Guide RNA design and complexes for V-type Cas systems

Abstract

We provide a novel gRNA-ligand binding complex that can be used to attract V-type Cas proteins and additional effectors to DNA for base editing. The design of this system allows for the production of efficient modular building blocks that provide flexibility in editing DNA.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of the filing date of U.S. Provisional Application Serial No. 63 / 133,945, filed January 5, 2021, the entire disclosure of which is incorporated by reference as if fully set forth herein.

[0002] FIELD OF THEINVENTION The present invention relates to the field of gene editing. [Background technology]

[0003] Researchers are actively exploring the use of the Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) system to modify DNA. To date, the majority of work in this field has been in the Cas9 system. In these systems, the trans-activating CRISPR RNA (tracrRNA) and the CRISPR RNA (crRNA) hybridize, either as two separate strands of nucleotides or as a single stranded region, to recruit the Cas9 protein and guide it to the DNA location that is complementary to the sequence in the crRNA. The complementary sequence in the DNA then becomes the target site, and the Cas9 protein can effect editing at this target site based on its functional domain.

[0004] Although the power of Cas9 systems is now well known, these systems are not effective for all applications.One of the limitations of the Cas9 system is that the functional domains over which the Cas9 system can act are defined by the functional domain of the Cas9 protein used.

[0005] Other Cas proteins are known. Among these other Cas proteins, those whose potential has not been fully explored are those in the V-type family. The use of V-type family enzymes is particularly unexplored when attempting to introduce multiple editing sites at or near a target site. This necessitates the development of improved gRNAs (guide RNAs) and the complexes and systems that incorporate and use them. Summary of the Invention [Problem to be solved by the invention]

[0006] The present invention provides novel and innovative gRNA-ligand binding complexes, base editing complexes, and methods for base editing and genome modification. The use of various embodiments of the present invention may allow efficient and effective base editing in vitro, in vitro, and in vivo. Furthermore, some embodiments of the present invention provide a modular design that allows the same V-type Cas protein to be directed to different target sites and, optionally, associated with various effector proteins at the same or different sites. [Means for solving the problem]

[0007] According to a first embodiment, the present invention provides a gRNA-ligand binding complex, wherein the gRNA-ligand binding complex comprises: (a) a gRNA, the gRNA comprising 60-210 nucleotides or 80-180 nucleotides, the gRNA comprising: (i) a crRNA sequence, the crRNA sequence being 36-60 nucleotides in length or 35-60 nucleotides in length, the crRNA sequence comprising a Cas association region, the Cas association region being 18-30 nucleotides in length, and a targeting region, the targeting region being 18-30 nucleotides in length; and (ii) a tracrRNA sequence, the tracrRNA sequence being 45-120 nucleotides in length, the tracrRNA sequence being 50-60 nucleotides in length, the tracrRNA sequence being 60-210 nucleotides in length, the gRNA comprising: a gRNA comprising a tracrRNA sequence, the tracrRNA sequence comprising an anti-repeat region and a distal region, the anti-repeat region being at least 80% complementary to the Cas association region over at least 18 consecutive nucleotides of the Cas association region, the Cas association region and the anti-repeat region forming a hybridization region capable of hybridizing, the hybridization region being capable of maintaining association with an RNA-binding domain of a V-type Cas protein; 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.

[0008] According to a second embodiment, the present invention provides a base editing complex comprising a gRNA-ligand binding complex of the present invention and a V-type Cas protein, wherein the Cas association region and the anti-repeat region of the gRNA-ligand binding complex associate with the V-type Cas protein. Optionally, the ligand binding moiety reversibly binds to a ligand that is bound to or part of an effector molecule.

[0009] According to a third embodiment, the present invention provides a method for base editing. 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 dsDNA or ssDNA under conditions that allow base editing.

[0010] According to a fourth embodiment, the gRNA-ligand binding complex comprises or encodes SEQ ID NO:28.

[0011] According to a fifth embodiment, the gRNA-ligand binding complex comprises or encodes any of SEQ ID NO:59 to SEQ ID NO:65.

[0012] According to a sixth embodiment, the gRNA-ligand binding complex comprises or encodes any of SEQ ID NO:67 to SEQ ID NO:71. Effect of the Invention

[0013] Where an 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 a corresponding ligand that is 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 bound or associated with the effector in a manner that allows the effector to reversibly bind to the ligand-binding moiety.

[0014] 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. When the base editing complex also contains a Cas protein, the Cas protein and the effector can be held in the same location, e.g., at or near the target site of interest.

[0015] Therefore, if one wishes to use a particular effector with a Cas protein, one simply associates that effector with a ligand that can reversibly bind to the ligand-binding moiety that is part of the base editing complex that contains that Cas protein. To change the effector from one system to the next, one simply changes 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 description of the drawings]

[0016] [Figure 1A] FIG. 1A depicts Cas12b gRNA modified with one or two ligand-binding moieties, where the tracrRNA and crRNA are separate oligonucleotide strands. [Figure 1B] FIG. 1B depicts Cas12b gRNA modified with one or two ligand-binding moieties, where the tracrRNA and crRNA are separate oligonucleotide strands. [Figure 1C] FIG. 1C depicts Cas12b gRNA modified with one or two ligand-binding moieties, where the tracrRNA and crRNA are separate oligonucleotide strands. [Figure 1D] FIG. 1D depicts Cas12b gRNA modified with one or two ligand-binding moieties, where the tracrRNA and crRNA are separate oligonucleotide strands. [Figure 2A] FIG. 2A shows Cas12b gRNA modified with one or two ligand-binding moieties, where the tracrRNA and crRNA are part of the same oligonucleotide strand. [Figure 2B]FIG. 2B depicts Cas12b gRNA modified with one or two ligand-binding moieties, where the tracrRNA and crRNA are part of the same oligonucleotide strand. [Figure 2C] FIG. 2C shows Cas12b gRNA modified with one or two ligand-binding moieties, where the tracrRNA and crRNA are part of the same oligonucleotide strand. [Figure 2D] FIG. 2D depicts Cas12b gRNA modified with one or two ligand-binding moieties, where the tracrRNA and crRNA are part of the same oligonucleotide strand. [Figure 3A] FIG. 3A shows Cas12e gRNA modified with ligand-binding moieties at different positions, where tracrRNA and crRNA are separate oligonucleotide strands. [Figure 3B] FIG. 3B shows Cas12e gRNA modified with ligand-binding moieties at different positions, where tracrRNA and crRNA are separate oligonucleotide strands. [Figure 3C] FIG. 3C shows Cas12e gRNA modified with ligand-binding moieties at different positions, where tracrRNA and crRNA are separate oligonucleotide strands. [Figure 3D] FIG. 3D shows Cas12e gRNA modified with ligand-binding moieties at different positions, where tracrRNA and crRNA are separate oligonucleotide strands. [Figure 4A] FIG. 4A depicts a Cas12e gRNA modified with a ligand-binding moiety, where the tracrRNA and crRNA are separate oligonucleotide strands. [Figure 4B] FIG. 4B depicts a Cas12e gRNA modified with a ligand-binding moiety, where the tracrRNA and crRNA are separate oligonucleotide strands. [Figure 4C] FIG. 4C depicts a Cas12e gRNA modified with a ligand-binding moiety, where the tracrRNA and crRNA are separate oligonucleotide strands. [Figure 5A] FIG. 5A shows Cas12e gRNA modified with ligand-binding moieties at different positions, where tracrRNA and crRNA are part of the same oligonucleotide strand. [Figure 5B] FIG. 5B shows Cas12e gRNA modified with ligand-binding moieties at different positions, where tracrRNA and crRNA are part of the same oligonucleotide strand. [Figure 5C] FIG. 5C shows Cas12e gRNA modified with ligand-binding moieties at different positions, where tracrRNA and crRNA are part of the same oligonucleotide strand. [Figure 5D] FIG. 5D shows Cas12e gRNA modified with ligand-binding moieties at different positions, where tracrRNA and crRNA are part of the same oligonucleotide strand. [Figure 6A] FIG. 6A shows Cas12f gRNA modified with ligand-binding moieties at different positions, where tracrRNA and crRNA are separate oligonucleotide strands. [Figure 6B] FIG. 6B shows Cas12f gRNA modified with ligand-binding moieties at different positions, where tracrRNA and crRNA are separate oligonucleotide strands. [Figure 6C] FIG. 6C shows Cas12f gRNA modified with ligand-binding moieties at different positions, where tracrRNA and crRNA are separate oligonucleotide strands. [Figure 7A] FIG. 7A shows Cas12f gRNA modified with one or two ligand-binding moieties, where the tracrRNA and crRNA are part of the same oligonucleotide strand. [Figure 7B] FIG. 7B shows Cas12f gRNA modified with one or two ligand-binding moieties, where the tracrRNA and crRNA are part of the same oligonucleotide strand. [Figure 7C]FIG. 7C shows Cas12f gRNA modified with one or two ligand-binding moieties, where the tracrRNA and crRNA are part of the same oligonucleotide strand. [Figure 8A] FIG. 8A is a bar graph showing the percentage of C to T mutations introduced by the Cas12b base editor in HEK-293T cell lines with various effectors. [Figure 8B] FIG. 8B is a bar graph showing the percentage of C to T mutations introduced by the Cas12b base editor in HEK-293T cell lines with various effectors. [Figure 9A] FIG. 9A is a bar graph showing the percentage of C to T mutations introduced by the Cas12b base editor at multiple sites in the HEK-293T cell line. [Figure 9B] FIG. 9B is a bar graph showing the percentage of C to T mutations introduced by the Cas12b base editor at multiple sites in the HEK-293T cell line. [Figure 9C] Figure 9C is a bar graph showing the percentage of C to T mutations introduced by the Cas12b base editor at multiple sites in the HEK-293T cell line. [Figure 9D] FIG. 9D is a bar graph showing the percentage of C to T mutations introduced by the Cas12b base editor at multiple sites in the HEK-293T cell line. [Figure 9E] FIG. 9E is a bar graph showing the percentage of C to T mutations introduced by the Cas12b base editor at multiple sites in the HEK-293T cell line. [Figure 9F] Figure 9F is a bar graph showing the percentage of C to T mutations introduced by the Cas12b base editor at multiple sites in the HEK-293T cell line. [Figure 9G]Figure 9G is a bar graph showing the percentage of C to T mutations introduced by the Cas12b base editor at multiple sites in the HEK-293T cell line. [Figure 10] FIG. 10 is a bar graph showing the percentage of C to T mutations introduced by the Cas12b base editor in the U2OS cell line. [Figure 11A] FIG. 11A is a bar graph showing the percentage of C to T mutations introduced by the CasMINI base editor in the HEK-293T cell line. [Figure 11B] FIG. 11B is a bar graph showing the percentage of C to T mutations introduced by the CasMINI base editor in the HEK-293T cell line. [Figure 11C] FIG. 11C is a bar graph showing the percentage of C to T mutations introduced by the CasMINI base editor in the HEK-293T cell line. [Figure 11D] FIG. 11D is a bar graph showing the percentage of C to T mutations introduced by the CasMINI base editor in the HEK-293T cell line. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0017] Detailed Description of the Invention Next, various embodiments of the present invention will 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 considered as limiting the scope of the present invention in any manner. Furthermore, features described in connection with various or specific embodiments are not to 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.

[0018] Headings are provided herein for the convenience of the reader and do not limit the scope of any embodiments disclosed herein.

[0019] definition

[0020] Unless otherwise stated or apparent from the context, the following terms shall have the meanings set forth below:

[0021] 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.

[0022] 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 located at the 2' position of the sugar and to the alkyl group. In various embodiments, the alkyl moiety consists of carbon(s) and hydrogen(s) or consists essentially of carbon and hydrogen. When the O moiety and the alkyl group to which it is attached are viewed as a group, they may be referred to as O-alkyl groups, e.g., -O-methyl, -O-ethyl, -O-propyl, -O-isopropyl, -O-butyl, -O-isobutyl, -O-ethyl-O-methyl (-OCH2CH2OCH3), and -O-ethyl-OH (-OCH2CH2OH). 2'-O-alkyl modified nucleotides may be substituted or unsubstituted.

[0023] 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., F, Cl, or Br.

[0024] "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 associate with that ligand reversibly or irreversibly.

