Cell-penetrating peptide compositions and uses thereof

Abstract

Provided herein are compositions and methods related to cell-penetrating peptides that enhance gene editing ex vivo. The compositions and methods include a site-directed modifying peptide conjugated to a cell-penetrating peptide in combination with cell-penetrating peptides that are not fused to the site-directed modifying peptide.

Description

CELL-PENETRATING PEPTIDE COMPOSITIONS AND USES THEREOFRELATED APPLICATIONSThis application claims priority to U.S. Provisional Application No. 63 / 519,998, filed on August 16, 2023. The content of the foregoing application is incorporated by reference herein in its entirety.SEQUENCE LISTINGThe instant application contains a Sequence Listing which has been submitted electronically in XML format, and which is hereby incorporated by reference in its entirety. Said XML copy, created on June 26, 2024, is named SI 06638 1320WO_SL.xml, and is 27,595 bytes in size.Field of Invention

[0001] The present disclosure relates generally to compositions that enhance gene editing. More specifically, the disclosure pertains to compositions of cell -penetrating peptides for use in ex vivo gene editing.Background

[0002] Despite significant advances in gene editing tools, the ability to efficiently edit cells is limited by existing delivery methods. Current methods for delivery rely on electroporation or viral transduction, which result in variable delivery efficiency, cytotoxicity, and viral transgene insertion. Accordingly, there exists a need in the art for methods to delivery gene editing tools that are highly-efficient and non-toxic.Summary of the Invention

[0003] In one aspect disclosed herein is a composition for ex vivo gene editing comprising (a) a site-directed modifying polypeptide, wherein the site-directed modifying polypeptide is conjugated to one or more cell-penetrating peptides (CPPs), and (b) a plurality of CPPs, wherein the plurality of CPPs comprise a thiol -reactive moiety.

[0004] In some embodiments, the CPP is a cationic CPP. In some embodiments, the cationic CPP is 5 to 40 amino acid residues and has an average net charge of at least +0.3 at pH 7. Insome embodiments, the cationic CPP is 5 to 20 amino acid residues and has an average net charge of at least +0.5 at pH 7. In some embodiments, the cationic CPP comprises at least five arginine residues.

[0005] In some embodiments, the CPP comprises a cationic arginine-rich peptide (CARP). In some embodiments, the CPP is a TAT -related CPP.

[0006] In some embodiments, the CPP further comprises a nuclear localization signal (NLS). In some embodiments, the NLS is an SV40 NLS (SEQ ID NO: 4).

[0007] In some embodiments, the thiol -reactive moiety is a cysteine. In some embodiments, the cysteine is further modified with a 3-nitro-2-pyridinesulfenyl group (NPyS).

[0008] In some embodiments, the CPP comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 11 (TAT -NLS). In some embodiments, the CPP comprises an amino acid sequence having at least 95% identity to SEQ ID NO: 11 (TAT -NLS). In some embodiments, the CPP comprises an amino acid sequence of SEQ ID NO: 11 (TAT -NLS).

[0009] In some embodiments, the CPP comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 12 (HIS-TAT-NLS). In some embodiments, the CPP comprises an amino acid sequence having at least 95% identity to SEQ ID NO: 12 (HIS-TAT-NLS). In some embodiments, the CPP comprises an amino acid sequence of SEQ ID NO: 12 (HIS- TAT-NLS).

[0010] In some embodiments, a cysteine of the CPP is further modified with a 3-nitro- pyridinesulfenyl group (NPyS).

[0011] In some embodiments, the site-directed modifying polypeptide is an RNA-guided nuclease. In some embodiments, the RNA-guided nuclease is a Class 2 Cas polypeptide. In some embodiments, the Class 2 Cas polypeptide is a Type II Cas polypeptide or a Type V Cas polypeptide. In some embodiments, the Type II Cas polypeptide is Cas9. In some embodiments, the Type V Cas polypeptide is Casl2.

[0012] In some embodiments, the site-directed modifying polypeptide is conjugated to the one or more CPPs via a cysteine residue.

[0013] In some embodiments, the plurality of CPPs is at a concentration between about 5 to about 15 pM. In some embodiments, the plurality of CPPs is at a concentration between about 10 to about 15 pM.

[0014] The present disclosure also provides a method of modifying a nucleic acid in a cell, the method comprising contacting the cell with any exemplary composition for ex vivo gene editing disclosed herein. In some embodiments, the method is performed ex vivo. In some embodiments, the cell is an immune cell. In some embodiments, the immune cell is a T-cell.Brief Description of the Drawings

[0015] FIG. 1 provides a graphical overview of a co-incubation assay to measure gene editing efficiency.

[0016] FIG. 2 shows the results of a screen for cell-penetrating peptides (CCPs).

[0017] FIG. 3 graphically depicts the results of an in vitro study evaluating the editing efficiency and cell viability using two cell -penetrating peptides (CPPs) from the screen of FIG. 2

[0018] FIG. 4A-B shows the gene editing efficiency of multiple Cas9 variants with cellpenetrating peptides (CPPs) provided in trans. FIG. 4A shows the results for an HTN with a thiol -reactive moiety. FIG. 4B shows the results for an HA-TAT peptide with no thiolreactive moiety.

[0019] FIG. 5A-C demonstrates the gene editing efficiency of multiple Cas9 variants with an HTN CPP. FIG. 5A shows the results for a WT Cas9 variant. FIG. 5B shows the results for a Cas9 with NPyS-activated cysteines. FIG. 5C shows the results for a cysteine-less Cas9.

[0020] FIG. 6 demonstrates the gene editing efficiency of Cas9 variants coupled to an HTN or NN CPP and mixed in trans with excess peptide containing or lacking a thiol -reactive moiety.Detailed Description

[0021] Provided herein are compositions and methods for enhancing the delivery and internalization of a site-directed modifying polypeptide. In order that the present disclosure may be more readily understood, certain terms are first defined. It should be noted that whenever a value or range of values of a parameter are recited, it is intended that values and ranges intermediate to the recited values are also part of the disclosure. The word “about” if not otherwise defined means ±5%. It is also noted that as used herein and in the claims, the singular forms “a,” “an”, and “the” include plural references unless the context clearly dictates otherwise.Definitions

[0022] As used herein, the term “internalization” refers to the entry, uptake, or transport of a macromolecule across a cell membrane. Internalization can occur via direct cell penetration (e.g., via cell -penetrating peptides) or via receptor-mediated endocytosis.

[0023] As used herein, the term “cell-penetrating peptide” (CPP) refers to a peptide, generally of about 5 to 60 amino acid residues in length, that can facilitate cellular uptake of amolecule, particularly one or more site-directed modifying polypeptides. In some embodiments, the CPP is conjugated to the one or more site-directed modifying polypeptides. A CPP can also be characterized by its ability to facilitate the movement or traversal of a molecular conjugate across or through one or more of a lipid bilayer, micelle, cell membrane, organelle membrane (e.g., a nuclear membrane or an endosomal membrane), vesicle membrane, or cell wall. CPPs can be classified as cationic, amphipathic, hydrophobic, or combinations thereof. Examples of CPPs suitable for use with the compositions and methods disclosed herein are described in Borrelli, Antonella, et al. Molecules 23.2 (2018): 295; Milletti, Francesca. Drug discovery today 17.15-16 (2012): 850-860, which are incorporated herein by reference. Further, there exists a database of experimentally validated CPPs (CPPsite, Gautam et al., 2012). The CPP can be any known CPP, such as a CPP shown in the CPPsite database. In some embodiments, the CPP comprises a nuclear localization signal or sequence (NLS).

[0024] As used herein, the term “conjugation” or “conjugated” refers to the physical or chemical complexation formed between two or more molecules (e.g., between a CPP and a site-directed modifying polypeptide). The chemical complexation constitutes specifically a bond or chemical moiety formed between a functional group of a first molecule with a functional group of a second molecule. In an exemplary embodiment, the functional group is a thiol -reactive group. Such bonds include, but are not limited to, covalent linkages and non- covalent bonds, while such chemical moieties include, but are not limited to, esters, carbonates, imines, phosphate esters, hydrazones, acetals, orthoesters, disulfides, peptide linkages, and oligonucleotide linkages.

[0025] Amino acid sequences described herein may include “conservative mutations,” including the substitution, deletion or addition of nucleic acids that alter, add or delete a single amino acid or a small number of amino acids in a coding sequence where the nucleic acid alterations result in the substitution of a chemically similar amino acid. A conservative amino acid substitution refers to the replacement of a first amino acid by a second amino acid that has chemical and / or physical properties (e.g., charge, structure, polarity, hydrophobicity / hydrophilicity) that are similar to those of the first amino acid. Conservative substitutions include replacement of one amino acid by another within the following groups: lysine (K), arginine (R) and histidine (H); aspartate (D) and glutamate (E); asparagine (N) and glutamine (Q); N, Q, serine (S), threonine (T), and tyrosine (Y); K, R, H, D, and E; D, E, N, and Q; alanine (A), valine (V), leucine (L), isoleucine (I), proline (P), phenylalanine (F), tryptophan (W), methionine (M), cysteine (C), and glycine (G); F, W, and Y; H, F, W, and Y;C, S and T; C and A; S and T; C and S; S, T, and Y; V, I, and L; V, I, and T. Other conservative amino acid substitutions are also recognized as valid, depending on the context of the amino acid in question. For example, in some cases, methionine (M) can substitute for lysine (K). In addition, sequences that differ by conservative variations are generally homologous.

[0026] As used herein, the term “genetically-modified cell” refers to a cell, or an ancestor thereof, in which a DNA sequence has been deliberately modified by a site-directed modifying polypeptide (e.g., a nucleic acid-guided nuclease). In an exemplary embodiment, the genetically-modified cell is produced ex vivo.

[0027] As used herein, the term “ex vivo gene editing” refers to the production of a genetically-modified cell in an ex vivo environment, for example using cells extracted from a subject such as autologous- or allogeneic-derived cells. In some embodiments, the cells are chimeric antigen receptor T (CAR-T) cells.

[0028] As used herein, the term “modifying a nucleic acid” refers to any modification to a nucleic acid targeted by a site-directed modifying polypeptide. Examples of such modifications include any changes to the amino acid sequence including, but not limited to, an insertion, deletion, or substitution of an amino acid residue in the nucleic acid sequence relative to a reference sequence (e.g., a wild-type or native sequence). Such amino acid changes may, for example, lead to a change in expression of a gene (e.g., an increase or decrease in expression) or replacement of a nucleic acid sequence). Modifications of nucleic acids can further include double stranded cleavage, single stranded cleavage, or binding of any RNA-guided endonuclease disclosed herein to a target site. Binding of an RNA-guided endonuclease can inhibit expression of the nucleic acid or can increase expression of any nucleic acid in operable linkage to the nucleic acid comprising the target site.

[0029] As used herein, the term “nuclear localization signal” or “NLS” refers to a peptide that, when conjugated or operably linked to a molecule (e.g., a CPP or a site-directed modifying polypeptide), is capable of promoting import of the molecule into the cell nuclease by nuclear transport. The NLS can, for example, direct transport of a protein with which it is associated from the cytoplasm of a cell across the nuclear envelope barrier. The NLS is intended to encompass not only the NLS of a particular peptide, but also derivatives thereof that are capable of directing translocation of a cytoplasmic polypeptide across the nuclear envelope barrier. In some embodiments, one or more NLSs (e.g., 1, 2, 3, 4, 5, 6 or more) can be conjugated or operably linked to a CPP and / or site-directed modifying polypeptide).

[0030] As used herein, the term “operably linked” refers to polynucleotide sequences or amino acid sequences placed into a functional relationship with one another. For example, regulatory sequences (e.g., a promoter or enhancer) are “operably linked” to a polynucleotide (c.g, encoding a guide RNA or nucleic acid-guided nuclease) if the regulatory sequences regulate or contribute to the modulation of the transcription or translation of the polynucleotide. Similarly, two polypeptide-encoding nucleotide sequences are operably linked if they are contiguous and capable of expression in the same reading frame so as to produce a “fusion protein” following transcription and translation.

[0031] As used herein, the term “site-directed modifying polypeptide” refers to a protein that is targeted to a specific nucleic acid sequence or set of similar sequences of a polynucleotide chain via recognition of the particular sequence(s) by the modifying polypeptide itself or an associated molecule (e.g., an RNA), wherein the polypeptide can modify the polynucleotide chain.

[0032] As used herein, the term “target cell” refers to a cell or population of cells, such as mammalian cells (e.g., human cells), which includes a nucleic acid sequence in which site- directed modification of the nucleic acid is desired (e.g., to produce a genetically-modified cell). In some embodiments, the target cell is a CAR-T cell.

[0033] As used herein, the term “TAT CPP” or “TAT-related CPP” refers to a CPP that is derived from the transactivator of transcription (TAT) of human immunodeficiency virus. The amino acid sequence of a TAT peptide comprises RKKRRQRRR (SEQ ID NO: 10). Thus, a TAT-related CPP includes any peptide comprising the amino acid sequence of RKKRRQRRR (SEQ ID NO: 10), or an amino acid sequence having conservative amino acid substitutions wherein the peptide retains its functionality (e.g., is still able to internalize into a cell). In some embodiments a TAT-related CPP includes 1, 2, or 3 amino acid substitutions. In some embodiments, the TAT-related CPP is operably linked to one or more NLSs and / or a site-directed modifying polypeptide. In some embodiments, the TAT-related CPP is further modified to comprise a cysteine at the N- or C-terminus.

