Guide RNA design and complexes for Tracr-free V-type Cas systems

Abstract

A novel gRNA-ligand binding complex is provided. The complex can be used to target V-type Cas proteins and additional effectors to DNA for base editing. The design of the system allows for the production of efficient modular components that provide flexibility in editing DNA.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of the filing date of U.S. Provisional Application Serial No. 63 / 133,942, filed January 5, 2021, the entire disclosure of which is incorporated by reference as if fully set forth herein.

[0003] FIELD OF THE INVENTION

[0004] The present invention relates to the field of gene editing. [Background technology]

[0005] Researchers are actively exploring the use of the Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) system to modify DNA. To date, the majority of research in this field has been in the Cas9 system. In these systems, trans-activating CRISPR RNA (tracrRNA) and CRISPR RNA (crRNA) hybridize to recruit the Cas9 protein, directing it to a DNA location complementary to the sequence in the crRNA. This complementary sequence in DNA then becomes the target site, and the Cas9 protein can initiate editing at this target site based on its functional domain.

[0006] Although the power of the Cas9 system is now well recognized, these systems are not effective for all applications. One of the limitations of the Cas9 system is that the functional domains in which the Cas9 system can act are defined by the functional domain of the Cas9 protein used, making the use of both tracrRNA and crRNA cumbersome.

[0007] Other Cas proteins are known, and although their capabilities have not yet been fully explored, some of these other Cas proteins, particularly those within the V-type family, do not require the presence of tracrRNA to function. These systems can use a single guide RNA (gRNA) containing a crRNA sequence. This crRNA sequence is capable of associating with the desired Cas protein without the need for tracrRNA binding. The lack of a tracrRNA requirement offers untapped potential for the development of improved gRNAs and complexes and systems that incorporate and use them. [Prior art documents] [Patent documents]

[0008] [Patent Document 1] U.S. Patent No. 3,687,808 [Non-patent literature]

[0009] [Non-Patent Document 1] Adams, The Biochemistry of the Nucleic Acids, 11th edition, 1992 [Non-patent document 2] Martin et al., Helv.Chim.Acta, 1995, 78, 486-504 [Non-patent document 3] The Concise Encyclopedia Of Polymer Science And Engineering, pages 858-859, [Non-patent document 4] Kroschwitz, JI, John Wiley & Sons, 1990 [Non-Patent Document 5] Englisch et al., Angewandte Chemie, International Edition, 1991, 30, 613, [Non-patent document 6] Sanghvi, YS, Chapter 15, Antisense Research and Applications, pp. 289-302, Crooke, ST and Lebleu, B., eds., CRC Press, 1993 Summary of the Invention [Problem to be solved by the invention]

[0010] The present invention provides novel and innovative gRNA-ligand binding complexes, base editing complexes, and methods for base editing. Using various embodiments of the present invention, it may be possible to efficiently and effectively perform base editing ex vivo, in vitro, and in vivo. Furthermore, some embodiments of the present invention provide a modular design that allows the same V-Cas protein to be directed to different target sites and, optionally, to associate with different effector proteins at the same or different sites. [Means for solving the problem]

[0011] According to a first embodiment, the present invention provides a gRNA-ligand-binding complex, comprising: (a) a gRNA, the gRNA being 35 to 60 or 36 to 60 nucleotides in length, the gRNA having a crRNA sequence, the crRNA sequence being 35 to 60 or 36 to 60 nucleotides in length, and the crRNA sequence comprising a Cas association region and a targeting region, the Cas association region being 14 to 37 or 18 to 30 nucleotides in length, the targeting region being 14 to 37, 18 to 30, or 18 to 20 nucleotides in length, and the Cas association region being capable of maintaining association with the RNA-binding domain of a V-type Cas protein in the absence of tracrRNA; and (b) a ligand-binding moiety, the ligand-binding moiety being (i) directly bound to the gRNA or (ii) bound to the gRNA via a linker. In one embodiment, the gRNA of the gRNA-ligand binding complex comprises or consists essentially of a chemically modified or chemically unmodified sequence that is or encodes SEQ ID NO: 137.

[0012] According to a second embodiment, the present invention provides a base editing complex, the base editing complex comprising a gRNA-ligand binding complex of the present invention and a V-type Cas protein, wherein the Cas association region of the gRNA-ligand binding complex associates with the V-type Cas protein. Optionally, the ligand binding moiety reversibly binds to a ligand that is connected to or part of an effector molecule.

[0013] According to a third embodiment, the present invention provides a base editing method. The method includes exposing a base editing complex of the present invention to double-stranded DNA ("dsDNA") or single-stranded DNA ("ssDNA"). The base editing complex may be exposed to the dsDNA or ssDNA under conditions that allow base editing. [Effects of the Invention]

[0014] When the effector is bound to (or contains) a ligand, the system has a modular design. The presence of a ligand-binding moiety in a gRNA-ligand binding complex allows the complex to bind to the corresponding ligand bound to (or contained within) the effector. Thus, the ligand-binding moiety is bound to the gRNA in a manner and orientation that allows it to bind to the ligand. Similarly, the ligand is connected or bound to the effector in a manner that allows the effector to reversibly bind to the ligand-binding moiety.

[0015] When the ligand and ligand-binding moiety are bound to each other, the effector bound to the ligand becomes part of any base editing complex, including the gRNA-ligand binding complex. If the base editing complex also contains a Cas protein, the Cas protein and effector can be held in the same location, e.g., at or near the intended target site.

[0016] Therefore, if you want to use a particular effector with a Cas protein, you simply associate that effector with a ligand that can reversibly bind to the ligand-binding moiety that is part of the base editing complex containing that Cas protein. Changing the effector from one system to the next simply involves changing the effector-ligand. As a result, the same gRNA-ligand complex and its associated Cas protein can be used with multiple different effectors. Multiple different effectors can be used sequentially in the same system, or simultaneously or sequentially in different systems, by binding and dissociating their ligands from the ligand-binding moiety. [Brief explanation of the drawings]

[0017] [Figure 1A]Figure 1A shows an example of a CasPhi guide RNA, showing the location of the direct repeat, spacer, and, if present, the MS2 ligand-binding moiety. The gRNA is shown bound to the DNA strand (SEQ ID NO: 66) at the spacer region. [Figure 1B] Figure 1B shows an example of a CasPhi guide RNA, showing the location of the direct repeat, spacer, and, if present, MS2 ligand-binding moiety. The gRNA is shown bound to the DNA strand (SEQ ID NO: 66) at the spacer region. [Figure 1C] Figure 1C shows an example of a CasPhi guide RNA, showing the location of the direct repeat, spacer, and, if present, MS2 ligand-binding moiety, where the gRNA binds to the DNA strand (SEQ ID NO: 66) at the spacer region. [Figure 1D] Figure ID shows an example of a CasPhi guide RNA, showing the location of the direct repeat, spacer, and, if present, the MS2 ligand-binding moiety, where the gRNA binds to the DNA strand (SEQ ID NO: 66) at the spacer region. [Figure 1E] Figure 1E shows an example of a CasPhi guide RNA, showing the location of the direct repeat, spacer, and, if present, MS2 ligand-binding moiety, where the gRNA binds to the DNA strand (SEQ ID NO: 66) at the spacer region. [Figure 1F] Figure IF shows an example of a CasPhi guide RNA, showing the location of the direct repeat, spacer, and, if present, the MS2 ligand-binding moiety. The gRNA is shown bound to the DNA strand (SEQ ID NO: 66) at the spacer region. [Figure 1G] Figure 1G shows an example of a CasPhi guide RNA, showing the location of the direct repeat, spacer, and, if present, the MS2 ligand-binding moiety, where the gRNA binds to the DNA strand (SEQ ID NO: 66) at the spacer region. [Figure 2A]Figure 2A is a bar graph illustrating the effect of positioning MS2 on dCasPhi base editing levels for two genomic target sites. [Figure 2B] Figure 2B is a bar graph illustrating the effect of positioning MS2 on dCasPhi base editing levels for two genomic target sites. [Figure 2C] Figure 2C illustrates the evaluation of the effect of MS2 placement on gene disruption levels using CasPhi. [Figure 2D] Figure 2D illustrates the evaluation of the effect of MS2 placement on gene disruption levels using CasPhi. [Figure 3A] Figure 3A is a bar graph illustrating base editing using multiple sets of inactivating CasPhi mutant guides. [Figure 3B] Figure 3B is a bar graph illustrating base editing using multiple sets of inactivating CasPhi mutant guides. [Figure 4A] Figure 4A is a bar graph summarizing dCasPhi base editing efficiency using different spacer lengths for the expressed gRNA. [Figure 4B] Figure 4B is a bar graph summarizing dCasPhi base editing efficiency using different spacer lengths for the expressed gRNA. [Figure 4C] Figure 4C is a schematic diagram of gRNAs with different spacer lengths. [Figure 4D] Figure 4D is a schematic diagram of gRNAs with different spacer lengths. [Figure 4E] Figure 4E is a schematic diagram of gRNAs with different spacer lengths. [Figure 4F] Figure 4F is a schematic diagram of gRNAs with different spacer lengths. [Figure 5A] Figure 5A is a bar graph illustrating dCasPhi base editing at multiple sites in HEK293T cells using chemically synthesized and chemically modified guides. [Figure 5B]Figure 5B is a bar graph illustrating dCasPhi base editing at multiple sites in HEK293T cells using chemically synthesized and chemically modified guides. [Figure 5C] Figure 5C is a bar graph illustrating dCasPhi base editing at multiple sites in HEK293T cells using chemically synthesized and chemically modified guides. [Figure 6] Figure 6 is a bar graph representation of two different codon-optimized dCasPhi base editing at HEK site 2 in HEK293T cells using chemically modified synthetic guides. [Figure 7A] Figure 7A shows a bar graph depicting the effect of synthetic guide chemical modifications on dCasPhi base editing levels at two different genomic target sites in HEK293T cells. [Figure 7B] Figure 7A shows a bar graph depicting the effect of synthetic guide chemical modifications on dCasPhi base editing levels at two different genomic target sites in HEK293T cells. [Figure 8A] Figure 8A shows a bar graph assessing the effect of linkers in the gRNA sequence on dCasPhi base editing levels. [Figure 8B] Figure 8B is a schematic diagram of a gRNA without a linker. [Figure 8C] Figure 8C is a schematic diagram of gRNA sequences with various linkers combined at different positions. [Figure 8D] Figure 8D is a schematic diagram of gRNA sequences with various linkers combined at different positions. [Figure 8E] Figure 8E is a schematic diagram of gRNA sequences with various linkers combined at different positions. [Figure 8F] Figure 8F is a schematic diagram of gRNA sequences with various linkers combined at different positions. [Figure 8G] Figure 8G is a schematic diagram of gRNA sequences with various linkers combined at different positions. [Figure 8H]Figure 8H is a schematic diagram of gRNA sequences with various linkers combined at different positions. [Figure 8I] Figure 8I is a schematic diagram of gRNA sequences with various linkers combined at different positions. [Figure 8J] Figure 8J is a schematic diagram of gRNA sequences with various linkers combined at different positions. [Figure 8K] Figure 8K is a schematic diagram of gRNA sequences with various linkers combined at different positions. [Figure 9] Figure 9 is a bar graph representation of dCasPhi aptamer-recruited base editing in T lymphocytes. [Figure 10] Figure 10 is a bar graph representation of dCas12a base editing at HEK site 2 in HEK293T cells using chemically modified guides. [Figure 11A] Figure 11A illustrates the evaluation of the efficacy of dCas12i2 base editing at HEK site 2 using multiple deaminases in HEK293T cells. [Figure 11B] Figure 11B illustrates the evaluation of the efficacy of dCas12i2 base editing at HEK site 2 using multiple deaminases in HEK293T cells. [Figure 12] Figure 12 is a diagram showing a base editing complex of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0018] Detailed Description of the Invention

[0019] DETAILED DESCRIPTION OF THE INVENTION

[0013] Various embodiments of the present invention will now be described in detail, examples of which are illustrated in the accompanying drawings. In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present invention. However, unless otherwise indicated or implied from the context, the details are intended to be illustrative and should not be construed as limiting the scope of the invention in any way. Furthermore, features described in connection with various embodiments or specific embodiments should not be construed as not being suitable for use in connection with other embodiments disclosed herein, unless such exclusivity is expressly stated or implied from the context.

[0020] Headings are provided herein for the convenience of the reader and do not limit the scope of any embodiments disclosed herein.

[0021] definition

[0022] Unless otherwise stated or apparent from the context, the following terms shall have the meanings set forth below:

[0023] The phrase "2'-modified" refers to a nucleotide unit having a sugar moiety that is modified at the 2' position of the sugar moiety. Examples of 2'-modifications are a 2'-O-alkyl modification to form a 2'-O-alkyl-modified nucleotide or a 2'-halogen modification to form a 2'-halogen-modified nucleotide.

[0024] The phrase "2'-O-alkyl modified nucleotide" refers to a nucleotide unit having a sugar moiety, e.g., a deoxyribosyl or ribosyl moiety, modified at the 2' position such that an oxygen atom is attached to both the carbon atom at the 2' position of the sugar and the alkyl group. In various embodiments, the alkyl moiety consists of, or consists essentially of, carbon(s) and hydrogen(s). When the O moiety and the alkyl group to which it is attached are viewed as a single group, they may be referred to as an O-alkyl group, e.g., -O-methyl, -O-ethyl, -O-propyl, -O-isopropyl, -O-butyl, -O-isobutyl, -O-ethyl-O-methyl (-OCH2CHOCH3), and -O-ethyl-OH (-OCH2CH2OH). 2'-O-alkyl modified nucleotides can be substituted or unsubstituted.

[0025] The phrase "2' halogen modified nucleotide" refers to a nucleotide unit having a sugar moiety, e.g., a deoxyribosyl moiety, modified at the 2' position such that the carbon at that position is directly bonded to a halogen species, e.g., Fl, Cl, or Br.

[0026] "Ligand-binding moiety" refers to a moiety such as an aptamer, e.g., an oligonucleotide, peptide, or other compound that can bind to a specific ligand and bind reversibly or irreversibly to that ligand.

