Composition for treating hemoglobinopathy and use thereof

Abstract

The present disclosure provides a composition for treating hemoglobinopathy (e.g. sickle cell anemia, hemophilia, β-thalassemia, etc.). The present composition comprises a nuclease for modifying the BCL11A gene and a CRISPR-Cas system comprising a guide RNA. Also provided is a method for treatment by administering, in a subject with a hemoglobinopathy-related disease, a system that targets the BCL11A gene or a nucleic acid that encodes such a system.

Description

Compositions for treating hemoglobinopathies and uses thereof Cross-reference to related applications This application claims the benefit of and priority to patent application CN2024113867954, filed September 30, 2024, entitled “Compositions for treating hemoglobinopathies and uses thereof,” the entire contents of which, including any sequence listing and drawings, are incorporated herein by reference in their entirety. Regarding electronic sequence listing In accordance with WIPO Standard ST.26, the symbol “t” is used to represent both T in DNA and U in RNA (the definition of the symbol “t” is “thymine in DNA / uracil in RNA (t / u)”). Thus, in a sequence listing prepared according to ST.26, in any instance where the sequence is RNA, all T’s in the sequence should be considered as U’s. BACKGROUND Hemoglobinopathies include inherited anemias that result in decreased production and / or increased destruction of red blood cells. Common hemoglobinopathies include sickle cell disease as well as alpha- and beta-thalassemia. Hemoglobinopathies are most common in populations in Africa, the Mediterranean basin, and Southeast Asia. Most hemoglobinopathies, including sickle cell anemia, are due to abnormalities in the structure of the globin proteins themselves. Sickle cell anemia is caused by a point mutation in the beta-globin structural gene HBB, leading to the production of abnormal hemoglobin (HbS), which results in decreased oxygen-carrying capacity of the blood, which can cause symptoms such as fatigue, dizziness, and shortness of breath, particularly when exercising. In contrast, thalassemia generally results in insufficient production of normal globin proteins, often through mutations in regulatory genes, leading to a deficiency or absence of adult hemoglobin (HbA). Both sickle cell disease and thalassemia can cause anemia. For patients diagnosed with a hemoglobinopathy, there are currently only a few palliative treatments available, such as blood transfusions, to increase blood oxygen levels. The expression of B-cell lymphoma / leukemia 11A (BCL11A) is downregulated during hematopoietic cell differentiation, and this gene has been found to play a role in suppressing fetal hemoglobin production. Gene editing of BCL11A to increase expression of gamma-globin beyond the remaining ~1% fetal hemoglobin has been proposed as an attractive therapeutic strategy in adults with hemoglobinopathies (Smith, E.C. et al., “Strict in vivo specificity of the Bcl11a erythroid enhancer.” Blood 128(19):2338 (2016)). There remains a need to develop safe and effective treatments for hemoglobinopathies, and modulation of BCL11A using CRISPR-Cas technology is a possible therapeutic avenue. SUMMARY In view of the above background, the present disclosure provides a nuclease variant and a composition targeting a BCL11A gene. In some aspects, the present disclosure provides a nuclease comprising an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identical to SEQ ID NO. 1. In some aspects, the present disclosure provides a composition targeting a BCL11A gene for gene editing of a BCL11A gene, the composition comprising: (i) a nuclease of the present disclosure or a first nucleic acid encoding the nuclease; (ii) a guide RNA or a second nucleic acid encoding the guide RNA, wherein the guide RNA comprises a spacer sequence specific to a target sequence within a BCL11A gene. In some aspects, the present disclosure provides a polynucleotide encoding the nuclease and / or the guide RNA of the composition of the present disclosure. In some aspects, the present disclosure provides a vector comprising the polynucleotide of the present disclosure. In some embodiments, the vector encodes a guide RNA as disclosed herein; in some embodiments, the vector is a plasmid vector, a recombinant AAV (rAAV) vector, or a recombinant lentivirus vector. In some aspects, the present disclosure provides a ribonucleoprotein (RNP) comprising the nuclease and the guide RNA of the present disclosure. In some aspects, the present disclosure provides a lipid nanoparticle (LNP) comprising the composition of the present disclosure. In some aspects, the present disclosure provides a pharmaceutical composition comprising the composition of the present disclosure, the polynucleotide of the present disclosure, the vector of the present disclosure, the ribonucleoprotein (RNP) of the present disclosure, or the lipid nanoparticle (LNP) of the present disclosure; and a pharmaceutically acceptable carrier or excipient. In some aspects, the present disclosure provides a kit comprising the composition of the present disclosure, the polynucleotide of the present disclosure, the vector of the present disclosure, the ribonucleoprotein (RNP) of the present disclosure, or the lipid nanoparticle (LNP) of the present disclosure. In some aspects, the present disclosure provides a cell comprising the composition of the present disclosure, the nucleic acid of the present disclosure, or the vector of the present disclosure. In some aspects, the present disclosure provides a formulation containing a composition of the present disclosure, a polynucleotide of the present disclosure, a vector of the present disclosure, a ribonucleoprotein of the present disclosure, a lipid nanoparticle of the present disclosure, a pharmaceutical composition of the present disclosure, or a cell of the present disclosure, and a pharmaceutically acceptable carrier, diluent, or excipient. In some embodiments, the formulation is a liquid formulation. In some embodiments, the dosage form of the formulation is an injection. In some aspects, the present disclosure provides a guide RNA comprising (i) a spacer sequence specific to a target sequence in a BCL11 A gene, wherein the target sequence is adjacent to a protospacer adjacent motif (PAM) located 5' of the target sequence comprising a motif of 5'-TTN-3'; and (ii) a direct repeat sequence. In some embodiments, the target sequence is selected from the sequences set forth in SEQ ID NO. 5, 11, 17, and / or 18; In some embodiments, the spacer sequence is selected from SEQ ID NO. 12, 13, 19, and / or 20; In some embodiments, the direct repeat sequence is selected from SEQ ID NO. 3 or 4; In some embodiments, the PAM is 5'-TTN-3'; optionally, the guide RNA sequence is selected from SEQ ID NO. 6, 14, 15, 16, 21, 22, 23, and / or 24. In some aspects, the present disclosure provides a method for editing a BCL11 A gene in a cell, the method comprising contacting a host cell with a composition of the present disclosure, a polynucleotide of the present disclosure, a vector of the present disclosure, or a ribonucleoprotein (RNP) of the present disclosure to genetically edit a BCL11 A gene in the host cell. In some aspects, the present disclosure provides a method for treating a hemoglobinopathy in a subject, the method comprising administering to a subject in need thereof a composition of the present disclosure, a polynucleotide of the present disclosure, a vector of the present disclosure, or a ribonucleoprotein (RNP) of the present disclosure, a lipid nanoparticle (LNP) of the present disclosure, a pharmaceutical composition of the present disclosure, or a cell of the present disclosure for editing a BCL11 A gene. BRIEF DESCRIPTION OF DRAWINGS An understanding of certain features and advantages of the present disclosure will be obtained by reference to the following detailed description and drawings, which sets forth illustrative embodiments in which the principles of the present disclosure can be utilized, and in which: FIG. 1 depicts cleavage activity of different CasY7 variants and guide RNAs targeting the BCL11 A gene. Figure 2 depicts cleavage activity of variant C10135 under mediation of BCL11A-crRNA-1 to 4, respectively. Figure 3 depicts a map of PHK09T plasmid. DETAILED DESCRIPTION The techniques and procedures described or referenced herein include techniques and procedures that are generally well understood and / or routinely practiced by those skilled in the art, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (3rd ed., 2001); Current Protocols in Molecular Biology (Ausubel et al. eds., 2003). TECHNICAL TERMS Each of the following terms has the meaning associated with it in this section, unless otherwise specified. The indefinite articles "a" and "an" mean at least one, and are used interchangeably with the phrases "at least one" and "one or more." For example, "a member" means at least one member, or one or more members. The conjunction "or" and "and / or" are used as nonexclusive alternatives. Also, the use of the term "including" as well as other forms such as "include," "includes," and "included" is not limiting. The terms "comprising," "having," "including," and "containing" and any form of these terms are open-ended and do not exclude additional, unrecited elements, or method steps. Any implementation discussed in this specification is considered to be implemented with any method or composition of this disclosure, and vice versa. The term "consisting essentially of as used herein with reference to a given embodiment, pertains to those elements required for the given embodiment. The term allows for the presence of additional elements that do not materially affect the basic and novel or functional character of the embodiments of this disclosure. The term "about" means within an acceptable error range for the particular value stated, as determined by one of ordinary skill in the art, which will depend on the particular context in which the value is being applied. If a particular value is specified, unless otherwise stated the term "about" means within an acceptable error range for that particular value. The term "subject" refers to a human, a mouse, or a non-human primate. The human subject can be of any age (e.g., infant, child, young adult, or adult) and can have a disease and can actually have a genetic alteration. As used herein, the term "BCL11A" refers to "B-cell lymphoma / leukemia 11A." BCL11A plays a role in hematopoietic development and can also play a role as a leukemic disease gene. The terms "treat," "treating," and "treatment" mean to treat a subject (e.g., a human subject) for a disease, including one or more of: inhibiting the disease, i.e., arresting or preventing its development or progression; relieving the disease, i.e., causing regression of the disease state; relieving one or more symptoms of the disease; and curing the disease. The terms "prevent," "preventing," and "prevention" mean to prevent a disease in a subject, including: (a) avoiding or precluding the disease; (b) affecting the predisposition toward the disease; or (c) preventing or delaying the onset of at least one symptom of the disease. As used herein, the term "complex" refers to a population of two or more molecules. In some embodiments, a complex comprises a polypeptide and a nucleic acid molecule that interact with each other (e.g., bind, contact, adhere). As used herein, the term "complex" can refer to a population of a guide RNA and a polypeptide (e.g., CasY7 or a variant thereof). As used herein, the term "complex" can refer to a population of a guide RNA, a polypeptide, and a target sequence. As used herein, the term "complex" can refer to a complex of a guide RNA targeting BCL11A and CasY7 / variant. As used herein, the terms“protospacer adjacent motif” and“PAM” are used interchangeably to refer to a DNA sequence adjacent to a target sequence (e.g., a BCL11 A target sequence) to which a complex of a guide RNA (e.g., a guide RNA targeting BCL11 A) and CasY7 / variant binds. In the case of a double-stranded target, the guide RNA binds to the first strand of the target (e.g., the target strand or spacer-complementary strand), and the PAM sequence as described herein is present in the second complementary strand (e.g., the non-target strand or non-spacer-complementary strand). As used herein, the term“adjacent” includes instances in which the guide RNA of the complex specifically binds to, interacts with, or associates with the target sequence immediately adjacent to the PAM. In such instances, there are no nucleotides between the target sequence and the PAM. The term“adjacent” also includes instances in which there are a small number (e.g., 1, 2, 3, 4, or 5) of nucleotides between the target sequence to which the guide RNA binds and the PAM. In some embodiments, the PAM sequence as described herein is present in the non-target strand (e.g., the non-spacer-complementary strand). As used herein, the term“guide RNA” refers to any RNA molecule that facilitates targeting of a nuclease polypeptide described herein (e.g., CasY7 / variant) to a target sequence (e.g., a sequence of the BCL11 A gene). A guide RNA can be designed as a molecule that includes a sequence that is complementary to a particular nucleic acid sequence (e.g., a sequence of the BCL11 A gene). A guide RNA can comprise a sequence that targets DNA (i.e., a spacer sequence) and a direct repeat (DR) sequence. The term“crRNA” is also used herein to refer to a guide RNA. In this document,“spacer sequence” and“spacer sequence” are used interchangeably. In some embodiments, the spacer sequence is complementary to the target sequence. As used herein, the term “complementary” refers to the ability of a nucleobase of a first nucleic acid molecule (e.g., a guide RNA) to base pair with a nucleobase of a second nucleic acid molecule (e.g., a target sequence). Two complementary nucleic acid molecules are capable of non-covalently binding under appropriate conditions of temperature and ionic strength of the solution. In some embodiments, a first nucleic acid molecule (e.g., a spacer sequence of a guide RNA) comprises 100% complementarity to a second nucleic acid (e.g., a target sequence). In some embodiments, a first nucleic acid molecule (e.g., a spacer sequence of a guide RNA) is complementary to a second nucleic acid molecule (e.g., a target sequence) if the first nucleic acid molecule comprises at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% complementarity to the second nucleic acid. As used herein, the term “substantially complementary” refers to a polynucleotide (e.g., a spacer sequence of a guide RNA) having a certain level of complementarity to a target sequence. In some embodiments, the level of complementarity is such that the polynucleotide can hybridize to the target sequence with sufficient affinity to allow a nuclease polypeptide (e.g., CasY7 or a variant thereof) complexed with the polynucleotide to act on (e.g., cleave) the target sequence. In some embodiments, a spacer sequence that is substantially complementary to a target sequence has less than 100% complementarity to the target sequence. In some embodiments, a spacer sequence that is substantially complementary to a target sequence has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% complementarity to the target sequence. In some embodiments, a guide RNA having a spacer sequence that is substantially complementary to a target sequence has 100% complementarity to the target sequence. As used herein, the terms “target” and “target sequence” refer to a nucleic acid sequence to which a guide RNA specifically binds. In some embodiments, a DNA-targeting sequence (e.g., a spacer) of a guide RNA binds to a target sequence. In the case of a double-stranded target, a guide RNA binds to a first strand of the target (i.e., the target strand or spacer-complementary strand), and a PAM sequence as described herein is present in the second complementary strand (i.e., the