WO2021158658A1 - Split crispr-cas for biological signal integration - Google Patents

Split crispr-cas for biological signal integration Download PDF

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WO2021158658A1
WO2021158658A1 PCT/US2021/016407 US2021016407W WO2021158658A1 WO 2021158658 A1 WO2021158658 A1 WO 2021158658A1 US 2021016407 W US2021016407 W US 2021016407W WO 2021158658 A1 WO2021158658 A1 WO 2021158658A1
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effector
casl2a
nucleic acid
crrna
cell
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WO2021158658A9 (en
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Lei S. QI
Hannah R. KEMPTON
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The Board Of Trustees Of The Leland Stanford Junior University
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    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/635—Externally inducible repressor mediated regulation of gene expression, e.g. tetR inducible by tetracyline
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    • 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
    • C12N15/102—Mutagenizing nucleic acids
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    • 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
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
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    • 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
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/20—Type of nucleic acid involving clustered regularly interspaced short palindromic repeats [CRISPRs]
    • C—CHEMISTRY; METALLURGY
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    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/16011—Human Immunodeficiency Virus, HIV
    • C12N2740/16041—Use of virus, viral particle or viral elements as a vector
    • C12N2740/16043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector

Definitions

  • the system is configured such that all components of the Casl2a/crRNA complex are present or capable of being expressed in the sample when the first input and the second input each have a desired state. In some embodiments, the system is configured such that all components of the Casl2a/crRNA complex are present or capable of being expressed in the sample when the first input is present and the second input is present.
  • the transcriptional activation domain is a tripartite VP64-p65-Rta (VPR) transcriptional activation domain or variant thereof.
  • VPR tripartite VP64-p65-Rta
  • targeting of the genomic DNA by the Casl2a system is capable of enhancing transcription of the mRNA.
  • the target gene encodes a target receptor.
  • the input cell is a target immune cell
  • the target receptor is a chimeric antigen receptor (CAR) targeting a disease-associated cell
  • one or more inputs of the system are associated with the disease-associated cell
  • the first output of the system increases the expression of the CAR in the engineered cell.
  • the engineered cell is a CAR T cell, a CAR NK cell, or a CAR macrophage cell.
  • FIG. 7A shows a schematic of cumate and Doxycycline (Dox)-controlled AND gate. Addition of cumate and Dox induce expression of N- and C-terms, respectively, in a stable HEK293T cell line. Reconstitution of a functional dCasl2a results in activation of a P2-GFP reporter.
  • CuO cumate operator
  • TetO Tetracycline operator
  • CymR CuO binding repressor
  • rtTA reverse Tetracycline-controlled transactivator.
  • FIG. 8D shows simultaneous activation of endogenous IFNg and IL2 using the transiently transfected split dCasl2a-miniVPR in HEK293T cells, as measured by ELISA two days post transfection. Bars represent mean values and dots represent three biological replicates. Dotted line represents the lower detection limit of ELISA kit (4 pg/mL).
  • FIG. 10D shows comparison of performance of U6- or TRE3G-driven crRNA variants for activating IFNg measured by ELISA two days post transfection with full length dCasl2a- miniVPR in HEK293T cells.
  • Minimal Dox-inducible crlFN ⁇ activation (DRspacer-DR) was initially observed.
  • Duplicating the crRNA (DR-spacer-DR-spacer-DR) significantly enhanced activation, suggesting the expression level of the crRNA may be a limiting factor.
  • crLacZ the non-targeting crRNA. Bars represent mean values and dots represent 3 biological replicates.
  • FIG. 12A shows constructs used to express the full dCasl2a activator and the anti- CRISPR AcrVAl.
  • FIG. 14A shows a schematic showing the application of split dCasl2a for detecting two tumor-relevant signals in breast cancer cells and activating a therapeutic response.
  • HREs hypoxia-responsive elements.
  • TFs transcription factors driving expression of the RRM2 promoter (RRM2p).
  • FIG. 14B shows performance of RRM2p for expressing N-term dCasl2a in MDA-MB- 231 breast cancer cells and MCF10A normal breast cells as measured by flow cytometry.
  • Top construct used for lentiviral transduction.
  • Fefit representative histogram for MDA-MB-231 and MCF 10A cells.
  • Right bars represent mean values and dots represent three biological replicates.
  • EFS Elongation Factor short, compact promoter.
  • FIG. 14H shows performance of the 3-input dCasl2a gate in MDA-MB-231 cells for activating endogenous IFN ⁇ as measured by ELISA two days post treatment.
  • the addition of Dox controls expression of AcrVAl. Bars represent mean values and dots represent three biological replicates.
  • the present invention relates to split CRISPR-Cas effector protein Casl2a and variants thereof, such as nuclease-deficient Casl2a (dCasl2a) and fusions thereof with an effector domain, such as a transcriptional activation domain, wherein the split Cas 12a or variant thereof comprises a plurality of effector subunits comprising fragments of a Cas 12a protein or variant thereof, wherein the plurality of effector subunits are capable of forming a split Cas 12a complex (such as by spontaneous assembly) retaining at least some of an effector activity of the Casl2a protein or variant thereof.
  • dCasl2a nuclease-deficient Casl2a
  • an effector domain such as a transcriptional activation domain
  • the split Cas 12a or variant thereof comprises a plurality of effector subunits comprising fragments of a Cas 12a protein or variant thereof, wherein the plurality of effector subunits are capable of
  • Applicant developed a split Casl2a system capable of spontaneously reassembling and demonstrated that it allows for construction of two, three, and four-input AND gates in mammalian cells.
  • Anti-CRISPR (Acr) proteins were also incorporated to further control circuit activity.
  • the potential therapeutic utility of such systems to activate anti-tumoral programs specifically in cancer cells was demonstrated.
  • Further aspects of the present disclosure are directed to methods of modulating gene expression from a target nucleic acid in a cell, or otherwise modifying the target nucleic acid.
  • the methods include providing to the cell (a) effector subunits of a split Casl2a protein or variant thereof, or nucleic acid encoding the effector subunits; and (b) a crRNA comprising a spacer sequence complementary to a target polynucleotide sequence in the target nucleic acid, or nucleic acid encoding the crRNA.
  • the method comprises providing to the cell nucleic acid encoding the effector subunits and nucleic acid encoding the crRNA.
  • the nucleic acids encoding the effector subunits and the crRNA are present on a single vector or on separate vectors, such as engineered DNA plasmid vectors or viral vectors.
  • the vector is then used to deliver the nucleic acids encoding the effector subunits and the crRNA into desired cells or tissues.
  • the effector subunits of the split Casl2a protein or variant thereof and the crRNA may be expressed depending on the state of certain inputs to the cell that modulate their expression.
  • the crRNA comprises a portion that is complementary to a sequence of a target site and can guide a split Casl2a complex to the target site. In this manner, expression or modification of the target nucleic acid sequence is modulated depending on the location of the target site and the state of each of the inputs that modulate expression of the split Casl2a system components.
  • Class 2 CRISPR-Cas systems endow microbes with diverse mechanisms for adaptive immunity.
  • CRISPR systems are generally divided into two classes, with class 1 systems using a complex of multiple Cas proteins to degrade foreign nucleic acids, and class 2 systems using a single, generally larger, Cas protein for the same purpose.
  • Class 1 is divided into types I, III, and IV, and class 2 is divided into types II, V, and VI.
  • the RNA-guided, nucleic acid-targeting CRISPR systems described herein are based on Cas 12a (such as Cas 12a from Lachnospiraceae bacterium), a class 2 Cas effector protein classified as Type V.
  • the systems include a split Cas 12a protein or variant thereof comprising two or more effector subunits comprising fragments of a Cas 12a protein or variant thereof, or nucleic acid encoding the two or more effector subunits, wherein the two or more effector subunits are capable of spontaneously assembling to form a split Cas 12a complex retaining at least some of the effector activity of the Cas 12a protein or variant thereof.
  • Embodiments of the present disclosure are directed to split Casl2a-based regulation of gene expression, as well as nucleic acid engineering (including genome editing and epigenetic modification).
  • the small size of Cas 12a proteins allows Cas 12a proteins and effector domain fusions thereof to be paired with a CRISPR array encoding multiple crRNAs while remaining under the packaging size limit of the versatile adeno-associated virus (AAV) delivery vehicle for primary cell and in vivo delivery.
  • AAV versatile adeno-associated virus
  • CRISPR-Cas 12a and engineered variants such as dCasl2a allow for flexible nucleic acid engineering, regulation of gene expression, and therapeutics, expanding the genome editing and regulation toolbox.
  • split Casl2a proteins or variants thereof comprising fragments of a Casl2a protein or variant thereof having an effector activity are contemplated by the present disclosure.
  • the split Casl2a protein or variant thereof comprises two or more effector subunits comprising fragments of the Casl2a protein or variant thereof, wherein the two or more effector subunits are capable of spontaneously assembling to form a split Casl2a complex retaining at least some of the effector activity of the Casl2a protein or variant thereof.
  • the two or more effector subunits comprise a first effector subunit and a second effector subunit.
  • the first effector subunit comprises an N-terminal fragment of the Casl2a protein or variant thereof up to and including a split point amino acid
  • the second effector subunit comprises a C-terminal fragment of the Casl2a protein or variant thereof following the split point amino acid.
  • the split point amino acid corresponds to A406 or G550 with respect to a parental Casl2a protein amino acid sequence of SEQ ID NO: 1.
  • the first effector subunit further comprises a first stabilization domain at its C-terminus and the second effector subunit further comprises a second stabilization domain at its N-terminus, wherein the first stabilization domain and the second stabilization domain are capable of interacting with one another to stabilize a complex comprising the first effector subunit and the second effector subunit.
  • the Casl2a protein or variant thereof comprises a nuclear localization signal (NLS).
  • the NLS comprises one or more repeats of a conventional NLS.
  • the Casl2a protein or variant thereof comprises a C-terminal NLS.
  • one or more of the two or more effector subunits that do not include the NLS of the Casl2a protein or variant thereof further comprise an additional NLS. In some embodiments, none of the two or more effector subunits that do not include the NLS of the Casl2a protein or variant thereof comprise an additional NLS.
  • the first effector subunit further comprises the first stabilization domain at its N-terminus. In some embodiments, the first effector subunit further comprises the first stabilization domain at its C-terminus. In some embodiments, the second effector subunit further comprises the second stabilization domain at its N-terminus. In some embodiments, the second effector subunit further comprises the second stabilization domain at its C-terminus. [0095] In some embodiments, according to any of the split Casl2a proteins or variants thereof described herein comprising a first stabilization domain and a second stabilization domain, the first stabilization domain and the second stabilization domain are derived from subunits of a heterodimerizing binding pair or a split-intein.
  • the CC heterodimer is a CC peptide heterodimer ( see Lebar, T., et al. (2020). Nature Chemical Biology, 1-7).
  • the first stabilization domain and the second stabilization domain are derived from subunits of a split-intein.
  • the split-intein subunits comprise DnaE N-intein and C-intein.
  • DnaE N-intein comprises the amino acid sequence encoded by SEQ ID NO: 7.
  • DnaE C-intein comprises the amino acid sequence encoded by SEQ ID NO: 8.
  • the split-intein is gp41-l, gp41-8, NrdJ-1, or IMPDH-1 ( see Dassa, B., et al. (2009). Nucleic acids research, 57(8), 2560-2573).
  • the split-intein comprises split portions of the Mxe GyrA intein (see Fine, E. J., et al. (2015). Scientific reports, 5, 10777).
  • the two or more effector subunits comprise a first effector subunit, a second effector subunit, and a third effector subunit.
  • the Casl2a protein or variant thereof is a fusion protein comprising an effector domain fused to a Casl2a domain, and the first effector subunit comprises an N-terminal fragment of the Casl2a domain up to and including a split point amino acid, the second effector subunit comprises a C-terminal fragment of the Cast 2a domain following the split point amino acid; and the third effector subunit comprises the effector domain.
  • the split point amino acid corresponds to A406 or G550 with respect to a parental Casl2a protein amino acid sequence of SEQ ID NO: 1.
  • the first effector subunit further comprises a first stabilization domain at its C-terminus and the second effector subunit further comprises a second stabilization domain at its N-terminus, wherein the first stabilization domain and the second stabilization domain are capable of interacting with one another to stabilize a complex comprising the first effector subunit and the second effector subunit; and/or the third effector subunit further comprises a third stabilization domain at either terminus and (i) the second effector subunit further comprises a fourth stabilization domain at its C-terminus or (ii) the first effector subunit further comprises the fourth stabilization domain at its N-terminus, wherein the third stabilization domain and the fourth stabilization domain are capable of interacting with one another to stabilize a complex comprising the first effector subunit, the second effector subunit, and the third effector subunit.
  • the third stabilization domain and the fourth stabilization domain are derived from subunits of a heterodimerizing binding pair or a split-intein and are orthogonal to the first stabilization domain and the second stabilization domain.
  • the first stabilization domain and the second stabilization domain are derived from subunits of a heterodimerizing binding pair and the third stabilization domain and the fourth stabilization domain are derived from subunits of a split-intein; or the first stabilization domain and the second stabilization domain are derived from subunits of a split-intein and the third stabilization domain and the fourth stabilization domain are derived from subunits of a heterodimerizing binding pair.
  • the heterodimerizing binding pair is a coiled-coil (CC) heterodimer.
  • the CC heterodimer is a leucine zipper.
  • the leucine zipper subunits comprise RR12EE345L and EE12RR345L.
  • RR12EE345L comprises the amino acid sequence encoded by SEQ ID NO: 6.
  • EE12RR345L comprises the amino acid sequence encoded by SEQ ID NO: 5.
  • the CC heterodimer is a CC peptide heterodimer ( see Lebar, T., et al. (2020). Nature Chemical Biology, 1-7).
  • the split-intein subunits comprise DnaE N-intein and C-intein.
  • DnaE N-intein comprises the amino acid sequence encoded by SEQ ID NO: 7.
  • DnaE C-intein comprises the amino acid sequence encoded by SEQ ID NO: 8.
  • the split-intein is gp41 -
  • the split-intein comprises split portions of the Mxe GyrA intein (see Fine, E. J., et al. (2015). Scientific reports, 5, 10777).
  • the Casl2a protein or variant thereof is derived from a parental Casl2a protein from Lachnospiraceae bacterium.
  • the parental Casl2a protein from Lachnospiraceae bacterium comprises the amino acid sequence of SEQ ID NO: 1.
  • the Casl2a protein or variant thereof is a nuclease-competent Cas protein comprising a Cas 12a domain having some, the same, or greater nuclease activity as compared to a parental Cas 12a protein from which it is derived.
  • the Cas 12a domain comprises the amino acid sequence of SEQ ID NO: 1 or a variant thereof having at least about 80% sequence identity to the amino acid sequence of SEQ ID NO: 1.
  • the Cas 12a protein or variant thereof is a fusion protein comprising an effector domain fused to a nuclease-deficient dCasl2a domain having reduced or eliminated nuclease activity as compared to a parental Cas 12a protein from which it is derived, and the first output is generated by activity of the effector domain.
  • the dCasl2a domain comprises a modification of amino acid D832 with respect to SEQ ID NO: 1 that reduces, substantially eliminates, or eliminates nuclease activity of the dCasl2a domain.
  • a Casl2a protein is altered or otherwise modified to inactivate its nuclease activity.
  • Such alteration or modification includes altering one or more amino acids to inactivate the nuclease activity or the nuclease domain, as well as removing the polypeptide sequence or polypeptide sequences exhibiting nuclease activity, i.e., the nuclease domain, such that the polypeptide sequence or polypeptide sequences exhibiting nuclease activity, i.e. nuclease domain, are absent from the Casl2a protein.
  • a nuclease-null Casl2a protein includes polypeptide sequences modified to inactivate nuclease activity or removal of a polypeptide sequence or sequences to inactivate nuclease activity.
  • the dCasl2a protein retains the ability to bind to target nucleic acid even though the nuclease activity has been inactivated. Accordingly, the dCasl2a protein includes the polypeptide sequence or sequences required for nucleic acid binding.
  • the dCasl2a protein includes homologs and orthologs thereof which retain the ability of the protein to be guided by RNA to bind to target nucleic acid.
  • the dCasl2a protein comprises the amino acid sequence of SEQ ID NO: 2 or a variant thereof having at least 80% (such as at least about 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater) sequence identity to the amino acid sequence of SEQ ID NO: 2.
  • the dCasl2a protein is attached to, bound to, or fused with an effector domain, such as a transcriptional regulatory domain or an epigenetic modifying domain.
  • a dCasl2a fusion protein comprising a dCasl2a protein fused to an effector domain.
  • the effector domain is fused to the C-terminus of the dCasl2a protein.
  • the effector domain is fused to the N-terminus of the dCasl2a protein.
  • the effector domain comprises a subcellular localization signal.
  • the subcellular localization signals is an organelle localization signal, such as a nuclear localization signal (NLS), nuclear export signal (NES), or mitochondrial localization signal.
  • the effector domain comprises a polypeptide that can (i) cleave a nucleic acid, (ii) edit a nucleotide, (iii) activate transcription, (iv) repress transcription, (v) methylate a nucleic acid, and/or (vi) demethylate a nucleic acid.
  • isolated nucleic acid encoding a split Casl2a protein subunit according to any of the embodiments described herein.
  • the isolated nucleic acid is part of a vector (such as a plasmid or viral vector), and can be operably linked to a promoter, such as an inducible promoter.
  • Nucleotide sequences encoding split Casl2a protein subunits can be generated based on the amino acid sequences provided herein.
  • the isolated nucleic acid encoding a split Casl2a protein subunit comprises a protein coding sequence that is codon optimized for expression in a eukaryotic cell (e.g., a mammalian cell, such as a human cell).
  • a eukaryotic cell e.g., a mammalian cell, such as a human cell.
  • nucleic acid molecules encoding the split Casl2a protein subunits disclosed herein can be designed to have codons that are preferentially used by a particular organism of interest.
  • the nucleotide sequence encoding split Casl2a subunits is optimized for expression in human cells.
  • Casl2a is a single crRNA-guided endonuclease.
  • Cas 12a processes crRNA arrays independent of tracrRNA, and Casl2a-crRNA complexes alone are sufficient to cleave target DNA molecules, without the requirement for any additional RNA species ( see Zetsche, B., et al. (2015). Cell, 163(3), 759-771).
  • guide RNA gRNA
  • crRNA are used interchangeably herein in the context of Cas 12a systems and derivatives thereof, such as the split Cas 12a systems described herein.
  • Embodiments ofthe present disclosure are directed to the use of a split CRISPR Casl2a system and, in particular, a crRNA that includes a spacer sequence.
  • the term spacer sequence is understood by those of skill in the art and may include any polynucleotide having sufficient complementarity with a target nucleic acid sequence to hybridize with the target nucleic acid sequence and direct sequence-specific binding of a CRISPR complex to the target sequence.
  • a mature crRNA of a split Casl2a system may begin with an appropriate direct repeat followed by the spacer sequence.
  • the crRNA is between about 20 and about 100 nucleotides in length.
  • the spacer sequence is between about 10 and about 50 nucleotides in length.
  • nucleic acid encoding a crRNA is provided, wherein the nucleic acid comprises an inducible promoter operably linked to a polynucleotide sequence encoding the crRNA.
  • the inducible promoter is a polymerase II promoter.
  • nucleic acid encoding a crRNA is provided, wherein the nucleic acid comprises a constitutive promoter operably linked to a polynucleotide sequence encoding the crRNA.
  • the crRNA may be delivered directly to a cell as a native species by methods known to those of skill in the art, including injection or lipofection, or delivered indirectly to a cell as transcribed from a cognate DNA, with the cognate DNA introduced into the cell through electroporation, transient or stable transfection (including lipofection), or viral transduction.
  • compositions herein comprise, or the methods herein comprise delivering, one or more components of a split Casl2a nucleic acid-targeting system (also referred to herein as a “split Casl2a system”).
  • a split Casl2a nucleic acid-targeting system also referred to herein as a “split Casl2a system”.
  • split Casl2a system refers collectively to elements involved in the activity of a split Casl2a protein in association with a compatible crRNA to be targeted to a particular nucleic acid sequence (also referred to herein as a “target sequence,” or “protospacer-like sequence” in the context of an endogenous CRISPR-Cas system) as guided by the spacer sequence of the crRNA, and can include nucleic acid encoding the split Casl2a protein and/or the crRNA.
  • the split Casl2a protein and/or crRNA are derived from a particular organism comprising an endogenous Casl2a system.
  • a split Casl2a system is characterized by elements that promote the formation of a nucleic acid-targeting complex including a split Casl2a protein and a crRNA at the site of a target sequence, which can be present in a DNA molecule.
  • target sequence refers to a sequence to which a guide sequence (also referred to herein as a “spacer” or “spacer sequence”) in a crRNA of the system is designed to have complementarity, where hybridization between the target sequence and the crRNA allows for localization of the split Casl2a protein to the target sequence.
  • Full complementarity is not necessarily required, provided there is sufficient complementarity to allow for hybridization between the target sequence and the crRNA.
  • a target sequence is located in the nucleus or cytoplasm of a cell.
  • the target sequence may be within an organelle of a eukaryotic cell, for example, a mitochondrion or a chloroplast.
  • a split Casl2a system for conditionally generating an output to a sample comprising the system, the system comprising: (a) (i) a Casl2a protein or variant thereof having an effector activity, or nucleic acid encoding the Casl2a protein or variant thereof; or (ii) two or more effector subunits comprising fragments of the Casl2a protein or variant thereof, or nucleic acid encoding the two or more effector subunits, wherein the two or more effector subunits are capable of spontaneously assembling to form a split Casl2a complex in the sample, wherein the split Casl2a complex retains at least some of the effector activity of the Casl2a protein or variant thereof; and (b) a first CRISPR RNA (crRNA) or nucleic acid encoding the first crRNA, wherein the first crRNA comprises a first spacer having sufficient complementary to a first target polynucleotide sequence
  • the system further comprises nucleic acid comprising a NOT inducible promoter operably linked to a polynucleotide sequence encoding an anti- CRISPR (Acr) protein capable of inhibiting activity of the split Casl2a complex in the sample, wherein a NOT input to the sample is capable of increasing the expression of Acr protein from the nucleic acid in the sample such that activity of split Casl2a complex in the sample is inhibited.
  • a “NOT inducible promoter” as used herein refers to an inducible promoter operably linked to a polynucleotide sequence encoding an Acr protein capable of inhibiting activity of a split Casl2a complex.
  • a “NOT input” as used herein refers to an input having a cognate inducible promoter, wherein the cognate inducible promoter is operably linked to a polynucleotide sequence encoding an Acr protein capable of inhibiting activity of a split Casl2a complex.
  • the Acr protein is AcrVAl or a variant thereof.
  • the AcrVAl or variant thereof comprises the amino acid sequence encoded by SEQ ID NO: 9 or a variant thereof having at least 80% sequence identity to the amino acid sequence encoded by SEQ ID NO: 9.
  • a split Casl2a complex that retains at least some of the effector activity of a Casl2a protein or variant thereof retains at least about 5% (such as at least about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or greater) of the effector activity of the Casl2a protein or variant thereof.
  • the crRNA comprises a spacer sequence selected from the polynucleotide sequence of any one of SEQ ID NOs: 15-43.
  • the inducible promoter is selected from an RRM2 promoter, a hypoxia-inducible promoter, a TRE3G promoter, a CMV5-CuO promoter, and a P2 inducible promoter.
  • the RRM2 promoter comprises the polynucleotide sequence of SEQ ID NO: 10.
  • the hypoxia-inducible promoter comprises the polynucleotide sequence of SEQ ID NO: 11.
  • the TRE3G promoter comprises the polynucleotide sequence of SEQ ID NO: 12.
  • the CMV5-CuO promoter comprises the polynucleotide sequence of SEQ ID NO: 13.
  • the P2 inducible promoter comprises the polynucleotide sequence of SEQ ID NO: 14.
  • the split Casl2a system comprises nucleic acid comprising a plurality of inducible promoters, each of the plurality of inducible promoters operably linked to a polynucleotide sequence encoding an effector subunit are different.
  • the split Casl2a system comprises nucleic acid comprising a plurality of inducible promoters, all of the plurality of inducible promoters operably linked to a polynucleotide sequence encoding a crRNA are the same. In some embodiments, where the split Casl2a system comprises nucleic acid comprising a plurality of inducible promoters, each of the plurality of inducible promoters are different.
  • the system is configured such that all components of the Casl2a/crRNA complex are present or capable of being expressed in the sample when the first input and the second input each have a desired state.
  • the state of an input to a sample is a quantitative measure of some property of the input, wherein the input property is indicative of whether, and to what extent, the expression of a gene under the control of a cognate inducible promoter will be modified.
  • the input is doxycycline, having a cognate inducible promoter including a TRE3G promoter
  • the state of the input includes the concentration of doxycycline in the sample.
  • the system comprises nucleic acid encoding the Casl2a protein or variant thereof, wherein the nucleic acid encoding the Cas 12a protein or variant thereof comprises a first inducible promoter operably linked to a polynucleotide sequence encoding the Cas 12a protein or variant thereof, and the first input is capable of modulating the expression of the Cas 12a protein or variant thereof in the sample; and nucleic acid encoding the first crRNA, wherein the nucleic acid encoding the first crRNA comprises a second inducible promoter operably linked to a polynucleotide sequence encoding the first crRNA, and the second input is capable of modulating the expression of the first crRNA in the sample.
  • the system comprises nucleic acid encoding a first effector subunit and a second effector subunit of the two or more effector subunits, wherein the nucleic acid comprises a first inducible promoter operably linked to a polynucleotide sequence encoding the first effector subunit and a second inducible promoter operably linked to a polynucleotide sequence encoding the second effector subunit, and the first input is capable of modulating the expression of the first effector subunit in the sample and the second input is capable of modulating the expression of the second effector subunit in the sample; or nucleic acid encoding a first effector subunit of the two or more effector subunits, wherein the nucleic acid comprises a first inducible promoter operably linked to a polyn
  • the expression of a third component of the Casl2a/crRNA complex is under the control of a third input to the sample, and the system is configured such that all components of the Casl2a/crRNA complex are present or capable of being expressed in the sample when the first input, the second input, and the third input each have a desired state.
  • the system is configured such that all components of the Casl2a/crRNA complex are present or capable of being expressed in the sample when the first input is present, the second input is present, and the third input is present.
  • the system comprises nucleic acid encoding a first effector subunit, a second effector subunit, and a third effector subunit of the two or more effector subunits, wherein the nucleic acid comprises a first inducible promoter operably linked to a polynucleotide sequence encoding the first effector subunit, a second inducible promoter operably linked to a polynucleotide sequence encoding the second effector subunit, and a third inducible promoter operably linked to a polynucleotide sequence encoding the third effector subunit, and the first input is capable of modulating the expression of the first effector subunit in the sample, the second input is capable of modulating the expression of the second effector subunit in the sample, and the third input is capable of modulating the expression of the third effector subunit in the sample; or nucleic acid encoding a first effector subunit and a second effector subunit of the two or more effector subunits, wherein the nucle
  • the expression of a third component of the Casl2a/crRNA complex is under the control of a third input to the sample and the expression of a fourth component of the Casl2a/crRNA complex is under the control of a fourth input to the sample, and the system is configured such that all components of the Casl2a/crRNA complex are present or capable of being expressed in the sample when the first input, the second input, the third input, and the fourth input each have a desired state.
  • the system is configured such that all components of the Casl2a/crRNA complex are present or capable of being expressed when the first input is present, the second input is present, the third input is present, and the fourth input is present.
  • the system comprises nucleic acid encoding a first effector subunit, a second effector subunit, and a third effector subunit of the two or more effector subunits, wherein the nucleic acid comprises a first inducible promoter operably linked to a polynucleotide sequence encoding the first effector subunit, a second inducible promoter operably linked to a polynucleotide sequence encoding the second effector subunit, and a third inducible promoter operably linked to a polynucleotide sequence encoding the third effector subunit, and the first input is capable of modulating the expression of the first effector subunit in the sample, the second input is capable of modulating the expression of the second effector subunit in the sample
  • the two or more effector subunits comprise a first effector subunit and a second effector subunit, wherein the first effector subunit comprises an N-terminal fragment of the Casl2a protein or variant thereof up to and including a split point amino acid, and the second effector subunit comprises a C-terminal fragment of the Casl2a protein or variant thereof following the split point amino acid.
  • the split point amino acid corresponds to A406 or G550 with respect to a parental Cas 12a protein amino acid sequence of SEQ ID NO: 1.
  • the first effector subunit further comprises a first stabilization domain at its C- terminus and the second effector subunit further comprises a second stabilization domain at its N-terminus, wherein the first stabilization domain and the second stabilization domain are capable of interacting with one another to stabilize a complex comprising the first effector subunit and the second effector subunit in the sample.
  • the two or more effector subunits comprise a first effector subunit and a second effector subunit, wherein the Cas 12a protein or variant thereof is a fusion protein comprising an effector domain fused to a Cas 12a domain, and the first effector subunit comprises the Cas 12a domain and the second effector subunit comprises the effector domain.
  • the first effector subunit further comprises a first stabilization domain at either terminus and the second effector subunit further comprises a second stabilization domain at either terminus, wherein the first stabilization domain and the second stabilization domain are capable of interacting with one another to stabilize a complex comprising the first effector subunit and the second effector subunit in the sample.
  • the first stabilization domain and the second stabilization domain are derived from subunits of a heterodimerizing binding pair or a split-intein.
  • the first stabilization domain and the second stabilization domain are derived from subunits of a heterodimerizing binding pair.
  • the heterodimerizing binding pair is a coiled-coil (CC) heterodimer.
  • the CC heterodimer is a leucine zipper.
  • the leucine zipper subunits comprise RR12EE345L and EE12RR345L.
  • RR12EE345L comprises the amino acid sequence encoded by SEQ ID NO: 6. In some embodiments, EE12RR345L comprises the amino acid sequence encoded by SEQ ID NO: 5.
  • the CC heterodimer is a CC peptide heterodimer ( see Lebar, T., et al. (2020). Nature Chemical Biology, 1-7).
  • the first stabilization domain and the second stabilization domain are derived from subunits of a split-intein.
  • the split-intein subunits comprise DnaEN-intein and C-intein.
  • DnaEN- intein comprises the amino acid sequence encoded by SEQ ID NO: 7.
  • DnaE C-intein comprises the amino acid sequence encoded by SEQ ID NO: 8.
  • the split-intein is gp41-l, gp41-8, NrdJ-1, or IMPDH-1 ( see Dassa, B., et al. (2009). Nucleic acids research, 37(8), 2560-2573).
  • the split-intein comprises split portions of the Mxe GyrA intein (see Fine, E. J., et al. (2015). Scientific reports, 5, 10777).
  • the two or more effector subunits comprise a first effector subunit, a second effector subunit, and a third effector subunit.
  • the Casl2a protein or variant thereof is a fusion protein comprising an effector domain fused to a Casl2a domain, and the first effector subunit comprises an N-terminal fragment of the Casl2a domain up to and including a split point amino acid, the second effector subunit comprises a C-terminal fragment of the Casl2a domain following the split point amino acid; and the third effector subunit comprises the effector domain.
  • the split point amino acid corresponds to A406 or G550 with respect to a parental Cas 12a protein amino acid sequence of SEQ ID NO: 1.
  • the first effector subunit further comprises a first stabilization domain at its C- terminus and the second effector subunit further comprises a second stabilization domain at its N-terminus, wherein the first stabilization domain and the second stabilization domain are capable of interacting with one another to stabilize a complex comprising the first effector subunit and the second effector subunit in the sample; and/or the third effector subunit further comprises a third stabilization domain at either terminus and (i) the second effector subunit further comprises a fourth stabilization domain at its C-terminus or (ii) the first effector subunit further comprises the fourth stabilization domain at its N-terminus, wherein the third stabilization domain and the fourth stabilization domain are capable of interacting with one another to stabilize a complex comprising the first effector subunit, the second effector subunit, and the third effector subunit in the sample.
  • the third stabilization domain and the fourth stabilization domain are derived from subunits of a heterodimerizing binding pair or a split-intein and are orthogonal to the first stabilization domain and the second stabilization domain.
  • the first stabilization domain and the second stabilization domain are derived from subunits of a heterodimerizing binding pair and the third stabilization domain and the fourth stabilization domain are derived from subunits of a split- intein; or the first stabilization domain and the second stabilization domain are derived from subunits of a split-intein and the third stabilization domain and the fourth stabilization domain are derived from subunits of a heterodimerizing binding pair.
  • the heterodimerizing binding pair is a coiled-coil (CC) heterodimer.
  • the CC heterodimer is a leucine zipper.
  • the leucine zipper subunits comprise RR12EE345L and EE12RR345L.
  • RR12EE345L comprises the amino acid sequence encoded by SEQ ID NO: 6.
  • EE12RR345L comprises the amino acid sequence encoded by SEQ ID NO: 5.
  • the CC heterodimer is a CC peptide heterodimer ( see Lebar, T., et al. (2020). Nature Chemical Biology, 1-7).
