WO2021062201A1 - Compositions et procédés pour le ciblage et l'expression de nucléoprotéines - Google Patents

Compositions et procédés pour le ciblage et l'expression de nucléoprotéines Download PDF

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WO2021062201A1
WO2021062201A1 PCT/US2020/052791 US2020052791W WO2021062201A1 WO 2021062201 A1 WO2021062201 A1 WO 2021062201A1 US 2020052791 W US2020052791 W US 2020052791W WO 2021062201 A1 WO2021062201 A1 WO 2021062201A1
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cell
conformation
specific
nucleic acid
binding agent
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PCT/US2020/052791
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Hariharan JAYARAM
Aaron CANTOR
Akshay TAMBE
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Spotlight Therapeutics
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6875—Nucleoproteins
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/22—Ribonucleases RNAses, DNAses
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K2035/11—Medicinal preparations comprising living procariotic cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/10—Fusion polypeptide containing a localisation/targetting motif containing a tag for extracellular membrane crossing, e.g. TAT or VP22
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/20—Fusion polypeptide containing a tag with affinity for a non-protein ligand
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/33—Fusion polypeptide fusions for targeting to specific cell types, e.g. tissue specific targeting, targeting of a bacterial subspecies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/40—Fusion polypeptide containing a tag for immunodetection, or an epitope for immunisation
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/914—Hydrolases (3)
    • G01N2333/916—Hydrolases (3) acting on ester bonds (3.1), e.g. phosphatases (3.1.3), phospholipases C or phospholipases D (3.1.4)
    • G01N2333/922—Ribonucleases (RNAses); Deoxyribonucleases (DNAses)

Definitions

  • the present invention generally relates to methods and compositions involving a conformational specific-nucleoprotein binding agent such as an Anti-CRISPR (Acr) protein, including methods for transporting nucleoproteins into cells, optimizing nucleoprotein expression, and isolating nucleoproteins.
  • a conformational specific-nucleoprotein binding agent such as an Anti-CRISPR (Acr) protein
  • RNA-guided endonucleases such as Cas9
  • Cas9 CRISPR-associated RNA-guided endonucleases
  • Cas9 CRISPR-associated RNA-guided endonucleases
  • a guide RNA such as a dual-RNA complex or a chimeric single-guide RNA
  • RNA-guided endonucleases e.g., Cas9
  • DSBs site-specific double strand breaks
  • SSBs single- stranded breaks
  • target nucleic acids e.g., double-stranded DNA (dsDNA), single- stranded DNA (ssDNA), or RNA).
  • RNA-guided endonucleases e.g., Cas9 alone or fused to transcriptional activator or repressor domains can be used to alter transcription levels at sites within target nucleic acids by binding to the target site without cleavage.
  • RNA-guided endonucleases associate with a guide RNA to form a ribonucleoprotein, which must be delivered to a cell and internalized within the nucleus to cleave a target nucleic acid.
  • RNA-guided nucleases to specific cells or tissues remains a challenge.
  • a variety of methods or vehicles for delivery of RNA-guided endonucleases have been utilized, such as electroporation, nucleofection, microinjection, adeno- associated vectors (AAV), lentivirus, and lipid nanoparticles (see, e.g., in Lino, C.A. et al., 2018.
  • RNA-guided endonucleases by lipid nanoparticles has several drawbacks, including endosomal degradation of cargo, specific cell tropism, and bioaccumulation in the liver (see, e.g., Lino et al., 2018; and Finn, J.D., et al., 2018. Cell reports,
  • Anti-CRISPR (Acr) proteins have been identified that bind RNPs.
  • Acr proteins were originally discovered in bacteriophage, which produce Acr proteins as a means to inhibit the native CRISPR/Cas systems of bacteria (Bondy-Denomy, Joe, et al. Nature 493.7432 (2013): 429-432; Pawluk, April, et al. MBio 5.2 (2014)). Accordingly, such Acr proteins have been proposed for use in systems that modulate Cas nuclease activity (US Publication No. US20200087354A1 , and International Publication Nos. WO2019076651 A1 , W02020068196A2, and WO2019034784A1 ).
  • Immobilized Acr proteins have additionally been tested as RNP capture ligands in bioanalytical assays, but these assays required use of anti-Cas9 antibodies as detection probes (Johnston, et al. Biosensors and Bioelectronics 141 (2019): 111361 ).
  • the anti-Cas9 antibodies tested in these assays bound to Cas9 with a higher affinity than Cas9 RNPs, and thus have limited usefulness for detection of RNPs (Johnston et al). Accordingly, there is a need in the art for methods and reagents that enable direct detection of RNPs in vitro. Further, Acr proteins have yet to be utilized in an in vivo or ex vivo context for RNP delivery, detection, or expression.
  • compositions and methods that utilize a conformation-specific nucleoprotein binding agent, such as an Anti-CRISPR (Acr) protein (e.g., AcrllA4 or AcrVAI) to enable delivery, detection, expression, or production of nucleoproteins (NPs).
  • a conformation-specific nucleoprotein binding agent such as an Anti-CRISPR (Acr) protein (e.g., AcrllA4 or AcrVAI) to enable delivery, detection, expression, or production of nucleoproteins (NPs).
  • Acr Anti-CRISPR
  • NPs nucleoproteins
  • nucleoprotein expression or production expression of both the nucleic acid-guided nuclease and guide nucleic acid in a cell must be tuned to promote optimal nucleoprotein formation in the cell. There is thus a need in the art for expression constructs, and methods for identifying expression constructs, that provide optimal expression of nucleoproteins or components thereof.
  • nucleic acid-guided nucleases Once expressed, there is further need in the art for methods to rapidly identify expression systems that express active nucleoproteins or cells containing such expression systems. Finally, there is a need in the art for means of targeting nucleic acid-guided nucleases to specific cells and tissues, for internalization of these nucleases into target cells, and for translocation to the nucleus.
  • the methods herein utilize a conformation-specific nucleoprotein binding agent, such as an Acr protein, for promoting delivery of nucleoproteins (i.e. , comprising a nucleic acid-guided nuclease, such as an RNA-guided nuclease, e.g., Cas9) and guide nucleic acid) to a target cell, promoting internalization of nucleoproteins into target cells, and/or promoting translocation of the nucleoprotein into the nucleus into a cell (see the Targeted Active Gene Editors (TAGE) described herein).
  • TAGE Targeted Active Gene Editors
  • nucleoproteins e.g., comprising a nucleic acid-guided nuclease and a guide nucleic acid
  • methods for rapidly identifying expression systems that express active nucleoproteins e.g., comprising a nucleic acid-guided nuclease and a guide nucleic acid
  • identifying cells containing such expression systems or measuring nucleoprotein levels without the need for additional purification steps based on a detectable interaction between nucleoproteins and a conformation-specific NP binding agent.
  • the methods herein further include affinity purification methods using a conformational specific-NP binding agent to isolate a nucleic acid-guided nuclease.
  • a method of measuring the level of nucleoproteins comprising combining (i) a conformation-specific NP binding agent comprising a first detectable label; (ii) an NP comprising a nucleic acid-guided nuclease and a guide nucleic acid (gNA), and (iii) a labelling moiety capable of specifically binding the nucleic acid-guided nuclease or a conjugation moiety attached thereto, wherein the labelling moiety comprises a second detectable label capable of generating a signal upon interaction with the first detectable label, and measuring the level of the signal generated by interaction between the first and second detectable labels, wherein the signal level corresponds to the level of NPs.
  • a conformation-specific NP binding agent comprising a first detectable label
  • an NP comprising a nucleic acid-guided nuclease and a guide nucleic acid (gNA)
  • gNA guide nucleic acid
  • a labelling moiety capable of specifically binding the
  • the labelling moiety comprises the second detectable label conjugated to an antibody capable of specifically binding the nucleic acid-guided nuclease, such that the second detectable label is stably associated with the NP.
  • the nucleic acid-guided nuclease comprises a first conjugation moiety and the labelling moiety comprises the second detectable label conjugated to a second conjugation moiety capable of binding the first conjugation moiety, such that the second detectable label is stably associated with the NP.
  • first or second conjugation moiety is selected from CBP, MBP, GST, poly(His), biotin, streptavidin, V5-tag, Myc-tag, HA-tag, NE-tag, His-tag, Flag tag, Halo-tag, Snap-tag, Fc-tag, Nus-tag, BCCP, Thioredoxin, SnooprTag, SpyTag, SpyCatcher, Isopeptag, SBP-tag, S- tag, AviTag, Protein A, or Calmodulin.
  • a method of measuring the level of nucleoproteins comprising combining (i) a conformation-specific NP binding agent comprising a first detectable label, and (ii) a NP comprising a nucleic acid-guided nuclease and a guide nucleic acid (gNA), wherein the nucleic acid-guided nuclease or the gNA comprises a second detectable label capable of generating a signal upon interaction with the first detectable label; and measuring the level of the signal generated by interaction between the first and second detectable labels, wherein the signal level corresponds to the level of NPs.
  • the gNA comprises the second detectable label.
  • the nucleic acid-guided nuclease comprises the second detectable label.
  • the second detectable label is attached to the nucleic acid- guided nuclease by a linker.
  • the first detectable label and/or second detectable label comprises a fluorophore.
  • the fluorophore is a fluorescent protein.
  • the first detectable label and second detectable label are a FRET pair
  • the method comprises measuring the level of NPs based on the level of fluorescence generated by interaction of the FRET pair as measured by FRET.
  • the first detectable label and second detectable label are a split luciferase pair
  • the method comprises measuring the level of NPs based on the level of luminescence generated by interaction of pair, e.g., in the presence of appropriate luciferase substrates.
  • the first detectable label and second detectable label are a split fluorescent protein (e.g., split GFP), and the method comprises measuring the level of NPs based on the level of fluorescence generated by formation of a functional fluorescent protein from combination of the split fragments.
  • split fluorescent protein e.g., split GFP
  • the first detectable label comprises a first oligonucleotide and the second detectable label comprises a second oligonucleotide capable of ligating to the first oligonucleotide to form a ligated polynucleotide when the first and second detectable labels are in close proximity
  • the first detectable label comprises a first oligonucleotide and the second detectable label comprises a second oligonucleotide
  • the method further comprises introducing a third oligonucleotide capable of hybridizing to both the first oligonucleotide and second oligonucleotide to form a ligated polynucleotide when the first and second oligonucleotides are in close proximity.
  • the measuring step comprises measuring the level of NPs based on the level of ligated polynucleotides.
  • the level of the ligated polynucleotide is measured by one or more methods selected from gel electrophoresis, PCR amplification, or sequencing.
  • the measuring step comprises amplifying the ligated polynucleotide by PCR amplification.
  • PCR amplification is rolling-circle amplification.
  • the measuring step further comprises introducing a detectable probe that binds the ligated polynucleotide and is capable of generating a detectable signal, and measuring the level of the signal generated by the bound detectable probe, wherein the signal level corresponds to the level of NPs.
  • a method of measuring the level of nucleoproteins comprising combining onto a surface comprising a surface-immobilized agent capable of binding a nucleoprotein, or a component thereof (e.g., a nucleic acid-guided nuclease): (i) a conformation-specific NP binding agent comprising a detectable label capable of generating a signal; and (ii) an NP comprising a nucleic acid-guided nuclease and a guide nucleic acid (gNA), such that the NP binds to the surface-immobilized agent and the conformation-specific NP binding agent binds to the nucleic acid-guided nuclease; washing the surface; and measuring the level of the signal generated by the detectable label on the surface, wherein the signal level corresponds to the level of NPs.
  • a surface-immobilized agent capable of binding a nucleoprotein, or a component thereof (e.g., a nucleic acid-guided nucle
  • the detectable label comprises a fluorophore.
  • the fluorophore is a fluorescent protein.
  • the detectable label comprises an enzyme, such as horseradish peroxidase or alkaline phosphatase.
  • the surface-immobilized agent capable of binding the nucleoprotein, or component thereof is an antibody, or antigen binding fragment thereof that specifically binds the nucleic acid-guided nuclease.
  • the nucleoprotein or component thereof e.g., nucleic acid-guided nuclease
  • the surface-immobilized agent capable of binding the nucleoprotein comprises a second conjugation moiety capable of binding the first binding moiety.
  • the surface is the surface of a plate or the surface of a bead.
  • the detectable label and the conformation-specific NP binding agent are conjugated by way of complementary conjugation moieties.
  • the method comprises providing a lysate from a host cell comprising the NPs. In some embodiments, the method comprises providing a lysate from a host cell comprising the nucleic acid-guided nucleases and adding a gNA to the lysate to form the NPs. In certain embodiments, the host cell further comprises the conformation-specific NP binding agent. In alternative embodiments, the method comprises adding the conformation-specific NP binding agent to the host cell lysate. In some embodiments, the host cell is a bacterial cell (e.g., E. coif). In some embodiments, the host cell is a eukaryotic cell.
  • the eukaryotic cell is a mammalian cell or a fungal cell.
  • the fungal cell is a yeast cell.
  • the yeast cell is Pichia pastoris or Saccharomyces cerevisiae.
  • the mammalian cell is a COP cell, an L cell, a C127 cell, an Sp2/0 cell, an NS-0 cell, an NIH3T3 cell, a PC12 cell, a PC12h cell, a BHK cell, a CHO cell, a COS1 cell, a COS3 cell, a COST cell, a CV1 cell, a Vero cell, a HeLa cell, an HEK-293 cell, a PER C6 cell, a cell derived from diploid fibroblasts, a myeloma cell, or HepG2.
  • a method of measuring the levels of an unlabeled nucleoprotein comprising combining into a solution (i) a conformation-specific NP binding agent comprising a first detectable label; and (ii) a labelled NP comprising a nucleic acid-guided nuclease and a guide nucleic acid (gNA), wherein the nucleic acid-guided nuclease, or a conjugation moiety attached thereto, comprises a second detectable label that is quenched by or quenches the first detectable label; such that complexes comprising the conformation-specific NP binding agent and the labelled NP are formed; adding to the solution an unlabeled NP capable of associating with the conformation-specific NP binding agent; and measuring the level of the signal generated by dissociation of the labelled NP from the complexes, wherein the degree of dissociation of the labelled NP and the resulting signal level corresponds to the level of unlabeled NP
  • the method comprises providing a lysate from a host cell comprising the nucleic acid-guided nucleases and adding a gNA to the lysate to form the unlabeled NPs.
  • the host cell is a bacterial cell (e.g., E. coif).
  • the host cell is a eukaryotic cell.
  • the eukaryotic cell is a mammalian cell or a fungal cell.
  • the fungal cell is a yeast cell.
  • the yeast cell is Pichia pastoris or Saccharomyces cerevisiae.
  • the mammalian cell is a COP cell, an L cell, a C127 cell, an Sp2/0 cell, an NS-0 cell, an NIH3T3 cell, a PC12 cell, a PC12h cell, a BHK cell, a CHO cell, a COS1 cell, a COS3 cell, a COST cell, a CV1 cell, a Vero cell, a HeLa cell, an HEK-293 cell, a PER C6 cell, a cell derived from diploid fibroblasts, a myeloma cell, or HepG2.
  • the nucleic acid-guided nuclease is a nucleic acid-guided nuclease fusion protein. In some such embodiments, the nucleic acid-guided nuclease fusion protein comprises a cell targeting agent.
  • the cell targeting agent is a peptide. In some embodiments, the peptide is a cell penetrating peptide.
  • the cell targeting agent is an antigen-binding agent.
  • the antigen binding agent is a nanobody, a domain antibody, an scFv, a Fab, a diabody, a BiTE, a diabody, a DART, a minibody, a F(ab’)2, an intrabody, or an antibody mimetic.
  • the antibody mimetic is an adnectin (i.e.
  • fibronectin based binding molecules an affilin, an affimer, an affitin, an alphabody, an affibody, a DARPin, an anticalin, an avimer, a fynomer, a Kunitz domain peptide, a monobody, a nanoCLAMP, a unibody, or a versabody, an aptamer, or a cyclotide.
  • the cell targeting agent is a cell-surface receptor ligand, or portion thereof.
  • the nucleoprotein is a ribonucleoprotein
  • the gNA is a guide RNA (gRNA)
  • the nucleic acid-guided nuclease is a RNA-guided nuclease.
  • the guide RNA is a single guide RNA (sgRNA) or a cntrRNA.
  • the RNA-guided nuclease is a Class 2 Cas polypeptide.
  • the Class 2 Cas polypeptide is a Type II Cas polypeptide.
  • the Type II Cas polypeptide is Cas9.
  • the Class 2 Cas polypeptide is a CRISPR Type V nuclease.
  • the RNA-guided DNA endonuclease is a Cas12 nuclease.
  • the conformation-specific NP binding agent is an anti-CRISPR (Acr) protein.
  • the Acr protein is AcrllA4 (e.g., SEQ ID NO: 1).
  • the Acr protein is AcrVAI (Uniprot Accession Nos. A0A5H1ZR47 or A0A5H1ZR46).
  • the conformation-specific NP binding agent is an antibody, or antigen binding fragment thereof, that specifically binds the NP.
  • the conformation-specific NP binding agent is an aptamer that specifically binds the NP.
  • the method further comprises isolating NPs from a solution having levels of NPs higher than a reference level.
  • the first detectable label and/or second detectable label comprises a fluorophore.
  • the fluorophore is a fluorescent protein.
  • the first detectable label and second detectable label are a FRET pair
  • the method comprises measuring the level of NPs based on the level of fluorescence generated by interaction of the FRET pair as measured by FRET.
  • the first detectable label and second detectable label are a split luciferase pair
  • the method comprises measuring the level of NPs based on the level of luminescence generated by interaction of pair, e.g., in the presence of appropriate luciferase substrates.
  • the first detectable label and second detectable label are a split fluorescent protein (e.g., split GFP), and the method comprises measuring the level of NPs based on the level of fluorescence generated by formation of a functional fluorescent protein from combination of the split fragments.
  • split fluorescent protein e.g., split GFP
  • the method prior to the step of providing the host cells, further comprises providing a library of test expression constructs comprising a polynucleotide encoding the NA-guided nuclease and transferring the library into the host cells.
  • the method prior to the step of providing the host cells, further comprises providing a library of test expression constructs comprising a polynucleotide encoding the gNA and transferring the library into the host cells.
  • the method further comprises providing a library of test expression constructs comprising a polynucleotide encoding the NP and transferring the library into the host cells. In some embodiments, prior to the step of providing the host cells, the method further comprises providing a library of test expression constructs comprising a polynucleotide encoding the conformation-specific NP binding agent and transferring the library into the host cells.
  • the one or more test expression constructs comprise a first expression construct comprising the polynucleotide encoding the NA-guided nuclease and a second expression construct comprising the polynucleotide encoding the gNA.
  • the first expression construct comprises a first selectable marker and the second expression construct comprises a second selectable marker.
  • the first expression construct comprises a first origin of replication and the second expression construct comprises a second origin of replication.
  • the one or more test expression constructs comprise a third expression construct comprising the polynucleotide encoding the conformation-specific NP binding agent.
  • the first expression construct comprises a third selectable marker.
  • the third expression construct comprises a third origin of replication.
  • the one or more test expression constructs comprise a first promoter operatively linked to the polynucleotide encoding the nucleic acid-guided nuclease and a second promoter operatively linked to the polynucleotide encoding the gNA.
  • the one or more test expression constructs comprise a third promoter operatively linked to the polynucleotide encoding the conformation-specific NP binding agent.
  • the first promoter, the second promoter, and/or the third promoter are an inducible promoter such that the expression level of the nucleic acid-guided nuclease and the expression level of the gNA can be modulated.
  • the inducible promoter is a L-arabinose-inducible promoter, a propionate-inducible promoter, a rhamnose-inducible promoter, a xylose-inducible promoter, a lactose-inducible promoter, an IPTG-inducible promoter, or a promoter inducible by phosphate depletion.
  • the inducible promoter is the araBAD promoter, the T7 promoter, the T5 promoter, the pLac promoter, pTac promoter, the rhaBAD promoter, the prpBCDE promoter, the rhaSR promoter, or the xlyA promoter.
  • the one or more test expression constructs comprise a first ribosome binding site operatively linked to the polynucleotide encoding the nucleic acid-guided nuclease and a second ribosome binding site operatively linked to the polynucleotide encoding the gNA. In some embodiments, the one or more test expression constructs comprise a third ribosome binding site operatively linked to the polynucleotide encoding the conformation-specific NP binding agent.
  • test expression construct comprising the polynucleotide encoding the gRNA further comprises a 3‘ hammerhead ribozyme.
  • test expression construct comprising the polynucleotide encoding the gRNA further comprises a polynucleotide encoding a ribozyme-guide-ribozyme.
  • the host cell is a bacterial cell. In certain embodiments, the bacterial cells are E. coli cells. In some embodiments, the host cell is a eukaryotic cell. In certain embodiments, the eukaryotic cell is a mammalian cell or a fungal cell. In some embodiments, the fungal cell is a yeast cell. In certain embodiments, the yeast cell is Pichia pastoris or Saccharomyces cerevisiae.
  • the mammalian cell is a COP cell, an L cell, a C127 cell, an Sp2/0 cell, an NS-0 cell, an NIH3T3 cell, a PC12 cell, a PC12h cell, a BHK cell, a CHO cell, a COS1 cell, a COS3 cell, a COST cell, a CV1 cell, a Vero cell, a HeLa cell, an HEK-293 cell, a PER C6 cell, a cell derived from diploid fibroblasts, a myeloma cell, or HepG2.
  • the nucleic acid-guided nuclease is a nucleic acid-guided nuclease fusion protein. In some such embodiments, the nucleic acid-guided nuclease fusion protein comprises a cell targeting agent.
  • the cell targeting agent is a peptide. In some embodiments, the peptide is a cell penetrating peptide.
  • the cell targeting agent is an antigen-binding agent.
  • the antigen binding agent is a nanobody, a domain antibody, an scFv, a Fab, a diabody, a BiTE, a diabody, a DART, a minibody, a F(ab’)2, an intrabody, or an antibody mimetic.
  • the antibody mimetic is an adnectin (i.e.
  • fibronectin based binding molecules an affilin, an affimer, an affitin, an alphabody, an affibody, a DARPin, an anticalin, an avimer, a fynomer, a Kunitz domain peptide, a monobody, a nanoCLAMP, a unibody, or a versabody, an aptamer, or a cyclotide.
  • the cell targeting agent is a ligand, or portion thereof.
  • the nucleoprotein is a ribonucleoprotein
  • the gNA is a guide RNA (gRNA)
  • the nucleic acid-guided nuclease is a RNA-guided nuclease.
  • the guide RNA is a single guide RNA (sgRNA) or a cntrRNA.
  • the RNA-guided nuclease is a Class 2 Cas polypeptide.
  • the Class 2 Cas polypeptide is a Type II Cas polypeptide.
  • the Type II Cas polypeptide is Cas9.
  • the Class 2 Cas polypeptide is a CRISPR Type V nuclease.
  • the RNA-guided DNA endonuclease is a Cas12 nuclease.
  • the conformation-specific NP binding agent is an anti-CRISPR (Acr) protein.
  • the Acr protein is AcrllA4 (e.g., SEQ ID NO: 1).
  • the Acr protein is AcrVAI (Uniprot Accession Nos. A0A5H1ZR47 or A0A5H1ZR46).
  • the conformation-specific NP binding agent is an antibody, or antigen binding fragment thereof, that specifically binds the NP.
  • the conformation-specific NP binding agent is an aptamer that specifically binds the NP.
  • the method further comprises isolating NPs from a solution having levels of NPs higher than a reference level.
  • a targeted active gene editor comprising a cell targeting agent conjugated to a conformation-specific nucleoprotein (NP) binding agent, and a nucleoprotein comprising a nucleic-guided nuclease and a guide nucleic acid (gNA), wherein the nucleoprotein interacts with the conformation-specific NP binding agent and is thereby stably associated with the cell targeting agent via the conformation-specific NP binding agent.
  • TAGE targeted active gene editor
  • the cell targeting agent is a peptide. In some embodiments, the peptide is a cell penetrating peptide. In certain embodiments of the TAGE, the cell targeting agent is an antigen-binding agent. In some embodiments, the antigen binding agent is a nanobody, a domain antibody, an scFv, a Fab, a diabody, a BiTE, a diabody, a DART, a minibody, a F(ab’)2, an intrabody, or an antibody mimetic. In some embodiments, the antibody mimetic is an adnectin (i.e.
  • fibronectin based binding molecules an affilin, an affimer, an affitin, an alphabody, an affibody, a DARPin, an anticalin, an avimer, a fynomer, a Kunitz domain peptide, a monobody, a nanoCLAMP, a unibody, or a versabody, an aptamer, or a cyclotide.
  • the cell targeting agent is a ligand, or portion thereof.
  • the nucleoprotein is a ribonucleoprotein
  • the gNA is a guide RNA (gRNA)
  • the nucleic acid-guided nuclease is a RNA-guided nuclease.
  • the guide RNA is a single guide RNA (sgRNA) or a cntrRNA.
  • the RNA-guided nuclease is a Class 2 Cas polypeptide.
  • the Class 2 Cas polypeptide is a Type II Cas polypeptide.
  • the Type II Cas polypeptide is Cas9.
  • the Class 2 Cas polypeptide is a CRISPR Type V nuclease.
  • the RNA-guided DNA endonuclease is a Cas12 nuclease.
  • the conformation-specific NP binding agent is an anti- CRISPR (Acr) protein.
  • the Acr protein is AcrllA4 (e.g., SEQ ID NO: 1).
  • the Acr protein is AcrVAI (Uniprot Accession Nos. A0A5H1ZR47 or A0A5H1ZR46).
  • the conformation-specific NP binding agent is an antibody, or antigen binding fragment thereof, that specifically binds the NP.
  • the conformation-specific NP binding agent is an aptamer that specifically binds the NP.
  • the conformation-specific NP binding agent is an anti- CRISPR (Acr) protein.
  • the Acr protein is AcrllA4 (e.g., SEQ ID NO: 1) or AcrVAI (Uniprot Accession Nos. A0A5H1ZR47 or A0A5H1ZR46).
  • the conformation-specific NP binding agent is an antibody, or antigen binding fragment thereof, that specifically binds the NP.
  • the conformation-specific NP binding agent is an aptamer that specifically binds the NP.
  • the conformation-specific NP binding agent further comprises a conjugation moiety that binds to the cell targeting agent.
  • the conformation-specific NP binding agent further comprises a first conjugation moiety and the cell targeting agent comprises a second conjugation moiety that binds the first conjugation moiety.
  • a method of separating nucleoproteins (NPs) from one or more other compounds in a liquid sample comprising contacting a surface comprising a conformation-specific NP binding agent with the liquid sample under conditions that allow binding of the NPs to the conformation-specific NP binding agent and removal of an unbound fraction comprising the one or more other compounds; and contacting the surface comprising the conformation-specific NP binding agent with an elution buffer, thereby eluting the NPs from the conformation-specific NP binding agents to provide an NP eluate lacking the one or more other compounds in the liquid sample.
  • NPs nucleoproteins
  • a method of separating nucleoproteins (NPs) from one or more other compounds in a liquid sample comprising contacting a surface comprising a conformation-specific NP binding agent with the liquid sample under conditions that allow binding of the NPs to the conformation-specific NP binding agent; contacting the surface comprising the conformation-specific NP binding agent with a wash buffer; and contacting the surface comprising the conformation-specific NP binding agent with an elution buffer, thereby eluting the NPs from the conformation-specific NP binding agents to provide an NP eluate lacking or having a reduction in the one or more other compounds in the liquid sample.
  • NPs nucleoproteins
  • the surface is a bead, plate, or a column.
  • the wash buffer is at a first conductivity and the elution buffer is at a second conductivity.
  • the wash buffer is at a first salt concentration and the elution buffer is at a second salt concentration.
  • the wash buffer is at a first pH and the elution buffer is at a second pH.
  • the elution buffer contains an NP binding agent that competes for binding the NP with the immobilized NP binding agent.
  • the nucleic acid-guided nuclease is a nucleic acid-guided nuclease fusion protein. In some such embodiments, the nucleic acid-guided nuclease fusion protein comprises a cell targeting agent.
  • the cell targeting agent is a peptide. In some embodiments, the peptide is a cell penetrating peptide.
  • the cell targeting agent is an antigen-binding agent.
  • the antigen binding agent is a nanobody, a domain antibody, an scFv, a Fab, a diabody, a BiTE, a diabody, a DART, a minibody, a F(ab’)2, an intrabody, or an antibody mimetic.
  • the antibody mimetic is an adnectin (i.e.
  • fibronectin based binding molecules an affilin, an affimer, an affitin, an alphabody, an affibody, a DARPin, an anticalin, an avimer, a fynomer, a Kunitz domain peptide, a monobody, a nanoCLAMP, a unibody, or a versabody, an aptamer, or a cyclotide.
  • the cell targeting agent is a ligand, or portion thereof.
  • the nucleoprotein is a ribonucleoprotein
  • the gNA is a guide RNA (gRNA)
  • the nucleic acid-guided nuclease is a RNA-guided nuclease.
  • the guide RNA is a single guide RNA (sgRNA) or a cntrRNA.
  • the RNA-guided nuclease is a Class 2 Cas polypeptide.
  • the Class 2 Cas polypeptide is a Type II Cas polypeptide.
  • the Type II Cas polypeptide is Cas9.
  • the Class 2 Cas polypeptide is a CRISPR Type V nuclease.
  • the RNA-guided DNA endonuclease is a Cas12 nuclease.
  • the conformation-specific NP binding agent is an anti-CRISPR (Acr) protein.
  • the Acr protein is AcrllA4 (e.g., SEQ ID NO: 1).
  • the Acr protein is AcrVAI (Uniprot Accession Nos. A0A5H1ZR47 or A0A5H1ZR46).
  • the conformation-specific NP binding agent is an antibody, or antigen binding fragment thereof, that specifically binds the NP.
  • the conformation-specific NP binding agent is an aptamer that specifically binds the NP.
  • the one or more other compounds is a host cell protein.
  • the liquid sample comprises a host cell lysate comprising the nucleoprotein.
  • a method of identifying a conformation-specific nucleoprotein (NP) binding agent that enables release of a nucleoprotein in the endosome of a target cell comprising providing a plurality of host cells each comprising one or more expression constructs comprising (i) a polynucleotide encoding a test conformation-specific NP binding agent, (ii) a polynucleotide encoding a nucleic acid-guided nuclease, and (iii) a polynucleotide encoding a unique identifying nucleic acid (uiNA) comprising a guide nucleic acid (gNA) and a sequence identifier, wherein at least the polynucleotide encoding the test conformation-specific NP binding agent and the polynucleotide encoding the guide nucleic acid are on the same expression construct; transferring the one or more expression constructs into a host cell suitable to express the test conform
  • the measuring step comprises: exposing the separated complexes to a first buffer having a first pH at about the pH of the cytoplasm in the target cell; testing the uiRNA of the separated complexes in the first buffer for the level of the identifier sequence; exposing the separated complexes to a second buffer having a second pH at about the pH of an endosome in the target cell; and measuring the degree of dissociation of the nucleoprotein and the test conformation- specific binding agent following exposure to the second buffer based on changes in the level of the identifier sequence, wherein a decrease in the level of the identifier sequence in the second buffer relative to the level of the identifier sequence in the first buffer identifies the test conformation-specific NP binding agent as one that enables release of a nucleoprotein in the endosome of the target cell.
  • the second pH is less than the first pH.
  • the first pH is about pH 6.5 to about pH 8.
  • the first pH is about pH 7.
  • the second pH is about pH 4.5 to about pH 6.4.
  • the second pH is about pH 5.
  • the method comprises sequencing portions of the expression construct encoding the uiNA and the test conformation-specific NP binding agent prior to the expression construct being transferred into the host cell, thereby providing a reference for identifying the test conformation-specific NP based on the identifier sequence.
  • the level of the identifier sequence is measured by polymerase chain reaction (PCR), RT-PCR, sequencing, or a nucleic acid microarray.
  • PCR polymerase chain reaction
  • RT-PCR RT-PCR
  • sequencing or a nucleic acid microarray.
  • the host cell is a eukaryotic cell. In other embodiments, the host cell is a bacterial cell (e.g., E. coif).
  • the nucleoprotein is a ribonucleoprotein
  • the gNA is a guide RNA (gRNA)
  • the nucleic acid-guided nuclease is a RNA-guided nuclease.
  • the guide RNA is a single guide RNA (sgRNA) or a cntrRNA.
  • the RNA-guided nuclease is a Class 2 Cas polypeptide.
  • the Class 2 Cas polypeptide is a Type II Cas polypeptide.
  • the Type II Cas polypeptide is Cas9.
  • the Class 2 Cas polypeptide is a CRISPR Type V nuclease.
  • the RNA-guided DNA endonuclease is a Cas12 nuclease.
  • the RNA-guided nuclease is a CRISPR Type V nuclease.
  • the RNA-guided DNA endonuclease is a Cas12 nuclease.
  • the conformation-specific NP binding agent is an anti-CRISPR (Acr) protein.
  • the Acr protein is AcrllA4 (e.g., SEQ ID NO: 1).
  • the Acr protein is AcrVAI (Uniprot Accession Nos. A0A5H1ZR47 or A0A5H1ZR46).
  • the conformation-specific NP binding agent is an antibody, or antigen binding fragment thereof, that specifically binds the NP.
  • the conformation-specific NP binding agent is an aptamer that specifically binds the NP.
  • a method of producing a conformation-specific binding agent comprising transferring to a host cell an expression construct comprising a polynucleotide encoding a conformation-specific NP binding agent identified by any of the methods of the invention.
  • the method further comprises separating the conformation-specific NP binding agent from the host cell.
  • a targeted active gene editor comprising: a cell targeting agent conjugated to a conformation-specific nucleoprotein (NP) binding agent that enables release of a nucleoprotein in the endosome of a target cell, wherein the conformation-specific NP binding agent is one identified by any of the methods of the invention, and a nucleoprotein comprising a nucleic-guided nuclease and a guide nucleic acid (gNA), wherein the nucleoprotein interacts with the conformation-specific NP binding agent and is thereby stably associated with the cell targeting agent via the conformation-specific NP binding agent.
  • the target cell is a mammalian cell.
  • the mammalian cells are hematopoietic stem cells (HSC), neutrophils, T cells, B cells, dendritic cells, macrophages, ocular cells, or fibroblasts.
  • the cell targeting agent is an antigen binding polypeptide that specifically binds to an extracellular cell membrane-bound molecule, a cell penetrating peptide, or a ligand that specifically binds to an extracellular cell membrane-bound molecule, or combinations thereof.
  • the nucleic acid-guided nuclease is an RNA-guided nuclease and the guide-nucleic acid is a guide RNA.
  • the RNA-guided nuclease is Cas9 or Cas12.
  • the RNA-guided nuclease is a Cas9 nuclease.
  • the Cas9 nuclease is wildtype Cas9 nuclease (e.g., Streptococcus pyogenes Cas9, SEQ ID NO: 32).
  • the Cas9 nuclease comprises an amino acid sequence having at least 85%, 90%, 95%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 32.
  • the Cas9 nuclease comprises the amino acid substitution C80A (e.g., SEQ ID NO: 33).
  • the Cas9 nuclease comprises an amino acid sequence having at least 85%, 90%, 95%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 33.
  • the RNA-guided nuclease is a nuclease other than Cas9 (e.g., such as one described in Section V).
  • the RNA-guided nuclease is a CRISPR Type V nuclease.
  • the RNA-guided DNA endonuclease is a Cas12 nuclease.
  • the Cas12 nuclease is wildtype Cas12a nuclease (e.g., Acidaminococcus sp. Cas12a, SEQ ID NO: 34).
  • the Cas12 nuclease comprises an amino acid sequence having at least 85%, 90%, 95%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 34.
  • Examples of Cas12a variants useful in the TAGE agents herein include, but are not limited to, Alt-R® Cas12a (Cpf1) Ultra (e.g., IDT Catalog No. 10001272) or Cas12a as described in Kleinstiver, et al. Nature biotechnology 37.3 (2019): 276-282, which is hereby incorporated by reference.
  • the conformation-specific NP binding agent is an anti-CRISPR (Acr) protein.
  • the Acr protein is an AcrllA4 protein.
  • the AcrllA4 protein is a wildtype AcrllA4 protein (e.g., Listeria monocytogenes AcrllA4, SEQ ID NO: 1).
  • the AcrllA4 protein comprises an amino acid sequence having at least 85%, 90%, 95%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 1 .
  • the Acr protein is an AcrVAI protein.
  • the AcrVAI protein is a wildtype AcrVAI protein (e.g., Uniprot Accession Nos. A0A5H1ZR47 or A0A5H1ZR46).
  • the AcrVAI protein comprises an amino acid sequence having at least 85%, 90%, 95%, 97%, 98%, 99%, or 100% identity to the amino acid sequences set forth Uniprot Accession Nos. A0A5H1ZR47 or A0A5H1ZR46.
  • the invention relates to compositions and methods for internalizing nucleoproteins comprising a nucleic acid-guided nuclease and guide nucleic acid into a cell.
  • the invention includes a targeted active gene editing (TAGE) agent that comprises a cell targeting agent conjugated to a conformation- specific nucleoprotein binding agent that can stably associate with a nucleoprotein comprising a nucleic acid-guided nuclease and guide nucleic acid.
  • TAGE targeted active gene editing
  • the cell targeting agent/conformation-specific NP binding agent stably associate with the nucleoprotein such that the nucleoprotein can be delivered and internalized into the target cell.
  • methods of identifying or isolating conformation-specific NP binding agents having characteristics (e.g., increased cytoplasmic or endosomal release) that optimize delivery of a nucleoprotein to a cell.
  • compositions for isolating one or more expression constructs that enable optimal expression of nucleoproteins comprising a nucleic acid-guided nuclease and a guide nucleic acid in cells.
  • methods for rapidly identifying expression systems that express active nucleoproteins comprising a nucleic acid-guided nuclease and a guide nucleic acid identifying cells containing such expression systems, or measuring nucleoprotein levels without the need for additional purification steps.
  • the methods of the invention further include affinity purification methods using a conformational specific-NP binding agent to isolate a nucleic acid-guided nuclease.
  • the term “conformation-specific nucleoprotein binding agent” refers to an agent (e.g., a protein or nucleic acid), such as an anti-CRISPR protein, an antibody, or an aptamer capable of binding a nucleoprotein (NP) comprising a nucleic acid-guided nuclease (e.g., an RNA- guided nuclease) complexed with a guide nucleic acid (gNA, e.g., a guide RNA), but has reduced or no detectable binding to either the nucleic acid-guided nuclease in the absence of a gNA, nor the gNA in the absence in the absence of the nuclease.
  • NP nucleoprotein
  • gNA guide nucleic acid
  • the conformation-specific NP binding agent can be one having one or more sequence modifications (e.g., amino acid substitutions, insertions, or deletions), such as ones that confer characteristics (e.g., increased cytoplasmic release of an NP or endosomal release of an NP) that optimize delivery of a nucleoprotein to a cell.
  • the conformation-specific NP binding agent has at least 50, 60, 70, 75, 80, 85, 90, 95,
  • nucleic acid-guided nuclease refers to a protein that is targeted to a specific nucleic acid sequence or set of similar sequences of a polynucleotide chain via recognition of the particular sequence(s) by the modifying polypeptide itself or an associated molecule (e.g., RNA), wherein the polypeptide can modify the polynucleotide chain.
  • a nucleic acid-guided nuclease is a RNA-guided endonuclease, such as Cas9.
  • nucleic acid-guided nuclease fusion protein refers to a complex of molecules including a cell targeting agent conjugated to a nucleic acid-guided nuclease (e.g., a RNA-guided nuclease or a DNA-guided nuclease) that recognizes a nucleic acid sequence.
  • a nucleic acid-guided nuclease e.g., a RNA-guided nuclease or a DNA-guided nuclease
  • the term “targeted active gene editor” or “TAGE” refers to a complex of molecules including a cell targeting agent (such as, but not limited to, an antigen binding polypeptide (e.g., an antibody or an antigen-binding portion thereof), a ligand, a cell penetrating peptide (CPP), or combinations thereof), that specifically binds to an extracellular target molecule (e.g., an extracellular protein or glycan, such as an extracellular protein on the cell surface) displayed on a cell membrane (e.g., a cell surface protein) or otherwise promotes cellular internalization, and a site-directed modifying polypeptide (such as, but not limited to, a nucleic acid-guided nuclease) that recognizes a nucleic acid sequence.
  • a cell targeting agent such as, but not limited to, an antigen binding polypeptide (e.g., an antibody or an antigen-binding portion thereof), a ligand, a cell penetrating peptid
  • the cell targeting agent of a TAGE can be associated with the site-directed modifying polypeptide indirectly or directly such that at least the site-directed modifying polypeptide is internalized by a target cell, e.g., a cell expressing an extracellular molecule bound by the cell targeting agent.
  • a target cell e.g., a cell expressing an extracellular molecule bound by the cell targeting agent.
  • the cell targeting agent can be conjugated to a conformation-specific nucleoprotein binding agent (e.g., an anti-CRISPR protein), which in turn binds to a nucleoprotein.
  • TAGE is an active CRISPR targeting or TAGE agent where the site directed polypeptide is a nucleic acid-guided nuclease (e.g., RNA-guided nuclease or DNA-nuclease), such as Cas9 or Cas12.
  • the TAGE includes at least one NLS (e.g., one, two, three, four, five, six, seven, eight, nine, ten, or more than ten NLSs).
  • a TAGE can target any nucleic acid within a cell, including, but not limited to, a gene.
  • cell targeting agent refers to a protein (e.g., a ligand, a cell penetrating peptide, or an antigen binding agent) that, when conjugated with a conformation-specific NP binding agent that stably associates with a nucleoprotein comprising a nucleic acid-guided nuclease and a guide nucleic acid, enables at least the nucleoprotein to be targeted to the surface of a target cell or internalized by a target cell, i.e., a cell targeted by the cell targeting agent.
  • a protein e.g., a ligand, a cell penetrating peptide, or an antigen binding agent
  • the cell targeting agent may be one that specifically binds to an extracellular target molecule (e.g., an extracellular protein, lipid, or glycan) displayed on a cell membrane.
  • the cell targeting agent can be associated with a nucleic acid-guided nuclease such that at least the nucleoprotein is internalized by a target cell, i.e., a cell expressing an extracellular molecule bound by the cell targeting agent.
  • polypeptide or “protein”, as used interchangeably herein, refer to any polymeric chain of amino acids.
  • polypeptide encompasses native or artificial proteins, protein fragments and polypeptide analogs of a protein sequence.
  • test protein refers to any protein capable of being assessed for cell targeting in accordance with the methods described herein (e.g., a test Conformation-specific NP Binding agent).
  • the test protein is a protein capable of being conjugated to a nucleic acid- guided nuclease.
  • the methods herein are further useful for identifying variants of a conformation- specific NP binding agent (e.g., mutagenized conformation-specific NP Binding agents, such as Acrl IA4) having desired cell targeting properties. In such cases, the conformation-specific NP binding agent is considered the test protein.
  • target cell refers to a cell or population of cells, such as mammalian cells (e.g., human cells), which includes a nucleic acid sequence in which site-directed modification of the nucleic acid is desired (e.g., to produce a genetically-modified cell in vivo or ex vivo).
  • a target cell displays on its cell membrane an extracellular molecule (e.g., an extracellular protein such as a receptor or a ligand, or glycan) specifically bound by an extracellular cell membrane binding moiety of the TAGE agent.
  • extracellular molecule e.g., an extracellular protein such as a receptor or a ligand, or glycan
  • nucleic acid refers to a molecule comprising nucleotides, including a polynucleotide, an oligonucleotide, or other DNA or RNA. In one embodiment, a nucleic acid is present in a cell and can be transmitted to progeny of the cell via cell division.
  • a nucleic acid is a gene (e.g., an endogenous gene) found within the genome of a cell within its chromosomes.
  • a nucleic acid is a mammalian expression vector that has been transfected into a cell. DNA that is incorporated into the genome of a cell using, e.g., transfection methods, is also considered within the scope of a “nucleic acid” as used herein, even if the incorporated DNA is not meant to be transmitted to progeny cells.
  • endosomal escape agent or “endosomal release agent” refers to an agent (e.g., a peptide) that, when conjugated to a molecule (e.g., a polypeptide, such as a site- directed modifying polypeptide), is capable of promoting release of the molecule from an endosome within a cell.
