Cell-penetrating peptide and use thereof

Abstract

Provided is a cell-penetrating peptide capable of delivering a variety of biological macromolecules such as proteins, antibodies, nucleic acids, etc. into cells across the membrane. In addition, a fusion protein, conjugate and complex containing the cell-penetrating peptide, and the use of the cell-penetrating peptide are also provided.

Description

Technical Field

[0001] This application relates to the field of biological technology, particularly the field of transmembrane delivery technology. Specifically, this application relates to a cell penetrating peptide (CPP) that can efficiently deliver various biological macromolecules, such as proteins, antibodies, and nucleic acids, across the membrane into cells. Furthermore, this application also relates to fusion proteins, conjugates, and complexes containing the said cell penetrating peptide, as well as the uses of the said cell penetrating peptide. Background Technology

[0002] Current biologics, especially antibodies, primarily target free targets and targets on the cell membrane surface. However, many potential targets exist intracellularly. Due to the accessibility issues of macromolecular drugs (such as antibodies) to these intracellular targets, current drugs targeting these intracellular targets are mainly chemical drugs. However, chemical drugs still lag behind antibodies or other bioactive macromolecules in areas such as the recognition of antigens overexpressed in certain diseases, the blocking of protein-protein interactions, and specificity. For example, intracellular tumor-associated antigens or signaling pathways that promote tumor development are potential intervention targets, provided that active biomolecules can be efficiently delivered into cells to intervene against these targets.

[0003] Cell-penetrating peptides (CPPs) are a class of short peptides capable of crossing cell membranes or tissue barriers. These peptides do not bind to specific receptors and cross tissue barriers and cell membranes through energy-dependent or energy-independent mechanisms. CPPs mainly enter cells through two mechanisms: endocytosis and direct penetration. They have advantages such as high transmembrane efficiency and low cytotoxicity. CPPs can be mainly classified into cationic, amphiphilic, and hydrophobic types. Since 1988, when two independent research teams reported that HIV-1's trans-activator of transcription (TAT) could effectively cross cell membranes in vitro, research on cell-penetrating peptides has made significant progress. It has been found that CPPs can carry bioactive molecules (also collectively referred to as cargoes, such as proteins, peptides, DNA, siRNAs, and small drugs) into cells; therefore, delivery systems utilizing CPPs are also called peptide delivery systems. Cell-penetrating peptides have been applied in basic research, such as as transfection tools targeting various cell types and for post-transfection translation studies. Furthermore, the membrane-penetrating properties of cell-penetrating peptides (CPPs) have been utilized to improve the delivery and therapeutic efficacy of drugs (including antibiotics, anti-inflammatory drugs, anti-tumor drugs, and some neuroprotective drugs) that are difficult to penetrate cells and tissues. Numerous preclinical studies of cell-penetrating peptides have shown promising therapeutic results in various disease models, and some of these drugs have already been advanced to the clinical stage. These preclinical and clinical studies have led to unprecedented advancements in human treatment methods.

[0004] However, current clinically applied cell-penetrating peptides / drugs still suffer from low delivery efficiency and poor targeting. Therefore, there is a continued need to develop new, highly efficient cell-penetrating peptides to further improve the efficiency of bioactive macromolecules (such as antibodies, protein molecules like those related to gene editing systems, and nucleic acid molecules) crossing the cell membrane, thereby better enabling them to perform their biological functions. These new, highly efficient cell-penetrating peptides will provide a more effective delivery method for therapeutic drugs targeting intracellular sites. Summary of the Invention

[0005] In this invention, unless otherwise stated, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, the cell culture, molecular genetics, nucleic acid chemistry, and immunology laboratory procedures used herein are all standard procedures widely used in their respective fields. To better understand this invention, definitions and explanations of relevant terms are provided below.

[0006] As used herein, the term "cell-penetrating peptide" refers to a peptide capable of delivering a linked target molecule across the cell membrane into the cell. For example, the cell-penetrating peptide of this application is capable of delivering a linked target biological molecule (e.g., a target peptide or a target nucleic acid) across the cell membrane into the cell. In this application, the cell-penetrating peptide can be linked to the target biological molecule (e.g., a target peptide or a target nucleic acid) via covalent or non-covalent linkage.

[0007] For example, the cell-penetrating peptide of this application can be covalently linked to a target peptide or target nucleic acid via a linker, such as a peptide linker. Therefore, in some embodiments, the cell-penetrating peptide of this application can optionally be fused to a target peptide via a peptide linker. In some embodiments, the cell-penetrating peptide of this application can optionally be conjugated to a target peptide or target nucleic acid via a linker (e.g., a peptide linker or a bifunctional linker). Methods for conjugating a peptide molecule to a target peptide or target nucleic acid are known in the art, and various known bifunctional linkers may be used, for example.

[0008] Furthermore, the cell-penetrating peptides of this application can be linked to target biological molecules (e.g., target peptides or target nucleic acids) via non-covalent linkage. Therefore, in some embodiments, the cell-penetrating peptides of this application can be linked to target biological molecules (e.g., target peptides or target nucleic acids) through specific intermolecular interactions / specific binding (e.g., interactions / binding between antigens and antibodies; interactions / binding between DNA-binding domains and DNA molecules).

[0009] As used herein, the terms "specific binding" or "specific interaction" refer to a non-random binding reaction between two molecules, such as the reaction between an antibody and an antigen, or the reaction between a DNA-binding domain and a DNA molecule. For example, a non-random binding reaction between an antibody and an antigen may have a binding frequency of ≤10. -6 The binding affinity (KD) of M. In this application, KD refers to the ratio of dissociation rate to binding rate (koff / kon), which can be determined by methods such as surface plasmon resonance, for example, using an instrument such as Biacore.

[0010] As used herein, the term "conservative substitution" means an amino acid substitution that does not adversely affect or alter the essential properties of a protein / peptide containing an amino acid sequence. For example, conservative substitutions can be introduced using standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions include substitutions of amino acid residues with amino acid residues having similar side chains, such as substitutions with residues that are physically or functionally similar to the corresponding amino acid residues (e.g., having similar size, shape, charge, chemical properties, including the ability to form covalent or hydrogen bonds). Families of amino acid residues with similar side chains have been defined in the art. These families include: amino acid families with basic side chains (e.g., lysine, arginine, and histidine); amino acid families with acidic side chains (e.g., aspartic acid and glutamic acid); amino acid families with uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, and tryptophan); amino acid families with nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, and methionine); amino acid families with β-branched side chains (e.g., threonine, valine, and isoleucine); and amino acid families with aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, and histidine). Therefore, it is preferable to replace the corresponding amino acid residue with another amino acid residue from the same side chain family. Methods for identifying conserved substitutions of amino acids are well known in the art (see, for example, Brummell et al., Biochem. 32:1180-1187 (1993); Kobayashi et al. Protein Eng. 12(10):879-884 (1999); and Burks et al. Proc. Natl Acad. Set USA 94:412-417 (1997), which are incorporated herein by reference). In this application, the term “conservative substitution” generally refers to the substitution of a corresponding amino acid residue with another amino acid residue from the same side chain family.

[0011] As used herein, the term "N-terminal truncated by X amino acid residues" means that the first X amino acid residues from the N-terminus of a peptide / polypeptide are deleted (X is an integer not less than 1). For example, the expression "N-terminal truncated by 5 amino acid residues" means that the first 5 amino acid residues from the N-terminus of a peptide / polypeptide are deleted.

[0012] As used herein, the term "C-terminal truncated by X amino acid residues" means that the last X amino acid residues at the C-terminus of a peptide / polypeptide have been deleted (X is an integer not less than 1). For example, the expression "C-terminal truncated by 8 amino acid residues" means that the last 8 amino acid residues at the C-terminus of a peptide / polypeptide have been deleted.

[0013] In this application, the terms "peptide," "polypeptide," and "protein" have the same meaning and are used interchangeably. Furthermore, in this application, amino acids are generally represented by single-letter and three-letter abbreviations known in the art. For example, alanine can be represented by A or Ala.

[0014] As used in this article, the term "separated" refers to a substance that has been artificially altered from its natural state. If a substance or component is found in nature that is "separated," then it has been altered or removed from its original state, or both. For example, a polynucleotide or polypeptide naturally present in a living animal may not be separated, but it can be considered "separated" if it is sufficiently separated from the substance that coexists with it in its natural state and exists in a sufficiently pure state.

[0015] As used herein, the term "vector" refers to a nucleic acid delivery vehicle into which polynucleotides can be inserted. When a vector enables the expression of a protein encoded by the inserted polynucleotide, it is called an expression vector. Vectors can be used to transform, transduce, or transfect host cells, allowing the expression of the genetic material elements they carry within the host cells. Examples of vectors include plasmids, phagemids, Cos plasmids, artificial chromosomes such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC), or P1-derived artificial chromosomes (PAC), bacteriophages such as λ phage or M13 phage, and animal viruses. Animal viruses used as vectors include retroviruses (including lentiviruses, adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papillomaviruses (such as SV40)). Vectors may contain various elements that control expression, including promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. Additionally, vectors may contain replication initiation sites. The carrier may also include components that facilitate its entry into the cell, including but not limited to viral particles, liposomes, or protein coats.

[0016] As used herein, the term "host cell" refers to a cell into which exogenous polynucleotides and / or vectors are introduced. Host cells described in this application include, but are not limited to, prokaryotic cells such as *Escherichia coli* or *Bacillus subtilis*, fungal cells such as yeast cells or *Aspergillus*, insect cells such as S2 *Drosophila* cells or Sf9, or animal cells such as fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK 293 cells, or human cells.

[0017] As used herein, the term "pharmaceutically acceptable carrier or excipient" means a carrier or excipient that is pharmacologically and / or physiologically compatible with the subject and the active ingredient, which is well known in the art (see, for example, Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995), and includes, but is not limited to: pH adjusters, surfactants, adjuvants, and ionic strength enhancers. For example, pH adjusters include, but are not limited to, phosphate buffers; surfactants include, but are not limited to, cationic, anionic, or nonionic surfactants, such as Tween-80; and ionic strength enhancers include, but are not limited to, sodium chloride.

[0018] In this application, after in-depth research, the inventors have developed a new class of cell-penetrating peptides having the motif PRRR***PRRRR*Q*PRRRR. It has been shown that cell-penetrating peptides containing this motif can effectively deliver target biological molecules (e.g., target peptides or target nucleic acids) across the membrane into cells and allow them to function within the cells.

[0019] Therefore, in one aspect, this application provides a cell-penetrating peptide or a truncated version thereof, said cell-penetrating peptide having the structure of Formula I:

[0020]

[0021] in,

[0022] X1-X3 are each independently selected from (i) amino acid residue R and (ii) amino acid residues that are conserved substitutions relative to (i) (e.g., K or H).

[0023] X4 is selected from (i) amino acid residues R, C, G and (ii) amino acid residues that are conserved substitutions relative to (i) (e.g., K, H, N, Q, S, T, Y, W).

[0024] X5 is selected from (i) amino acid residues R, N, G; and (ii) amino acid residues that are conserved substitutions relative to (i) (e.g., K, H, C, Q, S, T, Y, W).

[0025] X6 is selected from (i) amino acid residues R, G, P, S; and (ii) amino acid residues that are conserved substitutions relative to (i) (e.g., K, H, N, Q, C, T, Y, W, A, V, L, I, M).

[0026] X7 is selected from (i) amino acid residues R, D, Q; and (ii) amino acid residues that are conserved substitutions relative to (i) (e.g., K, H, N, G, C, S, T, Y, W, E).

[0027] X8 is selected from (i) amino acid residues R, A; and (ii) amino acid residues that are conserved substitutions relative to (i) (e.g., K, H, V, L, I, M).

[0028] X9 is selected from (i) amino acid residues G, P, T; and (ii) amino acid residues that are conserved substitutions relative to (i) (e.g., N, Q, S, C, Y, W, A, V, L, I, M);

[0029] X 10 Selected from (i) amino acid residue R and (ii) amino acid residues that are conserved substitutions relative to (i) (e.g., K or H);

[0030] X 11 Selected from (i) amino acid residues A, S, Q; and (ii) amino acid residues that are conserved substitutions relative to (i) (e.g., V, L, I, M, N, G, T, C, Y, W);

[0031] X 16 Selected from (i) amino acid residue T and (ii) amino acid residues that are conserved substitutions relative to (i) (e.g., N, Q, G, S, C, Y, W).

[0032] X 17 Selected from (i) amino acid residue P and (ii) amino acid residues that are conserved substitutions relative to (i) (e.g., A, V, L, I, M).

[0033] X 18 Selected from (i) amino acid residues S, Q and (ii) amino acid residues that are conserved substitutions relative to (i) (e.g. N, G, T, C, Y, W).

[0034] X 24 Selected from (i) amino acid residue S; and (ii) amino acid residues that are conserved substitutions relative to (i) (e.g., N, Q, G, T, C, Y, W).

[0035] X 26 Selected from (i) amino acid residues S, C, Q; and (ii) amino acid residues that are conserved substitutions relative to (i) (e.g., N, G, T, Y, W); X 32 Selected from (i) amino acid residues S, C; and (ii) amino acid residues that are conserved substitutions relative to (i) (e.g., N, Q, G, T, Y, W);

[0036] X 33Selected from (i) amino acid residues Q and K; and (ii) amino acid residues that are conserved substitutions relative to (i) (e.g., N, S, C, G, T, Y, W, R, H);

[0037] X 34 Selected from (i) amino acid residues S, C; and (ii) amino acid residues that are conserved substitutions relative to (i) (e.g., N, Q, G, T, Y, W);

[0038] X 35 Selected from (i) amino acid residues R, P; and (ii) amino acid residues that are conserved substitutions relative to (i) (e.g., K, H, A, V, L, I, M).

[0039] X 36 Selected from (i) amino acid residues E, A; and (ii) amino acid residues that are conserved substitutions relative to (i) (e.g., V, L, I, M, D).

[0040] X 37 Selected from (i) amino acid residues P, S, C; and (ii) amino acid residues that are conserved substitutions relative to (i) (e.g., A, V, L, I, M, N, Q, G, T, Y, W);

[0041] X 38 Selected from (i) amino acid residues Q, S; and (ii) amino acid residues that are conserved substitutions relative to (i) (e.g., N, C, G, T, Y, W); and

[0042] X 39 Selected from (i) amino acid residues C, S, N; and (ii) amino acid residues that are conserved substitutions relative to (i) (e.g., Q, G, T, Y, W);

[0043] Specifically, compared to the cell-penetrating peptide, the truncated form has its N-terminus shortened by 1-10 (e.g., 1-5, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) amino acid residues, and / or its C-terminus shortened by 1-14 (e.g., 1-8, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14) amino acid residues; and...

[0044] The cell-penetrating peptide or truncated form is capable of delivering biological molecules (such as target peptides or target nucleic acids) across the membrane into the cell.

[0045] In some preferred embodiments, X1-X3 are each independently selected from amino acid residues R, K, and H. In some preferred embodiments, X1-X3 are each independently selected from amino acid residues R and K. In some preferred embodiments, X1-X3 are each independently amino acid residue R.

[0046] In some preferred embodiments, X4 is selected from amino acid residues R, C, G, K, H, N, Q, S, T, Y, and W. In some preferred embodiments, X4 is selected from amino acid residues R, C, G, K, N, Q, S, and T. In some preferred embodiments, X4 is selected from amino acid residues R, C, G, and K. In some preferred embodiments, X4 is selected from amino acid residues R, C, and G.

[0047] In some preferred embodiments, X5 is selected from amino acid residues R, N, G, K, H, C, Q, S, T, Y, and W. In some preferred embodiments, X5 is selected from amino acid residues R, N, G, K, C, Q, S, and T. In some preferred embodiments, X5 is selected from amino acid residues R, N, G, K, C, and Q. In some preferred embodiments, X5 is selected from amino acid residues R, N, and G.

[0048] In some preferred embodiments, X6 is selected from amino acid residues R, G, P, S, K, H, N, Q, C, T, Y, W, A, V, L, I, and M. In some preferred embodiments, X6 is selected from amino acid residues R, G, P, S, K, N, Q, C, and T. In some preferred embodiments, X6 is selected from amino acid residues R, G, P, and S.

[0049] In some preferred embodiments, X7 is selected from amino acid residues R, D, Q, K, H, N, G, C, S, T, Y, W, and E. In some preferred embodiments, X7 is selected from amino acid residues R, D, Q, K, N, G, C, S, T, and E. In some preferred embodiments, X7 is selected from amino acid residues R, D, Q, K, N, and E. In some preferred embodiments, X7 is selected from amino acid residues R, D, and Q.

[0050] In some preferred embodiments, X8 is selected from amino acid residues R, A, K, H, V, L, I, and M. In some preferred embodiments, X8 is selected from amino acid residues R, A, K, V, L, and I. In some preferred embodiments, X8 is selected from amino acid residues R and A.

[0051] In some preferred embodiments, X9 is selected from amino acid residues G, P, T, N, Q, S, C, Y, W, A, V, L, I, and M. In some preferred embodiments, X9 is selected from amino acid residues G, P, T, N, Q, S, and C. In some preferred embodiments, X9 is selected from amino acid residues G, P, T, S, and C. In some preferred embodiments, X9 is selected from amino acid residues G, P, and T.

[0052] In some preferred embodiments, X10 Selected from amino acid residues R, K, and H. In some preferred embodiments, X 10 Selected from amino acid residues R and K. In some preferred embodiments, X 10 R is an amino acid residue.

[0053] In some preferred embodiments, X 11 The amino acid residues are selected from A, S, V, L, I, M, N, Q, G, T, C, Y, and W. In some preferred embodiments, X 11 Selected from amino acid residues A, S, V, L, I, N, Q, G, T, and C. In some preferred embodiments, X 11 Selected from amino acid residues A, S, V, L, I, and T. In some preferred embodiments, X 11 Selected from amino acid residues A and S.

[0054] In some preferred embodiments, X 16 The amino acid residues are selected from T, N, Q, G, S, C, Y, and W. In some preferred embodiments, X 16 Selected from amino acid residues T, N, Q, G, S, and C. In some preferred embodiments, X 16 Selected from amino acid residues T and S. In some preferred embodiments, X 16 It is the amino acid residue T.

[0055] In some preferred embodiments, X 17 Selected from amino acid residues P, A, V, L, I, and M. In some preferred embodiments, X 17 It is the amino acid residue P.

[0056] In some preferred embodiments, X 18 The amino acid residues are selected from S, N, Q, G, T, C, Y, and W. In some preferred embodiments, X 18 Selected from amino acid residues S, N, Q, G, T, and C. In some preferred embodiments, X 18 Selected from amino acid residues S, Q, and T. In some preferred embodiments, X 18 Selected from amino acid residues S and Q.

[0057] In some preferred embodiments, X 24 The amino acid residues are selected from S, N, Q, G, T, C, Y, and W. In some preferred embodiments, X 24 Selected from amino acid residues S, N, Q, G, T, and C. In some preferred embodiments, X 24 Selected from amino acid residues S and T. In some preferred embodiments, X 24 It is the amino acid residue S.

[0058] In some preferred embodiments, X 26 The amino acid residues are selected from S, N, Q, G, T, C, Y, and W. In some preferred embodiments, X 26 Selected from amino acid residues S, N, Q, G, T, and C. In some preferred embodiments, X 26 Selected from amino acid residues S, C, and Q.

[0059] In some preferred embodiments, X 32 Selected from amino acid residues S, C, N, Q, G, T, Y, and W. In some preferred embodiments, X 32 Selected from amino acid residues S, C, N, Q, G, and T. In some preferred embodiments, X 32 Selected from amino acid residues S, C, G, and T. In some preferred embodiments, X 32 Selected from amino acid residues S and C.

[0060] In some preferred embodiments, X 33 Selected from amino acid residues Q, K, N, S, C, G, T, Y, W, R, and H. In some preferred embodiments, X 33 Selected from amino acid residues Q, K, N, S, C, G, T, and R. In some preferred embodiments, X 33 Selected from amino acid residues Q, K, N, and R. In some preferred embodiments, X 33 Selected from amino acid residues Q and K.

[0061] In some preferred embodiments, X 34 Selected from amino acid residues S, C, N, Q, G, T, Y, and W. In some preferred embodiments, X 34 Selected from amino acid residues S, C, N, Q, G, and T. In some preferred embodiments, X 34 Selected from amino acid residues S, C, G, and T. In some preferred embodiments, X 34 Selected from amino acid residues S and C.

[0062] In some preferred embodiments, X 35 Selected from amino acid residues R, P, K, H, A, V, L, I, and M. In some preferred embodiments, X 35 Selected from amino acid residues R, P, K, and H. In some preferred embodiments, X 35 Selected from amino acid residues R, P, and K. In some preferred embodiments, X 35 Selected from amino acid residues R and P.

[0063] In some preferred embodiments, X36 Selected from amino acid residues E, A, V, L, I, M, and D. In some preferred embodiments, X 36 Selected from amino acid residues E, A, V, L, I, and D. In some preferred embodiments, X 36 Selected from amino acid residues E and A.

[0064] In some preferred embodiments, X 37 The amino acid residues are selected from P, S, C, A, V, L, I, M, N, Q, G, T, Y, and W. In some preferred embodiments, X 37 Selected from amino acid residues P, S, C, N, Q, G, and T. In some preferred embodiments, X 37 Selected from amino acid residues P, S, C, G, and T. In some preferred embodiments, X 37 Selected from amino acid residues P, S, and C.

[0065] In some preferred embodiments, X 38 The amino acid residues are selected from Q, S, N, C, G, T, Y, and W. In some preferred embodiments, X 38 Selected from amino acid residues Q, S, N, C, G, and T. In some preferred embodiments, X 38 Selected from amino acid residues Q, S, N, and T. In some preferred embodiments, X 38 Selected from amino acid residues Q and S.

[0066] In some preferred embodiments, X 39 Selected from amino acid residues C, S, N, Q, G, T, Y, and W. In some preferred embodiments, X 39 Selected from amino acid residues C, S, N, Q, G, and T. In some preferred embodiments, X 39 Selected from amino acid residues C, S, and N.

[0067] In some preferred embodiments, the cell-penetrating peptide has the structure of Formula II:

[0068]

[0069] in,

[0070] X4 is selected from (i) amino acid residues R, C, G and (ii) amino acid residues that are conserved substitutions relative to (i) (e.g., K, H, N, Q, S, T, Y, W).

[0071] X5 is selected from (i) amino acid residues R, N, G; and (ii) amino acid residues that are conserved substitutions relative to (i) (e.g., K, H, C, Q, S, T, Y, W).

[0072] X6 is selected from (i) amino acid residues R, G, P, S; and (ii) amino acid residues that are conserved substitutions relative to (i) (e.g., K, H, N, Q, C, T, Y, W, A, V, L, I, M).

[0073] X7 is selected from (i) amino acid residues R, D, Q; and (ii) amino acid residues that are conserved substitutions relative to (i) (e.g., K, H, N, G, C, S, T, Y, W, E).

[0074] X8 is selected from (i) amino acid residues R, A; and (ii) amino acid residues that are conserved substitutions relative to (i) (e.g., K, H, V, L, I, M).

[0075] X9 is selected from (i) amino acid residues G, P, T; and (ii) amino acid residues that are conserved substitutions relative to (i) (e.g., N, Q, S, C, Y, W, A, V, L, I, M);

[0076] X 11 Selected from (i) amino acid residues A, S, Q; and (ii) amino acid residues that are conserved substitutions relative to (i) (e.g., V, L, I, M, N, G, T, C, Y, W);

[0077] X 32 Selected from (i) amino acid residues S, C; and (ii) amino acid residues that are conserved substitutions relative to (i) (e.g., N, Q, G, T, Y, W);

[0078] X 33 Selected from (i) amino acid residues Q and K; and (ii) amino acid residues that are conserved substitutions relative to (i) (e.g., N, S, C, G, T, Y, W, R, H);

[0079] X 34 Selected from (i) amino acid residues S, C; and (ii) amino acid residues that are conserved substitutions relative to (i) (e.g., N, Q, G, T, Y, W);

[0080] X 35 Selected from (i) amino acid residues R, P; and (ii) amino acid residues that are conserved substitutions relative to (i) (e.g., K, H, A, V, L, I, M).