[0025] 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, a 5-position pyrimidine modification, an 8-position purine modification, a modification at the exocyclic amine of cytosine, and a substitution of 5-bromo-uracil 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, NH2, NHR, NR2 or CN).

[0026] 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 examples of modified bases include 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, and deazanucleotides such as 7-deazaadenosine, 6-azolidine, 6-azocytidine, and 6-azothymidine. Included among these are 5-methyl-2-thiouridine, other thio bases (e.g., 2-thiouridine, 4-thiouridine, 2-thiocytidine), dihydrouridine, pseudouridine, queosine, alkaeosine, 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, 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.

[0027] The phrases "codes for" and "encodes" 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 or encodes a cited 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 replaced by their deoxyribonucleotide counterparts or in which one or more deoxyribonucleotides are replaced by their ribonucleotide counterparts.

[0028] 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 base pairing. The percentage of 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 consecutive residues of a nucleic acid sequence will hydrogen bond with the same number of consecutive residues of a second nucleic acid sequence. As used herein, "substantially complementary" refers to a degree of complementarity of 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, e.g., 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 to two nucleic acids that hybridize under stringent conditions.

[0029] The terms "hybridization" and "hybridize" refer to the process by which fully, substantially, or partially complementary nucleic acid strands come together under specific hybridization conditions to form a double-stranded structure or region in which the two constituent strands are linked by hydrogen bonds. Unless otherwise specified, hybridization conditions may be naturally occurring or laboratory designed. Hydrogen bonds are usually formed between adenine and thymine or uracil (A and T or U) or between cytidine and guanine (C and G), although other base pairs may be formed (see, e.g., Adams et al., The Biochemistry of the Nucleic Acids, 11th ed., 1992).

[0030] The term "nucleotide" refers to ribonucleotides, deoxyribonucleotides or modifications thereof, and analogs thereof. Nucleotides include species consisting of purines (e.g., adenine, hypoxanthine, guanine) and their derivatives and analogs, and pyrimidines (e.g., cytosine, uracil, thymine) and their derivatives and analogs. Preferably, the nucleotide contains a cytosine, uracil, thymine, adenine, or guanine moiety. Additionally, the term nucleotide 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. By way of example, universal bases include, but are not limited to, 3-nitropyrrole, 5-nitroindole, nebularine, and the like. Nucleotide analogs are meant to include, for example, nucleotides having bases such as inosine, queosine, and xanthine, sugars such as 2'-methylribose, and non-natural phosphodiester internucleotide linkages, such as methylphosphonates, phosphorothioates, phosphoroacetates, and peptides.

[0031] 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, rats, monkeys, humans, farm animals, sport animals, pets such as dogs and cats, and the like. Tissues, cells, and the progeny thereof of organisms or other biological entities obtained in vivo or cultured in vitro are also encompassed by the terms subject and patient. Additionally, 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.

[0032] As used herein, "treatment", "treat", "alleviate" and "ameliorate" are used interchangeably. These terms refer to an approach to obtain beneficial or desired results, including but not limited to therapeutic benefit and / or preventive benefit. Therapeutic benefit refers to any therapeutically relevant improvement or effect in one or more diseases, conditions or symptoms being treated. For preventive benefit, the complex of the present invention can be administered to a subject at risk of developing a particular disease, condition or symptom, or to a subject's cells or tissues, or to another subject's cells or tissues prior to re-administration outside the body, or to a subject reporting one or more physiological symptoms of a disease, even though the disease, condition or symptom may not yet be manifest.

[0033] As disclosed herein, a number of ranges of values ​​are provided. Each intermediate value between the upper and lower limits of the range, to the tenth of the unit of the lower limit, is understood to be specifically disclosed, unless the context clearly indicates otherwise. Each subrange between any stated or intermediate value in a stated range and any other stated or intermediate value in that stated range is also encompassed by the present invention. The upper and lower limits of these subranges may each be independently included or excluded in the range, and each range in which either, neither, or both limits are included in the subrange is also encompassed by the present invention, subject to any limit specifically excluded in the stated range. When a stated range includes one or both limits, the range excluding one or both of those included limits is also encompassed by the present invention.

[0034] The term "about" generally refers to plus or minus 10% of the stated numerical value. For example, "about 10%" might indicate a range of 9% to 11%, and "about 20%" might mean a range of 18% to 22%. Other meanings of "about" may be clear from the context, such as rounding, for example, "about 1" might mean 0.5 to 1.4.

[0035] Consideration

[0036] According to a first embodiment, the present invention includes a gRNA-ligand binding complex that includes both a gRNA and a ligand binding moiety, the complex being capable of maintaining an association with a V-type Cas protein. In the gRNA-ligand binding complex, the gRNA may be directly covalently linked to the ligand binding moiety or may be linked to the ligand binding moiety via a linker.

[0037] gRNA

[0038] The gRNA in the gRNA-ligand complex is either a single strand of nucleotides with at least one region of self-complementarity or a double strand of nucleotides, each with at least one region complementary to a region of the other strand. Within the gRNA, whether it is a single strand of nucleotides or a double strand of nucleotides, there may be one or more loops. The gRNA contains two parts, the tracrRNA and the crRNA.

[0039] The nucleotides in a gRNA may be entirely RNA or a combination of other nucleotides, such as ribonucleotides and deoxyribonucleotides. 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 there is a modification pattern in which every second, third or fourth nucleotide is modified at its 2' position and all other nucleotides are 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.

[0040] In some embodiments, the crRNA is 35-60 nucleotides or 36-60 nucleotides or 40-55 nucleotides in length. Within the sequence of the crRNA is a Cas association region, sometimes also referred to as a repeat region, that is 18-30 nucleotides or 20-25 nucleotides in length, and a targeting region, sometimes also referred to as a spacer region, that is 18-30 nucleotides or 20-25 nucleotides in length.

[0041] The targeting region comprises a targeting sequence, which is a variable sequence that can be selected based on where the Cas protein and / or effector is desired to cause base editing. Thus, the targeting region may be designed to include a region that is complementary and hybridizable to 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 consecutive 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 consecutive nucleotides. The targeting region is a region that does not hybridize to tracrRNA and may be downstream of the Cas association region. The Cas association is designed based on the RNA-binding domain of the Cas protein with which it is intended to associate. (It is not necessary that all nucleotides in the Cas association region directly bind to the Cas protein.)

[0042] The tracrRNA sequence is, for example, 30-210 nucleotides long, or 45-120 nucleotides long, or 60-100 nucleotides long, or 70-90 nucleotides long. The tracrRNA sequence comprises an anti-repeat region and a distal region. In some embodiments, the anti-repeat region is 18-60 nucleotides long, or 25-50 nucleotides long, or 30-40 nucleotides long. The distal region is, for example, 18 to 60 nucleotides long, or 25 to 50 nucleotides long, or 30 to 40 nucleotides long. The distal region is a region that does not hybridize to the crRNA and may be located upstream of the anti-repeat region.

[0043] The anti-repeat region is at least 80%, at least 85%, at least 90%, at least 95%, or 100% complementary to the Cas association region over at least 18 consecutive nucleotides of the Cas association region, such that the Cas association region and the anti-repeat region can hybridize to form a hybridization region. When the tracrRNA and the crRNA hybridize on the hybridization region, the gRNA can maintain association with the RNA binding domain of the V-type Cas protein. Preferably, this association is possible both under naturally occurring conditions and under laboratory conditions using the complex.

[0044] When the tracrRNA and crRNA are part of a contiguous strand of nucleotides, there can be a loop region between the tracrRNA and the crRNA, e.g., of 4-20 or 8-15 nucleotides. In the 5' to 3' direction, the gRNA comprises, consists essentially of, or comprises a distal region, an anti-repeat region, a loop, a Cas association region, and a targeting region.

[0045] Examples of tracrRNAs and crRNAs that can be used in connection with the present invention are shown below (all in the 5'→3' orientation, where N is any nucleotide, e.g., A, C, G, or U). In each sequence below, a specific number of N nucleotides are shown. However, in any of these sequences, N can be 16-30, meaning that there can be 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides. Alicyclobacillus acidoterrestris, AacCas12b crRNA SEQ ID NO:1 5'-GUCGGAUCACUGAGCGAGCGAUCUGAGAAGUGGCACNNNNNNNNNNNNNNNNNNNN-3' SEQ ID NO:2 5'-CGAGCGAUCUGAGAAGUGGCACNNNNNNNNNNNNNNNNNNNN-3' SEQ ID NO:3 5'-CUGAGAAGUGGCACNNNNNNNNNNNNNNNNNNNN-3' AacCas12b tracrRNA SEQ ID NO:4 5'-GUCUAGAGGACAGAAUUUUUCAACGGGUGUGCCAAUGGCCACUUUCCAGGUGGCAAAGCCCGUUGAGCUUCUCAAAAAG-3' SEQ ID NO:5 5'-GUCUAGAGGACAGAAUUUUUCAACGGGUGUGCCAAUGGCCACUUUCCAGGUGGCAAAGCCCGUUGAGCUUCUCAAAAA-3' SEQ ID NO:6 5'-GUCUAGAGGACAGAAUUUUUCAACGGGUGUGCCAAUGGCCACUUUCCAGGUGGCAAAGCCCGUUGAG-3' AacCas12b gRNA SEQ ID NO:7 5'-GUCUAGAGGACAGAAUUUUUCAACGGGUGUGCCAAUGGCCACUUUCCAGGUGGCAAAGCCCGUUGAGCUUCUCAAAUCUGAGAAGUGGCACNNNNNNNNNNNNNNNNNNNN-3' AaCas12b artificial chimeric gRNA (artgRNA13) SEQ ID NO:8 5'-GUCGUCUAUAGGACGGCGAGGACAACGGGAGUGCAGUGCUCUUUCCAAGAGCAAACACCCCGUUGGCUUCAAGAGAAGUGGCACNNNNNNNNNNNNNNNNNNNN-3' Bacillus thermomylovorans, Bth Cas12b tracrRNA SEQ ID NO:9 5'-CGAGGUUCUGUCUUUUGGUCAGGACAACCGUCUAGCUAUAAGUGCUGCAGGGGUGAGAAACUCCUAUUGCUGGACGAUGUCUCUUUUAU-3' Bacillus thermomylovorans, Bth Cas12b crRNA SEQ ID NO:10 5'-GUCCAAGAAAAAAGAAAUGAUACGAGGCAUUAGCACNNNNNNNNNNNNNNNNNNNN-3' SEQ ID NO:11 5'-AAAUGAUACGAGGCAUUAGCACNNNNNNNNNNNNNNNNNNNN-3' SEQ ID NO:12 5'-CGAGGCAUUAGCACNNNNNNNNNNNNNNNNNNNN-3' Cas12e, CasX from Deltaproteobacteria Cas12e, DpbCasX, crRNA SEQ ID NO:13 5'-CCGAUAAGUAAAACGCAUCAAAGNNNNNNNNNNNNNNNNNNNNN-3' Cas12e, DpbCasX, tracrRNA SEQ ID NO:14 5'-GGCGCGUUUAUUCCAUUACUUUGGAGCCAGUCCCAGCGACUAUGUCGUAUGGACGAAGCGCUUAUUUAUCGGAGA-3' Cas12e, DpbCasX, gRNA (crRNA+tracrRNA fusion, short and compact mutant) SEQ ID NO:15 5'-GGCGCGUUUAUUCCAUUACUUUGGAGCCAGUCCCAGCGACUAUGUCGUAUGGACGAAGCGCUUAUUUAUCGGAGAGAAACCGAUAAGUAAAACGCAUCAAANNNNNNNNNNNNNNNNNNNN-3' Cas12e, DpbCasX, gRNA (crRNA+tracrRNA fusion, long mutant) SEQ ID NO:16 5'-ACAUCUGGCGCGUUUAUUCCAUUACUUUGGAGCCAGUCCCAGCGACUAUGUCGUAUGGACGAAGCGCUUAUUUAUCGGAGAGAAACCGAUAAGUAAAACGCAUCAAAGNNNNNNNNNNNNNNNNNNNN-3' Cas12f nuclease Un1Cas12f1(Cas14a1) gRNA SEQ ID NO:17 5'-GGGCUUCACUGAUAAAGUGGAGAACCGCUUCACCAAAAGCUGUCCCUUAGGGGAUUAGAACUUGAGUGAAGGUGGGCUGCUUGCAUCAGCCUAAUGUCGAGAAGUGCUUUCU UCGGAAAGUAACCCUCGAAACAAAUUCAUUUUUCCUCUCCAAUUCUGCACAAGAAAGUUGCAGAACCCGAAUAGACGAAUGAAGGAAUGCAACNNNNNNNNNNNNNNNNNNNN-3' Cas12f tracrRNA SEQ ID NO:18 5'-CUUCACCAAAAGCUGUCCCUUAGGGGAUUAGAACUUGAGUGAAGGUGGGCUGCUUGCAUCAGCCUAAUGUCGAGAAGUGCUUUCUUCGGAAAGUAACCCUCGAAACAAAUUCAUUU-3' Cas12f crRNA SEQ ID NO:19 5'-GAAUGAAGGAAUGCAACNNNNNNNNNNNNNNNNNNNN-3' SEQ ID NO:20 5'-GUUGCAGAACCCGAAUAGACGAAUGAAGGAAUGCAACNNNNNNNNNNNNNNNNNNNN-3' Cas12f gRNA (crRNA+tracrRNA fusion) SEQ ID NO:21 5'-CUUCACCAAAAGCUGUCCCUUAGGGGAUUAGAACUUGAGUGAAGGUGGGCUGCUUGCAUCAGCCUAAUGUCGAGAAGUGCUUUCUUCGGAAAGUAACCCUCGAAACAAAUUCAUUUGAAAGAAUGAAGGAAUGCAACNNNNNNNNNNNNNNNNNNNN-3'

