Novel small V-type RNA programmable endonuclease system

Abstract

Described herein are novel systems for targeting, editing or manipulating DNA in a cellular or cell-free environment using novel B-Gen.16 type V (SEQ ID NO: 1) and variants thereof, as well as methods and kits for manipulating DNA.

Description

Technical Field

[0001] The present disclosure relates generally to the field of molecular biology, and in particular to a novel CRISPR-Cas RNA programmable DNA endonuclease, designated B-GEn.16, for gene editing and other uses. Background Art

[0002] Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) genes, collectively referred to as CRISPR-Cas or CRISPR / Cas systems, are currently understood to provide immunity to bacteriophage infection in bacteria and archaea. The CRISPR-Cas system of prokaryotic adaptive immunity is an extremely diverse set of protein effector noncoding elements, and locus architectures, some examples of which have been engineered and adapted to produce important biotechnologies.

[0003] The components of the system involved in host defense include one or more effector proteins capable of modifying DNA or RNA, and RNA guide elements responsible for targeting these protein activities to specific sequences on phage DNA or RNA. The RNA guide consists of CRISPR RNA (crRNA), and may require additional trans-acting RNA (tracrRNA) to enable targeted nucleic acid manipulation by the effector protein. The crRNA consists of a segment called a "direct repeat" and a segment called a "spacer sequence", which is responsible for binding crRNA to the effector protein, and the "spacer sequence" is complementary to the desired nucleic acid target sequence. The CRISPR system can be reprogrammed to target alternative DNA or RNA targets by modifying the crRNA's spacer sequence.

[0004] CRISPR-Cas systems can be broadly divided into two categories: Class 1 systems are composed of multiple effector proteins, which together form a complex around crRNA, and Class 2 systems are composed of a single effector protein, which is complexed with crRNA guides to target DNA or RNA substrates. The single-subunit effector composition of the Class 2 system provides a simpler set of components for engineering and application, and is therefore an important source of programmable effectors to date. Therefore, the discovery, engineering and optimization of new Class 2 systems can produce widely used and powerful programmable technologies for genome engineering and other fields. The RNA-guided DNA targeting principle of CRISPR (Clustered Regularly interspaced Short Palindromic Repeats)-Cas (CRISPR-associated proteins) has been widely used in the past few years to edit genomes. Five types of CRISPR-Cas systems (Type I, Type II and Type IIb, Type Ill, Type V and Type VI) have been documented. The most commonly used CRISPR-Cas for genome editing is the Type II system. The main advantage provided by bacterial II type CRISPR-Cas system is the minimum requirement for programmable DNA interference: endonuclease Cas9 guided by customizable double-RNA structure.As confirmed in the original II type system of Streptococcus pyogenes (Streptococcus pyogenes), trans-activated CRISPR RNA (tracrRNA) is combined with the invariable repeat sequence of precursor CRISPR RNA (pre-crRNA) to form double-RNA, which is indispensable for the invading DNA cutting of crRNA co-maturation and Cas9 carried out by RNase III in the presence of Cas9.As demonstrated in Streptococcus pyogenes, Cas9 introduces site-specific double-stranded DNA (dsDNA) breakage in the homologous DNA of invasion under the guidance of the duplex formed between the mature activated tracrRNA and the targeting crRNA.Cas9 is a multi-domain enzyme, which uses HNH nuclease domain to cut target strand (defined as complementary to the spacer sequence of crRNA) and RuvC-like domain to cut non-target strand.

[0005] In addition to type II CRISPR Cas9 nucleases, many different type V CRISPR Cas9 nucleases have been described, such as Cas12a, Cas12b, Cas12e, Cas12f, Cas13a, Cas13b (Koonin et al., Curr Opin Microbiol. 2017 June; 37: 67-78, and Makarova et al., Nat Rev Microbiol. 2020 February; 18(2): 67-83.). Some of these systems do not require tracr RNA (Cas 12a, Cas 13a, Cas 13b), however, Cas 12b generally requires tracr RNA (Kooni et al., Curr Opin Microbiol. 2017 June; 37: 67-78).

[0006] Genome editing in mammalian cells has been limited so far, in part due to the size of various Cas9 proteins. Cas9 (SpyCas9) from Staphylococcus pyogenes is the most widely used enzyme to date, containing about 4.2 kb of DNA (WO2013 / 176722), and direct binding to homologous single guide RNA (sgRNA) further increases its size. Adeno-associated virus is one of the vectors used to deliver Cas9 enzymes in gene therapy applications. However, the cargo size of AAV is limited to about 4.5 kb. Due to size limitations, delivery of Cas9 with its sgRNA and potential DNA repair templates can become an obstacle to using this method. The characteristics of smaller Cas9 molecules have been characterized, but most of them have a prototype spacer adjacent motif (PAM) sequence that is not as well defined as the sequence used by SpyCas9. For example, Staphylococcus aureus (Sau) Cas9 uses a "NNGRR(T)" sequence, where R = A or G, and Campylobacter jejuni (Cja) Cas9 uses a "NNNACAC" / "NNNRYAC" PAM (where Y = T or G), respectively. The ambiguity of the PAM increases the likelihood that the enzyme will produce unwanted activity at off-target sequences that have high or complete sequence identity to the PAM. The specificity of these systems remains a concern, as accidental ("off-target") targeting of similar sites will increase the likelihood of adverse events.

[0007] Existing CRISPR-Cas systems generally have one or more of the following disadvantages:

[0008] a) They are too large to be carried within the genomes of established viral delivery systems suitable for therapeutics, such as adeno-associated virus (AAV).

[0009] b) Most of them have no appreciable activity in non-host environments, such as in eukaryotic cells, and particularly in mammalian cells.

[0010] c) Their nucleases can catalyze DNA strand cleavage when there is a mismatch between the spacer sequence and the protospacer sequence, leading to undesirable off-target effects, for example, making them unsuitable for gene therapy use or other applications requiring high precision.

[0011] d) They may elicit an immune response which may limit their use in mammals.

[0012] e) They require complex and / or long PAMs that limit target selection of the DNA targeting segment.

[0013] f) They show low expression on plasmid or viral vectors.

[0014] g) They require additional RNA sequences for activation or additional RNA sequences as part of the guide RNA.

[0015] The following references: Nucleic Acids Research, Vol. 48, Issue 9, 21 May 2020, pp5016-5023, WO2020123887, WO2017117395 disclose sequences that are very distantly related to SEQ ID NO: 1, having an amino acid identity of about 52% or less compared to SEQ ID NO: 1. Summary of the invention

[0016] The present invention relates to a novel V-type CRISPR Cas nuclease named B-GEn.16 (SEQ ID NO: 1). In addition, the present invention relates to a polypeptide having at least 60% identity, preferably at least 70%, more preferably at least 80%, even more preferably at least 90%, particularly preferably at least 95%, most preferably at least 99% amino acid identity at the amino acid level with the sequence according to SEQ ID NO: 1 over its entire length, or any nucleic acid encoding it. Unless otherwise specified, the name B-GEn.16 includes the polypeptide of SEQ ID NO: 1 and any polypeptide defined in this paragraph.

[0017] The present invention further relates to a CRISPR Cas system comprising B-GEn.16, a suitable guide RNA or tracrRNA (which comprises the sequence contained in SEQ ID NO: 8, 9 and 10), and a target DNA.

[0018] One of the main features of B-GE.16 is its particularly small size, which would make it an ideal candidate for integration into viral vectors (e.g., AAV).

[0019] Another beneficial feature of B-Gen. 16 is its favorable PAM sequence ("CCN").

[0020] Furthermore, as supported by the Examples herein, B-Gen.16 exhibits high activity in eukaryotic organisms, particularly in mammalian cells.

[0021] In one aspect, provided herein is a method for targeting, editing, modifying or manipulating a target DNA at one or more locations in a cell or in vitro, the method comprising (I) introducing a heterologous B-GEn.16 polypeptide disclosed herein or a nucleic acid encoding B-GEn.16 disclosed herein into a cell or in an in vitro environment; and (II) introducing one or more heterologous single guide RNAs (sgRNAs) or DNA encoding such one or more sgRNAs into a cell or in an in vitro environment, each sgRNA or DNA encoding an sgRNA comprising: (a) an engineered DNA targeting segment comprising RNA and capable of hybridizing to a target sequence in a polynucleotide locus, (b) a tracr mate sequence consisting of RNA, and (c) a tracr RNA sequence consisting of RNA, wherein the tracr mate sequence hybridizes to the tracr sequence, and wherein (a), (b) and (b) are arranged in a 5' to 3' direction; and (III) generating one or more nicks or cuts or base edits in the target DNA, wherein the B-GEn.16 polypeptide is guided to the target DNA by the sgRNA in its processed or unprocessed form.

[0022] In one aspect, provided herein is a composition for use in targeting, editing, modifying or manipulating a target DNA at one or more locations in a cell or in vitro, the composition comprising (I) a B-GEn.16 polypeptide disclosed herein or a nucleic acid encoding the same; and / or (II) one or more single heterologous guide RNAs (sgRNAs) or DNA suitable for in situ generation of such one or more sgRNAs, each comprising: (a) an engineered DNA targeting segment consisting of RNA and capable of hybridizing to such a target sequence in a polynucleotide locus, (b) a tracr pairing sequence consisting of RNA, and (c) a tracrRNA sequence consisting of RNA, wherein the tracr pairing sequence hybridizes to the tracr sequence, and wherein (c), (b) and (a) are arranged in a 5' to 3' direction, respectively.

[0023] In another aspect, provided herein is a cell comprising (I) a B-GEn.16 polypeptide as disclosed herein, or a nucleic acid encoding a B-GEn.16 polypeptide disclosed herein; and (II) one or more single heterologous guide RNAs (sgRNAs) or DNA suitable for in situ generation of such one or more sgRNAs, each comprising: (a) an engineered DNA targeting segment that can hybridize to a target sequence in a polynucleotide locus, (b) a tracr mate sequence, and (c) a tracr RNA sequence, wherein the tracr mate sequence can hybridize to the tracr sequence, and wherein (c), (b) and (a) are arranged in a 5' to 3' direction, respectively.

[0024] In yet another aspect, provided herein is a kit comprising (I) a nucleic acid sequence encoding a B-GEn.16 polypeptide as disclosed herein, wherein the nucleic acid sequence encoding B-GEn.16 is operably linked to a promoter; and (II) one or more single heterologous guide RNAs (sgRNAs) or DNA suitable for in situ generation of such one or more sgRNAs, each sgRNA comprising: (a) an engineered DNA targeting segment that can hybridize to a target sequence in a polynucleotide locus, (b) a tracr mate sequence, and (c) a tracr RNA sequence, wherein the tracr mate sequence can hybridize to the tracr sequence, and wherein (a), (b) and (c) are arranged in a 5' to 3' direction.

[0025] The entire disclosure of each patent document and scientific article cited herein, as well as the patent documents and scientific articles cited therein, are expressly incorporated into this specification by reference for all purposes.

[0026] Additional features and advantages of the invention are described in more detail below. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1a Illustrated are the results of experiments according to Example 1. The significant decrease in luminescence over time in experiments containing B-GEn.16 (SEQ ID NO: 1) compared to controls (non-targeting guide control; no nuclease control, target only control) indicates specific binding of the complex to the target DNA.

[0028] Figure 1b The assay used is described.

[0029] Figure 2 Provided are the average editing activities (% NHEJ) of B-GEn.16 (having the amino acid sequence SEQ ID NO: 1) in mammalian cells obtained from two independent experiments according to Example 2. The constructs used and shown on the x-axis have the following sequences:

[0030]

[0031]

[0032] Reference to a sequence listing

[0033] The 27 sequences SEQ ID NO: 1 to 27 disclosed herein are contained in the sequence listing named BHC221018-WO_Sequence_LIsting.xml (ST.26. Sequence of SEQ ID NO: 1 (B-GEn.16 protein)), which are also recorded herein.

[0034] Summary of sequences appearing in the sequence listing

[0035]

[0036]

[0037] SEQ ID NO: 1 (B-GEn.16) amino acid sequence

[0038] MTTKVVKIEIIKPINDTWNVLGQVLRDIQYQTWKFANKAIQMYWDFSNFSYSYKKRFGEELLSKGKTLPNGYRQVKSDILNEHKEIINKLNSNSKDALIDMVEKKWNADFNDII NGRKSIANFKRDLPIELHNKQFMNSKKQLMLYVENGDYYAEINLISRQYAKELGKKSTAFKLLLAVKDNYQKVIVDRLISGEYKLGMSKITKAKKGKTKWYLNLVYSFTEKKDN SLDPNKVMGIDLGVNIPAALAIYGDKYFKEFVGDRDEIENFRKQIEARKRKLQRQGKWCGEGRIGHGIKTRIKPLEKLAGKIANFKNTKNHCWSRYIVDAsAVKNKCGVIQMED LSGIAEENTFLKNWTYYDLQQKIKYKAEEKGIKVVFIDPSYTSARCNKCGHIHRSYEKKEYRPSQDQFICQSCGHKENADINAARNIATPNIEKIIKDQLEKQEKEMKQNLKYII DETAILED DESCRIPTION

[0039] The present application provides a new CRISPR-Cas nuclease and a gene editing system based on such a nuclease. The new nuclease is referred to herein as B-GEn nuclease or B-GEn.16 nuclease.

[0040] Very preferably, the B-GEn nuclease group includes the following members recorded in Table 1:

[0041] Table 1:

[0042] SEQ ID: NO Identifier describe 1 B-GEn.16 Type V CRISPR-Cas endonuclease B-GEn.16

[0043] One embodiment according to the invention is a polypeptide having at least 60% identity at the amino acid level compared to SEQ ID NO: 1 or a nucleic acid encoding the same.

[0044] A preferred embodiment according to the present invention is a polypeptide having at least 70% identity at the amino acid level compared to SEQ ID NO: 1 or a nucleic acid encoding the same.

[0045] A more preferred embodiment according to the present invention is a polypeptide having at least 80% identity at the amino acid level compared to SEQ ID NO: 1 or a nucleic acid encoding the same.

[0046] An even more preferred embodiment according to the invention is a polypeptide having at least 90% identity at the amino acid level compared to SEQ ID NO: 1 or a nucleic acid encoding the same.

[0047] A particularly preferred embodiment according to the invention is a polypeptide or a nucleic acid encoding the same which has at least 95% identity at the amino acid level compared to SEQ ID NO: 1.

[0048] A particularly preferred embodiment according to the invention is a polypeptide having at least 99% identity at the amino acid level compared to SEQ ID NO: 1 or a nucleic acid encoding the same.

[0049] An even more particularly preferred embodiment according to the invention is a polypeptide having at least 99.5% identity at the amino acid level compared to SEQ ID NO: 1 or a nucleic acid encoding the same.

[0050] Yet another embodiment according to the present invention is the following variant of B-GEn.16:

[0051] (I) variants having, in increasing order of preference, at least 60%, e.g., at least 70%, at least 80%, at least 90%, at least 95%, at least 99% amino acid identity with any sequence of SEQ ID NO: 1 over its entire length.

[0052] (II) a variant according to (I), comprising additional components as, for example, a nuclear localization signal, in order to obtain a proper activity of the B-GEn.16 CRISPR system not only in a cell-free reaction or in prokaryotic cells, but also in a eukaryotic cell environment (including in living organisms such as plants or animals);

[0053] (III) Codon-optimized variants of the corresponding polynucleotide sequences encoding B-GEn.16 and variants according to (I) and (II).

[0054] Unless otherwise specified, the term B-GEn.16 includes all variations specified under items (I), (II) and (III).

[0055] B-GEn.16-based CRISPR-Cas system

[0056] According to one embodiment of the present invention, a composition is presented, comprising:

[0057] (a) B-GEn.16 polypeptide, or a polynucleotide encoding such B-GEn.16;

[0058] (b) a single heterologous guide RNA (sgRNA) or a DNA that allows for the in situ generation of such an sgRNA, comprising:

[0059] i. an engineered DNA targeting segment consisting of RNA and capable of hybridizing to a target sequence in a polynucleotide locus;

[0060] ii. a tracr pairing sequence consisting of RNA, and

[0061] iii. a tracr RNA sequence consisting of RNA,

[0062] wherein the tracr mate sequence hybridizes to the tracr sequence, and wherein (i), (ii),

[0063] (iii) Arranged in a 5' to 3' direction.

[0064] In sgRNA, the tracr pairing sequence and the tracr sequence are usually connected by a suitable loop sequence to form a stem-loop structure.

[0065] Suitable PAM sequences for CRISPR-Cas systems including B-GEn.16

[0066] The function of B-GEn.16 generally requires a suitable protospacer adjacent motif ("PAM") 5' to the target sequence. Suitable PAM sequences are listed in Table 2, where the engineered DNA targeting segment is directly adjacent to the PAM sequence on the targeted DNA segment at its 3' end, or such a PAM sequence is part of the targeted DNA sequence in its 5' portion.

[0067] Table 2: Suitable PAM sequences for the corresponding B-GEn.16 endonucleases

[0068] B-GEn PAM Sequence B-GEn.16 "CYN", where "N" represents any of the four bases and "Y" represents a pyrimidine base (C or T) B-GEn.16 "CCN", where "N" represents any of the four bases

[0069] Suitable tracr sequences for B-GEn.16 in CRISPR-Cas systems

[0070] A suitable tracr sequence for B-GEn.16 in a CRISPR-Cas system is provided in SEQ ID NO: 10. Alternatively, a variant of the sequence may be employed. The variant may include a partial or truncated version of such a sequence and / or a sequence having a base modification at one or more positions of the sequence.

[0071] In some embodiments, the polynucleotides encoding B-GEn.16 and sgRNAs comprise a suitable promoter and / or a suitable nuclear localization signal for expression in cells or in an in vitro environment.

[0072] According to another embodiment of the present invention, a method for targeting, editing, modifying or manipulating target DNA in a cell or at one or more locations in vitro is presented, comprising the following steps:

[0073] (a) introducing a heterologous B-GEn.16 polypeptide or a nucleic acid encoding the same into a cell or in vitro environment; and

[0074] (b) introducing a single heterologous guide RNA (sgRNA) or a DNA suitable for generating such an sgRNA in situ, comprising:

[0075] i. an engineered DNA targeting segment consisting of RNA and capable of hybridizing to a target sequence in a polynucleotide locus;

[0076] ii. a tracr pairing sequence consisting of RNA, and

[0077] iii. a tracr RNA sequence consisting of RNA,

[0078] wherein the tracr mate sequence can hybridize to the tracr sequence, and wherein (i), (ii) and (iii) are arranged in a 5' to 3' direction;

[0079] (c) generating one or more nicks, cuts or edits in a target DNA, wherein the B-GEn.16 polypeptide is guided to the target DNA by a gRNA in its processed or unprocessed form.

[0080] Another embodiment according to the present invention is a composition for targeting, editing, modifying or manipulating target DNA at one or more locations in a cell or in vitro, the composition comprising:

[0081] (a) B-GEn.16 polypeptide, or a polynucleotide encoding such B-GEn.16;

[0082] (b) a single heterologous guide RNA (sgRNA) or a DNA suitable for generating such an sgRNA in situ, comprising:

[0083] i. an engineered DNA targeting segment consisting of RNA and capable of hybridizing to such a target sequence in a polynucleotide locus,

[0084] ii. a tracr pairing sequence consisting of RNA, and

[0085] iii. a tracr RNA sequence consisting of RNA,

[0086] Wherein the tracr mate sequence hybridizes to the tracr sequence, and wherein (i), (ii), and (iii) are arranged in a 5' to 3' direction.

[0087] Another embodiment according to the present invention is an ex vivo or in vitro cell comprising:

[0088] (a) Heterologous B-GEn.16 polypeptide or nucleic acid encoding the same

[0089] (b) a single heterologous guide RNA (sgRNA) or a DNA suitable for generating such an sgRNA in situ, comprising:

[0090] i. an engineered DNA targeting segment consisting of RNA and capable of hybridizing to such a target sequence in a polynucleotide locus,

[0091] ii. a tracr pairing sequence consisting of RNA, and

[0092] iii. a tracr RNA sequence consisting of RNA,

[0093] wherein the tracr mate sequence hybridizes to the tracr sequence, and wherein (i), (ii), and (iii) are arranged in a 5' to 3' direction;

[0094] Or such a cell whose genome has been targeted, edited, modified or manipulated using (a) and (b) above.

[0095] An additional embodiment according to the invention is a kit comprising:

[0096] (a) a nucleic acid sequence encoding B-GEn.16, wherein the nucleic acid sequence encoding B-GEn.16 is operably linked to a promoter or a ribosome binding site;

[0097] (b) a single heterologous guide RNA (sgRNA) or a DNA suitable for generating such an sgRNA in situ, comprising:

[0098] i. an engineered DNA targeting segment consisting of RNA and capable of hybridizing to such a target sequence in a polynucleotide locus,

[0099] ii. a tracr pairing sequence consisting of RNA, and

[0100] iii. a tracr RNA sequence consisting of RNA,

[0101] Wherein the tracr mate sequence hybridizes to the tracr sequence, and wherein (i), (ii), and (iii) are arranged in a 5' to 3' direction.

[0102] or

[0103] (a) B-GEn.16 protein;

[0104] (b) one or more single heterologous guide RNAs (sgRNAs), each sgRNA comprising:

[0105] iv. an engineered DNA targeting segment consisting of RNA and which is capable of hybridizing to such a target sequence in a polynucleotide locus,

[0106] v. a tracr-pairing sequence consisting of RNA, and

[0107] vi. tracr RNA sequence consisting of RNA,

[0108] Wherein the tracr mate sequence hybridizes to the tracr sequence, and wherein (i), (ii), and (iii) are arranged in a 5' to 3' direction.

[0109] Yet another embodiment according to the present invention includes compositions and methods for targeting, editing, modifying or manipulating one or more target DNAs in a cell or at one or more locations in vitro, comprising:

[0110] (a) B-GEn.16

[0111] (b) a guide RNA (gRNA) or a DNA suitable for generating such a gRNA in situ, comprising:

[0112] i. an engineered DNA targeting segment consisting of RNA and which is capable of hybridizing to such a target sequence in a polynucleotide locus,

[0113] ii. a tracr RNA sequence consisting of RNA;

[0114] wherein (i) and (ii) are a single RNA molecule, and (iii) is located on a separate RNA molecule.

[0115] Multiplexing

[0116] On the other hand, provided herein is a method for editing or modifying DNA at multiple locations in a cell, the method consisting essentially of the following steps: i) introducing a B-GEn.16 polypeptide or a nucleic acid encoding a B-GEn.16 polypeptide into a cell; and ii) introducing a single heterologous nucleic acid comprising two or more precursor CRISPR RNAs (pre-CRISPR RNA, pre-crRNA) in the form of RNA or encoded as DNA and under the control of a promoter into the cell, each pre-crRNA comprising an array of repeat sequence spacers or repeat sequence spacers, wherein the spacer comprises a nucleic acid sequence complementary to a target sequence in the DNA, and the repeat sequence comprises a stem-loop structure, wherein the B-GEn.16 polypeptide cuts two or more pre-crRNAs upstream of the stem-loop structure to generate two or more intermediate crRNAs, wherein the two or more intermediate crRNAs are processed into two or more mature crRNAs, and wherein each of the two or more mature crRNAs guides the B-GEn.16 polypeptide to generate two or more double-strand breaks (DSBs) in the DNA. For example, one advantage of B-GEn.16 is that it can introduce only one pre-crRNA containing multiple repeat sequence spacer units, which is processed by B-GEn.16 after introduction and converted into active repeat sequence spacer units that target multiple different sequences on DNA.

[0117] On the other hand, provided herein is a method for editing or modifying DNA at multiple locations in a cell, the method mainly consisting of the following steps: i) introducing a B-GEn.16 polypeptide with reduced endoribonuclease activity into a cell in the form of a polypeptide or in the form of a nucleic acid encoding the B-GEn.16 polypeptide; ii) introducing a single heterologous nucleic acid comprising two or more precursor CRISPR RNAs (pre-CRISPR RNA, pre-crRNA), intermediate crRNAs or mature crRNAs in the form of RNA or in the form of DNA encoded and under the control of one or more promoters, each crRNA comprising a repeat sequence-spacer array, wherein the spacer comprises a nucleic acid sequence complementary to a target sequence in the DNA, and the repeat sequence comprises a stem-loop structure, wherein the B-GEn.16 polypeptide binds to one or more regions of the single heterologous RNA, the single heterologous RNA having reduced or absent endoribonuclease activity and having complete endonuclease activity when guided by one or more spacer sequences in the single heterologous nucleic acid.

[0118] In some embodiments, the pre-crRNA sequences in a single heterologous nucleic acid are linked together in a specific position, orientation, sequence, or with specific chemical bonds to direct or differentially regulate the endonuclease activity of B-GEn.16 at each site specified by a different crRNA sequence.

[0119] In another aspect, provided herein is an example of a general method for editing or modifying the structure or function of DNA at multiple locations in a cell, which consists essentially of the following steps: i) introducing an RNA-guided endonuclease (such as B-GEn.16) into a cell as a polypeptide or in the form of a nucleic acid encoding the RNA-guided endonuclease; and ii) introducing a single heterologous nucleic acid comprising two or more guide RNAs or encoding two or more guide RNAs as RNA or in the form of DNA encoded and under the control of one or more promoters, wherein the activity or function of the RNA-guided endonuclease is guided by the guide RNA sequence in the single heterologous nucleic acid.

[0120] definition

[0121] The terms "polynucleotide" and "nucleic acid" are used interchangeably herein and refer to polymeric forms of nucleotides of any length, either ribonucleotides or deoxyribonucleotides. Thus, the term includes, but is not limited to, single-stranded, double-stranded, or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids / triple helices, or polymers comprising purine and pyrimidine bases or other natural, chemically or biochemically modified, non-natural or derived nucleotide bases.

[0122] "Oligonucleotide" generally refers to a polynucleotide between about 5 and about 100 nucleotides of single-stranded or double-stranded DNA. However, for the purposes of this disclosure, there is no upper limit to the length of the oligonucleotide. Oligonucleotides are also referred to as "oligomers" or "oligonucleotides," and can be isolated from genes or chemically synthesized by methods known in the art. The terms "polynucleotide" and "nucleic acid" are understood to include, when applied to the described embodiments, single-stranded (e.g., sense or antisense) and double-stranded polynucleotides.

[0123] "Genomic DNA" refers to the DNA of the genome of an organism, including but not limited to the DNA of the genome of bacteria, fungi, archaea, protists, viruses, plants or animals.

[0124] The term "manipulating" DNA includes binding, nicking a strand, or cleaving, such as cutting two DNA strands; or includes modifying or editing DNA or a polypeptide associated with the DNA. Manipulating DNA can silence, activate, or modulate (whether increasing or decreasing) the expression of a polypeptide encoded by RNA or DNA, or prevent or enhance the binding of a polypeptide to DNA.

[0125] "Stem-loop structure" refers to a nucleic acid with a secondary structure comprising a nucleotide region known or predicted to form a double strand (stem portion), which is connected on one side by a region of mainly single-stranded nucleotides (loop portion). The terms "hairpin" and "fold-back" structures are also used herein to refer to stem-loop structures. Such structures are well known in the art, and these terms are consistent with their use in the known meanings of the art. As is known in the art, stem-loop structures do not require accurate base pairing. Therefore, the stem structure may include one or more base mispairings. Alternatively, base pairing may be accurate, such as, without including any mispairing.

[0126] "Hybridizable" or "complementary" or "substantially complementary" refers to a nucleic acid (such as RNA or DNA) comprising a nucleotide sequence that enables it to non-covalently bind, for example, to form Watson-Crick base pairs and / or G / U base pairs, to "anneal" or "hybridize" with another nucleic acid in a sequence-specific, antiparallel manner (e.g., a nucleic acid specifically binds to a complementary nucleic acid) under appropriate in vitro and / or in vivo conditions of temperature and solution ionic strength. As known in the art, standard Watson-Crick base pairing includes: adenine (A) pairs with thymine (T), adenine (A) pairs with uracil (U), and guanine (G) pairs with cytosine (C) [DNA, RNA]. In addition, it is also known in the art that for hybridization between two RNA molecules (such as dsRNA), guanine (G) base pairs with uracil (U). For example, G / U base pairs are partially responsible for the degeneracy (e.g., redundancy) of the genetic code in the case of tRNA anticodons base pairing with codons in mRNA. In the context of the present disclosure, guanine (G) of the protein-binding segment (dsRNA duplex) of a guide RNA molecule is considered complementary to uracil (U), and vice versa. Thus, when a G / U base pair can be formed at a given nucleotide position, i.e., the protein-binding segment (dsRNA duplex) of a guide RNA molecule, the position is not considered non-complementary, but rather complementary.

[0127] Hybridization and washing conditions are well known and are exemplified in Sambrook, J., Fritsch, E F and Maniatis, T. Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor (1989), particularly Chapter 11 and Table 11.1 thereof; and Sambrook, J. and Russell, W., Molecular Cloning: A-Laboratory Manual, Third Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor (2001). Conditions of temperature and ionic strength determine the "stringency" of hybridization.

[0128] Hybridization requires that the two nucleic acids contain complementary sequences, although there may be mismatches between the bases. Suitable conditions for hybridization between two nucleic acids depend on the length of the nucleic acid and the degree of complementarity, which is a variable known in the art. The greater the degree of complementarity between the two nucleotide sequences, the greater the melting temperature (Tm) value of the hybrid nucleic acid with these sequences. For hybridization between nucleic acids with shorter complementary segments (e.g., complementarity exceeding 35 or less, 30 or less, 25 or less, 22 or less, 20 or less, or 18 or less nucleotides), the position of mismatch becomes important (see Sambrook et al., supra, 11.7-11.8). Typically, the length of the hybrid nucleic acid is at least 10 nucleotides. The exemplary minimum length of hybridizable nucleic acids is: at least 15 nucleotides; at least 20 nucleotides; at least 22 nucleotides; at least 25 nucleotides; and at least 30 nucleotides). In addition, the technician will recognize that, according to factors such as the length and degree of complementarity of the complementary region, the concentration of the salt of the temperature and washing solution can be adjusted as necessary.

[0129] It should be understood in the art that the sequence of a polynucleotide does not need to be 100% complementary to the sequence of its target nucleic acid in order to specifically hybridize. In addition, a polynucleotide may hybridize with one or more segments so that an intervening or adjacent segment does not participate in a hybridization event (e.g., a loop structure or a hairpin structure). A polynucleotide may comprise at least 70%, at least 80%, at least 90%, at least 95%, at least 99% or 100% sequence complementarity with a target region within a target nucleic acid sequence to which they are targeted. For example, an antisense nucleic acid in which 18 of the 20 nucleotides of an antisense compound are complementary to the target region and thus specifically hybridize, represents 90% complementarity. In this example, the remaining non-complementary nucleotides and complementary nucleotides may be clustered or interspersed, and the non-complementary nucleotides do not need to be continuous with each other or continuous with complementary nucleotides. The percent complementarity between specific stretches of nucleic acid sequences within a nucleic acid can be determined conventionally using the BLAST program (Search Tool Based on Local Sequence Alignment Algorithm) and the PowerBLAST program known in the art (Altschul et al., J. Mol. Biol. 1990, 215, 403-410; Zhang and Madden, Genome Res., 1997, 7, 649-656) or using the Gap program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, Madison Wis.), using default settings, which uses the algorithm of Smith and Waterman (Adv. Appl. Math. 1981 (2) 482-489).

[0130] The terms "peptide," "polypeptide," and "protein" are used interchangeably herein and refer to a polymeric form of amino acids of any length, which may include coded and non-coded amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides with modified peptide backbones.

[0131] As used herein, "binding" (e.g., referring to the RNA binding domain of a polypeptide) refers to a non-covalent interaction between macromolecules (e.g., between a protein and a nucleic acid). Macromolecules are said to be "associated" or "interacting" or "bound" when in a non-covalent interaction state (e.g., when molecule X is said to interact with molecule Y, this means that molecule X binds to molecule Y in a non-covalent manner). Not all components of a binding interaction need to be sequence specific (e.g., contacts with phosphate residues in a DNA backbone), but some portions of a binding interaction may be sequence specific. A binding interaction is generally characterized by a dissociation constant (Kd), which is less than 10 -6 M, less than 10 -7M, less than 10 -8 M, less than 10 -9 M, less than 10 -10 M, less than 10 -11 M, less than 10 -12 M, less than 10 -13 M, less than 10 -14 M or less than 10 -15 M. "Affinity" refers to the strength of binding, with increased binding affinity correlating to a lower Kd.

[0132] "Binding domain" refers to a protein domain that is capable of non-covalently binding to another molecule. A binding domain can bind to, for example, a DNA molecule (DNA binding protein), an RNA molecule (RNA binding protein), and / or a protein molecule (protein binding protein). For a protein domain binding protein, it can bind to itself (to form homodimers, homotrimers, etc.) and / or it can bind to one or more molecules of one or more different proteins.

[0133] The term "conservative amino acid substitution" refers to the interchangeability of amino acid residues with similar side chains in proteins. For example, a group of amino acids with aliphatic side chains consists of glycine, alanine, valine, leucine and isoleucine; a group of amino acids with aliphatic hydroxyl side chains consists of serine and threonine; a group of amino acids with amide-containing side chains consists of asparagine and glutamine; a group of amino acids with aromatic side chains consists of phenylalanine, tyrosine and tryptophan; a group of amino acids with basic side chains consists of lysine, arginine and histidine; a group of amino acids with acidic side chains consists of glutamic acid and aspartic acid; a group of amino acids with sulfur-containing side chains consists of cysteine ​​and methionine. Exemplary conservative amino acid substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine and asparagine-glutamine.