non-target strand or non-spacer-complementary strand). The term "kit" refers to any collection of two or more components that together constitute a functional unit useful for a particular purpose. By way of illustration (but not limitation), a kit according to the present disclosure can include a guide RNA complexed with or capable of complexing with an RNA-guided nuclease, and accompanied (e.g., suspended in, or suspendable in) a pharmaceutically acceptable carrier. In certain embodiments, a kit can include a boost element. The kit can be used to introduce the complex into, for example, a cell or a subject, for the purpose of causing a desired genomic alteration in such cell or subject. The components of the kit can be packaged together, or the components can be packaged separately. A kit according to the present disclosure also optionally includes a description of use (DFU) that describes, for example, use of the kit according to the methods of the present disclosure. The DFU can be packaged physically with the kit, or the DFU can be made available to a user of the kit, for example, electronically. The terms "polynucleotide," "nucleotide sequence," "nucleic acid," "nucleic acid molecule," "nucleic acid sequence," and "oligonucleotide" refer to a series of nucleotide bases (also referred to as "nucleotides") in DNA and RNA, and mean any chain of two or more nucleotides. Polynucleotides, nucleotide sequences, nucleic acids, etc. can be chimeric molecules or derivatives or modified forms thereof, single- or double-stranded. They can be modified at the base moiety, sugar moiety, or phosphate backbone, for example, to improve stability of the molecule, its hybridization properties, etc. Nucleotide sequences typically carry genetic information, including but not limited to information for cell organelles to make proteins and enzymes. The terms include both double- and single-stranded genomic DNA, RNA, any synthetic and genetically manipulated polynucleotides, and both sense and antisense polynucleotides. The terms also include nucleic acids containing modified bases. The term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a "plasmid", which refers to a circular double stranded DNA loop into which additional DNA segments can be ligated. Another type of vector is a viral vector, wherein additional DNA segments can be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) are integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as "recombinant expression vectors," or simply, "expression vectors." In general, expression vectors of utility in recombinant DNA techniques are often in the form of plasmids. In the present context, since a plasmid is the most commonly used form of vector for such purposes, "plasmid" and "vector" can be used interchangeably. However, the methods and compositions described herein can include other forms of expression vectors that serve equivalent functions in equivalent ways, such as viral vectors (e.g., replication defective retroviruses, lentivirus, adenovirus, and adeno-associated virus). The term "operably linked" means that the nucleotide sequence of interest is linked to the regulatory sequence(s) in a manner that allows for expression of the nucleotide sequence (e.g., in an in vitro transcription / translation system or in a target cell when the vector is introduced into the target cell). The term "regulatory sequence" is intended to include promoters, enhancers and other expression control elements (e.g., polyadenylation signals). Such regulatory sequences are described, for example, in Goeddel; Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA (1990). Regulatory sequences include those that direct constitutive expression of a nucleotide sequence in a variety of types of host cells, as well as those that direct expression of the nucleotide sequence only in certain host cells (e.g., tissue-specific regulatory sequences). Additionally, the DNA-targeting endonuclease can be delivered by way of a vector that includes a regulatory sequence that directs the synthesis of the DNA-targeting endonuclease at a particular interval of time or over a particular period of time. It will be appreciated by those skilled in the art that the design of the expression vector can depend on such factors as the choice of the target cell, the level of expression of the desired gene product, and the like. The term "treatment" includes reducing or alleviating at least one adverse effect or symptom of a condition, disease, or disorder. For example, the terms "treating" or "treatment" refer to the administration of an effective amount of a composition (e.g., an effective amount of a composition comprising a population of hematopoietic progenitor cells) to a subject to reduce at least one symptom of a disease or to improve the disease (e.g., beneficial or desired clinical results). For purposes of this disclosure, beneficial or desired clinical results include, but are not limited to, alleviation of one or more symptoms, diminishment of extent of disease, stabilized (e.g., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable. In some embodiments, treatment can refer to an increase in lifespan as compared to the expected lifespan without treatment. Thus, one of skill in the art will appreciate that treatment can improve the disease state, but not necessarily cure the disease. In some embodiments, treatment can include prophylaxis. In alternative embodiments, treatment does not include prophylaxis. The term "pharmaceutically acceptable" is used herein to refer to those compounds, materials, compositions, and / or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio. The terms "pharmaceutically acceptable," "physiologically tolerable," and grammatical variations thereof, as used herein, are interchangeable with respect to composition, carrier, diluent and reagent, and are used interchangeably with the term "physiologically tolerable" and refer to a material that does not produce an untoward, undesirable, or inappropriate in vivo response associated with its administration or use, such as nausea, dizziness, gastric upset, etc. A pharmaceutically acceptable carrier does not deliriously elicit an immune response to an agent with which it is combined unless so desired. The preparation / formulation of a pharmaceutical composition that includes the active ingredient dissolved or dispersed in a suitable carrier so as to allow the mixture to be injected can be well known in the art, and need not be limited according to formulation. Generally, such compositions can be prepared as injectable, liquid solutions or suspensions, however, solid forms suitable for solution or suspension in liquid prior to use can also be prepared. The preparation can also be emulsified or provided in liposome form. The active ingredient can be mixed with excipients that are pharmaceutically acceptable and compatible with the active ingredient in amounts suitable for use in the therapeutic methods described herein. Suitable excipients are, for example, water, saline, dextrose, glycerol, ethanol, or the like and combinations thereof. In addition, if desired, the composition can include minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents, and the like, which do not deliriously affect the efficacy of the active ingredient. The therapeutic compositions of the present disclosure can include pharmaceutically acceptable salts of the components therein. Pharmaceutically acceptable salts include the acid addition salts (formed with the free amino groups of the polypeptide) that are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, 2- ethylamino ethanol, histidine, procaine and the like. Physiologically tolerable carriers are well known in the art. An exemplary liquid carrier is a sterile aqueous solution that contains no substances other than the active ingredient and water, or a sterile aqueous solution containing a buffer, such as sodium phosphate at physiological pH, physiological saline or both, such as phosphate-buffered saline. Still further, the aqueous carrier can contain more than one buffer, as well as salts (such as sodium and potassium chlorides), dextrose, polyethylene glycol and other solutes. The liquid composition can also contain a liquid phase other than water, and can include a suspension of the active ingredient in a liquid. Exemplary such additional liquid phases are glycerol, vegetable oils such as cottonseed oil, and water-oil emulsions. The amount of active agent used in the methods described herein (which will be effective in the treatment of a particular disorder or condition) will depend on the nature of the disorder or condition, and can be determined by standard clinical techniques. As used herein, "prevention" or "preventing" when used in reference to a disease, disorder, or symptoms thereof, means reducing the likelihood that an individual will develop a disease or disorder (e.g., a hemoglobinopathy). For example, an individual having one or more risk factors for a disease or disorder does not develop the condition; or develops such a disease or disorder at a later time or with less severity, relative to a population having the same risk factors and not receiving a treatment described herein, has a reduced likelihood of developing the disease or disorder. A failure to develop symptoms of a disease, or a reduction in the development of symptoms (e.g., a reduction of at least 10% in a clinically accepted score for the disease or disorder) or delay (e.g., by days, weeks, months, or years) is considered effective prevention. With respect to contacting a cell with a DNA-targeting endonuclease to reduce BCL11 A gene expression, the term "increasing fetal hemoglobin levels in a cell" means that fetal hemoglobin in a cell or population of cells treated with a DNA-targeting endonuclease is at least 5% higher than a comparable control population in the absence of the DNA-targeting endonuclease. Preferably, fetal hemoglobin expression in a nuclease-treated cell is at least 10% higher, at least 20% higher, at least 30% higher, at least 40% higher, at least 50% higher, at least 60% higher, at least 70% higher, at least 80% higher, at least 90% higher, at least 1-fold higher, at least 2-fold higher, at least 5-fold higher, at least 10-fold higher, at least 100-fold higher, at least 1000-fold higher, or more, relative to a comparable control-treated population. The term "control-treated population" is used herein to describe a population of cells treated identically, with the exception of the addition of a BCL11 A inhibitor, to the nuclease-treated population. As used herein, the term "mammal" is intended to encompass both singular "mammals" and plural "mammals" and includes, but is not limited to, humans; primates such as apes, monkeys, orangutans, and chimpanzees; canids such as dogs and wolves; felines such as cats, lions, and tigers; equines such as horses, donkeys, and zebras; food animals such as cows, pigs, and sheep; ungulates such as deer and giraffes; rodents such as mice, rats, hamsters, and guinea pigs; and bears. In some preferred embodiments, the mammal is a human. In one embodiment, the mammal has been diagnosed with a hemoglobinopathy. In further embodiments, the hemoglobinopathy is a beta- hemoglobinopathy. In a preferred embodiment, the hemoglobinopathy is sickle cell disease. As used herein, "sickle cell disease" can be sickle cell anemia, sickle-hemoglobin C disease (HbSC), sickle beta+ thalassemia (HbS / β+), or sickle beta° thalassemia (HBS / β°). In another preferred embodiment, the hemoglobinopathy is beta- thalassemia. As used herein, the term "hemoglobinopathy" means any defect in the structure or function of any hemoglobin in an individual, including defects in the primary, secondary, tertiary, or quaternary structure of hemoglobin caused by any mutation, such as a deletion mutation or a substitution mutation in the coding region of the beta-globin gene, or a mutation or deletion in the promoter or enhancer of such gene that results in a decrease in the amount of hemoglobin produced as compared to normal or standard conditions. The term further includes any decrease in the amount or effectiveness of hemoglobin caused by external factors, such as disease, chemotherapy, toxins, poisons, and the like, whether the above-mentioned conditions are normal or abnormal. As used herein, the term "effective amount" in one embodiment means an amount of a cellular composition that is safe and sufficient to treat a hemoglobinopathy, reduce the likelihood of a hemoglobinopathy, or delay the progression of a hemoglobinopathy. Thus, the amount can cure a symptom of a hemoglobinopathy or ameliorate a symptom of a hemoglobinopathy, delay the progression of a disease process of a hemoglobinopathy, delay or inhibit a symptom of a hemoglobinopathy, delay or inhibit the establishment of a secondary symptom of a hemoglobinopathy, or inhibit the development of a secondary symptom of a hemoglobinopathy. The effective amount to treat a hemoglobinopathy depends on the type of hemoglobinopathy to be treated, the severity of the symptoms, the subject being treated, the age and general condition of the subject, the mode of administration, and the like. Thus, it is not possible to specify an exact "effective amount," or it is unwise to do so. However, for any given situation, an appropriate "effective amount" can be determined by one of ordinary skill in the art using only routine experimentation. I. Compositions The present disclosure provides compositions targeting the BCL11A gene, which can be used to edit the BCL11A gene target, illustratively, the compositions can disrupt the BCL11A gene. In some embodiments, the composition targeting the BCL11A gene for use in gene editing of the BCL11A gene, the composition comprises: (i) a nuclease of the present disclosure or a first nucleic acid encoding the nuclease; (ii) a guide RNA or a second nucleic acid encoding the guide RNA, wherein the guide RNA comprises a spacer sequence specific to a target sequence within the BCL11A gene. In some embodiments, the guide RNA is comprised of a direct repeat (DR) sequence and a spacer sequence. In some embodiments, the guide RNA binds to the nuclease to form a CRISPR-Cas complex targeting the BCL11A gene target sequence. In some embodiments, the spacer sequence is specific to a BCL11A target sequence, wherein the BCL11A target sequence is adjacent to a protospacer adjacent motif (PAM), in some embodiments, the PAM sequence is 5'-TTN-3' as described in the present disclosure. In the case of a dsDNA gene target, the guide RNA binds to the first strand (non-PAM strand) of the gene target, and the PAM sequence as described herein is present in the second complementary strand (i.e., PAM strand). In some embodiments, the present disclosure provides a complex comprising a guide RNA and a nuclease. In some embodiments, the guide RNA and the nuclease bind to each other in a molar ratio of about 1:1. In some embodiments, the complex comprising the guide RNA and the nuclease binds to a complementary region of a target sequence within the BCL11A gene. In some embodiments, the complex comprising the guide RNA and the nuclease targeting the BCL11A binds to a complementary region of a target sequence within the BCL11A gene in a molar ratio of about 1:1. In some embodiments, the complex of the guide RNA and the nuclease has enzymatic activity, such as nuclease activity, to cleave the BCL11A target sequence and / or the complementary sequence. In some embodiments, the guide RNA, the nuclease, and the complementary region of the BCL11A target sequence, whether alone or together, are not naturally occurring. In some embodiments, the guide RNA in the complex comprises a direct repeat (DR) sequence and / or a spacer sequence described herein. In some embodiments, the guide RNA sequence has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identity to the sequence of any one of SEQ ID NO. 6, 14, 15-16, 21-24. In some embodiments, the guide RNA has any one of SEQ ID NO. 6, 14, 15-16, 21-24. In some embodiments, the compositions provided by the present disclosure comprise a nuclease polypeptide as described herein and / or a nucleic acid (e.g., RNA) encoding a nuclease as described herein. In some embodiments, the compositions provided by the present disclosure comprise a nuclease polypeptide as described herein and / or a first nucleic acid (e.g., RNA) encoding a nuclease as described herein, and a guide RNA or a second nucleic acid encoding the guide RNA. In some embodiments, the nuclease polypeptide or the first nucleic acid encoding the nuclease is included within the same composition as the guide RNA or the second nucleic acid encoding the guide RNA. In some embodiments, the nuclease or the RNA encoding the nuclease polypeptide is included within a separate composition as the guide RNA or the second nucleic acid encoding the guide RNA. In some embodiments, the guide RNA comprises a direct repeat (DR) sequence and / or a spacer sequence described herein. In some embodiments, the DR sequence of the guide RNA has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identity to the sequence of any one of SEQ ID NO. 3 or 4. In some embodiments, the spacer sequence of the guide RNA has the sequence of any one of SEQ ID NO. 12-13, 19-20. 