Novel RNA-programmable endonuclease system and its uses

Abstract

Aspects of the invention relate particularly to novel systems for targeting, editing or manipulating DNA in cells using novel synthetic RNA-guided nucleases (sRGNs) derived from wild-type or parental small type II CRISPR Cas9 endonucleases.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of European Patent Application No. 18162681.3 filed on March 19, 2018, European Patent Application No. 18162683.9 filed on March 19, 2018, European Patent Application No. 18172625.8 filed on May 16, 2018, European Patent Application No. 18174707.2 filed on May 16, 2018, European Patent Application No. 18181680.2 filed on July 4, 2018, Priority to U.S. Patent Application No. 62 / 745,238 filed on October 12, 2018, U.S. Patent Application No. 62 / 745,239 filed on October 12, 2018, U.S. Patent Application No. 62 / 745,240 filed on October 12, 2018, and U.S. Patent Application No. 62 / 745,246 filed on October 12, 2018, the contents of each of which are expressly incorporated herein by reference in their entirety, including any drawings.

[0003] field

[0004] The present disclosure relates generally to the field of molecular biology, including compositions and methods, involving novel systems comprising RNA-programmable endonucleases, associated guide RNAs and / or target sequences, and methods of producing and using them in various applications, including methods of regulating transcription, and methods of targeting, editing and / or manipulating DNA in cells.

[0005] background

[0006] Endonucleases such as zinc finger endonucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and ribonucleases have been used as site-specific nucleases for genome targeting, genome editing, gene silencing, transcription regulation, promoted recombination, and other molecular biology techniques. The clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein (Cas) system provides a source of novel nucleases and endonucleases, including CRISPR-Cas9.

[0007] The CRISPR system is a prokaryotic system that provides prokaryotes with resistance to foreign genetic elements such as bacteriophages. Streptococcus pyogenes), Cas9 guided by the duplex formed between the mature activating tracr RNA and the targeting crRNA introduces site-specific double-stranded DNA (dsDNA) breaks in the invader-associated DNA. Cas9 is a multidomain enzyme that cleaves the target strand (defined as complementary to the spacer sequence of the crRNA) using the HNH nuclease domain and the non-target strand using the RuvC-like domain, enabling the conversion of the dsDNA-cleaving Cas9 into a nickase by selective motif inactivation. DNA cleavage specificity is determined by two parameters: the variable, spacer-derived sequence of the crRNA targeting the protospacer sequence (a sequence on the DNA target that is not complementary to the spacer of the crRNA) and a short sequence positioned immediately 3' (downstream) of the protospacer on the non-target DNA strand, the Protospacer Adjacent Motif (PAM).

[0008] The RNA-guided DNA targeting principle using CRISPR-Cas has been widely used to edit genomes in the past few years, as described in WO2013 / 176722. Studies have demonstrated that RNA-guided Cas9 can be used as a genome editing tool in human cells, mice, zebrafish, fruit flies, worms, plants, yeast and bacteria, as well as various other species. The system is versatile, enabling multiple genome engineering by programming Cas9 to edit several sites in the genome simultaneously using multiple guide RNAs. The conversion of Cas9 to a nickase has been shown to promote homology-guided repair in mammalian genomes with reduced mutagenic activity. In addition, for example, the DNA binding activity of Cas9 catalytically inactive mutants has been used to engineer RNA-programmable transcriptional silencing and activation devices or epigenetic modifiers.

[0009] Although the CRISPR-Cas 9 system is promising for gene editing, there are still many problems with the use of this system. For example, they have one or more of the following disadvantages:

[0010] a) They are too large to be carried within the genome of established therapeutically suitable viral delivery systems such as adeno-associated virus (AAV).

[0011] b) Their activity in heterologous environments is often too low for use in these environments, for example too low for effective use in eukaryotic and particularly mammalian environments.

[0012] c) Their nuclease activity lacks fidelity, leading to undesirable off-target effects, which would, for example, make them unsuitable for gene therapy use or other applications requiring high precision.

[0013] d) They may trigger an immune response, which may limit their usefulness in mammalian applications.

[0014] e) They require complex and / or long PAMs, which limits target selection for DNA targeting segments.

[0015] f) They show poor expression from plasmid or viral vectors. Summary of the invention

[0016] This section provides a general summary of the disclosure, and is not intended to encompass its full scope or all of its features.

[0017] In certain embodiments, provided herein are compositions and methods related to novel RNA-programmable endonucleases, and systems using such nucleases. The invention also relates to other components, including guide RNA and / or target sequences, and methods of producing and using them in various applications. Examples of such applications include methods for regulating transcription, and methods for targeting, editing and / or manipulating DNA using novel nucleases and other components such as nucleic acids and / or polypeptides. Some embodiments of the present disclosure also relate to recombinant cells and kits comprising one or more system elements disclosed herein.

[0018] In one aspect, provided herein is a synthetic RNA-guided nuclease (sRGN) polypeptide comprising, from N-terminus to C-terminus: 1) a small domain 1 comprising an amino acid sequence of any one of SEQ ID NOs: 34-37, or a variant thereof having at least about 90% sequence identity to any one of SEQ ID NOs: 34-37, 2) a small domain 2 comprising an amino acid sequence of any one of SEQ ID NOs: 38-41, or a variant thereof having at least about 90% sequence identity to any one of SEQ ID NOs: 38-41, 3) a small domain 3 comprising an amino acid sequence of any one of SEQ ID NOs: 42-45, or a variant thereof having at least about 90% sequence identity to any one of SEQ ID NOs: 42-45, 4) a small domain 4 comprising an amino acid sequence of any one of SEQ ID NOs: 46-49, or a variant thereof having at least about 90% sequence identity to any one of SEQ ID NOs: 46-49, 5) a small domain 5 comprising an amino acid sequence of any one of SEQ ID NOs: 47-49, or a variant thereof having at least about 90% sequence identity to any one of SEQ ID NOs: 47-49, NO: 50-53, or a variant thereof having at least about 90% sequence identity to any one of SEQ ID NO: 50-53, 6) a small domain 6 comprising an amino acid sequence of any one of SEQ ID NO: 54-57, or a variant thereof having at least about 90% sequence identity to any one of SEQ ID NO: 54-57, 7) a small domain 7 comprising an amino acid sequence of any one of SEQ ID NO: 58-61, or a variant thereof having at least about 90% sequence identity to any one of SEQ ID NO: 58-61, and 8) a small domain 8 comprising an amino acid sequence of any one of SEQ ID NO: 62-65, or a variant thereof having at least about 90% sequence identity to any one of SEQ ID NO: 62-65, wherein at least 2 of the small domains are derived from different parent Cas9 endonucleases selected from the group consisting of Staphylococcus lugdunensis ( Staphylococcus lugdunensis )Cas9 (SEQID NO: 30), Staphylococcus pasteurianus ( Staphylococcus pasteuri )Cas9 (SEQ ID NO: 31), Staphylococcus microti Cas9 (SEQ ID NO: 33), and Staphylococcus suis ( Staphylococcus hyicus ) Cas9 (SEQ ID NO: 32). In certain embodiments, at least three of the small domains are derived from different parent Cas9 endonucleases selected from the group consisting of Staphylococcus lugdunensis Cas9 (SEQ ID NO: 30), Staphylococcus pasteurianus Cas9 (SEQ ID NO: 31), Staphylococcus microtiCas9 (SEQ ID NO: 33) and Staphylococcus suis Cas9 (SEQ ID NO: 32). In certain embodiments, small domain 8 comprises the amino acid sequence of SEQ ID NO: 62 or a variant thereof having at least about 90% sequence identity with SEQ ID NO: 62. In certain embodiments, small domain 8 comprises the amino acid sequence of SEQ ID NO: 63 or a variant thereof having at least about 90% sequence identity with SEQ ID NO: 63. In certain embodiments, small domain 1 comprises the amino acid sequence of SEQ ID NO: 34 or a variant thereof having at least about 90% sequence identity with SEQ ID NO: 34.

[0019] In another aspect, provided herein is a sRGN polypeptide comprising, from N-terminus to C-terminus: 1) a small domain 1 comprising an amino acid sequence of any one of SEQ ID NOs: 66-69 or a variant thereof having at least about 90% sequence identity to any one of SEQ ID NOs: 66-69, 2) a small domain 2 comprising an amino acid sequence of any one of SEQ ID NOs: 70-73 or a variant thereof having at least about 90% sequence identity to any one of SEQ ID NOs: 70-73, 3) a small domain 3 comprising an amino acid sequence of any one of SEQ ID NOs: 74-77 or a variant thereof having at least about 90% sequence identity to any one of SEQ ID NOs: 74-77, 4) a small domain 4 comprising an amino acid sequence of any one of SEQ ID NOs: 78-81 or a variant thereof having at least about 90% sequence identity to any one of SEQ ID NOs: 78-81, 5) a small domain 5 comprising an amino acid sequence of any one of SEQ ID NOs: 8) a small domain 8 comprising an amino acid sequence of any one of SEQ ID NOs: 94-97 or a variant thereof having at least about 90% sequence identity to any one of SEQ ID NOs: 94-97, 9) a small domain 9 comprising an amino acid sequence of any one of SEQ ID NOs: 98-101 or a variant thereof having at least about 90% sequence identity to any one of SEQ ID NOs: 99-103, 10) a small domain 10 comprising an amino acid sequence of any one of SEQ ID NOs: 110-111 or a variant thereof having at least about 90% sequence identity to any one of SEQ ID NOs: 111-112, 11) a small domain 11 comprising an amino acid sequence of any one of SEQ ID NOs: 112-113 or a variant thereof having at least about 90% sequence identity to any one of SEQ ID NOs: 113-114, 11) a small domain 12 comprising an amino acid sequence of any one of SEQ ID NOs: 114-115 or a variant thereof having at least about 90% sequence identity to any one of SEQ ID NOs: 98-101, 10) a small domain 10 comprising an amino acid sequence of any one of SEQ ID NOs: 102-105 or a variant thereof having at least about 90% sequence identity with any one of SEQ ID NOs: 102-105, 11) a small domain 11 comprising an amino acid sequence of any one of SEQ ID NOs: 106-109 or a variant thereof having at least about 90% sequence identity with any one of SEQ ID NOs: 106-109, and 12) a small domain 12 comprising an amino acid sequence of any one of SEQ ID NOs: 110-113 or a variant thereof having at least about 90% sequence identity with any one of SEQ ID NOs: 110-113,wherein at least two of the small domains are derived from different parent Cas9 endonucleases selected from the group consisting of Staphylococcus lugdunensis Cas9 (SEQ ID NO: 30), Staphylococcus pasteurianus Cas9 (SEQ ID NO: 31), Staphylococcus microti Cas9 (SEQ ID NO: 33) and Staphylococcus suis Cas9 (SEQ ID NO: 32). In certain embodiments, at least three of the small domains are derived from different parent Cas9 endonucleases selected from the group consisting of Staphylococcus lugdunensis Cas9 (SEQ ID NO: 30), Staphylococcus pasteurianus Cas9 (SEQ ID NO: 31), Staphylococcus microti Cas9 (SEQ ID NO: 33) and Staphylococcus suis Cas9 (SEQ ID NO: 32). In certain embodiments, small domain 12 comprises an amino acid sequence of SEQ ID NO: 110 or a variant thereof having at least about 90% sequence identity with SEQ ID NO: 110. In certain embodiments, small domain 12 comprises an amino acid sequence of SEQ ID NO: 111 or a variant thereof having at least about 90% sequence identity with SEQ ID NO: 111. In certain embodiments, (i) small domain 1 comprises an amino acid sequence of SEQ ID NO: 66 or a variant thereof having at least about 90% sequence identity with SEQ ID NO: 66, and (ii) small domain 2 comprises an amino acid sequence of SEQ ID NO: 70 or a variant thereof having at least about 90% sequence identity with SEQ ID NO: 70.

[0020] In another aspect, provided herein is a sRGN polypeptide selected from the group consisting of: a) a Gib11 polypeptide comprising an amino acid sequence of SEQ ID NO: 1 or a variant thereof having at least about 90% sequence identity to SEQ ID NO: 1, b) a Gib11Spa-1 polypeptide comprising an amino acid sequence of SEQ ID NO: 2 or a variant thereof having at least about 90% sequence identity to SEQ ID NO: 2, c) a Gib11Spa-2 polypeptide comprising an amino acid sequence of SEQ ID NO: 3 or a variant thereof having at least about 90% sequence identity to SEQ ID NO: 3, d) a Gib11Spa-3 polypeptide comprising an amino acid sequence of SEQ ID NO: 4 or a variant thereof having at least about 90% sequence identity to SEQ ID NO: 4, e) a P2H12 polypeptide comprising an amino acid sequence of SEQ ID NO: 5 or a variant thereof having at least about 90% sequence identity to SEQ ID NO: 5, f) an E2 polypeptide comprising an amino acid sequence of SEQ ID NO: 6 or a variant thereof having at least about 90% sequence identity to SEQ ID NO: 6, g) an E2+K741D+L743K polypeptide comprising an amino acid sequence of SEQ ID NO: 7 or a variant thereof having at least about 90% sequence identity to SEQ ID NO: 7, h) an E2+S670T+N675D polypeptide comprising an amino acid sequence of SEQ ID NO: 8 or a variant thereof having at least about 90% sequence identity to SEQ ID NO: 8, i) an E2+K741N+L743N polypeptide comprising an amino acid sequence of SEQ ID NO: 9 or a variant thereof having at least about 90% sequence identity to SEQ ID NO: 9, j) an F8 polypeptide comprising an amino acid sequence of SEQ ID NO: 10 or a variant thereof having at least about 90% sequence identity to SEQ ID NO: 10; k) an F8+K737D+L739K polypeptide comprising an amino acid sequence of SEQ ID NO: 11 or a variant thereof having at least about 90% sequence identity to SEQ ID NO: NO:11 has a variant with at least about 90% sequence identity; and 1) F8+K737N+L739N polypeptide, which comprises the amino acid sequence of SEQ ID NO: 12 or a variant thereof with at least about 90% sequence identity with SEQ ID NO: 12.

[0021] In certain embodiments, the sRGN polypeptide is selected from: a) a Gib11 polypeptide comprising the amino acid sequence of SEQ ID NO: 1 or a variant thereof having at least about 90% sequence identity with SEQ ID NO: 1, b) a Gib11Spa-1 polypeptide comprising the amino acid sequence of SEQ ID NO: 2 or a variant thereof having at least about 90% sequence identity with SEQ ID NO: 2, and c) a Gib11Spa-3 polypeptide comprising the amino acid sequence of SEQ ID NO: 4 or a variant thereof having at least about 90% sequence identity with SEQ ID NO: 4. In certain embodiments, the sRGN polypeptide is selected from: a) a Gib11 polypeptide comprising the amino acid sequence of SEQ ID NO: 1, b) a Gib11Spa-1 polypeptide comprising the amino acid sequence of SEQ ID NO: 2, and c) a Gib11Spa-3 polypeptide comprising the amino acid sequence of SEQ ID NO: 4.

[0022] In another aspect, provided herein is a nucleic acid encoding a sRGN polypeptide according to any of the above embodiments. In certain embodiments, the nucleic acid is codon optimized for expression in a host cell. In certain embodiments, the nucleic acid comprises a nucleotide sequence of any one of SEQ ID NOs: 13-29 or a variant thereof having at least about 90% sequence identity to any one of SEQ ID NOs: 13-29.

[0023] In another aspect, provided herein is a system comprising: (a) a sRGN polypeptide or a nucleic acid encoding a sRGN polypeptide according to any one of claims 1-11; and (b) a guide RNA (gRNA) or a nucleic acid encoding the gRNA, wherein the gRNA is capable of guiding the sRGN polypeptide or a variant thereof to a target polynucleotide sequence. In certain embodiments, the system further comprises a donor template comprising a heterologous polynucleotide sequence, wherein the heterologous polynucleotide sequence is capable of being inserted into the target polynucleotide sequence.

[0024] In certain embodiments, according to any of the above systems, the nucleic acid encoding the sRGN polypeptide is codon-optimized for expression in a host cell and / or the heterologous polynucleotide sequence is codon-optimized for expression in a host cell.

[0025] In certain embodiments, according to any of the above systems, the nucleic acid encoding the sRGN polypeptide is deoxyribonucleic acid (DNA).

[0026] In certain embodiments, according to any of the above systems, the nucleic acid encoding the sRGN polypeptide is a ribonucleic acid (RNA). In certain embodiments, the RNA encoding the sRGN polypeptide is an mRNA.

[0027] In certain embodiments, according to any of the above systems, the donor template is encoded in an adeno-associated virus (AAV) vector.

[0028] In some embodiments, according to any of the above systems, the sRGN polypeptide or the nucleic acid encoding the sRGN polypeptide is formulated in a liposome or lipid nanoparticle. In some embodiments, the liposome or lipid nanoparticle also comprises the gRNA or the nucleic acid encoding the gRNA.

[0029] In certain embodiments, according to any of the above systems, the system comprises a sRGN polypeptide pre-complexed with a gRNA to form a ribonucleoprotein (RNP) complex.

[0030] In another aspect, provided herein is a method for targeting, editing, modifying or manipulating a target DNA at a target locus, the method comprising providing the following substances to the target DNA: (a) an sRGN polypeptide according to any one of the above embodiments or a nucleic acid encoding the sRGN polypeptide; and (b) a guide RNA (gRNA) or a nucleic acid encoding the gRNA, wherein the gRNA is capable of guiding the sRGN polypeptide to the target locus. In certain embodiments, the method further comprises providing the target DNA with a donor template comprising a heterologous polynucleotide sequence, wherein the heterologous polynucleotide sequence is capable of being inserted into the target locus.

[0031] In certain embodiments, according to any of the above methods, the nucleic acid encoding the sRGN polypeptide is codon-optimized for expression in a host cell and / or the heterologous polynucleotide sequence is codon-optimized for expression in a host cell.

[0032] In certain embodiments, according to any of the above methods, the nucleic acid encoding the sRGN polypeptide is deoxyribonucleic acid (DNA).

[0033] In certain embodiments, according to any of the above methods, the nucleic acid encoding the sRGN polypeptide is a ribonucleic acid (RNA). In certain embodiments, the RNA encoding the sRGN polypeptide is an mRNA.

[0034] In certain embodiments, according to any of the above methods, the donor template is encoded in an adeno-associated virus (AAV) vector.

[0035] In some embodiments, according to any of the above methods, the sRGN polypeptide or the nucleic acid encoding the sRGN polypeptide is formulated in a liposome or lipid nanoparticle. In some embodiments, the liposome or lipid nanoparticle also comprises the gRNA or the nucleic acid encoding the gRNA. In some embodiments, the method comprises providing the target DNA with a sRGN polypeptide, and the sRGN polypeptide is pre-complexed with the gRNA as an RNP complex.

[0036] In another aspect, provided herein is a modified cell comprising: (a) an sRGN polypeptide according to any one of the above embodiments or a nucleic acid encoding the sRGN polypeptide; and (b) a guide RNA (gRNA) or a nucleic acid encoding the gRNA, wherein the gRNA is capable of guiding the sRGN polypeptide or variant thereof to a target polynucleotide sequence. In certain embodiments, the modified cell further contains a donor template comprising a heterologous polynucleotide sequence, wherein the heterologous polynucleotide sequence is capable of being inserted into the target polynucleotide sequence.

[0037] In another aspect, provided herein is a genetically modified cell, wherein the genome of the cell is edited by a method according to any one of the above embodiments.

[0038] In another aspect, provided herein is a kit comprising: (a) an sRGN polypeptide according to any one of claims 1-11 or a nucleic acid encoding the sRGN polypeptide; and (b) a guide RNA (gRNA) or a nucleic acid encoding the gRNA, wherein the gRNA is capable of guiding the sRGN polypeptide or a variant thereof to a target polynucleotide sequence. In certain embodiments, the kit further contains a donor template comprising a heterologous polynucleotide sequence, wherein the heterologous polynucleotide sequence is capable of being inserted into the target polynucleotide sequence. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 A schematic representation of the gene family shuffling technique used to generate Gib11, P2H12, E2, and F8 is shown.

[0040] Figure 2A Results are shown for fluorescence polarization-based biochemical activity in disruption assays (in some cases, activity exceeding that of wild-type Cas9 such as SluCas9), and for Gib11. Diamonds depict conditions with sgRNA, and squares depict control conditions without sgRNA.

[0041] Figure 2BResults of a fluorescence polarization-based biochemical cleavage assay for SluCas9 are shown. Diamonds depict situations with sgRNA and squares depict control situations without sgRNA.

[0042] Figure 2C The relative activities of Gib11 and SluCas9 in the cleavage assay are shown, as shown by Figure 2A and 2B The initial slope of the curve for the case with sgRNA is shown.

[0043] Figure 3 Shown are the results of Gib11 recognizing the "NNGG" PAM motif as determined by a "live-dead" assay in liquid culture.

[0044] Figure 4 Results of a fluorescence polarization-based biochemical cleavage assay for P2H12 are shown. Diamonds depict situations with sgRNA and squares depict control situations without sgRNA.

[0045] Figure 5 Shown are the results of a BFP disruption-based cleavage assay for E2.

[0046] Figure 6 showed that the Staphylococcus aureus ) protein's domain organization. The brackets named PI indicate the PI domain of the base Gib11 sequence. The corresponding cRGN-A portion is shown as a shaded box (in this case, for Gib11), while the cRGN-B S. pasteurianus portion is shown as a white box. Amino acid positions refer to the Gib11 protein (SEQ ID NO: 1).

[0047] Figure 7 The results of the BFP disruption-based cleavage assay for Gib11Spa-1, Gib11Spa-2, and Gib11Spa-3 are shown.

[0048] Figure 8 Shown are the results of a BFP disruption-based cleavage assay for F8.

[0049] Fig. 9 Shown are the specificity profiles of Gib11, Gib11Spa-1, Gib11Spa-3, E2, and F8, as assessed using a biochemical cleavage assay.

[0050] Fig.10The overall specificity of Gib11, Gib11Spa-1, Gib11Spa-3, E2 and F8 based on their specificity profiles are shown.

[0051] Fig.11 Distribution of activity profiles of Gib11, Gib11Spa-1, Gib11Spa-3, E2 and F8, as assessed using a biochemical cleavage assay, is shown. On the x-axis, from left to right are the target DNA substrates, ranked from highest to lowest activity for each indicated nuclease. DETAILED DESCRIPTION

[0052] Most existing type II CRISPR Cas systems are based on enzymes from Streptococcus pyogenes, which have the particular disadvantage of being too large for packaging into viral vectors such as AAV (1638 amino acids). There is an alternative type II CRISPR Cas system based on a nuclease from Staphylococcus aureus (EP 2 898 075), which is significantly smaller in size. However, this nuclease requires a complex PAM, which greatly limits its application for gene editing purposes. In addition, small nucleases can be more useful for gene editing methods, but a limited number of such small nucleases have been identified, and their optimal features for gene editing are not fully defined for gene editing at least in most cases.

[0053] Provided herein are novel synthetic CRISPR-Cas polypeptides (also referred to herein as "sRGN polypeptides") derived from four different Staphylococcus species (S. lugdunensis, S. pasteurianus, Staphylococcus microti Artificially defined small domains of CRISPR-Cas endonucleases of (Staphylococcus aureus and Staphylococcus suis), and variants thereof with different and advantageous features and functionality. These sRGNs provide other opportunities for genome editing that did not exist before. Cas nucleases can have different activities, depending on the system in which they are used. A feature of the invention is the provision of additional engineered nucleases that increase the toolbox of nucleases that can be used for gene editing and related methods.

[0054] In many cases, known naturally occurring Cas nucleases target longer PAMs (such as NNAAAA), which can limit the utility of those nucleases. The present invention provides synthetic nucleases (e.g., small nucleases) derived from naturally occurring small nucleases but that recognize NNGG PAMs.

[0055] The invention further provides suitable PAM sequences and suitable guides, including single guide RNA (sgRNA), for use in prokaryotic, eukaryotic and in vitro settings.

[0056] The sRGN polypeptides provided herein can exhibit advantageous properties relative to reported CRISPR-Cas endonucleases, such as higher activity in prokaryotic, eukaryotic and / or in vitro environments, and / or greater expression of sRGN polypeptides from nucleic acids in eukaryotic environments (e.g., human host cells). In some cases, sRGNs combine one or more of the following: small size, high editing activity, and only requiring a short PAM sequence. Small size with respect to RNA-guided nucleases refers to nucleases that are no greater than about 1100 amino acids in length.

[0057] Also provided are systems for targeting, editing, or manipulating DNA in a cell, comprising a sRGN polypeptide or a nucleic acid encoding a sRGN polypeptide, and one or more guide RNAs (gRNAs), e.g., one or more single guide RNAs (sgRNAs), or nucleic acids encoding one or more gRNAs.

[0058] In the following detailed description, reference is made to the accompanying drawings which form a part thereof. In the accompanying drawings, similar symbols generally identify similar parts unless the context otherwise indicates. The exemplary alternatives described in the detailed description, drawings, and claims are not intended to be limiting. Other alternatives may be used and other changes may be made without departing from the spirit or scope of the subject matter presented herein. It should be readily understood that the various aspects as generally described herein and illustrated in the accompanying drawings may be arranged, permuted, combined, and designed in a variety of different configurations, all of which are expressly contemplated and constitute a part of this application.

[0059] Unless otherwise defined, all technical terms, symbols and other scientific terms or special terms used herein are intended to have the meanings commonly understood by those skilled in the art to which this application belongs. In some cases, for clarity and / or for ease of reference, terms with commonly understood meanings are defined herein, and the inclusion of such definitions herein is not necessarily interpreted as representing considerable differences compared to the common understanding of the art. Many techniques and procedures described or referenced herein are well understood and commonly employed by those skilled in the art using conventional methods.

[0060] definition

[0061] The terms "polynucleotide" and "nucleic acid" are used interchangeably herein to refer to a polymeric form 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 containing purine and pyrimidine bases or other natural, chemically or biochemically modified, non-natural or derived nucleotide bases.

[0062] "Oligonucleotide" generally refers to a polynucleotide of about 5 to 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 an oligonucleotide. Oligonucleotides are also referred to as "oligomers" or "oligos" and can be isolated from genes or chemically synthesized by methods known in the art. Where applicable to the embodiments described, the terms "polynucleotide" and "nucleic acid" should be understood to include single-stranded (such as sense or antisense) and double-stranded polynucleotides.

[0063] "Genomic DNA" means the DNA of the genome of an organism, including, but not limited to, the genome of a bacterium, fungus, archaea, protist, virus, plant, or animal.

[0064] "Manipulation" of DNA includes binding, nicking one strand, or cutting (i.e., severing) both strands of DNA, or includes modifying or editing DNA or a polypeptide that binds to DNA. Manipulation of DNA can silence, activate, or modulate (increase or decrease) the expression of RNA or polypeptides encoded by the DNA, or prevent or enhance the binding of polypeptides to DNA.

[0065] "Stem-loop structure" means a nucleic acid with a secondary structure comprising a nucleotide region (stem portion) known or predicted to form a double strand, which is connected on one side by a nucleotide region (loop portion) that is mainly single-stranded. The terms "hairpin" and "fold-back" structures are also used herein to represent stem-loop structures. Such structures are well known in the art, and these terms are used consistently with their known meanings in the art. As known in the art, stem-loop structures do not require accurate base pairing. Therefore, the stem can include one or more base mispairings. Alternatively, base pairing can be accurate, that is, without including any mispairings.

[0066] By "hybridizable" or "complementary" or "substantially complementary" is meant that a nucleic acid (e.g., RNA) comprises a nucleotide sequence that enables it to non-covalently bind to another nucleic acid in a sequence-specific, antiparallel manner (i.e., nucleic acid specifically binds to complementary nucleic acid) under appropriate in vitro and / or in vivo conditions of temperature and solution ionic strength, i.e., to form Watson-Crick base pairs and / or G / U base pairs, "anneal," or "hybridize." As is known in the art, standard Watson-Crick base pairing includes: adenine (A) pairs with thymidine (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 (e.g., dsRNA), guanine (G) base pairs with uracil (U). For example, in the case of tRNA anticodons that base pair with codons in mRNA, G / U base pairing is partially responsible for the degeneracy (i.e., redundancy) of the genetic code. In the context of the present disclosure, guanine (G) of the protein-binding segment (dsRNA duplex) of the guide RNA molecule is considered to be complementary to uracil (U), and vice versa. Thus, when a G / U base pair can be generated at a given nucleotide position of the protein-binding segment (dsRNA duplex) of the guide RNA molecule, the position is not considered to be non-complementary, but rather complementary.

[0067] Hybridization and washing conditions are well known and are exemplified in Sambrook, J. and Russell, W., Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor (2001); and Green, MR, and Sambrook, J., Molecular cloning: A Laboratory Manual, 4th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor (2012). Conditions of temperature and ionic strength determine the "stringency" of hybridization.

[0068] Hybridization requires that the two nucleic acids contain complementary sequences, although mismatches between bases are possible. Conditions suitable for hybridization between two nucleic acids depend on the length and degree of complementarity of the nucleic acids, variables well known in the art. The greater the degree of complementarity between two nucleotide sequences, the greater the value of the melting temperature (Tm) of the hybrid of nucleic acids with those sequences. For hybridization between nucleic acids with short complementary sequence segments (e.g., complementarity within the range of 35 or less, 30 or less, 25 or less, 22 or less, 20 or less or 18 or less nucleotides), the position of the mismatch becomes important (see Sambrook et al., supra, 11.7-11.8). Typically, the length of a hybridizable nucleic acid is at least 10 nucleotides. An exemplary minimum length of a hybridizable nucleic acid is: at least 15 nucleotides; at least 20 nucleotides; at least 22 nucleotides; at least 25 nucleotides; and at least 30 nucleotides). In addition, a skilled technician will recognize that the temperature and washing solution salt concentration can be adjusted as necessary, depending on factors such as the length and degree of complementarity of the complementary region.