[0027] The term "modified nucleotide" refers to a nucleotide having at least one modification in the chemical structure of the base, sugar, and / or phosphate, including, but not limited to, 5-position pyrimidine modifications, 8-position purine modifications, modifications at the exocyclic amine of cytosine, and substitution of 5-bromouracil or 5-iodouracil; as well as 2'-modifications (including, but not limited to, sugar-modified ribonucleotides in which the 2'-OH is replaced with a group such as H, OR, R, halo, SH, SR, NH, NHR, NR, or CN, where R is alkyl).

[0028] Modified bases refer to nucleotide bases, such as adenine, guanine, cytosine, thymine, uracil, xanthine, inosine, queosine, etc., that have been modified by the substitution or addition of one or more atoms or groups. Some examples of these types of modifications include, but are not limited to, alkylated, halogenated, thiolated, aminated, amidated, or acetylated bases, alone or in various combinations thereof. More specific modified bases include, for example, 5-propynyluridine, 5-propynylcytidine, 6-methyladenine, 6-methylguanine, N,N-dimethyladenine, 2-propyladenine, 2-propylguanine, 2-aminoadenine, 1-methylinosine, 3-methyluridine, 5-methylcytidine, 5-methyluridine and other nucleotides having modifications at the 5-position, 5-(2-amino)propyluridine, 5-halocytidine, 5-halolidine, 4-acetylcytidine, 1-methyladenosine, 2-methyladenosine, 3-methylcytidine, 6-methyluridine, 2-methylguanosine, 7-methylguanosine, 2,2-dimethylguanosine, 5-methylaminoethyluridine, 5-methyloxyuridine, deazanucleotides such as 7-deazaadenosine, 6-azolidine, 6-azocytidine, 6-azothymidine, Modified nucleotides include 5-methyl-2-thiouridine, other thio bases (e.g., 2-thiouridine, 4-thiouridine, 2-thiocytidine), dihydrouridine, pseudouridine, queusine, archaeosine, naphthyl and substituted naphthyl groups, O- and N-alkylated purines and pyrimidines (e.g., N6-methyladenosine), 5-methylcarbonylmethyluridine, uridine 5-oxyacetic acid, pyridin-4-one, pyridin-2-one, phenyl groups, modified phenyl groups (e.g., aminophenol, 2,4,6-trimethoxybenzene), modified cytosines that act as G-clamp nucleotides, 8-substituted adenines and guanines, 5-substituted uracils and thymines, azapyrimidines, carboxyhydroxyalkyl nucleotides, carboxyalkylaminoalkyl nucleotides, and alkylcarbonyl alkylated nucleotides. Modified nucleotides also include nucleotides modified with respect to the sugar moiety and nucleotides having non-ribosyl sugars or analogs thereof.For example, the sugar moiety can be or be based on mannose, arabinose, glucopyranose, galactopyranose, 4-thioribose, and other sugars, heterocycles, or carbocycles.

[0029] The phrases "code for" and "encoding" mean that a sequence includes either a sequence that is identical to a referenced nucleotide sequence, a DNA or RNA equivalent of the referenced nucleotide sequence, or a DNA or RNA sequence that is the DNA or RNA complement of the referenced nucleotide sequence. Thus, when referring to a sequence that codes for a referenced DNA sequence or encodes a referenced DNA sequence, unless otherwise specified, it refers to the same DNA sequence, the complement of the DNA sequence, the RNA equivalent of that sequence, or the RNA complement of that sequence, or any of the foregoing in which one or more ribonucleotides are substituted for their deoxyribonucleotide counterparts, or in which one or more deoxyribonucleotides are substituted for their ribonucleotide counterparts.

[0030] The term "complementary" refers to the ability of a nucleic acid to form one or more hydrogen bonds with another nucleic acid sequence, either by conventional Watson-Crick base pairing or other non-conventional methods. The percent complementarity indicates the percentage of residues in a nucleic acid molecule that can form hydrogen bonds (e.g., Watson-Crick base pairs) with a second nucleic acid sequence (e.g., 5, 6, 7, 8, 9, and 10 out of 10 are 50%, 60%, 70%, 80%, 90%, and 100% complementary, respectively). "Fully complementary" means that all contiguous residues of a nucleic acid sequence will hydrogen bond with the same number of contiguous residues in a second nucleic acid sequence. As used herein, "substantially complementary" refers to a degree of complementarity, e.g., at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% over a region of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, or more contiguous nucleotides, or refers to two nucleic acids that hybridize under stringent conditions.

[0031] The terms "hybridization" and "hybridize" refer to the process by which, under specific hybridization conditions, fully, substantially, or partially complementary nucleic acid strands come together to form a double-stranded structure or region in which the two component strands are joined by hydrogen bonds. Unless otherwise specified, hybridization conditions are naturally occurring or laboratory-designed. Hydrogen bonds typically form between adenine and thymine or uracil (A and T or U) or between cytidine and guanine (C and G), although other base pairs may also be formed (see, e.g., Adams et al., The Biochemistry of the Nucleic Acids, 11th ed., 1992).

[0032] The term "nucleotide" refers to ribonucleotides, deoxyribonucleotides, or modifications thereof, and their analogs. Nucleotides include species containing purines, such as adenine, hypoxanthine, and guanine, and their derivatives and analogs, as well as pyrimidines, such as cytosine, uracil, and thymine, and their derivatives and analogs. Preferably, a nucleotide contains a cytosine, uracil, thymine, adenine, or guanine moiety. Additionally, the term nucleotide also includes species having a detectable label, such as a radioactive or fluorescent moiety, or a mass label attached to the nucleotide. The term nucleotide also includes what is known in the art as a universal base. Exemplary universal bases include, but are not limited to, 3-nitropyrrole, 5-nitroindole, and nebularine. Nucleotide analogs are meant to include nucleotides having bases such as inosine, queosine, and xanthine, sugars such as 2'-methylribose, and non-natural phosphodiester internucleotide linkages such as methylphosphonates, phosphorothioates, and phosphoroacetates, as well as peptides.

[0033] The terms "subject" and "patient" are used interchangeably herein to refer to an organism (e.g., a vertebrate, preferably a mammal, more preferably a human). Mammals include, but are not limited to, rodents, monkeys, humans, farm animals, sport animals, and pets such as dogs and cats. Tissues, cells, and their progeny of organisms or other biological entities obtained in vivo or cultured in vitro are also encompassed by the terms subject and patient. Furthermore, in some embodiments, the subject may be an invertebrate, such as an insect or nematode, while in other embodiments, the subject may be a plant or fungus.

[0034] As used herein, the terms "treatment," "treat," "alleviate," and "ameliorate" are used interchangeably. These terms refer to an approach to obtaining beneficial or desired results, including, but not limited to, therapeutic benefit and / or prophylactic benefit. By therapeutic benefit, we mean any therapeutically relevant improvement or effect in one or more diseases, conditions, or symptoms being treated. For prophylactic benefit, the complexes of the present invention can be administered to a subject at risk of developing a particular disease, condition, or symptom, or to the cells or tissues of a subject, or to the cells or tissues of another subject before ex vivo re-administration, or to a subject reporting one or more physiological symptoms of a disease, although the disease, condition, or symptom may not yet be manifest.

[0035] As disclosed herein, a number of ranges of values ​​are provided. Each intervening value, to the tenth of the unit of the lower limit, between the upper and lower limits of that range is understood to be specifically disclosed, unless the context clearly dictates otherwise. Each subrange between any stated or intervening value within a stated range and any other stated or intervening value within that stated range is also encompassed within the invention. The upper and lower limits of these subranges may each independently be included or excluded within the range, and each range in which either, neither, or both limits are included within the scope of the invention, subject to any specifically excluded limit in the stated range. When a stated range includes one or both of the limits, ranges excluding either or both of those included limits are also encompassed within the invention.

[0036] The term "about" generally refers to plus or minus 10% of the stated numerical value. For example, "about 10%" indicates a range of 9% to 11%, and "about 20%" may mean a range of 18 to 22%. Other meanings of "about," such as rounding, may be apparent from the context; for example, "about 1" may mean 0.5 to 1.4.

[0037] Consideration

[0038] According to a first embodiment, the present invention comprises a gRNA-ligand binding complex comprising, consisting essentially of, or consisting of both a gRNA and a ligand binding moiety. In the absence of tracrRNA, this complex is capable of retaining association with a V-type Cas protein. Within the gRNA-ligand binding complex, the gRNA may be covalently linked directly to the ligand binding moiety or may be linked to the ligand binding moiety via a linker.

[0039] gRNA

[0040] The gRNA of the gRNA-ligand complex is a single strand of nucleotides. The nucleotides may be entirely RNA or a combination of ribonucleotides and other nucleotides, such as deoxynucleotides. Each nucleotide may be unmodified, or one or more nucleotides may be modified, for example, with one of the following modifications: 2'-O-methyl, 2'-fluoro, or 2'-aminopurine. In some embodiments, there are consecutive modified nucleotides or a modification pattern in which every second, third, or fourth nucleotide is modified at its 2' position, with all other nucleotides unmodified, over one or more ranges of 1 to 40 or 2 to 20, or 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 35, or 36 nucleotides. Additionally or alternatively, there may be modified or unmodified internucleotide linkages between one or more or all pairs of consecutive nucleotides.

[0041] In some embodiments, the gRNA is 35-60 nucleotides, 36-60 nucleotides, or 40-55 nucleotides in length. The gRNA has a sequence that includes, consists essentially of, or consists of the crRNA sequence. Within the crRNA is a Cas association region, sometimes referred to as a repeat region, that is 14-37 nucleotides, 18-30 nucleotides, or 20-25 nucleotides in length, and a targeting region, sometimes referred to as a spacer region, that is 14-37 nucleotides, 18-30 nucleotides, or 20-25 nucleotides in length.

[0042] The targeting region (targeting region) comprises a targeting sequence, which is a variable sequence that can be selected based on the desired location where the Cas protein and / or effector is to cause base editing. Thus, the targeting region can be designed to include a region that is complementary to and hybridizable with a preselected target site of interest. For example, the complementary region between the targeting region and the corresponding target site sequence is greater than about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 25 contiguous nucleotides, or is at least 80%, at least 85%, at least 90%, or at least 95% complementary to a region of DNA greater than 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 25 contiguous nucleotides.

[0043] The Cas-association region of the gRNA is designed to retain binding to the RNA-binding domain of the V-type Cas protein in the absence of tracrRNA. (Not all nucleotides in the Cas-association region need to directly bind to the Cas protein.) Preferably, this association is possible under both naturally occurring and laboratory conditions in which the complex is used. In some embodiments, the gRNA has or encodes one of the following sequences: SEQ ID NO: 1: UUAAUUUCUACUCUUGUAGAUN14―30 ; SEQ ID NO: 2: UGCUCGAUUAGUCGACACN 14―30 ; SEQ ID NO: 3GGAGAGAUCUCAAACGAUUGCUCGAUUAGUCGAGACN 14―30 ; SEQ ID NO: 4: GUCGGAACGCUCAACGAUUGCCCCUCACGAGGGGACN 14―30 ; SEQ ID NO: 5: ACCAAAACGACUAUUGAUUGCCCAGUACGCUGGGACN 14―30 ; SEQ ID NO: 6: AUGGCAACAGACUCUCAUUGCGCGGUACGCCGCGACN 14―30 ; SEQ ID NO: 7: GUCCCAACGAAUUGGGCAAUCAAAAAGGAUUGGAUCCN 14―30 ; SEQ ID NO: 8: CCUGCGAAACCUUUUGAUUGCUCAGUACGCUGAGACN 14―30 ; SEQ ID NO: 9: CUUUCAAGACUAAUAGAUUGCUCCUUACGAGGAGACN 14―30 ; SEQ ID NO: 10: GUAGAAGACCUCGCUGAUUGCUCGGUGCGCCGAGACN 14―30 ; SEQ ID NO: 11: UAAUUUCUACUCUUGUAGAUN 14―30 ; SEQ ID NO: 12: UAAUUUCUACUAAGUGUAGAUN 14―30 ; SEQ ID NO: 67: GCUUUCAAGACUAAUAGAUUGCUCCUUACGAGGAGACN 14―30 ; SEQ ID NO: 68: AGAAAUCCGUCUUUCAUUGACGGN 14―30 ; SEQ ID NO: 69: UAAUUUCUACUAAGUGUAGAUN 14―30 ; SEQ ID NO: 70: GCUUUCAAGACUAAUAGAUUGCUCCUUACGAGGAGACN 14―30 ; or SEQ ID NO: 71: AGAAAUCCGUCUUUCAUUGACGGN 14―30 ;

[0044] The downstream portion of the crRNA sequence is N in SEQ ID NO: 1 to SEQ ID NO: 12 or SEQ ID NO: 67 to SEQ ID NO: 71. 16~30 and corresponds to the targeting region, and the sequence upstream of that sequence corresponds to the Cas association region. N refers to any modified or unmodified nucleotide. In SEQ ID NOs: 1 to 12, N 14~30 means that there can be 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides. In some embodiments, rather than there being 14 to 40 nucleotides designated by N, there are 14 to 37 nucleotides. In some embodiments, N is 16 to 30. In some embodiments, the Cas association region has a sequence that is at least 80%, at least 85%, at least 90%, at least 95% similar or identical to the Cas association region (region upstream of the N(s)) of SEQ ID NO:1 to SEQ ID NO:12 or SEQ ID NO:67 to SEQ ID NO:71, or the Cas association region (region upstream of the N(s)) of a wild-type crRNA in a naturally occurring state or endogenous to a naturally occurring or genetically modified organism.

[0045] Ligand-binding moiety

[0046] A ligand-binding moiety is an entity that can reversibly bind to a ligand, for example, by forming a non-covalent interaction. In some embodiments, the ligand-binding moiety is an aptamer. The ligand-binding moiety can be directly attached to the gRNA, for example, via a covalent bond, or can be attached via a linker. The attachment of the ligand-binding moiety to the gRNA can be at any of a number of positions, whether directly attached via a covalent bond or via a linker. A ligand-binding moiety binds directly to the gRNA if it is attached to a nucleotide within the gRNA, for example, a phosphate in the backbone of the unit, a sugar moiety, or a nitrogenous base of the nucleotide.