[0034] As used herein, the term “thiol -reactive moiety” refers to a thiol-comprising moiety that is capable of reacting with another functional group to produce a covalent bond. In a preferred embodiment, the thiol -reactive moiety reacts with a second thiol -reactive moiety to produce a disulfide bond. In some embodiments, the thiol -reactive moiety is a cysteine. In this respect, the cysteine provides a thiol -reactive moiety via its thiol side chain. In some embodiments, the thiol -reactive moiety is treated with a thiol-protecting group and / or thiol- activating group (e.g., NPyS). An NPyS-capped cysteine can interact with, and readily form adisulfide bond with a free thiol. However, in certain embodiments an NPyS-capped cysteine cannot interact with, or form a disulfide bond with, another NPyS-capped cysteine. The use of thiol-protecting and activating groups are further described in Spears et al., Chem. Soc. Rev., 2021, the entirety of which is incorporated herein by reference.Cell-penetrating Peptides

[0035] In one aspect, disclosed herein are compositions and methods that comprise a plurality of cell-penetrating peptides (CPPs). The CPPs for use in the disclosed compositions and methods are provided both in cis and in trans with respect to the site-directed modifying polypeptide. That is, when one or more CPPs are conjugated to the site-directed modifying polypeptide, the CPPs are considered to be in cis with the site-directed modifying polypeptide. When one or more CPPs are provided without conjugation to the site-directed modifying polypeptide, the CPPs are considered to be in trans with the site-directed modifying polypeptide.

[0036] Accordingly, in one aspect provided herein are compositions that comprise a site- directed modifying polypeptide, wherein the site-directed modifying polypeptide is conjugated to one or more cell-penetrating peptides (e.g., in cis) and a plurality of CPPs in solution (e.g., in trans). Surprisingly, it was found that this combination resulted in an additive effect, affording enhanced internalization of the site-directed modifying polypeptide.

[0037] CPPs useful in the compositions and methods disclosed herein include, but are not limited to, protein-derived CPPs (e.g., TAT protein and penetratin), chimeric CPPs (e.g., Transportan derived from the binding of the neuropeptide galanin N-terminus to the Mastoparan toxin), and / or synthetic CPPs (e.g., oligoarginines or peptide nucleic acids formed by synthetic nucleic acid analogues bound to pseudopeptide backbone).

[0038] In some embodiments, the CPP is a cationic CPP. Cationic CPPs have a net positive charge, and accordingly have a high affinity for negatively charged cell membranes because of electrostatic interactions. As such, cationic CPPs may internalize into the cell through a receptor-independent mechanism. Key factors determining the activity of cationic CPPs are the number and position of positively charged amino acids in their structure. For example, a cationic or polycationic CPP may include an amino acid sequence having a high relative abundance (at least 60%) of positively charged amino acids such as lysine (K), arginine (R), and / or histidine (H). The CPPsite 2.0 database, which can be sorted by category according to cationic CPPs, includes the sequences of 714 known cationic CPPs. Examples of cationic cell-penetrating peptides include, but are not limited to, HIV-1 twinarginine translocation(TAT), diatos peptide vectors (DPV), arginine-rich peptides, polyarginine, penetratin, L5a, and protamine (See, e.g., Tarvirdipour, et al. International Journal of Molecular Sciences. 22.16 (2021)).

[0039] CPPs can be characterized by their net charge or net average charge. The term "net charge" refers to the summation of charges of ionizable groups of the constituent residues of a peptide such as at a pH in the range pH 6.0 to pH 7.0 including pH 7.0. In some embodiments, the cationic CPP has a net charge of +0.1 to +30 at neutral pH (e.g., pH 7) (Milletti, F.. "Cell-penetrating peptides: classes, origin, and current landscape." Drug discovery today 17.15-16 (2012): 850-860.) In some embodiments, the cationic CPP has a net charge of +1 to +30, +1 to +25, +1 to +20, +1 to +15, +1 to +10, +1 to +5, +5 to +30, +5 to +25, +5 to +20, +5 to +15, +5 to +10, +10 to +30, +10 to +25, +10 to +20, +10 to +15, +15 to +30, +15 to +25, +15 to +20, +20 to +30, +20 to +25, or +25 to +30. In some embodiments, the cationic CPP has a net charge of about +1, +2, +3, +4, +5, +6, +7, +8, +9, +10, +11, +12, +13, +14, +15, +16, +17, +18, +19, +20, +21, +22, +23, +24, +25, +26, +27, +28, +29, or +30.

[0040] Average net charge, as used herein, refers to the net charge of a peptide at pH 7 divided by the total number of residues. In some embodiments, the cationic CPP has a net average charge of +0.1 to +1 at neutral pH (e.g., pH 7) (Milletti, F. "Cell-penetrating peptides: classes, origin, and current landscape." Drug discovery today 17.15-16 (2012): 850- 860.) In some embodiments, the cationic CPP has a net average charge of +0.1 to +1, +0.1 to +0.8, +0.1 to +0.6, +0.1 to +0.4, +0.2 to +1, +0.2 to +0.8, +0.2 to +0.6, +0.2 to +0.4, +0.4 to +1, +0.4 to +0.8, +0.5 to +1, +0.5 to +0.8, +0.5 to +0.6, +0.6 to +1, +0.6 to +0.8, +0.8 to +1, +0.9 to +1. In some embodiments, the cationic CPP has a net average charge of about +0.1, +0.2, +0.3, +0.4, +0.5, +0.6, +0.7, +0.8, +0.9, or +1.

[0041] In some embodiments, the CPP comprises at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, or at least twelve positively charged residues. In some embodiments, the CPP comprises 3 to 15, 4 to 12, or 5 to 10 positively charged residues. In some embodiments, the CPP comprises three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, or fifteen positively charged residues.

[0042] In some embodiments, the positively charged residue is an arginine residue. In some embodiments, the CPP comprises at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, or at least twelve arginine residues. In some embodiments, the CPP comprises three to 15, four to 12, or five to tenarginine residues. In some embodiments, the CPP comprises three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, or fifteen arginine residues.

[0043] In some embodiments, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the amino acid residues of the CPP are arginine residues (e.g., before modifying the CPP with additional amino acids for conjugation). In some embodiments, 10-100%, 20-100%, 30-100%, 40-100%, 50-100%, 60- 100%, 70-100%, 80-100%, 90-100%, 10-90%, 20-90%, 30-90%, 40-90%, 50-90%, 60-90%, 70-90%, 80-90%, 10-80%, 20-80%, 30-80%, 40-80%, 50-80%, 60-80%, 70-80%, 10-70%,20-70%, 30-70%, 40-70%, 50-70%, 60-70%, 10-60%, 20-60%, 30-60%, 40-60%, 50-60%,10-50%, 20-50%, 30-50%, 40-50%, 10-40%, 20-40%, 30-40%, 10-30%, 20-30%, or 10-20% of the amino acid residues of the CPP are arginine residues (e.g., before modifying the CPP with additional amino acids for conjugation). In some embodiments, 100% of the amino acid residues of the CPP (e.g., before modifying the CPP with additional amino acids for conjugation) are arginine residues.

[0044] In some embodiments, the CPP is a cationic arginine-rich peptide (CARP). CARPs generally include the following properties: (i) range in size from 4 to 40 amino acids; (ii) have a positive net charge greater than or equal to +2 to +20; (iii) one or more positively charged arginine residues that comprise between 20% and 100% of the peptide; (iv) other positively charged amino acids namely lysine and histidine; and (v) amphiphilicity due to the presence of both hydrophilic (e.g., arginine, lysine) and hydrophobic (e.g., tryptophan, phenylalanine, tyrosine) amino acids. In some embodiments, the CARP has endocytic and / or non-endocytic cell membrane traversing properties, including the ability to cross the bloodbrain and blood-spinal cord barriers (BBB / BSCB). In some embodiments, the CARP comprises a CPP selected from TAT, penetratin, R5, R6, R7, R8, R9, R10, R11, R12, R13, R14, R15, R16, R17, R18, R19, R20, R21, R22, BEN2540, BN0540, XIP, R9 / X7 / R9, NCXBP3, LMWP, R10W4D, R10W8, R12W8a, R12F8, and R12Y8. Additional descriptions and examples of CARPs can be found, for example, at Meloni et al. Frontiers in Neurology. 11 (2020): 108, which is hereby incorporated by reference.

[0045] In some embodiments, the CPP is an amphiphilic or amphipathic CPP. Amphipathic CPPs may include polar and non-polar (rich in hydrophobic) amino acid regions that are able to fold into a-helical and P-sheet-like structures. In some embodiments, an amphipathic or amphiphilic CPP includes an amino acid sequence containing an alternating pattern of polar / charged residues and non-polar, hydrophobic residues. An amphipathic CPP can alternatively be characterized as possessing both hydrophilic and lipophilic properties. TheCPPsite 2.0 database, which can be sorted by category according to cationic CPPs, includes the sequences of 391 known cationic CPPs. Examples of amphipathic CPPs include, but are not limited to, MPG, transportan, NickFet (NF), PepFect (PF), MAP, croatmine, VP22, anatennapedia (Antp), Pep-1, CADY, FGF, pVEC (See, e.g., Tarvirdipour, et al. International Journal of Molecular Sciences. 22.16 (2021)).

[0046] In some embodiments, the CPP is a hydrophobic or lipophilic CPP. For example, a hydrophobic or lipophilic CPP may include an amino acid sequence having mostly, or only, non-polar residues with low net charge and / or hydrophobic amino acid groups. Examples of hydrophobic CPPs include, but are not limited to, C105Y, K-FGF, Bip, melittin-derived peptides (See, e.g., Tarvirdipour, et al. International Journal of Molecular Sciences. 22.16 (2021).

[0047] In particular embodiments, the CPP is a TAT -related CPP comprising the transactivator of transcription (TAT) of human immunodeficiency virus. The sequence of TAT is RKKRRQRRR (SEQ ID NO: 10). In some embodiments, the TAT-related cellpenetrating peptide, or variant thereof, may comprise one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) additional amino acids at the N-or C-terminus of SEQ ID NO: 10. In some embodiments, the TAT-related CPP includes one or more (e.g., 1, 2, 3, 4, 5 or more) amino acid insertions, deletions, or substitutions that do not disrupt the cell-penetrating properties of the TAT sequence. In some embodiments, the additional amino acid at the C-terminus is a cysteine.

[0048] In some embodiments, the CPP is or comprises an endosomal escape agent. In some embodiments, the CPP comprises a nuclear localization sequence or signal such as the SV40 large T antigen NLS comprising the sequence PKKKRKV as set forth in SEQ ID NO: 4 or the nucleoplasmin NLS comprising the sequence KRPAATKKAGQAKKKK as set forth in SEQ ID NO: 5. Other NLSs suitable for use in the disclosed compositions and methods are known in the art, see, e.g., Cokol et al., EMBO Rep. 2000 Nov. 15; 1(5): 411-415; Freitas and Cunha, Curr Genomics. 2009 December; 10(8): 550-557.

[0049] Further, CPPs suitable for use in the disclosed compositions and methods may comprise a fusion of one or more CPPs. In a preferred embodiment, the CPP comprises a TAT-related CPP and one or more nuclear localization signals, such as TAT-NLS (TN) as set forth in SEQ ID NO: 11. In some embodiments, the CPP comprises a TAT-related CPP and one or more nuclear localization signals in combination with one or more histidine residues (e.g., polyhistidines or His tags), such as HIS-TAT-NLS (HTN) as set forth in SEQ ID NO: 12.

[0050] A CPP herein can be about 5-40, 5-30, 5-25, 5-20, 5-15, 5-10,10-30, 10-25, 10-20, or 10-15 amino acid residues in length, for example. As other examples, a CPP can be about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acid residues in length. Yet in further aspects herein, a CPP can be up to about 35, 40, 45, 50, 55, or 60 amino acid residues in length.

[0051] A CPP suitable herein can alternatively comprise an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any of the CPP amino acid sequences disclosed herein. Such a variant CPP protein should have CPP activity, such as the ability to mediate cellular uptake of molecular cargo (e.g., an amino acid sequence comprising one or more site-specific modifying polypeptides (e.g., nuclease)). Testing the activity of a variant CPP can be done any number of ways, such as by covalently linking it with a fluorescent protein (e.g., GFP) and measuring the degree of fluorescence emitted from a cell contacted with a the CPP -fluorescent protein complex.

[0052] The CPPs disclosed herein may be used in cis and / or in trans with one or more site- directed modifying polypeptides.Site-directed Modifying Polypeptides

[0053] The cell-penetrating peptide compositions disclosed herein enhance the internalization of a side-directed modifying polypeptides. The site-directed modifying polypeptides used in the presently disclosed compositions and methods are site-specific, in that the polypeptide itself or an associated molecule recognizes and is targeted to a particular nucleic acid sequence or a set of similar sequences. In some embodiments, the site-directed modifying polypeptide (or its associated molecule) recognizes sequences that are similar in sequence, comprising conserved bases or motifs that can be degenerate at one or more positions.