[0069] It is understood in the art that the sequence of polynucleotides does not need to be 100% complementary to the sequence of the target nucleic acid of its specific hybridization. In addition, polynucleotides can be hybridized on one or more sections, so as not to involve intervening or adjacent sections (for example, loop structures or hairpin structures) in hybridization events. Polynucleotides can include at least 70%, at least 80%, at least 90%, at least 95%, at least 99% or 100% sequence complementarity with the target region in the target nucleic acid sequence targeted thereto. For example, wherein 18 of the 20 nucleotides of antisense compounds are complementary to the target region and therefore the antisense nucleic acid of specific hybridization will represent 90% complementarity. In this example, the remaining non-complementary nucleotides can be clustered or interspersed with complementary nucleotides, and do not need to be adjacent to each other or to complementary nucleotides. The percent complementarity between specific nucleic acid sequence segments within nucleic acids can be routinely determined using BLAST programs (basic local alignment search tools) and PowerBLAST programs known in the art (Altschul et al., J. Mol. Biol. 1990, 215, 403-410; Zhang and Madden, Genome Res., 1997, 7, 649-656) or by 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).

[0070] 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.

[0071] As used herein, "binding" (e.g., with respect to an RNA-binding domain of a polypeptide) refers to a non-covalent interaction between macromolecules (e.g., between a protein and a nucleic acid). When in a non-covalent interaction state, the macromolecules are considered to be "associated" or "interacting" or "bound" (e.g., when molecule X is considered to interact with molecule Y, it 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 parts of a binding interaction may be sequence-specific. Binding interactions are typically characterized by less than 10-6 M, less than 10 -7 M, 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 The dissociation constant (Kd) of M. “Affinity” indicates the strength of binding, with increased binding affinity being associated with a lower Kd.

[0072] "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). In the case of a protein domain-binding protein, it can bind to itself (to form a homodimer, homotrimer, etc.) and / or it can bind to one or more molecules of one or more different proteins.

[0073] The term "conservative amino acid substitution" refers to the interchangeability of amino acid residues with similar side chains in a protein. 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-hydroxy 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; and 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.

[0074] A polynucleotide or polypeptide has a certain percentage of "sequence identity" with another polynucleotide or polypeptide, which means that when the two sequences are compared, the percentage of bases or amino acids are the same and in the same relative position when aligned. Sequence identity can be determined in a variety of different ways. To determine sequence identity, sequences can be aligned using various methods and computer programs available on the world wide web at websites including ncbi.nlm.nili.gov / BLAST, ebi.ac.uk / Tools / msa / tcoffee, ebi.Ac.Uk!Tools / msa / muscle, mafft.cbrc / alignment / software (e.g., BLAST, T-COFFEE, MUSCLE, MAFFT, etc.). See, e.g., Altschul et al. (1990), J. Mol. Biol. 215:403-10. According to the present invention, standard sequence alignments in the art are used to determine the amino acid residues in one sRGN variant that "correspond to" the amino acid residues in another sRGN variant. Amino acid residues of sRGN variants that correspond to amino acid residues of other sRGN variants appear at the same position in a sequence alignment.

[0075] A DNA sequence that "encodes" a specific RNA is a DNA nucleic acid sequence that is transcribed into RNA. A DNA polynucleotide may encode an RNA (mRNA) that is translated into a protein, or a DNA polynucleotide may encode an RNA that is not translated into a protein (e.g., 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 (in the case of mRNA) into a polypeptide in vitro or in vitro when placed under the control of an appropriate regulatory sequence. The boundaries of the coding sequence are determined by a start codon at the 5' end (N-terminus) and a translation termination nonsense codon at the 3' end (C-terminus). The coding sequence may include, but is not limited to, cDNA from prokaryotic or eukaryotic mRNA, genomic DNA sequences from prokaryotic or eukaryotic DNA, and synthetic nucleic acids. The transcription termination sequence is usually located 3' of the coding sequence.

[0076] "Promoter sequence" or "promoter" used herein is a DNA regulatory region that can bind RNA polymerase and initiate transcription of downstream (3' direction) coding or non-coding sequences. For purposes of the present invention, the promoter sequence is bound by a transcription initiation site at its 3' end, and extends upstream (5' direction) to include the minimum number of bases or elements required to initiate transcription at a level detectable above background. A transcription initiation site will be found within the promoter sequence, as well as the protein binding domain responsible for RNA polymerase binding. Eukaryotic promoters often, but not always, contain "TATA" boxes and "CAAT" boxes. Various promoters, including inducible promoters, can be used to drive various vectors of the present invention. The promoter can be a constitutively active promoter (i.e., a promoter that is constitutively in an active "on" state), it can be an inducible promoter (i.e., a promoter whose state activity / "on" or inactivity / "off" is controlled by an external stimulus such as a specific temperature, a compound or the presence of an albumen), it can be a spatially restricted promoter (i.e., 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 (i.e., a promoter that is in an "on" state or a "off" state during a specific stage of embryonic development or during a specific stage of a biological process (e.g., the hair follicle cycle in mice). Suitable promoters can be derived from viruses, and therefore can be referred to as viral promoters, or they can be derived from any organism, including prokaryotes or eukaryotes. Suitable promoters can be used to drive expression by any RNA polymerase (e.g., pol I, pol II, pol III). Exemplary promoters include, but are not limited to, the SV40 early promoter, the mouse mammary tumor virus long terminal repeat (LTR) promoter; the adenovirus major late promoter (Ad MLP); the herpes simplex virus (HSV) promoter, the cytomegalovirus (CMV) promoter such as the CMV immediate early promoter region (CMVIE), the Rous sarcoma virus (RSV) promoter, the human U6 small nuclear promoter (U6) (Miyagishi et al., Nature Biotechnology 20, 497-500 (2002)), the enhanced U6 promoter (e.g., Xia et al., Nucleic Acids Res. 2003 Sep 1; 31(17)), the human H1 promoter (H1), etc. Examples of inducible promoters include, but are not limited to, T7 RNA polymerase promoter, T3 RNA polymerase promoter, isopropyl-β-D-thiogalactopyranoside (IPTG)-regulated promoter, lactose-inducible 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.

[0077] In certain embodiments, the promoter is a spatially restricted promoter (i.e., a cell type-specific promoter, a tissue-specific promoter, etc.), such that in a multicellular organism, the promoter is active (i.e., "on") in a specific cell subpopulation. A spatially restricted promoter may also be referred to as an enhancer, a transcriptional control element, a control sequence, etc. Any convenient 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 germline, a promoter that drives expression in the lung, a promoter that drives expression in muscle, a promoter that drives expression in 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. Thus, depending on the organism, a spatially restricted promoter can be used to regulate the expression of a nucleic acid encoding a sRGN polypeptide in a variety of different tissues and cell types. Some spatially restricted promoters are also temporally restricted, such that the promoter is in an "on" state or a "off" state during a specific stage of embryonic development or during a specific stage of a biological process (e.g., the hair follicle cycle in mice). For purposes 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, and the like.Neuron-specific spatially restricted promoters include, but are not limited to, neuron-specific enolase (NSE) promoter (e.g., EMBLHSENO2, X51956); aromatic amino acid decarboxylase (AADC) promoter; neurofilament promoter (e.g., GenBank HUMNFL, L04147); synapsin promoter (e.g., GenBank HUMSYNIB, M55301); thy-1 promoter (e.g., Chen et al. (1987) Cell 51:7-19; and Llewellyn, et al. (2010) Nat. Med. 16(10):1161-1166); serotonin receptor promoter (e.g., GenBank S62283); tyrosine hydroxylase promoter (TH) (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 6:583-594); GnRH promoter (e.g., Radovick et al. (1991) Proc. Natl. Acad. Sci. USA 88:3402-3406); L7 promoter (e.g., Oberdick et al. (1990) Science 248:223-226); DNMT promoter (e.g., Bartge et al. (1988) Proc. Natl. Acad. Sci. USA 85:3648-3652); enkephalin promoter (e.g., Comb et al. (1988) EMBO J. 17:3793-3805); myelin basic protein (MBP) promoter; Ca2+-calmodulin-dependent protein kinase 11-alpha (CamKIM) promoter (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-β promoter (e.g., Liu et al. (2004) Gene Therapy 11:52-60); etc.

[0078] 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 (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 (e.g., Knight et al. (2003) Proc. Natl. Acad. Sci. USA 100:14725); the fatty acid translocase (FAT / CD36) promoter (e.g., Kuriki et al. (2002) Biol. Pharm. Bull. 25:1476; and Sato et al. (2002) J. Biol. Chem. 277:15703); stearoyl-CoA desaturase-1 (SCD1) promoter (Tabor et al. (1999) J. Biol. Chem. 274:20603); leptin promoter (e.g., Mason et al. (1998) Endocrinol. 139:1013; and Chen et al. (1999) Biochem. Biophys. Res. Comm. 262:187); adiponectin promoter (e.g., Kita et al. (2005) Biochem. Biophys. Res. Comm. 331:484; and Chakrabarti (2010) Endocrinol. 151:2408); lipoprotein promoter (e.g., Platt et al. (1989) Proc. Natl. Acad. Sci. USA 86:7490); resistin promoter (e.g., Seo et al. (2003) Molec. Endocrinol. 17:1522); etc.

[0079] Cardiomyocyte-specific spatially restricted promoters include, but are not limited to, control sequences derived from the following genes: myosin light chain-2, α-myosin heavy chain, AE3, cardiac troponin C, cardiac myosin, 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.

[0080] Smooth muscle-specific spatially restricted promoters include, but are not limited to, the SM22a promoter (e.g., Akyiirek et al. (2000) Mol. Med. 6:983; and U.S. Pat. No. 7,169,874); smooth muscle-specific protein promoters (e.g., WO 2001 / 018048); α-smooth muscle actin promoter; etc. For example, a 0.4 kb region of the SM22a promoter in which two CArG elements are located has been shown to mediate vascular smooth muscle cell-specific expression (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).

[0081] Photoreceptor-specific spatially restricted promoters include, but are not limited to, the rhodopsin promoter; the rhodopsin kinase promoter (Young et al. (2003) Ophthalmol. Vis. Sci. 44:4076); the beta phosphodiesterase gene promoter (Nicoud et al. (2007) J. Gene Med. 9:1015); the retinitis pigmentosa gene promoter (Nicoud et al. (2007) supra); the interphotoreceptor retinoid-binding protein (IRBP) gene enhancer (Nicoud et al. (2007) supra); the IRBP gene promoter (Yokoyama et al. (1992) Exp Eye Res. 55:225); etc.

[0082] The terms "DNA regulatory sequence," "control element," and "regulatory element," used interchangeably herein, refer to transcriptional and translational control sequences, such as promoters, enhancers, polyadenylation signals, terminators, protein degradation signals, and the like, which provide for and / or regulate the transcription of a non-coding sequence (e.g., a guide RNA) or a coding sequence (e.g., a sRGN polypeptide) and / or regulate the translation of the encoded polypeptide.

[0083] As used herein, the terms "naturally occurring" or "unmodified" as applied to nucleic acids, polypeptides, cells or organisms refer to nucleic acids, polypeptides, cells or organisms found in nature. For example, a polypeptide or polynucleotide sequence present in an organism (including viruses) that can be isolated from a source in nature and which has not been intentionally modified by man in the laboratory is naturally occurring.

[0084] As used herein, the term "chimeric" when applied to nucleic acids or polypeptides refers to an entity composed of structures derived from different sources. For example, where "chimeric" is used in the context of a chimeric polypeptide (e.g., a chimeric sRGN polypeptide), the chimeric polypeptide comprises an amino acid sequence derived from a different polypeptide, such as a polypeptide having a function other than a Cas nuclease, including, for example, a polypeptide having DNA modification activity, transcription factor activity, and / or DNA-associated polypeptide modification activity. A chimeric polypeptide may comprise a modified or naturally occurring polypeptide sequence (e.g., a first amino acid sequence from a modified or unmodified sRGN protein; and a second amino acid sequence different from the sRGN protein). Similarly, "chimeric" in the context of a nucleic acid encoding a chimeric polypeptide includes nucleotide sequences derived from different coding regions (e.g., a first nucleotide sequence encoding a modified or unmodified sRGN protein; and a second nucleotide sequence encoding a polypeptide other than an sRGN protein).

[0085] The term "chimeric polypeptide" refers to a non-naturally occurring polypeptide, for example, one that is made by artificially combining (i.e., "fusing") two or more otherwise separate amino acid sequence 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."

[0086] As used herein, "heterologous" refers to a nucleotide or peptide that is not found in a natural nucleic acid or protein, respectively. For example, in a chimeric sRGN protein, the RNA binding domain of a naturally occurring bacterial sRGN polypeptide (or a variant thereof) can be fused to a heterologous polypeptide sequence (i.e., a polypeptide sequence from a protein other than sRGN, or a polypeptide sequence from another organism). The heterologous polypeptide can exhibit an activity (e.g., an enzymatic activity) also exhibited by the chimeric sRGN protein (e.g., methyltransferase activity, acetyltransferase activity, kinase activity, ubiquitination activity, etc.). A heterologous nucleic acid can be linked (e.g., by genetic engineering) to a naturally occurring nucleic acid (or a variant thereof) to produce a chimeric nucleic acid encoding a chimeric polypeptide. As another example, in a fusion variant sRGN localization polypeptide, a variant sRGN localization polypeptide can be fused to a heterologous polypeptide (i.e., a polypeptide other than sRGN) that exhibits an activity that will also be exhibited by the fusion variant sRGN localization polypeptide. A heterologous nucleic acid can be linked (e.g., by genetic engineering) to a variant sRGN localization polypeptide to produce a nucleic acid encoding a fusion variant sRGN localization polypeptide. As used herein, "heterologous" further refers to a nucleotide or polypeptide that is in a cell other than its native cell.

[0087] The term "cognate" refers to two biological molecules that typically interact or coexist in nature.

[0088] As used herein, "recombinant" means that a particular nucleic acid (DNA or RNA) or vector is the product of various combinations of cloning, restriction, polymerase chain reaction (PCR) and / or ligation steps, thereby producing a construct having a structural coding or non-coding sequence that is distinguishable from an endogenous nucleic acid found in a natural system. A DNA sequence encoding a polypeptide can be assembled from a cDNA fragment or from a series of synthetic oligonucleotides to provide a synthetic nucleic acid that can be expressed from a recombinant transcription unit contained in a cell or cell-free transcription and translation system. Genomic DNA containing relevant sequences can also be used for the formation of recombinant genes or transcription units. Sequences of non-translated DNA can be present at 5' or 3' of an open reading frame, where such sequences do not interfere with the operation or expression of the coding region, and can indeed act to regulate the production of the desired product by various mechanisms (see "DNA regulatory sequences" below). Alternatively, DNA sequences encoding untranslated RNA (e.g., guide RNA) can also be considered to be recombinant. Thus, for example, the term "recombinant" nucleic acid refers to a non-naturally occurring nucleic acid, for example, prepared by artificially combining two otherwise separate sequence segments by human intervention. This artificial combination can be achieved by chemical synthesis means or by artificial manipulation of isolated nucleic acid segments, such as by genetic engineering techniques. This is usually done to replace codons with codons encoding the same amino acid, conservative amino acids or non-conservative amino acids. Alternatively, it is performed to connect nucleic acid segments with desired functions together to produce a combination of desired functions. This artificial combination can be achieved by chemical synthesis means or by artificial manipulation of isolated nucleic acid segments, such as by genetic engineering techniques. When a recombinant polynucleotide encodes a polypeptide, the sequence of the encoded polypeptide may be naturally occurring ("wild type"), or may be a variant (e.g., mutant) of a naturally occurring sequence. Therefore, the term "recombinant" polypeptide does not necessarily mean a polypeptide whose sequence does not occur naturally. In contrast, 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 (e.g., variants, mutants, etc.). Therefore, a "recombinant" polypeptide is the result of human intervention, but may be a naturally occurring amino acid sequence. The term "non-naturally occurring" includes molecules that are significantly different from their naturally occurring counterparts, including chemically modified or mutated molecules.

[0089] A "vector" or "expression vector" is a replicon, such as a plasmid, phage, virus, or cosmid, to which another DNA segment, ie, an "insert," may be attached so as to achieve replication of the attached segment in a cell.

[0090] An "expression cassette" comprises a DNA coding sequence operably linked to a promoter. "Operably linked" means a juxtaposed connection wherein the components described are in a relationship that allows them to function in their intended manner. For example, if a promoter affects the transcription or expression of a coding sequence, the promoter is operably linked to the coding sequence. The terms "recombinant expression vector" or "DNA construct" are used interchangeably herein to represent a DNA molecule comprising a vector and at least one insert. Recombinant expression vectors are typically produced for the purpose of expressing and / or amplifying inserts or for the construction of 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.

[0091] A cell has been "genetically modified" or "transformed" or "transfected" by exogenous DNA, such as a recombinant expression vector, when the DNA has been introduced into the cell. The presence of the exogenous DNA results in a permanent or transient genetic change. The transforming DNA may or may not be integrated (covalently linked) into the genome of the cell.

[0092] In some embodiments, the present invention relates to a cell line or clone of a prokaryotic cell. For example, in prokaryotes, yeast and mammalian cells, the transforming DNA can be maintained on an additional element such as a plasmid. With respect to eukaryotic cells, stably transformed cells are such cells: wherein the transforming DNA has become integrated into the chromosome so that the transforming DNA is inherited by daughter cells through chromosome replication. The ability to establish a cell line or clone comprising a daughter cell colony containing the transforming DNA by eukaryotic cells has demonstrated this stability." Clone" is a cell colony derived from a single cell or a common ancestor by mitosis. "Cell line" is a primary cell clone that can stably grow multiple generations in vitro.

[0093] Suitable genetic modification methods (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, gene gun technology, calcium phosphate precipitation, direct microinjection, nanoparticle-mediated nucleic acid delivery (e.g., Panyam et al., Adv Drug Deliv Rev. 2012 Sep 13. pp: S0169-409X(12)00283-9. doi:10.1016 / j.addr.2012.09.023), etc.

[0094] The choice of genetic modification method generally depends on the type of cells 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 edition, Wiley & Sons, 1995.

[0095] As used herein, "host cell" refers to a eukaryotic cell, a prokaryotic cell (e.g., a bacterial or archaeal cell) in vivo or in vitro, or a cell from a multicellular organism (e.g., a cell line) cultured as a unicellular entity, which can or has been used as a recipient of nucleic acid, and includes the offspring of the original cell that has been transformed with nucleic acid. It should be understood that the offspring of a single cell are not necessarily 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 (e.g., an expression vector) has been introduced. For example, a bacterial host cell is a bacterial host cell genetically modified by the introduction of an exogenous nucleic acid (e.g., a plasmid or a recombinant expression vector) into a suitable bacterial host cell, while a eukaryotic host cell is a eukaryotic host cell (e.g., a mammalian germ cell) genetically modified by the introduction of an exogenous nucleic acid into a suitable eukaryotic host cell.

[0096] As used herein, a "target DNA" is a DNA polynucleotide comprising a "target site" or "target sequence". The terms "target site", "target sequence", "target protospacer DNA" or "protospacer-like sequence" are used interchangeably herein to refer to a nucleic acid sequence present in the target DNA to which the DNA-targeting segment of the guide RNA will bind if sufficient conditions for binding exist. For example, the target site (or target sequence) 5'GAGCATATC-3' within the target DNA is targeted (or bound by, or hybridized with, 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 (e.g., conditions in a cell-free system) are known in the art; for example, Sambrook, supra. The strand of the target DNA that is complementary to and hybridized with the guide RNA is referred to as the "complementary strand", and the strand of the target DNA that is complementary to the "complementary strand" (and therefore not complementary to the guide RNA) is referred to as the "non-complementary strand" or "non-complementary strand". "Location-modifying polypeptide" or "RNA-binding site-guided polypeptide" or "RNA-binding site-guided modifying polypeptide" or "location-modifying polypeptide" refers to a polypeptide that binds to RNA and is targeted to a specific DNA sequence. A location-modifying polypeptide as described herein is targeted to a specific DNA sequence by an RNA molecule to which it is bound. The RNA molecule comprises a sequence that binds, hybridizes, or is complementary to a target sequence within the target DNA, thereby targeting the bound polypeptide to a specific position (target sequence) within the target DNA. "Cutting" refers to the breaking of the covalent backbone of a 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 can occur as a result of two different single-stranded cutting events. DNA cutting can result in the production of blunt ends or staggered ends. In certain embodiments, a complex comprising a guide RNA and a location-modifying polypeptide is used for targeted double-stranded DNA cutting.

[0097] "Nuclease" and "endonuclease" are used interchangeably herein to refer to an enzyme with endonucleolytic catalytic activity for polynucleotide cleavage.

[0098] A "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 for DNA cleavage. A cleavage domain may be contained within a single polypeptide chain, or the cleavage activity may result from the association of two (or more) polypeptides. A single nuclease domain may consist of more than one isolated amino acid sequence segment within a given polypeptide.

[0099] "Localization polypeptide" or "RNA-binding site-guided polypeptide" or "RNA-binding site-guided polypeptide" refers to a polypeptide that binds RNA and is targeted to a specific DNA sequence. A localization polypeptide as described herein is targeted to a specific DNA sequence by an RNA molecule to which it is bound. The RNA molecule contains a sequence that is complementary to a target sequence within the target DNA, thereby targeting the bound polypeptide to a specific location (target sequence) within the target DNA.

[0100] The "guide sequence" or DNA-targeting segment (or "DNA-targeting sequence") comprises a nucleotide sequence that is complementary to a specific sequence within the target DNA, referred to herein as a "protospacer-like" sequence (the complementary strand of the target DNA). The protein-binding segment (or "protein-binding sequence") interacts with the localization-modifying polypeptide. When the localization-modifying polypeptide is a sRGN or sRGN-associated polypeptide (described in more detail below), site-specific cleavage of the target DNA occurs at a position determined by both (i) base-pairing complementarity between the guide RNA and the target DNA; and (ii) a short motif in the target DNA, referred to as a protospacer adjacent motif (PAM).

[0101] The protein-binding segment of the guide RNA comprises, in part, two complementary nucleotide sequence stretches that hybridize to each other to form a double-stranded RNA duplex (dsRNA duplex).

[0102] In certain 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 localization polypeptide; etc.) comprises modifications or sequences that provide additional desired characteristics (e.g., modified or regulated stability; subcellular targeting; tracking, such as fluorescent labeling; binding sites for proteins or protein complexes; etc.). Non-limiting examples include: a 5' cap (e.g., a 7-methylguanylate cap (m7G)); a 3' polyadenylation tail (i.e., a 3' poly(A) tail); a riboswitch sequence (e.g., to allow regulated stability and / or regulated accessibility of a protein and / or protein complex); a stability control sequence; a sequence that forms a dsRNA duplex (i.e., 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 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 binding site for a protein (e.g., a protein that acts on DNA, including transcriptional activators, transcriptional repressors, DNA methyltransferases, DNA demethylases, histone acetyltransferases, histone deacetylases, etc.); and combinations thereof.

[0103] In certain embodiments, the guide RNA comprises 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 (e.g., a 7-methylguanylate cap (m7G)); a 3' polyadenylation tail (i.e., a 3' polyadenylation tail); a riboswitch sequence (e.g., to allow for regulated stability and / or regulated accessibility of proteins and protein complexes); a stability control sequence; a sequence that forms a dsRNA duplex (i.e., a hairpin); a sequence that targets RNA to a subcellular location (e.g., nucleus, mitochondria, chloroplasts, etc.); a modification or sequence that provides tracking (e.g., direct conjugation to a fluorescent molecule, conjugation to a portion that promotes fluorescence detection, a sequence that allows fluorescence detection, etc.); a modification or sequence that provides a binding site for a protein (e.g., a protein that acts on DNA, including a transcriptional activator, a transcriptional repressor, a DNA methyltransferase, a DNA demethylase, a histone acetyltransferase, a histone deacetylase, etc.); and combinations thereof.

[0104] The guide RNA and the sRGN polypeptide form a complex (i.e., bind by non-covalent interactions). The guide RNA provides target specificity to the complex by comprising a nucleotide sequence complementary to the sequence of the target DNA. The sRGN polypeptide of the complex provides site-specific activity. In other words, the sRGN polypeptide is guided to the target DNA sequence (e.g., a target sequence in a chromosomal nucleic acid; a target sequence in an extrachromosomal nucleic acid, such as an episomal nucleic acid, a minicircle, etc.; a target sequence in a mitochondrial nucleic acid; a target sequence in a chloroplast nucleic acid; a target sequence in a plasmid; etc.) by virtue of its association with the protein binding segment of the guide RNA. RNA aptamers are known in the art and are generally synthetic forms of riboswitches. The terms "RNA aptamer" and "riboswitch" are used interchangeably herein to include synthetic and natural nucleic acid sequences that provide inducible regulation of the structure of the RNA molecule of which they are a part (and therefore the availability of a specific sequence). RNA aptamers generally comprise a sequence that folds into a specific structure (e.g., a hairpin) that specifically binds a specific drug (e.g., a small molecule). Binding of the drug results in a structural change in the RNA folding, which alters the characteristics of the nucleic acid of which the aptamer is a part. As non-limiting examples: (i) an activator-RNA having an aptamer may not bind to a corresponding targeter-RNA unless the aptamer is bound by an appropriate drug; (ii) a targeter-RNA having an aptamer may not bind to a corresponding activator-RNA unless the aptamer is bound by an appropriate drug; and (iii) a targeter-RNA and an activator-RNA each comprising a different aptamer that binds a different drug may not bind to each other unless both drugs are present. As illustrated by these examples, two guide RNAs can be designed to be inducible.

[0105] Examples of aptamers and riboswitches can be found, e.g., in Nakamura et al., Genes Cells. 2012 May;17(5):344-64; Vavalle et al., Future Cardiol. 2012 May;8(3):371-82; Citartan et al., Biosens Bioelectron. 2012 Apr 15;34(1):1-11; and Liberman et al., Wileylnterdiscip Rev RNA. 2012 May-Jun;3(3):369-84; all of which are incorporated herein by reference in their entirety.

[0106] The term "stem cell" is used herein to represent cells (e.g., plant stem cells, vertebrate stem cells) that have both self-renewal capacity and the ability to produce differentiated cell types (see Morrison et al. (1997) Cell 88:287-298). In the context of cell ontogeny, the adjective "differentiated" or "differentiation" is a relative term. "Differentiated cells" are cells that have further developed downstream of the developmental pathway than the cells compared therewith. Therefore, pluripotent stem cells (as described below) can be differentiated into ancestral cells (e.g., mesoderm stem cells) of lineage restriction, which in turn can be differentiated into further restricted cells (e.g., neuron ancestral cells), which can be differentiated into terminal stage cells (i.e., terminally differentiated cells, such as neurons, cardiomyocytes, etc.), which play a unique role in a certain tissue type, and may or may not retain the ability to further proliferate. Stem cells can be characterized by the presence of specific markers (e.g., proteins, RNA, etc.) and the absence of specific markers. Stem cells can also be identified by in vitro and in vivo functional assays, particularly assays related to the ability of stem cells to produce multiple differentiated offspring.

[0107] The stem cells of interest include pluripotent stem cells (PSC). The term "pluripotent stem cell" or "PSC" is used herein to refer to a stem cell that can produce all cell types of an organism. Thus, PSC can produce cells of all germ layers of an organism (e.g., endoderm, mesoderm, and ectoderm of vertebrates). Pluripotent cells can form teratomas and can affect ectoderm, mesoderm, or endodermal tissues in living organisms. Plant pluripotent stem cells can produce all cell types of a plant (e.g., cells of roots, stems, leaves, etc.).

[0108] Animal PSCs can be obtained in a variety of different ways. For example, embryonic stem cells (ESCs) are derived from the inner cell mass of the embryo (Thomson et al., Science. 1998 Nov 6;282(5391):1145-7), while induced pluripotent stem cells (iPSCs) are derived from somatic cells (Takahashi 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 Nov 20).

[0109] Because the term PSC means pluripotent stem cells, regardless of their origin, the term PSC includes the terms ESC and iPSC, as well as the term embryonic germ stem cell (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 obtained from somatic cells. PSCs can be target cells for the methods described herein.

[0110] "Embryonic stem cells" (ESC) refer to PSCs isolated from embryos, typically from the inner cell mass of a blastocyst. ESCs 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); Miz-hES1 (MizMedi Hospital-Seoul NationalUniversity); HSF-1, HSF-6 (University of California at San Francisco); and H1, H7, H9, H13, H14 (Wisconsin Alumni Research Foundation (WiCell Research Institute)). Stem cells of interest also include embryonic stem cells from other primates, such as rhesus monkey stem cells and marmoset stem cells. Stem cells can be obtained from any mammalian species, such as humans, equines, bovines, pigs, canines, felines, 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 typically grow as flat colonies with a large nuclear-cytoplasmic ratio, well-defined edges, 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 in, e.g., 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 propagating hESCs in an undifferentiated form are described in WO 99 / 20741, WO 01 / 51616, and WO 03 / 020920. "Embryonic germ stem cells" (EGSCs) or "embryonic germ cells" or "EG cells" refer to PSCs that are derived from germ cells and / or germ cell ancestors, such as primordial germ cells, i.e., those that will become sperm and eggs.Embryonic germ cells (EG cells) are believed to have properties similar to those of embryonic stem cells as described above. Examples of methods for producing 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.

[0111] "Induced pluripotent stem cells" or "iPSCs" refer to PSCs derived from cells that are not PSCs (i.e., from cells differentiated relative to PSCs). iPSCs can be derived from a variety of different cell types, including terminally differentiated cells. iPSCs have an ES cell-like morphology, growing as flat colonies with a large nuclear-cytoplasmic ratio, well-defined edges, and prominent nucleoli. In addition, iPSCs express one or more key pluripotency markers known to those of ordinary skill 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.

[0112] Examples of methods for producing 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. Typically, to produce iPSCs, reprogramming factors known in the art (e.g., Oct4, SOX2, KLF4, MYC, Nanog, Lin28, etc.) are provided to somatic cells to reprogram somatic cells to become pluripotent stem cells.

[0113] "Somatic cell" refers to any cell in an organism that does not normally give rise to all types of cells in the organism in the absence of experimental manipulation. In other words, somatic cells are cells that have differentiated sufficiently that they will not naturally give rise to cells of all three germ layers of the body (i.e., ectoderm, mesoderm, and endoderm). For example, somatic cells would include neurons and neural progenitors, which may be able to naturally give rise to all or some cell types of the central nervous system, but not to cells of the mesodermal or endodermal lineages.

[0114] "Mitotic cell" refers to a cell that undergoes mitosis. Mitosis is the process by which a eukaryotic cell splits the chromosomes in its nucleus into two identical sets in two separate nuclei. It is usually followed by cytokinesis, which splits the nucleus, cytoplasm, organelles, and cell membrane into two cells containing approximately equal portions of these cellular components.

