Anti-crispr delivery compositions and methods

Abstract

Described in certain example embodiments herein are engineered Anti-CRISPR (Acr) polypeptides having an Acr polypeptide and one or more cell -penetrating peptides (CPPs) operatively coupled to the Acr polypeptide. Also described in several example embodiments herein are methods of producing and purifying Acr polypeptides and delivering engineered Acr polypeptides to a cell or cell population.

Description

ANTI-CRISPR DELIVERY COMPOSITIONS AND METHODSCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Application Nos. 63 / 653,105, filed May 29, 2024 and 63 / 717,502, filed November 7, 2024. The contents of the above-identified applications are hereby incorporated herein by reference in their entireties.STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

[0002] This invention was made with government support under Grant No. GM137606 and GM1 32825 awarded by the National Institutes of Health. The government has certain rights in the invention.REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

[0003] The contents of the electronic sequence listing (“BROD-5665WP_ST26.xml”; Size is 313,798 bytes and it was created on May 23, 2025) is herein incorporated by reference in its entirety.TECHNICAL FIELD

[0004] The subject matter disclosed herein is generally directed to compositions, systems, and methods for delivering Anti-CRISPR molecules to targets, such as target cells.BACKGROUND

[0005] Clustered regularly interspaced short palindromic repeats (CRISPR)-associated systems (Cas) are in development for laboratory research, agricultural engineering, various biotechnology endeavors, and treatment of a variety of genetic disorders. In particular, Class 2 systems (e g., Cas9 and Casl2a systems) have been engineered to treat blood and muscle disorders, such as Sickle cell disease and Duchenne muscular dystrophy. Despite the rapid progress in basic research and clinical tests, some underlying problems present continuous, significant challenges, such as editing efficiency, relative difficulty in delivery, off-target effects, and immunogenicity, among others. Off-target effects observed with Cas systems include chromosomal rearrangements, disruption of essential genes in non-target cells, and others. It has been observed that Cas-systemsmay be more prone to these off-target effects than other conventional gene-editing methods because the Cas protein is a monomer that can facilitate recognition of shorter target sequences in comparison with transcription activator-like effector nucleases (TALENs) or Zinc Finger Nucleases (ZFNs), whose assemblies are dimeric.

[0006] To counter the off-target effects, several methods have been utilized, including optimization of the RNA guide sequences and engineering Cas proteins to improve system specificity and minimize the chances of causing off-target activity. Another approach is the inhibition of Cas protein activity, particularly by anti-CRISPR (Acr) proteins, small molecule inhibitors, and nucleic acid-based inhibitors. Acrs are being intensely developed as they offer a highly potent approach due to possessing several CRISPR-Cas interaction sites and function through a variety of mechanisms, such as binding of Cas with or without the target nucleic acid or modifying Cas to prevent its nuclease activity. These features of Acrs may make it possible to control Cas activity and improve targeting specificity. However, and like Cas proteins, delivery of Acrs has proved a significant challenge due to, inter alia, their ability to stimulate a host immune response and, due to their size, an inability to freely diffuse through a cell membrane. As such, there exists a need for alternative delivery compositions and methods of Acrs.

[0007] Citation or identification of any document in this application is not an admission that such a document is available as prior art to the present invention.SUMMARY

[0008] Described in an embodiment herein is an engineered Anti-CRISPR (Acr) polypeptide including: an Acr polypeptide; and one or more cell-penetrating peptides (CPPs) operatively coupled to the Acr polypeptide.

[0009] In an embodiment, the compositions described herein relate to an engineered Acr polypeptide, wherein one or more of the one or more cell-penetrating peptides includes or consists of one or more nuclear localization signals (NLSs).

[0010] In an embodiment, the compositions described herein relate to an engineered Acr polypeptide, wherein the one or more CPPs are operatively coupled to an N-terminus, a C- terminus, the N-terminus and the C-terminus of the Acr polypeptide, and / or to one or more amino acids between the N-terminus and C-terminus of the Acr polypeptide.

[0011] In an embodiment, the compositions described herein relate to an engineered Acr polypeptide, wherein 1-10 CPPs are operatively coupled to the Acr polypeptide.

[0012] In an embodiment, the compositions described herein relate to an engineered Acr polypeptide, wherein 6 CPPs are operatively coupled to the Acr polypeptide.

[0013] In an embodiment, the compositions described herein relate to an engineered Acr polypeptide, wherein (a) 4 CPPs are operatively coupled to the N-terminus of the Acr polypeptide, (b) wherein 2 CPPs are operatively coupled to the C-terminus of the Acr polypeptide, or (c) both (a) and (b).

[0014] In an embodiment, the compositions described herein relate to an engineered Acr polypeptide, wherein the one or more CPPs are operatively coupled to the Acr polypeptide via a linker.

[0015] In an embodiment, the compositions described herein relate to an engineered Acr polypeptide, wherein the linker is a Gly-Ser linker.

[0016] In an embodiment, the compositions described herein relate to an engineered Acr polypeptide, wherein the linker is G4CG4S (SEQ ID NO: 1).

[0017] In an embodiment, the compositions described herein relate to an engineered Acr polypeptide, wherein the Acr polypeptide inhibits a Type I, Type II, Type III, Type V, or Type VI CRISPR-Cas system or component or activity thereof.

[0018] In an embodiment, the compositions described herein relate to an engineered Acr polypeptide, wherein the Acr polypeptide is selected from AcrIEl, AcrIE2, AcrIE3, AcrIE4, AcrIE5, AcrIE6, AcrIE7, AcrIE8, AcrIE8.1, AcrIE8.2, AcrIE9, AcrIFI, AcrIF2, AcrIF3, AcrIF4, AcrIF5, AcrIF6, AcrIF7, AcrIF8, AcrIF9, AcrIFlO, AcrIFl l, AcrIFl l. l, AcrIF11.2, AcrIF12, AcrIF13, AcrIF14, AcrIF15, AcrIF16, AcrIF17, AcrIF18, AcrIF19, AcrIF20, AcrIF21, AcrIF22, AcrIF23, AcrIF24, AcrIE4-F7, AcrlAI, AcrIBI, AcrIB2, AcrIB3, AcrIB4, AcrIB5, AcrIB6, AcrIB7, AcrIB9, AcrICI, AcrIF2 / C2, AcrIC3, AcrIC4, AcrIC5, AcrIC6, AcrIC7, AcrIC8, AcrIC9, AcrIClO, AcrICI 1, AcrIDl, AcrIIAl, AcrIIA2, AcrIIA2-l, AcrIIA2-2, AcrIIA2b, AcrIIA3, AcrIIA4, AcrIIA4-2, AcrIIA4-3, AcrIIA4 variant Ins. 5, AcrIIA4 variant N39A, AcrIIA4 variant D14A / G38A, Acrobat-AcrIIA4, AcrIIA4-dTAG, SMASh-AcrIIA4, AcrIIA5, AcrIIA5-2, AcrIIA6, AcrIIA7, AcrIIA8, AcrIIA9, AcrIIAlO, AcrIIAl 1, AcrIIA12, AcrIIA13, AcrIIA13b, AcrIIA14, AcrIIA15, AcrIIA16, AcrIIA17, AcrIIA18, AcrIIA19, AcrIIA20, AcrIIA21, AcrIIA22,AcrIIA23, AcrIIA24, AcrIIA25, AcrIIA26, AcrIIA27, AcrIIA28, AcrIIA29, AcrIIA30, AcrIIA31, AcrIIA32, AcrIIA33, AcrIIA33(Seq), AcrIIA34, AcrIICl, AcrIICl-1, AcrIIC2, AcrIIC3, AcrIIC4, AcrIIC5, AcrIIC6, AcrIIC7, AcrIIC8, AcrIIC9, AcrIII-1, AcrIIIBl, AcrVAl, AcrVA2, AcrVA3, AcrVA3.1, AcrVA4, AcrVA5, AcrVIAl, AcrVIAl(Lwa), AcrVIA2, AcrVIA2(Lwa), AcrVIA3, AcrVIA3(Lwa), AcrVIA4, AcrVIA5, AcrVIA6, AcrVIA7, AcrVIBl, Csx27, enAcr-1, ErAcr-310, a homologue thereof, an orthologue thereof, or any combination thereof

[0019] In an embodiment, the compositions described herein relate to an engineered Acr polypeptide, further including a targeting moiety operatively coupled to the Acr polypeptide.

[0020] In an embodiment, the compositions described herein relate to an engineered Acr polypeptide, further including a reporter molecule operatively coupled to the Acr polypeptide.

[0021] In an embodiment, the compositions described herein relate to an engineered Acr polypeptide, further including a secondary delivery enhancer molecule.

[0022] In an embodiment, the compositions described herein relate to an engineered Acr polypeptide, wherein the secondary delivery enhancer molecule is an endosomal escape peptide.

[0023] In an embodiment, the compositions described herein relate to an engineered Acr polypeptide, wherein the engineered Acr polypeptide has increased penetration across a lipid bilayer as compared to a wild-type Acr polypeptide.

[0024] In an embodiment, the compositions described herein relate to an engineered Acr polypeptide, wherein the lipid bilayer is a cell membrane.

[0025] In an embodiment, the compositions described herein relate to an engineered Acr polypeptide, where penetration across a lipid bilayer is increased 1-1,000,000,000 fold or more.

[0026] Described in an embodiment herein is a polynucleotide encoding an engineered Acr polypeptide of the present disclosure.

[0027] Described in an embodiment herein is a vector or vector system including a polynucleotide encoding one or more engineered Acr polypeptides of the present disclosure.

[0028] In an embodiment, the compositions described herein relate to a vector or vector system, wherein the vector system includes one or more vectors having a polynucleotide encoding one or more engineered Acr polypeptides of the present disclosure.

[0029] In an embodiment, the compositions described herein relate to a vector or vector system, further including one or more regulatory elements, wherein the one or more regulatoryelements is operatively coupled to the polynucleotide encoding one or more engineered Acr polypeptides.

[0030] Described in an embodiment herein is a delivery vehicle including (a) an engineered Acr polypeptide of the present disclosure; (b) a polynucleotide of the present disclosure; (c) a vector or vector system of the present disclosure; or (d) any combination of (a)-(c).

[0031] Described in an embodiment herein is a cell or cell population including (a) an engineered Acr polypeptide; (b) a polynucleotide; (c) a vector or vector system; (d) a delivery vehicle of the present disclosure; or (e) any combination of (a)-(d).

[0032] Described in an embodiment herein is a pharmaceutical formulation including: (a) an engineered Acr polypeptide; (b) a polynucleotide; (c) a vector or vector system; (d) a delivery vehicle of the present disclosure; (e) a cell or cell population; or (f) any combination of (a)-(e); and a pharmaceutically acceptable carrier.

[0033] Described in an embodiment herein is a kit including: (a) an engineered Acr polypeptide; (b) a polynucleotide; (c) a vector or vector system; (d) a delivery vehicle of the present disclosure; (e) a cell or cell population; (f) the pharmaceutical formulation; or (g) any combination of (a)-(f)-

[0034] Described in an embodiment herein is a method of delivering an anti-CRISPR (Acr) polypeptide to a cell including providing, to a cell or cell population, (a) an engineered Acr polypeptide of the present disclosure; (b) a polynucleotide encoding the engineered Acr polypeptide; (c) a vector or vector system of the present disclosure including the polynucleotide encoding the engineered Acr polypeptide; (d) a delivery vehicle of the present disclosure; (e) a cell or cell population of including (a)-(d) or any combination thereof; (f) a pharmaceutical formulation including (a)-(e) or any combination thereof and a pharmaceutically acceptable carrier; or (g) any combination of (a)-(f).

[0035] In an embodiment, the techniques described herein relate to a method, wherein the cell or cell population includes a CRISPR-Cas system or component thereof.

[0036] In an embodiment, the techniques described herein relate to a method, wherein an activity of the CRISPR-Cas system or component thereof is reduced or inhibited.

[0037] Described in an embodiment herein is a method of inhibiting activity of a CRISPR-Cas system in a cell including providing, to a cell or cell population a) an engineered Acr polypeptideof the present disclosure; (b) a polynucleotide encoding the engineered Acr polypeptide; (c) a vector or vector system of the present disclosure including the polynucleotide encoding the engineered Acr polypeptide; (d) a cell or cell population of including (a)-(c) or any combination thereof; (e) a pharmaceutical formulation including (a)-(d) or any combination thereof and a pharmaceutically acceptable carrier; or (f) any combination of (a)-(e).

[0038] In an embodiment, the techniques described herein relate to a method, wherein activity of a CRISPR-Cas system or a component thereof is reduced or inhibited by any non-zero percent to 100 percent.

[0039] Described in an embodiment herein is a method of in vitro protein production including(a) expressing a protein by culturing, in a first cell culture media, a cell population comprising one or more cells comprising an expression vector or expression vector system that comprises a polynucleotide encoding a protein, wherein the first cell culture media comprises a first amount of glucose; and (b) subculturing, in a second cell culture media, the cell population of (a), wherein the second cell culture media comprises a second amount of glucose. In an embodiment, (b) subculturing is discontinued when the ODeoo reaches 0.6-1.3. In some embodiments, the protein is a CRISPR-Cas protein, an Acr, an engineered Anti-CRISPR (Acr) polypeptide, or any combination thereof.

[0040] The first amount of glucose may range from any non-zero percent weight / volume (w / v) to about 5 percent w / v, optionally to about 2 percent w / v. The percent w / v of glucose is defined as the mass of glucose in grams divided by the volume of solution in mL and multiplied by 100. The second amount of glucose may range from any non-zero percent w / v to about 5 percent w / v; optionally, the second amount of glucose is about 2 percent w / v. The first cell culture media may be Lysogeny Broth and the second culture media may be Terrific Broth. The expression vector may comprise or consist of a T7 promoter-based expression vector or expression vector system. The T7 promoter-based expression vector or expression vector system may comprise or consist of a pET, 2CT, pMBP, pML-2CT, and pMal-C2 expression vector or expression vector system containing an MBP-TEV protease cleavage site fusion or a SUMO fusion, optionally a 4xNLS- pMJ915v2, pKEW-MBP-TEV, pAV-MBP-TEV, or pAV-SUMO expression vector or expression vector system.

[0041] The expression vector or expression vector system comprises or consists of a pET or pKEW expression vector or expression vector system, optionally a pET-TEV, pET MBP-TEV, pET-SUMO, pKEW-TEV, or pKEW-MBP-TEV expression vector or expression vector system.

[0042] The cell population may be a bacterial cell population. The cell population may be an E. coli cell population, optionally an E. coli Rosetta2 (DE3) cell population or an E. coli BL21 (DE3) cell population.

[0043] The method may further include (c) inducing protein production after (b) and further culturing the cell population. The method further includes harvesting and / or lysing the cell population after (b) or (c).

[0044] In an embodiment, the method further includes (d) purifying the protein. In an embodiment, the protein comprises a purification tag and purifying the protein comprises purification tag-based affinity purification. The purification tag may then be subsequently removed.

[0045] In an embodiment, the method comprises ion exchange chromatography, size exclusion chromatography, or both.

[0046] In an embodiment, the method does not comprise ion exchange chromatography.

[0047] In an embodiment, the method does not comprise size exclusion chromatography.

[0048] In an embodiment, the protein is any protein set forth in an embodiment, a Cas protein, or an engineered Anti-CRISPR (Acr) polypeptide of the present description.

[0049] In an embodiment, the protein yield of the method is increased 1, 2, 3, 4, 5, 6 or morefold.

[0050] These and other aspects, objects, features, and advantages of the example embodiments will become apparent to those having ordinary skill in the art upon consideration of the following detailed description of example embodiments.BRIEF DESCRIPTION OF THE DRAWINGS

[0051] An understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention may be utilized, and the accompanying drawings of which:

[0052] FIG. 1 - Shows an exemplary embodiment of a method that uses nuclear localization signals (NLSs) to deliver an anti-CRISPR-(Acr) polypeptide into the cytosol and nucleus of a target cell via an endocytic or non-endocytic mechanism.

[0053] FIG. 2A-2B - An exemplary construct for the production and purification of an engineered, cell-permeable Acr polypeptide in E. colt. FIG. 2A - A protein fusion between a polyhistidine-tagged maltose-binding protein (MBP, for solubility and purification), a TEV protease cleavage site, an array of four SV40 NLS tags (to endow cell permeability), a glycine- cysteine-serine linker (G4CG4S, for flexibility and optional bioconjugation), AcrIIA4 (for Cas9 inhibition), a glycine-serine GS linker (for flexibility), and an array of two SV40 NLS tags (to endow cell permeability). After isolating the 6*His-MBP-TEV-4><NLS-G4CG4S-AcrIIA4-GS- 2xNLS protein (SEQ ID NO: 1) via metal affinity chromatography, the MBP tag is cleaved using TEV protease and separated from 4xNLS-G4CG4S-AcrIIA4-GS-2xNLS (henceforth referred to as 6*NLS-AcrIIA4) (SEQ ID NO: 1) via affinity chromatography, ion exchange chromatography, and optionally size-exclusion chromatography. FIG. 2B - The sequence that encodes 6*His-MBP- TEV-4*NLS-G4CG4S-AcrIIA4-GS-2xNLS is in uppercase, and its domains are underlined differently: 6xHis- BP tag, TEV_site, 4xSV40 NLS, GATES (SEQ ID NO: 1) linker. AcrIIA4, GS linker, 2xSV40 NLS. The MBP-TEV-4xNLS-GS-2xNLS backbone plasmid was a gift from Jennifer Doudna (Addgene plasmid # 88917, Staahl et al., Nat. Biotechnol. 2017, 35, 431-434). The AcrIIA4 sequence was originally reported in Rauch et al., Cell 2017, 168, 150-158.

[0054] FIG. 3A-3C - Purification and characterization of a representative cell-permeable Acr polypeptide. FIG. 3A - SDS-PAGE gel of increasing concentrations of the 6xNLS-AcrIIA4 polypeptide after purification by affinity, cation exchange, and size-exclusion chromatography. The 6xNLS-AcrIIA4 band appears between 15 and 25 kDa. FIG. 3B - A cartoon of 6xNLS- AcrIIA4 and some of its physical properties determined using the Expasy ProtParam tool. FIG. 3C - Q-TOF Liquid chromatography-mass spectrum of 6xNLS-AcrIIA4 (18,454 Da) after purification by affinity, cation exchange, and size-exclusion chromatography.

[0055] FIG. 4 - Assessing the delivery of an engineered, cell-permeable Acr (e.g., 6xNLS- AcrIIA4) via the EGF -disruption assay. A CRISPR-Cas system (e.g., a Streptococcus pyogenes Cas9, SpCas9, ribonucleoprotein, RNP, complex) targeting the EGFP gene is delivered via nucleofection into U2OS-EGFP.PEST cells (300,000 cells). The cells are then resuspended inDMEM medium (supplemented with 10% FBS, 1 mM sodium pyruvate, and 100 U / mL penicillinstreptomycin) and transferred to a 96-well plate (100 pL final volume, 100,000 cells / mL) where they are incubated (37 °C) with the cell-permeable Acr (e.g., 6*NLS-AcrIIA4) 0-24 h post- nucleofection / resuspension and imaged at 48 h. Live cells are stained with Hoechst 33342 and imaged via confocal microscopy. Cas-mediated cleavage disrupts EGFP production. Successful delivery of the Acr prevents Cas-mediated EGFP disruption.

[0056] FIG. 5A-5B - The FGFF-disruption assay confirms that an engineered, cell- permeable Acr (e.g., 6><NLS-AcrIIA4) enters human cells in a dose-dependent manner. FIG. 5A - C-NLS-SpCas9 RNP (20 pmol) targeting the EGFP gene is delivered via nucleofection into U2OS-EGFP.PEST cells, followed by resuspension in DMEM medium (supplemented with 10% FBS, 1 mM sodium pyruvate, and 100 U / mL penicillin-streptomycin), addition of 6><NLS-AcrIIA4 (0.03-4 pM) 2 h post-nucleofection / resuspension, and imaging at 48 h. Positive controls include nucleofection of Apo-Cas9 and co-nucleofection (NF) of Cas9 RNP with Cys-AcrIIA4 (Acr, 20 pmol) or 6><NLS-AcrIIA4 (6xNLS-Acr, 20 pmol). Negative controls include nucleofection of Cas9 RNP and nucleofection of Cas9 RNP followed by addition of Cys-AcrIIA4 (Acr, 4 pM) 2 h post-nucleofection / resuspension. The data are normalized to Cas9 RNP and are the mean ± s.d. of three independent replicates. The significance of Cas9 RNP + 6xNLS-AcrIIA4 additions is determined with an unpaired, two-tailed t-test versus Cas9 RNP, where *, **, ***, and **** stand for P < 0.05, P < 0.01, P < 0.001, and P < 0.0001, respectively. FIG. 5B - Representative fluorescence microscopy images from the FGFF-disruption assay of FIG. 5A demonstrating the dose-dependent delivery of an engineered, cell-permeable Acr polypeptide (e.g., 6xNLS-AcrIIA4) into human cells. The nucleus is blue due to Hoechst 33342, and the cytosol is green due to EGFP.

[0057] FIG. 6A-6B - The FGFF-disruption assay confirms that an engineered, cell-permeable Acr (e.g., 6xNLS-AcrIIA4) enters human cells in a time-dependent manner. FIG. 6A - C-NLS- SpCas9 RNP (20 pmol) targeting the EGFP gene is delivered via nucleofection into U2OS- EGFP.PEST cells, followed by resuspension in DMEM medium (supplemented with 10% FBS, 1 mM sodium pyruvate, and 100 U / mL penicillin-streptomycin), addition of 6xNLS-AcrIIA4 (6xNLS-Acr, 1.5 pM) 2-6 h post-nucleofection / resuspension, and imaging at 48 h. Nucleofection of Apo-Cas9 is a positive control. Negative controls include nucleofection of Cas9 RNP and nucleofection of Cas9 RNP followed by addition of AcrIIA4 (Acr, 4 pM) 2 h post-nucleofection / resuspension. The data are normalized to Cas9 RNP and are the mean ± s.d. of three independent replicates. The significance of Cas9 RNP + 6*NLS-AcrIIA4 additions is determined with an unpaired, two-tailed t-test versus Cas9 RNP, where *, **, ***, and **** stand for P < 0.05, P < 0.01, P < 0.001, and P < 0.0001, respectively. FIG. 6B - Representative fluorescence microscopy images from the AGFP-disruption assay of FIG. 6A demonstrating the timedependent delivery of an engineered, cell-permeable Acr polypeptide (e.g., 6xNLS-AcrIlA4) into human cells. The nucleus is blue due to Hoechst 33342, and the cytosol is green due to EGFP.

[0058] FIG. 7 - The AG77J-disruption assay confirms that an engineered, cell-permeable Acr (e g., 6><NLS-AcrIIA4) enters human cells in a time-dependent manner. C-NLS-SpCas9 RNP (20 pmol) targeting the EGFP gene is delivered via nucleofection into U2OS-EGFP.PEST cells, followed by resuspension in DMEM medium (supplemented with 10% FBS, 1 mM sodium pyruvate, and 100 U / mL penicillin-streptomycin), addition of 6xNLS-AcrIIA4 (6xNLS-Acr, 1 pM) 0-24 h post-nucleofection / resuspension, and imaging at 48 h. Nucleofection of Apo-Cas9 is a positive control. Negative controls include nucleofection of Cas9 RNP and nucleofection of Cas9 RNP followed by addition of Cys-AcrIIA4 (Acr, 4 pM) 2 h post-nucleofection / resuspension. The data are normalized to Cas9 RNP and are the mean ± s.d. of three independent replicates. The significance of Cas9 RNP + 6xNLS-AcrIIA4 additions is determined with an unpaired, two-tailed t-test versus Cas9 RNP, where *, **, ***, and **** stand for P < 0.05, P < 0.01, P < 0.001, and P < 0.0001, respectively.

[0059] FIG. 8A-8B - The EGFP-disruption assay confirms that an engineered, cell-permeable Acr (e.g., 6xNLS-AcrIIA4) enters human cells in a dose-dependent manner in the presence of a pore-forming protein (e.g., protective antigen, PA). FIG. 8A - C-NLS-SpCas9 RNP (20 pmol) targeting the EGFP gene is delivered via nucleofection into U2OS-EGFP.PEST cells, followed by resuspension in DMEM medium (supplemented with 10% FBS, 1 mM sodium pyruvate, and 100 U / mL penicillin-streptomycin), addition of 6xNLS-AcrIIA4 (6xNLS-Acr, 0.03-4 pM), Cys- AcrIIA4-NLS (Acr-NLS, 0.03-4 pM), or Cys-AcrIIA4 (Acr, 0.03-4 pM) and PA (20 nM) 2 h post- nucleofection / resuspension, and imaging at 48 h. Positive controls include nucleofection of Apo- Cas9 and co-nucleofection (NF) of Cas9 RNP with Cys-AcrIIA4-NLS (Acr-NLS, 20 pmol). Negative controls include nucleofection of Cas9 RNP and nucleofection of Cas9 RNP followed by addition of Cys-AcrIIA4-NLS (Acr-NLS, 4 pM) 2 h post-nucleofection / resuspension. The dataare normalized to Cas9 RNP and are the mean ± s.d. of three independent replicates. The significance of Cas9 RNP + PA + 6><NLS-AcrIIA4 additions is determined with an unpaired, two- tailed t-test versus Cas9 RNP, where *, **, ***, and **** stand for P < 0.05, P < 0.01, P < 0.001, and P < 0.0001, respectively. FIG. 8B - Representative fluorescence microscopy images from the EGFP-disruption assay of FIG. 8A demonstrating the dose-dependent delivery of an engineered, cell-permeable Acr polypeptide (e.g., 6><NLS-AcrIIA4) into human cells in the presence of PA. The nucleus is blue due to Hoechst 33342, and the cytosol is green due to EGFP.

[0060] FIG. 9A-9E - Exemplary NLS peptides for mediating Acr delivery. FIG. 9A - Chemical structure of the 4><NLS peptide. The 4><NLS peptide was synthesized via solid-phase peptide synthesis. FIG. 9B - Chemical structure of the 4 *NLS-mal eimide peptide. The 4*NLS- maleimide peptide was synthesized by conjugating the 4><NLS peptide to a DBCO-PEG4- maleimide moiety via a strain-promoted azide-alkyne cycloaddition. FIG. 9C - Abbreviated structures of the 4><NLS and 4><NLS-maleimide peptides. Amino acids were abbreviated using one-letter codes. FIG. 9D - MALDI-TOF mass spectrum of the 4*NLS peptide (4,379 Da) after purification by reversed-phase high-performance liquid chromatography. The observed m / z values of 4380 and 2191 are consistent with +1 and +2 charge states, respectively. FIG. 9E - Q-TOF Liquid chromatography-mass spectrum of the 4 xNLS-mal eimide peptide (4,983 Da) after purification by reversed-phase high-performance liquid chromatography.

[0061] FIG. 10A-10B - Synthesis of NLS-Acr conjugates. FIG. 10A - Synthesis of 4*NLS- AcrIIA4. A 2.9 mM stock solution of 4xNLS-maleimide peptide was prepared in water. Cys- AcrIIA4 (Acr) in 20 mM Tris-HCl buffer, pH 7.5, containing 150 mM NaCl, 1 mM TCEP, and 5% v / v glycerol was rapidly dialyzed into 20 mM HEPES-HC1 buffer, pH 7.5, containing 500 mM NaCl, using a Pierce microdialysis plate (3.5 KDa MWCO). Immediately afterward, 5 pL of the 2.9 mM 4xNLS-maleimide stock solution were added to 30 pL of 157 pM Cys-AcrIIA4. The reaction mixture was gently shaken (80 RPM) for 1.5 h at 22 °C. Afterward, an additional 6 pL of the 2.9 mM 4xNLS-maleimide stock were added to the reaction mixture. The reaction mixture was gently shaken for an additional 1.5 h at 37 °C. In total, 6.8 molar equivalents of 4xNLS-maleimide were added to 1 equivalent of Cys-AcrIIA4. Afterward, the reaction mixture was passed through a 0.5 mL Zeba spin desalting column (7 kDa MWCO), and the buffer was exchanged to 20 mM HEPES-HC1 buffer, pH 7.5, containing 500 mM NaCl, 1 mM TCEP, and 5% v / v glycerol. Theeluent from this column was used for genome editing experiments without further purification, and total protein concentration was determined using the Bradford assay. Q-TOF LC-MS confirmed that the reaction mixture contained 4><NLS-AcrIIA4, Cys-AcrIIA4, and 4*NLS- maleimide. FIG. 10B - Synthesis of 5xNLS-AcrIIA4. A 2.9 mM stock solution of 4*NLS- maleimide peptide was prepared in water. Cys-AcrIIA4-NLS (Acr-NLS) in 20 mM Tris-HCl buffer, pH 7.5, containing 150 mM NaCl, 1 mM TCEP, and 5% v / v glycerol was rapidly dialyzed into 20 mM HEPES-HC1 buffer, pH 7.5, containing 500 mM NaCl, using a Pierce microdialysis plate (3.5 KDa MWCO). Immediately afterward, 5 uL of the 2.9 mM 4 xNLS-mal eimide stock solution were added to 30 pL of 165 pM Cys-AcrIIA4-NLS. The reaction mixture was gently shaken (80 RPM) for 1.5 h at 22 °C. Afterward, an additional 6 pL of the 2.9 mM 4*NLS- maleimide stock were added to the reaction mixture. The reaction mixture was gently shaken for an additional 1.5 h at 37 °C. In total, 6.4 molar equivalents of 4*NLS-maleimide were added to 1 equivalent of Cys-AcrIIA4-NLS. The reaction mixture was passed through a 0.5 mL Zeba spin desalting column (7 kDa MWCO), and the buffer was exchanged to 20 mM HEPES-HC1 buffer, pH 7.5, containing 500 mMNaCl, 1 mM TCEP, and 5% v / v glycerol. The eluent from this column was used for genome editing experiments without further purification, and total protein concentration was determined using the Bradford assay. Q-TOF LC-MS confirmed that the reaction mixture contained 5*NLS-AcrIIA4, Cys-AcrIIA4-NLS, and 4 / NLS-maleimide.

[0062] FIG. 11A-11B - Q-TOF Liquid chromatography-mass spectrum of the NLS-Acr conjugates. FIG. 11A - Q-TOF Liquid chromatography-mass spectrum of the 4*NLS-AcrIIA4 reaction mixture after Zeba spin purification. LC-MS detected a 4 / NLS-AcrIIA4 product (m / z: 16,067) in the presence of starting material: Cys-AcrIIA4 (m / z: 11,093) and 4*NLS-maleimide (m / z: 4,983). The observed m / z for 4*NLS-AcrIIA4 (16,067) is 9 Da below the expected mass (16,076 Da). This difference could be due to the lower signal-to-noise ratio in the reaction mixture and / or an unexpected fragmentation or rearrangement. For simplicity, this crude product will be referred to as 4*NLS-AcrIIA4 or 4 / \LS-Acr going forward. Further characterization of 4 / NLS- AcrIIA4 is necessary. FIG. 11B - Q-TOF Liquid chromatography-mass spectrum of the 5*NLS- AcrIIA4 reaction mixture after Zeba spin purification. LC-MS detected a 5 / NLS-AcrIIA4 product (m / z: 17,060) in the presence of starting material: Cys-AcrIIA4-NLS (m / z: 12,102) and 4*NLS- maleimide (m / z: 4,983). The observed m / z for 5 NLS-AcrIIA4 (17,060) is 25 Da below theexpected mass (17,085 Da). This difference could be due to the lower signal -to-noise ratio in the reaction mixture and / or an unexpected fragmentation or rearrangement. For simplicity, this reaction mixture will be referred to as 5xNLS-AcrIIA4 going forward. Further characterization of 5><NLS-AcrIIA4 is necessary.

[0063] FIG. 12 - The EGFP-disruption assay confirms that an engineered, cell-permeable Acr (e.g., 4><NLS-AcrIIA4, 5xNLS-AcrlIA4, 6xNLS-AcrIIA4) enters human cells and that adding the 4xNLS peptide in trans enhances the delivery of a cell-permeable Acr. C-NLS-SpCas9 RNP (20 pmol) targeting the EGFP gene is delivered via nucleofection into U2OS-EGFP.PEST cells, followed by resuspension in DMEM medium (supplemented with 10% FBS, 1 mM sodium pyruvate, and 100 U / mL penicillin-streptomycin), addition of 4xNLS-AcrIIA4 (4xNLS-Acr, 2 pM), 5xNLS-AcrIIA4 (5xNLS-Acr, 2 pM), or 6xNLS-AcrIIA4 (6xNLS-Acr, 0.13-4 pM) ± 4xNLS peptide (4 pM, added as a separate, noncovalently-bound component) 0 h post- nucleofection / resuspension, and imaging at 48 h. Nucleofection of Apo-Cas9 is a positive control. Negative controls include nucleofection of Cas9 RNP and nucleofection of Cas9 RNP followed by addition of Cys-AcrIIA4 (Acr, 4 pM) or Cys-AcrIIA4-NLS (Acr-NLS, 4 pM) 0 h post- nucleofection / resuspension. The data are normalized to Cas9 RNP and are the mean ± s.d. of three independent replicates. The significance of Cas9 RNP + NLS-Acr additions is determined with an unpaired, two-tailed t-test versus Cas9 RNP, where *, **, ***, and **** stand for P < 0.05, P < 0.01, P < 0.001, and P < 0.0001, respectively.

[0064] FIG. 13 - The FGFF-disruption assay confirms that NLS-Acr conjugates (e.g., 4xNLS-AcrIIA4 and 5xNLS-AcrIIA4) enter human cells in a dose-dependent manner. C-NLS- SpCas9 RNP (20 pmol) targeting the EGFP gene is delivered via nucleofection into U2OS- EGFP.PEST cells, followed by resuspension in DMEM medium (supplemented with 10% FBS, 1 mM sodium pyruvate, and 100 U / mL penicillin-streptomycin), addition of 4xNLS-AcrIIA4 (4xNLS-Acr, 0.13-1 pM) or 5xNLS-AcrIIA4 (5xNLS-Acr, 0.13-1 pM) 0 h post- nucleofection / resuspension, and imaging at 48 h. Nucleofection of Apo-Cas9 is a positive control. Negative controls include nucleofection of Cas9 RNP and nucleofection of Cas9 RNP followed by addition of Cys-AcrIIA4 (Acr, 4 pM) or Cys-AcrIIA4-NLS (Acr-NLS, 4 pM) 0 h post- nucleofection / resuspension. The data are normalized to Cas9 RNP and are the mean ± s.d. of three independent replicates. The significance of Cas9 RNP + NLS-Acr additions is determined with anunpaired, two-tailed t-test versus Cas9 RNP, where *, **, ***, and **** stand for P < 0.05, P < 0.01, P < 0.001, and P < 0.0001, respectively.

[0065] FIG. 14A-14C. - A modified EGFP-disruption assay confirms that an engineered, cell- permeable Acr (e.g., 6*NLS-AcrIIA4) enters 3D cell cultures. FIG. 14A - C-NLS-SpCas9 RNP (20 pmol) targeting the EGFP gene is delivered via nucleofection into U2OS-EGFP PEST cells (300,000 cells). The cells are then resuspended in DMEM medium (supplemented with 10% FBS, 1 mM sodium pyruvate, and 100 U / mL penicillin-streptomycin), transferred to a 96-well spheroid plate (100 pL final volume, 850,000 cells / mL), and incubated at 37 °C. 6*NLS-AcrIIA4 (6*NLS- Acr, 2 LIM) is added 7-13 h post-nucleofection / resuspension, and the cells are imaged at 48 h. Nucleofection of Apo-Cas9 is a positive control. Negative controls include nucleofection of Cas9 RNP and nucleofection of Cas9 RNP followed by addition of Cys-AcrIIA4 (Acr, 2 pM) 7 h post- nucleofection / resuspension. White bars show EGFP disruption in the outer spheroid region (the area that is 0.20 mm in depth). Gray bars show EGFP disruption in the entire spheroid region, which has an average diameter of 1.25 ± 0.05 mm. The data are normalized to Cas9 RNP for each region and are the mean ± s.d. of three independent replicates. The significance of Cas9 RNP + 6*NLS-AcrIIA4 additions is determined with an unpaired, two-tailed t-test versus Cas9 RNP for each region, where *, **, ***, and **** stand for P < 0.05, P < 0.01, P < 0.001, and P < 0.0001, respectively. FIG. 14B - Visual representation of the spheroid regions defined to quantify EGFP disruption in FIG. 14A. Each region (outer and entire region) is shaded in light purple. FIG. 14C - Representative fluorescence microscopy images from the EGFP-disruption assay of FIG. 14A demonstrating the delivery of an engineered, cell-permeable Acr polypeptide (e.g., 6*NLS- AcrIIA4) into 3D cell cultures. The images represent maximum intensity projections from a Z- stack. The approximate diameter of these spheroids is 1.25 ± 0.05 mm. The cytosol is green due to EGFP.

[0066] FIG. 15 - Assessing the delivery of an engineered, cell-permeable Acr (e.g., 6*NLS- AcrIIA4) via the HiBiT assay. A CRISPR-Cas system (e.g., SpCas9 RNP) targeting the GAPDH gene and a single- stranded oligodeoxynucleotide (ssODN) donor encoding for the HiBiT tag are delivered via nucleofection into HEK293T cells (300,000 cells) or human embryonic stem cells (HUES 8, 450,000 cells). HEK293T cells are resuspended in DMEM medium (supplemented with 10% FBS, 1 mM sodium pyruvate, and 100 U / mL penicillin-streptomycin) and transferred to apoly(D-lysine)-coated 96-well plate (100 pL final volume, 150,000 cells / mL). HUES 8 cells are resuspended in mTeSRl medium (supplemented with 10 pM Y-27632) and transferred to a Matrigel-coated 96-well plate (100 pL final volume, 420,000 cells / mL). Cells are incubated (37 °C) with the engineered, cell-permeable Acr (e.g., 6><NLS-AcrIIA4) 0-16 h post- nucleofection / resuspension until 72 h (HEK293T) or 24 h (HUES 8). Cell viability is measured with the PrestoBlue reagent, followed by cell lysis, addition of LgBiT, and quantification of luminescence. Cas-mediated cleavage and HiBiT tag insertion via homology-directed repair lead to the formation of NanoLuc after LgBiT addition. Successful delivery of the Acr prevents Cas- mediated cleavage, HiBiT tag insertion, and NanoLuc formation after LgBiT addition.