[0054] In particular embodiments, the site-directed modifying polypeptide modifies the polynucleotide at particular location(s) (i.e., modification site(s)) outside of its target sequence. The modification site(s) modified by a particular site-directed modifying polypeptide are also generally specific to a particular sequence or set of similar sequences. In some of these embodiments, the site-directed modifying polypeptide modifies sequences that are similar in sequence, comprising conserved bases or motifs that can be degenerate at one or more positions. In other embodiments, the site-directed modifying polypeptide modifies sequences that are within a particular location relative to the target sequence(s). For example, the site-directed modifying polypeptide may modify sequences that are within a particular number of nucleic acids upstream or downstream from the target sequence(s).

[0055] As used herein with respect to site-directed modifying polypeptides, the term “modification” means any insertion, deletion, substitution, or chemical modification of at least one nucleotide the modification site or alternatively, a change in the expression of a gene that is adjacent to the target site. The substitution of at least one nucleotide in the modification site can be the result of the recruitment of a base editing domain, such as a cytidine deaminase or adenine deaminase domain (see, for example, Eid et al. (2018) Biochem J 475(11): 1955-1964, which is herein incorporated in its entirety).

[0056] The change in expression of a gene adjacent to a target site can result from the recruitment of a transcriptional activation domain or transcriptional repression domain to the promoter region of the gene or the recruitment of an epigenetic modification domain that covalently modifies DNA or histone proteins to alter histone structure and / or chromosomal structure without altering the DNA sequence, leading to changes in gene expression of an adjacent gene. The term “modification” also encompasses the recruitment to a target site of a detectable label that can be conjugated to the site-directed modifying polypeptide or an associated molecule (e.g., gRNA) that allows for the detection of a specific nucleic acid sequence (e.g., a disease-associated sequence).

[0057] In some embodiments, the site-directed modifying polypeptide is a nuclease or variant thereof and the agent comprising the nuclease or variant thereof. As used herein a “nuclease” refers to an enzyme which cleaves a phosphodiester bond in the backbone of a polynucleotide chain. Suitable nucleases for the presently disclosed compositions and methods can have endonuclease and / or exonuclease activity. An exonuclease cleaves nucleotides one at a time from the end of a polynucleotide chain. An endonuclease cleaves a polynucleotide chain by cleaving phosphodiester bonds within a polynucleotide chain, other than those at the two ends of a polynucleotide chain. The nuclease can cleave RNA polynucleotide chains (i.e., ribonuclease) and / or DNA polynucleotide chains (i.e., deoxyribonuclease).

[0058] Nucleases cleave polynucleotide chains, resulting in a cleavage site. As used herein, the term “cleave” refers to the hydrolysis of phosphodiester bonds within the backbone of a polynucleotide chain. Cleavage by nucleases can be single-stranded or double-stranded. In some embodiments, a double-stranded cleavage of DNA is achieved via cleavage with two nucleases wherein each nuclease cleaves a single strand of the DNA. Cleavage by the nuclease can result in blunt ends or staggered ends.

[0059] Non-limiting examples of nucleases suitable for the presently disclosed compositions and methods include meganucleases, such as homing endonucleases; restriction endonucleases, such as Type IIS endonucleases (e.g., FokI)); zinc finger nucleases;transcription activator-like effector nucleases (TALENs), and nucleic acid-guided nucleases (e.g., RNA-guided endonuclease, DNA-guided endonuclease, or DNA / RNA-guided endonuclease).

[0060] As used herein, a “meganuclease” refers to an endonuclease that binds DNA at a target sequence that is greater than 12 base pairs in length. Meganucleases bind to doublestranded DNA as heterodimers. Suitable meganucleases for the presently disclosed compositions and methods include homing endonucleases, such as those of the LAGLID ADG (SEQ ID NO: 14) family comprising this amino acid motif or a variant thereof.

[0061] As used herein, a “zinc finger nuclease” or “ZFN” refers to a chimeric protein comprising a zinc finger DNA-binding domain fused to a nuclease domain from an exonuclease or endonuclease, such as a restriction endonuclease or meganuclease. The zinc finger DNA-binding domain is bound by a zinc ion that serves to stabilize the unique structure.

[0062] As used herein, a “transcription activator-like effector nuclease” or “TALEN” refers to a chimeric protein comprising a DNA-binding domain comprising multiple TAL domain repeats fused to a nuclease domain from an exonuclease or endonuclease, such as a restriction endonuclease or meganuclease. TAL domain repeats can be derived from the TALE family of proteins from the Xanthomonas genus of Proteobacteria. TAL domain repeats are 33-34 amino acid sequences with hypervariable 12th and 13th amino acids that are referred to as the repeat variable diresidue (RVD). The RVD imparts specificity of target sequence binding. The TAL domain repeats can be engineered through rational or experimental means to produce variant TALENs that have a specific target sequence specificity (see, for example, Boch et al. (2009) Science 326(5959): 1509-1512 and Moscou and Bogdanove (2009) Science 326(5959): 1501, each of which is incorporated by reference in its entirety). DNA cleavage by a TALEN requires two DNA target sequences flanking a nonspecific spacer region, wherein each DNA target sequence is bound by a TALEN monomer. In some embodiments, the TALEN comprises a compact TALEN, which refers to an endonuclease comprising a DNA- binding domain with one or more TAL domain repeats fused in any orientation to any portion of a homing endonuclease (e.g., I-TevI, Mmel, EndA, Endl, I-BasI, LTevII, I-TevIII, I- Twol, MspI, Mval, NucA, and NucM). Compact TALENs are advantageous in that they do not require dimerization for DNA processing activity, thus only requiring a single target site.

[0063] As used herein, a “nucleic acid-guided nuclease” refers to a nuclease that is directed to a specific target sequence based on the complementarity (full or partial) between a guidenucleic acid (i.e., guide RNA or gRNA, guide DNA or gDNA, or guide DNA / RNA hybrid) that is associated with the nuclease and a target sequence. The binding between the guide RNA and the target sequence serves to recruit the nuclease to the vicinity of the target sequence. Non-limiting examples of nucleic acid-guided nucleases suitable for the presently disclosed compositions and methods include naturally-occurring Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-associated (Cas) polypeptides from a prokaryotic organism (e.g., bacteria, archaea) or variants thereof. CRISPR sequences found within prokaryotic organisms are sequences that are derived from fragments of polynucleotides from invading viruses and are used to recognize similar viruses during subsequent infections and cleave viral polynucleotides via CRISPR-associated (Cas) polypeptides that function as an RNA-guided nuclease to cleave the viral polynucleotides. As used herein, a “CRISPR-associated polypeptide” or “Cas polypeptide” refers to a naturally-occurring polypeptide that is found within proximity to CRISPR sequences within a naturally-occurring CRISPR system. Certain Cas polypeptides function as RNA-guided nucleases.

[0064] There are at least two classes of naturally-occurring CRISPR systems, Class 1 and Class 2. In general, the nucleic acid-guided nucleases of the presently disclosed compositions and methods are Class 2 Cas polypeptides or variants thereof given that the Class 2 CRISPR systems comprise a single polypeptide with nucleic acid-guided nuclease activity, whereas Class 1 CRISPR systems require a complex of proteins for nuclease activity. There are at least three known types of Class 2 CRISPR systems, Type II, Type V, and Type VI, among which there are multiple subtypes (subtype II-A, II-B, II-C, V-A, V-B, V-C, VI-A, VI-B, and VI-C, among other undefined or putative subtypes). In general, Type II and Type V-B systems require a tracrRNA, in addition to crRNA, for activity. In contrast, Type V-A and Type VI only require a crRNA for activity. All known Type II and Type V RNA-guided nucleases target double-stranded DNA, whereas all known Type VI RNA- guided nucleases target single-stranded RNA. The RNA-guided nucleases of Type II CRISPR systems are referred to as Cas9 herein and in the literature. In some embodiments, the nucleic acid-guided nuclease of the presently disclosed compositions and methods is a Type II Cas9 protein or a variant thereof. Type V Cas polypeptides that function as RNA-guided nucleases do not require tracrRNA for targeting and cleavage of target sequences. The RNA- guided nuclease of Type VA CRISPR systems are referred to as Cpfl; of Type VB CRISPR systems are referred to as C2C1; of Type VC CRISPR systems are referred to as Casl2C or C2C3; of Type VIA CRISPR systems are referred to as C2C2 or Casl3Al; of Type VLBCRISPR systems are referred to as Casl3B; and of Type VIC CRISPR systems are referred to as Casl3A2 herein and in the literature. In certain embodiments, the nucleic acid-guided nuclease of the presently disclosed compositions and methods is a Type VA Cpfl protein or a variant thereof. Naturally-occurring Cas polypeptides and variants thereof that function as nucleic acid-guided nucleases are known in the art and include, but are not limited to Streptococcus pyogenes Cas9 (SpCas9), Staphylococcus aureus Cas9 (SaCas9), Streptococcus thermophilus Cas9 (StCas9), Francisella novicida Cpfl, or those described in Shmakov et al. (2017) Nat Rev Microbiol 15(3): 169-182; Makarova et al. (2015) Nat Rev Microbiol 13(11):722-736; and U.S. Pat. No. 9790490, each of which is incorporated herein in its entirety. Class 2 Type V CRISPR nucleases include Casl2 and any subtypes of Casl2, such as Cas 12a, Cas 12b, Cas 12c, Cas 12d, Casl2e, Casl2f, Cas 12g, Casl2h, and Casl2i. Class 2 Type VI CRISPR nucleases including Cas 13 can be used as the site-directed modifying polypeptide in order to cleave RNA target sequences.

[0065] The nucleic acid-guided nuclease of the presently disclosed compositions and methods can be a naturally-occurring nucleic acid-guided nuclease (e.g., S. pyogenes Cas9) or a variant thereof. Variant nucleic acid-guided nucleases can be engineered or naturally occurring variants that contain substitutions, deletions, or additions of amino acids that, for example, alter the activity of one or more of the nuclease domains, fuse the nucleic acid- guided nuclease to a heterologous domain that imparts a modifying property (e.g., transcriptional activation domain, epigenetic modification domain, detectable label), modify the stability of the nuclease, or modify the specificity of the nuclease.

[0066] In some embodiments, a nucleic acid-guided nuclease includes one or more mutations to improve specificity for a target site and / or stability in the intracellular microenvironment. For example, where the protein is Cas9 (e.g., SpCas9) or a modified Cas9, it may be beneficial to delete any or all residues from N175 to R307 (inclusive) of the Rec2 domain. It may be found that a smaller, or lower-molecular mass, version of the nuclease is more effective. In some embodiments, the nuclease comprises at least one substitution relative to a naturally-occurring version of the nuclease. For example, where the protein is Cas9 or a modified Cas9, it may be beneficial to mutate C80 or C574 (or homologs thereof, in modified proteins with indels). In Cas9, desirable substitutions may include any of C80A, C80L, C80I, C80V, C80K, C574E, C574D, C574N, C574Q (in any combination) and in particular C80A. Substitutions may be included to reduce intracellular protein binding of the nuclease and / or increase target site specificity. Additionally or alternatively, substitutions may be included to reduce off-target toxicity of the composition.

[0067] In some embodiments, the RNA-guided DNA endonuclease is a Cas9 nuclease. In some embodiments, the Cas9 nuclease is wildtype Cas9 nuclease (e.g., Streptococcus pyogenes Cas9, SEQ ID NO: 1). In some embodiments, the Cas9 nuclease comprises an amino acid sequence having at least 85%, 90%, 95%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 1. In some embodiments, the Cas9 nuclease comprises an amino acid sequence having at least 85%, 90%, 95%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 1 (e.g., an engineered version of Cas9, such as variants of Cas9 described herein or otherwise known in the art).

[0068] In some embodiments, the RNA-guided DNA endonuclease is a nuclease other than Cas9 (e.g., such as a RNA-guided DNA endonuclease described herein). In some embodiments, the RNA-guided DNA endonuclease is a CRISPR Type V nuclease. In specific embodiments, the RNA-guided DNA endonuclease is a Casl2 nuclease. In some embodiments, the Casl2 nuclease is wildtype Casl2a nuclease (e.g., Acidaminococcus sp. Casl2a, SEQ ID NO: 2). In some embodiments, the Casl2 nuclease comprises an amino acid sequence having at least 85%, 90%, 95%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 2 (e.g., an engineered version of Casl2a, such as variants of Casl2a described herein or otherwise known in the art). Examples of Casl2a variants useful in the compositions disclosed herein include, but are not limited to, Alt-R® Cast 2a (Cpfl) Ultra (e.g., IDT Catalog No. 10001272) or Cast 2a as described in KI einstiver, et al. Nature biotechnology 37.3 (2019): 276-282, which is hereby incorporated by reference.

