Guide RNA molecule, nucleic acid, vector, composition, ribonucleoprotein complex, cells, methods for modifying a cell and for treating an individual, and gene editing system.

Description

1 / 111 “Guide RNA Molecule, Nucleic Acid, Vector, Composition, Ribonucleoprotein Complex, Cells, Methods for Modifying a Cell and for Treating an Individual, and Gene Editing System” Cross-reference to related orders

[0001] The application claims priority over U.S. Provisional Application No. 63 / 458017, filed April 7, 2023, the contents of which are incorporated by reference in their entirety herein. Field

[0002] This disclosure refers to guide RNAs that have been modified to improve stability and performance. Background

[0003] Clusters of regularly interspaced short palindromic repeat (CRISPR) / CRISPR-associated protein (Cas) systems are widely used to edit the genomes of various cell types. CRISPR / Cas systems can be categorized into two classes (class I and class II), which can be further subdivided into at least six different types: Type I, Type II, Type III, Type IV, Type V, and Type VI. In a Type II CRISPR / Cas system, a Cas protein (such as Cas9) forms a complex with a guide RNA (gRNA) molecule and binds to a target nucleic acid that has an adjacent protospacer motif (PAM) and a spacer. The gRNA molecule includes a sequence that is complementary to the spacer in the target nucleic acid and acts as a guide for 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 discovered that gRNA molecules can be degraded in cells by cleavage by nucleases, for example, cleavage by endonuclease or exonuclease, which can affect the effectiveness of a system. Petition 870250090770, dated 06 / 10 / 2025, pp. 178 / 369 2 / 111 CRISPR-Cas. Therefore, there is a need in the state of the art for gRNA molecules with improved gene editing stability and efficiency. Brief Description

[0005] The present disclosure provides modified guide RNA molecules. In certain embodiments, the guide RNA molecule includes (i) a phosphorodithioate linkage between the first and second nucleotides at the 5' end of the guide RNA molecule, (ii) a phosphorodithioate linkage between the nucleotide at the 3' end (n) and the n-1 nucleotide of the guide RNA molecule, or (iii) a phosphorodithioate linkage between the first and second nucleotides at the 5' end of the guide RNA molecule and a phosphorodithioate linkage between the n-n1 nucleotides of the guide RNA molecule.

[0006] In certain embodiments, the guide RNA molecule additionally includes phosphorodithioate linkages between the second and third nucleotides and the third and fourth nucleotides at the 5' end of the molecule. Guide RNA. In certain embodiments, the guide RNA molecule includes phosphorodithioate linkages between the first and second nucleotides, the second and third nucleotides, and the third and fourth nucleotides at the 5' end of the molecule. Guide RNA. In certain embodiments, the guide RNA molecule includes phosphorodithioate linkages 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' terminus of the guide RNA molecule.

[0007] In certain embodiments, the guide RNA molecule comprises phosphorodithioate linkages between the n-1 ne nucleotides and the n-1 and n-2 nucleotides or the n-1 ne nucleotides and the n-2 and n-3 nucleotides of the guide RNA molecule. In certain embodiments, the guide RNA molecule comprises phosphorodithioate linkages between the n-1 ne nucleotides, the n-1 and n-2 nucleotides, and the n-2 and n-3 nucleotides of the guide RNA molecule. In certain embodiments, the guide RNA molecule comprises linkages Petition 870250090770, dated 06 / 10 / 2025, pp. 179 / 369 3 / 111 phosphorodithioate between the n-1, n-1 and n-2, n-2 and n-3, and n-3 and n-4 nucleotides of the guide RNA molecule.

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

[0009] In certain embodiments, (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 (vii) the second, third, and fourth nucleotides at the 5' terminus of the guide RNA molecule include a modification selected from the group consisting of (a) a 2'-fluoro-modified nucleotide, (b) a 2'-O-methyl-modified nucleotide, (c) a 2'-O-(2-methoxyethyl)-modified nucleotide, (d) a blocked nucleic acid (LNA), (e) a deoxyribose nucleotide; and a combination of two or more of (a)-(e).

[0010] In certain embodiments, the guide RNA molecule includes three nucleotides modified with consecutive 2'-fluoro in the first three Petition 870250090770, dated 06 / 10 / 2025, pp. 180 / 369 4 / 111 nucleotides at the 5' end of the guide RNA molecule. In certain embodiments, the guide RNA molecule includes three consecutive 2'-O-methyl-modified nucleotides in the first three nucleotides at the 5' end of the guide RNA molecule. In certain embodiments, the guide RNA molecule includes four consecutive 2'-O-methyl-modified nucleotides in the first four nucleotides at the 5' end of the guide RNA molecule. In certain embodiments, the guide RNA molecule includes three consecutive 2'-O-(2-methoxyethyl)-modified nucleotides in the first three nucleotides at the 5' end of the guide RNA molecule. In certain embodiments, the guide RNA molecule includes three consecutive LNAs in the first three nucleotides at the 5' end of the guide RNA molecule. In certain embodiments, the guide RNA molecule includes three consecutive deoxyribose nucleotides in the first three nucleotides at the 5' end of the guide RNA molecule.

[0011] In certain embodiments, the 3'-terminal (n) nucleotide of the guide RNA molecule includes a modification selected from the group consisting of (a) a 2'-fluoro-modified nucleotide, (b) a 2'-O-methyl-modified nucleotide, (c) a 2'-O-(2-methoxyethyl)-modified nucleotide, (d) a blocked nucleic acid (LNA), (e) a deoxyribose nucleotide and a combination of two or more of (a)-(e). In certain embodiments, the n nucleotide of the guide RNA molecule comprises a 2'-fluoro-modified nucleotide. In certain embodiments, the n nucleotide of the guide RNA molecule comprises a 2'-O-methyl-modified nucleotide. In certain embodiments, the n nucleotide of the guide RNA molecule comprises a 2'-O-(2-methoxyethyl)-modified nucleotide. In certain embodiments, the n nucleotide of the guide RNA molecule comprises an LNA. In certain embodiments, the n nucleotide of the guide RNA molecule comprises a deoxyribose nucleotide.

[0012] In certain embodiments, (i) the n-1 nucleotide, (ii) the Petition 870250090770, dated 06 / 10 / 2025, pp. 181 / 369 5 / 111 nucleotide n-2, (iii) nucleotide n-3, (iv) nucleotide n-4, (v) nucleotides n and n-1, (vi) nucleotides n and n-2, (vii) nucleotides n-1 and n-2, (viii) nucleotides n, n-1 and n-2, (ix) nucleotides n-1, n-2 and n-3, (x) nucleotides n, n-1, n-2 and n-3, or (xi) nucleotides n, n-1, n-2, n-3 and n-4 at the 3' terminus of the guide RNA molecule, each comprising a modification selected from the group consisting of (a) a nucleotide modified with 2'-fluoro, (b) a nucleotide modified with 2'-O-methyl, (c) a nucleotide modified with 2'-O-(2-methoxyethyl), (d) a blocked nucleic acid (LNA), (e) a deoxyribose nucleotide and a combination of two or more of (a)-(e). In certain embodiments, the n-1 nucleotides, the n-2 nucleotides, the n1 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 nucleotides modified with 2'-fluoro.In certain embodiments, the ne n-1 nucleotides, the ne n-2 nucleotides, the n-1 and n-2 nucleotides, the n, n1 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 nucleotides modified with 2'O-methyl. In certain embodiments, the ne n-1 nucleotides, the ne 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 nucleotides modified with 2'-O-(2-methoxyethyl). In certain embodiments, the nucleotides ne n-1, ne n-2, n1 and n-2, n, n-1 and n-2, n-1, n-2, n-3 and n-4, or n, n-1, n-2, n-3 and n-4 of the guide RNA molecule are each LNAs.In certain embodiments, the nucleotides ne n-1, nucleotides ne n-2, nucleotides n-1 and n-2, nucleotides n, n-1 and n-2, nucleotides n-1, n-2, n3 and n-4, or nucleotides n, n-1, n-2, n-3 and n-4 of the guide RNA molecule are each deoxyribose nucleotides.

[0013] Additionally, this disclosure provides acids Petition 870250090770, dated 06 / 10 / 2025, p. 182 / 369 6 / 111 nucleic acids that include a polynucleotide encoding a guide RNA molecule disclosed in this document. In certain embodiments, the nucleic acid additionally includes a polynucleotide encoding an RNA-guided nuclease. In certain embodiments, the RNA-guided nuclease is a Cas protein. In certain embodiments, the Cas protein is selected from the group consisting of a Cas9, a Cas12, a Cas13, and a combination thereof. This disclosure provides vectors that include a nucleic acid disclosed in this document.

[0014] This disclosure provides compositions that include a guide RNA molecule disclosed in this document. In certain embodiments, a composition of this disclosure includes a vector disclosed in this document. In certain embodiments, the composition additionally includes an RNA-guided nuclease. Alternatively or additionally, the composition additionally includes a nucleic acid encoding the RNA-guided nuclease. In certain embodiments, the RNA-guided nuclease is a Cas protein, for example, a Cas9, a Cas12, and / or a Cas13. In certain embodiments, the composition includes a nucleic acid disclosed in this document, for example, a nucleic acid that includes a polynucleotide encoding a guide RNA molecule.

[0015] The present disclosure further includes a ribonucleoprotein (RNP) complex comprising a modified guide RNA molecule of the present disclosure and an RNA-guided nuclease. In certain embodiments, the RNA-guided nuclease is a Cas protein. In certain embodiments, the Cas protein is selected from the group consisting of a Cas9, a Cas12, a Cas13, and a combination thereof.

[0016] This disclosure additionally includes a cell comprising an RNP composition or complex disclosed in this document. Petition 870250090770, dated 06 / 10 / 2025, pp. 183 / 369 7 / 111

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

[0018] This disclosure provides methods for treating an individual in need thereof. In certain embodiments, the method includes modifying an individual's cell ex vivo by contacting the cell with a composition or RNP complex disclosed herein and returning the modified cell to the individual.

[0019] This disclosure further provides gene editing systems comprising one or more modified guide RNAs from this disclosure, one or more nucleic acids, one or more vectors, one or more compositions and / or one or more RNP complexes disclosed herein. Brief Description of the Figures

[0020] FIGURE 1 provides an illustrative structure of a gRNA of the present disclosure.

[0021] FIGURE 2 provides illustrative modified profiles of the present disclosure.

[0022] FIGURE 3 provides illustrative modified profiles of the present disclosure.

[0023] FIGURE 4 provides illustrative modified profiles of the present disclosure.

[0024] FIGURE 5 provides illustrative examples of modified gRNA elution profiles for the present disclosure.

[0025] FIGURE 6 shows the editing efficiency of exemplary modified gRNAs from this disclosure in different Petition 870250090770, dated 06 / 10 / 2025, pp. 184 / 369 8 / 111 concentrations, compared to reference gRNAs.

[0026] FIGURE 7 shows the editing efficiency of exemplary modified gRNAs from this disclosure at different concentrations.

[0027] FIGURE 8 shows the editing efficiency of exemplary modified gRNAs from this disclosure compared to reference gRNAs, using cells from different donors.

[0028] FIGURE 9 shows the editing efficiency of exemplary modified gRNAs from this disclosure at different concentrations, compared to a reference gRNA.

[0029] FIGURE 10 shows the editing efficiency of exemplary modified gRNAs from this disclosure at different concentrations, compared with reference gRNAs.

[0030] FIGURE 11 shows the editing efficiency of exemplary modified gRNAs from this disclosure compared to a reference gRNA.

[0031] FIGURE 12 shows the editing efficiency of exemplary modified gRNAs from this disclosure compared to reference gRNAs.

[0032] FIGURE 13 provides the percentage of cell viability and the total number of cells in the presence of exemplary modified gRNAs from this disclosure under knockout conditions compared to a reference gRNA.

[0033] FIGURE 14 provides the percentage of cell viability and the total number of cells in the presence of exemplary modified gRNAs from this disclosure under knockout conditions, compared with reference gRNAs.

[0034] FIGURE 15 provides the percentage of cell viability. Petition 870250090770, dated 06 / 10 / 2025, pp. 185 / 369 9 / 111 and the total number of cells in the presence of exemplary modified gRNAs from this disclosure under knock-in conditions, compared to a reference gRNA.

[0035] FIGURE 16 provides the percentage of cell viability and the total number of cells in the presence of exemplary modified gRNAs from this disclosure, under knock-out conditions, compared with reference gRNAs.

[0036] FIGURE 17 provides elution profiles of modified gRNAs exemplified in this disclosure.

[0037] FIGURE 18 provides the elution profiles of reference gRNA 3 under basic stress (pH 11), acidic stress (pH 5) and oxidative stress (0.3% H2O2) conditions.

[0038] FIGURE 19 provides elution profiles of reference gRNA 3 under conditions of basic stress (pH 11), acidic stress (pH 5) and oxidative stress (0.3% H2O2) over specified time periods.

[0039] FIGURE 20 provides elution profiles of modified gRNAs exemplifying the present disclosure under acid stress conditions (pH 5).

[0040] FIGURE 21 provides elution profiles of modified gRNAs exemplifying the present disclosure under acid stress conditions (pH 5).

[0041] FIGURE 22 provides elution profiles of modified gRNAs exemplifying the present disclosure under acid stress conditions (pH 5).

[0042] FIGURE 23 provides elution profiles of modified gRNAs exemplifying the present disclosure under acid stress conditions (pH 5).

[0043] FIGURE 24 provides elution profiles of gRNAs Petition 870250090770, dated 06 / 10 / 2025, pp. 186 / 369 10 / 111 modified examples of the present disclosure under basic stress conditions (pH 11).

[0044] FIGURE 25 provides elution profiles of modified gRNAs exemplifying the present disclosure under basic stress conditions (pH 11).

[0045] FIGURE 26 provides elution profiles of modified gRNAs exemplifying the present disclosure under basic stress conditions (pH 11).

[0046] FIGURE 27 provides elution profiles of modified gRNAs exemplifying the present disclosure under basic stress conditions (pH 11).

[0047] FIGURE 28 provides elution profiles of modified gRNAs exemplifying the present disclosure under thermal stress.

[0048] FIGURE 29 provides elution profiles of modified gRNAs exemplifying the present disclosure under thermal stress.

[0049] FIGURE 30 provides elution profiles of modified gRNAs exemplifying the present disclosure under thermal stress.

[0050] FIGURE 31 provides elution profiles of modified gRNAs exemplifying the present disclosure under thermal stress.

[0051] FIGURE 32 provides elution profiles of modified gRNAs exemplifying the present disclosure under oxidative stress (0.3% H2O2).

[0052] FIGURE 33 provides elution profiles of modified gRNAs exemplifying the present disclosure under oxidative stress (0.3% H2O2).

[0053] FIGURE 34 provides elution profiles of modified gRNAs exemplifying the present disclosure under oxidative stress (0.3% H2O2). Petition 870250090770, dated 06 / 10 / 2025, pp. 187 / 369 11 / 111

[0054] FIGURE 35 provides elution profiles of modified gRNAs exemplifying the present disclosure under oxidative stress (0.3% H2O2).

[0055] FIGURE 36 provides elution profiles of modified gRNAs exemplifying the present disclosure under oxidative stress (0.3% H2O2) over a period of 5 days.

[0056] FIGURE 37 provides the changes in purity of exemplary modified gRNAs from this disclosure over a period of 3 days under acid stress conditions (pH 5).

[0057] FIGURE 38 provides the changes in purity of exemplary modified gRNAs from this disclosure over a 24-hour period under basic stress (pH 11).

[0058] FIGURE 39 provides the changes in purity of exemplary modified gRNAs from this disclosure over a period of 7 days under thermal stress.

[0059] FIGURE 40 provides the changes in purity of exemplary modified gRNAs from this disclosure over a period of 5 days under oxidative stress (0.3% H2O2).

[0060] FIGURE 41 provides elution profiles of modified gRNAs exemplified in this disclosure.

[0061] FIGURE 42 provides elution profiles of modified gRNAs exemplifying the present disclosure under acid stress conditions (pH 5).

[0062] FIGURE 43 provides elution profiles of modified gRNAs exemplifying the present disclosure under acid stress conditions (pH 5).

[0063] FIGURE 44 provides elution profiles of modified gRNAs exemplifying the present disclosure under stress conditions. Petition 870250090770, dated 06 / 10 / 2025, pp. 188 / 369 12 / 111 acid (pH 5).

[0064] FIGURE 45 provides elution profiles of modified gRNAs exemplifying the present disclosure under acid stress conditions (pH 5).

[0065] FIGURE 46 provides elution profiles of modified gRNAs exemplifying the present disclosure under basic stress conditions (pH 11).

[0066] FIGURE 47 provides elution profiles of modified gRNAs exemplifying the present disclosure under basic stress conditions (pH 11).

[0067] FIGURE 48 provides elution profiles of modified gRNAs exemplifying the present disclosure under basic stress conditions (pH 11).

[0068] FIGURE 49 provides elution profiles of modified gRNAs exemplifying the present disclosure under basic stress conditions (pH 11).

[0069] FIGURE 50 provides elution profiles of modified gRNAs exemplifying the present disclosure under thermal stress.

[0070] FIGURE 51 provides elution profiles of modified gRNAs exemplifying the present disclosure under thermal stress.

[0071] FIGURE 52 provides elution profiles of modified gRNAs exemplifying the present disclosure under thermal stress.

[0072] FIGURE 53 provides elution profiles of modified gRNAs exemplifying the present disclosure under thermal stress.

[0073] FIGURE 54 provides elution profiles of modified gRNAs exemplifying the present disclosure under oxidative stress (0.3% H2O2).

[0074] FIGURE 55 provides gRNA elution profiles Petition 870250090770, dated 06 / 10 / 2025, pp. 189 / 369 13 / 111 modified examples of the present disclosure under oxidative stress (0.3% H2O2).

[0075] FIGURE 56 provides the changes in purity of exemplary modified gRNAs from this disclosure over a period of 7 days under thermal stress.

[0076] FIGURE 57 provides the changes in purity of exemplary modified gRNAs from this disclosure under acid stress conditions (pH 5).

[0077] FIGURE 58 provides the changes in purity of exemplary modified gRNAs from this disclosure under basic stress conditions (pH 11).

[0078] FIGURE 59 provides the changes in purity of exemplary modified gRNAs from this disclosure under oxidative stress (0.3% H2O2).

[0079] FIGURE 60 shows the editing efficiency of exemplary modified gRNAs from this disclosure at an average time point, compared to a reference gRNA.

[0080] FIGURE 61 shows the editing efficiency of modified gRNAs exemplifying the present disclosure at the final time point, compared to a reference gRNA.

[0081] FIGURE 62 shows the expansion of donor cells 1 on day 5 and from day 5 to day 12 after transfection (Tfx) with the modified gRNAs exemplifying the present disclosure.

[0082] FIGURE 63 shows the phenotype of modified gRNAs exemplifying the present disclosure at the final time point, compared to a reference gRNA.

[0083] FIGURE 64 shows the editing efficiency of modified gRNAs exemplifying the present disclosure at a point in time. Petition 870250090770, dated 06 / 10 / 2025, pp. 190 / 369 14 / 111 average, compared to a reference gRNA.

[0084] FIGURE 65 shows the editing efficiency of exemplary modified gRNAs from this disclosure at the final time point, compared to a reference gRNA.

[0085] FIGURE 66 shows the cell expansion of donor 2 cells on day 5 and from day 5 to day 12 after transfection (Tfx) with the exemplary modified gRNAs of this disclosure.

[0086] FIGURE 67 shows the phenotype of exemplary modified gRNAs from this disclosure at the final time point, compared to a reference gRNA.

[0087] FIGURE 68 shows the editing efficiency of exemplary modified gRNAs from this disclosure at an average time point, compared to a reference gRNA.

[0088] FIGURE 69 shows the editing efficiency of exemplary modified gRNAs from this disclosure at the final time point, compared to a reference gRNA.

[0089] FIGURE 70 shows the expansion of donor cells 3 on day 5 and from day 5 to day 12 after transfection (Tfx) with the modified gRNAs exemplifying the present disclosure. Detailed Description

[0090] This disclosure relates to modified guide RNA molecules, pharmaceutical compositions including such modified guide RNAs, and methods for modifying a cell by administering the modified guide RNA molecules. This disclosure is based, in part, on the discovery that the introduction of phosphorothioate linkages into a gRNA molecule eliminates the chiral centers generated by phosphorothioate linkages, resulting in stable gRNA molecules, gRNA compositions that do not include diastereomers at these modified phosphate positions, and more defined gRNA populations. Petition 870250090770, dated 06 / 10 / 2025, pp. 191 / 369 15 / 111 As shown in Figures 37, 38, 57, and 58, gRNA molecules that include phosphorodithioate linkages at the 5' and / or 3' ends result in greater stability under forced degradation conditions, such as under basic stress and acid stress conditions, compared to gRNA molecules that do not include phosphorodithioate linkages. Furthermore, as shown in Figures 61, 65, and 69, gRNA molecules that include phosphorodithioate linkages at the 5' and / or 3' ends exhibit comparable or increased editing efficiency compared to gRNA molecules that do not include phosphorodithioate linkages.

[0091] For clarity, but not as a limitation, the detailed description of the subject matter disclosed here is divided into the following subsections: I. Definitions; II. Modified guide RNAs; III. RNA-Guided Nucleases; IV. Compositions; V. Methods of Use; VI. Gene Editing Systems; and VII. Illustrative Achievements. I. Definitions

[0092] Unless defined otherwise, all technical and scientific terms used in this document have the meaning commonly understood by a person skilled in the art to which the subject matter of this disclosure pertains. The following references provide a person skilled in the art with a general definition of many of the terms used in this disclosure: 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). Petition 870250090770, dated 06 / 10 / 2025, pp. 192 / 369 16 / 111 used in this document, the following terms have the meanings assigned to them below, unless otherwise specified.

[0093] As used in this document, the use of the word "a" or "an" when used in conjunction with the term "comprising" in the claims and / or descriptive report may mean "one," but is also consistent with the meaning of "one or more," "at least one," and "one or more of one."

[0094] The term about or approximately means within an acceptable range of error for the particular value, as determined by a person skilled in the art, which depends in part on how the value is measured or determined, i.e., the limitations of the measuring system. For example, about may mean within 3 or more standard deviations, as is practice in the art. Alternatively, about may mean a range up to 20%, preferably up to 10%, more preferably up to 5%, and most preferably up to 1% of a given value. Alternatively, particularly in relation to biological systems or processes, the term may mean within an order of magnitude, preferably within 5 times, and more preferably within 2 times, of a value.

[0095] The terms comprise, include, having, has, may, contains and their variants, as used in this document, are intended to be open transitional phrases, terms or words that do not exclude additional acts or structures. This disclosure also includes other realizations comprising, consisting of and consisting essentially of, the realizations or elements presented in this document, whether explicitly stated or not.

[0096] The term “coupled” can refer to the connection or union of two or more components by an interaction, link, bond, force, or connection, in order to hold two or more components together. In certain embodiments, the term Petition 870250090770, dated 06 / 10 / 2025, pp. 193 / 369 17 / 111 coupled encompasses both direct and indirect bonding, where, for example, a first component is directly bonded to a second component, or one or more intermediate molecules are arranged between the first component and the second component. Exemplary bonds include covalent bonds, ionic bonds, van der Waals interactions, and other bonds identifiable by a person skilled in the art.

[0097] The terms detect or detection, as used in this document, indicate the determination of the existence and / or presence of a target, for example, a target nucleic acid, in a limited portion of space, including, without limitation, a sample. The terms detect or detection, as used in this document, may encompass the determination of chemical and / or biological properties of the target, including, without limitation, the ability to interact with, and in particular bind to, other compounds, and the ability to activate another compound and additional properties identifiable by a person skilled in the art upon reading this disclosure. Detection may be quantitative or qualitative. A detection is “quantitative” when it refers to, relates to, or involves the measurement of the quantity or sum of the target or signal (also called quantification), which includes, among other things, any analysis intended to determine the quantities or proportions of the target or signal.A detection is considered "qualitative" when it refers to, relates to, or involves the identification of a quality or type of target or signal in terms of its relative abundance to another target or signal, which is not quantified.

[0098] As used in this document, the term domain refers to a segment of a protein or nucleic acid, for example, a gRNA molecule. Unless otherwise indicated, a domain need not have any specific functional properties.

[0099] The term editing efficiency, as used in this document, refers to the total number of sequence reads with Petition 870250090770, dated 06 / 10 / 2025, pp. 194 / 369 18 / 111 nucleotide insertions or deletions in a target region of interest over the total number of sequence reads after cleavage by an RNA-guided nuclease.

[00100] The terms guide RNA, gRNA, or gRNA molecule, as used interchangeably in this document, refer to a nucleic acid that promotes the specific targeting or homing of an RNA-guided nuclease to a target nucleic acid.

[00101] As used in this document, the term hybridization refers to the process in which two single-stranded polynucleotides non-covalently link to form a stable double-stranded polynucleotide.