[0081] X 36 Selected from (i) amino acid residues E, A; and (ii) amino acid residues that are conserved substitutions relative to (i) (e.g., V, L, I, M, D).

[0082] X 37Selected from (i) amino acid residues P, S, C; and (ii) amino acid residues that are conserved substitutions relative to (i) (e.g., A, V, L, I, M, N, Q, G, T, Y, W);

[0083] X 38 Selected from (i) amino acid residues Q, S; and (ii) amino acid residues that are conserved substitutions relative to (i) (e.g., N, C, G, T, Y, W); and

[0084] X 39 The amino acid residues selected are (i) amino acid residues C, S, N; and (ii) amino acid residues that are conserved substitutions relative to (i) (e.g., Q, G, T, Y, W).

[0085] In some preferred embodiments, X4, X5, X6, X7, X8, X9, X 11 X 32 X 33 X 34 X 35 X 36 X 37 X 38 X 39 Each is defined independently as described above.

[0086] In some preferred embodiments, the cell-penetrating peptide has the structure of Formula II:

[0087]

[0088] X4 is selected from amino acid residues R, C, and G;

[0089] X5 is selected from amino acid residues R, N, and G;

[0090] X6 is selected from amino acid residues R, G, P, and S;

[0091] X7 is selected from amino acid residues R, D, and Q;

[0092] X8 is selected from amino acid residues R and A;

[0093] X9 is selected from amino acid residues G, P, and T;

[0094] X 11 Selected from amino acid residues A, S, and Q;

[0095] X 32 Selected from amino acid residues S and C;

[0096] X 33 Selected from amino acid residues Q and K;

[0097] X 34 Selected from amino acid residues S and C;

[0098] X 35 Selected from amino acid residues R and P;

[0099] X 36 Selected from amino acid residues E and A;

[0100] X 37 Selected from amino acid residues P, S, and C;

[0101] X 38 Selected from amino acid residues Q, S; and

[0102] X 39 Selected from amino acid residues C, S, and N.

[0103] In some preferred embodiments, the truncated form is shortened by 1-10 amino acid residues (e.g., 1-5 amino acid residues) at the N-terminus and / or 1-14 amino acid residues (e.g., 1-8 amino acid residues) at the C-terminus compared to the cell-penetrating peptide. In some preferred embodiments, the truncated form is shortened by 1-10 amino acid residues at the N-terminus compared to the cell-penetrating peptide, for example, by 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid residues at the N-terminus. In some preferred embodiments, the truncated form is shortened by 1-14 amino acid residues at the C-terminus compared to the cell-penetrating peptide, for example, by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 amino acid residues at the C-terminus. In some preferred embodiments, the truncated form is shortened by 1-10 amino acid residues (e.g., 1-5 amino acid residues) at the N-terminus and 1-14 amino acid residues (e.g., 1-8 amino acid residues) at the C-terminus compared to the cell-penetrating peptide. In some embodiments, the truncated form is shortened by 5 or 10 amino acid residues at the N-terminus and 1-8 amino acid residues (e.g., 1, 3, 6, or 8 amino acid residues) at the C-terminus compared to the cell-penetrating peptide. In some embodiments, the truncated form is shortened by 1-10 amino acid residues (e.g., 2, 3, 5, or 10 amino acid residues) at the N-terminus compared to the cell-penetrating peptide, and the C-terminus is either untruncated or shortened by 1-8 amino acid residues (e.g., 1, 3, 6, or 8 amino acid residues). In some preferred embodiments, the truncated form is shortened by 5 amino acid residues at the N-terminus and 1-14 amino acid residues (e.g., 1, 3, 6, 8, or 14 amino acid residues) at the C-terminus compared to the cell-penetrating peptide. In some preferred embodiments, the truncated form is shortened by 1-5 amino acid residues (e.g., 2, 3, or 5 amino acid residues) at the N-terminus compared to the cell-penetrating peptide, and the C-terminus is either untrunculated or shortened by 1-14 amino acid residues (e.g., 1, 3, 6, 8, or 14 amino acid residues). In some preferred embodiments, the truncated form is shortened by 14 amino acid residues at the C-terminus compared to the cell-penetrating peptide, and X... 26 The amino acid residue is C. In some preferred embodiments, the truncated form does not have the amino acid sequence shown in SEQ ID NO:14.

[0104] In some preferred embodiments, the cell-penetrating peptide has the structure of Formula III:

[0105]

[0106] in,

[0107] X5 is selected from (i) amino acid residues R, N, G; and (ii) amino acid residues that are conserved substitutions relative to (i) (e.g., K, H, C, Q, S, T, Y, W).

[0108] X6 is selected from (i) amino acid residues R, G, P, S; and (ii) amino acid residues that are conserved substitutions relative to (i) (e.g., K, H, N, Q, C, T, Y, W, A, V, L, I, M).

[0109] X8 is selected from (i) amino acid residues R, A; and (ii) amino acid residues that are conserved substitutions relative to (i) (e.g., K, H, V, L, I, M).

[0110] X9 is selected from (i) amino acid residues G, P, T; and (ii) amino acid residues that are conserved substitutions relative to (i) (e.g., N, Q, S, C, Y, W, A, V, L, I, M);

[0111] X 32 and X 34 Each is independently selected from (i) amino acid residues S and C; and (ii) amino acid residues that are conserved substitutions relative to (i) (e.g., N, Q, G, T, Y, W);

[0112] X 35 Selected from (i) amino acid residues R, P; and (ii) amino acid residues that are conserved substitutions relative to (i) (e.g., K, H, A, V, L, I, M).

[0113] X 36 Selected from (i) amino acid residues E, A; and (ii) amino acid residues that are conserved substitutions relative to (i) (e.g., V, L, I, M, D); and

[0114] X 37 Selected from (i) amino acid residues P, S, C; and (ii) amino acid residues that are conserved substitutions relative to (i) (e.g., A, V, L, I, M, N, Q, G, T, Y, W).

[0115] In some preferred embodiments, X5 is selected from amino acid residues R, N, G, K, H, C, Q, S, T, Y, and W. In some preferred embodiments, X5 is selected from amino acid residues N, G, C, Q, S, and T. In some preferred embodiments, X5 is selected from amino acid residues N, G, C, and Q. In some preferred embodiments, X5 is selected from amino acid residues N and G.

[0116] In some preferred embodiments, X6 is selected from amino acid residues R, G, P, S, K, H, N, Q, C, T, Y, W, A, V, L, I, and M. In some preferred embodiments, X6 is selected from amino acid residues P, S, N, Q, C, and T. In some preferred embodiments, X6 is selected from amino acid residues P and S.

[0117] In some preferred embodiments, X8 is selected from amino acid residues R, A, K, H, V, L, I, and M. In some preferred embodiments, X8 is selected from amino acid residues R, A, K, V, L, and I. In some preferred embodiments, X8 is selected from amino acid residues R and A. In some preferred embodiments, X8 is amino acid residue A.

[0118] In some preferred embodiments, X9 is selected from amino acid residues G, P, T, N, Q, S, C, Y, W, A, V, L, I, and M. In some preferred embodiments, X9 is selected from amino acid residues G, P, T, N, Q, S, and C. In some preferred embodiments, X9 is selected from amino acid residues P, T, and S. In some preferred embodiments, X9 is selected from amino acid residues P and T.

[0119] In some preferred embodiments, X 32 and X 34 Each amino acid residue is independently selected from S, C, N, Q, G, T, Y, and W. In some preferred embodiments, X 32 and X 34 Each amino acid residue is independently selected from S, C, N, Q, G, and T. In some preferred embodiments, X 32 and X 34 Each amino acid residue is independently selected from S, C, G, and T. In some preferred embodiments, X 32 and X 34 Each is independently selected from amino acid residues S and C. In some preferred embodiments, X 32 and X 34 Each is an amino acid residue S.

[0120] In some preferred embodiments, X 35 Selected from amino acid residues R, P, K, H, A, V, L, I, and M. In some preferred embodiments, X 35 Selected from amino acid residues R, P, K, and H. In some preferred embodiments, X 35 Selected from amino acid residues R, P, and K. In some preferred embodiments, X 35 Selected from amino acid residues R and P. In some preferred embodiments, X 35 It is the amino acid residue P.

[0121] In some preferred embodiments, X 36 Selected from amino acid residues E, A, V, L, I, M, and D. In some preferred embodiments, X 36 Selected from amino acid residues E, A, V, L, I, and D. In some preferred embodiments, X 36 Selected from amino acid residues E and A. In some preferred embodiments, X 36 It is amino acid residue A.

[0122] In some preferred embodiments, X 37 The amino acid residues are selected from P, S, C, A, V, L, I, M, N, Q, G, T, Y, and W. In some preferred embodiments, X 37 Selected from amino acid residues P, S, C, N, Q, G, and T. In some preferred embodiments, X 37 Selected from amino acid residues P, S, C, G, and T. In some preferred embodiments, X 37 Selected from amino acid residues P, S, and C. In some preferred embodiments, X 37 Selected from amino acid residues P and S.

[0123] In some preferred embodiments, the cell-penetrating peptide has an amino acid sequence selected from the following: SEQ ID NO: 20-21.

[0124] In some preferred embodiments, the truncated body includes the structure of form IV:

[0125]

[0126] in,

[0127] X 11 The amino acid residues selected are (i) amino acid residues S, Q, A; and (ii) amino acid residues that are conserved substitutions relative to (i) (e.g., V, L, I, M, N, G, T, C, Y, W).

[0128] X 18 Selected from (i) amino acid residues S, Q; and (ii) amino acid residues that are conserved substitutions relative to (i) (e.g., C, N, G, T, Y, W).

[0129] X 26 The amino acid residues selected are (i) amino acid residues S, C, Q; and (ii) amino acid residues that are conserved substitutions relative to (i) (e.g., N, G, T, Y, W).

[0130] In some preferred embodiments, X 11The amino acid residues are selected from A, S, V, L, I, M, N, Q, G, T, C, Y, and W. In some preferred embodiments, X 11 Selected from amino acid residues A, S, V, L, I, N, Q, G, T, and C. In some preferred embodiments, X 11 Selected from amino acid residues A, Q, S, V, L, I, and T. In some preferred embodiments, X 11 Selected from amino acid residues A, Q, and S.

[0131] In some preferred embodiments, X 18 The amino acid residues are selected from S, N, Q, G, T, C, Y, and W. In some preferred embodiments, X 18 Selected from amino acid residues S, N, Q, G, T, and C. In some preferred embodiments, X 18 Selected from amino acid residues S, Q, and T. In some preferred embodiments, X 18 Selected from amino acid residues S and Q.

[0132] In some preferred embodiments, X 26 The amino acid residues are selected from S, N, Q, G, T, C, Y, and W. In some preferred embodiments, X 26 Selected from amino acid residues S, N, Q, G, T, and C. In some preferred embodiments, X 26 Selected from amino acid residues S, C, and Q.

[0133] In some preferred embodiments, the truncated body includes a structure of form V:

[0134] in,

[0135] X6 is selected from (i) amino acid residues R, G; and (ii) amino acid residues that are conserved substitutions relative to (i) (e.g., K, H, C, Q, S, T, Y, W, N); X7 is selected from (i) amino acid residues R, D, Q; and (ii) amino acid residues that are conserved substitutions relative to (i) (e.g., K, H, N, G, C, S, T, Y, W, E);

[0136] X 11 Selected from (i) amino acid residues S, A, Q; and (ii) amino acid residues that are conserved substitutions relative to (i) (e.g., V, L, I, M, N, G, T, C, Y, W);

[0137] X 18 and X 26 Each is independently selected from (i) amino acid residues S, Q; and (ii) amino acid residues that are conserved substitutions relative to (i) (e.g., N, G, T, C, Y, W).

[0138] X 32 Selected from (i) amino acid residues S, C; and (ii) amino acid residues that are conserved substitutions relative to (i) (e.g., N, Q, G, T, Y, W).

[0139] In some preferred embodiments, X6 is selected from amino acid residues R, N, G, K, H, C, Q, S, T, Y, and W. In some preferred embodiments, X6 is selected from amino acid residues R, N, G, K, C, Q, S, and T. In some preferred embodiments, X6 is selected from amino acid residues R, N, G, K, C, and Q. In some preferred embodiments, X6 is selected from amino acid residues R and G. In some preferred embodiments, X6 is selected from amino acid residue R.

[0140] In some preferred embodiments, X7 is selected from amino acid residues R, D, Q, K, H, N, G, C, S, T, Y, W, and E. In some preferred embodiments, X7 is selected from amino acid residues R, D, Q, K, N, G, C, S, T, and E. In some preferred embodiments, X7 is selected from amino acid residues R, D, and Q. In some preferred embodiments, X7 is selected from amino acid residue R.

[0141] In some preferred embodiments, X 11 The amino acid residues are selected from A, S, V, L, I, M, N, Q, G, T, C, Y, and W. In some preferred embodiments, X 11 Selected from amino acid residues A, S, V, L, I, N, Q, G, T, and C. In some preferred embodiments, X 11 Selected from amino acid residues A, Q, S, V, L, I, and T. In some preferred embodiments, X 11 Selected from amino acid residues A, Q, and S. In some preferred embodiments, X 11 Selected from amino acid residues Q and S.

[0142] In some preferred embodiments, X 18 and X 26 Each amino acid residue is independently selected from S, N, Q, G, T, C, Y, and W. In some preferred embodiments, X 18 and X 26 Each amino acid residue is independently selected from S, N, Q, G, T, and C. In some preferred embodiments, X 18 and X 26 Each is independently selected from amino acid residues S, Q, and T. In some preferred embodiments, X 18 and X 26 Each is independently selected from amino acid residues Q and S.

[0143] In some preferred embodiments, X 32 Selected from amino acid residues S, C, N, Q, G, T, Y, and W. In some preferred embodiments, X 32 Selected from amino acid residues S, C, N, Q, G, and T. In some preferred embodiments, X 32 Selected from amino acid residues S, C, G, and T. In some preferred embodiments, X 32 Selected from amino acid residues S and C.

[0144] In some preferred embodiments, the truncated body includes the structure of form VI:

[0145]

[0146] in,

[0147] X6 is selected from (i) amino acid residues R and G; and (ii) amino acid residues that are conserved substitutions relative to (i) (e.g., K, H, C, Q, S, T, Y, W, N);

[0148] X7 is selected from (i) amino acid residues R, D, Q; and (ii) amino acid residues that are conserved substitutions relative to (i) (e.g., K, H, N, G, C, S, T, Y, W, E);

[0149] X 11 Selected from (i) amino acid residues S, A, Q; and (ii) amino acid residues that are conserved substitutions relative to (i) (e.g., V, L, I, M, N, G, T, C, Y, W);

[0150] X 18 and X 26 Each is independently selected from (i) amino acid residues S, Q; and (ii) amino acid residues that are conserved substitutions relative to (i) (e.g., N, G, T, C, Y, W).

[0151] X 33 Selected from (i) amino acid residues Q and K; and (ii) amino acid residues that are conserved substitutions relative to (i) (e.g., N, S, C, G, T, Y, W, R, H);

[0152] X 36 Selected from (i) amino acid residues E and A; and (ii) amino acid residues that are conserved substitutions relative to (i) (e.g., V, L, I, M, D);

[0153] X 37 Selected from (i) amino acid residues S, C; and (ii) amino acid residues that are conserved substitutions relative to (i) (e.g., N, Q, G, T, Y, W).

[0154] In some preferred embodiments, X6 is selected from amino acid residues R, N, G, K, H, C, Q, S, T, Y, and W. In some preferred embodiments, X6 is selected from amino acid residues R, N, G, K, C, Q, S, and T. In some preferred embodiments, X6 is selected from amino acid residues R, N, G, K, C, and Q. In some preferred embodiments, X6 is selected from amino acid residues R and G. In some preferred embodiments, X6 is selected from amino acid residue R.

[0155] In some preferred embodiments, X7 is selected from amino acid residues R, D, Q, K, H, N, G, C, S, T, Y, W, and E. In some preferred embodiments, X7 is selected from amino acid residues R, D, Q, K, N, G, C, S, T, and E. In some preferred embodiments, X7 is selected from amino acid residues R, D, and Q. In some preferred embodiments, X7 is selected from amino acid residue R.

[0156] In some preferred embodiments, X 11 The amino acid residues are selected from A, S, V, L, I, M, N, Q, G, T, C, Y, and W. In some preferred embodiments, X 11 Selected from amino acid residues A, S, V, L, I, N, Q, G, T, and C. In some preferred embodiments, X 11 Selected from amino acid residues A, Q, S, V, L, I, and T. In some preferred embodiments, X 11 Selected from amino acid residues A, Q, and S. In some preferred embodiments, X 11 Selected from amino acid residues Q and S.

[0157] In some preferred embodiments, X 18 and X 26 Each amino acid residue is independently selected from S, N, Q, G, T, C, Y, and W. In some preferred embodiments, X 18 and X 26 Each amino acid residue is independently selected from S, N, Q, G, T, and C. In some preferred embodiments, X 18 and X 26 Each is independently selected from amino acid residues S, Q, and T. In some preferred embodiments, X 18 and X 26 Each is independently selected from amino acid residues Q and S.

[0158] In some preferred embodiments, X 33 Selected from amino acid residues Q, K, N, S, C, G, T, Y, W, R, and H. In some preferred embodiments, X33 Selected from amino acid residues Q, K, N, S, C, G, T, and R. In some preferred embodiments, X 33 Selected from amino acid residues Q, K, N, and R. In some preferred embodiments, X 33 Selected from amino acid residues Q and K. In some preferred embodiments, X 33 Selected from amino acid residue Q.

[0159] In some preferred embodiments, X 36 Selected from amino acid residues E, A, V, L, I, M, and D. In some preferred embodiments, X 36 Selected from amino acid residues E, A, V, L, I, and D. In some preferred embodiments, X 36 Selected from amino acid residues E and A. In some preferred embodiments, X 36 Selected from amino acid residue E.

[0160] In some preferred embodiments, X 37 Selected from amino acid residues S, C, N, Q, G, T, Y, and W. In some preferred embodiments, X 37 Selected from amino acid residues S, C, N, Q, G, and T. In some preferred embodiments, X 37 Selected from amino acid residues S, C, G, and T. In some preferred embodiments, X 37 Selected from amino acid residues S and C.

[0161] In some preferred embodiments, the truncated body has the structure of formula VII:

[0162]

[0163] in,

[0164] X7 is selected from (i) amino acid residues R, D, Q; and (ii) amino acid residues that are conserved substitutions relative to (i) (e.g., K, H, N, G, C, S, T, Y, W, E).

[0165] X 11 Selected from (i) amino acid residues A and S; and (ii) amino acid residues that are conserved substitutions relative to (i) (e.g., V, L, I, M, N, Q, G, T, C, Y, W);

[0166] X 32 X 34 X 37 and X 39 Each is independently selected from (i) amino acid residues S and C; and (ii) amino acid residues that are conserved substitutions relative to (i) (e.g., N, Q, G, T, Y, W);

[0167] X 33 Selected from (i) amino acid residues Q, K; and (ii) amino acid residues that are conserved substitutions relative to (i) (e.g., N, S, C, G, T, Y, W, R, H); and

[0168] X 36 Selected from (i) amino acid residues E, A; and (ii) amino acid residues that are conserved substitutions relative to (i) (e.g., V, L, I, M, D).

[0169] In some preferred embodiments, X7 is selected from amino acid residues R, D, Q, K, H, N, G, C, S, T, Y, W, and E. In some preferred embodiments, X7 is selected from amino acid residues R, D, Q, K, N, G, C, S, T, and E. In some preferred embodiments, X7 is selected from amino acid residues R, D, and Q. In some preferred embodiments, X7 is selected from amino acid residues R and Q.

[0170] In some preferred embodiments, X 11 The amino acid residues are selected from A, S, V, L, I, M, N, Q, G, T, C, Y, and W. In some preferred embodiments, X 11 Selected from amino acid residues A, S, V, L, I, N, Q, G, T, and C. In some preferred embodiments, X 11 Selected from amino acid residues A, S, V, L, I, and T. In some preferred embodiments, X 11 Selected from amino acid residues A and S.

[0171] In some preferred embodiments, X 32 X 34 X 37 and X 39 Each amino acid residue is independently selected from S, C, N, Q, G, T, Y, and W. In some preferred embodiments, X 32 X 34 X 37 and X 39 Each amino acid residue is independently selected from S, C, N, Q, G, and T. In some preferred embodiments, X 32 X 34 X 37 and X 39 Each amino acid residue is independently selected from S, C, G, and T. In some preferred embodiments, X 32 X 34 X 37 and X 39 Each is independently selected from amino acid residues S and C.

[0172] In some preferred embodiments, X 33 Selected from amino acid residues Q, K, N, S, C, G, T, Y, W, R, and H. In some preferred embodiments, X 33 Selected from amino acid residues Q, K, N, S, C, G, T, and R. In some preferred embodiments, X 33 Selected from amino acid residues Q, K, N, and R. In some preferred embodiments, X 33 Selected from amino acid residues Q and K.

[0173] In some preferred embodiments, X 36 Selected from amino acid residues E, A, V, L, I, M, and D. In some preferred embodiments, X 36 Selected from amino acid residues E, A, V, L, I, and D. In some preferred embodiments, X 36 Selected from amino acid residues E and A.

[0174] In some preferred embodiments, the cell-penetrating peptide has an amino acid sequence selected from the following: SEQ ID NO: 20-21.

[0175] In some preferred embodiments, the cell-penetrating peptide or a truncated form thereof has an amino acid sequence selected from the following: SEQ ID NO: 10-13, 15, 17-18, 20-21, 23-29 and 32-37.

[0176] In some preferred embodiments, the cell-penetrating peptide or truncated form is isolated.

[0177] In another aspect, this application provides a fusion protein comprising the cell-penetrating peptide or a truncated form thereof as described above, and a target peptide. The cell-penetrating peptide or truncated form thereof of this application is capable of delivering the target peptide (the fusion protein) across the membrane into the cell.

[0178] In some preferred embodiments, the target peptide is directly covalently linked to the cell-penetrating peptide or its truncated form (i.e., directly linked via peptide bonds). In some preferred embodiments, the target peptide is covalently linked to the cell-penetrating peptide or its truncated form via a peptide linker (e.g., a flexible peptide linker). In some preferred embodiments, the peptide linker has the amino acid sequence shown in SEQ ID NO: 39. However, it will be readily understood that various known peptide linkers can be used in this application to fuse the target peptide with the cell-penetrating peptide or its truncated form.

[0179] In some preferred embodiments, the cell-penetrating peptide or a truncated form thereof is attached to the N-terminus of the target peptide (optionally, via a peptide linker). In some preferred embodiments, the cell-penetrating peptide or a truncated form thereof is attached to the C-terminus of the target peptide (optionally, via a peptide linker).