[0047] As a non-limiting example, the ligand-binding moiety can be directly attached (e.g., via a covalent bond) to the 3' or 5' end of the gRNA. Thus, the ligand-binding moiety is the first or last nucleotide of the gRNA. If the ligand-binding moiety is a nucleotide sequence and it is directly attached to the 5' or 3' end of the gRNA, it may be in the same 5' to 3' orientation as the gRNA. In this situation, there is a continuous strand of nucleotides that includes both the ligand-binding moiety and the gRNA, and the gRNA is 5'-[gRNA]-[ligand-binding portion]-3' or 5'-[ligand-binding moiety]-[gRNA]-3' In other embodiments, the ligand binding moiety may be directly attached to the gRNA in the opposite orientation, thus 5'-[gRNA]-3'-3'-[ligand-binding moiety]-5' or 3'-[ligand-binding moiety]-5'-5'-[gRNA]-3'. When the ligand binding moiety is not a nucleotide sequence, it is attached to a phosphate moiety, for example, to a sugar at the 2', 3', or 5' position, or to a nitrogen group at either the 5' or 3' end of the gRNA.

[0048] The ligand-binding moiety can also be attached to the gRNA at a position other than the 5' or 3' end. If the ligand-binding moiety is a nucleotide sequence, it can be inserted into the gRNA, such that, for example, there is a first section of the gRNA that is 5' to the ligand-binding moiety and a second section of the gRNA that is 3' to the ligand-binding moiety, and there is one oligonucleotide sequence. 5'-[first section of gRNA]-[ligand-binding portion]-[second section of gRNA]-3' exists. In some embodiments, the first section of the gRNA comprises the entire Cas association region, and the second section of the gRNA comprises the entire targeting region (region to be targeted). In other embodiments, the first section of the gRNA comprises the entire Cas association region and a portion of the targeting region, and the second section of the gRNA comprises the remainder of the targeting region. In other embodiments, the first section of the gRNA comprises a portion of the Cas association region, while the second section of the gRNA comprises the remainder of the Cas association region and the entire targeting region. Compared to a gRNA that does not include a ligand-binding moiety, a complex comprising a gRNA and an inserted ligand-binding moiety may have no nucleotide deletions from either the Cas association region or the targeting region. Alternatively, one or more nucleotides (e.g., 1-10 nucleotides) may be deleted on one or both sides of the insertion site.

[0049] In some embodiments, if the ligand-binding moiety is not a nucleotide sequence, it may be attached to a phosphorus moiety, e.g., the 2', 3', or 5' position of a sugar, or a nitrogenous base at either the 5' or 3' end of the gRNA. In other embodiments, if the ligand-binding moiety is not a nucleotide sequence, it may be attached to the gRNA at a position other than the 5' or 3' end of the gRNA, or may be attached between two consecutive nucleotides, for example, as follows: 5'-[first section of gRNA]-[ligand-binding portion]-[second section of gRNA]-3'.

[0050] In some embodiments, one or more linkers connect the ligand-binding moiety to the gRNA. In these embodiments, each of the linker and the ligand-binding moiety may independently comprise, consist essentially of, or consist of nucleotides. In some embodiments, each of the linker and the ligand-binding moiety may independently comprise, consist substantially of, or consist of non-nucleotide moieties. In some embodiments, one of the linker and the ligand-binding moiety comprises, consists substantially of, or consists of non-nucleotide moieties, and the other of the linker and the ligand-binding moiety comprises, consists substantially of, or consists of nucleotides.

[0051] When the ligand-binding moiety is a nucleotide sequence and is attached to the 5' or 3' end of the gRNA via a linker that is also a nucleotide sequence, the gRNA, linker, and ligand-binding moiety may each be in the same 5' to 3' orientation. In this situation, there is a continuous stretch of nucleotides that includes both the ligand-binding moiety and the gRNA, which are 5'-[gRNA]-[linker]-[ligand-binding moiety]-3', or 5'-[ligand binding moiety]-[linker]-[gRNA]-3' Either: In other embodiments, the ligand binding moiety and / or linker may be directly attached to the gRNA in the opposite orientation, thus 5'-[gRNA]-3'-3'-[linker]-5'-3'-[ligand-binding moiety]-5', or 5'-[gRNA]-3'-5'-[linker]-3'-3'-[ligand-binding moiety]-5', or 3'-[ligand-binding moiety]-5'-3'-[linker]-5'-5'-[ligand-binding moiety]-3', or 3'-[ligand binding moiety]-5'-5'-[linker]-3'-5'-[gRNA]-3' is.

[0052] The ligand-binding moiety can also be attached to the gRNA at a position other than the 5' or 3' end via a linker or two linkers. If the ligand-binding moiety and linker are nucleotide sequences, they can be inserted into the gRNA, so that, for example, there can be a first section of the gRNA that is 5' to the ligand-binding moiety and a second section of the gRNA that is 3' to the ligand-binding moiety. There can be one or two linker sequences.

[0053] If there is only one linker sequence, it is either 5' or 3' to the ligand-binding moiety. 5'-[first section of gRNA]-[linker]-[ligand-binding portion]-[second section of gRNA]-3', or 5'-[first section of gRNA]-[ligand-binding portion]-[linker]-[second section of gRNA]-3' is.

[0054] When there are two linker sequences, the first linker may be 5' to the ligand binding moiety and the second linker may be 3' to the ligand binding moiety. 5'-[first section of gRNA]-[first linker]-[ligand-binding portion]-[second linker]-[second section of gRNA]-3' is.

[0055] In some embodiments, the first section of the gRNA, the first linker, the ligand-binding moiety, the second linker, and the second section of the gRNA are nucleotide sequences of the same orientation (facility orientation). In other embodiments, one or more of the first linker, the ligand-binding moiety, and the second linker are oriented in the opposite direction from the first section of the gRNA and the second section of the gRNA (which sections are oriented in the same way).

[0056] When the ligand-binding moiety is located between the first and second gRNA sections (if one or two linkers are present, they are also located between the first and second gRNA sections), in some embodiments, the first gRNA section includes the entire Cas association region and the second gRNA section includes the entire targeting region. In other embodiments, the first gRNA section includes the entire Cas association region and a portion of the targeting region, while the second gRNA section includes the remainder of the targeting region. In other embodiments, the first gRNA section includes a portion of the Cas association region, while the second gRNA section includes the remainder of the Cas association region and the entire targeting region. Compared to a gRNA without a ligand-binding moiety, a complex containing a gRNA and an inserted ligand-binding moiety may have no nucleotide deletions from either the Cas association region or the targeting region. Alternatively, one or more nucleotides (e.g., 1-10 nucleotides) may be deleted at the insertion site.

[0057] When two linkers are present, they may be sufficiently complementary to hybridize with each other, e.g., each linker may be 1-20 nucleotides in length, the linkers may be at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or 100% complementary, and may have no bulges, one or more bulges.

[0058] If the linker is not a nucleotide sequence, it can be attached to the 5'-most nucleotide in the gRNA, the 3'-most nucleotide in the gRNA, or a nucleotide other than the 5'-most nucleotide or the 3'-most nucleotide in the gRNA. Furthermore, the non-nucleotide or non-oligonucleotide linker can be attached to any position of the sugar or nitrogenous base, or to an internucleotide linkage. Furthermore, in some embodiments, there are two non-nucleotide linkers, or one nucleotide linker and one non-nucleotide linker.

[0059] In some embodiments, the gRNA forms a loop and, if currently bound to the loop, forms a ligand-binding moiety or linker. When bound to the loop of the gRNA, either directly or via a linker, the linkage may be, for example, the first nucleotide in the loop, the second nucleotide in the loop, the third nucleotide in the loop, the fourth nucleotide in the loop, the central nucleotide in the loop if the loop has an odd number of nucleotides, one of the two central-most nucleotides in the loop if the loop has an even number of nucleotides, or the last nucleotide in the loop. Any one or more of the aforementioned nucleotides and / or their corresponding 5' and / or 3' internucleotide linkages may be modified. These modifications may occur, for example, at the point where the ligand-binding moiety is bound to the gRNA (directly or via a linker), or may occur only at a location other than the point where the ligand-binding moiety is bound to the gRNA (directly or via a linker). For example, the ligand-binding moiety may be attached to the 2' position of the sugar or to a nitrogenous base in the gRNA oligonucleotide sequence.

[0060] In some embodiments, the ligand-binding moiety is unmodified, or comprises, consists essentially of, or consists of an oligonucleotide sequence containing one or more modified nucleotides. For example, the ligand-binding moiety is 10-50 or 18-50 nucleotides in length. In one embodiment, the ligand-binding moiety comprises, consists essentially of, or consists of SEQ ID NO: 13 (ACAUGAGGAUCACCCAUGU) or a sequence substantially similar to SEQ ID NO: 13. In some embodiments, the ligand-binding moiety forms a stem-loop structure. In the absence of a linker, the ligand-binding moiety can appear as an extension of the gRNA sequence immediately 5' or 3' of the gRNA, or as an insertion into the gRNA sequence.

[0061] In some embodiments, the ligand binding moiety comprises, consists essentially of, or consists of biotin or streptavidin.

[0062] In some embodiments, the ligand binding moiety is covalently attached or non-covalently attached.

[0063] In some embodiments, the ligand-binding moiety is selected from the group consisting of moieties that bind to the following ligands: MS2 coat protein (MCP), Ku, PP7 coat protein (PCP), Com RNA-binding protein or its binding domain, SfMu, Sm7, Tat, glutathione S-transferase (GST), CSY4, Qbeta, COM, pumilio, anti-His tag (6H7), SNAP-tag, lambda N22, lectin (in which case the ligand-binding moiety may be a carbohydrate or sugar chain or oligosaccharide), and PDGF beta chain. In some embodiments, the ligand-binding moiety is an aptamer comprising deoxyribonucleotides, ribonucleotides, or a combination of both. Thus, by way of non-limiting example, DNA aptamers, RNA aptamers, DNA aptamers with modified nucleosides in the backbone, RNA aptamers with modified nucleosides in the backbone, and combinations thereof can be used.

[0064] In some embodiments, naturally occurring MS2 aptamers are used as ligand-binding moieties. In other embodiments, MS2 C-5 mutants, MS2 F-5 mutants, or modified MS2 (e.g., MS2 with one or more, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12, modified nucleotides such as aminopurines at position 10 (position 10 being the 10th nucleotide from the 5' end of the aptamer)) are used. 2-aminopurines are, for example, 2'-deoxy-2-aminopurine or 2'-ribose-2-aminopurine. Modifications at any one position can be in addition to modifications at another position or can be exclusive of modifications at any or all other positions.

[0065] In some embodiments, the ligand-binding moiety is an aptamer comprising a 5'-modified nucleotide, wherein the 5'-modified nucleotide comprises at least one of a 2'-modification, a 5' PO4 group, or a modification of a nitrogenous base.

[0066] In some embodiments, the ligand-binding moiety is an aptamer, which is or comprises one part of an aptamer-ligand pair. As described below, the effector is linked to or comprises the other part of the aptamer-ligand pair. For example, the aptamer may comprise an MS2 operator motif that specifically binds to the MS2 coat protein MCP. Additionally, as will be appreciated by those skilled in the art, the aptamer can comprise an MCP moiety (or other ligand), in which case the effector comprises or is linked to an MS2 operator motif (or other corresponding ligand-binding moiety).

[0067] Linker

[0068] When present, the linker may be a single linker connecting the ligand-binding moiety and the gRNA. The linker may be attached to both the ligand-binding moiety and the gRNA at a single site, or to either or both the gRNA and the ligand-binding moiety at multiple sites. Attachment at multiple sites may allow for more precise spatial control of the ligand-binding moiety, and thus the effector used.

[0069] As non-limiting examples, the linker may be attached to the gRNA at one site and to the ligand-binding moiety at two or more sites; or the linker may be attached to the ligand-binding moiety at one site and to the gRNA at two or more sites. When the linker is attached to the gRNA at two or more sites, the linker may be attached to the gRNA only at the targeting region, only at the Cas association region, or both.

[0070] In some embodiments, the linker comprises, consists essentially of, or consists of an oligonucleotide sequence, and optionally, the linker includes at least one or more 2' modifications, e.g., all nucleotides in the linker are 2'-modified nucleotides. The nucleotide sequence may be random or may be intentionally designed to avoid undesired complementarity with sequences within the target site of the aptamer, gRNA, or DNA. In some embodiments where two linkers are present, the two linkers are adjacent to the linker junction.

[0071] In some embodiments, the linker comprises, consists essentially of, or consists of at least one phosphorothioate linkage.

[0072] In some embodiments, the linker comprises, consists essentially of, or consists of a levulinyl moiety.

[0073] In some embodiments, the linker comprises, consists essentially of, or consists of ethylene glycol moieties.

[0074] In some embodiments, the linker comprises or is selected from the group consisting of 18S, 9S, or C3.

[0075] In some embodiments, the linker is a nucleotide sequence that is 1 to 60, 1 to 24, 2 to 20, or 5 to 15 nucleotides in length. Furthermore, in some embodiments, the linker is GC-rich, e.g., having at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% GC nucleotides. When the linker comprises nucleotides, it may be, for example, single-stranded or double-stranded, or partially single-stranded and partially double-stranded. Furthermore, when the linker is an oligonucleotide, it may be exclusively RNA, exclusively DNA, or a combination thereof.

[0076] In some embodiments, the linker is a nucleotide sequence located upstream or downstream of the ligand-binding moiety.If the linker is located upstream of the ligand-binding moiety and the gRNA is located upstream of the linker, there may be another sequence complementary to the linker downstream of the ligand-binding moiety.Similarly, if the linker is located downstream of the ligand-binding moiety and the gRNA is located downstream of the linker, there may be another sequence complementary to the linker upstream of the ligand-binding moiety.As those skilled in the art will recognize, complementarity is determined when the oligonucleotide folds on itself and the strand aligns with each corresponding moiety in the 5' to 3' direction.