[00102] As used in this document, the term patient or individual refers to a vertebrate or an invertebrate, such as a human or non-human animal, for example, a mammal. Mammals include, without limitation, humans, non-human primates, farm animals, sporting animals, rodents, and pets. Non-limiting examples of non-human animal individuals include rodents such as mice, rats, hamsters, guinea pigs, rabbits, dogs, cats, sheep, pigs, goats, cattle, horses, monkeys, and apes. In certain embodiments, the patient or individual is a human.

[00103] As used in this document, the term in vitro refers to an artificial environment and to processes or reactions that occur within an artificial environment. In vitro environments include, but are not limited to, test tubes and cell cultures.

[00104] As used in this document, the term in vivo refers to the natural environment (e.g., an animal or a cell) and to the processes or reactions that occur within a natural environment, such as embryonic development, cell differentiation, neural tube formation, Petition 870250090770, dated 06 / 10 / 2025, pp. 195 / 369 19 / 111 etc.

[00105] As used in this document, a marker refers to an agent that allows direct or indirect detection. Markers include, without limitation, fluorescent markers, chromogenic markers, electron-dense markers, chemiluminescent markers, and radioactive markers. Non-limiting examples of markers 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) incorporated by reference herein,32P,14C,125I,3H and131I), fluorophores (such as Rare Earth Chelate or yellow luciferin and its derivatives), Rhodamine and its derivatives, dansyl, umbelliferone, luciferase (such as firefly luciferase and simple bacterial fluorescence enzyme) (U.S. Patent No. 4,737).456), fluorescein, 2,3-dihydrophthalazine diketone, as well as enzymes that produce detectable signals, for example, horseradish peroxidase (HRP), alkaline phosphorus acid enzyme, beta galactosidase, glucoamylase, lysozyme, carbohydrate oxidase (such as glucose oxidase, galactose oxidase and glucose-6-phosphate dehydrogenase (G6PD)) and heterocyclic oxidases (such as uricase and xanthine oxidase).

[00106] The term nucleic acid or polynucleotide includes any compound and / or substance comprising a polymer of nucleotides. Each nucleotide is composed 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. Frequently, the nucleic acid molecule is described by the base sequence, where said bases represent the primary structure (linear structure) of a nucleic acid molecule. The base sequence is commonly represented from 5' to 3'. The term nucleic acid encompasses nucleic acids. Petition 870250090770, dated 06 / 10 / 2025, pp. 196 / 369 20 / 111 deoxyribonucleic acid (DNA), including, for example, complementary DNA (cDNA) and genomic DNA, ribonucleic acid (RNA), for example, messenger RNA (mRNA), synthetic forms of DNA or RNA, and mixed polymers comprising two or more of these molecules. The nucleic acid molecule may be linear or circular. Furthermore, the term nucleic acid includes both sense and antisense strands, as well as single-stranded and double-stranded forms. In addition, the nucleic acid described herein may contain naturally occurring or unnaturally occurring nucleotides. Examples of unnaturally occurring nucleotides include nucleotide bases modified with derivatized sugars or phosphate backbone linkages or chemically modified residues.

[00107] The term “nucleoside” refers to a compound that includes 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).

[00108] An “isolated” nucleic acid refers to a nucleic acid molecule that has been separated from a component of its natural environment. An isolated nucleic acid includes a nucleic acid molecule contained in cells that normally contain the nucleic acid molecule, but the nucleic acid molecule is present extrachromosomally or in a chromosomal location different from its natural chromosomal location.

[00109] The term plurality refers to a number greater than one. In certain embodiments, the term plurality of guide RNAs refers to a number of guide RNAs greater than one. For example, but not as a limitation, a plurality of guide RNAs includes at least two guide RNAs. In certain embodiments, the term plurality of nucleic acids refers to a number of nucleic acids greater than one. For example, but not as a limitation, a plurality of nucleic acids includes at least two nucleic acids.

[00110] The term reference molecule or molecule of Petition 870250090770, dated 06 / 10 / 2025, pp. 197 / 369 21 / 111 control, for example, a reference or control gRNA molecule, as used herein, refers to a molecule to which an individual molecule, for example, an individual gRNA molecule, is compared. For example, but not as a limitation, the individual gRNA molecule includes one or more modifications, for example, nucleotide modifications, compared to a reference molecule.

[00111] The term binds specifically, as used in this document, refers to preferential binding to a target molecule, for example, a protein or nucleic acid, over other molecules, for example, proteins or nucleic acids, in a sample.

[00112] As used in this document, treatment (and its grammatical variations, such as treat or treating) refers to clinical intervention in an attempt to alter the natural course of a disease in the individual being treated, and may be performed both for prophylaxis and during the course of the clinical pathology. The desirable effects of treatment include, without limitation, prevention of the occurrence or recurrence of the disease, relief of symptoms, reduction of any direct or indirect pathological consequences of the disease, prevention of metastasis, reduction of the rate of disease progression, improvement or palliation of the disease state, and remission or improved prognosis. The reduction may be at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 99% in the severity of complications, signs or symptoms, or in the likelihood of progression to another grade."Treatment" may also refer to inhibiting the proliferation of a cancer or its progression to a higher grade 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 development of a disease or to slow the progression of a disease.

[00113] The term therapeutic effect, as used herein Petition 870250090770, dated 06 / 10 / 2025, pp. 198 / 369 22 / 111 document, refers to a local or systemic effect in an individual caused by a pharmacologically active substance.

[00114] The terms therapeutically effective amount and effective amount, as used herein, refer to the amount of a composition of this disclosure that is effective in producing some desired therapeutic effect in at least one subpopulation of cells in an individual, with a reasonable benefit / risk ratio applicable to any medical treatment.

[00115] The term “variation,” when referring to a variation at a given position of a polypeptide residue, refers to any modifications to those residues. For example, but not as a limitation, the term “variation” may refer to the substitution of an amino acid at a given position by a different amino acid.

[00116] The term vector, as used in this document, refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is attached. The term includes the vector as a self-replicating nucleic acid structure, as well as the vector incorporated into the genome of a host cell into which it has been introduced. Certain vectors have the ability to operatively direct the expression of nucleic acids to which they are attached. These vectors are referred to in this document as expression vectors. II. Modified Guide RNA Molecules

[00117] This disclosure provides gRNAs that include one or more modifications. In certain embodiments, one or more modifications increase the stability of the gRNA compared to a gRNA that does not include one or more modifications, for example, a control or reference gRNA. For example, but not as a limitation, the modifications enhance the stability of the disclosed gRNA molecules by preventing the degradation of the molecules. Petition 870250090770, dated 06 / 10 / 2025, pp. 199 / 369 23 / 111 gRNA by nucleases, for example, such as endonucleases and / or exonucleases. This enhanced stability can improve the therapeutic efficacy of modified gRNA molecules. Furthermore, the modifications can provide better validity stability and result in higher quality gRNA compositions, as diastereomers would not be present in these compositions. The modifications can also improve editing efficiency and reduce potential off-target effects.

[00118] In certain embodiments, the modified gRNAs of the present disclosure exhibit an increase in stability of about 1% or more, for example, 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, 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, compared to a reference gRNA under degradative conditions, for example, basic stress conditions and / or acid stress conditions. In certain embodiments, the modified gRNAs of the present disclosure exhibit an increase in stability of about 1% to about 60% compared to a reference gRNA under degradative conditions, for example, basic stress conditions and / or acid stress conditions.In certain embodiments, the modified gRNAs of the present disclosure exhibit an increase in stability of about 1% or more, about 2% or more, about 3% or more, about 4% or more, or about 5% or more under acid stress conditions. In certain embodiments, the modified gRNAs of the present disclosure exhibit an increase in stability of about 1% to about 5% compared to a reference gRNA under acid stress conditions. In certain embodiments, the modified gRNAs of the present disclosure exhibit an increase in stability of about 5% or more. Petition 870250090770, dated 06 / 10 / 2025, pp. 200 / 369 24 / 111 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 the present disclosure exhibit an increase in stability of about 1% to about 60% compared to a reference gRNA under basic stress conditions.In certain embodiments, the modified gRNAs of the present disclosure exhibit a reduction in degradation of about 1% or more, for example, 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, 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, compared to a reference gRNA under degradative conditions, for example, basic stress conditions and / or acid stress conditions. In certain embodiments, the modified gRNAs of the present disclosure exhibit a reduction in degradation of about 1% to about 60% compared to a reference gRNA under degradative conditions, for example, basic stress conditions and / or acid stress conditions.In certain embodiments, the modified gRNAs of this disclosure exhibit a reduction in degradation of about 1% or more, about 2% or more, about 3% or more, about 4% or more, or about 5% or more under acid stress conditions. In certain embodiments, the modified gRNAs of this disclosure exhibit a reduction in degradation of about 1% to about 5% compared to a reference gRNA under acid stress conditions. In certain embodiments, the modified gRNAs of this disclosure exhibit a reduction in degradation of about 5% or more, about 10% or more, about 15% or more, or about 20% or more. Petition 870250090770, dated 06 / 10 / 2025, p. 201 / 369 25 / 111 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 the present disclosure exhibit a reduction in degradation of approximately 1% to approximately 60% compared to a reference gRNA under basic stress conditions. In certain embodiments, the increase in stability and / or reduction in degradation is observed approximately 24 hours, 48 ​​hours, 3 days, 4 days, 5 days, 6 days, or 7 days after being subjected to degradation conditions. In certain embodiments, thermal stress conditions include subjecting the gRNA to high temperatures, for example, temperatures above approximately 37°C. In certain embodiments, oxidative stress conditions include subjecting the gRNA to free radicals and / or compounds (e.g., H2O2).In certain embodiments, the basic stress conditions include subjecting the gRNA to a basic pH, for example, a pH greater than about 8, e.g., about 11. In certain embodiments, the acidic stress conditions include subjecting the gRNA to an acidic pH, for example, a pH less than about 6, e.g., about 5. In certain embodiments, the reference gRNA may be a gRNA that has the same nucleotide sequence as the modified gRNA (e.g., without the same modifications as the modified gRNA). In certain embodiments, the reference gRNA may be a gRNA that has a target domain with the same sequence as the modified gRNA (e.g., without the same modifications as the modified gRNA). In certain embodiments, the reference gRNA may be a gRNA that has the same number of nucleotides as the modified gRNA (e.g., without the same modifications as the modified gRNA).

[00119] In certain embodiments, the gRNAs of this disclosure include 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 Petition 870250090770, dated 06 / 10 / 2025, page 202 / 369 26 / 111 plus, twelve or more, thirteen or more, fourteen or more, or fifteen or more modifications. In certain embodiments, the gRNAs of this disclosure include 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 gRNAs of this disclosure include at least about eleven modifications.

[00120] In certain embodiments, a gRNA of the present disclosure has 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 from about 200, from about 80 to about 200, from about 90 to about 200, from about 50 to about 150, from about 80 to about 120, or from about 90 to about 100 nucleotides. In certain embodiments, a gRNA of the present disclosure has a length of about 80 to about 120 nucleotides.In certain embodiments, a gRNA of the present disclosure has about 5, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, about 100, about 105, about 110, about 115, about 120, about 125, about 130, about 135, about 140, about 145, about 150, about 155, about 160, about. Petition 870250090770, dated 06 / 10 / 2025, pp. 203 / 369 27 / 111 of 165, approximately 170, approximately 175, approximately 180, approximately 185, approximately 190, approximately 195, approximately 200 or more nucleotides in length.

[00121] In certain embodiments, about 1% to about 20% of the nucleotides present in the gRNAs of the present disclosure are modified, for example, from about 1% to about 19%, about 1% to about 18%, about 1% to about 17%, about 1% to about 16%, about 1% to about 15%, about 1% to about 14%, about 1% to about 13%, about 1% to about 12%, about 1% to about 11%, about 1% to about 10%, 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 20%, about 3% to about 20%, about 4% to about 20%, about 5% to about 20%, about 6% to about 20%, about 7% to about 20%, about 8% to about 20%, about 9% to about 20%, about 10% to about 20%, about 11% to about 20%, about 12% to about 20%, about 13% to about 20%,Approximately 14% to approximately 20%, approximately 15% to approximately 20%, approximately 16% to approximately 20%, approximately 17% to approximately 20%, approximately 18% to approximately 20%, approximately 19% to approximately 20%, approximately 5% to approximately 15%, or approximately 10% to approximately 15% of the nucleotides present in the gRNAs of this disclosure are modified. In certain embodiments, approximately 1% to approximately 15% of the nucleotides present in the gRNAs of this disclosure are modified. In certain embodiments, approximately 1% to approximately 10% of the nucleotides present in the gRNAs of this disclosure are modified. In certain embodiments, approximately 1% to approximately 6% of the nucleotides present in the gRNAs of this disclosure are modified. In certain realizations, approximately 1%, approximately 2%, approximately 3%, approximately 4%, approximately 5%, approximately 6%, approximately 7%, approximately 8%, approximately 9%, approximately 10%, approximately 11%, approximately 12%, approximately 13%, approximately 14%, approximately 15%, approximately 16%, approximately, Petition 870250090770, dated 06 / 10 / 2025, p. 204 / 369 28 / 111 17%, approximately 18%, approximately 19%, or approximately 20% or more of the nucleotides present in gRNAs are modified.

[00122] In certain embodiments, the gRNA molecules of the present disclosure have the structure shown in FIGURE 1. For example, but not as a limitation, a gRNA molecule of the present disclosure includes a spacer region (blue region in FIGURE 1) at its 5' terminus. A further exemplary structure of a gRNA molecule of the present disclosure is 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 indicated in blue and red in FIGURE 1A in Daniel et al.).

[00123] In certain embodiments, a gRNA molecule of the present disclosure includes one or more modifications in the gRNA molecule's spacer region. In certain embodiments, the spacer region of a currently disclosed gRNA molecule is complementary, for example, at least about 80%, about 85%, about 90%, about 95%, about 98%, about 99%, or about 100% complementary, to a target nucleic acid sequence.In certain embodiments, the spacer region includes the first 30 nucleotides, for example, the first 29 nucleotides, the first 28 nucleotides, the first 27 nucleotides, the first 26 nucleotides, the first 25 nucleotides, the first 24 nucleotides, the first 23 nucleotides, the first 22 nucleotides, the first 21 nucleotides, the first 20 nucleotides, the first 19 nucleotides, the first 18 nucleotides, the first 17 nucleotides, the first 16 nucleotides, or the first 15 nucleotides, present at the 5' terminus of a gRNA molecule. In certain embodiments, the spacer region includes the first 20 nucleotides present at the 5' terminus of a gRNA molecule. In certain implementations, modifications to the spacer region do not interfere with editing efficiency, which... Petition 870250090770, dated 06 / 10 / 2025, pp. 205 / 369 29 / 111 can be evaluated using techniques known in the art or described herein. In certain embodiments, modifications to the spacer region can improve editing efficiency and reduce off-target effects. In certain embodiments, the spacer region includes 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 modifications. In certain embodiments, the spacer region includes 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 modifications within the first 5 nucleotides at its 5' terminus. In certain embodiments, the spacer region includes 1, 2, 3, 4, 5, or 6 modifications within the first 5 nucleotides at its 5' terminus. In certain embodiments, the spacer region includes 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 modifications within the first 3 nucleotides at its 5' terminus. In certain embodiments, the spacer region includes 1, 2, 3, 4, 5, or 6 modifications within the first 3 nucleotides at its 5' terminus.In certain embodiments, the spacer region includes at least one chemically modified nucleotide within the first 5 nucleotides at its 5' terminus. In certain embodiments, the spacer region includes 1, 2, 3, 4, or 5 chemically modified nucleotides within the first 5 nucleotides at its 5' terminus. In certain embodiments, the spacer region includes 1 chemically modified nucleotide within the first 5 nucleotides at its 5' terminus. In certain embodiments, the spacer region includes 2 chemically modified nucleotides within the first 5 nucleotides at its 5' terminus. In certain embodiments, the spacer region includes 3 chemically modified nucleotides within the first 5 nucleotides at its 5' terminus. In certain embodiments, the spacer region includes 4 chemically modified nucleotides within the first 5 nucleotides at its 5' terminus.In certain embodiments, the spacer region includes 5 chemically modified nucleotides within the first 5 nucleotides at its 5' terminus. In certain embodiments, the spacer region includes at least one chemically modified nucleotide within the first 3 nucleotides at its 5' terminus. In certain embodiments, the spacer region... Petition 870250090770, dated 06 / 10 / 2025, pp. 206 / 369 30 / 111 includes 1, 2, or 3 chemically modified nucleotides within the first 3 nucleotides at its 5' end. In certain embodiments, the spacer region includes 1 chemically modified nucleotide within the first 3 nucleotides at its 5' end. In certain embodiments, the spacer region includes 2 chemically modified nucleotides within the first 3 nucleotides at its 5' end. In certain embodiments, the spacer region includes 3 chemically modified nucleotides within the first 3 nucleotides at 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 a 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 modification in the phosphate backbone, as described below. In certain embodiments, a chemically modified nucleotide may include three modifications, for example, two sugar modifications and a modification in the phosphate backbone, as described below.

[00124] In certain embodiments, a gRNA molecule of the present disclosure includes one or more modifications at its 5' terminus. In certain embodiments, the term 5' terminus, as used herein in relation to a modification, refers to the first 5 nucleotides at the 5' terminus of a gRNA molecule. For example, but not as a limitation, one or more nucleotides present at the 5' terminus of a gRNA molecule of the present disclosure are modified. In certain embodiments, the first nucleotide present at the 5' terminus of a gRNA molecule of the present disclosure is modified. In certain embodiments, the second nucleotide present Petition 870250090770, dated 06 / 10 / 2025, pp. 207 / 369 In certain embodiments, the third nucleotide present at the 5' end of a gRNA molecule of the present disclosure is modified. In certain embodiments, the fourth nucleotide present at the 5' end of a gRNA molecule of the present disclosure is modified. In certain embodiments, the fifth nucleotide present at the 5' end of a gRNA molecule of the present disclosure is modified. In certain embodiments, a gRNA molecule of the present disclosure includes 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 modifications within the first 5 nucleotides at its 5' end.

[00125] In certain embodiments, a gRNA molecule of the present disclosure includes modifications in two consecutive nucleotides, for example, two consecutive nucleotides that are within the first 5 nucleotides of the 5' terminus of the gRNA molecule. For example, but not as a limitation, 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' terminus of the gRNA molecule are modified. In certain embodiments, the third and fourth nucleotides at the 5' terminus of the gRNA molecule are modified. In certain embodiments, the fourth and fifth nucleotides at the 5' terminus of the gRNA molecule are modified.

[00126] In certain embodiments, a gRNA molecule of the present disclosure includes modifications in three consecutive nucleotides, for example, three consecutive nucleotides that are within the first 5 nucleotides of the 5' terminus of the gRNA molecule. For example, but not as a limitation, the first, second, and third nucleotides at the 5' terminus of the gRNA molecule are modified. In certain embodiments, the second, third, and fourth nucleotides at the 5' terminus of the gRNA molecule are modified. In certain embodiments, the third, fourth, and fifth nucleotides at the 5' terminus of the molecule Petition 870250090770, dated 06 / 10 / 2025, pp. 208 / 369 32 / 111 of gRNA are modified.

[00127] In certain embodiments, a gRNA molecule of the present disclosure includes modifications in four consecutive nucleotides, for example, four consecutive nucleotides that are within the first 5 nucleotides of the 5' terminus of the gRNA molecule. For example, but not as a limitation, the first, second, third, and fourth nucleotides at the 5' terminus of the gRNA molecule are modified. In certain embodiments, the second, third, fourth, and fifth nucleotides at the 5' terminus of the gRNA molecule are modified.

[00128] In certain embodiments, a gRNA molecule of the present disclosure includes modifications in five consecutive nucleotides, for example, five consecutive nucleotides that are within the first 5 nucleotides of the 5' terminus of the gRNA molecule. For example, but not as a limitation, the first, second, third, fourth, and fifth nucleotides in the 5' terminus of the gRNA molecule are modified.

[00129] In certain embodiments, a gRNA molecule of the present disclosure includes one or more modifications at its 3' terminus. In certain embodiments, the term 3' terminus, as used herein in relation to a modification, refers to the last 5 nucleotides (n, n-1, n-2, n3, and n-4 nucleotides) at the 3' terminus of a gRNA molecule. For example, but not as a limitation, one or more nucleotides present at the 3' terminus of a gRNA molecule of the present disclosure are modified. In certain embodiments, the last nucleotide (n) present at the 3' terminus of a gRNA molecule of the present disclosure is modified. In certain embodiments, the penultimate nucleotide (n-1) present at the 3' terminus of a gRNA molecule of the present disclosure is modified. In certain embodiments, the third nucleotide from the end (n-2) present at the 3' terminus of a gRNA molecule of the present disclosure is modified. In certain achievements, the fourth until the last Petition 870250090770, dated 06 / 10 / 2025, pp. 209 / 369 33 / 111 nucleotide (n-3) present at the 3' terminus of a gRNA molecule of the present disclosure is modified. In certain embodiments, the fifth to last nucleotide (n-4) present at the 3' terminus of a gRNA molecule of the present disclosure is modified. In certain embodiments, a gRNA molecule of the present disclosure includes 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 modifications in the last 5 nucleotides at its 3' terminus.

[00130] In certain embodiments, a gRNA molecule of the present disclosure includes modifications in two consecutive nucleotides, for example, two consecutive nucleotides that are within 5 nucleotides of the 3' terminal of the gRNA molecule. For example, but not as a limitation, the n-1 nucleotides of the 3' terminal of the gRNA molecule are modified. In certain embodiments, the n-2 nucleotides of the 3' terminal of the gRNA molecule are modified. In certain embodiments, the n-1 and n-2 nucleotides of the 3' terminal of the gRNA molecule are modified. In certain embodiments, the n-2 and n-3 nucleotides of the 3' terminal of the gRNA molecule are modified. In certain embodiments, the n-3 and n-4 nucleotides of the 3' terminal 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.

[00131] In certain embodiments, a gRNA molecule of the present disclosure includes modifications in three consecutive nucleotides, for example, three consecutive nucleotides that are within the last 5 nucleotides of the 3' terminal of the gRNA molecule. For example, but not as a limitation, 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 of the 3' terminal of the gRNA molecule are modified. In certain embodiments, the n-2, n-3 and n-4 nucleotides of the 3' terminal of the gRNA molecule are modified.

[00132] In certain embodiments, a gRNA molecule of the present disclosure includes modifications in four consecutive nucleotides, by Petition 870250090770, dated 06 / 10 / 2025, pp. 210 / 369 34 / 111 example, four consecutive nucleotides that are within 5 nucleotides of the 3' terminal of the gRNA molecule. For example, but not as a limitation, 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.

[00133] In certain embodiments, a gRNA molecule of the present disclosure includes modifications in five consecutive nucleotides, for example, five consecutive nucleotides that are within 5 nucleotides of the 3' terminal of the gRNA molecule. For example, but not as a limitation, the n, n-1, n-2, n-3 and n-4 nucleotides of the gRNA molecule are modified.

[00134] In certain embodiments, a gRNA molecule of the present disclosure includes one or more modifications at its 5' terminus and one or more modifications at its 3' terminus. In certain embodiments, a gRNA molecule of the present disclosure includes two or more modifications at its 5' terminus and two or more modifications at its 3' terminus. In certain embodiments, a gRNA molecule of the present disclosure includes three or more modifications at its 5' terminus and three or more modifications at its 3' terminus. In certain embodiments, a gRNA molecule of the present disclosure includes four or more modifications at its 5' terminus and four or more modifications at its 3' terminus. In certain embodiments, a gRNA molecule of the present disclosure includes five or more modifications at its 5' terminus and five or more modifications at its 3' terminus.

[00135] Non-limiting examples of modifications that may be included in a gRNA molecule of the present disclosure are disclosed herein. In certain embodiments, a gRNA of the present disclosure may include one or more of the following modifications: (i) a modification of the phosphate backbone and (ii) a sugar modification. Petition 870250090770, dated 06 / 10 / 2025, pp. 211 / 369 35 / 111 Modifications to the Phosphate Main Chain

[00136] In certain embodiments, the phosphate group of a nucleotide present in a gRNA molecule can be modified. For example, but not as a limitation, the phosphate group of a nucleotide can be modified by replacing one or more oxygens, for example, bridging or non-bridging oxygens, in a phosphodiester bond with a different substituent. Non-limiting examples of substituents include sulfur (S), nitrogen (N), hydrogen (H), and carbon (C). In certain embodiments, one or more oxygens in a phosphodiester bond are replaced by S.

[00137] In certain embodiments, a gRNA molecule can be modified with one or more phosphorothioate (PS) linkages. In certain embodiments, a PS linkage or bond refers to a linkage in which a sulfur is replaced by a non-bridged phosphate oxygen in a phosphodiester internucleotide bond. In certain embodiments, * is used herein to indicate a nucleotide that is linked to the adjacent 3' nucleotide with a PS linkage. In certain embodiments, the phosphorus of an unmodified phosphodiester bond is achiral, and the substitution of a non-bridged phosphate oxygen with sulfur causes the chiral phosphorus to become chiral.