  • a TAGE agent comprises an endosomal escape agent.
  • the term “stably associated” when used in the context of a TAGE agent refers to the ability of the cell targeting agent or a conformation specific-NP binding agent and the site-directed modifying polypeptide to complex in such a way that the complex can be internalized into a target cell such that nucleic acid editing can occur within the cell.
  • ways to determine if a TAGE agent is stably associated include in vitro assays whereby association of the complex is determined following exposure of a cell to the TAGE agent, e.g., by determining whether gene editing occurred using a standard gene editing systems.
  • Examples of such assays are known in the art, such as SDS-PAGE, Western blot, size exclusion chromatography (SEC), and electrophoretic mobility shift assay to determine protein complexes; PCR amplification, direct sequencing (e.g., next-generation sequencing or Sanger sequencing), enzymatic cleavage of a locus with a nuclease (e.g., Celery) of the gene locus to confirm editing; and indirect phenotypic assays that measure the downstream effects of editing a specific gene, such as loss of a protein as measured by Western blot or flow cytometry or generation of a functional protein, as measured by functional assays.
  • SEC size exclusion chromatography
  • electrophoretic mobility shift assay to determine protein complexes
  • PCR amplification direct sequencing (e.g., next-generation sequencing or Sanger sequencing), enzymatic cleavage of a locus with a nuclease (e.g., Celery) of the gene locus to
  • modifying a nucleic acid refers to any modification to a nucleic acid targeted by a site-directed modifying polypeptide (e.g., a nucleic acid-guided nuclease).
  • modifications include any changes to the amino acid sequence including, but not limited to, any insertion, deletion, or substitution of an amino acid residue in the nucleic acid sequence relative to a reference sequence (e.g., a wild-type or a native sequence).
  • Such amino acid changes may, for example, may lead to a change in expression of a gene (e.g., an increase or decrease in expression) or replacement of a nucleic acid sequence.
  • Modifications of nucleic acids can further include double stranded cleavage, single stranded cleavage, or binding of any RNA-guided endonuclease disclosed herein to a target site. Binding of a RNA-guided endonuclease can inhibit expression of the nucleic acid or can increase expression of any nucleic acid in operable linkage to the nucleic acid comprising the target site.
  • conjugation moiety refers to a moiety that is capable of conjugating two more or more molecules, such as a cell targeting agent, a conformation-specific NP binding agent, or a detectable label and a protein (e.g., conformation-specific NP binding agent or nucleic acid-guided nuclease).
  • conjugation refers to the physical or chemical complexation formed between a molecule and the second molecule.
  • the chemical complexation constitutes specifically a bond or chemical moiety formed between a functional group of a first molecule with a functional group of a second molecule.
  • bonds include, but are not limited to, covalent linkages and non-covalent bonds
  • chemical moieties include, but are not limited to, esters, carbonates, imines phosphate esters, hydrazones, acetals, orthoesters, peptide linkages, and oligonucleotide linkages.
  • conjugation is achieved via a physical association or non-covalent complexation.
  • ligand refers to a molecule that is capable of specifically binding to another molecule on or in a cell, such as one or more cell surface receptors, and includes molecules such as proteins, hormones, neurotransmitters, cytokines, growth factors, cell adhesion molecules, or nutrients. Generally, a ligand that binds to another specific molecule or molecules. For example, a ligand may bind to a receptor.
  • a nucleic acid-guided nuclease can be associated with one or more ligands through covalent or non-covalent linkage. Examples of ligands useful herein, or targets bound by ligands, and further description of ligands in general, are disclosed in Bryant & Stow (2005).
  • an antigen binding polypeptide that specifically binds to an antigen binds to an antigen with an Kd of at least about 1 x10- 4 , 1 x10 5 , 1 c10- 6 M, 1 c10- 7 M, 1 c10- 8 M, 1 c10- 9 M, 1 c10- 10 M, 1 c10 ⁇ 11 M, 1 c10 ⁇ 12 M, or more as determined by surface plasmon resonance or other approaches known in the art (e.g., filter binding assay, fluorescence polarization, isothermal titration calorimetry), including those described further herein.
  • an antigen binding polypeptide specifically binds to an antigen if the antigen binding polypeptide binds to an antigen with an affinity that is at least two-fold greater as determined by surface plasmon resonance than its affinity for a nonspecific antigen.
  • CPP cell-penetrating peptide
  • a peptide generally of about 5-60 amino acid residues (e.g., 5-10, 10-15, 15-20, 20-25, 25-30, 30-35, 35-40, 40-45, 45-50, 50-55, or 55-60 amino acid resides) in length, that can facilitate cellular uptake of a conjugated molecule, particularly a nucleoprotein comprising a nucleic acid-guided nuclease and a guide nucleic acid.
  • a CPP can also be characterized in certain embodiments as being able to facilitate the movement or traversal of a molecular conjugate across/through one or more of a lipid bilayer, micelle, cell membrane, organelle membrane (e.g., nuclear membrane), vesicle membrane, or cell wall.
  • a CPP herein can be cationic, amphipathic, or hydrophobic in certain embodiments. Examples of CPPs useful herein, and further description of CPPs in general, are disclosed in Borrelli, Antonella, et al. Molecules 23.2 (2016): 295; Milletti, Francesca. Drug discovery today 17.15-16 (2012): 850-860, which are incorporated herein by reference. Further, there exists a database of experimentally validated CPPs (CPPsite, Gautam et al., 2012). The CPP can be any known CPP, such as a CPP shown in the CPPsite database.
  • nuclear localization signal refers to a peptide that, when conjugated to a molecule (e.g., a polypeptide, such as a site-directed modifying polypeptide), is capable of promoting import of the molecule into the cell nucleus by nuclear transport.
  • the NLS can, for example, direct transport of a protein with which it is associated from the cytoplasm of a cell across the nuclear envelope barrier.
  • the NLS is intended to encompass not only the nuclear localization sequence of a particular peptide, but also derivatives thereof that are capable of directing translocation of a cytoplasmic polypeptide across the nuclear envelope barrier.
  • one or more NLSs can be attached to the N-terminus, the C-terminus, or both the N- and C-termini of the polypeptide of a TAGE agent herein.
  • TAT-related peptide refers to a CPP that is derived from the transactivator of transcription (TAT) of human immunodeficiency virus.
  • the amino acid sequence of a TAT peptide comprises RKKRRQRRR (SEQ ID NO: 3).
  • a TAT-related peptide includes any peptide comprising the amino acid sequence of RKKRRQRRR (SEQ ID NO: 3), or an amino acid sequence having conservative amino acid substitutions wherein the peptide is still able to internalize into a cell.
  • a TAT-related peptide includes 1 , 2, or 3 amino acid substitutions, wherein the TAT-related peptide is able to internalize into a target cell.
  • an antigen binding agent refers to an agent, such as a protein or nucleic acid, that binds to a specified target antigen, such as an extracellular cell-membrane bound protein (e.g., cell surface protein).
  • a specified target antigen such as an extracellular cell-membrane bound protein (e.g., cell surface protein).
  • an antigen binding agent include an antibody, antigen-binding fragments of an antibody, and an antibody mimetic.
  • an antigen-binding agent is an antigen binding peptide.
  • an antigen binding polypeptide that specifically binds to an antigen binds to an antigen with an Kd of at least about 1 x10 -4 , 1 x10 -5 , 1 x10 -6 M, 1 x10 -7 M,
  • an antigen binding polypeptide specifically binds to an antigen if the antigen binding polypeptide binds to an antigen with an affinity that is at least two-fold greater as determined by surface plasmon resonance than its affinity for a nonspecific antigen.
  • the term “specifically binds” refers to the ability of a ligand to recognize and bind to its respective receptor(s).
  • the term “specifically binds” refers to the ability of CPPs to translocate a cell’s membrane.
  • the TAGE agent may display the specific binding properties of both the antibody or ligand and the CPP(s).
  • the antibody or ligand of the TAGE agent may confer specific binding to an extracellular cell surface molecule, such as a cell surface protein, while the CPP(s) confers enhanced ability of the TAGE agent to translocate across a cell membrane.
  • antibody is used herein in the broadest sense and encompasses various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), nanobodies, monobodies, and antibody fragments so long as they exhibit the desired antigen-binding activity.
  • antibody includes an immunoglobulin molecule comprising four polypeptide chains, two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds, as well as multimers thereof (e.g., IgM).
  • Each heavy chain (HC) comprises a heavy chain variable region (or domain) (abbreviated herein as HCVR or VH) and a heavy chain constant region (or domain).
  • the heavy chain constant region comprises three domains, CH1 , CH2 and CH3.
  • Each light chain (LC) comprises a light chain variable region (abbreviated herein as LCVR or VL) and a light chain constant region.
  • the light chain constant region comprises one domain (CL1 ).
  • Each VH and VL is composed of three complementarity determining regions (CDRs) and four framework (FRs), arranged from amino- terminus to carboxy-terminus in the following order: FR1 , CDR1 , FR2, CDR2, 1 -R3, CDR3, FR4
  • Immunoglobulin molecules can be of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g.,
  • VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDRs), interspersed with regions that are more conserved, termed framework regions (FR).
  • CDRs complementarity determining regions
  • FR framework regions
  • Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1 , CDR1 , FR2, CDR2, FR3, CDR3, FR4.
  • CDR complementarity determining region
  • CDR refers to the noncontiguous antigen combining sites found within the variable region of both heavy and light chain polypeptides. These particular regions have been described by Kabat et al. , J. Biol. Chem. 252, 6609- 6616 (1977) and Kabat et al., Sequences of protein of immunological interest. (1991 ), and by Chothia et al., J. Mol. Biol. 196:901 -917 (1987) and by MacCallum et al., J. Mol. Biol. 262:732-745 (1996) where the definitions include overlapping or subsets of amino acid residues when compared against each other. The amino acid residues which encompass the CDRs as defined by each of the above cited references are set forth for comparison.
  • the term “CDR” is a CDR as defined by Kabat, based on sequence comparisons.
  • the term “Fc domain” is used to define the C-terminal region of an immunoglobulin heavy chain, which may be generated by papain digestion of an intact antibody.
  • the Fc domain may be a native sequence Fc domain or a variant Fc domain.
  • the Fc domain of an immunoglobulin generally comprises two constant domains, a CH2 domain and a CH3 domain, and optionally comprises a CH4 domain Replacements of amino acid residues in the Fc portion to alter antibody effector function are known in the art (Winter, et al. U.S. Pat. Nos. 5,648,260; 5,624,821 ).
  • the Fc domain of an antibody mediates several important effector functions e.g.
  • At least one amino acid residue is altered (e.g., deleted, inserted, or replaced) in the Fc domain of an Fc domain-containing binding agent such that effector functions of the binding agent are altered.
  • an “intact” or a “full length” antibody refers to an antibody comprising four polypeptide chains, two heavy (H) chains and two light (L) chains.
  • an intact antibody is an intact IgG antibody.
  • monoclonal antibody refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e. , the individual antibodies comprising the population are identical and/or bind the same epitope, except for possible variant antibodies, e.g., containing naturally occurring mutations or arising during production of a monoclonal antibody preparation, such variants generally being present in minor amounts.
  • polyclonal antibody preparations typically include different antibodies directed against different determinants (epitopes)
  • each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on an antigen.
  • the modifier "monoclonal” indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies and is not to be construed as requiring production of the antibody by any particular method.
  • the monoclonal antibodies to be used in accordance with the present invention may be made by a variety of techniques, including but not limited to the hybridoma method, recombinant DNA methods, phage- display methods, and methods utilizing transgenic animals containing all or part of the human immunoglobulin loci, such methods and other exemplary methods for making monoclonal antibodies being described herein.
  • human antibody refers to an antibody having variable regions in which both the framework and CDR regions are derived from human germline immunoglobulin sequences. Furthermore, if the antibody contains a constant region, the constant region also is derived from human germline immunoglobulin sequences.
  • the human antibodies of the invention may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo).
  • the term “human antibody”, as used herein is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences.
  • humanized antibody is intended to refer to antibodies in which CDR sequences derived from the germline of one mammalian species, such as a mouse, have been grafted onto human framework sequences. Additional framework region modifications may be made within the human framework sequences.
  • a "humanized form" of an antibody e.g., a non-human antibody, refers to an antibody that has undergone humanization.
  • chimeric antibody is intended to refer to antibodies in which the variable region sequences are derived from one species and the constant region sequences are derived from another species, such as an antibody in which the variable region sequences are derived from a mouse antibody and the constant region sequences are derived from a human antibody.
  • An "antibody fragment”, “antigen-binding fragment” or “antigen-binding portion” of an antibody refers to a molecule other than an intact antibody that comprises a portion of an intact antibody and that binds the antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2; diabodies; linear antibodies; single-chain antibody molecules (e.g. scFv); and multispecific antibodies formed from antibody fragments.
  • a “multispecific antigen binding polypeptide” or “multispecific antibody” is an antigen binding polypeptide that targets and binds to more than one antigen or epitope.
  • a “bispecific,” “dual-specific” or “bifunctional” antigen binding polypeptide or antibody is a hybrid antigen binding polypeptide or antibody, respectively, having two different antigen binding sites.
  • Bispecific antigen binding polypeptides and antibodies are examples of a multispecific antigen binding polypeptide or a multispecific antibody and may be produced by a variety of methods including, but not limited to, fusion of hybridomas or linking of Fab' fragments. See, e.g., Songsivilai and Lachmann, 1990, Clin. Exp. Immunol.
  • antibody mimetic or “antibody mimic” refers to a molecule that is not structurally related to an antibody but is capable of specifically binding to an antigen.
  • antibody mimetics include, but are not limited to, an adnectin (i.e.
  • fibronectin based binding molecules an affilin, an affimer, an affitin, an alphabody, an affibody, DARPins, an anticalin, an avimer, a fynomer, a Kunitz domain peptide, a monobody, a nanoCLAMP, a nanobody, a unibody, a versabody, an aptamer, a cyclotide, and a peptidic molecule all of which employ binding structures that, while they mimic traditional antibody binding, are generated from and function via distinct mechanisms.
  • Amino acid sequences described herein may include “conservative mutations,” including the substitution, deletion or addition of nucleic acids that alter, add or delete a single amino acid or a small number of amino acids in a coding sequence where the nucleic acid alterations result in the substitution of a chemically similar amino acid.
  • a conservative amino acid substitution refers to the replacement of a first amino acid by a second amino acid that has chemical and/or physical properties (e.g., charge, structure, polarity, hydrophobicity/hydrophilicity) that are similar to those of the first amino acid.
  • Conservative substitutions include replacement of one amino acid by another within the following groups: lysine (K), arginine (R) and histidine (H); aspartate (D) and glutamate (E); asparagine (N) and glutamine (Q); N, Q, serine (S), threonine (T), and tyrosine (Y); K, R, H, D, and E; D, E, N, and Q; alanine (A), valine (V), leucine (L), isoleucine (I), proline (P), phenylalanine (F), tryptophan (W), methionine (M), cysteine (C), and glycine (G); F, W, and Y; H, F, W, and Y; C, S and T; C and A; S and T; C and S; S, T, and Y; V, I, and L; V, I, and T.
  • Other conservative amino acid substitutions are also recognized as valid, depending on the context of
  • operably linked refers to polynucleotide sequences or amino acid sequences placed into a functional relationship with one another.
  • regulatory sequences e.g., a promoter or enhancer
  • a polynucleotide e.g., encoding a guide RNA or nucleic acid-guided nuclease
  • two polypeptide-encoding nucleotide sequences are operably linked if they are contiguous and capable of expression in the same reading frame so as to produce a "fusion protein" following transcription and translation.
  • the term “conformation-specific nucleoprotein binding agent” refers to an agent (e.g., a protein or nucleic acid), such as an anti-CRISPR protein, an antibody, or an aptamer capable of binding a nucleoprotein (NP) comprising a nucleic acid-guided nuclease (e.g., an RNA- guided nuclease) complexed with a guide nucleic acid (gNA, e.g., a guide RNA), but has reduced or no detectable binding to either the nucleic acid-guided nuclease in the absence of a gNA, nor the gNA in the absence in the absence of the nuclease.
  • a nucleoprotein e.g., a protein or nucleic acid
  • gNA guide nucleic acid
  • the conformation-specific NP binding agent can be one having one or more sequence modifications (e.g., amino acid substitutions, insertions, or deletions), such as ones that confer characteristics (e.g., increased cytoplasmic release of an NP or endosomal release of an NP) that optimize delivery of a nucleoprotein to a cell.
  • the conformation-specific NP binding agent has at least 50, 60, 70, 75, 80, 85, 90, 95,
  • the conformation-specific NP-binding agent is an anti-CRISPR (Acr) protein.
  • anti-CRISPR proteins or “Acr proteins” refer to proteins capable of binding a nucleic acid-guided nuclease (e.g., a RNA-guided nuclease) when bound to a guide nucleic acid that inhibit NA-guided nuclease activity (e.g., by preventing recognition or cleavage of target DNA) or variants of naturally occurring Acr proteins that lack inhibitory activity but maintain their ability to bind to gNA-bound nucleic acid-guided nucleases.
  • Acr proteins include those naturally encoded by bacteriophage or variants thereof (e.g., at least 50, 60, 70, 75, 80, 85, 90, 95, or 98% sequence identity) that retain nucleoprotein binding capabilities.
  • the Acr protein e.g., AcrllA4
  • the Acr protein can be a variation of a naturally occurring Acr protein comprising one or more modifications (e.g., amino acid substitutions, insertions, or deletions) that confer characteristics (e.g., increased cytoplasmic of an NP or endosomal release of an NP) that optimize delivery of a nucleoprotein to a cell.
  • any anti-CRISPR proteins known in the art can be used in the present methods and compositions capable of binding a nucleoprotein comprising a nucleic acid-guided nuclease (see, for example, W02018093990A1 and Bondy-Denomy, Joseph. "Protein inhibitors of CRISPR-Cas9.” ACS chemical biology 13.2 (2016): 417-423; Zhu et al. "Structural insights into the inactivation of CRISPR- Cas systems by diverse anti-CRISPR proteins.” BMC biology 16.1 (2016): 1 -11 ; and Kim et al. "Solution structure and dynamics of anti-CRISPR AcrllA4, the Cas9 inhibitor.” Scientific reports 8.1 (2016): 3883, which are hereby incorporated by reference).
  • the anti-CRISPR protein is a type IF Acr Protein, such as AcrIFI (YP_007392342.1 ), AcrlF2 (YP_002332454.1 ), AcrlF3 (YP_007392440.1 ), AcrlF4 (WP_016068584.1 ), AcrlF5 (YP_007392740.1 ), AcrlF6 (WP_043884810.1 ).
  • AcrlF7 (YP_009146150.1 ), AcrlF8 (YP_007006940.1 ), AcrlF9 (WP_031500045), or AcrIFI 0 (WP_037415910.1 ).
  • the anti-CRISPR protein is a type l-E Acr Protein, such as AcrIEI (YP_007392738.1 ), AcrlE2 (YP_007392439.1 ), AcrlE3 (YP_950454.1 ), and AcrlE4 (NP_938238.1 ).
  • the anti-CRISPR protein is a type ll-A Acr Protein, such as AcrIIAI (WP_003722518.1 ), AcrllA2 (WP_003722517.1 ), AcrllA3 (WP_014930691 .1 ), AcrllA4 (WP_003723290.1 ), or AcrllA5.
  • the anti-CRISPR protein is a type ll-A Acr protein and the nucleic acid-guided nuclease is Cas9.
  • the anti-CRISPR protein is AcrllA4 and the nucleic acid-guided nuclease is Cas9.
  • the AcrllA4 protein is a wildtype AcrllA4 protein (e.g., Listeria monocytogenes Acrl I A4, SEQ ID NO: 1 ). In some embodiments, the AcrllA4 protein comprises an amino acid sequence having at least 85%, 90%, 95%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 1
  • the anti-CRISPR protein is a type ll-C Acr protein, such as Acrl IC1 (WP_049360089.1 ), AcrllC2 (WP_042743678.1 ), or AcrllC3 (WP_042743676.1 ).
  • Anti-CRISPR proteins that inhibit Cas12a are also known in the art (Zhang, Heng, et ai. "Structural basis for the inhibition of GR!SPR-Cas12a by anti-CRISPR proteins.” Cell host & microbe 25.6 (2019): 815-826, which is hereby Incorporated by reference). Accordingly, in some embodiments (e.g., where the nucleic-guided nuclease is Cas12a), the anti-CRISPR protein is AcrVAI (Uniprot Accession Nos. A0A5H1ZR47 or A0A5H1 ZR46, which are hereby incorporated by reference).
  • the conformation-specific NP-binding agent may be an aptamer that binds a nucleoprotein comprising a nucleic acid-guided nuclease and a guide nucleic acid.
  • the conformation-specific NP-binding agent may be an antibody, or antigen-binding fragment thereof, that binds a nucleoprotein comprising a nucleic acid-guided nuclease and a guide nucleic acid.
  • Binding or affinity between a conformation-specific NP binding agent and a nucleoprotein can be assessed using a variety of techniques known in the art, for example but not limited to, equilibrium methods (e.g., enzyme-linked immunosorbent assay (ELISA); KinExA, Rathanaswami et al. Analytical Biochemistry, Vol. 373:52-60, 2008; or radioimmunoassay (RIA)), or by a surface plasmon resonance assay or other mechanism of kinetics-based assay (e.g., BIACORE.RTM. analysis or Octet. RTM.
  • equilibrium methods e.g., enzyme-linked immunosorbent assay (ELISA); KinExA, Rathanaswami et al. Analytical Biochemistry, Vol. 373:52-60, 2008; or radioimmunoassay (RIA)
  • a surface plasmon resonance assay e.g., BIACORE.RTM. analysis or Octet. RTM.
  • forteBIO indirect binding assays
  • competitive binding assays fluorescence resonance energy transfer (FRET)
  • FRET fluorescence resonance energy transfer
  • chromatography e.g., gel filtration
  • detection methods including but not limited to chromogenic, fluorescent, luminescent, or isotopic labels.
  • the conformation-specific NP binding agent is one identified by the methods described herein (see Section III. iv) that enables a nucleoprotein with which it associates to be delivered to a target cell and released (e.g., dissociated from the conformation-specific nucleoprotein (NP) binding agent) in a target cell, e.g., in the cytoplasm or an endosome of the target cell.
  • the conformation-specific binding protein and/or the nucleic acid-guided nuclease have been engineered to promote release of the nucleoprotein in a target cell, e.g., in the cytoplasm or an endosome of the target cell.
  • Such conformation-specific NP binding agents may be useful in the methods and compositions provided herein, for example, in the Targeted Active Gene Editors (TAGE) further described in section IV for delivering a nucleoprotein to a target cell.
  • cysteines can be rationally introduced into the conformation-specific NP binding protein (e.g., AcrllA4) and the nucleic acid-guided nuclease (e.g., Cas9) interface based on the crystal structure (see, e.g., the crystal structure of AcrllA4 in Kim, et al. Scientific reports 8.1 (2016): 3883., which is hereby incorporated by reference in its entirety) such that a disulfide bridge is formed between the conformation-specific NP binding protein (e.g., AcrllA4) and a nucleoprotein comprising the nucleic acid-guided nuclease (e.g., Cas9).
  • the conformation-specific NP binding protein e.g., AcrllA4
  • the nucleic acid-guided nuclease e.g., Cas9
  • the disulfide bonds can be reduced in the cytoplasm of the cell. In certain embodiments, the reduction of the disulfide bond relieves the affinity of and promotes dissociation of the nucleoprotein and conformation-specific NP binding protein.
  • Such engineered complexes can be used throughout the methods and compositions described herein.
  • nucleoprotein comprising a nucleic acid-guided nuclease in a host cell based on a detectable interaction between the nucleoprotein and a conformation-specific NP binding agent.
  • the methods of the invention are useful in isolating or identifying expression constructs, such as those in a library of expression constructs, that provide optimal expression of a nucleic-acid- guided nuclease, guide nucleic acid, or both for nucleoprotein formation.
  • the methods provided can be used to isolate or identify one or more expression constructs, or portions thereof (e.g., promoters, ribosome binding sites, origins of replication) that enable tunable expression of both the nucleic acid-guided nuclease and the guide nucleic acid for improved expression of nucleoproteins.
  • expression constructs e.g., promoters, ribosome binding sites, origins of replication
  • nucleoprotein comprising a nucleic acid-guided nuclease in cell lysates based on a detectable interaction between the nucleoprotein and a conformation-specific NP binding agent.
  • in-process functional assays enable rapid identification and isolation of expression constructs capable of expressing active nucleoproteins comprising a nucleic acid-guided nuclease having nuclease activity in host cells without further purification steps.
  • the expression constructs isolated by the methods herein can subsequently be used in any context where optimized expression of a nucleoprotein, or component thereof, is desired.
  • the expression constructs isolated by the present methods can be used for the production and purification of a nucleic acid-guided nuclease or associated nucleoprotein, or for expressing of the nucleoprotein in a target cell for genome editing.
  • a method of isolating one or more expression constructs that expresses a nucleoprotein (e.g., a ribonucleoprotein) or a component thereof (e.g., a nucleic acid-guided nuclease, such as an RNA-guided nuclease, or a guide nucleic acid, such as a guide RNA) in a host cell based on a detectable interaction between the nucleoprotein and a conformation-specific NP binding agent.
  • the expression construct is selected from a library of test expression constructs (i.e., a plurality of different expression constructs).
  • the method may involve transferring a library of test expression constructs encoding a nucleoprotein, or component thereof, into host cells and screening or selecting for host cells that express the nucleoprotein or component thereof.
  • the host cells further comprise a polynucleotide encoding a conformation-specific NP binding agent.
  • the one or more expression vectors comprise a polynucleotide encoding a nucleic acid-guided nuclease operably linked to a detectable label or a guide nucleic acid operably linked to a polynucleotide encoding a detectable label.
  • polynucleotide encoding the conformation-specific NP binding agent is operably linked to a polynucleotide encoding a detectable label.
  • the nucleoprotein (e.g., the nucleic acid-guided nuclease or the guide nucleic acid) comprises a first detectable label and the conformation-specific NP binding agent comprises a second detectable label, wherein the first detectable label and second detectable label generate a signal or detectable product upon interaction.
  • the method involves selecting or screening for host cells with a level of nucleic acid-guided nuclease expression or nucleoprotein expression above a reference level (e.g., above a pre-determined level, above a level of expression from a control construct, or above a level of expression in a control cell or population of cells), indicating that the expression constructs contained therein provide a threshold level of expression.
  • a reference level e.g., above a pre-determined level, above a level of expression from a control construct, or above a level of expression in a control cell or population of cells
  • the host cells may be screened for nuclease activity by a nucleoprotein comprising a nucleic acid-guided nuclease in cells or cell lysates, where nuclease activity above a threshold level indicates that the cell expression constructs within the host cell express an active nucleoprotein.
  • Expression constructs can subsequently be isolated from host cells that express the nucleoprotein or the component thereof.
  • the one or more expression constructs isolated herein are an inducible co-expression system that co-expresses multiple components of a nucleoprotein, such as a guide nucleic acid (e.g., gRNA) and a nucleic acid-guided nuclease (e.g., a RNA-guided nuclease), which then assemble to form nucleoproteins in the host cell.
  • a nucleoprotein such as a guide nucleic acid (e.g., gRNA) and a nucleic acid-guided nuclease (e.g., a RNA-guided nuclease), which then assemble to form nucleoproteins in the host cell.
  • the inducible co-expression system can further include a conformation-specific NP binding agent.
  • the conformation-specific NP binding agent is an anti-CRISPR (Acr) protein, such as one further described herein.
  • the Acr protein can be AcrllA4.
  • the conformation-specific NP binding agent may be an antibody, or antigen binding fragment thereof, that specifically binds the NP.
  • the conformation-specific NP binding agent may be an aptamer that specifically binds the NP.
  • the conformation-specific NP binding agent can comprise one or more modifications (e.g., an amino acid substitution, deletion, or insertion) relative to a naturally occurring conformation-specific NP binding agent (e.g., Acr protein, such as AcrllA4) or one further described herein.
  • the conformation-specific NP binding agent has at least 50, 60, 70, 75, 80, 85, 90, 95, 98%, or 99% sequence identity to a conformation-specific NP binding agent described herein.
  • Methods for isolating or identifying one or more expression constructs that express a nucleoprotein comprising a nucleic acid-guided nuclease at or above a desired level in a host cell are further outlined below.
  • the methods herein involve providing one or more test expression constructs comprising a polynucleotide encoding a nucleic acid-guided nuclease (e.g., an RNA- guided nuclease), a polynucleotide encoding a guide nucleic acid (gNA, e.g., a guide RNA), or both.
  • the method involves providing a polynucleotide encoding a conformation-specific NP binding agent.
  • the polynucleotide encoding the nucleic acid-guided nuclease and the polynucleotide encoding the gNA may be on a single test expression construct.
  • the polynucleotide encoding the nucleic acid-guided nuclease and the polynucleotide encoding the gNA may be on separate test expression constructs.
  • a single test expression construct encodes the nucleic acid-guided nuclease and a gNA is provided separately (e.g., prior to, concomitant with, or subsequent to expression of the nuclease) to associate with the expressed nucleic acid-guided nuclease in the host cell.
  • the polynucleotide encoding the conformation-specific NP binding agent may be on the same or a different construct than the nucleic acid-guided nuclease or the guide nucleic acid.
  • a variety of regulatory elements can be used to control expression levels of the nucleic acid-guided nuclease, the gNA, or the conformation-specific NP binding agent. Examples of expression constructs and regulatory elements therein are further described in Section VI. Examples of nucleic acid-guided nucleases and gNAs, are described in further detail in Section V.
  • the step of providing the test expression construct can include possession of the expression construct, such as an expression construct in a container, in a solution (e.g., a plasmid preparation), or in a cell carrying the expression construct.
  • the expression construct can be one isolated from a single clone or can be in the form of a library of expression constructs (e.g., isolated from multiple clones).
  • Expression constructs can be assembled from DNA encoding components of interest (e.g., a nucleic acid-guided nuclease, a gNA, a conformation-specific NP binding agent or a regulatory element), such as construct components described in Section VI.
  • the DNA can be obtained from any source, such as through amplification of sequences of interest from genomic DNA or through synthesis.
  • DNA encoding a component of interest can be amplified and cloned using a known technique, such as PCR using appropriately-selected primers, in order to produce sufficient quantities of the DNA and to modify the DNA in such a manner (e.g., by addition of appropriate restriction sites) that it can be introduced as an insert into an expression construct (such as those described in Section VI).
  • Amplified and cloned DNA can be further diversified, using mutagenesis, such as PCR, in order to produce a greater diversity or wider repertoire of expression constructs.
  • the method involves producing or providing a plurality of different test expression constructs in the form of a library of expression constructs.
  • library refers to a mixture of heterogeneous polypeptides or nucleic acids.
  • the library is composed of individual members and sequence variations between library members, such as sequence variations between different regulatory elements and/or nuclease variants, are responsible for the diversity present in the library.
  • multiple members e.g., two or more members
  • the library can encode the same sequence variants or a member can be represented more than once.
  • the library may take the form of a simple mixture of polypeptides or nucleic acids, or may be in the form organisms or cells, for example bacteria, viruses, animal or plant cells and the like, transformed with a library of nucleic acids, such as expression constructs.
  • each individual organism or cell contains only one member of the library.
  • a library of expression constructs having a plurality of different expression or regulatory elements (e.g., promoter, ribosome binding site, origins or replication, or a combination thereof) but otherwise share the same backbone and encode the same nucleic acid-guided nuclease or gNA.
  • the methods may be able to isolate an expression construct from the library that enables higher expression levels of the nucleoprotein or component thereof relative to a reference level.
  • a library of expression constructs is provided where the library of expression constructs encodes different nucleic acid- guided nuclease variants or gNA variants.
  • some nucleic acid-guided nuclease variants or gNA variants may form unstable or inactive nucleoproteins.
  • the methods provide herein enable isolation of constructs encoding stable or active nucleoproteins.
  • the method further involves preparing the library of expression constructs by cloning nucleic acids encoding a plurality of different test expression elements, such as a test promoter or a test ribosome binding site, into a construct to prepare a library of constructs, wherein each construct in the library encodes one test expression element.
  • the test expression element is inserted into the construct so as to be operatively linked to a polynucleotide encoding a nucleic acid-guided nuclease or a gNA.
  • a single expression construct includes two or more test expression elements, such as a test promoter and a test ribosome binding site.
  • the method involves cloning nucleic acids encoding a plurality of different test nucleic acid-guided nucleases (e.g., variants of nucleic acid-guided nucleases that have been codon-optimized or variants comprising different cell targeting agents) into the same construct backbone to prepare a library of constructs, wherein each construct in the library encodes one test nucleic acid-guided nuclease.
  • the method involves preparing the library of expression constructs by cloning nucleic acids encoding a plurality of different test gNAs into a construct to prepare a library of constructs, wherein each construct in the library encodes one test guide nucleic acid.
  • One or more expression constructs can be introduced into host cells for expression of components encoded on the constructs (e.g., a nucleic acid-guided nuclease, a gNA, and/or a conformation-specific NP binding agent). Transfer of the construct into host cells (e.g., by infection, transformation, or transfection) can be carried out using known techniques. In cases where the method requires two constructs, both libraries can be introduced into appropriate host cells either simultaneously or sequentially. Methods of introducing polynucleotides (e.g., an expression constructs) into host cells are typically selected based on the kind of host cell.
  • Such methods include, for example, heat shock, viral or bacteriophage infection, transfection, conjugation, electroporation, calcium phosphate precipitation, polyethyleneimine-mediated transfection, DEAE-dextran mediated transfection, protoplast fusion, lipofection, liposome-mediated transfection, particle gun technology, direct microinjection, and nanoparticle-mediated delivery.
  • the two expression constructs can either be transferred simultaneously (e.g., co-transformed) or in series.
  • the method involves transferring one or more test expression constructs into a host cell suitable to express the nucleic acid-guided nuclease, the gNA, or both the nucleic acid-guided nuclease and the gNA.
  • the method further involves transferring one or more test expressions into a host cell suitable to express a conformation- specific NP binding agent.
  • the expression construct is in a plurality of expression constructs and the plurality of expression constructs is transferred into host cells under conditions such that the average expression construct per host cell is 1 .
  • the expression construct is in a plurality of expression constructs and the plurality of expression constructs are transferred into host cells under conditions such that the average construct per host cell is less than 1 .
  • each expression construct are transferred into host cells under conditions such that the average expression construct per host cell is about 2.
  • the nucleic acid-guided nuclease and/or the gNA can be expressed from the expression construct in the host cell, such that nucleoproteins (NP: e.g., DNPs or RNPs) are formed, wherein the nucleoprotein comprises the nucleic acid-guided nuclease and the gNA encoded on the construct.
  • NP nucleoproteins
  • the method involves further expressing the conformation-specific NP binding agent from an expression construct in the host cell comprising NPs such that the conformation-specific NP binding agent can stably associate with the NPs. Compartmentalized Nucleoprotein Expression
  • nucleoprotein comprising a nucleic acid-guided nuclease and a guide nucleic acid in a host cell based on a detectable interaction between the nucleoprotein and a conformation-specific NP binding agent.
  • methods for isolating one or more expression constructs that enable expression of a nucleoprotein (e.g., a ribonucleoprotein), or a component thereof (e.g., a nucleic acid-guided nuclease such as an RNA-guided nuclease or a guide nucleic acid such as a guide RNA) in a host cell.
  • a nucleoprotein e.g., a ribonucleoprotein
  • a component thereof e.g., a nucleic acid-guided nuclease such as an RNA-guided nuclease or a guide nucleic acid such as a guide RNA
  • host cell refers a cell that can express proteins, protein fragments, or peptides of interest from an expression construct.
  • the term encompasses not only the particular subject cell but also the progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not be identical to the parent cell, but are still included within the scope of the term “host cell.”
  • the host cell may be a prokaryotic cell or eukaryotic cell, such as a bacterial cell (e.g., E. coli), an animal cell, a plant cell, an insect cell, or a fungal cell (e.g., a S. cerevisiae cell, Pichia pastoris, or the like).
  • a prokaryotic cell such as E. coli
  • E. coli E. coli
  • an animal cell e.g., E. coli
  • a plant cell e.g., an insect cell
  • a fungal cell e.g., a S. cerevisiae cell, Pichia pastoris, or the like.
  • the host cells are prokaryotic host cells.
  • Prokaryotic host cells can include archaea (such as Haloferax volcanii, Sulfolobus solfataricus), gram-positive bacteria (such as Bacillus subtilis, Bacillus licheniformis, Brevibacillus choshinensis, Lactobacillus brevis, Lactobacillus buchneri, Lactococcus lactis, and Streptomyces lividans), or gram-negative bacteria, including Alphaproteobacteria ( Agrobacterium tumefaciens, Caulobacter crescentus, Rhodobacter sphaeroides, and Sinorhizobium meliloti), Betaproteobacteria ( Alcaligenes eutrophus), and Gammaproteobacteria (Acinetobacter calcoaceticus, Azotobacter vinelandii, Escherichia coli, Pseudomonas
  • the host cells include Gammaproteobacteria of the family Enterobacteriaceae, such as Enterobacter, Erwinia, Escherichia (including E. coli), Klebsiella, Proteus, Salmonella (including Salmonella typhimurium), Serratia (including Serratia marcescans), and Shigella.
  • the bacteria can be gram-positive bacteria or gram-negative bacteria.
  • the host cells are E. coli.
  • the host cells are eukaryotic host cells, such as yeast ( Candida shehatae, Kluyveromyces lactis, Kluyveromyces fragilis, other Kluyveromyces species, Pichia pastoris, Saccharomyces cerevisiae, Saccharomyces pastorianus also known as Saccharomyces carlsbergensis, Schizosaccharomyces pombe, Dekkera/Brettanomyces species, and Yarrowia lipolytica ); other fungi ( Aspergillus nidulans, Aspergillus niger, Neurospora crassa, Penicillium, Tolypocladium, Trichoderma reesia) insect cell lines ( Drosophila melanogaster Schneider 2 cells and Spodoptera frugiperda Sf9 cells); and mammalian cell lines including immortalized cell lines (Chinese hamster ovary (CHO) cells, HeLa cells, baby hamster kidney (
  • the host cell is a mammalian cultured cell derived from rodents (rats, mice, guinea pigs, or hamsters) such as CHO, BHK, NSO, SP2/0, YB2/0; or human tissues or hybridoma cells, yeast cells, or insect cells.
  • rodents rats, mice, guinea pigs, or hamsters
  • rodents such as CHO, BHK, NSO, SP2/0, YB2/0
  • human tissues or hybridoma cells yeast cells, or insect cells.
  • the mammalian cell is a COP cell, an L cell, a C127 cell, an Sp2/0 cell, an NS-0 cell, an NIH3T3 cell, a PC12 cell, a PC12h cell, a BHK cell, a CHO cell, a COS1 cell, a COS3 cell, a COST cell, a CV1 cell, a Vero cell, a HeLa cell, an HEK-293 cell, a PER C6 cell, a cell derived from diploid fibroblasts, a myeloma cell, or HepG2.
  • the method may involve transferring the one or more expression constructs to a cell-free system or non-cellular compartment (e.g., an emulsion droplet) suitable to express the nucleic acid-guided nuclease, the gNA, and the conformation-specific NP binding agent, and expressing the nucleic acid-guided nuclease, the gNA, and the conformation-specific NP binding agent in the non-cellular compartment (e.g., the emulsion droplet), such that nucleoproteins (NPs) each comprising the nucleic acid-guided nuclease and the gNA are formed along with the conformation-specific NP binding agent.
  • a cell-free system or non-cellular compartment e.g., an emulsion droplet
  • NPs nucleoproteins
  • the cell-free system is one based on wheat germ extracts or on bacterial cell extracts, such as a continuous-exchange cell-free (CECF) protein synthesis system using E. coli extracts and an incubation apparatus such as the RTS ProteoMaster (Roche Diagnostics GmbH; Mannheim, Germany) (Jun et al., “Continuous-exchange cell-free protein synthesis using PCR-generated DNA and an RNase E-deficient extract”, Biotechniques 2008 March; 44(3): 387-391).
  • CECF continuous-exchange cell-free
  • the non-cellular compartment is a droplet, such as a droplet in an emulsion and/or a microfluidic droplet.
  • the droplet may contain an acellular system, such as a cell-free extract.
  • the droplet may contain a single cell.
  • An emulsion, cell-free extract, or a single cell can be contained in a well or a plurality of wells, such as a multi-well plate (e.g., 6, 12, 24, 48, 96, or 384-well microplate).
  • Emulsification can be used in the methods of the disclosure to separate or segregate a sample or set of samples into a series of compartments, for example a compartment having a single cell or a discrete portion of an acellular sample, such as a cell-free extract or a cell-free transcription and/or cell-free translation mixture.
  • an emulsion will include a plurality of droplets, each droplet including an expression construct.
  • An emulsion can include various compounds, enzymes, or reagents in addition to the expression construct (e.g., compounds, enzymes, or reagents in addition to the expression construct to achieve cell-free transcription or translation). These additives may be included in the emulsion solution prior to emulsification. Alternatively, the additives may be added to individual droplets after emulsification.
  • Droplets in an emulsion can be sorted and/or isolated according to methods well known in the art. For example, double emulsion droplets containing a fluorescence signal can be analyzed and/or sorted using conventional fluorescence-activated cell sorting (FACS) machines at rates of >10 4 droplets s 1 , and have been used to improve the activity of enzymes produced by single cells or by in vitro translation of single genes (Aharoni et al., Chem Biol 12(12): 1281-1289, 2005; Mastrobattista et al., Chem Biol 2(12): 1291- 1300, 2005).
  • FACS fluorescence-activated cell sorting
  • Emulsion may be achieved by a variety of methods known in the art (see, for example, US 2006/0078888 Al, of which paragraphs [0139]-[0143] are incorporated by reference herein).
  • An exemplary emulsion is a water-in-oil emulsion.
  • the continuous phase of the emulsion includes a fluorinated oil.
  • An emulsion can contain a surfactant or emulsifier (for example, a detergent, anionic surfactant, cationic surfactant, or amphoteric surfactant) to stabilize the emulsion.
  • a surfactant or emulsifier for example, a detergent, anionic surfactant, cationic surfactant, or amphoteric surfactant
  • Other oil/surfactant mixtures for example, silicone oils, may also be utilized in particular embodiments.
  • An emulsion can be a monodisperse emulsion or a polydisperse emulsion.
  • the expression construct encodes a nucleoprotein (e.g., a nucleic acid- guided nuclease and a guide nucleic acid) or a conformation-specific NP binding agent comprising a detectable label
  • a population of host cells containing the expression constructs can be screened for nucleoprotein levels based on the level of the signal generated by the detectable label.
  • one or more expression constructs are provided that encode a nucleoprotein (e.g., a nucleic acid-guided nuclease and a guide nucleic acid) comprising a first detectable label and a conformation-specific NP binding agent comprising a second detectable label capable of generating a signal upon interaction with the first detectable label.
  • a population of host cells comprising the one or more expression constructs can then be screened for nucleoprotein levels based on the level of the signal generated by the interaction between the first and second detectable labels.
  • a detectable label is a molecule that can be visualized or otherwise observed, measured, or detected.
  • the detectable label may be encoded by a polynucleotide that is operably linked to the polynucleotide encoding the nucleic acid-guided nuclease.
  • the expression construct will encode a nucleic acid-guided nuclease fusion protein.
  • Detectable labels include any detectable protein domain, including but not limited to, a fluorescent protein (or a split version thereof, e.g., split-GFP), or a protein domain that can be detected with a specific antibody.
  • Non-limiting examples of fluorescent proteins include green fluorescent proteins (e.g., GFP, sfGFP, EGFP, ZsGreenl ), yellow fluorescent proteins (e.g., YFP, EYFP, ZsYellowl ), or red fluorescent protein (e.g., RFP).
  • fluorescent proteins include green fluorescent proteins (e.g., GFP, sfGFP, EGFP, ZsGreenl ), yellow fluorescent proteins (e.g., YFP, EYFP, ZsYellowl ), or red fluorescent protein (e.g., RFP).
  • Non-limiting examples of small molecule detectable labels include radioactive labels, such as 3 FI and 35 S.
