Precision lifespan control of intracellularly delivered therapeutic proteins

Abstract

An isolated peptide for regulating intracellular protein degradation that includes a linker sequence fused to a C-end degron peptide and, optionally, a caging molecule bonded to the carboxyl group of a C -terminal alanine residue. Also disclosed is a method for temporal control of protein degradation that relies on fusing a protein of interest at its C- terminus to the degron peptide having a caging molecule, and a method for high-fidelity gene editing that utilizes an RNA-dependent endonuclease modified at the C -terminus with the caged isolated peptide. A fusion protein for high-fidelity gene editing is further provided.

Description

[0001] BC2025.003.niu

[0002] PRECISION LIFESPAN CONTROL OF INTRACELLULARLY DELIVERED THERAPEUTIC PROTEINS

[0003] CROSS-REFERENCE TO RELATED APPLICATIONS

[0004] This application claims priority to and the benefit of U.S. Provisional Application No. 63 / 691,177, filed September 5, 2024. The entire teachings of the above application are incorporated herein by reference.

[0005] GOVERNMENT SUPPORT

[0006] This invention was made with government support under HG011027 awarded by the National Institutes of Health. The government has certain rights in the invention.

[0007] BACKGROUND

[0008] Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) form the basis of a revolutionary gene-editing technology that allows precise modification of the genome within living organisms. One of the main drawbacks associated with CRISPR technology is the possibility of off-target effects. Off-target effects occur when the CRISPR system unintentionally modifies genomic sequences that are similar but not identical to the target sequence. These off-target effects pose a risk as they may lead to changes in the organism’s genetic information that can potentially lead to adverse side effects.

[0009] Several strategies have been developed to increase the specificity of CRISPR technology and minimize unintended modifications, for example using high-fidelity CRISPR-associated protein (Cas) variants, enhancing the specificity of single guide RNA sequences (sgRNAs), and modifying protospacer adjacent motif (PAM) recognition sites. However, these techniques are labor intensive, making them impractical in a clinical setting.

[0010] A small-molecule-controlled system has been reported that relies on fusing Cas9 to the destabilizing domains (DDs) of E. coli dihydrofolate reductase (DHFR) to achieve dosage and temporal control of CRISPR-Cas9. This system exhibited significant

[0011] 1

[0012] 4914-1459-3110, v. 1 BC2025.003.niu background activity in the absence of the small molecule trimethoprim (TMP), thus limiting the ability of this system to achieve precise temporal control of Cas9 activity.

[0013] The need exists to develop compositions and methods for precision temporal regulation of the CRISPR system to reduce off-target effects that can be universally applied to regulate any desired intracellular protein target.

[0014] SUMMARY

[0015] To meet the need set forth above, an isolated peptide for regulating intracellular protein degradation is provided. The isolated peptide comprises, from N-terminus to C -terminus, a linker sequence G4X fused to a C-end degron peptide in which X is S(GS)n, n being 0 to 4, the C-end degron peptide comprising 4 to 6 consecutive alanine residues and the isolated peptide being free of any lysine residues. The isolated peptide can include a caging molecule bonded to the carboxyl group of the C-terminal alanine residue.

[0016] Also disclosed is a method for temporal control of protein degradation that is carried out by (i) fusing a protein of interest at its C-terminus to a sortase recognition sequence to form a fusion protein, (ii) reacting the fusion protein in vitro with a sortase enzyme and the isolated peptide having a caging molecule described above to form a C-terminal caged fusion protein, (iii) introducing the C-terminal caged fusion protein into a cell, and (iv) exposing the cell to a stimulus, thereby removing the caging molecule to form an uncaged fusion protein. The uncaged fusion protein is degraded by cellular protein degradation machinery.

[0017] Further, provided is a method for high-fidelity gene editing by (i) obtaining a modified RNA-dependent endonuclease having at the C-terminus the caged isolated peptide described, supra, (ii) introducing the modified RNA-dependent endonuclease and a guide RNA into a cell, the guide RNA having a sequence that hybridizes to a target gene, (iii) culturing the cell for a time period that allows gene editing of the target gene; and (iv) exposing the cultured cell to a stimulus that removes the caging molecule from the modified RNA-dependent endonuclease, thereby inducing degradation of the modified RNA-dependent endonuclease and reducing or eliminating off-target gene editing events.

[0018] 2

[0019] 4914-1459-3110, v. 1 BC2025.003.niu

[0020] Finally, disclosed is a fusion protein for high-fidelity gene editing, the fusion protein comprising an RNA-dependent endonuclease fused to the caged isolated peptide described above.