[0115] A "post-mitotic cell" refers to a cell that has exited mitosis, ie, it is "quiescent," ie, it no longer undergoes division. This state of quiescence may be temporary, ie, reversible, or it may be permanent.

[0116] "Meiotic cell" refers to a cell that is undergoing meiosis. Meiosis is the process by which a cell divides its nuclear material in order to produce gametes or spores. Unlike mitosis, in meiosis the chromosomes undergo a recombination step, which shuffles the genetic material between chromosomes. Furthermore, the result of meiosis is four (genetically unique) haploid cells, compared to the two (genetically identical) diploid cells that result from mitosis.

[0117] "Recombination" refers to the process of exchanging genetic information between two polynucleotides. "Homology-directed repair (HDR)" as used herein refers to a specialized form of DNA repair that occurs, for example, during double-strand break repair in a cell. This process requires nucleotide sequence homology, uses a "donor" molecule to provide a template for the repair of a "target" molecule (i.e., 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 result in changes in the sequence of the target molecule (e.g., insertions, deletions, mutations). In certain embodiments, the donor polynucleotide, a portion of the donor polynucleotide, a copy of the donor polynucleotide, or a portion of a copy of the donor polynucleotide is integrated into the target DNA.

[0118] "Non-homologous end joining (NHEJ)" refers to the repair of double-strand breaks in DNA by directly joining the broken ends to each other without the need for a homologous template (in contrast to homology-directed repair which requires a homologous sequence to direct the repair). NHEJ often results in the loss (deletion) of nucleotide sequence near the site of the double-strand break.

[0119] The terms "treatment", "treating" and the like are generally used herein to refer to obtaining a desired pharmacological and / or physiological effect. The effect may be preventive in terms of completely or partially preventing a disease or its symptoms, and / or may be therapeutic in terms of partial or complete cure of a disease and / or side effects attributable to the disease. "Treatment" as used herein includes any treatment of a disease or symptom in a mammal, and includes: (a) preventing the disease or symptom from occurring in a subject who may be predisposed to acquiring the disease or symptom but has not yet been diagnosed as having the disease or symptom; (b) inhibiting the disease or symptom, i.e., arresting its development; or (c) alleviating the disease, i.e., causing regression of the disease. The therapeutic agent may be administered before, during or after the onset of the disease or injury. Of particular importance is the treatment of an ongoing disease, wherein the treatment stabilizes or alleviates the patient's undesirable clinical symptoms. It is desirable to perform such treatment prior to complete loss of function in the affected tissue. It would be desirable to administer the treatment during the symptomatic stage of the disease and in some cases after the symptomatic stage of the disease.

[0120] 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.

[0121] General methods in molecular and cellular biochemistry can be found in such standard textbooks as Molecular Cloning: A Laboratory Manual, 3rd edition (Sambrook et al., Harboor Laboratory Press 2001); Short Protocols in Molecular Biology, 4th edition (Ausubel et al. ed., John Wiley & Sons 1999); Protein Methods (Bollag et al., John Wiley & Sons 1996); Nonviral Vectors for Gene Therapy (Wagner et al. ed., Academic Press 1999); Viral Vectors (Kaplift & Loewy ed., Academic Press 1995); Immunology Methods Manual (I. Lefkovits ed., Academic Press 1997); and Cell and Tissue Culture: Laboratory Procedures in Biotechnology (Doyle & Griffiths, John Wiley & Sons 1998), the disclosures of which are incorporated herein by reference.

[0122] In the case of providing a range of values, it should be understood that each intermediate value between the upper and lower limits of the range (up to one tenth of the lower limit unit, unless the context clearly indicates otherwise) and any other described value or intermediate value in the described range are included in the present invention. The upper and lower limits of these smaller ranges can be independently included in the described smaller range, and are also included in the present invention, subject to the constraints of any specific exclusion limits in the described range. In the case where the described range includes one or two limits, the scope excluding any one or two limits in those included limits is also included in the present invention.

[0123] The phrase "consisting essentially of" is meant herein to exclude everything that is not one or more specified active components of a system or that is not one or more specified active moieties of a molecule.

[0124] Certain ranges of numerical values ​​presented herein are preceded by the term "about". The term "about" is used herein to provide literal support for the exact number that follows it, as well as numbers that are near or close to the number that follows the term. In determining whether a number is near or close to a specifically mentioned number, the near or close unmentioned number may be a number that provides a substantial numerical equivalent to the specifically mentioned number in the context in which it is presented.

[0125] It should be understood that certain features of the invention described in the context of separate embodiments for clarity may also be provided in combination in a single embodiment. Conversely, various features of the invention described in the context of a single embodiment for brevity may also be provided individually or in any suitable sub-combination. All combinations of embodiments related to the present invention are specifically included by the present invention and disclosed herein, just as each and every combination is individually and explicitly disclosed. In addition, all sub-combinations of various embodiments and elements thereof are also specifically included by the present invention and disclosed herein, just as each and every such sub-combination is individually and explicitly disclosed herein.

[0126] Synthetic RNA-guided nucleases (sRGNs)

[0127] Synthetic RNA-guided nucleases (sRGNs) can be generated, for example, using homology-based gene family shuffling of artificially defined small domains of CRISPR-Cas9 endonucleases from different species, e.g., four different Staphylococcus species (S. lugdunensis, S. pasteurianus, Staphylococcus microti and Staphylococcus suis). Figure 1 One such gene family shuffling scheme is schematically depicted in . 1. Briefly, the sequences of different Cas9 endonucleases are compared to identify regions with high homology as anchors, and each Cas9 endonuclease is decomposed into multiple (such as 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more) corresponding small domains. A library of sRGNs for gene family shuffling is prepared by randomly assigning each small domain to be derived from one of the original Cas9 endonucleases. In some cases, it may be desirable to keep the PAM-interaction (PI) domain fixed so that a single PAM sequence is recognized by each library construct. For example, a library can be prepared by decomposing each Cas9 endonuclease into 8 small domains, keeping the C-terminal small domain fixed, resulting in a theoretical complexity of 8192. In another embodiment, a library can be prepared by dividing each Cas9 endonuclease into 12 small domains, keeping the C-terminal small domain fixed, resulting in a theoretical complexity of 1.3 x 10 5Libraries can be screened using known assays for RNA-guided endonuclease activity (e.g., bacterial live / dead assays and reporter gene disruption assays) to identify sRGNs with high activity.

[0128] In one aspect, candidate sRGNs designed according to a homology-based gene family shuffling scheme are provided herein. Candidate sRGNs have not been shown to have any activity. Also provided are sRGNs that have been shown to have at least one activity. In certain embodiments, at least one activity includes, for example, activity in a cleavage assay (such as a fluorescence polarization-based biochemical cleavage assay), a gene editing assay (such as conversion from GFP to BFP) (Glaser et al., 2016, see below), a gene insertion assay, or an assay of other suitable parameters. In certain embodiments, on-target cleavage is assayed, off-target cleavage is assayed, and / or on-target editing (e.g., correction of a mutation or insertion of a gene or a portion of a gene) is assayed, which can be compared to off-target editing. In some cases, the activity is compared to a reference or control nuclease (such as wild-type Cas9). Assays are described in the art and are available from commercial resources. Suitable assays can be performed in an organism, such as an animal with a deleterious mutation, and the efficacy of the sRGN can be determined by the improvement of one or more symptoms associated with the mutation in an animal to which the sRGN system is administered. In certain embodiments, at least one activity is greater than the corresponding activity in one or more parent Cas9 endonucleases from which the sRGN is derived. In certain embodiments, at least one activity is greater than the corresponding activity in another wild-type Cas9 such as SluCas9.

[0129] In certain embodiments, provided herein are candidate sRGNs comprising 8 small domains, wherein the small domains are, from N-terminus to C-terminus, 1) small domain 1 comprising an amino acid sequence of any one of SEQ ID NOs: 34-37, or a variant thereof having at least about 90% (such as at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to any one of SEQ ID NOs: 34-37, 2) small domain 2 comprising an amino acid sequence of any one of SEQ ID NOs: 38-41, or a variant thereof having at least about 90% (such as at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to any one of SEQ ID NOs: 38-41, 3) small domain 3 comprising an amino acid sequence of any one of SEQ ID NOs: 42-45, or a variant thereof having at least about 90% (such as at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to any one of SEQ ID NOs: 4) a small domain 4 comprising an amino acid sequence of any one of SEQ ID NOs: 46-49 or a variant thereof having at least about 90% (such as at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to any one of SEQ ID NOs: 46-49, 5) a small domain 5 comprising an amino acid sequence of any one of SEQ ID NOs: 50-53 or a variant thereof having at least about 90% (such as at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to any one of SEQ ID NOs: 50-53, 6) a small domain 6 comprising an amino acid sequence of any one of SEQ ID NOs: 51-54 or a variant thereof having at least about 90% (such as at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to any one of SEQ ID NOs: 51-54, 54-57, or a variant thereof having at least about 90% (such as at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to any one of SEQ ID NOs: 54-57, 7) a small domain 7 comprising an amino acid sequence of any one of SEQ ID NOs: 58-61, or a variant thereof having at least about 90% (such as at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to any one of SEQ ID NOs: 58-61,and 8) a small domain 8 comprising an amino acid sequence of any one of SEQ ID NOs: 62-65 or a variant thereof having at least about 90% (such as at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to any one of SEQ ID NOs: 62-65. In certain embodiments, the candidate sRGN comprises a small domain 8 from Staphylococcus lugdunensis, Staphylococcus pasteurianus, Staphylococcus microti and a small domain of Staphylococcus suis Cas9. In certain embodiments, the candidate sRGN comprises a small domain of Staphylococcus lugdunensis, Staphylococcus pasteurianus, Staphylococcus microti In some embodiments, the small domain 8 comprises an amino acid sequence of SEQ ID NO: 62 or a variant thereof having at least about 90% (such as at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity with SEQ ID NO: 62. In some embodiments, the small domain 8 comprises an amino acid sequence of SEQ ID NO: 63 or a variant thereof having at least about 90% (such as at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity with SEQ ID NO: 63. In certain embodiments, small domain 1 comprises the amino acid sequence of SEQ ID NO: 34 or a variant thereof having at least about 90% (such as at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to SEQ ID NO: 34.

[0130] In certain embodiments, provided herein are candidate sRGNs comprising 12 small domains, wherein the small domains are, from N-terminus to C-terminus, 1) small domain 1 comprising an amino acid sequence of any one of SEQ ID NOs: 66-69, or a variant thereof having at least about 90% (such as at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to any one of SEQ ID NOs: 66-69, 2) small domain 2 comprising an amino acid sequence of any one of SEQ ID NOs: 70-73, or a variant thereof having at least about 90% (such as at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to any one of SEQ ID NOs: 70-73, 3) small domain 3 comprising an amino acid sequence of any one of SEQ ID NOs: 74-77, or a variant thereof having at least about 90% (such as at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to any one of SEQ ID NOs: 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to any one of SEQ ID NOs: 74-77, 4) a small domain 4 comprising an amino acid sequence of any one of SEQ ID NOs: 78-81 or a variant thereof having at least about 90% (such as at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to any one of SEQ ID NOs: 78-81, 5) a small domain 5 comprising an amino acid sequence of any one of SEQ ID NOs: 82-85 or a variant thereof having at least about 90% (such as at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to any one of SEQ ID NOs: 82-85, 6) a small domain 6 comprising SEQ ID NO: 86-89, or a variant thereof having at least about 90% (such as at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity with any one of SEQ ID NOs: 86-89, 7) a small domain 7 comprising an amino acid sequence of any one of SEQ ID NOs: 90-93, or a variant thereof having at least about 90% (such as at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity with any one of SEQ ID NOs: 90-93,8) a small domain 8 comprising an amino acid sequence of any one of SEQ ID NOs: 94-97 or a variant thereof having at least about 90% (such as at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to any one of SEQ ID NOs: 94-97, 9) a small domain 9 comprising an amino acid sequence of any one of SEQ ID NOs: 98-101 or a variant thereof having at least about 90% (such as at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to any one of SEQ ID NOs: 98-101, 10) a small domain 10 comprising an amino acid sequence of any one of SEQ ID NOs: 102-105 or a variant thereof having at least about 90% (such as at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to any one of SEQ ID NOs: 102-105 having at least about 90% (such as at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to any one of SEQ ID NOs: 102-105, 11) small domain 11, which comprises the amino acid sequence of any one of SEQ ID NOs: 106-109 or a variant thereof having at least about 90% (such as at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to any one of SEQ ID NOs: 106-109, and 12) small domain 12, which comprises the amino acid sequence of any one of SEQ ID NOs: 110-113 or a variant thereof having at least about 90% (such as at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to any one of SEQ ID NOs: 110-113. In certain embodiments, the candidate sRGNs include those from Staphylococcus lugdunensis, Staphylococcus pasteurianus, Staphylococcus microti and a small domain of Staphylococcus suis Cas9. In certain embodiments, the candidate sRGN comprises a small domain of Staphylococcus lugdunensis, Staphylococcus pasteurianus, Staphylococcus microtiAnd Staphylococcus suis less than all 4 (such as 2 or 3) Cas9 small domains. In certain embodiments, small domain 12 comprises an amino acid sequence of SEQ ID NO: 110 or a variant thereof having at least about 90% (such as at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity with SEQ ID NO: 110. In certain embodiments, small domain 12 comprises an amino acid sequence of SEQ ID NO: 111 or a variant thereof having at least about 90% (such as at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity with SEQ ID NO: 111. In certain embodiments, (i) small domain 1 comprises the amino acid sequence of SEQ ID NO: 66, or a variant thereof having at least about 90% (such as at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to SEQ ID NO: 66 and (ii) small domain 2 comprises the amino acid sequence of SEQ ID NO: 70, or a variant thereof having at least about 90% (such as at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to SEQ ID NO: 70.

[0131] Using such a gene family shuffling approach, the sRGN polypeptides Gib11 (SEQ ID NO: 1), P2H12 (SEQ ID NO: 5), E2 (SEQ ID NO: 6), and F8 (SEQ ID NO: 10) disclosed herein were identified, and Gib11Spa variants (SEQ ID NOs: 2-4) were generated by replacing different C-terminal portions of Gib11 with the corresponding portions of Staphylococcus pasteurianus Cas9.

[0132] The sRGN polypeptides disclosed herein are smaller than Cas9 from Streptococcus pyogenes (also referred to herein as "SpCas9" or "SpyCas9") and interact with the NNGG PAM sequence.

[0133] In one aspect, provided herein is a sRGN polypeptide comprising an amino acid sequence of any one of SEQ ID NOs: 1-12, or a variant thereof having at least about 90% (such as at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to any one of SEQ ID NOs: 1-12. In certain embodiments, the sRGN polypeptide comprises an amino acid sequence of any one of SEQ ID NOs: 1-12.

[0134] In certain embodiments, provided herein is a sRGN polypeptide comprising an amino acid sequence having at least about 90% (such as at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to a subsequence of any one of SEQ ID NOs: 1-12, wherein the subsequence comprises one or more functional sRGN domains. In certain embodiments, the sRGN polypeptide comprises (or consists of) a subsequence of any one of SEQ ID NOs: 1-12. In certain embodiments, the one or more sRGN domains comprise a nuclease domain (e.g., a HNH or RuvC domain). In certain embodiments, the one or more sRGN domains comprise an RNA-binding domain (e.g., a Rec I domain). In certain embodiments, the one or more sRGN domains comprise a PAM-interaction domain.

[0135] In certain embodiments, provided herein is a Gib11 variant comprising:

[0136] (I) a variant having at least 90% (such as at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity with the amino acid sequence of SEQ ID NO: 1 over its entire length or over the sequence from position 787 to position 1057 of SEQ ID NO: 1;

[0137] (II) a variant according to (I), which further comprises a component such as a nuclear localization signal to obtain proper activity of the Gib11 CRISPR system not only in a cell-free reaction or prokaryotic cells but also in a eukaryotic cell environment (e.g., a living organism such as a plant or an animal);

[0138] (III) codon-optimized variants of the corresponding polynucleotide sequences encoding Gib11 and variants according to (I) and (II);

[0139] (IV) The variant according to any one of (I) to (III), further comprising:

[0140] (a) one or more modifications or mutations that result in a sRGN with significantly reduced or undetectable nuclease activity, e.g., having an alanine at position 10 (e.g., D10A), position 562 (e.g., H562A), and / or position 585 (e.g., N585A) with respect to SEQ ID NO: 1, or any other exchange that results in significantly reduced nuclease activity; and

[0141] (b) A C- or N-terminally linked polypeptide having nucleotide base editing activity or deaminase activity, such as via a linker peptide.

[0142] In certain embodiments, provided herein is a P2H12 variant comprising:

[0143] (I) a variant having at least 90% (such as at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity with the amino acid sequence of SEQ ID NO: 5 over its entire length or over the sequence from position 784 to position 1055 of SEQ ID NO: 5;

[0144] (II) a variant according to (I), which further comprises a component such as a nuclear localization signal to obtain appropriate activity of the P2H12 CRISPR system not only in a cell-free reaction or prokaryotic cells but also in a eukaryotic cell environment (e.g., a living organism such as a plant or an animal);

[0145] (III) codon-optimized variants of the corresponding polynucleotide sequences encoding P2H12 and variants according to (I) and (II);

[0146] (IV) The variant according to any one of (I) to (III), further comprising:

[0147] (a) one or more modifications or mutations that result in a sRGN with significantly reduced or undetectable nuclease activity, e.g., having an alanine at position 10 (e.g., D10A), position 559 (e.g., H559A), and / or position 582 (e.g., N582A) with respect to SEQ ID NO: 5, or any other exchange that results in significantly reduced nuclease activity; and

[0148] (b) A C- or N-terminally linked polypeptide having nucleotide base editing activity or deaminase activity, such as via a linker peptide.

[0149] In certain embodiments, provided herein is an E2 variant comprising:

[0150] (I) a variant having at least 90% (such as at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity with the amino acid sequence of SEQ ID NO: 6 over its entire length or over the sequence from position 1 to position 789 of SEQ ID NO: 6;

[0151] (II) A variant according to (I), which further comprises a component such as a nuclear localization signal to obtain proper activity of the E2 CRISPR system not only in a cell-free reaction or prokaryotic cells but also in a eukaryotic cell environment (e.g., a living organism such as a plant or an animal);

[0152] (III) codon-optimized variants of the corresponding polynucleotide sequences encoding E2 and variants according to (I) and (II);

[0153] (IV) The variant according to any one of (I) to (III), further comprising:

[0154] (a) one or more modifications or mutations that result in a sRGN with significantly reduced or undetectable nuclease activity, e.g., having an alanine at position 10 (e.g., D10A), position 562 (e.g., H562A), and / or position 585 (e.g., N585A) with respect to SEQ ID NO: 6, or any other exchange that results in significantly reduced nuclease activity; and

[0155] (b) A C- or N-terminally linked polypeptide having nucleotide base editing activity or deaminase activity, such as via a linker peptide.

[0156] In certain embodiments, provided herein is a F8 variant comprising:

[0157] (I) a variant having at least 90% (such as at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity with the amino acid sequence of SEQ ID NO: 10 over its entire length or over the sequence from position 1 to position 789 of SEQ ID NO: 10;

[0158] (II) a variant according to (I), which further comprises a component such as a nuclear localization signal to obtain proper activity of the F8 CRISPR system not only in a cell-free reaction or prokaryotic cells but also in a eukaryotic cell environment (e.g., a living organism such as a plant or an animal);

[0159] (III) codon-optimized variants of the corresponding polynucleotide sequences encoding F8 and variants according to (I) and (II);

[0160] (IV) The variant according to any one of (I) to (III), further comprising:

[0161] (a) one or more modifications or mutations that result in a sRGN with significantly reduced or undetectable nuclease activity, e.g., having an alanine at position 10 (e.g., D10A), position 562 (e.g., H562A), and / or position 585 (e.g., N585A) with respect to SEQ ID NO: 10, or any other exchange that results in significantly reduced nuclease activity; and

[0162] (b) A C- or N-terminally linked polypeptide having nucleotide base editing activity or deaminase activity, such as via a linker peptide.

[0163] In certain embodiments, provided herein is a Gib11Spa-1 variant comprising:

[0164] (I) a variant having at least about 90% (such as at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to the amino acid sequence of SEQ ID NO: 2;

[0165] (II) A variant according to (I), which further comprises a component such as a nuclear localization signal to obtain appropriate activity of the Gib11Spa-1 CRISPR system not only in a cell-free reaction or prokaryotic cells but also in a eukaryotic cell environment (e.g., a living organism such as a plant or an animal);

[0166] (III) codon-optimized variants of the corresponding polynucleotide sequences encoding Gib11Spa and variants according to (I) and (II);

[0167] (IV) The variant according to any one of (I) to (III), further comprising:

[0168] (a) one or more modifications or mutations that result in a sRGN with significantly reduced or undetectable nuclease activity, e.g., having an alanine at position 10 (e.g., D10A), position 562 (e.g., H562A), and / or position 585 (e.g., N585A) with respect to SEQ ID NO: 2, or any other exchange that results in significantly reduced nuclease activity; and

[0169] (b) A C- or N-terminally linked polypeptide having nucleotide base editing activity or deaminase activity, such as via a linker peptide.

[0170] In certain embodiments, provided herein is a Gib11Spa-2 variant comprising:

[0171] (I) a variant having at least about 90% (such as at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to the amino acid sequence of SEQ ID NO: 3;

[0172] (II) A variant according to (I), which further comprises a component such as a nuclear localization signal to obtain appropriate activity of the Gib11Spa-2 CRISPR system not only in a cell-free reaction or prokaryotic cells but also in a eukaryotic cell environment (e.g., a living organism such as a plant or an animal);

[0173] (III) codon-optimized variants of the corresponding polynucleotide sequences encoding Gib11Spa and variants according to (I) and (II);

[0174] (IV) The variant according to any one of (I) to (III), further comprising:

[0175] (a) one or more modifications or mutations that result in a sRGN with significantly reduced or undetectable nuclease activity, e.g., having an alanine at position 10 (e.g., D10A), position 562 (e.g., H562A), and / or position 585 (e.g., N585A) with respect to SEQ ID NO: 3, or any other exchange that results in significantly reduced nuclease activity; and

[0176] (b) A C- or N-terminally linked polypeptide having nucleotide base editing activity or deaminase activity, such as via a linker peptide.

[0177] In certain embodiments, provided herein is a Gib11Spa-3 variant comprising:

[0178] (I) a variant having at least about 90% (such as at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to the amino acid sequence of SEQ ID NO: 4;

[0179] (II) A variant according to (I), which further comprises a component such as a nuclear localization signal to obtain appropriate activity of the Gib11Spa-3 CRISPR system not only in a cell-free reaction or prokaryotic cells but also in a eukaryotic cell environment (e.g., a living organism such as a plant or an animal);

[0180] (III) codon-optimized variants of the corresponding polynucleotide sequences encoding Gib11Spa and variants according to (I) and (II);

[0181] (IV) The variant according to any one of (I) to (III), further comprising:

[0182] (a) one or more modifications or mutations that result in a sRGN with significantly reduced or undetectable nuclease activity, e.g., having an alanine at position 10 (e.g., D10A), position 562 (e.g., H562A), and / or position 585 (e.g., N585A) with respect to SEQ ID NO: 4, or any other exchange that results in significantly reduced nuclease activity; and

[0183] (b) A C- or N-terminally linked polypeptide having nucleotide base editing activity or deaminase activity, such as via a linker peptide.

[0184] For example, the term "significantly reduced" used in the context of an enzyme activity means that such enzyme activity is less than about 10% of the activity of a reference protein (e.g., any one of SEQ ID NOs: 1-12), for example, less than 5%, less than 2%, less than 1%, or less than 0.1% of the activity of such a reference enzyme.

[0185] Suitable polypeptides under (IV) (b) and their linkage to the C-terminal domains of sRGNs under (I), (II) and (III) are described, for example, in WO 2017 / 070632, Gaudelli et al. et al., Nature 551 (November 23, 2017), 464-471; Komor et al., Sci. Adv. 2017; 3eaao4774; Kim et al., Nature Biotechnol. 2017 Apr; 35(4) 371-376; Komor et al., Nature 533(7603);420-424.

[0186] The sRGN variants under (IV) can convert any base pair to any possible other base pair, rather than introducing double-strand breaks in the target DNA sequence. A non-limiting example is a deaminase activity that can act on cytosine, guanine, or adenine bases, and then replicate and repair the deamination site within the cell to generate guanine, thymine, and guanine, respectively.

[0187] If not otherwise indicated, the term sRGN includes the sRGN polypeptide of any one of SEQ ID NOs: 1-12 and all variants thereof described herein, such as those specified under (I), (II), (III) and (IV).

[0188] In certain embodiments, provided herein is a mutant of E2 comprising, relative to SEQ ID NO: 6, a) an aspartic acid residue at position 741, and a lysine residue at position 743 (E2+K741D+L743K, SEQ ID NO: 7); or b) a threonine residue at position 670, and an aspartic acid residue at position 675 (E2+S670T+N675D, SEQ ID NO: 8); or c) an asparagine residue at position 741, and an asparagine residue at position 743 (E2+K741N+L743N, SEQ ID NO: 9); or d) any combination of (a) to (c) above.

[0189] In certain embodiments, provided herein is a mutant of E2 comprising i) having at least about 90% (such as at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) amino acid identity over its entire length with the sequence according to SEQ ID NO: 6 or with the sequence from position 1 to position 789 of SEQ ID NO: 6; and ii) exhibiting the amino acid residue at position a1 as shown in Table 1 when aligned with a continuous stretch starting from 5 positions before a1 in Table 1 and ending at 5 positions after a1; and iii) exhibiting the amino acid residue at position a2 as shown in Table 1 when aligned with a continuous stretch starting from 5 positions before a2 in Table 1 and ending at 5 positions after a2; wherein alignment refers to aligning a stretch of 11 amino acid residues with the sequence according to SEQ ID NO: 6 using a standard local alignment tool or algorithm, including, for example, Protein BLAST. (https: / / blast.ncbi.nlm.nih.gov, the National Center for Biotechnology [cited May 17, 2018] Information; Altschul, SF, Gish, W., Miller, W., Myers, EW & Lipman, DJ (1990) "Basic local alignment search tool." J. Mol. Biol. 215:403-410.), using standard parameters, and wherein at least 4 of the 5 preceding amino acids of a1 or a2 and 4 of the 5 following amino acids of a1 or a2 are identical.

[0190] Table 1

[0191] According to the position of SEQ ID NO: 6 The amino acid residue at position a1 The amino acid residue at position a2 a1=670, a2=675 Threonine Aspartic acid a1=741, a2=743 Aspartic acid Lysine a1=741, a2=743 Asparagine Asparagine

[0192] If not otherwise indicated, the term E2 includes E2 mutants including E2+K741D+L743K; E2+S670T+N675D; and E2+K741N+L743N.

[0193] In certain embodiments, provided herein are mutants of F8 comprising, relative to SEQ ID NO: 10, a) an aspartic acid residue at position 737, and a lysine residue at position 739 (F8+K737D+L739K, SEQ ID NO: 11); or b) an asparagine residue at position 737, and an asparagine residue at position 739 (F8+K737N+L739N, SEQ ID NO: 12).

[0194] In certain embodiments, provided herein is a mutant of F8 comprising i) having at least about 90% (such as at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) amino acid identity over its entire length with the sequence according to SEQ ID NO: 10 or with the sequence from position 1 to position 787 of SEQ ID NO: 10; and ii) exhibiting an amino acid residue at position a1 as shown in Table 2 when aligned with a continuous stretch starting from 5 positions before a1 in Table 2 and ending at 5 positions after a1; and iii) exhibiting an amino acid residue at position a2 as shown in Table 2 when aligned with a continuous stretch starting from 5 positions before a2 in Table 2 and ending at 5 positions after a2; wherein alignment refers to aligning a stretch of 11 amino acid residues with the sequence according to SEQ ID NO: 10 using a standard local alignment tool or algorithm, including, for example, Protein BLAST. (https: / / blast.ncbi.nlm.nih.gov, the National Center for Biotechnology [cited May 17, 2018] Information; Altschul, SF, Gish, W., Miller, W., Myers, EW & Lipman, DJ (1990) "Basic local alignment search tool." J. Mol. Biol. 215:403-410.), using standard parameters, and wherein at least 4 of the 5 preceding amino acids of a1 or a2 and 4 of the 5 following amino acids of a1 or a2 are identical.

[0195] Table 2

[0196] According to the position of SEQ ID NO: 10 The amino acid residue at position a1 The amino acid residue at position a2 a1=737, a2=739 Aspartic acid Lysine a1=737, a2=739 Asparagine Asparagine

[0197] If not otherwise indicated, the term F8 includes F8 mutants including F8+K737D+L739K; and F8+K737N+L739N.

[0198] Other sRGNs can be generated by identifying paired combinations of small domains in highly active sRGNs generated by gene family shuffling using a given library, and including at least one of these paired combinations in a rationally designed sRGN, wherein the remaining sRGN small domains are selected from the library. The remaining small domains can be selected from those that originate from the same parent Cas9 as the small domains in the paired combination. The rationally designed sRGN can further include one or more other paired combinations of small domains in the highly active sRGN. For example, a rationally designed sRGN can include 2, 3, 4, 5 or more paired combinations of small domains present in a highly active sRGN.

[0199] Chimeric RNA-guided nucleases (cRGNs)

[0200] Also provided herein are chimeric RNA-guided nucleases (cRGNs) comprising: a) an N-terminal portion (cRGN-A) taken from a Type II CRISPR RNA-guided endonuclease Cas9 from Staphylococcus, or a synthetic RNA-guided nuclease produced by DNA shuffling sequences of two or more Type II CRISPR RNA-guided endonuclease Cas proteins from Staphylococcus, such as any of the sRGNs described herein; and b) a C-terminal portion (cRGN-B) taken from a Type II CRISPR RNA-guided endonuclease Cas9 from Staphylococcus pasteurianus (NCBI Refseq WP_023374365.1); wherein cRGN-B comprises i) the entire PI domain of a Type II CRISPR RNA-guided endonuclease Cas9 from Staphylococcus pasteurianus (NCBI Refseq WP_023374365.1), which is obtained from a sRGN according to NCBI Refseq WP_023374365.1. WP_023374365.1); or ii) the entire PI domain and the entire WED domain of a type II CRISPR RNA-guided endonuclease Cas9 from Staphylococcus pasteurianus (NCBI Refseq WP_023374365.1), the latter being located at amino acid position 784 to amino acid position 904 of the sequence according to NCBI Refseq WP_023374365.1; cRGN-A comprises the first 700 amino acids of the corresponding type II CRISPR RNA-guided endonuclease Cas9 sequence, such as the first 739 amino acids or the first 783 amino acids, except that the amino acid level is identical to the NCBI Refseq WP_023374365.1 from Staphylococcus pasteurianus. cRGN-A is linked to cRGN-B directly or through a linker peptide sequence. In certain embodiments, cRGN-A comprises more than the first 700 amino acids of the respective type II CRISPR RNA-guided endonuclease Cas9 sequence, such as the first 739 amino acids or the first 783 amino acids.