  • the first detectable label and second detectable label are fragments of a split fluorescent protein (e.g., split-GFP) or split luciferase protein
  • the method comprises measuring the level of NPs based on the level of fluorescence or luminescence generated by the activity of the complemented fragments under appropriate illumination conditions in the case of spit fluorescent proteins or in the presence of appropriate substrates known in the art in the case of split luciferase proteins.
  • detectable labels and methods of preparing fusion proteins and split derivatives comprising detectable labels are known in the art, e.g., see Thorn, K. (2017). Genetically encoded fluorescent tags. Molecular biology of the cell, 28(7), 848-857, S. Cabantous, T.C. Terwilliger, G.S. Waldo (2005) Protein tagging and detection with engineered self-assembling fragments of green fluorescent protein. Nature Biotechnology 23(1 ), 102-107, and D. Kamiyama, et al. (2016) Versatile protein tagging in cells with split fluorescent protein. Nature Communinations 7, 11046; and Pedelacq et al.
  • the nucleoprotein expression levels can be measured by use of a conformation-specific NP binding protein comprising an enzyme (e.g., by way of conjugation) that enables introduction of a detectable label to the nucleoprotein, e.g., by a proximity labeling approach.
  • the conformation-specific NP binding protein includes a promiscuous biotin ligase (e.g., BiolD, see, e.g., Roux et al.
  • the promiscuous biotin ligase on the conformation-specific NP binding protein labels a nucleic acid-guided nuclease of the nucleoprotein with biotin at one or more sites, wherein the nucleoprotein comprises a first fluorophore positioned elsewhere on the nucleoprotein, such as on the guide nucleic acid or on a second site on the nucleic acid-guided nuclease.
  • streptavidin carrying a fluorophore that acts as a FRET donor/acceptor or quencher for the first fluorophore is contacted with the nucleoprotein such that the levels of nucleoprotein can be measured by FRET.
  • Other enzymes known in the art capable of generating a detectable label can be used in the methods herein, such as engineered ascorbate peroxidase (APEX; see, e.g., Rhee et al. Science 339.6125 (2013): 1328-1331).
  • host cells comprising an expression construct e.g., a population of host cells comprising a library of expression constructs
  • a nucleoprotein or a conformation-specific NP binding agent comprising a second detectable label can be sorted by fluorescence activated cell sorting (FACS).
  • FACS fluorescence activated cell sorting
  • one or more expression constructs are provided that encode a nucleoprotein (e.g., a nucleic acid-guided nuclease and a guide nucleic acid) comprising a first detectable label and a conformation-specific NP binding agent comprising a second detectable label capable of generating a signal upon interaction with the first detectable label (e.g., by FRET or complementation of split fluorescent protein fragments), and the cells are sorted based on the level of the signal generated by the interaction between the first and second detectable labels.
  • the FACS sorting not only measures fluorescence signals in cells at a rapid rate, but also collects cells that have specified fluorescence properties. This enables enrichment of the initial library population for expression constructs that have desired characteristics.
  • some expression constructs may not produce fluorescent proteins inside cells, indicating that the encoded nucleic acid-guided nuclease express at low levels, is folded incorrectly, or is digested rapidly by proteases within the cell.
  • Flost cells having expression constructs that result in non-fluorescent protein may be easily eliminated from the cell population by only collecting cells on the cell sorter which express levels of fluorescence above a predetermined threshold.
  • Such a selection procedure improves the quality of the expression constructs isolated by removing constructs that do not produce functional proteins or express proteins at low level.
  • the selection experiments can be performed in a variety of host cells such as yeast, bacteria, plant, insect, or mammalian cells depending on the requirements of the experiment and the capabilities of the expression vectors being used.
  • nucleic acid-guided nuclease and/or guide nucleic acid e.g., above a threshold expression level.
  • the library of expression constructs may generate a wide range of expression levels in cells due to different stabilities, folding tendencies, etc. This can be visualized on the flow sorter as a broadening of the distribution of fluorescence intensities.
  • cells may be collected on the flow sorter that fall near the extreme right ("bright") end of the fluorescence intensity distribution. This process can be repeated in order to further skew the host cell population toward those that contain expression constructs that lead to the highest levels of expression.
  • Host cells with the highest level of expression can be collected and the expression constructs located in the collected host cells can be isolated for identification and for further use, e.g., to produce and isolate the encoded nucleoprotein or component thereof.
  • a spectrophotometer In cases, where individual members of a host cell library are assayed, such as in an array, other methods can be used to detect fluorescence as a measurement of expression. For example, a spectrophotometer, a microtitre plate reader, a CCD, a fluorescence microscope, or other similar device may be used to detect fluorescence in an array of cells.
  • Host cells can be lysed using any methods known in the art to generate a cell lysate (see, e.g., Shehadul Islam et al ., (2017). A review on macroscale and microscale cell lysis methods. Micromachines, 8(3), 83, which is hereby incorporated by reference). The methods and reagents necessary for lysing the host cells can be determined by one skilled in the art based on the type of host cell.
  • Cells can be lysed by one or more methods of physical disrupting the cells (e.g., mechanical disruption, liquid homogenization, sonication, freeze-thaw, or manual grinding) or by incubating the cells with a cell lysis reagent.
  • the method can include, for example, lysing the cells with a sonicator, a French press, a Dounce homogenizer, a Potter-Elvehjem homogenizer, exposing the cells to freeze-thaw cycles, or by heating the cells.
  • Methods of physical disruption can also involve the use of additives, such as hypotonic buffers, or facilitators, such as glass beads, to promote lysis.
  • the cell lysis reagent can include one or more of an enzyme (e.g., lysozyme for bacterial cells), detergents, a hypotonic solution (i.e., for osmotic lysis), chelating agents, or commercially available buffers.
  • an enzyme e.g., lysozyme for bacterial cells
  • detergents e.g., a hypotonic solution
  • chelating agents i.e., for osmotic lysis
  • reagents such as enzymes that help prevent proteolysis (e.g., a protease inhibitor) or stabilize proteins can be added to the cell lysates.
  • nucleoprotein activity or levels can be assessed in the cell lysates.
  • Nucleoprotein levels in host cell lysates can be measured in accordance with the methods (e.g., FRET) further described herein.
  • NP nucleoprotein
  • a solution e.g., a cell lysate
  • the methods provided herein may be used to measure any nucleoprotein (e.g., a ribonucleoprotein (RNP) or a deoxyribonucleoprotein (DNP)) recognized by a conformation-specific nucleoprotein (NP) binding agent.
  • RNP ribonucleoprotein
  • DNP deoxyribonucleoprotein
  • the nucleoprotein is a ribonucleoprotein
  • the nucleic acid-guided nuclease is an RNA-guided nuclease, such as Cas9
  • the guide nucleic acid is a guide RNA (gRNA).
  • the nucleic acid-guided nuclease of the nucleoprotein is a fusion protein, such as one comprising a cell targeting agent (e.g., a peptide, ligand, antigen-binding agent). Examples of nucleic acid-guided nucleases are further described in Section V.
  • the method involves measuring the level of NPs (e.g., in a solution) by combining (i) a conformation-specific NP binding agent comprising a first detectable label; (ii) an NP comprising a nucleic acid-guided nuclease and a guide nucleic acid (gNA), and (iii) a labelling moiety capable of specifically binding the nucleic acid-guided nuclease or a conjugation moiety attached thereto, wherein the labelling moiety comprises a second detectable label capable of generating a signal upon interaction with the first detectable label.
  • a conformation-specific NP binding agent comprising a first detectable label
  • an NP comprising a nucleic acid-guided nuclease and a guide nucleic acid (gNA)
  • gNA guide nucleic acid
  • the method involves measuring the level of the signal generated by interaction between the first and second detectable labels, wherein the signal level corresponds to the level of NPs.
  • Any pairs of detectable labels known in the art that generate a measurable signal or product upon interaction can be used in the present methods, including those further described herein.
  • the labelling moiety can be any compound comprising a second detectable label that is capable of directly binding the NP or binding a conjugation moiety on the NP.
  • the labelling moiety comprises a second detectable label conjugated (e.g., conjugated by way of a linker or conjugation moiety) to an antibody capable of specifically binding the nucleic acid-guided nuclease.
  • the labelling moiety may comprise a second detectable label conjugated to a conjugation moiety that in turn binds a conjugation moiety on the NP.
  • the nucleic acid-guided nuclease may comprise a first conjugation moiety and the labelling moiety may comprise the second detectable label conjugated to a second conjugation moiety capable of binding the first conjugation moiety, such that the detectable label is stably associated with the NP.
  • the first conjugation moiety is a first protein and the second conjugation moiety is a second protein capable of binding the first protein.
  • conjugation moieties with complementary binding agents include, but are not limited to, CBP, MBP, GST, poly(His), biotin, streptavidin, V5-tag, Myc-tag, HA-tag, NE-tag, His-tag, Flag tag, Halo-tag, Snap-tag, Fc-tag, Nus-tag, BCCP, Thioredoxin, SnooprTag, SpyTag, SpyCatcher, Isopeptag, SBP-tag, S- tag, AviTag, Protein A, or Calmodulin.
  • the method involves measuring the level of NPs (e.g., in a solution) by combining (i) a conformation-specific NP binding agent comprising a first detectable label, and (ii) a NP comprising a nucleic acid-guided nuclease and a guide nucleic acid (gNA), wherein the nucleic acid -guided nuclease or the gNA comprises a second detectable label capable of generating a signal upon interaction with the first detectable label, and measuring the level of the signal generated by interaction between the first and second detectable labels, wherein the signal level corresponds to the level of NPs.
  • the second detectable label may be associated with the gNA.
  • the second detectable label may be associated with the nucleic acid-guided nuclease, such as by a linker (e.g., a chemical or peptide linker).
  • the nucleic acid-guided nuclease fusion comprising a detectable label, such as one encoded by an expression vector comprising a polynucleotide that encodes the nucleic acid-guided nuclease operably linked to a polynucleotide encoding the detectable label.
  • the method involves measuring the level of the signal generated by interaction between the first and second detectable labels, wherein the signal level corresponds to the level of NPs.
  • the method of detection will depend on the detectable labels selected.
  • the detectable label e.g., the first and/or second detectable label
  • the first detectable label and second detectable label are a FRET pair
  • the method comprises measuring the level of NPs based on the level of fluorescence generated by interaction of the FRET pair as measured by FRET.
  • the first detectable label and second detectable label are fragments of a split fluorescent protein (e.g., split-GFP) or split luciferase protein
  • the method comprises measuring the level of NPs based on the level of fluorescence or luminescence generated by the activity of the complemented fragments under appropriate illumination conditions in the case of spit fluorescent proteins or in the presence of appropriate substrates known in the art in the case of split luciferase proteins.
  • detectable labels and methods of preparing fusion proteins and split derivatives comprising detectable labels are known in the art, e.g., see Thorn, K. (2017). Genetically encoded fluorescent tags. Molecular biology of the cell, 28(7), 848-857, S. Cabantous, T.C. Terwilliger, G.S. Waldo (2005) Protein tagging and detection with engineered self-assembling fragments of green fluorescent protein. Nature Biotechnology 23(1 ), 102-107, and D. Kamiyama, et al. (2016) Versatile protein tagging in cells with split fluorescent protein. Nature Communinations 7, 11046; and Pedelacq et al. "Development and Applications of Superfolder and Split Fluorescent Protein Detection Systems in Biology.” International journal of molecular sciences 20.14 (2019): 3479. which are hereby incorporated by reference in their entirety.
  • the nucleoprotein levels can be measured by use of a conformation- specific NP binding protein comprising an enzyme (e.g., by way of conjugation) that enables introduction of a detectable label to the nucleoprotein, e.g., by a proximity labeling approach.
  • the conformation-specific NP binding protein includes a promiscuous biotin ligase (e.g., BiolD, see, e.g., Roux et al. J Cell Biol 196.6 (2012): 801 -810, which is hereby incorporated by reference).
  • the promiscuous biotin ligase on the conformation-specific NP binding protein labels a nucleic acid-guided nuclease of the nucleoprotein with biotin at one or more sites, wherein the nucleoprotein comprises a first fluorophore positioned elsewhere on the nucleoprotein, such as on the guide nucleic acid or on a second site on the nucleic acid-guided nuclease. Then, streptavidin carrying a fluorophore that acts as a FRET donor/acceptor or quencher for the first fluorophore is contacted with the nucleoprotein such that the levels of nucleoprotein can be measured by FRET.
  • APEX engineered ascorbate peroxidase
  • detectable labels such as those found in commercially available kits can be used in the methods provided herein.
  • the detectable labels include AlphaScreen reagents (see, e.g., Yasgar et al. "AlphaScreen-based assays: ultra-high- throughput screening for small-molecule inhibitors of challenging enzymes and protein-protein interactions.” High Throughput Screening. Humana Press, New York, NY, 2016. 77-98).
  • AlphaScreen is a bead-based, non-radioactive Amplified Luminescent Proximity Homogeneous Assay.
  • the nucleic acid-guided nuclease and the nucleic acid can include epitope tags or conjugation moieties, offering means of coupling to donor and acceptor beads/labels.
  • the detectable label is an oligonucleotide.
  • the first detectable label may comprise a first oligonucleotide and the second detectable label may comprise a second oligonucleotide capable of ligating to the first oligonucleotide to form a ligated polynucleotide when the first and second detectable labels are in close proximity.
  • the first detectable label comprises a first oligonucleotide and the second detectable label comprises a second oligonucleotide
  • the method further comprises introducing a third oligonucleotide capable of hybridizing to both the first oligonucleotide and second oligonucleotide to form a ligated polynucleotide when the first and second oligonucleotides are in close proximity.
  • the measuring step comprises measuring the level of NPs based on the level of ligated polynucleotides. In some embodiments, the measuring step comprises amplifying the ligated polynucleotide by PCR amplification. In some embodiments, PCR amplification is rolling- circle amplification. In some embodiments, the measuring step further comprises introducing a detectable probe that binds the ligated polynucleotide and is capable of generating a detectable signal, and measuring the level of the signal generated by the bound detectable probe, wherein the signal level corresponds to the level of NPs.
  • the level of the ligated polynucleotide is measured by one or more methods selected from gel electrophoresis, PCR amplification, or sequencing, as further described herein.
  • the ligated polynucleotide can be detected using commercially available assays, such as the DUOLINK® Proximity Ligation Assay (Sigma Aldrich).
  • NPs nucleoproteins
  • a method of measuring the level of nucleoproteins by combining onto a surface comprising a surface- or bead-immobilized agent capable of specifically binding a nucleoprotein or a component thereof (e.g., a nucleic acid-guided nuclease) (i) a conformation-specific NP binding agent comprising a detectable label capable of generating a signal; (ii) an NP comprising a nucleic acid-guided nuclease and a guide nucleic acid (gNA), such that the NP binds to the surface-immobilized agent and the conformation-specific NP binding agent binds to the nucleoprotein.
  • gNA guide nucleic acid
  • the surface may be the surface of a plate or a surface of a bead.
  • the surface-immobilized agent capable of binding the nucleoprotein, or component thereof e.g., nucleic acid-guided nuclease
  • the surface-immobilized agent capable of binding the nucleoprotein, or component thereof is an antibody, or antigen binding fragment thereof that specifically binds the nucleic acid-guided nuclease.
  • the nucleoprotein or component thereof e.g., nucleic acid-guided nuclease
  • the surface-immobilized agent capable of binding the nucleoprotein comprises a second conjugation moiety capable of binding the first binding moiety.
  • the first and second conjugation moieties can be selected from any complementary conjugation moieties known in the art (e.g., streptavidin and biotin, Strep-Tactin and Strep-tag, or others pairs described herein).
  • binding moiety pairings include (i) streptavidin-binding peptide (streptavidin binding peptide; SBP) and streptavidin (STV), (ii) biotin and EMA (enhanced monomeric avidin), (iii) SpyTag (ST) and SpyCatcher (SC), (iv) Flalo-tag and Flalo-tag ligand, (v) SNAP-Tag and an anti-SNAP antibody, (vi) Myc tag and anti-Myc antibody (vii) FLAG tag and anti-FLAG antibodies, and (ix) ybbFt tag and coenzyme A groups.
  • the method further comprises washing the surface (e.g., with a solution or buffer) to remove unbound materials.
  • the method comprises measuring the level of the signal generated by the detectable label on the surface, wherein the signal level corresponds to the level of NPs.
  • the method of detection will depend on the detectable labels selected.
  • the detectable label comprises a fluorophore, such as a fluorescent protein (e.g., GFP).
  • the detectable label comprises an enzyme, that converts a substrate into a colored or chemiluminescent product (e.g., alkaline phosphatase or horseradish peroxidase).
  • the method may alternatively use other commercially available labelling reagents, such as AlphaScreen reagents, as described in, e.g., Yasgar et al. "AlphaScreen-based assays: ultra-high-throughput screening for small-molecule inhibitors of challenging enzymes and protein-protein interactions.” High Throughput Screening. Humana Press, New York, NY, 2016. 77-98; and “A Practical Guide to working with AlphaScreen”, Perkin Elmer Life and Analytical Sciences, 2004, which are hereby incorporated by reference.
  • AlphaScreen reagents as described in, e.g., Yasgar et al. "AlphaScreen-based assays: ultra-high-throughput screening for small-molecule inhibitors of challenging enzymes and protein-protein interactions.” High Throughput Screening. Humana Press, New York, NY, 2016. 77-98; and “A Practical Guide to working with AlphaScreen”, Perkin Elmer Life and Analytical Sciences, 2004, which are hereby incorporated by reference.
  • a method of measuring the levels of an unlabeled nucleoprotein by combining into a solution (i) a conformation-specific NP binding agent comprising a first detectable label; and (ii) a labelled NP comprising a nucleic acid-guided nuclease and a guide nucleic acid (gNA), wherein the nucleic acid-guided nuclease, or a conjugation moiety attached thereto, comprises a second detectable label that is quenched by or quenches the first detectable label, such that complexes comprising the conformation-specific NP binding agent and the labelled NP are formed.
  • gNA guide nucleic acid
  • the first detectable label and the second detectable label can be a quenching pair detectable by FRET.
  • the first detectable label is a fluorophore and the second detectable label is a quencher.
  • the first detectable label can be a quencher and the second detectable label can be a fluorophore.
  • an unlabeled NP capable of associating with the conformation-specific NP binding agent is added to the solution.
  • the level of the signal generated by dissociation of the labelled NP from the complexes is then measured, wherein the degree of dissociation of the labelled NP and the resulting signal level corresponds to the level of unlabeled NPs in the solution.
  • the signal is measured by FRET.
  • the detectable label can be attached to the conformation-specific NP binding agent by way of complementary conjugation moieties, wherein the conformation-specific NP binding agent comprises a first conjugation moiety and the detectable label comprises a second conjugation moiety that binds to the first conjugation moiety, thereby stably associating the detectable label with the conformation-specific NP binding agent.
  • the methods provided herein can be used to measure NPs, or components thereof, as expressed in a host cell lysate. Accordingly, in some embodiments, the method comprises providing a lysate from a host cell comprising the NPs. In alternative embodiments, the method comprises providing a lysate from a host cell comprising the nucleic acid- guided nucleases and adding a gNA to the lysate to form the NPs. Further, a host cell from which a lysate is prepared (e.g., either the same or different host cell expressing the NP or component thereof) may express the conformation-specific NP binding agent.
  • the conformation- specific NP binding agent may be added to a composition comprising the host cell lysate.
  • the host cell is a bacterial cell.
  • the host cell may be a eukaryotic cell, such as a mammalian cell or a fungal cell.
  • the fungal cell may be a yeast cell (e.g., Pichia pastoris or Saccharomyces cerevisiae).
  • the mammalian cell may be a COP cell, an L cell, a C127 cell, an Sp2/0 cell, an NS-0 cell, an NIH3T3 cell, a PC12 cell, a PC12h cell, a BHK cell, a CFIO cell, a COS1 cell, a COS3 cell, a COST cell, a CV1 cell, a Vero cell, a FleLa cell, an FIEK-293 cell, a PER C6 cell, a cell derived from diploid fibroblasts, a myeloma cell, or FlepG2.
  • Flost cells can be lysed using any methods known in the art to generate a cell lysate (see, e.g., Shehadul Islam et al ., (2017). A review on macroscale and microscale cell lysis methods. Micromachines, 8(3), 83, which is hereby incorporated by reference).
  • the methods and reagents necessary for lysing the host cells can be determined by one skilled in the art based on the type of host cell.
  • Cells can be lysed by one or more methods of physical disrupting the cells (e.g., mechanical disruption, liquid homogenization, sonication, freeze-thaw, or manual grinding) or by incubating the cells with a cell lysis reagent.
  • the method can include, for example, lysing the cells with a sonicator, a French press, a Dounce homogenizer, a Potter-Elvehjem homogenizer, exposing the cells to freeze-thaw cycles, or by heating the cells.
  • Methods of physical disruption can also involve the use of additives, such as hypotonic buffers, or facilitators, such as glass beads, to promote lysis.
  • the cell lysis reagent can include one or more of an enzyme (e.g., lysozyme for bacterial cells), detergents, a hypotonic solution (i.e., for osmotic lysis), chelating agents, or commercially available buffers.
  • an enzyme e.g., lysozyme for bacterial cells
  • detergents e.g., a hypotonic solution
  • chelating agents i.e., for osmotic lysis
  • reagents such as enzymes that help prevent proteolysis (e.g., a protease inhibitor) or stabilize proteins can be added to the cell lysates.
  • nucleoprotein activity or levels can be assessed in the cell lysates.
  • nucleoprotein levels or activity is measured by fluorescence resonance energy transfer (FRET).
  • FRET fluorescence resonance energy transfer
  • FRET refers to an energy transfer mechanism occurring between two signaling molecules: a fluorescent donor and a fluorescent acceptor (i.e., a FRET pair) or a signaling partner and a quencher (i.e., a quenching pair) positioned within a range of about 1 to about 10 nanometers of each other.
  • a FRET donor moiety e.g., donor fluorophore
  • FRET acceptor moiety e.g., acceptor fluorophore
  • FRET Fluorescence Activated FRET
  • a FRET signal detected from a FRET pair serves as a proximity gauge of the donor and acceptor, such that a signal is only generated when they are in close proximity.
  • FRET may involve analysis of quenching of a signal exhibited by one signal partner (a fluorophore) by another signal partner (a quencher).
  • quenching pair Such a signal pair is referred to herein as a “quenching pair.”
  • quenching refers to partial or full absorption of energy emitted in form of fluorescence by a fluorescent component.
  • one signal partner is a fluorophore that produces a detectable signal that is quenched by the second signal partner (e.g., a quencher).
  • the second signal partner e.g., a quencher
  • label or “detectable label” means a molecule that can be directly (i.e. , a primary label) or indirectly (i.e., a secondary label) detected.
  • a label can be visualized and/or measured and/or otherwise identified so that its presence, absence, or a parameter or characteristic thereof can be measured and/or determined.
  • fluorescent label refers to any molecule that can be detected via its inherent fluorescent properties, which include fluorescence detectable upon excitation. Examples of fluorescent labels are described herein and elsewhere in the art (e.g., see The Tenth Edition of Haugland, RP. The Handbook: A Guide to Fluorescent Probes and Labeling Technologies. 10th. Invitrogen/Molecular Probes; Carlsbad, CA: 2005, hereby incorporated by reference).
  • fluorophores that can be used as FRET pairs in FRET biosensors include small organic dyes, fluorescent proteins (FPs), lanthanide chelates, and quantum dots (QDs).
  • FPs fluorescent proteins
  • QDs quantum dots
  • a broad range of fluorescent proteins have been developed that feature fluorescence emission spectral profiles spanning almost the entire visible light spectrum.
  • green Fluorescent Protein include EGFP, GFP10, Emerald, Superfolder GFP, Azami Green, mWasabi, TagGFP, TurboGFP, AcGFP, ZsGreen and T-Sapphire.
  • Non-limiting examples of blue fluorescent protein include EBFP, EBFP2, Azurite and mTagBFP.
  • Non-limiting examples of Cyan Fluorescent proteins include ECFP, mECFP, Cerulean, mTurquoise, CyPet, AmCyanl, Midori-lshi Cyan, TagCFP, mTFP1 (Teal).
  • Non-limiting examples of Yellow fluorescent proteins include EYFP, Topaz, Venus, mVenus, mCitrine, mAmetrine, YPet, TagYFP, PhiYFP, ZsYellowl and mBanana.
  • Non-limiting examples of orange fluorescent proteins include Kusabira Orange, Kusabira Orange2, mOrange, mOrange2, dTomato, dTomato-Tandem, TagRFP, DsRed, DsRed2, DsRed-Express (T1), DsRed- Monomer and mTangerine.
  • Non-limiting Examples of red fluorescent proteins include mRuby, mApple, mStrawberry, AsRed2, mRFP1 , JRed, mCherry, HcRedl, mRaspberry, dKeima-Tandem, HcRed-Tandem, mPlum and AQ143.
  • FRET pairs include cyan FP- yellow FP (CFP-YFP), green FP-red FP (CFP- YFP), far-red FP-infrared FP (FFP-IFP), large Stokes shift (LSS) FP-based FRET pairs, and dark FP- based FRET pairs.
  • a blue-green FRET pair can be used to detect an interaction between a gNA and a nucleic acid-guided nuclease.
  • an orange-red FRET pair can be used to detect an interaction between a nucleoprotein and a test polynucleotide containing a target site recognized by the gNA of the nucleoprotein.
  • multiple FRET pairs can be included in a single cell.
  • fluorophores that can be used in the methods provided herein include Alexa Fluor® 350; Marina Blue®; Atto 390; Alexa Fluor® 405; Pacific Blue ⁇ ; Pacific Green ⁇ ; Atto 425; Alexa Fluor® 430; Atto 465; DY-485XL; DY-475XL; FAMTM 494; Alexa Fluor® 488; DY-495-05; Atto 495; Oregon Green® 488; DY-480XL 500; Atto 488; Alexa Fluor® 500; Rhodamin Green®; DY- 505-05; DY-500XL; DY-510XL; Oregon Green® 514; Atto 520; Alexa Fluor® 514; JOE 520; TETTM 521 ; CAL Fluor® Gold 540; DY-521 XL; Rhodamin 6G@; Yakima Yellow® 526; Atto 532; Alexa Fluor®53
  • a quencher is a molecule which reduces the fluorescence intensity of a fluorophore by absorbing the excitation energy from the fluorophore.
  • quenchers that can be used in the methods herein include, but are not limited to an ATTO quencher (e.g., ATTO 540Q, ATTO 580Q, and ATTO 612Q), Dabcyl (4-(dimethylaminoazo)benzene-4-carboxylic acid), TAMRA, a Black Hole Quencher (e.g., BHQ-0, BHQ-1 , BHQ-2, BHQ-3), BBQ-650, DDQ-1 , Iowa Black RQTM, Iowa Black FQTM, IRDye QC-1 , a QSY dye (e.g., QSY- 21®; QSY-35®; QSY-7®; QSY-9®); a Qxl quencher (e.g., QXLTM 490; QXLTM 570; QX
  • fluorophore and quencher pairs must be chosen such that the fluorophore's optimal excitation and emission spectra are matched to the quencher's effective range.
  • ODN probes linear fluorescence resonance energy transfer (FRET) probes
  • FRET fluorescence resonance energy transfer
  • dual-labeled oligonucleotide hairpin probes e.g., molecular beacons
  • FRET molecular beacons dual FRET molecular beacons
  • autoligation FRET probes and probes using fluorescent proteins as reporters (see, e.g., Bao et al. "Fluorescent probes for live-cell RNA detection.” Annual review of biomedical engineering 11 (2009): 25-47).
  • the methods provided herein are used to measure nucleoprotein levels or activity in a cell lysate derived from a single clone in a library of host cell clones, wherein the library of host cell clones comprise a library of expression vectors encoding a nucleic acid-guided nuclease and/or a gNA.
  • the host cells can optionally be arranged in an array of host cells, wherein each member of the array can be tested for RNP levels or activity separately from other members of the array. Methods of arranging cells into an array, such as representatives of a library of host cells, are known in the art.
  • individual host cell clones comprising different constructs are sorted into a multi-well plate (e.g., 6, 12, 24, 48, 96, or 384-well microplate), with one host cell clone per well, prior to lysis and further analysis.
  • the cells may be sorted into single-cell compartments comprising a single cell, or a population of cells derived therefrom.
  • the FRET assay is Homogeneous Time Resolved Fluorescence (HTRF), as described in, e.g., Degorce et al. "HTRF: a technology tailored for drug discovery-a review of theoretical aspects and recent applications.” Current chemical genomics 3 (2009): 22.
  • HTRF Homogeneous Time Resolved Fluorescence
  • detectable labels and methods of preparing fusion proteins and split derivatives comprising detectable labels are known in the art and can be used in any of the methods described herein, e.g., see Thorn, K. (2017). Genetically encoded fluorescent tags. Molecular biology of the cell, 28(7), 848-857, S. Cabantous, T.C. Terwilliger, G.S. Waldo (2005) Protein tagging and detection with engineered self-assembling fragments of green fluorescent protein. Nature Biotechnology 23(1 ), 102-107, and D. Kamiyama, et al. (2016) Versatile protein tagging in cells with split fluorescent protein. Nature Communications 7, 11046; and Pedelacq et al. "Development and Applications of Superfolder and Split Fluorescent Protein Detection Systems in Biology.” International journal of molecular sciences 20.14 (2019): 3479. which are hereby incorporated by reference in their entirety.
  • detectable labels such as those found in commercially available kits can be used in any of the methods provided herein.
  • the detectable labels are AlphaScreen reagents (see, e.g., Yasgar et al. "AlphaScreen-based assays: ultra-high-throughput screening for small-molecule inhibitors of challenging enzymes and protein- protein interactions.” High Throughput Screening. Humana Press, New York, NY, 2016. 77-98).
  • AlphaScreen is a bead-based, non-radioactive Amplified Luminescent Proximity Homogeneous Assay.
  • the nucleic acid-guided nuclease and the nucleic acid can include epitope tags or conjugation moieties, offering means of coupling to donor and acceptor beads/labels.
  • the methods provided herein can use a variety of conformation-specific NP binding agents capable of binding a nucleoprotein.
  • the conformation-specific NP binding agent is an anti-CRISPR (Acr) protein, such as one further described herein.
  • the Acr protein can be AcrllA4.
  • the conformation-specific NP binding agent may be an antibody, or antigen binding fragment thereof, that specifically binds the NP.
  • the conformation-specific NP binding agent may be an aptamer that specifically binds the NP.
  • the conformation-specific NP binding agent can comprise one or more modifications (e.g., an amino acid substitution, deletion, or insertion) relative to a naturally occurring conformation- specific NP binding agent (e.g., Acr protein, such as AcrllA4) or one further described herein.
  • a naturally occurring conformation- specific NP binding agent e.g., Acr protein, such as AcrllA4
  • the conformation-specific NP binding agent has at least 50, 60, 70, 75, 80, 85, 90, 95, 98%, or 99% sequence identity to a conformation-specific NP binding agent described herein.
  • the conformation-specific NP binding agent can comprise one or more modifications (e.g., an amino acid substitution, deletion, or insertion).
  • cysteines can be rationally introduced into the conformation-specific NP binding protein (e.g., AcrllA4) and the nucleic acid-guided nuclease (e.g., Cas9) interface based on the crystal structure (see, e.g., the crystal structure of AcrllA4 in Kim, et al.
  • a disulfide bridge is formed between the conformation-specific NP binding protein (e.g., AcrllA4) and a nucleoprotein comprising the nucleic acid-guided nuclease (e.g., Cas9).
  • the solution comprising the engineered conformation-specific NP binding agent may, in some instances, be treated with an oxidizing agent so the disulfide remains intact.
  • the disulfide bond may promote affinity between the conformation-specific NP binding protein and nucleoprotein in any of the methods described herein.
  • the method further involves isolating the nucleoproteins from a host cell comprising an expression construct described herein, wherein each nucleoprotein comprises a nucleic acid-guided nuclease and a guide nucleic acid (gNA). Any purification methods can be used to isolate nucleoproteins from a host cell.
  • Exemplary isolation techniques include, without limitation, affinity capture, immunoprecipitation, chromatography (for example, size exclusion chromatography, hydrophobic interaction chromatography, reverse-phase chromatography, ion exchange chromatography, affinity chromatography, metal binding chromatography, immunoaffinity chromatography, high performance liquid chromatography (HPLC), and liquid chromatography-mass spectrometry (LC-MS)), electrophoresis, hybridization to a capture oligonucleotide, phenol-chloroform extraction, minicolumn purification, or ethanol or isopropanol precipitation. Chromatography methods are described in detail, for example, in Fledhammar et al.
  • Such techniques can utilize a capture molecule that recognizes a nucleoprotein or a component thereof (e.g., a nucleic acid-guided nuclease or gNA).
  • a capture molecule that recognizes a nucleoprotein or a component thereof (e.g., a nucleic acid-guided nuclease or gNA).
  • the capture molecule used for isolating the nucleoprotein is a conformation-specific nucleoprotein binding agent (e.g., an Acr protein, such as AcrllA4).
  • a conformation-specific NP binding agent such as one immobilized on a surface (e.g., a column, a bead, or a plate) is used to specifically bind the nucleoprotein in a solution while allowing compounds that are not the nucleoprotein to remain in solution (e.g., in a supernatant or flow through).
  • the unbound compounds can be removed, for example, by collecting the supernatant or flow-through.
  • the surface comprising the conformation-specific NP binding agent can optionally be washed with a wash buffer to promote removal of compounds other than nucleoproteins.
  • the nucleoproteins are eluted by contacting the surface comprising the conformation-specific NP binding agent with an elution buffer that enables the nucleoprotein to disassociate from the conformation-specific NP binding agent.
  • a method of separating nucleoproteins (NPs) from one or more other compounds (e.g., a compound other than the nucleoprotein) in a liquid sample comprising contacting a surface, such as a bead, plate, or column, comprising a conformation-specific NP binding agent (e.g., an Acr protein, such as AcrllA4) with the liquid sample under conditions that allow binding of the NPs to the conformation-specific NP binding agent and removal of an unbound fraction (e.g., flow-through or supernatant) comprising the one or more other compounds.
  • a conformation-specific NP binding agent e.g., an Acr protein, such as AcrllA4
  • the method may additionally involve contacting the surface comprising the conformation-specific NP binding agent with a wash buffer.
  • the method involves contacting the surface comprising the conformation-specific NP binding agent with an elution buffer, thereby eluting the NPs from the conformation-specific NP binding agents to provide a nucleoprotein eluate lacking the one or more other compounds in the liquid sample.
  • the liquids sample can be any sample comprising a nucleoprotein and one or more additional compounds (e.g., polypeptides, carbohydrate, lipids, polynucleotides, or inorganic compounds) that are not the nucleoprotein.
  • the liquid sample comprises a host cell lysate that includes the nucleoprotein, such as a lysate that would be obtained following expression of a nucleoprotein in a host cell and subsequent lysis of the host cell.
  • the cell lysate may be one obtained from lysis of host cell expressing a nucleic acid-guided nuclease followed by addition of a gNA to the lysate to form nucleoproteins.
  • the one or more other compounds in the liquid sample is a host cell protein.
  • conformation-specific NP binding agent is an anti-CRISPR (Acr) protein, such as one further described herein.
  • the Acr protein can be AcrllA4.
  • the Acr protein can be used to capture and isolate it’s cognate CRISPR-Cas RNP.
  • AcrllA4 can be used to capture and isolate an RNP comprising Cas9.
  • AcrVAI can be used to capture and isolate an RNP comprising Cas12a.
  • the conformation-specific NP binding agent may be an antibody, or antigen binding fragment thereof, that specifically binds the NP.
  • the conformation-specific NP binding agent may be an aptamer that specifically binds the NP.
  • the conformation-specific NP binding agent can comprise one or more modifications (e.g., an amino acid substitution, deletion, or insertion) relative to a naturally occurring conformation-specific NP binding agent (e.g., Acr protein, such as AcrllA4 or AcrVAI ) or one further described herein.
  • a naturally occurring conformation-specific NP binding agent e.g., Acr protein, such as AcrllA4 or AcrVAI
  • the conformation-specific NP binding agent has at least 50, 60, 70, 75, 80, 85, 90, 95, 98%, or 99% sequence identity to a conformation-specific NP binding agent described herein.
  • the conformation-specific NP binding agent can be associated with a surface, such as a column, plate, or bead, using any conjugation or attachment methods known in the art.
  • the conformation-specific NP binding agent comprises conjugation moieties to promote attachment of the conformation-specific NP binding agent to the surface.
  • the surface may also include a conjugation moiety as deemed appropriate for the attachment method selected.
  • the method may involve the use of one or more buffers to promote washing and/or elution of the nucleoprotein.
  • the different buffers may have different properties so as to promote optimal removal of other compounds or to promote elution and recovery of the nucleoprotein.
  • the wash buffer may be at a first conductivity and the elution buffer may at a second (i.e. , different) conductivity.
  • the wash buffer may be at a first salt concentration and the elution buffer may be at a second (i.e., different) salt concentration.
  • the wash buffer may be at a first pH and the elution buffer may be at a second (i.e., different pH).
  • the elution buffer may also comprise an un-immobilized NP binding agent, such as an Acr protein (e.g., AcrllA4 or AcrVAI) that competes with the immobilized conformation-specific NP binding agent for binding to the NP.
  • an Acr protein e.g., AcrllA4 or AcrVAI
  • the purification methods provided herein may be used to isolate any nucleoprotein bound by a conformation-specific NP binding agent.
  • the nucleoprotein is a ribonucleoprotein
  • the nucleic acid-guided nuclease is an RNA-guided nuclease, such as Cas9
  • the guide nucleic acid is a guide RNA (gRNA).
  • the nucleic acid- guided nuclease of the nucleoprotein is a fusion protein, such as one comprising a cell targeting agent (e.g., a peptide, ligand, antigen-binding agent). Examples of nucleoproteins and nucleic acid- guided nucleases are further described in Section V.
  • NP conformation-specific nucleoprotein
  • the methods herein are further useful for identifying variants of a conformation-specific NP binding agent (e.g., mutagenized conformation- specific NP binding agents that have retained the ability to bind a nucleoprotein).
  • the mutagenized conformation-specific NP binding agent is a mutagenized anti- CRISPR protein, such as AcrllA4 or AcrVAI .
  • the conformation-specific NP binding agent can comprise one or more modifications (e.g., an amino acid substitution, deletion, or insertion) relative to a naturally occurring conformation-specific NP binding agent (e.g., Acr protein, such as AcrllA4 or AcrVAI) or one further described herein.
  • the method involves providing one or more expressing constructs comprising (1 ) a polynucleotide encoding a test conformation-specific NP binding agent encoding; (2) a polynucleotide encoding a nucleic acid-guided nuclease; and (3) a polynucleotide encoding a unique identifying nucleic acid (uiNA) (e.g., uiRNA or uiDNA) comprising a guide nucleic acid (e.g., gRNA or gDNA) and a sequence identifier, wherein at least the polynucleotide encoding the test conformation-specific NP binding agent and the polynucleotide encoding the guide nucleic acid are on the same expression construct.
  • uiNA unique identifying nucleic acid
  • gRNA or gDNA guide nucleic acid
  • the polynucleotide encoding a test conformation-specific NP binding agent encoding; and a polynucleotide encoding a unique identifying nucleic acid (uiNA) (e.g., uiRNA or uiDNA) comprising a guide nucleic acid (e.g., gRNA or gDNA) and a sequence identifier are on the same expression construct, while the polynucleotide encoding the nucleic acid-guided nuclease is on a separate construct.
  • uiNA unique identifying nucleic acid
  • uiNA unique identifying nucleic acid
  • gRNA or uiDNA e.gRNA or uiDNA
  • the method further comprises sequencing portions of the expression construct encoding the sequence identifier of the uiNA and the test conformation-specific NP binding agent, thereby establishing an association between the test conformation-specific NP binding agent and identifier sequence.
  • This association can be used to provide a reference or index for identifying the test conformation-specific NP binding agent based on the presence of the identifier sequence, for example, at later steps in the method.
  • the method further involves transferring the one or more expression constructs to a host cell suitable to express the test conformation-specific NP binding agent, the nucleic acid-guided nuclease, and the uiNA.
  • the expression construct is in a plurality of expression constructs and the plurality of expression constructs is transferred into host cells under conditions such that the average construct per host cell is 1 or more.
  • the expression construct is in a plurality of expression constructs and the plurality of expression constructs are transferred into host cells under conditions such that the average expression construct per host cell is less than 1 .
  • test conformation-specific NP binding agent, the nucleic acid-guided nuclease, and the uiNA can be expressed from the one or more expression constructs in the host cell, such that complexes are formed comprising the test conformation-specific NP binding agent and a nucleoprotein comprising the nucleic acid-guided nuclease and the uiRNA encoded on the one or more expression constructs.
  • test conformation-specific NP binding agent, the nucleic acid-guided nuclease, and the uiNA can be expressed from the one or more expression constructs in the host cell, such that complexes are formed comprising the test conformation-specific NP binding agent and a nucleoprotein comprising the nucleic acid-guided nuclease and the uiRNA encoded on the one or more expression constructs.
  • the test conformation-specific NP binding agent, the nucleic acid- guided nuclease, and/or the uiNA can each be operatively linked to a promoter, such as an inducible promoter, including those further described herein,
  • the one or more expression constructs comprises a first promoter operatively linked to a nucleic acid sequence encoding the RNA-guided nuclease, and a second promoter operatively linked to a nucleic acid sequence encoding the uiNA.
  • the first and second promoter are each inducible (e.g., T7 or T5) such that the expression level of the nucleic acid-guided nuclease and the expression level of the uiNA can be controlled to obtain nucleoproteins.
  • the first and/or second promoter is a constitutive promoter.
  • the one or more expression constructs comprises a third promoter operatively linked to a conformation-specific NP binding agent.
  • the third promoter may be an inducible promoter such that the expression level of the conformation-specific NP binding agent may be controlled.
  • the method comprises expressing the test conformation-specific NP binding agent, the nucleic acid-guided nuclease, and the uiNA in the host cell, such that complexes are formed comprising the test conformation-specific NP binding agent and a nucleoprotein comprising the nucleic acid-guided nuclease and the uiNA.
  • the complexes are then separated (e.g., by affinity purification) from the host cell, e.g., such that the conformation-specific NP binding agent and nucleoprotein remain stably associated following co-purification.
  • the separated complexes can optionally be pooled together and further assessed as a pooled library of complexes (e.g., comprising a plurality of different conformation-specific NP binding agents, e.g., mutagenized variants of a conformation-specific NP binding agent), or the conformation-specific NP binding agents can be assessed individually, e.g., in an array.
  • a pooled library of complexes e.g., comprising a plurality of different conformation-specific NP binding agents, e.g., mutagenized variants of a conformation-specific NP binding agent
  • the conformation-specific NP binding agents can be assessed individually, e.g., in an array.
  • Release or dissociation of nucleoproteins from the complexes can then be measured following exposure of the complexes to a condition of interest, such as a buffer having similar conditions to the interior of an endosome (e.g., pH inside an endosome) of a cell (e.g., a target cell).
  • a condition of interest such as a buffer having similar conditions to the interior of an endosome (e.g., pH inside an endosome) of a cell (e.g., a target cell).
  • release of nucleoproteins from the complexes can be measured based on changes in the level of the uiNA following exposure to a buffer having the pH of an endosome, wherein a decreased level of the uiNA relative to a reference level (e.g., the level of complex formation at a cytoplasmic pH) identifies the test conformation-specific NP binding agent as one that enables release of a nucleoprotein in the endosome of the target cell.
  • identification of the test conformation-specific NP binding agent may be based on a previously established reference or index establishing an association between the uiNA and the test conformation-specific NP binding agent.
  • a conformation-specific NP binding agent that releases nucleoproteins above a threshold level or at a level greater than a control is subsequently isolated for further use in delivery of a nucleoprotein to a cell, e.g., in a Targeted Active Gene Editor (TAGE) described herein.
  • TAGE Targeted Active Gene Editor
  • the method may involve exposing the complexes to a first buffer having a first pH at about the pH of the cytoplasm in the target cell (e.g., a eukaryotic cell), and testing the uiNA of the complexes in the first buffer for the presence of the identifier sequence.