[0021] The details of one or more embodiments of the invention are set forth in the drawings and description below. Other features, objects, and advantages of the invention will be apparent from the drawings and description and from the claims.

[0022] BRIEF DESCRIPTION OF THE DRAWINGS

[0023] The foregoing and other objects, features and advantages will be apparent from the following description of particular embodiments of the disclosure, as illustrated in the accompanying drawings.

[0024] FIG. 1 A is a diagram showing how a protein fused to a caged C-degron can undergo controlled protein degradation upon activation by light. AAAA (SEQ ID NO: 1)

[0025] FIG. IB shows diagrammatically the lifespan control of an intracellularly delivered Cas9 protein by intracellular protein degradation machinery. IPD can be achieved through fusion with caged C-degrons.

[0026] FIG. 1C shows the removal by exposure to light of a 4-methoxy-7-nitroindolinyl (MNI) caging group from an alanine residue of a C-degron peptide.

[0027] FIG. 2A is a bar graph of relative light units (RLU) from Luciferase assays of HEK293T cells transfected with luciferase-degron fusion constructs and treated with vehicle (DMSO) or with the proteasome inhibitor MG-132. RRRG (SEQ ID NO: 2); ARRG (SEQ ID NO: 3); RARG (SEQ ID NO: 4); RRAG (SEQ ID NO: 5); RRRA (SEQ ID NO: 6); RAAG (SEQ ID NO: 7), AAAG (SEQ ID NO: 8), AAAA (SEQ ID NO: 1).

[0028] FIG. 2B shows a Western blot analysis of degron candidate screening in which constructs including the indicated degron sequence fused to the C-terminus of FL AG- tagged Cas9 were transfected into HEK293T cells and extracts analyzed with anti -FLAG and anti-GAPDH. RRRG (SEQ ID NO: 2); ARRG (SEQ ID NO: 3); RARG (SEQ ID NO: 4); RRAG (SEQ ID NO: 5); RRRA (SEQ ID NO: 6); AAAA (SEQ ID NO: 1), KGGR (SEQ ID NO: 9); RKRG (SEQ ID NO: 10); RRKG (SEQ ID NO: 11); RRRK (SEQ ID NO: 12); and KKKK (SEQ ID NO: 13).

[0029] 3

[0030] 4914-1459-3110, v. 1 BC2025.003.niu

[0031] FIG. 2C shows fluorescence images of HEK293T cells transfected with constructs expressing wild-type green fluorescent protein (GFP(wt)) or GFP fused to AAAA (SEQ ID NO: 1) (GFP AAAA).

[0032] FIG. 2D shows flow-cytometry results of the same cells described in FIG. 2C.

[0033] FIG. 3 A shows a Western blot analysis of HEK293T cells transfected with the indicated FLAG-tagged Cas9 constructs in which a linker region between Cas9 and the C-terminal AAAA sequence was varied. G4SA4 (SEQ ID NO: 14), G4SGSA4 (SEQ ID NO: 15), G4SGSGSA4 (SEQ ID NO: 16), G4SGSGSGSA4 (SEQ ID NO: 17), G4SGSGSGSGSA4 (SEQ ID NO: 18).

[0034] FIG. 3B shows a Western blot analysis of HEK293T cells transfected with the indicated FLAG-tagged Cas9 constructs in which the length of the C-terminal poly alanine was varied (a linker region between Cas9 and the C-terminal AAAA sequence was varied. G4SA4 (SEQ ID NO: 14), G4SGSGSA4 (SEQ ID NO: 16), G4SGSGSA5 (SEQ ID NO: 19), G4SGSGSA6 (SEQ ID NO: 20).

[0035] FIG. 3C shows a Western blot analysis of HEK293T cells transfected with the indicated FLAG-tagged Cas9 constructs in which lysine residues were included in the degron sequence. Cell extracts were analyzed at the indicated times after transfection. G4SK4A4 (SEQ ID NO: 21), G4SA4 (SEQ ID NO: 14).

[0036] FIG. 4 shows a synthetic route to protect the C-terminal arginine with MNI as the caging molecule.

[0037] FIG. 5 shows Liquid Chromatographs of MNLcaged peptide G4SK4A5 (SEQ ID NO: 32) after exposure to UV light for 1 min. or 5 min.

