 to 8, 11, and 14, wherein the guide RNA molecule comprises three consecutive 2'-O-(2-methoxyethyl) modified nucleotides in the first three nucleotides of the 5' end of the guide RNA molecule.

19. The guide RNA molecule according to any one of claims 1 to 8, 12, and 14, wherein the guide RNA molecule comprises three consecutive LNAs in the first three nucleotides of the 5' end of the guide RNA molecule.

20. The guide RNA molecule according to any one of claims 1 to 8, 13, and 14, wherein the guide RNA molecule comprises three consecutive deoxyribose nucleotides in the first three nucleotides of the 5' end of the guide RNA molecule.

21. The nucleotide at the 3' end ("n") of the guide RNA molecule is (a) 2'-fluoromodified nucleotides, (b) 2'-O-methyl modified nucleotide, (c) 2'-O-(2-methoxyethyl) modified nucleotide, (d) Locked nucleic acid (LNA), (e) Deoxyribose nucleotides, and (f) Two or more combinations of (a) to (e) A guide RNA molecule according to any one of claims 1 to 20, comprising a modification selected from the group consisting of the following.

22. The guide RNA molecule according to claim 21, wherein the n nucleotide of the guide RNA molecule includes a 2'-fluoromodified nucleotide.

23. The guide RNA molecule according to claim 21, wherein the n nucleotide of the guide RNA molecule comprises a 2'-O-methyl modified nucleotide.

24. The guide RNA molecule according to claim 21, wherein the n nucleotide of the guide RNA molecule comprises a 2'-O-(2-methoxyethyl) modified nucleotide.

25. The guide RNA molecule according to claim 21, wherein the n nucleotide of the guide RNA molecule comprises LNA.

26. The guide RNA molecule according to claim 21, wherein the n nucleotide of the guide RNA molecule comprises a deoxyribose nucleotide.

27. The (i) n-1 nucleotide, (ii) n-2 nucleotide, (iii) n-3 nucleotide, (iv) n-4 nucleotide, (v) n and n-1 nucleotides, (vi) n and n-2 nucleotides, (vii) n-1 and n-2 nucleotides, (viiii) n, n-1 and n-2 nucleotides, (ix) n-1, n-2 and n-3 nucleotides, (x) n, n-1, n-2 and n-3 nucleotides, or (xi) n, n-1, n-2, n-3 and n-4 nucleotides of the guide RNA molecule, each, (a) 2'-fluoromodified nucleotides, (b) 2'-O-methyl modified nucleotide, (c) 2'-O-(2-methoxyethyl) modified nucleotide, (d) Locked nucleic acid (LNA), (e) Deoxyribose nucleotides, and (f) Two or more combinations of (a) to (e) A guide RNA molecule according to any one of claims 1 to 26, comprising a modification selected from the group consisting of the following.

28. The guide RNA molecule according to any one of claims 1 to 22 and 27, wherein the n and n-1 nucleotides, the n and n-2 nucleotides, the n-1 and n-2 nucleotides, the n, n-1 and n-2 nucleotides, the n-1, n-2, n-3 and n-4 nucleotides, or the n, n-1, n-2, n-3 and n-4 nucleotides are each 2'-fluoromodified nucleotides.

29. The guide RNA molecule according to any one of claims 1 to 21, 23, and 27, wherein the n and n-1 nucleotides, the n and n-2 nucleotides, the n-1 and n-2 nucleotides, the n, n-1 and n-2 nucleotides, the n-1, n-2, n-3, and n-4 nucleotides, or the n, n-1, n-2, n-3, and n-4 nucleotides are each 2'-O-methyl modified nucleotides.

30. The guide RNA molecule according to any one of claims 1 to 21, 23, 27, and 29, wherein the n, n-1, n-2, n-3, and n-4 nucleotides of the guide RNA molecule are each 2'-O-methyl modified nucleotides.

31. The guide RNA molecule according to any one of claims 1 to 21, 24, and 27, wherein the n and n-1 nucleotides, the n and n-2 nucleotides, the n-1 and n-2 nucleotides, the n, n-1 and n-2 nucleotides, the n-1, n-2, n-3, and n-4 nucleotides, or the n, n-1, n-2, n-3, and n-4 nucleotides are each 2'-O-(2-methoxyethyl) modified nucleotides.

32. The guide RNA molecule according to any one of claims 1 to 21, 25, and 27, wherein the n and n-1 nucleotides, the n and n-2 nucleotides, the n-1 and n-2 nucleotides, the n, n-1 and n-2 nucleotides, the n-1, n-2, n-3, and n-4 nucleotides, or the n, n-1, n-2, n-3, and n-4 nucleotides are each LNA.

33. The guide RNA molecule according to any one of claims 1 to 21, 26, and 27, wherein the n and n-1 nucleotides, the n and n-2 nucleotides, the n-1 and n-2 nucleotides, the n, n-1 and n-2 nucleotides, the n-1, n-2, n-3, and n-4 nucleotides, or the n, n-1, n-2, n-3, and n-4 nucleotides are each deoxyribose nucleotides.

34. A nucleic acid comprising a polynucleotide encoding a guide RNA molecule according to any one of claims 1 to 33.

35. The nucleic acid according to claim 34, further comprising a polynucleotide encoding an RNA-inducible nuclease.

36. The nucleic acid according to claim 35, wherein the RNA-induced nuclease is a Cas protein.

37. The nucleic acid according to claim 36, wherein the Cas protein is selected from the group consisting of Cas9, Cas12, Cas13 and combinations thereof.

38. A vector comprising the nucleic acid described in any one of claims 34 to 37.

39. A composition comprising the guide RNA molecule described in any one of claims 1 to 33.

40. The composition according to claim 39, further comprising an RNA-induced nuclease.

41. The composition according to claim 39, further comprising a nucleic acid encoding the RNA-induced nuclease.

42. The composition according to claim 40 or 41, wherein the RNA-induced nuclease is a Cas protein.

43. A composition comprising the nucleic acid according to any one of claims 34 to 37.

44. A composition comprising the vector described in claim 38.

45. A ribonucleoprotein (RNP) complex comprising a guide RNA molecule and an RNA-inducible nuclease according to any one of claims 1 to 33.

46. The RNP complex according to claim 45, wherein the RNA-induced nuclease is a Cas protein.

47. The RNP complex according to claim 46, wherein the Cas protein is selected from the group consisting of Cas9, Cas12, Cas13 and combinations thereof.

48. Cells comprising the composition according to any one of claims 39 to 44.

49. A cell comprising the RNP complex according to any one of claims 45 to 47.

50. A method for modifying cells, comprising contacting the cells with the composition according to any one of claims 39 to 44 or the RNP complex according to any one of claims 45 to 47.

51. The method according to claim 50, wherein the contact includes introducing the composition into the cells by electroporation.

52. A method of treating an object that requires treatment, (a) Ex vivo modification of the target cells by contacting them with the composition according to any one of claims 39 to 44 or the RNP complex according to any one of claims 45 to 47, (b) Returning the modified cells to the target Methods that include...

53. It is a gene editing system, (a) One or more guide RNA molecules according to any one of claims 1 to 33, (b) One or more nucleic acids according to any one of claims 34 to 37 (c) One or more vectors according to claim 38, (d) One or more compositions according to any one of claims 39 to 44, and / or (e) One or more RNP composites according to any one of claims 45 to 47 Gene editing systems, including those mentioned above.

Images