[0201] The PI domain and WED domain of the type II CRISPR RNA-guided endonuclease Cas9 from Staphylococcus aureus are described in Nishimasu et al., Cell. 2015 Aug 27;162(5):1113-1126. Figure 6 The potential positioning of the two domains is further shown in FIG.

[0202] Any reference to the NCBI Refseq database in this patent application means a reference to: National Center for Biotechnology Information (NCBI) [Internet]. Bethesda (MD): National Library of Medicine (US), National Center for Biotechnology Information;

[1988] - [cited May 11, 2018]. Available from: https: / / www.ncbi.nlm.nih.gov / protein. For example, with respect to NCBI Refseq WP_048803085, this is https: / / www.ncbi.nlm.nih.gov / protein / WP_048803085.

[0203] Any reference to Staphylococcus alone refers to the genus Staphylococcus.

[0204] In the context of the present invention, DNA shuffling refers to exchanging sequence segments between different (e.g., from different Staphylococcus species) Type II CRISPR Cas9 endonuclease DNA sequences to generate chimeric DNA sequences encoding synthetic proteins with RNA-guided endonuclease activity (such as the activity of a Type II CRISPR Cas9 endonuclease). Such techniques are described in Joern JM (2003) DNA Shuffling. In: Arnold FH, Georgiou G. (eds.) Directed Evolution Library Creation. Methods in Molecular Biology™, vol231. Humana Press, and Gibbs et al., Gene. 2001 Jun 13; 271(1):13-20.

[0205] In certain embodiments, the type II CRISPR RNA-guided endonuclease Cas9 from Staphylococcus is identified by one of the following entries in the NCBI Refseq database:

[0206]

[0207]

[0208]

[0209] In certain embodiments, cRGN-A is from Gib11 (SEQ ID NO: 1). In certain embodiments, the cRGN is Gib11Spa-1 (SEQ ID NO: 2), Gib11Spa-2 (SEQ ID NO: 3), or Gib11Spa-3 (SEQ ID NO: 4).

[0210] Other small Cas nucleases

[0211] In one aspect, provided herein is a Cas9 from Staphylococcus pasteurianus, Staphylococcus microti or Staphylococcus suis. In certain embodiments, the Cas9 is from Staphylococcus pasteurianus and comprises an amino acid sequence of SEQ ID NO: 31 or a variant thereof with SEQ ID NO: 31 having at least about 90% (such as at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity. Staphylococcus pasteurianus Cas9 or "SpaCas9" recognizes the PAM sequence NNGG. In certain embodiments, the Cas9 is from Staphylococcus microti and comprises an amino acid sequence of SEQ ID NO: 33 or a variant thereof with SEQ ID NO: 33 having at least about 90% (such as at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity. Staphylococcusmicroti Cas9 or "SmiCas9" will recognize the PAM sequence NNGG. In certain embodiments, the Cas9 is from Staphylococcus suis and comprises an amino acid sequence of SEQ ID NO: 32 or a variant thereof having at least about 90% (such as at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to SEQ ID NO: 32. Staphylococcus suis Cas9 or "ShyCas9" will recognize the PAM sequence NNAAAA. In some cases, these small Cas9s can be used as alternatives to sRGNs in any of the systems, compositions and methods described herein.

[0212] sRGN peptide-based CRISPR-Cas system

[0213] In certain embodiments, provided herein is a system comprising: (a) a sRGN polypeptide or a variant thereof having at least about 90% (such as at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity with any one of SEQ ID NOs: 1-12, or a nucleic acid encoding the sRGN polypeptide or variant thereof; and (b) a guide RNA (gRNA) or a nucleic acid encoding the gRNA, wherein the gRNA is capable of guiding the sRGN polypeptide or variant thereof to a target polynucleotide sequence. In certain embodiments, the system comprises a sRGN polypeptide or a variant thereof. In certain embodiments, the sRGN polypeptide comprises (or consists of) the amino acid sequence of any one of SEQ ID NOs: 1-12. In certain embodiments, the system comprises a nucleic acid encoding sRGN. In certain embodiments, the nucleic acid has at least about 90% (such as at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity with SEQ ID NO: 13-29. In certain embodiments, the nucleic acid comprises (or consists of) the sequence of SEQ ID NO: 13-29. In certain embodiments, the system comprises a gRNA. In certain embodiments, the system comprises a nucleic acid encoding a gRNA. In certain embodiments, the gRNA is a single guide RNA (sgRNA). In certain embodiments, the system further comprises one or more additional gRNAs or nucleic acids encoding one or more additional gRNAs.

[0214] In certain embodiments, according to any nucleic acid comprising a polynucleotide sequence encoding a sRGN polypeptide or variant thereof described herein, the polynucleotide sequence is operably linked to (i) a suitable promoter for expression in a cell or in vitro environment; and / or (ii) a suitable nuclear localization signal.

[0215] In certain embodiments, according to any nucleic acid encoding a gRNA described herein, the nucleic acid comprises a polynucleotide sequence encoding the gRNA, which is operably linked to (i) a suitable promoter for expression in a cell or in vitro environment; and / or (ii) a suitable nuclear localization signal.

[0216] In certain embodiments, provided herein is a system comprising: (a) a sRGN polypeptide comprising an amino acid sequence of any one of SEQ ID NO: 1-12 or a variant thereof having at least 90% sequence identity to any one of SEQ ID NO: 1-12; and (b) a gRNA, wherein the gRNA is capable of directing the sRGN polypeptide or variant thereof to a target polynucleotide sequence. In certain embodiments, the gRNA is a sgRNA.

[0217] In certain embodiments, provided herein is a system comprising: (a) a sRGN polypeptide comprising an amino acid sequence of any one of SEQ ID NO: 1-12 or a variant thereof having at least 90% sequence identity to any one of SEQ ID NO: 1-12; and (b) a nucleic acid encoding a gRNA, wherein the gRNA is capable of directing the sRGN polypeptide or variant thereof to a target polynucleotide sequence. In certain embodiments, the gRNA is a sgRNA.

[0218] In certain embodiments, provided herein is a system comprising: (a) a nucleic acid encoding a sRGN polypeptide comprising an amino acid sequence of any one of SEQ ID NOs: 1-12 or a variant thereof having at least 90% sequence identity to any one of SEQ ID NOs: 1-12; and (b) a gRNA, wherein the gRNA is capable of directing the sRGN polypeptide or variant thereof to a target polynucleotide sequence. In certain embodiments, the nucleic acid comprises a polynucleotide sequence of any one of SEQ ID NOs: 13-29 or a variant thereof having at least 90% sequence identity to a polynucleotide sequence of any one of SEQ ID NOs: 13-29. In certain embodiments, the gRNA is an sgRNA.

[0219] In certain embodiments, provided herein is a system comprising: (a) a nucleic acid encoding a sRGN polypeptide comprising an amino acid sequence of any one of SEQ ID NOs: 1-12 or a variant thereof having at least 90% sequence identity to any one of SEQ ID NOs: 1-12; and (b) a nucleic acid encoding a gRNA, wherein the gRNA is capable of directing the sRGN polypeptide or variant thereof to a target polynucleotide sequence. In certain embodiments, the nucleic acid comprises a polynucleotide sequence of any one of SEQ ID NOs: 13-29 or a variant thereof having at least 90% sequence identity to a polynucleotide sequence of any one of SEQ ID NOs: 13-29. In certain embodiments, the gRNA is an sgRNA. In certain embodiments, the system comprises a nucleic acid comprising (i) a nucleic acid encoding a sRGN polypeptide or a variant thereof; and (ii) a nucleic acid encoding a gRNA.

[0220] In certain embodiments, according to any system described herein, the system further includes a donor template. In certain embodiments, the donor template includes a donor sequence for inserting a target polynucleotide sequence. In certain embodiments, the donor sequence is an exogenous sequence, such as a transgenic for expression in a target. In certain embodiments, the donor template includes a donor sequence for modifying one or more bases in a target polynucleotide sequence. In certain embodiments, the system further includes one or more additional donor templates. In certain embodiments, the donor template is physically connected to the gRNA in the system.

[0221] According to one embodiment of the present invention, there is provided a composition comprising:

[0222] (a) a sRGN polypeptide or a nucleic acid encoding such a sRGN;

[0223] (b) a single heterologous guide RNA (sgRNA) or a nucleic acid (e.g., DNA) that allows for the in situ generation of such an sgRNA (e.g., a nucleic acid encoding the sgRNA), wherein the sgRNA comprises:

[0224] i. a DNA-targeting segment comprising RNA and capable of hybridizing to a target sequence in a polynucleotide locus,

[0225] ii. a tracr partner sequence comprising an RNA, and

[0226] iii. a tracr RNA sequence comprising RNA,

[0227] wherein the tracr partner sequence is capable of hybridizing to the tracr sequence, and wherein (i), (ii) and (iii) are arranged in a 5' to 3' orientation.

[0228] Within the sgRNA, the tracr partner sequence and the tracr sequence are usually connected by a suitable loop sequence to form a stem-loop structure.

[0229] Further, in certain embodiments, the nucleic acid encoding the sRGN polypeptide and / or the sgRNA contains a suitable promoter and / or a suitable nuclear localization signal for expression in a cell or in vitro environment.

[0230] According to another embodiment of the present invention, there is provided a method for targeting, editing, modifying or manipulating a target DNA at one or more locations in a cell or in vitro, comprising:

[0231] (a) introducing a heterologous sRGN polypeptide or a nucleic acid encoding a sRGN polypeptide into a cell or in vitro environment; and

[0232] (b) introducing a single heterologous guide RNA (sgRNA) or a nucleic acid (e.g., DNA) suitable for generating such an sgRNA in situ (e.g., a nucleic acid encoding the sgRNA), wherein the sgRNA comprises:

[0233] i. a DNA-targeting segment comprising RNA and capable of hybridizing to a target sequence in a polynucleotide locus,

[0234] ii. a tracr partner sequence comprising an RNA, and

[0235] iii. a tracr RNA sequence comprising RNA,

[0236] wherein the tracr partner sequence is capable of hybridizing to the tracr sequence, and wherein (i), (ii) and (iii) are arranged in a 5' to 3' orientation; and

[0237] (c) generating one or more cuts, nicks or edits in the target DNA, wherein the sRGN polypeptide is guided to the target DNA by a gRNA in a processed or unprocessed form thereof.

[0238] Another embodiment according to the present invention is the use of a composition comprising the following for targeting, editing, modifying or manipulating target DNA at one or more locations in a cell (e.g., in vitro or in vivo) or in a cell-free system:

[0239] (a) a sRGN polypeptide or a nucleic acid encoding such a sRGN;

[0240] (b) a single heterologous guide RNA (sgRNA) or a nucleic acid (e.g., DNA) suitable for generating such an sgRNA in situ (e.g., a nucleic acid encoding the sgRNA), wherein the sgRNA comprises:

[0241] i. a DNA-targeting segment comprising RNA and capable of hybridizing to such a target sequence in a polynucleotide locus,

[0242] ii. a tracr partner sequence comprising an RNA, and

[0243] iii. a tracr RNA sequence comprising RNA,

[0244] wherein the tracr partner sequence is capable of hybridizing to the tracr sequence, and wherein (i), (ii) and (iii) are arranged in a 5' to 3' orientation.

[0245] Another embodiment according to the invention is an ex vivo or in vitro cell comprising:

[0246] (a) a heterologous sRGN polypeptide or a nucleic acid encoding the same;

[0247] (b) a single heterologous guide RNA (sgRNA) or a nucleic acid (e.g., DNA) suitable for generating such an sgRNA in situ (e.g., a nucleic acid encoding the sgRNA), wherein the sgRNA comprises:

[0248] i. a DNA-targeting segment comprising RNA and capable of hybridizing to such a target sequence in a polynucleotide locus,

[0249] ii. a tracr partner sequence comprising an RNA, and

[0250] iii. a tracr RNA sequence comprising RNA,

[0251] wherein the tracr partner sequence is capable of hybridizing to the tracr sequence, and wherein (i), (ii) and (iii) are arranged in a 5' to 3' orientation;

[0252] Or such a cell whose genome has been targeted, edited, modified or manipulated using (a) and (b) above.

[0253] Additional embodiments according to the present invention include kits comprising:

[0254] (a) a nucleic acid encoding a sRGN polypeptide, wherein the nucleic acid is operably linked to a promoter or a ribosome binding site; and

[0255] (b) a single heterologous guide RNA (sgRNA) or a nucleic acid (e.g., DNA) suitable for generating such an sgRNA in situ (e.g., a nucleic acid encoding the sgRNA), wherein the sgRNA comprises:

[0256] i. a DNA-targeting segment comprising RNA and capable of hybridizing to such a target sequence in a polynucleotide locus,

[0257] ii. a tracr partner sequence comprising an RNA, and

[0258] iii. a tracr RNA sequence comprising RNA,

[0259] wherein the tracr partner sequence is capable of hybridizing to the tracr sequence, and wherein (i), (ii) and (iii) are arranged in a 5' to 3' orientation.

[0260] or

[0261] (a) sRGN polypeptide; and

[0262] (b) one or more single heterologous guide RNAs (sgRNAs), each of which comprises:

[0263] iv. a DNA-targeting segment comprising RNA and capable of hybridizing to such a target sequence in a polynucleotide locus,

[0264] v. a tracr partner sequence comprising an RNA, and

[0265] vi. a tracr RNA sequence comprising RNA,

[0266] wherein the tracr partner sequence is capable of hybridizing to the tracr sequence, and wherein (iv), (v) and (vi) are arranged in a 5' to 3' orientation.

[0267] Yet another embodiment according to the present invention comprises compositions and methods for targeting, editing, modifying or manipulating one or more target DNAs at one or more locations in a cell (e.g., in vitro or in vivo) or in a cell-free system, comprising:

[0268] (a) a sRGN polypeptide or a nucleic acid encoding a sRGN polypeptide; and

[0269] (b) a guide RNA (gRNA) or a nucleic acid (e.g., DNA) suitable for producing such a gRNA in situ (e.g., a nucleic acid encoding the gRNA), wherein the gRNA comprises:

[0270] i. a DNA-targeting segment comprising RNA and capable of hybridizing to such a target sequence in a polynucleotide locus,

[0271] ii. a tracr RNA sequence comprising RNA;

[0272] Where (i) and (ii) are on a single RNA molecule, and (iii) is on a separate RNA molecule.

[0273] Mutants

[0274] In certain embodiments, the sRGN polypeptide is a mutant polypeptide with altered endoribonuclease activity or associated half-life of pre-crRNA, intermediate crRNA, or mature crRNA. In certain embodiments, the sRGN polypeptide is a mutant polypeptide with altered or abolished DNA endoribonuclease activity without substantially reduced or enhanced endoribonuclease activity or binding affinity to DNA. Such modifications may facilitate sequence-specific DNA targeting of sRGN polypeptides for transcriptional regulation, activation, or repression; epigenetic modification or chromatin modification by methylation, demethylation, acetylation, or deacetylation, or any other modification of DNA binding and / or DNA modifying proteins known in the art.

[0275] In certain embodiments, the sRGN polypeptide is a mutant polypeptide that has no DNA endonuclease activity.

[0276] In certain embodiments, the cell is a bacterial cell, a fungal cell, an archaeal cell, a protist, a plant cell, or an animal cell.

[0277] In certain embodiments, a sRGN polypeptide and one or more guide RNAs (gRNAs), such as a single heterologous guide RNA (sgRNA), are introduced into a cell via the same or different recombinant vectors encoding the polypeptide and the nucleic acid.

[0278] In certain embodiments, the nucleic acid encoding the polypeptide, the nucleic acid, or both the polypeptide and the nucleic acid is modified.

[0279] In certain embodiments, the method or system further comprises adding a donor DNA sequence, and wherein the target DNA sequence is edited by homology-directed repair. In certain embodiments, the polynucleotide donor template is physically linked to the crRNA or guide RNA.

[0280] In another aspect, provided herein are methods for modifying or editing double-stranded or single-stranded target DNA without activity towards single-stranded RNA (ssRNA), double-stranded RNA (dsRNA), or heteroduplexes of RNA and DNA.

[0281] Multiplexing

[0282] In another aspect, provided herein is a method for editing or modifying DNA at multiple locations in a cell, consisting essentially of: i) introducing a sRGN polypeptide or a nucleic acid encoding a sRGN polypeptide into a cell; and ii) introducing a single heterologous nucleic acid comprising two or more pre-CRISPR RNAs (pre-crRNAs) as RNA or encoded as DNA and under the control of a promoter into the cell, each pre-crRNA comprising an array of repeat spacers or a repeat spacer, 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 sRGN polypeptide cleaves the two or more pre-crRNAs upstream of the stem-loop structure to produce 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 directs the sRGN polypeptide to create two or more double-strand breaks (DSBs) into the DNA. For example, one advantage of sRGN polypeptides is that it is possible to introduce only one pre-crRNA comprising several repeat spacer units, which upon introduction, is processed by the sRGN polypeptide into active repeat spacer units that target several different sequences on DNA.

[0283] In certain embodiments, the pre-crRNA sequences in a single heterologous nucleic acid are linked together in a specific position, orientation, order, or with specific chemical bonds to direct or differentially regulate the endonuclease activity of the sRGN polypeptide at each site specified by a different crRNA sequence.

[0284] In another aspect, provided herein are examples of overall methods for editing or modifying the structure or function of DNA at multiple locations in a cell, consisting essentially of: i) introducing an RNA-guided endonuclease, such as an sRGN, into a cell as a polypeptide or a nucleic acid encoding the RNA-guided endonuclease; and ii) introducing a single heterologous nucleic acid comprising or encoding two or more guide RNAs as RNA or encoded as DNA and under the control of one or more promoters, wherein the activity or function of the RNA-guided endonuclease is directed by the guide RNA sequence in the single heterologous nucleic acid.

[0285] Codon-optimized DNA sequences of sRGN polypeptides

[0286] In some embodiments of the methods, the nucleic acid sequence encoding the sRGN polypeptide is a modified nucleic acid, e.g., codon-optimized. In some embodiments of the methods, the single heterologous nucleic acid is a modified nucleic acid. In some embodiments of the methods, the methods further comprise introducing a polynucleotide donor template into the cell. In certain embodiments, the polynucleotide donor template is physically linked to the crRNA or guide RNA. In some embodiments of the methods, the DNA is repaired at DSBs by homology-directed repair, non-homologous end joining, or microhomology-mediated end joining.

[0287] In some embodiments of the methods, the sRGN polypeptide is more readily complexed with mature crRNA in the local environment, and thus more readily available to direct DNA endonuclease activity due to crRNA being processed by the same sRGN polypeptide from pre-crRNA in the local environment.

[0288] In some embodiments of the methods, the sRGN polypeptide is used to cleave, isolate or purify one or more mature crRNA sequences from a modified pre-crRNA oligonucleotide sequence in which a heterologous sequence is incorporated 5' or 3' to one or more crRNA sequences in an RNA oligonucleotide or DNA expression construct. Heterologous sequences can be incorporated to modify stability, half-life, expression level or temporal control, interaction with the sRGN polypeptide or target DNA sequence, or any other physical or biochemical characteristic known in the art.

[0289] In some embodiments of the methods, the pre-crRNA sequence is modified to provide differential regulation of two or more mature crRNA sequences within the pre-crRNA sequence to differentially modify stability, half-life, expression level or temporal control, interaction with a sRGN polypeptide or target DNA sequence, or any other physical or biochemical characteristic known in the art.

[0290] In certain embodiments, the sRGN polypeptide (or nucleic acid encoded variant thereof) is modified to improve desired characteristics, such as function, activity, kinetics, half-life, etc. One such non-limiting example of such a modification is replacement of the 'cleavage domain' of the sRGN polypeptide with a homologous or heterologous cleavage domain from a different nuclease (e.g., a RuvC or HNH domain from the type II CRISPR-associated nuclease Cas9).

[0291] In one aspect, provided herein are methods for targeting, editing or manipulating DNA in a cell comprising attaching a complete or partially or completely defective sRGN polypeptide or pre-crRNA or crRNA portion to a dimeric FOK1 nuclease to direct endonuclease cleavage when directed to one or more specific DNA target sites by one or more crRNA molecules. In another embodiment, the FOK1 nuclease system is a nickase or a temperature-sensitive mutant or any other variant known in the art.

[0292] In certain embodiments, a sRGN polypeptide linked to a dimeric FOK1 nuclease is introduced into a cell along with a single heterologous nucleic acid comprising two or more pre-crISPR RNAs (pre-crRNAs) as RNA or encoded as DNA and under the control of a promoter, each pre-crRNA comprising an array of repeat sequence spacers, wherein the spacers comprise a nucleic acid sequence complementary to a target sequence in the DNA, and the repeat sequence comprises a stem-loop structure, wherein the sRGN polypeptide cleaves the two or more pre-crRNAs upstream of the stem-loop structure to produce two or more intermediate crRNAs.

[0293] In one aspect, provided herein are methods for targeting, editing or manipulating DNA in a cell, comprising ligating a complete or partially or completely defective sRGN polypeptide or pre-crRNA, an intermediate crRNA, a mature crRNA portion or a gRNA (collectively referred to as crRNA) to a donor single-stranded or double-stranded DNA donor template to promote homologous recombination of an exogenous DNA sequence when guided to one or more specific DNA target sites by one or more guide RNA or crRNA molecules.

[0294] In yet another aspect, provided herein is a method for directing a DNA template to a specific site of gene editing for homologous recombination or homology-directed repair. In this regard, a single-stranded or double-stranded DNA template is chemically or otherwise connected to a crRNA or guide RNA as known in the art. In certain embodiments, the DNA template remains connected to the crRNA or guide RNA; in yet other examples, the sRGN polypeptide cuts the crRNA or guide RNA, thereby releasing the DNA template to enable or promote homologous recombination.

[0295] In yet another aspect, provided herein are methods for targeting, editing or manipulating DNA in a cell comprising linking a complete or partially or completely defective sRGN polypeptide or pre-crRNA or crRNA portion to a transcriptional activator or repressor protein, or an epigenetic modifier, such as a methylase, demethylase, acetyltransferase or deacetylase, or signal transduction or detection, when directed to one or more specific DNA target sites by one or more crRNA molecules, all aspects of which have been previously described with respect to the Cas9 system.

[0296] In another aspect, compositions are provided herein, comprising a polynucleotide donor template linked to a crRNA or guide RNA. A method for targeting, editing or manipulating DNA in a cell, comprising linking a pre-crRNA or crRNA or guide RNA to a donor single-stranded or double-stranded polynucleotide donor template, such that the donor template is cleaved from the pre-crRNA or crRNA or guide RNA by a sRGN polypeptide, thereby promoting homology-directed repair by the donor template when directed to one or more specific DNA target sites by one or more guide RNA or crRNA molecules.

[0297] sRGN polypeptides can also be used to form chimeric binding proteins into which other domains and activities are introduced. By way of illustration, a Fok1 domain can be fused to a sRGN protein that can contain a catalytically active endonuclease domain, or a Fok1 domain can be fused to a sRGN protein that has been modified to render the sRGN polypeptide endonuclease domain inactive. Other domains that can be fused to sRGN polypeptides to prepare chimeric proteins include transcriptional regulators, epigenetic modifiers, tags and other markers or imaging agents, histones, and / or other modalities known in the art that regulate or modify the structure or activity of a gene sequence.

[0298] Nucleic acid and / or amino acid modifications

[0299] In certain embodiments, the polynucleotide introduced into the cell comprises one or more modifications that can be used, for example, to enhance activity, stability or specificity, to alter delivery, to reduce innate immune responses in the host cell, to further reduce protein size, or for other enhancements, as further described herein and known in the art.

[0300] In certain embodiments, modified polynucleotides are used in sRGN-based CRISPR-Cas systems, in which case the guide RNA and / or the DNA or RNA encoding the sRGN polypeptide introduced into the cell can be modified, as described and exemplified below. Such modified polynucleotides can be used in CRISPR-Cas systems to edit any one or more genomic loci.

[0301] Components of the CRISPR-Cas system

[0302] A. Guide RNA / sgRNA

[0303] In aspects of the invention, the terms "chimeric RNA", "chimeric guide RNA", "guide RNA", "single guide RNA" and "synthetic guide RNA" are used interchangeably and refer to a polynucleotide sequence comprising a DNA-targeting segment, a tracr sequence and a tracr partner sequence. The term "guide sequence" or "DNA-targeting sequence" refers to a sequence of about 20 bp within a guide RNA that specifies a target site and can be used interchangeably with the terms "guide" or "spacer". The term "tracr partner sequence" can also be used interchangeably with the term "one or more direct repeat sequences".

[0304] In some aspects, a single guide RNA (sgRNA) according to the invention comprises:

[0305] i. a DNA-targeting segment or DNA-targeting sequence comprising RNA and capable of hybridizing to a target sequence in a polynucleotide locus,

[0306] ii. a tracr partner sequence comprising an RNA, and

[0307] iii. a tracr RNA sequence comprising RNA,

[0308] wherein the tracr partner sequence is capable of hybridizing to the tracr sequence, and wherein (i), (ii) and (iii) are arranged in a 5' to 3' orientation. In certain embodiments, components (i), (ii) and (iii) are on a single chimeric RNA.

[0309] Within the sgRNA, the tracr partner sequence and the tracr sequence can be connected by a suitable loop sequence and form a stem-loop structure.

[0310] Typically, a tracr partner sequence includes any sequence that has sufficient complementarity to a tracr sequence to promote one or more of: (1) excision of a DNA-targeting segment flanking the tracr partner 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 the tracr partner sequence hybridized to the tracr sequence. Typically, the degree of complementarity is based on the optimal alignment of the tracr partner sequence and the tracr sequence along the length of the shorter of the two sequences. The optimal alignment can be determined by any suitable alignment algorithm and can further account for secondary structure, such as self-complementarity within the tracr sequence or the tracr partner sequence. In certain embodiments, the degree of complementarity between the tracr sequence and the tracr partner sequence is about or greater than 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97.5%, 99% or more along the 30 nucleotide length of the shorter of the two when optimally aligned. In certain embodiments, the tracr sequence is about or greater 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 length. In certain embodiments, the tracr sequence and the tracr partner 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 embodiments of the invention, the transcript or transcribed polynucleotide sequence has at least two or more hairpins.

[0311] In certain embodiments, the transcript has two, three, four or five hairpins. In further embodiments of the invention, the transcript has up to five hairpins. In the hairpin structure, the sequence portion 5' of the last "N" and upstream of the loop corresponds to the tracr partner sequence, while the sequence portion 3' of the loop corresponds to the tracr sequence. Further non-limiting examples of single polynucleotides comprising a DNA-targeting segment, a tracr partner sequence, and a tracr sequence are as follows (listed 5' to 3'), wherein "N" represents a base of the DNA-targeting segment, the first group of lowercase letters represents the tracr partner sequence, and the second group of lowercase letters represents the tracr sequence, and the last poly-thymidylic acid (poly-T) sequence represents a transcription terminator:

[0312] The DNA-targeting segment of the guide RNA comprises a nucleotide sequence that is complementary to a sequence in the target DNA. In other words, the DNA-targeting segment of the guide RNA interacts with the target DNA in a sequence-specific manner by hybridization (i.e., base pairing). Therefore, the nucleotide sequence of the DNA-targeting segment can vary and determine the position within the target DNA where the guide RNA and the target DNA will interact. The DNA-targeting segment of the guide RNA can be modified (e.g., by genetic engineering) to hybridize with any desired sequence within the target DNA.

[0313] The DNA-targeting segment can have a length of 10 nucleotides to 30 nucleotides.

[0314] In certain embodiments, the DNA-targeting segment has a length of 13 nucleotides to 25 nucleotides.

[0315] In certain embodiments, the DNA-targeting segment has a length of 15 nucleotides to 23 nucleotides.

[0316] In certain embodiments, the DNA-targeting segment has a length of 18 nucleotides to 22 nucleotides, eg, 20 to 22 nucleotides.

[0317] The percent complementarity between the DNA-targeting sequence of the DNA-targeting segment and the target sequence of the target DNA can be 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 a range of 20-22 nucleotides.

[0318] The protospacer sequence complementary to the target sequence in the target DNA may be adjacent at its 3' end to a suitable PAM sequence for the sRGN polypeptide, or such a PAM sequence may be part of the 3' portion of the DNA-targeting segment.

[0319] To illustrate this use for non-limiting purposes using the CRISPR-Cas system, modifications of guide RNAs can be used to enhance the formation or stability of a CRISPR-Cas genome editing complex comprising a guide RNA and a Cas endonuclease, such as an sRGN. Modifications of guide RNAs can also or alternatively be used to enhance the initiation, stability, or kinetics of the interaction between the genome editing complex and a target sequence in the genome, which can, for example, be used to enhance on-target activity. Modifications of guide RNAs can also or alternatively be used to enhance specificity, for example, the relative ratio of genome editing at an on-target site compared to the effect at other (off-target) sites.

[0320] Modifications can also or alternatively be used to increase the stability of the guide RNA, for example, by increasing its resistance to degradation by ribonucleases (RNases) present in the cell, thereby resulting in an increase in its half-life in the cell. Modifications that enhance the half-life of the guide RNA are particularly useful in embodiments in which a Cas endonuclease, such as an sRGN polypeptide, is introduced into the cell to be edited via an RNA that needs to be translated to produce the sRGN polypeptide, because increasing the half-life of a guide RNA that is introduced simultaneously with 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.

[0321] B. Protospacer-adjacent motif (PAM or PAM sequence)

[0322] Suitable protospacer adjacent motif (PAM) sequences for sRGN polypeptides include "ANGG" and "NNGG".