[0038] FIG. 6 is a fluorescence photograph of an SDS-PAGE gel showing successful in vitro ligation of Cas9_LPETG_6His to FAM-labelled degron peptide GGGSRRO(Fam)G using recombinant eSortaseA enzyme purified from E. coli.

[0039] FIG. 7A shows a Western blot analysis of HEK293T cells not transfected (N.T.) or transfected with constructs expressing FLAG-tagged wild-type Cas9, catalytically inactive Cas9 (dCas9), or the indicated Cas9-degron fusion constructs. G4SGSA4 (SEQ ID NO: 15), G4SGSA3T (SEQ ID NO: 22).

[0040] FIG. 7B shows flow cytometry of U2OS GFP cells not transfected (N.T.) or transfected with the indicated Cas9 construct plus an sgRNA targeting GFP performed

[0041] 4

[0042] 4914-1459-3110, v. 1 BC2025.003.niu

[0043] 24 h or 48 h after transfection. G4SGSA4(SEQ ID NO: 15), G4SGSA3T (SEQ ID NO: 22).

[0044] FIG. 8A shows a Western blot analysis of the indicated FLAG-tagged Cas9 proteins in U2OS GFP cells and in HEK293T cells. G4SA4 (SEQ ID NO: 14), G4SGSA4(SEQ ID NO: 15).

[0045] FIG. 8B shows flow cytometry of the transfected U2OS GFP cells. G4SGSA4 (SEQ ID NO: 15).

[0046] DETAILED DESCRIPTION

[0047] As summarized above, an isolated peptide is provided for regulating intracellular protein degradation. The peptide can be, but is not limited to, 9 to 19 amino acids in length, e.g., 9, 10, 11, 12, 13, 14 ,15, 16, 17, 18, and 19 amino acids.

[0048] The isolated peptide includes at its C-terminus a C-end degron peptide having 4 to 6 consecutive alanine residues, i.e., 4, 5, or 6 consecutive alanine residues extending to the C-terminus. A preferred isolated peptide has 4 consecutive alanine residues at its C- terminus.

[0049] The isolated peptide has at its N-terminus a linker sequence fused to the C-end degron peptide and having the sequence G4X in which X is S(GS)n, n being 0 to 4. Exemplary linker sequences include G4S (SEQ ID NO: 23), G4SGS (SEQ ID NO: 24), G4SGSGS (SEQ ID NO: 25), G4SGSGSGS (SEQ ID NO: 26), and G4SGSGSGSGS (SEQ ID NO: 27). Preferably, the linker sequence is G4S (SEQ ID NO: 23) or G4SGS (SEQ ID NO: 24).

[0050] Preferred isolated peptides have the amino acid sequence G4SA4 (SEQ ID NO: 14) and G4SGSA4 (SEQ ID NO: 15) and are free of any lysine residues.

[0051] Any of the above-described isolated peptides can be a caged peptide by including a caging molecule bonded to the carboxyl group of the C-terminal alanine residue. The caging molecule can be removed from the isolated peptide by exposure to light, to reactive oxygen species, to low or high pH, or to glycosidase digestion.

[0052] Caging molecules that can be removed by light contain, among other chemical groups, a 4-methoxy-7-nitroindolinyl (MNI) caging group, a 7-N,N-diethyl

[0053] 5

[0054] 4914-1459-3110, v. 1 BC2025.003.niu aminocoumarin (DECM) caging group, or a boron-dipyrromethane (BODIPY)-derived caging group. Preferably, the caging group is MNI.

[0055] Other isolated peptides include caging molecules that are compounds of

[0056] Formula (I): in which R is sensitive to reactive oxygen species (ROS), pH, or glycosidases.

[0057] Exemplary R moieties include the following:

[0058] ROS responsive

[0059] Also disclosed is a method for temporal control of protein degradation that relies on fusing a protein of interest at its C-terminus to one of the isolated peptides described above containing a caging molecule.

[0060] To produce this fusion protein, the protein of interest is first fused to a sortase recognition sequence. The sortase recognition sequence is LPXTG (SEQ ID NO: 28), X being any amino acid except proline. In a specific example, the sortase recognition sequence is LPETG (SEQ ID NO: 29). The fusion protein can be produced by any methods known in the art, e.g., by constructing a plasmid DNA that encodes the fusion protein and expressing and purifying the fusion protein from E. coli.

[0061] The protein of interest fused to the sortase recognition site is reacted in vitro with a sortase enzyme and any of the isolated peptides having a caging molecule described above to form a C-terminal caged fusion protein. The sortase can be, e.g., Sortase A (SrtA) or eSortase (eSrtA), preferably eSrtA.