[0077] Thus, in some embodiments, the ligand-binding moiety, e.g., MS2, has an upstream sequence that is 1-12 nucleotides in length and a downstream sequence that is 1-12 nucleotides in length, where these upstream and downstream sequences are immediately adjacent to the ligand-binding moiety (i.e., there are no other nucleotides between the ligand-binding moiety and each of the upstream and downstream sequences), and the upstream sequence is complementary to the downstream sequence. In some embodiments, each of the upstream and downstream sequences is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 nucleotides in length. In one embodiment, each of the upstream and downstream sequences comprises or is GC. When both an upstream and downstream sequence are present, they may be referred to as extended sequences.

[0078] In some embodiments, both the upstream and downstream sequences are 2 nucleotides in length, or 3 nucleotides in length, or 4 nucleotides in length. In some embodiments, one of the upstream and downstream sequences is 2 nucleotides in length, and the other of the upstream and downstream sequences is 4 nucleotides in length. In some embodiments, one or both of the linker sequences is GC or GCGC or encodes GC or GCGC. In some embodiments, one of the upstream or downstream linker is GC, and the other of the upstream or downstream linker is GCGC.

[0079] qualification

[0080] In some embodiments, at least one of the gRNA or the ligand-binding portion is modified, or, if a linker is present, at least one of the gRNA, the ligand-binding portion, or the linker is modified. Modifications include the introduction of non-naturally occurring sites or species. Modifications can be used to enhance stability and / or specificity. In some embodiments, stability is improved with respect to resistance to the activation domain of a Cas protein (e.g., the RuvC domain) and / or the activation domain of one or more other enzymes in the system into which the complex of the invention is introduced (including, but not limited to, any effectors). Resistance, in some embodiments, is caused by steric hindrance. In some embodiments, modifications are located within one or more, if not all, nucleotides and / or between nucleotides within the targeted region.

[0081] Specificity is improved when the modification reduces the possibility of off-target effects and / or increases the likelihood that the base editing complex of the present invention will reach its target site. Nucleotides can be modified at the ribose, phosphate linkage, and / or base moiety. For example, phosphorothioate backbones can be used at one, several, or all positions within the gRNA, targeting region, or Cas association region and / or ligand binding portion and / or linker (if present).

[0082] In some embodiments, the modification is the presence of one or more 2'-modified nucleotides (e.g., 2'-O-methyl or 2'-fluoro) and / or the presence of phosphorothioate internucleotide linkages, or the introduction of a 5'-PO4 group in the gRNA and / or ligand binding moiety.

[0083] When more than one modification is present, the modifications may, for example, all be in the targeting region; all be in the Cas association region; all be in the ligand binding moiety; all be in the linker (if present); in both the targeting region and the Cas association region; in both the Cas association region and the ligand binding moiety; in both the Cas association region and the linker (if present); in both the targeting region and the ligand binding moiety; in both the targeting region and the linker (if present); in both the ligand binding moiety and the linker (if present); in all three of the Cas association region, targeting region, and ligand binding moiety; in the Cas association region, targeting region, and linker (if present); in the Cas association region, ligand binding moiety, and linker (if present); in the targeting region, ligand binding moiety, and linker (if present); or in each of the Cas association region, targeting region, ligand binding moiety, and linker (if present).

[0084] In some embodiments, there are 1 to 60, 1 to 30, 1 to 10, 10 to 20, 20 to 30, 30 to 40, 40 to 50, or 50 to 60 2' modifications. As non-limiting examples, the set of 2' modifications can be located in the targeting region, or, if the ligand-binding portion is or includes an oligonucleotide sequence, the set of 2' modifications can be located in the ligand-binding portion; or, the set of 2' modifications can be located in the Cas association region. The modifications can be on consecutive nucleotides, or there can be one or more pairs of unmodified nucleotides between modified nucleotides in a regular or irregular pattern. As a further non-limiting example, any one or more of positions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 within the gRNA contain a 2'-O-alkyl group, where the positions are counted from the 5' or 3' end of the gRNA.

[0085] In some embodiments, in addition to or in the absence of 2' modified nucleotides, modified internucleotide linkages such as phosphorothioate linkages are present. Examples of modifications to the backbone of the gRNA, aptamer (in the oligonucleotide), and linker (if present and of the oligonucleotide) include, but are not limited to, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates including 3'-alkylene phosphonates, 5'-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3'-amino phosphoramidates and aminoalkyl phosphoramidates, phosphorodiamidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, selenophosphates and boranophosphates with normal 3'-5' linkages, 2'-5' linked analogs thereof, and those with reversed polarity where one or more internucleotide linkages are 3'-3', 5'-5', or 2'-2' linkages. Preferred oligonucleotides having inverted polarity contain a single 3' to 3' linkage at the 3'-most internucleotide linkage, i.e., a single inverted nucleoside residue that may be abasic (one lacking a nucleobase or having a hydroxyl group instead). Various salts (e.g., potassium or sodium), mixed salts, and free acid forms of the foregoing internucleotide linkages are also within the scope of the present invention.

[0086] Also within the scope of the present invention is the use of polynucleotide backbones that do not contain phosphorus atoms and instead are formed by short-chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkyl or cycloalkyl internucleoside linkages, or one or more short-chain heteroatom or heterocyclic internucleoside linkages. These modifications include those with morpholino linkages (formed in part from the sugar portion of the nucleoside); siloxane backbones; sulfide, sulfoxide, and sulfone backbones; formacetyl and thioformacetyl backbones; methyleneformacetyl and thioformacetyl backbones; riboacetyl backbones; alkene-containing backbones; sulfamate backbones; methyleneimino and methylenehydrazino backbones; sulfonate and sulfonamide backbones; amide backbones; and other backbones containing a mixture of N, O, S, and CH2 moieties.

[0087] In some embodiments, one or more moieties of the conjugate have 1 to 60, or 1 to 20, or 1 to 10, or 10 to 20, or 20 to 30, or 30 to 40, or 40 to 50, or 50 to 60 phosphorothioate linkages. These phosphorothioate linkages may be all in the Cas association region; all in the ligand binding region; all in the linker; in both the targeting region and the Cas association region; in both the Cas association region and the ligand binding moiety; in both the Cas association region and the linker (if present); in both the targeting region and the ligand binding moiety; in both the targeting region and the linker (if present); in both the ligand binding moiety and the linker (if present); in all three of the Cas association region, targeting region, and ligand binding moiety; in the Cas association region, targeting region, and linker (if present); in the Cas association region, ligand binding moiety, and linker (if present); in the targeting region, ligand binding moiety, and linker (if present); in the Cas association region, targeting region, ligand binding moiety, and linker (if present); in the Cas association region, targeting region, ligand binding moiety, and linker (if present); in each of the Cas association region, targeting region, ligand binding moiety, and linker (if present).

[0088] Any nucleotide in the complexes of the present invention can contain one or more substituted sugar moieties. These nucleotides may contain a sugar substituent selected from the following: OH; H; F; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S-, or N-alkynyl; or O-alkyl-Co-alkyl, where alkyl, alkenyl, and alkynyl are substituted or unsubstituted C1-C10 alkyl or C2-C10 alkenyl and alkynyl. Particularly preferred are O((CH2) n O)mCH3, O(CH2) n OCH3, O(CH2) n NH2, O(CH2) n CH3, O(CH2) n ONH2, and O(CH2) n ON((CH2) nCH3)2, where n and m are 1 to about 10. Other suitable nucleotides contain sugar substituents selected from C1-C10 lower alkyl, substituted lower alkyl, alkenyl, alkynyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH3, OCN, Cl, Br, CN, CF3, OCF3, SOCH3, SO2CH3, ONO2, NO2, N3, NH2, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, RNA cleaving group, reporter group, intercalator, group for improving the pharmacokinetic properties of an oligonucleotide, or group for improving the pharmacodynamic properties of an oligonucleotide, and other substituents with similar properties. As non-limiting examples, suitable modifications include 2'-methoxyethoxy (2'-O-CH2CHOCH3, also known as 2'-O-(2-methoxyethyl) or 2'-MOE) (Martin et al., Helv. Chim. Acta, 1995, 78, 486-504) or another alkoxyalkoxy group. Further suitable modifications include the 2'-dimethylaminooxyethoxy, i.e., O(CH2)2ON(CH3)2 group (also known as 2'-DMAOE), and 2'-dimethylaminoethoxyethoxy (also known in the art as 2'-O-dimethyl-amino-ethoxy-ethyl or 2'-DMAEOE), i.e., 2'-O-CH2-O-CH2-N(CH3)2.

[0089] Other suitable sugar substituents include methoxy (-O-CH), aminopropoxy (-O-CHCHCHNH), allyl (-CH-CH=CH), -O-allyl (CH-CH=CH), and fluoro (F). The 2'-sugar substituent may be at the arabino position (up) or the ribo position (down). A suitable 2'-arabino modification is 2'-F. Similar modifications are also made at other positions in oligomeric compounds, particularly the 3' position of the sugar on the 3'-terminal nucleoside or in 2'-5' linked oligonucleotides, and at the 5' position of the 5'-terminal nucleotide.

[0090] Any nucleotide in the complexes of the invention may also contain modifications or substitutions of the nucleobase (often simply referred to in the art as "base"). Modified nucleobases include, but are not limited to, other synthetic and natural nucleobases such as 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl (-C=C-CH3) uracil and cytosine, and other alkyl derivatives of pyrimidine bases. alkynyl derivatives, 6-azouracil, cytosine, thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl, and other 8-substituted adenines and guanines, 5-halo, especially 5-bromo, 5-trifluoromethyl, and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 2-F-adenine, 2-aminoadenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine, and 3-deazaguanine and 3-deazaadenine. Additional modified nucleobases include, but are not limited to, tricyclic pyrimidines such as, for example, phenoxazine cytidine (1H-pyrimido(5,4-b)(1,4)benzoxazin-2(3H)-one)), phenothiazine cytidine (1H-pyrimido(5,4-b)(1,4)benzothiazin-2(3H)-one), G-clamps such as substituted phenoxazine cytidines (e.g., 9-(2-aminoethoxy)-H-pyrimido(5,4-(b)(1,4)benzoxazin-2(3H)-one), carbazole cytidine (2H-pyrimido(4,5-b)indol-2-one), pyridoindole cytidine (H-pyrido(3',2':4,5)pyrrolo(2,3-d)pyrimidin-2-one), and 5-methoxyuracil.

[0091] Heterocyclic base moieties include, but are not limited to, those in which the purine or pyrimidine base is replaced with other heterocycles, such as 7-deazaadenine, 7-deazaguanosine, 2-aminopyridine, 2-pyridone, etc. Other exemplary nucleobases include those disclosed in U.S. Pat. No. 3,687,808, those disclosed in The Concise Encyclopedia of Polymer Science and Engineering, pp. 858-859, Kroschwitz, JI, ed., John Wiley & Sons, 1990, those disclosed by Englisch et al., Angewandte Chemie, International Edition, 1991, 30, 613, and those disclosed by Sanghvi, YS, Chapter 15, Antisense Research and Applications, pp. 289-302, Crooke, ST and Lebleu, B. eds., CRC Press, 1993. These nucleobases are proven useful in increasing the binding affinity of the following oligomeric compounds: 5-substituted pyrimidines, 6-azapyrimidines, N-2, N-6, O-6 substituted purines (including 2-aminopropyladenine), 5-propynyluracil, and 5-propynylcytosine. Additionally, 5-methylcytosine substitutions are advantageous in combination with 2'-O-methoxyethyl sugar modifications.

[0092] In some embodiments, there are two ligand-binding moieties attached to the gRNA: a first ligand-binding moiety and a second ligand-binding moiety. Optionally, when there are two ligand-binding moieties, there are two linkers: a first linker and a second linker, with the first ligand-binding moiety attached to the first linker and the second ligand-binding moiety attached to the second linker. In these embodiments, the first linker and the second linker may each be attached to a Cas association region; or the first linker and the second linker may each be attached to a targeting region; or one of the first linker and the second linker may be attached to a Cas association region and the other of the first linker and the second linker may be attached to a targeting region.

[0093] Base editing complex

[0094] According to another embodiment of the present invention, there is a base editing complex. The base editing complex comprises, consists essentially of, or consists of a gRNA-ligand binding complex and a V-type Cas protein of the present invention, wherein the Cas association region of the gRNA-ligand binding complex associates with the V-type Cas protein. In this way, the gRNA can associate with the gRNA bearing the Cas protein, and deliver the Cas protein to the target nucleic acid without the need for a tracrRNA.

[0095] An example of a base editing complex of the invention is shown in Figure 12. A gRNA 310 is bound to a Cas protein 340. A ligand-binding moiety 320 is attached to the 5' end of the gRNA. An effector 350, which may be, for example, a deaminase, is recruited at 360 to a ligand 330 via an RNA-ligand binding interaction.

[0096] V-type Cas proteins

[0097] Typically, Cas proteins contain at least one RNA-binding domain, which interacts with the gRNA at the Cas association region. The V-type Cas proteins used in the present invention are capable of associating with the gRNA-ligand complex in the absence of tracrRNA. In some embodiments, the V-type Cas protein is an endonuclease containing a RuvC domain. The RuvC domain may be mutated to inactivate the endonuclease activity. In some embodiments, the protein is a nickase containing an activated or inactivated RuvC domain.

[0098] Examples of V-type Cas proteins that may be used in connection with the present invention include, but are not limited to, activated or inactivated Cas12a, MAD7 (an engineered variant of ErCas12a), Cas12h, Cas12i, and Cas12j (also known as CasPhi, CasΦ).

[0099] In some embodiments, the type V Cas protein comprises a fusion protein having (a) an activated, partially inactivated, or inactivated type V Cas protein and (b) a uracil DNA glycosylase (UNG) inhibitor peptide (UGI). The UGI peptide can be fused directly to the type V Cas protein or via a linker peptide consisting of 1 to 100 amino acid residues. In some embodiments, the UGI comprises the wild-type UGI sequence from Bacillus phage PBS2 (https: / / www.ncbi.nlm.nih.gov / protein / P14739): MTNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTAYDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKML (SEQ ID NO: 140). In some embodiments, the UGI comprises a fragment of the wild-type UGI peptide or a variant of SEQ ID NO: 140 comprising an amino acid sequence homologous to SEQ ID NO: 28. In some embodiments, the UGI fragment of the homologous sequence comprises at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or at least 99.5% homology to the wild-type UGI peptide sequence (SEQ ID NO: 140).