[0067] FIG. 16 - The HiBiT assay confirms that an engineered, cell-permeable Acr (e.g., 6><NLS-AcrIIA4) enters human cells in a dose-dependent manner. C-NLS-SpCas9 RNP (20 pmol) targeting the GAPDH gene and the HiBiT ssODN (80 pmol) are co-delivered via nucleofection into HEK293T cells, followed by resuspension in DMEM medium (supplemented with 10% FBS, 1 mM sodium pyruvate, and 100 U / mL penicillin-streptomycin), addition of 6><NLS-AcrIIA4 (6*NLS-Acr, 0.4-1.5 pM) 0 h post-nucleofection / resuspension, and quantification of luminescence at 72 h. Positive controls include nucleofection of Cas9 RNP and co-nucleofection (NF) of Cas9 RNP and ssODN with AcrIIA4 (Acr, 20 pmol) or 6xNLS-AcrIIA4 (6xNLS-Acr, 20 pmol). Negative controls include co-nucleofection of Cas9 RNP and ssODN and co-nucleofection of Cas9 RNP and ssODN followed by addition of AcrIIA4 (Acr, 1.5 pM). The data are normalized to Cas9 RNP + ssODN and are the mean ± s.d. of three independent replicates. The significance of Cas9 RNP + ssODN + 6xNLS-AcrIIA4 additions is determined with an unpaired, two-tailed t- test versus Cas9 RNP + ssODN, where *, **, ***, and **** stand for P < 0.05, P < 0.01, P < 0.001, and P < 0.0001, respectively.

[0068] FIG. 17 - The HiBiT assay confirms that an engineered, cell-permeable Acr (e.g., 6xNLS-AcrIIA4) enters human embryonic stem cells in a dose-dependent manner. C-NLS- SpCas9 RNP (20 pmol) targeting the GAPDH gene and the HiBiT ssODN (80 pmol) are codelivered via nucleofection into HUES 8 cells, followed by resuspension in mTeSRl medium (supplemented with 10 pM Y-27632), addition of 6xNLS-AcrIIA4 (6xNLS-Acr, 0.25-4 pM) 0 h post-nucleofection / resuspension, and quantification of luminescence at 24 h. Nucleofection of Cas9 RNP is a positive control. Negative controls include co-nucleofection of Cas9 RNP andssODN and co-nucleofection of Cas9 RNP and ssODN followed by addition of Cys-AcrIIA4 (Acr, 4 pM). The data are normalized to Cas9 RNP + ssODN and are the mean ± s.d. of three independent replicates. The significance of Cas9 RNP + ssODN + 6><NLS-AcrIIA4 additions is determined with an unpaired, two-tailed t-test versus Cas9 RNP + ssODN, where *, **, ***, and **** stand for P < 0.05, P < 0.01, P < 0.001, and P < 0.0001, respectively.

[0069] FIG. 18 - The HiBiT assay confirms that an engineered, cell-permeable Acr (e.g., 6*NLS-AcrIIA4) enters human embryonic stem cells in a time-dependent manner. C-NLS-SpCas9 RNP (20 pmol) targeting the GAPDH gene and the HiBiT ssODN (80 pmol) are co-delivered via nucleofection into HUES 8 cells, followed by resuspension in mTeSRl medium (supplemented with 10 pM Y-27632), addition of 6*NLS-AcrIIA4 (6xNLS-Acr, 1 pM) 0-16 h post- nucleofection / resuspension, and quantification of luminescence at 24 h. Nucleofection of Cas9 RNP is a positive control. Negative controls include co-nucleofection of Cas9 RNP and ssODN and co-nucleofection of Cas9 RNP and ssODN followed by addition of Cys-AcrIIA4 (Acr, 4 pM). The data are normalized to Cas9 RNP + ssODN and are the mean ± s.d. of three independent replicates. The significance of Cas9 RNP + ssODN + 6*NLS-AcrIIA4 additions is determined with an unpaired, two-tailed t-test versus Cas9 RNP + ssODN, where *, **, ***, and **** stand for P < 0.05, P < 0.01, P < 0.001, and P < 0.0001, respectively.

[0070] FIG. 19 - Next-generation sequencing (NGS) to assess Cas9 specificity after Acr delivery. HEK293T cells in DMEM (10% FBS) are seeded at 150,000 cells per well (600 pL final volume) on a 24-well poly-(D-lysine) plate. After 20 h, the cells are transfected with Cas9 and sgRNA plasmids targeting the EMX1 gene via lipofection. The cells are then incubated with 6*NLS-AcrIIA4 (4 pM) for 72 h. Genomic DNA is extracted and the target and off-target sites are PCR amplified and barcoded. The amplicons are sequenced via NGS and analyzed using the CRISPResso2 software pipeline to determine insertions, deletions, and substitutions (% modification).

[0071] FIG. 20A-20B - NGS confirms that delivering an engineered Acr polypeptide (e.g., 6*NLS-AcrIIA4) increases genome-editing specificity in human cells. FIG. 20A - NLS-SpCas9- NLS (750 ng) and sgRNA (250 ng) targeting the EMX1 gene are delivered via lipofection into HEK293T cells. After 1 h, the cells are incubated with 6*NLS-AcrIIA4 (6*NLS-Acr, 4 pM) until 72 h. Genomic DNA is sequenced via NGS and analyzed using CRISPResso2 to determine %modification. Lipofection of Cas9-sgRNA plasmids, followed by addition of AcrIIA4 (Acr, 4 pM), is a negative control. The data are normalized to Cas9-sgRNA + AcrIIA4 (On-target) and are the mean ± s.d. of two independent replicates. The significance of Cas9-sgRNA + 6*NLS-AcrIIA4 is determined with an unpaired, two-tailed t-test versus Cas9-sgRNA + AcrIIA4, where *, **, ***, and **** stand for P < 0.05, P < 0.01, P < 0.001, and P < 0.0001, respectively. FIG. 20B - 6*NLS- AcrIlA4 delivery increases the on-target / off-target ratio (i.e., the specificity) of Cas9. The specificity of Cas9-sgRNA + 6*NLS-AcrIIA4 is calculated as a ratio of on-target to off-target % modification of FIG. 20A, normalizing against the specificity of Cas9-sgRNA + AcrIIA4 (specificity = 1). The significance of the calculated specificity of Cas9-sgRNA + 6*NLS-AcrIIA4 is determined with an unpaired, two-tailed t-test versus the specificity of Cas9-sgRNA + AcrIIA4, where *, **, ***, and **** stand for P < 0.05, P < 0.01, P < 0.001, and P < 0.0001, respectively.

[0072] FIG. 21A-21C - Synthesis of Cy5-Acr conjugates. FIG. 21A - Synthesis of Cy5- AcrIIA4. An 8.3 mM stock solution of sulfo-Cyanine5 maleimide was prepared in water. 14.5 pL of the 8.3 mM sulfo-Cyanine5 maleimide stock solution (14.7 equivalents) were added to 40 pL of 204 pM Cys-AcrIIA4 (Acr, 1 equivalent) in 20 mM Tris-HCl buffer, pH 7.5, containing 150 mM NaCl, 1 mM TCEP, and 5% v / v glycerol. The reaction mixture was gently shaken (80 RPM) for 30 min at 22 °C. Afterward, the reaction mixture was passed through a 0.5 mb Zeba spin desalting column (7 kDa MWCO), and the buffer was exchanged to 20 mM HEPES-HC1 buffer, pH 7.5, containing 500 mMNaCl, 1 mM TCEP, and 5% v / v glycerol. The eluent from this column was used for genome editing experiments, and total protein concentration was determined using the Bradford assay. FIG. 21B - Synthesis of Cy5-AcrIIA4-NLS. An 8.3 mM stock solution of sulfo-Cyanine5 maleimide was prepared in water. 12 pL of the 8.3 mM sulfo-Cyanine5 maleimide stock solution (12.8 equivalents) were added to 40 pL of 195 pM Cys-AcrIIA4-NLS (Acr-NLS, 1 equivalent) in 20 mM Tris-HCl buffer, pH 7.5, containing 150 mM NaCl, 1 mM TCEP, and 5% v / v glycerol. The reaction mixture was gently shaken (80 RPM) for 30 min at 22 °C. Afterward, the reaction mixture was passed through a 0.5 mb Zeba spin desalting column (7 kDa MWCO), and the buffer was exchanged to 20 mM HEPES-HC1 buffer, pH 7.5, containing 500 mM NaCl, 1 mM TCEP, and 5% v / v glycerol. The eluent from this column was used for genome editing experiments, and total protein concentration was determined using the Bradford assay. FIG. 21C - Synthesis of Cy5-6><NLS-AcrIIA4. An 8.3 mM stock solution of sulfo-Cyanine5 maleimide wasprepared in water. 4 pL of the 8.3 mM sulfo-Cyanine5 maleimide stock solution (6.9 equivalents) were added to 27.8 pL of 172 pM 6><NLS-AcrIIA4 (6><NLS-Acr, 1 equivalent) in 20 mM HEPES- HC1 buffer, pH 7.5, containing 500 mM NaCl, 1 mM TCEP, and 5% v / v glycerol. The reaction mixture was gently shaken (80 RPM) for 30 min at 22 °C. Afterward, the reaction mixture was passed through a 0.5 mL Zeba spin desalting column (7 kDa MWCO) and kept in 20 mM HEPES- HC1 buffer, pH 7.5, containing 500 mM NaCl, 1 mM TCEP, and 5% v / v glycerol. The eluent from this column was used for genome editing experiments, and total protein concentration was determined using the Bradford assay.

[0073] FIG. 22A-22C - Q-TOF Liquid chromatography-mass spectrum of Cy5-Acr conjugates. FIG. 22A - Q-TOF Liquid chromatography-mass spectrum of Cy5-AcrIIA4. LC-MS confirmed that Cys-AcrIIA4 (11,093 Da) was almost entirely converted to Cy5-AcrIIA4 (11,858 Da). A smaller peak with an m / z of 11,095 was also detected in the mass spectrum. This m / z is close to the mass of Cys-AcrIIA4 (11,093 Da), suggesting a small amount of unreacted Cys- AcrIIA4 is present. FIG. 22B - Q-TOF Liquid chromatography-mass spectrum of Cy5-AcrIIA4- NLS. LC-MS confirmed that Cys-AcrIIA4-NLS (12, 102 Da) was fully converted to Cy5-AcrIIA4- NLS (12,867 Da). FIG. 22C - Q-TOF Liquid chromatography-mass spectrum of Cy5-6*NLS- AcrIIA4. LC-MS confirmed that 6><NLS-AcrIIA4 (18,454 Da) was fully converted to Cy5- 6*NLS-AcrIIA4 (19,219 Da).

[0074] FIG. 23A-23D - Confocal microscopy confirms that an engineered, cell-permeable Acr conjugated to a Cy5 dye (e g., Cy5-6xNLS-AcrIIA4) enters mammalian cells in a dosedependent manner. INS-1E rat [3-cells (100 pL final volume, 750,000 cells / mL) in RPMI medium (supplemented with 10% FBS, 1 mM pyruvate, penicillin-streptomycin, and 50 pM of 2- mercaptoethanol) were seeded on an ECM-coated 96-well plate and incubated for 25 h at 37 °C. Then, 1.25-5 pL of Cy5-6*NLS-AcrIIA4 (Cy5-6*NLS-Acr) or Cy5-AcrIIA4 (Cy5-Acr) were directly added to the cells to a final concentration of 1-4 pM, and the cells were incubated for 1.5 h at 37 °C. The cells were washed with FluoroBrite DMEM (100 pL) and stained with Hoechst33342 (100 pL, 4 pg / mL, 10 min, 37 °C) and WGA555 (100 pL, 2.5 pg / mL, 5 min, 22 °C) in FluoroBrite DMEM. The cells were imaged via confocal microscopy using a 60* objective. FIG. 23A - Representative fluorescence images obtained using the 647 nm excitation channel (Cy5). Each image corresponds to a different well or replicate, while keeping the field of viewwithin the well constant. FIG. 23B - Representative fluorescence images obtained using the 568 nm excitation channel (WGA555, cell membrane stain). Each image corresponds to a different well or replicate, while keeping the field of view within the well constant. FIG. 23C - Representative fluorescence images obtained using the 405 nm excitation channel (Hoechst33342, nuclear stain). Each image corresponds to a different well or replicate, while keeping the field of view within the well constant. FIG. 23D - Representative fluorescence images obtained using the digital phase contrast channel. Each image corresponds to a different well or replicate, while keeping the field of view within the well constant. Images at the different panels (A, B, C or D) correspond to the same well and field of view, with varying excitation wavelengths.

[0075] FIG. 24A-24D - Confocal microscopy confirms that an engineered, cell-permeable Acr conjugated to a Cy5 dye (e.g., Cy5-6*NLS-AcrIIA4) enters human cells. U2OS cells (100 pL final volume, 100,000 cells / mL) in DMEM medium (supplemented with 10% FBS, 1 mM sodium pyruvate, and 100 U / mL penicillin-streptomycin) were seeded on a 96-well plate and incubated for 18.5 h at 37 °C. Then, 2 pL of Cy5-6xNLS-AcrIIA4 (Cy5-6*NLS-Acr) or Cy5-AcrIIA4 (Cy5- Acr) were directly added to the cells to a final concentration of 1.5 pM, and the cells were incubated for 20 min at 37 °C. The cells were washed with FluoroBrite DMEM (100 pL) and stained with Hoechst33342 (100 pL, 4 pg / mL, 10 min, 37 °C) and WGA555 (100 pL, 2.5 pg / mL, 5 min, 22 °C) in FluoroBrite DMEM. The cells were imaged via confocal microscopy using a 60* objective. FIG. 24A - Representative fluorescence images obtained using the 647 nm excitation channel (Cy5). Each image corresponds to a different well or replicate, while keeping the field of view within the well constant. FIG. 24B - Representative fluorescence images obtained using the 568 nm excitation channel (WGA555, cell membrane stain). Each image corresponds to a different well or replicate, while keeping the field of view within the well constant. FIG. 24C - Representative fluorescence images obtained using the 405 nm excitation channel (Hoechst33342, nuclear stain). Each image corresponds to a different well or replicate, while keeping the field of view within the well constant. FIG. 24D - Representative fluorescence images obtained using the digital phase contrast channel. Each image corresponds to a different well or replicate, while keeping the field of view within the well constant. Images at the different panels (A, B, C or D) correspond to the same well and field of view, with varying excitation wavelengths.

[0076] FIG. 25A-25D - Confocal microscopy confirms that an engineered, cell-permeable Acr conjugated to a Cy5 dye (e.g., Cy5-6*NLS-AcrIIA4) enters human cells at 4 °C. U2OS cells (100 pL final volume, 100,000 cells / mL) in DMEM medium (supplemented with 10% FBS, 1 mM sodium pyruvate, and 100 U / mL penicillin-streptomycin) were seeded on a 96-well plate and incubated for 18.5 h at 37 °C. After cooling the cells to 4 °C, 2 pL of Cy5-6><NLS-AcrIIA4 (Cy5- 6*NLS-Acr) or Cy5-AcrIIA4 (Cy5-Acr) were directly added to the cells to a final concentration of 1.5 pM, and the cells were incubated for 20 min at 4 °C. Then, the cells were washed with FluoroBrite DMEM (100 pL) and stained with Hoechst33342 (100 pL, 4 pg / mL) and WGA555 (100 pL, 2.5 pg / mL) for 10 min at 4 °C in FluoroBrite DMEM. The cells were imaged via confocal microscopy using a 60* objective. FIG. 25A - Representative fluorescence images obtained using the 647 nm excitation channel (Cy5). Each image corresponds to a different well or replicate, while keeping the field of view within the well constant. FIG. 25B - Representative fluorescence images obtained using the 568 nm excitation channel (WGA555, cell membrane stain). Each image corresponds to a different well or replicate, while keeping the field of view within the well constant. FIG. 25C - Representative fluorescence images obtained using the 405 nm excitation channel (Hoechst33342, nuclear stain). Each image corresponds to a different well or replicate, while keeping the field of view within the well constant. FIG. 25D - Representative fluorescence images obtained using the digital phase contrast channel. Each image corresponds to a different well or replicate, while keeping the field of view within the well constant. Images at the different panels (A, B, C or D) correspond to the same well and field of view, with varying excitation wavelengths.

[0077] FIG. 26A-26D - Confocal microscopy confirms that an engineered, cell-permeable Acr conjugated to a Cy5 dye (e.g., Cy5-6*NLS-AcrIIA4) enters human cells in the presence of small-molecule regulators of endocytosis (20* objective). U2OS cells (100 pL final volume, 125,000 cells / mL) in DMEM medium (supplemented with 10% FBS, 1 mM sodium pyruvate, and 100 U / mL penicillin-streptomycin) were seeded on a 96-well plate and incubated for 16.5 h at 37 °C. Then, 2 pL of water or an aqueous solution of amiloride hydrocholoride (final concentration: 50 pM), bafilomycin Al (final concentration: 25 nM), or chloroquine diphosphate (final concentration: 100 pM) were directly added to the cells. The cells were incubated for 3 h at 37 °C. Then, 2 pL of Cy5-6*NLS-AcrIIA4 (Cy5-6*NLS-Acr) or Cy5-AcrIIA4 (Cy5-Acr) were directlyadded to the cells to a final concentration of 1.5 pM, and the cells were incubated for 20 min at 37 °C. The cells were washed with FluoroBrite DMEM (100 pL) and stained with Hoechst33342 (100 |1L, 4 pg / mL, 10 min, 37 °C) and WGA555 (100 pL, 2.5 pg / mL, 5 min, 22 °C) in FluoroBrite DMEM. The cells were imaged via confocal microscopy using a 20* objective. FIG. 26A - Representative fluorescence images obtained using the 647 nm excitation channel (Cy5). Each image corresponds to a different well or replicate, while keeping the field of view within the well constant. FIG. 26B - Representative fluorescence images obtained using the 568 nm excitation channel (WGA555, cell membrane stain). Each image corresponds to a different well or replicate, while keeping the field of view within the well constant. FIG. 26C - Representative fluorescence images obtained using the 405 nm excitation channel (Hoechst33342, nuclear stain). Each image corresponds to a different well or replicate, while keeping the field of view within the well constant. FIG. 26D - Representative fluorescence images obtained using the digital phase contrast channel. Each image corresponds to a different well or replicate, while keeping the field of view within the well constant. Images at the different panels (A, B, C or D) correspond to the same well and field of view, with varying excitation wavelengths.

[0078] FIG. 27A-27D - Confocal microscopy confirms that an engineered, cell-permeable Acr conjugated to a Cy5 dye (e.g., Cy5-6*NLS-AcrIIA4) enters human cells in the presence of small-molecule regulators of endocytosis (60* objective). U2OS cells (100 pL final volume, 125,000 cells / mL) in DMEM medium (supplemented with 10% FBS, 1 mM sodium pyruvate, and 100 U / mL penicillin-streptomycin) were seeded on a 96-well plate and incubated for 16.5 h at 37 °C. Then, 2 pL of water or an aqueous solution of amiloride hydrochloride (final concentration: 50 pM), bafilomycin Al (final concentration: 25 nM), or chloroquine diphosphate (final concentration: 100 pM) were directly added to the cells. The cells were incubated for 3 h at 37 °C. Then, 2 pL of Cy5-6><NLS-AcrIIA4 (Cy5-6><NLS-Acr) or Cy5-AcrIIA4 (Cy5-Acr) were directly added to the cells to a final concentration of 1.5 pM, and the cells were incubated for 20 min at 37 °C. The cells were washed with FluoroBrite DMEM (100 pL) and stained with Hoechst33342 (100 pL, 4 pg / mL, 10 min, 37 °C) and WGA555 (100 pL, 2.5 pg / mL, 5 min, 22 °C) in FluoroBrite DMEM. The cells were imaged via confocal microscopy using a 60* objective. FIG. 27A - Representative fluorescence images obtained using the 647 nm excitation channel (Cy5). Each image corresponds to a different well or replicate, while keeping the field of view within the wellconstant. FIG. 27B - Representative fluorescence images obtained using the 568 nm excitation channel (WGA555, cell membrane stain). Each image corresponds to a different well or replicate, while keeping the field of view within the well constant. FIG. 27C - Representative fluorescence images obtained using the 405 nm excitation channel (Hoechst33342, nuclear stain). Each image corresponds to a different well or replicate, while keeping the field of view within the well constant. FIG. 27D - Representative fluorescence images obtained using the digital phase contrast channel. Each image corresponds to a different well or replicate, while keeping the field of view within the well constant. Images at the different panels (A, B, C or D) correspond to the same well and field of view, with varying excitation wavelengths. The cell plate imaged in FIG. 27A-27D is the same one imaged in FIG. 26A-26D but with a different magnification.

[0079] FIG. 28A-28D - Confocal microscopy confirms that an engineered, cell-permeable Acr conjugated to a Cy5 dye (e.g., Cy5-6*NLS-AcrIIA4) enters human cells in the presence of small-molecule regulators of the cell cycle. U2OS cells (100 pL final volume, 125,000 cells / mL) in DMEM medium (supplemented with 10% FBS, 1 mM sodium pyruvate, and 100 U / mL penicillin-streptomycin) were seeded on a 96-well plate and incubated for 10 min at 37 °C. Then, 2 pL of water or 2 pL of lovastatin (final concentration: 40 pM) in water: ethanol (1 :1) or 4 pL of thymidine (final concentration: 5 mM) in water were directly added to the cells. The cells were incubated for 18.5 h at 37 °C. Then, 2 pL of Cy5-6*NLS-AcrIIA4 (Cy5-6*NLS-Acr) or Cy5- AcrIIA4 (Cy5-Acr) were directly added to the cells to a final concentration of 1.5 pM, and the cells were incubated for 20 min at 37 °C. The cells were washed with FluoroBrite DMEM (100 pL) and stained with Hoechst33342 (100 pL, 4 pg / mL, 10 min, 37 °C) and WGA555 (100 pL, 2.5 pg / mL, 5 min, 22 °C) in FluoroBrite DMEM. The cells were imaged via confocal microscopy using a 60* objective. FIG. 28A - Representative fluorescence images obtained using the 647 nm excitation channel (Cy5). Each image corresponds to a different well or replicate, while keeping the field of view within the well constant. FIG. 28B - Representative fluorescence images obtained using the 568 nm excitation channel (WGA555, cell membrane stain). Each image corresponds to a different well or replicate, while keeping the field of view within the well constant. FIG. 28C - Representative fluorescence images obtained using the 405 nm excitation channel (Hoechst33342, nuclear stain). Each image corresponds to a different well or replicate, while keeping the field of view within the well constant. FIG. 28D - Representative fluorescenceimages obtained using the digital phase contrast channel. Each image corresponds to a different well or replicate, while keeping the field of view within the well constant. Images at the different panels (A, B, C or D) correspond to the same well and field of view, with varying excitation wavelengths.

[0080] FIG. 29 - SDS-PAGE gel of equivalent dilutions of (1) MBP-MbCasl2a (- glucose), (2) MBP-MbCasl2a (+ glucose), (3) MBP-LbCasl2a (- glucose), (4) MBP-LbCasl2a (+ glucose), (5) MBP-LbCasl2a-2xNLS (- glucose), and MBP-LbCasl2a-2xNLS (+ glucose). The MBP- Casl2a bands appear between 260 and 140 kDa.

[0081] FIG. 30 - SDS-PAGE gel of equivalent dilutions of the (1) MbCasl2a (- glucose), (2) MbCasl2a (+ glucose), (3) LbCasl2a (- glucose), (4) LbCasl2a (+ glucose), (5) LbCasl2a- 2xNLS (- glucose), and LbCasl2a-2xNLS (+ glucose) Ni-NTA flowthrough. The Casl2a bands appear around 140 kDa.

[0082] FIG. 31A-31B - SDS-PAGE gel of increasing concentrations of (FIG. 31A) LbCasl2a and (FIG. 31B) LbCasl2a-2xNLS after purification by affinity, anion exchange, and sizeexclusion chromatography. The Casl2a bands appear around 140 kDa.

[0083] FIG. 32 - SDS-PAGE gel of (1) MBP-AcrVAl (- glucose, 2 / 3-diluted), (2) MBP- AcrVAl (+ glucose, 1 / 6-diluted), (3) SUMO-AcrVAl (- glucose, undiluted), (4) SUMO-AcrVAl (+ glucose, undiluted). The MBP-AcrVAl and SUMO-AcrVAl bands appear around 70 and 50 kDa, respectively.

[0084] FIG 33 - SDS-PAGE gel of increasing concentrations of AcrVAl after purification by affinity and anion exchange chromatography. The AcrVAl band appears between 25 and 35 kDa.

[0085] FIG. 34 - SDS-PAGE gel of decreasing concentrations of AcrVAl after purification by affinity and anion exchange chromatography. The AcrVAl band appears between 25 and 35 kDa.

[0086] FIG. 35 - SDS-PAGE gel of increasing concentrations of AcrVA5 after purification by affinity and anion exchange chromatography. The AcrVA5 band appears between 10 and 15 kDa.

[0087] FIG. 36 - SDS-PAGE gel of equivalent dilutions of (5) MBP-AcrIIA5 (- glucose) and (6) MBP-AcrIIA5 (+ glucose). The MBP-AcrIIA5 band appears between 50 and 70 kDa. Lanes 1-4 are identified in FIG. 15 and are not relevant in the context of AcrIIA5.

[0088] FIG. 37 - SDS-PAGE gel of MBP-AcrIIA5 batches 1-8. All samples are undiluted except batches 5 (1 / 4-diluted) and 7 (1 / 2-diluted). The MBP-AcrIIA5 band appears between 50 and 70 k a.

[0089] FIG. 38 - SDS-PAGE gel of increasing concentrations of AcrIIA5 after purification by affinity and cation exchange chromatography. The AcrIIA5 band appears between 15 and 25 kDa.

[0090] FIG. 39A-39B - SDS-PAGE gel of increasing concentrations of (FIG. 39A) Cys- AcrIIA4 and (FIG. 39B) Cys-AcrIIA4-NLS after purification by affinity, anion exchange, and size-exclusion chromatography. The Cys-AcrIIA4 and Cys-AcrIIA4-NLS bands appear between 10 and 15 kDa.

[0091] FIG. 40A-40B - Q-TOF Liquid chromatography-mass spectrum of (FIG. 40A) Cys- AcrIIA4 (11,093 Da) and (FIG. 40B) Cys-AcrIIA4-NLS (12,102 Da) after purification by affinity, anion exchange, and size-exclusion chromatography.

[0092] FIG. 41A-41B - SDS-PAGE gel of increasing concentrations of (FIG. 41A) LFN- AcrIIA4 and (FIG. 41B) LF -AcrVAl after purification by affinity, anion exchange, and sizeexclusion chromatography. The LFN-AcrIIA4 band appears between 40 and 50 kDa. The LFN- AcrVAl band appears between 50 and 70 kDa.

[0093] FIG. 42 - SDS-PAGE gel of increasing concentrations of the 6><NLS-AcrIIA4 polypeptide after purification by affinity and cation exchange chromatography. The 6*NLS- AcrIIA4 band appears between 15 and 25 kDa.

[0094] The figures herein are for illustrative purposes only and are not necessarily drawn to scale.DETAILED DESCRIPTION OF THE EXAMPLE EMBODIMENTSGeneral Definitions

[0095] Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosurepertains. Definitions of common terms and techniques in molecular biology may be found in Molecular Cloning: A Laboratory Manual, 2ndedition, published by Cold Spring Harbor Laboratory Press (Cold Spring Harbor, N.Y. 1989) (Sambrook, Fritsch, and Maniatis); Molecular Cloning: A Laboratory Manual, 4thedition, published by Cold Spring Harbor Laboratory Press (Cold Spring Harbor, N.Y. 2012) (Green and Sambrook); Current Protocols in Molecular Biology, published by John Wiley & Sons (New York, N.Y. 1987) (Ausubel et al., eds.); the series Methods in Enzymology, published by Academic Press, Inc. (Cambridge, M.A. 1955-2023) (Pyle and Christianson, eds.); PCR 2: A Practical Approach, published by Oxford University Press, (New York, N.Y. 1995) (MacPherson, Hames, and Taylor eds.); Antibodies, A Laboratory Manual, published by Cold Spring Harbor Laboratory Press (Cold Spring Harbor, N.Y. 1988) (Harlow and Lane, eds.); Antibodies, A Laboratory Manual, 2ndedition, published by Cold Spring Harbor Laboratory Press (Cold Spring Harbor, N.Y. 2014) (E.A. Greenfield ed.); Animal Cell Culture, published by IRL Press (Oxford, U.K. 1987) (Freshney, ed ); Lewin’s Essential Genes, 4thedition, published by Jones and Bartlett Learning (Burlington, M.A. 2021) (ed.); The Encyclopedia of Molecular Biology, published by Blackwell Science Ltd. (Oxford, U.K. 1994) (Kendrew ed.); Molecular Biology and Biotechnology: A Comprehensive Desk Reference, published by VCH Publishers, Inc., (Hoboken, N.J. 1995) (Meyers ed.); Dictionary of Microbiology and Molecular Biology 2nd ed., published by J. Wiley & Sons (New York, N.Y. 1994) (Singleton et al.); Advanced Organic Chemistry: Reactions, Mechanisms and Structure 4th edition, published by John Wiley & Sons (New York, N.Y. 1992) (March); and Transgenic Mouse Methods and Protocols, 2ndedition, published by Humana Press (New York, N. Y. 2011) (Hofker and van Deursen).

[0096] As used herein, the singular forms “a”, “an”, and “the” include both singular and plural referents unless the context clearly dictates otherwise.

[0097] The term “optional” or “optionally” means that the subsequent described event, circumstance or substituent may or may not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not.

[0098] The recitation of numerical ranges by endpoints includes all numbers and fractions subsumed within the respective ranges, as well as the recited endpoints.

[0099] The terms “about” or “approximately” as used herein when referring to a measurable value such as a parameter, an amount, a temporal duration, and the like, are meant to encompass variations of and from the specified value, such as variations of ±10% or less, ±5% or less, ±1% or less, and ±0.1% or less of and from the specified value, insofar such variations are appropriate to perform in the disclosed invention. It is to be understood that the value to which the modifier “about” or “approximately” refers is itself also specifically, and preferably, disclosed.

[0100] As used herein, a “biological sample” may contain whole cells and / or live cells and / or cell debris. The biological sample may contain (or be derived from) a “bodily fluid”. The present invention encompasses embodiments wherein the bodily fluid is selected from amniotic fluid, aqueous humour, vitreous humour, bile, blood serum, breast milk, cerebrospinal fluid, cerumen (earwax), chyle, chyme, endolymph, perilymph, exudates, feces, female ejaculate, gastric acid, gastric juice, lymph, mucus (including nasal drainage and phlegm), pericardial fluid, peritoneal fluid, pleural fluid, pus, rheum, saliva, sebum (skin oil), semen, sputum, synovial fluid, sweat, tears, urine, vaginal secretion, vomit and mixtures of one or more thereof. Biological samples include cell cultures, bodily fluids, cell cultures from bodily fluids. Bodily fluids may be obtained from a mammal organism, for example by puncture, or other collecting or sampling procedures.

[0101] The terms “subject,” “individual,” and “patient” are used interchangeably herein to refer to a vertebrate, preferably a mammal, more preferably a human. Mammals include, but are not limited to, murines, simians, humans, farm animals, sport animals, and pets. Tissues, cells and their progeny of a biological entity obtained in vivo or cultured in vitro are also encompassed.

[0102] As used interchangeably herein, “operatively coupled” and “operatively linked”, in the context of engineered polynucleotide or polypeptide constructs and compositions, including but not limited to DNA, RNA, DNA-RNA, vectors, peptides, polypeptides (including, but not limited to proteins), and the like, refers to the attachment or association of one or more nucleic acids, oligonucleotides, polynucleotides, amino acids, peptides, and / or polypeptides to one or more other molecules or compositions. The attachment can be covalent or non-covalent between any two or more molecules. Such non-covalent interactions include by are not limited to, entrapment by the surface substrate, ionic bonds, electrostatic interactions, van der Walls forces, dipole-dipole interactions, dipole-induced-dipole interactions, London dispersion forces, hydrogen bonding, halogen bonding, electromagnetic interactions, n-n. interactions, cation-7i interactions, anion-ninteractions, polar ^-interactions, and hydrophobic effects. The attachment may be direct, such as a direct fusion between two polynucleotide molecules or two polypeptide molecules. The attachment may be indirect, such as via a linking molecule. It will be appreciated that the linking molecule may be directly fused to each of the other two molecules so as to indirectly link the two molecules together. Associations are other interactions that are not covalent or non-covalent attachments that associate the one or more of the molecules with each other such that they can interact or otherwise form a system and / or perform a function. Thus, these terms can refer to an arrangement of two or more molecules such that they can perform a function or have a particular activity. Specifically in the context of engineered polynucleotide molecules in constructs and vectors, “operatively coupled” and “operatively linked” refers to the regulatory and other sequences useful for expression, stabilization, replication, and the like of the coding and transcribed non-coding sequences of a nucleic acid that are placed in the nucleic acid molecule in the appropriate positions relative to the coding sequence so as to effect expression or other characteristic of the coding sequence or transcribed non-coding sequence. Also in this context, these same terms can be applied to the arrangement of coding sequences, non-coding and / or transcription control elements (e.g., promoters, enhancers, and termination elements), and / or selectable markers in an expression vector. Specifically, in the context of a system, the term can refer to the interaction or attachment, even if transient, of molecules or other components of the system such that they perform a function.

[0103] Various embodiments are described hereinafter. It should be noted that the specific embodiments are not intended as an exhaustive description or as a limitation to the broader aspects discussed herein. One aspect described in conjunction with a particular embodiment is not necessarily limited to that embodiment and can be practiced with any other embodiment(s). Reference throughout this specification to “one embodiment”, “an embodiment,” “an example embodiment,” means that a particular feature, structure or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention. Thus, appearances of the phrases “in one embodiment,” “in an embodiment,” or “an example embodiment” in various places throughout this specification are not necessarily all referring to the same embodiment, but may. Furthermore, the particular features, structures or characteristics may be combined in any suitable manner, as would be apparent to a person skilled in the art from thisdisclosure, in one or more embodiments. Furthermore, while some embodiments described herein include some but not other features included in other embodiments, combinations of features of different embodiments are meant to be within the scope of the invention. For example, in the appended claims, any of the claimed embodiments can be used in any combination.

[0104] All publications, published patent documents, and patent applications cited herein are hereby incorporated by reference to the same extent as though each individual publication, published patent document, or patent application was specifically and individually indicated as being incorporated by reference.OVERVIEW

[0105] The present disclosure provides compositions and methods for regulating the activity of a CRISPR-Cas system. Described herein are engineered compositions and methods of delivering Anti-CRISPR (Acr) compositions. In certain example embodiments herein are engineered Acr polypeptides that include an Acr polypeptide that is operatively coupled to one or more cell penetrating peptides (CPPs). In an embodiment, the one or more CPPs are one or more nuclear localization signals (NLSs). Without being bound by theory and as shown in FIG. 1, the CPPs facilitate delivery of the Acr across a lipid membrane, such as a cell membrane. When delivered to a target cell containing a CRISPR-Cas system, the Acr can inhibit activity of the CRISPR-Cas system.

[0106] Other compositions, compounds, methods, features, and advantages of the present disclosure will be or become apparent to one having ordinary skill in the art upon examination of the following drawings, detailed descriptions, and examples. It is intended that all such additional compositions, compounds, methods, features, and advantages be included within this description, and be within the scope of the present disclosure.ACR DELIVERY COMPOSITIONS AND SYSTEMSEngineered Acr Delivery Polypeptides

[0107] Described herein are engineered Acr delivery polypeptides (also referred to herein as “engineered Acr polypeptides”) that include an Acr polypeptide, and one or more cell-penetrating peptides (CPPs) operatively coupled to the Acr polypeptides. Without being bound by theory, coupling of one or more CPPs can enhance delivery of an Acr polypeptide to a target cell. In an embodiment, the engineered Acr polypeptide has increased penetration across a lipid bilayer ascompared to a wild-type Acr polypeptide or other suitable control. As shown in the Working Examples, wild-type Acr polypeptide was not observed to enter the cells. Without being bound by theory, operatively coupling one or more CPPs can provide the Acr the ability to enter cells across the cell membrane, which can be extrapolated to lipid bilayers. In an embodiment, the lipid bilayer is a cell membrane. In an embodiment, penetration across a lipid bilayer is increased 1- 1,000, 000, OOO-fold or more as compared to a suitable control, such as a wild-type Acr. As used throughout this specification, “suitable control” is a control that will be instantly appreciated by one of ordinary skill in the art as one that is included such that it can be determined if the variable being evaluated an effect, such as a desired effect or hypothesized effect. One of ordinary skill in the art will also instantly appreciate based on inter alia, the context, the variable(s), the desired or hypothesized effect. In an embodiment, the one or more CPPs can facilitate transport of an Acr polypeptide across the cell or nuclear membrane.Acr Polypeptides

[0108] The engineered Acr polypeptides contain one or more Acr polypeptides. Acr polypeptides are capable of inhibiting Cas system activity by a variety of mechanisms, including but not limited to inhibiting target nucleic acid binding, target nucleic acid cleavage, gRNA binding, and / or Cas complex formation. In an embodiment, the Acr polypeptide(s) prevent or inhibit target nucleic acid binding by the CRISPR-Cas system or component thereof. In an embodiment, the Acr polypeptide(s) prevent or inhibit Cas nuclease or nickase activity. In an embodiment, the Acr polypeptide(s) prevent or inhibit target nucleic acid cleavage. In an embodiment, the Acr polypeptide(s) prevent or inhibit target nucleic acid (e.g., DNA or RNA) binding or interaction with a Cas or Cas complex. In an embodiment, the Acr polypeptide(s) prevent or inhibit crRNA or gRNA loading into a Cas. In an embodiment, the Acr polypeptide(s) prevent or inhibit Cascade complex recruitment, formation and / or activity. For example, AcrIE2 inhibits Cas3 recruitment to the Cascade complex (see, e.g., Mejdani et al., J. Mol. Biol. 2021, 433(3), 166759). In an embodiment, the Acr polypeptide is capable of cleaving gRNAs. See, e.g., Knott et al., Nat. Struct. Mol. Biol. 2019, 26, 315-321, Wang et al., Nuc. Acid. Res. 2022, 50, 512- 521. In an embodiment, the Acr polypeptide is capable of homo or heterodimerization (see, e.g., Wiegand et al., Annu. Rev. Microbiol. 2020, 74, 21-37, particularly at Fig. 4). In an embodiment, the Acr polypeptide(s) inhibit or prevent Cas system activity by allosteric or steric inhibition. Inan embodiment, the Acr polypeptide(s) inhibit or prevent Cas system activity by preventing or inhibiting Cas system enzyme recycling (see, e.g., Peng et al., Proc. Natl. Acad. Sci. USA 2019, 776(38), 18928-18936).