[0062] To achieve temporal control of protein degradation, the C-terminal caged fusion protein is introduced into a cell by means known in the art, for example, transfection or electroporation. To initiate protein degradation, the cell carrying the C-terminal caged fusion protein is exposed to a stimulus that removes the caging molecule. The stimulus, which is given at a desired time after introducing the C-terminal caged fusion protein into the cell, can be any of those mentioned above, for example, light, reactive oxygen species, low pH, high pH, or glycosidase activity. The stimulus is selected based on

[0063] 6

[0064] 4914-1459-3110, v. 1 BC2025.003.niu which caging group was used to form the C-terminal caged fusion protein. In a particular embodiment, the caging molecule contains an MNI group and the stimulus is ultraviolet light.

[0065] The stimulus leads to the removal of the caging molecule to form an uncaged fusion protein. The uncaged fusion protein is degraded by cellular protein degradation machinery.

[0066] Based on the above-described embodiments, a method is provided for high- fidelity gene editing by an RNA-dependent endonuclease. The RNA-dependent endonuclease is modified at the C-terminus to include any of the caged isolated peptides described, supra. In a particular method, the RNA-dependent endonuclease is CRISPR- associated Protein 9 (Cas9) and the caged isolated peptide is G4SA4-MNI or G4SGSA4- MNI.

[0067] To achieve high-fidelity gene editing, the modified RNA-dependent endonuclease and a guide RNA are introduced into a cell, the guide RNA having a sequence that hybridizes to a target gene, followed by culturing the cell for a time period that allows gene editing of the target gene. The time period can be 24 h to 48 h (e.g., 24 h, 30 h, 36 h, 42 h, and 48 h).

[0068] To reduce possible off-target effects by degrading the RNA-dependent endonuclease, the cultured cell is exposed to a stimulus that removes the caging molecule from the modified RNA-dependent endonuclease. As set forth above, the stimulus can be light, e.g., ultraviolet light, reactive oxygen species, low pH, high pH, or glycosidase activity. In a preferred method, the caging molecule contains an MNI group and the stimulus is ultraviolet light.

[0069] Exposing the cell to the stimulus induces de-caging and subsequent degradation of the modified RNA-dependent endonuclease by intracellular protein degradation pathways, thereby reducing or eliminating off-target gene editing events.

[0070] Finally, a fusion protein for high-fidelity gene editing is provided. The fusion protein includes an RNA-dependent endonuclease, for example, Cas9, fused to the caged isolated peptide described above, preferably fused to G4SA4-MNI or G4SGSA4-MNI.

[0071] Without further elaboration, it is believed that one skilled in the art can, based on the above description, utilize the present invention to its fullest extent. The following

[0072] 7

[0073] 4914-1459-3110, v. 1 BC2025.003.niu specific examples are, therefore, to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever. All publications cited herein are incorporated by reference.

[0074] EXAMPLES

[0075] Example 1: Cloning and site mutagenesis.

[0076] Expression constructs for Cas9-degron fusions were cloned for mammalian expression via Gibson cloning using HiFi Assembly mix (NEB) according to the manufacturer’s instructions. For all Gibson cloning, 2 pl of assembled reaction was transformed into 20 pl of competent DH5alpha cells and plated on agar plates supplemented with appropriate antibiotics. After growth overnight at 37°C, colonies were picked into LB medium and incubated with shaking at 37°C for 24 h. Cultures were collected using a QIAprep Spin Miniprep kit (Qiagen) according to the manufacturer’s instructions.

[0077] Example 2: Cell culture.

[0078] All cells were cultured with standard protocols at 37°C with 5% CO2. HEK293T cells (ATCC) used in transfection experiments were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) (Gibco) supplemented with 10% Fetal Bovine Serum (Corning) and 1% penicillin-streptomycin-glutamine (Thermo Fisher).

[0079] U2OS GFP cells stably integrated with the GFP gene were maintained in DMEM (GIBCO) supplemented with 10% FBS, l x penicillin-streptomycin-glutamax (Thermo Fisher). Cells were continuously maintained at <90% confluency. Example 3: Transfection.

[0080] Cells were plated at 5,000-15,000 cells the day before transfection in a 6-well plate for western blot or 48-well for flow cytometry. HEK293T cells were transfected with polyethyleneimine and U2OS GFP cells were transfected with Lipofectamine 3000 (Thermo Fisher Scientific), respectively, according to the manufacturer’s specifications. Example 4: Western blot analyses.

