Compositions and methods for modification of target molecules

Abstract

To provide a method for chemoselective modification of a target molecule, and a modified target molecule.SOLUTION: A subject method includes contacting a target molecule comprising a thiol moiety with a biomolecule including a reactive moiety, where the reactive moiety is generated by reaction of a biomolecule including a phenol moiety or a catechol with an enzyme capable of oxidizing the phenol or the catechol moiety. The contacting is carried out under conditions sufficient for conjugation of the target molecule to the biomolecule, thereby producing a modified target molecule.SELECTED DRAWING: None

Description

[Technical field]

[0001] cross reference This application claims the benefit of U.S. Provisional Patent Application No. 62 / 822,616, filed March 22, 2019, and U.S. Provisional Patent Application No. 62 / 910,836, filed October 4, 2019, which are incorporated by reference in their entireties.

[0002] INCORPORATION BY REFERENCE OF SEQUENCE LISTING PROVIDED AS A TEXT FILE A sequence listing is provided as a text file "BERK-405WO_SEQ_LISTING_ST25.txt" of size 8,056 KB, created on March 17, 2020. The contents of the text file are incorporated herein by reference in their entirety.

[0003] Statement on Federally Sponsored Research This invention was made with Government support under Grant Nos. 1059083 and 1808189 awarded by the National Science Foundation. The Government has certain rights in this invention. [Background technology]

[0004] preface Linking biomolecules to target molecules while maintaining their functions to produce conjugates has long been a goal of chemical biology and biopharmaceutical research. Examples of conjugates include protein-peptide conjugates for vaccine development, therapeutic antibodies, and antibody-protein conjugates for immunotherapy.

[0005] Although many techniques have been developed to attach molecules of modest size to proteins, it has been difficult to develop simple biomolecular modification procedures that allow the site-specific attachment of proteins or biomolecules to any location on the surface of a protein.

[0006] There is a need for improved targeted molecule modification procedures that are simple yet capable of modifying target molecules in a site-specific manner. Summary of the Invention

[0007] overview The present disclosure provides a method for chemoselectively modifying a target molecule. The subject method includes contacting a target molecule that includes a thiol moiety with a biomolecule that includes a reactive moiety, where the reactive moiety is generated by reaction of the biomolecule that includes a phenol moiety or a catechol moiety with an enzyme that can oxidize the phenol or catechol moiety. The contacting is performed under conditions sufficient to complex the target molecule with the biomolecule, thereby generating a modified target molecule. The present disclosure provides a composition that includes the subject target molecule that includes a thiol moiety and a biomolecule that includes a phenol moiety or a catechol moiety. The present disclosure provides a kit for carrying out the subject method. The present disclosure also provides modified target molecules and methods of use thereof. [Brief description of the drawings]

[0008] The invention is best understood from the following detailed description when read in conjunction with the accompanying drawings. It is emphasized that, according to common practice, the various features of the drawings are not to scale. Conversely, the dimensions of various features may be increased or decreased for clarity. The drawings include the following figures: It should be understood that the figures described below are for illustrative purposes only. The figures are not intended to limit the scope of the present teachings in any way.

[0009] [Figure 1A] FIG. 1A shows the activation of phenol and catechol moieties by tyrosinase enzyme to provide a quinone intermediate, and the subsequent reaction of the quinone intermediate with a potential nucleophile. [Figure 1B]FIG. 1B shows an exemplary subject chemoselective modification reaction of a solvent-exposed thiol-bearing target protein (A) and a tyrosine / phenol-bearing coupling partner (B) to yield a covalently linked conjugate product (C). [Figure 2-1] Figure 2, panel A shows ESI-TOF data showing MS2 N87C modified with α-endorphin peptide, as well as a maleimide blocking experiment demonstrating that capping the thiol on the protein via maleimide blocks addition via the tyrosinase-catalyzed reaction, and that performing tyrosinase treatment first also blocks the maleimide reaction. This figure shows that the surface cysteine ​​is the residue that is modified. [Figure 2-2] Figure 2, panel B shows the stability test results of the protein-peptide complexes under various conditions. All samples were stored in 50 mM phosphate buffer at the conditions indicated. [Figure 3-1] FIG. 3 shows illustrative examples of biomolecules containing phenol moieties that are compatible in the subject methods. [Figure 3-2] A continuation of Figure 3-1 is shown. [Figure 4] Figure 4 shows ESI-TOF data showing the coupling of various peptides to a cysteine-containing mutant of the MS2 viral capsid. The peptides consisted of the following sequences with an acylated N-terminus: 2NLS: Ac-YGPKKKRKVGGSPKKKRKV (SEQ ID NO: 943); IL13: Ac-GYACGEMGWVRCGGSK (SEQ ID NO: 944); R8: Ac-YGRRRRRRRR (SEQ ID NO: 945); and HIV-Tat: Ac-YGRKKRRQRRRPPQ (SEQ ID NO: 946). [Figure 5-1]Figure 5, panel A shows ESI-TOF data demonstrating that Cas9 (C80,C574) is modified twice by endorphins. Figure 5, panel B shows an in vitro DNA cleavage assay, which demonstrates that Cas9 (RNP) modified with a peptide (End) retains cleavage activity even when modified prior to attachment to the guide RNA (apo). For each treatment, RNP was added across a concentration gradient and activity against the target DNA strand was measured. [Figure 5-2] Figure 5, panel C shows ESI-TOF data showing successful Cas9-GFP complex. Sequences are listed as follows: GYGGS (SEQ ID NO: 1021), MYGGS (SEQ ID NO: 1022). Figure 5, panel D shows an in vitro cleavage assay showing that GFP-modified Cas9 retains activity compared to the control. Sequences are listed as follows: MYGGS (SEQ ID NO: 1022), SGGGGY (SEQ ID NO: 1040). [Figure 6] FIG. 6 shows that Cas9 modified with a peptide containing two copies of the SV40 nuclear localization sequence was able to enter and edit neural progenitor cells, increasing the editing efficiency by 20-fold. [Figure 7] FIG. 7 shows ESI-TOF data demonstrating the modification of phenol-containing proteins with small molecule thiols. [Figure 8] Figure 8 shows the amino acid sequence of mushroom tyrosinase. The sequence is set forth in SEQ ID NO:971. [Figure 9] Figure 9 shows the amino acid sequence of mushroom tyrosinase. The sequence is set forth in SEQ ID NO:972. [Figure 10-1] Figures 10A to 10Z and 10AA to 10VV show the amino acid sequences of tyrosinase from Bacillus megaterium. The sequences of Figures 10A to 10Z are set forth in SEQ ID NOs: 973 to 998. The sequences of Figures 10AA to 10VV are set forth in SEQ ID NOs: 999 to 1020. [Figure 10-2] A continuation of Figure 10-1 is shown. [Figure 10-3]A continuation of Figure 10-2 is shown. [Figure 10-4] A continuation of Figure 10-3 is shown. [Figure 10-5] A continuation of Figure 10-4 is shown. [Figure 10-6] A continuation of Figure 10-5 is shown. [Figure 10-7] A continuation of Figure 10-6 is shown. [Figure 10-8] A continuation of Figure 10-7 is shown. [Figure 10-9] A continuation of Figure 10-8 is shown. [Figure 10-10] A continuation of Figure 10-9 is shown. [Figure 10-11] Continuation of Figure 10-10 is shown. [Figure 10-12] A continuation of Figure 10-11 is shown. [Figure 11-1] Figure 11 shows abTYR-peptide charge screening: A 5-mer tyrosine-containing peptide was coupled to Y182C GFP and pAF MS2 using abTYR. The resulting reaction mixture was analyzed using Q-TOF mass spectrometry. Reaction conditions: 50 μM GFP, 250 μM peptide, 0.167 μM tyrosinase, 10 mM buffer pH 6.5, 30 min at room temperature, all reactions were quenched with 10 mM tropolone. The sequences are as follows: GGGGY (SEQ ID NO:1024), RGGGY (SEQ ID NO:1025), RGRGY (SEQ ID NO:1026), RRRGY (SEQ ID NO:1027), RRRRY (SEQ ID NO:1028), EGGGY (SEQ ID NO:1029), EGEGY (SEQ ID NO:1030), EEEGY (SEQ ID NO:1031), EEEEY (SEQ ID NO:1032), GGGWY (SEQ ID NO:1033), GGWGY (SEQ ID NO:1034), RRRWY (SEQ ID NO:1035), RRWRY (SEQ ID NO:1036), EEEWY (SEQ ID NO:1037), EEWEY (SEQ ID NO:1038). [Figure 11-2] A continuation of Figure 11-1 is shown. [Figure 12]12A-12B show the abTYR and bmTYR models: abTYR (a) has an overall negative charge (red residues) around its active site due to the abundance of glutamic and aspartic acid residues, whereas bmTYR (b) has only a small positive charge (blue residues) around its active site. [Figure 13-1] Figure 13 shows bmTYR charge screening. 5-Mer tyrosine-containing peptides were coupled to Y182CGFP using bmTYR. The resulting reaction mixture was analyzed using Q-TOF mass spectrometry, showing that bmTYR prefers negatively charged substrates. Reaction conditions: 50M μM GFP, 250 μM peptide, 0.2 μM tyrosinase, 10 mM buffer pH 6.5, 30 min at 37°C, all reactions were quenched with 10 mM tropolone. The sequences are as follows: GGGGY (SEQ ID NO: 1024), GGGWY (SEQ ID NO: 1033), EEEGY (SEQ ID NO: 1031), RRRGY (SEQ ID NO: 1027). [Figure 13-2] A continuation of Figure 13-1 is shown. [Figure 14-1] Figure 14 shows a comparison between abTYR and bmTYR for EGGGY (SEQ ID NO: 1029) and EEEEY (SEQ ID NO: 1032) peptides. Reaction conditions (abTYR): 50 μM GFP, 250 μM peptide, 0.167 μM tyrosinase, 10 mM buffer pH 6.5, 30 min at room temperature. Reaction conditions (bmTYR): 10 μM GFP, 50 μM peptide, 0.8 μM tyrosinase, 10 mM buffer pH 6.5, 1 hr at room temperature, all reactions were quenched with 10 mM tropolone. [Figure 14-2] A continuation of Figure 14-1 is shown. [Figure 15]15A-15C show oxidative coupling strategies for protein modification: a) chemical and physical methods to access o-quinones and o-iminoquinones for coupling to N-terminal proline residues and aminophenyl groups; b) tyrosinase-mediated oxidation of phenols for coupling to N-terminal proline residues; c) selective tyrosinase-mediated generation of o-quinones at the N- or C-terminus of proteins followed by tyrosine-tagged proteins for coupling with exogenous amine nucleophiles. [Figure 16] Figures 16A-16B show the binding of Tyr-containing peptides to p-aminophenylalanine-containing MS2 (pAF-MS2). N-Ac-α-endorphin has an accessible tyrosine residue at its N-terminus. This site can be oxidized by tyrosinase and coupled to the pAF-MS2 capsid, which contains an aniline group introduced using the Schultz amber codon suppression method. The sequence is written as follows: GGFMTSEKSQTPLVT (SEQ ID NO: 1039). (b) The position of the aniline group at residue 180 is shown in pink on the intact viral capsid (PDB ID: 2MS2). ESI-TOF MS analysis showed virtually complete conversion to the expected product (predicted: 15589 Da). No overmodification was observed. [Figure 17A] Figures 17A-17E show A. bisporus tyrosinase mediated coupling with amine nucleophiles. a) C-terminally-GGY tagged trastuzumab scFv was used as a model coupling partner. [Figure 17B] b) Crystal structure of the heavy and light chain variable domains of trastuzumab that make up the scFv. [Figure 17C] c) Representative mass spectra of the starting scFv-GGY before and after coupling with 150 μM aniline. [Figure 17D]d) 4-Aminophenyl derived nucleophiles were screened at concentrations ranging from 25 μM to 750 μM. e) Pyrrolidine and piperazine derived nucleophiles were screened at concentrations ranging from 100 μM to 5000 μM. Conversions were estimated by TOF-LCMS integration. See Supplementary Figure X for representative spectra. [Figure 17E] See legend to Figure 17D. [Figure 18-1] Figure 18 shows tyrosine tagged protein substrates successfully coupled using A. bisporus tyrosinase. The C-terminus is highlighted in red and the internal tyrosine residues are highlighted in orange. The reaction was performed with tyrosinase and aniline in phosphate buffer pH 6.5. a) N-terminally tagged ubiquitin. b) C-terminally-(GGGGS)2GGY tagged sfGFP. (SEQ ID NO: 947) c) C-terminally-GGY tagged trastuzumab scFv. The sequence is as follows: SGGGGY (SEQ ID NO: 1040). [Figure 18-2] A continuation of Figure 18-1 is shown. [Figure 19A] Figures 19A-19C show flow cytometry studies of fluorophore-conjugated trastuzumab scFv binding to SKBR3 (HER2+) cells. a) Oxidative coupling of GGY-tagged scFv with 12 U / L A. bisporus tyrosinase and 50 μM aniline-Oregon Green 488. b) ESI TOF-MS shows that scFv-GGY was coupled with 85% conversion. An untagged version of the scFv was not modified. [Figure 19B] See legend to Figure 19A. [Figure 19C] See legend to Figure 19A. [Figure 20-1]Figures 20A-20B show the exploration of C-terminal linkers and the utility of B. megaterium tyrosinase. a) Linkers of various types and lengths were added to the C-terminus of Protein L, including two that utilized the native interdomain linker sequences of domains 4 and 5. Protein L variants were subjected to standard coupling reactions with B. megaterium tyrosinase. The sequences are set forth in SEQ ID NO: 1041. [Figure 20-2] b) Conversion observed by TOF-LCMS after treatment with B. megaterium tyrosinase. None of the variants could be modified by A. bisporus tyrosinase. The sequences are listed as follows: (G4S)2GGY (SEQ ID NO: 947), (G4S)3GGY (SEQ ID NO: 1042), A(EAAAK)2AGGY (SEQ ID NO: 1043), (AP)3GGY (SEQ ID NO: 1044), AN20GGY (SEQ ID NO: 1045), EIKRTGGY (SEQ ID NO: 1046), G4SGGY (SEQ ID NO: 968). [Figure 21A] 21A-21C show B. megaterium-mediated oxidative coupling of C-terminal tyrosine-tagged MBP. a) Crystal structure of MBP with the C-terminus highlighted in red. Tyrosine residues are shown in orange. Bound maltose is shown in yellow. [Figure 21B] b) Data for MBP-SSGGGGY (SEQ ID NO: 948). [Figure 21C] c) Data using MBP-GGY. [Figure 22A] 22A-22D show detection of HER2+ cells using Protein LO.G.488-conjugated "secondary" affinity reagents. a) Detection scheme: Non-tyrosine tagged trastuzumab scFv binds to HER2+ SK-BR-3 cells and is recognized by OG488-modified Protein L. [Figure 22B]b) A secondary affinity reagent was made from the -AN20GGY-terminated protein L variant using 25 μM OG488-anilin and B. megaterium tyrosinase. [Figure 22C] c) Mass spectra of protein L-AN20GGY before and after modification. [Figure 22D] d) Flow cytometry fluorescence data of SK-BR-3 cells and negative control treated according to the above scheme. MDA-MB-468 cells were used as HER2 control. The sequence is as follows: AN20GGY (SEQ ID NO: 1045). [Diagram 23] FIG. 23 shows C-terminal-GGY tyrosine tagged and untagged trastuzumab scFv subjected to oxidative coupling conditions (12 U / mL abTYR, 150 μM aniline, 20 mM sodium phosphate buffer, pH 6.5, 1 hour). [Figure 24A] Figures 24A-24D show the change in abTyr and aniline concentrations upon conversion of -GGY tagged trastuzumab scFv. a) Reaction scheme. [Figure 24B-1] b) Representative mass spectra: 1000 M aniline at various abTYR concentrations. [Figure 24B-2] A continuation of Figure 24B-1 is shown. [Figure 24C] c) Table showing the conversion rate of trastuzumab ("Tras.") scFv-GGY to aniline coupled products. [Figure 24D] d) Graphical representation of the conversion of Tras.scFv-GGY to the aniline coupled product. [Figure 25-1] Figure 25 shows an alate nucleophile addition experiment. Aniline was added 5, 10, 20, 40, or 60 minutes after the tyrosinase enzyme for oxidative coupling via abTYR. [Figure 25-2] A continuation of Figure 25-1 is shown. [Figure 26-1]Figure 26 shows representative spectra for oxidative coupling reactions with 4-aminophenyl derived nucleophiles: a) o-toluidine, b) 2,6-dimethylaniline, c) 4-aminophenyl-N-methylamide. [Figure 26-2] A continuation of Figure 26-1 is shown. [Figure 26-3] A continuation of Figure 26-1 is shown. [Figure 27-1] FIG. 27 shows a representative spectrum of the oxidative coupling reaction. [Figure 27-2] A continuation of Figure 27-1 is shown. [Figure 27-3] A continuation of Figure 27-1 is shown. [Figure 28-1] Figure 28 shows the oxidative coupling reaction of Protein L variants, with sequences as set forth below: (G4S)2GGY (SEQ ID NO:947);(G4S)3GGY (SEQ ID NO:1042), A(EAAAK)2AGGY (SEQ ID NO:1043), (AP)3GGY (SEQ ID NO:1044). [Figure 28-2] A continuation of Figure 28-1 is shown. [Figure 28-3] A continuation of Figure 28-1 is shown. [Figure 28-4] A continuation of Figure 28-1 is shown. [Figure 28-5] A continuation of Figure 28-1 is shown. [Figure 29A-1] Figures 29A-29B show a stability study of trastuzumab scFv-GGY in protein storage buffer (20 mM Na2HPO4, 150 mM NaCl, 15% glycerol, pH 7.4) + 10 mM dithiothreitol (DTT) stored at 4 °C. The TOF-LCMS spectra in each row were from the same aliquot of protein sampled at the indicated time points. The uncoupled and coupled proteins, disulfide reduced scFv-GGY, have predicted masses of 26,337.2 Da and 26,442.2 Da, respectively. Aniline coupling + reduction + DTT = 26,594.45 Da a) subjected to oxidative coupling with aniline via abTYR. b) not subjected to oxidative coupling reaction. [Figure 29A-2]A continuation of Figure 29A-1 is shown. [Figure 29B] See description of Figure 29A-1. [Figure 30A-1] Figures 30A-30B show stability studies of trastuzumab scFv-GGY in protein storage buffer (20 mM Na2HPO4, 150 mM NaCl, 15% glycerol, pH 7.4) stored at 4 °C. The TOF-LCMS spectra in each row were from the same aliquot of protein sampled at the indicated time points. The uncoupled and coupled protein disulfide-reduced scFv-GGY have predicted masses of 26,337.2 Da and 26,442.2 Da, respectively. a) Subjected to oxidative coupling with aniline via abTYR and buffer-exchanged into protein storage buffer. b) Not subjected to oxidative coupling reaction. [Figure 30A-2] A continuation of Figure 30A-1 is shown. [Figure 30B] See description of Figure 30A-1. [Figure 31A-1] Figures 31A-31B show stability studies of trastuzumab scFv-GGY in protein storage buffer (20 mM Na2HPO4, 150 mM NaCl, 15% glycerol, pH 7.4) + 10 mM glutathione stored at 4 °C. The TOF-LCMS spectra in each row were from the same aliquot of protein sampled at the indicated time points. The uncoupled and coupled proteins disulfide reduced scFv-GGY have predicted masses of 26,337.2 Da and 26,442.2 Da, respectively. Aniline coupling + reduction + 1 x glutathione = 26,747.58 Da; aniline coupling + reduction + 2 x glutathione = 27,052.89 Da a) subjected to oxidative coupling with aniline via abTYR. b) not subjected to oxidative coupling reaction. [Figure 31A-2] A continuation of Figure 31A-1 is shown. [Figure 31B] See description of Figure 31A-1. [Figure 32-1]Figure 32 shows a study of thiol exchange in the oxidative coupling reaction products. Trastuzumab scFv-GGY was buffer exchanged into protein storage buffer (20 mM Na2HPO4, 150 mM NaCl, 15% glycerol, pH 7.4) + 10 mM glutathione and stored at 4°C. After 24 hours, a portion of the sample was subjected to TOF-LCMS analysis, and the remainder was buffer exchanged into protein storage buffer + 10 mM DTT and stored at 4°C for an additional 24 hours. A second sample was then subjected to TOF-LCMS analysis. [Figure 32-2] A continuation of Figure 32-1 is shown. [Diagram 33] Figure 33 shows the average mass of the protein constructs. The sequences are as follows: GGGGSGGY (SEQ ID NO: 968); (GGGGS)2GGY (SEQ ID NO: 947); (AP)4GGY (SEQ ID NO: 1061); AN20GGY (SEQ ID NO: 1045), SSGGGGY (SEQ ID NO: 948), (GGGGS)3GGY (SEQ ID NO: 1042), AEAAAKEAAAKAGGY (SEQ ID NO: 1043), (AP)3GGY (SEQ ID NO: 1044), EIKRTGGY (SEQ ID NO: 1046), GGGGSGGY (SEQ ID NO: 968). [Figure 34A] Figures 34A-34E show the amino acid sequences of the protein constructs. The sequences of Figures 34A-34E are set forth in SEQ ID NOs: 1049-1053. [Figure 34B] See legend to Figure 34A. [Figure 34C] See legend to Figure 34A. [Fig. 34D] See legend to Figure 34A. [Figure 34E] See legend to Figure 34A. [Diagram 35] FIG. 35 shows the use of the D55K mutant of Bacillus megaterium tyrosinase (bmTYR) to couple phenol-labeled nucleic acids to cysteine-containing proteins. [Diagram 36] 36A-36C show coupling of a nucleic acid to a polypeptide using the methods of the present disclosure. [Figure 37]Figures 37A-37C show the effect of various mutations in bmTYR on its preference for charged substrates. [Figure 38] FIG. 38 shows the lack of activity of abTYR towards activation of negatively charged substrates. [Figure 39A] 39A-39G show schematic diagrams of protein ligation using the methods of the present disclosure. [Figure 39B] See legend to Figure 39A. [Figure 39C] See legend to Figure 39A. [Figure 39D] See legend to Figure 39A. [Figure 39E] See legend to Figure 39A. [Figure 39F] See legend to Figure 39A. [Figure 39G] See legend to Figure 39A. [Figure 40A] 40A-40C show the stability of target molecule-biomolecule complexes in human serum. [Figure 40B] See legend to Figure 40A. [Figure 40C] See legend to Figure 40A. [Diagram 41] FIG. 41 shows the coupling of Cas9 to: i) an Ig Fc polypeptide; ii) and a nanobody using the methods of the disclosure. [Diagram 42] Figure 42 shows time-of-flight mass spectrometry data of Cas9-nanobody complexes. [Diagram 43] 43A-43B show the use of the methods of the present disclosure to: i) directly label the surface of live mammalian cells (FIG. 43A); and ii) couple a polypeptide to the cell surface. [Diagram 44] 44A-44B show reactions where the target molecule contains two thiol moieties. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0010] definition Before the present invention is further described, it is to be understood that this invention is not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.

[0011] Where a range of values ​​is provided, it is understood that each intervening value between the upper and lower limit of that range, to one-tenth of the unit of the lower limit, as well as any other stated or intervening value in that stated range, is encompassed within the invention, unless the context clearly dictates otherwise. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also encompassed within the invention, subject to any specific excluded limits in the stated range. Where a stated range includes one or both of its limits, ranges excluding either or both of those included limits are also encompassed within the invention.

[0012] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention, the preferred methods and materials are described herein. All publications mentioned herein are incorporated by reference to disclose and describe the methods and / or materials in connection with which the publications are cited.

[0013] It should be noted that, as used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, a reference to "thiol groups" includes a reference to a plurality of such thiol groups, and a reference to "thiol groups" includes a reference to one or more thiol groups and equivalents thereof known to those of skill in the art, and so forth. It should be further noted that the claims may be drafted to exclude any optional element. Thus, this statement is intended to serve as a predicate for the use of exclusive language such as "solely," "only," or "negative" limitations in connection with the recitation of claim elements.

[0014] It is understood that certain features of the invention that are described for clarity in the context of separate embodiments may also be provided in combination in a single embodiment. Conversely, various features of the invention that are described for brevity in the context of a single embodiment may also be provided separately or in any suitable subcombination. All combinations of the embodiments related to the present invention are expressly embraced by the present invention and are disclosed herein as if each and every combination was individually and explicitly disclosed. Moreover, all subcombinations of the various embodiments and elements thereof are also expressly embraced by the present invention and are disclosed herein as if each and every such subcombination was individually and explicitly disclosed herein.

[0015] The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein should be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates, which may need to be independently confirmed.

[0016] As used herein, the term "affinity tag" refers to a member of a specific binding pair, i.e., two molecules that specifically bind one of the molecules to the other molecule through chemical or physical means. The complementary member of the affinity tag may be immobilized (e.g., on a chromatographic support, bead, or planar surface) to generate an affinity chromatography support that specifically binds to the affinity tag. Once a compound of interest is attached with an affinity tag, the compound can be separated from a mixture of untagged compounds by affinity, for example, using affinity chromatography. Examples of specific binding pairs include biotin and streptavidin (or avidin), and antigens and antibodies, but binding pairs such as nucleic acid hybrids, polyhistidine and nickel, and azide and alkynyl (e.g., cyclooctynyl) or phosphino groups are also contemplated. Specific binding pairs may include analogs, derivatives, and fragments of the original specific binding members.

[0017] As used herein, the term "biotin moiety" refers to an affinity tag that includes biotin or a biotin analog, such as desthiobiotin, oxybiotin, 2'-iminobiotin, diaminobiotin, biotin sulfoxide, biocytin, etc. The biotin moiety is attached to streptavidin at least 10 -8 The biotin moiety can also be linked with a linker, such as -LC biotin, -LC-LC-biotin, -SLC biotin, or -PEG. n 1 -biotin may be included, where n 1 is 3 to 12.

[0018] "Linkage" or "linker" as in "linking group", "linker moiety" and the like means a linking moiety that connects two groups via a covalent bond. The linker may be linear, branched, cyclic or a single atom. Examples of such linking groups include alkyl, alkenylene, alkynylene, arylene, alkarylene, aralkylene, and linking moieties that contain functional groups including, but not limited to, amide (-NH-CO-), ureylene (-NH-CO-NH-), imide (-CO-NH-CO-), epoxy (-O-), epithio (-S-), epidioxy (-OO-), epidithio (-SS-), carbonyldioxy (-O-CO-O-), alkyldioxy (-O-(CH2)nO-), epoxyimino (-O-NH-), epimino (-NH-), carbonyl (-CO-), and the like. In certain cases, one, two, three, four, five, or more carbon atoms of the linker backbone may be optionally replaced with sulfur, nitrogen, or oxygen heteroatoms. The bonds between the backbone atoms may be saturated or unsaturated, and often there will be one, two, or no more than three unsaturated bonds in the linker backbone. The linker may include one or more substituents, such as alkyl, aryl, or alkenyl groups. Linkers include, but are not limited to, poly(ethylene glycol) unit(s) (e.g., -(CH 2 -CH 2 -O)-; ethers, thioethers, amines, alkyls which may be linear or branched (e.g., (C 1 -C 12 ) alkyl), such as methyl, ethyl, n-propyl, 1-methylethyl (isopropyl), n-butyl, n-pentyl, 1,1-dimethylethyl (t-butyl), and the like. The linker backbone may include a cyclic group, such as an aryl, heterocyclic, or cycloalkyl group, and the backbone includes two or more atoms, such as 2, 3, or 4 atoms, of the cyclic group. The linker may be cleavable or non-cleavable. Any convenient orientation and / or connection of the linker to the linking group may be used.

[0019] "Alkyl" refers to a monovalent saturated aliphatic hydrocarbyl group having 1 to 10 carbon atoms, e.g., 1 to 6 carbon atoms. This term includes, by way of example, straight-chain and branched-chain hydrocarbyl groups, such as methyl (CH 3 -), ethyl (CH 3 CH 2 -), n-propyl (CH 3 CH 2 CH 2 -), isopropyl ((CH 3 ) 2 CH-), n-Butyl (CH 3 CH 2 CH 2 CH 2 -), isobutyl ((CH 3 ) 2 CHCH 2 -), sec-butyl ((CH 3 )(CH 3 CH 2 )CH-), t-butyl ((CH 3 ) 3 C-), n-pentyl (CH 3 CH 2 CH 2 CH 2 CH 2 -), and neopentyl ((CH 3 ) 3 CCH 2 -) and others.

[0020] The term "substituted alkyl" refers to an alkyl group, as defined herein, which has one or more carbon atoms (C 1 carbon atom) may optionally be -O-, -N-, -S-, -S(O) n 2 -(in the formula, n 2is 0 to 2), -NR- (wherein R is hydrogen or alkyl), and is substituted with a heteroatom such as alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino, acyloxy, amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azido, cyano, halogen, hydroxyl, oxo, thioketo, carboxyl, carboxyalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclooxy, thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclyl, heterocyclooxy, hydroxyamino, alkoxyamino, nitro, -SO-alkyl, -SO-aryl, -SO-heteroaryl, -SO 2 -Alkyl, -SO 2 -Aryl, -SO 2 -heteroaryl and -NR a R b wherein R ’ and R ” may be the same or different and are selected from hydrogen, optionally substituted alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, aryl, heteroaryl, and heterocycle.

[0021] "Aryl" or "Ar" refers to a monovalent aromatic carbocyclic group of 6 to 18 carbon atoms having a single ring (as in a phenyl group) or a ring system having multiple condensed rings (examples of such aromatic ring systems include naphthyl, anthryl, and indanyl), which may or may not be aromatic, provided that the point of attachment is through an atom of the aromatic ring. This term includes, for example, phenyl and naphthyl. Unless otherwise limited by the definition of the aryl substituent, such aryl groups are optionally substituted or unsubstituted with any of the following radicals: acyloxy, hydroxy, thiol, acyl, alkyl, alkoxy, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, substituted alkyl, substituted alkoxy, substituted alkenyl, substituted alkynyl, substituted cycloalkyl, substituted cycloalkenyl, amino, substituted amino, aminoacyl, acylamino, alkaryl, aryl, aryloxy, azido, carboxyl, carboxyalkyl, cyano, halogen, nitro, heteroaryl, heteroaryloxy, heterocyclyl, heterocyclooxy, aminoacyloxy, oxyacylamino, thioalkoxy, substituted thioalkoxy, thioaryloxy, thioheteroaryloxy, -SO-alkyl, -SO-substituted alkyl, -SO-aryl, -SO-heteroaryl, -SO 2 -Alkyl, -SO 2 -substituted alkyl, -SO 2 -Aryl, -SO 2 -can be substituted by 1 to 5 substituents, or 1 to 3 substituents selected from heteroaryl and trihalomethyl.

[0022] "Amino" means -NH 2 Refers to the base.

[0023] The term "substituted amino" refers to the group -NRR, where each R is independently selected from the group consisting of hydrogen, alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, alkenyl, substituted alkenyl, cycloalkenyl, substituted cycloalkenyl, alkynyl, substituted alkynyl, aryl, heteroaryl, and heterocyclyl, provided that at least one R is not hydrogen.

[0024] Further to the disclosure herein, the term "substituted," when used to modify a particular group or radical, can also mean that one or more hydrogen atoms of the particular group or radical are replaced, each independently of the other, with the same or different substituents as defined below.

[0025] In addition to the groups disclosed for each individual term herein, substituents to replace one or more hydrogens on a saturated carbon atom in a particular group or radical (such as =O, =NR 70 , =N-OR 70 , =N 2 or =S) are -R, unless otherwise stated. 60 , halo, =O, -OR 70 , -SR 70 , -NR 80 R 80 , trihalomethyl, -CN, -OCN, -SCN, -NO, -NO 2 , =N 2 , -N 3 , -SO 2 R 70 , -SO 2 O - M + , -SO 2 OR 70 , -OSO 2 R 70 , -OSO 2 O - M + , -OSO 2 OR 70 , -P(O)(O - ) 2 (M + ) 2 , -P(O)(OR 70 )O - M + , -P(O)(OR 70 ) 2 , -C(O)R 70 , -C(S)R 70 , -C(NR 70 )R 70 , -C(O)O - M + , -C(O)OR70 , -C(S)OR 70 , -C(O)NR 80 R 80 , -C(NR 70 )NR 80 R 80 , -OC(O)R 70 , -OC(S)R 70 , -O-C(O)O - M + , -OC(O)OR 70 , -OC(S)OR 70 , -NR 70 C(O)R 70 , -NR 70 C(S)R 70 , -NR 70 CO 2 - M + , -NR 70 CO 2 R 70 , -NR 70 C(S)OR 70 , -NR 70 C(O)NR 80 R 80 , -NR 70 C(NR 70 )R 70 and -NR 70 C(NR 70 )NR 80 R 80 where R 60 is selected from the group consisting of optionally substituted alkyl, cycloalkyl, heteroalkyl, heterocycloalkylalkyl, cycloalkylalkyl, aryl, arylalkyl, heteroaryl, and heteroarylalkyl; each R 70 are independently hydrogen or R 60 and each R 80 is independently R 70 or two R 80 together with the nitrogen atom to which they are attached form a 5-, 6-, or 7-membered heterocycloalkyl, which is optionally selected from the group consisting of O, N, and S, where N is -H or C 1 -C 3each M + is a counterion with a net single positive charge. + are independently, e.g., K + , Na + , Li + Alkaline ions such as; e.g. + N(R 60 ) 4 or ammonium ions such as [Ca 2+ ] 0.5 , [Mg 2+ ] 0.5 , or [Ba 2+ ] 0.5 (The "subscript 0.5" means that one of the counterions to such divalent alkaline earth ions can be the ionized form of a compound of the invention and the other a typical counterion such as chloride, or a two ionized compound disclosed herein can act as a counterion to such divalent alkaline earth ion, or a doubly ionized compound of the invention can act as a counterion to such divalent alkaline earth ion.) As a specific example, -NR 80 R 80 is -NH 2 , -NH-alkyl, N-pyrrolidinyl, N-piperazinyl, 4N-methyl-piperazin-1-yl and N-morpholinyl.

[0026] Further to the disclosure herein, the substitution of hydrogen on an unsaturated carbon atom in a "substituted" alkene, alkyne, aryl, and heteroaryl group is, unless otherwise indicated, -R 60 , halo, -O - M + , -OR 70 , -SR 70 , -S - M + , -NR 80 R 80 , trihalomethyl, -CF 3 , -CN, -OCN, -SCN, -NO, -NO 2 , -N3 、-SO 2 R 70 、-SO 3 - M + 、-SO 3 R 70 、-OSO 2 R 70 、-OSO 3 - M + 、-OSO 3 R 70 、-PO 3 -2 (M + ) 2 、-P(O)(OR 70 )O - M + 、-P(O)(OR 70 ) 2 、-C(O)R 70 、-C(S)R 70 、-C(NR 70 )R 70 、-CO 2 - M + 、-CO 2 R 70 、-C(S)OR 70 、-C(O)NR 80 R 80 、-C(NR 70 )NR 80 R 80 、-OC(O)R 70 、-OC(S)R 70 、-OCO 2 - M + 、-OCO 2 R 70 、-OC(S)OR 70 、-NR 70 C(O)R 70 、-NR 70 C(S)R 70 、-NR 70 CO 2 - M + 、-NR 70 CO 2 R 70 、-NR 70 C(S)OR 70 、-NR 70 C(O)NR80 R 80 , -NR 70 C(NR 70 )R 70 and -NR 70 C(NR 70 )NR 80 R 80 where R 60 , R 70 , R 80 and M + is as previously defined, except that in the case of a substituted alkene or alkyne, the substituent is -O - M + , -OR 70 , -SR 70 , or -S - M + isn't it.