  • the split-intein subunits comprise DnaE N-intein and C-intein.
  • DnaE N-intein comprises the amino acid sequence encoded by SEQ ID NO: 7.
  • DnaE C-intein comprises the amino acid sequence encoded by SEQ ID NO: 8.
  • the split-intein is gp41-l, gp41-8, NrdJ-1, or IMPDH-1 ( see Dassa, B., et al. (2009). Nucleic acids research, 37(8), 2560-2573).
  • the split-intein comprises split portions of the Mxe GyrA intein (see Fine, E. J., et al. (2015). Scientific reports, 5, 10777).
  • the system further comprises one or more additional crRNAs or nucleic acid encoding the one or more additional crRNAs, wherein Casl2a/crRNA complexes formed by association of the one or more additional crRNAs with (I) the Casl2a protein or variant thereof or (II) the two or more effector subunits are capable of generating one or more additional outputs to the sample comprising effector activity of the Casl2a/crRNA complexes at target sites directed by the one or more additional crRNAs.
  • the system comprises nucleic acid encoding a crRNA array comprising one or more of the first crRNA and the one or more additional crRNAs.
  • the Casl2a protein or variant thereof is derived from a parental Casl2a protein from Lachnospiraceae bacterium.
  • the parental Casl2a protein from Lachnospiraceae bacterium comprises the amino acid sequence of SEQ ID NO: 1.
  • the target nucleic acid is a target dsDNA.
  • a protospacer in the target dsDNA targeted by the crRNA spacer has a 5' PAM selected from 5'-TTTG-3', 5'- TTTA-3', and 5'-TTTC-3'.
  • the Casl2a protein or variant thereof is a nuclease-competent Cas protein comprising a Casl2a domain having some, the same, or greater nuclease activity as compared to a parental Cas 12a protein from which it is derived, and the first output comprises cleavage (e.g., double-stranded cleavage) of the first target nucleic acid near the first target polynucleotide sequence.
  • a split Casl2a/crRNA complex cleaves the first target nucleic acid distal from the PAM, with cleavage after the 18th base on the non-targeted (+) strand and after the 23rd base on the targeted (-) strand.
  • the Cas 12a domain comprises the amino acid sequence of SEQ ID NO: 1.
  • the Cas 12a protein or variant thereof is a fusion protein comprising an effector domain fused to a nuclease-deficient dCasl2a domain having reduced or eliminated nuclease activity as compared to a parental Cas 12a protein from which it is derived, and the first output is generated by activity of the effector domain.
  • the dCasl2a domain comprises a modification of amino acid D832 with respect to SEQ ID NO: 1 that reduces, substantially eliminates, or eliminates nuclease activity of the dCasl2a domain.
  • the dCasl2a domain comprises a D832A substitution with respect to SEQ ID NO: 1.
  • the dCasl2a domain comprises the amino acid sequence of SEQ ID NO: 2.
  • the effector domain comprises a polypeptide that can (i) cleave a nucleic acid, (ii) edit a nucleotide, (iii) activate transcription, (iv) repress transcription, (v) methylate a nucleic acid, and/or (vi) demethylate a nucleic acid.
  • the effector domain comprises a transcriptional activation domain
  • the first output comprises transcriptional activation of a first gene targeted by the first crRNA.
  • the transcriptional activation domain is a tripartite VP64-p65-Rta (VPR) transcriptional activation domain or variant thereof.
  • the sample is a cell or a composition comprising a cell.
  • the sample is a biological sample comprising a cell, including, without limitation, a tissue, fluid, or other composition in an organism.
  • the inputs to the sample are selected from physical inputs, small molecule inputs, and biologic inputs.
  • the physical inputs are selected from oxygen levels, pH, ion concentrations, temperature, light, mechanical force, ultrasound, shock wave, magnetic field, and electric field; the small molecule inputs are selected from drugs, metabolites, and adjuvants; and/or the biologic inputs are selected from polypeptides, polypeptide derivatives, and nucleic acids.
  • the system comprises a TRE3G-specific crRNA targeting the TRE3G promoter, or nucleic acid encoding the TRE3G-specific crRNA
  • the Casl2a protein or variant thereof is a fusion protein comprising an effector domain fused to a Casl2a domain, wherein the effector domain comprises a transcriptional activation domain, such that association of the TRE3G-specific crRNA with the split Casl2a complex allows for increasing the expression of a gene operably linked to the TRE3G promoter, and an output of the system comprises increased expression of the gene.
  • the TRE3G-specific crRNA comprises a spacer comprising the polynucleotide sequence of SEQ ID NO: 15.
  • the system comprises nucleic acid comprising the TRE3G promoter operably linked to a gene encoding a component of the split Casl2a system, such as an effector subunit or crRNA, and an input of the system is concentration of doxycycline in the sample, wherein a concentration of doxycycline in the sample within a certain range (including, e.g., above a certain threshold) allows for increased expression of the split Casl2a system component.
  • a concentration of doxycycline in the sample within a certain range (including, e.g., above a certain threshold) allows for increased expression of the split Casl2a system component.
  • a concentration of doxycycline in the sample within the certain range allows for sufficient expression of the split Casl2a system component such that it can participate in the formation of a split Casl2a/crRNA complex capable of mediating an output of the system.
  • the sample is a composition comprising a cell comprising the split Casl2a system.
  • the system comprises nucleic acid comprising the RRM2 promoter operably linked to a gene encoding a component of the split Casl2a system, such as an effector subunit or crRNA, and an input of the system is expression of one or more transcription factors capable of driving the expression of the RRM2 promoter in the sample, wherein expression of the one or more transcription factors in the sample within a certain range (including, e.g., above a certain threshold) allows for increased expression of the split Casl2a system component.
  • a certain range including, e.g., above a certain threshold
  • expression of the one or more transcription factors in the sample within the certain range allows for sufficient expression of the split Casl2a system component such that it can participate in the formation of a split Casl2a/crRNA complex capable of mediating an output of the system.
  • the sample is a cell, such as a cancer cell, wherein the cancer cell is characterized by increased expression of at least one of the one or more transcription factors capable of driving the expression of the RRM2 promoter.
  • the cancer cell is a breast cancer cell.
  • the system comprises nucleic acid comprising the hypoxia-inducible promoter operably linked to a gene encoding a component of the split Casl2a system, such as an effector subunit or crRNA, and an input of the system is expression of one or more hypoxia-responsive elements (HREs) capable of driving the expression of the hypoxia-inducible promoter in the sample, wherein expression of the one or more HREs in the sample within a certain range (including, e.g., above a certain threshold) allows for increased expression of the split Casl2a system component.
  • HREs hypoxia-responsive elements
  • expression of the one or more HREs in the sample within the certain range allows for sufficient expression of the split Casl2a system component such that it can participate in the formation of a split Casl2a/crRNA complex capable of mediating an output of the system.
  • the sample is a cell, such as a cancer cell, wherein the cancer cell is characterized by hypoxia leading to increased expression of at least one of the one or more HREs capable of driving the expression of the hypoxia-inducible promoter.
  • the cancer cell is a breast cancer cell.
  • the HREs include, without limitation, stabilized cellular HIFl ⁇ .
  • the system comprises nucleic acid comprising the CMV5- CuO promoter operably linked to a gene encoding a component of the split Casl2a system, such as an effector subunit or crRNA, and an input of the system is concentration of cumate in the sample, wherein a concentration of cumate in the sample within a certain range (including, e.g., above a certain threshold) allows for increased expression of the split Casl2a system component.
  • a concentration of cumate in the sample within the certain range allows for sufficient expression of the split Casl2a system component such that it can participate in the formation of a split Casl2a/crRNA complex capable of mediating an output of the system.
  • the sample is a composition comprising a cell comprising the split Casl2a system.
  • the system comprises a P2-specific crRNA targeting the P2 promoter, or nucleic acid encoding the P2-specific crRNA
  • the Casl2a protein or variant thereof is a fusion protein comprising an effector domain fused to a Casl2a domain, wherein the effector domain comprises a transcriptional activation domain, such that association of the P2-specific crRNA with the split Casl2a complex allows for increasing the expression of a gene operably linked to the P2 promoter, and an output of the system comprises increased expression of the gene.
  • the P2-specific crRNA comprises a spacer comprising the polynucleotide sequence of SEQ ID NO: 17.
  • the system comprises a CXCR4-specific crRNA targeting a CXCR4 gene, or nucleic acid encoding the CXCR4-specific crRNA.
  • the CXCR4-specific crRNA comprises a spacer comprising a polynucleotide sequence selected from any one of SEQ ID NOs: 18-23.
  • the Casl2a protein or variant thereof is a fusion protein comprising an effector domain fused to a Casl2a domain, wherein the effector domain comprises a transcriptional activation domain, such that association of the CXCR4-specific crRNA with the split Cas 12a complex allows for increasing the expression of the CXCR4 gene, and an output of the system comprises increased expression of the CXCR4 gene.
  • the CXCR4-specific crRNA comprises a spacer comprising a polynucleotide sequence selected from any one of SEQ ID NOs: 18-22.
  • the Casl2a protein or variant thereof is a nuclease-competent Cas 12a protein or variant thereof, such that association of the CXCR4-specific crRNA with the split Cas 12a complex allows for cleavage of the CXCR4 gene, and an output of the system comprises cleavage of the CXCR4 gene.
  • the CXCR4-specific crRNA comprises a spacer comprising the polynucleotide sequence of SEQ ID NO: 23.
  • the system comprises an IFN ⁇ -specific crRNA targeting an IFNg gene, or nucleic acid encoding the IFN ⁇ -specific crRNA.
  • the IFN ⁇ -specific crRNA comprises a spacer comprising a polynucleotide sequence selected from any one of SEQ ID NOs: 24-29.
  • the Cas 12a protein or variant thereof is a fusion protein comprising an effector domain fused to a Cas 12a domain, wherein the effector domain comprises a transcriptional activation domain, such that association of the IFN ⁇ -specific crRNA with the split Cas 12a complex allows for increasing the expression of the IFNg gene, and an output of the system comprises increased expression of the IFNg gene.
  • the system comprises an interleukin 2 (IL2)-specific crRNA targeting an IL2 gene, or nucleic acid encoding the IL2-specific crRNA.
  • IL2 interleukin 2
  • the IL2-specific crRNA comprises a spacer comprising a polynucleotide sequence selected from any one of SEQ ID NOs: 30-43.
  • the Casl2a protein or variant thereof is a fusion protein comprising an effector domain fused to a Casl2a domain, wherein the effector domain comprises a transcriptional activation domain, such that association of the IL2-specific crRNA with the split Casl2a complex allows for increasing the expression of the IL2 gene, and an output of the system comprises increased expression of the IL2 gene.
  • the system comprises a plurality of IL2-specific crRNAs targeting the IL2 gene, or nucleic acid encoding the plurality of IL2-specific crRNAs.
  • the system comprises a first crRNA targeting the IL2 gene comprising a first spacer comprising the polynucleotide sequence of SEQ ID NO: 31, or nucleic acid encoding the first crRNA, and a second crRNA targeting the IL2 gene comprising a second spacer comprising the polynucleotide sequence of SEQ ID NO: 35, or nucleic acid encoding the second crRNA.
  • the sample comprises a cancer cell comprising the system, and one or more outputs of the system comprise increased expression in the cancer cell of one or more immunostimulatory cytokines, including, without limitation, IFNg and IL2.
  • the system comprises nucleic acid comprising an RRM2 promoter operably linked to a gene encoding a first component of the split Casl2a system, such as an effector subunit or crRNA, and an input of the system is expression of one or more transcription factors capable of driving the expression of the RRM2 promoter in the cancer cell, wherein expression of the one or more transcription factors in the cancer cell within a certain range (including, e.g., above a certain threshold) allows for increased expression of the first component, and the cancer cell is characterized by increased expression of at least one of the one or more transcription factors capable of driving the expression of the RRM2 promoter.
  • a certain range including, e.g., above a certain threshold
  • expression of the one or more transcription factors in the cancer cell within the certain range allows for sufficient expression of the first component such that it can participate in the formation of a split Casl2a/crRNA complex capable of mediating a first output of the system, such as increased expression in the cancer cell of one or more immunostimulatory cytokines, including, without limitation, IFNg and IL2.
  • the system comprises nucleic acid comprising a hypoxia-inducible promoter operably linked to a gene encoding a second component of the split Cast 2a system, such as an effector subunit or crRNA, and an input of the system is expression of one or more hypoxia-responsive elements (HREs) capable of driving the expression of the hypoxia-inducible promoter in the sample, wherein expression of the one or more HREs in the sample within a certain range (including, e.g., above a certain threshold) allows for increased expression of the second component, wherein the cancer cell is characterized by hypoxia leading to increased expression of at least one of the one or more HREs capable of driving the expression of the hypoxia-inducible promoter.
  • HREs hypoxia-responsive elements
  • expression of the one or more HREs in the sample within the certain range allows for sufficient expression of the second component such that it can participate in the formation of a split Casl2a/crRNA complex capable of mediating a second output of the system, such as increased expression in the cancer cell of one or more immunostimulatory cytokines, including, without limitation, IFNg and IL2.
  • the HREs include, without limitation, stabilized cellular HIFl ⁇ .
  • the system comprises the nucleic acid comprising an RRM2 promoter operably linked to a gene encoding the first component and the nucleic acid comprising a hypoxia-inducible promoter operably linked to a gene encoding the second component, wherein the first and second outputs comprise increased expression of IFN ⁇ and IL2.
  • the Casl2a protein or variant thereof is a fusion protein comprising an effector domain fused to a Casl2a domain, wherein the effector domain comprises a transcriptional activation domain.
  • the system comprises an IFN ⁇ -specific crRNA targeting an IFNg gene, or nucleic acid encoding the IFN ⁇ -specific crRNA.
  • the IFN ⁇ -specific crRNA comprises a spacer comprising a polynucleotide sequence selected from any one of SEQ ID NOs: 24-29.
  • the system comprises an interleukin 2 (IL2)-specific crRNA targeting an IL2 gene, or nucleic acid encoding the IL2-specific crRNA.
  • the IL2-specific crRNA comprises a spacer comprising a polynucleotide sequence selected from any one of SEQ ID NOs: 30-43.
  • the system comprises a first crRNA targeting the IL2 gene comprising a first spacer comprising the polynucleotide sequence of SEQ ID NO: 31 , or nucleic acid encoding the first crRNA, and a second crRNA targeting the IL2 gene comprising a second spacer comprising the polynucleotide sequence of SEQ ID NO: 35, or nucleic acid encoding the second crRNA.
  • the cancer cell is a breast cancer cell.
  • An aspect of the disclosure is one or more nucleic acids that encode one or more components of a split Cast 2a system as described herein.
  • nucleic acids encoding one or more of: (a) (i) a Casl2a protein or variant thereof having an effector activity; or (ii) two or more effector subunits comprising fragments of the Casl2a protein or variant thereof, wherein the two or more effector subunits are capable of spontaneously assembling to form a split Cas 12a complex, wherein the split Cas 12a complex retains at least some of the effector activity of the Cas 12a protein or variant thereof; and (b) a first crRNA, wherein the first crRNA comprises a first spacer having sufficient complementary to a first target polynucleotide sequence in a first target nucleic acid such that it can hybridize to the first target nucleic acid, wherein a Casl2a/crRNA complex formed by association of the first crRNA with (I) the Cas 12a protein or variant thereof or (II) the two or more effector subunits is capable of
  • nucleic acid and “polynucleotide” are used interchangeably herein, and refer to both RNA and DNA molecules, including nucleic acid molecules comprising cDNA, genomic DNA, and/or synthetic DNA, and DNA or RNA molecules containing nucleic acid analogs.
  • a nucleic acid can be double-stranded or single-stranded (for example, a sense strand or an antisense strand).
  • a nucleic acid may contain unconventional or modified nucleotides.
  • polynucleotide sequence and “nucleic acid sequence” as used herein interchangeably refer to the sequence of a nucleic acid molecule. The nomenclature for nucleotide bases set forth in 37 CFR ⁇ 1.822 is used herein.
  • Nucleic acids of the invention include multicistronic nucleic acids, wherein two or more coding sequences are separated by sequences encoding an IRES (internal ribosome entry site) or a self-cleaving polypeptide sequence, such as a 2A sequence, providing for expression of each protein encoded by the two or more coding sequences separately, or for the immediate cleavage into two separate proteins upon expression.
  • IRES internal ribosome entry site
  • 2A sequences include T2A, P2A, E2A, and F2A.
  • the nucleic acid is operably linked to a heterologous nucleic acid sequence, such as, for example a structural gene that encodes a protein of interest or a regulatory sequence (e.g., a promoter sequence).
  • a heterologous nucleic acid sequence such as, for example a structural gene that encodes a protein of interest or a regulatory sequence (e.g., a promoter sequence).
  • the nucleic acid is an expression cassette or a vector.
  • the vector is a lentiviral vector, an adeno virus vector, an adeno-associated virus vector, or a retroviral vector.
  • Some embodiments disclosed herein relate to vectors or expression cassettes including a nucleic acid molecule as disclosed herein.
  • An expression cassettes is a construct of genetic material that contains coding sequences and enough regulatory information to direct proper transcription and/or translation of the coding sequences in a recipient cell, in vivo and/or ex vivo.
  • the expression cassette may be inserted into a vector for targeting to a desired host cell.
  • expression cassette may be used interchangeably with the term “expression construct.”
  • the nucleic acid molecules described herein can be contained within a vector that is capable of directing their expression in, for example, a cell that has been transduced with the vector.
  • Suitable vectors for use in eukaryotic cells are known in the art and are commercially available or readily prepared by a skilled artisan. Additional vectors can also be found, for example, in Ausubel, F. M., et al , Current Protocols in Molecular Biology, (Current Protocol, 1994) and Sambrook et al., “Molecular Cloning: A Laboratory Manual,” 2nd Ed. (1989).
  • the vectors are useful for autonomous replication in a host cell or may be integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome (e.g., non-episomal mammalian vectors).
  • Expression vectors are capable of directing the expression of coding sequences to which they are operably linked.
  • expression vectors of utility in recombinant DNA techniques are often in the form of plasmids (vectors).
  • viral vectors e.g., replication defective retroviruses, adenoviruses, and adeno-associated viruses
  • DNA vectors can be introduced into eukaryotic cells via conventional transformation or transfection techniques. Suitable methods for transforming or transfecting host cells can be found in Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual (2nd ed., Cold Spring Harbor Laboratory Press, Plainview, N.Y.) and other standard molecular biology laboratory manuals.
  • nucleic acid inserts which encode a split Casl2a system component in such vectors, can be operably linked to a promoter, which is selected based on, for example, the cell type in which expression is sought.
  • Viral vectors that can be used in the disclosure include, for example, retroviral, adenoviral, and adeno-associated vectors, herpes virus, simian virus 40 (SV40), and bovine papilloma virus vectors (see, for example, Gluzman (Ed.), Eukaryotic Viral Vectors, CSH Laboratory Press, Cold Spring Harbor, N.Y.).
  • the expression vector can be a viral vector.
  • viral vector is widely used to refer either to a nucleic acid molecule that includes virus-derived nucleic acid elements that typically facilitate transfer of the nucleic acid molecule or integration into the genome of a cell, or to a viral particle that mediates nucleic acid transfer. Viral particles typically include viral components, and sometimes also host cell components, in addition to nucleic acid(s).
  • Retroviral vectors used herein contain structural and functional genetic elements, or portions thereof, that are primarily derived from a retrovirus.
  • Retroviral lentivirus vectors contain structural and functional genetic elements, or portions thereof including LTRs, that are primarily derived from a lentivirus (a sub-type of retrovirus).
  • Viral vectors that can be used in the disclosure include, for example, retrovirus vectors (including lentivirus vectors), adenovirus vectors, and adeno-associated virus vectors, herpes virus, simian virus 40 (SV40), and bovine papilloma virus vectors (see, for example, Gluzman (Ed.), Eukaryotic Viral Vectors, CSH Laboratory Press, Cold Spring Harbor, N.Y.).
  • the nucleic acid molecules are delivered by viral or non-viral delivery vehicles known in the art.
  • the nucleic acid molecule can be stably integrated in the host genome, or can be episomally replicating, or present in the recombinant host cell as a mini-circle expression vector for stable or transient expression.
  • the nucleic acid molecule is maintained and replicated in the recombinant host cell as an episomal unit.
  • the nucleic acid molecule is stably integrated into the genome of the recombinant cell.
  • Stable integration can also be accomplished using classical random genomic recombination techniques or with more precise genome editing techniques such as using guide RNA-directed CRISPR/Cas9, DNA-guided endonuclease genome editing NgAgo ( Natronobacterium gregoryi Argonaute ), or TALENs genome editing (transcription activator-like effector nucleases).
  • the nucleic acid molecule is present in the recombinant host cell as a mini-circle expression vector for stable or transient expression.
  • the nucleic acid molecules can be encapsulated in a viral capsid or a lipid nanoparticle.
  • introduction of nucleic acids into cells may be achieved using viral transduction methods.
  • adeno-associated virus AAV is a non-enveloped virus that can be engineered to deliver nucleic acids to target cells via viral transduction.
  • AAV serotypes have been described, and all of the known serotypes can infect cells from multiple diverse tissue types. AAV is capable of transducing a wide range of species and tissues in vivo with no evidence of toxicity, and it generates relatively mild innate and adaptive immune responses.
  • Lentiviral systems are also useful for nucleic acid delivery and gene therapy via viral transduction.
  • Lentiviral vectors offer several attractive properties as gene-delivery vehicles, including: (i) sustained gene delivery through stable vector integration into the host cell genome; (ii) the ability to infect both dividing and non-dividing cells; (iii) broad tissue tropisms, including important gene- and cell-therapy-target cell types; (iv) no expression of viral proteins after vector transduction; (v) the ability to deliver complex genetic elements, such as polycistronic or intron-containing sequences; (vi) a potentially safer integration site profile (e.g., by targeting a site for integration that has little or no oncogenic potential); and (vii) a relatively easy system for vector manipulation and production.
  • a split Casl2a system as described herein.
  • Such methods can include contacting one or more target nucleic acid molecules with a non-naturally occurring or engineered (e.g., does not naturally occur in the cell or system into which it is introduced) split Casl2a complex and a crRNA.
  • a spacer sequence within the crRNA molecule is not naturally occurring, and has been modified to be complementary to a target nucleic acid molecule.
  • the target nucleic acid molecule is a DNA molecule, such as a genomic DNA.
  • a method of conditionally targeting e.g., binding to, modifying, detecting, etc.
  • a target nucleic acid in a sample e.g., a biological sample, such as a cell
  • a sample e.g., a biological sample, such as a cell
  • the expression of each of the components is individually under the control of different inputs to the sample such that targeting of the target nucleic acid by a split Casl2a/crRNA complex only occurs when each of the different inputs have a desired state.
  • the split Casl2a system comprises (a) nucleic acid encoding two or more effector subunits comprising fragments of a Casl2a protein or variant thereof, wherein the two or more effector subunits are capable of spontaneously assembling to form a split Casl2a complex retaining at least some of the effector activity of the Casl2a protein or variant thereof; and (b) a CRISPR RNA (crRNA) or nucleic acid encoding the crRNA, wherein the crRNA comprises a spacer having sufficient complementary to a target polynucleotide sequence in the target nucleic acid in the cell such that it can hybridize to the target nucleic acid.
  • crRNA CRISPR RNA
  • the split Casl2a system comprises nucleic acid encoding the first crRNA.
  • the split Casl2a system further comprises nucleic acid comprising a NOT inducible promoter operably linked to a polynucleotide sequence encoding an anti-CRISPR (Acr) protein capable of inhibiting activity of the split Casl2a complex, wherein a NOT input to the cell is capable of increasing the expression of Acr protein in the cell such that activity of split Cas 12a complex in the cell is inhibited.
  • the sample is a cell or a composition comprising a cell.
  • the cell is a mammalian cell, e.g., a human cell.
  • Targeting a nucleic acid molecule can include one or more of cutting or nicking the target nucleic acid molecule; modulating the expression of a gene present in the target nucleic acid molecule (such as by regulating transcription of the gene from a target DNA, e.g., to downregulate or upregulate expression of the gene); visualizing, labeling, or detecting the target nucleic acid molecule; binding the target nucleic acid molecule, editing the target nucleic acid molecule, trafficking the target nucleic acid molecule, and masking the target nucleic acid molecule.
  • modifying the target nucleic acid molecule includes introducing one or more of a nucleobase substitution, a nucleobase deletion, a nucleobase insertion, a break in the target nucleic acid molecule, methylation of the target nucleic acid molecule, and demethylation of the nucleic acid molecule.
  • such methods are used to treat a disease, such as a disease in a human.
  • one or more target nucleic acid molecules are associated with the disease.
  • a split Casl2a system as described herein is introduced into a sample (e.g., a biological sample, such as a cell) to allow for the conditional generation of an output to the sample, wherein for two or more components of the split Cas 12a system, the expression of each of the components is individually under the control of different inputs to the sample.
  • the inputs are selected from physical inputs, small molecule inputs, and biologic inputs.
  • the output comprises modulating gene expression from a target nucleic acid in the sample, or otherwise modifying the target nucleic acid.
  • a method of conditionally generating an output to a sample comprising introducing into the sample the components of a split Cas 12a system as described herein, wherein for two or more components of the split Cas 12a system, the expression of each of the components is individually under the control of different inputs to the sample such that an output mediated by a split Cas 12a/ crRNA complex is only generated when each of the different inputs have a desired state.
  • the split Cas 12a system comprises (a) nucleic acid encoding two or more effector subunits comprising fragments of a Cas 12a protein or variant thereof, wherein the two or more effector subunits are capable of spontaneously assembling to form a split Cas 12a complex retaining at least some of the effector activity of the Cas 12a protein or variant thereof; and (b) a CRISPR RNA (crRNA) or nucleic acid encoding the crRNA, wherein the crRNA comprises a spacer having sufficient complementary to a target polynucleotide sequence in the target nucleic acid in the sample such that it can hybridize to the target nucleic acid.
  • crRNA CRISPR RNA
  • the split Cas 12a system comprises nucleic acid encoding the first crRNA.
  • the split Cas 12a system further comprises nucleic acid comprising a NOT inducible promoter operably linked to a polynucleotide sequence encoding an anti-CRISPR (Acr) protein capable of inhibiting activity of the split Cas 12a complex, wherein a NOT input to the sample is capable of increasing the expression of Acr protein in the cell such that activity of split Cas 12a complex in the cell is inhibited.
  • the sample is a cell or a composition comprising a cell.
  • the cell is a mammalian cell, e.g., a human cell.
  • provided herein are methods of conditionally editing a target nucleic acid in a sample using a split Cas 12a system as described herein.
  • the methods generally involve contacting the target nucleic acid with a nuclease-competent split Casl2a/crRNA complex comprising activity that cleaves the target nucleic acid.
  • the split Casl2a/crRNA complex has nucleobase-editing activity.
  • the target nucleic acid molecule is a DNA molecule, such as a genomic DNA.
  • a method of conditionally editing a target nucleic acid in a sample comprising introducing into the sample the components of a split Cas 12a system as described herein, wherein for two or more components of the split Cas 12a system, the expression of each of the components is individually under the control of different inputs to the sample such that editing of the target nucleic acid by a split Casl2a/crRNA complex only occurs when each of the different inputs have a desired state.
  • the split Cas 12a system comprises (a) nucleic acid encoding two or more effector subunits comprising fragments of a Cas 12a protein or variant thereof having a nucleic acid-editing effector activity, wherein the two or more effector subunits are capable of spontaneously assembling to form a split Cas 12a complex retaining at least some of the effector activity of the Cas 12a protein or variant thereof; and (b) a CRISPR RNA (crRNA) or nucleic acid encoding the crRNA, wherein the crRNA comprises a spacer having sufficient complementary to a target polynucleotide sequence in the target nucleic acid in the sample such that it can hybridize to the target nucleic acid.
  • crRNA CRISPR RNA
  • the split Cas 12a system comprises nucleic acid encoding the first crRNA.
  • the split Cas 12a system further comprises nucleic acid comprising a NOT inducible promoter operably linked to a polynucleotide sequence encoding an anti-CRISPR (Acr) protein capable of inhibiting activity of the split Cas 12a complex, wherein a NOT input to the sample is capable of increasing the expression of Acr protein in the sample such that activity of split Cas 12a complex in the sample is inhibited.
  • the sample is a cell or a composition comprising a cell.
  • the cell is a mammalian cell, e.g., a human cell.
  • the Acr protein is AcrVAl or a variant thereof.
  • the AcrVAl or variant thereof comprises the amino acid sequence encoded by SEQ ID NO: 9 or a variant thereof having at least 80% sequence identity to the amino acid sequence encoded by SEQ ID NO: 9.
  • a split Cas 12a complex that retains at least some of the effector activity of a Cas 12a protein or variant thereof retains at least about 5% (such as at least about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or greater) of the effector activity of the Cas 12a protein or variant thereof.
  • the crRNA comprises a spacer sequence selected from the polynucleotide sequence of any one of SEQ ID NOs: 15-43.
  • the inducible promoter is selected from an RRM2 promoter, a hypoxia-inducible promoter, a TRE3G promoter, a CMV5-CuO promoter, and a P2 inducible promoter.
  • the RRM2 promoter comprises the polynucleotide sequence of SEQ ID NO: 10.
  • the hypoxia-inducible promoter comprises the polynucleotide sequence of SEQ ID NO: 11.
  • the TRE3G promoter comprises the polynucleotide sequence of SEQ ID NO: 12.
  • the CMV5-CuO promoter comprises the polynucleotide sequence of SEQ ID NO: 13.
  • the P2 inducible promoter comprises the polynucleotide sequence of SEQ ID NO: 14.
  • the split Cas 12a system comprises nucleic acid comprising a plurality of inducible promoters, each of the plurality of inducible promoters operably linked to a polynucleotide sequence encoding an effector subunit are different.
  • the split Cas 12a system comprises nucleic acid comprising a plurality of inducible promoters
  • each of the plurality of inducible promoters operably linked to a polynucleotide sequence encoding a crRNA are different.
  • the split Cas 12a system comprises nucleic acid comprising a plurality of inducible promoters, at least some (such as at least about any of 2, 3, 4, or more) of the plurality of inducible promoters operably linked to a polynucleotide sequence encoding a crRNA are the same.
  • the split Cas 12a system comprises nucleic acid comprising a plurality of inducible promoters, all of the plurality of inducible promoters operably linked to a polynucleotide sequence encoding a crRNA are the same. In some embodiments, where the split Cas 12a system comprises nucleic acid comprising a plurality of inducible promoters, each of the plurality of inducible promoters are different.
  • the system is configured such that all components of the Casl2a/crRNA complex are present or capable of being expressed in the sample when the first input and the second input each have a desired state. In some embodiments, the system is configured such that all components of the Casl2a/crRNA complex are present or capable of being expressed in the sample when the first input is present and the second input is present.
  • the system comprises (a) nucleic acid encoding a first effector subunit and a second effector subunit of the two or more effector subunits, wherein the nucleic acid comprises a first inducible promoter operably linked to a polynucleotide sequence encoding the first effector subunit and a second inducible promoter operably linked to a polynucleotide sequence encoding the second effector subunit, and the first input is capable of modulating the expression of the first effector subunit in the sample and the second input is capable of modulating the expression of the second effector subunit in the sample; or (b) nucleic acid encoding a first effector subunit of the two or more effector subunits, wherein the nucleic acid comprises a first inducible promoter operably linked to a polynucleotide sequence encoding the first effector sub
  • modulating the expression of each of the Casl2a/crRNA complex components comprises increasing their expression.
  • the expression of a third component of the Casl2a/crRNA complex is under the control of a third input to the sample, and the system is configured such that all components of the Casl2a/crRNA complex are present or capable of being expressed in the sample when the first input, the second input, and the third input each have a desired state.
  • the system is configured such that all components of the Casl2a/crRNA complex are present or capable of being expressed in the sample when the first input is present, the second input is present, and the third input is present.
  • the system comprises (a) nucleic acid encoding a first effector subunit, a second effector subunit, and a third effector subunit of the two or more effector subunits, wherein the nucleic acid comprises a first inducible promoter operably linked to a polynucleotide sequence encoding the first effector subunit, a second inducible promoter operably linked to a polynucleotide sequence encoding the second effector subunit, and a third inducible promoter operably linked to a polynucleotide sequence encoding the third effector subunit, and the first input is capable of modulating the expression of the first effector subunit in the sample, the second input is capable of modulating the expression of the second effector subunit in the sample
  • modulating the expression of each of the Casl2a/crRNA complex components comprises increasing their expression.
  • the expression of a third component of the Casl2a/crRNA complex is under the control of a third input to the sample and the expression of a fourth component of the Casl2a/crRNA complex is under the control of a fourth input to the sample, and the system is configured such that all components of the Casl2a/crRNA complex are present or capable of being expressed in the sample when the first input, the second input, the third input, and the fourth input each have a desired state.