[0081] Cells transiently expressing different Cas9_degron constructs were incubated for 24 h or 48 h with or without the proteasome inhibitor MG-132 for 8 h before harvesting.

[0082] 8

[0083] 4914-1459-3110, v. 1 BC2025.003.niu

[0084] Cell suspensions were spun down at 1,000 x g for 5 min. and processed according to standard Western Blot protocols.

[0085] Briefly, cells were resuspended in RIPA buffer (Thermo Fisher) with protease inhibitor cocktail (Thermo Fisher) and incubated at 4°C for 10 min. Then cell suspensions were agitated for 10 min. at 4°C and spun down at 16,000 x g for 15 min. at 4°C. The supernatants were transferred to fresh tubes and processed for immunoblotting.

[0086] An anti -Flag M2 monoclonal antibody (Fl 804, Sigma- Aldrich) was used to detect Cas9-FLAG expression followed by horseradish peroxidase (HRP)-conjugated antimouse HRP. The HRP antibody was detected with the Signal detection kit (Bio-Rad) according to the manufacturers protocol.

[0087] Example 5: Bacterial Expression of eSortase A

[0088] E. coli BL21(DE3) transformed with pET29 sortase expression plasmids (Addgene #75144) were cultured at 37°C in LB medium with 100 pg / mL kanamycin until OD600 = 0.6. IPTG was added to a final concentration of 0.4 mM and protein expression was induced overnight at room temperature. The cells were harvested by centrifugation and resuspended in lysis buffer (50 mM Tris pH 8.0, 300 mM NaCl supplemented with 1 mM MgCh, 2 units / mL DNAsel (NEB), 260 nM aprotinin, 1.2 pM leupeptin, and 1 mM PMSF). Cells were lysed by sonication and the clarified supernatant was purified on Ni-NTA agarose following the manufacturer’s instructions. Fractions that were >95% pure, as judged by SDS-PAGE, were consolidated and dialyzed against Tris-buffered saline (25 mM Tris pH 7.5, 150 mM NaCl).

[0089] Example 6: Analysis of Cas9 nuclease activity via disruption of genomic GFP with U2OS GFP stable cell line.

[0090] Approximately 200,000 U2OS GFP cells were transfected using Lipofectamine 3000 in duplicate with 100 ng of Cas9 (with or without degron) and sgRNA targeting GFP according to the manufacturer’s protocol. Approximately 30,000 transfected cells per well in five replicates were plated in a 48-well plate (Coming 3904 clear-bottom) and incubated for 24 or 48 h. Cells were collected and the GFP signal was quantified by flow cytometry.

[0091] 9

[0092] 4914-1459-3110, v. 1 BC2025.003.niu

[0093] Results

[0094] Degron Candidate Screening

[0095] C-terminal degrons (C-degrons) play a crucial role in maintaining cellular homeostasis by ensuring the timely degradation of proteins that are no longer needed or are damaged. The regulation of protein degradation through C-degrons has functional implications for various cellular processes, including cell cycle progression, signal transduction, and response to cellular. The core recognition motif for the so-called C-end rules pathway is the R / KXXR / K peptide sequence, where R is arginine, K is lysine, and X can be any amino acid. To investigate the ideal C-degron for protein lifetime control and rapidly select the best degron candidates, a luciferase reporter system was developed by fusing the luciferase gene with various C-degron motifs. The luciferase reporter plasmids were transfected into HEK293T cells and luminescence was quantified and compared between different degron candidates. The results are shown in FIG. 2A.

[0096] Surprisingly, a short amino acid sequence consisting of four alanine residues (tetra-alanine; AAAA SEQ ID NO: 1) was the most effective for degrading the fused luciferase protein. The degradation was blocked in the presence of proteasome inhibitor MG132, demonstrating that the degradation of degron-fused luciferase proteins indeed occurs through a proteasome-mediated pathway.

[0097] The potency of the tetra-alanine sequence in facilitating the degradation of fused- luciferase proteins was tested in the context of Cas9-degron constructs. HEK293T cells were transiently transfected with various Cas9-degron constructs harboring the tetraalanine sequence and other potential degron sequences and subsequently collected for analysis. Immunoblotting assays were then performed to assess the protein levels and degradation dynamics of the Cas9_degron constructs. The results are shown in FIG. 2B.