[0069] The nucleic acid-guided nuclease is directed to a particular target sequence through its association with a guide nucleic acid (e.g., guideRNA (gRNA), guideDNA (gDNA)). The nucleic acid-guided nuclease is bound to the guide nucleic acid via non-covalent interactions, thus forming a complex. The polynucleotide-targeting nucleic acid provides target specificity to the complex by comprising a nucleotide sequence that is complementary to a sequence of a target sequence. The nucleic acid-guided nuclease of the complex or a domain or label fused or otherwise conjugated thereto provides the site-specific activity. In other words, the nucleic acid-guided nuclease is guided to a target polynucleotide sequence (e.g. a target sequence in a chromosomal nucleic acid; a target sequence in an extrachromosomal nucleic acid, e.g. an episomal nucleic acid, a minicircle; a target sequence in a mitochondrial nucleic acid; a target sequence in a chloroplast nucleic acid; a target sequence in a plasmid) by virtue of its association with the protein-binding segment of the polynucleotide-targeting guide nucleic acid.

[0070] Thus, the guide nucleic acid comprises two segments, a "polynucleotide-targeting segment" and a "polypeptide-binding segment." By "segment" it is meant a segment / section / region of a molecule (e.g., a contiguous stretch of nucleotides in an RNA). A segment can also refer to a region / section of a complex such that a segment may comprise regions of more than one molecule. For example, in some cases the polypeptide-binding segment (described below) of a polynucleotide-targeting nucleic acid comprises only one nucleic acid molecule and the polypeptide-binding segment therefore comprises a region of that nucleic acid molecule. In other cases, the polypeptide-binding segment (described below) of a DNA-targeting nucleic acid comprises two separate molecules that are hybridized along a region of complementarity.

[0071] The polynucleotide-targeting segment (or "polynucleotide-targeting sequence" or “guide sequence”) comprises a nucleotide sequence that is complementary (fully or partially) to a specific sequence within a target sequence (for example, the complementary strand of a target DNA sequence). The polypeptide-binding segment (or "polypeptide-binding sequence") interacts with a nucleic acid-guided nuclease. In general, site-specific cleavage or modification of the target DNA by a nucleic acid-guided nuclease occurs at locations determined by both (i) base-pairing complementarity between the polynucleotide-targeting sequence of the nucleic acid and the target DNA; and (ii) a short motif (referred to as the protospacer adjacent motif (PAM)) in the target DNA.

[0072] A protospacer adjacent motif can be of different lengths and can be a variable distance from the target sequence, although the PAM is generally within about 1 to about 10 nucleotides from the target sequence, including about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, or about 10 nucleotides from the target sequence. The PAM can be 5' or 3' of the target sequence. Generally, the PAM is a consensus sequence of about 3-4 nucleotides, but in particular embodiments, can be 2, 3, 4, 5, 6, 7, 8, 9, or more nucleotides in length. Methods for identifying a preferred PAM sequence or consensus sequence for a given RNA-guided nuclease are known in the art and include but are not limited to the PAM depletion assay described by Karvelis et al. (2015) Genome Biol 16:253, or the assay disclosed in Pattanayak et al. (2013) Nat Biotechnol 31 (9): 839-43 , each of which is incorporated by reference in its entirety.

[0073] The polynucleotide-targeting sequence (i.e., guide sequence) is the nucleotide sequence that directly hybridizes with the target sequence of interest. The guide sequence is engineered to be fully or partially complementary with the target sequence of interest. In various embodiments, the guide sequence can comprise from about 8 nucleotides to about 30nucleotides, or more. For example, the guide sequence can be about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, or more nucleotides in length. In some embodiments, the guide sequence is about 10 to about 26 nucleotides in length, or about 12 to about 30 nucleotides in length. In particular embodiments, the guide sequence is about 30 nucleotides in length. In some embodiments, the degree of complementarity between a guide sequence and its corresponding target sequence, when optimally aligned using a suitable alignment algorithm, is about or more than about 50%, about 60%, about 70%, about 75%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more. In particular embodiments, the guide sequence is free of secondary structure, which can be predicted using any suitable polynucleotide folding algorithm known in the art, including but not limited to mFold (see, e.g., Zuker and Stiegler (1981) Nucleic Acids Res. 9: 133-148) and RNAfold (see, e.g., Gruber et al. (2008) Cell 106(l):23-24).

[0074] In some embodiments, a guide nucleic acid comprises two separate nucleic acid molecules (an "activator-nucleic acid" and a "targeter-nucleic acid", see below) and is referred to herein as a "double-molecule guide nucleic acid" or a "two-molecule guide nucleic acid." In other embodiments, the subject guide nucleic acid is a single nucleic acid molecule (single polynucleotide) and is referred to herein as a "single-molecule guide nucleic acid," a "single-guide nucleic acid," or an "sgNA." The term "guide nucleic acid” or "gNA" is inclusive, referring both to double-molecule guide nucleic acids and to single-molecule guide nucleic acids (i.e., sgNAs). In those embodiments wherein the guide nucleic acid is an RNA, the gRNA can be a double-molecule guide RNA or a single-guide RNA (sgRNA). Likewise, in those embodiments wherein the guide nucleic acid is a DNA, the gDNA can be a doublemolecule guide DNA or a single-guide DNA.

[0075] An exemplary two-molecule guide nucleic acid comprises a crRNA-like ("CRISPR RNA" or "targeter-RNA" or "crRNA" or "crRNA repeat") molecule and a corresponding tracrRNA-like ("trans-acting CRISPR RNA" or "activator-RNA" or "tracrRNA") molecule. A crRNA-like molecule (targeter-RNA) comprises both the polynucleotide-targeting segment (single stranded) of the guide RNA and a stretch ("duplex-forming segment") of nucleotides that forms one half of the dsRNA duplex of the polypeptide-binding segment of the guide RNA, also referred to herein as the CRISPR repeat sequence.

[0076] The term "activator-nucleic acid" or “activator-NA” is used herein to mean a tracrRNA-like molecule of a double-molecule guide nucleic acid. The term "targeter-nucleic acid" or “targeter-NA” is used herein to mean a crRNA-like molecule of a double-molecule guide nucleic acid. The term "duplex-forming segment" is used herein to mean the stretch of nucleotides of an activator-NA or a targeter-NA that contributes to the formation of the dsRNA duplex by hybridizing to a stretch of nucleotides of a corresponding activator-NA or targeter-NA molecule. In other words, an activator-NA comprises a duplex-forming segment that is complementary to the duplex-forming segment of the corresponding targeter-NA. As such, an activator-NA comprises a duplex -forming segment while a targeter-NA comprises both a duplex-forming segment and the DNA-targeting segment of the guide nucleic acid. Therefore, a subject double-molecule guide nucleic acid can be comprised of any corresponding activator-NA and targeter-NA pair.

[0077] The activator-NA comprises a CRISPR repeat sequence comprising a nucleotide sequence that comprises a region with sufficient complementarity to hybridize to an activator-NA (the other part of the polypeptide-binding segment of the guide nucleic acid). In various embodiments, the CRISPR repeat sequence can comprise from about 8 nucleotides to about 30 nucleotides, or more. For example, the CRISPR repeat sequence can be about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, or more nucleotides in length. In some embodiments, the degree of complementarity between a CRISPR repeat sequence and the antirepeat region of its corresponding tracr sequence, when optimally aligned using a suitable alignment algorithm, is about or more than about 50%, about 60%, about 70%, about 75%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more.

[0078] A corresponding tracrRNA-like molecule (i.e., activator-NA) comprises a stretch of nucleotides (duplex-forming segment) that forms the other part of the double-stranded duplex of the polypeptide-binding segment of the guide nucleic acid. In other words, a stretch of nucleotides of a crRNA-like molecule (i.e., the CRISPR repeat sequence) are complementary to and hybridize with a stretch of nucleotides of a tracrRNA-like molecule (i.e., the antirepeat sequence) to form the double-stranded duplex of the polypeptide-binding domain of the guide nucleic acid. The crRNA-like molecule additionally provides the single stranded DNA-targeting segment. Thus, a crRNA-like and a tracrRNA-like molecule (as acorresponding pair) hybridize to form a guide nucleic acid. The exact sequence of a given crRNA or tracrRNA molecule is characteristic of the CRISPR system and species in which the RNA molecules are found. A subject double-molecule guide RNA can comprise any corresponding crRNA and tracrRNA pair.

[0079] A trans-activating-like CRISPR RNA or tracrRNA-like molecule (also referred to herein as an “activator-NA”) comprises a nucleotide sequence comprising a region that has sufficient complementarity to hybridize to a CRISPR repeat sequence of a crRNA, which is referred to herein as the anti-repeat region. In some embodiments, the tracrRNA-like molecule further comprises a region with secondary structure (e.g., stem-loop) or forms secondary structure upon hybridizing with its corresponding crRNA. In particular embodiments, the region of the tracrRNA-like molecule that is fully or partially complementary to a CRISPR repeat sequence is at the 5' end of the molecule and the 3' end of the tracrRNA-like molecule comprises secondary structure. This region of secondary structure generally comprises several hairpin structures, including the nexus hairpin, which is found adjacent to the anti-repeat sequence. The nexus hairpin often has a conserved nucleotide sequence in the base of the hairpin stem, with the motif UNANNC found in many nexus hairpins in tracrRNAs. There are often terminal hairpins at the 3' end of the tracrRNA that can vary in structure and number, but often comprise a GC-rich Rho-independent transcriptional terminator hairpin followed by a string of U’s at the 3' end. See, for example, Briner et al. (2014) Molecular Cell 56:333-339, Briner and Barrangou (2016) Cold Spring Harb Protoc; doi: 10.1101 / pdb.top090902, and U.S. Publication No. 2017 / 0275648, each of which is herein incorporated by reference in its entirety.

[0080] In various embodiments, the anti-repeat region of the tracrRNA-like molecule that is fully or partially complementary to the CRISPR repeat sequence comprises from about 8 nucleotides to about 30 nucleotides, or more. For example, the region of base pairing between the tracrRNA-like anti-repeat sequence and the CRISPR repeat sequence can be about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, or more nucleotides in length. In some embodiments, the degree of complementarity between a CRISPR repeat sequence and its corresponding tracrRNA-like anti-repeat sequence, when optimally aligned using a suitable alignment algorithm, is about or more than about 50%, about 60%, about 70%, about 75%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more.

[0081] In various embodiments, the entire tracrRNA-like molecule can comprise from about 60 nucleotides to more than about 140 nucleotides. For example, the tracrRNA-like molecule can be about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, about 100, about 105, about 110, about 115, about 120, about 125, about 130, about 135, about 140, or more nucleotides in length. In particular embodiments, the tracrRNA-like molecule is about 80 to about 100 nucleotides in length, including about 80, about 81, about 82, about 83, about 84, about 85, about 86, about 87, about 88, about 89, about 90, about 91, about 92, about 93, about 94, about 95, about 96, about 97, about 98, about 99, and about 100 nucleotides in length.

[0082] A subject single-molecule guide nucleic acid (i.e., sgNA, e.g., sgRNA) comprises two stretches of nucleotides (a targeter-NA and an activator-NA) that are complementary to one another, are covalently linked by intervening nucleotides ("linkers" or "linker nucleotides"), and hybridize to form the double stranded nucleic acid duplex of the protein-binding segment, thus resulting in a stem-loop structure. The targeter-NA and the activator-NA can be covalently linked via the 3' end of the targeter-NA and the 5' end of the activator-NA.Alternatively, the targeter-NA and the activator-NA can be covalently linked via the 5' end of the targeter-NA and the 3' end of the activator-NA.

[0083] The linker of a single-molecule DNA-targeting nucleic acid can have a length of from about 3 nucleotides to about 100 nucleotides. For example, the linker can have a length of from about 3 nucleotides (nt) to about 90 nt, from about 3 nt to about 80 nt, from about 3 nt to about 70 nt, from about 3 nt to about 60 nt, from about 3 nt to about 50 nt, from about 3 nt to about 40 nt, from about 3 nt to about 30 nt, from about 3 nt to about 20 nt or from about 3 nt to about 10 nt, including but not limited to about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, or more nucleotides. In some embodiments, the linker of a single-molecule DNA-targeting nucleic acid is 4 nt.

[0084] An exemplary single-molecule DNA-targeting nucleic acid comprises two complementary stretches of nucleotides that hybridize to form a double-stranded duplex, along with a guide sequence that hybridizes to a specific target sequence.

[0085] Appropriate naturally-occurring cognate pairs of crRNAs (and, in some embodiments, tracrRNAs) are known for most Cas proteins that function as nucleic acid-guided nucleases that have been discovered or can be determined for a specific naturally-occurring Cas proteinthat has nucleic acid-guided nuclease activity by sequencing and analyzing flanking sequences of the Cas nucleic acid-guided nuclease protein to identify tracrRNA-coding sequence, and thus, the tracrRNA sequence, by searching for known antirepeat-coding sequences or a variant thereof. Antirepeat regions of the tracrRNA comprise one-half of the ds protein-binding duplex. The complementary repeat sequence that comprises one-half of the ds protein-binding duplex is called the CRISPR repeat. CRISPR repeat and antirepeat sequences utilized by known CRISPR nucleic acid-guided nucleases are known in the art and can be found, for example, at the CRISPR database on the world wide web at crispr.i2bc.paris-saclay.fr / crispr / .