[0100] In some embodiments, an activated or inactivated Type V Cas protein comprises a fusion with two or more UGI peptides or variants. A UGI peptide or variant of a UGI peptide can be linked to another UGI peptide or Type V Cas protein directly or via a linker of 1 to 100 amino acid residues.

[0101] The Cas protein or Cas protein fusion can be provided in purified or isolated form, or can be part of a composition or complex. Preferably, when in a composition, the protein is first purified to some degree, and more preferably to a high level of purity (e.g., about 80%, 90%, 95%, or 99% or greater). The compositions in which the complexes and components of the invention can be stored and transferred can be any type of composition desired, such as an aqueous composition suitable for use as, or inclusion in, a composition for RNA-guided targeting.

[0102] effector

[0103] The base editing complexes of the present invention may include an effector attached to a ligand. The ligand can bind reversibly or irreversibly to the ligand-binding moiety. Thus, the ligand-binding moiety can retain its binding to the ligand and recruit an effector (e.g., a base editing enzyme) that is fused to or otherwise bound to the ligand. This design is particularly advantageous because it provides a modular design in which the nucleic acid sequence targeting function of the gRNA and the effector function reside on different molecules. For example, to introduce sequential modifications at the same site, different effectors that bind to the same ligand can be used. Conversely, to introduce the same modifications at different sites, the same effector-ligand can be used with the same ligand-binding moiety on different gRNAs. This design therefore allows for multiplexing of the system without the undesirable burden of fusing effectors to either the gRNA or the Cas protein.

[0104] Examples of effectors that can be used in connection with the present invention include deaminases, such as those with cytidine deamination or adenine deamination activity, as well as transcriptional regulators, repair enzymes, epigenetic modifiers, histone acetylases, deacetylases, histone and nucleotide methylases, and histone and nucleotide demethylases. In some embodiments, the effector is selected from the group consisting of AID, CDA, APOBEC1, APOBEC3A, APOBEC3B, APOBEC3C, APOBEC3D, APOBEC3F, ADA, ADAR, and tRNA adenosine deaminase. Examples of effectors and the types of genetic changes they cause are listed in Table 1. [Table 1] Effector protein full name: AID: Activation-induced cytidine deaminase, also known as AICDA APOBEC1: Apolipoprotein B mRNA-editing enzyme, polypeptide-like catalytic 1. APOBEC3A: Apolipoprotein B mRNA-editing enzyme, polypeptide-like catalytic 3A APOBEC3B: Apolipoprotein B mRNA-editing enzyme, polypeptide-like catalytic 3B APOBEC3C: Apolipoprotein B mRNA-editing enzyme, polypeptide-like catalytic 3C APOBEC3D: Apolipoprotein B mRNA editing enzyme, polypeptide-like catalytic 3D APOBEC3F: Apolipoprotein B mRNA-editing enzyme, polypeptide-like catalytic 3F APOBEC3G: Apolipoprotein B mRNA-editing enzyme, polypeptide-like catalytic 3G APOBEC3H: Apolipoprotein B mRNA editing enzyme, polypeptide-like catalytic 3H ADA: adenosine deaminase ADAR1: RNA-acting adenosine deaminase 1 ADAR2: RNA-acting adenosine deaminase 2 ADAR3: RNA-acting adenosine deaminase 3 Dnmt1: DNA (cytosine-5)-methyltransferase 1 Dnmt3a: DNA (cytosine-5)-methyltransferase 3 alpha TadA: a tRNA-specific adenosine deaminase TADA: tRNA (adenine (34)) deaminase, chloroplast-specific TAD3: tRNA-specific adenosine deaminase TAD3 TET1: Methylcytosine dioxygenase TET1 TET2: Methylcytosine dioxygenase TET2 TDG: G / T mismatch-specific thymine DNA glycosylase

[0105] In some embodiments, the base editing complex comprises two or more effectors. When there are two effectors, they may be referred to as a first effector and a second effector. Each effector may be attached to a different ligand-binding moiety via a different ligand. Alternatively, when there are two effectors, one is attached to a ligand and binds to the gRNA through a ligand-binding moiety, and the other is attached directly to the Cas protein. Examples of deaminase sequences that may be incorporated into the present invention include, but are not limited to, the following: hA3A (SEQ ID NO: 149): ATGGAGGCATCTCCAGCATCCGGTCCAAGGCATCTCATGGATCCCCATATCTTCACCTCCAATTTTAATAACGGAATCGGGCGCCACAAGACATACTTGTGCTATGAGGTGGAACGACTGGACAACGGTACCTCCGTGAAAATGGACCAACATCGCGGATTTCTGCATAATCAGGCTAAAAACCTTCTGTGTGGATTTTATGGGAGACACGCTGAGCTGAGATTTCTTGACCTGGTCCCGAGCTTACAGCTGGACCCAGCCCAAATCTATCGCGTAACTTGGTTCATCAGCTGGAGCCCCTGCTTTTCCGCCGGGTGCGCTGGAGAAGTGCGGGCGTTCCTGCAGGAAAACACCCACGTCAGACTGAGGATTTTTGCAGCACGCATCTACGACTATGATTATCTTTACAAGGAGGCATTACAGATGTTGCGCGATGCCGGAGCCCAAGTAAGCATTATGACTTATGATGAGTTCAAACACTGTTGGGACACCTTTGTAGACCACCAGGGCTGCCCCTTTCAGCCTTGGGATGGGCTCGACGAGCACAGCCAGGCACTCAGCGGACGCCTCCGCGCTATCCTCCAGAACCAGGGTAAC · rat APO (SEQ ID NO: 33): ATGTCCTCAGAGACTGGGCCTGTCGCCGTCGATCCAACCCTGCGCCGCCGGATTGAACCTCACGAGTTTGAAGTGTTCTTTGACCCCCGGGAGCTGAGAAAGGAGACATGCCTGCTGTACGAGATCAACTGGGGAGGCAGGCACTCCATCTGGAGGCACACCTCTCAGAAC ACAAATAAGCACGTGGAGGTGAACTTCATCGAGAAGTTTACCACAGAGCGGTACTTCTGCCCCAATCCAGATGTAGCATCACATGGTTTCTGAGCTGGTCCCCTTGCGGAGAGTGTAGCAGGCCATCACCGAGTTCCTGTCCAGATATCCACACGTGACACTGTTTATCT ACATCGCCAGGCTGTATCACCACGCAGACCCAAGGAATAGGCAGGGCCTGCGCGATCTGATCAGCTCCGGCGTGACCATCCAGATCATGACAGAGCAGGAGTCCGGCTACTGCTGGCGGAACTTCGTGAATTATTCTCCTAGCAACGAGGCCCACTGGCCTAGGTACCCACA CCTGTGGGTGGCCCTGTACGTGCTGGAGCTGTATTGCATCATCCTGGGCCTGCCCCCTTGCTCTGAATATCCTGCGGAGAAAGCAGCCCCAGCTGACCTTCTTTTACAATCGCCCTGCAGTCTTGTCACTATCAGAGGCTGCCACCCCCACATCCTGTGGGCCACAGGCCTGAAG ·GgAID sequence (sequence number 34): ATGGATTCTCTGCTGATGAAGAGGAAGCTGTTTCTGTACAATTTTAAGAATCTGAGGTGGGCCAAGGGCAGAAGGGAGACCTATCTGTGCTACGTGGTGAAGAGAAGGGACAGCGCCACCAGCTGCAGCCTCGATTTCGGCTATCTGAGGAACAAGATGGGCTGTCACGTGGAGGTGCTGTTTCTGAGATACATCTCCGCTTGGGATCTGGATCCCGGCAGATGCTATAGAATCACATGGTTCACCAGCTGGAGCCCTTGTTACGACTGTGCTAGACATGTGGCCGACTTTCTGAGGGCCTATCCCAATCTGACACTGAGAATCTTCACCGCTAGACTGTACTTCTGCGAGGACAGAAAGGCTGAGCCCGAGGGACTGAGAAGGCTGCACAGAGCCGGCGCCCAGATCGCCATCATGACCTTTAAGGACTTTTTCTATTGCTGGAACACCTTCGTGGAGAATAGAGAGAAGACCTTCAAGGCTTGGGAGGGACTGCACGAGAACTCCGTGCATCTGTCTAGAAAGCTGAGGAGAATTCTGCTGCCTCTGTATGAGGTGGACGATCTGAGAGATGCCTTCAAGACCCTCGGACTG ·hAID (SEQ ID NO: 141): ATGGATAGCCTGCTGATGAACCGGAGAAAGTTCCTGTATCAGTTTAAGAATGTGCGCTGGGCAAAGGGCAGGCGCGAGACCTACCTGTGCTATGTGGTGAAGCGGAGAGATTCCGCCACATCCTTCTCTCTGGACTT TGGCTACCTGCGGAACAAGAATGGCTGCCACGTGGAGCTGCTGTTCCTGAGATACATCTCTGACTGGGATCTGGACCCAGGCAGGTGTTATCGCGTGACCTGGTTCACAAGCTGGTCCCCTGCTACGATTGTGCAAGGCACGTGGCAGACT TTCTGAGGGGAAACCCAAATCTGTCCCTGCGGATCTTCACCGCCAGACTGTATTTTTGCGAGGATAGGAAGGCAGAGCCAGAGGGACTGAGGCGCCTGCACAGGGCCGGCGTGCAGATCGCCATCATGACCTTCAAGGACTACTTTTATTGT TGGAACACCTTCGTGGAGAATCACGAGCGGACCTTCAAGGCCTGGGAGGGACTGCACGAGAACTCCGTGCGGCTGTCTAGACAGCTGCGGAGAATCCTGCTGCCTCTGTACGAGGTGGACGATCTGAGGGATGCCTTCCGCACCCTGGGACTG

[0106] Ligand

[0107] As noted above, the effector is bound to the ligand, e.g., by one or more covalent bonds. A non-exhaustive list of examples of ligand-binding moiety-ligand pairs that may be used in various embodiments of the invention is provided in Table 2. Both unmodified and chemically modified forms, or ligand-binding moieties and ligands, are within the scope of the invention.

[0108] [Table 2] Some of the sequences of the above binding pairs are shown below. 1. Telomerase Ku-binding motif / Ku heterodimer a. Ku-binding hairpin 5'-UUCUUGUCGUACUUAUAGAUCGCUACGUUAUUUCAAUUUUGAAAAUCUGAGUCCUGGGAGUGCGGA-3' (SEQ ID NO: 14) b. Ku heterodimer (SEQ ID NO: 15) MVRSGNKAAVVLCMDVGFTMSNSIPGIESPFEQAKKVITMFVQRQVFAENKDEIALVLFGTDGTDNPLSGGDQYQNITVHRHLMLPDFDLLEDIESKIQPGSQQADFLDALIVSMDVIQHETI GKKFEKRHIEIFTDLSRFSKSQLDIIIHSLKKCDISERHSIHWPCRLTIGSNLSIRIAAYKSILQERVKKTWTVVDAKTLKKEDIQKETVYCLNDDDETEVLKEDIIQGFRYGSDIVPFSKV DEEQMKYKSEGKCFSVLGFCKSSQVQRRFFMGNQVLKVFAARDDEAAAVALSSLIHALDDLDMVAIVRYAYDKRANPQVGVAFPHIKHNYECLVYVQLPFMEDLRQYMFSSLKNSKKYAPTEAQLNAVDALIDSMSLAKKDEKTDTLEDLFPTTKIPNPRFQRLFQCLLHRALHPREPLPPIQQHIWNMLNPPAEVTTKSQIPLSKIKTLFPLIEAKKKDQVTAQEIFQDNHEDGPTAK (SEQ ID NO: 16) 2. Telomerase Sm7 binding motif / Sm7 homoheptamer a. Sm consensus site (single-stranded) 5'-AAUUUUUGGA-3' (SEQ ID NO: 17) b. Monomeric Sm-like protein (archaea) GSVIDVSSQRVNVQRPLDALGNSLNSPVIIKLKGDREFRGVLKSFDLHMNLVLNDAEELEDGEVTRRLGTVLIRGDNIVYISP (SEQ ID NO: 18) 3. MS2 phage operator stem loop / MS2 coat protein a. MS2 phage operator stem loop 5'-ACAUGAGGAUCACCCAUGU-3' (SEQ ID NO: 19) B MS2 coat protein MASNFTQFVLVDNGGTGDVTVAPSNFANGIAEWISSNSRSQAYKVTCSVRQSSAQNRKYTIKVEVPKGAWRSYLNMELTIPIFATNSDCELIVKAMQGLLKDGNPIPSAIAANSGIY (SEQ ID NO: 20) 4. PP7 phage operator stem loop / PP7 coat protein a. PP7 phage operator stem loop 5'-AUAAGGAGUUUAUAUGGAAACCCUUA-3' (SEQ ID NO: 21) b. PP7 coat protein (PCP) MSKTIVLSVGEATRTLTEIQSTADRQIFEEKVGPLVGRLRLTASLRQNGAKTAYRVNLKLDQADVVDCSTSVCGELPKVRYTQVWSHDVTIVANSTEASRKSLYDLTKSLVATSQVEDLVVNLVPLGR (SEQ ID NO: 22) 5. SfMu Com stem-loop / SfMu Com binding protein a.SfMu Com stem loop 5'-CUGAAUGCCUGCGAGCAUC-3' (SEQ ID NO: 23) b. SfMu Com-binding protein MKSIRCKNCNKLLFKADSFDHIEIRCPRCKRHIIMLNACEHPTEKHCGKREKITHSDETVRY (SEQ ID NO: 24) 6.BoxB aptamer / lambda N22plus a.BoxB aptamer 5'-GCCCUGAAGAAGGGC-3' (SEQ ID NO: 25) b. Lambda N22plus protein MNARTRRRERRAEKQAQWKAAN (SEQ ID NO: 26) 7. Csy4-binding stem-loop / Csy4[H29A] a.Csy4 binding motif 5'-CUGCCGUAUAGGCAGC-3' (SEQ ID NO: 27) b.Csy4[H29A] MDHYLDIRLRPDPEFPPAQLMSVLFGKLAQALVAQGGDRIGVSFPDLDESRSRLGERLRIHASADDLRALLARPWLEGLRDHLQFGEPAVVPHPTPYRQVSRVQAKSNPERLRRRLMRRHDLSEEEARKRIPDTVARALDLPFVTLRSQSTGQHFRLFIRHGPLQVTAEEGGFTCYGLSKGGFVPWF (SEQ ID NO: 28)

[0109] For each of the foregoing sequences, for example, identical sequences or sequences with one or more insertions, deletions, or substitutions in one or both sequences of the binding pair can be used. As non-limiting examples, sequences at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identical to the foregoing sequences can be used in one or both members of the binding pair.