[0134] A polynucleotide or polypeptide has a certain percentage of "sequence identity" with another polynucleotide or polypeptide, which means that when the two sequences are aligned, the percentage of bases or amino acids are the same and are located in the same relative position when the two sequences are compared. Sequence identity can be determined in many different ways. To determine the identity of the sequence, various methods and computer programs (such as BLAST, T-COFFEE, MUSCLE, MAFFT, etc.) can be used to align the sequences, which are available on the World Wide Web, including ncbi.nlm.nili.gov / BLAST, ebi.ac.uk / Tools / msa / tcoffee, ebi.Ac.Uk / Tools / msa / muscle, mafft.cbrc / alignment / software. See, e.g., Altschul et al. (1990), J.Mol.Biol.215:403-10. In some embodiments of the present disclosure, sequence alignment standards in the art are used according to the present disclosure to determine the amino acid residues in the B-GEn.16 polypeptide or its variants that "correspond" to the amino acid residues in another Cas9 endonuclease. The amino acid residues of the B-GEn.16 polypeptide or its variants corresponding to the amino acid residues of other Cas9 endonucleases appear in the same position in the sequence alignment.

[0135] A DNA sequence that "encodes" a specific RNA is a DNA nucleic acid sequence that is transcribed into RNA. A polydeoxyribonucleotide may encode an RNA (mRNA) that is translated into a protein, or a polydeoxyribonucleotide may encode an RNA that is not translated into a protein (such as tRNA, rRNA, or guide RNA; also referred to as "non-coding" RNA or "ncRNA"). A "protein coding sequence" or a sequence encoding a specific protein or polypeptide is a nucleic acid sequence that is transcribed into mRNA (in the case of DNA) and translated into a polypeptide (in the case of mRNA) in vitro or in vivo when placed under the control of an appropriate regulatory sequence. The boundaries of the coding sequence are determined by the start codon at the 5' end (N-terminus) and the translation termination nonsense codon at the 3' end (C-terminus). The coding sequence may include, but is not limited to, cDNA of prokaryotic or eukaryotic mRNA, genomic DNA sequences of prokaryotic or eukaryotic DNA, and synthetic nucleic acids. The transcription termination sequence is generally located at the 3' of the coding sequence.

[0136] As used herein, a "promoter sequence" or "promoter" is a DNA regulatory region that is capable of binding RNA polymerase and initiating transcription of downstream (3' direction) coding or non-coding sequences. As used herein, the promoter sequence is bounded by the transcription start site at its 3' end and extends upstream (5' direction) to contain the minimum number of bases or elements necessary to initiate transcription at a level detectable above background. A transcription start site can be found within the promoter sequence, as well as protein binding domains responsible for RNA polymerase binding. Eukaryotic promoters typically, but not always, contain a "TATA" box and a "CAAT" box. Various promoters, including inducible promoters, can be used to drive various vectors of the present disclosure. A promoter may be a constitutively active promoter (e.g., a promoter that is constitutively in an activated "on" state), it may be an inducible promoter (e.g., a promoter whose state, activated / "on" or inactivated / "off", is controlled by an external stimulus, such as the presence of a specific temperature, compound, or protein), it may be a spatially restricted promoter (e.g., a transcriptional regulatory element, an enhancer, etc.) (e.g., a tissue-specific promoter, a cell type-specific promoter, etc.), and it may be a temporally restricted promoter (e.g., a promoter that is in an "on" state or an "off" state at a specific stage of embryonic development or at a specific stage of a biological process, such as the hair follicle cycle of a mouse). Suitable promoters may be derived from viruses, and therefore may be referred to as viral promoters, or they may be derived from any organism, including prokaryotes or eukaryotes. Suitable promoters may be used to drive the expression of any RNA polymerase (e.g., pol I, pol II, pol III).Typical promoters include, but are not limited to, SV40 early promoter, mouse mammary tumor virus long terminal repeat (LTR) promoter; adenovirus major late promoter (Ad MLP); herpes simplex virus (HSV) promoter, cytomegalovirus (CMV) promoter, such as CMV immediate early promoter region (CMVIE), Rous sarcoma virus (RSV) promoter, human U6 small nuclear promoter (U6) (Miyagishi et al., Nature Biotechnology 20, 497-500 (2002)), enhanced U6 promoter (e.g., Xia et al., Nucleic Acids Res. 2003 Sep 1; 31 (17)), human H1 promoter (e.g., human HER2 promoter, human HER3 promoter, human HER4 promoter, human HER5 promoter, human HER6 promoter (e.g., H1 promoter, H1), etc. Examples of inducible promoters include, but are not limited to, T7 RNA polymerase promoter, T3 RNA polymerase promoter, isopropyl-beta-D-thiogalactopyranoside-regulated promoter, lactose induced promoter, heat shock promoter, tetracycline-regulated promoter, steroid-regulated promoter, metal-regulated promoter, estrogen receptor-regulated promoter, etc.Thus, inducible promoters can be regulated by molecules including, but not limited to, doxycycline; RNA polymerases, such as T7 RNA polymerase; estrogen receptors; estrogen receptor fusions, and the like.

[0137] In some embodiments, the promoter is a spatially restricted promoter (e.g., a cell type-specific promoter, a tissue-specific promoter, etc.) such that in a multicellular organism, the promoter is active (e.g., "ON") in a subset of specific cells. Spatially restricted promoters may also be referred to as enhancers, transcriptional regulatory elements, regulatory sequences, and the like. Any suitable spatially restricted promoter may be used, and the selection of a suitable promoter (e.g., a brain-specific promoter, a promoter that drives expression in a subset of neurons, a promoter that drives expression in the reproductive system, a promoter that drives expression in the lungs, a promoter that drives expression in muscles, a promoter that drives expression in pancreatic islet cells, and the like) will depend on the organism. For example, various spatially restricted promoters for known plants, flies, worms, mammals, mice, and the like. Therefore, spatially restricted promoters may be used to regulate the expression of nucleic acids encoding site-specific modification enzymes in a variety of different tissues and cell types, depending on the organism. Some spatially restricted promoters are also temporally restricted, such that the promoter is "on" or "off" at a specific stage of embryonic development or a specific stage of a biological process (such as the hair follicle cycle in mice). For the purpose of illustration, examples of spatially restricted promoters include, but are not limited to, neuron-specific promoters, adipocyte-specific promoters, cardiomyocyte-specific promoters, smooth muscle-specific promoters, photoreceptor-specific promoters, etc.Neuron-specific spatially restricted promoters include, but are not limited to, neuron-specific enolase (NSE) promoter (see, e.g., EMBL HSEN02, X51956); aromatic amino acid decarboxylase (AADC) promoter; neurofilament promoter (see, e.g., GenBank HUMNFL, L04147); synapsin promoter (see, e.g., GenBank HUMSYNIB, M55301); thy-1 promoter (see, e.g., Chen et al. (1987) Cell 51:7-19; and Llewellyn et al. (2010) Nat. Med. 16(10):1161-1166); serotonin receptor promoter (see, e.g., GenBank S62283); tyrosine hydroxylase promoter (see, e.g., GenBank S62284); promoter, TH) (see, e.g., Oh et al. (2009) Gene Ther. 16:437; Sasaoka et al. (1992) Mol. Brain Res. 16:274; Boundy et al. (1998) J. Neurosci. 18:9989; Kaneda et al. (1991) Neuron:583-594); GnRH promoter (see, e.g., Radovick et al. (1991) Proc. Natl. Acad. Sci. USA 88:3402-3406); L7 promoter (see, e.g., Oberdick et al. (1990) Science 248:223-226); DNMT promoter (see, e.g., Bartge et al. (1988) Proc. Natl. Acad. Sci. USA 85:3648-3652); enkephalin promoter (see, e.g., Comb et al. (1988) EMBO J. 17:3793-3805); myelin basic protein (MBP) promoter; Ca2+ / calmodulin-dependent kinase II-α (Ca2+). 2+-calmodulin-dependentprotein kinase II-alpha, CaMKIM) promoter (see, e.g., Mayford et al. (1996) Proc. Natl. Acad. Sci. USA 93:13250; and Casanova et al. (2001) Genesis 31:37); CMV enhancer / platelet-derived growth factor-p promoter (CMV enhancer / platelet-derived growth factor-p promoter) (see, e.g., Liu et al. (2004) Gene Therapy 11:52-60), etc.

[0138] The terms "DNA regulatory sequence," "control element," and "regulatory element" are used interchangeably herein and refer to transcriptional and translational control sequences, such as promoters, enhancers, polyadenylation signals, terminators, protein degradation signals, etc., which provide and / or regulate the transcription of non-coding sequences (such as guide RNA) or coding sequences (such as B-GEn.16 polypeptide or its variants) and / or regulate the translation of the encoded polypeptide.

[0139] The term "naturally occurring" or "unmodified" as used herein for nucleic acids, polypeptides, cells or organisms refers to nucleic acids, polypeptides, cells or organisms found in nature. For example, a polypeptide or polynucleotide sequence present in an organism (including a virus) that can be isolated from a source in nature and has not been artificially modified in a laboratory is naturally occurring.

[0140] The term "chimeric" as used herein for nucleic acids or polypeptides refers to an entity composed of structures from different sources. For example, when "chimeric" is used in the context of a chimeric polypeptide (such as a chimeric B-GEn.16 protein), the chimeric polypeptide includes amino acid sequences derived from different polypeptides. A chimeric polypeptide may include a modified or naturally occurring polypeptide sequence (such as a first amino acid sequence from a modified or unmodified B-GEn.16 protein; and a second amino acid sequence other than the B-GEn.16 protein). Similarly, "chimeric" in the context of a polynucleotide encoding a chimeric polypeptide includes nucleotide sequences derived from different coding regions (such as a first nucleotide sequence encoding a modified or unmodified B-GEn.16 protein; and a second nucleotide sequence encoding a polypeptide other than the B-GEn.16 protein).

[0141] The term "chimeric polypeptide" refers to a non-naturally occurring polypeptide, such as one that is made by artificially combining (e.g., "fusion") two or more otherwise separate amino acid segments through human intervention. A polypeptide comprising a chimeric amino acid sequence is a chimeric polypeptide. Some chimeric polypeptides may be referred to as "fusion variants."

[0142] As used herein, "heterologous" refers to a nucleic acid or protein that cannot be found in a natural nucleic acid or protein, respectively. The B-GEn.16 fusion protein described herein may comprise an RNA binding domain of a B-GEn.16 polypeptide (or a variant thereof) fused to a heterologous polypeptide sequence (e.g., a polypeptide sequence from a protein other than B-GEn.16). A heterologous polypeptide may exhibit activity (e.g., enzymatic activity), and the B-GEn.16 fusion protein also exhibits this activity (e.g., methyltransferase activity, acetyltransferase activity, kinase activity, ubiquitinating activity, etc.). A heterologous nucleic acid may be linked to a naturally occurring nucleic acid (or a variant thereof) (e.g., by genetic engineering) to produce a fusion polynucleotide encoding a fusion polypeptide. In another example, in a B-GEn.16 polypeptide fusion variant, a B-GEn.16 polypeptide variant may be fused to a heterologous polypeptide (e.g., a polypeptide other than B-GEn.16), which exhibits the activity exhibited by the B-GEn.16 polypeptide fusion variant. A heterologous nucleic acid can be linked to a B-GEn.16 polypeptide variant (eg, by genetic engineering) to generate a polynucleotide encoding a B-GEn.16 polypeptide fusion variant. "Heterologous," as used herein, further refers to a nucleotide or polypeptide in a cell other than a native cell.

[0143] The term "cognate" refers to two biological molecules that normally interact or coexist in nature.

[0144] As used herein, "recombinant" refers to a specific nucleic acid (DNA or RNA) or vector that is a product of various combinations of cloning, restriction, polymerase chain reaction (PCR) and / or ligation steps, resulting in a construct having a structural coding or non-coding sequence that is distinguished from an endogenous nucleic acid found in a natural system. The DNA sequence encoding the polypeptide can be assembled from cDNA fragments or a series of synthetic oligonucleotides to provide a synthetic nucleic acid that can be expressed in a recombinant transcription unit or a cell-free transcription and translation system contained in a cell. Genomic DNA containing related sequences can also be used to form a recombinant gene or transcription unit. Non-translated DNA sequences may appear at 5' or 3' of the open reading frame, where these sequences do not interfere with the operation or expression of the coding region, and can indeed regulate the production of the desired product by various mechanisms (see "DNA regulatory sequence" below). In addition or alternatively, DNA sequences encoding untranslated RNA (such as guide RNA) can also be considered to be recombinant. Therefore, the term "recombinant" nucleic acid refers to a non-naturally occurring nucleic acid, such as an artificial combination of two other separated sequence segments by human intervention. This artificial combination is usually accomplished by chemical synthesis, or by artificial manipulation of separated nucleic acid segments, such as by genetic engineering technology. This is usually done by replacing codons with codons encoding the same amino acid, conservative amino acids or non-conservative amino acids. In addition or alternatively, the desired functional nucleic acid segments are connected together to produce the desired functional combination. This artificial combination is usually accomplished by chemical synthesis, or by artificial manipulation of separated nucleic acid segments, such as by genetic engineering technology. When a recombinant polynucleotide encodes a polypeptide, the sequence of the encoded polypeptide may be naturally occurring ("wild type"), or may be a variant (such as a mutant) of a naturally occurring sequence. Therefore, the term "recombinant" polypeptide does not necessarily refer to a polypeptide whose sequence is not naturally occurring. On the contrary, a "recombinant" polypeptide is encoded by a recombinant DNA sequence, but the sequence of the polypeptide may be naturally occurring ("wild type") or non-naturally occurring (such as a variant, mutant, etc.). Therefore, a "recombinant" polypeptide is the result of human intervention, but may also be a naturally occurring amino acid sequence. The term "non-naturally occurring" includes molecules that are significantly different from naturally occurring counterparts, including chemically modified or mutated molecules.

[0145] A "vector" or "expression vector" is a replicon, such as a plasmid, phage, virus or cosmid, to which another DNA segment, such as an "insert," can be attached so as to replicate the attached segment in the cell.

[0146] An "expression cassette" includes a DNA coding sequence operably linked to a promoter. "Operably linked" refers to a juxtaposition in which the components are in a relationship that allows them to function in an intended manner. For example, if a promoter affects its transcription or expression, the promoter is operably linked to a coding sequence. The terms "recombinant expression vector" or "DNA construct" are used interchangeably herein and refer to a DNA molecule comprising a vector and at least one insert. Recombinant expression vectors are typically used for the purpose of expressing and / or propagating the insert, or for constructing other recombinant nucleotide sequences. Nucleic acids may or may not be operably linked to a promoter sequence, and may or may not be operably linked to a DNA regulatory sequence.

[0147] As used herein, the term "operably connected" refers to a physical or functional connection between two or more elements, such as a polypeptide sequence or a polynucleotide sequence, which allows them to operate in their intended manner. For example, an operably connected between a polynucleotide of interest and a regulatory sequence (e.g., a promoter) is a functional connection that allows the expression of the polynucleotide of interest. In this sense, the term "operably connected" refers to positioning a regulatory region with a coding sequence to be transcribed so that the regulatory region can effectively regulate the transcription or translation of the coding sequence of interest. In some embodiments disclosed herein, the term "operably connected" refers to a conformation in which a regulatory sequence is placed in an appropriate position relative to a sequence encoding a polypeptide or a functional RNA, so that the control sequence directs or regulates the expression or cellular localization of the mRNA encoding the polypeptide, the polypeptide and / or the functional RNA. Therefore, if a promoter can mediate the transcription of a nucleic acid sequence, the promoter is in operably connected to the nucleic acid sequence. Operably connected elements can be continuous or non-continuous.

[0148] When exogenous DNA (e.g., a recombinant expression vector) has been introduced into a cell, the cell has been "genetically modified," "transformed," or "transfected" by such DNA. The presence of the exogenous DNA results in permanent or transient genetic changes. The transforming DNA may or may not be integrated (covalently linked) into the genome of the cell.

[0149] In prokaryotes, yeast and mammalian cells, for example, transforming DNA can be maintained on episomal elements, such as plasmids. For eukaryotic cells, stably transformed cells are cells in which the transformed DNA has been integrated into the chromosome, thereby being inherited by daughter cells through chromosome replication. This stability is demonstrated by the ability of eukaryotic cells to establish cell lines or clones that include daughter cell groups containing transforming DNA. A "clone" is a cell group derived from a single cell or a common ancestor that is produced by mitosis. A "cell line" is a clone in which primary cells can stably grow for many generations in vitro.

[0150] Suitable methods of genetic modification (also referred to as "transformation") include, but are not limited to, viral or bacteriophage infection, transfection, conjugation, protoplast fusion, lipofection, electroporation, calcium phosphate precipitation, polyethyleneimine (PEI)-mediated transfection, DEAE-dextran mediated transfection, liposome-mediated transfection, particle gun technology, calcium phosphate precipitation, direct microinjection, nanoparticle-mediated nucleic acid delivery (see, e.g., Panyam et al., Adv Drug Delivery). Rev. 2012.09.13, pp:S0169-409X(12)00283-9.doi:10.1016 / j.addr.2012.09.023) etc.

[0151] As used herein, "host cell" refers to a eukaryotic cell, a prokaryotic cell (such as a bacterial or archaeal cell) in vivo or in vitro, or a cell from a multicellular organism (such as a cell line) cultured as a unicellular entity, wherein the eukaryotic cell or prokaryotic cell can or has been used as a recipient of nucleic acids, and includes the descendants of the original cell that has been transformed with nucleic acids. It should be understood that the descendants of a single cell may not necessarily be completely identical to the original parent in morphology or genome or total DNA complement due to natural, accidental or deliberate mutations. A "recombinant host cell" (also referred to as a "genetically modified host cell") is a host cell into which a heterologous nucleic acid (such as an expression vector) has been introduced. For example, a bacterial host cell is a genetically modified bacterial host cell by introducing an exogenous nucleic acid (such as a plasmid or a recombinant expression vector) into a suitable bacterial host cell, and a eukaryotic host cell is a genetically modified eukaryotic host cell (such as a mammalian germ cell) by introducing an exogenous nucleic acid into a suitable eukaryotic host cell.

[0152] As used herein, "target DNA" is a polydeoxyribonucleotide comprising a "target site" or "target sequence". The terms "target site", "target sequence", "target protospacer DNA" or "protospacer-like sequence" are used interchangeably herein and refer to a nucleic acid sequence present in the target DNA to which the DNA targeting segment (also referred to as a "spacer") of the guide RNA will bind, provided sufficient binding conditions are provided. For example, the target site (or target sequence) 5'-GAGCATATC-3' within the target DNA is targeted (or bound to, hybridized or complementary to) by the RNA sequence 5'-GAUAUGCUC-3'. Suitable DNA / RNA binding conditions include physiological conditions that are normally present in cells. Other suitable DNA / RNA binding conditions known in the art (such as conditions in a cell-free system); see, Sambrook, as described above. The target DNA strand that is complementary and hybridized to the guide RNA is referred to as the "complementary strand", and the target DNA strand that is complementary to the "complementary strand" (and therefore not complementary to the guide RNA) is referred to as the "non-complementary strand".

[0153] "Site-specific modifying enzyme" or "RNA binding site-specific modifying enzyme" refers to a polypeptide that binds to RNA and is targeted to a specific DNA sequence, such as the B-GEn.16 polypeptide. The site-specific modifying enzyme described herein is targeted to a specific DNA sequence through the RNA molecule to which it binds. The RNA molecule includes a sequence that binds, hybridizes, or is complementary to a target sequence in the target DNA, thereby targeting the bound polypeptide to a specific position (target sequence) in the target DNA. By "cutting" is meant the breaking of the covalent backbone of the DNA molecule. Cutting can be initiated by a variety of methods, including, but not limited to, enzymatic or chemical hydrolysis of phosphodiester bonds. Both single-stranded and double-stranded cutting are possible, and double-stranded cutting may be the result of two different single-stranded cutting events. DNA cutting can produce blunt ends or staggered ends. In certain embodiments, a complex comprising a guide RNA and a site-specific modifying enzyme is used for targeted double-stranded DNA cutting.

[0154] "Nuclease" and "endonuclease" are used interchangeably herein to refer to an enzyme with catalytic endonucleolytic activity for polynucleotide cleavage.

[0155] The "cleavage domain" or "active domain" or "nuclease domain" of a nuclease refers to a polypeptide sequence or domain within a nuclease that has catalytic activity that causes DNA cleavage. The cleavage domain may be contained in a single polypeptide chain, or the cleavage activity may be generated by the combination of two (or more) polypeptides. Within a given polypeptide, a single nuclease domain may consist of more than one isolated amino acid stretch.

[0156] A "guide sequence" or "DNA targeting segment" or "DNA targeting sequence" or "spacer" includes a nucleotide sequence that is complementary to a specific sequence within a target DNA (the complementary strand of the target DNA), designated herein as a "protospacer-like" sequence. The protein binding segment (or "protein binding sequence") interacts with a site-specific modifying enzyme. When the site-specific modifying enzyme is B-GEn.16 or a B-GEn.16-related polypeptide (described in more detail below), site-specific cleavage of the target DNA occurs at a location determined by: (i) base pairing complementarity between the guide RNA and the target DNA; (ii) a short motif in the target DNA (called a protospacer adjacent motif (PAM)). The protein binding segment portion of the guide RNA comprises two complementary nucleotide segments that hybridize to each other to form a double-stranded RNA duplex (dsRNA duplex). In some embodiments, a nucleic acid (e.g., a guide RNA, a nucleic acid comprising a nucleotide sequence encoding a guide RNA; a nucleic acid encoding a site-specific modifying enzyme; etc.) comprises modifications or sequences that provide additional desired properties (e.g., modified or regulated stability; subcellular targeting; tracking, such as a fluorescent tag; a binding site for a protein or protein complex; etc.). Non-limiting examples include: a 5' cap (e.g., a 7-methylguanylate cap (m7G)); a 3' polyadenylic acid tail (e.g., a 3' poly(A) tail); a riboswitch sequence (e.g., one that allows regulated stability and / or regulated accessibility of proteins and / or protein complexes); a stability control sequence; a sequence that forms a dsRNA duplex (e.g., a hairpin sequence); a modification or sequence that targets RNA to a subcellular location (e.g., the nucleus, mitochondria, chloroplasts, etc.); a modification or sequence that provides tracking (e.g., direct conjugation to a fluorescent molecule, conjugation to a moiety that facilitates fluorescence detection, a sequence that allows fluorescence detection, etc.); a modification or sequence that provides a protein binding site (e.g., proteins that act on DNA, including transcription activators, transcription repressors, DNA methyltransferases, DNA demethylases, histone acetyltransferases, histone deacetylases, etc.); and combinations thereof.

[0157] In some embodiments, the guide RNA includes an additional segment at the 5' or 3' end that provides any of the above features. For example, a suitable third segment may include a 5' cap (such as a 7-methylguanosine cap (m7G); a 3' polyadenylation tail (such as a 3' poly (A) tail); a riboswitch sequence (such as a stability that allows regulation and / or accessibility of regulated proteins and / or protein complexes); a stability control sequence; a sequence that forms a dsRNA duplex (such as a hairpin sequence); a sequence that targets RNA to a subcellular location (such as a nucleus, mitochondria, chloroplasts, etc.); a modification or sequence that provides tracking (such as direct conjugation to a fluorescent molecule, conjugation to a portion that facilitates fluorescence detection, a sequence that allows fluorescence detection, etc.); a modification or sequence that provides a protein binding site (such as a protein that acts on DNA, including a transcription activator, a transcription repressor, a DNA methyltransferase, a DNA demethylase, a histone acetyltransferase, a histone deacetylase, etc.); and combinations thereof.

[0158] Guide RNA and site-specific modification enzymes, such as B-GEn.16 polypeptides or variants thereof, can form ribonucleoprotein complexes (e.g., bound by non-covalent interactions). Guide RNA provides the targeting specificity of the complex by comprising a nucleotide sequence complementary to the target DNA sequence. The site-specific modification enzyme of the complex provides endonuclease activity. In other words, the site-specific modification enzyme is guided to the target DNA sequence (e.g., target sequence in chromosomal nucleic acid; target sequence in extrachromosomal nucleic acid, such as free nucleic acid, minicircle, etc.; target sequence in mitochondrial nucleic acid; target sequence in chloroplast nucleic acid; target sequence in plasmid; etc.) by means of its combination with the protein binding segment of the guide RNA. RNA aptamers are known in the art and are generally synthetic versions of riboswitches. The terms "RNA aptamers" and "riboswitches" are used interchangeably herein to include synthetic and natural nucleic acid sequences that provide inducible regulation of the structure of the RNA molecule to which they belong (and thus provide the availability of specific sequences). RNA aptamers typically contain sequences that fold into a specific structure (such as a hairpin) that specifically bind to a specific drug (such as a small molecule). The binding of the drug causes structural changes in the RNA folding, which changes the characteristics of the nucleic acid to which the aptamer belongs. As non-limiting examples: (i) an activator RNA with an aptamer may not bind to a cognate target RNA unless the aptamer is bound by an appropriate drug; (ii) a target RNA with an aptamer may not bind to a cognate activator RNA unless the aptamer is bound by an appropriate drug; (iii) a target RNA and an activator RNA, each of which contains different aptamers that bind different drugs, may not bind to each other unless both drugs are present. As these examples illustrate, bimolecular guide RNAs can be designed to be inducible.

[0159] Examples of aptamers and riboswitches can be found in, e.g., Nakamura et al., Genes Cells. 2012 May; 17(5):344-64; Vavalle et al., Future Cardiol. 2012 May; 8(3):371-82; Citarta et al., Biosens Bioelectron. 2012 Apr 15; 34(1):1-11; and Liberman et al., Wiley Interdiscip Rev RNA. 2012 May-Jun; 3(3):369-84; all of which are incorporated herein by reference in their entirety.

[0160] The choice of gene modification method generally depends on the type of cell to be transformed and the environment in which the transformation occurs (e.g., in vitro, ex vivo, or in vivo). A general discussion of these methods can be found in Ausubel, et al., Short Protocols in Molecular Biology, 3rd ed., Wiley & Sons, 1995.

[0161] Examples of aptamers and riboswitches can be found in, e.g., Nakamura et al., Genes Cells. 2012 May; 17(5):344-64; Vavalle et al., Future Cardiol. 2012 May; 8(3):371-82; Citarta et al., Biosens Bioelectron. 2012 Apr 15; 34(1):1-11; and Liberman et al., Wiley Interdiscip Rev RNA. 2012 May-Jun; 3(3):369-84; all of which are incorporated herein by reference in their entirety.

[0162] The term "stem cell" as used herein refers to a cell (e.g., plant stem cells, vertebrate stem cells) that has the ability to self-renew and generate differentiated cell types (see Morrison et al. (1997) Cell 88: 287-298). In the context of cell ontogeny, the adjective "differentiated" or "differentiating" is a relative term. A "differentiated cell" is a cell that has developed further in a developmental pathway than a comparable cell. Thus, pluripotent stem cells (as described below) can differentiate into lineage-restricted progenitor cells (e.g., mesodermal stem cells), which in turn can differentiate into further restricted cells (e.g., neuronal progenitor cells), which can differentiate into terminal cells (e.g., terminally differentiated cells, such as neurons, cardiomyocytes, etc.), which play a characteristic role in a certain tissue type and may or may not retain the ability to further proliferate. Stem cells may be characterized by the presence or absence of specific markers (e.g., proteins, RNA, etc.). Stem cells can also be identified by in vitro and in vivo functional assays, particularly assays related to the ability of stem cells to give rise to multiple differentiated progeny.

[0163] Target stem cells include pluripotent stem cells (PSCs). The term "pluripotent stem cell" or "PSC" is used herein to refer to stem cells that can produce all cell types of an organism. Therefore, PSC can produce cells of all germ layers of an organism (such as the endoderm, mesoderm, and ectoderm of vertebrates). Pluripotent cells can form teratomas and form ectoderm, mesoderm, or endoderm tissues in an organism. Plant pluripotent stem cells can produce all cell types of plants (such as cells of roots, stems, leaves, etc.).

[0164] Animal PSCs can be obtained in many different ways. For example, embryonic stem cells (ESCs) are derived from the inner cell mass of the embryo (Thomson et al., Science. 1998 November 6; 282(5391): 1145-7), while induced pluripotent stem cells (iPSCs) are derived from somatic cells (Takahash et al., Cell. 2007 Nov 30; 131(5): 861-72; Takahashi et al., Nat Protoc. 2007; 2(12): 3081-9; Yu et al., Science. 2007 Dec 21; 318(5858): 1917-20. Epub 2007 Nove 20).

[0165] Because the term PSC refers to pluripotent stem cells (regardless of their origin), the term PSC includes the terms ESC and iPSC, as well as the term embryonic germ stem cells (EGSC), which is another example of PSC. PSCs can be in the form of established cell lines, they can be obtained directly from primary embryonic tissue, or they can be derived from somatic cells. PSCs can be target cells for the methods described herein.

[0166] "Embryonic stem cells" (ESC) refer to PSCs isolated from embryos, usually from the inner cell mass of blastocysts. ESC cell lines are listed in the NIH Human Embryonic Stem Cell Registry, such as hESBGN-01, hESBGN-02, hESBGN-03, hESBGN-04 (BresaGen, Inc.); HES-1, HES-2, HES-3, HES-4, HES-5, HES-6 (ES Cell International); MizMedi (MizMedi Hospital-Seoul National University); HSF-1, HSF-6 (University of California at San Francisco); H1, H7, H9, H13, H14 (Wisconsin Alumni Research Foundation (WiCell Research Institute)). Target stem cells also include embryonic stem cells from other primates, such as rhesus stem cells and marmoset stem cells. The stem cells can be obtained from any mammalian species, such as humans, horses, cattle, pigs, dogs, cats, rodents, such as mice, rats, hamsters, primates, etc. (Thomson et al. (1998) Science 282: 1145; Thomson et al. (1995) Proc. Natl. Acad. Sci. USA 92: 7844; Thomson et al. (1996) Biol. Reprod. 55: 254; Shamblott et al., Proc. Natl. Acad. Sci. USA 95: 13726, 1998). In culture, ESCs usually grow as flat colonies with a large nuclear-cytoplasmic ratio, clear borders and prominent nucleoli. In addition, ESCs express SSEA-3, SSEA-4, TRA-1-60, TRA-1-81, and alkaline phosphatase, but do not express SSEA-1. Examples of methods for generating and characterizing ESCs can be found, for example, in U.S. Pat. No. 7,029,913, U.S. Pat. No. 5,843,780, and U.S. Pat. No. 6,200,806, the disclosures of which are incorporated herein by reference. Methods for the proliferation of undifferentiated forms of hESCs are described in WO 99 / 20741, WO 01 / 51616, and WO 03 / 020920."Embryonic germ stem cells" (EGSC) or "embryonic germ cells" or "EG cells" refer to PSCs derived from germ cells and / or germ cell progenitors, such as primordial germ cells, such as those that will become sperm and eggs. Embryonic germ cells (EG cells) are believed to have properties similar to those of the embryonic stem cells described above. Examples of methods for generating and characterizing EG cells can be found in, for example, U.S. Patent No. 7,153,684; Matsui, Y., et al., (1992) Cell 70:841; Shamblott, M., et al. (2001) Proc. Natl. Acad. Sci. USA 98:113; Shamblott, M., et al. (1998) Proc. Natl. Acad. Sci. USA, 95:13726; and Koshimizu, U., et al. (1996) Development, 122:1235, the disclosures of which are incorporated herein by reference.

[0167] "Induced pluripotent stem cells" or "iPSCs" refer to PSCs derived from non-PSC cells (e.g., PSCs derived from cells differentiated relative to PSCs). Pluripotent stem cells can be derived from a variety of different cell types, including terminally differentiated cells. iPSCs have an ES cell-like morphology, grow as flat colonies, have a large nuclear-to-cytoplasmic ratio, have clear boundaries, and have prominent nuclei. In addition, iPSCs express one or more key pluripotency markers known to those skilled in the art, including but not limited to alkaline phosphatase, SSEA3, SSEA4, Sox2, Oct3 / 4, Nanog, TRA160, TRA181, TDGF 1, Dnmt3b, Fox03, GDF3, Cyp26al, TERT, and zfp42.

[0168] Examples of methods for generating and characterizing iPSCs can be found in, for example, U.S. Patent Publication Nos. US20090047263, US20090068742, US20090191159, US20090227032, US20090246875, and US20090304646, the disclosures of which are incorporated herein by reference. Generally, in order to generate iPSCs, it is necessary to provide somatic cells with reprogramming factors known in the art (such as Oct4, SOX2, KLF4, MYC, Nanog, Lin28, etc.), and reprogram somatic cells to become pluripotent stem cells.

[0169] "Somatic cell" refers to any cell in an organism that, in the absence of experimental manipulation, does not normally give rise to all cell types in the organism. In other words, somatic cells are cells that have differentiated sufficiently that they do not naturally give rise to cells of all three germ layers of the body, such as ectoderm, mesoderm, and endoderm. For example, somatic cells may include neurons and neural progenitor cells, which may be able to naturally give rise to all or some cell types of the central nervous system, but not to give rise to mesodermal or endodermal cell lineages.

[0170] A "mitotic cell" is a cell that is undergoing mitosis.

[0171] A "post-mitotic cell" refers to a cell that has exited mitosis, i.e., it is "quiescent," i.e., it no longer divides. This quiescent state may be temporary, i.e., reversible, or it may be permanent.

[0172] A "meiotic cell" is a cell that is undergoing meiosis.