  • the testing step involves exposing the complexes to a second buffer having a second pH at about the pH of an endosome in the target cell (e.g., a eukaryotic cell), and measuring the changes in the levels of each complex based on changes in the corresponding uiNA levels.
  • the first pH is about pH 6.5 to about pH 8 (e.g., about pH 6.6 to about pH 7.8, to about pH 6.8 to about pH 7.6, about pH 6.8 to about pH 7.4, about pH 6.8 to about pH 7.2, about pH 6.9 to about pH 7.1 ).
  • the first pH is about 6.5, about 6.6, about 6.7, about 6.8, about 6.9, about 7, about 7.1 about 7.2, about 7.3, about 7.4, about 7.5, about 7.6, about 7.6, about 7.7, and about 7.8.
  • the first pH is about pH 7.
  • the second pH will be less than the first pH.
  • the second pH is about pH 4.5 to about pH 6.4 (e.g., about pH 4.7 to about pH 6.2, about pH 4.8 to about pH 6, about pH 4.8 to about pH 5.8, about pH 4.8 to about pH 5.6, about pH 4.8 to about pH 5.4, about pH 4.8 to about pH 5.2, about pH 4.9 to about pH 5.1 .
  • the second pH is about pH 5.
  • the method may involve testing the levels of uiNA following pull-down or separation of a component of the complex (e.g., the conformation-specific NP binding agent) from the second buffer. Finally, the method may involve measuring for release or dissociation of nucleoproteins from the complexes after exposure to the second buffer at the second pH.
  • a component of the complex e.g., the conformation-specific NP binding agent
  • release of nucleoproteins from the complexes is measured based on changes in the presence or level of a identifier sequence (e.g., a identifier sequence previously associated with a conformation-specific NP binding agent encoded on the same expression construct in a library), wherein a decrease in the level of the identifier sequence in the second buffer relative to the level of the identifier sequence in the first buffer identifies the associated test conformation-specific NP binding agent as one that enables release of a nucleoprotein in the endosome of the target cell.
  • a identifier sequence e.g., a identifier sequence previously associated with a conformation-specific NP binding agent encoded on the same expression construct in a library
  • test conformation-specific NP binding agent can be any conformation-specific NP binding agent known in the art, such as one described herein (See section II).
  • Test conformation-specific NP binding agents can be natural, recombinant, or synthetic.
  • the test conformation-specific NP binding agent is one selected from a library of test conformation-specific NP binding agents.
  • the test conformation-specific NP binding agent can be selected from a library of randomly mutated conformation-specific NP binding agents.
  • the method can include mutagenizing a test conformation-specific NP binding agent (e.g., through random mutagenesis or site-directed mutagenesis) and preparing a library of mutagenized conformation-specific NP binding agents.
  • the mutagenized test conformation-specific NP binding agents can then be assessed for the ability to enable an associated nucleoprotein to be delivered to a target cell and released (e.g., dissociated from the conformation-specific NP binding agent) in an endosome of the target cell.
  • a test conformation-specific NP binding agent is one found in a protein or peptide database (for example, SWISS-PROT, TrEMBL, SBASE, PFAM, or others known in the art), or a fragment or variant thereof.
  • a test conformation-specific NP binding agent may be a protein or peptide that may be derived (for example, by transcription and/or translation) from a nucleic acid sequence known in the art, such as a nucleic acid sequence found in a nucleic acid database (for example, GenBank, TIGR, or others known in the art), or a fragment or variant thereof.
  • the test conformation-specific NP binding agent may be a fusion protein, such as those described herein (see, e.g., Section VI).
  • An expression construct encoding a test conformation-specific nucleoprotein binding agent can be designed to encode molecular moieties that are fused to or integrated with the protein to form a conformation-specific nucleoprotein binding fusion protein. For example, such moieties can aid in the purification and/or detection of the test conformation-specific nucleoprotein binding agent.
  • the conformation-specific nucleoprotein binding agent further comprises a cell targeting agent, as described in the targeted active gene editors of the invention, to be targeted to the surface of a target cell or internalized by a target cell, i.e. , a cell targeted by the cell targeting agent.
  • uiNA unique identifying nucleic acid
  • a guide nucleic acid e.g., DNA or RNA that is capable of stably associating with a nucleic acid-guided nuclease
  • a unique sequence identifier e.g., barcode
  • the sequence identifier can be any portion of the guide sequence that differentiates it from other guide sequences, including the guide sequence itself.
  • the uiNA is on the same expression construct as a polynucleotide a test conformation-specific nucleoprotein binding agent.
  • the identifier in the uiNA can be used to identify associated test conformation-specific binding agents that are on the same expression construct as the uiNA.
  • the sequence identifier can be located anywhere on or adjacent to the guide nucleic acid (e.g., in or adjacent to crRNA, tracrRNA, or in the tetraloop between the crRNA / trRNA on a single guide RNA).
  • the unique identifier is a randomized guide nucleic acid.
  • the randomized guide sequence may be one that is not capable of hybridizing with a target sequence yet can still stably associate with a nucleic acid-guided nuclease.
  • the guide nucleic acid retains its ability to hybridize with a complementary nucleic acid sequence.
  • Sequence identifiers can be any nucleic acid sequence that uniquely identifies the guide nucleic acid, and may be generated from a variety of different formats, including bulk synthesized polynucleotide barcodes, randomly synthesized barcode sequences, microarray based barcode synthesis, native nucleotides, a partial complement with an N-mer, a random N-mer, a pseudo random N-mer, or combinations thereof.
  • the sequence identifier can be a non-naturally occurring sequence.
  • the sequence identifier can comprise, for example less than 10, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 88, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, or more than 200 nucleotides. Further, the sequence identifier can be located anywhere on or adjacent to the guide nucleic acid (e.g., in or adjacent to crRNA, tracrRNA, or in the tetraloop between the crRNA / trRNA on a single guide RNA). In some instances, the unique identifier is a randomized guide nucleic acid.
  • the randomized guide sequence may be one that is not capable of hybridizing with a target sequence yet can still stably associate with a nucleic acid-guided nuclease.
  • the guide nucleic acid retains its ability to hybridize with a complementary nucleic acid sequence.
  • the uiNA may also include additional sequence segments.
  • additional sequence segments may include functional sequences, such as primer sequences, primer annealing site sequences, immobilization sequences, or other recognition or binding sequences useful for subsequent processing, e.g., a sequencing primer or primer binding site for use in sequencing of samples to which the uiNA oligonucleotide is attached.
  • the method involves producing a plurality (e.g., a library) of expression constructs, the method comprising cloning nucleic acids encoding a plurality of test conformation- specific NP binding agents into an expression construct such that each expression construct comprises a polynucleotide encoding a single test conformation-specific NP binding agent and a unique identifying nucleic acid (uiRNA or uiDNA), wherein the uiNA comprises a guide nucleic acid (e.g., RNA or DNA) and a sequence identifier.
  • a plurality e.g., a library
  • the method involves providing a plurality (e.g., a library) of expression constructs each encoding a test conformation-specific NP binding agent, a nucleic acid-guided nuclease (e.g., RNA-guided nuclease or DNA-guided nuclease), and a unique identifying nucleic acid (uiNA) (e.g., uiRNA or uiDNA) comprising a guide nucleic acid (e.g., gRNA or gDNA) and a sequence identifier.
  • a nucleic acid-guided nuclease e.g., RNA-guided nuclease or DNA-guided nuclease
  • uiNA unique identifying nucleic acid
  • the plurality of expression constructs may be a library of constructs.
  • the library may be composed of a plurality of different library members, which each encode a single conformation- specific NP binding agent. Sequence differences, between library members, such as sequence differences between different test conformation-specific NP binding agents or uiNAs, are responsible for the diversity present in the library.
  • the library may take the form of a simple mixture of expression constructs, or may be in the form organisms or cells, for example bacteria, viruses, animal or plant cells and the like, transformed with a library of expression constructs. Preferably, each individual organism or cell contains only one member of the library.
  • Expression constructs can be assembled from DNA encoding components of interest (e.g., a test conformation-specific NP binding agent, a nucleic acid-guided nuclease, a uiNA, and/or a regulatory element).
  • the DNA can be obtained from any source, such as through amplification of sequences of interest from genomic DNA or through synthesis.
  • DNA encoding a component of interest can be amplified and cloned using a known technique, such as PCR using appropriately- selected primers, in order to produce sufficient quantities of the DNA and to modify the DNA in such a manner (e.g., by addition of appropriate restriction sites) that it can be introduced as an insert into an expression construct (such as those described in Section VI).
  • Amplified and cloned DNA can be further diversified, using mutagenesis, such as PCR, in order to produce a greater diversity or wider repertoire of test proteins, as well as novel test proteins.
  • a cloned polynucleotide encoding any construct component described herein e.g., a test conformation-specific NP binding agent, a nucleic acid-guided nuclease, a uiNA, and/or a regulatory element
  • an expression construct e.g., a plasmid
  • the polynucleotide is inserted into the expression construct in such a manner that the protein will be expressed as protein in appropriate host cells.
  • the method further comprises sequencing one or more portions of the expression construct.
  • the method may further include sequencing one or more portions of the expression construct encoding the nucleic acid sequence identifier and/or the test conformation-specific NP binding agent, thereby establishing an association between the test conformation-specific NP binding agent and identifier sequence. This association can be used to provide a reference or index for identifying the test protein based on the presence of the identifier sequence, for example, at later steps in the method.
  • sequencing can be performed using automated Sanger sequencing (ABI 3730x1 genome analyzer), pyrosequencing on a solid support (454 sequencing, Roche), sequencing-by-synthesis with reversible terminations (ILLUMINA® Genome Analyzer), sequencing-by-ligation (ABI SOLiD®) or sequencing-by-synthesis with virtual terminators (HELISCOPE®); Moleculo sequencing (see Voskoboynik et al. eLife 20132:e00569 and US Patent Application No. 13/608,778, filed Sep 10, 2012); DNA nanoball sequencing; Single molecule real time (SMRT) sequencing; Nanopore DNA sequencing; Sequencing by hybridization; Sequencing with mass spectrometry; and Microfluidic Sanger sequencing.
  • automated Sanger sequencing (ABI 3730x1 genome analyzer), pyrosequencing on a solid support (454 sequencing, Roche), sequencing-by-synthesis with reversible terminations (ILLUMINA® Genome Analyzer), sequencing-by-ligation (ABI SOLiD®)
  • Exemplary next generating sequencing methods known to those of skill in the art include Massively parallel signature sequencing (MPSS), Polony sequencing, pyrosequencing (454), lllumina (Solexa) sequencing by synthesis, SOLiD sequencing by ligation, Ion semiconductor sequencing (Ion Torrent sequencing), DNA nanoball sequencing, chain termination sequencing (Sanger sequencing), Heliscope single molecule sequencing, Single molecule real time (SMRT) sequencing ( Pacific Biosciences) and nanopore sequencing such as is described at world wide website nanoporetech.com.
  • the library of expression constructs can then be introduced into host cells, which can be eukaryotic or prokaryotic, for expression of one or more components encoded on the construct (e.g., a test conformation-specific NP binding agent, a nucleic acid-guided nuclease, a uiNA, and/or a regulatory element).
  • host cells can be eukaryotic or prokaryotic, for expression of one or more components encoded on the construct (e.g., a test conformation-specific NP binding agent, a nucleic acid-guided nuclease, a uiNA, and/or a regulatory element).
  • Transfer of the expression construct into host cells e.g., by infection, transformation, or transfection
  • electroporation protoplast fusion
  • calcium phosphate co-precipitation can be carried out using known techniques, such as electroporation, protoplast fusion, or calcium phosphate co-precipitation.
  • both constructs can be introduced into appropriate host
  • the method further involves introducing the expression construct into a host cell suitable to express the test conformation-specific NP binding agent, the nucleic acid-guided nuclease, and the uiNA in the host cell, such that complexes form comprising the test conformation- specific NP binding agent stably associated with the expressed nucleoproteins (NPs; RNP or DNP) comprising a nucleic acid-guided nuclease and the corresponding uiNA.
  • NPs expressed nucleoproteins
  • the host cell may be a prokaryotic cell or eukaryotic cell, such as a bacterial cell, an animal cell, a plant cell, or a fungal cell.
  • the eukaryotic cell is a yeast cell (e.g., a S. cerevisiae cell, Pichia pastoris, or the like), a plant cell, or mammalian cell.
  • the bacterial cell is an E. coli cell.
  • the host cell is a mammalian cultured cell derived from rodents (rats, mice, guinea pigs, or hamsters) such as CHO, BHK, NSO, SP2/0, YB2/0; or human tissues or hybridoma cells, yeast cells, or insect cells.
  • rodents rats, mice, guinea pigs, or hamsters
  • CHO BHK, NSO, SP2/0, YB2/0
  • human tissues or hybridoma cells yeast cells, or insect cells.
  • yeast cells or insect cells.
  • the term encompasses not only the particular subject cell but also the progeny of such a cell.
  • the mammalian cell is a COP cell, an L cell, a C127 cell, an Sp2/0 cell, an NS-0 cell, an NIH3T3 cell, a PC12 cell, a PC12h cell, a BHK cell, a CHO cell, a COS1 cell, a COS3 cell, a COST cell, a CV1 cell, a Vero cell, a HeLa cell, an HEK-293 cell, a PER C6 cell, a cell derived from diploid fibroblasts, a myeloma cell, or HepG2.
  • polynucleotides e.g., an expression construct
  • methods of introducing polynucleotides are known in the art and are typically selected based on the kind of host cell.
  • Such methods include, for example, viral or bacteriophage infection, transfection, conjugation, electroporation, calcium phosphate precipitation, polyethyleneimine-mediated transfection, DEAE-dextran mediated transfection, protoplast fusion, lipofection, liposome-mediated transfection, particle gun technology, direct microinjection, and nanoparticle-mediated delivery.
  • the method may involve transferring the construct to a non-cellular compartment (e.g., an emulsion droplet) suitable to express the test conformation-specific NP binding agent, the nucleic acid-guided nuclease, and the uiNA, and expressing the test conformation-specific NP binding agent, the nucleic acid-guided nuclease, and the uiNA in the non-cellular compartment (e.g., the emulsion droplet).
  • a non-cellular compartment e.g., an emulsion droplet
  • the non-cellular compartment is a droplet, such as a droplet in an emulsion and/or a microfluidic droplet.
  • Emulsification can be used in the methods of the disclosure to separate or segregate a sample or set of samples into a series of compartments, for example a compartment having a single cell or a discrete portion of an acellular sample, such as a cell-free extract or a cell-free transcription and/or cell- free translation mixture, such as those described herein.
  • the method further involves separating the complexes comprising the test conformation-specific NP binding agent and the nucleoprotein from a host cell comprising an expression construct described herein, wherein each nucleoprotein comprises a nucleic acid-guided nuclease and a unique identifying nucleic acid (uiNA) (e.g., comprising a guide nucleic acid and a sequence identifier).
  • uiNA unique identifying nucleic acid
  • Any purification methods can be used to isolate the complexes from a host cell.
  • Exemplary isolation techniques include, without limitation, affinity capture, immunoprecipitation, chromatography (for example, size exclusion chromatography, hydrophobic interaction chromatography, reverse-phase chromatography, ion exchange chromatography, affinity chromatography, metal binding chromatography, immunoaffinity chromatography, high performance liquid chromatography (HPLC), and liquid chromatography-mass spectrometry (LC-MS)), electrophoresis, hybridization to a capture oligonucleotide, phenol-chloroform extraction, minicolumn purification, or ethanol or isopropanol precipitation. Chromatography methods are described in detail, for example, in Hedhammar et al.
  • Such techniques can utilize a capture molecule that recognizes a labeled component of the complex, such as the test conformation-specific NP binding agent or a component of the nucleoprotein (e.g., a nucleic acid- guided nuclease or a uiNA).
  • a capture molecule that recognizes a labeled component of the complex, such as the test conformation-specific NP binding agent or a component of the nucleoprotein (e.g., a nucleic acid- guided nuclease or a uiNA).
  • Complexes comprising the test conformation-specific NP binding agent and a nucleoprotein (e.g., comprising a nucleic acid-guided nuclease, and a unique identifying nucleic acid (uiNA)), can be assessed for release of the nucleoprotein in a cellular compartment or in a solution having similar conditions (e.g., pH) to a cellular compartment, such as the lumen of an endosome.
  • the dissociation of the test conformation-specific NP binding agent and the nucleoprotein is assessed using any methods known in the art to test for protein-protein dissociation.
  • the release of the nucleoprotein from the complexes i.e.
  • dissociation from the test conformation-specific NP binding agent is measured based on the level of the uiNA following exposure to a buffer having similar conditions (e.g., pH) to the lumen of the endosome, wherein a decreased level of the uiNA relative to a reference level identifies the test conformation-specific NP binding agent as one that enables release of a nucleoprotein in the endosome of a cell (e.g., a target cell).
  • a buffer having similar conditions e.g., pH
  • the uiNA, or the identifier sequence therein can be amplified for further analysis following any amplification methods known in the art.
  • An example of amplification is the polymerase chain reaction (PCR), in which a sample is contacted with a pair of oligonucleotide primers under conditions that allow for the hybridization of the primers to a nucleic acid template in the sample.
  • the primers are extended under suitable conditions, dissociated from the template, re-annealed, extended, and dissociated to amplify the number of copies of the nucleic acid. This cycle can be repeated.
  • the product of amplification can be characterized by such techniques as electrophoresis, restriction endonuclease cleavage patterns, oligonucleotide hybridization or ligation, and/or nucleic acid sequencing.
  • in vitro amplification techniques include quantitative real-time PCR; reverse transcriptase PCR (RT-PCR); real-time PCR (rt PCR); realtime reverse transcriptase PCR (rt RT-PCR); nested PCR; strand displacement amplification (see U.S. Patent No. 5,744,311 ); transcription-free isothermal amplification (see U.S. Patent No. 6,033,881 , repair chain reaction amplification (see WO 90/01069); ligase chain reaction amplification (see European patent publication EP-A-320308); gap filling ligase chain reaction amplification (see U.S. Patent No. 5,427,930); coupled ligase detection and PCR (see U.S.
  • the testing step comprises reverse-transcribing the identifier sequence to produce cDNA, and sequencing the cDNA. In some embodiments, the testing step comprises sequencing the uiNA to determine the presence of the identifier sequence.
  • PCR polymerase chain reaction
  • RACE ligation chain reaction
  • LCR ligation chain reaction
  • Patent Nos. 6,391 ,544, 6,365,375, 6,294,323, 6,261 ,797, 6,124,090 and 5,612, 199 isothermal amplification (e.g., rolling circle amplification (RCA), hyperbranched rolling circle amplification (HRCA), strand displacement amplification (SDA), helicase-dependent amplification (HDA), PWGA) or any other nucleic acid amplification method using techniques well known to those of skill in the art.
  • isothermal amplification e.g., rolling circle amplification (RCA), hyperbranched rolling circle amplification (HRCA), strand displacement amplification (SDA), helicase-dependent amplification (HDA), PWGA
  • RCA rolling circle amplification
  • HRCA hyperbranched rolling circle amplification
  • SDA strand displacement amplification
  • HDA helicase-dependent amplification
  • PWGA helicase-dependent amplification
  • the nucleic acid (e.g., isolated nucleic acids) obtained can be tested for the presence of the identifier sequence by a variety of methods, including any sequencing or microarray methods known in the art.
  • the identity of a unique identifying nucleic acid is determined by DNA or RNA sequencing (e.g., RNA-seq).
  • the sequencing can be performed using automated Sanger sequencing (ABI 3730x1 genome analyzer), pyrosequencing on a solid support (454 sequencing, Roche), sequencing-by-synthesis with reversible terminations (ILLUMINA®
  • Genome Analyzer sequencing-by-ligation (ABI SOLiD®) or sequencing-by-synthesis with virtual terminators (HELISCOPE®); Moleculo sequencing (see Voskoboynik et al. eLife 20132:e00569 and US Patent Application No. 13/608,778, filed Sep 10, 2012); DNA nanoball sequencing; Single molecule real time (SMRT) sequencing; Nanopore DNA sequencing; Sequencing by hybridization; Sequencing with mass spectrometry; and Microfluidic Sanger sequencing.
  • Exemplary next generating sequencing methods known to those of skill in the art include Massively parallel signature sequencing (MPSS), Polony sequencing, pyrosequencing (454), lllumina (Solexa) sequencing by synthesis,
  • the uiNA is sequenced using a template-switch reaction (e.g., with MaximaH-Minus reverse transcriptase, derived from SMART seq, 10x Genomics), ssRNA ligation (e.g., with T4 RNA ligase K227Q, derived from microRNA seq), ssDNA ligation (e.g., with cricLigase, derived from SHAPE-seq), homopolymer tailing (e.g., with terminal transferase, derived from HTL- PCR), or splinted ligation (e.g., with T4 DNA ligase, derived from SRSLY-seq).
  • a template-switch reaction e.g., with MaximaH-Minus reverse transcriptase, derived from SMART seq, 10x Genomics
  • ssRNA ligation e.g., with T4 RNA ligase K227Q,
  • the decrease or absence of the identifier sequence in a condition indicates that in the lumen of an endosome of a cell relative to a reference level (e.g., the level or presence of the identifier sequence in a condition (e.g., a pH) similar to a cytoplasmic pH of a cell) indicates that an associated test conformation-specific binding agent enables release of a nucleoprotein in the endosome of a cell (e.g., a target cell).
  • Identification of the test conformation-specific binding agent may be based on a previously established reference or index establishing an association between the uiNA and the test conformation-specific binding agent.
  • the target cell(s) is a eukaryotic cell, such as a mammalian cell (e.g., a human cell).
  • the target cells are hematopoietic stem cells (HSCs), hematopoietic progenitor stem cells (HPSCs), natural killer cells, macrophages, DC cells, non-DC myeloid cells, B cells, T cells (e.g., activated T cells), fibroblasts, ocular cells, stromal cells, or other cells.
  • the target cells are T cells.
  • the T cells are CD4 or CD8 T cells.
  • the T cells are regulatory T cells (T regs) or effector T cells.
  • the T cells are tumor infiltrating T cells.
  • the target cell is a hematopoietic stem cell (HSC) or a hematopoietic progenitor cells (HPSCs).
  • the macrophages are M0, M1 , or M2 macrophages.
  • the target cells are diseased cells. In certain embodiments, the target cells are tumor cells.
  • complexes comprising a conformation-specific NP binding agent and a nucleoprotein can be assessed for cell targeting capacity and/or nuclear internalization capacity by contacting (e.g., co incubating) the nucleoproteins with multiple (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) target cells, such as multiple target cells selected from hematopoietic stem cells (HSCs), hematopoietic progenitor stem cells (HPSCs), natural killer cells, macrophages (e.g., M0, M1 , or M2 macrophages), DC, non-DC myeloid cells, B cells, T cells (e.g., activated T cells, CD4 T cells, CD8 T cells, T regs, effector T cells, and/or tumor infiltrating T cells), fibroblasts, ocular cells, stromal
  • isolated nucleoproteins comprising a nucleic acid- guided nuclease fusion protein and a uiNA, can be assessed for cell targeting capacity and/or nuclear internalization capacity by contacting, such as co-incubating the nucleoproteins with multiple populations of target cells, such as a population of T cells and a population of macrophages.
  • the cells can be in any conditions or cell media suitable for cell viability. Further, the cells may be attached to a surface or suspended in cell media. After contacting nucleoproteins with a target cell, nucleic acids inside the target cell can then be assessed to identify internalized uiNAs.
  • the present invention includes a targeted active gene editor (TAGE) that is useful for delivering a gene editing polypeptide (i.e. , a nucleic acid-guided nuclease) to a target cell.
  • TAGE targeted active gene editor
  • the TAGE provided herein include a conformation-specific nucleoprotein (NP) binding agent comprising a cell targeting agent, alternatively referred to herein as an extracellular cell membrane binding moiety.
  • NP nucleoprotein
  • the nucleoprotein is stably associated with the cell targeting agent via the conformation-specific NP binding agent.
  • the conformation-specific NP binding agent can be any such protein known in the art, such as those described herein in Section II.
  • the conformation-specific NP binding agent is an anti-CRISPR (Acr) protein, such as one further described herein.
  • the Acr protein can be AcrllA4 or AcrVAI .
  • the conformation-specific NP binding agent may be an antibody, or antigen binding fragment thereof, that specifically binds the NP.
  • the conformation-specific NP binding agent may be an aptamer that specifically binds the NP.
  • the conformation-specific NP binding agent can comprise one or more modifications (e.g., an amino acid substitution, deletion, or insertion) relative to a naturally occurring conformation-specific NP binding agent (e.g., Acr protein, such as AcrllA4 (SEQ ID NO: 1) or AcrVAI ) or one further described herein.
  • a naturally occurring conformation-specific NP binding agent e.g., Acr protein, such as AcrllA4 (SEQ ID NO: 1) or AcrVAI
  • the conformation-specific NP binding agent has at least 50, 60, 70, 75, 80, 85, 90, 95, 98%, or 99% sequence identity to a conformation-specific NP binding agent described herein (e.g., SEQ ID NO: 1).
  • the conformation-specific NP binding agent e.g., a mutagenized variant of a conformation-specific NP binding agent, e.g., an anti-CRISPR (ACR) protein, such as AcrllA4 or AcrVAI (Uniprot Accession Nos. A0A5H1 ZR47 or A0A5H1 ZR46)
  • ACR anti-CRISPR
  • the TAGE can be a biologic.
  • the conformation-specific NP binding agent may be conjugated to the cell targeting agent by a variety of means, such as those described herein.
  • the conformation-specific NP binding agent includes a conjugation moiety that allows the protein to be conjugated to a cell targeting agent (i.e., an extracellular cell membrane binding moiety, such as an antigen binding agent, ligand, or cell penetrating peptide (CPP), or combinations thereof).
  • a conjugation moiety that allows the protein to be conjugated to a cell targeting agent (i.e., an extracellular cell membrane binding moiety, such as an antigen binding agent, ligand, or cell penetrating peptide (CPP), or combinations thereof).
  • the cell targeting agent may be one that specifically binds to an extracellular target molecule (e.g., an extracellular protein, lipid, or glycan) displayed on a cell membrane or cell surface, such as an antigen associated with the extracellular region of a cell membrane or otherwise increases cellular or nuclear internalization of the site-directed modifying polypeptide.
  • an extracellular target molecule e.g., an extracellular protein, lipid, or glycan
  • this target specificity allows for delivery of the site-directed modifying polypeptide only to cells displaying the antigen (e.g., hematopoietic stem cells (HSCs), hematopoietic progenitor stem cells (HPSCs), natural killer cells, macrophages, DC cells, non-DC myeloid cells, B cells, T cells (e.g., activated T cells), fibroblasts, or other cells).
  • HSCs hematopoietic stem cells
  • HPSCs hematopoietic progenitor stem cells
  • natural killer cells e.g., macrophages, DC cells, non-DC myeloid cells, B cells, T cells (e.g., activated T cells), fibroblasts, or other cells.
  • TAGE thus provides a means by which the genome of a target cell can be modified.
  • a TAGE comprises a nucleic acid-guided endonuclease (e.g., RNA- guided endonuclease or DNA-guided endonuclease), such as Cas9, that recognizes a CRISPR sequence, a conformation-specific NP binding agent, and an antigen binding agent that specifically binds to an extracellular molecule (e.g., protein, glycan, lipid) localized on a target cell membrane.
  • nucleic acid-guided endonuclease e.g., RNA- guided endonuclease or DNA-guided endonuclease
  • Cas9 RNA- guided endonuclease
  • an antigen binding agent that specifically binds to an extracellular molecule localized on a target cell membrane.
  • antigen binding agents include, but are not limited to, an antibody, an antigen-binding portion of an antibody, or an antibody mimetic. The types of antigen binding agents that can be used
  • Proteins within the TAGE are stably associated via a conformation- specific NP binding agent such that the cell targeting agent directs the site-directed modifying polypeptide (e.g., nucleic acid-guided nuclease) to the cell surface and the site-directed modifying polypeptide (e.g., nucleic acid-guided nuclease) is internalized into the target cell.
  • site-directed modifying polypeptide e.g., nucleic acid-guided nuclease
  • the site-directed modifying polypeptide e.g., nucleic acid-guided nuclease
  • the cell targeting agent binds to the antigen on the cell surface such that the site- directed modifying polypeptide (e.g., nucleic acid-guided nuclease) is internalized by the target cell but the antigen binding agent is not internalized.
  • the site-directed modifying polypeptide e.g., nucleic acid-guided nuclease
  • the conformation-specific NP binding agent, and the cell targeting agent are internalized into the target cell.
  • cell targeting agents include, but are not limited to, an antigen binding polypeptide such as an antibody or fragment thereof, a ligand, or a CPP.
  • a TAGE includes a two or more cell membrane binding agents, e.g., a CPP and an antibody.
  • Such class pairings can, in certain embodiments, improve internalization of the site-directed modifying polypeptide.
  • a class pairing includes a TAGE comprising a CPP, an antigen binding polypeptide (e.g., an antibody), a conformation-specific NP binding agent, and a site-directed modifying polypeptide, in any arrangement.
  • a TAGE comprises an antibody, a peptide cell surface TCR, a conformation-specific NP binding agent, and a site-directed modifying polypeptide, in any arrangement.
  • the site-directed modifying polypeptide is a nucleic acid-guided endonuclease, such as Cas9
  • the nucleic acid-guided endonuclease is associated with a guide nucleic acid to form a nucleoprotein.
  • the guide RNA binds to an RNA-guided nuclease to form a ribonucleoprotein (RNP) or a guide DNA binds to a DNA-guided nuclease to form a deoxyribonucleoprotein (DNP).
  • the nucleic acid-guided endonuclease is associated with a guide nucleic acid that comprises a DNA:RNA hybrid.
  • the ribonucleoprotein i.e., the RNA-guided endonuclease and the guide RNA
  • deoxyribonucleoprotein i.e., the DNA-guided endonuclease and the guide DNA
  • the nucleic acid-guided endonuclease bound to a DNA:RNA hybrid guide are internalized into the target cell.
  • the guide nucleic acid (e.g., RNA, DNA, or DNA:RNA hybrid) is delivered to the target cell separately from the nucleic acid-guided endonuclease into the same cell.
  • the guide nucleic acid e.g., RNA, DNA, or DNA:RNA hybrid
  • a TAGE specifically binds to an extracellular molecule (e.g., protein, glycan, lipid) localized on a target cell membrane.
  • the target molecule can be, for example, an extracellular membrane-bound protein, but can also be a non-protein molecule such as a glycan or lipid.
  • the extracellular molecule is an extracellular protein that is expressed on the surface of the target cell, such as a ligand or a receptor.
  • the extracellular target molecule may be associated with a specific disease condition or a specific tissue within in an organism. Examples of extracellular molecular targets associated with the cell membrane are described in the sections below.
  • the conformation-specific NP binding agent comprises a conjugation moiety such that a cell targeting agent can stably associate with the conformation-specific NP binding agent, which can in turn associate with a site-directed modifying polypeptide, such as a nucleic acid-guided nuclease (thus forming a TAGE).
  • the conjugation moiety provides for either a covalent or a non-covalent linkage between the cell targeting agent and the conformation-specific NP binding agent.
  • the conjugation moiety useful for the present TAGEs are stable extracellularly, prevent aggregation of TAGE molecules, and/or keep TAGEs freely soluble in aqueous media and in a defined oligomeric state.
  • the TAGE Before transport or delivery into a cell, the TAGE is stable and remains intact, e.g., the cell targeting agent remains linked to the conformation-specific NP binding agent.
  • the conjugation moiety is Protein A, wherein the conformation-specific NP binding agent comprises Protein A and the cell targeting agent, e.g., an antigen binding agent, comprises an Fc region that can be bound by Protein A, e.g., an antibody comprising an Fc domain.
  • a conformation-specific NP binding agent comprises Protein A as described in the Sequence Table, or an Fc binding portion thereof.
  • conjugation moieties include, but are not limited to, a Spycatcher tag, Snoop tag, Halo- tag (e.g., derived from haloalkane dehalogenase), Sortase, mono-avidin, ACP tag, a SNAP tag, or any other conjugation moieties known in the art.
  • the conjugation moiety is selected from Protein A, CBP, MBP, GST, poly(His), biotin/streptavidin, V5-tag, Myc-tag, HA-tag, NE- tag, His-tag, Flag tag, Halo-tag, Snap- tag, Fc-tag, Nus-tag, BCCP, thioredoxin, SnooprTag, SpyTag, SpyCatcher, Isopeptag, SBP-tag, S- tag, AviTag, and calmodulin.
  • the conjugation moiety is a chemical tag.
  • a chemical tag may be SNAP tag, a CLIP tag, a HaloTag (e.g., derived from haloalkane dehalogenase) or a TMP- tag.
  • the chemical tag is a SNAP-tag or a CLIP-tag.
  • SNAP and CLIP fusion proteins enable the specific, covalent attachment of virtually any molecule to a protein of interest.
  • the chemical tag is a HaloTag.
  • HaloTag involves a modular protein tagging system that allows different molecules to be linked onto a single genetic fusion, either in solution, in living cells, or in chemically fixed cells.
  • the chemical tag is a TMP-tag.
  • the conjugation moiety is an epitope tag.
  • an epitope tag may be a poly-histidine tag such as a hexahistidine tag or a dodecahistidine, a FLAG tag, a Myc tag, a HA tag, a GST tag or a V5 tag.
  • the conformation-specific NP binding agent and the cell targeting agent may each be engineered to comprise complementary binding pairs that enable stable association upon contact.
  • Exemplary binding moiety pairings include (i) streptavidin-binding peptide (streptavidin binding peptide; SBP) and streptavidin (STV), (ii) biotin and EMA (enhanced monomeric avidin), (iii) SpyTag (ST) and SpyCatcher (SC), (iv) Halo-tag and Halo-tag ligand, (v) and SNAP-Tag , (vi) Myc tag and anti-Myc immunoglobulins (vii) FLAG tag and anti-FLAG immunoglobulins, and (ix) ybbR tag and coenzyme A groups.
  • the conjugation moiety is selected from SBP, biotin, SpyTag, SpyCatcher, halo-tag, SNAP-tag, Myc tag, or FLAG tag.
  • the conformation-specific NP binding agent can alternatively be associated with a cell targeting agent via one or more linkers as described herein.
  • linker means a divalent chemical moiety comprising a covalent bond or a chain of atoms that covalently attaches a cell targeting agent to a conformation-specific NP binding agent. Any known method of conjugation of peptides or macromolecules can be used in the context of the present disclosure. Generally, covalent attachment of the cell targeting agent and the conformation-specific NP binding agent requires the linker to have two reactive functional groups, i.e. , bivalency in a reactive sense.
  • Bivalent linker reagents which are useful to attach two or more functional or biologically active moieties, such as peptides, nucleic acids, drugs, toxins, antibodies, haptens, and reporter groups are known, and methods for such conjugation have been described in, for example, Hermanson, G. T. (1996) Bioconjugate Techniques; Academic Press: New York, p234- 242, the disclosure of which is incorporated herein by reference as it pertains to linkers suitable for covalent conjugation. Further linkers are disclosed in, for example, Tsuchikama, K. and Zhiqiang, A. Protein and Cell, 9(1), p.33-46, (2016), the disclosure of which is incorporated herein by reference as it pertains to linkers suitable for covalent conjugation.
  • linkers suitable for use in the compositions and methods disclosed are stable in circulation, but allow for release of the cell targeting agent and/or the site-directed modifying polypeptide in the target cell or, alternatively, in close proximity to the target cell.
  • Linkers suitable for the present disclosure may be broadly categorized as non-cleavable or cleavable, as well as intracellular or extracellular, each of which is further described herein below.
  • the linker conjugating the cell targeting agent and the conformation- specific NP binding agent is non-cleavable.
  • Non-cleavable linkers comprise stable chemical bonds that are resistant to degradation (e.g., proteolysis). Generally, non-cleavable linkers require proteolytic degradation inside the target cell, and exhibit high extracellular stability.
  • heteroatoms e.g., S, N, or O
  • Non-limiting examples of non-cleavable linker utilized in antibody-drug conjugation include those based on maleimidomethylcyclohexanecarboxylate, caproylmaleimide, and acetylphenylbutanoic acid.
  • the linker conjugating the cell targeting agent and the conformation- specific NP binding agent is cleavable, such that cleavage of the linker (e.g., by a protease, such as metalloproteases) releases the conformation-specific NP binding agent from the TAGE in the intracellular or extracellular (e.g., upon binding of the molecule to the cell surface) environment.
  • a protease such as metalloproteases
  • Cleavable linkers are designed to exploit the differences in local environments, e.g., extracellular and intracellular environments, for example, pH, reduction potential or enzyme concentration, to trigger the release of an TAGE component (i.e., the cell targeting agent, the conformation-specific NP binding agent, or the nucleic acid-guided nuclease) in the target cell.
  • TAGE component i.e., the cell targeting agent, the conformation-specific NP binding agent, or the nucleic acid-guided nuclease
  • cleavable linkers are relatively stable in circulation in vivo, but are particularly susceptible to cleavage in the intracellular environment through one or more mechanisms (e.g., including, but not limited to, activity of proteases, peptidases, and glucuronidases).
  • Cleavable linkers used herein are stable outside the target cell and may be cleaved at some efficacious rate inside the target cell or in close proximity to the extracellular membrane of the target cell.
  • An effective linker will: (i) maintain the specific binding properties of the cell targeting agent;
  • TAGE intra- or extracellular delivery of the TAGE or a component thereof (i.e., the conformation- specific NP binding agent and the nucleic acid-guided nuclease); (iii) remain stable and intact, i.e. not cleaved, until the TAGE has been delivered or transported to its targeted site; and (iv) maintain the gene targeting effect of the nucleic acid-guided nuclease.
  • Stability of the TAGE may be measured by standard analytical techniques such as mass spectroscopy, size determination by size exclusion chromatography or diffusion constant measurement by dynamic light scattering, HPLC, and the separation/analysis technique LC/MS.
  • Suitable cleavable linkers include those that may be cleaved, for instance, by enzymatic hydrolysis, photolysis, hydrolysis under acidic conditions, hydrolysis under basic conditions, oxidation, disulfide reduction, nucleophilic cleavage, or organometallic cleavage (see, for example, Leriche et al. , Bioorg. Med. Chem., 20:571-582, 2012, the disclosure of which is incorporated herein by reference as it pertains to linkers suitable for covalent conjugation).
  • Suitable cleavable linkers may include, for example, chemical moieties such as a hydrazine, a disulfide, a thioether or a peptide.
  • Linkers hydrolyzable under acidic conditions include, for example, hydrazones, semicarbazones, thiosemicarbazones, cis-aconitic amides, orthoesters, acetals, ketals, or the like.
  • hydrazones include, for example, hydrazones, semicarbazones, thiosemicarbazones, cis-aconitic amides, orthoesters, acetals, ketals, or the like.
  • linkers are relatively stable under neutral pH conditions, such as those in the blood, but are unstable at below pH 5.5 or 5.0, the approximate pH of the lysosome.
  • linkers including such acid-labile functionalities tend to be relatively less stable extracellularly. This lower stability may be advantageous where extracellular cleavage is desired.
  • Linkers cleavable under reducing conditions include, for example, a disulfide.
  • a variety of disulfide linkers are known in the art, including, for example, those that can be formed using SATA (N- succinimidyl-S-acetylthioacetate), SPDP (N-succinimidyl-3-(2-pyridyldithio)propionate), SPDB (N- succinimidyl-3-(2-pyridyldithio)butyrate) and SMPT (N-succinimidyl-oxycarbonyl-alpha-methyl-alpha-(2- pyridyl-dithio)toluene), SPDB and SMPT (See, e.g., Thorpe et al., 1987, Cancer Res.
  • Linkers susceptible to enzymatic hydrolysis can be, e.g., a peptide-containing linker that is cleaved by an intracellular peptidase or protease enzyme, including, but not limited to, a lysosomal or endosomal protease.
  • the peptidyl linker is at least two amino acids long or at least three amino acids long.
  • Exemplary amino acid linkers include a dipeptide, a tripeptide, a tetrapeptide or a pentapeptide.
  • suitable peptides include those containing amino acids such as Valine, Alanine, Citrulline (Cit), Phenylalanine, Lysine, Leucine, and Glycine.
  • Amino acid residues which comprise an amino acid linker component include those occurring naturally, as well as minor amino acids and non-naturally occurring amino acid analogs, such as citrulline.
  • Exemplary dipeptides include valine-citrulline (vc or val-cit) and alanine-phenylalanine (af or ala-phe).
  • Exemplary tripeptides include glycine-valine-citrulline (gly-val-cit) and glycine-glycine-glycine (gly-gly-gly).
  • the linker includes a dipeptide such as Val-Cit, Ala-Val, or Phe-Lys, Val-Lys, Ala-Lys, Phe- Cit, Leu-Cit, lle-Cit, Phe-Arg, or Trp-Cit.
  • Linkers containing dipeptides such as Val-Cit or Phe-Lys are disclosed in, for example, U.S. Pat. No. 6,214,345, the disclosure of which is incorporated herein by reference in its entirety as it pertains to linkers suitable for covalent conjugation.
  • the linker includes a dipeptide selected from Val-Ala and Val-Cit.
  • linkers comprising a peptide moiety may be susceptible to varying degrees of cleavage both intra- and extracellularly. Accordingly, in some embodiments, the linker comprises a dipeptide, and the TAGE is substantially cleaved extracellularly. Accordingly, in some embodiments, the linker comprises a dipeptide, and the TAGE is stable extracellularly and is cleaved intracellularly.
  • Linkers suitable for conjugating the cell targeting agent as disclosed herein to a conformation- specific NP binding agent, as disclosed herein, include those capable of releasing the cell targeting agent by a 1 ,6-elimination process.
  • Chemical moieties capable of this elimination process include the p- aminobenzyl (PAB) group, 6-maleimidohexanoic acid, pH-sensitive carbonates, and other reagents as described in Jain et al. , Pharm. Res. 32:3526-3540, 2015, the disclosure of which is incorporated herein by reference in its entirety as it pertains to linkers suitable for covalent conjugation.
  • PAB p- aminobenzyl
  • the linker includes a "self-immolative" group such as the afore mentioned PAB or PABC (para-aminobenzyloxycarbonyl), which are disclosed in, for example, Carl et al., J. Med. Chem. (1981 ) 24:479-480; Chakravarty et al (1983) J. Med. Chem.
  • a dipeptide is used in combination with a self-immolative linker.
  • Linkers suitable for use herein further may include one or more groups selected from C1-C6 alkylene, C1-C6 heteroalkylene, C2-C6 alkenylene, C2-C6 heteroalkenylene, C2-C6alkynylene, C2-C6 heteroalkynylene, C3-C6cycloalkylene, heterocycloalkylene, arylene, heteroarylene, and combinations thereof, each of which may be optionally substituted.
  • the linker includes a p-aminobenzyl group (PAB).
  • PAB p-aminobenzyl group
  • the p-aminobenzyl group is disposed between the cytotoxic drug and a protease cleavage site in the linker.
  • the p-aminobenzyl group is part of a p-aminobenzyloxycarbonyl unit.
  • the p-aminobenzyl group is part of a p-aminobenzylamido unit.
  • the linker comprises PAB, Val-Cit-PAB, Val-Ala- PAB, Val-Lys(Ac)-PAB, Phe-Lys-PAB, Phe-Lys(Ac)-PAB, D-Val-Leu-Lys, Gly-Gly-Arg, Ala-Ala-Asn-PAB, or Ala-PAB.
  • the linker comprises a combination of one or more of a peptide, oligosaccharide, -(CH 2 ) P -, -(CH 2 CH 2 0) P -, PAB, Val-Cit-PAB, Val-Ala-PAB, Val- Lys(Ac)-PAB, Phe-Lys-PAB, Phe-Lys(Ac)-PAB, D-Val-Leu-Lys, Gly-Gly-Arg, Ala-Ala-Asn-PAB, or Ala- PAB.
