Use of nucleosome-interacting protein domains to enhance targeted genome modifications

Abstract

To provide compositions and methods for increasing efficiency of targeted genome / epigenetic modification in eukaryotic cells.SOLUTION: A fusion protein comprises at least one nucleosome interacting protein domain linked to a programmable DNA modification protein. A method for increasing efficiency of targeted genome or epigenetic modification in an eukaryotic cell comprises introducing, into the eukaryotic cell, the fusion protein or a nucleic acid encoding the fusion protein.SELECTED DRAWING: None

Description

[Technical Field]

[0001] This disclosure relates to compositions and methods for increasing the efficiency of targeted genomic modifications, targeted transcriptional regulation, or targeted epigenetic modifications. [Background technology]

[0002] Programmable endonucleases are becoming an important tool for targeted genome manipulation or modification in eukaryotes. Recently, RNA-guided clustering of regularly scattered short palindromic repeats ( c stestered r egularly i nterspersed s hort p alindromic r The CRISPR system emerged as a new generation of genome modification tools. These novel programmable endonucleases offered unprecedented simplicity and versatility compared to previous generations of nucleases such as zinc finger nucleases (ZFNs) and activator-like effector nucleases (TALENs). However, chromatin barriers in eukaryotic cells can hinder access to and cleavage of targets by prokaryotic CRISPR systems (Hinz et al, Biochemistry, 2015, 54:7063-66; Horlbeck et al., eLife, 2016, 5:e12677).

[0003] In fact, when using Streptococcus pyogenes Cas9 (SpCas9), considered the most active CRISPR nuclease to date, no or low editing activity was observed at specific mammalian genomic sites. Furthermore, many characterized CRISPR nucleases, while active in bacteria or on purified DNA substrates, have so far shown no activity in mammalian cells. Therefore, to increase the efficiency of targeted genomic or epigenetic modifications in eukaryotes and to overcome chromatin inhibition, the capabilities of CRISPR nuclease systems and other programmable DNA modification proteins need to be improved.

[0004] summary Various aspects of this disclosure include the provision of fusion proteins, each of which comprises at least one nucleosome-interacting protein domain linked to a programmable DNA-modifying protein.

[0005] At least one nucleosome-interacting protein domain may be a DNA-binding domain from a high-mobility group (HMG) box (HMGB) protein selected from HMGB1, HMGB2, or HMGB3; an HMG nucleosome-binding (HMGN) protein selected from HMGN1, HMGN2, HMGN3a, HMGN3b, HMGN4, or HMGN5; a central globule domain from a histone H1 variant; or a DNA-binding domain from a chromatin remodeling complex protein selected from a switch / sucrose non-fermentable (SWI / SNF) complex, a mimic switch (ISWI) complex, a chromodomain-helicase-DNA-binding (CHD) complex, a nucleosome remodeling and deacetylase (NuRD) complex, an INO80 complex, an SWR1 complex, an RSC complex, or a combination thereof. In some embodiments, at least one nucleosome-interacting protein domain may be an HMGB1 box A domain, an HMGN1 protein, an HMGN2 protein, an HMGN3a protein, an HMGN3b protein, a histone H1 central globule domain, an ISWI protein DNA-binding domain, a CHD1 protein DNA-binding domain, or a combination thereof.

[0006] In some embodiments, the programmable DNA-modified protein has nuclease activity, and the programmable DNA-modified protein may be a chimeric protein containing a programmable DNA-binding domain ligated to a clustered regularly scattered short palindromic repeat (CRISPR) nuclease or nickas, zinc finger nuclease (ZFN), transcription activator-like effector nuclease (TALEN), meganuclease, or nuclease domain.

[0007] In other embodiments, the programmable DNA-modified protein may have non-nuclease activity and may be a chimeric protein comprising a programmable DNA-binding domain ligated to a non-nuclease domain. The programmable DNA-binding domain of the chimeric protein may be a CRISPR protein, zinc finger protein, or transcription activator-like effector modified to lack all nuclease activity, and the non-nuclease domain of the chimeric protein may have: acetyltransferase activity, deacetylase activity, methyltransferase activity, demethylase activity, kinase activity, phosphatase activity, ubiquitin ligase activity, deubiquitination activity, adenylation activity, deenylation activity, SUMOylation activity, deSUMOylation activity, ribosylation activity, deribosylation activity, myristoylation activity, demyristoylation activity, citrullination activity, helicase activity, amination activity, deamination activity, alkylation activity, dealkylation activity, oxidative activity, transcription activating activity, or transcription repressor activity. In certain embodiments, the non-nuclease domain of the chimeric protein may have cytosine deaminase activity, histone acetyltransferase activity, transcriptional activating activity, or transcriptional repressor activity.

[0008] At least one nucleosome-interacting protein domain can be ligated to a programmable DNA-modified protein directly via a chemical bond, indirectly via a linker, or a combination thereof. The at least one nucleosome-interacting protein domain can be ligated to the N-terminus, C-terminus, and / or internal position of the programmable DNA-modified protein. In some embodiments, the fusion protein comprises at least two nucleosome-interacting protein domains ligated to the programmable DNA-modified protein.

[0009] The fusion proteins disclosed herein may further comprise at least one nuclear localization signal, at least one cell permeability domain, at least one marker domain, or a combination thereof.

[0010] Another aspect of this disclosure is the inclusion of fusion proteins comprising a CRISPR protein linked to at least one nucleosome interacting protein domain.

[0011] Generally, the CRISPR protein in the aforementioned fusion protein can be either a type II CRISPR / Cas9 protein or a type V CRISPR / Cpf1 protein. In certain embodiments, the CRISPR protein may be Streptococcus pyogenes Cas9 (SpCas9), Streptococcus thermophilus Cas9 (StCas9), Streptococcus pasteurianus (SpaCas9), Campylobacter jejuni Cas9 (CjCas9), Staphylococcus aureus (SaCasFvics), Francisella novicida Cas9 (FnCas9), Neisseria cinerea Cas9 (NcCas9), Neisseria meningitis Cas9 (NmCas9), Francisella novicida Cpf1 (FnCpf1), Acidaminococcus sp. Cpf1 (AsCpf1), or Lachnospiraceae bacterium ND2006 Cpf1 (LbCpf1).

[0012] In some embodiments, the CRISPR protein has nuclease or nickase activity. For example, the CRISPR protein may be a type II CRISPR / Cas9 nuclease or nickase, or a type V CRISPR / Cpf1 nuclease or nickase. In other embodiments, the CRISPR protein has non-nuclease activity. In such iterations, the CRISPR protein may be a type II CRISPR / Cas9 protein modified to lack all nuclease activity and linked to a non-nuclease domain, or a type V CRISPR / Cpf1 protein modified to lack all nuclease activity and linked to a non-nuclease domain, where the non-nuclease domain may have cytosine deaminase activity, histone acetyltransferase activity, transcriptional activating activity, or transcriptional repressor activity.

[0013] At least one nucleosome-interacting protein domain of a CRISPR fusion protein can be a high mobility group (HMG) box (HMGB) DNA-binding domain, an HMG nucleosome-binding (HMGN) protein, a central globule domain from a histone H1 variant, a DNA-binding domain from a chromatin remodeling complex protein, or a combination thereof. In certain embodiments, at least one nucleosome-interacting protein domain of the CRISPR fusion protein can be an HMGB1 box A domain, an HMGN1 protein, an HMGN2 protein, an HMGN3a protein, an HMGN3b protein, a histone H1 central globule domain, an ISWI protein DNA-binding domain, a chromodomain-helicase-DNA protein 1 (CHD1) DNA-binding domain, or a combination thereof.

[0014] At least one nucleosome-interacting protein domain can be ligated to a CRISPR protein directly via a chemical bond, indirectly via a linker, or a combination thereof. This at least one nucleosome-interacting protein domain can be ligated to the N-terminus, C-terminus, and / or internal position of the CRISPR protein. In some embodiments, the fusion protein comprises at least two nucleosome-interacting protein domains ligated to the CRISPR protein.

[0015] The CRISPR fusion proteins disclosed herein may further comprise at least one nuclear localization signal, at least one cell permeability domain, at least one marker domain, or a combination thereof.

[0016] In certain embodiments, the CRISPR fusion protein may have an amino acid sequence that has at least about 90% sequence identity with SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78, or SEQ ID NO: 79.

[0017] In other embodiments, the CRISPR fusion protein may have the amino acid sequence described in SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78, or SEQ ID NO: 79.

[0018] Another aspect of this disclosure is the inclusion of a protein-RNA complex comprising at least one CRISPR-containing fusion protein and at least one guide RNA as disclosed herein.

[0019] A further aspect of the present disclosure provides a nucleic acid encoding any of the fusion proteins disclosed herein. The nucleic acid can be codon-optimized for translation in eukaryotic cells. In some embodiments, the nucleic acid can be part of a vector, such as a viral vector, a plasmid vector, or a self-replicating RNA.

[0020] Yet another aspect of the present disclosure provides a method for increasing the efficiency of targeted genomic or epigenetic modifications in eukaryotic cells. The method includes introducing into a eukaryotic cell (a) at least one fusion protein disclosed herein or a nucleic acid encoding the fusion protein, wherein the at least one nucleosome interaction protein domain of the at least one fusion protein alters the nucleosome or chromatin structure, such that the at least one fusion protein has increased access to the target chromosomal sequence, thereby increasing the efficiency of the targeted genomic or epigenetic modification.

[0021] In some iterations, the method comprises introducing into a eukaryotic cell: (a) at least one CRISPR fusion protein disclosed herein or a nucleic acid encoding the CRISPR fusion protein, wherein the CRISPR protein has (i) nuclease or nickase activity or (ii) is modified to lack all nuclease activity and is linked to a non-nuclease domain; and (b) at least one guide RNA or a nucleic acid encoding the at least one guide RNA; wherein the CRISPR protein of the at least one CRISPR fusion protein is targeted to a target chromosomal sequence, and the at least one nucleosome interaction protein domain of the at least one CRISPR fusion protein alters the nucleosome or chromatin structure, such that the at least one CRISPR fusion protein has increased access to the target chromosomal sequence, thereby increasing the efficiency of targeted genomic or epigenetic modification.

[0022] In certain embodiments, the method can further comprise introducing into the eukaryotic cell at least one donor polynucleotide, the donor polynucleotide comprising at least one donor sequence.

[0023] The eukaryotic cells used in the methods disclosed herein can be mammalian cells. In some embodiments, the cells can be human cells. The cells can be in vitro or in vivo.

[0024] Other aspects and features of the disclosure are detailed below. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] [Figure 1]Figure 1 shows the cleavage efficiency (as a percentage of indels) of wild-type CjCas9 (CjeCas9), a fusion protein containing CjCas9 ligated to HMGN1 and HMGB1 box A (CjeCas9-HN1HB1), and a fusion protein containing CjCas9 ligated to HMGN1 and histone H1 central globule domain (CjeCas9-HN1H1G) in the presence of a wild-type or modified sgRNA backbone.

[0026] Detailed explanation This disclosure provides compositions and methods for increasing the accessibility of chromosomal DNA to programmable DNA modification proteins, including those involving the CRISPR system. In particular, this disclosure provides a fusion protein comprising at least one nucleosome interacting protein domain ligated to a programmable DNA modification protein. The nucleosome interacting protein domain alters or modifies the nucleosome and / or chromatin structure, resulting in increased access for the programmable DNA modification protein to the targeted chromosomal sequence, thereby increasing the efficiency of targeted genomic modification, targeted transcriptional regulation, or targeted epigenetic modification.

[0027] (I) Fusion protein One aspect of this disclosure provides fusion proteins, each of which comprises at least one nucleosome-interacting protein domain linked to a programmable DNA-modified protein. The programmable DNA-modified protein may have nuclease activity (see Section (I)(b)(i) below) or non-nuclease activity (see Section (I)(b)(ii) below). The nucleosome-interacting protein domains are described in Section (I)(a) below, and the linkages between these domains are described in Section (I)(c) below.

[0028] (a) Nucleosome interacting protein domain The nucleosome-interacting protein domain refers to a chromosomal protein or fragment thereof that interacts with nucleosomes and / or chromosomal proteins to facilitate nucleosome rearrangement and / or chromatin remodeling. In some embodiments, the nucleosome-interacting protein domain may be derived from a high-mobility group (HMG) box (HMGB) protein. In other embodiments, the nucleosome-interacting protein domain may be an HMG nucleosome-binding (HMGN) protein or fragment thereof. In yet another embodiment, the nucleosome-interacting protein domain may be derived from a linker histone H1 variant. In yet another embodiment, the nucleosome-interacting protein domain may be derived from a chromatin remodeling complex protein.

[0029] (i) HMGB protein In some embodiments, at least one nucleosome-interacting protein domain may be derived from an HMGB protein. HMGB proteins interact with nucleosomes and other chromosomal proteins to regulate chromatin structure and function. Suitable HMGB proteins include mammalian HMGB1, mammalian HMGB2, and mammalian HMGB3. For example, the nucleosome-interacting protein domain may be derived from human HMGB1 (RefSeq gene, U51677), human HMGB2 (RefSeq gene, M83665), or human HMGB3 (RefSeq gene, NM_005342). In other embodiments, the nucleosome-interacting protein domain may be derived from an HMGB protein or HMGB-like protein from another vertebrate, invertebrate (e.g., Drosophila DSP1), plant, yeast, or other single-cell eukaryote.

[0030] In specific embodiments, at least one nucleosome-interacting protein domain can be a fragment of the HMGB protein. In particular, the HMGB protein fragment is a DNA-binding domain. Typically, the HMGB protein contains two DNA-binding domains called box A and box B. In some embodiments, the nucleosome-interacting domain can be either the box A domain or the box B domain from the HMGB protein. In specific embodiments, the nucleosome-interacting domain can be the HMGB1 box A domain, the HMGB2 box A domain, or the HMGB3 box A domain.