1. CasY7 polypeptides / variants The compositions of the present disclosure comprise CasY7 or variants thereof as described in PCT / CN2024 / 092707, the relevant disclosures of which are incorporated by reference for the subject matter and purposes cited herein. The nucleotide sequence of the CasY7 polypeptide parent is shown in SEQ ID NO. 1, and the encoding nucleotide is shown in SEQ ID NO. 2. In some embodiments, the nuclease of the present disclosure has an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identical to SEQ ID NO. 1. In some embodiments, the nuclease of the present disclosure comprises an amino acid sequence that differs from the amino acid sequence of SEQ ID NO. 1 by no more than 70 amino acid residues, e.g., differs by 69, 68, 67, 66, 65, 64, 63, 62, 61, 60, 59, 58, 57, 56, 55, 54, 53, 52, 51, 50, 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 amino acid residues. In some embodiments, the nuclease of the present disclosure comprises one or more mutations at positions 2, 3, 5, 8, 165, 166, 175, 176, 228, 229, 240, 241, 242, 243, 282, 283, 285, 308, 309, 316, 317, 318, 319, 416, 417, 418, 419, 420, 431, 435, 441, 552, 564, 574, 638, 639, 640, 641, 642, 648, 651, 652, 681, 682, 683, 684, 748, 749, 751, 829, 865, 866, 867, 868, 788, 896, 915, 916, 918, and / or 919 of SEQ ID NO. 1. In some embodiments, the nuclease of the present disclosure comprises one or more mutations relative to the amino acid sequence set forth in SEQ ID NO. 1 that are amino acid substitutions, optionally comprising the following mutation patterns: (i) Y282 + D283 + A285; or (ii) G416 + I417 + E418 + F419 + D420. In some embodiments, the nuclease of the present disclosure comprises one or more mutations relative to the amino acid sequence set forth in SEQ ID NO. 1 that are amino acid substitutions, optionally comprising the following mutation patterns: (a) Y282 + D283 + A285 + N682 + E683 + S684 + E748 + G749 + S751 + K865 + P866 + Y867 + N868; (b) F228 + L229 + Y282 + D283 + A285 + G416 + I417 + E418 + F419 + D420; (c) Y282 + D283 + A285 + G416 + I417 + E418 + F419 + D420 + K865 + P866 + Y867 + N868; (d) Y165 + S166 + G416 + I417 + E418 + F419 + D420 + K865 + P866 + Y867 + N868; (e) Y282 + D283 + A285 + Y308 + S309 + T681 + N682 + E683 + S684 + E748 + G749 + S751; (f) A175 + E176 + Y282 + D283 + A285 + G416 + I417 + E418 + F419 + D420 + T681 + N682 + E683 + S684; (g) A175 + E176 + S240 + S241 + Q242 + E243 + Y282 + D283 + A285 + G416 + I417 + E418 + F419 + D420 + T681 + N682 + E683 + S684; (h) Y165 + S166 + E176 + G416 + I417 + E418 + F419 + D420 + T681 S + N682 L + E683 + S684 + E748 + G749 + S751 + E788; (i) E176 + S240 + S241 + Q242 + E243 + Y282 + D283 + A285 + G416 + I417 + E418 + F419 + D420 + E748 + G749 + S751 + E788; (j) Y282 + D283 + A285 + G416 + I417 + E418 + F419 + D420 + T681 + N682 + E683 + S684 + K865 + P866 + Y867 + N868; (k) Y165 + S166 + G416 + I417 + E418 + F419 + D420 + E748 + G749 + S751 + K865 + P866 + Y867 + N868; (l) E176 + Y282 + D283 + A285 + G416 + I417 + E418 + F419 + D420 + E748 + G749 + S751 + E788; (m) E176 + Y282 + D283 + A285 + N682 + E683 + S684 + E748 + G749 + S751 + E788; (n) E176 + Y282 + D283 + A285 + G416 + I417 + E418 + F419 + D420 + N682 + E683 + S684 + E788; (o) Y165 + S166 + E176 + G416 + I417 + E418 + F419 + D420 + N682 + E683 + S684 + E788; (p) Y165 + S166 + E176 + G416 + I417 + E418 + F419 + D420 + T681 + N682 + E683 + S684 + E748 + G749 + S751 + V915 + L916 + S918 + I919 + E788; (q) E176 + Y282 + D283 + A285 + G416 + I417 + E418 + F419 + D420 + T681 + N682 + E683 + S684 + E829; (r) Y282 + D283 + A285 + N682 + E683 + S684 + E748 + G749 + S751 ; (s) Y282 + D283 + A285 + E316 + T317 + I318 + I319 + T681 + N682 + E683 + S684 + E748 + G749 + S751 ; (t) Y282 + D283 + A285 + G416 + I417 + E418 + F419 + D420 + N682 + E683 + S684 + K865 + P866 + Y867 + N868; (u) Y282 + D283 + A285 + T681 + N682 + E683 + S684 + E748 + G749 + S751 + K865 + Y867 + N868; (v) Y165 + S166 + G416 + I417 + E418 + F419 + D420 + K865 + Y867 + N868; (w) Y282 + D283 + A285 + G416 + I417 + E418 + F419 + D420 + T681 + N682 + E683 + S684 + K865 + Y867 + N868; (x) Y165 + S166 + G416 + I417 + E418 + F419 + D420 + E748 + G749 + S751 + K865 + Y867 + N868; (y) Y282 + D283 + A285 + G416 + I417 + E418 + F419 + D420 + E748 + G749 + S751 + K865 + Y867 + N868; (z) Y165 + S166 + G416 + I417 + E418 + F419 + D420 + N682 + E683 + S684 + E748 + G749 + S751 + K865 + Y867 + N868 + D896; (aa) Y165 + S166 + G416 + I417 + E418 + F419 + D420 + E748 + G749 + S751 + K865 + Y867 + N868; (bb) Y282 + D283 + A285 + G416 + I417 + E418 + F419 + D420 + D638 + R639 + G640 + E641 + F642 + T681 + N682 + E683 + S684; (cc) A175 + E176 + Y282 + D283 + A285 + G416 + I417 + E418 + F419 + D420 + E748 + G749 + S751; (dd) A175 + E176 + Y282 + D283 + A285 + G416 + I417 + E418 + F419 + D420 + E648 + A651 + Y652; (ee) Y165 + S166 + G416 + I417 + E418 + F419 + D420 + K441 + E748 + G749 + S751; (ff) Y165 + S166 + G416 + I417 + E418 + F419 + D420 + Y431R + E748 + G749 + S751; (gg) A2 + Y165 + S166 + G416 + I417 + E418 + F419 + D420 + E748 + G749 + S751; (hh) Y165 + S166 + G416 + I417 + E418 + F419 + D420 + K574 + E748 + G749 + S751; (ii) T3 + Y165 + S166 + G416 + I417 + E418 + F419 + D420 + E748 + G749 + S751; (jj) Y165 + S166 + G416 + I417 + E418 + F419 + D420 + P552 + E748 + G749 + S751; (kk) R8 + Y165 + S166 + G416 + I417 + E418 + F419 + D420 + E748 + G749 + S751; (ll) Y165 + S166 + G416 + 1417 + E418 + F419 + D420 + N564 + E748 + G749 + S751 ; (mm) Y165 + S166 + G416 + 1417 + E418 + F419 + D420 + T435 + E748 + G749 + S751 ; or (nn) T5 + Y165 + S166 + G416 + 1417 + E418 + F419 + D420 + E748 + G749 + S751. In some embodiments, the nuclease of the present disclosure comprises the following pattern of mutations relative to the amino acid sequence set forth in SEQ ID NO. 1 : (oo) Y282F + D283Q + A285T + N682G + E683A + S684R + E748A + G749S + S751G + K865G + P866A + Y867A + N868Y; (pp) F228W + L229R + Y282F + D283Q + A285T + G416V + 1417H + E418Q + F419R + D420L; (qq) Y165W + S166R + G416R + 1417E + E418Q + F419V + D420A + K865G + P866A + Y867A + N868Y; (rr) A175R + E176R + Y282F + D283Q + A285T + G416V + 1417H + E418Q + F419R + D420L + T681E + N682G + E683L + S684A; (ss) A175R + E176R + S240T + S241A + Q242L + E243D + Y282F + D283Q + A285T + G416V + 1417H + E418Q + F419R + D420L + T681E + N682G + E683L + S684A; (tt) Y165W + S166R + E176R + G416R + 1417V + E418Q + F419V + D420T + T681S + N682L + E683A + S684R + E748A + G749S + S751G + E788R; (uu) E176R + S240T + S241A + Q242L + E243D + Y282F + D283Q + A285T + G416V + 1417H + E418Q + F419R + D420L + E748A + G749S + S751G + E788R; (vv) Y282F + D283Q + A285T + G416V + I417H + E418Q + F419R + D420L + T681E + N682G + E683L + S684A + K865G + P866A + Y867A + N868Y; (ww) Y165W + S166R + G416R + I417V + E418Q + F419V + D420T + E748A + G749S + S751G + K865G + P866A + Y867A + N868Y; (xx) E176R + Y282F + D283Q + A285T + G416V + I417H + E418Q + F419R + D420L + E748A + G749S + S751G + E788R; (yy) E176R + Y282F + D283Q + A285T + N682G + E683A + S684R + E748A + G749S + S751G + E788R; (zz) E176R + Y282F + D283Q + A285T + G416V + I417H + E418Q + F419R + D420L + N682G + E683A + S684R + E788R; (aaa) Y165W + S166R + E176R + G416R + I417E + E418Q + F419V + D420A + N682G + E683A + S684R + E788R; (bbb) Y165W + S166R + E176R + G416R + I417V + E418Q + F419V + D420T + T681E + N682G + E683L + S684A + E748A + G749S + S751G + V915I + L916V + S918K + I919V + E788R; (ccc) E176R + Y282F + D283Q + A285T + G416V + I417H + E418Q + F419R + D420L + T681E + N682G + E683L + S684A + E829R; (ddd) Y282F + D283Q + A285T + N682G + E683A + S684R + E748A + G749S + S751G; (eee) Y282F + D283Q + A285T + E316T + T317L + I318R + I319V + T681E + N682G + E683L + S684A + E748A + G749S + S751G; (fff) Y282F + D283Q + A285T + G416V + I417H + E418Q + F419R + D420L + N682G + E683A + S684R + K865G + P866A + Y867A + N868Y; (ggg) Y282F + D283Q + A285T + T681E + N682G + E683L + S684A + E748A + G749S + S751G + K865S + Y867N + N868M; (hhh) Y165W + S166R + G416R + I417E + E418Q + F419V + D420A + K865S + Y867N + N868M; (iii) Y282F + D283Q + A285T + G416V + I417H + E418Q + F419R + D420L + T681E + N682G + E683L + S684A + K865S + Y867N + N868M; (jjj) Y165W + S166R + G416R + I417V + E418Q + F419V + D420T + E748A + G749S + S751G + K865S + Y867N + N868M; (kkk) Y282F + D283Q + A285T + G416V + I417H + E418Q + F419R + D420L + E748A + G749S + S751G + K865S + Y867N + N868M; (lll) Y165W + S166R + G416R + I417V + E418Q + F419V + D420T + N682G + E683A + S684R + E748A + G749S + S751G + K865S + Y867N + N868M + D896N; (mmm) Y165W + S166R + G416R + I417E + E418Q + F419V + D420A + E748A + G749S + S751G + K865S + Y867N + N868M; (nnn) Y282F + D283Q + A285T + G416V + I417H + E418Q + F419R + D420L + D638S + R639A + G640D + E641Y + F642I + T681E + N682G + E683L + S684A; (ooo) A175R+E176R+Y282F+D283Q+A285T+G416V+I417H+E418Q+F419R+D420L+E748A+ G749S+S751G; (ppp) A175R+E176R+Y282F+D283Q+A285T+G416V+I417H+E418Q+F419R+D420L+E648T+A651 S+Y652F; (qqq) Y165W+S166R+G416R+I417V+E418Q+F419V+D420T+K441R+E748A+G749S+S751G; (rrr) Y165W+S166R+G416R+I417V+E418Q+F419V+D420T+Y431R+E748A+G749S+S751G; (sss) A2R+Y165W+S166R+G416R+I417V+E418Q+F419V+D420T+E748A+G749S+S751G; (ttt) Y165W+S166R+G416R+I417V+E418Q+F419V+D420T+K574R+E748A+G749S+S751G; (uuu) T3R+Y165W+S166R+G416R+I417V+E418Q+F419V+D420T+E748A+G749S+S751G; (vvv) Y165W+S166R+G416R+I417V+E418Q+F419V+D420T+P552R+E748A+G749S+S751G; (www) R8K+Y165W+S166R+G416R+I417V+E418Q+F419V+D420T+E748A+G749S+S751G; (xxx) Y165W+S166R+G416R+I417V+E418Q+F419V+D420T+N564R+E748A+G749S+S751G; (yyy) Y165W+S166R+G416R+I417V+E418Q+F419V+D420T+T435R+E748A+G749S+S751G; (zzz) T5R + Y165W + S166R + G416R + I417V + E418Q + F419V + D420T + E748A + G749S + S751G; (aaaa) Y282F + D283Q + A285T + G416V + I417H + E418Q + F419R + D420L + K865G + P866A + Y867A + N868Y; or (bbbb) Y282F + D283Q + A285T + Y308F + S309A + T681E + N682G + E683L + S684A + E748A + G749S + S751G. The amino acid mutations of the nucleases described herein relative to the amino acid sequence set forth in SEQ ID NO. 1 can be one or more amino acid changes, in some cases, the changes to the nucleases of the disclosure can also be substantial, for example, the nucleases are part of an amino-terminal extension and / or carboxy-terminal extension of a fusion protein. In some embodiments, the nucleases can contain additional proteins or peptides, for example, one or more proteins or polypeptides. In some cases, the additional proteins or polypeptides can be selected from a nuclear localization signal (NLS), a nuclear export signal (NES), a reporter protein (e.g., a fluorescent protein), a Cas protein targeting moiety, a DNA binding domain (e.g., Lex A DBD, Gal4 DBD, Sp1 DBD), an epitope tag (e.g., His, myc, V5, FLAG, HA, VSV-G, etc.), a transcriptional activation domain (e.g., VP64, VPR, p65, Rta), a transcriptional repression domain (e.g., KRAB domain, SID domain, NuE domain, NcoR domain, or SID4X domain), a nuclease, a deaminase (e.g., an adenosine deaminase or a cytidine deaminase), a methylase (e.g., a DNA methylase DNMT), a demethylase, a transcriptional release factor, an HDAC, a lytic activity polypeptide, a ligase, an integrase, a transposase, a recombinase, a polymerase, an exonuclease (e.g., T5E), and a base excision repair inhibitor (e.g., a uracil-DNA glycosylase inhibitor (UGI)). In some cases, the additional protein or polypeptide can be selected from a protein or peptide having a methylase activity, a demethylase activity, an acetyltransferase activity, a deacetylase activity, a kinase activity, a phosphatase activity, a ubiquitin ligase activity, a deubiquitinating activity, an adenylation activity, a deadenylation activity, a SUMOylation activity, a desumoylation activity, a ribosylation activity, a deribosylation activity, a myristoylation activity, a demyristoylation activity, a glycosylation activity (e.g., from an O-GlcNAc transferase), and a deglycosylation activity. In some embodiments, the functional domain is selected from an adenosine deaminase catalytic domain or a cytidine deaminase catalytic domain. In some embodiments, the adenosine deaminase catalytic domain or cytidine deaminase catalytic domain comprises one or more of ADAR1, ADAR2, APOBEC, AID, or TAD. In some embodiments, the additional protein or peptide can comprise an epitope peptide for labeling, such as a His tag, Myc, and FLAG. In some embodiments, the nuclease described in the present disclosure can be fused to a detectable moiety, such as a fluorescent protein (e.g., green fluorescent protein (GFP) or yellow fluorescent protein (YFP)). In some embodiments, the nuclease of the present disclosure comprises at least 1 nuclear localization signal (NLS) and / or nuclear export signal (NES), e.g., comprises 2, 3, 4, 5, or more NLS, and / or comprises at least 1 nuclear export signal (NES) (e.g., 2, 3, 4, 5, or more. In some embodiments, the nuclease of the present disclosure comprises 2-4 nuclear localization signals (NLS). In some embodiments, the nuclease of the present disclosure comprises at least 1 (e.g., 2, 3, 4, 5, or more) NLS and at least 1 (e.g., 2, 3, 4, 5, or more) NES. In some embodiments, the nuclease of the present disclosure is substantially inactive, e.g., the nuclease comprises amino acid mutations selected from the group consisting of D592A, D643A, E820A, and / or D992A, relative to the amino acid sequence set forth in SEQ ID NO. 1. The nuclease of the present disclosure (e.g., CasY7 parent polypeptide and variants thereof) is smaller relative to other nucleases (e.g., CasY7 parent polypeptide and variants thereof has only 1022 amino acids, while spCas9 is 1368 amino acids, and LbCpf1 is 1246 amino acids), and thus, the CasY7 parent polypeptide and variants thereof of the present disclosure is advantageous for delivery, and has low off-target, high specificity properties. 