[0027] In addition to the groups disclosed for each individual term herein, the substituents of the hydrogen on the nitrogen atom in "substituted" heteroalkyl and cycloheteroalkyl groups are, unless otherwise specified, -R 60 , -O - M + , -OR 70 , -SR 70 , -S - M + , -NR 80 R 80 , trihalomethyl, -CF 3 , -CN, -NO, -NO 2 , -S(O) 2 R 70 , -S(O) 2 O - M + , -S(O) 2 OR 70 , -OS(O) 2 R 70 , -OS(O) 2 O - M + , -OS(O) 2 OR 70 , -P(O)(O - ) 2 (M + ) 2 , -P(O)(OR 70 )O - M +, -P(O)(OR 70 )(OR 70 ), -C(O)R 70 , -C(S)R 70 , -C(NR 70 )R 70 , -C(O)OR 70 , -C(S)OR 70 , -C(O)NR 80 R 80 , -C(NR 70 )NR 80 R 80 , -OC(O)R 70 , -OC(S)R 70 , -OC(O)OR 70 , -OC(S)OR 70 , -NR 70 C(O)R 70 , -NR 70 C(S)R 70 , -NR 70 C(O)OR 70 , -NR 70 C(S)OR 70 , -NR 70 C(O)NR 80 R 80 , -NR 70 C(NR 70 )R 70 and -NR 70 C(NR 70 )NR 80 R 80 where R 60 , R 70 , R 80 and M + is as previously defined.

[0028] Further to the disclosure herein, in certain embodiments a substituted group has 1, 2, 3, or 4 substituents, 1, 2, or 3 substituents, 1 or 2 substituents, or 1 substituent.

[0029] It should be understood that in all of the above defined substituents, polymers arrived at by defining the substituent with a further substituent on itself (e.g., a substituted aryl having as a substituent a substituted aryl group which is itself substituted with a substituted aryl group, which is further substituted with a substituted aryl group, etc.) are not intended to be encompassed herein. In such cases, the maximum number of such substitutions is 3. For example, the sequential substitution of substituted aryl groups specifically contemplated herein is limited to substituted aryl-(substituted aryl)-substituted aryl.

[0030] With respect to any of the groups disclosed herein that contain one or more substituents, it is of course understood that such groups do not include any substitutions or substitution patterns that are sterically impractical and / or synthetically impractical. Moreover, the compounds of this invention include all stereochemical isomers arising from the substitution of these compounds.

[0031] In certain embodiments, the substituents may contribute to the optical isomerism and / or stereoisomerism of the compound. The salts, solvates, hydrates, and prodrug forms of the compound are also intended. All such forms are included in the present disclosure. Thus, the compounds described herein include their salts, solvates, hydrates, prodrugs, and isomeric forms, including their pharma- ceutically acceptable salts, solvates, hydrates, prodrugs, and isomeric forms. In certain embodiments, the compounds may be metabolized to pharma- ceutically active derivatives.

[0032] Unless otherwise specified, a reference to an atom is intended to include isotopes of that atom. For example, a reference to H includes 1 H, 2 H (i.e., D) and 3 H (i.e., T) is intended to be included, and references to C include 12 C and all isotopes of carbon ( 13 C, etc.) are intended to be included.

[0033] As used herein, the term "cleavable linker" or "cleavably linked" refers to a linker or bond that can be selectively cleaved using a stimulus (e.g., physical, chemical, or enzymatic stimulus) while the connecting portion of the bond remains intact. Several cleavable bonds have been described in the literature (e.g., Brown (1997) Contemporary Organic Synthesis 4(3);216-237). and Guillier et al. (Chem.Rev.2000 1000:2091-2157). Disulfide bonds (which can be cleaved by DDT) and photocleavable linkers are examples of cleavable bonds.

[0034] The term "fluorophore" refers to any molecular entity that can absorb energy at a first wavelength and re-emit the energy at a different second wavelength. In certain embodiments, the biomolecule of interest comprises a fluorophore attached to one end or a central location of the biomolecule. In some embodiments, the fluorophore may be attached to one end of the biomolecule. The fluorophore attached to the biomolecule does not have to be a single molecule, but may comprise multiple molecules.

[0035] As known to those skilled in the art, fluorophores can be synthetic or biological in nature. More generally, any fluorophore that is stable under coupling conditions and can be sufficiently suppressed when in close proximity to a quencher can be used, so that a significant change in the fluorescence intensity of the fluorophore can be detected in response to specific binding of the target to the probe. Examples of suitable fluorophores include, but are not limited to, Oregon Green 488 dye, rhodamine and rhodamine derivatives, fluorescein isothiocyanate, fluorescein, 6-carboxyfluorescein (6-FAM), coumarin and coumarin derivatives, cyanine and cyanine derivatives, Alexa Fluor, DyLight Fluor, etc.

[0036] In certain embodiments, the biomolecule comprises a metal chelator. As used herein, "chelate" in reference to the complex between a metal and a chelating ligand refers to a combination of a metal ion bound to one or more ligands to form a heterocyclic structure. The chelate formation by neutralization of the positive charge(s) of the metal ion can be by the formation of ionic, covalent, or coordinate covalent bonds. In certain embodiments, the metal chelator includes, but is not limited to, 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA, also called tetraxetane).

[0037] The terms "polynucleotide" and "nucleic acid," used interchangeably herein, refer to a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides. Thus, the terms include, but are not limited to, single-stranded, double-stranded, or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or polymers containing purine and pyrimidine bases or other natural, chemically or biochemically modified, non-natural, or derivatized nucleotide bases.

[0038] The terms "polypeptide" and "protein" are used interchangeably herein to refer to polymeric forms of amino acids of any length, which may include coded and non-coded amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides with modified peptide backbones. The term "fusion protein" or grammatical equivalents is meant to include proteins consisting of multiple polypeptide components that are not normally linked in their native state, but are generally linked at their respective amino and carboxyl termini via peptide bonds to form a single continuous polypeptide. A fusion protein may be a combination of two, three, four or more different proteins.

[0039] Generally, a polypeptide can be of any length, e.g., 2 or more amino acids, more than 4 amino acids, more than about 10 amino acids, more than about 20 amino acids, more than about 50 amino acids, more than about 100 amino acids, more than about 300 amino acids, often up to about 500 or 1000 amino acids or more. A "peptide" is generally 2 or more amino acids in length, e.g., 4 or more amino acids, more than about 10 amino acids, more than about 20 amino acids, often up to about 50 amino acids. In some embodiments, a peptide is 2-30 amino acids in length.

[0040] As used herein, the term "target protein" refers to all members of the target family, as well as fragments and enantiomers thereof, and proteomimetics thereof. Target proteins of interest as described herein are intended to include all members of the target family, as well as fragments and enantiomers thereof, and proteomimetics thereof, unless otherwise specified. Target proteins can be any protein of interest, such as therapeutic or diagnostic targets, including, but not limited to, hormones, growth factors, receptors, enzymes, cytokines, osteoinductive factors, colony stimulating factors, and immunoglobulins. The term "target protein" is intended to include recombinant and synthetic molecules that can be prepared using any convenient recombinant expression method or any convenient synthetic method, or can be purchased commercially, as well as fusion proteins that include the target molecule.

[0041] The term "physiological conditions" refers to conditions compatible with living cells, eg, primarily aqueous conditions of temperature, pH, salinity, etc., compatible with living cells.

[0042] "Solid support", "support", and "solid phase support" are used interchangeably and refer to a material or group of materials having a rigid or semi-rigid surface. In many embodiments, at least one surface of the solid support is substantially flat, although in some embodiments it may be desirable to physically separate synthesis regions of different compounds, e.g., by wells, raised areas, pins, etched trenches, etc. According to other embodiments, the solid support(s) will take the form of beads, resins, gels, microspheres, or other geometric configurations.

[0043] The terms "antibody" and "immunoglobulin" include antibodies or immunoglobulins of any isotype that retain specific binding to an antigen, including, but not limited to, Fab, Fv, scFv, and Fd fragments, chimeric antibodies, humanized antibodies, single chain antibodies (scAbs), single domain antibodies (dAbs), single domain heavy chain antibodies, single domain light chain antibodies, nanobodies, bispecific antibodies, multispecific antibodies, and antigen-binding (also referred to herein as antigen binding) portions of antibodies and fusion proteins such as non-antibody proteins. Antibodies can be detectably labeled, for example, with a radioisotope, an enzyme that generates a detectable product, a fluorescent protein, and the like. Antibodies can be further conjugated to other moieties, such as members of a specific binding pair, for example, biotin (a member of the biotin-avidin specific binding pair). Antibodies can also be bound to solid supports, including, but not limited to, polystyrene plates or beads. The terms include Fab', Fv, F(ab') 2, and / or other antibody fragments that retain specific binding to the antigen, and monoclonal antibodies. As used herein, a monoclonal antibody is an antibody produced by an identical population of cells, all of which have been produced from a single cell by repeated cell replication. That is, a clone of cells produces only a single antibody species. Monoclonal antibodies can be produced using hybridoma production techniques, although other production methods known to those skilled in the art can also be used (e.g., antibodies derived from antibody phage display libraries). Antibodies can be monovalent or bivalent. Antibodies can be Ig monomers, which are "Y-shaped" molecules consisting of four polypeptide chains, two heavy and two light chains connected by disulfide bonds.

[0044] The term "humanized immunoglobulin" as used herein refers to an immunoglobulin that contains portions of immunoglobulins of different origins, at least one portion containing amino acid sequences of human origin. For example, a humanized antibody may contain a portion derived from an immunoglobulin of non-human origin, such as a mouse, with the required specificity, chemically coupled by conventional techniques (e.g., synthesis) or prepared as a continuous polypeptide using genetic engineering techniques, and a portion derived from an immunoglobulin sequence of human origin (e.g., chimeric immunoglobulin) (e.g., DNA encoding the protein portion of a chimeric antibody can be expressed to produce a continuous polypeptide chain). Another example of a humanized immunoglobulin is an immunoglobulin that contains one or more immunoglobulin chains that contain complementarity determining regions (CDRs) derived from an antibody of non-human origin and framework regions derived from light and / or heavy chains of human origin (e.g., CDR-grafted antibodies with or without framework changes). Chimeric or CDR-grafted single chain antibodies are also encompassed by the term humanized immunoglobulin. See, e.g., Cabilly et al., U.S. Pat. No. 4,816,567; Cabilly et al., European Patent No. 0,125,023B1; Boss et al., U.S. Pat. No. 4,816,397; Boss et al., European Patent No. 0,120,694B1; Neuberger, MS et al., WO86 / 01533; Neuberger, MS et al., European Patent No. 0,194,276B1; Winter, U.S. Pat. No. 5,225,539; Winter, European Patent No. 0,239,400B1; Padlan, EA et al., European Patent Application No. 0,519,596A1. Regarding single chain antibodies, see also Ladner et al., U.S. Patent No. 4,946,778; Huston, U.S. Patent No. 5,476,786; and Bird, RE et al., Science, 242:423-426 (1988).

[0045] As used herein, the term "nanobody" (Nb) refers to the smallest antigen-binding fragment or single variable domain (V) derived from a naturally occurring heavy chain antibody. HH), which are known to those skilled in the art. They are derived from heavy chain only antibodies found in camels (Hamers-Casterman et al., (1993) Nature 363:446; Desmyter et al., (1996) Nature Struct. Biol. 3:803). In the "Camelidae" family, immunoglobulins that lack light chain polypeptides are found. "Camelidae" includes Old World camelids (Camelus bactrianus and Camelus dromedarius) and New World camelids (e.g., Llama paccos, Llama glama, Llama guanicoe, and Llama vicugna). Single variable domain heavy chain antibodies are referred to herein as nanobodies or V HH are called antibodies.

[0046] An "antibody fragment" includes a portion of an intact antibody, such as the antigen-binding or variable region of an intact antibody. Examples of antibody fragments include Fab, Fab', F(ab') 2 and Fv fragments; diabodies; linear antibodies (Zapata et al. Protein Eng. 8(10):1057-1062 (1995)); domain antibodies (dAbs; Holt et al. (2003) Trends Biotechnol. 21:484); single-chain antibody molecules; and multispecific antibodies formed from antibody fragments. Papain digestion of antibodies produces two identical antigen-binding fragments, called "Fab" fragments, each with a single antigen-binding site, and a remaining "Fc" fragment, a name reflecting the ability to crystallize readily. Pepsin treatment produces F(ab') fragments that have two antigen-binding sites and are still capable of cross-linking antigen. 2 A fragment is generated.

[0047] "Fv" is the minimum antibody fragment that contains a complete antigen recognition and binding site. This region consists of a dimer of one heavy chain variable domain and one light chain variable domain in tight non-covalent association. The three CDRs of each variable domain are V H -V LIt is in this configuration that they interact to define an antigen-binding site on the surface of the dimer. Together, the six CDRs confer antigen-binding specificity to the antibody. However, even a single variable domain (i.e., half of an Fv containing only three CDRs specific for an antigen) has the ability to recognize and bind antigen, albeit with lower affinity than the entire binding site.

[0048] The Fab fragment also contains the constant domain of the light chain and the first constant domain of the heavy chain (CH 1 ) The Fab fragment also contains the heavy chain CH 1 Fab' fragments differ from Fab' fragments by the addition of a few residues at the carboxyl terminus of the constant domains including one or more cysteines from the antibody hinge region. Fab'-SH is the designation herein for Fab' in which the cysteine ​​residue(s) of the constant domains bear a free thiol group. F(ab') 2 Antibody fragments originally were produced as pairs of Fab' fragments which have hinge cysteines between them, although other chemical couplings of antibody fragments are also known.

[0049] The "light chains" of antibodies (immunoglobulins) from any vertebrate species can be assigned to one of two clearly distinct types, called kappa and lambda, based on the amino acid sequence of their constant domains. Depending on the amino acid sequence of the constant domains of their heavy chains, immunoglobulins can be assigned to different types. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and several of these classes can be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA, and IgA2. The subclasses can be further divided into types, such as IgG2a and IgG2b.

[0050] "Single-chain Fv" or "sFv" or "scFv" antibody fragments are fragments of the V H and V L In some embodiments, the Fv polypeptide comprises a V domain, and these domains are present in a single polypeptide chain. H Domain and VL It further comprises a polypeptide linker between the domains, which enables the sFv to form the desired structure for antigen binding. For a review of sFv, see The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994).

[0051] The term "diabody" refers to a diabody comprising a light chain variable domain (V L ) linked to a heavy chain variable domain (V H A small antibody fragment (V) with two antigen-binding sites H -V L By using a linker that is too short to allow pairing between the two domains in the same chain, the domains pair with the complementary domains of another chain and create two antigen binding sites. Diabodies are described in further detail in, for example, EP 404,097; WO 93 / 11161; and Hollinger et al. (1993) Proc. Natl. Acad. Sci USA 90:6444-6448.

[0052] As used herein, the term "affinity" refers to the equilibrium constant for the reversible binding of two agents (e.g., an antibody and an antigen) and is expressed as the dissociation constant (K D) The affinity can be at least 1-fold, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 20-fold, at least 30-fold, at least 40-fold, at least 50-fold, at least 60-fold, at least 70-fold, at least 80-fold, at least 90-fold, at least 100-fold, or at least 1,000-fold, or more, compared to the affinity of the antibody for an unrelated amino acid sequence. The affinity of the antibody for the target protein can be, for example, from about 100 nanomolar (nM) to about 0.1 nM, from about 100 nM to about 1 picomolar (pM), or from about 100 nM to about 1 femtomolar (fM), or more. As used herein, the term "binding activity" refers to the resistance of a complex of two or more agents to dissociation after dilution. With respect to antibodies and / or antigen-binding fragments, the terms "immunoreactive" and "preferentially bind" are used interchangeably herein.

[0053] The term "binding" refers to a direct association between two molecules, for example, by covalent, electrostatic, hydrophobic, and ionic bonds and / or hydrogen bonding interactions, including interactions such as salt bridges and water bridges. "Specific binding" refers to binding with an affinity of at least about 10 -7 M or more, for example, affinity is 5×10 -7 M, 10 -8 M, 5×10 -8 M and higher. "Non-specific binding" refers to binding with an affinity of about 10 -7 Binding of less than M, e.g., affinity of 10 -6 M, 10 -5 M, 10 -4 This refers to bonds such as M.

[0054] An "isolated" polypeptide is one that has been identified, separated, and / or recovered from a component of its natural environment. Contaminating components of its natural environment are substances that would interfere with diagnostic or therapeutic uses of the polypeptide and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. In some embodiments, the polypeptide is purified (1) to greater than 90%, greater than 95%, or greater than 98%, e.g., greater than 99%, by weight of protein as determined by the Lowry method, (2) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence using a spinning cup sequenator, or (3) to homogeneity by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) under reducing or non-reducing conditions using Coomassie blue or silver staining. Isolated polypeptides include polypeptides in situ within recombinant cells, since at least one component of the polypeptide's natural environment is not present. In some examples, isolated polypeptides are prepared by at least one purification step.

[0055] As will be apparent to one of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has distinct components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the invention. Any method described can be carried out in the order of events described or in any other order which is logically possible.

[0056] Where a range of values ​​is provided, it is understood that each intervening value between the upper and lower limits of that range, to one-tenth of the unit of the lower limit, as well as any other stated or intervening value in that stated range, is encompassed within the invention, unless the context clearly dictates otherwise. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also encompassed within the invention, subject to any specific excluded limits in the stated range. Where a stated range includes one or both of its limits, ranges excluding either or both of those included limits are also encompassed within the invention.

[0057] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention, the preferred methods and materials are described herein. All publications mentioned herein are incorporated by reference to disclose and describe the methods and / or materials in connection with which the publications are cited.

[0058] It should be noted that, as used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, a reference to a "target molecule" includes a plurality of such target molecules, a reference to a "biomolecule" includes a reference to one or more biomolecules and their equivalents known to those skilled in the art, and so forth. It should be further noted that the claims may be drafted to exclude any optional element. Thus, this statement is intended to serve as a predicate for the use of exclusive language such as "solely," "only," or "negative" limitations in connection with the recitation of claim elements.

[0059] It is understood that certain features of the invention that are described for clarity in the context of separate embodiments may also be provided in combination in a single embodiment. Conversely, various features of the invention that are described for brevity in the context of a single embodiment may also be provided separately or in any suitable subcombination. All combinations of the embodiments related to the present invention are expressly embraced by the present invention and are disclosed herein as if each and every combination was individually and explicitly disclosed. Moreover, all subcombinations of the various embodiments and elements thereof are also expressly embraced by the present invention and are disclosed herein as if each and every such subcombination was individually and explicitly disclosed herein.

[0060] The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein should be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates, which may need to be independently confirmed.

[0061] Detailed Description The present disclosure provides a method for chemoselectively modifying a target molecule. The present disclosure provides a composition comprising a subject target molecule comprising a thiol moiety and a biomolecule comprising a phenolic or catechol moiety. The present disclosure provides a kit providing a first container comprising a subject composition and a second container comprising an enzyme capable of oxidizing the phenolic or catechol moiety. The present disclosure also provides modified target molecules that may find use in gene therapy, novel immunotherapy via antibody conjugates, biomaterial construction and delivery of biomolecules for vaccine development.

[0062] method As summarized above, aspects of the present disclosure include methods for chemoselectively modifying a target molecule. The subject method includes contacting a target molecule that includes a thiol moiety with a biomolecule that includes a reactive moiety, where the reactive moiety is generated by reaction of the biomolecule that includes a phenolic or catechol moiety with an enzyme that can oxidize the phenolic or catechol moiety. The contacting is performed under conditions sufficient to complex the target molecule with the biomolecule, thereby generating a modified target molecule.

[0063] In some cases, the subject method for chemoselective modification of a target molecule includes contacting i) a target molecule comprising a thiol moiety; ii) a biomolecule comprising a phenolic or catechol moiety; and iii) an enzyme capable of oxidizing the phenolic or catechol moiety; the enzyme oxidizes the phenolic or catechol moiety of the biomolecule to generate a reactive moiety, thereby generating a biomolecule comprising the reactive moiety, which reacts with the thiol moiety, thereby linking the target molecule and the biomolecule to one another, thereby generating a modified target molecule. In some cases, the target molecule comprises a single thiol moiety. In some cases, the target molecule comprises two thiol moieties.

[0064] The target molecule can be any of a variety of molecules (e.g., polypeptides; nucleic acids; small molecules, etc.). Similarly, the biomolecule can be any of a variety of molecules (e.g., polypeptides; nucleic acids; small molecules, etc.). In some cases, the target molecule is a polypeptide; the biomolecule is a nucleic acid. In some cases, the target molecule is a nucleic acid; the biomolecule is a polypeptide. In some cases, the target molecule is a polypeptide; the biomolecule is a small molecule (e.g., a cancer chemotherapeutic agent). In some cases, the target molecule is a first polypeptide; the biomolecule is a second polypeptide, and the first polypeptide and the second polypeptide can be the same or different.

[0065] The subject method provides a simple coupling procedure that allows a biomolecule of interest to be attached in a site-specific manner to any location on the surface of a target molecule, thereby generating a modified target molecule of interest. In some embodiments, the target molecule is a second biomolecule (e.g., as described herein). In some embodiments, the second biomolecule is a polypeptide.

[0066] Biomolecules of interest include, but are not limited to, polypeptides, polynucleotides, carbohydrates, lipids, fatty acids, steroids, purines, pyrimidines, derivatives thereof, structural analogs, and combinations thereof. In certain examples, the biomolecule of interest is an antibody. In some cases, the biomolecule of interest is an antibody fragment or a conjugated derivative thereof. In some cases, the antibody fragment or a conjugated derivative thereof is selected from the group consisting of a Fab fragment, a F(ab')2 fragment, a single chain Fv (scFv), a diabody, a nanobody, and a triabody. Suitable biomolecules include, for example, small molecules (e.g., cancer chemotherapeutic agents, etc.), cytokines, hormones, immunomodulatory polypeptides, etc. In some cases, the biomolecule is a nucleic acid; the target molecule is an antibody (e.g., scFv; nanobody, etc.). In some cases, the biomolecule is a small molecule (e.g., cancer chemotherapeutic agents); the target molecule is an antibody (e.g., scFv; nanobody, etc.). In some cases, for example, when the target molecule is an antibody, the biomolecule is conjugated to the Fc portion of the target molecule. In some cases, the target molecule is an immunoglobulin (Ig) Fc polypeptide.

[0067] In certain embodiments, the biomolecule comprising a phenol or catechol moiety further comprises one or more moieties selected from a fluorophore, an active small molecule, an affinity tag, and a metal chelator (e.g., as described herein). In certain examples, the biomolecule of interest is a fluorescent protein. In certain cases, the fluorescent protein is green fluorescent protein (GFP). In certain cases, the biomolecule is an enzyme. In certain cases, the biomolecule is a ligand for a receptor. In certain cases, the biomolecule is a receptor.

[0068] In some embodiments, the enzyme capable of oxidizing a phenolic or catechol moiety is a phenol oxidase or a catechol oxidase. In certain cases, the enzyme is tyrosinase.

[0069] The term "tyrosinase" is used herein to refer to monophenol monooxygenase (EC 1.14.18.1; CAS number: 9002-10-2), an enzyme that catalyzes the oxidation of phenols (such as tyrosine). It is a copper-containing enzyme present in plant and animal tissues that catalyzes the production of melanin and other pigments from tyrosine by oxidation. All tyrosinases have in common a dinuclear type 3 copper center in their active site, where two copper atoms are coordinated with three histidine residues each. Matoba et al., "Crystallographic evidence that the dinuclear copper center of tyrosinase is flexible during catalysis," J Biol Chem. 2006 Mar 31; 281(13):8981-90. Epub 2006 Jan 25, discloses a three-dimensional model of the tyrosinase catalytic center.

[0070] In certain embodiments, the subject enzymes are immobilized on a solid support system, e.g., beads, resins, gels, microspheres, or other geometric configurations. In certain cases, the solid support is a glass bead. In some cases, the solid support is a resin bead. The use of enzymes attached to a solid support system can facilitate purification of the subject target molecules by allowing multiple uses of the subject enzymes and allowing the enzyme to be easily removed from the reaction mixture. In certain embodiments, the subject enzymes attached to a solid support system can enable the subject methods to be performed in a continuous flow system. In certain embodiments, the subject enzymes attached to a solid support system can facilitate large-scale batch processing of the subject methods.

[0071] In certain cases, the subject phenolic moiety is present in a tyrosine residue. In certain cases, the tyrosine residue is part of a biomolecule of interest. In certain cases, the tyrosine residue is synthetically introduced into the biomolecule of interest. In some other cases, the tyrosine residue is linked to the biomolecule of interest via a linker (e.g., as described herein). The tyrosine residue can be introduced using standard recombinant techniques, for example, by modifying the nucleotide sequence encoding the polypeptide biomolecule such that the tyrosine residue is introduced into the polypeptide biomolecule.

[0072] In some cases, the phenol or catechol moiety is part of the non-natural (non-genetically encoded) amino acid to be introduced into the biomolecule of interest. For example, amber codon (TAG) suppression can be used to incorporate a non-genetically encoded amino acid residue that includes a phenol or catechol moiety. See, for example, Chin et al. (2002) J. Am. Chem. Soc. 124:9026; Chin and Schultz (2002) Chem. Biol. Chem. 3:1135; Chin et al. (2002) Proc. Natl. Acad. Sci. USA 99:11020; US2015 / 0240249; and US2018 / 0171321. As another example, orthogonal RNA synthetases and / or orthogonal tRNAs can be used to introduce a non-genetically encoded amino acid into a biomolecule, where the non-genetically encoded amino acid includes a phenol or catechol moiety.

[0073] In some embodiments of the subject methods, the thiol moiety present in the target molecule is part of a cysteine ​​residue. In certain cases, the cysteine ​​residue is a naturally occurring cysteine ​​residue. In certain cases, the cysteine ​​residue is a residue that is synthetically introduced into the target molecule.

[0074] In certain embodiments, the reactive moiety is an orthoquinone or semiquinone radical, or a combination thereof. In certain embodiments, the subject method provides for the reaction between an orthoquinone reactive intermediate and a thiol moiety, as shown in Scheme 1 below. TIFF2025029024000001.tif80158 Scheme 1 In the formula, Y 1 is any convenient biomolecule, optionally including one or more moieties selected from active small molecules, cleavable probes, fluorophores, and metal chelators; L is an optional linker (e.g., as described herein); X 1 is selected from hydrogen and hydroxyl; Y 2 is any convenient biomolecule; and n is an integer from 1 to 3.

[0075] As shown in Scheme 1, in certain embodiments, a biomolecule comprising a phenol or catechol moiety (e.g., of formula (I)) undergoes activation with an enzyme capable of oxidizing the phenol or catechol moiety. In some cases, activation is accomplished using a tyrosinase enzyme in the presence of oxygen to generate an intermediate comprising a reactive moiety (e.g., an orthoquinone radical of formula (II) and / or a semiquinone radical of formula (IIA)), which reacts with a target molecule comprising a thiol-based nucleophile (e.g., of formula (III)) to result in the conjugation of the target molecule with the biomolecule, thereby generating a modified target molecule (e.g., of formula (gen IV)). In certain embodiments, the target molecule of formula (III) can include any convenient biomolecule, for example, as described herein. In certain cases, Y in formula (III) can be a thiol-based nucleophile, which ... 2 is a polypeptide. In certain cases, the modified molecule is represented by formula (IV). In some cases, the modified target molecule is represented by formula (IVA). In certain cases, the modified target molecule is described by any one of formulas (IV)-(IVL), as described herein.

[0076] In certain embodiments, the subject method can be carried out according to the following Scheme 2: As shown in TIFF2025029024000002.tif29165, the reaction between an orthoquinone reactive intermediate and a thiol moiety is provided.

[0077] As shown in Scheme 2, in certain embodiments, a biomolecule comprising a phenolic moiety (e.g., of formula (IB)) undergoes activation by a tyrosinase enzyme in the presence of oxygen to generate an intermediate comprising a reactive moiety (e.g., an orthoquinone of formula (II)), which reacts with a target molecule comprising a thiol-based nucleophile (e.g., of formula (III)) to result in conjugation of the target molecule with the biomolecule, thereby generating a modified target molecule (e.g., of formula (IVM)). In certain embodiments, the target molecule of formula (III) can include any convenient biomolecule, for example, as described herein. In certain cases, Y in formula (III) can be a thiol-based nucleophile, which ... 2 is a polypeptide. In the particular case of a modified molecule of formula (IVM), a thiol group is present at the 3-position of the catechol ring. In the particular case of a modified molecule of formula (IVM), a thiol group is present at the 5-position of the catechol ring. In the particular case of a modified molecule of formula (IVM), a thiol group is present at the 6-position of the catechol ring.

[0078] In certain embodiments, the biomolecule of formula (I) can be any one of formulas (IA)-(IDb), for example, as described herein and discussed in more detail below. In certain embodiments, the modified target molecule can be any one of formulas (IV)-(IVL), for example, as described herein and discussed in more detail below. In certain embodiments, the modified target molecule is the product of a single complexation, for example, as shown in formulas (IV1)-(IV3), (IVA1)-(IVA3), (IVB1)-(IVB3), (IVC1)-(IVC3), (IVD1)-(IVD3), (IVE1)-(IVE3), (IVF1)-(IVF3), (IVG1)-(IVG3), (IVH1)-(IVH3), and (IVJ1)-(IVJ3). In certain cases, the modified target molecule is the product of double complexation, for example, as shown in formulas (IV4)-(IV5), (IVA4)-(IVA5), (IVB4)-(IVB5), (IVC4)-(IVC5), (IVD4)-(IVD5), (IVE4)-(IVE5), (IVF4)-(IVF5), (IVG4)-(IVG5), (IVH4)-(IVH5), and (IVJ4)-(IVJ5).

[0079] In certain embodiments, the subject method is carried out at a pH between 4 and 9, e.g., 4.2, 4.5, 4.8, 5.0, 5.2, 5.5, 5.8, 6.0, 6.2, 6.5, 6.8, 7.0, 7.2, 7.5, 7.8, 8.0, 8.2, 8.5, 8.8, or 9. In certain embodiments, the subject method is carried out at a pH between 5 and 8, e.g., 5.2, 5.5, 5.8, 6.0, 6.2, 6.5, 6.8, 7.0, 7.2, 7.5, 7.8, or 8.0. In certain cases, the subject method is carried out at a pH between 6 and 7.5, e.g., 6.0, 6.3, 6.4, 6.5, 6.6, 6.8, 7.0, 7.2, 7.4, or 7.5. In certain embodiments, the subject method is carried out at neutral pH. As used herein, the expression "neutral pH" refers to a pH of about 7.0 to about 7.4. The expression "neutral pH" includes pH values ​​of about 7.0, 7.05, 7.1, 7.15, 7.2, 7.25, 7.3, 7.35, and 7.4.

[0080] In certain embodiments, the subject methods may be performed under physiological conditions. In some embodiments, the methods are performed on living cells in vitro. In other embodiments, the methods are performed on living cells ex vivo.

[0081] In certain embodiments, the subject method may be carried out in an aqueous medium in the presence of one or more buffers. Buffers of interest include, but are not limited to, phosphate buffer, 2-amino-2-(hydroxymethyl)propane-1,3-diol (TRIS), 4-[4-(2-hydroxyethyl)piperzin-1-yl]ethanesulfonic acid (HEPES), and the like. In certain embodiments, the subject method may be carried out in an organic solvent. In certain cases, the organic solvent is a water-miscible solvent. In certain cases, the organic solvent is a dipolar aprotic solvent. In certain cases, the organic solvent is selected from acetonitrile, dimethylformamide, methanol, and acetone. In certain cases, the organic solvent is present in an amount of 1-20% relative to water, e.g., 2%, 5%, 10%, 15%, or 20%. In some cases, the subject method is carried out with 1%-20%, e.g., 5%, 10%, 15%, or 20% acetonitrile. In some cases, the subject method is carried out with 1%-20%, e.g., 5%, 10%, 15%, or 20% dimethylformamide. In some cases, the subject method is carried out with 1%-20%, e.g., 5%, 10%, 15%, or 20% methanol. In some cases, the subject method is carried out with 1%-20%, e.g., 5%, 10%, 15%, or 20% acetone.

[0082] In certain embodiments of the subject methods, the modified target molecule is a product of double or triple conjugation (e.g., see formula (IV) when n is 2 or 3 (collectively referred to herein as "multiple conjugation products")). In certain embodiments of the subject methods, the multiple conjugation products are present at less than 10-fold, e.g., less than 20-fold, less than 25-fold, less than 50-fold, less than 75-fold, less than 100-fold, or less, parts by weight, compared to the single conjugation products (e.g., see formula (IV) when n is 1). In certain embodiments of the subject methods, multiple conjugation products are not observed.

[0083] In certain embodiments of the method, the modified target molecule is stable over a range of pH and temperature values, as well as in the presence of some additional molecules. In some cases, the modified target molecule is stable at 0°C to 50°C, e.g., 4°C to 40°C, e.g., 4°C to 37°C. In certain cases, the modified target molecule is stable over a pH range of 4 to 9, e.g., pH 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, or 9. In certain cases, the modified target molecule is stable in the presence of biologically relevant molecules. In certain cases, the modified target molecule is stable in the presence of molecules such as guanidinium groups of arginine residues, primary amines of lysine residues, and aniline moieties. In some cases, the modified target molecule is stable at physiological conditions; for example, in some cases, the modified target molecule is stable in human serum. In some cases, the modified target molecule (also referred to herein as a "target molecule-biomolecular complex") is stable in human serum for a period of at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 10 days, or at least 14 days at 37° C. In some cases, the modified target molecule (also referred to herein as a "target molecule-biomolecular complex") is stable in human serum for a period of about 2 days to about 7 days, about 7 days to about 10 days, or about 10 days to about 14 days at 37° C.

[0084] As noted above, in some cases, the target molecule contains a single thiol moiety. In other instances, the target molecule contains two thiol moieties. In addition to being eligible for the initial oxidative coupling reaction, nucleophiles that have a second thiol in close proximity to the newly formed catechol may add again in the event of reoxidation. The intramolecular nature of the second addition may prevent or minimize a second addition by glutathione or other molecules with free thiols in the biological environment. An example of the use of dithiol nucleophiles (dithiol target molecules) is shown diagrammatically in Figure 44A.

[0085] Another embodiment of this strategy can be a protein coupling partner with two cysteine ​​residues. The cysteine ​​residues can be adjacent to each other by their position in the amino acid sequence of the protein, or by their position in the three-dimensional structure of the protein. The polypeptide can include a dithiol, for example, the polypeptide includes a CC, CGC, CGGC (SEQ ID NO: 1055), or CGGGC (SEQ ID NO: 1056) sequence. For example, the polypeptide can be of the general formula: X n1 C(X) n2 CX n3 (SEQ ID NO: 1057), where X is any naturally occurring (encoded) or non-naturally occurring (non-encoded) amino acid, n1 and n3 are each independently zero or an integer between 1 and 5000 (or greater than 5000), and n2 is zero or an integer between 1 and about 10. An example of the use of such a dithiol targeting molecule is shown diagrammatically in Figure 44B.

[0086] Tyrosinase Polypeptides Tyrosinase polypeptides suitable for use in generating reactive moieties (e.g., orthoquinones) include tyrosinase polypeptides having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to any one of the amino acid sequences of tyrosinases shown in Figures 8, 9, 10A-10Z, and 10AA-10VV. In some cases, the tyrosinase polypeptide is an Agricus bisporus tyrosinase polypeptide. In some cases, the tyrosinase polypeptide is a Bacillus megaterium tyrosinase polypeptide. In some cases, the tyrosinase polypeptide is a Streptomyces castaneoglobisporus tyrosinase polypeptide. In some cases, the tyrosinase polypeptide is a Citrobacter freundii tyrosinase polypeptide. In some cases, the tyrosinase polypeptide is a Homo sapiens tyrosinase polypeptide. In some cases, the tyrosinase polypeptide is a Malus domestica tyrosinase polypeptide. In some cases, the tyrosinase polypeptide is an Aspergillus oryzae tyrosinase polypeptide. In some cases, the tyrosinase polypeptide is a Solanum lycopersicum tyrosinase polypeptide. In some cases, the tyrosinase polypeptide is a Burkholderia thailandensis tyrosinase polypeptide. In some cases, the tyrosinase polypeptide is a Juglans regia tyrosinase polypeptide.See, e.g., Pretzler et al. Sci.Rep.2017,7(1),1810; Ren et al. BMC Biotechnol.2013,13,18; Faccio et al. Process Biochem.2012,47(12),1749-1760; Fairhead et al. FEBS J.2010,277(9),2083-2095; Do et al. Sci.Rep.2017,7(1),17267; Elsayed and Danial J.Appl. Pharm.Sci.2018,8(09),93-101; Lopez-Tejedor and Palomo Protein Expr. Purif.2018,145,64-70; and Fairhead et al. Nature Biotechnol.2012,29(2),183-191.