[0046] Ligand-binding moiety

[0047] The ligand-binding moiety is an element 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 may be directly bound to the gRNA, for example, via a covalent bond, or may be bound via a linker. The binding between the ligand-binding moiety and the gRNA, whether directly bound via a covalent bond or via a linker, may be at any of a number of locations, for example, the tracrRNA in either the anti-repeat or distal region, or the crRNA in the targeting or Cas-association region, or in the loop between the tracrRNA and the crRNA, if present. A ligand-binding moiety is said to be directly bound to the gRNA if it is bound to a nucleotide in the gRNA, for example, to a backbone phosphate of a unit, a sugar moiety, or a nitrogenous base of a nucleotide.

[0048] As a non-limiting example, the ligand-binding moiety can be directly attached (e.g., via a covalent bond) to the 3'-end or 5'-end of the gRNA (corresponding to the 5'-end of the tracrRNA and the 3'-end of the crRNA), e.g., if the gRNA is single stranded, or to the 3'-end of the crRNA, the 3'-end of the tracrRNA, the 5'-end of the tracrRNA, or the 5'-end of the crRNA (if they are separate strands). Thus, the ligand-binding moiety can bind to the first or last nucleotide of the gRNA, or the first or last nucleotide of either strand of the gRNA. Alternatively, the ligand-binding moiety may bind to a nucleotide that is not the first or last nucleotide of the gRNA (if single stranded) or the tracrRNA or crRNA (if separate strands).

[0049] 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 stretch of nucleotides that includes both the ligand binding moiety and the gRNA, and thus 5'-[gRNA]-[ligand-binding portion]-3' or 5'-[ligand-binding portion]-[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 portion]-5' or 3'-[ligand-binding portion]-5'-5'-[gRNA]-3'.

[0050] 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, so 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 moiety]-[second section of gRNA]-3'. Compared to a gRNA that does not contain a ligand-binding moiety, a complex that includes a gRNA and an inserted ligand-binding moiety may not have any nucleotides deleted from either the Cas association region or the targeting region. Alternatively, there may be a deletion of one or more nucleotides (e.g., 1-10 nucleotides) at the insertion position.

[0051] In some embodiments, the ligand binding moiety forms a stem and loop complex that may be located at a bulge or another stem and loop complex in the absence of the ligand binding moiety.

[0052] In some embodiments, the ligand binding moiety is not a nucleotide sequence and may be attached to the gRNA at a position other than the 5' or 3' end, for example, between two consecutive nucleotides: 5'-[first section of gRNA]-[ligand-binding portion]-[second section of gRNA]-3', Alternatively, the ligand binding moieties may be attached to phosphorus moieties, e.g., the 2', 3' or 5' positions of sugars, or nitrogenous bases at either the 5' or 3' end of the gRNA. These ligand binding moieties may also be attached, for example, to bulges, stem-loop positions, or positions without bulges or stem-loops on the gRNA.

[0053] In other embodiments, the ligand binding moiety is attached to a linker that is attached to the 3' end of the gRNA, the 5' end of the gRNA, or another location or locations within the gRNA. In some 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 essentially of, or consist of moieties other than nucleotides.

[0054] When the ligand-binding moiety is a nucleotide sequence and is attached to the 5' or 3' end of the gRNA via a linker, 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 portion]-3', or 5'-[ligand binding portion]-[linker]-[gRNA]-3'. 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', 5'-[gRNA]-3'-5'-[linker]-3'-3'-[ligand binding moiety]-5', 3'-[ligand-binding moiety]-5'-3'-[linker]-5'-5'-[gRNA]-3', or 3'-[ligand binding moiety]-5'-5'-[linker]-3'-5'-[gRNA]-3'.

[0055] The ligand binding moiety can also be attached to the gRNA via a linker at a position other than the 5' or 3' end. When the ligand binding moiety and linker are nucleotide sequences, they 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 linker, and a second section of the gRNA that is 3' to the ligand binding moiety and linker. In some embodiments, there are two linkers that are adjacent to the ligand binding moiety.

[0056] 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'.

[0057] 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'.

[0058] In some embodiments, the first section of the gRNA, the linker, the ligand binding portion, the second linker, and the second section of the gRNA are each nucleotide sequences of the same orientation, while in other embodiments, one or more of the first linker, the ligand binding portion, and the second linker are oriented in the opposite direction to the first section of the gRNA and the second section of the gRNA, which sections are oriented in the same direction.

[0059] In some embodiments, when the ligand binding moiety is between the first section of the gRNA and the second section of the gRNA (if one or two linkers are present, they are also between the first section of the gRNA and the second section of the gRNA): · The first section of the gRNA contains part of the distal region, and the second section of the gRNA contains the remainder of the distal region and the entire anti-repeat region; · The first section of gRNA contains part of the distal region, and the second section of gRNA contains the rest of the distal region, the entire anti-repeat region and the crRNA; · The first section of the gRNA includes the entire distal region and the second section of the gRNA includes the entire anti-repeat region; · The first section of gRNA contains the entire distal region, and the second section of gRNA contains the entire anti-repeat region and the crRNA; · The first section of the gRNA contains the entire distal region and part of the anti-repeat region, and the second section of the gRNA contains the remainder of the anti-repeat region; · The first section of the gRNA contains the entire distal region and part of the anti-repeat region, and the second section of the gRNA contains the remainder of the anti-repeat region and the crRNA; The first section of the gRNA contains the tracrRNA and the second section of the gRNA contains the crRNA; · The first section of the gRNA contains the tracrRNA and part of the Cas association region, and the second section of the gRNA contains the rest of the Cas association region and the entire targeting region; · The first section of the gRNA contains part of the Cas association region, and the second section of the gRNA contains the remainder of the Cas association region and the entire targeting region; · The first section of the gRNA contains the entire tracrRNA and Cas association region, and the second section of the gRNA contains the entire targeting region; · The first section of the gRNA contains the entire Cas association region, and the second section of the gRNA contains the entire targeting region; a first section of the gRNA that includes the entire tracrRNA and Cas association region and a portion of the targeting region, and a second section of the gRNA that includes the remainder of the targeting region; and The first section of the gRNA contains the entire Cas association region and part of the targeting region, and the second section of the gRNA contains the remainder of the targeting region. Compared to a gRNA that does not include a ligand-binding moiety (and optionally one or more linkers), a complex that includes a gRNA and a ligand-binding moiety inserted therein may not have any nucleotides deleted from or surrounding the region of insertion, or may have one or more nucleotides (e.g., 1-10 nucleotides) deleted at or surrounding the insertion site.

[0060] When two linkers are present, they may be sufficiently complementary to hybridize with each other. For example, 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 or one or more bulges. In some embodiments, the linkers are the same size, and in other embodiments, the linkers are different sizes from each other.

[0061] When the ligand-binding moiety is attached to a loop of the gRNA, either directly or through a linker, the attachment 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 most central 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, for example, occur at the point where the ligand-binding moiety is attached to the gRNA (directly or through a linker) or may occur only at a location other than where the ligand-binding moiety is attached to the gRNA (directly or through a linker). For example, the ligand-binding moiety may be attached to the 2' position of a sugar or to a nitrogenous base of the gRNA oligonucleotide sequence.

[0062] In some embodiments, the ligand-binding moiety comprises, consists essentially of, or consists of an oligonucleotide sequence that is unmodified or includes one or more modified nucleotides. For example, the ligand-binding moiety is 10-50 or 18-50 nucleotides in length. In some embodiments, the ligand-binding moiety forms a stem-loop structure. In the absence of a linker, the ligand-binding moiety may appear as an extension or insertion of the gRNA sequence immediately 5' or 3' of the gRNA, or 5' or 3' of the tracrRNA or crRNA.

[0063] In some embodiments, the ligand binding moiety comprises, consists essentially of, or consists of biotin or streptavidin.

[0064] 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 binding domain thereof, SfMu, Sm7, Tat, glutathione S-transferase (GST), CSY4, Qbeta, COM, pumilio, anti-His tag (6H7), SNAP-tag, lambda (λ)N22, lectin (wherein the ligand-binding moiety may be a carbohydrate or sugar chain or an oligosaccharide), and PDGF beta chain. In some embodiments, the ligand-binding moiety is an aptamer consisting of deoxyribonucleotides, ribonucleotides, or a combination of both. Thus, as non-limiting examples, 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.

[0065] 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 (e.g., aminopurine at position 10), where position 10 is the 10th nucleotide from the 5' end of the aptamer) are used. The 2-aminopurine is, for example, 2'deoxy-2-aminopurine or 2'ribose 2-aminopurine. The modification at any one position may be in addition to the modification at another position or may be exclusive of the modification at any or all other positions.

[0066] In some embodiments, the ligand-binding moiety is an aptamer comprising a 5'-modified nucleotide, where the 5'-modified nucleotide comprises at least one of a 2' modification, a 5' PO4 group, or a modification of a nitrogenous base.

[0067] 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. Also, as will be appreciated by those skilled in the art, the aptamer may comprise an MCP moiety (or other ligand), in which case the effector comprises or is linked to the MS2 operator motif (or other corresponding ligand-binding moiety).

[0068] Linker

[0069] The linker, if present, may be a type that connects the ligand-binding moiety and the gRNA. It may be attached at one site to the ligand-binding moiety and one site to the gRNA, or it may be attached at multiple sites to either the gRNA or the ligand-binding moiety or both. Attachment at multiple sites may allow for more precise control in three-dimensional space of the ligand-binding moiety, and therefore the effector used.

[0070] As non-limiting examples, the linker binds to the gRNA at one site and to the ligand binding moiety at two or more sites; or the linker binds to the ligand binding moiety at one site and to the gRNA at two or more sites. When the linker binds to the gRNA at two or more sites, the linker may bind to the gRNA only at the targeting region, only at the Cas association region, or at both the targeting region and the Cas association region, only at the anti-repeat region, only at the distal region, both at the anti-repeat region and the distal region, both at the distal region and the targeting region, both at the anti-repeat region and the Cas association region, both at the anti-repeat region and the targeting region, or both at the Cas association region and the distal region.

[0071] In some embodiments, the linker comprises, consists essentially of, or consists of an oligonucleotide sequence, and optionally, the linker comprises 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 purposefully designed to have no unwanted complementarity with sequences in the target site of the aptamer, gRNA, or DNA.

[0072] In some embodiments, the linker comprises, consists essentially of, or consists of at least one phosphorothioate linkage.

[0073] In some embodiments, the linker comprises, consists essentially of, or consists of a levulinyl moiety.

[0074] In some embodiments, the linker comprises, consists essentially of, or consists of an ethylene glycol moiety.

[0075] In some embodiments, the linker comprises or is selected from the group consisting of 18S, 9S, or C3.