  • the system is configured such that all components of the Casl2a/crRNA complex are present or capable of being expressed when the first input is present, the second input is present, the third input is present, and the fourth input is present.
  • the system comprises nucleic acid encoding a first effector subunit, a second effector subunit, and a third effector subunit of the two or more effector subunits, wherein the nucleic acid comprises a first inducible promoter operably linked to a polynucleotide sequence encoding the first effector subunit, a second inducible promoter operably linked to a polynucleotide sequence encoding the second effector subunit, and a third inducible promoter operably linked to a polynucleotide sequence encoding the third effector subunit, and the first input is capable of modulating the expression of the first effector subunit in the sample, the second input is capable of modulating the expression of the second effector subunit in the sample
  • the two or more effector subunits comprise a first effector subunit and a second effector subunit, wherein the first effector subunit comprises an N-terminal fragment of the Casl2a protein or variant thereof up to and including a split point amino acid, and the second effector subunit comprises a C-terminal fragment of the Casl2a protein or variant thereof following the split point amino acid.
  • the split point amino acid corresponds to A406 or G550 with respect to a parental Cas 12a protein amino acid sequence of SEQ ID NO: 1.
  • the first effector subunit further comprises a first stabilization domain at its C-terminus and the second effector subunit further comprises a second stabilization domain at its N-terminus, wherein the first stabilization domain and the second stabilization domain are capable of interacting with one another to stabilize a complex comprising the first effector subunit and the second effector subunit in the sample.
  • the two or more effector subunits comprise a first effector subunit and a second effector subunit, wherein the Casl2a protein or variant thereof is a fusion protein comprising an effector domain fused to a Casl2a domain, and the first effector subunit comprises the Casl2a domain and the second effector subunit comprises the effector domain.
  • the first effector subunit further comprises a first stabilization domain at either terminus and the second effector subunit further comprises a second stabilization domain at either terminus, wherein the first stabilization domain and the second stabilization domain are capable of interacting with one another to stabilize a complex comprising the first effector subunit and the second effector subunit in the sample.
  • the two or more effector subunits comprise a first effector subunit, a second effector subunit, and a third effector subunit.
  • the Casl2a protein or variant thereof is a fusion protein comprising an effector domain fused to a Casl2a domain
  • the first effector subunit comprises an N-terminal fragment of the Casl2a domain up to and including a split point amino acid
  • the second effector subunit comprises a C-terminal fragment of the Casl2a domain following the split point amino acid
  • the third effector subunit comprises the effector domain.
  • the split point amino acid corresponds to A406 or G550 with respect to a parental Cas 12a protein amino acid sequence of SEQ ID NO: 1.
  • the first effector subunit further comprises a first stabilization domain at its C-terminus and the second effector subunit further comprises a second stabilization domain at its N-terminus, wherein the first stabilization domain and the second stabilization domain are capable of interacting with one another to stabilize a complex comprising the first effector subunit and the second effector subunit in the sample; and/or the third effector subunit further comprises a third stabilization domain at either terminus and (i) the second effector subunit further comprises a fourth stabilization domain at its C-terminus or (ii) the first effector subunit further comprises the fourth stabilization domain at its N-terminus, wherein the third stabilization domain and the fourth stabilization domain are capable of interacting with one another to stabilize a complex comprising the first effector subunit, the second effector subunit, and the third effector subunit in the sample.
  • the first stabilization domain and the second stabilization domain are derived from subunits of a heterodimerizing binding pair or a split-intein.
  • the method further employs a third stabilization domain and a fourth stabilization domain
  • the third stabilization domain and the fourth stabilization domain are derived from subunits of a heterodimerizing binding pair or a split-intein and are orthogonal to the first stabilization domain and the second stabilization domain.
  • the first stabilization domain and the second stabilization domain are derived from subunits of a heterodimerizing binding pair and the third stabilization domain and the fourth stabilization domain are derived from subunits of a split-intein; or the first stabilization domain and the second stabilization domain are derived from subunits of a split-intein and the third stabilization domain and the fourth stabilization domain are derived from subunits of a heterodimerizing binding pair.
  • the heterodimerizing binding pair is a coiled-coil (CC) heterodimer.
  • the CC heterodimer is a leucine zipper.
  • the leucine zipper subunits comprise RR12EE345L and EE12RR345L.
  • RR12EE345L comprises the amino acid sequence encoded by SEQ ID NO: 6.
  • EE12RR345L comprises the amino acid sequence encoded by SEQ ID NO: 5.
  • the CC heterodimer is a CC peptide heterodimer ( see Lebar, T., et al. (2020). Nature Chemical Biology, 1-7).
  • the split-intein subunits comprise DnaE N-intein and C-intein.
  • DnaE N-intein comprises the amino acid sequence encoded by SEQ ID NO: 7.
  • DnaE C-intein comprises the amino acid sequence encoded by SEQ ID NO: 8.
  • the split-intein is gp41-l, gp41-8, NrdJ-1, or IMPDH-1 ( see Dassa, B., et al. (2009). Nucleic acids research, 57(8), 2560-2573).
  • the split-intein comprises split portions of the Mxe GyrA intein (see Fine, E. J., et al. (2015). Scientific reports, 5, 10777).
  • the system further comprises one or more additional crRNAs or nucleic acid encoding the one or more additional crRNAs, wherein Casl2a/crRNA complexes formed by association of the one or more additional crRNAs with (I) the Casl2a protein or variant thereof or (II) the two or more effector subunits are capable of generating one or more additional outputs to the sample comprising effector activity of the Casl2a/crRNA complexes at target sites directed by the one or more additional crRNAs.
  • the system comprises nucleic acid encoding a crRNA array comprising one or more of the first crRNA and the one or more additional crRNAs.
  • the Cas 12a protein or variant thereof is derived from a parental Cas 12a protein from Lachnospiraceae bacterium.
  • the parental Cas 12a protein from Lachnospiraceae bacterium comprises the amino acid sequence of SEQ ID NO: 1.
  • the target nucleic acid is a target dsDNA.
  • a protospacer in the target dsDNA targeted by the crRNA spacer has a 5' PAM selected from 5'-TTTG-3', 5'-TTTA-3', and 5'-TTTC-3'.
  • the Cas 12a protein or variant thereof is a nuclease- competent Cas protein comprising a Cas 12a domain having some, the same, or greater nuclease activity as compared to a parental Cas 12a protein from which it is derived, and the first output comprises cleavage (e.g., double-stranded cleavage) of the first target nucleic acid near the first target polynucleotide sequence.
  • the Cas 12a domain comprises the amino acid sequence of SEQ ID NO: 1.
  • the Cas 12a protein or variant thereof is a fusion protein comprising an effector domain comprising a polypeptide that can edit a nucleotide fused to a nuclease-deficient dCasl2a domain having reduced or eliminated nuclease activity as compared to a parental Cas 12a protein from which it is derived, and the first output is generated by activity of the effector domain to edit a nucleotide.
  • the dCasl2a domain comprises a modification of amino acid D832 with respect to SEQ ID NO: 1 that reduces, substantially eliminates, or eliminates nuclease activity of the dCasl2a domain.
  • the dCasl2a domain comprises a D832A substitution with respect to SEQ ID NO: 1.
  • the dCasl2a domain comprises the amino acid sequence of SEQ ID NO: 2.
  • the sample is a cell or a composition comprising a cell.
  • the inputs to the sample are selected from physical inputs, small molecule inputs, and biologic inputs.
  • the physical inputs are selected from oxygen levels, pH, ion concentrations, temperature, light, mechanical force, ultrasound, shock wave, magnetic field, and electric field;
  • the small molecule inputs are selected from drugs, metabolites, and adjuvants; and/or the biologic inputs are selected from polypeptides, polypeptide derivatives, and nucleic acids.
  • RNA molecules are a DNA molecule, such as a genomic DNA.
  • the split Casl2a complex of the split Casl2a system has activity that modulates the transcription of target DNA.
  • a split Casl2a fusion protein or variant thereof comprising an effector subunit comprising a heterologous polypeptide that exhibits the ability to increase or decrease transcription is used to increase or decrease the transcription of target DNA at a specific location in a target DNA, which is guided by the spacer of the crRNA.
  • source polypeptides for providing a split Casl2a fusion protein or variant thereof with transcription modulatory activity include, but are not limited to, light-inducible transcription regulators, small molecule/drug-responsive transcription regulators, transcription factors, transcription repressors, etc.
  • the method is used to control the transcription of a targeted gene-coding RNA (protein-encoding mRNA) and/or a targeted non-coding RNA (e.g., tRNA, rRNA, snoRNA, siRNA, miRNA, long ncRNA. etc.).
  • the split Casl2a fusion protein or variant has enzymatic activity that modifies a polypeptide associated with DNA (e.g., histone).
  • the enzymatic activity is methyltransferase activity, demethylase activity, acetyltransferase activity, deacetylase activity, kinase activity, phosphatase activity, ubiquitin ligase activity (e.g., ubiquitination activity), deubiquitinating activity, adenylation activity, deadenylation activity, SUMOylating activity, deSUMOylating activity, ribosylation activity, deribosylation activity, myristoylation activity, demyristoylation activity, glycosylation activity (e.g., from GlcNAc transferase), or deglycosylation activity.
  • ubiquitin ligase activity e.g., ubiquitination activity
  • deubiquitinating activity e.g., ubiquitinating activity
  • adenylation activity e.g., deadenylation activity
  • SUMOylating activity e.g., deSUMOy
  • Histone proteins are known in the art to bind DNA and form complexes known as nucleosomes. Histones can be modified (e.g., by methylation, acetylation, ubiquitination, phosphorylation) to elicit structural changes in the surrounding DNA, thus controlling the accessibility of potentially large portions of DNA to interacting factors such as transcription factors, polymerases, and the like.
  • a single histone can be modified in many different ways and in many different combinations (e.g., trimethylation of lysine 27 of histone 3, H3K27, is associated with DNA regions of repressed transcription while trimethylation of lysine 4 of histone 3, H3K4, is associated with DNA regions of active transcription).
  • a split Casl2a fusion protein or variant thereof with histone-modifying activity finds use in the site-specific control of chromosomal structure and can be used to alter the histone modification pattern in a selected region of target DNA. Such methods find use in both research and clinical applications.
  • a method of conditionally modulating transcription of a target nucleic acid in a sample comprising introducing into the sample the components of a split Casl2a system as described herein, wherein for two or more components of the split Casl2a system, the expression of each of the components is individually under the control of different inputs to the sample such that modulation of transcription of the target nucleic acid by a split Casl2a/crRNA complex only occurs when each of the different inputs have a desired state.
  • the split Casl2a system comprises (a) nucleic acid encoding two or more effector subunits comprising fragments of a dCasl2a fusion protein or variant thereof comprising an effector domain having a transcription-modulating effector activity, wherein the two or more effector subunits are capable of spontaneously assembling to form a split Casl2a complex retaining at least some of the effector activity of the Casl2a protein or variant thereof; and (b) a CRISPR RNA (crRNA) or nucleic acid encoding the crRNA, wherein the crRNA comprises a spacer having sufficient complementary to a target polynucleotide sequence in the target nucleic acid in the sample such that it can hybridize to the target nucleic acid.
  • crRNA CRISPR RNA
  • the split Casl2a system comprises nucleic acid encoding the first crRNA.
  • the split Casl2a system further comprises nucleic acid comprising a NOT inducible promoter operably linked to a polynucleotide sequence encoding an anti-CRISPR (Acr) protein capable of inhibiting activity of the split Casl2a complex, wherein a NOT input to the sample is capable of increasing the expression of Acr protein in the sample such that activity of split Casl2a complex in the sample is inhibited.
  • the sample is a cell or a composition comprising a cell.
  • the cell is a mammalian cell, e.g., a human cell.
  • a transcription modulation method described herein allows for conditional modulation (e.g., reduction or increase) of transcription of a target DNA in a cell when each of two or more inputs to the cell have a desired state.
  • conditional reduction of transcription of a target DNA in a cell wherein each of the two or more inputs to the cell have the desired state, reduces transcription of the target DNA by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or greater than 90%, compared to the level of transcription of the target DNA in a corresponding cell that does not contain the split Casl2a system or where at least one of the two or more inputs to the corresponding cell does not have the desired state.
  • conditional reduction of transcription of the target DNA in the cell reduces transcription of the target DNA, but does not substantially reduce transcription of a non- target DNA, e.g., transcription of a non-target DNA is reduced, if at all, by less than 10% compared to the level of transcription of the non-target DNA in the absence of the split Casl2a system or where at least one of the two or more inputs to the cell does not have the desired state.
  • conditional increase of transcription of a target DNA in a cell increases transcription of the target DNA by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or greater than 90%, compared to the level of transcription of the target DNA in a corresponding cell that does not contain the split Cas 12a system or where at least one of the two or more inputs to the corresponding cell does not have the desired state.
  • conditional increase of transcription of the target DNA in the cell increases transcription of the target DNA, but does not substantially increase transcription of a non-target DNA, e.g., transcription of a non-target DNA is increased, if at all, by less than 10% compared to the level of transcription of the non-target DNA in the absence of the split Cas 12a system or where at least one of the two or more inputs to the cell does not have the desired state.
  • the Acr protein is AcrVAl or a variant thereof.
  • the AcrVAl or variant thereof comprises the amino acid sequence encoded by SEQ ID NO: 9 or a variant thereof having at least 80% sequence identity to the amino acid sequence encoded by SEQ ID NO: 9.
  • a split Casl2a complex that retains at least some of the effector activity of a Casl2a protein or variant thereof retains at least about 5% (such as at least about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or greater) of the effector activity of the Casl2a protein or variant thereof.
  • the crRNA comprises a spacer sequence selected from the polynucleotide sequence of any one of SEQ ID NOs: 15-43.
  • the inducible promoter is selected from an RRM2 promoter, a hypoxia-inducible promoter, a TRE3G promoter, a CMV5-CuO promoter, and a P2 inducible promoter.
  • the RRM2 promoter comprises the polynucleotide sequence of SEQ ID NO: 10.
  • the hypoxia-inducible promoter comprises the polynucleotide sequence of SEQ ID NO: 11.
  • the TRE3G promoter comprises the polynucleotide sequence of SEQ ID NO: 12.
  • the CMV5- CuO promoter comprises the polynucleotide sequence of SEQ ID NO: 13.
  • the P2 inducible promoter comprises the polynucleotide sequence of SEQ ID NO: 14.
  • the split Casl2a system comprises nucleic acid comprising a plurality of inducible promoters, each of the plurality of inducible promoters operably linked to a polynucleotide sequence encoding an effector subunit are different. In some embodiments, where the split Casl2a system comprises nucleic acid comprising a plurality of inducible promoters, each of the plurality of inducible promoters operably linked to a polynucleotide sequence encoding a crRNA are different.
  • the split Casl2a system comprises nucleic acid comprising a plurality of inducible promoters, at least some (such as at least about any of 2, 3, 4, or more) of the plurality of inducible promoters operably linked to a polynucleotide sequence encoding a crRNA are the same. In some embodiments, where the split Casl2a system comprises nucleic acid comprising a plurality of inducible promoters, all of the plurality of inducible promoters operably linked to a polynucleotide sequence encoding a crRNA are the same. In some embodiments, where the split Cas 12a system comprises nucleic acid comprising a plurality of inducible promoters, each of the plurality of inducible promoters are different.
  • the system is configured such that all components of the Casl2a/crRNA complex are present or capable of being expressed in the sample when the first input and the second input each have a desired state. In some embodiments, the system is configured such that all components of the Casl2a/crRNA complex are present or capable of being expressed in the sample when the first input is present and the second input is present.
  • the system comprises (a) nucleic acid encoding a first effector subunit and a second effector subunit of the two or more effector subunits, wherein the nucleic acid comprises a first inducible promoter operably linked to a polynucleotide sequence encoding the first effector subunit and a second inducible promoter operably linked to a polynucleotide sequence encoding the second effector subunit, and the first input is capable of modulating the expression of the first effector subunit in the sample and the second input is capable of modulating the expression of the second effector subunit in the sample; or (b) nucleic acid encoding a first effector subunit of the two or more effector subunits, wherein the nucleic acid comprises a first inducible promoter operably linked to a polynucleotide sequence encoding the first
  • the expression of a third component of the Casl2a/crRNA complex is under the control of a third input to the sample, and the system is configured such that all components of the Casl2a/crRNA complex are present or capable of being expressed in the sample when the first input, the second input, and the third input each have a desired state.
  • the system is configured such that all components of the Casl2a/crRNA complex are present or capable of being expressed in the sample when the first input is present, the second input is present, and the third input is present.
  • the system comprises (a) nucleic acid encoding a first effector subunit, a second effector subunit, and a third effector subunit of the two or more effector subunits, wherein the nucleic acid comprises a first inducible promoter operably linked to a polynucleotide sequence encoding the first effector subunit, a second inducible promoter operably linked to a polynucleotide sequence encoding the second effector subunit, and a third inducible promoter operably linked to a polynucleotide sequence encoding the third effector subunit, and the first input is capable of modulating the expression of the first effector subunit in the sample, the second input is capable of modulating the expression of the second effector subunit in the sample, and the third input is capable of modulating the expression of the third effector subunit in the sample; or (b) nucleic acid encoding a first effector subunit and a second effector subunit of the two or more effect
  • modulating the expression of each of the Casl2a/crRNA complex components comprises increasing their expression.
  • the expression of a third component of the Casl2a/crRNA complex is under the control of a third input to the sample and the expression of a fourth component of the Casl2a/crRNA complex is under the control of a fourth input to the sample, and the system is configured such that all components of the Casl2a/crRNA complex are present or capable of being expressed in the sample when the first input, the second input, the third input, and the fourth input each have a desired state.
  • the system is configured such that all components of the Casl2a/crRNA complex are present or capable of being expressed when the first input is present, the second input is present, the third input is present, and the fourth input is present.
  • the system comprises nucleic acid encoding a first effector subunit, a second effector subunit, and a third effector subunit of the two or more effector subunits, wherein the nucleic acid comprises a first inducible promoter operably linked to a polynucleotide sequence encoding the first effector subunit, a second inducible promoter operably linked to a polynucleotide sequence encoding the second effector subunit, and a third inducible promoter operably linked to a polynucleotide sequence encoding the third effector subunit, and the first input is capable of modulating the expression of the first effector subunit in the sample, the second input is capable of modulating the expression of the second effector subunit in the sample
  • the two or more effector subunits comprise a first effector subunit and a second effector subunit, wherein the first effector subunit comprises an N-terminal fragment of the Casl2a protein or variant thereof up to and including a split point amino acid, and the second effector subunit comprises a C- terminal fragment of the Cas 12a protein or variant thereof following the split point amino acid.
  • the split point amino acid corresponds to A406 or G550 with respect to a parental Cas 12a protein amino acid sequence of SEQ ID NO: 1.
  • the first effector subunit further comprises a first stabilization domain at its C-terminus and the second effector subunit further comprises a second stabilization domain at its N-terminus, wherein the first stabilization domain and the second stabilization domain are capable of interacting with one another to stabilize a complex comprising the first effector subunit and the second effector subunit in the sample.
  • the two or more effector subunits comprise a first effector subunit and a second effector subunit, wherein the Cas 12a protein or variant thereof is a fusion protein comprising an effector domain fused to a Cas 12a domain, and the first effector subunit comprises the Cas 12a domain and the second effector subunit comprises the effector domain.
  • the first effector subunit further comprises a first stabilization domain at either terminus and the second effector subunit further comprises a second stabilization domain at either terminus, wherein the first stabilization domain and the second stabilization domain are capable of interacting with one another to stabilize a complex comprising the first effector subunit and the second effector subunit in the sample.
  • the two or more effector subunits comprise a first effector subunit, a second effector subunit, and a third effector subunit.
  • the Casl2a protein or variant thereof is a fusion protein comprising an effector domain fused to a Casl2a domain, and the first effector subunit comprises an N- terminal fragment of the Cas 12a domain up to and including a split point amino acid, the second effector subunit comprises a C-terminal fragment of the Cas 12a domain following the split point amino acid; and the third effector subunit comprises the effector domain.
  • the split point amino acid corresponds to A406 or G550 with respect to a parental Casl2a protein amino acid sequence of SEQ ID NO: 1.
  • the first effector subunit further comprises a first stabilization domain at its C-terminus and the second effector subunit further comprises a second stabilization domain at its N-terminus, wherein the first stabilization domain and the second stabilization domain are capable of interacting with one another to stabilize a complex comprising the first effector subunit and the second effector subunit in the sample; and/or the third effector subunit further comprises a third stabilization domain at either terminus and (i) the second effector subunit further comprises a fourth stabilization domain at its C-terminus or (ii) the first effector subunit further comprises the fourth stabilization domain at its N-terminus, wherein the third stabilization domain and the fourth stabilization domain are capable of interacting with one another to stabilize a complex comprising the first effector subunit, the second effector subunit, and the third effector subunit in the sample.
  • the first stabilization domain and the second stabilization domain are derived from subunits of a heterodimerizing binding pair or a split- intein.
  • the method further employs a third stabilization domain and a fourth stabilization domain
  • the third stabilization domain and the fourth stabilization domain are derived from subunits of a heterodimerizing binding pair or a split-intein and are orthogonal to the first stabilization domain and the second stabilization domain.
  • the first stabilization domain and the second stabilization domain are derived from subunits of a heterodimerizing binding pair and the third stabilization domain and the fourth stabilization domain are derived from subunits of a split-intein; or the first stabilization domain and the second stabilization domain are derived from subunits of a split-intein and the third stabilization domain and the fourth stabilization domain are derived from subunits of a heterodimerizing binding pair.
  • the heterodimerizing binding pair is a coiled-coil (CC) heterodimer.
  • the CC heterodimer is a leucine zipper.
  • the leucine zipper subunits comprise RR12EE345L and EE12RR345L.
  • RR12EE345L comprises the amino acid sequence encoded by SEQ ID NO: 6.
  • EE12RR345L comprises the amino acid sequence encoded by SEQ ID NO: 5.
  • the CC heterodimer is a CC peptide heterodimer ( see Lebar, T., et al. (2020). Nature Chemical Biology, 1-7).
  • the split-intein subunits comprise DnaE N-intein and C-intein.
  • DnaE N-intein comprises the amino acid sequence encoded by SEQ ID NO: 7.
  • DnaE C-intein comprises the amino acid sequence encoded by SEQ ID NO: 8.
  • the split-intein is gp41-l, gp41-8, NrdJ-1, or IMPDH-1 ( see Dassa, B., et al. (2009). Nucleic acids research, 57(8), 2560-2573).
  • the split-intein comprises split portions of the Mxe GyrA intein (see Fine, E. J., et al. (2015). Scientific reports, 5, 10777).
  • the system further comprises one or more additional crRNAs or nucleic acid encoding the one or more additional crRNAs, wherein Casl2a/crRNA complexes formed by association of the one or more additional crRNAs with (I) the Casl2a protein or variant thereof or (II) the two or more effector subunits are capable of generating one or more additional outputs to the sample comprising effector activity of the Casl2a/crRNA complexes at target sites directed by the one or more additional crRNAs.
  • the system comprises nucleic acid encoding a crRNA array comprising one or more of the first crRNA and the one or more additional crRNAs.
  • the Casl2a protein or variant thereof is derived from a parental Casl2a protein from Lachnospiraceae bacterium.
  • the parental Casl2a protein from Lachnospiraceae bacterium comprises the amino acid sequence of SEQ ID NO: 1.
  • the target nucleic acid is a target dsDNA.
  • a protospacer in the target dsDNA targeted by the crRNA spacer has a 5' PAM selected from 5'-TTTG-3', 5'-TTTA-3', and 5'-TTTC-3'.
  • the Casl2a protein or variant thereof is a fusion protein comprising an effector domain fused to a nuclease-deficient dCas 12a domain having reduced or eliminated nuclease activity as compared to a parental Casl2a protein from which it is derived, and the first output is generated by activity of the effector domain.
  • the dCasl2a domain comprises a modification of amino acid D832 with respect to SEQ ID NO: 1 that reduces, substantially eliminates, or eliminates nuclease activity of the dCasl2a domain.
  • the dCasl2a domain comprises a D832A substitution with respect to SEQ ID NO: 1.
  • the dCasl2a domain comprises the amino acid sequence of SEQ ID NO: 2.
  • the effector domain comprises a polypeptide that can (i) activate transcription, (ii) repress transcription, (iii) methylate a nucleic acid, and/or (iv) demethylate a nucleic acid.
  • the effector domain comprises a transcriptional activation domain, and the first output comprises transcriptional activation of a first gene targeted by the first crRNA.
  • the transcriptional activation domain is a tripartite VP64-p65-Rta (VPR) transcriptional activation domain or variant thereof.
  • the effector domain has enzymatic activity that modifies a polypeptide associated with DNA (e.g., histone).
  • the enzymatic activity is methyltransferase activity, demethylase activity, acetyltransferase activity, deacetylase activity, kinase activity, phosphatase activity, ubiquitin ligase activity (e.g., ubiquitination activity), deubiquitinating activity, adenylation activity, deadenylation activity, SUMOylating activity, deSUMOylating activity, ribosylation activity, deribosylation activity, myristoylation activity, demyristoylation activity, glycosylation activity (e.g., from GlcNAc transferase), or deglycosylation activity.
  • ubiquitin ligase activity e.g., ubiquitination activity
  • deubiquitinating activity e.g., ubiquitinating activity
  • adenylation activity e.g., deadenylation activity
  • SUMOylating activity e.g., deSUMOy
  • the sample is a cell or a composition comprising a cell.
  • the inputs to the sample are selected from physical inputs, small molecule inputs, and biologic inputs.
  • the physical inputs are selected from oxygen levels, pH, ion concentrations, temperature, light, mechanical force, ultrasound, shock wave, magnetic field, and electric field;
  • the small molecule inputs are selected from drugs, metabolites, and adjuvants; and/or the biologic inputs are selected from polypeptides, polypeptide derivatives, and nucleic acids.
  • kits for conditionally editing an epigenome of a target genomic DNA in a cell using a split Casl2a system as described herein generally involve contacting the target nucleic acid with a nuclease-deficient split dCasl2a/crRNA complex comprising activity that modifies the epigenome of the target genomic DNA.
  • the split Cas 12a system comprises (a) nucleic acid encoding two or more effector subunits comprising fragments of a dCasl2a fusion protein or variant thereof comprising an effector domain having an epigenetic-editing effector activity, wherein the two or more effector subunits are capable of spontaneously assembling to form a split Cas 12a complex retaining at least some of the effector activity of the Cas 12a protein or variant thereof; and (b) a CRISPR RNA (crRNA) or nucleic acid encoding the crRNA, wherein the crRNA comprises a spacer having sufficient complementary to a target polynucleotide sequence in the target nucleic acid in the cell such that it can hybridize to the target nucleic acid.
  • crRNA CRISPR RNA
  • the split Cas 12a system comprises nucleic acid encoding the first crRNA.
  • the split Cas 12a system further comprises nucleic acid comprising a NOT inducible promoter operably linked to a polynucleotide sequence encoding an anti-CRISPR (Acr) protein capable of inhibiting activity of the split Cas 12a complex, wherein a NOT input to the cell is capable of increasing the expression of Acr protein in the cell such that activity of split Cas 12a complex in the cell is inhibited.
  • the cell is a mammalian cell, e.g., a human cell.
  • the Acr protein is AcrVAl or a variant thereof.
  • the AcrVAl or variant thereof comprises the amino acid sequence encoded by SEQ ID NO: 9 or a variant thereof having at least 80% sequence identity to the amino acid sequence encoded by SEQ ID NO: 9.
  • a split Cas 12a complex that retains at least some of the effector activity of a Cas 12a protein or variant thereof retains at least about 5% (such as at least about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or greater) of the effector activity of the Cas 12a protein or variant thereof.
  • the crRNA comprises a spacer sequence selected from the polynucleotide sequence of any one of SEQ ID NOs: 15-43.
  • the inducible promoter is selected from an RRM2 promoter, a hypoxia-inducible promoter, a TRE3G promoter, a CMV5-CuO promoter, and a P2 inducible promoter.
  • the RRM2 promoter comprises the polynucleotide sequence of SEQ ID NO: 10.
  • the hypoxia-inducible promoter comprises the polynucleotide sequence of SEQ ID NO: 11.
  • the TRE3G promoter comprises the polynucleotide sequence of SEQ ID NO: 12.
  • the CMV5- CuO promoter comprises the polynucleotide sequence of SEQ ID NO: 13.
  • the P2 inducible promoter comprises the polynucleotide sequence of SEQ ID NO: 14.
  • the split Casl2a system comprises nucleic acid comprising a plurality of inducible promoters, each of the plurality of inducible promoters operably linked to a polynucleotide sequence encoding an effector subunit are different.
  • the split Casl2a system comprises nucleic acid comprising a plurality of inducible promoters
  • each of the plurality of inducible promoters operably linked to a polynucleotide sequence encoding a crRNA are different.
  • the split Casl2a system comprises nucleic acid comprising a plurality of inducible promoters, at least some (such as at least about any of 2, 3, 4, or more) of the plurality of inducible promoters operably linked to a polynucleotide sequence encoding a crRNA are the same.
  • the split Casl2a system comprises nucleic acid comprising a plurality of inducible promoters, all of the plurality of inducible promoters operably linked to a polynucleotide sequence encoding a crRNA are the same. In some embodiments, where the split Cas 12a system comprises nucleic acid comprising a plurality of inducible promoters, each of the plurality of inducible promoters are different.
  • the system is configured such that all components of the Casl2a/crRNA complex are present or capable of being expressed in the cell when the first input and the second input each have a desired state. In some embodiments, the system is configured such that all components of the Casl2a/crRNA complex are present or capable of being expressed in the cell when the first input is present and the second input is present.
  • the system comprises (a) nucleic acid encoding a first effector subunit and a second effector subunit of the two or more effector subunits, wherein the nucleic acid comprises a first inducible promoter operably linked to a polynucleotide sequence encoding the first effector subunit and a second inducible promoter operably linked to a polynucleotide sequence encoding the second effector subunit, and the first input is capable of modulating the expression of the first effector subunit in the cell and the second input is capable of modulating the expression of the second effector subunit in the cell; or (b) nucleic acid encoding a first effector subunit of the two or more effector subunits, wherein the nucleic acid comprises a first inducible promoter operably linked to a polynucleotide sequence encoding the first
  • the expression of a third component of the Casl2a/crRNA complex is under the control of a third input to the cell, and the system is configured such that all components of the Casl2a/crRNA complex are present or capable of being expressed in the cell when the first input, the second input, and the third input each have a desired state.
  • the system is configured such that all components of the Casl2a/crRNA complex are present or capable of being expressed in the cell when the first input is present, the second input is present, and the third input is present.
  • the system comprises (a) nucleic acid encoding a first effector subunit, a second effector subunit, and a third effector subunit of the two or more effector subunits, wherein the nucleic acid comprises a first inducible promoter operably linked to a polynucleotide sequence encoding the first effector subunit, a second inducible promoter operably linked to a polynucleotide sequence encoding the second effector subunit, and a third inducible promoter operably linked to a polynucleotide sequence encoding the third effector subunit, and the first input is capable of modulating the expression of the first effector subunit in the cell, the second input is capable of modulating the expression of the second effector subunit in the cell, and the third input is capable of modulating the expression of the third effector subunit in the cell; or (b) nucleic acid encoding a first effector subunit and a second effector subunit of the two or more effect
  • the expression of a third component of the Casl2a/crRNA complex is under the control of a third input to the cell and the expression of a fourth component of the Casl2a/crRNA complex is under the control of a fourth input to the cell, and the system is configured such that all components of the Casl2a/crRNA complex are present or capable of being expressed in the cell when the first input, the second input, the third input, and the fourth input each have a desired state.
  • the system is configured such that all components of the Casl2a/crRNA complex are present or capable of being expressed when the first input is present, the second input is present, the third input is present, and the fourth input is present.
  • the system comprises nucleic acid encoding a first effector subunit, a second effector subunit, and a third effector subunit of the two or more effector subunits, wherein the nucleic acid comprises a first inducible promoter operably linked to a polynucleotide sequence encoding the first effector subunit, a second inducible promoter operably linked to a polynucleotide sequence encoding the second effector subunit, and a third inducible promoter operably linked to a polynucleotide sequence encoding the third effector subunit, and the first input is capable of modulating the expression of the first effector subunit in the cell, the second input is capable of modulating the expression of the second effector subunit in the cell
  • the two or more effector subunits comprise a first effector subunit and a second effector subunit, wherein the first effector subunit comprises an N-terminal fragment of the Casl2a protein or variant thereof up to and including a split point amino acid, and the second effector subunit comprises a C- terminal fragment of the Cas 12a protein or variant thereof following the split point amino acid.
  • the split point amino acid corresponds to A406 or G550 with respect to a parental Cas 12a protein amino acid sequence of SEQ ID NO: 1.