[0087] In some cases, a tyrosinase polypeptide selectively acts on a substrate (biomolecule) that contains a phenolic (e.g., tyrosine) or catechol moiety (e.g., produces a reactive moiety such as an orthoquinone), where the substrate is neutral or positively charged within 50 Å (e.g., within 50 Å, within 40 Å, within 30 Å, or within 20 Å) of the phenolic or catechol moiety. A tyrosinase having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to any one of the amino acid sequences of tyrosinases shown in FIG. 8 or FIG. 9 can selectively modify a phenolic or catechol moiety on a substrate, where the substrate is neutral or positively charged within 50 Å (e.g., within 50 Å, within 40 Å, within 30 Å, or within 20 Å) of the phenolic or catechol moiety. For example, when the biomolecule is a polypeptide, in some cases the biomolecule comprises at least two neutral or positively charged amino acids within 10 amino acids of the phenolic (e.g., tyrosine) or catechol moiety. For example, when the biomolecule is a polypeptide, in some cases the biomolecule comprises 2, 3, 4, 5, 6, 7, 8, 9, or 10 neutral or positively charged amino acids within 10 amino acids of the phenolic (e.g., tyrosine) or catechol moiety. For example, when the biomolecule is a polypeptide, in some cases the biomolecule comprises the amino acid sequence RRRY (SEQ ID NO: 949), YRRR (SEQ ID NO: 950), RRRRY (SEQ ID NO: 951), or YRRRR (SEQ ID NO: 952).

[0088] In some cases, a tyrosinase polypeptide selectively acts on a substrate (biomolecule) that contains a phenolic (e.g., tyrosine) or catechol moiety (e.g., produces a reactive moiety such as an orthoquinone), where the substrate is negatively charged within 50 Å (e.g., within 50 Å, within 40 Å, within 30 Å, or within 20 Å) of the phenolic or catechol moiety. A tyrosinase having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to any one of the tyrosinase amino acid sequences shown in Figures 10A-10Z and 10AA-10VV can selectively modify a phenolic or catechol moiety on a substrate, where the substrate is negatively charged within 50 Å (e.g., within 50 Å, within 40 Å, within 30 Å, or within 20 Å) of the phenolic or catechol moiety. For example, when the biomolecule is a polypeptide, in some cases the biomolecule comprises at least two negatively charged amino acids within 10 amino acids of a phenolic moiety (e.g., tyrosine) or a catechol moiety. For example, when the biomolecule is a polypeptide, in some cases the biomolecule comprises 2, 3, 4, 5, 6, 7, 8, 9, or 10 negatively charged amino acids within 10 amino acids of a phenolic moiety (e.g., tyrosine) or a catechol moiety. For example, when the biomolecule is a polypeptide, in some cases the biomolecule comprises the amino acid sequence EEEY (SEQ ID NO: 953), YEEEE (SEQ ID NO: 954), EEEEY (SEQ ID NO: 955), or YEEEE (SEQ ID NO: 956).

[0089] In some cases, the tyrosinase polypeptide comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the amino acid sequence of tyrosinase shown in FIG. 10M, and the tyrosinase polypeptide comprises an amino acid substitution of D55, e.g., D55 is replaced with Lys. Such tyrosinase polypeptides are particularly useful when the biomolecule has a net negative charge and / or the region surrounding the phenol or catechol moiety has a net negative charge (e.g., when the phenol group is Tyr, Tyr can be present in the EEEEY (SEQ ID NO: 955) or EEEY (SEQ ID NO: 953) peptide). Such tyrosinase polypeptides are particularly useful when the biomolecule is a nucleic acid.

[0090] In some cases, the tyrosinase polypeptide has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the amino acid sequence of tyrosinase shown in Figure 10C, and the tyrosinase polypeptide includes an amino acid substitution of R209, e.g., R209 is replaced with His. Such tyrosinase polypeptides are particularly useful when the biomolecule has a net positive charge and / or when the region surrounding the phenol or catechol moiety has a net positive charge (e.g., when the phenol group is Tyr, Tyr can be present in the RRRY (SEQ ID NO:949) or RRRRY (SEQ ID NO:951) peptide).

[0091] Cell surface modification In some embodiments, the subject method is used to modify the surface of a cell. Thus, in one aspect, the invention features a method of modifying the surface of a cell in vitro. The method generally includes reacting a thiol group of a target molecule with a biomolecule that includes a reactive moiety to provide chemoselective conjugation at the cell surface. In some embodiments, the method includes modifying a target molecule on the cell surface with a thiol moiety; reacting the thiol moiety of the target molecule with a biomolecule that includes a reactive moiety (e.g., an orthoquinone moiety). In other embodiments, the method includes activating a biomolecule that includes a phenol moiety on the cell surface to produce a biomolecule that includes a reactive moiety; and reacting the reactive moiety of the biomolecule with a target molecule that includes a thiol moiety.

[0092] Modification of target molecules with detectable labels, drugs, and other molecules In some embodiments, the present disclosure provides for the attachment of a biomolecule of interest to a target molecule that includes a thiol moiety. The method generally involves reacting a thiol-containing target molecule with a subject biomolecule that includes a reactive moiety (e.g., an orthoquinone moiety). Target molecules and biomolecules of interest include, but are not limited to, polypeptides, polynucleotides, carbohydrates, fatty acids, steroids, purines, pyrimidines, derivatives, and the like.

[0093] Attachment of a biomolecule of interest to a support Biomolecules that include reactive moieties may also include one or more hydrocarbon linkers (e.g., alkyl groups or derivatives thereof, e.g., alkyl esters or PEG) conjugated to a moiety that provides binding to a solid substratum (e.g., to facilitate assays) or that provides easy separation (e.g., a hapten recognized by an antibody bound to a magnetic bead). In one embodiment, the methods of the invention are used to provide for the attachment of a protein (or other molecule that contains a thiol or can be modified to contain a thiol) to a chip in a defined orientation. For example, the disclosed methods and compositions can be used to deliver a tag or other moiety (e.g., as described herein) to a thiol of a target molecule, e.g., a polypeptide that has a thiol moiety at a selected site (e.g., at or proximal to the N-terminus). The tag or other moiety can then be used as a binding site to immobilize the molecule to a support (e.g., a solid or semi-solid support, e.g., a support suitable for use as a microchip in a high throughput assay).

[0094] Conjugation of biomolecules for delivery to targeted sites The biomolecule comprising the reactive moiety, in some embodiments, comprises a small molecule drug, a toxin, or other molecule for delivery to a cell. The small molecule drug, toxin, or other molecule, in some embodiments, provides pharmacological activity. The small molecule drug, toxin, or other molecule, in some embodiments, serves as a target for delivery of the other molecule.

[0095] Small molecule drugs can be small organic or inorganic compounds with a molecular weight of more than 50 Daltons and less than about 2500 Daltons. Small molecule drugs can contain functional groups necessary for structural interaction with proteins, especially hydrogen bonding, and can contain at least amine, carbonyl, hydroxyl or carboxyl groups, and can contain at least two functional chemical groups. The drug contains cyclic carbon or heterocyclic structures and / or aromatic or polyaromatic structures substituted with one or more of the above functional groups. Small molecule drugs are also found among biomolecules, including peptides, sugars, fatty acids, steroids, purines, pyrimidines, derivatives, structural analogs, or combinations thereof.

[0096] In another embodiment, the subject biomolecules that include a reactive moiety include a pair of binding partners (e.g., a ligand; a ligand-binding portion of a receptor; an antibody; an antigen-binding fragment of an antibody; an antigen; a hapten; a lectin; a lectin-binding carbohydrate, etc.). For example, the biomolecule can include a polypeptide that functions as a viral receptor and, upon binding to a viral envelope protein or a viral capsid protein, promotes binding of the virus to the cell surface on which the biomolecule is displayed. Alternatively, the biomolecule includes an antigen that specifically binds to an antibody (e.g., a monoclonal antibody) to facilitate detection and / or separation of host cells that display the antigen on their cell surface. In another example, the biomolecule includes a ligand-binding portion of a receptor, or a receptor-binding portion of a ligand.

[0097] compound Biomolecules containing phenolic or catechol moieties In certain embodiments of the subject method, a biomolecule comprising a phenol or catechol moiety is provided having formula (I): TIFF2025029024000003.tif17128, in which Y 1 is a biomolecule, optionally comprising one or more moieties selected from an active small molecule, an affinity tag, a fluorophore, and a metal chelator; X 1 is selected from hydrogen and hydroxyl; L is an optional linker.

[0098] In certain embodiments of formula (I), X 1 is hydrogen, such that the biomolecule contains a phenol moiety. In another embodiment of formula (I), X 1 is a hydroxyl, so that the biomolecule contains a catechol moiety.

[0099] In some embodiments of formula (I), the phenol moiety is present on a tyrosine residue. In certain cases, the biomolecule comprising the phenol moiety of formula (I) has formula (IB) or (IC): TIFF2025029024000004.tif37128, wherein R 2 is selected from alkyl, and substituted alkyl; R 3 is selected from hydrogen, alkyl, substituted alkyl, peptide, and polypeptide.

[0100] In certain embodiments of the subject methods, the biomolecule comprising a phenol moiety or a catechol (e.g., of formula (I)) comprises a linker (e.g., as described herein). Suitable linkers include, but are not limited to, carboxylic acids, alkyl esters, aryl esters, substituted aryl esters, aldehydes, amides, aryl amides, alkyl halides, thioesters, sulfonyl esters, alkyl ketones, aryl ketones, substituted aryl ketones, halosulfonyl, nitrile, nitro, PEG, and peptide linkers.

[0101] Exemplary linkers for use in linking the phenol moiety to the subject biomolecule of interest include, in some embodiments, PEG linkers. As used herein, the term "PEG" refers to polyethylene glycol or modified polyethylene glycol. Modified polyethylene glycol polymers include methoxypolyethylene glycol and polymers that are unsubstituted or substituted at one end with alkyl, substituted alkyl, or functional groups (e.g., as described herein). Any convenient linking group can be utilized at the end of the PEG to connect the group to the moiety of interest, including, but not limited to, alkyl, aryl, hydroxyl, amino, acyl, acyloxy, carboxyl ester, and amide termini and / or substituents. In certain examples, the linker comprises more than one PEG unit, e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 PEG units. In certain examples, the linker comprises less than 10 PEG units, e.g., 9, 8, 7, 6, 5, 4, 3, 2, or 1 PEG unit. In certain cases, the linker consists of four or fewer PEG units.

[0102] In certain cases, the biomolecule comprising a phenol moiety has the formula (IA): TIFF2025029024000005.tif26128, wherein: Y 1 is a biomolecule that optionally includes one or more groups selected from an active small molecule, an affinity tag, a fluorophore, and a metal chelator. Each R 1 is independently selected from hydrogen, acyl, substituted acyl, alkyl, and substituted alkyl; X 1 is selected from hydrogen and hydroxyl; and L 1 is a linker selected from linear or branched alkyl, linear or branched substituted alkyl, polyethylene glycol (PEG), substituted PEG, and one or more peptides.

[0103] In certain embodiments, X 1is hydrogen, such that the compound of formula (IA) has the formula (IAa): The compound is TIFF2025029024000006.tif20128.

[0104] In certain embodiments of any of the above formulas (IA)-(IAa), at least one R 1 is hydrogen. In certain cases, both R 1 The R groups are hydrogen. In certain cases, one R 1 group is hydrogen, and the other R 1 The groups are selected from alkyl, substituted alkyl, acyl, and substituted acyl. In certain cases, one R 1 group is hydrogen, and the other R 1 The group is alkyl. In some cases, one of the R 1 group is hydrogen, and the other R 1 The group is a substituted alkyl. In certain cases, one R 1 group is hydrogen, and the other R 1 The group is acyl. In certain cases, one R 1 group is hydrogen, and the other R 1 The group is a substituted acyl. In some cases, the acyl group has the formula -C(O)R 4 and R 4 is a lower alkyl group, e.g., methyl, ethyl, propyl, butyl, pentyl, or hexyl. In some cases, the substituted acyl group has the formula -C(O)R 4 NH 2 and R 4 is a lower alkyl group, e.g., methyl, ethyl, propyl, butyl, pentyl, or hexyl. In some cases, the substituted acyl group has the formula -C(O)CH 2 NH 2 It is.

[0105] In certain embodiments of any of Formulas (IA)-(IAa), L 1 is a straight or branched alkyl. In certain cases, L 1 is a lower alkyl group such as methyl, ethyl, propyl, butyl, pentyl, or hexyl. 1is a substituted alkyl group. In certain cases, L 1 is a substituted lower alkyl group. In certain cases, L 1 is PEG or substituted PEG (e.g., as described herein). In certain other cases, L 1 is a peptide. In certain other cases, L 1 is a polypeptide. In certain cases, L 1 is a linear linker having a length of 1 to 12 atoms, e.g., a length of 1 to 10, 1 to 8, or 1 to 6 atoms, e.g., a length of 1, 2, 3, 4, 5, or 6 atoms. 1 is (C 1-6 ) alkyl linker or substituted (C 1-6 ) alkyl linker, optionally with an ester (-CO 2 In certain cases, the linker L is substituted with a heteroatom or linking functional group, such as -, amide (CONH), carbamate (OCONH), ether (-O-), thioether (-S-) and / or amino group (-NR-, where R is H or alkyl). 1 may contain a keto (C=O) group. In certain cases, the keto group can be combined with an amino, thiol, or ether group of the linker chain to provide an amide, ester, or thioester group bond.

[0106] In certain embodiments, the linking group L or L 1 is a cleavable linker, e.g., as described herein.

[0107] In certain embodiments, the biomolecule comprising a phenol or catechol moiety is represented by formula (ID): TIFF2025029024000007.tif23128, where Y 1 is a biomolecule, optionally including one or more groups selected from an active small molecule, an affinity tag, a fluorophore, and a metal chelator; X 1is selected from hydrogen and hydroxyl; and n is an integer from 0 to 20. In certain cases, n is 10 or less, e.g., 9, 8, 7, 6, 5, 4, 3, 2, 1, or 0. In certain cases, n is 5. In certain cases, n is 4. In certain cases, n is 3. In certain cases, n is 2. In certain cases, n is 1. In certain cases, n is 0.

[0108] In certain embodiments, n is 1, such that the compound of formula (ID) has the formula (IDa): The compound is TIFF2025029024000008.tif23128.

[0109] In certain embodiments of formula (ID) or (IDa), X 1 is hydrogen, such that the biomolecule contains a phenol moiety. In other embodiments of formula (ID) or (IDa), X 1 is a hydroxyl, so that the biomolecule contains a catechol moiety.

[0110] In certain cases, the compound of formula (IDa) has the formula (IDb): The compound is TIFF2025029024000009.tif17128.

[0111] Compounds of any of formulae (ID)-(IDb) may be prepared by reacting tyramine, or the corresponding phenol- or catechol-containing amine, with a biomolecule containing an N-hydroxysuccinimide (NHS) ester or maleimide group in a suitable solvent. For example, compounds of formula ( ... with an NHS-ester (Y 1 —NHS) with tyramine in dry dimethylformamide (DMF) to provide compound (IDb). TIFF2025029024000010.tif26148 Scheme 3

[0112] It will be understood that biomolecules containing phenol or catechol moieties (e.g., any of formulae (I)-(IDb)) can be prepared by any convenient method. Many general references are available that provide commonly known chemical synthesis schemes and conditions useful for synthesizing the subject phenol and catechol-containing moieties (see, for example, Smith and March, March's Advanced Organic Chemistry: Reactions, Mechanisms, and Structure, Fifth Edition, Wiley-Interscience, 2001; or Vogel, A Textbook of Practical Organic Chemistry, Including Qualitative Organic Analysis, Fourth Edition, New York: Longman, 1978). As disclosed herein, in certain cases, the subject phenol moieties are present in tyrosine residues. The tyrosine residues can be part of the biomolecule of interest. In other cases, the tyrosine moiety may be synthetically introduced into the biomolecule of interest. For example, if the biomolecule is a peptide or polypeptide, a tyrosine residue may be introduced by standard solid-phase Fmoc peptide chemistry (Fields GB, Noble RL. Solid phase peptide synthesis utilizing 9-fluorenylmethoxycarbonyl amino acids. Int J Pept Protein Res 35:161-214, 1990). In some cases, a phenol or catechol moiety is part of the unnatural (non-genetically encoded) amino acid that is introduced into the biomolecule of interest. For example, amber codon (TAG) suppression can be used to incorporate a non-genetically encoded amino acid residue that contains a phenol or catechol moiety.See, e.g., Chin et al. (2002) J. Am. Chem. Soc. 124:9026; Chin and Schultz (2002) Chem. Biol. Chem. 3:1135; Chin et al. (2002) Proc. Natl. Acad. Sci. USA 99:11020; US2015 / 0240249; and US2018 / 0171321. As another example, orthogonal RNA synthetases and / or orthogonal tRNAs can be used to introduce non-genetically encoded amino acids into biomolecules, where the non-genetically encoded amino acids include phenolic or catechol moieties.

[0113] In some embodiments of any one of formulas (I)-(IDb), the biomolecule of interest comprises one or more groups selected from an active small molecule, an affinity tag, a fluorophore, and a metal chelator. In certain cases, the fluorophore is a rhodamine dye. In certain cases, the fluorophore is a xanthene dye. In certain cases, the fluorophore is Oregon Green 488. In certain cases, the metal chelator is 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA, also known as tetraxetane). In certain cases, the affinity tag is a biotin moiety (e.g., as described herein).

[0114] In certain cases, biomolecules that contain a phenolic moiety are described by structures such as those shown in FIG.

[0115] Target molecules containing thiol moieties Molecules that contain thiol moieties and are suitable for use in the subject methods, and methods for generating thiol-containing molecules suitable for use in the subject methods, are well known in the art.

[0116] The target molecule may be naturally occurring or synthetically or recombinantly produced, and may be isolated, substantially purified, or present within the natural environment of the unmodified molecule on which the thiol-containing target molecule is based (e.g., on the cell surface or within a host animal, including a mammal, such as a murine host (e.g., rat, mouse), hamster, dog, cat, cow, pig, etc.). In some embodiments, the target molecule is present in an in vitro cell-free reaction. In other embodiments, the target molecule is present within a cell and / or is displayed on a cell surface. In many embodiments of interest, the target molecule is within a living cell; on the surface of a living cell; within an organism, e.g., a living multicellular organism. Suitable living cells include cells that are part of a living multicellular organism; cells isolated from a multicellular organism; immortalized cell lines, and the like.

[0117] A target molecule may be composed of D-amino acids, L-amino acids, or both, and may be further modified to contain other moieties, either naturally, synthetically, or recombinantly, For example, a target molecule may be a lipoprotein, glycoprotein, or other such modified protein.

[0118] Generally, a target molecule will contain at least one thiol moiety for reaction with a biomolecule comprising a reactive moiety according to the invention, but may contain two or more, three or more, five or more, ten or more thiol moieties. The number of thiol moieties that may be present on a target molecule will vary depending on the intended use of the reactive modified target molecule, the nature of the target molecule itself, and other considerations that will be readily apparent to one of skill in the art when practicing the methods disclosed herein.

[0119] Target molecules can be modified to include thiol moieties at the sites where they are desired to be bound to biomolecules that include reactive moieties.For example, when target molecules are peptides or polypeptides, target molecule substrates can be modified to include N-terminal thiol moieties, thereby producing the subject target peptides or polypeptides that include thiol moieties.It is understood that any convenient position on a peptide or polypeptide substrate can be modified to include thiol moieties, thereby producing the subject target peptides or polypeptides for use in the subject methods.

[0120] In certain embodiments, the target molecule comprising a thiol moiety is a CRISPR-Cas effector polypeptide.

[0121] In certain cases, the thiol moiety is present in a cysteine ​​residue. In certain cases, the cysteine ​​residue is native to the CRISPR-Cas effector polypeptide. In other cases, the cysteine ​​residue is introduced into the CRISPR-Cas effector polypeptide. For example, the cysteine ​​residue may be introduced by standard solid-phase Fmoc peptide chemistry (Fields GB, Noble RL Solid phase peptide synthesis utilizing 9-fluorenylmethoxycarbonyl amino acids. Int J Pept Protein Res 35:161-214,1990).

[0122] Modified target molecule In certain embodiments of the subject method, the modified target molecule produced is a molecule of formula (IV) or (IVA), or a combination thereof. Thus, an aspect of the present disclosure is a compound of formula (IV) or (IVA): TIFF2025029024000011.tif29128, wherein Y1 is a biomolecule and optionally comprises one or more moieties selected from an active small molecule, an affinity tag, a fluorophore, and a metal chelator; L is an optional linker; Y2 is a second biomolecule; and n is an integer from 1 to 3.

[0123] In certain embodiments of formula (IV) or (IVA), n is less than 3, e.g., 2 or 1. In certain cases, n is 2. In certain cases, n is 1. In certain cases, the subject modified target molecule is a compound of formula (IV). In certain cases, the subject modified target molecule is a compound of formula (IVA).

[0124] In some embodiments, n in the modified target molecule of formula (IV) is 1 and the compound has the formula (IV1)-(IV3): It is described by either TIFF2025029024000012.tif32146.

[0125] In some embodiments, n in the modified targeting molecule of formula (IV) is 2, and the compound has the formula (IV4)-(IV5): It is described by either TIFF2025029024000013.tif32128.

[0126] In some embodiments, the modified targeting molecule of formula (IVA) has n=1 and the compound has formula (IV1)-(IV3): It is described by either TIFF2025029024000014.tif32131.

[0127] In some embodiments, the modified targeting molecule of formula (IVA) has n=2 and the compound has formula (IV4)-(IV5): It is described by either TIFF2025029024000015.tif32128.

[0128] In certain embodiments, the modified target molecule comprises a linker (e.g., as described herein). Suitable linkers include, but are not limited to, carboxylic acids, alkyl esters, aryl esters, substituted aryl esters, aldehydes, amides, aryl amides, alkyl halides, thioesters, sulfonyl esters, alkyl ketones, aryl ketones, substituted aryl ketones, halosulfonyl, nitrile, nitro, and peptide linkers.

[0129] Exemplary linkers (Y 1 ) is, in some embodiments, -(CR 1 2 ) m NHC(O)-, where R 1 is selected from hydrogen, or a substituent (e.g., as described herein), and m is an integer from 1 to 20. Exemplary linkers can also include PEG or substituted PEG linkers, e.g., as described herein.

[0130] In certain embodiments, the linker is a cleavable linker, e.g., as described herein.

[0131] In certain embodiments, the modified target molecule has formula (IVB) or (IVC): TIFF2025029024000016.tif31128, where Y 1 is a biomolecule that optionally includes one or more groups selected from an active small molecule, an affinity tag, a fluorophore, and a metal chelator; 1 is independently selected from hydrogen, acyl, substituted acyl, alkyl, and substituted alkyl; Y 2 is a second biomolecule; L 1 is a linker selected from linear or branched alkyl, linear or branched substituted alkyl, polyethylene glycol (PEG), substituted PEG, and one or more peptides; and n is an integer from 1 to 3.

[0132] In certain embodiments of formula (IVB) or (IVC), n is less than 3, e.g., 2 or 1. In certain cases, n is 2. In certain cases, n is 1. In certain cases, the subject modified target molecule is a compound of formula (IVB). In certain cases, the subject modified target molecule is a compound of formula (IVC).

[0133] In some embodiments, n in the modified target molecule of formula (IVB) is 1 and the compound has the formula (IVB1)-(IVB3): It is described by either TIFF2025029024000017.tif34137.

[0134] In some embodiments, n in the modified targeting molecule of formula (IVB) is 2 and the compound has the formula (IVB4)-(IVB5): It is described by either TIFF2025029024000018.tif34128.

[0135] In some embodiments, n in the modified target molecule of formula (IVC) is 1 and the compound has the formula (IVC1)-(IVC3): It is described by either TIFF2025029024000019.tif34148.

[0136] In some embodiments, the modified targeting molecule of formula (IVC) has n=2 and the compound has formula (IVC4)-(IVC5): It is described by either TIFF2025029024000020.tif34128.

[0137] In certain embodiments of any of the above formulas (IVB)-(IVC5), at least one R 1 is hydrogen. In certain cases, both R 1 The R groups are hydrogen. In certain cases, one R 1 The group is hydrogen and the other R 1 The groups are selected from alkyl, substituted alkyl, acyl, and substituted acyl. In certain cases, one R 1group is hydrogen, and the other R 1 The group is alkyl. In some cases, one of the R 1 group is hydrogen, and the other R 1 The group is a substituted alkyl. In certain cases, one R 1 group is hydrogen, and the other R 1 The group is acyl. In certain cases, one R 1 group is hydrogen, and the other R 1 The group is a substituted acyl. In some cases, the acyl group has the formula -C(O)R 4 and R 4 is a lower alkyl group, e.g., methyl, ethyl, propyl, butyl, pentyl, or hexyl. In some cases, the substituted acyl group has the formula -C(O)R 4 NH 2 and R 4 is a lower alkyl group, e.g., methyl, ethyl, propyl, butyl, pentyl, or hexyl. In some cases, the substituted acyl group has the formula -C(O)CH 2 NH 2 It is.

[0138] In certain embodiments of any of Formulas (IVB)-(IVC5), L 1 is a straight or branched alkyl. In certain cases, L 1 is a lower alkyl group such as methyl, ethyl, propyl, butyl, pentyl, or hexyl. 1 is a substituted alkyl group. In certain cases, L 1 is a substituted lower alkyl group. In certain cases, L 1 is PEG or substituted PEG (e.g., as described herein). In certain other cases, L 1 is a peptide. In certain other cases, L 1 is a polypeptide. In certain cases, L 1 is a linear linker having a length of 1 to 12 atoms, e.g., a length of 1 to 10, 1 to 8, or 1 to 6 atoms, e.g., a length of 1, 2, 3, 4, 5, or 6 atoms. 1 is (C 1-6 ) alkyl linker or substituted (C1-6 ) alkyl linker, optionally with an ester (-CO 2 In certain cases, the linker L is substituted with a heteroatom or linking functional group, such as -, amide (CONH), carbamate (OCONH), ether (-O-), thioether (-S-) and / or amino group (-NR-, where R is H or alkyl). 1 may contain a keto (C=O) group. In certain cases, the keto group can be combined with an amino, thiol, or ether group of the linker chain to provide an amide, ester, or thioester group bond.

[0139] In certain embodiments, the linking group L 1 is a cleavable linker, e.g., as described herein.

[0140] In certain embodiments, the modified target molecule has the formula (IVD)-(IVG): TIFF2025029024000021.tif74128, where R 2 is selected from alkyl, and substituted alkyl; R 3 is selected from hydrogen, alkyl, substituted alkyl, peptide, and polypeptide; and n is an integer from 1 to 3.

[0141] In certain embodiments of any of formulae (IVD)-(IVG), n is less than 3, e.g., 2 or 1. In certain cases, n is 2. In certain cases, n is 1. In certain cases, the subject modified target molecule is a compound of formula (IVD). In certain cases, the subject modified target molecule is a compound of formula (IVE). In certain cases, the subject modified target molecule is a compound of formula (IVF). In certain cases, the subject modified target molecule is a compound of formula (IVG).

[0142] In certain embodiments, any of Formulas (IVD)-(IVG) has the following structure: It may have the relative stereochemical structure as shown in TIFF2025029024000022.tif64128.

[0143] In some embodiments, n in the modified targeting molecule of formula (IVD) is 1 and the compound has the formula (IVD1)-(IVD3): It is described by either TIFF2025029024000023.tif37138.

[0144] In some embodiments, n in the modified targeting molecule of formula (IVD) is 2 and the compound has the formula (IVD4)-(IVD5): It is described by either TIFF2025029024000024.tif39128.

[0145] In some embodiments, n in the modified target molecule of formula (IVE) is 1 and the compound has the formulas (IVE1)-(IVE3): It is described by either TIFF2025029024000025.tif37138.

[0146] In some embodiments, n in the modified targeting molecule of formula (IVE) is 2 and the compound has the formula (IVE4)-(IVE5): It is described by either TIFF2025029024000026.tif38128.

[0147] In some embodiments, n of the modified target molecule of formula (IVF) is 1 and the compound has the formula (IVF1)-(IVF3): It is described by either TIFF2025029024000027.tif37135.

[0148] In some embodiments, the modified target molecule of formula (IVF) has n=2 and the compound has formula (IVF4)-(IVF5): It is described by either TIFF2025029024000028.tif39128.

[0149] In some embodiments, n in the modified targeting molecule of formula (IVG) is 1 and the compound has the formula (IVG1)-(IVG3): It is described by either TIFF2025029024000029.tif37135.

[0150] In some embodiments, n in the modified targeting molecule of formula (IVG) is 2 and the compound has the formula (IVG4)-(IVG5): It is described by either TIFF2025029024000030.tif38128.

[0151] In certain embodiments of the targeting molecules described herein, R 2 is an alkyl group. In certain cases, L 2 is a substituted alkyl group. In certain cases, the alkyl group is a lower alkyl group, for example, methyl, ethyl, propyl, butyl, pentyl, or hexyl.

[0152] In certain embodiments of the targeting molecules described herein, R 3 is hydrogen. In certain cases, R 3 is an alkyl group. In certain cases, R 3 is a substituted alkyl group. In certain cases, the alkyl group is a lower alkyl group, e.g., methyl, ethyl, propyl, butyl, pentyl, or hexyl. In certain cases, R 3 is a peptide. In certain cases, R 3 is a polypeptide.

[0153] In certain embodiments, the modified target molecule has the formula (IVH) or (IVJ): TIFF2025029024000031.tif31128, where Y 1 is a biomolecule, optionally comprising one or more groups selected from an active small molecule, an affinity tag, a fluorophore, and a metal chelator; Y 2is a second biomolecule; n is an integer from 1 to 3; and m is an integer from 0 to 20. In certain cases, m is 10 or less, e.g., 9, 8, 7, 6, 5, 4, 3, 2, 1, or 0. In certain cases, m is 5. In certain cases, m is 4. In certain cases, m is 3. In certain cases, m is 2. In certain cases, m is 1. In certain cases, m is 0.

[0154] In certain embodiments of the subject methods, the modified target molecule has the formula (IVK) or (IVL): Described by TIFF2025029024000032.tif32128.

[0155] In certain embodiments of any of formulas (IVH)-(IVJ), n is less than 3, such as 2 or 1. In certain cases, n is 2. In certain cases, n is 1.

[0156] In some embodiments, n in the modified targeting molecule of formula (IVH) is 1 and the compound has the formula (IVH1)-(IVH3): It is described by either TIFF2025029024000033.tif29154.

[0157] In some embodiments, n in the modified targeting molecule of formula (IVH) is 2 and the compound has the formula (IVH4)-(IVH5): It is described by either TIFF2025029024000034.tif32128.

[0158] In some embodiments, n of the modified targeting molecule of formula (IVJ) is 1 and the compound has the formula (IVJ1)-(IVJ3): It is described by either TIFF2025029024000035.tif29152.

[0159] In some embodiments, the n of the modified targeting molecule of formula (IVJ) is 2 and the compound has the formula (IVJ4)-(IVJ5): It is described by either TIFF2025029024000036.tif32128.

[0160] In certain embodiments, the target molecule containing a thiol group is a CRISPR-Cas effector polypeptide (e.g., as described herein).

[0161] In certain embodiments of any one of formulas (IV)-(IVJ5), Y 1 is a polypeptide. In certain cases, Y 1 The polypeptide is selected from a fluorescent protein, an antibody, and an enzyme. In certain cases, the fluorescent protein is green fluorescent protein. Other suitable polypeptides are described elsewhere herein.

[0162] Cleavable Linkers Cleavable linkers that may be used in the subject molecules of interest include electrophilically cleavable linkers, nucleophilically cleavable linkers, photocleavable linkers, metal cleavable linkers, electrolytically cleavable, and linkers cleavable under reductive and oxidative conditions. In certain cases, the cleavable linker is cleaved under acidic conditions. In certain cases, the cleavable linker is cleaved by enzymes. In certain cases, the cleavable linker is a linker that is cleaved under reducing conditions. In certain cases, the cleavable linker is rapidly cleaved by glutathione reduction. In certain cases, the cleavable linker includes a disulfide bond. In certain cases, the cleavable linker is cleaved by a physical stimulus. In certain cases, the cleavable linker is photocleavable.

[0163] In certain cases, L or L 1is an acid labile linker. In certain cases, the linker cleaves at a pH of 6 or less, e.g., 6.0, 5.95, 5.9, 5.85, 5.8, 5.75, 5.7, 5.65, 5.6, 5.55, 5.5, 5.45, 5.4, 5.35, 5.3, 5.25, 5.2, 5.15, 5.1, 5.05, 5.0, 4.9, 4.85, 4.80, 4.75, 4.7, 4.65, 4.6, 4.55, 4.5 or less.

[0164] In certain cases, L or L 1 is a photocleavable linker. Suitable photocleavable linkers include ortho-nitrobenzyl-based linkers, phenacyl linkers, alkoxybenzoin linkers, chromarene complex linkers, NpSSMpact linkers, and pivaloyl glycol linkers, as described in Guillier et al. (Chem. Rev. 2000 1000:2091-2157).

[0165] In some cases, L or L 1 is a proteolytically cleavable linker.

[0166] Proteolytically cleavable linkers include alanine carboxypeptidase, Armillaria mellea astacin, bacterial leucyl aminopeptidase, cancer coagulant promoter, cathepsin B, clostripain, cytosolic alanyl aminopeptidase, elastase, endoproteinase Arg-C, enterokinase, gastricin, gelatinase, Gly-X carboxypeptidase, glycyl endopeptidase, human rhinovirus 3C protease, hypodermin C, IgA-specific serine endopeptidase, leucyl aminopeptidase, leucyl endopeptidase, lysC, lysosomal pro-X carboxypeptidase, lysyl aminopeptidase, methionyl aminopeptidase, and myxobacter er), nardilysin, pancreatic endopeptidase E, picornain 2A, picornain 3C, proendopeptidase, prolyl aminopeptidase, proprotein convertase I, proprotein convertase II, russerylisin, saccharopepsin, semenogelase, T-plasminogen activator, thrombin, tissue kallikrein, tobacco etch virus (TEV), togavirin, tryptophanyl aminopeptidase, U-plasminogen activator, V8, venombin A, venombin AB, and Xaa-pro aminopeptidase.