[0323] The cleavage site is typically present within one to three base pairs upstream of the PAM sequence, such as within one or three base pairs upstream of the PAM sequence, within three base pairs of the PAM sequence, or within three base pairs upstream of the PAM sequence "NNGG." Other PAMs are known in the art, and sRGNs can be engineered to target such PAMs.

[0324] C. Use in eukaryotic cells

[0325] In one embodiment according to the invention, the sRGN system described herein can be used in eukaryotic cells, such as mammalian cells. In certain embodiments, the cell is not a human fetal cell or is not derived from a human fetal cell. In certain embodiments, the cell is not a human embryonic cell or is not derived from a human embryonic cell. In certain embodiments, the compositions and methods disclosed herein do not involve the destruction of a human fetus or human embryo. In certain embodiments, the subject (or individual, regardless of which is used in the specification) is not a human fetus or embryo.

[0326] Other features of the present invention

[0327] Modifications can also or alternatively be used to reduce the likelihood or extent to which RNA introduced into a cell elicits an innate immune response. As described below and in the art, such responses, which have been well characterized in the context of RNA interference (RNAi), including small interfering RNA (siRNA), tend to be associated with reduced half-life of the RNA and / or the eduction of cytokines or other factors associated with an immune response.

[0328] The RNA encoding the endonuclease (e.g., sRGN polypeptide) introduced into the cell may also be subjected to one or more types of modifications, including, but not limited to, modifications that enhance the stability of the RNA (e.g., by reducing its degradation by RNases present in the cell), modifications that enhance translation of the resulting product (i.e., the endonuclease), and / or modifications that reduce the likelihood or extent to which the RNA introduced into the cell elicits an innate immune response. Combinations of modifications, such as those described above and others, may also be used. For example, in the case of CRISPR-Cas, one or more types of modifications may be made to the guide RNA (including those exemplified above), and / or one or more types of modifications may be made to the RNA encoding the CAS endonuclease (including those exemplified above).

[0329] As an illustration, guide RNAs or other smaller RNAs used in CRISPR-Cas systems can be readily synthesized by chemical methods, enabling the easy incorporation of many modifications, as exemplified below and described in the art. Although chemical synthesis procedures continue to advance, as polynucleotide lengths increase significantly beyond a hundred or so nucleotides, purification of such RNAs by procedures such as high performance liquid chromatography (HPLC, which avoids the use of gels such as PAGE) tends to become more challenging. One method for producing chemically modified RNAs of greater length is to produce two or more molecules linked together. Longer RNAs (e.g., those encoding sRGN polypeptides) are more easily produced enzymatically. Although there are fewer types of modifications that are generally available for use with RNAs produced enzymatically, there are modifications that can be used, for example, 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 novel types of modifications are often being developed. As an illustration of various types of modifications, especially those often used with smaller chemically synthesized RNAs, modifications can include one or more nucleotides modified at the 2' position of the sugar, in certain embodiments 2'-O-alkyl, 2'-O-alkyl-O-alkyl or 2'-fluoro modified nucleotides. In certain embodiments, RNA modifications include 2'-fluoro, 2'-amino and 2'O-methyl modifications on the ribose sugar, basic residues of pyrimidines or the reverse base at the 3' end of the RNA. Such modifications are routinely incorporated into oligonucleotides, and these oligonucleotides have been shown to have higher Tm (i.e., higher target binding affinity) for a given target than 2'-deoxy oligonucleotides.

[0330] It has been demonstrated that many nucleotide and nucleoside modifications make the oligonucleotides into which they are incorporated more resistant to nuclease digestion than natural oligonucleotides; these modified oligonucleotides survive longer intact than unmodified oligonucleotides. Specific examples of modified oligonucleotides include those comprising modified backbones, for example, phosphorothioate, phosphotriester, methylphosphonate, short chain alkyl or cycloalkyl sugar bonds or short chain heteroatoms or heterocyclic sugar bonds. Some oligonucleotides are those with a phosphorothioate backbone, as well as those with a heteroatom backbone, particularly CH2-NH-O-CH2, CH,-N(CH3)-O-CH2 (referred to as a methylene (methylimino) or MMI backbone), CH2-O-N(CH3)-CH2, CH2-N(CH3)-N(CH3)-CH2, and O-N(CH3)-CH2-CH2 backbones; amide backbones [see De Mesmaeker et al., Ace. Chem. Res., 28:366-374 (1995)]; morpholino 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, with the nucleotides being bound directly or indirectly 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 alkyl phosphonates including 3' alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3'-aminophosphoramidates and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphates having normal 3'-5' linkages, 2'-5' linked analogs of these, and those having reversed polarity. some, wherein adjacent pairs of nucleoside units are linked 3'-5' to 5'-3' or 2'-5' to 5'-2'; see U.S. Patent Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,196; 5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131; 5,399,676; 5,405,939; 5,453,496; 5,455, and 5,625,050.

[0331] Morpholino-based oligomeric compounds are described in Braasch and David Corey, Biochemistry, 41(14): 4503-4510 (2002); Genesis, Vol. 30, No. 3, (2001); Heasman, Dev. Biol., 243:209-214 (2002); Nasevicius et al., Nat. Genet., 26:216-220 (2000); Lacenra et al., Proc. Nat / . Acad. Sci., 97: 9591-9596 (2000); and U.S. Pat. No. 5,034,506, issued July 23, 1991. Cyclohexenyl nucleic acid oligonucleotide mimetics are described in Wang et al., J. Am. Chem. Soc., 122:8595-8602 (2000).

[0332] The modified oligonucleotide backbones which do not contain a phosphorus atom have backbones formed from short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatom or heterocyclic internucleoside linkages. These include those having morpholino linkages (formed in part 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; sulfamate backbones; methyleneimino and methylenehydrazine backbones; sulfonate and sulfamoyl backbones; amide backbones; and others having mixed N, O, S and CH2 component moieties; see U.S. Patent Nos. 5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033; 5,264, 5,623, 070; 5,663,312; 5,633,360; 5,677,437; and 5,677,439, each of which is incorporated herein by reference.

[0333] One or more substituted sugar moieties may also be included, for example, one of the following at the 2' position: OH, SH, SCH3, F, OCN, OCH3, OCH3 O(CH2)n CH3, O(CH2)n NH2, or O(CH2) n CH3, wherein n is 1 to 10; C1 to C 10 Lower alkyl, alkoxyalkoxy, substituted lower alkyl, alkaryl or aralkyl; Cl; Br; CN; CF3; OCF3; O-, S- or N-alkyl; O-, S- or N-alkenyl: SOCH3; SO2 CH3; ONO2; NO2; N3; NH2; heterocycloalkyl; heterocycloalkylaryl; aminoalkylamino; polyalkylamino; substituted silyl; RNA cleavage group; reporter group; intercalator; group for improving the pharmacokinetic properties of oligonucleotides; or group for improving the pharmacokinetic properties of oligonucleotides and other substituents with similar properties. In certain embodiments, the modification includes 2'-methoxyethoxy (2'-O-CH2CH2OCH3, also known as 2'-O-(2-methoxyethyl)) (Martin et al., Helv. Chim. Acta, 1995, 78, 486). Other modifications include 2'-methoxy (2'-O-CH3), 2'-propoxy (2'-OCH2 CH2CH3) and 2'-fluorine (2'-F). Similar modifications can also be made at other positions of the oligonucleotide, particularly 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 cyclobutyl replacing furanopentosyl. In certain embodiments, both the sugar and the internucleoside bond of the nucleotide unit, i.e., the main chain, are replaced by novel groups. The base unit is maintained for hybridization with appropriate nucleic acid target compounds. One such oligomeric compound, an oligonucleotide mimic with excellent hybridization properties, is referred to as peptide nucleic acid (PNA). In PNA compounds, the sugar-main chain of the oligonucleotide is replaced by a main chain containing amides, such as an aminoethylglycine main chain. The nucleoside base is retained and directly or indirectly bonded to the aza nitrogen atom of the amide portion of the main chain. Representative US patents that teach the preparation of PNA compounds include, but are not limited to, US Patent Nos. 5,539,082; 5,714,331; and 5,719,262. Further teachings of PNA compounds can be found in Nielsen et al., Science, 254: 1497-1500 (1991).

[0334] The guide RNA may also additionally or alternatively include nucleobase (alternatively referred to as "base") modifications or substitutions. "Unmodified" or "natural" nucleobases as used herein include adenine (A), guanine (G), thymine (T), cytosine (C), and uracil (U). Modified nucleobases include nucleobases that are only occasionally or transiently found in natural nucleic acids, such as hypoxanthine, 6-methyladenine, 5-Me pyrimidine, particularly 5-methylcytosine (also known as 5-methyl-2'deoxycytosine, and may be referred to in the art as 5-Me-C), 5-hydroxymethylcytosine (HMC), glycosyl HMC and gentiobiosyl HMC, as well as synthetic nucleobases such as 2-aminoadenine, 2-(methylamino)adenine, 2-(imidazolylalkyl)adenine, 2-(aminoalkylamino)adenine or other heterosubstituted alkyladenines, 2-thiouracil, 2-thiothymine, 5-bromouracil, 5-hydroxymethyluracil, 8-azaguanine, 7-deazaguanine, N6 (6-aminohexyl)adenine and 2.6-diaminopurine. Kornberg, A, DNA Replication, WH Freeman & Co., San Francisco, pp. 75-77 (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 substitutions have 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 are embodiments of base substitutions.

[0335] Modified nucleoside bases include other synthetic and natural nucleoside bases, 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-thioalkyl, 8-hydroxy and other α-substituted adenines and guanines, 5-halo, especially 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine and 3-deazaguanine and 3-deazaadenine.

[0336] Further, nucleoside bases include those disclosed in U.S. Pat. No. 3,687,808, those disclosed in 'The Concise Encyclopedia of Polymer Science And Engineering', pp. 858-859, Kroschwitz, Jl, ed. John Wiley & Sons, 1990, those disclosed by Englisch et al., Angewandte Chemie, International Edition', 1991, 30, p. 613, and those disclosed by Sanghvi, YS, Chapter 15, Antisense Research and Applications', pp. 289- 302, Crooke, ST and Lebleu, B.ea., CRC Press, 1993. Certain of these nucleoside bases are particularly useful for increasing the binding affinity of the oligomeric compounds of the present invention. 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 in U.S. Patent Application Publication 20030158403.

[0337] It is not necessary that all positions in a given oligonucleotide be uniformly modified, and in fact more than one of the above modifications may be incorporated into a single oligonucleotide, or even at a single nucleoside within an oligonucleotide.

[0338] In certain embodiments, the guide RNA and / or mRNA (or DNA) encoding an endonuclease such as a sRGN polypeptide is chemically linked to one or more moieties or conjugates that enhance the activity, cellular distribution, or cellular uptake of the oligonucleotide. Such moieties include, but are not limited to, lipid moieties, such as a cholesterol moiety [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. NY 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 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)]; polyamines or polyethylene glycol chains [Mancharan et al., Nucfeosides & Nucleotides, 14: 969-973 (1995)]; adamantane acetic acid [Manoharan et al., Tetrahedron Lett., 36: 3651-3654 (1995)]; a palmityl moiety [(Mishra et al., Biochim. Biophys. Acta, 1264: 229-237 (1995)]; or an octadecylamine or hexylamino-carbonyl-t-hydroxycholesterol moiety [Crooke et al., J. Pharmacal. 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, and 5,688,941.

[0339] Sugars and other moieties can be used to target proteins and complexes containing nucleotides (e.g., cationic polysomes and liposomes) to specific sites. For example, hepatocyte-directed metastasis can be mediated by asialoglycoprotein receptors (ASGPRs); e.g., Hu, et al., Protein Pept Lett. 21(1 0): 1025-30 (2014). Other systems known in the art and often developed can be used to target the biomolecules and / or their complexes used in this case to specific target cells of interest.

[0340] These targeting moieties or conjugates can include conjugate groups covalently bound to functional groups such as primary or secondary hydroxyl groups. The conjugate groups of the present invention include intercalators, reporters, polyamines, polyamides, polyethylene glycols, polyethers, groups that enhance the pharmacokinetic properties of oligomers, and groups that enhance the pharmacokinetic properties of oligomers. Typical conjugate groups include cholesterol, lipids, phospholipids, biotin, phenazine, folic acid, phenanthridine, anthraquinone, acridine, fluorescein, rhodamine, coumarin and dyes. In the context of the present invention, the group that enhances the pharmacokinetic properties includes groups that improve uptake, enhance resistance to degradation and / or enhance sequence-specific hybridization with target nucleic acids. In the context of the present invention, the group that enhances the pharmacokinetic properties includes groups that improve the uptake, distribution, metabolism or excretion of the compounds of the present invention. Representative conjugate 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. Conjugate moieties include, but are not limited to, lipid moieties, such as cholesterol moieties, cholic acid, thioethers, such as hexyl-5-tritylthiol, thiocholesterol, aliphatic chains, such as dodecandiol or undecyl residues, phospholipids, such as di-hexadecyl-rac-glycerol or 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonic acid triethylammonium, polyamines or polyethylene glycol chains, or adamantaneacetic acid, a palmityl moiety, or an octadecylamine or hexylamino-carbonyl-hydroxycholesterol moiety.See, e.g., 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, and 5,688,941.

[0341] Longer polynucleotides that are less amenable to chemical synthesis and are typically produced by enzymatic synthesis can be modified in a variety of ways. Such modifications can include, for example, the introduction of certain nucleotide analogs, the incorporation of specific sequences or other moieties at the 5' or 3' end of the molecule, and other modifications. By way of illustration, an mRNA encoding a sRGN polypeptide has a length of approximately 3 kb to 3.3 kb and can be synthesized by in vitro transcription. Modifications to the mRNA can be applied, for example, to increase its translation or stability (e.g., by increasing its resistance to cellular degradation), or to reduce the propensity of the RNA to elicit an innate immune response, which may occur following the introduction of exogenous RNA (particularly longer RNAs such as those encoding sRGNs).

[0342] Many such modifications have been described in the art, such as a poly-A tail, a 5' cap analog (e.g., Anti Reverse Cap Analog (ARCA) or m7G(5')ppp(5')G (mCAP)), modified 5' or 3' untranslated regions (UTRs), use of modified bases (e.g., pseudo-UTP, 2-thio-UTP, 5-methylcytidine-5'-triphosphate (5-methyl-CTP) or N6-methyl-ATP), or treatment with a phosphatase to remove the 5' terminal phosphate. These and other modifications are known in the art, and new modifications of RNA are being developed.

[0343] Modified RNA can be obtained, for example, from commercial suppliers, 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 confer desired characteristics, such as increased nuclease stability, increased translation, or reduced interaction of innate immune receptors with in vitro transcribed RNA. 5'-methylcytidine-5'-triphosphate (5-methyl-CTP), N6-methyl-ATP, and pseudo-UTP and 2-sulfur-UTP have also been shown to reduce innate immune stimulation in culture and in vivo while enhancing translation, as exemplified by the publications by Konmann et al. and Warren et al., referenced below.

[0344] It has been demonstrated that chemically modified mRNA delivered in vivo can be used to achieve improved therapeutic effects; see, e.g., Kormann et al., Nature Biotechnology 29, 154-157 (2011). Such modifications can be used, for example, to increase the stability of RNA molecules and / or reduce their immunogenicity. Using chemical modifications, such as pseudo-U, N6-methyl-A, 2-thio-U and 5-methyl-C, it was found that replacing only one-quarter of the uridine and cytidine residues with 2-thio-U and 5-methyl-C, respectively, resulted in a significant reduction in the toll-like receptor (TLR)-mediated recognition of mRNA in mice. These modifications can therefore be used to effectively improve the stability and lifespan of mRNA in vivo by reducing the activation of the innate immune system; e.g., Konmann et al., supra.

[0345] Repeated administration of synthetic messenger RNAs incorporating modifications designed to bypass the innate antiviral response has also been shown to reprogram differentiated human cells to pluripotency. See, e.g., Warren et al., Cell Stem Cell, 7(5):618-30 (2010). Such modified mRNAs acting as primary reprogramming proteins may be an effective means of reprogramming a variety of human cell types. Such cells are called induced pluripotent stem cells (iPSCs), and enzymatically synthesized RNAs incorporating 5-methyl-CTP, pseudo-UTP, and anti-reverse cap analogs (ARCA) have been found to be effective in evading the antiviral response of cells; e.g., Warren et al., supra. Other modifications of polynucleotides described in the art include, e.g., the use of poly(A) tails, the addition of 5' cap analogs (e.g., m7G(5')ppp(5')G (mCAP)), modification of 5' or 3' untranslated regions (UTRs), or treatment with phosphatases to remove the 5' terminal phosphate - and new methods are being developed frequently.

[0346] Many compositions and techniques suitable for producing modified RNA for use herein have been developed in conjunction with modifications of RNA interference (RNAi), including small interfering RNA (siRNA). siRNAs pose special challenges in vivo because their effects on gene silencing through mRNA interference are generally transient, which may require repeated administration. In addition, siRNAs are double-stranded RNAs (dsRNAs), and mammalian cells have immune responses that have evolved to detect and neutralize dsRNAs, which are often byproducts of viral infections. Thus, there are mammalian enzymes such as PKR (dsRNA-responsive kinase), and possibly retinoic acid-inducible gene I (RIG-I), which can mediate cellular responses to dsRNA, as well as Toll-like receptors (e.g., TLR3, TLR7, and TLR8) that can trigger the induction of cytokines in response to such molecules; e.g., 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); reviews by Judge and Maclachlan, Hum Gene Ther 19(2): 111-24 (2008); and references cited therein.

[0347] Modifications described in the art have been developed and will be applied to enhance RNA stability, reduce innate immune responses and / or obtain other benefits that may be useful in introducing polynucleotides into human cells as described herein; 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., Front Genet 3:154 (2012) for a review.

[0348] As noted above, there are many commercial suppliers of modified RNA, many of which are designed specifically to improve the effectiveness of siRNA. Based on the various findings reported in the literature, a variety of methods are provided. For example, Dharmacon points out that the replacement of non-bridging oxygen (phosphorothioate, PS) with sulfur has been widely used to improve the nuclease resistance of siRNA, as reported by Kale, Nature Reviews Drug Discovery 11:125-140 (2012). It has been reported that the modification of the ribose 2'-position improves the nuclease resistance of the phosphate bond between nucleotides while increasing duplex stability (Tm), which has also been shown to provide protection from immune activation. The combination of medium PS backbone modification with small, well-tolerated 2'-substitutions (2'-O-methyl, 2'-fluoro, 2'-hydrogen) has been associated with highly stable siRNAs for in vivo applications, as reported by Soutschek et al. Nature 432: 173-178 (2004); and 2'-O-methyl modification has 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, modification of specific sequences with 2'-O-methyl, 2'-fluoro, 2'-hydrogen has been reported to reduce TLR7 / TLR8 interactions while generally maintaining silencing activity; 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 immunity mediated by TLR3, TLR7, and TLR8; eg, Kariko, K. et al., Immunity 23:165-175 (2005).

[0349] As is also known in the art and commercially available, a number of conjugates can be applied to polynucleotides, such as RNA for use herein, which can enhance their delivery and / or uptake by cells, including, for example, cholesterol, tocopherol and folic acid, lipids, peptides, polymer linkers, and aptamers; reviewed, for example, by Winkler, Ther Deliv. 4:791-809 (2013), and references cited therein.

[0350] Simulants

[0351] Nucleic acid can be a nucleic acid mimic. The term "mimetic" when applied to polynucleotides is intended to include polynucleotides in which only the furanose ring or both the furanose ring and the internucleotide bond are replaced by non-furanose groups, and only the replacement of the furanose ring is also referred to as sugar substitution in the art. The heterocyclic base moiety or the modified heterocyclic base moiety is maintained for hybridization with an appropriate target nucleic acid. A kind of such nucleic acid, a polynucleotide mimic that has been shown to have excellent hybridization properties, is referred to as a peptide nucleic acid (PNA). In PNA, the sugar-backbone of the polynucleotide is replaced by an amide-containing backbone, particularly an aminoethylglycine backbone. Nucleotides are retained and directly or indirectly bonded to the aza nitrogen atoms of the amide moiety of the backbone.

[0352] One polynucleotide mimetic that has been reported to have excellent hybridization properties is peptide nucleic acid (PNA). The backbone in a PNA compound is two or more linked aminoethylglycine units, which employ an amide-containing backbone of a PNA. The heterocyclic base moiety is directly or indirectly bound to an aza nitrogen atom of the amide portion of the backbone. 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.

[0353] Another class of polynucleotide mimics studied is based on morpholino units (morpholino nucleic acids) connected with heterocyclic bases attached to the morpholino ring. Many linking groups connecting morpholino monomer units in morpholino nucleic acids have been reported. A class of linking groups has been selected to produce nonionic oligomeric compounds. Nonionic oligomeric compounds based on morpholinos are unlikely to have undesirable interactions with cell proteins. Polynucleotides based on morpholinos are nonionic mimics of oligonucleotides, which are unlikely to form undesirable interactions with cell proteins (Braasch, DA, and Corey, DR, Biochemistry, 2002, 41 (14), 4503-4510). Polynucleotides based on morpholinos are disclosed in U.S. Patent No. 5,034,506. Various compounds in the morpholino class of polynucleotides have been prepared, which have a variety of different linking groups connecting monomer subunits.

[0354] 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. GeNA DMT-protected phosphoramidite monomers have been prepared according to classical phosphoramidite chemistry principles and used in 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, 8595-8602). Generally, incorporating GeNA monomers into DNA chains increases the stability of DNA / RNA hybrids. GeNA oligoadenylates form complexes with RNA and DNA complements that have similar stability to natural complexes. NMR and circular dichroism show that the study of incorporating GeNA structures into natural nucleic acid structures starts with easy conformational adaptation.

[0355] Further modifications include locked nucleic acids (LNA), in which the 2'-hydroxyl group is linked to the 4' carbon atom of the sugar ring to form a 2'-C, 4'-C-oxymethylene bond, thereby forming a bicyclic sugar moiety. The bond can be a methylene group (-CHz-), 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 show very high duplex thermal stability (T) with complementary DNA and RNA. m = +3 to +10°C), stability to 3'-exonucleolytic degradation and good solubility properties. Effective and non-toxic antisense oligonucleotides containing LNA have been described (Wahlestedt et al., Proc. Natl. Acad. Sci. USA, 2000, 97, 5633-5638).

[0356] 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). LNA and its preparation are also described in WO 98 / 39352 and WO 99 / 14226.

[0357] Modified sugar moiety

[0358] The nucleic acid may also contain one or more substituted sugar moieties. Suitable polynucleotides contain a sugar substituent group selected from the group consisting of OH; F; O-, S- or N-alkyl; O-, S- or N-alkenyl; O-, S- or N-alkynyl; or O-alkyl-O-alkyl, wherein alkyl, alkenyl and alkynyl groups may be substituted or unsubstituted C1-C 10 Alkyl or C2-C 10 Particularly suitable are O((CH2)nO)mCH3, O(CH2)nOCH3, O(CH2)nNH2, O(CH2)CH3, O(CH2)nONH2 and O(CH2)nON((CH2)nCH3)2, wherein n and m are from 1 to about 10. Other suitable polynucleotides contain a sugar substituent selected from the group consisting of: 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, ONO z , NO2, N3, NH2, heterocycloalkyl, heterocycloalkylaryl, aminoalkylamino, polyalkylamino, substituted silyl, RNA cleavage group, reporter group, intercalator, group for improving the pharmacokinetic properties of oligonucleotides or group for improving the pharmacokinetic properties of oligonucleotides and other substituents with similar properties. Suitable modifications include 2'-methoxyethoxy 2'-O-CH2 CHzOCH3, also referred to as -2'-O-(2-methoxyethyl) or 2'-MOE) (Martin et al., Hely.Chim.Acta, 1995, 78, 486-504), i.e., alkoxyalkoxy groups. Further suitable modifications include 2'-dimethylaminooxyethoxy, i.e., O(CHz)zON(CH3)z group, also known as 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), i.e., 2'-O-CHz-O-CHz-N(CH3)z.

[0359] Other suitable sugar substituent groups include methoxy (-O-CH3), aminopropoxy (-O-CH z CH z CH z NH z ), allyl (-CH z -CH=CH z ), -O-allyl (-O-CH z -CH=CH z) and fluorine (F). The 2'-sugar substituent group can be 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 on the oligomeric compound, particularly the 3' position of the sugar on the 3' terminal nucleoside or in a 2'-5' linked oligonucleotide and the 5' position of the 5' terminal nucleotide. The oligomeric compound can also have a sugar mimetic, such as a cyclobutyl moiety in place of the furanosyl sugar.

[0360] Base modification and substitution

[0361] Nucleic acids can also include modifications or substitutions of nucleoside bases (often referred to in the art as "bases"). "Unmodified" or "natural" nucleoside bases used herein include the purine bases adenine (A) and guanine (G) and the pyrimidine bases thymine (T), cytosine (C) and uracil (U). Modified nucleoside bases include other synthetic and natural nucleoside bases, such as 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl (-C=C-CH3) uracil and other alkynyl derivatives of cytosine and pyrimidine bases, 6-azouracil, 2-hydroxymethylcytosine ... Pyrimidine, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 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-amino-adenine, 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 cytidine (1H-pyrimido(5,4-b)(1,4)benzothiazin-2(3H)-one), G-seals such as substituted phenoxazine cytidines (e.g. 9-(2-aminoethoxy)-H-pyrimido(5,4-(b)(1,4)benzoxazin-2(3H)-one), carbazole cytidine (2H-pyrimido(4,5-b)indol-2-one), pyridoindole cytidine (H-pyrido(3',2':4,5)pyrrolo(2,3-d)pyrimidin-2-one).

[0362] Heterocyclic base moiety can also include those wherein purine or pyrimidine base are replaced by other heterocycles, such as 7-deaza-adenine, 7-deazaguanosine, 2-aminopyridine and 2-pyridone. Further, nucleoside base is included in those disclosed in U.S. Patent number 3,687,808, at The Concise Encyclopedia Of Polymer Science And Engineering, 858-859 pages, Kroschwitz, J.I., compile John Wiley & Sons, disclosed in 1990, by Englisch et al., Angewandte Chemie, International Edition, 1991, 30, 613 disclosed ones, and by Sanghvi, YS, Chapter 15, Antisense Research and Applications, 289- 302 pages, Crooke, ST and Lebleu, B. compile, CRC Press, disclosed in 1993. Some of these nucleoside bases are useful for increasing 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-propynyluracil and 5-propynylcytosine. 5-methylcytosine substitution has been shown to improve nucleic acid duplex stability by 0.6-1.2 oc (Sanghvi et al., ed., Antisense Research and Applications, CRC Press, Boca Raton, 1993, pp. 276-278) and is a suitable base substitution, for example, when combined with 2'-O-methoxyethyl sugar modification.

[0363] "Complementary" refers to the ability to pair between two sequences comprising naturally or non-naturally occurring (e.g., modified as described above) bases (nucleosides) or their analogs by base stacking and specific hydrogen bonding. For example, if a base at one position of a nucleic acid is capable of hydrogen bonding with a base at a corresponding position of a target, the bases are considered to be complementary to each other at that position. Nucleic acids may include universal bases, or inert abasic spacers that do not provide positive or negative contributions to hydrogen bonding. Base pairing may include typical Watson-Crick base pairing and non-Watson-Crick base pairing (e.g., wobble base pairing and Hoogsteen base pairing).

[0364] It is understood that for complementary base pairing, an adenosine-type base (A) is complementary to a thymidine-type base (T) or a uracil-type base (U), a cytosine-type base (C) is complementary to a guanosine-type base (G), and a universal base such as 3-nitropyrrole or 5-nitroindole can hybridize with any A, C, U, or T and be considered complementary thereto. Nichols et al., Nature, 1994;369:492-493 and Loakes et al., Nucleic Acids Res., 1994;22:4039-4043. Inosine (I) has also been recognized in the art as a universal base and is considered complementary to any A, C, U, or T. See Watkins and Santalucia, Nucl. Acids Research, 2005;33(19):6258-6267.

[0365] Conjugate

[0366] Another possible modification of nucleic acid includes chemically connecting one or more parts or conjugates that enhance the activity, cell distribution or cellular uptake of oligonucleotides to polynucleotides. These parts or conjugates can include conjugate groups covalently bound to functional groups such as primary or secondary hydroxyl groups. Conjugate groups include, but are not limited to, intercalators, reporters, polyamines, polyamides, polyethylene glycols, polyethers, groups that enhance the pharmacokinetic properties of oligomers and groups that enhance the pharmacokinetic properties of oligomers. Suitable conjugate 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 pharmacokinetic properties include groups that improve uptake, enhance resistance to degradation and / or enhance sequence-specific hybridization with target nucleic acids. Groups that enhance pharmacokinetic properties include groups that improve uptake, distribution, metabolism or excretion of nucleic acids.

[0367] Conjugate 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, for example dodecanediol 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, for example di-hexadecyl-rac-glycerol or 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), a polyamine or polyethylene glycol chain (Manoharan et al., Nucleosides & Nucleotides, 1995, 14, 969-97), or adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36, 36513654), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264, 229-237), or an octadecylamine or hexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J. Pharmacal. Exp. Ther., 1996, 277, 923-937).

[0368] The conjugate may include a "protein transduction domain" or PTD (also known as 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 ranging from a small polar molecule to a large macromolecule and / or nanoparticle facilitates the passage of the molecule through a membrane, such as from the extracellular space to the intracellular space, or from the cytosol to an organelle. In certain embodiments, the PTD is covalently attached to the amino terminus of an exogenous polypeptide (e.g., a sRGN polypeptide). In certain embodiments, the PTD is covalently attached to the C-terminus or N-terminus of an exogenous polypeptide (e.g., a sRGN polypeptide). In certain embodiments, the PTD is covalently attached to a nucleic acid (e.g., a guide RNA, a nucleic acid encoding a guide RNA, a nucleic acid encoding a sRGN polypeptide, etc.). Exemplary PTDs include, but are not limited to, a minimal undecapeptide protein transduction domain (e.g., corresponding to residues 47-57 of HIV-1 TAT containing the YGRKKRRQRRR (SEQ ID NO: 126) motif (for identification but not for disclosure in this application); a polyarginine sequence containing a sufficient number of arginines to direct entry into 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 antennapedia 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. USA97:13003-13008); 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 (e.g., Arg9 or "R9") connected to a matching polyanion (e.g., Glu9 or "E9") via a cleavable linker, which reduces the net charge to almost zero and thereby inhibits adhesion and uptake into cells. Upon cleavage of the linker, the polyanion is released, thereby locally exposing the polyarginine and its inherent adhesiveness, thereby "activating" the ACPP to pass through 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 6(11):1195-205.