[0031] (ii) HMGN Protein In other embodiments, at least one nucleosome-interacting protein domain may be an HMGN protein or a fragment thereof. HMGN proteins are chromosomal proteins that regulate the structure and function of chromatin. Suitable mammalian HMGN proteins include HMGN1, HMGN2, HMGN3, HMGN4, and HMGN5. In various embodiments, the nucleosome-interacting protein domain may be human HMGN1 (RefSeq gene, M21339), human HMGN2 (RefSeq gene, X13546), human HMGN3a or human HMGN3b (RefSeq gene, L40357), human HMGN4 (RefSeq gene, NM_030763), human HMGN5 (RefSeq gene, NM_016710), a fragment thereof, or a derivative thereof. In other embodiments, the nucleosome-interacting protein domain may be a non-human HMGN protein, a fragment thereof, or a derivative thereof. HMGN proteins are relatively small proteins. Therefore, the entire HMGN protein can be ligated to a programmable DNA modification protein. However, in some embodiments, a fragment of the HMGN protein (e.g., the centrally located nucleosome-binding domain) can be ligated to the programmable DNA modification protein.

[0032] (iii) Histone H1 variant In yet another embodiment, at least one nucleosome-interacting protein domain may be derived from a linker histone H1 variant. For example, the nucleosome-interacting protein domain may be a central globule domain from a histone H1 variant. The histone H1 variant binds to internucleosome linker DNA, and the central globule domain (about 80 amino acids) binds to the linker DNA at the nucleosome entrance and exit sites near the nucleosome dyad. Histone H1 variants comprise a large family of related proteins that have distinct specificity for tissues, developmental stages, and the organisms in which they are expressed. For example, humans and mice contain 11 histone H1 variants, chickens have 6 variants (called histone H5), frogs have 5 variants, nematodes have 8 variants, fruit fly species have 1 to 3 variants, and tobacco has 6 variants. In some embodiments, the histone H1 variant may be a human variant as shown below. [Table A]

[0033] (iv) Chromatin remodeling complex protein In further embodiments, at least one nucleosome-interacting protein domain may be derived from a chromatin remodeling complex protein. For example, the nucleosome-interacting protein domain may be a DNA-binding domain from a chromatin remodeling complex protein. The chromatin remodeling complex is a multi-subunit enzyme complex that has the ability to remodel the structure of chromatin. These remodeling complexes use the energy of ATP hydrolysis to move, destabilize, evacuate, or remodel nucleosomes.

[0034] An example of a chromatin remodeling complex is SWI / SNF( SWI tch / S ucrose N on- F (ermentable), ISWI ( I mitation SWI tch), CHD( C hromodomain- H elicase- D NA binding), Mi-2 / NuRD( Nu cleosome R emodeling and D The complex includes eacetylase, INO80, SWR1, and RSC complexes. In various embodiments, the nucleosome interacting protein domain may be derived from ATPases, helicases, and / or DNA-binding proteins in the chromatin remodeling complex. In some embodiments, the nucleosome interacting protein domain may be derived from ATPase ISWI from the ISWI complex, DNA-binding protein CHD1 from the CHD complex, ATP-dependent helicase SMARCA4 or ATPase Snf2 from the SWI / SNF complex, ATPase Mi-2α or ATPase Mi2-β from the Mi-1 / NuRD complex, RuvB-like AAAATPase 1 or RuvB-like AAAATPase 2 from the INO80 complex, ATPase Swr1 from the SWR1 complex, or ATPase Rsc1 or ATPase Rcs2 from the RSC complex. In specific embodiments, the nucleosome interacting protein domain may be a DNA-binding domain from the ISWI protein or a DNA-binding domain from the CHD1 protein.

[0035] (b) Programmable DNA modification proteins Programmable DNA modification proteins are proteins targeted to bind to specific sequences in chromosomal DNA, modifying DNA or proteins associated with the targeted sequence, either at or near the target sequence. Therefore, programmable DNA modification proteins include a programmable DNA-binding domain and a catalytically active modification domain.

[0036] The DNA-binding domain of a programmable DNA-modified protein is programmable, meaning it can be designed or manipulated to recognize and bind to different DNA sequences. In some embodiments, for example, DNA binding is mediated by an interaction between the DNA-modified protein and the target DNA. Thus, the DNA-binding domain can be programmed to bind to a desired DNA sequence through protein manipulation. In other embodiments, for example, DNA binding is mediated by a guide RNA that interacts with the DNA-modified protein and the target DNA. In such cases, the programmable DNA-binding protein can be targeted to a desired DNA sequence by designing an appropriate guide RNA.

[0037] Various modification domains can be included in programmable DNA modification proteins. In some embodiments, the modification domain has nuclease activity and can cleave one or both strands of a double-stranded DNA sequence. The DNA disruption can then be repaired by cellular DNA repair processes such as non-homologous end joining (NHEJ) or homology-directed repair (HDR), resulting in the DNA sequence being modified by deletions, insertions, and / or substitutions of at least one base pair. Examples of programmable DNA modification proteins with nuclease activity include, but are not limited to, CRISPR nucleases (or nickases), zinc finger nucleases, transcription activator-like effector nucleases, meganucleases, and programmable DNA-binding domains ligated to nuclease domains. Programmable DNA modification proteins with nuclease activity are described in detail in the following sections (I)(b)(i).

[0038] In other embodiments, the modification domain of a programmable DNA modification protein has non-nuclease activity (e.g., epigenetic modification activity or transcriptional regulatory activity), and as a result, the programmable DNA modification protein modifies the structure and / or activity of DNA and / or related DNA and / or proteins. Thus, a programmable DNA modification protein may include a programmable DNA-binding domain ligated to a non-nuclease domain. Such proteins are described in detail in the following sections (I)(b)(ii).

[0039] Programmable DNA-modified proteins may include wild-type or native DNA-binding and / or modification domains, modified versions of native DNA-binding and / or modification domains, synthetic or artificial DNA-binding and / or modification domains, and combinations thereof.

[0040] (i) Programmable DNA modification protein with nuclease activity Examples of programmable DNA-modified proteins with nuclease activity include, but are not limited to, CRISPR nucleases, zinc finger nucleases, activator-like effector nucleases, meganucleases, and programmable DNA-binding domains ligated to nuclease domains.

[0041] CRISPR nucleaseThe CRISPR nuclease can be derived from type I, type II (i.e., Cas9), type III, type V (i.e., Cpf1), or type VI (i.e., Cas13) CRISPR proteins present in various bacteria and archaea. In further embodiments, the CRISPR nuclease can be derived from the archaeal CRISPR system, CRISPR / CasX system, or CRISPR / CasY system (Burstein et al., Nature, 2017, 542(7640):237-241).In the present study, CRISPR genes from Streptococcus sp. pasteurianus) Campylobacter sp. (Campylobacter jejuni) Francisella sp. (Francisella novicida) Acaryochloris sp., Acetohalobium sp., Acidaminococcus sp., Acidithiobacillus sp., Alicyclobacillus sp., Allochromatium sp., Ammonifex sp., Anabaena sp., Arthrospira sp. Bacillus sp. Burkholderiales sp. Caldicellulosiruptor sp. Candidatus sp. Clostridium sp. Crocosphaera sp. Cyanothece sp. Exiguobacterium sp. Finegoldia sp. Ctedonobacter sp. Lachnospiraceae sp. Lactobacillus sp. Lyngbya sp. Marinobacter sp. Methanohalobium sp. Microscilla sp. Microcoleus sp. Microcystis sp. Natranaerobius sp. Neisseria sp. Nitrosococcus sp sp. Nodularia sp. Nostoc sp. Oscillatoria sp. Polaromonas sp. Pelotomaculum sp. Pseudoalteromonas sp. Petrotoga sp. Prevotella sp. Staphylococcus sp. Streptomyces sp. Streptomyces sp sp., Synechococcus sp., Thermosipho sp., Verrucomicrobia sp.

[0042] CRISPR nucleases can be wild-type or naturally occurring proteins. Alternatively, CRISPR nucleases can be engineered to have improved specificity, altered PAM specificity, reduced off-target effects, increased stability, etc.

[0043] In some embodiments, the CRISPR nuclease can be a type II CRISPR / Cas9 protein. For example, the CRISPR nuclease can be Streptococcus pyogenes Cas9 (SpCas9), Streptococcus thermophilus Cas9 (StCas9), Streptococcus pasteurianus (SpaCas9), Campylobacter jejuni Cas9 (CjCas9), Staphylococcus aureus (SaCas9), Francisella novicida Cas9 (FnCas9), Neisseria cinerea Cas9 (NcCas9), or Neisseria meningitis Cas9 (NmCas9). In other embodiments, the CRISPR nuclease may be a type V CRISPR / Cpf1 protein, such as Francisella novicida Cpf1 (FnCpf1), Acidaminococcus sp. Cpf1 (AsCpf1), or Lachnospiraceae bacterium ND2006 Cpf1 (LbCpf1). In further embodiments, the CRISPR nuclease may be a type VI CRISPR / Cas13 protein, such as Leptotrichia wadei Cas13a (LwaCas13a) or Leptotrichia shahii Cas13a (LshCas13a).

[0044] Generally, CRISPR nucleases contain at least one nuclease domain having endonuclease activity. For example, Cas9 nuclease contains an HNH domain that cleaves the complementary guide RNA strand and a RuvC domain that cleaves the non-complementary strand; Cpf1 protein contains a RuvC domain and a NUC domain; and Cas13a nuclease contains two HNEPN domains. In some embodiments, both nuclease domains are active, and the CRISPR nuclease has double-strand cleavage activity (i.e., cleaves both strands of a double-stranded nucleic acid sequence). In other embodiments, one of the nuclease domains is inactivated by one or more mutations and / or deletions, and the CRISPR variant is a nickasase that cleaves one strand of a double-stranded nucleic acid sequence. For example, one or more mutations in the RuvC domain of the Cas9 protein (e.g., D10A, D8A, E762A, and / or D986A) result in HNH nickases that nick the complementary guide RNA strand; one or more mutations in the HNH domain of the Cas9 protein (e.g., H840A, H559A, N854A, N856A, and / or N863A) result in RuvC nickases that nick the non-complementary guide RNA strand. Equivalent mutations can convert Cpf1 and Cas13a nucleases into nickases.

[0045] Zinc finger nuclease In yet another embodiment, a programmable DNA-modified protein having nuclease activity may be a pair of zinc finger nucleases (ZFNs). A ZFN comprises a DNA-binding zinc finger region and a nuclease domain. The zinc finger region may contain about 2 to 7 zinc fingers, for example, about 4 to 6 zinc fingers, where each zinc finger binds to three consecutive base pairs. The zinc finger region can be engineered to recognize and bind to any DNA sequence. Zinc finger design tools or algorithms are available on the internet or from commercial sources. Zinc fingers can be linked together using a suitable linker sequence.

[0046] ZFNs also include nuclease domains that can be obtained from any endonuclease or exonuclease. Non-limiting examples of endonucleases from which the nuclease domain can be derived include, but are not limited to, restriction endonucleases and homing endonucleases. In some embodiments, the nuclease domain can be derived from a type II-S restriction endonuclease. Type II-S endonucleases cleave DNA at a site typically several base pairs away from the recognition / binding site and therefore have separable binding and cleavage domains. These enzymes are generally monomers that transiently bind to form a dimer and cleave each strand of DNA at alternating positions. Non-limiting examples of suitable type II-S endonucleases include BfiI, BpmI, BsaI, BsgI, BsmBI, BsmI, BspMI, FokI, MboII, and SapI. In some embodiments, the nuclease domain may be a FokI nuclease domain or a derivative thereof. The type II-S nuclease domain may be modified to promote the dimerization of two different nuclease domains. For example, the cleavage domain of FokI may be modified by mutating specific amino acid residues. As a non-limiting example, amino acid residues at positions 446, 447, 479, 483, 484, 486, 487, 490, 491, 496, 498, 499, 500, 531, 534, 537, and 538 of the FokI nuclease domain are targets for modification. In specific embodiments, the FokI nuclease domain may include a first FokI half-domain containing the Q486E, I499L, and / or N496D mutations, and a second FokI half-domain containing the E490K, I538K, and / or H537R mutations. In some embodiments, the ZFN has double-strand cleavage activity. In other embodiments, the ZFN has nickase activity (i.e., one of the nuclease domains is inactivated).

[0047] Transcription activator-like effector nucleaseIn an alternative embodiment, a programmable DNA modification protein having nuclease activity may be a transcription activator-like effector nuclease (TALEN). TALEN contains a DNA-binding domain consisting of highly conserved repeats derived from a transcription activator-like effector (TALE) ligated to a nuclease domain. TALE is a protein secreted by the plant pathogen Xanthomonas that alters gene transcription in host plant cells. TALE repeat arrays can be manipulated via modular protein design to target any desired DNA sequence. The nuclease domain of TALEN can be any nuclease domain, as described above in the subsection describing ZFNs. In a specific embodiment, the nuclease domain is derived from FokI (Sanjana et al., 2012, Nat Protoc, 7(1):171-192). TALEN may have double-strand cleavage activity or nickase activity.

[0048] Meganuclease or rare endonucleaseIn further embodiments, the programmable DNA modification protein having nuclease activity may be a meganuclease or a derivative thereof. A meganuclease is an endodeoxyribonuclease characterized by a long recognition sequence, i.e., generally ranging from about 12 to about 45 base pairs. As a result of this requirement, the recognition sequence generally occurs only once in any given genome. Among meganucleases, the family of homing endonucleases named LAGLIDADG has become a valuable tool for genome research and genome manipulation. In some embodiments, the meganuclease may be I-SceI, I-TevI, or variants thereof. The meganuclease may be targeted to a specific chromosomal sequence by modifying its recognition sequence using techniques well known to those skilled in the art. In alternative embodiments, the programmable DNA modification protein having nuclease activity may be a rare cleavage endonuclease or a derivative thereof. Rare cleavage endonucleases are site-specific endonucleases whose recognition sequences rarely occur in the genome, preferably only once in the genome. Rare cleavage endonucleases may recognize 7-nucleotide sequences, 8-nucleotide sequences, or longer recognition sequences. Non-limiting examples of rare cleavage endonucleases include NotI, AscI, PacI, AsiSI, SbfI, and FseI.