[0086] The single guide nucleic acid or dual-guide nucleic acid can be synthesized chemically or via in vitro transcription. Assays for determining sequence-specific binding between a nucleic acid-guided nuclease and a guide nucleic acid are known in the art and include, but are not limited to, in vitro binding assays between an expressed nucleic acid- guided nuclease and the guide nucleic acid, which can be tagged with a detectable label (e.g., biotin) and used in a pull-down detection assay in which the nucleoprotein complex is captured via the detectable label (e.g., with streptavidin beads). A control guide nucleic acid with an unrelated sequence or structure to the guide nucleic acid can be used as a negative control for non-specific binding of the nucleic acid-guided nuclease to nucleic acids.

[0087] In some embodiments, the DNA-targeting RNA, gRNA, or sgRNA or nucleotide sequence encoding the DNA-targeting RNA, gRNA, or sgRNA comprises modifications of the nucleotide sequence. In some cases, the sgRNA (e.g., truncated sgRNA) comprises a first nucleotide sequence that is complementary to the target nucleic acid and a second nucleotide sequence that interacts with a Cas polypeptide. In other instances, the sgRNA comprises one or more modified nucleotides. In some cases, one or more of the nucleotides in the first nucleotide sequence and / or the second nucleotide sequence are modified nucleotides.

[0088] In some embodiments, the modified nucleotides comprise a modification in a ribose group, a phosphate group, a nucleobase, or a combination thereof. In some instances, the modification in the ribose group comprises a modification at the 2' position of the ribose group. In some cases, the modification at the 2' position of the ribose group is selected from the group consisting of 2'-O-methyl, 2'-fluoro, 2'-deoxy, 2'-O-(2-methoxy ethyl), and a combination thereof. In other instances, the modification in the phosphate group comprises a phosphorothioate modification. In other embodiments, the modified nucleotides are selected from the group consisting of a 2’ -ribo 3 ’-phosphorothioate (S), 2'-O-methyl (M) nucleotide, a2'-0-m ethyl 3'-phosphorothioate (MS) nucleotide, a 2'-O-m ethyl 3 '-thioPACE (MSP) nucleotide, and a combination thereof.

[0089] In certain embodiments, the site-directed modifying polypeptide of the presently disclosed compositions and methods comprise a nuclease variant that functions as a nickase. In one embodiment, the nuclease comprises a mutation in comparison to the wild-type nuclease that results in the nuclease only being capable of cleaving a single strand of a double-stranded nucleic acid molecule, or lacks nuclease activity altogether (i.e., nuclease- dead).

[0090] A nuclease, such as a nucleic acid-guided nuclease, that functions as a nickase only comprises a single functioning nuclease domain. In some of these embodiments, additional nuclease domains have been mutated such that the nuclease activity of that particular domain is reduced or eliminated.

[0091] In other embodiments, the nuclease (e.g., RNA-guided nuclease) lacks nuclease activity completely and is referred to herein as nuclease-dead. In some of these embodiments, all nuclease domains within the nuclease have been mutated such that all nuclease activity of the polypeptide has been eliminated. Any method known in the art can be used to introduce mutations into one or more nuclease domains of a site-directed nuclease, including those set forth in U.S. Publ. Nos. 2014 / 0068797 and U.S. Pat. No. 9,790,490, each of which is incorporated by reference in its entirety.

[0092] Any mutation within a nuclease domain that reduces or eliminates the nuclease activity can be used to generate a nucleic acid-guided nuclease having nickase activity or a nuclease-dead nucleic acid-guided nuclease. Such mutations are known in the art and include, but are not limited to the D10A mutation within the RuvC domain or H840A mutation within the HNH domain of the S. pyogenes Cas9 or at similar position(s) within another nucleic acid-guided nuclease when aligned for maximal homology with the S. pyogenes Cas9. Other positions within the nuclease domains of S. pyogenes Cas9 that can be mutated to generate a nickase or nuclease-dead protein include G12, G17, E762, N854, N863, H982, H983, and D986. Other mutations within a nuclease domain of a nucleic acid-guided nuclease that can lead to nickase or nuclease-dead proteins include a D917A, El 006 A, E1028A, D1227A, D1255A, N1257A, D917A, E1006A, E1028A, D1227A, D1255A, and N1257A of the Francisella novicida Cpfl protein or at similar position(s) within another nucleic acid-guided nuclease when aligned for maximal homology with the F. novicida Cpfl protein (U.S. Pat. No. 9,790,490, which is incorporated by reference in its entirety).

[0093] Site-directed modifying polypeptides comprising a nuclease-dead domain can further comprise a domain capable of modifying a polynucleotide. Non-limiting examples of modifying domains that may be fused to a nuclease-dead domain include but are not limited to, a transcriptional activation or repression domain, a base editing domain, and an epigenetic modification domain. In other embodiments, the site-directed modifying polypeptide comprising a nuclease-dead domain further comprises a detectable label that can aid in detecting the presence of the target sequence.

[0094] The epigenetic modification domain that can be fused to a nuclease-dead domain serves to covalently modify DNA or histone proteins to alter histone structure and / or chromosomal structure without altering the DNA sequence itself, leading to changes in gene expression. Non-limiting examples of epigenetic modifications that can be induced by site- directed modifying polypeptides include the following alterations in histone residues and the reverse reactions thereof: sumoylation, methylation of arginine or lysine residues, acetylation or ubiquitination of lysine residues, phosphorylation of serine and / or threonine residues; and the following alterations of DNA and the reverse reactions thereof: methylation or hydroxymethylation of cystosine residues. Non-limiting examples of epigenetic modification domains thus include histone acetyltransferase domains, histone deacetylation domains, histone methyltransferase domains, histone demethylase domains, DNA methyltransferase domains, and DNA demethylase domains.

[0095] In some embodiments, the site-directed polypeptide comprises a transcriptional activation domain that activates the transcription of at least one adjacent gene through the interaction with transcriptional control elements and / or transcriptional regulatory proteins, such as transcription factors or RNA polymerases. Suitable transcriptional activation domains are known in the art and include, but are not limited to, VP 16 activation domains.

[0096] In other embodiments, the site-directed polypeptide comprises a transcriptional repressor domain, which can also interact with transcriptional control elements and / or transcriptional regulatory proteins, such as transcription factors or RNA polymerases, to reduce or terminate transcription of at least one adjacent gene. Suitable transcriptional repression domains are known in the art and include, but are not limited to, IKB and KRAB domains.

[0097] In still other embodiments, the site-directed modifying polypeptide comprising a nuclease-dead domain further comprises a detectable label that can aid in detecting the presence of the target sequence. A detectable label is a molecule that can be visualized or otherwise observed. The detectable label may be fused to the nucleic-acid guided nuclease asa fusion protein (e.g., fluorescent protein) or may be a small molecule conjugated to the nuclease polypeptide that can be detected visually or by other means. Detectable labels that can be fused to the presently disclosed nucleic-acid guided nucleases as a fusion protein include any detectable protein domain, including but not limited to, a fluorescent protein or a protein domain that can be detected with a specific antibody. Non-limiting examples of fluorescent proteins include green fluorescent proteins (e.g., GFP, EGFP, ZsGreenl) and yellow fluorescent proteins (e.g., YFP, EYFP, ZsYellowl). Non-limiting examples of small molecule detectable labels include radioactive labels, such as3H and35S.

[0098] In some embodiments, the site-directed modifying polypeptide comprises a base editor comprised of a nickase domain or a nuclease-dead domain and a deaminase domain. The deaminase domain is capable of catalyzing the deamination of a nucleobase, such as cytosine or adenine, and can thus be referred to as a nucleobase deaminase. Deamination of a nucleobase by a deaminase can lead to a point mutation. The deaminase domain may be active on single-stranded nucleic acids or on double-stranded nucleic acids, but in some embodiments, the deaminase is only capable of deaminating single-stranded nucleic acids, including a single-stranded region of a double-stranded nucleic acid molecule (e.g., dsDNA) that is present in an R-loop formed by the binding of a nucleic acid-guided nickase or nuclease-dead domain to the double-stranded nucleic acid molecule (e.g., dsDNA). In certain embodiments, the nickase cleaves one strand of a double-stranded molecule and the complementary, non-target strand is acted upon by the deaminase.

[0099] In some embodiments, the deaminase domain of the site-directed modifying polypeptide is an adenine deaminase and the site-directed modifying polypeptide is referred to herein as an “A-base editor,” “adenine base editor” or “ABE”. The adenine deaminase domain is capable of deaminating an adenine, adenosine, or deoxyadenosine, yielding inosine which is recognized as guanine by polymerases and allows for the incorporation of a cytosine on the complementary nucleic acid strand, resulting in an A:T to G:C base pair change. While there are no known naturally-occurring DNA adenine deaminases, methods known in the art can be used to evolve and optimize adenine deaminase acting on tRNA (AD AT) proteins to have activity on DNA molecules, including a bacterial selection assay (see, e.g. Gaudelli et al, 2017; Koblan, L. W. et al, 2018, Nat Biotechnol 36, 843-846; Richter, M. F. et al, 2020, Nat Biotechnol, doi: 10.1038 / s41587-020-0562-8).

[0100] In other embodiments, the deaminase domain of the site-directed modifying polypeptide is a cytosine deaminase and the site-directed modifying polypeptide is referred to herein as a “C-base editor,” “cytosine base editor” or “CBE”. The cytosine deaminasedomain is capable of deaminating a cytosine, cytidine, or deoxycytidine to uracil, which can then be subsequently converted to thymine through DNA replication or repair. In particular embodiments, the deamination of a cytosine can result in the conversion of the cytosine to a guanine or adenine due to the activity of an uracil DNA glycosylase during base excision repair of the uracil residue. Thus, in some embodiments wherein the site-directed modifying polypeptide is a C-base editor and the desired nucleotide modification is a OT mutation, the site-directed modifying polypeptide further comprises an uracil DNA glycosylase inhibitor which prevents uracil DNA glycosylase from cleaving the N-glycosidic bond of uracil and initiating the base-excision repair pathway, leading to the stabilization of the uracil residue. Uracil glycosylase inhibitors are known in the art and include the small protein from Bacillus subtilis bacteriophage PBS1, the sequence of which is set forth as SEQ ID NO: 3.

[0101] Cytosine deaminases are known in the art and include apolipoprotein B mRNA- editing complex (APOB EC) family deaminases, such as deaminases from the APOBEC1 family, the activation-induced cytidine deaminase (AID), and the ACF1 / ASE deaminase. The cytosine deaminase may be a naturally-occurring protein or an active variant or fragment thereof.

[0102] The nucleic acid-guided nuclease can be delivered into a cell as a nucleoprotein complex comprising the nucleic acid-guided nuclease bound to its guide nucleic acid. Alternatively, the nucleic acid-guided nuclease is delivered and the guide nucleic acid is provided separately. In certain embodiments, a guide RNA can be introduced into a target cell as an RNA molecule. The guide RNA can be transcribed in vitro or chemically synthesized. In other embodiments, a nucleotide sequence encoding the guide RNA is introduced into the cell. In some of these embodiments, the nucleotide sequence encoding the guide RNA is operably linked to a promoter (e.g., an RNA polymerase III promoter), which can be a native promoter or heterologous to the guide RNA-encoding nucleotide sequence.

[0103] In certain embodiments, the site-directed polypeptide can comprise additional amino acid sequences, such as at least one nuclear localization sequence (NLS). Nuclear localization sequences enhance transport of the site-directed polypeptide into the nucleus of a cell. Proteins that are imported into the nucleus bind to one or more of the proteins within the nuclear pore complex, such as importin / karypherin proteins, which generally bind best to lysine and arginine residues. The best characterized pathway for nuclear localization involves short peptide sequence which binds to the importin-a protein. These nuclear localization sequences often comprise stretches of basic amino acids and given that there aretwo such binding sites on importin-a, two basic sequences separated by at least 10 amino acids can make up a bipartite NLS. The second most characterized pathway of nuclear import involves proteins that bind to the importin-pi protein, such as the HIV-TAT and HIV- REV proteins, which use the sequences RKKRRQRRR (SEQ ID NO: 10) and RQARRNRRRRWR (SEQ ID NO: 13), respectively to bind to importin-pi. Other nuclear localization sequences are known in the art (see, e.g., Lange et al., J. Biol. Chem. (2007) 282:5101-5105). The NLS can be the naturally-occurring NLS of the site-directed polypeptide or a heterologous NLS. As used herein, “heterologous” in reference to a sequence is a sequence that originates from a foreign species, or, if from the same species, is substantially modified from its native form in composition and / or genomic locus by deliberate human intervention. Non-limiting examples of NLS sequences that can be used to enhance the nuclear localization of the site-directed polypeptides include the NLS of the SV40 Large T-antigen and c-Myc. In certain embodiments, the NLS comprises the amino acid sequence PKKKRKV (SEQ ID NO: 4).

[0104] The site-directed polypeptide can comprise more than one NLS, such as two, three, four, five, six, or more NLS sequences. Each of the multiple NLSs can be unique in sequence or there can be more than one of the same NLS sequence used. The NLS can be on the amino-terminal (N-terminal) end of the site-directed polypeptide, the carboxy-terminal (C-terminal) end, or both the N-terminal and C-terminal ends of the polypeptide. In certain embodiments, the site-directed polypeptide comprises four NLS sequences on its N-terminal end. In other embodiments, the site-directed polypeptide comprises two NLS sequences on the C-terminal end of the site-directed polypeptide. In still other embodiments, the site- directed polypeptide comprises four NLS sequences on its N-terminal end and two NLS sequences on its C-terminal end.