[0369] Nucleic acids encoding guide RNA and / or sRGN polypeptides

[0370] The present disclosure provides nucleic acids comprising nucleotide sequences encoding guide RNAs and / or sRGN polypeptides. In certain embodiments, the nucleic acid encoding the guide RNA is an expression vector, such as a recombinant expression vector.

[0371] In certain embodiments, the methods comprise contacting the target DNA or introducing one or more nucleic acids comprising a nucleotide sequence encoding a guide RNA and / or a sRGN polypeptide into a cell (or cell population). In certain embodiments, the cell comprising the target DNA is in vitro. In certain embodiments, the cell comprising the target DNA is in vivo. Suitable nucleic acids comprising a nucleotide sequence encoding a guide RNA and / or a sRGN polypeptide include expression vectors, wherein an expression vector comprising a nucleotide sequence encoding a guide RNA and / or a sRGN polypeptide is a "recombinant expression vector".

[0372] In certain embodiments, the recombinant expression vector is a viral construct, such as a recombinant adeno-associated virus construct (eg, U.S. Pat. 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., vaccinia virus-based viral vectors; poliovirus; adenovirus (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 5:1088 1097, 1999; WO 94 / 12649, WO 93 / 03769; WO 93 / 19191; WO 94 / 28938; WO 95 / 11984 and WO 95 / 00655); adeno-associated virus (e.g., Ali et al., Hum Gene Ther 9:8186, 1998, Flannery et al., PNAS 94:6916 6921, 1997; Bennett et al., Invest Opthalmol Vis Sci 38:2857 2863, 1997; Jomary et al., Gene Ther 4:683 690, 1997, Rolling et al., Hum Gene Ther 10:641 648, 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; Mendelson et al., Viral. (1988) 166:154-165; and Flotte et al., PNAS (1993) 90:10613-10617); SV40; herpes simplex virus; human immunodeficiency virus (e.g., Miyoshi et al., PNAS 94:10319 23, 1997; Takahashi et al., J Viral 73:7812 7816, 1999); retroviral vectors (e.g., murine leukemia virus, spleen necrosis virus, and vectors derived from retroviruses, such as Rous sarcoma virus, Harvey sarcoma virus, avian leukemia virus, lentivirus, human immunodeficiency virus, myeloproliferative sarcoma virus, and mammary tumor virus); etc.

[0373] Many suitable expression vectors are known to those skilled in the art, and many are commercially available. The following vectors are provided as examples; for eukaryotic host cells: pXT1, pSGS (Stratagene), pSVK3, pBPV, pMSG, and pSVLSV40 (Phanmacia). However, any other vector may be used as long as it is compatible with the host cell. Depending on the host / vector system used, any of a number of suitable transcription and translation control elements may be used in the expression vector, including constitutive and inducible promoters, transcription enhancer elements, transcription terminators, etc. (e.g., Bitter et al. (1987) Methods in Enzymology, 153:516-544).

[0374] In certain embodiments, the nucleotide sequence encoding the guide RNA and / or sRGN polypeptide is operably linked to a control element, such as a transcriptional control element, such as a promoter. The transcriptional control element can function in a eukaryotic cell, such as a mammalian cell; or a prokaryotic cell, such as a bacterial or archaeal cell. In certain embodiments, the nucleotide sequence encoding the guide RNA and / or sRGN polypeptide is operably linked to a plurality of control elements that allow expression of the nucleotide sequence encoding the guide RNA and / or sRGN polypeptide in both prokaryotic and eukaryotic cells.

[0375] Non-limiting examples of suitable eukaryotic promoters (promoters that function in eukaryotic cells) include those from cytomegalovirus (CMV) immediate early, herpes simplex virus (HSV) thymidine kinase, early and late SV40, long terminal repeats (LTRs) from retroviruses, and mouse metallothionein-1. The selection of suitable vectors and promoters is within the ordinary skill level in the art. The expression vector may also contain a ribosome binding site and a transcription terminator for translation initiation. The expression vector may also include a suitable sequence for amplifying expression. The expression vector may also include a nucleotide sequence encoding a protein tag (e.g., a 6xHis tag, a hemagglutinin tag, a green fluorescent protein, etc.) fused to the sRGN polypeptide, resulting in the production of a chimeric polypeptide.

[0376] In certain embodiments, the nucleotide sequence encoding the guide RNA and / or sRGN polypeptide is operably linked to an inducible promoter. In certain embodiments, the nucleotide sequence encoding the guide RNA and / or sRGN polypeptide is operably linked to a constitutive promoter.

[0377] Methods for introducing nucleic acids into host cells are known in the art, and such methods can be used to introduce nucleic acids (e.g., expression constructs) into cells. Suitable methods include, for example, viral or bacteriophage infection, transfection, conjugation, protoplast fusion, lipofection, electroporation, calcium phosphate precipitation, polyethyleneimine (PEI)-mediated transfection, DEAE-dextran-mediated transfection, liposome-mediated transfection, gene gun technology, calcium phosphate precipitation, direct microinjection, nanoparticle-mediated nucleic acid delivery (e.g., Panyam et al., Adv Drug Deliv Rev. 2012 Sep 13. pii: S0169-409X(12)00283-9.doi: 10.1016 / j.addr.2012.09.023), etc.

[0378] Chimeric polypeptide

[0379] The present disclosure provides chimeric localization modifying polypeptides comprising a sequence derived from an sRGN polypeptide. The chimeric localization modifying polypeptide interacts (e.g., binds) with a guide RNA (described above). The guide RNA guides the chimeric localization modifying polypeptide to a target sequence within a target DNA (e.g., a chromosomal sequence or an extrachromosomal sequence, such as an additional sequence, a minicircle sequence, a mitochondrial sequence, a chloroplast sequence, a cell-free polynucleotide, etc.). The chimeric localization modifying polypeptide modifies the target DNA (e.g., cleavage or methylation of the target DNA) and / or a polypeptide associated with the target DNA (e.g., methylation or acetylation of a histone tail). The chimeric localization modifying polypeptide is also referred to herein as a "chimeric localization polypeptide" or a "chimeric RNA binding site-guided modified polypeptide."

[0380] Chimeric localization modifying polypeptides comprise two portions, an RNA-binding portion and an active portion. Chimeric localization modifying polypeptides comprise amino acid sequences derived from at least two different polypeptides. Chimeric localization modifying polypeptides may comprise modified and / or naturally occurring polypeptide sequences (e.g., a first amino acid sequence from a modified or unmodified sRGN protein; and a second amino acid sequence other than a sRGN protein).

[0381] RNA-binding moiety

[0382] In certain embodiments, the RNA-binding portion of the chimeric localization-modifying polypeptide comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the RNA-binding portion of the polypeptide set forth in any one of SEQ ID NOs: 1-12.

[0383] Active part

[0384] In addition to the RNA-binding portion, the chimeric localization modifying polypeptide comprises an "active portion". In certain embodiments, the active portion of the chimeric localization modifying polypeptide comprises an active portion of a localization modifying polypeptide (e.g., a Cas9 endonuclease or sRGN). In other embodiments, the active portion of the subject chimeric localization modifying polypeptide comprises a modified amino acid sequence (e.g., substitution, deletion, insertion) of a naturally occurring active portion of a localization modifying polypeptide. The naturally occurring active portions of interest are derived from localization modifying polypeptides known in the art. The active portion of the chimeric localization modifying polypeptide is variable and may comprise any heterologous polypeptide sequence that can be used in the methods disclosed herein. In certain embodiments, the active portion of the chimeric localization modifying polypeptide comprises a portion of a sRGN polypeptide that is at least 90% identical to the active portion of any one of SEQ ID NOs: 1-12. In certain embodiments, a chimeric localization-modifying polypeptide comprises: (i) an RNA-binding portion that interacts with a guide RNA, wherein the guide RNA comprises a nucleotide sequence that is complementary to a sequence in a target DNA; (ii) an active portion that exhibits localization-enzyme activity (e.g., RNA cleavage activity), wherein the site of enzymatic activity is determined by a palindromic hairpin structure formed by the repeat sequence of the pre-crRNA and cleaves the pre-crRNA 4 nucleotides upstream of the hairpin, thereby generating an intermediate form of crRNA consisting of a repeat sequence spacer region (5'-3'); and (iii) an active portion that exhibits localization-enzyme activity (e.g., DNA cleavage activity), wherein the site of enzymatic activity is determined by the guide RNA.

[0385] Exemplary Chimeric Localization Modified Polypeptides

[0386] In certain embodiments, the active portion of the chimeric localization-modified polypeptide comprises a modified form of a sRGN protein, including a modified form of any sRGN ortholog. In some cases, the modified form of the sRGN protein comprises an amino acid change (e.g., a deletion, insertion, or substitution) that reduces or increases the naturally occurring nuclease activity of the sRGN protein. For example, in some cases, the modified form of the sRGN 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 the corresponding wild-type sRGN polypeptide. In some cases, the modified form of the sRGN polypeptide has no significant nuclease activity. In other cases, it may have 50%, 2-fold, 4-fold, or up to 10-fold more nuclease activity.

[0387] In some cases, the chimeric localization modifying polypeptide comprises 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 any one of SEQ ID NOs: 1-12. In some embodiments, the active portion of the localization modifying polypeptide comprises a heterologous polypeptide having DNA-modifying activity and / or transcription factor activity and / or DNA-related polypeptide modifying activity. In some cases, the heterologous polypeptide replaces the portion of the sRGN polypeptide that provides the nuclease activity. In other embodiments, the localization modifying polypeptide comprises both a portion of the sRGN polypeptide that normally provides the nuclease activity (and the portion may be fully active, or may be modified to have less than 100% of the corresponding wild-type activity instead) and a heterologous polypeptide. In other words, in some cases, the chimeric localization modifying polypeptide is a fusion polypeptide that comprises both a portion of the sRGN polypeptide that normally provides the nuclease activity and a heterologous polypeptide. In other cases, the chimeric localization modifying polypeptide is a fusion polypeptide that comprises a modified variant (e.g., amino acid change, deletion, insertion) of the active portion of the sRGN polypeptide and a heterologous polypeptide. In still other cases, a chimeric localization modifying polypeptide is a fusion polypeptide comprising a heterologous polypeptide and an RNA-binding portion of a naturally occurring or modified localization modifying polypeptide.

[0388] For example, in a chimeric sRGN protein, a naturally occurring (or modified, e.g., mutated, deleted, inserted) sRGN polypeptide can be fused to a heterologous polypeptide sequence (i.e., a polypeptide sequence from a protein other than sRGN or a polypeptide sequence from another organism). The heterologous polypeptide sequence can exhibit an activity (e.g., an enzymatic activity) that will also be exhibited by the chimeric sRGN protein (e.g., methyltransferase activity, acetyltransferase activity, kinase activity, ubiquitination activity, etc.). A heterologous nucleic acid sequence can be linked to another nucleic acid sequence (e.g., by genetic engineering) to produce a chimeric nucleotide sequence encoding a chimeric polypeptide. In certain embodiments, a chimeric sRGN polypeptide is produced by fusing an sRGN polypeptide (e.g., a wild-type sRGN or an sRGN variant, e.g., an sRGN with reduced or inactivated nuclease activity) 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 the mitochondria; a chloroplast localization signal for targeting the chloroplast: an ER retention signal; etc.). In certain embodiments, the heterologous sequence can provide a tag for easy tracking or purification (e.g., 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 certain embodiments, the heterologous sequence can provide increased or decreased stability. In certain embodiments, the heterologous sequence can provide a binding domain (e.g., to provide the chimeric sRGN polypeptide with the ability to bind to another protein of interest, such as a DNA or histone modifying protein, a transcription factor or transcription repressor protein, a recruitment protein, etc.) or a nucleotide of interest (e.g., a target site for an aptamer or nucleotide binding protein).

[0389] Nucleic acid encoding a chimeric localization-modified polypeptide

[0390] The present disclosure provides nucleic acids comprising a nucleotide sequence encoding a chimeric localization modifying polypeptide. In certain embodiments, the nucleic acid comprising a nucleotide sequence encoding a chimeric localization modifying polypeptide is an expression vector, such as a recombinant expression vector.

[0391] In certain embodiments, the method comprises contacting the target DNA or introducing one or more nucleic acids comprising a chimeric localization modifying polypeptide into a cell (or cell population). Suitable nucleic acids comprising a nucleotide sequence encoding a chimeric localization modifying polypeptide include expression vectors, wherein an expression vector comprising a nucleotide sequence encoding a chimeric localization modifying polypeptide is a "recombinant expression vector".

[0392] In certain embodiments, the recombinant expression vector is a viral construct, such as a recombinant adeno-associated virus construct (eg, US Pat. No. 7,078,387), a recombinant adenovirus construct, a recombinant lentivirus construct, or the like. Suitable expression vectors include, but are not limited to, viral vectors (e.g., vaccinia virus-based viral vectors; poliovirus; adenovirus (e.g., Li et al., Invest Opthalmol Vis Sci 35:2543 2549, 1994; Borras et al., Gene Ther 6:515524, 1999; Li and Davidson, PNAS 92:7700 7704, 1995; Sakamoto et al., H Gene Ther 5:1088 1097, 1999; WO 94 / 12649, WO 93 / 03769: WO 93 / 19191; WO 94 / 28938; WO 95 / 11984 and WO 95 / 00655); adeno-associated virus (e.g., Ali et al., Hum Gene Ther 9:81 86, 1998, Flannery et al., PNAS 94:6916-6921, 1997; Bennett et al., Invest Opthalmol Vis Sci 38:2857-2863, 1997; Jomary et al., Gene Ther 4:683-690, 1997, Rolling et al., Hum Gene Ther 10:641-648, 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; Mendelson et al., Viral. (1988) 166:154-165; and Flotte et al., PNAS (1993) 90:10613-10617); SV40; herpes simplex virus; human immunodeficiency virus (e.g., Miyoshi et al., PNAS 94:10319 23, 1997; Takahashi et al., J Viral 73:7812 7816, 1999); retroviral vectors (e.g., murine leukemia virus, spleen necrosis virus, and vectors derived from retroviruses, such as Rous sarcoma virus, Harvey sarcoma virus, avian leukemia virus, lentivirus, human immunodeficiency virus, myeloproliferative sarcoma virus, and mammary tumor virus); etc.

[0393] Many suitable expression vectors are known to those skilled in the art, and many are commercially available. The following vectors are provided as examples; for eukaryotic host cells: pXT1, pSGS (Stratagene), pSVK3, pBPV, pMSG, and pSVLSV40 (Pharmacia). However, any other vector may be used as long as it is compatible with the host cell.

[0394] Depending on the host / vector system utilized, any of a number of suitable 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).

[0395] In certain embodiments, the nucleotide sequence encoding the chimeric localization modifying polypeptide is operably linked to a control element, such as a transcriptional control element, such as a promoter. The transcriptional control element can function in a eukaryotic cell, such as a mammalian cell; or a prokaryotic cell, such as a bacterial or archaeal cell. In certain embodiments, the nucleotide sequence encoding the chimeric localization modifying polypeptide is operably linked to a plurality of control elements that allow the nucleotide sequence encoding the chimeric localization modifying polypeptide to be expressed in both prokaryotic and eukaryotic cells.

[0396] The non-limiting examples of suitable eukaryotic promoters (promoters that work in eukaryotic cells) include those from cytomegalovirus (CMV) immediate early, herpes simplex virus (HSV) thymidine kinase, early and late SV40, long terminal repeats (LTRs) from retroviruses, and mouse metallothionein-1. The selection of suitable vectors and promoters is completely within the ordinary skill level of the art. The expression vector may also contain a ribosome binding site and a transcription terminator for translation initiation. The expression vector may also include a suitable sequence for amplification of expression. The expression vector may also include a nucleotide sequence encoding a protein tag (e.g., 6xHis tag, hemagglutinin (HA) tag, fluorescent protein (e.g., green fluorescent protein; yellow fluorescent protein, etc.) fused to a chimeric localization modified polypeptide.

[0397] In certain embodiments, the nucleotide sequence encoding the chimeric localization modifying polypeptide is operably linked to an inducible promoter (e.g., a heat shock promoter, a tetracycline-regulated promoter, a steroid-regulated promoter, a metal-regulated promoter, an estrogen receptor-regulated promoter, etc.). In certain embodiments, the nucleotide sequence encoding the chimeric localization modifying polypeptide is operably linked to a spatially restricted and / or temporally restricted promoter (e.g., a tissue-specific promoter, a cell type-specific promoter, etc.). In certain embodiments, the nucleotide sequence encoding the chimeric localization modifying polypeptide is operably linked to a constitutive promoter.

[0398] Methods for introducing nucleic acids into host cells are known in the art, and any known method can be used to introduce nucleic acids (e.g., expression constructs) into stem cells or ancestral cells. Suitable methods include, for example, viral or bacteriophage infection, transfection, conjugation, protoplast fusion, lipofection, electroporation, calcium phosphate precipitation, polyethyleneimine (PEI)-mediated transfection, DEAE-dextran-mediated transfection, liposome-mediated transfection, gene gun technology, calcium phosphate precipitation, direct microinjection, nanoparticle-mediated nucleic acid delivery (e.g., Panyam et al. Adv Drug Deliv Rev. 2012.09.13. pp:50169-409X(12)00283-9.doi: 10.1016 / j.addr.2012.09.023), etc.

[0399] method

[0400] The present disclosure provides methods for modifying target DNA and / or target DNA-associated polypeptides. Generally, the method comprises contacting the target DNA with a complex ("targeting complex") comprising a guide RNA and a sRGN polypeptide.

[0401] As discussed above, the gRNA or sgRNA and the sRGN polypeptide form a complex. The guide RNA provides target specificity to the complex by comprising a nucleotide sequence complementary to the sequence of the target DNA. The sRGN polypeptide of the complex provides site-specific activity. In certain embodiments, the complex modifies the target DNA, resulting in, for example, DNA cleavage, DNA methylation, DNA damage, DNA repair, etc. In other embodiments, the complex modifies a target polypeptide associated with the target DNA (e.g., histones, DNA-binding proteins, etc.), resulting in, for example, histone methylation, histone acetylation, histone ubiquitination, etc. The target DNA can be, for example, naked DNA in vitro (i.e., not bound by DNA-associated proteins), chromosomal DNA in in vitro cells, chromosomal DNA in in vivo cells, etc.

[0402] sRGN proteins from various species may require different PAM sequences in the target DNA. Therefore, for a particular sRGN protein selected, the PAM sequence requirements may differ from the PAM sequences described above.

[0403] Exemplary methods for providing features that utilize sRGN orthologs include the following.

[0404] Nuclease activity cuts the target DNA to produce double-strand breaks. These breaks are then repaired by the cell in one of two ways: non-homologous end joining and homology-guided repair. In non-homologous end joining (NHEJ), double-strand breaks are repaired by directly connecting the broken ends to each other. In the process, a few base pairs can be inserted or deleted at the cut site. In homology-guided repair, a donor polynucleotide having homology to the cut target DNA sequence is used as a template for repairing the cut target DNA sequence, thereby causing genetic information to be transferred from the donor polynucleotide to the target DNA. Therefore, new nucleic acid material can be inserted / copied into the site. In some cases, the target DNA is contacted with a donor polynucleotide. In some cases, the donor polynucleotide is introduced into the cell. Modification of the target DNA due to NHEJ and / or homology-guided repair leads to, for example, gene correction, gene replacement, gene tagging, transgenic insertion, nucleotide deletion, nucleotide insertion, gene disruption, gene mutation, sequence replacement, etc. Thus, cleavage of DNA by sRGN polypeptides can be used to delete nucleic acid material from a target DNA sequence (e.g., to disrupt a gene that renders a cell susceptible to infection (e.g., CCRS or CXCR4 genes, which render T cells susceptible to HIV infection, to remove disease-causing trinucleotide repeat sequences in neurons, to generate gene knockouts and mutations that serve as disease models under study, etc.) by cleaving the target DNA sequence and allowing the cell to repair the sequence in the absence of an exogenously provided donor polynucleotide. Thus, the methods can be used to knock out a gene (thereby resulting in a complete lack of transcription or altered transcription) or to knock genetic material into a selected locus in the target DNA.

[0405] Alternatively, if the guide RNA and sRGN polypeptide are co-administered to a cell having a donor polynucleotide sequence that includes at least a segment with homology to a target DNA sequence, the subject methods can be used to add, i.e., insert or replace, nucleic acid material to the target DNA sequence (e.g., to "knock in" a nucleic acid encoding a protein, siRNA, miRNA, etc.), add a tag (e.g., 6xHis, a fluorescent protein (e.g., green fluorescent protein; yellow fluorescent protein, etc.), hemagglutinin (HA), FLAG, etc.), add a regulatory sequence to a gene (e.g., a promoter, a polyadenylation signal, an internal ribosome entry sequence (IRES), a 2A peptide, a start codon, a stop codon, a splicing signal, a localization signal, etc.), modify a nucleic acid sequence (e.g., introduce a mutation), etc. Thus, complexes comprising guide RNAs and sRGN polypeptides can be used in any in vitro or in vivo application in which it is desired to modify DNA in a site-specific (i.e., "targeted") manner, such as gene knockout, gene knockin, gene editing, gene tagging, sequence replacement, and the like, as used, for example, in gene therapy, e.g., to treat disease or as an antiviral, antipathogenic, or anticancer treatment, production of genetically modified organisms in agriculture, large-scale production of proteins by cells for therapeutic, diagnostic, or research purposes, induction of iPS cells, biological research, targeting of pathogen genes for deletion or replacement, and the like.

[0406] In certain embodiments, the sRGN polypeptide comprises a modified form of a sRGN protein. In some cases, the modified form of a sRGN protein comprises an amino acid change (e.g., a deletion, insertion, or substitution) that reduces the naturally occurring nuclease activity of the sRGN protein. For example, in some cases, the modified form of a sRGN 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 the corresponding wild-type sRGN polypeptide. In some cases, the modified form of a sRGN polypeptide has no significant nuclease activity. When the sRGN polypeptide is a modified form of a sRGN protein that has no significant nuclease activity, it can be referred to as a "dsRGN".

[0407] In certain embodiments, the sRGN polypeptide comprises a heterologous sequence (e.g., a fusion). In certain embodiments, the heterologous sequence can provide subcellular localization of the sRGN polypeptide (e.g., a nuclear localization signal (NLS) for targeting the nucleus; a mitochondrial localization signal for targeting the mitochondria; a chloroplast localization signal for targeting the chloroplast; an ER retention signal; etc.). In certain embodiments, the heterologous sequence can provide a tag for easy tracking or purification (e.g., 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 certain embodiments, the heterologous sequence can provide increased or decreased stability.

[0408] In certain embodiments, modified polynucleotides are used in the CRISPR-sRGN system described herein, wherein guide RNA and / or DNA or RNA comprising a polynucleotide sequence encoding an sRGN polypeptide or a variant thereof can be modified, as described and shown below. Such modified polynucleotides can be used in the CRISPR-sRGN system, for example, to edit any one or more genomic loci. In certain 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 to be expressed. A skilled artisan will appreciate that any nucleotide sequence and / or recombinant nucleic acid of the present disclosure can be codon optimized for expression in any target species. Codon optimization is well known in the art and involves the use of a species-specific codon selection table to modify the codon selection bias of a nucleotide sequence. A codon selection table is generated based on sequence analysis of the highest expressed genes of a target species. In a non-limiting example, when a nucleotide sequence is to be translated in a host cell, a codon selection table is generated based on sequence analysis of a highly expressed nuclear gene of a target species. The modification of the nucleotide sequence is determined by comparing the species-specific codon selection table with the codons present in the natural polynucleotide sequence. For example, if the intended target cell is a human cell, a human codon-optimized polynucleotide sequence encoding a sRGN (or a sRGN variant, e.g., an enzymatically inactive variant) would be appropriate. As another non-limiting example, if the intended host cell is a mouse cell, a mouse codon-optimized polynucleotide sequence encoding a sRGN (or a sRGN variant, e.g., an enzymatically inactive variant) would be appropriate.

[0409] 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 E. coli (Feng et al., Biochemistry, 39(50):15399-15409 (2000)). In certain embodiments, codon optimization is performed by using GeneGPS® expression optimization technology (ATUM) and using the expression optimization algorithm recommended by the manufacturer. In certain embodiments, the polynucleotides of the present disclosure are codon optimized to increase expression in human cells. In certain embodiments, the polynucleotides of the present disclosure are codon optimized to increase expression in E. coli cells. In certain embodiments, the polynucleotides of the present disclosure are codon optimized to increase expression in insect cells. In certain embodiments, the expression optimization algorithm used in the codon optimization program is defined to avoid putative poly-A signals (e.g., AATAAA and ATTAAA) and long (greater than 4) A segments, which may cause polymerase slippage. The polyadenylation signal may also be selected to optimize expression in the intended host.The polyadenylation signal may also be selected to optimize expression in the intended host.

[0410] As is well understood in the art, codon optimization of a nucleotide sequence produces a nucleotide sequence that is less than 100% identical (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%) to a native nucleotide sequence, but that still encodes a polypeptide having the same function as the polypeptide encoded by the original native nucleotide sequence. Thus, in representative embodiments of the present disclosure, nucleotide sequences and / or recombinant nucleic acids of the present disclosure may be codon optimized for expression in a particular target species.

[0411] In certain embodiments, the codon-optimized polynucleotide sequence encoding a sRGN has at least 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.2%, 99.5%, 99.8%, 99.9%, or 100% sequence identity to any one of SEQ ID NOs: 15, 18, 20, 22, and 27. In certain embodiments, the polynucleotides of the disclosure are codon-optimized to increase expression of the encoded sRGN polypeptide in target cells. In certain embodiments, the polynucleotides of the disclosure are codon-optimized to increase expression in human cells. In certain embodiments, the polynucleotides of the disclosure are codon-optimized to increase expression in Escherichia coli Expression in cells. In certain embodiments, the polynucleotides of the disclosure are codon optimized for increased expression in insect cells.

[0412] In certain embodiments, the sRGN polypeptide may be codon optimized. This type of optimization is known in the art and requires mutation of exogenous DNA to mimic the codon preference of the intended host organism or cell while encoding the same protein. Thus, the codons are changed, but the encoded protein remains unchanged. For example, if the intended target cell is a human cell, a human codon optimized sRGN (or variant, e.g., an enzymatically inactive variant) would be suitable. Any suitable sRGN polypeptide (e.g., any sRGN, such as the sequence shown in any one of SEQ ID NOs: 1-12) may be codon optimized. As another non-limiting example, if the intended host cell is a mouse cell, a mouse codon optimized sRGN (or variant, e.g., an enzymatically inactive variant) would be suitable.

[0413] In one aspect, provided herein is a nucleic acid comprising a codon-optimized polynucleotide sequence encoding a sRGN polypeptide (e.g., a polypeptide comprising an amino acid sequence of any one of SEQ ID NOs: 1-12) or a variant thereof having at least about 90% (such as at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to any one of SEQ ID NOs: 1-12. Such codon-optimized nucleic acids encoding sRGN or variants thereof are also referred to herein as "codon-optimized sRGN polypeptides" or "codon-optimized sRGNs". In certain embodiments, the codon-optimized sRGN encodes an sRGN polypeptide of any one of SEQ ID NOs: 1-12. In certain embodiments, the codon-optimized sRGN is codon-optimized for expression in prokaryotic cells, such as bacterial cells. In certain embodiments, the codon-optimized sRGN is codon-optimized for expression in eukaryotic cells, such as insect or mammalian cells. In certain embodiments, the codon-optimized sRGN is codon-optimized for expression in human cells. In certain embodiments, codon optimization is performed according to GeneGPS® optimization (ATUM). In certain embodiments, codon optimization is performed according to the GeneOptimizer method (ThermoFisher Scientific). In certain embodiments, the codon optimized sRGN has at least about 90% (such as at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity with any one of SEQ ID NOs: 15, 18, 20, 22, and 27. In certain embodiments, the codon optimized sRGN comprises (or consists of) the sequence of any one of SEQ ID NOs: 15, 18, 20, 22, and 27.

[0414] In some embodiments according to any of the nucleic acids described herein comprising a codon-optimized polynucleotide sequence encoding a sRGN polypeptide or variant thereof, the nucleic acid further comprises a nucleotide sequence encoding a gRNA. In certain embodiments, the nucleic acid is an expression vector, such as a recombinant expression vector. In certain embodiments, the nucleotide sequence encoding a sRGN polypeptide or variant thereof comprises a codon-optimized polynucleotide sequence of any one of SEQ ID NOs: 15, 18, 20, 22, and 27, or a variant thereof having at least 90% sequence identity to a codon-optimized polynucleotide sequence of any one of SEQ ID NOs: 15, 18, 20, 22, and 27.

[0415] In certain embodiments, provided herein is a nucleic acid comprising a polynucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to any one of SEQ ID NOs: 15, 18, 20, 22, and 27, wherein the polynucleotide sequence encodes a sRGN polypeptide or a variant thereof as described herein. In certain embodiments, the polynucleotide sequence comprises (or consists of) a codon-optimized polynucleotide sequence of any one of SEQ ID NOs: 15, 18, 20, 22, and 27.

[0416] In certain embodiments, provided herein is a nucleic acid comprising a polynucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to a subsequence of any one of SEQ ID NOs: 15, 18, 20, 22 and 27, wherein the polynucleotide sequence encodes one or more functional sRGN domains. In certain embodiments, the polynucleotide sequence comprises (or consists of) a subsequence of any one of SEQ ID NOs: 15, 18, 20, 22 and 27. In certain embodiments, the one or more sRGN domains include a nuclease domain (e.g., a HNH or RuvC domain). In certain embodiments, the one or more sRGN domains include an RNA-binding domain (e.g., a Rec I domain). In certain embodiments, the one or more sRGN domains include a PAM-interaction domain.