[0049] A programmable DNA-binding domain that is linked to the nuclease domain.In further embodiments, a programmable DNA-modified protein having nuclease activity may be a chimeric protein comprising a programmable DNA-binding domain ligated to a nuclease domain. The nuclease domain may be any of those described above in the subsection describing nuclease domains derived from meganucleases or rare cleavage endonucleases, such as a ZFN (e.g., the nuclease domain may be a FokI nuclease domain), a CRISPR nuclease (e.g., the RuvC or HNH nuclease domain of Cas9), or a meganuclease or rare cleavage endonuclease.

[0050] The programmable DNA-binding domain of a chimeric protein can be any programmable DNA-binding protein, such as a zinc finger protein or a transcription activator-like effector. Alternatively, the programmable DNA-binding domain can be a catalytically inactive (dead) CRISPR protein modified by deletion or mutation to lack all nuclease activity. For example, the catalytically inactive CRISPR protein could be a catalytically inactive (dead) Cas9 (dCas9) whose RuvC domain contains D10A, D8A, E762A, and / or D986A mutations, and whose HNH domain contains H840A, H559A, N854A, N865A, and / or N863A mutations. Alternatively, the catalytically inactive CRISPR protein could be a catalytically inactive (dead) Cpf1 protein containing equivalent mutations in the nuclease domain. In yet another embodiment, the programmable DNA-binding domain may be a catalytically inactive meganuclease, which may include, for example, a C-terminal truncation, in which nuclease activity has been removed by mutation and / or deletion.

[0051] (ii) Programmable DNA modification protein with non-nuclease activity In an alternative embodiment, the programmable DNA-modified protein may be a chimeric protein containing a programmable DNA-binding domain linked to a non-nuclease domain. The programmable DNA-binding domain may be a zinc finger protein, a transcription activator-like effector, a catalytically inactive (dead) CRISPR protein, or a catalytically inactive (dead) meganuclease. For example, the catalytically inactive CRISPR protein may be a catalytically inactive (dead) Cas9 (dCas9) in which the RuvC domain contains D10A, D8A, E762A, and / or D986A mutations and the HNH domain contains H840A, H559A, N854A, N865A, and / or N863A mutations. Alternatively, the catalytically inactive CRISPR protein may be a catalytically inactive (dead) Cpf1 protein containing equivalent mutations in the nuclease domain.

[0052] In some embodiments, the non-nuclease domain of a chimeric protein can be an epigenetic modification domain that alters (and may or may not alter) the DNA or chromatin structure and DNA sequence. Non-limiting examples of suitable epigenetic modification domains include DNA methyltransferase activity (e.g., cytosine methyltransferase), DNA demethylase activity, DNA deamination (e.g., cytosine deaminase, adenosine deaminase, guanine deaminase), DNA amination, DNA oxidation activity, DNA helicase activity, histone acetyltransferase (HAT) activity (e.g., E1A-binding protein p300 derived from the HAT domain), histone deacetylase activity, histone methyltransferase activity, and histone methyltransferase activity. This includes those having lanceferase activity, histone demethylase activity, histone kinase activity, histone phosphatase activity, histone ubiquitin ligase activity, histone deubiquitination activity, histone adenylation activity, histone dedenylation activity, histone SUMOylation activity, histone deSUMOylation activity, histone ribosylation activity, histone deribosylation activity, histone myristoylation activity, histone demyristoylation activity, histone citrustone alkylation activity, histone dealkylation activity, or histone oxidation activity. In specific embodiments, the epigenetic modification domain may include cytidine deaminase activity, histone acetyltransferase activity, or DNA methyltransferase activity.

[0053] In other embodiments, the non-nuclease-modified domain of the chimeric protein may be a transcriptional activation domain or a transcriptional repressor domain. Suitable transcriptional activation domains include, but are not limited to, the herpes simplex virus VP16 domain, VP64 (a tetrameric derivative of VP16), VP160, the NFκBp65 activation domain, p53 activation domains 1 and 2, the CREB (cAMP response element binding protein) activation domain, the E2A activation domain, the activation domain from human heat shock factor 1 (HSF1), or the NFAT (nuclear factor of activated T cells) activation domain. Non-limited examples of suitable transcriptional repressor domains include the inducible cAMP initial repressor (ICER) domain, the Kruppel-associated box (KRAB) repressor domain, the YY1 glycine-rich repressor domain, the Sp1-like repressor, the E(spl) repressor, the IκB repressor, or the methyl-CpG binding protein 2 (MeCP2) repressor. Transcriptional activator or repressor domains can be genetically fused to or bound to DNA-binding proteins via non-covalent protein-protein, protein-RNA, or protein-DNA interactions.

[0054] In certain embodiments, the non-nuclease domain of the chimeric protein may include cytidine deaminase activity, histone acetyltransferase activity, transcriptional activating activity, or transcriptional repressor activity.

[0055] In some embodiments, the non-nuclease activity chimeric protein may further include at least one detectable label. The detectable label may be a fluorophore (e.g., FAM, TMR, Cy3, Cy5, Texas Red, Oregon Green, Alexa Fluor, Halo tag, or appropriate fluorescent dye), a detection tag (e.g., biotin, digoxigenin, etc.), a quantum dot, or a gold particle.

[0056] (c) Consolidation The fusion proteins disclosed herein include at least one nucleosome-interacting protein domain linked to a programmable DNA-modified protein. The linkage between the at least one nucleosome-interacting protein domain and the programmable DNA-modified protein may be direct via a chemical bond, or it may be indirect via a linker.

[0057] In some embodiments, at least one nucleosome-interacting protein domain can be directly linked to a programmable DNA-modified protein by a covalent bond (e.g., peptide bond, ester bond, etc.). Alternatively, the chemical bond can be non-covalent (e.g., ionic, electrostatic, hydrogen, hydrophobic, van der interaction, or π effect).

[0058] In other embodiments, at least one nucleosome-interacting protein domain can be linked to a DNA-modified protein programmable by a linker. The linker is a chemical group that connects one or more other chemical groups via at least one covalent bond. Suitable linkers include amino acids, peptides, nucleotides, nucleic acids, organic linker molecules (e.g., maleimide derivatives, N-ethoxybenzylimidazole, biphenyl-3,4',5-tricarboxylic acid, p-aminobenzyloxycarbonyl, etc.), disulfide linkers, and polymer linkers (e.g., PEG). The linker may contain one or more spacing groups, including but not limited to alkylene, alkenylene, alkynylene, alkyl, alkenyl, alkynyl, alkoxy, aryl, heteroaryl, aralkyl, aralkenyl, aralquinyl, etc. The linker may be neutral or carry a positive or negative charge. In addition, linkers are cleavable, and the covalent bonds of linkers that connect them to other chemical groups can be broken or cleaved under certain conditions, including pH, temperature, salt concentration, light, catalysts, or enzymes.

[0059] In yet another embodiment, at least one nucleosome-interacting protein domain can be linked to a DNA-modified protein programmable by a peptide linker. The peptide linker can be a flexible amino acid linker (e.g., containing small, nonpolar, or polar amino acids). Non-limiting examples of flexible linkers include LEGGGS (SEQ ID NO: 1), TGSG (SEQ ID NO: 2), GGSGGGSG (SEQ ID NO: 3), and (GGGGS). 1-4 (Sequence ID: 4), and (Gly) 6-8 (Sequence ID: 5) is included. Alternatively, the peptide linker can be a rigid amino acid linker. Such a linker is (EAAAK) 1-4 (Sequence ID: 6), A(EAAAK) 2-5 A (Sequence ID: 7), PAPAP (Sequence ID: 8), and (AP) 6-8 (Sequence ID: 9) is included. Examples of suitable linkers are well known in the art, and programs for designing linkers are readily available (Crasto et al., Protein Eng., 2000, 13(5):309-312).

[0060] At least one nucleosome-interacting protein domain can be ligated to the N-terminus, C-terminus, and / or internal position of a programmable DNA-modified protein. In some embodiments, at least one nucleosome-interacting protein domain can be ligated to the N-terminus of a programmable DNA-modified protein. In other embodiments, at least one nucleosome-interacting protein domain can be ligated to the C-terminus of a programmable DNA-modified protein. In yet another embodiment, at least one nucleosome-interacting protein domain can be ligated to the N-terminus, and at least one nucleosome-interacting protein domain can be ligated to the C-terminus of a programmable DNA-modified protein.

[0061] In some embodiments, the fusion protein may contain one nucleosome-interacting protein domain. In other embodiments, the fusion protein may contain two nucleosome-interacting protein domains. In yet another embodiment, the fusion protein may contain three nucleosome-interacting protein domains. In an additional embodiment, the fusion protein may contain four, five, or more than five nucleosome-interacting protein domains. One or more nucleosome-interacting protein domains may be the same or different.

[0062] In embodiments in which the fusion protein comprises two or more nucleosome-interacting protein domains, the two or more nucleosome-interacting domains can be ligated to any end, both ends, and / or internal position of a programmable DNA-modified protein. The two or more nucleosome-interacting protein domains may be the same or different. For example, the complex may include at least two HMGDNA-binding domains, at least two HMGN proteins, at least one HMGDNA-binding domain and at least one HMGN protein, at least one central domain from an HMGDNA-binding domain or HMGN protein and a histone H1 variant, at least one HMGDNA-binding domain or HMGN protein, at least one histone H1 variant central domain, and at least one domain from a chromatin remodeling complex protein, and at least one domain from a chromatin remodeling complex protein, etc.

[0063] (d) Desired nuclear localization signal, cell permeability domain, and / or marker domain The fusion proteins disclosed herein may further comprise at least one nuclear localization signal, a cell permeability domain, and / or a marker domain.

[0064] Non-exclusive examples of nuclear localization signals include: PKKKRKV (SEQ ID NO: 10), PKKKRRV (SEQ ID NO: 11), KRPAATKKAGQAKKKK (SEQ ID NO: 12), YGRKKRRQRRR (SEQ ID NO: 13), RKKRRQRRR (SEQ ID NO: 14), PAAKRVKLD (SEQ ID NO: 15), RQRRNELKRSP (SEQ ID NO: 16), VSRKRPRP (SEQ ID NO: 17), PPKKARED (SEQ ID NO: 18), PQPKKKPL (SEQ ID NO: 19), SALIKKKKKMA P (SEQ ID NO: 20), PKQKKRK (SEQ ID NO: 21), RKLKKKIKKL (SEQ ID NO: 22), REKKKFLKRR (SEQ ID NO: 23), KRKGDEVDGVDEVAKKKSKK (SEQ ID NO: 24), RKCLQAGMNLEARKTKK (SEQ ID NO: 25), NQSSNFGPMKGGNFGGRSSGPYGGGGQYFAKPRNQGGY (SEQ ID NO: 26), and RMRIZFKNKGKDTAELRRRRVEVSVELRKAKKDEQILKRRNV (SEQ ID NO: 27).

[0065] Examples of suitable cell permeability domains include, but are not limited to, GRKKRRQRRRPPQPKKKRKV (SEQ ID NO: 28), PLSSIFSRIGDPPKKKRKV (SEQ ID NO: 29), GALFLGWLGAAGSTMGAPKKKRKVV (SEQ ID NO: 30), GALFLGFLGAAGSTMGAWSQPKKKRKV (SEQ ID NO: 31), KETWWET (SEQ ID NO: 32), YARAAARQARA (SEQ ID NO: 33), THRLPRRRRRR (SEQ ID NO: 34), GGRRARRRRRR (SEQ ID NO: 35), RRQRRTSKLMKR (SEQ ID NO: 36), GWTLNSAGYLLGKINLKALAALAKKIL (SEQ ID NO: 37), KALAWEAKLAKALAKALAKHLAKA (SEQ ID NO: 38), and RQIKIWFQNRRMKWKK (SEQ ID NO: 39).

[0066] The marker domain includes a fluorescent protein and a purified or epitope tag. Suitable fluorescent proteins include, but are not limited to, the following: green fluorescent proteins (e.g., GFP, eGFP, GFP-2, tagged GFP, turboGFP, Emerald, Azami Green, Monomeric Azami) Green, CopGFP, AceGFP, ZsGreen1), yellow fluorescent proteins (e.g., YFP, EYFP, Citrine, Venus, YPet, PhiYFP, ZsYellow1), blue fluorescent proteins (e.g., BFP, EBFP, EBFP2, Azurite, mKalama1, GFPuv, Sapphire, T-sapphire), cyan fluorescent proteins (e.g., ECFP, Cerulean, CyPet, AmCyan1, Midoriishi-Cyan), red fluorescent proteins (e.g., mKate, mKate2, mPlum, DsRed monomer, mCherry, mRFP1, DsRed-Express, DsRed2, DsRed-Monomer, HcRed-Tandem, HcRed1, AsRed2, eqFP611, mRaspberry, mStrawberry, Jred), and orange fluorescent proteins (e.g., mOrange, mKO, Kusabira-Orange, Monomermeric) (Kusabira-Orange, mTangerine, tdTomato). Appropriate purification or non-exclusive examples of epitope tags include 6xHis, FLAG®, HA, GST, Myc, etc.

[0067] At least one nuclear localization signal, a cell permeability domain, and / or a marker domain can be located at the N-terminus, C-terminus, and / or internal position of the fusion protein.

[0068] (e) Specific fusion protein Generally, at least one nucleosome-interacting protein domain of a fusion protein is selected from the HMGB1 box A domain, HMGN1 protein, HMGN2 protein, HMGN3a protein, HMGN3b protein, histone H1 central globule domain, mimic switch (ISWI) protein DNA-binding domain, chromodomain-helicase-DNA protein 1 (CHD1) DNA-binding domain, or a combination thereof.

[0069] In specific embodiments, the programmable DNA modification protein of the fusion protein is a CRISPR protein. For example, the CRISPR protein may be Streptococcus pyogenes Cas9 (SpCas9), Streptococcus thermophilus Cas9 (StCas9), Streptococcus pasteurianus (SpaCas9), Campylobacter jejuni Cas9 (CjCas9), Staphylococcus aureus (SaCas9), Francisella novicida Cas9 (FnCas9), Neisseria cinerea Cas9 (NcCas9), Neisseria meningitis Cas9 (NmCas9), Francisella novicida Cpf1 (FnCpf1), Acidaminococcus sp. Cpf1 (AsCpf1), or Lachnospiraceae bacterium ND2006 Cpf1 (LbCpf1).