  • the first effector subunit further comprises a first stabilization domain at its C-terminus and the second effector subunit further comprises a second stabilization domain at its N-terminus, wherein the first stabilization domain and the second stabilization domain are capable of interacting with one another to stabilize a complex comprising the first effector subunit and the second effector subunit in the cell.
  • the two or more effector subunits comprise a first effector subunit and a second effector subunit, wherein the Cas 12a protein or variant thereof is a fusion protein comprising an effector domain fused to a Cas 12a domain, and the first effector subunit comprises the Cas 12a domain and the second effector subunit comprises the effector domain.
  • the first effector subunit further comprises a first stabilization domain at either terminus and the second effector subunit further comprises a second stabilization domain at either terminus, wherein the first stabilization domain and the second stabilization domain are capable of interacting with one another to stabilize a complex comprising the first effector subunit and the second effector subunit in the cell.
  • the two or more effector subunits comprise a first effector subunit, a second effector subunit, and a third effector subunit.
  • the Cas 12a protein or variant thereof is a fusion protein comprising an effector domain fused to a Cas 12a domain
  • the first effector subunit comprises an N- terminal fragment of the Cas 12a domain up to and including a split point amino acid
  • the second effector subunit comprises a C-terminal fragment of the Cast 2a domain following the split point amino acid
  • the third effector subunit comprises the effector domain.
  • the split point amino acid corresponds to A406 or G550 with respect to a parental Casl2a protein amino acid sequence of SEQ ID NO: 1.
  • the first effector subunit further comprises a first stabilization domain at its C-terminus and the second effector subunit further comprises a second stabilization domain at its N-terminus, wherein the first stabilization domain and the second stabilization domain are capable of interacting with one another to stabilize a complex comprising the first effector subunit and the second effector subunit in the cell; and/or the third effector subunit further comprises a third stabilization domain at either terminus and (i) the second effector subunit further comprises a fourth stabilization domain at its C-terminus or (ii) the first effector subunit further comprises the fourth stabilization domain at its N-terminus, wherein the third stabilization domain and the fourth stabilization domain are capable of interacting with one another to stabilize a complex comprising the first effector subunit, the second effector subunit, and the third effector subunit in the cell
  • the first stabilization domain and the second stabilization domain are derived from subunits of a heterodimerizing binding pair or a split- intein.
  • the method further employs a third stabilization domain and a fourth stabilization domain
  • the third stabilization domain and the fourth stabilization domain are derived from subunits of a heterodimerizing binding pair or a split-intein and are orthogonal to the first stabilization domain and the second stabilization domain.
  • the first stabilization domain and the second stabilization domain are derived from subunits of a heterodimerizing binding pair and the third stabilization domain and the fourth stabilization domain are derived from subunits of a split-intein; or the first stabilization domain and the second stabilization domain are derived from subunits of a split-intein and the third stabilization domain and the fourth stabilization domain are derived from subunits of a heterodimerizing binding pair.
  • the heterodimerizing binding pair is a coiled-coil (CC) heterodimer.
  • the CC heterodimer is a leucine zipper.
  • the leucine zipper subunits comprise RR12EE345L and EE12RR345L.
  • RR12EE345L comprises the amino acid sequence encoded by SEQ ID NO: 6.
  • EE12RR345L comprises the amino acid sequence encoded by SEQ ID NO: 5.
  • the CC heterodimer is a CC peptide heterodimer (see Lebar, T., et al. (2020). Nature Chemical Biology, 1-7).
  • the split-intein subunits comprise DnaE N-intein and C-intein.
  • DnaE N-intein comprises the amino acid sequence encoded by SEQ ID NO: 7.
  • DnaE C-intein comprises the amino acid sequence encoded by SEQ ID NO: 8.
  • the split-intein is gp41-l, gp41-8, NrdJ-1, or IMPDH-1 ( see Dassa, B., et al. (2009). Nucleic acids research, 57(8), 2560-2573).
  • the split-intein comprises split portions of the Mxe GyrA intein (see Fine, E. J., et al. (2015). Scientific reports, 5, 10777).
  • the system further comprises one or more additional crRNAs or nucleic acid encoding the one or more additional crRNAs, wherein Casl2a/crRNA complexes formed by association of the one or more additional crRNAs with (I) the Casl2a protein or variant thereof or (II) the two or more effector subunits are capable of generating one or more additional outputs to the cell comprising effector activity of the Cas 12a/crRNA complexes at target sites directed by the one or more additional crRNAs.
  • the system comprises nucleic acid encoding a crRNA array comprising one or more of the first crRNA and the one or more additional crRNAs.
  • the Cas 12a protein or variant thereof is derived from a parental Cas 12a protein from Lachnospiraceae bacterium.
  • the parental Cas 12a protein from Lachnospiraceae bacterium comprises the amino acid sequence of SEQ ID NO: 1.
  • the target nucleic acid is a target dsDNA.
  • a protospacer in the target dsDNA targeted by the crRNA spacer has a 5' PAM selected from 5'-TTTG-3', 5'-TTTA-3', and 5'-TTTC-3'.
  • the Cas 12a protein or variant thereof is a fusion protein comprising an effector domain fused to a nuclease-deficient dCas 12a domain having reduced or eliminated nuclease activity as compared to a parental Cas 12a protein from which it is derived, and the first output is generated by activity of the effector domain.
  • the dCasl2a domain comprises a modification of amino acid D832 with respect to SEQ ID NO: 1 that reduces, substantially eliminates, or eliminates nuclease activity of the dCasl2a domain.
  • the dCasl2a domain comprises a D832A substitution with respect to SEQ ID NO: 1.
  • the dCasl2a domain comprises the amino acid sequence of SEQ ID NO: 2.
  • the effector domain comprises a polypeptide that can (i) methylate a nucleic acid, and/or (ii) demethylate a nucleic acid.
  • the effector domain has enzymatic activity that modifies a polypeptide associated with DNA (e.g., histone).
  • the enzymatic activity is methyltransferase activity, demethylase activity, acetyltransferase activity, deacetylase activity, kinase activity, phosphatase activity, ubiquitin ligase activity (e.g., ubiquitination activity), deubiquitinating activity, adenylation activity, deadenylation activity, SUMOylating activity, deSUMOylating activity, ribosylation activity, deribosylation activity, myristoylation activity, demyristoylation activity, glycosylation activity (e.g., from GlcNAc transferase), or deglycosylation activity.
  • ubiquitin ligase activity e.g., ubiquitination activity
  • deubiquitinating activity e.g., ubiquitinating activity
  • adenylation activity e.g., deadenylation activity
  • SUMOylating activity e.g., deSUMOy
  • the inputs to the cell are selected from physical inputs, small molecule inputs, and biologic inputs.
  • the physical inputs are selected from oxygen levels, pH, ion concentrations, temperature, light, mechanical force, ultrasound, shock wave, magnetic field, and electric field;
  • the small molecule inputs are selected from drugs, metabolites, and adjuvants;
  • the biologic inputs are selected from polypeptides, polypeptide derivatives, and nucleic acids.
  • a split Casl2a system as described herein is employed to modify target gene expression from a target nucleic acid, or otherwise modify the target nucleic acid, for purposes of treating a disease or condition in a subject.
  • the split Casl2a system components can be incorporated into a variety of formulations. More particularly, the split Casl2a system components of the present disclosure can be formulated into pharmaceutical compositions by combination with appropriate pharmaceutically acceptable carriers or diluents.
  • compositions comprising components of a Casl2a system including (a) nucleic acid encoding two or more effector subunits comprising fragments of a Casl2a protein or variant thereof, wherein the two or more effector subunits are capable of spontaneously assembling to form a split Casl2a complex retaining at least some of the effector activity of the Casl2a protein or variant thereof; and (b) a CRISPR RNA (crRNA) or nucleic acid encoding the crRNA, wherein the crRNA comprises a spacer having sufficient complementary to a target polynucleotide sequence in a target nucleic acid such that it can hybridize to the target nucleic acid, present in a pharmaceutically acceptable vehicle.
  • crRNA CRISPR RNA
  • the split Casl2a system comprises nucleic acid encoding the first crRNA. In some embodiments, the split Casl2a system further comprises nucleic acid comprising a NOT inducible promoter operably linked to a polynucleotide sequence encoding an anti-CRISPR (Acr) protein capable of inhibiting activity of the split Casl2a complex, wherein a NOT input to the cell is capable of increasing the expression of Acr protein in the cell such that activity of split Casl2a complex in the cell is inhibited.
  • Acr anti-CRISPR
  • “Pharmaceutically acceptable vehicles” may be vehicles approved by a regulatory agency of the Federal or a state government or listed in the US Pharmacopeia or other generally recognized pharmacopeia for use in mammals, such as humans.
  • vehicle refers to a diluent, adjuvant, excipient, or carrier with which a compound of the disclosure is formulated for administration to a mammal.
  • Such pharmaceutical vehicles can be lipids, e.g., liposomes, e.g., liposome dendrimers; liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like, saline; gum acacia, gelatin, starch paste, talc, keratin, colloidal silica, urea, and the like.
  • auxiliary, stabilizing, thickening, lubricating and coloring agents may be used.
  • compositions may be formulated into preparations in solid, semisolid, liquid or gaseous forms, such as tablets, capsules, powders, granules, ointments, solutions, suppositories, injections, inhalants, gels, microspheres, and aerosols.
  • administration of the Casl2a system components can be achieved in various ways, including oral, buccal, rectal, parenteral, intraperitoneal, intradermal, transdermal, intra-tracheal, intraocular, etc., administration.
  • the active agent may be systemic after administration or may be localized by the use of regional administration, intramural administration, or use of an implant that acts to retain the active dose at the site of implantation.
  • the active agent may be formulated for immediate activity or it may be formulated for sustained release.
  • compositions can include, depending on the formulation desired, pharmaceutically acceptable, non-toxic carriers of diluents, which are defined as vehicles commonly used to formulate pharmaceutical compositions for animal or human administration.
  • the diluent is selected so as not to affect the biological activity of the combination. Examples of such diluents are distilled water, buffered water, physiological saline, PBS, Ringer's solution, dextrose solution, and Hank's solution.
  • the pharmaceutical composition or formulation can include other carriers, adjuvants, or non-toxic, nontherapeutic, nonimmunogenic stabilizers, excipients and the like.
  • the compositions can also include additional substances to approximate physiological conditions, such as pH adjusting and buffering agents, toxicity adjusting agents, wetting agents and detergents.
  • the composition can also include any of a variety of stabilizing agents, such as an antioxidant for example.
  • the pharmaceutical composition includes a polypeptide
  • the polypeptide can be complexed with various well-known compounds that enhance the in vivo stability of the polypeptide, or otherwise enhance its pharmacological properties (e.g., increase the half-life of the polypeptide, reduce its toxicity, and/or enhance solubility or uptake). Examples of such modifications or complexing agents include sulfate, gluconate, citrate and phosphate.
  • the nucleic acids or polypeptides of a composition can also be complexed with molecules that enhance their in vivo attributes. Such molecules include, for example, carbohydrates, polyamines, amino acids, other peptides, ions (e.g., sodium, potassium, calcium, magnesium, manganese), and lipids.
  • a method of treating a disease or condition in a subject in need thereof comprising: 1) modulating the expression of a target gene in input cells according to any of the methods described herein, thereby producing engineered cells and administering the engineered cells to the subject; or 2) modulating the expression of a target gene in input cells in the subject according to any of the methods described herein, thereby producing engineered cells in the subject.
  • the input cells of 1) are autologous to the subject.
  • the input cells of 1) are allogeneic to the subject.
  • the subject is human.
  • a method of treating a disease or condition associated with a disease-associated cell in a subject in need thereof comprising administering to the subject an engineered cell comprising a split Casl2a system as described herein, wherein the engineered cell is capable of mediating killing of the disease-associated cell to treat the disease or condition.
  • the engineered cell is capable of expressing a target gene
  • the first target nucleic acid comprises the target gene
  • the first output of the system modulates the expression of the target gene in a target cell.
  • one or more NOT inputs of the split Casl2a system capable of increasing the expression of Acr protein in the engineered cell are associated with off-target cells and not the target cell.
  • the engineered cell is capable of expressing a target receptor
  • the first target nucleic acid comprises a polynucleotide sequence encoding the target receptor
  • the first output of the system mediates expression of the target receptor.
  • the target receptor is a chimeric antigen receptor (CAR) targeting the disease- associated cell
  • the engineered cell is a CAR immune cell
  • one or more inputs of the system are associated with the disease-associated cell.
  • the CAR immune cell is a CAR T cell, a CAR NK cell, or a CAR macrophage cell.
  • the target receptor is a T cell receptor (TCR) targeting the disease-associated cell
  • the engineered cell is a T cell
  • one or more inputs of the system are associated with the disease-associated cell.
  • one or more NOT inputs of the system capable of increasing the expression of Acr protein in the cell are associated with healthy cells and not the disease- associated cell.
  • the engineered cells are autologous to the subject.
  • the engineered cells are allogeneic to the subject.
  • the subject is human. The number of administrations of treatment to a subject may vary.
  • introducing the engineered cells into the subject is a one-time event.
  • the method further comprises one or more additional administrations of engineered cells.
  • a method of treating a disease or condition associated with a disease-associated cell in a subject in need thereof comprising modifying the disease-associated cell in the subject by introduction of a split Casl2a system as described herein to produce an engineered cell, wherein the engineered cell is capable of expressing a target gene, the first target nucleic acid comprises the target gene, one or more inputs of the system are disease-associated inputs characteristic of the disease-associated cell, and the first output of the system modulates expression of the target gene in the engineered cell such that the disease or condition is treated.
  • the first output of the system modulates expression of the target gene in the engineered cell such that it is rendered benign.
  • the first output of the system modulates expression of the target gene in the engineered cell such that it is killed.
  • the disease-associated cell is a cancer cell, and one or more inputs to the cell are cancer-associated inputs characteristic of the cancer cell.
  • one or more NOT inputs of the system capable of increasing the expression of Acr protein in the engineered cell are associated with non-disease-associated cells and not the disease-associated cell.
  • a method of treating a disease or condition associated with a target gene in a target cell in a subject in need thereof comprising modifying the target cell in the subject by introduction of a split Casl2a system as described herein to produce an engineered cell, wherein the engineered cell is capable of expressing the target gene, the first target nucleic acid comprises the target gene, and the first output of the system modulates the expression of the target gene in the target cell, and wherein the expression of the target gene in the engineered cell is modulated such that the disease or condition is treated.
  • one or more NOT inputs of the split Casl2a system capable of increasing the expression of Acr protein in the engineered cell are associated with off-target cells and not the target cell.
  • the Acr protein is AcrVAl or a variant thereof.
  • the AcrVAl or variant thereof comprises the amino acid sequence encoded by SEQ ID NO: 9 or a variant thereof having at least 80% sequence identity to the amino acid sequence encoded by SEQ ID NO: 9.
  • a split Casl2a complex that retains at least some of the effector activity of a Casl2a protein or variant thereof retains at least about 5% (such as at least about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or greater) of the effector activity of the Casl2a protein or variant thereof.
  • the crRNA comprises a spacer sequence selected from the polynucleotide sequence of any one of SEQ ID NOs: 15-43.
  • the inducible promoter is selected from an RRM2 promoter, a hypoxia-inducible promoter, a TRE3G promoter, a CMV5-CuO promoter, and a P2 inducible promoter.
  • the RRM2 promoter comprises the polynucleotide sequence of SEQ ID NO: 10.
  • the hypoxia-inducible promoter comprises the polynucleotide sequence of SEQ ID NO: 11.
  • the TRE3G promoter comprises the polynucleotide sequence of SEQ ID NO: 12.
  • the CMV5-CuO promoter comprises the polynucleotide sequence of SEQ ID NO: 13.
  • the P2 inducible promoter comprises the polynucleotide sequence of SEQ ID NO: 14.
  • the split Cas 12a system comprises nucleic acid comprising a plurality of inducible promoters, each of the plurality of inducible promoters operably linked to a polynucleotide sequence encoding an effector subunit are different.
  • the split Cas 12a system comprises nucleic acid comprising a plurality of inducible promoters
  • each of the plurality of inducible promoters operably linked to a polynucleotide sequence encoding a crRNA are different.
  • the split Casl2a system comprises nucleic acid comprising a plurality of inducible promoters, at least some (such as at least about any of 2, 3, 4, or more) of the plurality of inducible promoters operably linked to a polynucleotide sequence encoding a crRNA are the same.
  • the split Casl2a system comprises nucleic acid comprising a plurality of inducible promoters, all of the plurality of inducible promoters operably linked to a polynucleotide sequence encoding a crRNA are the same. In some embodiments, where the split Casl2a system comprises nucleic acid comprising a plurality of inducible promoters, each of the plurality of inducible promoters are different. [0230] In some embodiments, according to any of the methods of treating a disease or condition described herein, the system is configured such that all components of the Casl2a/crRNA complex are present or capable of being expressed in the sample when the first input and the second input each have a desired state.
  • the system is configured such that all components of the Casl2a/crRNA complex are present or capable of being expressed in the sample when the first input is present and the second input is present.
  • the system comprises (a) nucleic acid encoding a first effector subunit and a second effector subunit of the two or more effector subunits, wherein the nucleic acid comprises a first inducible promoter operably linked to a polynucleotide sequence encoding the first effector subunit and a second inducible promoter operably linked to a polynucleotide sequence encoding the second effector subunit, and the first input is capable of modulating the expression of the first effector subunit in the sample and the second input is capable of modulating the expression of the second effector subunit in the sample; or (b) nucleic acid encoding a first effector subunit of the two or more effector subunit
  • modulating the expression of each of the Casl2a/crRNA complex components comprises increasing their expression.
  • the expression of a third component of the Casl2a/crRNA complex is under the control of a third input to the sample, and the system is configured such that all components of the Casl2a/crRNA complex are present or capable of being expressed in the sample when the first input, the second input, and the third input each have a desired state.
  • the system is configured such that all components of the Casl2a/crRNA complex are present or capable of being expressed in the sample when the first input is present, the second input is present, and the third input is present.
  • the system comprises (a) nucleic acid encoding a first effector subunit, a second effector subunit, and a third effector subunit of the two or more effector subunits, wherein the nucleic acid comprises a first inducible promoter operably linked to a polynucleotide sequence encoding the first effector subunit, a second inducible promoter operably linked to a polynucleotide sequence encoding the second effector subunit, and a third inducible promoter operably linked to a polynucleotide sequence encoding the third effector subunit, and the first input is capable of modulating the expression of the first effector subunit in the sample, the second input is capable of modulating the expression of the second effector subunit in the sample
  • the expression of a third component of the Casl2a/crRNA complex is under the control of a third input to the sample and the expression of a fourth component of the Casl2a/crRNA complex is under the control of a fourth input to the sample, and the system is configured such that all components of the Casl2a/crRNA complex are present or capable of being expressed in the sample when the first input, the second input, the third input, and the fourth input each have a desired state.
  • the system is configured such that all components of the Casl2a/crRNA complex are present or capable of being expressed when the first input is present, the second input is present, the third input is present, and the fourth input is present.
  • the system comprises nucleic acid encoding a first effector subunit, a second effector subunit, and a third effector subunit of the two or more effector subunits, wherein the nucleic acid comprises a first inducible promoter operably linked to a polynucleotide sequence encoding the first effector subunit, a second inducible promoter operably linked to a polynucleotide sequence encoding the second effector subunit, and a third inducible promoter operably linked to a polynucleotide sequence encoding the third effector subunit, and the first input is capable of modulating the expression of the first effector subunit in the sample, the second input is capable of modulating the expression of the second effector subunit in the sample
  • the two or more effector subunits comprise a first effector subunit and a second effector subunit, wherein the first effector subunit comprises an N-terminal fragment of the Casl2a protein or variant thereof up to and including a split point amino acid, and the second effector subunit comprises a C-terminal fragment of the Casl2a protein or variant thereof following the split point amino acid.
  • the split point amino acid corresponds to A406 or G550 with respect to a parental Casl2a protein amino acid sequence of SEQ ID NO: 1.
  • the first effector subunit further comprises a first stabilization domain at its C-terminus and the second effector subunit further comprises a second stabilization domain at its N-terminus, wherein the first stabilization domain and the second stabilization domain are capable of interacting with one another to stabilize a complex comprising the first effector subunit and the second effector subunit in the sample.
  • the two or more effector subunits comprise a first effector subunit and a second effector subunit, wherein the Casl2a protein or variant thereof is a fusion protein comprising an effector domain fused to a Casl2a domain, and the first effector subunit comprises the Casl2a domain and the second effector subunit comprises the effector domain.
  • the first effector subunit further comprises a first stabilization domain at either terminus and the second effector subunit further comprises a second stabilization domain at either terminus, wherein the first stabilization domain and the second stabilization domain are capable of interacting with one another to stabilize a complex comprising the first effector subunit and the second effector subunit in the sample.
  • the two or more effector subunits comprise a first effector subunit, a second effector subunit, and a third effector subunit.
  • the Casl2a protein or variant thereof is a fusion protein comprising an effector domain fused to a Casl2a domain
  • the first effector subunit comprises anN-terminal fragment of the Casl2a domain up to and including a split point amino acid
  • the second effector subunit comprises a C-terminal fragment of the Casl2a domain following the split point amino acid
  • the third effector subunit comprises the effector domain.
  • the split point amino acid corresponds to A406 or G550 with respect to a parental Casl2a protein amino acid sequence of SEQ ID NO: 1.
  • the first effector subunit further comprises a first stabilization domain at its C-terminus and the second effector subunit further comprises a second stabilization domain at its N-terminus, wherein the first stabilization domain and the second stabilization domain are capable of interacting with one another to stabilize a complex comprising the first effector subunit and the second effector subunit in the sample; and/or the third effector subunit further comprises a third stabilization domain at either terminus and (i) the second effector subunit further comprises a fourth stabilization domain at its C-terminus or (ii) the first effector subunit further comprises the fourth stabilization domain at its N-terminus, wherein the third stabilization domain and the fourth stabilization domain are capable of interacting with one another to stabilize a complex comprising the first effector subunit, the second effector subunit, and the third effector subunit in the sample
  • the first stabilization domain and the second stabilization domain are derived from subunits of a heterodimerizing binding pair or a split-intein.
  • the method further employs a third stabilization domain and a fourth stabilization domain
  • the third stabilization domain and the fourth stabilization domain are derived from subunits of a heterodimerizing binding pair or a split-intein and are orthogonal to the first stabilization domain and the second stabilization domain.
  • the first stabilization domain and the second stabilization domain are derived from subunits of a heterodimerizing binding pair and the third stabilization domain and the fourth stabilization domain are derived from subunits of a split-intein; or the first stabilization domain and the second stabilization domain are derived from subunits of a split-intein and the third stabilization domain and the fourth stabilization domain are derived from subunits of a heterodimerizing binding pair.
  • the heterodimerizing binding pair is a coiled-coil (CC) heterodimer.
  • the CC heterodimer is a leucine zipper.
  • the leucine zipper subunits comprise RR12EE345L and EE12RR345L.
  • RR12EE345L comprises the amino acid sequence encoded by SEQ ID NO: 6.
  • EE12RR345L comprises the amino acid sequence encoded by SEQ ID NO: 5.
  • the CC heterodimer is a CC peptide heterodimer ( see Lebar, T., et al. (2020). Nature Chemical Biology, 1-7).
  • the split-intein subunits comprise DnaE N-intein and C-intein.
  • DnaE N-intein comprises the amino acid sequence encoded by SEQ ID NO: 7.
  • DnaE C-intein comprises the amino acid sequence encoded by SEQ ID NO: 8.
  • the split-intein is gp41 - 1, gp41-8, NrdJ-1, or IMPDH-1 ( see Dassa, B., et al. (2009). Nucleic acids research, 57(8), 2560-2573).
  • the split-intein comprises split portions of the Mxe GyrA intein (see Fine, E. J., et al. (2015). Scientific reports, 5, 10777).
  • the system further comprises one or more additional crRNAs or nucleic acid encoding the one or more additional crRNAs, wherein Casl2a/crRNA complexes formed by association of the one or more additional crRNAs with (I) the Casl2a protein or variant thereof or (II) the two or more effector subunits are capable of generating one or more additional outputs to the sample comprising effector activity of the Casl2a/crRNA complexes at target sites directed by the one or more additional crRNAs.
  • the system comprises nucleic acid encoding a crRNA array comprising one or more of the first crRNA and the one or more additional crRNAs.
  • the Casl2a protein or variant thereof is derived from a parental Casl2a protein from Lachnospiraceae bacterium.
  • the parental Casl2a protein from Lachnospiraceae bacterium comprises the amino acid sequence of SEQ ID NO: 1.
  • the target nucleic acid is a target dsDNA.
  • a protospacer in the target dsDNA targeted by the crRNA spacer has a 5' PAM selected from 5'- TTTG-3', 5'-TTTA-3', and 5'-TTTC-3'.
  • the Cast 2a protein or variant thereof is a nuclease-competent Cas protein comprising a Cas 12a domain having some, the same, or greater nuclease activity as compared to a parental Cas 12a protein from which it is derived, and the first output comprises cleavage (e.g., double-stranded cleavage) of the first target nucleic acid near the first target polynucleotide sequence.
  • the Cas 12a domain comprises the amino acid sequence of SEQ ID NO: 1.
  • the Cas 12a protein or variant thereof is a fusion protein comprising an effector domain fused to a nuclease-deficient dCasl2a domain having reduced or eliminated nuclease activity as compared to a parental Cas 12a protein from which it is derived, and the first output is generated by activity of the effector domain.
  • the dCasl2a domain comprises a modification of amino acid D832 with respect to SEQ ID NO: 1 that reduces, substantially eliminates, or eliminates nuclease activity of the dCasl2a domain.
  • the dCasl2a domain comprises a D832A substitution with respect to SEQ ID NO: 1.
  • the dCasl2a domain comprises the amino acid sequence of SEQ ID NO: 2.
  • the effector domain comprises a polypeptide that can (i) cleave a nucleic acid, (ii) edit a nucleotide, (iii) activate transcription, (iv) repress transcription, (v) methylate a nucleic acid, and/or (vi) demethylate a nucleic acid.
  • the effector domain comprises a transcriptional activation domain
  • the first output comprises transcriptional activation of a first gene targeted by the first crRNA.
  • the transcriptional activation domain is a tripartite VP64-p65-Rta (VPR) transcriptional activation domain or variant thereof.
  • the sample is a cell or a composition comprising a cell.
  • the inputs to the sample are selected from physical inputs, small molecule inputs, and biologic inputs.
  • the physical inputs are selected from oxygen levels, pH, ion concentrations, temperature, light, mechanical force, ultrasound, shock wave, magnetic field, and electric field;
  • the small molecule inputs are selected from drugs, metabolites, and adjuvants;
  • the biologic inputs are selected from polypeptides, polypeptide derivatives, and nucleic acids.
  • components of a split Cas 12a system are introduced into a cell to generate an engineered cell.
  • components of a split Casl2a system include (a) two or more effector subunits comprising fragments of a Casl2a protein or variant thereof, or nucleic acid encoding the two or more effector subunits, wherein the two or more effector subunits are capable of spontaneously assembling to form a split Casl2a complex retaining at least some of the effector activity of the Casl2a protein or variant thereof; and (b) a first CRISPR RNA (crRNA) or nucleic acid encoding the first crRNA, wherein the first crRNA comprises a first spacer having sufficient complementary to a first target polynucleotide sequence in a first target nucleic acid in the cell such that it can hybridize to the first target nucleic acid.
  • the engineered cells can be incorporated into a variety of formulations. More
  • provided herein is a method of generating an engineered cell comprising introducing into an input cell one or more components of a split Casl2a system as described herein. In some embodiments, the method comprises introducing into the input cell all of the components of the split Casl2a system.
  • the split Casl2a system comprises (a) nucleic acid encoding two or more effector subunits comprising fragments of a Casl2a protein or variant thereof, wherein the two or more effector subunits are capable of spontaneously assembling to form a split Casl2a complex retaining at least some of the effector activity of the Casl2a protein or variant thereof; and (b) a first CRISPR RNA (crRNA) or nucleic acid encoding the first crRNA, wherein the first crRNA comprises a first spacer having sufficient complementary to a first target polynucleotide sequence in a first target nucleic acid in the cell such that it can hybridize to the first target nucleic acid.
  • crRNA CRISPR RNA
  • the split Casl2a system comprises nucleic acid encoding the first crRNA.
  • the split Casl2a system further comprises nucleic acid comprising a NOT inducible promoter operably linked to a polynucleotide sequence encoding an anti-CRISPR (Acr) protein capable of inhibiting activity of the split Casl2a complex, wherein a NOT input to the sample is capable of increasing the expression of Acr protein from the nucleic acid in the sample such that activity of Casl2a complex in the sample is inhibited.
  • Acr anti-CRISPR
  • the split Casl2a systems described herein are used in eukaryotic cells, such as mammalian cells, for example, human cells, to produce engineered cells with modulated expression of a target gene. Any human cell is contemplated for use with the split Casl2a systems disclosed herein.
  • an engineered cell ex vivo or in vitro includes: (a) nucleic acid encoding two or more effector subunits comprising fragments of a Casl2a protein or variant thereof, wherein the two or more effector subunits are capable of spontaneously assembling to form a split Casl2a complex retaining at least some of the effector activity of the Casl2a protein or variant thereof; and (b) a CRISPR RNA (crRNA) or nucleic acid encoding the crRNA, wherein the crRNA comprises a spacer having sufficient complementary to a target polynucleotide sequence in a target nucleic acid in the cell such that it can hybridize to the target nucleic acid.
  • crRNA CRISPR RNA
  • some embodiments disclosed herein relate to a method of engineering an input cell that includes introducing into the input cell, such as an animal cell, one or more components of a split Casl2a system as described herein, and selecting or screening for an engineered cell transformed by the one or more system components.
  • engineered cell refers not only to the particular subject cell but also to the progeny or potential progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein. Techniques for transforming a wide variety of cell are known in the art.
  • engineered cells for example, engineered animal cells that include a heterologous nucleic acid and/or polypeptide as described herein.
  • the nucleic acid can be stably integrated in the host genome, or can be episomally replicating, or present in the engineered cell as a mini-circle expression vector for stable or transient expression.
  • an engineered cell e.g., an isolated engineered cell, prepared by modulating the expression of a target gene in a target nucleic acid or otherwise modifying the target nucleic acid in an input cell according to any of the methods described herein, thereby producing the engineered cell.
  • an engineered cell prepared by a method comprising providing to an input cell a split Casl2a system as described herein, wherein for two or more components of the split Casl2a system, the expression of each of the components is individually under the control of different inputs to the engineered cell such that an output mediated by a split Casl2a/crRNA complex is only generated when each of the different inputs have a desired state.
  • the split Casl2a system comprises (a) nucleic acid encoding two or more effector subunits comprising fragments of a Cast 2a protein or variant thereof, wherein the two or more effector subunits are capable of spontaneously assembling to form a split Casl2a complex retaining at least some of the effector activity of the Casl2a protein or variant thereof; and (b) a CRISPR RNA (crRNA) or nucleic acid encoding the crRNA, wherein the crRNA comprises a spacer having sufficient complementary to a target polynucleotide sequence in the target nucleic acid in the engineered cell such that it can hybridize to the target nucleic acid.
  • crRNA CRISPR RNA
  • the split Casl2a system comprises nucleic acid encoding the first crRNA.
  • the split Casl2a system further comprises nucleic acid comprising a NOT inducible promoter operably linked to a polynucleotide sequence encoding an anti-CRISPR (Acr) protein capable of inhibiting activity of the split Casl2a complex, wherein a NOT input to the sample is capable of increasing the expression of Acr protein in the engineered cell such that activity of split Casl2a complex in the engineered cell is inhibited.
  • the engineered cell is a mammalian cell, e.g., a human cell.
  • the engineered cell is capable of expressing a target gene
  • the first target nucleic acid comprises the target gene
  • the first output of the system modulates the expression of the target gene in a target cell.
  • one or more NOT inputs of the system capable of increasing the expression of Acr protein in the engineered cell are associated with off-target cells and not the target cell.
  • the engineered cell is capable of expressing a target receptor
  • the first target nucleic acid comprises a polynucleotide sequence encoding the target receptor
  • the first output of the system mediates expression of the target receptor.
  • the target receptor is a chimeric antigen receptor (CAR)
  • the engineered cell is a CAR immune cell
  • one or more inputs of the system are associated with a target cell targeted by the CAR immune cell.
  • the CAR immune cell is a CAR T cell, a CAR NK cell, or a CAR macrophage cell.
  • the target receptor is a T cell receptor (TCR)
  • the engineered cell is a T cell
  • one or more inputs of the system are associated with a target cell targeted by the TCR.
  • one or more NOT inputs of the system capable of increasing the expression of Acr protein in the engineered cell are associated with off-target cells and not the target cell.
  • the engineered cell is a disease-associated cell capable of expressing a target gene
  • the first target nucleic acid comprises the target gene
  • one or more inputs of the system are disease-associated inputs characteristic of the disease-associated cell
  • the first output of the system mediates expression of the target gene in the disease-associated cell.