[0180] It is readily understood that the target peptide in this application can be any desired protein or polypeptide. For example, in some cases, it may be desirable to deliver an antibody into cells (e.g., tumor cells, immune cells, etc.) and to perform its function (e.g., inhibiting viral infection, replication, and / or assembly, inhibiting or enhancing intracellular signaling, etc.). In this case, the cell-penetrating peptide of this application or a truncated version thereof can be fused with the antibody of interest to facilitate the transmembrane delivery of the antibody. Therefore, in some preferred embodiments, the target peptide is an antibody. In some preferred embodiments, the antibody is selected from anti-hepatitis B virus antibodies (e.g., anti-HBsAg antibody, anti-HBcAg antibody, anti-HBeAg antibody, etc.), anti-influenza virus antibodies (e.g., anti-HA1 antibody, anti-HA2 antibody), anti-tumor antigen antibodies (e.g., anti-p53 antibody, anti-kras antibody, anti-PRL-3 antibody), anti-immune checkpoint antibodies (e.g., anti-PD1 or PDL1 antibody), anti-melanin synthesis-related antibodies (e.g., tyrosinase-associated protein TYRP1 antibody), and anti-SARS-CoV-2 antibodies (e.g., anti-SARS-CoV-2 S protein antibody, such as anti-S protein RBD or S1 or S2 antibody).

[0181] In some cases, it may be desirable to deliver gene-editing-related proteins into cells and allow them to perform their functions (e.g., editing a target gene within the cell, or inhibiting gene editing within the cell). In such cases, the cell-penetrating peptide of this application or a truncated version thereof can be fused with a gene-editing-related protein to facilitate the transmembrane delivery of the protein. Therefore, in some preferred embodiments, the target peptide is a gene-editing-related protein. In some preferred embodiments, the protein is selected from Cas9 protein, AcrIIA4 protein, Cas13 protein, Cre recombinase, and Flip recombinase.

[0182] In some cases, it may be desirable to deliver an active or traceable protein into a cell to perform its function (e.g., to study or alter signaling pathways, to localize cells, etc.). In such cases, the cell-penetrating peptide of this application or a truncated version thereof can be fused with such a protein to facilitate its transmembrane delivery. Therefore, in some preferred embodiments, the target peptide is an active or traceable protein. In some preferred embodiments, the protein is selected from fluorescent proteins (e.g., green fluorescent protein), toxin proteins (e.g., endotoxins), cytokines (e.g., interleukins or interferons, such as IL10 or IFNγ), immunomodulatory proteins (e.g., PD1), enzymes (e.g., luciferases, nucleases, recombinases, methyltransferases, protein kinases, etc.), signaling pathway-related molecules (e.g., β-catenin protein), cell cycle proteins (e.g., Cyclin D1 protein), transcription activators, and transcription repressors.

[0183] In some cases, it may be desirable to deliver DNA molecules into cells and allow them to perform their functions (e.g., express exogenous proteins, interfere with endogenous gene expression, etc.). In such cases, the cell-penetrating peptide of this application or a truncated version thereof can be fused with a protein capable of binding DNA molecules (hereinafter also referred to as a DNA-binding protein) to facilitate the transmembrane delivery of the protein and the DNA molecule. Therefore, in some preferred embodiments, the target peptide is a protein capable of binding DNA molecules. In some preferred embodiments, the protein is selected from zinc finger proteins and transcription activator-like effector nucleases (TALEN proteins).

[0184] In some preferred embodiments, the target peptide has an amino acid sequence selected from the following: SEQ ID NO:1, 6-7, 43-44 and 46-53.

[0185] In some preferred embodiments, the fusion protein further comprises additional domains. In some preferred embodiments, the fusion protein further comprises a tag domain (e.g., a 6*His tag, HA tag, hapten tag, Strep tag, MBP tag, GST tag, etc.) to facilitate the preparation and purification of the fusion protein. In some preferred embodiments, the fusion protein further comprises an antibody heavy chain constant region. In some preferred embodiments, the additional domain (e.g., the tag domain) is located at the N-terminus of the fusion protein. In some preferred embodiments, the additional domain (e.g., the tag domain) is located at the C-terminus of the fusion protein.

[0186] In another aspect, this application provides a conjugate comprising the cell-penetrating peptide or a truncated form thereof as described above, and a target molecule (e.g., a target protein or a target nucleic acid).

[0187] In some preferred embodiments, the target molecule is directly covalently linked to the cell-penetrating peptide or its truncated form (i.e., directly linked via a chemical bond). In some preferred embodiments, the target peptide and the cell-penetrating peptide or its truncated form are covalently linked via a linker (e.g., a bifunctional linker or a peptide linker). In some preferred embodiments, the peptide linker has the amino acid sequence shown in SEQ ID NO: 39. However, it will be readily understood that various known linkers can be used in this application to covalently link the target molecule to the cell-penetrating peptide or its truncated form. In some preferred embodiments, the target molecule and the cell-penetrating peptide or its truncated form are linked non-covalently.

[0188] It is readily understood that the cell-penetrating peptides or truncated forms thereof in this application can be linked to a variety of target molecules. These target molecules can be linked to the cell-penetrating peptides or truncated forms thereof through covalent binding, affinity binding, intercalation, coordinate binding, complexation, binding, mixing, or addition, or other means. In some embodiments, the cell-penetrating peptides or truncated forms thereof disclosed in this application can be engineered to contain additional specific sites that can be used to bind one or more target molecules. For example, such sites may contain one or more reactive amino acid residues, such as cysteine ​​and histidine residues, for assisting covalent linking with the target molecule. In some embodiments, the cell-penetrating peptides or truncated forms thereof may be indirectly linked to target molecules. For example, the cell-penetrating peptides or truncated forms thereof may bind biotin and then indirectly bind a second target molecule linked to avidin.

[0189] It is readily understood that the target molecule in this application can be any desired molecule. For example, in some embodiments, the target molecule can be a detectable marker. Examples of detectable markers may include fluorescent markers (e.g., fluorescein, rhodamine, dansyl, phycoerythrin, or Texas red), enzyme substrate markers (e.g., horseradish peroxidase, alkaline phosphatase, luciferase, glucosylamylase, lysozyme, sugar oxidase, or β-D-galactosidase), stable isotopes or radioisotopes, chromophores, digoxigenin, biotin / avidin, DNA molecules, or gold, to facilitate detection. In some embodiments, the target molecule can be a cytotoxic agent. A "cytotoxic agent" can be any agent that is harmful to cells or may damage or kill cells. Examples of cytotoxic agents include, but are not limited to, paclitaxel, cytochalasin B, bacitracin D, ethamsylate, ipecacine, mitomycin, etoposide, teniposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, anthraquinone, mitoxantrone, styromycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, puromycin and its analogues, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-mercapguanine, cytarabine, 5-fluorouracil). Pyrimidine dacarbazine), alkylating agents (such as nitrogen mustard, thiotepa chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C and cis-dichlorodiamineplatin (DDP) cisplatin), anthracycline antibiotics (such as daunorubicin (formerly doxorubicin) and doxorubicin), antibiotics (such as dermatomycin (formerly actinomycin), bleomycin, photomycin and ammoniac (AMC)), and antimitotic agents (such as vincristine and vinblastine).

[0190] In some embodiments, the target molecule can be any desired protein or polypeptide. For example, in some cases, it may be desirable to deliver the antibody into cells (e.g., tumor cells, immune cells, etc.) and to perform its function (e.g., inhibiting viral infection, replication, and / or assembly, inhibiting or enhancing intracellular signaling, etc.). Therefore, in some preferred embodiments, the target molecule is an antibody. In some preferred embodiments, the antibody is selected from anti-hepatitis B virus antibodies (e.g., anti-HBsAg antibodies, anti-HBcAg antibodies, anti-HBeAg antibodies, etc.), anti-influenza virus antibodies (e.g., anti-HA1 antibodies, anti-HA2 antibodies), anti-tumor antigen antibodies (e.g., anti-p53 antibodies, anti-kras antibodies, anti-PRL-3 antibodies), anti-immune checkpoint antibodies (e.g., anti-PD1 or PDL1 antibodies), anti-melanin synthesis-related antibodies (e.g., tyrosinase-associated protein TYRP1 antibodies), and anti-SARS-CoV-2 antibodies (e.g., anti-SARS-CoV-2 S protein antibodies, such as antibodies against S protein RBD or S1 or S2).

[0191] In some cases, it may be desirable to deliver gene-editing-related proteins into cells and allow them to perform their functions (e.g., editing a target gene within the cell, or inhibiting gene editing within the cell). Therefore, in some preferred embodiments, the target molecule is a gene-editing-related protein. In some preferred embodiments, the protein is selected from Cas9 protein, AcrIIA4 protein, Cas13 protein, Cre recombinase, and Flip recombinase.

[0192] In some cases, it may be desirable to deliver an active or traceable protein into cells to perform its function (e.g., to study or alter signaling pathways, to localize cells, etc.). Therefore, in some preferred embodiments, the target molecule is an active or traceable protein. In some preferred embodiments, the protein is selected from fluorescent proteins (e.g., green fluorescent protein), toxic proteins (e.g., endotoxins), cytokines (e.g., interleukins or interferons, such as IL10 or IFNγ), immunomodulatory proteins (e.g., PD1), enzymes (e.g., luciferases, nucleases, recombinases, methyltransferases, protein kinases, etc.), signaling pathway-related proteins (e.g., β-catenin protein), cell cycle proteins (e.g., Cyclin D1 protein), transcription activators, and transcription repressors.

[0193] In some cases, it may be desirable to deliver DNA molecules into cells and allow them to perform their functions (e.g., express exogenous proteins, interfere with endogenous gene expression, etc.). Therefore, in some preferred embodiments, the target molecule is a protein capable of binding to DNA molecules. In some preferred embodiments, the protein is selected from zinc finger proteins and transcription activator-like effector nucleases (TALEN proteins).

[0194] In some preferred embodiments, the target molecule is a polypeptide having an amino acid sequence selected from the following: SEQ ID NO: 1, 6-7, 43-44 and 46-53.

[0195] In another aspect, this application provides a multimer comprising two or more fusion proteins or conjugates as described above. Without being limited by theory, in certain circumstances, the multimer form may be advantageous, as it can further facilitate the cellular delivery of the fusion protein or conjugate.

[0196] In another aspect, this application provides a complex comprising the fusion protein, conjugate, or polymer as described above, and a component non-covalently bound to or complexed with the fusion protein, conjugate, or polymer. In some preferred embodiments, the fusion protein, conjugate, or polymer comprises a protein capable of binding DNA molecules, which is linked to the cell-penetrating peptide or its truncated form of this application; and the complex comprises a DNA molecule, which is non-covalently bound to or complexed with the protein capable of binding DNA molecules. In some preferred embodiments, the protein capable of binding DNA molecules is covalently linked to the cell-penetrating peptide or its truncated form (optionally, via a linker). In some preferred embodiments, the protein capable of binding DNA molecules is non-covalently linked to the cell-penetrating peptide or its truncated form (optionally, via a linker). In some preferred embodiments, the protein capable of binding DNA molecules is linked to the N-terminus of the cell-penetrating peptide or its truncated form. In some preferred embodiments, the protein capable of binding DNA molecules is linked to the C-terminus of the cell-penetrating peptide or its truncated form. In some preferred embodiments, the DNA molecule comprises a nucleotide sequence recognized and bound by the protein capable of binding DNA molecules. In some preferred embodiments, the protein capable of binding to DNA molecules is selected from zinc finger proteins and transcription activator-like effector nucleases (TALEN proteins). In some preferred embodiments, the nucleotide sequence is a binding sequence of a zinc finger protein (e.g., SEQ ID NO: 54).

[0197] In another aspect, this application provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding the cell-penetrating peptide or a truncated form thereof or the fusion protein.

[0198] In another aspect, this application provides a vector (e.g., a cloning vector or an expression vector) containing isolated nucleic acid molecules as described above. In some embodiments, the vector of this application is selected from plasmids, granules, artificial chromosomes such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC) or P1-derived artificial chromosomes (PAC), bacteriophages such as λ phage or M13 phage, and animal viruses, etc.

[0199] In another aspect, this application provides a host cell comprising the isolated nucleic acid molecules or vectors as described above. In some embodiments, the host cell is a prokaryotic cell, including but not limited to Gram-negative or Gram-positive bacteria, such as Enterobacteriaceae (e.g., *Escherichia coli*), *Enterobacter* spp., *Erwinia* spp., *Klebsiella* spp., *Proteus* spp., *Salmonella* spp., such as *Salmonella typhimurium*, *Serratia* spp., such as *Serratia marcescens*, and *Shigella* spp., and *Bacillus* spp. such as *Bacillus subtilis* and *Bacillus licheniformis*, and *Pseudomonas* spp. such as *Pseudomonas aeruginosa* and *Streptomyces*. In some embodiments, the host cell is, for example, *Escherichia coli* or *Bacillus subtilis* cells.

[0200] In some embodiments, the host cell is a eukaryotic cell, such as a fungal cell like yeast or Aspergillus, an insect cell like S2 ​​Drosophila or Sf9, or an animal cell like fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK 293 cells, or human cells. In some embodiments, the host cell is a mammalian cell. In some embodiments, the host cell is a human, mouse, sheep, horse, dog, or cat cell. In some embodiments, the host cell is a Chinese hamster ovary cell.

[0201] In another aspect, a method for preparing the cell-penetrating peptide or a truncated form or fusion protein of the present application is provided, comprising culturing host cells as described above under conditions that allow expression of the cell-penetrating peptide or a truncated form or fusion protein, and recovering the cell-penetrating peptide or a truncated form or fusion protein from the cultured host cell culture.

[0202] In another aspect, this application provides a composition comprising the fusion protein or conjugate or polymer or complex or nucleic acid molecule or carrier as described above.

[0203] In another aspect, this application provides a pharmaceutical composition comprising the fusion protein, conjugate, polymer, or complex as described above, and a pharmaceutically acceptable carrier or excipient, wherein the fusion protein, conjugate, polymer, or complex comprises a therapeutically active polypeptide linked to a cell-penetrating peptide or a truncated form thereof of this application. In some preferred embodiments, the therapeutically active polypeptide is covalently linked to the cell-penetrating peptide or a truncated form thereof (optionally, via a linker). In some preferred embodiments, the therapeutically active polypeptide is non-covalently linked to the cell-penetrating peptide or a truncated form thereof (optionally, via a linker). In some preferred embodiments, the therapeutically active polypeptide is linked to the N-terminus of the cell-penetrating peptide or a truncated form thereof. In some preferred embodiments, the therapeutically active polypeptide is linked to the C-terminus of the cell-penetrating peptide or a truncated form thereof. It is readily understood that the therapeutically active polypeptide can be any polypeptide desired to be delivered transmembrane into cells, such as toxic proteins (e.g., endotoxins), cytokines (e.g., interleukins or interferons, such as IL10 or IFNγ), immunomodulatory proteins (e.g., PD1), antibodies, etc. In some preferred embodiments, the antibody is selected from anti-hepatitis B virus antibodies (e.g., anti-HBsAg antibody, anti-HBcAg antibody, anti-HBeAg antibody, etc.), anti-influenza virus antibodies (e.g., anti-HA1 antibody, anti-HA2 antibody), anti-tumor antigen antibodies (e.g., anti-p53 antibody, anti-kras antibody, anti-PRL-3 antibody), anti-immune checkpoint antibodies (e.g., anti-PD1 or PDL1 antibody), anti-melanin synthesis-related antibodies (e.g., tyrosinase-associated protein TYRP1 antibody), and anti-SARS-CoV-2 antibodies (e.g., anti-SARS-CoV-2 S protein antibody, such as anti-S protein RBD or S1 or S2 antibody).

[0204] In another aspect, this application provides a method for delivering a target molecule across a membrane into a cell, comprising linking a cell-penetrating peptide or a truncated form thereof as described above to the target molecule, and then contacting the cell.

[0205] In some preferred embodiments, the method includes (1) linking the target molecule to the cell-penetrating peptide or a truncated form thereof to obtain a conjugate; and (2) contacting the conjugate with a cell to deliver the target molecule transmembrane into the cell.

[0206] It is readily understood that in this application, cell-penetrating peptides or their truncated forms can be linked to target molecules in various ways. For example, the target molecule can be linked to the cell-penetrating peptide or its truncated form through covalent bonding, affinity bonding, embedding, coordinate binding, complexation, binding, mixing, or addition. In some preferred embodiments, in step (1), the target molecule is directly covalently linked to the cell-penetrating peptide or its truncated form (i.e., directly linked by a chemical bond). In some preferred embodiments, in step (1), the target peptide is covalently linked to the cell-penetrating peptide or its truncated form through a connector (e.g., a bifunctional connector or a peptide connector). In some preferred embodiments, in step (1), the target molecule is non-covalently linked to the cell-penetrating peptide or its truncated form. In some preferred embodiments, the target molecule and the cell-penetrating peptide or its truncated form are respectively as defined above.

[0207] In some preferred embodiments, the target molecule is a target peptide, and the method includes (1) linking the target molecule to the cell-penetrating peptide or a truncated form thereof to obtain a fusion protein; and (2) contacting the fusion protein with a cell to deliver the target molecule transmembrane into the cell.

[0208] It is readily understood that in this application, cell-penetrating peptides or their truncated forms can be linked to a target peptide in various ways to obtain a fusion protein. In some preferred embodiments, in step (1), the target peptide is directly covalently linked to the cell-penetrating peptide or its truncated form (i.e., directly linked via peptide bonds) to obtain a fusion protein. In some preferred embodiments, in step (1), the target peptide is covalently linked to the cell-penetrating peptide or its truncated form via a peptide linker (e.g., a flexible peptide linker) to obtain a fusion protein. In some preferred embodiments, the peptide linker has the amino acid sequence shown in SEQ IDNO: 39. However, it is readily understood that in this application, various known peptide linkers can be used to fuse the target peptide with the cell-penetrating peptide or its truncated form. In some preferred embodiments, in step (1), the cell-penetrating peptide or its truncated form is linked to the N-terminus of the target peptide (optionally, via a peptide linker). In some preferred embodiments, in step (1), the cell-penetrating peptide or its truncated form is linked to the C-terminus of the target peptide (optionally, via a peptide linker). In some preferred embodiments, the target peptide and the cell-penetrating peptide or their truncated form are as defined above.

[0209] In some preferred embodiments, the target molecule is a target nucleic acid, and the method includes (1) linking a protein capable of binding the target nucleic acid to the cell-penetrating peptide or a truncated form thereof; (2) contacting the product of step (1) with the target nucleic acid to obtain a complex; and (3) contacting the complex with a cell to deliver the target nucleic acid across the membrane into the cell.

[0210] In some preferred embodiments, the target molecule is a target nucleic acid, and the method includes: (1) adding a nucleotide sequence to the target nucleic acid that can be recognized and bound by a DNA-binding protein; (2) linking the DNA-binding protein to the cell-penetrating peptide or a truncated form thereof; (3) contacting the product of step (1) with the product of step (2) to obtain a complex; and (4) contacting the complex with a cell to deliver the target nucleic acid across the membrane into the cell. It will be readily understood that various known DNA-binding proteins and the nucleotide sequences they recognize and bind can be used in this application. In some preferred embodiments, the DNA-binding protein, the nucleotide sequence it recognizes and binds, and the cell-penetrating peptide or a truncated form thereof are as defined above.

[0211] In another aspect, this application relates to the use of the cell-penetrating peptide or a truncated form thereof as described above for the transmembrane delivery of a target molecule into a cell. In yet another aspect, this application relates to the use of the cell-penetrating peptide or a truncated form thereof as described above for the preparation of a kit for the transmembrane delivery of a target molecule into a cell.

[0212] Beneficial effects of the invention

[0213] The cell-penetrating peptide or its truncated form disclosed in this application can deliver target biological molecules (such as target peptides or target nucleic acids) across the membrane into cells and allow them to function within the cells. Compared with existing technologies, the cell-penetrating peptide or its truncated form disclosed in this application has a high delivery efficiency (significantly higher than TAT peptides derived from HIV), and therefore has good application prospects and value.

[0214] The embodiments of the present invention will now be described in detail with reference to the accompanying drawings and examples. However, those skilled in the art will understand that the following drawings and examples are for illustrative purposes only and are not intended to limit the scope of the invention. Various objects and advantages of the present invention will become apparent to those skilled in the art from the following detailed description of the drawings and preferred embodiments. Attached Figure Description

[0215] Figure 1 A schematically illustrates the working principle of the reporting system constructed in Example 1.

[0216] Figure 1 B schematically illustrates the main structural elements contained in the plasmid EHIPS-U2MG constructed in Example 1.

[0217] Figure 1 C shows the fluorescence microscopy observations of engineered cell lines transfected with the pcDNA3.1-GFP plasmid or the negative control plasmid.

[0218] Figure 2 The results of SDS-PAGE analysis of the purified target proteins (GFP, GFP-CPP28, and GFP-TAT) are shown. M: Protein molecular weight marker.

[0219] Figures 3A-3B The results show the observations of 293β5 cells cultured for 36 h in media containing or without the target protein (GFP, GFP-CPP28, or GFP-TAT) using a laser confocal high-content imaging analysis system. Figure 3A The results of quantitative analysis of the mCherry fluorescence intensity of the cells ( ) and ( ) Figure 3B The cells were transfected with the EHIPS-U2MG plasmid.

[0220] Figures 4A-4B The results of observations using a laser confocal high-content imaging analysis system on 293β5 cells cultured for 36 h in medium containing GFP-CPP28 (30, 10, 5, 2.5 μg / mL) are shown. Figure 4A The results of quantitative analysis of the mCherry fluorescence intensity of the cells ( ) and ( ) Figure 4B The cells were transfected with the EHIPS-U2MG plasmid.

[0221] Figure 5 The results show the analysis of various cell types (MDCK, VERO, and Hacat cells) and their culture supernatants cultured for 36 h in a medium containing the target protein (GFP, GFP-CPP28, or GFP-TAT) using a laser confocal high-content imaging analysis system and a luciferase detection system, respectively. The various cell types were transfected with the EHIPS-U2MG plasmid.

[0222] Figure 6A The results of Hela-p63mRb3 cells observed by confocal microscopy at specified time points are shown. These Hela-p63mRb3 cells were cultured in a medium containing GFP-CPP28 (60 μg / mL).

[0223] Figure 6BThe results show the GFP protein content in Hela-p63mRb3 cells, which were cultured in a medium containing GFP-CPP28 (60 μg / mL), at specified time points using a double-antibody sandwich assay.

[0224] Figures 7A-7B The confocal microscopy observation results of Hela-p63mRb3 cells are shown. Figure 7A ) and GFP fluorescence intensity analysis results ( Figure 7B The Hela-p63mRb3 cells were first treated with a specified inhibitor of endocytosis pathway or at low temperature (4°C) for 2 h, and then treated with 30 μg / ml GFP-cpp28 protein for 1 h.

[0225] Figures 8A-8B The results of observations using a laser confocal high-content imaging analysis system on 293β5 cells cultured for 36 h in a medium containing various recombinant proteins at specified concentrations, wherein the cells were transfected with the EHIPS-U2MG plasmid, are shown.

[0226] Figures 9A-9B The results of GFP fluorescence intensity analysis of Hela-p63mRb3 cells at specified time points are shown. Figure 9A ) and confocal microscopy observation results ( Figure 9B The cells were cultured in a medium containing GFP-cpp28, GFP-cpp28a, GFP-cpp28b, GFP-cpp28c, GFP-cpp28d and GFP-TAT (60 μg / mL).

[0227] Figures 10A-10B The results show the intracellular GFP fluorescence spot counts of HeLa-p63mRb3 cells cultured for 1 h in a medium containing GFP-cpp28, GFP-cpp28oS, GFP-cpp28a, GFP-cpp28aC, GFP-cpp28b, and GFP-cpp28bC (60 μg / mL). Figure 10A ) and confocal microscopy observation results ( Figure 10B ).