[0070] In some embodiments, the fusion protein has an amino acid sequence that has at least about 80% sequence identity with any of SEQ ID NOs: 61-79. Generally, any amino acid substitutions are conserved, i.e., limited to substitutions within members of Group 1: glycine, alanine, valine, leucine, and isoleucine; Group 2: serine, cysteine, threonine, and methionine; Group 3: proline; Group 4: phenylalanine, tyrosine, and tryptophan; and Group 5: aspartic acid, glutamic acid, asparagine, and glutamine. In various embodiments, the amino acid sequence of the fusion protein has at least about 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with any of SEQ ID NOs: 61-79. In some embodiments, the fusion protein has the amino acid sequence described in SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78, or SEQ ID NO: 79.

[0071] (II) Complex Another aspect of this disclosure encompasses a complex comprising at least one CRISPR system (i.e., a CRISPR protein and a guide RNA) and at least one nucleosome-interacting protein domain. In some embodiments, at least one nucleosome-interacting protein domain can be ligated to the CRISPR protein of the CRISPR system (i.e., a complex comprising the CRISPR fusion protein described above in Section (I)). In other embodiments, at least one nucleosome-interacting protein domain can be ligated to the guide RNA of the CRISPR system. Such ligation can be essentially direct or indirect, as described above in Section (I)(c). For example, a nucleosome-interacting protein domain can be ligated to an RNA aptamer-binding protein, and the guide RNA can contain an aptamer sequence, so that the binding of the aptamer-binding protein to the RNA aptamer sequence causes the nucleosome-interacting protein domain to be ligated to the guide RNA.

[0072] Nucleosome-interacting protein domains are described above in section (I)(a), and CRISPR proteins are described above in detail in section (I)(b). CRISPR proteins can have nuclease or nickase activity (e.g., they can be type II CRISPR / Cas9, type V CRISPR / Cpf1, or type VI CRISPR / Cas13). For example, a complex can contain a CRISPR nuclease, or a complex can contain two CRISPR nickases. Alternatively, CRISPR proteins can be modified to lack all nuclease activity and can be linked to a non-nuclease-active domain (e.g., a domain having cytosine deaminase activity, histone acetyltransferase activity, transcriptional activating activity, or transcriptional repressor activity). In some embodiments, the non-nuclease domain can also be linked to an RNA aptamer-binding protein.

[0073] Guide RNAs contain (i) a CRISPRRNA (crRNA) with a guide sequence at its 5' end that hybridizes with the target sequence, and (ii) a transacting crRNA (tracrRNA) sequence that interacts with the CRISPR protein. The crRNA guide sequence for each guide RNA is different (i.e., sequence-specific). The tracrRNA sequence is generally the same in guide RNAs designed to complex with CRISPR proteins from specific bacterial species.

[0074] The crRNA guide sequence is designed to hybridize with a target sequence (i.e., a protospacer) adjacent to a protospacer-adjacent motif (PAM) in the double-stranded sequence. The PAM sequence for the Cas9 protein includes 5'-NGG, 5'-NGGNG, 5'-NNAGAAW, and 5'-ACAY, while the PAM sequence for Cpf1 includes 5'-TTN (where N is defined as any nucleotide, W is defined as either A or T, and Y is defined as either C or T). Generally, the complementarity between the crRNA guide sequence and the target sequence is at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%. In specific embodiments, the complementarity is perfect (i.e., 100%). In various embodiments, the length of the crRNA guide sequence can range from about 15 nucleotides to about 25 nucleotides. For example, the crRNA guide sequence can be about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides long. In a specific embodiment, the crRNA can be about 19, 20, 21, or 22 nucleotides long.

[0075] crRNA and tracrRNA contain repetitive sequences that form one or more stem-loop structures capable of interacting with CRISPR proteins. The length of each loop and stem can vary. For example, one or more loops can range in length from about 3 to about 10 nucleotides, and one or more stems can range in length from about 6 to about 20 base pairs. One or more stems can contain one or more bulges ranging from 1 to about 10 nucleotides.

[0076] crRNA can be in the range of about 25 to about 100 nucleotides in length. In various embodiments, crRNA can be in the range of about 25 to about 50 nucleotides, about 590 to about 75 nucleotides, or about 75 to about 100 nucleotides in length. tracrRNA can be in the range of about 50 to about 300 nucleotides in length. In various embodiments, tracrRNA can be in the range of about 50 to about 90 nucleotides, about 90 to about 110 nucleotides, about 110 to about 130 nucleotides, about 130 to about 150 nucleotides, about 150 to about 170 nucleotides, about 170 to about 200 nucleotides, about 200 to about 250 nucleotides, or about 250 to about 300 nucleotides in length.

[0077] Generally, the tracrRNA sequence in the guide RNA is based on the coding sequence of the wild-type tracrRNA in the target bacterial species. In some embodiments, the wild-type tracrRNA sequence (or the constant crRNA repeat region and the corresponding 5' region of the tracrRNA that forms a duplex with the constant crRNA repeat region) can be modified to promote secondary structure formation, increase secondary structure stability, enhance expression in eukaryotic cells, increase editing efficiency, etc. For example, one or more nucleotide changes can be introduced into the constant guide RNA sequence (see Example 8 below).

[0078] The guide RNA can be a single molecule (i.e., a single guide RNA or sgRNA), where the crRNA sequence is ligated to a tracrRNA sequence. Alternatively, the guide RNA can be two distinct molecules. The first molecule contains a crRNA guide sequence at its 5' end and an additional sequence at its 3' end that can base pair with the 5' end of the second molecule, where the second molecule contains a 5' sequence that can base pair with the 3' end of the first molecule, as well as an additional tracrRNA sequence. In some embodiments, the guide RNA for a type V CRISPR / Cpf1 system may contain only crRNA.

[0079] In some embodiments, one or more stem-loop regions of the guide RNA can be modified to include one or more aptamer sequences (Konermann et al., Nature, 2015, 517(7536):583-588; Zalatan et al., Cell, 2015, 160(1-2):339-50). Examples of suitable RNA aptamer protein domains include proteins derived from bacteriophages such as MS2 coat protein (MCP), PP7 bacteriophage coat protein (PCP), Mu bacteriophage Com protein, lambda bacteriophage N22 protein, stem-loop binding protein (SLBP), fragile X intellectual disability syndrome-associated protein 1 (FXR1), AP205, BZ13, f1, f2, fd, fr, ID2, JP34 / GA, JP501, JP34, JP500, KU1, M11, M12, MX1, NL95, PP7, φCb5, φCb8r, φCb12r, φCb23r, Qβ, R17, SP-β, TW18, TW19, and VK, as well as their fragments or derivatives. The length of the additional aptamer sequence can range from approximately 20 to approximately 200 nucleotides.

[0080] Guide RNA may include standard ribonucleotides, modified ribonucleotides (e.g., pseudouridine), ribonucleotide isomers, and / or ribonucleotide analogs. In some embodiments, the guide RNA may further include at least one detectable label. The detectable label may be a fluorophore (e.g., FAM, TMR, Cy3, Cy5, Texas Red, Oregon Green, Alexa Fluor, Halo tag, or appropriate fluorescent dye), a detection tag (e.g., biotin, digoxigenin, etc.), a quantum dot, or a gold particle. Those skilled in the art are familiar with the design and construction of gRNAs; for example, gRNA design tools are available on the internet or from commercial sources.

[0081] Guide RNA can be chemically synthesized, enzymatically synthesized, or a combination of both. For example, guide RNA can be synthesized using a standard phosphoramidite-based solid-phase synthesis method. Alternatively, guide RNA can be synthesized in vitro by operably ligating the DNA encoding the guide RNA to a promoter control sequence recognized by phage RNA polymerase. Examples of suitable phage promoter sequences include the T7, T3, SP6 promoter sequences, or variations thereof. In embodiments in which the guide RNA comprises two distinct molecules (i.e., crRNA and tracrRNA), crRNA can be chemically synthesized and tracrRNA can be enzymatically synthesized.

[0082] (III) Nucleic acid Further aspects of this disclosure provide nucleic acids encoding the fusion proteins described above in Section (I) and the CRISPR complexes described above in Section (II). The CRISPR complex may be encoded by a single nucleic acid or multiple nucleic acids. The nucleic acids may be DNA or RNA, linear or round, single-stranded or double-stranded. The RNA or DNA may be codon-optimized for efficient translation into proteins in the eukaryotic cell of interest. Codon optimization programs are available as freeware or from commercial sources.

[0083] In some embodiments, the nucleic acid encoding the fusion protein or protein component of the CRISPR complex can be RNA. RNA can be enzymatically synthesized in vitro. Therefore, the DNA encoding the protein of interest can be operably ligated to a promoter sequence recognized by phage RNA polymerase for in vitro RNA synthesis. For example, the promoter sequence can be a T7, T3, or SP6 promoter sequence or a variation thereof. As detailed below, the protein-encoding DNA can be part of a vector. In such embodiments, the in vitro transcribed RNA can be purified, capped, and / or polyadenylated. In other embodiments, the RNA encoding the fusion protein or protein component of the complex can be part of a self-replicating RNA (Yoshioka et al., Cell Stem Cell, 2013, 13:246-254). Self-replicating RNA can be derived from non-infectious, self-replicating Venezuelan equine encephalitis (VEE) virus RNA replicons, which are self-replicating positive-strand single-strand RNAs capable of self-replicating for a limited number of cell divisions and can be modified to encode a protein of interest (Yoshioka et al., Cell Stem Cell, 2013, 13:246-254).

[0084] In other embodiments, the nucleic acids encoding the CRISPR protein and guide RNA of the fusion protein or complex may be DNA. The DNA coding sequence may be operably ligated to at least one promoter control sequence for expression in the cell of interest. In certain embodiments, the DNA coding sequence may be operably ligated to a promoter sequence for the expression of a protein or RNA in bacterial (e.g., E. coli) cells or eukaryotes (e.g., yeast, insects, or mammals). Suitable bacterial promoters include, but are not limited to, the T7 promoter, the lac operon promoter, the trp promoter, the tac promoter (a hybrid of the trp and lac promoters), any variation of the foregoing, and any combination thereof. Non-limiting examples of suitable eukaryotic PolII promoters include constitutive, regulated, or cell- or tissue-specific promoters. Appropriate eukaryotic constitutive promoter regulatory sequences include, but are not limited to, the cytomegalovirus early promoter (CMV), Simian virus (SV40) promoter, adenovirus major late promoter, Rous sarcoma virus (RSV) promoter, mouse mammary tumor virus (MMTV) promoter, phosphoglycerate kinase (PGK) promoter, elongation factor (ED1)-alpha promoter, ubiquitin promoter, actin promoter, tubulin promoter, immunoglobulin promoter, fragments thereof, or any combination thereof. Examples of appropriate eukaryotic regulatory promoter regulatory sequences include, but are not limited to, those regulated by heat shock, metals, steroids, antibiotics, or alcohol.Non-limiting examples of tissue-specific promoters include the B29 promoter, CD14 promoter, CD43 promoter, CD45 promoter, CD68 promoter, desmin promoter, elastase-1 promoter, endoglin promoter, fibronectin promoter, Flt-1 promoter, GFAP promoter, GPIIb promoter, ICAM-2 promoter, INF-β promoter, Mb promoter, NphsI promoter, OG-2 promoter, SP-B promoter, SYN1 promoter, and WASP promoter. Promoter sequences may be wild-type or modified for more efficient or effective expression. In some embodiments, the DNA coding sequence may also be ligated to a polyadenylation signal (e.g., SV40 polyA signal, bovine growth hormone (BGH) polyA signal, etc.) and / or at least one transcription termination sequence. The sequence encoding the guide RNA is operably ligated to a Pol III promoter regulatory sequence for expression in eukaryotic cells. Examples of suitable Pol III promoters include, but are not limited to, the mammalian U6, U3, H1, and 7SL RNA promoters. In some cases, the fusion proteins or components of the complex can be purified from bacteria or eukaryotic cells.

[0085] In various embodiments, the nucleic acid encoding the complex fusion protein or CRISPR protein and guide RNA can be present in the vector. Suitable vectors include plasmid vectors, viral vectors, and self-replicating RNA (Yoshioka et al., Cell Stem Cell, 2013, 13:246-254). In some embodiments, the nucleic acid encoding the complex fusion protein or component can be present in a plasmid vector. Non-limiting examples of suitable plasmid vectors include pUC, pBR322, pET, pBluescript, and their variants. In other embodiments, the nucleic acid encoding the complex fusion protein or component can be part of a viral vector (e.g., lentiviral vector, adeno-associated virus vector, adenovirus vector, etc.). The plasmid or viral vector may include additional expression control sequences (e.g., enhancer sequences, Kozak sequences, polyadenylation sequences, transcription termination sequences, etc.), selectable marker sequences (e.g., antibiotic resistance genes), replication origin, etc. Additional information regarding the vectors and their use can be found in "Current Protocols in Molecular Biology" Ausubel et al., John Wiley & Sons, New York, 2003, or "Molecular Cloning: A Laboratory Manual" Sambrook & Russell, Cold Spring Harbor Press, Cold Spring Harbor, NY, 3rd edition, 2001.

[0086] (IV) Kit Further aspects of this disclosure provide a kit comprising at least one of the fusion proteins detailed above in Section (I), at least one of the CRISPR complexes described above in Section (II), and / or at least one of the nucleic acids described above in Section (III). The kit may further include nucleic acid delivery reagents, cell growth media, selective media, in vitro transcription reagents, nucleic acid purification reagents, protein purification reagents, buffers, etc. The kits provided herein generally include instructions for performing the methods detailed below. The instructions included in the kit may be attached to the packaging or included as accompanying documentation. The instructions are typically written or printed, but are not limited thereto. Any medium on which such instructions can be stored and communicated to end users is intended by this disclosure. Such mediums include, but are not limited to, electronic storage media (e.g., magnetic disks, tapes, cartridges, chips), optical media (e.g., CD-ROMs), etc. The term “instructions” as used herein may include the address of an internet site providing the instructions.

[0087] (V) Cell The disclosure also provides a cell comprising at least one of the fusion proteins detailed above in Section (I), at least one of the CRISPR complexes described above in Section (II), and / or at least one of the nucleic acids described above in Section (III). Generally, the cell is a eukaryotic cell. For example, the cell may be a human cell, a non-human mammalian cell, a non-mammalian vertebrate cell, an invertebrate cell, an insect cell, a plant cell, a yeast cell, or a single-cell eukaryote.