  • the first output of the system mediates expression of the target gene in the engineered cell such that it is rendered benign.
  • the first output of the system mediates expression of the target gene in the engineered cell such that it is killed.
  • the disease- associated cell is a cancer cell
  • one or more inputs to the cell are cancer-associated inputs characteristic of the cancer cell.
  • one or more NOT inputs of the system capable of increasing the expression of Acr protein in the cell are associated with non-disease- associated cells and not the disease-associated cell.
  • an engineered cell can be prepared from an input cell from any organism, e.g., a bacterial cell, an archaeal cell, a cell of a single-cell eukaryotic organism, a plant cell, an algal cell (e.g., Botryococcus braunii, Chlamydomonas reinhardtii, Nannochloropsis gaditana, Chlorela pyrenoidosa, Sargassum patens C.
  • any organism e.g., a bacterial cell, an archaeal cell, a cell of a single-cell eukaryotic organism, a plant cell, an algal cell (e.g., Botryococcus braunii, Chlamydomonas reinhardtii, Nannochloropsis gaditana, Chlorela pyrenoidosa, Sargassum patens C.
  • a fungal cell e.g., a yeast cell
  • an animal cell e.g., a cell from an invertebrate animal (e.g., fruit fly, cnidarian, echinoderm, nematode, etc.), a cell from a vertebrate animal (e.g., fish, amphibian, reptile, bird, mammal), a cell from a mammal (e.g., a pig, a cow, a goat, a sheep, a rodent a rat, a mouse, a non-human primate, a human, etc.).
  • the engineered cell can be prepared from any input cell from a human.
  • an engineered cell is in vitro. In some embodiments, an engineered cell is in vivo. In some embodiments, an engineered cell is a prokaryotic cell or is derived from a prokaryotic cell. In some embodiments, an engineered cell is a bacterial cell or is derived from a bacterial cell. In some embodiments, an engineered cell is an archaeal cell or is derived from an archaeal cell. In some embodiments, an engineered cell is a eukaryotic cell or is derived from a eukaryotic cell. In some embodiments, an engineered cell is a plant cell or is derived from a plant cell.
  • an engineered cell is an animal cell or is derived from an animal cell. In some embodiments, an engineered cell is an invertebrate cell or is derived from an invertebrate cell. In some embodiments, an engineered cell is a vertebrate cell or is derived from a vertebrate cell. In some embodiments, an engineered cell is a mammalian cell or is derived from a mammalian cell. In some embodiments, an engineered cell is a rodent cell or is derived from a rodent cell. In some embodiments, an engineered cell is a human cell or is derived from a human cell. In some embodiments, the engineered cell is a human cell or is derived from a human cell. In some embodiments, the engineered cell is a human cell or is derived from a human cell.
  • the present disclosure further provides progeny of an engineered cell, where the progeny can include the same exogenous nucleic acid or polypeptide as the engineered cell from which it was derived.
  • the present disclosure further provides, in some embodiments, a composition comprising an engineered cell.
  • the Acr protein is AcrVAl or a variant thereof.
  • the AcrVAl or variant thereof comprises the amino acid sequence encoded by SEQ ID NO: 9 or a variant thereof having at least 80% sequence identity to the amino acid sequence encoded by SEQ ID NO: 9.
  • a split Casl2a complex that retains at least some of the effector activity of a Casl2a protein or variant thereof retains at least about 5% (such as at least about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or greater) of the effector activity of the Casl2a protein or variant thereof.
  • the crRNA comprises a spacer sequence selected from the polynucleotide sequence of any one of SEQ ID NOs: 15-43.
  • the inducible promoter is selected from an RRM2 promoter, a hypoxia-inducible promoter, a TRE3G promoter, a CMV5-CuO promoter, and a P2 inducible promoter.
  • the RRM2 promoter comprises the polynucleotide sequence of SEQ ID NO: 10.
  • the hypoxia-inducible promoter comprises the polynucleotide sequence of SEQ ID NO: 11.
  • the TRE3G promoter comprises the polynucleotide sequence of SEQ ID NO: 12.
  • the CMV5-CuO promoter comprises the polynucleotide sequence of SEQ ID NO: 13.
  • the P2 inducible promoter comprises the polynucleotide sequence of SEQ ID NO: 14.
  • the split Casl2a system comprises nucleic acid comprising a plurality of inducible promoters, each of the plurality of inducible promoters operably linked to a polynucleotide sequence encoding an effector subunit are different.
  • the split Casl2a system comprises nucleic acid comprising a plurality of inducible promoters
  • each of the plurality of inducible promoters operably linked to a polynucleotide sequence encoding a crRNA are different.
  • the split Casl2a system comprises nucleic acid comprising a plurality of inducible promoters, at least some (such as at least about any of 2, 3, 4, or more) of the plurality of inducible promoters operably linked to a polynucleotide sequence encoding a crRNA are the same.
  • the split Casl2a system comprises nucleic acid comprising a plurality of inducible promoters, all of the plurality of inducible promoters operably linked to a polynucleotide sequence encoding a crRNA are the same. In some embodiments, where the split Casl2a system comprises nucleic acid comprising a plurality of inducible promoters, each of the plurality of inducible promoters are different.
  • the system is configured such that all components of the Casl2a/crRNA complex are present or capable of being expressed in the sample when the first input and the second input each have a desired state. In some embodiments, the system is configured such that all components of the Casl2a/crRNA complex are present or capable of being expressed in the sample when the first input is present and the second input is present.
  • the system comprises (a) nucleic acid encoding a first effector subunit and a second effector subunit of the two or more effector subunits, wherein the nucleic acid comprises a first inducible promoter operably linked to a polynucleotide sequence encoding the first effector subunit and a second inducible promoter operably linked to a polynucleotide sequence encoding the second effector subunit, and the first input is capable of modulating the expression of the first effector subunit in the sample and the second input is capable of modulating the expression of the second effector subunit in the sample; or (b) nucleic acid encoding a first effector subunit of the two or more effector subunits, wherein the nucleic acid comprises a first inducible promoter operably linked to a polynucleotide sequence encoding the first effector subunit; and nucleic acid encoding the first cr
  • the expression of a third component of the Casl2a/crRNA complex is under the control of a third input to the sample, and the system is configured such that all components of the Casl2a/crRNA complex are present or capable of being expressed in the sample when the first input, the second input, and the third input each have a desired state.
  • the system is configured such that all components of the Casl2a/crRNA complex are present or capable of being expressed in the sample when the first input is present, the second input is present, and the third input is present.
  • the system comprises (a) nucleic acid encoding a first effector subunit, a second effector subunit, and a third effector subunit of the two or more effector subunits, wherein the nucleic acid comprises a first inducible promoter operably linked to a polynucleotide sequence encoding the first effector subunit, a second inducible promoter operably linked to a polynucleotide sequence encoding the second effector subunit, and a third inducible promoter operably linked to a polynucleotide sequence encoding the third effector subunit, and the first input is capable of modulating the expression of the first effector subunit in the sample, the second input is capable of modulating the expression of the second effector subunit in the sample, and the third input is capable of modulating the expression of the third effector subunit in the sample; or (b) nucleic acid encoding a first effector subunit and a second effector subunit of the two or more effect
  • the expression of a third component of the Casl2a/crRNA complex is under the control of a third input to the sample and the expression of a fourth component of the Casl2a/crRNA complex is under the control of a fourth input to the sample, and the system is configured such that all components of the Casl2a/crRNA complex are present or capable of being expressed in the sample when the first input, the second input, the third input, and the fourth input each have a desired state.
  • the system is configured such that all components of the Casl2a/crRNA complex are present or capable of being expressed when the first input is present, the second input is present, the third input is present, and the fourth input is present.
  • the system comprises nucleic acid encoding a first effector subunit, a second effector subunit, and a third effector subunit of the two or more effector subunits, wherein the nucleic acid comprises a first inducible promoter operably linked to a polynucleotide sequence encoding the first effector subunit, a second inducible promoter operably linked to a polynucleotide sequence encoding the second effector subunit, and a third inducible promoter operably linked to a polynucleotide sequence encoding the third effector subunit, and the first input is capable of modulating the expression of the first effector subunit in the sample, the second input is capable of modulating the expression of the second effector subunit in the sample
  • the two or more effector subunits comprise a first effector subunit and a second effector subunit, wherein the first effector subunit comprises an N-terminal fragment of the Casl2a protein or variant thereof up to and including a split point amino acid, and the second effector subunit comprises a C-terminal fragment of the Casl2a protein or variant thereof following the split point amino acid.
  • the split point amino acid corresponds to A406 or G550 with respect to a parental Cas 12a protein amino acid sequence of SEQ ID NO: 1.
  • the first effector subunit further comprises a first stabilization domain at its C- terminus and the second effector subunit further comprises a second stabilization domain at its N-terminus, wherein the first stabilization domain and the second stabilization domain are capable of interacting with one another to stabilize a complex comprising the first effector subunit and the second effector subunit in the sample.
  • the two or more effector subunits comprise a first effector subunit and a second effector subunit, wherein the Cas 12a protein or variant thereof is a fusion protein comprising an effector domain fused to a Cas 12a domain, and the first effector subunit comprises the Cas 12a domain and the second effector subunit comprises the effector domain.
  • the first effector subunit further comprises a first stabilization domain at either terminus and the second effector subunit further comprises a second stabilization domain at either terminus, wherein the first stabilization domain and the second stabilization domain are capable of interacting with one another to stabilize a complex comprising the first effector subunit and the second effector subunit in the sample.
  • the two or more effector subunits comprise a first effector subunit, a second effector subunit, and a third effector subunit.
  • the Casl2a protein or variant thereof is a fusion protein comprising an effector domain fused to a Casl2a domain
  • the first effector subunit comprises an N-terminal fragment of the Cas 12a domain up to and including a split point amino acid
  • the second effector subunit comprises a C-terminal fragment of the Cas 12a domain following the split point amino acid
  • the third effector subunit comprises the effector domain.
  • the split point amino acid corresponds to A406 or G550 with respect to a parental Casl2a protein amino acid sequence of SEQ ID NO: 1.
  • the first effector subunit further comprises a first stabilization domain at its C- terminus and the second effector subunit further comprises a second stabilization domain at its N-terminus, wherein the first stabilization domain and the second stabilization domain are capable of interacting with one another to stabilize a complex comprising the first effector subunit and the second effector subunit in the sample; and/or the third effector subunit further comprises a third stabilization domain at either terminus and (i) the second effector subunit further comprises a fourth stabilization domain at its C-terminus or (ii) the first effector subunit further comprises the fourth stabilization domain at its N-terminus, wherein the third stabilization domain and the fourth stabilization domain are capable of interacting with one another to stabilize a complex comprising the first effector subunit, the second effector subunit, and the third effector subunit in the sample
  • the first stabilization domain and the second stabilization domain are derived from subunits of a heterodimerizing binding pair or a split-intein.
  • the method further employs a third stabilization domain and a fourth stabilization domain
  • the third stabilization domain and the fourth stabilization domain are derived from subunits of a heterodimerizing binding pair or a split-intein and are orthogonal to the first stabilization domain and the second stabilization domain.
  • the first stabilization domain and the second stabilization domain are derived from subunits of a heterodimerizing binding pair and the third stabilization domain and the fourth stabilization domain are derived from subunits of a split-intein; or the first stabilization domain and the second stabilization domain are derived from subunits of a split-intein and the third stabilization domain and the fourth stabilization domain are derived from subunits of a heterodimerizing binding pair.
  • the heterodimerizing binding pair is a coiled-coil (CC) heterodimer.
  • the CC heterodimer is a leucine zipper.
  • the leucine zipper subunits comprise RR12EE345L and EE12RR345L.
  • RR12EE345L comprises the amino acid sequence encoded by SEQ ID NO: 6.
  • EE12RR345L comprises the amino acid sequence encoded by SEQ ID NO: 5.
  • the CC heterodimer is a CC peptide heterodimer ( see Lebar, T., et al. (2020). Nature Chemical Biology, 1-7).
  • the split-intein subunits comprise DnaE N-intein and C-intein.
  • DnaE N-intein comprises the amino acid sequence encoded by SEQ ID NO: 7.
  • DnaE C-intein comprises the amino acid sequence encoded by SEQ ID NO: 8.
  • the split-intein is gp41-l, gp41-8, NrdJ-1, or IMPDH-1 ( see Dassa, B., et al. (2009). Nucleic acids research, 57(8), 2560-2573).
  • the split-intein comprises split portions of the Mxe GyrA intein (see Fine, E. J., et al. (2015). Scientific reports, 5, 10777).
  • the system further comprises one or more additional crRNAs or nucleic acid encoding the one or more additional crRNAs, wherein Casl2a/crRNA complexes formed by association of the one or more additional crRNAs with (I) the Cas 12a protein or variant thereof or (II) the two or more effector subunits are capable of generating one or more additional outputs to the sample comprising effector activity of the Casl2a/crRNA complexes at target sites directed by the one or more additional crRNAs.
  • the system comprises nucleic acid encoding a crRNA array comprising one or more of the first crRNA and the one or more additional crRNAs.
  • the Cas 12a protein or variant thereof is derived from a parental Cas 12a protein from Lachnospiraceae bacterium.
  • the parental Cas 12a protein from Lachnospiraceae bacterium comprises the amino acid sequence of SEQ ID NO: 1.
  • the target nucleic acid is a target dsDNA.
  • a protospacer in the target dsDNA targeted by the crRNA spacer has a 5' PAM selected from 5'-TTTG-3', 5'- TTTA-3', and 5'-TTTC-3'.
  • the Casl2a protein or variant thereof is a nuclease-competent Cas protein comprising a Casl2a domain having some, the same, or greater nuclease activity as compared to a parental Cas 12a protein from which it is derived, and the first output comprises cleavage (e.g., double-stranded cleavage) of the first target nucleic acid near the first target polynucleotide sequence.
  • the Casl2a domain comprises the amino acid sequence of SEQ ID NO: 1.
  • the Cas 12a protein or variant thereof is a fusion protein comprising an effector domain fused to a nuclease-deficient dCasl2a domain having reduced or eliminated nuclease activity as compared to a parental Cas 12a protein from which it is derived, and the first output is generated by activity of the effector domain.
  • the dCasl2a domain comprises a modification of amino acid D832 with respect to SEQ ID NO: 1 that reduces, substantially eliminates, or eliminates nuclease activity of the dCasl2a domain.
  • the dCasl2a domain comprises a D832A substitution with respect to SEQ ID NO: 1.
  • the dCasl2a domain comprises the amino acid sequence of SEQ ID NO: 2.
  • the effector domain comprises a polypeptide that can (i) cleave a nucleic acid, (ii) edit a nucleotide, (iii) activate transcription, (iv) repress transcription, (v) methylate a nucleic acid, and/or (vi) demethylate a nucleic acid.
  • the effector domain comprises a transcriptional activation domain
  • the first output comprises transcriptional activation of a first gene targeted by the first crRNA.
  • the transcriptional activation domain is a tripartite VP64-p65-Rta (VPR) transcriptional activation domain or variant thereof.
  • the inputs to the cell are selected from physical inputs, small molecule inputs, and biologic inputs.
  • the physical inputs are selected from oxygen levels, pH, ion concentrations, temperature, light, mechanical force, ultrasound, shock wave, magnetic field, and electric field;
  • the small molecule inputs are selected from drugs, metabolites, and adjuvants;
  • the biologic inputs are selected from polypeptides, polypeptide derivatives, and nucleic acids.
  • the split Cast 2a system comprises (a) nucleic acid encoding two or more effector subunits comprising fragments of a Casl2a protein or variant thereof, wherein the two or more effector subunits are capable of spontaneously assembling to form a split Casl2a complex retaining at least some of the effector activity of the Casl2a protein or variant thereof; and (b) a CRISPR RNA (crRNA) or nucleic acid encoding the crRNA, wherein the crRNA comprises a spacer having sufficient complementary to a target polynucleotide sequence in a target nucleic acid such that it can hybridize to the target nucleic acid.
  • crRNA CRISPR RNA
  • the composition is a pharmaceutical composition and further comprises a pharmaceutically acceptable excipient and/or carrier.
  • the composition is used for carrying out a method of the present disclosure, e.g., a method for site-specific modification of a target DNA; etc.
  • kits for carrying out a method described herein can include one or more components of a split Cas 12a system as described herein.
  • the split Cas 12a system comprises (a) nucleic acid encoding two or more effector subunits comprising fragments of a Cas 12a protein or variant thereof, wherein the two or more effector subunits are capable of spontaneously assembling to form a split Cas 12a complex retaining at least some of the effector activity of the Cas 12a protein or variant thereof; and (b) a CRISPR RNA (crRNA) or nucleic acid encoding the crRNA, wherein the crRNA comprises a spacer having sufficient complementary to a target polynucleotide sequence in a target nucleic acid such that it can hybridize to the target nucleic acid.
  • crRNA CRISPR RNA
  • a kit as described herein can further include one or more additional reagents, where such additional reagents can be selected from: a buffer for introducing one or more components of a split Cas 12a system into a cell; a dilution buffer; a reconstitution solution; a wash buffer; a control reagent; a control expression vector or polyribonucleotide; a reagent for in vitro production of one or more components of a split Cas 12a system, and the like.
  • kits can be in separate containers; or can be combined in a single container.
  • a kit can further include instructions for using the components of the kit to practice the methods.
  • the instructions for practicing the methods are generally recorded on a suitable recording medium.
  • the instructions may be printed on a substrate, such as paper or plastic, etc.
  • the instructions may be present in the kits as a package insert, in the labeling of the container of the kit or components thereof (e.g., associated with the packaging or sub-packaging) etc.
  • the instructions are present as an electronic storage data file present on a suitable computer readable storage medium, e.g., CD-ROM, diskette, flash drive, etc.
  • the actual instructions are not present in the kit, but means for obtaining the instructions from a remote source, e.g., via the internet, are provided.
  • An example of this embodiment is a kit that includes a web address where the instructions can be viewed and/or from which the instructions can be downloaded. As with the instructions, this means for obtaining the instructions is recorded on a suitable substrate.
  • Example 1A Design and characterization of a split Cas 12a system in mammalian cells
  • This example demonstrates the design and characterization of a split Casl2a system in mammalian cells, allowing the construction of AND gates.
  • the split Casl2a system was based on a nuclease-dead dCasl2a (D832A) from Lachnospiraceae bacterium fused with a tripartite VPR activator for targeted gene activation (Tak, Y.E. et al , 2017. Nature Methods, 14(12), pp.l 163-1166).
  • a minimized VPR miniVPR was used (Vora, S., et al. (2018). bioRxiv, 298620), which showed comparable activation in a TRE3G-GFP HEK293T reporter cell line (FIGS. 1A and IB, Tables 1-3).
  • Table 2 Plasmids used in experiments corresponding to indicated FIGS.
  • Table 3 Fold-changes and statistical analyses related to indicated FIGS.
  • a stable cell line was generated with the N- and C- terminal halves under the control of cumate and Doxycycline (Dox)-inducible promoters, respectively.
  • Dox cumate and Doxycycline
  • the amount of each molecule was titrated and the resulting activation of a P2- GFP reporter was measured (FIGS. 7A, 7B, and 7C) (Nissim, L. et al, 2014. Molecular Cell, 54(4), pp.698-710). Strong GFP activation was found to occur only in the presence of high levels of both inputs (FIG. 7D).
  • Example IB Construction of multi-input logic using split dCas 12a
  • This example demonstrates the design and characterization of a split Casl2a system in mammalian cells with multiple-input logic.
  • Casl2a can process crRNAs from a Pol II promoter-driven transcript (FIG. 10A) (Zhong, G. et al., 2017. Nature Chemical Biology, 13(8), pp.839-841; Campa, C.C. et al, 2019. Nature Methods, pp.1-7).
  • the ability to express crRNAs under the wide variety of inducible Pol II promoters, rather than being limited to a constitutive Pol III promoter like U6, provides another means of controlling the split dCasl2a system activity. It was verified that constitutive CAG and Dox-inducible TRE3G Pol II crRNA cassettes could efficiently activate IFNg (FIGS. 10B-D).
  • a 3-way split of dCasl2a- miniVPR was created by using the 406 leucine zipper split further to separate the fused activator using an orthogonal intein system (Truong, D.-J.J. et al., 2015. Nucleic Acids Research, 43(13), pp.6450-6458).
  • the DnaEN-intein and C-intein domains were placed in the linker between dCasl2a and miniVPR, and it was verified that this 3 -way split protein could reconstitute and activate reporter gene expression, to 43% of the full-length dCasl2al l6 miniVPR (FIG. 11A and 1 IB).
  • the 3 -way split system was then used to generate a tight 3- input Boolean AND gate (FIGS. 11C and 1 ID).
  • This example demonstrates the design and characterization of an exemplary split Casl2a system in mammalian cells for detection and integration of cancer-specific inputs for output of therapeutic gene activation.
  • the split dCasl2a system architecture was used to construct a proof-of-concept anti- tumoral circuit with biologically relevant inputs and outputs. Cancer cells exhibit a number of altered genetic signatures and receive distinctive external signals from the surrounding tumor microenvironment (TME) (Hanahan & Weinberg 2011). However, individual signatures are rarely unique and may also be present at varying levels in non-cancerous tissues. Multi-input sensing and integration can provide the control to ensure specificity of therapeutic programs. As a demonstration, a split dCasl2a was expressed with elements under the control of promoters responsive to intracellular and extracellular signals indicative of breast cancer, and the system was used to activate endogenous cytokines (FIG. 14A).
  • RRM2 ribonucleotide reductase subunit 2
  • RRM2 converts ribonucleotides to deoxyribonucleotides, catalyzing the rate limiting step for DNA synthesis, and has been reported to be overexpressed in breast cancer (Y un, H J. et al, 2008. Experimental & molecular medicine, 40(3), pp.345-53).
  • RRM2 promoter activity was significantly higher in the cancer cells, allowing RRM2 promoter activity to be used as a first tumor input (FIG.
  • split dCasl2a systems demonstrate the scalability of the split dCasl2a architecture to build multi- input therapeutic circuits, where in this example an orthogonal input (Dox) could either turn on or turn off circuit behavior to provide additional control.
  • Dox orthogonal input
  • the third component could alternatively be controlled by additional tumor signatures or by signals characteristic of healthy tissue, as could additional components, to build a variety of cancer-targeting systems.
  • Examples 1A-1C the development and repurposing of a split dCasl2a system for implementing versatile, robust, and scalable logic gates is described. It was shown that a simpler 2-input system can be combined with inducible crRNAs and additional split strategies to generate higher-order AND gates (FIG. 15).
  • the availability of anti-CRISPR systems provides a means of implementing “NOT” logic.
  • the exemplified systems behaved robustly in carrying out Boolean logic operations, with a high dynamic range between ON and OFF states. Extending to biologically relevant inputs and outputs, it was demonstrated that elements of the systems can be linked to tumor-relevant promoters, allowing for the detection of combinations of tumor cues for logic integration to control therapeutic targets.
  • the systems described herein can serve as a scaffold for future modular engineering of logic gates with diverse inputs and outputs.
  • tissue-specific and disease-relevant promoters that can be used to limit split protein expression to cells of interest.
  • the split dCasl2a components could be incorporated into systems that control protein localization in response to soluble or cell-surface signals (Kipniss, N.H. et al, 2017. Nature Communications, 8(1), p.2212; Chung, H.K. et al., 2019. Science (New York, N.Y.), 364(6439), p.eaat6982; Morsut, L., et al. (2016).
  • the facile programmability of the dCasl2a systems could be leveraged to effectively activate various endogenous or exogenous gene programs.
  • Activation of secreted factors including cytokines or antibodies, could be used for localized delivery of cell-manufactured therapeutics.
  • Modulation of targets like transcription factors or noncoding RNAs could change cell behavior or differentiation.
  • changing the fused activator to a repressor, such as KRAB could expand the output to include repression of endogenous gene targets.
  • Casl2a-mediated gene editing or base editing systems could be used to permanently alter the genome, for example, for protein knockout, lineage tracing, or genome recording upon encountering combinations of cellular inputs.
  • Genome engineering tools are emerging as an avenue to enrich mammalian synthetic biology approaches for treatment of complex diseases. While the performance of the split dCasl2a platform was demonstrated in cancer cells, it can easily be implemented in other therapeutically relevant cell types, including, for example, immune cells and stem cells, to create novel sensor-computation-actuator systems with a variety of inputs and outputs for more effective and safer cell engineering and cell therapy.
  • HEK293T cells (Clontech) and MDA-MB-231 cells (Sigma- Aldrich) were cultured in DMEM + GlutaMAX (Thermo Fisher) supplemented with 10% Tet System Approved FBS (Clontech) and lOOU/mL of penicillin and streptomycin (Life Technologies).
  • MCF10A cells were a gift from Mingyu Chung (Tobias Meyer lab) and were cultured in MEGM Bullet Kit Growth Media (Fisher) supplemented with lOOng/mL Choleratoxin (Sigma). Cells were maintained at 37°C and 5% C02 and passaged using standard cell culture techniques. Cells were not tested for mycoplasma contamination.
  • transient transfection of HEK293Ts cells were seeded the day before transfection at lxl 0 5 cells/mL. Transient transfections were performed using 3pL of TransIT-LTl transfection reagent (Mirus) per pg of plasmid. Cells were analyzed 2 or 3 days post transfection, as indicated. F or crRNA testing, 25 Ong of dCas 12a construct and 250ng of crRNA plasmid were transfected into 24-well plates. For other activation experiments, 166ng of each plasmid were transfected into 24-well plates.
  • Stable MDA-MB-231 and MCF10A cell lines were generated using lentiviral transduction.
  • F1EK293T cells were transfected with 1.51 pg of pHR vector with construct of interest, 1.32 pg of dR8.91 and 165 ng ofpMD2 with 7.5 pL ofMirus TransIT-LTl (for 6 well plate format).
  • One day post transfection media was replaced with fresh media, and two days post transfection lentivirus was harvested and filtered through a 0.45 pm polyvinylidene fluoride filter (Millipore). Following filtration, 1 volume lentivirus was mixed with 4 volumes Lentivirus Precipitation Solution (Alstem) and refrigerated overnight.
  • Cells were seeded for transduction at 1 x 10 5 cells/well of a 12-well plate. The next day, lentivirus was pelleted at 1500xg for 30 minutes at 4°C and resuspended in 1/100 of original volume before transduction of target cells. Transduced cells were selected for using 200 pg/mL Zeocin (Thermofisher) and 2 pg/mL Puromycin (Gold Biotech).
  • the stable F1EK293T cell line for Dox/cumate dose-response was generated as follows.
  • the cumate-inducible construct was introduced by lentivirus transduction, as described above, and selected for using 2 pg/mL Puromycin.
  • the Dox-inducible construct was introduced using the PiggyBac system. Briefly, 250ng of the construct of interest was transfected with lOOng of PiggyBac Transposase vector in cells in 12-well plate. Cells were expanded for 3 days then selected for with 400 pg/mL Zeocin.
  • cells were stimulated with both 60 pg/mL cumate (System Biosciences) and lOOOng/mL Dox (Gold Biotech) and sorted for the mCherry+/BFP+ cell population using the SONY SF1800 Cell Sorter to select for cells that were able to successfully turn on both constructs in response to stimulation.
  • the P2 reporter was modified from a plasmid obtained from Addgene (#55198) (Nissim, L. et al., 2014. Molecular Cell, 54(4), pp.698-710).
  • the RRM2 promoter was amplified from HEK293T genomic DNA extracted using the DNeasy Blood & Tissue kit (Qiagen).
  • the intein and AcrVAl DNA was cloned from ordered gBlocks (Integrated DNA Technologies). Plasmids were cloned using InFusion (Takara Bio) and Stellar Competent cells (Takara Bio). DNA sequences and plasmids used can be found in Table 2 and the Sequence Listing.
  • the crRNA plasmids were cloned using ligation cloning. Oligos were annealed and phosphorylated with PNK (NEB) and inserted into backbone using T4 DNA Ligase (NEB).
  • the scaffold used (N’s denote spacer sequence): 5’-
  • T7E1 endonuclease activity assessed via T7E1 endonuclease activity (NEB).

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Abstract

The present disclosure relates to systems, compositions, and methods for modulating the expression of a target nucleic acid or engineering a target nucleic acid employing a split Cas12a CRISPR-Cas system based on the detection and integration of multiple biological inputs.

Description

Split CRISPR-Cas for Biological Signal Integration
CROSS-REFERENCE TO RELATED APPLICATIONS [0001] This application claims priority to U.S. Provisional Patent Application No. 62/970,150, filed February 4, 2020, the disclosure of which is incorporated by reference herein in its entirety, including any drawings.
INCORPORATION OF THE SEQUENCE LISTING [0002] This application contains a Sequence Listing which is hereby incorporated by reference in its entirety. The accompanying Sequence Listing text file, named “078430- 515001WO_SequenceListing_ST25,” was created on February 1, 2021 and is 44,198 bytes in size.
FIELD
[0001] The present disclosure generally relates to systems, compositions, and methods for effecting a biological output in a cell based on the detection and integration of multiple biological inputs. The systems include a split Casl2a Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) effector protein or variant thereof, or nucleic acid encoding the split Casl2a protein or variant thereof, and a CRISPR RNA (crRNA) comprising a spacer sequence complementary to a target polynucleotide sequence in the cell or nucleic acid encoding the crRNA, wherein expression of all components of the split Casl2a protein or variant thereof and the crRNA in the cell allows for formation of a functional split Casl2a/crRNA complex that can bind to the target polynucleotide sequence in a site specific manner to effect a biological output, such as modulated expression of a gene associated with the target polynucleotide sequence.
BACKGROUND
[0002] The successful application of synthetic biology to human disease requires the integration of information about diverse biological inputs and the modulation of various genomic programs in response. Such multi-input/multi-output, sensor-driven actuator systems have the potential to transform therapeutics in a variety of fields including cancer, immune disorders, and regenerative medicine (Ruder, W.C., et a , 2011. Science (New York, N.Y.), 333(6047), pp.1248-52). For example, a recent RNA-based AND logic gate strategy has been implemented to detect the presence of two intracellular tumor signals and direct expression of therapeutic transgenes (Nissim, L. et al., 2017. Cell, 171(5), p.l 138-1150. el5). RNA-based circuity has also been utilized to activate apoptosis in cancer cells in response to combinations of miRNAs (Xie, Z. et al, 2011. Science (New York, N.Y.), 333(6047), pp.1307-11). Despite these successes, engineering versatile, scalable systems in mammalian cells that can detect diverse types of disease-relevant cues and execute customizable therapeutic programs remains difficult, especially when extending beyond two inputs. Here, we aim to employ synthetic biology and molecular engineering to adapt the CRISPR-Cas system for such scalable system design.
[0003] CRISPR-based effectors provide an easily programmable platform for multiplexed control of genes of interest (Dominguez, A. A., et al., 2016. Nature reviews. Molecular cell biology, 17(1), pp.5-15). Using a nuclease-dead dCas protein fused to effector domains in combination with a guide RNA generates an artificial transcription factor for the modulation of endogenous genes (Gilbert, L.A. et al, 2013. Cell, 154(2), pp.442-451; Chavez, A. et al, 2015. Nature Methods, 12(4), pp.326-328; Konermann, S. et al., 2015. Nature, 517(7536), pp.583-8; Perez-Pinera, P. et al., 2013. Nature Methods, 10(10), pp.973-976). The Type V Casl2a (Cpfl) system is of particular interest for complex circuit design as it can process its own CRISPR RNAs (crRNAs) from a single CRISPR array, which allows for easy multiplexed control of many genes (Zetsche, B. et al, 2015. Cell, 163(3), pp.759-71; Fonfara, I. et al, 2016. Nature, 532(7600), pp.517-521; Zetsche, B. et al., 2016. Nature Biotechnology, 35(1), pp.31- 34). By splitting the Casl2a protein into distinct components that can be individually controlled, it theoretically could provide a functional core for a variety of synthetic circuits. Despite recent efforts to develop dimerizing variants of Casl2a (Tak, Y.E. et al., 2017. Nature Methods, 14(12), pp.l 163-1166; Nihongaki, Y. et al., 2019. Nature Chemical Biology, pp.l- 7), there is limited work on repurposing this system for complex cellular circuits that can sense, integrate and respond (Liu, Y. et al., 2014. Nature Communications, 5, p.5393). Prior work has engineered split Cas9 proteins, and a functional split Casl2a could utilize its unique RNA processing features to expand the toolbox for complex synthetic Cas-based circuits (Ma, D., et al., 2016. Nature Communications, 7, p.13056).
SUMMARY
[0004] This section provides a general summary of the disclosure, and is not comprehensive of its full scope or all of its features.