[0076] In some embodiments, the linker is a nucleotide sequence that is 1-60, 1-24, 2-20, or 5-15 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, the linker may be exclusively RNA, exclusively DNA, or a combination thereof.

[0077] In some embodiments, the linker is a nucleotide sequence that is upstream or downstream of the ligand-binding portion. If the linker is upstream of the ligand-binding portion and the gRNA is upstream of the linker, there may be another sequence that is complementary to the linker downstream of the ligand-binding portion. Similarly, if the linker is downstream of the ligand-binding portion and the gRNA is downstream of the linker, there may be another sequence that is complementary to the linker upstream of the ligand-binding portion. As those skilled in the art will recognize, the complementarity is determined when the oligonucleotide folds on itself and the strand aligns with each corresponding portion in the 5' to 3' direction.

[0078] Thus, in some embodiments, the ligand-binding moiety, e.g., MS2, has an upstream sequence that is 1-12 nucleotides long and a downstream sequence that is 1-12 nucleotides long, where the 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 long. In one embodiment, each of the upstream and downstream sequences comprises or is GC. When there are both upstream and downstream sequences, they may be referred to as extended sequences.

[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. A modification is the introduction of a site or species that does not occur in nature. Modifications can be used to increase one or both of stability and specificity. In some embodiments, stability is improved with respect to resistance to one or both of the active domain of the Cas protein (e.g., the RuvC domain) and the active domain of one or more other enzymes in the system into which the complex of the present invention is introduced (including, but not limited to, any effector). Specificity is improved if 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, multiple, or all positions in the gRNA, anti-repeat region, distal region, targeting region, or Cas association region, and / or the ligand binding portion, and / or the linker (if present).

[0081] 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 of the gRNA and / or ligand binding moiety.

[0082] In some embodiments, the modification or set of modifications is selected to confer resistance to the RuvC active nuclease domain compared to a gRNA-ligand binding complex lacking that modification or set of modifications. In some embodiments, the resistance may be caused by steric hindrance. In some embodiments, the modification(s) are located within and / or between one or more, if not all, of the nucleotides in the targeted region.

[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 anti-repeat region; all be in the distal region; all be in the ligand binding portion; 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 portion; in both the Cas association region and the linker (if present); in both the targeting region and the ligand binding portion; in both the targeting region and the linker (if present); in both the ligand binding portion and the linker (if present); in all three of the Cas association region, the targeting region and the ligand binding portion; in the Cas association region, the targeting region and the linker (if present); in the Cas association region, the ligand binding portion and the linker (if present); in the targeting region, the ligand binding portion and the linker (if present); in the Cas association region and the target It may be present in each of the targeting region, the ligand binding portion, and the linker (if present); in the anti-repeat region and the distal region; in the anti-repeat region and the Cas association region; in the anti-repeat region and the targeting region; in the distal region and the Cas association region; in the distal region and the targeting region; in the entire region of the gRNA except the distal region; in the entire region of the gRNA except the anti-repeat region; in the entire region of the gRNA except the Cas association region; in the entire region of the gRNA except the targeting region; in each of the targeting region, the Cas association region, the anti-repeat region, and the distal region; in the entire region of the gRNA except the ligand binding portion and the anti-repeat region; in the entire region of the gRNA except the ligand binding portion and the Cas association region; in the entire region of the gRNA except the ligand binding portion and the targeting region; and in each of the ligand binding portion, the targeting region, the Cas association region, the anti-repeat region, and the distal region.

[0084] In some embodiments, there are 1-60 or 1-30 or 1-10 or 10-20 or 20-30 or 30-40 or 40-50 or 50-60 2' modifications. As non-limiting examples, the set of 2' modifications may be located in the targeting region; the anti-repeat region; the distal region; the ligand binding portion if the ligand binding portion is or comprises an oligonucleotide sequence; or the Cas association region, or a combination thereof. The modifications may be on consecutive nucleotides, or there may be one or more pairs of unmodified nucleotides between modified nucleotides in a regular or irregular pattern. As further non-limiting examples, 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 in the gRNA contain a 2'-O-alkyl group, the positions being counted from the 5' or 3' end of the gRNA, tracrRNA, or crRNA.

[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, the ligand binding moiety (in the oligonucleotide), and the linker (if present and of the oligonucleotide) include, but are not limited to, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkyl phosphotriesters, 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, thionoalkyl phosphonates, thionoalkyl phosphotriesters, selenophosphates and borane phosphates having normal 3'-5' linkages, 2'-5' linked analogs of these, and those having inverted 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 terminal 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 therein, but 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, sulfone backbones; formacetyl and thioformacetyl backbones; methyleneformacetyl and thioformacetyl backbones; riboacetyl backbones; backbones containing alkenes; sulfamic acid backbones; methyleneimino and methylenehydrazino backbones; sulfonate and sulfonamide backbones; amide backbones; and other backbones with a mixture of N, O, S, CH2 moieties.

[0087] In some embodiments, one or more portions of the conjugate have 1-60, or 1-20, or 1-10, or 10-20, or 20-30, or 30-40, or 40-50, or 50-60 phosphorothioate linkages.These phosphorothioate linkages may all be in the targeting region; all in the Cas association region; all in the anti-repeat region; all in the distal region; all in the ligand binding portion; all 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 portion; in both the Cas association region and the linker (if present); in both the targeting region and the ligand binding portion; in both the targeting region and the linker (if present); in both the ligand binding portion and the linker (if present); in all three of the Cas association region, the targeting region and the ligand binding portion; in the Cas association region, the targeting region and the linker (if present); in the Cas association region, the ligand binding portion and the linker (if present); in the targeting region, the ligand binding portion and the linker (if present); in the Cas association region and the targeting region. and the ligand binding portion and the linker (if present); in the anti-repeat region and the distal region; in the anti-repeat region and the Cas association region; in the anti-repeat region and the targeting region; in the distal region and the Cas association region; in the distal region and the targeting region; in the entire region of the gRNA except the distal region; in the entire region of the gRNA except the anti-repeat region; in the entire region of the gRNA except the Cas association region; in the entire region of the gRNA except the targeting region; in each of the targeting region, Cas association region, anti-repeat region and distal region; in the entire region of the gRNA except the ligand binding portion and the anti-repeat region; in the entire region of the gRNA except the ligand binding portion and the Cas association region; in the entire region of the gRNA except the ligand binding portion and the targeting region; and in each of the ligand binding portion, targeting region, Cas association region, anti-repeat region and distal region.

[0088] Any nucleotide in the complex of the invention may contain one or more substituted sugar moieties. These nucleotides may contain a sugar substituent selected from: 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 may be 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 from 1 to about 10. Other suitable nucleotides include 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 groups, reporter groups, intercalators, groups for improving the pharmacodynamic properties of oligonucleotides, or groups for improving the pharmacokinetic properties of oligonucleotides, and other substituents with similar properties. As non-limiting examples, suitable modifications include 2'-methoxyethoxy (also known as 2'-O-CH2CHOCH3, 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 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-CH3), aminopropoxy (-O-CH2CH2CH2NH2), allyl (-CH2-CH=CH2), -O-allyl (CH2-CH=CH2) and fluoro (F). The 2'-sugar substituent may be at the arabino position (up) or ribo position (down). A suitable 2'-arabino modification is 2'-F. Similar modifications can also be made at other positions in the oligomeric compound, 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 complex of the invention may also contain modifications or substitutions of the nucleobase (often referred to in the art simply 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-azo-uracil, 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, 3-deazaguanine and 3-deazaadenine. Further modified nucleobases include, but are not limited to, tricyclic pyrimidines (e.g., 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 examples of nucleobases include those disclosed in U.S. Pat. No. 3,687,808, those disclosed in The Concise Encyclopedia Of Polymer Science And Engineering, pages 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, pages 289-302, Crooke, ST and Lebleu, B. eds., CRC Press, 1993. Some of these nucleobases are useful for increasing the binding affinity of 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, there may be two linkers: a first linker and a second linker, where the first ligand binding moiety is attached to the first linker and the second ligand binding moiety is attached to the second linker. In these embodiments, the first linker and the second linker may each be attached to the Cas association region; or the first linker and the second linker may each be attached to the targeting region; or one of the first linker and the second linker may be attached to the Cas association region and the other of the first linker and the second linker may be attached to the targeting region, or the first linker and the second linker may each be attached to the anti-repeat region; or the first linker and the second linker may each be attached to the distal region; or one of the first linker and the second linker may be attached to the distal region and the other of the first linker and the second linker may be attached to the anti-repeat region; or one of the first linker and the second linker may be attached to the distal region and the first linker. and the other of the second linker may be attached to the Cas association region; or one of the first and second linkers may be attached to the distal region and the other of the first and second linker may be attached to the targeting region; or one of the first and second linkers may be attached to the anti-repeat region and the other of the first and second linker may be attached to the Cas association region; or one of the first and second linkers may be attached to the anti-repeat region and the other of the first and second linker may be attached to the targeting region; or one of the first and second linkers may be attached to the loop region (if present) and the other of the first and second linker may be attached to one of the targeting region, the Cas association region, the anti-repeat region, or the distal region.

[0093] FIG. 1A is a diagram of a gRNA-ligand binding complex of the invention in which a ligand binding moiety 114 is attached to the 5' end of tracrRNA112 in Cas12b gRNA (wherein crRNA110 is a separate strand that hybridizes to a portion of tracrRNA). FIG. 1B is a diagram of a gRNA-ligand binding complex of the invention in which a ligand binding moiety 124 is attached to the 3' end of tracrRNA122. Also shown is crRNA120. FIG. 1C is a diagram of a gRNA-ligand binding complex of the invention in which a ligand binding moiety 134 is attached to the 5' end of crRNA130. Also shown is tracrRNA132. FIG. 1D is a diagram of a gRNA-ligand binding complex of the invention in which a first ligand binding moiety 146 is attached to the 5' end of tracrRNA142 and a second ligand binding moiety 144 is attached to the 5' end of crRNA140. As non-limiting examples, each of the ligand binding moieties shown in Figures 1A-1D and all other figures may be, for example, a DNA or RNA aptamer, a carbohydrate or oligosaccharide group, a benzylguanine or benzylcytosine group for SNAP / CLIP tagging, a bioconjugation group (e.g., an azide-alkyne group for click chemistry), biotin, or other functional groups used for affinity binding or covalent attachment. In a dual ligand binding moiety system such as that shown in Figure 1D, different or similar functional groups can be attached to each molecule to further enhance functionality.

[0094] FIG. 2A is a diagram of a gRNA-ligand binding complex of the invention, in which a ligand binding moiety 214 is bound to the 5' end of tracrRNA 212 in Cas12b gRNA (wherein crRNA 210 is part of the same strand of nucleotides as tracrRNA). FIG. 2B is a diagram of a gRNA-ligand binding complex of the invention, in which a ligand binding moiety 224 is bound to the loop between tracrRNA 222 and crRNA 220 in Cas12b gRNA. FIG. 2C is a diagram of a gRNA-ligand binding complex of the invention, in which a first ligand binding moiety 234 is bound to the loop region between tracrRNA 232 and crRNA 230, and a second ligand binding moiety 236 is bound to the 5' end of tracrRNA in Cas12b gRNA. FIG. 2D is a diagram of a gRNA-ligand binding complex of the invention, in which a ligand binding moiety 244 is bound to a loop in tracrRNA region 242, and crRNA 240 forms the 3' portion of the gRNA in Cas12b gRNA.

[0095] FIG. 3A is a diagram of a gRNA-ligand binding complex of the invention in which a ligand binding moiety 314 is attached to the 3' end of tracrRNA 312 in a Cas12e gRNA (with separate crRNA 310). FIG. 3B is a diagram of a gRNA-ligand binding complex of the invention in which a ligand binding moiety 324 is attached to the 5' end of tracrRNA 322 in a Cas12e gRNA (with separate crRNA 320). FIG. 3C is a diagram of a gRNA-ligand binding complex of the invention in which a ligand binding moiety 334 is attached to the 5' end of crRNA 330 in a Cas12e gRNA (with separate tracrRNA 332). FIG. 3D is a diagram of a gRNA-ligand binding complex of the invention in which a ligand binding moiety 334 is attached to the 3' end of crRNA 340 in a Cas12e gRNA (with separate tracrRNA 342).