[0105] Along with or as an alternative to an NLS, the site-directed polypeptide can comprise additional heterologous amino acid sequences, such as a detectable label (e.g., fluorescent protein) described elsewhere herein, or a purification tag, to form a fusion protein. A purification tag is any molecule that can be utilized to isolate a protein or fused protein from a mixture (e.g., biological sample, culture medium). Non-limiting examples of purification tags include biotin, myc, maltose binding protein (MBP), and glutathione-S-transferase (GST).

[0106] The presently disclosed compositions and methods can be used to edit genomes through the introduction of a sequence-specific, double-stranded break that is repaired (via e.g., error-prone non-homologous end-joining (NHEJ), microhomology -mediated end joining(MMEJ), or alternative end-joining (alt-EJ) pathway) to introduce a mutation at a specific genomic location. Due to the error-prone nature of repair processes, repair of the doublestranded break can result in a modification to the target sequence. Alternatively, a donor template polynucleotide may be integrated into or exchanged with the target sequence during the course of repair of the introduced double-stranded break, resulting in the introduction of the exogenous donor sequence. Accordingly, the compositions and methods can further comprise a donor template polynucleotide that may comprise flanking homologous ends. In some of these embodiments, the donor template polynucleotide is tethered to the site-directed modifying polypeptide via a linker as described elsewhere herein (e.g., the donor template polynucleotide is bound to the site-directed polypeptide via a cleavable linker).

[0107] In some embodiments, the donor sequence alters the original target sequence such that the newly integrated donor sequence will not be recognized and cleaved by the nucleic acid- guided nuclease. The donor sequence may comprise flanking sequences that have substantial sequence identity with the sequences flanking the target sequence to enhance the integration of the donor sequence via homology-directed repair. In particular embodiments wherein the nucleic acid-guided nuclease generates double-stranded staggered breaks, the donor polynucleotide can be flanked by compatible overhangs, allowing for incorporation of the donor sequence via a non-homologous repair process during repair of the double-stranded break.

[0108] The site-directed modifying polypeptides described herein may be conjugated to one or more CPPs to enhance internalization.Expression Vectors

[0109] In another aspect, provided herein is a cell expression vector comprising: a nucleic acid encoding a site-directed modifying polypeptide. In some embodiments, the expression construct(s) herein is an inducible co-expression system that co-expresses multiple components of a nucleoprotein, such as a guide nucleic acid (e.g., gRNA) and a site-directed modifying polypeptide (e.g., a RNA-guided nuclease), which then assemble to form nucleoproteins in the host cell. The gRNA and site-directed modifying polypeptide of the coexpression system can be encoded on the same construct or on separate constructs.

[0110] “Expression construct”, “construct” or “vector”, as used herein, refers to a polynucleotide vehicle that can be used to introduce genetic material into a cell. Constructs can be linear or circular. Constructs useful as expression constructs herein include plasmids, viral vectors (including phage), and integratable DNA fragments (i.e., fragments integratableinto the host genome by homologous recombination). The four major types of vectors are plasmids, viral vectors, cosmids, and artificial chromosomes. Expression constructs can contain a replication sequence capable of effecting replication of the construct in a suitable host cell (i.e., an origin of replication). Typically, expression constructs comprise an origin of replication, a multicloning site, and / or a selectable marker. Upon transformation of a suitable host, the expression construct may replicate and function independently of the host genome or integrate into the host genome. For example, methods for integrating expression constructs for stable transformation of prokaryotes are known in the art (see, e.g., Heap, J. T., et al., "Integration of DNA into bacterial chromosomes from plasmids without a counterselection marker," Nucleic Acids Res. (2012) 40:e59). Construct design depends, among other things, on the intended use and host cell for the expression construct, and the design of an expression construct for a particular use and host cell is within the level of skill in the art. [OHl] General methods for construction of expression constructs are known in the art. Expression constructs for most host cells are commercially available. There are several commercial software products designed to facilitate selection of appropriate constructs and construction thereof, such as bacterial plasmids for bacterial transformation and gene expression in bacterial cells, yeast plasmids for cell transformation and gene expression in yeast and other fungi, mammalian vectors for mammalian cell transformation and gene expression in mammalian cells or mammals, viral vectors (including retroviral, lentiviral, and adenoviral vectors) for cell transformation and gene expression and methods to easily enable cloning of such polynucleotides.

[0112] Expression constructs typically comprise regulatory sequences that are involved in one or more of the following: regulation of transcription, post-transcriptional regulation, and regulation of translation. Expression constructs can be introduced into a wide variety of organisms including bacterial cells, yeast cells, mammalian cells, and plant cells. Expression constructs typically comprise functional regulatory sequences set forth in the host cells or organism(s) into which they are being introduced. Further, expression constructs can include polynucleotides encoding protein tags (e.g., poly-His tags, hemagglutinin tags, fluorescent protein tags, quenchers, bioluminescent tags, nuclear localization tags). The coding sequences for such protein tags can be fused to the coding sequences (e.g., a sequence encoding a site directed modifying polypeptide). The expression construct can further comprise additional elements to promote nucleoprotein formation, such as 3’ hammerhead ribozyme or a ribozyme-guide-ribozyme system (see e.g., Gao, Y. and Yunde, Z. “Self-processing of ribozyme-flanked RNAs into guide RNAs in vitro and in vivo for CRISPR-mediated genomeediting,” J. Integr. Plant Biol. (2013) 56:343-349), to produce uniform gRNA termini compatible with nucleoprotein formation. In further embodiments, the expression vector can include restrict site cassettes to facilitate insertion of additional sequences, such as spacer sequences on an expression construct encoding a gRNA.

[0113] Expression constructs can be designed for expression in prokaryotic or eukaryotic cells. Examples of regulatory elements and expression constructs suitable for use in a variety of host cells are described further herein.Prokaryotic Expression Constructs

[0114] In some embodiments, the expression construct comprising the site-directed modifying polypeptide and / or CPP is an expression construct that can be introduced into, propagated in, and / or expressed in a prokaryotic cell (i.e., a prokaryotic expression construct). Prokaryotic expression constructs are well known in the art. For example, expression constructs and co-expression systems for use in prokaryotic systems are described in, e.g., US20160160203 Al, US20160376602A1, US20170159061A1, US20180282405 Al, WO2017106583A1, and WO2014025663A1, which are hereby incorporated by reference.

[0115] To be maintained with the host cell as independently replicating polynucleotides, typically a prokaryotic expression construct comprises an origin of replication, also called a replicon, suitable for the target host cell (e.g., oriC derived from E. coli, pUC derived from pBR322, pSClOl derived from Salmonella), or 15A origin (derived from pl5A) or bacterial artificial chromosomes). Origins of replication can be selected for use in expression constructs on the basis of incompatibility group, copy number, and / or host range, among other criteria.

[0116] The average number of copies of an expression construct in the cell, relative to the number of host chromosome molecules, is determined by the origin of replication contained in that expression construct. Copy number can range from a few copies per cell to several hundred.

[0117] Expression constructs can further include a selectable marker. A “selectable marker gene” refers to a gene that upon expression confers a phenotype by which successfully transformed cells carrying the expression construct can be identified. For example, the selectable marker can encode a protein necessary for the survival or growth of host cells in a selective culture medium. In such instances, host cells not containing the expression construct comprising the selection gene will not survive in the culture medium. Typical selection genes for prokaryotic expression systems encode proteins that confer resistance to antibiotics or other toxins, or that complement auxotrophic deficiencies of the host cell. One example of aselection scheme utilizes a drug such as an antibiotic to arrest growth of a host cell. Those cells that contain an expression construct comprising the selectable marker produce a protein conferring drug resistance and survive the selection regimen. Some examples of antibiotics that are commonly used for the selection of selectable markers (and abbreviations indicating genes that provide antibiotic resistance phenotypes) are: ampicillin (AmpR), chloramphenicol (CmlR or CmR), kanamycin (KanR), spectinomycin (SpcR), streptomycin (StrR), tetracycline (TetR), gentamicin (GenR). The native promoter region for a selection gene is usually included, along with the coding sequence for its gene product, as part of a selectable marker portion of an expression construct. Alternatively, the coding sequence for the selection gene can be expressed from a constitutive promoter. As another example, Zeocin™ (Life Technologies, Grand Island, NY) can be used as a selection in bacteria, fungi (including yeast), plants and mammalian cell lines. In some embodiments, the selectable marker is a gene that upon expression confers an identifiable phenotype. For example, the selectable marker may be a fluorescent marker that confers fluorescence in cells carrying the expression construct that can be identified visually or by machine, e.g., flow cytometry.

[0118] Useful promoters are known for expression of proteins in prokaryotes, for example, include those inducible or regulated by rhamnose, arabinose, xylose, lactose, IPTG, or phosphate. In some embodiments, the inducible promoter is an L-arabinose-inducible promoter, a propionate-inducible promoter, a rhamnose-inducible promoter, a xylose- inducible promoter, a lactose-inducible promoter, an IPTG-inducible promoter, or a promoter inducible by phosphate depletion. In certain embodiments, the inducible promoter is the araBAD promoter (see, e.g., Guzman et al. J of bacteriology 177.14 (1995): 4121-4130.), the T7 promoter, the T5 promoter, the pLac promoter, pTac promoter, the rhaBAD promoter (see, e.g., Kelly et al. ACS synthetic biology 5.10 (2016): 1136-1145), the prpBCDE promoter, the rhaSR promoter, or the xlyA promoter. For additional examples of promoters suitable for use in bacteria, see, for example, U.S. Publication No. US20160376602A1, which is hereby incorporated by reference in its entirety.

[0119] For example, in some embodiments the gRNA can be under a tunable promoter system such as AraC-ParaBAD or RhamS-PrhaBAD to generated optimal levels of expression for maximizing nucleoprotein formation. Alternatively, in other embodiments, the gRNA can be under a T7 promoter for maximum expression. In yet another embodiment the nucleic acid-guided nuclease or gRNA can be under an IPTG-inducible promoter such as T5, pLac, or pTac.

[0120] Expression constructs can also comprise coding sequences that are expressed from constitutive promoters. Unlike inducible promoters, constitutive promoters initiate continual gene product production under most growth conditions. Examples of constitutive promoters includes the promoter of the Tn3 bla gene, the promoter for the E. coli lipoprotein gene, Ipp; or the trpLEDCBA promoter. Constitutive promoters can be used in expression constructs for the expression of selectable markers, as described herein, and also for the constitutive expression of other gene products useful for the expression of the desired product. For example, transcriptional regulators of the inducible promoters, such as AraC, PrpR, RhaR, and XylR, if not expressed from a bidirectional inducible promoter, can alternatively be expressed from a constitutive promoter, on either the same expression construct as the inducible promoter they regulate, or a different expression construct. Similarly, gene products useful for the production or transport of the inducer, such as PrpEC, AraE, or Rha, or proteins that modify the reduction-oxidation environment of the cell, as a few examples, can be expressed from a constitutive promoter within an expression construct.

[0121] Expression of proteins in prokaryotes is often carried out in bacteria, such as Escherichia coli with expression constructs containing constitutive or inducible promoters directing the expression of the expressed components of the expression construct. Examples of inducible promoters and related genes include those that function in Escherichia coli (E. coli) strain MG1655 (American Type Culture Collection deposit ATCC 700926), which is a substrain of E. coli K-12 (American Type Culture Collection deposit ATCC 10798). For example, Table 1 in US Patent Application No. US20160376602 lists the genomic locations, in E. coli MG1655, of the nucleotide sequences for these examples of inducible promoters and related genes, which is hereby incorporated by reference. Additional information about E. coli promoters, genes, and strains described herein can be found in many public sources, including the online EcoliWiki resource, located at ecoliwiki.net.

[0122] Prokaryotic expression constructs can also include ribosome binding sites of varying strength, and secretion signals (e.g., mal, sec, tat, ompC, and pelB). For polypeptide gene products, the nucleotide sequence of the region between the transcription initiation site and the initiation codon of the coding sequence of the gene product that is to be inducibly expressed corresponds to the 5' untranslated region (‘UTR’) of the mRNA for the polypeptide gene product. In some embodiments, the region of the expression construct that corresponds to the 5' UTR comprises a polynucleotide sequence similar to the consensus ribosome binding site (RBS, also called the Shine-Dalgarno sequence) that is found in the species of the host cell. In prokaryotes, the RBS consensus sequence is GGAGG or GGAGGU, and inbacteria such as E. coli, the RBS consensus sequence is AGGAGG or AGGAGGU. The RBS is typically separated from the initiation codon by 5 to 10 intervening nucleotides. In some embodiments, the RBS sequence is at least 55% identical to the AGGAGGU consensus sequence, at least 70% identical, or at least 85% identical, and is separated from the initiation codon by 5 to 10 intervening nucleotides, by 6 to 9 intervening nucleotides, or by 6 or 7 intervening nucleotides.Cell-penetrating Peptides Compositions

[0123] The compositions disclosed herein comprise one or more CPPs provided in cis (with respect to the site-directed modifying polypeptide) and a plurality of CPPs provided in solution in trans (with respect to the site-directed modifying polypeptide).CPPs provided in cis

[0124] In one aspect, disclosed herein are compositions comprising a site-directed modifying peptide conjugated to one or more CPPs. In some embodiments, the one or more CPPs are conjugated to the site-directed modifying polypeptide via a disulfide bond formed between a thiol -reactive moiety on the site-directed modifying polypeptide and a thiol -reactive moiety on the CPP. The thiol -reactive moiety can be naturally present in the site-directed modifying peptide, or can be introduced. In some embodiments, the thiol -reactive moiety is from a cysteine. The CPP provided in cis can be conjugated to the site-directed modifying polypeptide via any method known in the art. In other embodiments, the CPP can be operably linked to the site-directed modifying polypeptide. In some embodiments, the CPP is an HTN (SEQ ID NO: 12) and the site-directed modifying polypeptide is a Cas9 (SEQ ID NO: 1). In some embodiments, the CPP is a TN (SEQ ID NO: 11) and the site-directed modifying polypeptide is a Cas9 (SEQ ID NO: 1).