[0005] In one aspect, provided herein is a system for conditionally generating an output to a sample comprising the system, the system comprising: (a) (i) a Casl2a protein or variant thereof having an effector activity, or nucleic acid encoding the Casl2a protein or variant thereof; or (ii) two or more effector subunits comprising fragments of the Cast 2a protein or variant thereof, or nucleic acid encoding the two or more effector subunits, wherein the two or more effector subunits are capable of spontaneously assembling to form a split Casl2a complex in the sample, and the split Casl2a complex retains at least some of the effector activity of the Casl2a protein or variant thereof; and (b) a first CRISPR RNA (crRNA) or nucleic acid encoding the first crRNA, wherein the first crRNA comprises a first spacer having sufficient complementary to a first target polynucleotide sequence in a first target nucleic acid in the sample such that it can hybridize to the first target nucleic acid, wherein a Casl2a/crRNA complex formed in the sample by association of the first crRNA with (I) the Casl2a protein or variant thereof or (II) the two or more effector subunits is capable of generating a first output to the sample comprising the effector activity of the Casl2a/crRNA complex at the first target nucleic acid, and wherein the expression of a first component of the Casl2a/crRNA complex is under the control of a first input to the sample and the expression of a second component of the Casl2a/crRNA complex is under the control of a second input to the sample.
[0006] In some embodiments, according to any of the systems described above, the system further comprises nucleic acid comprising a NOT inducible promoter operably linked to a polynucleotide sequence encoding an anti-CRISPR (Acr) protein capable of inhibiting activity of the split Casl2a complex in the sample, wherein a NOT input to the sample is capable of increasing the expression of Acr protein from the nucleic acid in the sample such that activity of split Casl2a complex in the sample is inhibited.
[0007] In some embodiments, according to any of the systems described above, the system is configured such that all components of the Casl2a/crRNA complex are present or capable of being expressed in the sample when the first input and the second input each have a desired state. In some embodiments, the system is configured such that all components of the Casl2a/crRNA complex are present or capable of being expressed in the sample when the first input is present and the second input is present.
[0008] In some embodiments, according to any of the systems described above, the system comprises (a) nucleic acid encoding the Casl2a protein or variant thereof, wherein the nucleic acid encoding the Casl2a protein or variant thereof comprises a first inducible promoter operably linked to a polynucleotide sequence encoding the Casl2a protein or variant thereof, and the first input is capable of modulating the expression of the Casl2a protein or variant thereof in the sample; and (b) nucleic acid encoding the first crRNA, wherein the nucleic acid encoding the first crRNA comprises a second inducible promoter operably linked to a polynucleotide sequence encoding the first crRNA, and the second input is capable of modulating the expression of the first crRNA in the sample. In some embodiments, modulating the expression of each of the Casl2a/crRNA complex components comprises increasing their expression.
[0009] In some embodiments, according to any of the systems described above, the system comprises (a) nucleic acid encoding a first effector subunit and a second effector subunit of the two or more effector subunits, wherein the nucleic acid comprises a first inducible promoter operably linked to a polynucleotide sequence encoding the first effector subunit and a second inducible promoter operably linked to a polynucleotide sequence encoding the second effector subunit, and the first input is capable of modulating the expression of the first effector subunit in the sample and the second input is capable of modulating the expression of the second effector subunit in the sample; or (b) nucleic acid encoding a first effector subunit of the two or more effector subunits, wherein the nucleic acid comprises a first inducible promoter operably linked to a polynucleotide sequence encoding the first effector subunit; and nucleic acid encoding the first crRNA, wherein the nucleic acid comprises a second inducible promoter operably linked to a polynucleotide sequence encoding the first crRNA, and the first input is capable of modulating the expression of the first effector subunit in the sample and the second input is capable of modulating the expression of the first crRNA in the sample. In some embodiments, modulating the expression of each of the Casl2a/crRNA complex components comprises increasing their expression.
[0010] In some embodiments, according to any of the systems described above, the expression of a third component of the Casl2a/crRNA complex is under the control of a third input to the sample, and the system is configured such that all components of the Casl2a/crRNA complex are present or capable of being expressed in the sample when the first input, the second input, and the third input each have a desired state. In some embodiments, the system is configured such that all components of the Casl2a/crRNA complex are present or capable of being expressed in the sample when the first input is present, the second input is present, and the third input is present. In some embodiments, the system comprises (a) nucleic acid encoding a first effector subunit, a second effector subunit, and a third effector subunit of the two or more effector subunits, wherein the nucleic acid comprises a first inducible promoter operably linked to a polynucleotide sequence encoding the first effector subunit, a second inducible promoter operably linked to a polynucleotide sequence encoding the second effector subunit, and a third inducible promoter operably linked to a polynucleotide sequence encoding the third effector subunit, and the first input is capable of modulating the expression of the first effector subunit in the sample, the second input is capable of modulating the expression of the second effector subunit in the sample, and the third input is capable of modulating the expression of the third effector subunit in the sample; or (b) nucleic acid encoding a first effector subunit and a second effector subunit of the two or more effector subunits, wherein the nucleic acid comprises a first inducible promoter operably linked to a polynucleotide sequence encoding the first effector subunit and a second inducible promoter operably linked to a polynucleotide sequence encoding the second effector subunit; and nucleic acid encoding the first crRNA, wherein the nucleic acid comprises a third inducible promoter operably linked to a polynucleotide sequence encoding the first crRNA, and the first input is capable of modulating the expression of the first effector subunit in the sample, the second input is capable of modulating the expression of the second effector subunit in the sample, and the third input is capable of modulating the expression of the first crRNA in the sample. In some embodiments, modulating the expression of each of the Casl2a/crRNA complex components comprises increasing their expression. [0011] In some embodiments, according to any of the systems described above, the expression of a third component of the Casl2a/crRNA complex is under the control of a third input to the sample and the expression of a fourth component of the Casl2a/crRNA complex is under the control of a fourth input to the sample, and the system is configured such that all components of the Casl2a/crRNA complex are present or capable of being expressed in the sample when the first input, the second input, the third input, and the fourth input each have a desired state. In some embodiments, the system is configured such that all components of the Cas 12a/crRNA complex are present or capable of being expressed when the first input is present, the second input is present, the third input is present, and the fourth input is present. In some embodiments, the system comprises (a) nucleic acid encoding a first effector subunit, a second effector subunit, and a third effector subunit of the two or more effector subunits, wherein the nucleic acid comprises a first inducible promoter operably linked to a polynucleotide sequence encoding the first effector subunit, a second inducible promoter operably linked to a polynucleotide sequence encoding the second effector subunit, and a third inducible promoter operably linked to a polynucleotide sequence encoding the third effector subunit, and the first input is capable of modulating the expression of the first effector subunit in the sample, the second input is capable of modulating the expression of the second effector subunit in the sample, and the third input is capable of modulating the expression of the third effector subunit in the sample; and (b) nucleic acid encoding the first crRNA, wherein the nucleic acid comprises a fourth inducible promoter operably linked to a polynucleotide sequence encoding the first crRNA, and the fourth input is capable of modulating the expression of the first crRNA. In some embodiments, modulating the expression of each of the Casl2a/crRNA complex components comprises increasing their expression.
[0012] In some embodiments, according to any of the systems described above, the two or more effector subunits consist of a first effector subunit and a second effector subunit. In some embodiments, the first effector subunit comprises an N-terminal fragment of the Cas 12a protein or variant thereof up to and including a split point amino acid, and the second effector subunit comprises a C-terminal fragment of the Cas 12a protein or variant thereof following the split point amino acid. In some embodiments, the split point amino acid corresponds to A406 or G550 with respect to a parental Cas 12a protein amino acid sequence of SEQ ID NO: 1. In some embodiments, the first effector subunit further comprises a first stabilization domain at its C- terminus and the second effector subunit further comprises a second stabilization domain at its N-terminus, wherein the first stabilization domain and the second stabilization domain are capable of interacting with one another to stabilize a complex comprising the first effector subunit and the second effector subunit in the sample.
[0013] In some embodiments, according to any of the systems described above, the Cas 12a protein or variant thereof is a fusion protein comprising an effector domain fused to a Cas 12a domain, and the first effector subunit comprises the Cas 12a domain and the second effector subunit comprises the effector domain. In some embodiments, the first effector subunit further comprises a first stabilization domain at either terminus and the second effector subunit further comprises a second stabilization domain at either terminus, wherein the first stabilization domain and the second stabilization domain are capable of interacting with one another to stabilize a complex comprising the first effector subunit and the second effector subunit in the sample.
[0014] In some embodiments, according to any of the systems described above comprising a first stabilization domain and a second stabilization domain, the first stabilization domain and the second stabilization domain are derived from subunits of a heterodimerizing binding pair or a split-intein. In some embodiments, the first stabilization domain and the second stabilization domain are derived from subunits of a leucine zipper. In some embodiments, the leucine zipper subunits comprise RR12EE345L and EE12RR345L. In some embodiments, the first stabilization domain and the second stabilization domain are derived from subunits of a split- intein, and the split-intein subunits comprise DnaE N-intein and C-intein. [0015] In some embodiments, according to any of the systems described above, the two or more effector subunits consist of a first effector subunit, a second effector subunit, and a third effector subunit. In some embodiments, the Cas 12a protein or variant thereof is a fusion protein comprising an effector domain fused to a Cas 12a domain, and (a) the first effector subunit comprises an N-terminal fragment of the Cas 12a domain up to and including a split point amino acid, (b) the second effector subunit comprises a C-terminal fragment of the Cas 12a domain following the split point amino acid; and (c) the third effector subunit comprises the effector domain. In some embodiments, the split point amino acid corresponds to A406 or G550 with respect to a parental Casl2a protein amino acid sequence of SEQ ID NO: 1. In some embodiments, (a) the first effector subunit further comprises a first stabilization domain at its C-terminus and the second effector subunit further comprises a second stabilization domain at its N-terminus, wherein the first stabilization domain and the second stabilization domain are capable of interacting with one another to stabilize a complex comprising the first effector subunit and the second effector subunit in the sample; and/or (b) the third effector subunit further comprises a third stabilization domain at either terminus and (i) the second effector subunit further comprises a fourth stabilization domain at its C-terminus or (ii) the first effector subunit further comprises the fourth stabilization domain at its N-terminus, wherein the third stabilization domain and the fourth stabilization domain are capable of interacting with one another to stabilize a complex comprising the first effector subunit, the second effector subunit, and the third effector subunit in the sample. In some embodiments, the first stabilization domain and the second stabilization domain are derived from subunits of a heterodimerizing binding pair or a split-intein, and the third stabilization domain and the fourth stabilization domain are derived from subunits of a heterodimerizing binding pair or a split-intein and are orthogonal to the first stabilization domain and the second stabilization domain. In some embodiments, the heterodimerizing binding pair is a leucine zipper. In some embodiments, (a) the first stabilization domain and the second stabilization domain are derived from subunits of a leucine zipper and the third stabilization domain and the fourth stabilization domain are derived from subunits of a split-intein; or (b) the first stabilization domain and the second stabilization domain are derived from subunits of a split-intein and the third stabilization domain and the fourth stabilization domain are derived from subunits of a leucine zipper. In some embodiments, the leucine zipper subunits comprise RR12EE345L and EE12RR345L and/or the split-intein subunits comprise DnaE N-intein and C-intein.
[0016] In some embodiments, according to any of the systems described above, the system further comprises one or more additional crRNAs or nucleic acid encoding the one or more additional crRNAs, wherein Casl2a/crRNA complexes formed by association of the one or more additional crRNAs with (I) the Casl2a protein or variant thereof or (II) the two or more effector subunits are capable of generating one or more additional outputs to the sample comprising effector activity of the Casl2a/crRNA complexes at target sites directed by the one or more additional crRNAs. In some embodiments, the system comprises nucleic acid encoding a crRNA array comprising one or more of the first crRNA and the one or more additional crRNAs.
[0017] In some embodiments, according to any of the systems described above, the Casl2a protein or variant thereof is derived from a parental Casl2a protein from Lachnospiraceae bacterium comprising the amino acid sequence of SEQ ID NO: 1.
[0018] In some embodiments, according to any of the systems described above, the target nucleic acid is a target dsDNA, such as genomic DNA. In some embodiments, a protospacer in the target dsDNA targeted by the crRNA spacer has a 5' PAM selected from 5'-TTTG-3', 5'- TTTA-3', and 5'-TTTC-3'.
[0019] In some embodiments, according to any of the systems described above, the Casl2a protein or variant thereof is a nuclease-competent Cas protein comprising a Casl2a domain having some, the same, or greater nuclease activity as compared to a parental Cas 12a protein from which it is derived, and the first output comprises cleavage of the first target nucleic acid near the first target polynucleotide sequence. In some embodiments, the Cas 12a domain comprises the amino acid sequence of SEQ ID NO: 1.
[0020] In some embodiments, according to any of the systems described above, the Cas 12a protein or variant thereof is a fusion protein comprising an effector domain fused to a nuclease- deficient dCasl2a domain having reduced or eliminated nuclease activity as compared to a parental Cas 12a protein from which it is derived, and the first output is generated by activity of the effector domain. In some embodiments, the dCasl2a domain comprises a modification of amino acid D832 with respect to SEQ ID NO: 1 that reduces, substantially eliminates, or eliminates nuclease activity of the dCasl2a domain. In some embodiments, the dCasl2a domain comprises a D832A substitution with respect to SEQ ID NO: 1. In some embodiments, the dCasl2a domain comprises the amino acid sequence of SEQ ID NO: 2. In some embodiments, the effector domain comprises a polypeptide that can (i) cleave a nucleic acid, (ii) edit a nucleotide, (iii) activate transcription, (iv) repress transcription, (v) methylate a nucleic acid, and/or (vi) demethylate a nucleic acid. In some embodiments, the effector domain comprises a transcriptional activation domain, and the first output comprises transcriptional activation of a first gene targeted by the first crRNA. In some embodiments, the transcriptional activation domain is a tripartite VP64-p65-Rta (VPR) transcriptional activation domain or variant thereof. In some embodiments, targeting of the genomic DNA by the Casl2a system is capable of enhancing transcription of the mRNA.
[0021] In some embodiments, according to any of the systems described above, the sample is a cell or a composition comprising a cell, such as a mammalian cell. In some embodiments, the cell is a human cell.
[0022] In some embodiments, according to any of the systems described above, the inputs to the sample are selected from physical inputs, small molecule inputs, and biologic inputs. In some embodiments, (a) the physical inputs are selected from oxygen levels, pH, ion concentrations, temperature, light, mechanical force, ultrasound, shock wave, magnetic field, and electric field; (b) the small molecule inputs are selected from drugs, metabolites, and adjuvants; and/or (c) the biologic inputs are selected from polypeptides, polypeptide derivatives, and nucleic acids.
[0023] In another aspect, provided herein is an engineered cell prepared by introducing a system according to any of the embodiments described above into an input cell. In some embodiments, the engineered cell is capable of expressing a target gene, the first target nucleic acid comprises the target gene, and the first output of the system modulates the expression of the target gene in the engineered cell. In some embodiments, the input cell is a target cell, and one or more NOT inputs of the system capable of increasing the expression of Acr protein in the engineered cell are associated with off-target cells and not the target cell.
[0024] In some embodiments, according to any of the engineered cells described above, the target gene encodes a target receptor. In some embodiments, the input cell is a target immune cell, the target receptor is a chimeric antigen receptor (CAR) targeting a disease-associated cell, one or more inputs of the system are associated with the disease-associated cell, and the first output of the system increases the expression of the CAR in the engineered cell. In some embodiments, the engineered cell is a CAR T cell, a CAR NK cell, or a CAR macrophage cell. In some embodiments, the input cell is a target T cell, the target receptor is a T cell receptor (TCR) targeting a disease-associated cell, one or more inputs of the system are associated with the disease-associated cell, and the first output of the system increases the expression of the TCR in the engineered cell. In some embodiments, one or more NOT inputs of the system capable of increasing the expression of Acr protein in the engineered cell are associated with healthy cells and not the disease-associated cell.
[0025] In some embodiments, according to any of the engineered cells described above, the input cell is a target disease-associated cell, and one or more inputs of the system are associated with the disease-associated cell. In some embodiments, the first output of the system modulates the expression of the target gene in the engineered cell such that it is rendered benign. In some embodiments, the first output of the system modulates the expression of the target gene in the engineered cell such that it is killed. In some embodiments, the target disease-associated cell is a cancer cell, and one or more inputs to the cell are cancer-associated inputs characteristic of the cancer cell. In some embodiments, one or more NOT inputs of the system capable of increasing the expression of Acr protein in the engineered cell are associated with healthy cells and not the disease-associated cell.
[0026] In another aspect, provided herein is a method for conditionally generating an output to a sample, the method comprising providing to the sample a system according to any of the embodiments described above, thereby generating an output to the sample mediated by a split Casl2a/crRNA complex when all components of the split Casl2a/crRNA complex are expressed. In some embodiments, the output comprises targeting of a target nucleic acid by the split Casl2a/crRNA complex. In some embodiments, the Casl2a protein or variant thereof has a nucleic acid-editing effector activity and the output comprises editing of a target nucleic acid by the split Casl2a/crRNA complex. In some embodiments, the Casl2a protein or variant thereof is a dCasl2a fusion protein or variant thereof comprising an effector domain having a transcription-modulating effector activity and the output comprises modulation of transcription of a target nucleic acid by the split Casl2a/crRNA complex. In some embodiments, the Casl2a protein or variant thereof is a dCasl2a fusion protein or variant thereof comprising an effector domain having an epigenetic-editing effector activity and the output comprises editing of the epigenome of a target genomic DNA by the split Casl2a/crRNA complex.
[0027] In some embodiments, according to any of the methods for conditionally generating an output to a sample described above, the sample is a cell or a composition comprising a cell. In some embodiments, the cell is a mammalian cell. In some embodiments, the cell is a human cell.
[0028] In some embodiments, according to any of the methods for conditionally generating an output to a sample described above, the inputs to the sample are selected from physical inputs, small molecule inputs, and biologic inputs. In some embodiments, (a) the physical inputs are selected from oxygen levels, pH, ion concentrations, temperature, light, mechanical force, ultrasound, shock wave, magnetic field, and electric field; (b) the small molecule inputs are selected from drugs, metabolites, and adjuvants; and/or (c) the biologic inputs are selected from polypeptides, polypeptide derivatives, and nucleic acids.
[0029] In another aspect, provided herein is a method for treating a disease or condition associated with a target gene in a subject in need thereof, the method comprising: modifying a target cell in the subject to produce an engineered cell according to any of the embodiments described above, wherein the expression of the target gene in the engineered cell is modulated such that the disease or condition is treated.
[0030] In another aspect, provided herein is a method for treating a disease or condition associated with a disease-associated cell in a subject in need thereof, the method comprising: providing to the subject an engineered cell according to any of the embodiments described above, wherein the engineered cell is capable of mediating killing of the disease-associated cell to treat the disease or condition.
[0031] In another aspect, provided herein is a method for treating a disease or condition associated with a disease-associated cell in a subject in need thereof, the method comprising: modifying the disease-associated cell in the subject to produce an engineered cell according to any of the embodiments described above, wherein the expression of the target gene in the engineered cell is modulated such that the disease or condition is treated.
[0032] In another aspect, provided herein is a kit comprising one or more components of a system according to any of the embodiments described above.
[0033] The foregoing summary is illustrative only and is not intended to be in any way limiting. In addition to the illustrative embodiments and features described herein, further aspects, embodiments, objects and features of the disclosure will become fully apparent from the drawings and the detailed description and the claims.
BRIEF DESCRIPTION OF THE DRAWINGS [0034] FIG. 1A shows a schematic of the plasmids used for transient transfection for testing dCasl2a-VPR or dCasl2a-miniVPR. The TRE3G-GFP reporter assay used throughout this paper is shown on the bottom.
[0035] FIG. IB shows activation of TRE3G-GFP in HEK293T reporter cells using dCasl2a- VPR and dCasl2aminiVPR as measured by flow cytometry two days post- transfection. Casl2a plasmids were transiently transfected with a crRNA targeting the TetO repeats (crTet). Bars represent mean values and dots represent 2 biological replicates.
[0036] FIG. 2A shows the crystal structure of LbCasl2a in complex with its guide, showing the 5 selected split sites.
[0037] FIG. 2B shows a schematic of constructs used for testing split dCasl2a in mammalian cells. N-dCasl2a, N-terminal half; C-dCasl2a, C-terminal half; Zl, EE12RR345L; Z2, RR12EE345L; pA, polyA sequence. The dot in dCasl2a indicates the nuclease-deactivating mutation D832A.
[0038] FIG. 2C shows performance for GFP reporter activation using different pairs of split proteins at selected sites shown in FIG. 2A as measured by flow cytometry. Pairs of plasmids encoding the N- and C-term halves of dCasl2a-miniVPR split at selected sites with added leucine zippers were transiently transfected in HEK293T reporter cells with crTet and compared to the activity of the full length protein. The percentages of activities of each split compared to the full length protein are shown above the bars. Fold change calculated relative to nontargeting crLacZ control. Bars represent mean values and dots represent 3 biological replicates.
[0039] FIG. 2D shows activation of the TRE3G-GFP HEK293T reporter using the split dCasl2a with and without the leucine zipper, as measured by flow cytometry. Bars represent mean values and dots represent 3 biological replicates.
[0040] FIG. 3A shows possible explanations for intrinsic spontaneous pairing of the 406-split dCas 12a without the heterodimerizing leucine zipper. The first inset box on the crystal structure of dCasl2a highlights the bundling of α-helices in the helical domains of the N-term and C- term. The second inset box highlights potential scaffolding of the crRNA to bring the N-term and C-term halves together. The crRNA interacts with the major RNA binding pocket of the oligo binding domain (OBD) on the C-term half, as well as potential interaction between the negatively charged crRNA and positively charged K15, R18, and K20 in the Helical I domain of the N-term half.
[0041] FIG. 3B shows testing of three additional split sites identified for rapamycin-induced Casl2a dimerization for their ability to spontaneously dimerize using leucine zippers, as measured by flow cytometry two days post transfection. The percentages of activities of each split compared to the full length protein are shown above the bars. Fold change calculated relative to non-targeting crLacZ control. Bars represent mean values and dots represent 3 biological replicates.
[0042] FIG. 4 shows comparison of GFP reporter activation as measured by flow cytometry two days post transfection using the original dCasl2a-miniVPR with a single C term NLS compared to additional NLS at the N-term (right) or additional NLS at the C-term (left). An additional N-term NLS was added in an effort to ensure both halves were independently nuclear localized but surprisingly worsened performance and was not included in the final design. The constructs with an additional C-term NLS performed similarly, but this extra C-term NLS was included to ensure maximal nuclear localization. Bars represent mean values and dots represent 3 biological replicates.
[0043] FIG. 5 A shows performance of 2-input AND gate using split dCasl2a for activating TRE3G-GFP or endogenous CXCR4 in HEK293T cells, as measured by flow cytometry two days post-transfection. Left, truth table; right, bars represent mean values and dots represent n = three biological replicates. Listed fold changes calculated relative to non-targeting crLacZ. [0044] FIG. 5B shows representative flow cytometry histograms of the full length vs 406-split dCasl2aminiVPR activating TRE3G-GFP reporter (left) or endogenous CXCR4 (right) in HEK293Ts. Cells were analyzed 2 days post-transfection by flow cytometry. For CXCR4, cell were stained with APC-conjugated CXCR4 antibody prior to analysis.
[0045] FIG. 6 shows testing crRNAs for CXCR4 activation. Plasmids encoding different crRNAs and the full dCasl2a-miniVPR were transiently transfected into HEK293T cells and CXCR4 protein expression was measured by flow cytometry of anti-CXCR4-APC stained cells 2 days post transfection. Fold changes were calculated relative to non-targeting crLacZ. Given the low activation of single guides, the best two guides (#1 and 3, indicated in red) were used together in following experiments. Bars represent mean values and dots represent biological replicates.
[0046] FIG. 7A shows a schematic of cumate and Doxycycline (Dox)-controlled AND gate. Addition of cumate and Dox induce expression of N- and C-terms, respectively, in a stable HEK293T cell line. Reconstitution of a functional dCasl2a results in activation of a P2-GFP reporter. CuO, cumate operator; TetO, Tetracycline operator; CymR, CuO binding repressor; rtTA, reverse Tetracycline-controlled transactivator. Full constructs used in Table 2.
[0047] FIG. 7B shows dose-response curve of cumate driving expression of N-term half of dCasl2a with a bicistronic BFP. Stable cumate/Dox inducible HEK293T cells were stimulated with indicated cumate concentration for 24 hours before analysis by flow cytometry. Dots represent average of 3 biological replicates, and error bars indicate standard deviation.
[0048] FIG. 7C shows dose-response curve of Dox driving expression of C-term half of dCasl2a with fused mCherry. Stable cumate/dox inducible HEK293T cells were stimulated with indicated Dox concentration for 24 hours before analysis by flow cytometry. Dots represent average of 3 biological replicates, and error bars indicate standard deviation. [0049] FIG. 7D shows activation of the P2-GFP reporter by split dCasl2a-miniVPR at varying levels of cumate and Dox. The cells were analyzed by flow cytometry two days post stimulation/transfection· Heatmap values represent mean of two biological replicates.
[0050] FIG. 8A shows testing of crRNAs for IFNg activation. Plasmids encoding different crRNAs and the full dCasl2a-miniVPR were transiently transfected into HEK293T cells and IFNg activation was measured by ELISA two days post transfection. The best guide (#5) was used in following experiments. Bars represent mean values and dots represent biological replicates. Fold change labeled above bar calculated relative to nontargeting (NT) guide crLacZ or lower detection limit of ELISA (4pg/mL) when appropriate.
[0051] FIG. 8B shows testing crRNAs for IL2 activation. Pairs of crRNAs for IL2 were tested after minimal activation was observed using single guides (left), and certain pairs showed strong synergistic effects (right). The best pair (#2 and 6, indicated in red) was used in following experiments. Bars represent mean values and dots represent n = 2 biological replicates. Fold change labeled above bar calculated relative to non-targeting (NT) guide crLacZ or lower detection limit of ELISA (4pg/mL) when appropriate.
[0052] FIG. 8C shows the relative positioning of different crRNAs targeting the IL2 locus, with the most effective pair (#2 and 6) highlighted.
[0053] FIG. 8D shows simultaneous activation of endogenous IFNg and IL2 using the transiently transfected split dCasl2a-miniVPR in HEK293T cells, as measured by ELISA two days post transfection. Bars represent mean values and dots represent three biological replicates. Dotted line represents the lower detection limit of ELISA kit (4 pg/mL).
[0054] FIG. 9A shows a schematic of constructs used for testing split catalytically active Casl2a in mammalian cells.
[0055] FIG. 9B shows split Casl2a-mediated gene editing at the genomic CXCR4 locus of HEK293T cells as measured by the T7E1 assay three days post transfection. The uncut band is 960 bp, and the expected cut bands are 748 bp and 212 bp. A representative gel is shown from two independent replicates. NT, non-targeting crLacZ.
[0056] FIG. 10A shows a schematic of Pol II-expressed crRNA processing by the Casl2a. CAG, a strong constitutive Pol II promoter.
[0057] FIG. 10B shows comparison of the performance of Pol III U6- or Pol II CAG-driven crRNA constructs for activating IFNg as measured by ELISA two days post transfection with full length dCasl2a-miniVPR in HEK293T cells. The design with the crRNA in the 3’ UTR of a longer luciferase (Flue) Pol II transcript activated more efficiently than the crRNA-only Pol II transcript, and thus this architecture was used moving forward. crLacZ, nontargeting crRNA; Flue, 1.6kb long firefly luciferase coding sequence. Bars represent mean values and dots represent 3 biological replicates.
[0058] FIG. IOC shows a schematic of Pol II-expressed “2X” crRNA array processed by Casl2a to generate higher levels of crRNA from a single mRNA transcript. TRE3G, Dox- inducible Pol II promoter.
[0059] FIG. 10D shows comparison of performance of U6- or TRE3G-driven crRNA variants for activating IFNg measured by ELISA two days post transfection with full length dCasl2a- miniVPR in HEK293T cells. Minimal Dox-inducible crlFNγ activation (DRspacer-DR) was initially observed. Duplicating the crRNA (DR-spacer-DR-spacer-DR) significantly enhanced activation, suggesting the expression level of the crRNA may be a limiting factor. crLacZ, the non-targeting crRNA. Bars represent mean values and dots represent 3 biological replicates. [0060] FIG. 10E shows a 3-input “A AND B AND C” logic gate using the split dCasl2a system and Dox-inducible crRNA expression for activating IFNg in HEK293T cells, as measured by ELISA three days post transfection. Bars represent mean values and dots represent three biological replicates.
[0061] FIG. 11A shows constructs used to express the 3-way split dCasl2a activator and a constitutive U6 crRNA. The N- and C- inteins were introduced into the linker between dCas 12a and miniVPR such that the C-intein was followed by a serine for efficient protein splicing. [0062] FIG. 11B shows performance for HEK293T GFP reporter activation using the full dCasl2a-miniVPR and 3 -way split dCasl2a-miniVPR as measured by flow cytometry two days post transfection. The percentage indicates performance compared to the full-length protein. Fold change calculated relative to non-targeting crLacZ. Bars represent mean values and dots represent 3 biological replicates. Representative histograms are included on the right. The reduction in activation may be due to efficiency of simultaneous zipper binding and intein splicing, or due to the reduction in transfection efficiency with the increasing number of plasmids and inability to gate for all components.
[0063] FIG. l lC shows a 3-input “A AND B AND C” AND logic gate using a 3-way split dCasl2a system for activating TRE3G-GFP in HEK293T cells, as measured by flow cytometry two days post transfection. Bars represent mean values and dots represent three biological replicates.
[0064] FIG. 1 ID shows representative histograms of the 3 -input A and B and C AND gate in FIG. llC as measured by flow cytometry two days post transfection in TRE3G-GFP reporter HEK293T cells.
[0065] FIG. 12A shows constructs used to express the full dCasl2a activator and the anti- CRISPR AcrVAl.
[0066] FIG. 12B shows performance for HEK293T GFP reporter activation using the full dCasl2a-miniVPR with and without AcrVAl, as measured by flow cytometry two days post transfection. Fold change calculated relative to non-targeting crLacZ. Bars represent mean values and dots represent 3 biological replicates. Representative histograms are included on the right.
[0067] FIG. 12C shows a 3-input “A AND B AND (NOT C)” logic gate using the split dCasl2a and AcrVAl for activating TRE3G-GFP in HEK293T cells, as measured by flow cytometry two days post transfection. Bars represent mean values and dots represent three biological replicates.
[0068] FIG. 12D shows representative histograms of the 3-input A and B and (NOT C) AND gate in FIG. 12C as measured by flow cytometry two days post transfection in TRE3G-GFP reporter HEK293T cells.
[0069] FIG. 13 shows a 4-input “A AND B and C AND D” logic gate using the 3-way split dCasl2a system and dox-inducible crRNA expression for activating IFNγ as measured by EFISA three days post transfection. Bars represent mean values and dots represent two biological replicates. The dotted line shows the detection limit of the EFISA assay.
[0070] FIG. 14A shows a schematic showing the application of split dCasl2a for detecting two tumor-relevant signals in breast cancer cells and activating a therapeutic response. HREs, hypoxia-responsive elements. TFs, transcription factors driving expression of the RRM2 promoter (RRM2p).
[0071] FIG. 14B shows performance of RRM2p for expressing N-term dCasl2a in MDA-MB- 231 breast cancer cells and MCF10A normal breast cells as measured by flow cytometry. Top, construct used for lentiviral transduction. Fefit, representative histogram for MDA-MB-231 and MCF 10A cells. Right, bars represent mean values and dots represent three biological replicates. EFS, Elongation Factor short, compact promoter.
[0072] FIG. 14C shows performance of the synthetic hypoxia promoter for driving expression of C-term dCasl2a in MDA-MB-231 breast cancer cells treated with DMSO or hypoxia agonist DMOG. Top, construct used for lentiviral transduction. Feft, representative histogram for the presence and absence of DMOG. Right, bars represent mean values and dots represent three biological replicates. [0073] FIG. 14D shows performance of the 2-input dCasl2a AND gate in MDA-MB-231 cells for activating endogenous IFNg as measured by ELISA two days post treatment. To control expression of the C-term half, cells were transduced with the hypoxia-inducible construct and treated with or without DMOG. To control expression of the N-term half, cells were transduced with or without the RRM2p-driven N term. Bars represent mean values and dots represent three biological replicates.
[0074] FIG. 14E shows performance of the dCas 12a AND gate in MCF 10A cells for activating endogenous IFNg as measured by ELISA two days post treatment. The dotted line shows the detection limit of the assay. Bars represent mean values and dots represent three biological replicates.
[0075] FIG. 14F shows performance of the 2-input dCasl2a AND gate in MDA-MB-231 breast cancer cells for activating endogenous IL2 measured as by ELISA two days post treatment. Bars represent mean values and dots represent three biological replicates.
[0076] FIG. 14G shows performance of the 3-input dCasl2a gate in MDA-MB-231 cells for activating endogenous IFNg as measured by ELISA two days post treatment. The addition of Dox controls expression of crlFNγ. Bars represent mean values and dots represent three biological replicates.