[00138] In certain embodiments, a gRNA molecule can be modified with one or more phosphorodithioate (PS2) linkages. In certain embodiments, a PS2 linkage or bond refers to a linkage in which both non-bridge oxygens in a phosphodiester internucleotide bond are replaced by sulfur. In certain embodiments, ** is used herein to indicate a nucleotide that is linked to the adjacent 3' nucleotide with a PS2 linkage. Similar to a naturally occurring phosphodiester backbone, a PS2 linkage is achiral at the phosphorus, resulting in gRNA molecules that are not diastereomeric at the PS2 linkage. In certain embodiments, the phosphorodithioate linkages are resistant to Petition 870250090770, dated 06 / 10 / 2025, pp. 212 / 369 36 / 111 degradation by nucleases and the presence of one or more phosphorodithioate linkages in a gRNA of the present disclosure may increase the stability of the gRNA, for example, compared to an unmodified gRNA. Sugar Modifications

[00139] In certain embodiments, the sugar group of a nucleotide present in a gRNA of the present disclosure may be modified. For example, but not as a limitation, a nucleotide of a gRNA of the present disclosure may include one or more modifications in its sugar group, for example, ribose.

[00140] In certain embodiments, a sugar group may be modified at the hydroxyl group 2' (OH). In certain embodiments, the hydroxyl group 2' may be replaced by a different substituent. Non-limiting examples of substituents include hydrogen (H), a halogen, an alkyl or an alkoxy (OR, where R may be an alkyl, a cycloalkyl or an alkoxy).

[00141] In certain embodiments, the 2'-hydroxyl group is replaced by an alkoxy group. In certain embodiments, the 2'-hydroxyl group is replaced by a methoxy group. In certain embodiments, m is used in this document to indicate a nucleotide that is modified with a 2'-O-methyl (i.e., a nucleotide modified with 2'-O-methyl).

[00142] In certain embodiments, the hydrogen (H) of the 2' hydroxyl group is replaced by a methoxyethyl group. In certain embodiments, M is used herein to indicate a nucleotide that is modified with a 2'O-(2-methoxyethyl) (i.e., nucleotide modified with 2'-O-(2-methoxyethyl)).

[00143] In certain embodiments, the 2' hydroxyl group may be replaced by 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 by a fluorine. In certain embodiments, f is used in this document to indicate a nucleotide that is modified with a 2'. Petition 870250090770, dated 06 / 10 / 2025, page 213 / 369 37 / 111 fluoro (i.e., a nucleotide modified with 2'-fluoro).

[00144] In certain embodiments, the 2' hydroxyl group can be replaced by a hydrogen (H) to generate a deoxyribose sugar. For example, but not as a limitation, a nucleotide present in a gRNA of the present disclosure may have a deoxyribose sugar. In certain embodiments, d is used in the present document to indicate a nucleotide that has a deoxyribose sugar.

[00145] In certain embodiments, modification of the 2' hydroxyl group may include blocked nucleic acids (LNAs) in which the 2' hydroxyl group is connected to the 4' carbon of the same ribose sugar. In certain embodiments, the 2' hydroxyl group is connected to the 4' carbon by a bridge, for example, an alkylene (e.g., methylene), ether, or amino bridge. In certain embodiments, LNA is used herein to indicate a nucleotide that is an LNA. Modified gRNAs Examples

[00146] In certain embodiments, a gRNA of the present disclosure may have at least one modification in the phosphate backbone, for example, a phosphorodithioate linkage. In certain embodiments, a gRNA may have at least two modifications in the phosphate backbone, for example, phosphorodithioate linkages. In certain embodiments, a gRNA may have at least three modifications in the phosphate backbone, for example, phosphorodithioate linkages. In certain embodiments, a gRNA may have at least four modifications in the phosphate backbone, for example, phosphorodithioate linkages. In certain embodiments, a gRNA may have at least five modifications in the phosphate backbone, for example, phosphorodithioate linkages. In certain embodiments, a gRNA may have at least six modifications in the phosphate backbone, for example, phosphorodithioate linkages. In certain embodiments, a gRNA may have at least seven Petition 870250090770, dated 06 / 10 / 2025, pp. 214 / 36938 / 111 modifications to the phosphate backbone, for example, phosphorodithioate linkages. In certain embodiments, a gRNA may have at least eight modifications to the phosphate backbone, for example, phosphorodithioate linkages. In certain embodiments, a gRNA may have at least nine modifications to the phosphate backbone, for example, phosphorodithioate linkages. In certain embodiments, a gRNA may have at least ten modifications to the phosphate backbone, for example, phosphorodithioate linkages.In certain embodiments, a gRNA may have from about 1 to about 10 modifications in the phosphate backbone, for example, from about 1 to about 9, from about 1 to about 8, from about 1 to about 7, from about 1 to about 6, from about 1 to about 5, from about 1 to about 4, from about 1 to about 3, from about 1 to about 2, from about 2 to about 10, from about 3 to about 10, from about 4 to about 10, from about 5 to about 10, from about 6 to about 10, from about 7 to about 10, from about 8 to about 10, from about 9 to about 10, from about 2 to about 8, from about 2 to about 6, from about 3 to about 8, from about 3 to about 6 or from 3 to 5 modifications in the phosphate backbone, for example, phosphorodithioate linkages. In certain embodiments, a gRNA may have from about 2 to about 10 modifications in the phosphate backbone, for example, phosphorodithioate linkages.In certain embodiments, a gRNA may have from about 2 to about 9 modifications in the phosphate backbone, for example, phosphorodithioate linkages. In certain embodiments, a gRNA may have from about 3 to about 8 modifications in the phosphate backbone, for example, phosphorodithioate linkages. In certain embodiments, a gRNA may have from about 3 to about 5 modifications in the phosphate backbone, for example, phosphorodithioate linkages.

[00147] In certain embodiments, a gRNA of the present disclosure may include at least one phosphorodithioate linkage and at least one linkage Petition 870250090770, dated 06 / 10 / 2025, pp. 215 / 369 39 / 111 phosphorothioate, for example, as shown in Table 10. For example, but not as a limitation, a gRNA of the present disclosure may include at least two phosphorothioate linkages and at least one phosphorothioate linkage. In certain embodiments, a gRNA of the present disclosure may include at least three phosphorothioate linkages and at least one phosphorothioate linkage. In certain embodiments, a gRNA of the present disclosure may include at least four phosphorothioate linkages and at least one phosphorothioate linkage. In certain embodiments, a gRNA of the present disclosure may include at least two phosphorothioate linkages and at least two phosphorothioate linkages. In certain embodiments, a gRNA of the present disclosure may include at least two phosphorothioate linkages and at least three phosphorothioate linkages.

[00148] In certain embodiments, a gRNA may have at least one sugar modification. In certain embodiments, a gRNA may have at least two sugar modifications. In certain embodiments, a gRNA may have at least three sugar modifications. In certain embodiments, a gRNA may have at least four sugar modifications. In certain embodiments, a gRNA may have at least five sugar modifications. In certain embodiments, a gRNA may have at least six sugar modifications. In certain embodiments, a gRNA may have at least seven sugar modifications. In certain embodiments, a gRNA may have at least eight sugar modifications. In certain embodiments, a gRNA may have at least nine sugar modifications. In certain embodiments, a gRNA may have at least ten sugar modifications.In certain embodiments, a gRNA may have from about 1 to about 10 sugar modifications, for example, from about 1 to about 9, from about 1 to about 8, from about 1 to about 7, from about 1 to about 6, from about 1 to about 5, from about 1 to about 4, from about 1 to about 3, from about 1 to about 2, from about 2 to about 10, from about 3 to about 10, from about 4 to about 10, from about 5 to about 10, from about 6 to... Petition 870250090770, dated 06 / 10 / 2025, pp. 216 / 369 40 / 111 approximately 10, approximately 7 to approximately 10, approximately 8 to approximately 10, approximately 9 to approximately 10, approximately 2 to approximately 8, approximately 2 to approximately 6, approximately 3 to approximately 8, or approximately 3 to approximately 6 sugar modifications. In certain embodiments, a gRNA may have from approximately 2 to approximately 10 sugar modifications. In certain embodiments, a gRNA may have from approximately 3 to approximately 8 sugar modifications. In certain embodiments, a gRNA molecule of the present disclosure may have from approximately 3 to approximately 6 sugar modifications.

[00149] In certain embodiments, a 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 sugar 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 sugar modifications).

[00150] In certain embodiments, a gRNA may have at least two phosphate backbone modifications and at least two sugar backbone modifications. In certain embodiments, a gRNA may have at least three phosphate backbone modifications and at least three sugar backbone modifications. In certain embodiments, a gRNA may have at least four phosphate backbone modifications and at least four sugar backbone modifications. In certain embodiments, a gRNA may have at least five phosphate backbone modifications and at least five sugar backbone modifications. In certain embodiments, a gRNA may have at least five phosphate backbone modifications and at least six sugar backbone modifications. In certain embodiments, a gRNA may have at least six phosphate backbone modifications and at least six sugar backbone modifications. In certain embodiments, a Petition 870250090770, dated 06 / 10 / 2025, pp. 217 / 369 41 / 111 gRNA can have at least seven phosphate backbone modifications and at least seven sugar backbone modifications. In certain embodiments, a gRNA can have at least eight phosphate backbone modifications and at least eight sugar backbone modifications. In certain embodiments, a gRNA can have at least eight phosphate backbone modifications and at least nine sugar backbone modifications. In certain embodiments, a gRNA can have at least nine phosphate backbone modifications and at least nine sugar backbone modifications. In certain embodiments, a gRNA can have at least ten phosphate backbone modifications and at least ten sugar backbone modifications.

[00151] In certain embodiments, a gRNA molecule of the present disclosure includes at least one phosphorodithioate linkage at the 5' terminus of the gRNA molecule. In certain embodiments, a gRNA molecule of the present disclosure includes a phosphorodithioate linkage between the first and second nucleotides at the 5' terminus of the gRNA molecule. In certain embodiments, a gRNA molecule of the present disclosure includes a phosphorodithioate linkage between the second and third nucleotides at the 5' terminus of the gRNA molecule. In certain embodiments, a gRNA molecule of the present disclosure includes a phosphorodithioate linkage between the third and fourth nucleotides at the 5' terminus of the gRNA molecule. In certain embodiments, a gRNA molecule of the present disclosure includes a phosphorodithioate linkage between the fourth and fifth nucleotides at the 5' terminus of the gRNA molecule.

[00152] In certain embodiments, a gRNA molecule of the present disclosure includes at least one phosphorodithioate linkage at the 3' terminus of the gRNA molecule. In certain embodiments, a gRNA molecule of the present disclosure includes a phosphorodithioate linkage between the 3'-terminal nucleotide (n) and the n-1 nucleotide of the gRNA molecule. In certain embodiments, a gRNA molecule of the present disclosure includes a phosphorodithioate linkage between the n-1 and n-2 nucleotides at the 3' terminus of the gRNA molecule. In certain embodiments Petition 870250090770, dated 06 / 10 / 2025, pp. 218 / 369 In 42 / 111 embodiments, a gRNA molecule of the present disclosure includes a phosphorodithioate linkage between the n-2 and n-3 nucleotides at the 3' terminus of the gRNA molecule. In certain embodiments, a gRNA molecule of the present disclosure includes a phosphorodithioate linkage between the n-3 and n-4 nucleotides at the 3' terminus of the gRNA molecule.

[00153] In certain embodiments, a gRNA molecule of the present disclosure includes a phosphorodithioate linkage between the first and second nucleotides at the 5' terminus of the gRNA molecule and a phosphorodithioate linkage between the n-1 nucleotides of the gRNA molecule.

[00154] In certain embodiments, a gRNA molecule of the present disclosure includes phosphorodithioate linkages between the first and second nucleotides and the second and third nucleotides at the 5' terminus of the gRNA molecule.

[00155] In certain embodiments, a gRNA molecule of the present disclosure includes phosphorodithioate linkages between the first and second nucleotides and the third and fourth nucleotides at the 5' terminus of the gRNA molecule.

[00156] In certain embodiments, a gRNA molecule of the present disclosure includes phosphorodithioate linkages between the second and third nucleotides and the third and fourth nucleotides at the 5' terminus of the gRNA molecule.

[00157] In certain embodiments, a gRNA molecule of the present disclosure includes phosphorodithioate linkages between the first and second nucleotides, the second and third nucleotides, and the third and fourth nucleotides at the 5' terminus of the gRNA molecule.

[00158] In certain embodiments, a gRNA molecule of the present disclosure includes phosphorodithioate linkages between the first and second nucleotides, the second and third nucleotides, the third and fourth nucleotides. Petition 870250090770, dated 06 / 10 / 2025, pp. 219 / 369 43 / 111 nucleotides and the fourth and fifth nucleotides at the 5' end of the gRNA molecule.

[00159] In certain embodiments, a gRNA molecule of the present disclosure includes a phosphorothioate linkage between the first and second nucleotides.

[00160] In certain embodiments, a gRNA molecule of the present disclosure includes a phosphorothioate linkage between the third and fourth nucleotides.

[00161] In certain embodiments, a gRNA molecule of the present disclosure includes a phosphorothioate linkage between the first and second nucleotides, the second and third nucleotides, and the third and fourth nucleotides.

[00162] In certain embodiments, a gRNA molecule of the present disclosure includes phosphorodithioate linkages between the n-1 nucleotides and the n-1 and n-2 nucleotides.

[00163] In certain embodiments, a gRNA molecule of the present disclosure includes phosphorodithioate linkages between the n-1 ne nucleotides and the n-2 and n-3 nucleotides of the gRNA molecule.

[00164] In certain embodiments, a gRNA molecule of the present disclosure includes phosphorodithioate linkages between the n-1 and n-2 nucleotides and the n-2 and n-3 nucleotides of the gRNA molecule.

[00165] In certain embodiments, a gRNA molecule of the present disclosure includes phosphorodithioate linkages between the n-1, n-1 and n-2, n-2 and n-3, and n-3 and n-4 nucleotides of the gRNA molecule.

[00166] In certain embodiments, a gRNA molecule of the present disclosure includes phosphorodithioate linkages between the n-1 and n-2, n-2 and n-3, n-3 and n-4, and n-4 and n-5 nucleotides of the gRNA molecule.

[00167] In certain embodiments, a gRNA molecule of the present Petition 870250090770, dated 06 / 10 / 2025, pp. 220 / 369 44 / 111 disclosure includes a phosphorothioate linkage between the nucleotides ne n-1.

[00168] In certain embodiments, a gRNA molecule of the present disclosure includes a phosphorothioate linkage between nucleotides n-1 and n-2.

[00169] In certain embodiments, a gRNA molecule of the present disclosure includes a phosphorothioate linkage between nucleotides n-1 and n-2 and a phosphorodithioate linkage between nucleotides n and n-1. In certain embodiments, the gRNA additionally includes phosphorothioate linkages between the first and second nucleotides, the second and third nucleotides, and the third and fourth nucleotides.

[00170] In certain embodiments, a gRNA molecule of the present disclosure includes a phosphorothioate linkage between the third and fourth nucleotides and phosphorodithioate linkages between the first and second nucleotides and between the second and third nucleotides. In certain embodiments, the gRNA additionally includes phosphorodithioate linkages between nucleotides n and n-1 and between n-1 and n-2.

[00171] In certain embodiments, a gRNA molecule of the present disclosure includes phosphorothioate linkages between the first and second nucleotides, the second and third nucleotides, and the third and fourth nucleotides. In certain embodiments, the gRNA additionally includes phosphorothioate linkages between nucleotides n and n-1 and between n-1 and n-2.

[00172] In certain embodiments, a gRNA molecule of the present disclosure includes one or more of a 2'-fluoro-modified nucleotide, a 2'-O-methyl-modified nucleotide, a 2'-O(2-methoxyethyl)-modified nucleotide, a deoxyribose nucleotide, and a 5'-terminus-blocked nucleic acid (LNA), for example, within the first 5 nucleotides at the 5' end. For example, but not as a limitation, 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-modified nucleotide Petition 870250090770, dated 06 / 10 / 2025, pp. 221 / 369 45 / 111 (2-methoxyethyl), a deoxyribose nucleotide, or a blocked nucleic acid (LNA). In certain embodiments, the first nucleotide at the 5' end of the gRNA molecule is a nucleotide modified with 2'-fluoro. In certain embodiments, the first nucleotide at the 5' end of the guide RNA molecule is a nucleotide modified with 2'-O-methyl. In certain embodiments, the first nucleotide at the 5' end of the gRNA molecule is a nucleotide modified with 2'-O-(2-methoxyethyl). In certain embodiments, the first nucleotide at the 5' end of the gRNA molecule is an LNA. In certain embodiments, the first nucleotide at the 5' end of the gRNA molecule has a deoxyribose sugar.

[00173] In certain embodiments, a gRNA of the present disclosure includes two or more nucleotides modified with 2'-fluoro, nucleotides modified with 2'-O-methyl, nucleotides modified with 2'-O-(2-methoxyethyl), deoxyribose nucleotides and / or LNAs at its 5' terminus, for example, within the first 5 nucleotides at the 5' terminus. For example, but not as a limitation, the first and second nucleotides at the 5' terminus of the gRNA molecule are nucleotides modified with 2'-fluoro, nucleotides modified with 2'-O-methyl, nucleotides modified with 2'-O-(2-methoxyethyl), deoxyribose nucleotides, LNAs or a combination thereof. In certain embodiments, the first and second nucleotides at the 5' end of the gRNA molecule are nucleotides modified with 2'-fluoro. In certain embodiments, the first and second nucleotides at the 5' end of the guide RNA molecule are nucleotides modified with 2'-O-methyl.In certain embodiments, the first and second nucleotides at the 5' end of the gRNA molecule are nucleotides modified with 2'-O-(2-methoxyethyl). In certain embodiments, the first and second nucleotides at the 5' end of the gRNA molecule are LNAs. In certain embodiments, the first and second nucleotides at the 5' end of the gRNA molecule have a deoxyribose sugar.

[00174] In certain realizations, a gRNA of the present disclosure Petition 870250090770, dated 06 / 10 / 2025, pp. 222 / 369 46 / 111 includes three or more nucleotides modified with 2'-fluoro, 2'-O-methyl, 2'-O-(2-methoxyethyl), deoxyribose nucleotides and / or LNAs at its 5' terminus, for example, within the first 5 nucleotides at the 5' terminus. For example, but not as a limitation, the first, second and third nucleotides at the 5' terminus of the gRNA molecule are nucleotides modified with 2'-fluoro, 2'-O-methyl, 2'-O-(2-methoxyethyl), deoxyribose nucleotides, LNAs or a combination thereof. In certain embodiments, the first, second, and third nucleotides at the 5' end of the gRNA molecule are nucleotides modified with 2'-fluoro. In certain embodiments, the first, second, and third nucleotides at the 5' end of the gRNA molecule are nucleotides modified with 2'-O-methyl.In certain embodiments, the first, second, and third nucleotides at the 5' end of the gRNA molecule are nucleotides modified with 2'-O-(2-methoxyethyl). In certain embodiments, the first, second, and third nucleotides at the 5' end of the gRNA molecule are LNAs. In certain embodiments, the first, second, and third nucleotides at the 5' end of the gRNA molecule have a deoxyribose sugar.

[00175] In certain embodiments, a gRNA of the present disclosure includes four or more nucleotides modified with 2'-fluoro, nucleotides modified with 2'-O-methyl, nucleotides modified with 2'-O-(2-methoxyethyl), deoxyribose nucleotides and / or LNAs at its 5' terminus, for example, within the first 5 nucleotides at the 5' terminus. For example, but not as a limitation, the first, second, third and fourth nucleotides at the 5' terminus of the gRNA molecule are nucleotides modified with 2'-fluoro, nucleotides modified with 2'-O-methyl, nucleotides modified with 2'-O(2-methoxyethyl), deoxyribose nucleotides, LNAs or a combination thereof. In certain embodiments, the first, second, third and fourth Petition 870250090770, dated 06 / 10 / 2025, pp. 223 / 369 47 / 111 nucleotides at the 5' end of the gRNA molecule are 2'-fluoro-modified nucleotides. In certain embodiments, the first, second, third, and fourth nucleotides at the 5' end of the gRNA molecule are 2'-O-methyl-modified nucleotides. In certain embodiments, the first, second, third, and fourth nucleotides at the 5' 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.

[00176] In certain embodiments, a gRNA of the present disclosure includes five or more nucleotides modified with 2'-fluoro, nucleotides modified with 2'-O-methyl, nucleotides modified with 2'-O-(2-methoxyethyl), deoxyribose nucleotides and / or LNAs at its 5' terminus, for example, within the first 5 nucleotides at the 5' terminus. For example, but not as a limitation, the first, second, third, fourth and fifth nucleotides at the 5' terminus of the gRNA molecule are nucleotides modified with 2'-fluoro, nucleotides modified with 2'-O-methyl, nucleotides modified with 2'-O(2-methoxyethyl), deoxyribose nucleotides, LNAs or a combination thereof. In certain embodiments, the first, second, third, fourth, and fifth nucleotides at the 5' end of the gRNA molecule are nucleotides modified with 2'-fluoro. In certain embodiments, the first, second, third, fourth, and fifth nucleotides at the 5' end of the gRNA molecule are nucleotides modified with 2'-O-methyl.In certain embodiments, the first, second, third, fourth, and fifth nucleotides at the 5' end of the gRNA molecule are modified nucleotides with 2'-O-(2-methoxyethyl). 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... Petition 870250090770, dated 06 / 10 / 2025, pp. 224 / 369 48 / 111 a deoxyribose sugar.

[00177] In certain embodiments, a gRNA of the present disclosure includes one or more of a 2'-fluoro-modified nucleotide, a 2'-O-methyl-modified nucleotide, a 2'-O-(2-methoxyethyl)-modified nucleotide, a deoxyribose nucleotide and / or a locked nucleic acid (LNA) at its 3' terminus, for example, within the last 5 nucleotides at the 3' terminus. For example, but not as a limitation, the 3'-terminal (n) nucleotide 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 a locked nucleic acid (LNA). In certain embodiments, the n nucleotide of the gRNA molecule is a nucleotide modified with 2'-fluoro. In certain embodiments, the n nucleotide of the gRNA molecule is a nucleotide modified with 2'-O-methyl. In certain embodiments, the n nucleotide of the gRNA molecule is a nucleotide modified with 2'-O-(2-methoxyethyl).In certain embodiments, the n nucleotide of the gRNA molecule is an LNA. In certain embodiments, the n nucleotide of the gRNA molecule has a deoxyribose sugar.

[00178] In certain embodiments, a gRNA of the present disclosure includes two or more nucleotides modified with 2'-fluoro, nucleotides modified with 2'-O-methyl, nucleotides modified with 2'-O-(2-methoxyethyl), deoxyribose nucleotides and / or LNAs at its 3' terminus, for example, within the last 5 nucleotides at the 3' terminus. For example, but not as a limitation, the n-1 nucleotides of the gRNA molecule are nucleotides modified with 2'-fluoro, nucleotides modified with 2'-O-methyl, nucleotides modified with 2'-O-(2-methoxyethyl), deoxyribose nucleotides, LNAs or a combination thereof. In certain embodiments, the n-n1 nucleotides of the gRNA molecule are nucleotides modified with 2'-fluoro. In certain embodiments, the n-n1 nucleotides of the guide RNA molecule are nucleotides. Petition 870250090770, dated 06 / 10 / 2025, pp. 225 / 369 49 / 111 modified with 2'-O-methyl. In certain embodiments, the n-1 nucleotides at the terminal of the gRNA molecule are nucleotides modified with 2'-O-(2-methoxyethyl). In certain embodiments, the n-1 nucleotides of the gRNA molecule are LNAs. In certain embodiments, the n-1 nucleotides of the gRNA molecule have a deoxyribose sugar.

[00179] In certain embodiments, a gRNA of the present disclosure includes three or more nucleotides modified with 2'-fluoro, nucleotides modified with 2'-O-methyl, nucleotides modified with 2'-O-(2-methoxyethyl), deoxyribose nucleotides and / or LNAs at its 3' terminus, for example, within the last 5 nucleotides at the 3' terminus. For example, but not as a limitation, the n, n-1 and n-2 nucleotides of the gRNA molecule are nucleotides modified with 2'-fluoro, nucleotides modified with 2'-O-methyl, nucleotides modified with 2'-O-(2-methoxyethyl), deoxyribose nucleotides, LNAs or a combination thereof. In certain embodiments, the n, n-1, and n-2 nucleotides of the gRNA molecule are nucleotides modified with 2'-fluoro. In certain embodiments, the n, n-1, and n-2 nucleotides of the guide RNA molecule are nucleotides modified with 2'-O-methyl. In certain embodiments, the n, n-1, and n-2 nucleotides at the terminal of the gRNA molecule are nucleotides modified with 2'-O-(2-methoxyethyl).In certain embodiments, the n, n-1, and n-2 nucleotides of the gRNA molecule are LNAs. In certain embodiments, the n, n-1, and n-2 nucleotides of the gRNA molecule have a deoxyribose sugar.