[0109] In an embodiment, the Acr polypeptide(s) binds a Cas protein or a domain thereof. In an embodiment, the Acr polypeptide(s) bind an HNH domain of a Cas (see, e.g., Liu et al., Nuc. Acid. Res. 2021, 46(\ 1), 6587-6595 and Harrington et al., Cell 2017, 770(6), 1224-1223.el5). In an embodiment, the Acr polypeptide(s) bind a RuvC domain of a Cas (see, e.g., Song et al., Cell Rep. 2019, 29, 2579-2589; Kim et al., Sci. Rep. 2018, 8, 3883; Knott et al., eLife 2019, 8, e49110). In an embodiment, the Acr polypeptide(s) bind or interact with a bridge-helix domain of a Cas (see, e.g., Knott et al., eLife 2019, 9, e49110). In an embodiment, the Acr polypeptide(s) bind one or more nucleic acid binding sites in a Cas or Cas complex (see, e.g., Zhang et al., 2020. Proc. Natl. Acad. Sci. USA 2020, 777(13), 7176-7182). In an embodiment, the Acr polypeptide(s) bind and / or interact with one or more REC domains (see, e.g., Harrington et al., Cell 2017, 770(6), 1224-1233. el5). In an embodiment, the Acr polypeptide(s) bind and / or interact with a PAM- interacting domain (see, e.g., Dong et al., Nature 2017, 546(7658), 436-439; Yang et al., Mol. Cell 2017, 67(1), 117-127. e5; and Zhang et al., Cell Host Microbe 2019, 25(6), 815-826.e4). In an embodiment, the Acr polypeptide(s) mimic dsDNA (see, e.g., Wiegand et al., Annu. Rev. Microbiol. 2020, 74, 21-37, particularly at Fig. 3). Without being bound by theory, mimicking dsDNA structure facilitates the interaction of the Acr with a Cas and inhibits Cas activity on a dsDNA target.

[0110] In certain example embodiments, the Acr polypeptide inhibits or is capable of inhibiting a Type I, Type II, Type III, Type V, or Type VI CRISPR-Cas system, component thereof, and / or activity thereof. CRISPR-Cas systems that can be inhibited by the Acr polypeptide also include derivatives and variants thereof, such as, without limitation, CAST systems, prime editor systems, and base editors systems. Exemplary CRISPR-Cas systems that can be inhibited by the Acr polypeptide are described in greater detail elsewhere herein, such as in connection with co-therapies. In certain example embodiments, the Acr polypeptide inhibits or is capable of inhibiting a catalytically inactive Cas polypeptide. In an embodiment, the Acr polypeptide is capable of inhibiting a Cas lacking double-stranded nucleic acid cleavage activity. In an embodiment, the Acr polypeptide is capable of inhibiting a Cas lacking nucleic acid single-stranded cleavage activity. In an embodiment, the Acr polypeptide is capable of inhibiting a Cas lacking nuclease activity. See, e.g., Liu et al., Cell 2018, 772(5), 979-992. e6. In an embodiment, the Acr polypeptide is capable of inhibiting a Cas lacking nuclease activity but having nickase activity (see, e.g., Liang et al., Cells 2020, 9, 1786 and Song et al., Cell Rep. 2019, 29, 2579-2589). In an embodiment, the Acr polypeptide inhibits or is capable of inhibiting a dead Cas polypeptide (dCas) (see, e.g., Knott et al., Nat. Struct. Mol. Biol. 2019, 26, 315-321).

[0111] In an embodiment, the Acr polypeptide is a Type I Acr polypeptide. In an embodiment, the Acr polypeptide is a Type II Acr polypeptide. In an embodiment, the Acr polypeptide is a Type III Acr polypeptide. In an embodiment, the Acr polypeptide is a Type V Acr polypeptide. In an embodiment, the Acr polypeptide is a Type VI Acr polypeptide. See also e.g., Marshall et al., Mol. Cell 2018, 69, 146-157, particularly at Fig. 5b.

[0112] In certain example embodiments, the one or more Acr polypeptides are selected from an AcrIEl, AcrIE2, AcrIE3, AcrIE4, AcrIE5, AcrIE6, AcrIE7, AcrIE8, AcrIE8.1, AcrIE8.2, AcrIE9, AcrIFI, AcrIF2, AcrIF3, AcrIF4, AcrIF5, AcrIF6, AcrIF7, AcrIF8, AcrIF9, AcrIFlO, AcrIFl l, AcrIFl l.1, AcrIF11.2, AcrIF12, AcrIF13, AcrIF14, AcrIF15, AcrIF16, AcrIF17, AcrIF18, AcrIF19, AcrIF20, AcrIF21, AcrIF22, AcrIF23, AcrIF24, AcrIE4-F7, AcrlAI, AcrIBI, AcrIB2, AcrIB3, AcrIB4, AcrIB5, AcrIB6, AcrIB7, AcrIB9, AcrICI, AcrIF2 / C2, AcrIC3, AcrIC4, AcrIC5, AcrIC6, AcrIC7, AcrIC8, AcrIC9, AcrIClO, AcrICI 1, AcrIDl, AcrIIAl, AcrIIA2, AcrIIA2-l, AcrIIA2-2, AcrIIA2b, AcrIIA3, AcrIIA4, AcrIIA4-2, AcrIIA4-3, AcrIIA4 variant Ins. 5, AcrIIA4 variant N39A, AcrIIA4 variant D14A / G38A, Acrobat-AcrIIA4, AcrIIA4-dTAG, SMASh-AcrIIA4, AcrIIA5, AcrIIA5-2, AcrIIA6, AcrIIA7, AcrIIA8, AcrIIA9, AcrIIAl 0, AcrIIAl 1, AcrIIAl 2, AcrIIAl 3, AcrIIAl 3b, AcrIIAl 4, AcrIIAl 5, AcrIIAl 6, AcrIIAl 7, AcrIIA18, AcrIIA19, AcrIIA20, AcrIIA21, AcrIIA22, AcrIIA23, AcrIIA24, AcrIIA25, AcrIIA26, AcrIIA27, AcrIIA28, AcrIIA29, AcrIIA30, AcrIIA31, AcrIIA32, AcrIIA33, AcrIIA33(Seq), AcrIIA34, AcrIICl, AcrIICl-1, AcrIIC2, AcrIIC3, AcrIIC4, AcrIIC5, AcrIIC6, AcrIIC7, AcrIIC8, AcrIIC9, AcrIII-1, AcrIIIBl, AcrVAl, AcrVA2, AcrVA3, AcrVA3.1, AcrVA4, AcrVA5, AcrVIAl, AcrVIAl(Lwa), AcrVIA2, AcrVIA2(Lwa), AcrVIA3, AcrVIA3(Lwa), AcrVIA4, AcrVIA5, AcrVIA6, AcrVIA7, AcrVIBl, Csx27, enAcr-1, ErAcr-310, a homologue thereof, an orthologue thereof, or any combination thereof.

[0113] In an embodiment, the one or more Acr polypeptides comprise or contain only the functional domain(s) of one or more Acr polypeptides selected from an AcrIEl, AcrIE2, AcrIE3, AcrIE4, AcrIE5, AcrIE6, AcrIE7, AcrIE8, AcrIE8.1, AcrIE8.2, AcrIE9, AcrIFI, AcrIF2, AcrIF3, AcrIF4, AcrIF5, AcrIF6, AcrIF7, AcrIF8, AcrIF9, AcrIFlO, AcrIFI 1, AcrIFI 1.1, AcrIFI 1.2, AcrIF12, AcrIF13, AcrIF14, AcrIF15, AcrIF16, AcrIF17, AcrIF18, AcrIF19, AcrIF20, AcrIF21, AcrIF22, AcrIF23, AcrIF24, AcrIE4-F7, AcrlAI, AcrIBI, AcrIB2, AcrIB3, AcrIB4, AcrIB5, AcrIB6, AcrIB7, AcrIB9, AcrICI, AcrIF2 / C2, AcrIC3, AcrIC4, AcrIC5, AcrIC6, AcrIC7, AcrIC8, AcrIC9, AcrIClO, AcrICI 1, AcrIDl, AcrIIAl, AcrIIA2, AcrIIA2-l, AcrIIA2-2, AcrIIA2b, AcrIIA3, AcrIIA4, AcrIIA4-2, AcrIIA4-3, AcrIIA4 variant Ins. 5, AcrIIA4 variant N39A, AcrIIA4 variant DI 4A / G38A, Acrobat-AcrIIA4, AcrIIA4-dTAG, SMASh-AcrIIA4, AcrIIA5, AcrIIA5-2, AcrIIA6, AcrIIA7, AcrIIA8, AcrIIA9, AcrIIAlO, AcrIIAl 1, AcrIIA12, AcrIIA13, AcrIIA13b, AcrIIA14, AcrIIA15, AcrIIA16, AcrIIA17, AcrIIA18, AcrIIA19, AcrIIA20, AcrIIA21, AcrIIA22, AcrIIA23, AcrIIA24, AcrIIA25, AcrIIA26, AcrIIA27, AcrIIA28, AcrIIA29, AcrIIA30, AcrIIA31, AcrIIA32, AcrIIA33, AcrIIA33(Seq), AcrIIA34, AcrIICl, AcrIICl-1, AcrIIC2, AcrIIC3, AcrIIC4, AcrIIC5, AcrIIC6, AcrIIC7, AcrIIC8, AcrIIC9, AcrIII-1, AcrIIIBl, AcrVAl, AcrVA2, AcrVA3, AcrVA3.1, AcrVA4, AcrVA5, AcrVIAl, AcrVIAl(Lwa), AcrVIA2, AcrVIA2(Lwa), AcrVIA3, AcrVIA3(Lwa), AcrVIA4, AcrVIA5, AcrVIA6, AcrVIA7, AcrVIBl, Csx27, enAcr-1, ErAcr-310, a homologue thereof, an orthologue thereof, or any combination thereof.

[0114] In an embodiment, the one or more Acr polypeptides are variants, derivatives, homologs, orthologs, or paralogues of one or more Acr polypeptides selected from an AcrIEl, AcrIE2, AcrIE3, AcrIE4, AcrIE5, AcrIE6, AcrIE7, AcrIE8, AcrIE8.1, AcrIE8.2, AcrIE9, AcrIFI, AcrIF2, AcrIF3, AcrIF4, AcrIF5, AcrIF6, AcrIF7, AcrIF8, AcrIF9, AcrIFlO, AcrIFI 1, AcrIFI 1.1, AcrIFI 1.2, AcrIF12, AcrIF13, AcrIF14, AcrIF15, AcrIF16, AcrIF17, AcrIF18, AcrIF19, AcrIF20, AcrIF21, AcrIF22, AcrIF23, AcrIF24, AcrIE4-F7, AcrlAI, AcrIBI, AcrIB2, AcrIB3, AcrIB4, AcrIB5, AcrIB6, AcrIB7, AcrIB9, AcrICI, AcrIF2 / C2, AcrIC3, AcrIC4, AcrIC5, AcrIC6, AcrIC7, AcrIC8, AcrIC9, AcrIClO, AcrICI 1, AcrIDl, AcrIIAl, AcrIIA2, AcrIIA2-l, AcrIIA2-2, AcrIIA2b, AcrIIA3, AcrIIA4, AcrIIA4-2, AcrIIA4-3, AcrIIA4 variant Ins. 5, AcrIIA4 variant N39A, AcrIIA4 variant D14A / G38A, Acrobat- AcrIIA4, AcrIIA4-dTAG, SMASh-AcrIIA4, AcrIIA5, AcrIIA5-2, AcrIIA6, AcrIIA7, AcrIIA8, AcrIIA9, AcrIIAlO, AcrIIAl 1, AcrIIA12, AcrIIA13, AcrIIA13b, AcrIIA14, AcrIIA15, AcrIIA16, AcrIIA17, AcrIIA18, AcrIIA19,AcrIIA20, AcrIIA21, AcrIIA22, AcrIIA23, AcrIIA24, AcrIIA25, AcrIIA26, AcrIIA27, AcrIIA28, AcrIIA29, AcrIIA30, AcrIIA31, AcrIIA32, AcrIIA33, AcrIIA33(Seq), AcrIIA34, AcrIICl, AcrIICl-1, AcrIIC2, AcrIIC3, AcrIIC4, AcrIIC5, AcrIIC6, AcrIIC7, AcrIIC8, AcrIIC9, AcrIII-1, AcrIIIBl, AcrVAl, AcrVA2, AcrVA3, AcrVA3.1, AcrVA4, AcrVA5, AcrVIAl, AcrVIAl(Lwa), AcrVIA2, AcrVIA2(Lwa), AcrVIA3, AcrVIA3(Lwa), AcrVIA4, AcrVIA5, AcrVIA6, AcrVIA7, AcrVIBl, Csx27, enAcr-1, ErAcr-310, a homologue thereof, an orthologue thereof, or any combination thereof.

[0115] Table 1 provides exemplary GenBank Accession numbers, polypeptide sequences, and / or references for Acr polypeptides suitable for use in the engineered Acr polypeptides of the present disclosure.

[0116] Additional exemplary Acr polypeptides and / or anti-CRISPR functional domains thereof that can be included in the engineered Acr polypeptides of the present invention are discussed in e.g., Zhang and Marchisio, RNA Biol. 2021 75(8), 1085-1098; Liu et al., FEBS J.2020, 287(4), 626-644, particularly at Table 1; Yu and Marchisio, Front. Bioeng. Biotechnol. 2020, 8, 575393, particularly at Table 2; Zhu et al., BMC Biol. 2018, 76(1), 32; an Acr in any one or more of the following databases: AcrDB (Acr Database) (Huang et al., Nucleic Acids Res. 2021, 9(D1), D622-D629), Anti-CRISPRdb (see, e.g., Dong et al., Nucleic Acids Res. 2018, 6(D1), D393-D398, Anti-CRISPRdb V2.2 (available at guolab.whu.edu.cn / anti-CRISPRdb / ), the Acr Registry (anti-CRISPR assembly spreadsheet, available at tinyurl.com / anti-CRISPR), CRISPRimer (see, e.g., Zhang et al., Commun. Biol. 2018, 1, 180; Acr nomenclature (see, e.g., Bondy-Denomy et al., CRISPR J. 2018, 7(5), 304-305), AcrCatalog (see, e g., Gussow et al., Nat. Commun. 2020, 77, 3784), AcRanker (see, e.g., Eitzinger et al., Nucleic Acids Res. 2020, 48(9), 4698-4708 and those identified using the method described therein), AcrFinder (see, e.g., Yi et al., Nucleic Acids Res. 2020, 45(W1), W358-W365), PaCRISPR (see, e.g., Wang et al., Nucleic Acids Res. 2020, 45(W1), W348-W357), AcrDetector (see, e.g., Dong et al., bioRxiv2020, doi.org / 10.1101 / 2020.05.23.112011), AcrHub (see, e.g., Wang et al., Nucleic Acids Res. 2021, 49(D1), D630-D638); Rauch et al., Cell 2017, 765(1-2), 150-158; Hwang and Maxwell, CRISPR I. 2019, 2(1), 23-30, particularly at Table 1; Pinilla-Redondo et al., Nat. Commun. 2020, 6(11), 5652, particularly at Figures 1 and 2; Forsberg et al., PLoS Biol. 2021, J 9(10), e3001428; Meeske et al., Science 2020, 369(6499), 54-59; Davidson et al., Annu. Rev. Biochem. 2020, 89, 309-332; Stanley et al., Cell 2019, 775(6), 1452-1464; Pawluk et al., Nat. Rev. Microbiol. 2018, 76(1), 12- 17; Peng et al., Trends Microbiol. 2020, 25(11), 913-921; Osuna et al., Cell Host Microbe 2020, 25(1), 31-40; Thavalingam et al., Nat. Comm. 2019, JO, 2806-2811; Liu et al., Nucleic Acids Res.2021, 49(11), 6587-6595; Watters et al., Proc. Natl. Acad. Sci. USA 2020, 777(12), 6531-6539;Mahendra et al., Nat. Microbiol. 2020, 5(4), 620-629; Varble et al., bioRxiv, 2020, doi.org / 10.1101 / 2020.10.09.333658; Leon et al., Nucleic Acids Res. 2021, 49(4), 2114-2125; Jiang et al., Mol. Cell 2019, 73(3), 601-610; Liu, et al., Mol. Cell 73(3), 611-620; Dong et al., Nature 2017, 546, 429-432; Stanley, S.Y., An Investigation of Bacteriophage Anti-CRISPR and Anti-CRISPR Associated Proteins, 2018, Doctoral Dissertation in the Department of Molecular Genetics of the University of Toronto); Yang et al., Nat. Commun. 2022, 13, 1931; European Patent Publication No. EP3615552; EP3615665; and EP3429635; International Patent Publication No. WO 2017 / 160689; WO 2018 / 197495; WO 2019 / 185751; WO 2020 / 059708; WO 2020 / 043148; WO 2019 / 076651; WO 2018 / 197520; and WO 2021 / 108442; and U.S. PatentPublication No. US20200190492; US20200040328; US20190382741; US20210198328; US20210317480; and US20210095004; the disclosures of which can be adapted for use with the present invention.

[0117] In an embodiment, the Acr polypeptide has 80%-100%, such as 80%, to / or 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, sequence identity to any one of the Acr polypeptides described herein. One of ordinary skill in the art, in view of the description herein, will be able to determine a reference sequence of an Acr polypeptide and, using conventional alignment tools, determine Acr polypeptides having 80% -100% such as 80%, to / or 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, sequence identity to any one of the Acr polypeptides described herein. In an embodiment, the Acr polypeptide is a homologue or orthologue of any one of the Acr polypeptides described herein. The terms “orthologue” (also referred to as “ortholog” herein) and “homologue” (also referred to as “homolog” herein) are well known in the art. By means of further guidance, a “homologue” of a protein as used herein is a protein of the same species which performs the same or a similar function as the protein it is a homologue of. Homologous proteins may but need not be structurally related, or are only partially structurally related. An “orthologue” of a protein as used herein is a protein of a different species which performs the same or a similar function as the protein it is an orthologue of. Orthologous proteins may but need not be structurally related, or are only partially structurally related. Homologs and orthologs may be identified by homology modelling (see, e.g., J. Greer, Science 1985, 225(4703), 1055-1060, and Blundell et al., Eur. J. Biochem. 1988, 772, 513-520, or "structural BLAST" (Dey et al., Protein Sci. 2013, 22(4), 359-366). Homologous proteins may but need not be structurally related, or are only partially structurally related.

[0118] In an embodiment, the Acr polypeptide is an engineered Acr polypeptide. In an embodiment, the Acr polypeptide is an engineered polypeptide with improved potency. In an embodiment, the engineered Acr polypeptide with improved potency is any of those set forth and as described in Mathony et al., Nat. Chem. Biol. 2020, 76(7): 725-730. In an embodiment, the Acr polypeptide is engineered to be responsive to an external stimulus (e.g., a chemical stimulus, light stimulus, radiation stimulus, magnetic stimulus, temperature stimulus, or other physical or energy stimulus. Such engineered Acr polypeptides can allow for further temporal or spatial control ofAcr activity. In some example embodiments, the engineered Acr polypeptide is coupled to a light- sensitive molecule. See, e.g., Bubeck et al., Nat. Methods 2018, 15, 924-927. In some example embodiments, the engineered Acr polypeptide is coupled to a molecule or polypeptide that is degraded in a particular phase of the cell cycle. See, e.g., Matsumoto et al., Commun. Biol. 2020, 3, 601.

[0119] In an embodiment, the Acr polypeptide is about 12 to about 1,000 amino acids in length. See, e.g., Cui et al., Genome Biol. 2020, 21, 51. In an embodiment, the Acr polypeptide is about 12, to / or 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63. 64. 65. 66. 67. 68. 69. 70. 71. 72. 73. 74. 75. 76. 77. 78. 79. 80. 81. 82. 83. 84. 85. 86. 87. 88.89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101 , 102, 103, 104, 105, 106, 107, 108, 109, 110,111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129,130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, 383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457 458, 459, 460, 461, 462 463, 464, 465, 466, 467, 468, 469, 470, 471472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 486, 487, 488, 489, 490,491, 492, 493, 494, 495, 496, 497, 498, 499, 500 500, 501, 502, 503, 504, 505, 506, 507, 508, 509,510, 511, 512, 513, 514, 515, 516, 517, 518, 519, 520, 521, 522, 523, 524, 525, 526, 527, 528, 529, 530, 531, 532, 533, 534, 535, 536, 537, 538, 539, 540, 541, 542, 543, 544, 545, 546, 547, 548, 549, 550, 551, 552, 553, 554, 555, 556, 557, 558, 559, 560, 561, 562, 563, 564, 565, 566, 567, 568, 569, 570, 571, 572, 573, 574, 575, 576, 577, 578, 579, 580, 581, 582, 583, 584, 585, 586, 587, 588, 589, 590, 591, 592, 593, 594, 595, 596, 597, 598, 599, 600, 601, 602, 603, 604, 605, 606, 607, 608, 609, 610, 611, 612, 613, 614, 615, 616, 617, 618, 619, 620, 621, 622, 623, 624, 625, 626, 627, 628, 629, 630, 631, 632, 633, 634, 635, 636, 637, 638, 639, 640, 641, 642, 643, 644, 645, 646, 647, 648, 649, 650, 651, 652, 653, 654, 655, 656, 657, 658, 659, 660, 661, 662, 663, 664, 665, 666, 667, 668, 669, 670, 671, 672, 673, 674, 675, 676, 677, 678, 679, 680, 681, 682, 683, 684, 685, 686, 687, 688, 689, 690, 691, 692, 693, 694, 695, 696, 697, 698, 699, 700, 701, 702, 703, 704, 705, 706, 707, 708, 709, 710, 711, 712, 713, 714, 715, 716, 717, 718, 719, 720, 721, 722, 723, 724, 725, 726, 727, 728, 729, 730, 731, 732, 733, 734, 735, 736, 737, 738, 739, 740, 741, 742, 743, 744, 745, 746, 747, 748, 749, 750, 751, 752, 753, 754, 755, 756, 757, 758, 759, 760, 761, 762, 763, 764, 765, 766, 767, 768, 769, 770, 771, 772, 773, 774, 775, 776, 777, 778, 779, 780, 781, 782, 783, 784, 785, 786, 787, 788, 789, 790, 791, 792, 793, 794, 795, 796, 797, 798, 799, 800, 801, 802, 803, 804, 805, 806, 807, 808, 809, 810, 811, 812, 813, 814, 815, 816, 817, 818, 819, 820, 821, 822, 823, 824, 825, 826, 827, 828, 829, 830, 831, 832, 833, 834, 835, 836, 837, 838, 839, 840, 841, 842, 843, 844, 845, 846, 847, 848, 849, 850, 851, 852, 853, 854, 855, 856, 857, 858, 859, 860, 861, 862, 863, 864, 865, 866, 867, 868, 869, 870, 871, 872, 873, 874, 875, 876, 877, 878, 879, 880, 881, 882, 883, 884, 885, 886, 887, 888, 889, 890, 891, 892, 893, 894, 895, 896, 897, 898, 899, 900, 901, 902, 903, 904, 905, 906, 907, 908, 909, 910, 911, 912, 913, 914, 915, 916, 917, 918, 919, 920, 921, 922, 923, 924, 925, 926, 927, 928, 929, 930, 931, 932, 933, 934, 935, 936, 937, 938, 939, 940, 941, 942, 943, 944, 945, 946, 947, 948, 949, 950, 951, 952, 953, 954, 955, 956, 957, 958, 959, 960, 961, 962, 963, 964, 965, 966, 967, 968, 969, 970, 971, 972, 973, 974, 975, 976, 977, 978, 979, 980, 981, 982, 983, 984, 985, 986, 987, 988, 989, 990, 991, 992, 993, 994, 995, 996, 997, 998, 999, 1000 or more amino acids in length.

[0120] In an embodiment, the Acr polypeptide(s) reduce one or more CRISPR-Cas system activities by 1 to 1,000 fold or more. In an embodiment, the Acr polypeptide(s) reduce one or more CRISPR-Cas system activities by 1 to / or 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45,46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71,72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97,98. 99. 100. 101. 102. 103. 104. 105. 106. 107. 108. 109. 110. 111. 112. 113. 114. 115. 116. 117.118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, 383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 486, 487, 488, 489, 490, 491, 492, 493, 494, 495, 496, 497, 498, 499, 500, 501, 502, 503, 504, 505, 506, 507, 508, 509, 510, 511, 512, 513, 514, 515, 516, 517, 518, 519, 520, 521, 522, 523, 524, 525, 526, 527, 528, 529, 530, 531, 532, 533, 534, 535, 536, 537, 538, 539, 540 541, 542, 543, 544, 545 546, 547, 548, 549, 550, 551, 552, 553, 554555, 556, 557, 558, 559, 560, 561, 562, 563, 564, 565, 566, 567, 568, 569, 570, 571, 572, 573,574, 575, 576, 577, 578, 579, 580, 581, 582, 583, 584, 585, 586, 587, 588, 589, 590, 591, 592,593, 594, 595, 596, 597, 598, 599, 600, 601, 602, 603, 604, 605, 606, 607, 608, 609, 610, 611,612, 613, 614, 615, 616, 617, 618, 619, 620, 621, 622, 623, 624, 625, 626, 627, 628, 629, 630,631, 632, 633, 634, 635, 636, 637, 638, 639, 640, 641, 642, 643, 644, 645, 646, 647, 648, 649,650, 651, 652, 653, 654, 655, 656, 657, 658, 659, 660, 661, 662, 663, 664, 665, 666, 667, 668,669, 670, 671, 672, 673, 674, 675, 676, 677, 678, 679, 680, 681, 682, 683, 684, 685, 686, 687,688, 689, 690, 691, 692, 693, 694, 695, 696, 697, 698, 699, 700, 701, 702, 703, 704, 705, 706,707, 708, 709, 710, 711, 712, 713, 714, 715, 716, 717, 718, 719, 720, 721, 722, 723, 724, 725,726, 727, 728, 729, 730, 731, 732, 733, 734, 735, 736, 737, 738, 739, 740, 741, 742, 743, 744,745, 746, 747, 748, 749, 750, 751, 752, 753, 754, 755, 756, 757, 758, 759, 760, 761, 762, 763,764, 765, 766, 767, 768, 769, 770, 771, 772, 773, 774, 775, 776, 777, 778, 779, 780, 781, 782,783, 784, 785, 786, 787, 788, 789, 790, 791, 792, 793, 794, 795, 796, 797, 798, 799, 800, 801,802, 803, 804, 805, 806, 807, 808, 809, 810, 811, 812, 813, 814, 815, 816, 817, 818, 819, 820,821, 822, 823, 824, 825, 826, 827, 828, 829, 830, 831, 832, 833, 834, 835, 836, 837, 838, 839,840, 841, 842, 843, 844, 845, 846, 847, 848, 849, 850, 851, 852, 853, 854, 855, 856, 857, 858,859, 860, 861, 862, 863, 864, 865, 866, 867, 868, 869, 870, 871, 872, 873, 874, 875, 876, 877,878, 879, 880, 881, 882, 883, 884, 885, 886, 887, 888, 889, 890, 891, 892, 893, 894, 895, 896,897, 898, 899, 900, 901, 902, 903, 904, 905, 906, 907, 908, 909, 910, 911, 912, 913, 914, 915,916, 917, 918, 919, 920, 921, 922, 923, 924, 925, 926, 927, 928, 929, 930, 931, 932, 933, 934,935, 936, 937, 938, 939, 940, 941, 942, 943, 944, 945, 946, 947, 948, 949, 950, 951, 952, 953,954, 955, 956, 957, 958, 959, 960, 961, 962, 963, 964, 965, 966, 967, 968, 969, 970, 971, 972,973, 974, 975, 976, 977, 978, 979, 980, 981, 982, 983, 984, 985, 986, 987, 988, 989, 990, 991,992, 993, 994, 995, 996, 997, 998, 999, 1000 fold or more.

[0121] In an embodiment, the Acr polypeptide(s) reduce one or more CRISPR-Cas system activities by any non-zero number to / or 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%,30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%,47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%,64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%,81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%.Cell Penetrating Peptides

[0122] In an embodiment, the engineered Acr polypeptide includes one or more cellpenetrating peptides (CPPs) operatively coupled to an Acr polypeptide. In general, CPPs are short peptides that facilitate cellular uptake of various molecular cargo. In an embodiment, the CPP is a nuclear localization signal. In an embodiment, 1-10 or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) CPPs are operatively coupled to the Acr polypeptide. In an embodiment, one or more CPPs are coupled to each of the N-terminus and the C-terminus of the Acr polypeptide, and / or one or more amino acids between the N-terminus and C-terminus of the Acr polypeptide. In an embodiment, one or more CPPs are directly fused or linked via a suitable linker to each of the N- terminus and the C-terminus of the Acr polypeptide, and / or one or more amino acids between the N-terminus and C-terminus of the Acr polypeptide. In an embodiment, one or more CPPs are chemically conjugated to the N-terminal amino acid, the C-terminal amino acid, and / or one or more amino acids between the N-terminal amino acid and the C-terminal amino acid of the Acr polypeptide. Suitable chemical conjugations will be appreciated by one of ordinary skill in the art. In an embodiment, site-specific bioconjugation of a CPP at one or more of the amino acids of the Acr polypeptide as previously described can be achieved using a cysteine bond formation (e.g., a tyrosine cysteine bond. See, e.g., Lobba et al., ACS Cent. Sci. 2020, 6(9), 1564-1571, which is incorporated by reference as if expressed in its entirety herein and can be adapted for use with the present invention.

[0123] In an embodiment, 6 CPPs are operatively coupled to the Acr polypeptide. In an embodiment, 4 CPPs are operatively coupled to the N-terminus of the Acr polypeptide, (b) wherein 2 CPPs are operatively coupled to the C-terminus of the Acr polypeptide, or (c) both (a) and (b).

[0124] CPPs may be of different sizes, amino acid sequences, and charges. In some examples, CPPs can translocate the plasma membrane and facilitate the delivery of various molecular cargoes to the cytoplasm or an organelle. CPPs may be introduced into cells via different mechanisms, e.g., direct penetration in the membrane, endocytosis-mediated entry, and translocation through the formation of a transitory structure.

[0125] In an embodiment, one or more of the CPPs has an amino acid composition that either contains a high (e g., greater than 30%, greater than 40%, greater than 50%, greater than 60%, greater than 70%, greater than 80%, greater than 90%, greater than 95%, or greater than 99%) relative abundance of positively charged amino acids such as lysine or arginine. In an embodiment, one or more of the CPPs has sequences that contain an alternating pattern of polar / charged amino acids and non-polar, hydrophobic amino acids. These two types of structures are referred to as polycationic or amphipathic, respectively. In an embodiment, one or more of the CPPs is a hydrophobic peptide, containing only apolar residues, with low net charge or have hydrophobic amino acid groups that are important for cellular uptake. In an embodiment, one or more of the CPPs is a trans-activating transcriptional activator (Tat) from Human Immunodeficiency Virus 1 (HIV-1). Examples of CPPs include Penetratin, Tat (48-60), Transportan, and (R-AhX-R4) (Ahx refers to aminohexanoyl), Kaposi fibroblast growth factor (FGF) signal peptide sequence, integrin P3 signal peptide sequence, polyarginine peptide Args sequence, Guanine rich-molecular transporters, sweet arrow peptide, penetratin 1 (CRQIKIWFQNRRMKWKK (SEQ ID NO: 112)), Transportan (CLIKKALAALAKLNIKLLYGASNLTWG (SEQ ID NO: 113)), Cholesteryl oligo- d-arginine (Cholesteryl-RRRRRRRRR (SEQ ID NO: 114)), EB1(LIRLWSHLIHIWFQNRRLKWKKK (SEQ ID NO: 115)), Bovine Prp (1-30) (MVKSKIGSWILVLFVAMWSDVGLCKKRPKP (SEQ ID NO: 116)), Stearyl-R8(Stearyl- RRRRRRRR (SEQ ID NO: 117)), TP 10 (AGYLLGKINLKALAALAKKIL (SEQ ID NO: 118)), Stearyl-TPIO (Stearyl-AGYLLGKINLKALAALAKKIL (SEQ ID NO: 119)), MPGANLS(GALFLGWLGAAGSTMGAPKSKRKV (SEQ ID NO: 120)), Stearyl-R9(Stearyl- RRRRRRRRR (SEQ ID NO: 114)), R9(RRRRRRRRR (SEQ ID NO: 114)), Penetratin2 RQIKIWFQNRRMKWKK (SEQ ID NO: 121), Stearyl-Penetratin2 (Stearyl- RQIKIWFQNRRMKWKK (SEQ ID NO: 121)), MPG(GALFLGFLGAAGSTMGAWSQPKKKRKV (SEQ ID NO: 122)), TAT-U1A RNA binding domain (GRKKRRQRRRPPQC-U1 A (SEQ ID NO: 123)), PTD-dsRNA binding domain (dsRBD) (MGRKKRRQRRRGHSGRKKRRQRRRGHIYPYDVPDYAGDPGRKKRRQRRR-dsRBD (SEQ ID NO: 124)), CADY-1 (Acetyl-GLWRALWRLLRSLWRLLWRA-cysteamide (SEQ ID NO: 125)), CADY-2 (GLWRALWRLLRSLWRLLWRA (SEQ ID NO: 125)), CADY-K (GLWRALWRLLRSLWRLLWK (SEQ ID NO: 126)), D-Cady-K (glwralwrllrslwrllwk (SEQ IDNO: 126)), S-CADY (GWRALWRLWRSLWRA (SEQ ID NO: 127)), CADY-H (GLWHALWHLLHSLWHLLWHA (SEQ ID NO: 128)), a Stearoyl peptide, (e g, Stearyl- CHHRRRRHHC (SEQ ID NO: 129) and Stearyl-GHHRRRRHHG (SEQ ID NO: 130)), PepFect6(Stear l-AGYLLGK(K(K2(trifluoromethylquinoline4)))INLKALAALAKKIL (SEQ ID NO: 131)), PepFectl4 (Stearyl-AGYLLGKLLOOLAAAALOOLL (SEQ ID NO: 132)), PLL-CA (Poly(L-ly sine-cholic acid), NickFect (Stearyl-TPIO analogs), Myr-TP-Transferrin targeting peptide (Tf) (Myristyl-GWTLNSAGYLLGKINLKALAALAKKIL-Tf (SEQ ID NO: 133)),Hphl-Hphl-dsRBD (YARVRRRGPRRGHYARVRRRGPRR-dsRBD (SEQ ID NO: 134)), TAT-TAT-dsRBD (RKKRRQRRRGHYPYDVPDYAGDRKKRRQRRR-dsRBD (SEQ ID NO: 135)), TAT-TAT-TAT-dsRBD(RKKRRQRRRGHYP YD VPD YAGDRKKRRQRRRGDPAGSRKKRRQRRRR-dsRBD ( SEQID NO: 136)), PTD4-PTD4-dsRBD(YARAAARQ ARARS YARAAARQARALQYP YD VPD YA-dsRBD (SEQ ID NO: 137)), PTD4- PTD4-MPG-MPG (YARAAARQ ARARS YARAAARQ AR-dsRBD (SEQ ID NO: 138)), Hphl- Hphl-dsRBD (YARVRRRGPRRGHYARVRRRGPRRRR-dsRBD (SEQ ID NO: 139)), Hphl-Hphl-dsRBD (YARVRRRGPRRGHYARVRRRGPRRRR-dsRBD (SEQ ID NO: 139)), CA-R8(Capryl-RRRRRRRR (SEQ ID NO: 117)), OA-R8(Oleyl-RRRRRRRR (SEQ ID NO: 117)), LA-R8(Linolyl-RRRRRRRR (SEQ ID NO: 117)), RGD10-10R(DGARYCRGDCFDGRRRRRRRRRR (SEQ ID NO: 140)), PSW(GLWRALWRLWRSLWRLLWKA (SEQ ID NO: 141)), PSR(GLWRALWRLLRSLWRLWRKA (SEQ ID NO: 142)), PG09(GLWRALWRALWRSLWRLKRKV (SEQ ID NO: 143)), PG16(GLWRALWRGLRSLWRLLWKV (SEQ ID NO: 144)), STR-KV (Stearyl-HHHKKKVVVVVV(SEQ ID NO: 145)), CKRRMKWKK (SEQ ID NO: 146), LMWP-PEG (VSRRRRGGRRRRRR- PEG (SEQ ID NO: 147)), NickFect51 (Stearyl-AGYLLGoOlNLKALAALAKKIL (SEQ ID NO: 148)), NickFect57 (Stearyl-AGYLLG8OINLKALAALAKAIL (SEQ ID NO: 149)), PepFect3 (Stearyl-AGYLLGKINLKALAALAKKI (SEQ ID NO: 150)), PL9R (l-myristoyl-2-(14- carboxymyristoyl)- n-glycero-3-phosphocholine-RRRRRRRRR (SEQ ID NO: 114)), RICK (re / ro-inverso form of CADY-K) (kwllrwlsrllrwlarwlg (SEQ ID NO: 151)), PEG-RICK (PEG- kwllrwlsrllrwlarwlg (SEQ ID NO: 151)), RALA(WEARLARALARALARHLARALARALRACEA (SEQ ID NO: 152)), gH625(HGLASTLTRWAHYNALIRAFGGG (SEQ ID NO: 153)), NickFect71 (Stearyl-HHYHHGO8ILLKALKALAKAIL (SEQ ID NO: 154)), NickFect700(HHHHYHHGO8ILLKALKALAKAIL (SEQ ID NO: 155)), NickFect704 (Stearyl-HHHHHHGO8ILLKALKALAKAIL (SEQ ID NO: 156)), NickFect707 (Stearyl- HHHHHHYLLGO8ILLKALKALAKAIL (SEQ ID NO: 157)), NickFect721 (Stearyl-HHHHHHYHHGO8ILLKALKALAKAIL (SEQ ID NO: 158)), KL4(AAKLLLLKLLLLKLLLLKLLLLK (SEQ ID NO: 159)), PEGI2KL4 (PEG- AAKLLLLKLLLLKLLLLKLLLLK (SEQ ID NO: 160)), LAH4(KKALLALALHHLAHLALHLALALKKA (SEQ ID NO: 161)), LAH4-L I (I< I<ALLAHALHLLALLALHLAHALI<I< A (SEQ ID NO: 162)) Melittin (GIGAVLKVLTTGLPALISWIKRKRQQ (SEQ ID NO: 163)), C 12-H5-S4I3-PV (Lauroyl-HHHHHALWKTLLKKVLKAPKKKRKVC (SEQ ID NO: 164)), H5-S4I3-PV-C12 (Acetyl-HHHHHALWKTLLKKVLKAPKKKRKVCK-Lauroyl (SEQ ID NO: 165)), HALA1(WEAHLAHALARALARHLARALARALRACEA (SEQ ID NO: 166)), HALA2 (WEARL ARAL ARAL ARHL ARAL AHALHACEA (SEQ ID NO: 167)), HALA3 (WEAHLAHALAHAL ARHL ARAL ARALRACEA (SEQ ID NO: 168)), HALA4(WEARLARALARALARHLAHALAHALHACEA (SEQ ID NO: 169)), OligoR (RRRRRRRRR (SEQ ID NO: 114)), OligoR-Aib (RRXRRXRRXRRXRRX (SEQ ID NO: 170)), (CP)6(Cyclo- DPDPDP (SEQ ID NO: 171)), Protein transduction domain (PTD)l (PFVYLI (SEQ ID NO: 172)), PTD2 (WSYGLRPG (SEQ ID NO: 173)), LMWP (VSRRRRRRGGRRRR (SEQ ID NO: 174)), P5RHH (VLTTGLPALISWIRRRHRRHC (SEQ ID NO: 175)), Pep-1 (KETWWETWWTEWSQPKKKRKV (SEQ ID NO: 176)), Tamra-LK (LKKLLKLLKKLLKLG(SEQ ID NO: 177)), Acetyl-LK (Acetyl-LKKLLKLLKKLLKLG (SEQ ID NO: 177)), and PFC- PR (RRRRR-perfluorocarbon-RGFLGR-perfluorocarbon-RRRRR (SEQ ID NO: 178)), CL (KVRVRVRVpPTRVRERVK (SEQ ID NO: 179)), BFPD (Crosslinked fluorinated poly(L- lysine) dendrimers), cRIO (see, e.g., Herce et al., Nat. Chem. 2017, 9, 762-771), and L17E (see, e g., Akishiba and Futaki, Mol. Pharm. 2019, 76(6), 2540-2548). Examples of other CPPs that may be used in the context of the present disclosure also include those described in U.S. Patent No. 8,372,951 as well as any other described herein in this specification.