[0110] Other Chemistry

[0111] In some embodiments, the base editing complexes of the present invention are combined with additional chemical technologies. For example, in some embodiments, the base editing complexes further comprise a cysteine / selenocysteine ​​tag. In some embodiments, the base editing complexes comprise or are linked to elements for cycloaddition via click chemistry.

[0112] Base editing methods

[0113] In another embodiment, the present invention provides base editing methods. In these methods, a base editing complex of the present invention is exposed to double-stranded DNA, a solution containing dsDNA, a cell containing dsDNA, or a subject. The method may be performed in vitro, in vivo, or ex vivo, and may include a step of delivering the base editing complex to a subject as part of a therapeutic medicament.

[0114] These methods include, for example, the isolation and characterization of T cells (including primary T cells), natural killer (NK) cells, B cells, CD34 + The methods can be used to modify immune cells selected from hematopoietic stem and progenitor cells (HSPCs). The immune cells may be modified (engineered) immune cells, such as T cells containing a CAR or TCR. Thus, the methods herein can be applied to further engineer (modify) cells already modified to contain therapeutically useful CARs and / or TCRs. As a further example, primary immune cells naturally occurring in a host animal or patient or derived from stem cells or induced pluripotent stem cells (iPSCs) can be genetically modified using the methods and complexes provided herein. Suitable stem cells include, but are not limited to, mammalian stem cells, such as human stem cells, including, but not limited to, hematopoietic stem cells, neural stem cells, embryonic stem cells, induced pluripotent stem cells (iPSCs), mesenchymal stem cells, mesodermal stem cells, liver stem cells, pancreatic stem cells, muscle stem cells, and retinal stem cells. Other stem cells include, but are not limited to, mammalian stem cells, such as mouse stem cells, e.g., mouse embryonic stem cells.

[0115] Also provided herein are methods for genome engineering (e.g., altering or manipulating the expression of one or more genes or one or more gene products) in prokaryotic or eukaryotic cells in vitro, in vivo, or ex vivo. In particular, the methods provided herein are useful for the generation of primary human T cells, natural killer (NK) cells, CD34 +It may be useful for targeted base editing disruption in mammalian cells, including hematopoietic stem and progenitor cells (HSPCs) (e.g., HSPCs isolated from umbilical cord blood or bone marrow and cells differentiated therefrom, as well as HSPCs isolated from mobilized peripheral blood).

[0116] Also provided herein are genetically modified cells derived from hematopoietic stem cells, such as T cells, that have been modified according to the methods described herein.

[0117] In some cases, the methods are configured to produce genetically engineered T cells derived from HSCs or iPSCs that are suitable as "universally acceptable" cells for therapeutic applications. Hematopoietic stem cells (HSCs) arise from hemangioblasts, which can give rise to hematopoietic stem cells, vascular smooth muscle cells, and hemangioblasts, which differentiate into vascular endothelial cells. Hematopoietic stem cells give rise to common myeloid progenitor cells and common lymphoid progenitor cells, which give rise to T cells, natural killer (NK) cells, B cells, myeloblasts, erythroblasts, and other cells involved in cell production in the blood, bone marrow, spleen, lymph nodes, and thymus. Such methods are also applicable to the production of natural killer (NK) cells, CD34 + It is applicable to hematopoietic stem and progenitor cells (HSPCs), such as HSPCs isolated from umbilical cord blood or bone marrow and cells differentiated therefrom, as well as HSPCs isolated from mobilized peripheral blood.

[0118] In another aspect, provided herein is a method for targeting a disease for correction by base editing. In some methods, the base editing complex is delivered to a subject for treatment. The target sequence can be any disease-related polynucleotide or gene. Examples of useful applications of mutation or correction of endogenous gene sequences according to the present invention include, but are not limited to, modifying disease-related gene mutations, modifying sequences encoding splice sites, modifying regulatory sequences, modifying sequences that cause gain-of-function mutations and / or modifying sequences that cause loss-of-function mutations, and targeted modification of sequences encoding structural features of proteins.

[0119] Delivery of components into cells

[0120] The base editing complex or its components can be delivered to target cells or organisms by various methods, in various formats (DNA, RNA, protein), or a combination of these different formats. Base editing components can be delivered as (a) a DNA polynucleotide encoding a sequence associated with a protein effector or guide RNA; (b) a synthetic RNA encoding the sequence of the protein effector (messenger RNA) or guide RNA; or (c) a purified effector protein. When delivered in protein format, the V-type Cas protein assembles with the guide RNA to form a ribonucleoprotein complex (RNP) that can be delivered to target cells or organisms.

[0121] For example, the assembled components or complexes can be delivered together or separately by electroporation, by nucleofection, by transfection, via nanoparticles, by viral-mediated RNA delivery, by non-viral-mediated delivery, via extracellular vesicles (e.g., exosomes and microvesicles), eukaryotic cell transfer (e.g., via recombinant yeast), and other methods that can package molecules for delivery to target living cells without altering the overall genome landscape.

[0122] Other methods include, but are not limited to, non-integrative transient transfer of DNA polynucleotides containing sequences related to protein recruitment, such that the molecules are transcribed into the desired RNA molecules and the amino acid-containing components are translated into proteins or protein fragments. These include, but are not limited to, DNA-only vehicles (e.g., plasmids, minicircles, minivectors, ministrings, protelomerase-generated DNA molecules (e.g., doggybone™), artificial chromosomes (e.g., HACs), and cosmids), nanoparticles, extracellular vesicles (e.g., exosomes and microvesicles), eukaryotic cell transfer (e.g., recombinant yeast), transient viral transfer with AAV, non-integrative viral particles (e.g., lentivirus- or retrovirus-based systems), cell-penetrating peptides, and other techniques that can introduce DNA into cells without directly integrating into the entire genome. Another method for the introduction of RNA components is the use of integrative gene transfer technologies to stably introduce the machinery for RNA transcription into the genome of target cells. This can be controlled via constitutive or promoter-inducible systems to attenuate RNA expression, and can also be designed to allow removal of the system after its intended purpose (e.g., by introducing a Cre-Lox recombination system). Such techniques for stable gene transfer include, but are not limited to, integration via viral particles (e.g., lentivirus-, adenovirus-, or retrovirus-based systems), transposase-mediated transfer (e.g., Sleeping Beauty or PiggyBac™), the use of non-homologous repair pathways induced by DNA breaks (e.g., using CRISPR or TALEN), surrogate DNA molecules, and other techniques that promote integration of target DNA into the cells of interest.

[0123] The various components of the complexes of the present invention, if not enzymatically synthesized in cells or in solution, can be produced chemically or, if naturally occurring, isolated and purified from naturally occurring sources. Methods for chemically and enzymatically synthesizing various embodiments of the present invention are well known to those skilled in the art. Similarly, methods for linking or introducing covalent bonds between components of the present invention are well known to those skilled in the art.

[0124] Application Examples

[0125] As non-limiting examples, the complexes of the present invention can be used to recruit transcriptional activators, such as p65 and V64, or moieties that introduce epigenetic modifications or affect HDR. The complexes of the present invention can also be used for the following applications: base editing, genome editing, genome screening, therapeutic cell generation, genome tagging, epigenome editing, karyotype modification, chromatin imaging, transcriptome and metabolic pathway modification, genetic circuit modification, cell signal sensing, cellular event recording, lineage reconstruction, genetic manipulation, DNA genotyping, miRNA quantification, in vivo cloning, site-directed mutagenesis, genome diversification, and in situ proteome analysis. In some embodiments, a cell or cell population is exposed to the base editing complex of the present invention, and the cell or cell population is introduced into a subject by intravenous infusion.

[0126] Applications include research into human diseases such as cancer immunotherapy, antiviral therapy, bacteriophage therapy, cancer diagnosis, pathogen screening, microbiota remodeling, stem cell reprogramming, immunogenomic engineering, vaccine development, and antibody production.

[0127] Example

[0128] Example 1: Transfection of dCasPhi base editing plasmid components:

[0129] Vector construction:

[0130] The coding sequence of the inactivated version of CasPhi and the 2xUGI fusion (dCasPhi-2xUGI) was obtained and cloned into an expression vector under the control of the mouse CMV promoter in a T2A polycistronic cassette with a red fluorescent protein-puromycin fusion. The coding sequence of the MS2 coat protein lizard Anolis Apobec fusion (MCP-lizAnoA1) ("AnoA1") is as follows: ATGGCCCCCAAGAAGAAGCGGAAAGTGATGGAGCCGGAGGCTTTTCAGCGCAACTTTGACCCTCGGGAATTTGCCGCCTGTACACTCCTCTTGTATGAGATCCACTGGGACAATAACACATCTAGAAATTGGTGTACGAATAAGCCTGGGCTCCACGCTGAGGAGAATTTCTTGCAGATATTTAATGAGAAAATTGACATTAAACAGGATACGCCGTGCTCTATAACATGGTTCCTTTCTTGGAGCCCCTGTTACCCTTGTAGCCAAGCAATAATAAAATTCTTGGAGGCACACCCGAATGTCAGTCTGGAGATTAAGGCTGCGCGGCTGTATATGCATCAAATAGACTGTAACAAGGAGGGACTCAGAAATCTGGGCCGGAATCGAGTGTCAATAATGAACCTGCCTGATTATAGGCATTGCTGGACTACGTTTGTTGTGCCAAGGGGAGCAAACGAAGATTACTGGCCACAAGACTTTCTGCCTGCGATCACAAATTACTCCCGAGAACTCGACTCCATACTGCAGGATGAGCTGAAGACACCCCTGGGCGACACCACACACACCTCTCCACCTTGCCCAGCACCAGAGCTGCTGGGAGGCCCTATGGCCAGCAACTTCACACAGTTTGTGCTGGTGGATAATGGAGGAACCGGCGACGTGACAGTGGCACCATCTAACTTTGCCAATGGCATCGCCGAGTGGATCAGCTCCAACTCTCGGAGCCAGGCCTATAAGGTGACCTGTAGCGTGCGGCAGTCTAGCGCCCAGAATAGAAAGTATACAATCAAGGTGGAGGTGCCTAAGGGCGCCTGGAGATCCTACCTGAACATGGAGCTGACCATCCCAATCTTTGCCACAAATTCTGATTGCGAGCTGATCGTGAAGGCCATGCAGGGCCTGCTGAAGGACGGCAACCCTATCCCAAGCGCCATCGCCGCCAATAGCGGAATCTACACGCGTAAAATCAGCCTCGACTGTGCCTTCTAG (SEQ ID NO: 139). This sequence was obtained and cloned into an expression vector under the control of the mouse CMV promoter. The sequence of the crRNA containing the MS2 ligand-binding portion and a unique spacer region was cloned into an expression vector under the control of the hU6 promoter.

[0131] HEK293T cells (ATCC, #CRL-11268) were seeded at 20,000 cells per well in 96-well plates the day before transfection. Cells were co-transfected with DharmaFECT™ Duo Transfection Reagent (Horizon Discovery, #T-2010) and 200 ng of dCasPhi-2xUGI plasmid, 100 ng of AnoA1 plasmid, and 100 ng of crRNA plasmid. The plasmid crRNA consisted of a 35-nucleotide direct repeat and a different 18- or 20-nucleotide spacer sequence targeting transcripts within the Site 2 or B2M gene target. Additionally, the MS2 ligand-binding moiety was located at the 5' end, 3' end, internally (not at the 5' or 3' end), or a combination thereof. See SEQ ID NOs: 35-58 in Table 3 below.

[0132] Cell treatment

[0133] Cells were lysed in 100 μL of a buffer containing proteinase K (Thermo Scientific, #FEREO0492), RNase A (Thermo Scientific, #FEREN0531), and Phusion® HF buffer (Thermo Scientific, #F-518L) at 56°C for 30 minutes, followed by heat inactivation at 95°C for 5 minutes. This cell lysate was used to generate PCR amplicons spanning the region containing the base-edited site(s). PCR amplicons ranging in length from 400 to 1000 bp were sequenced by Sanger sequencing.

[0134] Editorial Analysis

[0135] Base editing efficiency was calculated using the Chimera analysis tool, a modified version of the open-source tool BEAT. Chimera first determines editing efficiency by subtracting background noise and defining the expected variability across samples. This allows us to estimate editing efficiency without normalizing to a control sample. Chimera then uses the Median Absolute Deviation (MAD) method to remove any outliers from the noise and evaluates the editing efficiency of base editors across a range of 18–20 bp of input guide sequences.

[0136] Table 3 provides a list of plasmid guide sequences. The sequences of the spacer region are in bold. The direct repeat sequences are underlined. The sequences of the ligand binding moieties are in italics.

[0137] [Table 3] TIFF2024502348000005.tif220152TIFF2024502348000006.tif124153

[0138] Figure 1A-Figure 1G provide guide RNA fold predictions using Geneious software for CasPhi guide RNAs, with parameters as follows: No MS2 aptamer, SEQ ID NO: 59, Figure 1A ; The MS2 aptamer is 5' of pre-cr, SEQ ID NO: 60, Figure 1B ; MS2 aptamer incorporated into pre-cr, SEQ ID NO: 61, Figure 1C ; The MS2 aptamer is within the loop, SEQ ID NO: 62, Figure 1D ; The MS2 aptamer is at the 3′ end of the guide, SEQ ID NO: 63, Figure 1E ; No MS2 sequence, no pre-cr, SEQ ID NO: 64, Figure 1F; and The MS2 sequence is 5' of the crRNA, SEQ ID NO: 65, Figure 1G. In these figures, the spacer sequence is shown attached to an oligonucleotide that does not constitute a gRNA: TAAAGGAAACTGAACAC (SEQ ID NO: 66).