[0228] Figure 11A-11BThe confocal microscopy results of Hela-p63mRb3 cells cultured for 1 h in medium containing different concentrations (6.25 μg / mL, 12.5 μg / mL, 25 μg / mL, 50 μg / mL, 100 μg / mL, 200 μg / mL) of GFP-cpp28, GFP-cpp28aC, GFP-cpp28bC, GFP-cpp28cC, GFP-cpp28dC, GFP-cpp28dREC, and GFP-cpp-oS are shown. Figure 11A ) and intracellular GFP fluorescence intensity analysis results ( Figure 11B ).

[0229] Figure 12A-12B The confocal microscopy results of Hela-p63mRb3 cells cultured for 1 h in medium containing different concentrations (6.25 μg / mL, 12.5 μg / mL, 25 μg / mL, 50 μg / mL, 100 μg / mL, 200 μg / mL) of GFP-cpp28, GFP-cpp28a, GFP-cpp28b, GFP-cpp28c, GFP-cpp28d, GFP-cpp28oNT-1, GFP-cpp28oNT-2, and GFP-cpp28oNT-3 are shown. Figure 12A ) and intracellular GFP fluorescence intensity analysis results ( Figure 12B ).

[0230] Figures 13A-13B The confocal microscopy results of HeLa-p63mRb3 cells cultured for 1 h in medium containing different concentrations (6.25 μg / mL, 12.5 μg / mL, 25 μg / mL, 50 μg / mL, 100 μg / mL, 200 μg / mL) of GFP-cpp28, GFP-cpp28aCQ1, GFP-cpp28aCQ2, GFP-cpp28aCQ3, GFP-cpp28aCNT, GFP-cpp28bCNT, GFP-cpp28cCNT, and GFP-cpp28dCNT are shown. Figure 13A ) and intracellular GFP fluorescence intensity analysis results ( Figure 13B ).

[0231] Figure 14A The results of high-performance liquid chromatography (HPLC) analysis of GFP, GFP-cpp28e and GFP-TAT are shown.

[0232] Figure 14BThe results of analytical ultracentrifuge analysis using an Optima XL-100 (AUC) are shown for GFP-cpp28, GFP, and GFP-TAT.

[0233] Figure 15A The purification results of preparative molecular sieves for GFP-TAT, GFP-cpp28, GFP-cpp28e, GFP-cpp28g, GFP-cpp28h, and GFP-cpp28j are shown.

[0234] Figure 15B The results of SDS-PAGE analysis of various protein fractions (polymer fractions and monomer fractions) obtained by preparative molecular sieve purification methods are shown.

[0235] Figure 15C The results of quantitative analysis of mCherry fluorescence intensity in 293β5 cells using a laser confocal high-content imaging analysis system are shown. The cells were transfected with the EHIPS-U2MG plasmid and cultured in a medium containing specified concentrations of specified protein fractions (multimeric fractions and monomer fractions).

[0236] Figure 16A The main structural elements contained in the Reporter plasmid constructed in Example 6 are shown.

[0237] Figure 16B-16C The image shows 293β5 cells transfected with a specified plasmid or plasmid combination, captured using a laser confocal high-content imaging analysis system. Figure 16B ) and fluorescence intensity analysis ( Figure 16C The result of ).

[0238] Figure 17A The results of observations on 293β5 cells using a laser confocal high-content imaging analysis system are shown, wherein the cells were transfected with a specified plasmid or plasmid combination and treated for 12 h with a culture medium containing or not containing a specified concentration of various recombinant proteins.

[0239] Figure 17B The results of detecting intracellular EGFP protein content using Native PAGE (native gel electrophoresis) are shown, wherein the cells were transfected with a specified plasmid or combination of plasmids and treated for 12 h with a culture medium containing or not containing a specified concentration of various recombinant proteins.

[0240] Figure 17C The results of Western blotting analysis of intracellular EGFP protein levels are shown, wherein the cells were transfected with a specified plasmid or combination of plasmids and treated for 12 h with a culture medium containing or not containing a specified concentration of various recombinant proteins.

[0241] Figure 17D The sequencing analysis results of the PCR amplification products obtained in Example 6 are shown, where Cas9: plasmid expressing Cas9 protein; Vector con: control vector (empty plasmid); aCRISPR-pl: plasmid expressing anti-CRISPR protein; aCRISPR-pro: anti-CRISPR protein; aCRISPR-cpp28-pro: anti-CRISPR-cpp28ori protein.

[0242] Figure 18 The results of SDS-PAGE analysis of the purified antibody or recombinant protein in Example 7 are shown. Figure 18 A) and Western blot analysis results ( Figure 18 B).

[0243] Figure 19 Immunofluorescence assays of cells (HepG2-N10, HepG2-C3A, Hela, MNT-1, and A375) treated for 6 h with a culture medium containing 100 μg / ml of the specified antibody or recombinant protein are shown.

[0244] Figure 20 The results of detection of TYRP-1 antigen in MNT-1 cell lysates by Western blot are shown. The MNT-1 cells were treated with a specified concentration of 2A7-cpp28ori, TA99, or TA99-cpp28ori for 6 h.

[0245] Figure 21A The results show the levels of HBcAg, TRIM21, and antibodies in HepG2-N10 cell lysates detected by Western blot. The HepG2-N10 cells were treated with 100 μg / ml of 2A7-cpp28ori, 2A7, 16D5-cpp28ori, 16D5, TA99-cpp28ori, or TA99 antibody for 6 h.

[0246] Figure 21B The results show the detection results of HBeAg antigen and HBV DNA levels in the culture supernatant of HepG2-N10 cells, which were treated with 100 μg / ml of 2A7-cpp28ori, 2A7, 16D5-cpp28ori, 16D5, TA99-cpp28ori, or TA99 antibody for 6 h.

[0247] Figure 22The results show the observation of mRuby3 fluorescence in 293β5 cells using a laser confocal high-content imaging analysis system, wherein the cells were transfected with plasmid pTT5-mRuby3-ZF motif using ZF-cpp28ori or PEI transfection reagent.

[0248] Sequence information

[0249] Information about the sequences involved in this application is summarized in Table 1.

[0250] Table 1: Sequence Information

[0251]

[0252] 1. Amino acid sequence of GFP (SEQ ID NO: 1)

[0253] MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDT LVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYK

[0254] 2. Amino acid sequence of the DNA-binding domain of Gal4 (SEQ ID NO: 2)

[0255] MKLLSSIEQACDICRLKKLKCSKEKPKCAKCLKNNWECRYSPKTKRSPLTRAHLTEVESRLERLEQLFLLIFPREDLDMILKMDSLQDIKALLTGLFVQDNVNKDAVTDRLASVETDMPLTLRQHRISATSSSEESSNKGQRQLTVS

[0256] 3. Amino acid sequence of Anti-GFP VHH2 (SEQ ID NO: 3)

[0257] MADVQLQESGGGSVQAGEALRLSCVGSGYTSINPYMAWFRQAPGKEREGVAAISSGGQYTYYADSVKGRFTISRDNAKNTMYLQMPSLKPDDSAKYYCAADFRRGGSWNVDPLRYDYQHWGQGTQVTVSS

[0258] 4. Amino acid sequence of the VP16 activation domain (SEQ ID NO: 4)

[0259] APPTDVSLGDELHLDGEDVAMAHADALDDFDLDMLGDGDSPGPGFTPHDSAPYGALDMADFEFEQMFTDALGIDEYGG

[0260] 5. Amino acid sequence of Anti-GFP VHH6 (SEQ ID NO: 5)

[0261] MADVQLQESGGGSVQTGGSLRLSCAVSPYIGSRISLGWFRQAPGKVREGVALINSRDGSTYYADTVKGRFTISQGDANTVYLQMNSLKPEDTAIYYCAARWGQITDIQALAVASFPDWGQGTQVTVSS

[0262] 6. Amino acid sequence of RFP (SEQ ID NO: 6)

[0263] MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGTQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGVVTVTQDSSL QDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKGEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAEGRHSTGGMDGCTS

[0264] 7. Amino acid sequence of luciferase (SEQ ID NO: 7)

[0265] MGVKVLFALICIAVAEAKPTENNEDFNIVAVASNFATTDLDADRGKLPGKKLPLEVLKELEANARKAGCTRGCLICLSHIKCTPKMKKFIPGRCHTYEGDKESAQGGIGEAIVDIPEIPGFKDLEPLEQFIAQVDLCVDCTTGCLKGLANVQCSDLLKKWLPQRCATFASKIQGQVDKIKGAGGD

[0266] 8. UAS sequence (SEQ ID NO: 8)

[0267] AGCTTGCATGCCTGCAGGTCGGAGTACTGTCCTCCGAGCGGAGTACTGTCCTCCGAGCGGAGTACTGTCCTCCGAGCGGAGTACTGTCCTCCGAGCGGAGTACTGTCCTCCGAGCGGAGACTCTAGCGAGCGCCGGAGTATAAATAGAGGCGCTTCGTCTACGGAGCGACAATTCAATTCAAACAAGCAAAGTGAACACGTCGCTAAGCGAAAGCTAAGCAAATAAACAAGCGCAGCTGAACAAGCTAAACAATCTGCAGTAAAGTGCAAGTTAAAGTGAATCAATTAAAAGTAACCAGCAACCAAGTAAATCAACTGCAACTACTGAAATCTGCCAAGAAGTAATTATTGAATACAA

[0268] 9. Amino acid sequence of iRFP670 (SEQ ID NO: 9)

[0269] MARKVDLTSCDREPIHIPGSIQPCGCLLACDAQAVRITRITENAGAFFGRETPRVGELLADYFGETEAHALRNALAQSSDPKRPALIFGWRDGLTGRTFDISLHRHDGTSIIEFEPAAAEQADNPLRLTRQIIARTKELKSLEEMAARVPRYLQA MLGYHRVMLYRFADDGSGMVIGEAKRSDLESFLGQHFPASLVPQQARLLYLKNAIRVVSDSRGISSRIVPEHDASGAALDLSFAHLRSISPCHLEFLRNMGVSASMSLSIIIDGTLWGLIICHHYEPRAVPMAQRVAAEMFADFLSLHFTAAHHQR

[0270] The amino acid sequence of 10.cpp28 (SEQ ID NO: 10)

[0271] RRRGRSPRRRTPSPRRRRSQSPRRRRSQSRESQC

[0272] 11. Amino acid sequence of cpp28a (SEQ ID NO: 11)

[0273] RRRGRSPRRRTPSPRRRRSQSPRRRRSQSRES

[0274] The amino acid sequence of 12.cpp28b (SEQ ID NO: 12)

[0275] RRRGRSPRRRTPSPRRRRSQSPRRRRSQS

[0276] The amino acid sequence of 13.cpp28c (SEQ ID NO: 13)

[0277] RRRGRSPRRRTPSPRRRRSQSPRRRRS

[0278] The amino acid sequence of 14.cpp28d (SEQ ID NO: 14)

[0279] RRRGRSPRRRTPSPRRRRSQS

[0280] The amino acid sequence of 15.cpp28e (SEQ ID NO: 15)

[0281] RRRDRGRSPRRRTPSPRRRRSQSPRRRRSQSRESQC

[0282] The amino acid sequence of 16.cpp28f (SEQ ID NO: 16)

[0283] RRRGRSPRRRTPSPRRRRSKSPRRRRSKSRESQC

[0284] The amino acid sequence of 17.cpp28g (SEQ ID NO: 17)

[0285] RQRGRAPRRRTPSPRRRRSQSPRRRRSQSRASQC

[0286] The amino acid sequence of 18.cpp28h (SEQ ID NO: 18)

[0287] RCRGRRGRSPRRRTPSPRRRRSQSPRRRRKSRESQC

[0288] The amino acid sequence of 19.cpp28i (SEQ ID NO: 19)

[0289] RRRGGARASRSPRRRTPSPRRRRSQSPRRRRRSQSRSANC

[0290] The amino acid sequence of 20.cpp28j (SEQ ID NO: 20)

[0291] RRRGNPRAPRSPRRRTPSPRRRRSQSPRRRRSQSPAPSNC

[0292] The amino acid sequence of 21.cpp28k (SEQ ID NO: 21)

[0293] RRRGGSRATRSPRRRTPSPRRRRSQSPRRRRRSQSPASSNC

[0294] The amino acid sequence of 22.cpp28L (SEQ ID NO: 22)

[0295] RRRPASRRSTPSPRRRRSQSPRRRRSPSPRPASNC

[0296] The amino acid sequence of 23.cpp28-Os (SEQ ID NO: 23)

[0297] RRRGRSPRRRTPSPRRRRSQSPRRRRSQSRESQS

[0298] The amino acid sequence of 24.cpp28aC (SEQ ID NO: 24)

[0299] RRRGRSPRRRTPSPRRRRSQSPRRRRSQSREC

[0300] The amino acid sequence of 25.cpp28bC (SEQ ID NO: 25)

[0301] RRRGRSPRRRTPSPRRRRSQSPRRRRSQC

[0302] The amino acid sequence of 26.cpp28cC (SEQ ID NO: 26)

[0303] RRRGRSPRRRTPSPRRRRSQSPRRRRC

[0304] The amino acid sequence of 27.cpp28dC (SEQ ID NO: 27)

[0305] RRRGRSPRRRTPSPRRRRSQC

[0306] The amino acid sequence of 28.cpp28dREC (SEQ ID NO: 28)

[0307] RRRGRSPRRRTPSPRRRRSQRREC

[0308] The amino acid sequence of 29.cpp28oNT-1 (SEQ ID NO: 29)

[0309] SPRRRTPSPRRRRSQSPRRRRSQSRESQC

[0310] The amino acid sequence of 30.cpp28oNT-2 (SEQ ID NO: 30)

[0311] SPRRRRSQSPRRRRSQSRESQC

[0312] The amino acid sequence of 31.cpp28oNT-3 (SEQ ID NO: 31)

[0313] SPRRRRSQSRESQC

[0314] The amino acid sequence of 32.cpp28aCQ1 (SEQ ID NO: 32)

[0315] RRRGRQPRRRTPSPPRRRRSQSPRRRRSQSREC

[0316] The amino acid sequence of 33.cpp28aCQ2 (SEQ ID NO: 33)

[0317] RRRGRQPRRRTPQPRRRRSQSPRRRRSQSREC

[0318] The amino acid sequence of 34.cpp28aCQ3 (SEQ ID NO: 34)

[0319] RRRGRQPRRRTPQPRRRRSQQPRRRRSQSREC

[0320] The amino acid sequence of 35.cpp28aCNT (SEQ ID NO: 35)

[0321] SPRRRTPSPRRRRSQSPRRRRSQSREC

[0322] The amino acid sequence of 36.cpp28bCNT (SEQ ID NO: 36)

[0323] SPRRRTPSPRRRRSQSPRRRRSQC

[0324] The amino acid sequence of 37.cpp28cCNT (SEQ ID NO: 37)

[0325] SPRRRTPSPRRRRSQSPRRRRC

[0326] The amino acid sequence of 38.cpp28dCNT (SEQ ID NO: 38)

[0327] SPRRRTPSPRRRRSQC

[0328] 39. Amino acid sequence of the flexible peptide linker (SEQ ID NO: 39)

[0329] GGGGSGGGGSGGGGS

[0330] 40. Amino acid sequence of GFP-cpp28 (SEQ ID NO: 40)

[0331] MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYKGGGGSGGGGSGGGGSRRRGRSPRRRTPSPRRRRSQSPRRRRSQSRESQC

[0332] 41. Amino acid sequence of GFP-TAT (SEQ ID NO: 41)

[0333] MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYKGGGGSGGGGSGGGGSGRKKRRQRRRPPQ

[0334] 42. Amino acid sequence of cell-penetrating peptide TAT (SEQ ID NO: 42)

[0335] GRKKRRQRRRPPQ

[0336] 43. Amino acid sequence of mRuby3 protein (SEQ ID NO: 43)

[0337] MVSKGEELIKENMRMKVVMEGSVNGHQFKCTGEGEGRPYEGVQTMRIKVIEGGPLPFAFDILATSFMYGSRTFIKYPADIPDFFKQSFPEGFTWERVTRYEDGGVVTVTQDTSLEDGE LVYNVKVRGVNFPSNGPVMQKKTKGWEPNTEMMYPADGGLRGYTDIALKVDGGGHLHCNFVTTYRSKKTVGNIKMPGVHAVDHRLERIEESDNETYVVQREVAVAKYSNLGGGMDELYK

[0338] 44. Amino acid sequence of anti-CRISPR protein (SEQ ID NO: 44)

[0339] MNINDLIREIKNKDYTVKLSGTDSNSITQLIIRVNNDGNEYVISESENESIVEKFISAFKNGWNQEYEDEEEFYNDMQTITLKSELN

[0340] 45. Amino acid sequence of anti-CRISPR-cpp28ori protein (SEQ ID NO: 45)

[0341] MNINDLIREIKNKDYTVKLSGTDSNSITQLIIRVNNDGNEYVISESENESIVEKFISAFKNGWNQEYEDEEEFYNDMQTITLKSELNGGGGSGGGGSGGGGSRRRGRSPRRRTPSPRRRRSQSPRRRRSQSRESQC

[0342] 46. ​​Amino acid sequence of cas9 protein (SEQ ID NO: 46)

[0343]

[0344] 47. Amino acid sequence of the light chain variable region of antibody 2A7 (SEQ ID NO: 47)

[0345] DIQMTQTSSSLSASPGDRVTISCRASQGINNYLNWYKQKTDGTFKLLIYYTSYLHSGVPSRFSGRGSGTDYSLTISNLEPEDVATYYCQQYGKLPWTFGGGTKLEIK

[0346] 48. Amino acid sequence of the heavy chain variable region of antibody 2A7 (SEQ ID NO: 48)

[0347] QVQLQQPGAELVKPGASVKLSCKASGYTFTRYWMHWVMQRPGQDLEWIGEINPINGRTNYNEKFRRKATLTVDKSSSTVYIQFSSLTSEDSAVYFCTREGYRNDYYYAMDFWGRGTSVTVSS

[0348] 49. Amino acid sequence of the light chain variable region of antibody 16D5 (SEQ ID NO: 49)

[0349] DIVLTQSPGSLAVFLGQRATISCRASQSVSGSIYSYMHWYQQKPGQPPKLLIKFASNLESGVPARFSGGGSGTDFTLNIHPVEEEDAATYYCQHSWEIPYTFGGGTKLEIK

[0350] 50. Amino acid sequence of the heavy chain variable region of antibody 16D5 (SEQ ID NO: 50)

[0351] EVQLQQSGAEVVKPGASVKLSCTASGFKIEDTYIHWVKQRPEQGLEWIGRIDPANGNSRYDPNFQGKATIIADTSSYTIYLQLSSLTSEDTAVYYCSSPLSLLRLGGFAYWGQGTLITVSA

[0352] 51. Amino acid sequence of the light chain variable region of antibody TA99 (SEQ ID NO: 51)

[0353] AIQMSQSPASLSSASVGETVTITCRASGNIYNYLAWYQQKQGKSPHLLVYDAKTLADGVPSRFSGSGSGTQYSLKISSLQTEDSGNYYCQHFWSLPFTFGSGTKLEIK

[0354] 52. Amino acid sequence of the heavy chain variable region of antibody TA99 (SEQ ID NO: 52)

[0355] VQLQQSGAELVRPGALVKLSCKTSGFNIKDYFLHWVRQRPDQGLEWIGWINPDNGNTVYDPKFQGTASLTADTSSNTVYLQLSGLTSEDTAVYFCTRRDYTYEKAALDYWGQGASVIVSS

[0356] 53. Amino acid sequence of ZF protein (SEQ ID NO: 53)

[0357] VSRPGERPFQCRICMRNFSDKTKLRVHTRTHTGEKPFQCRICMRNFSVRHNLTRHLRTHTGEKPFQCRICMRNFSQSTSLQRHLKTHLRGS

[0358] 54. Binding sequence of ZF protein (SEQ ID NO: 54)

[0359] tGTAGATGGAg Detailed Implementation

[0360] The invention will now be described with reference to the following embodiments, which are intended to illustrate the invention (and not limit it).

[0361] Unless otherwise specified, the molecular biology experimental methods and immunoassays used in this invention are substantially in accordance with the methods described in J. Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press, 1989, and F.M. Usubel et al., A Concise Guide to Laboratory Molecular Biology, 3rd Edition, John Wiley & Sons, Inc., 1995; the use of restriction endonucleases is in accordance with the manufacturer's recommendations. Those skilled in the art will appreciate that the examples illustrate the invention by way of illustration and are not intended to limit the scope of the invention as claimed.

[0362] Example 1: Establishment of a reporter system for evaluating the efficiency of cell-penetrating peptide delivery protein entry into cells.

[0363] In this embodiment, in order to easily and efficiently evaluate the efficiency of cell-penetrating peptides in mediating the entry of target proteins into cells, the inventors used green fluorescent protein (GFP; SEQ ID NO: 1) as a model protein to establish a report system that can quantify and display the efficiency of cell-penetrating peptides in delivering proteins into cells.

[0364] The working principle of the reporting system constructed in this embodiment is as follows: Figure 1As shown in Figure A. In short, GFP is used as the cargo molecule and fused with the cell-penetrating peptide to be tested. Furthermore, the DNA-binding domain (DBD; SEQ ID NO: 2) of the transcriptional regulator Gal4 is fused with Anti-GFP VHH2 (first anti-GFP single-domain antibody; SEQ ID NO: 3) to form the first fusion protein DBD-VHH2; the VP16 activation domain (AD; SEQ ID NO: 4) is fused with Anti-GFP VHH6 (second anti-GFP single-domain antibody; SEQ ID NO: 5) to form the second fusion protein AD-VHH6; and the mCherry gene encoding Red fluorescent protein (RFP; SEQ ID NO: 6) and the Luc gene encoding luciferase (Luc; SEQ ID NO: 7) are placed under the regulation of the Gal4-recognized UAS sequence (upstream activating sequence; SEQ ID NO: 8). Therefore, when the cell-penetrating peptide to be tested can deliver the cargo molecule GFP into the cell, the DBD-VHH2 and AD-VHH6 expressed in the cell will specifically recognize and bind to the GFP molecules that have entered the cell, causing DBD and AD to approach each other and form a complex that can recognize the UAS sequence and initiate transcription. This complex will initiate the transcription and expression of downstream genes (mCherry gene and Luc gene) regulated by the UAS sequence (Cell. 2013 Aug 15; 154(4): 928-939). Therefore, by detecting the intensity of fluorescence emitted by the mCherry protein and / or the activity level of luciferase Luc, the number of GFP molecules entering the cell can be determined, thereby determining the efficiency of the cell-penetrating peptide delivered by the GFP-fused protein. In this reporter system, the transcriptional activation of the mCherry gene and Luc gene depends on the presence of GFP in the cell. Therefore, the intracellular GFP level can be determined by detecting the intensity of mCherry fluorescence and / or the activity of luciferase Luc. The stronger the fluorescence emitted by the mCherry protein or the higher the activity of luciferase Luc, the more GFP molecules are present in the cell, and the higher the delivery efficiency of the transmembrane peptide to be tested.

[0365] To simplify and expedite the evaluation, the inventors constructed the components involved in the aforementioned system into a single plasmid, named EHIPS-U2MG. The main structural elements contained in plasmid EHIPS-U2MG are as follows: Figure 1As shown in B, where UAS: upstream activation sequence recognized by Gal4; RFP: nucleotide sequence encoding red fluorescent protein; 2A: linker nucleotide sequence; Luc: nucleotide sequence encoding luciferase; BGH: transcription termination sequence; TMP / CAG / EF1p: promoter sequence; DBD: nucleotide sequence encoding DNA-binding domain; AD: nucleotide sequence encoding VP16 activation domain; VHH2 / VHH6: nucleotide sequence encoding anti-GFP single-domain antibody VHH2 / VHH6; H2B: nucleotide sequence encoding nuclear localization signal; iRFP670: nucleotide sequence encoding far-red fluorescent protein 670 (SEQ ID NO: 9); PurR: puromycin resistance gene; ins: insulating sequence; TR: transposon sequence.