[00180] In certain embodiments, a gRNA of the present disclosure includes four or more nucleotides modified with 2'-fluoro, nucleotides modified with 2'-O-methyl, nucleotides modified with 2'-O-(2-methoxyethyl), deoxyribose nucleotides and / or LNAs at its 3' terminus, for example, within the last 5 nucleotides at the 3' terminus. For example, but not as a limitation, the n, n-1, n-2 and n-3 nucleotides of the gRNA molecule are nucleotides modified with 2'-fluoro, nucleotides modified with 2'-O-methyl, Petition 870250090770, dated 06 / 10 / 2025, pp. 226 / 369 50 / 111 nucleotides modified with 2'-O-(2-methoxyethyl), deoxyribose nucleotides, LNAs, or a combination thereof. In certain embodiments, the n-1, n-2, n-3, and n-4 nucleotides of the gRNA molecule are nucleotides modified with 2'-fluoro, nucleotides modified with 2'-O-methyl, nucleotides modified with 2'-O-(2-methoxyethyl), LNAs, or a combination thereof. In certain embodiments, the n, n-1, n-2, and n-3 nucleotides of the gRNA molecule are nucleotides modified with 2'-fluoro. In certain embodiments, the n, n-1, n-2, and n-3 nucleotides of the guide RNA molecule are nucleotides modified with 2'O-methyl. In certain embodiments, the n, n-1, n-2, and n-3 nucleotides at the terminal of the gRNA molecule are nucleotides modified with 2'-O-(2-methoxyethyl). In certain embodiments, the n, n-1, n-2, and n-3 nucleotides of the gRNA molecule are LNAs. 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 n1, n-2, n-3, and n-4 nucleotides of the gRNA molecule are nucleotides modified with 2'-O-methyl.

[00181] In certain embodiments, a gRNA of the present disclosure includes five or more nucleotides modified with 2'-fluoro, nucleotides modified with 2'-O-methyl, nucleotides modified with 2'-O-(2-methoxyethyl), deoxyribose nucleotides and / or LNAs at its 3' terminus, for example, within the last 5 nucleotides at the 3' terminus. For example, but not as a limitation, the n, n-1, n-2, n-3 and n-4 nucleotides of the gRNA molecule are nucleotides modified with 2'-fluoro, nucleotides modified with 2'-O-methyl, nucleotides modified with 2'-O-(2-methoxyethyl), deoxyribose nucleotides, LNAs or a combination thereof. In certain embodiments, the n, n-1, n-2, n-3, and n-4 nucleotides of the gRNA molecule are nucleotides modified with 2'-fluoro. In certain embodiments, the n, n-1, n-2, n-3, and n-4 nucleotides of the guide RNA molecule are nucleotides modified with 2'-O-methyl. In certain embodiments, the n, n-1, n-2, n-3, and n-4 nucleotides at the terminal of the molecule of Petition 870250090770, dated 06 / 10 / 2025, pp. 227 / 369 51 / 111 gRNAs are nucleotides modified with 2'-O-(2-methoxyethyl). 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.

[00182] In certain embodiments, a gRNA of the present disclosure includes (i) one or more nucleotides modified with 2'-fluoro, nucleotides modified with 2'-O-methyl, nucleotides modified with 2'-O-(2-methoxyethyl), deoxyribose nucleotide and / or LNAs at its 5' terminus and (ii) one or more phosphorodithioate linkages at the 5' terminus 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 a blocked nucleic acid (LNA), and the gRNA includes a phosphorodithioate linkage 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 nucleotide modified with 2'-O-methyl, and the gRNA includes a phosphorodithioate linkage between the first and second nucleotides at the 5' end of the gRNA molecule.

[00183] In certain embodiments, a gRNA of the present disclosure includes (i) two or more nucleotides modified with 2'-fluoro, nucleotides modified with 2'-O-methyl, nucleotides modified with 2'-O-(2-methoxyethyl), deoxyribose nucleotides and / or LNAs at its 5' terminus and (ii) two or more phosphorodithioate linkages at the 5' terminus 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 includes a phosphorodithioate linkage between the first and second nucleotides and between the second and third nucleotides. Petition 870250090770, dated 06 / 10 / 2025, pp. 228 / 369 52 / 111 nucleotides 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 nucleotides modified with 2'-O-methyl, and the gRNA molecule includes phosphorodithioate linkages between the first and second nucleotides and the second and third nucleotides at the 5' end of the gRNA molecule.

[00184] In certain embodiments, a gRNA of the present disclosure includes (i) three or more nucleotides modified with 2'-fluoro, nucleotides modified with 2'-O-methyl, nucleotides modified with 2'-O-(2-methoxyethyl), deoxyribose nucleotides and / or LNAs at its 5' terminus and (ii) three or more phosphorodithioate linkages at the 5' terminus of the gRNA molecule. In certain embodiments, the first, second, and third nucleotides at the 5' end of the gRNA molecule are nucleotides modified with 2'-fluoro, nucleotides modified with 2'-O-methyl, nucleotides modified with 2'-O-(2-methoxyethyl), deoxyribose nucleotides, or LNAs, and the gRNA includes phosphorodithioate linkages between the first and second nucleotides, the second and third nucleotides, and 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 nucleotides modified with 2'-O-methyl, and the gRNA includes phosphorodithioate linkages between the first and second nucleotides, the second and third nucleotides, and the third and fourth nucleotides at the 5' end of the gRNA molecule.

[00185] In certain embodiments, a gRNA of the present disclosure includes (i) four or more nucleotides modified with 2'-fluoro, nucleotides modified with 2'-O-methyl, nucleotides modified with 2'-O-(2-methoxyethyl), deoxyribose nucleotides and / or LNAs at its 5' terminus and (ii) four or more phosphorodithioate linkages at the 5' terminus of the gRNA molecule. In certain embodiments, the first, second, third and fourth nucleotides at the 5' terminus of the gRNA molecule are nucleotides modified with 2'-fluoro, nucleotides Petition 870250090770, dated 06 / 10 / 2025, pp. 229 / 369 53 / 111 modified with 2'-O-methyl, nucleotides modified with 2'-O-(2-methoxyethyl), deoxyribose nucleotides or LNAs, and the gRNA includes phosphorodithioate linkages 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. In certain embodiments, the first, second, third, and fourth nucleotides at the 5' end of the gRNA molecule are nucleotides modified with 2'-O-methyl, and the gRNA includes phosphorodithioate linkages 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.

[00186] In certain embodiments, a gRNA of the present disclosure includes (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 3' terminus and (ii) one or more phosphorodithioate linkages at the 3' terminus of the gRNA molecule. In certain embodiments, the n nucleotide of the gRNA molecule is a 2'-fluoro-modified nucleotide, a 2'-O-methyl-modified nucleotide, a 2'-O-(2-methoxyethyl)-modified nucleotide, deoxyribose nucleotides or a blocked nucleic acid (LNA) and the gRNA includes a phosphorodithioate linkage between the n-1 ne nucleotides of the gRNA molecule. In certain embodiments, the n nucleotide of the gRNA molecule is a nucleotide modified with 2'-O-methyl, and the gRNA includes a phosphorodithioate linkage between the n-1 ne nucleotides of the gRNA molecule.

[00187] In certain embodiments, a gRNA of the present disclosure includes (i) two or more nucleotides modified with 2'-fluoro, nucleotides modified with 2'-O-methyl, nucleotides modified with 2'-O-(2-methoxyethyl), deoxyribose nucleotides and / or LNAs at its 3' terminus and (ii) two or more phosphorodithioate linkages at the 3' terminus of the gRNA molecule. In certain Petition 870250090770, dated 06 / 10 / 2025, pp. 230 / 369 54 / 111 realizations, the ne n-1 or ne n-2 nucleotides of the gRNA molecule are nucleotides modified with 2'-fluoro, nucleotides modified with 2'-O-methyl, nucleotides modified with 2'-O-(2-methoxyethyl) or LNAs, and the gRNA molecule includes a phosphorodithioate linkage between the ne n-1 nucleotides and the n-1 and n-2 nucleotides or between the ne n-1 nucleotides and the n-2 and n-3 nucleotides of the gRNA molecule. In certain embodiments, the ne n-1 or ne n-2 nucleotides of the gRNA molecule are nucleotides modified with 2'-O-methyl, and the gRNA molecule includes a phosphorodithioate linkage between the ne n-1 nucleotides and the n-1 and n-2 nucleotides or between the ne n-1 nucleotides and the n-2 and n-3 nucleotides of the gRNA molecule.

[00188] In certain embodiments, a gRNA of the present disclosure includes (i) three or more nucleotides modified with 2'-fluoro, nucleotides modified with 2'-O-methyl, nucleotides modified with 2'-O-(2-methoxyethyl), deoxyribose nucleotides and / or LNAs at its 3' terminus and (ii) two or more phosphorodithioate linkages at the 3' terminus of the gRNA molecule. In certain embodiments, the n, n-1, and n-2 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 includes a phosphorodithioate linkage between the n-1 ne nucleotide and the n-1 and n-2 nucleotides or between the n-1 ne nucleotide and the n-2 and n-3 nucleotides of the gRNA molecule.In certain embodiments, the n, n-1, and n-2 nucleotides of the gRNA molecule are nucleotides modified with 2'-O-methyl, and the gRNA molecule includes a phosphorodithioate linkage between the n-1 ne nucleotides and the n-1 and n-2 nucleotides, or between the n-1 ne nucleotides and the n-2 and n-3 nucleotides of the gRNA molecule. In certain embodiments, the n, n-1, and n-2 nucleotides of the gRNA molecule are nucleotides modified with 2'-O-methyl, and the gRNA molecule includes a phosphorodithioate linkage between the n-1 ne nucleotides and the n-1 and n-2 nucleotides, or between the n and n-3 nucleotides. Petition 870250090770, dated 06 / 10 / 2025, pp. 231 / 369 55 / 111 n-1 of the gRNA molecule.

[00189] In certain embodiments, a gRNA of the present disclosure includes (i) four or more nucleotides modified with 2'-fluoro, nucleotides modified with 2'-O-methyl, nucleotides modified with 2'-O-(2-methoxyethyl), deoxyribose nucleotides and / or LNAs at its 3' terminus and (ii) four or more phosphorodithioate linkages at the 3' terminus 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 includes a phosphorodithioate linkage between the n-1 nucleotides, between the n-1 and n-2 nucleotides, between the n-2 and n-3 nucleotides, and between the n-3 and n-4 nucleotides of the gRNA molecule.In certain embodiments, the n, n-1, n-2, and n-3 nucleotides of the gRNA molecule are nucleotides modified with 2'-O-methyl, and the gRNA molecule includes a phosphorodithioate linkage between the n-1 nucleotides, between the n-1 and n-2 nucleotides, between the n-2 and n-3 nucleotides, and between the n-3 and n-4 nucleotides of the gRNA molecule.

[00190] In certain embodiments, a gRNA of the present disclosure includes (i) five or more nucleotides modified with 2'-fluoro, nucleotides modified with 2'-O-methyl, nucleotides modified with 2'-O-(2-methoxyethyl), deoxyribose nucleotides and / or LNAs at its 3' terminus and (ii) four or more phosphorodithioate linkages at the 3' terminus 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 includes a phosphorodithioate linkage between the n-1 nucleotides, between the n-1 and n-2 nucleotides, between the n-2 and n-3 nucleotides, and between the n-3 and n-4 nucleotides of the gRNA molecule. Petition 870250090770, dated 06 / 10 / 2025, pp. 232 / 369 56 / 111 In certain embodiments, the n, n-1, n-2, n-3, and n-4 nucleotides of the gRNA molecule are nucleotides modified with 2'-O-methyl, and the gRNA molecule includes a phosphorodithioate linkage between the n-1 nucleotides, between the n-1 and n-2 nucleotides, between the n-2 and n-3 nucleotides, and between the n-3 and n-4 nucleotides of the gRNA molecule.

[00191] In certain embodiments, a gRNA of the present disclosure includes (i) one or more nucleotides modified with 2'-fluoro, nucleotides modified with 2'-O-methyl, nucleotides modified with 2'-O-(2-methoxyethyl), deoxyribose nucleotides and / or LNAs at the 5'-terminus, (ii) one or more phosphorodithioate linkages at the 5'-terminus, (iii) one or more nucleotides modified with 2'fluoro, nucleotides modified with 2'-O-methyl, nucleotides modified with 2'-O-(2-methoxyethyl), deoxyribose nucleotides and LNAs at the 3'-terminus, and (iv) one or more phosphorodithioate linkages at the 3'-terminus. In certain embodiments, a gRNA of the present disclosure includes (i) one or more nucleotides modified with a 5'-term 2'-O-methyl group, (ii) one or more 5'-term phosphorodithioate linkages, (iii) one or more nucleotides modified with a 3'-term 2'-O-methyl group, and (iv) one or more 3'-term phosphorodithioate linkages.

[00192] In certain embodiments, a gRNA of the present disclosure includes (i) two or more nucleotides modified with 2'-fluoro, nucleotides modified with 2'-O-methyl, nucleotides modified with 2'-O-(2-methoxyethyl), deoxyribose nucleotides and / or LNAs at the 5'-terminus, (ii) two or more phosphorodithioate linkages at the 5'-terminus, (iii) two or more nucleotides modified with 2'-fluoro, nucleotides modified with 2'-O-methyl, nucleotides modified with 2'-O-(2-methoxyethyl), deoxyribose nucleotides and LNAs at the 3'-terminus, and (iv) two or more phosphorodithioate linkages at the 3'-terminus. In certain embodiments, a gRNA of the present disclosure includes (i) two or more nucleotides modified with a 2'-O-methyl group at the 5' end, (ii) two or more phosphorodithioate linkages at the 5' end, (iii) two or more nucleotides modified with a 2'-O-methyl group. Petition 870250090770, dated 06 / 10 / 2025, page 233 / 369 57 / 111 (iv) two or more phosphorodithioate linkages at the 3' end.

[00193] In certain embodiments, a gRNA of the present disclosure includes (i) three or more nucleotides modified with 2'-fluoro, nucleotides modified with 2'-O-methyl, nucleotides modified with 2'-O-(2-methoxyethyl), deoxyribose nucleotides and / or LNAs at the 5'-terminus, (ii) two or more phosphorodithioate linkages at the 5'-terminus, (iii) three or more nucleotides modified with 2'-fluoro, nucleotides modified with 2'-O-methyl, nucleotides modified with 2'-O-(2-methoxyethyl), deoxyribose nucleotides and LNAs at the 3'-terminus, and (iv) two or more phosphorodithioate linkages at the 3'-terminus. In certain embodiments, a gRNA of the present disclosure includes (i) three or more nucleotides modified with a 5'-term 2'-O-methyl group, (ii) two or more 5'-term phosphorodithioate linkages, (iii) three or more nucleotides modified with a 3'-term 2'-O-methyl group, and (iv) two or more 3'-term phosphorodithioate linkages.

[00194] In certain embodiments, a gRNA of the present disclosure includes (i) three or more nucleotides modified with 2'-fluoro, nucleotides modified with 2'-O-methyl, nucleotides modified with 2'-O-(2-methoxyethyl), deoxyribose nucleotides and / or LNAs at the 5'-terminus, (ii) three or more phosphorodithioate linkages at the 5'-terminus, (iii) three or more nucleotides modified with 2'fluoro, nucleotides modified with 2'-O-methyl, nucleotides modified with 2'-O-(2-methoxyethyl), deoxyribose nucleotides and LNAs at the 3'-terminus, and (iv) one or more phosphorodithioate linkages at the 3'-terminus. In certain embodiments, a gRNA of the present disclosure includes (i) three or more nucleotides modified with a 5'-term 2'-O-methyl group, (ii) three or more 5'-term phosphorodithioate linkages, (iii) three or more nucleotides modified with a 3'-term 2'-O-methyl group, and (iv) one or more 3'-term phosphorodithioate linkages.

[00195] In certain embodiments, a gRNA of the present disclosure includes (i) three or more nucleotides modified with 2'-fluoronucleotides Petition 870250090770, dated 06 / 10 / 2025, pp. 234 / 369 58 / 111 modified with 2'-O-methyl, nucleotides modified with 2'-O-(2-methoxyethyl), deoxyribose nucleotides and / or LNAs at the 5' end, (ii) three or more phosphorodithioate linkages at the 5' end, (iii) three or more nucleotides modified with 2'fluoro, nucleotides modified with 2'-O-methyl, nucleotides modified with 2'-O-(2-methoxyethyl), deoxyribose nucleotides and LNAs at the 3' end and (iv) two or more phosphorodithioate linkages at the 3' end. In certain embodiments, a gRNA of the present disclosure includes (i) three or more nucleotides modified with a 5'-term 2'-O-methyl group, (ii) three or more 5'-term phosphorodithioate linkages, (iii) three or more nucleotides modified with a 3'-term 2'-O-methyl group, and (iv) two or more 3'-term phosphorodithioate linkages.

[00196] In certain embodiments, a gRNA of the present disclosure includes (i) four or more nucleotides modified with 2'-fluoro, nucleotides modified with 2'-O-methyl, nucleotides modified with 2'-O-(2-methoxyethyl), deoxyribose nucleotides and / or LNAs at the 5'-terminus, (ii) four or more phosphorodithioate linkages at the 5'-terminus, (iii) four or more nucleotides modified with 2'-fluoro, nucleotides modified with 2'-O-methyl, nucleotides modified with 2'-O-(2-methoxyethyl), deoxyribose nucleotides and LNAs at the 3'-terminus, and (iv) four or more phosphorodithioate linkages at the 3'-terminus. In certain embodiments, a gRNA of the present disclosure includes (i) four or more nucleotides modified with a 5'-term 2'-O-methyl group, (ii) four or more 5'-term phosphorodithioate linkages, (iii) four or more nucleotides modified with a 3'-term 2'-O-methyl group, and (iv) four or more 3'-term phosphorodithioate linkages.

[00197] In certain embodiments, a gRNA of the present disclosure includes (i) four or more nucleotides modified with 2'-fluoro, nucleotides modified with 2'-O-methyl, nucleotides modified with 2'-O-(2-methoxyethyl), deoxyribose nucleotides and LNAs at the 5' terminus, (ii) four or more phosphorodithioate linkages at the 5' terminus, (iii) five or more nucleotides modified with Petition 870250090770, dated 06 / 10 / 2025, pages 235 / 369 59 / 111 2'-fluoro, 2'-O-methyl modified nucleotides, 2'-O-(2-methoxyethyl) modified nucleotides, deoxyribose nucleotides and LNAs at the 3' terminus and (iv) four or more 3'-term phosphorodithioate linkages. In certain embodiments, a gRNA of the present disclosure includes (i) four or more 5'-O-methyl modified nucleotides, (ii) four or more 5'-term phosphorodithioate linkages, (iii) five or more 3'-O-methyl modified nucleotides and (iv) four or more 3'-term phosphorodithioate linkages.

[00198] In certain embodiments, a gRNA of the present disclosure includes (i) a nucleotide modified with a 5'-term 2'-O-methyl group (e.g., first nucleotide), (ii) a 5'-term phosphorodithioate linkage (e.g., between the first and second nucleotides), (iii) a 3'-term 2'-O-methyl group modified (e.g., nucleotide n), and (iv) a 3'-term phosphorodithioate linkage (e.g., between nucleotides n and n-1).

[00199] In certain embodiments, a gRNA of the present disclosure includes (i) two nucleotides modified with 2'-O-methyl at the 5' terminus (e.g., first and second nucleotides), (ii) two phosphorodithioate linkages at the 5' terminus (e.g., between the first and second nucleotides and between the second and third nucleotides), (iii) two nucleotides modified with 2'-O-methyl (e.g., the n-1 nucleotides) at the 3' terminus, and (iv) two phosphorodithioate linkages at the 3' terminus (e.g., between the n-1 nucleotides and between the n-1 and n-2 nucleotides).

[00200] In certain embodiments, a gRNA of the present disclosure includes (i) three nucleotides modified with 2'-O-methyl (e.g., first, second, and third nucleotides) at the 5'-terminus, (ii) three phosphorodithioate linkages at the 5'-terminus (e.g., between the first and second nucleotides, between the second and third nucleotides, and between the third and fourth nucleotides), (iii) three nucleotides modified with 2'-O-methyl (e.g., the n nucleotides, Petition 870250090770, dated 06 / 10 / 2025, pp. 236 / 369 60 / 111 n-1 and n-2) at the 3' end and (iv) two phosphorodithioate bonds at the 3' end (e.g., between nucleotides n and n-1 and between nucleotides n-1 and n-2).

[00201] In certain embodiments, a gRNA of the present disclosure includes (i) three 2'-O-methyl-modified nucleotides (e.g., first, second, and third nucleotides) at the 5'-terminus, (ii) three phosphorothioate linkages at the 5'-terminus (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 (e.g., n, n-1, and n-2 nucleotides) at the 3'-terminus, and (iv) two phosphorothioate linkages at the 3'-terminus (e.g., between n and n-1 nucleotides and between n-1 and n-2 nucleotides).

[00202] In certain embodiments, a gRNA of the present disclosure includes (i) three nucleotides modified with 2'-O-methyl (e.g., first, second, and third nucleotides) at the 5'-terminus, (ii) two phosphorodithioate linkages at the 5'-terminus (e.g., between the first and second nucleotides and between the second and third nucleotides or between the second and third nucleotides and the third and fourth nucleotides), (iii) three nucleotides modified with 2'O-methyl (e.g., nucleotides n, n-1, and n-2) at the 3'-terminus, and (iv) two phosphorodithioate linkages at the 3'-terminus (e.g., between nucleotides n and n-1 and between nucleotides n-1 and n-2 or between nucleotides n-1 and n-2 and between nucleotides n-2 and n-3).

[00203] In certain embodiments, a gRNA of the present disclosure includes (i) three nucleotides modified with 2'-O-methyl (e.g., first, second, and third nucleotides) at the 5' terminus, (ii) two phosphorothioate linkages at the 5' terminus (e.g., between the first and second nucleotides and between the second and third nucleotides or between the second and third nucleotides and the third and fourth nucleotides), (iii) one phosphorothioate linkage at the 5' terminus (e.g., between the first and second nucleotides, between the second and third nucleotides, or between the third and fourth nucleotides), (iv) three Petition 870250090770, dated 06 / 10 / 2025, pp. 237 / 369 61 / 111 nucleotides modified with 2'-O-methyl (e.g., n, n-1 and n-2 nucleotides) at the 3' end and (v) two phosphorodithioate linkages at the 3' end (e.g., between n and n-1 nucleotides and between n-1 and n-2 nucleotides or between n-1 and n-2 nucleotides and between n-2 and n-3 nucleotides).

[00204] In certain embodiments, a gRNA of the present disclosure includes (i) three 2'-O-methyl-modified nucleotides (e.g., first, second, and third nucleotides) at the 5'-terminus, (ii) three phosphorodithioate linkages at the 5'-terminus (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 (e.g., n, n-1, and n-2 nucleotides) at the 3'-terminus, and (iv) a phosphorodithioate linkage at the 3'-terminus (e.g., between n and n-1 nucleotides, between n-1 and n-2 nucleotides, or between n-2 and n-3 nucleotides).

[00205] In certain embodiments, a gRNA of the present disclosure includes (i) three nucleotides modified with 2'-O-methyl (e.g., first, second, and third nucleotides) at the 5'-terminus, (ii) three phosphorothioate linkages at the 5'-terminus (e.g., between the first and second nucleotides, between the second and third nucleotides, and between the third and fourth nucleotides), (iii) three nucleotides modified with 2'-O-methyl (e.g., nucleotides n, n-1, and n-2) at the 3'-terminus, (iv) a phosphorothioate linkage at the 3'-terminus (e.g., between nucleotides n and n-1, between nucleotides n-1 and n-2, or between nucleotides n-2 and n-3), and (v) a phosphorothioate linkage at the 3'-terminus (e.g., between the nucleotides n and n-1, between nucleotides n-1 and n-2, or between nucleotides n-2 and n-3).

[00206] In certain embodiments, a gRNA of the present disclosure includes (i) four nucleotides modified with 2'-O-methyl (e.g., first, second, third, and fourth nucleotides) at the 5'-terminus, (ii) four phosphorodithioate linkages at the 5'-terminus (e.g., between the first and second nucleotides). Petition 870250090770, dated 06 / 10 / 2025, pp. 238 / 369 62 / 111 nucleotides, between the second and third nucleotides, between the third and fourth nucleotides and between the fourth and fifth nucleotides), (iii) four nucleotides modified with 2'-O-methyl (for example, nucleotides n, n-1, n-2 and n-3) at the 3' end and (iv) four phosphorodithioate linkages at the 3' end (for example, between nucleotides n and n-1, between nucleotides n-1 and n-2, between nucleotides n-2 and n-3 and between nucleotides n-3 and n-4).