[0167] For example, the proteolytically cleavable linker may include a matrix metalloproteinase cleavage site, e.g., a cleavage site for an MMP selected from collagenase-1, -2, and -3 (MMP-1, -8, and -13), gelatinase A and B (MMP-2 and -9), stromelysin 1, 2, and 3 (MMP-3, -10, and -11), matrilysin (MMP-7), and membrane metalloproteinases (MT1-MMP and MT2-MMP). For example, the cleavage sequence of MMP-9 is Pro-XX-Hy (SEQ ID NO: 1054) (wherein X represents any residue; Hy, hydrophobic residue), such as Pro-XX-Hy-(Ser / Thr) (SEQ ID NO: 847), such as Pro-Leu / Gln-Gly-Met-Thr-Ser (SEQ ID NO: 848) or Pro-Leu / Gln-Gly-Met-Thr (SEQ ID NO: 849). Another example of a protease cleavage site is a plasminogen activator cleavage site, such as a uPA or tissue plasminogen activator (tPA) cleavage site. In some cases, the cleavage site is a furin cleavage site. Particular examples of cleavage sequences for uPA and tPA include sequences that include Val-Gly-Arg. Another example of a protease cleavage site that can be included in a proteolytically cleavable linker is the Tobacco Etch Virus (TEV) protease cleavage site, e.g., ENLYTQS (SEQ ID NO: 850), where the protease cleaves between glutamine and serine. The TEV protease has the general formula EX 1 X 2 YX 3 Q(G / S) linear amino acid sequence (SEQ ID NO:), 1 , X 2 , and X 3are any amino acid, with cleavage occurring between Q and G or Q and S. TEV protease cleavable linkers can include ENLYFQG (SEQ ID NO: 957); ENLYTQS (SEQ ID NO: 958); ENLYFQGGY (SEQ ID NO: 959); ENLYFQS (SEQ ID NO: 960), and the like. Another example of a protease cleavage site that can be included in a proteolytically cleavable linker is an enterokinase cleavage site, e.g., DDDDK (SEQ ID NO: 851), with cleavage occurring after the lysine residue. Another example of a protease cleavage site that can be included in a proteolytically cleavable linker is a thrombin cleavage site, e.g., LVPR (SEQ ID NO: 852). Additional suitable linkers that include a protease cleavage site include linkers that include one or more of the following amino acid sequences: LEVLFQGP (SEQ ID NO: 853) (a fusion protein comprising human rhinovirus 3C protease and glutathione-S-transferase) cleaved by PreScission protease; Walker et al. al. (1994) Biotechnol. 12:601; thrombin cleavage sites, e.g., CGLVPAGSGP (SEQ ID NO: 854); SLLKSRMVPNFN (SEQ ID NO: 855) or SLLIARRMPNFN (SEQ ID NO: 856) cleaved by cathepsin B; SKLVQASASGVN (SEQ ID NO: 857) or SSYLKASDAPDN (SEQ ID NO: 858) cleaved by Epstein-Barr virus protease; RPKPQQFFGLMN (SEQ ID NO: 859) cleaved by MMP-3 (stromelysin); MMP-7 ( SLRPLALWRSFN (SEQ ID NO: 860) cleaved by matrilysin; SPQGIAGQRNFN (SEQ ID NO: 861) cleaved by MMP-9; DVDERDVRGFASFL (SEQ ID NO: 862) cleaved by thermolysin-like MMPs; SLPLGLWAPNFN (SEQ ID NO: 863) cleaved by matrix metalloproteinase 2 (MMP-2); SLLIFRSWANFN (SEQ ID NO: 864) cleaved by cathepsin L; SGVVIATVIVIT (SEQ ID NO: 865) cleaved by cathepsin D;SLGPQGIWGQFN (SEQ ID NO: 866), cleaved by matrix metalloproteinase 1 (MMP-1); KKSPGRVVGGSV (SEQ ID NO: 867), cleaved by urokinase-type plasminogen activator; PQGLLGAPGILG (SEQ ID NO: 868), cleaved by membrane-type matrix metalloproteinase 1 (MT-MMP); HGPEGLRVGFYESDVMGRGHARLVHVEEPHT (SEQ ID NO: 869), cleaved by stromelysin 3 (or MMP-11), thermolysin, fibroblast collagenase, and stromelysin-1; GPQGLAGQRGIV (SEQ ID NO: 870), cleaved by collagenase-13 (collagenase-3); GGSGQRGRKALE (SEQ ID NO: 871), cleaved by tissue-type plasminogen activator (tPA); SLSALLSSDIFN (SEQ ID NO: 872), cleaved by human prostate-specific antigen; SLPRFKIIGGFN (SEQ ID NO: 873), cleaved by kallikrein (hK3); SLLGIAVPGNFN (SEQ ID NO: 874), cleaved by neutrophil elastase; and FFKNIVTPRTPP (SEQ ID NO: 875), cleaved by calpain (calcium-activated neutral protease).

[0168] In some cases, the linker contains a disulfide bond and is cleavable under reducing conditions, for example, using β-mercaptoethanol, cysteine-HCl, tris(2-carboxyethyl)phosphine hydrochloride, or another reducing agent.

[0169] In some cases, the linker comprises a dipeptide, such as a valine-citrulline dipeptide or a valine-lysine dipeptide.

[0170] Biomolecules Biomolecules suitable for use in the methods or conjugates of the present disclosure include polypeptides, polynucleotides, carbohydrates, lipids, fatty acids, steroids, purines, pyrimidines, derivatives, structural analogs thereof, and combinations thereof.

[0171] Suitable biomolecules include, but are not limited to, polypeptides, nucleic acids, glycoproteins, small molecules, carbohydrates, lipids, glycolipids, lipoproteins, lipopolysaccharides, sugars, amino acids, organic dyes, synthetic polymers, and the like.

[0172] Suitable lipids include, for example, 3-N-[(methoxypoly(ethylene glycol)2000)carbamoyl]-1,2-dimyristyloxypropylamine (PEG-C-DMA), 1,2-dilinoleyloxy-N,N-dimethyl-3-aminopropane (DLinDMA), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), cholesterol, dipalmitoylphosphatidylcholine, 3-N-[(w-methoxypoly(ethylene glycol)2000)carbamoyl]-1,2-dimyristyloxypropylamine, 1,2-dilinoleyloxy-3-N,N-dimethylaminopropane, 1,2-distearoyl-sn-glycero-3-phosphocholine, PEG-cDMA, 1,2-dilinoleyloxy-3-(N;N-dimethyl)aminopropane (DLinDMA), ), 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA), etc.

[0173] Suitable biomolecules include affinity moieties. Suitable affinity moieties include His5 (HHHHH) (SEQ ID NO: 876); HisX6 (HHHHHH) (SEQ ID NO: 877); c-myc (EQKLISEEDL) (SEQ ID NO: 878); Flag (DYKDDDDK) (SEQ ID NO: 879); Strep tag (WSHPQFEK) (SEQ ID NO: 880); hemagglutinin, e.g., HA tag (YPYDVPDYA) (SEQ ID NO: 881); glutathione-S-transferase (GST); thioredoxin; cellulose binding domain, RYIRS (SEQ ID NO: 882); Phe-His-His-Thr ( SEQ ID NO: 883); chitin binding domain; S-peptide; T7 peptide; SH2 domain; C-terminal RNA tag, WEAAAREACCRECCARA (SEQ ID NO: 884); metal binding domain, e.g., zinc binding domain or calcium binding domain, such as from calcium binding proteins, e.g., calmodulin, troponin C, calcineurin B, myosin light chain, recoverin, S-modulin, visinin, VILIP, neurocalcin, hippocalcin, flekenin, caltractin, calpain large subunit. S100 proteins. Parvalbumin, calbindin D9K, calbindin D28K, and calretinin; biotin; streptavidin; MyoD; leucine zipper polypeptide; and maltose binding protein. In some cases, the preferred biomolecule is biotin.

[0174] In some cases, a suitable biomolecule for complexing with a target polypeptide is a dimerization domain. Non-limiting examples of suitable dimerization domains include the following dimerization pairs of polypeptides: a) FK506 binding protein (FKBP) and FKBP; b) FKBP and calcineurin catalytic subunit A (CnA); c) FKBP and cyclophilin; d) FKBP and FKBP-rapamycin-related protein (FRB); e) gyrase B (GyrB) and GyrB; f) dihydrofolate reductase (DHFR) and DHFR; g) DmrB and DmrB; h) PYL and ABI; i) Cry2 and CIB1; and j) GAI and GID1.

[0175] For example, in some cases, the biomolecule is a polypeptide having at least about 85%, at least about 90%, at least about 95%, at least about 98%, or 100% amino acid sequence identity to the following amino acid FKBP amino acid sequence: MGVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDSSRDRNKPFKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELLKLE (SEQ ID NO: 885).

[0176] In some cases, a biomolecule suitable for complexing with a target polypeptide is a member of a specific binding pair, including, for example, i) antibody-antigen; ii) cell adhesion molecule-extracellular matrix; iii) ligand-receptor; iv) biotin-avidin, etc.

[0177] Suitable synthetic polymers include polyalkylenes, such as polyethylene and polypropylene and polyethylene glycol (PEG); polychloroprene; polyvinyl ethers, such as poly(vinyl acetate); polyvinyl halides, such as poly(vinyl chloride); polysiloxanes; polystyrene; polyurethanes; polyacrylates, such as poly(methyl(meth)acrylate), poly(ethyl(meth)acrylate), poly(n-butyl(meth)acrylate), poly(isobutyl(meth)acrylate), poly(tert-butyl(meth)acrylate), poly(hexyl(meth)acrylate), poly(methyl(meth)acrylate), poly(ethyl ... Polyacrylamides, such as, but not limited to, poly(isodecyl (meth)acrylate), poly(lauryl (meth)acrylate), poly(phenyl (meth)acrylate), poly(methyl acrylate), poly(isopropyl acrylate), poly(isobutyl acrylate), and poly(octadecyl acrylate); polyacrylamides, such as poly(acrylamide), poly(methacrylamide), poly(ethyl acrylamide), poly(ethyl methacrylamide), poly(N-isopropyl acrylamide), poly(n, iso, and tert-butyl acrylamide); and copolymers and mixtures thereof.

[0178] In some cases, the biomolecule that is conjugated to the target polypeptide is a polypeptide. Suitable polypeptides include, for example, fluorescent proteins, receptors, enzymes, structural proteins, affinity tags, and the like.

[0179] Suitable fluorescent proteins include green fluorescent protein (GFP) or variants thereof, blue fluorescent variants of GFP (BFP), cyan fluorescent variants of GFP (CFP), yellow fluorescent variants of GFP (YFP), enhanced GFP (EGFP), enhanced CFP (ECFP), enhanced YFP (EYFP), GFPS65T, Emerald, Topaz (TYFP), Venus, Citrine, mCitrine, GFPuv, destabilized EGFP (dEGFP), destabilized ECFP (dECFP), destabilized EYFP (dEYFP), mCFPm, Cerulean, T-Sapphire, CyPet, YPet, mKO, HcRed, t-HcRed, DsRed, DsRed2, DsRed-monomer, J-Red, dimer2, t-dimer2(12), mRFP1, pocilloporin, Renilla GFP, Monster GFP, paGFP, Kaede protein, and kindling. Examples of fluorescent proteins include, but are not limited to, phycobiliproteins, phycobiliproteins, and phycobiliprotein complexes including B-phycoerythrin, R-phycoerythrin, and allophycocyanin. Other examples of fluorescent proteins include mHoneydew, mBanana, mOrange, dTomato, tdTomato, mTangerine, mStrawberry, mCherry, mGrape1, mRaspberry, mGrape2, mPlum (Shaner et al. (2005) Nat. Methods 2:905-909), and the like. Any of a variety of fluorescent and chromatic proteins from anthozoan species, such as those described in Matz et al. (1999) Nature Biotechnol. 17:969-973, are suitable for use.

[0180] In some cases, the biomolecule is an antibody. Suitable antibodies are described elsewhere herein. The antibody can be any antigen-binding antibody-based polypeptide, and a wide variety of antibodies are known in the art. In some examples, the antibody is a single chain Fv (scFv). Other antibody-based antigen-binding domains are suitable for use, including cAb VHH (camelized antibody variable domain) and humanized versions, IgNAR VH (shark antibody variable domain) and humanized versions, sdAb VH (single domain antibody variable domain) and "camelized" antibody variable domains. In some examples, T cell receptor (TCR)-based recognition domains, such as single chain TCRs (scTv, single chain two domain TCRs including VαVβ), are also suitable for use.

[0181] The antibody can be specific for an antigen, such as CD19, CD20, CD38, CD30, Her2 / neu, ERBB2, CA125, MUC-1, prostate specific membrane antigen (PSMA), CD44 surface adhesion molecule, mesothelin, carcinoembryonic antigen (CEA), epidermal growth factor receptor (EGFR), EGFRvIII, vascular endothelial growth factor receptor-2 (VEGFR2), high molecular weight melanoma associated antigen (HMW-MAA), MAGE-A1, IL-13R-a2, GD2, etc. In some cases, the antibody is specific for a cytokine. In some cases, the antibody is specific for a cytokine receptor. In some cases, the antibody is specific for a growth factor. In some cases, the antibody is specific for a growth factor receptor. In some cases, the antibody is specific for a cell surface receptor. In some cases, the antibody is an anti-CD3 antibody.

[0182] In some cases, both the target molecule and the biomolecule are antibodies. In some cases, the target molecule is a first antibody specific to a first antigen, and the biomolecule is a second antibody specific to a second antigen. The first and second antigens can be completely separate molecules. For example, the first antigen can be a first polypeptide, and the second antigen can be a second polypeptide. The first antigen can be a first epitope presented by an antigen, and the second antigen can be a second epitope presented by the same antigen. The resulting complex can be a bispecific antibody.

[0183] In some cases, the biomolecule confers properties to the target biomolecule, such as: i) increased serum half-life; ii) increased immunogenicity; iii) enhanced pharmacokinetic properties; iv) increased transport across the blood-brain barrier, etc. For example, in some cases, the biomolecule that extends serum half-life is human serum albumin. In some cases, the biomolecule that extends serum half-life is an albumin binding domain. In some cases, the biomolecule that extends serum half-life is transthyretin. In some cases, the biomolecule that extends serum half-life is a thyroxine binding protein. In some cases, the biomolecule is an immunoglobulin Fc polypeptide. In some cases, the biomolecule that enhances transport across the blood-brain barrier is a transferrin receptor (TR), insulin receptor (HIR), insulin-like growth factor receptor (IGFR), low density lipoprotein receptor-related protein 1 and 2 (LPR-1 and 2), diphtheria toxin receptor, llama single domain antibody, protein transduction domain, TAT, penetratin, or a polyarginine peptide.

[0184] Suitable biomolecules include small molecules such as cancer chemotherapeutic agents. Suitable cancer chemotherapeutic agents include, for example, alkylating agents such as nitrogen mustards (e.g., chlorambucil, chlormethine, cyclophosphamide, ifosfamide, and melphalan); nitrosoureas (e.g., carmustine, fotemustine, lomustine, and streptozocin); platinum compounds (e.g., carboplatin, cisplatin, oxaliplatin, and BBR3464); busulfan; dacarbazine; mechlorethamine; procarbazine; temozolomide; thiotepa; uramustine; phenylalanine ... antagonists, such as folates (e.g., methotrexate, pemetrexed, and raltitrexate); purines (e.g., cladribine, clofarabine, fludarabine, mercaptopurine, and thioguanine); pyrimidines (e.g., capecitabine); cytarabine; fluorouracil; gemcitabine; plant alkaloids, such as podophyllum (e.g., etoposide, and teniposide), taxanes (e.g., docetaxel and paclitaxel), vincas (e.g., vinblastine, vincristine, vindesine, and vinorelbine); cytotoxic / antitumor antibiotics such as anthracycline family members (e.g., daunorubicin, doxorubicin, epirubicin, idarubicin, mitoxantrone, and barbicin), bleomycin, rifampicin, hydroxyurea, and mitomycin; topoisomerase inhibitors such as topotecan and irinotecan; photosensitizers such as aminolevulinic acid, methyl aminolevulinate, porfimer sodium, and verteporfin; and other agents such as , alitretinoin, altretamine, amsacrine, anagrelide, arsenic trioxide, asparaginase, axitinib, bexarotene, bevacizumab, bortezomib, celecoxib, denileukin diftitox, erlotinib, estramustine, gefitinib, hydroxycarbamide, imatinib, lapatinib, pazopanib, pentostatin, masoprocol, mitotane, pegaspargase, tamoxifen, sorafenib, sunitinib, vemurafenib, vandetanib, and tretinoin. For example, in some cases, the target molecule is an antibody; the biomolecule is a cancer chemotherapeutic agent.

[0185] Suitable biomolecules include cytokines, chemokines, peptide hormones, etc. Suitable biomolecules include, for example, interferons (e.g., IFN-γ); interleukins (e.g., IL-1α, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-7, IL-9, IL-10, IL-12p40, IL-12p70, IL-13, IL-15, IL-17, etc.); IP-10, KC, MCP-1, MIP-1α, MIP-1β, M-CSF, etc. These include MIP-2, MIG; α chemokines (e.g., CXC chemokines; e.g., CXC-1 to CXC-17); β chemokines (CC chemokines), e.g., RANTES or CCL20 (also known as MIP-3α); tumor necrosis factor-α (TNF-α); eotaxin; granulocyte colony-stimulating factor (G-CSF); granulocyte-macrophage-colony-stimulating factor (GM-CSF); erythropoietin; insulin; Gro-α; Groβ; Gro-γ; stromal-derived factors; platelet-derived growth factor (PDGF); vascular endothelial growth factor (VEGF); insulin-like growth factor (IGF); fibroblast growth factor (FGF); epidermal growth factor (EGF); leukemia inhibitory factor (LIF); hepatocyte growth factor (HGF); thrombopoietin, and the like.

[0186] Suitable biomolecules include nucleic acids. In some cases, the nucleic acid is a DNA molecule. In some cases, the nucleic acid is an RNA molecule. In some cases, the nucleic acid includes both deoxyribonucleotides and ribonucleotides. In some cases, the nucleic acid is a single-stranded DNA molecule. In some cases, the nucleic acid is a double-stranded DNA molecule. In some cases, the nucleic acid is a single-stranded RNA molecule. Suitable nucleic acids include, for example, small interfering RNA (siRNA), short hairpin RNA (shRNA), microRNA (miRNA), ribozymes, and the like. Suitable nucleic acids include nucleic acids that are or act as siRNA or other RNA interference agents (RNAi agents or iRNA agents), shRNA, antisense oligonucleotides, self-cleaving RNA, ribozymes, fragments and / or variants thereof (e.g., peptidyl transferase 23S rRNA, RNase P, group I and group II introns, GIR1 branched ribozyme, leadzyme, hairpin ribozyme, hammerhead ribozyme, HDV ribozyme, mammalian CPEB3 ribozyme, VS ribozyme, glmS ribozyme, CoTC ribozyme, etc.), microRNA, microRNA mimics, supermir, aptamers, antimir, antagomir, Ul adaptor, triplex forming oligonucleotides, RNA activators, long non-coding RNA, short non-coding RNA (e.g., piRNA), immune modulatory oligonucleotides (e.g., immune stimulatory oligonucleotides, immune suppressive oligonucleotides), GNA, LNA, ENA, PNA, TNA, HNA, TNA, XNA, HeNA, CeNA, morpholino, G-quadruplexes (RNA and DNA), antiviral oligonucleotides, and decoy oligonucleotides. The nucleic acids can be of any length and can contain one or more of modified ribonucleotide bases, modified deoxyribonucleotide bases, modified deoxyribose, modified ribose, and modified backbone linkages (eg, phosphorothioate linkages).

[0187] Biomolecules for complexing with CRISPR-Cas effector polypeptides In some cases, the biomolecule that is complexed to the target polypeptide is a biomolecule suitable for complexing to a CRISPR-Cas effector polypeptide.

[0188] In some cases, a biomolecule suitable for complexing to a CRISPR-Cas effector polypeptide is a biomolecule that can modulate (e.g., inhibit transcription, increase transcription) transcription of a target DNA. For example, in some cases, the biomolecule is a protein (or a protein-derived domain) that inhibits transcription (e.g., a transcription repressor, i.e., a protein that functions via recruitment of transcription inhibitory proteins, modification of target DNA such as methylation, recruitment of DNA modifiers, regulation of histones associated with target DNA, recruitment of histone modifiers such as factors that modify histone acetylation and / or methylation, etc.). In some cases, the biomolecule is a protein (or a protein-derived domain) that increases transcription (e.g., a transcription activator, i.e., a protein that functions via recruitment of transcription activator proteins, modification of target DNA such as demethylation, recruitment of DNA modifiers, regulation of histones associated with target DNA, recruitment of histone modifiers such as factors that modify histone acetylation and / or methylation, etc.).

[0189] In some cases, a biomolecule suitable for binding to a CRISPR-Cas effector polypeptide is a polypeptide having an enzymatic activity that modifies a target nucleic acid (e.g., a nuclease activity such as FokI nuclease activity, a methyltransferase activity, a demethylase activity, a DNA repair activity, a DNA damage activity, a deamination activity, a dismutase activity, an alkylation activity, a depurination activity, an oxidation activity, a pyrimidine dimer formation activity, an integrase activity, a transposase activity, a recombinase activity, a polymerase activity, a ligase activity, a helicase activity, a photolyase activity, or a glycosylase activity).

[0190] In some cases, a biomolecule suitable for conjugation to a CRISPR-Cas effector polypeptide is a polypeptide that has an enzymatic activity (e.g., methyltransferase activity, demethylase activity, acetyltransferase activity, deacetylase activity, kinase activity, phosphatase activity, ubiquitin ligase activity, deubiquitinating activity, adenylating activity, deadenylating activity, SUMOylating activity, deSUMOylating activity, ribosylation activity, deribosylation activity, myristoylating activity or demyristoylating activity) that modifies a polypeptide (e.g., a histone) associated with a target nucleic acid.

[0191] Examples of proteins (or fragments thereof) that can be used to increase transcription and are suitable as biomolecules for conjugation to CRISPR-Cas effector polypeptides include transcription activators such as VP16, VP64, VP48, VP160, the p65 subdomain (e.g., from NFkB), and the activation domain of EDLL, and / or the TAL activation domain (e.g., for activity in plants); histone lysine methyltransferases such as SET1A, SET1B, MLL1-5, ASH1, SYMD2, NSD1; histone lysine demethylases such as JHDM2a / b, UTX, JMJD3; histone acetyltransferases such as GCN5, PCAF, CBP, p300, TAF1, TIP60 / PLIP, MOZ / MYST3, MORF / MYST4, SRC1, ACTR, P160, CLOCK; and Ten-Eleven Examples of DNA demethylases include, but are not limited to, DNA translocation (TET) dioxygenase 1 (TET1CD), TET1, DME, DML1, DML2, and ROS1.

[0192] Examples of proteins (or fragments thereof) that can be used to reduce transcription and that are suitable as biomolecules for conjugation to a CRISPR-Cas effector polypeptide include transcriptional repressors such as Kruppel-associated box (KRAB or SKD); the KOX1 repression domain; mSIN3 interacting domain (SID); ERF repression domain (ERD), SRDX repression domain (e.g., for repression in plants), etc.; histone lysine methyltransferases such as Pr-SET7 / 8, SUV4-20H1, RIZ1; histone lysine demethylases such as JMJD2A / JHDM3A, JMJD2B, JMJD2C / GASC1, JMJD2D, JARID1A / RBP2, JARID1B / PLU-1, JARID1C / SMCX, JARID1D / SMCY; histone lysine deacetylases such as HDAC1, HDAC2, HDAC3, HDAC8, HDAC4, HDAC5, HDAC7, HDAC9, SIRT1, SIRT2, HDAC11; HhaI DNA These include, but are not limited to, DNA methylases such as m5c-methyltransferase (M.HhaI), DNA methyltransferase 1 (DNMT1), DNA methyltransferase 3a (DNMT3a), DNA methyltransferase 3b (DNMT3b), METI, DRM3 (plants), ZMET2, CMT1, CMT2 (plants); and peripheral mobilization elements such as Lamin A and Lamin B.

[0193] In some cases, the biomolecule complexed to the CRISPR-Cas effector polypeptide has an enzymatic activity that modifies a target nucleic acid (e.g., ssRNA, dsRNA, ssDNA, dsDNA). Examples of enzymatic activities that can be provided by the biomolecule include nuclease activity, such as that provided by a restriction enzyme (e.g., FokI nuclease), methyltransferase activity, such as that provided by a methyltransferase (e.g., HhaI DNA m5c-methyltransferase (M.HhaI), DNA methyltransferase 1 (DNMT1), DNA methyltransferase 3a (DNMT3a), DNA methyltransferase 3b (DNMT3b), METI, DRM3 (plants), ZMET2, CMT1, CMT2 (plants), etc.); demethylase (e.g., Ten-Eleven demethylase activity, such as that provided by TET dioxygenase 1 (TET1CD), TET1, DME, DML1, DML2, ROS1, etc.), DNA repair activity, DNA damage activity, deamination activity, such as that provided by deaminases (e.g., cytosine deaminase enzymes such as rat APOBEC1), dismutase activity, alkylation activity, depurination activity, oxidation activity, pyrimidine dimer formation activity, integrases and / or resolvases (e.g., G These include, but are not limited to, integrase activity such as that provided by a hyperactive mutant of Gin invertase, Gin invertase such as GinH106Y; human immunodeficiency virus type 1 integrase (IN); Tn3 resolvase, etc.; transposase activity; recombinase activity such as that provided by a recombinase (e.g., the catalytic domain of Gin recombinase); polymerase activity, ligase activity, helicase activity, photolyase activity, and glycosylase activity.

[0194] In some cases, the biomolecule that is complexed to the CRISPR-Cas effector polypeptide has an enzymatic activity that modifies a protein (e.g., histone, RNA-binding protein, DNA-binding protein, etc.) associated with a target nucleic acid (e.g., ssRNA, dsRNA, ssDNA, dsDNA).Examples of enzymatic activities (which modify proteins associated with target nucleic acids) that can be provided by biomolecules include methyltransferases (HMTs) (e.g., methyltransferases such as those provided by suppressor of variegation 3-9 homolog 1 (SUV39H1, also known as KMT1A), euchromatin histone lysine methyltransferase 2 (G9A, also known as KMT1C and EHMT2), SUV39H2, ESET / SETDB1, etc., SET1A, SET1B, MLL1-5, ASH1, SYMD2, NSD1, DOT1L, Pr-SET7 / 8, SUV4-20H1, EZH2, RIZ1). histone acetyltransferase activity, demethylase activity such as that provided by histone demethylases (e.g., lysine demethylase 1A (KDM1A, also known as LSD1), JHDM2a / b, JMJD2A / JHDM3A, JMJD2B, JMJD2C / GASC1, JMJD2D, JARID1A / RBP2, JARID1B / PLU-1, JARID1C / SMCX, JARID1D / SMCY, UTX, JMJD3, etc.), histone acetyltransferase activity, acetyltransferase activity, such as that provided by enzyme transferases (e.g., catalytic cores / fragments of human acetyltransferases p300, GCN5, PCAF, CBP, TAF1, TIP60 / PLIP, MOZ / MYST3, MORF / MYST4, HBO1 / MYST2, HMOF / MYST1, SRC1, ACTR, P160, CLOCK, etc.), histone deacetylases (e.g., HDAC1, HDAC2, HDAC3, These include, but are not limited to, deacetylase activity, such as that provided by HDAC8, HDAC4, HDAC5, HDAC7, HDAC9, SIRT1, SIRT2, HDAC11, etc., kinase activity, phosphatase activity, ubiquitin ligase activity, deubiquitinating activity, adenylating activity, deadenylating activity, sumoylating activity, desumoylating activity, ribosylation activity, deribosylation activity, myristoylating activity, and demyristoylating activity.

[0195] In some cases, the biological molecule that is complexed with CRISPR-Cas effector polypeptide is a catalytically active endonuclease.For example, in some cases, the target polypeptide is a CRISPR-Cas effector polypeptide that is catalytically inactive (e.g., does not show endonuclease activity) and retains target nucleic acid binding activity (when complexed with guide RNA); the biological molecule that is complexed with CRISPR-Cas effector polypeptide is a catalytically active endonuclease.For example, in some cases, the catalytically active endonuclease is a FokI polypeptide. As a non-limiting example, in some cases, the biomolecule complexed to the CRISPR-Cas effector polypeptide is a FokI nuclease comprising an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the FokI amino acid sequence shown below; the length of the FokI nuclease is about 195 amino acids to about 200 amino acids.

[0196] FokI nuclease amino acid sequence: QLVKSELEEKKSELRHKLKYVPHEYIELIEIARNSTQDRILEMKVMEFFMKVYGYRGKHLGGSRKPDGAIYTVGSPIDYGVIVDTKAYSGGYNLPIGQADEMQRYVEENQTRNKHINPNEWWKVYPSSVTEFKFLFVSGHFKGNYKAQLTRLNHITNCNGAVLSVEELLIGGEMIKAGTLTLEEVRRKFNNGEINF (SEQ ID NO: 886).

[0197] In some cases, the biomolecule that is complexed to the CRISPR-Cas effector polypeptide is a deaminase. In some cases, the target CRISPR / Cas effector polypeptide is catalytically inactive. Suitable deaminases include cytidine deaminase and adenosine deaminase.

[0198] A suitable adenosine deaminase is any enzyme capable of deaminating adenosine in DNA. In some cases, the deaminase is TadA deaminase.

[0199] In some cases, a suitable adenosine deaminase has the following amino acid sequence: MSEVEFSHEYWMRHALTLAKRAWDEREVPVGAVLVHNNRVIGEGWNRPIGRHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTLEPCVMCAGAMIHSRIGRVVFGARDAKTGAAGSLMDVLHHPGMNHRVEITEGILADECAALLSDFFRMRRQEIKAQKKAQSSTD (SEQ ID NO: 887) The amino acid sequence may have at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to

[0200] In some cases, a suitable adenosine deaminase has the following amino acid sequence: MRRAFITGVFFLSEVEFSHEYWMRHALTLAKRAWDEREVPVGAVLVHNNRVIGEGWNRPIGRHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTLEPCVMCAGAMIHSRIGRVVFGARDAKTGAAGSLMDVLHHPGMNHRVEITEGILADECAALLSDFFRMRRQEIKAQKKAQSSTD (SEQ ID NO: 888) The amino acid sequence may have at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to

[0201] In some cases, a suitable adenosine deaminase has the following Staphylococcus aureus TadA amino acid sequence: MGSHMTNDIYFMTLAIEEAKKAAQLGEVPIGAIITKDDEVIARAHNLRETLQQPTAHAEHIAIERAAKVLGSWRLEGCTLYVTLEPCVMCAGTIVMSRIPRVVYGADDPKGGCSGSLMNLLQQSNFNHRAIVDKGVLKEACSTLLTTFFK NLRANKKSTN: (SEQ ID NO: 889) The amino acid sequence may have at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to

[0202] In some cases, a suitable adenosine deaminase has the following Bacillus subtilis TadA amino acid sequence: MTQDELYMKEAIKEAKKAEEKGEVPIGAVLVINGEIIARAHNLRETEQRSIAHAEMLVIDEACKALGTWRLEGATLYVTLEPCPMCAGAVVLSRVEKVVFGAFDPKGGCSGTLMNLLQEERFNHQAEVVSGVLEEECGGMLSAFFRELRKKKKAARKNLSE (SEQ ID NO: 890) The amino acid sequence may have at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to

[0203] In some cases, a suitable adenosine deaminase has the following Salmonella typhimurium TadA amino acid sequence: MPPAFITGVTSLSDVELDHEYWMRHALTLAKRAWDEREVPVGAVLVHNHRVIGEGWNRPIGRHDPTAHAEIMALRQGGLVLQNYRLLDTTLYVTLEPCVMCAGAMVHSRIGRVVFGARDAKTGAAGSLIDVLHHPGMNHRVEIIEGVLRDECATLLSDFFRMRRQEIKALKKADRAEGAGPAV (SEQ ID NO: 891) The amino acid sequence may have at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to

[0204] In some cases, a suitable adenosine deaminase has the following Shewanella putrefaciens TadA amino acid sequence: MDEYWMQVAMQMAEKAEAAGEVPVGAVLVKDGQQIATGYNLSISQHDPTAHAEILCLRSAGKKLENYRLLDATLYITLEPCAMCAGAMVHSRIARVVYGARDEKTGAAGTVVNLLQHPAFNHQVEVTSGVLAEACSAQLSRFFKRRRDEKKALKLAQRAQQGIE (SEQ ID NO: 892) The amino acid sequence may have at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to

[0205] In some cases, a suitable adenosine deaminase has the following Haemophilus influenzae F3031 TadA amino acid sequence: MDAAKVRSEFDEKMMRYALELADKAEALGEIPVGAVLVDDARNIIGEGWNLSIVQSDPTAHAEIIALRNGAKNIQNYRLLNSTLYVTLEPCTMCAGAILHSRIKRLVFGASDYKTGAIGSRFHFFDDYKMNHTLEITSGVLAEECSQKLS TFFQKRREEKKIEKALLKSLSDK (sequence number 893) The amino acid sequence may have at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to

[0206] In some cases, a suitable adenosine deaminase has the following Caulobacter crescentus TadA amino acid sequence: MRTDESEDQDHRMMRLALDAARAAAEAGETPVGAVILDPSTGEVIATAGNGPIAAHDPTAHAEIAAMRAAAAKLGNYRLTDLTLVVTLEPCAMCAGAISHARIGRVVFGADDPKGGAVVHGPKFFAQPTCHWRPEVTGGVLADESADLLRGFFRARRKAKI (SEQ ID NO: 894) The amino acid sequence may have at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to

[0207] In some cases, a suitable adenosine deaminase has the following Geobacter sulfurreducens TadA amino acid sequence: MSSLKKTPIRDDAYWMGKAIREAAKAAARDEVPIGAVIVRDGAVIGRGHNLREGSNDPSAHAEMIAIRQAARRSANWRLTGATLYVTLEPCLMCMGAIILARLERVVFGCYDPKGGAAGSLYDLSADPRLNHQVRLSPGVCQEECGTMLSDFFRDLRRRKKAKATPALFIDERKVPPEP (SEQ ID NO: 895) The amino acid sequence may have at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to

[0208] Cytidine deaminases suitable as biomolecules to be conjugated to a CRISPR-Cas effector polypeptide include any enzyme capable of deaminating cytidine in DNA.

[0209] In some cases, the cytidine deaminase is a deaminase from the apolipoprotein B mRNA editing complex (APOBEC) family of deaminases. In some cases, the APOBEC family deaminase is selected from the group consisting of APOBEC1 deaminase, APOBEC2 deaminase, APOBEC3A deaminase, APOBEC3B deaminase, APOBEC3C deaminase, APOBEC3D deaminase, APOBEC3F deaminase, APOBEC3G deaminase, and APOBEC3H deaminase. In some cases, the cytidine deaminase is an activation-induced deaminase (AID).

[0210] In some cases, a suitable cytidine deaminase has the following amino acid sequence: MDSLLMNRRKFLYQFKNVRWAKGRRETYLCYVVKRRDSATSFSLDFGYLRNKNGCHVELLFLRYISDWDLDPGRCYRVTWFTSWSPCYDCARHVADFLRGNPNLSLRIFTARLYFCEDRKAEPEGLRRLHRAGVQIAIMTFKDYFYCWNTFVENHERTFKAWEGLHENSVRLSRQLRRILLPLYEVDDLRDAFRTLGL (SEQ ID NO: 896) The amino acid sequence may have at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to

[0211] In some cases, a suitable cytidine deaminase is AID, which has the following amino acid sequence: MDSLLMNRRK FLYQFKNVRW AKGRRETYLC YVVKRRDSAT SFSLDFGYLR NKNGCHVELL FLRYISDWDL DPGRCYRVTW FTSWSPCYDC ARHVADFLRG NPNLSLRIFT ARLYFCEDRK AEPEGLRRLH RAGVQIAIMT FKENHERTFK AWEGLHENSV RLSRQLRRIL LPLYEVDDLRDAFRTLGL (sequence number 897) The amino acid sequence may have at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to

[0212] In some cases, a suitable cytidine deaminase is AID, which has the following amino acid sequence: MDSLLMNRRK FLYQFKNVRW AKGRRETYLC YVVKRRDSAT SFSLDFGYLR NKNGCHVELL FLRYISDWDL DPGRCYRVTW FTSWSPCYDC ARHVADFLRG NPNLSLRIFT ARLYFCEDRK AEPEGLRRLH RAGVQIAIMT FKDYFYCWNT FVENHERTFK AWEGLHENSV RLSRQLRRIL LPLYEVDDLR DAFRTLGL (sequence number 898) The amino acid sequence may have at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to

[0213] In some cases, the disclosed method for complexing a biomolecule to a CRISPR-Cas effector polypeptide is carried out in the presence of trehalose. The concentration of trehalose can be 25 mM to about 100 mM (e.g., 25 mM to 50 mM, 50 mM to 100 mM). For example, in some cases, the disclosed method for complexing a biomolecule to a CRISPR-Cas effector polypeptide is carried out under the following conditions: 20 mM Tris HCl, 300 mM KCl, 50 mM trehalose pH 7.0, 4° C., 1 hour; 10 μM CRISPR-Cas effector polypeptide.

[0214] target molecule Target molecules suitable for modification include, but are not limited to, polypeptides, polynucleotides, carbohydrates, lipids, glycolipids, glycopolypeptides, etc. Target molecules modified according to the methods of the present disclosure contain, or are modified to contain, a phenolic or catechol moiety.

[0215] In some cases, the target molecule is a polypeptide (a "target polypeptide").