[0173] "Recombination" refers to the process of exchanging genetic information between two polynucleotides. As used herein, "homology-directed repair (HDR)" refers to, for example, a special form of DNA repair that occurs during the repair process of double-strand breaks in cells. This process requires nucleotide sequence homology, and a "donor" molecule is used to perform template repair on a "target" molecule (e.g., a molecule that undergoes a double-strand break), and causes genetic information to be transferred from the donor to the target. If the donor polynucleotide is different from the target molecule, and part or all of the sequence of the donor polynucleotide is incorporated into the target DNA, homology-directed repair may cause changes in the target molecule sequence (e.g., insertion, deletion, mutation). In some embodiments, a donor polynucleotide, a partial donor polynucleotide, a copy of a donor polynucleotide, or a portion of a copy of a donor polynucleotide is integrated into the target DNA.

[0174] "Non-homologous end joining (NHEJ)" refers to the repair of DNA double-strand breaks by directly joining the broken ends together without the need for a homologous template (in contrast to homology-directed repair, which requires a homologous sequence to guide the repair). NHEJ typically results in the loss (deletion) of nucleotide sequence adjacent to the site of the double-strand break.

[0175] The terms "treatment", "treating", and the like are used herein to generally refer to obtaining a desired pharmacological and / or physiological effect. The effect may be preventive for the complete or partial prevention of a disease or its symptoms, and / or may be therapeutic for the partial or complete cure of a disease and / or adverse effects attributable to the disease. "Treatment" as used herein encompasses any treatment of a disease or symptom in a mammal, and includes: (a) preventing a disease or symptom from occurring in a subject who may be predisposed to acquiring the disease or symptom but has not yet been diagnosed with the disease or symptom; (b) inhibiting a disease or symptom, such as preventing its development; or (c) alleviating a disease, such as causing the disease to regress. The therapeutic agent may be administered before, during, or after the onset of the disease or injury. The treatment of persistent diseases is particularly noteworthy, wherein the treatment stabilizes or reduces adverse clinical symptoms of the subject. Such treatment needs to be performed before the function of the affected tissue is completely lost. The therapy will need to be administered during the symptomatic stage of the disease, and in some cases after the symptomatic stage of the disease.

[0176] The terms "individual," "subject," "host," and "patient" are used interchangeably herein and refer to any mammalian subject, particularly a human, for whom diagnosis, treatment, or therapy is desired.

[0177] General methods of molecular and cellular biochemistry can be found in such standard textbooks as Molecular Cloning: A Laboratory Manual, 3rd Ed. (Sambrook et al., Harbor Laboratory Press 2001); Short Protocols in Molecular Biology, 4th Ed. (Ausubel et al. eds., John Wiley & Sons 1999); Protein Methods (Bollag et al., John Wiley & Sons 1996); Nonviral Vectors for Gene Therapy (Wagner et al. eds., Academic Press 1999); Viral Vectors (Kaplift & Loewy eds., Academic Press 1995); Immunology Methods Manual (1. Lefkovits ed., Academic Press 1997); and Cell and Tissue Culture: Laboratory Procedures in Biotechnology (Doyle & Griffiths, John Wiley & Sons 1998), the disclosure of which is incorporated herein by reference.

[0178] Where a numerical range is provided, it is understood that, unless the context clearly dictates otherwise, each intervening value between the upper and lower limits of the range, up to one tenth of the lower limit, and any other intervening values ​​specified or within the specified range, are included in the disclosure. The upper and lower limits of these smaller ranges may be independently included in the smaller range, and are also included in the disclosure, subject to any specific non-inclusive limits within the specified range. Where a specified range includes one or both of the limits, ranges that do not include one or both of the limits are also included in the disclosure.

[0179] The phrase "consisting essentially of" is intended herein to exclude any component or components that are not a specific activity of the system, or a moiety that is not a specific activity or activities of the molecule.

[0180] Certain ranges appearing herein have numerical values ​​preceded by the term "about". The term "about" is used herein to provide written support for the exact number preceding it as well as numbers that are close to or approximately preceding the term. In determining whether a number is close to or approximately a specifically recited number, a close or approximate unrecited number may be a number that, in the context in which it appears, provides a substantial equivalence to the specifically recited number.

[0181] It should be understood that certain features of the present disclosure, which for clarity are described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the present disclosure, which for brevity are described in the context of a single embodiment, may also be provided separately or in any suitable subcombination. The present disclosure expressly encompasses all combinations of embodiments related to the present disclosure and are disclosed herein, as if each and every combination were individually and expressly disclosed. In addition, all subcombinations of the various embodiments and elements thereof are also expressly encompassed by the present disclosure and are disclosed herein, as if each and every such subcombination were individually and expressly disclosed herein.

[0182] B-GEn.16 fusion polypeptide

[0183] B-GEn.16 can be used to form fusion proteins with additional domains and activity compared to the B-GEn.16 nuclease. By way of non-limiting illustration, the Fok1 domain can be fused to a B-GEn.16 polypeptide or variant thereof, which can include a catalytically active endonuclease domain, or the Fok1 domain can be fused to a B-GEn.16 polypeptide or variant thereof, which has been modified to render the B-GEn.16 endonuclease domain inactive. Other domains that can be fused to B-GEn.16 to form fusion proteins include transcriptional regulators, epigenetic modifiers, tags and other markers or imaging agents, histones and / or other patterns of structure or activity that regulate or modify gene sequences known in the art.

[0184] In some embodiments, the B-GEn.16 polypeptides described herein or variants thereof are fused to a transcriptional activator or inhibitor, or an epigenetic modifier, such as a methylase, demethylase, acetylase, or deacetylase.

[0185] In some embodiments, the B-GEn.16 polypeptides or variants thereof described herein are fused into functional protein components for detection, intermolecular interaction, translation activation, modification, or any other manipulation known in the art.

[0186] Exemplary B-GEn.16 variant polypeptides

[0187] In some embodiments, the B-GEn.16 polypeptide described herein or a variant thereof retains a) the ability to bind to the targeted site, and optionally, b) retains its activity. In some embodiments, the retained activity is endonuclease activity. In certain embodiments, endonuclease activity does not require tracrRNA.

[0188] In some embodiments, the active portion of the B-GEn.16 polypeptide or its variant is modified. In some embodiments, the modification includes an amino acid change (such as a deletion, insertion, or substitution) that reduces or increases the nuclease activity of the B-GEn.16 polypeptide or its variant. For example, in some embodiments, the modified B-GEn.16 polypeptide or its variant has less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, less than 5%, or less than 1% of the nuclease activity of the corresponding unmodified B-GEn.16 polypeptide or its variant. In some embodiments, the modified B-GEn.16 polypeptide or its variant has no substantial nuclease activity. In some embodiments, it may have 50%, 2 times, 4 times, or up to 10 times more nuclease activity.

[0189] In some embodiments, the active portion of the B-GEn.16 polypeptide or its variant comprises a heterologous polypeptide having DNA modification activity and / or transcription factor activity and / or DNA-related polypeptide modification activity. In some embodiments, the heterologous polypeptide replaces the portion of the B-GEn.16 polypeptide or its variant that provides nuclease activity. In some embodiments, the B-GEn.16 polypeptide or its variant comprises a portion of the B-GEn.16 polypeptide or its variant that generally provides nuclease activity (and the portion may be fully activated or may be modified to have less than 100% of the corresponding unmodified activity instead) and a heterologous polypeptide. In other words, in some embodiments, the B-GEn.16 polypeptide or its variant may be a fusion polypeptide comprising a portion of the B-GEn.16 polypeptide or its variant that generally provides nuclease activity and a heterologous polypeptide.

[0190] For example, in the B-GEn.16 fusion protein, the B-GEn.16 polypeptide or its variant can be fused to a heterologous polypeptide sequence (e.g., a polypeptide sequence derived from a protein other than B-GEn.16). The heterologous polypeptide sequence can exhibit activity (e.g., enzymatic activity), which is also exhibited by the B-GEn.16 fusion protein (e.g., methyltransferase activity, acetyltransferase activity, kinase activity, ubiquitination activity, etc.). A heterologous nucleic acid sequence can be connected to another nucleic acid sequence (e.g., by genetic engineering) to produce a fusion nucleotide sequence encoding a fusion polypeptide. In some embodiments, the B-GEn.16 fusion polypeptide is produced by fusion of a B-GEn.16 polypeptide or its variant with a heterologous sequence that provides subcellular localization (e.g., a nuclear localization signal (NLS) for targeting the nucleus; a mitochondrial localization signal for targeting mitochondria; a chloroplast localization signal for targeting chloroplasts; an ER retention signal; etc.). In some embodiments, the heterologous sequence can provide a tag that is easy to track or purify (such as a fluorescent protein, such as green fluorescent protein (GFP), YFP, RFP, CFP, mCherry, tdTomato, etc.; a HIS tag, such as a 6XHis tag; a hemagglutinin (HA) tag; a FLAG tag; a Myc tag; etc.). In some embodiments, the heterologous sequence can provide increased or decreased stability. In some embodiments, the heterologous sequence can provide a binding domain (such as providing the ability of the B-GEn.16 fusion polypeptide to bind to another target protein (such as a DNA or histone modifying protein, a transcription factor or a transcription repressor, a recruitment protein, etc.), or a target nucleotide (such as an aptamer or target site of a nucleic acid binding protein)).

[0191] In some embodiments, according to any of the B-GEn.16 polypeptide variants described herein, the B-GEn.16 polypeptide variant has reduced endodeoxyribonuclease activity. For example, a B-GEn.16 polypeptide variant suitable for use in the transcriptional regulation methods of the present disclosure exhibits less than about 20%, less than about 15%, less than about 10%, less than about 5%, less than about 1%, or less than about 0.1% of the endodeoxyribonuclease activity of the unmodified B-GEn.16 polypeptide.

[0192] In some embodiments, the B-GEn.16 polypeptide variant has substantially no detectable endodeoxyribonuclease activity (dB-GEn.16). In some embodiments, when the B-GEn.16 polypeptide variant has reduced catalytic activity, the polypeptide can still bind to the target DNA in a site-specific manner (because it is still guided to the target DNA sequence by the guide RNA) as long as the polypeptide retains the ability to interact with the guide RNA. In some embodiments, the B-GEn.16 polypeptide variant is a nickase, which can cut the complementary strand of the target DNA, but has a reduced ability to cut the non-complementary strand of the target DNA.

[0193] In some embodiments, the B-GEn.16 polypeptide variant in the nickase can cleave the non-complementary strand of the target DNA, but has a reduced ability to cleave the complementary strand of the target DNA.

[0194] In some embodiments, the B-GEn.16 polypeptide variant has a reduced ability to cleave both the complementary and non-complementary strands of a target DNA. For example, alanine substitution is contemplated.

[0195] In some embodiments, the variant B-GEn.16 polypeptide is a fusion polypeptide ("variant B-GEn.16 fusion polypeptide"), for example, a fusion polypeptide comprising: i) a variant B-GEn.16 polypeptide; and ii) a covalently linked heterologous polypeptide (also referred to as a "fusion partner").

[0196] Heterologous polypeptide sequences can exhibit activity (e.g., enzymatic activity), which is also exhibited by B-GEn.16 fusion protein variants (e.g., methyltransferase activity, acetyltransferase activity, kinase activity, ubiquitination activity, etc.). Heterologous nucleic acid sequences can be linked to another nucleic acid sequence (e.g., by genetic engineering) to produce a fusion nucleotide sequence encoding a fusion polypeptide. In some embodiments, B-GEn.16 fusion polypeptide variants are produced by fusion of B-GEn.16 polypeptide variants with a heterologous sequence that provides subcellular localization (e.g., the heterologous sequence is a subcellular localization sequence, such as a nuclear localization signal (NLS) for targeting the nucleus; a mitochondrial localization signal for targeting mitochondria; a chloroplast localization signal for targeting chloroplasts; an ER retention signal; etc.). In some embodiments, the heterologous sequence can provide a tag (e.g., the heterologous sequence is a detectable marker) for easy tracking and / or purification (e.g., a fluorescent protein, such as green fluorescent protein (GFP), YFP, RFP, CFP, mCherry, tdTomato, etc.; a histidine tag, such as a 6XHis tag; a hemagglutinin (HA) tag; a FLAG tag; a Myc tag; etc.). In some embodiments, the heterologous sequence can provide increased or decreased stability (e.g., the exogenous sequence is a stability control peptide, such as a degron, which is controllable in some cases (e.g., a temperature-sensitive or drug-controllable degron sequence, see below). In some embodiments, the heterologous sequence can provide increased or decreased transcription from the target DNA (e.g., the exogenous sequence is a transcription regulatory sequence, such as a transcription factor / activator or a fragment thereof, a protein or a fragment thereof that recruits a transcription factor / activator, a transcription repressor or a fragment thereof, a protein or a fragment thereof that recruits a transcription repressor, a small molecule / drug-responsive transcription regulator, etc.). In some embodiments, the heterologous sequence can provide a binding domain (e.g., the exogenous sequence is a protein binding sequence, such as providing the ability of the dB-GEn.16 fusion polypeptide to bind to another protein of interest (e.g., a DNA or histone modifying protein, a transcription factor or transcription repressor, a recruiting protein, etc.)

[0197] Suitable fusion partners that increase or decrease stability are provided include, but are not limited to, degron sequences. It will be readily appreciated by those skilled in the art that degron is an amino acid sequence that controls the stability of the protein to which it belongs. For example, the stability of a protein comprising a degron sequence is at least partially controlled by the degron sequence. In some embodiments, suitable degron is constitutive, so that the degron exerts its influence independent of experimental control on protein stability (e.g., degron is not drug-induced, temperature-induced, etc.). In some embodiments, degron provides B-GEn.16 polypeptide variants with controlled stability, so that B-GEn.16 polypeptide variants can be "opened" (e.g., stable) or "closed" (e.g., unstable, degraded) according to desired conditions. For example, if the degradation determinant is a temperature-sensitive degradation determinant, the B-GEn.16 polypeptide variant may be functional (e.g., "on", stable) below a threshold temperature (e.g., 42°C, 41°C, 40°C, 39°C, 38°C, 37°C, 36°C, 35°C, 34°C, 33°C, 32°C, 31°C, 30°C, etc.), but not functional (e.g., "off", degraded) above the threshold temperature. As another example, if the degradation determinant is a drug-induced degradation determinant, the presence or absence of the drug can switch the protein from a "off" (e.g., unstable) state to an "on" (e.g., stable) state, and vice versa. An example of a drug-induced degradation determinant is derived from the FKBP12 protein. The stability of the degradation determinant is controlled by the presence or absence of a small molecule that binds to the degradation determinant.

[0198] Examples of suitable degrons include, but are not limited to, degrons controlled by Shield-1, DHFR, auxin, and / or temperature.Non-limiting examples of suitable degron-containing substrates are known in the art (e.g., Dohmen et al., Science, 1994. 263(5151): p. 1273-1276: Heat-inducible degron: a method for constructing temperature-sensitive mutants; Schoeber et al., Am J Physiol Renal Physiol. 2009 Jan; 296(1): F204-11: Conditional fast expression and function of multimeric TRPV5 channels using Shield-1; Chu et al., Bioorg Med Chem Lett. 2008 Nov 15; 18(22): 5941-4: Recent progress with FKBP-derived destabilizing domains; Kanemaki, Pflugers Arch. 2012 Dec 28: Frontiers of protein expression control with conditional degrons; Yang et al., Mol Cell. 2012 Nov 30; 48(4): 487-8: Titivated for destruction: the methyl degron; Barbour et al., Biosci Rep. 2013 Jan 18; 33(1).: Characterization of the bipartite degron that regulates ubiquitin-independent degradation of thymidylate synthase; and Greussing et al., J Vis Exp. 2012 Nov 10; (69): Monitoring of ubiquitin-proteasome activity in living cells using a Degron (dgn)-destabilized green fluorescent protein (GFP)-based reporter protein; which are incorporated herein by reference in their entirety).

[0199] Exemplary degradation determinant sequences have been well characterized and tested in cells and animals. Therefore, B-GEn.16 is fused to a degradation determinant sequence to produce a "tunable" and "inducible" B-GEn.16 polypeptide. Any fusion partner described herein can be used in any desired combination. As a non-limiting example to illustrate this point, the B-GEn.16 fusion protein can include a YFP sequence for detection, a degradation determinant sequence for stability, and a transcriptional activator sequence for increasing target DNA transcription. In addition, the number of fusion partners that can be used in the B-GEn.16 fusion protein is unlimited. In some embodiments, the B-GEn.16 fusion protein includes one or more (such as two or more, three or more, four or more, or five or more) heterologous sequences.

[0200] Suitable fusion partners include, but are not limited to, providing methyltransferase activity, demethylase activity, acetyltransferase activity, deacetylase activity, kinase activity, phosphatase activity, ubiquitin ligase activity, deubiquitinating activity, adenylylation activity, deadenylation activity, SUMOylation activity, deSUMOylation activity, ribosylation activity, deribosylation activity, crotonylation activity, decrotonylation activity, propionylation activity, depropionylation activity, myristoylation activity or myristoylation activity polypeptide, wherein any activity can be directed to directly modify DNA (e.g., DNA methylation) or modify DNA-related polypeptides (e.g., histones or DNA binding proteins). Further suitable fusion partners include, but are not limited to, boundary elements (e.g., CTCF), proteins and fragments thereof (e.g., lamin A, lamin B, etc.) provided for peripheral recruitment, and protein docking elements (e.g., FKBP / FRB, Pil 1 / Aby 1, etc.).

[0201] B-GEn.16 polypeptide or its variant can also be separated and purified according to the conventional method of recombinant synthesis. Lysate of expression host can be prepared, and lysate is purified using HPLC, exclusion chromatography, gel electrophoresis, affinity chromatography or other purification techniques. For most cases, the composition used will include at least 20% weight of the desired product, at least about 75% weight, at least about 95% weight, and for therapeutic purposes, usually at least 99.5% weight, which relates to the pollutants related to the product preparation method and its purification. Usually, the percentage is based on total protein. To induce DNA cleavage and recombination, or to make any desired modifications to the target DNA, or to make any desired modifications to the polypeptide associated with the target DNA, the guide RNA and / or B-GEn.16 polypeptide or its variant and / or donor polynucleotide, whether introduced as a nucleic acid or polypeptide, is provided to the cell for about 30 minutes to about 24 hours, such as 1 hour, 1.5 hours, 2 hours, 2.5 hours, 3 hours, 3.5 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 12 hours, 16 hours, 18 hours, 20 hours, or any other time period from about 30 minutes to about 24 hours, which can be repeated at a frequency of about every day to about every 4 days, such as every 1.5 days, every 2 days, every 3 days, or any other frequency of about every day to about every four days. The agent can be provided to the cell once or more, such as once, twice, three times or more, and after each contact event (such as 16-24 hours), the cell can be incubated with the agent for a period of time, after which the culture medium is replaced with fresh culture medium and the cell is further cultured. In the case of providing two or more different targeting complexes to a cell (e.g., two different guide RNAs complementary to different sequences in the same or different target DNAs), the complexes can be provided simultaneously (e.g., as two polypeptides and / or nucleic acids), or delivered simultaneously. Alternatively, they can be provided sequentially, e.g., first providing a target complex, then providing a second target complex, etc., or vice versa.

[0202] Nucleic Acids

[0203] Guide RNA / sgRNA

[0204] The systems, compositions and methods described in some embodiments use genome targeting nucleic acids that can guide the activity of related polypeptides (such as B-GEn.16 polypeptides or variants thereof) to specific target sequences within target nucleic acids. In some embodiments, genome targeting nucleic acids are RNA. Genome targeting RNA is referred to herein as "guide RNA" or "gRNA". Guide RNA has a spacer sequence that can hybridize at least with the target nucleic acid sequence and CRISPR repeat sequence (such CRISPR repeat sequence is also referred to as "tracr partner sequence"). In the type II system, gRNA also has a second RNA, called a tracrRNA sequence. In type II guide RNA (gRNA), CRISPR repeat sequence and tracrRNA sequence hybridize with each other to form a duplex. In type V guide RNA (gRNA), crRNA forms a duplex. In both systems, the duplex is combined with a site-specific polypeptide so that the guide RNA and the site guide polypeptide form a complex. Genome targeting nucleic acids provide targeting specificity for the complex by virtue of their association with site-specific polypeptides. Genome targeting nucleic acids therefore guide the activity of site-specific polypeptides.

[0205] In some embodiments, the genome targeting nucleic acid is a bimolecular guide RNA. In some embodiments, the genome targeting nucleic acid is a single molecule guide RNA or a single guide RNA (sgRNA). The bimolecular guide RNA has two RNA chains. The first chain has an optional spacer extension sequence, a spacer sequence and a minimum CRISPR repeat sequence in the 5' to 3' direction. The second chain has a minimum tracrRNA sequence (complementary to the minimum CRISPR repeat sequence), a 3'tracrRNA sequence and an optional tracrRNA extension sequence. The single molecule guide RNA (sgRNA) in the type II system has an optional spacer extension sequence, a spacer sequence, a minimum CRISPR repeat sequence, a single molecule guide linker, a minimum tracrRNA sequence, a 3'tracrRNA sequence and an optional tracrRNA extension sequence in the 5' to 3' direction. The optional tracrRNA extension may have an element that provides additional functions (e.g., stability) for the guide RNA. The single molecule guide linker connects the minimum CRISPR repeat sequence and the minimum tracrRNA sequence to form a hairpin structure. The optional tracrRNA extension has one or more hairpins.

[0206] The single-molecule guide RNA (sgRNA) in the V-type system has an optional tracr extension sequence, a tracr RNA sequence, a single-molecule guide linker, a minimal CRISPR repeat sequence, a spacer sequence, and an optional spacer extension sequence in the 5' to 3' direction.

[0207] Alternatively, the single-molecule guide RNA (sgRNA) in the V-type system has an optional extension sequence, a minimal CRISPR repeat sequence, a spacer sequence, and an optional spacer extension sequence in the 5' to 3' direction.

[0208] In yet another alternative, the single-molecule guide RNA (sgRNA) in the V-type system includes an optional extension sequence, an artificial nuclease binding RNA sequence and a spacer sequence in the 5' to 3' direction, and an optional spacer extension sequence.

[0209] Exemplary genome targeting nucleic acids are described, for example, in WO2018002719.

[0210] Typically, a CRISPR repeat sequence includes any sequence that has sufficient complementarity with a tracr sequence to facilitate one or more of the following: (1) excision of a DNA targeting segment flanking the CRISPR repeat sequence in a cell containing the corresponding tracr sequence; and (2) formation of a CRISPR complex at the target sequence, wherein the CRISPR complex comprises a CRISPR repeat sequence hybridized to the tracr sequence. Typically, the degree of complementarity refers to the optimal alignment of the CRISPR repeat sequence and the tracr sequence along the shorter length of the two sequences. The optimal alignment can be determined by any suitable alignment algorithm, and can further take into account secondary structures, such as self-complementarity within the tracr sequence or the CRISPR repeat sequence. In some embodiments, when the tracr sequence and the CRISPR repeat sequence are optimally aligned, the degree of complementarity along the shorter 30 nucleotide length of the two is about or greater than 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97.5%, 99% or more. In some embodiments, the length of the tracr sequence is about or more than 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 40, 50 or more nucleotides. In some embodiments, the tracr sequence and the CRISPR repeat sequence are contained in a single transcript such that hybridization between the two produces a transcript having a secondary structure, such as a hairpin. In some embodiments, the transcript or transcribed polynucleotide sequence has at least two or more hairpins.

[0211] The spacer of the guide RNA includes a nucleotide sequence that is complementary to a sequence in the target DNA. In other words, the spacer of the guide RNA interacts with the target DNA in a sequence-specific manner by hybridization (such as base pairing). Therefore, the nucleotide sequence of the spacer can vary and determines the position in the target DNA where the guide RNA and the target DNA interact. The DNA targeting segment of the guide RNA can be modified (e.g., by genetic engineering) to hybridize with any desired sequence in the target DNA.

[0212] In some embodiments, the spacer has a length of 10 nucleotides to 30 nucleotides. In some embodiments, the spacer has a length of 13 nucleotides to 25 nucleotides. In some embodiments, the spacer has a length of 15 nucleotides to 23 nucleotides. In some embodiments, the spacer has a length of 18 nucleotides to 22 nucleotides, such as 20 to 22 nucleotides.

[0213] In some embodiments, the percent complementarity between the DNA targeting sequence of the spacer and the protospacer of the target DNA is at least 60% (e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100%) over 20-22 nucleotides.

[0214] In some embodiments, the protospacer is directly adjacent to a suitable PAM sequence at its 3' end, or such a PAM sequence is part of a DNA targeting sequence in its 3' portion.

[0215] The modification of guide RNA can be used to enhance the formation or stabilization of CRISPR-Cas genome editing complexes comprising guide RNA and Cas endonucleases (e.g., B-GEn.16). The modification of guide RNA can also or alternatively be used to enhance the initiation, stability or kinetics of the interaction between the genome editing complex and the target sequence in the genome, for example, can be used to enhance on-target activity. The modification of guide RNA can also or alternatively be used to enhance specificity, such as, compared to the effect of other (off-target) sites, the relative ratio of genome editing at the target site.

[0216] Modifications can also or alternatively be used to increase the stability of the guide RNA, such as by increasing its resistance to degradation by ribonucleases (RNases) present in the cell, thereby extending its half-life in the cell. Modifications that enhance the half-life of the guide RNA are particularly useful in embodiments where a Cas endonuclease such as B-GEn.16 is introduced into the edited cell via an RNA that needs to be translated in order to produce the B-GEn.16 endonuclease, because the half-life of the introduced guide RNA is increased while the RNA encoding the endonuclease can be used to increase the time that the guide RNA and the encoded Cas endonuclease coexist in the cell.

[0217] Donor DNA or donor template

[0218] Site-specific polypeptides, such as DNA endonucleases, can introduce double-strand breaks or single-strand breaks in nucleic acids, such as genomic DNA. Double-strand breaks can stimulate the cell's endogenous DNA repair pathways (such as homology-dependent repair (HDR) or non-homologous end joining or selective non-homologous end joining (A-NHEJ) or microhomology-mediated end joining (MMEJ)). NHEJ can repair the broken target nucleic acid without the need for a homologous template. This sometimes results in small deletions or insertions (indels) of the target nucleic acid at the cleavage site, which can lead to interruptions or changes in gene expression. HDR, also known as homologous recombination (HR), can occur when a homologous repair template or donor is available.

[0219] Homologous donor templates have sequences homologous to sequences flanking the target nucleic acid cleavage site. Sister chromatids are typically used by cells as repair templates. However, for the purpose of genome editing, repair templates are typically provided in the form of exogenous nucleic acids, such as plasmids, duplex oligonucleotides, single-stranded oligonucleotides, double-stranded oligonucleotides, or viral nucleic acids. For exogenous donor templates, additional nucleic acid sequences (such as transgenes) or modifications (such as single or multiple base changes or deletions) are typically introduced between homologous flanking regions so that additional or altered nucleic acid sequences are also incorporated into the target locus. The genetic results caused by MMEJ are similar to NHEJ, with small deletions and insertions occurring at the cleavage site. MMEJ utilizes homologous sequences of several base pairs flanking the cleavage site to drive favorable end-joining DNA repair results. In some embodiments, possible repair results can be predicted based on an analysis of potential microhomologies in the nuclease target region.

[0220] Therefore, in some cases, homologous recombination is used to insert an exogenous polynucleotide sequence into the target nucleic acid cleavage site. Exogenous polynucleotide sequences are referred to herein as donor polynucleotides (or donors or donor sequences or polynucleotide donor templates). In some embodiments, a donor polynucleotide, a part of a donor polynucleotide, a copy of a donor polynucleotide or a part of a copy of a donor polynucleotide is inserted into the target nucleic acid cleavage site. In some embodiments, the donor polynucleotide is an exogenous polynucleotide sequence, i.e., a sequence that is not naturally present in the target nucleic acid cleavage site.

[0221] When the exogenous DNA molecule has sufficient concentration in the nucleus where double-strand breaks occur, the exogenous DNA can be inserted into the double-strand breaks during the NHEJ repair process, thereby permanently adding to the genome. These exogenous DNA molecules are referred to as donor templates in some embodiments. If the donor template contains a coding sequence for one or more system components described herein and related regulatory sequences, such as a promoter, an enhancer, a polyA sequence, and / or a splice acceptor sequence, one or more system components can be expressed from the integrated nucleic acid in the genome, thereby permanently expressing during the life cycle of the cell. In addition, when the cell divides, the integrated nucleic acid of the donor DNA template can be passed to the daughter cells.

[0222] In the presence of a sufficient concentration of donor DNA template, the donor DNA template contains flanking DNA sequences (called homology arms) with homology to the DNA sequences on either side of the double-strand break, and the donor DNA template can be integrated via the HDR approach. The homology arms act as substrates for homologous recombination between the donor template and the sequences on either side of the double-strand break. This can result in error-free insertion of the donor template, wherein the sequences on either side of the double-strand break are unchanged from the sequences in the unmodified genome.

[0223] The donors provided for HDR editing vary significantly, but generally contain the expected sequence with small or large flanking homology arms to allow annealing to genomic DNA. The homology regions flanking the introduced genetic changes can be 30 bp or smaller, or as large as a cassette of several kilobases containing promoters, cDNAs, etc. Single-stranded and double-stranded oligonucleotide donors can be used. These oligonucleotides range in size from less than 100nt to more than many kb, although longer ssDNA can also be generated and used. Double-stranded donors are generally used, including PCR amplicons, plasmids, and mini-circles. In general, AAV vectors have been found to be a very effective means of delivering donor templates, although the packaging limit for a single donor is less than 5kb. Active transcription of the donor increased HDR by 3 times, indicating that the inclusion of a promoter can increase conversion. Conversely, CpG methylation of the donor can reduce gene expression and HDR.

[0224] In some embodiments, donor DNA can be supplied with a nuclease or independently by a variety of different methods, such as by transfection, nanoparticles, microinjection, or viral transduction. In some embodiments, a range of bundling options can be used to increase the availability of HDR donors. Examples include linking the donor to a nuclease, to a DNA binding protein that binds nearby, or to a protein involved in DNA end binding or repair.

[0225] In addition to genome editing via NHEJ or HDR, it is also possible to use both the NHEJ pathway and the HR pathway to insert site-specific genes. Combination approaches may be applicable in certain settings, potentially including intron / exon boundaries. NHEJ is effective for intron junctions, while error-free HDR is more suitable for coding regions.

[0226] Carrier

[0227] On the other hand, provided herein are nucleic acids, gRNAs and / or any nucleic acid or protein molecules necessary for carrying out the embodiments of the present disclosure that comprise codon-optimized polynucleotide sequences encoding B-GEn.16 polypeptides or variants thereof. In some embodiments, such nucleic acids are vectors (e.g., recombinant expression vectors).

[0228] Contemplated expression vectors include, but are not limited to, vectors based on vaccinia virus, poliovirus, adenovirus, adeno-associated virus, SV40, herpes simplex virus, human immunodeficiency virus, retroviruses (e.g., Murine Leukemia Virus, spleen necrosis virus, and vectors derived from retroviruses (e.g., Rous Sarcoma Virus, Harvey Sarcoma Virus, avian leukosis virus, lentivirus, human immunodeficiency virus, myeloproliferative sarcoma virus, and mammary tumor virus)) and other recombinant vectors. Other vectors contemplated for use in eukaryotic target cells include, but are not limited to, vectors pXT1, pSG5, pSVK3, pBPV, pMSG, and pSVLSV40 (Pharmacia). Additional vectors contemplated for use with eukaryotic target cells include, but are not limited to, vectors pCTx-1, pCTx-2, and pCTx-3. Other vectors may be used so long as they are compatible with the host cell.

[0229] In some embodiments, the vector has one or more transcription and / or translation control elements. Depending on the host / vector system used, a number of suitable transcription and translation control elements can be used in the expression vector, including constitutive and inducible promoters, transcription enhancer elements, transcription terminators, etc. In some embodiments, the vector is a self-inactivating vector that inactivates viral sequences or components or other elements of the CRISPR machinery.

[0230] Non-limiting examples of suitable eukaryotic promoters (i.e., promoters that function in eukaryotic cells) include those derived from cytomegalovirus (CMV) immediate early, herpes simplex virus (HSV) thymidine kinase, early and late SV40, retroviral long terminal repeats (LTRs), human elongation factor-1 promoter (EF1), a hybrid construct of the cytomegalovirus (CMV) enhancer fused to the chicken beta-actin promoter (CAG), mouse stem cell virus promoter (MSCV), phosphoglycerate kinase-1 locus promoter (PGK), and mouse metallothionein-I.

[0231] For expression of small RNAs, including guide RNAs for connection to Cas endonucleases, various promoters, such as RNA polymerase III promoters, including, for example, U6 and H1, can be advantageous. Descriptions and parameters for enhancing the use of such promoters are known in the art, and additional information and methods are frequently described; see, e.g., Ma, H. et al., Molecular Therapy-Nucleic Acids 3, e161 (2014) doi: 10.1038 / mtna.2014.12.

[0232] The expression vector may also include a ribosome binding site and a transcription terminator for translation initiation. The expression vector may also include an appropriate sequence for amplification of expression. The expression vector may also include a nucleotide sequence encoding a non-natural tag (such as a histidine tag, a hemagglutinin tag, a green fluorescent protein, etc.), which is fused to a site-specific polypeptide to form a fusion protein.

[0233] In some embodiments, the promoter is an inducible promoter (e.g., heat shock promoter, tetracycline regulated promoter, steroid regulated promoter, metal regulated promoter, estrogen receptor regulated promoter, etc.). In some embodiments, the promoter is a constitutive promoter (e.g., CMV promoter, UBC promoter). In some embodiments, the promoter is a spatially restricted and / or temporally restricted promoter (e.g., tissue-specific promoter, cell type-specific promoter, etc.). In some embodiments, if the gene will be expressed under an endogenous promoter present in the genome after insertion into the genome, the vector does not have a promoter for at least one gene to be expressed in the host cell.