[00207] In certain embodiments, a gRNA of the present disclosure includes (i) four 2'-O-methyl-modified nucleotides (e.g., first, second, third, and fourth nucleotides) at the 5'-terminus, (ii) four phosphorodithioate linkages at the 5'-terminus (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 (e.g., n, n-1, n-2, n-3, and n-4 nucleotides) at the 3'-terminus, and (iv) four phosphorodithioate linkages at the 3'-terminus (e.g., between n and n-1 nucleotides, between n-1 and n-2 nucleotides, between n-2 and n-3 nucleotides, and between the n-3 and n-4 nucleotides).

[00208] In certain embodiments, a gRNA of the present disclosure includes (i) four 2'-O-methyl-modified nucleotides (e.g., first, second, third, and fourth nucleotides) at the 5'-terminus, (ii) four phosphorodithioate linkages at the 5'-terminus (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 (e.g., nucleotides n, n-1, n-2, n-3, and n-4) at the 3'-terminus, and (iv) four phosphorodithioate linkages at the 3'-terminus (e.g., between nucleotides n-1 and n-2, between nucleotides n-2 and n-3, between nucleotides n-3 and n-4 and between the nucleotides n-4 and n-5).

[00209] In certain embodiments, a modified gRNA of the present disclosure may be modified according to a modification scheme. Petition 870250090770, dated 06 / 10 / 2025, pp. 239 / 369 63 / 111 disclosed in Tables 1 and 10. For example, but not as a limitation, a modified gRNA may be modified according to modification scheme A of Table 1. In certain embodiments, a modified gRNA may be modified according to modification scheme A of Table 1. In certain embodiments, a modified gRNA may be modified according to modification scheme B of Table 1. In certain embodiments, a modified gRNA may be modified according to modification scheme C of Table 1. In certain embodiments, a modified gRNA may be modified according to modification scheme D of Table 1. In certain embodiments, a modified gRNA may be modified according to modification scheme E of Table 1. In certain embodiments, a modified gRNA may be modified according to modification scheme F of Table 1. In certain embodiments, a modified gRNA may be modified according to modification scheme G of Table 1.In certain embodiments, a modified gRNA can be modified according to the H modification scheme of Table 1. In certain embodiments, a modified gRNA can be modified according to the I modification scheme of Table 1. In certain embodiments, a modified gRNA can be modified according to the J modification scheme of Table 1. In certain embodiments, a modified gRNA can be modified according to the K modification scheme of Table 1. In certain embodiments, a modified gRNA can be modified according to the L modification scheme of Table 1. In certain embodiments, a modified gRNA can be modified according to the M modification scheme of Table 10. In certain embodiments, a modified gRNA can be modified according to the N modification scheme of Table 10. In certain embodiments, a modified gRNA can be modified according to the O modification scheme of Table 10.In certain embodiments, a modified gRNA can be modified according to the... Petition 870250090770, dated 06 / 10 / 2025, pp. 240 / 369 64 / 111 modification scheme P of Table 10. In certain embodiments, a modified gRNA can be modified according to modification scheme Q of Table 10. In certain embodiments, a modified gRNA can be modified according to modification scheme R of Table 10. In certain embodiments, a modified gRNA can be modified according to modification scheme S of Table 10. In certain embodiments, a modified gRNA can be modified according to modification scheme T of Table 10. In certain embodiments, a modified gRNA can be modified according to modification scheme U of Table 10. In certain embodiments, a modified gRNA can be modified according to modification scheme V of Table 10.

[00210] In certain embodiments, a modified gRNA of the present disclosure may have an editing efficiency of about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 26%, about 27%, about 28%, about 29%, about 30%, about 31%, about 32%, about 33%, about 34%, about 35%, about 36%, about 37%, about 38%, about 39%, about 40%, about 41%, about 42%, about 43%, about 44%, about 45%, about 46%, about 47%, about 48%, about 49%, about 50%, about 51%, about 52%, about 53%, about 54%, about 55%, about 56%, about 57%, about 58%, about 59%, about 60%, about 61%, about 62%, about 63%, about 64%,about 65%, about 66%, about 67%, about 68%, about 69%, about 70%, about 71%, about 72%, about 73%, about 74%, about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about, Petition 870250090770, dated 06 / 10 / 2025, pp. 241 / 369 65 / 111 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%.

[00211] In certain embodiments, a modified gRNA of the present 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, a modified gRNA of the present disclosure has an editing efficiency of about 60% or more. In certain embodiments, a modified gRNA from this disclosure has an editing efficiency of about 65% or more. In certain embodiments, a modified gRNA from this disclosure has an editing efficiency of about 70% or more.In certain embodiments, a modified gRNA from this disclosure has an editing efficiency of about 75% or more. In certain embodiments, a modified gRNA from this disclosure has an editing efficiency of about 80% or more. In certain embodiments, a modified gRNA from this disclosure has an editing efficiency of about 85% or more. In certain embodiments, a modified gRNA from this disclosure has an editing efficiency of about 90% or more. In certain embodiments, a modified gRNA from this disclosure has an editing efficiency of about 95% or more.

[00212] In certain embodiments, a modified gRNA of the present disclosure may have an editing efficiency of about 5% to about 100%. For example, but not as a limitation, a modified gRNA of the present disclosure may have an editing efficiency of about 5% to about 100%, Petition 870250090770, dated 06 / 10 / 2025, pp. 242 / 369 66 / 111 close to 10% to close to 100%, close to 20% to close to 100%, close to 30% to close to 100%, close to 40% to close to 100%, close to 45% to close to 100%, close to 50% to close to 100%, close to 55% to close to 100%, close to 60% to nearly 100%, nearly 65% ​​to nearly 100%, nearly 70% to nearly 100%, approximately 75% to approximately 100%, approximately 80% to approximately 100%, approximately 85% to about 100%, about 90% to about 100%, or about 95% to about 100%. In certain embodiments, a modified gRNA from this disclosure may have an editing efficiency of about 50% to about 100%. In certain embodiments, a modified gRNA from this disclosure may have an editing efficiency of about 60% to about 100%. In certain embodiments, a modified gRNA from this disclosure may have an editing efficiency of about 70% to about 100%. In certain embodiments, a modified gRNA from this disclosure may have an editing efficiency of about 80% to about 100%. In certain embodiments, a modified gRNA from this disclosure may have an editing efficiency of about 90% to about 100%.

[00213] In certain embodiments, a modified gRNA of the present disclosure may maintain the editing efficiency of a reference gRNA or have improved editing efficiency compared to a reference gRNA.For example, but not as a limitation, a modified gRNA of the present 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 a reference gRNA. In certain embodiments, a modified gRNA of the present disclosure may retain about 50% or more of the editing efficiency of a reference gRNA. In certain embodiments, a modified gRNA of the present disclosure may retain about 60% or more of the editing efficiency of a reference gRNA. In certain embodiments, a modified gRNA of the present disclosure... Petition 870250090770, dated 06 / 10 / 2025, pp. 243 / 369 67 / 111 may retain approximately 70% or more of the editing efficiency of a reference gRNA. In certain embodiments, a gRNA modified from this disclosure may retain approximately 80% or more of the editing efficiency of a reference gRNA. In certain embodiments, a gRNA modified from this disclosure may retain approximately 90% or more of the editing efficiency of a reference gRNA. In certain embodiments, a gRNA modified from this disclosure may retain from approximately 50% to approximately 100% of the editing efficiency of a reference gRNA, for example, from approximately 55% to approximately 100%, approximately 60% to approximately 100%, approximately 65% ​​to approximately 100%, approximately 70% to approximately 100%. 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 nearly 100%, nearly 95% to nearly 100%, nearly 96% to nearly 100%, approximately 97% to approximately 100%, approximately 98% to approximately 100%, or approximately 99% to approximately 100%. In certain embodiments, a modified gRNA from this disclosure may retain approximately 60% to approximately 100% of the editing efficiency of a reference gRNA. In certain embodiments, a modified gRNA from this disclosure may retain approximately 70% to approximately 100% of the editing efficiency of a reference gRNA. In certain embodiments, a modified gRNA from this disclosure may retain approximately 80% to approximately 100% of the editing efficiency of a reference gRNA. In certain embodiments, a modified gRNA from this disclosure may retain approximately 90% to approximately 100% of the editing efficiency of a reference gRNA.In certain embodiments, a modified gRNA of the present disclosure may exhibit an increase in editing efficiency compared with a reference gRNA, for example, an increase in editing efficiency 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. Petition 870250090770, dated 06 / 10 / 2025, pp. 244 / 369 68 / 111 of 9% or more or about 10% or more. In certain embodiments, a modified gRNA of the present disclosure may exhibit an increase in editing efficiency of about 1% to about 10%. In certain embodiments, the reference gRNA may be a gRNA having the same nucleotide sequence as the modified gRNA (e.g., without the same modifications as the modified gRNA). In certain embodiments, the reference gRNA may be a gRNA that has a target domain with the same sequence as the modified gRNA (e.g., without the same modifications as the modified gRNA). In certain embodiments, the reference gRNA may be a gRNA that has the same number of nucleotides as the modified gRNA (e.g., without the same modifications as the modified gRNA). III. RNA-guided nucleases

[00214] RNA-guided nucleases of various types and species may be used in this disclosure. RNA-guided nucleases are for use in combination with one or more modified gRNAs disclosed in this document, for example, in Section II.

[00215] In certain embodiments, the RNA-guided 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, 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 a Cas9, a Cas12a, a Cas13, and a combination thereof. In certain embodiments, the Cas protein is a Cas9 protein. Petition 870250090770, dated 06 / 10 / 2025, pp. 245 / 369 69 / 111

[00216] In certain embodiments, the Cas protein is an engineered Cas protein that differs from a reference Cas protein, for example, a wild-type Cas protein. In certain embodiments, the reference Cas protein is a naturally occurring Cas protein. In certain embodiments, the Cas protein includes one or more amino acid variations compared to a reference Cas protein, for example, a wild-type Cas protein. In certain embodiments, an 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, an engineered Cas protein has no cleavage activity or no substantial cleavage activity.In certain embodiments, a Cas protein may have no cleavage activity or substantially less, for example, less than 20%, about 10%, about 5%, or about 1% of the cleavage activity of a reference Cas protein.

[00217] In certain embodiments, the engineered Cas protein includes one or more deletions that reduce the size of the Cas protein while retaining, at least partially, the nuclease activity of the Cas protein. In certain embodiments, the reduced size of the engineered Cas protein may allow flexibility regarding the methods for delivering such engineered Cas proteins.

[00218] In certain embodiments, a Cas protein interacts with a gRNA molecule of the present disclosure and, together with the gRNA molecule, localizes to a target nucleic acid that includes a target sequence (e.g., a sequence that is complementary to a sequence of the gRNA molecule) and a PAM sequence. In certain embodiments, the ability to Petition 870250090770, dated 06 / 10 / 2025, pp. 246 / 369 70 / 111 The ability of a Cas protein to interact with and cleave a target nucleic acid depends on the PAM sequence. In certain embodiments, the cleavage of the target nucleic acid occurs upstream of the PAM sequence. In certain embodiments, the cleavage of the target nucleic acid occurs downstream of the PAM sequence. Cas molecules from different species, for example, bacterial species, may recognize different PAM sequences.

[00219] In certain embodiments, a Cas protein for use in the present disclosure may be derived from any 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 bacterium, 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.

[00220] In certain embodiments, a Cas protein for use in the present disclosure directs the cleavage of one or both strands at the location of a target nucleic acid. For example, but not as a limitation, a Cas protein for use in the present disclosure directs the cleavage of one or both strands within the target nucleic acid. Alternatively, the protein Petition 870250090770, dated 06 / 10 / 2025, pp. 247 / 369 71 / 111 Cas directs the cleavage of one or both strands within about 500 base pairs (e.g., within about 400, about 300, about 200, about 100, about 80, about 60, about 40, about 20, about 10, or about 5 base pairs) of the target nucleic acid.

[00221] In certain embodiments, a Cas protein, for example, a Cas9, which includes functional RuvC and HNH nuclease domains, can cleave both strands of a target nucleic acid sequence. In certain embodiments, the Cas protein, for example, Cas9, comprises a functional endonuclease domain that allows the Cas protein to cleave only one strand (i.e., cut) of a target nucleic acid sequence. For example, but not as a limitation, a Cas9 nicase may include (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 nicase may comprise (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 nicase comprises a functional HNH-like molecule and includes a mutation in D10, for example, D10A.In certain embodiments, a Cas9 nicase comprises a functional RuvC domain and includes a mutation in H840, for example, H840A. In certain embodiments, a Cas9 nicase comprises a functional RuvC domain and includes a mutation in N863, for example, N863A.

[00222] In certain embodiments, the nucleotide sequence encoding a Cas protein is codon-optimized. For example, but not as a limitation, the nucleotide sequence encoding a Cas protein may be codon-optimized, for example, where at least one uncommon codon or least common codon has been replaced by a common codon, for optimized expression in a particular cell type, for example, a mammalian cell.

[00223] In certain embodiments, the Cas protein is a protein of Petition 870250090770, dated 06 / 10 / 2025, pp. 248 / 369 72 / 111 Fusion protein that includes one or more heterologous protein domains. In certain embodiments, a Cas fusion protein may include any additional protein domains, for example, epitope tags, reporter sequences, and protein domains that have one or more of the following activities: methylase activity, demethylase activity, transcription activation activity, transcription repression activity, transcription release factor activity, histone modification activity, RNA cleavage activity, and nucleic acid binding activity.

[00224] In certain embodiments, the Cas protein includes one or more nuclear localization sequences to promote the accumulation of the Cas protein in a detectable quantity in the nucleus of a cell. Nuclear localization sequences are known in the art. For example, but not as a limitation, a Cas protein may include a nuclear localization sequence (e.g., from SV40) at its N-terminus and / or C-terminus. IV. Compositions

[00225] This disclosure further provides compositions that include one or more modified gRNAs disclosed in this document. In certain embodiments, a composition in this document may include 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 in this document.

[00226] In certain embodiments, a composition of this document includes a gRNA comprising one or more modifications disclosed in this document, for example, as described in Section II. In certain embodiments, the composition may include a nucleic acid encoding a gRNA molecule disclosed in this document. Alternatively or additionally, the composition includes the gRNA molecule as a transcribed or synthesized RNA molecule. Petition 870250090770, dated 06 / 10 / 2025, pp. 249 / 369 73 / 111

[00227] In certain embodiments, a composition of the present disclosure may additionally include an RNA-guided nuclease. In certain embodiments, the composition includes a nucleic acid encoding an RNA-guided nuclease. Alternatively or additionally, the composition includes RNA-guided nuclease as a protein. In certain embodiments, the RNA-guided nuclease is a Cas protein, for example, a Cas9 protein.

[00228] This disclosure further provides nucleic acid compositions that include one or more modified gRNAs disclosed in this document. In certain embodiments, a nucleic acid composition includes a polynucleotide encoding a gRNA molecule described in this document. In certain embodiments, the nucleic acid composition may further include a polynucleotide encoding an RNA-guided nuclease, for example, a Cas protein. In certain embodiments, a nucleic acid composition of this disclosure may include a nucleic acid encoding both the gRNA molecule and the RNA-guided nuclease, for example, a nucleic acid that includes a polynucleotide encoding a gRNA molecule (e.g., coupled to a first promoter) and a polynucleotide encoding an RNA-guided nuclease (e.g., coupled to a second promoter).In certain embodiments, a nucleic acid composition of the present disclosure may include a first nucleic acid encoding the gRNA molecule and a second nucleic acid encoding the RNA-guided nuclease.

[00229] In certain embodiments, nucleic acid compositions encoding one or more gRNA molecules and / or one or more RNA-guided nucleases may be delivered to individuals or delivered into cells by a method known in the art or as described herein. For example, but not as a limitation, a nucleic acid encoding a gRNA molecule and / or an RNA-guided nuclease may be Petition 870250090770, dated 06 / 10 / 2025, pp. 250 / 369 74 / 111 delivered to an individual or delivered into a cell by non-vector-based methods (e.g., using DNA complexes or naked DNA), by vector-based methods, or a combination thereof. In certain embodiments, the vector may be a viral vector. In certain embodiments, the virus is 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, a vector of the present disclosure includes a polynucleotide sequence encoding a gRNA molecule and / or an RNA-guided nuclease. In certain embodiments, the vector further includes a sequence encoding a nuclear location fused to the RNA-guided nuclease, for example, at the N-terminus and / or C-terminus of the RNA-guided nuclease.

[00230] This disclosure further provides compositions comprising ribonucleoprotein (RNP) complexes. In certain embodiments, an RNP complex includes a gRNA molecule, for example, as a transcribed or synthesized RNA, and an RNA-guided nuclease. In certain embodiments, the gRNA molecule forms an RNP complex with RNA-guided nuclease under suitable conditions prior to delivery to target cells.

[00231] This disclosure further provides cells that include one or more compositions from this disclosure. For example, but not as a limitation, a cell may include a nucleic acid composition from this disclosure. In certain embodiments, a cell may include an RNP complex from this disclosure. A variety of cells may be modified using the disclosed compositions. For example, but not as a limitation, the cell may be an immune cell, for example, a T cell. In certain embodiments, the T cell may be a CD8+ T cell and / or a CD4+ T cell. In certain embodiments, the cell may be a stem cell. In certain Petition 870250090770, dated 06 / 10 / 2025, pp. 251 / 369 In 75 / 111 embodiments, the cell may be an induced pluripotent stem cell (iPS). In certain embodiments, the cell may be a cell derived from a stem cell or an iPS, for example, an immune cell derived from a stem cell or an iPS.

[00232] In certain embodiments, the compositions of the present disclosure, for example, nucleic acid compositions of the present disclosure, can be delivered to target cells by methods known in the art or as described herein. For example, but not as a limitation, a composition of the present disclosure can be delivered to a cell by microinjection, electroporation, transient cell compression or squeezing, lipid-mediated transfection, peptide-mediated delivery, or a combination thereof. In certain embodiments, a composition, for example, comprising an RNP complex, is delivered to target cells by electroporation. V. Methods of Use

[00233] This document further provides methods for the use of the gRNAs disclosed herein. In certain embodiments, gRNA molecules provided herein can be used to modify a target nucleic acid in a cell. For example, but not as a limitation, a gRNA molecule from this disclosure can be used to modify a target nucleic acid in a cell, for example, ex vivo or in vivo.

[00234] In certain embodiments, a gRNA molecule of the present disclosure can be used in a method to reduce the expression of the target nucleic acid in a cell. In certain embodiments, a gRNA molecule of the present disclosure can be used in a method to increase the expression of the target nucleic acid in a cell. In certain embodiments, a gRNA molecule of the present disclosure can be used in a method to introduce Petition 870250090770, dated 06 / 10 / 2025, pp. 252 / 369 76 / 111 an insertion or deletion of one or more nucleotides in proximity to the target nucleic acid. In certain embodiments, a gRNA molecule of the present disclosure can be used in a method to introduce a deletion in proximity to the target nucleic acid. In certain embodiments, a gRNA molecule of the present disclosure can be used in a method to introduce an insertion in proximity to the target nucleic acid. In certain embodiments, a gRNA molecule of the present disclosure can be used in a method to introduce one or more breaks (e.g., double-strand breaks or single-strand breaks) in proximity to the target nucleic acid.

[00235] In certain embodiments, a method for modifying a target nucleic acid in a cell includes bringing the cell into contact with a gRNA molecule that includes a spacer (e.g., a domain that is targeted) specific to a sequence in the target nucleic acid. In certain embodiments, the method includes bringing the cell into contact with a composition disclosed herein, for example, a composition comprising a modified gRNA molecule and an RNA-guided nuclease.

[00236] The present disclosure further provides methods for treating an individual. For example, but not as a limitation, a composition comprising a gRNA molecule of the present disclosure may be used in a therapeutic method. In certain embodiments, the present disclosure provides a gRNA molecule for use in treating an individual in need thereof. In certain embodiments, the present disclosure provides a gRNA molecule for use in treating an individual with a disease. In certain embodiments, the gRNA molecule comprises a spacer that is complementary to a target nucleic acid, for example, a gene that is associated with the disease to be treated. In certain embodiments, the method includes modifying a cell of the individual ex vivo by placing the cell in contact with a composition comprising an effective amount of a gRNA molecule. Petition 870250090770, dated 06 / 10 / 2025, pp. 253 / 369 77 / 111 gRNA disclosed herein or an RNP complex disclosed herein, for example, by any of the delivery methods disclosed herein or known in the art. In certain embodiments, the method may additionally include returning the modified cell to the individual.

[00237] In certain embodiments, a gRNA molecule of the present disclosure may be used as a medicament. In certain embodiments, the present document provides for the use of a gRNA molecule disclosed herein in the manufacture or preparation of a medicament. In certain embodiments, the medicament is for the treatment of a disease. In certain embodiments, the medicament is for use in a method of treating a disease that includes administering an effective amount of the medicament to an individual with the disease.

[00238] In certain embodiments, compositions comprising any of the gRNA molecules provided herein, for example, can be used in any of the above therapeutic methods. In certain embodiments, a composition comprising any of the gRNA molecules and / or RNA-guided nucleases provided herein can be used in a therapeutic method, for example, by modifying cells ex vivo.

[00239] In certain embodiments, one or more compositions disclosed in this document, for example, nucleic acid compositions, may be administered to an individual or brought into contact with a cell of an individual, for example, ex vivo. In certain embodiments, the composition may include a gRNA from the present disclosure and an RNA-guided nuclease. Alternatively, the composition includes a gRNA from the present disclosure and does not include an RNA-guided nuclease. In certain embodiments, the composition, for example, the nucleic acid composition, which includes gRNA is Petition 870250090770, dated 06 / 10 / 2025, pp. 254 / 369 78 / 111 administered concurrently with a composition that includes an RNA-guided nuclease. In certain embodiments, the composition, for example, the nucleic acid composition that includes gRNA, is administered after the composition that includes the RNA-guided nuclease. In certain embodiments, the composition, for example, the nucleic acid composition that includes gRNA, is administered before the composition that includes the RNA-guided nuclease. In certain embodiments, the nucleic acid composition may be delivered by any of the delivery methods described in this document, for example, a nucleic acid encoding gRNA may be delivered by a viral vector. In certain embodiments, the composition that includes the RNA-guided nuclease may be delivered to a cell by electroporation. VI. Gene Editing Systems

[00240] This disclosure also provides gene editing systems comprising one or more compositions disclosed herein and / or materials useful for carrying out the methods described herein.

[00241] In certain embodiments, a gene-editing system of the present document may comprise one or more modified gRNA molecules disclosed in the present document. For example, but not as a limitation, the gene-editing system of the present disclosure may comprise one or more gRNA molecules disclosed in Section II.

[00242] In certain embodiments, the gRNA molecule of the gene-editing system of the present disclosure comprises: (a) a phosphorodithioate linkage between the first and second nucleotides at the 5' end of the guide RNA molecule; (b) a phosphorodithioate linkage between the 3' (n)-terminal nucleotide and the n-1 nucleotide of the guide RNA molecule; or (c) a phosphorodithioate linkage between the first and second nucleotides at the 5' end of the guide RNA molecule and a phosphorodithioate linkage between the n-terminal nucleotides. Petition 870250090770, dated 06 / 10 / 2025, pp. 255 / 369 79 / 111 of the guide RNA molecule. In certain embodiments, phosphorodithioate linkages are present between the first and second nucleotides, the second and third nucleotides, and the third and fourth nucleotides at the 5' end of the gRNA molecule. In certain embodiments, phosphorodithioate linkages are present between the n-1 nucleotide and the n-1 and n-2 nucleotides or the n-1 nucleotide and the n-2 and n-3 nucleotides of the gRNA molecule.

[00243] In certain embodiments, the first, second, and / or third nucleotide at the 5' end of the gRNA molecule comprises a modification selected from the group consisting of: a 2'-fluoro-modified nucleotide; a 2'-O-methyl-modified nucleotide; a 2'-O-(2-methoxyethyl)-modified nucleotide; a locking nucleic acid (LNA); a deoxyribose nucleotide; and a combination of two or more of the aforementioned modifications. In certain embodiments, the first, second, third, and / or fourth nucleotide at the 5' end of the gRNA molecule comprises a modification selected from the group consisting of: a 2'-fluoro-modified nucleotide; a 2'-O-methyl-modified nucleotide; a 2'-O-(2-methoxyethyl)-modified nucleotide; a locking nucleic acid (LNA); a deoxyribose nucleotide; and a combination of two or more of the aforementioned modifications.In certain embodiments, the first, second, third, fourth, and / or fifth nucleotide at the 5' terminus of the gRNA molecule comprises a modification selected from the group consisting of: a 2'-fluoro-modified nucleotide; a 2'-O-methyl-modified nucleotide; a 2'-O-(2-methoxyethyl)-modified nucleotide; a blocked nucleic acid (LNA); a deoxyribose nucleotide; and a combination of two or more of the aforementioned modifications.