[0216] Target polypeptides that can be modified using the methods of the present disclosure include, but are not limited to, enzymes, antibodies, structural polypeptides, ligands for receptors, receptors, etc. Target polypeptides include, but are not limited to, structural proteins; receptors; enzymes; cell surface proteins; proteins essential to the function of a cell; proteins involved in catalytic activity; proteins involved in motor activity; proteins involved in helicase activity; proteins involved in metabolic processes (anabolism and catabolism); proteins involved in antioxidant activity; proteins involved in protein degradation; proteins involved in biosynthesis; proteins with kinase activity; proteins with oxidoreductase activity; proteins with transferase activity; proteins with hydrolase activity; proteins with lyase activity; proteins with isomerase activity; proteins with ligase activity; proteins with enzyme regulatory activity; proteins with signaling activity; structural polypeptides; polypeptides with binding activity; receptor These may include somatic polypeptides; proteins involved in cell motility; proteins involved in membrane fusion; proteins involved in cell communication; proteins involved in the regulation of biological processes; proteins involved in development; proteins involved in cell differentiation; proteins involved in response to stimuli; behavioral proteins; cell adhesion proteins; proteins involved in cell death; proteins involved in transport (including protein transporter activity, nuclear transport, ion transporter activity, channel transporter activity, etc.); proteins involved in secretory activity; proteins involved in electron transporter activity; proteins involved in pathogenesis; proteins involved in chaperone regulatory activity; proteins with nucleic acid binding activity; proteins with transcriptional regulatory activity; proteins involved in extracellular organization; proteins involved in biosynthesis; proteins involved in translational regulation, etc.

[0217] In some cases, the target polypeptide is an antibody. The antibody can be any antigen-binding antibody-based polypeptide, and a wide variety of antibodies are known in the art. In some examples, the antibody is a single chain Fv (scFv). Other antibody-based antigen-binding domains (cAb VHH (camelized antibody variable domain) and humanized versions, IgNAR VH (shark antibody variable domain) and humanized versions, sdAb VH (single domain antibody variable domain) and "camelized" antibody variable domains are suitable for use. In some examples, T cell receptor (TCR)-based recognition domains such as single chain TCR (scTv, single chain two domain TCR including VαVβ) are also suitable for use.

[0218] The antibody can be specific for an antigen, such as CD19, CD20, CD38, CD30, Her2 / neu, ERBB2, CA125, MUC-1, prostate specific membrane antigen (PSMA), CD44 surface adhesion molecule, mesothelin, carcinoembryonic antigen (CEA), epidermal growth factor receptor (EGFR), EGFRvIII, vascular endothelial growth factor receptor-2 (VEGFR2), high molecular weight melanoma associated antigen (HMW-MAA), MAGE-A1, IL-13R-a2, GD2, etc. In some cases, the antibody is specific for a cytokine. In some cases, the antibody is specific for a cytokine receptor. In some cases, the antibody is specific for a growth factor. In some cases, the antibody is specific for a growth factor receptor. In some cases, the antibody is specific for a cell surface receptor. In some cases, the antibody is an anti-CD3 antibody.

[0219] In some cases, the antibodies are: 806, 9E10, 3F8, 81C6, 8H9, abagovomab, abatacept, abciximab, avituzumab, abrilumab, actoxumab, adalimumab, adecatuzumab, aducanumab, afelimomab, afutuzumab, alacizumab pegol, ALD518, alefacept, alemtuzumab, alirocumab, altumomab pentetate, amatuximab, AMG102, anatumomab mafenatox, anetumab Ravtansine, Anifrolumab, Anrukinzumab, Apolizumab, Arcitumomab, Asclinbacumab, Acelizumab, Atacicept, Atezolizumab, Atinumab, Atolizumab / Tocilizumab, Atollimumab, AVE1642, Bapineuzumab, Basiliximab, Bavituximab, Bectumomab, Begelomab, Belimumab, Benralizumab, Bertilimumab, Besilesomab, Bevacizumab, Bezlotoxumab, Biciromab, Bimagrumab, Bimekizumab, Bivatuzumab Mertansine, Blinatumomab, Brosozumab, BMS-936559, Bococizumab, Brentuximab Vedotin, briakinumab, brodalumab, brolucizumab, brontixituzumab, canakinumab, cantuzumab mertansine, cantuzumab ravtansine, caplacizumab, capromab pendetide, carlumab, catumaxomab, cBR96-doxorubicin immunoconjugate, CC49, CDP791, cedelizumab, certolizumab pegol, cetuximab, cG250, Ch.14.18, sitatuzumab bogatox, cixutumumab, clazakizumab, clenoliximab, clivatuzumab tetraxetan, codrituzumab, coltuximab Ravtansine, conatumumab, concizumab, CP751871, CR6261, crenezumab, CS-1008, dacetuzumab, daclizumab, dalotuzumab, dapirorizumab pegol, daratumumab, dectrecumab, demcizumab, denintuzumab mafodotin, denosumab, delrotuximab biotin, detumomab, dinutuximab, dilidabumab, dorlimomabAritux, drotizumab, durigotumab, dupilumab, durvalumab, dusigitumab, ecromeximab, eculizumab, edovacomab, edrecolomab, efalizumab, efalizumab, eldelumab, elgemtumab, elotuzumab, ersilimomab, emactuzumab, emibetuzumab, enabatuzumab, enfortumab vedotin, enlimomab pegol, enoblitzumab, enokizumab, enoticumab, ensituximab, epitumomab Sitaxetan, epratuzumab, erlizumab, ertumaxomab, etanercept, etaracizumab, etrolizumab, evinacumab, evolocumab, exbivirumab, F19, fanolesomab, faralimomab, faretuzumab, fasinumab, FBTA05, felvizumab, fezakinumab, ficlatuzumab, figitumumab, filibumab, framvotumab, fretikumab, fontolizumab, foralumab, foravirumab, fresolimumab, furanumab, futuximab, galiximab, ganitumab, gantenerumab, gavilimomab, gemtuzumab ozogamicin, gevokizumab, girentuximab, glembatumumab Vedotin, golimumab, gomilikumab, guselkumab, HGS-ETR2, hu3S193, huA33, ibalizumab, ibritumomab tiuxetan, icrucumab, idarucizumab, IGN101, IgN311, igovomab, IIIA4, IM-2C6, IMAB362, imalumab, IMC-A12, imciromab, imgatuzumab, inlacumab, indatuximab ravtansine, indusatumab vedotin, infliximab, inolimomab, inotuzumab Ozogamicin, Intetumumab, Ipilimumab, Iratumumab, Isatuximab, Itolizumab, Ixekizumab, J591, KB004, Keliximab, KW-2871, Labetuzumab, Lambrolizumab, Lampalizumab, Lebrikizumab, Remaresomab, Lenzilumab, Lerdelimumab, Lexatumumab, Ribivirumab, Rifastuzumab vedotin, Ligelizumab, Rilotomab satetratetan, Lintuzumab, Lirilumab, Roderucizumab, Lokivetomab, Lorvotuzumab mertansine, Lucatumumab, LulizumabPegol, lumiliximab, lumuletuzumab, mapatumumab, margetuximab, maslimomab, matuzumab, mavrilimumab, MEDI4736, mepolizumab, metelimuab, METMAB, milatuzumab, minletumomab, mirvetuximab soravtansine, mitumomab, MK-0646, MK-3475, MM-121, mogamulizumab, MORAb-003, morolimumab, motavizumab, MOv18, moxetumomab passudotox, MPDL33280A, muromonab-CD3, nacolomab tafenatox, namilumab, naptumomab Estafenatox, Narutuzumab, Natalizumab, Nebacumab, Necitumumab, Nemolizumab, Nerelimomab, Nesbacumab, Nimotuzumab, Nivolumab, Nofetumomab Merpentan, Oviltoxaximab, Obinutuzumab, Ocaratuzumab, Ocrelizumab, Ozlimomab, Ofatumumab, Olaratumab, Olokizumab, Omalizumab, Onartuzumab, Ontuxizumab, Opicinumab, Oportuzumab Monatox, oregovomab, olticumab, otelixizumab, otlertuzumab, oxelumab, ozanezumab, ozoralizumab, pagibaximab, palivizumab, panitumumab, pancomab, panobacumab, palsatuzumab, pascolizumab, pasotuxizumab, pateclizumab, patritumab, pembrolizumab, pemtumomab, perakizumab, pertuzumab, pexelizumab, pidilizumab, pinatuzumab vedotin, pintumomab, placumab, polatuzumab Vedotin, ponezumab, priliximab, plitoxaximab, pritumumab, PRO140, kirisumab, R1507, racotumomab, radletumab, rafivirumab, ralpancizumab, ramucirumab, ranibizumab, raxibacumab, refanezumab, regavirumab, reslizumab, rilotumumab, rinukumab, rituximab, lobatumumab, loredumab, romosozumab, rontalizumab, rovelizumab, ruplizumab, sacituzumab govitecan, samalizumab, sarilumab, satumomab Pendetide, SCH900105, Secukinumab, Seribantumab, Setoxaximab, Sevirumab, SGN-CD19A, SGN-CD33A, Sibrotuzumab, Sifalimumab, Siltuximab, Simtuzumab, Siplizumab, Sirukumab, SofituzumabVedotin, solanezumab, solitomab, sonepcizumab, sontuzumab, stamulumab, sulesomab, subizumab, tabalumab, tacatuzumab tetraxetan, tadocizumab, talizumab, tanezumab, taplitumab paptox, tarectumab, tefibazumab, terimomab Aritox, tenatumomab, teneliximab, teplizumab, teprotumumab, tesidolumab, tetulomab, TGN1412, ticilimumab / tremelimumab, tigatuzumab, tildrakizumab, TNX-650, tocilizumab, toralizumab, tosatoxumab, tositumomab, tobetumab, tralokinumab, trastuzumab, TRBS07, tregalizumab, tremelimumab, trevoglumab, tucotuzumab celmoleukin, tuvilumab, ublituximab, urocupulumab, urelumab, ustekinumab, ustekinumab, vandorutuzumab and selected from vedotin, vanticutumab, vanucizumab, bapaliximab, varlilumab, batelizumab, vedolizumab, veltuzumab, beparimomab, besenkumab, visilizumab, volociximab, borsetuzumab mafodotin, votumumab, zalutumumab, zanolimumab, zatuximab, diralimumab, and zolimomab alitox.

[0220] In some cases, the target polypeptide is a CRISPR-Cas effector polypeptide. The suitable CRISPR-Cas effector polypeptide is a class 2 CRISPR / Cas endonuclease, for example, type II, type V, or type VI CRISPR / Cas effector polypeptide. In some cases, the suitable RNA-guided endonuclease is a class 2 CRISPR / Cas endonuclease. In some cases, the suitable RNA-guided endonuclease is a class 2 type II CRISPR / Cas endonuclease (for example, Cas9 protein). In some cases, the CRISPR-Cas guided effector polypeptide is a class 2 type V CRISPR / Cas effector polypeptide (for example, Cpf1 protein, C2c1 protein, or C2c3 protein). In some cases, the preferred CRISPR-Cas effector polypeptide is a class 2 type VI CRISPR / Cas effector polypeptide (e.g., C2c2 protein; also referred to as "Cas13a" protein). CasX protein is also preferred. CasY protein is also preferred.

[0221] In some cases, the CRISPR / Cas effector polypeptide is a type II CRISPR / Cas effector polypeptide. In some cases, the CRISPR / Cas effector polypeptide is a Cas9 polypeptide. The Cas9 protein is directed to (e.g., stabilized at) a target site within a target nucleic acid sequence (e.g., a chromosomal or extrachromosomal sequence, such as an episomal sequence, a minicircle sequence, a mitochondrial sequence, a chloroplast sequence, etc.) by association with a protein-binding segment of the Cas9 guide RNA. In some cases, the Cas9 polypeptide comprises an amino acid sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or more than 99% amino acid sequence identity to the Streptococcus pyogenes Cas9 set forth in SEQ ID NO: 753. In some cases, the Cas9 polypeptide comprises an amino acid sequence set forth in any one of SEQ ID NOs: 5-816. In some cases, the Cas9 polypeptide comprises an amino acid sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or greater than 99% amino acid sequence identity to an amino acid sequence set forth in any one of SEQ ID NOs:5-816.

[0222] In some cases, the Cas9 polypeptide is a Staphylococcus aureus Cas9 (saCas9) polypeptide. In some cases, the saCas9 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the saCas9 amino acid sequence set forth in SEQ ID NO:249.

[0223] In some cases, the Cas9 polypeptide is a Campylobacter jejuni Cas9 (CjCas9) polypeptide. CjCas9 recognizes 5'-NNNVRYM-3' as a protospacer adjacent motif (PAM). The amino acid sequence of CjCas9 is set forth in SEQ ID NO: 55. In some cases, a suitable Cas9 polypeptide comprises an amino acid sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or more than 99% amino acid sequence identity to the CjCas9 amino acid sequence set forth in SEQ ID NO: 55.

[0224] In some cases, a suitable Cas9 polypeptide is a high fidelity (HF) Cas9 polypeptide. Kleinstiver et al. (2016) Nature 529:490. For example, the amino acids N497, R661, Q695, and Q926 of the Streptococcus pyogenes Cas9 amino acid sequence (e.g., SEQ ID NO: 5) are substituted, for example, with alanine. For example, the HF Cas9 polypeptide may have at least 90%, at least 95%, at least 98%, at least 99%, or more than 100% amino acid sequence identity to Streptococcus pyogenes Cas9 (e.g., SEQ ID NO: 5), and the amino acids N497, R661, Q695, and Q926 are substituted, for example, with alanine. In some cases, a suitable Cas9 polypeptide exhibits altered PAM specificity. See, for example, Kleinstiver et al. (2015) Nature 523:481.

[0225] In some cases, a suitable Cas9 polypeptide has the following Cas9-HF1 sequence: The amino acid sequence may have at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to

[0226] In some cases, the suitable CRISPR / Cas effector polypeptide is a type V CRISPR / Cas effector polypeptide. In some cases, the type V CRISPR / Cas effector polypeptide is a Cpf1 protein. In some cases, the Cpf1 protein comprises an amino acid sequence having at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 90%, or 100% amino acid sequence identity to the Cpf1 amino acid sequence set forth in any one of SEQ ID NOs: 818-822.

[0227] In some cases, a suitable CRISPR / Cas effector polypeptide is a CasX or CasY polypeptide. CasX and CasY polypeptides are described in Burstein et al. (2017) Nature 542:237.

[0228] In some cases, a suitable CRISPR / Cas effector polypeptide is a fusion protein that includes a CRISPR / Cas effector polypeptide fused to a heterologous polypeptide (also referred to as a "fusion partner"). In some cases, the CRISPR / Cas effector polypeptide is fused to an amino acid sequence (the fusion partner) that provides subcellular localization, i.e., the fusion partner is a subcellular localization sequence (e.g., one or more nuclear localization signals (NLS) for targeting to the nucleus, two or more NLSs, three or more NLSs, etc.).

[0229] A nucleic acid that binds to a class 2 CRISPR / Cas effector polypeptide (e.g., Cas9 protein; type V or type VI CRISPR / Cas protein; Cpf1 protein, etc.) and targets the complex to a specific location within a target nucleic acid is referred to herein as a "guide RNA" or "CRISPR / Cas guide nucleic acid" or "CRISPR / Cas guide RNA." Guide RNAs provide target specificity to the complex (RNP complex) by including a targeting segment that includes a guide sequence (also referred to herein as a targeting sequence), which is a nucleotide sequence complementary to the target nucleic acid sequence.

[0230] In some cases, the guide RNA comprises two separate nucleic acid molecules: an "activator" and a "targeter," and is referred to herein as a "dual guide RNA," "dual molecule guide RNA," "two molecule guide RNA," or "dgRNA." In some cases, the guide RNA is one molecule (e.g., for some class 2 CRISPR / Cas proteins, the corresponding guide RNA is a single molecule; in some cases, the activator and targeter are covalently linked to each other, e.g., via an intervening nucleotide), and the guide RNA is referred to as a "single guide RNA," "single molecule guide RNA," "one molecule guide RNA," or simply "sgRNA."

[0231] Class 2 CRISPR / Cas effector polypeptides In class 2 CRISPR systems, the function of the effector complex (e.g., cleavage of target DNA) is performed by a single endonuclease (see, e.g., Zetsche et al., Cell. 2015 Oct 22; 163(3):759-71; Makarova et al., Nat Rev Microbiol. 2015 Nov; 13(11):722-36; Shmakov et al., Mol Cell. 2015 Nov 5; 60(3):385-97); and Shmakov et al. (2017) Nature Reviews Microbiology 15:169. Thus, the term "class 2 CRISPR / Cas protein" is used herein to encompass CRISPR / Cas effector polypeptides (e.g., target nucleic acid cleavage proteins) from class 2 CRISPR systems. Thus, as used herein, the term "Class 2 CRISPR / Cas effector polypeptide" refers to a type II CRISPR / Cas effector polypeptide (e.g., Cas9); a type VA CRISPR / Cas effector polypeptide (e.g., Cpf1 (also called "Cas12a")); a type VB CRISPR / Cas effector polypeptide (e.g., C2c1 (also called "Cas12b")); a type VC CRISPR / Cas effector polypeptide (e.g., C2c3 (also called "Cas12c")); a type V-U1 CRISPR / Cas effector polypeptide (e.g., C2c4); a type V-U2 CRISPR / Cas effector polypeptide (e.g., C2c5 (also called "Cas12c"); V-U5 type CRISPR / Cas effector polypeptides (e.g., C2c5); V-U4 type CRISPR / Cas proteins (e.g., C2c9); V-U3 type CRISPR / Cas effector polypeptides (e.g., C2c10); Type VI-A CRISPR / Cas effector polypeptides (e.g., C2c2 (also known as "Cas13a")); Type VI-B CRISPR / Cas effector polypeptides (e.g., Cas13b (also known as C2c4)); and Type VI-C CRISPR / Cas effector polypeptides (e.g., Cas13c (also known as C2c7)).To date, Type II, Type V, and Type VI CRISPR / Cas effector polypeptides are encompassed by Class 2 CRISPR / Cas effector polypeptides, but the term is also meant to encompass any Class 2 CRISPR / Cas effector polypeptide suitable for binding to a corresponding guide RNA to form an RNP complex.

[0232] Type II CRISPR / Cas endonucleases (e.g., Cas9) In the native type II CRISPR / Cas system, Cas9 functions as an RNA-guided endonuclease and uses dual guide RNAs with crRNA and transactivating crRNA (tracrRNA) to recognize and cleave targets by a mechanism in which the two nuclease active sites of Cas9 work together to generate double-stranded DNA breaks (DSBs) or individually to generate single-stranded DNA breaks (SSBs). The type II CRISPR endonuclease Cas9 and modified dual guide RNAs (dgRNAs) or single guide RNAs (sgRNAs) form a ribonucleoprotein (RNP) complex that can target desired DNA sequences. Guided by the dual RNA complex or chimeric single guide RNA, Cas9 generates site-specific DSBs or SSBs within double-stranded DNA (dsDNA) target nucleic acids, which are repaired by either nonhomologous end joining (NHEJ) or homologous recombination (HDR).

[0233] The type II CRISPR / Cas effector polypeptide is a type of class 2 CRISPR / Cas endonuclease. In some cases, the type II CRISPR / Cas endonuclease is a Cas9 protein. The Cas9 protein forms a complex with the Cas9 guide RNA. The guide RNA provides target specificity to the Cas9-guide RNA complex by having a nucleotide sequence (guide sequence) that is complementary to the sequence (target site) of the target nucleic acid (as described elsewhere herein). The Cas9 protein of the complex provides site-specific activity. In other words, the Cas9 protein is directed to (e.g., stabilized at) the target site in the target nucleic acid sequence (e.g., chromosomal or extrachromosomal sequence, e.g., episomal sequence, minicircle sequence, mitochondrial sequence, chloroplast sequence, etc.) by association with the protein-binding segment of the Cas9 guide RNA.

[0234] The Cas9 protein can bind to and / or modify (e.g., cleave, nick, methylate, demethylate, etc.) a target nucleic acid and / or a polypeptide associated with the target nucleic acid (e.g., methylate or acetylate histone tails) (e.g., when the Cas9 protein comprises an active fusion partner). In some cases, the Cas9 protein is a naturally occurring protein (e.g., naturally occurring in bacterial and / or archaeal cells). In other cases, the Cas9 protein is not a naturally occurring polypeptide (e.g., the Cas9 protein is a variant Cas9 protein, a chimeric protein, etc.).

[0235] Examples of suitable Cas9 proteins include, but are not limited to, those set forth in SEQ ID NOs: 5-816. A native Cas9 protein binds to a Cas9 guide RNA, thereby being directed to a specific sequence within a target nucleic acid (target site), and cleaves the target nucleic acid (e.g., cleaves dsDNA to generate double-stranded breaks, cleaves ssDNA, cleaves ssRNA, etc.). A chimeric Cas9 protein is a fusion protein that includes a Cas9 polypeptide fused to a heterologous protein (called a fusion partner), where the heterologous protein provides an activity (e.g., an activity not provided by the Cas9 protein). The fusion partner can provide, for example, an enzymatic activity (e.g., a nuclease activity, a DNA and / or RNA methylation activity, a DNA and / or RNA cleavage activity, a histone acetylation activity, a histone methylation activity, an RNA modification activity, an RNA binding activity, an RNA splicing activity, etc.). In some cases, a portion of the Cas9 protein (e.g., the RuvC domain and / or the HNH domain) exhibits reduced nuclease activity compared to the corresponding portion of a wild-type Cas9 protein (e.g., in some cases, the Cas9 protein is a nickase). In some cases, the Cas9 protein is enzymatically inactive or has reduced enzymatic activity compared to a wild-type Cas9 protein (e.g., compared to Streptococcus pyogenes Cas9).

[0236] In some cases, the fusion protein comprises: a) a catalytically inactive Cas9 protein (or other catalytically inactive CRISPR effector polypeptide); and b) a catalytically active endonuclease. For example, in some cases, the catalytically active endonuclease is a FokI polypeptide. As a non-limiting example, in some cases, the fusion protein comprises: a) a catalytically inactive Cas9 protein (or other catalytically inactive CRISPR effector polypeptide); and b) a FokI nuclease comprising an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the FokI amino acid sequence shown below; the length of the FokI nuclease is about 195 amino acids to about 200 amino acids.

[0237] FokI nuclease amino acid sequence: QLVKSELEEKKSELRHKLKYVPHEYIELIEIARNSTQDRILEMKVMEFFMKVYGYRGKHLGGSRKPDGAIYTVGSPIDYGVIVDTKAYSGGYNLPIGQADEMQRYVEENQTRNKHINPNEWWKVYPSSVTEFKFLFVSGHFKGNYKAQLTRLNHITNCNGAVLSVEELLIGGEMIKAGTLTLEEVRRKFNNGEINF (sequence number 900).

[0238] The assay for determining whether a given protein interacts with the Cas9 guide RNA can be any convenient binding assay that tests for binding between a protein and a nucleic acid. Suitable binding assays (e.g., gel shift assays) will be known to those skilled in the art (e.g., assays that include adding the Cas9 guide RNA and protein to a target nucleic acid).

[0239] The assay to determine whether a protein has activity (e.g., to determine whether a protein has nuclease activity and / or some heterologous activity that cleaves a target nucleic acid) can be any convenient assay (e.g., any convenient nucleic acid cleavage assay that tests for nucleic acid cleavage). Suitable assays (e.g., cleavage assays) are known to those of skill in the art and can include adding a Cas9 guide RNA and a protein to a target nucleic acid.

[0240] In some cases, a suitable Cas9 protein comprises an amino acid sequence having 60% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 99% or more, or 100% amino acid sequence identity to amino acids 7-166 or 731-1003 of the Cas9 amino acid sequence set forth in SEQ ID NO:5, or to a corresponding portion of any of the amino acid sequences set forth in SEQ ID NOs:6-816.

[0241] Examples of various Cas9 proteins (and Cas9 domain structures) and Cas9 guide RNAs (and information regarding the requirements related to the protospacer adjacent motif (PAM) sequence present in the target nucleic acid) can be found in the art, e.g., Jinek et al., Science. 2012 Aug 17; 337(6096):816-21; Chylinski et al., RNA Biol. 2013 May; 10(5):726-37; Ma et al., Biomed Res Int. 2013; 2013:270805; Hou et al., Proc Natl Acad Sci USA. 2013 Sep 24; 110(39):15644-9; Jinek et al., Elife. 2013; 2:e00471; Pattanayak et al., Nat Biotechnol. 2013 Sep;31(9):839-43, Qi et.al.,Cell.2013 Feb 28;152(5):1173-83;Wang et.al.,Cell.2013 May 9;153(4):910-8, Auer et.al.,Genome Res.2013 Oct 31, Chen et.al.,Nucleic Acids Res.2013 Nov 1;41(20):e19, Cheng et.al., Cell Res.2013 Oct;23(10):1163-71, Cho et.al., Genetics.2013 Nov;195(3):1177-80, DiCarlo et al., Nucleic Acids Res.2013 Apr;41(7):4336-43, Dickinson et.al., Nat Methods.2013 Oct;10(10):1028-34, Ebina et.al., Sci Rep.2013;3:2510, Fujii et.al, Nucleic Acids Res.2013 Nov 1;41(20):e187, Hu et.al., Cell Res.2013 Nov;23(11):1322-5, Jiang et.al., Nucleic Acids Res.2013 Nov 1;41(20):e188, Larson et.al., Nat Protoc.2013 Nov;8(11):2180-96、Mali et.at.,Nat Methods.2013 Oct;10(10):957-63、Nakayama et.al.,Genesis.2013 Dec;51(12):835-43、Ran et.al.,Nat Protoc.2013 Nov;8(11):2281-308、Ran et.al.,Cell.2013 Sep 12;154(6):1380-9、Upadhyay et.al.,G3(Bethesda).2013 Dec 9;3(12):2233-8、Walsh et.al.,Proc Natl Acad Sci USA.2013 Sep 24;110(39):15514-5、Xie et.al.,Mol Plant.2013 Oct 9、Yang et.al.,Cell.2013 Sep 12;154(6):1370-9、Briner et al.,Mol Cell.2014 Oct 23;56(2):333-9;Shmakov et al.,Nat Rev Microbiol.2017 Mar;15(3):169-182, and U.S. Patents and Patent Applications Nos. 8,906,616, 8,895,308, 8,889,418, 8,889,356, 8,871,445, 8,865,406, 8,795,965, 8,771,945, 8,697,359, 20140068797, 20140170753, 20140179006, 20140179770, 20140186843, 2014018799, 2014018798, 201401879 ... No. 0140186919, No. 20140186958, No. 20140189896, No. 20140227787, No. 20140234972, No. 20140242664, No. 20140242699, No. 201402427 No. 00, No. 20140242702, No. 20140248702, No. 20140256046, No. 20140273037, No. 20140273226, No. 20140273230, No. 20140273231, No. 2014 No. 0273232, No. 20140273233, No. 20140273234, No. 20140273235, No. 20140287938, No. 20140295556, No. 20140295557, No. 20140298547 , No. 20140304853, No. 20140309487, No. 20140310828, No. 20140310830, No. 20140315985, No. 20140335063, No. 20140335620, No. 201403 42456, 20140342457, 20140342458, 20140349400, 20140349405, 20140356867, 20140356956, 20140356958, 20140356959, 20140357523, 20140357530, 20140364333, and 20140377868; each of which is incorporated by reference herein in its entirety.

[0242] Variant Cas9 proteins - Nickase and dCas9 In some cases, the Cas9 protein is a variant Cas9 protein. The variant Cas9 protein has an amino acid sequence that differs (e.g., has a deletion, insertion, substitution, fusion) by at least one amino acid when compared to the amino acid sequence of the corresponding wild-type Cas9 protein. In some cases, the variant Cas9 protein has an amino acid change (e.g., a deletion, insertion, or substitution) that reduces the nuclease activity of the Cas9 protein. For example, in some cases, the variant Cas9 protein has 50% or less, 40% or less, 30% or less, 20% or less, 10% or less, 5% or less, or 1% or less of the nuclease activity of the corresponding wild-type Cas9 protein. In some cases, the variant Cas9 protein does not have substantial nuclease activity. When the Cas9 protein is a variant Cas9 protein that does not have substantial nuclease activity, the Cas9 protein may be referred to as a "dCas9" for nuclease-deficient Cas9 protein or "dead" Cas9. Proteins that cleave one strand of a double-stranded target nucleic acid but not the other (e.g., class 2 CRISPR / Cas proteins, e.g., Cas9 proteins) are referred to herein as "nickases" (e.g., "nickase Cas9").

[0243] In some cases, the variant Cas9 protein can cleave the complementary strand of the target nucleic acid (sometimes referred to in the art as the target strand), but has a reduced ability to cleave the non-complementary strand of the target nucleic acid (sometimes referred to in the art as the non-target strand). For example, the variant Cas9 protein can have a mutation (amino acid substitution) that reduces the function of the RuvC domain. Thus, the Cas9 protein can be a nickase that cleaves the complementary strand but not the non-complementary strand. As a non-limiting example, in some embodiments, a variant Cas9 protein has a mutation at an amino acid position corresponding to residue D10 (e.g., D10A, aspartic acid to alanine) of SEQ ID NO:5 (or any corresponding position in the proteins set forth in SEQ ID NOs:6-261 and 264-816) and is thus capable of cleaving the complementary strand of a double-stranded target nucleic acid, but has a reduced ability to cleave the non-complementary strand of a double-stranded target nucleic acid (and thus, when the variant Cas9 protein cleaves a double-stranded target nucleic acid, it generates a single-stranded break (SSB) instead of a double-stranded break (DSB)) (see, e.g., Jinek et al., Science. 2012 Aug 17;337(6096):816-21). See, e.g., SEQ ID NO:262.

[0244] In some cases, a variant Cas9 protein can cleave a non-complementary strand of a target nucleic acid, but has a reduced ability to cleave a complementary strand of the target nucleic acid. For example, a variant Cas9 protein can have a mutation (amino acid substitution) that reduces the function of the HNH domain. Thus, the Cas9 protein can be a nickase that cleaves a non-complementary strand, but not a complementary strand. As a non-limiting example, in some embodiments, a variant Cas9 protein has a mutation at an amino acid position corresponding to residue H840 (e.g., H840A mutation, histidine to alanine) of SEQ ID NO:5 (or the corresponding position in any of the proteins shown as SEQ ID NOs:6-261 and 264-816), and thus can cleave a non-complementary strand of a target nucleic acid, but has a reduced ability to cleave (e.g., does not cleave) a complementary strand of the target nucleic acid. Such a Cas9 protein has a reduced ability to cleave a target nucleic acid (e.g., a single-stranded target nucleic acid), but retains the ability to bind to a target nucleic acid (e.g., a single-stranded target nucleic acid). See, for example, SEQ ID NO:263.

[0245] In some cases, the variant Cas9 protein has a reduced ability to cleave both complementary and non-complementary strands of a double-stranded target nucleic acid. As a non-limiting example, in some cases, the variant Cas9 protein has mutations (e.g., D10A and H840A) at amino acid positions corresponding to residues D10 and H840 of SEQ ID NO:5 (or the corresponding residues of any of the proteins set forth in SEQ ID NOs:6-261 and 264-816), such that the polypeptide has a reduced ability to cleave (e.g., does not cleave) both complementary and non-complementary strands of a target nucleic acid. Such Cas9 proteins have a reduced ability to cleave a target nucleic acid (e.g., a single-stranded or double-stranded target nucleic acid), but retain the ability to bind to the target nucleic acid. A Cas9 protein that is unable to cleave a target nucleic acid (e.g., due to one or more mutations in the catalytic domain of the RuvC and HNH domains) is referred to as a "dead" Cas9 or simply "dCas9". See, e.g., SEQ ID NO:264.

[0246] Type V and Type VI CRISPR / Cas endonucleases In some cases, a suitable CRISPR / Cas effector polypeptide is a type V or type VI CRISPR / Cas endonuclease (i.e., the CRISPR / Cas effector polypeptide is a type V or type VI CRISPR / Cas endonuclease) (e.g., Cpf1, C2c1, C2c2, C2c3). Type V and type VI CRISPR / Cas endonucleases are a type of class 2 CRISPR / Cas endonucleases. Examples of type V CRISPR / Cas endonucleases include, but are not limited to, Cpf1, C2c1, and C2c3. One example of a type VI CRISPR / Cas effector polypeptide is C2c2. In some cases, a suitable CRISPR / Cas effector polypeptide is a type V CRISPR / Cas endonuclease (e.g., Cpf1, C2c1, C2c3). In some cases, the type V CRISPR / Cas effector polypeptide is a Cpf1 protein. In some cases, a suitable CRISPR / Cas effector polypeptide is a type VI CRISPR / Cas endonuclease (e.g., Cas13a).

[0247] Like type II CRISPR / Cas endonucleases, type V and type VI CRISPR / Cas endonucleases form a complex with a corresponding guide RNA. The guide RNA provides target specificity to the endonuclease-guide RNA RNP complex by having a nucleotide sequence (guide sequence) that is complementary to the sequence (target site) of the target nucleic acid (as described elsewhere herein). The endonuclease of the complex provides site-specific activity. In other words, the endonuclease is guided to (e.g., stabilized at) a target site in a target nucleic acid sequence (e.g., a chromosomal or extrachromosomal sequence, such as an episomal sequence, a minicircle sequence, a mitochondrial sequence, a chloroplast sequence, etc.) by association with the protein-binding segment of the guide RNA.

[0248] Examples and guidance relating to Type V and Type VI CRISPR / Cas proteins (e.g., Cpf1, C2c1, C2c2, and C2c3 guide RNAs) can be found in the art, see, e.g., Zetsche et al., Cell. 2015 Oct 22; 163(3):759-71; Makarova et al., Nat Rev Microbiol. 2015 Nov; 13(11):722-36; Shmakov et al., Mol Cell. 2015 Nov 5; 60(3):385-97; and Shmakov et al. (2017) Nature Reviews Microbiology 15:169.

[0249] In some cases, the type V or type VI CRISPR / Cas endonuclease (e.g., Cpf1, C2c1, C2c2, C2c3) is enzymatically active, for example, the type V or type VI CRISPR / Cas polypeptide cleaves the target nucleic acid when bound to the guide RNA. In some cases, the type V or type VI CRISPR / Cas endonuclease (e.g., Cpf1, C2c1, C2c2, C2c3) exhibits reduced enzymatic activity and retains DNA binding activity compared to the corresponding wild-type type V or type VI CRISPR / Cas endonuclease (e.g., Cpf1, C2c1, C2c2, C2c3).

[0250] In some cases, the type V CRISPR / Cas endonuclease is a Cpf1 protein. In some cases, the Cpf1 protein comprises an amino acid sequence having at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 90%, or 100% amino acid sequence identity to the Cpf1 amino acid sequence set forth in any of SEQ ID NOs: 818-822. In some cases, the Cpf1 protein comprises an amino acid sequence having at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 90%, or 100% amino acid sequence identity to a contiguous stretch of 100 amino acids to 200 amino acids (aa), 200 aa to 400 aa, 400 aa to 600 aa, 600 aa to 800 aa, 800 aa to 1000 aa, 1000 aa to 1100 aa, 1100 aa to 1200 aa, or 1200 aa to 1300 aa of the Cpf1 amino acid sequence set forth in any of SEQ ID NOs:818-822.

[0251] In some cases, the Cpf1 protein comprises an amino acid sequence having at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 90%, or 100% amino acid sequence identity to the RuvCI domain of a Cpf1 amino acid sequence set forth in any of SEQ ID NOs:818-822. In some cases, the Cpf1 protein comprises an amino acid sequence having at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 90%, or 100% amino acid sequence identity to the RuvCII domain of a Cpf1 amino acid sequence set forth in any of SEQ ID NOs:818-822. In some cases, the Cpf1 protein comprises an amino acid sequence having at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 90%, or 100% amino acid sequence identity to the RuvCIII domain of the Cpf1 amino acid sequence set forth in any of SEQ ID NOs:818-822. In some cases, the Cpf1 protein comprises an amino acid sequence having at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 90%, or 100% amino acid sequence identity to the RuvCI, RuvCII, and RuvCIII domains of the Cpf1 amino acid sequence set forth in any of SEQ ID NOs:818-822.