[0234] Modification of nucleic acids and peptides

[0235] In some embodiments, the polynucleotides described herein comprise one or more modifications as further described herein and known in the art, such as can be used to enhance activity, stability or specificity, alter delivery, reduce innate immune response in host cells, further reduce protein size or for other enhancements. In some embodiments, such modifications will produce a B-GEn.16 polypeptide comprising an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% amino acid sequence identity to the SEQ ID NO:2 sequence.

[0236] Codon optimization

[0237] In certain embodiments, the modified polynucleotides used in the CRISPR-B-GEn.16 system described herein, wherein the guide RNA and / or the DNA or RNA comprising the polynucleotide sequence encoding the B-GEn.16 polypeptide or its variant can be modified, as described and illustrated below. Such modified polynucleotides can be used in the CRISPR-B-GEn.16 system to edit any one or more genomic loci. In some embodiments, such modifications in the polynucleotides of the present disclosure are achieved by codon optimization, such as codon optimization based on a specific host cell in which the encoded polypeptide is expressed. It will be appreciated by those skilled in the art that any nucleotide sequence and / or recombinant nucleic acid of the present disclosure can be codon optimized so as to be expressed in any target species. Codon optimization is well known in the art and involves the use of species-specific codon usage tables to modify the codon usage bias of nucleotide sequences. The codon usage table is generated based on sequence analysis of the highest expressed genes of the target species. In a non-limiting example, when the nucleotide sequence is to be expressed in the nucleus, the codon usage table is generated based on sequence analysis of highly expressed nuclear genes of the target species. Modifications of the nucleotide sequence are determined by comparing species-specific codon usage tables to the codons present in the native polynucleotide sequence.

[0238] In some embodiments, the B-GEn.16 polypeptides or variants thereof described herein are expressed by codon-optimized polynucleotide sequences. For example, if the intended target cell is a human cell, a human codon-optimized polynucleotide sequence encoding B-GEn.16 (or a B-GEn.16 variant, such as an enzyme-inactivating variant) will be suitable. As another non-limiting example, if the intended host cell is a mouse cell, a mouse codon-optimized polynucleotide sequence encoding B-GEn.16 (or a B-GEn.16 variant, such as an enzyme-inactivating variant) will be suitable.

[0239] Strategies and methods for codon optimization are known in the art and have been described for various systems, including but not limited to yeast (Outchkourov et al., Protein Expr Purif, 24(1):18-24 (2002)) and Escherichia coli (E. coli) (Feng et al., Biochemistry, 39(50):15399-15409 (2000)). In some embodiments, codon optimization is performed by using Expression optimization technology (ATUM) and expression optimization algorithms recommended by the manufacturer are used. In some embodiments, the polynucleotides of the present disclosure are codon optimized to increase expression in human cells. In some embodiments, the polynucleotides of the present disclosure are codon optimized to increase expression in E. coli cells. In some embodiments, the polynucleotides of the present disclosure are codon optimized to increase expression in insect cells. In some embodiments, the polynucleotides of the present disclosure are codon optimized to increase expression in Sf9 insect cells. In some embodiments, the expression optimization algorithm used in the codon optimization process is defined to avoid putative polyA signals (e.g., AATAAA and ATTAAA) and long (greater than 4) A segments that may cause polymerase slippage.

[0240] As is well known in the art, codon optimization of a nucleotide sequence results in a nucleotide sequence having less than 100% identity (e.g., less than 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) with a native nucleotide sequence, but the polypeptide encoded thereby is still functionally identical to the polypeptide encoded by the original native nucleotide sequence. Therefore, in representative embodiments of the present disclosure, the nucleotide sequence and / or recombinant nucleic acid of the present disclosure may be codon optimized to be expressed in a specific species of interest.

[0241] In some embodiments, the codon-optimized polynucleotide sequence has at least 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.2%, 99.5%, 99.8%, 99.9% or 100% sequence identity with SEQ ID NO: 1. In some embodiments, the polynucleotides of the present disclosure are codon-optimized to increase the expression of the encoded B-GEn.16 polypeptide in the target cell. In some embodiments, the polynucleotides of the present disclosure are codon-optimized to increase expression in human cells. Typically, the polynucleotides of the present disclosure are codon-optimized to increase expression in any human cell. In some embodiments, the polynucleotides of the present disclosure are codon-optimized to increase expression in Escherichia coli cells. In some embodiments, the polynucleotides of the present disclosure are codon-optimized to increase expression in insect cells. Typically, the polynucleotides of the present disclosure are codon-optimized to increase expression in any insect cell. In some embodiments, the polynucleotides of the present disclosure are codon optimized for increased expression in the Sf9 insect cell expression system.

[0242] The polyadenylation signal may also be selected to optimize expression in the intended host.

[0243] Other Modifications

[0244] Modifications may also or alternatively be used to reduce the likelihood or extent that the RNA introduced into the cell elicits an innate immune response. Such responses, as described below and in the art, have been well characterized in the context of RNA interference (RNAi), including small interfering RNA (siRNA), which are often associated with shortened RNA half-life and / or the stimulation of cytokines or other factors associated with an immune response.

[0245] The RNA encoding the endonuclease (e.g., B-GEn.16) introduced into the cell may also be subjected to one or more types of modifications, including, but not limited to, modifications that enhance RNA stability (e.g., by reducing its degradation by RNases present in the cell), modifications that enhance translation of the resulting product (e.g., endonucleases), and / or modifications that reduce the likelihood or extent that the RNA introduced into the cell triggers an innate immune response. Combinations such as the above and other modifications may also be used. In the case of CRISPR-B-GEn.16, for example, one or more types of modifications (including those exemplified above) may be performed on the guide RNA, and / or one or more types of modifications (including those exemplified above) may be performed on the RNA encoding the B-GEn.16 endonuclease.

[0246] By way of example, the guide RNA or other smaller RNA used in the CRISPR-B-GEn.16 system can be easily synthesized by chemical means, so that many modifications can be easily incorporated, as described below and in the art. Although chemical synthesis procedures continue to expand, due to the significant increase in polynucleotide length, more than 100 nucleotides, it often becomes more challenging to purify these RNAs using procedures such as high performance liquid chromatography (HPLC, which avoids the use of gels such as PAGE). One method for producing longer chemically modified RNAs is to produce two or more molecules linked together. Longer RNAs, such as RNA encoding the B-GEn.16 endonuclease, are more easily produced enzymatically. Although there are fewer types of modifications that can be used for enzymatically produced RNAs, there are still some modifications that can be used to enhance stability, reduce the likelihood or extent of innate immune responses, and / or enhance other properties, as further described below and in the art; and new modifications are also being developed. By way of illustration of various types of modifications, particularly those frequently used in smaller chemically synthesized RNAs, modifications can include one or more nucleotides modified at the 2' position of the sugar, in some embodiments 2'-O-alkyl, 2'-O-alkyl-O-alkyl or 2'-fluoro modified nucleotides. In some embodiments, RNA modifications include 2'-fluoro, 2'-amino and 2'-O-methyl modifications on pyrimidine ribose, base residues or anti-bases on the 3' end of the RNA. Such modifications are often incorporated into oligonucleotides, and these oligonucleotides have been shown to have higher Tm (e.g., higher target binding affinity) than 2'-deoxy oligonucleotides for a given target.

[0247] Many nucleotide and nucleoside modifications have been shown to render the oligonucleotides incorporating them more resistant to nuclease digestion than natural oligonucleotides; these modified oligonucleotides survive intact longer than unmodified oligonucleotides. Specific examples of modified oligonucleotides include those comprising modified backbones, for example, phosphosulfate, phosphotriester, methylphosphonate, short chain alkyl or cycloalkyl sugar bonds or short chain heteroatom or heterocyclic sugar bonds. Some oligonucleotides are oligonucleotides with phosphorus-based backbones and oligonucleotides with heteroatom backbones, particularly CH2-NH-O-CH2, CH,-N(CH3)-O-CH2 (called methylene(methylimino) or MMI backbone), CH2-ON(CH3)-CH2, CH2-N(CH3)-N(CH3)-CH2 and ON(CH3)-CH2-CH2; amide backbones [see De Mesmaeker et al., Ace. Chem. Res., 28:366-374 (1995)]; morpholine backbone structures (see Summerton and Weller, U.S. Pat. No. 5,034,506); peptide nucleic acid (PNA) backbones (in which the phosphodiester backbone of the oligonucleotide is replaced by a polyamide backbone, and the nucleotides are directly or indirectly linked to the aza nitrogen atoms of the polyamide backbone, see Nielsen et al., Science 1991, 254, 1497). Phosphorus-containing linkages include, but are not limited to, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkylphosphonates, including 3' alkylphosphonates and chiral phosphonates, phosphonates, phosphoamidates, including 3'-phosphoramidates and aminoalkylphosphonates, thiophosphoramidates, thioalkylphosphonates, thioalkylphosphotriesters, and borophosphates having normal 3'-5' linkages, 2'-5' linked analogs of these, as well as analogs with reversed polarity wherein adjacent pairs of nucleoside units are linked 3'-5' to 5'-3' or 2'-5' to 5'-2'; see U.S. Pat. Nos. 3,687,808; 4,469,808. and 5,625,050.

[0248] Morpholinyl-based oligomeric compounds are described in Braasch and Corey, Biochemistry, 41(14):4503-4510 (2002); Genesis, Volume 30, Issue 3, (2001); Heasman, Dev. Biol., 243:209-214 (2002); Nasevicius et al., Nat. Genet., 26:216-220 (2000); Lacenra etc., Proc. Nat / . Acad. Sci., 97:9591-9596 (2000); and U.S. Pat. No. 5,034,506, issued Jul. 23, 1991. Cyclohexenyl nucleic acid oligonucleotide mimetics are described in Wang et al., J. Am. Chem. Soc., 122:8595-8602 (2000).

[0249] Modified oligonucleotide backbones that do not include phosphorus atoms have backbones formed by short-chain alkyl or cycloalkyl nucleoside bonds, mixed heteroatoms and alkyl or cycloalkyl nucleoside bonds, or one or more short-chain heteroatoms or heterocyclic nucleoside bonds. These include those backbones with morpholine bonds (partially formed by the sugar portion of the nucleoside); siloxane backbones; sulfide, sulfoxide, and sulfone backbones; formacetyl and thioformacetyl backbones; methyleneformacetyl and thioformacetyl backbones; olefin-containing backbones; aminosulfonate backbones; methyleneimine and methylenehydrazine backbones; sulfonate and sulfonamide backbones; amide backbones; and other backbones with mixed N, O, S, and CH2 component parts; see U.S. Patent Nos. 5,034,506; 5,166,315; 5,185,444; 5,2 14,134; 5,216,141; 5,235,033; 5,264,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967; 5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,677,437; and 5,677,439, each of which is incorporated herein by reference.

[0250] One or more substituted sugar groups may also be included, such as one of the following at the 2' position: OH, SH, SCH3, F, OCN, OCH3, OCH3O(CH2) n CH3, O(CH2) n NH2 or O(CH2) n CH3, wherein n is 1 to 10; C1-C 10 Lower alkyl, alkoxyalkoxy, substituted lower alkyl, alkylaryl or arylalkyl; Cl; Br; CN; CF3; OCF3; O-, S- or N-alkyl; O-, S- or N-alkenyl; SOCH3; SO2CH3; ONO2; NO2; N3; NH2; heterocycloalkyl; heterocycloalkylaryl; aminoalkylamino; polyalkylamino; substituted silyl; RNA cleavage group; reporter group; intercalator; groups for improving the pharmacokinetic properties of oligonucleotides; or groups for improving the pharmacodynamic properties of oligonucleotides and other substituents with similar properties. In some embodiments, the modification includes 2'-methoxyethoxy (2'-O-CH2CH2OCH3, also known as 2'-O-(2-methoxyethyl)) (Martinet a / , Helv.Chim.Acta, 1995, 78, 486). Other modifications include 2'-methoxy (2'-O-CH3), 2'-propoxy (2'-OCH2CH2CH3) and 2'-fluoro (2'-F). Similar modifications can also be made at other positions of the oligonucleotide, particularly at the 3' position of the sugar on the 3' terminal nucleotide and the 5' position of the 5' terminal nucleotide. Oligonucleotides can also have sugar mimics, such as cyclobutyls that replace the pentofuranosyl group. In some embodiments, the sugar and internucleoside bonds of the nucleotide units, such as the backbone, are replaced by new groups. The retained base units are used for hybridization with suitable nucleic acid target compounds. One such oligomeric compound (an oligonucleotide mimic that has been shown to have excellent hybridization properties) is referred to as peptide nucleic acid (PNA). In PNA compounds, the sugar backbone of the oligonucleotide is replaced by a backbone containing amides, such as an aminoethylglycine backbone. The core base is retained and is directly or indirectly attached to the aza nitrogen atom of the amide portion of the backbone. Representative U.S. patents that teach the preparation of PNA compounds include, but are not limited to, U.S. Patent Nos. 5,539,082; 5,714,331; and 5,719,262. Further teachings of PNA compounds are described in Nielsen et al., Science, 254: 1497-1500 (1991).

[0251] The guide RNA may also additionally or alternatively comprise modifications or substitutions of nucleobases (commonly referred to in the art as "bases"). As used herein, "unmodified" or "natural" nucleobases include adenine (A), guanine (G), thymine (T), cytosine (C) and uracil (U). Modified nucleobases include nucleobases that are only rarely or transiently present in natural nucleic acids, such as hypoxanthine, 6-methyladenine, 5-Me pyrimidines, particularly 5-methylcytosine (5-methylcytosine) (also known as 5-methyl-2'deoxycytosine, commonly referred to in the art as 5-Me-C), 5-hydroxymethylcytosine (5-hydroxymethylcytosine, HMC), glycosyl HMC and gentiobiosyl HMC (gentobiosyl HMC), and synthetic nucleobases such as 2-aminoadenine, 2-(methylamino)adenine, 2-(imidazolidinyl)adenine, 2-(aminoalkyl)adenine or other heterosubstituted alkyladenines, 2-thiouracil, 2-thiothymine, 5-bromopyrimidine, 5-hydroxymethyluracil, 8-azaguanine, 7-deazaguanine, N6 (6-aminohexyl)adenine and 2,6-diaminopurine. Kornberg, A, DNA Replication, WH Freeman & Co., San Francisco, pp75-77 (1980); Gebeyehu et al., Nucl. Acids Res. 15:4513 (1997). "Universal" bases known in the art, such as inosine, may also be included. 5-Me-C substitution has been shown to increase nucleic acid duplex stability by 0.6-1.2 degrees Celsius (Sanghvi, YS, in Crooke, ST and Lebleu, B., eds., Antisense Research and Applications, CRC Press, Boca Raton, 1993, pp. 276-278) and is an embodiment of base substitution.

[0252] Modified nucleobases include other synthetic and natural nucleobases such as 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyluracil and cytosine, 6-azouracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-sulfanyl, 8-hydroxy and other α-substituted adenines and guanines, 5-halo, especially 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylquinone and 7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine, and 3-deazaguanine and 3-deazaadenine

[0253] Other useful nucleobases include those disclosed in U.S. Pat. No. 3,687,808, "The Concise Encyclopedia of Polymer Science And Engineering", pages 858-859, Kroschwitz, Jl, ed. John Wiley & Sons, 1990, Englisch et al., Angewandte Chemie, International Edition, 1991, 30, page 613, and Sanghvi, YS, Chapter 15, Antisense Research and Applications, pages 289-302, Crooke, ST and Lebleu, B.ea., CRC Press, 1993. Certain of these nucleobases are particularly useful for increasing the binding affinity of the oligomeric compounds of the present disclosure. These include 5-substituted pyrimidines, 6-azapyrimidines, and N-2, N-6, and -O-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil, and 5-propynylcytosine. 5-Methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2°C (Sanghvi, YS, Crooke, ST and Lebleu, B., eds, "Antisense Research and Applications", CRC Press, Boca Raton, 1993, pp. 276-278), and are embodiments of base substitutions, even more particularly when combined with 2'-O-methoxyethyl sugar modifications. Modified nucleobases are described in U.S. Patent Nos. 3,687,808 and 4,845,205; 5,130,302; 5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,459,255; 5,484,908; 5,502,177; 5,525,711; 5,552,540; 5,587,469; 5,596,091; 5,614,617; 5,681,941; 5,750,692; 5,763,588; 5,830,653; 6,005,096; and U.S. Patent Application Publication 20030158403.

[0254] All positions in a given oligonucleotide need not be uniformly modified, and, in fact, more than one of the above modifications may be incorporated in a single oligonucleotide, or even within a single nucleoside within an oligonucleotide.

[0255] In some embodiments, the guide RNA and / or the mRNA encoding the endonuclease (e.g., B-GEn.16 of the present disclosure) is capped using any of the current capping methods, such as mCAP, ARCA, or enzymatic capping methods, to create a viable mRNA construct that maintains biological activity and avoids self / non-self intracellular responses. In some embodiments, the guide RNA and / or the mRNA encoding the endonuclease (e.g., B-GEn.16 of the present disclosure) is capped using CleanCap TM (TriLink) co-transcriptional capping method was used for capping.

[0256] In some embodiments, the guide RNA and / or the mRNA encoding the endonuclease of the present disclosure comprises a moiety selected from pseudouracil, N 1 In some embodiments, one or more N 1 -methyl pseudouracil is incorporated into the guide RNA and / or mRNA encoding the endonuclease of the present disclosure to provide enhanced RNA stability and / or protein expression and reduced immunogenicity in animal cells, e.g., mammalian cells (e.g., humans and mice). 1 -methylpseudouracil modifications are incorporated in combination with one or more 5-methylcytosines.

[0257] In some embodiments, the guide RNA and / or mRNA (or DNA) encoding an endonuclease such as B-GEn.16 is chemically linked to one or more moieties or conjugates that enhance the activity, cellular distribution, or cellular uptake of the oligonucleotide. These moieties include, but are not limited to, lipid moieties, such as cholesterol moieties [Letsinger et al., Proc. Nat / . Acad. Sci. USA, 86:6553-6556 (1989)]; cholic acid [Manoharan et al., Bioorg. Med. Chem. Let., 4:1053-1060 (1994)]; thioethers, such as hexyl-S-tritylthiol [Manoharan et al., Ann. N. Y. Acad. Sci., 660:306-309 (1992) and Manoharan et al., Bioorg. Med. Chem. Let., 3.·2765-2770 (1993)]; thiocholesterol [Oberhauser et al., Nucl. Acids Res., 20:533-538 (1992)]; aliphatic chains, such as dodecandiol or undecyl residues [Kabanov et al., FEBS Lett., 259:327-330 (1990) and Svinarchuk et al., Biochimie, 75:49-54 (1993)]; phospholipids, such as di-hexadecyl-rac-glycerol or triethylammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonic acid triethylammonium [Manoharan et al., Tetrahedron Lett., 36:3651-3654 (1995) and Shea et al., Nucl. Acids Res., 18:3777-3783 (1990)]; polyamine or polyethylene glycol chains [Mancharan etc., Nucleosides & Nucleotides, 14:969-973 (1995)]; adamantane acetic acid [Manoharan et al., Tetrahedron Lett., 36:3651-3654 (1995)]; a palmityl moiety [(Mishra etc., Biochim. Biophys. Acta, 1264:229-237 (1995)]; or an octadecylamine or hexylamino-carbonyl-t-oxycholesterol moiety [Crooke et al., J. Pharmacol. Exp. Ther., 277:923-937 (1996)].See also U.S. Patent Nos. 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313; 5,545,730; 5,552,538; 5,578,717; 5,580,731; 5,580,731; 5,591,584; 5,109,124; 5,118,802; 5,138,045 ; 5,414,077; 5,486,603; 5,512,439; 5,578,718; 5,608,046; 4,587,044; 4,605,735; 4,667,025; 4,762,779; 4,789,737; 4,824,941; 4,835,263; 4,876,335; 4,904,582; 4,958,013 ; 5,082,830; 5,112,963; 5,214,136; 5,082,830; 5,112,963; 5,214,136; 5,245,022; 5,254,469; 5,258,506; 5,262,536; 5,272,250; 5,292,873; 5,317,098; 5,371,241; 5,391,723 ; 5,416,203; 5,451,463; 5,510,475; 5,512,667; 5,514,785; 5,565,552; 5,567,810; 5,574,142; 5,585,481; 5,587,371; 5,595,726; 5,597,696; 5,599,923; 5,599,928 and 5,688,941.

[0258] Sugars and other moieties can be used to target proteins and nucleotide-containing complexes (such as cationic polysomes and liposomes) to specific sites. For example, hepatocyte directed metastasis can be mediated by asialoglycoprotein receptors (ASGPR); see, e.g., Hu et al., Protein Pept Lett. 21(10): 1025-30 (2014). Other systems known in the art and continuously developed can be used to target the biomolecules and / or their complexes used in this application to specific target cells of interest.

[0259] These targeting moieties or conjugates may include conjugated groups covalently linked to functional groups such as primary or secondary hydroxyl groups. Suitable conjugated groups include intercalators, reporter molecules, polyamines, polyamides, polyethylene glycols, polyethers, groups that enhance the pharmacodynamic properties of oligomers, and groups that enhance the pharmacokinetic properties of oligomers. Typical conjugated groups include cholesterol, lipids, phospholipids, biotin, phenazine, folic acid, phenanthridine, anthraquinone, acridine, fluorescein, rhodamine, coumarin, and dyes. Groups that can enhance pharmacodynamic properties include groups that improve uptake, enhance degradation resistance, and / or strengthen specific hybridization with target nucleic acid sequences. Groups that can enhance pharmacokinetic properties include groups that improve the uptake, distribution, metabolism, or secretion of compounds of the present disclosure. Representative conjugated groups are disclosed in International Patent Application No. PCT / US92 / 09196, filed on October 23, 1992, and U.S. Patent No. 6,287,860, which are incorporated herein by reference. Conjugated moieties include, but are not limited to, lipid groups such as cholesterol moieties, cholic acid, thioethers such as hexyl-5-tritylthiol, mercaptocholesterol, aliphatic chains such as dodecandiol or undecyl residues, phospholipids such as dihexadecyl-rac-glycerol or triethylammonium 1,2-bis-O-hexadecyl-rac-glycero-3-H-phosphonate, polyamines or polyethylene glycol chains, or adamantaneacetic acid, palmityl moieties or octadecylamine or hexylamino-carbonyl-oxycholesterol moieties.See U.S. Patent Nos. 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313; 5,545,730; 5,552,538; 5,578,717; 5,580,731; 5,580,731; 5,591,584; 5,109,124; 5,118,802; 5,138,045; 5,414,077; 5,486,603; 5,512,439; 5,578,718; 5,608,046; 4,587,044; 4,605,735; 4,667,025; 4,762,779; 4,789,737; 4,824,941; 4,835,263; 4,876,335; 4,904,582; 4,958,013; 5,082,830; 5,112,963; 5,214,136; 5,082,830; 5,112,963; 5,214,136; 5,245,022; 5,254,469; 5,258,506; 5,262,536; 5,272,250; 5,292,873; 5,317,098; 5,371,241, 5,391,723; and 5,688,941.

[0260] Longer polynucleotides that are not well suited for chemical synthesis and are typically produced by enzymatic synthesis can also be modified in various ways. Such modifications may include, for example, the introduction of certain nucleotide analogs, the inclusion of specific sequences or other parts at the 5' or 3' end of the molecule, and other modifications. By way of example, the mRNA encoding B-GEn.16 is about 4 kb in length and can be synthesized by in vitro transcription. Modification of mRNA can be used, for example, to increase its translation or stability (e.g., by increasing its resistance to degradation in cells), or to reduce the tendency of RNA to trigger an innate immune response, which is typically observed in cells after the introduction of exogenous RNA, particularly longer RNA, such as RNA encoding B-GEn.16.

[0261] Many such modifications have been described in the art, such as poly A tails, 5' cap analogs (such as AntiReverse Cap Analog (ARCA) or m7G(5')ppp(5')G (mCAP)), modified 5' or 3' untranslated regions (UTRs), use of modified bases (such as pseudo-UTP, 2-thio-UTP, 5-methylcytidine-5'-triphosphate (5-methyl-CTP) or N6-methyl-ATP), or treatment with phosphatase to remove the 5' terminal phosphate. These and other modification methods are known in the art, and new RNA modification methods are constantly being developed.

[0262] There are many commercial suppliers of modified RNA, including, for example, TriLink Biotech, Axolabs, Bio-Synthesis Inc., Dharmacon, and many others. As described by TriLink, for example, 5-methyl-CTP can be used to impart desired properties, such as increased nuclease stability, increased translation, or reduced interaction of innate immune receptors with in vitro transcribed RNA. 5'-methylcytosine-5'-triphosphate (5-methyl-CTP), N6-methyl-ATP, as well as pseudo-UTP and 2-thio-UTP have also been shown to reduce innate immune stimulation in culture and in vivo while increasing translational capacity, as described in the publications by Konmann et al. and Warren et al. mentioned below.

[0263] It has been shown that chemical modifications can be used to deliver mRNA in vivo to achieve better therapeutic effects; see, for example, Kormann et al., Nature Biotechnology 29, 154-157 (2011). Such modifications can be used, for example, to improve the stability of RNA molecules and / or reduce their immunogenicity. By using chemical modifications such as pseudo-U, N6-methyl-A, 2-thio-U and 5-methyl-C, it has been found that as long as 2-thio-U and 5-methyl-C replace a quarter of the uridine and cytidine residues, respectively, the recognition of mRNA mediated by toll-like receptors (TLRs) in mice can be significantly reduced. Therefore, by reducing the activation of the innate immune system, these modifications can be used to effectively improve the stability and lifespan of mRNA in vivo; see, for example, Konmann et al., supra.

[0264] It has also been shown that repeated administration of modified messenger RNA designed to bypass the innate antiviral response can reprogram differentiated human cells into pluripotent cells. See, for example, Warren et al., Cell Stem Cell, 7(5):618-30 (2010). This modified mRNA as a primary reprogramming protein is an effective means of reprogramming a variety of human cell types. Such cells are called induced pluripotent stem cells (iPSCs). It has also been found that enzymatically synthesized RNA incorporates 5-methyl-CTP, pseudo-UTP, and anti-reverse cap analogs (ARCA) that can be used to effectively escape the antiviral response of cells; see, for example, Warren et al., supra. Other modifications to polynucleotides described in the art include, for example, the use of poly A tails, the addition of 5' end cap analogs (such as m7G(5')ppp(5')G (mCAP)), modifications of the 5' or 3' untranslated regions (UTRs), or treatment with phosphatases to remove 5' end phosphates - and new methods are constantly being developed.

[0265] Many compositions and techniques suitable for generating modified RNA for use herein have been developed, which are related to modifications of RNA interference (RNAi), including small interfering RNA (siRNA). siRNAs face particular challenges in vivo because their effects on gene silencing through mRNA interference are often transient, which may require repeated administration. In addition, siRNAs are double-stranded RNA (dsRNA), and mammalian cells have immune responses that have evolved to detect and neutralize dsRNA, which are often a byproduct of viral infection. Thus, there are some mammalian enzymes, such as PKR (dsRNA responsive kinase) and potentially retinoic acid-inducible gene I (RIG-I), which can mediate cellular responses to dsRNA, and there are also toll-like receptors (such as TLR3, TLR7 and TLR8) that can trigger cytokine induction of these molecules; see, for example, Angart et al., Pharmaceuticals (Basel) 6(4):440-468 (2013); Kanasty et al., Molecular Therapy 20(3):513-524 (2012); Burnett et al., Biotechnol J. 6(9):1130-46 (2011); Judge and Maclachlan, Hum Gene Ther 19(2):111-24 (2008) for review; and references cited therein.

[0266] A large number of modification methods have been developed and applied to improve RNA stability, reduce innate immune responses and / or achieve other benefits that may be useful in introducing polynucleotides into human cells as described herein; see, for example, Whitehead KA et al., Annual Review of Chemical and Biomolecular Engineering, 2:77-96 (2011); Gaglione and Messere, Mini Rev Med Chem, 10(7):578-95 (2010); Chernolovskaya et al, Curr Opin Mol Ther., 12(2):158-67 (2010); Deleavey et al, Curr Protoc Nucleic Acid Chem Chapter 16:Unit 16.3 (2009); Behlke, Oligonucleotides 18(4):305-19 (2008); Fucini et al, Nucleic Acid Ther 22(3):205-210 (2012); Bremsen et al, Curr Opin Mol Ther, 12(2):158-67 (2010); Deleavey et al, Curr Protoc Nucleic Acid Chem Chapter 16:Unit 16.3 (2009); Behlke, Oligonucleotides 18(4):305-19 (2008); Fucini et al, Nucleic Acid Ther 22(3):205-210 (2012); et al., Front Genet 3:154 (2012).

[0267] As mentioned above, there are many commercial suppliers of modified RNA, many of which specialize in modifications intended to improve siRNA effectiveness. According to the various findings reported in the literature, a variety of methods are provided. For example, Dharmacon notes that replacing non-bridging oxygen with sulfur (phosphorothioate (phosphorothioate, PS)) has been widely used to improve the nuclease resistance of siRNA, as reported by Kale, Nature Reviews Drug Discovery 11:125-140 (2012). The modification of ribose 2' position has been reported to improve the nuclease resistance of phosphate bonds between nucleotides, while increasing duplex stability (Tm), which also shows that immune activation protection can be provided. Moderate PS backbone modifications in combination with small, well-tolerated 2'-substitutions (2'-O-, 2'-fluoro, 2'-hydrogen) have been associated with highly stable siRNA applications in vivo, as reported by Soutschek et al. Nature 432: 173-178 (2004); and 2'-O-methyl modifications have been reported to be effective in improving stability, as reported by Volkov, Oligonucleotides 19: 191-202 (2009). With regard to reducing the induction of innate immune responses, it has been reported that modifying specific sequences with 2'-O-methyl, 2'-fluoro, 2'-hydrogen can reduce TLR7 / TLR8 interactions while generally maintaining silencing activity; see, e.g., Judge et al., Mol. Ther. 13: 494-505 (2006); and Cekaite et al., J. Mol. Biol. 365: 90-108 (2007). Additional modifications, such as 2-thiouracil, pseudouracil, 5-methylcytosine, 5-methyluracil, and N6-methyladenosine have also been shown to minimize TLR3-, TLR7-, and TLR8-mediated immune effects; see, e.g., Kariko et al., Immunity 23:165-175 (2005).

[0268] As is also known in the art and commercially available, a number of conjugates are applicable to the polynucleotides used herein, such as RNA, which can enhance their delivery and / or uptake by cells, including, for example, cholesterol, tocopherol and folic acid, lipids, peptides, polymers, linkers and aptamers; see, e.g., the review by Winkler, Ther. Deliv. 4:791-809 (2013), and references cited therein.

[0269] Additional Sequences

[0270] In certain embodiments, the RNA includes at least one additional segment at the 5' end or the 3' end. For example, suitable additional segments may include a 5' cap (such as a 7-methylguanylate cap (m7G)); a 3' polyadenylation tail (such as a 3' poly (A) tail); a riboswitch sequence (such as a sequence that allows for regulation of stability and / or regulation of accessibility by proteins and protein complexes); a sequence that forms a dsRNA duplex (such as a hairpin); a sequence that targets RNA to a subcellular location (such as a nucleus, mitochondria, chloroplasts, etc.); a modification or sequence that provides a tracking function (such as direct conjugation with a fluorescent molecule, conjugation with a moiety that promotes fluorescence detection, a sequence that allows fluorescence detection, etc.); a modification or sequence that provides a protein binding site (such as a protein that acts on DNA, including a transcriptional activator, a transcriptional inhibitor, a DNA methyltransferase, a DNA demethylase, a histone acetyltransferase, a histone deacetylase, etc.); a modification or sequence that provides increased, decreased and / or controllable stability; and combinations thereof.

[0271] Stability Control Sequence

[0272] Stability control sequences affect the stability of RNA (such as guide RNA). Non-limiting examples of suitable stability control sequences are transcription terminator segments (such as transcription termination sequences). The total length of the transcription terminator segment of guide RNA can be from 10 nucleotides to 100 nucleotides, such as from 10 nucleotides (nt) to 20nt, from 20nt to 30nt, from 30nt to 40nt, from 40nt to 50nt, from 50nt to 60nt, from 60nt to 70nt, from 70nt to 80nt, from 80nt to 90nt, or from 90nt to 100nt. For example, the length of the transcription terminator segment can be 15 nucleotides (nt) to 80nt, from 15nt to 50nt, from 15nt to 40nt, from 15nt to 30nt or from 15nt to 25nt.

[0273] In some embodiments, the transcription termination sequence is a sequence that functions in eukaryotic cells. In some embodiments, the transcription termination sequence is a sequence that functions in prokaryotic cells.

[0274] Nucleotide sequences that may be included in a stability control sequence (such as a transcription termination segment, or any segment of a guide RNA to provide increased stability) include, for example, a Rho-independent trp termination site.

[0275] Simulants

[0276] In some embodiments, nucleic acid can be a nucleic acid mimic.When the term "simulation" is applied to polynucleotides, it is intended to include polynucleotides in which only the furanose ring or the furanose ring and the internucleotide connection are replaced by non-furanose groups, and only the replacement of the furanose ring is also referred to as a sugar substitute in the art. The heterocyclic base part or the modified heterocyclic base part keeps hybridizing with the appropriate target nucleic acid. Such a nucleic acid, a polynucleotide mimic that has been proven to have excellent hybridization characteristics, is referred to as peptide nucleic acid (PNA). In PNA, the sugar backbone of polynucleotides is replaced by an amide-containing backbone (especially an aminoethylglycine backbone). Nucleotide is retained and is directly or indirectly combined with the nitrogen-nitrogen atom of the backbone amide part.