  • Suitable linkers may be substituted with groups which modulate solubility or reactivity. Suitable linkers may contain groups having solubility enhancing properties. Linkers including the (CH 2 CH 2 0) unit (polyethylene glycol, PEG), for example, can enhance solubility, as can alkyl chains substituted with amino, sulfonic acid, phosphonic acid or phosphoric acid residues. Linkers including such moieties are disclosed in, for example, U.S. Patent Nos. 8,236,319 and 9,504,756, the disclosure of each of which is incorporated herein by reference as it pertains to linkers suitable for covalent conjugation. Linkers containing such groups are described, for example, in U.S. Patent No. 9,636,421 and U.S. Patent Application Publication No. 2017/0298145, the disclosures of which are incorporated herein by reference as they pertain to linkers suitable for covalent conjugation.
  • Suitable linkers for covalently conjugating moiety cell targeting agent and a conformation-specific NP binding agent as disclosed herein can have two reactive functional groups (i.e. , two reactive termini), one for conjugation to the cell targeting agent, and the other for conjugation to the conformation-specific NP binding agent.
  • Suitable sites for conjugation on the cell targeting agent are, in certain embodiments, nucleophilic, such as a thiol, amino group, or hydroxyl group.
  • Reactive (e.g., nucleophilic) sites that may be present within an cell targeting agent as disclosed herein include, without limitation, nucleophilic substituents on amino acid residues such as (i) N-terminal amine groups, (ii) side chain amine groups, e.g.
  • lysine (iii) side chain thiol groups, e.g. cysteine, (iv) side chain hydroxyl groups, e.g. serine; or (iv) sugar hydroxyl or amino groups where the cell targeting agent is glycosylated.
  • Suitable sites for conjugation on the cell targeting agent include, without limitation, hydroxyl moieties of serine, threonine, and tyrosine residues; amino moieties of lysine residues; carboxyl moieties of aspartic acid and glutamic acid residues; and thiol moieties of cysteine residues, as well as propargyl, azido, haloaryl (e.g., fluoroaryl), haloheteroaryl (e.g., fluoroheteroaryl), haloalkyl, and haloheteroalkyl moieties of non-naturally occurring amino acids.
  • the antibody conjugation reactive terminus on the linker is, in certain embodiments, a thiol-reactive group such as a double bond (as in maleimide), a leaving group such as a chloro, bromo, iodo, or an R-sulfanyl group, or a carboxyl group.
  • a thiol-reactive group such as a double bond (as in maleimide)
  • a leaving group such as a chloro, bromo, iodo, or an R-sulfanyl group, or a carboxyl group.
  • Suitable sites for conjugation on the conformation-specific NP binding agent can also be, in certain embodiments, nucleophilic.
  • Reactive (e.g., nucleophilic) sites that may be present within conformation-specific NP binding agent as disclosed herein include, without limitation, nucleophilic substituents on amino acid residues such as (i) N-terminal amine groups, (ii) side chain amine groups, e.g. lysine, (iii) side chain thiol groups, e.g. cysteine, (iv) side chain hydroxyl groups, e.g. serine; or (iv) sugar hydroxyl or amino groups where the antibody is glycosylated.
  • Suitable sites for conjugation on the conformation-specific NP binding agent include, without limitation, hydroxyl moieties of serine, threonine, and tyrosine residues; amino moieties of lysine residues; carboxyl moieties of aspartic acid and glutamic acid residues; and thiol moieties of cysteine residues, as well as propargyl, azido, haloaryl (e.g., fluoroaryl), haloheteroaryl (e.g., fluoroheteroaryl), haloalkyl, and haloheteroalkyl moieties of non-naturally occurring amino acids.
  • haloaryl e.g., fluoroaryl
  • haloheteroaryl e.g., fluoroheteroaryl
  • haloalkyl e.g., fluoroheteroaryl
  • haloheteroalkyl e.g.,
  • the conformation-specific NP binding agent conjugation reactive terminus on the linker is, in certain embodiments, a thiol-reactive group such as a double bond (as in maleimide), a leaving group such as a chloro, bromo, iodo, or an R-sulfanyl group, or a carboxyl group.
  • the reactive functional group attached to the linker is a nucleophilic group which is reactive with an electrophilic group present on a cell targeting agent, the conformation-specific NP binding agent, or both.
  • Useful electrophilic groups on a cell targeting agent or conformation-specific NP binding agent include, but are not limited to, aldehyde and ketone carbonyl groups.
  • the heteroatom of a nucleophilic group can react with an electrophilic group on a cell targeting agent or conformation- specific NP binding agent and form a covalent bond to the cell targeting agent or conformation-specific NP binding agent.
  • nucleophilic groups include, but are not limited to, hydrazide, oxime, amino, hydroxyl, hydrazine, thiosemicarbazone, hydrazine carboxylate, and arylhydrazide.
  • the TAGE as disclosed herein comprises a nucleoside or a nucleotide. Suitable sites for conjugation on such nucleosides or nucleotides include -OH or phosphate groups, respectively.
  • Linkers and conjugation methods suitable for use in such embodiments are disclosed in, for example, Wang, T.P., et al. , Bioconj. Chem. 21 (9), 1642-55, 2010, and Bernardinelli, G. and Hogberg, B. Nucleic Acids Research, 45(18), p. e160; published online 16 August, 2017, the disclosure of each of which is incorporated herein by reference as it pertains to linkers suitable for covalent conjugation.
  • linker When the term "linker” is used in describing the linker in conjugated form, one or both of the reactive termini will be absent, (having been converted to a chemical moiety) or incomplete (such as being only the carbonyl of a carboxylic acid) because of the formation of the bonds between the linker and the cell targeting agent, and/or between the linker and the conformation-specific NP binding agent.
  • linkers useful herein include, without limitation, linkers containing a chemical moiety formed by a coupling reaction between a reactive functional group on the linker and a nucleophilic group or otherwise reactive substituent on the cell targeting agent, and a chemical moiety formed by a coupling reaction between a reactive functional group on the linker and a nucleophilic group on the conformation- specific NP binding agent.
  • Examples of chemical moieties formed by these coupling reactions result from reactions between chemically reactive functional groups, including a nucleophile/electrophile pair (e.g., a thiol/haloalkyl pair, an amine/carbonyl pair, or a thiol/DaDpD-unsaturated carbonyl pair, and the like), a diene/dienophile pair (e.g., an azide/alkyne pair, or a diene/ aDpDunsaturated carbonyl pair, among others), and the like.
  • a nucleophile/electrophile pair e.g., a thiol/haloalkyl pair, an amine/carbonyl pair, or a thiol/DaDpD-unsaturated carbonyl pair, and the like
  • a diene/dienophile pair e.g., an azide/alkyne pair, or a diene/ aD
  • Coupling reactions between the reactive functional groups to form the chemical moiety include, without limitation, thiol alkylation, hydroxyl alkylation, amine alkylation, amine or hydroxylamine condensation, hydrazine formation, amidation, esterification, disulfide formation, cycloaddition (e.g., [4+2] Diels-Alder cycloaddition, [3+2] Huisgen cycloaddition, among others), nucleophilic aromatic substitution, electrophilic aromatic substitution, and other reactive modalities known in the art or described herein.
  • Suitable linkers may contain an electrophilic functional group for reaction with a nucleophilic functional group on the cell targeting agent, the conformation-specific NP binding agent, or both.
  • the reactive functional group present within the cell targeting agent, the conformation-specific NP binding agent, or both as disclosed herein are amine or thiol moieties.
  • Certain cell targeting agents have reducible interchain disulfides, i.e. cysteine bridges.
  • Cell targeting agents may be made reactive for conjugation with linker reagents by treatment with a reducing agent such as DTT (dithiothreitol). Each cysteine bridge will thus form, theoretically, two reactive thiol nucleophiles.
  • Additional nucleophilic groups can be introduced into the cell targeting agent through the reaction of lysines with 2-iminothiolane (Traut's reagent) resulting in conversion of an amine into a thiol.
  • Reactive thiol groups may be introduced into the cell targeting agent by introducing one, two, three, four, or more cysteine residues (e.g., preparing mutant antibodies comprising one or more non-native cysteine amino acid residues).
  • U.S. Pat. No. 7,521 ,541 teaches engineering antibodies by introduction of reactive cysteine amino acids.
  • Linkers suitable for the synthesis of the covalent conjugates as disclosed herein include, without limitation, reactive functional groups such as maleimide or a haloalkyl group. These groups may be present in linkers or cross linking reagents such as succinimidyl 4-(N-maleimidomethyl)-cyclohexane-L- carboxylate (SMCC), N-succinimidyl iodoacetate (SIA), sulfo-SMCC, m-maleimidobenzoyl-A/- hydroxysuccinimidyl ester (MBS), sulfo-MBS, and succinimidyl iodoacetate, among others described, in for instance, Liu et al., 18:690-697, 1979, the disclosure of which is incorporated herein by reference as it pertains to linkers for chemical conjugation.
  • SMCC succinimidyl 4-(N-maleimidomethyl)-cyclohexane-L- carboxylate
  • one or both of the reactive functional groups attached to the linker is a maleimide, azide, or alkyne.
  • a maleimide-containing linker is the non-cleavable maleimidocaproyl-based linker.
  • linkers are described by Doronina et al., Bioconjugate Chem. 17:14-24, 2006, the disclosure of which is incorporated herein by reference as it pertains to linkers for chemical conjugation.
  • the reactive functional group is an N-maleimidyl group, halogenated N- alkylamido group, sulfonyloxy N-alkylamido group, carbonate group, sulfonyl halide group, thiol group or derivative thereof, alkynyl group comprising an internal carbon-carbon triple bond, (het-ero)cycloalkynyl group, bicyclo[6.1 0]non-4-yn-9-yl group, alkenyl group comprising an internal carbon-carbon double bond, cycloalkenyl group, tetrazinyl group, azido group, phosphine group, nitrile oxide group, nitrone group, nitrile imine group, diazo group, ketone group, (O-alkyl)hydroxylamino group, hydrazine group, halogenated N-maleimidyl group, 1 ,1 -bis (sulfonylmethyl)methylcarbonyl
  • Suitable bivalent linker reagents suitable for preparing conjugates as disclosed herein include, but are not limited to, N-succinimidyl 4-(maleimidomethyl)cyclohexanecarboxylate (SMCC), N-succinimidyl-4-(N-maleimidomethyl)-cyclohexane-1-carboxy-(6-amidocaproate), which is a “long chain” analog of SMCC (LC-SMCC), k-maleimidoundecanoic acid N-succinimidyl ester (KMUA), y- maleimidobutyric acid N-succinimidyl ester (GMBS), e-maleimidocaproic acid N-hydroxysuccinimide ester (EMCS), m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS), N-(a-maleimidoacetoxy)- succinimide ester (AMAS), succinimidyl-6
  • Cross-linking reagents comprising a haloacetyl-based moiety include N-succinimidyl-4-(iodoacetyl)-aminobenzoate (SIAB), N-succinimidyl iodoacetate (SIA), N-succinimidyl bromoacetate (SBA), and N-succinimidyl 3- (bromoacetamido)propionate (SBAP).
  • SIAB N-succinimidyl-4-(iodoacetyl)-aminobenzoate
  • SIA N-succinimidyl iodoacetate
  • SBA N-succinimidyl bromoacetate
  • SBAP N-succinimidyl 3- (bromoacetamido)propionate
  • any one or more of the chemical groups, moieties and features disclosed herein may be combined in multiple ways to form linkers useful for conjugation of the cell targeting agent as disclosed herein to a conformation-specific NP binding agent, as disclosed herein.
  • Further linkers useful in conjunction with the compositions and methods described herein, are described, for example, in U.S. Patent Application Publication No. 2015/0218220, the disclosure of which is incorporated herein by reference as is pertain to linkers suitable for covalent conjugation.
  • agents that can be used as the cell targeting agent of a TAGE include, but are not limited to, an antigen binding polypeptide, such as an antibody, a cell penetrating peptide (CPP), a ligand, or any combinations thereof. More detail regarding these cell targeting agents is provided below. Further, extracellular cell membrane binding moieties, such as ligands and antigen-binding polypeptides, not only allow for receptor-mediated entry of TAGE agents, but in certain instances, the moieties also mediate the biology of the cell (e.g., by altering intracellular signal transduction pathways), which can be exploited for therapeutic uses. (i) Antigen Binding Polypeptides
  • An antigen binding polypeptide targets an extracellular antigen associated with a cell membrane and provide specificity with which to deliver a conformation-specific NP binding agent stably associated with a nucleic acid-guided nuclease and a guide nucleic acid.
  • antigen binding polypeptides that may be included in the TAGE described herein include, but are not limited to, an antibody, an antigen-binding fragment of an antibody, or an antibody mimetic.
  • a TAGE as provided herein comprises an antigen binding polypeptide that is an antibody, or an antigen-binding fragment thereof, that specifically binds to an extracellular molecule (e.g., protein, glycan, lipid) localized on a target cell membrane or associated with a specific tissue.
  • the extracellular molecule specifically bound by the antibody, or antigen binding fragment thereof can be an antigen, such as, but not limited to, HLA-DR, CD3, CD20, CD22, CD25, CD32, CD33, CD44, CD47, CD54, CD59, CD70, CD74, AchR, CTLA4, CXCR4, EGFR, Her2, EpCam, PD-1 , or FAP1 .
  • the antigen is CD22.
  • the antibody or antigen binding portion thereof specifically binds to CD3.
  • Other exemplary targets for the antibody, antigen-binding fragment thereof, in the TAGE of the present invention include: (i) tumor- associated antigens; (ii) cell surface receptors, (iii) CD proteins and their ligands, such as CD3, CD4, CD8, CD19, CD20, CD22, CD25, CD32, CD33, CD34, CD40, CD44, CD47, CD54, CD59, CD70, CD74, CD79a (CD79a), and CD79P (CD79b); (iv) members of the ErbB receptor family such as the EGF receptor, HER2, HER3 or HER4 receptor; (v) cell adhesion molecules such as LFA-1 , Mac1 , p150,95, VLA-4, ICAM-1 , VCAM and an/b3 integrin including either alpha or beta subunits thereof (e.g.
  • anti-CD11 a, anti-CD18 or anti-CD11 b antibodies include growth factors such as VEGF; IgE; blood group antigens; flk2/flt3 receptor; obesity (OB) receptor; mpl receptor; CTLA4; protein C, BR3, c-met, tissue factor, b7 etc.
  • growth factors such as VEGF; IgE; blood group antigens; flk2/flt3 receptor; obesity (OB) receptor; mpl receptor; CTLA4; protein C, BR3, c-met, tissue factor, b7 etc.
  • antigens that can be targeted by the antibody, or an antigen-binding fragment thereof, include cell surface receptors such as those described in Chen and Flies. Nature reviews immunology. 13.4 (2013): 227, which is incorporated herein by reference.
  • Antigen binding polypeptides used in the TAGE agents described herein may also be specific to a certain cell type.
  • an antigen binding polypeptide such as an antibody or antigen binding portion thereof, may bind to an antigen present on the cell surface of a hematopoietic cell (HSC).
  • HSC hematopoietic cell
  • antigens found on HSCs include, but are not limited to, CD34, EMCN, CD59, CD90, c-KIT, CD45, or CD49F.
  • cell types that may be bound by the antigen binding polypeptide via an antigen expressed or displayed on the cell’s extracellular surface, and thus gene edited by the TAGE agent, include a neutrophil, a T cell, a B cell, a dendritic cell, a macrophage, and a fibroblast.
  • Exemplary antibodies include those selected from, and without limitation, an anti-HLA-DR antibody, an anti-CD3 antibody, an anti-CD20 antibody, an anti- CD22 antibody, an anti-CD1a antibody, an anti-CD25 antibody, an anti-CD32 antibody, an anti-CD33 antibody, an anti-CD44 antibody, an anti-CD47 antibody, an anti-CD54 antibody, an anti-CD59 antibody, an anti-CD70 antibody, an anti-CD74 antibody, an anti-AchR antibody, an anti-CTLA4 antibody, an anti-CXCR4 antibody, an anti-EGFR antibody, an anti-Her2 antibody, an anti-EpCam antibody, an-anti-PD-1 antibody, or an anti-FAP1 antibody.
  • the TAGE includes an antigen binding polypeptide that is anti-CD22 antibody, or antigen-binding fragment thereof.
  • the anti-CD22 antibody is selected from epratuzumab (also known as hl_22, see, e.g., US Pat. No. 5789554; US. App. No. 20120302739; sold by Novus Biologicals, Cat No. NBP2-75189 (date March 3, 2019), bectumomab (see, e.g., US Pat. No. US8420086), RFB4 (see, e.g., US Pat. No. US7355012), SM03(see, e.g.,
  • NCI m972 see, e.g., US8591889, US9279019, US9598492
  • NCI m971 see, e.g., US7456260, US8591889, US9279019, US9598492.
  • the TAGE includes an antigen binding polypeptide that is an anti- FAP antibody, or fragment thereof.
  • Fibroblast activation protein also known as Seprase, is a membrane-bound serine protease of the prolyl oligopeptidase family with post-prolyl endopeptidase activity. FAP’s restricted expression to the tumor microenvironment (e.g., tumor stroma) makes it an attractive therapeutic candidate to target in the treatment of various tumors.
  • the anti-FAP antibody is selected from Sibrotuzumab/BIBH1 (described in WO 99/57151 , Mersmann et al., Int J Cancer 92, 240-248 (2001 ); Schmidt et al., Eur J Biochem 268, 1730-1738 (2001 ); WO 01/68708, WO 2007/077173), F19 (described in WO 93/05804, ATCC Number HB 8269, sold by R&D systems, Catalog No.: MAB3715), OS4 (described in WOest et al., J Biotech 92, 159-168 (2001 )).
  • Other anti-FAP antibodies are described, for example, in US Pat. No. 8568727; US Pat. No. 8999342, US. App. No. 20160060356; US. App. No. 20160060357, and US Pat. No. US9011847, each of which is incorporated by reference herein.
  • the TAGE includes an antigen binding polypeptide that is an anti- CTLA4 antibody, or fragment thereof.
  • CTLA-4 cytotoxic T-lymphocyte-associated protein 4
  • CD152 cluster of differentiation 152
  • the anti-CTLA4 antibody is selected from Ipilimumab (trade name: YERVOY®, described in US Pat. No. 6984720; US Pat. No. 605238, US Pat. No. 8017114, US Pat. No. 8318916, and US Pat. No. 8784815.)
  • Ipilimumab trade name: YERVOY®
  • Other anti-CTLA4 antibodies are described, for example, in US Pat. No. 9714290; US Pat. No. 10202453, and US. Publication No. 20170216433, each of which is incorporated by reference herein.
  • the TAGE includes an antigen binding polypeptide that is an anti- CD44 antibody, or fragment thereof.
  • CD44 is a ubiquitous cell surface glycoprotein that is highly expressed in many cancers and regulates metastasis via recruitment of CD44 to the cell surface.
  • the anti-CD44 antibody is selected from RG7356 (described in PCT Publication: WO2013063498A1).
  • Other anti-CTLA4 antibodies are described, for example, in US. Publication No.. 20170216433, US. Publication No. 20070237761 A1 , and US. Publication No. US20100092484, each of which is incorporated by reference herein.
  • the TAGE includes an antigen binding polypeptide that is an anti- CD54 antibody, or fragment thereof.
  • the CD54 is a cell surface glycoprotein that binds to the leucocyte function-associated antigen-1 (CD11 a/CD18 [LFA-1]). CD54 modulates both LFA-1- dependent adhesion of leucocytes to endothelial cells and immune functions involving cell-to-cell contact.
  • Anti-CD54 antibodies are described, for example, in US. Pat No. 7943744, US. Pat No. 5773293, US. Pat No. 8623369, PCT Publication No. W091/16928, and US. Publication No.
  • the TAGE includes an antigen binding polypeptide that is an anti- CD33 antibody, or fragment thereof.
  • CD33 or Siglec-3 sialic acid binding Ig-like lectin 3, SIGLEC3, SIGLEC-3, gp67, p67
  • the anti-CD33 antibody is selected from lintuzumab (also known as clone HuM195, described in US Pat. No. 9079958,) 2H12 (described in US Pat. No. 9587019).
  • lintuzumab also known as clone HuM195, described in US Pat. No. 9079958,
  • 2H12 described in US Pat. No. 9587019.
  • Other CD33 antibodies have been described in, for example, U.S. Pat.
  • the TAGE includes an antigen binding polypeptide that is an anti-CD22 antibody, or fragment thereof.
  • the anti-CD22 antibody is the anti-CD22 antibody epratuzumab (also known as hl_22, see, e.g., US Pat. No. 5789554; US. App. No. 20120302739; sold by Novus Biologicals, Cat No.
  • Epratuzumab antibody is a humanized antibody derived from antibody LL2 (EPB-2), a murine anti-CD22 lgG2a raised against Raji Burkitt lymphoma cells.
  • the anti-CD22 antibody comprises a heavy chain comprising a CDR1 , CDR2 and CDR3 of anti-CD22 antibody epratuzumab, and a light chain variable region comprising a CDR1 , CDR2 and CDR3 of anti-CD22 antibody epratuzumab.
  • the TAGE includes an antigen binding polypeptide that is an anti-CD3 antibody, or antigen binding fragment thereof.
  • the anti-CD3 antibody is muromonab (also known as OKT3; sold by BioLegend, Cat. No. 317301 or 317302 (date March 3,
  • visilizumab see, e.g., US Pat. No. 5834597, US Pat. No. 7381803, US App. No. 20080025975
  • otelixizumab see, e.g., W02007145941
  • Dow2 see, e.g., WO2014129270.
  • the TAGE comprises an anti-CD3 antibody, wherein the anti-CD3 antibody is the anti-CD3 antibody muromonab (also known as OKT3; sold by BioLegend, Cat. No.
  • the anti-CD3 antibody comprises a heavy chain comprising a CDR1 , CDR2 and CDR3 of anti-CD3 antibody muromonab, and a light chain variable region comprising a CDR1 , CDR2 and CDR3 of anti-CD3 antibody muromonab.
  • the antibody, antigen binding fragment thereof comprises variable regions having an amino acid sequence that is at least 95%, 96%, 97% or 99% identical to the SEQ ID Nos disclosed herein, including sequences in the cited references.
  • the antibody, or antigen binding fragment thereof comprises CDRs comprising the SEQ ID Nos disclosed herein with framework regions of the variable regions described herein having an amino acid sequence that is at least 95%, 96%, 97% or 99% identical to the SEQ ID Nos disclosed herein, including sequences in the cited references.
  • the sequences and disclosure specifically recited herein are expressly incorporated by reference.
  • the TAGE agent comprises an antigen binding polypeptide that binds to a protein expressed on the surface of cells selected from hematopoietic stem cells (HSCs), hematopoietic progenitor stem cells (HPSCs), natural killer cells, macrophages, DC cells, non-DC myeloid cells, B cells, T cells (e.g., activated T cells), fibroblasts, or other cells.
  • HSCs hematopoietic stem cells
  • HPSCs hematopoietic progenitor stem cells
  • natural killer cells macrophages
  • DC cells DC cells
  • non-DC myeloid cells B cells
  • T cells e.g., activated T cells
  • fibroblasts fibroblasts
  • the T cells are CD4 or CD8 T cells.
  • the T cells are regulatory T cells (T regs) or effector T cells.
  • the T cells are tumor infiltrating T cells.
  • the cell is a hematopoietic stem cell (HSCs or a hematopoietic progenitor cells (HPSCs).
  • the macrophages are M1 or M2 macrophages.
  • the antigen binding agent of the TAGE is an antigen-binding fragment.
  • antigen-binding fragments include, but are not limited to, a domain antibody, a nanobody, a unibody, an scFv, a Fab, a BiTE, a diabody, a DART, a minibody, a F(ab’)2, or an intrabody.
  • the antigen binding polypeptide of the TAGE agent is a nanobody.
  • a TAGE comprises a domain antibody, a conformation-specific NP binding agent, and a nucleoprotein comprising a nucleic acid-guided nuclease and a guide nucleic acid.
  • Domain antibodies are small functional binding units of antibodies, corresponding to the variable regions of either the heavy (VH) or light (VL) chains of human antibodies. Domain Antibodies have a molecular weight of approximately 13 kDa. Domantis has developed a series of large and highly functional libraries of fully human VH and VL dAbs (more than ten billion different sequences in each library), and uses these libraries to select dAbs that are specific to therapeutic targets.
  • domain antibodies are well expressed in bacterial, yeast, and mammalian cell systems. Further details of domain antibodies and methods of production thereof may be obtained by reference to U.S. Pat. Nos. 6,291 ,158; 6,582,915; 6,593,081 ; 6,172,197; 6,696,245; U.S. Serial No. 2004/0110941 ; European patent application No. 1433846 and European Patents 0368684 & 0616640; WO05/035572, WO04/101790, W004/081026, W004/058821 , W004/003019 and W003/002609, each of which is herein incorporated by reference in its entirety.
  • a TAGE comprises a nanobody, a conformation-specific NP binding agent, and a nucleoprotein comprising a nucleic acid-guided nuclease and guide nucleic acid.
  • Nanobodies are antibody-derived therapeutic proteins that contain the unique structural and functional properties of naturally-occurring heavy-chain antibodies. These heavy-chain antibodies contain a single variable domain (VHH) and two constant domains (CH2 and CH3). Importantly, the cloned and isolated VHH domain is a perfectly stable polypeptide harbouring the full antigen-binding capacity of the original heavy-chain antibody. Nanobodies have a high homology with the VH domains of human antibodies and can be further humanized without any loss of activity. Importantly, nanobodies have a low immunogenic potential, which has been confirmed in primate studies with Nanobody lead compounds.
  • Nanobodies combine the advantages of conventional antibodies with important features of small molecule drugs. Like conventional antibodies, nanobodies show high target specificity, high affinity for their target and low inherent toxicity. However, like small molecule drugs they can inhibit enzymes and readily access receptor clefts. Furthermore, nanobodies are extremely stable, can be administered by means other than injection (see, e.g., WO 04/041867, which is herein incorporated by reference in its entirety) and are easy to manufacture. Other advantages of nanobodies include recognizing uncommon or hidden epitopes as a result of their small size, binding into cavities or active sites of protein targets with high affinity and selectivity due to their unique 3-dimensional, drug format flexibility, tailoring of half-life and ease and speed of drug discovery.
  • Nanobodies are encoded by single genes and are efficiently produced in almost all prokaryotic and eukaryotic hosts, e.g., E. coli (see, e.g., U.S. Pat. No. 6,765,087, which is herein incorporated by reference in its entirety), molds (for example Aspergillus or Trichoderma) and yeast (for example Saccharomyces, Kluyveromyces, Hansenula or Pichia) (see, e.g., U.S. Pat. No. 6,838,254, which is herein incorporated by reference in its entirety).
  • the production process is scalable and multi-kilogram quantities of nanobodies have been produced. Because nanobodies exhibit a superior stability compared with conventional antibodies, they can be formulated as a long shelf-life, ready-to-use solution.
  • the nanoclone method (see, e.g., WO 06/079372, which is herein incorporated by reference in its entirety) is a proprietary method for generating nanobodies against a desired target, based on automated high-throughput selection of B-cells and could be used in the context of the instant invention.
  • a TAGE comprises a unibody, a conformation-specific NP binding agent, and a nucleoprotein comprising a nucleic acid-guided nuclease and a guide nucleic acid.
  • UniBodies are another antibody fragment technology, however this technology is based upon the removal of the hinge region of lgG4 antibodies. The deletion of the hinge region results in a molecule that is essentially half the size of traditional lgG4 antibodies and has a univalent binding region rather than the bivalent binding region of lgG4 antibodies. It is also well known that lgG4 antibodies are inert and thus do not interact with the immune system, which may be advantageous for the treatment of diseases where an immune response is not desired, and this advantage is passed onto UniBodies.
  • unibodies may function to inhibit or silence, but not kill, the cells to which they are bound. Additionally, unibody binding to cancer cells do not stimulate them to proliferate. Furthermore, because unibodies are about half the size of traditional lgG4 antibodies, they may show better distribution over larger solid tumors with potentially advantageous efficacy. UniBodies are cleared from the body at a similar rate to whole lgG4 antibodies and are able to bind with a similar affinity for their antigens as whole antibodies. Further details of UniBodies may be obtained by reference to patent application W02007/059782, which is herein incorporated by reference in its entirety.
  • a TAGE comprises an affibody, a conformation-specific NP binding agent, and a nucleoprotein comprising a nucleic acid-guided nuclease and a guide nucleic acid.
  • Affibody molecules represent a class of affinity proteins based on a 58-amino acid residue protein domain, derived from one of the IgG-binding domains of staphylococcal protein A.
  • This three helix bundle domain has been used as a scaffold for the construction of combinatorial phagemid libraries, from which affibody variants that target the desired molecules can be selected using phage display technology (Nord K, Gunneriusson E, Ringdahl J, Stahl S, Uhlen M, Nygren P A, Binding agents selected from combinatorial libraries of an a-helical bacterial receptor domain, Nat Biotechnol 1997;
  • Affibody molecules in combination with their low molecular weight (6 kDa), make them suitable for a wide variety of applications, for instance, as detection reagents (Ronmark J, Harmon M, Nguyen T, et al, Construction and characterization of affibody-Fc chimeras produced in Escherichia coli, J Immunol Methods 2002; 261 :199-211 ) and to inhibit receptor interactions (Sandstorm K, Xu Z, Forsberg G, Nygren P A, Inhibition of the CD28-CD80 co-stimulation signal by a CD28-binding Affibody ligand developed by combinatorial protein engineering, Protein Eng 2003; 16:691 -7). Further details of Affibodies and methods of production thereof may be obtained by reference to U.S. Pat. No. 5,831 ,012 which is herein incorporated by reference in its entirety.
  • the antibody, antigen-binding fragment thereof, or antibody mimetic may specifically bind to an extracellular molecule (e.g., protein, glycan, lipid) localized on a target cell membrane or associated with a specific tissue with an Kd of at least about 1 x10 -4 , 1 x10 -5 , 1 x10 -6 M,
  • an extracellular molecule e.g., protein, glycan, lipid
  • the binding agent may be capable of specifically binding to two or more antigens which are related in sequence.
  • the binding polypeptides of the invention can specifically bind to both human and a non human (e.g., mouse or non-human primate) orthologs of an antigen.
  • the antibody, antigen-binding fragment thereof, or antibody mimetic binds to a hapten which in turn specifically binds an extracellular cell surface (e.g., a Cas9-antibody- hapten targeting a cell receptor).
  • a hapten which in turn specifically binds an extracellular cell surface (e.g., a Cas9-antibody- hapten targeting a cell receptor).
  • Binding or affinity between an antigen and an antibody can be determined using a variety of techniques known in the art, for example but not limited to, equilibrium methods (e.g., enzyme-linked immunosorbent assay (ELISA); KinExA, Rathanaswami et al. Analytical Biochemistry, Vol. 373:52-60, 2008; or radioimmunoassay (RIA)), or by a surface plasmon resonance assay or other mechanism of kinetics-based assay (e.g., BIACORE.RTM. analysis or Octet.RTM.
  • equilibrium methods e.g., enzyme-linked immunosorbent assay (ELISA); KinExA, Rathanaswami et al. Analytical Biochemistry, Vol. 373:52-60, 2008; or radioimmunoassay (RIA)
  • a surface plasmon resonance assay e.g., BIACORE.RTM. analysis or Octet.RTM.
  • binding affinities and kinetics can be found in Paul, W. E., ed., Fundamental Immunology, 4th Ed., Lippincott-Raven, Philadelphia (1999), which focuses on antibody-immunogen interactions.
  • a competitive binding assay is a radioimmuno assay comprising the incubation of labeled antigen with the antibody of interest in the presence of increasing amounts of unlabeled antigen, and the detection of the antibody bound to the labeled antigen.
  • the affinity of the antibody of interest for a particular antigen and the binding off-rates can be determined from the data by scatchard plot analysis.
  • Competition with a second antibody can also be determined using radioimmunoassays.
  • the antigen is incubated with antibody of interest conjugated to a labeled compound in the presence of increasing amounts of an unlabeled second antibody.
  • the antibody or antigen-binding fragment thereof, described herein can be in the form of full- length antibodies, bispecific antibodies, dual variable domain antibodies, multiple chain or single chain antibodies, and/or binding fragments that specifically bind an extracellular molecule, including but not limited to Fab, Fab', (Fab')2, Fv), scFv (single chain Fv), surrobodies (including surrogate light chain construct), single domain antibodies, camelized antibodies and the like. They also can be of, or derived from, any isotype, including, for example, IgA (e.g., lgA1 or lgA2), IgD, IgE, IgG (e.g.
  • the antibody is an IgG (e.g. lgG1 , lgG2, lgG3 or lgG4).
  • the antibody is Abciximab (ReoPro; CD41 ), alemtuzumab (Lemtrada, Campath; CD52), abrilumab (integrin a4b7), alacizumab pegol (VEGFR2), alemtuzumab (Lemtrada, Campath; CD52), anifrolumab (interferon a/b receptor), apolizumab (HLA-DR), aprutumab (FGFR2); aselizumab (L-selectin or CD62L), atezolizumab (Tecentriq; PD-L1 ), avelumab (Bavencio; PD-L1 ), azintuxizumab (CD319); basiliximab (Simulect; CD25), BCD-100 (PD-1 ), bectummomab (LymphoScan; CD22), belantamab (BCMA); belimumab (
  • HER3 ziralimumab (CD147, basigin); zolbetuximab (Claudin 18 Isoform 2), zolimomab (CD5), 3F8 (GD2 ganglioside), adecatumumab (EpCAM), altumomab (Hybri-ceaker; CEA), amatuximab (mesothelin), anatumomab mafenatox (TAG-72), anetumab (MSLN), arcitumomab (CEA), atorolimumab (Rhesus factor); bavituximab (phosphatidylserine), besilesomab (Scintimun; CEA- related antigen), cantuzumab (MUC1), caplacizumab (Cablivi; VWF), clivatuzumab tetraxetan (hPAM4-Cide; MUC1), codrituzuma
  • an antibody that can be used in the compositions and methods disclosed herein is an antibody known to internalize in cells.
  • examples of such antibodies, which can be used in TAGE agents described herein includes, but are not limited to, anetumab (mesothelin), aorutumab (FGFR2), azintuxizumab (SLAMF7), belantamab (TNFRSF17), bivatuzumab (CD44v6), brentuximab (CD30), camidanlumab (CD25), cantuzumab (CanAg), cantuzumab (CanAg), clivatuzumab (MUC1), cofetuzumab (PTK7), coltuximab (CD19), denintuzumab (CD19), depatuxizumab (EGFR), enapotamab (AXL), enfortumab (Nectin-4), epratuzumab (CD22), gem
  • DLK1 DLK1 , ENPP3, FLT3, ADAM-9, CD248, endothelin receptor ETB, HER3, TM4SF1 , SLC44A4, 5T4, AXL, Ror2, CA9, CFC1 B, MT1 -MMP, HGFR, CXCR4, TIM-1 , CD166, CD163, GPC2, S.
  • aeruginosa antigen CD38, H-Ferritin, SLeA, NKA, CD147, OFP, SLITRK5, EphrinA4, VEGFR2, GCL, CEACAM1 , CEACAM6, or NaPi2b.
  • the antibody, or antigen-binding fragment thereof, described herein can be in the form of full- length antibodies, bispecific antibodies, dual variable domain antibodies, multiple chain or single chain antibodies, and/or binding fragments that specifically bind an extracellular molecule, including but not limited to Fab, Fab', (Fab')2, Fv), scFv (single chain Fv), surrobodies (including surrogate light chain construct), single domain antibodies, camelized antibodies and the like. They also can be of, or derived from, any isotype, including, for example, IgA (e.g., lgA1 or lgA2), IgD, IgE, IgG (e.g.
  • the antibody is an IgG (e.g. lgG1 , lgG2, lgG3 or lgG4).
  • the antigen binding polypeptide is a multispecific protein, such as a multispecific (e.g., bispecific) antibody.
  • the antigen binding protein is a bispecific molecule comprising a first antigen binding site from a first antibody that binds to a target on the extracellular cell membrane of a cell and a second antigen binding site with a different binding specificity, such as a binding specificity for a second target on the extracellular cell membrane of the cell, i.e. a bispecific antibody wherein the first and second antigen binding sites do not cross-block each other for binding to either the first or the second antigen.
  • target antigens are provided above.
  • a TAGE comprises a bispecific molecule that binds to two antigens, including those described herein, e.g., CTLA4 and CD44.
  • Exemplary bispecific antibody molecules comprise (i) two antibodies, one with a specificity to a first antigen and another to a second target that are conjugated together, (ii) a single antibody that has one chain or arm specific to a first antigen and a second chain or arm specific to a second antigen, (iii) a single chain antibody that has specificity to a first antigen and a second antigen, e.g., via two scFvs linked in tandem by an extra peptide linker; (iv) a dual-variable-domain antibody (DVD- Ig), where each light chain and heavy chain contains two variable domains in tandem through a short peptide linkage (Wu et al.
  • Examples of platforms useful for preparing bispecific antibodies include but are not limited to BITE (Micromet), DART (MacroGenics), Fcab and Mab.sup.2 (F-star), Fc-engineered IgG 1 (Xencor) or DuoBody (based on Fab arm exchange, Genmab).
  • bispecific antibodies include but are not limited to asymmetric IgG-like molecules, wherein the one side of the molecule contains the Fab region or part of the Fab region of at least one antibody, and the other side of the molecule contains the Fab region or parts of the Fab region of at least one other antibody; in this class, asymmetry in the Fc region could also be present, and be used for specific linkage of the two parts of the molecule; symmetric IgG-like molecules, wherein the two sides of the molecule each contain the Fab region or part of the Fab region of at least two different antibodies; IgG fusion molecules, wherein full length IgG antibodies are fused to extra Fab regions or parts of Fab regions; Fc fusion molecules, wherein single chain Fv molecules or stabilized diabodies are fused to Fcgamma regions or parts thereof; Fab fusion molecules, wherein different Fab-fragments are fused together; ScFv-and diabody-based molecules wherein different single chain Fv molecules or different diabodies
  • asymmetric IgG-like molecules include but are not limited to the Triomab/Quadroma (Trion Pharma/Fresenius Biotech), the Knobs-into-Holes (Genentech),
  • Example of symmetric IgG-like molecules include but are not limited to Dual Targeting (DT)-lg (GSK/Domantis), Two-in-one Antibody (Genentech), Cross-linked Mabs (Karmanos Cancer Center), mAb2 (F-Star) and CovX-body (CovX/Pfizer).
  • DT Dual Targeting
  • Genentech Two-in-one Antibody
  • Cross-linked Mabs Karmanos Cancer Center
  • mAb2 F-Star
  • CovX-body CovX/Pfizer
  • IgG fusion molecules include but are not limited to Dual Variable Domain (DVD)- Ig (Abbott), IgG-like Bispecific (ImClone/Eli Lilly), Ts2Ab (Medlmmune/AZ) and BsAb (Zymogenetics), HERCULES (Biogen pout) and TvAb (Roche).
  • DVD Dual Variable Domain
  • IgG-like Bispecific ImClone/Eli Lilly
  • Ts2Ab Medlmmune/AZ
  • BsAb Zymogenetics
  • HERCULES Biogen personal
  • TvAb Roche.
  • Fc fusion molecules include but are not limited to ScFv/Fc Fusions (Academic Institution), SCORPION (Emergent BioSolutions/Trubion, Zymogenetics/BMS), Dual Affinity Retargeting Technology (Fc-DART) (MacroGenics) and Dual(ScFv) 2-Fab (National Research Center for Antibody Medicine-China).
  • class V bispecific antibodies include but are not limited to F(ab)2 (Medarex/Amgen), Dual-Action or Bis-Fab (Genentech), Dock-and-Lock (DNL) (ImmunoMedics), Bivalent Bispecific (Biotecnol) and Fab-Fv (UCB-Celltech).
  • ScFv-and diabody-based molecules include but are not limited to Bispecific T Cell Engager (BITE) (Micromet), Tandem Diabody (Tandab) (Affimed), Dual Affinity Retargeting Technology (DART) (MacroGenics), Single-chain Diabody (Academic), TCR-like Antibodies (AIT, ReceptorLogics), Human Serum Albumin ScFv Fusion (Merrimack) and COM BODY (Epigen Biotech).
  • BITE Bispecific T Cell Engager
  • Tandab Tandem Diabody
  • DART Dual Affinity Retargeting Technology
  • AIT TCR-like Antibodies
  • AIT Human Serum Albumin ScFv Fusion
  • COM BODY Epigen Biotech
  • Antibodies, antigen-binding fragments, or an antibody mimetic that may be used in conjunction with the compositions and methods described herein include the above-described antibodies and antigen-binding fragments thereof, as well as humanized variants of those non-human antibodies and antigen-binding fragments described above and antibodies or antigen-binding fragments that bind the same epitope as those described above, as assessed, for instance, by way of a competitive antigen binding assay.
  • the antibodies or binding fragments described herein may also include modifications and/or mutations that alter the properties of the antibodies and/or fragments.
  • Methods of engineering antibodies to include any modifications are well known in the art. These methods include, but are not limited to, preparation by site-directed (or oligonucleotide-mediated) mutagenesis, PCR mutagenesis, and cassette mutagenesis of a prepared DNA molecule encoding the antibody or at least the constant region of the antibody.
  • Site-directed mutagenesis is well known in the art (see, e.g., Carter et al. , Nucleic Acids Res., 13:4431-4443 (1985) and Kunkel et al., Proc. Natl. Acad. Sci.
  • PCR mutagenesis is also suitable for making amino acid sequence variants of the starting polypeptide. See Higuchi, in PCR Protocols, pp. 177-183 (Academic Press, 1990); and Vallette et al., Nuc. Acids Res. 17:723-733 (1989). Another method for preparing sequence variants, cassette mutagenesis, is based on the technique described by Wells et al., Gene, 34:315-323 (1985).
  • Antibodies or fragments thereof may be produced using recombinant methods and compositions, e.g., as described in U.S. Pat. No. 4,816,567.
  • isolated nucleic acid encoding an antibody described herein is provided. Such nucleic acid may encode an amino acid sequence comprising the VL and/or an amino acid sequence comprising the VH of the antibody (e.g., the light and/or heavy chains of the antibody).
  • one or more vectors e.g., expression vectors
  • a host cell comprising such nucleic acid is provided.
  • a host cell comprises (e.g., has been transformed with): (1) a vector comprising a nucleic acid that encodes an amino acid sequence comprising the VL of the antibody and an amino acid sequence comprising the VH of the antibody, or (2) a first vector comprising a nucleic acid that encodes an amino acid sequence comprising the VL of the antibody and a second vector comprising a nucleic acid that encodes an amino acid sequence comprising the VH of the antibody.
  • the host cell is eukaryotic, e.g. a Chinese Hamster Ovary (CHO) cell or lymphoid cell (e.g., Y0, NS0, Sp20 cell).
  • a method of making an anti-CLL-1 antibody comprises culturing a host cell comprising a nucleic acid encoding the antibody, as provided above, under conditions suitable for expression of the antibody, and optionally recovering the antibody from the host cell (or host cell culture medium).
  • nucleic acid encoding an antibody is isolated and inserted into one or more vectors for further cloning and/or expression in a host cell.
  • nucleic acid may be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of the antibody).
  • Suitable host cells for cloning or expression of antibody-encoding vectors include prokaryotic or eukaryotic cells described herein.
  • antibodies may be produced in bacteria, in particular when glycosylation and Fc effector function are not needed.
  • For expression of antibody fragments and polypeptides in bacteria see, e.g., U.S. Pat. Nos. 5,648,237, 5,789,199, and 5,840,523. (See also Charlton, Methods in Molecular Biology, Vol. 248 (B.K.C. Lo, ed., Humana Press, Totowa, N.J., 2003), pp. 245-254, describing expression of antibody fragments in E. coli.)
  • the antibody may be isolated from the bacterial cell paste in a soluble fraction and can be further purified.
  • Vertebrate cells may also be used as hosts.
  • mammalian cell lines that are adapted to grow in suspension may be useful.
  • Other examples of useful mammalian host cell lines are monkey kidney CV1 line transformed by SV40 (COS-7); human embryonic kidney line (293 or 293 cells as described, e.g., in Graham et al. , J. Gen Virol. 36:59 (1977)); baby hamster kidney cells (BHK); mouse sertoli cells (TM4 cells as described, e.g., in Mather, Biol. Reprod.