[0077] FIG. 14H shows performance of the 3-input dCasl2a gate in MDA-MB-231 cells for activating endogenous IFNγ as measured by ELISA two days post treatment. The addition of Dox controls expression of AcrVAl. Bars represent mean values and dots represent three biological replicates.
[0078] FIG. 15 shows potential applications of Casl2a synthetic circuits for cell engineering and cell therapy. The circuit can integrate information about multiple biological signals to control expression of split Casl2a, effector, crRNA, and optional anti-CRISPR. The desired inputs can control these components on the transcriptional level (promoters) or the protein level (protein localization or degradation). The circuit can then drive a variety of outputs, including activation/repression of target genes or permanent modification of genomic DNA.
DETAILED DESCRIPTION OF THE DISCLOSURE [0079] The present invention relates to split CRISPR-Cas effector protein Casl2a and variants thereof, such as nuclease-deficient Casl2a (dCasl2a) and fusions thereof with an effector domain, such as a transcriptional activation domain, wherein the split Cas 12a or variant thereof comprises a plurality of effector subunits comprising fragments of a Cas 12a protein or variant thereof, wherein the plurality of effector subunits are capable of forming a split Cas 12a complex (such as by spontaneous assembly) retaining at least some of an effector activity of the Casl2a protein or variant thereof. Applicant developed a split Casl2a system capable of spontaneously reassembling and demonstrated that it allows for construction of two, three, and four-input AND gates in mammalian cells. Anti-CRISPR (Acr) proteins were also incorporated to further control circuit activity. In one example, by coupling a split Casl2a system to cancer relevant inputs, the potential therapeutic utility of such systems to activate anti-tumoral programs specifically in cancer cells was demonstrated.
[0080] The invention further relates to CRISPR-Cas systems comprising such split Casl2a proteins that allow for detection and integration of multiple biological inputs to effect one or more biological outputs, as well as polynucleotide sequences encoding such split Casl2a proteins or systems and vectors or vector systems comprising such and delivery systems comprising such. The invention further relates to cells or cell lines or organisms comprising such split Casl2a proteins, CRISPR-Cas systems, polynucleotide sequences, vectors, vector systems, and/or delivery systems. The invention further relates to medical and non-medical uses of such split Casl2a proteins, CRISPR-Cas systems, polynucleotide sequences, vectors, vector systems, delivery systems, cells, cell lines, and/or organisms, such as for targeted genome regulation. The split Casl2a protein may be derived from a wild type Casl2a protein or a mutated (such as comprising point mutation(s) and/or truncations) Casl2a protein. In one aspect, embodiments disclosed herein are directed to split engineered Casl2a proteins that comprise at least one modification compared to an unmodified Casl2a protein that reduces or eliminates its endonuclease activity.
[0081] Further aspects of the present disclosure are directed to methods of modulating gene expression from a target nucleic acid in a cell, or otherwise modifying the target nucleic acid. The methods include providing to the cell (a) effector subunits of a split Casl2a protein or variant thereof, or nucleic acid encoding the effector subunits; and (b) a crRNA comprising a spacer sequence complementary to a target polynucleotide sequence in the target nucleic acid, or nucleic acid encoding the crRNA. In some embodiments, the method comprises providing to the cell nucleic acid encoding the effector subunits and nucleic acid encoding the crRNA. In some embodiments, the nucleic acids encoding the effector subunits and the crRNA are present on a single vector or on separate vectors, such as engineered DNA plasmid vectors or viral vectors. The vector is then used to deliver the nucleic acids encoding the effector subunits and the crRNA into desired cells or tissues. Once delivered into a cell, the effector subunits of the split Casl2a protein or variant thereof and the crRNA may be expressed depending on the state of certain inputs to the cell that modulate their expression. The crRNA comprises a portion that is complementary to a sequence of a target site and can guide a split Casl2a complex to the target site. In this manner, expression or modification of the target nucleic acid sequence is modulated depending on the location of the target site and the state of each of the inputs that modulate expression of the split Casl2a system components.
[0082] In the following detailed description, reference is made to the accompanying drawings, which form a part hereof. In the drawings, similar symbols generally identify similar components, unless context dictates otherwise. The illustrative alternatives described in the detailed description, drawings, and claims are not meant to be limiting. Other alternatives may be used and other changes may be made without departing from the spirit or scope of the subject matter presented here. It will be readily understood that the aspects, as generally described herein, and illustrated in the Figures, can be arranged, substituted, combined, and designed in a wide variety of different configurations, all of which are explicitly contemplated and make part of this application.
[0083] Unless otherwise defined, all terms of art, notations, and other scientific terms or terminology used herein are intended to have the meanings commonly understood by those of skill in the art to which this application pertains. In some cases, terms with commonly understood meanings are defined herein for clarity and/or for ready reference, and the inclusion of such definitions herein should not necessarily be construed to represent a substantial difference over what is generally understood in the art. Many of the techniques and procedures described or referenced herein are well understood and commonly employed using conventional methodology by those skilled in the art. Definitions of common terms and techniques in molecular biology may be found in Molecular Cloning: A Laboratory Manual, 2nd edition (1989) (Sambrook, Fritsch, and Maniatis); Molecular Cloning: A Laboratory Manual, 4th edition (2012) (Green and Sambrook); Current Protocols in Molecular Biology (1987) (F.M. Ausubel et al. eds.); the series Methods in Enzymology (Academic Press, Inc.): PCR 2: A Practical Approach (1995) (M.J. MacPherson, B.D. Hames, and G.R. Taylor eds.): Antibodies, A Laboraotry Manual (1988) (Harlow and Lane, eds.): Antibodies A Laboraotry Manual, 2nd edition 2013 (E.A. Greenfield ed.); Animal Cell Culture (1987) (R.I. Freshney, ed.); Benjamin Lewin, Genes IX, published by Jones and Bartlet, 2008 (ISBN 0763752223); Kendrew et al. (eds.), The Encyclopedia of Molecular Biology, published by Blackwell Science Ltd., 1994 (ISBN 0632021829); Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995 (ISBN 9780471185710); Singleton et al., Dictionary of Microbiology and Molecular Biology 2nd ed., J. Wiley & Sons (New York, N.Y. 1994), March, Advanced Organic Chemistry Reactions, Mechanisms and Structure 4th ed., John Wiley & Sons (New York, N.Y. 1992); and Marten H. Hofker and Jan van Deursen, Transgenic Mouse Methods and Protocols, 2nd edition (2011).
Definitions
[0084] As used herein, the singular forms “a,” “an,” and “the” include both singular and plural referents unless the context clearly dictates otherwise.
[0085] The term “optional” or “optionally” means that the subsequent described event, circumstance or substituent may or may not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not.
[0086] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the disclosure. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the disclosure, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure.
[0087] Certain ranges are presented herein with numerical values being preceded by the term “about.” The term “about” is used herein to provide literal support for the exact number that it precedes, as well as a number that is near to or approximately the number that the term precedes, such as variations of +/- 10% or less, +/- 1-5% or less, +/- 1% or less, and +/- 0.1% or less from the specified value. In determining whether a number is near to or approximately a specifically recited number, the near or approximating unrecited number may be a number which, in the context in which it is presented, provides the substantial equivalent of the specifically recited number.
[0088] The terms “subject” and “individual” are used interchangeably herein to refer to a vertebrate, preferably a mammal, more preferably a human. In some cases, a subject is a patient. Mammals include, but are not limited to, murines, simians, humans, farm animals, sport animals, and pets. Tissues, cells and their progeny of a biological entity obtained in vivo or cultured in vitro are also encompassed.
[0089] It is appreciated that certain features of the disclosure, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the disclosure, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable sub combination. All combinations of the embodiments pertaining to the disclosure are specifically embraced by the present disclosure and are disclosed herein just as if each and every combination was individually and explicitly disclosed. In addition, all sub-combinations of the various embodiments and elements thereof are also specifically embraced by the present disclosure and are disclosed herein just as if each and every such sub- combination was individually and explicitly disclosed herein.
[0090] All publications, published patent documents, and patent applications cited herein are hereby incorporated by reference to the same extent as though each individual publication, published patent document, or patent application was specifically and individually indicated as being incorporated by reference.
CAS12 NUCLEIC ACID-TARGETING SYSTEMS
[0091] Class 2 CRISPR-Cas systems endow microbes with diverse mechanisms for adaptive immunity. CRISPR systems are generally divided into two classes, with class 1 systems using a complex of multiple Cas proteins to degrade foreign nucleic acids, and class 2 systems using a single, generally larger, Cas protein for the same purpose. Class 1 is divided into types I, III, and IV, and class 2 is divided into types II, V, and VI. The RNA-guided, nucleic acid-targeting CRISPR systems described herein are based on Cas 12a (such as Cas 12a from Lachnospiraceae bacterium), a class 2 Cas effector protein classified as Type V. In particular, the systems include a split Cas 12a protein or variant thereof comprising two or more effector subunits comprising fragments of a Cas 12a protein or variant thereof, or nucleic acid encoding the two or more effector subunits, wherein the two or more effector subunits are capable of spontaneously assembling to form a split Cas 12a complex retaining at least some of the effector activity of the Cas 12a protein or variant thereof.
[0092] Embodiments of the present disclosure are directed to split Casl2a-based regulation of gene expression, as well as nucleic acid engineering (including genome editing and epigenetic modification). The small size of Cas 12a proteins allows Cas 12a proteins and effector domain fusions thereof to be paired with a CRISPR array encoding multiple crRNAs while remaining under the packaging size limit of the versatile adeno-associated virus (AAV) delivery vehicle for primary cell and in vivo delivery. CRISPR-Cas 12a and engineered variants such as dCasl2a allow for flexible nucleic acid engineering, regulation of gene expression, and therapeutics, expanding the genome editing and regulation toolbox.
Split Casl2a
[0093] Split Casl2a proteins or variants thereof comprising fragments of a Casl2a protein or variant thereof having an effector activity are contemplated by the present disclosure. In some embodiments, the split Casl2a protein or variant thereof comprises two or more effector subunits comprising fragments of the Casl2a protein or variant thereof, wherein the two or more effector subunits are capable of spontaneously assembling to form a split Casl2a complex retaining at least some of the effector activity of the Casl2a protein or variant thereof. In some embodiments, the two or more effector subunits comprise a first effector subunit and a second effector subunit. In some embodiments, the first effector subunit comprises an N-terminal fragment of the Casl2a protein or variant thereof up to and including a split point amino acid, and the second effector subunit comprises a C-terminal fragment of the Casl2a protein or variant thereof following the split point amino acid. In some embodiments, the split point amino acid corresponds to A406 or G550 with respect to a parental Casl2a protein amino acid sequence of SEQ ID NO: 1. In some embodiments, the first effector subunit further comprises a first stabilization domain at its C-terminus and the second effector subunit further comprises a second stabilization domain at its N-terminus, wherein the first stabilization domain and the second stabilization domain are capable of interacting with one another to stabilize a complex comprising the first effector subunit and the second effector subunit. In some embodiments, the Casl2a protein or variant thereof comprises a nuclear localization signal (NLS). In some embodiments, the NLS comprises one or more repeats of a conventional NLS. In some embodiments, the Casl2a protein or variant thereof comprises a C-terminal NLS. In some embodiments, one or more of the two or more effector subunits that do not include the NLS of the Casl2a protein or variant thereof further comprise an additional NLS. In some embodiments, none of the two or more effector subunits that do not include the NLS of the Casl2a protein or variant thereof comprise an additional NLS.
[0094] In some embodiments, according to any of the split Casl2a proteins or variants thereof described herein, the Casl2a protein or variant thereof is a fusion protein comprising an effector domain fused to a Casl2a domain, and the split Casl2a protein or variant thereof comprises a first effector subunit comprising the Casl2a domain and a second effector subunit comprising the effector domain. In some embodiments, the first effector subunit further comprises a first stabilization domain at either terminus and the second effector subunit further comprises a second stabilization domain at either terminus, wherein the first stabilization domain and the second stabilization domain are capable of interacting with one another to stabilize a complex comprising the first effector subunit and the second effector subunit. In some embodiments, the first effector subunit further comprises the first stabilization domain at its N-terminus. In some embodiments, the first effector subunit further comprises the first stabilization domain at its C-terminus. In some embodiments, the second effector subunit further comprises the second stabilization domain at its N-terminus. In some embodiments, the second effector subunit further comprises the second stabilization domain at its C-terminus. [0095] In some embodiments, according to any of the split Casl2a proteins or variants thereof described herein comprising a first stabilization domain and a second stabilization domain, the first stabilization domain and the second stabilization domain are derived from subunits of a heterodimerizing binding pair or a split-intein. In some embodiments, the first stabilization domain and the second stabilization domain are derived from subunits of a heterodimerizing binding pair. In some embodiments, the heterodimerizing binding pair is a coiled-coil (CC) heterodimer. In some embodiments, the CC heterodimer is a leucine zipper. In some embodiments, the leucine zipper subunits comprise RR12EE345L and EE12RR345L. In some embodiments, RR12EE345L comprises the amino acid sequence encoded by SEQ ID NO: 6. In some embodiments, EE12RR345L comprises the amino acid sequence encoded by SEQ ID NO: 5. In some embodiments, the CC heterodimer is a CC peptide heterodimer ( see Lebar, T., et al. (2020). Nature Chemical Biology, 1-7). In some embodiments, the first stabilization domain and the second stabilization domain are derived from subunits of a split-intein. In some embodiments, the split-intein subunits comprise DnaE N-intein and C-intein. In some embodiments, DnaE N-intein comprises the amino acid sequence encoded by SEQ ID NO: 7. In some embodiments, DnaE C-intein comprises the amino acid sequence encoded by SEQ ID NO: 8. In some embodiments, the split-intein is gp41-l, gp41-8, NrdJ-1, or IMPDH-1 ( see Dassa, B., et al. (2009). Nucleic acids research, 57(8), 2560-2573). In some embodiments, the split-intein comprises split portions of the Mxe GyrA intein (see Fine, E. J., et al. (2015). Scientific reports, 5, 10777).
[0096] In some embodiments, according to any of the split Casl2a proteins or variants thereof described herein, the two or more effector subunits comprise a first effector subunit, a second effector subunit, and a third effector subunit. In some embodiments, the Casl2a protein or variant thereof is a fusion protein comprising an effector domain fused to a Casl2a domain, and the first effector subunit comprises an N-terminal fragment of the Casl2a domain up to and including a split point amino acid, the second effector subunit comprises a C-terminal fragment of the Cast 2a domain following the split point amino acid; and the third effector subunit comprises the effector domain. In some embodiments, the split point amino acid corresponds to A406 or G550 with respect to a parental Casl2a protein amino acid sequence of SEQ ID NO: 1. In some embodiments, the first effector subunit further comprises a first stabilization domain at its C-terminus and the second effector subunit further comprises a second stabilization domain at its N-terminus, wherein the first stabilization domain and the second stabilization domain are capable of interacting with one another to stabilize a complex comprising the first effector subunit and the second effector subunit; and/or the third effector subunit further comprises a third stabilization domain at either terminus and (i) the second effector subunit further comprises a fourth stabilization domain at its C-terminus or (ii) the first effector subunit further comprises the fourth stabilization domain at its N-terminus, wherein the third stabilization domain and the fourth stabilization domain are capable of interacting with one another to stabilize a complex comprising the first effector subunit, the second effector subunit, and the third effector subunit.
[0097] In some embodiments, according to any of the split Casl2a proteins or variants thereof described herein comprising a third stabilization domain and a fourth stabilization domain, the third stabilization domain and the fourth stabilization domain are derived from subunits of a heterodimerizing binding pair or a split-intein and are orthogonal to the first stabilization domain and the second stabilization domain. In some embodiments, the first stabilization domain and the second stabilization domain are derived from subunits of a heterodimerizing binding pair and the third stabilization domain and the fourth stabilization domain are derived from subunits of a split-intein; or the first stabilization domain and the second stabilization domain are derived from subunits of a split-intein and the third stabilization domain and the fourth stabilization domain are derived from subunits of a heterodimerizing binding pair. In some embodiments, the heterodimerizing binding pair is a coiled-coil (CC) heterodimer. In some embodiments, the CC heterodimer is a leucine zipper. In some embodiments, the leucine zipper subunits comprise RR12EE345L and EE12RR345L. In some embodiments, RR12EE345L comprises the amino acid sequence encoded by SEQ ID NO: 6. In some embodiments, EE12RR345L comprises the amino acid sequence encoded by SEQ ID NO: 5. In some embodiments, the CC heterodimer is a CC peptide heterodimer ( see Lebar, T., et al. (2020). Nature Chemical Biology, 1-7). In some embodiments, the split-intein subunits comprise DnaE N-intein and C-intein. In some embodiments, DnaE N-intein comprises the amino acid sequence encoded by SEQ ID NO: 7. In some embodiments, DnaE C-intein comprises the amino acid sequence encoded by SEQ ID NO: 8. In some embodiments, the split-intein is gp41 -
1, gp41-8, NrdJ-1, or IMPDH-1 ( see Dassa, B., et al. (2009). Nucleic acids research, 57(8), 2560-2573). In some embodiments, the split-intein comprises split portions of the Mxe GyrA intein (see Fine, E. J., et al. (2015). Scientific reports, 5, 10777).
[0098] In some embodiments, according to any of the split Casl2a proteins or variants thereof described herein, the Casl2a protein or variant thereof is derived from a parental Casl2a protein from Lachnospiraceae bacterium. In some embodiments, the parental Casl2a protein from Lachnospiraceae bacterium comprises the amino acid sequence of SEQ ID NO: 1. In some embodiments, the Casl2a protein or variant thereof is a nuclease-competent Cas protein comprising a Cas 12a domain having some, the same, or greater nuclease activity as compared to a parental Cas 12a protein from which it is derived. In some embodiments, the Cas 12a domain comprises the amino acid sequence of SEQ ID NO: 1 or a variant thereof having at least about 80% sequence identity to the amino acid sequence of SEQ ID NO: 1.
[0099] In some embodiments, according to any of the split Cas 12a proteins or variants thereof described herein, the Cas 12a protein or variant thereof is a fusion protein comprising an effector domain fused to a nuclease-deficient dCasl2a domain having reduced or eliminated nuclease activity as compared to a parental Cas 12a protein from which it is derived, and the first output is generated by activity of the effector domain. In some embodiments, the dCasl2a domain comprises a modification of amino acid D832 with respect to SEQ ID NO: 1 that reduces, substantially eliminates, or eliminates nuclease activity of the dCasl2a domain. In some embodiments, according to any of the split Cas 12a proteins or variants thereof described herein, the dCasl2a domain comprises a D832A substitution with respect to SEQ ID NO: 1. In some embodiments, according to any of the split Cas 12a proteins or variants thereof described herein, the dCasl2a domain comprises the amino acid sequence of SEQ ID NO: 2 or a variant thereof having at least about 80% sequence identity to the amino acid sequence of SEQ ID NO:
2.
[0100] In some embodiments, according to any of the split Cas 12a proteins or variants thereof comprising an effector domain fused to a nuclease-deficient dCasl2a domain described herein, the effector domain comprises a polypeptide that can (i) cleave a nucleic acid, (ii) edit a nucleotide, (iii) activate transcription, (iv) repress transcription, (v) methylate a nucleic acid, and/or (vi) demethylate a nucleic acid. In some embodiments, the effector domain comprises a transcriptional activation domain. In some embodiments, the transcriptional activation domain is a tripartite VP64-p65-Rta (VPR) transcriptional activation domain or variant thereof. In some embodiments, the VPR domain comprises the amino acid sequence encoded by SEQ ID NO: 4 or a variant thereof having at least about 80% sequence identity to the amino acid sequence encoded by SEQ ID NO: 4. In some embodiments, the effector domain comprises a transcriptional repression domain. dCasl2a
[0101] Modifications to Casl2a proteins are contemplated by the present disclosure. In some embodiments, a Casl2a protein is altered or otherwise modified to inactivate its nuclease activity. Such alteration or modification includes altering one or more amino acids to inactivate the nuclease activity or the nuclease domain, as well as removing the polypeptide sequence or polypeptide sequences exhibiting nuclease activity, i.e., the nuclease domain, such that the polypeptide sequence or polypeptide sequences exhibiting nuclease activity, i.e. nuclease domain, are absent from the Casl2a protein. Thus, in some embodiments, a nuclease-null Casl2a protein (dCasl2a) includes polypeptide sequences modified to inactivate nuclease activity or removal of a polypeptide sequence or sequences to inactivate nuclease activity. The dCasl2a protein retains the ability to bind to target nucleic acid even though the nuclease activity has been inactivated. Accordingly, the dCasl2a protein includes the polypeptide sequence or sequences required for nucleic acid binding.
[0102] In some embodiments, the dCasl2a protein includes homologs and orthologs thereof which retain the ability of the protein to be guided by RNA to bind to target nucleic acid. In some embodiments, the dCasl2a protein comprises the amino acid sequence of SEQ ID NO: 2 or a variant thereof having at least 80% (such as at least about 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater) sequence identity to the amino acid sequence of SEQ ID NO: 2. dCas!2a Fusion Protein
[0103] In some embodiments, the dCasl2a protein is attached to, bound to, or fused with an effector domain, such as a transcriptional regulatory domain or an epigenetic modifying domain.
[0104] In some embodiments, provided herein is a dCasl2a fusion protein comprising a dCasl2a protein fused to an effector domain. In some embodiments, the effector domain is fused to the C-terminus of the dCasl2a protein. In some embodiments, the effector domain is fused to the N-terminus of the dCasl2a protein. In some embodiments, the effector domain comprises a subcellular localization signal. In some embodiments, the subcellular localization signals is an organelle localization signal, such as a nuclear localization signal (NLS), nuclear export signal (NES), or mitochondrial localization signal. In some embodiments, the effector domain comprises a polypeptide that can (i) cleave a nucleic acid, (ii) edit a nucleotide, (iii) activate transcription, (iv) repress transcription, (v) methylate a nucleic acid, and/or (vi) demethylate a nucleic acid.
Nucleic Acid
[0105] Also provided herein is isolated nucleic acid encoding a split Casl2a protein subunit according to any of the embodiments described herein. In some embodiments, the isolated nucleic acid is part of a vector (such as a plasmid or viral vector), and can be operably linked to a promoter, such as an inducible promoter. Nucleotide sequences encoding split Casl2a protein subunits can be generated based on the amino acid sequences provided herein. In some embodiments, the isolated nucleic acid encoding a split Casl2a protein subunit comprises a protein coding sequence that is codon optimized for expression in a eukaryotic cell (e.g., a mammalian cell, such as a human cell). For example, nucleic acid molecules encoding the split Casl2a protein subunits disclosed herein can be designed to have codons that are preferentially used by a particular organism of interest. In some embodiments, the nucleotide sequence encoding split Casl2a subunits is optimized for expression in human cells.
Guide RNA
[0106] In general, bacterial and archaeal CRISPR-Cas systems rely on short guide RNAs in complex with Cas proteins to direct degradation of complementary sequences present within invading foreign nucleic acid. See Deltcheva, E. et al. Nature 471, 602-607 (2011); Gasiunas, G., et al. Proceedings of the National Academy of Sciences of the United States of America 109, E2579-2586 (2012); Jinek, M. et al. Science 337, 816-821 (2012); Sapranauskas, R. et al. Nucleic acids research 39, 9275- 9282 (2011); and Bhaya, D., Davison, M. & Barrangou, R. Annual review of genetics 45, 273-297 (2011). Unlike Cas9, Casl2a is a single crRNA-guided endonuclease. Cas 12a processes crRNA arrays independent of tracrRNA, and Casl2a-crRNA complexes alone are sufficient to cleave target DNA molecules, without the requirement for any additional RNA species ( see Zetsche, B., et al. (2015). Cell, 163(3), 759-771). Accordingly, the terms “guide RNA,” “gRNA,” and “crRNA” are used interchangeably herein in the context of Cas 12a systems and derivatives thereof, such as the split Cas 12a systems described herein.
[0107] Embodiments ofthe present disclosure are directed to the use of a split CRISPR Casl2a system and, in particular, a crRNA that includes a spacer sequence. The term spacer sequence is understood by those of skill in the art and may include any polynucleotide having sufficient complementarity with a target nucleic acid sequence to hybridize with the target nucleic acid sequence and direct sequence-specific binding of a CRISPR complex to the target sequence. A mature crRNA of a split Casl2a system may begin with an appropriate direct repeat followed by the spacer sequence. In some embodiments, the crRNA is between about 20 and about 100 nucleotides in length. In some embodiments, the spacer sequence is between about 10 and about 50 nucleotides in length.
[0108] In some embodiments, methods of making a crRNA as described herein are employed, such as by expressing constructs encoding the crRNA using promoters and terminators and optionally other genetic elements known in the art for such purposes. In some embodiments, nucleic acid encoding a crRNA is provided, wherein the nucleic acid comprises an inducible promoter operably linked to a polynucleotide sequence encoding the crRNA. In some embodiments, the inducible promoter is a polymerase II promoter. In some embodiments, nucleic acid encoding a crRNA is provided, wherein the nucleic acid comprises a constitutive promoter operably linked to a polynucleotide sequence encoding the crRNA. In some embodiments, the constitutive promoter is a polymerase III promoter (e.g., U6 or HI). In some embodiments, nucleic acid encoding a crRNA is provided, wherein the nucleic acid encodes an array of crRNAs capable of being processed by a split Casl2a complex into individual mature crRNAs.
[0109] In some embodiments, the crRNA may be delivered directly to a cell as a native species by methods known to those of skill in the art, including injection or lipofection, or delivered indirectly to a cell as transcribed from a cognate DNA, with the cognate DNA introduced into the cell through electroporation, transient or stable transfection (including lipofection), or viral transduction.
Systems
[0110] In some embodiments, the compositions herein comprise, or the methods herein comprise delivering, one or more components of a split Casl2a nucleic acid-targeting system (also referred to herein as a “split Casl2a system”). In general, “split Casl2a system” as used in the present application refers collectively to elements involved in the activity of a split Casl2a protein in association with a compatible crRNA to be targeted to a particular nucleic acid sequence (also referred to herein as a “target sequence,” or “protospacer-like sequence” in the context of an endogenous CRISPR-Cas system) as guided by the spacer sequence of the crRNA, and can include nucleic acid encoding the split Casl2a protein and/or the crRNA. In some embodiments, the split Casl2a protein and/or crRNA are derived from a particular organism comprising an endogenous Casl2a system. In general, a split Casl2a system is characterized by elements that promote the formation of a nucleic acid-targeting complex including a split Casl2a protein and a crRNA at the site of a target sequence, which can be present in a DNA molecule. In the context of a split Casl2a system, “target sequence” refers to a sequence to which a guide sequence (also referred to herein as a “spacer” or “spacer sequence”) in a crRNA of the system is designed to have complementarity, where hybridization between the target sequence and the crRNA allows for localization of the split Casl2a protein to the target sequence. Full complementarity is not necessarily required, provided there is sufficient complementarity to allow for hybridization between the target sequence and the crRNA. In some embodiments, a target sequence is located in the nucleus or cytoplasm of a cell. In some embodiments, the target sequence may be within an organelle of a eukaryotic cell, for example, a mitochondrion or a chloroplast.
[0111] In some embodiments, provided herein is a split Casl2a system for conditionally generating an output to a sample comprising the system, the system comprising: (a) (i) a Casl2a protein or variant thereof having an effector activity, or nucleic acid encoding the Casl2a protein or variant thereof; or (ii) two or more effector subunits comprising fragments of the Casl2a protein or variant thereof, or nucleic acid encoding the two or more effector subunits, wherein the two or more effector subunits are capable of spontaneously assembling to form a split Casl2a complex in the sample, wherein the split Casl2a complex retains at least some of the effector activity of the Casl2a protein or variant thereof; and (b) a first CRISPR RNA (crRNA) or nucleic acid encoding the first crRNA, wherein the first crRNA comprises a first spacer having sufficient complementary to a first target polynucleotide sequence in a first target nucleic acid in the sample such that it can hybridize to the first target nucleic acid, wherein a Casl2a/crRNA complex formed in the sample by association of the first crRNA with (I) the Casl2a protein or variant thereof or (II) the two or more effector subunits is capable of generating a first output to the sample comprising the effector activity of the Casl2a/crRNA complex at the first target nucleic acid, and wherein the expression of a first component of the Casl2a/crRNA complex is under the control of a first input to the sample and the expression of a second component of the Casl2a/crRNA complex is under the control of a second input to the sample. In some embodiments, the system further comprises nucleic acid comprising a NOT inducible promoter operably linked to a polynucleotide sequence encoding an anti- CRISPR (Acr) protein capable of inhibiting activity of the split Casl2a complex in the sample, wherein a NOT input to the sample is capable of increasing the expression of Acr protein from the nucleic acid in the sample such that activity of split Casl2a complex in the sample is inhibited. A “NOT inducible promoter” as used herein refers to an inducible promoter operably linked to a polynucleotide sequence encoding an Acr protein capable of inhibiting activity of a split Casl2a complex. A “NOT input” as used herein refers to an input having a cognate inducible promoter, wherein the cognate inducible promoter is operably linked to a polynucleotide sequence encoding an Acr protein capable of inhibiting activity of a split Casl2a complex.
[0112] In some embodiments, according to any of the split Casl2a systems described herein comprising nucleic acid comprising a polynucleotide sequence encoding an Acr protein, the Acr protein is AcrVAl or a variant thereof. In some embodiments, the AcrVAl or variant thereof comprises the amino acid sequence encoded by SEQ ID NO: 9 or a variant thereof having at least 80% sequence identity to the amino acid sequence encoded by SEQ ID NO: 9. [0113] In some embodiments, according to any of the split Casl2a systems described herein, a split Casl2a complex that retains at least some of the effector activity of a Casl2a protein or variant thereof retains at least about 5% (such as at least about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or greater) of the effector activity of the Casl2a protein or variant thereof.
[0114] In some embodiments, according to any of the split Casl2a systems described herein, the crRNA comprises a spacer sequence selected from the polynucleotide sequence of any one of SEQ ID NOs: 15-43.
[0115] In some embodiments, according to any of the split Casl2a systems described herein comprising nucleic acid comprising an inducible promoter, the inducible promoter is selected from an RRM2 promoter, a hypoxia-inducible promoter, a TRE3G promoter, a CMV5-CuO promoter, and a P2 inducible promoter. In some embodiments, the RRM2 promoter comprises the polynucleotide sequence of SEQ ID NO: 10. In some embodiments, the hypoxia-inducible promoter comprises the polynucleotide sequence of SEQ ID NO: 11. In some embodiments, the TRE3G promoter comprises the polynucleotide sequence of SEQ ID NO: 12. In some embodiments, the CMV5-CuO promoter comprises the polynucleotide sequence of SEQ ID NO: 13. In some embodiments, the P2 inducible promoter comprises the polynucleotide sequence of SEQ ID NO: 14. In some embodiments, where the split Casl2a system comprises nucleic acid comprising a plurality of inducible promoters, each of the plurality of inducible promoters operably linked to a polynucleotide sequence encoding an effector subunit are different. In some embodiments, where the split Casl2a system comprises nucleic acid comprising a plurality of inducible promoters, each of the plurality of inducible promoters operably linked to a polynucleotide sequence encoding a crRNA are different. In some embodiments, where the split Casl2a system comprises nucleic acid comprising a plurality of inducible promoters, at least some (such as at least about any of 2, 3, 4, or more) of the plurality of inducible promoters operably linked to a polynucleotide sequence encoding a crRNA are the same. In some embodiments, where the split Casl2a system comprises nucleic acid comprising a plurality of inducible promoters, all of the plurality of inducible promoters operably linked to a polynucleotide sequence encoding a crRNA are the same. In some embodiments, where the split Casl2a system comprises nucleic acid comprising a plurality of inducible promoters, each of the plurality of inducible promoters are different.
[0116] In some embodiments, according to any of the split Casl2a systems described herein, the system is configured such that all components of the Casl2a/crRNA complex are present or capable of being expressed in the sample when the first input and the second input each have a desired state. The state of an input to a sample is a quantitative measure of some property of the input, wherein the input property is indicative of whether, and to what extent, the expression of a gene under the control of a cognate inducible promoter will be modified. For example, where the input is doxycycline, having a cognate inducible promoter including a TRE3G promoter, the state of the input includes the concentration of doxycycline in the sample. A desired state for an input may include ranges of the measure, e.g., greater than 0.2 M doxycycline. In some embodiments, the system is configured such that all components of the Casl2a/crRNA complex are present or capable of being expressed in the sample when the first input is present and the second input is present.