2. Guide RNA In some embodiments, the compositions described herein include a guide RNA targeting the BCL11 A gene. The guide RNA can direct a CasY7 polypeptide or a variant thereof included in the compositions as described herein to a BCL11 A gene target sequence. Two or more guide RNAs can direct two or more separate CasY7 and / or variants thereof to 2 or more (e.g., 3, 4, 5, 6, 7, 8, 9, or more) BCL11 A gene target sequences. In some embodiments, the compositions of the present disclosure include a guide RNA targeting a BCL11 A gene regulatory sequence (e.g., an enhancer sequence). In some embodiments, the compositions of the present disclosure include 1 guide RNA targeting the BCL11 A gene, in some cases, the compositions of the present disclosure include 2 or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) guide RNAs. In some embodiments, the guide RNA included in the compositions herein comprises: (1) a direct repeat (DR) sequence capable of forming a complex with a nuclease of the present disclosure or a variant thereof, and (2) a spacer sequence capable of hybridizing to a target sequence of a BCL11 A gene, thereby directing the complex to the target DNA of the BCL11 A gene. In some embodiments, the guide RNA does not comprise a tracrRNA. In some embodiments, the direct repeat (DR) sequence is at the 5' end of the spacer sequence, which is capable of forming a complex with a Cas protein or a variant polypeptide thereof of the present disclosure, or a fusion protein of the present disclosure. In some embodiments, the guide RNA comprises, or consists essentially of, or consists of a direct repeat sequence (DR) and a spacer sequence. In some embodiments, the guide RNA is a single molecule nucleic acid, the direct repeat (DR) sequence is linked to the spacer sequence. In some embodiments, the guide RNA comprises multiple tandemly arranged spacer sequences, optionally separated by nucleotide sequences, e.g., direct repeat (DR) sequences as defined herein. The positions of the different spacer sequences are tandem without affecting activity. In some embodiments, the guide RNA comprises multiple (e.g., 2 or more) identical direct repeat (DR) sequences. In some embodiments, the guide RNA comprises multiple (e.g., 2 or more) different direct repeat (DR) sequences. In some embodiments, the guide RNA comprises spacer sequences and direct repeat sequences that are directly connected to each other. In some embodiments, the guide RNA comprises spacer sequences and direct repeat sequences that are connected to each other by a linker (e.g., an RNA linker comprising multiple nucleotides). (i) direct repeat (DR) sequence In some embodiments, the guide RNA described herein comprises a direct repeat (DR) sequence. In some embodiments, the direct repeat (DR) sequence of the guide RNA has a sequence consisting of multiple nucleotides, in some cases, the guide RNA described herein comprises a direct repeat (DR) sequence consisting of 15-90 (e.g., 16-80, 17-75, 18-70, 19-65, 20-60, 23-55, 25-50, 23, or 27) nucleotides. In some embodiments, the direct repeat (DR) sequence can comprise the nucleotide sequence set forth in any one of SEQ ID NO. 3 or 4, or a nucleotide sequence having at least about 50% (e.g., at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the nucleotide sequence set forth in any one of SEQ ID NO. 3 or 4. In some embodiments, the DR is a “functional variant” (e.g., a “functionally truncated version,” a “functionally extended version,” or a “functionally replaced version”) of the nucleotide sequence set forth in SEQ ID NO. 3 or 4, but still has the function of a DR, still retains at least a portion (e.g., at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or higher) of the function of the reference DR (parental DR). In some embodiments, the DR sequence comprises a stem-loop structure near the 3' end (immediately adjacent to the spacer sequence). A "stem-loop structure" refers to a nucleic acid having a secondary structure, including a region of nucleotides known or predicted to form a double-stranded (stem) portion, connected at one end by a substantially single-stranded nucleotide connection (loop). The term "hairpin" structure is also used herein to refer to a stem-loop structure. These structures are well known in the art, and the terms are used according to their meanings generally known in the art. The stem-loop structure does not require exact base pairing. Thus, the stem can comprise one or more base mismatches. Alternatively, the base pairing can be exact, i.e., does not include any mismatches. In one embodiment, the guide RNA of the present disclosure comprises a direct repeat (DR) sequence comprising a stem-loop structure near the 3' end of the DR. In some embodiments, the stem comprised in the DR consists of 5 pairs of complementary bases that hybridize to each other, and the loop is 6, 7, 8, or 9 nucleotides in length. In some embodiments, the loop is 7 nucleotides in length. In some embodiments, the stem can comprise at least 2, at least 3, at least 4, or at least 5 base pairs. In some embodiments, the DR comprises two stretches of complementary nucleotides of about 5 nucleotides in length separated by about 7 nucleotides. In some embodiments, the stem-loop structure comprises a first stem nucleotide strand of 5 nucleotides in length; a second stem nucleotide strand of 5 nucleotides in length, wherein the first and second stem nucleotide strands can hybridize to each other; and a loop nucleotide strand arranged between the first and second stem nucleotide strands, wherein the loop nucleotide strand comprises 6, 7, or 8 nucleotides. As used herein, two or more guide RNAs having substantially identical or no substantial difference in secondary structure means that the stems and / or loops comprised in the crRNAs differ in length by no more than 1, 2, or 3 nucleotides; differ in nucleotide type (A, U, G, or C) by no more than 1, 2, 3, 4, 5, 6, 7, or 8 nucleotides when the nucleotide sequences of the guide RNAs are compared by sequence alignment. In some embodiments, two or more guide RNAs having substantially identical or no substantial difference in secondary structure means that the stems comprised in the crRNAs differ by at most one pair of complementary bases, and / or the loops differ by at most one nucleotide in length, and / or comprise stems of identical length but with mismatched bases. In some embodiments, the stem loop structure comprises 5'-X1X2X3X4X5NNNNNNNX6X7X8X9X10-3'; X1, X2, X3, X4, X5, X6, X7, X8, X9, X10 are any base comprising A, T, C, or G, N is any base comprising A, T, C, or G; wherein X1, X2, X3, X4, X5and X6, X7, X8, X9, X10 can hybridize to each other to form a stem and such that NNNNNNN forms a loop; more preferably wherein the DR sequence comprises any one of the following stem loop structure near the 3' end of the DR sequence: 5'-CCGTCNNNNNNNGACGG-3' (SEQ ID NO. 8); wherein, wherein N is any base comprising A, T, C, or G. In some embodiments, a DR sequence that can direct any nuclease or variant polypeptide of the disclosure to a target site comprises one or more nucleotide changes selected from nucleotide additions, insertions, deletions, and substitutions that do not result in a substantial difference in secondary structure compared to the DR sequence set forth in SEQ ID NO 3 or 4 or a functionally truncated version thereof. In some embodiments, a direct repeat (DR) sequence herein has at least 90% identity (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity) to the sequence set forth in SEQ ID NO. 3 or 4. In some embodiments, a direct repeat (DR) sequence described herein has at least 95% identity (e.g., at least 95%, 96%, 97%, 98%, or 99% identity) to a portion of the sequence set forth in SEQ ID NO. 3 or 4. In some embodiments, a direct repeat (DR) sequence described herein has at least 90% identity (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity) to the reverse complement of SEQ ID NO. 3 or 4. In some embodiments, a direct repeat (DR) sequence described herein has at least 90% identity (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity) to a portion of the reverse complement of SEQ ID NO. 3 or 4. In some embodiments, a direct repeat (DR) sequence herein is set forth in SEQ ID NO. 3 or 4. In some embodiments, a direct repeat (DR) sequence herein is the reverse complement of SEQ ID NO. 3 or 4. When a sequence listing is expressed in RNA, "T" or "t" is to be considered "U". (ii) a spacer sequence In some embodiments, the guide RNA of the present disclosure comprises a spacer sequence. In some embodiments, the spacer sequence is at least about 15 nucleotides in length, preferably from about 15 to about 100 nucleotides, more preferably from about 15 to about 50 nucleotides (e.g., any of about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 nucleotides). In certain embodiments, the spacer sequence is from about 16 to about 27 nucleotides, e.g., from about 17 to about 24 nucleotides, from about 18 to about 24 nucleotides, or from about 18 to about 22 nucleotides. In some embodiments, the spacer sequence of the guide RNA is complementary to a non-PAM strand sequence. In some embodiments, the spacer sequence is designed to be complementary to a particular DNA strand, e.g., a DNA strand of a genomic locus. In some embodiments, the spacer sequence is at least about 70% complementary (e.g., at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) to a target sequence of a BCL11 A gene. In certain embodiments, the spacer sequence is at least about 15 (e.g., at least about 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, or more) nucleotides of match between the spacer sequence and a target sequence of a nucleic acid of interest (e.g., DNA). For the spacer sequence, full complementarity is not required, as long as there is sufficient complementarity for the guide RNA to function (i.e., to guide the nuclease to the target site). In some embodiments, the efficiency of nuclease-mediated cleavage can be tuned by introducing one or more mismatches between the spacer sequence and the target sequence (e.g., 1 or 2 mismatches between the spacer sequence and the target sequence, including the position of the mismatch along the spacer / target sequence). The effect of the mismatch (e.g., doublet mismatch) on the cleavage efficiency is greater when the mismatch is located more centrally in the spacer sequence (i.e., not at the 3’ or 5’ end of the spacer sequence). Thus, by selecting the position of the mismatch along the spacer sequence, the efficiency of the nuclease cleavage targeted to the BCL11 A gene target sequence can be tuned. For example, if it is desired that the cleavage rate of the target sequence is less than 100% (e.g., in a population of cells), 1 or 2 mismatches between the spacer sequence and the target sequence can be introduced in the spacer sequence. In some embodiments, the spacer sequence of the guide RNA of the present disclosure comprises at least 15 contiguous nucleotides of any one of the nucleotide sequences set forth in SEQ ID NOs. 12-13, 19-20. In some embodiments, the spacer sequence comprises the nucleotide sequence set forth in any one of SEQ ID NO. 12-13, 19-20. (iii) Modifications of guide RNA In some embodiments, a guide RNA herein comprises one or more nucleotide modifications. Exemplary modifications can include any modification to a sugar, a nucleobase, an internucleoside linkage (e.g., to a linking phosphate / to a phosphodiester bond / to a phosphodiester backbone), and any combination thereof. In some embodiments, a guide RNA can comprise any available modification to a sugar, a nucleobase, or an internucleoside linkage (e.g., to a linking phosphate, to a phosphodiester bond, to a phosphodiester backbone). One or more atoms of a pyrimidine nucleobase can be replaced or substituted with an optionally substituted amino group, an optionally substituted thiol, an optionally substituted alkyl group (e.g., methyl or ethyl), or a halo group (e.g., chloro or fluoro). In certain embodiments, a modification (e.g., one or more modifications) is present in each of a sugar and an internucleoside linkage. The modification can be a modification of ribonucleic acid (RNA) to deoxyribonucleic acid (DNA), threose nucleic acid (TNA), glycol nucleic acid (GNA), peptide nucleic acid (PNA), locked nucleic acid (LNA), or a hybrid thereof. In some embodiments, a modification of a guide RNA can comprise a chemical modification or a cell-induced modification. For example, Lewis and Pan describe some non-limiting examples of RNA modifications within a cell in “RNA modifications and structures cooperate to RNA guide-protein interactions,” Nat Reviews Mol Cell Biol, 2017, 18:202-210. Different sugar modifications, nucleotide modifications, and / or internucleoside linkages (e.g., backbone structures) can be present at different positions in the sequence. One of ordinary skill in the art will appreciate that nucleotide analogs or other modifications can be located at any position in the sequence such that the function of the sequence is not substantially reduced. A sequence can comprise from about 1% to about 100% modified nucleotides (relative to the total nucleotide content, or relative to one or more types of nucleotides, i.e., any one or more of A, G, U, or C), or any intermediate percentage (e.g., 1-100%). In some embodiments, a functional nucleotide analog comprises at least one chemical modification to a nucleobase, a sugar group, and / or a phosphate group. Thus, a payload nucleic acid molecule comprising at least one functional nucleotide analog contains at least one chemical modification to a nucleobase, a sugar group, and / or a nucleoside linkage. Exemplary chemical modifications to a nucleobase, a sugar group, or a nucleoside linkage of a nucleic acid molecule are provided herein. In some embodiments, functional nucleotide analogs comprise non-standard nucleobases. In some embodiments, standard nucleobases (e.g., adenine, guanine, uracil, thymine, and cytosine) in a nucleotide can be modified or replaced to provide one or more functional analogs of that nucleotide. Exemplary modifications of nucleobases include, but are not limited to, one or more substitutions or modifications including, but not limited to, alkyl, aryl, halogen, oxo, hydroxyl, alkoxy, and / or thio substitutions; one or more fused or open rings, oxidations, and / or reductions. In some embodiments, the non-standard nucleobase is a modified uracil. Exemplary nucleobases and nucleosides having modified uracil include pseudouridine (ψ), pyridin-4-one ribonucleoside, 5-azauracil, 6-azauracil, 2-thio-5-azauracil, 2-thiouracil (s2U), 4-thio-uracil (s4U), 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxy-uracil (ho5U), 5-aminoallyl-uracil, 5-halo-uracil (e.g., 5-iodo-uracil or 5-bromo uracil), 3-methyluracil (m3U), 5-methoxyuracil (mo5U), uracil 5-oxyacetic acid (cmo5U), uracil 5-oxyacetic acid methyl ester (mcmo5U), 5-carboxymethyl-uracil (cm5U), 1-carboxymethyl-pseudouridine, 5-carboxyhydroxymethyl-uracil (chm5U), 5-carboxyhydroxymethyl-uracil methyl ester (mchm5U), 5-methoxycarbonylmethyluracil (mcm5U), 5-methoxycarbonylmethyl-2-thiouracil (mcm5s2U), 5-aminomethyl-2-thiouracil (nm5s2U), 5-methylaminomethyl-2-uracil (mnm5U), 5-methylaminomethyl-2-thiouracil (mnm5s2U), 5-methylaminomethyl-2-selenouracil (mnm5se2U), 5-carbamoylmethyluracil (ncm5U), 5-carboxymethylaminomethyluracil (cmnm5U), 5-carboxymethylaminomethyl-2-thiouracil (cmnm5s2U), 5-propynyluracil, 1-propynyl-pseudouracil, 5-taurinomethyluracil (τm5U), 1-taurinomethyl-pseudouridine, 5-taurinomethyl-2-thiouracil (τm5s2U), 1-taurinomethyl-4-thio-pseudouridine, 5-methyl-uracil (m5U, i.e., having the nucleobase deoxythymine), 1-methyl-pseudouridine (m1ψ), 1-ethyl-pseudouridine (Et1ψ), 5-methyl-2-thio-uracil (m5s2U), 1-methyl-4-thio-guanylate (m1s4ψ), 4-thio-1-methyl-guanylate, 3-methyl-guanylate (m3ψ), 2-thio-1-methyl-guanylate, 1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-1-deaza-pseudouridine, dihydrouracil (D), dihydropseudouridine, 5,6-dihydrouracil, 5-methyl-dihydrouracil (m5D), 2-thio-dihydrouracil, 2-thio- dihydrouridine, 2-methoxy-uracil, 2-methoxy-4-thiouracil, 4-methoxy-uridine, 4- methoxy-2-thiouridine, N1-methyl-uridine, 3-(3-amino-3-carboxypropyl)uracil (acp3U), 1-methyl-3-(3-amino-3-carboxypropyl)uridine (acp3y), 5-(isopentenylaminomethyl)uracil (m5U), 5-(isopentenyl)aminomethyl)-2-thiouracil (m5s2U), 5,2'-O-dimethyluridine (m5Um), 2-thio-2'-O-methyluridine (s2Um), 5-methoxycarbonylmethyl-2'-O-methyluridine (mcm5Um), 5- carbamoylmethyl-2'-O-methyluridine (ncm5Um), 5-carboxymethylaminomethyl-2'-O- methyluridine (cmnm5Um), 3,2'-O-dimethyluridine (m3Um), and 5-(isopentenylaminomethyl)-2'-O-methyl-uridine (inm5Umm), 1-thio-uracil, deoxythymidine, 5-(2-carbomethoxyethenyl)-uracil, 5-(carbamoyloxymethyl)-uracil, 5- carbamoylmethyl-2-thiouracil, 5-carboxymethyl-2-thiouracil, 5-cyanomethyluracil, 5- methoxy-2-thiouracil, and 5-3-(1-E-propenylamino)uracil.