[00244] In certain embodiments, the n, ne n-1 nucleotides, the ne n-2 nucleotides, the n-1 and n-2 nucleotides, or the n, n-1 and n-2 nucleotides of the gRNA molecule comprise a selected modification from the Petition 870250090770, dated 06 / 10 / 2025, pp. 256 / 369 80 / 111 group consisting of: a 2'-fluoro-modified nucleotide; a 2'-O-methyl-modified nucleotide; a 2'-O-(2-methoxyethyl)-modified nucleotide; a blocked nucleic acid (LNA); a deoxyribose nucleotide; and a combination of two or more of the aforementioned modifications. In certain embodiments, the n, n-1, n-2 and n-3 nucleotides or the n-1, n-2, n-3 and n-4 nucleotides of the gRNA molecule comprise(s) a modification selected from the group consisting of: a 2'-fluoro-modified nucleotide; a 2'-O-methyl-modified nucleotide; a 2'-O(2-methoxyethyl)-modified nucleotide; a blocked nucleic acid (LNA); a deoxyribose nucleotide; and a combination of two or more of the aforementioned modifications.In certain embodiments, the n, n-1, n-2, n-3 and n-4 nucleotides of the gRNA molecule comprise(s) a modification selected from the group consisting of: a 2'-fluoro-modified nucleotide; a 2'-O-methyl-modified nucleotide; a 2'-O-(2-methoxyethyl)-modified nucleotide; a blocked nucleic acid (LNA); a deoxyribose nucleotide; and a combination of two or more of the aforementioned modifications.

[00245] In certain embodiments, the gene editing systems of the present disclosure may include one or more modified gRNAs, for example, as disclosed in Section II, and an RNA-guided nuclease, for example, an RNA-guided nuclease as disclosed in Section III. In certain embodiments, the RNA-guided nuclease is a Cas protein. In certain embodiments, the Cas protein is selected from the group consisting of a Cas9, a Cas12, a Cas13, and combinations thereof.

[00246] In certain embodiments, the gene editing systems of this document comprise one or more nucleic acids encoding a gRNA as described herein, for example, as disclosed in Section II. In certain embodiments, the one or more nucleic acids additionally comprise a polynucleotide encoding a nuclease. Petition 870250090770, dated 06 / 10 / 2025, pp. 257 / 369 81 / 111 guided by RNA, for example, an RNA-guided nuclease as disclosed in Section III. In certain embodiments, the RNA-guided nuclease is a Cas protein. In certain embodiments, the Cas protein is selected from the group consisting of a Cas9, a Cas12, a Cas13, and combinations thereof.

[00247] In certain embodiments, the gene editing system comprises one or more vectors containing a nucleic acid as described herein, for example, as disclosed in Section IV.

[00248] In certain embodiments, the gene editing system comprises one or more compositions that include a gRNA disclosed herein, for example, as disclosed in Section II. In certain embodiments, the composition further comprises an RNA-guided nuclease. In certain embodiments, the composition comprises a nucleic acid described herein, for example, as disclosed in Section IV.

[00249] In certain embodiments, the gene editing system comprises one or more RNP complexes that include a gRNA from Section II and disclosed in this document and an RNA-guided nuclease. In certain embodiments, the RNA-guided nuclease is a Cas protein. In certain embodiments, the Cas protein is selected from the group consisting of a Cas9, a Cas12, a Cas13, and combinations thereof.

[00250] Exemplary Achievements: A1. The subject matter disclosed here provides a guide RNA molecule comprising: (a) a phosphorodithioate linkage between the first and second nucleotides at the 5' end of the guide RNA molecule; (b) a phosphorodithioate linkage between the 3' terminal nucleotide (“n”) and the n-1 nucleotide of the guide RNA molecule; or (c) a phosphorodithioate linkage between the first and the second Petition 870250090770, dated 06 / 10 / 2025, pp. 258 / 369 82 / 111 nucleotides at the 5' end of the guide RNA molecule and a phosphorodithioate linkage between the n-1 nucleotides of the guide RNA molecule; A2. Guide RNA molecule, according to A1, which additionally comprises phosphorodithioate linkages between the second and third nucleotides and the third and fourth nucleotides at the 5' end of the guide RNA molecule; A3. Guide RNA molecule, according to A1 or A2, comprising phosphorodithioate linkages between the first and second nucleotides, the second and third nucleotides, and the third and fourth nucleotides at the 5' end of the guide RNA molecule; A4. Guide RNA molecule, according to any one of A1 to A3, comprising phosphorodithioate linkages 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' terminus of the guide RNA molecule; A5. Guide RNA molecule, according to any one of A1 to A4, comprising phosphorodithioate linkages between nucleotides n-1 and n-1 and n-2 or nucleotides n-1 and n-2 and n-3 of the guide RNA molecule; A6. Guide RNA molecule, according to any one of A1 to A5, comprising phosphorodithioate linkages between the n-1 nucleotides, the n-1 and n-2 nucleotides, and the n-2 and n-3 nucleotides of the guide RNA molecule; A7. Guide RNA molecule, according to any one of A1A6, comprising phosphorodithioate linkages between the n-1 nucleotides, the n-1 and n-2 nucleotides, the n-2 and n-3 nucleotides, and the n-3 and n-4 nucleotides of the guide RNA molecule; A8. Guide RNA molecule, according to any one of A1A7, in which the first nucleotide is at the 5' end of the guide RNA molecule. Petition 870250090770, dated 06 / 10 / 2025, pp. 259 / 369 83 / 111 comprises a selected modification of the group consisting of: (a) a nucleotide modified with 2'-fluoro; (b) a nucleotide modified with 2'-O-methyl; (c) a nucleotide modified with 2'-O-(2-methoxyethyl); (d) a blocked nucleic acid (LNA); (e) a deoxyribose nucleotide; and (f) a combination of two or more of (a)-(e); A9. Guide RNA molecule, according to A8, wherein the first nucleotide at the 5' end of the guide RNA molecule comprises a nucleotide modified with 2'-fluoro; A10. Guide RNA molecule, according to A8, in which the first nucleotide at the 5' end of the guide RNA molecule comprises a nucleotide modified with 2'-O-methyl; A11. Guide RNA molecule, according to A8, wherein the first nucleotide at the 5' end of the guide RNA molecule comprises a nucleotide modified with 2'-O-(2-methoxyethyl); A12. Guide RNA molecule, according to A8, wherein the first nucleotide at the 5' end of the guide RNA molecule comprises an LNA; A13. Guide RNA molecule, according to A8, wherein the first nucleotide at the 5' end of the guide RNA molecule comprises a deoxyribose nucleotide; A14. Guide RNA molecule, according to any one of A1 to A13, wherein (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 at the 5' end of the guide RNA molecule comprises a modification selected from the group consisting of: Petition 870250090770, dated 06 / 10 / 2025, pp. 260 / 369 84 / 111 (a) a nucleotide modified with 2'-fluoro; (b) a nucleotide modified with 2'-O-methyl; (c) a nucleotide modified with 2'-O-(2-methoxyethyl); (d) a blocked nucleic acid (LNA); (e) a deoxyribose nucleotide; and (f) a combination of two or more of (a)-(e); A15. Guide RNA molecule, according to any one of A1 to A9 and A14, wherein the guide RNA molecule comprises three nucleotides modified with consecutive 2'-fluoro in the first three nucleotides at the 5' terminus of the guide RNA molecule; A16. Guide RNA molecule, according to any one of A1 to A8, A10 and A14, wherein the guide RNA molecule comprises three consecutive nucleotides modified with 2'-O-methyl in the first three nucleotides at the 5' end of the guide RNA molecule; A17. Guide RNA molecule, according to any one of A1 to A8, A10 and A14, wherein the guide RNA molecule comprises four consecutive nucleotides modified with 2'-O-methyl on the first four nucleotides at the 5' end of the guide RNA molecule; A18. Guide RNA molecule, according to any one of A1 to A8, A11 and A14, wherein the guide RNA molecule comprises three consecutive nucleotides modified with 2'-O-(2-methoxyethyl) in the first three nucleotides at the 5' terminus of the guide RNA molecule; A19. Guide RNA molecule, according to any one of A1 to A8, A12 and A14, wherein the guide RNA molecule comprises three consecutive LNAs in the first three nucleotides at the 5' terminus of the guide RNA molecule; A20. Guide RNA molecule, according to any one of A1 to A8, A13 and A14, where the guide RNA molecule comprises three nucleotides. Petition 870250090770, dated 06 / 10 / 2025, pp. 261 / 369 85 / 111 consecutive deoxyribose groups in the first three nucleotides at the 5' end of the guide RNA molecule; A21. Guide RNA molecule, according to any one of A1 to A20, in which the 3' terminal nucleotide (n) of the guide RNA molecule comprises a modification selected from the group consisting of: (a) a nucleotide modified with 2'-fluoro; (b) a nucleotide modified with 2'-O-methyl; (c) a nucleotide modified with 2'-O-(2-methoxyethyl); (d) a blocked nucleic acid (LNA); (e) a deoxyribose nucleotide; and (f) a combination of two or more of (a)-(e); A22. Guide RNA molecule, according to A21, wherein the n nucleotide of the guide RNA molecule comprises a nucleotide modified with 2'-fluoro; A23. Guide RNA molecule, according to A21, wherein the n nucleotide of the guide RNA molecule comprises a nucleotide modified with 2'-O-methyl; A24. Guide RNA molecule, according to A21, wherein the n nucleotide of the guide RNA molecule comprises a nucleotide modified with 2'-O-(2-methoxyethyl); A25. Guide RNA molecule, according to A21, wherein the n nucleotide of the guide RNA molecule comprises an LNA; A26. Guide RNA molecule, according to A21, wherein the n nucleotide of the guide RNA molecule comprises a deoxyribose nucleotide; A27. Guide RNA molecule, according to any one of A1 to A26, wherein (i) nucleotide n-1, (ii) nucleotide n-2, (iii) nucleotide n-3, (iv) nucleotide n-4, (v) nucleotides ne n-1, (vi) nucleotides ne n-2, Petition 870250090770, dated 06 / 10 / 2025, pp. 262 / 369 86 / 111 (vii) the n-1 and n-2 nucleotides, (viii) the n, n-1 and n-2 nucleotides, (ix) the n-1, n-2 and n-3 nucleotides, (x) the n, n-1, n-2 and n-3 nucleotides, or (xi) the n, n-1, n-2, n-3 and n-4 nucleotides of the guide RNA molecule each comprise a modification selected from the group consisting of: (a) a nucleotide modified with 2'-fluoro; (b) a nucleotide modified with 2'-O-methyl; (c) a nucleotide modified with 2'-O-(2-methoxyethyl); (d) a blocked nucleic acid (LNA); (e) a deoxyribose nucleotide; and (f) a combination of two or more of (a)-(e); A28. Guide RNA molecule, according to any one of A1 to A22 and A27, wherein the nucleotides n and n-1, the nucleotides n and n-2, the nucleotides n-1 and n-2, the nucleotides n, n-1 and n-2, the nucleotides n-1, n-2, n-3 and n-4 or the nucleotides n, n-1, n-2, n-3 and n-4 of the guide RNA molecule are each nucleotides modified with 2'-fluoro; A29. Guide RNA molecule, according to any one of A1 to A21, A23 and A27, in which the nucleotides ne n-1, the nucleotides ne n-2, the nucleotides n-1 and n-2, the nucleotides n, n-1 and n-2, the nucleotides n-1, n-2, n3 and n-4 or the nucleotides n, n-1, n-2, n-3 and n-4 of the guide RNA molecule are each nucleotides modified with 2'-O-methyl; A30. Guide RNA molecule, according to any one of A1 to A21, A23, A27 and A29, where the n, n-1, n-2, n-3 and n-4 nucleotides of the guide RNA molecule are each modified nucleotides with 2'-O-methyl; A31. Guide RNA molecule, according to any one of A1 to A21, A24 and A27, wherein the nucleotides ne n-1, the nucleotides ne n-2, the nucleotides n-1 and n-2, the nucleotides n, n-1 and n-2, the nucleotides n-1, n-2, n3 and n-4 or the nucleotides n, n-1, n-2, n-3 and n-4 of the guide RNA molecule are each nucleotides modified with 2'-O-(2-methoxyethyl); Petition 870250090770, dated 06 / 10 / 2025, pp. 263 / 369 87 / 111 A32. Guide RNA molecule, according to any one of A1 to A21, A25 and A27, in which the nucleotides n and n-1, the nucleotides n and n-2, the nucleotides n-1 and n-2, the nucleotides n, n-1 and n-2, the nucleotides n-1, n-2, n-3 and n-4 or the nucleotides n, n-1, n-2, n-3 and n-4 of the guide RNA molecule are each LNAs; A33. Guide RNA molecule, according to any one of A1A21, A26 and A27, in which the nucleotides n and n-1, the nucleotides n and n-2, the nucleotides n-1 and n-2, the nucleotides n, n-1 and n-2, the nucleotides n-1, n-2, n-3 and n-4 or the nucleotides n, n-1, n-2, n-3 and n-4 of the guide RNA molecule are each deoxyribose nucleotides; B. The subject matter disclosed here provides a nucleic acid comprising a polynucleotide encoding a guide RNA molecule according to any one from A1 to A33; B1. Nucleic acid, according to B, further comprising a polynucleotide encoding an RNA-guided nuclease; B2. Nucleic acid, in accordance with B1, wherein the RNA-guided nuclease is a Cas protein; B3. Nucleic acid, according to B2, in which the Cas protein is selected from the group consisting of a Cas9, a Cas12, a Cas13 and a combination thereof; C. The subject matter disclosed here provides a vector comprising a nucleic acid, according to any one from B to B3; D. The subject matter disclosed here provides a composition comprising a guide RNA molecule, conforming to any one of A1 to A33; D1. Composition, according to D, which additionally comprises an RNA-guided nuclease; D2. Composition, according to D or D1, comprising Petition 870250090770, dated 06 / 10 / 2025, pp. 264 / 369 88 / 111 additionally a nucleic acid that encodes the RNA-guided nuclease; D3. Composition, according to D1 or D2, where the RNA-guided nuclease is a Cas protein; E. The subject matter disclosed here provides a composition comprising a nucleic acid, according to B to B3; F. The subject matter disclosed here provides a composition comprising a vector, according to C; G. The subject disclosed here provides a ribonucleoprotein (RNP) complex comprising a guide RNA molecule, corresponding to any one from A1 to A33, and an RNA-guided nuclease; G1. RNP complex, according to G, in which the RNA-guided nuclease is a Cas protein; G2. RNP complex, according to G1, in which the Cas protein is selected from the group consisting of a Cas9, a Cas12, a Cas13, and a combination thereof; H. The subject matter disclosed here provides a cell comprising a composition, according to any one of D to D3, E or F; I. The subject matter disclosed here provides a cell comprising an RNP complex, according to any of G1 to G2; J. The subject matter disclosed here provides a method for modifying a cell, comprising bringing the cell into contact with a composition, according to any one from D to D3, E or F, or an RNP complex, according to any one from G to G2; J1. Method, according to J, in which the aforementioned contact comprises the introduction of the composition into the cell by electroporation; K. The subject matter disclosed here provides a method for treating an individual in need of such treatment, which includes: (a) modifying an individual's cell ex vivo through contact Petition 870250090770, dated 06 / 10 / 2025, pp. 265 / 369 89 / 111 of the cell with a composition of any one from D to D3, E or F, or an RNP complex, according to any one from G to G2; and (b) return the modified cell to the individual; L. The subject matter disclosed here provides a gene-editing system that includes: (a) one or more guide RNA molecules, according to any one from A1 to A33; (b) one or more nucleic acids, according to any one from B to B3; (c) one or more vectors of C; (d) one or more compositions of D to D3, E or F; and / or (e) one or more RNP complexes, according to any one of G to G2. Examples

[00251] The subject discussed here will be better understood by reference to the following Example, which is provided as an illustration of the subject discussed here, and not as a form of limitation. Example 1: Modifications of gRNA Molecules

[00252] This Example provides gRNA molecules that have been chemically synthesized to include multiple modifications at the 5' and 3' ends of the gRNA molecules. As shown in Table 1, this Example provides AL modification strategies. Table 1 provides a summary of the modifications for each of these strategies compared to the reference gRNA molecule.

[00253] Nucleotide modifications are indicated in Table 1 as follows: *: phosphorothioate (PS) linkage; **: phosphorodithioate (PS2) linkage; m: 2'-OMe; ln: blocked nucleic acid (LNA); f: 2'-fluoro; M: 2'-O-MOE; ed: deoxyribonucleotide. For example, mA represents 2'-O-methyl adenosine. Petition 870250090770, dated 06 / 10 / 2025, pp. 266 / 369 90 / 111 and dA represents an adenosine deoxyribonucleotide. ON recited in the sequences of Table 1 represents any nucleotide base, for example, guanine (G), adenine (A), thymine (T), or cytosine (C). The reference gRNAs disclosed in Table 1, named Reference gRNA 1, Reference gRNA 2, and Reference gRNA 3, have the same nucleotide sequence as the modified gRNAs of the AL strategies. Reference gRNAs 1 and 3 have the same modification strategy shown in Table 1. Reference gRNA 2 has 3 phosphorothioate linkages at the 3' end and terminates with rU compared to Reference gRNAs 1 and 3. Table 1 gRNA Name 5' Terminal Modification Strategy 3' Terminal Modification Strategy Reference gRNA Sequence 1 • 2'-O-methyl modifications for the first 3 consecutive nucleotides • 3 phosphorothioate linkages • 2'-O-methyl modifications for the terminal 3 nucleotides • 2 phosphorothioate linkages 5'- mN*mN*mN*rNrNrNrNrNr NrNrNrNrN rNrNrNrNrNrNrNrGrUrUrU rUrArGrArGrCrUrArGrArA rArUrArGrCrArArGrUrUrAr ArArArUrArArG rGrCrUrAr GrUrCrCrGrUrUrArUrCrAr ArCrUrUrG rArArArArArArG r Reference RNA (mU) • 2'-O-methyl modifications for the first 3 consecutive nucleotides • 3 phosphorothioate bonds • 2'-O-methyl modifications for nucleotides n-1 and n-2 • 3 phosphorothioate bonds 5'- mN*mN*mN*rNrNrNrNrNr NrNrNrNrN rNrNrNrNrNrNrNrGrUrUrU rUrArGrArGrCrUrArGrArA rArUrArGrCrArArGrUrUrAr ArArArUrArArG rGrCrUrAr GrUrCrCrGrUrUrArUrCrAr ArCrUrUrG rArArArArArG r UrGrGrCrArCrCrGrArGrUr CrGrGrUrGrCrU*mU*mU* rU-3' Petition 870250090770, dated 06 / 10 / 2025, pp. 267 / 369 91 / 111 gRNA Name 5' Terminal Modification Strategy 3' Terminal Modification Strategy Reference gRNA Sequence 3 • 2'-O-methyl modifications for the first 3 consecutive nucleotides • 3 phosphorothioate linkages • 2'-O-methyl modifications for the terminal 3 nucleotides • 2 phosphorothioate linkages 5'- mN*mN*mN*rNrNrNrNrNr NrNrNrNrN rNrNrNrNrNrNrNrGrUrUrU rUrArGrArGrCrUrArGrArA rArUrArGrCrArArGrUrUrAr ArArArUrArArG rGrCrUrAr GrUrCrCrGrUrUrArUrCrAr ArCrUrUrG rArArArArArArG r UrGrGrCrArCrCrGrArGrUr CrGrGrUrGrCrUmU*mU* mU-3' A • Replace nucleotides modified with 2'O-methyl with LNAs • Replace nucleotides modified with 2'-O-methyl with LNAs 5'- lnN*lnN*lnN*rNrNrNrNrNr NrNrNrN rNrNrNrNrNrNrNrNrGrUrU rUrUrArGrArGrCrUrArGrA rArArUrArG rCrArArGrUrUr ArArArArUrArArG rGrCrUr ArGrUrCrCrGrUrUrArUrCr ArArCrUrUrGrArArArArArAr GrUrGrGrCrArCrCrGrArGr UrCrGrGrUrGrCrUlnU*lnU *lnU-3' B • Replace phosphorothioate bonds with phosphorodithioate bonds • Replace bondsphosphorothioate by phosphorodithioate linkages 5'- mN**mN**mN**rNrNrNrNr NrNrNrNr NrNrNrNrNrNrNrNrNrGrUr UrUrUrArGrArGrCrUrArGr ArArArUrArG rCrArArGrUr UrArArArArUrArArGrGrCr UrArGrUrCrCrGrUrUrArUr CrArArCrUrUrGrArArArArAr ArGrUrGrGrCrArCrCrGrAr GrUrCrGrGrUrGrCrUmU** mU**mU-3' C • Replace the nucleotides modified with 2'O-methyl by nucleotides modified with 2'O-(2-methoxyethyl) • Replace the nucleotides modified with 2'-O-methyl by nucleotides modified with 2'-O(2-methoxyethyl) 5'- MN*MN*MN*rNrNrNrNrNr NrNrNrNr NrNrNrNrNrNrNrNrGrUrUr UrUrArGrArGrCrUrArGrAr ArArUrArG rCrArArGrUrUr ArArArArUrArArG rGrCrUr ArGrUrCrCrGrUrUrArUrCr ArArCrUrUrGrArArArArArAr GrUrGrGrCrArCrCrGrArGr UrCrGrUrGrCrUMU*MU *MU-3' Petition 870250090770, dated 06 / 10 / 2025, pp. 268 / 369 92 / 111 gRNA Name 5'-Terminal Modification Strategy 3'-Terminal Modification Strategy D Sequence • Replace nucleotides modified with 2'O-methyl with nucleotides modified with 2'fluoro • Replace nucleotides modified with 2'O-methyl with nucleotides modified with 5'fluoro fN*fN*fN*rNrNrNrNrNrNrN rNrNrNrNrNrNrGrUrUrUrUr ArGrArGrCrUrArGrArArArAr UrArGrCrArArGrUrUrArArAr ArArUrArArGrGrCrUrArGr UrCrCrGrUrUrArUrCrArArAr CrUrUrG rArArArArArArG rUr GrGrCrArCrCrGrArGrUrCr GrGrUrGrCrUfU*fU*fU-3' E • No change in reference gRNA 1 • No change in reference gRNA 1 5'mN*mN*mN*fNfNfNfNfNf NfNfNfNfN fNfNfNfNfNfNfNfGfUfUfUf UfAfGfAfGfCfUfAfGfAfAfA fUfAfGfCfAfAfGfUfUfAfAfA fAfUfAfAfGfGfCfUfAfGfUf CfCfGfUfUfAfUfCfAfAfCfUfGfAfAfAfAfGfUfGfGf CfAfCfCfGfAfGfUfCfGfGf UfGfCfUmU*mU*mU-3' F • Replace nucleotides modified with 2'O-methyl with deoxyribose nucleotides • Replace phosphorothioate linkages with phosphorodithioate linkages • ReplaceModified nucleotides with 2'-O-methyl by deoxyribose nucleotides • Replace phosphorothioate linkages with 5'-phosphorodithioate linkages dN**dN**dN**rNrNrNrNrNr NrNrNrNr NrNrNrNrNrNrNrNrGrUrUr UrUrArGrArGrCrUrArGrAr ArArUrArG rCrArArGrUrUr ArArArArUrArArG rGrCrUr ArGrUrCrCrGrUrUrArUrCr ArArCrUrUrGrArArArArArArGrUrGrGrGrCrGrArCrGrArGr UrCrGrUrGrUdU**dU **dU-3' G • Remove phosphorothioate linkages • Remove linkages phosphorothioate 5'- mNmNmNrNrNrNrNrNrNr NrNrNrNr NrNrNrNrNrNrNrGrUrUrUr UrArGrArGrCrUrArGrArAr ArUrArGrCrArArGrUrUrAr ArArArUrArArG rGrCrUrAr GrUrCrCrGrUrUrArUrCrAr ArCrUrUrG rArArArArArG r UrGrGrCrArCrCrGrArGrUr CrGrGrUrGrCrUmUmU mU-3' Petition 870250090770, dated 06 / 10 / 2025, pp. 269 / 369 93 / 111 gRNA Name 5' Terminal Modification Strategy 3' Terminal Modification Strategy H Sequence • No change to reference gRNA 1 • No change to reference gRNA 1 5'- mN*mN*mN*rNrNrNrNrNr NrNrNrNr NrNrNrNrNrNrNrNrGrU*rU *rU*rU*rA*rG*rArGrCrUrAr GrArArArUrArGrCrArArG* rU*rU*rArA*rArA*rUrA*rAr GrGrCrUrArG*rU*rCrCrG* rU*rU*rA*rUrCrA*rA*rCrUr UrGrArArArArArGrUrGrG* rCrArCrCrGrArGrUrCrGrG rUrGrCrUmU*mU*mU-3' I • No change in reference gRNA 1 • No change in reference gRNA 1 5'- mN*mN*mN*dNdNdNdNd NdNdNdN dNdNdNdNdNdNdNdNdN rGrUrUrUrUrArGrArGrCrU rArGrArArArUrArGrCrArAr GrUrUrArArArArUrArArG r GrCrUrArGrUrCrCrGrUrUr ArUrCrArArCrUrUrGrArArArArGrUrGrGrCrArCrCr GrArGrUrCrGrGrUrGrUrUr mU*mU*mU-3' J • Remove modifications 2'-Omethyl • Remove modifications 2'-O-methyl 5'- rN*rN*rN*rNrNrNrNrNrNrN rNrNrNrN rNrNrNrNrNrNrGrUrUrUrU rArGrArGrCrUrArGrArArArAr UrArGrCrArArGrUrUrArArAr ArArUrArArArGrGrCrUrArGr UrCrCrGrUrUrArUrCrArArAr CrUrUrG rArArArArArArGrUr GrGrCrArCrCrGrArGrUrCr GrGrUrGrCrUrU*rU*rU-3' K • Replace modified nucleotides with 2'O-methyl with deoxyribose nucleotides • 3 phosphorodithioate linkages • No alteration in the reference gRNA 1 5'- dN**dN**dN**rNrNrNrNrNr NrNrNrN rNrNrNrNrNrNrNrNrGrUrU rUrUrArGrArGrCrUrArGrA rArArUrArG rCrArArGrUrUr ArArArArUrArArG rGrCrUr ArGrUrCrCrGrUrUrArUrCrArArCrUrUrGrArArArArArAr GrUrGrGrCrArCrCrGrArGr UrCrGrGrUrGrCrUmU*mU *mU-3' Petition 870250090770, dated 06 / 10 / 2025, pp. 270 / 369 94 / 111 gRNA Name 5' Terminal Modification Strategy 3' Terminal Modification Strategy L Sequence • Remove all modifications • No changes to the reference gRNA 1 5'- rNrNrNrNrNrNrNrNrNrNrN rNrNrNrN rNrNrNrNrNrGrUrUrUrUrA rGrArGrCrUrArGrArArArU rArGrCrArArGrUrUrArArArAr ArUrArArGrGrCrUrArGrUr CrCrGrUrUrArUrCrArArCr UrUrG rArArArArArG rUrGr GrCrArCrCrGrArGrUrCrGr GrUrGrCrUmU*mU*mU-3'

[00254] The identity of the synthesized modified gRNAs was confirmed by mass spectrometry with a mass error of less than 200 ppm between the experimental and theoretical masses. As shown in Tables 2 and 3, the experimental masses were consistent with the calculated masses, confirming the correct length and composition of the nucleobases.