[0417] In some embodiments according to any of the nucleic acids described herein comprising a codon-optimized polynucleotide sequence, expression of a sRGN polypeptide or variant thereof from the nucleic acid in a host cell is increased compared to expression of a sRGN polypeptide or variant thereof from a corresponding nucleic acid comprising a reference polynucleotide sequence from which the codon-optimized polynucleotide sequence is derived in a host cell. In certain embodiments, the codon-optimized polynucleotide sequence comprises a codon-optimized polynucleotide sequence of any one of SEQ ID NOs: 15, 18, 20, 22, and 27, or a variant thereof having at least 90% sequence identity to a codon-optimized polynucleotide sequence of any one of SEQ ID NOs: 15, 18, 20, 22, and 27. In certain embodiments, the reference polynucleotide sequence comprises a polynucleotide sequence encoding a sRGN from any one of SEQ ID NOs: 13, 14, 16, 17, 19, 21, 23-26, 28, and 29, or a variant thereof having at least 90% sequence identity to a polynucleotide sequence encoding a sRGN from any one of SEQ ID NOs: 13, 14, 16, 17, 19, 21, 23-26, 28, and 29. In certain embodiments, expression of a sRGN polypeptide or variant thereof from the codon-optimized polynucleotide sequence in a host cell is increased by at least about 10% (e.g., at least about any of 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, or more) compared to expression of the sRGN polypeptide or variant thereof from the reference polynucleotide sequence in the host cell.

[0418] In certain embodiments, guide RNAs and sRGN polypeptides are used as induction systems for shutting down gene expression in bacterial cells. In some cases, nucleic acids encoding suitable guide RNAs and / or suitable sRGN polypeptides are incorporated into the chromosomes of target cells and are under the control of an inducible promoter. When the guide RNA and / or sRGN polypeptide are induced, the target DNA is cut (or otherwise modified) at a location of interest (e.g., a target gene on an independent plasmid) when both the guide RNA and the sRGN polypeptide are present and form a complex. Thus, in some cases, bacterial expression strains are engineered to contain nucleic acid sequences encoding appropriate sRGN polypeptides in the bacterial genome and / or appropriate guide RNAs on plasmids (e.g., under the control of an inducible promoter), thereby allowing experiments in which the expression of any targeted gene (expressed by an independent plasmid introduced into the strain) can be controlled by inducing the expression of guide RNAs and sRGN polypeptides. In some cases, the sRGN polypeptide has enzymatic activity that modifies the target DNA in a manner other than introducing double-strand breaks. Enzyme activities of interest that can be used to modify target DNA (e.g., by fusing a heterologous polypeptide with enzymatic activity to a sRGN polypeptide to produce a chimeric sRGN polypeptide) include, but are 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 considered in the art to be important modes of epigenetic gene regulation, while DNA damage and repair activities are essential for cell survival and for proper genome maintenance in response to environmental stress. Therefore, the methods herein are applied to epigenetic modification of target DNA and can be used to control epigenetic modification of target DNA at any position in the target DNA by genetically engineering the desired complementary nucleic acid sequence into the DNA-targeting segment of the guide RNA. The methods herein are also applied to the intentional and controlled damage of DNA at any desired position within the target DNA. The methods herein also have application to sequence-specific and controlled repair of DNA at any desired position within the target DNA.Methods of targeting DNA-modifying enzyme activity to specific locations in target DNA have application in both research and clinical applications.

[0419] In some cases, the sRGN polypeptide has the activity of regulating the transcription of the target DNA (e.g., in the case of a chimeric sRGN polypeptide, etc.). In some cases, a chimeric sRGN polypeptide comprising a heterologous polypeptide (e.g., a transcription activator or transcription repressor 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 DNA-targeting segment of the guide RNA. Examples of source polypeptides for providing chimeric sRGN polypeptides having transcriptional regulatory activity include, but are not limited to, light-induced transcriptional regulators, small molecule / drug-responsive transcriptional regulators, transcription factors, transcriptional repressors, etc. In some cases, the method is used to control the expression of targeted coding-RNA (protein-coding genes) and / or targeted non-coding RNA (e.g., tRNA, rRNA, snoRNA, siRNA, miRNA, long ncRNA, etc.). In some cases, the sRGN polypeptide has an enzymatic activity that modifies polypeptides associated with DNA (e.g., histones). In certain 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 (i.e., ubiquitination activity), a deubiquitinating activity, an adenylation activity, a deadenylation activity, a sumoylation activity, a desumoylation activity, a ribosylation activity, a deribosylation activity, a myristoylation activity, a demyristoylation activity, a glycosylation activity (e.g., from a GlcNAc transferase) or a deglycosylation activity. The enzymatic activities listed herein catalyze the covalent modification of proteins. Such modifications are known in the art to alter the stability or activity of a target protein (e.g., because phosphorylation of kinase activity may depend on target protein stimulation or silencing protein activity). Particularly interesting as a protein target are histones. Histones are known in the art to bind DNA and form a complex called a nucleosome. Histones can be modified (e.g., by methylation, acetylation, ubiquitination, phosphorylation) to elicit structural changes in the surrounding DNA, thereby controlling the accessibility of potentially large portions of the DNA to interacting factors such as 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 that are repressed from transcription, while trimethylation of lysine 4, H3K4, of histone 3 is associated with regions of DNA that are actively transcribed). Thus, localized modifying polypeptides having histone modifying activity are applied to site-specific control of DNA structure and can be used to alter histone modification patterns in selected regions of target DNA. This approach is applied in both research and clinical applications.

[0420] In certain embodiments, multiple guide RNAs are used simultaneously to modify different positions on the same target DNA or different target DNAs simultaneously. In certain embodiments, two or more guide RNAs target the same gene or transcript or locus. In certain embodiments, two or more guide RNAs target different unrelated loci. In certain embodiments, two or more guide RNAs target different but related loci.

[0421] In some cases, the sRGN polypeptide is provided directly as a protein. As a non-limiting example, fungi (e.g., yeast) can be transformed with exogenous proteins and / or nucleic acids using spheroplast transformation (see Kawai et al., BioengBugs. 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 are incorporated herein by reference in their entirety). Thus, the sRGN polypeptide can be incorporated into spheroplasts (with or without nucleic acids encoding guide RNAs and with or without donor polynucleotides), and the spheroplasts can be used to introduce the contents into yeast cells. The sRGN polypeptide can be introduced into cells (provided to cells) by any convenient method; such methods are known to those of ordinary skill in the art. As another non-limiting example, a sRGN polypeptide can be injected directly into a cell (e.g., with or without a nucleic acid encoding a guide RNA and with or without a donor polynucleotide), such as a cell of a zebrafish embryo, a pronucleus of a fertilized mouse oocyte, etc.

[0422] Target cells of interest

[0423] In some of the above applications, the methods 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). Because the guide RNA provides specificity by hybridizing to the target DNA, in the disclosed methods, the mitotic and / or post-mitotic cells of interest may include cells from any organism (e.g., bacterial cells, archaeal cells, cells of unicellular eukaryotes, plant cells, algal cells, such as Botryococcus braunii, Chlamydomonas reinhardtii, Nannochloropsis gaditana, Chlorella pyrenoidosa, Sargassum patens C. Agardh, etc., fungal cells (e.g., yeast cells), animal cells, cells from invertebrates (e.g., Drosophila, Cnidaria, Echinodermata, nematodes, etc.), cells from vertebrates (e.g., fish, amphibians, reptiles, birds, mammals), cells from mammals, cells from rodents, cells from primates, cells from humans, etc.).

[0424] Any type of cell may be meaningful (e.g., stem cells, such as embryonic stem (ES) cells, induced pluripotent stem (iPS) cells, germ cells; somatic cells, such as fibroblasts, hematopoietic cells, neurons, myocytes, osteocytes, hepatocytes, pancreatic cells; embryonic cells in vitro or in vivo in embryos of any stage, such as zebrafish embryos at stages such as 1-cell, 2-cell, 4-cell, 8-cell, etc.; etc.). Cells may be from established cell lines, or they may be primary cells, wherein "primary cells," "primary cell lines," and "primary cultures" are used interchangeably herein to refer to cells and cell cultures that have been derived from organisms and allowed to grow a limited number of cultures in vitro, i.e., to divide. For example, a primary culture is a culture that may have been passed down 0, 1, 2, 4, 5, 10, or 15 times but has not been passed down through a sufficient number of transition stages. Typically, primary cell lines of the present invention maintain less than 10 passages in vitro. The target cell is in many embodiments a unicellular organism, or is grown in culture.

[0425] If the cells are primary cells, they can be harvested from an individual by any convenient method. For example, leukocytes can be easily harvested by apheresis, leukocytapheresis, density gradient separation, etc., and cells from tissues such as skin, muscle, bone marrow, spleen, liver, pancreas, lung, intestine, stomach, etc. are most conveniently harvested by biopsy. The harvested cells can be dispersed or suspended using an appropriate solution. This solution will generally be a balanced salt solution, such as physiological saline, phosphate buffered saline (PBS), Hank's balanced salt solution, etc., conveniently supplemented with fetal bovine serum or other naturally occurring factors, with an acceptable low concentration buffer, typically 5-25 mM. Convenient buffers include HEPES, phosphate buffer, lactate buffer, etc. The cells can be used immediately, or they can be frozen for a long period of time, thawed and can be reused. In this case, the cells are typically frozen in 10% DMSO, 50% serum, 40% buffered culture medium, or some other such solution as is commonly used in the art, to preserve the cells at such freezing temperatures, and thawed in a manner as is commonly known in the art for thawing frozen cultured cells.

[0426] Nucleic acids encoding guide RNA and / or sRGN polypeptides

[0427] In certain embodiments, the methods comprise contacting the target DNA or introducing one or more nucleic acids and / or donor polynucleotides comprising a nucleotide sequence encoding a guide RNA and / or sRGN polypeptide into a cell (or a population of cells). Suitable nucleic acids comprising a nucleotide sequence encoding a guide RNA and / or sRGN polypeptide include expression vectors, wherein an expression vector comprising a nucleotide sequence encoding a guide RNA and / or sRGN polypeptide is a "recombinant expression vector."

[0428] In certain embodiments, the recombinant expression vector is a viral construct, such as a recombinant adeno-associated virus construct (eg, US Pat. No. 7,078,387), a recombinant adenovirus construct, a recombinant lentivirus construct, or the like.

[0429] Suitable expression vectors include, but are not limited to, viral vectors (e.g., vaccinia virus-based viral vectors; poliovirus; adenovirus (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 5:1088 1097, 1999; WO 94112649, WO 93 / 03769; WO 93 / 19191; WO 94 / 28938; WO 95 / 11984 and WO 95 / 00655); adeno-associated virus (e.g., Ali et al., Hum Gene Ther 9:81 86, 1998, Flannery et al., PNAS 94:6916 6921, 1997; Bennett et al., Invest Opthalmol Vis Sci 38:2857 2863, 1997; Jomary et al., Gene Ther 4:683 690, 1997, Rolling et al., Hum Gene Ther 10:641 648, 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; Mendelson et al., Viral. (1988) 166:154-165; and Flotte et al., PNAS (1993) 90:10613-10617); SV40; herpes simplex virus; human immunodeficiency virus (e.g., Miyoshi et al., PNAS 94:10319 23,1997; Takahashi et al., J Viral 73:7812 7816, 1999); retroviral vectors (e.g., murine leukemia virus, spleen necrosis virus, and vectors derived from retroviruses, such as Rous sarcoma virus, Harvey sarcoma virus, avian leukemia virus, lentivirus, human immunodeficiency virus, myeloproliferative sarcoma virus, and mammary tumor virus); etc.

[0430] Many suitable expression vectors are known to those skilled in the art, and many are commercially available. The following vectors are provided as examples; 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.

[0431] In certain embodiments, the nucleotide sequence encoding the guide RNA and / or sRGN polypeptide is operably linked to a control element, such as a transcriptional control element, such as a promoter. The transcriptional control element can function in a eukaryotic cell, such as a mammalian cell, or a prokaryotic cell, such as a bacterial or archaeal cell. In certain embodiments, the nucleotide sequence encoding the guide RNA and / or sRGN polypeptide is operably linked to a plurality of control elements that allow expression of the nucleotide sequence encoding the guide RNA and / or sRGN polypeptide in both prokaryotic and eukaryotic cells.

[0432] Depending on the host / vector system utilized, any of a number of suitable 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 (e.g., U6 promoter, H1 promoter, etc.; see above) (see, e.g., Bitter et al. (1987) Methods in Enzymology, 153:516-544).

[0433] In certain embodiments, the guide RNA and / or sRGN polypeptide may be provided as RNA. In such cases, the guide RNA and / or RNA encoding the sRGN polypeptide may be produced by direct chemical synthesis, or may be transcribed in vitro from DNA encoding the guide RNA. Methods for synthesizing RNA from a DNA template are well known in the art. In certain cases, the guide RNA and / or RNA encoding the sRGN polypeptide will be synthesized in vitro using an RNA polymerase (e.g., T7 polymerase, T3 polymerase, SP6 polymerase, etc.). Once synthesized, the RNA may be directly contacted with the target DNA, or may be introduced into the cell by any well-known technique for introducing nucleic acids into cells (e.g., microinjection, electroporation, transfection, etc.).

[0434] Nucleotides and / or donor polynucleotides encoding guide RNAs (introduced as DNA or RNA) and / or sRGN polypeptides (introduced as DNA or RNA) can be provided to cells using well-established transfection techniques; for example, Angel and Yanik (2010) PLoS ONE 5(7): e 11756, and the commercially available TransMessenger from Qiagen. ®Reagents, Stemfect™ RNA Transfection Kit from Stemgent, and TransiT from Mims Bio ® -mRNA transfection kit. See also Beumer et al. (2008) Efficient gene targeting in Drosophila by direct embryo injection with zinc-finger nucleases. PNAS 105(50):19821-19826. On the other hand, nucleic acids and / or donor polynucleotides encoding guide RNAs and / or sRGN polypeptides and / or chimeric sRGN polypeptides can be provided on DNA vectors. Many vectors are available for transferring nucleic acids into target cells, such as plasmids, cosmids, minicircles, phages, viruses, etc. Vectors containing one or more nucleic acids can be maintained in an episomal manner, for example as plasmids, minicircle DNA, viruses, such as cytomegalovirus, adenovirus, etc., or they can 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.

[0435] The vector can be provided directly to the cell. In other words, the cell is contacted with a vector comprising a nucleic acid encoding a guide RNA and / or a sRGN polypeptide and / or a chimeric sRGN polypeptide and / or a donor polynucleotide 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 lipofection are well known in the art. For viral vector delivery, the cell is contacted with a viral particle comprising a nucleic acid encoding a guide RNA and / or a sRGN polypeptide and / or a chimeric sRGN polypeptide and / or a donor polynucleotide. Retroviruses, such as lentiviruses, are particularly suitable for the methods of the present invention. Commonly used retroviral vectors are "defective", that is, they cannot produce the viral proteins required for productive infection. Instead, the replication of the vector requires growth in a packaging cell line. In order to produce viral particles containing the nucleic acid of interest, the retroviral nucleic acid containing the nucleic acid is packaged into a viral capsid by a packaging cell line. Different packaging cell lines provide different envelope proteins (ecotropic, amphoteric or heterotropic) to be incorporated into the capsid, which determines the specificity of the viral particles to the cell (ecotropic for mice and rats; amphoteric for most mammalian cell types including humans, dogs and mice; and heterotropic for most mammalian cell types except mouse cells). Appropriate packaging cell lines can be used to ensure that cells are targeted by packaged viral particles. Methods for introducing retroviral vectors containing nucleic acids encoding reprogramming factors into packaging cell lines and collecting viral particles produced by packaging lines are well known in the art. Nucleic acids can also be introduced by direct microinjection (e.g., injecting RNA into zebrafish embryos).

[0436] Vectors for providing nucleic acids encoding guide RNAs and / or sRGN polypeptides and / or chimeric sRGN polypeptides and / or donor polynucleotides to cells will typically contain a suitable promoter for driving expression of the nucleic acid of interest, i.e., transcriptional activation. In other words, the nucleic acid of interest will be operably linked to a promoter. This may include ubiquitous promoters, such as the CMV-13-actin promoter, or inducible promoters, such as promoters that are active in a particular cell population or that respond to the presence of a drug such as tetracycline. By transcriptional activation, it is intended that transcription will increase in the target cell to at least 10 times, at least 100 times, more typically at least 1000 times the basal level. In addition, vectors for providing guide RNAs and / or sRGN polypeptides and / or chimeric sRGN polypeptides and / or donor polynucleotides to cells may include a nucleic acid sequence encoding a selectable marker in the target cell to identify cells that have taken up the guide RNAs and / or sRGN polypeptides and / or chimeric sRGN polypeptides and / or donor polynucleotides.

[0437] Guide RNA and / or sRGN polypeptides and / or chimeric sRGN polypeptides can instead be used to contact DNA or introduced into cells as RNA. Methods for introducing RNA into cells are known in the art and can include, for example, direct injection, transfection, or any other method for introducing DNA.

[0438] The sRGN polypeptide can be provided to the cell as a polypeptide. Such a polypeptide can optionally be fused to a polypeptide domain that increases the solubility of the product. The domain can be connected to the polypeptide through a defined protease cleavage site, such as a TEV sequence, which can be cleaved by a TEV protease. The linker can also include one or more flexible sequences, for example, 1 to 10 glycine residues. In certain embodiments, the cleavage of the fusion protein is performed in a buffer that maintains the solubility of the product, for example, in the presence of 0.5 to 2M urea, in the presence of polypeptides and / or polynucleotides that increase solubility, etc. Domains of interest include endosomolytic domains, for example, influenza HA domains; and other polypeptides that help production, such as IF2 domains, GST domains, GRPE domains, etc. The polypeptide can be formulated for increased stability. For example, the peptide can be PEGylated, where the polyethyleneoxy group provides an increased survival period in the bloodstream.

[0439] Additionally or alternatively, the sRGN polypeptide can be fused to a polypeptide permeant domain to facilitate uptake by cells. Many permeant domains are known in the art and can be used in the non-integrating polypeptides of the present invention, including peptides, peptidomimetics, and non-peptide vectors. For example, a permeant peptide can be derived from the third alpha helix of the Drosophila melanogaster transcription factor Antennapaedia, called penetratin, which contains the amino acid sequence RQIKIWFQNRRMKWKK (SEQ ID NO: 127) motif also disclosed in J. Biol. Chem., 271 (1996), pp. 18188-18193 (for identification but not for disclosure in this application). As another example, a permeant peptide contains an HIV-1 tat basic region amino acid sequence, which can include, for example, amino acids 49-57 of the naturally occurring tat protein.

[0440] Other permeabilized domains include polyarginine motifs, such as the region of amino acids 34-56 of the HIV-1 rev protein, nona-arginine, acta-arginine, etc. (See, e.g., Futaki et al. (2003) Curr Protein Pept Sci. 2003 April; 4(2): 87-9 and 446; and Wender et al. (2000) Proc. Natl. Acad. Sci. USA 2000 Nov 21;97(24):13003-8; published U.S. Patent Application Publication Nos. 20030220334; 20030083256; 20030032593; and 20030022831, the teachings of which regarding translocation peptides and peptidomimetics are specifically incorporated herein by reference. The nona-arginine (R9) sequence is one of the more effective PTDs that have 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 characteristics of the polypeptide. The optimal site will be determined by routine experimentation. In certain embodiments, the polypeptide permeabilizing 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.

[0441] sRGN polypeptides can be produced in vitro or by eukaryotic cells, by prokaryotic cells or by in vitro transcription and translation (IVTT), and they can be further processed by unfolding, such as heat denaturation, OTT reduction, etc., and can be further refolded using methods known in the art.

[0442] Modifications of interest 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, for example, those produced by modifying the glycosylation pattern of the polypeptide during synthesis and processing of the polypeptide or in a further processing step; for example, by exposing the polypeptide to an enzyme that affects glycosylation, such as a mammalian glycosylation or deglycosylation enzyme. Also included are sequences with phosphorylated amino acid residues, for example, phosphotyrosine, phosphoserine or phosphothreonine.

[0443] The present invention also includes guide RNAs and sRGN polypeptides that have been modified using common molecular biology techniques and synthetic chemistry to increase their resistance to proteolytic degradation, alter target sequence specificity, optimize solubility properties, alter protein activity (e.g., transcriptional regulatory activity, enzymatic activity, etc.), or make them more suitable as therapeutic agents. Analogs of such polypeptides include those containing residues other than naturally occurring L-amino acids, such as O-amino acids or non-naturally occurring synthetic amino acids. D-amino acids may replace some or all of the amino acid residues. sRGN polypeptides can be prepared by in vitro synthesis using conventional methods known in the art. Various commercial synthesis apparatus are available, for example, automated synthesizers from Applied Biosystems, Inc., Beckman, etc. By using a synthesizer, naturally occurring amino acids can be replaced by non-natural amino acids. The specific sequence and preparation method will be determined by convenience, economy, desired purity, etc.

[0444] If desired, various groups can be introduced into the peptide during synthesis or during expression which allow attachment to other molecules or surfaces. Thus, cysteine ​​can be used to prepare thioethers, histidine for attachment to metal ion complexes, carboxyl groups for forming amides or esters, amino groups for forming amides, etc.

[0445] sRGN polypeptides can also be isolated and purified according to conventional methods of recombinant synthesis. Lysates of the expression host can be prepared and purified using HPLC, size exclusion chromatography, gel electrophoresis, affinity chromatography or other purification techniques. Generally, the composition used will contain at least 20% by weight of the desired product, such as at least about 75% by weight or at least about 95% by weight, and for therapeutic purposes, at least 99.5% by weight, relative to contaminants associated with the preparation of the product and its purification method. Typically, the percentages will be based on total protein. To induce DNA cleavage and recombination, or any desired modification of the target DNA, or any desired modification of a polypeptide associated with the target DNA, the guide RNA and / or sRGN polypeptide and / or donor polynucleotide, whether introduced as a nucleic acid or as a 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 from about every day to about every four days. One or more agents can be provided to the cell once or more times, such as once, twice, three times, or more than three times, and after each contact event, the cell is allowed to incubate with the one or more agents for some amount of time, such as 16-24 hours, after which the culture medium is replaced with fresh culture medium and the cells are 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 within the same or different target DNA), the complexes can be provided simultaneously (e.g., as two polypeptides and / or nucleic acids), or delivered simultaneously. On the other hand, they can be provided sequentially, e.g., providing the targeting complex first, followed by the second targeting complex, etc., or vice versa.

[0446] Typically, an effective amount of a guide RNA and / or sRGN polypeptide and / or donor polynucleotide is provided to a target DNA or cell to induce target modification. An effective amount of a guide RNA and / or sRGN polypeptide and / or donor polynucleotide is an amount that induces a 2-fold increase or more in the amount of target modification observed between two homologous sequences relative to a negative control (e.g., a cell contacted with an empty vector or an irrelevant polypeptide). That is, an effective amount or dose of a guide RNA and / or sRGN polypeptide and / or donor polynucleotide will induce a 2-fold increase, 3-fold increase, 4-fold increase or more in the amount of target modification observed in the target DNA region, in some cases a 5-fold increase, 6-fold increase or more in the amount of recombination observed, sometimes a 7-fold or 8-fold increase or more, such as a 10-fold, 50-fold or 100-fold increase or more in the amount of recombination observed, in some cases a 200-fold, 500-fold, 700-fold or 1000-fold increase or more, such as a 5000-fold or 10,000-fold increase. The amount of target modification can be measured by any convenient method. For example, a silencing reporter construct comprising a sequence complementary to the targeting segment of the guide RNA flanked by repeat sequences (targeting sequence) which upon recombination will reconstitute a nucleic acid encoding an active reporter gene can be co-transfected into the cell, and the amount of reporter protein assessed after contact with the guide RNA and / or sRGN polypeptide and / or donor polynucleotide, e.g., 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 sRGN polypeptide and / or donor polynucleotide. As another more sensitive assay, the extent of recombination in a genomic DNA region of interest comprising a target DNA sequence can be assessed, for example, by PCR or Southern hybridization of said region after contact with the guide RNA and / or sRGN polypeptide and / or donor polynucleotide, e.g., 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 sRGN polypeptide and / or donor polynucleotide.

[0447] Contacting the cells with the guide RNA and / or sRGN polypeptide and / or donor polynucleotide can occur in any culture medium and under any culture conditions that promote cell survival. For example, the cells can be suspended in any suitable convenient nutrient medium, such as lscove's modified DMEM or RPMI1640, supplemented with fetal bovine serum or heat-inactivated goat serum (about 5-10%), L-glutamine, thiols, particularly 2-mercaptoethanol, and antibiotics, such as penicillin and streptomycin. The culture can contain growth factors to which the cells are responsive. As defined herein, growth factors are molecules that are capable of promoting cell survival, growth and / or differentiation in culture or in intact tissues by specific actions on transmembrane receptors. Growth factors include polypeptide 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 the donor sequence to be inserted is also provided to the cell. "Donor sequence" or "donor polynucleotide" refers to a nucleic acid sequence that is inserted at a cleavage site induced by an sRGN polypeptide. The donor polynucleotide will contain sufficient homology to the genomic sequence at the cleavage site, such as 70%, 80%, 85%, 90%, 95% or 100% homology 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 immediately flanking the cleavage site, to support homology-directed repair between it and the genomic sequence with homology thereto. About 25, 50, 100 or 200 nucleotides or more than 200 nucleotides (or any integer value between 10 and 200 nucleotides, or more) of sequence homology between the donor and the genomic sequence will support homology-directed repair. The donor sequence can be of any length, e.g., 10 nucleotides or more, 50 nucleotides or more, 100 nucleotides or more, 250 nucleotides or more, 500 nucleotides or more, 1000 nucleotides or more, 5000 nucleotides or more, etc.

[0448] The donor sequence is usually different from the genomic sequence it replaces. On the contrary, the donor sequence may contain at least one or more single base changes, insertions, deletions, inversions or rearrangements relative to the genomic sequence, as long as there is enough homology to support homology-guided repair. In certain embodiments, the donor sequence comprises a non-homologous sequence flanking two homology regions, so that the homology-guided repair between the target DNA region and the two flanking sequences causes the non-homologous sequence to be inserted into the target region. The donor sequence may also comprise a vector backbone that contains a sequence that is not homologous to the DNA region of interest and is not intended to be inserted into the DNA region of interest. Typically, one or more homologous regions of the donor sequence will have at least 50% sequence identity with the genomic sequence with which it is desired to be recombined. 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, any value between 1% and 100% sequence identity may be present. Compared to the genomic sequence, the donor sequence may contain certain sequence differences, such as restriction sites, nucleotide polymorphisms, selection 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., to indicate expression at a targeted genomic locus). In some cases, if located in the coding region, such nucleotide sequence differences will not change the amino acid sequence, or will produce silent amino acid changes (i.e., changes that do not affect protein structure or function). On the other hand, 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.

[0449] The donor sequence can be provided to the cell as 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 end of the donor sequence can be protected by methods known to those skilled in the art (e.g., from exonucleolytic degradation). For example, one or more dideoxynucleotide residues are added to the 3' end of the linear molecule and / or a self-complementary oligonucleotide is connected to one or both ends. See, for example, Chang et al. (1987) Proc. Natl. Acad. Sci. USA 84: 4959-4963; Nehls et al. (1996) Science 272: 886-889. Additional methods for protecting exogenous polynucleotides from degradation include, but are not limited to, adding one or more terminal amino groups and using modified internucleotide bonds, such as, for example, phosphorothioates, phosphoramidates and O-methyl ribose or deoxyribose residues. As an alternative to protecting the ends of linear donor sequences, sequences of additional length that can be degraded without affecting recombination can be included outside the region with homology. Donor sequences can be introduced into cells as part of a vector molecule having additional sequences such as, for example, an origin of replication, a promoter, and genes encoding antibiotic resistance. In addition, donor sequences can be introduced as naked (i.e., unmodified) nucleic acids, as nucleic acids complexed with agents such as liposomes or poloxamers, or can be delivered by viruses (e.g., adenovirus, AAV), as described above for nucleic acids encoding guide RNAs and / or sRGN polypeptides and / or donor polynucleotides.

[0450] According to the above method, the DNA region of interest can be cut and modified in vitro, that is, "genetically modified". In certain embodiments, when a selection marker has been inserted into the DNA region of interest, the enriched cell population containing genetic modification can be separated from the remaining population by separating the genetically modified cells. Before enrichment, "genetically modified" cells may only account for about 1% or more (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) of the cell population. The separation of "genetically modified" cells can be achieved by any convenient separation technique suitable for the selected 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 affinity separation techniques, for example, magnetic separation, affinity chromatography, "elutriation" or other convenient techniques with affinity reagents attached to a solid matrix. The technology providing accurate separation includes a fluorescence-activated cell sorter, which can have varying degrees of complexity. For example, multiple color channels, low angle and obtuse light scattering detection channels, impedance channels, etc. By using a dye (such as propidium iodide) associated with dead cells, cells can be selected relative to dead cells. Any technology that is not excessively harmful to the viability of genetically modified cells can be adopted. In this way, a cell composition highly enriched for the cell comprising the modified DNA is obtained. "Highly enriched" refers to that the genetically modified cell will be 70% or more, 75% or more, 80% or more, 85% or more, 90% or more of the cell composition, for example, about 95% or more or 98% or more of the cell composition. In other words, the composition can be a substantially pure composition of a genetically modified cell.

[0451] Genetically modified cells produced by the methods described herein can be used immediately. On the other hand, cells can be frozen at liquid nitrogen temperatures and stored for a long period of time, thawed and can be reused. In this case, cells are usually frozen in 10% dimethyl sulfoxide (DMSO), 50% serum, 40% buffered culture medium or some other such solution as commonly used in the art to preserve cells at such freezing temperatures and thawed in a manner as commonly known in the art for thawing frozen cultured cells.

[0452] Genetically modified cells can be cultured in vitro using methods known in the art. Cells can be expanded in culture, that is, grown under conditions that promote their proliferation. The culture medium can be liquid or semisolid, for example containing agar, methylcellulose, etc. The cell population can be suspended in a suitable nutrient medium, such as lscove's modified DMEM or RPMI 1640, normally supplemented with fetal bovine serum (about 5-10%), L-glutamine, thiols, especially 2-mercaptoethanol, and antibiotics, such as penicillin and streptomycin. The culture can contain various growth factors to which cells are responsive. As defined herein, growth factors are molecules that can promote cell survival, growth and / or differentiation in culture or in intact tissues by specific effects on transmembrane receptors. Growth factors include polypeptides and non-polypeptide factors. Cells that have been genetically modified in this way can be transplanted into subjects for purposes such as gene therapy, for example to treat diseases or as antiviral, antipathogenic or anticancer treatments, for the production of genetically modified organisms in agriculture, or for biological research. In some cases, cells for humans are cultured in Xeno-free culture medium. The subject may be a neonate, juvenile or adult. Of particular interest are mammalian subjects. Mammalian species that may be treated with the present method include canines and felines; equines; bovines; sheep; etc. and primates, particularly humans. Animal models, particularly small mammals (e.g., mice, rats, guinea pigs, hamsters, lagomorpha (e.g., rabbits), etc.) may be used for experimental studies.