[0366] The plasmid EHIPS-U2MG carries the piggyBac transposon system and the puromycin resistance gene (PurR). After transfecting 293T cells with this plasmid, puromycin resistance selection can be performed to obtain engineered cell lines that stably integrate these structural elements into their genome. The pcDNA3.1-GFP plasmid (carrying the nucleotide sequence encoding GFP) or the negative control plasmid was transfected into these engineered cell lines, and observation was performed using a fluorescence microscope. The results showed that green fluorescence from GFP and red fluorescence from mCherry were observed in cells transfected with the pcDNA3.1-GFP plasmid; no green or red fluorescence signals were detected in the negative control cells. Figure 1 C).

[0367] Therefore, using the reporter system and engineered cell line constructed in this embodiment, the presence and amount of GFP protein in the cell can be determined by detecting the red fluorescence signal and intensity of mCherry.

[0368] Example 2: Design of cell-penetrating peptide cpp28 and preparation of recombinant protein containing cpp28 and GFP

[0369] In this embodiment, the inventors designed a novel cell-penetrating peptide and prepared a recombinant protein containing the cell-penetrating peptide and green fluorescent protein.

[0370] The cell-penetrating peptide cpp28 designed in this embodiment has the following amino acid sequence: RRRGRSPRRRTPSPRRRRSQSPRRRRSQSRESQC (SEQ ID NO: 10). Based on this, a recombinant protein GFP-CPP28 (SEQ ID NO: 40) was designed, wherein the C-terminus of GFP is linked to the cell-penetrating peptide cpp28 via a flexible linker GGGGS-GGGGS-GGGGS (SEQ ID NO: 39). The nucleotide sequence encoding GFP-CPP28 was cloned into the pTO-T7 prokaryotic expression vector (which carries a nucleotide sequence encoding a 6×His tag; Luo Wenxin et al., Chinese Journal of Biotechnology, 2000, 16:53-57) to obtain the expression plasmid pTO-T7-GFP-CPP28.

[0371] In addition, a recombinant protein GFP-TAT (SEQ ID NO: 41) was designed, in which the C-terminus of GFP was linked to the cell-penetrating peptide TAT (a cell-penetrating peptide derived from HIV; SEQ ID NO: 42) via a flexible linker (SEQ ID NO: 39). The nucleotide sequence encoding GFP-TAT was cloned into the pTO-T7 prokaryotic expression vector to obtain the expression plasmid pTO-T7-GFP-TAT.

[0372] The expression plasmids constructed above were transformed into *E. coli* Shuffle T7 strain for recombinant protein expression. Subsequently, the recombinant proteins expressed in *E. coli* were purified by nickel column affinity chromatography to obtain the purified target proteins GFP-CPP28 and GFP-TAT. In addition, GFP protein was expressed and purified using a similar method. SDS-PAGE analysis of the purified target proteins (GFP, GFP-CPP28, and GFP-TAT) yielded the following results: Figure 2 As shown in the figure. The results show that the purity of the obtained purified proteins is greater than 95%.

[0373] Example 3: Evaluation of the efficiency of cpp28 in delivering proteins into cells

[0374] In this embodiment, the efficiency of cell-penetrating peptide cpp28 in delivering green fluorescent protein into cells was evaluated using the reporting system constructed in Example 1.

[0375] 293β5 cells were seeded in 96-well plates at a density of 20,000 cells per well. After 12 h, the EHIPS-U2MG plasmid constructed in Example 1 was transfected into the cells using Lipofectamine™ 2000 (Thermo Fisher, 11668019) transfection reagent. After 12 h of transfection, the culture medium was removed, and culture medium containing different recombinant proteins (DMEM, Gibco + 10% Gibco FBS) was added, with the concentration of recombinant protein (GFP, GFP-CPP28, or GFP-TAT) at 60 μg / mL. After culturing for another 36 h, the mCherry fluorescence of the cells was captured and analyzed using a laser confocal high-content imaging system. In addition, negative and positive controls were set up as follows: In the negative control group, cells were transfected with the EHIPS-U2MG plasmid, but no recombinant protein was added to the culture medium; In the positive control group, cells were transfected with both the EHIPS-U2MG and pcDNA3.1-GFP plasmids, and no recombinant protein was added to the culture medium.

[0376] Experimental results are as follows Figures 3A-3B As shown. The results showed that: (1) iRFP670 fluorescence could be observed in cells of all groups, indicating that the EHIPS-U2MG plasmid had been successfully transfected into the cells; (2) in the negative control group, mCherry fluorescence was basically undetectable. Figure 3B Furthermore, the strongest mCherry fluorescence was detected in the positive control group. Figures 3A-3B (3) GFP alone cannot cross the membrane into the cell and cannot induce the cell to express mCherry (mCherry fluorescence can not be detected in this group of cells); (4) cpp28 and TAT can both deliver GFP into the cell and then activate the expression of downstream mCherry genes through the fluorescent reporter system, so that the cell emits mCherry fluorescence; (5) cpp28 delivers GFP across the membrane into the cell with significantly higher efficiency than TAT.

[0377] Furthermore, a concentration gradient experiment was performed on cpp28 to evaluate its transmembrane efficiency. In short, 293β5 cells were seeded in 96-well plates at a density of 20,000 cells per well. After 12 h, the EHIPS-U2MG plasmid constructed in Example 1 was transfected into the cells using Lipofectamine™ 2000 (ThermoFisher, 11668019) transfection reagent. After 12 h of transfection, the culture medium was removed, and culture medium containing GFP-CPP28 (DMEM, Gibco + 10% Gibco FBS) was added, with GFP-CPP28 concentrations of 30, 10, 5, and 2.5 μg / mL. After culturing for another 36 h, the mCherry fluorescence of the cells was captured and analyzed using a laser confocal high-content imaging system.

[0378] Experimental results are as follows Figures 4A-4B As shown in the figure. The results showed that cpp28 can effectively deliver GFP protein into cells and activate the expression of downstream mCherry genes through a fluorescent reporter system, causing cells to emit mCherry fluorescence; and, as the concentration of GFP-cpp28 protein increases, the expression level of mCherry gradually increases, and the fluorescence intensity of mCherry gradually increases.

[0379] Furthermore, the efficiency of cpp28 in delivering GFP molecules into cells was evaluated across multiple cell types. In short, specified cell types (MDCK, VERO, or Hacat) were seeded at a density of 20,000 cells per well in 96-well plates. After 12 h, the EHIPS-U2MG plasmid constructed in Example 1 was transfected into the cells using Lipofectamine™ 2000 (Thermo Fisher, 11668019) transfection reagent. After 12 h of transfection, the culture medium was removed, and culture medium containing different recombinant proteins (DMEM, Gibco + 10% Gibco FBS) was added, with a concentration of 60 μg / mL for each recombinant protein (GFP, GFP-CPP28, or GFP-TAT). After culturing for another 36 h, the mCherry fluorescence of the cells was captured and analyzed using a laser confocal high-content imaging system; and the enzyme activity of gLuc protein secreted into the cell supernatant was detected using a Pierce™ Gaussia Luciferase assay system.

[0380] Experimental results are as follows Figure 5As shown in the figure. The results showed that for all cell types (MDCK, VERO, and Hacat cells), cells cultured in medium containing GFP-CPP28 exhibited significantly higher mCherry fluorescence intensity and significantly higher luciferase activity. This indicates that CPP28 can deliver GFP molecules into all cell types (MDCK, VERO, and Hacat cells), and its delivery efficiency is significantly higher than that of TAT.

[0381] Example 4: Dynamic process and pathway of cpp28 protein delivery into cells

[0382] In this embodiment, the inventors studied the dynamic process and pathway of cpp28 delivery protein into cells.

[0383] To investigate the dynamic process of cpp28 protein delivery into cells, the inventors constructed a HeLa cell line (named HeLa-p63mRb3) that expresses mRuby3 protein (SEQ ID NO: 43; emitting red fluorescence) on the cell membrane and iRFP670 protein (emitting far-red fluorescence) in the nucleus. By acquiring and analyzing images of this cell line using a laser confocal high-content imaging analysis system, the green, red, and far-red fluorescence within the cells can be detected, located, and quantified, allowing for direct observation and analysis of GFP protein, the cell membrane, and the cell nucleus.

[0384] In short, Hela-p63mRb3 cells were seeded at a density of 10,000 cells per well in 24-well PerkinElmer microplates. After cell attachment, the culture medium was removed, and DMEM (Gibco + 10% Gibco FBS) containing 60 μg / mL GFP-cpp28 was added. After 5 min, 10 min, 20 min, 30 min, 40 min, 1 h, 2 h, 3 h, 4 h, 6 h, 8 h, 10 h, 12 h, and 24 h, the cells were washed with 50 μM heparin (to remove non-specifically adsorbed GFP protein on the cell membrane), then fixed with 3.7% paraformaldehyde, and subsequently imaged using a laser confocal microscope to observe green, red, and far-red fluorescence to determine the intracellular localization of GFP protein. The experimental results are as follows: Figure 6A As shown.

[0385] In addition, another batch of cells that underwent the same treatment were washed with 50 μM heparin and then lysed with DDM lysis buffer (n-Dodecyl β-D-maltoside (Sigma) 4 mg / mL, 20 mM Hepes, 1 mM EGTA, 100 mM NaCl, 5 mM MgCl2). The intracellular GFP protein content was then detected using a double-antibody sandwich assay. In short, ELISA plates coated with 400 ng / well of the GFP-binding protein GBP4 and HRP-labeled anti-GFP antibody were used to detect GFP protein in serially diluted cell lysates (100 μL), and the GFP protein content in the cell lysates was calculated based on a pre-plotted standard curve. The experimental results are as follows: Figure 6B As shown.

[0386] Figure 6A The results showed that GFP protein could be detected in cells within minutes of GFP-cpp28 exposure, and the intensity of green fluorescence reached its peak (plateau phase) within 1-4 hours. The amount of GFP protein in the cells decreased over time. Furthermore, the colocalization results of green and red fluorescence indicated that for a period after GFP entry into the cell, most of the GFP protein colocalized with the cytoplasmic membrane labeled with mRuby3, suggesting that cpp28 carrying GFP was primarily located in the endosome for a period after entry. However, over time, the colocalization signal between GFP and mRuby3 decreased, indicating that the GFP protein escaped from the endosome and entered the cytoplasm. Figure 6B The detection results and the observation results of confocal microscopy ( Figure 6A (Same as above)

[0387] In addition, the Hela-p63mRb3 cells were used to analyze the pathway of cpp28 delivery of GFP protein into cells. In short, Hela-p63mRb3 cells were seeded at a density of 10,000 cells per well in a 24-well PerkinElmer black plate. After cell attachment, cells were treated with various inhibitors of endocytosis (amiloride, chlorpromazine, filipin, cytochalasin D, dansylcadaverine, dysore, nystatin) or individually at 4°C for 2 h. Subsequently, 30 μg / ml of GFP-cpp28 protein was added, and the cells were treated for 1 h. After treatment, cells were washed three times with heparin, fixed with 3.7% paraformaldehyde, and photographed and observed using a laser confocal microscope. The experimental results are as follows: Figures 7A-7BAs shown.

[0388] From fluorescence imaging results ( Figure 7A ) and the results of analysis on the intracellular GFP fluorescence intensity ( Figure 7B It can be seen that low-temperature treatment (4℃) significantly inhibits GFP-cpp28 entry into cells, indicating that the entry of cpp28 carrying GFP protein into cells is energy-dependent. Furthermore, cytochalasin D and monodansylpentanediamine, two inhibitors, also inhibited the transmembrane delivery of cpp28 to some extent, but the inhibitory effect was weaker than that of low-temperature treatment. This suggests that the transmembrane delivery of cpp28 involves clathrin-mediated endocytosis.

[0389] Example 5: Evaluation of cpp28 variants and their efficiency in delivering proteins into cells.

[0390] In this embodiment, the inventors designed several variants of cpp28 (also referred to as cpp28ori in this application) (cpp28 variants listed in Table 2) and evaluated the efficiency of these variants in delivering the protein into cells.

[0391] Table 2: Amino acid sequences of cpp28 and its variants

[0392]

[0393] Among the cpp28 variants listed in Table 2, cpp28a, cpp28b, cpp28c, and cpp28d are truncated versions of cpp28, with their C-termini truncated by 2, 5, 7, and 13 amino acid residues, respectively. Compared to cpp28, cpp28e, cpp28h, cpp28i, cpp28j, and cpp28k have 2-5 amino acid residues added to their N-termini. cpp28oNT-1, cpp28oNT-2, and cpp28oNT-3 are N-terminal truncated versions of cpp28, with their N-termini truncated by 5, 12, and 20 amino acids, respectively. cpp28aC, cpp28bC, cpp28cC, and cpp28dC are variants of cpp28a, cpp28b, cpp28c, and cpp28d, respectively, with the C-terminus serine... The following are variants of cpp28aC: cpp28aC, cpp28dREC, cpp28dC, cpp28oS, cpp28oS, cpp28aC, cpp28oS, cpp28aC, cpp28oS, cpp28oS, cpp28aC, cpp28aC, cpp28aC, cpp28aC, cpp28aC, cpp28aC, cpp28aC, cpp28aC, cpp28aC, cpp28aC, cpp28aC, cpp28aC, cpp28aC, cpp28aC, cpp28bC, cpp28cC, cpp28d ...aC, cpp28dC, cpp28aC, cpp28bC, cpp28aC, cpp28dC, cpp28aC, cpp28bC,

[0394] Following the method described in Example 2, based on the cpp28 variants designed in Table 2, a variety of recombinant proteins (GFP-cpp28a, GFP-cpp28b, GFP-cpp28c, GFP-cpp28d, GFP-cpp28e, GFP-cpp28f, GFP-cpp28g, GFP-cpp28h, GFP-cpp28i, GFP-cpp28j, GFP-cpp28k, GFP-cpp28L, GFP-cpp28oS, GFP-cpp28aC, GFP-cpp28k, GFP-cpp28L, GFP-cpp28oS, GFP-cpp28aC, GFP-cpp28k, GFP-cpp28e, GFP-cpp28f, GFP-cpp28g, GFP-cpp28h, GFP-cpp28i, GFP-cpp28j ...oS, GFP-cpp28aC, GFP-cpp28k, GFP-cpp28k, GFP-cpp28e, GFP-cpp28oS, GFP-cpp28aC, GFP-cpp28k, GFP-cpp28k, GFP-cpp28e 8bC, GFP-cpp28cC, GFP-cpp28dC, GFP-cpp28dREC, GFP-cpp28oNT-1, GFP-cpp28oNT-2, GFP-cpp28oNT-3, GFP-cpp28aCQ1, GFP-cpp28aCQ2, GFP-cpp28aCQ3, GFP-cpp28aCNT, GFP-cpp28bCNT, GFP-cpp28cCNT and GFP-cpp28dCNT), wherein each cpp28 variant is linked to the C-terminus of GFP via a linker (SEQ ID NO: 39).

[0395] Then, as described in Example 3, the efficiency of cpp28 variants (cpp28a, cpp28b, cpp28c, cpp28d, cpp28e, cpp28f, cpp28g, cpp28h, cpp28i, cpp28j, cpp28k, and cpp28L) in delivering green fluorescent protein into cells was evaluated using the report system constructed in Example 1. Experimental results are as follows: Figures 8A-8B As shown.

[0396] Figure 8A The results showed that cpp28a, cpp28b, and cpp28c all possessed the ability to deliver GFP into cells, but their delivery efficiency was lower than that of cpp28. Furthermore, cpp28d essentially lost its ability to deliver GFP into cells. These results indicate that cpp28 can tolerate C-terminal deletion to some extent; in particular, truncated cpp28 variants with a C-terminus shortened by 1-7 amino acid residues can still deliver GFP molecules across the membrane.

[0397] Figure 8B The results showed that cpp28, cpp28e, cpp28g, cpp28h, cpp28j, and cpp28k all had the ability to deliver GFP into cells. Among them, cpp28e, cpp28g, cpp28h, and cpp28j had even better delivery efficiency than cpp28. cpp28f, cpp28i, and cpp28L, on the other hand, essentially lost their ability to deliver GFP into cells.

[0398] also, Figures 8A-8B The results also showed that cpp28a, cpp28b, cpp28c, cpp28e, cpp28g, cpp28h, cpp28j, and cpp28k could deliver GFP into cells in a dose-dependent manner.

[0399] Furthermore, using the method described in Example 4, the process of GFP protein delivery into cells by cpp28, cpp28a, cpp28b, cpp28c, cpp28d, and TAT was observed in a constructed Hela-p63mRb3 cell model. The experimental results are as follows: Figures 9A-9B As shown.

[0400] Figures 9A-9B The results showed that cpp28a, cpp28b, and cpp28c all possessed the ability to deliver GFP into cells, and although their delivery efficiency was lower than that of cpp28, it was significantly higher than that of cpp28d and TAT. This result is consistent with... Figure 8A The results were basically the same.

[0401] In addition, using the method described in Example 4, the delivery of GFP protein into cells by cpp28, cpp28-oS, cpp28a, cpp28aC, cpp28b, cpp28bC, cpp28cC, cpp28dC, and cpp28dREC was observed using the constructed Hela-p63mRb3 cell model, and their efficiency and dose-dependent characteristics of protein delivery into cells were evaluated. The experimental results are as follows: Figures 10A-10B and Figure 11A-11B As shown.

[0402] Figures 10A-10B and Figure 11A-11B The results showed that after mutating the C-terminal cysteine ​​to serine of cpp28, cpp28-oS could still deliver GFP into cells, but the delivery efficiency decreased. Furthermore, mutating the C-terminal serine to cysteine ​​of cpp28a, cpp28b, cpp28c, and cpp28d significantly enhanced the GFP protein delivery activity of cpp28aC, cpp28bC, cpp28cC, and cpp28dC. These results indicate that the C-terminal cysteine ​​is beneficial for cpp28 to perform its transmembrane delivery function. Figure 11A-11B The results showed that cpp28dREC had a certain improvement in the activity of delivering GFP protein into cells compared with cpp28dC, and still maintained good membrane penetration activity at a low concentration of 12.5 μg / mL.

[0403] Meanwhile, using the method described in Example 4, the delivery of GFP protein into cells by cpp28, cpp28a, cpp28b, cpp28c, cpp28d, cpp28oNT-1, cpp28oNT-2, and cpp28oNT-3 was observed and evaluated using the constructed Hela-p63mRb3 cell model. The efficiency and dose-dependent characteristics of their protein delivery into cells were also evaluated. Experimental results are as follows: Figure 12A-12B As shown.

[0404] Figure 12A-12B The results showed that cpp28a, cpp28b, cpp28c, and cpp28oNT-1 all possessed the ability to deliver GFP into cells in a dose-dependent manner, with cpp28oNT-1 and cpp28 exhibiting essentially equivalent delivery efficiencies. Furthermore, cpp28d, cpp28oNT-2, and cpp28oNT-3 essentially lost their ability to deliver GFP into cells. These results indicate that cpp28 can tolerate N-terminal or C-terminal deletions to some extent; truncated cpp28 molecules with a C-terminus of 1-7 amino acid residues can still deliver GFP across the membrane, while the deletion of 1-5 amino acids at the N-terminus has minimal impact on its transmembrane delivery efficiency. However, cpp28oNT-2 and cpp28oNT-3, lacking one or two SPRRR structures, essentially lost their ability to deliver GFP into cells.

[0405] Furthermore, using the method described in Example 4, the delivery of GFP protein into cells by cpp28, cpp28aCQ1, cpp28aCQ2, cpp28aCQ3, cpp28aCNT, cpp28bCNT, cpp28cCNT, and cpp28dCNT was observed using the constructed HeLa-p63mRb3 cell model, and the efficiency and dose-dependent characteristics of their protein delivery into cells were evaluated. The experimental results are as follows: Figures 13A-13B As shown.

[0406] Figures 13A-13B The results showed that cpp28aCQ1, cpp28aCQ2, and cpp28aCQ3 all had the ability to deliver GFP into cells, and their delivery efficiency was comparable to, or even slightly better than, that of cpp28. cpp28aCNT, cpp28bCNT, cpp28cCNT, and cpp28dCNT, which combined N-terminal and C-terminal truncation, also had the ability to deliver GFP into cells, and the delivery efficiency of cpp28aCNT, cpp28bCNT, and cpp28cCNT was comparable to that of cpp28 at low concentrations (12.5 μg / mL).

[0407] Example 6: Analysis of recombinant proteins containing GFP and cpp28 or their variants

[0408] Recombinant proteins containing GFP and cpp28 or their variants were analyzed by high-performance liquid chromatography (HPLC). The results are as follows: Figure 14A As shown in the figure, the elution times of individual GFP and GFP-TAT proteins are approximately 14 minutes. Taking GFP-cpp28e as an example, this recombinant protein has two peaks, with elution times of approximately 11 minutes and 14 minutes, respectively. Furthermore, fluorescence detection analysis shows that the fluorescence peaks of GFP-cpp28e also appear at their respective elution times, with the peak at approximately 11 minutes exhibiting a stronger fluorescence value. This result indicates that GFP-cpp28e forms a multimer.

[0409] Furthermore, taking GFP-cpp28, GFP, and GFP-TAT as examples, these proteins were analyzed using an analytical ultracentrifuge Optima XL-100 (AUC) to determine their sedimentation coefficients. The results are as follows: Figure 14B As shown in the figure. The results show that the sedimentation coefficient of the multimer-forming protein GFP-cpp28 is approximately 34S, with a relative molecular mass of approximately 2200 kD; while the sedimentation coefficients of the pure GFP and GFP-TAT proteins are approximately 2.4S, with a relative molecular mass of approximately 30 kD. This result indicates that GFP-cpp28 forms a large molecule with approximately 70 aggregates compared to the pure GFP protein molecule.

[0410] Based on the above conclusions, the inventors also used Sephacryl™ High Resolution resins HiPrep TM Sephacryl HR columns 26 / 60 (320 ml) preparative molecular sieves were used to purify recombinant proteins (GFP-TAT, GFP-cpp28, GFP-cpp28e, GFP-cpp28g, GFP-cpp28h, GFP-cpp28j) obtained by nickel column affinity chromatography. Results are as follows: Figure 15A As shown in the figure. The results indicate that for the protein GFP-TAT, it mainly exists in monomeric form; for proteins GFP-cpp28, GFP-cpp28e, GFP-cpp28g, GFP-cpp28h, and GFP-cpp28j, the target protein can be collected at two different elution times, and the percentage of proteins with shorter elution times (multimers) is much higher than that of proteins with longer elution times (monomers). This result is consistent with... Figure 14A and 14B The results are consistent. Further, the collected protein fractions (multimeric fractions and monomer fractions) were analyzed by SDS-PAGE. The results are as follows: Figure 15BAs shown in the figure. The results showed that after denaturation (i.e., the multimers dissociated into monomers), the size of these proteins was approximately 30 kDa. Additionally, as described in Example 3, the efficiency of the collected protein fractions (multimer fractions and monomer fractions) in delivering green fluorescent protein into cells was evaluated using the reporter system constructed in Example 1. The experimental results are as follows. Figure 15C As shown in the figure. The results indicate that the cell entry efficiency of multimeric proteins is significantly higher than that of monomeric proteins.

[0411] Example 7: Evaluation of the cell entry efficiency of Anti-CRISPR-cpp28ori protein

[0412] In this embodiment, the inventors verified whether cpp28ori can deliver anti-CRISPR protein (AcrIIA4) into cells and exert its gene-editing effect by inhibiting Cas9.