[0277] One polynucleotide mimetic that has been reported to have excellent hybridization properties is peptide nucleic acid (PNA). The backbone of the PNA compound is two or more linked aminoethylglycine units, which makes PNA an amide-containing backbone. The heterocyclic moiety is directly or indirectly bound to the aza nitrogen atom of the backbone amide portion. Representative U.S. patents describing the preparation of PNA compounds include, but are not limited to: U.S. Patent Nos. 5,539,082; 5,714,331; and 5,719,262.

[0278] Another class of polynucleotide mimics that have been studied is based on the morpholino unit (morpholino nucleic acid) connected, and its heterocyclic base is connected to the morpholino ring. It is reported that there are many linking groups that can be connected to the morpholino monomer unit in the morpholino nucleic acid. A class of linking groups has been selected to obtain non-ionic oligomeric compounds. Non-ionic oligomeric compounds based on morpholino are unlikely to produce undesirable interactions with cell proteins. Morpholino-based polynucleotides are non-ionic mimics of oligonucleotides, which are unlikely to form undesirable interactions with cell proteins (Dwaine A. Braasch and David R. Corey, Biochemistry, 2002, 41 (14), 45034510). Morpholino-based polynucleotides are disclosed in U.S. Patent No. 5,034,506. Various compounds within the morpholino class of polynucleotides have been prepared, which have various different linking groups connecting monomer subunits.

[0279] Another class of polynucleotide mimics is called cyclohexenyl nucleic acid (GeNA). The furanose ring usually present in DNA / RNA molecules is replaced by a cyclohexenyl ring. According to classical phosphoramidite chemistry, phosphoramidite monomers protected by GeNA DMT have been prepared and used for the synthesis of oligomeric compounds. Fully modified GeNA oligomeric compounds and oligonucleotides modified by GeNA at specific positions have been prepared and studied (see Wang et al., J.Am.Chem.Soc., 2000, 122, 85958602). Generally, the inclusion of GeNA monomers in DNA chains increases the stability of DNA / RNA hybridization. GeNA oligomeric adenosine forms a complex with RNA and DNA, which has a stability similar to that of natural complexes. NMR and circular dichroism spectra show that the inclusion of GeNA structure in the study of natural nucleic acid structure is easy to conformational adaptation.

[0280] Further modifications include locked nucleic acids (LNAs), in which the 2'-hydroxyl group is linked to the 4' carbon atom of the sugar ring to form a 2'-C, 4'-C-methoxy bond, thereby forming a bicyclic sugar moiety. The connection can be a methylene (-CH2-) group bridging the 2' oxygen atom and the 4' carbon atom, where n is 1 or 2 (Singh et al., Chem. Commun., 1998, 4, 455-456). LNA and LNA analogs exhibit very high duplex thermal stability (Tm = +3 to +10°C) with complementary DNA and RNA, stability to 3'-exonuclease degradation and good solubility. Potential and non-toxic antisense oligonucleotides containing LNAs have been described (Wahlestedt et al., Proc. Natl. Acad. Sci. USA, 2000, 97, 5633-5638).

[0281] The synthesis and preparation of the LNA monomers adenine, cytosine, guanine, 5-methylcytosine, thymine and uracil, as well as their oligomerization and nucleic acid recognition properties have been described (Koshkin et al., Tetrahedron, 1998, 54, 3607-3630). LNAs and their preparation are also described in WO 98 / 39352 and WO 99 / 14226.

[0282] Modified sugar moiety

[0283] The nucleic acid may also include one or more substituted sugar moieties. Suitable polynucleotides include sugar substituents selected from: OH; F; O-, S- or N-alkyl; O-, S- or N-alkenyl; O-, S- or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl, alkenyl and alkynyl groups may be substituted or unsubstituted C1 to C10 alkyl or C2 to C10 alkenyl and alkynyl groups. Particularly suitable is O((CH2) n O) m CH3, O(CH2) n OCH3, O(CH2) n NH2, O(CH2)CH3, O(CH2) n ONH2 and O(CH2) n ON((CH2) n CH3)2, wherein n and m range from 1 to about 10. Other suitable polynucleotides contain sugar substituents selected from: C1 to C 10 Lower alkyl, substituted lower alkyl, alkenyl, alkynyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH3, OCN, Cl, Br, CN, CF3, OCF3, SOCH3, SO2CH3, ONO2, NO2, N3, NH2, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, RNA cleavage group, reporter group, intercalator, group for improving pharmacokinetic properties of oligonucleotide, or group for improving pharmacodynamic properties of oligonucleotide and other substituents with similar properties. Suitable modifications include 2'-methoxyethoxy (2'-O-CH2-CH2OCH3, also known as 2'-O-(2-methoxyethyl) or 2'-MOE) (Martin et al., Hely. Chim. Acta, 1995, 78, 486-504), for example, alkoxyalkoxy groups. Other suitable modifications include 2'-dimethylaminooxyethoxy, such as O(CH2)2ON(CH3)2 groups (2'-DMAOE), as described in the Examples below, and 2'-dimethylaminoethoxyethoxy (also known in the art as 2'-O-dimethyl-amino-ethoxy-ethyl or 2'-DMAEOE), for example 2'-O-CH2-O-CH2-N(CH3)2.

[0284] Other suitable sugar substituents include methoxy (-O-CH3), aminopropoxy (-O-CH2CH2CH2NH2), allyl (-CH2-CH=CH2), -O-allyl (-O-CH2-CH=CH2) and fluorine (F). The 2'-sugar substituent can be located at the arabinose (upper) position or the ribose (lower) position. A suitable 2'-arabinose modification is 2'-F. Similar modifications can also be made at other positions of the oligomeric compound, particularly the sugar at the 3' position on the 3' terminal nucleoside or at the 5' position of the 2'-5' linked oligonucleotide and the 5' terminal nucleotide. The oligomeric compound can also have a sugar mimetic, such as a cyclobutyl moiety replacing the pentofuranosyl sugar.

[0285] Base modification and substitution

[0286] Nucleic acids may also contain modifications or substitutions of nucleobases (often referred to in the art as "bases"). As used herein, "unmodified" or "natural" nucleobases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C), and uracil (U). Modified nucleobases include other synthetic and natural nucleobases, such as 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine (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-halouracil and cytosine, 5-propynyl (-C=C -CH3) uracil and cytosine and other alkynyl derivatives of pyrimidine bases, 6-azouracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-sulfanyl, 8-hydroxy and other 8-substituted adenines and guanines, 5-halo, especially 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 2-F-adenine, 2-aminoadenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine and 3-deazaguanine and 3-deazaadenine. Further modified nucleobases include tricyclic pyrimidines, such as phenoxazine cytidine (1H-pyrimido(5,4-b)(1,4)benzoxazin-2(3H)-one), phenothiazine pyrimidine (1H-pyrimido(5,4-b)(1,4)benzothiazin-2(3H)-one), G-clamps, such as substituted phenoxazine pyrimidines (such as 9-(2-aminoethoxy)-H-pyrimido(5,4-(b)(1,4)benzoxazin-2(3H)-one), carbazole cytidine (2H-pyrimido(4,5-b)indol-2-one), pyridoindole cytidine (H-pyrido(3',2':4,5)pyrrolo(2,3-d)pyrimidin-2-one).

[0287] The heterocyclic base moiety can also include those in which purine or pyrimidine bases are substituted by other heterocycles, such as 7-deaza-adenine, 7-deazaguanosine, 2-aminopyridine and 2-pyridone. Further nucleobases include those disclosed in U.S. Patent No. 3,687,808, The Concise Encyclopedia Of Polymer Science And Engineering, pages 858-859, Kroschwitz, J.I., ed. John Wiley & Sons, 1990, Englisch et al., Angewandte Chemie, International Edition, 1991, 30, those disclosed in 613 and Sanghvi, YS, Chapter 15, Antisense Research and Applications, pages 289-302, Crooke, ST and Lebleu, B., ed., CRC Press, 1993. Some of these nucleobases can be used to increase the binding affinity of oligomeric compounds. These include 5-substituted pyrimidines, 6-azapyrimidines, and N-2, N-6, and O-6 substituted purines, including 2-aminopropyladenine, 5-propyluracil, and 5-propylcytosine. 5-methylcytosine substitution has been shown to increase nucleic acid duplex stability by 0.6-1.2°C (Sanghvi et al., eds., Antisense Research and Applications, CRC Press, Boca Raton, 1993, pp. 276-278), and is a suitable base substitution, such as when combined with 2'-O-methoxyethyl sugar modification.

[0288] "Complementary" refers to the ability to pair between two sequences by base stacking and specific hydrogen bonds, and these sequences include naturally or non-naturally occurring (e.g., modified as described above) bases (nucleosides) or their analogs. For example, if a base at a certain position of a nucleic acid can form a hydrogen bond with a base at a corresponding position of a target, the base is considered to be complementary at that position. Nucleic acids can contain universal bases, or inert base spacers that have no positive or negative effects on hydrogen bonds. Base pairing can include typical Watson-Crick base pairing and non-Watson-Crick base pairing (such as Wobble base pairing and Hoogsteen base pairing).

[0289] It should be understood that for complementary base pairing, adenosine-type bases (A) are complementary to thymine-type bases (T) or uracil-type bases (U), cytosine-type bases (C) are complementary to guanosine-type bases (G), and universal bases such as 3-nitropyrrole or 5-nitroindole can hybridize with any A, C, U or T and can be considered complementary. Nichols et al., Nature, 1994; 369: 492-493 and Loakes et al., Nucleic Acids Res., 1994; 22: 4039-4043. Inosine (I) is also considered a universal base in the art and is considered complementary to any A, C, U or T. See Watkins and Santalucia, Nucl. Acids Research, 2005; 33(19): 6258-6267.

[0290] Conjugate

[0291] Another possible modification of nucleic acid involves chemical connection to one or more parts or conjugates of polynucleotides, which enhance the activity, cellular distribution or cellular uptake of oligonucleotides. These parts or conjugates may include covalently bound conjugated groups to functional groups, such as primary hydroxyl or secondary hydroxyl, etc. Conjugated groups include but are not limited to intercalators, reporter molecules, polyamines, polyamides, polyethylene glycols, polyethers, groups that enhance oligomer pharmacodynamic properties and groups that enhance oligomer pharmacokinetic properties. Suitable conjugated groups include but are not limited to cholesterol, lipids, phospholipids, biotin, phenazine, folic acid, phenanthridine, anthraquinone, acridine, fluorescein, rhodamine, coumarin and dyes. Groups that enhance pharmacodynamic properties include groups that improve uptake, enhance degradation resistance and / or strengthen specific hybridization with target nucleic acid sequences. Groups that enhance pharmacokinetic properties include groups that improve uptake, distribution, metabolism or secretion of nucleic acids.

[0292] Conjugated moieties include, but are not limited to, lipid moieties, such as cholesterol moieties (Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86, 6553-6556); cholic acid (Manoharan et al., Bioorg. Med. Chem. Let., 1994, 4, 1053-1060); thioethers, such as hexyl-S-tritylthiol (Manoharan et al., Ann. NY Acad. Sci., 1992, 660, 306-309; Manoharan et al., Bioorg. Med. Chem. Let., 1993, 3, 2765-2770); thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20, 533-538); aliphatic chains, such as dodecandiol or undecyl residues (Saison-Behmoaras et al., EMBO J., 1991, 10, 1111-1118; Kabanov et al., FEBS Lett., 1990, 259, 327-330; Svinarchuk et al., Biochimie, 1993, 75, 49-54); phospholipids, such as di-hexadecyl-rac-glycerol or triethylammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonic acid triethylammonium (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651-3654; Shea et al., Nucl. Acids Res., 1990, 18, 3777-3783); polyamine or polyethylene glycol chain (Manoharan et al., Nucleosides & Nucleotides, 1995, 14, 969-973); adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36, 36513654); palmityl moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264, 229-237); or octadecylamine or hexylamino-carbonyl-t-oxycholesterol moiety (Crooke et al., J. Pharmacal. Exp. Ther., 1996, 277, 923-937).

[0293] The conjugate may comprise a "Protein Transduction Domain" or PTD (also known as a CPP-cell penetrating peptide), which may refer to a polypeptide, polynucleotide, carbohydrate, or organic or inorganic compound that facilitates passage through a lipid bilayer, micelle, cell membrane, organelle membrane, or vesicle membrane. The PTD attached to another molecule, which may range from small polar molecules to large macromolecules and / or nanoparticles, may facilitate the passage of molecules through a membrane, for example, from the extracellular space to the intracellular space, or from the cytosol to the organelle. In some embodiments, the PTD is covalently linked to the amino terminus of an exogenous polypeptide (such as a B-GEn.16 polypeptide or a variant thereof). In some embodiments, the PTD is covalently linked to the C-terminus or N-terminus of an exogenous polypeptide (such as a B-GEn.16 polypeptide or a variant thereof). In some embodiments, the PTD is covalently linked to a nucleic acid (e.g., a guide RNA, a polynucleotide encoding a guide RNA, a polynucleotide encoding a B-GEn.16 polypeptide or a variant thereof, etc.). Exemplary PTDs include, but are not limited to, a minimal eleven amino acid polypeptide protein transduction domain (corresponding to residues 47-57 of HIV-1 TAT comprising YGRKKRRQRRR; a polyarginine sequence comprising sufficient arginines to directly enter cells (e.g., 3, 4, 5, 6, 7, 8, 9, 10, or 10-50 arginines); a VP22 domain (Zender et al. (2002) Cancer Gene Ther. 9(6):489-96); a Drosophila antennal protein transduction domain (Noguchi et al. (2003) Diabetes 52(7):1732-1737); a truncated human calcitonin peptide (Trehin et al. (2004) Pharm. Research 21:1248-1256); a polylysine (Wender et al. (2000) Proc. Natl. Acad. Sci. USA 97:13003-13008). In some embodiments, the PTD is an activatable CPP (ACPP) (Aguilera et al. (2009) Integr Biol (Camb) June; 1(5-6):371-381). The ACPP comprises a polycationic CPP (such as Arg9 or "R9"), connected to a matching polyanion (such as Glu9 or "E9") via a cleavable linker, which reduces the net charge to near zero and thereby inhibits adhesion and uptake into cells. When the linker is cleaved, the polyanion is released, locally uncovering the polyarginine and its inherent adhesive properties, thereby "activating" the ACPP to cross the membrane.In some embodiments, the PTD is chemically modified to increase the bioavailability of the PTD. Exemplary modifications are disclosed in Expert Opin Drug Deliv. 2009 Nov; 6(11): 1195-205.

[0294] Peptide modification

[0295] The B-GEn.16 polypeptide or its variant expressed from the codon-optimized polynucleotide sequence can be produced in vitro or by eukaryotic cells, prokaryotic cells or by in vitro transcription and translation (IVTT), and can be further treated by unfolding, such as heat denaturation, OTT reduction, etc., and can be further refolded using methods known in the art.

[0296] Intended modifications that do not alter the primary sequence include chemical derivatization of the polypeptide, such as acylation, acetylation, carboxylation, amidation, etc. Also included are modifications of glycosylation, such as modifications by changing the glycosylation pattern of the polypeptide during the synthesis and processing of the polypeptide or in further processing steps; for example, exposing the polypeptide to an enzyme that affects glycosylation, such as a mammalian glycosylase or deglycosylase. Also included are sequences with phosphorylated amino acid residues, such as phosphotyrosine, phosphoserine, or phosphothreonine.

[0297] In some embodiments, the B-GEn.16 polypeptide or its variant is modified using common molecular biology techniques and synthetic chemistry to increase its resistance to proteolytic degradation, change target sequence specificity, optimize solubility characteristics, change protein activity (such as transcriptional regulatory activity, enzyme activity, etc.) or make it more suitable as a therapeutic agent. Analogs of this polypeptide include those containing residues other than naturally occurring L-amino acids, such as O-amino acids or non-natural synthetic amino acids. D-amino acids can be substituted for some or all of the amino acid residues. B-GEn.16 polypeptide or its variant can be prepared by in vitro synthesis using conventional methods known in the art. Various commercial synthesis equipment is available, for example, by automatic synthesizers such as Applied Biosystems, Inc., Beckman, etc. Using a synthesizer, natural amino acids can be replaced by non-natural amino acids. The specific sequence and preparation method can be determined by factors such as convenience, economy, and required purity.

[0298] If desired, various groups can be introduced into the peptide during synthesis or expression to allow attachment to other molecules or surfaces. Thus, cysteine ​​can be used to make thioethers, histidine for attachment to metal ion complexes, carboxyl groups for amide or ester formation, amino groups for amide formation, etc.

[0299] Recombinant cells

[0300] In some embodiments, the codon-optimized B-GEn.16 system described herein can be used in eukaryotic organisms, such as mammalian cells, such as human cells.Any human cell is suitable for use with the codon-optimized B-GEn.16 system disclosed herein.

[0301] In some embodiments, the ex vivo or in vitro cell comprises: (a) a nucleic acid comprising a codon-optimized polynucleotide sequence encoding a B-GEn.16 polypeptide or variant described herein, or a B-GEn.16 polypeptide or variant thereof expressed by a nucleic acid; and (b) a gRNA or nucleic acid encoding the gRNA, wherein the gRNA is capable of guiding the B-GEn.16 polypeptide or variant thereof to a target polynucleotide sequence. In some embodiments, the cell comprises a nucleic acid comprising a codon-optimized polynucleotide sequence. In some embodiments, the cell comprises a gRNA. In some embodiments, the cell comprises a nucleic acid encoding a gRNA. In some embodiments, the gRNA is a single guide RNA (sgRNA). In some embodiments, the cell comprises one or more additional gRNAs or nucleic acids encoding one or more additional gRNAs. In some embodiments, the cell further comprises a donor template.

[0302] On the one hand, some embodiments disclosed herein relate to a method for transforming cells, which method includes introducing a nucleic acid provided herein into a host cell, such as an animal cell, and selecting or screening the transformed cell. The terms "host cell" and "recombinant host cell" are used interchangeably herein. It should be understood that such terms refer not only to specific subject cells, but also to the progeny or potential progeny of such cells. Because, due to mutations or environmental influences, certain modifications may occur in offspring, such offspring may actually be different from the parental cell, but are still included in the scope of the terms used herein. The techniques for transforming a variety of the above-mentioned host cells and species are known in the art and are recorded in the technical and scientific literature. Therefore, a cell culture comprising at least one recombinant cell disclosed herein is also within the scope of the present application. Methods and systems suitable for producing and maintaining cell cultures are known in the art.

[0303] In related aspects, some embodiments relate to recombinant host cells, for example, recombinant animal cells comprising nucleic acids described herein. The nucleic acid can be stably integrated into the host genome, or can be replicated alone, or present in a recombinant host cell as a minicircle expression vector for stable or transient expression. Therefore, in some embodiments disclosed herein, the nucleic acid is maintained and replicated in a recombinant host cell as a free unit. In some embodiments, the nucleic acid is stably integrated into the genome of the recombinant cell. In some embodiments, the nucleic acid is present in a recombinant host cell as a minicircle expression vector for stable or transient expression.

[0304] In some embodiments, host cells can be genetically engineered (e.g., transduced or transformed or transfected) using, for example, a vector construct of the present application, which can be, for example, a vector for homologous recombination, comprising a nucleic acid sequence homologous to a portion of the host cell genome, or can be an expression vector for any expression or combination of target genes. For example, the vector can be in the form of a plasmid, a viral particle, a bacteriophage, etc. In some embodiments, the vector for expression of the target polypeptide can also be designed to be integrated into the host, such as by homologous recombination.

[0305] In some embodiments, the disclosure provides a genetically modified host cell, such as an isolated genetically modified host cell, wherein the genetically modified host cell comprises: 1) an exogenous guide RNA; 2) an exogenous nucleic acid comprising a nucleotide sequence encoding the guide RNA; 3) an exogenous nucleic acid comprising a codon-optimized polynucleotide sequence encoding a B-GEn.16 polypeptide or a variant thereof; 4) an exogenous B-GEn.16 polypeptide or a variant thereof, expressed by a nucleic acid comprising a codon-optimized polynucleotide sequence; or 5) any combination of the above. In some embodiments, a genetically modified cell is produced by genetically modifying a host cell, such as: 1) an exogenous guide RNA; 2) an exogenous nucleic acid comprising a nucleotide sequence encoding the guide RNA; 3) an exogenous nucleic acid comprising a codon-optimized polynucleotide sequence encoding a B-GEn.16 polypeptide or a variant thereof; 4) an exogenous B-GEn.16 polypeptide or a variant thereof, expressed by a nucleic acid comprising a codon-optimized polynucleotide sequence; or 5) any combination of the above.

[0306] As mentioned above, all cells that are suitable as target cells are also suitable as host cells for genetic modification. For example, the genetically modified host cell of interest can be a cell from any organism, such as a bacterial cell, an archaeal cell, a cell of a unicellular eukaryote, a plant cell, an algal cell (e.g., Botryococcus braunii, Chlamydomonas reinhardtii, Nannochloropsis gaditana, Chlorela pyrenoidosa, Sargassum patens (C. Agardh), etc.), a fungal cell (e.g., a yeast cell), an animal cell, a cell from an invertebrate (e.g., a fruit fly, a cnidarian, an echinoderm, a nematode, etc.), a cell from a vertebrate (e.g., a fish, an amphibian, a reptile, a bird, a mammal), a cell from a mammal (e.g., a pig, a cow, a goat, a sheep, a rodent rat, a mouse, a non-human primate, a human, etc.). In some embodiments, the genetically modified host cell can be any cell from a human.

[0307] In some embodiments, the genetically modified host cells of the present disclosure have been genetically modified using an exogenous nucleic acid containing a nucleotide sequence encoding a B-GEn.16 polypeptide or a variant thereof. In some embodiments, the genetically modified host cells are genetically modified using an exogenous nucleic acid containing a nucleotide sequence encoding a B-GEn.16 polypeptide or a variant as described herein. The DNA of the genetically modified host cell can be targeted to be modified by introducing a guide RNA (or DNA encoding a guide RNA, which determines the genomic position / sequence to be modified) and optionally a donor nucleic acid into the cell. In certain embodiments, the nucleotide sequence encoding a B-GEn.16 polypeptide or a variant thereof is operably linked to an inducible promoter (such as a heat shock promoter, a tetracycline-regulated promoter, a steroid-regulated promoter, a metal-regulated promoter, an estrogen receptor-regulated promoter, etc.). In some embodiments, the codon-optimized nucleotide sequence encoding the B-GEn.16 polypeptide or its variant is operably linked to a spatially restricted and / or temporally restricted promoter (e.g., a tissue-specific promoter, a cell type-specific promoter, a cell cycle-specific promoter). In some embodiments, the codon-optimized nucleotide sequence encoding the B-GEn.16 polypeptide or its variant is operably linked to a constitutive promoter.

[0308] In some embodiments, the genetically modified host cell is in vitro. In some embodiments, the genetically modified host cell is in vivo. In some embodiments, the genetically modified host cell is a prokaryotic cell or is derived from a prokaryotic cell. In some embodiments, the genetically modified host cell is a bacterial cell or is derived from a bacterial cell. In some embodiments, the genetically modified host cell is an ancient cell or is derived from an ancient cell. In some embodiments, the genetically modified host cell is a eukaryotic cell or is derived from a eukaryotic cell. In some embodiments, the genetically modified host cell is a plant cell or is derived from a plant cell. In some embodiments, the genetically modified host cell is an animal cell or is derived from an animal cell. In some embodiments, the genetically modified host cell is an invertebrate cell or is derived from an invertebrate cell. In some embodiments, the genetically modified host cell is a vertebrate cell or is derived from a vertebrate cell. In some embodiments, the genetically modified host cell is a mammalian cell or is derived from a mammalian cell. In some embodiments, the genetically modified host cell is a rodent cell or is derived from a rodent cell. In some embodiments, the genetically modified host cell is a human cell or is derived from a human cell. In some embodiments, the genetically modified host cell is a human cell or is derived from a human cell.

[0309] The present disclosure further provides progeny of the genetically modified cell, wherein the progeny may include the same exogenous nucleic acid or polypeptide as the genetically modified cell from which it was derived.In some embodiments, the present disclosure further provides compositions comprising a genetically modified host cell.

[0310] In some embodiments, the genetically modified host cell is a genetically modified stem cell or progenitor cell. Suitable host cells include, such as stem cells (adult stem cells, embryonic stem cells, iPS cells, etc.) and progenitor cells (such as cardiac progenitor cells, neural progenitor cells, etc.). Other suitable host cells include mammalian stem cells and progenitor cells, such as rodent stem cells, rodent progenitor cells, human stem cells, human progenitor cells, etc. Other suitable host cells include in vitro host cells, such as isolated host cells. In some embodiments, the genetically modified host cell comprises an exogenous guide RNA nucleic acid. In some embodiments, the genetically modified host cell comprises an exogenous nucleic acid comprising a nucleotide sequence encoding a guide RNA. In some embodiments, the genetically modified host cell comprises an exogenous B-GEn.16 polypeptide or a variant thereof expressed by a codon-optimized nucleotide sequence. In some embodiments, the genetically modified host cell comprises an exogenous nucleic acid comprising a codon-optimized nucleotide sequence encoding a B-GEn.16 polypeptide or a variant thereof. In some embodiments, the genetically modified host cell includes an exogenous nucleic acid comprising 1) a nucleotide sequence encoding a guide RNA and 2) a codon-optimized nucleotide sequence encoding a B-GEn.16 polypeptide or a variant thereof.

[0311] Non-human genetically modified organisms

[0312] In some embodiments, the genetically modified host cell has been genetically modified with an exogenous nucleic acid comprising a codon-optimized nucleotide sequence encoding a B-GEn.16 polypeptide or a variant thereof. If such a cell is a eukaryotic unicellular organism, the modified cell can be considered a genetically modified organism. In some embodiments, the non-human genetically modified organism is a multicellular organism transgenic for B-GEn.16.

[0313] In some embodiments, a genetically modified non-human host cell (e.g., a cell genetically modified with an exogenous nucleic acid comprising a codon-optimized nucleotide sequence encoding a B-GEn.16 polypeptide or a variant thereof) can produce a genetically modified non-human organism (e.g., a mouse, a fish, a frog, a fly, a worm, etc.). For example, if the genetically modified host cell is a pluripotent stem cell (e.g., a PSC) or a germ cell (e.g., a sperm, an egg cell, etc.), the entire genetically modified organism can be derived from the genetically modified host cell. In some embodiments, the genetically modified host cell is a pluripotent stem cell (e.g., an ESC, an iPSC, a pluripotent plant stem cell, etc.) or a germ cell (e.g., a sperm cell, an egg cell, etc.) in vivo or in vitro, which can produce a genetically modified organism. In some embodiments, the genetically modified host cell is a vertebrate PSC (such as ESC, iPSC, etc.), and is used to produce a genetically modified organism (such as injecting PSC into a blastocyst to produce a chimeric / mosaic animal, and then mating to produce a non-chimeric / non-integrated genetically modified organism; plant grafting, etc.). Any suitable method / scheme for producing a genetically modified organism, including the method described herein, is suitable for producing a genetically modified host cell comprising an exogenous nucleic acid comprising a codon-optimized nucleotide sequence encoding a B-GEn.16 polypeptide or a variant thereof. Methods for producing genetically modified organisms are known in the art. See, for example, Cho et al., Curr Protoc Cell Biol. 2009 Mar; Chapter 19: Unit 19.11: Generation of transgenic mice; Gama et al., Brain Struct Funct. 2010 Mar; 214(2-3):91-109. Epub2009 Nov 25: Animal transgenesis: an overview; Husaini et al., GM Crops.2011 Jun-Dec;2(3):150-62.Epub 2011 Jun 1:Approaches for gene targeting and targetedgene expression in plants.

[0314] In some embodiments, the genetically modified organism comprises a target cell for the method of the present disclosure, and therefore can be regarded as the source of the target cell. For example, if the genetically modified cell comprising an exogenous nucleic acid is used to produce a genetically modified organism, the exogenous nucleic acid comprises a codon-optimized nucleotide sequence encoding a B-GEn.16 polypeptide or its variant, then the cell of the genetically modified organism comprises an exogenous nucleic acid, the exogenous nucleic acid comprises a codon-optimized nucleotide sequence encoding a B-GEn.16 polypeptide or its variant. In some such embodiments, the DNA of one or more cells of the genetically modified organism can be targeted, so that it can be modified by introducing a guide RNA (or DNA encoding a guide RNA) and optionally a donor nucleic acid into the cell. For example, a guide RNA (or DNA encoding a guide RNA) is introduced into a subset of cells (such as brain cells, intestinal cells, kidney cells, lung cells, blood cells, etc.) of a genetically modified organism, and the DNA of these cells can be targeted and modified, and its genomic position depends on the DNA targeting sequence of the guide RNA introduced.

[0315] In some embodiments, a genetically modified organism is a target cell source for the methods of the present disclosure. For example, a genetically modified organism comprising cells genetically modified with an exogenous nucleic acid, wherein the exogenous nucleic acid comprises a codon-optimized nucleotide sequence encoding a B-GEn.16 polypeptide or a variant thereof, can provide a genetically modified cell source, such as PSCs (such as ESCs, iPSCs, sperm, egg cells, etc.), neurons, progenitor cells, cardiomyocytes, etc.

[0316] In some embodiments, the genetically modified cell is a PSC comprising an exogenous nucleic acid, which comprises a codon-optimized nucleotide sequence encoding a B-GEn.16 polypeptide or a variant thereof. Therefore, the PSC can be a target cell so that the DNA of the PSC can be targeted to be modified by introducing a guide RNA (or DNA encoding a guide RNA) and optionally a donor nucleic acid into the PSC, and the genomic position of the modification will depend on the DNA targeting sequence of the guide RNA introduced. Therefore, in some embodiments, the methods described herein can be used to modify the DNA of the PSC derived from a genetically modified organism (such as deleting and / or replacing any desired genomic position). The PSC modified in this way can then be used to produce an organism having both of the following characteristics: (i) an exogenous nucleic acid comprising a codon-optimized nucleotide sequence encoding a B-GEn.16 polypeptide or a variant thereof; (ii) a DNA modification introduced into the PSC.

[0317] In some embodiments, the exogenous nucleic acid can be controlled by an unknown promoter (e.g., operably linked to an unknown promoter) (e.g., when the nucleic acid is randomly integrated into the host cell genome), or by a known promoter (e.g., operably linked to a known promoter). Suitable known promoters can be any known promoters, including constitutively active promoters (e.g., CMV promoters), inducible promoters (e.g., heat shock promoters, tetracycline-regulated promoters, steroid-regulated promoters, metal-regulated promoters, estrogen receptor-regulated promoters, etc.), spatially restricted and / or temporally restricted promoters (e.g., tissue-specific promoters, cell type-specific promoters, etc.), etc.

[0318] The genetically modified organism (e.g., an organism whose cells contain a codon-optimized nucleotide sequence encoding a B-GEn.16 polypeptide or a variant thereof) can be any organism, including, for example, plants; algae; invertebrates (e.g., cnidarians, echinoderms, worms, flies, etc.); vertebrates (e.g., fish (e.g., zebrafish, pufferfish, goldfish, etc.); amphibians (e.g., salamanders, frogs, etc.); reptiles, birds, mammals, etc.); ungulates (e.g., goats, pigs, sheep, cattle, etc.); rodents (e.g., mice, rats, hamsters, guinea pigs, etc.); long-tailed animals (e.g., rabbits), etc.

[0319] In some embodiments, the active portion is a plurality of RNase domains. In some embodiments, the active portion is a DNase domain.

[0320] Genetically modified non-human animals

[0321] As described above, in some embodiments, a nucleic acid (e.g., a codon-optimized nucleotide sequence encoding a B-GEn.16 polypeptide or a variant thereof) or a recombinant expression vector is used as a transgene to generate a transgenic animal producing a B-GEn.16 polypeptide or a variant thereof. Therefore, the present disclosure further provides a transgenic non-human animal comprising a transgene comprising a nucleic acid comprising a codon-optimized nucleotide sequence encoding a B-GEn.16 polypeptide or a variant thereof, as described above. In some embodiments, the genome of the transgenic non-human animal comprises a codon-optimized nucleotide sequence encoding a B-GEn.16 polypeptide or a variant thereof. In some embodiments, the transgenic non-human animal is homologous to the genetic modification. In some embodiments, the transgenic non-human animal is heterologous to the genetic modification. In some embodiments, the transgenic non-human animal is a vertebrate, such as fish (e.g., zebrafish, goldfish, pufferfish, cave fish, etc.), amphibians (frogs, salamanders, etc.), birds (e.g., chickens, turkeys, etc.), reptiles (e.g., snakes, lizards, etc.), mammals (e.g., ungulates, such as pigs, cattle, goats, sheep, etc.; long-tailed animals (e.g., rabbits); rodents (e.g., rats, mice); non-human primates, etc.), etc.

[0322] In some embodiments, the nucleic acid is an exogenous nucleic acid comprising a codon-optimized nucleotide sequence encoding a B-GEn.16 polypeptide or a variant thereof. In some embodiments, the exogenous nucleic acid may be controlled by an unknown promoter (e.g., operably linked to an unknown promoter) (e.g., when the nucleic acid is randomly integrated into the host cell genome), or may be controlled by a known promoter (e.g., operably linked to a known promoter). Suitable known promoters may be any known promoter, including constitutively active promoters (e.g., CMV promoters), inducible promoters (e.g., heat shock promoters, tetracycline-regulated promoters, steroid-regulated promoters, metal-regulated promoters, estrogen receptor-regulated promoters, etc.), spatially restricted and / or temporally restricted promoters (e.g., tissue-specific promoters, cell type-specific promoters, etc.), etc.