[0096] FIG 4A is a diagram of a gRNA for the Cas12e system, in which a first ligand binding moiety 414 is attached to the 5' end of crRNA 410 and a second ligand binding moiety 416 is attached to the 5' end of tracrRNA 412. FIG 4B is a diagram of a gRNA for the Cas12e system, in which a first ligand binding moiety 426 is attached to the 3' end of crRNA 420 and a second ligand binding moiety 424 is attached to the 3' end of tracrRNA 422. FIG 4C is a diagram of a gRNA for the Cas12e system, in which a first ligand binding moiety 434 is attached to the 5' end of tracrRNA 432 and a second ligand binding moiety 436 is attached to the 3' end of crRNA 430.

[0097] FIG. 5A is a diagram of a gRNA for the Cas12e system in which a first ligand binding moiety 514 binds to the 5' end of tracrRNA 512, and crRNA 510 is part of the same nucleotide strand as tracrRNA. FIG. 5B is a diagram of a gRNA for the Cas12e system in which a ligand binding moiety 524 binds to the loop between tracrRNA 522 and crRNA 520. FIG. 5C is a diagram of a gRNA for the Cas12e system in which a ligand binding moiety 534 binds to the 3' end of crRNA 530 (downstream of tracrRNA 532 and forming a hairpin within it). FIG. 5D is a diagram of a gRNA for the Cas12e system in which a ligand binding moiety 544 binds to a loop within tracrRNA 542, and crRNA 540 forms a hairpin with tracrRNA.

[0098] Figure 6A is a diagram of a gRNA for the Cas12f system, where a ligand binding moiety 614 binds to the 5' end of tracrRNA 612, and crRNA 610 is a separate strand that forms a hybridization region with tracrRNA. Figure 6B is a diagram of a gRNA for the Cas12f system, where a ligand binding moiety 624 binds to the 3' end of tracrRNA 622, and crRNA 620 is a separate strand that forms a hybridization region with tracrRNA. Figure 6C is a diagram of a gRNA for the Cas12f system, where a ligand binding moiety 634 binds to the 5' end of crRNA 630, and tracrRNA 632 is shown.

[0099] Figure 7A shows a gRNA that is a single nucleotide strand containing both tracrRNA and crRNA for the Cas12f system, where a ligand binding moiety 714 is attached to the 5' end of tracrRNA 712, which forms a continuous nucleotide strand with crRNA 710. Figure 7B shows a gRNA that is a single nucleotide strand containing both tracrRNA 722 and crRNA 720 for the Cas12f system, where a ligand binding moiety 724 is attached to the loop between tracrRNA and crRNA. Figure 7C shows a gRNA for Cas12f, where a first ligand binding moiety 736 is located at the 5' end of tracrRNA 732 of the gRNA, and a second ligand binding moiety 734 is located in the loop between tracrRNA and crRNA 730 of the gRNA.

[0100] Base editing complex

[0101] 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 the gRNA-ligand binding complex of the present invention and a V-type Cas protein, and the Cas association region and anti-repeat region of the gRNA-ligand binding complex associate with the V-type Cas protein. In this way, the gRNA can associate with the Cas protein. Once associated, the Cas protein finds the target site based on the identity of the targeting sequence.

[0102] V-type Cas proteins

[0103] Generally, Cas proteins contain at least one RNA binding domain. The RNA binding domain interacts with the guide RNA at the Cas association region. The V-type Cas proteins used in the present invention are capable of associating with a gRNA-ligand binding complex in the presence of a tracrRNA that contains an anti-repeat region with sufficient complementarity to the Cas association region. In some embodiments, the V-type Cas protein is an endonuclease that contains a RuvC domain. The RuvC domain may be mutated to inactivate the endonuclease activity. In some embodiments, the protein is a nickase that contains an activated or inactivated RuvC domain.

[0104] Examples of V-type Cas proteins that may be used in connection with the present invention include, but are not limited to, active or inactive forms of Cas12b, Cas12e, CasMINI and Cas12f.

[0105] The Cas protein can be provided in purified or isolated form and may be part of a composition or complex. Preferably, when in a composition, the protein is first purified to some degree, more preferably to a high level of purity (e.g., about 80%, 90%, 95%, or 99% or more). The composition in which the complexes and components of the invention can be stored and transferred can be any type of composition desired, for example, an aqueous composition suitable for use as or inclusion in a composition for RNA-guided targeting, which one of skill in the art would understand to be useful in the context of the present invention.

[0106] In some embodiments, the V-type Cas protein comprises a fusion protein having (a) an active, partially inactivated, or inactivated V-type Cas protein, and (b) a uracil DNA glycosylase (UNG) inhibitor peptide (UGI). The UGI peptide can be fused to the V-type Cas protein directly or via a linker peptide consisting of 1-100 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: 22). In some embodiments, the UGI comprises a fragment of the wild-type UGI peptide or a variant of SEQ ID NO: 22 comprising an amino acid sequence homologous to SEQ ID NO: 22. In some embodiments, a UGI fragment of a 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:22).

[0107] 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 1-100 amino acid residues.

[0108] The Cas protein or Cas protein fusion may be provided in purified or isolated form and may be part of a composition or complex.

[0109] effector

[0110] The base editing complex of the present invention may include an effector bound to a ligand. The ligand can bind reversibly or irreversibly to the ligand-binding moiety. In this way, the ligand-binding moiety can retain the ligand binding and thus recruit an effector (e.g., a base editing enzyme) that is fused to or otherwise bound to the ligand. This design is believed to be particularly advantageous because it provides a modular design in which the nucleic acid sequence targeting function of the gRNA and the effector function are present on different molecules. For example, different effectors that bind to the same ligand can be used to introduce sequential modifications at the same site. Conversely, the same ligand-binding moiety can be used on different gRNAs while using the same effector-ligand to introduce the same modifications at different sites. This design therefore allows for multiplexing of the system without the undesirable burden of fusing effectors to either the gRNA or the Cas protein.

[0111] Examples of effectors that can be used in the context of the present invention include deaminase, such as those with cytidine deamination or adenine deamination activity, as well as transcription 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 shown in Table 1.

[0112] Table 1. Examples of effector proteins

[0113] [Table 1]

[0114] 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: 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

[0115] 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 bound to a different ligand-binding moiety via a different ligand. Alternatively, when there are two effectors, one binds to a ligand and binds to the gRNA through a ligand-binding moiety, and the other binds directly to the Cas protein.

[0116] Ligand

[0117] As discussed above, the effector is bound to the ligand, for example, 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.

[0118] [Table 2]

[0119] Some of the sequences of the above binding pairs are shown below.

[0120] 1. Telomerase Ku-binding motif / Ku heterodimer a. Ku-binding hairpin 5'-UUCUUGUCGUACUUAUAGAUCGCUACGUUAUUUCAAUUUUGAAAAUCUGAGUCCUGGGAGUGCGGA-3' (SEQ ID NO: 23) b Ku heterodimer >(Sequence number:24) MVRSGNKAAVVLCMDVGFTMSNSIPGIESPFEQAKKVITMFVQRQVFAENKDEIALVLFGTDGTDNPLSGGDQYQNITVHRHLMLPDFDLLEDIESKIQPGSQQADFLDALIVSMDVIQHETI GKKFEKRHIEIFTDLSRFSKSQLDIIIHSLKKCDISERHSIHWPCRLTIGSNLSIRIAAYKSILQERVKKTWTVVDAKTLKKEDIQKETVYCLNDDDETEVLKEDIIQGFRYGSDIVPFSKVD EEQMKYKSEGKCFSVLGFCKSSQVQRRFFMGNQVLKVFAARDDEAAAVALSSLIHALDDLDMVAIVRYAYDKRANPQVGVAFPHIKHNYECLVYVQLPFMEDLRQYMFSSLKNSKKYAPTEAQLNAVDALIDSMSLAKKDEKTDTLEDLFPTTKIPNPRFQRLFQCLLHRALHPREPLPPIQQHIWNMLNPPAEVTTKSQIPLSKIKTLFPLIEAKKKDQVTAQEIFQDNHEDGPTAK (SEQ ID NO: 25)

[0121] 2. Telomerase Sm7 binding motif / Sm7 homoheptamer a.Sm consensus site (single stranded) 5'-AAUUUUUGGA-3' (SEQ ID NO: 26) B Monomeric Sm-like protein (Archaea) GSVIDVSSQRVNVQRPLDALGNSLNSPVIIKLKGDREFRGVLKSFDLHMNLVLNDAELEDGEVTRRLGTVLIRGDNIVYISP (SEQ ID NO: 27)

[0122] 3. MS2 phage operator stem loop / MS2 coat protein a. MS2 phage operator stem loop 5'-GCACAUGAGGAUCACCCAUGUGC-3' (SEQ ID NO: 28) B MS2 coat protein MASNFTQFVLVDNGGTGDVTVAPSNFANGIAEWISSNSRSQAYKVTCSVRQSSAQNRKYTIKVEVPKGAWRSYLNMELTIPIFATNSDCELIVKAMQGLLKDGNPIPSAIAANSGIY (SEQ ID NO: 29)

[0123] 4. PP7 phage operator stem loop / PP7 coat protein a. PP7 phage operator stem loop 5'-AUAAGGAGUUUAUAUGGAAACCCUUA-3' (SEQ ID NO: 30) b. PP7 coat protein (PCP) MSKTIVLSVGEATRTLTEIQSTADRQIFEEKVGPLVGRLRLTASLRQNGAKTAYRVNLKLDQADVVDCSTSVCGELPKVRYTQVWSHDVTIVANSTEASRKSLYDLTKSLVATSQVEDLVVNLVPLGR (SEQ ID NO: 31)

[0124] 5. SfMu Com stem-loop / SfMu Com binding protein a.SfMu Com stem loop 5'-CUGAAUGCCUGCGAGCAUC-3' (SEQ ID NO: 32) b. SfMu Com binding protein MKSIRCKNCNKLLFKADSFDHIEIRCPRCKRHIIMLNACEHPTEKHCGKREKITHSDETVRY (SEQ ID NO: 33)

[0125] 6.BoxB aptamer / λN22plus a.BoxB aptamer 5'-GCCCUGAAGAAGGGC-3' (SEQ ID NO: 34) b. λN22plus protein MNARTRRRERRAEKQAQWKAAN (SEQ ID NO: 35)

[0126] 7. Csy4-binding stem loop / Csy4[H29A] Csy4 binding motif 5'-CUGCCGUAUAGGCAGC-3' (SEQ ID NO: 36) b.Csy4[H29A] MDHYLDIRLRPDPEFPPAQLMSVLFGKLAQALVAQGGDRIGVSFPDLDESRSRLGERLRIHASADDLRALLARPWLEGLRDHLQFGEPAVVPHPTPYRQVSRVQAKSNPERLRRRLMRRHDLSEEEARKRIPDTVARALDLPFVTLRSQSTGQHFRLFIRHGPLQVTAEEGGFTCYGLSKGGFVPWF (SEQ ID NO: 37)

[0127] 8. Qβ-binding stem loop [Q65H] a.Qβ phage operator stem loop 5'-ATGCTGTCTAAGACAGCAT-3' (SEQ ID NO: 96) b. Qβ coat protein [Q65H] MAKLETVTLGNIGKDGKQTLVLNPRGVNPTNGVASLSQAGAVPALEKRVTVSVSQPSRNRKNYKVHVKIQNPTACTANGSCDPSVTRQAYADVTFSFTQYSTDEERAFVRTELAALLASPLLIDAIDQLNPAY (SEQ ID NO: 97)

[0128] For each of the above 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 above sequences can be used in one or both members of the binding pair.

[0129] Other Chemistry

[0130] In some embodiments, the base editing complexes of the present invention are combined with additional chemistry techniques. 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 attached to elements for cycloaddition via click chemistry.

[0131] Base editing methods

[0132] In another embodiment, the present invention provides a base editing method. In these methods, the base editing complex of the present invention is exposed to double-stranded DNA, or a solution containing dsDNA, or 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 medicament for treatment.

[0133] These methods include, for example, the detection and / or differentiation of T cells (including primary T cells), natural killer (NK) cells, B cells, CD34 + The immune cells can be used to modify immune cells selected from hematopoietic stem progenitor cells (HSPCs). The immune cells can be modified immune cells, such as T cells, that contain a CAR or TCR. Thus, the methods herein can be applied to further modify cells that have already been modified to contain a therapeutically useful CAR and / or TCR. As a further example, primary immune cells naturally present 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, retinal stem cells, and the like. Other stem cells include, but are not limited to, mammalian stem cells, such as mouse stem cells, e.g., mouse embryonic stem cells.