[0139] We evaluated the effect of MS2 placement on base editing levels at the two indicated genomic target sites using the sequences in Table 3. Editing results are shown for the 11 targeted C residues, indicated by their positions in Figures 2A and 2B.

[0140] In these experiments, we varied the placement of the MS2 aptamer and compared the C>T base editing efficiency of guide RNAs targeting (A) Site 2 gRNA4, SEQ ID NOs: 35, 37, 39, 45, 47, and 49 (see Figure 2A), and (B) Site 2 gRNA5, SEQ ID NOs: 36, 38, 40, 46, 48, and 50 (see Figure 2B). HEK293T cells were co-transfected with the aforementioned guide RNA plasmids, dCasPhi plasmid, and deaminase plasmid (liz AnoA1). Cells were analyzed for base editing using Chimera software. Data show the % C>T conversion rate at the indicated cytosines along the spacer sequence.

[0141] Data indicate that the 5' MS2 pre-cr configuration results in higher levels of base editing at both sites compared to other MS2 configurations. Therefore, for the CasPhi system, gRNAs may encode SEQ ID NO: 137 and / or SEQ ID NO: 138 (see Figure 4E and Figure 4F).

[0142] Example 2: Transfection of CasPhi (Cas12j) DNA cleavage plasmid components

[0143] Vector construction

[0144] The coding sequence of CasPhi was obtained and cloned into an expression vector under the control of the mouse CMV promoter (mCMV) in a T2A polycistronic cassette carrying a red fluorescent protein-puromycin fusion. The crRNA and unique spacer region sequences were cloned into an expression vector under the control of the hU6 promoter.

[0145] HEK293T cells (ATCC, #CRL-11268) were seeded at 20,000 cells per well in a 96-well plate the day before transfection. Cells were co-transfected with DharmaFECT™ Duo transfection reagent (Horizon Discovery, #T-2010) and 200 ng of CasPhi plasmid and 100 ng of crRNA plasmid. The plasmid crRNA consists of a 35-nucleotide direct repeat.

[0146] Cell treatment :

[0147] Cells were lysed in 100 μL of a buffer containing Proteinase K (Thermo Scientific, #FEREO0492), RNase A (Thermo Scientific, #FEREN0531), and Phusion® HF buffer (Thermo Scientific, #F-518L) at 56°C for 30 minutes, followed by heat inactivation at 95°C for 5 minutes. This cell lysate can be used to generate PCR amplification products spanning the region containing the base-edited site(s). PCR amplification products between 400-1000 bp in length can be digested with T7 endonuclease I (T7EI) enzyme (NEB, M0302S) in the presence of NEBuffer2® (NEB, B7002S) for 25 minutes. Digested PCR products can be run on a 2% agarose gel at 80 volts for 90 minutes, imaged, and analyzed using Horizon Discovery's online T7EI calculator (https: / / horizondiscovery.com / en / ordering-and-calculation-tools / t7ei-calculator).

[0148] In this example, SEQ ID NOs: 36, 38, 40, 42, 44, and 46 from Table 3 were used.

[0149] The percentage of C to T edits is shown in Figure 2C and Figure 2D. These figures demonstrate that the inclusion of MS2 at the 5' pre-cr position, embedded within the 5' pre-cr, and 3' position of site 2 gRNA5 does not affect the ability of CasPhi to induce indel formation.

[0150] Example 3: Guided base editing for multiple sets of inactivated CasPhi mutants

[0151] Different inactivating mutations of CasPhi (D369A, E566A, and D369A / E566A / D658A) were compared for base editing efficiency. HEK293T cells were transfected with the dCasPhi-2xUGI plasmid, the AnoA1 plasmid, and the indicated plasmid gRNAs (35, 37, 39, and 45 for site 2 gRNA4 (Figure 3A), and 36, 38, 40, and 46 for site 2 gRNA5 (Figure 3B)). Cells were harvested and base editing levels were analyzed using Chimera software. Data summarized in Figure 3A and Figure 3B show the C>T conversion rate (%) at the indicated cytosine positions along the spacer.

[0152] These data demonstrate the ability to edit multiple distinct target C residues in HEK Site 2 using the dCasPhi 5'MS2 pre-cr guide with different inactivating mutations. Table 4 shows the inactivated CasPhi sequences.

[0153] [Table 4] TIFF2024502348000008.tif231152TIFF2024502348000009.tif231152TIFF2024502348 000010.tif231153TIFF2024502348000011.tif231157TIFF2024502348000012.tif41154

[0154] Example 4: Base editing with different spacer lengths

[0155] In one experiment, HEK293T cells were electroporated with dCasPhi-2xUGI mRNA, AnoA1 mRNA, and the indicated extended spacer synthetic gRNA for Site 2 gRNA5 (SEQ ID NOS: 101 and 102) (see Table 5). In the second experiment, HEK293T cells were transfected with dCasPhi-2xUGI plasmid, AnoA1 plasmid, and the indicated gRNA for Site 2 gRNA5 (SEQ ID NOS: 36, 38, 54, and 57) (see Table 3). Cells were harvested and analyzed for base editing levels using Chimera software. Data show the percentage of C>T conversion at the indicated cytosine positions along the spacer. The percentage of C to T editing for each experiment is shown in Figure 4A and Figure 4B, respectively.

[0156] The gRNAs used in this example can be more generally represented by the schematic diagrams in Figures 4C to 4F: Figure 4C is a schematic diagram of a synthetic gRNA with an 18 nt spacer, where the spacer is represented by multiple Ns (SEQ ID NO: 135). Figure 4D is a schematic diagram of a synthetic gRNA with a 20 nt spacer represented by multiple Ns (SEQ ID NO: 136). Figure 4E is a schematic diagram of the plasmid gRNA with an 18 nt spacer (SEQ ID NO: 137). Figure 4F is a schematic diagram of the plasmid gRNA with a 20 nt spacer. (SEQ ID NO: 138)

[0157] These data demonstrate that base editing is successful when different spacer lengths are used in gRNAs containing ligand-binding moieties.

[0158] Example 5: Electroporation of mRNA and synthetic guides for dCasPhi (Cas12j) base editing

[0159] Preparation of mRNA:

[0160] Messenger RNA was prepared from a DNA vector carrying a T7 promoter and the coding sequences of dCasPhi-2xUGI and AnoA1 according to standard protocols for mRNA in vitro transcription.

[0161] RNA synthesis:

[0162] All crRNAs were synthesized by Horizon Discovery or Agilent using either 2'-acetoxyethyl orthoester (2'-ACE) or 2'-tert-butyldimethylsilyl (2'-TBDMS) protection chemistry. Chemical modifications were included where noted. RNA oligos were 2'-deprotected, desalted, and purified by either high-performance liquid chromatography (HPLC) or polyacrylamide gel electrophoresis (PAGE). Oligos were resuspended in 10 mM Tris pH 7.5 buffer before electroporation.

[0163] HEK293T cells (ATCC, #CRL-11268) were electroporated using a 10 μl kit from the Invitrogen™ Neon™ Transfection System. A mixture of 650 ng of dCasPhi-2xUGI mRNA, 200 ng of AnoA1 mRNA, and 6 μM synthetic crRNA was electroporated at 1150 V for 20 ms with two pulses. The chemically synthesized crRNA consisted of different direct repeats ranging in length from 21 to 35 nucleotides, different spacer sequences targeting transcripts within the Site 2 or B2M gene targets, and MS2 ligand-binding moieties at the 5' end, 3' end, internally at neither the 5' nor 3' end, or combinations thereof. Each sequence optionally contained chemical modifications at one or more bases and within one or more junctions. Cells were plated in 96-well plates using full-serum medium and harvested after 48–72 hours for further processing.

[0164] Cell treatment

[0165] Cells were lysed in 100 μL of a buffer containing proteinase K (Thermo Scientific, #FEREO0492), RNase A (Thermo Scientific, #FEREN0531), and Phusion™ HF buffer (Thermo Scientific, #F-518L) at 56°C for 30 minutes, followed by heat inactivation at 95°C for 5 minutes. This cell lysate was used to generate PCR amplification products spanning the region containing the base-edited site(s). PCR amplification products between 400 and 1000 bp in length can be sequenced by Sanger sequencing. PCR amplification products may be purified (Qiagen, #28181) and subjected to NGS sequencing.

[0166] Edit Analysis

[0167] Base editing efficiency was calculated using the Chimera analysis tool, a modified version of the open-source tool BEAT. Chimera first determines editing efficiency by subtracting background noise and defining the expected variability across samples. This allows us to estimate editing efficiency without normalizing to a control sample. Next, Chimera uses the Median Absolute Deviation (MAD) method to remove any outliers from the noise and evaluates the editing efficiency of the base editor over a range of 18–20 bp of input guide sequence. High-throughput sequencing data analysis, particularly single nucleotide polymorphism (SNP) and insertion / deletion (indel) frequency analysis, was performed as follows: barcoded samples were demultiplexed, and the demultiplexed paired-end reads were combined using a custom Python script. This removed any reads with mismatches in the overlapping regions and retained the highest Phred score for each overlapping base. Non-overlapping portions of the reads were then trimmed, and combined reads containing any bases with a Phred score <30 were removed. The resulting reads were aligned using Bowtie2, and a mpileup file was created using SAMtools.

[0168] Table 5 provides examples of chemically synthesized guides for use with CasPhi (Cas12j) that were successfully delivered by electroporation.

[0169] Chemical modifications are indicated (m = 2'-O-methyl; * = phosphorothioate). The sequence of the spacer region is in bold. The direct repeat sequence is underlined. The sequence of the ligand-binding moiety is in italics.

[0170] [Table 5] TIFF2024502348000014.tif226152TIFF2024502348000015.tif232153TIFF2024502348000016.tif232153TIFF20245023480 00017.tif233151TIFF2024502348000018.tif232156TIFF2024502348000019.tif219152TIFF2024502348000020.tif156152

[0171] Example 6: dCasPhi base editing at multiple sites in HEK293T cells using chemically synthesized and chemically modified guides.

[0172] HEK293T cells were electroporated with dCasPhi-2xUGI mRNA, AnoA1 mRNA, and the indicated synthetic gRNAs for (Figure 5A) B2M_gRNA4, (Figure 5B) B2M_gRNA6, and (Figure 5C) Site2_gRNA5. SEQ ID NOs: 119 and 120 for B2M_gRNA4; 84 and 85 for B2M_gRNA6; and 73 and 73 for Site2_gRNA5. Cells were harvested and analyzed for base editing levels using Chimera software. Data show the % C>T conversion rate at the indicated cytosine positions along the spacer. These data demonstrate base editing for the 5' MS2 guide at a total of 11 targeted C residues across the three spacers / genomic target sites.

[0173] Example 7: dCasPhi base editing at HEKsite 2 in HEK293T cells using chemically synthesized and modified synthetic guides.

[0174] HEK293T cells were electroporated with dCasPhi-2xUGI mRNA, AnoA1 mRNA, and the indicated synthetic gRNA. SEQ ID NO: 75. The different codon-optimized dCasPhi mRNAs are shown in Table 6 below. Cells were harvested, and base editing levels were analyzed using Chimera software. The data summarized in Figure 6 show the C>T conversion rate (%) at the indicated cytosine positions along the spacer. These data reveal additional DNA targeting sequences that exhibit base editing and demonstrate that the 5' MS2 pre-cr guide can be used to perform base editing in conjunction with multiple codon optimization of dCasPhi.

[0175] Table 6 provides CasPhi (Cas12j) mRNA.

[0176] [Table 6] TIFF2024502348000022.tif231155TIFF2024502348000023.tif231158TIFF2024502348 000024.tif231158TIFF2024502348000025.tif231157TIFF2024502348000026.tif54156

[0177] Example 8: Effect of chemical modifications of synthetic guides on base editing levels.

[0178] Guide RNAs for Site 2 gRNA5 (SEQ ID NOs: 107, 108, 109, 110, 111, and 112 (Table 5)) and B2M gRNA6 (SEQ ID NOs: 113, 114, 115, 86, 116, 117, and 118 (Table 5)) were synthesized with varying combinations of chemical modifications at the 5' and 3' ends (see Sequence Listing for details). HEK293T cells were electroporated with dCasPhi-2xUGI mRNA, AnoA1 mRNA, and synthetic gRNA. Base editing levels were measured using Chimera or NGS and compared with gRNAs with mN*mN*...mN*mN*N modifications. Data show the C>T conversion rate (%) at the indicated cytosines along the spacer. The results are shown in Figures 7A and 7B, respectively. The mN*mN*...mN*mN*N guides were chemically synthesized in a different batch from the remaining guides.

[0179] These data demonstrate that several chemical modification patterns significantly improve base editing levels over unmodified chemically synthesized guides, such as one 2'-O-methyl modification and phosphorothioate linkage at both the 5' and 3' ends, and the additional incorporation of two or three 2'-O-methyl modifications and phosphorothioate linkages.

[0180] Example 9: Evaluation of the effect of linkers in gRNA sequences on base editing levels.

[0181] Guide RNAs were designed with (UC) linkers at the 5' end of the guide, between MS2 and the spacer, and / or at the 3' end. SEQ ID NOS: 73, 75, 79, 92, 93, 94, 95, 96, 97, and 98 (Table 5). RNAs were synthesized using the same method with the same chemical modifications. HEK293T cells were electroporated with dCasPhi-2xUGI mRNA, AnoA1 deaminase mRNA, and synthetic gRNA. Base editing levels were measured using Chimera or next-generation sequencing (NGS) and compared to gRNAs without linkers. Data represent C>T conversion rates at the indicated cytosines along the spacer sequence.