[0126] In an embodiment, all the one or more CPPs are the same. In an embodiment, at least two, at least 3, at least 4, at least 5, or at least 6 of the one or more CPPs are different. In an embodiment, all of the one or more CPPs are different.Nuclear Localization Signals

[0127] In an embodiment, the one or more CPPs comprise one or more NLSs. In an embodiment, the engineered Acr polypeptide includes one or more nuclear localization signals (NLSs) at the C-terminus, the N-terminus, or both the N- and C- terminus of the Acr polypeptide. Without being bound by theory, such sequences may increase the transport of the Acr polypeptide to the nucleus of a cell. In an embodiment, 1-10 or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) NLSs are operatively coupled to the Acr polypeptide. In an embodiment, 6 NLSs are operatively coupled to the Acr polypeptide. In an embodiment, 4 NLSs are operatively coupled to the N- terminus of the Acr polypeptide, (b) wherein 2 NLSs are operatively coupled to the C-terminus of the Acr polypeptide, or (c) both (a) and (b). In an embodiment, all of the one or more CPPs are NLSs. In an embodiment, all the one or more NLSs are the same. In an embodiment, at least two, at least 3, at least 4, at least 5, or at least 6 of the one or more NLSs are different. In an embodiment, all of the one or more NLSs are different.

[0128] In an embodiment, the NLSs used in the context of the present disclosure are heterologous to the proteins. Non-limiting examples of NLSs include an NLS sequence derived from: the NLS of the SV40 virus large T-antigen, having the amino acid sequence PKKKRKV (SEQ ID NO: 180) or PKKKRKVEAS (SEQ ID NO: 181); the NLS from nucleoplasmin (e.g., the nucleoplasmin bipartite NLS with the sequence KRPAATKKAGQAKKKK (SEQ ID NO: 182)); the c-myc NLS having the amino acid sequence PAAKRVKLD (SEQ ID NO: 183) or RQRRNELKRSP (SEQ ID NO: 184); the hRNPAl M9 NLS having the sequence NQSSNFGPMKGGNFGGRSSGPYGGGGQYFAKPRNQGGY (SEQ ID NO: 185); the sequence RMRIZFKNKGKDTAELRRRRVEVSVELRKAKKDEQILKRRNV (SEQ ID NO: 186) of the IBB domain from importin-alpha; the sequences VSRKRPRP (SEQ ID NO: 187) and PPKKARED (SEQ ID NO: 188) of the myoma T protein; the sequence PQPKKKPL (SEQ ID NO: 189) of human p53; the sequence SALIKKKKKMAP (SEQ ID NO: 190) of mouse c-abl IV; the sequences DRLRR (SEQ ID NO: 191) and PKQKKRK (SEQ ID NO: 192) of the influenza virus NS1; the sequence RKLKKKIKKL (SEQ ID NO: 193) of the Hepatitis virus delta antigen;the sequence REKKKFLKRR (SEQ ID NO: 194) of the mouse Mxl protein; the sequence KRKGDEVDGVDEVAKKKSKK (SEQ ID NO: 195) of the human poly(ADP-ribose) polymerase; and the sequence RKCLQAGMNLEARKTKK (SEQ ID NO: 196) of the steroid hormone receptors (human) glucocorticoid, TAT peptide (GRKKRRQRRRPQ (SEQ ID NO: 197)), and RIO or any other polyarginine peptides, or any combination thereof. Additional NLSs that are suitable for use with the present invention as described herein are any of those in Srivaths et al., Bioinformation 2018, 7-7(3), 132-139; Bbhmova et al. Physiol. Res. 2018, 67(Suppl. 2), S267-S279; Lange et al., J. Biol. Chem. 2007, 252(8), 5101-5105; and Negi et al., Database 2015, 2015, bav003-bav003.Coupling the Acr Polypeptide and CPP

[0129] As previously discussed, the Acr polypeptide and the CPP(s) are operatively coupled. In an embodiment, the one or more CPPs are operatively coupled to the Acr polypeptide by fusing, in-frame, the Acr polypeptide and the one or more CPPs. In an embodiment, they are directly fused such that one or more of the CPP(s) is contiguous with the N-terminal amino acid of the Acr polypeptide, the C-terminal amino acid of the Acr polypeptide, or both. In an embodiment, one or more of the one or more CPPs are operatively coupled to the Acr polypeptide via a linker. In an embodiment, the linker is a peptide linker. In an embodiment, the peptide linker is a flexible peptide linker. In an embodiment, the peptide linker is a rigid peptide linker. In an embodiment, the linker is a non-cleavable linker. In an embodiment, the linker is a cleavable linker. In an embodiment, the cleavable linker is cleaved by an enzyme, light, radiation, a chemical reaction, and / or the like. In an embodiment, the linker is configured or otherwise capable of bioconjugation. For example, the linker can be used with or include a cysteine, which can allow for bioconjugation. In an embodiment, the one or more CPPs are operatively coupled to the Acr polypeptide via a GlySer (GS) linker with a cysteine between the GS linker and the cargo delivery molecule and / or Acr polypeptide. The term of art “Gly-Ser linker” refers to a group of linkers that contain at least 20 percent, at least 30 percent at least 40 percent, at least 50 percent, at least 60 percent, at least 70 percent, at least 80 percent, at least 90 percent, or 100 percent Glycine and Serine amino acid residues. In an embodiment, the linker includes one or more residues for bioconjugation reactions.

[0130] In an embodiment, the peptide linker has a sequence of GGGLLK (SEQ ID NO: 198). In an embodiment, the peptide linker has a sequence of GGGLLK (SEQ ID NO: 199), wherein L4and / or L5 are D-Leu. In an embodiment, the peptide linker has a sequence of GGG[GGS]?K (SEQ ID NO: 200). In an embodiment, the peptide linker has a sequence of GGG[GGS]?K (SEQ ID NO: 201), where S is L-Ser and / or K is L-Lys. In an embodiment, the peptide linker contains an Ne- linked a-bromoacetyl group. In an embodiment, the peptide linker contains an NMinked maleimide group. In an embodiment, the peptide linker is linker peptide 1, 2, or 3 of Lu et al., ACS Cent. Sci. 2021, 7, 365-378. In an embodiment, the peptide linker comprises LPSTGGK (SEQ ID NO: 202). In an embodiment, the linker is or contains GGGGCGGGGS (SEQ ID NO: 203). In an embodiment, the linker is a GlySer linker. Additional exemplary linkers include those set forth in Chen et al., Adv. Drug Deliv. Rev. 2013, 65(10), 1357-1369; van Rosmalen et al., Biochemistry 2017, 56(50), 6565-6574e; a Proline 9 (P9) linker,GAAPAAAPAKQEAAAPAPAAKAEAPAAAPAAKA (SEQ ID NO: 204), (GGGGS)s (SEQ ID NO: 205), (G)8(SEQ ID NO: 206), (G)6(SEQ ID NO: 207), (EAAAK)3(SEQ ID NO: 208), (EAAAK)n (n=l-3) (SEQ ID NO: 209-210, 208), A(EAAAK)4ALEA(EAAAK)4A (SEQ ID NO: 211), GGGGS (SEQ ID NO: 212), PAPAP (SEQ ID NO: 213), AEAAAKEAAAKA (SEQ ID NO: 214), (GGGGS)n (n=l-10) (SEQ ID NO: 212, 215-223), (Ala-Pro)n (n=10-32) (SEQ ID NO: 224-246), disulfide, VSQTSKLTR^AETVFPDV (SEQ ID NO: 247), PLGJ.LWA (SEQ ID NO: 248), RVL^AEA (SEQ ID NO: 249); EDVVCQSMSY (SEQ ID NO: 250); GGIEGR^GS (SEQ ID NO: 251), TRHRQPRjGWE (SEQ ID NO: 252); AGNRVRR|SVG (SEQ ID NO: 253); RRRRRRR R|R (SEQ ID NO: 254), GFLG| (SEQ ID NO: 255), LE, LEAGCKNFFPR|SFTSCGSLE (SEQ ID NO: 256), CRRRRRREAEAC (SEQ ID NO: 257), a TEV site linker, e.g., ENLYFQ|(S, G, A, M, C, or H) (SEQ ID NO: 258), (Protease-sensitive cleavage sites are indicated with “J,”) or any combination thereof. In an embodiment, the linker is or comprises LPSTGGK (SEQ ID NO: 202). Other suitable linkers will be appreciated by those of ordinary skill in the art in view of the description herein.

[0131] In an embodiment, the one or more CPPs are operatively coupled to the N-terminus of the Acr polypeptide, the C-terminus of the Acr polypeptide, between the N- and C-terminus of the Acr polypeptide, or any combination thereof. In an embodiment, the one or more CPPs are operatively coupled to the N-terminus of the Acr polypeptide, the C-terminus of the Acr polypeptide, or both the N-terminus of the Acr polypeptide and the C-terminus of the Acr polypeptide. In an embodiment, the one or more CPPs are cleavably coupled to the Acrpolypeptide. In an embodiment, the one or more CPPs are cleavably coupled to the Acr polypeptide such that cleavage occurs via protease, deubiquitinase, small molecule, reducing agent, metabolite, an external stimulus (e.g., a light energy, pH, osmolarity, a magnetic energy, a thermal energy, an acoustic energy, etc.) etc. In an embodiment, the protease, deubiquitinase, small molecule, reducing agent, metabolite, is native to a cytosol. In an embodiment, the protease, deubiquitinase, small molecule, reducing agent, metabolite, is exogenous to a cytosol. In an embodiment, the one or more CPPs have a cleavable domain. In an embodiment, one or more CPPs that contain a cleavable domain is fused, in frame, to the Acr polypeptide. In an embodiment, the one or more CPPs are linked via a cleavable linker to the Acr polypeptide. Exemplary cleavable linkers are described elsewhere herein.Targeting Moieties

[0132] As previously discussed, in an embodiment, the engineered Acr polypeptide and / or Acr polypeptide contains or is operatively coupled (e.g., fused to or linked) to a targeting moiety, which includes without limitation targeting molecules and targeting domains. Without being bound by theory, the targeting moiety is capable of recognizing, binding, attaching to, or otherwise interacting with a binding partner that can be present on the surface of a target cell. This can direct where the engineered Acr polypeptide is delivered. In an embodiment, the target cell contains a CRISPR-Cas system or a component thereof. In an embodiment, the targeting moiety targets a receptor that is present on all cell types. In an embodiment, the targeting moiety targets a receptor that is present on multiple cell types. In an embodiment, the targeting moiety targets a receptor that is present on a single cell type. In an embodiment, the targeting moiety targets a specific cell or tissue type and / or cell state. As used herein, “cell state” is used to describe transient elements of a cell’s identity. Cell state can be thought of as the transient characteristic profile or phenotype of a cell. Cell states arise transiently during time-dependent processes, either in a temporal progression that is unidirectional (e.g., during differentiation, or following an environmental stimulus) or in a state vacillation that is not necessarily unidirectional and in which the cell may return to the origin state. Vacillating processes can be oscillatory (e.g., cell-cycle or circadian rhythm) or can transition between states with no predefined order (e.g., due to stochastic, or environmentally controlled, molecular events). These time-dependent processes may occur transiently within a stable cell type (as in a transient environmental response), or may lead to anew, distinct type (as in differentiation). See, e.g., Wagner et al., Nat. Biotechnol. 2016, 34(11), 1145-1160. Exemplary targeting moieties and binding partners are discussed below.

[0133] In an embodiment, the targeting moiety is or includes a peptide or a polypeptide. In an embodiment, the targeting moiety is or includes an antibody or fragment thereof. In an embodiment, the targeting moiety is or includes an aptamer. In an embodiment, the targeting moiety is or includes a small molecule. In an embodiment, the targeting moiety is or includes a nucleic acid (e.g., DNA or RNA). In an embodiment, the targeting moiety is or includes a receptor. In an embodiment, the targeting moiety is or includes a receptor ligand. In an embodiment, the targeting moiety is or includes a carbohydrate (e g., a sugar). In an embodiment, the targeting moiety is or includes a lipid. In an embodiment, the targeting moiety is an engineered protein scaffold. In an embodiment, the targeting moiety is an affibody. In an embodiment, the targeting moiety is an antibody mimetic. In an embodiment, the targeting moiety is an engineered binding protein, such as a designed ankyrin repeat proteins (DARPins) (see, e.g., Pliickthun et al., Annu. Rev. Pharmacol. Toxicol. 2015, 55(1), 489-511), avimers (Silverman et al., Nat. Biotechnol. 2005, 23(12), 1556-1561and Jeong et al., Nat. Biotechnol., 2005, 23(12), 1493-1494), or affibodies (see, e.g., Nord et al., Nat. Biotechnol. 1997, 15, 772-777). In an embodiment, the targeting moiety is a receptor ligand or binding protein.

[0134] In an embodiment, the targeting moiety targets an anthrax receptor. In an embodiment, the targeting moiety targets a cell adhesion molecule, selectin, or syndecan. In an embodiment, the targeting moiety targets an integrin.

[0135] The term “antibody” is used interchangeably with the term “immunoglobulin” herein, and includes intact antibodies, fragments of antibodies, e.g., Fab, F(ab')2 fragments, and intact antibodies and fragments that have been mutated either in their constant and / or variable region (e.g., mutations to produce chimeric, partially humanized, or fully humanized antibodies, as well as to produce antibodies with a desired trait, e.g., enhanced binding and / or reduced Immunoglobulin Fc receptor (FcR) binding). “Antibody” includes monovalent and multivalent antibodies. The term “fragment” refers to a part or portion of an antibody or antibody chain comprising fewer amino acid residues than an intact or complete antibody or antibody chain. Fragments can be obtained via chemical or enzymatic treatment of an intact or complete antibodyor antibody chain. Fragments can also be obtained by recombinant means. Exemplary fragments include Fab, Fab', F(ab')2, Fabc, Fd, dAb, VHH and scFv and / or Fv fragments.

[0136] As used herein, a preparation of antibody protein having less than about 50% of nonantibody protein (also referred to herein as a “contaminating protein”), or of chemical precursors, is considered to be “substantially free.” In an embodiment, a preparation of antibody protein having less than about 40%, 30%, 20%, 10% and more preferably 5% (by dry weight), of nonantibody protein, or of chemical precursors is considered to be substantially free. When the antibody protein or biologically active portion thereof is recombinantly produced, it is also preferably substantially free of culture medium, i.e., culture medium represents less than about 30%, preferably less than about 20%, more preferably less than about 10%, and most preferably less than about 5% of the volume or mass of the protein preparation.

[0137] As used herein, “nanobody” refers to a single-domain antibody fragment that is capable of specifically binding an antigen. Nanobodies can be engineered to have desired antigen-binding capabilities. Nanobodies can be based on heavy-chain or light-chain domains. See, e.g., Arbabi Ghahroudi et al., FEBS Lett. 1997, 414, 521-526; Ward et al., Nature 1989, 341, 544-546; Holt et al., Trends Biotechnol. 2003, 2 / (11), 484-490; Borrebaeck et al., Nat. Biotechnol. 2002, 20, 1189- 1190; Van de Broek et al., ACS Nano 2011, 5(6), 4319-4328.

[0138] As used herein, the term “antigen-binding fragment” refers to a polypeptide fragment of an immunoglobulin or antibody that binds antigen or competes with intact antibody (i.e., with the intact antibody from which they were derived) for antigen binding (i.e., specific binding). As such these antibodies or fragments thereof are included in the scope of the invention, provided that the antibody or fragment binds specifically to a target molecule.

[0139] It is intended that the term “antibody” encompass any Ig class or any Ig subclass (e.g., the IgGl, IgG2, IgG3, and IgG4 subclasses of IgG) obtained from any source (e.g., humans and non-human primates, and in rodents, lagomorphs, caprines, bovines, equines, ovines, etc.).

[0140] The term “Ig class” or “immunoglobulin class”, as used herein, refers to the five classes of immunoglobulin that have been identified in humans and higher mammals, IgG, IgM, IgA, IgD, and IgE. The term “Ig subclass” refers to the two subclasses of IgM (H and L), three subclasses of IgA (IgAl, IgA2, and secretory IgA), and four subclasses of IgG (IgGl, IgG2, IgG3, and IgG4) that have been identified in humans and higher mammals. The antibodies can exist in monomericor polymeric form; for example, IgM antibodies exist in pentameric form, and IgA antibodies exist in monomeric, dimeric, or multimeric form.

[0141] The term “IgG subclass” refers to the four subclasses of immunoglobulin class IgG — IgGl, IgG2, IgG3, and IgG4 that have been identified in humans and higher mammals by the heavy chains of the immunoglobulins, VI — y4, respectively. The term “single-chain immunoglobulin” or “single-chain antibody” (used interchangeably herein) refers to a protein having a two- polypeptide chain structure consisting of a heavy and a light chain, said chains being stabilized, for example, by interchain peptide linkers, which has the ability to specifically bind the antigen. The term “domain” refers to a globular region of a heavy or light chain polypeptide comprising peptide loops (e.g., comprising 3 to 4 peptide loops) stabilized, for example, by a P pleated sheet and / or intrachain disulfide bond. Domains are further referred to herein as “constant” or “variable,” based on the relative lack of sequence variation within the domains of various class members in the case of a “constant” domain, or the significant variation within the domains of various class members in the case of a “variable” domain. Antibody or polypeptide “domains” are often referred to interchangeably in the art as antibody or polypeptide “regions.” The “constant” domains of an antibody light chain are referred to interchangeably as “light chain constant regions,” “light chain constant domains,” “CL” regions, or “CL” domains. The “constant” domains of an antibody heavy chain are referred to interchangeably as “heavy chain constant regions,” “heavy chain constant domains,” “CH” regions, or “CH” domains). The “variable” domains of an antibody light chain are referred to interchangeably as “light chain variable regions,” “light chain variable domains,” “VL” regions, or “VL” domains). The “variable” domains of an antibody heavy chain are referred to interchangeably as “heavy chain variable regions,” “heavy chain variable domains,” “VH” regions, or “VH” domains). In an embodiment, the VH domain is a human VH domain.

[0142] The term “region” can also refer to a part or portion of an antibody chain or antibody chain domain (e.g., a part or portion of a heavy or light chain or a part or portion of a constant or variable domain, as defined herein), as well as more discrete parts or portions of said chains or domains. For example, light and heavy chains or light and heavy chain variable domains include “complementarity determining regions” or “CDRs” interspersed among “framework regions” or “FRs”, as defined herein.

[0143] The term “conformation” refers to the tertiary structure of a protein or polypeptide (e.g., an antibody, antibody chain, domain, or region thereof). For example, the phrase “light (or heavy) chain conformation” refers to the tertiary structure of a light (or heavy) chain variable region, and the phrase “antibody conformation” or “antibody fragment conformation” refers to the tertiary structure of an antibody or fragment thereof.

[0144] As used herein, “affibody” refers to small (typically around 6.5 kDa) non- immunoglobulin-engineered proteins based on a three-helix bundle domain framework that is based on a 58-amino-acid Z-domain scaffold, derived from one of the IgG-binding domains of staphylococcal protein A and can be engineered for desired target recognition. See, e g., Frejd et al., Exp. Mol. Med. 2017, 49, e306-e306; Lofblom et al., FEBS Lett. 2010, 584, 2670-2680; and Nygren, P. FEBS J. 2008, 275(11), 2668-2676.

[0145] The term “antibody-like protein scaffolds” or “engineered protein scaffolds” broadly encompasses proteinaceous non-immunoglobulin specific-binding agents, typically obtained by combinatorial engineering (such as site-directed random mutagenesis in combination with phage display or other molecular selection techniques). Usually, such scaffolds are derived from robust and small soluble monomeric proteins (such as Kunitz inhibitors or lipocalins) or from a stably folded extra-membrane domain of a cell surface receptor (such as protein A, fibronectin, or the ankyrin repeat).

[0146] Such scaffolds have been extensively reviewed in Binz et al., Nat. Biotechnol. 2005, 23(10), 1257-1268; Gebauer et al., Curr. Opin. Chem. Biol. 2009, 13, 245-255; Gill et al., Curr. Opin. Chem. Biol. 2006, 77(6), 653-658; Skerra et al., J. Mol. Recognit. 2000, 73(4), 167-187; and Skerra et al., Curr. Opin. Biotechnol. 2007, 75(4), 295-304; and include without limitation affibodies, based on the Z-domain of staphylococcal protein A, a three-helix bundle of 58 residues providing an interface on two of its alpha-helices (Nygren et al., FEBS J. 2008, 275, 2668-2676); engineered Kunitz domains based on a small (ca. 58 residues) and robust, disulfide-crosslinked serine protease inhibitor, typically of human origin (e.g., LACI-D1), which can be engineered for different protease specificities (Nixon et al., Curr. Opin. Drug Discovery Dev. 2006, 9(2), 261- 268); monobodies or adnectins based on the 10thextracellular domain of human fibronectin III (10Fn3), which adopts an Ig-like beta-sandwich fold (94 residues) with 2-3 exposed loops, but lacks the central disulfide bridge (Koide et al., Methods Mol. Biol. 2007, 352, 95-109); anticalinsderived from the lipocalins, a diverse family of eight-stranded beta-barrel proteins (ca. 180 residues) that naturally form binding sites for small ligands by means of four structurally variable loops at the open end, which are abundant in humans, insects, and many other organisms (Skerra et al., FEBS J. 2008, 275, 2677-2683); DARPins, designed ankyrin repeat domains (166 residues), which provide a rigid interface arising from typically three repeated beta-turns (Stumpp et al., Drug Discov. Today 2008, 13, 695-701); avimers (multimerized LDLR-A module) (Silverman et al., Nat. Biotechnol. 2005, 23, 1556-1561); and cysteine-rich knottin peptides (Kolmar et al., FEBS J. 2008, 275, 2684-2690).

[0147] In certain embodiments, the targeting moiety is an aptamer. Nucleic acid aptamers are nucleic acid species that have been engineered through repeated rounds of in vitro selection or equivalently, SELEX (systematic evolution of ligands by exponential enrichment) to bind to various molecular targets such as small molecules, proteins, nucleic acids, cells, tissues, and organisms. Nucleic acid aptamers have a specific binding affinity to molecules through interactions other than classic Watson-Crick base pairing. Aptamers are useful in biotechnological and therapeutic applications as they offer molecular recognition properties similar to antibodies. In addition to their discriminate recognition, aptamers offer advantages over antibodies as they can be engineered completely in a test tube, are readily produced by chemical synthesis, possess desirable storage properties, and elicit little or no immunogenicity in therapeutic applications. In certain embodiments, RNA aptamers may be expressed from a DNA construct. In other embodiments, a nucleic acid aptamer may be linked to another polynucleotide sequence. The polynucleotide sequence may be a double- stranded DNA polynucleotide sequence. The aptamer may be covalently linked to one strand of the polynucleotide sequence. The aptamer may be ligated to the polynucleotide sequence. The polynucleotide sequence may be configured, such that the polynucleotide sequence may be linked to a solid support or ligated to another polynucleotide sequence.

[0148] Aptamers, like peptides generated by phage display or monoclonal antibodies (“mAbs”), are capable of specifically binding to selected targets and modulating the target’s activity, e g., through binding, aptamers may block their target’s ability to function. A typical aptamer is 10-15 kDa in size (30-45 nucleotides), binds its target with sub-nanomolar affinity, and discriminates against closely related targets (e.g., aptamers will typically not bind other proteinsfrom the same gene family). Structural studies have shown that aptamers are capable of using the same types of binding interactions (e.g., hydrogen bonding, electrostatic complementarity, hydrophobic contacts, steric exclusion) that drives affinity and specificity in antibody-antigen complexes.

[0149] Aptamers have a number of desirable characteristics for use in research and as therapeutics and diagnostics including high specificity and affinity, biological efficacy, and excellent pharmacokinetic properties. In addition, they offer specific competitive advantages over antibodies and other protein biologies. Aptamers are chemically synthesized and are readily scaled as needed to meet production demand for research, diagnostic, or therapeutic applications. Aptamers are chemically robust. They are intrinsically adapted to regain activity following exposure to factors such as heat and denaturants and can be stored for extended periods (>1 yr) at room temperature as lyophilized powders. Not being bound by theory, aptamers bound to a solid support or beads may be stored for extended periods.

[0150] Oligonucleotides in their phosphodiester form may be quickly degraded by intracellular and extracellular enzymes such as endonucleases and exonucleases. Aptamers can include modified nucleotides conferring improved characteristics on the ligand, such as improved in vivo stability or improved delivery characteristics. Examples of such modifications include chemical substitutions at the ribose and / or phosphate and / or base positions. SELEX-identified nucleic acid ligands containing modified nucleotides are described, e.g., in U.S. Patent No. 5,660,985, which describes oligonucleotides containing nucleotide derivatives chemically modified at the 2’ position of ribose, 5 position of pyrimidines, and 8 position of purines, U.S. Patent No. 5,756,703, which describes oligonucleotides containing various 2’-modified pyrimidines, and U.S. Patent No. 5,580,737, which describes highly specific nucleic acid ligands containing one or more nucleotides modified with 2’-amino (2’-NH2), 2’-fluoro (2’-F), and / or 2’-O-methyl (2’-0me) substituents. Modifications of aptamers may also include, modifications at exocyclic amines, substitution of 4- thiouridine, substitution of 5-bromo or 5 -iodo-uracil; backbone modifications, phosphorothioate or allyl phosphate modifications, methylations, and unusual base-pairing combinations such as the isobases isocytidine and isoguanosine. Modifications can also include 3’ and 5’ modifications such as capping. As used herein, the term phosphorothioate encompasses one or more non-bridging oxygen atoms in a phosphodiester bond replaced by one or more sulfur atoms. In furtherembodiments, the oligonucleotides comprise modified sugar groups, for example, one or more of the hydroxyl groups is replaced with halogen, aliphatic groups, or functionalized as ethers or amines. In one embodiment, the 2’ -position of the furanose residue is substituted by any of an O- methyl, O-alkyl, O-allyl, S-alkyl, S-allyl, or halo group. Methods of synthesis of 2’ -modified sugars are described, e.g., in Sproat et al., Nucleic Acids Res. 1991, 79(4), 733-738; Tumour et al., Nucleic Acids Res. 1991, 79(10), 2629-2635; and Hobbs et al., Biochemistry 1973, 72(25), 5138-5145. Other modifications are known to one of ordinary skill in the art. In certain embodiments, aptamers include aptamers with improved off-rates as described in International Patent Publication No. WO 2009 / 012418, which is incorporated herein by reference in its entirety. In certain embodiments, aptamers are chosen from a library of aptamers. Such libraries include, but are not limited to, those described in Rohloff et al., Mol. Ther. Nucleic Acids 2014, 3, e201. Aptamers are also commercially available (see, e.g., SomaLogic, Inc., Boulder, Colorado). In certain embodiments, the present invention may utilize any aptamer containing any modification as described herein.

[0151] In an embodiment, the targeting moiety is a small molecule, such as a small molecule receptor ligand. The term “small molecule” refers to compounds, preferably organic compounds, with a size comparable to those organic molecules generally used in pharmaceuticals. The term excludes biological macromolecules (e.g., proteins, peptides, nucleic acids, etc.). Preferred small organic molecules range in size up to about 5000 Da, e.g., up to about 4000, preferably up to 3000 Da, more preferably up to 2000 Da, even more preferably up to about 1000 Da, e.g., up to about 900, 800, 700, 600 or up to about 500 Da. In certain embodiments, the small molecule may act as an antagonist or agonist (e.g., blocking an enzyme active site or activating a receptor by binding to a ligand binding site).

[0152] The targeting moiety can be capable of specifically binding, attaching, or otherwise interacting with a binding partner (also referred to herein as a “specific binding partner”) on a target cell. In an embodiment, the specific binding partner, and thus the target cell, is predetermined.

[0153] As used herein, the term “specific binding” refers to non-covalent physical association of a first and a second moiety wherein the association between the first and second moieties is at least 2 times as strong, at least 5 times as strong as, at least 10 times as strong as, at least 50 timesas strong as, at least 100 times as strong as, or stronger than the association of either moiety with most or all other moieties present in the environment in which binding occurs. The binding of two or more entities may be considered specific if the equilibrium dissociation constant, Kd, is 103M or less, 104M or less, I O5M or less, 106M or less, 107M or less, 108M or less, 109M or less, 1010M or less, 10 " M or less, or 1012M or less under the conditions employed, e.g., under physiological conditions such as those inside a cell or consistent with cell survival. In an embodiment, specific binding can be accomplished by a plurality of weaker interactions (e.g., a plurality of individual interactions, wherein each individual interaction is characterized by a Kd of greater than 103M). In an embodiment, specific binding, which can be referred to as “molecular recognition,” is a saturable binding interaction between two entities that is dependent on complementary orientation of functional groups on each entity. Examples of specific binding interactions include primer-polynucleotide interaction, aptamer-aptamer target interactions, antibody-antigen interactions, avidin-biotin interactions, ligand-receptor interactions, metalchelate interactions, hybridization between complementary nucleic acids, etc.

[0154] Exemplary target cells include, but are not limited to liver cells, pancreatic cells, muscle cells (e.g., skeletal, cardiac, and / or smooth muscle cells), brain cells, neurons, nerve support cells (e.g., glial cells, Schwann cells, astrocytes, dendrites, etc.), immune cells (T-cells, B-cells, monocytes, macrophages, dendritic cells, NK cells, neutrophils, plasma cells, etc.), kidney cells, thyroid cells, bone cells, gastrointestinal tract cells, auditory cells (e.g., hair cells), eye cells (e.g., retinal cells, corneal cells, etc.), skin cells, lung cells, adipocytes, bladder cells, olfactory cells, vasculature cells, cancer cells, tumour cells, cancer stem cells, and / or the like. In an embodiment, the target cells are diseased. In an embodiment, the target cells are normal (non-diseased). In an embodiment, the target cells are progenitor cells. In an embodiment, the target cells are differentiated cells. In an embodiment, the target cells contain a CRISPR-Cas system or component thereof. In an embodiment, the target cell is also a target cell of a CRISPR-Cas system (i.e., a cell in which it is desirable that a CRISPR-Cas system be active in). In an embodiment, the target cell is not an intended target cell of a CRISPR-Cas system (i.e., a cell in which it is not desirable that a CRISPR-Cas system be active in). Exemplary targeting moieties are described in Table 2. Other suitable targeting moieties will be appreciated by those of skill in the art in view of the description herein.Reporter Molecules and Tags

[0155] In certain example embodiments, the engineered Acr polypeptide further comprises a reporter molecule operatively coupled to the Acr polypeptide and / or any other component of the engineered Acr polypeptide (e.g., the one or more CPPs, reporter molecule or tags, etc.).

[0156] Exemplary reporter proteins and tags include, but are not limited to, affinity tags, such as chitin binding protein (CBP), maltose-binding protein (MBP), glutathione-S-transferase (GST), poly(His) tag; solubilization tags such as thioredoxin (TRX) and poly(NANP), MBP, and GST; chromatography tags such as those consisting of polyanionic amino acids, such as FLAG-tag; epitope tags such as V5-tag, Myc-tag, HA-tag and NE-tag; protein tags that can allow specific enzymatic modification (such as biotinylation by biotin ligase) or chemical modification (such as reaction with FlAsH-EDT2 for fluorescence imaging), DNA and / or RNA segments that contain restriction enzyme or other enzyme cleavage sites; DNA segments that encode products that provide resistance against otherwise toxic compounds including antibiotics, such as spectinomycin, ampicillin, kanamycin, tetracycline, Basta, neomycin phosphotransferase II (NEO), hygromycin phosphotransferase (HPT) and the like; DNA and / or RNA segments that encode products that are otherwise lacking in the recipient cell (e g., tRNA genes, auxotrophic markers); DNA and / or RNA segments that encode products which can be readily identified (e.g., phenotypic markers such as P-galactosidase, P-glucuronidase (GUS)); fluorescent proteins such as green fluorescent protein (GFP), cyan fluorescent protein (CFP), yellow fluorescent protein (YFP), red fluorescent protein (RFP), mCherry, or other optically active proteins e g., luciferase, and cell surface proteins); optically active dyes (e.g., fluorescent, UV, IR, and NIR dyes), polynucleotides that can generate one or more new primer sites for PCR (e.g., the juxtaposition of two DNA sequences not previously juxtaposed), DNA sequences not acted upon or acted upon by a restriction endonuclease or other DNA modifying enzyme, chemical, etc.; epitope tags (e.g., GFP, FLAG- and His-tags), and, DNA sequences that make a molecular barcode or unique molecularidentifier (UMI), DNA sequences required for a specific modification (e.g., methylation) that allows its identification. Other suitable markers will be appreciated by those of skill in the art.Secondary Delivery Enhancers

[0157] In an embodiment, the engineered Acr polypeptide includes one or more secondary delivery enhancer molecules operatively coupled to the Acr polypeptide or other component of the engineered Acr polypeptide (e.g., the one or more CPPs, reporter molecule or tags, etc.). The term “secondary delivery enhancers” as used herein, refers to any molecule (e.g., chemical, biological (e.g., DNA, RNA, peptide, lipid, etc.)) that is in addition to the one or more CPPs that can further enhance delivery of the Acr polypeptide to a target cell. In an embodiment, the secondary delivery enhancer is an endosomal escape molecule. As used herein, “endosomal escape molecule” refers to any molecule (e.g., chemical, biological (e.g., DNA, RNA, peptide, lipid, etc.)) that increases endosomal escape of one or more molecules to which it is operatively coupled to. In an embodiment, the one or more endosomal escape molecules are or include one or more endosomal escape peptides. In an embodiment, the endosomal one or more escape peptides is individually selected from HA2, GALA (SEQ ID NO: 259), GALA3 (SEQ ID NO: 259), and aurein (GLFDIKKIAESF (SEQ ID NO: 260)) or a CPP. In an embodiment, the one or more endosomal escape peptides are capable of disrupting a cell membrane at an acidic pH. In an embodiment, the endosomal escape peptide is a CPP. Exemplary CPPs are described elsewhere herein. In an embodiment, a covalent endosomal escape molecule and a non-covalent endosomal escape molecule can be attached to the engineered Acr polypeptide of the present invention, similar to that set forth in Zhang et al., Nat. Biotechnol. 2024, 42(2), 305-315, which can be adapted for use with the present invention.