[0088] (VI) Methods for increasing the efficiency of targeted genome, transcription, or epigenetic modifications Another aspect of this disclosure encompasses methods for increasing the efficiency of targeted genomic modifications, targeted transcriptional modifications, or targeted epigenetic modifications in eukaryotic cells by increasing the accessibility of programmable DNA modification proteins to their target sequences in chromosomal DNA. In some embodiments, the method comprises introducing at least one of the fusion proteins described above in Section (I), at least one of the CRISPR complexes described above in Section (II), or a nucleic acid encoding at least one of the fusion proteins or CRISPR complexes described above in Section (III), and optionally a donor polynucleotide into a target eukaryotic cell.

[0089] The programmable DNA modification protein of the fusion protein is engineered to recognize and bind to a target sequence of chromosomal DNA, and one or more nucleosome-interacting protein domains of the fusion protein interact with nucleosomes at or near the target sequence, altering or modifying the nucleosome and / or chromatin structure. As a result, the DNA modification protein has increased access to the target chromosomal sequence, and consequently the efficiency of modification by the DNA modification protein is increased. In specific embodiments, the fusion protein includes at least one nucleosome-interacting protein domain linked to a CRISPR nuclease so that the nucleosome-interacting protein domain at or near the target sequence and the interaction between nucleosomes / chromatin increase the efficiency of targeted genome modification (see Examples 1-8).

[0090] Therefore, the methods disclosed herein can increase the efficiency of targeted genome editing (e.g., gene proofreading, gene knockout, gene knock-in, etc.), targeted epigenetic modification, and targeted transcriptional regulation.

[0091] (a) Introduction into cells As described above, the method involves introducing at least one fusion protein, at least one CRISPR complex, or a nucleic acid (and optionally a donor polynucleotide) encoding the fusion protein or CRISPR complex into a cell. The at least one fusion protein, CRISPR complex, or nucleic acid can be introduced into the target cell by various means.

[0092] In some embodiments, cells can be transfused with nucleic acids using appropriate molecules (i.e., proteins, DNA, and / or RNA). Appropriate nucleic acid transfusion methods include nucleofection (or electroporation), nucleic acid transfusion via calcium phosphate, cationic polymer nucleic acid transfusion (e.g., DEAE-dextran or polyethyleneimine), viral transfusion, virosomal nucleic acid transfusion, virion nucleic acid transfusion, liposomal nucleic acid transfusion, cationic liposomal nucleic acid transfusion, immunoliposomal nucleic acid transfusion, non-liposomal lipid nucleic acid transfusion, dendrimer nucleic acid transfusion, heat shock nucleic acid transfusion, magnetofection, lipofection, gene gun delivery, impalefection, sonoporation, phototransfusion, and proprietary drug-enhanced nucleic acid uptake. Nucleic acid delivery methods are well known in the art (see, for example, "Current Protocols in Molecular Biology," Ausubel et al., John Wiley & Sons, New York, 2003, or "Molecular Cloning: A Laboratory Manual," Sambrook & Russell, Cold Spring Harbor Press, Cold Spring Harbor, NY, 3rd edition, 2001). In other embodiments, molecules can be introduced into cells by microinjection. For example, molecules can be injected into the cytoplasm or nucleus of the cell of interest. The amount of each molecule introduced into the cell can vary, but those skilled in the art are familiar with means of determining appropriate amounts.

[0093] Various molecules can be introduced into cells simultaneously or sequentially. For example, a fusion protein or CRISPR complex (or coding nucleic acid) and a donor polynucleotide can be introduced at the same time. Alternatively, one can be introduced first, and then the other later into the cell.

[0094] Generally, cells are maintained under conditions suitable for cell growth and / or maintenance. Appropriate cell culture conditions are well known in the art and are described, for example, in Santiago et al., Proc. Natl. Acad. Sci. USA, 2008, 105:5809-5814; Moehle et al. Proc. Natl. Acad. Sci. USA, 2007, 104:3055-3060; Urnov et al., Nature, 2005, 435:646-651; and Lombardo et al., Nat. Biotechnol., 2007, 25:1298-1306. Those skilled in the art will understand that methods for culturing cells are known in the art, can and will vary depending on the cell type. In all cases, periodic optimization may be used to determine the best technique for a particular cell type.

[0095] (b) Targeted genomic or epigenetic modifications One or more nucleosome-interacting protein domains of a fusion protein or CRISPR complex interact with nucleosomes and / or chromosomal DNA at or near the target chromosomal sequence, resulting in alteration / modification of the nucleosome and / or chromatin structure, thereby increasing the accessibility of the programmable DNA modification protein of the fusion protein or the CRISPR protein of the CRISPR complex to the target chromosomal sequence. This increased access to the target chromosomal sequence leads to an increased frequency / efficiency of targeted genomic, transcriptional, or epigenetic modifications.

[0096] In embodiments where the fusion protein comprises a programmable DNA modification protein having nuclease activity, the fusion protein can cleave one or both strands of a targeted chromosomal sequence. Double-strand disruption can be repaired by a non-homologous end-joining (NHEJ) repair process. Because NHEJs are error-prone, at least one base pair indel (i.e., deletion or insertion), at least one base pair substitution, or a combination thereof can occur during the repair of the disruption. Thus, the targeted chromosomal sequence can be modified, mutated, or inactivated. For example, a deletion, insertion, or substitution in the reading frame of a coding sequence can lead to an altered protein product or the absence of a protein product (referred to as "knockout"). In some iterations, the method may further include introducing a donor polynucleotide (see below) into cells, which contains a donor sequence adjacent to a sequence located on either side of the target chromosome sequence, thereby allowing the donor sequence in the donor polynucleotide to be exchanged for or incorporated into the chromosome sequence in the target chromosome sequence during the repair of double-strand disruption by homology-directed repair (HDR). The incorporation of an exogenous sequence is called "knock-in".

[0097] Therefore, in various iterations, the efficiency of targeted genome modifications can be increased by at least about 0.1 times, at least about 0.5 times, at least about 1 time, at least about 2 times, at least about 5 times, at least about 10 times, or at least about 20 times, at least about 50 times, at least about 100 times, or more than about 100 times compared to parental programmable DNA modification proteins that are not linked to at least one nucleosome interacting protein domain.

[0098] In embodiments where the fusion protein comprises a programmable DNA modification protein having non-nuclease activity, the fusion protein can modify DNA or related proteins in a target chromosome sequence, or modify the expression of the target chromosome sequence. For example, if the programmable DNA modification protein includes epigenetic modification activity, the state of histones such as acetylation, methylation, phosphorylation, and adenylation can be modified, or the state of DNA such as methylation and amination can be modified. For example, in embodiments where the programmable DNA modification protein includes cytidine deaminase activity, one or more cytidine residues in the target chromosome sequence can be converted to uridine residues. Alternatively, if the programmable DNA modification protein includes transcriptional activation or repressor activity, transcription in the target chromosome sequence can be increased or decreased.

[0099] The resulting epigenetic modifications or transcriptional regulation can be increased by at least about 0.1 times, at least about 0.5 times, at least about 1 time, at least about 2 times, at least about 5 times, at least about 10 times, or at least about 20 times, at least about 50 times, at least about 100 times, or about 100 times or more compared to the parental programmable DNA modification protein that is not linked to at least one nucleosome interacting protein domain.

[0100] The targeted genome, transcription, and epigenetic modifications detailed above can be performed individually or in multiples (i.e., two or more chromosomal sequences can be targeted simultaneously).

[0101] (c) Desired donor polynucleotide In embodiments in which the fusion protein comprises a programmable DNA-modified protein having nuclease activity, the method may further include introducing at least one donor polynucleotide into a cell. The donor polynucleotide may be single-stranded or double-stranded, linear or round, and / or RNA or DNA. In some embodiments, the donor polynucleotide may be a vector, such as a plasmid vector.

[0102] A donor polynucleotide contains at least one donor sequence. In some aspects, the donor sequence of a donor polynucleotide can be a modified version of an endogenous or intrinsic chromosomal sequence. For example, a donor sequence may be essentially identical to a portion of a chromosomal sequence at or near the sequence targeted by a DNA modification protein, but it contains at least one nucleotide change. Thus, upon incorporation or exchange with an intrinsic sequence, the sequence at the targeted chromosomal location contains at least one nucleotide change. For example, this change may be one or more nucleotide insertions, one or more nucleotide deletions, one or more nucleotide substitutions, or a combination thereof. As a result of the “genetic proofreading” incorporation of the modified sequence, the cell can produce a modified gene product from the targeted chromosomal sequence.

[0103] In other contexts, the donor sequence of a donor polynucleotide can be an exogenous sequence. As used herein, “exogenous” sequence refers to a sequence that is not cell-specific, or a sequence whose specific location is different from that of the cell’s genome. For example, an exogenous sequence may include a protein-coding sequence that can be operably ligated to an exogenous promoter-regulating sequence, so that upon integration into the genome, the cell can express the protein encoded by the integrated sequence. Alternatively, an exogenous sequence may be integrated into a chromosomal sequence such that its expression is regulated by an endogenous promoter-regulating sequence. In other repetitions, an exogenous sequence may be a transcriptional regulatory sequence, another expression regulatory sequence, an RNA-coding sequence, etc. As described above, the integration of an exogenous sequence into a chromosomal sequence is called “knock-in.”

[0104] As those skilled in the art will understand, the length of a donor sequence can and will vary. For example, a donor sequence can vary in length from a few nucleotides to several hundred nucleotides to several hundred thousand nucleotides.

[0105] Typically, the donor sequence in a donor polynucleotide is adjacent to upstream and downstream sequences that have substantial sequence identity with respect to the sequences located upstream and downstream, respectively, of the sequence targeted by a programmable DNA modification protein. Due to the similarity of these sequences, the upstream and downstream sequences of the donor polynucleotide enable homologous recombination between the donor polynucleotide and the targeted chromosomal sequence, and as a result, the donor sequence can be incorporated into (or replaced by) the chromosomal sequence.

[0106] As used herein, an upstream sequence refers to a nucleic acid sequence that shares substantial sequence identity with a chromosomal sequence upstream of a sequence targeted by a programmable DNA-modifying protein. Similarly, a downstream sequence refers to a nucleic acid sequence that shares substantial sequence identity with a chromosomal sequence downstream of a sequence targeted by a programmable DNA-modifying protein. As used herein, the term "substantial sequence identity" refers to sequences having at least about 75% sequence identity. Thus, upstream and downstream sequences in a donor polynucleotide may have about 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% sequence identity with the upstream or downstream sequence relative to the target sequence. In exemplary embodiments, the upstream and downstream sequences in a donor polynucleotide can have approximately 95% or 100% sequence identity with the upstream or downstream chromosomal sequences of the sequence targeted by the programmable DNA modification protein.

[0107] In some embodiments, the upstream sequence shares substantial sequence identity with a chromosomal sequence located immediately upstream of the sequence targeted by the programmable DNA modification protein. In other embodiments, the upstream sequence shares substantial sequence identity with a chromosomal sequence located within approximately 100 nucleotides upstream of the target sequence. Thus, for example, the upstream sequence can share substantial sequence identity with chromosomal sequences located approximately 1 to 20, 21 to 40, 41 to 60, 61 to 80, or 81 to 100 nucleotides upstream of the target sequence. In some embodiments, the downstream sequence shares substantial sequence identity with a chromosomal sequence located immediately downstream of the sequence targeted by the programmable DNA modification protein. In other embodiments, the downstream sequence shares substantial sequence identity with a chromosomal sequence located approximately 100 nucleotides downstream of the target sequence. Therefore, for example, downstream sequences can share substantial sequence identity with chromosomal sequences located downstream of the target sequence at approximately 1 to 20, 21 to 40, 41 to 60, 61 to 80, or 81 to 100 nucleotides.

[0108] Each upstream or downstream sequence can be in the range of approximately 20 to 5000 nucleotides in length. In some embodiments, the upstream and downstream sequences may contain approximately 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2800, 3000, 3200, 3400, 3600, 3800, 4000, 4200, 4400, 4600, 4800, or 5000 nucleotides. In a specific embodiment, the lengths of the upstream and downstream sequences can range from about 50 to about 1500 nucleotides.

[0109] (d) Cell type Various cells are suitable for use in the methods disclosed herein. Generally, the cells are eukaryotic cells. For example, the cells may be human cells, non-human mammalian cells, non-mammalian vertebrate cells, invertebrate cells, insect cells, plant cells, yeast cells, or single-cell eukaryotes. In some embodiments, the cells may also be single-cell embryos. For example, non-human mammalian embryos include those of rats, hamsters, rodents, rabbits, cats, dogs, sheep, pigs, cattle, horses, and primates. In yet another embodiment, the cells may be stem cells such as embryonic stem cells, ES-like stem cells, fetal stem cells, or adult stem cells. In some embodiments, the stem cells are not human embryonic stem cells. Furthermore, the stem cells may include those produced by techniques disclosed in WO2003 / 046141 or Chung et al. (Cell Stem Cell, 2008, 2:113-117), which are entirely incorporated herein. The cells may be in vitro or in vivo (i.e., within a living organism). In exemplary embodiments, the cells are mammalian cells or mammalian cell lines. In specific embodiments, the cells are human cells or human cell lines.