[0252] In some cases, the Cpf1 protein exhibits reduced enzymatic activity and retains DNA binding activity compared to a wild-type Cpf1 protein (e.g., compared to a Cpf1 protein comprising an amino acid sequence set forth in any of SEQ ID NOs: 818-822). In some cases, the Cpf1 protein comprises an amino acid sequence having at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 90%, or 100% amino acid sequence identity to the Cpf1 amino acid sequence set forth in any of SEQ ID NOs: 818-822; and comprises an amino acid substitution (e.g., a D→A substitution) at an amino acid residue corresponding to amino acid 917 of the Cpf1 amino acid sequence set forth in SEQ ID NO: 818. In some cases, the Cpf1 protein comprises an amino acid sequence having at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 90%, or 100% amino acid sequence identity to the Cpf1 amino acid sequence set forth in any of SEQ ID NOs:818-822; and comprises an amino acid substitution (e.g., an E→A substitution) at an amino acid residue corresponding to amino acid 1006 of the Cpf1 amino acid sequence set forth in SEQ ID NO:818. In some cases, the Cpf1 protein comprises an amino acid sequence having at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 90%, or 100% amino acid sequence identity to the Cpf1 amino acid sequence set forth in any of SEQ ID NOs:818-822; and comprises an amino acid substitution (e.g., a D→A substitution) at an amino acid residue corresponding to amino acid 1255 of the Cpf1 amino acid sequence set forth in SEQ ID NO:818.

[0253] In some cases, a suitable Cpf1 protein comprises an amino acid sequence having at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 90%, or 100% amino acid sequence identity to a Cpf1 amino acid sequence set forth in any of SEQ ID NOs:818-822.

[0254] In some cases, the Type V CRISPR / Cas endonuclease is a C2c1 protein (e.g., as set forth in SEQ ID NOs: 823-830). In some cases, the C2c1 protein comprises an amino acid sequence having at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 90%, or 100% amino acid sequence identity to the C2c1 amino acid sequence set forth in any of SEQ ID NOs: 823-830. In some cases, the C2c1 protein comprises an amino acid sequence having at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 90%, or 100% amino acid sequence identity to a contiguous stretch of 100 amino acids to 200 amino acids (aa), 200aa to 400aa, 400aa to 600aa, 600aa to 800aa, 800aa to 1000aa, 1000aa to 1100aa, 1100aa to 1200aa, or 1200aa to 1300aa of the C2c1 amino acid sequence set forth in any of SEQ ID NOs:823-830.

[0255] In some cases, the C2c1 protein comprises an amino acid sequence having at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 90%, or 100% amino acid sequence identity to the RuvCI domain of a C2c1 amino acid sequence set forth in any of SEQ ID NOs: 823-830. In some cases, the C2c1 protein comprises an amino acid sequence having at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 90%, or 100% amino acid sequence identity to the RuvCII domain of a C2c1 amino acid sequence set forth in any of SEQ ID NOs: 823-830. In some cases, the C2c1 protein comprises an amino acid sequence having at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 90%, or 100% amino acid sequence identity to the RuvCIII domain of a C2c1 amino acid sequence set forth in any of SEQ ID NOs: 823-830. In some cases, the C2c1 protein comprises an amino acid sequence having at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 90%, or 100% amino acid sequence identity to the RuvCI, RuvCII, and RuvCIII domains of the C2c1 amino acid sequence set forth in any of SEQ ID NOs: 823-830.

[0256] In some cases, the Type V CRISPR / Cas endonuclease is a C2c3 protein (e.g., as set forth in SEQ ID NOs: 831-834). In some cases, the C2c3 protein comprises an amino acid sequence having at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 90%, or 100% amino acid sequence identity to the C2c3 amino acid sequence set forth in any of SEQ ID NOs: 831-834. In some cases, the C2c3 protein comprises an amino acid sequence having at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 90%, or 100% amino acid sequence identity to a contiguous stretch of 100 amino acids to 200 amino acids (aa), 200aa to 400aa, 400aa to 600aa, 600aa to 800aa, 800aa to 1000aa, 1000aa to 1100aa, 1100aa to 1200aa, or 1200aa to 1300aa of the C2c3 amino acid sequence set forth in any of SEQ ID NOs: 831-834.

[0257] In some cases, the C2c3 protein comprises an amino acid sequence having at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 90%, or 100% amino acid sequence identity to the RuvCI domain of a C2c3 amino acid sequence set forth in any of SEQ ID NOs: 831-834. In some cases, the C2c3 protein comprises an amino acid sequence having at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 90%, or 100% amino acid sequence identity to the RuvCII domain of a C2c3 amino acid sequence set forth in any of SEQ ID NOs: 831-834. In some cases, the C2c3 protein comprises an amino acid sequence having at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 90%, or 100% amino acid sequence identity to the RuvCIII domain of a C2c3 amino acid sequence set forth in any of SEQ ID NOs: 831-834. In some cases, the C2c3 protein comprises an amino acid sequence having at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 90%, or 100% amino acid sequence identity to the RuvCI, RuvCII, and RuvCIII domains of a C2c3 amino acid sequence set forth in any of SEQ ID NOs: 831-834.

[0258] In some cases, the C2c3 protein exhibits reduced enzymatic activity and retains DNA binding activity compared to a wild-type C2c3 protein (e.g., compared to a C2c3 protein comprising an amino acid sequence set forth in any of SEQ ID NOs: 831-834). In some cases, a suitable C2c3 protein comprises an amino acid sequence having at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 90%, or 100% amino acid sequence identity to the C2c3 amino acid sequence set forth in any of SEQ ID NOs: 831-834.

[0259] In some cases, the Type VI CRISPR / Cas endonuclease is a C2c2 protein (e.g., as set forth in SEQ ID NOs: 835-846). In some cases, the C2c2 protein comprises an amino acid sequence having at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 90%, or 100% amino acid sequence identity to a C2c2 amino acid sequence set forth in any of SEQ ID NOs: 835-846. In some cases, the C2c2 protein comprises an amino acid sequence having at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 90%, or 100% amino acid sequence identity to a contiguous stretch of 100 amino acids to 200 amino acids (aa), 200aa to 400aa, 400aa to 600aa, 600aa to 800aa, 800aa to 1000aa, 1000aa to 1100aa, 1100aa to 1200aa, or 1200aa to 1300aa of a C2c2 amino acid sequence set forth in any of SEQ ID NOs:835-846.

[0260] In some cases, the C2c2 protein comprises an amino acid sequence having at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 90%, or 100% amino acid sequence identity to the RuvCI domain of a C2c2 amino acid sequence set forth in any of SEQ ID NOs: 835-846. In some cases, the C2c2 protein comprises an amino acid sequence having at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 90%, or 100% amino acid sequence identity to the RuvCII domain of a C2c2 amino acid sequence set forth in any of SEQ ID NOs: 835-846. In some cases, the C2c2 protein comprises an amino acid sequence having at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 90%, or 100% amino acid sequence identity to the RuvCIII domain of a C2c2 amino acid sequence set forth in any of SEQ ID NOs: 835-846. In some cases, the C2c2 protein comprises an amino acid sequence having at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 90%, or 100% amino acid sequence identity to the RuvCI, RuvCII, and RuvCIII domains of a C2c2 amino acid sequence set forth in any of SEQ ID NOs: 835-846.

[0261] In some cases, the C2c2 protein exhibits reduced enzymatic activity and retains DNA binding activity compared to a wild-type C2c2 protein (e.g., compared to a C2c2 protein comprising an amino acid sequence set forth in any of SEQ ID NOs: 835-846). In some cases, a suitable C2c2 protein comprises an amino acid sequence having at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 90%, or 100% amino acid sequence identity to the C2c2 amino acid sequence set forth in any of SEQ ID NOs: 835-846.

[0262] Examples and guidance relating to Type V or Type VI CRISPR / Cas endonucleases (including domain structures) and guide RNAs (as well as information regarding requirements relating to protospacer adjacent motif (PAM) sequences present in target nucleic acids) can be found in the art, e.g., Zetsche et al., Cell. 2015 Oct 22; 163(3):759-71; Makarova et al., Nat Rev Microbiol. 2015 Nov; 13(11):722-36; Shmakov et al., Mol Cell. 2015 Nov 5; 60(3):385-97; and Shmakov et al., Nat Rev Microbiol. 2017 Mar.; 15(3): 169-182; and U.S. Patents and Patent Applications: 9,580,701; 20170073695, 20170058272, 20160362668, 20160362667, 20160298078, 20160289637, 20160215300, 20160208243, and 20160208241, each of which is incorporated by reference in its entirety.

[0263] CasX and CasY proteins Suitable CRISPR / Cas effector polypeptides include CasX and CasY polypeptides. See, for example, Burstein et al. (2017) Nature 542:237. Suitable CasX polypeptides include those described in WO2018 / 064371. Suitable CasY polypeptides include those described in WO2018 / 064352.

[0264] CRISPR / Cas effector fusion polypeptides In some cases, the CRISPR / Cas effector polypeptide is: i) a CRISPR / Cas effector polypeptide; and ii) a CRISPR / Cas effector fusion polypeptide that comprises a heterologous fusion partner.

[0265] In some cases, the fusion partner can modulate (e.g., inhibit transcription, increase transcription) transcription of the target DNA. For example, in some cases, the fusion partner is a protein (or protein-derived domain) that inhibits transcription (e.g., a transcription repressor, a protein that functions via recruitment of transcription inhibitor proteins, modification of the target DNA such as methylation, recruitment of DNA modifiers, regulation of histones associated with the target DNA, recruitment of histone modifiers, e.g., factors that modify histone acetylation and / or methylation, etc.). In some cases, the fusion partner is a protein (or protein-derived domain) that increases transcription (e.g., a transcription activator, a protein that functions via recruitment of transcription activator proteins, modification of the target DNA such as demethylation, recruitment of DNA modifiers, regulation of histones associated with the target DNA, recruitment of histone modifiers, e.g., factors that modify histone acetylation and / or methylation, etc.).

[0266] In some cases, the CRISPR / Cas effector fusion polypeptide comprises a heterologous polypeptide having an enzymatic activity that modifies a target nucleic acid (e.g., a nuclease activity such as FokI nuclease activity, a methyltransferase activity, a demethylase activity, a DNA repair activity, a DNA damage activity, a deamination activity, a dismutase activity, an alkylation activity, a depurination activity, an oxidation activity, a pyrimidine dimer formation activity, an integrase activity, a transposase activity, a recombinase activity, a polymerase activity, a ligase activity, a helicase activity, a photolyase activity, or a glycosylase activity).

[0267] In some cases, a CRISPR / Cas effector fusion polypeptide comprises a heterologous polypeptide having an enzymatic activity (e.g., methyltransferase activity, demethylase activity, acetyltransferase activity, deacetylase activity, kinase activity, phosphatase activity, ubiquitin ligase activity, deubiquitinating activity, adenylating activity, deadenylating activity, SUMOylating activity, deSUMOylating activity, ribosylation activity, deribosylation activity, myristoylating activity, or demyristoylating activity) that modifies a polypeptide (e.g., a histone) associated with a target nucleic acid.

[0268] Examples of proteins (or fragments thereof) that can be used to increase transcription and are suitable as heterologous fusion partners include transcription activators such as VP16, VP64, VP48, VP160, the p65 subdomain (e.g., from NFkB), and the activation domain of EDLL and / or the TAL activation domain (e.g., for activity in plants); histone lysine methyltransferases such as SET1A, SET1B, MLL1-5, ASH1, SYMD2, NSD1; histone lysine demethylases such as JHDM2a / b, UTX, JMJD3; histone acetyltransferases such as GCN5, PCAF, CBP, p300, TAF1, TIP60 / PLIP, MOZ / MYST3, MORF / MYST4, SRC1, ACTR, P160, CLOCK; and Ten-Eleven Examples of DNA demethylases include, but are not limited to, DNA translocation (TET) dioxygenase 1 (TET1CD), TET1, DME, DML1, DML2, and ROS1.

[0269] Examples of proteins (or fragments thereof) that can be used to reduce transcription and are suitable as heterologous fusion partners include transcriptional repressors such as Kruppel-associated box (KRAB or SKD); the KOX1 repression domain; mSIN3 interacting domain (SID); ERF repression domain (ERD), SRDX repression domain (e.g., for repression in plants), etc.; histone lysine methyltransferases such as Pr-SET7 / 8, SUV4-20H1, RIZ1; histone lysine demethylases such as JMJD2A / JHDM3A, JMJD2B, JMJD2C / GASC1, JMJD2D, JARID1A / RBP2, JARID1B / PLU-1, JARID1C / SMCX, JARID1D / SMCY; histone lysine deacetylases such as HDAC1, HDAC2, HDAC3, HDAC8, HDAC4, HDAC5, HDAC7, HDAC9, SIRT1, SIRT2, HDAC11; HhaI DNA These include, but are not limited to, DNA methylases such as m5c-methyltransferase (M.HhaI), DNA methyltransferase 1 (DNMT1), DNA methyltransferase 3a (DNMT3a), DNA methyltransferase 3b (DNMT3b), METI, DRM3 (plants), ZMET2, CMT1, CMT2 (plants); and peripheral mobilization elements such as Lamin A and Lamin B.

[0270] In some cases, the fusion partner has an enzymatic activity that modifies the target nucleic acid (e.g., ssRNA, dsRNA, ssDNA, dsDNA). Examples of enzymatic activities that can be provided by the fusion partner include: nuclease activity, such as that provided by a restriction enzyme (e.g., FokI nuclease), methyltransferase activity, such as that provided by a methyltransferase (e.g., HhaI DNA m5c-methyltransferase (M.HhaI), DNA methyltransferase 1 (DNMT1), DNA methyltransferase 3a (DNMT3a), DNA methyltransferase 3b (DNMT3b), METI, DRM3 (plants), ZMET2, CMT1, CMT2 (plants), etc.); demethylase (e.g., Ten-Eleven These include, but are not limited to, demethylase activity such as that provided by TET dioxygenase 1 (TET1CD), TET1, DME, DML1, DML2, ROS1, etc.), DNA repair activity, DNA damage activity, deamination activity such as that provided by deaminases (e.g., cytosine deaminase enzymes such as rat APOBEC1), dismutase activity, alkylation activity, depurination activity, oxidation activity, pyrimidine dimer formation activity, integrase activity such as that provided by integrases and / or resolvases (e.g., Gin invertase, such as a hyperactive mutant of Gin invertase, GinH106Y; human immunodeficiency virus type 1 integrase (IN); Tn3 resolvase, etc.), transposase activity, recombinase activity such as that provided by recombinases (e.g., the catalytic domain of Gin recombinase), polymerase activity, ligase activity, helicase activity, photolyase activity, and glycosylase activity.

[0271] In some cases, the fusion partner has an enzymatic activity that modifies a protein (e.g., histone, RNA-binding protein, DNA-binding protein, etc.) associated with a target nucleic acid (e.g., ssRNA, dsRNA, ssDNA, dsDNA).Examples of enzymatic activities (which modify proteins associated with target nucleic acids) that can be provided by a fusion partner include methyltransferases (HMTs) such as those provided by suppressor of variegation 3-9 homolog 1 (SUV39H1, also known as KMT1A), euchromatin histone lysine methyltransferase 2 (G9A, also known as KMT1C and EHMT2), SUV39H2, ESET / SETDB1, etc., SET1A, SET1B, MLL1-5, ASH1, SYMD2, NSD1, DOT1L, Pr-SET7 / 8, SUV4-20H1, EZH2, RIZ1, etc. histone demethylase activity, such as that provided by histone demethylases (e.g., lysine demethylase 1A (KDM1A, also known as LSD1), JHDM2a / b, JMJD2A / JHDM3A, JMJD2B, JMJD2C / GASC1, JMJD2D, JARID1A / RBP2, JARID1B / PLU-1, JARID1C / SMCX, JARID1D / SMCY, UTX, JMJD3, etc.), histone acetyltransferase activity, acetyltransferase activity, such as that provided by acetyltransferases (e.g., catalytic cores / fragments of human acetyltransferases p300, GCN5, PCAF, CBP, TAF1, TIP60 / PLIP, MOZ / MYST3, MORF / MYST4, HBO1 / MYST2, HMOF / MYST1, SRC1, ACTR, P160, CLOCK, etc.), histone deacetylases (e.g., HDAC1, HDAC2, HDAC3, These include, but are not limited to, deacetylase activity, such as that provided by HDAC8, HDAC4, HDAC5, HDAC7, HDAC9, SIRT1, SIRT2, HDAC11, etc., kinase activity, phosphatase activity, ubiquitin ligase activity, deubiquitinating activity, adenylating activity, deadenylating activity, sumoylating activity, desumoylating activity, ribosylation activity, deribosylation activity, myristoylating activity, and demyristoylating activity.

[0272] In some cases, the fusion protein comprises: a) a catalytically inactive CRISPR / Cas effector polypeptide (e.g., a catalytically inactive Cas9 polypeptide); and b) a catalytically active endonuclease. For example, in some cases, the catalytically active endonuclease is a FokI polypeptide. As a non-limiting example, in some cases, the fusion protein comprises: a) a catalytically inactive Cas9 protein (or other catalytically inactive CRISPR effector polypeptide); and b) a FokI nuclease comprising an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the FokI amino acid sequence shown below; the length of the FokI nuclease is about 195 amino acids to about 200 amino acids.

[0273] FokI nuclease amino acid sequence: QLVKSELEEKKSELRHKLKYVPHEYIELIEIARNSTQDRILEMKVMEFFMKVYGYRGKHLGGSRKPDGAIYTVGSPIDYGVIVDTKAYSGGYNLPIGQADEMQRYVEENQTRNKHINPNEWWKVYPSSVTEFKFLFVSGHFKGNYKAQLTRLNHITNCNGAVLSVEELLIGGEMIKAGTLTLEEVRRKFNNGEINF (SEQ ID NO: 901)

[0274] In some cases, the fusion partner is a deaminase. Thus, in some cases, the CRISPR / Cas effector polypeptide fusion polypeptide comprises: a) a CRISPR / Cas effector polypeptide; and b) a deaminase. In some cases, the CRISPR / Cas effector polypeptide is catalytically inactive. Suitable deaminases include cytidine deaminase and adenosine deaminase.

[0275] A suitable adenosine deaminase is any enzyme capable of deaminating adenosine in DNA. In some cases, the deaminase is TadA deaminase.

[0276] In some cases, a suitable adenosine deaminase has the following amino acid sequence: MSEVEFSHEYWMRHALTLAKRAWDEREVPVGAVLVHNNRVIGEGWNRPIGRHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTLEPCVMCAGAMIHSRIGRVVFGARDAKTGAAGSLMDVLHHPGMNHRVEITEGILADECAALLSDFFRMRRQEIKAQKKAQSSTD (SEQ ID NO: 902) The amino acid sequence may have at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to

[0277] In some cases, a suitable adenosine deaminase has the following amino acid sequence: MRRAFITGVFFLSEVEFSHEYWMRHALTLAKRAWDEREVPVGAVLVHNNRVIGEGWNRPIGRHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTLEPCVMCAGAMIHSRIGRVVFGARDAKTGAAGSLMDVLHHPGMNHRVEITEGILADECAALLSDFFRMRRQEIKAQKKAQSSTD (SEQ ID NO: 903) The amino acid sequence may have at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to

[0278] In some cases, a suitable adenosine deaminase has the following Staphylococcus aureus TadA amino acid sequence: MGSHMTNDIYFMTLAIEEAKKAAQLGEVPIGAIITKDDEVIARAHNLRETLQQPTAHAEHIAIERAAKVLGSWRLEGCTLYVTLEPCVMCAGTIVMSRIPRVVYGADDPKGGCSGSLMNLLQQSNFNHRAIVDKGVLKEACSTLLTTFFK NLRANKKSTN: (SEQ ID NO: 904) The amino acid sequence may have at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to

[0279] In some cases, a suitable adenosine deaminase has the following Bacillus subtilis TadA amino acid sequence: MTQDELYMKEAIKEAKKAEEKGEVPIGAVLVINGEIIARAHNLRETEQRSIAHAEMLVIDEACKALGTWRLEGATLYVTLEPCPMCAGAVVLSRVEKVVFGAFDPKGGCSGTLMNLLQEERFNHQAEVVSGVLEEECGGMLSAFFRELRKKKKAARKNLSE (SEQ ID NO: 905) The amino acid sequence may have at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to

[0280] In some cases, a suitable adenosine deaminase has the following Salmonella typhimurium TadA amino acid sequence: MPPAFITGVTSLSDVELDHEYWMRHALTLAKRAWDEREVPVGAVLVHNHRVIGEGWNRPIGRHDPTAHAEIMALRQGGLVLQNYRLLDTTLYVTLEPCVMCAGAMVHSRIGRVVFGARDAKTGAAGSLIDVLHHPGMNHRVEIIEGVLRDECATLLSDFFRMRRQEIKALKKADRAEGAGPAV (SEQ ID NO: 906) The amino acid sequence may have at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to

[0281] In some cases, a suitable adenosine deaminase has the following Shewanella putrefaciens TadA amino acid sequence: MDEYWMQVAMQMAEKAEAAGEVPVGAVLVKDGQQIATGYNLSISQHDPTAHAEILCLRSAGKKLENYRLLDATLYITLEPCAMCAGAMVHSRIARVVYGARDEKTGAAGTVVNLLQHPAFNHQVEVTSGVLAEACSAQLSRFFKRRRDEKKALKLAQRAQQGIE (SEQ ID NO: 907) The amino acid sequence may have at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to

[0282] In some cases, a suitable adenosine deaminase has the following Haemophilus influenzae F3031 TadA amino acid sequence: MDAAKVRSEFDEKMMRYALELADKAEALGEIPVGAVLVDDARNIIGEGWNLSIVQSDPTAHAEIIALRNGAKNIQNYRLLNSTLYVTLEPCTMCAGAILHSRIKRLVFGASDYKTGAIGSRFHFFDDYKMNHTLEITSGVLAEECSQKLS TFFQKRREEKKIEKALLKSLSDK (sequence number 908) The amino acid sequence may have at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to

[0283] In some cases, a suitable adenosine deaminase has the following Caulobacter crescentus TadA amino acid sequence: MRTDESEDQDHRMMRLALDAARAAAEAGETPVGAVILDPSTGEVIATAGNGPIAAHDPTAHAEIAAMRAAAAKLGNYRLTDLTLVVTLEPCAMCAGAISHARIGRVVFGADDPKGGAVVHGPKFFAQPTCHWRPEVTGGVLADESADLLRGFFRARRKAKI (SEQ ID NO: 909) The amino acid sequence may have at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to

[0284] In some cases, a suitable adenosine deaminase has the following Geobacter sulfurreducens TadA amino acid sequence: MSSLKKTPIRDDAYWMGKAIREAAKAAARDEVPIGAVIVRDGAVIGRGHNLREGSNDPSAHAEMIAIRQAARRSANWRLTGATLYVTLEPCLMCMGAIILARLERVVFGCYDPKGGAAGSLYDLSADPRLNHQVRLSPGVCQEECGTMLSDFFRDLRRRKKAKATPALFIDERKVPPEP (SEQ ID NO: 910) The amino acid sequence may have at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to

[0285] Cytidine deaminases suitable for inclusion in a CRISPR / Cas effector polypeptide fusion polypeptide include any enzyme capable of deaminating cytidine in DNA.

[0286] In some cases, the cytidine deaminase is a deaminase from the apolipoprotein B mRNA editing complex (APOBEC) family of deaminases. In some cases, the APOBEC family deaminase is selected from the group consisting of APOBEC1 deaminase, APOBEC2 deaminase, APOBEC3A deaminase, APOBEC3B deaminase, APOBEC3C deaminase, APOBEC3D deaminase, APOBEC3F deaminase, APOBEC3G deaminase, and APOBEC3H deaminase. In some cases, the cytidine deaminase is an activation-induced deaminase (AID).

[0287] In some cases, a suitable cytidine deaminase has the following amino acid sequence: MDSLLMNRRKFLYQFKNVRWAKGRRETYLCYVVKRRDSATSFSLDFGYLRNKNGCHVELLFLRYISDWDLDPGRCYRVTWFTSWSPCYDCARHVADFLRGNPNLSLRIFTARLYFCEDRKAEPEGLRRLHRAGVQIAIMTFKDYFYCWNTFVENHERTFKAWEGLHENSVRLSRQLRRILLPLYEVDDLRDAFRTLGL (SEQ ID NO: 911) The amino acid sequence may have at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to

[0288] In some cases, a suitable cytidine deaminase is AID, which has the following amino acid sequence: MDSLLMNRRK FLYQFKNVRW AKGRRETYLC YVVKRRDSAT SFSLDFGYLR NKNGCHVELL FLRYISDWDL DPGRCYRVTW FTSWSPCYDC ARHVADFLRG NPNLSLRIFT ARLYFCEDRK AEPEGLRRLH RAGVQIAIMT FKENHERTFK AWEGLHENSV RLSRQLRRIL LPLYEVDDLRDAFRTLGL (SEQ ID NO: 912) The amino acid sequence may have at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to

[0289] In some cases, a suitable cytidine deaminase is AID, which has the following amino acid sequence: MDSLLMNRRK FLYQFKNVRW AKGRRETYLC YVVKRRDSAT SFSLDFGYLR NKNGCHVELL FLRYISDWDL DPGRCYRVTW FTSWSPCYDC ARHVADFLRG NPNLSLRIFT ARLYFCEDRK AEPEGLRRLH RAGVQIAIMT FKDYFYCWNT FVENHERTFK AWEGLHENSV RLSRQLRRIL LPLYEVDDLR DAFRTLGL (SEQ ID NO: 913) The amino acid sequence may have at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to

[0290] In some cases, the CRISPR / Cas effector polypeptide fusion polypeptide comprises a CRISPR / Cas effector polypeptide that exhibits nickase activity. Suitable nickases are described elsewhere herein.

[0291] In some cases, the fusion CRISPR / Cas effector polypeptide comprises one or more localization signal peptides.Suitable localization signals ("subcellular localization signals") include, for example, nuclear localization signal (NLS) for targeting the nucleus; a sequence for moving the fusion protein away from the nucleus, such as a nuclear export sequence (NES); a sequence for retaining the fusion protein in the cytoplasm; a mitochondrial localization signal for targeting the mitochondria; a chloroplast localization signal for targeting the chloroplast; an endoplasmic reticulum (ER) retention signal; and an ER export signal.In some cases, the fusion polypeptide does not comprise an NLS, so that the protein is not targeted to the nucleus (this may be advantageous, for example, when the target nucleic acid is an RNA present in the cytosol).

[0292] In some cases, the fusion polypeptide comprises (is fused to) a nuclear localization signal (NLS) (e.g., in some cases, two or more, three or more, four or more, or five or more NLSs). Thus, in some cases, the fusion polypeptide comprises one or more NLSs (e.g., two or more, three or more, four or more, or five or more NLSs). In some cases, one or more NLSs (two or more, three or more, four or more, or five or more NLSs) are positioned at or proximal to (e.g., within 50 amino acids of) the N-terminus and / or C-terminus. In some cases, one or more NLSs (two or more, three or more, four or more, or five or more NLSs) are positioned at or proximal to (e.g., within 50 amino acids of) the N-terminus. In some cases, one or more NLSs (two or more, three or more, four or more, or five or more NLSs) are positioned at or proximal to (e.g., within 50 amino acids of) the C-terminus. In some cases, one or more NLSs (three or more, four or more, or five or more NLSs) are located at or proximal to (e.g., within 50 amino acids of) both the N-terminus and the C-terminus. In some cases, one NLS is located at the N-terminus and one NLS is located at the C-terminus.

[0293] In some cases, the fusion polypeptide comprises (fuses) 1-10 NLSs (e.g., 1-9, 1-8, 1-7, 1-6, 1-5, 2-10, 2-9, 2-8, 2-7, 2-6, or 2-5 NLSs). In some cases, the fusion polypeptide comprises (fuses) 2-5 NLSs (e.g., 2-4, or 2-3 NLSs).

[0294] Non-limiting examples of NLSs include: the NLS of the SV40 virus large T antigen having the amino acid sequence PKKKRKV (SEQ ID NO: 914); an NLS from nucleoplasmin (e.g., the nucleoplasmin bipartite NLS having the sequence KRPAATKKAGQAKKKK (SEQ ID NO: 915); a c-myc NLS having the amino acid sequence PAAKRVKLD (SEQ ID NO: 916) or RQRRNELKRSP (SEQ ID NO: 917); a hRNPA1 M9 NLS having the sequence NQSSNFGPMKGGNFGGRSSGPYGGGGQYFAKPRNQGGY (SEQ ID NO: 918); the IBB domain from importin-alpha having the sequence RMRIZFKNKGKDTAELRRRRVEVSVELRKAKKDEQILKRRNV (SEQ ID NO: 919); the sequences VSRKRPRP (SEQ ID NO: 920) and PPKKARED (SEQ ID NO: 921) of the sarcoma T protein; the sequence PQPKKKPL (SEQ ID NO: 922) of human p53; the mouse c-abl IV sequence SALIKKKKKMAP (SEQ ID NO: 923); influenza virus NS1 sequences DRLRR (SEQ ID NO: 924) and PKQKKRK (SEQ ID NO: 925); hepatitis virus delta antigen sequence RKLKKKIKKL (SEQ ID NO: 926); mouse Mx1 protein sequence REKKKFLKRR (SEQ ID NO: 927); human poly(ADP-ribose) polymerase sequence KRKGDEVDGVDEVAKKKSKK (SEQ ID NO: 928); and steroid hormone receptor (human) glucocorticoid sequence RKCLQAGMNLEARKTKK (SEQ ID NO: 929). In some cases, the NLS comprises the amino acid sequence MDSLLMNRRKFLYQFKNVRWAKGRRETYLC (SEQ ID NO: 930). In general, the NLS (or NLSs) is strong enough to drive the accumulation of a detectable amount of the fusion polypeptide in the nucleus of a eukaryotic cell. Detection of accumulation in the nucleus can be performed by any suitable technique. For example, a detectable marker may be fused to the fusion polypeptide, thereby allowing visualization of its subcellular location, and the cell nucleus may be isolated from the cell and its contents may then be analyzed by any suitable process for detecting proteins, such as immunohistochemistry, Western blot, or enzyme activity assays.Accumulation in the nucleus may also be measured indirectly.

[0295] In some cases, the CRISPR / Cas effector polypeptide comprises a "protein transduction domain" or PTD (also known as CPP-cell penetrating peptide), which refers to a polypeptide, polynucleotide, carbohydrate, or organic or inorganic compound that facilitates crossing of a lipid bilayer, micelle, cell membrane, organelle membrane, or vesicle membrane. A PTD attached to another molecule and / or nanoparticle, which can range from a small polar molecule to a large macromolecule, facilitates membrane crossing of a molecule, for example, moving from the extracellular space to the intracellular space or from the cytosol into an organelle. In some embodiments, the PTD is covalently attached to the amino terminus of the polypeptide. In some embodiments, the PTD is covalently attached to the carboxy terminus of the polypeptide. In some cases, the PTD is inserted internally into the fusion polypeptide at a suitable insertion site (i.e., not at the N-terminus or C-terminus of the fusion polypeptide). In some cases, the fusion polypeptide of the present subject matter comprises (complexed, fused to) one or more PTDs (e.g., two or more, three or more, four or more PTDs). In some cases, the PTD comprises a nuclear localization signal (NLS) (e.g., in some cases, two or more, three or more, four or more, or five or more NLSs). Thus, in some cases, the fusion polypeptide comprises one or more NLSs (e.g., two or more, three or more, four or more, or five or more NLSs). In some embodiments, the PTD is covalently linked to a nucleic acid (e.g., a guide nucleic acid, a polynucleotide encoding a guide nucleic acid, a polynucleotide encoding a fusion polypeptide, a donor polynucleotide, etc.).Examples of PTDs include a minimal undecapeptide protein transduction domain (corresponding to residues 47-57 of HIV-1 TAT with YGRKKRRQRRR (SEQ ID NO: 931)); a polyarginine sequence containing a number of arginines sufficient for direct entry into a cell (e.g., 3, 4, 5, 6, 7, 8, 9, 10, or 10-50 arginine residues); a VP22 domain (Zender et al. (2002) Cancer Gene Ther. 9(6):489-96); a Drosophila Antennapedia protein transduction domain (Noguchi et al. (2003) Diabetes 52(7):1732-1737); a truncated human calcitonin peptide (Trehin et al. (2004) Pharm. Research 21:1248-1256); polylysine (Wender et al. (2000) Proc. Natl. Acad. Sci. USA 97:13003-13008;RRQRRTSKLMKR (SEQ ID NO:932);transportan GWTLNSAGYLLGKINLKALAALAKKIL (SEQ ID NO:933);KALAWEAKLAKALAKALAKHLAKALAKALKCEA (SEQ ID NO:934);and RQIKIWFQNRRMKWKK (SEQ ID NO:935).Exemplary PTDs include, but are not limited to, YGRKKRRQRRR (SEQ ID NO:936), RKKRRQRRR (SEQ ID NO:937);arginine homopolymers of 3 to 50 arginine residues; exemplary PTD domain amino acid sequences include, but are not limited to, any of the following:YGRKKRRQRRR (SEQ ID NO:938), RKKRRQRR (SEQ ID NO:939), YARAAARQARA (SEQ ID NO:940), THRLPRRRRRR (SEQ ID NO:941), and GGRRARRRRRR (SEQ ID NO:942). In some embodiments, the PTD is an activatable CPP (ACPP) (Aguilera et al. (2009) Integr Biol (Camb) June;1(5-6):371-381).ACPPs contain a polycationic CPP (e.g., Arg9 or "R9") connected to a matching polyanion (e.g., Glu9 or "E9") via a cleavable linker, which reduces the net charge to near zero, thereby inhibiting adhesion and uptake into cells. Upon cleavage of the linker, the polyanion is released, locally unmasking the polyarginine and its inherent adhesive properties, thus "activating" the ACPP to cross membranes.

[0296] Guide RNA When the target polypeptide is a CRISPR / Cas effector polypeptide, in some cases, the CRISPR / Cas effector polypeptide is complexed with a CRISPR / Cas effector polypeptide guide RNA (also referred to as a "CRISPR-Cas guide RNA").

[0297] A nucleic acid molecule that binds to a CRISPR / Cas effector polypeptide protein and targets the complex to a specific location within a target nucleic acid is referred to herein as a "CRISPR / Cas effector polypeptide guide RNA" or simply "guide RNA."

[0298] A guide RNA can be said to include two segments: a first segment (herein referred to as a "targeting segment"); and a second segment (herein referred to as a "protein-binding segment"). By "segment" is meant a segment / section / region of a molecule, e.g., a contiguous stretch of nucleotides in a nucleic acid molecule. A segment can also mean a complex region / section, such that a segment can include multiple molecular regions. The "targeting segment" is also referred to herein as the "variable region" of the guide RNA. The "protein-binding segment" is also referred to herein as the "constant region" of the guide RNA. In some cases, the guide RNA is a Cas9 guide RNA.

[0299] The first segment of the guide RNA (the targeting segment) comprises a nucleotide sequence (the guide sequence) that is complementary to (and therefore hybridizes with) a specific sequence (the target site) within the target nucleic acid (e.g., the target ssRNA, the target ssDNA, the complementary strand of a double-stranded target DNA, etc.). The protein-binding segment (or "protein-binding sequence") interacts with (binds to) a CRISPR / Cas effector polypeptide. The protein-binding segment of the guide RNA comprises two complementary stretches of nucleotides that hybridize to each other to form a double-stranded RNA duplex (dsRNA duplex). Site-specific binding and / or cleavage of the target nucleic acid (e.g., genomic DNA) can occur at a location (e.g., the target sequence of the target locus) that is determined by the base-pairing complementarity between the guide RNA (the guide sequence of the guide RNA) and the target nucleic acid.

[0300] The guide RNA and the CRISPR / Cas effector polypeptide form a complex (e.g., bind via non-covalent interactions). The guide RNA provides target specificity to the complex by including a targeting segment that includes a guide sequence (a nucleotide sequence that is complementary to a sequence of a target nucleic acid). The CRISPR / Cas effector polypeptide of the complex provides a site-specific activity (e.g., cleavage activity, or activity provided by the CRISPR / Cas effector polypeptide when the CRISPR / Cas effector polypeptide is a CRISPR / Cas effector polypeptide fusion polypeptide, i.e., has a fusion partner). In other words, the CRISPR / Cas effector polypeptide is guided to a target nucleic acid sequence (e.g., a target sequence in a chromosomal nucleic acid, e.g., a chromosome; a target sequence in an extrachromosomal nucleic acid, e.g., an episomal nucleic acid, a minicircle, ssRNA, ssDNA, etc.; a target sequence in a mitochondrial nucleic acid; a target sequence in a chloroplast nucleic acid; a target sequence in a plasmid; a target sequence in a viral nucleic acid, etc.) by association with the guide RNA.