[0158] In an embodiment, the endosomal escape molecule is a protein-based endosomal enhancer. Exemplary protein-based endosomal enhancers that can be incorporated into the engineered Acr polypeptide of the present disclosure include, without limitation, Perfringolysin O, Listeriolysin O, Phospholipase A2, and Phospholipase C. In an embodiment, the endosomal escape molecule is a pore-forming protein. Exemplary pore-forming protein endosomal enhancers that can be incorporated into the engineered Acr polypeptide of the present disclosure include perfringolysin O (PFO), listeriolysin O (LLO), and protective antigen (see, e.g., Sharma and Collier, Biochem. 2014, 53(44), 6934-6940). In an embodiment where the endosomal enhancer isor includes protective antigen, a polylysine tag can be operatively coupled to the engineered Acr and / or other cargo to facilitate interaction between the protective antigen pore and translocation of the engineered Acr and / or other cargo to the cytosol. In an embodiment, the polylysine tag is an NLS, including but not limited to an SV40 NLS. Other exemplary NLSs that can be used in this context are described elsewhere herein.Polynucleotides and Vectors

[0159] Described in certain example embodiments herein are polynucleotides encoding an engineered Acr polypeptide of the present invention. As used herein, “nucleic acid,” “nucleotide sequence,” and “polynucleotide” can be used interchangeably herein and can generally refer to a string of at least two base-sugar-phosphate combinations and refers to, among others, single- and double-stranded DNA, DNA that is a mixture of single-and double-stranded regions, single- and double-stranded RNA, and RNA that is a mixture of single- and double-stranded regions, hybrid molecules comprising DNA and RNA that may be single-stranded or, more typically, doublestranded or a mixture of single- and double-stranded regions. In addition, polynucleotide as used herein can refer to triple-stranded regions comprising RNA or DNA or both RNA and DNA. The strands in such regions can be from the same molecule or from different molecules. The regions may include all of one or more of the molecules, but more typically involve only a region of some of the molecules. One of the molecules of a triple-helical region often is an oligonucleotide. “Polynucleotide” and “nucleic acids” also encompass such chemically, enzymatically, or metabolically modified forms of polynucleotides, as well as the chemical forms of DNA and RNA characteristic of viruses and cells, including simple and complex cells, inter alia. For instance, the term polynucleotide as used herein can include DNAs or RNAs as described herein that contain one or more modified bases. Thus, DNAs or RNAs including unusual bases, such as inosine, or modified bases, such as tritylated bases, to name just two examples, are polynucleotides as the term is used herein. “Polynucleotide”, “nucleotide sequences” and “nucleic acids” also includes PNAs (peptide nucleic acids), phosphorothioates, phosphorodiamidate morpholino oligomers, and other variants of the phosphate backbone of native nucleic acids. Natural nucleic acids have a phosphate backbone, artificial nucleic acids can contain other types of backbones, but contain the same bases. Thus, DNAs or RNAs with backbones modified for stability or for other reasons are “nucleic acids” or “polynucleotides” as that term is intended herein. As used herein, “nucleic acidsequence” and “oligonucleotide” also encompass a nucleic acid and polynucleotide as defined elsewhere herein. As used herein “encode”, “encoding,” and / or the like refers to the general principle that DNA is transcribed into an RNA product, which in some cases is translated into amino acids that form polypeptides. Thus, a protein-encoding polynucleotide is a polynucleotide that encodes an RNA product that is translated into the protein. In an embodiment, the polynucleotides are codon-optimized. Codon optimization of polynucleotides is described elsewhere herein, see, e.g., below with respect to “vector polynucleotides”. In an embodiment, the polynucleotides are included in a vector or vector system. In an embodiment, the polynucleotides are not included in a vector or vector system. In an embodiment, the polynucleotides are contained in a delivery vehicle. Delivery vehicles are described in greater detail elsewhere herein.Vectors and Vector systems

[0160] Described in certain example embodiments herein are vector systems comprising one or more vectors comprising one or more polynucleotides of the present invention encoding an engineered Acr polypeptide of the present invention or component thereof of the present invention; and optionally one or more regulatory elements operatively coupled to the one or more polynucleotides.

[0161] In certain embodiments, the vector can contain one or more polynucleotides encoding one or more elements of an engineered Acr polypeptide (e.g., an Acr polypeptide, a CPP, targeting moiety, reporter molecule, etc.) described herein. The vectors can be useful in producing bacterial, fungal, yeast, plant cells, animal cells, and transgenic animals that can express one or more components of the engineered Acr polypeptide described herein. Within the scope of this disclosure are vectors containing one or more of the polynucleotide sequences described herein. One or more of the polynucleotides that are part of the engineered Acr polypeptide described herein can be included in a vector or vector system. The vectors and / or vector systems can be used, for example, to express one or more of the polynucleotides in a cell, such as a producer cell, to produce virus particles containing an engineered Acr polypeptide described elsewhere herein. Other uses for the vectors and vector systems described herein are also within the scope of this disclosure. In general, and throughout this specification, the term “vector” refers to a tool that allows or facilitates the transfer of an entity from one environment to another. In some contexts which will be appreciated by those of ordinary skill in the art, “vector” can be a term of art to referto a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. A vector can be a replicon, such as a plasmid, phage, or cosmid, into which another DNA segment may be inserted so as to bring about the replication of the inserted segment. Generally, a vector is capable of replication when associated with the proper control elements.

[0162] Vectors include, but are not limited to, nucleic acid molecules that are single-stranded, double-stranded, or partially double-stranded; nucleic acid molecules that comprise one or more free ends, no free ends (e.g., circular); nucleic acid molecules that comprise DNA, RNA, or both; and other varieties of polynucleotides known in the art. One type of vector is a “plasmid,” which refers to a circular double-stranded DNA loop into which additional DNA segments can be inserted, such as by standard molecular cloning techniques. Another type of vector is a viral vector, wherein virally-derived DNA or RNA sequences are present in the vector for packaging into a virus (e.g., retroviruses, replication defective retroviruses, adenoviruses, replication defective adenoviruses, and adeno-associated viruses (AAVs)). Viral vectors also include polynucleotides carried by a virus for transfection into a host cell. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) are integrated into the genome of a host cell upon introduction into the host cell and, thereby, are replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as “expression vectors.” Common expression vectors of utility in recombinant DNA techniques are often in the form of plasmids.

[0163] Recombinant expression vectors can be composed of a nucleic acid (e.g., a polynucleotide) of the invention in a form suitable for expression of the nucleic acid in a host cell, which means that the recombinant expression vectors include one or more regulatory elements, which can be selected on the basis of the host cells to be used for expression, that is operatively- linked to the nucleic acid sequence to be expressed. Within a recombinant expression vector, “operatively linked” and “operatively-linked” are used interchangeably herein and further defined elsewhere herein. In the context of a vector, the term “operatively linked” is intended to mean that the nucleotide sequence of interest is linked to the regulatory element(s) in a manner that allows for expression of the nucleotide sequence (e.g., in an in vitro transcription / translation system or ina host cell when the vector is introduced into the host cell). Advantageous vectors include lentiviruses and adeno-associated viruses, and types of such vectors can also be selected for targeting particular types of cells. These and other embodiments of the vectors and vector systems are described elsewhere herein.

[0164] In an embodiment, the vector can be a bicistronic vector. In an embodiment, a bicistronic vector can be used for one or more elements of the engineered Acr polypeptide described herein. In an embodiment, expression of element(s) of the engineered Acr polypeptide described herein can be driven by a ubiquitous Pol II promoter, such as beta-actin, CMV, SV40, or another ubiquitous promoter. In an embodiment, expression of element(s) of the engineered Acr polypeptide described herein can be driven by a tissue-specific Pol II promoter. Where the element of the engineered Acr polypeptide is an RNA, its expression can be driven by a Pol III promoter, such as a U6 promoter. In an embodiment, the two are combined.

[0165] These and others are further detailed and described elsewhere herein.Cell-based Vector Amplification and Expression

[0166] Vectors may be introduced and propagated in a prokaryote or prokaryotic cell. In an embodiment, a prokaryote is used to amplify copies of a vector to be introduced into a eukaryotic cell or as an intermediate vector in the production of a vector to be introduced into a eukaryotic cell (e.g., amplifying a plasmid as part of a viral vector packaging system). The vectors can be viral-based or non-viral-based. In an embodiment, a prokaryote is used to amplify copies of a vector and express one or more nucleic acids, such as to provide a source of one or more proteins for delivery to a host cell or host organism.

[0167] Vectors can be designed for expression of one or more elements of the engineered Acr polypeptide described herein (e.g., nucleic acid transcripts, proteins, enzymes, and combinations thereof) in a suitable host cell. In an embodiment, the suitable host cell is a prokaryotic cell. Suitable host cells include, but are not limited to, bacterial cells, yeast cells, insect cells, and mammalian cells. In an embodiment, the suitable host cell is a eukaryotic cell.

[0168] In an embodiment, the suitable host cell is a suitable bacterial cell. Suitable bacterial cells include, but are not limited to bacterial cells from the bacteria of the species Escherichia coli. Many suitable strains of E. coli are known in the art for expression of vectors. These include, but are not limited to Pirl, Stbl2, Stbl3, Stbl4, TOP10, XL1 Blue, XLIO Gold, Rosetta 2(DE3)(Novagen), NEB® 5-alpha Competent E. coli (High Efficiency) (New England Biolabs), and BL21(DE3) Competent E. coli (New England Biolabs). In an embodiment, the host cell is a suitable insect cell. Suitable insect cells include those from Spodoptera frugiperda. Suitable strains of S. frugiperda cells include, but are not limited to Sf9 and Sf21. In an embodiment, the host cell is a suitable yeast cell. In an embodiment, the yeast cell can be from Saccharomyces cerevisiae. In an embodiment, the host cell is a suitable mammalian cell. Many types of mammalian cells have been developed to express vectors. Suitable mammalian cells include, but are not limited to, HEK293, HEK293T, HEK293FT, Chinese Hamster Ovary Cells (CHOs), mouse myeloma cells, HeLa, U2OS, A549, HT1080, CAD, P19, NIH 3T3, L929, N2a, MCF-7, Y79, SO-Rb50, HepG G2, DIKX-X11, J558L, Baby hamster kidney cells (BHK), and chicken embryo fibroblasts (CEFs). Suitable host cells are discussed further in Gene Expression Technology: Methods in Enzymology, 185, published by Academic Press (San Diego, C.A. 1990) (Goeddel).

[0169] In an embodiment, the vector can be a yeast expression vector. Examples of vectors for expression in yeast Saccharomyces cerevisiae include pYepSecl (Baldari et al., The EMBO J. 1987, 6, 229-234), pMFa (Kuijan et al., Cell 1982, 30, 933-943), pJRY88 (Schultz et al., Gene 1987, 5- / (l), 113-123), pYES2 (Invitrogen Corporation, San Diego, Calif.), and picZ (InVitrogen Corp, San Diego, Calif). As used herein, a “yeast expression vector” refers to a nucleic acid that contains one or more sequences encoding an RNA and / or polypeptide and may further contain any desired elements that control the expression of the nucleic acid(s), as well as any elements that enable the replication and maintenance of the expression vector inside the yeast cell. Many suitable yeast expression vectors and features thereof are known in the art; for example, various vectors and techniques are illustrated in Yeast Protocols, 3rdedition (2014), published by Humana Press (New York, N.Y. 2014) (Wei, ed.); and Buckholz and Gleeson, Biotechnol. 1991, 9, 1067-1072. Yeast vectors can contain, without limitation, a centromeric (CEN) sequence, an autonomous replication sequence (ARS), a promoter, such as an RNA Polymerase III promoter, operatively linked to a sequence or gene of interest, a terminator such as an RNA polymerase III terminator, an origin of replication, and a marker gene (e.g., auxotrophic, antibiotic, or other selectable markers). Examples of expression vectors for use in yeast may include plasmids, yeast artificial chromosomes, 2p plasmids, yeast integrative plasmids, yeast replicative plasmids, shuttle vectors, and episomal plasmids.

[0170] In an embodiment, the vector is a baculovirus vector or expression vector and can be suitable for expression of polynucleotides and / or proteins in insect cells. In an embodiment, the suitable host cell is an insect cell. Baculovirus vectors available for expression of proteins in cultured insect cells (e.g., SF9 cells) include the pAc series (Smith et al., Mol. Cell. Biol. 1983, 3(12), 2156-2165) and the pVL series (Lucklow et al., Virology 1989, 770(1), 31-39). rAAV (recombinant Adeno-associated viral) vectors are preferably produced in insect cells, e.g., Spodoptera frugiperda Sf9 insect cells, grown in serum-free suspension culture. Serum-free insect cells can be purchased from commercial vendors, e g., Sigma Aldrich (EX-CELL 405).

[0171] In an embodiment, the vector is a mammalian expression vector. In an embodiment, the mammalian expression vector is capable of expressing one or more polynucleotides and / or polypeptides in a mammalian cell. Examples of mammalian expression vectors include, but are not limited to, pCDM8 (Seed et al., Nature 1987, 329, 840-842) and pMT2PC (Kaufman et al., EMBO J. 1987, 6, 187-195). The mammalian expression vector can include one or more suitable regulatory elements capable of controlling expression of the one or more polynucleotides and / or proteins in the mammalian cell. For example, commonly used promoters are derived from polyoma, adenovirus 2, cytomegalovirus, simian virus 40, and others disclosed herein and known in the art. More details on suitable regulatory elements are described elsewhere herein.

[0172] For other suitable expression vectors and vector systems for both prokaryotic and eukaryotic cells see, e.g., Molecular Cloning: A Laboratory Manual 4thedition, published by Cold Spring Harbor Laboratory Press, (New York, N.Y. 1989) (Sambrook and Green, eds.), particularly at chapters 16 and 17.

[0173] In an embodiment, the recombinant mammalian expression vector is capable of directing expression of the nucleic acid preferentially in a particular cell type (e.g., tissue-specific regulatory elements are used to express the nucleic acid). Tissue-specific regulatory elements are known in the art. Non-limiting examples of suitable tissue-specific promoters include the albumin promoter (liver-specific; Pinkert et al., Genes Dev. 1987, 1, 268-276), lymphoid-specific promoters (Calame et al., Adv Immunol. 1988, 73, 235-275), in particular promoters of T cell receptors (Winoto et al., The EMBO J. 1989, S, 729-733) and immunoglobulins (Baneiji, et al., Cell 1983, 33(3), 729-740; Queen et al., Cell 1983, 33(3), 741-748), neuron-specific promoters (e.g., the neurofilament promoter; Byrne et al., Proc. Natl. Acad. Sci. USA. 1989, 86, 5473-5477),pancreas-specific promoters (Edlund et al., Science 1985, 230(4728), 912-916), and mammary gland-specific promoters (e.g., milk whey promoter; U.S. Patent No. 4,873,316). Developmentally-regulated promoters are also encompassed, e.g., the murine hox promoters (Kessel et al., Science, 1990, 249, 374-379) and the a-fetoprotein promoter (Campes et al., Genes Dev. 1989, 3(4), 537-546). With regards to these prokaryotic and eukaryotic vectors, mention is made of U.S. Patent No. 6,750,059, the contents of which are incorporated by reference herein in their entirety. Other embodiments can utilize viral vectors, with regards to which mention is made of U.S. Patent Pub. No. US20120003201, the contents of which are incorporated by reference herein in their entirety. Tissue-specific regulatory elements are known in the art and in this regard, mention is made ofU.S. Patent No. 7,776,321, the contents of which are incorporated by reference herein in their entirety. In an embodiment, a regulatory element can be operatively linked to one or more elements of the engineered Acr polypeptide so as to drive expression of the one or more elements of the engineered Acr polypeptide described herein.

[0174] In an embodiment, the vector can be a fusion vector or fusion expression vector. In an embodiment, fusion vectors add a number of amino acids to a protein encoded therein, such as to the amino terminus, carboxy terminus, or both of a recombinant protein. Such fusion vectors can serve one or more purposes, such as: (i) to increase expression of recombinant protein; (ii) to increase the solubility of the recombinant protein; and (iii) to aid in the purification of the recombinant protein by acting as a ligand in affinity purification. In an embodiment, expression of polynucleotides (such as non-coding polynucleotides) and proteins in prokaryotes can be carried out in Escherichia coli with vectors containing constitutive or inducible promoters directing the expression of either fusion or non-fusion polynucleotides and / or proteins. In an embodiment, the fusion expression vector can include one or more proteolytic cleavage sites, which can be introduced at the junction of the fusion vector backbone or other fusion moiety and the recombinant polynucleotide or protein to enable separation of the recombinant polynucleotide or protein from the fusion vector backbone or other fusion moiety subsequent to purification of the fusion polynucleotide or protein. Such enzymes, and their cognate recognition sequences, include Factor Xa, thrombin and enterokinase, and TEV protease sites. Example fusion expression vectors include pGEX (Pharmacia Biotech Inc; Smith et al., Gene 1988, 67(1), 31-40), pMAL (New England Biolabs, Beverly, Mass.) and pRIT5 (Pharmacia, Piscataway, N.J.) that fuse glutathioneS-transferase (GST), maltose-binding protein, or protein A, respectively, to the target recombinant protein. Examples of suitable inducible non-fusion E. coli expression vectors include pTrc (Amrann et al., Gene 1988, 69(2), 301-315) and pET l id (Studier et al., in Gene Expression Technology: Methods in Enzymology, 185, published by Academic Press (San Diego, C.A. 1990) (Goeddel, ed ), 60-89).

[0175] In an embodiment, one or more vectors driving expression of one or more elements of an engineered Acr polypeptide described herein are introduced into a host cell such that expression of the elements of the engineered Acr polypeptide described herein direct formation of an engineered Acr polypeptide delivery complex at one or more target cells. For example, an engineered Acr polypeptide described herein and targeting moiety, CRISPR-Cas system or component, etc. component can each be operatively linked to separate regulatory elements on separate vectors. DNAs and / or RNA(s) of different elements of an engineered Acr polypeptide described herein can be delivered to an animal, plant, microorganism or cell thereof to produce an animal (e.g., a mammal, reptile, avian, etc.), plant, microorganism or cell thereof that constitutively, inducibly, or conditionally expresses different elements of the engineered Acr polypeptides described herein that incorporates one or more elements of the engineered Acr polypeptide herein or contains one or more cells that incorporate and / or express one or more elements of the engineered Acr polypeptide of the present disclosure.

[0176] In an embodiment, two or more of the elements expressed from the same or different regulatory element(s) can be combined in a single vector, with one or more additional vectors providing any components of the system not included in the first vector. Engineered Acr polypeptide polynucleotides that are combined in a single vector may be arranged in any suitable orientation, such as one element located 5’ with respect to (“upstream” of) or 3’ with respect to (“downstream” of) a second element. The coding sequence of one element may be located on the same or opposite strand of the coding sequence of a second element, and oriented in the same or opposite direction. In an embodiment, a single promoter drives expression of a transcript encoding one or more engineered Acr polypeptides, embedded within one or more intron sequences (e.g., each in a different intron, two or more in at least one intron, or all in a single intron). In an embodiment, the engineered Acr polypeptide polynucleotides can be operatively linked to and expressed from the same promoter.Cell-Free Vector and Polynucleotide Expression

[0177] In an embodiment, the polynucleotide encoding one or more features of the engineered Acr polypeptide can be expressed from a vector or suitable polynucleotide in a cell-free in vitro system. In other words, the polynucleotide can be transcribed and optionally translated in vitro. In vitro transcription / translation systems and appropriate vectors are generally known in the art and commercially available. Generally, in vitro transcription and in vitro translation systems replicate the processes of RNA and protein synthesis, respectively, outside of the cellular environment. Vectors and suitable polynucleotides for in vitro transcription can include T7, SP6, and T3 promoters or other regulatory sequences that can be recognized and acted upon by an appropriate polymerase to transcribe the polynucleotide or vector.

[0178] In vitro translation can be stand-alone (e.g., translation of a purified polyribonucleotide) or linked / coupled to transcription. In an embodiment, the cell-free (or in vitro) translation system can include extracts from rabbit reticulocytes, wheat germ, and / or E. coli. The extracts can include various macromolecular components that are needed for translation of exogenous RNA (e.g., 70S or 80S ribosomes, tRNAs, aminoacyl-tRNA, synthetases, initiation, elongation factors, termination factors, etc.). Other components can be included or added during the translation reaction, including but not limited to, amino acids, energy sources (ATP, GTP), energy regenerating systems (e.g., creatine phosphate and creatine phosphokinase for use in eukaryotic systems) and phosphoenolpyruvate and pyruvate kinase for use in bacterial systems), and other co-factors (e.g., Mg2+, K+, etc.). As previously mentioned, in vitro translation can be based on RNA or DNA starting material. Some translation systems can utilize an RNA template as starting material (e.g., reticulocyte lysates and wheat germ extracts). Some translation systems can utilize a DNA template as a starting material (e.g., E. co / z-based systems). In these systems, transcription and translation are coupled and DNA is first transcribed into RNA, which is subsequently translated. Suitable standard and coupled cell-free translation systems are generally known in the art and are commercially available.Vector Features

[0179] The vectors can include additional features that can confer one or more functionalities to the vector, the polynucleotide to be delivered, a virus particle produced therefrom, or polypeptide expressed thereof. Such features include, but are not limited to, regulatory elements,selectable markers, molecular identifiers (e.g., molecular barcodes), stabilizing elements, and the like. It will be appreciated by those skilled in the art that the design of the expression vector and additional features included can depend on such factors as the choice of the host cell to be transformed, the level of expression desired, etc.Regulatory Elements

[0180] In certain embodiments, the polynucleotides and / or vectors thereof described herein (such as the engineered Acr polypeptide polynucleotides of the present invention) can include one or more regulatory elements that can be operatively linked to the polynucleotide. The term “regulatory element” is intended to include promoters, enhancers, internal ribosomal entry sites (IRES), other expression control elements (e.g., transcription termination signals, such as polyadenylation signals and poly-U sequences), and cellular localization signals (e.g., nuclear localization signals). Such regulatory elements are described, for example, in Gene Expression Technology: Methods in Enzymology, 185, published by Academic Press (San Diego, C.A. 1990) (Goeddel, ed.). Regulatory elements include those that direct constitutive expression of a nucleotide sequence in many types of host cells and those that direct expression of the nucleotide sequence only in certain host cells (e.g., tissue-specific regulatory sequences). A tissue-specific promoter can direct expression primarily in a desired tissue of interest, such as muscle, neuron, bone, skin, blood, specific organs (e.g., liver, pancreas), or particular cell types (e.g., lymphocytes). Regulatory elements may also direct expression in a temporal-dependent manner, such as in a cell cycle-dependent or developmental stage-dependent manner, which may or may not also be tissue or cell-type specific. In an embodiment, a vector comprises one or more pol III promoters (e.g., 1, 2, 3, 4, 5, or more pol III promoters), one or more pol II promoters (e.g., 1, 2, 3, 4, 5, or more pol II promoters), one or more pol I promoters (e.g., 1, 2, 3, 4, 5, or more pol I promoters), or combinations thereof. Examples of pol III promoters include, but are not limited to, U6 and Hl promoters. Examples of pol II promoters include, but are not limited to, the retroviral Rous sarcoma virus (RSV) long terminal repeat (LTR) promoter (optionally with the RSV enhancer), the cytomegalovirus (CMV) promoter (optionally with the CMV enhancer) (see, e.g., Boshart et al., Cell 1985, 4 / (2), 521-530), the SV40 promoter, the dihydrofolate reductase promoter, the P- actin promoter, the phosphoglycerol kinase (PGK) promoter, and the EFla promoter. Also encompassed by the term “regulatory element” are enhancer elements, such as woodchuckhepatitis virus post-transcriptional regulator element (WPRE); CMV enhancers; the R-U5’ segment in the LTR of HTLV-I (Takebe et al., Mol. Cell. Biol. 1988, 5(1), 466-472); SV40 enhancer; and the intron sequence between exons 2 and 3 of rabbit P-globin (O’Hare et al., Proc. Natl. Acad. Sci. USA. 1981, 75(3), 1527-1531).

[0181] In an embodiment, the regulatory sequence can be a regulatory sequence described in U.S. Patent No. 7,776,32 and 8,927,807; and U.S. Patent Application Publication No. US20110027239, the contents of which are incorporated by reference herein in their entirety. In an embodiment, the vector can contain a minimal promoter. In an embodiment, the minimal promoter is the Mecp2 promoter, tRNA promoter, or U6. In a further embodiment, the minimal promoter is tissue-specific. In an embodiment, the length of the vector polynucleotide, the minimal promoters, and polynucleotide sequences is less than 4.4 kb.

[0182] To express a polynucleotide, the vector can include one or more transcriptional and / or translational initiation regulatory sequences, e.g., promoters, that direct the transcription of the gene and / or translation of the encoded protein in a cell. In an embodiment, a constitutive promoter may be employed. Suitable constitutive promoters for mammalian cells are generally known in the art and include, but are not limited to SV40, CAG, CMV, EF-la, 0-actin, RSV, and PGK. Suitable constitutive promoters for bacterial cells, yeast cells, and fungal cells are generally known in the art, such as a T7 promoter for bacterial expression and an alcohol dehydrogenase promoter for expression in yeast.

[0183] In an embodiment, the regulatory element can be a regulated promoter. “Regulated promoter” refers to promoters that direct gene expression not constitutively but in a temporally- and / or spatially-regulated manner, and includes tissue-specific, tissue-preferred, and inducible promoters. Regulated promoters include conditional promoters and inducible promoters. In an embodiment, conditional promoters can be employed to direct expression of a polynucleotide in a specific cell type, under certain environmental conditions, and / or during a specific state of development. Suitable tissue-specific promoters can include, but are not limited to, liver-specific promoters (e.g., APOA2, SERPIN Al (hAAT), CYP3A4, and MIR122), pancreatic cell promoters (e g., INS, IRS2, Pdxl, Alx3, Ppy), cardiac-specific promoters (e.g., Myh6 (alpha MHC), MYL2 (MLC-2v), TNI3 (cTnl), NPPA (ANF), Slc8al (Next)), central nervous system cell promoters (SYN1, GFAP, INA, NES, MOBP, MBP, TH, FOXA2 (HNF3 beta)), skin cell-specific promoters(e g., FLG, K14, TGM3), immune cell-specific promoters, (e.g., ITGAM, CD43 promoter, CD14 promoter, CD45 promoter, CD68 promoter), urogenital cell-specific promoters (e.g., Pbsn, Upk2, Sbp, Ferll4), endothelial cell-specific promoters (e.g., ENG), pluripotent and embryonic germ layer cell-specific promoters (e.g., Oct4, NANOG, Synthetic Oct4, T brachyury, NES, SOX17, F0XA2, MIR122), and muscle cell-specific promoter (e.g., Desmin). Other tissue and / or cellspecific promoters are generally known in the art and are within the scope of this disclosure.

[0184] Inducible / conditional promoters can be positively inducible / conditional promoters (e.g., a promoter that activates transcription of the polynucleotide upon appropriate interaction with an activated activator, or an inducer compound, environmental condition, or another stimulus) or a negative / conditional inducible promoter (e.g., a promoter that is repressed by e.g., being bound by a repressor) until the repressor condition of the promotor is removed e.g., when inducer binds a repressor bound to the promoter, stimulating release of the promoter by the repressor or removal of a chemical repressor from the promoter environment. The inducer can be a compound, environmental condition, or another stimulus. Thus, inducible / conditional promoters can be responsive to any suitable stimuli such as chemical, biological, or other molecular agents, temperature, light, and / or pH. Suitable inducible / conditional promoters include, but are not limited to, Tet-On, Tet-Off, Lac promoter, pBad, AlcA, LexA, Hsp70 promoter, Hsp90 promoter, pDawn, XVE / OlexA, GVG, and pOp / LhGR.

[0185] Where expression in a plant cell is desired, the components of the engineered Acr polypeptide described herein are typically placed under control of a plant promoter, i .e., a promoter operable in plant cells. The use of different types of promoters is envisaged.

[0186] A constitutive plant promoter is a promoter that is able to express the open reading frame (ORF) that it controls in all or nearly all of the plant tissues during all or nearly all developmental stages of the plant (referred to as “constitutive expression”). One non-limiting example of a constitutive promoter is the cauliflower mosaic virus 35S promoter. Different promoters may direct the expression of a gene in different tissues or cell types, or at different stages of development, or in response to different environmental conditions. In particular embodiments, one or more of the engineered Acr polypeptide components are expressed under the control of a constitutive promoter, such as the cauliflower mosaic virus 35S promoter tissue-preferred promoters can be utilized to target enhanced expression in certain cell types within a particularplant tissue, for instance, vascular cells in leaves or roots or in specific cells of the seed. Examples of particular promoters for use with the engineered Acr polypeptide encoding polynucleotides are found in Kawamata et al., Plant Cell Physiol. 1997, 3S(7), 792-803; Yamamoto et al., Plant J. 1997, 72(2), 255-265; Hire et al., Plant Mol. Biol. 1992, 20, 207-218; Kuster et al., Plant Mol. Biol. 1995, 29, 759-772, and Capana et al., Plant Mol. Biol. 1994, 25, 681-691.

[0187] Examples of promoters that are inducible and that can allow for spatiotemporal control of gene editing or gene expression may use a form of energy. The form of energy may include but is not limited to sound energy, electromagnetic radiation, chemical energy, and / or thermal energy. Examples of inducible systems include tetracycline-inducible promoters (Tet-On or Tet-Off), small molecule two-hybrid transcription activation systems (FKBP, ABA, etc.), or light-inducible systems (Phytochrome, Light-oxygen-voltage-sensing (LOV) domains, or cryptochrome, such as a Light Inducible Transcriptional Effector (LITE) that directs changes in transcriptional activity in a sequence-specific manner. The components of a light-inducible system may include one or more elements of the engineered Acr polypeptide described herein, a light-responsive cytochrome heterodimer (e.g., from Arabidopsis thaliana), and a transcriptional activation / repression domain. In an embodiment, the vector can include one or more of the inducible DNA binding proteins provided in U. S. Patent Publication No. US20190390204; US20150291966; US20170166903 ; and US20190203212, which describe e.g., embodiments of inducible DNA binding proteins and methods of use and can be adapted for use with the present invention.

[0188] In an embodiment, transient or inducible expression can be achieved by including, for example, chemical-regulated promoters, i.e., whereby the application of an exogenous chemical induces gene expression. Modulation of gene expression can also be obtained by including a chemical-repressible promoter, where application of the chemical represses gene expression. Chemical-inducible promoters include, but are not limited to, the maize ln2-2 promoter, activated by benzene sulfonamide herbicide safeners (see, e.g., De Veylder et al., Plant Cell Physiol. 1997, 35(5), 568-577), the maize GST promoter (GST-11-27, see, e.g., International Patent Publication No. WO 1993 / 001294), activated by hydrophobic electrophilic compounds used as pre-emergent herbicides, and the tobacco PR-la promoter (Ono et al., Biosci., Biotechnol., Biochem. 2004, 68, 803-807) activated by salicylic acid. Promoters that are regulated by antibiotics, such astetracycline-inducible and tetracycline-repressible promoters (see e.g., Gatz et al., Mol. Gen Genet. 1991, 227, 229-237; U.S. Patent No. 5,814,618 and 5,789,156) can also be used herein.

[0189] In an embodiment, the polynucleotide, vector or system thereof can include one or more elements capable of translocating and / or expressing an engineered Acr polypeptide polynucleotide to / in a specific cell component or organelle. Such organelles can include, but are not limited to, nucleus, ribosome, endoplasmic reticulum, Golgi apparatus, chloroplast, mitochondria, vacuole, lysosome, cytoskeleton, plasma membrane, cell wall, peroxisome, centrioles, etc. Such regulatory elements can include, but are not limited to, nuclear localization signals (examples of which are described in greater detail elsewhere herein), any such as those that are annotated in the LocSigDB database (see, e.g., Negi et al., Database, 2015, 2015, bav003-bav003), nuclear export signals (e.g., LXXXLXXLXL (SEQ ID NO: 261) and others described elsewhere herein), endoplasmic reticulum localization / retention signals (e.g., KDEL (SEQ ID NO: 262), KDXX, KKXX, KXX, and others described elsewhere herein; and see, e.g., Liu et al., Mol. Biol. Cell 2007, 18(3), 1073- 1082 and Gorleku et al., J. Biol. Chem. 2011, 286(45), 39573-39584), mitochondria (see, e.g., Chin et al., Cell Rep. 2018, 22, 2818-2826, particularly at Fig. 2; Doyle et al., PLoS ONE, 2013, 8, e67938; Funes et al., J. Biol. Chem. 2002, 277, 6051-6058; Matouschek et al., Proc. Natl. Acad. Sci. USA 2010, 107(11), 5047-5052; Oca-Cossio et al., Genetics, 2003, 165(2), 707-720; Waltner et al., J. Biol. Chem. 1996, 271, 21226-21230; Wilcox et al., Proc. Natl. Acad. Sci. USA 2005, 102(43), 15435-15440; Galanis et al., FEBS Lett. 1991, 282(2), 425-430), peroxisome (e.g., (S / A / C)-(K / R / H)-(L / A), SLK, (R / K)-(L / V / I)-XXXXX-(H / Q)-(L / A / F). Suitable protein targeting motifs can also be designed or identified using any suitable database or prediction tool, including but not limited to Minimotif Miner (minimotifminer.org, mitominer.mrc-mbu.cam.ac.uk / release- 4.0 / embodiment.do?name=Protein%20MTS), LocSigDB (see above), PTSs predictor, TargetP- 2.0 (www.cbs.dtu.dk / services / TargetP / ), ChloroP (www.cbs.dtu.dk / services / ChloroP / ); NetNES (www.cbs.dtu.dk / services / NetNES / ), Predotar (urgi.versailles.inra.fr / predotar / ), and SignalP (www.cbs.dtu.dk / services / SignalP / ).Selectable Markers and Tags

[0190] One or more of the engineered Acr polypeptide polynucleotides can be operatively linked, fused to, or otherwise modified to include a polynucleotide that encodes or is a selectable marker or tag, which can be a polynucleotide or polypeptide. In an embodiment, the polypeptideselectable marker can be incorporated in the engineered Acr polypeptide polynucleotide such that the selectable marker polypeptide, when translated, is inserted between two amino acids between the N- and C-terminus of the engineered Acr polypeptide polypeptide or at the N- and / or C- terminus of the engineered Acr polypeptide polypeptide. In an embodiment, the selectable marker or tag is a polynucleotide barcode or unique molecular identifier (UMI).

[0191] It will be appreciated that the polynucleotide encoding such selectable markers or tags can be incorporated into a polynucleotide encoding one or more components of the engineered Acr polypeptide described herein in an appropriate manner to allow expression of the selectable marker or tag. Such techniques and methods are described elsewhere herein and will be instantly appreciated by one of ordinary skill in the art in view of this disclosure. Many such selectable markers and tags are generally known in the art and are intended to be within the scope of this disclosure.

[0192] Suitable selectable markers and tags include, but are not limited to, affinity tags, such as chitin binding protein (CBP), maltose-binding protein (MBP), glutathione-S-transferase (GST), poly(His) tag; solubilization tags such as thioredoxin (TRX) and poly(NANP), MBP, and GST; chromatography tags such as those consisting of polyanionic amino acids, such as FLAG-tag; epitope tags such as V5-tag, Myc-tag, HA-tag and NE-tag; protein tags that can allow specific enzymatic modification (such as biotinylation by biotin ligase) or chemical modification (such as reaction with FlAsH-EDT2 for fluorescence imaging), DNA and / or RNA segments that contain restriction enzyme or other enzyme cleavage sites; DNA segments that encode products that provide resistance against otherwise toxic compounds including antibiotics, such as, spectinomycin, ampicillin, kanamycin, tetracycline, Basta, neomycin phosphotransferase II (NEO), hygromycin phosphotransferase (HPT) and the like; DNA and / or RNA segments that encode products that are otherwise lacking in the recipient cell (e.g., tRNA genes, auxotrophic markers); DNA and / or RNA segments that encode products which can be readily identified (e.g., phenotypic markers such as P-galactosidase, GUS; fluorescent proteins such as green fluorescent protein (GFP), cyan (CFP), yellow (YFP), red (RFP), luciferase, and cell surface proteins); polynucleotides that can generate one or more new primer sites for PCR (e.g., the juxtaposition of two DNA sequences not previously juxtaposed), DNA sequences not acted upon or acted upon by a restriction endonuclease or other DNA modifying enzyme, chemical, etc.; epitope tags (e.g.,GFP, FLAG- and His-tags), and, DNA sequences that make a molecular barcode or unique molecular identifier (UMI), DNA sequences required for a specific modification (e.g., methylation) that allows its identification. Other suitable markers will be appreciated by those of skill in the art.

[0193] Selectable markers and tags can be operatively linked to one or more components of the engineered Acr polypeptide described herein via suitable linkers. In an embodiment, the linker is a glycine, glycine serine, or glycine-cysteine-serine linker. The linker can be as short as GS or GG up to (GGGGG)3(SEQ ID NO: 263)or (GGGGS)3(SEQ ID NO: 216). Other linkers are described elsewhere herein.