[0134] 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 directed to the expression of primary human T cells, natural killer (NK) cells, CD34 + These 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).

[0135] 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.

[0136] In some cases, the methods are configured to produce genetically modified 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 hemangioblasts that differentiate into hematopoietic stem cells, vascular smooth muscle cells, and 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 can be used to generate natural killer (NK) cells, CD34 + It is also applicable to hematopoietic stem and progenitor cells (HSPCs), such as HSPCs isolated from umbilical cord blood or bone marrow, and cells differentiated therefrom.

[0137] In another aspect, provided herein is a method for targeting disease for modification 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 the mutation or modification of endogenous gene sequence according to the present invention include, but are not limited to, modifying disease-related gene mutations, modifying sequences that code 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 that code structural features of proteins.

[0138] Delivery of components into cells

[0139] The base editing complex or its components can be delivered to the target cell or organism by various methods, in various formats (DNA, RNA, protein), or a combination of these different formats. The base editing components can be delivered as (a) a DNA polynucleotide encoding a sequence corresponding to the 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 protein of the effector. When delivered in protein format, the V-type Cas protein can assemble with the guide RNA to form a ribonucleoprotein complex (RNP) and be delivered to the target cell or organism.

[0140] 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 by which molecules can be packaged for delivery to target living cells without altering the overall genome landscape.

[0141] Other methods include, but are not limited to, non-integrative transient introduction 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. This includes, but is not limited to, via DNA-only vehicles (e.g., plasmids, Minicircles, minivectors, ministrings, protelomerase-generated DNA molecules (e.g., doggybone™), artificial chromosomes (e.g., HACs), and cosmids), nanoparticles, DNA vehicles with extracellular vesicles (e.g., exosomes and microvesicles), eukaryotic cell transfer (e.g., by recombinant yeast), transient viral transfer with AAV, non-integrative viral particles (e.g., lentivirus and retrovirus-based systems), cell-penetrating peptides, and other techniques that can introduce DNA into cells without directly integrating into the overall genome. Another method for the introduction of RNA components is the use of integrative gene transfer techniques to stably introduce the machinery for RNA transcription into the genome of the target cell. 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 the utility is met (e.g., by introducing a Cre-Lox recombination system). Such techniques for stable gene transfer include, but are not limited to, integration by viral particles (e.g., lentivirus, adenovirus, retrovirus-based systems), transposase-mediated transfer (e.g., Sleeping Beauty and PiggyBac™), use of non-homologous repair pathways introduced by DNA breaks (e.g., using CRISPR and TALEN) techniques, surrogate DNA molecules, and other techniques that promote integration of target DNA into the cells of interest.

[0142] The various components of the complex of the present invention, if not enzymatically synthesized in a cell or in solution, can be produced chemically, or, if naturally occurring, can be isolated and purified from naturally occurring sources. Methods for chemically and enzymatically synthesizing the various embodiments of the present invention are well known to those skilled in the art. Similarly, methods for linking or introducing covalent bonds between the components of the present invention are well known to those skilled in the art.

[0143] Application Examples

[0144] As non-limiting examples, the complexes of the present invention can be used to recruit transcriptional activators such as p65 and V64, as well as 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, generating therapeutic cells, genome tagging, epigenome editing, karyotype modification, chromatin imaging, transcriptome and metabolic pathway modification, genetic circuit modification, cell signal sensing, cell event recording, lineage information 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 infusion.

[0145] Applications also include human disease research (e.g., cancer immunotherapy, antiviral therapy, bacteriophage therapy, cancer diagnostics, pathogen screening, microbiota remodeling, stem cell reprogramming, immunogenomic engineering, vaccine development, and antibody production). EXAMPLES

[0146] Working Example

[0147] Example 1: Transfection of plasmid constructs for Cas12e base editing in mammalian cells (projected)

[0148] Vector construction

[0149] The coding sequence for Cas12e can be obtained synthetically and cloned into a vector under the control of the mouse CMV promoter (mCMV) in a T2A polycistronic cassette with a red fluorescent protein-puromycin fusion. Inactive versions of Cas12e and 2xUGI fusions to inactive Cas12e mutants can also be obtained and cloned into the aforementioned vectors. The coding sequence for the MS2 coat protein-APOBEC fusion (MCP-APOBEC) can be obtained and cloned into an expression vector under the control of the mouse CMV promoter. The sequence of the gRNA containing the MS2 ligand binding portion and the unique spacer region can be cloned into an expression vector under the control of the hU6 promoter.

[0150] Transfection:

[0151] HEK293T cells (ATCC, #CRL-11268) can be seeded at 20,000 cells per well in 96-well plates the day before transfection. Cells can be co-transfected with DharmaFECT™ Duo transfection reagent (Horizon Discovery, #T-2010) and 200 ng of Cas12e plasmid or its inactivated mutants fused or not fused with UGI, 50 ng of MCP-APOBEC plasmid, and 50 ng of gRNA plasmid. The gRNA plasmid consists of a constant region length of 101 nucleotides, various spacer sequences targeting transcripts within the PPIB or EMX1 gene targets, and may have an MS2 ligand binding moiety at the 5' end, 3' end, internally at neither the 5' nor 3' end, or a combination thereof.

[0152] Cells are selected in puromycin-containing medium, harvested 48 hours post-transfection, and further processed as described below. The following sequences are available:

[0153] Cas12e gRNA sequence:

[0154] 5'-GCGCACATGAGGATCACCCATGTGCGGCGCGUUUAUUCCAUUACUUUGGAGCCAGUCCCAGCGACUAUGUCGUAUGGACGAAGCGCUUAUUUAUCGGAGAGAAACCGAUAAGUAAAACGCAUCAAANNNNNNNNNNNNNNNNNNNNN-3' (SEQ ID NO: 38)

[0155] 5'-GGCGCGUUUAUUCCAUUACUUUGGAGCCAGUCCCAGCGACUGCGCACATGAGGATCACCCATGTGCUGUCGUAUGGACGAAGCGCUUAUUUAUCGGAGAGAAACCGAUAAGUAAAACGCAUCAAANNNNNNNNNNNNNNNNNNNNN-3' (SEQ ID NO: 39)

[0156] 5'-GGCGCGUUUAUUCCAUUACUUUGGAGCCAGUCCCAGCGACUAUGUCGUAUGGACGAAGCGCUUAUUUAUCGGAGAGCGCACATGAGGATCACCCATGTGCAAACCGAUAAGUAAAACGCAUCAAANNNNNNNNNNNNNNNNNNNNN-3' (SEQ ID NO: 40)

[0157] 5'-GGCGCGUUUAUUCCAUUACUUUGGAGCCAGUCCCAGCGACUAUGUCGUAUGGACGAAGCGCUUAUUUAUCGGAGAGAAACCGAUAAGUAAAACGCAUCAAANNNNNNNNNNNNNNNNNNNNNGCGCACATGAGGATCACCCATGTGC-3' (SEQ ID NO: 41)

[0158] 5'-GCGCACATGAGGATCACCCATGTGCGGCGCGUUUAUUCCAUUACUUUGGAGCCAGUCCCAGCGACUAUGUCGUAUGGACGAAGCGCUUAUUUAUCGGAGAGCGCACATGAGGATCACCCATGTGCAAACCGAUAAGUAAAACGCAUCAAANNNNNNNNNNNNNNNNNNNN-3' (SEQ ID NO: 42)

[0159] EMX1 target sequence:

[0160] 5'-AGAACCGGAGGACAAAGTAC-3' (SEQ ID NO: 43)

[0161] 5'-TGGCAATGCGCCACCGGTTG-3' (SEQ ID NO: 44)

[0162] 5'-TCTTCTGCTCGGACTCAGGC-3' (SEQ ID NO: 45)

[0163] 5'-TCTGCTCGGACTCAGGCCCT-3' (SEQ ID NO: 46)

[0164] 5'-CCAGCTTCTGCCGTTTGTAC-3' (SEQ ID NO: 47)

[0165] PPIB target sequence

[0166] 5'-AAAAACAGTGGATAATTTTG-3' (SEQ ID NO: 48)

[0167] 5'-GAAGAGACCAAAGATCACCC-3' (SEQ ID NO: 49)

[0168] 5'-CCTCCGCCTGTGGATGCTGC-3' (SEQ ID NO: 50)

[0169] 5'-TCCTGCTGCTGCCGGGACCT-3' (SEQ ID NO: 51)

[0170] 5'-GCGGCCGATGAGAAGAAGAA-3' (SEQ ID NO: 52)

[0171] Example 2: Electroporation of mRNA and synthetic guides for Cas12e base editing in mammalian cells (predicted)

[0172] Preparation of mRNA : Messenger mRNA can be prepared from DNA vectors carrying the T7 promoter and the coding sequences of Cas12e, dCas12e-UGI and MCP-APOBEC according to standard protocols for mRNA in vitro transcription.

[0173] RNA synthesis:

[0174] crRNAs can be synthesized by Horizon Discovery using either 2'-acetoxyethyl orthoester (2'-ACE) or 2'-tert-butyldimethylsilyl (2'-TBDMS) protection chemistries. RNA oligos can be 2'-deprotected / desalted and purified by either high performance liquid chromatography (HPLC) or polyacrylamide gel electrophoresis (PAGE). Oligos can be resuspended in 10 mM Tris pH 7.5 buffer prior to electroporation.

[0175] HEK293T cells (ATCC, #CRL-11268) can be electroporated using the Invitrogen™ Neon™ Transfection System 10 μl kit. 50,000 cells, 1 μg of Cas12e or dCas12e-UGI mRNA and MCP-APOBEC mRNA, and a mixture of 3 μM synthetic crRNA and tracrRNA can be electroporated at 1150V for 20 ms and 2 pulses. Chemically synthesized crRNAs consist of a constant region length of 23 nucleotides, various spacer sequences targeting transcripts within the PPIB or EMX1 gene targets, and may have MS2 ligand binding moieties at the 5' end, 3' end, internally at neither the 5' nor 3' end, or combinations thereof. Each sequence may contain chemical modifications within one or more bases and one or more junctions. Cells may be plated in 96-well plates with full serum medium and harvested after 72 hours for further processing. The following sequences can be used:

[0176] Cas12e crRNA sequence (N, target sequence):

[0177] 5'-GCGCACATGAGGATCACCCATGTGCCCGAUAAGUAAAACGCAUCAAAGNNNNNNNNNNNNNNNNNNNNN-3' (SEQ ID NO: 53)

[0178] 5'-CCGAUAAGUAAAACGCAUCAAAGNNNNNNNNNNNNNNNNNNNNNNGCGCACATGAGGATCACCCATGTGC-3' (SEQ ID NO: 54)

[0179] Cas12e tracrRNA sequence (N, target sequence):

[0180] 5'-GCGCACATGAGGATCACCCATGTGCGGCGCGUUUAUUCCAUUACUUUGGAGCCAGUCCCAGCGACUAUGUCGUAUGGACGAAGCGCUUAUUUAUCGGAGA-3' (SEQ ID NO: 55)

[0181] 5'-GGCGCGUUUAUUCCAUUACUUUGGAGCCAGUCCCAGCGACUGCGCACATGAGGATCACCCATGTGCUGUCGUAUGGACGAAGCGCUUAUUUAUCGGAGA-3' (SEQ ID NO: 56)

[0182] 5'-GGCGCGUUUAUUCCAUUACUUUGGAGCCAGUCCCAGCGACUAUGUCGUAUGGACGAAGCGCUUAUUUAUCGGAGAGCGCACATGAGGATCACCCATGTGC-3' (SEQ ID NO: 57)

[0183] For both Example 1 and Example 2:

[0184] Cell treatment

[0185] Cells may be lysed in 100 μL of buffer containing Proteinase K (Thermo Scientific, #FEREO0492), RNase A (Thermo Scientific, #FEREN0531), Phusion® GC 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 amplicons spanning the region containing the base-edited site(s). Unpurified PCR amplicons of 500-1000 bp in length can be sequenced by Sanger sequencing.