[0267] In some embodiments, the non-standard nucleobase is a modified cytosine. Exemplary nucleobases and nucleosides having a modified cytosine include 5-azacytosine, 6-azacytosine, pseudisocytidine, 3-methylcytosine (m3C), N4-acetylcytosine (ac4C), 5-formylcytosine (f5C), N4-methyl-cytosine (m4C), 5-methyl-cytosine (m5C), 5-halo-cytosine (e.g., 5-iodo-cytosine), 5-hydroxymethyl-cytosine (hm5C), 1-methyl-pseudisocytidine, pyrrolocytosine, pyrrolo pseudisocytosine nucleoside, 2-thiocytosine nucleoside (s2C), 2-thio-5-methylcytosine nucleoside, 4-thio-pseudisocytosine nucleoside, 4-thio-1-methyl-pseudisocytosine nucleoside, 4-sulfanyl-1-methyl-1-deaza-pseudisocytidine, 1-methyl-1-deaza-pseudisocytidine, zebularine, 5-aza-zebularine, 5-methyl-zebularine, 5-aza-2-thio-zebularine, 2-thio-zebularine, 2-methoxy-cytosine, 2-methoxy-5-methylcytosine, 4-methoxy-pseudisocytosine nucleoside, 4-methoxy-1-methyl-pseudisocytosine nucleoside, lysidine (k2C), 5,2'-0-dimethylcytosine nucleoside (m5Cm), N4-acetyl-2'-0-methylcytidine (ac4Cm), N4,2'-0-dimethylcytidine (m4Cm), 5-formyl-2'-0-methylcytidine (f5Cm), N4,N4,2'-0-trimethylcytidine (m42Cm), 1-thiocytosine, 5-hydroxy-cytosine, 5-(3-azidopropyl)-cytosine, and 5-(2-azidoethyl)-cytosine. In some embodiments, the non-standard nucleobase is a modified adenine. Exemplary nucleobases and nucleosides having a substitution for adenine include 2-aminopurine, 2,6-diaminopurine, 2-amino-6-halopurine (e.g., 2-amino-6-chloropurine), 6-halopurine (e.g., 6-chloropurine), 2-amino-6-methylpurine, 8-azidopurine, 7-deazapurine, 7-deaza-8-azapurine, 7-deaza-2-aminopurine, 7-deaza-8-aza-2-aminopurine, 7-deaza-2,6-diaminopurine, 7-deaza-8-aza-2,6-diaminopurine, 1-methyladenine (mlA), 2-methyladenine (m2A), N6-methyladenine (m6A), 2-methylthio-N6-methyladenine (ms2m6A), N6-isopentenyladenine (i6A), 2-methylthio-N6-isopentenyladenine (ms2i6A), N6-(cis-hydroxyisopentenyl)adenine (i06A), 2-methylthio-N6-(cis-hydroxyisopentenyl)adenine (ms2i06A), N6-glycinamidoformyl-adenine (g6A), N6-threoninamidoformyl-adenine (t6A), N6-methyl-N6-threoninamidoformyl-adenine (m6t6A), 2-methylthio-N6-threoninamidoformyl-adenine (ms2g6A), N6,N6-dimethyl-adenine (m62A), N6-hydroxy-n-pentylaminoformyl-adenine (hn6A), 2-methylthio-N6-hydroxy-n-pentylaminoformyl-adenine (ms2hn6A), N6-acetyl-adenine (ac6A), 7-methyladenine, 2-methylthioadenine, 2-methoxyadenine, N6,2'-O-dimethyladenosine (m6Am), N6,N6,2'-O-trimethyladenosine (m62Am), 1,2'-O-dimethyladenosine (mlAm), 2-amino-N6-methylpurine, l-thioadenine, 8-azidopurine, N6-(19-amino-pentaoxadodecyl)-adenine, 2,8-dimethyl-adenine, N6-formyl-adenine, and N6-hydroxymethyl-adenine In some embodiments, the non-standard nucleobase is a modified guanine. Exemplary nucleobases and nucleosides having modified guanines include inosine (I), 1-methyl inosine (ml), inosine (imG), methyl inosine (mimG), 4-demethyl inosine (imG-14), isodeoxytyrosine (imG2), wybutosine (yW), peroxytyrosine (o2yW), hydroxytyrosine (OHyW), undermodified hydroxytyrosine (OHyW*), 7-deazaguanine, queuosine (Q), epoxyqueuosine (oQ), galactosylqueuosine (galQ), mannosylqueosine, 7-cyano-7-deazaguanine (preQO), 7-aminomethyl-7-deazaguanine (preQl), geobiosine (G+), 7-deaza-8-azaguanine, 6-thioguanine, 6-thio-7-deaza-guanine, 6-thio-7-deaza-8-azaguanine, 7-methyl-guanine (m7G), 6-thio-7-methylguanine, 7-methyl-inosine, 6-methoxy-guanine, 1-methylguanine (mlG), N2-methylguanine (m2G), N2,N2-dimethylguanine (m22G), N2,7-dimethylguanine (m2,7G), N2,N2,7-dimethylguanine (m2,2,7G), 8-oxoguanine, 7-methyl-8-oxoguanine, 1-methyl-6-thioguanine, N2-methyl-6-thioguanine, N2,N2-dimethyl-6-thioguanine, N2-methyl-2'-0-methyl-guanine (m2Gm), N2,N2-dimethyl-2'-0-methylguanosine (m22Gm), 1-methyl-2'-0-methylguanosine (mlGm), N2,7-dimethyl-2'-0-methylguanosine (m2,7Gm), 2'-0-methyl inosine (Im), 1,2'-0-dimethyl inosine (mIm), 1-thioguanine, and O-6-methylguanine. In some embodiments, the non-standard nucleobases of the functional nucleotide analogs can independently be purine, pyrimidine, purine or pyrimidine analogs. For example, in some embodiments, the non-canonical nucleobases can be modified adenine, cytosine, guanine, uracil, or hypoxanthine. In other embodiments, the non-canonical nucleobases can also include, for example, naturally occurring and synthetic derivatives of bases, including pyrazolo[3,4-d]pyrimidine, 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine, 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-propynyl uracil and cytosine, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo (e.g., 8-bromo), 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo particularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 8-azaguanine and 8-azadenine, deazaguanine, 7-deazaguanine, 3-deazaguanine, deazauridine, 7-deazauridine, 3-deazauridine, pyrazolo[3,4-d]pyrimidine, imidazo[l,5-a]l,3,5 triazinone, 9-deazapurine, imidazo[4,5-d]pyrazine, thiazolo[4,5-d]pyrimidine, pyrazin-2-one, 1,2,4-triazine, pyridazine, or 1,3,5 triazine. In some embodiments, the functional nucleotide analogs comprise non-standard sugar groups. In various embodiments, the non-standard sugar groups can be 5-carbon or 6-carbon sugars (e.g., pentose, ribose, arabinose, xylose, glucose, galactose, or deoxy derivatives thereof) having one or more substituents which can be halogen, hydroxyl, thiol, alkyl, alkoxy, alkenyloxy, alkynyloxy, cycloalkyl, aminoalkoxy, alkoxyalkoxy, hydroxyalkoxy, amino, azido groups, aryl, aminoalkyl, aminoalkenyl, aminoalkynyl, and the like. Generally, RNA molecules comprise a ribose sugar group, which is a five-membered ring with an oxygen. Exemplary, non-limiting alternative nucleotides include replacement of the oxygen in ribose (e.g., with S, Se, or an alkylene group, such as methylene or ethylene); addition of a double bond (e.g., replacing ribose with a cyclopentenyl or cyclohexenyl group); annulation of the ring of ribose (e.g., forming a four-membered ring of cyclobutane or oxetane); expansion of the ring of ribose (e.g., forming a 6- or 7-membered ring with additional carbon or heteroatoms, such as a deoxyhexitol, arabitol, mannitol, cyclohexyl, cyclohexenyl, and morpholino (also with phosphoramidate backbones)); polycyclic forms (e.g., tricyclic and “unlocked” forms, such as glycol nucleic acids (GNA) (e.g., R-GNA or S-GNA, in which ribose is replaced with glycol units attached to the phosphodiester linkage), threose nucleic acids (TNA, in which ribose is replaced with a-L-threofuranosyl-(3’→2’)), and peptide nucleic acids (PNA, in which 2-amino-ethyl-glycine linkages replace ribose and the phosphodiester backbone). In some embodiments, the sugar group comprises one or more carbons having the opposite stereochemical configuration as the corresponding carbon in ribose. Thus, nucleic acid molecules can include nucleotides containing, for example, arabinose or L-ribose as the sugar. In some embodiments, the nucleic acid molecule includes at least one nucleoside in which the sugar is L-ribose, 2’-O-methyl ribose, 2’-fluoro ribose, arabinose, a hexitol, LNA, or PNA. In some embodiments, the payload nucleic acid molecules of the present disclosure can comprise one or more modified nucleoside linkages (e.g., phosphate backbones). The phosphate groups of the backbone can be altered by substituting one or more of the oxygen atoms with different substituents. In some embodiments, functional nucleotide analogs can include another nucleoside linkage in place of the unaltered phosphate moiety. Examples of alternative phosphate groups include, but are not limited to, phosphorothioates, phosphoroselenoates, phosphoroborates, phosphoroboranoates, phosphonooxidic hydrogens, phosphoramidates, phosphorodiamidates, alkyl or aryl phosphonates, and phosphotriesters. Both non-linking oxygens of phosphorodithioates are replaced with sulfur. Altered phosphate linkages can also be linked by replacing oxygen with nitrogen (bridging phosphoramidates), sulfur (bridging phosphorothioates), and carbon (bridging methylenephosphonates). Alternative nucleosides and nucleotides include boranophosphate (BH3), thio, methyl, ethyl and / or methoxy groups in place of one or more non-bridging oxygens. As non-limiting examples, two non-bridging oxygens at the same position (e.g., alpha, beta or gamma position) can be replaced with a thio and a methoxy group. The stability of RNA and DNA is enhanced (e.g., against exonucleases and endonucleases) by the substitution of one or more oxygen atoms at the position of the phosphate moiety (e.g., alpha-thio-phosphates). Thio-phosphate DNA and RNA have enhanced nuclease resistance and thus have longer half-lives in cellular environments. Other nucleoside linkages for use in accordance with the present disclosure include nucleoside linkages that do not include a phosphorus atom. When the compositions disclosed herein include a nucleic acid (e.g., an mRNA molecule) encoding a nuclease disclosed herein, the nucleic acid molecule can contain any of the modifications disclosed herein, if applicable. (iv) Exemplary guide RNAs In some embodiments, the guide RNA in the compositions of the present disclosure is as set forth in any one of SEQ ID NO. 6, 14-16, 21-24. In some embodiments, the guide RNA with chemical modifications is as set forth in the following table. II. Preparation and expression of compositions of BCL11A 1. Preparation of CasY7 polypeptides and variants thereof In some embodiments, the nuclease of the present disclosure can be prepared in a variety of ways. In some embodiments, the nuclease of the present disclosure is expressed by a host cell by constructing an expression vector capable of expressing the nuclease, isolated, and purified. In some embodiments, it is obtained by in vitro coupled transcription-translation system. In some embodiments, the host cell for expressing the nuclease of the present disclosure can be selected from any cell that can be used for nuclease expression. Exemplarily, the host cell can be selected from E. coli, yeast (budding yeast, Saccharomyces cerevisiae and Schizosaccharomyces, Schizosaccharomyces pombe), nematode (Caenorhabditis elegans), Xenopus oocyte, and animal cells (e.g., CHO cells, COS cells, and HEK293 cells). The method for in vivo expression of the nuclease of the present disclosure or its variant in a host cell comprises providing a polynucleotide encoding the nuclease or its variant to the host cell, expressing the nuclease polypeptide or its variant in the host cell, and then being able to obtain the nuclease polypeptide or its variant from the host cell. The method for transferring the above-mentioned expression vector into the host cell (i.e., transformation method) is not particularly limited, and known methods such as electroporation, calcium phosphate method, liposome method, and DEAE dextran method can be used. In some embodiments, following transformation of a host with an expression vector, the host cell is cultured, incubated, or propagated to produce the nuclease. In some embodiments, following expression of the nuclease, the host cell is collected and the nuclease polypeptide is purified, illustratively, the nuclease can be purified from the culture and the like according to conventional methods (e.g., filtration, centrifugation, cell disruption, gel filtration chromatography, ion exchange chromatography, and the like). Various methods can be used to determine the level of nuclease production in a host cell, illustratively, methods employing polyclonal or monoclonal antibodies specific for the nuclease polypeptides of the present disclosure and variants thereof or a marker tag as described elsewhere herein, for example, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (MA), fluorescence immunoassay (FIA), and fluorescence-activated cell sorting (FACS) can be employed. These and other assays are well known in the art (see, e.g., Maddox et al., J. Exp. Med. 158:1211