  • monkey kidney cells (CV1 ); African green monkey kidney cells (VERO-76); human cervical carcinoma cells (HELA); canine kidney cells (MDCK; buffalo rat liver cells (BRL 3A); human lung cells (W138); human liver cells (Hep G2); mouse mammary tumor (MMT 060562); TRI cells, as described, e.g., in Mather et al., Annals N.Y. Acad. Sci. 383:44-68 (1982); MRC 5 cells; and FS4 cells.
  • Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells, including DHFR- CHO cells (Urlaub et al., Proc. Natl. Acad. Sci.
  • the TAGE may include an antibody mimetic capable of binding an antigen of interest.
  • an antibody mimetic capable of binding an antigen of interest.
  • an antibody mimetic described herein, are not structurally related to an antibody, and include adnectins, affibodies, DARPins, anticalins, avimers, versabodies, aptamers and SMIPS.
  • An antibody mimetic uses binding structures that, while mimicking traditional antibody binding, are generated from and function via distinct mechanisms. Some of these alternative structures are reviewed in Gill and Damle (2006) 17: 653-658.
  • a TAGE comprises an adnectin molecule, a conformation-specific NP binding agent, and a nucleoprotein comprising a nucleic acid-guided nuclease and a guide nucleic acid.
  • Adnectin molecules are engineered binding agents derived from one or more domains of the fibronectin protein. Fibronectin exists naturally in the human body. It is present in the extracellular matrix as an insoluble glycoprotein dimer and also serves as a linker protein. It is also present in soluble form in blood plasma as a disulphide linked dimer.
  • fibronectin The plasma form of fibronectin is synthesized by liver cells (hepatocytes), and the ECM form is made by chondrocytes, macrophages, endothelial cells, fibroblasts, and some cells of the epithelium.
  • fibronectin may function naturally as a cell adhesion molecule, or it may mediate the interaction of cells by making contacts in the extracellular matrix.
  • fibronectin is made of three different protein modules, type I, type II, and type III modules.
  • adnectin molecules are derived from the fibronectin type III domain by altering the native protein which is composed of multiple beta strands distributed between two beta sheets.
  • fibronectin may contain multiple type III domains which may be denoted, e.g., 1 Fn3, 2Fn3, 3Fn3, etc.
  • the 10Fn3 domain contains an integrin binding motif and further contains three loops which connect the beta strands. These loops may be thought of as corresponding to the antigen binding loops of the IgG heavy chain, and they may be altered by methods discussed below to specifically bind a target of interest.
  • a fibronectin type III domain useful for the purposes of this invention is a sequence which exhibits a sequence identity of at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% to the sequence encoding the structure of the fibronectin type III molecule which can be accessed from the Protein Data Bank (PDB, rcsb.org/pdb/home/home.do) with the accession code:
  • PDB Protein Data Bank
  • Adnectin molecules may also be derived from polymers of 10Fn3 related molecules rather than a simple monomeric 10Fn3 structure.
  • the native 10Fn3 domain typically binds to integrin
  • 10Fn3 proteins adapted to become adnectin molecules are altered so to bind antigens of interest.
  • the alteration to the 10Fn3 molecule comprises at least one mutation to a beta strand.
  • the loop regions which connect the beta strands of the 10Fn3 molecule are altered to bind to the antigen of interest.
  • the alterations in the 10Fn3 may be made by any method known in the art including, but not limited to, error prone PCR, site-directed mutagenesis, DNA shuffling, or other types of recombinational mutagenesis which have been referenced herein.
  • variants of the DNA encoding the 10Fn3 sequence may be directly synthesized in vitro, and later transcribed and translated in vitro or in vivo.
  • a natural 10Fn3 sequence may be isolated or cloned from the genome using standard methods (as performed, e.g., in U.S. Pat. Application No. 20070082365), and then mutated using mutagenesis methods known in the art.
  • a target antigen may be immobilized on a solid support, such as a column resin or a well in a microtiter plate.
  • the target is then contacted with a library of potential binding agents.
  • the library may comprise 10Fn3 clones or adnectin molecules derived from the wild type 10Fn3 by mutagenesis/randomization of the 10Fn3 sequence or by mutagenesis/randomization of the 10Fn3 loop regions (not the beta strands).
  • the library may be an RNA-protein fusion library generated by the techniques described in Szostak et al. , U.S. Ser. No.
  • the library may also be a DNA-protein library (e.g., as described in Lohse, U.S. Ser. No.
  • Adnectin molecules for use in the present invention may be engineered using the PROfusionTM technology employed by Adnexus, a Briston-Myers Squibb company.
  • fibronectin type III domains such as 10Fn3, followed by a selection step may be accomplished using other methods known in the art such as phage display, ribosome display, or yeast surface display, e.g., Lipovsek et al. (2007) Journal of Molecular Biology 368: 1024-1041 ; Sergeeva et al. (2006) Adv Drug Deliv Rev. 58:1622-1654; Petty et al. (2007) Trends Biotechnol. 25: 7-15; Rothe et al. (2006) Expert Opin Biol Ther. 6:177-187; and Hoogenboom (2005) Nat Biotechnol. 23:1105-1116.
  • exemplary proteins having immunoglobulin-like folds include N-cadherin, ICAM-2, titin, GCSF receptor, cytokine receptor, glycosidase inhibitor, E-cadherin, and antibiotic chromoprotein.
  • Further domains with related structures may be derived from myelin membrane adhesion molecule P0, CD8, CD4, CD2, class I MHC, T-cell antigen receptor, CD1 , C2 and l-set domains of VCAM-1 , l-set immunoglobulin fold of myosin-binding agent C, l-set immunoglobulin fold of myosin-binding agent H, l-set immunoglobulin-fold of telokin, telikin, NCAM, twitchin, neuroglian, growth hormone receptor, erythropoietin receptor, prolactin receptor, GC-SF receptor, interferon-gamma receptor, beta-galactosidase/glucuronidase, beta-glucuronidase, and transglutaminase.
  • any other protein that includes one or more immunoglobulin-like folds may be utilized to create a adnecting like binding moiety.
  • Such proteins may be identified, for example, using the program SCOP (Murzin et al ., J. Mol. Biol. 247:536 (1995); Lo Conte et al. ,
  • a TAGE comprises an aptamer, a conformation-specific NP binding agent, and a nucleoprotein comprising a nucleic acid-guided nuclease and a guide nucleic acid.
  • An “aptamer” used in the compositions and methods disclosed herein includes aptamer molecules made from either peptides or nucleotides. Peptide aptamers share many properties with nucleotide aptamers (e.g., small size and ability to bind target molecules with high affinity) and they may be generated by selection methods that have similar principles to those used to generate nucleotide aptamers, for example Baines and Colas. 2006. Drug Discov Today. 11 (7-8):334-41 ; and Bickle et al. 2006. Nat Protoc. 1 (3):1066-91 which are incorporated herein by reference.
  • an aptamer is a small nucleotide polymer that binds to specific molecular targets.
  • Aptamers may be single or double stranded nucleic acid molecules (DNA or RNA), although DNA based aptamers are most commonly double stranded. There is no defined length for an aptamer nucleic acid; however, aptamer molecules are most commonly between 15 and 40 nucleotides long.
  • Aptamers often form complex three-dimensional structures which determine their affinity for target molecules. Aptamers can offer many advantages over simple antibodies, primarily because they can be engineered and amplified almost entirely in vitro. Furthermore, aptamers often induce little or no immune response.
  • Aptamers may be generated using a variety of techniques, but were originally developed using in vitro selection (Ellington and Szostak. (1990) Nature. 346 (6287) :818-22) and the SELEX method (systematic evolution of ligands by exponential enrichment) (Schneider et al. 1992. J Mol Biol. 228 (3):862-9) the contents of which are incorporated herein by reference. Other methods to make and uses of aptamers have been published including Klussmann. The Aptamer Handbook Functional Oligonucleotides and Their Applications. ISBN: 978-3-527-31059-3; Ulrich et al. 2006. Comb Chem High Throughput Screen 9 (8):619-32; Cerchia and de Franciscis. 2007.
  • the SELEX method is clearly the most popular and is conducted in three fundamental steps. First, a library of candidate nucleic acid molecules is selected from for binding to specific molecular target. Second, nucleic acids with sufficient affinity for the target are separated from non-binders. Third, the bound nucleic acids are amplified, a second library is formed, and the process is repeated. At each repetition, aptamers are chosen which have higher and higher affinity for the target molecule. SELEX methods are described more fully in the following publications, which are incorporated herein by reference: Bugaut et al. 2006. 4 (22):4082-8; Stoltenburg et al. 2007 Biomol Eng. 200724 (4):381- 403; and Gopinath. 2007. Anal Bioanal Chem. 2007. 387 (1 ):171 -82.
  • a TAGE comprises a DARPin, a conformation-specific NP binding agent, and a nucleoprotein comprising a nucleic acid-guided nuclease and a guide nucleic acid.
  • DARPins Designed Ankyrin Repeat Proteins
  • Repeat proteins such as ankyrin or leucine-rich repeat proteins, are ubiquitous binding molecules, which occur, unlike antibodies, intra- and extracellularly.
  • Their unique modular architecture features repeating structural units (repeats), which stack together to form elongated repeat domains displaying variable and modular target-binding surfaces. Based on this modularity, combinatorial libraries of polypeptides with highly diversified binding specificities can be generated. This strategy includes the consensus design of self-compatible repeats displaying variable surface residues and their random assembly into repeat domains.
  • DARPins can be produced in bacterial expression systems at very high yields and they belong to the most stable proteins known. Highly specific, high-affinity DARPins to a broad range of target proteins, including human receptors, cytokines, kinases, human proteases, viruses and membrane proteins, have been selected. DARPins having affinities in the single-digit nanomolar to picomolar range can be obtained.
  • DARPins have been used in a wide range of applications, including ELISA, sandwich ELISA, flow cytometric analysis (FACS), immunohistochemistry (IHC), chip applications, affinity purification or Western blotting. DARPins also proved to be highly active in the intracellular compartment for example as intracellular marker proteins fused to green fluorescent protein (GFP). DARPins were further used to inhibit viral entry with IC50 in the pM range. DARPins are not only ideal to block protein-protein interactions, but also to inhibit enzymes. Proteases, kinases and transporters have been successfully inhibited, most often an allosteric inhibition mode. Very fast and specific enrichments on the tumor and very favorable tumor to blood ratios make DARPins well suited for in vivo diagnostics or therapeutic approaches.
  • a TAGE comprises an anticalin, a conformation-specific NP binding agent, and a nucleoprotein comprising a nucleic acid-guided nuclease and a guide nucleic acid.
  • Anticalins are an additional antibody mimetic technology, however in this case the binding specificity is derived from lipocalins, a family of low molecular weight proteins that are naturally and abundantly expressed in human tissues and body fluids. Lipocalins have evolved to perform a range of functions in vivo associated with the physiological transport and storage of chemically sensitive or insoluble compounds. Lipocalins have a robust intrinsic structure comprising a highly conserved b-barrel which supports four loops at one terminus of the protein.
  • a TAGE comprises a lipocalin, a conformation-specific NP binding agent, and a nucleoprotein comprising a nucleic acid-guided nuclease and a guide nucleic acid.
  • Lipocalins are cloned and their loops are subjected to engineering in order to create anticalins. Libraries of structurally diverse anticalins have been generated and anticalin display allows the selection and screening of binding function, followed by the expression and production of soluble protein for further analysis in prokaryotic or eukaryotic systems. Studies have successfully demonstrated that anticalins can be developed that are specific for virtually any human target protein can be isolated and binding affinities in the nanomolar or higher range can be obtained.
  • Anticalins can also be formatted as dual targeting proteins, so-called duocalins.
  • duocalins binds two separate therapeutic targets in one easily produced monomeric protein using standard manufacturing processes while retaining target specificity and affinity regardless of the structural orientation of its two binding domains.
  • Modulation of multiple targets through a single molecule is particularly advantageous in diseases known to involve more than a single causative factor.
  • bi- or multivalent binding formats such as duocalins have significant potential in targeting cell surface molecules in disease, mediating agonistic effects on signal transduction pathways or inducing enhanced internalization effects via binding and clustering of cell surface receptors.
  • the high intrinsic stability of duocalins is comparable to monomeric Anticalins, offering flexible formulation and delivery potential for Duocalins.
  • Avimers are evolved from a large family of human extracellular receptor domains by in vitro exon shuffling and phage display, generating multidomain proteins with binding and inhibitory properties. Linking multiple independent binding domains has been shown to create avidity and results in improved affinity and specificity compared with conventional single-epitope binding agents. Other potential advantages include simple and efficient production of multitarget-specific molecules in Escherichia coli, improved thermostability and resistance to proteases. Avimers with sub-nanomolar affinities have been obtained against a variety of targets.
  • a TAGE comprises a versabody, a conformation-specific NP binding agent, and a nucleoprotein comprising a nucleic acid-guided nuclease and a guide nucleic acid.
  • Versabodies are another antibody mimetic technology that could be used in the context of the instant invention. Versabodies are small proteins of 3-5 kDa with >15% cysteines, which form a high disulfide density scaffold, replacing the hydrophobic core that typical proteins have.
  • the inspiration for versabodies comes from the natural injectable biopharmaceuticals produced by leeches, snakes, spiders, scorpions, snails, and anemones, which are known to exhibit unexpectedly low immunogenicity.
  • Starting with selected natural protein families by design and by screening the size, hydrophobicity, proteolytic antigen processing, and epitope density are minimized to levels far below the average for natural injectable proteins.
  • versabodies Given the structure of versabodies, these antibody mimetics offer a versatile format that includes multi-valency, multi-specificity, a diversity of half-life mechanisms, tissue targeting modules and the absence of the antibody Fc region. Furthermore, versabodies are manufactured in E. coli at high yields, and because of their hydrophilicity and small size, Versabodies are highly soluble and can be formulated to high concentrations. Versabodies are exceptionally heat stable (they can be boiled) and offer extended shelf-life.
  • a TAGE comprises an SMIP, a conformation-specific NP binding agent, and a nucleoprotein comprising a nucleic acid-guided nuclease and a guide nucleic acid.
  • SMIPsTM Small Modular ImmunoPharmaceuticals-Trubion Pharmaceuticals
  • SMIPS consist of three distinct modular domains. First they contain a binding domain which may consist of any protein which confers specificity (e.g., cell surface receptors, single chain antibodies, soluble proteins, etc).
  • SMIPs contain a hinge domain which serves as a flexible linker between the binding domain and the effector domain, and also helps control multimerization of the SMIP drug.
  • SMIPS contain an effector domain which may be derived from a variety of molecules including Fc domains or other specially designed proteins. The modularity of the design, which allows the simple construction of SMIPs with a variety of different binding, hinge, and effector domains, provides for rapid and customizable drug design.
  • antibody fragment and antibody mimetic technologies are not intended to be a comprehensive list of all technologies that could be used in the context of the instant specification.
  • additional technologies including alternative polypeptide-based technologies, such as fusions of complimentary determining regions as outlined in Qui et al. , Nature Biotechnology, 25 (8) 921-929 (2007), which is hereby incorporated by reference in its entirety, as well as nucleic acid-based technologies, such as the RNA aptamer technologies described in U.S. Pat. Nos.
  • a ligand binds to an extracellular molecule associated with a cell membrane and provides specificity with which to deliver a conformation-specific NP binding agent stably associated with a nucleoprotein comprising a nucleic acid-guided nuclease and a guide nucleic acid. Examples of ligands that may be included in the TAGE are described below.
  • the TAGE provided herein includes one or more ligands, which refers to a molecule that is capable of binding to another molecule on or in a cell, including one or more cell surface receptors, and includes molecules such as proteins, hormones, neurotransmitters, cytokines, growth factors, cell adhesion molecules, or nutrients. Also contemplated are binding fragments of the ligands described herein, e.g., where the fragment binds to the corresponding receptor of the ligand.
  • Ligands useful in TAGEs herein include any molecules capable of binding a cell surface receptor.
  • ligands useful herein include, for example, interferons (such as type I, II, III); peptides; lymphokines such as IL-2, IL-3, IL-4, IL-6, GM-CSF (Granulocyte-macrophage colony- stimulating factor, also known as CSF-3), MCSF (Macrophage colony-stimulating factor 1 , also known as CSF-1), interferon-gamma (IFN-.gamma.); hormones such as insulin, TRH (thyrotropin releasing hormones), MSH (melanocyte-stimulating hormone), steroid hormones, such as androgens and estrogens, melanocyte-stimulating hormone (MSH); growth factors and colony-stimulating factors such as epidermal growth factors (EGF), granulocyte-macrophage colony-stimulating factor (GM- CSF), transforming growth factors
  • the ligand included in the TAGEs herein is selected from, IL2, CSF-1 , CSF-2, CSF-3, CCL2, CCL5, CCL7, CCL8, CCL13, CCL16, IGF2, IL7,
  • the ligand is IL-2, or a functional fragment thereof.
  • the ligand is IFNy.
  • the ligand is CSF-2, or a functional fragment thereof.
  • the ligand is a mutant and/or recombinant version of a ligand described herein.
  • the ligand is a mammalian ligand, e.g., a human ligand, non-human primate ligand, or a mouse ligand.
  • Ligands useful in the TAGEs herein include those capable of specifically binding cell surface receptors or cell surface molecules.
  • the extracellular molecule specifically bound by the ligand can include, but is not limited to, IL2Ra (CD25), IFNgR (CD119), CCR2 (CD192), Li (CD74), or PD-1 (CD279).
  • the ligand binds surface molecules with demonstrated cytoplasmic release and/or nuclear trafficking.
  • the extracellular molecule targeted by the ligand is HLA-DR, CD3, CD20, CD22, CD25, CD32, CD33, CD44, CD47, CD54, CD59, CD70, CD74, AchR, CTLA-4, CXCR4, EGFR, Her2, EpCam, PD-1 , or FAP1 .
  • exemplary targets for the ligand in the TAGE of the present invention include: (i) tumor-associated surface molecules; (ii) cell surface receptors, (iii) CD proteins and their ligands, such as CD3, CD4, CD8, CD19, CD20, CD22, CD25, CD32, CD33, CD34, CD40, CD44, CD47, CD54, CD59, CD70, CD74, CD79a (CD79a), and CD79P (CD79b); (iv) members of the ErbB receptor family such as the EGF receptor, HER2, HER3 or HER4 receptor; (v) cell adhesion molecules such as LFA-1 , Mac1 , p150,95, VLA-4, ICAM-1 , VCAM and an/b3 integrin including either alpha or beta subunits thereof; and (vi) growth factors such as VEGF; IgE; blood group antigens; flk2/flt3 receptor; obesity (OB) receptor; mpl receptor; CTLA-4; protein C,
  • cell surface receptors such as those described in Chen and Flies. Nature reviews immunology. 13.4 (2013): 227, which is incorporated herein by reference.
  • cell surface receptors that may be bound by a ligand include an immunoglobulin gene superfamily member (e.g. CD2, CD3,
  • CD4 CD8, CD19, CD20, CD22, CD28, CD30, CD37, CD38, CD56, CD70, CD79, CD90, CD125,
  • CD152/CTLA-4, PD-1 , or ICOS a TNF receptor superfamily member
  • a TNF receptor superfamily member e.g. CD27, CD40, CD95/Fas, CD134/0X40, CD137/4-1 BB, INF-R1 , TNFR-2, RANK, TACI, BCMA, osteoprotegerin, Apo2/TRAIL- R1 , TRAIL-R2, TRAIL-R3, TRAIL-R4, and APO-3
  • an integrin e.g. CD27, CD40, CD95/Fas, CD134/0X40, CD137/4-1 BB, INF-R1 , TNFR-2, RANK, TACI, BCMA, osteoprotegerin, Apo2/TRAIL- R1 , TRAIL-R2, TRAIL-R3, TRAIL-R4, and APO-3
  • an integrin e.g. CD27, CD40, CD95/Fas, CD134/0
  • a ligand suitable herein can alternatively comprise an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any of the ligand amino acid sequences disclosed herein.
  • Such a variant ligand protein should have ligand activity, such as the ability to retain binding activity to its corresponding receptor and/or the ability to mediate cellular uptake of molecular cargo (e.g., a conformation-specific NP binding agent stably associated with a nucleoprotein comprising a nucleic acid-guided nuclease and guide nucleic acid).
  • Testing the activity of a variant ligand can be done any number of ways, such as by covalently linking it with a fluorescent protein (e.g., GFP) and measuring the degree of fluorescence emitted from a cell contacted with the ligand-fluorescent protein complex, or by testing for binding of the ligand to a receptor or cell using binding assays known in the art.
  • a fluorescent protein e.g., GFP
  • the TAGE comprises a ligand that binds to a protein expressed on the surface of cells selected from hematopoietic stem cells (HSCs), hematopoietic progenitor stem cells (HPSCs), natural killer cells, macrophages, DC cells, non-DC myeloid cells, B cells, T cells (e.g., activated T cells), fibroblasts, or other cells.
  • HSCs hematopoietic stem cells
  • HPSCs hematopoietic progenitor stem cells
  • natural killer cells macrophages
  • DC cells DC cells
  • non-DC myeloid cells B cells
  • T cells e.g., activated T cells
  • fibroblasts fibroblasts
  • the T cells are CD4 or CD8 T cells.
  • the T cells are regulatory T cells (T regs) or effector T cells.
  • the T cells are tumor infiltrating T cells.
  • the cell is a hematopoietic stem cell (HSCs or a hematopoietic progenitor cells (HPSCs).
  • the macrophages are M1 or M2 macrophages.
  • the TAGE can include a nuclear localization sequence, e.g., SV40 large T antigen NLS (PKKKRKV; SEQ ID NO: 2) and nucleoplasmin NLS (KRPAATKKAGQAKKKK (SEQ ID NO: 31).
  • SV40 large T antigen NLS PKKRKV; SEQ ID NO: 2
  • KRPAATKKAGQAKKKK SEQ ID NO: 31
  • Other NLSs are known in the art; see, e.g., Cokol et al ., EMBO Rep. 2000 Nov. 15; 1 (5): 411 -415; Freitas and Cunha, Curr Genomics. 2009 December; 10(8): 550-557.
  • the TAGE includes one or more NLS such as 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 NLSs.
  • the TAGE includes one or more (e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 NLSs) C-terminal NLSs and one or more (e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 NLSs) N- terminal NLSs.
  • One or more ligands can be located at the N-terminus or C-terminus of a conformation- specific NP binding agent. Alternatively, one or more ligands can be located at both the N- and C- termini of the conformation-specific NP binding agent. Alternatively still, one or more ligands can be located within the amino acid sequence of the conformation-specific NP binding agent.
  • Embodiments herein comprising more than one ligand can comprise at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14,
  • the ligands fused to the conformation-specific NP binding agent can be the same or different (e.g., 2, 3, 4, or more different types of ligands).
  • One or more ligands can be fused directly to the amino acid sequence of a conformation-specific NP binding agent, and/or can be fused to a heterologous domain(s) (e.g., NLS) that is fused with a conformation-specific NP binding agent.
  • Ligands can be linked with a conformation-specific NP binding agent through covalent or non- covalent strategies.
  • Methods for covalently joining a ligand and a conformation-specific NP binding agent are known in the art, e.g. chemical cross-linking or cloning a fusion protein, as further described herein.
  • Non-covalent coupling between the cargo and short amphipathic ligands comprising polar and non-polar domains is established through electrostatic and hydrophobic interactions.
  • a fusion between a ligand and a conformation-specific NP binding agent can be directly through a peptide bond.
  • a fusion between a ligand and a conformation-specific NP binding agent can be via an intermediary amino acid sequence.
  • an intermediary amino acid sequence include suitable linker sequences comprising at least 1 , 2, 3, 4, 5,
  • amino acid residues such as glycine, serine, alanine and/or proline.
  • Suitable amino acid linkers are disclosed in U.S. Pat. Nos. 8,580,922 and 5,990,275, for example, which are incorporated herein by reference.
  • intermediary amino acid sequences can comprise one or more other types of proteins and/or domains.
  • a marker protein e.g., a fluorescent protein such as any of those disclosed herein
  • NLS peptide can be comprised in an intermediary amino acid sequence.
  • a conformation-specific NP binding agent and at least one ligand can be covalently linked in a TAGE via crosslinking (chemical crosslinking).
  • Crosslinking herein refers to a process of chemically joining two or more molecules (a conformation-specific NP binding agent and at least one ligand, in this case) by a covalent bond(s).
  • Crosslinking can be performed using any number of processes known in the art, such as those disclosed in U.S. Patent Appl. Publ. No. 2011 /0190813, U.S. Pat. No. 8,642,744, and Bioconjugate Techniques, 2nd Edition (G. T. Flermanson, Academic Press, 2008), which are all incorporated herein by reference.
  • a ligand and/or a conformation-specific NP binding agent can be modified and/or synthesized to contain a suitable protein linking group at its N-terminus, C-terminus, and/or an amino acid side group, for the purpose of crosslinking the ligand to a conformation-specific NP binding agent.
  • suitable protein linking group at its N-terminus, C-terminus, and/or an amino acid side group. Examples of chemical crosslinkers are further described herein.
  • a conformation-specific NP binding agent and at least one ligand herein can be non- covalently linked to each other in a TAGE using a variety of approaches known in the art. Though not intending to be held to any particular theory or mechanism, it is contemplated that a non-covalent linkage between the conformation-specific NP binding agent and at least one ligand can be due to electrostatic, Van der Waals, and/or hydrophobic forces.
  • the ligand e.g., IL-2, IFNy, or CSF-2
  • the ligand is delivered in trans with the site-directed modifying polypeptide.
  • a conformation-specific NP binding agent and at least one ligand herein can be non- covalently or covalently linked to each other in a TAGE using a variety of bioconjugation tools known in the art (see, e.g., Rabuka, David. Current opinion in chemical biology 14.6 (2010): 790-796, which is hereby incorporated by reference).
  • a conformation-specific NP binding agent can be complexed with a ligand via a bio-conjugation molecule.
  • bio-conjugation molecules include, without limitation, Spycatcher tag, Halo-tag, Sortase, mono-avidin, or a SNAP tag.
  • the bio-conjugation moleucle is selected from CBP, MBP, GST, poly(His), biotin/streptavidin, V5-tag, Myc-tag, HA-tag, NE-tag, His-tag, Flag tag, Halo-tag, Snap- tag, Fc-tag, Nus-tag, BCCP, Thioredoxin, SnooprTag, SpyTag, SpyCatcher, Isopeptag, SBP-tag, S- tag, AviTag, Calmodulin.
  • the ligand or conformation-specific NP binding agent includes a chemical tag.
  • a chemical tag may be SNAP tag, a CLIP tag, a HaloTag or a TMP-tag.
  • the chemical tag is a SNAP-tag or a CLIP-tag.
  • SNAP and CLIP fusion proteins enable the specific, covalent attachment of virtually any molecule to a protein of interest.
  • the chemical tag is a HaloTag.
  • HaloTag involves a modular protein tagging system that allows different molecules to be linked onto a single genetic fusion, either in solution, in living cells, or in chemically fixed cells.
  • the chemical tag is a TMP-tag.
  • the ligand or conformation-specific NP binding agent includes an epitope tag.
  • an epitope tag may be a poly-histidine tag such as a hexahistidine tag or a dodecahistidine, a FLAG tag, a Myc tag, a HA tag, a GST tag or a V5 tag.
  • the ligand or conformation-specific NP binding agent may each be engineered to comprise complementary binding pairs that enable stable association of the ligand and conformation-specific NP binding agent upon contact.
  • exemplary conjugation moiety pairings include (i) streptavidin-binding peptide (streptavidin binding peptide; SBP) and streptavidin (STV), (ii) biotin and EMA (enhanced monomeric avidin), (iii) SpyTag (ST) and SpyCatcher (SC), (iv) Halo-tag and Halo-tag ligand, (v) and SNAP-Tag , (vi) Myc tag and anti-Myc immunoglobulins (vii) FLAG tag and anti-FLAG immunoglobulins, and (ix) ybbR tag and coenzyme A groups.
  • More than one type of ligand can be covalently or non-covalently linked to a conformation-specific NP binding agent in certain embodiments.
  • the ratio (molar ratio) of ligand(s) to conformation-specific NP binding agent that can be used to prepare such a complex can be at least about 1 :1 , 2:1 , 3:1 , 4:1 , 5:1 , 6:1 , 7:1 , 8:1 , 9:1 , 10:1 15:1 , 20:1 , 30:1 , 40:1 , or 50:1 , for example.
  • the average number of ligands non- covalently linked to the conformation-specific NP binding agent may be at least 1 , 2, 3, 4, 5, 6, 7, 8, 9,
  • the provided herein is a method of modifying the genome of a target cell, the method comprising contacting the target cell with a targeted active gene editing (TAGE) agent comprising a ligand and confirmation specific-NP binding agent, as described herein.
  • the target cell is a eukaryotic cell.
  • the eukaryotic cell is a mammalian cell.
  • the mammalian cell is a mouse cell, a non-human primate cell, or a human cell.
  • the site-directed modifying polypeptide of the TAGE agent comprising the ligand produces a cleavage site at the target region of the genome, thereby modifying the genome.
  • the target region of the genome is a target gene.
  • a method comprising the use of a TAGE agent (e.g., a TAGE agent comprising a ligand and confirmation specific-NP binding agent) described herein is effective to modify expression of the target gene.
  • the method is effective to increase expression of the target gene relative to a reference level.
  • the method is effective to decrease expression of the target gene relative to a reference level.
  • the TAGE provided herein includes one or more cell penetrating peptides (CPPs).
  • CPPs cell penetrating peptides
  • Proteins within the TAGE are stably associated such that the CPP directs the conformation- specific NP binding agent and a nucleoprotein comprising a nucleic acid-guided nuclease and a guide nucleic acid to the cell surface and at least the nucleoprotein is internalized into the target cell.
  • the CPP binds to the cell surface such that the conformation-specific NP binding agent and the nucleoprotein comprising a nucleic acid-guided nuclease and a guide nucleic acid are internalized by the target cell as is the CPP.
  • internalization refers to at least 0.01%, at least 0.05%, at least 0.1%, at least 0.5%, at least 1%, at least 2%, at least 5% at least 10%, at least 15%, or at least 20% of the peptides or compositions internalized localize into the cytoplasm of a cell (e.g., within 1 hr, 2 hrs, 3 hrs, 4 hrs, or more).
  • the TAGE provided herein includes one or more cell penetrating peptides (CPPs), which refers to a peptide, typically of about 5-60 amino acid residues in length, that can facilitate cellular uptake of molecular cargo.
  • CPPs cell penetrating peptides
  • a conformation-specific NP binding agent can be associated with one or more CPPs through covalent or non-covalent linkage.
  • a CPP can also be characterized in certain embodiments as being able to facilitate the movement or traversal of molecular cargo across/through one or more of a lipid bilayer, micelle, cell membrane, organelle membrane, vesicle membrane, or cell wall.
  • a CPP herein can be cationic, amphipathic, or hydrophobic in certain embodiments.
  • CPPs useful in the TAGEs herein include, but are not limited to, protein-derived CPPs, including the Tat protein and Penetratin; chimeric CPPs, such the Transportan derived from the binding of the neuropeptide galanin N-terminus to the Mastoparan toxin; and synthetic CPPs, including oligoarginines or peptide nucleic acids (PNAs) formed by synthetic nucleic acid analogues bound to pseudopeptide backbone.
  • protein-derived CPPs including the Tat protein and Penetratin
  • chimeric CPPs such the Transportan derived from the binding of the neuropeptide galanin N-terminus to the Mastoparan toxin
  • synthetic CPPs including oligoarginines or peptide nucleic acids (PNAs) formed by synthetic nucleic acid analogues bound to pseudopeptide backbone.
  • the CPP is an amphiphilic or amphipathic CPP.
  • an amphipathic or amphiphilic CPP may include an amino acid sequence containing an alternating pattern of polar/charged residues and non-polar, hydrophobic residues.
  • An amphipathic CPP can alternatively be characterized as possessing both hydrophilic and lipophilic properties.
  • the CPP is a cationic or polycationic CPP.
  • a cationic or polycationic CPP may include an amino acid sequence having a high relative abundance (at least 60%) of positively charged amino acids such as lysine (K), arginine (R), and/or histidine (H).
  • the CPP is a hydrophobic or lipophilic CPP.
  • a hydrophobic or lipophilic CPP may include an amino acid sequence having mostly, or only, non-polar residues with low net charge and/or hydrophobic amino acid groups.
  • the TAGEs described herein can include one or more CPPS selected from NLS, Tat, Tat-HA, S19-Tat, CM18, hPFH , L17E, IMT-P8, TDP, TDP-KDEL (SEQ ID NO: 9), penetratin, polyR, or Aurein.
  • the TAGE includes one or more CPPs selected from Table 2.
  • the CPP is a TAT-related peptide comprising the transactivator of transcription (TAT) of human immunodeficiency virus (e.g., Tat, Tat-HA, S19-Tat).
  • TAT transactivator of transcription
  • a TAT-related peptide, or variant thereof may comprise one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, or more) additional amino acids at the N- or C-terminus of the sequence RKKRRQRRR (SEQ ID NO: 3).
  • the TAT-related peptide may include one or more (e.g., 1 , 2, 3, 4, 5, or more) amino acid insertions, deletions, or substitutions (e.g., conservative amino acid substitutions) that do not disrupt the cell penetrating properties of the TAT sequence.
  • the CPP may be a non-TAT-related peptide, such as NLS, hPH1 , penetratin, TDP, TDP-KDEL (SEQ ID NO: 9), Aurein, IMT-P8, L17E, or a polyR CPP.
  • the TAGE includes a TAT peptide and one or more additional CPPs, such as an NLS.
  • the TAGE may include a TAT peptide and one or more NLS, optionally in combination with one or more His tags, thereby forming a HIS-TAT-NLS (HTN) fusion.
  • the TAGE comprises the HTN peptide of SEQ ID NO: 17.
  • the CPP is an endosomal escape agent.
  • the endosomal escape agent may be TDP or TDP-KDEL (SEQ ID NO: 9).
  • the TAGE can include CPPs that act as a nuclear localization sequence, e.g., SV40 large T antigen NLS (PKKKRKV; SEQ ID NO: 2) and nucleoplasmin NLS (KRPAATKKAGQAKKKK; SEQ ID NO: 31 ).
  • CPPs that act as a nuclear localization sequence
  • PKKRKV SV40 large T antigen NLS
  • KRPAATKKAGQAKKKK SEQ ID NO: 31
  • Other NLSs are known in the art; see, e.g., Cokol et al., EMBO Rep. 2000 Nov. 15; 1 (5): 411 -415; Freitas and Cunha, Curr Genomics. 2009 December; 10(8): 550-557.
  • the TAGE includes one or more NLS such as 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 NLSs.
  • the TAGE includes one or more (e.g., 1 , 2, 3, 4,
  • NLSs 5, 6, 7, 8, 9, 10, or more than 10 NLSs
  • C-terminal NLSs C-terminal NLSs and one or more (e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 NLSs) C-terminal NLSs and one or more (e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 NLSs) C-terminal NLSs and one or more (e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 NLSs) C-terminal NLSs and one or more (e.g., 1 , 2, 3, 4, 5, 6, 7, 8,
  • NLSs 9, 10, or more than 10 NLSs.
  • a CPP herein can be about 5-30, 5-25, 5-20, 10-30, 10-25, or 10-20 amino acid residues in length, for example.
  • a CPP can be about 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16,
  • a CPP can be up to about 35, 40, 45, 50, 55, or 60 amino acid residues in length.
  • a CPP suitable herein can alternatively comprise an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any of the CPP amino acid sequences disclosed herein (e.g., CPP sequences selected from Table 1 ).
  • Such a variant CPP protein should have CPP activity, such as the ability to mediate cellular uptake of molecular cargo (e.g., a conformation-specific NP binding agent stably associated with a nucleic acid-guided nuclease).
  • Testing the activity of a variant CPP can be done any number of ways, such as by covalently linking it with a fluorescent protein (e.g., GFP) and measuring the degree of fluorescence emitted from a cell contacted with a the CPP-fluorescent protein complex.
  • a fluorescent protein e.g., GFP
  • One or more CPPs can be located at the N-terminus or C-terminus of a conformation-specific NP binding agent which can then be associated with a nucleic acid-guided nuclease to form a TAGE herein.
  • one or more CPPs can be located at both the N- and C-termini of the conformation-specific NP binding agent.
  • one or more CPPs can be located within the amino acid sequence of the conformation-specific NP binding agent.
  • Embodiments herein comprising more than one CPP can comprise at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, or 20 CPPs, or 5-10, 5-20, or 10-20 CPPs.
  • the CPPs fused to the conformation-specific NP binding agent can be the same or different (e.g., 2, 3, 4, or more different types of CPPs).
  • One or more CPPs can be fused directly to the amino acid sequence of a conformation-specific NP binding agent, and/or can be fused to a heterologous domain(s) (e.g., NLS) that is fused with a conformation- specific NP binding agent.
  • CPPs can be linked with a conformation-specific NP binding agent through covalent or non- covalent strategies.
  • Methods for covalently joining a CPP and a conformation-specific NP binding agent are known in the art, e.g. chemical cross-linking or cloning a fusion protein, as further described herein.
  • Non-covalent coupling between the cargo and short amphipathic CPPs comprising polar and non-polar domains is established through electrostatic and hydrophobic interactions.
  • a fusion between a CPP and a conformation-specific NP binding agent can be directly through a peptide bond or an iso-peptide bond.
  • a fusion between a CPP and a conjugation moiety (e.g., SpyTag) of the TAGE herein can be directly through a peptide bond or an iso-peptide bond.
  • a fusion between a CPP and a conformation-specific NP binding agent can be via an intermediary amino acid sequence. Examples of an intermediary amino acid sequence include suitable linker sequences comprising at least 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13,
  • amino acid residues such as glycine, serine, alanine and/or proline.
  • Suitable amino acid linkers are disclosed in U.S. Pat. Nos. 8,580,922 and 5,990,275, for example, which are incorporated herein by reference.
  • intermediary amino acid sequences can comprise one or more other types of proteins and/or domains.
  • a marker protein e.g., a fluorescent protein such as any of those disclosed herein
  • an intermediary amino acid sequence can be comprised in an intermediary amino acid sequence.
  • a conformation-specific NP binding agent and at least one CPP can be covalently linked in a TAGE via crosslinking (chemical crosslinking).
  • Crosslinking herein refers to a process of chemically joining two or more molecules (a conformation-specific NP binding agent and at least one CPP, in this case) by a covalent bond(s).
  • Crosslinking can be performed using any number of processes known in the art, such as those disclosed in U.S. Patent Appl. Publ. No. 2011 /0190813, U.S. Pat. No. 8,642,744, and Bioconjugate Techniques, 2nd Edition (G. T. Flermanson, Academic Press, 2008), which are all incorporated herein by reference.
  • a CPP and/or a conformation-specific NP binding agent can be modified and/or synthesized to contain a suitable protein linking group at its N-terminus, C-terminus, and/or an amino acid side group, for the purpose of crosslinking the CPP to a conformation-specific NP binding agent.
  • suitable protein linking group at its N-terminus, C-terminus, and/or an amino acid side group, for the purpose of crosslinking the CPP to a conformation-specific NP binding agent.
  • Examples of chemical crosslinkers are further described herein.
  • a conformation-specific NP binding agent and at least one CPP herein can be non-covalently linked to each other in a TAGE in certain aspects herein using a variety of approaches known in the art. Though not intending to be held to any particular theory or mechanism, it is contemplated that a non-covalent linkage between a conformation-specific NP binding agent and at least one CPP can be due to electrostatic, Van der Waals, and/or hydrophobic forces.
  • More than one type of CPP can be covalently or non-covalently linked to a conformation-specific NP binding agent in certain embodiments.
  • the ratio (molar ratio) of CPP(s) to conformation-specific NP binding agent that can be used to prepare such an agent can be at least about 1 :1 , 2:1 , 3:1 , 4:1 , 5:1 , 6:1 , 7:1 , 8:1 , 9:1 , 10:1 15:1 , 20:1 , 30:1 ,
  • the average number of CPPs non-covalently linked to the conformation-specific NP binding agent may be at least 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15,
  • the provided herein is a method of modifying the genome of a target cell, the method comprising contacting the target cell with a targeted active gene editing (TAGE) agent comprising a CPP and a confirmation specific-NP binding agent, as described herein.
  • the target cell is a eukaryotic cell.
  • the eukaryotic cell is a mammalian cell.
  • the mammalian cell is a mouse cell, a non-human primate cell, or a human cell.
  • the site-directed modifying polypeptide of the TAGE agent comprising the CPP produces a cleavage site at the target region of the genome, thereby modifying the genome.
  • the target region of the genome is a target gene.
  • a method comprising the use of a TAGE agent (e.g., a TAGE agent comprising a CPP and a confirmation specific-NP binding agent) described herein is effective to modify expression of the target gene.
  • the method is effective to increase expression of the target gene relative to a reference level.
  • the method is effective to decrease expression of the target gene relative to a reference level.
  • the TAGE includes a two or more cell targeting agents in addition to the conformation specific NP binding agent, e.g., a CPP and an antibody, a CPP and a ligand, or a ligand and antibody.
  • Such pairings can include agents that bind to target molecules on the surface of a cell, e.g., an antibody / ligand pairing.
  • Class pairings can, in certain embodiments, improve internalization of the site-directed modifying polypeptide.
  • a class pairing includes a TAGE agent comprising a CPP, an antigen binding polypeptide (e.g., an antibody), and a site-directed modifying polypeptide, in any arrangement.
  • a TAGE agent comprises an antibody, a peptide cell surface TCR, and a site-directed modifying polypeptide, in any arrangement.
  • the TAGE agent comprises one or more CPPs and one or more antigen-binding polypeptides. In certain embodiments, the TAGE agent comprises two or more CPPs and one or more antigen-binding polypeptides. In other embodiments, the TAGE agent comprises four or more CPPs and one or more antigen-binding polypeptides. In some embodiments, the TAGE agent comprises six or more CPPs and one or more antigen-binding polypeptides. In some embodiments, the TAGE agent comprises eight or more CPPs and one or more antigen-binding polypeptides.
  • the TAGE agent comprises one or more CPPs and one or more ligands. In certain embodiments, the TAGE agent comprises two or more CPPs and one or more ligands. In other embodiments, the TAGE agent comprises four or more CPPs and one or more ligands. In some embodiments, the TAGE agent comprises six or more CPPs and one or more ligands. In some embodiments, the TAGE agent comprises eight or more CPPs and one or more ligands.
  • the TAGE agent comprises one or more antigen-binding polypeptides and one or more ligands.
  • the TAGE agent comprises one or more antigen-binding polypeptides, one or more CPPs, and one or more ligands.
  • a TAGE described herein can be used to modify the genome of a target cell.
  • the method comprises contacting the target cell with a TAGE disclosed herein, such that at least a nucleoprotein (e.g., a nucleoprotein comprising a nucleic acid-guided nuclease and a guide nucleic acid) associated with the conformation-specific NP binding agent is internalized into the cell and subsequently modifies the genome (or target nucleic acid) of the targeted cell.
  • a nucleoprotein e.g., a nucleoprotein comprising a nucleic acid-guided nuclease and a guide nucleic acid
  • Such methods may be used in an in vitro setting or in vivo, including for therapeutic use where the modification of the genome of a subject in need thereof results in treatment of a disease or disorder.
  • a TAGE described herein can be used to target a nucleoprotein (e.g., a nucleoprotein comprising a nucleic acid-guided nuclease and a guide nucleic acid) to any cell displaying an antigen of interest.
  • the cell can be a eukaryotic cell, including, but not limited to, a mammalian cell.
  • mammalian cells that can be targeted (and have their genome’s modified) by the TAGE of the invention include, but are not limited to, a mouse cell, a non-human primate cell, or a human cell.
  • a TAGE agent in certain instances, can be used to edit specific cell types ex vivo or in vivo, such as hematopoietic stem cells (HSCs), hematopoietic progenitor stem cells (HPSCs), natural killer cells, macrophages, DC cells, non-DC myeloid cells, B cells, T cells (e.g., activated T cells), fibroblasts, or other cells.
  • the T cells are CD4 or CD8 T cells.
  • the T cells are regulatory T cells (T regs) or effector T cells.
  • the T cells are tumor infiltrating T cells.
  • the cell is a hematopoietic stem cell (HSC) or a hematopoietic progenitor cells (HPSCs).
  • the macrophages are M0, M1 , or M2 macrophages.