[0125] To enhance efficiency of the conjugation of the CPP to the site-directed modifying polypeptide, a thiol-protecting group and / or a thiol-activating group may be utilized. In a preferred embodiment, the thiol-protecting group and / or thiol-activating group is a 3-nitro-2- pyridinesulfenyl (NPyS) group, shown below:-nitro-2-pyridinesulfenyl).

[0126] In some embodiments, the thiol-protecting group and / or thiol-activating group is a 5- nitro-2-pyridinesulfenyl group.

[0127] In some embodiments, the thiol-protecting group and / or thiol-activating group is a 2- pyridinesulfenyl group.

[0128] The NPyS group can selectively interact with a free thiol on a cysteine to form a disulfide bond, resulting in an NPyS-capped cysteine:-capped cysteine)

[0129] As used herein, the term “NPyS-capped cysteine” or “NPyS-capped thiol” refers to a disulfide bond formed between an NPyS group and a thiol group (e.g., the free thiol of a cysteine). Importantly, NPyS functions as both a protecting and activating group. An NPyS- capped cysteine can interact with, and readily form a disulfide bond with a free thiol.However, an NPyS-capped cysteine cannot interact with, or form a disulfide bond with, another NPyS-capped cysteine. The use of thiol-protecting and activating groups are further described in Spears et al., Chem. Soc. Rev., 2021, the entirety of which is incorporated herein by reference.CPPs provided in trans

[0130] In one aspect, disclosed herein are compositions comprising a plurality of CPPs in solution that are provided in trans. The addition of CPPs in solution that are provided in trans were found to enhance the internalization of the CPP-conjugated site-directed modifying polypeptide provided in cis.

[0131] The plurality of CPPs in solution that are provided in trans were found to enhance the internalization of a site-directed modifying polypeptide relative to the site-directed modifying polypeptide alone, even if the site-directed modifying polypeptide was conjugated to one or more CPPs. Without wishing to be bound by any particular theory, the enhanced internalization afforded by the CPPs in solution is thought to be due to the presence of a thiol -reactive moiety (see e.g., Schneider et al., 2021; Guo et al., 2021; and Abegg et al., 2017). Accordingly, in one aspect, the CPPs in solution provided in trans comprise a thiolreactive moiety. In some embodiments, the thiol -reactive moiety is a cysteine (e.g., the thiolside chain residue of a cysteine). In some embodiments, the CPP is HTN (SEQ ID NO: 12). In some embodiments, the CPP is TN (SEQ ID NO: 11). In some embodiments, the thiolreactive moiety is treated with a thiol-protecting group and / or a thiol-activating group (e.g., NPyS). As such, in some embodiments, the CPP comprises an NPyS-capped cysteine.

[0132] CPPs suitable for use in the disclosed compositions can be determined by screening for enhanced internalization. In particular, a screen may be utilized to identify CPPs that confer an additive effect when provided in trans. Using the screen as described in Example 1, it was determined that CPPs with multiple stretches of cationic residues interspersed with non-polar residues afforded high degrees of additivity, but also had a high tendency for aggregation and cellular toxicity. Less hydrophobic CPPs, such as the HTN and TN peptides disclosed herein, were highly effective at enhancing internalization with minimal cytotoxicity.Effect of CPPs provided in cis and in trans

[0133] It was determined that certain CPPs, when provided both a) in cis (with respect to the site-directed modifying polypeptide) and b) in trans (with respect to the site-directed modifying polypeptide) afforded enhanced internalization relative to either a) or b) alone. Accordingly, the CPPs disclosed herein provide an additive effect to internalization of the site-directed modifying peptide when provided both in cis and in trans. This additive effect is further enhanced when the CPP comprises a thiol -reactive moiety, such as a cysteine or an NPyS-capped cysteine. In some embodiments, the effect of providing the CPPs disclosed herein both in cis and in trans along with a site-directed modifying polypeptide can be greater than the sum of the effects of providing the CPPs in cis and in trans. That is, in specific embodiment the effect of providing the CPPs disclosed herein both in cis and in trans along with a site-directed modifying polypeptide can be synergistic.Methods of Use

[0134] The compositions disclosed herein (i.e., the composition comprising a CPP- conjugated site-directed modifying polypeptide in cis and a plurality of CPPs in solution provided in trans) can be used to modify the genome of a target cell. The method comprises contacting a cell with a gene editing agent disclosed herein, such that at least the site-directed modifying polypeptide is internalized into the cell and subsequently modifies the genome (or target nucleic acid) of the targeted cell. Such methods may be used in an in vitro setting or an ex vivo setting, including the modification of a cell or population of cells that are derived from a subject (e.g., an autologous or allogeneic cell or gene therapy setting).

[0135] Accordingly, in one aspect disclosed herein is a method of modifying a nucleic acid sequence within a cell, the method comprising contacting the cell with the compositions disclosed herein, including a) a site-directed modifying polypeptide, wherein the site-directed modifying polypeptide is conjugated to one or more CPPs; and b) a plurality of CPPs in solution, wherein the plurality of CPPs comprise a thiol -reactive moiety.

[0136] The cell can be a eukaryotic cell, such as a mammalian cell (e.g., a human cell). Examples of mammalian cells that can be targeted (and have their genome’s modified) by the DNA-targeting systems herein include, but are not limited to, a mouse cell, a non-human primate cell, or a human cell.

[0137] The compositions disclosed herein can be used to edit a range of specific cell types ex vivo, including cancer cells, immune cells, monocytes, macrophages, endothelial cells, epithelial cells, natural killer cells, pericytes, DC cells, non-DC myeloid cells, B cells, T cells (e.g., activated T cells; human T cells), retinal endothelial cells, fibroblasts, or other cells.

[0138] In some embodiments, the cells are cancer cells. In some embodiments, the cells are immune cells. In some embodiments, the cells are T cells. In some embodiments, the T cells are CD4+ or CD8+ T cells. In some embodiments, the T cells are regulatory T cells (Tregs) or effector T cells. In some embodiments, the T cells are tumor infiltrating T cells. In some embodiments, the cell is a hematopoietic stem cell (HSC) or a hematopoietic progenitor cell (HPSC). In some embodiments, the cells are macrophages. In some embodiments, the macrophages are MO, Ml, or M2 macrophages. In some embodiments, the cells are endothelial cells. In some embodiments, the cells are epithelial cells. In some embodiments, the cells are natural killer cells. In some embodiments, the cells are pericytes.

[0139] In some embodiments, the compositions disclosed herein can be used to edit multiple (e.g., two or more) cell types from cancer cells, monocytes, macrophages (e.g., Ml macrophages, M2 macrophages), endothelial cells, epithelial cells, retinal epithelial cells, natural killer cells, pericytes, DC cells, non-DC myeloid cells, T cells (e.g., activated T cells) and fibroblasts. The cell can be one that exists in a tissue or group of cells ex vivo or in an in vitro state. In certain instances, the eukaryotic cell herein can be as it exists in an isolated state (e.g., an in vitro state or ex vivo state).

[0140] In certain embodiments, the site-directed modifying polypeptide can be delivered in combination with a guide RNA (gRNA) ) into a cell as a nucleoprotein complex comprising the site-directed modifying polypeptide bound to the gRNA. Alternatively, the site-directed modifying polypeptide is delivered and the guide RNA is provided separately. In certain embodiments, a guide RNA can be introduced into a target cell as an RNA molecule. Theguide RNA can be transcribed in vitro or chemically synthesized. In other embodiments, a nucleotide sequence encoding the guide RNA is introduced into the cell. In some of these embodiments, the nucleotide sequence encoding the guide RNA is operably linked to a promoter (e.g., an RNA polymerase III promoter), which can be a native promoter or heterologous to the guide RNA-encoding nucleotide sequence.

[0141] In certain embodiments, the site-directed modifying polypeptide produces a cleavage site at the target region of the genome of the target cell, subsequently modifying the genome of the cell and impacting gene expression. Thus, in one embodiment, the target region of the genome is a target gene. The site-directed modifying polypeptide’s ability to modify the genome of the target cell provides, in certain embodiments, a way to modify expression of the target gene. Expression levels of a target nucleic acid, e.g., a gene, can be determined according to standard methods. In certain circumstances, the method disclosed herein is effective to increase expression of the target gene relative to a reference level. Alternatively, in other circumstances, the method disclosed herein is able to decrease expression of the target gene relative to a reference level. Reference levels can be determined in standard assays using a non-specific guide RNA / site-directed modifying polypeptide, where increases or decreases in the target nucleic acid, e.g., gene, may be measured relative to the control.

[0142] The ability of the site-directed modifying polypeptide to target a nucleic acid , e.g., gene, in a target cell can be determined according to methods known in the art, including, for example, phenotypic assays or sequencing assays. Such assays may determine the presence or absence of a modified gene, or a marker associated with the gene or nucleic acid, in a cell or in a population of cells that have been contacted by the site-directed modifying polypeptide.

[0143] The percentage of cells that include a modified gene may be used to assess the editing rate. For example, the target gene may be modified in at least about 0.10%, 0.11%, 0.12%, 0.13%, 0.14%, 0.15%, 0.16%, 0.17%, 0.18%, 0.19%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%, 4%, 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more cells in the population of cells. In certain embodiments, the methods herein are effective to modify a target gene in at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or atleast about 99% of cells in a population of cells. Ranges of the foregoing percentages are also contemplated herein.

[0144] In some embodiments, editing of a target gene can be determined by measuring the levels of the corresponding mRNA transcript or protein in a population of cells and compared to a control site-directed modifying polypeptide, e.g., where a non-targeting guide RNA is used as a negative control in the same type of target cell. In some embodiments, decreases in the level of a target gene for example, relative to the control indicates editing of the corresponding gene. In certain instances, a decrease of at least 0.10%, at least 0.11%, at least 0.12%, at least 0.13%, at least 0.14%, at least 0.15%, at least 0.16%, at least 0.17%, at least 0.18%, at least 0.19%, at least 0.2%, at least 0.3%, at least 0.4%, at least 0.5%, at least 0.6%, at least 0.7%, at least 0.8%, at least 0.9%, at least 1%, at least 2%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, and so forth, relative to a control in a testing assay indicates editing of a target gene. Ranges of the foregoing percentages are also contemplated herein.

[0145] Other ways in which nucleic acid (e.g., gene) editing activity of a site-directed modifying polypeptide can be determined include sequence-based assays, e.g., amplicon sequencing, known in the art.

[0146] The compositions disclosed herein enhance the internalization of the site-directed modifying polypeptide. In one aspect, disclosed herein are compositions that increase the level of internalization of the site-directed modifying polypeptide into the cell, e.g., relative to a reference level.

[0147] Accordingly, in one aspect, provided herein is a method of increasing internalization of a site-directed modifying polypeptide within a cell, the method comprising contacting the cell with a composition comprising a site-directed modifying peptide conjugated to one or more CPPs and a plurality of CPPs in solution wherein the CPPs comprise a thiol -reactive group, in concentrations effective to increase internalization of the site-directed modifying polypeptide into the cell relative to a reference level. In some embodiments, the reference level is the level of internalization of the site-directed modifying polypeptide in the absence of the plurality of CPPs provided in solution. In some embodiments, the reference level is a predetermined threshold level. In some embodiments, the reference level is the level of internalization of the site-directed modifying polypeptide fused to one or more alternative cell-penetrating peptides.

[0148] Internalization of the site-directed modifying polypeptide can be determined using standard internalization assays. In one embodiment, at least 1%, at least 2%, at least 3%, atleast 4%, at least 5%, at least 8%, at least 9%, at least 10%, or at least 15% of the site- directed modifying polypeptide is internalized by the cell within a given time (e.g., one hour, two hours, three hours, or more than three hours) of contact with the target cell. For instance, in certain embodiments, the site-directed modifying polypeptide is internalized by a target cell within a given amount of time of contact at a higher efficiency versus a control agent.