[00255] The purity of the synthesized modified gRNAs was analyzed. As shown in Figures 2 and 3, similar purity profiles were observed for gRNAs with modifications according to the AF modification schemes. 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 a gRNA targeting Reference Target 1 and modified according to the AL modification schemes, and Figures 3 and 5 and Table 3 provide the purity profiles of a gRNA targeting Reference Target 2 and modified according to the AL modification schemes. As shown in Figures 2 and 3, the gRNA sequence has a negligible effect on the purity profiles and ion-pairing reverse-phase liquid chromatography (IPRP) of gRNAs modified with modifications according to the AF modification schemes.Similar results were observed for gRNAs that target these two target sequences and with modifications according to the GL modification schemes (FIGURES 4 to 5 and Tables 2 and 3). Petition 870250090770, dated 06 / 10 / 2025, pp. 271 / 369 95 / 111 3) In addition, it was observed that the impurities were mainly shortmers.

[00256] As observed in FIGURE 4, the 2'-fluoro modification significantly increased the hydrophobicity of the sgRNA. Without being tied to a particular theory, increased hydrophobicity may allow an oligonucleotide to bind to interact with certain domains of a protein and modulate its activity (see Crooke et al., Nucleic Acids Research 48(10):5235-5253 (2020), the contents of which are incorporated herein by reference in their entirety). Table 2 gRNA Modification Strategy Targeting Reference Target 1 Purity (% w / w) Theoretical Mass (Da) Average Experimental Mass (Da) Difference in Mass (Da) A 98.7 32428.7 32426.8 -1.9 B 95.8 32465.0 32464.0 -1.0 C 99.5 32705.1 32705.0 -0.1 D 98.7 32312.5 32312.5 0.0 E 80.1 32571.9 32572.0 0.1 F 98.5 32327.0 32326.4 -0.6 G 85.3 32304.3 32304.5 0.2 H 54.2 32722.0 32720.4 -1.6 Table 3 gRNA modification strategy targeting Reference Target 2 Purity (% w / w) Theoretical Mass (Da) Average Experimental Mass (Da) Difference in Mass (Da) A 98.8 32379.7 32374.3 -5.4 B 98.6 32416.0 32415.5 -0.5 C 97.9 32656.1 32656.4 0.3 D 99.0 32263.5 32263.0 -0.5 E 87.9 32522.8 32523.0 0.2 F 98.2 32292.0 32291.5 -0.5 G 79.8 32255.3 32255.0 -0.3 H 88.0 32673.0 32673.0 0.0 I 87.6 32063.6 32063.6 0.0 Petition 870250090770, dated 06 / 10 / 2025, pp. 272 / 369 96 / 111 gRNA modification strategy targeting Reference Target 2 Purity (% w / w) Theoretical Mass (Da) Average Experimental Mass (Da) Difference in Mass (Da) J 73.7 32251.5 32251.6 0.1 K 85.1 32307.8 32307.6 -0.2 L 79.1 32245.4 32245.5 0.1

[00257] The efficacy of the gRNA molecules described above targeting Reference Target 1 and modified as shown in Table 1 was analyzed as follows. Cells were seeded in Prime-XV (IL7 (25 ng / ml), IL-15 (50 ng / ml), TransAct 1:100) (= Day 0). 4 mg of each sgRNA were resuspended in 611-624 μM (depending on MW) of IDT Duplex buffer (Catalog #1072570) to obtain a 200 μM solution. RNPs were generated using a 3:1 sgRNA:Cas9 ratio. For knockout conditions, 1, 11, 3, 33, 10 and 30 pmols of RNP were titrated. For knock-in conditions, 30 pmols of RNP were used with the addition of 2 μg of a 1-mNeon nanoplasmid from the Reference Target. The components were added in the following order: 1 - RNP (sgRNA and Cas9 pre-incubated for 15 min in TA first), 2 - Template (3 μg), and 3 - Cells (2 million). The cells were electroporated 46 hours post-stimulation in P3 buffer on the Lonza 4D electroporator.After electroporation, the cells were incubated at 37°C for 15 min. 75 μL of plain Prime-XV were added, and the cells were transferred to 1 ml of complete medium without TransAct in 48-well plates. 3 ml of complete medium were added on day 5, followed by transfer of the cells to a 12-well plate. 0.5 ml of the culture was removed for FAC reading on day 5 (KO samples only). 0.5 ml of the culture was removed for FAC reading on day 6 (KI samples only). The cells were subsequently stained on days 5 and 6 post-stimulation to detect the reduction or increase in the expression of Reference Target 1.

[00258] The editing efficiencies of modified gRNAs Petition 870250090770, dated 06 / 10 / 2025, pp. 273 / 369 The 97 / 111 shown in FIGURES 6-12 are summarized in Tables 4 and 5. As shown in FIGURES 9 and 11 and Table 4, gRNA B performed very similarly to the reference gRNA under knockout and knockout conditions. gRNA D outperformed gRNA C, and gRNA C outperformed gRNA A. gRNAs E and F showed very little activity compared to the reference gRNAs under knockout and knockout conditions.

[00259] As shown in FIGURES 6-8, 10 and 12 and in Table 5, gRNAs G, J and L exhibited a moderate level of knockout activity compared to the reference gRNAs, but showed knockout activity similar to the two reference gRNAs. gRNAs H and I did not result in knockout or knockin activity. Furthermore, gRNA K produced a very small amount of knockout or knockin activity compared to the reference gRNAs.

[00260] Cell viability under knockout and knockin conditions in the presence of the modified gRNAs was tested. As shown in FIGURES 13 to 16, cell viability was very similar for all modified gRNAs tested under knockout (FIGURES 13-14) and knockin (FIGURES 15-16) conditions. Similarly, the total number of cells was very similar for most gRNAs tested under knockout (FIGURES 13-14) and knockin (FIGURES 15-16) conditions. However, for the knockin experiment, more total cells were measured for gRNA E and gRNA F, which may be due to the failure of these gRNAs to efficiently cleave the target nucleic acid (FIGURE 15). Table 4 gRNA Name Average KO Activity (10 pmols) Average KI Activity (30 pmols) Reference gRNA 2 89% 50% A 58% 29% B 91% 47% C 80% 35% Petition 870250090770, dated 06 / 10 / 2025, pp. 274 / 369 98 / 111 gRNA Name Average KO Activity (10 pmols) Average KI Activity (30 pmols) D 83% 43% E 2% 0% F 4% 1% Table 5 gRNA Name Average KO Activity (10 pmols) Average KI Activity (30 pmols) Reference gRNA 2 77% 37% Reference gRNA 3 70% 37% G 52% 39% H 11% 0% I 6% 0% J 46% 34% K 8% 1% L 38% 34%

[00261] As summarized in Tables 4 and 5, gRNAs “B”, “C”, and “D” exhibited the highest editing effectiveness compared to the other gRNAs tested and were most comparable to the control. Example 2: Forced Degradation of Modified gRNA Molecules

[00262] This example shows the degradation of the exemplary gRNA molecules described in Example 1 under forced conditions. The gRNA molecules from Example 1 were subjected to forced degradation conditions, including acid stress (pH 5), basic stress (pH 11), oxidative stress, and thermal stress. Forced Degradation Conditions:

[00263] Acid Stress (pH 5): Acid buffer: 20 mM sodium acetate, pH 5.0. Temperature: 40°C. Time Points: 0, 1, 2, and 3 days (gRNAs “A” to “L”); 0, 1, 2, 3, 5, and 7 days (gRNAs “M” to “V”).

[00264] 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”).

[00265] Oxidative Stress: 0.3% H2O2. Temperature: 40oC. Petition 870250090770, dated 06 / 10 / 2025, pp. 275 / 369 99 / 111 Time Points: 0, 1, 2, 3 and 5 days (gRNAs “A” to “V”).

[00266] Thermal Stress: Tris-EDTA (TE) buffer, pH 8.0. Temperature: 40°C. Time Points: 0, 1, 3, 5 and 7 days (gRNAs “A” to “V”). Sample Preparation Methods:

[00267] Acid Stress Analysis (pH 5): To prepare the acid stress solution (pH 5), 164 mg of sodium acetate were added to a 100 ml volumetric flask. 80 ml of LC grade water were added to the flask and the pH was adjusted to 5.0 with acetic acid. The solution was made up to volume with water. 10 µl of sgRNA were pipetted into an HPLC insert and 55 µl of acid stress solution (pH 5) were added followed by gentle shaking for 5 s.

[00268] Basic Stress Analysis (pH 11): To prepare the basic stress solution (pH 11), 212 mg of sodium carbonate were weighed and placed in a 100 ml volumetric flask. Approximately 80 ml of LC-grade water were added to the flask to dissolve the sodium carbonate, and the pH was adjusted to 11.0 with 1N HCl. The solution was made up to volume with water. 10 µl of sgRNA were pipetted into an HPLC insert, and 55 µl of basic stress solution (pH 11) were added, followed by gentle stirring for 5 s.

[00269] Thermal (liquid) analysis: 10 µl of sgRNA were pipetted into an HPLC insert and 55 µl of TE buffer (pH 8) were added, followed by gentle stirring for 5s.

[00270] Oxidation Analysis: To prepare the oxidative solution, 0.59 ml of 3% H2O2 was added to a 5 ml volumetric flask and the volume of TE buffer was made up to the mark, followed by mixing. 10 µl of sgRNA was pipetted into an HPLC insert and 55 µl of 0.35% H2O2 was added, followed by gentle shaking for 5 s.

[00271] HPLC: HPLC was performed as shown in Table 6. Petition 870250090770, dated 06 / 10 / 2025, pp. 276 / 369 100 / 111 Table 6 Parameters Condition Sampler temperature 40°C Column DNAPac RP, 4 pm, 2.1 χ 100 mm Flow rate 0.40 ml / min Column temperature 70°C Mobile Phase A: 400 mM HFIP + 16.3 mM TEA in water B: 40% MPA + 60% Methanol Injection volume 2 pl UV wavelength 260 nm Gradient (min) MPA: MPB: 0 90 10 20 40 60 21 0 100 23 0 100 23.5 90 10 30 90 10

[00272] FIGURE 17 shows the elution profiles of the modified gRNAs under normal conditions. As shown in FIGURE 17, the gRNAs with the AL modification strategies had an initial purity ranging from 47% to 92% at Time Point 0 (To). The results of the forced degradation studies of these modified gRNAs are shown in Tables 7 to 9 and FIGURES 18 to 40. Table 7. Purity Summary under Forced Degradation Name of gRNA Purity (% in area) T(0) 0.3% H2O2, 40oC, T(5d) pH 8, 40oC, T(7d) pH 5, 40oC, T(3d) pH 11, 40oC, T(1d) reference gRNA 3 91.4 91.2 86.0 81.9 11.2 A 66.6 63.7 62.8 61.0 11.8 B 89.3 78.3 82.2 81.6 23.0 C 64.6 22.4 61.0 59.9 16.6 D 93.4 85.0 88.9 85.1 75.9 E 67.7 61.0 - 66.5 67.0 F 63.5 61.3 60.6 59.8 52.6 Petition 870250090770, dated 06 / 10 / 2025, pp. 277 / 369 101 / 111 Name of gRNA Purity (% in area) T(0) 0.3% H2O2, 40oC, T(5d) pH 8, 40oC, T(7d) pH 5, 40oC, T(3d) pH 11, 40oC, T(1d) G 65.3 59.4 59.5 60.2 0.8 H 52.3 3.9 47.7 49.6 9.9 I 66.9 64.4 63.8 64.2 9.3 J 56.3 55.2 52.3 52.9 38.2 K 47.4 45.8 42.6 44.5 11.7 L 47.8 47.7 45.5 45.5 7.0

[00273] FIGURE 18 shows the chromatograms of reference gRNA 3 under basic stress (pH 11), acid stress (pH 5), and oxidative stress (0.3% H2O2). FIGURE 19 shows an overlay of the chromatograms of reference gRNA 3 under basic stress (pH 11), acid stress (pH 5), and oxidative stress (0.3% H2O2) over specific time periods. As shown in FIGURES 18 and 19, reference gRNA 3 was more resistant to acid hydrolysis (i.e., resists degradation by exposure to acid) compared to basic stress (pH 11), which led to the formation of shortmers due to rapid degradation.

[00274] FIGURE 20 shows the chromatograms of gRNAs A, B, and C under acid stress (pH 5). FIGURE 21 shows the chromatograms of modified gRNAs D, E, and F under acid stress (pH 5). FIGURE 22 shows the chromatograms of modified gRNAs G, H, and I under acid stress (pH 5). FIGURE 23 shows the chromatograms of gRNAs J, K, and L under acid stress (pH 5). As shown in FIGURES 20 to 23, gRNAs E, F, H, I, and J were more resistant to acid stress.

[00275] FIGURE 24 shows the chromatograms of gRNAs A, B, and C under basic stress (pH 11). FIGURE 25 shows the chromatograms of modified gRNAs D, E, and F under basic stress (pH 11). FIGURE 26 shows the chromatograms of modified gRNAs G, H, and I under basic stress (pH 11). Petition 870250090770, dated 06 / 10 / 2025, pp. 278 / 369 102 / 111 FIGURE 27 shows the chromatograms of the modified gRNAs J, K, and L under basic stress (pH 11). As shown in FIGURES 24 to 27, the modified gRNAs D, E, F, H, I, J, and K were resistant to basic stress (pH 11).

[00276] FIGURE 28 shows the chromatograms of gRNAs A, B, and C under thermal stress. FIGURE 29 shows the chromatograms of gRNAs D, F, and G modified under thermal stress. FIGURE 30 shows the chromatograms of gRNAs H, I, and J modified under thermal stress. FIGURE 31 shows the chromatograms of gRNAs K and L modified under thermal stress. As shown in FIGURES 28 to 31, most gRNAs were resistant to thermal degradation.

[00277] FIGURE 32 shows the chromatograms of gRNAs A, B, and C modified under oxidative stress (0.3% H2O2). FIGURE 33 shows the chromatograms of gRNAs D, E, and F modified under oxidative stress (0.3% H2O2). FIGURE 34 shows the chromatograms of gRNAs G, H, and I modified under oxidative stress (0.3% H2O2). FIGURE 35 shows the chromatograms of gRNAs J, K, and L modified under oxidative stress (0.3% H2O2). FIGURE 36 shows an overlay of the chromatograms of AL gRNAs modified under oxidative stress (0.3% H2O2) over a period of 5 days. As shown in Figures 32 to 36, gRNAs E, G, I, J, K, and L were more resistant to oxidative stress, except for gRNAs C and H.

[00278] FIGURE 37 shows the changes in purity over a 3-day period during acid stress (pH 5). FIGURE 38 shows the changes in purity over a 24-hour period during basic stress (pH 11). FIGURE 39 shows the changes in purity over a 7-day period during thermal stress. As shown in FIGURE 39, there was not enough gRNA E available for the thermal stress study. FIGURE 40 shows the changes in purity over a Petition 870250090770, dated 06 / 10 / 2025, pp. 279 / 369 103 / 111 time period of 5 days under oxidative stress (0.3% H2O2).

[00279] As shown in Table 7, many of the modified gRNAs resisted degradation under forced degradation conditions. For example, gRNAs D, E, F, and J resisted degradation conditions, including basic stress (pH 11), compared to the reference gRNA (Table 7).

[00280] Tables 8 and 9 provide a summary of the changes in purity of the modified gRNAs under acid hydrolysis (Table 8) and under thermal stress (Table 9). The slope shows the decrease in purity per day, the intercept shows the number of days in which the purity decreases to 0, and R is the correlation coefficient of the linearity curve. Table 8 gRNA Name | Linear Function | Correction Coefficient R | Reference gRNA 3 | y = -3.165x + 91.6 | 0.998 | A | y = -1.692x + 65.588 | 0.967 | B | y = -2.708x + 89.262 | 0.986 | C | y = -1.376x + 64.214 | 0.962 | D | y = -2.702x + 93.073 | 0.998 | E | y = -0.125x + 66.970 | 0.965 | F | y = -1.416x + 63.684 | 0.970 | G | y = -1.828x + 64.972 | 0.951 | H | y = -0.65x + 51.575 | 0.990 | I | y = -0.533x + 66.437 0.986 J y = -0.450x + 55.455 0.966 K y = -0.754x + 46.876 0.964 L y = -1.335x + 49.32 0.986 Table 9 gRNA Name | Linear Function | Correction Coefficient R | Reference gRNA 3 | y = -0.7998x + 91.763 | 0.990 Petition 870250090770, dated 06 / 10 / 2025, pp. 280 / 369 104 / 111 gRNA Name Linear Function Correction Coefficient RA y = -0.5379x + 66.753 0.991 B y = -1.0456x + 89.982 0.979 C y = -0.4806x + 64.432 0.990 D y = -0.6165x + 93.653 0.978 F y = -0.4057x + 63.624 0.987 G y = -0.6451x + 65.244 0.988 H y = ​​-0.6665x + 52.715 0.981 I y = -0.4537x + 67.004 0.998 J y = -0.5459x + 56.121 0.996 K y = -0.6445x + 47.372 0.980 L y = -0.2826x + 47.982 0.963 Example 3: Modifications of gRNA Molecules

[00281] This example provides additional gRNA molecules that have been chemically synthesized to include multiple modifications at the 5' and 3' ends of the gRNA molecules. As shown in Table 10, this example provides the B and MV modification strategies. The B modification strategy provided in Table 10 is the same strategy as the B modification strategy shown in Example 1 and Table 1. Table 10 provides a summary of the modifications for each of these strategies, and Table 11 provides a comparison of the modifications for each of these strategies with the reference gRNA 3.