[0323] Introducing nucleic acids into host cells

[0324] In some embodiments, the methods of the present disclosure include introducing one or more nucleic acids into a host cell (or a host cell population), the nucleic acid comprising a nucleotide sequence encoding a guide RNA and / or a codon-optimized nucleotide sequence encoding a B-GEn.16 polypeptide or a variant thereof. In some embodiments, the cell comprising the target DNA is an in vitro cell. In some embodiments, the cell comprising the target DNA is an in vivo cell. In some embodiments, the nucleotide sequence encoding the guide RNA and / or the B-GEn.16 polypeptide or a variant thereof is operably connected to an inducible promoter. In some embodiments, the nucleotide sequence encoding the guide RNA and / or the B-GEn.16 polypeptide or a variant thereof is operably connected to a constitutive promoter.

[0325] The guide RNA or nucleic acid comprising a nucleotide sequence encoding the guide RNA can be introduced into a host cell by any of various known methods. Similarly, when the method involves introducing a nucleic acid comprising a codon-optimized nucleotide sequence encoding a B-GEn.16 polypeptide or a variant thereof into a host cell, such nucleic acid can be introduced into a host cell by any of various known methods. The guide polynucleotide (RNA or DNA) and / or the B-GEn.16 polynucleotide (RNA or DNA) can be delivered by viral or non-viral delivery vectors known in the art.

[0326] Methods for introducing nucleic acids into host cells are known in the art, and any known method can be used to introduce nucleic acids (such as expression constructs) into stem cells or progenitor cells. Suitable methods include, for example, viral or bacteriophage infection, transfection, conjugation, protoplast fusion, lipid infection, electroporation, calcium phosphate precipitation, polyethyleneimine (PEI)-mediated transfection, diethylamineethyl dextran (DEAE-dextran)-mediated transfection, liposome-mediated transfection, particle gun technology, calcium phosphate precipitation, direct microinjection, nanoparticle-mediated nucleic acid delivery (see, e.g., Panyam et al., al Adv Drug Deliv Rev. 2012 Sep 13. pii: 50169-409X (12) 00283-9. doi: 10.1016 / j.addr.2012.09.023), etc., including but not limited to exosome delivery.

[0327] Polynucleotides can be delivered by non-viral delivery vectors, including but not limited to nanoparticles, liposomes, ribonucleoproteins, positively charged peptides, small molecule RNA conjugates, aptamers-RNA chimeras and RNA fusion protein complexes. Some exemplary non-viral delivery vectors are described in Peer and Lieberman, Gene Therapy, 18: 1127-1133 (2011) (which focuses on non-viral delivery vectors of siRNA, which can also be used to deliver other polynucleotides).

[0328] Suitable systems and techniques for delivering nucleic acids (such as mRNA and sgRNA) of the present disclosure for gene editing include lipid nanoparticles (LNPs). As used herein, the term "lipid nanoparticle" includes liposomes, regardless of their lamellarity, shape or structure, and also includes lipid complexes describing the introduction of nucleic acids and / or polypeptides into cells. These lipid nanoparticles can be compounded with biologically active compounds (such as nucleic acids and / or polypeptides) and can be used as in vivo delivery vectors. Generally, any method known in the art can be used to prepare lipid nanoparticles containing one or more nucleic acids of the present disclosure, as well as to prepare complexes of biologically active compounds and the lipid nanoparticles. Examples of such methods have been widely disclosed, for example, in Biochim Biophys Acta 1979, 557:9; Biochim et Biophys Acta 1980, 601:559; Liposomes: A practical approach (Oxford University Press, 1990); Pharmaceutica Acta Helvetiae 1995, 70:95; Current Science 1995, 68:715; Pakistan Journal of Pharmaceutical Sciences 1996, 19:65; Methods in Enzymology 2009, 464:343. Particularly suitable systems and technologies for preparing LNP formulations comprising one or more nucleic acids and / or polypeptides of the present disclosure include, but are not limited to, those developed by Intellia (see, e.g., WO2017173054A1), Alnylam (see, e.g., WO2014008334A1), Modernatx (see, e.g., WO2017070622A1 and WO2017099823A1), TranslateBio, Acuitas (see, e.g., WO2018081480A1), Genevant Sciences, Arbutus Biopharma, Tekmira, Arcturus, Merck (see, e.g., WO2015130584A2), Novartis (see, e.g., WO2015095340A1), and Dicerna; all of which are incorporated herein by reference in their entirety.

[0329] Suitable nucleic acids comprising nucleotide sequences encoding guide RNA and / or B-GEn.16 polypeptides or variants thereof include expression vectors, wherein the expression vector comprises a nucleotide sequence encoding a guide. In some embodiments, the expression vector is a viral construct, such as a recombinant adeno-associated virus construct (see, e.g., U.S. Patent No. 7,078,387), a recombinant adenovirus construct, a recombinant lentivirus construct, a recombinant retrovirus construct, and the like.Suitable expression vectors include, but are not limited to, viral vectors (e.g., viral vectors based on vaccinia virus; viral vectors based on polio virus; viral vectors based on adenovirus (see, e.g., Li et al., Invest Opthalmol Vis Sci 35:2543 2549, 1994; Borras et al., Gene Ther 6:515 524, 1999; Li and Davidson, PNAS 92:7700 7704, 1995; Sakamoto et al., H Gene Ther 5:10881097, 1999; WO 94 / 12649, WO 93 / 03769; WO 93 / 19191; WO 94 / 28938; WO 95 / 11984 and WO 95 / 00655); adeno-associated virus (see, e.g., Ali et al., Hum Gene Ther 9:81 86,1998,Flannery et al.,PNAS 94:6916 6921,1997;Bennett et al.,Invest OpthalmolVis Sci 38:2857 2863,1997;Jomary et al.,Gene Ther 4:683-690,1997,Rolling etal.,Hum Gene Ther 10:641648,1999; Ali et al., Hum Mol Genet 5:591 594,1996; Srivastava in WO 93 / 09239, Samulski et al., J. Vir. (1989) 63:3822-3828; Mendelsonet al.,Viral.(1988)166:154-165; and Flotte et al. al., PNAS (1993) 90:10613-10617); SV40; herpes simplex virus; human immunodeficiency virus (see, e.g., Miyoshi et al., PNAS 94:10319 23, 1997; Takahashi et al., J Virol 73:7812 7816, 1999); retroviral vectors (e.g., vectors derived from murine leukemia virus, spleen necrosis virus, retroviruses (e.g., Rous sarcoma virus, Harvey sarcoma virus, avian leukosis virus, lentivirus, human immunodeficiency virus, myeloproliferative sarcoma virus, and mammary tumor virus)), etc.

[0330] Adeno-associated virus (AAV)

[0331] Recombinant adeno-associated virus (AAV) vectors can be used for delivery. The known technology for producing rAAV particles in the art is to provide cells with polynucleotides to be delivered between two AAV inverted terminal repeats (ITRs), AAV rep and cap genes, and helper virus functions. The production of rAAV requires the following components in a single cell (referred to herein as a packaging cell): the target polynucleotide between the two ITRs, the AAV rep and cap genes separated from the AAV genome (i.e., not in the AAV genome), and helper virus functions. The AAV rep and cap genes can be derived from any AAV serotype from which the recombinant virus can be derived, or from an AAV serotype different from the ITR on the packaging polynucleotide, including but not limited to AAV serotypes AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-7, AAV-8, AAV-9, AAV-10, AAV-11, AAV-12, AAV-13, and AAV rh.74. For example, WO 01 / 83692 discloses the production of pseudovirus rAAV.

[0332]

[0333] The method for producing packaging cells is to create a cell line that stably expresses all the components required for the production of AAV particles. For example, a plasmid (or multiple plasmids) is integrated into the genome of the cell, and the plasmid contains a plurality of AAV ITRs between the target polynucleotides, AAV rep and cap genes independent of the AAV genome, and selectable markers, such as neomycin resistance genes. The AAV genome has been introduced into bacterial plasmids by procedures such as GC tailing (Samulski et al., 1982, Proc. Natl. Acad. Sci. USA, 79: 2077-2081), adding synthetic linkers containing restriction endonuclease cleavage sites (Laughlin et al., 1983, Gene, 23: 65-73) or direct blunt end connection (Senapathy & Carter, 1984, J. Bioi. Chem., 259: 4661-4666). The packaging cell line is then infected with a helper virus, such as an adenovirus. The advantage of this method is that the cell has selectivity and is suitable for large-scale production of rAAV. Other examples of suitable methods are the use of adenovirus or baculovirus rather than plasmids to introduce the rAAV genome and / or the rep and cap genes into packaging cells.

[0334] General principles of rAAV production are reviewed in, e.g., Carter, 1992, Current Opinions in Biotechnology, 1533-539; and Muzyczka, 1992, Curr. Topics in Microbial. and Immunol., 158:97-129). Various methods are described in Ratschin et al., Mol. Cell. Biol. 4:2072 (1984); Hermonat et al., Proc. Natl. Acad. Sci. USA, 81:6466 (1984); Tratschin et al., Mol. Cell. Biol. 5:3251 (1985); Mclaughlin et al., J. Virol., 62:1963 (1988); and Lebkowski et al., 1988 Mol. Cell. Biol., 7:349 (1988). Samulski et al. (1989, J. Virol., 63:3822-3828); U.S. Patent No. 5,173,414; WO 95 / 13365 and corresponding U.S. Patent No. 5,658.776; WO 95 / 13392; WO 96 / 17947; PCT / US98 / 18600; WO97 / 09441 (PCT / US96 / 14423); WO97 / 08298 (PCT / US96 / 13872); WO 97 / 21825 (PCT / US96 / 20777); WO 97 / 06243(PCT / FR96 / 01064); WO 99 / 11764; Perrin et al. (1995) Vaccine 13:1244-1250; Paul et al. (1993) Human Gene Therapy 4:609-615; Clark et al. (1996) Gene Therapy 3:1124-1132; U.S. Patent No. 5,786,211; U.S. Patent No. 5,871,982; and U.S. Patent No. 6,258,595.

[0335] The AAV vector serotype used for transduction depends on the target cell type. For example, the following exemplary cell types are known to be transducible by the indicated AAV serotypes, etc.

[0336] Tissue / cell type Serotype liver AAV8, AAV9 Skeletal muscle AAV1, AAV7, AAV6, AAV8, AAV9 Central nervous system AAV5, AAV1, AAV4 RPE AAV5, AAV4 Photoreceptor cells AAV5 lung AAV9 Heart AAV8 pancreas AAV8 kidney AAV2

[0337] Many suitable expression vectors are known to those skilled in the art, and many are commercially available. The following vectors are provided by way of example; for eukaryotic host cells: pXT1, pSG5 (Stratagene), pSVK3, pBPV, pMSG, and pSVLSV40 (Pharmacia). However, any other vector may be used as long as it is compatible with the host cell.

[0338] Depending on the host / vector system utilized, a number of appropriate transcription and translation control elements may be used in the expression vector, including constitutive and inducible promoters, transcription enhancer elements, transcription terminators, and the like (see, e.g., Bitter et al. (1987) Methods in Enzymology, 153:516-544).

[0339] In some embodiments, guide RNA and / or B-GEn.16 polypeptide or its variants may be provided as RNA. In this case, guide RNA and / or RNA encoding B-GEn.16 polypeptide or its variants may be produced by direct chemical synthesis, or may be transcribed in vitro from DNA encoding the guide RNA. Methods for synthesizing RNA from DNA templates are well known in the art. In some embodiments, guide RNA and / or RNA encoding B-GEn.16 polypeptide or its variants will be synthesized in vitro using RNA polymerase (such as T7 polymerase, T3 polymerase, SP6 polymerase, etc.). Once synthesized, RNA may directly contact the target DNA, or may be introduced into the cell by any known technique for introducing nucleic acid into the cell (such as microinjection, electroporation, transfection, etc.).

[0340] Nucleotides encoding guide RNA (introduced as DNA or RNA) and / or B-GEn.16 polypeptide or variants thereof (introduced as DNA or RNA) and / or donor polynucleotides can be provided to cells using established transfection techniques; see, e.g., Angeland Yanik (2010) PLoS ONE 5(7):e 11756, and Qiagen's commercially available Reagents, stemeffect from Stemgent TM RNA transfection kit and from Mims Bio -mRNA transfection kit. See also Beumer et al. (2008) Efficient gene targeting in Drosophila bydirect embryo injection with zinc-finger nucleases. PNAS 105 (50): 19821-19826. Additionally or alternatively, nucleic acids encoding guide RNA and / or B-GEn.16 polypeptides or variants thereof and / or B-GEn.16 fusion polypeptides or variants thereof and / or donor polynucleotides can be provided on DNA vectors. Many vectors are available, such as plasmids, cosmids, minicircles, phages, viruses, etc., which can be used to transfer nucleic acids to target cells. The vector containing nucleic acid can remain in a free state, such as plasmids, minicircle DNA, cytomegalovirus, adenovirus, etc., or it can also be integrated into the target cell genome by homologous recombination or random integration, such as retrovirus-derived vectors, such as MMLV, HIV-1, ALV, etc.

[0341] The vector can be provided directly to the cell. In other words, the cell is contacted with a vector containing nucleic acid and / or donor polynucleotides encoding guide RNA and / or B-GEn.16 polypeptide or its variant and / or B-GEn.16 fusion polypeptide or its variant, so that the vector is taken up by the cell. Methods for contacting cells with nucleic acid vectors as plasmids, including electroporation, calcium chloride transfection, microinjection and liposome infection, are well known in the art. For the delivery of viral vectors, cells are contacted with viral particles containing nucleic acid and / or donor polynucleotides encoding guide RNA and / or B-GEn.16 polypeptide or its variant and / or B-GEn.16 fusion polypeptide or its variant. Retroviruses, such as lentiviruses, are particularly suitable for the methods of the present disclosure. Commonly used retroviral vectors are "defective", such as, they cannot produce viral proteins required for effective infection. In contrast, the replication of the vector needs to be grown in a packaging cell line. In order to produce viral particles containing the target nucleic acid, the retroviral nucleic acid containing the nucleic acid is packaged into a viral capsid by the packaging cell line. Different packaging cell lines provide different envelope proteins (ecotype, amphoteric or heterotropism) to be integrated into the capsid, which determines the specificity of the virus particles of the cell (ecotype of mouse and rat; for most mammalian cell types, including humans, dogs and mice, they are amphoteric; and except for mouse cells, most mammalian cell types are heterotropes). Appropriate packaging cell lines can be used to ensure that the cells are targeted by the packaged virus particles. Methods for introducing retroviral vectors containing nucleic acids encoding reprogramming factors into packaging cell lines and collecting virus particles produced by the packaging cell lines are well known in the art. Nucleic acids can also be introduced by direct microinjection (such as injecting RNA into zebrafish embryos).

[0342] The vector for providing nucleic acids encoding guide RNA and / or B-GEn.16 polypeptides or variants thereof and / or B-GEn.16 fusion polypeptides or variants thereof and / or donor polynucleotides to cells generally comprises a suitable promoter for driving the expression (i.e., transcriptional activation) of the target nucleic acid. In other words, the target nucleic acid will be operably linked to the promoter. This may include ubiquitous active promoters, such as the CMV-13-actin promoter, or inducible promoters, such as promoters active in a particular cell population or promoters that respond to the presence of a drug (e.g., tetracycline). Through transcriptional activation, transcription is expected to increase at least 10 times, at least 100 times, and more typically at least 1000 times over the basal level of the target cell. In addition, the vector used to provide guide RNA and / or B-GEn.16 polypeptide or its variant and / or B-GEn.16 fusion polypeptide or its variant and / or donor polynucleotide to cells may include a nucleic acid sequence encoding a marker that can be selected in the target cell to identify cells that have taken up the guide RNA and / or B-GEn.16 polypeptide or its variant and / or B-GEn.16 fusion polypeptide or its variant and / or donor polynucleotide.

[0343] Guide RNA and / or B-GEn.16 polypeptide or variant thereof and / or B-GEn.16 fusion polypeptide or variant thereof may be used to contact DNA or introduced into cells as RNA instead. Methods for introducing RNA into cells are known in the art and may include, for example, direct injection, transfection or any other method for introducing DNA. B-GEn.16 polypeptide or variant thereof may be provided to cells as a polypeptide instead. Such a polypeptide may optionally be fused to a polypeptide domain that increases the solubility of the product. The domain may be connected to the polypeptide via a defined protease cleavage site, such as a TEV sequence that is cleaved by a TEV protease. The linker may also include one or more flexible sequences, such as from 1 to 10 glycine residues. In some embodiments, cleavage of the fusion protein is performed in a buffer that maintains the solubility of the product, such as in the presence of 0.5 to 2M urea, in the presence of polypeptides and / or polynucleotides that increase solubility, etc. Target domains include endosomolytic domains, such as the influenza virus HA domain; and other polypeptides that contribute to production, such as IF2 domains, GST domains, GRPE domains, etc. Polypeptides can be formulated to improve stability. For example, peptides can be PEGylated, where the polyethyleneoxy groups provide for extended life in the blood.

[0344] Additionally, or alternatively, the B-GEn.16 polypeptide or a variant thereof may be fused to a polypeptide penetration domain to facilitate cellular uptake. A variety of penetration domains are known in the art and can be used for non-integrating polypeptides of the present disclosure, including polypeptides, peptide mimics, and non-peptide vectors. For example, a penetration peptide may be derived from the third alpha helix of the Drosophila melanogaster transcription factor Antennapaedia gene, called penetratin, which comprises the amino acid sequence RQIKIWFQNRRMKWKK (this sequence is not disclosed under this patent application). As another example, the penetration peptide comprises the amino acid sequence of the HIV-1 tat basic region, which may include, for example, amino acids 49-57 of the naturally occurring tat protein.

[0345] Other penetration peptide domains include polyarginine motifs, e.g., the amino acid 34-56 region of the HIV-1rev protein, nona-arginine, acta-arginine, etc. (see, e.g., Futaki et al. (2003) Curr Protein Pept Sci. 2003 Apr; 4(2): 87-9 and 446; and Wender et al. (2000) Proc. Natl. Acad. Sci. USA 2000 November 21; 97(24): 13003-8; published U.S. Patent Application Publication Nos. 20030220334; 20030083256; 20030032593; and 20030022831, specifically incorporated herein by reference to teach translocation peptides and peptides). The nona-arginine (R9) sequence is one of the more effective PTDs that has been characterized (Wender et al., 2000; Uemura et al. 2002). The site for fusion can be selected to optimize the biological activity, secretion or binding properties of the polypeptide. The optimal site can be determined by routine experiments. In some embodiments, the polypeptide penetration domain is chemically modified to increase the bioavailability of the PTD. Exemplary modifications are disclosed in Expert Opin Drug Deliv. 2009 Nov; 6(11): 1195-205.

[0346] Typically, an effective amount of guide RNA and / or B-GEn.16 polypeptide or variant thereof and / or donor polynucleotide is provided to the target DNA or cell to induce targeted modification. The effective amount of guide RNA and / or B-GEn.16 polypeptide or variant thereof and / or donor polynucleotide is relative to a negative control, for example, a cell contacted with an empty vector or an unrelated polypeptide, inducing a 2-fold increase or more in the amount of targeted modification observed with gRNA. That is, an effective amount or dosage of guide RNA and / or B-GEn.16 polypeptide or variant thereof and / or donor polynucleotide will induce a 2-fold increase, a 3-fold increase, a 4-fold increase or more in the amount of target modification observed at the target DNA region, in some embodiments, a 5-fold increase, a 6-fold increase or more, sometimes a 7-fold or 8-fold increase or more in the amount of recombination observed, such as an increase of 10 times, 50 times or 100 times or more, in some embodiments, an increase of 200 times, 500 times, 700 times or 1000 times or more, such as an observed recombination amount increase of 5000 times or 10,000 times. The amount of target modification can be measured by any suitable method. For example, a split reporter construct comprising a sequence complementary to the spacer of a guide RNA (which is flanked by homologous sequences) can be co-transfected into a cell when recombined to reconstruct a nucleic acid encoding an active reporter and to assess the amount of the reporter protein after contact with the guide RNA and / or the B-GEn.16 polypeptide or its variant and / or the donor polynucleotide, for example, 2 hours, 4 hours, 8 hours, 12 hours, 24 hours, 36 hours, 48 ​​hours, 72 hours or more after contact with the guide RNA and / or the B-GEn.16 polypeptide or its variant and / or the donor polynucleotide. As another more sensitive assay, for example, the extent of recombination at the target region of genomic DNA containing the target DNA sequence can be assessed by PCR or Southern hybridization of the region after contact with the guide RNA and / or B-GEn.16 polypeptide or its variants and / or donor polynucleotides, for example 2 hours, 4 hours, 8 hours, 12 hours, 24 hours, 36 hours, 48 ​​hours, 72 hours or longer after contact with the guide RNA and / or B-GEn.16 polypeptide or its variants and / or donor polynucleotides.

[0347] Contacting cells with guide RNA and / or B-GEn.16 polypeptide or variants thereof and / or donor polynucleotides can be performed in any culture medium and any culture conditions that promote cell survival. For example, cells can be suspended in any suitable nutrient medium, such as Iscove's modified DMEM or RPMI1640, supplemented with fetal bovine serum or heat-inactivated fetal bovine serum (about 5-10%), L-glutamine, thiols, particularly 2-mercaptoethanol, and antibiotics, such as penicillin and streptomycin. The culture medium may contain growth factors to which the cells respond. As defined herein, growth factors are molecules that can promote the survival, growth and / or differentiation of cells in culture or intact tissues through specific effects on transmembrane receptors. Growth factors include polypeptide factors and non-polypeptide factors. Conditions that promote cell survival generally allow non-homologous end joining and homology-directed repair. In applications where it is desired to insert a polynucleotide sequence into a target DNA sequence, a polynucleotide comprising a donor sequence to be inserted is also provided to the cell. "Donor sequence" or "donor polynucleotide" refers to a nucleic acid sequence inserted at the cleavage site induced by a B-GEn.16 polypeptide or its variant. The donor polynucleotide has enough sequence identity to the flanking genomic region of the cleavage site, such as to the nucleotide sequence flanking the cleavage site, such as within about 50 bases or less of the cleavage site, such as within about 30 bases, within about 15 bases, within about 10 bases, within about 5 bases, or the flank next to the cleavage site has 70%, 80%, 85%, 90%, 95% or 100% sequence identity to support homology-directed repair between it and the genomic sequence with homology thereto. About 25, 50, 100 or 200 nucleotides of the homologous sequence between the donor and the genomic sequence, or more than 200 nucleotides (or any integer value between 10 and 200 nucleotides, or more) will support homology-directed repair. The donor sequence can be of any length, such as 10 or more nucleotides, 50 or more nucleotides, 100 or more nucleotides, 250 or more nucleotides, 500 or more nucleotides, 1000 or more nucleotides, 5000 or more nucleotides, etc.

[0348] The donor sequence is usually not completely the same as the genomic sequence it replaces. On the contrary, the donor sequence may include at least one or more single base substitutions, insertions, deletions, inversions or rearrangements relative to the genomic sequence, as long as there is enough sequence identity to support homology directed repair. In some embodiments, the donor sequence includes non-homologous sequences flanked by two regions (also referred to as homology arms) homologous to the target DNA region, so that the homology directed repair between the target DNA region and the two flanking homology arms causes the insertion of non-homologous sequences at the target region. The donor sequence may also include a vector backbone, which includes sequences that are not homologous to the target DNA region and are not intended to be inserted into the target DNA region. Typically, the homology region of the donor sequence has at least 50% sequence identity with the genomic sequence of the desired recombination. In certain embodiments, there is 60%, 70%, 80%, 90%, 95%, 98%, 99% or 99.9% sequence identity. Depending on the length of the donor polynucleotide, there may be any value between 1% and 100% sequence identity. The donor sequence may include certain sequence differences compared to the genomic sequence, such as restriction sites, nucleotide polymorphisms, selectable markers (e.g., drug resistance genes, fluorescent proteins, enzymes, etc.), etc., which can be used to evaluate the successful insertion of the donor sequence at the cleavage site, or in some cases can be used for other purposes (e.g., indicating expression at the targeted genomic locus). In some embodiments, if located in the coding region, such nucleotide sequence differences will not change the amino acid sequence, or will make the amino acid changes not substantially affect the structure or function of the protein. Alternatively, these sequence differences can include flanking recombination sequences (such as FLPs, loxP sequences, etc.), which can be activated at a later time to remove the marker sequence.

[0349] The donor sequence can be provided to the cell in the form of single-stranded DNA, single-stranded RNA, double-stranded DNA or double-stranded RNA. It can be introduced into the cell in a linear or circular form. If introduced in a linear form, the ends of the donor sequence can be protected by methods known to those skilled in the art (such as preventing exonuclease degradation). For example, one or more dideoxynucleotide residues are added to the 3' end of the linear molecule and / or self-complementary oligonucleotides are connected to one or both ends. For example, see Chang et al. (1987) Proc. Natl. Acad. Sci. USA 84: 4959-4963; Nehls et al. (1996) Science 272: 886-889. For protecting the exogenous polynucleotide from degradation, the extra method includes but is not limited to, adding terminal amino groups and using modified internucleotide connections, such as phosphorothioates, phosphoramidates and O-methyl ribose or deoxyribose residues. As a substitute for protecting the end of the linear donor sequence, the sequence of extra length can be included outside the homology arm, which can be degraded without affecting the reorganization. The donor sequence can be introduced into the cell as a part of a vector molecule, and the vector molecule has extra sequences, such as replication origin, promoter and genes encoding antibiotic resistance. In addition, the donor sequence can be introduced as naked (such as unmodified) nucleic acid, as a nucleic acid that forms a complex with a reagent such as liposomes or poloxamer, or can be delivered by a virus (such as adenovirus, AAV), as described above for nucleic acids encoding guide RNA and / or B-GEn.16 polypeptide or its variants and / or donor polynucleotides.

[0350] According to the above method, the target DNA region can be cut and modified in vitro, such as "gene modification". In some embodiments, when a selectable marker has been inserted into the target DNA region, the cell population containing the gene modification can be enriched by separating the gene-modified cells from the remaining population. Before enrichment, the "genetically modified" cells may only account for about 1% or more of the cell population (e.g., 2% or more, 3% or more, 4% or more, 5% or more, 6% or more, 7% or more, 8% or more, 9% or more, 10% or more, 15% or more, or 20% or more). The separation of "genetically modified" cells can be achieved by any suitable separation technique suitable for the selectable marker used. For example, if a fluorescent marker has been inserted, cells can be separated by fluorescence-activated cell sorting, and if a cell surface marker has been inserted, cells can be separated from a heterogeneous population by the following techniques: affinity separation techniques, such as magnetic separation, affinity chromatography, "panning" with affinity reagents attached to a solid matrix, or other suitable techniques. The technology that provides accurate separation includes fluorescence-activated cell sorters, which may have varying degrees of complexity. For example, multicolor channels, low-angle and obtuse light scattering detection channels, impedance channels, etc. Cells relative to dead cells can be selected by using dyes associated with dead cells (e.g., propidium iodide). Any technology can be used as long as the vitality of genetically modified cells is not unduly damaged. In this way, a cell composition highly enriched for cells containing modified DNA is obtained. So-called "highly enriched" means that the genetically modified cells will be 70% or more of the cell composition, 75% or more, 80% or more, 85% or more, 90% or more, for example, about 95% or more of the cell composition, or 98% or more. In other words, this composition can be a substantially pure composition of genetically modified cells.

[0351] Genetically modified cells produced by the methods described herein can be used immediately. In addition or alternatively, cells can be frozen at liquid nitrogen temperature and stored for a long time and reused after thawing. In this case, cells are usually frozen in 10% dimethyl sulfoxide (DMSO), 50% serum, 40% buffered medium or some other such solution commonly used in the art to preserve cells at such freezing temperatures and thawed in a manner commonly known in the art to thaw frozen culture cells.

[0352] Genetically modified cells can be cultured in vitro under various culture conditions. Cells can be expanded in a culture medium, such as growing under conditions that promote cell proliferation. The culture medium can be liquid or semisolid, such as containing agar, methylcellulose, etc. The cell population can be suspended in an appropriate nutrient medium, such as lscove's modified DMEM or RPMI 1640, usually supplemented with fetal bovine serum (about 5-10%), L-glutamine, thiols (especially 2-mercaptoethanol) and antibiotics (such as penicillin and streptomycin). The culture may contain growth factors to which the corresponding cells respond. Growth factors, as defined herein, are molecules that can promote cell survival, growth and / or differentiation in culture or intact tissues through specific effects on transmembrane receptors. Growth factors include polypeptide and non-polypeptide factors. Cells genetically modified in this manner can be transplanted into subjects for purposes such as gene therapy, such as treating diseases or as antiviral, antipathogenic or anticancer therapies; for the production of genetically modified organisms in agriculture, or for biological research. The subject can be a newborn, a teenager or an adult. Of particular concern is mammals. The mammals that can be treated by the method include canines and felines, equines, bovines, ovines, etc., as well as primates, especially humans. Animal models, especially small mammals (such as mice, rats, guinea pigs, hamsters, long-tailed animals (such as rabbits), etc.) can be used for experimental research.

[0353] The cells may be provided to the subject alone or in combination with an appropriate culture medium or matrix, such as to support the growth and / or organization of the cells in the tissue into which they are transplanted. Typically, at least 1 x 10 3 cells, for example 5x10 3 cells, 1x10 4 cells, 5x10 4 cells, 1x10 5 cells, 1x10 6Cells can be introduced into the subject by any of the following routes: parenteral, subcutaneous, intravenous, intracranial, intraspinal, intraocular, or by injection into the cerebrospinal fluid. Cells can be introduced by injection, catheter, etc. Examples of methods for local delivery, i.e., delivery to the site of injury, include, e.g., via an Ommaya capsule, e.g., for intracapsular delivery (see, e.g., U.S. Pat. Nos. 5,222,982 and 5,385,582, incorporated herein by reference); by bolus injection, e.g., by syringe, e.g., into a joint; by continuous infusion, e.g., by cannulation, e.g., by convection (see, e.g., U.S. Pat. Nos. 20070254842, incorporated herein by reference); or by implanting a device for reversibly adhering cells (see, e.g., U.S. Application Nos. 20080081064 and 20090196903, incorporated herein by reference). Cells may also be introduced into embryos (e.g., blastocysts) for use in generating transgenic animals (e.g., transgenic mice).

[0354] In some embodiments, the nucleotide sequence encoding the guide RNA and / or B-GEn.16 polypeptide or its variant is operably linked to a control element, such as a transcriptional control element, such as a promoter. Transcriptional control elements typically function in eukaryotic cells, such as mammalian cells (such as human cells) or prokaryotic cells, such as bacteria or ancient cells. In some embodiments, the nucleotide sequence encoding the guide RNA and / or B-GEn.16 polypeptide or its variant is operably linked to a plurality of control elements, which allow the nucleotide sequence encoding the guide RNA and / or B-GEn.16 polypeptide or its variant to be expressed in both prokaryotic and eukaryotic cells.

[0355] A promoter can be a constitutively active promoter (e.g., a constitutive promoter that is in an active "on" state), it can be an inducible promoter (e.g., a promoter whose activity / "on" or inactive / "off" state is controlled by an external stimulus, such as the presence of a specific temperature, compound, or protein), it can be a spatially restricted promoter (e.g., a transcriptional control element, an enhancer, etc.) (e.g., a tissue-specific promoter, a cell type-specific promoter, etc.), and it can be a temporally restricted promoter (e.g., a promoter that is in an "on" state or an "off" state at a specific stage of embryonic development or a specific stage of a biological process, such as the hair follicle cycle in mice).

[0356] Suitable promoters can be derived from viruses and therefore can be referred to as viral promoters, or they can also be derived from any organism, including prokaryotes or eukaryotic organisms. Suitable promoters can be used to drive the expression of any RNA polymerase (such as pol I, pol II, pol III). Exemplary promoters include, but are not limited to, SV40 early promoter, mouse mammary tumor virus long terminal repeat (LTR) promoter, adenovirus major late promoter (Ad MLP); herpes simplex virus (HSV) promoter, cytomegalovirus (CMV) promoter, such as CMV immediate early promoter region (CMVIE), Rous sarcoma virus (RSV) promoter, human U6 small nuclear promoter (U6) (Miyagishi et al., Nature Biotechnology 20, 497-500 (2002)), enhanced U6 promoter (such as, Xia et al., Nucleic Acids Res. 2003 September 1; 31 (17)), human H1 promoter (H1), etc.

[0357] Examples of inducible promoters include, but are not limited to, T7 RNA polymerase promoter, T3 RNA polymerase promoter, isopropyl-β-D-thiogalactoside (IPTG) regulated promoter, lactose inducible promoter, heat shock promoter, tetracycline regulated promoter (such as Tet-ON, Tet-OFF, etc.), steroid regulated promoter, metal regulated promoter, estrogen receptor regulated promoter, etc. Therefore, inducible promoters can be regulated by the following molecules, which include, but are not limited to doxycycline; RNA polymerase (such as T7 RNA polymerase); estrogen receptor; estrogen receptor fusion protein, etc.

[0358] In some embodiments, the promoter is a spatially restricted promoter (e.g., a cell type-specific promoter, a tissue-specific promoter, etc.) such that in a multicellular organism, the promoter is active (e.g., "on") in a specific cell subpopulation. Spatially restricted promoters may also be referred to as enhancers, transcriptional control elements, control sequences, etc. Any suitable spatially restricted promoter may be used, and the selection of a suitable promoter (e.g., a brain-specific promoter, a promoter that drives expression in a neuronal subpopulation, a promoter that drives expression in the reproductive system, a promoter that drives expression in the lungs, a promoter that drives expression in the muscles, a promoter that drives expression in the islet cells of the pancreas, etc.) will depend on the organism. For example, various spatially restricted promoters are known for plants, flies, worms, mammals, mice, etc. Therefore, spatially restricted promoters may be used to regulate the expression of nucleic acids encoding B-GEn.16 polypeptides or variants thereof in a variety of different tissues and cell types, depending on the organism. Some spatially restricted promoters are also temporally restricted, such that the promoter is "on" or "off" at specific stages of embryonic development or at specific stages of a biological process (such as the hair follicle cycle in mice).