[0301] The "guide sequence", also called the "targeting sequence" of the guide RNA, can be modified so that the guide RNA can target the CRISPR / Cas effector polypeptide to any desired sequence of any desired target nucleic acid, provided that the protospacer adjacent motif (PAM) sequence can be taken into consideration. Thus, for example, the guide RNA can have a targeting segment with a sequence (guide sequence) that has complementarity (e.g., can hybridize) to a sequence in a nucleic acid in a eukaryotic cell, such as a viral nucleic acid, a eukaryotic nucleic acid (e.g., a eukaryotic chromosome, a chromosomal sequence, a eukaryotic RNA, etc.).

[0302] In some embodiments, the guide RNA comprises two separate nucleic acid molecules: an "activator" and a "targeter," and is referred to herein as a "dual guide RNA," "dual molecule guide RNA," or "two molecule guide RNA," "dual guide RNA," or "dgRNA." In some embodiments, the activator and targeter are covalently linked to each other (e.g., via an intervening nucleotide), and the guide RNA is referred to as a "single guide RNA," "Cas9 single guide RNA," "single molecule Cas9 guide RNA," or "single molecule Cas9 guide RNA," or simply "sgRNA."

[0303] Guide RNAs include crRNA-like ("CRISPR RNA" / "targeter" / "crRNA" / "crRNA repeat") molecules and corresponding tracrRNA-like ("trans-acting CRISPR RNA" / "activator" / "tracrRNA") molecules. A crRNA-like molecule (targeter) comprises both the targeting segment (single strand) of the guide RNA and a stretch of nucleotides ("duplex-forming segment") that forms one half of the dsRNA duplex of the protein-binding segment of the guide RNA. A corresponding tracrRNA-like molecule (activator / tracrRNA) comprises a stretch of nucleotides (duplex-forming segment) that forms the other half of the dsRNA duplex of the protein-binding segment of the guide nucleic acid. In other words, the stretch of nucleotides of the crRNA-like molecule is complementary to and hybridizes with the stretch of nucleotides of the tracrRNA-like molecule to form the dsRNA duplex of the protein-binding domain of the guide RNA. Each targeter molecule can therefore be referred to as having a corresponding activator molecule (having a region that hybridizes with the targeter). The targeter molecule further provides a targeting segment. Thus, the targeter and activator molecules (as a corresponding pair) hybridize to form a guide RNA. The exact sequence of a given crRNA or tracrRNA molecule is unique to the species in which the RNA molecule is found. A dual guide RNA may contain a corresponding activator and targeter pair.

[0304] The term "activator" or "activator RNA" is used herein to refer to a tracrRNA-like molecule (tracrRNA: "trans-acting CRISPR RNA") of a dual guide RNA (and thus a single guide RNA when the "activator" and "targeter" are linked to each other, e.g., by an intervening nucleotide). Thus, for example, a guide RNA (dgRNA or sgRNA) comprises an activator sequence (e.g., a tracrRNA sequence). A tracr molecule (tracrRNA) is a naturally occurring molecule that hybridizes with a CRISPR RNA molecule (crRNA) to form a dual guide RNA. The term "activator" is used herein to encompass naturally occurring tracrRNA, but also tracrRNAs with modifications (e.g., truncations, sequence mutations, base modifications, backbone modifications, linkage modifications, etc.) where the activator retains at least one function of the tracrRNA (e.g., contributes to the dsRNA duplex to which the Cas9 protein binds). In some cases, the activator provides one or more stem loops that can interact with the Cas9 protein. An activator can be referred to as having a tracr sequence (tracrRNA sequence), and in some cases the activator is a tracrRNA, although the term "activator" is not limited to naturally occurring tracrRNA.

[0305] The term "targeter" or "targeter RNA" is used herein to refer to a crRNA-like molecule (crRNA: "CRISPR RNA") of a dual guide RNA (and thus of a single guide RNA, where the "activator" and the "targeter" are linked together, for example, by an intervening nucleotide). Thus, for example, a guide RNA (dgRNA or sgRNA) comprises a targeting segment (comprising nucleotides that hybridize (are complementary to) the target nucleic acid), and a duplex-forming segment (e.g., a duplex-forming segment of the crRNA, which may also be referred to as a crRNA repeat). The sequence of the targeter's targeting segment (the segment that hybridizes with the target sequence of the target nucleic acid) is modified by the user to hybridize with the desired target nucleic acid, and therefore the sequence of the targeter is often a non-natural sequence. However, the duplex-forming segment of the targeter (described in more detail below) that hybridizes with the duplex-forming segment of the activator may comprise a natural sequence (e.g., may comprise the sequence of the duplex-forming segment (also referred to as a crRNA repeat) of a natural crRNA). Thus, the term targeter is used herein to distinguish it from a natural crRNA, despite the fact that a part of the targeter (e.g., a duplex-forming segment) often comprises a natural sequence derived from the crRNA. However, the term "targeter" includes a natural crRNA.

[0306] A guide RNA may also be referred to as comprising three portions: (i) a targeting sequence (a nucleotide sequence that hybridizes with a sequence of a target nucleic acid); (ii) an activator sequence (as described above) (in some cases, referred to as a tracr sequence); and (iii) a sequence that hybridizes to at least a portion of the activator sequence to form a duplex. A targeter has (i) and (iii); whereas an activator has (ii).

[0307] A guide RNA (e.g., dual guide RNA or single guide RNA) may be composed of a corresponding activator and targeter pair. In some cases, the duplex-forming segment may be exchanged between the activator and the targeter. In other words, in some cases, the targeter contains a nucleotide sequence from the duplex-forming segment of the tracrRNA (this sequence would normally be part of the activator), while the activator contains a nucleotide sequence from the duplex-forming segment of the crRNA (this sequence would normally be part of the targeter).

[0308] As mentioned above, the targeter comprises both the targeting segment (single strand) of the guide RNA and a stretch of nucleotides ("duplex forming segment") that forms one half of the dsRNA duplex of the protein binding segment of the guide RNA. The corresponding tracrRNA-like molecule (activator) comprises a stretch of nucleotides (duplex forming segment) that forms the other half of the dsRNA duplex of the protein binding segment of the guide RNA. In other words, the stretch of nucleotides of the targeter is complementary to the stretch of nucleotides of the activator and hybridizes with it to form the dsRNA duplex of the protein binding segment of the guide RNA. Thus, each targeter may be referred to as having a corresponding activator (having a region that hybridizes with the targeter). The targeter molecule further provides a targeting segment. Thus, the targeter and activator (as a corresponding pair) hybridize to form the guide RNA. The specific sequence of a given naturally occurring crRNA or tracrRNA molecule is specific to the species in which that RNA molecule is found. Examples of suitable activators and targeters are well known in the art.

[0309] Nucleic acid modification In some cases, the CRISPR-Cas guide RNA has one or more modifications, e.g., base modifications, backbone modifications, sugar modifications, etc., to provide the nucleic acid with new or enhanced properties (e.g., improved stability).

[0310] Suitable nucleic acid modifications include, but are not limited to, 2'O-methyl modified nucleotides, 2'fluoro modified nucleotides, locked nucleic acid (LNA) modified nucleotides, peptide nucleic acid (PNA) modified nucleotides, nucleotides having phosphorothioate linkages, and 5' caps (e.g., 7-methylguanylate cap (m7G)).

[0311] Suitable modified nucleic acid backbones containing an internal phosphate atom include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkyl phosphotriesters, methyl and other alkyl phosphonates (including 3'-alkylene phosphonates, 5'-alkylene phosphonates, and chiral phosphonates), phosphinates, phosphoramidates (including 3'-amino phosphoramidates and aminoalkyl phosphoramidates), phosphorodiamidates, thionophosphoramidates, thionoalkyl phosphonates, thionoalkyl phosphotriesters, selenophosphates, and boranophosphates having normal 3'-5' linkages, their 2'-5' linked analogs, and those with inverted polarity where one or more internucleotide linkages are 3'-3', 5'-5' or 2'-2' linkages. Preferred oligonucleotides with inverted polarity contain a single 3'-3' linkage at the 3'-most internucleotide linkage, i.e., a single inverted nucleoside residue that may be basic (either lacking a nucleobase or having a hydroxyl group instead).Various salts (e.g., potassium or sodium), mixed salts, and free acid forms are also included.

[0312] In some cases, the CRISPR-Cas guide RNA has one or more nucleotides linked by phosphorothioate bonds (i.e., the subject nucleic acid has one or more phosphorothioate linkages). Phosphorothioate (PS) linkages (i.e., phosphorothioate linkages) replace non-bridging oxygen in the phosphate backbone of a nucleic acid (e.g., an oligo) with a sulfur atom. This modification renders the linkage between nucleotides resistant to nuclease degradation. Phosphorothioate linkages can be introduced between the last 3-5 nucleotides at the 5' or 3' end of an oligo to inhibit exonuclease degradation. Including phosphorothioate linkages in an oligo (e.g., throughout the oligo) also helps reduce attack by endonucleases.

[0313] Also suitable is the CRISPR-Cas guide RNA with morpholino backbone structure, for example as described in US Patent No. 5,034,506.For example, in some embodiments, the CRISPR-Cas guide RNA comprises a 6-membered morpholino ring instead of a ribose ring.In some of these embodiments, phosphorodiamidate or other non-phosphodiester internucleoside linkages replace phosphodiester linkages.

[0314] CRISPR-Cas guide RNAs can also include one or more substituted sugar moieties. Suitable polynucleotides include sugar substituents selected from: OH; F; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S-, or N-alkynyl; or O-alkyl-O-alkyl, where the alkyl, alkenyl, and alkynyl are substituted or unsubstituted C. 1 ~C 10 Alkyl or C 2 ~C 10 It may be alkenyl or alkynyl. O((CH 2 ) n O) m CH 3 , O(CH 2 ) n OCH 3 , O(CH2 ) n NH 2 , O(CH 2 ) n CH 3 , O(CH 2 ) n O.N.H. 2 , and O(CH 2 ) n ON((CH 2 ) n CH 3 ) 2 is particularly preferred, where n and m are from 1 to about 10. Other preferred polynucleotides include 1 ~C 10 Lower alkyl, substituted lower alkyl, alkenyl, alkynyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH 3 , OCN, Cl, Br, CN, CF 3 , OCF 3 , SOCH 3 , S.O. 2 CH 3 , O.N.O. 2 , NO 2 , N 3 , N.H. 2 , heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, RNA cleaving groups, reporter groups, intercalators, groups for improving the pharmacokinetic properties of oligonucleotides, or groups for improving the pharmacological properties of oligonucleotides, and other substituents with similar properties. Suitable modifications include 2'-methoxyethoxy (2'-O-CH 2 CH 2 OCH 3 , also known as 2'-O-(2-methoxyethyl) or 2'-MOE (Martin et al., Helv. Chim. Acta, 1995, 78, 486-504), i.e., alkoxyalkoxy groups. Further suitable modifications include 2'-dimethylaminooxyethoxy, i.e., O(CH 2 ) 2 ON(CH 3 ) 2The groups 2'-dimethylaminoethoxyethoxy (also known in the art as 2'-O-dimethyl-amino-ethoxy-ethyl or 2'-DMAEOE), i.e., 2'-O-CH 2 -O-CH 2 -N(CH 3 ) 2 Examples include:

[0315] Methods for coupling two proteins via a coupling protein The present disclosure provides a method for chemoselective coupling of a first polypeptide and a second polypeptide via a coupling polypeptide. The product of the chemoselective coupling may comprise, in order from N-terminus to C-terminus: i) the first polypeptide; ii) the coupling polypeptide; and iii) the second polypeptide. The method exploits the substrate selectivity of the tyrosinase polypeptide, as described above.

[0316] For example, in some cases, the disclosure provides a method for chemoselectively coupling a first polypeptide and a second polypeptide to a coupling polypeptide, the method comprising: a) contacting a first polypeptide with a coupling polypeptide to generate a first polypeptide-coupling polypeptide complex, the first polypeptide comprising a thiol moiety (e.g., Cys, where Cys can be present at any solvent accessible position within the first polypeptide), the coupling polypeptide comprising an N-terminal reactive moiety that forms a covalent bond with a thiol moiety present on the first polypeptide, the coupling polypeptide comprising the N-terminal reactive moiety being generated by reacting a polypeptide comprising an N-terminal phenol or catechol moiety and a C-terminal phenol or catechol moiety with a first enzyme capable of oxidizing the N-terminal phenol or catechol moiety but not the C-terminal phenol or catechol moiety to generate the N-terminal reactive moiety (a "coupling precursor polypeptide"); the ring polypeptide comprises two or more positively charged or neutral amino acids within 10 amino acids of the N-terminal phenol or catechol moiety and two or more negatively charged amino acids within 10 amino acids of the C-terminal phenol or catechol moiety; the method also comprises b) contacting a second polypeptide with the first polypeptide-coupling polypeptide complex, the second polypeptide comprising a thiol moiety (e.g., Cys, where Cys can be present at any solvent accessible position within the second polypeptide), the first polypeptide-coupling polypeptide complex comprising a C-terminal reactive moiety that forms a covalent bond with the thiol moiety present on the second polypeptide, the first polypeptide-coupling polypeptide complex comprising a C-terminal reactive moiety being generated by reaction of the first polypeptide-coupling polypeptide complex with a second enzyme capable of oxidizing the C-terminal phenol or catechol moiety to generate the C-terminal reactive moiety; said contacting generates a first polypeptide-coupling polypeptide-second polypeptide complex.In some cases, the first enzyme is a tyrosinase polypeptide comprising an amino acid sequence having at least 75% amino acid sequence identity to the amino acid sequence of abTYR shown in FIG. 8 or FIG. 9; and the second enzyme is a tyrosinase polypeptide comprising an amino acid sequence having at least 75% amino acid sequence identity to any one of the amino acid sequences shown in any one of FIGs. 10A-10Z and 10AA-10VV.

[0317] As another example, in some cases, the disclosure provides a method of chemoselectively coupling a first polypeptide and a second polypeptide to a coupling polypeptide, the method comprising: a) contacting a first polypeptide with a coupling polypeptide to generate a first polypeptide-coupling polypeptide complex, wherein the first polypeptide comprises a thiol moiety, and the coupling polypeptide comprises an N-terminal reactive moiety that forms a covalent bond with a thiol moiety present on the first polypeptide, and the coupling polypeptide comprising the N-terminal reactive moiety is generated by reacting a polypeptide comprising an N-terminal phenol or catechol moiety and a C-terminal phenol or catechol moiety with a first enzyme capable of oxidizing the N-terminal phenol or catechol moiety but not the C-terminal phenol or catechol moiety to generate the N-terminal reactive moiety; comprises two or more negatively charged amino acids within 10 amino acids of an N-terminal phenol or catechol moiety and two or more positively charged or neutral amino acids within 10 amino acids of a C-terminal phenol or catechol moiety; the method also comprises b) contacting a second polypeptide with the first polypeptide-coupling polypeptide complex, the second polypeptide comprising a thiol moiety, the first polypeptide-coupling polypeptide complex comprising a C-terminal reactive moiety that forms a covalent bond with a thiol moiety present on the second polypeptide, the first polypeptide-coupling polypeptide complex comprising a C-terminal reactive moiety being produced by reaction of the first polypeptide-coupling polypeptide complex with a second enzyme capable of oxidizing the C-terminal phenol or catechol moiety to produce a C-terminal reactive moiety; said contacting produces a first polypeptide-coupling polypeptide-second polypeptide complex.In some cases, the first enzyme is a tyrosinase polypeptide comprising an amino acid sequence having at least 75% amino acid sequence identity to any one of the amino acid sequences depicted in any one of Figures 10A-10Z and 10AA-10VV; and the second enzyme is a tyrosinase polypeptide comprising an amino acid sequence having at least 75% amino acid sequence identity to the amino acid sequence of abTYR depicted in Figure 8 or Figure 9.

[0318] The coupling polypeptide may have a length of 10 amino acids to 100 amino acids, or more than 100 amino acids. In some cases, the coupling polypeptide has a length of 10 amino acids to 25 amino acids. In some cases, the coupling polypeptide has a length of 25 amino acids to 50 amino acids. In some cases, the coupling polypeptide has a length of 50 amino acids to 100 amino acids. In some cases, the coupling polypeptide has a length of more than 100 amino acids; for example, in some cases, the coupling polypeptide has a length of 100 amino acids to 200 amino acids, 200 amino acids to 500 amino acids, or more than 500 amino acids (e.g., 500 to 1000, 1000 to 2000, or more than 2000 amino acids). In some cases, both the N-terminal phenolic moiety and the C-terminal phenolic moiety are tyrosine, and the enzyme that generates the reactive moiety is tyrosinase.

[0319] As noted above, in some cases, the coupling polypeptide comprises: a) two or more negatively charged amino acids within 10 amino acids of the N-terminal phenolic or catechol moiety; or b) two or more negatively charged amino acids within 10 amino acids of the C-terminal phenolic or catechol moiety. In such cases, the coupling polypeptide may comprise: a) 2, 3, 4, 5, 6, 7, 8, 9, or 10 negatively charged amino acids within 10 amino acids of the N-terminal phenolic or catechol moiety; or b) 2, 3, 4, 5, 6, 7, 8, 9, or 10 negatively charged amino acids within 10 amino acids of the C-terminal phenolic or catechol moiety. As a non-limiting example, the coupling polypeptide may comprise the amino acid sequence: YEEEE(X) n RRRRY (SEQ ID NO: 961), where X is any amino acid and n is an integer from 0 to 40 (e.g., where n is an integer from 0 to 5, 5 to 10, 10 to 15, 15 to 20, 20 to 25, 25 to 30, 30 to 35, or 35 to 40). n KKKKY (SEQ ID NO: 962), where X is any amino acid and n is an integer from 0 to 40 (e.g., where n is an integer from 0 to 5, 5 to 10, 10 to 15, 15 to 20, 20 to 25, 25 to 30, 30 to 35, or 35 to 40).

[0320] As noted above, in some cases, the coupling polypeptide comprises: a) two or more positively charged amino acids within 10 amino acids of the N-terminal phenolic or catechol moiety; or b) two or more positively charged amino acids within 10 amino acids of the C-terminal phenolic or catechol moiety. In such cases, the coupling polypeptide may comprise: a) 2, 3, 4, 5, 6, 7, 8, 9, or 10 positively charged amino acids within 10 amino acids of the N-terminal phenolic or catechol moiety; or b) 2, 3, 4, 5, 6, 7, 8, 9, or 10 positively charged amino acids within 10 amino acids of the C-terminal phenolic or catechol moiety. As a non-limiting example, the coupling polypeptide may comprise the amino acid sequence: YKKKK(X)n DDDDY (SEQ ID NO: 963), where X is any amino acid and n is an integer from 0 to 40 (e.g., where n is an integer from 0 to 5, 5 to 10, 10 to 15, 15 to 20, 20 to 25, 25 to 30, 30 to 35, or 35 to 40). n EEEEY (SEQ ID NO: 964), where X is any amino acid and n is an integer from 0 to 40 (e.g., where n is an integer from 0 to 5, 5 to 10, 10 to 15, 15 to 20, 20 to 25, 25 to 30, 30 to 35, or 35 to 40).

[0321] The present disclosure provides a coupling polypeptide as described above. The present disclosure provides a composition comprising a coupling polypeptide of the present disclosure. The present disclosure provides a composition comprising: a) a coupling polypeptide of the present disclosure; and b) a buffer.

[0322] Suitable first and second polypeptides include any of the polypeptides described above. For example, in some cases, the first and / or second polypeptide is an antibody (e.g., a single chain antibody). As another example, in some cases, the first and / or second polypeptide is a CRISPR / Cas effector polypeptide. As an example, in some cases, the first polypeptide is a CRISPR / Cas effector polypeptide; the second polypeptide is an Ig Fc polypeptide. As another example, in some cases, the first polypeptide is a CRISPR / Cas effector polypeptide; the second polypeptide is a nanobody. As another example, in some cases, the first polypeptide is a CRISPR / Cas effector polypeptide; the second polypeptide is an scFv polypeptide.

[0323] Methods for linking two or more polypeptides The present disclosure provides a method for sequentially coupling two or more polypeptides to each other. The method utilizes the substrate selectivity of tyrosinase polypeptides as described above. The method can be carried out on an immobilized surface such as an insoluble substrate, i.e., a bead. The method of the present disclosure for sequentially coupling two or more polypeptides to each other is shown in schematic form in Figures 39A-39G.

[0324] Accordingly, the disclosure provides a method of covalently linking a first polypeptide to a second polypeptide, the method comprising: a) contacting the first polypeptide with an immobilized reactive moiety, wherein the immobilized reactive moiety is generated by reaction of an immobilized phenolic or catechol moiety with a first enzyme; the first enzyme is capable of oxidizing the immobilized phenolic or catechol moiety, thereby generating the immobilized reactive moiety, the first polypeptide comprising: i) a thiol moiety; and ii) a phenolic or catechol moiety, wherein the first polypeptide comprises two or more negatively charged amino acids within 10 amino acids of the phenolic or catechol moiety, and the immobilized reactive moiety forms a covalent bond with a thiol moiety present on the first polypeptide, thereby generating the immobilized first polypeptide; the method also comprises: b) contacting the first polypeptide with an immobilized reactive moiety; and c) contacting the immobilized first polypeptide with a second enzyme, which oxidizes a phenolic or catechol moiety present on the first polypeptide to generate an immobilized first polypeptide comprising a reactive moiety; the method also includes c) contacting the immobilized first polypeptide comprising a reactive moiety with a second polypeptide, the second polypeptide comprising: i) a thiol moiety; and ii) a phenolic or catechol moiety, the second polypeptide having two or more neutral or positive charges within 10 amino acids of the phenolic or catechol moiety, the reactive moiety present on the immobilized first polypeptide forming a covalent bond with the thiol moiety present on the second polypeptide, thereby generating an immobilized complex comprising the first polypeptide covalently bound to the second polypeptide. In some cases, the first enzyme is a tyrosinase polypeptide comprising an amino acid sequence having at least 75% amino acid sequence identity to any one of the amino acid sequences depicted in any one of Figures 8, 9, 10A-10Z, and 10AA-10VV. In some cases, the thiol moiety present in the first polypeptide is present at a Cys (e.g., a solvent-accessible Cys; e.g., an N-terminal Cys) and the phenol moiety present in the first polypeptide is present at a Tyr residue.In some cases, the Tyr residue is present in a stretch of amino acids comprising EEEY (SEQ ID NO:953), EEEEY (SEQ ID NO:955), DDDDY (SEQ ID NO:965), or DDDDY (SEQ ID NO:965). In some cases, the second enzyme is a tyrosinase polypeptide comprising an amino acid sequence having at least 75% amino acid sequence identity to any one of the amino acid sequences depicted in any one of Figures 10A-10Z and 10AA-10VV. In some cases, the second enzyme is a tyrosinase polypeptide comprising amino acids having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the amino acid sequence depicted in Figure 10M, wherein the tyrosinase polypeptide comprises a D55 substitution (e.g., comprises a D55K substitution).

[0325] This method can be used to sequentially link any number of polypeptides. For example, in some cases, the method further comprises c) contacting the immobilized complex with a third enzyme, which can oxidize a phenolic or catechol moiety present in the second polypeptide to generate an immobilized complex comprising a reactive moiety; the method also comprises d) contacting the immobilized complex comprising a reactive moiety with a third polypeptide, the third polypeptide comprising: i) a thiol moiety; and ii) a phenolic or catechol moiety, the third polypeptide having two or more negative charges within 10 amino acids of the phenolic or catechol moiety, the reactive moiety present in the immobilized complex forms a covalent bond with the thiol moiety present in the second polypeptide, thereby generating an immobilized complex comprising the third polypeptide covalently linked to the second polypeptide. In some cases, the third enzyme is a tyrosinase polypeptide comprising an amino acid sequence having at least 75% amino acid sequence identity to the amino acid sequence shown in FIG. 8 or FIG. 9.

[0326] A polypeptide substrate can be sequentially added to an immobilized complex comprising one, two, three, or more polypeptides by alternating between a) a tyrosinase enzyme that preferentially modifies Tyr residues that are present in a negatively charged environment (e.g., the polypeptide has two or more negative charges within 10 amino acids of the Tyr residue); and b) a tyrosinase enzyme that preferentially modifies Tyr residues that are present in a neutral or positively charged environment (e.g., the polypeptide has two or more neutral or positive charges within 10 amino acids of the Tyr residue). The above method can be modified, for example, such that a first polypeptide comprises: i) a thiol moiety; and ii) a phenolic or catechol moiety, where the first polypeptide comprises two or more neutral or positively charged amino acids within 10 amino acids of the phenolic or catechol moiety; in such a case, a second polypeptide comprises: i) a thiol moiety; and ii) a phenolic or catechol moiety, where the first polypeptide comprises two or more negatively charged amino acids within 10 amino acids of the phenolic or catechol moiety.

[0327] In some cases, between any two steps of the method, the tyrosinase enzyme is inactivated or removed before further addition of the tyrosinase enzyme. For example, in some cases, between step (b) and step (c) of the above method, the second enzyme is inactivated or removed. In some cases, the thiol moiety present in the second polypeptide is present in a Cys and the phenol moiety present in the second polypeptide is present in a Tyr residue. In some cases, the Tyr residue is present in a stretch of amino acids that includes RRRY (SEQ ID NO: 949), RRRRY (SEQ ID NO: 951), KKKY (SEQ ID NO: 966), or KKKKY (SEQ ID NO: 967).

[0328] As shown generally in FIG. 39A, abTYR is used to link biotin-phenol to a first polypeptide ("protein A") that includes a thiol group and an EEEEY (SEQ ID NO: 955) sequence to generate a biotin-first polypeptide complex. The biotin-first polypeptide complex can be contacted with streptavidin beads to immobilize the biotin-first polypeptide complex. A second polypeptide ("protein B") that includes a thiol group and an RRRRY (SEQ ID NO: 951) sequence can be conjugated to the first polypeptide of the immobilized biotin-first polypeptide complex by the action of bmTYR(D55K) (e.g., bmTYR(D55K) shown in FIG. 10M) to generate an immobilized first polypeptide-second polypeptide complex. In some cases, two different polypeptides (e.g., "protein A" and "protein B") are added alternately to the concatemer, as shown in FIG. 39A, FIG. 39B, and FIG. 39C. Alternatively, multiple copies of a single polypeptide are concatenated, as shown in Figures 39D and 39E. As yet another possibility, in some cases, each of the polypeptides to be concatenated is different from the others (e.g., "protein A," "protein B," and "protein C"), as shown in Figures 39F and 39G.

[0329] composition An embodiment of the present disclosure relates to a method for the preparation of a thiol-containing targeting molecule of formula (III) and a thiol-containing targeting molecule of formula (I): and a biomolecule comprising a phenolic or catechol moiety of TIFF2025029024000037.tif17128; wherein Y1 is a biomolecule, optionally comprising one or more moieties selected from an active small molecule, an affinity tag, a fluorophore, and a metal chelator; L is an optional linker; X1 is selected from hydrogen and hydroxyl; and Y2 is a second biomolecule.

[0330] In certain embodiments, compositions are provided of a targeting molecule comprising a thiol of formula (III) and a pharma- ceutically acceptable excipient.

[0331] In certain embodiments, compositions of biomolecules comprising a phenolic or catechol moiety of formula (I) and a pharma- ceutically acceptable excipient are provided.

[0332] In certain embodiments of the subject compositions, Y 2 is a CRISPR-Cas effector polypeptide, e.g., as described herein.

[0333] In certain embodiments of the subject compositions, formula (I) is described by any of formulas (IA), (IAa), (IB), (IC), (ID), (IDa) and (IDb), as disclosed herein.

[0334] The subject compositions generally include a subject target molecule comprising a thiol of formula (III); a biomolecule comprising a phenolic or catechol moiety of formula (I); and at least one additional compound. Suitable additional compounds include: salts, such as magnesium salts, sodium salts, and the like, e.g., NaCl, MgCl 2 , KCl, MgSO 4 etc.; buffering agents, such as Tris buffer, N-(2-hydroxyethyl)piperazine-N'-(2-ethanesulfonic acid) (HEPES), 2-(N-morpholino)ethanesulfonic acid (MES), 2-(N-morpholino)ethanesulfonic acid sodium salt (MES), 3-(N-morpholino)propanesulfonic acid (MOPS), N-tris[hydroxymethyl]methyl-3-aminopropanesulfonic acid (TAPS), etc.; solubilizing agents; surfactants, such as non-ionic surfactants such as Tween-20; protease inhibitors, etc.

[0335] In some embodiments, the subject compositions include a subject target molecule comprising a thiol of formula (III); a biomolecule comprising a phenolic or catechol moiety of formula (I); and a pharmaceutically acceptable excipient. A wide variety of pharmaceutically acceptable excipients are known in the art and need not be described in detail herein. Pharmaceutically acceptable excipients can be found, for example, in A. Gennaro (2000) "Remington: The Science and Practice of Pharmacy," 2000, 1999, 144:131-135, 1999. th edition, Lippincott, Williams, & Wilkins;Pharmaceutical Dosage Forms and Drug Delivery Systems(1999)HCAnsel et al.,eds.,7 th ed., Lippincott, Williams, & Wilkins; and Handbook of Pharmaceutical Excipients (2000) AHKibbe et al., eds., 3 rd These are described in detail in various publications, including ed. Amer. Pharmaceutical Assoc.

[0336] Pharmaceutically acceptable excipients, such as vehicles, adjuvants, carriers or diluents, are generally readily available. Moreover, pharma- ceutically acceptable auxiliary substances, such as pH adjusting and buffering agents, isotonicity agents, stabilizers, wetting agents, and the like, are generally readily available.

[0337] kit The compounds and compositions described herein can be packaged as kits, which can optionally include instructions for using the compounds or compositions in various exemplary applications. Non-limiting examples include kits that include, for example, a powder or lyophilized form of the compound or composition, and instructions for use, including reconstitution, dosage information, and storage information for use in the subject methods. The kits can optionally include containers of the compounds or compositions in a ready-to-use liquid form, or that require further mixing with a solution for administration.

[0338] An embodiment of the present disclosure includes a kit comprising a first container comprising a composition comprising a subject target molecule comprising a thiol of formula (III) and a biomolecule comprising a phenolic moiety of formula (I); and a second container comprising an enzyme capable of oxidizing the phenolic moiety or the catechol moiety. In certain cases, the enzyme is a tyrosinase enzyme.

[0339] In certain embodiments, the subject kits include a first container comprising a subject target molecule comprising a thiol of formula (III), a second container comprising a biomolecule comprising a phenolic or catechol moiety of formula (I), and a third container comprising an enzyme capable of oxidizing the phenolic or catechol moiety. In certain cases, the enzyme is a tyrosinase enzyme.

[0340] The kits can include optional components to aid in the subject methods, such as vials for reconstituting powder forms. The kits may be provided in either containers with a seal (e.g., a crimped septum seal closure) suitable for single or multiple puncture by a hypodermic needle while maintaining sterile integrity. The kit components may be assembled into cartons, blister packs, bottles, tubes, and the like.

[0341] In addition to the above-mentioned components, the kit of the present invention may further include instructions for carrying out the method of the present invention. These instructions may be present in the subject kit in various forms, one or more of which may be present in the kit. One form in which these instructions may be present is instructions printed on a suitable medium or material, such as one or more pieces of paper on which the information is printed, placed in the kit packaging, packaging insert, etc. Another means would be a computer readable medium on which the information is recorded and stored, such as a CD, DVD, Blu-Ray, computer readable memory, etc. Yet another means that may be present is a website address, such as a link to a website to download a suitable smartphone app for use in detecting the functional dye, which may be utilized to access the information on the site relocated via the Internet. Any convenient means may be present in the kit.

[0342] usefulness The subject compounds, compositions, kits, and subject modification methods are useful in a variety of applications, including research and diagnostic applications.

[0343] Research applications of interest include any application involving the selective manipulation of target molecules, biomolecules, cells, particles or surfaces, including the manipulation, tagging and tracking of biomolecules (e.g. proteins) in vitro.

[0344] The subject methods and compositions can also be used in therapeutic applications, for example therapeutic applications of interest include those applications where antibody drug conjugates (ADCs) are available (e.g., novel immunotherapies), protein delivery for gene therapy, and vaccine development.

[0345] Method for screening tyrosinase variants The present disclosure provides a method for identifying tyrosinase variants with a preference for a particular substrate, which can provide for the identification of tyrosinase variants with a preference for phenols or catechols present in a particular sequence in a negatively or positively charged environment. The method generally includes: a) contacting a peptide with a test tyrosinase and thiol-modified biotin (biotin-thiol), where the peptide has a length of about 4 amino acids to about 25 amino acids (e.g., about 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15-20, or 20-25 amino acids) and where the peptide has a C-terminal Tyr residue, a biotin-peptide complex is formed; the method also includes b) contacting the biotin-peptide complex with streptavidin-conjugated beads (e.g., streptavidin conjugated to magnetic beads) to form a streptavidin-biotin-peptide complex; and c) determining the amino acid sequence of the peptide in the streptavidin-biotin-peptide complex. In some cases, the method further includes washing the streptavidin-biotin-peptide complex to remove unbound peptides (peptides not conjugated to biotin). In some cases, the method further comprises releasing the peptide from the streptavidin-biotin-peptide complex before determining the amino acid sequence of the peptide. The peptide can be released (eluted) from the streptavidin-biotin-peptide complex by incubating the streptavidin-biotin-peptide complex in a mixture containing an excess of free biotin, acetonitrile, and formic acid (e.g., 80% acetonitrile, 5% formic acid, and 2 mM biotin). The amino acid sequence of the peptide (e.g., eluted peptide) present in the streptavidin-biotin-peptide complex can be determined using any of a variety of well-known methods, including, for example, mass spectrometry (MS) (e.g., tandem MS). A library of peptides can be used to determine whether the test tyrosinase has a preference for a particular amino acid sequence, a negatively charged environment, or a positively charged environment.

[0346] Examples of Non-Limiting Aspects of the Disclosure Aspect A The aspects including the embodiments of the subject matter described above may be effective alone or in combination with one or more other aspects or embodiments. Without being limited to the above description, certain non-limiting aspects of the present disclosure numbered 1 to 39 are set forth below. As will be apparent to one skilled in the art upon reading this disclosure, each of the individually numbered aspects may be used or combined with any of the preceding or succeeding individually numbered aspects. This is intended to provide support for all such combinations of aspects, and is not intended to be limited to the combinations of aspects explicitly provided below.

[0347] Embodiment 1. A method for chemoselectively modifying a target molecule, the method comprising: contacting a target molecule comprising a thiol moiety with a biomolecule comprising a reactive moiety; wherein the biomolecule comprising the reactive moiety is produced by reaction of a biomolecule comprising a phenol or catechol moiety with an enzyme capable of oxidizing the phenol or catechol moiety; and wherein the contacting is performed under conditions sufficient for complexation of the target molecule with the biomolecule, thereby producing a modified target molecule.

[0348] The method of embodiment 1, wherein the target molecule is a polypeptide.

[0349] Embodiment 3 The method of embodiment 1 or 2, wherein the enzyme is a tyrosinase enzyme.

[0350] Embodiment 4. The method of any one of embodiments 1 to 3, wherein the enzyme is bound to a solid support.

[0351] Embodiment 5. The method of any one of embodiments 1 to 4, wherein the phenol moiety is present in a tyrosine residue.

[0352] Embodiment 6 The method of any one of embodiments 1 to 5, wherein the thiol moiety is present in a cysteine ​​residue.

[0353] The method of claim 6, wherein the cysteine ​​residue is a naturally occurring cysteine ​​residue.

[0354] Embodiment 8. The method of any one of embodiments 1 to 7, wherein the biomolecule comprises one or more moieties selected from a fluorophore, an active small molecule, an affinity tag, and a metal chelator.