[0413] The CRISPR-Cas9 system is currently a very popular gene editing system, but its high gene editing efficiency is accompanied by off-target effects that have troubled researchers. A paper published in Cell in 2017 (Cell. 2017 Jan 12;168(1-2): 150-158.e10) reported the protein AcrIIA4 (also referred to as anti-CRISPR protein in this application; SEQ ID NO: 44), which can inhibit CRISPR activity, and this protein can also exert an inhibitory effect in mammalian cells. In this embodiment, the inventors prepared and purified an anti-CRISPR protein (anti-CRISPR-cpp28ori; SEQ ID NO: 45) fused with the cell-penetrating peptide cpp28 using a prokaryotic expression system, and evaluated its cell penetration efficiency.

[0414] In short, the inventors constructed a fluorescent reporter system to verify the membrane-penetrating effect of the anti-CRISPR-cpp28ori protein. For example... Figure 16A As shown, this fluorescent reporter system uses a plasmid (hereinafter referred to as the Reporter plasmid) carrying the red fluorescent mRuby3 gene, the sgRNA sequence targeting the EGFP protein, and the EGFP gene initiated by the TRE promoter. The sequence is as follows: EF1p / U6 / TREp: promoter sequence; H2B: nucleotide sequence encoding the nuclear localization signal; mRuby3: nucleotide sequence of the mRuby3 gene; 2A: linker nucleotide sequence; BGH: transcription termination sequence; sgRNA: nucleotide sequence encoding sgRNA; EGFP: nucleotide sequence encoding the EGFP protein; PurR: puromycin resistance gene; ins: insulating sequence; TR: transposon sequence.

[0415] When the Reporter plasmid and a plasmid carrying the Cas9 gene (addgene ID: 52961) (hereinafter referred to as the Cas9 plasmid) are co-transfected in cells, the cells will express the Cas9 protein (SEQ ID NO: 46). This Cas9 protein will edit the EGFP gene through an sgRNA sequence targeting the EGFP protein, thereby preventing the cells from expressing the EGFP protein normally. As a result, the cells will not emit or will only emit a very weak green fluorescence.

[0416] In addition, the inventors constructed the pTT5-anti-CRISPR-cpp28ori plasmid (which is used to transfect eukaryotic cells and express the anti-CRISPR-cpp28ori protein in eukaryotic cells) and the plasmids pTO-T7-his-anti-CRISPR and pTO-T7-his-anti-CRISPR-cpp28ori (which are used to transfect prokaryotic cells and express the anti-CRISPR and anti-CRISPR-cpp28ori proteins carrying the 6*His tag in prokaryotic cells, wherein the anti-CRISPR protein and cpp28ori are linked by a flexible linker SEQ ID NO: 39).

[0417] The function of the fluorescent reporter system was validated in 293β5 cells using the Reporter plasmid, Cas9 plasmid, and pTT5-anti-CRISPR-cpp28ori plasmid constructed above. In short, 293β5 cells were transfected with the specified plasmids or plasmid combinations. Forty-eight hours after transfection, the cells were cultured for another 12 hours in DMEM medium containing doxorubicin. Intracellular EGFP fluorescence was then imaged and analyzed using a laser confocal high-content imaging system. Experimental results are as follows: Figure 16B-16C As shown in the figure. The results showed that: obvious EGFP fluorescence expression was observed in cells transfected with Reporter plasmid alone; only very weak green fluorescence was observed in cells transfected with both Reporter and Cas9 plasmids; only very weak green fluorescence was observed in cells transfected with Reporter plasmid, Cas9 plasmid, and empty pTT5 plasmid; and more obvious green fluorescence was observed in cells transfected with pTT5-anti-CRISPR-cpp28ori plasmid, Reporter plasmid, and Cas9 plasmid. These results indicate that Cas9 protein can exert gene editing function in cells and inhibit EGFP protein expression; while anti-CRISPR protein can inhibit the gene editing function of Cas9 protein and restore EGFP protein expression. Average fluorescence intensity analysis results ( Figure 16CThe results showed that anti-CRISPR restored approximately 40% of EGFP fluorescence. Therefore, the constructed fluorescent reporter system can indicate the function of anti-CRISPR through EGFP fluorescence intensity.

[0418] Following the method described in Example 2, anti-CRISPR and anti-CRISPR-cpp28ori proteins were expressed in E. coli and purified using nickel column affinity chromatography. SDS-PAGE analysis showed that the purified anti-CRISPR protein had a purity of over 95%, and the purified anti-CRISPR-cpp28ori protein had a purity of over 90%, suitable for further cell experiments.

[0419] 293β5 cells were simultaneously transfected with Reporter and Cas9 plasmids. Twelve hours after transfection, the culture medium was replaced with DMEM (Gibco + 10% Gibco FBS) containing a specified concentration (20, 40, 80, or 100 µg / ml) of anti-CRISPR or anti-CRISPR-cpp28ori protein, and the cells were cultured at 37°C for another 12 hours. Subsequently, the culture medium was replaced with normal DMEM (Gibco + 10% Gibco FBS), and 48 hours after transfection, the medium was replaced with DMEM containing doxorubicin. After another 12 hours of culture, EGFP fluorescence in 293β5 cells was imaged and analyzed using a laser confocal high-content imaging system. The experimental results are as follows: Figure 17A As shown in the figure, the results indicated that adding anti-CRISPR-cpp28ori protein to the culture medium partially restored intracellular EGFP fluorescence intensity, and the percentage of EGFP fluorescence restoration increased with increasing protein concentration. In contrast, adding anti-CRISPR protein to the culture medium did not restore intracellular EGFP fluorescence intensity. This result suggests that cpp28ori can deliver anti-CRISPR protein into cells, enabling it to inhibit Cas9 within the cell.

[0420] In another experiment, 293β5 cells were simultaneously transfected with Reporter and Cas9 plasmids. Twelve hours after transfection, the culture medium was replaced with DMEM (Gibco + 10% Gibco FBS) containing a specified concentration (100, 50, or 25 µg / ml) of anti-CRISPR, anti-CRISPR-cpp28ori, or ctrl-protein-cpp28ori protein, and the cells were cultured at 37°C for another 12 hours. Subsequently, the culture medium was replaced with normal medium (DMEM, Gibco + 10% Gibco FBS), and 48 hours after transfection, the medium was replaced with DMEM containing doxorubicin, and the cells were cultured for another 12 hours. In this experiment, negative and positive controls were also set up as follows: In the negative control group, the culture medium used did not contain recombinant protein, and the cells were transfected with Reporter plasmid, or Reporter plasmid and cas9 plasmid, or Reporter plasmid and anti-CRISPR plasmid, or Reporter plasmid and empty plasmid, or Reporter plasmid, cas9 plasmid and empty plasmid; In the positive control group, the culture medium used did not contain recombinant protein, and the cells were transfected with Reporter plasmid, cas9 plasmid and anti-CRISPR plasmid.

[0421] After culture, cells were lysed using DDM lysis buffer, and the intracellular EGFP protein content was detected by Native PAGE and Western Blot. In Native PAGE analysis, the mRuby3 fluorescent gene on the Reporter plasmid was used as an internal control; in Western Blot analysis, the Tubulin gene was used as an internal control. Experimental results are as follows: Figure 17B-17C As shown.

[0422] Results of Native PAGE analysis ( Figure 17B The results showed that when the anti-CRISPR-cpp28ori plasmid was transfected or the anti-CRISPR-cpp28ori protein was added to the cell culture medium, a significant EGFP protein signal (green fluorescence) could be detected in the cells; and the EGFP protein signal (green fluorescence) increased with the increase of the anti-CRISPR-cpp28ori protein concentration. However, when anti-CRISPR protein or ctrl-protein-cpp28ori protein was added to the cell culture medium, the EGFP protein signal was basically undetectable or only very weak. Western blot analysis results ( Figure 17CThe results are consistent with those of the Native PAGE analysis. Figure 17B-17C The results showed that cpp28ori can deliver anti-CRISPR proteins into cells, enabling them to inhibit Cas9 within the cells.

[0423] In addition, cellular genomic DNA was extracted from cells using the QIAamp DNA blood Mini Kit (QIAGEN) and amplified by PCR. The two primers used targeted positions approximately 200 bp upstream and downstream of the sgRNA binding site of the EGFP gene sequence, respectively. The PCR amplification products were recovered and sequenced using next-generation sequencing technology. Experimental results are as follows: Figure 17D As shown. The results showed that: (1) compared with the original EGFP gene sequence, the gene editing effect of Cas9 could cause mutations in the target sequence (EGFP gene sequence), with a mutation rate of about 15%; (2) co-transfection with anti-CRISPR-cpp28ori plasmid or the addition of anti-CRISPR-cpp28ori protein in the culture medium could inhibit the function of Cas9 and reduce the mutation rate of the target sequence (below 10%); (3) the effect of anti-CRISPR-cpp28ori protein was significantly better than that of anti-CRISPR protein (p<0.001). This result indicates that cpp28ori can deliver anti-CRISPR protein into cells, enabling it to inhibit the function of Cas9 in cells.

[0424] Example 8: Evaluation of the efficiency of cpp28ori in delivering antibody into cells

[0425] In this embodiment, the inventors verified whether cpp28ori could deliver antibodies into cells and exert antibody functions.

[0426] Using a flexible linker (SEQ ID NO: 39), cpp28ori was linked to the C-terminus of the Fc of three chimeric antibodies: anti-HBeAg 2A7 (whose VL and VH sequences are shown in SEQ ID NO: 47 and 48, respectively), anti-HBcAg 16D5 (whose VL and VH sequences are shown in SEQ ID NO: 49 and 50, respectively), and anti-tyrosinase-associated protein (TYRP1) TA99 (whose VL and VH sequences are shown in SEQ ID NO: 51 and 52, respectively), thereby obtaining recombinant proteins 2A7-cpp28ori, 16D5-cpp28ori, and TA99-cpp28ori. Specifically, antibody TA99 can specifically target intracellular TYRP-1 antigen and reduce its expression; antibody 16D5 can specifically target and clear intracellular HBcAg antigen and reduce HBV DNA levels; and antibody 2A7 can specifically target HBeAg (but not HBcAg).

[0427] Antibodies 2A7, 16D5, and TA99, as well as recombinant proteins 2A7-cpp28ori, 16D5-cpp28ori, and TA99-cpp28ori, were expressed in ExpiCHO cells via transient transfection. After 12 days, cell supernatant was collected, and the expressed antibodies or recombinant proteins were purified using a Protein A column. The purified antibodies or recombinant proteins were detected by SDS-PAGE and Western blot (anti-human IgG). Experimental results are shown below. Figure 18 As shown in the figure. The results indicate that the purified antibody or recombinant protein has a purity of over 95% and can be used for the next step of cell experiments.

[0428] Five cell lines—HepG2-N10, HepG2-C3A, HeLa, MNT-1, and A375—were seeded in 96-well plates. After 12 h, the culture medium was removed, and DMEM (Gibco + 10% Gibco FBS) containing 100 μg / ml of 2A7-cpp28ori, 2A7, 16D5-cpp28ori, 16D5, TA99-cpp28ori, or TA99 was added, and the cells were cultured at 37°C for another 6 h. Subsequently, the cells were washed three times with heparin, and intracellular antibodies were detected by immunofluorescence. The experimental results are as follows: Figure 19 As shown in the figure, the results indicate that antibodies fused with cpp28ori (2A7-cpp28ori, 16D5-cpp28ori, and TA99-cpp28ori) can be detected in various cell types, while antibodies without cpp28ori fusion (2A7, 16D5, and TA99) cannot be detected intracellularly. This demonstrates that cpp28ori can effectively carry antibodies into various cell types.

[0429] To verify whether the antibody delivered into cells by cpp28ori could function, the following experiment was also conducted.

[0430] MNT-1 cells were seeded at a density of 30,000 cells per well in 24-well plates. The following day, the medium was removed, and DMEM (with or without 10% Gibco FBS) containing 2A7-cpp28ori (100 μg / mL; used as a control antibody), TA99 (100 μg / mL and 50 μg / mL), or TA99-cpp28ori (100 μg / mL and 50 μg / mL) was added, and the cells were cultured at 37°C for 6 h. Subsequently, the medium was replaced with normal medium (DMEM, Gibco + 10% Gibco FBS), and the cells were cultured for another 48 h. Cells were then lysed with DDM lysis buffer, and the lysates were analyzed by Western blot to detect the level of TYRP-1 antigen. The experimental results are as follows: Figure 20 As shown in the figure. The results showed that the expression level of TYRP-1 antigen was significantly reduced in cells treated with TA99-cpp28ori. This indicates that cpp28ori can deliver TA99 antibody into cells, and that the TA99-cpp28ori that enters the cells can perform its biological function normally (i.e., it can specifically target intracellular TYRP-1 antigen and reduce its expression level).

[0431] HepG2-N10 cells were seeded in 24-well plates at a density of 30,000 cells per well. The next day, the medium was removed, and DMEM (Gibco + 10% Gibco FBS) containing 100 μg / ml of 2A7-cpp28ori, 2A7, 16D5-cpp28ori, 16D5, TA99-cpp28ori, or TA99 antibody was added, and the cells were cultured at 37°C for 6 h. Subsequently, the medium was replaced with normal DMEM (Gibco + 10% Gibco FBS), and the cells were cultured for another 48 h. Cells were then lysed with DDM lysis buffer, and the lysis buffer was analyzed by Western blot to detect the levels of HBcAg, TRIM21, and antibodies. Additionally, the cell culture supernatant was collected, and the levels of HBeAg antigen and HBV DNA were detected. The experimental results are as follows: Figures 21A-21B As shown.

[0432] Figure 21A The results showed that 2A7-cpp28ori, 16D5-cpp28ori, and TA99-cpp28ori were detected in the cell lysate, indicating that these proteins were delivered into the cells. Furthermore, Figure 21AThe results showed that 16D5-cpp28ori significantly cleared HBcAg antigen from HepG2-N10 cells, reducing its intracellular level. Previous studies have shown that TRIM21 is an intracellular antibody receptor that mediates antibody ubiquitination and degradation by binding to the Fc of antibodies. Detection of TRIM21 protein levels in cell lysates suggests that 16D5-cpp28ori may clear HBcAg by reducing intracellular TRIM21 levels after entering the cell. Figure 21A ).in addition, Figure 21A The results also showed that 2A7-cpp28ori had little effect on intracellular HBcAg levels, but still significantly reduced TRIM21 levels.

[0433] Figure 21B The results showed that HBeAg levels in cell supernatants were significantly reduced by 2A7-cpp28ori treatment, and 16D5-cpp28ori treatment also inhibited HBeAg levels to some extent. Furthermore, Figure 21B The results also showed that treatment with 16D5-cpp28ori could suppress HBV DNA levels in cell supernatants, which is consistent with previous findings (i.e., 16D5 antibody can reduce HBV DNA levels in HBV transgenic mice).

[0434] Figures 21A-21B The results demonstrate that cpp28ori can not only deliver various antibodies across the membrane into cells, but also precisely perform the specific function of each antibody. For example, the delivered antibodies can accurately recognize and target specific antigens (even HBeAg and HBcAg, two antigens with highly overlapping sequences).

[0435] Example 9: Evaluation of the efficiency of cpp28ori in delivering DNA into cells

[0436] In this embodiment, the inventors verified whether cpp28ori can bind to DNA and deliver it into cells to perform its function.

[0437] Previous research (Nat Methods. 2015 Nov;12(11):1085-90) has shown that zinc finger proteins (hereinafter referred to as ZF) can effectively bind to DNA. Therefore, the recombinant protein ZF-cpp28ori was constructed by linking cpp28ori to the C-terminus of ZF protein (SEQ ID NO: 53) using a flexible linker (SEQ ID NO: 39).

[0438] Following the method described in Example 2, ZF-cpp28ori was expressed in *E. coli* and purified using nickel column affinity chromatography. SDS-PAGE analysis showed that the purified ZF-cpp28ori protein had a purity of over 90% and could be used for further cell experiments. Additionally, a binding sequence for this zinc finger protein (SEQ ID NO: 54) was inserted downstream of the mRuby3 gene into the pTT5-mRuby3 plasmid to obtain the plasmid pTT5-mRuby3-ZF motif.

[0439] 293β5 cells were seeded in 96-well plates at a density of 20,000 cells per well. The following day, ZF-cpp28ori protein (60 µg / mL) and plasmid pTT5-mRuby3-ZFmotif (0.8 µg) were diluted in vitro with culture medium (DMEM, Gibco + 10% Gibco FBS) and pre-reacted at 37°C for 1 h. Then, the culture medium in the 96-well plates was removed, and the pre-reacted mixture was added to the cells. After 6 h, the pre-reacted mixture was removed, and normal culture medium (DMEM, Gibco + 10% Gibco FBS) was added, and the cells were cultured for another 48 h. Additionally, plasmid pTT5-mRuby3-ZF motif was transfected into 293β5 cells using PEI transfection reagent as a positive control. The fluorescence of mRuby3 within 293β5 cells was then imaged and analyzed using a laser confocal high-content imaging system. The experimental results are as follows: Figure 22 As shown in the figure. The results showed that ZF-cpp28ori could effectively deliver the pTT5-mRuby3-ZFmotif plasmid into 293β5 cells and express the red fluorescent protein mRuby3, and its delivery efficiency was higher than that of PEI transfection reagent.