[0149] In specific embodiments, internalization refers to at least 0.01%, at least 0.05%, at least 0.1%, at least 0.5%, at least 1%, at least 2%, at least 5% at least 10%, at least 15%, or at least 20% of the agents or compositions internalized into a cell (e.g., within 10 minutes, 15 minutes, 20 minutes, 30 minutes, 40 minutes, 50 minutes, 1 hr, 2 hrs, 3 hrs, 4 hrs, or more). Internalization of an agent into a cell can refer to agents that have internalized, for example, into the cytoplasm of the cell or have been internalized as cargo in vesicles (e.g., endocytic vesicles).

[0150] Internalization of the site-directed modifying polypeptide, or a component thereof, can be assessed using any internalization assays known in the art. For example, internalization can be assessed by attaching a detectable label (e.g. a fluorescent dye) to the peptide (and / or to the cargo to be transfected) or by fusing the peptide with a reporter molecule, thus enabling detection once cellular uptake has occurred, e.g., by means of FACS analysis or via specific antibodies. In some embodiments, one or more components of the site-directed modifying polypeptide is conjugated to a reporter molecule having a quenchable signal (e.g, for FRET analysis).

[0151] In some embodiments, a flow cytometry-based internalization assay can be utilized based on the detection of a site-directed modifying polypeptide having one or more components labeled with a reporter molecule (e.g, a fluorescent protein). Following incubation of the labeled site-directed modifying polypeptide with cells for a given period of time, the cells are treated with a compound that quenches the signal and the results achieved with or without quenching are compared. Labeled molecules that are internalized by a target cell are protected from quenching (provided the quenching agent cannot permeate the target cell) and therefore retain a stronger signal relative to a control. In contrast, labeled molecules that are not internalized are susceptible to quenching and therefore display a reduced signal relative to an unquenched control.

[0152] In some embodiments, internalization can be assessed indirectly based on assays to assess gene editing. For instance, an increase in internalization is expected to correspond to an increase in the percentage of cells that undergo gene editing relative to a control condition.For example, without wishing to be bound to theory, cells contacted with the compositions disclosed herein may show increased gene editing relative to controls.

[0153] Once a site-directed modifying polypeptide is inside a cell, it may traverse an organelle membrane such as a nuclear membrane or mitochondrial membrane, for example. In certain embodiments, the site-specific modifying polypeptide includes at least one (e.g., at least 1, 2, 3, 4, or more) nuclear-targeting sequence (e.g., NLS). In other embodiments, the ability to traverse an organelle membrane such as a nuclear membrane or mitochondrial membrane does not depend on the presence of a nuclear-targeting sequence. Accordingly, in some embodiments, the site-specific modifying polypeptide does not include an NLS.

[0154] The compositions disclosed herein can be formulated to be compatible with the intended method for use. Solutions, suspensions, dispersions, or emulsions may be used for such administrations and may include a sterile diluent, such as water for injection, saline solution, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methylparabens; antioxidants such as ascorbic acid or sodium bi sulfate; buffers such as acetates, citrates or phosphates, and agents for the adjustment of tonicity such as sodium chloride or dextrose. The pH may be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. Preparations may be enclosed in ampules, disposable syringes or multiple dose vials made of glass or plastic. In certain embodiments, a pharmaceutical composition comprises the compositions disclosed herein and a pharmaceutically acceptable carrier.Exemplification

[0155] The disclosure will be more fully understood by reference to the following examples. They should not, however, be construed as limiting the scope of the disclosure.Example 1

[0156] The following example describes a screen for identifying high-activity cellpenetrating peptides. CPPs were incubated with either a wild-type Cas9 (“Cas9wt”)(SEQ ID NO: 1) or a cysteine-less Cas9 variant (“Cas9C80’574S”)(SEQ ID NO: 17). In both instances, the Cas9 was complexed with CD47 knockout sgRNA. The CPPs in conjunction with the Cas9 complexes were tested in the assay as shown in FIG. 1. Primary T-cells were first stimulated, and then the Cas9 complexes were separately co-incubated with or without 8 pM of a selected CPP. The cells were washed and assessed for viability after 24 hours using live / dead staining. The editing efficiency was evaluated following a 5 day incubation periodusing flow cytometry (assessing the % of cells with a CD47 knockout amongst CD3+ cells). The results of the screen are shown in FIG. 2 as the fold change of % editing with CPP versus without CPP. Higher activity of a peptide with wild-type Cas9 indicates that some of the activity is driven by the Cas9-CPP conjugate and not purely trans peptide-mediated delivery.

[0157] CPPs identified from this screen were tested for their ability to mediate high- efflciency editing while maintaining viability. Human T-cells were co-incubated with wildtype Cas9 (3 pM) complexed with a CD47 knockout sgRNA and either HTN{NPyS-Cys} (SEQ ID NO: 15) or TN{NPyS-Cys}(SEQ ID NO: 16). HTN{NPyS-Cys} and TN{NPyS- Cys} were tested across a range of concentrations from 2 pM to 18 pM. Cell viability and % editing were measured using live / dead staining and flow cytometry, respectively, approximately 6 days post-incubation. As shown in FIG. 3, both HTN{NPyS-Cys} and TN{NPyS-Cys}permitted high editing efficiency, up to -80% (black circles and triangles), without compromising cell viability (grey circle and triangles).Example 2

[0158] To further understand the additivity of the CPPs provided in cis and in trans, various combinations of CPP and site-directed modifying polypeptide were tested using a coincubation protocol as shown in FIG. 1. Three Cas9 constructs were generated: 1) a WT Cas9 (SEQ ID NO: 20) [square]; 2) a WT Cas9 (SEQ ID NO: 20)activated with 3-nitro-2- pyridinesulfenyl (NPyS) [circle]; and 3) a cysteine-less Cas9 (SEQ ID NO: 19), wherein the native cysteines are substituted with serine [cross]. Two CPPs were tested: 1) HTN with a C- terminal cysteine (SEQ ID NO: 15) (a cationic peptide); and 2) HA16d-TAT (SEQ ID NO: 18) (an amphipathic peptide). The indicated Cas9 constructs were complexed with sgRNA and then incubated with a cationic peptide (FIG. 4A) or an amphipathic peptide (FIG. 4B) and allowed to react at room temperature for two hours. The mixtures were added to cells such that the final concentration of Cas9 was 1 pM and the peptide concentrations were as indicated on the x-axis in FIG. 4A-4B

[0159] As shown in FIG. 4A,the HTN CPP has an activated S-(3-nitro-2-pyridinesulfenyl) cysteine (NPyS-Cys) group, which can react with other free thiols, but not other NPyS- capped thiols. When the HTN CPP is able to conjugate with the Cas9 complex, there is an additive effect associated with higher concentrations of CPP provided in trans. The HA16d- TAT, which lacks a free thiol -reactive moiety, but contains an amphipathic helix-forming element, does not exhibit any additive effect regardless of Cas9 complex tested (FIG. 4B).Thus, for the cationic peptide (FIG. 4A), editing depended on the presence of matched activated and free thiols between the CPP and site-directed modifying polypeptide, while for the amphipathic peptide (FIG. 4B), editing was independent of thiol reactivity.

[0160] The above Cas9 constructs were further tested with two CPPs: the HTN with a C- terminal cysteine (as described above)(”HTN{Cys-SH}”) (SEQ ID NO: 15) and the same CPP capped with NPyS (“HTN{NpyS-Cys}”) (SEQ ID NO: 15). The results for each Cas9 construct are shown in FIG. 5A-5C. FIG. 5A provides the results for WT Cas9 and the NPyS-capped CPP. This combination provides an in cv.s-conjugated CPP and the in trans CPP is capable of reacting with free thiols. FIG. 5B provides the results for WT Cas9 activated with NPyS and the HTN with a C-terminal cysteine. This combination provides an in cv.s-conjugated CPP and the in trans CPP is capable of reacting with free thiols, though with a slower rate. FIG. 5C provides the results for the cysteine-less Cas9 with each CPP. This combination does not afford any in cv.s-conjugated CPP.Example 3

[0161] To further examine the additivity of the CPPs provided in cis and in trans in the presence of excess CCP (“added peptide”) on editing, a Cas9WTconstruct containing two native cysteines was coupled via disulfides (“SS”) to a C-terminal cysteine on one of two peptides, a His-Tat-NLS peptide (“HTN”) or an NLS-NLS (“NN”), or left uncoupled (with free thiols) (“unconjugated”), as shown in FIG. 6. These doubly coupled conjugates were purified to remove uncoupled peptide and then complexed with a CD47 knockout sgRNA. The resulting ribonucleoproteins (RNPs) were then mixed with a 3.3-fold excess of each of the indicated peptides bearing an activated cysteine (“NPyS-Cys”) or no cysteine giving 15 pM RNP, 50 pM peptide. This mixture was incubated at room temperature for 5 minutes and then diluted 1 / 5 in media with activated human T cells, yielding 3 pM RNP and 10 pM added peptide with cells. As outlined in the assay diagram of FIG. 1, after 1 hour at 37 °C, cells were washed with media to remove RNP and peptide. Cells were incubated for an additional 5 days with one media change, and then editing was determined by flow cytometry, with staining for T cell markers and CD47. As shown in FIG. 6, the activated version of HTN (“HTN{NPyS-Cys}”) (SEQ ID NO: 15) yielded much higher editing than the non-cysteine containing version (“HTN”) (SEQ ID NO: 21) for all three conjugates. The unconjugated RNP (top set of bars) was presumably able to react with the activated forms of the added peptides (“HTN {NPyS-Cys}” and “NN{NPyS-Cys}”), so this condition also reports on cis and trans additivity. Specifically, these results show that a plurality of CPPs provided in trans enhance the internalization of a site-directed modifying polypeptide relative to the site-directed modifying polypeptide alone, even when the site-directed modifying polypeptide is conjugated to at least one CPPs.Table 1: Summary of Sequences

Claims

ClaimsWe claim:

1. A composition for ex vivo gene editing comprising:(a) a site-directed modifying polypeptide, wherein the site-directed modifying polypeptide is conjugated to one or more cell-penetrating peptides (CPPs); and(b) a plurality of CPPs in solution, wherein the plurality of CPPs comprise a thiolreactive moiety.

2. The composition of claim 1, wherein the CPP is a cationic CPP.

3. The composition of claim 2, wherein the cationic CPP is 5 to 40 amino acid residues and has an average net charge of at least +0.3 at pH 7.

4. The composition of any one of claims 2 to 3, wherein the cationic CPP is 5 to 20 amino acid residues and has an average net charge of at least +0.5 at pH 7.

5. The composition of any one of claims 2 to 4, wherein the cationic CPP comprises at least five arginine residues.

6. The composition of claim 1, wherein the CPP comprises a cationic arginine-rich peptide (CARP).

7. The composition of claim 1, wherein the CPP is a TAT-related CPP.

8. The composition of any one of claims 2 to 7, wherein the CPP further comprises a nuclear localization signal (NLS).

9. The composition of claim 8, wherein the NLS is an SV40 NLS (SEQ ID NO: 4).

10. The composition of any one of claims 1-9, wherein the thiol -reactive moiety is a cysteine.

11. The composition of claim 10, wherein the cysteine is further modified with a 3-nitro- 2-pyridinesulfenyl group (NPyS).

12. The composition of claim 1, wherein the CPP comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 11 (TAT-NLS).

13. The composition of claim 1, wherein the CPP comprises an amino acid sequence having at least 95% identity to SEQ ID NO: 11 (TAT-NLS).

14. The composition of claim 1, wherein the CPP comprises an amino acid sequence of SEQ ID NO: 11 (TAT-NLS).

15. The composition of claim 1, wherein the CPP comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 12 (HIS-TAT-NLS).

16. The composition of claim 1, wherein the CPP comprises an amino acid sequence having at least 95% identity to SEQ ID NO: 12 (HIS-TAT-NLS).

17. The composition of claim 1, wherein the CPP comprises an amino acid sequence of SEQ ID NO: 12 (HIS-TAT-NLS).

18. The composition of any one of claims 12 to 17, wherein a cysteine of the CPP is further modified with a 3-nitro-pyridinesulfenyl group (NPyS).

19. The composition of any one of claims 1 to 18, wherein the site-directed modifying polypeptide is an RNA-guided nuclease.

20. The composition of claim 19, wherein the RNA-guided nuclease is a Class 2 Cas polypeptide.

21. The composition of claim 20, wherein the Class 2 Cas polypeptide is a Type II Cas polypeptide or a Type V Cas polypeptide.

22. The composition of claim 21, wherein the Type II Cas polypeptide is Cas9.

23. The composition of claim 21, wherein the Type V Cas polypeptide is Casl2.

24. The composition of claim 1, wherein the site-directed modifying polypeptide is conjugated to the one or more CPPs via a cysteine residue.

25. The composition of claim 1, wherein the plurality of CPPs is at a concentration between about 5 to about 15 pM.

26. The composition of claim 25, wherein the plurality of CPPs is at a concentration between about 10 to about 15 pM.

27. A method of modifying a nucleic acid in a cell, the method comprising contacting the cell with the composition of any one of claims 1-26.

28. The method of claim 27, wherein the method is performed ex vivo.

29. The method of claim 27 or 28, wherein the cell is an immune cell.

30. The method of claim 29, wherein the immune cell is a T-cell.

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