[1983] ). 2. Preparation of guide RNA Various methods can be employed to prepare the guide RNA. In some embodiments, the guide RNA can be synthesized chemically, enzymatically, or in combinations thereof, for example, standard phosphoramidite-based solid phase synthesis methods can be employed, or the guide RNA can be synthesized in vitro by operably linking DNA encoding the guide RNA to a promoter control sequence recognized by a bacteriophage RNA polymerase. Examples of suitable bacteriophage promoter sequences include T7, T3, SP6 promoter sequences, or variants thereof. In some embodiments, the guide RNA is expressed from DNA encoding it, for example, a DNA vector comprising a sequence encoding the guide RNA. The guide RNA can be encoded separately or with the nuclease. Such DNA sequences can be introduced into an expression system (e.g., a cell) together or separately. For example, DNA sequences encoding the nuclease and DNA sequences encoding the guide RNA can be introduced into a cell, each DNA sequence can be part of a separate molecule (e.g., one vector containing the nuclease-encoding sequence and a second vector containing the guide RNA-encoding sequence), or both can be part of the same molecule (e.g., one vector containing the encoding (and regulatory) sequences for both the nuclease and the guide RNA). The RNA can be transcribed from a synthetic DNA molecule (e.g., a gene fragment). The guide RNA molecule can be transcribed in vitro. In some embodiments, the guide RNA is synthesized using one or more modified nucleotides, e.g., as described above. In some embodiments, the guide RNA is prepared by expressing the guide RNA sequence in a cell transfected with a plasmid comprising a sequence encoding the guide RNA. In some embodiments, the plasmid encodes multiple different guide RNAs. In some embodiments, multiple different plasmids each encoding a different guide RNA are transfected into the cell. In some embodiments, the guide RNA is expressed from a recombinant vector (e.g., a plasmid) that encodes the guide RNA and also encodes a nuclease. In some embodiments, the guide RNA is expressed from a recombinant vector (e.g., a plasmid) that expresses the guide RNA but does not express a nuclease. In some embodiments, the guide RNA can be provided by a vendor. 3. Preparation of a ribonucleic acid complex (RNP) Provided herein is a ribonucleic acid complex (RNP) comprising a nuclease of the disclosure and a guide RNA of the disclosure. In some embodiments, the guide RNA targeting the BCL11A gene is complexed with the nuclease to form a ribonucleoprotein (RNP). In some embodiments, the guide RNA and the nuclease complex to form the RNP at a temperature (e.g., at 20-50 °C, specifically, at about 20 °C, 25 °C, 30 °C, 35 °C, 36 °C, 37 °C, 38 °C, 39 °C, 40 °C, 41 °C, 40 °C, 41 °C, 42 °C, 43 °C, 44 °C, 45 °C, 46 °C, 47 °C, 48 °C, 49 °C, 50 °C). In some embodiments, the guide RNA does not dissociate from the nuclease at 37 °C for an incubation period (e.g., at least about 10 minutes, 15 minutes, 20 minutes, 25 minutes, 30 minutes, 35 minutes, 40 minutes, 45 minutes, 50 minutes, 55 minutes, 1 hour, 2 hours, 3 hours, 4 hours, or more). In some embodiments, the guide RNA and the nuclease are complexed in a complexation buffer. In some embodiments, the complexation buffer has a pH value in a range. In some embodiments, the range is about 7.3 to 8.6 (e.g., about 7.3, about 7.4, about 7.5, about 7.6, about 7.7, about 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6). In some embodiments, the nuclease can be overexpressed in a host cell and complexed with the guide RNA prior to purification as described herein. In some embodiments, the mRNA or DNA encoding the nuclease is introduced into the cell such that the nuclease is expressed in the cell. In some embodiments, the guide RNA is also introduced into the cell, simultaneously, separately, or sequentially, from a single mRNA or DNA construct, such that the RNP complex is formed in the cell. III. Methods of targeting the BCL11A gene The present disclosure also provides methods of targeting a target sequence of the BCL11A gene in a cell. In some embodiments, the methods comprise introducing into a cell a guide RNA and a nuclease targeting the BCL11A gene, e.g., an RNP formed by a guide RNA and a nuclease targeting the BCL11A gene can be introduced into a cell; or a nucleic acid vector expressing a guide RNA and a nuclease targeting the BCL11A gene is introduced into a cell; or an mRNA expressing a nuclease, a guide RNA is introduced into a cell, optionally, also a template DNA. In some embodiments, a BCL11A gene in a cell / tissue / human is disrupted by delivering a composition described herein to the cell / tissue / human. 1. Design of target sequence In some embodiments, the guide RNA disclosed herein is designed to be complementary to a target sequence adjacent to a PAM (5'-TTN-3' sequence) of a CasY7 polypeptide or a variant thereof. In some embodiments, the target sequence is within a regulatory sequence of the BCL11A gene, to which the guide RNA can bind through base pairing. In some embodiments, the cell comprises one or more (e.g., 1, 2, 3, 4, 5, 6, or more) copies of the target sequence. In some embodiments, the BCL11A gene targeted by the guide RNA of the present disclosure is located in a mammalian cell. In some embodiments, the BCL11A gene is located in a primate cell, preferably, a human cell. In some embodiments, the target sequence of the BCL11A gene is present in the nucleus of the cell. In some embodiments, the target sequence is endogenous to the cell. In some embodiments, the target sequence is located in genomic DNA. In some embodiments, the target sequence is located in chromosomal DNA. In some embodiments, the target sequence is located in a regulatory region of the BCL11A gene, such as a promoter, an enhancer, a 5' or 3' untranslated region, etc. In some embodiments, the target sequence is adjacent to a 5'-TTN-3' PAM sequence, where N is any nucleotide. The 5'-TTN-3' sequence can be immediately adjacent to the target sequence, or within a small number (e.g., 1, 2, 3, 4, or 5) of nucleotides of the target sequence, for example. In some embodiments, the target sequence is ssDNA (single-stranded DNA). In some embodiments, the target sequence is dsDNA (double-stranded DNA). In some embodiments, the target sequence comprises both single-stranded and / or double-stranded regions. In some embodiments, the guide RNA is designed to bind to a first strand (non-PAM strand) of a double-stranded target nucleic acid, and a 5'-TTN-3' PAM sequence is present in the second complementary strand (PAM strand). In some embodiments, the guide RNA binds to a target sequence on the non-PAM strand that is complementary to a target sequence on the PAM strand adjacent to a 5'-TTN-3' sequence. In some embodiments, the target sequence of the BCL11A gene is set forth in SEQ ID NO. 5, 11, 17-18. 2. Gene Editing The present disclosure also provides methods of modifying a target sequence of a BCL11A gene. In some embodiments, the method comprises introducing a guide RNA targeting BCL11A and a nuclease into a cell. In some embodiments, the target sequence of the BCL11A gene is set forth in SEQ ID NO. 5, 11, 17-18, or the reverse complement thereof. Exemplarily, the target sequence is set forth in the following table: In some embodiments, the nuclease has nuclease activity. In some embodiments, CasY7 or a variant thereof induces one or more DNA double-strand breaks in a cell. In some embodiments, CasY7 or a variant thereof induces one or more DNA single-strand breaks in a cell. In some embodiments, CasY7 or a variant thereof induces one or more DNA nicks in a cell. In some embodiments, the DNA breaks and / or nicks result in formation of one or more indels (e.g., one or more deletions). In some embodiments, the guide RNA disclosed herein forms a complex with CasY7 or a variant thereof and directs it to a target sequence adjacent to a 5'-TTN-3' sequence. In some embodiments, the complex induces a deletion (e.g., a nucleotide deletion or a DNA deletion) adjacent to a 5'-TTN-3' sequence. In some embodiments, the complex induces a deletion adjacent to a PAM sequence of 5'-TTA-3', 5'-TTT-3', 5'-TTG-3', or 5'-TTC-3'. In some embodiments, the deletion is downstream of a 5'-TTN-3' sequence, e.g., the deletion is downstream of a sequence of 5'-TTA-3', 5'-TTT-3', 5'-TTG-3', or 5'-TTC-3'. In some embodiments, the deletion results in altered expression of the BCL11A gene. In some embodiments, the deletion results in altered function of the BCL11A gene. In some embodiments, the deletion results in reduced or inactivated function of the BCL11A gene. In some embodiments, the deletion is a frameshift deletion or a non-frameshift deletion. In some embodiments, the deletion begins within about 5 to about 10 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 nucleotides) downstream of a 5'-TTN-3' sequence. In some embodiments, the methods described herein are used to engineer cells comprising a deletion in the BCL11A gene as described herein. The compositions, vectors, nucleic acids, guide RNAs, and cells disclosed herein can be used in the treatment of hemoglobinopathies. Any suitable delivery or administration method known in the art can be used to deliver the compositions, vectors, nucleic acids, guide RNAs, and cells disclosed herein. Such methods can involve contacting a target sequence with a composition, vector, nucleic acid, or guide RNA disclosed herein. Such methods can involve methods of editing a BCL11A sequence as disclosed herein. In some embodiments, cells engineered using the guide RNAs of the disclosure are used for ex vivo gene therapy. In some embodiments, the compositions, vectors, nucleic acids, guide RNAs, and cells of the disclosure are used to treat sickle cell anemia. In some embodiments, the compositions, vectors, nucleic acids, guide RNAs, and cells of the disclosure are used to treat beta-thalassemia. In some embodiments, wherein one or more guide RNAs target the enhancer region of the BCL11A gene, the one or more guide RNAs are used to treat sickle cell anemia or beta-thalassemia. 3. Delivery The compositions of the disclosure can be formulated. In some aspects, the disclosure provides a polynucleotide encoding a nuclease of the disclosure (e.g., CasY7 or a variant thereof). In yet another aspect, the disclosure provides a delivery composition comprising (1) a nuclease of the disclosure, a polynucleotide of the disclosure, or a composition of the disclosure; and (2) a delivery vehicle. In yet another aspect, the disclosure also provides a vector comprising a polynucleotide of the disclosure. In some embodiments, the vector encodes a guide nucleic acid as defined in the disclosure. In some embodiments, the vector is a plasmid vector, a recombinant AAV (rAAV) vector (vector genome), or a recombinant lentivirus vector. In yet another aspect, the present disclosure provides a recombinant AAV (rAAV) viral particle comprising the rAAV vector genome of the present disclosure. A brief introduction to AAV for delivery can be found at “Adeno-Associated Virus (AAV) Guide” (addgene.org / guides / aav / ). In some embodiments, the compositions of the present disclosure comprise a delivery vehicle, such as a liposome, and are delivered to cells (e.g., prokaryotic, eukaryotic, plant, mammalian, etc.) by known methods. These methods include, but are not limited to, transfection (e.g., lipid-mediated cationic polymers, calcium phosphate, dendrimers); electroporation or other membrane-disrupting methods (e.g., nucleofection), viral delivery (e.g., lentivirus, retrovirus, adenovirus, adeno-associated virus (AAV)), microinjection, microparticle bombardment (“gene gun”), direct sonication, cell squeezing, optical transfection, protoplast fusion, impalefection, magnetofection, exosomes, lipid nanoparticle (LNP)-mediated transfer, and any combination thereof. In some embodiments, the nuclease and the guide RNA of the compositions of the present disclosure are delivered together, illustratively, packaged together in a single AAV particle. In another example, the nuclease component and the guide RNA component are delivered together via LNP. In some embodiments, the nuclease component and the guide RNA component are delivered separately, illustratively, packaged into separate AAV particles. In some cases, the nuclease component and the guide RNA are delivered differently, e.g., the nuclease component is delivered via an AAV particle while the guide RNA is delivered via an LNP. In some embodiments, the present disclosure provides an LNP comprising an mRNA encoding a nuclease, a guide RNA, or an mRNA encoding both a nuclease and a guide RNA. In some embodiments, the transcription template for the mRNA encoding a CasY7 polypeptide is set forth in SEQ ID NO: 10. In some embodiments, the transcription template for the mRNA encoding a CasY7 polypeptide is adapted to yield the mRNA transcription template for each variant by modifying the mutation pattern of the control CasY7 variant. Correspondingly, in some embodiments, the present application further provides cells produced by such methods, as well as organisms (e.g., animals, plants, or fungi) comprising such cells or produced from such cells. 4. Cells The compositions or complexes of the present disclosure can be delivered to various cells. In some embodiments, the cell is an isolated cell. In some embodiments, the cell is in a cell culture or in a co-culture of two or more cell types. In some embodiments, the cell is ex vivo. In some embodiments, the cell is derived from a living organism and maintained in a cell culture. In some embodiments, the cell is a unicellular organism. In some embodiments, the cell is a prokaryotic cell. In some embodiments, the cell is a bacterial cell. In some embodiments, the cell is an archaeal cell. In some embodiments, the cell is a eukaryotic cell. In some embodiments, the cell is selected from an animal cell, a vertebrate cell, a mammalian cell, a non-human mammalian cell, a non-human primate cell, a rodent (e.g., a mouse or a rat) cell, a human cell, a plant cell, or a yeast cell) or a prokaryotic cell (e.g., a bacterial cell). In some embodiments, the cell is a plant cell. In some embodiments, the cell is a fungal cell. In some embodiments, the cell is an animal cell. In some embodiments, the cell is an invertebrate cell. In some embodiments, the cell is a vertebrate cell. In some embodiments, the cell is a mammalian cell. In some embodiments, the cell is a human cell. In some embodiments, the cell is a rodent cell. In some embodiments, the cell is synthetically made, sometimes referred to as an artificial cell. In some embodiments, the plant cell is derived from a monocotyledonous plant, e.g., rice, corn, wheat, barley, oat, sorghum, millet, grasses, Poaceae, Zizania, Avena, Coix, Hordeum, Oryza, Panicum (e.g., Panicum miliaceum), Secale, Setaria (e.g., Setaria italica), Sorghum, Triticum, Zea, Cymbopogon, Saccharum (e.g., Saccharum officinarum), Phyllostachys, Dendrocalamus, Bambusa, Yushania. In some embodiments, the cell is derived from an animal, e.g., a pig, an ox, a sheep, a goat, a mouse, a rat, a llama, a monkey, a rabbit, a chicken, a duck, a goose, a fish (e.g., a zebrafish). In some embodiments, the cells are derived from a cell line. A wide variety of cell lines for use in tissue culture are known in the art. Examples of cell lines include, but are not limited to, 293T, MF7, K562, HeLa, CHO. In some embodiments, the cells are immortal or immortalized cells. In some embodiments, the cells are primary cells. In some embodiments, the cells are selected from hematopoietic progenitor cells (HPCs), hematopoietic stem cells (HSCs), CD34+ cells, CD117+ cells, mesenchymal stem cells (MSCs), induced pluripotent stem cells (iPSCs), common myeloid progenitor cells, proerythroblasts, or erythroblasts. In some embodiments, the population of modified cells are animal cells; for example, derived from a rodent, rat, mouse, rabbit, dog, or non-human primate cell; for example, a cynomolgus monkey cell. In some embodiments, the cells are human cells. Any genetically modified cells produced using the compositions disclosed herein are also within the scope of the present disclosure, which can include a disrupted BCL11A gene. The compositions, vectors, nucleic acids, guide RNAs, and cells of the present disclosure can be used in therapy. The compositions, vectors, nucleic acids, guide RNAs, and cells disclosed herein can be used in a method of treating a disease or condition in a subject. Any suitable delivery or administration method known in the art can be used to deliver the compositions, vectors, nucleic acids, guide RNAs, and cells disclosed herein. Such methods can involve contacting a target sequence with a composition, vector, nucleic acid, or guide RNA of the present disclosure. Such methods can involve a method of editing a BCL11A gene target sequence as disclosed herein. In some embodiments, cells engineered using the guide RNAs disclosed herein are used for ex vivo gene therapy. IV. Therapeutic Applications Any of the compositions of the present disclosure or modified cells produced using the compositions of the present disclosure can be used to treat a disease associated with the BCL11A gene. In some embodiments, the disease associated with the BCL11A gene is a hemoglobinopathy, illustratively including sickle cell disease or beta-thalassemia. Any suitable delivery or administration method known in the art can be used to deliver the compositions, vectors, nucleic acids, guide RNAs, and cells of the present disclosure. Such methods can involve contacting a target sequence with a composition, vector, nucleic acid, or guide RNA of the present disclosure. Such methods can involve a method of editing a BCL11A gene target sequence as disclosed herein. In some embodiments, cells engineered using the compositions disclosed herein can be used for ex vivo gene therapy. In some embodiments, provided herein is a method for treating a target disease disclosed herein (e.g., a hemoglobinopathy, specifically, for example, sickle cell disease or beta-thalassemia), the method comprising administering to a subject (e.g., a human patient) in need of treatment any of the compositions of the disclosure. The composition can be delivered to a specific tissue or a specific type of cell in need of gene editing. The gene editing system can include an LNP encompassing one or more of the components, one or more vectors (e.g., viral vectors) encoding one or more of the components, or a combination thereof. The compositions of the disclosure can be formulated to form a pharmaceutical composition, which can further include one or more pharmaceutically acceptable carriers. In some embodiments, a modified cell produced using any of the compositions of the disclosure can be administered to a subject (e.g., a human patient) in need of treatment. The modified cell can include substitutions, insertions, and / or deletions described herein. Illustratively, the modified cell can comprise any cell modified by a composition or system comprising a nucleic acid of the disclosure. In some cases, the modified cell can be a heterogeneous population of cells comprising cells with different types of gene edits. In some cases, the modified cell can comprise a substantially homogenous population of cells comprising one particular gene edit in the BCL11A gene. In some embodiments, provided herein is a pharmaceutical composition comprising a composition of the disclosure. The pharmaceutical compositions of the disclosure can be administered using any route, for example, orally, rectally, vaginally, parenterally, topically, pulmonarily, intranasally, intralesionally, buccally, ocularly, intravenously, or intraorgan. The pharmaceutical compositions of the disclosure can be prepared, packaged, or sold in bulk, as single unit doses, or as a plurality of single unit doses. As used herein, a “unit dose” is discrete amount of the pharmaceutical composition to be administered to a subject, or a convenient fraction of such a dose, for example, one-half, one- fourth or other fraction of a dose. Formulations suitable for parenteral administration can include an active agent (e.g., a composition or modified cell of the disclosure) in combination with a pharmaceutically acceptable carrier, such as sterile water or sterile isotonic saline. Such formulations can be prepared, packaged, or sold in unit- dose or multi-dose containers, for example, in ampoules or in multi-dose vials. Some injectable formulations can be prepared, packaged, or sold as a dry powder for reconstitution in sterile water or another suitable diluent. Some formulations for parenteral administration can include but are not limited to suspensions, solutions, emulsions, or pastes in either oily or aqueous vehicles, gels, creams, and implants. Some formulations can further include one or more additional ingredients including but not limited to suspending, stabilizing, or dispersing agents. In some embodiments, the pharmaceutical compositions of the present disclosure can be in the form of a sterile injectable aqueous or oleaginous suspension or solution. This suspension or solution can be formulated according to known techniques, and can include, in addition to the cells, additional ingredients such as dispersing, wetting, or suspending agents as described herein. Such sterile injectable formulations can be prepared