[0440] Although specific embodiments of the invention have been described in detail, those skilled in the art will understand that various modifications and variations can be made to the details based on all the teachings disclosed, and all such changes are within the scope of protection of the invention. The full scope of the invention is given by the appended claims and any equivalents thereof. SEQUENCE LISTING <110> Yangshengtang Co., Ltd.; Xiamen University <120> A cell-penetrating peptide and its applications <130> IDC220337 <160> 54 <170> PatentIn version 3.5 <210> 1 <211> 239 <212> PRT <213> Artificial Sequence <220> <223> Amino acid sequence of GFP <400> 1 Met Val Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu 1 5 10 15 Val Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Ser Gly 20 25 30 Glu Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Phe Ile 35 40 45 Cys Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr 50 55 60 Leu Thr Tyr Gly Val Gln Cys Phe Ser Arg Tyr Pro Asp His Met Lys 65 70 75 80 Gln His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu 85 90 95 Arg Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu 100 105 110 Val Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly 115 120 125 Ile Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr 130 135 140 Asn Tyr Asn Ser His Asn Val Tyr Ile Met Ala Asp Lys Gln Lys Asn 145 150 155 160 Gly Ile Lys Val Asn Phe Lys Ile Arg His Asn Ile Glu Asp Gly Ser 165 170 175 Val Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly 180 185 190 Pro Val Leu Leu Pro Asp Asn His Tyr Leu Ser Thr Gln Ser Ala Leu 195 200 205 Ser Lys Asp Pro Asn Glu Lys Arg Asp His Met Val Leu Leu Glu Phe 210 215 220 Val Thr Ala Ala Gly Ile Thr Leu Gly Met Asp Glu Leu Tyr Lys 225 230 235 <210> 2 <211> 147 <212> PRT <213> Artificial Sequence <220> <223> Amino acid sequence of the DNA binding domain of Gal4 <400> 2 Met Lys Leu Leu Ser Ser Ile Glu Gln Ala Cys Asp Ile Cys Arg Leu 1 5 10 15 Lys Lys Leu Lys Cys Ser Lys Glu Lys Pro Lys Cys Ala Lys Cys Leu 20 25 30 Lys Asn Asn Trp Glu Cys Arg Tyr Ser Pro Lys Thr Lys Arg Ser Pro 35 40 45 Leu Thr Arg Ala His Leu Thr Glu Val Glu Ser Arg Leu Glu Arg Leu 50 55 60 Glu Gln Leu Phe Leu Leu Ile Phe Pro Arg Glu Asp Leu Asp Met Ile 65 70 75 80 Leu Lys Met Asp Ser Leu Gln Asp Ile Lys Ala Leu Leu Thr Gly Leu 85 90 95 Phe Val Gln Asp Asn Val Asn Lys Asp Ala Val Thr Asp Arg Leu Ala 100 105 110 Ser Val Glu Thr Asp Met Pro Leu Thr Leu Arg Gln His Arg Ile Ser 115 120 125 Ala Thr Ser Ser Ser Glu Glu Ser Ser Asn Lys Gly Gln Arg Gln Leu 130 135 140 Thr Val Ser 145 <210> 3 <211> 130 <212> PRT <213> Artificial Sequence <220> <223> Anti-GFP VHH2's amino acid sequence <400> 3 Met Ala Asp Val Gln Leu Gln Glu Ser Gly Gly Gly Ser Val Gln Ala 1 5 10 15 Gly Glu Ala Leu Arg Leu Ser Cys Val Gly Ser Gly Tyr Thr Ser Ile 20 25 30 Asn Pro Tyr Met Ala Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu 35 40 45 Gly Val Ala Ala Ile Ser Ser Gly Gly Gln Tyr Thr Tyr Tyr Ala Asp 50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr 65 70 75 80 Met Tyr Leu Gln Met Pro Ser Leu Lys Pro Asp Asp Ser Ala Lys Tyr 85 90 95 Tyr Cys Ala Ala Asp Phe Arg Arg Gly Gly Ser Trp Asn Val Asp Pro 100 105 110 Leu Arg Tyr Asp Tyr Gln His Trp Gly Gln Gly Thr Gln Val Thr Val 115 120 125 Ser Ser 130 <210> 4 <211> 78 <212> PRT <213> Artificial Sequence <220> <223> Amino acid sequence of the VP16 activation domain <400> 4 Ala Pro Pro Thr Asp Val Ser Leu Gly Asp Glu Leu His Leu Asp Gly 1 5 10 15 Glu Asp Val Ala Met Ala His Ala Asp Ala Leu Asp Asp Phe Asp Leu 20 25 30 Asp Met Leu Gly Asp Gly Asp Ser Pro Gly Pro Gly Phe Thr Pro His 35 40 45 Asp Ser Ala Pro Tyr Gly Ala Leu Asp Met Ala Asp Phe Glu Phe Glu 50 55 60 Gln Met Phe Thr Asp Ala Leu Gly Ile Asp Glu Tyr Gly Gly 65 70 75 <210> 5 <211> 128 <212> PRT <213> Artificial Sequence <220> <223> Amino acid sequence of Anti-GFP VHH6 <400> 5 Met Ala Asp Val Gln Leu Gln Glu Ser Gly Gly Gly Ser Val Gln Thr 1 5 10 15 Gly Gly Ser Leu Arg Leu Ser Cys Ala Val Ser Pro Tyr Ile Gly Ser 20 25 30 Arg Ile Ser Leu Gly Trp Phe Arg Gln Ala Pro Gly Lys Val Arg Glu 35 40 45 Gly Val Ala Leu Ile Asn Ser Arg Asp Gly Ser Thr Tyr Tyr Ala Asp 50 55 60 Thr Val Lys Gly Arg Phe Thr Ile Ser Gln Gly Asp Ala Asn Thr Val 65 70 75 80 Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Ile Tyr Tyr 85 90 95 Cys Ala Ala Arg Trp Gly Gln Ile Thr Asp Ile Gln Ala Leu Ala Val 100 105 110 Ala Ser Phe Pro Asp Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 125 <210> 6 <211> 236 <212> PRT <213> Artificial Sequence <220> <223> Amino acid sequence of RFP <400> 6 Met Val Ser Lys Gly Glu Glu Asp Asn Met Ala Ile Ile Lys Glu Phe 1 5 10 15 Met Arg Phe Lys Val His Met Glu Gly Ser Val Asn Gly His Glu Phe 20 25 30 Glu Ile Glu Gly Glu Gly Glu Gly Arg Pro Tyr Glu Gly Thr Gln Thr 35 40 45 Ala Lys Leu Lys Val Thr Lys Gly Gly Pro Leu Pro Phe Ala Trp Asp 50 55 60 Ile Leu Ser Pro Gln Phe Met Tyr Gly Ser Lys Ala Tyr Val Lys His 65 70 75 80 Pro Ala Asp Ile Pro Asp Tyr Leu Lys Leu Ser Phe Pro Glu Gly Phe 85 90 95 Lys Trp Glu Arg Val Met Asn Phe Glu Asp Gly Gly Val Val Thr Val 100 105 110 Thr Gln Asp Ser Ser Leu Gln Asp Gly Glu Phe Ile Tyr Lys Val Lys 115 120 125 Leu Arg Gly Thr Asn Phe Pro Ser Asp Gly Pro Val Met Gln Lys Lys 130 135 140 Thr Met Gly Trp Glu Ala Ser Ser Glu Arg Met Tyr Pro Glu Asp Gly 145 150 155 160 Ala Leu Lys Gly Glu Ile Lys Gln Arg Leu Lys Leu Lys Asp Gly Gly 165 170 175 His Tyr Asp Ala Glu Val Lys Thr Thr Tyr Lys Ala Lys Lys Pro Val 180 185 190 Gln Leu Pro Gly Ala Tyr Asn Val Asn Ile Lys Leu Asp Ile Thr Ser 195 200 205 His Asn Glu Asp Tyr Thr Ile Val Glu Gln Tyr Glu Arg Ala Glu Gly 210 215 220 Arg His Ser Thr Gly Gly Met Asp Gly Cys Thr Ser 225 230 235 <210> 7 <211> 185 <212> PRT <213> Artificial Sequence <220> <223> Amino acid sequence of luciferase <400> 7 Met Gly Val Lys Val Leu Phe Ala Leu Ile Cys Ile Ala Val Ala Glu 1 5 10 15 Ala Lys Pro Thr Glu Asn Asn Glu Asp Phe Asn Ile Val Ala Val Ala 20 25 30 Ser Asn Phe Ala Thr Thr Asp Leu Asp Ala Asp Arg Gly Lys Leu Pro 35 40 45 Gly Lys Lys Leu Pro Leu Glu Val Leu Lys Glu Leu Glu Ala Asn Ala 50 55 60 Arg Lys Ala Gly Cys Thr Arg Gly Cys Leu Ile Cys Leu Ser His Ile 65 70 75 80 Lys Cys Thr Pro Lys Met Lys Lys Phe Ile Pro Gly Arg Cys His Thr 85 90 95 Tyr Glu Gly Asp Lys Glu Ser Ala Gln Gly Gly Ile Gly Glu Ala Ile 100 105 110 Val Asp Ile Pro Glu Ile Pro Gly Phe Lys Asp Leu Glu Pro Leu Glu 115 120 125 Gln Phe Ile Ala Gln Val Asp Leu Cys Val Asp Cys Thr Thr Gly Cys 130 135 140 Leu Lys Gly Leu Ala Asn Val Gln Cys Ser Asp Leu Leu Lys Lys Trp 145 150 155 160 Leu Pro Gln Arg Cys Ala Thr Phe Ala Ser Lys Ile Gln Gly Gln Val 165 170 175 Asp Lys Ile Lys Gly Ala Gly Gly Asp 180 185 <210> 8 <211> 358 <212> DNA <213> Artificial Sequence <220> <223> UAS sequence <400> 8 agcttgcatg cctgcaggtc ggagtactgt cctccgagcg gagtactgtc ctccgagcgg 60 agtactgtcc tccgagcgga gtactgtcct ccgagcggag tactgtcctc cgagcggaga 120 ctctagcgag cgccggagta taaatagagg cgcttcgtct acggagcgac aattcaattc 180 aaacaagcaa agtgaacacg tcgctaagcg aaagctaagc aaataaacaa gcgcagctga 240 acaagctaaa caatctgcag taaagtgcaa gttaaagtga atcaattaaa agtaaccagc 300 aaccaagtaa atcaactgca actactgaaa tctcccaaga agtatatt gatataca 358 <210> 9 <211> 311 <212> PRT <213> Artificial Sequence <220> <223> iRFP670's Facebook page <400> 9 Met Ala Arg Lys Val Asp Leu Thr Ser Cys Asp Arg Glu Pro Ile His 1 5 10 15 Pro Gly Ile Ser Gln Pro Cys Gly Cys Leu Leu Ala Cys Asp Ala 20 25 30 Gln with Val Arg and Thr Arg with Thr Glu Asn and Gly with Phe Phe 35 40 45 Gly Arg Glu Thr Pro Arg Val Gly Glu Leu Leu Ala Asp Tyr Phe Gly 50 55 60 Glu Thr Glu Ala His Ala Leu Arg Asn Ala Leu Ala Gln Ser Ser Asp 65 70 75 80 Pro Lys Arg Pro Ala Leu Ile Phe Gly Trp Arg Asp Gly Leu Thr Gly 85 90 95 Arg Thr Phe Asp Ser Ile Leu His Arg His Asp Ser Ile Thr Gly 100 105 110 Glu Phe Glu Pro Ala Ala Ala Glu Gln Ala Asp Asn Pro Leu Arg Leu 115 120 125 Thr Arg Gln Ile Ile Ala Arg Thr Lys Glu Leu Lys Ser Leu Glu Glu 130 135 140 Met Ala Ala Arg Val Pro Arg Tyr Leu Gln Ala Met Leu Gly Tyr His 145 150 155 160 Arg Val Met Leu Tyr Arg Phe Ala Asp Asp Gly Ser Gly Met Val Ile 165 170 175 Gly Glu Ala Lys Arg Ser Asp Leu Glu Ser Phe Leu Gly Gln His Phe 180 185 190 Pro Ala Ser Leu Val Pro Gln Gln Ala Arg Leu Leu Tyr Leu Lys Asn 195 200 205 Ala Ile Arg Val Val Ser Asp Ser Arg Gly Ile Ser Ser Arg Ile Val 210 215 220 Pro Glu His Asp Ala Ser Gly Ala Ala Leu Asp Leu Ser Phe Ala His 225 230 235 240 Leu Arg Ser Ile Ser Pro Cys His Leu Glu Phe Leu Arg Asn Met Gly 245 250 255 Val Ser Ala Ser Met Ser Leu Ser Ile Ile Ile Asp Gly Thr Leu Trp 260 265 270 Gly Leu Ile Ile Cys His His Tyr Glu Pro Arg Ala Val Pro Met Ala 275 280 285 Gln Arg Val Ala Ala Glu Met Phe Ala Asp Phe Leu Ser Leu His Phe 290 295 300 Thr Ala Ala His His Gln Arg 305 310 <210> 10 <211> 34 <212> PRT <213> Artificial Sequence <220> <223> cpp28 in the <400> 10 Arg Arg Arg Gly Arg Ser Pro Arg Arg Arg Thr Pro Ser Pro Arg Arg 1 5 10 15 Arg Arg Ser Gln Ser Pro Arg Arg Arg Ser Gln Ser Arg Glu Ser 20 25 30 Gln Cys <210> 11 <211> 32 <212> PRT <213> Artificial Sequence <220> <223> cpp28a <400> 11 Arg Arg Arg Gly Arg Ser Pro Arg Arg Arg Thr Pro Ser Pro Arg Arg 1 5 10 15 Arg Arg Ser Gln Ser Pro Arg Arg Arg Arg Ser Gln Ser Arg Glu Ser 20 25 30 <210> 12 <211> 29 <212> PRT <213> Artificial Sequence <220> <223> The amino acid sequence of cpp28b <400> 12 Arg Arg Arg Gly Arg Ser Pro Arg Arg Arg Thr Pro Ser Pro Arg Arg 1 5 10 15 Arg Arg Ser Gln Ser Pro Arg Arg Arg Arg Ser Gln Ser 20 25 <210> 13 <211> 27 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence of cpp28c <400> 13 Arg Arg Arg Gly Arg Ser Pro Arg Arg Arg Thr Pro Ser Pro Arg Arg 1 5 10 15 Arg Arg Ser Gln Ser Pro Arg Arg Arg Arg Ser 20 25 <210> 14 <211> twenty one <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence of cpp28d <400> 14 Arg Arg Arg Gly Arg Ser Pro Arg Arg Arg Thr Pro Ser Pro Arg Arg 1 5 10 15 Arg Arg Ser Gln Ser 20 <210> 15 <211> 36 <212> PRT <213> Artificial Sequence <220> <223> cpp28e <400> 15 Arg Arg Arg Asp Arg Gly Arg Ser Pro Arg Arg Arg Thr Pro Ser Pro 1 5 10 15 Arg Arg Arg Arg Ser Gln Ser Pro Arg Arg Arg Arg Ser Gln Ser Arg 20 25 30 Glu Ser Gln Cys 35 <210> 16 <211> 34 <212> PRT <213> Artificial Sequence <220> <223> cpp28f <400> 16 Arg Arg Arg Gly Arg Ser Pro Arg Arg Arg Thr Pro Ser Pro Arg Arg 1 5 10 15 Arg Arg Ser Lys Ser Pro Arg Arg Arg Ser Lys Ser Arg Glu Ser 20 25 30 Gln Cys <210> 17 <211> 34 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence of cpp28g <400> 17 Arg Gln Arg Gly Arg Ala Pro Arg Arg Arg Thr Pro Ser Pro Arg Arg 1 5 10 15 Arg Arg Ser Gln Ser Pro Arg Arg Arg Arg Ser Gln Ser Arg Ala Ser 20 25 30 Gln Cys <210> 18 <211> 37 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence of cpp28h <400> 18 Arg Cys Arg Gly Arg Arg Gly Arg Ser Pro Arg Arg Arg Thr Pro Ser 1 5 10 15 Pro Arg Arg Arg Arg Ser Gln Ser Pro Arg Arg Arg Arg Ser Lys Ser 20 25 30 Arg Glu Ser Gln Cys 35 <210> 19 <211> 39 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence of cpp28i <400> 19 Arg Arg Arg Gly Gly Ala Arg Ala Ser Arg Ser Pro Arg Arg Arg Thr 1 5 10 15 Pro Ser Pro Arg Arg Arg Arg Ser Gln Ser Pro Arg Arg Arg Arg Ser 20 25 30 Gln Ser Arg Ser Ala Asn Cys 35 <210> 20 <211> 40 <212> PRT <213> Artificial Sequence <220> <223> cpp28j's amino acid sequence <400> 20 Arg Arg Arg Gly Asn Pro Arg Ala Pro Arg Ser Pro Arg Arg Arg Thr 1 5 10 15 Pro Ser Pro Arg Arg Arg Arg Ser Gln Ser Pro Arg Arg Arg Arg Ser 20 25 30 Gln Ser Pro Ala Pro Ser Asn Cys 35 40 <210> 21 <211> 40 <212> PRT <213> Artificial Sequence <220> <223> cpp28k's amino acid sequence <400> 21 Arg Arg Arg Gly Gly Ser Arg Ala Thr Arg Ser Pro Arg Arg Arg Thr 1 5 10 15 Pro Ser Pro Arg Arg Arg Arg Ser Gln Ser Pro Arg Arg Arg Arg Ser 20 25 30 Gln Ser Pro Ala Ser Ser Asn Cys 35 40 <210> twenty two <211> 35 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence of cpp28L <400> twenty two Arg Arg Arg Pro Ala Ser Arg Arg Ser Thr Pro Ser Pro Arg Arg Arg 1 5 10 15 Arg Ser Gln Ser Pro Arg Arg Arg Arg Ser Pro Ser Pro Arg Pro Ala 20 25 30 Ser Asn Cys 35 <210> twenty three <211> 34 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence of cpp28-Os <400> twenty three Arg Arg Arg Gly Arg Ser Pro Arg Arg Arg Thr Pro Ser Pro Arg Arg 1 5 10 15 Arg Arg Ser Gln Ser Pro Arg Arg Arg Arg Ser Gln Ser Arg Glu Ser 20 25 30 Gln Ser <210> twenty four <211> 32 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence of cpp28aC <400> twenty four Arg Arg Arg Gly Arg Ser Pro Arg Arg Arg Thr Pro Ser Pro Arg Arg 1 5 10 15 Arg Arg Ser Gln Ser Pro Arg Arg Arg Ser Gln Ser Arg Glu Cys 20 25 30 <210> 25 <211> 29 <212> PRT <213> Artificial Sequence <220> <223> cpp28bC <400> 25 Arg Arg Arg Gly Arg Ser Pro Arg Arg Arg Thr Pro Ser Pro Arg Arg 1 5 10 15 Arg Arg Ser Gln Ser Pro Arg Arg Arg Arg Ser Gln Cys 20 25 <210> 26 <211> 27 <212> PRT <213> Artificial Sequence <220> <223> cpp28cC <400> 26 Arg Arg Arg Gly Arg Ser Pro Arg Arg Arg Thr Pro Ser Pro Arg Arg 1 5 10 15 Arg Arg Ser Gln Ser Pro Arg Arg Arg Arg Cys 20 25 <210> 27 <211> 21 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence of cpp28dC <400> 27 Arg Arg Arg Gly Arg Ser Pro Arg Arg Arg Thr Pro Ser Pro Arg Arg 1 5 10 15 Arg Arg Ser Gln Cys 20 <210> 28 <211> twenty four <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence of cpp28dREC <400> 28 Arg Arg Arg Gly Arg Ser Pro Arg Arg Arg Thr Pro Ser Pro Arg Arg 1 5 10 15 Arg Arg Ser Gln Arg Arg Glu Cys 20 <210> 29 <211> 29 <212> PRT <213> Artificial Sequence <220> <223> The amino acid sequence of cpp28oNT-1 <400> 29 Ser Pro Arg Arg Arg Thr Pro Ser Pro Arg Arg Arg Arg Ser Gln Ser 1 5 10 15 Pro Arg Arg Arg Arg Ser Gln Ser Arg Glu Ser Gln Cys 20 25 <210> 30 <211> twenty two <212> PRT <213> Artificial Sequence <220> <223> The amino acid sequence of cpp28oNT-2 <400> 30 Ser Pro Arg Arg Arg Arg Ser Gln Ser Pro Arg Arg Arg Arg Ser Gln 1 5 10 15 Ser Arg Glu Ser Gln Cys 20 <210> 31 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> The amino acid sequence of cpp28oNT-3 <400> 31 Ser Pro Arg Arg Arg Arg Ser Gln Ser Arg Glu Ser Gln Cys 1 5 10 <210> 32 <211> 32 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence of cpp28aCQ1 <400> 32 Arg Arg Arg Gly Arg Gln Pro Arg Arg Arg Thr Pro Ser Pro Arg Arg 1 5 10 15 Arg Arg Ser Gln Ser Pro Arg Arg Arg Arg Ser Gln Ser Arg Glu Cys 20 25 30 <210> 33 <211> 32 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence of cpp28aCQ2 <400> 33 Arg Arg Arg Gly Arg Gln Pro Arg Arg Arg Thr Pro Gln Pro Arg Arg 1 5 10 15 Arg Arg Ser Gln Ser Pro Arg Arg Arg Ser Gln Ser Arg Glu Cys 20 25 30 <210> 34 <211> 32 <212> PRT <213> Artificial Sequence <220> <223> cpp28aCQ3 <400> 34 Arg Arg Arg Gly Arg Gln Pro Arg Arg Arg Thr Pro Gln Pro Arg Arg 1 5 10 15 Arg Arg Ser Gln Gln Pro Arg Arg Arg Ser Gln Ser Arg Glu Cys 20 25 30 <210> 35 <211> 27 <212> PRT <213> Artificial Sequence <220> <223> cpp28aCNT in the <400> 35 Ser Pro Arg Arg Arg Thr Pro Ser Pro Arg Arg Arg Arg Ser Gln Ser 1 5 10 15 Pro Arg Arg Arg Ser Gln Ser Arg Glu Cys 20 25 <210> 36 <211> 24 <212> PRT <213> Artificial Sequence <220> <223> The amino acid sequence of cpp28bCNT <400> 36 Ser Pro Arg Arg Arg Thr Pro Ser Pro Arg Arg Arg Arg Ser Gln Ser 1 5 10 15 Pro Arg Arg Arg Arg Ser Gln Cys 20 <210> 37 <211> twenty two <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence of cpp28cCNT <400> 37 Ser Pro Arg Arg Arg Thr Pro Ser Pro Arg Arg Arg Arg Ser Gln Ser 1 5 10 15 Pro Arg Arg Arg Arg Cys 20 <210> 38 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence of cpp28dCNT <400> 38 Ser Pro Arg Arg Arg Thr Pro Ser Pro Arg Arg Arg Arg Ser Gln Cys 1 5 10 15 <210> 39 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> Amino acid sequence of the flexible peptide linker <400> 39 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 1 5 10 15 <210> 40 <211> 288 <212> PRT <213> Artificial Sequence <220> <223> Amino acid sequence of GFP-cpp28 <400> 40 Met Val Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu 1 5 10 15 Val Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Ser Gly 20 25 30 Glu Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Phe Ile 35 40 45 Cys Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr 50 55 60 Leu Thr Tyr Gly Val Gln Cys Phe Ser Arg Tyr Pro Asp His Met Lys 65 70 75 80 Gln His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu 85 90 95 Arg Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu 100 105 110 Val Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly 115 120 125 Ile Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr 130 135 140 Asn Tyr Asn Ser His Asn Val Tyr Ile Met Ala Asp Lys Gln Lys Asn 145 150 155 160 Gly Ile Lys Val Asn Phe Lys Ile Arg His Asn Ile Glu Asp Gly Ser 165 170 175 Val Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly 180 185 190 Pro Val Leu Leu Pro Asp Asn His Tyr Leu Ser Thr Gln Ser Ala Leu 195 200 205 Ser Lys Asp Pro Asn Glu Lys Arg Asp His Met Val Leu Leu Glu Phe 210 215 220 Val Thr Ala Ala Gly Ile Thr Leu Gly Met Asp Glu Leu Tyr Lys Gly 225 230 235 240 Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Arg Arg 245 250 255 Arg Gly Arg Ser Pro Arg Arg Arg Thr Pro Ser Pro Arg Arg Arg Arg 260 265 270 Ser Gln Ser Pro Arg Arg Arg Arg Ser Gln Ser Arg Glu Ser Gln Cys 275 280 285 <210> 41 <211> 267 <212> PRT <213> Artificial Sequence <220> <223> Amino acid sequence of GFP-TAT <400> 41 Met Val Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu 1 5 10 15 Val Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Ser Gly 20 25 30 Glu Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Phe Ile 35 40 45 Cys Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr 50 55 60 Leu Thr Tyr Gly Val Gln Cys Phe Ser Arg Tyr Pro Asp His Met Lys 65 70 75 80 Gln His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu 85 90 95 Arg Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu 100 105 110 Val Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly 115 120 125 Ile Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr 130 135 140 Asn Tyr Asn Ser His Asn Val Tyr Ile Met Ala Asp Lys Gln Lys Asn 145 150 155 160 Gly Ile Lys Val Asn Phe Lys Ile Arg His Asn Ile Glu Asp Gly Ser 165 170 175 Val Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly 180 185 190 Pro Val Leu Leu Pro Asp Asn His Tyr Leu Ser Thr Gln Ser Ala Leu 195 200 205 Ser Lys Asp Pro Asn Glu Lys Arg Asp His Met Val Leu Leu Glu Phe 210 215 220 Val Thr Ala Ala Gly Ile Thr Leu Gly Met Asp Glu Leu Tyr Lys Gly 225 230 235 240 Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Arg 245 250 255 Lys Lys Arg Arg Gln Arg Arg Arg Pro Pro Gln 260 265 <210> 42 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> Amino acid sequence of cell-penetrating peptide TAT <400> 42 Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg Pro Pro Gln 1 5 10 <210> 43 <211> 237 <212> PRT <213> Artificial Sequence <220> <223> The amino acid sequence of the mRuby3 protein <400> 43 Met Val Ser Lys Gly Glu Glu Leu Ile Lys Glu Asn Met Arg Met Lys 1 5 10 15 Val Val Met Glu Gly Ser Val Asn Gly His Gln Phe Lys Cys Thr Gly 20 25 30 Glu Gly Glu Gly Arg Pro Tyr Glu Gly Val Gln Thr Met Arg Ile Lys 35 40 45 Val Ile Glu Gly Gly Pro Leu Pro Phe Ala Phe Asp Ile Leu Ala Thr 50 55 60 Ser Phe Met Tyr Gly Ser Arg Thr Phe Ile Lys Tyr Pro Ala Asp Ile 65 70 75 80 Pro Asp Phe Phe Lys Gln Ser Phe Pro Glu Gly Phe Thr Trp Glu Arg 85 90 95 Val Thr Arg Tyr Glu Asp Gly Gly Val Val Thr Val Thr Gln Asp Thr 100 105 110 Ser Leu Glu Asp Gly Glu Leu Val Tyr Asn Val Lys Val Arg Gly Val 115 120 125 Asn Phe Pro Ser Asn Gly Pro Val Met Gln Lys Lys Thr Lys Gly Trp 130 135 140 Glu Pro Asn Thr Glu Met Met Tyr Pro Ala Asp Gly Gly Leu Arg Gly 145 150 155 160 Tyr Thr Asp Ile Ala Leu Lys Val Asp Gly Gly Gly His Leu His Cys 165 170 175 Asn Phe Val Thr Thr Tyr Arg Ser Lys Lys Thr Val Gly Asn Ile Lys 180 185 190 Met Pro Gly Val His Ala Val Asp His Arg Leu Glu Arg Ile Glu Glu 195 200 205 Ser Asp Asn Glu Thr Tyr Val Val Gln Arg Glu Val Ala Val Ala Lys 210 215 220 Tyr Ser Asn Leu Gly Gly Gly Met Asp Glu Leu Tyr Lys 225 230 235 <210> 44 <211> 87 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence of anti-CRISPR protein <400> 44 Met Asn Ile Asn Asp Leu Ile Arg Glu Ile Lys Asn Lys Asp Tyr Thr 1 5 10 15 Val Lys Leu Ser Gly Thr Asp Ser Asn Ser Ile Thr Gln Leu Ile Ile 20 25 30 Arg Val Asn Asn Asp Gly Asn Glu Tyr Val Ile Ser Glu Ser Glu Asn 35 40 45 Glu Ser Ile Val Glu Lys Phe Ile Ser Ala Phe Lys Asn Gly Trp Asn 50 55 60 Gln Glu Tyr Glu Asp Glu Glu Glu Phe Tyr Asn Asp Met Gln Thr Ile 65 70 75 80 Thr Leu Lys Ser Glu Leu Asn 85 <210> 45 <211> 136 <212> PRT <213> Artificial Sequence <220> <223> The amino acid sequence of the anti-CRISPR-cpp28ori protein <400> 45 Met Asn Ile Asn Asp Leu Ile Arg Glu Ile Lys Asn Lys Asp Tyr Thr 1 5 10 15 Val Lys Leu Ser Gly Thr Asp Ser Asn Ser Ile Thr Gln Leu Ile Ile 20 25 30 Arg Val Asn Asn Asp Gly Asn Glu Tyr Val Ile Ser Glu Ser Glu Asn 35 40 45 Glu Ser Ile Val Glu Lys Phe Ile Ser Ala Phe Lys Asn Gly Trp Asn 50 55 60 Gln Glu Tyr Glu Asp Glu Glu Glu Phe Tyr Asn Asp Met Gln Thr Ile 65 70 75 80 Thr Leu Lys Ser Glu Leu Asn Gly Gly Gly Gly Ser Gly Gly Gly Gly 85 90 95 Ser Gly Gly Gly Gly Ser Arg Arg Arg Gly Arg Ser Pro Arg Arg Arg 100 105 110 Thr Pro Ser Pro Arg Arg Arg Arg Ser Gln Ser Pro Arg Arg Arg Arg 115 120 125 Ser Gln Ser Arg Glu Ser Gln Cys 130 135 <210> 46 <211> 1368 <212> PRT <213> Artificial Sequence <220> <223> Amino acid sequence of cas9 protein <400> 46 Met Asp Lys Lys Tyr Ser Ile Gly Leu Asp Ile Gly Thr Asn Ser Val 1 5 10 15 Gly Trp Ala Val Ile Thr Asp Glu Tyr Lys Val Pro Ser Lys Lys Phe 20 25 30 Lys Val Leu Gly Asn Thr Asp Arg His Ser Ile Lys Asn Leu Ile 35 40 45 Gly Ala Leu Phe Asp Ser Gly Glu Thr Ala Glu Ala Thr Arg Leu 50 55 60 Lys Arg Thr Ala Arg Arg Tyr Thr Arg Arg Lys Asn Arg With Cys 65 70 75 80 Tyr Leu Gln Glu Ile Phe Ser Asn Glu Met Ala Asp Ser 85 90 95 Phe Phe His Arg Leu Glu Glu Ser Phe Leu Val Glu Glu Asp Lys Lys 100 105 110 His Glu Arg His Pro Ile Phe Gly Asn Ile Val Asp Glu Val Ala Tyr 115 120 125 His Glu Lys Tyr Pro Thr Ile Tyr His Leu Arg Lys Leu Val Asp 130 135 140 Ser Thr Asp Lys Ala Asp Leu Arg Leu Ile Tyr Leu Ala Leu Ala His 145 150 155 160 Met Ile Lys Phe Arg Gly His Phe Leu Ile Glu Gly Asp Leu Asn Pro 165 170 175 Asp Asn Ser Asp Val Asp Lys Leu Phe Ile Gln Leu Val Gln Thr Tyr 180 185 190 Asn Gln Leu Phe Glu Glu Asn Pro Ile Asn Ala Ser Gly Val Asp Ala 195 200 205 Lys Ala Ile Leu Ser Ala Arg Leu Ser Lys Ser Arg Arg Leu Glu Asn 210 215 220 Leu Ile Ala Gln Leu Pro Gly Glu Lys Lys Asn Gly Leu Phe Gly Asn 225 230 235 240 Leu Ile Ala Leu Ser Leu Gly Leu Thr Pro Asn Phe Lys Ser Asn Phe 245 250 255 Asp Leu Ala Glu Asp Ala Lys Leu Gln Leu Ser Lys Asp Thr Tyr Asp 260 265 270 Asp Asp Leu Asp Asn Leu Leu Ala Gln Ile Gly Asp Gln Tyr Ala Asp 275 280 285 Leu Phe Leu Ala Ala Lys Asn Leu Ser Asp Ala Ile Leu Leu Ser Asp 290 295 300 Ile Leu Arg Val Asn Thr Glu Ile Thr Lys Ala Pro Leu Ser Ala Ser 305 310 315 320 Met Ile Lys Arg Tyr Asp Glu His His Gln Asp Leu Thr Leu Leu Lys 325 330 335 Ala Leu Val Arg Gln Gln Leu Pro Glu Lys Tyr Lys Glu Ile Phe Phe 340 345 350 Asp Gln Ser Lys Asn Gly Tyr Ala Gly Tyr Ile Asp Gly Gly Ala Ser 355 360 365 Gln Glu Glu Phe Tyr Lys Phe Ile Lys Pro Ile Leu Glu Lys Met Asp 370 375 380 Gly Thr Glu Glu Leu Leu Val Lys Leu Asn Arg Glu Asp Leu Leu Arg 385 390 395 400 Lys Gln Arg Thr Phe Asp Asn Gly Ser Ile Pro His Gln Ile His Leu 405 410 415 Gly Glu Leu His Ala Ile Leu Arg Arg Gln Glu Asp Phe Tyr Pro Phe 420 425 430 Leu Lys Asp Asn Arg Glu Lys Ile Glu Lys Ile Leu Thr Phe Arg Ile 435 440 445 Pro Tyr Tyr Val Gly Pro Leu Ala Arg Gly Asn Ser Arg Phe Ala Trp 450 455 460 Met Thr Arg Lys Ser Glu Glu Thr Ile Thr Pro Trp Asn Phe Glu Glu 465 470 475 480 Val Val Asp Lys Gly Ala Ser Ala Gln Ser Phe Ile Glu Arg Met Thr 485,490,495 Asn Phe Asp Lys Asn Leu Pro Asn Glu Lys Val Leu Pro Lys His Ser 500 505 510 Leu Tyr Glu Tyr Phe Thr Val Tyr Asn Glu Thr Lys Val Lys 515,520,525 Tyr Val Thr Glu Gly Met Arg Lys Pro Ala Phe Leu Ser Gly Glu Gln 530 535 540 Lys Lys Ala Ile Val Asp Leu Leu Phe Lys Thr Asn Arg Lys Val Thr 545 550 555 560 Val Lys Gln Leu Lys Glu Asp Tyr Phe Lys Ile Glu Cys Phe Asp 565,570,575 Ser Val Glu Ile Ser Gly Val Glu Asp Arg Phe Asn Ala Ser Leu Gly 580,585,590 Thr Tyr His Asp Leu Leu Lys Ile Ile Lys Asp Lys Asp Phe Leu Asp 595,600,605 Asn Glu Glu Asn Glu Asp With Glu Asp With Val With Thr With Thr 610 615 620 Leu Phe Glu Asp Arg Glu Met Ile Glu Glu Arg Leu Lys Thr Tyr Ala 625 630 635 640 His Leu Phe Asp Asp Lys Val Met Lys Gln Leu Lys Arg Arg Arg Tyr 645 650 655 Thr Gly Trp Gly Arg Leu Ser Arg Lys Leu Ile Asn Gly Ile Arg Asp 660 665 670 Lys Gln Ser Gly Lys Thr Ile Leu Asp Phe Leu Lys Ser Asp Gly Phe 675 680 685 Ala Asn Arg Asn Phe Met Gln Leu Ile His Asp Asp Ser Leu Thr Phe 690 695 700 Lys Glu Asp Ile Gln Lys Ala Gln Val Ser Gly Gln Gly Asp Ser Leu 705 710 715 720 His Glu His Ile Ala Asn Leu Ala Gly Ser Pro Ala Ile Lys Lys Gly 725 730 735 Ile Leu Gln Thr Val Lys Val Val Asp Glu Leu Val Lys Val Met Gly 740 745 750 Arg His Lys Pro Glu Asn Ile Val Ile Glu Met Ala Arg Glu Asn Gln 755 760 765 Thr Thr Gln Lys Gly Gln Lys Asn Ser Arg Glu Arg Met Lys Arg Ile 770 775 780 Glu Glu Gly Ile Lys Glu Leu Gly Ser Gln Ile Leu Lys Glu His Pro 785 790 795 800 Val Glu Asn Thr Gln Leu Gln Asn Glu Lys Leu Tyr Leu Tyr Tyr Leu 805 810 815 Gln Asn Gly Arg Asp Met Tyr Val Asp Gln Glu Leu Asp Ile Asn Arg 820 825 830 Leu Ser Asp Tyr Asp Val Asp His Ile Val Pro Gln Ser Phe Leu Lys 835 840 845 Asp Asp Ser Ile Asp Asn Lys Val Leu Thr Arg Ser Asp Lys Asn Arg 850 855 860 Gly Lys Ser Asp Asn Val Pro Ser Glu Glu Val Val Lys Lys Met Lys 865 870 875 880 Asn Tyr Trp Arg Gln Leu Leu Asn Ala Lys Leu Ile Thr Gln Arg Lys 885 890 895 Phe Asp Asn Leu Thr Lys Ala Glu Arg Gly Gly Leu Ser Glu Leu Asp 900 905 910 Lys Ala Gly Phe Ile Lys Arg Gln Leu Val Glu Thr Arg Gln Ile Thr 915 920 925 Lys His Val Ala Gln Ile Leu Asp Ser Arg Met Asn Thr Lys Tyr Asp 930 935 940 Glu Asn Asp Lys Leu Ile Arg Glu Val Lys Val Ile Thr Leu Lys Ser 945 950 955 960 Lys Leu Val Ser Asp Phe Arg Lys Asp Phe Gln Phe Tyr Lys Val Arg 965 970 975 Glu Ile Asn Asn Tyr His His Ala His Asp Ala Tyr Leu Asn Ala Val 980 985 990 Val Gly Thr Ala Leu Ile Lys Lys Tyr Pro Lys Leu Glu Ser Glu Phe 995 1000 1005 Val Tyr Gly Asp Tyr Lys Val Tyr Asp Val Arg Lys Met Ile Ala 1010 1015 1020 Lys Ser Glu Gln Glu Ile Gly Lys Ala Thr Ala Lys Tyr Phe Phe 1025 1030 1035 Tyr Ser Asn Ile Met Asn Phe Phe Lys Thr Glu Ile Thr Leu Ala 1040 1045 1050 Asn Gly Glu Ile Arg Lys Arg Pro Leu Ile Glu Thr Asn Gly Glu 1055 1060 1065 Thr Gly Glu Ile Val Trp Asp Lys Gly Arg Asp Phe Ala Thr Val 1070 1075 1080 Arg Lys Val Leu Ser Met Pro Gln Val Asn Ile Val Lys Lys Thr 1085 1090 1095 Glu Val Gln Thr Gly Gly Phe Ser Lys Glu Ser Ile Leu Pro Lys 1100 1105 1110 Arg Asn Ser Asp Lys Leu Ile Ala Arg Lys Lys Asp Trp Asp Pro 1115 1120 1125 Light Light Tyr Gly Gly Phe Asp Ser Pro Thr Val Ala Tyr Ser Val 1130 1135 1140 Leu Val Val Ala Lys Val Glu Lys Gly Lys Ser Lys Lys Leu Lys 1145 1150 1155 Ser Val Lys Glu Leu Leu Gly Ile Thr Ile Met Glu Arg Ser Ser 1160 1165 1170 Phe Glu Lys Asn Pro Ile Asp Phe Leu Glu Ala Lys Gly Tyr Lys 1175 1180 1185 Glu Val Lys Lys Asp Leu Ile Ile Lys Leu Pro Lys Tyr Ser Leu 1190 1195 1200 Phe Glu Leu Glu Asn Gly Arg Lys Arg Met Leu Ala Ser Ala Gly 1205 1210 1215 Glu Leu Gln Lys Gly Asn Glu Leu Ala Leu Pro Ser Lys Tyr Val 1220 1225 1230 Asn Phe Leu Tyr Leu Ala Ser His Tyr Glu Lys Leu Lys Gly Ser 1235 1240 1245 Pro Glu Asp Asn Glu Gln Lys Gln Leu Phe Val Glu Gln His Lys 1250 1255 1260 His Tyr Leu Asp Glu Ile Ile Glu Gln Ile Ser Glu Phe Ser Lys 1265 1270 1275 Arg Val Ile Leu Ala Asp Ala Asn Leu Asp Lys Val Leu Ser Ala 1280 1285 1290 Tyr Asn Lys His Arg Asp Lys Pro Ile Arg Glu Gln Ala Glu Asn 1295 1300 1305 Ile Ile His Leu Phe Thr Leu Thr Asn Leu Gly Ala Pro Ala Ala 1310 1315 1320 Phe Lys Tyr Phe Asp Thr Thr Ile Asp Arg Lys Arg Tyr Thr Ser 1325 1330 1335 Thr Lys Glu Val Leu Asp Ala Thr Leu Ile His Gln Ser Ile Thr 1340 1345 1350 Gly Leu Tyr Glu Thr Arg Ile Asp Leu Ser Gln Leu Gly Gly Asp 1355 1360 1365 <210> 47 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> The amino acid sequence of the light chain variable region of antibody 2A7 <400> 47 Asp Ile Gln Met Thr Gln Thr Ser Ser Ser Leu Ser Ala Ser Pro Gly 1 5 10 15 Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln Gly Ile Asn Asn Tyr 20 25 30 Leu Asn Trp Tyr Lys Gln Lys Thr Asp Gly Thr Phe Lys Leu Leu Ile 35 40 45 Tyr Tyr Thr Ser Tyr Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Arg Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser Asn Leu Glu Pro 65 70 75 80 Glu Asp Val Ala Thr Tyr Tyr Cys Gln Gln Tyr Gly Lys Leu Pro Trp 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 48 <211> 122 <212> PRT <213> Artificial Sequence <220> <223> Amino acid sequence of the heavy chain variable region of antibody 2A7 <400> 48 Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Arg Tyr 20 25 30 Trp Met His Trp Val Met Gln Arg Pro Gly Gln Asp Leu Glu Trp Ile 35 40 45 Gly Glu Ile Asn Pro Ile Asn Gly Arg Thr Asn Tyr Asn Glu Lys Phe 50 55 60 Arg Arg Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Val Tyr 65 70 75 80 Ile Gln Phe Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys 85 90 95 Thr Arg Glu Gly Tyr Arg Asn Asp Tyr Tyr Tyr Ala Met Asp Phe Trp 100 105 110 Gly Arg Gly Thr Ser Val Thr Val Ser Ser 115 120 <210> 49 <211> 111 <212> PRT <213> Artificial Sequence <220> <223> The amino acid sequence of the light chain variable region of antibody 16D5 <400> 49 Asp Ile Val Leu Thr Gln Ser Pro Gly Ser Leu Ala Val Phe Leu Gly 1 5 10 15 Gln Arg Ala Thr Ile Ser Cys Arg Ala Ser Gln Ser Val Ser Gly Ser 20 25 30 Ile Tyr Ser Tyr Met His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro 35 40 45 Lys Leu Leu Ile Lys Phe Ala Ser Asn Leu Glu Ser Gly Val Pro Ala 50 55 60 Arg Phe Ser Gly Gly Gly Ser Gly Thr Asp Phe Thr Leu Asn Ile His 65 70 75 80 Pro Val Glu Glu Glu Asp Ala Ala Thr Tyr Tyr Cys Gln His Ser Trp 85 90 95 Glu Ile Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 110 <210> 50 <211> 121 <212> PRT <213> Artificial Sequence <220> <223> Amino acid sequence of the heavy chain variable region of antibody 16D5 <400> 50 Glu Val Gln Leu Gln Gln Ser Gly Ala Glu Val Val Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Leu Ser Cys Thr Ala Ser Gly Phe Lys Ile Glu Asp Thr 20 25 30 Tyr Ile His Trp Val Lys Gln Arg Pro Glu Gln Gly Leu Glu Trp Ile 35 40 45 Gly Arg Ile Asp Pro Ala Asn Gly Asn Ser Arg Tyr Asp Pro Asn Phe 50 55 60 Gln Gly Lys Ala Thr Ile Ile Ala Asp Thr Ser Ser Tyr Thr Ile Tyr 65 70 75 80 Leu Gln Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ser Ser Pro Leu Ser Leu Leu Arg Leu Gly Gly Phe Ala Tyr Trp Gly 100 105 110 Gln Gly Thr Leu Ile Thr Val Ser Ala 115 120 <210> 51 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> Amino acid sequence of the light chain variable region of antibody TA99 <400> 51 Ala Ile Gln Met Ser Gln Ser Pro Ala Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Glu Thr Val Thr Ile Thr Cys Arg Ala Ser Gly Asn Ile Tyr Asn Tyr 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Gln Gly Lys Ser Pro His Leu Leu Val 35 40 45 Tyr Asp Ala Lys Thr Leu Ala Asp Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Gln Tyr Ser Leu Lys Ile Ser Ser Leu Gln Thr 65 70 75 80 Glu Asp Ser Gly Asn Tyr Tyr Cys Gln His Phe Trp Ser Leu Pro Phe 85 90 95 Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 52 <211> 120 <212> PRT <213> Artificial Sequence <220> <223> The amino acid sequence of the heavy chain variable region of antibody TA99 <400> 52 Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Arg Pro Gly Ala Leu 1 5 10 15 Val Lys Leu Ser Cys Lys Thr Ser Gly Phe Asn Ile Lys Asp Tyr Phe 20 25 30 Leu His Trp Val Arg Gln Arg Pro Asp Gln Gly Leu Glu Trp Ile Gly 35 40 45 Trp Ile Asn Pro Asp Asn Gly Asn Thr Val Tyr Asp Pro Lys Phe Gln 50 55 60 Gly Thr Ala Ser Leu Thr Ala Asp Thr Ser Ser Ser Asn Thr Val Tyr Leu 65 70 75 80 Gln Leu Ser Gly Leu Thr Ser Glu Asp Thr Ala Val Tyr Phe Cys Thr 85 90 95 Arg Arg Asp Tyr Thr Tyr Glu Lys Ala Ala Leu Asp Tyr Trp Gly Gln 100 105 110 Gly Ala Ser Val Ile Val Ser Ser 115 120 <210> 53 <211> 91 <212> PRT <213> Artificial Sequence <220> <223> Amino acid sequence of ZF protein <400> 53 Val Ser Arg Pro Gly Glu Arg Pro Phe Gln Cys Arg Ile Cys Met Arg 1 5 10 15 Asn Phe Ser Asp Lys Thr Lys Leu Arg Val His Thr Arg Thr His Thr 20 25 30 Gly Glu Lys Pro Phe Gln Cys Arg Ile Cys Met Arg Asn Phe Ser Val 35 40 45 Arg His Asn Leu Thr Arg His Leu Arg Thr His Thr Gly Glu Lys Pro 50 55 60 Phe Gln Cys Arg Ile Cys Met Arg Asn Phe Ser Gln Ser Thr Ser Leu 65 70 75 80 Gln Arg His Leu Lys Thr His Leu Arg Gly Ser 85 90 <210> 54 <211> 11 <212> DNA <213> Artificial Sequence <220> <223> ZF protein binding sequence <400> 54 tgtagatgga g 11