[0194] The vector or vector system can include one or more polynucleotides encoding one or more targeting moieties. In an embodiment, the targeting moiety encoding polynucleotides can be included in the vector or vector system, such as a viral vector system, such that they are expressed within and / or on the virus particle(s) produced such that the virus particles can be targeted to specific cells, tissues, organs, etc. In an embodiment, the targeting moiety encoding polynucleotides can be included in the vector or vector system such that the engineered Acr polypeptide polynucleotide(s) and / or products expressed therefrom include the targeting moiety and can be targeted to specific cells, tissues, organs, etc. In an embodiment, such as non-viral carriers, the targeting moiety can be attached to the carrier (e.g., a polymer, lipid, inorganic molecule, etc.) and can be capable of targeting the carrier and any attached or associated engineered Acr polypeptide polynucleotide(s) to specific cells, tissues, organs, etc.Codon Optimization of Vector Polynucleotides

[0195] As described elsewhere herein, the polynucleotide encoding one or more embodiments of the engineered Acr polypeptide described herein can be codon optimized. In an embodiment, one or more polynucleotides contained in a vector (“vector polynucleotides”) that are not polynucleotides encoding an engineered Acr polypeptide or component thereof of the present invention are codon optimized. In general, codon optimization refers to a process of modifying a nucleic acid sequence for enhanced expression in the host cells of interest by replacing at least one codon (e.g., about or more than about 1, 2, 3, 4, 5, 10, 15, 20, 25, 50, or more codons) of the native sequence with codons that are more frequently or most frequently used in the genes of that host cell while maintaining the native amino acid sequence. Various species exhibit a particular biasfor certain codons of a particular amino acid. Codon bias (differences in codon usage between organisms) often correlates with the efficiency of translation of messenger RNA (mRNA), which is in turn believed to be dependent on, among other things, the properties of the codons being translated and the availability of particular transfer RNA (tRNA) molecules. The predominance of selected tRNAs in a cell is generally a reflection of the codons used most frequently in peptide synthesis. Accordingly, genes can be tailored for optimal gene expression in a given organism based on codon optimization. Codon usage tables are readily available, for example, at the “Codon Usage Database” available at www.kazusa.or.jp / codon / and these tables can be adapted in a number of ways. See Nakamura et al., Nucleic Acids Res. 2000, 25(1), 292-292. Computer algorithms for codon optimizing a particular sequence for expression in a particular host cell are also available, such as Gene Forge (Aptagen; Jacobus, PA). In an embodiment, one or more codons (e.g., 1, 2, 3, 4, 5, 10, 15, 20, 25, 50, or more, or all codons) in a sequence encoding an engineered Acr polypeptide or component thereof of the present invention and any additional protein described herein (e.g., a DNA / RNA-targeting Cas protein) corresponds to the most frequently used codon for a particular amino acid. As to codon usage in yeast, reference is made to the online Yeast Genome database available at www.yeast enome.org / community / codon_usage.shtml, or Bennetzen et al., J. Biol. Chem. 1982, 257(6), 3026-3031. As to codon usage in plants including algae, reference is made to Campbell et al., Plant Physiol. 1990, 92(1), 1-11; Murray et al., Nucleic Acids Res. 1989, 77(2), 477-498; and Morton, J Mol. Evol. 1998, 46(4), 449-459.

[0196] The vector polynucleotide can be codon optimized for expression in a specific cell type, tissue type, organ type, and / or subject type. In an embodiment, a codon-optimized sequence is a sequence optimized for expression in a eukaryote, e.g., humans (i.e., being optimized for expression in a human or human cell), or for another eukaryote, such as another animal (e.g., a mammal or avian) as is described elsewhere herein. Such codon-optimized sequences are within the ambit of the ordinary skilled artisan in view of the description herein. In an embodiment, the polynucleotide is codon optimized for a specific cell type. Such cell types can include, but are not limited to, epithelial cells (including skin cells, cells lining the gastrointestinal tract, cells lining other hollow organs), nerve cells (nerves, brain cells, spinal column cells, nerve support cells (e.g., astrocytes, glial cells, Schwann cells etc.)), muscle cells (e.g., cardiac muscle cells, smooth muscle cells, and skeletal muscle cells), connective tissue cells (fat and other soft tissue padding cells,bone cells, tendon cells, cartilage cells), blood cells, stem cells and other progenitor cells, immune system cells, germ cells, and combinations thereof. Such codon-optimized sequences are within the ambit of the ordinary skilled artisan in view of the description herein. In an embodiment, the polynucleotide is codon optimized for a specific tissue type. Such tissue types can include, but are not limited to, muscle tissue, connective tissue, nervous tissue, and epithelial tissue. Such codon- optimized sequences are within the ambit of the ordinary skilled artisan in view of the description herein. In an embodiment, the polynucleotide is codon optimized for a specific organ. Such organs include, but are not limited to, muscles, skin, intestines, liver, spleen, brain, lungs, stomach, heart, kidneys, gallbladder, pancreas, bladder, thyroid, bone, blood vessels, blood, and combinations thereof. Such codon-optimized sequences are within the ambit of the ordinary skilled artisan in view of the description herein.

[0197] In an embodiment, a vector polynucleotide is codon optimized for expression in particular cells, such as prokaryotic or eukaryotic cells. The eukaryotic cells may be those of or derived from a particular organism, such as a plant or a mammal, including but not limited to human, or non-human eukaryote or animal or mammal as discussed herein, e.g., mouse, rat, rabbit, dog, livestock, or non-human mammal or primate.Vector Construction

[0198] The vectors described herein can be constructed using any suitable process or technique. In an embodiment, one or more suitable recombination and / or cloning methods or techniques can be used to design the vector(s) described herein. Suitable recombination and / or cloning techniques and / or methods can include, but not limited to, those described in U.S. Patent Publication No. US20040171156. Other suitable methods and techniques are described elsewhere herein.

[0199] Construction of recombinant AAV vectors is described in a number of publications, including U.S. Patent No. 5,173,414; Tratschin et al., Mol. Cell. Biol. 1985, 5(11), 3251-3260; Tratschin, et al., Mol. Cell. Biol. 1984, 4(10), 2072-2081; Hermonat and Muzyczka, Proc. Natl. Acad. Sci. USA 1984, 57(20), 6466-6470; and Samulski et al., J. Virol. 1989, 63(9), 3822-3828. Any of the techniques and / or methods can be used and / or adapted for constructing an AAV or other vector described herein. nullAAV (nAAV) vectors are discussed elsewhere herein.

[0200] In an embodiment, a vector comprises one or more insertion sites, such as a restriction endonuclease recognition sequence (also referred to as a “cloning site”). In an embodiment, one or more insertion sites (e.g., about or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more insertion sites) are located upstream and / or downstream of one or more sequence elements of one or more vectors. When multiple different guide polynucleotides are used, such in the context of a CRISPR- Cas system, a single expression construct may be used to target multiple different, corresponding target sequences within a cell. For example, a single vector may comprise about or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or more guide polynucleotides. In an embodiment, about or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more guide-polynucleotide-containing vectors may be provided, and optionally delivered to a cell.

[0201] Delivery vehicles, vectors, particles, nanoparticles, formulations, and components thereof for expression of one or more elements of an engineered Acr polypeptide described herein are as used in the foregoing documents, such as International Patent Publication No. WO 2014093622 and are discussed in greater detail herein.Viral Vectors

[0202] In an embodiment, the vector is a viral vector. The term of art “viral vector” and as used herein in this context, refers to polynucleotide-based vectors that contain one or more elements from or based upon one or more elements of a virus that can be capable of expressing and packaging a polynucleotide, such as an engineered Acr polypeptide encoding polynucleotide of the present invention, into a virus particle and producing said virus particle when used alone or with one or more other viral vectors (such as in a viral vector system). Viral vectors and systems thereof can be used for producing viral particles for delivery of and / or expression of one or more components of the engineered Acr polypeptide described herein. The viral vector can be part of a viral vector system involving multiple vectors. In an embodiment, systems incorporating multiple viral vectors can increase the safety of these systems. Suitable viral vectors can include retroviralbased vectors, lentiviral-based vectors, adenoviral-based vectors, adeno-associated vectors, helper-dependent adenoviral (HdAd) vectors, hybrid adenoviral vectors, herpes simplex virusbased vectors, poxvirus-based vectors, and Epstein-Barr virus-based vectors. Other embodiments of viral vectors and viral particles produced therefrom are described elsewhere herein. In anembodiment, the viral vectors are configured to produce replication incompetent viral particles for improved safety of these systems.

[0203] In certain embodiments, the virus structural component, which can be encoded by one or more polynucleotides in a viral vector or vector system, comprises one or more capsid proteins including an entire capsid. In certain embodiments, such as wherein a viral capsid comprises multiple copies of different proteins, the viral delivery system can provide one or more of the same protein or a mixture of such proteins. For example, AAV comprises 3 capsid proteins, VP1, VP2, and VP3, thus the viral delivery systems of the invention can comprise one or more of VP1, and / or one or more of VP2, and / or one or more of VP3. Accordingly, the present invention is applicable to a virus within the family Adenoviridae, such as Atadenovirus, e.g., Ovine atadenovirus D, Aviadenovirus, e.g., Fowl aviadenovirus A, Ichtadenovirus, e.g., Sturgeon ichtadenovirus A, Mastadenovirus (which includes adenoviruses such as all human adenoviruses), e.g., Human mastadenovirus C, and Siadenovirus, e.g., Frog siadenovirus A. Target-specific AAV capsid variants can be used or selected. Non-limiting examples include capsid variants selected to bind to chronic myelogenous leukemia cells, human CD34 PBPC cells, breast cancer cells, cells of lung, heart, dermal fibroblasts, melanoma cells, stem cells, glioblastoma cells, coronary artery endothelial cells, and keratinocytes. See, e.g., Biining et al., Curr. Opin. Pharmacol. 2015, 24, 94- 104. From teachings herein and knowledge in the art as to modifications of adenovirus (see, e.g., U.S. Patent No. 9,410,129; 7,344,872; 7,256,036; 6,911,199; and 6,740,525; and Matthews, Mol. Pharmacol. 2011, S(l), 3-11), as well as regarding modifications of AAV, the skilled person can readily obtain a modified adenovirus that has a large payload protein. Such modified adenovirus systems may be advantageous for embodiments of an engineered Acr polypeptide or one or more components thereof that may, when considered alone or together, be payload larger than the capacity of a native AAV. As to the viruses related to adenovirus mentioned herein, as well as to the viruses related to AAV mentioned elsewhere herein, the teachings herein as to modifying adenovirus and AAV, respectively, can be applied to those viruses without undue experimentation from this disclosure and the knowledge in the art.

[0204] In an embodiment, the viral vector is configured such that when the cargo is packaged the cargo(s) (e.g., one or more components of the engineered Acr polypeptide or engineered Acr polypeptide polynucleotide) is external to the capsid or virus particle. In the sense that it is notinside the capsid (enveloped or encompassed with the capsid) but is externally exposed so that it can contact the target cellular component (e.g., DNA, RNA, proteins). In an embodiment, the viral vector is configured such that all the cargo(s) are contained within the capsid after packaging.Split Viral Vector Systems

[0205] In an embodiment, the engineered Acr polypeptide viral vector or vector system (be it a retroviral (e.g., AAV) or lentiviral vector) is designed so as to position the cargo(s) (e.g., one or more engineered Acr polypeptide components) at the internal surface of the capsid. Once formed the cargo(s) will fill most or all of the internal volume of the capsid. In other embodiments, the engineered Acr polypeptide or component thereof may be modified or divided so as to occupy less of the capsid internal volume. Accordingly, in certain embodiments, the engineered Acr polypeptide or component thereof (e.g., an engineered Acr polypeptide) can be divided in two portions, one portion comprised in one viral particle or capsid and the second portion comprised in a second viral particle or capsid. In certain embodiments, by splitting the engineered Acr polypeptide or component thereof in two portions, space is made available to link one or more additional domains to one or both of the engineered Acr polypeptide components (e.g., Acr polypeptide, CPP, targeting moiety, and / or the like) portions. Such systems can be referred to as “split vector systems” or in the context of the present disclosure a “split system” a “split protein” and the like. This split protein approach is also described elsewhere herein. When the concept is applied to a vector system, it thus describes putting pieces of the split proteins on different vectors, thus reducing the payload of any one vector. This approach can facilitate delivery of systems where the total system size is close to or exceeds the packaging capacity of the vector. This is independent of any regulation of the engineered Acr polypeptide or component thereof that can be achieved with a split system or split protein design.

[0206] Split-engineered Acr polypeptides that can be incorporated into the AAV or other vectors described herein are set forth elsewhere herein and in documents incorporated herein by reference in further detail herein. In certain embodiments, each part of a split-engineered Acr polypeptide is attached to a member of a specific binding pair, and when bound with each other, the members of the specific binding pair maintain the parts of the engineered Acr polypeptide in proximity. In certain embodiments, each part of a split-engineered Acr polypeptide is associated with an inducible binding pair. An inducible binding pair is one which is capable of being switched“on” or “off’ by a protein or small molecule that binds to both members of the inducible binding pair. In general, according to the invention, engineered Acr polypeptides may preferably split between domains, leaving domains intact. Exemplary engineered Acr polypeptides are described in greater detail elsewhere herein.Retroviral and Lentiviral Vectors

[0207] Retroviral vectors can be composed of cis-acting long terminal repeats (LTRs) with packaging capacity for up to 6-10 kb of foreign sequence. The minimum cis-acting LTRs are those sufficient for replication and packaging of the vectors, which are then used to integrate the therapeutic gene into the target cell to provide permanent transgene expression. Suitable retroviral vectors for the engineered Acr polypeptide and / or components thereof can include those based upon murine leukemia virus (MuLV), gibbon ape leukemia virus (GaLV), Simian immunodeficiency virus (SIV), human immunodeficiency virus (HIV), and combinations thereof (see, e.g., Buchschacher, Jr. and Panganiban, Virol. 1992, 66(5), 2731-2739; Johann et al., J. Virol. 1992, 66(3), 1635-1640; Sommnerfelt et al., J. Virol., 1990, 776, 58-59; Wilson et al., J. Virol., 1989, 63(5), 2374-2378; Miller et al., J. Virol., 1991, 65(5), 2220-2224; and International Patent Application Publication No. WO 1994 / 026877). The selection of a retroviral gene transfer system may therefore depend on the target tissue.

[0208] The tropism of a retrovirus can be altered by incorporating foreign envelope proteins, expanding the potential target population of target cells. Lentiviral vectors are retroviral vectors that are able to transduce or infect non-dividing cells and are described in greater detail elsewhere herein. A retrovirus can also be engineered to allow for conditional expression of the inserted transgene, such that only certain cell types are infected by the lentivirus.

[0209] Lentiviruses are complex retroviruses that have the ability to infect and express their genes in both mitotic and post-mitotic cells. Advantages of using a lentiviral approach can include the ability to transduce or infect non-dividing cells and their ability to typically produce high viral titers, which can increase efficiency or efficacy of production and delivery. Suitable lentiviral vectors include, but are not limited to, human immunodeficiency virus (HlV)-based lentiviral vectors, feline immunodeficiency virus (FlV)-based lentiviral vectors, simian immunodeficiency virus (SlV)-based lentiviral vectors, Moloney Murine Leukemia Virus (Mo-MLV), Visna-maedi virus (VMV)-based lentiviral vector, caprine arthritis-encephalitis virus (CAEV)-based lentiviralvector, bovine immune deficiency virus (BlV)-based lentiviral vector, and Equine infectious anemia (EIAV)-based lentiviral vector. In an embodiment, an HIV-based lentiviral vector system can be used. In an embodiment, an FIV-based lentiviral vector system can be used.

[0210] In an embodiment, the lentiviral vector is an EIAV-based lentiviral vector or vector system. EIAV vectors have been used to mediate expression, packaging, and / or delivery in other contexts, such as for ocular gene therapy (see, e.g., Balagaan et al., J. Gene. Med. 2005, 8, 275- 285). In another embodiment, RetinoStat®, (see, e.g., Binley et al., Hum. Gene Ther. 2012, 23(9), 980-991, which describes an equine infectious anemia virus-based lentiviral gene therapy vector that expresses angiostatic proteins endostatin and angiostatin that is delivered via a subretinal injection for the treatment of the wet form of age-related macular-degeneration. Any of these vectors described in these publications can be modified for the elements of the engineered Acr polypeptide described herein.

[0211] In an embodiment, the lentiviral vector or vector system thereof can be a first- generation lentiviral vector or vector system thereof. First-generation lentiviral vectors can contain a large portion of the lentivirus genome, including the gag and pol genes, other additional viral proteins (e.g., VSV-G) and other accessory genes (e.g., vif, vprm vpu, nef, and combinations thereof), regulatory genes (e.g., tat and / or rev) as well as the gene of interest between the LTRs. First-generation lentiviral vectors can result in the production of virus particles that can be capable of replication in vivo, which may not be appropriate for some instances or applications.

[0212] In an embodiment, the lentiviral vector or vector system thereof can be a second- generation lentiviral vector or vector system thereof. Second-generation lentiviral vectors do not contain one or more accessory virulence factors and do not contain all components necessary for virus particle production on the same lentiviral vector. This can result in the production of a replication-incompetent virus particle and thus increase the safety of these systems over first- generation lentiviral vectors. In an embodiment, the second-generation vector lacks one or more accessory virulence factors (e.g., vif, vprm, vpu, nef, and combinations thereof). Unlike the first- generation lentiviral vectors, no single second-generation lentiviral vector includes all features necessary to express and package a polynucleotide into a virus particle. In an embodiment, the envelope and packaging components are split between two different vectors with the gag, pol, rev, and tat genes being contained on one vector and the envelope proteins (e.g., VSV-G) are containedon a second vector. The gene of interest, its promoter, and LTRs can be included on a third vector that can be used in conjunction with the other two vectors (packaging and envelope vectors) to generate a replication-incompetent virus particle.

[0213] In an embodiment, the lentiviral vector or vector system thereof can be a third- generation lenti viral vector or vector system thereof. Third-generation lentiviral vectors and vector systems thereof have increased safety over first- and second-generation lentiviral vectors and systems thereof because, for example, the various components of the viral genome are split between two or more different vectors but used together in vitro to make virus particles, they can lack the tat gene (when a constitutively active promoter is included upstream of the LTRs), and they can include one or more deletions in the 3’LTR to create self-inactivating (SIN) vectors having disrupted promoter / enhancer activity of the LTR. In an embodiment, a third-generation lentiviral vector system can include (i) a vector plasmid that contains the polynucleotide of interest and upstream promoters that are flanked by the 5’ and 3’ LTRs, which can optionally include one or more deletions present in one or both of the LTRs to render the vector self-inactivating; (ii) a “packaging vector(s)” that can contain one or more genes involved in packaging a polynucleotide into a virus particle that is produced by the system (e.g., gag, pol, and rev) and upstream regulatory sequences (e.g., promoter(s)) to drive expression of the features present on the packaging vector, and (iii) an “envelope vector” that contains one or more envelope protein genes and upstream promoters. In certain embodiments, the third-generation lentiviral vector system can include at least two packaging vectors, with the gag-pol being present on a different vector than the rev gene.

[0214] In an embodiment, self-inactivating lentiviral vectors with an siRNA targeting a common exon shared by HIV tat / rev, a nucleolar-localizing TAR decoy, and an anti-CCR5- specific hammerhead ribozyme (see, e.g., DiGiusto et al., Sci. Transl. Med. 2010, 2(36), 36ra43) can be used / and or adapted to the engineered Acr polypeptide of the present invention.

[0215] In an embodiment, the pseudotype and infectivity or tropism of a lentivirus particle can be tuned by altering the type of envelope protein(s) included in the lentiviral vector or system thereof. As used herein, an “envelope protein” or “outer protein” means a protein exposed at the surface of a viral particle that is not a capsid protein. For example, envelope or outer proteins typically comprise proteins embedded in the envelope of the virus. In an embodiment, a lentiviral vector or vector system thereof can include a VSV-G envelope protein. VSV-G mediates viralattachment to a low-density lipoprotein (LDL) receptor (LDLR) or an LDLR family member present on a host cell, which triggers endocytosis of the viral particle by the host cell. Because LDLR is expressed by a wide variety of cells, viral particles expressing the VSV-G envelope protein can infect or transduce a wide variety of cell types. Other suitable envelope proteins can be incorporated based on the host cell that a user desires to be infected by a virus particle produced from a lentiviral vector or system thereof described herein and can include, but are not limited to, feline endogenous virus envelope protein (RD114) (see, e.g., Hanawa et al., Molec. Ther. 2002, 5(3), 242-251), modified Sindbis virus envelope proteins (see, e.g., Morizono et al., J. Virol. 2010, 54(14), 6923-6934; Morizono et al., J. Virol. 2001, 75(17), 8016-8020; Morizono et al., J. Gene Med. 2009, 77(7), 549-558; Morizono et al., Virology 2006, 555(1), 71-81; Morizono et al., J. Gene Med. 2009, 77, 655-663; and Morizono et al., Nat. Med. 2005, 77, 346-352), baboon retroviral envelope protein (see, e.g., Girard-Gagnepain et al., Blood 2014, 724(8), 1221-1231); Tupaia paramyxovirus glycoproteins (see, e.g., Enkirch T. et al., Gene Ther. 2012, 20, 16-23); measles virus glycoproteins (see, e.g., Funke et al., Molec. Ther. 2008, 76(8), 1427-1436), rabies virus envelope proteins, MLV envelope proteins, Ebola envelope proteins, baculovirus envelope proteins, filovirus envelope proteins, hepatitis El and E2 envelope proteins, gp41 and gpl20 of HIV, hemagglutinin, neuraminidase, M2 proteins of influenza virus, and combinations thereof.

[0216] In an embodiment, the tropism of the resulting lentiviral particle can be tuned by incorporating cell-targeting peptides into a lentiviral vector such that the cell -targeting peptides are expressed on the surface of the resulting lentiviral particle. In an embodiment, a lentiviral vector can contain an envelope protein that is fused to a cell-targeting protein (see, e.g., Buchholz et al., Trends Biotechnol. 2015, 55(12), 777-790; Bender et al., PLoS Pathog. 2016, 72, el005641; and Friedrich et al., Mol. Ther. 2013, 27(4), 849-859.

[0217] In an embodiment, a split-intein-mediated approach to target lentiviral particles to a specific cell type can be used (see, e.g., Chamoun-Emaneulli et al., Biotechnol. Bioeng. 2015, 772(12), 2611-2617; and Ramirez et al., Protein. Eng. Des. Sei. 2012, 26(3), 215-223). In these embodiments, a lentiviral vector can contain one-half of a splicing-deficient variant of the naturally split intein from Nostoc punctiforme fused to a cell -targeting peptide and the same or different lentiviral vector can contain the other half of the split intein fused to an envelope protein, such as a binding-deficient, fusion-competent virus envelope protein. This can result in production of avirus particle from the lentiviral vector or vector system that includes a split intein that can function as a molecular Velcro linker to link the cell-binding protein to the pseudotyped lentivirus particle. This approach can be advantageous for use where surface-incompatibilities can restrict the use of, e.g., cell-targeting peptides.

[0218] In an embodiment, a covalent-bond-forming protein-peptide pair can be incorporated into one or more of the lentiviral vectors described herein to conjugate a cell-targeting peptide to the virus particle (see, e.g., Kasaraneni et al., Sci. Rep. 2018, 8, 10990). In an embodiment, a lentiviral vector can include an N-terminal PDZ domain of InaD protein (PDZ1) and its pentapeptide ligand (TEFCA (SEQ ID NO: 264)) from NorpA, which can conjugate the celltargeting peptide to the virus particle via a covalent bond (e.g., a disulfide bond). In an embodiment, the PDZ1 protein can be fused to an envelope protein, which can optionally be binding deficient and / or fusion competent virus envelope protein and included in a lentiviral vector. In an embodiment, the TEFCA (SEQ ID NO: 264) can be fused to a cell-targeting peptide and the TEFCA-CPT (SEQ ID NO: 264) fusion construct can be incorporated into the same or a different lentiviral vector as the PDZ 1 -envelope protein construct. During virus production, specific interaction between the PDZ1 and TEFCA (SEQ ID NO: 264) facilitates producing virus particles covalently functionalized with the cell targeting peptide and thus capable of targeting a specific cell type based upon a specific interaction between the cell targeting peptide and cells expressing its binding partner. This approach can be advantageous for use where surfaceincompatibilities can restrict the use of, e g., cell -targeting peptides.

[0219] Lentiviral vectors have been disclosed as in the treatment for Parkinson’s Disease, see, e.g., U.S. Patent Publication No. US20120295960 and U.S. Patent No. 7,303,910 and 7,351,585. Lentiviral vectors have also been disclosed for the treatment of ocular diseases, see, e.g., U.S. Patent Publication No. US20060281180; US20090007284; US20110117189; US20090017543; US20070054961; and US20100317109. Lentiviral vectors have also been disclosed for delivery to the brain, see, e.g., U.S. Patent Publication No. US20110293571; US20110293571; US20040013648; US20070025970; and US20090111106; and U.S. Patent No. 7,259,015. Any of these systems or a variant thereof can be used to deliver an engineered Acr polypeptide polynucleotide described herein to a cell.

[0220] In an embodiment, a lentiviral vector system can include one or more transfer plasmids. Transfer plasmids can be generated from various other vector backbones and can include one or more features that can work with other retroviral and / or lentiviral vectors in the system that can, for example, improve safety of the vector and / or vector system, increase virial titers, and / or increase or otherwise enhance expression of the desired insert to be expressed and / or packaged into the viral particle. Suitable features that can be included in a transfer plasmid can include, but are not limited to, 5’LTR, 3’LTR, SIN / LTR, origin of replication (Ori), selectable marker genes (e.g., antibiotic resistance genes), Psi (T), RRE (rev response element), cPPT (central polypurine tract), promoters, WPRE (woodchuck hepatitis post-transcriptional regulatory element), SV40 polyadenylation signal, pUC origin, SV40 origin, Fl origin, and combinations thereof.

[0221] In another embodiment, Cocal vesiculovirus envelope pseudotyped retroviral or lentiviral vector particles are contemplated (see, e.g., U.S. Patent Publication No. US20120164118). Cocal virus is in the Vesiculovirus genus, and is a causative agent of vesicular stomatitis in mammals. Cocal virus was originally isolated from mites in Trinidad (Jonkers et al., Am. J. Vet. Res. 1964, 25, 236-242), and infections have been identified in Trinidad, Brazil, and Argentina from insects, cattle, and horses. Many of the vesiculoviruses that infect mammals have been isolated from naturally infected arthropods, suggesting that they are vector-borne. Antibodies to vesiculoviruses are common among people living in rural areas where the viruses are endemic and laboratory-acquired; infections in humans usually result in influenza-like symptoms. The Cocal virus envelope glycoprotein shares 71.5% identity at the amino acid level with VSV-G Indiana, and phylogenetic comparison of the envelope gene of vesiculoviruses shows that Cocal virus is serologically distinct from, but most closely related to, VSV-G Indiana strains among the vesiculoviruses. See, e.g., Jonkers et al., Am. J. Vet. Res. 1964, 25, 236-242 and Travassos da Rosa et al., Am. J. Tropical Med. & Hygiene 1984, 33, 999-1006. The Cocal vesiculovirus envelope pseudotyped retroviral vector particles may include for example, lentiviral, alpharetroviral, betaretroviral, gammaretroviral, deltaretroviral, and epsilonretroviral vector particles that may comprise retroviral Gag, Pol, and / or one or more accessory protein(s) and a Cocal vesiculovirus envelope protein. In certain embodiments, the Gag, Pol, and accessory proteins are lentiviral and / or gammaretroviral. In an embodiment, a retroviral vector can containencoding polypeptides for one or more Cocal vesiculovirus envelope proteins such that the resulting viral or pseudoviral particles are Cocal vesiculovirus envelope pseudotyped.Adenoviral vectors, Helper-dependent Adenoviral vectors, and Hybrid Adenoviral Vectors

[0222] In an embodiment, the vector can be an adenoviral vector. In an embodiment, the adenoviral vector can include elements such that the virus particle produced using the vector or system thereof can be serotype 2 or serotype 5. In an embodiment, the polynucleotide to be delivered via the adenoviral particle can be up to about 8 kb. Thus, in an embodiment, an adenoviral vector can include a DNA polynucleotide to be delivered that can range in size from about 0.001 kb to about 8 kb. Adenoviral vectors have been used successfully in several contexts (see, e.g., Teramato et al., Lancet. 2000, 355, 1911-1912; Lai et al., DNA Cell. Biol. 2002, 72(12), 895-913; Flotte et al., Hum. Gene. Ther. 1996, 7(9), 1145-1159; and Kay et al., Nat. Genet. 2000, 24, 257-261.

[0223] In an embodiment, the vector can be a helper-dependent adenoviral vector or system thereof. These are also referred to in the art as “gutless” or “gutted” vectors and are a modified generation of adenoviral vectors (see, e.g., Thrasher et al., Nature 2006, 443, E5-E6). In certain embodiments of the helper-dependent adenoviral vector system, one vector (the helper) can contain all the viral genes required for replication but contains a conditional gene defect in the packaging domain. The second vector of the system can contain only the ends of the viral genome, one or more engineered Acr polypeptide polynucleotides, and the native packaging recognition signal, which can allow selective packaged release from the cells (see, e.g., Cideciyan et al., N. Engl. J. Med. 2009, 361, 725-727). Helper-dependent adenoviral vector systems have been successful for gene delivery in several contexts (see, e.g., Simonelli et al., Mol. Ther. 2010, 18, 643-650; Cideciyan et al., N. Engl. J. Med. 2009, 361, 725-727; Crane et al., Gene. Ther. 2011, 19, 443-452; Alba et al., Gene. Ther. 2005, 72, S18-S27; Croyle et al., Gene. Ther. 2005, 72, 579- 587; Amalfitano et al., J. Virol. 1998, 72, 926-933; and Morral et al., PNAS.1999, 96, 12816- 12821). The techniques and vectors described in these publications can be adapted for inclusion and delivery of the engineered Acr polypeptide polynucleotides described herein. In an embodiment, the polynucleotide to be delivered via the viral particle produced from a helperdependent adenoviral vector or system thereof can be up to about 37 kb. Thus, in an embodiment, an adenoviral vector can include a DNA polynucleotide to be delivered that can range in size fromabout 0.001 kb to about 37 kb (see, e.g., Rosewell et al., J. Genet. Syndr. Gene Ther. 2011, 2, s5- 001).

[0224] In an embodiment, the vector is a hybrid-adenoviral vector or system thereof. Hybrid adenoviral vectors are composed of the high transduction efficiency of a gene-deleted adenoviral vector and the long-term genome-integrating potential of adeno-associated retroviruses, lentiviruses, and transposon-based gene transfer. In an embodiment, such hybrid vector systems can result in stable transduction and limited integration sites. See, e.g., Balague et al., Blood 2000, 95, 820-828; Morral et al., Hum. Gene Ther. 1998, 9, 2709-2716; Kubo et al., J. Virol. 2003, 77, 2964-2971; Zhang et al., PLoS ONE 2013, 8, e76771; and Cooney et al., Mol. Ther. 2015, 23, 667-674, whose techniques and vectors described therein can be modified and adapted for use in the engineered Acr polypeptide of the present invention. In an embodiment, a hybrid-adenoviral vector can include one or more features of a retrovirus and / or an adeno-associated virus. In an embodiment, the hybrid-adenoviral vector can include one or more features of a spuma retrovirus or foamy virus (FV). See, e.g., Ehrhardt et al., Mol. Ther. 2007, 15, 146-156 and Liu et al., Mol. Ther. 2007, 75(10), 1834-1841, whose techniques and vectors described therein can be modified and adapted for use in the Acr of the present invention. Advantages of using one or more features from the FVs in the hybrid-adenoviral vector or system thereof can include the ability of the viral particles produced therefrom to infect a broad range of cells, a large packaging capacity as compared to other retroviruses, and the ability to persist in quiescent (non-dividing) cells. See, e.g., Ehrhardt et al., Mol. Ther. 2007, 75, 146-156 and Shuji et al., Mol. Ther. 2011, 79, 76-82, whose techniques and vectors described therein can be modified and adapted for use with the engineered Acr polypeptides and encoding polynucleotides of the present invention.Adeno Associated Viral (A A V) Vectors

[0225] In an embodiment, the vector, such as a vector that can include an engineered Acr polypeptide of the present invention and / or a CRISPR-Cas system can be an adeno-associated virus (AAV) vector. See, e.g., U.S. Patent No. 4,797,368; International Patent Application No. WO 1993 / 024641; West et al., Virology 1987, 760(1), 38-47; Kotin et al., Hum. Gene Ther. 1994, 5(7), 793-801; and Muzyczka et al., J. Clin. Invest. 94(4), 1351-1351. Although similar to adenoviral vectors in some of their features, AAVs have some deficiency in their replication and / or pathogenicity and thus can be safer than adenoviral vectors. In an embodiment, the AAV canintegrate into a specific site on chromosome 19 of a human cell with no observable side effects. In an embodiment, the capacity of the AAV vector, system thereof, and / or AAV particles can be up to about 4.7 kb. In an embodiment, such as those where a CRISPR-Cas system is delivered as a co-therapy, homologs of the Cas effector protein that are shorter than e.g., SpCas9 (-4104 bp) can be utilized, such as those in Table 3.

[0226] The AAV vector or system thereof can include one or more regulatory molecules. In an embodiment, the regulatory molecules can be promoters, enhancers, repressors, and the like, which are described in greater detail elsewhere herein. In an embodiment, the AAV vector or system thereof can include one or more polynucleotides that can encode one or more regulatory proteins. In an embodiment, the one or more regulatory proteins can be selected from Rep78, Rep68, Rep52, Rep40, variants thereof, and combinations thereof.

[0227] The AAV vector or system thereof can include one or more polynucleotides that can encode one or more capsid proteins. The capsid proteins can be selected from VP1, VP2, VP3, and combinations thereof. The capsid proteins can be capable of assembling into a protein shell of the AAV virus particle. In an embodiment, the AAV capsid can contain 60 capsid proteins. In an embodiment, the ratio of VP1 :VP2:VP3 in a capsid can be about 1 : 1 : 10.

[0228] In an embodiment, the AAV vector or system thereof can include one or more adenovirus helper factors or polynucleotides that can encode one or more adenovirus helper factors. Such adenovirus helper factors can include, but are not limited to, E1A, E1B, E2A,E4ORF6, and VA RNAs. In an embodiment, a producing host cell line expresses one or more of the adenovirus helper factors.

[0229] The AAV vector or system thereof can be configured to produce AAV particles having a specific serotype. In an embodiment, the serotype can be AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-7, AAV-8, AAV-9 or any combinations thereof. In an embodiment, the AAV can be AAV-1, AAV-2, AAV-5 or any combination thereof. One can select the AAV serotype of the AAV with regard to the cells to be targeted; e.g., one can select AAV serotypes 1, 2, 5 or a hybrid capsid AAV-1, AAV-2, AAV-5 or any combination thereof for targeting brain and / or neuronal cells; and one can select AAV-4 for targeting cardiac tissue; and one can select AAV-8 for delivery to the liver. Thus, in an embodiment, an AAV vector or system thereof capable of producing AAV particles capable of targeting the brain and / or neuronal cells can be configured to generate AAV particles having serotypes 1, 2, 5 or a hybrid capsid AAV-1, AAV-2, AAV-5 or any combination thereof. In an embodiment, an AAV vector or system thereof capable of producing AAV particles capable of targeting cardiac tissue can be configured to generate an AAV particle having an AAV-4 serotype. In an embodiment, an AAV vector or system thereof capable of producing AAV particles capable of targeting the liver can be configured to generate an AAV having an AAV-8 serotype. In an embodiment, the AAV vector is a hybrid AAV vector or system thereof. Hybrid AAVs are AAVs that include genomes with elements from one serotype that are packaged into a capsid derived from at least one different serotype. For example, if it is the recombinant AAV2 / 5 (rAAV2 / 5) that is to be produced, and if the production method is based on the helper-free, transient transfection method discussed elsewhere herein, all plasmids but the RepCap (pRepCap) plasmid will be the same. In the RepCap plasmid, called pRep2 / Cap5, the Rep gene is still derived from AAV-2, while the Cap gene is derived from AAV-5. The production scheme is the same as the above-mentioned approach for AAV-2 production. The resulting rAAV is called rAAV2 / 5, in which the genome is based on recombinant AAV-2, while the capsid is based on AAV-5. It is assumed the cell or tissue-tropism displayed by this AAV2 / 5 hybrid virus should be the same as that of AAV-5. This can be applied to generate other hybrid serotypes.

[0230] A tabulation of certain AAV serotypes as to these cells can be found in Grimm et al., I. Virol. 2008, 82, 5887-5911 at Table 3.

[0231] In an embodiment, the AAV vector or system thereof is configured as a “gutless” vector, similar to that described in connection with a retroviral vector. In an embodiment, the “gutless” AAV vector or system thereof can have the cis-acting viral DNA elements involved in genome amplification and packaging in linkage with the heterologous sequences of interest (e.g., the engineered Acr polypeptide or component thereof, a CRISPR-Cas system polynucleotide(s) co-therapy, or any combination thereof).

[0232] In an embodiment, the AAV vectors are produced in insect cells, e.g., Spodoptera frugiperda Sf9 insect cells, grown in serum-free suspension culture. Serum-free insect cells can be purchased from commercial vendors, e.g., Sigma Aldrich (EX-CELL 405).

[0233] In an embodiment, an AAV vector or vector system can contain or consists essentially of one or more polynucleotides encoding one or more components of a CRISPR system, such as when included as a co-therapy. In an embodiment, the AAV vector or vector system can contain a plurality of cassettes comprising or consisting a first cassette comprising or consisting essentially of a promoter, a nucleic acid molecule encoding a CRISPR-associated (Cas) protein (putative nuclease or helicase proteins), e.g., a Cas protein and a terminator, and a two, or more, advantageously up to the packaging size limit of the vector, e.g., in total (including the first cassette) five, cassettes comprising or consisting essentially of a promoter, nucleic acid molecule encoding guide RNA (gRNA) and a terminator (e.g., each cassette schematically represented as Promoter-gRNAl -terminator, Promoter-gRNA2-terminator, ... Promoter-gRNA(N)-terminator; where N is a number that can be inserted that is at an upper limit of the packaging size limit of the vector), or two or more individual rAAVs, each containing one or more than one cassette of a CRISPR system, e.g., a first rAAV containing the first cassette comprising or consisting essentially of a promoter, a nucleic acid molecule encoding Cas, e.g., a Cas and a terminator, and a second rAAV containing a plurality of cassettes comprising or consisting essentially of a promoter, nucleic acid molecule encoding guide RNA (gRNA) and a terminator (e.g., each cassette schematically represented as Promoter-gRNAl -terminator, Promoter-gRNA2-terminator, ... Promoter-gRNA(N)-terminator; where N is a number that can be inserted that is at an upper limit of the packaging size limit of the vector). As rAAV is a DNA virus, the nucleic acid molecules in the herein discussion concerning AAV or rAAV are advantageously DNA. In an embodiment, the promoter is a tissue-specific promoter or another tissue-specific regulatory element. Suitabletissue-specific regulatory elements, including promoters, are described in greater detail elsewhere herein.