[0355] Embodiment 9. The method of any one of embodiments 1 to 8, wherein the reactive moiety is an orthoquinone or semiquinone radical, or a combination thereof.

[0356] Aspect 10. The method according to any one of aspects 1 to 9, wherein the biomolecule is a polypeptide.

[0357] Aspect 11. The method of aspect 10, wherein the biological molecule is a polypeptide selected from a fluorescent protein, an antibody, an enzyme, a ligand for a receptor, and a receptor.

[0358] Embodiment 12. The biomolecule comprising a phenolic or catechol moiety is a molecule of formula (I) and the biomolecule comprising a reactive moiety is a molecule of formula (II) or (IIA) or a combination thereof: TIFF2025029024000038.tif25130 formula: Y 1 is a biomolecule, optionally comprising one or more moieties selected from an active small molecule, an affinity tag, a fluorophore, and a metal chelator; X 1 is selected from hydrogen and hydroxyl; and The method of any one of embodiments 1-11, wherein L is an optional linker.

[0359] Embodiment 13. The targeting molecule comprising a thiol moiety is a molecule of formula (III) and the modified targeting molecule is a molecule of formula (IV) or (IVA), or a combination thereof: TIFF2025029024000039.tif29128 formula: Y 1 is a biomolecule, optionally comprising one or more moieties selected from an active small molecule, an affinity tag, a fluorophore, and a metal chelator; Y 2 is a second biological molecule; L is an optional linker; and The method according to any one of aspects 1 to 12, wherein n is an integer from 1 to 3.

[0360] Aspect 14. The modified target molecule of formula (IV) is represented by the formulas (IV1) to (IV3): TIFF2025029024000040.tif32146; The modified target molecule of formula (IVA) is represented by the formulas (IVA1) to (IVA3): 14. The method of claim 13, wherein the molecule is any one of TIFF2025029024000041.tif32131.

[0361] Aspect 15. The modified target molecule of formula (IV) is represented by formula (IV5) to (IV6): TIFF2025029024000042.tif32128; The modified target molecule of formula (IVA) is represented by the formulas (IVA4) to (IVA5): 14. The method of claim 13, wherein the molecule is any of the molecules in TIFF2025029024000043.tif32128.

[0362] Embodiment 16. The biomolecule comprising a phenol or catechol moiety has the formula (IA): Described by TIFF2025029024000044.tif26128, During the ceremony: Y 1 is a biomolecule, optionally comprising one or more groups selected from an active small molecule, an affinity tag, a fluorophore, and a metal chelator; Each R 1is independently selected from hydrogen, acyl, substituted acyl, alkyl, and substituted alkyl; X 1 is selected from hydrogen and hydroxyl; and L 1 is a linker selected from linear or branched alkyl, linear or branched substituted alkyl, polyethylene glycol (PEG), substituted PEG, and one or more peptides; The method according to any one of aspects 1 to 15.

[0363] The method of claim 16, wherein the fluorophore is a rhodamine dye or a xanthene dye.

[0364] Embodiment 18. The modified target molecule has formula (IVB) or (IVC): TIFF2025029024000045.tif29128 or a combination thereof, During the ceremony: Y 1 is a biomolecule, optionally comprising one or more groups selected from an active small molecule, an affinity tag, a fluorophore, and a metal chelator; Each R 1 is independently selected from hydrogen, acyl, substituted acyl, alkyl, and substituted alkyl; Y 2 is a second biological molecule; L 1 is a linker selected from linear or branched alkyl, linear or branched substituted alkyl, polyethylene glycol (PEG), substituted PEG, and one or more peptides; and The method according to any one of aspects 1 to 17, wherein n is an integer from 1 to 3.

[0365] Aspect 19. The modified target molecule of formula (IVB) is represented by the formulas (IVB1) to (IVZB3): TIFF2025029024000046.tif34150 is one of the molecules; The modified target molecule of formula (IVC) is represented by the formulas (IVC1) to (IVC3): 20. The method of embodiment 18, wherein the molecule is any of those set forth in TIFF2025029024000047.tif34148.

[0366] Aspect 20. The modified target molecule of formula (IVB) is represented by the formulas (IVB5) to (IVB6): TIFF2025029024000048.tif34128 is one of the molecules; The modified target molecule of formula (IVC) is represented by the formulas (IVC4) to (IVC5): 20. The method of claim 18, wherein the molecule is any of the molecules in TIFF2025029024000049.tif34128.

[0367] Embodiment 21. The method according to any one of embodiments 1 to 20, which is carried out at a pH of 4 to 9.

[0368] Embodiment 22. The method according to embodiment 21, wherein the method is carried out at neutral pH.

[0369] Embodiment 23 The method according to any one of embodiments 1 to 22, wherein the target molecule comprising a thiol group is a CRISPR-Cas effector polypeptide.

[0370] Aspect 24. Formula (III): A thiol-containing target molecule of TIFF2025029024000050.tif6128, Formula (I): TIFF2025029024000051.tif17128 and biomolecules containing phenolic or catechol moieties A composition comprising: During the ceremony: Y 1 is a biomolecule, optionally comprising one or more moieties selected from an active small molecule, an affinity tag, a fluorophore, and a metal chelator; X 1 is selected from hydrogen and hydroxyl; L is an optional linker; and Y 2 is a second biomolecule, The composition.

[0371] Aspect 25.Y 2 is a CRISPR-Cas effector polypeptide.

[0372] Aspect 26. Formula (I) is a compound of formula (IA): Described by TIFF2025029024000052.tif26128, During the ceremony: Y 1 is a biomolecule, optionally comprising one or more groups selected from an active small molecule, an affinity tag, a fluorophore, and a metal chelator; Each R 1 is independently selected from hydrogen, acyl, substituted acyl, alkyl, and substituted alkyl; X 1 is selected from hydrogen and hydroxyl; and L 1 is a linker selected from linear or branched alkyl, linear or branched substituted alkyl, polyethylene glycol (PEG), substituted PEG, and one or more peptides; 26. The composition of embodiment 24 or 25.

[0373] Embodiment 27. A first container comprising the composition according to any one of embodiments 24 to 26; and a second vessel containing an enzyme capable of oxidizing the phenol or catechol moiety; Including the kit.

[0374] 28. The kit of claim 27, wherein the enzyme is a tyrosinase enzyme.

[0375] Embodiment 29. Formula (IV) or (IVA): The compound of TIFF2025029024000053.tif29128, During the ceremony: Y 1is a biomolecule, optionally comprising one or more moieties selected from an active small molecule, an affinity tag, a fluorophore, and a metal chelator; L is an optional linker; Y 2 is a second biological molecule; and n is an integer from 1 to 3; The compound.

[0376] Aspect 30. The modified target molecule of formula (IV) is represented by the formulas (IV1) to (IV5): 30. The compound according to embodiment 29, which is any of the molecules of TIFF2025029024000054.tif67146.

[0377] Aspect 31. The modified target molecule of formula (IVA) is represented by the formulas (IVA1) to (IVA5): 30. The compound according to embodiment 29, which is any molecule of TIFF2025029024000055.tif67131.

[0378] Embodiment 32. A compound according to any one of embodiments 29 to 31, wherein L is a cleavable linker.

[0379] Aspect 33.Y 1 The compound according to any one of aspects 29 to 32, wherein is a polypeptide.

[0380] Aspect 34. Y 34. The compound according to embodiment 33, wherein 1 is selected from a fluorescent protein, an antibody, and an enzyme.

[0381] Embodiment 35. The compound has formula (IVB) or (IVC): Described by TIFF2025029024000056.tif31128, During the ceremony: Y 1 is a biomolecule, optionally comprising one or more groups selected from an active small molecule, an affinity tag, a fluorophore, and a metal chelator; Each R 1is independently selected from hydrogen, acyl, substituted acyl, alkyl, and substituted alkyl; Y 2 is a second biomolecule; L 1 is a linker selected from linear or branched alkyl, linear or branched substituted alkyl, polyethylene glycol (PEG), substituted PEG, and one or more peptides; and n is an integer from 1 to 3; A compound according to any one of aspects 29 to 34.

[0382] Aspect 36. The modified target molecule of formula (IVB) is represented by the formulas (IVB1) to (IVZB5): 36. The compound according to embodiment 35, which is any of the molecules of TIFF2025029024000057.tif72150.

[0383] Aspect 37. The modified target molecule of formula (IV) is represented by the formulas (IVC1) to (IVC5): 36. The compound according to embodiment 35, which is any one of the molecules of TIFF2025029024000058.tif78148.

[0384] Aspect 38. The compound has the formula (IVD)-(IVG): TIFF2025029024000059.tif74128, During the ceremony: R 2 is selected from alkyl and substituted alkyl; R 3 is selected from hydrogen, alkyl substituted alkyl, peptide, and polypeptide; and n is an integer from 1 to 3; A compound according to any one of aspects 29 to 37.

[0385] Aspect 39.Y 2 is a CRISPR-Cas effector polypeptide.

[0386] Aspect B The aspects including the embodiments of the subject matter described above may be effective alone or in combination with one or more other aspects or embodiments. Without being limited to the above description, certain non-limiting aspects of the present disclosure numbered 1 to 71 are set forth below. As will be apparent to one skilled in the art upon reading this disclosure, each of the individually numbered aspects may be used or combined with any of the preceding or succeeding individually numbered aspects. This is intended to provide support for all such combinations of aspects, and is not intended to be limited to the combinations of aspects explicitly provided below.

[0387] Embodiment 1. A method for chemoselectively modifying a target molecule, the method comprising: contacting a target molecule comprising a thiol moiety with a biomolecule comprising a reactive moiety; wherein the biomolecule comprising the reactive moiety is produced by reaction of a biomolecule comprising a phenol or catechol moiety with an enzyme capable of oxidizing the phenol or catechol moiety; and wherein the contacting is performed under conditions sufficient to complex the target molecule with the biomolecule, thereby producing a modified target molecule.

[0388] The method of embodiment 1, wherein the target molecule is a polypeptide or a polynucleotide.

[0389] The method of claim 1 or 2, wherein the enzyme is a tyrosinase polypeptide.

[0390] Aspect 4. The method of any one of aspects 1 to 3, wherein the tyrosinase polypeptide is an Agaricus bisporus tyrosinase (abTYR) polypeptide.

[0391] Embodiment 5. The method of any one of embodiments 1 to 3, wherein said tyrosinase polypeptide comprises an amino acid sequence having at least 75% amino acid sequence identity to the amino acid sequence of abTYR shown in FIG. 8 or FIG. 9.

[0392] Embodiment 6 The method of embodiment 4 or embodiment 5, wherein the biomolecule comprising a phenol or catechol moiety is neutral or positively charged within 50 angstroms (Å) of the phenol or catechol moiety.

[0393] Aspect 7. The method of any one of aspects 1 to 3, wherein the tyrosinase polypeptide is a Bacillus megaterium tyrosinase (bmTYR) polypeptide.

[0394] Aspect 8. The method of any one of aspects 1-3, wherein the tyrosinase polypeptide comprises an amino acid sequence having at least 75% amino acid sequence identity to any one of the amino acid sequences depicted in any one of Figures 10A-10Z and 10AA-10VV.

[0395] Embodiment 9 The method of embodiment 7 or embodiment 8, wherein the biomolecule comprising a phenol or catechol moiety is negatively charged within 50 Å of the phenol or catechol moiety.

[0396] Embodiment 10. The method of any one of embodiments 1 to 9, wherein the target molecule is a polynucleotide.

[0397] The method of claim 10, wherein the target molecule is a DNA molecule.

[0398] Embodiment 12 The method of embodiment 10, wherein the target molecule is an RNA molecule.

[0399] Aspect 13. The method according to any one of aspects 10 to 12, wherein the biomolecule is a polypeptide.

[0400] Embodiment 14 The method according to any one of embodiments 1 to 13, wherein the enzyme is bound to a solid support.

[0401] Embodiment 15 The method of any one of embodiments 1 to 14, wherein the phenol moiety is present in a tyrosine residue.

[0402] Embodiment 16 The method of any one of embodiments 1 to 15, wherein the thiol moiety is present on a cysteine ​​residue.

[0403] Aspect 17. The method of aspect 16, wherein the cysteine ​​residue is a naturally occurring cysteine ​​residue.

[0404] Embodiment 18. The method of any one of embodiments 1 to 17, wherein the biomolecule comprises one or more moieties selected from a fluorophore, an active small molecule, an affinity tag, and a metal chelator.

[0405] Embodiment 19. The method of any one of embodiments 1 to 18, wherein the reactive moiety is an orthoquinone or semiquinone radical, or a combination thereof.

[0406] Aspect 20. The method according to any one of aspects 1 to 19, wherein the biomolecule is a polypeptide.

[0407] Aspect 21. The method of aspect 20, wherein the biomolecule is a polypeptide selected from a fluorescent protein, an antibody, an enzyme, a ligand for a receptor, and a receptor.

[0408] 22. The biomolecule comprising a phenolic or catechol moiety is a molecule of formula (I) and the biomolecule comprising a reactive moiety is a molecule of formula (II) or (IIA) or a combination thereof: TIFF2025029024000060.tif26131 formula: Y 1 is a biomolecule, optionally comprising one or more moieties selected from an active small molecule, an affinity tag, a fluorophore, and a metal chelator; X 1 is selected from hydrogen and hydroxyl; and L is an optional linker; 22. The method according to any one of aspects 1 to 21.

[0409] Embodiment 23. The targeting molecule comprising a thiol moiety is a molecule of formula (III) and the modified targeting molecule is a molecule of formula (IV) or (IVA), or a combination thereof: TIFF2025029024000061.tif30128 formula: Y 1 is a biomolecule, optionally comprising one or more moieties selected from an active small molecule, an affinity tag, a fluorophore, and a metal chelator; Y 2 is a second biological molecule; L is an optional linker; and n is an integer from 1 to 3; 23. The method according to any one of aspects 1 to 22.

[0410] Aspect 24. The modified target molecule of formula (IV) is represented by the formulas (IV1) to (IV3): TIFF2025029024000062.tif30144; and The modified target molecule of formula (IVA) is represented by the formulas (IVA1) to (IVA3): 24. The method of claim 23, wherein the molecule is any one of TIFF2025029024000063.tif32131.

[0411] Aspect 25. The modified target molecule of formula (IV) is represented by the formulas (IV5) to (IV6): TIFF2025029024000064.tif32128; and The modified target molecule of formula (IVA) is represented by the formulas (IVA4) to (IVA5): 24. The method of claim 23, wherein the molecule is any of the molecules in TIFF2025029024000065.tif32128.

[0412] 26. The biomolecule comprising a phenol or catechol moiety has the formula (IA): Described by TIFF2025029024000066.tif26128, During the ceremony: Y 1 is a biomolecule, optionally comprising one or more groups selected from an active small molecule, an affinity tag, a fluorophore, and a metal chelator; Each R 1 is independently selected from hydrogen, acyl, substituted acyl, alkyl, and substituted alkyl; X 1 is selected from hydrogen and hydroxyl; and L 1 is a linker selected from linear or branched alkyl, linear or branched substituted alkyl, polyethylene glycol (PEG), substituted PEG, and one or more peptides; The method according to any one of aspects 1 to 25.

[0413] Aspect 27. The method of aspect 26, wherein the fluorophore is a rhodamine dye or a xanthene dye.

[0414] Embodiment 28. The modified target molecule has formula (IVB) or (IVC): TIFF2025029024000067.tif32128 or a combination thereof, During the ceremony: Y 1 is a biomolecule, optionally comprising one or more groups selected from an active small molecule, an affinity tag, a fluorophore, and a metal chelator; Each R 1 is independently selected from hydrogen, acyl, substituted acyl, alkyl, and substituted alkyl; Y 2 is a second biological molecule; L 1 is a linker selected from linear or branched alkyl, linear or branched substituted alkyl, polyethylene glycol (PEG), substituted PEG, and one or more peptides; and n is an integer from 1 to 3; A method according to any one of aspects 1 to 27.

[0415] Aspect 29. The modified target molecule of formula (IVB) is represented by the formulas (IVB1) to (IVZB3): TIFF2025029024000068.tif32137; and The modified target molecule of formula (IVC) is represented by the formulas (IVC1) to (IVC3): 29. The method of embodiment 28, wherein the molecule is any of TIFF2025029024000069.tif31133.

[0416] Aspect 30. The modified target molecule of formula (IVB) is represented by the formulas (IVB5) to (IVB6): TIFF2025029024000070.tif35128; and The modified target molecule of formula (IVC) is represented by the formulas (IVC4) to (IVC5): 29. The method of embodiment 28, wherein the molecule is any of TIFF2025029024000071.tif35128.

[0417] Embodiment 31. The method according to any one of embodiments 1 to 30, which is carried out at a pH of 4 to 9.

[0418] Embodiment 32. The method according to embodiment 31, wherein the method is carried out at neutral pH.

[0419] Embodiment 33 The method according to any one of embodiments 1 to 32, wherein the target molecule comprising a thiol group is a CRISPR-Cas effector polypeptide.

[0420] Aspect 34. Formula (III): A thiol-containing target molecule of TIFF2025029024000072.tif6128, Formula (I): TIFF2025029024000073.tif17128 and biomolecules containing phenolic or catechol moieties A composition comprising: During the ceremony: Y 1is a biomolecule, optionally comprising one or more moieties selected from an active small molecule, an affinity tag, a fluorophore, and a metal chelator; X 1 is selected from hydrogen and hydroxyl; L is an optional linker; and Y 2 is a second biomolecule, The composition.

[0421] Embodiment 35. The composition of embodiment 34, wherein the biomolecule comprising a phenol or catechol moiety is neutral or positively charged within 50 Å of the phenol or catechol moiety.

[0422] Embodiment 36 The composition of embodiment 34, wherein the biomolecule comprising a phenol or catechol moiety is negatively charged within 50 Å of the phenol or catechol moiety.

[0423] Aspect 37. The Y 1 is a polypeptide and Y 2 The composition according to any one of aspects 34 to 36, wherein is a polypeptide.

[0424] Aspect 38.Y 1 is a polynucleotide, and Y 2 The composition according to any one of aspects 34 to 36, wherein is a polypeptide.

[0425] Aspect 39.Y 2 The composition of any one of aspects 34 to 38, wherein said polypeptide is a CRISPR-Cas effector polypeptide.

[0426] Embodiment 40. Formula (I) is a compound of formula (IA): Described by TIFF2025029024000074.tif26128, During the ceremony: Y 1 is a biomolecule, optionally comprising one or more groups selected from an active small molecule, an affinity tag, a fluorophore, and a metal chelator; Each R 1 is independently selected from hydrogen, acyl, substituted acyl, alkyl, and substituted alkyl; X 1 is selected from hydrogen and hydroxyl; and L 1 is a linker selected from linear or branched alkyl, linear or branched substituted alkyl, polyethylene glycol (PEG), substituted PEG, and one or more peptides; 40. The composition according to any one of embodiments 34 to 39.

[0427] Embodiment 41. A first container comprising the composition of any one of embodiments 34 to 40; and a second vessel containing an enzyme capable of oxidizing the phenol or catechol moiety; Including the kit.

[0428] Embodiment 42. The kit according to embodiment 41, wherein the enzyme is a tyrosinase polypeptide.

[0429] Aspect 43. The kit of aspect 42, wherein the tyrosinase enzyme is Agaricus bisporus tyrosinase enzyme (abTYR).

[0430] Embodiment 44. The kit according to embodiment 42, wherein said tyrosinase polypeptide comprises an amino acid sequence having at least 75% amino acid sequence identity to the amino acid sequence of abTYR shown in FIG. 8 or FIG. 9.

[0431] Aspect 45. The kit described in Aspect 42, wherein the tyrosinase enzyme is Bacillus megaterium tyrosinase enzyme (bmTYR).

[0432] Aspect 46. The kit of aspect 42, wherein the tyrosinase polypeptide comprises an amino acid sequence having at least 75% amino acid sequence identity to any one of the amino acid sequences depicted in any one of Figures 10A-10Z and 10AA-10VV.

[0433] Embodiment 47. Formula (IV) or (IVA): The compound of TIFF2025029024000075.tif30128, During the ceremony: Y 1 is a biomolecule, optionally comprising one or more moieties selected from an active small molecule, an affinity tag, a fluorophore, and a metal chelator; L is an optional linker; Y 2 is a second biological molecule; and n is an integer from 1 to 3; The compound.

[0434] Aspect 48. The modified target molecule of formula (IV) is represented by the formulas (IV1) to (IV5): 48. The compound according to embodiment 47, which is any of the molecules of TIFF2025029024000076.tif66144.

[0435] Aspect 49. The modified target molecule of formula (IVA) is represented by the formulas (IVA1) to (IVA5): 48. The compound according to embodiment 47, which is any of the molecules of TIFF2025029024000077.tif68131.

[0436] Embodiment 50. A compound according to any one of embodiments 47 to 49, wherein L is a cleavable linker.

[0437] Aspect 51.Y 1 The compound according to any one of aspects 47 to 50, wherein is a polypeptide.

[0438] Aspect 52. Y52. The compound according to embodiment 51, wherein 1 is selected from a fluorescent protein, an antibody, and an enzyme.

[0439] Embodiment 53. Formula (IVB) or (IVC): Described by TIFF2025029024000078.tif32128, During the ceremony: Y 1 is a biomolecule, optionally comprising one or more groups selected from an active small molecule, an affinity tag, a fluorophore, and a metal chelator; Each R 1 is independently selected from hydrogen, acyl, substituted acyl, alkyl, and substituted alkyl; Y 2 is a second biological molecule; L 1 is a linker selected from linear or branched alkyl, linear or branched substituted alkyl, polyethylene glycol (PEG), substituted PEG, and one or more peptides; and n is an integer from 1 to 3; A compound according to any one of aspects 47 to 52.

[0440] Embodiment 54. The modified target molecule of formula (IVB) is represented by the formulas (IVB1) to (IVZB5): 54. The compound according to embodiment 53, which is any of the molecules of TIFF2025029024000079.tif71139.

[0441] 55. The modified target molecule of formula (IV) is represented by the formulas (IVC1) to (IVC5): 54. The compound according to embodiment 53, which is any one of the molecules of TIFF2025029024000080.tif77134.

[0442] 56. The compound has the formula (IVD)-(IVG): TIFF2025029024000081.tif75128, During the ceremony: R 2is selected from alkyl and substituted alkyl; R 3 is selected from hydrogen, alkyl substituted alkyl, peptide, and polypeptide; and n is an integer from 1 to 3; A compound according to any one of aspects 47 to 55.

[0443] Aspect 57.Y 2 is a CRISPR-Cas effector polypeptide.

[0444] 58. A method for chemoselectively coupling a first polypeptide and a second polypeptide to a coupling polypeptide, the method comprising: a) contacting said first polypeptide with said coupling polypeptide to form a first polypeptide-coupling polypeptide complex, the first polypeptide comprises a thiol moiety; the coupling polypeptide comprises an N-terminal reactive moiety that forms a covalent bond with a thiol moiety present on the first polypeptide; the coupling polypeptide comprising the N-terminal reactive moiety is produced by reacting a polypeptide comprising an N-terminal phenol or catechol moiety and a C-terminal phenol or catechol moiety with a first enzyme capable of oxidizing the N-terminal phenol or catechol moiety but not the C-terminal phenol or catechol moiety to produce the N-terminal reactive moiety; the coupling polypeptide comprises two or more positively charged or neutral amino acids within 10 amino acids of the N-terminal phenol or catechol moiety and two or more negatively charged amino acids within 10 amino acids of the C-terminal phenol or catechol moiety; the steps; and b) contacting said second polypeptide with said first polypeptide-coupling polypeptide complex, the two polypeptides each comprise a thiol moiety; the first polypeptide-coupling polypeptide complex comprises a C-terminal reactive moiety that forms a covalent bond with a thiol moiety present on the second polypeptide; the first polypeptide-coupling polypeptide complex comprising the C-terminal reactive moiety is produced by reacting the first polypeptide-coupling polypeptide complex with a second enzyme capable of oxidizing the C-terminal phenolic or catechol moiety to produce a C-terminal reactive moiety; said contacting producing a first polypeptide-coupling polypeptide-second polypeptide complex. The above steps The method comprising:

[0445] Aspect 59. a) the first enzyme is a tyrosinase polypeptide comprising an amino acid sequence having at least 75% amino acid sequence identity to the amino acid sequence of abTYR shown in FIG. 8 or FIG. 9; and b) the second enzyme is a tyrosinase polypeptide comprising an amino acid sequence having at least 75% amino acid sequence identity to any one of the amino acid sequences shown in any one of Figures 10A to 10Z and Figures 10AA to 10VV; The method according to embodiment 58.

[0446] Embodiment 60. A method for chemoselectively coupling a first polypeptide and a second polypeptide to a coupling polypeptide, the method comprising: a) contacting said first polypeptide with said coupling polypeptide to form a first polypeptide-coupling polypeptide complex, the first polypeptide comprises a thiol moiety; the coupling polypeptide comprises an N-terminal reactive moiety that forms a covalent bond with a thiol moiety present on the first polypeptide; the coupling polypeptide comprising the N-terminal reactive moiety is produced by reacting a polypeptide comprising an N-terminal phenol or catechol moiety and a C-terminal phenol or catechol moiety with a first enzyme capable of oxidizing the N-terminal phenol or catechol moiety but not the C-terminal phenol or catechol moiety to produce the N-terminal reactive moiety; the coupling polypeptide comprises two or more negatively charged amino acids within 10 amino acids of the N-terminal phenol or catechol moiety and two or more positively charged or neutral amino acids within 10 amino acids of the C-terminal phenol or catechol moiety; the steps; and b) contacting said second polypeptide with said first polypeptide-coupling polypeptide complex, the two polypeptides each comprise a thiol moiety; the first polypeptide-coupling polypeptide complex comprises a C-terminal reactive moiety that forms a covalent bond with a thiol moiety present on the second polypeptide; the first polypeptide-coupling polypeptide complex comprising the C-terminal reactive moiety is produced by reacting the first polypeptide-coupling polypeptide complex with a second enzyme capable of oxidizing the C-terminal phenolic or catechol moiety to produce a C-terminal reactive moiety; said contacting producing a first polypeptide-coupling polypeptide-second polypeptide complex. The above steps The method comprising:

[0447] Aspect 61. a) the first enzyme is a tyrosinase polypeptide comprising an amino acid sequence having at least 75% amino acid sequence identity to any one of the amino acid sequences depicted in any one of Figures 10A-10Z and 10AA-10VV; and b) the second enzyme is a tyrosinase polypeptide comprising an amino acid sequence having at least 75% amino acid sequence identity to the amino acid sequence of abTYR shown in FIG. 8 or FIG. 9; The method according to embodiment 60.

[0448] Embodiment 62. A method for covalently linking a first polypeptide to a second polypeptide, the method comprising: a) contacting said first polypeptide with an immobilized reactive moiety, the immobilized reactive moiety is generated by reaction of an immobilized phenolic or catechol moiety with a first enzyme, the first enzyme being capable of oxidizing the immobilized phenolic or catechol moiety, thereby generating the immobilized reactive moiety; the first polypeptide comprises: i) a thiol moiety; and ii) a phenol moiety or a catechol moiety, the first polypeptide comprising two or more negatively charged amino acids within 10 amino acids of the phenol moiety or the catechol moiety; the immobilized reactive moiety forms a covalent bond with the thiol moiety present on the first polypeptide, thereby generating an immobilized first polypeptide. The above steps; b) contacting the immobilized first polypeptide with a second enzyme, wherein the second enzyme is capable of oxidizing the phenol or catechol moiety present on the first polypeptide to generate an immobilized first polypeptide comprising a reactive moiety; and c) contacting the immobilized first polypeptide comprising said reactive moiety with a second polypeptide, the second polypeptide comprises: i) a thiol moiety; and ii) a phenol moiety or a catechol moiety, the second polypeptide having two or more neutral or positive charges within 10 amino acids of the phenol moiety or the catechol moiety; the reactive moiety present on the immobilized first polypeptide forms a covalent bond with the thiol moiety present on the second polypeptide, thereby generating an immobilized complex comprising the first polypeptide covalently bound to the second polypeptide. The above steps The method comprising:

[0449] Embodiment 63. The method of embodiment 62, wherein the first enzyme is a tyrosinase polypeptide comprising an amino acid sequence having at least 75% amino acid sequence identity to any one of the amino acid sequences shown in any one of Figures 8, 9, 10A-10Z, and 10AA-10VV.

[0450] Embodiment 64 The method according to embodiment 62 or embodiment 63, wherein the thiol moiety present in the first polypeptide is present on a Cys residue and the phenol moiety present in the first polypeptide is present on a Tyr residue.

[0451] Embodiment 65. The method of embodiment 64, wherein said Tyr residue is present in a stretch of amino acids comprising EEEY (SEQ ID NO: 953), EEEEY (SEQ ID NO: 955), DDDDY (SEQ ID NO: 965), or D...

Claims

1. 1. A method for chemoselectively modifying a target molecule to produce a modified target molecule, comprising: The method comprises the step of: contacting the target molecule with a first biomolecule comprising a reactive moiety; a first biomolecule comprising said reactive moiety is produced by reaction of a biomolecule comprising a phenolic or catechol moiety with a tyrosinase polypeptide; the contacting step is carried out under conditions sufficient to complex the target molecule with the biomolecule, thereby producing a modified target molecule; and The modified target molecule is a molecule of formula (IV) or (IVA): During the ceremony: Y １ is the first biological molecule; Y ２ is a second biological molecule; L is an optional linker; and n is an integer from 1 to 3; Y １ and Y ２ may be the same or different, Optionally, Y ２ is a polypeptide or polynucleotide, and / or The tyrosinase polypeptide is an Agaricus bisporus tyrosinase (abTYR) polypeptide; The method.

2. The method of claim 1 , wherein the target molecule is a polypeptide or a polynucleotide.

3. 3. The method of claim 1 or 2, wherein the tyrosinase polypeptide comprises an amino acid sequence having at least 98% amino acid sequence identity to an amino acid sequence set forth in any one of SEQ ID NOs: 973-1020.

4. The method of any one of claims 1 to 3, wherein the phenol moiety is present in a tyrosine residue and / or the thiol moiety is present in a cysteine ​​residue.

5. The method according to any one of claims 1 to 4, wherein the first biological molecule and / or the second biological molecule is a polypeptide selected from a fluorescent protein, an antibody, an enzyme, a receptor ligand, and a receptor.

6. The first biomolecule comprising a phenolic or catechol moiety is a molecule of formula (I) below, and the first biomolecule comprising a reactive moiety is a molecule of formula (II) or (IIA) below, or a combination thereof: The method according to any one of claims 1 to 5.

7. A) The modified target molecule of formula (IV) is represented by the following formulae (IV1) to (IV3): and The modified target molecule of formula (IVA) is represented by the following formulas (IVA1) to (IVA3): or B) The modified target molecule of formula (IV) is represented by the following formulas (IV4) to (IV5): and The modified target molecule of formula (IVA) is represented by the following formulas (IVA4) to (IVA5): A molecule of either The method according to any one of claims 1 to 6.

8. The modified target molecule has the following formula (IVB) or (IVC): or a combination thereof, During the ceremony: Y １ is a first biomolecule, optionally further comprising one or more groups selected from a small molecule, an affinity tag, a fluorophore, and a metal chelator; Each R １ is independently selected from hydrogen, acyl, substituted acyl, alkyl, and substituted alkyl; Y ２ is a second biological molecule; L １ is a linker selected from linear or branched alkyl, linear or branched substituted alkyl, polyethylene glycol (PEG), substituted PEG, and one or more peptides; and n is an integer from 1 to 3; The method according to any one of claims 1 to 7.

9. The modified target molecule of formula (IVB) is represented by the following formulas (IVB1) to (IVB3): and The modified target molecule of formula (IVC) is represented by the following formulas (IVC1) to (IVC3): The method according to claim 8, wherein the molecule is any one of the following:

10. The modified target molecule of formula (IVB) is represented by the following formulas (IVB4) to (IVB5): and The modified target molecule of formula (IVC) may be represented by the following formulas (IVC4) to (IVC5): The method according to claim 8, wherein the molecule is any one of the following:

11. The following formula (IV) or (IVA): A modified target molecule of During the ceremony: Y １ is a first biomolecule, optionally further comprising one or more moieties selected from a small molecule, an affinity tag, a fluorophore, and a metal chelator; L is an optional linker; Y ２ is the second biological molecule; and n is an integer from 1 to 3; Y １ and Y ２ may be the same or different, and Y １ , Y ２ , or Y １ and Y ２ are both antibodies or antibody fragments; The modified target molecule.

12. The following formula (IV) or (IVA): A modified target molecule of During the ceremony: Y １ is the first polypeptide; Y ２ is the second polypeptide; L is an optional linker; and n is an integer from 1 to 3; Y １ and Y ２ are the same polypeptide or are different polypeptides, The modified target molecule.

13. The following formula (IV) or (IVA): A modified target molecule of During the ceremony: Y １ is the first biological molecule; Y ２ is a second biological molecule; L is an optional linker; and n is an integer from 1 to 3; and Y １ comprises a polypeptide, and Y ２ comprises at least one of a polynucleotide, a glycopolypeptide, a protein-derived domain, a peptide, a carbohydrate, a lipid, a glycolipid, a nucleic acid, a sugar, one or more amino acids, or one or more synthetic polymers; or Y ２ comprises a polypeptide, and Y １ comprises at least one of a polynucleotide, a glycopolypeptide, a protein-derived domain, a peptide, a carbohydrate, a lipid, a glycolipid, a nucleic acid, a sugar, one or more amino acids, or one or more synthetic polymers; The modified target molecule.

14. The modified target molecule of formula (IV) is represented by the following formulas (IV1) to (IV5): The modified target molecule according to any one of claims 11 to 13, which is any one of the molecules represented by the formula (I).

15. The modified target molecule of formula (IVA) is represented by the following formulas (IVA1) to (IVA5): The modified target molecule according to any one of claims 11 to 13, which is any one of the molecules represented by the formula (I).

16. The modified targeting molecule of any one of claims 11 to 13, wherein L is a cleavable linker.

17. Y １ The modified target molecule of any one of claims 11 to 16, wherein comprises an antibody or an antibody fragment.

18. Y １ The modified target molecule of any one of claims 11 to 16, wherein said target molecule comprises a polypeptide.

19. Y １ The modified target molecule of any one of claims 12 to 16, wherein said modified target molecule comprises at least one of a fluorescent protein, an antibody or antibody fragment, a polynucleotide, a glycopolypeptide, a protein-derived domain, a peptide, a cytokine, a chemokine, a peptide hormone, and an enzyme.

20. The modified target molecule of claim 11, wherein the first biomolecule or the second biomolecule comprises a polypeptide, a nucleic acid or a small molecule, and optionally further comprises at least one of a small molecule, an affinity tag, a fluorophore, and a metal chelator.

21. The antibody or antibody fragment comprises a single variable domain (V ＨＨ 18. The modified target molecule of any one of claims 11 to 17, which is a IgG antibody, optionally a camelid antibody.

22. The modified targeting molecule of any one of claims 11 to 17 and 21, wherein the antibody or antibody fragment comprises a HER2 antibody.

23. 21. The modified target molecule of claim 13 or 20, wherein the nucleic acid comprises at least one of a DNA molecule, an RNA molecule, or a molecule containing both deoxyribonucleotides and ribonucleotides.

24. 24. The modified target molecule of claim 23, wherein the nucleic acid comprises at least one of a single-stranded DNA molecule, a double-stranded DNA molecule, a single-stranded RNA molecule, a small interfering RNA (siRNA), a short hairpin RNA (shRNA), a microRNA (miRNA), a ribozyme, an shRNA, an antisense oligonucleotide, a self-cleaving RNA, a microRNA, a microRNA mimic, a supermir, an aptamer, an antimir, an antagomir, an Ul adaptor, a triplex forming oligonucleotide, an RNA activator, a long non-coding RNA, a short non-coding RNA, a piRNA, an immune modulatory oligonucleotide, an antiviral oligonucleotide, or a decoy oligonucleotide.

25. Y ２ The modified target molecule of any one of claims 11 to 18, wherein said CRISPR-Cas effector polypeptide is a CRISPR-Cas effector polypeptide.

26. A first container comprising a modified target molecule according to any one of claims 11 to 25; and a second container containing a tyrosinase polypeptide. Including the kit.

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