[0110] Non-limiting examples of suitable mammalian cells or cell lines include: human fetal kidney cells (HEK293, HEK293T); human cervical cancer cells (HELA); human lung cells (W138); human hepatocytes (HepG2); human U2-OS osteosarcoma cells, human A549 cells, human A-431 cells, and human K562 cells; Chinese hamster ovary (CHO) cells, baby hamster kidney (BHK) cells; mouse myeloma NS0 cells, mouse embryonic fibroblast 3T3 cells (NIH3T3), mouse B lymphoma A20 cells; mouse melanoma B16 cells; mouse myoblast C2C12 cells; mouse myeloma SP2 / 0 cells; mouse germinal mesenchymal C3H-10T1 / 2 cells; mouse cancer CT26 cells, mouse prostate DuCuP cells; Mouse mammary EMT6 cells; mouse hepatocarcinoma Hepa1c1c7 cells; mouse myeloma J5582 cells; mouse epithelial MTD-1A cells; mouse cardiomyocyte MyEnd cells; mouse renal RenCa cells; mouse pancreatic RIN-5F cells; mouse melanoma X64 cells; mouse lymphoma YAC-1 cells; rat glioblastoma 9L cells; rat B lymphoma RBL cells; rat neuroblastoma B35 cells; rat hepatocarcinoma cells (HTC); buffalo rat liver BRL3A cells; canine kidney cells (MDCK); canine mammary gland (CMT) cells; rat osteosarcoma D17 cells; rat monocyte / macrophage DH82 cells; monkey kidney SV-40 transformed fibroblast (COS7) cells; monkey kidney CVI-76 cells; African green monkey kidney (VERO-76) cells. A broad list of mammalian cell lines may be found in the Catalogue of the American Cell Culture and Cell Lineage Preservation Organization (ATCC, Manassas, VA).

[0111] (VI) Methods for detecting specific gene loci In embodiments in which the fusion protein contains a programmable DNA modification having non-DNA activity, or the CRISPR complex contains a catalytically inactive CRISPR protein having non-nuclease activity, the fusion protein or CRISPR complex can be used in a method for detecting or visualizing specific genomic loci in eukaryotic cells. In such embodiments, the fusion protein or CRISPR protein further comprises at least one detectable label, such as a fluorophore (e.g., FAM, TMR, Cy3, Cy5, Texas Red, Oregon Green, Alexa Fluor, Halo tag, or appropriate fluorescent dye), a detection tag (e.g., biotin, digoxigenin, etc.), a quantum dot, or a gold particle. Alternatively, the guide RNA of the CRISPR complex may further comprise a detectable label for in situ detection (e.g., FISH or CISH). At least one nucleosome-interacting protein domain of a fusion protein or CRISPR complex increases access to target chromosomal sequences for programmable DNA-modifying proteins or CRISPR proteins with non-nuclease activity, thereby enhancing the detection of specific genomic loci or targeted chromosomal sequences.

[0112] The method involves introducing a detectably labeled fusion protein, a detectably labeled CRISPR complex, or an encoding nucleic acid into eukaryotic cells and detecting a labeled programmable DNA-modified protein or labeled CRISPR protein bound to a target chromosome sequence. This detection can be performed via dynamic live-cell imaging, fluorescence microscopy, confocal microscopy, immunofluorescence, immunodetection, RNA-protein binding, protein-protein binding, etc. The detection step can be performed in live or fixed cells.

[0113] In embodiments of the method, which includes detecting chromatin structure dynamics in living cells, a detectably labeled fusion protein or a detectably labeled CRISPR complex can be introduced into the cells as a protein or nucleic acid. In embodiments of the method, which includes detecting targeted chromosomal sequences in fixed cells, a detectably labeled fusion protein or a detectably labeled CRISPR complex can be introduced into the cells as a protein (or protein-RNA complex). Means for fixing and permeabilizing cells are well known in the art. In some embodiments, fixed cells can undergo chemical and / or thermal denaturation processes to convert double-stranded chromosomal DNA to single-stranded DNA. In other embodiments, fixed cells do not undergo chemical and / or thermal denaturation processes.

[0114] (VIII) Application The compositions and methods disclosed herein can be used in a variety of therapeutic, diagnostic, industrial, and research applications. In some embodiments, the disclosure can be used to modify any chromosomal sequence of interest in cells, animals, or plants; to model and / or study gene function; to study genetic or epigenetic conditions of interest; or to study biochemical pathways involved in various diseases or disorders. For example, a transgenic organism can be created to model a disease or disorder, in which the expression of one or more nucleic acid sequences associated with the disease or disorder is altered. Disease models can be used to study the effects of mutations on organisms; to study the onset and / or progression of diseases; to study the effects of pharmaceutically active compounds on diseases; and / or to evaluate the effectiveness of potential gene therapy strategies.

[0115] In other embodiments, compositions and methods can be used to conduct efficient and cost-effective functional genome screening, which can be used to study the function of genes involved in specific biological processes and how any changes in gene expression can affect those biological processes, or to perform saturation or deep-scanning mutagenesis of genomic loci in combination with cellular phenotypes. Saturation or deep-scanning mutagenesis can be used, for example, to determine critical minimum features and individual vulnerabilities of functional elements necessary for gene expression, drug resistance, or disease reversal.

[0116] In further embodiments, the compositions and methods disclosed herein may be used for diagnostic tests to determine the presence of a disease or disorder and / or for use in determining treatment options. Examples of appropriate diagnostic tests include the detection of specific mutations in cancer cells (e.g., specific mutations in EGFR, HER2, etc.), the detection of specific mutations associated with a particular disease (e.g., trinucleotide repeats, mutations in β-globin associated with sickle cell disease, specific SNPs, etc.), the detection of hepatitis, the detection of viruses (e.g., Zika), etc.

[0117] In additional embodiments, the compositions and methods disclosed herein can be used to correct genetic mutations associated with specific diseases or disorders, for example, to correct globin gene mutations associated with sickle cell disease or thalassemia, to correct mutations in the adenosine deaminase gene associated with severe combined immunodeficiency (SCID), to reduce the expression of genes causing HTT, Huntington's disease, or to correct mutations in the rhodopsin gene for the treatment of retinitis pigmentosa. Such modifications may be carried out ex vivo in cells.

[0118] In further embodiments, the compositions and methods disclosed herein can be used to produce crops having improved properties or increased resistance to environmental stress. The disclosure can also be used to produce livestock or production animals having improved properties. For example, pigs have many characteristics that result in attr activity as a biomedical model, particularly in regenerative medicine or xenotransplantation.

[0119] (IX) Enumerated embodiments The embodiments listed below are provided to demonstrate specific aspects of the invention and are not intended to limit its scope.

[0120] 1. A fusion protein comprising at least one nucleosome-interacting protein domain linked to a programmable DNA-modified protein.

[0121] 2. A fusion protein of Embodiment 1, wherein the at least one nucleosome interacting protein domain is a DNA-binding domain from a high-mobility group (HMG) box (HMGB) protein selected from HMGB1, HMGB2, or HMGB3; an HMG nucleosome-binding (HMGN) protein selected from HMGN1, HMGN2, HMGN3a, HMGN3b, HMGN4, or HMGN5; a central globule domain from a histone H1 variant; and a DNA-binding domain from a chromatin remodeling complex protein selected from a switch / sucrose non-fermentable (SWI / SNF) complex, a mimic switch (ISWI) complex, a chromodomain-helicase-DNA binding (CHD) complex, a nucleosome remodeling and deacetylase (NuRD) complex, an INO80 complex, an SWR1 complex, an RSC complex, or a combination thereof.

[0122] 3. A fusion protein of Embodiment 2, wherein the at least one nucleosome interacting protein domain is an HMGB1 box A domain, an HMGN1 protein, an HMGN2 protein, an HMGN3a protein, an HMGN3b protein, a histone H1 central globule domain, an ISWI protein DNA-binding domain, a CHD1 protein DNA-binding domain, or a combination thereof.

[0123] 4. A fusion protein in any one of embodiments 1 to 3, wherein the programmable DNA-modified protein has nuclease activity.

[0124] 5. A fusion protein of Embodiment 4, wherein the programmable DNA-modified protein is a chimeric protein comprising a programmable DNA-binding domain ligated to a clustered regularly dispersed short palindromic repeat (CRISPR) nuclease or nickase, zinc finger nuclease (ZFN), transcription activator-like effector nuclease (TALEN), meganuclease, or nuclease domain.

[0125] 6. A fusion protein in any one of Embodiments 1 to 3, wherein the programmable DNA-modified protein has non-nuclease activity.

[0126] 7. A fusion protein of Embodiment 6, wherein the programmable DNA-modified protein is a chimeric protein comprising a programmable DNA-binding domain linked to a non-nuclease domain.

[0127] 8. A fusion protein of Embodiment 7, wherein the programmable DNA-binding domain is a CRISPR protein, zinc finger protein, or transcription activator-like effector modified to lack all nuclease activity.

[0128] 9. A fusion protein of Embodiment 7, wherein the non-nuclease domain has acetyltransferase activity, deacetylase activity, methyltransferase activity, demethylase activity, kinase activity, phosphatase activity, ubiquitin ligase activity, deubiquitination activity, adenylation activity, deadenylation activity, SUMOylation activity, deSUMOylation activity, ribosylation activity, deribosylation activity, myristoylation activity, demyristoylation activity, citrullination activity, helicase activity, amination activity, deamination activity, alkylation activity, dealkylation activity, oxidation activity, transcription activation activity, or transcription repressor activity.

[0129] 10. A fusion protein of Embodiment 9, wherein the non-nuclease domain has cytosine deaminase activity, histone acetyltransferase activity, transcriptional activating activity, or transcriptional repressor activity.

[0130] 11. A fusion protein according to any one of Embodiments 1 to 10, wherein at least one nucleosome interacting protein domain is linked to the programmable DNA-modified protein directly via a chemical bond, indirectly via a linker, or a combination thereof.

[0131] 12. A fusion protein according to any one of Embodiments 1 to 11, wherein at least one nucleosome interacting protein domain is ligated to the programmable DNA-modified protein at its N-terminus, C-terminus, internal position, or a combination thereof.

[0132] 13. A fusion protein according to any one of Embodiments 1 to 12, further comprising at least one nuclear localization signal, at least one cell permeability domain, at least one marker domain, or a combination thereof.

[0133] 14. A fusion protein comprising a clustered, regularly dispersed, short palindromic repeat (CRISPR) protein linked to at least one nucleosome-interacting protein domain.

[0134] 15. A fusion protein of Embodiment 14, wherein the CRISPR protein is a type II CRISPR / Cas9 nuclease or nickase, or the CRISPR protein is a type V CRISPR / Cpf1 nuclease or nickase.

[0135] 15. A fusion protein of Embodiment 14, wherein the CRISPR protein is a type II CRISPR / Cas9 protein modified to lack all nuclease activity and linked to a non-nuclease domain, or a type V CRISPR / Cpf1 protein modified to lack all nuclease activity and linked to a non-nuclease domain.

[0136] 17. A fusion protein according to Embodiment 16, wherein the non-nuclease domain has cytosine deaminase activity, histone acetyltransferase activity, transcriptional activating activity, or transcriptional repressor activity.

[0137] 18. A fusion protein according to any one of embodiments 14 to 17, wherein the at least one nucleosome interacting protein domain is a high mobility group (HMG) box (HMGB) DNA binding domain, an HMG nucleosome binding (HMGN) protein, a central globule domain from a histone H1 variant, a DNA binding domain from a chromatin remodeling complex protein, or a combination thereof.

[0138] 19. A fusion protein of Embodiment 18, wherein at least one nucleosome interacting protein domain is an HMGB1 box A domain, an HMGN1 protein, an HMGN2 protein, an HMGN3a protein, an HMGN3b protein, a histone H1 central globule domain, a mimetic switch (ISWI) protein DNA binding domain, a chromodomain-helicase-DNA protein 1 (CHD1) DNA binding domain, or a combination thereof.

[0139] 20. A fusion protein, one of embodiments 14 to 19, wherein at least one nucleosome interacting protein domain is linked to the CRISPR protein directly via a chemical bond, indirectly via a linker, or a combination thereof.

[0140] 21. A fusion protein according to any one of embodiments 14 to 20, wherein at least one nucleosome interacting protein domain is ligated to the CRISPR protein at its N-terminus, C-terminus, internal position, or a combination thereof.

[0141] 22. A fusion protein according to any one of embodiments 14 to 21, further comprising at least one nuclear localization signal, at least one cell permeability domain, at least one marker domain, or a combination thereof.

[0142] 23. A fusion protein according to any one of Embodiments 14 to 22, wherein the CRISPR protein is Streptococcus pyogenes Cas9 (SpCas9), Streptococcus thermophilus Cas9 (StCas9), Streptococcus pasteurianus (SpaCas9), Campylobacter jejuni Cas9 (CjCas9), Staphylococcus aureus (SaCas9), Francisella novicida Cas9 (FnCas9), Neisseria cinerea Cas9 (NcCas9), Neisseria meningitis Cas9 (NmCas9), Francisella novicida Cpf1 (FnCpf1), Acidaminococcus sp. Cpf1 (AsCpf1), or Lachnospiraceae bacterium ND2006 Cpf1 (LbCpf1).

[0143] 24. A fusion protein according to any one of embodiments 14 to 23, wherein the fusion protein has an amino acid sequence that has at least about 90% sequence identity with SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78, or SEQ ID NO: 79.

[0144] 25. A fusion protein according to any one of embodiments 14 to 24, wherein the fusion protein has the amino acid sequence described in SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78, or SEQ ID NO: 79.

[0145] 26. A complex comprising at least one fusion protein and at least one guide RNA from any one of embodiments 14 to 25.

[0146] 27. A nucleic acid encoding one of the fusion proteins of Embodiments 1 to 25.

[0147] 28. A nucleic acid of Embodiment 27, which is codon-optimized for translation in eukaryotic cells.

[0148] 29. A nucleic acid of Embodiment 27 or 28, which is part of a viral vector, a plasmid vector, or a self-replicating RNA.

[0149] 30. A method for increasing the efficiency of targeted genomic or epigenetic modifications in eukaryotic cells, comprising introducing into the eukaryotic cells a nucleic acid encoding at least one fusion protein described in any one of Embodiments 1 to 25, or at least one fusion protein described in any one of Embodiments 27 to 29, wherein the programmable DNA modification protein of the at least one fusion protein is targeted to a target chromosome sequence, and the at least one nucleosome interacting protein domain of the at least one fusion protein alters the nucleosome or chromatin structure, thereby giving the at least one fusion protein increased access to the target chromosome sequence, thereby increasing the efficiency of targeted genomic or epigenetic modifications.

[0150] 31. A method according to Embodiment 30, wherein the DNA-modified protein of the at least one fusion protein comprises a CRISPR protein, and the method further comprises introducing at least one guide RNA or a nucleic acid encoding the at least one guide RNA into the eukaryotic cell.