[0234] In another embodiment, the invention provides a non-naturally occurring or engineered Acr polypeptide or component(s) thereof and / or CRISPR-Cas system protein or polynucleotide associated with Adeno Associated Virus (AAV), e.g., an AAV comprising an engineered Acr polypeptide or component(s) thereof and / or CRISPR-Cas system protein or polynucleotide as a fusion, with or without a linker, to or with an AAV capsid protein such as VP1, VP2, and / or VP3. Incorporation of proteins in viral capsids is described in, e.g., Rybniker et al., J. Virol. 2012, 56(24), 13800-13804; Lux et al., J. Virol. 2005, 79(18), 11776-11787; Munch et al., Mol. Ther. 2013, 27(1), 109-118; Warrington et al., J. Virol. 2004, 75(12), 6595-6609, which can each be adapted for use with the present invention. It will be understood by those skilled in the art that the modifications described herein, if inserted into the AAV capsid gene (cap gene), may result in modifications in the VP1, VP2 and / or VP3 capsid subunits. Alternatively, the capsid subunits can be expressed independently to achieve modification in only one or two of the capsid subunits (VP1, VP2, VP3, VP1+VP2, VP1+VP3, or VP2+VP3). One can modify the cap gene to have expressed at a desired location a non-capsid protein, advantageously a large payload protein, such as a CRISPR-protein. Likewise, these can be fusions, with the protein, e.g., a large payload protein such as a CRISPR-protein fused in a manner analogous to prior art fusions. See, e.g., U.S. Patent Publication No. US20090215879; and Nance et al., Hum. Gene Ther. 2015, 26(12), 786-800 and documents cited therein, which are incorporated herein by reference. The skilled person, from this disclosure and the knowledge in the art, can make and use modified AAV or AAV capsid as in the herein invention, and through this disclosure, one knows now that large payload proteins can be fused to the AAV capsid. In an embodiment, the AAV-capsid recombinant AAVs contain proteins and / or nucleic acid molecule(s) encoding or providing an engineered Acr polypeptide or component thereof and / or a CRISPR-Cas system or component thereof co-therapy to a cell. In an embodiment, the engineered Acr polypeptide and / or CRISPR-Cas system co-therapy is / are assembled from the nucleic acid molecule(s) contained in the AAV and a protein component on a surface of the capsid, such as outer or inner surface. The instant invention is also applicable to a virus in the genus Dependoparvovirus or in the family Parvoviridae, for instance, AAV, or a virus of Amdoparvovirus, e.g., Carnivore amdoparvovirus 1, a virus of Aveparvovirus, e.g., Galliformaveparvovirus 1, a virus of Bocaparvovirus, e.g., Ungulate bocaparvovirus 1, a virus of Copiparvovirus, e.g., Ungulate copiparvovirus 1, a virus of Dependoparvovirus, e.g., Adeno- associated dependoparvovirus A, a virus of Erythroparvovirus, e.g., Primate erythroparvovirus 1, a virus of Protoparvovirus, e.g., Rodent protoparvovirus 1, a virus of Tetraparvovirus, e.g., Primate tetraparvovirus 1. Thus, a virus within the family Parvoviridae or the genus Dependoparvovirus or any of the other foregoing genera within Parvoviridae is contemplated as within the invention with discussion herein as to AAV applicable to such other viruses.

[0235] In an embodiment, an engineered Acr polypeptide component and / or CRISPR-Cas system co-therapy component is external to the capsid or virus particle in the sense that it is not inside the capsid (enveloped or encompassed with the capsid) but is externally exposed so that it can contact the target cellular component (e.g., DNA, RNA, and / or protein). In an embodiment, an engineered Acr polypeptide and / or component hereof and / or CRISPR-Cas system co-therapy component is associated with the AAV VP2 domain by way of a fusion protein. In an embodiment, the association may be considered to be a modification of the VP2 domain. In an embodiment, the AAV VP2 domain may be associated (or tethered) to an engineered Acr polypeptide and / or component thereof and / or CRISPR-Cas system co-therapy component via a connector protein, for example, using a system such as the streptavidin-biotin system. In an embodiment, the engineered Acr polypeptide and / or component thereof and / or CRISPR-Cas system co-therapy component and associated AAV VP2 domain are encoded by a polynucleotide. In one embodiment, the invention provides a non-naturally occurring modified AAV having a VP2-engineered Acr polypeptide and / or component thereof and / or CRISPR-Cas system co-therapy component capsid protein, wherein the engineered Acr polypeptide and / or component thereof and / or CRISPR-Cas system co- therapy component is part of or tethered to the VP2 domain. In an embodiment, the engineered Acr polypeptide and / or component thereof and / or CRISPR-Cas system co-therapy component is fused to the VP2 domain to produce a modified AAV having a VP2-engineered Acr polypeptide and / or component thereof and / or CRISPR-Cas system co-therapy component fusion capsid protein. In an embodiment, the VP2-engineered Acr polypeptide and / or component thereof and / or CRISPR-Cas system co-therapy component capsid protein further comprises a linker, whereby the VP2-engineered Acr polypeptide and / or component thereof and / or CRISPR-Cas system co- therapy component is distanced from the remainder of the AAV. In an embodiment, the VP2-engineered Acr polypeptide and / or component thereof and / or CRISPR-Cas system co-therapy component capsid protein further comprises at least one protein complex, e.g., CRISPR complex, such as a CRISPR-Cas complex guide RNA that targets a particular cellular polynucleotide target (e.g., a DNA or an RNA molecule), such as in a co-therapy.

[0236] In one embodiment, the invention provides a non-naturally occurring or engineered composition comprising an engineered Acr polypeptide and / or component thereof and / or CRISPR-Cas system co-therapy component. In some of such embodiments, the engineered Acr polypeptide and / or component thereof and / or CRISPR-Cas system co-therapy is part of or tethered to an AAV capsid domain, i.e., VP1, VP2, or VP3 domain of Adeno- Associated Virus (AAV) capsid. In an embodiment, part of an engineered Acr polypeptide and / or component thereof and / or CRISPR-Cas system co-therapy component tethered to an AAV capsid domain is associated with an AAV capsid domain. In an embodiment, an engineered Acr polypeptide and / or component thereof and / or CRISPR-Cas system co-therapy component may be fused to the AAV capsid domain. In an embodiment, the fusion may be to the N-terminal end of the AAV capsid domain. As such, in an embodiment, the C-terminal end of the engineered Acr polypeptide and / or component thereof component and / or CRISPR-Cas system co-therapy component is fused to the N-terminal end of the AAV capsid domain. In an embodiment, an NLS and / or a linker (such as a GlySer linker) may be positioned between the C-terminal end of the engineered Acr polypeptide and / or component thereof and / or CRISPR-Cas system co-therapy component and the N-terminal end of the AAV capsid domain. In an embodiment, the fusion may be to the C-terminal end of the AAV capsid domain. In an embodiment, this is not preferred due to the fact that the VP1, VP2, and VP3 domains of AAV are alternative splices of the same RNA and so a C-terminal fusion may affect all three domains. In an embodiment, the AAV capsid domain is truncated. In an embodiment, some or all of the AAV capsid domain is removed. In an embodiment, some of the AAV capsid domain is removed and replaced with a linker (such as a GlySer linker), typically leaving the N-terminal and C-terminal ends of the AAV capsid domain intact, such as the first 2, 5, or 10 amino acids. In this way, the internal (non-terminal) portion of the VP3 domain may be replaced with a linker. In an embodiment, the linker is fused to the engineered Acr polypeptide and / or component thereof and / or CRISPR-Cas system co-therapy component. A branched linker may be used. In such embodiments, an engineered Acr polypeptide and / or component thereofand / or CRISPR-Cas system co-therapy component is fused to the end of one of the branches. Without being bound by theory, this allows for some degree of spatial separation between the capsid and the polypeptide and / or component(s) thereof and / or CRISPR-Cas protein. In this way, the polypeptide and / or component(s) thereof and / or CRISPR-Cas protein is part of (or fused to) the AAV capsid domain.

[0237] In other embodiments, the engineered Acr polypeptide and / or component thereof and / or CRISPR-Cas system co-therapy component may be fused in frame within, e.g., internal to, the AAV capsid domain. Thus, in an embodiment, the AAV capsid domain again preferably retains its N-terminal and C-terminal ends. In this case, a linker is preferred, in an embodiment, either at one or both ends of the engineered Acr polypeptide and / or component thereof and / or CRISPR-Cas system co-therapy component. In this way, the engineered Acr polypeptide and / or component thereof and / or CRISPR-Cas system co-therapy component is again part of (or fused to) the AAV capsid domain. In certain embodiments, the positioning of the CRISPR enzyme is such that the engineered Acr polypeptide and / or component thereof and / or CRISPR-Cas system co-therapy component is at the external surface of the viral capsid once formed. In one embodiment, the invention provides a non-naturally occurring or engineered composition comprising an engineered Acr polypeptide and / or component thereof and / or CRISPR-Cas system co-therapy component associated with an AAV capsid domain of the AAV capsid. In this context, “associated” refers, in an embodiment to fused, or in an embodiment bound to, or in an embodiment tethered to. The engineered Acr polypeptide and / or component thereof and / or CRISPR-Cas system co-therapy component may, in an embodiment, be tethered to the VP1, VP2, or VP3 domain. This may be via a connector protein or tethering system such as the biotin-streptavidin system. In one example, a biotinylation sequence (15 amino acids) could therefore be fused to an engineered Acr polypeptide and / or component thereof and / or CRISPR-Cas system co-therapy component protein. When a fusion of the AAV capsid domain, especially the N-terminus of the AAV capsid domain with streptavidin, is also provided, the two will therefore associate with very high affinity. Thus, in an embodiment, provided is a composition or system comprising an engineered Acr polypeptide and / or component thereof and / or CRISPR-Cas system co-therapy component-biotin fusion and a streptavidin-AAV capsid domain arrangement, such as a fusion. The engineered Acr polypeptide and / or component thereof and / or CRISPR-Cas system co-therapy component-biotin andstreptavidin- AAV capsid domain forms a single complex when the two parts are brought together. NLSs may also be incorporated between the engineered Acr polypeptide and / or component thereof and / or CRISPR-Cas system co-therapy component and the biotin; and / or between the streptavidin and the AAV capsid domain.

[0238] As such, provided is a fusion of an engineered Acr polypeptide and / or component thereof and / or CRISPR-Cas system co-therapy component with a connector protein specific for a high-affinity ligand for that connector, whereas the AAV VP2 domain is bound to said high- affinity ligand. For example, streptavidin may be the connector fused to the engineered Acr polypeptide and / or component thereof and / or CRISPR-Cas system co-therapy component, while biotin may be bound to the AAV VP2 domain. Upon co-localization, the streptavidin will bind to the biotin, thus connecting the engineered Acr polypeptide and / or component thereof and / or CRISPR-Cas system co-therapy component to the AAV VP2 domain. The reverse arrangement is also possible. In an embodiment, a biotinylation sequence (15 amino acids) could therefore be fused to the AAV VP2 domain, especially the N-terminus of the AAV VP2 domain. A fusion of an engineered Acr polypeptide and / or component thereof and / or CRISPR-Cas system co-therapy component with streptavidin is also preferred, in an embodiment. In an embodiment, the biotinylated AAV capsids with streptavidin-engineered Acr polypeptide and / or component(s) thereof and / or CRISPR-Cas system co-therapy component(s) are assembled in vitro. This way, the AAV capsids should assemble in a straightforward manner, and the engineered Acr polypeptide and / or component thereof and / or CRISPR-Cas system co-therapy component-streptavidin fusion can be added after assembly of the capsid. In other embodiments, a biotinylation sequence (15 amino acids) could therefore be fused to the engineered Acr polypeptide and / or component thereof and / or CRISPR-Cas system co-therapy component, together with a fusion of the AAV VP2 domain, especially the N-terminus of the AAV VP2 domain, with streptavidin. For simplicity, a fusion of the engineered Acr polypeptide and / or component thereof and / or CRISPR-Cas system co-therapy component and the AAV VP2 domain is preferred in an embodiment. In an embodiment, the fusion may be to the N-terminal end of the engineered Acr polypeptide and / or component thereof and / or CRISPR-Cas system co-therapy component. In other words, in an embodiment, the AAV and engineered Acr polypeptide and / or component thereof and / or CRISPR- Cas system co-therapy component are associated via fusion. In an embodiment, the AAV andengineered Acr polypeptide and / or component thereof and / or CRISPR-Cas system co-therapy component are associated via fusion including a linker. Suitable linkers are discussed herein, but include Gly Ser linkers. Fusion to the N-terminus of AAV VP2 domain is preferred in an embodiment. In an embodiment, an engineered Acr polypeptide and / or component thereof and / or CRISPR-Cas system co-therapy component comprises at least one Nuclear Localization Signal (NLS). In a further embodiment, the present invention provides compositions comprising the engineered Acr polypeptide and / or component thereof and / or CRISPR-Cas system co-therapy component and associated AAV VP2 domain or the polynucleotides or vectors described herein. Such compositions and formulations are discussed elsewhere herein.

[0239] An alternative tether may be to fuse or otherwise associate the AAV capsid domain to an adaptor protein which binds to or recognizes to a corresponding RNA sequence or motif. In an embodiment, the adaptor is or comprises a binding protein which recognizes and binds (or is bound by) an RNA sequence specific for said binding protein. In an embodiment, a preferred example is the MS2 (see, e.g., Konermann et al., Nature 2014, 517, 583-588, which is incorporated herein by reference) binding protein which recognizes and binds (or is bound by) an RNA sequence specific for the MS2 protein. In an embodiment, the RNA sequence specific for a binding protein is a gRNA that can bind to a Cas protein.

[0240] With the AAV capsid domain associated with the adaptor protein, an engineered Acr polypeptide and / or component thereof and / or CRISPR-Cas system co-therapy component may, in an embodiment, be tethered to the adaptor protein of the AAV capsid domain. The engineered Acr polypeptide and / or component thereof and / or CRISPR-Cas system co-therapy component may, in an embodiment, be tethered to the adaptor protein of the AAV capsid domain via the engineered Acr polypeptide and / or component thereof and / or CRISPR-Cas system co-therapy component being in a complex with a modified guide, see Konermann et al., Nature 2014, 517, 583-588. The modified guide is, in an embodiment, an sgRNA. In an embodiment, the modified guide comprises a distinct RNA sequence; see, e.g., International Patent Application Publication No. WO 2015 / 089486, which is incorporated herein by reference. In an embodiment, the distinct RNA sequence is an aptamer. Thus, corresponding aptamer-adaptor protein systems are preferred. One or more functional domains may also be associated with the adaptor protein. An example of apreferred arrangement would be: [AAV capsid domain - adaptor protein] - [modified guide - CRISPR protein and / or Acr polypeptide and / or component thereof],

[0241] In certain embodiments, the positioning of the engineered Acr polypeptide and / or component thereof and / or CRISPR-Cas system co-therapy component is such that the engineered Acr polypeptide and / or component thereof and / or CRISPR-Cas system co-therapy component is at the internal surface of the viral capsid once formed. In one embodiment, the invention provides a non-naturally occurring or engineered composition comprising an engineered Acr polypeptide and / or component thereof and / or CRISPR-Cas system co-therapy component associated with an internal surface of an AAV capsid domain. Here again, associated may mean in an embodiment fused, or in an embodiment bound to, or in an embodiment tethered to. The engineered Acr polypeptide and / or component thereof and / or CRISPR-Cas system co-therapy component may, in an embodiment, be tethered to the VP1, VP2, or VP3 domain such that it locates to the internal surface of the viral capsid once formed. This may be via a connector protein or tethering system such as the biotin-streptavidin system as described above and / or elsewhere herein.

[0242] In one embodiment, a co-therapy can include a non-naturally occurring CRISPR-Cas system comprising an AAV-Cas protein and a guide RNA that targets a DNA molecule encoding a gene product in a cell, whereby the guide RNA targets the DNA molecule encoding the gene product and the Cas protein cleaves the DNA molecule encoding the gene product, whereby expression of the gene product is altered; and, wherein the Cas protein and the guide RNA do not naturally occur together. The invention comprehends the guide RNA comprising a guide sequence fused to a Trans-activating CRISPR (tracr) sequence. In a preferred embodiment, the Cas protein is a Cas9, a Casl3, or a Casl2 protein. Other suitable Cas proteins are described elsewhere herein. In an embodiment, the polynucleotide encoding the Cas protein is codon optimized for expression in a eukaryotic cell. In an embodiment, the eukaryotic cell is a mammalian cell and in a more preferred embodiment the mammalian cell is a human cell. In a further embodiment, the expression of the gene product is decreased.

[0243] In another embodiment, a co-therapy comprises non-naturally occurring vector system comprising one or more vectors comprising a first regulatory element operatively linked to a CRISPR-Cas system guide RNA that targets a DNA molecule encoding a gene product and an AAV-Cas protein. The components may be located on same or different vectors of the system ormay be the same vector whereby the AAV-Cas protein also delivers the RNA of the CRISPR system. The guide RNA targets the DNA molecule encoding the gene product in a cell and the AAV-Cas protein may cleave the DNA molecule encoding the gene product (it may cleave one or both strands or have substantially no nuclease activity), whereby expression of the gene product is altered; and, wherein the AAV-Cas protein and the guide RNA do not naturally occur together. The invention comprehends the guide RNA comprising a guide sequence fused to a tracr sequence. In an embodiment of the invention, the AAV-Cas protein is a type II AAV-CRISPR-Cas protein and in a preferred embodiment the AAV-Cas protein is an AAV-Cas9, AAV-Casl2, or AAV- Casl3 protein. The invention further comprehends the coding for the AAV-Cas protein being codon optimized for expression in a eukaryotic cell. In a preferred embodiment, the eukaryotic cell is a mammalian cell and in a more preferred embodiment, the mammalian cell is a human cell. In a further embodiment of the invention, the expression of the gene product is decreased.

[0244] In one embodiment, the invention provides a vector system comprising one or more vectors. In an embodiment, the system comprises a CRISPR-Cas co-therapy that comprises: (a) a first regulatory element operatively linked to a tracr mate sequence and one or more insertion sites for inserting one or more guide sequences upstream of the tracr mate sequence, wherein when expressed, the guide sequence directs sequence-specific binding of an AAV-CRISPR complex to a target sequence in a eukaryotic cell, wherein the CRISPR complex comprises an AAV-CRISPR enzyme complexed with (1) the guide sequence that is hybridized to the target sequence, and (2) the tracr mate sequence that is hybridized to the tracr sequence; and (b) said AAV-CRISPR enzyme comprising at least one nuclear localization signal and / or at least one nuclear export signal (NES); wherein components (a) and (b) are located on or in the same or different vectors of the system. In an embodiment, component (a) further comprises the tracr sequence downstream of the tracr mate sequence under the control of the first regulatory element. In an embodiment, component (a) further comprises two or more guide sequences operatively linked to the first regulatory element, wherein when expressed, each of the two or more guide sequences direct sequence-specific binding of an AAV-CRISPR complex to a different target sequence in a eukaryotic cell. In an embodiment, the system comprises the tracr sequence under the control of a third regulatory element, such as a polymerase III promoter. In an embodiment, the tracr sequence exhibits at least 50%, 60%, 70%, 80%, 90%, 95%, or 99% of sequence complementarity along the length of thetracr mate sequence when optimally aligned. Determining optimal alignment is within the purview of one of skill in the art. For example, there are publicly and commercially available alignment algorithms and programs such as, but not limited to, ClustalW, Smith-Waterman in matlab, Bowtie, Geneious, Biopython and SeqMan. In an embodiment, the AAV-CRISPR complex comprises one or more nuclear localization signals of sufficient strength to drive accumulation of said CR1SPR complex in a detectable amount in the nucleus of a eukaryotic cell. Without wishing to be bound by theory, it is believed that a nuclear localization signal is not necessary for AAV- CRISPR complex activity in eukaryotes, but that including such sequences enhances activity of the system, especially as to targeting nucleic acid molecules in the nucleus and / or having molecules exit the nucleus. In an embodiment, the AAV-CRISPR enzyme is an AAV-Cas enzyme. In an embodiment, the AAV-Cas enzyme is derived from S. pneumoniae, S. pyogenes, S. thermophiles, F. novicida or S. aureus Cas9, Casl2 (e.g., Casl2a), Casl3, etc. (e.g., a Cas protein of one of these organisms modified to have or be associated with at least one AAV) and may include further mutations or alterations or be a chimeric Cas9. The enzyme may be an AAV-Cas9 homolog or ortholog. In an embodiment, the AAV-CRISPR enzyme is codon-optimized for expression in a eukaryotic cell. In an embodiment, the AAV-CRISPR enzyme directs the cleavage of one or two strands at the location of the target sequence. In an embodiment, the AAV-CRISPR enzyme lacks DNA strand cleavage activity. In an embodiment, the first regulatory element is a polymerase III promoter. In an embodiment, the second regulatory element is a polymerase II promoter. In an embodiment, the guide sequence is at least 15, 16, 17, 18, 19, 20, 25 nucleotides, or between 10-30, or between 15-25, or between 15-20 nucleotides in length.

[0245] In general, in an embodiment, the AAV further comprises a repair template. It will be appreciated that comprises in the phrase “the virus comprises...”, “the AAV comprises...”, “the lentiviral vector LVV0 comprises”, “the LVV comprises,” and / or the like may mean encompassed within the viral capsid or that the virus encodes the comprised protein or polynucleotide such as a repair template, gRNA, mRNA, and / or the like. In an embodiment, one or more, preferably two or more guide RNAs, may be comprised / encompassed within the AAV vector. Two may be preferred in an embodiment, as it allows for multiplexing or dual nickase approaches. Particularly for multiplexing, two or more guides may be used. In fact, in an embodiment, three or more, four or more, five or more, or even six or more guide RNAs may be comprised / encompassed within theAAV. More space has been freed up within the AAV by virtue of the fact that the AAV no longer needs to comprise / encompass the CRISPR enzyme. In each of these instances, a repair template may also be provided comprised / encompassed within the AAV. In an embodiment, the repair template corresponds to or includes the DNA target.

[0246] It will be appreciated that where a CRISPR-Cas system is delivered as part of a therapy, it can be delivered via any suitable manner or delivery vehicle, including but not limited to AAVs. Exemplary delivery of a CRISPR-Cas system or component thereof are described elsewhere herein and include those described in paragraphs

[0117] to

[0278] of International Patent Publication No. WO 2016 / 10623), and pages 1241-1251 and Table 1 of Lino et al., Drug Deliv. 2018, 25(1), 1234-1257, which are incorporated by reference herein in their entireties.Herpes Simplex Viral Vectors

[0247] In an embodiment, the vector can be a Herpes Simplex Viral (HSV)-based vector or system thereof. HSV systems can include the disabled infections single copy (DISC) viruses, which are composed of a glycoprotein H defective mutant HSV genome. When the defective HSV is propagated in complementing cells, virus particles can be generated that are capable of infecting subsequent cells, permanently replicating their own genome but are not capable of producing more infectious particles. See, e.g., Trobridge et al., Exp. Opin. Biol. Ther. 2009, 9(11), 1427-1436, whose techniques and vectors described therein can be modified and adapted for use with the engineered Acr polypeptide and / or component thereof and / or CRISPR-Cas co-therapy and their encoding polynucleoitdes. In an embodiment where an HSV vector or system thereof is utilized, the host cell can be a complementing cell. In an embodiment, the HSV vector or system thereof can be capable of producing virus particles capable of delivering a polynucleotide cargo of up to 150 kb. Thus, in some embodiment, the engineered Acr polypeptide and / or component thereof and / or CRISPR-Cas co-therapy polynucleotide(s) included in the HSV-based viral vector or system thereof can sum from about 0.001 to about 150 kb. HSV-based vectors and systems thereof have been successfully used in several contexts including various models of neurologic disorders. See, e.g., Cockrell et al., Mol. Biotechnol. 2007, 36, 184-204; Kafri et al., Mol. Biol. 246, 367- 390; Balaggan et al., Gene Ther. 2011, 19, 145-153; Wong et al., Hum. Gen. Ther. 2006, 77(1), 1- 9; Azzouz et al., J. Neurosci. 2002, 22, 10302-10312; and Betchen et al., Curr. Opin. Neurol. 2003,16(4), 487-493, whose techniques and vectors described therein can be modified and adapted for use in the engineered Acr polypeptide and / or component thereof and / or CRISPR-Cas co-therapy.Poxvirus Vectors

[0248] In an embodiment, the vector can be a poxvirus vector or system thereof. In an embodiment, the poxvirus vector can result in cytoplasmic expression of one or more engineered Acr polypeptides and / or component(s) thereof and / or CRISPR-Cas co-therapy polynucleotides described herein. In an embodiment, the capacity of a poxvirus vector or system thereof can be about 25 kb or more. In an embodiment, a poxvirus vector or system thereof can include one or more CRISPR-Cas system polynucleotides described herein.Viral Vectors for delivery to plants

[0249] The systems and compositions may be delivered to plant cells using viral vehicles. In particular embodiments, the compositions and systems may be introduced in the plant cells using a plant viral vector (e.g., as described in Scholthof et al., Annu. Rev. Phytopathol. 1996, 34, 299- 323). Such viral vector may be a vector from a DNA virus, e.g., geminivirus (e.g., cabbage leaf curl virus, bean yellow dwarf virus, wheat dwarf virus, tomato leaf curl virus, maize streak virus, tobacco leaf curl virus, or tomato golden mosaic virus) or nanovirus (e.g., Faba bean necrotic yellow virus). The viral vector may be a vector from an RNA virus, e.g., tobravirus (e.g., tobacco rattle virus, tobacco mosaic virus), potexvirus (e.g., potato virus X), or hordeivirus (e.g., barley stripe mosaic virus). The replicating genomes of plant viruses may be non-integrative vectors.Virus-Like Particles and Vectors

[0250] In an embodiment, the vector is a vector that is capable of generating virus-like particles (VLPs). VLPs is a term of art that refers to particles produced from virus proteins, such as capsid or other proteins, but that do not contain the native viral genetic materials. Exemplary VLPs and their production systems and vectors for delivery of an engineered Acr polypeptide and / or component thereof described herein are described in e.g., Bhat et al., Viruses 2022, 14(2), 383; Hill et al., Curr. Protein Pept. Sci. 2018, 79(1), 112-127; Schwarz B et al., Adv. Virus Res. 2017, 97, 1-60; Banskota et al., Cell 2022, 185, 250-265. el6; Ikwuagwu et al., Curr. Opin. Biotechnol. 2022, 78, 102785; Zdanowicz et al., Acta Biochim Pol. 2016, 63(3), 469-473; Suffian et al., Adv. Drug Deliv. Rev. 2022, 180, 114030; and Segel et al., Science 2021, 373, 882-889.Virus Particle Production from Viral VectorsRetroviral Production

[0251] In an embodiment, one or more viral vectors and / or systems thereof can be delivered to a suitable cell line for production of virus particles containing the polynucleotide or other payload to be delivered to a host cell. Suitable host cells for virus production from viral vectors and systems thereof described herein are known in the art and are commercially available. For example, suitable host cells include HEK293 cells and its variants (HEK293T and HEK293TN cells). In an embodiment, the suitable host cell for virus production from viral vectors and systems thereof described herein can stably express one or more genes involved in packaging (e.g., pol, gag, and / or VSV-G) and / or other supporting genes.

[0252] In an embodiment, after delivery of one or more viral vectors to the suitable host cells for virus production from viral vectors and systems thereof, the cells are incubated for an appropriate length of time to allow for viral gene expression from the vectors, packaging of the polynucleotide to be delivered (e.g., an engineered Acr polypeptide and / or component thereof and / or CRISPR-Cas co-therapy polynucleotide), and virus particle assembly, and secretion of mature virus particles into the culture media. Various other methods and techniques are generally known to those of ordinary skill in the art.

[0253] Mature virus particles can be collected from the culture media by a suitable method. In an embodiment, this can involve centrifugation to concentrate the virus. The titer of the composition containing the collected virus particles can be obtained using a suitable method. Such methods can include transducing a suitable cell line (e.g., NIH 3T3 cells) and determining transduction efficiency and infectivity in that cell line by a suitable method. Suitable methods include PCR-based methods, flow cytometry, and antibiotic selection-based methods. Various other methods and techniques are generally known to those of ordinary skill in the art. The concentration of virus particles can be adjusted as needed. In an embodiment, the resulting composition containing virus particles can contain 1 x 101-! x IO20particles / mL.

[0254] Lentiviruses may be prepared from any lentiviral vector or vector system described herein. In one example embodiment, after cloning pCasESlO (which contains a lentiviral transfer plasmid backbone), HEK293FT at low passage (p=5) can be seeded in a T-75 flask to 50% confluence the day before transfection in DMEM with 10% fetal bovine serum and withoutantibiotics. After 20 hours, the media can be changed to OptiMEM (serum-free) media and transfection of the lentiviral vectors can be done 4 hours later. Cells can be transfected with 10 pg of lentiviral transfer plasmid (pCasESlO) and the appropriate packaging plasmids (e.g., 5 pg of pMD2.G (VSV-g pseudotype), and 7.5 pg of psPAX2 (gag / pol / rev / tat)). Transfection can be carried out in 4 mb OptiMEM with a cationic lipid delivery agent (50 pL Lipofectamine 2000 and 100 pL Plus reagent). After 6 hours, the media can be changed to antibiotic-free DMEM with 10% fetal bovine serum. These methods can use serum during cell culture, but serum-free methods are preferred.

[0255] Following transfection and allowing the producing cells (also referred to as packaging cells) to package and produce virus particles with packaged cargo, the lentiviral particles can be purified. In an exemplary embodiment, virus-containing supernatants can be harvested after 48 hours. Collected virus-containing supernatants can first be cleared of debris and filtered through a 0.45 pm low protein binding (PVDF) filter. They can then be spun in an ultracentrifuge for 2 hours at 24,000 rpm. The resulting virus-containing pellets can be resuspended in 50 pL of DMEM overnight at 4 degrees C. They can be then aliquoted and used immediately or immediately frozen at -80 degrees C for storage.AA V Particle Production

[0256] There are two main strategies for producing AAV particles from AAV vectors and systems thereof, such as those described herein, which depend on how the adenovirus helper factors are provided (helper- v. helper-free). In an embodiment, a method of producing AAV particles from AAV vectors and systems thereof can include adenovirus infection into cell lines that stably harbor AAV replication and capsid encoding polynucleotides along with AAV vector containing the polynucleotide to be packaged and delivered by the resulting AAV particle (e.g., the engineered Acr polypeptide and / or component thereof and / or CRISPR-Cas system polynucleotide(s)). In an embodiment, a method of producing AAV particles from AAV vectors and systems thereof can be a “helper-free” method, which includes co-transfection of an appropriate producing cell line with three vectors (e.g., plasmid vectors): (1) an AAV vector that contains a polynucleotide of interest (e.g., the engineered Acr polypeptide and / or component thereof and / or CRISPR-Cas system polynucleotide(s)) between 2 inverted terminal repeats (ITRs); (2) a vector that carries the AAV Rep-Cap encoding polynucleotides; and (3) a vector that carrieshelper polynucleotides. One of skill in the art will appreciate various methods and variations thereof that are both helper- and helper-free and as well as the different advantages of each system.Non- Viral Vectors

[0257] In an embodiment, the vector is a non-viral vector or vector system. The term of art “Non-viral vector” and as used herein in this context refers to molecules and / or compositions that are vectors but that are not based on one or more components of a virus or virus genome (excluding any nucleotide to be delivered and / or expressed by the non-viral vector) that can be capable of incorporating engineered Acr polypeptide and / or component thereof polynucleotide(s) and / or CRISPR-Cas polynucleotide(s) and delivering said engineered Acr polypeptide and / or component thereof polynucleotide(s) and / or CRISPR-Cas polynucleotide(s) to a cell and / or expressing the polynucleotide in the cell. It will be appreciated that this does not exclude vectors containing a polynucleotide designed to target a virus-based polynucleotide that is to be delivered. For example, if a gRNA to be delivered is directed against a virus component and it is inserted or otherwise coupled to an otherwise non-viral vector or carrier, this would not make said vector a “viral vector.” Non-viral vectors can include, without limitation, naked polynucleotides and polynucleotide (non-viral) based vector and vector systems.Naked Polynucleotides

[0258] In an embodiment, one or more engineered Acr polypeptide polynucleotides and / or component thereof polynucleotide(s) and / or CRISPR-Cas system polynucleotides described elsewhere herein can be included in a naked polynucleotide. The term of art “naked polynucleotide” as used herein refers to polynucleotides that are not associated with another molecule (e.g., proteins, lipids, and / or other molecules) that can often help protect it from environmental factors and / or degradation. As used herein, associated with includes, but is not limited to, linked to, adhered to, adsorbed to, enclosed in, enclosed in or within, mixed with, and the like. Naked polynucleotides that include one or more of the engineered Acr polypeptide polynucleotide and / or component thereof polynucleotide(s) and / or CRISPR-Cas system polynucleotides described herein can be delivered directly to a host cell and optionally expressed therein. The naked polynucleotides can have any suitable two- and three-dimensional configurations. By way of non-limiting examples, naked polynucleotides can be single- stranded molecules, double-stranded molecules, circular molecules (e.g., plasmids and artificialchromosomes), molecules that contain portions that are single-stranded and portions that are double-stranded (e.g., ribozymes), and the like. In an embodiment, the naked polynucleotide contains only the engineered Acr polypeptide polynucleotide(s) and / or component thereof polynucleotide(s) and / or CRISPR-Cas system polynucleotide(s) of the present invention. In an embodiment, the naked polynucleotide can contain other nucleic acids and / or polynucleotides in addition to the engineered Acr polypeptide polynucleotide(s) and / or component thereof polynucleotide(s) and / or CRISPR-Cas system polynucleotide(s) of the present invention. The naked polynucleotides can include one or more elements of a transposon system. Transposons and systems thereof are described in greater detail elsewhere herein.Non- Viral Polynucleotide Vectors

[0259] In an embodiment, one or more of the engineered Acr polypeptide polynucleotide(s) and / or component thereof polynucleotide(s) and / or CRISPR-Cas system polynucleotides can be included in a non-viral polynucleotide vector. Suitable non-viral polynucleotide vectors include, but are not limited to, transposon vectors and vector systems, plasmids, bacterial artificial chromosomes, yeast artificial chromosomes, AR (antibiotic resistance)-free plasmids and miniplasmids, circular covalently closed vectors (e.g., minicircles, minivectors, miniknots), linear covalently closed vectors (“dumbbell-shaped”), MIDGE (minimalistic immunologically defined gene expression) vectors, MiLV (micro-linear vector) vectors, Ministrings, mini-intronic plasmids, PSK systems (post-segregationally killing systems), ORT (operator repressor titration) plasmids, and the like. See, e.g., Hardee et al., Genes, 2017, 8(2), 65.

[0260] In an embodiment, the non-viral polynucleotide vector can have a conditional origin of replication. In an embodiment, the non-viral polynucleotide vector can be an ORT plasmid. In an embodiment, the non-viral polynucleotide vector can have a minimalistic immunologically defined gene expression. In an embodiment, the non-viral polynucleotide vector can have one or more post-segregationally killing system genes. In an embodiment, the non-viral polynucleotide vector is AR-free. In an embodiment, the non-viral polynucleotide vector is a minivector. In an embodiment, the non-viral polynucleotide vector includes a nuclear localization signal. In an embodiment, the non-viral polynucleotide vector can include one or more CpG motifs. In an embodiment, the non-viral polynucleotide vectors can include one or more scaffold / matrix attachment regions (S / MARs). See, e.g., Mirkovitch et al., Cell 1984, 39, 223-232; Wong et al.,Adv. Genet. 2015, 59, 113-152; whose techniques and vectors can be adapted for use in the present invention. S / MARs are AT -rich sequences that play a role in the spatial organization of chromosomes through DNA loop base attachment to the nuclear matrix. S / MARs are often found close to regulatory elements such as promoters, enhancers, and origins of DNA replication. The inclusion of one or more S / MARs can facilitate a once-per-cell-cycle replication to maintain the non-viral polynucleotide vector as an episome in daughter cells. In certain embodiments, the S / MAR sequence is located downstream of an actively transcribed polynucleotide (e.g., one or more Acr polypeptide polynucleotide(s) and / or component thereof polynucleotide(s) and / or CRISPR-Cas system polynucleotide(s) co-therapy of the present invention) included in the non- viral polynucleotide vector. In an embodiment, the S / MAR can be a S / MAR from the betainterferon gene cluster. See, e.g., Verghese et al., Nucleic Acids Res. 2014, 42(7), e53; Xu et al., Sci. China Life Sci. 2016, 59, 1024-1033; Jin et al., EMBO Mol. Med. 2016, 8, 702-711; Koirala et al., Adv. Exp. Med. Biol. 2014, 801, 703-709; and Nehlsen et al., Gene Ther. Mol. Biol. 2006, 10, 233-244, whose techniques and vectors can be adapted for use in the present invention.

[0261] In an embodiment, the non-viral vector is a transposon vector or system thereof. As used herein, “transposon” (also referred to as transposable element) refers to a polynucleotide sequence that is capable of moving from one location in a genome to another. There are several classes of transposons. Transposons include retrotransposons and DNA transposons. Retrotransposons require the transcription of the polynucleotide that is moved (or transposed) in order to transpose the polynucleotide to a new genome or polynucleotide. DNA transposons are those that do not require reverse transcription of the polynucleotide that is moved (or transposed) in order to transpose the polynucleotide to a new genome or polynucleotide. In an embodiment, the non-viral polynucleotide vector can be a retrotransposon vector. In an embodiment, the retrotransposon vector includes long terminal repeats. In an embodiment, the retrotransposon vector does not include long terminal repeats. In an embodiment, the non-viral polynucleotide vector can be a DNA transposon vector. DNA transposon vectors can include a polynucleotide sequence encoding a transposase. In an embodiment, the transposon vector is configured as a non- autonomous transposon vector, meaning that the transposition does not occur spontaneously on its own. In some of these embodiments, the transposon vector lacks one or more polynucleotidesequences encoding proteins required for transposition. In an embodiment, the non-autonomous transposon vectors lack one or more Ac transposable elements.