[00282] Nucleotide modifications are indicated in Table 10 as follows: *: phosphorothioate (PS) linkage; **: phosphorodithioate (PS2) linkage; m: 2'-OMe; ln: blocked nucleic acid (LNA); f: 2'-fluoro; M: 2'-O-MOE; ed: deoxyribonucleotide. ON recited in the sequences of Table 10 represents any nucleotide base, for example, guanine (G), adenine Petition 870250090770, dated 06 / 10 / 2025, pp. 281 / 369 105 / 111 (A), thymine (T) or cytosine (C). Table 10 gRNA Name 5' Terminal Modification Strategy 3' Terminal Modification Strategy Sequence B • 2-O-methyl modifications for the first 3 consecutive nucleotides • 3 phosphorodithioate linkages • 2'-O-methyl modifications for the 3 terminal nucleotides • 2 phosphorodithioate linkages (between n-1 and n-2) 5'- mN**mN**mN**rNrNrNrNr NrNrNrNrNrNrNrNrNrNrNrNr NrNrGrUrUrUrUrArGrArGr CrUrArGrArArArUrArGrCr ArArG rUrUrArArArArUrArA rGrGrCrUrArGrUrCrCrGrU rUrArUrCrArArCrUrUrGrAr ArArArArGrUrGrGrCrArCr CrGrArGrUrCrGrGrUrGrCr UmU**mU**mU-3' M • Modifications with 2'-O-methyl for the first 3 consecutive nucleotides • 3 phosphorodithioate linkages • 3 deoxyribose nucleotides for the 3 terminal nucleotides • 2 phosphorodithioate linkages (between n and n-1 and between n-1 and n-2) 5'- mN**mN**mN**rNrNrNrNr NrNrNrNrNrNrNrNrNrNrNrNr NrNrGrUrUrUrUrArGrArGr CrUrArGrArArArUrArGrCr ArArGrUrUrArArArArUrArA rGrGrCrUrArGrUrCrCrGrU rUrArUrCrArArCrUrUrGrAr ArArArArGrUrGrGrCrArCrCrGrArGrUrCrGrGrUrGrCr UdU**dU**dU-3' N • Deoxyribose nucleotides for the first 3 consecutive nucleotides • 3 phosphorothioate linkages • 2'-O-methyl modifications for the 3 terminal nucleotides • 2 phosphorothioate linkages (between n and n-1 and between n-1 and n-2) 5'- dN**dN**dN**rNrNrNrNrNr NrNrNrNrNrNrNrNrNrNrNrNr NrGrUrUrUrUrArGrArGrCr UrArGrArArArUrArGrCrArA rGrUrUrArArArArUrArArG r GrCrUrArGrUrCrCrGrUrUr ArUrCrArArCrUrUrGrArArA rArArGrUrGrGrCrArCrCrGr ArGrUrCrGrGrUrGrCrUmU *mU*mU-3' O • Modifications with 2'-O-methyl for the first 3 consecutive nucleotides • 3 phosphorothioate linkages • 3 deoxyribose nucleotides for the 3 terminal nucleotides • 2 phosphorodithioate linkages (between n and n-1 and between n-1 and n-2) 5'- mN*mN*mN*rNrNrNrNrNr NrNrNrNrNrNrNrNrNrNrNr NrGrUrUrUrUrArGrArGrCr UrArGrArArArUrArGrCrArA rGrUrUrArArArArUrArArG r GrCrUrArGrUrCrCrGrUrUr ArUrCrArArCrUrUrGrArArA rArArGrUrGrGrCrArCrCrGr ArGrUrCrGrGrUrGrCrUdU* *dU**dU-3' Petition 870250090770, dated 06 / 10 / 2025, pp. 282 / 369 106 / 111 gRNA Name 5' Terminal Modification Strategy 3' Terminal Modification Strategy P Sequence • 2'-O-methyl modifications for the first 3 consecutive nucleotides • 3 phosphorothioate linkages • 2'-O-methyl modifications for the 3 terminal nucleotides • 2 phosphorothioate linkages (between n-1 and n-2) 5'- mN**mN**mN**rNrNrNrNr NrNrNrNrNrNrNrNrNrNrNrNr NrNrGrUrUrUrUrArGrArGr CrUrArGrArArArUrArGrCr ArArGrUrUrArArArArUrArA rGrGrCrUrArGrUrCrCrGrU rUrArUrCrArArCrUrUrGrAr ArArArArGrUrGrGrCrArCr CrGrArGrUrCrGrGrUrGrCr UmU*mU*mU-3' Q • 2'-O-methyl modifications for the first 3 consecutive nucleotides • 3 phosphorothioate linkages • 2'-O-methyl modifications for the 3 terminal nucleotides • 2 phosphorothioate linkages (between n and n-1 and between n-1 and n-2) 5'- mN*mN*mN*rNrNrNrNrNr NrNrNrNrNrNrNrNrNrNrNr NrGrUrUrUrUrArGrArGrCr UrArGrArArArUrArGrCrArA rGrUrUrArArArArUrArArG r GrCrUrArGrUrCrCrGrUrUr ArUrCrArArCrUrUrGrArArA rArArGrUrGrGrCrArCrCrGrArGrUrCrGrGrUrGrCrUmU **mU**mU-3' R • 2'-O-methyl modifications for the first 3 consecutive nucleotides • 1 phosphorothioate linkage between the first and second nucleotides • 2 phosphorodithioate linkages (between the second and third and the third and fourth) • 2'-O-methyl modifications for the 3 terminal nucleotides • 2 phosphorodithioate linkages (between n-1 and n-2) 5'- mN*mN**mN**rNrNrNrNrN NrNrNrNrNrNrNrNrNrNrNr NrGrUrUrUrUrArGrArGrCr UrArGrArArArUrArGrCrArA rGrUrUrArArArArUrArArG r GrCrUrArGrUrCrCrGrUrUr ArUrCrArArCrUrUrGrArArA rArArGrUrGrGrCrArCrCrGr ArGrUrCrGrGrUrGrCrUmU **mU**mU-3' S • 2'-O-methyl modifications for the first 3 consecutive nucleotides • 2 phosphorodithioate linkages (between the first and second and between the second and third) • 1 phosphorodithioate linkage between the third and fourth nucleotides • 2'-O-methyl modifications for the 3 terminal nucleotides • 2 phosphorodithioate linkages (between n-1 and n-2) 5'-mN**mN**mN*rNrNrNrNrNrNr NrNrNrNrNrNrNrNrNrNrNrGrU rUrUrUrArGrArGrCrUrArGrAr ArArUrArGrCrArArGrUrUrArAr ArArUrArArGrGrCrUrArGrUrC rCrGrUrUrArUrCrArArCrUrUr GrArArArArArGrUrGrGrCrArC rCrGrArGrUrCrGrGrUrGrCrU mU**mU**mU-3' Petition 870250090770, dated 06 / 10 / 2025, pp. 283 / 369 107 / 111 gRNA Name 5' Terminal Modification Strategy 3' Terminal Modification Strategy T Sequence • 2'-O-methyl modifications for the first 3 consecutive nucleotides • 3 phosphorodithioate linkages • 2'-O-methyl modifications for the 3 terminal nucleotides • 1 phosphorodithioate linkage (between n and n-1) • 1 phosphorodithioate linkage (between n-1 and n-2) 5'- mN**mN**mN**rNrNrNrNr NrNrNrNrNrNrNrNrNrNrNrNr NrNrGrUrUrUrUrArGrArGr CrUrArGrArArArUrArGrCr ArArGrUrUrArArArArUrArA rGrGrCrUrArGrUrCrCrGrU rUrArUrCrArArCrUrUrGrAr ArArArArGrUrGrGrCrArCr CrGrArGrUrCrGrGrUrGrCr UmU*mU**mU-3' U • 2'-O-methyl modifications for the first 4 consecutive nucleotides • 4 phosphorodithioate linkages • 2'-O-methyl modifications for the 5 terminal nucleotides • 4 phosphorodithioate linkages (between n and n-1, between n-1 and n-2,between n-2 and n-3 and between n-3 and n-4) 5'- mN**mN**mN**mN**rNrNr NrNrNrNrNrNrNrNrNrNrNr NrNrNrGrUrUrUrUrArGrAr GrCrUrArG rArArArUrArG r CrArArG rUrUrArArArArUrA rArGrGrCrUrArGrUrCrCrGr UrUrArUrCrArArCrUrUrGr ArArArArArArGrUrGrGrCrAr CrCrGrArGrUrCrGrGrUrG mC**mU**mU**mU**mU-3' V • Modifications with 2'-O-methyl for the first 3 consecutive nucleotides • 3 phosphorodithioate linkages • modifications with 2'-O-methyl for n-1, n-2, n-3 and n-4. • phosphorodithioate bonds (between n and n-1, between n-1 and n-2 and between n-2 and n-3) 5'- mN**mN**mN**rNrNrNrNr NrNrNrNrNrNrNrNrNrNrNr NrNrGrUrUrUrUrArGrArGr CrUrArGrArArArUrArGrCr ArArG rUrUrArArArArUrArA rGrGrCrUrArGrUrCrCrGrU rUrArUrCrArArCrUrUrGrAr ArArArArGrUrGrGrCrArCr CrGrArGrUrCrGrGrUrGrC mU**mU**mU**U-3', Table 11 gRNA Name | 5'-Terminal Modification Strategy Compared to Reference gRNA 3 | 3'-Terminal Modification Strategy Compared to Reference gRNA 3 | B • Replace phosphorothioate bonds with phosphorodithioate bonds • Replace phosphorothioate bonds with phosphorodithioate bonds | M • Replace phosphorothioate bonds with phosphorodithioate bonds • Replace phosphorothioate bonds with phosphorodithioate bonds • Replace 2'-O-methyl modified nucleotides with deoxyribose nucleotides Petition 870250090770, dated 06 / 10 / 2025, pp. 284 / 369 108 / 111 gRNA Name | 5'-Terminal Modification Strategy Compared to Reference gRNA | 3 | 3'-Terminal Modification Strategy Compared to Reference gRNA | 3 | N • Replace phosphorothioate linkages with phosphorodithioate linkages • Replace nucleotides modified with 2'-O-methyl with deoxyribose nucleotides • No changes | O • No changes | Replace phosphorothioate linkages with phosphorodithioate linkages • Replace nucleotides modified with 2'-O-methyl with deoxyribose nucleotides | P • Replace phosphorothioate linkages with phosphorodithioate linkages • No changes | Q • No changes | Replace phosphorothioate linkages with phosphorodithioate linkages | R • Replace phosphorothioate linkages between the second and third and the third and fourth with phosphorodithioate linkages • Replace phosphorothioate linkages with phosphorodithioate linkages | S • Replace linkages phosphorothioate bonds between the first and second, and between the second and third, through phosphorodithioate bonds.Replace phosphorothioate linkages with phosphorodithioate linkages T • Replace phosphorothioate linkages with phosphorodithioate linkages • Replace phosphorothioate linkage between n and n-1 with a phosphorodithioate linkage U • Replace phosphorothioate linkages with phosphorodithioate linkages and add an additional phosphorodithioate linkage (the first four nucleotides have phosphorodithioate linkages) • Add two additional 2'-O-methyl modified nucleotides (at n-3 and n-4) • Replace phosphorothioate linkages with phosphorodithioate linkages and add two additional phosphorodithioate linkages (the last four nucleotides have phosphorodithioate linkages) V • Replace phosphorothioate linkages with phosphorodithioate linkages • Replace 2'-O-methyl modified nucleotides with RNA at the n-3 position Replace RNA at the n-3 position with modified nucleotides 2'-O-methyl • Replace phosphorothioate bonds with phosphorodithioate bonds and add additional phosphorodithioate bonds. Petition 870250090770, dated 06 / 10 / 2025, pp. 285 / 369 109 / 111

[00283] FIGURE 41 and Table 12 show the initial purity profiles of reference gRNA 3 and the gRNAs modified according to modification schemes B and MV. Similar purity profiles were observed for gRNAs with modifications according to modification schemes B and MV (Table 12 and FIGURE 41). The results of the forced degradation studies of these modified gRNAs are shown in Table 12 and FIGURES 41 to 59. Experimental details of the conditions for the forced degradation studies are provided in Example 2. Table 12. Purity Summary under Forced Degradation Name of gRNA Purity (% in area) T(0) 0.3% H2O2, 40oC, T(5d) PPH 8, 40oC, T(7d) PPH 5, 40oC, T(7d) PPH 11, 40oC, T(2d) Reference gRNA 3 91.2 86.8 86.8 72.6 18.4 B 92.5 83.8 89.7 78.8 40.6 M 88.1 76.0 85.1 73.5 49.2 N 82.7 79.1 79.1 69.5 44.6 W 93.4 78.4 89.8 75.7 59.5 P 88.9 77.1 86.0 74.3 73.1 Q 90.4 74.1 87.3 75.2 49.8 R 83.9 79.1 80.9 71.5 51.6 S 92.7 79.7 89.8 76.5 40.9 T 90.3 82.7 86.9 75.2 34.7 U 84.2 74.2 81.2 69.4 42.4 V 93.3 78.5 89.0 77.6 64.6

[00284] FIGURES 42-45 show the chromatograms of gRNAs B and MV under acid stress (pH 5). As shown in FIGURE 44, the gRNA modified according to scheme “T” appears to be the most resistant to acid stress conditions (pH 5). These data show that the inclusion of phosphorodithioate linkages at the 3' ends of the gRNA results in greater stability. Petition 870250090770, dated 06 / 10 / 2025, pp. 286 / 369 110 / 111 under acid stress conditions.

[00285] FIGURES 46 to 49 show the chromatograms of gRNAs B and MV under basic stress (pH 11). As shown in FIGURE 47, the gRNA modified according to scheme “P” appears to be the most resistant to basic stress conditions (pH 11). These data show that the inclusion of phosphorodithioate linkages at the 5' end of the gRNA results in greater stability under basic stress conditions.

[00286] FIGURES 50 to 53 show the chromatograms of gRNAs B and MV under thermal stress. Most gRNAs were resistant to thermal degradation.

[00287] FIGURES 54 to 55 show the chromatograms of gRNAs B and MV under oxidative stress (0.3% H2O2). Most gRNAs were susceptible to oxidative degradation.

[00288] FIGURE 56 shows the changes in purity over a period of 7 days under thermal stress. FIGURE 57 shows the changes in purity over a period of 7 days under acidic stress (pH 5). FIGURE 58 shows the changes in purity over a period of 48 hours under basic stress (pH 11). FIGURE 59 shows the changes in purity over a period of 5 days under oxidative stress (0.3% H2O2).

[00289] Further analysis of the characteristics of gRNAs targeting Reference Target 1 and modified according to modification schemes B and MV was performed in three donor cell lines, donor 1, donor 2, and donor 3. Using the Lonza 4D system, the editing efficiency, cell expansion, and cell phenotype of these gRNAs were analyzed using approximately 3.0 x 107 cells / tfx on a 100 μL scale. In donor 1 (FIGURES 60 and 61), donor 2 (FIGURES 64 and 65), and donor 3 (FIGURES 68 and 69), low editing was observed with the modified gRNA of Petition 870250090770, dated 06 / 10 / 2025, pp. 287 / 369 111 / 111 according to scheme “N” at the midpoint and at the endpoint. Furthermore, similar percentages were observed for knock-in (KI) and knockout (KO) editing for each modified gRNA in all 3 donors, except for the gRNA modified using scheme “N”. As shown in FIGURES 61, 65, and 69, improved editing efficiency was observed with gRNAs modified according to the “S” and “T” modification schemes compared to the reference gRNA. In particular, the number of wild-type cells for the reference gRNA (i.e., cells that were not edited by the gRNA) was observed to be 22% to 39%, while the number of wild-type cells for the “S” and “T” modified gRNAs was between 18% and 36% at the final time point of the study.Furthermore, the percentages for KO editing ranged from 51% to 78% for reference gRNA 3, compared to 64% to 82% for the “S” and “T” modified gRNAs, and the percentages for KI editing ranged from 16% to 23% for reference gRNA 3, compared to 13% to 28% for the “S” and “T” modified gRNAs, at the final time point of the study. These results show that the “S” and “T” modified gRNAs exhibited comparable or improved editing efficiency compared to reference gRNA 3.

[00290] Similar cell expansion rates were observed for all modified gRNAs in donor 1 (FIGURE 62), donor 2 (FIGURE 66), and donor 3 (FIGURE 70).

[00291] All publications, patents and patent applications mentioned herein are hereby incorporated by reference in their entirety, as if each individual publication, patent or patent application were specifically and individually designated for incorporation by reference. In case of conflict, this application, including any definitions herein, shall prevail. Petition 870250090770, dated 06 / 10 / 2025, pp. 288 / 369

Claims

1 / 9 Claims 1. GUIDE RNA MOLECULE, characterized by comprising: (a) a phosphorodithioate linkage between the first and second nucleotides at the 5' end of the guide RNA molecule; (b) a phosphorodithioate linkage between the 3' (n)-terminal nucleotide and the n-1 nucleotide of the guide RNA molecule; or (c) a phosphorodithioate linkage between the first and second nucleotides at the 5' end of the guide RNA molecule and a phosphorodithioate linkage between the n and n-1 nucleotides of the guide RNA molecule.

2. GUIDE RNA MOLECULE, according to claim 1, characterized by further comprising phosphorodithioate linkages between the second and third nucleotides and the third and fourth nucleotides at the 5' terminus of the guide RNA molecule.

3. GUIDE RNA MOLECULE, according to any one of claims 1 to 2, characterized by comprising phosphorodithioate linkages between the first and second nucleotides, the second and third nucleotides, and the third and fourth nucleotides at the 5' terminus of the guide RNA molecule.

4. GUIDE RNA MOLECULE, according to any one of claims 1 to 3, characterized by comprising phosphorodithioate linkages 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' terminus of the guide RNA molecule.

5. GUIDE RNA MOLECULE, according to any one of claims 1 to 4, characterized by comprising phosphorodithioate linkages between nucleotides n-1 and n-1 and n-2 or nucleotides n-1 and n-2 and n-3 of the guide RNA molecule.

6. GUIDE RNA MOLECULE, according to any one of claims 1 to 5, characterized by comprising phosphorodithioate linkages between the n-1 nucleotides, the n-1 and n-2 nucleotides, and the n-2 and n-3 nucleotides of the guide RNA molecule.

7. GUIDE RNA MOLECULE, according to any one of claims 1 to 6, characterized by comprising phosphorodithioate linkages between the n-1 nucleotides, the n-1 and n-2 nucleotides, the n-2 and n-3 nucleotides, and the n-3 and n-4 nucleotides of the guide RNA molecule.

8. GUIDE RNA MOLECULE, according to any one of claims 1 to 7, characterized in that the first nucleotide at the 5' terminus of the guide RNA molecule comprises a modification selected from the group consisting of: (a) a 2'-fluoro-modified nucleotide; (b) a 2'-O-methyl-modified nucleotide; (c) a 2'-O-(2-methoxyethyl)-modified nucleotide; (d) a blocked nucleic acid (LNA); (e) a deoxyribose nucleotide; and (f) a combination of two or more of (a)-(e).

9. GUIDE RNA MOLECULE, according to claim 8, characterized in that the first nucleotide at the 5' end of the guide RNA molecule comprises a nucleotide modified with 2'-fluoro.

10. GUIDE RNA MOLECULE, according to claim 8, characterized in that the first nucleotide at the 5' end of the guide RNA molecule comprises a nucleotide modified with 2'-O-methyl.

11. GUIDE RNA MOLECULE, according to claim 8, characterized by the first nucleotide at the 5' end of the guide RNA molecule comprising a nucleotide modified with 2'-O-(2-methoxyethyl).

12. GUIDE RNA MOLECULE, according to claim 8, characterized by the first nucleotide at the 5' terminus of the guide RNA molecule. Petition 870250090770, dated 06 / 10 / 2025, p. 290 / 369 3 / 9 13. GUIDE RNA MOLECULE, according to claim 8, characterized by the first nucleotide at the 5' terminus of the guide RNA molecule comprising a deoxyribose nucleotide.

14. GUIDE RNA MOLECULE, according to any one of claims 1 to 13, characterized in that (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 (vii) the second, third and fourth nucleotides at the 5' terminus of the guide RNA molecule comprise a modification selected from the group consisting of: (a) a 2'-fluoro-modified nucleotide; (b) a 2'-O-methyl-modified nucleotide; (c) a 2'-O-(2-methoxyethyl)-modified nucleotide; (d) a blocked nucleic acid (LNA); (e) a deoxyribose nucleotide; and (f) a combination of two or more of (a)-(e).

15. GUIDE RNA MOLECULE, according to any one of claims 1 to 9 and 14, characterized by the guide RNA molecule comprising three consecutive nucleotides modified with 2'-fluoro in the first three nucleotides at the 5' terminus of the guide RNA molecule.

16. GUIDE RNA MOLECULE, according to any one of claims 1 to 8, 10 and 14, characterized by the guide RNA molecule comprising three consecutive nucleotides modified with 2'-O-methyl in the first three nucleotides at the 5' end of the guide RNA molecule.

17. GUIDE RNA MOLECULE, according to any one of claims 1 to 8, 10 and 14, characterized by the guide RNA molecule comprising four consecutive nucleotides modified with 2'-O-methyl in the first four nucleotides at the 5' end of the guide RNA molecule.

18. GUIDE RNA MOLECULE, according to any one of claims 1 to 8, 11 and 14, characterized by the guide RNA molecule comprising three consecutive nucleotides modified with 2'-O-(2-methoxyethyl) in the first three nucleotides at the 5' terminus of the guide RNA molecule.

19. GUIDE RNA MOLECULE, according to any one of claims 1 to 8, 12 and 14, characterized by the guide RNA molecule comprising three consecutive LNAs in the first three nucleotides at the 5' terminus of the guide RNA molecule.

20. GUIDE RNA MOLECULE, according to any one of claims 1 to 8, 13 and 14, characterized by the guide RNA molecule comprising three consecutive deoxyribose nucleotides in the first three nucleotides at the 5' terminus of the guide RNA molecule.

21. GUIDE RNA MOLECULE, according to any one of claims 1 to 20, characterized in that the terminal 3'(n) nucleotide of the guide RNA molecule comprises a modification selected from the group consisting of: (a) a 2'-fluoro-modified nucleotide; (b) a 2'-O-methyl-modified nucleotide; (c) a 2'-O-(2-methoxyethyl)-modified nucleotide; (d) a blocked nucleic acid (LNA); (e) a deoxyribose nucleotide; and (f) a combination of two or more of (a)-(e).

22. GUIDE RNA MOLECULE, according to claim 21, characterized in that the n nucleotide of the guide RNA molecule comprises a nucleotide modified with 2'-fluoro.

23. GUIDE RNA MOLECULE, according to claim Petition 870250090770, dated 06 / 10 / 2025, page 292 / 369 5 / 9 21, characterized in that the n nucleotide of the guide RNA molecule comprises a nucleotide modified with 2'-O-methyl.

24. GUIDE RNA MOLECULE, according to claim 21, characterized in that the n nucleotide of the guide RNA molecule comprises a nucleotide modified with 2'-O-(2-methoxyethyl).

25. GUIDE RNA MOLECULE, according to claim 21, characterized in that the nucleotide n of the guide RNA molecule comprises an LNA.

26. GUIDE RNA MOLECULE, according to claim 21, characterized in that the n nucleotide of the guide RNA molecule comprises a deoxyribose nucleotide.

27. GUIDE RNA MOLECULE, according to any one of claims 1 to 26, characterized in that (i) the n-1 nucleotide, (ii) the n-2 nucleotide, (iii) the n-3 nucleotide, (iv) the n-4 nucleotide, (v) the n-1 nucleotides, (vi) the n-2 nucleotides, (vii) the n-1 and n-2 nucleotides, (viii) the n, n-1 and n-2 nucleotides, (ix) the n-1, n-2 and n-3 nucleotides, (x) the n, n-1, n-2 and n-3 nucleotides, or (xi) the n, n-1, n-2, n-3 and n-4 nucleotides of the guide RNA molecule each comprising a modification selected from the group consisting of: (a) a modified nucleotide with 2'-fluoro; (b) a nucleotide modified with 2'-O-methyl; (c) a nucleotide modified with 2'-O-(2-methoxyethyl); (d) a blocked nucleic acid (LNA); (e) a deoxyribose nucleotide; and (f) a combination of two or more of (a)-(e).

28. GUIDE RNA MOLECULE, according to any one of claims 1 to 22 and 27, characterized by the nucleotides n-1, n-2, n-1 and n-2, n, n-1 and n-2, n, n-1 and n-2, n-1, n-2, n-3 and n-4 or n, n-1, n-2, n-3 and n-4 of the guide RNA molecule, each being nucleotides modified with 2'-fluoro.

29. GUIDE RNA MOLECULE, according to any one of claims 1 to 21, 23 and 27, characterized by the nucleotides ne n-1, the nucleotides ne n-2, the nucleotides n-1 and n-2, the nucleotides n, n-1 and n-2, the nucleotides n-1, n-2, n-3 and n-4 or the nucleotides n, n-1, n-2, n-3 and n-4 of the guide RNA molecule, each being nucleotides modified with 2'-O-methyl.

30. GUIDE RNA MOLECULE, according to any one of claims 1 to 21, 23, 27 and 29, characterized in that the n, n-1, n-2, n-3 and n-4 nucleotides of the guide RNA molecule are each modified nucleotides with 2'-O-methyl.

31. GUIDE RNA MOLECULE, according to any one of claims 1 to 21, 24 and 27, characterized by the nucleotides ne n-1, the nucleotides ne n-2, the nucleotides n-1 and n-2, the nucleotides n, n-1 and n-2, the nucleotides n-1, n-2, n-3 and n-4 or the nucleotides n, n-1, n-2, n-3 and n-4 of the guide RNA molecule, each being nucleotides modified with 2'-O-(2-methoxyethyl).

32. GUIDE RNA MOLECULE, according to any one of claims 1 to 21, 25 and 27, characterized by the nucleotides n and n-1, the nucleotides n and n-2, the nucleotides n-1 and n-2, the nucleotides n, n-1 and n-2, the nucleotides n-1, n-2, n-3 and n-4 or the nucleotides n, n-1, n-2, n-3 and n-4 of the guide RNA molecule being, each, LNAs.

33. GUIDE RNA MOLECULE, according to any one of claims 1 to 21, 26 and 27, characterized in that the nucleotides n and n-1, the nucleotides n and n-2, the nucleotides n-1 and n-2, the nucleotides n, n-1 and n-2, the nucleotides n-1, n-2, n-3 and n-4 or the nucleotides n, n-1, n-2, n-3 and n-4 of the guide RNA molecule are each deoxyribose nucleotides. Petition 870250090770, dated 06 / 10 / 2025, p. 294 / 369 7 / 9 34. NUCLEIC ACID, characterized by comprising a polynucleotide encoding a guide RNA molecule, as defined in any one of claims 1 to 33.

35. NUCLEIC ACID, according to claim 34, characterized by further comprising a polynucleotide encoding an RNA-guided nuclease.

36. NUCLEIC ACID, according to claim 35, characterized by the RNA-guided nuclease being a Cas protein.

37. NUCLEIC ACID, according to claim 36, characterized in that the Cas protein is selected from the group consisting of a Cas9, a Cas12, a Cas13 and a combination thereof.

38. VECTOR, characterized by comprising a nucleic acid, as defined in any one of claims 34 to 37.

39. COMPOSITION, characterized by comprising a guide RNA molecule, as defined in any one of claims 1 to 33.

40. COMPOSITION, according to claim 39, characterized by further comprising an RNA-guided nuclease.

41. COMPOSITION, according to claim 39, characterized by further comprising a nucleic acid encoding RNA-guided nuclease.

42. COMPOSITION, according to any one of claims 40 to 41, characterized in that the RNA-guided nuclease is a Cas protein.

43. COMPOSITION, characterized by comprising a nucleic acid, as defined in any one of claims 34 to 37.

44. COMPOSITION, characterized by comprising a vector, as defined in claim 38. Petition 870250090770, dated 06 / 10 / 2025, pp. 295 / 369 8 / 9 45. RIBONUCLEOPROTEIN (RNP) COMPLEX, characterized by comprising a guide RNA molecule, as defined in any one of claims 1 to 33, and an RNA-guided nuclease.

46. ​​RNP COMPLEX, according to claim 45, characterized in that the RNA-guided nuclease is a Cas protein.

47. RNP COMPLEX, according to claim 46, characterized in that the Cas protein is selected from the group consisting of a Cas9, a Cas12, a Cas13 and a combination thereof.

48. CELL, characterized by comprising a composition, as defined in any one of claims 39 to 44.

49. CELL, characterized by comprising an RNP complex, as defined in any one of claims 45 to 47.

50. METHOD FOR MODIFYING A CELL, characterized by comprising bringing the cell into contact with a composition, as defined in any one of claims 39 to 44, or an RNP complex, as defined in any one of claims 45 to 47.

51. METHOD, according to claim 50, characterized in that said contact comprises the introduction of the composition into the cell by electroporation.

52. METHOD FOR TREATING AN INDIVIDUAL in need thereof, characterized by comprising: (a) modifying a cell of the individual ex vivo by contacting the cell with a composition, as defined in any one of claims 39 to 44, or an RNP complex, as defined in any one of claims 45 to 47; and (b) returning the modified cell to the individual.

53. GENETIC EDITING SYSTEM, characterized by Petition 870250090770, dated 06 / 10 / 2025, p. 296 / 369 9 / 9, comprises: (a) one or more guide RNA molecules, as defined in any one of claims 1 to 33; (b) one or more nucleic acids, as defined in any one of claims 34 to 37; (c) one or more vectors as defined in claim 38; (d) one or more compositions, as defined in any one of claims 39 to 44; and / or (e) one or more RNP complexes, as defined in any one of claims 45 to 47. Petition 870250090770, dated 06 / 10 / 2025, p. 297 / 369

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