[0151] 32. A method according to Embodiment 30 or 31, wherein the method further comprises introducing a donor polynucleotide comprising at least one donor sequence into a eukaryotic cell.

[0152] 33. A method according to any one of embodiments 30 to 32, wherein the eukaryotic cell is in vitro.

[0153] 34. A method according to any one of embodiments 30 to 32, wherein the eukaryotic cells are in vivo.

[0154] 35. A method according to any one of embodiments 30 to 34, wherein the eukaryotic cell is a mammalian cell.

[0155] 36. A method according to any one of embodiments 30 to 35, wherein the eukaryotic cell is a human cell.

[0156] 37. A method for increasing the efficiency of targeted genomic or epigenetic modifications in eukaryotic cells, the method comprising introducing into the eukaryotic cells (a) at least one fusion protein or nucleic acid encoding at least one fusion protein, each fusion protein comprising a CRISPR protein ligated to at least one nucleosome interacting protein domain, wherein the CRISPR protein is (i) modified to have nuclease or nickase activity or (ii) modified to lack all nuclease activity and ligated to a non-nuclease domain; and (b) at least one guide RNA or nucleic acid encoding at least one guide RNA, wherein the CRISPR protein of the at least one fusion protein is targeted to a target chromosome sequence, and the at least one nucleosome interacting protein domain of the at least one fusion protein alters the nucleosome or chromatin structure, thereby the at least one fusion protein having increased access to the target chromosome sequence, thereby increasing the efficiency of targeted genomic or epigenetic modifications.

[0157] 38. The method of Embodiment 37, wherein the CRISPR protein is a type II CRISPR / Cas9 protein or a type V CRISPR / Cpf1 protein.

[0158] 39. A method according to Embodiment 37 or 38, wherein the non-nuclease domain has cytosine deaminase activity, histone acetyltransferase activity, transcriptional activating activity, or transcriptional repressor activity.

[0159] 40. A method according to any one of embodiments 37 to 39, wherein the at least one nucleosome interacting protein domain is a high mobility group (HMG) box (HMGB) DNA binding domain, an HMG nucleosome binding (HMGN) protein, a central globule domain from a histone H1 variant, a DNA binding domain from a chromatin remodeling complex protein, or a combination thereof.

[0160] 41. A method according to any one of embodiments 37 to 40, wherein at least one nucleosome interacting protein domain is linked to the CRISPR protein directly via a chemical bond, indirectly via a linker, or a combination thereof.

[0161] 42. A method according to any one of embodiments 37 to 41, wherein at least one nucleosome interacting protein domain is ligated to the N-terminus, C-terminus, and / or internal position of a CRISPR protein.

[0162] 43. A method according to any one of embodiments 37 to 42, wherein the at least one fusion protein further comprises at least one nuclear localization signal, at least one cell permeability domain, at least one marker domain, or a combination thereof.

[0163] 44. A method according to any one of embodiments 37 to 43, wherein the nucleic acid encoding the at least one fusion protein is codon-optimized for translation in a eukaryotic cell.

[0164] 45. A method according to any one of embodiments 37 to 44, wherein the nucleic acid encoding the at least one fusion protein is part of a viral vector, a plasmid vector, or a self-replicating RNA.

[0165] 46. ​​A method according to any one of embodiments 37 to 45, wherein the method further comprises introducing a donor polynucleotide comprising at least one donor sequence into a eukaryotic cell.

[0166] 47. A method according to any one of embodiments 37 to 46, wherein the eukaryotic cell is in vitro.

[0167] 48. A method according to any one of embodiments 37 to 46, wherein the eukaryotic cells are in vivo.

[0168] 49. A method according to any one of embodiments 37 to 48, wherein the eukaryotic cell is a mammalian cell.

[0169] 50. A method according to any one of embodiments 37 to 48, wherein the eukaryotic cell is a human cell.

[0170] definition Unless otherwise defined, all technical and scientific terms used herein have the meanings generally understood by those skilled in the art to which the invention pertains. The following references provide those skilled in the art with many general definitions of terms used in this invention: Singleton et al., Dictionary of Microbiology and Molecular Biology (2nd edition, 1994); The Cambridge Dictionary of Science and Technology (Walker ed., 1988); The Glossary of Genetics, 5th edition, R. Rieger et al. (eds.), Springer Verlag (1991); and Hale & Marham, The Harper Collins Dictionary of Biology (1991). Where used herein, the following terms have the meanings attributed to them unless otherwise specified.

[0171] Where introducing elements of this disclosure or its preferred embodiments, the articles “a,” “an,” “the,” and “said” are intended to mean that there is one or more of the elements. The terms “comprising,” “including,” and “having” are intended to mean comprehensive and that there may be additional elements other than those listed.

[0172] The term "approximately," when used in relation to a numerical value x, means, for example, x ± 5%.

[0173] As used herein, the terms “complementary” or “complementarity” refer to the association of double-stranded nucleic acids by base pairing via specific hydrogen bonds. Base pairing may be standard Watson-Crick base pairing (e.g., 5'-AGTC-3' pairs with the complementary sequence 3'-TCAG-5'). Base pairing may also be Hougsteen or reverse Hougsteen hydrogen bonds. Complementarity is typically measured with respect to a bilayer region and therefore, excluding, for example, overhangs. Complementarity between the two strands of a bilayer region may be partial, and if only some of the bases (e.g., 70%) are complementary, it may be expressed as a percentage (e.g., 70%). Non-complementary bases are “mismatched”. If all bases within a bilayer region are complementary, the complementarity may be complete (i.e., 100%).

[0174] As used herein, the term “CRISPR system” refers to a complex comprising a CRISPR protein (i.e., a nuclease, nicasse, or catalytically dead protein) and guide RNA.

[0175] As used herein, the term “endogenous sequence” refers to a chromosomal sequence unique to a cell.

[0176] As used herein, the term “exogenous” refers to a sequence that is not specific to the cell, or a chromosomal sequence that is located at a chromosomal position different from its specific position in the cell’s genome.

[0177] As used herein, “gene” refers to the DNA region (including exons and introns) that codes for a gene product, regardless of whether such regulatory sequences are adjacent to the coded and / or transcribed sequences, as well as all DNA regions that regulate the production of the gene product. Thus, a gene includes, but is not limited to, the promoter sequence, terminator, translation regulatory sequences such as ribosome binding sites and internal ribosome entry sites, enhancers, silencers, insulators, boundary elements, origins of replication, matrix attachment sites, and locus regulatory regions.

[0178] The term "heterogeneous" refers to entities that are not endogenous or inherent to the target cell. For example, heterogeneous proteins refer to proteins such as nucleic acid sequences that are exogenously introduced, either from an exogenous source or originally from an exogenous source. In some cases, heterogeneous proteins are not typically produced by the target cell.

[0179] The term "nickase" refers to an enzyme that cleaves one strand of a double-stranded nucleic acid sequence (i.e., inserts a nick into the double-stranded sequence). For example, a nuclease with double-strand cleavage activity can function as a nickase and can be modified by mutation and / or deletion to cleave only one strand of a double-stranded sequence.

[0180] As used herein, the term "nuclease" refers to an enzyme that cleaves both strands of a double-stranded nucleic acid sequence.

[0181] The terms “nucleic acid” and “polynucleotide” refer to deoxyribonucleotide or ribonucleotide polymers in linear or cyclic conformations and in single or double-stranded forms. For the purposes of this disclosure, these terms should not be construed as limiting with respect to the length of the polymer. The terms may include known analogues of natural nucleotides, as well as nucleotides modified in base, sugar, and / or phosphate moieties (e.g., phosphorothioate skeletons). Generally, analogues of a particular nucleotide have the same base-pairing specificity; that is, an analogue of A base-pairs with T.

[0182] The term "nucleotide" refers to deoxyribonucleotide or ribonucleotide. A nucleotide may be a standard nucleotide (i.e., adenosine, guanosine, cytidine, thymidine, and uridine), a nucleotide isomer, or a nucleotide analog. A nucleotide analog refers to a nucleotide having a modified purine or pyrimidine base or a modified ribose moiety. A nucleotide analog may be a natural nucleotide (e.g., inosine, pseudouridine, etc.) or a non-natural nucleotide. Non-limiting examples of modifications to the sugar or base moiety of a nucleotide include the addition (or removal) of acetyl, amino, carboxyl, carboxymethyl, hydroxyl, methyl, phosphoryl, and thiol groups, as well as the substitution of carbon and nitrogen atoms of the base with other atoms (e.g., 7-deazapurine). Nucleotide analogs also include dideoxynucleotides, 2′-O-methylnucleotides, locked nucleic acids (LNAs), peptide nucleic acids (PNAs), and morpholinos.

[0183] The terms "polypeptide" and "protein" are used interchangeably to refer to polymers of amino acid residues.

[0184] As used herein, the term “programmable DNA-modifying protein” refers to a protein that is programmed to bind to a specific target sequence in chromosomal DNA and modifies the DNA or DNA-related proteins at or near the target sequence.

[0185] As used herein, the term “sequence identity” refers to a quantitative measure of the degree of identity between two sequences of substantially equal length. Whether nucleic acid sequences or amino acid sequences, the percentage identity of two sequences is calculated by dividing the number of exact matches between the two aligned sequences by the length of the shorter sequence and multiplying by 100. Approximate alignment of nucleic acid sequences is provided by the local homology algorithm of Smith and Waterman, Advances in Applied Mathematics 2:482-489 (1981). This algorithm can be applied to amino acid sequences using a scoring matrix developed by Dayhoff, Atlas of Protein Sequences and Structure, MO Dayhoff ed., 5 suppl. 3:353-358, National Biomedical Research Foundation, Washington, DC, USA, and normalized by Gribskov, Nucl. Acids Res. 14(6):6745-6763 (1986). An exemplary implementation of the algorithm for determining the percent identity of sequences is provided by Genetics Computer Group (Madison, Wis.) in the “BestFit” utility application. Other suitable programs for calculating the percent identity or similarity between sequences are generally known in the art, and another alignment program used with default parameters is BLAST, for example. For example, BLASTN and BLASTP can be used with the following default parameters: genetic code=standard; filter=none; strand=both; cutoff=60; expect=10; Matrix=BLOSUM62; Descriptions=50 sequences; sort by=HIGH SCORE; Databases=non-redundant, GenBank+EMBL+DDBJ+PDB+GenBank CDS translations+Swiss protein+Spupdate+PIR.Details of these programs can be found on the GenBank website. Generally, substitutions are conservative amino acid substitutions and are limited to substitutions within the following groups: Group 1: glycine, alanine, valine, leucine, and isoleucine; Group 2: serine, cysteine, threonine, and methionine; Group 3: proline; Group 4: phenylalanine, tyrosine, and tryptophan; Group 5: aspartic acid, glutamic acid, asparagine, and glutamine.

[0186] The terms “target sequence,” “target chromosome sequence,” and “target site” are used interchangeably to refer to the specific sequence in chromosomal DNA that a programmable DNA modification protein targets, and the site on which the programmable DNA modification protein modifies the DNA or a protein associated with that DNA.

[0187] Techniques for determining nucleic acid and amino acid sequence identity are known in the art. Typically, such techniques involve determining the nucleotide sequence of mRNA for a gene and / or the amino acid sequence encoded thereby, and comparing these sequences with a second nucleotide or amino acid sequence. Genomic sequences can also be determined and compared by these methods.

[0188] Generally, identity refers to the exact nucleotide-to-nucleotide or amino acid-to-amino acid correspondence between two polynucleotide or polypeptide sequences. Two or more sequences (polynucleotides or amino acids) can be compared by determining their percentage identity.

[0189] Since various modifications can be made to the cells and methods described above without departing from the scope of the present invention, all matters contained in the above description and the examples given below are intended to be interpreted as illustrative, not restrictive.

[0190] Examples The following examples demonstrate specific aspects of this disclosure. Table 1 lists the peptide sequences of the nucleosome interaction domain, and Table 2 shows the target chromosome sequences used in Examples 1-8 shown below. [Table 1] [Table 2]

[0191] Example 1. Improvement of Streptococcus pyogenes Cas9 (SpCas9) activity using human HMGB1 box A domain. The human HMGB1 box A domain (SEQ ID NO: 40) was fused with SpCas9 (+NLS) at its nuclease carboxyl terminus using the linker LEGGGS (SEQ ID NO: 1) between Cas9 and the HMGB1 box A domain. Human K562 cells (1 x 10⁻¹⁰) 6 Nucleotransduction was performed using a DNA extraction solution (QuickExtract) containing molar equivalents of plasmid DNA encoding the fusion protein or wild-type SpCas9 protein (5.2 and 5.0 μg for the fusion protein and 5.0 μg for the wild-type Cas9 protein, respectively), combined with a 3 μg sgRNA plasmid to target a genomic site (#1) at the human cytochrome p450 oxidoreductase (POR) locus. Nucleotransduction was performed using nucleofection with an Amaxi nucleofector. Three days after nucleotransduction, the cells were extracted using a DNA extraction solution (QuickExtract). TM The target genomic region was lysed using ) and amplified by PCR. Cas9 nuclease target cleavage activity (% indel) was measured using the Cel-I assay. As shown in Table 3, fusion of the human HMGB1 box A domain with the antigen increased the SpCas9 cleavage efficiency at the target site. [Table 3]

[0192] Example 2. Improvement of Streptococcus pyogenes Cas9 (SpCas9) activity using human HMGN1, HMGN2, HMGN3a, and HMGN3b Human HMGN1, HMGN2, HMGN3a, and HMGN3b (SEQ ID NOs: 41 - 44 respectively) were each fused to SpCas9(+NLS) at the nuclease carboxyl terminus using the linker LEGGGS (SEQ ID NO: 1) between each of Cas9 and the HMGN peptide. Human K562 cells (1x10 6 ) were combined with 3 μg of an sgRNA plasmid targeting the genomic site (#1) at the human cytochrome p450 oxidoreductase (POR) locus and transfected with a molar equivalent of plasmid DNA encoding each of the fusion proteins or wild - type SpCas9 protein (5.2 and 5.0 μg respectively for the fusion protein and wild - type Cas9 protein) using Amaxi nucleofector with nucleofection. Three days after transfection, the cells were lysed using a DNA extraction solution (QuickExtract TM ) and the targeted genomic region was PCR - amplified. Cas9 target cleavage activity (% indel) was measured using the Cel - I assay. The results show that the fusion of each human HMGN peptide with the nuclease increased the SpCas9 cleavage efficiency at the target site, as summarized in Table 4.