using a non-toxic parenterally acceptable diluent or solvent such as water or saline. Other acceptable diluents and solvents include, but are not limited to, Ringer's solution, isotonic sodium chloride solution, and fixed oils such as synthetic mono- or diglycerides. Other formulations for parenteral administration include those which can include the cells as components in packaged form, in liposome formulations, or as components of biodegradable polymer systems. Some compositions for sustained release or implantation can include a pharmaceutically acceptable polymer or hydrophobic material such as an emulsion, ion exchange resin, sparingly soluble polymer, or sparingly soluble salt. In some embodiments, the median survival of a subject having a BCL11 A gene-related disease but receiving the administration is longer than the median survival of a subject or population of subjects having the disease and not receiving the administration by 5 days, 10 days, 20 days, 30 days, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 1.5 years, 2 years, 2.5 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10 years, or more. The therapeutically effective dose can be via a single dose or multiple doses. It will be understood by those skilled in the art that the actual dose can vary significantly depending on a variety of factors such as carrier selection, target cell, organism, tissue, general condition of the subject to be treated, degree of transformation / modification sought, route of administration, mode of administration, type of transformation / modification sought, etc. V. Detection methods The present disclosure also provides a method of detecting a target DNA, the method comprising contacting the target DNA with a system of the present disclosure, wherein the target DNA is modified by the complex, and wherein the modification detects the target DNA. In some embodiments, the modification generates a detectable signal, e.g., a fluorescent signal. VI. Kits and uses thereof The present disclosure also provides kits, e.g., kits that can be used, e.g., to perform the methods described herein for genetically modifying the BCL11 A gene. In some embodiments, a kit of the present disclosure comprises a guide RNA and a nuclease (CasY7 or a variant thereof). In some embodiments, a kit of the present disclosure comprises a guide RNA, a template DNA, and a nuclease (CasY7 or a variant thereof). In some embodiments, a kit comprises a polynucleotide encoding a nuclease of the present disclosure (CasY7 or a variant thereof), and optionally the polynucleotide is included within, e.g., any of the vectors described herein. In some embodiments, a kit of the present disclosure comprises a polynucleotide encoding a guide RNA disclosed herein. In some embodiments, the nuclease (or polynucleotide encoding CasY7 or a variant thereof) and the guide RNA (e.g., as a ribonucleoprotein) can be packaged within the same container (e.g., vial) within the kit, or can be packaged in separate containers (e.g., vials), the contents of which can be mixed prior to use. In some embodiments, a kit of the present disclosure can additionally comprise instructions for use of the optional buffers, guide RNA, template DNA, and / or nuclease. In some embodiments, a kit includes one or more buffers that can be used to solubilize any of the one or more components contained therein, and / or to provide suitable reaction conditions for one or more of the one or more components. Illustratively, the buffers can include one or more of the following: PBS, HEPES, Tris, MOPS, Na2CO3, NaHCO3, NaB, or combinations thereof. In some embodiments, the reaction conditions include an appropriate pH, such as an alkaline pH. In some embodiments, the pH is between 7-10. In some embodiments, any one or more of the kit components can be stored in a suitable container or at a suitable temperature, e.g., 4°C. Further embodiments are illustrated in the following examples, which are for illustrative purposes only and are not intended to limit the scope of the present disclosure. All references and publications cited herein are hereby incorporated by reference. Examples The following examples are provided to further illustrate some embodiments of the present disclosure, but are not intended to limit the scope of the present disclosure; it being understood by those skilled in the art that other procedures, methods, or techniques, known to those skilled in the art, can alternatively be used. Example 1 Construction of variants of CasY7 To identify more nucleases, the applicant annotated and obtained CasY7 protein (amino acid sequence as shown in SEQ ID NO. 1, nucleotide coding sequence as shown in SEQ ID NO. 2), DR sequence (SEQ ID NO. 3) and PAM preference (5'-TTN-3'), optimized DR-1 (SEQ ID NO. 4) and the like (PCT / CN2024 / 092707, the contents of which are hereby incorporated by reference in their entirety) using a bioinformatics approach. After protein function prediction, it was considered that the 2-920th engineering mutation of the CasY7 polypeptide amino acid sequence might affect its cleavage activity. Therefore, the CasY7 expression plasmid was used as a template, and the predicted mutation sites were used as the center to design PCR primers, and the nucleotide sequence after introducing mutations on the PCR primers was used to perform engineering mutations. The CasY7 and each variant are shown in Table 1: Table 1 Mutation method of CasY7 mutant (PAM after mutation is 5'-TTN-3') Example 2 CasY7 and its variants mediated BCL11A target editing in eukaryotic cells 1. The mRNA transcription template of each variant of CasY7 synthesized by Nanjing Kingsray (the sequence of CasY7 WT mRNA transcription template is shown in SEQ ID NO. 10, and the transcription template of the variant is changed according to the adaptability of the codon after mutation) was used T7 High Yield RNA Synthesis Kit (NEB, E2040S) kit for in vitro transcription reaction to obtain the mRNA of each nuclease. 2. According to the PAM (5'-TTN-3') of CasY7 and its variants, the spacer sequence was designed under the target sequence downstream of the PAM sequence meeting 22 bp, and the following crRNA sequence was designed using the optimized DR sequence (DR-1, SEQ ID NO. 4): BCL11A-crRNA-1 was synthesized by Nanjing Kingsray, and was delivered to HepG2 cells using LNP, as follows: The four-component LNP lipid (purchased from Avitide (Shanghai) Pharmaceutical Technology Co., Ltd.) was used for delivery, specifically, Yoltech Lipid1 (compound 10), DSPC, cholesterol, PEG-DMG were dissolved in anhydrous ethanol at a molar ratio of 50:10:38.5:1.5. Each CasY7 variant mRNA and BCL11A-crRNA-1 targeting the BCL11A gene (mass ratio 1:1) were dissolved in 100 mM pH 4 enzyme-free citric acid buffer (RNA concentration 0.2 mg / mL). The ethanol solution of the lipid carrier was mixed with the buffer solution of the mRNA at a ratio of 1:3 (volume / volume) (where the mass ratio of total lipid and mRNA was 40:1), and nucleic acid lipid nanoparticles were obtained by a microfluidic nanomedicine manufacturing system (NanoAssemblr Ignite, Canada) at a flow rate of 12 ml / min. The obtained nucleic acid lipid nanoparticles were immediately diluted 40 times in 1x DPBS buffer. HepG2 cells (purchased from ATCC) were inoculated in DMEM medium (Gibco, 11965092) added with 10% FBS (v / v) containing 1% Penicillin Streptomycin (v / v) (Gibco, 15140122) and cultured in a 37°C cell incubator containing 5% CO2. The cells for transfection were inoculated in a 96-well cell culture plate the day before and observed the next day. When the cells grew to about 80% of the cell density, LNP transfection was performed. LNP@mRNA (transfection dose of 2 ng / well, 4 ng / well) was added to the HepG2 cells, and the cells were collected 48 hours after transfection. The collected cells were subjected to genomic extraction (TIANGEN, DP304-03) and cleavage activity detection, and sequencing detection by PCR amplification by Beijing Qikong Biological Company. Analysis showed that each CasY7 variant and guide RNA (BCL11A-crRNA-1) mediated significant targeted cleavage activity (Figure 1). wherein Yoltech Lipid1 (compound 10, PCT / CN2024 / 104304, the contents of which are hereby incorporated in their entirety) is synthesized as follows: 5-[(2-Butyl-1-oxoalkyl)oxy]pentanoic acid-7-butyl-21-(10-butyl-3,9-dioxo-2,8-dioxahexadecan-1-yl)-19-[3-(diethylamino)propyl]-8-oxo-19-aza-9-oxadocosan-22-yl ester Step 1: Synthesis of compound 1-2 In a 500 mL round bottom flask was added cyclohexyl acetate (25.00 g, 249.70 mmol, 1.0 eq), distilled water (20 mL), ethanol (200 mL), sodium hydroxide (10.99 g, 274.67 mmol, 1.1 eq). After 3 hours of reaction at 70 °C, the solvent was removed by concentration under reduced pressure, 200 mL of acetone was added slowly into the flask, tetrabutylammonium iodide (4.61 g, 12.48 mmol, 0.05 eq), benzyl bromide (51.25 g, 299.64 mmol, 1.2 eq) was added, followed by overnight reaction at 70 °C. The reaction was quenched by adding 500 mL of water, extracted twice with 500 mL of ethyl acetate each, the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and column chromatography to give compound benzyl 5-hydroxypentanoate (37.00 g, yield 71.2%). Step 2: Synthesis of compound 1-4 In a 500 mL round bottom flask was added benzyl 5-hydroxypentanoate (37.00 g, 177.67 mmol, 1.0 eq), 2-butyloctanoic acid (35.59 g, 177.67 mmol, 1.0 eq), 250 mL of dichloromethane and 4-dimethylaminopyridine (21.70 g, 177.67 mmol, 1.0 eq), and finally 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (51.09 g, 266.50 mmol, 1.5 eq). The reaction was carried out at room temperature for 4 hours, diluted with 500 mL of water, extracted twice with 500 mL of dichloromethane each, the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and column chromatography to give compound 2-butyloctanoic acid-5-(benzyloxy)-5- oxopentyl ester (64.00 g, yield 92.2%). Step 3: Synthesis of compound 1-5 In a 250 mL round bottom flask was added 2-butyloctanoic acid-5-(benzyloxy)-5- oxopentyl ester (64.00 g, 163.87 mmol, 1.0 eq), methanol (75 mL), tetrahydrofuran (75 mL), and finally Pd / C (3.49 g, 32.78 mmol, 0.2 eq, 10% purity). The reaction was carried out at room temperature for 16 hours under an atmosphere of one atmosphere of hydrogen, filtered and concentrated to give compound 5-[(2-butyloxy-1- oxido octyl)oxy]pentanoic acid (45.00 g, yield 91.4%). Step 4: Synthesis of compound 1-7 To a round bottom flask containing 100 mL of dichloromethane was added 5-[(2- butyl-l-oxoacetyl)oxy]pentanoic acid (10.00 g, 33.29 mmol, 1.0 eq), 2-hydroxymethylpropane- 1,3-diol (3.53 g, 33.29 mmol, 1.0 eq), 4-dimethylaminopyridine (0.81 g, 6.66 mmol, 0.2 eq), N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (9.57 g, 49.94 mmol, 1.5 eq), and N,N-diisopropylethylamine (8.60 g, 66.58 mmol, 2.0 eq) at room temperature and stirred for 4 hours. The reaction was quenched by adding 200 mL of water and extracted twice with 200 mL of dichloromethane each time. The organic phases were combined, washed with brine, dried over anhydrous sodium sulfate, filtered, and concentrated. Purification by column chromatography yielded 2-butylacetic acid-18-butyl-8- (hydroxymethyl)-5, 11, 17-trioxo-6, 10, 16-trioxa-24-azatetracosan-1-yl ester (7.80 g, 69.9% yield). Step 5: Synthesis of compound 1-8 To a round bottom flask containing 30 mL of dichloromethane was added compound 2-butylacetic acid-18-butyl-8-(hydroxymethyl)-5, 11, 17-trioxo-6, 10, 16-trioxa-24- azatetracosan-1-yl ester (3.90 g, 5.81 mmol, 1.0 eq), triethylamine (1.76 g, 17.43 mmol, 3.0 eq), and methanesulfonic anhydride (2.02 g, 11.62 mmol, 2.0 eq) at room temperature and slowly added at 0 °C. The reaction was slowly allowed to warm to room temperature and stirred for 4 hours. The reaction was quenched by adding 30 mL of water and extracted twice with 50 mL of dichloromethane each time. The organic phases were combined, washed with brine, dried over anhydrous sodium sulfate, filtered, and concentrated. Purification by column chromatography yielded methane sulfonic acid-12-butyl-2-(10-butyl-3, 9-dioxo-2, 8-dioxadecan-1-yl)-5, 11-dioxo-4, 10-dioxaoctadecan-1-yl ester (3.85 g, 88.4% yield). Step 6: Synthesis of compound 1-10 Compound 1-8 (600.0 mg, 0.80 mmol, 1.0 eq), 3-amino-1-propanol (300.0 mg, 3.99 mmol, 5.0 eq), potassium carbonate (280.0 mg, 2.00 mmol, 2.5 eq), potassium iodide (130.0 mg, 0.80 mmol, 1.0 eq) were added into 10 mL of acetonitrile, protected by nitrogen, heated to 90 degrees Celsius, and reacted for 16 hours at room temperature. The reaction solution was concentrated, diluted with water, extracted with ethyl acetate three times, the organic phase was combined, washed with saturated brine, dried over anhydrous sodium sulfate, concentrated, and purified by column chromatography to obtain compound 5-[(2-butyl-1-oxoacetyl)oxy]pentanoic acid-12-butyl-2-{[(3-hydroxypropyl)amino]methyl}-5,11-dioxo-4,10-dioxadodecan-1-yl ester (210.0 mg, 36.11%). MS: m / z [M+H] = 728.6. + = 728.6. Step 7: Synthesis of compound 10 Compound 2-butyl octanoic acid-8-(10-butyl-3,9-dioxo-2,8-dioxahexadecan-1-yl)-14-ethyl-5-oxo-10,14-diaza-6-oxahexadecan-1-yl ester (500.0 mg, 0.64 mmol, 1.0 eq), 2-butyl octanoic acid-9-bromononyl ester (390.0 mg, 0.96 mmol, 1.5 eq), potassium carbonate (270.0 mg, 1.92 mmol, 3.0 eq), potassium iodide (110.0 mg, 0.64 mmol, 1.0 eq) were added into 20 mL of acetonitrile, protected by nitrogen, heated to 90 degrees Celsius, and reacted overnight. The reaction solution was concentrated, diluted with water, extracted with dichloromethane three times, the organic phase was combined, washed with saturated brine, dried over anhydrous sodium sulfate, concentrated, and purified by column chromatography to obtain 5-[(2-butyl-1-oxoacetyl)oxy]pentanoic acid-7-butyl-21-(10-butyl-3,9-dioxo-2,8-dioxahexadecan-1-yl)-19-[3-(diethylamino)propyl]-8-oxo-19-aza-9-oxadocosan-22-yl ester (132.8 mg, yield 18.8%). MS: m / z [M+H] = 1107.9. + = 728.6. 1 HNMR (300MHz, CDCl3) δ 4.15-4.01 (m, 10H), 3.44-3.20 (m, 4H), 2.71-2.50 (m, 6H), 2.39-2.23 (m, 12H), 2.02-1.40 (m, 28H), 1.38-1.22 (m, 48H), 0.92-0.75 (m, 18H). 3. To study the mediation effect of BCL11A-crRNA-2, BCL11A-crRNA-3, and BCL11A-crRNA-4, the C10135 variant was selected as the research object, and BCL11A-crRNA-1 was used as a control for the experiment: (1) The coding nucleotide sequence of C10135 was constructed into the ABE8e plasmid (Addgene, Plasmid #138489) at positions 466-5160 to construct a C10135 expression plasmid. BCL11A-crRNA (BCL11A-crRNA-1, BCL11A-crRNA-2, BCL11A-crRNA-3, and BCL11A-crRNA-4) expression plasmids were constructed. A CACC sequence was added to the 5' end of the upstream sequence of BCL11A-crRNA, and an AAAA sequence was added to the 5' end of the downstream sequence, and oligos were synthesized as follows: (2) After the synthesis of the aforementioned BCL11A-crRNA upstream and downstream sequences, annealing was performed by a pre-set program (95°C for 5 min; -2°C / s from 95°C to 85°C; -0.1°C / s from 85°C to 25°C), and then the annealing product was ligated to the PHK09T vector (the sequence of the PHK09T vector is shown in SEQ ID NO. 25, and the plasmid map is shown in FIG. 3) linearized by BsmBI (NEB, #R0580L). The linearization of the PHK09T vector and the ligation of the BCL11A-crRNA annealing product are as follows: The PHK09T vector was first linearized, and the linearization system was as follows: 3 μg of PHK09T vector; 6 μL of buffer (NEB, #R0539L); 2 μL of BsmBI; ddH2O was added to 60 μL, and enzyme digestion was performed at 50°C overnight. The BCL11A-crRNA annealing product and the linearized vector ligation system were as follows: 1 μL of T4 ligase buffer (NEB, #M0202L), 20 ng of linearized vector, 5 μL of annealed oligo fragment, 0.5 μL of T4 ligase (NEB, #M0202L), and ddH2O was added to 10 μL, and ligation was performed at 16°C overnight. (3) The BCL11A-crRNA expression plasmid obtained in step (2) was transformed into E. coli DH5a competent cells (Weidi Biology, DL1001) for amplification, and the plasmid was extracted (using an endotoxin-free plasmid large extraction kit, TIANGEN: DP120-01). After determining the plasmid concentration, it was saved for later use, and the BCL11A-crRNA expression plasmid was obtained. (4)HEK293T cells (purchased from ATCC) were seeded in DMEM medium (Gibco, 11965092) added with 10% FBS (v / v) containing 1% Penicillin Streptomycin (v / v) (Gibco, 15140122) and cultured in a 37°C cell incubator containing 5% CO2. The cells for transfection were seeded in a 24-well cell culture plate and cultured for one day, and the cells were observed the next day. When the cells grew to a cell density of about 80%, transfection was performed. C10135 expression plasmid and BCL11A-crRNA expression plasmid were transfected into HEK293T cells, and the amount of plasmid transfected into cells in each well of a 24-well plate was 0.3 ug of C10135 expression plasmid, 0.3 ug of BCL11A-crRNA expression plasmid, and 0.3 ug of EGFP-C1 plasmid (control plasmid). The specific transfection operation is as follows: C10135 expression plasmid, BCL11A-crRNA expression plasmid, and EGFP-C1 plasmid were mixed respectively, and 25 ul of The serum-reduced medium (Source Bioscience, L530KJ) was diluted and 2 ul of Lipofectamine 3000 (Invitrogen, L3000015) reagent was added and mixed to serve as reagent A, and it was allowed to stand for 5 minutes. At the same time, 2 ul of Lipofectamine 3000 transfection reagent (Invitrogen, L3000015) was diluted with 25 ul of The serum-reduced medium (Source Bioscience, L530KJ) was diluted and mixed to serve as reagent B, and it was allowed to stand for 5 minutes. The reagent A and reagent B were mixed and uniformly blown, and allowed to stand for 20 minutes. After the standing was completed, the mixed reagent was added dropwise to the 24-well plate cells to be transfected, and it was placed back into a 37°C, 5% CO2 incubator for culture. After 6 hours of transfection, the culture medium was replaced with DMEM medium containing 10% FBS. (5) After 48 hours of transfection, the expression of EGFP fluorescent protein indicated that the cells were successfully transfected, and the cells expressing EGFP were sorted for detection of editing efficiency. The cells were subjected to genomic extraction (using a genomic DNA extraction kit, TIANGEN, DP304-03), and primers (upstream and downstream primers are shown in SEQ ID NO. 7 and 9) were designed for PCR. The PCR amplification system is as follows: 2x Taq Master Mix (Vazyme, P112-03) 25 μL; Primer-F (Bcl11a-F, SEQ ID NO. 7) (10 pmol / μL) 1 μL; Primer-R (Bcl11a-R, SEQ ID NO. 9) (10 pmol / μL) 1 μL; Template 1 μL; ddH2O up to 50 μL. After PCR amplification, the obtained PCR product was used for high-throughput deep sequencing (Qingke Biotechnology Co., Ltd.) or Sanger sequencing (Platinum Biotechnology (Shanghai) Co., Ltd.) to identify the editing efficiency, and the identification results are shown in FIG. 2. Analysis shows that both BCL11A-crRNA-1 and BCL11A-crRNA-3 mediate an editing efficiency of more than 40%. Example 3 Off-target identification of CasY7 variants, guide RNAs To evaluate the application of CasY7 variants in human cells, according to the PAM (5'-TTN-3') sequence, a spacer sequence BCL11A-crRNA-1 target sequence (SEQ ID NO. 5) was designed for the BCL11A gene target, and a plurality of gene sites and off-target protospacers that may occur off-target cleavage were predicted, as shown in Table 2: Table 2 Prediction of possible off-target sites and corresponding spacer sequences The C10135 and C10731 encoding nucleotides were constructed into the ABE8e plasmid (Plasmid #138489) backbone to obtain expression plasmids, and each variant expression plasmid was obtained. The expression plasmid of BCL11A-crRNA-1 (SEQ ID NO. 6) was constructed in the manner of Example 2 (the map is shown in FIG. 3). Each variant plasmid and crRNA expression plasmid was co-transfected into HEK293T cells by Lipofectamine 3000 transfection. Through high-throughput deep sequencing, it was found that each variant basically showed no significant off-target cleavage activity at the predicted sites (the net editing efficiency at each predicted site was less than 0.4%). This shows that the above-mentioned variants and the BCL11A-targeting crRNA-mediated targeted editing do not have obvious off-targets and have high fidelity. In summary, the compositions of the present disclosure have robust editing activity and high specificity for therapeutic applications. Various modifications and variations to the described products, methods, and uses of the disclosure will be apparent to those skilled in the art without departing from the scope and spirit of the disclosure. Although the disclosure has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any variations, uses, or adaptations of the disclosure following, in general, the principles of the disclosure and including such departures from the present disclosure that come within known use of the setrespective arts to which the disclosure pertains. It is intended to include all such modifications and alterations insofar as they come within the scope of the present disclosure. All documents referred to in this disclosure are incorporated herein by reference as if each were individually incorporated. In addition, it is to be understood that the disclosure is not limited to particular examples described, and is intended to cover numerous modifications, equivalents, and alternatives.