[0098] The results confirmed that inclusion of the short peptide tetra-alanine at the C-terminus led to efficient intracellular degradation of the Cas9 protein and unexpectedly showed that canonical C-end degron sequences such as KGGR (SEQ ID NO: 9), RRRK (SEQ ID NO: 12), and KKKK (SEQ ID NO: 13) were less efficient for inducing protein degradation than the tetra-alanine sequence. See FIG. 2B.

[0099] These results was confirmed in another experimental system by fusing the tetraalanine degron to GFP and transfecting the resulting construct into HEK293T cells. The

[0100] 10

[0101] 4914-1459-3110, v. 1 BC2025.003.niu results, shown in Figs 2C and 2D, confirmed that the tetra-alanine sequence effectively led to intracellular degradation of the fused GFP protein.

[0102] Cas9 Degron Construct Optimization

[0103] After determining the efficacy of the tetra-alanine sequence in enhancing the degradation of fused proteins, i.e., luciferase, GFP, and Cas9, further optimization was undertaken by systematically evaluating a range of glycine-serine (GS) linkers inserted between Cas9 and the tetra-alanine sequence. The results are shown in FIG. 3 A.

[0104] Varying the lengths of GS linkers exerted diverse effects on the degradation efficiency of Cas9. Unexpectedly, the insertion of an additional GS linker between Cas9G4S and tetra-alanine (construct Cas9G4SGSA4) resulted in the lowest protein levels observed, indicative of the constructs most sensitive to protein degradation.

[0105] These results highlight the importance of fine-tuning linker composition in Cas9_degron fusion strategies to achieve desired outcomes in various applications.

[0106] The influence of the alanine repeat length was examined next. Cas9_degron fusion proteins were prepared having 4, 5, or 6 alanine resides at the C-terminus and separated from the G4 by the optimal SGSGS (SEQ ID NO: 30) linker sequence. The results are shown in FIG. 3B. The tetra-alanine sequence was the most efficient at inducing protein degradation, although degradation was still observed with 5 or 6 alanine residues. However, increasing the length of the alanine repeat could significantly lower protein solubility.

[0107] Further, the effect of inserting lysine residues on Cas9 degradation efficiency was examined, as the lysine insertion should significantly increase the solubility of the fusion proteins. Surprisingly, insertion of four lysine residues between Cas9G4S and tetraalanine led to significantly lower degradation efficiency, as compared to the Cas9G4SA4 fusion protein. See FIG. 3C.

[0108] Caged Degron and De-caging

[0109] For the purpose of temporally or conditionally inhibiting the function of a C- terminal degron, blocking the end with a caging molecule prevents the targeted degradation of the therapeutic protein until the cage is removed, allowing control over the timing or circumstances under which a therapeutic protein is targeted for degradation. For example, photo-caging groups, which can be activated by light, can be used for this

[0110] 11

[0111] 4914-1459-3110, v. 1 BC2025.003.niu purpose. The caged C-degron is inactive, preventing its recognition by the intracellular protein degradation machinery, until the caging group is removed by light. For C-degron tetra-alanine, the C-terminal free carboxylic acid was found crucial for the degron activity. Thus, caging strategies were developed to control the activity of the tetraalanine C-degrons.

[0112] The 4-methoxy-7-nitroindolinyl (MNI) group was tested to cage the C-terminal free carboxylic acid of alanine. The MNI and related nitroindoline caging groups have been used previously to generate caged derivatives of glutamate, glycine, and alanine and the mechanism and kinetics of product release from these caging groups have been studied. The rate constant for amino acid release is 5 * 106s'1. The reaction of the MNI group with a C-terminal alanine residue is shown in FIG. 4.

[0113] The de-caging reaction was tested in vitro on MNI-caged degron peptides in PBS buffer and monitored by LCMS. The results are shown in FIG. 5. The degron peptides were fully uncaged, i.e., the MNI group was removed, after 5 min. exposure to a handheld UV light. eSrtA Catalyzed Ligation of MNI-Caged Degron and Cas9

[0114] To take advantage of the caged degron to implement a photocaged Cas9 system, the caged degron must be fused to the Cas9 protein.

[0115] Sortase A, a transpeptidase enzyme catalyzes peptide ligation reactions. This enzyme functions by recognizing a specific amino acid sequence motif, LPXTG (SEQ ID NO: 28) where X can be any amino acid except proline. It cleaves the peptide bond between the threonine (T) and glycine (G) residues of the LPXTG motif and subsequently attaches the carboxyl group of the threonine residue to the amino group of the peptide of interest.