[0453] The cells may be provided to the subject alone or together with a suitable substrate or matrix, for example to support their growth and / or organization in the tissue into which they are to be transplanted. Typically, at least 1 x 10^3 cells will be administered, for example 5 x 10^3 cells. 3 cells, 1x10 4 cells, 5x10 4 cells, 1x10 5 of cells, 1 x 10 6Cells or more. Cells can be introduced into a subject by any of the following routes: parenteral, subcutaneous, intravenous, intracranial, intraspinal, intraocular or in 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, for example, by Ommaya reservoir, for example, for intrathecal delivery (e.g., U.S. Patent Nos. 5,222,982 and 5,385,582, incorporated herein by reference); by rapid bolus injection, for example, by syringe, for example, into a joint; by continuous infusion, for example, by cannulation, for example, with convection (e.g., U.S. Application No. 20070254842, incorporated herein by reference); or by implantation of a device to which cells have been reversibly attached (e.g., U.S. Application Nos. 20080081064 and 20090196903, incorporated herein by reference). For the purpose of producing transgenic animals (e.g., transgenic mice), cells can also be introduced into embryos (e.g., blastocysts).

[0454] Delivery of sRGN system

[0455] Guide RNA polynucleotides (RNA or DNA) and / or sRGN polypeptides (RNA or DNA) can be delivered by viral or non-viral delivery vehicles known in the art.

[0456] Polynucleotides can be delivered by non-viral delivery vehicles, including, but not limited to, nanoparticles, liposomes, ribonucleoproteins, positively charged peptides, small molecule RNA-conjugates, aptamer-RNA chimeras, and RNA-fusion protein complexes. Peer and Lieberman, Gene Therapy, 18: 1127-1133 (2011) describe some exemplary non-viral delivery vehicles (which focus on non-viral delivery vehicles for siRNA, which can also be used to deliver other polynucleotides).

[0457] Recombinant adeno-associated virus (AAV) vectors can be used for delivery. The technology for producing rAAV particles is standard in the art, wherein the AAV genome to be packaged, including the polynucleotide to be delivered, the rep and cap genes, and the helper virus function are provided to the cell. The production of rAAV requires the presence of the following components in a single cell (represented herein as a packaging cell): the rAAV genome, the AAV rep and cap genes separated from the rAAV genome (i.e., not in the rAAV genome), and the helper virus function. The AAV rep and cap genes may be from any AAV serotype from which recombinant viruses can be derived, and may be from an AAV serotype different from the rAAV genome ITRs, 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. The generation of pseudotyped rAAV is disclosed in, for example, WO 01 / 83692.

[0458] Table 3

[0459]

[0460] The method of producing packaging cells is to produce a cell line that stably expresses all the necessary components for AAV particle production. For example, a plasmid (or plasmids) containing a rAAV genome lacking AAV rep and cap genes, AAV rep and cap genes separated from the rAAV genome, and a selection marker (e.g., a neomycin resistance gene) is integrated into the genome of the cell. The AAV genome has been introduced into a bacterial plasmid by procedures such as GC tailing (Samulski et al., 1982, Proc. Natl. Acad. S6. USA, 79: 2077-2081), adding a synthetic linker containing a restriction endonuclease cleavage site (Laughlin et al., 1983, Gene, 23: 65-73) or by direct blunt end ligation (Senapathy & Carter, 1984, J. Biol. Chem., 259: 4661-4666). The packaging cell line is then infected with a helper virus such as adenovirus. The advantage of this method is that the cells are selectable and suitable for large-scale production of rAAV.Other examples of suitable methods employ adenovirus or baculovirus rather than plasmids to introduce the rAAV genome and / or the rep and cap genes into packaging cells.

[0461] The general principles of rAAV production are reviewed in, e.g., Carter, 1992, Current Opinions in Biotechnology, 1533-539; and Muzyczka, 1992, Curr. Topics in Micro Biol. and Immunol., 158:97-129. Various methods are described in Ratschin et al., Mol. Cell. Bio. 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. Pat. No. 5,173,414; WO 95 / 13365 and corresponding U.S. Pat. No. 5,658.776; WO 95 / 13392; WO 96 / 17947; PCT / US98 / 18600; WO 97 / 09441 (PCT / US96 / 14423); WO 97 / 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. Pat. No. 5,786,211; U.S. Pat. No. 5,871,982; and U.S. Pat. No. 6,258,595.

[0462] The AAV vector serotype can be matched to the target cell type. For example, the following exemplary cell types were transduced by the indicated AAV serotypes, among others.

[0463] Table 4

[0464] 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

[0465] The number of treatments administered to a subject may vary. Introducing the genetically modified cells into a subject may be a one-time event; however, in some cases, such treatment may elicit improvement for a limited period of time and require an ongoing series of repeated treatments. In other cases, multiple administrations of the genetically modified cells may be required before an effect is observed. The exact protocol depends on the disease or condition, the stage of the disease, and the parameters of the individual subject being treated.

[0466] In other aspects of the present disclosure, guide RNAs and / or sRGN polypeptides and / or donor polynucleotides are employed to modify cellular DNA in vivo, e.g., for purposes such as gene therapy, e.g., to treat disease or as an antiviral, antipathogenic, or anticancer therapy, for the production of genetically modified organisms in agriculture, or for biological research. In these in vivo embodiments, the guide RNAs and / or sRGN polypeptides and / or donor polynucleotides are administered directly to an individual. The guide RNAs and / or sRGN polypeptides and / or donor polynucleotides may be administered by any of a number of methods known in the art for administering peptides, small molecules, and nucleic acids to a subject. The guide RNAs and / or sRGN polypeptides and / or donor polynucleotides may be incorporated into a variety of formulations. More particularly, the guide RNAs and / or sRGN polypeptides and / or donor polynucleotides of the present invention may be formulated into pharmaceutical compositions by combining with a suitable pharmaceutically acceptable carrier or diluent. In some cases, the guide RNAs and sRGNs are formulated for ex vivo delivery.

[0467] A pharmaceutical formulation is a composition comprising one or more guide RNAs and / or sRGN polypeptides and / or donor polynucleotides in a pharmaceutically acceptable carrier. A "pharmaceutically acceptable carrier" may be a carrier approved by a regulatory agency of a federal or state government or listed in the US Pharmacopeia or other generally recognized pharmacopeia for use in mammals, such as humans. The term "carrier" refers to a diluent, adjuvant, excipient, or carrier with which the compounds of the invention are formulated for administration to a mammal. Such pharmaceutical carriers may be lipids, such as liposomes, such as liposome dendrimers; liquids, such as water and oils, including those of petroleum, animal, plant, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, etc., saline; gum arabic, gelatin, starch paste, talc, keratin, colloidal silica, urea, etc. In addition, adjuvants, stabilizers, thickeners, lubricants, and colorants may be used. The pharmaceutical composition can be formulated into a solid, semisolid, liquid or gaseous form, such as tablets, capsules, powders, granules, ointments, solutions, suppositories, injections, inhalants, gels, microspheres and aerosols. Thus, administration of the guide RNA and / or sRGN polypeptide and / or donor polynucleotide can be achieved in a variety of ways, including oral, buccal, rectal, parenteral, intraperitoneal, intradermal, transdermal, intratracheal, intraocular, etc. The active agent can be systemic after administration, or can be localized by using regional administration, intramural administration, or using an implant that acts to maintain the active dose at the implantation site. The active agent can be formulated for immediate activity, or it can be formulated for sustained release.

[0468] For some conditions, particularly central nervous system conditions, it may be necessary to formulate agents to cross the blood-brain barrier (BBB). One strategy for drug delivery across the BBB requires biochemical disruption of the BBB by osmotic means (e.g., mannitol or leukotrienes) or by the use of vasoactive substances such as bradykinin. The potential to target specific agents to brain tumors using BBB openings is also an option. When the composition is administered by intravascular injection, a BBB disruptor can be co-administered with the therapeutic composition of the present invention. Other strategies for delivery across the BBB may require the use of endogenous transport systems, including caveolin-1-mediated transcytosis, carrier-mediated transporters (e.g., glucose and amino acid carriers), receptor-mediated transcytosis for insulin or transferrin, and active efflux transporters (e.g., p-glycoprotein). Active transport moieties can also be conjugated to therapeutic compounds for use in the present invention to facilitate transport across the vascular endothelial wall. On the other hand, drug delivery of therapeutic agents behind the BBB can be by local delivery, such as by intrathecal delivery, such as through an Ommaya reservoir (see, e.g., U.S. Pat. Nos. 5,222,982 and 5385582, incorporated herein by reference); by bolus injection, such as by syringe, such as intravitreal or intracranial; by continuous infusion, such as by cannulation, such as with convection (see, e.g., U.S. App. No. 20070254842, incorporated herein by reference); or by implantation of a device to which an agent has been reversibly attached (see, e.g., U.S. App. Nos. 20080081064 and 20090196903, incorporated herein by reference).

[0469] Typically, an effective amount of guide RNA and / or sRGN polypeptide and / or donor polynucleotide is provided. As discussed above with respect to the ex vivo method, an effective amount or effective dose of guide RNA and / or sRGN polypeptide and / or donor polynucleotide in vivo is an amount that induces a 2-fold increase or more in the amount of recombination observed between two homologous sequences relative to a negative control (e.g., cells contacted with an empty vector or an irrelevant polypeptide). The amount of recombination can be measured by any convenient method, such as described above and known in the art. Calculation of the effective amount or effective dose of guide RNA and / or sRGN polypeptide and / or donor polynucleotide is within the skill of one of ordinary skill in the art and will be routine to one of ordinary skill in the art. The final amount to be administered will depend on the route of administration and the nature of the disorder or condition to be treated.

[0470] The effective dose of using a specific patient will depend on multiple factors, several of which will be different from one patient to another. A competent clinician will be able to determine the effective dose of the therapeutic agent, to be used to the patient to stop or reverse the progress of the disease condition, as required. Utilizing LD50 animal data and other information available for the reagent, the clinician can determine the maximum safe dose for an individual depending on route of administration. For example, considering the larger fluid entity of the therapeutic composition being used therein, the dosage of intravenous administration can have a greater amount of therapeutic agent than the dosage used in the sheath. Similarly, the compositions removed quickly from health can be used with higher doses or with repeated doses to maintain therapeutic concentrations. Utilizing common techniques, a competent clinician will be able to optimize the dosage of a specific therapeutic agent in a conventional clinical trial process.

[0471] For inclusion in a medicament, guide RNA and / or sRGN polypeptide and / or donor polynucleotide can be obtained from suitable commercial sources. As a general suggestion, the total pharmaceutically effective amount of guide RNA and / or sRGN polypeptide and / or donor polynucleotide administered per parenteral dose will be within the range that can be measured by a dose response curve.

[0472] Therapies based on guide RNA and / or sRGN polypeptides and / or donor polynucleotides, i.e., formulations of guide RNA and / or sRGN polypeptides and / or donor polynucleotides to be used for therapeutic administration must be sterile. Sterility can be achieved by filtration through a sterile filtration membrane (e.g., a 0.2 1-1 m membrane). The therapeutic composition is typically placed in a container with a sterile access port, for example, an intravenous solution bag or vial with a stopper that can be pierced by a hypodermic needle. Therapies based on guide RNA and / or sRGN polypeptides and / or donor polynucleotides can be stored as an aqueous solution or as a lyophilized formulation for reconstitution in a unit or multi-dose container, for example, a sealed ampoule or vial. As an example of a lyophilized formulation, a 10-ml vial is filled with 5 ml of a sterile filtered 1% (w / v) aqueous solution of the compound, and the resulting mixture is lyophilized. The infusion solution is prepared by reconstituting the lyophilized compound using bacteriologically inhibited water for injection (Water-for-Injection).

[0473] Depending on the desired formulation, the pharmaceutical composition may include a pharmaceutically acceptable non-toxic carrier of a diluent, which is defined as a vehicle commonly used to formulate a pharmaceutical composition for animal or human administration. The diluent is selected so as not to affect the biological activity of the combination. Examples of such diluents are distilled water, buffered water, saline, PBS, Ringer's solution, dextrose solution, and Hank's solution. In addition, the pharmaceutical composition or formulation may include other carriers, adjuvants or non-toxic, non-therapeutic, non-immunogenic stabilizers, excipients, etc. The composition may also include additional substances to approximate physiological conditions, such as pH adjustment and buffering agents, toxicity regulators, wetting agents, and detergents.

[0474] The composition can also include any of a variety of stabilizers, such as, for example, antioxidants. When the pharmaceutical composition includes a polypeptide, the polypeptide can be compounded with various well-known compounds that enhance the in vivo stability of the polypeptide, or otherwise enhance its pharmacological properties (e.g., increase the half-life of the polypeptide, reduce its toxicity, improve solubility or uptake). Examples of such modifications or compounding agents include sulfates, gluconates, citrates, and phosphates. The nucleic acid or polypeptide of the composition can also be compounded with molecules that enhance its in vivo properties. Such molecules include, for example, carbohydrates, polyamines, amino acids, other peptides, ions (e.g., sodium, potassium, calcium, magnesium, manganese), and lipids.

[0475] Further guidance on formulations suitable for various types of administration is known in the art, for example, Remington's Pharmaceutical Sciences, Mace Publishing Company, Philadelphia, Pa., 21st edition (2006). For a brief review of methods for drug delivery, see, Langer, Science 249:1527-1533 (1990).

[0476] The pharmaceutical composition can be administered for preventive and / or therapeutic treatment. The toxicity and therapeutic efficacy of the active ingredient can be determined according to standard pharmaceutical procedures in cell culture and / or experimental animals, including, for example, determining the LD50 (the dose that is lethal to 50% of the population) and the ED50 (the dose that is therapeutically effective in 50% of the population). The dose ratio between toxicity and therapeutic effect is the therapeutic index, and it can be expressed as the ratio LD50 / ED50. Treatments that exhibit a large therapeutic index may be preferred.

[0477] The data obtained from cell culture and / or animal studies can be used to formulate a dosage range for humans. The dosage of the active ingredient is generally within a circulating concentration range including an ED50 with low toxicity. Depending on the dosage form adopted and the route of administration used, the dosage can vary within this range. The components used to prepare the pharmaceutical composition have high purity and are substantially free of potentially harmful contaminants (e.g., at least National Food (NF) grade, generally at least analytical grade, including at least pharmaceutical grade) in certain embodiments. In addition, the compositions intended for use in vivo are generally sterile. In terms of having to synthesize a given compound before use, the resulting product is generally substantially free of any potential toxic agent, particularly any endotoxin, which may exist during synthesis or purification. Compositions for parenteral administration are also sterile, substantially isotonic, and prepared under GMP conditions.

[0478] The effective amount of the therapeutic composition to be used for a particular patient will depend on multiple factors, several of which will be different from one patient to another. A competent clinician will be able to determine the effective amount of the therapeutic agent, to be used to the patient to stop or reverse the progress of the disease condition, as required. Utilizing LD50 animal data and other information available for the reagent, the clinician can determine the maximum safe dose for an individual depending on route of administration. For example, considering the larger fluid entity of the therapeutic composition being used therein, the dosage of intravenous administration can be greater than the dosage of intrathecal administration. Similarly, the compositions removed quickly from health can be used with higher doses or with repeated doses to maintain therapeutic concentrations. Utilizing common techniques, a competent clinician will be able to optimize the dosage of a specific therapeutic agent in a conventional clinical trial process.

[0479] Genetically modified host cells

[0480] The present disclosure provides genetically modified host cells, including isolated genetically modified host cells, wherein the genetically modified host cell comprises (has been genetically modified with:

[0481] 1) exogenous guide RNA; 2) exogenous nucleic acid comprising a nucleotide sequence encoding the guide RNA; 3) exogenous sRGN polypeptide (e.g., naturally occurring sRGN; modified, i.e., mutated or variant, sRGN; chimeric sRGN; etc.); 4) exogenous nucleic acid comprising a nucleotide sequence encoding an sRGN polypeptide; or 5) any combination of the above. Genetically modified cells are produced by using, for example, a genetically modified host cell with: 1) exogenous guide RNA; 2) exogenous nucleic acid comprising a nucleotide sequence encoding the guide RNA; 3) exogenous sRGN polypeptide; 4) exogenous nucleic acid comprising a nucleotide sequence encoding an sRGN polypeptide; or 5) any combination of the above).

[0482] All cells suitable as target cells are also suitable as genetically modified host cells. For example, the genetically modified host cell of interest can be a cell from any organism (e.g., bacterial cell, archaebacterial cell, cell of a unicellular eukaryotic organism, plant cell, algae cell, such as Botryococcus braunii, Chlamydomonas reinhardtii, Nannochloropsis gaditana, Chlorel / a pyrenoidosa, Sargassum patens C.Agardh, etc., fungal cell (e.g., yeast cell), animal cell, cell from invertebrates (e.g., Drosophila, Cnidaria, Echinodermata, nematodes, etc.), cell from vertebrates (e.g., fish, amphibians, reptiles, birds, mammals), cell from mammals (e.g., pigs, cattle, goats, sheep, rodents, rats, mice, non-human primates, humans, etc.), etc.

[0483] In certain embodiments, the genetically modified host cell has been genetically modified with an exogenous nucleic acid comprising a nucleotide sequence encoding a sRGN polypeptide (e.g., a naturally occurring sRGN; a modified, i.e., mutated or variant, sRGN; a chimeric sRGN; etc.). The DNA of the genetically modified host cell can be targeted for modification by introducing a guide RNA (or DNA encoding the guide RNA, which determines the genomic location / sequence to be modified) and optionally a common nucleic acid into the cell. In certain embodiments, the nucleotide sequence encoding the sRGN polypeptide is operably linked to an inducible promoter (e.g., a heat shock promoter, a tetracycline-regulated promoter, a steroid-regulated promoter, a metal-regulated promoter, an estrogen receptor-regulated promoter, etc.). In certain embodiments, the nucleotide sequence encoding the sRGN polypeptide 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 certain embodiments, the nucleotide sequence encoding the sRGN polypeptide is operably linked to a constitutive promoter.

[0484] In certain embodiments, the genetically modified host cell is in vitro. In certain embodiments, the genetically modified host cell is in vivo. In certain embodiments, the genetically modified host cell is a prokaryotic cell or is derived from a prokaryotic cell. In certain embodiments, the genetically modified host cell is a bacterial cell or is derived from a bacterial cell. In certain embodiments, the genetically modified host cell is an archaeal cell or is derived from an archaeal cell. In certain embodiments, the genetically modified host cell is a eukaryotic cell or is derived from a eukaryotic cell. In certain embodiments, the genetically modified host cell is a plant cell or is derived from a plant cell. In certain embodiments, the genetically modified host cell is an animal cell or is derived from an animal cell. In certain embodiments, the genetically modified host cell is an invertebrate cell or is derived from an invertebrate cell. In certain embodiments, the genetically modified host cell is a vertebrate cell or is derived from a vertebrate cell. In certain embodiments, the genetically modified host cell is a mammalian cell or is derived from a mammalian cell. In certain embodiments, the genetically modified host cell is a rodent cell or is derived from a rodent cell. In certain embodiments, the genetically modified host cell is a human cell or is derived from a human cell.

[0485] The disclosure further provides progeny of the genetically modified cell, wherein the progeny may comprise the same exogenous nucleic acid or polypeptide as the genetically modified cell from which it was derived. The disclosure further provides compositions comprising the genetically modified host cell.

[0486] Genetically modified stem cells and genetically modified progenitor cells

[0487] In certain embodiments, the genetically modified host cell is a genetically modified stem cell or ancestral cell. Suitable host cells include, for example, stem cells (adult stem cells, embryonic stem cells, iPS cells, etc.) and ancestral cells (e.g., cardiac ancestral cells, neural ancestral cells, etc.). Suitable host cells include mammalian stem cells and ancestral cells, including, for example, rodent stem cells, rodent ancestral cells, human stem cells, human ancestral cells, etc. Suitable host cells include in vitro host cells, such as isolated host cells.

[0488] In certain embodiments, the genetically modified host cell comprises an exogenous guide RNA nucleic acid. In certain embodiments, the genetically modified host cell comprises an exogenous nucleic acid comprising a nucleotide sequence encoding a guide RNA. In certain embodiments, the genetically modified host cell comprises an exogenous sRGN polypeptide (e.g., a naturally occurring sRGN; a modified, i.e., mutated or variant, sRGN; a chimeric sRGN; etc.). In certain embodiments, the genetically modified host cell comprises an exogenous nucleic acid comprising a nucleotide sequence encoding a sRGN polypeptide. In certain embodiments, the genetically modified host cell comprises an exogenous nucleic acid comprising a nucleotide sequence encoding 1) a guide RNA and 2) a sRGN polypeptide.

[0489] In some cases, the sRGN polypeptide comprises 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 any of SEQ ID NOs: 1-12, or an active portion thereof having a length of at least 100, 150, 200, 300, 350, 400, or 500 amino acids. In certain embodiments, the active portion comprises an RNase domain. In other embodiments, the active portion comprises a DNase domain.

[0490] Composition

[0491] The present disclosure provides compositions comprising guide RNA and / or localization-modifying polypeptides. In some cases, the sRGN polypeptide is a chimeric polypeptide. The compositions can be used to perform the methods of the present disclosure, such as methods for site-specific modification of target DNA; methods for site-specific modification of polypeptides associated with target DNA; and the like.

[0492] Compositions comprising guide RNA

[0493] The present disclosure provides a composition comprising a guide RNA. In addition to the guide RNA, the composition may include one or more of the following: a salt, such as NaCl, MgCl, KCl, MgSO4, etc.; a buffer, such as Tris 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.; a solubilizing agent; a detergent, such as a non-ionic detergent such as Tween ® 20, etc.; nuclease inhibitors; etc. For example, in some cases, the composition comprises a guide RNA and a buffer for stabilizing the nucleic acid.

[0494] In certain 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 more than 99% pure, wherein "% purity" refers to the guide RNA being the stated percentage free of other macromolecules or contaminants that may be present during the guide RNA production process.

[0495] Compositions comprising chimeric polypeptides

[0496] The present disclosure provides compositions of chimeric polypeptides. In addition to the guide RNA, the composition may include one or more of the following: salts, e.g., NaCl, MgCl, KCl, MgSO4, etc.; buffers, e.g., Tris buffer, HEPES, MES, MES sodium salt, MOPS, TAPS, etc.; solubilizers; detergents, e.g., non-ionic deter...

Claims

1. A synthetic RNA-guided nuclease (sRGN) polypeptide comprising, from N-terminus to C-terminus: 1) a small domain 1 comprising an amino acid sequence of any one of SEQ ID NOs: 34-37, 2) a small domain 2 comprising an amino acid sequence of any one of SEQ ID NOs: 38-41, 3) small domain 3, which comprises the amino acid sequence of any one of SEQ ID NOs: 42-45, 4) a small domain 4 comprising an amino acid sequence of any one of SEQ ID NOs: 46-49, 5) small domain 5, which comprises the amino acid sequence of any one of SEQ ID NOs: 50-53, 6) small domain 6, which comprises the amino acid sequence of any one of SEQ ID NOs: 54-57, 7) small domain 7 comprising the amino acid sequence of any one of SEQ ID NOs: 58-61, and 8) small domain 8, which comprises the amino acid sequence of any one of SEQ ID NOs: 62-65, wherein at least 2 of the small domains are derived from different parent Cas9 endonucleases selected from the group consisting of: Staphylococcus lugdunensis Cas9 as shown in SEQ ID NO:30, Staphylococcus pasteurianus Cas9 as shown in SEQ ID NO:31, Staphylococcus microti Cas9 as shown in SEQ ID NO:33, and Staphylococcus suis Cas9 as shown in SEQ ID NO:32; and wherein the sRGN polypeptide consists of an amino acid sequence selected from the group consisting of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 and 12.

2. The sRGN polypeptide of claim 1, wherein at least three of the small domains are derived from different parent Cas9 endonucleases selected from the group consisting of Staphylococcus lugdunensis Cas9 as shown in SEQ ID NO:30, Staphylococcus pasteurianus Cas9 as shown in SEQ ID NO:31, Staphylococcus microti Cas9 as shown in SEQ ID NO:33, and Staphylococcus suis Cas9 as shown in SEQ ID NO:

32.

3. The sRGN polypeptide of claim 1 or 2, wherein small domain 8 comprises the amino acid sequence of SEQ ID NO:

62.

4. The sRGN polypeptide of claim 1 or 2, wherein small domain 8 comprises the amino acid sequence of SEQ ID NO:

63.

5. The sRGN polypeptide of claim 1 or 2, wherein small domain 1 comprises the amino acid sequence of SEQ ID NO:

34.

6. The sRGN polypeptide of claim 1, selected from the group consisting of: a) a Gib11 polypeptide consisting of the amino acid sequence of SEQ ID NO: 1, b) a Gib11Spa-1 polypeptide consisting of the amino acid sequence of SEQ ID NO: 2, and c) a Gib11Spa-3 polypeptide consisting of the amino acid sequence of SEQ ID NO:

4.

7. A nucleic acid encoding the sRGN polypeptide of any one of claims 1-6.

8. The nucleic acid of claim 7, wherein the nucleic acid is codon optimized for expression in a host cell.

9. The nucleic acid of claim 7 or 8, comprising the nucleotide sequence of any one of SEQ ID NOs: 13-29.

10. A system comprising: (a) an sRGN polypeptide according to any one of claims 1 to 6, or a nucleic acid encoding the sRGN polypeptide; and (b) a guide RNA (gRNA) or a nucleic acid encoding the gRNA, wherein the gRNA is capable of guiding the sRGN polypeptide or variant thereof to a target polynucleotide sequence.

11. The system of claim 10, further comprising a donor template comprising a heterologous polynucleotide sequence, wherein the heterologous polynucleotide sequence is capable of being inserted into the target polynucleotide sequence.

12. The system of claim 10 or 11, wherein the nucleic acid encoding the sRGN polypeptide is codon optimized for expression in a host cell.

13. The system of claim 11, wherein the heterologous polynucleotide sequence is codon optimized for expression in a host cell.

14. The system of claim 10 or 11, wherein the nucleic acid encoding the sRGN polypeptide is deoxyribonucleic acid (DNA).

15. The system of claim 10 or 11, wherein the nucleic acid encoding the sRGN polypeptide is ribonucleic acid (RNA).

16. The system of claim 15, wherein the RNA encoding the sRGN polypeptide is mRNA.

17. The system of any one of claims 11 and 16, wherein the donor template is encoded in an adeno-associated virus (AAV) vector.

18. The system of any one of claims 10-11 and 16, wherein the sRGN polypeptide or nucleic acid encoding the sRGN polypeptide is formulated in a liposome or lipid nanoparticle.

19. The system of claim 18, wherein the liposome or lipid nanoparticle also comprises a gRNA or a nucleic acid encoding the gRNA.

20. The system of any one of claims 10-11 and 16, comprising a sRGN polypeptide pre-complexed with a gRNA to form a ribonucleoprotein (RNP) complex.

21. A method of targeting, editing, modifying or manipulating a target DNA at a target locus, the method comprising providing the following to the target DNA: (a) an sRGN polypeptide according to any one of claims 1 to 6, or a nucleic acid encoding the sRGN polypeptide; and (b) a guide RNA (gRNA) or a nucleic acid encoding the gRNA, wherein the gRNA is capable of guiding the sRGN polypeptide to the target locus.

22. The method of claim 21, further comprising providing the target DNA with a donor template comprising a heterologous polynucleotide sequence, wherein the heterologous polynucleotide sequence is capable of being inserted into the target locus.

23. The method of claim 21 or 22, wherein the nucleic acid encoding the sRGN polypeptide is codon optimized for expression in a host cell.

24. The method of claim 22, wherein the heterologous polynucleotide sequence is codon optimized for expression in a host cell.

25. The method of claim 21 or 22, wherein the nucleic acid encoding the sRGN polypeptide is deoxyribonucleic acid (DNA).

26. The method of claim 21 or 22, wherein the nucleic acid encoding the sRGN polypeptide is a ribonucleic acid (RNA).

27. The method of claim 26, wherein the RNA encoding the sRGN polypeptide is mRNA.

28. The method of any one of claims 21-22 and 27, wherein the donor template is encoded in an adeno-associated virus (AAV) vector.

29. The method of any one of claims 21-22 and 27, wherein the sRGN polypeptide or nucleic acid encoding the sRGN polypeptide is formulated in a liposome or lipid nanoparticle.

30. The method of claim 29, wherein the liposome or lipid nanoparticle also comprises a gRNA or a nucleic acid encoding the gRNA.

31. The method of any one of claims 21-22 and 27, comprising providing the target DNA with a sRGN polypeptide, the sRGN polypeptide being pre-complexed with the gRNA as an RNP complex.

32. A modified cell comprising: (a) an sRGN polypeptide according to any one of claims 1 to 6, or a nucleic acid encoding the sRGN polypeptide; and (b) a guide RNA (gRNA) or a nucleic acid encoding the gRNA, wherein the gRNA is capable of guiding the sRGN polypeptide or variant thereof to a target polynucleotide sequence; The modified cell is a bacterial cell, a fungal cell, an archaeal cell, a protist or an animal cell.

33. The modified cell of claim 32, further comprising a donor template comprising a heterologous polynucleotide sequence, wherein the heterologous polynucleotide sequence is capable of being inserted into the target polynucleotide sequence.

34. A genetically modified cell, wherein the genome of the cell is edited by the method according to any one of claims 21-31, wherein the genetically modified cell is a bacterial cell, a fungal cell, an archaeal cell, a protist or an animal cell.

35. A kit comprising: (a) an sRGN polypeptide according to any one of claims 1 to 6, or a nucleic acid encoding the sRGN polypeptide; and (b) a guide RNA (gRNA) or a nucleic acid encoding the gRNA, wherein the gRNA is capable of guiding the sRGN polypeptide or variant thereof to a target polynucleotide sequence.

36. The kit of claim 35, further comprising a donor template comprising a heterologous polynucleotide sequence, wherein the heterologous polynucleotide sequence is capable of being inserted into the target polynucleotide sequence.

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