Table 4

[0193] Example 3. Improvement of Streptococcus pyogenes Cas9 (SpCas9) activity using the human histone H1 central globular domain The human histone H1 central globule domain (SEQ ID NO: 45) was fused with SpCas9 (+NLS) at its nuclease carboxyl terminus using the linker LEGGGS (SEQ ID NO: 1) between Cas9 and the globule domain. Human K562 cells (1 x 10⁻¹⁰) 6 The sgRNA plasmid (3 μg) was combined with 3 μg of plasmid DNA encoding either the fusion protein or the wild-type SpCas9 protein (5.2 μg and 5.0 μg, respectively) to target a genomic site (#1) at the human cytochrome p450 oxidoreductase (POR) gene locus, and nucleic acid transduction was performed using nucleofection with Amaxinucleofector. Three days after nucleic acid transduction, the cells were subjected to DNA extraction (QuickExtract). TM The target genomic region was lysed using () and amplified by PCR. Cas9 targeted cleavage activity (% indel) was measured using the Cel-I assay. The results are shown in Table 5. Fusion of the human histone H1 central globule domain with a nuclease increased the efficiency of SpCas9 cleavage at the target site. [Table 5]

[0194] Example 4. Improvement of Streptococcus pyogenes Cas9 (SpCas9) activity using a chromatin remodeling protein DNA binding domain. SpCas9(+NLS) was fused at the nuclease amino terminus of the DNA-binding domain of yeast ISWI chromatin remodeling complex ATPase ISW1 (SEQ ID NO: 46) using the linker TGSG (SEQ ID NO: 2) between Cas9 and its DNA-binding domain. Independently, wild-type SpCas9 was combined with 3 μg of an sgRNA plasmid targeting a genomic site (#1) at the human cytochrome p450 oxidoreductase (POR) locus and fused at the nuclease carboxyl terminus of the DNA-binding domain of yeast chromodomain-containing protein 1 (CHD1) (SEQ ID NO: 47) using the linker LEGGGS (SEQ ID NO: 1) between Cas9 and its DNA-binding domain. Human K562 cells (1 x 10⁻¹⁰ 6 The cells were transfused using plasmid DNA in molar equivalents encoding either the fusion protein or the wild-type SpCas9 protein (6.0 μg and 5.0 μg, respectively, for the fusion protein and wild-type Cas9 protein). Nucleofusion was performed using nucleofection with an Amaxi nucleofector. Three days after nucleic acid transfusion, the cells were subjected to DNA extraction (QuickExtract). TM The target genomic region was lysed using ) and amplified by PCR. Cas9 targeted cleavage activity (% indel) was measured using the Cel-I assay. The results, summarized in Table 6, show that fusion of the DNA-binding domain with each nuclease increased the SpCas9 cleavage efficiency at the target site. [Table 6]

[0195] Example 5. Improvement of Streptococcus pyogenes Cas9 (SpCas9) activity using a combination of nucleosome interaction domains. SpCas9(+NLS) was combined with 3 μg of sgRNA plasmids to target genomic sites (#1, #2, #3) at the human cytochrome p450 oxidoreductase (POR) locus, or genomic site (#1) at the human nuclear receptor subfamily 1 group I member 3 (CAR) locus, or genomic sites (#1, #2) at the human blank spiracle homeobox 1 (EMX1) locus. The fused molecules were then combined with human HMGB1 (SEQ ID NO: 41) at the nuclease amino terminus using the linker TGSG (SEQ ID NO: 2) between Cas9 and HMGN1, and with human HMGB1 box A domain (SEQ ID NO: 40), human histone H1 central globule domain (SEQ ID NO: 45), or yeast chromodomain-containing protein 1 (CHD1) DNA-binding domain (SEQ ID NO: 47) at the nuclease carboxyl terminus using the linker LEGGGS (SEQ ID NO: 1) between Cas9 and the respective protein domains. Human K562 cells (1 x 10⁻¹⁰ 6 The cells were transfused using molar equivalents of plasmid DNA encoding each of the fusion proteins or the wild-type SpCas9 protein (5.4 μg for the HMGB1 box A and H1 central globule domain fusion protein, 6.0 μg for the CHD1 DNA-binding domain fusion protein, and 5.0 μg for the wild-type Cas9 protein). Nucleotransduction was performed using nucleofection with an Amaxi nucleofector. Five days after nucleotransduction, the cells were extracted using a DNA extraction solution (QuickExtract). TM The proteins were lysed using ) and each targeted genomic region was amplified by PCR. Cas9 target cleavage activity (% indel) was measured using the Cel-I assay. The results, summarized in Table 7, indicate that the combined fusion of these protein domains with nucleases increased the efficiency of SpCas9 cleavage at the target site. [Table 7]

[0196] Example 6. Improvement of Streptococcus pasteurianus Cas9 (SpaCas9) activity using a combination of nucleosome interaction domains. Streptococcus pasteurianus Cas9 (SpaCas9) (+NLS) was combined with 3 μg of sgRNA plasmid to target genomic sites (#1, #2) at the human cytochrome p450 oxidoreductase (POR) locus. The Cas9 was fused to human HMGN1 (SEQ ID NO: 41) at the nuclease amino terminus using the linker TGSG (SEQ ID NO: 2) between Cas9 and HMGN1, and to human HMGN1 (SEQ ID NO: 41) at the nuclease amino terminus using the linker LEGGGS (SEQ ID NO: 1) between Cas9 and the respective protein domains, to human HMGB1 box A domain (SEQ ID NO: 41), human histone H1 central globule domain (SEQ ID NO: 45), or yeast chromodomain-containing protein 1 (CHD1) DNA-binding domain (SEQ ID NO: 47) at the nuclease carboxyl terminus. Human K562 cells (1 x 10⁶) 6 The cells were transfused using plasmid DNA in molar equivalents encoding either the fusion protein or the wild-type SpaCas9 protein (5.4 μg and 5.0 μg, respectively, for the fusion protein and wild-type Cas9 protein). Nucleofusion was performed using nucleofection with an Amaxi nucleofector. Three days after nucleic acid transfusion, the cells were subjected to DNA extraction (QuickExtract). TM The proteins were lysed using [a specific method], and the targeted genomic region was amplified by PCR. Cas9 targeted cleavage activity (% indel) was measured using the Cel-I assay. As summarized in Table 8, combined fusion of these protein domains with nucleases increased SpaCas9 cleavage efficiency at the target site. [Table 8]

[0197] Example 7. Improvement of Francisella novicida Cpf1 (FnCpf1) activity using a combination of nucleosome interaction domains. Francisella novicida Cpf1 (FnCpf1) (+NLS) was fused to human aminoHMGN1 (SEQ ID NO: 41) at the nuclease amino terminus using the linker TGSG (SEQ ID NO: 2) between Cpf1 and HMGN1, and to human HMGB1 box A domain (SEQ ID NO: 40), human histone H1 central globule domain (SEQ ID NO: 45), or yeast chromodomain-containing protein 1 (CHD1) DNA-binding domain (SEQ ID NO: 47) at the nuclease carboxyl terminus using the linker LEGGGS (SEQ ID NO: 1) between Cpf1 and the protein domain, respectively. Human K562 cells (1 x 10⁻¹⁰ 6 The cells were transfused using 3 μg of sgRNA plasmid to target genomic sites (#1, #2, #3) in the human cytochrome p450 oxidoreductase (POR) gene locus, with molar equivalents of plasmid DNA encoding either the fusion protein or the wild-type FnCpf1 protein (5.4 and 5.0 μg for the fusion protein and 5.0 μg for the wild-type Cas9 protein, respectively). Nucleotransduction was performed using nucleofection with an Amaxi nucleofector. Three days after nucleotransduction, the cells were extracted using a DNA extraction solution (QuickExtract). TM The proteins were lysed using ) and the targeted genomic region was amplified by PCR. Cas9 targeted cleavage activity (% indel) was measured using the Cel-I assay. The results, summarized in Table 9, indicate that the combined fusion of these protein domains with nucleases increased the efficiency of FnCpf1 cleavage at the target site. [Table 9]

[0198] Example 8. Improvement of Campylobacter jejuni Cas9 (CjCas9) gene editing efficiency. Campylobacter jejuni Cas9 (CjCas9) (+NLS) was fused with human aminoHNG1 (SEQ ID NO: 41) at the nuclease amino terminus using the linker TGSG (SEQ ID NO: 2) between Cas9 and HMGN1, and with the human HMGB1 box A domain (SEQ ID NO: 40) or human histone H1 central globule domain (SEQ ID NO: 45) at the nuclease carboxyl terminus using the linker LEGGGS (SEQ ID NO: 1) between Cas9 and the protein domain, respectively. Wild-type CjCas9 gRNA was modified by introducing a U-to-C mutation in the constant repeat region of the crRNA and a corresponding A-to-G mutation in the 5' region of the tracrRNA sequence. The modified sgRNA sequences are as follows: [ka] [Here, the mutated nucleotides in the crRNA and tracrRNA regions are underlined.] Guide sequences targeting two different sites (#1 and #2) in the human cytochrome p450 oxidoreductase gene (POR) were cloned into wild-type and modified CjCas9 sgRNA skeletons, respectively. sgRNA expression was controlled under the U6 promoter. (Human K562 cells (1x10)) 6 The cells were transfused using 4 μg of CjCas9 plasmid DNA and 3 μg of sgRNA plasmid DNA. Nucleofusion was performed using nucleofection with an Amaxi nucleofector. Three days after transfusion, the cells were lysed using QuickExtract, and the targeted genomic region was amplified by PCR. CjCas9 targeted DNA cleavage activity (% indel) was measured using the Cel-I assay. The results are shown in Figure 1, demonstrating that the fusion protein increased cleavage efficiency at the target site, and that the modified CjCas9 sgRNA backbone effectively increased CjCas9 cleavage efficiency at the target site.

[0199] Table 10 shows the amino acid sequences of the specific fusion proteins. Nucleosome interacting protein domains are shown in bold, linkers in italics, and NLS are underlined.

[0200] [Table 10-1]

[0201] [Table 10-2]

[0202] [Table 10-3]

[0203] [Table 10-4]

[0204] [Table 10-5]

[0205] [Table 10-6]

[0206] [Table 10-7]

[0207] [Table 10-8]

[0208] Table 10-9

[0209] Table 10-10

Claims

1. A method for increasing the efficiency of targeted genomic or epigenetic modifications in non-human eukaryotic cells or in vitro human cells, the method being: (a) at least one fusion protein or nucleic acid encoding at least one fusion protein, each fusion protein comprising a CRISPR protein linked to at least one nucleosome interacting protein domain, wherein the CRISPR protein is derived from Streptococcus pyogenes Cas9 (SpCas9), Streptococcus thermophilus Cas9 (StCas9), Streptococcus pasteurianus (SpaCas9), Campylobacter jejuni Cas9 (CjCas9), Staphylococcus aureus (SaCas9), Francisella novicida Cas9 (FnCas9), Neisseria cinerea Cas9 (NcCas9), Neisseria meningitis Cas9 (NmCas9), Francisella novicida Cpfl (FnCpf1), Acidaminococcus sp. Cpfl (AsCpf1), or Lachnospiraceae bacterium ND2006 Cpfl (LbCpf1), where the CRISPR protein is modified to (i) have nuclease or nickase activity or (ii) lack all nuclease activity and is linked to a non-nuclease domain, and where at least one fusion protein further comprises at least one nuclear localization signal, at least one cell permeability domain, at least one marker domain, or a combination thereof; (b) at least one guide RNA or nucleic acid encoding at least one guide RNA This includes introducing the above into the cells; A method wherein at least one CRISPR protein of the fusion protein is targeted to a target chromosome sequence, at least one nucleosome interacting protein domain of the fusion protein alters the nucleosome or chromatin structure, resulting in at least one fusion protein having increased access to the target chromosome sequence, thereby increasing the efficiency of the targeted genomic or epigenetic modification, and wherein the at least one nucleosome interacting protein domain is an HMGB1 box A domain, an HMGN1 protein, an HMGN2 protein, an HMGN3a protein, an HMGN3b protein, a histone H1 central globule domain, an ISMILK switch (ISWI) protein DNA-binding domain, a chromodomain-helicase-DNA protein 1 (CHD1) DNA-binding domain, or a combination thereof.

2. The method according to claim 1, wherein the non-nuclease domain has cytosine deaminase activity, histone acetyltransferase activity, transcriptional activating activity, or transcriptional repressor activity.

3. The method according to either claim 1 or 2, wherein at least one nucleosome interacting protein domain is ligated to the CRISPR protein directly via a chemical bond, indirectly via a linker, or a combination thereof; and / or at least one nucleosome interacting protein domain is ligated to the CRISPR protein at its N-terminus, C-terminus, internal position, or a combination thereof.

4. The method according to any one of claims 1 to 3, wherein at least one fusion protein comprises at least one nuclear localization signal.

5. The method according to any one of claims 1 to 4, wherein the nucleic acid encoding at least one fusion protein is codon-optimized for translation in eukaryotic cells, and / or the nucleic acid encoding at least one fusion protein is part of a viral vector, a plasmid vector, or a self-replicating RNA.

6. The method according to any one of claims 1 to 5, further comprising introducing at least one donor polynucleotide into the cell, wherein the donor polynucleotide comprises at least one donor sequence.

7. The fusion protein has an amino acid sequence that has at least 90% sequence identity with SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78, or SEQ ID NO: 79; or The fusion protein has the amino acid sequence described in SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78, or SEQ ID NO:

79. The method according to claim 1.

8. A fusion protein having an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78, or SEQ ID NO: 79, or a nucleic acid encoding a fusion protein having an amino acid sequence described in SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78, or SEQ ID NO: 79, which is codon-optimized for translation in eukaryotic cells and / or is part of a viral vector, plasmid vector, or self-replicating RNA, and the fusion protein has increased access to a target chromosomal sequence when introduced into a cell.

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