Claims

1. A cell-penetrating peptide, wherein, The amino acid sequence of the cell-penetrating peptide is shown in SEQ ID NO:

27.

2. A fusion protein comprising the cell-penetrating peptide of claim 1, and, a peptide of interest; wherein, The cell-penetrating peptide is attached to the C-terminus of the target peptide.

3. The fusion protein of claim 2, wherein, The target peptide is directly covalently linked to the cell-penetrating peptide; or, they are covalently linked through a peptide linker.

4. The fusion protein of claim 3, wherein, The peptide linker is a flexible peptide linker.

5. The fusion protein of claim 2, wherein, The target peptide is an antibody, a gene-editing-related protein, an active or traceable protein, or a protein that can bind to DNA molecules.

6. The fusion protein of claim 2, wherein, The fusion protein also contains additional structural domains.

7. The fusion protein of claim 6, wherein, The additional structural field is the label structural field.

8. The fusion protein according to claim 6 or 7, wherein, The additional domain is located at the N-terminus or C-terminus of the fusion protein.

9. A conjugate comprising the cell-penetrating peptide of claim 1, and a target molecule; wherein, The target molecule is the target protein.

10. The conjugate of claim 9, wherein, The target molecule is directly covalently linked to the cell-penetrating peptide or covalently linked through a linker; or, the target molecule is non-covalently linked to the cell-penetrating peptide.

11. The conjugate of claim 9, wherein, The target molecule is an antibody, a gene-editing-related protein, an active or traceable protein, or a protein that can bind to DNA molecules.

12. A polymer comprising the fusion protein of any one of claims 2-8 or the conjugate of any one of claims 9-11.

13. A complex comprising the fusion protein of any one of claims 2-8, the conjugate of any one of claims 9-11, or the polymer of claim 12, and a component non-covalently bound to said fusion protein, conjugate, or polymer; wherein, The fusion protein, conjugate, or polymer comprises a protein capable of binding DNA molecules, which is linked to the cell-penetrating peptide; and the complex comprises DNA molecules, which are non-covalently bound to the protein capable of binding DNA molecules.

14. A complex comprising the fusion protein of any one of claims 2-8, the conjugate of any one of claims 9-11, or the polymer of claim 12, and a component complexed with said fusion protein, conjugate, or polymer; wherein, The fusion protein, conjugate, or polymer comprises a protein capable of binding DNA molecules, which is linked to the cell-penetrating peptide; and the complex comprises a DNA molecule, which is complexed with a protein capable of binding DNA molecules.

15. The complex of claim 13 or 14, wherein, The DNA molecule contains a nucleotide sequence that is recognized and bound by the protein capable of binding to the DNA molecule.

16. An isolated nucleic acid molecule comprising a nucleotide sequence encoding the cell-penetrating peptide of claim 1 or the fusion protein of any one of claims 2-8.

17. A carrier comprising the isolated nucleic acid molecule of claim 16.

18. A host cell comprising the isolated nucleic acid molecule of claim 16 or the vector of claim 17.

19. A method for preparing the cell-penetrating peptide of claim 1 or the fusion protein of any one of claims 2-8, comprising culturing the host cell of claim 18 under conditions that allow expression of the cell-penetrating peptide or the fusion protein, and recovering the cell-penetrating peptide or the fusion protein from the cultured host cell culture.

20. A composition comprising the fusion protein of any one of claims 2-8, the conjugate of any one of claims 9-11, the polymer of claim 12, the complex of any one of claims 13-15, the isolated nucleic acid molecule of claim 16, or the carrier of claim 17.

21. A pharmaceutical composition comprising the fusion protein of any one of claims 2-8, the conjugate of any one of claims 9-11, the polymer of claim 12, or the complex of any one of claims 13-15, and a pharmaceutically acceptable carrier, wherein, The fusion protein, conjugate, polymer, or complex contains a therapeutically active polypeptide linked to the cell-penetrating peptide.

22. A method for non-therapeutic delivery of a target molecule across a membrane into a cell, comprising: linking the cell-penetrating peptide of claim 1 to the target molecule, and then contacting the cell; wherein, (a) The target molecule is a target peptide, and the method includes: (a-1) linking the target molecule to the cell-penetrating peptide to obtain a fusion protein; wherein the cell-penetrating peptide is linked to the C-terminus of the target molecule; (a-2) contacting the fusion protein with a cell to deliver the target molecule across the membrane into the cell; or, (b) The target molecule is a target peptide, and the method includes: (b-1) linking the target molecule to the cell-penetrating peptide to obtain a conjugate; (b-2) contacting the conjugate with a cell to deliver the target molecule across the membrane into the cell; or, (c) The target molecule is a target nucleic acid, and the method includes: (c-1) linking a protein capable of binding the target nucleic acid to the cell membrane-penetrating peptide; (c-2) contacting the product of step (c-1) with the target nucleic acid to obtain a complex; and (c-3) contacting the complex with a cell to deliver the target nucleic acid across the membrane into the cell; or, (d) The target molecule is a target nucleic acid, and the method comprises: (d-1) adding a nucleotide sequence to the target nucleic acid that can be recognized and bound by a DNA-binding protein; (d-2) linking the DNA-binding protein to the cell-penetrating peptide; (d-3) contacting the product of step (d-1) with the product of step (d-2) to obtain a complex; and (d-4) contacting the complex with a cell to deliver the target nucleic acid across the membrane into the cell.

23. The method of claim 22, wherein, In step (b-1), the target molecule is directly covalently linked to the cell-penetrating peptide; or, the target peptide is covalently linked to the cell-penetrating peptide through a connector.

24. The method of claim 22, wherein, In step (b-1), the target molecule is linked to the cell-penetrating peptide in a non-covalent manner.

25. The method of claim 22, wherein, In step (a-1), the target peptide is directly covalently linked to the cell-penetrating peptide; or, the target peptide is covalently linked to the cell-penetrating peptide through a peptide linker to obtain a fusion protein.

26. The method of claim 25, wherein, The peptide linker is a flexible peptide linker.

27. The method of claim 22, wherein, The target peptide is an antibody, a gene-editing-related protein, an active or traceable protein, or a protein that can bind to DNA molecules.

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