[0116] Evolved sortase A (eSrtA) pentamutant with improved kinetics and activity was chosen to ligate a degron peptide to the Cas9 protein. First, a Cas9 fusion protein that includes the amino acid sequence LPETG was created using standard recombinant techniques. The C-terminal sequence of the Cas9 / sortase site fusion protein (Cas9_LPETG) is TGLYETRIDLSQLGGDLPETGGHHHHHH (SEQ ID NO: 31; SortaseA recognition site in bold). The terminal 6 histidine residues were added to facilitate purification of the fusion protein.

[0117] 12

[0118] 4914-1459-3110, v. 1 BC2025.003.niu

[0119] Initially, a FAM-modified degron peptide was used for a ligation reaction to facilitate the visualization and tracking of the ligation process. The FAM-modified degron peptide was subjected to ligation with recombinant purified Cas9_LPETG protein, with the reaction allowed to proceed at room temperature. Following the completion of the ligation process, the reaction products were subjected to analysis using SDS-PAGE. The result is shown in FIG. 6. The analysis confirmed the successful ligation of the FAM-modified degron peptide to the Cas9_LPETG protein, as evidenced by the appearance of the desired fluorescent band corresponding to the ligated product.

[0120] Ligation of the MNI-caged tetra-alanine degron (G4SGSGSA4-MNI) to Cas9_LPETG was also carried out successfully under the same ligation conditions, as confirmed by mass spectrometry.

[0121] Analysis of Cas9 Degron Nuclease Activity via Disruption of Genomic GFP

[0122] Following the successful ligation of the degron peptide to the Cas9 protein, the activity and efficiency of Cas9_degron-mediated genome editing process was evaluated. To this end, U2OS GFP cells engineered to harbor a GFP reporter gene were transfected with Cas9_Degron proteins, along with a single guide RNA (sgRNA) specifically designed to target the genomic GFP constructs. By precisely directing the Cas9_Degron to the desired genomic loci containing the GFP gene, targeted DNA cleavage and subsequent genetic modifications could be monitored.

[0123] First, constructs expressing wild-type Cas9, dCas9 (enzymatically inactive) Cas9- G4SGSA4 degron fusion, and Cas9-G4SGSA3T were transfected into HEK293T cells to assess protein stability by immunoblot analysis. The results are shown in FIG. 7A. As expected, the Cas9_Degron fusion was the least stable. Notably, changing the last alanine residue to a threonine resulted in diminished degradation of the resulting Cas9 fusion protein.

[0124] The same constructs were introduced into the U2OS GFP cells together with the sgRNA targeting the integrated GFP gene. The results are shown in FIG. 7B. As expected, non -transfected control cells and cells transfected with dCas9 showed no reduction in the number of GFP-expressing cells, as measured by flow cytometry. Cells transfected with Cas9_degron construct Cas9-G4SGSA4 also failed to show a reduction in GFP-expressing cells, likely due to the lower stability of this fusion protein.

[0125] 13

[0126] 4914-1459-3110, v. 1 BC2025.003.niu

[0127] Expression of both wild type Cas9 and Cas9-G4SGSA3T resulted in a significant decrease in GFP-expressing cells, as evidenced by the appearance of a second population of cells having lower fluorescence.

[0128] In addition, certain Cas9 constructs were transfected in parallel into U2OS GFP cells and HEK293T cells to assess protein stability in these two different cell lines. The results are shown in FIG. 8A. The stabilities of Cas9 and Cas9-Degron constructs were similar in the two cell lines, with an overall higher expression level in the HEK293T cells. Transfection into U2OS GFP cells of the constructs together with the sgRNA targeting GFP was also performed and the results shown in FIG. 8B. The reduction in GFP-expressing cells was higher in the Cas9-expressing cells, as compared to cells expressing Cas9G4SGSA4.

[0129] The development and evaluation of precision longevity control of intracellularly delivered Cas9 by light should be a generally applicable platform for any therapeutic proteins of interest. Photo-uncaging provides precise spatial and temporal control over the release of the active degron. By focusing the light on specific regions of interest, such as individual cells or subcellular compartments, the target therapeutic proteins degradation can be manipulated with high precision. The ability to precisely regulate the lifespan of therapeutic proteins like Cas9 within cells enables tailored modulation of protein activity, minimizing off-target effects and enhancing therapeutic efficacy.