  • the TAGE agent is used to edit multiple (e.g., two or more) cell types selected from hematopoietic stem cells, hematopoietic progenitor stem cells (HPSCs), natural killer cells, macrophages, DC cells, non-DC myeloid cells, B cells, T cells (e.g., activated T cells), and fibroblasts.
  • a TAGE in certain instances, can be used to edit specific cell types ex vivo or in vivo, such as hematopoietic stem cells (HSCs), hematopoietic progenitor stem cells (HPSCs), natural killer cells, macrophages, DC cells, non-DC myeloid cells, B cells, T cells (e.g., activated T cells), fibroblasts, or other cells.
  • the T cells are CD4 or CD8 T cells.
  • the T cells are regulatory T cells (T regs) or effector T cells.
  • the T cells are tumor infiltrating T cells.
  • the cell is a hematopoietic stem cell (HSC) or a hematopoietic progenitor cells (HPSCs).
  • the macrophages are M0, M1 , or M2 macrophages.
  • the TAGE agent is used to edit multiple (e.g., two or more) cell types selected from hematopoietic stem cells, hematopoietic progenitor stem cells (HPSCs), natural killer cells, macrophages, DC cells, non-DC myeloid cells, B cells, T cells (e.g., activated T cells), and fibroblasts.
  • the TAGE agent comprises a CPP and the method comprises contacting a T cell (e.g., a human T cell) with the TAGE agent.
  • the TAGE agent comprises a CPP and the method comprises contacting a macrophage (e.g., a human macrophage) with the TAGE agent.
  • the TAGE agent comprises a CPP and the method comprises contacting an HSC (e.g., a human HSC) with the TAGE agent.
  • the TAGE agent comprises CPP and the method comprises contacting a cell in the bone marrow of a subject with the TAGE agent.
  • the cell is not a hematopoietic stem cell (e.g., fibroblast, macrophages, osteoblasts, osteoclasts, or endothelial cells).
  • the TAGE agent comprises at least four NLSs and the method comprises contacting a T cell (e.g., a human T cell) with the TAGE agent.
  • the TAGE agent comprises at least four NLSs and the method comprises contacting a macrophage (e.g., a human macrophage) with the TAGE agent.
  • the TAGE agent comprises a CPP and the method comprises contacting an HSC (e.g., a human HSC) with the TAGE agent.
  • the TAGE agent comprises at least six NLSs and the method comprises contacting a T cell (e.g., a human T cell) with the TAGE agent.
  • the TAGE agent comprises at least four NLSs and the method comprises contacting a macrophage (e.g., a human macrophage) with the TAGE agent.
  • the TAGE agent comprises at least six NLSs and the method comprises contacting an HSC (e.g., a human HSC) with the TAGE agent.
  • the TAGE agent comprises at least six NLSs and the method comprises contacting a fibroblast (e.g., a human fibroblast) with the TAGE agent.
  • the TAGE agent comprises a His-TAT-NLS (HTN) peptide and the method comprises contacting a T cell (e.g., a human T cell) with the TAGE agent.
  • the TAGE agent comprises an HTN peptide and the method comprises contacting a macrophage (e.g., a human macrophage) with the TAGE agent.
  • the TAGE agent comprises an HTN peptide and the method comprises contacting an HSC (e.g., a human HSC) with the TAGE agent.
  • the TAGE agent comprises an HTN peptide and the method comprises contacting a fibroblast (e.g., a human fibroblast) with the TAGE agent.
  • the TAGE agent comprises a IMT-P8 peptide and the method comprises contacting a T cell (e.g., a human T cell) with the TAGE agent.
  • the TAGE agent comprises IMT-P8 and the method comprises contacting a macrophage (e.g., a human macrophage) with the TAGE agent.
  • the TAGE agent comprises IMT-P8 and the method comprises contacting an HSC (e.g., a human HSC) with the TAGE agent.
  • the TAGE agent comprises a IMT-P8 peptide and the method comprises contacting a fibroblast (e.g., a human fibroblast) with the TAGE agent.
  • the TAGE agent comprises a ligand and the method comprises contacting a T cell (e.g., a human T cell) with the TAGE agent.
  • the TAGE agent comprises a ligand and the method comprises contacting a macrophage (e.g., a human macrophage) with the TAGE agent.
  • the TAGE agent comprises a ligand and the method comprises contacting an HSC (e.g., a human HSC) with the TAGE agent.
  • the TAGE agent comprises a ligand and the method comprises contacting a fibroblast (e.g., a human fibroblast) with the TAGE agent.
  • the TAGE agent comprises an IL-2 ligand and the method comprises contacting a T cell (e.g., a human T cell, such as a CD4+ T cell or a CD8+ T cell) with the TAGE agent.
  • a T cell e.g., a human T cell, such as a CD4+ T cell or a CD8+ T cell
  • the TAGE agent comprises IFNy and the method comprises contacting a macrophage (e.g., a human macrophage) with the TAGE agent.
  • a macrophage e.g., a human macrophage
  • the TAGE agent comprises MCS-F and the method comprises contacting a macrophage (e.g., a human macrophage) with the TAGE agent.
  • a macrophage e.g., a human macrophage
  • the TAGE agent comprises an antibody and the method comprises contacting a T cell (e.g., a human T cell) with the TAGE agent.
  • the TAGE agent comprises an antibody and the method comprises contacting a macrophage (e.g., a human macrophage) with the TAGE agent.
  • the TAGE agent comprises an antibody and the method comprises contacting an HSC (e.g., a human HSC) with the TAGE agent.
  • the TAGE agent comprises an antibody and the method comprises contacting a fibroblast (e.g., a human fibroblast) with the TAGE agent.
  • the TAGE agent comprises an anti-FAP antibody and the method comprises contacting a fibroblast (e.g., a human fibroblast) with the TAGE.
  • the TAGE agent comprises an anti-CTLA-4 antibody and the method comprises contacting a T cell (e.g., a human T cell) with the TAGE agent.
  • a T cell e.g., a human T cell
  • the TAGE agent comprises an anti-CD25 antibody and the method comprises contacting a T cell (e.g., a human T cell) with the TAGE agent.
  • a T cell e.g., a human T cell
  • the TAGE agent comprises an anti-CD11 a antibody and the method comprises contacting a T cell (e.g., a human T cell) with the TAGE agent.
  • a T cell e.g., a human T cell
  • the nucleic acid-guided nuclease produces a cleavage site at the target region of the genome of the target cell, subsequently modifying the genome of the cell and impacting gene expression.
  • the target region of the genome is a target gene.
  • the ability of the nucleic acid-guided nuclease to modify the genome of the target cell provides, in certain embodiments, a way to modify expression of the target gene.
  • Expression levels of a target nucleic acid, e.g., a gene can be determined according to standard methods, where in certain circumstances, the method disclosed herein is effective to increase expression of the target gene relative to a reference level.
  • the method disclosed herein is able to decrease expression of the target gene relative to a reference level.
  • Reference levels can be determined in standard assays using a non-specific guide RNA/site-directed modifying polypeptide, where increases or decreases in the target nucleic acid, e.g., gene, may be measured relative to the control.
  • Internalization of the TAGE, or a component thereof can be determined according to standard internalization assays. In one embodiment, at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 8%, at least 9%, at least 10%, or at least 15% of the TAGE or component thereof (e.g., nucleic acid-guided nuclease) is internalized by the cell within a given time (e.g., one hour, two hours, three hours, or more than three hours) of contact of the TAGE with a cell, such as a cell comprising an extracellular cell-bound antigen.
  • a given time e.g., one hour, two hours, three hours, or more than three hours
  • the TAGE is internalized by a target cell within one hour of contact of the TAGE with the cell such as a cell comprising an extracellular cell-bound antigen, at a higher efficiency versus a control agent, e.g., an unconjugated (i.e., without the cell targeting agent) conformation-specific NP binding agent stably associated with a nucleoprotein comprising a nucleic acid-guided nuclease and a guide nucleic acid).
  • a control agent e.g., an unconjugated (i.e., without the cell targeting agent) conformation-specific NP binding agent stably associated with a nucleoprotein comprising a nucleic acid-guided nuclease and a guide nucleic acid).
  • Internalization of the TAGE, or a component thereof can be assessed using any internalization assays known in the art.
  • internalization of a TAGE, or a component thereof can be assessed by attaching a detectable label (e.g. a fluorescent dye) to the peptide (and/or to the cargo to be transfected) or by fusing the peptide with a reporter molecule, thus enabling detection once cellular uptake has occurred, e.g., by means of FACS analysis or via specific antibodies.
  • a detectable label e.g. a fluorescent dye
  • one or more components of the TAGE is conjugated to a reporter molecule having a quenchable signal.
  • a FACS-based internalization assay can be utilized based on the detection of Alexa-488 labeled TAGE components (e.g., a nucleic acid-guided nuclease component or nucleic acid guide) following incubation of the labeled component with cells for a given period of time, after which the results achieved with or without quenching with an anti- A488 antibody are compared.
  • Labeled molecules that are internalized by a target cell are protected from quenching by the anti-A488 antibody and therefore retain a stronger Alexa488 signal relative to a control following quenching.
  • labeled molecules that are not internalized, and therefore remain on the cell surface are susceptible to quenching by the anti-A488 antibody and therefore display a reduced Alexa488 signal relative to an unquenched control.
  • the TAGE described herein can be used to target a nucleoprotein (e.g., a nucleoprotein comprising a nucleic acid-guided nuclease and a guide nucleic acid) to any cell that can be targeted by a given cell targeting agent.
  • the cell can be a eukaryotic cell, including, but not limited to, a mammalian cell.
  • mammalian cells that can be targeted (and have their genome’s modified) by the TAGE of the invention include, but are not limited to, a mouse cell, a non-human primate cell, or a human cell.
  • the eukaryotic cell can be one that exists (i) in an organism/tissue in vivo, (ii) in a tissue or group of cells ex vivo, or (iii) in an in vitro state.
  • the eukaryotic cell herein can be as it exists in an isolated state (e.g., in vitro cells, cultured cells) or a non-isolated state (e.g., in a subject, e.g., a mammal, such as a human, non-human primate, or a mouse).
  • a eukaryotic cell in certain embodiments is a mammalian cell, such as a human cell.
  • a TAGE agent to edit a target nucleic acid, e.g., gene, in a target cell can be determined according to methods known in the art, including, for example, phenotypic assays or sequencing assays. Such assays may determine the presence or absence of a marker associated with the gene or nucleic acid of the target cell that is being edited by the TAGE agent.
  • a CD47 flow cytometry assay can be used to determine the efficacy of a TAGE agent for gene editing.
  • an endogenous CD47 gene sequence in the target cell is targeted by the TAGE agent, where editing is evidenced by a lack of CD47 expression on the cell surface of the target cell.
  • Levels of CD47 can be measured in a population of cells and compared to a control TAGE agent where a non-targeting guide RNA is used as a negative control in the same type of target cell. Decreases in the level of CD47, for example, relative to the control indicates gene editing of the TAGE agent. In certain instances, a decrease of at least 1%, at least 2%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, and so forth, relative to a control in a testing assay indicates nucleic acid, e.g., gene, editing by the TAGE agent. Ranges of the foregoing percentages are also contemplated herein. Other ways in which nucleic acid, e.g., gene, editing activity of a TAGE agent can be determined include sequence based assays, e.g., amplicon sequencing, known in the art.
  • an endogenous sequence in the target cell is targeted by the TAGE agent, where editing is evidenced by an increase in expression of a marker on the cell surface of the target cell or intracellular to account for tDtomato, fluorescent (GFP), etc., reporters.
  • increases in the level of a marker as detected by flow cytometry, for example, relative to the control indicates gene editing of the TAGE agent.
  • an increase of the cell surface marker of at least 1 %, at least 2%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, and so forth, relative to a control in a testing assay indicates nucleic acid, e.g., gene, editing by the TAGE agent.
  • an increase in expression of the cell surface marker of at least 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, or more, and so forth, relative to a control in a testing assay indicates nucleic acid, e.g., gene, editing by the TAGE agent.
  • nucleic acid e.g., gene
  • an increase in the expression of a fluorescent marker e.g., TdTomato fluorescent system
  • Ranges of the foregoing percentages are also contemplated herein.
  • Other ways in which nucleic acid, e.g., gene, editing activity of a TAGE agent can be determined include sequence based assays, e.g., amplicon sequencing, known in the art.
  • the TAGE agent targets an endogenous gene sequence (e.g., CD47) encoding a cell surface protein in the target cell, and editing is evidenced by the percentage of target cells that lack expression of the cell surface protein on the cell surface of the target cell. In some embodiments, the percentage of target cells that lack expression of the cell surface protein, as detected by flow cytometry, for example, relative to the control indicates gene editing of the TAGE agent.
  • endogenous gene sequence e.g., CD47
  • absence of a cell surface protein in at least 0.05%, at least 0.1%, at least 0.2%, at least 0.3%, at least 0.4%, at least 0.5%, at least 0.6%, at least 0.7%, at least 0.8%, at least 0.9%, at least 1%, at least 2%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, and so forth of target cells in a population of target cells as detected by a testing assay indicates nucleic acid, e.g., gene, editing by the TAGE agent. Ranges of the foregoing percentages are also contemplated herein.
  • the percentage of target cells with an absence of a cell surface protein is increased by at least 2-fold, 3-fold, 4- fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, or more, relative to a control in a testing assay indicates nucleic acid, e.g., gene, editing by the TAGE agent.
  • nucleic acid e.g., gene, editing by the TAGE agent.
  • sequence based assays e.g., amplicon sequencing, known in the art.
  • an endogenous sequence in the target cell is targeted by the TAGE agent, where editing is evidenced by a change in fold of the level of gene editing relative to a control (e.g., a non-edited target cell).
  • a control e.g., a non-edited target cell.
  • a certain fold increase or decrease of a cell surface marker as detected by flow cytometry would indicate nucleic acid, e.g., gene, editing relative to a control, e.g., a TAGE agent with a non-targeting guide RNA, or a TAGE agent which lacks the antigen binding polypeptide as a negative control.
  • the fold increase of the cell surface marker is at least 1 fold, at least 2 fold, at least 3 fold, at least 4 fold, at least 5 fold, at least 1 -5 fold, at least 1 -4 fold, at least 2-5 fold higher in level, and so forth, relative to a control.
  • an increase in expression of the cell surface marker of at least 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, or more, and so forth, relative to a control in a testing assay indicates nucleic acid, e.g., gene, editing by the TAGE agent.
  • a decrease in expression of the cell surface marker of at least 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9- fold, 10-fold, or more, and so forth, relative to a control in a testing assay indicates nucleic acid, e.g., gene, editing by the TAGE agent.
  • nucleic acid e.g., gene, editing by the TAGE agent.
  • Such a fold increase or decrease would indicate nucleic acid, e.g., gene, editing by the TAGE agent.
  • an increase in expression of the cell surface marker of at least 2- fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, or more, and so forth, relative to a control in a testing assay indicates nucleic acid, e.g., gene, editing by the TAGE agent. Ranges of the foregoing fold changes are also contemplated herein. Other ways in which nucleic acid, e.g., gene, editing activity of a TAGE agent can be determined include sequence based assays, e.g., amplicon sequencing, known in the art.
  • the protein components of the TAGE can be produced using any method known in the art, e.g., through covalent or non-covalent linkages, or expression in a suitable host cell from nucleic acid encoding components of the TAGE.
  • a number of methods are known in the art for producing proteins.
  • the components of the TAGE can be produced in and isolated from yeast, bacteria, insect cell lines, plants, transgenic animals, or cultured mammalian cells; see, e.g., Palomares et al., "Production of Recombinant Proteins: Challenges and Solutions," Methods Mol Biol. 2004; 267:15-52.
  • the cell targeting agents can be linked to a moiety that facilitates transfer into a cell, e.g., a lipid nanoparticle, optionally with a linker that is cleaved once the protein is inside the cell.
  • the cell targeting agent may deliver a conformation-specific NP binding agent stably associated with a nucleoprotein (e.g., a nucleoprotein comprising a nucleic acid- guided nuclease and a guide nucleic acid) into a cell via an endocytic process.
  • a nucleoprotein e.g., a nucleoprotein comprising a nucleic acid- guided nuclease and a guide nucleic acid
  • a conformation-specific NP binding agent stably associated with a nucleoprotein (e.g., a nucleoprotein comprising a nucleic acid- guided nuclease and a guide nucleic acid) into a cell via an endocytic process.
  • a conformation-specific NP binding agent stably associated with a nucleoprotein (e.g., a nucleoprotein comprising a nucleic acid- guided nuclease and a guide nucleic acid) into a cell
  • nucleoprotein e.g., a nucleoprotein comprising a nucleic acid-guided nuclease and a guide nucleic acid
  • a nucleoprotein e.g., a nucleoprotein comprising a nucleic acid-guided nuclease and a guide nucleic acid
  • the nucleoprotein includes at least one (e.g., at least 1 , 2, 3, 4, or more) nuclear-targeting sequence (e.g., NLS).
  • the ability to traverse an organelle membrane such as a nuclear membrane or mitochondrial membrane does not depend on the presence of a nuclear-targeting sequence.
  • the nucleoprotein e.g., a nucleoprotein comprising a nucleic acid-guided nuclease and a guide nucleic acid
  • the nucleoprotein does not include an NLS.
  • the TAGE agent is administered to cells ex vivo, such as hematopoietic stem cells (HSCs) or hematopoietic progenitor stem cells (HSPCs).
  • HSCs hematopoietic stem cells
  • HSPCs hematopoietic progenitor stem cells
  • TAGE- edited HSCs may then be transplanted into a subject in need of a hematopoietic stem cell transplant.
  • the TAGE agent described herein may be administered to a subject, e.g., by local administration.
  • the TAGE agent may be administered to the subject transdermally, subcutaneously, intravenously, intramuscularly, intraocularly, intraosseously, or intratumorally.
  • the TAGE agent may be administered to a subject in a therapeutically effective amount (e.g., in an amount to achieve a level of genome editing that treats or prevents a disease in a subject).
  • a therapeutically effective amount of a TAGE agent may be administered to a subject having a cancer (e.g., a colon carcinoma or a melanoma), an eye disease, or a stem cell disorder.
  • a therapeutically effective amount may depend on the mode of delivery, e.g., whether the TAGE agent is administered locally (e.g., by intradermal (e.g., via the flank or ear in the case of a mouse), intratumoral, intraosseous, intraocular, or intramuscular injection) or systemically.
  • TAGE agents described herein may be formulated to be compatible with the intended route of administration, such as by intradermal, intratumoral, intraosseous, intraocular, or intramuscular injection.
  • Solutions, suspensions, dispersions, or emulsions may be used for such administrations and may include a sterile diluent, such as water for injection, saline solution, polyethylene glycols, glycerin, propylene glycol or other synthetic solvents; anti-bacterial agents such as benzyl alcohol or methylparabens; antioxidants such as ascorbic acid or sodium bisulfate; buffers such as acetates, citrates or phosphates, and agents for the adjustment of tonicity such as sodium chloride or dextrose.
  • a sterile diluent such as water for injection, saline solution, polyethylene glycols, glycerin, propylene glycol or other synthetic solvents
  • anti-bacterial agents such as benzyl alcohol or methylparab
  • a pharmaceutical composition comprises a TAGE agent and a pharmaceutically acceptable carrier.
  • the TAGE agents can be included in a kit, container, pack or dispenser, together with medical devices suitable for delivering the compositions to a subject, such as by intradermal, intratumoral, intraosseous, intraocular, or intramuscular injection.
  • kits may be supplied in containers of any sort such that the life of the different components may be preserved and may not be adsorbed or altered by the materials of the container.
  • sealed glass ampules or vials may contain the compositions described herein that have been packaged under a neutral non-reacting gas, such as nitrogen.
  • Ampules may consist of any suitable material, such as glass, organic polymers, such as polycarbonate, polystyrene, etc., ceramic, metal or any other material typically employed to hold reagents.
  • suitable containers include bottles that are fabricated from similar substances as ampules, and envelopes that consist of foil-lined interiors, such as aluminum or an alloy.
  • Other containers include test tubes, vials, flasks, bottles, syringes, etc.
  • Some containers may have a sterile resealable access port, such as a bottle having a stopper that may be pierced repeatedly by a hypodermic injection needle.
  • a TAGE agent may be administered to a subject by a route in accordance with the therapeutic goal.
  • routes may be used to deliver a TAGE agent to desired cells or tissues, including systemic or local delivery.
  • a TAGE agent is administered to a subject having a cancer, such as a colon carcinoma or a melanoma.
  • the cancer is, for example, a melanoma, a urogenetical cancer, a non-small cell lung cancer, a small-cell lung cancer, a lung cancer, a leukemia, a hepatocarcinoma, a retinoblastoma, an astrocytoma, a glioblastoma, a gum cancer, a tongue cancer, a neuroblastoma, a head cancer, a neck cancer, a breast cancer, a pancreatic cancer, a prostate cancer, a renal cancer, a bone cancer, a testicular cancer, an ovarian cancer, a mesothelioma, a cervical cancer, a gastrointestinal cancer, a lymphoma, a myeloma, a brain cancer, a colon cancer, a sarcom
  • the cancer may be a primary cancer or a metastasized cancer.
  • the TAGE agent may be injected directly into a tumor (i.e. , by intratumoral injection) in a subject, for instance, in an amount effective to edit one or more cell types in the tumor (e.g., macrophages, CD4+ T cells, CD8+ T cells, or fibroblasts).
  • TAGE agents of the present disclosure may be used to treat a solid tumor in subject (e.g., a human) by administering the TAGE agent intratumorally.
  • a TAGE agent may be injected directly into a solid tumor with a needle, such as a Turner Biopsy Needle or a Chiba Biopsy Needle.
  • a TAGE agent may be administered within the thorax using a bronchoscope or other device capable of cannulating the bronchial. Masses accessible via the bronchial tree may be directly injected by using a widely available transbronchial aspiration needles.
  • a TAGE agent may also be implanted within a solid tumor using any suitable method known to those skilled in the art of penetrating tumor tissue. Such techniques may include creating an opening into the tumor and positioning a TAGE agent in the tumor.
  • a TAGE agent may be injected into the bone marrow (i.e., intraosseous injection) of a subject.
  • Intraosseous delivery may be used to edit bone marrow cells (e.g., hematopoietic stem cells (HSCs)) in a subject.
  • a TAGE agent of the present disclosure may be used to treat a stem cell disorder in a subject (e.g., a human) where bone marrow cells, e.g., HSCS, are modified in such a way as to provide treatment for a stem cell disorder.
  • stem cell disorders include, but are not limited to, disorders treatable with autologous transplantation, including Hodgkin lymphoma, non-Hodgkin lymphoma, or multiple myeloma.
  • disorders treatable with autologous transplantation including Hodgkin lymphoma, non-Hodgkin lymphoma, or multiple myeloma.
  • Another example of a stem cell disorder that may be treated with the compositions and methods disclosed herein is a hemoglobinopathy.
  • a TAGE agent may be injected directly into the ocular compartment of a subject, e.g., a human, in an amount effective to edit subretinal cells (e.g., retinal pigment epithelium (RPE) or photoreceptors).
  • subretinal cells e.g., retinal pigment epithelium (RPE) or photoreceptors
  • TAGE agents of the present disclosure may be used to treat an eye disease in a subject (e.g., a human) by administering the TAGE agent intraocularly (e.g., by subretinal injection).
  • CPP based TAGE agents are particularly advantageous for local delivery as described in the examples below, e.g., Examples 48 (intraosseous), Example 49 (intraocular), and Example 51 (intratumoral).
  • a TAGE agent comprising a CPP, or a class paired TAGE agent containing a CPP and a ligand or an antigen binding polypeptide (e.g., antibody), may be administered to a human subject via local delivery.
  • Local delivery refers to delivery to a specific location on a body where the TAGE agent will act within the region it is delivered to, and not systemically.
  • Examples of local delivery for a TAGE agent containing a CPP as an extracellular membrane binding moiety include topical administration, ocular delivery, intra-articular delivery, intra cardiac delivery, intradermal, intracutaneous delivery, intraosseous delivery, intrathecal delivery, or inhalation.
  • a TAGE agent comprising a ligand or an antigen binding polypeptide (e.g., an antibody or an antigen-binding fragment thereof), or a class paired TAGE agent comprising a ligand or an antigen binding polypeptide (e.g., an antibody or an antigen-binding fragment thereof), is administered to a human subject via systemic administration.
  • systemic delivery for a TAGE agent containing a ligand or an antigen binding polypeptide (e.g., an antibody or an antigen binding fragment thereof), extracellular membrane binding moiety include intravenous injection or intraperitoneal injection.
  • nucleic acid-guided nuclease refers to a nuclease that is directed to a specific target sequence based on the complementarity (full or partial) between a guide nucleic acid (i.e., guide RNA or gRNA, guide DNA or gDNA, or guide DNA/RNA hybrid) that is associated with the nuclease and a target sequence.
  • the nucleic acid-guided nuclease is a RNA guided nuclease. The binding between the guide RNA and the target sequence serves to recruit the nuclease to the vicinity of the target sequence.
  • Non-limiting examples of nucleic acid-guided nucleases suitable for the presently disclosed compositions and methods include naturally-occurring Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-associated (Cas) polypeptides from a prokaryotic organism (e.g., bacteria, archaea) or variants thereof.
  • CRISPR Clustered Regularly Interspaced Short Palindromic Repeats
  • Cas Clustered Regularly Interspaced Short Palindromic Repeats
  • CRISPR sequences found within prokaryotic organisms are sequences that are derived from fragments of polynucleotides from invading viruses and are used to recognize similar viruses during subsequent infections and cleave viral polynucleotides via CRISPR-associated (Cas) polypeptides that function as an RNA-guided nuclease to cleave the viral polynucleotides.
  • CRISPR-associated polypeptide or “Cas polypeptide” refers to a naturally-occurring polypeptide that is found within proximity to CRISPR sequences within a naturally-occurring CRISPR system. Certain Cas polypeptides function as RNA- guided nucleases.
  • CRISPR systems There are at least two classes of naturally-occurring CRISPR systems, Class 1 and Class 2.
  • the nucleic acid-guided nucleases of the presently disclosed compositions and methods are Class 2 Cas polypeptides or variants thereof given that the Class 2 CRISPR systems comprise a single polypeptide with nucleic acid-guided nuclease activity, whereas Class 1 CRISPR systems require a complex of proteins for nuclease activity.
  • Class 2 CRISPR systems There are at least three known types of Class 2 CRISPR systems, Type II, Type V, and Type VI, among which there are multiple subtypes (subtype II- A, ll-B, ll-C, V-A, V-B, V-C, Vl-A, Vl-B, and Vl-C, among other undefined or putative subtypes).
  • Type II and Type V-B systems require a tracrRNA, in addition to crRNA, for activity.
  • Type V-A and Type VI only require a crRNA for activity.
  • All known Type II and Type V RNA- guided nucleases target double-stranded DNA, whereas all known Type VI RNA-guided nucleases target single-stranded RNA.
  • the RNA-guided nucleases of Type II CRISPR systems are referred to as Cas9 herein and in the literature.
  • the nucleic acid-guided nuclease of the presently disclosed compositions and methods is a Type II Cas9 protein or a variant thereof.
  • Type V Cas polypeptides that function as RNA-guided nucleases do not require tracrRNA for targeting and cleavage of target sequences.
  • the RNA-guided nuclease of Type VA CRISPR systems are referred to as Cpf1 ; of Type VB CRISPR systems are referred to as C2C1 ; of Type VC CRISPR systems are referred to as Cas12C or C2C3; of Type VIA CRISPR systems are referred to as C2C2 or Cas13A1 ; of Type VIB CRISPR systems are referred to as Cas13B; and of Type VIC CRISPR systems are referred to as Cas13A2 herein and in the literature.
  • the nucleic acid-guided nuclease of the presently disclosed compositions and methods is a Type VA Cpf1 protein or a variant thereof.
  • Naturally-occurring Cas polypeptides and variants thereof that function as nucleic acid- guided nucleases are known in the art and include, but are not limited to Streptococcus pyogenes Cas9, Staphylococcus aureus Cas9, Streptococcus thermophilus Cas9, Francisella novicida Cpf1 , or those described in Shmakov et al. (2017) Nat Rev Microbiol 15(3) :169-182; Makarova et al.
  • Class 2 Type V CRISPR nucleases include Cas12 and any subtypes of Cas12, such as Cas12a, Cas12b, Cas12c, Cas12d, Cas12e, Cas12f, Cas12g, Cas12h, and Cas12i.
  • Class 2 Type VI CRISPR nucleases including Cas13 can be used in order to cleave RNA target sequences.
  • nucleic acid-guided nuclease of the presently disclosed compositions and methods can be a naturally-occurring nucleic acid-guided nuclease (e.g., S. pyogenes Cas9) or a variant thereof.
  • a naturally-occurring nucleic acid-guided nuclease e.g., S. pyogenes Cas9
  • a variant thereof e.g., S. pyogenes Cas9
  • Variant nucleic acid-guided nucleases can be engineered or naturally occurring variants that contain substitutions, deletions, or additions of amino acids that, for example, alter the activity of one or more of the nuclease domains, fuse the nucleic acid-guided nuclease to a heterologous domain that imparts a modifying property (e.g., transcriptional activation domain, epigenetic modification domain, detectable label), modify the stability of the nuclease, or modify the specificity of the nuclease.
  • a modifying property e.g., transcriptional activation domain, epigenetic modification domain, detectable label
  • a nucleic acid-guided nuclease includes one or more mutations to improve specificity for a target site and/or stability in the intracellular microenvironment.
  • the protein is Cas9 (e.g., SpCas9) or a modified Cas9
  • the nuclease comprises at least one substitution relative to a naturally-occurring version of the nuclease.
  • substitutions may include any of C80A, C80L, C80I, C80V, C80K, C574E, C574D, C574N, C574Q (in any combination) and in particular C80A. Substitutions may be included to reduce intracellular protein binding of the nuclease and/or increase target site specificity. Additionally or alternatively, substitutions may be included to reduce off-target toxicity of the composition.
  • the nucleic acid-guided nuclease is directed to a particular target sequence through its association with a guide nucleic acid (e.g., guideRNA (gRNA), guideDNA (gDNA)).
  • a guide nucleic acid e.g., guideRNA (gRNA), guideDNA (gDNA)
  • the nucleic acid- guided nuclease is bound to the guide nucleic acid via non-covalent interactions, thus forming a complex.
  • the polynucleotide-targeting nucleic acid provides target specificity to the complex by comprising a nucleotide sequence that is complementary to a sequence of a target sequence.
  • the nucleic acid-guided nuclease of the complex or a domain or label fused or otherwise conjugated thereto provides the site-specific activity.
  • the nucleic acid-guided nuclease is guided to a target polynucleotide sequence (e.g. a target sequence in a chromosomal nucleic acid; a target sequence in an extrachromosomal nucleic acid, e.g. an episomal nucleic acid, a minicircle; a target sequence in a mitochondrial nucleic acid; a target sequence in a chloroplast nucleic acid; a target sequence in a plasmid) by virtue of its association with the protein-binding segment of the polynucleotide-targeting guide nucleic acid.
  • a target polynucleotide sequence e.g. a target sequence in a chromosomal nucleic acid; a target sequence in an extrachromosomal nucleic acid, e.g. an episomal nucleic acid, a minicircle; a target sequence in a mitochondrial nucleic acid; a target sequence in a chloroplast nucleic
  • the guide nucleic acid comprises two segments, a “polynucleotide-targeting segment” and a “polypeptide-binding segment.”
  • segment it is meant a segment/section/region of a molecule (e.g., a contiguous stretch of nucleotides in an RNA).
  • a segment can also refer to a region/section of a complex such that a segment may comprise regions of more than one molecule.
  • the polypeptide-binding segment (described below) of a polynucleotide targeting nucleic acid comprises only one nucleic acid molecule and the polypeptide-binding segment therefore comprises a region of that nucleic acid molecule.
  • the polypeptide-binding segment (described below) of a DNA-targeting nucleic acid comprises two separate molecules that are hybridized along a region of complementarity.
  • the polynucleotide-targeting segment (or "polynucleotide-targeting sequence” or “guide sequence”) comprises a nucleotide sequence that is complementary (fully or partially) to a specific sequence within a target sequence (for example, the complementary strand of a target DNA sequence).
  • the polypeptide-binding segment (or "polypeptide-binding sequence") interacts with a nucleic acid-guided nuclease (e.g., RNA-guided nuclease).
  • site-specific cleavage or modification of the target DNA by a nucleic acid-guided nuclease occurs at locations determined by both (i) base-pairing complementarity between the polynucleotide-targeting sequence of the nucleic acid and the target DNA; and (ii) a short motif (referred to as the protospacer adjacent motif (PAM)) in the target DNA.
  • PAM protospacer adjacent motif
  • a protospacer adjacent motif can be of different lengths and can be a variable distance from the target sequence, although the PAM is generally within about 1 to about 10 nucleotides from the target sequence, including about 1 , about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, or about 10 nucleotides from the target sequence.
  • the PAM can be 5' or 3' of the target sequence.
  • the PAM is a consensus sequence of about 3-4 nucleotides, but in particular embodiments, can be 2, 3, 4, 5, 6, 7, 8, 9, or more nucleotides in length.
  • RNA-guided nuclease Methods for identifying a preferred PAM sequence or consensus sequence for a given RNA-guided nuclease are known in the art and include, but are not limited to the PAM depletion assay described by Karvelis et al. (2015) Genome Biol 16:253, or the assay disclosed in Pattanayak et al. (2013) Nat Biotechnol ⁇ (9):839-43, each of which is incorporated by reference in its entirety.
  • the polynucleotide-targeting sequence is the nucleotide sequence that directly hybridizes with the target sequence of interest.
  • the guide sequence is engineered to be fully or partially complementary with the target sequence of interest.
  • the guide sequence can comprise from about 8 nucleotides to about 30 nucleotides, or more.
  • the guide sequence can be about 8, about 9, about 10, about 11 , about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21 , about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, or more nucleotides in length.
  • the guide sequence is about 10 to about 26 nucleotides in length, or about 12 to about 30 nucleotides in length. In particular embodiments, the guide sequence is about 30 nucleotides in length.
  • the degree of complementarity between a guide sequence and its corresponding target sequence, when optimally aligned using a suitable alignment algorithm is about or more than about 50%, about 60%, about 70%, about 75%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91 %, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more.
  • the guide sequence is free of secondary structure, which can be predicted using any suitable polynucleotide folding algorithm known in the art, including but not limited to mFold (see, e.g., Zuker and Stiegler (1981 ) Nucleic Acids Res. 9:133-148) and RNAfold (see, e.g., Gruber et al. (2008) Ce// 106(1 ):23-24).
  • a guide nucleic acid comprises two separate nucleic acid molecules (an “activator-nucleic acid” and a “targeter-nucleic acid”, see below) and is referred to herein as a “double-molecule guide nucleic acid” or a "two-molecule guide nucleic acid.”
  • the subject guide nucleic acid is a single nucleic acid molecule (single polynucleotide) and is referred to herein as a “single-molecule guide nucleic acid,” a “single-guide nucleic acid,” or an “sgNA.”
  • the term “guide nucleic acid” or “gNA” is inclusive, referring both to double-molecule guide nucleic acids and to single-molecule guide nucleic acids (i.e., sgNAs).
  • the gRNA can be a double-molecule guide RNA or a single-guide RNA.
  • the gDNA can be a double molecule guide DNA or a single-guide DNA.
  • An exemplary two-molecule guide nucleic acid comprises a crRNA-like (“CRISPR RNA” or “targeter-RNA” or “crRNA” or “crRNA repeat”) molecule and a corresponding tracrRNA-like (“trans acting CRISPR RNA” or “activator-RNA” or “tracrRNA”) molecule.
  • a crRNA-like molecule comprises both the polynucleotide-targeting segment (single stranded) of the guide RNA and a stretch ("duplex-forming segment") of nucleotides that forms one half of the dsRNA duplex of the polypeptide-binding segment of the guide RNA, also referred to herein as the CRISPR repeat sequence.
  • activator-nucleic acid or “activator-NA” is used herein to mean a tracrRNA-like molecule of a double-molecule guide nucleic acid.
  • targeter-nucleic acid or “targeter-NA” is used herein to mean a crRNA-like molecule of a double-molecule guide nucleic acid.
  • duplex-forming segment is used herein to mean the stretch of nucleotides of an activator-NA or a targeter-NA that contributes to the formation of the dsRNA duplex by hybridizing to a stretch of nucleotides of a corresponding activator-NA or targeter-NA molecule.
  • an activator-NA comprises a duplex-forming segment that is complementary to the duplex-forming segment of the corresponding targeter-NA.
  • an activator-NA comprises a duplex-forming segment while a targeter-NA comprises both a duplex-forming segment and the DNA-targeting segment of the guide nucleic acid. Therefore, a subject double-molecule guide nucleic acid can be comprised of any corresponding activator-NA and targeter-NA pair.
  • the activator-NA comprises a CRISPR repeat sequence comprising a nucleotide sequence that comprises a region with sufficient complementarity to hybridize to an activator-NA (the other part of the polypeptide-binding segment of the guide nucleic acid).
  • the CRISPR repeat sequence can comprise from about 8 nucleotides to about 30 nucleotides, or more.
  • the CRISPR repeat sequence can be about 8, about 9, about 10, about 11 , about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21 , about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, or more nucleotides in length.
  • the degree of complementarity between a CRISPR repeat sequence and the antirepeat region of its corresponding tracr sequence when optimally aligned using a suitable alignment algorithm, is about or more than about 50%, about 60%, about 70%, about 75%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more.
  • a corresponding tracrRNA-like molecule comprises a stretch of nucleotides (duplex-forming segment) that forms the other part of the double-stranded duplex of the polypeptide binding segment of the guide nucleic acid.
  • a stretch of nucleotides of a crRNA-like molecule i.e., the CRISPR repeat sequence
  • a stretch of nucleotides of a tracrRNA-like molecule i.e., the anti-repeat sequence
  • the crRNA-like molecule additionally provides the single stranded DNA-targeting segment.
  • a crRNA-like and a tracrRNA- like molecule hybridize to form a guide nucleic acid.
  • the exact sequence of a given crRNA or tracrRNA molecule is characteristic of the CRISPR system and species in which the RNA molecules are found.
  • a subject double-molecule guide RNA can comprise any corresponding crRNA and tracrRNA pair.
  • a trans-activating-like CRISPR RNA or tracrRNA-like molecule (also referred to herein as an “activator-NA”) comprises a nucleotide sequence comprising a region that has sufficient complementarity to hybridize to a CRISPR repeat sequence of a crRNA, which is referred to herein as the anti-repeat region.
  • the tracrRNA-like molecule further comprises a region with secondary structure (e.g ., stem-loop) or forms secondary structure upon hybridizing with its corresponding crRNA.
  • the region of the tracrRNA-like molecule that is fully or partially complementary to a CRISPR repeat sequence is at the 5' end of the molecule and the 3' end of the tracrRNA-like molecule comprises secondary structure.
  • This region of secondary structure generally comprises several hairpin structures, including the nexus hairpin, which is found adjacent to the anti-repeat sequence.
  • the nexus hairpin often has a conserved nucleotide sequence in the base of the hairpin stem, with the motif UNANNC found in many nexus hairpins in tracrRNAs.
  • terminal hairpins at the 3' end of the tracrRNA that can vary in structure and number, but often comprise a GC-rich Rho-independent transcriptional terminator hairpin followed by a string of U’s at the 3' end. See, for example, Briner et al. (2014) Molecular Cell 56:333-339, Briner and Barrangou (2016) Cold Spring Harb Protoc ; doi: 10.1101/pdb.top090902, and U.S. Publication No. 2017/0275648, each of which is herein incorporated by reference in its entirety.
  • the anti-repeat region of the tracrRNA-like molecule that is fully or partially complementary to the CRISPR repeat sequence comprises from about 8 nucleotides to about 30 nucleotides, or more.
  • the region of base pairing between the tracrRNA-like anti repeat sequence and the CRISPR repeat sequence can be about 8, about 9, about 10, about 11 , about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21 , about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, or more nucleotides in length.
  • the degree of complementarity between a CRISPR repeat sequence and its corresponding tracrRNA-like anti-repeat sequence when optimally aligned using a suitable alignment algorithm, is about or more than about 50%, about 60%, about 70%, about 75%, about 80%, about 81 %, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more.
  • the entire tracrRNA-like molecule can comprise from about 60 nucleotides to more than about 140 nucleotides.
  • the tracrRNA-like molecule can be about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, about 100, about 105, about 110, about 115, about 120, about 125, about 130, about 135, about 140, or more nucleotides in length.
  • the tracrRNA-like molecule is about 80 to about 100 nucleotides in length, including about 80, about 81 , about 82, about 83, about 84, about 85, about 86, about 87, about 88, about 89, about 90, about 91 , about 92, about 93, about 94, about 95, about 96, about 97, about 98, about 99, and about 100 nucleotides in length.
  • a subject single-molecule guide nucleic acid comprises two stretches of nucleotides (a targeter-NA and an activator-NA) that are complementary to one another, are covalently linked by intervening nucleotides ("linkers” or "linker nucleotides”), and hybridize to form the double stranded nucleic acid duplex of the protein-binding segment, thus resulting in a stem-loop structure.
  • the targeter-NA and the activator-NA can be covalently linked via the 3' end of the targeter- NA and the 5' end of the activator-NA.
  • the targeter-NA and the activator-NA can be covalently linked via the 5' end of the targeter-NA and the 3' end of the activator-NA.
  • the linker of a single-molecule DNA-targeting nucleic acid can have a length of from about 3 nucleotides to about 100 nucleotides.
  • the linker can have a length of from about 3 nucleotides (nt) to about 90 nt, from about 3 nt to about 80 nt, from about 3 nt to about 70 nt, from about 3 nt to about 60 nt, from about 3 nt to about 50 nt, from about 3 nt to about 40 nt, from about 3 nt to about 30 nt, from about 3 nt to about 20 nt or from about 3 nt to about 10 nt, including but not limited to about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11 , about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, or more nucleotides.
  • the linker of a single-molecule DNA-targeting nucleic acid is 4 nt.
  • An exemplary single-molecule DNA-targeting nucleic acid comprises two complementary stretches of nucleotides that hybridize to form a double-stranded duplex, along with a guide sequence that hybridizes to a specific target sequence.
  • tracrRNAs Appropriate naturally-occurring cognate pairs of crRNAs (and, in some embodiments, tracrRNAs) are known for most Cas proteins that function as nucleic acid-guided nucleases that have been discovered or can be determined for a specific naturally-occurring Cas protein that has nucleic acid-guided nuclease activity by sequencing and analyzing flanking sequences of the Cas nucleic acid-guided nuclease protein to identify tracrRNA-coding sequence, and thus, the tracrRNA sequence, by searching for known antirepeat-coding sequences or a variant thereof.
  • Antirepeat regions of the tracrRNA comprise one-half of the ds protein-binding duplex.
  • CRISPR repeat The complementary repeat sequence that comprises one-half of the ds protein-binding duplex is called the CRISPR repeat.
  • CRISPR repeat and antirepeat sequences utilized by known CRISPR nucleic acid-guided nucleases are known in the art and can be found, for example, at the CRISPR database on the world wide web at crispr.i2bc.paris-saclay.fr/crispr/.
  • the single guide nucleic acid or dual-guide nucleic acid can be synthesized chemically or via in vitro transcription.
  • Assays for determining sequence-specific binding between a nucleic acid- guided nuclease and a guide nucleic acid are known in the art and include, but are not limited to, in vitro binding assays between an expressed nucleic acid-guided nuclease and the guide nucleic acid, which can be tagged with a detectable label (e.g., biotin) and used in a pull-down detection assay in which the nucleoprotein complex is captured via the detectable label (e.g., with streptavidin beads).
  • a control guide nucleic acid with an unrelated sequence or structure to the guide nucleic acid can be used as a negative control for non-specific binding of the nucleic acid-guided nuclease to nucleic acids.
  • the DNA-targeting RNA, gRNA, or sgRNA or nucleotide sequence encoding the DNA-targeting RNA, gRNA, or sgRNA comprises modifications of the nucleotide sequence.
  • the sgRNA e.g., truncated sgRNA
  • the sgRNA comprises a first nucleotide sequence that is complementary to the target nucleic acid and a second nucleotide sequence that interacts with a Cas polypeptide.