Claims

1. A nuclease comprising an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identical to SEQ ID NO.

1.

2. The nuclease of claim 1, comprising a substitution at one or more of the following positions: positions 2, 3, 5, 8, 165, 166, 175, 176, 228, 229, 240, 241, 242, 243, 282, 283, 285, 308, 309, 316, 317, 318, 319, 416, 417, 418, 419, 420, 431, 435, 441, 552, 564, 574, 638, 639, 640, 641, 642, 648, 651, 652, 681, 682, 683, 684, 748, 749, 751, 829, 865, 866, 867, 868, 788, 896, 915, 916, 918, and 919 of SEQ ID NO. 1; Preferably, the substitution at the one or more positions is an amino acid substitution, optionally comprising the following mutation patterns: (i) Y282 + D283 + A285; or (ii) G416 + I417 + E418 + F419 + D420; Further preferably, the nuclease comprises the following mutation patterns based on the amino acid sequence set forth in SEQ ID NO. 1: (a) Y282 + D283 + A285 + N682 + E683 + S684 + E748 + G749 + S751 + K865 + P866 + Y867 + N868; (b) F228 + L229 + Y282 + D283 + A285 + G416 + I417 + E418 + F419 + D420; (c) Y282 + D283 + A285 + G416 + I417 + E418 + F419 + D420 + K865 + P866 + Y867 + N868; (d) Y165 + S166 + G416 + I417 + E418 + F419 + D420 + K865 + P866 + Y867 + N868; (e) Y282 + D283 + A285 + Y308 + S309 + T681 + N682 + E683 + S684 + E748 + G749 + S751; (f) A175 + E176 + Y282 + D283 + A285 + G416 + I417 + E418 + F419 + D420 + T681 + N682 + E683 + S684; (g) A175 + E176 + S240 + S241 + Q242 + E243 + Y282 + D283 + A285 + G416 + I417 + E418 + F419 + D420 + T681 + N682 + E683 + S684; (h) Y165 + S166 + E176 + G416 + I417 + E418 + F419 + D420 + T681 S + N682L + E683 + S684 + E748 + G749 + S751 + E788; (i) E176 + S240 + S241 + Q242 + E243 + Y282 + D283 + A285 + G416 + I417 + E418 + F419 + D420 + E748 + G749 + S751 + E788; (j) Y282 + D283 + A285 + G416 + I417 + E418 + F419 + D420 + T681 + N682 + E683 + S684 + K865 + P866 + Y867 + N868; (k) Y165 + S166 + G416 + I417 + E418 + F419 + D420 + E748 + G749 + S751 + K865 + P866 + Y867 + N868; (l) E176 + Y282 + D283 + A285 + G416 + I417 + E418 + F419 + D420 + E748 + G749 + S751 + E788; (m) E176 + Y282 + D283 + A285 + N682 + E683 + S684 + E748 + G749 + S751 + E788; (n) E176 + Y282 + D283 + A285 + G416 + I417 + E418 + F419 + D420 + N682 + E683 + S684 + E788; (o) Y165 + S166 + E176 + G416 + I417 + E418 + F419 + D420 + N682 + E683 + S684 + E788; (p) Y165 + S166 + E176 + G416 + I417 + E418 + F419 + D420 + T681 + N682 + E683 + S684 + E748 + G749 + S751 + V915 + L916 + S918 + I919 + E788; (q) E176 + Y282 + D283 + A285 + G416 + I417 + E418 + F419 + D420 + T681 + N682 + E683 + S684 + E829; (r) Y282 + D283 + A285 + N682 + E683 + S684 + E748 + G749 + S751; (s) Y282 + D283 + A285 + E316 + T317 + I318 + I319 + T681 + N682 + E683 + S684 + E748 + G749 + S751; (t) Y282 + D283 + A285 + G416 + I417 + E418 + F419 + D420 + N682 + E683 + S684 + K865 + P866 + Y867 + N868; (u) Y282 + D283 + A285 + T681 + N682 + E683 + S684 + E748 + G749 + S751 + K865 + Y867 + N868; (v) Y165 + S166 + G416 + I417 + E418 + F419 + D420 + K865 + Y867 + N868; (w) Y282 + D283 + A285 + G416 + I417 + E418 + F419 + D420 + T681 + N682 + E683 + S684 + K865 + Y867 + N868; (x) Y165 + S166 + G416 + I417 + E418 + F419 + D420 + E748 + G749 + S751 + K865 + Y867 + N868; (y) Y282 + D283 + A285 + G416 + I417 + E418 + F419 + D420 + E748 + G749 + S751 + K865 + Y867 + N868; (z) Y165 + S166 + G416 + I417 + E418 + F419 + D420 + N682 + E683 + S684 + E748 + G749 + S751 + K865 + Y867 + N868 + D896; (aa) Y165 + S166 + G416 + I417 + E418 + F419 + D420 + E748 + G749 + S751 + K865 + Y867 + N868; (bb) Y282 + D283 + A285 + G416 + I417 + E418 + F419 + D420 + D638 + R639 + G640 + E641 + F642 + T681 + N682 + E683 + S684; (cc) A175 + E176 + Y282 + D283 + A285 + G416 + I417 + E418 + F419 + D420 + E748 + G749 + S751; (dd) A175 + E176 + Y282 + D283 + A285 + G416 + I417 + E418 + F419 + D420 + E648 + A651 + Y652; (ee) Y165 + S166 + G416 + I417 + E418 + F419 + D420 + K441 + E748 + G749 + S751; (ff) Y165 + S166 + G416 + I417 + E418 + F419 + D420 + Y431R + E748 + G749 + S751; (gg) A2 + Y165 + S166 + G416 + I417 + E418 + F419 + D420 + E748 + G749 + S751; (hh) Y165 + S166 + G416 + I417 + E418 + F419 + D420 + K574 + E748 + G749 + S751; (ii) T3+Y165+S166+G416+I417+E418+F419+D420+E748+G749+S751; (jj) Y165+S166+G416+I417+E418+F419+D420+P552+E748+G749+S751; (kk) R8+Y165+S166+G416+I417+E418+F419+D420+E748+G749+S751; (ll) Y165+S166+G416+I417+E418+F419+D420+N564+E748+G749+S751; (mm) Y165+S166+G416+I417+E418+F419+D420+T435+E748+G749+S751; or (nn) T5+Y165+S166+G416+I417+E418+F419+D420+E748+G749+S751; More preferably, the substitutions of the nuclease compared to the amino acid sequence set forth in SEQ ID NO. 1 are selected from the following mutation patterns: (oo) Y282F+D283Q+A285T+N682G+E683A+S684R+E748A+G749S+S751G+K865G+P866A+Y867A+N868Y; (pp) F228W+L229R+Y282F+D283Q+A285T+G416V+I417H+E418Q+F419R+D420L; (qq) Y165W+S166R+G416R+I417E+E418Q+F419V+D420A+K865G+P866A+Y867A+N868Y; (rr) A175R+E176R+Y282F+D283Q+A285T+G416V+I417H+E418Q+F419R+D420L+T681E+N682G+E683L+S684A; (ss) A175R+E176R+S240T+S241A+Q242L+E243D+Y282F+D283Q+A285T+G416V+I417H+E418Q+F419R+D420L+T681E+N682G+E683L+S684A; (tt) Y165W+S166R+E176R+G416R+I417V+E418Q+F419V+D420T+T681S+N682L+E683A+S684R+E748A+G749S+S751G+E788R; (uu) E176R+S240T+S241A+Q242L+E243D+Y282F+D283Q+A285T+G416V+I417H+E418Q+F419R+D420L+E748A+G749S+S751G+E788R; (vv) Y282F+D283Q+A285T+G416V+I417H+E418Q+F419R+D420L+T681E+N682G+E683L+S684A+K865G+P866A+Y867A+N868Y; (ww) Y165W+S166R+G416R+I417V+E418Q+F419V+D420T+E748A+G749S+S751G+K865G+P866A+Y867A+N868Y; (xx) E176R+Y282F+D283Q+A285T+G416V+I417H+E418Q+F419R+D420L+E748A+G749S+S751G+E788R; (yy) E176R+Y282F+D283Q+A285T+N682G+E683A+S684R+E748A+G749S+S751G+E788R; (zz) E176R+Y282F+D283Q+A285T+G416V+I417H+E418Q+F419R+D420L+N682G+E683A+S684R+E788R; (aaa) Y165W+S166R+E176R+G416R+I417E+E418Q+F419V+D420A+N682G+E683A+S684R+E788R; (bbb) Y165W+S166R+E176R+G416R+I417V+E418Q+F419V+D420T+T681E+N682G+E683L+S684A+E748A+G749S+S751G+V915I+L916V+S918K+I919V+E788R; (ccc) E176R+Y282F+D283Q+A285T+G416V+I417H+E418Q+F419R+D420L+T681E+N682G+E683L+S684A+E829R; (ddd) Y282F+D283Q+A285T+N682G+E683A+S684R+E748A+G749S+S751G; (eee) Y282F+D283Q+A285T+E316T+T317L+I318R+I319V+T681E+N682G+E683L+S684A+E748A+G749S+S751G; (fff) Y282F+D283Q+A285T+G416V+I417H+E418Q+F419R+D420L+N682G+E683A+ S684R+K865G+P866A+Y867A+N868Y; (ggg) Y282F+D283Q+A285T+T681E+N682G+E683L+S684A+E748A+G749S+S751G+ K865S+Y867N+N868M; (hhh) Y165W+S166R+G416R+I417E+E418Q+F419V+D420A+K865S+Y867N+N868M; (iii) Y282F+D283Q+A285T+G416V+I417H+E418Q+F419R+D420L+T681E+N682G+E683 L+S684A+K865S+Y867N+N868M; (jjj) Y165W+S166R+G416R+I417V+E418Q+F419V+D420T+E748A+G749S+S751G+ K865S+Y867N+N868M; (kkk) Y282F+D283Q+A285T+G416V+I417H+E418Q+F419R+D420L+E748A+G749S+ S751G+K865S+Y867N+N868M; (lll) Y165W+S166R+G416R+I417V+E418Q+F419V+D420T+N682G+E683A+S684R+ E748A+G749S+S751G+K865S+Y867N+N868M+D896N; (mmm) Y165W+S166R+G416R+I417E+E418Q+F419V+D420A+E748A+G749S+S751G+ K865S+Y867N+N868M; (nnn) Y282F+D283Q+A285T+G416V+I417H+E418Q+F419R+D420L+D638S+R639A+ G640D+E641Y+F642I+T681E+N682G+E683L+S684A; (ooo) A175R+E176R+Y282F+D283Q+A285T+G416V+I417H+E418Q+F419R+D420L+ E748A+G749S+S751G; (ppp) A175R+E176R+Y282F+D283Q+A285T+G416V+I417H+E418Q+F419R+D420L+ E648T+A651S+Y652F; (qqq) Y165W+S166R+G416R+I417V+E418Q+F419V+D420T+K441R+E748A+G749S+S751G; (rrr) Y165W+S166R+G416R+I417V+E418Q+F419V+D420T+Y431R+E748A+G749S+S751G; (sss) A2R+Y165W+S166R+G416R+I417V+E418Q+F419V+D420T+E748A+G749S+S751G; (ttt) Y165W+S166R+G416R+I417V+E418Q+F419V+D420T+K574R+E748A+G749S+S751G; (uuu) T3R+Y165W+S166R+G416R+I417V+E418Q+F419V+D420T+E748A+G749S+S751G; (vvv) Y165W+S166R+G416R+I417V+E418Q+F419V+D420T+P552R+E748A+G749S+S751G; (www) R8K+Y165W+S166R+G416R+I417V+E418Q+F419V+D420T+E748A+G749S+S751G; (xxx) Y165W+S166R+G416R+I417V+E418Q+F419V+D420T+N564R+E748A+G749S+S751G; (yyy) Y165W+S166R+G416R+I417V+E418Q+F419V+D420T+T435R+E748A+G749S+S751G; (zzz) T5R+Y165W+S166R+G416R+I417V+E418Q+F419V+D420T+E748A+G749S+S751G; (aaaa) Y282F+D283Q+A285T+G416V+I417H+E418Q+F419R+D420L+K865G+P866A+Y867A+N868Y; or (bbbb) Y282F+D283Q+A285T+Y308F+S309A+T681E+N682G+E683L+S684A+E748A+G749S+S751G.

3. A composition for gene editing of a BCL11A gene, the composition comprising: (i) the nuclease of claim 1 or 2 or a first nucleic acid encoding the nuclease; (ii) a guide RNA or a second nucleic acid encoding the guide RNA, wherein the guide RNA comprises a spacer sequence specific for a target sequence within the BCL11A gene.

4. The composition of claim 3, comprising the first nucleic acid encoding the nuclease, the first nucleic acid being codon-optimized for expression in a eukaryotic cell.

5. The composition of claim 3 or 4, wherein the target sequence is within a regulatory sequence of the BCL11 A gene; optionally, the target sequence is in an enhancer sequence of the BCL11 A gene.

6. The composition of any one of claims 3-5, wherein the spacer sequence is 15-100 nucleotides in length, preferably 16 to 50 nucleotides, more preferably 17 to 30 nucleotides, more preferably 18 to 22 nucleotides, more preferably 20 nucleotides.

7. The composition of any one of claims 3-6, wherein the target sequence is adjacent to a protospacer adjacent motif (PAM) comprising the sequence 5’-TTN-3’.

8. The composition of any one of claims 3-7, wherein the target sequence is at a GATA1 binding site of an erythroid enhancer of the BCL11 A gene; optionally, the target sequence is selected from the group consisting of: SEQ ID NO. 5, 11, 17, and / or 18.

9. The composition of any one of claims 3-8, the spacer sequence is optionally selected from the group consisting of: SEQ ID NO. 12, 13, 19, and / or 20.

10. The composition of any one of claims 3-9, wherein the guide RNA comprises the spacer sequence and a direct repeat (DR) sequence.

11. The composition of claim 10, wherein the direct repeat sequence comprises a nucleotide sequence that is at least about 50% (e.g., at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the nucleotide sequence of any one of SEQ ID NO. 3 or 4.

12. The composition of claim 3-11, comprising a guide RNA set forth in the following sequences: SEQ ID NO. 6, 14, 15, 16, 21, 22, 23, and / or 24.

13. The composition of any one of claims 3-12, wherein the second nucleic acid encoding the guide RNA has been codon-optimized for expression in a eukaryotic cell.

14. A polynucleotide encoding the nuclease and / or guide RNA of any one of claims 3-13.

15. A vector comprising the polynucleotide of claim 14; optionally, wherein the vector encodes the guide RNA as described in any one of claims 3-13; optionally, wherein the vector is a plasmid vector, a recombinant AAV (rAAV) vector, or a recombinant lentivirus vector.

16. A ribonucleoprotein (RNP) comprising the nuclease of claim 1 or 2 and optionally the guide RNA of any one of claims 3-13.

17. A lipid nanoparticle (LNP) comprising the composition of any one of claims 3-13.

18. A pharmaceutical composition comprising the composition of any one of claims 3-13, the polynucleotide of claim 14, the vector of claim 15, the ribonucleoprotein (RNP) of claim 16, or the lipid nanoparticle (LNP) of claim 17; and a pharmaceutically acceptable carrier or excipient.

19. A kit comprising the composition of any one of claims 3-13, the polynucleotide of claim 14, the vector of claim 15, the ribonucleoprotein (RNP) of claim 16, or the lipid nanoparticle (LNP) of claim 17.

20. A cell comprising the composition of any one of claims 3-13, the polynucleotide of claim 14, or the vector of claim 15, and obtained after editing of the cell by the composition, nucleic acid, or vector.

21. The cell of claim 20, which is a eukaryotic cell; optionally, the cell is a rodent cell (e.g., a mouse cell, a rat cell), a non-human primate cell, or a human cell; further optionally, the cell is a hematopoietic stem cell (HSC), a hematopoietic progenitor cell (HPC), a CD34+ cell, a mesenchymal stem cell (MSC), an induced pluripotent stem cell (iPSC), a common myeloid progenitor cell, a proerythroblast, or an erythroblast.

22. A guide RNA comprising: (i) a spacer sequence specific for a target sequence in a BCL11A gene, wherein the target sequence is adjacent to a protospacer adjacent motif (PAM) located 5' of the target sequence comprising the motif 5'-TTN-3'; and (ii) a direct repeat sequence; optionally, the target sequence is selected from SEQ ID NO. 5, 11, 17, and / or 18; optionally, the spacer sequence is SEQ ID NO. 12, 13, 19, and / or 20; optionally, the direct repeat sequence is selected from SEQ ID NO. 3 or 4; optionally, the PAM is 5'-TTN-3'; optionally, the guide RNA sequence is selected from SEQ ID NO. 6, 14, 15, 16, 21, 22, 23, and / or 24.

23. A method for editing a BCL11A gene in a cell, the method comprising contacting a host cell with the composition of any one of claims 3-13, the polynucleotide of claim 14, the vector of claim 15, or the ribonucleoprotein (RNP) of claim 16, to genetically edit a BCL11A gene in the host cell.

24. The method of claim 23, wherein the contacting occurs ex vivo, in vivo, or in vitro.

25. A method for treating a hemoglobinopathy in a subject, the method comprising administering to a subject in need thereof a composition of any one of claims 3-13, a polynucleotide of claim 14, a vector of claim 15, or a ribonucleoprotein (RNP) of claim 16, a lipid nanoparticle (LNP) of claim 17, a pharmaceutical composition of claim 18, or a cell according to any one of claims 20-21.

26. The method of claim 25, wherein the subject is a human patient having a hemoglobinopathy; optionally, the hemoglobinopathy is a sickle cell disease or a beta-thalassemia disease.

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