[0182] These data, summarized in Figure 8A, suggest that adding an additional UC linker outside the existing GC linker allows for similar levels of base editing compared to no additional linker. Figure 8B (SEQ ID NO: 29) shows a chemically modified template gRNA without a ligand-binding moiety. Figures 8C to 8K show templates with different modification patterns and one or two linkers (SEQ ID NOs: 30-32 and 86-91).

[0183] Example 10: CasPhi aptamer recruits base editing in T lymphocytes

[0184] CD3 from fresh blood sources + T cell isolation and culture: PBMCs were isolated from blood sources (e.g., CPD blood bags, Apheresis Cones, leukopaks) by layering onto Lymphoprep™ using SepMap columns (STEMCELL Technologies). Subsequently, negative selection was performed using the EasySep™ Human T Cell Isolation Kit (STEMCELL Technologies) to isolate total CD3 + T cells were isolated, identified by flow cytometry, and cultured in ImmunoCult™ XT medium (STEMCELL Technologies) supplemented with 1x penicillin / streptomycin (Thermo Fisher Scientific) at 37°C and 5% CO2.

[0185] T cell electroporation: 48-72 hours after activation, T cells were electroporated using a Neon™ electroporator (Thermofisher). The Neon™ electroporator conditions were 1600V / 10ms / 3 pulses, 250k cells per 10µl tip, a total mRNA concentration of 100ng / µl for both Deaminase-MCP and nCasPhi-UGI-UGI (synthesized by Trilink), and a final crRNA concentration of 2µM. After electroporation, cells were transferred to Immunocult™ XT medium (STEMCELL Technologies) supplemented with 100U of IL-2, 100U of IL-7, and 100U of IL-15 and cultured at 37°C and 5% CO2 for 48-72 hours.

[0186] CD3 + T cell activation: T cells were activated using Dynabeads® human T activator CD3 / CD28 beads (Thermofisher) at a bead:cell ratio of 1:1 in Immunocult™ XT medium (STEMCELL Technologies) in the presence of 100 U / ml IL-2 (STEMCELL Technologies) and 1x penicillin / streptomycin (Thermofisher) at 37°C and 5% CO2 for 48–72 hours. After activation, the beads were removed by placing on a magnet, and the cells were returned to culture.

[0187] Genomic DNA analysis: Genomic DNA was released from lysed cells 48-72 hours after electroporation. The locus of interest was amplified by PCR, and the products were then subjected to Sanger sequencing. Data were analyzed using Chimera.

[0188] Synthetic crRNA sequence (without aptamer) for B2M position: SEQ ID NO: 84 in Table 5.

[0189] Synthetic crRNA sequence for B2M position (with 1xMS2 aptamer): SEQ ID NO: 85 in Table 5.

[0190] T lymphocytes were stimulated and then electroporated in the presence of different aptamers designed with the same deaminase. The data summarized in Figure 9 demonstrate that the tracr-less Type V family can be used to recruit specific aptamers for base editing in T lymphocytes.

[0191] Example 11: Electroporation of mRNA and synthetic guides for dCas12a-UGI base editing

[0192] Preparation of mRNA:

[0193] Messenger mRNA was prepared from a DNA vector carrying a T7 promoter and the coding sequences of dCas12a-UGI and AnoA1 according to standard protocols for mRNA in vitro transcription.

[0194] RNA synthesis:

[0195] All crRNAs were synthesized by Horizon Discovery or Agilent using either 2'-acetoxyethyl orthoester (2'-ACE) or 2'-tert-butyldimethylsilyl (2'-TBDMS) protection chemistry. Chemical modifications were included where noted. RNA oligos were 2'-deprotected, desalted, and purified by either high-performance liquid chromatography (HPLC) or polyacrylamide gel electrophoresis (PAGE). Oligos were resuspended in 10 mM Tris pH 7.5 buffer before electroporation.

[0196] HEK293T cells (ATCC, #CRL-11268) were electroporated using a 10 μL kit from the Invitrogen™ Neon™ Transfection System. 50,000 cells, 1 μg of mRNA, and 6 μM synthetic crRNA were electroporated at 1150 V for 20 ms with two pulses. The mRNA was mixed at a molar ratio of 3:1 between dCas12a-2xUGI and AnoAl. Cells were seeded in 96-well plates using full-serum growth medium and harvested after 72 hours for further processing.

[0197] Cell treatment

[0198] Cells may be lysed in 100 μL of a buffer containing Proteinase K (Thermo Scientific, #FEREO0492), RNase A (Thermo Scientific, #FEREN0531), and Phusion™ HF Buffer (Thermo Scientific, #F-518L) at 56°C for 30 minutes, followed by heat inactivation at 95°C for 5 minutes. This cell lysate can be used to generate PCR amplification products spanning the region containing the base-edited site(s). PCR amplification products of 400-1000 bp in length can be sequenced by Sanger sequencing.

[0199] Edit Analysis

[0200] Base editing efficiency can be calculated using the Chimera analysis tool, an adaptation of the open-source tool BEAT. Chimera first determines editing efficiency by subtracting background noise and defining the expected variability across samples. This allows for estimation of editing efficiency without normalizing to a control sample. Chimera then uses the Median Absolute Deviation (MAD) method to remove any outliers from the noise and evaluates the editing efficiency of the base editor across a 23-nt input guide sequence.

[0201] Table 7 shows chemically synthesized gRNAs for use with Cas12a and the gRNAs used in this example. Chemically modified synthetic guides SEQ ID NOS: 142-144 demonstrated desirable levels of base editing. The sequences of the spacer regions are in bold. Direct repeat sequences are underlined. The sequences of the ligand binding moieties are in italics.

[0202] [Table 7]

[0203] Example 12: dCas12a base editing at HEK site 2 using chemical modification guides

[0204] HEK293T cells were electroporated with dCas12a-2xUGI mRNA, AnoA1 mRNA, and the indicated synthetic gRNA. Cells were harvested and analyzed for base editing levels by next-generation sequencing. The data summarized in Figure 10 show the percentage of C>T conversions at the indicated cytosine positions along the spacer. This data demonstrates that Cas12a and the corresponding guide are effective for base editing.

[0205] Example 13: Transfection of dCas12i2 base editing plasmid components

[0206] The coding sequence for the inactivated version of Cas12i2 and the 2xUGI fusion (dCas12i2-2xUGI) was obtained and cloned into an expression vector under the control of the mouse CMV promoter in a T2A polycistronic cassette with a red fluorescent protein-puromycin fusion. The coding sequence for the MS2 coat protein-Anolis Apobec fusion (AnoA1) was obtained and cloned into an expression vector under the control of the mouse CMV promoter. The coding sequence for the MS2 coat protein-human APOBEC3A (hA3A) fusion (MCP-hA3A) was obtained and cloned into an expression vector under the control of the mouse CMV promoter. The sequence of the crRNA containing the MS2 ligand-binding portion and unique spacer region was cloned into an expression vector under the control of the hU6 promoter.

[0207] HEK293T cells (ATCC, #CRL-11268) were seeded at 20,000 cells per well in 96-well plates the day before transfection. Cells were co-transfected with DharmaFECT™ Duo Transfection Reagent (Horizon Discovery, #T-2010) using 75–200 ng of dCas12i2-UGI plasmid, 75–100 ng of AnoA1 or hA3A plasmid, and 50–100 ng of crRNA plasmid. The plasmid crRNA consisted of a 31-nucleotide direct repeat, a different 31-nucleotide spacer sequence targeting transcripts within the Site 2 or B2M gene target, and contained an MS2 ligand-binding moiety at the 5' end, 3' end, internally at neither the 5' nor 3' end, or a combination thereof. The sequences are provided in Table 8 below.

[0208] Cells were grown for 72 hours after transfection and harvested for further processing.

[0209] Cell treatment

[0210] Cells were lysed in 100 μL of a buffer containing proteinase K (Thermo Scientific, #FEREO0492), RNase A (Thermo Scientific, #FEREN0531), and Phusion™ HF buffer (Thermo Scientific, #F-518L) at 56°C for 30 minutes, followed by heat inactivation at 95°C for 5 minutes. This cell lysate was used to generate PCR amplification products spanning the region containing the base-edited site(s). PCR amplification products ranging in length from 400 to 1000 bp were sequenced by Sanger sequencing.

[0211] Edit Analysis

[0212] Base editing efficiency was calculated using the Chimera analysis tool, a modified version of the open-source tool BEAT. Chimera first determines editing efficiency by subtracting background noise and defining the expected variability across samples. This allows us to estimate editing efficiency without normalizing to a control sample. Chimera then uses the Median Absolute Deviation (MAD) method to remove any outliers from the noise and evaluates the editing efficiency of the base editor across a 31-bp input guide sequence.

[0213] Table 8 provides a guide to plasmids generated for use with Cas12i2:

[0214] [Table 8] TIFF2024502348000029.tif86164

[0215] Example 14: Transfection of dCas12i2 base editing plasmid components at HEK site 2 using multiple deaminases

[0216] In this example, SEQ ID NOs: 128, 129, and 130 from Table 8 were used.

[0217] HEK293T cells were transfected with dCas12i2-2xUGI + AnoA1 or hA3A + the indicated gRNA plasmid. Cells were harvested and base editing levels were analyzed using Chimera. The data summarized in Figure 11 show the % C>T conversion rate at the indicated cytosine positions along the spacer. This data demonstrates that Cas12i2 and the corresponding guide are effective in base editing at either the 5' MS2 or 3' MS2 aptamer positions within gRNAs using either the AnoA1 (Figure 11A) or hA3A (Figure 11B) deaminase.

Claims

1. A gRNA-ligand binding complex, comprising: (a) a gRNA, the gRNA is 35-60 nucleotides in length, the gRNA having a crRNA sequence; The crRNA sequence is 35-60 nucleotides in length, and the crRNA sequence is (i) a Cas association region that is 14-37 nucleotides in length; (ii) a targeting region that is 14-37 nucleotides in length; The Cas association region can maintain association with the RNA binding domain of a V-type Cas protein in the absence of tracrRNA; gRNA; and (b) a ligand-binding moiety, said ligand-binding moiety comprising: (i) directly linked to the gRNA; or (ii) linked to the gRNA via a linker; a ligand-binding moiety, A gRNA-ligand binding complex comprising:

2. at least one of the gRNA and the ligand binding moiety comprises at least one modification; The at least one modification confers resistance to an active nuclease domain of the V-type Cas protein compared to a gRNA-ligand binding complex lacking the at least one modification. The gRNA-ligand binding complex according to claim 1.

3. the ligand binding moiety binds directly to the gRNA; (i) the gRNA has a 3' end and the ligand binding moiety is directly attached to the 3' end of the gRNA; or (ii) The gRNA-ligand binding complex of claim 1 or 2, wherein the gRNA has a 5' end and the ligand binding moiety is directly attached to the 5' end of the gRNA.

4. The ligand-binding moiety binds directly to the gRNA, the ligand-binding moiety is located between the Cas association region and the targeting region; The gRNA-ligand binding complex according to any one of claims 1 to 3.

5. the gRNA-ligand binding complex comprises a linker; the ligand binding moiety is attached to the gRNA via the linker; (i) the gRNA has a 3' end and the linker is attached to the 3' end of the gRNA; or (ii) the gRNA has a 5' end, and the linker is attached to the 5' end of the gRNA; The gRNA-ligand binding complex according to claim 1 or 2.

6. The gRNA-ligand binding complex having the linker, wherein the ligand binding moiety binds to the gRNA via the linker; the linker is a first linker, the gRNA-ligand binding complex, the ligand binding portion further comprising a second linker; the ligand-binding moiety is located between the first linker and the second linker, each of the first linker and the second linker being directly adjacent to the ligand-binding moiety; The gRNA-ligand binding complex according to claim 1 or 2.

7. The gRNA-ligand binding complex of any one of claims 1 to 6, wherein the ligand-binding moiety is capable of binding to a ligand selected from the group consisting of MS2, Ku, PP7, SfMu, Sm7, Tat, glutathione S-transferase (GST), CSY4, Qbeta, COM, pumilio, anti-His tag (6H7), lambda N22plus, SNAP-Tag, lectin, and PDGF beta chain.

8. a. a gRNA-ligand binding complex according to any one of claims 1 to 3; and b. A type V Cas protein, wherein the Cas association region of the gRNA-ligand binding complex is associated with the type V Cas protein; A base editing complex comprising:

9. The base editing complex of claim 8, wherein the V-type Cas protein comprises an active RuvC domain or an inactive RuvC domain.

10. The base editing complex further comprises an effector. the effector is linked to a ligand; the ligand is capable of binding to the ligand-binding moiety; The base editing complex described in claim 8.

11. The base editing complex of claim 10, wherein the effector is a deaminase.

12. A base editing method comprising a step of exposing the base editing complex described in claim 8 to double-stranded DNA.

13. The method of claim 12, wherein the method is performed in vitro.

14. The method of claim 12, wherein the method is performed in vivo.

15. The method of claim 12, wherein the method is performed ex vivo.

16. A base editing method comprising a step of exposing the base editing complex described in claim 8 to single-stranded DNA.

17. The method of claim 16 , wherein the method is performed in vitro.

18. The method of claim 16 , wherein the method is performed in vivo.

19. 17. The method of claim 16, wherein the method is performed ex vivo.

20. 10. A method for editing DNA in a cell, comprising exposing the base editing complex of claim 8 to the cell.

21. A genetically edited cell or cell population obtained by the method of claim 20.

22. A use of the base editing complex of any one of claims 8 to 11 in a subject susceptible to a base editing modification treatment, comprising: exposing the base editing complex to double-stranded DNA (dsDNA), a solution containing dsDNA, or a cell containing dsDNA outside the subject; After said exposure, a use of the base editing complex comprising injecting said dsDNA, a solution containing said dsDNA, or cells containing said dsDNA into said subject.

23. Use of the genetically edited cell or cell population of claim 21 in a subject susceptible to a base editing modification treatment, comprising: exposing the base editing complex to double-stranded DNA (dsDNA), a solution containing dsDNA, or a cell containing dsDNA outside the subject; Following said exposing, using the cells or cell population comprising injecting said dsDNA, a solution containing said dsDNA or cells containing said dsDNA into said subject.

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