  • the sgRNA comprises one or more modified nucleotides.
  • one or more of the nucleotides in the first nucleotide sequence and/or the second nucleotide sequence are modified nucleotides.
  • the modified nucleotides comprise a modification in a ribose group, a phosphate group, a nucleobase, or a combination thereof.
  • the modification in the ribose group comprises a modification at the 2' position of the ribose group.
  • the modification at the 2' position of the ribose group is selected from the group consisting of 2'-0-methyl, 2'-fluoro, 2'-deoxy, 2'-0-(2-methoxyethyl), and a combination thereof.
  • the modification in the phosphate group comprises a phosphorothioate modification.
  • the modified nucleotides are selected from the group consisting of a 2’-ribo 3’- phosphorothioate (S), 2'-0-methyl (M) nucleotide, a 2'-0-methyl 3'-phosphorothioate (MS) nucleotide, a 2'-0-methyl 3'-thioPACE (MSP) nucleotide, and a combination thereof.
  • the nucleic acid-guided nuclease of the presently disclosed compositions and methods comprise a nuclease variant that functions as a nickase, wherein the nuclease comprises a mutation in comparison to the wild-type nuclease that results in the nuclease only being capable of cleaving a single strand of a double-stranded nucleic acid molecule, or lacks nuclease activity altogether (i.e. , nuclease-dead).
  • a nuclease such as a nucleic acid-guided nuclease, that functions as a nickase only comprises a single functioning nuclease domain.
  • additional nuclease domains have been mutated such that the nuclease activity of that particular domain is reduced or eliminated.
  • the nuclease e.g., RNA-guided nuclease
  • the nuclease lacks nuclease activity completely and is referred to herein as nuclease-dead.
  • all nuclease domains within the nuclease have been mutated such that all nuclease activity of the polypeptide has been eliminated.
  • nucleic acid-guided nuclease Any method known in the art can be used to introduce mutations into one or more nuclease domains of a nucleic acid-guided nuclease, including those set forth in U.S. Publ. Nos. 2014/0068797 and U.S. Pat. No. 9,790,490, each of which is incorporated by reference in its entirety.
  • any mutation within a nuclease domain that reduces or eliminates the nuclease activity can be used to generate a nucleic acid-guided nuclease having nickase activity or a nuclease-dead nucleic acid-guided nuclease.
  • Such mutations are known in the art and include, but are not limited to the D10A mutation within the RuvC domain or H840A mutation within the HNH domain of the S. pyogenes Cas9 or at similar position(s) within another nucleic acid-guided nuclease when aligned for maximal homology with the S. pyogenes Cas9. Other positions within the nuclease domains of S.
  • pyogenes Cas9 that can be mutated to generate a nickase or nuclease-dead protein include G12, G17, E762, N854, N863, H982, H983, and D986.
  • Other mutations within a nuclease domain of a nucleic acid-guided nuclease that can lead to nickase or nuclease-dead proteins include a D917A, E1006A, E1028A, D1227A, D1255A, N1257A, D917A, E1006A, E1028A, D1227A, D1255A, and N1257A of the Francisella novicida Cpf1 protein or at similar position(s) within another nucleic acid- guided nuclease when aligned for maximal homology with the F. novicida Cpf1 protein (U.S. Pat. No. 9,790,490, which is incorporated by reference in its
  • Nucleic acid-guided nucleases comprising a nuclease-dead domain can further comprise a domain capable of modifying a polynucleotide.
  • modifying domains that may be fused to a nuclease-dead domain include but are not limited to, a transcriptional activation or repression domain, a base editing domain, and an epigenetic modification domain.
  • the nucleic acid-guided nuclease comprising a nuclease-dead domain further comprises a detectable label that can aid in detecting the presence of the target sequence.
  • An epigenetic modification domain that can be fused to a nuclease-dead domain can serve to covalently modify DNA or histone proteins to alter histone structure and/or chromosomal structure without altering the DNA sequence itself, leading to changes in gene expression (upregulation or downregulation).
  • Non-limiting examples of epigenetic modifications that can be induced by nucleic acid-guided nuclease include the following alterations in histone residues and the reverse reactions thereof: sumoylation, methylation of arginine or lysine residues, acetylation or ubiquitination of lysine residues, phosphorylation of serine and/or threonine residues; and the following alterations of DNA and the reverse reactions thereof: methylation or hydroxymethylation of cystosine residues.
  • Non limiting examples of epigenetic modification domains thus include histone acetyltransferase domains, histone deacetylation domains, histone methyltransferase domains, histone demethylase domains, DNA methyltransferase domains, and DNA demethylase domains.
  • the nucleic acid-guided nuclease comprises a transcriptional activation domain that activates the transcription of at least one adjacent gene through the interaction with transcriptional control elements and/or transcriptional regulatory proteins, such as transcription factors or RNA polymerases.
  • transcriptional activation domains are known in the art and include, but are not limited to, VP16 activation domains.
  • the nucleic acid-guided nuclease comprises a transcriptional repressor domain, which can also interact with transcriptional control elements and/or transcriptional regulatory proteins, such as transcription factors or RNA polymerases, to reduce or terminate transcription of at least one adjacent gene.
  • transcriptional repression domains are known in the art and include, but are not limited to, IKB and KRAB domains.
  • the nucleic acid-guided nuclease comprising a nuclease-dead domain further comprises a detectable label that can aid in detecting the presence of the target sequence, which may be a disease-associated sequence.
  • a detectable label is a molecule that can be visualized or otherwise observed.
  • the detectable label may be fused to the nucleic acid-guided nuclease as a fusion protein (e.g ., fluorescent protein) or may be a small molecule conjugated to the nuclease polypeptide that can be detected visually or by other means.
  • Detectable labels that can be fused to the presently disclosed nucleic acid-guided nucleases as a fusion protein include any detectable protein domain, including but not limited to, a fluorescent protein or a protein domain that can be detected with a specific antibody.
  • fluorescent proteins include green fluorescent proteins (e.g., GFP, EGFP, ZsGreenl ) and yellow fluorescent proteins (e.g., YFP, EYFP, ZsYellowl ).
  • Non-limiting examples of small molecule detectable labels include radioactive labels, such as 3 H and 35 S.
  • the nucleic acid-guided nuclease can be delivered as part of a fusion protein (e.g., RNA- guided nuclease fusion protein) into a cell as a nucleoprotein complex comprising the nucleic acid- guided nuclease bound to its guide nucleic acid.
  • a fusion protein e.g., RNA- guided nuclease fusion protein
  • the nucleic acid-guided nuclease is delivered as a fusion protein and the guide nucleic acid is provided separately.
  • a guide RNA can be introduced into a target cell as an RNA molecule.
  • the guide RNA can be transcribed in vitro or chemically synthesized.
  • a nucleotide sequence encoding the guide RNA is introduced into the cell.
  • the nucleotide sequence encoding the guide RNA is operably linked to a promoter (e.g., an RNA polymerase III promoter), which can be a native promoter or heterologous to the guide RNA-encoding nucleotide sequence.
  • a nucleic acid sequence encoding the guide RNA and RNA- guided nuclease operably linked to a promoter can be delivered on a vector, such as the expression vector described in detail herein.
  • the nucleic acid-guided nuclease fusion protein can comprise additional amino acid sequences, such as at least one nuclear localization sequence (NLS).
  • Nuclear localization sequences enhance transport of the nucleic acid-guided nuclease into the nucleus of a cell.
  • Proteins that are imported into the nucleus bind to one or more of the proteins within the nuclear pore complex, such as importin/karypherin proteins, which generally bind best to lysine and arginine residues.
  • the best characterized pathway for nuclear localization involves short peptide sequence which binds to the importin-a protein.
  • nuclear localization sequences often comprise stretches of basic amino acids and given that there are two such binding sites on importin-a, two basic sequences separated by at least 10 amino acids can make up a bipartite NLS.
  • the second most characterized pathway of nuclear import involves proteins that bind to the importin-b ⁇ protein, such as the HIV-TAT and HIV-REV proteins, which use the sequences RKKRRQRRR (SEQ ID N: 3) and RQARRNRRRRWR (SEQ ID NO: 35), respectively to bind to importin-b ⁇ .
  • Other nuclear localization sequences are known in the art (see, e.g., Lange et al., J. Biol. Chem. (2007) 282:5101 -5105).
  • the NLS can be the naturally-occurring NLS of the nucleic acid-guided nuclease or a heterologous NLS.
  • heterologous in reference to a sequence is a sequence that originates from a foreign species, or, if from the same species, is substantially modified from its native form in composition and/or genomic locus by deliberate human intervention.
  • Non-limiting examples of NLS sequences that can be used to enhance the nuclear localization of the nucleic acid-guided nuclease or nucleic acid-guided nuclease fusion protein include the NLS of the SV40 Large T-antigen and c- Myc.
  • the NLS comprises the amino acid sequence PKKKRKV (SEQ ID NO: 2).
  • a nucleic acid-guided nuclease fusion protein can comprise more than one NLS, such as two, three, four, five, six, or more NLS sequences. Each of the multiple NLSs can be unique in sequence or there can be more than one of the same NLS sequence used.
  • the NLS can be on the amino- terminal (N-terminal) end of the nucleic acid-guided nuclease fusion protein, the carboxy-terminal (C- terminal) end, or both the N-terminal and C-terminal ends of the fusion protein.
  • the nucleic acid-guided nuclease fusion protein comprises two NLS sequences on its N-terminal end.
  • the nucleic acid-guided nuclease fusion protein comprises two NLS sequences on the C-terminal end of the site-directed polypeptide.
  • the site-directed polypeptide comprises four NLS sequences on its N-terminal end and two NLS sequences on its C-terminal end.
  • the nucleic acid-guided nuclease fusion protein can comprise an epitope tag.
  • an epitope tag may be a poly-histidine tag such as a hexahistidine tag or a dodecahistidine, a FLAG tag, a Myc tag, a HA tag, a GST tag or a V5 tag.
  • the nucleic acid-guided nuclease fusion protein comprises a cell targeting agent, or variant thereof.
  • the nucleic acid-guided nuclease fusion protein comprises from 5' to 3' a hexahistidine tag (6xHis), a cell targeting agent (e.g., CPP, antigen-binding agent, or ligand), Cas9, and one or more NLS.
  • the cell targeting agent is a cell penetrating peptide (CPP), which induces the absorption of a linked protein or peptide through the plasma membrane of a cell.
  • CPPs induce entry into the cell because of their general shape and tendency to either self- assemble into a membrane-spanning pore, or to have several positively charged residues, which interact with the negatively charged phospholipid outer membrane inducing curvature of the membrane, which in turn activates internalization.
  • Exemplary permeable peptides include, but are not limited to, transportan, PEP1 , MPG, p-VEC, MAP, CADY, polyR, HIV-TAT, HIV-REV, Penetratin, R6W3, P22N, DPV3, DPV6, K-FGF, and C105Y, and are reviewed in van den Berg and Dowdy (2011 ) Current Opinion in Biotechnology 22:888-893 and Farkhani et al. (2014) Peptides 57:78-94, each of which is herein incorporated by reference in its entirety.
  • the nucleic acid-guided nuclease fusion protein can comprise additional heterologous amino acid sequences, such as a detectable label (e.g., fluorescent protein) described elsewhere herein, or a purification tag, to form a fusion protein.
  • a purification tag is any molecule that can be utilized to isolate a protein or fused protein from a mixture (e.g., biological sample, culture medium).
  • purification tags include biotin, myc, maltose binding agent (MBP), and glutathione-S-transferase (GST).
  • the cell targeting agent is a ligand, or portion thereof.
  • the antigen-binding agent is a nanobody, a domain antibody, an scFv, a Fab, a diabody, a BiTE, a diabody, a DART, a minibody, a F(ab’)2, an intrabody, or an antibody mimetic.
  • the antibody mimetic is an adnectin (i.e., fibronectin based binding molecules), an affilin, an affimer, an affitin, an alphabody, an affibody, a DARPin, an anticalin, an avimer, a fynomer, a Kunitz domain peptide, a monobody, a nanoCLAMP, a unibody, or a versabody, an aptamer, or a cyclotide.
  • adnectin i.e., fibronectin based binding molecules
  • a cell expression vector comprising a nucleic acid encoding a nucleic acid-guided nuclease and/or a guide nucleic acid (gNA).
  • an expression vector comprising a conformation-specific nucleoprotein (NP) binding agent (e.g., an anti-CRISPR (Acr) protein, such as AcrllA4 or AcrVAI).
  • NP conformation-specific nucleoprotein
  • Acr anti-CRISPR
  • the nucleic acid-guided nuclease and the gNA can, in some instances, be provided on separate expression constructs.
  • the nucleic acid-guided nuclease can be provided to a host cell on an expression construct while the gNA is provided to the cell following expression of the nucleic acid-guided nuclease.
  • the cell expression vector is one isolated by the methods provided herein.
  • “Expression construct”, “construct” or “vector”, as used herein, refers to a polynucleotide vehicle that can be used to introduce genetic material into a cell. Constructs can be linear or circular. Constructs useful as expression constructs herein include plasmids, viral vectors (including phage), and integratable DNA fragments (i.e., fragments integratable into the host genome by homologous recombination). The four major types of vectors are plasmids, viral vectors, cosmids, and artificial chromosomes. Expression constructs can contain a replication sequence capable of effecting replication of the construct in a suitable host cell (i.e., an origin of replication).
  • expression constructs comprise an origin of replication, a multicloning site, and/or a selectable marker.
  • the expression construct may replicate and function independently of the host genome or integrate into the host genome.
  • methods for integrating expression constructs for stable transformation of prokaryotes are known in the art (see, e.g., Heap,
  • Construct design depends, among other things, on the intended use and host cell for the expression construct, and the design of an expression construct for a particular use and host cell is within the level of skill in the art.
  • Expression constructs for most host cells are commercially available. There are several commercial software products designed to facilitate selection of appropriate constructs and construction thereof, such as bacterial plasmids for bacterial transformation and gene expression in bacterial cells, yeast plasmids for cell transformation and gene expression in yeast and other fungi, mammalian vectors for mammalian cell transformation and gene expression in mammalian cells or mammals, viral vectors (including retroviral, lentiviral, and adenoviral vectors) for cell transformation and gene expression and methods to easily enable cloning of such polynucleotides.
  • bacterial plasmids for bacterial transformation and gene expression in bacterial cells
  • yeast plasmids for cell transformation and gene expression in yeast and other fungi
  • mammalian vectors for mammalian cell transformation and gene expression in mammalian cells or mammals
  • viral vectors including retroviral, lentiviral, and adenoviral vectors
  • Expression constructs typically comprise regulatory sequences that are involved in one or more of the following: regulation of transcription, post-transcriptional regulation, and regulation of translation.
  • Expression constructs can be introduced into a wide variety of organisms including bacterial cells, yeast cells, mammalian cells, and plant cells.
  • Expression constructs typically comprise functional regulatory sequences corresponding to the host cells or organism(s) into which they are being introduced.
  • expression constructs can include polynucleotides encoding protein tags (e.g., poly-His tags, hemagglutinin tags, fluorescent protein tags, quenchers, bioluminescent tags, nuclear localization tags).
  • the coding sequences for such protein tags can be fused to the coding sequences (e.g., a sequence encoding a nucleic acid-guided nuclease).
  • the expression construct can additionally comprise additional elements to promote nucleoprotein formation, such as 3’ hammerhead ribozyme or a ribozyme-guide-ribozyme system (see e.g., Gao, Y. and Yunde, Z. “Self processing of ribozyme-flanked RNAs into guide RNAs in vitro and in vivo for CRISPR-mediated genome editing,” J. Integr. Plant Biol. (2013) 56:343-349), to produce uniform gNA termini compatible with nucleoprotein formation.
  • the expression vector can include restrict site cassettes to facilitate insertion of additional sequences, such as spacer sequences on an expression construct encoding a gNA.
  • the expression vector can further include a unique sequence identifier or barcode.
  • Sequence identifiers can be any nucleic acid sequence that uniquely identifies the expression construct or source of the expression construct, and may be generated from a variety of different formats, including bulk synthesized polynucleotide barcodes, randomly synthesized barcode sequences, microarray based barcode synthesis, native nucleotides, a partial complement with an N-mer, a random N-mer, a pseudo random N-mer, or combinations thereof.
  • the sequence identifier can be a non-naturally occurring sequence.
  • the sequence identifier can comprise, for example less than 10, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 88, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, or more than 200 nucleotides.
  • polynucleotides encoding one or more of the various components of the expression construct are operably linked to a promoter.
  • the operably linked promoter can be an inducible promoter, a repressible promoter, or a constitutive promoter.
  • the nucleic acid-guided nuclease and the gNA are on the same construct.
  • the nucleic acid-guided nuclease is on a first expression construct and the gNA is on a second expression construct.
  • the nucleic acid- guided nuclease and gNA may be operatively linked to the same or different regulatory elements (e.g., promoters, ribosome binding sites).
  • the polynucleotide encoding the nucleic acid-guided nuclease is operatively linked to a first promoter and the polynucleotide encoding the gNA is operatively linked to a second promoter.
  • the first and second promoters are different.
  • the first and second promoters may be the same.
  • the first and second promoter each comprise an inducible element such that the expression level of the nucleic acid-guided nuclease protein and the expression level of the gNA can be controlled to obtain nucleoproteins.
  • the first and second promoters are orthogonal to each other (see, e.g., Kelly et al. ACS synthetic biology 5.10 (2016): 1136-1145, which is hereby incorporated by reference).
  • the first and second promoters are the same.
  • the first promoter and/or second promoter is a constitutive promoter.
  • the expression construct comprises a first ribosome binding site operatively linked to a polynucleotide encoding the nucleic acid-guided nuclease and a second ribosome binding site operatively linked to a polynucleotide encoding the gNA.
  • the first and second ribosome binding site are different.
  • the first and second promoters may be the same.
  • Expression constructs can be designed for expression in prokaryotic or eukaryotic cells. Examples of regulatory elements and expression constructs suitable for use in a variety of host cells are described further herein.
  • the one or more test expression constructs can be introduced into and propagated in a prokaryotic host cell.
  • Prokaryotic expression constructs are well known in the art. For example, expression constructs and co-expression systems for use in prokaryotic systems are described in, e.g., US20160160203A1 , US20160376602A1 , US20170159061 A1 , US20180282405A1 , WO2017106583A1 , and WO2014025663A1 , which are hereby incorporated by reference.
  • a prokaryotic expression construct comprises an origin of replication, also called a replicon, suitable for the target host cell (e.g., oriC derived from E. coli, pUC derived from pBR322, pSC101 derived from Salmonella), or 15A origin (derived from p15A) or bacterial artificial chromosomes). Origins of replication can be selected for use in expression constructs on the basis of incompatibility group, copy number, and/or host range, among other criteria.
  • origin of replication also called a replicon, suitable for the target host cell (e.g., oriC derived from E. coli, pUC derived from pBR322, pSC101 derived from Salmonella), or 15A origin (derived from p15A) or bacterial artificial chromosomes). Origins of replication can be selected for use in expression constructs on the basis of incompatibility group, copy number, and/or host range, among other criteria.
  • nucleic acid-guided nuclease and guide RNA are expressed on different expression constructs, different replicons that use the same mechanism for replication cannot be maintained together in a single host cell through repeated cell divisions.
  • plasmids can be categorized into incompatibility groups depending on the origin of replication that they contain. See, for example, U.S. Publication No. US20160376602A1 , which is hereby incorporated by reference in its entirety.
  • one plasmid when the nucleic acid-guided nuclease and gNA are encoded on separate plasmids, one plasmid can have a p15a origin and the second plasmid can have a pBR322 origin.
  • the two plasmids can also have different selective resistance markers for stable plasmid propagation.
  • the average number of copies of an expression construct in the cell, relative to the number of host chromosome molecules, is determined by the origin of replication contained in that expression construct. Copy number can range from a few copies per cell to several hundred.
  • different expression constructs are used to express the nucleic acid-guided nuclease and the gNA, which comprise inducible promoters that are activated by the same inducer, but which have different origins of replication.
  • Expression constructs can further include a selectable marker.
  • a “selectable marker gene” refers to a gene that upon expression confers a phenotype by which successfully transformed cells carrying the expression construct can be identified.
  • the selectable marker can encode a protein necessary for the survival or growth of host cells in a selective culture medium. In such instances, host cells not containing the expression construct comprising the selection gene will not survive in the culture medium.
  • Typical selection genes for prokaryotic expression systems encode proteins that confer resistance to antibiotics or other toxins, or that complement auxotrophic deficiencies of the host cell.
  • a selection scheme utilizes a drug such as an antibiotic to arrest growth of a host cell.
  • antibiotics that are commonly used for the selection of selectable markers (and abbreviations indicating genes that provide antibiotic resistance phenotypes) are: ampicillin (Amp R ), chloramphenicol (Cml R or Cm R ), kanamycin (Kan R ), spectinomycin (Spc R ), streptomycin (Str R ), tetracycline (Tet R ), gentamicin (Gen R ).
  • the native promoter region for a selection gene is usually included, along with the coding sequence for its gene product, as part of a selectable marker portion of an expression construct.
  • the coding sequence for the selection gene can be expressed from a constitutive promoter.
  • ZeocinTM Life Technologies, Grand Island, NY
  • the selectable marker is a gene that upon expression confers an identifiable phenotype.
  • the selectable marker may be a fluorescent marker that confers fluorescence in cells carrying the expression construct that can be identified visually or by machine, e.g., flow cytometry.
  • Useful promoters are known for expression of proteins in prokaryotes, for example, include those inducible or regulated by rhamnose, arabinose, xylose, lactose, IPTG, or phosphate.
  • the inducible promoter is an L-arabinose-inducible promoter, a propionate-inducible promoter, a rhamnose-inducible promoter, a xylose-inducible promoter, a lactose-inducible promoter, an IPTG-inducible promoter, or a promoter inducible by phosphate depletion.
  • the inducible promoter is the araBAD promoter (see, e.g., Guzman et al. J of bacteriology 177.14 (1995): 4121-4130.), the T7 promoter, the T5 promoter, the pLac promoter, pTac promoter, the rhaBAD promoter (see, e.g., Kelly et al. ACS synthetic biology 5.10 (2016): 1136-1145), the prpBCDE promoter, the rhaSR promoter, or the xlyA promoter.
  • promoters suitable for use in bacteria see, for example, U.S. Publication No. US20160376602A1 , which is hereby incorporated by reference in its entirety.
  • the nucleic acid-guided nuclease or the gNA can be under a tunable promoter system such as AraC-ParaBAD or FthamS-PrhaBAD to generated optimal levels of expression for maximizing nucleoprotein formation.
  • the nucleic acid-guided nuclease or gNA can be under a T7 promoter for maximum expression.
  • the nucleic acid-guided nuclease or gNA can be under an IPTG-inducible promoter such as T5, pLac, or pTac.
  • protein expression is tunable using a lac permease-deficient strain of E. coli, such as NOVAGEN TUNER.
  • Expression constructs can also comprise coding sequences that are expressed from constitutive promoters. Unlike inducible promoters, constitutive promoters initiate continual gene product production under most growth conditions. Examples of constitutive promoters includes the promoter of the Tn3 bla gene, the promoter for the E. co// lipoprotein gene, Ipp; or the trpLEDCBA promoter. Constitutive promoters can be used in expression constructs for the expression of selectable markers, as described herein, and also for the constitutive expression of other gene products useful for the expression of the desired product.
  • transcriptional regulators of the inducible promoters such as AraC, PrpR, RhaR, and XylR
  • transcriptional regulators of the inducible promoters can alternatively be expressed from a constitutive promoter, on either the same expression construct as the inducible promoter they regulate, or a different expression construct.
  • gene products useful for the production or transport of the inducer such as PrpEC, AraE, or Rha, or proteins that modify the reduction-oxidation environment of the cell, as a few examples, can be expressed from a constitutive promoter within an expression construct.
  • Expression of proteins in prokaryotes is often carried out in bacteria, such as Escherichia coli with expression constructs containing constitutive or inducible promoters directing the expression of the expressed components of the expression construct (e.g., a gNA and nucleic acid-guided nuclease fusion protein).
  • constitutive or inducible promoters directing the expression of the expressed components of the expression construct (e.g., a gNA and nucleic acid-guided nuclease fusion protein).
  • inducible promoters and related genes include those that function in Escherichia coli (E. coli) strain MG1655 (American Type Culture Collection deposit ATCC 700926), which is a substrain of E. coli K-12 (American Type Culture Collection deposit ATCC 10798).
  • Table 1 in US Patent Application No. US20160376602 lists the genomic locations, in E.
  • E. coli MG1655 of the nucleotide sequences for these examples of inducible promoters and related genes, which is hereby incorporated by reference. Additional information about E. coli promoters, genes, and strains described herein can be found in many public sources, including the online EcoliWiki resource, located at ecoliwiki.net.
  • Prokaryotic expression constructs can also include ribosome binding sites of varying strength, and secretion signals (e.g., mal, sec, tat, ompC, and pelB).
  • secretion signals e.g., mal, sec, tat, ompC, and pelB.
  • the nucleotide sequence of the region between the transcription initiation site and the initiation codon of the coding sequence of the gene product that is to be inducibly expressed corresponds to the 5' untranslated region (‘UTR’) of the mRNA for the polypeptide gene product.
  • UTR 5' untranslated region
  • the region of the expression construct that corresponds to the 5' UTR comprises a polynucleotide sequence similar to the consensus ribosome binding site (RBS, also called the Shine-Dalgarno sequence) that is found in the species of the host cell.
  • RBS consensus ribosome binding site
  • the RBS consensus sequence is GGAGG or GGAGGU
  • bacteria such as E. coli
  • the RBS consensus sequence is AGGAGG or AGGAGGU.
  • the RBS is typically separated from the initiation codon by 5 to 10 intervening nucleotides.
  • the RBS sequence is at least 55% identical to the AGGAGGU consensus sequence, at least 70% identical, or at least 85% identical, and is separated from the initiation codon by 5 to 10 intervening nucleotides, by 6 to 9 intervening nucleotides, or by 6 or 7 intervening nucleotides.
  • the one or more test expression constructs can be introduced into and propagated in a eukaryotic host cell.
  • the host cell is a yeast and the expression construct is a yeast expression construct.
  • yeast expression constructs for expression in Saccharomyces cerivisae include, but are not limited to, the following: pYepSed , pMFa, pJRY88, pYES2, and picZ.
  • Methods for gene expression in yeast cells are known in the art (see, e.g., Methods in Enzymology, Volume 194, "Guide to Yeast Genetics and Molecular and Cell Biology, Part A,” (2004) Christine Guthrie and Gerald R. Fink (eds.), Elsevier Academic Press, San Diego, CA).
  • promoters include, but are not limited to, promoters of genes encoding the following yeast proteins: alcohol dehydrogenase 1 (ADH1) or alcohol dehydrogenase 2 (ADH2), phosphoglycerate kinase (PGK), triose phosphate isomerase (TPI), glyceraldehyde-3-phosphate dehydrogenase (GAPDH; also known as TDH3, or triose phosphate dehydrogenase), galactose-1 -phosphate uridyl-transferase (GAL7), UDP-galactose epimerase (GAL10), cytochrome ci (CYC1), acid phosphatase (PH05) and glycerol-3-phosphate
  • ADH1 alcohol dehydrogenase 1
  • ADH2 alcohol dehydrogenase 2
  • PGK phosphoglycerate kinase
  • TPI triose phosphate isomerase
  • Hybrid promoters such as the ADH2/GAPDH, CYC1/GAL10 and the ADH2/GAPDH promoter (which is induced at low cellular-glucose concentrations, e.g., about 0.1 percent to about 0.2 percent) also may be used.
  • suitable promoters include the thiamine-repressed nmtl promoter and the constitutive cytomegalovirus promoter in pTL2M.
  • Yeast RNA polymerase III promoters e.g., promoters from 5S, U6 or RPR1 genes
  • polymerase III termination sequences are known in the art (see, e.g., www.yeastgenome.org; Harismendy, O., et al., (2003) "Genome-wide location of yeast RNA polymerase III transcription machinery," The EMBO Journal. 22(18):4738-4747.)
  • upstream activation sequences may be used to enhance polypeptide expression.
  • upstream activation sequences for expression in yeast include the UASs of genes encoding these proteins: CYC1 , ADH2, GAL1 , GAL7, GAL10, and ADH2.
  • Exemplary transcription termination sequences for expression in yeast include the termination sequences of the a-factor, CYC1 , GAPDH, and PGK genes. One or multiple termination sequences can be used.
  • Suitable promoters, terminators, and coding regions may be cloned into E. coli- yeast shuttle expression constructs and transformed into yeast cells. These expression constructs allow strain propagation in both yeast and E. coli strains. Typically, the expression construct contains a selectable marker and sequences enabling autonomous replication or chromosomal integration in each host. Examples of plasmids typically used in yeast are the shuttle vectors pRS423, pRS424, pRS425, and pRS426 (American Type Culture Collection, Manassas, VA). These plasmids contain a yeast 2 micron origin of replication, an E. coli replication origin (e.g., pMB1), and a selectable marker.
  • the various components can also be expressed in insects or insect cells.
  • Suitable expression control sequences for use in such cells are well known in the art.
  • it is desirable that the expression control sequence comprises a constitutive promoter.
  • suitable strong promoters include, but are not limited to, the following: the baculovirus promoters for the piO, polyhedrin (polh), p 6.9, capsid, UAS (contains a Gal4 binding site), Ac5, cathepsin-like genes, the B.
  • baculovirus promoters for the iel, ie2, ieO, etl, 39K (aka pp31), and gp64 genes. If it is desired to increase the amount of gene expression from a weak promoter, enhancer elements, such as the baculovirus enhancer element, hr5, may be used in conjunction with the promoter.
  • RNA polymerase III promoters are known in the art, for example, the U6 promoter.
  • conserveed features of RNA polymerase III promoters in insects are also known (see, e.g., Hernandez, G., (2007) "Insect small nuclear RNA gene promoters evolve rapidly yet retain conserved features involved in determining promoter activity and RNA polymerase specificity," Nucleic Acids Res. 2007 Jan; 35(1):21-34).
  • the various components are incorporated into mammalian expression constructs for use in mammalian cells.
  • mammalian expression constructs suitable for use with the systems of the present invention are commercially available (e.g., from Life Technologies, Grand Island, NY; NeoBiolab, Cambridge, MA; Promega, Madison, Wl; DNA2.0, Menlo Park, CA; Addgene, Cambridge, MA).
  • Expression constructs derived from mammalian viruses can also be used for expressing the various components of the present methods in mammalian cells.
  • viruses such as adenovirus, papovirus, herpesvirus, polyomavirus, cytomegalovirus, lentivirus, retrovirus, vaccinia and Simian Virus 40 (SV40)
  • SV40 Simian Virus 40
  • Regulatory sequences operably linked to the components can include activator binding sequences, enhancers, introns, polyadenylation recognition sequences, promoters, repressor binding sequences, stem-loop structures, translational initiation sequences, translation leader sequences, transcription termination sequences, translation termination sequences, primer binding sites, and the like.
  • Commonly used promoters are constitutive mammalian promoters CMV, EF1a, SV40, PGK1 (mouse or human), Ubc, CAG, CaMKIla, and beta-Act. and others known in the art (Khan, K. H. (2013) "Gene Expression in Mammalian Cells and its Applications," Advanced Pharmaceutical Bulletin 3(2), 257-263).
  • mammalian RNA polymerase III promoters including HI and U6, can be used.
  • HEK 293 Human embryonic kidney
  • CHO Chiinese hamster ovary
  • These cell lines can be transfected by standard methods (e.g., using calcium phosphate or polyethyleneimine (PEI), or electroporation).
  • PKI polyethyleneimine
  • Other typical mammalian cell lines include, but are not limited to: HeLa, U20S, 549, HT 1080, CAD, P19, NIH 3T3, L929, N2a, Human embryonic kidney 293 cells, MCF-7, Y79, SO-Rb50, Hep G2, DUKX-X11 , J558L, and Baby hamster kidney (BHK) cells.
  • the mammalian cell is a COP cell, an L cell, a C127 cell, an Sp2/0 cell, an NS-0 cell, an NIH3T3 cell, a PC12 cell, a PC12h cell, a BHK cell, a CHO cell, a COS1 cell, a COS3 cell, a COST cell, a CV1 cell, a Vero cell, a HeLa cell, an HEK-293 cell, a PER C6 cell, a cell derived from diploid fibroblasts, a myeloma cell, or HepG2.
  • an expression construct encoding a nucleic acid-guided nuclease can be designed to encode molecular moieties that are fused to the nucleic acid-guided nuclease to form a nucleic acid-guided nuclease fusion protein.
  • an expression construct encoding a conformation-specific nucleoprotein binding agent can be designed to encode molecular moieties that are fused to the conformation-specific nucleoprotein binding agent to form conformation-specific nucleoprotein binding fusion proteins.
  • such moieties can aid in the purification and/or detection of the nucleic acid-guided nuclease or conformation-specific nucleoprotein binding agent.
  • the expression construct can encode a protein (e.g., nucleic acid-guided nuclease or conformation-specific nucleoprotein binding agent), operably linked to a polyhistidine ‘tag’ sequence (e.g., six to ten histidine residues) at its N- or C-terminus.
  • a polyhistidine ‘tag’ sequence e.g., six to ten histidine residues
  • the polyhistidine tag sequence can be removed by endo- or exopeptidases.
  • the polynucleotide encoding the nucleic acid-guided nuclease can be operably linked to a cloning site for inserting a nucleic acid encoding a cell targeting agent.
  • insertion of a polynucleotide encoding a cell targeting agent at the cloning site enables formation of a nucleic acid-guided nuclease fusion protein comprising the nucleic acid-guided nuclease and the cell targeting agent.
  • the polynucleotide encoding the nucleic acid- guided nuclease is operably linked to a cloning site encoding a conjugation moiety that enables the nucleic acid-guided nuclease to stably associate with a cell targeting agent comprising a complementary binding moiety.
  • the polynucleotide encoding the conformation-specific nucleoprotein binding agent can be operably linked to a cloning site for inserting a nucleic acid encoding a cell targeting agent.
  • insertion of a polynucleotide encoding a cell targeting agent at the cloning site enables formation of a conformation-specific nucleoprotein binding fusion protein comprising the conformation-specific nucleoprotein binding agent and the cell targeting agent.
  • the polynucleotide encoding the conformation-specific nucleoprotein binding agent is operably linked to a cloning site encoding a conjugation moiety that enables the conformation-specific nucleoprotein binding agent to stably associate with a cell targeting agent comprising a complementary binding moiety.
  • Conjugation moieties include, but are not limited to Protein A, Spycatcher tag, Halo-tag, Sortase, mono-avidin, ACP tag, a SNAP tag, or any other conjugation moieties known in the art.
  • the conjugation moiety is selected from Protein A, CBP, MBP, GST, poly(His), biotin/streptavidin, V5-tag, Myc-tag, HA-tag, NE-tag, His-tag, Flag tag, Halo-tag, Snap- tag, Fc-tag, Nus-tag, BCCP, thioredoxin, SnooprTag, SpyTag, SpyCatcher, Isopeptag, SBP-tag, S- tag, AviTag, and calmodulin.
  • Exemplary binding moiety pairings include (i) streptavidin-binding peptide (streptavidin binding peptide; SBP) and streptavidin (STV), (ii) biotin and EMA (enhanced monomeric avidin), (iii) SpyTag (ST) and SpyCatcher (SC), (iv) Halo-tag and Halo-tag ligand, (v) and SNAP-Tag, (vi) Myc tag and anti-Myc immunoglobulins (vii) FLAG tag and anti-FLAG immunoglobulins, and (ix) ybbFt tag and coenzyme A groups.
  • nucleic acid-guided nuclease or conformation-specific nucleoprotein binding agent comprises the self-cleaving N-terminal portions (NP ro ) of polyproteins from pestiviruses such as Hog cholera virus (strain Alfort), also called classical swine fever virus (CSFV), from border disease virus (BDV), bovine viral diarrhea virus (BVDV), or fragments thereof; (2) the N-terminal portion of carboxypeptidase B (‘CPB’) precursor (amino acids 21-110 of Sus scrofa CPB, SwissProt P09955.5), and fragments thereof; and/or (3) small ubiquitin-related modifier (SUMO) (SwissProt P55853.1).
  • NP ro N-terminal portions
  • CSFV border disease virus
  • BDV border disease virus
  • BVDV bovine viral diarrhea virus
  • SUMO small ubiquitin-related modifier
  • Any N-terminal tag may itself be further tagged at its N-terminus with a polyhistidine tag such as 6xHis, allowing for initial purification of the tagged polypeptide on a nickel column, followed by self-cleavage of tags such as NP ro , or enzymatic cleavage of the CPB or SUMO N-terminal tag by trypsin or SUMO protease, respectively, and elution of the freed polypeptide from the column.
  • a polyhistidine tag such as 6xHis
  • the SUMO protease polypeptides are also fusion proteins comprising 6xHis tags, allowing for a two-step purification: in the first step, the expressed 6xHis-SUMO-tagged nucleic acid- guided nuclease is purified by binding to a nickel column, followed by elution from the column.
  • the SUMO tags on the purified polypeptides are cleaved by the 6xHis-tagged SUMO protease, and the SUMO protease-nucleic acid-guided nuclease reaction mixture is run through a second nickel column, which retains the SUMO protease but allows the now untagged nucleic acid- guided nuclease to flow through.
  • fluorescent protein sequences can be expressed as part of a polypeptide gene product, with the amino acid sequence for the fluorescent protein preferably added at the N- or C-terminal end of the amino acid sequence of the polypeptide gene product.
  • the resulting fusion protein fluoresces when exposed to light of certain wavelengths, allowing the presence of the fusion protein to be detected visually.
  • a well-known fluorescent protein is the green fluorescent protein of Aequorea victoria, and many other fluorescent proteins are commercially available, along with nucleotide sequences encoding them.
  • the expression vectors herein comprise from 5’ to 3’ a promoter, a ribosome binding site, a nucleic acid-guided nuclease, and a detectable label, conjugation moiety, or other tag described herein. In other embodiments, the expression vectors herein comprise from 5’ to 3’ a promoter, a ribosome binding site; a detectable label, conjugation moiety, or other tag described herein; and a nucleic acid-guided nuclease.
  • the detectable label or other tag is operably linked to the nucleic acid-guided nuclease by a cleavable linker (e.g., a SUMO protease cleavable linker).
  • a cleavable linker e.g., a SUMO protease cleavable linker
  • the polynucleotide encoding the nucleic acid-guided nuclease is additionally operably linked to a polynucleotide encoding one or more cell targeting agents, such as a ligand, antigen-binding agent, or cell penetrating peptide (e.g., one or more nuclear localization signals).
  • the expression vectors herein comprise from 5’ to 3’ a promoter, a ribosome binding site, a conformation-specific nucleoprotein binding agent, and a detectable label, conjugation moiety, or other tag described herein.
  • the expression vectors herein comprise from 5’ to 3’ a promoter, a ribosome binding site; a detectable label, conjugation moiety, or other tag described herein; and a conformation-specific nucleoprotein binding agent.
  • the detectable label or other tag is operably linked to the conformation-specific nucleoprotein binding agent by a cleavable linker (e.g., a SUMO protease cleavable linker).
  • the polynucleotide encoding the conformation-specific nucleoprotein binding agent is additionally operably linked to a polynucleotide encoding one or more cell targeting agents, such as a ligand, antigen-binding agent, or cell penetrating peptide (e.g., one or more nuclear localization signals).
  • cell targeting agents such as a ligand, antigen-binding agent, or cell penetrating peptide (e.g., one or more nuclear localization signals).
  • Conformation-specific nucleoprotein binding fusion proteins e.g., as found in a TAGE
  • nucleic acid-guided fusion proteins are described in Section V.

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  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

La présente invention concerne des compositions et des procédés mettant en œuvre un agent de liaison de nucléoprotéine spécifique à une conformation. L'invention concerne des compositions et des procédés de transport de nucléoprotéines comprenant une nucléase guidée par acide nucléique et un acide nucléique guide dans une cellule. L'invention concerne en outre des compositions et des procédés pour isoler des constructions d'expression qui expriment une nucléoprotéine comprenant une nucléase guidée par acide nucléique et un acide nucléique guide dans une cellule hôte. De plus, la présente invention concerne des compositions et des procédés pour identifier des cellules, ou des constructions d'expression contenues dans celles-ci, qui expriment des nucléoprotéines actives comprenant une nucléase guidée par acide nucléique et un acide nucléique guide. Les procédés comprennent en outre des procédés de purification par affinité utilisant un agent de liaison de nucléoprotéine spécifique à une conformation pour isoler une nucléoprotéine à partir d'une cellule hôte.
PCT/US2020/052791 2019-09-25 2020-09-25 Compositions et procédés pour le ciblage et l'expression de nucléoprotéines WO2021062201A1 (fr)

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113698496A (zh) * 2021-08-27 2021-11-26 中国科学院深圳先进技术研究院 临近标记复合物、临近标记方法、分子间互作分析方法
CN114592038A (zh) * 2022-03-03 2022-06-07 重庆医科大学 一种检测Dam甲基转移酶的荧光生物传感系统及其构建与应用
WO2023049774A1 (fr) * 2021-09-21 2023-03-30 University Of Washington Ligature de protéine-protéine à déclenchement exogène et génétiquement codée
WO2024059773A3 (fr) * 2022-09-15 2024-04-25 The Children's Medical Center Corporation Détection et quantification d'analyte ultra-sensible à l'aide d'une capture et d'une libération avec détection de proximité

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20150010932A1 (en) * 2013-07-02 2015-01-08 Salk Institute For Biological Studies Methods for assaying protein-protein interactions
WO2016123578A1 (fr) * 2015-01-30 2016-08-04 The Regents Of The University Of California Livraison de protéines dans des cellules hématopoïétiques primaires
US20180201956A1 (en) * 2016-12-05 2018-07-19 Editas Medicine, Inc. SYSTEMS AND METHODS FOR ONE-SHOT GUIDE RNA (ogRNA) TARGETING OF ENDOGENOUS AND SOURCE DNA
US20180208976A1 (en) * 2016-06-16 2018-07-26 The Regents Of The University Of California Methods and compositions for detecting a target rna

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20150010932A1 (en) * 2013-07-02 2015-01-08 Salk Institute For Biological Studies Methods for assaying protein-protein interactions
WO2016123578A1 (fr) * 2015-01-30 2016-08-04 The Regents Of The University Of California Livraison de protéines dans des cellules hématopoïétiques primaires
US20180208976A1 (en) * 2016-06-16 2018-07-26 The Regents Of The University Of California Methods and compositions for detecting a target rna
US20180201956A1 (en) * 2016-12-05 2018-07-19 Editas Medicine, Inc. SYSTEMS AND METHODS FOR ONE-SHOT GUIDE RNA (ogRNA) TARGETING OF ENDOGENOUS AND SOURCE DNA

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
TURK ET AL.: "Characterization of a Novel pH-Sensitive Peptide that Enhances Drug Release from Folate-Targeted Liposomes at Endosomal pHs", BIOCHIMICA ET BIOPHYSICA ACTA (BBA) - BIOMEMBRANES, vol. 1559, no. Iss. 1, 10 February 2002 (2002-02-10), pages 56 - 68, XP004334733, DOI: 10.1016/S0005-2736(01)00441-2 *

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113698496A (zh) * 2021-08-27 2021-11-26 中国科学院深圳先进技术研究院 临近标记复合物、临近标记方法、分子间互作分析方法
CN113698496B (zh) * 2021-08-27 2023-03-28 中国科学院深圳先进技术研究院 临近标记复合物、临近标记方法、分子间互作分析方法
WO2023049774A1 (fr) * 2021-09-21 2023-03-30 University Of Washington Ligature de protéine-protéine à déclenchement exogène et génétiquement codée
CN114592038A (zh) * 2022-03-03 2022-06-07 重庆医科大学 一种检测Dam甲基转移酶的荧光生物传感系统及其构建与应用
CN114592038B (zh) * 2022-03-03 2023-06-06 重庆医科大学 一种检测Dam甲基转移酶的荧光生物传感系统及其构建与应用
WO2024059773A3 (fr) * 2022-09-15 2024-04-25 The Children's Medical Center Corporation Détection et quantification d'analyte ultra-sensible à l'aide d'une capture et d'une libération avec détection de proximité

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