[0186] Editorial Analysis

[0187] Base editing efficiency can be calculated using the Chimera analysis tool, an adaptation of the open source tool BEAT (Xu et al., 2019, BEAT: A Python Program to Quantify Base Editing from Sanger Sequencing. The CRISPR Journal 2,223-229). Chimera determines the editing efficiency by first subtracting background noise and defining the expected variability within a sample. This allows us to estimate the 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 evaluate the editing efficiency of the base editor over a range of 20 bp of the input guide sequence.

[0188] Examples 3 to 6

[0189] In Examples 3 to 6, the following materials and methods were used.

[0190] gRNA sequence

[0191] In Examples 3-5 (using the Cas12b base editor), gRNA sequences encoded by the sequences shown in Table 3 were used. All gRNA designs were based on the A. acidoterrestris Cas12b gRNA, consisting of a 91 nt constant gRNA sequence, a target-specific 20 nt spacer sequence, and a 7 nt polyT U6 termination signal. All modifications were made to the constant region of the gRNA and consisted of the inclusion of an RNA aptamer hairpin. One copy of the MS2 hairpin sequence (C5 mutant) was incorporated into either the 5', 3' stem loop of the gRNA or an internal polyU (an internal stretch of UUUUU within the gRNA). The corresponding gRNA sequences were cloned into separate expression vectors under the control of the hU6 promoter.

[0192] In Table 3, N denotes the 20 nt target-specific spacer sequence. The constant gRNA sequences mentioned above are highlighted in bold. MS2 (C5 mutant) is shown in italics, and the aptamer extensions are italicized and underlined. Mutations introduced into the internal polyU stretch are in bold and underlined.

[0193] Table 3: gRNA sequences for use with Cas12b

[0194] [Table 3]

[0195] In Example 6 (using the CasMINI base editor), the gRNA sequence encoded by the sequence shown in Table 4 was used. All gRNA designs were based on the Acidibacillus sulfuroxidans Cas12f gRNA, which consists of a 162 nt constant gRNA sequence, a target-specific 23 nt spacer sequence, and a 7 nt polyT U6 termination signal. All modifications were made to the constant components of the gRNA and consisted of the incorporation of an RNA aptamer hairpin and a stem-loop shortening. One copy of the MS2 hairpin sequence (C5 mutant) was incorporated either at the 5', 5' and stem-loop 1 shortening, stem-loop 1 extension, stem-loop 2 replacement and stem-loop 1 shortening, or repeat:anti-repeat replacement. The corresponding gRNA sequences were cloned into separate expression vectors under the control of the hU6 promoter.

[0196] In Table 4, N represents the 23 nt target-specific spacer sequence. Constant gRNA sequences are highlighted in bold. MS2 (C5 variant) is italicized, and aptamer extensions are italicized and underlined. Design 1 corresponds to the 5' MS2, design 2 corresponds to the 5' MS2 and a shortened portion of stem-loop 1, design 3 corresponds to the MS2 located in the extension of stem-loop 1, design 4 corresponds to the MS2 located in the replacement of stem-loop 2 and a shortened portion of stem-loop 1, and design 5 corresponds to the MS2 located in the replacement of the repeat:anti-repeat region.

[0197] Table 4: gRNA sequences for use with CasMINI

[0198] [Table 4]

[0199] Plasmid design

[0200] Apart from the gRNA, all components of the system used were encoded in two vectors and expressed from a CMV promoter. The first vector encoded an enhanced human Apobec3A-MCP or Anolis carolinensis Apobec1a-MCP fusion protein (deaminase vector). The second vector encoded dCas12b (D569A, E847A, D976A) or dCasMINI (D325A; D509A) fused to two copies of UGI through its C-terminus (Cas vector). dCasMINI is a previously described version of dUn1Cas12f1. The dCas12b-UGI-UGI and dCasMINI-UGI-UGI fusion proteins were flanked by two copies of SV40 NLS at the N-terminus of the Cas sequence and the C-terminus of the UGI sequence. Additionally, the vector encoded the expression of turboRFP to allow monitoring of transfection efficiency.

[0201] The corresponding gRNA sequence was cloned into another expression vector under the control of the hU6 promoter (gRNA expression vector).

[0202] One plasmid, shown below as SEQ ID NO:72 and used in Examples 3, 4 and 5, encodes an inactive Cas12b fused to two UGIs (:dCas12b-UGI-UGI). The following font is used: JPEG2024502114000006.jpg205155JPEG2024502114000007.jpg228154JPEG2024502114000008.jpg228153JPEG2024502114000009.jpg29152

[0203] The amino acid sequence corresponding to SEQ ID NO:72 is set forth below as SEQ ID NO:73, with the following font used in that sequence: JPEG2024502114000010.jpg206154JPEG2024502114000011.jpg62160

[0204] The second plasmid, shown below as SEQ ID NO:74 and used in Example 6, encodes an inactive CasMINI fused to two UGIs (:dCasMINI-UGI-UGI). The following font is used: JPEG2024502114000012.jpg215157JPEG2024502114000013.jpg204153

[0205] The amino acid sequence corresponding to SEQ ID NO:74 is set forth below as SEQ ID NO:75, with the following font used in that sequence: JPEG2024502114000014.jpg174156

[0206] The third plasmid, shown below as SEQ ID NO:76, encodes the deaminase fused to MCP (enhanced human Apobec3A-MCP). The following font is used: JPEG2024502114000015.jpg205154JPEG2024502114000016.jpg22156

[0207] The amino acid sequence corresponding to SEQ ID NO:76 is set forth below as SEQ ID NO:77, which uses the following font: JPEG2024502114000017.jpg118156

[0208] The fourth plasmid is shown below as SEQ ID NO:78 and encodes a deaminase fused to MCP (Anolis carolinensis Apobecla-MCP). The following font is used: JPEG2024502114000018.jpg207160JPEG2024502114000019.jpg25154

[0209] The amino acid sequence corresponding to SEQ ID NO:78 is set forth below as SEQ ID NO:79, which uses the following font: JPEG2024502114000020.jpg111160

[0210] The gRNA components of the base editing system were expressed in a separate vector (gRNA expression vector) whose expression was driven by the RNA polymerase III U6 promoter. The gRNA was expressed as a single unit containing the A. acidoterrestris Cas12b or Acidibacillus sulfuroxidans Cas12f crRNA and tracrRNA components linked by an artificial tetraloop as described previously. A list of gRNA target sequences for each Cas protein is shown in Table 5.

[0211] Table 5: gRNA target site sequences for base editing.

[0212] [Table 5]

[0213] Cell culture and transfection

[0214] HEK293 cells were cultured in 10% fetal bovine serum (FBS) and 100 U ml -1 The cells were cultured in Dulbecco's Modified Eagle Medium (DMEM) supplemented with penicillin / streptomycin. 24 hours prior to transfection, 10,000 cells were seeded per well of a 96-well plate to achieve approximately 70% confluency for transfection. After 24 hours, the cells were lipid transfected with 200 ng of plasmid DNA (75 ng of Cas vector, 75 ng of deaminase vector, 50 ng of gRNA expression vector) using 0.7 μl of DharmaFECT™ DUO (Horizon discovery) per well of a 96-well plate.

[0215] cell lysis

[0216] 72 hours after transfection, the medium was removed, the cells were washed once with PBS, and 50 μl of TrypLE express enzyme (ThermoFisher scientific) was added to each well. After disaggregating the cells, 100 μl of fresh DMEM was added, and 20 μl of the resuspended cells were transferred to a 96-well plate and incubated with 60 μl of DirectPCR lysis reagent (Viagen biotech) under the following conditions (55°C for 45 minutes, then 95°C for 15 minutes), and then the cell lysate was stored at -20°C.

[0217] PCR amplification of target regions

[0218] One μl of cell lysate obtained using DirectPCR lysis reagent was used per PCR reaction. For NGS analysis, Q5 high-fidelity 2x master mix (NEB) was used for amplification of sgRNA target sites, and the reaction mix was set up as follows:

[0219] JPEG2024502114000022.jpg92156

[0220] The PCR reactions were carried out under the following thermocycling conditions:

[0221] JPEG2024502114000023.jpg74160

[0222] For Sanger sequencing analysis, the regions of interest were PCR amplified using GoTaq Hot Start polymerase (Promega). The reaction mix was set up as follows:

[0223] JPEG2024502114000024.jpg54161

[0224] Details of the primers used are shown in Table 4.

[0225] Table 6: Primers [5'→3']

[0226] [Table 6]

[0227] Analysis of base editing

[0228] PCR products were subjected to Sanger sequencing (Genewiz). Data were analyzed with proprietary in-house software (Chimera).

[0229] Example 3: Base editing using Cas12b

[0230] In this example, next-generation sequencing analysis of the amplicon region of site 2 shows a specific C to T mutation introduced by introduction of the Cas12b base editor in the HEK-293T cell line. Two different effectors were used: hApobec3A (Apobec3A-MCP) or AnoApobec (Anolis carolinensis Apobec1a-MCP). They were introduced by transfection of a plasmid expressing the sequence highlighted in SEQ ID NO:76 and SEQ ID NO:78, respectively. At the same time, dCas12b-UGI-UGI was introduced by transfection of a plasmid expressing the sequence highlighted in SEQ ID NO:72. The gRNA was introduced by transfection of a plasmid expressing a gRNA backbone corresponding to SEQ ID NO:58 to 65. The sequence of the target site corresponds to SEQ ID NO:80. The constructs were introduced by DNA plasmid lipid transfection, and after lysis of the cells 72 hours after transfection, the target locus was amplified by PCR and analyzed by next-generation sequencing.

[0231] The results are reported in Figure 8A and Figure 8B, respectively. These figures show the level of C to T mutations achieved by delivery of base editing constructs by DNA plasmid introduction into HEK-293 cells. The X-axis indicates the various positions of the MS2 aptamer in the gRNA and corresponds to the sequences highlighted in Table 3. U->G indicates the change in the internal poly-U stretch present in the Cas12b gRNA backbone introduced to increase transcription efficiency under the hU6 promoter. The Y-axis displays the percentage of C to T edits at the indicated C residues in the protospacer as measured by NGS. Each bar represents a targeted C residue in the targeted protospacer and the numbers in the figure legend indicate the position of the corresponding C residue, with 1 being PAM-proximal and 20 being PAM-distal. no-tf=no transfection control; hApobec3A=Apobec3A-MCP; AnoApobec=Anolis carolinensis Apobec1a-MCP. Error bars indicate standard error of the mean of duplicates.

[0232] Figure 8A and Figure 8B show that the Cas12b base editor is functional in HEK-293T cell lines, with editing observed in both hApobec3A and AnoApobec at site 2. Editing is observed in both the 5'MS2 and PolyU-MS2 designs, with two gRNA designs functional. Furthermore, no editing is observed in the absence of the MS2 aptamer (no MS2), indicating that the observed editing is dependent on the recruitment of deaminase through the interaction of the MCP ligand with the MS2 ligand-binding moiety, followed by the assembly of the base editing complex. Editing is observed in a broad window, unlike that observed with the Cas9 base editor, where up to five C residues are edited in a 14 nt window. The shifted editing window highlights the general applicability of the tool and its potential to broaden the scope of sites that can be targeted for base editing.

[0233] Example 4: Base editing at multiple sites

[0234] This example demonstrates that the Cas12b base editor functions in multiple genomic loci and sequence contexts. Base editing analysis shows that the Cas12b base editor introduces C to T mutations at various sites in the HEK-293T cell line. Seven different regions were targeted by sgRNAs: VEGFA_sgRNA1, SEQ ID NO:82; VEGFA_sgRNA6, SEQ ID NO:83; FANCF_sgRNA3, SEQ ID NO:84; Site2_sgRNA2, SEQ ID NO:93; Site2_sgRNA4, SEQ ID NO:81; B2M_sgRNA2, SEQ ID NO:94; and B2M_sgRNA3, SEQ ID NO:95. Two different effectors, hApobec3A or AnoApobec, were used and introduced by transfection with a plasmid expressing sequences corresponding to SEQ ID NO:76 and SEQ ID NO:78, respectively. Cas12b was introduced by transfection with a plasmid expressing sequences corresponding to SEQ ID NO:72. The 5' MS2 version of gRNA (SEQ ID NO: 59) was used in all conditions. The sequences of the target sites correspond to SEQ ID NOs: 93-95 and 81-84. Constructs were introduced by DNA plasmid lipid transfection, and after lysis of cells 72 h post-transfection, the target locus was amplified by PCR and analyzed by Sanger sequencing. The spacer sequences are shown in Table 5 and the primers amplifying the edited region are shown in Table 6.