[0130] OTHER EMBODIMENTS

[0131] All of the features disclosed in this Specification may be combined in any combination. Each feature disclosed in this Specification may be replaced by an alternative feature serving the same, equivalent, or similar purpose. Thus, unless expressly stated otherwise, each feature disclosed is only an example of a generic series of equivalent or similar features.

[0132] From the above description, one skilled in the art can easily ascertain the essential characteristics of the present invention, and without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various usages and conditions. Thus, other embodiments are also within the scope of the following claims.

[0133] 14

[0134] 4914-1459-3110, v. 1

Claims

BC2025.003.niuCLAIMS1. An isolated peptide for regulating intracellular protein degradation, the isolated peptide comprising, from N-terminus to C-terminus, a linker sequence G4X fused to a C-end degron peptide, wherein X is S(GS)n, n being 0 to 4, the C-end degron peptide comprises 4 to 6 consecutive alanine residues and the isolated peptide is free of any lysine residues.

2. The isolated peptide of claim 1, wherein the C-end degron peptide has the amino acid sequence AAAA and the linker sequence is G4S (SEQ ID NO: 23) or G4SGS (SEQ ID NO: 24).

3. The isolated peptide of claim 1 or 2, wherein the isolated peptide is 9 to 19 amino acids in length.

4. The isolated peptide of any one of claims 1 to 3, wherein the isolated peptide has at the C-terminus an alanine residue that is modified by a caging molecule bonded to the carboxyl group of the C-terminal alanine residue.

5. The isolated peptide of claim 4, wherein the caging molecule can be removed by exposure to light, to reactive oxygen species, to low or high pH, or to glycosidase digestion.

6. The isolated peptide of claim 5, wherein the caging molecule contains a 4- methoxy-7-nitroindolinyl (MNI) caging group, a 7-N,N-diethyl aminocoumarin (DECM) caging group, or a boron-dipyrromethane (BODIPY)-derived caging group.154914-1459-3110, v. 1BC2025.003.niu7. The isolated peptide of claim 5, wherein the caging molecule is a compound of Formula (I):

8. A method for temporal control of protein degradation, the method comprising: fusing a protein of interest at its C-terminus to a sortase recognition sequence to form a fusion protein; reacting the fusion protein in vitro with a sortase enzyme and the isolated peptide of any one of claims 4 to 7 to form a C-terminal caged fusion protein; introducing the C-terminal caged fusion protein into a cell; and exposing the cell to a stimulus, thereby removing the caging molecule to form an uncaged fusion protein, whereby the uncaged fusion protein is degraded by cellular protein degradation machinery.

9. The method of claim 8, wherein the sortase is Sortase A (SrtA) or eSortase (eSrtA).

10. The method of claim 8 or 9, wherein the sortase recognition sequence is LPXTG (SEQ ID NO: 28), X being any amino acid except proline.

11. The method of any one of claims 8 to 10, wherein the stimulus is light, reactive oxygen species, low pH, high pH, or glycosidase activity.4914-1459-3110, v. 1BC2025.003.niu12. The method of any one of claims 8 to 11, wherein the caging molecule contains an MNI group and the stimulus is ultraviolet light.

13. A method for high-fidelity gene editing, the method comprising: obtaining a modified RNA-dependent endonuclease having at the C-terminus the isolated peptide of any one of claims 4 to 7; introducing the modified RNA-dependent endonuclease and a guide RNA into a cell, the guide RNA having a sequence that hybridizes to a target gene; culturing the cell for a time period that allows gene editing of the target gene; and exposing the cultured cell to a stimulus that removes the caging molecule from the modified RNA-dependent endonuclease, thereby inducing degradation of the modified RNA-dependent endonuclease and reducing or eliminating off-target gene editing events.

14. The method of claim 13, wherein the RNA-dependent endonuclease is CRISPR-associated Protein 9 (Cas9).

15. The method of claim 13 or 14, wherein the culturing time period is 24 to 48 hours.

16. The method of any one of claims 13 to 15, wherein the stimulus is light, reactive oxygen species, low pH, high pH, or glycosidase activity.

17. The method of any one of claims 13 to 16, wherein the caging molecule contains an MNI group and the stimulus is ultraviolet light.

18. A fusion protein for high-fidelity gene editing, comprising an RNA- dependent endonuclease fused to the isolated peptide of any one of claims 4 to 7.

19. The fusion protein of claim 18, wherein the RNA-dependent endonuclease is Cas9.174914-1459-3110, v. 1BC2025.003.niu20. The fusion protein of claim 19, wherein the caging molecule contains an MNI group.4914-1459-3110, v. 1

Images