[0359] For the purpose of illustration, examples of spatially restricted promoters include, but are not limited to, neuron-specific promoters, adipocyte-specific promoters, cardiomyocyte-specific promoters, smooth muscle-specific promoters, photoreceptor-specific promoters, etc.Neuron-specific spatially restricted promoters include, but are not limited to, neuron-specific enolase (NSE) promoter (see, e.g., EMBL HSEN02, X51956); aromatic amino acid decarboxylase (AADC) promoter; neurofilament promoter (see, e.g., GenBank HUMNFL, L04147); synapsin promoter (see, e.g., GenBank HUMSYNIB, M55301); thy-1 promoter (see, e.g., Chen et al. (1987) Cell 51:7-19; and Llewellyn et al. (2010) Nat. Med. 16(10):1161-1166); serotonin receptor promoter (see, e.g., GenBank S62283); tyrosine hydroxylase promoter (see, e.g., GenBank S62284); promoter (see, e.g., Oh et al. (2009) Gene Ther. 16:437; Sasaoka et al. (1992) Mol. Brain Res. 16:274; Boundy et al. (1998) J. Neurosci. 18:9989; and Kaneda et al. (1991) Neuron:583-594); GnRH promoter (see, e.g., Radovick et al. (1991) Proc. Natl. Acad. Sci. USA 88:3402-3406); L7 promoter (see, e.g., Oberdick et al. (1990) Science 248:223-226); DNMT promoter (see, e.g., Bartge et al. (1988) Proc. Natl. Acad. Sci. USA 85:3648-3652); enkephalin promoter (see, e.g., Comb et al. (1988) EMBO J. 17:3793-3805); myelin basic protein (MBP) promoter; Ca2+ / calmodulin-dependent kinase II-α (Ca2+). 2+-calmodulin-dependent protein kinase II-alpha, CaMKIIa) promoter (see, e.g., Mayford et al. (1996) Proc. Natl. Acad. Sci. USA 93:13250; and Casanova et al. (2001) Genesis 31:37); CMV enhancer / platelet-derived growth factor-p promoter (CMV enhancer / platelet-derived growth factor-p promoter) (see, e.g., Liu et al. (2004) Gene Therapy 11:52-60), etc.

[0360] Adipocyte-specific spatially restricted promoters include, but are not limited to, the aP2 gene promoter / enhancer, such as the region from -5.4 kb to +21 bp of the human aP2 gene (see, e.g., Tozzo et al. (1997) Endocrinol. 138:1604; Ross et al. (1990) Proc. Natl. Acad. Sci. USA 87:9590; and Pavjani et al. (2005) Nat. Med. 11:797); the glucose transporter-4 (GLUT4) promoter (see, e.g., Knight et al. (2003) Proc. Nat / . Acad. Sci. USA 100:14725); the fatty acid transporter (FAT / CD36) promoter (see, e.g., Kuriki et al. (2002) Biol. Pharm. Bull. 25:1476; and Sato et al. (2003) Proc. Natl. Acad. Sci. USA 100:14725). al. (2002) J. Biol. Chem. 277:15703); stearoyl-CoA desaturase-1 (SCD1) promoter (Tabor et al. (1999) J. Biol. Chem. 274:20603); leptin promoter (see, e.g., Mason et al. (1998) Endocrinol. 139:1013; and Chen et al. (1999) Biochem. Biophys. Res. Comm. 262:187); adiponectin promoter (see, e.g., Kita et al. (2005) Biochem. Biophys. Res. Comm. 331:484; and Chakrabarti (2010) Endocrinol. 151:2408); adipocyte promoter (see, e.g., Platt et al. (2003) Endocrinol. 151:2408); al. (1989) Proc. Nat / . Acad. Sci. USA 86:7490); resistin promoter (see, e.g., Seo et al. (2003) Malec. Endocrinol. 17:1522), etc.

[0361] Cardiomyocyte-specific spatially restricted promoters include, but are not limited to, control sequences from the following genes: myosin light chain-2, α-myosin heavy chain, AE3, cardiac troponin C, cardiac actin, etc. Franz et al. (1997) Cardiovasc. Res. 35:560-566; Robbins et al. (1995) Ann. NY Acad. Sci. 752:492-505; Linn et al. (1995) Circ. Res. 76:584-591; Parmacek et al. (1994) Mol. Cell. Biol. 14:1870-1885; Hunter et al. (1993) Hypertension 22:608-617; and Sartorelli et al. (1992) Proc. Natl. Acad. Sci. USA 89:4047-4051.

[0362] Smooth muscle-specific spatially restricted promoters include, but are not limited to, the SM22a promoter (see, e.g., Akyilrek et al. (2000) Mol. Med. 6:983; and U.S. Pat. No. 7,169,874); the smooth muscle actin promoter (see, e.g., WO 2001 / 018048); the α-smooth muscle actin promoter, and the like. For example, a 0.4 kb region of the SM22a promoter, which contains two CArG elements, has been shown to mediate vascular smooth muscle cell-specific expression (see, e.g., Kim et al. (1997) Mol. Cell. Biol. 17, 2266-2278; Li et al., (1996) J. Cell Biol. 132, 849-859; and Moessler, et al. (1996) Development 122, 2415-2425).

[0363] Photoreceptor-specific spatially restricted promoters include, but are not limited to, rhodopsin promoter, rhodopsin kinase promoter (Young et al. (2003) Ophthalmol. Vis. Sci. 44: 4076); β-phosphodiesterase gene promoter (Nicoud et al. (2007) J. Gene Med. 9: 1015); retinitis pigmentosa gene promoter (Nicoud et al. (2007), supra); interphotoreceptor retinol binding protein (IRBP) gene enhancer (Nicoud et al. (2007), supra); IRBP gene promoter (Yokoyamae et al. (1992), Exp Eye Res. 55: 225), etc.

[0364] Compositions containing guide RNA

[0365] In some embodiments, compositions comprising guide RNA are provided herein. In addition to guide RNA, the composition may also include one or more of the following: salts, such as NaCl, MgCl2, KCl, MgSO4, etc.; buffers, such as tris hydroxyethyl buffer, N-(2-hydroxyethyl) piperazine-N'-(2-ethanesulfonic acid) (HEPES), 2-(N-morpholino) ethanesulfonic acid (MES), MES sodium salt, 3-(N-morpholino) propanesulfonic acid (MOPS), N-tris[hydroxymethyl]methyl-3-aminopropanesulfonic acid (TAPS), etc.; solubilizing agents; detergents, such as non-ionic detergents, such as Tween-20, etc.; nuclease inhibitors, etc. For example, in some embodiments, the composition comprises guide RNA and a buffer for stabilizing nucleic acids.

[0366] In some embodiments, the guide RNA present in the composition is pure, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or greater than 99% pure, wherein "% purity" refers to the percentage of the guide RNA that is free of other macromolecules or contaminants that may be present during the guide RNA production process.

[0367] Compositions containing B-GEn.16 polypeptides

[0368] In some embodiments, a composition is provided herein, comprising a B-GEn.16 polypeptide or a variant thereof expressed by a codon-optimized polynucleotide sequence. In addition to the B-GEn.16 polypeptide or a variant thereof, the composition further comprises one or more of the following: salts, such as NaCl, MgCl2, KCl, MgSO4, etc.; buffers, such as Tris buffer, HEPES, MES, MES sodium salt, MOPS, TAPS, etc.; solubilizers; detergents, such as non-ionic detergents, such as Tween-20, etc.; nuclease inhibitors; reducing agents (such as dithiothreitol), etc.

[0369] In some embodiments, the B-GEn.16 polypeptide or its variant present in the composition is pure, for example, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or more pure, wherein "% purity" refers to the percentage of B-GEn.16 polypeptide or its variant that does not contain other proteins, other macromolecules, or contaminants that may be present in the production process of B-GEn.16 polypeptide or its variant.

[0370] Composition comprising guide RNA and site-directed modifying polypeptide

[0371] In some embodiments, a composition is provided herein, comprising: (i) a guide RNA or a polynucleotide encoding a guide RNA; and ii) a nucleic acid comprising a codon-optimized polynucleotide sequence encoding a B-GEn.16 polypeptide or a variant thereof, or a B-GEn.16 polypeptide or a variant thereof expressed by the nucleic acid. In some embodiments, the B-GEn.16 polypeptide or a variant thereof exhibits an enzymatic activity that modifies a target DNA. In some embodiments, the B-GEn.16 polypeptide or a variant thereof exhibits an enzymatic activity that modifies a polypeptide encoded by a target DNA. In some embodiments, the B-GEn.16 polypeptide or a variant thereof can regulate the transcription of a target DNA.

[0372] In some embodiments, the components of the composition are individually pure, such as each component is at least 75%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or at least 99% pure. In some embodiments, the components of the composition are pure before being added to the composition.

[0373] Reagent test kit

[0374] In some embodiments, a kit is provided for implementing the methods described herein. The kit may include one or more of the following: a B-GEn.16 polypeptide or a variant thereof expressed by, for example, a codon-optimized polynucleotide sequence; a guide RNA; a nucleic acid comprising a nucleotide sequence encoding the guide RNA. The kit may include a complex comprising two or more of the following: a B-GEn.16 polypeptide or a variant thereof; a nucleic acid comprising nucleotides encoding the B-GEn.16 polypeptide or a variant thereof; a guide RNA; a nucleic acid comprising a nucleotide sequence encoding the guide RNA. In some embodiments, the kit comprises a B-GEn.16 polypeptide or a variant thereof, or a polynucleotide encoding the same polypeptide. In some embodiments, an active portion of a B-GEn.16 polypeptide or a variant thereof exhibits reduced or inactivated nuclease activity. In some embodiments, the B-GEn.16 polypeptide or a variant thereof is a B-GEn.16 fusion protein.

[0375] In some embodiments, the kit includes (a) a nucleic acid comprising a codon-optimized polynucleotide sequence encoding a B-GEn.16 polypeptide or a variant thereof, or encoding a B-GEn.16 polypeptide or a variant thereof expressed by a nucleic acid; and (b) a gRNA or a nucleic acid encoding a gRNA, wherein the gRNA is capable of guiding the B-GEn.16 polypeptide or a variant thereof to a target polynucleotide sequence. In some embodiments, the kit includes a nucleic acid comprising a codon-optimized polynucleotide sequence. The kit comprising a B-GEn.16 polypeptide or a variant thereof expressed by a codon-optimized polynucleotide sequence, or a nucleic acid comprising a codon-optimized polynucleotide sequence may further include one or more additional reagents, wherein such additional reagents may be selected from: a buffer for introducing a B-GEn.16 polypeptide or a variant thereof into a cell; a wash buffer; a control reagent; a control expression vector or polynucleotide; a reagent for producing a B-GEn.16 polypeptide or a variant thereof in vitro from DNA, etc.

[0376] In some embodiments of any of the kits described herein, the kit includes an sgRNA. In some embodiments, the kit includes two or more sgRNAs.

[0377] In some embodiments of any kit described herein, gRNA (including two or more guide RNAs) can be provided as an array (e.g., an RNA molecule array, a DNA molecule array encoding a guide RNA, etc.). For example, such a kit can be used in combination with a genetically modified host cell comprising a B-GEn.16 polypeptide or a variant thereof as described above.

[0378] In some embodiments of any of the kits described herein, the kit further comprises a donor polynucleotide to achieve a desired genetic modification.

[0379] The components of the kit may be provided in separate containers or combined in a single container.

[0380] Any kit described herein may further include one or more additional reagents, wherein such additional reagents may be selected from: dilution buffer; reconstitution solution; wash buffer; control reagent; control expression vector or polynucleotide; reagent for in vitro production of B-GEn.16 polypeptide or its variants from DNA, etc.

[0381] In addition to the above components, the kit may further include instructions for using the kit components to carry out the method. The instructions for carrying out the method are usually recorded on a suitable recording medium. For example, the instructions can be printed on a substrate such as paper or plastic. Therefore, the instructions can appear in the kit as a package insert, in the label of the kit container or its components (such as, related to packaging or sub-packaging), etc. In some embodiments, the instructions are present in a suitable computer-readable storage medium in the form of an electronic storage data file, such as a CD-ROM, a disk, a flash drive, etc. In other embodiments, the actual instructions are not present in the kit, but a method for obtaining instructions from a remote source (such as via the Internet) is provided. An example of this embodiment is a kit comprising a website, on which the instructions can be browsed and / or downloaded. Like the instructions, this method for obtaining the instructions is also recorded on a suitable substrate.

[0382] Methods of the present disclosure

[0383] Methods for modifying target DNA and / or polypeptides encoded by target DNA

[0384] In some embodiments, provided herein are methods for modifying a target DNA and / or a polypeptide encoded by a target DNA. In some embodiments, the method involves providing (i) a nucleic acid encoding SEQ ID NO: 1 or a variant thereof, the variant having at least 90% sequence identity with SEQ ID NO: 1, SEQ ID NO: 1 encoding a B-GEn.16 polypeptide or a variant thereof or encoding a B-GEn.16 polypeptide or a variant thereof expressed by the nucleic acid; and (ii) a gRNA or a nucleic acid encoding the gRNA, wherein the gRNA is capable of guiding the B-GEn.16 polypeptide or a variant thereof to a target polynucleotide sequence, thereby forming a complex comprising the B-GEn.16 polypeptide or a variant thereof and the gRNA ("targeting complex"), and contacting the target DNA comprising the target polynucleotide sequence.

[0385] In some embodiments, the method involves providing (i) a nucleic acid encoding SEQ ID NO:2 or a variant thereof having at least 90% sequence identity with SEQ ID NO:2, SEQ ID NO:2 encoding a B-GEn.16 polypeptide or a variant thereof or encoding a B-GEn.16 polypeptide or a variant thereof expressed by the nucleic acid; and (ii) a gRNA or a nucleic acid encoding the gRNA, wherein the gRNA is capable of guiding the B-GEn.16 polypeptide or a variant thereof to a target polynucleotide sequence, thereby forming a complex comprising the B-GEn.16 polypeptide or a variant thereof and the gRNA (a "targeting complex"), and contacting a target DNA comprising the target polynucleotide sequence.

[0386] In some embodiments, the method involves providing (i) a nucleic acid encoding SEQ ID NO:3 or a variant thereof having at least 90% sequence identity with SEQ ID NO:3, SEQ ID NO:3 encoding a B-GEn.16 polypeptide or a variant thereof or encoding a B-GEn.16 polypeptide or a variant thereof expressed by the nucleic acid; and (ii) a gRNA or a nucleic acid encoding the gRNA, wherein the gRNA is capable of guiding the B-GEn.16 polypeptide or a variant thereof to a target polynucleotide sequence, thereby forming a complex comprising the B-GEn.16 polypeptide or a variant thereof and the gRNA (a "targeting complex"), and contacting a target DNA comprising the target polynucleotide sequence.

[0387] In some embodiments, the method involves providing (i) a nucleic acid encoding SEQ ID NO:4 or a variant thereof having at least 90% sequence identity with SEQ ID NO:4, SEQ ID NO:4 encoding a B-GEn.16 polypeptide or a variant thereof or encoding a B-GEn.16 polypeptide or a variant thereof expressed by the nucleic acid; and (ii) a gRNA or a nucleic acid encoding the gRNA, wherein the gRNA is capable of guiding the B-GEn.16 polypeptide or a variant thereof to a target polynucleotide sequence, thereby forming a complex comprising the B-GEn.16 polypeptide or a variant thereof and the gRNA (a "targeting complex"), and contacting a target DNA comprising the target polynucleotide sequence.

[0388] In some embodiments, the method involves providing (i) a nucleic acid encoding SEQ ID NO:5 or a variant thereof having at least 90% sequence identity with SEQ ID NO:5, SEQ ID NO:5 encoding a B-GEn.16 polypeptide or a variant thereof or encoding a B-GEn.16 polypeptide or a variant thereof expressed by the nucleic acid; and (ii) a gRNA or a nucleic acid encoding the gRNA, wherein the gRNA is capable of guiding the B-GEn.16 polypeptide or a variant thereof to a target polynucleotide sequence, thereby forming a complex comprising the B-GEn.16 polypeptide or a variant thereof and the gRNA (a "targeting complex"), and contacting a target DNA comprising the target polynucleotide sequence.

[0389] In some embodiments, the method involves providing (i) a nucleic acid encoding SEQ ID NO:6 or a variant thereof having at least 90% sequence identity with SEQ ID NO:6, SEQ ID NO:6 encoding a B-GEn.16 polypeptide or a variant thereof or encoding a B-GEn.16 polypeptide or a variant thereof expressed by the nucleic acid; and (ii) a gRNA or a nucleic acid encoding the gRNA, wherein the gRNA is capable of guiding the B-GEn.16 polypeptide or a variant thereof to a target polynucleotide sequence, thereby forming a complex comprising the B-GEn.16 polypeptide or a variant thereof and the gRNA (a "targeting complex"), and contacting a target DNA comprising the target polynucleotide sequence.

[0390] In some embodiments, the method involves providing (i) a nucleic acid encoding SEQ ID NO:7 or a variant thereof having at least 90% sequence identity with SEQ ID NO:7, SEQ ID NO:7 encoding a B-GEn.16 polypeptide or a variant thereof or encoding a B-GEn.16 polypeptide or a variant thereof expressed by the nucleic acid; and (ii) a gRNA or a nucleic acid encoding the gRNA, wherein the gRNA is capable of guiding the B-GEn.16 polypeptide or a variant thereof to a target polynucleotide sequence, thereby forming a complex comprising the B-GEn.16 polypeptide or a variant thereof and the gRNA (a "targeting complex"), and contacting a target DNA comprising the target polynucleotide sequence.

[0391] In some embodiments, the method involves providing (i) a nucleic acid encoding SEQ ID NO:8 or a variant thereof having at least 90% sequence identity with SEQ ID NO:8, SEQ ID NO:8 encoding a B-GEn.16 polypeptide or a variant thereof or encoding a B-GEn.16 polypeptide or a variant thereof expressed by the nucleic acid; and (ii) a gRNA or a nucleic acid encoding the gRNA, wherein the gRNA is capable of guiding the B-GEn.16 polypeptide or a variant thereof to a target polynucleotide sequence, thereby forming a complex comprising the B-GEn.16 polypeptide or a variant thereof and the gRNA (a "targeting complex"), and contacting a target DNA comprising the target polynucleotide sequence.

[0392] In some embodiments, the method involves providing (i) a nucleic acid encoding SEQ ID NO:9 or a variant thereof having at least 90% sequence identity with SEQ ID NO:9, SEQ ID NO:9 encoding a B-GEn.16 polypeptide or a variant thereof or encoding a B-GEn.16 polypeptide or a variant thereof expressed by the nucleic acid; and (ii) a gRNA or a nucleic acid encoding the gRNA, wherein the gRNA is capable of guiding the B-GEn.16 polypeptide or a variant thereof to a target polynucleotide sequence, thereby forming a complex comprising the B-GEn.16 polypeptide or a variant thereof and the gRNA (a "targeting complex"), and contacting a target DNA comprising the target polynucleotide sequence.

[0393] In some embodiments, the method involves providing (i) a nucleic acid encoding SEQ ID NO: 133 or a variant thereof having at least 90% sequence identity with SEQ ID NO: 133, SEQ ID NO: 133 encoding a B-GEn.16 polypeptide or a variant thereof or encoding a B-GEn.16 polypeptide or a variant thereof expressed by said nucleic acid; and (ii) a gRNA or a nucleic acid encoding the gRNA, wherein the gRNA is capable of guiding the B-GEn.16 polypeptide or a variant thereof to a target polynucleotide sequence, thereby forming a complex comprising the B-GEn.16 polypeptide or a variant thereof and the gRNA (a "targeting complex"), and contacting a target DNA comprising the target polynucleotide sequence.

[0394] In some embodiments, provided herein are methods for targeting, editing, modifying or manipulating a target DNA at one or more locations in a cell or in an in vitro environment, comprising introducing into a cell or in vitro environment (a) a nucleic acid comprising, for example, a codon-optimized polynucleotide sequence encoding a B-GEn.16 polypeptide or a variant thereof, or a B-GEn.16 polypeptide or a variant thereof expressed by the nucleic acid; and (b) a gRNA or a nucleic acid encoding the gRNA, wherein the gRNA is capable of guiding the B-GEn.16 polypeptide or a variant thereof to a target polynucleotide sequence in the target DNA. In some embodiments, the method comprises introducing a nucleic acid comprising a codon-optimized polynucleotide sequence into a cell or in vitro environment. In some embodiments, the method comprises introducing a B-GEn.16 polypeptide or a variant thereof expressed by the nucleic acid into a cell or in vitro environment. In some embodiments, the B-GEn.16 polypeptide comprises (or consists of) an amino acid sequence of SEQ ID NO: 1. In some embodiments, the method comprises introducing a gRNA into a cell or in vitro environment. In some embodiments, the method comprises introducing a nucleic acid encoding a gRNA into a cell or in vitro environment. In some embodiments, the gRNA is a single guide RNA (sgRNA). In some embodiments, the method includes introducing one or more additional gRNAs or nucleic acids encoding one or more additional gRNAs targeting the target DNA into the cell or in vitro environment. In some embodiments, the method further includes introducing a donor template into the cell or in vitro environment.

[0395] In some embodiments, provided herein are methods for targeting, editing, modifying or manipulating a target DNA at one or more locations in a cell or in an in vitro environment, comprising introducing into a cell or in vitro environment (a) a nucleic acid encoding a B-GEn.16 polypeptide or a variant thereof, or a B-GEn.16 polypeptide or a variant thereof expressed by such a nucleic acid; and (b) a gRNA or a nucleic acid encoding the gRNA, wherein the gRNA is capable of guiding the B-GEn.16 polypeptide or a variant thereof to a target polynucleotide sequence in the target DNA. In some embodiments, the method comprises introducing a B-GEn.16 polypeptide or a variant thereof expressed by the nucleic acid into a cell or in vitro environment. In some embodiments, the B-GEn.16 polypeptide or a variant thereof comprises an amino acid sequence of SEQ ID NO: 1 or a variant having at least 95% sequence identity with these amino acid sequences. In some embodiments, the method comprises introducing a gRNA into a cell or in vitro environment. In some embodiments, the method comprises introducing a nucleic acid encoding a gRNA into a cell or in vitro environment. In some embodiments, the gRNA is a single guide RNA (sgRNA). In some embodiments, the method includes introducing one or more additional gRNAs or nucleic acids encoding one or more additional gRNAs targeting the target DNA into a cell or in vitro environment. In some embodiments, the method further includes introducing a donor template into a cell or in vitro environment.

[0396] As described above, gRNA or sgRNA and B-GEn.16 polypeptide or its variant can form a ribonucleoprotein complex. The guide RNA provides targeting specificity for the complex by comprising a nucleotide sequence complementary to the target DNA sequence. The B-GEn.16 polypeptide or its variant in the complex has endonuclease activity. In some embodiments, the complex modifies the target DNA, resulting in DNA cutting, DNA methylation, DNA damage, DNA repair, etc. In some embodiments, the complex modifies the target polypeptide (such as histone, DNA binding protein, etc.) associated with the target DNA, resulting in, for example, histone methylation, histone acetylation, histone ubiquitination, etc. For example, the target DNA can be naked DNA in vitro (such as not bound by DNA-related proteins), chromosomal DNA in in vitro cells, chromosomal DNA in in vivo cells, etc.

[0397] The nuclease activity of B-GEn.16 polypeptide or its variant described herein can cut the target DNA to produce double-strand breaks. These breaks are then repaired by cells in one of the following two ways: non-homologous end joining and homology-directed repair. In non-homologous end joining (NHEJ), double-strand breaks are repaired by directly connecting the broken ends to each other. In this process, the cleavage site can insert or lack several base pairs. In homology-directed repair, a donor polynucleotide with homology to the cut target DNA sequence is used as a template for repairing the cut target DNA sequence, resulting in genetic information being transferred from the donor polynucleotide to the target DNA. Therefore, new nucleic acid material can be inserted / copied to the site. In some embodiments, the target DNA is contacted with a donor polynucleotide. In some embodiments, the donor polynucleotide is introduced into the cell. Due to the modification of the target DNA by NHEJ and / or homology-directed repair, it can result in, for example, gene correction, gene replacement, gene marking, transgenic insertion, nucleotide deletion, nucleotide insertion, gene disruption, gene mutation, sequence replacement, etc. Thus, cleavage of DNA by B-GEn.16 polypeptides or variants thereof can be used to delete nucleic acid material from a target DNA sequence (e.g., by cleaving the target DNA sequence and allowing the cell to repair the sequence in the absence of exogenously provided donor polynucleotides to interfere with genes that make the cell susceptible to infection (such as CCRS or CXCR4 genes, which make T cells susceptible to HIV infection, facilitate the removal of pathogenic trinucleotide repeat sequences in neurons, facilitate the creation of gene knockouts and mutations as disease models in research, etc.). Thus, the method can be used to knock out genes (resulting in complete loss of transcription / translation or altered transcription / translation), or to knock genetic material into a selected locus in the target DNA.

[0398] Additionally or alternatively, if the guide RNA and B-GEn.16 polypeptide or its variant are co-administered into the cell with a donor polynucleotide sequence comprising at least one segment homologous to the target DNA sequence, the subject method can be used to add to the target DNA sequence, such as inserting or replacing nucleic acid material (e.g., "knocking in" protein-encoding nucleic acid, siRNA, miRNA, etc.), adding tags (such as 6xHis, fluorescent proteins (such as green fluorescent protein, yellow fluorescent protein, etc.), hemagglutinin (HA), FLAG, etc.), adding regulatory sequences to genes (such as promoters, polyadenylation signals, internal ribosome entry sequences (IRES), 2A peptides, start codons, stop codons, splicing signals, localization signals, etc.), modifying nucleic acid sequences (such as introducing mutations), etc. Therefore, the complex comprising the guide RNA and the B-GEn.16 polypeptide or its variant can be used in any in vitro or in vivo application, in which it is desired to modify the DNA in a site-specific (e.g., "targeted") manner, such as gene knockout, gene knockin, gene editing, gene tagging, sequence replacement, etc., for example, for gene therapy, such as for the treatment of diseases or as an antiviral, antipathogenic or anticancer therapy, the production of genetically modified organisms in agriculture, large-scale production of cellular proteins for therapeutic, diagnostic or research purposes, induction of iPS cells, biological research, targeted deletion or replacement of pathogen genes, etc.

[0399] In some embodiments, the methods described herein employ a B-GEn.16 polypeptide or variant thereof comprising a heterologous sequence (e.g., a B-GEn.16 fusion polypeptide). In some embodiments, the heterologous sequence may provide subcellular localization of the B-GEn.16 polypeptide or variant thereof (e.g., a nuclear localization signal (NLS) targeting the nucleus; a mitochondrial localization signal targeting the mitochondria; a chloroplast localization signal targeting the chloroplast; an ER retention signal, etc.). In some embodiments, the heterologous sequence may provide a tag that facilitates tracking or purification (e.g., a fluorescent protein, such as green fluorescent protein (GFP), YFP, RFP, CFP, mCherry, tdTomato, etc.; a histidine tag, such as a 6XHis tag; a hemagglutinin (HA) tag; a FLAG tag; a Myc tag, etc.). In some embodiments, the heterologous sequence may provide increased or decreased stability.

[0400] In some embodiments, the methods described herein use guide RNA and B-GEn.16 polypeptide or its variant, which is used as an inducible system for shutting down gene expression in target cells. In some embodiments, nucleic acids encoding appropriate guide RNA and / or appropriate B-GEn.16 polypeptide or its variant are integrated into the chromosome of the target cell and are controlled by an inducible promoter. When guide RNA and / or B-GEn.16 polypeptide or its variant are induced, when guide RNA and B-GEn.16 polypeptide or its variant are present and form a complex, target DNA is cut (or otherwise modified) at the target location (such as the target gene on a separate plasmid). Therefore, in some embodiments, target cells are engineered to include in the genome encoding a nucleic acid sequence of appropriate B-GEn.16 polypeptide or its variant and / or an appropriate guide RNA on a plasmid (such as, under the control of an inducible promoter), thereby allowing experiments in which the expression of any targeted gene (expressed by a separate plasmid introduced into a strain) is controlled by inducing the expression of guide RNA and B-GEn.16 polypeptide or its variant. In some embodiments, the B-GEn.16 polypeptide or its variant has an enzymatic activity that modifies the target DNA in a manner other than introducing double-strand breaks. The enzymatic activity of interest that can be used to modify the target DNA (e.g., by fusing a heterologous polypeptide with enzymatic activity with a B-GEn.16 polypeptide or its variant to produce a B-GEn.16 fusion polypeptide or its variant) includes, but is not limited to, methyltransferase activity, demethylase activity, DNA repair activity, DNA damage activity, deamination activity, dismutase activity, alkylation activity, depurination activity, oxidation activity, pyrimidine dimer formation activity, integrase activity, transposase activity, recombinase activity, polymerase activity, ligase activity, helicase activity, photolyase activity or glycosylase activity. Methylation and demethylation are important epigenetic gene regulation patterns recognized in the art, and DNA damage repair is essential for cell survival and the correct genome maintenance in response to environmental stress. Therefore, the method herein can be used for the epigenetic modification of the target DNA, and can be used to control the epigenetic modification at any position of the target DNA by introducing the desired sequence into the spacer of the guide RNA. The method herein can also be used for any desired position in the target DNA to carry out intentional and controllable damage to DNA. The method described herein can also be used for the DNA at any desired position in the target DNA to carry out sequence-specific and controllable repair. The method for specific position in the DNA modification enzyme activity targeting target DNA can be used for research and clinical applications.

[0401] In some embodiments, multiple guide RNAs are used to modify different locations on the same target DNA or different target DNAs simultaneously. In some embodiments, two or more guide RNAs target the same gene or transcript or locus. In some embodiments, two or more guide RNAs target different unrelated loci. In some embodiments, two or more guide RNAs target different but related loci.

[0402] In some embodiments, the B-GEn.16 polypeptide or its variant is provided directly as a protein. As a non-limiting example, fungi (such as yeast) can use protoplast transformation to transform exogenous proteins and / or nucleic acids (see Kawai et al., Bioeng Bugs. 2010 Nov-Dec; 1(6): 395-403, "Transformation of Saccharomyces cerevisiae and other fungi: methods and possible underlying mechanism"; and Tanka et al., Nature. 2004 Mar 18; 428(6980): 323-8: "Conformational variations in an infectious protein determine prion strain differences"; both documents are incorporated herein by reference in their entirety). Therefore, the B-GEn.16 polypeptide or its variant can be integrated into protoplasts (with or without nucleic acids encoding guide RNAs, with or without donor polynucleotides), and the protoplasts can be used to introduce the contents into yeast cells. B-GEn.16 polypeptide or its variant can be introduced into cells (provided to cells) by any suitable method; these methods are known to those of ordinary skill in the art. As another non-limiting example, B-GEn.16 polypeptide or its variant can be directly injected into cells (e.g., with or without nucleic acid encoding guide RNA, and with or without donor polynucleotide), such as cells of zebrafish embryos, pronuclei of fertilized mouse oocytes, etc.

[0403] Methods for regulating transcription

[0404] In some embodiments, methods for regulating transcription of a target nucleic acid in a host cell are provided herein. These methods generally involve contacting the target nucleic acid with a B-GEn.16 polypeptide without enzymatic activity and a guide RNA. The methods can be used in a variety of applications, and these applications are also provided herein.

[0405] The transcriptional regulation method of the present disclosure overcomes some shortcomings of the RNAi method. The transcriptional regulation method of the present disclosure is widely used, including research applications, drug discovery (such as high-throughput screening), target verification, industrial applications (such as crop engineering, microbial engineering, etc.), diagnostic applications, therapeutic applications and imaging technology.

[0406] In some embodiments, provided herein is a method for selectively regulating transcription of a target DNA in a host cell (such as a human cell). The method generally comprises: a) introducing into a host cell: i) a guide RNA, or a nucleic acid comprising a nucleotide sequence encoding the guide RNA; and ii) a B-GEn.16 polypeptide or a variant thereof, or a nucleic acid comprising a nucleotide sequence encoding the B-GEn.16 polypeptide or a variant thereof, wherein the B-GEn.16 polypeptide or a variant thereof exhibits reduced endodeoxyribonuclease activity. The guide RNA and the B-GEn.16 polypeptide or a variant thereof form a complex in the host cell; the complex selectively regulates transcription of the target DNA in the host cell.

[0407] In some embodiments, the methods described herein employ a modified form of a B-GEn.16 protein. In some embodiments, the modified form of a B-GEn.16 protein comprises an amino acid change (such as a deletion, insertion, or substitution) that reduces the nuclease activity of the B-GEn.16 protein. For example, in some embodiments, the modified form of a B-GEn.16 protein has less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, less than 5%, or less than 1% of the nuclease activity of a corresponding unmodified B-GEn.16 polypeptide. In some embodiments, the modified form of a B-GEn.16 polypeptide has no substantial nuclease activity. When a B-GEn.16 polypeptide or a variant thereof is a modified form of a B-GEn.16 polypeptide that has no substantial nuclease activity, it may be referred to as "dB-GEn.16".

[0408] In some embodiments, the transcriptional regulation methods described herein allow for selective regulation (e.g., reduction or increase) of a target nucleic acid in a host cell. For example, a "selective" reduction in target nucleic acid transcription reduces the transcription of the target nucleic acid by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or greater than 90%, compared to the transcription level of the target nucleic acid in the absence of a guide RNA / B-GEn.16 polypeptide or its variant complex. Selective reduction of the transcription of a target nucleic acid reduces the transcription of the target nucleic acid, but does not substantially reduce the transcription of a non-target nucleic acid, such as a reduction in the transcription of a non-target nucleic acid (if any) by less than 10%, compared to the transcription level of the non-target nucleic acid in the absence of a guide RNA / B-GEn.16 polypeptide or its variant complex.