[0235] Figure 9A (VEGFA_sgRNA1), Figure 9B (VEGFA_sgRNA6), Figure 9C (FANCF_sgRNA3), Figure 9D (site2_sgRNA2), Figure 9E (site2_sgRNA4), Figure 9F (B2M_sgRNA2), and Figure 9G (B2M_sgRNA3). Levels of C to T mutations achieved by delivery of base editing constructs by DNA plasmid introduction into HEK-293 cells at multiple genomic sites and sequence contexts. X-axis indicates reagents used (used with dCas12b-UGI-UGI); hApobec3A = 5'MS2 gRNA + Apobec3A-MCP; AnoApobec = 5'MS2 gRNA + Anolis carolinensis Apobec1a-MCP; no-MS2-hApobec3A = same as hApobec3A except gRNA does not contain MS2 aptamer; no-MS2-AnoApobec = same as AnoApobec except gRNA does not contain MS2 aptamer; no-tf (no tf) = no transfection control. Y-axis displays percentage of C to T editing at the indicated C residues in the protospacer as measured by Sanger sequencing followed by Chimera analysis. Each bar represents a targeted C residue in the targeted protospacer and numbers in the figure legend indicate the position of the corresponding C residue, with 1 being PAM-proximal and 20 being PAM-distal. Error bars indicate standard error of the mean of duplicates.

[0236] Figure 9A-9G show that the Cas12b base editor is viable as a general base editing tool, with editing observed at multiple genomic loci and sequence contexts. Again, editing was observed at multiple C residues within the protospacer, and the size and location of the editing window differed from those observed in the Cas9 base editing system, thus further reinforcing the potential for unique functional applications of this tool. Furthermore, little editing was observed in the absence of the MS2 aptamer (no MS2). This indicates that the observed editing is dependent on the recruitment of the deaminase via interactions between the MCP ligand and the MS2 ligand-binding moiety, followed by the assembly of the base editing complex.

[0237] Example 5: Base editing in various cell lines

[0238] This example demonstrates that the Cas12b base editor is functional in different cell lines. Sanger sequencing and Chimera analysis show that the Cas12b base editor introduces a C to T mutation in the U2OS cell line. One region targeted by one sgRNA is shown, VEGFA_sgRNA1, SEQ ID NO:83. Two different effectors, hApobec3A or AnoApobec, were introduced by transfection with plasmids expressing sequences corresponding to SEQ ID NO:76 and SEQ ID NO:78, respectively. Cas12b was introduced by transfection with a plasmid expressing a sequence corresponding to SEQ ID NO:72. A plasmid expressing the 5'MS2 version of the sgRNA (SEQ ID NO:59) was transfected in all conditions. Constructs were introduced by DNA plasmid lipid transfection and cells were lysed 72 hours after transfection, after which the target locus was amplified by PCR and analyzed by Sanger sequencing.

[0239] Figure 10 shows the level of C to T mutations achieved by delivery of base editing constructs by DNA plasmid introduction into U2OS cells. Data is shown for guide VEGFA_sgRNA1, SEQ ID NO: 83. X-axis indicates reagents used (combined with dCas12b-UGI-UGI); hApobec3A = 5' MS2 gRNA + Apobec3A-MCP; AnoApobec = 5' MS2 gRNA + Anolis carolinensis Apobec1a-MCP; no MS2-hApobec3A = same as hApobec3A except gRNA does not contain MS2 aptamer; no MS2-AnoApobec = same as AnoApobec except gRNA does not contain MS2 aptamer; no-tf (no tf) = no transfection control. The y-axis displays the percentage of C to T editing at the indicated C residues in the protospacer as measured by Sanger sequencing followed by Chimera analysis. Each bar represents a targeted C residue in the target protospacer, and the numbers in the figure legend indicate the position of the corresponding C residue, with 1 being PAM-proximal and 20 being PAM-distal. Error bars indicate the standard error of the mean of two replicates.

[0240] Figure 10 shows that for the same sgRNAs as in Figure 9A, the Cas12b base editing tool works in the U2OS cell line, further demonstrating the general applicability of the base editing tool. Editing is observed with both deaminases, and as observed in HEK-293 cells, editing is seen across a broad window.

[0241] Example 6: Base editing using CasMINI

[0242] This example demonstrates that the gRNA-ligand base editing system can be applied to V-type enzymes other than Cas12b. Here, the CasMINI base editor is shown to introduce C to T mutations at various sites in HEK-293T cells. Two different effectors, hApobec3A or AnoApobec, were introduced by transfection with plasmids expressing sequences corresponding to SEQ ID NO:76 and SEQ ID NO:78, respectively. CasMINI was introduced by transfection with a plasmid expressing a sequence corresponding to SEQ ID NO:74. gRNA was introduced by transfection with a plasmid expressing a gRNA backbone corresponding to SEQ ID NO:66 to 71. Two different regions targeted by two different sgRNAs, VEGFA_sgRNA1, SEQ ID NO:85, and VEGFA_sgRNA2, SEQ ID NO:86, are shown. Constructs were introduced by DNA plasmid lipid transfection, and cells were lysed 72 hours post-transfection, after which the target loci were amplified by PCR and analyzed by Sanger sequencing.

[0243] 11A-11D: Levels of C to T mutations achieved by delivery of CasMINI base editing components by DNA plasmid introduction into HEK-293 cells. The x-axis indicates the various positions of the MS2 aptamer in the gRNA and corresponds to the sequences highlighted in Table 4. sgRNA design 1, SEQ ID NO:67 corresponds to the 5' MS2; sgRNA design 2, SEQ ID NO:68 corresponds to the 5' MS2 and truncated stem-loop 1; sgRNA design 3, SEQ ID NO:69 corresponds to the MS2 located on the extension of stem-loop 1; sgRNA design 4, SEQ ID NO:70 corresponds to the MS2 located at the replacement of stem-loop 2 and the truncated part of stem-loop 1; sgRNA design 5, SEQ ID NO:71 corresponds to the MS2 located at the replacement of the repeat:anti-repeat region. The absence of MS-2 in these figures corresponds to SEQ ID NO:66. The y-axis displays the percentage of C to T editing at the indicated C residues in the protospacer as measured by Sanger sequencing followed by Chimera analysis. Each bar represents a targeted C residue in the target protospacer, and the numbers in the figure legend indicate the position of the corresponding C residue, with 1 being PAM-proximal and 20 being PAM-distal. Error bars indicate the standard error of the mean of two replicates.

[0244] Figures 11A, 11B, 11C and 11D show that the CasMINI base editor is functional in HEK-293T cells. As demonstrated with Cas12b, both AnoApobec and hApobec3a function as part of the CasMINI base editor, and three different gRNA designs (designs 2, 3 and 4) function consistently. Interestingly, the placement of the ligand binding moiety has a distinct effect on the editing behavior, with design 4 exhibiting a wide editing window and a broad range of elevated editing levels, while designs 2 and 3 showed a pronounced preference for a specific C residue within the protospacer. This ability to vary the editing window (both positionally and in size) by varying the placement of the ligand binding moiety within the gRNA further illustrates the unique utility and flexibility of the gRNA-ligand base editing system. Again, little editing was observed in the absence of the MS2 aptamer (no MS2). This indicates that the observed editing depends on the recruitment of the deaminase via interactions between the MCP ligand and the MS2 ligand-binding moiety, followed by assembly of a base-editing complex.

Claims

1. A gRNA-ligand binding complex, comprising: a. a gRNA, said gRNA comprising 60-210 nucleotides, said gRNA comprising: i. a crRNA sequence, the crRNA sequence being 35-60 nucleotides in length, the crRNA sequence comprising: (i) a Cas association region, the Cas association region being 18-30 nucleotides in length; and (ii) a targeting region, the targeting region being 18-30 nucleotides in length; ii. a gRNA comprising a tracrRNA sequence, the tracrRNA sequence being 45-120 nucleotides in length, the tracrRNA sequence comprising an anti-repeat region and a distal region, the anti-repeat region being at least 80% complementary to the Cas association region over at least 18 consecutive nucleotides of the Cas association region, the Cas association region and the anti-repeat region forming a hybridization region capable of hybridizing, the hybridization region being capable of maintaining association with an RNA binding domain of a V-type Cas protein; and b. A gRNA-ligand binding complex comprising a ligand-binding moiety that is either (i) directly bound to the gRNA or (ii) bound to the gRNA via a linker.

2. 2. The gRNA-ligand binding complex of claim 1, wherein the gRNA is formed by a single strand of nucleotides, the single strand of nucleotides comprises the crRNA sequence and the tracrRNA sequence, and a loop sequence is present between the Cas association region and the anti-repeat region.

3. The gRNA-ligand binding complex of claim 1 or 2, wherein the gRNA is composed of a first strand of nucleotides and a second strand of nucleotides, the first strand of nucleotides comprising the tracrRNA sequence, and the second strand of nucleotides comprising the crRNA sequence.

4. 4. The gRNA-ligand binding complex of claim 1, wherein at least one of the gRNA and the ligand binding moiety comprises at least one modification. (i) the gRNA has a 5' end and the ligand-binding moiety is directly attached to the gRNA at the 5' end of the gRNA; or (ii) the gRNA has a 3' end and the ligand binding moiety is directly attached to the gRNA at the 3' end of the gRNA; or (iii) The gRNA-ligand binding complex of claim 2, wherein the gRNA has a 3' end and a 5' end, and the ligand binding moiety is directly attached to the gRNA at a nucleotide other than the 3' end or the 5' end.

6. The gRNA-ligand binding complex of claim 2, wherein the ligand binding moiety is directly bound to the gRNA at the loop sequence.

7. 2. The gRNA-ligand binding complex of claim 1, wherein the gRNA comprises a loop sequence and the ligand binding moiety is directly bound to the gRNA at the loop sequence.

8. The gRNA-ligand binding complex of any one of claims 1 to 3, wherein the ligand-binding moiety is capable of binding to a ligand from the group consisting of MS2, Ku, PP7, SfMu, Sm7, Tat, glutathione S-transferase (GST), CSY4, Qbeta, COM, pumilio, anti-His tag (6H7), λN22plus, SNAP-Tag, lectin, and PDGF β chain.

9. The gRNA-ligand binding complex of claim 8, wherein the ligand is MS2.

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

11. The base editing complex of claim 10, wherein the V-type Cas protein comprises an active RuvC domain or an inactive RuvC domain.

12. The base editing complex of Claim 10, further comprising an effector, wherein the effector binds to a ligand of the ligand binding moiety, and the ligand of the ligand binding moiety is capable of binding to the ligand binding moiety.

13. The base editing complex of claim 12, wherein the effector is selected from the group consisting of a deaminase, a reverse transcriptase, a transcriptional regulator and a repair enzyme.

14. 13. The base editing complex of claim 12, wherein the effector is selected from the group consisting of AID, CDA, APOBEC1, APOBEC3A, APOBEC3B, APOBEC3C, APOBEC3D, APOBEC3F, APOBEC3G, APOBEC3H, ADA, ADAR1, TadA, TADA, TAD3, ADAR2, ADAR3, Dnmt1, Dnmt3a, TET1, TET2, and TDG.

15. A base editing method comprising a step of exposing the base editing complex described in claim 10 to double-stranded DNA.

16. The method of claim 15, wherein the method is performed in vitro.

17. The method of claim 15 , wherein the method is performed in vivo.

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

19. A base editing method comprising a step of exposing the base editing complex described in claim 10 to single-stranded DNA.

20. 20. The method of claim 19, wherein the method is performed in vitro.

21. 20. The method of claim 19, wherein the method is performed in vivo.

22. 20. The method of claim 19, wherein the method is performed ex vivo.

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

24. A genetically edited cell or cell population obtained by the method of claim 23.

25. A use of the base editing complex of any one of claims 10 to 14 in a subject susceptible to a modification process by base editing, 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 a cell containing said dsDNA into said subject.

26. Use of the cell or cell population of claim 24 in a subject susceptible to a base editing modification process, 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.

Images