[0262] In an embodiment, a non-viral polynucleotide transposon vector system can include a first polynucleotide vector that contains the Acr polypeptide polynucleotide(s) and / or component thereof polynucleotide(s) and / or CRISPR-Cas system co-therapy polynucleotide(s) of the present invention flanked on the 5’ and 3’ ends by transposon terminal inverted repeats (TIRs) and a second polynucleotide vector that includes a polynucleotide capable of encoding a transposase coupled to a promoter to drive expression of the transposase. When both are expressed in the same cell the transposase can be expressed from the second vector and can transpose the material between the TIRs on the first vector (e.g., the Acr polypeptide and / or component thereof polynucleotide(s) and / or CRISPR-Cas system polynucleotide(s) of the present invention) and integrate it into one or more positions in the host cell’s genome. In an embodiment, the transposon vector or system thereof can be configured as a gene trap. In an embodiment, the TIRs can be configured to flank a strong splice acceptor site followed by a reporter and / or another gene (e.g., one or more of the Acr polypeptide and / or component thereof polynucleotide(s) and / or CRISPR- Cas system polynucleotide(s) of the present invention) and a strong poly A tail. When transposition occurs while using this vector or system thereof, the transposon can insert into an intron of a gene and the inserted reporter or another gene can provoke a mis-splicing process and as a result, it inactivates the trapped gene.

[0263] Any suitable transposon system can be used. Suitable transposon and systems thereof can include, Sleeping Beauty transposon system (Tcl / mariner superfamily) (see, e.g., Ivies et al., Cell 1997, 9 / (4), 501-510), piggyBac (piggyBac superfamily) (see, e.g., Li et al., Proc. Natl. Acad. Sci. USA 2013, 7 / 0(25), E2279-E2287 and Yusa et al., Proc. Natl. Acad. Sci. USA 2011, 705(4), 1531-1536), Tol2 (superfamily hAT), Frog Prince (Tcl / mariner superfamily) (see, e g., Miskey et al., Nucleic Acids Res. 2003, 3 / (23), 6873-6881) and variants thereof.Delivery Vehicles

[0264] Described in an example embodiment herein are delivery vehicles comprising (a) an engineered Acr polypeptide of the present invention; (b) one or more polynucleotides of the present invention; (c) one or more vectors of the present invention; or any combination of (a)-(c). In an embodiment, the delivery vehicle comprises a co-therapy, including but not limited to aCRISPR-Cas system or component thereof. In an embodiment, the delivery vehicle comprises a ribonucleoprotein (RNP) complex of a CRISPR-Cas system.

[0265] The delivery vehicles may deliver the engineered Acr polypeptide of the present invention, encoding polynucleotides, vectors, etc., of the present invention into and / or within effective proximity of cells, tissues, organs, or organisms (e.g., humans, animals, or plants).

[0266] In connection with delivery vehicles herein, the engineered Acr polypeptide, encoding polynucleotides, vectors, etc., of the present invention that are carried by the delivery vehicle are referred to as “cargos” for simplicity. The cargos may be packaged, carried, or otherwise associated with the delivery vehicles. The delivery vehicles may be selected based on the types of cargo to be delivered, and / or the mode of delivery (e.g., in vitro and / or in vivo). Examples of delivery vehicles include vectors, viruses (e.g., virus particles), non-viral vehicles, and other delivery reagents described herein.

[0267] The delivery vehicles described herein can have the greatest dimension or greatest average dimension (e.g., diameter or greatest average diameter) of less than 100 microns (pm). In an embodiment, the delivery vehicles have the greatest dimension or greatest average dimension of less than 10 pm. In an embodiment, the delivery vehicles have a greatest dimension or greatest average dimension of less than 2000 nanometers (nm). In an embodiment, the delivery vehicles have a greatest dimension or greatest average dimension of less than 1000 nanometers (nm). In an embodiment, the delivery vehicles have a greatest dimension or greatest average dimension (e.g., diameter or average diameter) of less than 900 nm, less than 800 nm, less than 700 nm, less than 600 nm, less than 500 nm, less than 400 nm, less than 300 nm, less than 200 nm, less than 150nm, less than lOOnm, or less than 50nm. In an embodiment, the delivery vehicles have a greatest dimension or greatest average dimension that ranges between 25 nm and 200 nm.

[0268] In an embodiment, the delivery vehicles may be or comprise particles. For example, the delivery vehicle may be or comprise nanoparticles (e.g., particles with a greatest dimension or greatest average dimension (e.g., diameter or greatest average diameter) no greater than 1000 nm. The particles may be provided in different forms, e.g., as solid particles (e.g., a metal such as silver, gold, iron, titanium), non-metal, lipid-based solids, polymers, suspensions of particles, or combinations thereof. Metal, dielectric, and semiconductor particles may be prepared, as well as hybrid structures (e.g., core-shell particles).

[0269] Nanoparticles may also be used to deliver the compositions and systems to cells, as described in International Patent Publication No. WO 2008 / 042156 and WO 2015 / 089419; and U.S. Patent Publication No. US20130185823. In general, a “nanoparticle” refers to any particle having a diameter of less than 1000 nm. In certain embodiments, nanoparticles of the invention have a greatest dimension or greatest average dimension (e.g., diameter or average diameter) of 500 nm or less. In other embodiments, nanoparticles of the invention have a greatest dimension or greatest average dimension ranging between 25 nm and 200 nm. In other embodiments, nanoparticles of the invention have a greatest dimension or greatest average dimension of 100 nm or less. In other embodiments, nanoparticles of the invention have a greatest dimension or greatest average dimensions ranging between 35 nm and 60 nm. It will be appreciated that reference made herein to particles or nanoparticles can be interchangeable, where appropriate. Nanoparticles made of semiconducting material may also be labeled quantum dots if they are small enough (typically sub 10 nm) that quantization of electronic energy levels occurs. Such nanoscale particles are used in biomedical applications as drug carriers or imaging agents and may be adapted for similar purposes in the present invention. Semi-solid and soft nanoparticles have been manufactured and are within the scope of the present invention. Nanoparticles with one-half hydrophilic and the other half hydrophobic are termed Janus particles and are particularly effective for stabilizing emulsions. They can self-assemble at water / oil interfaces and act as solid surfactants. In an embodiment, the nanoparticles of the present invention are Janus particles.

[0270] Particle characterization (including e.g., characterizing morphology, dimension, etc.) is done using a variety of different techniques. Common techniques are electron microscopy (TEM, SEM), atomic force microscopy (AFM), dynamic light scattering (DLS), X-ray photoelectron spectroscopy (XPS), powder X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR), matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF MS), ultraviolet-visible spectroscopy, dual polarization interferometry and nuclear magnetic resonance (NMR). Characterization (dimension measurements) may be made as to native particles (i.e., preloading) or after loading of the cargo (herein cargo refers to e.g., one or more components of the engineered Acr delivery system or any other system described herein e.g., CRISPR-Cas system e.g., CRISPR enzyme or mRNA or guide RNA, or any combination thereof, and may include additional carriers and / or excipients) to provide particles of an optimal size fordelivery for any in vitro, ex vivo and / or in vivo application of the present invention. In certain preferred embodiments, particle dimension (e.g., diameter) characterization is based on measurements using dynamic laser scattering (DLS). See, e.g., U.S. Patent No. 8,709,843; 6,007,845; 5,855,913; 5,985,309; and 5,543,158; and Dahlman et al. 2014, Nat. Nanotech. 9(8), 648-655, which describe particles, methods of making and using them, and measurements thereof and can be used with the present invention.Vectors and Vector systems

[0271] In an embodiment, the delivery vehicle is a vector or vector system. Vectors and vector systems of the present invention are described in greater detail elsewhere herein.Non-Vector Delivery Vehicles

[0272] The delivery vehicles may be or comprise non-viral vehicles. In general, the methods and vehicles capable of delivering nucleic acids and / or proteins may be used for delivering the engineered Acr polypeptide of the present invention, and / or encoding polynucleotides, vectors, etc., of the present invention. Non-limiting examples of non-viral vehicles include lipid nanoparticles, cell-penetrating peptides (CPPs), DNA nanoclews, metal nanoparticles, streptolysin O, multifunctional envelope-type nanodevices (MENDs), lipid-coated mesoporous silica particles, and other inorganic nanoparticles, and those systems described in Hirschenberger et al., Front. Pharmacol. 2021, 12, 770283 and Tian et al., Cell. Rep. 2022, 35(10), 110476.Lipid Particles

[0273] The delivery vehicles may comprise lipid particles, e.g., lipid nanoparticles (LNPs) and liposomes. Lipofection is described in e.g., U.S. Patent No. 5,049,386; 4,946,787; and 4,897,355. Additionally, lipofection reagents are sold commercially (e.g., Transfectam™ and Lipofectin™). Cationic and neutral lipids that are suitable for efficient receptor-recognition lipofection of polynucleotides include those described in International Patent Publication No. WO 91 / 17424 and WO 91 / 16024. The preparation of lipid and nucleic acid complexes, including targeted liposomes such as immunolipid complexes, is well known to one of skill in the art (see, e.g., Crystal, Science 1995, 270, 404-410; Blaese et al., Cancer Gene Ther. 1995, 2, 291-297; Behr et al., Bioconjugate Chem. 1994, 5, 382-389; Remy et al., Bioconjugate Chem. 1994, 5:647-654; Gao et al., Gene Therapy 1995, 2, 710-722; Ahmad et al., Cancer Res. 1992, 52, 4817-4820; and U.S. Patent No.4,186,183; 4,217,344; 4,235,871; 4,261,975; 4,485,054; 4,501,728; 4,774,085; 4,837,028; and 4,946,787).Lipid nanoparticles (LNPs)

[0274] In an embodiment, the delivery vehicle is or comprises an LNP. LNPs may encapsulate nucleic acids within cationic lipid particles (e.g., liposomes) and may be delivered to cells with relative ease. In some examples, lipid nanoparticles do not contain any viral components, which helps minimize safety and immunogenicity concerns. Lipid particles may be used for in vitro, ex vivo, and in vivo deliveries. Lipid particles may be used for various scales of cell populations.

[0275] In some examples, LNPs may be used for delivering DNA molecules (e.g., those comprising coding sequences of the engineered Acr polypeptide-encoding polynucleotides of the present invention, Cas, and / or gRNA) and / or RNA molecules (e.g., mRNA of an Acr polypeptide of the present invention or a Cas, gRNAs, etc.). In an embodiment, LNPs can include and be used to deliver the engineered Acr polypeptide and / or encoding polynucleotides of the present invention and / or a co-therapy, including but not limited to a CRISPR-Cas system or component thereof. In certain cases, LNPs may be used for delivering RNP complexes of the engineered Acr polypeptide, (or encoding polynucleotides) and / or a Cas polypeptide and / or gRNA.

[0276] The LNPs may comprise cationic lipids l,2-dilineoyl-3-dimethylammonium-propane (DLinDAP), l,2-dilinoleyloxy-3-N,N-dimethylaminopropane (DLinDMA), 1,2-dilinoleyloxyketo- N,N-dimethyl-3 -aminopropane (DLinK-DMA), l,2-dilinoleyl-4-(2-dimethylaminoethyl)-[l,3]- dioxolane (DLinKC2-DMA), (3- o-[2"-(methoxypolyethyleneglycol 2000) succinoyl]-l,2- dimyristoyLsn-glycol (PEG-S-DMG), R-3-[(ro-methoxy-poly(ethylene glycol)2000) carbamoyl]- l,2-dimyristyloxlpropyl-3-amine (PEG-C-DOMG), and any combination thereof. Preparation of LNPs and encapsulation may be adapted from Rosin et al., 2011, Mol. Ther. 19(12), pages 1286- 2200.

[0277] In an embodiment, an LNP delivery vehicle can be used to deliver virus particles, viruslike particles, proteins, and / or polynucleotides (e.g., DNA, RNA (e.g., mRNA)), or ribonucleoprotein (RNP) complex, that encodes or contains an engineered Acr polypeptide of the present invention, and / or a CRISPR-Cas system co-therapy and / or component(s) thereof. In an embodiment, the virus particle(s), virus-like particle(s), protein(s) polynucleotide(s), and / or RNPcomplex(es) can be adsorbed to the lipid particle, such as through electrostatic interactions, and / or can be attached to the lipid particle via a linker.

[0278] In an embodiment, the LNP contains a nucleic acid, where the charge ratio of nucleic acid backbone phosphates to cationic lipid nitrogen atoms is about 1 : 1.5-7 or about 1 :4.

[0279] In an embodiment, the LNP also includes a shielding compound, which is removable from the lipid composition under in vivo conditions. In an embodiment, the shielding compound is a biologically-inert compound. In an embodiment, the shielding compound does not carry any charge on its surface or on the molecule as such. In an embodiment, the shielding compounds are or comprise polyethylenglycoles (PEGs), hydroxyethylglucose (HEG) based polymers, polyhydroxyethyl starch (polyHES), and / or polypropylene. In an embodiment, the PEG, HEG, polyHES, and polypropylene weigh between about 500 to 10,000 Da or between about 2000 to 5000 Da. In an embodiment, the shielding compound is PEG2000 or PEG5000.

[0280] In an embodiment, the LNP can include one or more helper lipids. In an embodiment, the helper lipid can be a phospholipid or a steroid. In an embodiment, the helper lipid is between about 20 mol % to 80 mol % of the total lipid content of the composition. In an embodiment, the helper lipid component is between about 35 mol % to 65 mol % of the total lipid content of the LNP. In an embodiment, the LNP includes lipids at 50 mol% of the LNP, of which the helper lipid is present at 50 mol% of the total lipid content of the LNP.

[0281] Other non-limiting, exemplary LNP delivery vehicles are described in International Patent Publication No. WO 2012 / 135025; WO 2005 / 105152; WO 2006 / 069782; WO 2007 / 121947; U.S. Patent Publication No. US20160174546; US20140301951; US20150105538; US20150250725; US20140348900; US20140328759; US20140308304; US2015 / 082080; U.S. Patent No. 7,982,027; 7,799,565; 8,058,069; 8,283,333; 7,901,708; 7,745,651; 7,803,397; 8,101,741; 8,188,263; 7,915,399; 8,236,943 and 7,838,658; European Patent No. 1766035; 1519714; 1781593; and 1664316; Wang et al., J. Control. Release 2017, 263, 39-45; Altinoglu et al., Biomater Sci., 2016, 4(12), 1773-1780; Wang et al., Proc. Natl. Acad. Sci USA, 2016, 773(11), 2868-2873; Wang et al., PloS One, 2015, 70(11), e0141860; Takeda et al., Neural Regen. Res. 2015, 70(5), 689-690; Wang et al., Adv. Health. Mater. 2014, 3(9), 1398-1403; Wang et al., Angew Chem. Int. Ed. Engl., 2014, 53(11), 2893-2898; Dahlman et al. Nat. Nanotech. 2014, 9(8), 648- 655; Coelho et al., N. Engl. J. Med. 2013, 369, 819-829; Aleku et al., 2008, Cancer Res. 2008,68(23), 9788-9798; Strumberg et al., Int. J. Clin. Pharmacol. Ther. 2012, 50(1), 76-78; Schultheis et al., J. Clin. Oncol. 2014, 32(36): 4141-4148; Fehring et al., Mol. Ther. 2014, 22(4), 811-820; and Novobrantseva et al., Mol. Ther. Nucleic Acids 2012, 7(1), e4.Liposomes

[0282] In an embodiment, a lipid particle delivery vehicle may be a liposome. Liposomes are spherical vesicle structures composed of a uni- or multi-lamellar lipid bilayer surrounding internal aqueous compartments and a relatively impermeable outer lipophilic phospholipid bilayer. In an embodiment, liposomes are biocompatible, nontoxic, can deliver both hydrophilic and lipophilic drug molecules, protect their cargo from degradation by plasma enzymes, and transport their load across biological membranes and the blood-brain barrier (BBB).

[0283] Liposomes can be made from several different types of lipids, e.g., phospholipids. A liposome may comprise natural phospholipids and lipids such as l,2-distearoryl-sn-glycero-3- phosphatidyl choline (DSPC), sphingomyelin, egg phosphatidylcholines, monosial oganglioside, or any combination thereof.

[0284] Several other additives may be added to liposomes in order to modify their structure and properties. For instance, liposomes may further comprise cholesterol, sphingomyelin, and / or l,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), e.g., to increase stability and / or to prevent the leakage of the liposomal inner cargo.

[0285] In an embodiment, a liposome delivery vehicle can be used to deliver a virus particle, virus-like particle, vector, polynucleotide and / or protein, and / or complex thereof (e,g., an RNP) containing an engineered Acr polypeptide of the present invention and / or a CRISPR-Cas system and / or component(s) thereof. In an embodiment, the virus particle(s) can be adsorbed to the liposome, such as through electrostatic interactions, and / or can be attached to the liposomes via a linker.

[0286] In an embodiment, the liposome can be a Trojan Horse liposome (also known in the art as Molecular Trojan Horses). Methods of making Trojan Horse liposomes are generally known in the art. See e g., Pardridge. Front. Med. Technol. 2020, 2, 602236, the teachings of which can be applied and / or adapted to generate and / or deliver the engineered Acr delivery polypeptides or encoding polynucleotides, vectors etc. of the present invention or component(s) thereof and / or a CRISPR-Cas system and / or component s) thereof described herein.

[0287] Other non-limiting, exemplary liposomes can be those described in International Patent Publication No. WO 2008 / 042973; WO 2014 / 186366; and WO2013 / 093648; U.S. Patent Publication No. US20160129120; US20160244761; US20160257951; and US20120251618; U.S. Patent No. 8,071,082; Wang et al., ACS Synth. Biol. 2012, 1, 403-407; Wang et al., Proc. Natl. Acad. Sci. USA 2016, 773(11) 2868-2873; Spuch and Navarro, J. Drug Deliv., 2011, 2011, 469679; or are Lipofectin (a combination of DOTMA and DOPE), Lipofectase, LIPOFECTAMINE.RTM. (e.g., LIPOFECTAMINE.RTM. 2000, LIPOFECTAMINE.RTM. 3000, LIPOFECTAMINE.RTM. RNAiMAX, LIPOFECTAMINE.RTM. LTX), SAINT-RED (Synvolux Therapeutics, Groningen Netherlands), DOPE, Cytofectin (Gilead Sciences, Foster City, Calif.), or Eufectins (JBL, San Luis Obispo, Calif.).Stable nucleic-acid-lipid particles (SNALPs)

[0288] In an embodiment, the lipid particles may be stable nucleic-acid-lipid particles (SNALPs). SNALPs may comprise an ionizable lipid (e.g., DLinDMA, which is cationic at low pH), a neutral helper lipid (e.g., cholesterol), a diffusible polyethylene glycol (PEG)-lipid, or any combination thereof. In some examples, SNALPs may comprise synthetic cholesterol, dipalmitoylphosphatidylcholine, 3-N-[(w-methoxy polyethylene glycol)2000)carbamoyl]-l,2- dimyrestyloxypropylamine, and cationic l,2-dilinoleyloxy-3-N,Ndimethylaminopropane. In some examples, SNALPs may comprise synthetic cholesterol, l,2-distearoyl-sn-glycero-3- phosphocholine, PEG-cDMA, and l,2-dilinoleyloxy-3-(N,N-dimethyl)aminopropane (DLinDMAo).

[0289] Other non-limiting, exemplary SNALPs that can be used to deliver the engineered Acr polypeptides, encoding polynucleotides, vectors, etc. of the present invention and / or a CRISPR- Cas system and / or component(s) thereof described herein can be any such SNALP as described in Morrissey et al., Nat. Biotechnol. 2005, 23(8), 1002-1007; Zimmerman et al., Nat. Lett. 2006, Vol. 441, 111-114; Geisbert et al., Lancet 2010; 375, 1896-1905; Judge, J. Clin. Invest. 2009, 119, 661- 673; and Semple et al., Nat. Biotechnol. 2010, 28(2), 172-177. In an embodiment, the engineered Acr polypeptides, encoding polynucleotides, vectors, etc. of the present invention and / or a CRISPR-Cas system and / or component(s) thereof is included as an RNP. In other embodiments, the engineered Acr polypeptide of the present invention and / or a CRISPR-Cas system and / or component s) thereof is included as mRNA, such as an encoding mRNA.Other Lipids

[0290] The lipid particles may also comprise one or more other types of lipids, e.g., cationic lipids, such as amino lipid 2,2-dilinoleyl-4-dimethylaminoethyl-[l,3]-dioxolane (DLin-KC2- DMA), DLin-KC2-DMA4, C 12-200, and co-lipids disteroylphosphatidyl choline, cholesterol, and PEG-DMG.

[0291] In an embodiment, the delivery vehicle can be or include a lipidoid, such as any of those set forth in, for example, U.S. Patent Publication No. US20110293703.

[0292] In an embodiment, the delivery vehicle can be or include an amino lipid, such as any of those set forth in, for example, Jayaraman, Angew. Chem. Int. Ed. 2012, 51, 8529-8533.

[0293] In an embodiment, the delivery vehicle can be or include a lipid envelope, such as any of those set forth in, for example, Korman et al., Nat. Biotechol. 2011, 29, 154-157.Lipoplexes / polyplexes

[0294] In an embodiment, the delivery vehicles are or comprise lipoplexes and / or polyplexes. Lipoplexes may bind to negatively charged cell membranes and induce endocytosis into the cells. Examples of lipoplexes may be complexes comprising lipid(s) and non-lipid components. Examples of lipoplexes and polyplexes include FuGENE-6 reagent, a non-liposomal solution containing lipids and other components, zwitterionic amino lipids (ZALs), Ca2[? (e.g., forming DNA / Ca2+microcomplexes), polyethenimine (PEI) (e.g., branched PEI), and poly(L-lysine) (PLL).Susar-Based Particles

[0295] In an embodiment, the delivery vehicle is or comprises a sugar-based particle. In an embodiment, the sugar-based particles can be or include GalNAc, such as any of those described in International Patent Publication No. WO 2014 / 118272; U.S. Patent Publication No. US20020150626; Nair et al., J. Am. Chem. Soc. 2014, 736(49), 16958-16961; Ostergaard et al., Bioconjugate Chem. 2015, 26(8), 1451-1455.Cell-Penetratins Peptides

[0296] In an embodiment, the delivery vehicles are or comprise cell-penetrating peptides (CPPs). CPPs are short peptides that facilitate cellular uptake of various molecular cargos (e.g., from nanosized particles to small chemical molecules and large fragments of DNA).

[0297] CPPs may be of different sizes, amino acid sequences, and charges. In some examples, CPPs can translocate the plasma membrane and facilitate the delivery of various molecular cargosto the cytosol or an organelle. CPPs may be introduced into cells via different mechanisms, e.g., direct penetration in the membrane, endocytosis-mediated entry, and translocation through the formation of a transitory structure.

[0298] CPPs may have an amino acid composition that either contains a high relative abundance of positively charged amino acids such as lysine or arginine or has sequences that contain an alternating pattern of polar / charged amino acids and non-polar, hydrophobic amino acids. These two types of structures are referred to as polycationic or amphipathic, respectively. A third class of CPPs is the hydrophobic peptides, containing only apolar residues, with low net charge or with hydrophobic amino acid groups that are crucial for cellular uptake. Another type of CPPs is the trans-activating transcriptional activator (Tat) from Human Immunodeficiency Virus 1 (HIV-1). Examples of CPPs include Penetratin, Tat (48-60), Transportan, and (R-A11X-R4) (Ahx refers to aminohexanoyl), Kaposi fibroblast growth factor (FGF) signal peptide sequence, integrin P3 signal peptide sequence, polyarginine peptide (poly-Arg) sequence, Guanine rich-molecular transporters, and sweet arrow peptide. In an embodiment, the CPP is a cyclic CPP (see, e.g., Herce et al., Nat. Chem. 2017, 9, 762-771). Examples of CPPs and related applications also include those described in U.S. Patent No. 8,372,951, which can be incorporated into the polypeptides of the present invention.

[0299] CPPs can be used for in vitro and ex vivo work quite readily, and extensive optimization for each cargo and cell type is usually required. In some examples, CPPs may be covalently attached to the engineered Acr polypeptide and / or Cas protein and / or gRNA directly. In an embodiment where a Cas co-therapy is delivered, the Cas attached to a CPP is then complexed with a gRNA and delivered to cells. See e.g., Ramakrishna et al., Genome Res. 2014, 24, 1020- 1027 and Staahl et al., Nat. Biotechnol. 2017, 35, 431-434. In some examples, separate delivery of CPP-Cas and CPP-gRNA to multiple cells may be performed. CPPs may also be used to deliver RNPs.

[0300] CPPs may be used to deliver the compositions and systems to plants. In some examples, CPPs may be used to deliver the components to plant protoplasts, which are then regenerated to plant cells and further to plants.DNA Nanoclews

[0301] In an embodiment, the delivery vehicle is or comprises DNA nanoclews. A DNA nanoclew refers to a sphere-like structure of DNA (e.g., with a shape of a ball of yam). The nanoclew may be synthesized by rolling circle amplification with palindromic sequences that aid in the self-assembly of the structure. The sphere may then be loaded with a payload, such as an Acr polypeptide, encoding polynucleotide, vectors, ect. of the present invention. An example of DNA nanoclew is described in Sun et al., J. Am. Chem. Soc. 2014, 736(42), 14722-14725; and Sun et al., Angew Chem. Int. Ed. Engl. 2015, 54(41), 12029-12033. A DNA nanoclew may have a palindromic sequence to be partially complementary to the gRNA within the Cas:gRNA ribonucleoprotein complex. A DNA nanoclew may be coated, e.g., coated with PEI to induce endosomal escape.Metal Nanoparticles

[0302] In an embodiment, the delivery vehicles are or comprise gold nanoparticles (also referred to AuNPs or colloidal gold). Gold nanoparticles may form a complex with cargos (e.g., an engineered Acr polypeptide of the present invention, a Cas:gRNA RNP, etc.). Gold nanoparticles may be coated, e.g., coated in a silicate and an endosomal disruptive polymer, PAsp(DET). Examples of gold nanoparticles include AuraSense Therapeutics’ Spherical Nucleic Acid (SNA™) constructs and those ...

Claims

CLAIMSWhat is claimed is:

1. An engineered Anti-CRISPR (Acr) polypeptide comprising: an Acr polypeptide; and one or more cell-penetrating peptides (CPPs) operatively coupled to the Acr polypeptide.

2. The engineered Acr polypeptide of claim 1, wherein one or more of the one or more cellpenetrating peptides comprises or consists of one or more nuclear localization signals (NLSs).

3. The engineered Acr polypeptide of any one of claims 1 to 2, wherein the one or more CPPs are operatively coupled to an N-terminus, a C-terminus, both the N-tenninus and the C- terminus of the Acr polypeptide, and / or to one or more amino acids between the N-terminus and C-terminus of the Acr polypeptide.

4. The engineered Acr polypeptide of any one of claims 1 to 3, wherein 1-10 CPPs are operatively coupled to the Acr polypeptide.

5. The engineered Acr polypeptide of any one of claims 1 to 4, wherein 6 CPPs are operatively coupled to the Acr polypeptide.

6. The engineered Acr polypeptide of any one of claims 3 to 5, wherein (a) 4 CPPs are operatively coupled to the N-terminus of the Acr polypeptide, (b) wherein 2 CPPs are operatively coupled to the C-terminus of the Acr polypeptide, or (c) both (a) and (b).

7. The engineered Acr polypeptide of any one of claims 1 to 6, wherein the one or more CPPs are operatively coupled to the Acr polypeptide via a linker.

8. The engineered Acr polypeptide of claim 7, wherein the linker is a Gly-Ser linker.

9. The engineered Acr polypeptide of any one of claims 7-8, wherein the linker is G4CG4S (SEQ ID NO: 1).

10. The engineered Acr polypeptide of any one of claims 1 to 9, wherein the Acr polypeptide inhibits a Type I, Type II, Type III, Type V, or Type VI CRISPR-Cas system or component or activity thereof.

11. The engineered Acr polypeptide of any one of claims 1 to 10, wherein the Acr polypeptide is selected from AcrIEl, AcrIE2, AcrIE3, AcrIE4, AcrIE5, AcrIE6, AcrIE7, AcrIE8, AcrIE8.1, AcrIE8.2, AcrIE9, AcrIFI, AcrIF2, AcrIF3, AcrIF4, AcrIF5, AcrIF6, AcrIF7, AcrIF8, AcrIF9, AcrIFlO, AcrIFI 1, AcrIFI 1.1, AcrIFI 1.2, AcrIF12, AcrIF13, AcrIF14, AcrIF15, AcrIF16, AcrIF17, AcrIF18, AcrIF19, AcrIF20, AcrIF21, AcrIF22, AcrIF23, AcrIF24, AcrIE4-F7, AcrlAI, AcrIBI, AcrIB2, AcrIB3, AcrIB4, AcrIB5, AcrIB6, AcrIB7, AcrIB9, AcrICI, AcrIF2 / C2, AcrIC3, AcrIC4, AcrIC5, AcrIC6, AcrIC7, AcrIC8, AcrIC9, AcrIClO, AcrICI 1, AcrIDl, AcrIIAl, AcrIIA2, AcrIIA2-l, AcrIIA2-2, AcrIIA2b, AcrIIA3, AcrIIA4, AcrIIA4-2, AcrIIA4-3, AcrIIA4 variant Ins. 5, AcrIIA4 variant N39A, AcrIIA4 variant D14A / G38A, Acrobat- AcrIIA4, AcrIIA4-dTAG, SMASh-AcrIIA4, AcrIIA5, AcrIIA5-2, AcrIIA6, AcrIIA7, AcrIIA8, AcrIIA9, AcrIIAlO, AcrIIAl 1, AcrIIA12, AcrIIA13, AcrIIA13b, AcrIIA14, AcrIIA15, AcrIIA16, AcrIIA17, AcrIIA18, AcrIIAl 9, AcrIIA20, AcrIIA21, AcrIIA22, AcrIIA23, AcrIIA24, AcrIIA25, AcrIIA26, AcrIIA27, AcrIIA28, AcrIIA29, AcrIIA30, AcrIIA31, AcrIIA32, AcrIIA33, AcrIIA33(Seq), AcrIIA34, AcrIICl, AcrIICl-1, AcrIIC2, AcrIIC3, AcrIIC4, AcrIIC5, AcrIIC6, AcrIIC7, AcrIIC8, AcrIIC9, AcrIII-1, AcrIIIBl, AcrVAl, AcrVA2, AcrVA3, AcrVA3.1, AcrVA4, AcrVA5, AcrVIAl, AcrVIAl(Lwa), AcrVIA2, AcrVIA2(Lwa), AcrVIA3, AcrVIA3(Lwa), AcrVIA4, AcrVIA5, AcrVIA6, AcrVIA7, AcrVIBl, Csx27, enAcr-1, ErAcr-310, Acai, Aca2, Aca3, Aca4, Aca5, Aca6, Aca7, Aca8, Aca9, AcalO, Acal l, Acal2, Acal3, a homologue thereof, an orthologue thereof, or any combination thereof.

12. The engineered Acr polypeptide of any one of claims 1 to 11, further comprising a targeting moiety operatively coupled to the Acr polypeptide.

13. The engineered Acr polypeptide of any one of claims 1 to 12, further comprising a reporter molecule operatively coupled to the Acr polypeptide.

14. The engineered Acr polypeptide of any one of claims 1-13, further comprising a secondary delivery enhancer molecule operatively coupled to the Acr polypeptide.

15. The engineered Acr polypeptide of claim 14, wherein the secondary delivery enhancer molecule is an endosomal escape peptide.

16. The engineered Acr polypeptide of any one of claims 1-15, wherein the engineered Acr polypeptide has increased penetration across a lipid bilayer as compared to a wild-type Acr polypeptide.

17. The engineered Acr polypeptide of claim 16, wherein the lipid bilayer is a cell membrane.

18. The engineered Acr polypeptide of any one of claims 16-17, where penetration across a lipid bilayer is increased 1-1,000,000,000 fold or more.

19. The engineered Acr polypeptide of any one of claims 1-18, wherein the one or more CPPs localize the engineered Acr polypeptide to an intracellular compartment, optionally a cytosol or a nucleus.

20. A polynucleotide encoding the engineered Acr polypeptide of any one of claims 1-19.

21. A vector or vector system comprising:a polynucleotide encoding one or more engineered Acr polypeptides of any one of claims 1-19.

22. The vector or vector system of claim 21, wherein the vector system comprises one or more vectors, wherein at least one of the one or more vectors comprises the polynucleotide encoding one or more engineered Acr polypeptides of any one of claims 1-19.

23. The vector or vector system of any one of claims 21-22, further comprising one or more regulatory elements, wherein the one or more regulatory elements is operatively coupled to the polynucleotide encoding one or more engineered Acr polypeptides.

24. A delivery vehicle comprising:(a) an engineered Acr polypeptide of any one of claims 1-19;(b) a polynucleotide of claim 20;(c) a vector or vector system of any one of claims 21-23; or(d) any combination of (a)-(c).

25. A cell or cell population comprising:(a) an engineered Acr polypeptide of any one of claims 1-19;(b) a polynucleotide of claim 20;(c) a vector or vector system of any one of claims 21-23;(d) a delivery vehicle of claim 24; or(e) any combination of (a)-(d).

26. A pharmaceutical formulation comprising:(a) an engineered Acr polypeptide of any one of claims 1-19;(b) a polynucleotide of claim 20;(c) a vector or vector system of any one of claims 21-23;(d) a delivery vehicle of claim 24;(e) a cell or cell population of claim 25; or(f) any combination of (a)-(e); and a pharmaceutically acceptable carrier.

27. A kit comprising:(a) an engineered Acr polypeptide of any one of claims 1-19;(b) a polynucleotide of claim 20;(c) a vector or vector system of any one of claims 21-23;(d) a delivery vehicle of claim 24;(e) a cell or cell population of claim 25;(f) the pharmaceutical formulation of claim 26; or(g) any combination of (a)-(f).

28. A method of delivering an anti-CRISPR (Acr) polypeptide to a cell comprising: providing, to a cell or cell population,(a) an engineered Acr polypeptide of any one of claims 1-19;(b) a polynucleotide of claim 20;(c) a vector or vector system of any one of claims 21-23;(d) a delivery vehicle of claim 24;(e) a cell or cell population of claim 25;(f) the pharmaceutical formulation of claim 26; or(g) any combination of (a)-(f).

29. The method of claim 28, wherein the cell or cell population comprises a CRISPR-Cas system or component thereof.

30. The method of claim 29, wherein an activity of the CRISPR-Cas system or component thereof is reduced or inhibited.

31. A method of inhibiting activity of a CRISPR-Cas system in a cell comprising: providing, to a cell or cell population,(a) an engineered Acr polypeptide of any one of claims 1-19;(b) a polynucleotide of claim 20;(c) a vector or vector system of any one of claims 21-23;(d) a delivery vehicle of claim 24;(e) a cell or cell population of claim 25;(f) the pharmaceutical formulation of claim 26; or(g) any combination of (a)-(f), whereby the engineered Acr polypeptide inhibits activity of a CRISPR-Cas system or a component thereof in the cell.

32. The method of claim 31, wherein activity of a CRISPR-Cas system or a component thereof is reduced or inhibited by any non-zero percent to 100 percent.

33. A method of in vitro protein production comprising:(a) expressing or producing a protein by culturing, in a first cell culture media, a cell population comprising one or more cells comprising an expression vector or expression vector system that comprises a polynucleotide encoding a protein, wherein the first cell culture media comprises a first amount of glucose; and(b) subculturing, in a second cell culture media, the cell population of (a), wherein the second cell culture media comprises a second amount of glucose.

34. The method of claim 33, wherein the protein is a CRISPR-Cas protein, an Acr, an engineered Anti-CRISPR (Acr) polypeptide, or any combination thereof.

35. The method of any one of claims 33-34, further comprising(c) inducing protein production after (b) and further culturing the cell population.

36. The method of any one of claims 33-35, further comprising harvesting and / or lysing the cell population after (b) or (c).

37. The method of any one of claims 33-36, further comprising (d) purifying the protein.

38. The method of any one of claims 33-37, wherein the first amount of glucose ranges from any non-zero percent w / v to about 5 percent w / v, optionally wherein the first amount of glucose is about 2 percent w / v.

39. The method of any one of claims 33-38, wherein the second amount of glucose ranges from any non-zero percent w / v to about 5 percent w / v, optionally wherein the second amount of glucose is about 2 percent w / v.

40. The method of any one of claims 33-39, wherein the first cell culture media is Lysogeny Broth.

41. The method of any one of claims 33-40, wherein the second cell culture media is Terrific Broth.

42. The method of any one of claims 33-41, wherein the expression vector or expression vector system comprises or consists of a T7 promoter-based expression vector or expression vector system.

43. The method of claim 42, wherein the T7 promoter-based expression vector or expression vector system comprises or consists of a pET, 2CT, pMBP, pML-2CT, and pMal-C2 expression vector or expression vector system containing an MBP-TEV protease cleavage site fusion or a SUMO fusion, optionally a 4xNLS-pMJ915v2, pKEW -MBP-TEV, pAV- MBP-TEV, or pAV-SUMO expression vector or expression vector system.

44. The method of claims 36-43, wherein the protein comprises a purification tag and (d) purifying the protein comprises purification tag-based affinity purification.

45. The method of claim 44, further comprising removing the purification tag.

46. The method of any one of claims 35-45, wherein harvesting comprises centrifugation.

47. The method of any one of claims 33-46, wherein (b) subculturing is discontinued when the ODeoo reaches 0.6- 1.3.

48. The method of any one of claims 33-47, wherein the bacterial cell population is an E. coll cell population, optionally an E. coli Rosetta2 (DE3) cell population or an E. coli BL21 (DE3) cell population.

49. The method of any one of claims 33-48, wherein the method comprises ion exchange chromatography, size exclusion chromatography, or both.

50. The method of any one of claims 33-49, wherein the method does not comprise size exclusion chromatography and / or does not comprise ion exchange chromatography.

51. The method of any one of claims 33-50, wherein the protein is any protein set forth in Table 1, a Cas protein, or a protein of any one of claims 1-19.

52. The method of any one of claims 33-51, wherein the protein production and purification yield is increased 1, 2, 3, 4, 5, 6 or more-fold.

53. The method of any one of claims 36-52, wherein the cell population is a bacterial cell population.

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