[0409] In some embodiments, the B-GEn.16 polypeptide or its variant has the activity of regulating the transcription of the target DNA (such as in the case of the B-GEn.16 fusion polypeptide or its variant, etc.). In some embodiments, the B-GEn.16 fusion polypeptide or its variant comprising a heterologous polypeptide (such as a transcription activator or transcription inhibitor polypeptide) that exhibits the ability to increase or decrease transcription is used to increase or decrease the transcription of the target DNA at a specific position in the target DNA, which is guided by the spacer region of the guide RNA. Examples of source polypeptides that provide B-GEn.16 fusion polypeptides or their variants with transcriptional regulatory activity include, but are not limited to, light-induced transcriptional regulators, small molecule / drug-responsive transcriptional regulators, transcription factors, transcriptional inhibitors, etc. In some embodiments, the method is used to control the transcription of targeted gene-encoding RNA (mRNA encoding protein) and / or targeted non-coding RNA (such as tRNA, rRNA, snoRNA, siRNA, miRNA, long ncRNA, etc.). In some embodiments, the B-GEn.16 polypeptide or its variant has the enzymatic activity of modifying polypeptides associated with DNA (such as histones). In some embodiments, the enzymatic activity is a methyltransferase activity, a demethylase activity, an acetyltransferase activity, a deacetylase activity, a kinase activity, a phosphatase activity, an ubiquitin ligase activity (e.g., ubiquitination activity), a deubiquitinating activity, adenylating activity, deadenylating activity, SUMOylating activity, deSUMOylating activity, ribosylating activity, deribosylating activity, myristoylation activity, demyristoylation activity glycosylation activity (e.g., from a GlcNAc transferase) or a deglycosylation activity. The enzymatic activities listed herein can catalyze the covalent modification of proteins. As is known in the art, such modifications can alter the stability or activity of the target protein (e.g., depending on the target protein, phosphorylation caused by kinase activity can stimulate or silence the activity of the protein). Histones are protein targets of particular interest. As is known in the art, histones can bind to DNA and form a complex called a nucleosome. Histones can be modified (e.g., by methylation, acetylation, ubiquitination, phosphorylation) to cause structural changes in the surrounding DNA, thereby controlling the accessibility of potentially large portions of the DNA to interacting factors (e.g., transcription factors, polymerases, etc.). Individual histones can be modified in many different ways and in many different combinations (e.g., trimethylation of lysine 27 (H3K27) of histone 3 is associated with regions of DNA where transcription is repressed, while trimethylation of lysine 4 (H3K4) of histone 3 is associated with regions of DNA where transcription is active). Therefore, B-GEn.16 fusion polypeptides or variants thereof having histone modification activity can be used for site-specific control of chromosome structure and can be used to alter the histone modification pattern in selected regions of target DNA. This approach can be used in research and clinical applications.

[0410] Increased transcription

[0411] The "selective" increase in transcription of the target DNA can increase the transcription of the target DNA by at least 1.1 times (such as at least 1.2 times, at least 1.3 times, at least 1.4 times, at least 1.5 times, at least 1.6 times, at least 1.7 times, at least 1.8 times, at least 1.9 times, at least 2 times, at least 2.5 times, at least 3 times, at least 3.5 times, at least 4 times, at least 4.5 times, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times, at least 10 times, at least 12 times, at least 15 times or at least 20 times) compared to the transcription level of the target DNA in the absence of the guide RNA / B-GEn.16 polypeptide or its variant complex. Selectively increasing transcription of a target DNA can increase transcription of the target DNA without substantially increasing transcription of a non-target DNA, e.g., transcription of the non-target DNA is increased (if at all) by less than about 5-fold (e.g., less than about 4-fold, less than about 3-fold, less than about 2-fold, less than about 1.8-fold, less than about 1.6-fold, less than about 1.4-fold, less than about 1.2-fold, or less than about 1.1-fold) compared to the transcription level of the non-target DNA in the absence of the guide RNA / B-GEn.16 polypeptide or variant thereof complex.

[0412] As a non-limiting example, dB-GEn.16 can be fused to a heterologous sequence to increase transcription. Suitable fusion partners include, but are not limited to, polypeptides that provide an active increase in transcription indirectly by acting directly on the target DNA or a polypeptide associated with the target DNA (such as a histone or other DNA binding proteins). Suitable fusion partners include, but are not limited to, polypeptides that provide methyltransferase activity, demethylase activity, acetyltransferase activity, deacetylase activity, kinase activity, phosphatase activity, ubiquitin ligase activity, deubiquitinating activity, adenylation activity, deadenylation activity, SUMOylation activity, deSUMOylation activity, ribosylation activity, deribosylation activity, crotonylation, decrotonylation, propionylation, depropionylation, myristoylation activity or demyristoylation activity.

[0413] Other suitable fusion partners include, but are not limited to, polypeptides that directly increase transcription of the target nucleic acid (eg, transcriptional activators or fragments thereof, proteins or fragments thereof that recruit transcriptional activators, small molecule / drug-responsive transcriptional regulators, etc.).

[0414] Non-limiting examples of methods for using dB-GEn.16 fusion proteins to increase prokaryotic transcription include modifications of bacterial one-hybrid (B1H) or two-hybrid (B2H) systems. In the B1H system, a DNA binding domain (BD) is fused to a bacterial transcription activation domain (AD, such as the α subunit of Escherichia coli RNA polymerase (RNAPa)). Thus, dB-GEn.16 can be fused to a heterologous sequence comprising an AD. When the dB-GEn.16 fusion protein reaches the upstream region of the promoter (targeted there by the guide RNA), the AD (such as RNAPa) of the dB-GEn.16 fusion protein recruits the RNAP holoenzyme, thereby activating transcription. In the B2H system, the BD is not directly fused to the AD; instead, the interaction between them is mediated by protein-protein interactions (such as the GAL11P-GAL4 interaction). To modify this system for use in the present method, dB-GEn.16 can be fused to a first protein sequence that provides a protein-protein interaction (e.g., yeast GAL11P and / or GAL4 protein), and RNAa can be fused to a second protein sequence that completes a protein-protein interaction (e.g., if GAL11P is fused to dB-GEn.16, it is fused to GAL4; if GAL4 is fused to dB-GEn.16, it is fused to GAL11P, etc.). The binding affinity between GAL11P and GAL4 increases the binding efficiency and the transcription initiation rate (firing rate).

[0415] Non-limiting examples of methods for increasing eukaryotic transcription using dB-GEn.16 fusion proteins include fusing dB-GEn.16 to an activation domain (AD) (such as GAL4, herpes virus activation protein VP16 or VP64, human nuclear factor NF-KB p65 subunit, etc.). To make the system inducible, the expression of the dB-GEn.16 fusion protein can be controlled by an inducible promoter (such as TetON, Tet-OFF, etc.). Guide RNA can be designed to target known transcriptional response elements (such as promoters, enhancers, etc.), known upstream activation sequences (UAS), sequences of unknown or known functions that may be able to control the expression of target DNA, etc.

[0416] Additional Fusion Partners

[0417] Non-limiting examples of fusion partners that achieve increased or decreased transcription include, but are not limited to, transcription activator and transcription inhibitor domains (such as Krüppel-associated box (KRAB or SKD); Mad mSIN3 interaction domain (SID); ERF inhibitor domain (ERD), etc.). In some such cases, the dB-GEn.16 fusion protein is targeted to a specific location (such as a sequence) in the target DNA by a guide RNA and exhibits site-specific regulation, such as blocking the binding of RNA polymerase to the promoter (which selectively inhibits transcription activator function), and / or changing the local chromatin state (such as when a fusion sequence that can modify the target DNA or modify a polypeptide associated with the target DNA is used). In some embodiments, the change is temporary (such as transcriptional inhibition or activation). In some embodiments, the change is heritable (such as when epigenetic modifications are performed on the target DNA or proteins associated with the target DNA, such as ribosomal histones). In some embodiments, a heterologous sequence can be fused to the C-terminus of the dB-GEn.16 polypeptide. In some embodiments, the heterologous sequence can be fused to the N-terminus of the dB-GEn.16 polypeptide. In some embodiments, the heterologous sequence can be fused to an internal portion of the dB-GEn.16 polypeptide (e.g., a portion other than the N- or C-terminus). The biological effects of the methods using the dB-GEn.16 fusion protein can be detected by any suitable method (e.g., gene expression assays; chromatin-based assays such as chromatin immunoprecipitation (ChIP), in vivo chromatin assays (CiA), etc.).

[0418] In some embodiments, the methods involve the use of two or more different guide RNAs. For example, two different guide RNAs can be used in a single host cell, wherein the two different guide RNAs target two different target sequences in the same target nucleic acid. In some embodiments, the use of two different guide RNAs targeting two different target sequences in the same target nucleic acid provides for increasing the regulation (e.g., reduction or increase) of transcription of the target nucleic acid.

[0419] As another example, two different guide RNAs can be used in a single host cell, where the two different guide RNAs target two different target nucleic acids. Thus, for example, the transcriptional regulation method can further include introducing a second guide RNA into the host cell, or introducing a nucleic acid comprising a nucleotide sequence encoding the second guide RNA.

[0420] In some embodiments, the nucleic acid (e.g., a guide RNA, such as a single-molecule guide RNA; a donor polynucleotide; a nucleic acid encoding a B-GEn.16 polypeptide or a variant thereof, etc.) contains modifications or sequences that provide additional desired properties (e.g., modified or regulated stability; subcellular targeting; tracking, such as a fluorescent tag; a binding site for a protein or protein complex, etc.). Non-limiting examples include: a 5' cap (e.g., a 7-methylguanylate cap (m7G)); a 3' polyadenylation tail (e.g., a 3' poly(A) tail); a riboswitch sequence or an adapter sequence (e.g., to allow regulation of stability and / or regulation of accessibility of proteins and / or protein complexes); a termination sequence; a sequence that forms a dsRNA duplex (e.g., a hairpin); a modification or sequence that targets RNA to a subcellular location (e.g., the nucleus, mitochondria, chloroplasts, etc.); a modification or sequence that provides a tracking function (e.g., direct conjugation to a fluorescent molecule, conjugation to a portion that facilitates fluorescence detection, a sequence that allows fluorescence detection, etc.); a modification or sequence that provides a protein binding site (e.g., a protein that acts on DNA, including transcription activators, transcription inhibitors, DNA methyltransferases, DNA demethylases, histone acetyltransferases, histone deacetylases, etc.); an RNA modification that changes the structure of such RNA, and is therefore a B-GEn.16 ribonucleoprotein; and combinations thereof.

[0421] Multiple simultaneous guide RNAs

[0422] In some embodiments, multiple guide RNAs are used simultaneously in the same cell to simultaneously regulate transcription at different locations on the same target DNA or on different target DNAs. In some embodiments, two or more guide RNAs target the same gene or transcript or locus. In some embodiments, two or more guide RNAs target different unrelated loci. In some embodiments, two or more guide RNAs target different but related loci.

[0423] Because guide RNA is small and robust, they can appear on the same expression vector at the same time, and if desired, can even be under the same transcriptional control. In some embodiments, two or more (such as 3 or more, 4 or more, 5 or more, 10 or more, 15 or more, 20 or more, 25 or more, 30 or more, 35 or more, 40 or more, 45 or more or 50 or more) guide RNAs are expressed in target cells at the same time (from the same or different vectors / from the same or different promoters). In some embodiments, multiple guide RNAs can be encoded in a CRISPR targeting RNA array that mimics naturally occurring. The targeting segment is encoded as a sequence of about 30 nucleotides long (can be about 16 to about 100nt) and separated by CRISPR repeats. The array can be introduced into the cell by DNA encoding RNA or as RNA.

[0424] To express multiple guide RNAs, an artificial RNA processing system mediated by Csy4 endoribonuclease can be used. For example, multiple guide RNAs can be connected into a tandem array on a precursor transcript (such as expressed by a U6 promoter) and separated by a Csy4 specific RNA sequence. The co-expressed Csy4 protein cuts the precursor transcript into multiple guide RNAs. The advantages of using an RNA processing system include: first, there is no need to use multiple promoters; second, since all guide RNAs are processed from a precursor transcript, their concentrations can be standardized due to similar dB-GEn.16 binding.

[0425] Csy4 is a small endoribonuclease (RNase) protein derived from Pseudomonas aeruginosa. Csy4 can specifically recognize a minimum 17-bp RNA hairpin and exhibits rapid (<1 minute) and efficient (>99.9%) RNA cleavage. Unlike most RNases, the cut RNA fragments remain stable and functionally active. Csy4-based RNA cleavage can be modified for artificial RNA processing systems. In this system, a 17-bp RNA hairpin is inserted between multiple RNA fragments, which are transcribed into precursor transcripts from a single promoter. Co-expression of Csy4 can effectively generate single RNA fragments.

[0426] Host cells

[0427] In some embodiments, the methods of the present disclosure can be used to induce transcriptional regulation of mitotic or post-mitotic cells in vivo and / or ex vivo and / or in vitro. In some embodiments, the methods of the present disclosure can be used to induce DNA cleavage, DNA modification and / or transcriptional regulation in mitotic or post-mitotic cells in vivo and / or ex vivo and / or in vitro (e.g., to produce genetically modified cells that can be reintroduced into an individual).

[0428] Since the guide RNA provides specificity by hybridizing with the target DNA, the mitotic and / or post-mitotic cells can be any of a variety of host cells, wherein suitable host cells include, but are not limited to, bacterial cells; archaeal cells; unicellular eukaryotic organisms; plant cells; algal cells, such as, Botryococcus braunii, Chlamydomonas reinhardtii, Nannochloropsis gaditana, Chlorela pyrenoidosa, Sargassum patens (C. Agardh), etc.; fungal cells; animal cells; cells derived from invertebrates (such as insects, cnidarians, echinoderms, nematodes, etc.); eukaryotic parasites (such as malarial parasites, such as Plasmodium, worms, etc.); cells derived from vertebrates (such as fish, amphibians, reptiles, birds, mammals); mammalian cells, such as rodent cells, human cells, non-human primate cells, etc. In some embodiments, the host cell can be any human cell. Suitable host cells include naturally occurring cells; genetically modified cells (eg, cells that have been genetically modified in a laboratory, such as by "human hands"); and cells manipulated in vitro in any manner. In some embodiments, the host cell is isolated.

[0429] Any type of cell may be a cell of interest (e.g., stem cells, such as embryonic stem cells (ES), induced pluripotent stem cells (iPS), germ cells; somatic cells, such as fibroblasts, hematopoietic cells, neural cells, muscle cells, bone cells, liver cells, pancreatic cells; embryonic cells of any stage of embryos in vitro or in vivo, such as embryonic cells of zebrafish embryos at the 1-cell, 2-cell, 4-cell, 8-cell, etc. stages). Cells may be derived from established cell lines, or they may be primary cells, wherein "primary cells", "primary cell lines" and "primary cultures" are used interchangeably herein and refer to cells and cell cultures derived from a subject and allowed to grow in vitro for a limited number of passages (e.g., division of cultures). For example, primary cultures include cultures that have been passaged 0, 1, 2, 4, 5, 10, or 15 times, but have not undergone a sufficient number of crisis stages. Primary cell lines may be maintained in vitro for less than 10 passages. In some embodiments, the target cell is a unicellular organism, or is grown in culture.

[0430] If the cell is a primary cell, such cells can be harvested from an individual by any suitable method. For example, leukocytes can be harvested by methods such as apheresis, leukocytapheresis, density gradient separation, and cells from tissues (such as skin, muscle, bone marrow, spleen, liver, pancreas, lung, intestine, stomach, etc.) are most suitable for harvesting by biopsy. Suitable solutions can be used to disperse or suspend harvested cells. This solution is usually a balanced salt solution, such as physiological saline, phosphate buffered saline (PBS), Hank's balanced salt solution, etc., appropriately supplemented with fetal bovine serum or other naturally occurring factors, combined with acceptable low concentrations such as 5-25mM buffer. Suitable buffers include HEPES, phosphate buffer, lactate buffer, etc. Cells can be used immediately, or they can be stored for a long time, frozen, and can be reused after thawing. In this case, the cells are typically frozen in 10% dimethyl sulfoxide (DMSO), 50% serum, 40% buffered medium, or some other such solution commonly used in the art to preserve the cells at such a freezing temperature, and thawed in the manner commonly used in the art for thawing frozen cultured cells.

[0431] use

[0432] The methods for regulating transcription according to the present disclosure can be used in various applications, which are also provided in the present disclosure. The applications include research applications; diagnostic applications; industrial applications and therapeutic applications.

[0433] Research applications include, for example, determining the effects of reducing or increasing transcription of a target nucleic acid on, for example, development, metabolism, downstream gene expression, etc. High-throughput genomic analysis can be performed using transcription modulation methods, in which only the spacer of the guide RNA needs to be changed, while the protein binding segment and the transcription termination segment (in some cases) can remain unchanged. A library containing multiple nucleic acids used in genomic analysis includes: a promoter operably linked to a guide RNA encoding nucleotide sequence, wherein each nucleic acid contains a common protein binding segment, a different spacer, and a common transcription termination segment. The chip can contain more than 5x 104 unique guide RNAs. Its applications include large-scale phenotypic analysis, gene function mapping, and metagenomic analysis.

[0434] The methods disclosed herein can be used in the field of metabolic engineering. As disclosed herein, since the transcription level can be effectively and predictably controlled by designing appropriate guide RNA, the activity of metabolic pathways (such as biosynthetic pathways) can be precisely controlled and adjusted by controlling the level of specific enzymes in the target metabolic pathway (such as by increasing or decreasing transcription). Target metabolic pathways include pathways for the production of chemicals (fine chemicals, fuels, antibiotics, toxins, agonists, antagonists, etc.) and / or drugs.

[0435] Biosynthetic pathways of interest include, but are not limited to, (1) the mevalonate pathway (e.g., the HMG-CoA reductase pathway) (converting acetyl-CoA to dimethylallyl pyrophosphate (DMAPP) and isopentenyl pyrophosphate (IPP), which are used in the biosynthesis of a variety of biomacromolecules, including terpenes / isopentenoids), (2) non-mevalonate pathways (e.g., the "2-C-methyl-D-erythritol-4-phosphate / 1-deoxy-D-xylulose-5-phosphate pathway" or the "MEP / DOXP pathway" or the "DXP pathway") (also producing DMAPP and IPP, rather than converting pyruvate and glyceraldehyde 3-phosphate via an alternative pathway to the mevalonate pathway) DMAPP and IPP) (3) Polyketide synthesis pathway (various polyketides are produced by various polyketide synthases. Polyketide compounds include natural small molecule compounds used in chemotherapy (such as tetracycline and macrolide, etc.) and industrially important polyketides including rapamycin (immunosuppressant), erythromycin (antibiotic), lovastatin (anti-cholesterol drug) and asilon B (anti-cancer drug), (4) fatty acid synthesis pathway; (5) DAHP (3-deoxy-D-arabinoheptulose acid?-phosphate) synthesis pathway, (6) pathways for producing potential biofuels (such as short-chain alcohols and alkanes, fatty acid methyl esters and fatty alcohols, isopentene, etc.), etc.

[0436] Networking and Cascading

[0437] The methods disclosed herein can be used to design integrated networks (such as one cascade or multiple cascades). For example, guide RNA and B-GEn.16 polypeptide or its variant can be used to control (such as regulating, such as increasing, decreasing) the expression of another DNA targeting RNA or another B-GEn.16 polypeptide or its variant. For example, the first guide RNA can be designed to target and regulate the transcription of the second fusion dB-GEn.16 polypeptide, which has a function different from the first B-GEn.16 polypeptide or its variant (such as methyltransferase activity, demethylase activity, acetyltransferase activity, deacetylase activity, etc.). In some embodiments, the second fusion dB-GEn.16 polypeptide can be selected so that it does not interact with the first guide RNA. In some embodiments, the second fusion dB-GEn.16 polypeptide can be selected so that it interacts with the first guide RNA. In some such cases, the activity of two (or more) dB-GEn.16 proteins can produce competition (such as, if the polypeptides have opposite activities) or can produce synergy (such as, if the polypeptides have similar or synergistic activities). Similarly, as described above, any complex in the network (e.g., guide RNA / dB-GEn.16 polypeptide) can be designed to control other guide RNAs or dB-GEn.16 polypeptides. Since guide RNAs and dB-GEn.16 polypeptides or variants thereof can be targeted to any desired DNA sequence, the methods described herein can be used to control and regulate the expression of any desired target. Integrated networks (e.g., cascades of interactions) can be designed from very simple to very complex, without limitation.

[0438] In a network, in which two or more components (such as a guide RNA and a dB-GEn.16 polypeptide) are each regulated by another guide RNA / dB-GEn.16 polypeptide complex, the expression level of one component in the network can affect the expression level of another element in the network (such as increasing or decreasing expression). Through this mechanism, the expression of one component can affect the expression of different components in the same network, and the network can include a mixture of components that increase the expression of other components and components that decrease the expression of other components. It is easy for those skilled in the art to understand that in the above examples, the expression level of one component can affect the expression level of one or more different components for illustrative purposes and is not restrictive. When one or more components are modified (as described above) to be operable (e.g., under experimental control, such as temperature control; drug control, such as drug-induced control; light control, etc.), additional layers of complexity can be selectively introduced into the network.

[0439] As a non-limiting example, a first guide RNA can bind to the promoter of a second guide RNA, which controls the expression of a target therapeutic / metabolic gene. In this case, conditional expression of the first guide RNA would indirectly activate the therapeutic / metabolic gene. This type of RNA cascade is useful, for example, for easily converting a repressor into an activator, and can be used to control the logic or kinetics of expression of a target gene.

[0440] Transcriptional regulation methods can also be used for drug discovery and target validation.

[0441] Methods of treating a disease or condition

[0442] In some aspects of the present disclosure, guide RNA and / or B-GEn.16 polypeptides or variants thereof and / or donor polynucleotides are used to modify cellular DNA in vivo for gene therapy, such as treatment of disease or as antiviral, antipathogenic or anticancer therapy, for production of genetically modified organisms in agriculture, or for biological research. In these in vivo embodiments, the components of the CRISPR / B-GEn.16 system include: (i) guide RNA or nucleic acid encoding gRNA; (ii) a nucleic acid comprising a codon-optimized polynucleotide sequence encoding a B-GEn.16 polypeptide or variant thereof, or encoding a B-GEn.16 polypeptide or variant thereof expressed by the nucleic acid; and / or (iii) a donor polynucleotide, which is administered to an individual. The method of administration may be any well-known method of administering polypeptides, small molecules and nucleic acids to a subject in the art. The CRISPR / B-GEn.16 system components may be added to a variety of preparations. More specifically, the CRISPR / B-GEn.16 system components of the present disclosure can be formulated into pharmaceutical compositions by combining with appropriate pharmaceutically acceptable carriers or diluents.

[0443] In some embodiments, provided herein are pharmaceutical preparations or compositions comprising components of a CRISPR / B-GEn.16 system, the system comprising (i) a guide RNA or a nucleic acid encoding a gRNA; (ii) a nucleic acid comprising a codon-optimized polynucleotide sequence encoding a B-GEn.16 polypeptide or a variant thereof, or encoding a B-GEn.16 polypeptide or a variant thereof expressed by the nucleic acid; and / or (iii) a donor polynucleotide present in a pharmaceutically acceptable carrier. A "pharmaceutically acceptable vehicle" may be a carrier approved by a federal or state government regulatory agency, or a carrier for mammals (such as humans) listed in the US Pharmacopeia or other recognized pharmacopeia. The term "carrier" refers to a diluent, adjuvant, excipient, or carrier with which the compounds of the present disclosure formulated can be used in mammals. Such drug carriers can be lipids, such as liposomes, such as liposome dendrimers; liquids, such as water and oils, including oils of petroleum, animal, plant or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, etc.; physiological saline; gum arabic, gelatin, starch paste, talc, keratin, colloidal silica, urea, etc. In addition, adjuvants, stabilizers, thickeners, lubricants and colorants can also be used. The pharmaceutical composition can be formulated into solid, semisolid, liquid or gaseous preparations, such as tablets, capsules, powders, granules, ointments, solutions, suppositories, injections, inhalants, gels, microspheres and aerosols. Therefore, the administration of CRISPR / B-GEn.16 system components can be achieved in various ways, including oral, oral, rectal, parenteral, intraperitoneal, intradermal, transdermal, intratracheal, intraocular, etc. After administration, the active agent can be systemic or localized by regional administration, in vivo administration, or by using an implant to retain the active dose at the implantation site. The active agent may be formulated to be immediately active or may be formulated for sustained release.

[0444] For some conditions, particularly central nervous system conditions, it may be necessary to formulate drugs to cross the blood-brain barrier (BBB). One strategy for delivering drugs across the blood-brain barrier is to disrupt the BBB, either by osmotic means such as mannitol or leukotrienes, or by biochemical disruption using vasoactive substances such as bradykinin. Targeting specific drugs to brain tumors using BBB opening is also a potential option. When the composition is administered by intravascular injection, a BBB disruptor may be administered simultaneously with the therapeutic composition of the present disclosure. Other strategies for passing the BBB may require the use of endogenous transport systems, including Caveolin-1-mediated transport, carrier-mediated transport, such as glucose and amino acid carriers, receptor-mediated insulin or transferrin transport, and active efflux transport, such as glycoproteins. Active transport moieties may also be conjugated to therapeutic compounds used in the present disclosure to facilitate transport across the endothelial wall of blood vessels. In addition or alternatively, drug delivery of therapeutic agents behind the BBB can be by local delivery, such as by intrathecal delivery, such as through an Ommaya capsule (see U.S. Pat. Nos. 5,222,982 and 5385582, incorporated herein by reference); by suppository injection, such as by syringe, such as intravitreal or intracranial injection; by continuous infusion, such as by cannulation, such as by convection (see U.S. Application No. 20070254842, incorporated herein by reference); or by implantation of a device that reversibly adheres to the agent (see, e.g., U.S. Application Nos. 20080081064 and 20090196903, incorporated herein by reference).

[0445] Typically, the components of the CRISPR / B-GEn.16 system effective amount include: (i) guide RNA or nucleic acid encoding gRNA; (ii) a nucleic acid comprising a codon-optimized polynucleotide sequence encoding a B-GEn.16 polypeptide or a variant thereof, or encoding a B-GEn.16 polypeptide or a variant thereof expressed by the nucleic acid; and / or (iii) a donor polynucleotide provided. As described above with respect to the ex vivo method, the effective amount or effective dose of the CRISPR / B-GEn.16 system component in vivo refers to an amount that induces an increase of 2 times or more in the amount of recombination observed between two homologous sequences relative to a negative control, such as a cell contacted with an empty vector or an unrelated polypeptide. The amount of recombination can be measured by any suitable method, such as those described above and known in the art. Calculating the effective amount or effective dose of the CRISPR / B-GEn.16 system component to be administered is within the capabilities of a person of ordinary skill in the art and is a conventional method for those skilled in the art. The final amount to be administered will depend on the route of administration and the nature of the disease or condition to be treated.

[0446] The effective dosage for a particular subject depends on a variety of factors, some of which vary from subject to subject. A competent clinician can determine the effective dosage of the therapeutic agent administered to the subject as needed to prevent or reverse the development of a disease condition. The clinician can determine the maximum safe dosage for an individual based on the route of administration, using animal data of LD50 and other information about the agent. For example, the dose for intravenous administration may be larger than the dose for intradermal administration because the therapeutic composition is administered into a larger volume of liquid. Similarly, for compositions that can be rapidly removed from the body, the composition can be administered in larger doses or in repeated doses to maintain therapeutic concentrations. Using common skills, a competent clinician can optimize the dosage of a particular therapeutic agent during routine clinical trials.

[0447] For inclusion in a medicament, CRISPR / B-GEn.16 system components can be obtained from suitable commercial sources. As a general proposition, the total pharmaceutically effective amount of CRISPR / B-GEn.16 system components per dose of parenteral administration will be within the range that can be measured by a dose response curve.

[0448] Therapies based on CRISPR / B-GEn.16 system components, such as preparations comprising the following components: (i) guide RNA or nucleic acid encoding gRNA; (ii) a nucleic acid comprising a codon-optimized polynucleotide sequence encoding a B-GEn.16 polypeptide or a variant thereof, or encoding a B-GEn.16 polypeptide or a variant thereof expressed by the nucleic acid; and / or (iii) a donor polynucleotide for therapeutic administration must be sterile. Sterility is easily achieved by filtration through a sterile filter membrane (such as a 0.2 micron filter membrane). The therapeutic composition is typically placed in a container with a sterile access port, for example, an intravenous bag or vial with a stopper that can be pierced by a hypodermic needle. Therapies based on CRISPR / B-GEn.16 system components can be stored in unit or multi-dose containers, such as sealed ampoules or vials, as aqueous solutions or lyophilized preparations for reconstitution. Taking the lyophilized preparation as an example, 5 ml of a sterile filtered 1% (w / v) compound aqueous solution is injected into a 10 ml vial, and the resulting mixture is lyophilized. The infusion is prepared by reconstituting the lyophilized compound with bacteriostatic water for injection.

[0449] Depending on the desired formulation, the pharmaceutical composition may include a pharmaceutically acceptable, non-toxic diluent carrier, which is defined as a carrier commonly used in the formulation of pharmaceutical compositions for administration to animals or humans. The diluent is selected so that it does not affect the biological activity of the combination. Examples of such diluents are distilled water, buffered water, physiological saline, PBS, Ringer's solution, dextrose solution, and Hank's solution. In addition, the pharmaceutical composition or formulation may also include other carriers, adjuvants, or non-toxic, non-therapeutic, non-immunogenic stabilizers, excipients, etc. The compos...

Claims

1. A polypeptide according to SEQ ID NO: 1, or any polypeptide sequence having at least 60% identity to any of the above, or any nucleic acid encoding the same.

2. A polypeptide according to SEQ ID NO: 1, or any polypeptide sequence having at least 80% identity to any of the above, or any nucleic acid encoding the same.

3. A polypeptide according to SEQ ID NO: 1, or any polypeptide sequence having at least 95% identity to any of the above, or any nucleic acid encoding the same.

4. A composition comprising (i) a polypeptide according to any one of claims 1 to 3, and (ii) one or more single heterologous guide RNAs (sgRNAs) or DNA that allows for the in situ generation of such one or more sgRNAs, each sgRNA or DNA encoding a sgRNA comprising: a. an engineered DNA targeting segment that can hybridize to a target sequence in a polynucleotide locus, b. tracr mate sequence, and c.tracr RNA sequence, wherein the tracr mate sequence can hybridize to the tracr sequence, and wherein (a), (b) and (c) are arranged in a 5' to 3' direction.

5. The composition of claim 4, wherein the engineered DNA targeting segment is directly adjacent to a PAM sequence on the targeted DNA segment at its 3' end, or such PAM sequence is a part of the targeted DNA sequence in its 5' portion, wherein the PAM sequence comprises a sequence motif selected from "CYN" or "CNN", wherein "N" represents any base and "Y" represents "C" or "T".

6. A method for targeting, editing, modifying or manipulating target DNA at one or more locations in a cell or in vitro, the method comprising (i) introducing the polypeptide according to any one of claims 1 to 3 or the nucleic acid encoding the same into a cell or in vitro environment; and (ii) introducing one or more single heterologous guide RNAs (sgRNAs) or DNA encoding such one or more sgRNAs into a cell or in vitro environment, each sgRNA or DNA encoding sgRNA comprising: a. an engineered DNA targeting segment comprising RNA and capable of hybridizing to a target sequence in a polynucleotide locus, b. a tracr pairing sequence consisting of RNA, and c. The tracr RNA sequence composed of RNA, wherein the tracr mate sequence hybridizes to the tracr sequence, and wherein (a), (b) and (c) are arranged in a 5' to 3' direction; and (iii) generating one or more gaps or nicks or base edits in the target DNA, wherein the polypeptide is guided to the target DNA by the sgRNA in its processed or unprocessed form.

7. A composition for targeting, editing, modifying or manipulating target DNA at one or more locations in a cell or in vitro, the composition comprising i. A polypeptide according to any one of claims 1 to 3 or a nucleic acid encoding the same; and / or ii. one or more single heterologous guide RNAs (sgRNAs) or DNA suitable for generating such one or more sgRNAs in situ, each comprising: a. an engineered DNA targeting segment consisting of RNA and capable of hybridizing to such a target sequence in a polynucleotide locus, b. a tracr pairing sequence consisting of RNA, and c. The tracr RNA sequence composed of RNA, wherein the tracr mate sequence hybridizes to the tracr sequence, and wherein (a), (b) and (c) are arranged in a 5' to 3' direction.

8. A cell comprising (i) a polypeptide according to any one of claims 1 to 3, or a nucleic acid encoding the same; and (ii) one or more single heterologous guide RNAs (sgRNAs) or DNA suitable for generating such one or more sgRNAs in situ, each comprising: a. an engineered DNA targeting segment that can hybridize to a target sequence in a polynucleotide locus, b. tracr mate sequence, and c.tracr RNA sequence, wherein the tracr mate sequence can hybridize to the tracr sequence, and wherein (a), (b) and (c) are arranged in a 5' to 3' direction.

9. A kit comprising i. a nucleic acid encoding a polypeptide according to any one of claims 1 to 3, wherein the nucleic acid encoding such a polypeptide is operably linked to a promoter; and ii. one or more single heterologous guide RNAs (sgRNAs) or DNA suitable for generating such one or more sgRNAs in situ, each sgRNA comprising: a. an engineered DNA targeting segment that can hybridize to a target sequence in a polynucleotide locus, b. tracr mate sequence, and c.tracr RNA sequence, wherein the tracr mate sequence can hybridize to the tracr sequence, and wherein (a), (b) and (c) are arranged in a 5' to 3' direction.

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