[0117] In some embodiments, according to any of the split Casl2a systems described herein, the system comprises nucleic acid encoding the Casl2a protein or variant thereof, wherein the nucleic acid encoding the Cas 12a protein or variant thereof comprises a first inducible promoter operably linked to a polynucleotide sequence encoding the Cas 12a protein or variant thereof, and the first input is capable of modulating the expression of the Cas 12a protein or variant thereof in the sample; and nucleic acid encoding the first crRNA, wherein the nucleic acid encoding the first crRNA comprises a second inducible promoter operably linked to a polynucleotide sequence encoding the first crRNA, and the second input is capable of modulating the expression of the first crRNA in the sample. In some embodiments, modulating the expression of each of the Casl2a/crRNA complex components comprises increasing their expression. [0118] In some embodiments, according to any of the split Casl2a systems described herein, the system comprises nucleic acid encoding a first effector subunit and a second effector subunit of the two or more effector subunits, wherein the nucleic acid comprises a first inducible promoter operably linked to a polynucleotide sequence encoding the first effector subunit and a second inducible promoter operably linked to a polynucleotide sequence encoding the second effector subunit, and the first input is capable of modulating the expression of the first effector subunit in the sample and the second input is capable of modulating the expression of the second effector subunit in the sample; or nucleic acid encoding a first effector subunit of the two or more effector subunits, wherein the nucleic acid comprises a first inducible promoter operably linked to a polynucleotide sequence encoding the first effector subunit; and nucleic acid encoding the first crRNA, wherein the nucleic acid comprises a second inducible promoter operably linked to a polynucleotide sequence encoding the first crRNA, and the first input is capable of modulating the expression of the first effector subunit in the sample and the second input is capable of modulating the expression of the first crRNA in the sample. In some embodiments, modulating the expression of each of the Casl2a/crRNA complex components comprises increasing their expression.
[0119] In some embodiments, according to any of the split Casl2a systems described herein, the expression of a third component of the Casl2a/crRNA complex is under the control of a third input to the sample, and the system is configured such that all components of the Casl2a/crRNA complex are present or capable of being expressed in the sample when the first input, the second input, and the third input each have a desired state. In some embodiments, the system is configured such that all components of the Casl2a/crRNA complex are present or capable of being expressed in the sample when the first input is present, the second input is present, and the third input is present. In some embodiments, the system comprises nucleic acid encoding a first effector subunit, a second effector subunit, and a third effector subunit of the two or more effector subunits, wherein the nucleic acid comprises a first inducible promoter operably linked to a polynucleotide sequence encoding the first effector subunit, a second inducible promoter operably linked to a polynucleotide sequence encoding the second effector subunit, and a third inducible promoter operably linked to a polynucleotide sequence encoding the third effector subunit, and the first input is capable of modulating the expression of the first effector subunit in the sample, the second input is capable of modulating the expression of the second effector subunit in the sample, and the third input is capable of modulating the expression of the third effector subunit in the sample; or nucleic acid encoding a first effector subunit and a second effector subunit of the two or more effector subunits, wherein the nucleic acid comprises a first inducible promoter operably linked to a polynucleotide sequence encoding the first effector subunit and a second inducible promoter operably linked to a polynucleotide sequence encoding the second effector subunit; and nucleic acid encoding the first crRNA, wherein the nucleic acid comprises a third inducible promoter operably linked to a polynucleotide sequence encoding the first crRNA, and the first input is capable of modulating the expression of the first effector subunit in the sample, the second input is capable of modulating the expression of the second effector subunit in the sample, and the third input is capable of modulating the expression of the first crRNA in the sample. In some embodiments, modulating the expression of each of the Casl2a/crRNA complex components comprises increasing their expression.
[0120] In some embodiments, according to any of the split Casl2a systems described herein, the expression of a third component of the Casl2a/crRNA complex is under the control of a third input to the sample and the expression of a fourth component of the Casl2a/crRNA complex is under the control of a fourth input to the sample, and the system is configured such that all components of the Casl2a/crRNA complex are present or capable of being expressed in the sample when the first input, the second input, the third input, and the fourth input each have a desired state. In some embodiments, the system is configured such that all components of the Casl2a/crRNA complex are present or capable of being expressed when the first input is present, the second input is present, the third input is present, and the fourth input is present. In some embodiments, the system comprises nucleic acid encoding a first effector subunit, a second effector subunit, and a third effector subunit of the two or more effector subunits, wherein the nucleic acid comprises a first inducible promoter operably linked to a polynucleotide sequence encoding the first effector subunit, a second inducible promoter operably linked to a polynucleotide sequence encoding the second effector subunit, and a third inducible promoter operably linked to a polynucleotide sequence encoding the third effector subunit, and the first input is capable of modulating the expression of the first effector subunit in the sample, the second input is capable of modulating the expression of the second effector subunit in the sample, and the third input is capable of modulating the expression of the third effector subunit in the sample; and nucleic acid encoding the first crRNA, wherein the nucleic acid comprises a fourth inducible promoter operably linked to a polynucleotide sequence encoding the first crRNA, and the fourth input is capable of modulating the expression of the first crRNA. In some embodiments, modulating the expression of each of the Casl2a/crRNA complex components comprises increasing their expression.
[0121] In some embodiments, according to any of the split Casl2a systems described herein, the two or more effector subunits comprise a first effector subunit and a second effector subunit, wherein the first effector subunit comprises an N-terminal fragment of the Casl2a protein or variant thereof up to and including a split point amino acid, and the second effector subunit comprises a C-terminal fragment of the Casl2a protein or variant thereof following the split point amino acid. In some embodiments, the split point amino acid corresponds to A406 or G550 with respect to a parental Cas 12a protein amino acid sequence of SEQ ID NO: 1. In some embodiments, the first effector subunit further comprises a first stabilization domain at its C- terminus and the second effector subunit further comprises a second stabilization domain at its N-terminus, wherein the first stabilization domain and the second stabilization domain are capable of interacting with one another to stabilize a complex comprising the first effector subunit and the second effector subunit in the sample.
[0122] In some embodiments, according to any of the split Cas 12a systems described herein, the two or more effector subunits comprise a first effector subunit and a second effector subunit, wherein the Cas 12a protein or variant thereof is a fusion protein comprising an effector domain fused to a Cas 12a domain, and the first effector subunit comprises the Cas 12a domain and the second effector subunit comprises the effector domain. In some embodiments, the first effector subunit further comprises a first stabilization domain at either terminus and the second effector subunit further comprises a second stabilization domain at either terminus, wherein the first stabilization domain and the second stabilization domain are capable of interacting with one another to stabilize a complex comprising the first effector subunit and the second effector subunit in the sample.
[0123] In some embodiments, according to any of the split Cas 12a systems described herein comprising a first stabilization domain and a second stabilization domain, the first stabilization domain and the second stabilization domain are derived from subunits of a heterodimerizing binding pair or a split-intein. In some embodiments, the first stabilization domain and the second stabilization domain are derived from subunits of a heterodimerizing binding pair. In some embodiments, the heterodimerizing binding pair is a coiled-coil (CC) heterodimer. In some embodiments, the CC heterodimer is a leucine zipper. In some embodiments, the leucine zipper subunits comprise RR12EE345L and EE12RR345L. In some embodiments, RR12EE345L comprises the amino acid sequence encoded by SEQ ID NO: 6. In some embodiments, EE12RR345L comprises the amino acid sequence encoded by SEQ ID NO: 5. In some embodiments, the CC heterodimer is a CC peptide heterodimer ( see Lebar, T., et al. (2020). Nature Chemical Biology, 1-7). In some embodiments, the first stabilization domain and the second stabilization domain are derived from subunits of a split-intein. In some embodiments, the split-intein subunits comprise DnaEN-intein and C-intein. In some embodiments, DnaEN- intein comprises the amino acid sequence encoded by SEQ ID NO: 7. In some embodiments, DnaE C-intein comprises the amino acid sequence encoded by SEQ ID NO: 8. In some embodiments, the split-intein is gp41-l, gp41-8, NrdJ-1, or IMPDH-1 ( see Dassa, B., et al. (2009). Nucleic acids research, 37(8), 2560-2573). In some embodiments, the split-intein comprises split portions of the Mxe GyrA intein (see Fine, E. J., et al. (2015). Scientific reports, 5, 10777).
[0124] In some embodiments, according to any of the split Casl2a systems described herein, the two or more effector subunits comprise a first effector subunit, a second effector subunit, and a third effector subunit. In some embodiments, the Casl2a protein or variant thereof is a fusion protein comprising an effector domain fused to a Casl2a domain, and the first effector subunit comprises an N-terminal fragment of the Casl2a domain up to and including a split point amino acid, the second effector subunit comprises a C-terminal fragment of the Casl2a domain following the split point amino acid; and the third effector subunit comprises the effector domain. In some embodiments, the split point amino acid corresponds to A406 or G550 with respect to a parental Cas 12a protein amino acid sequence of SEQ ID NO: 1. In some embodiments, the first effector subunit further comprises a first stabilization domain at its C- terminus and the second effector subunit further comprises a second stabilization domain at its N-terminus, wherein the first stabilization domain and the second stabilization domain are capable of interacting with one another to stabilize a complex comprising the first effector subunit and the second effector subunit in the sample; and/or the third effector subunit further comprises a third stabilization domain at either terminus and (i) the second effector subunit further comprises a fourth stabilization domain at its C-terminus or (ii) the first effector subunit further comprises the fourth stabilization domain at its N-terminus, wherein the third stabilization domain and the fourth stabilization domain are capable of interacting with one another to stabilize a complex comprising the first effector subunit, the second effector subunit, and the third effector subunit in the sample.
[0125] In some embodiments, according to any of the split Cas 12a systems described herein comprising a third stabilization domain and a fourth stabilization domain, the third stabilization domain and the fourth stabilization domain are derived from subunits of a heterodimerizing binding pair or a split-intein and are orthogonal to the first stabilization domain and the second stabilization domain. In some embodiments, the first stabilization domain and the second stabilization domain are derived from subunits of a heterodimerizing binding pair and the third stabilization domain and the fourth stabilization domain are derived from subunits of a split- intein; or the first stabilization domain and the second stabilization domain are derived from subunits of a split-intein and the third stabilization domain and the fourth stabilization domain are derived from subunits of a heterodimerizing binding pair. In some embodiments, the heterodimerizing binding pair is a coiled-coil (CC) heterodimer. In some embodiments, the CC heterodimer is a leucine zipper. In some embodiments, the leucine zipper subunits comprise RR12EE345L and EE12RR345L. In some embodiments, RR12EE345L comprises the amino acid sequence encoded by SEQ ID NO: 6. In some embodiments, EE12RR345L comprises the amino acid sequence encoded by SEQ ID NO: 5. In some embodiments, the CC heterodimer is a CC peptide heterodimer ( see Lebar, T., et al. (2020). Nature Chemical Biology, 1-7). In some embodiments, the split-intein subunits comprise DnaE N-intein and C-intein. In some embodiments, DnaE N-intein comprises the amino acid sequence encoded by SEQ ID NO: 7. In some embodiments, DnaE C-intein comprises the amino acid sequence encoded by SEQ ID NO: 8. In some embodiments, the split-intein is gp41-l, gp41-8, NrdJ-1, or IMPDH-1 ( see Dassa, B., et al. (2009). Nucleic acids research, 37(8), 2560-2573). In some embodiments, the split-intein comprises split portions of the Mxe GyrA intein (see Fine, E. J., et al. (2015). Scientific reports, 5, 10777).
[0126] In some embodiments, according to any of the split Casl2a systems described herein, the system further comprises one or more additional crRNAs or nucleic acid encoding the one or more additional crRNAs, wherein Casl2a/crRNA complexes formed by association of the one or more additional crRNAs with (I) the Casl2a protein or variant thereof or (II) the two or more effector subunits are capable of generating one or more additional outputs to the sample comprising effector activity of the Casl2a/crRNA complexes at target sites directed by the one or more additional crRNAs. In some embodiments, the system comprises nucleic acid encoding a crRNA array comprising one or more of the first crRNA and the one or more additional crRNAs.
[0127] In some embodiments, according to any of the split Casl2a systems described herein, the Casl2a protein or variant thereof is derived from a parental Casl2a protein from Lachnospiraceae bacterium. In some embodiments, the parental Casl2a protein from Lachnospiraceae bacterium comprises the amino acid sequence of SEQ ID NO: 1. In some embodiments, the target nucleic acid is a target dsDNA. In some embodiments, a protospacer in the target dsDNA targeted by the crRNA spacer has a 5' PAM selected from 5'-TTTG-3', 5'- TTTA-3', and 5'-TTTC-3'.
[0128] In some embodiments, according to any of the split Casl2a systems described herein, the Casl2a protein or variant thereof is a nuclease-competent Cas protein comprising a Casl2a domain having some, the same, or greater nuclease activity as compared to a parental Cas 12a protein from which it is derived, and the first output comprises cleavage (e.g., double-stranded cleavage) of the first target nucleic acid near the first target polynucleotide sequence. For example, in some embodiments, a split Casl2a/crRNA complex cleaves the first target nucleic acid distal from the PAM, with cleavage after the 18th base on the non-targeted (+) strand and after the 23rd base on the targeted (-) strand. In some embodiments, the Cas 12a domain comprises the amino acid sequence of SEQ ID NO: 1.
[0129] In some embodiments, according to any of the split Cas 12a systems described herein, the Cas 12a protein or variant thereof is a fusion protein comprising an effector domain fused to a nuclease-deficient dCasl2a domain having reduced or eliminated nuclease activity as compared to a parental Cas 12a protein from which it is derived, and the first output is generated by activity of the effector domain. In some embodiments, the dCasl2a domain comprises a modification of amino acid D832 with respect to SEQ ID NO: 1 that reduces, substantially eliminates, or eliminates nuclease activity of the dCasl2a domain. In some embodiments, the dCasl2a domain comprises a D832A substitution with respect to SEQ ID NO: 1. In some embodiments, the dCasl2a domain comprises the amino acid sequence of SEQ ID NO: 2. In some embodiments, the effector domain comprises a polypeptide that can (i) cleave a nucleic acid, (ii) edit a nucleotide, (iii) activate transcription, (iv) repress transcription, (v) methylate a nucleic acid, and/or (vi) demethylate a nucleic acid. In some embodiments, the effector domain comprises a transcriptional activation domain, and the first output comprises transcriptional activation of a first gene targeted by the first crRNA. In some embodiments, the transcriptional activation domain is a tripartite VP64-p65-Rta (VPR) transcriptional activation domain or variant thereof.
[0130] In some embodiments, according to any of the split Cas 12a systems described herein, the sample is a cell or a composition comprising a cell. For example, in some embodiments, the sample is a biological sample comprising a cell, including, without limitation, a tissue, fluid, or other composition in an organism. In some embodiments, the inputs to the sample are selected from physical inputs, small molecule inputs, and biologic inputs. In some embodiments, the physical inputs are selected from oxygen levels, pH, ion concentrations, temperature, light, mechanical force, ultrasound, shock wave, magnetic field, and electric field; the small molecule inputs are selected from drugs, metabolites, and adjuvants; and/or the biologic inputs are selected from polypeptides, polypeptide derivatives, and nucleic acids. [0131] In some embodiments, according to any of the split Casl2a systems described herein employing a TRE3G promoter, the system comprises a TRE3G-specific crRNA targeting the TRE3G promoter, or nucleic acid encoding the TRE3G-specific crRNA, the Casl2a protein or variant thereof is a fusion protein comprising an effector domain fused to a Casl2a domain, wherein the effector domain comprises a transcriptional activation domain, such that association of the TRE3G-specific crRNA with the split Casl2a complex allows for increasing the expression of a gene operably linked to the TRE3G promoter, and an output of the system comprises increased expression of the gene. In some embodiments, the TRE3G-specific crRNA comprises a spacer comprising the polynucleotide sequence of SEQ ID NO: 15.
[0132] In some embodiments, according to any of the split Casl2a systems described herein employing a TRE3G promoter, the system comprises nucleic acid comprising the TRE3G promoter operably linked to a gene encoding a component of the split Casl2a system, such as an effector subunit or crRNA, and an input of the system is concentration of doxycycline in the sample, wherein a concentration of doxycycline in the sample within a certain range (including, e.g., above a certain threshold) allows for increased expression of the split Casl2a system component. In some embodiments, a concentration of doxycycline in the sample within the certain range allows for sufficient expression of the split Casl2a system component such that it can participate in the formation of a split Casl2a/crRNA complex capable of mediating an output of the system. In some embodiments, the sample is a composition comprising a cell comprising the split Casl2a system.
[0133] In some embodiments, according to any of the split Casl2a systems described herein employing an RRM2 promoter, the system comprises nucleic acid comprising the RRM2 promoter operably linked to a gene encoding a component of the split Casl2a system, such as an effector subunit or crRNA, and an input of the system is expression of one or more transcription factors capable of driving the expression of the RRM2 promoter in the sample, wherein expression of the one or more transcription factors in the sample within a certain range (including, e.g., above a certain threshold) allows for increased expression of the split Casl2a system component. In some embodiments, expression of the one or more transcription factors in the sample within the certain range allows for sufficient expression of the split Casl2a system component such that it can participate in the formation of a split Casl2a/crRNA complex capable of mediating an output of the system. In some embodiments, the sample is a cell, such as a cancer cell, wherein the cancer cell is characterized by increased expression of at least one of the one or more transcription factors capable of driving the expression of the RRM2 promoter. In some embodiments, the cancer cell is a breast cancer cell.
[0134] In some embodiments, according to any of the split Casl2a systems described herein employing a hypoxia-inducible promoter, the system comprises nucleic acid comprising the hypoxia-inducible promoter operably linked to a gene encoding a component of the split Casl2a system, such as an effector subunit or crRNA, and an input of the system is expression of one or more hypoxia-responsive elements (HREs) capable of driving the expression of the hypoxia-inducible promoter in the sample, wherein expression of the one or more HREs in the sample within a certain range (including, e.g., above a certain threshold) allows for increased expression of the split Casl2a system component. In some embodiments, expression of the one or more HREs in the sample within the certain range allows for sufficient expression of the split Casl2a system component such that it can participate in the formation of a split Casl2a/crRNA complex capable of mediating an output of the system. In some embodiments, the sample is a cell, such as a cancer cell, wherein the cancer cell is characterized by hypoxia leading to increased expression of at least one of the one or more HREs capable of driving the expression of the hypoxia-inducible promoter. In some embodiments, the cancer cell is a breast cancer cell. In some embodiments, the HREs include, without limitation, stabilized cellular HIFlα.
[0135] In some embodiments, according to any of the split Casl2a systems described herein employing a CMV5-CuO promoter, the system comprises nucleic acid comprising the CMV5- CuO promoter operably linked to a gene encoding a component of the split Casl2a system, such as an effector subunit or crRNA, and an input of the system is concentration of cumate in the sample, wherein a concentration of cumate in the sample within a certain range (including, e.g., above a certain threshold) allows for increased expression of the split Casl2a system component. In some embodiments, a concentration of cumate in the sample within the certain range allows for sufficient expression of the split Casl2a system component such that it can participate in the formation of a split Casl2a/crRNA complex capable of mediating an output of the system. In some embodiments, the sample is a composition comprising a cell comprising the split Casl2a system.
[0136] In some embodiments, according to any of the split Casl2a systems described herein employing a P2 promoter, the system comprises a P2-specific crRNA targeting the P2 promoter, or nucleic acid encoding the P2-specific crRNA, the Casl2a protein or variant thereof is a fusion protein comprising an effector domain fused to a Casl2a domain, wherein the effector domain comprises a transcriptional activation domain, such that association of the P2-specific crRNA with the split Casl2a complex allows for increasing the expression of a gene operably linked to the P2 promoter, and an output of the system comprises increased expression of the gene. In some embodiments, the P2-specific crRNA comprises a spacer comprising the polynucleotide sequence of SEQ ID NO: 17.
[0137] In some embodiments, according to any of the split Casl2a systems described herein, the system comprises a CXCR4-specific crRNA targeting a CXCR4 gene, or nucleic acid encoding the CXCR4-specific crRNA. In some embodiments, the CXCR4-specific crRNA comprises a spacer comprising a polynucleotide sequence selected from any one of SEQ ID NOs: 18-23. In some embodiments, the Casl2a protein or variant thereof is a fusion protein comprising an effector domain fused to a Casl2a domain, wherein the effector domain comprises a transcriptional activation domain, such that association of the CXCR4-specific crRNA with the split Cas 12a complex allows for increasing the expression of the CXCR4 gene, and an output of the system comprises increased expression of the CXCR4 gene. In some embodiments, the CXCR4-specific crRNA comprises a spacer comprising a polynucleotide sequence selected from any one of SEQ ID NOs: 18-22. In some embodiments, the Casl2a protein or variant thereof is a nuclease-competent Cas 12a protein or variant thereof, such that association of the CXCR4-specific crRNA with the split Cas 12a complex allows for cleavage of the CXCR4 gene, and an output of the system comprises cleavage of the CXCR4 gene. In some embodiments, the CXCR4-specific crRNA comprises a spacer comprising the polynucleotide sequence of SEQ ID NO: 23.
[0138] In some embodiments, according to any of the split Cas 12a systems described herein, the system comprises an IFNγ-specific crRNA targeting an IFNg gene, or nucleic acid encoding the IFNγ-specific crRNA. In some embodiments, the IFNγ-specific crRNA comprises a spacer comprising a polynucleotide sequence selected from any one of SEQ ID NOs: 24-29. In some embodiments, the Cas 12a protein or variant thereof is a fusion protein comprising an effector domain fused to a Cas 12a domain, wherein the effector domain comprises a transcriptional activation domain, such that association of the IFNγ-specific crRNA with the split Cas 12a complex allows for increasing the expression of the IFNg gene, and an output of the system comprises increased expression of the IFNg gene. [0139] In some embodiments, according to any of the split Cas 12a systems described herein, the system comprises an interleukin 2 (IL2)-specific crRNA targeting an IL2 gene, or nucleic acid encoding the IL2-specific crRNA. In some embodiments, the IL2-specific crRNA comprises a spacer comprising a polynucleotide sequence selected from any one of SEQ ID NOs: 30-43. In some embodiments, the Casl2a protein or variant thereof is a fusion protein comprising an effector domain fused to a Casl2a domain, wherein the effector domain comprises a transcriptional activation domain, such that association of the IL2-specific crRNA with the split Casl2a complex allows for increasing the expression of the IL2 gene, and an output of the system comprises increased expression of the IL2 gene. In some embodiments, the system comprises a plurality of IL2-specific crRNAs targeting the IL2 gene, or nucleic acid encoding the plurality of IL2-specific crRNAs. For example, in some embodiments, the system comprises a first crRNA targeting the IL2 gene comprising a first spacer comprising the polynucleotide sequence of SEQ ID NO: 31, or nucleic acid encoding the first crRNA, and a second crRNA targeting the IL2 gene comprising a second spacer comprising the polynucleotide sequence of SEQ ID NO: 35, or nucleic acid encoding the second crRNA. [0140] In some embodiments, according to any of the split Casl2a systems described herein, the sample comprises a cancer cell comprising the system, and one or more outputs of the system comprise increased expression in the cancer cell of one or more immunostimulatory cytokines, including, without limitation, IFNg and IL2. In some embodiments, the system comprises nucleic acid comprising an RRM2 promoter operably linked to a gene encoding a first component of the split Casl2a system, such as an effector subunit or crRNA, and an input of the system is expression of one or more transcription factors capable of driving the expression of the RRM2 promoter in the cancer cell, wherein expression of the one or more transcription factors in the cancer cell within a certain range (including, e.g., above a certain threshold) allows for increased expression of the first component, and the cancer cell is characterized by increased expression of at least one of the one or more transcription factors capable of driving the expression of the RRM2 promoter. In some embodiments, expression of the one or more transcription factors in the cancer cell within the certain range allows for sufficient expression of the first component such that it can participate in the formation of a split Casl2a/crRNA complex capable of mediating a first output of the system, such as increased expression in the cancer cell of one or more immunostimulatory cytokines, including, without limitation, IFNg and IL2. In some embodiments, the system comprises nucleic acid comprising a hypoxia-inducible promoter operably linked to a gene encoding a second component of the split Cast 2a system, such as an effector subunit or crRNA, and an input of the system is expression of one or more hypoxia-responsive elements (HREs) capable of driving the expression of the hypoxia-inducible promoter in the sample, wherein expression of the one or more HREs in the sample within a certain range (including, e.g., above a certain threshold) allows for increased expression of the second component, wherein the cancer cell is characterized by hypoxia leading to increased expression of at least one of the one or more HREs capable of driving the expression of the hypoxia-inducible promoter. In some embodiments, expression of the one or more HREs in the sample within the certain range allows for sufficient expression of the second component such that it can participate in the formation of a split Casl2a/crRNA complex capable of mediating a second output of the system, such as increased expression in the cancer cell of one or more immunostimulatory cytokines, including, without limitation, IFNg and IL2. In some embodiments, the HREs include, without limitation, stabilized cellular HIFlα. In some embodiments, the system comprises the nucleic acid comprising an RRM2 promoter operably linked to a gene encoding the first component and the nucleic acid comprising a hypoxia-inducible promoter operably linked to a gene encoding the second component, wherein the first and second outputs comprise increased expression of IFNγ and IL2. In some embodiments, the Casl2a protein or variant thereof is a fusion protein comprising an effector domain fused to a Casl2a domain, wherein the effector domain comprises a transcriptional activation domain. In some embodiments, the system comprises an IFNγ-specific crRNA targeting an IFNg gene, or nucleic acid encoding the IFNγ-specific crRNA. In some embodiments, the IFNγ-specific crRNA comprises a spacer comprising a polynucleotide sequence selected from any one of SEQ ID NOs: 24-29. In some embodiments, the system comprises an interleukin 2 (IL2)-specific crRNA targeting an IL2 gene, or nucleic acid encoding the IL2-specific crRNA. In some embodiments, the IL2-specific crRNA comprises a spacer comprising a polynucleotide sequence selected from any one of SEQ ID NOs: 30-43. In some embodiments, the system comprises a first crRNA targeting the IL2 gene comprising a first spacer comprising the polynucleotide sequence of SEQ ID NO: 31 , or nucleic acid encoding the first crRNA, and a second crRNA targeting the IL2 gene comprising a second spacer comprising the polynucleotide sequence of SEQ ID NO: 35, or nucleic acid encoding the second crRNA. In some embodiments, the cancer cell is a breast cancer cell.
Nucleic Acids
[0141] An aspect of the disclosure is one or more nucleic acids that encode one or more components of a split Cast 2a system as described herein.
[0142] Thus, in some embodiments, provided herein is one or more nucleic acids encoding one or more of: (a) (i) a Casl2a protein or variant thereof having an effector activity; or (ii) two or more effector subunits comprising fragments of the Casl2a protein or variant thereof, wherein the two or more effector subunits are capable of spontaneously assembling to form a split Cas 12a complex, wherein the split Cas 12a complex retains at least some of the effector activity of the Cas 12a protein or variant thereof; and (b) a first crRNA, wherein the first crRNA comprises a first spacer having sufficient complementary to a first target polynucleotide sequence in a first target nucleic acid such that it can hybridize to the first target nucleic acid, wherein a Casl2a/crRNA complex formed by association of the first crRNA with (I) the Cas 12a protein or variant thereof or (II) the two or more effector subunits is capable of being directed to the first target nucleic acid to mediate the effector activity of Cas 12a protein or variant thereof.
[0143] The terms “nucleic acid “ and “polynucleotide” are used interchangeably herein, and refer to both RNA and DNA molecules, including nucleic acid molecules comprising cDNA, genomic DNA, and/or synthetic DNA, and DNA or RNA molecules containing nucleic acid analogs. A nucleic acid can be double-stranded or single-stranded (for example, a sense strand or an antisense strand). A nucleic acid may contain unconventional or modified nucleotides. The terms “polynucleotide sequence” and “nucleic acid sequence” as used herein interchangeably refer to the sequence of a nucleic acid molecule. The nomenclature for nucleotide bases set forth in 37 CFR §1.822 is used herein.
[0144] Nucleic acids of the invention include multicistronic nucleic acids, wherein two or more coding sequences are separated by sequences encoding an IRES (internal ribosome entry site) or a self-cleaving polypeptide sequence, such as a 2A sequence, providing for expression of each protein encoded by the two or more coding sequences separately, or for the immediate cleavage into two separate proteins upon expression. Examples of 2A sequences include T2A, P2A, E2A, and F2A.
[0145] In some embodiments, the nucleic acid is operably linked to a heterologous nucleic acid sequence, such as, for example a structural gene that encodes a protein of interest or a regulatory sequence (e.g., a promoter sequence). In some embodiments, the nucleic acid is an expression cassette or a vector. In some embodiments, the vector is a lentiviral vector, an adeno virus vector, an adeno-associated virus vector, or a retroviral vector.
[0146] Some embodiments disclosed herein relate to vectors or expression cassettes including a nucleic acid molecule as disclosed herein. An expression cassettes is a construct of genetic material that contains coding sequences and enough regulatory information to direct proper transcription and/or translation of the coding sequences in a recipient cell, in vivo and/or ex vivo. The expression cassette may be inserted into a vector for targeting to a desired host cell. As such, the term “expression cassette” may be used interchangeably with the term “expression construct.”
[0147] Also provided herein are vectors, plasmids or viruses containing one or more of the nucleic acid molecules encoding any of the split Casl2a system components disclosed herein. The nucleic acid molecules described herein can be contained within a vector that is capable of directing their expression in, for example, a cell that has been transduced with the vector. Suitable vectors for use in eukaryotic cells are known in the art and are commercially available or readily prepared by a skilled artisan. Additional vectors can also be found, for example, in Ausubel, F. M., et al , Current Protocols in Molecular Biology, (Current Protocol, 1994) and Sambrook et al., “Molecular Cloning: A Laboratory Manual,” 2nd Ed. (1989).
[0148] The vectors are useful for autonomous replication in a host cell or may be integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome (e.g., non-episomal mammalian vectors). Expression vectors are capable of directing the expression of coding sequences to which they are operably linked. In general, expression vectors of utility in recombinant DNA techniques are often in the form of plasmids (vectors). However, other forms of expression vectors, such as viral vectors (e.g., replication defective retroviruses, adenoviruses, and adeno-associated viruses) are also included.
[0149] DNA vectors can be introduced into eukaryotic cells via conventional transformation or transfection techniques. Suitable methods for transforming or transfecting host cells can be found in Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual (2nd ed., Cold Spring Harbor Laboratory Press, Plainview, N.Y.) and other standard molecular biology laboratory manuals.
[0150] In some embodiments nucleic acid inserts, which encode a split Casl2a system component in such vectors, can be operably linked to a promoter, which is selected based on, for example, the cell type in which expression is sought. Viral vectors that can be used in the disclosure include, for example, retroviral, adenoviral, and adeno-associated vectors, herpes virus, simian virus 40 (SV40), and bovine papilloma virus vectors (see, for example, Gluzman (Ed.), Eukaryotic Viral Vectors, CSH Laboratory Press, Cold Spring Harbor, N.Y.). [0151] In some embodiments, the expression vector can be a viral vector. The term “viral vector” is widely used to refer either to a nucleic acid molecule that includes virus-derived nucleic acid elements that typically facilitate transfer of the nucleic acid molecule or integration into the genome of a cell, or to a viral particle that mediates nucleic acid transfer. Viral particles typically include viral components, and sometimes also host cell components, in addition to nucleic acid(s). Retroviral vectors used herein contain structural and functional genetic elements, or portions thereof, that are primarily derived from a retrovirus. Retroviral lentivirus vectors contain structural and functional genetic elements, or portions thereof including LTRs, that are primarily derived from a lentivirus (a sub-type of retrovirus).
[0152] Viral vectors that can be used in the disclosure include, for example, retrovirus vectors (including lentivirus vectors), adenovirus vectors, and adeno-associated virus vectors, herpes virus, simian virus 40 (SV40), and bovine papilloma virus vectors (see, for example, Gluzman (Ed.), Eukaryotic Viral Vectors, CSH Laboratory Press, Cold Spring Harbor, N.Y.).
[0153] In some embodiments, the nucleic acid molecules are delivered by viral or non-viral delivery vehicles known in the art. For example, the nucleic acid molecule can be stably integrated in the host genome, or can be episomally replicating, or present in the recombinant host cell as a mini-circle expression vector for stable or transient expression. Accordingly, in some embodiments disclosed herein, the nucleic acid molecule is maintained and replicated in the recombinant host cell as an episomal unit. In some embodiments, the nucleic acid molecule is stably integrated into the genome of the recombinant cell. Stable integration can also be accomplished using classical random genomic recombination techniques or with more precise genome editing techniques such as using guide RNA-directed CRISPR/Cas9, DNA-guided endonuclease genome editing NgAgo ( Natronobacterium gregoryi Argonaute ), or TALENs genome editing (transcription activator-like effector nucleases). In some embodiments, the nucleic acid molecule is present in the recombinant host cell as a mini-circle expression vector for stable or transient expression.
[0154] The nucleic acid molecules can be encapsulated in a viral capsid or a lipid nanoparticle. For example, introduction of nucleic acids into cells may be achieved using viral transduction methods. In a non-limiting example, adeno-associated virus (AAV) is a non-enveloped virus that can be engineered to deliver nucleic acids to target cells via viral transduction. Several AAV serotypes have been described, and all of the known serotypes can infect cells from multiple diverse tissue types. AAV is capable of transducing a wide range of species and tissues in vivo with no evidence of toxicity, and it generates relatively mild innate and adaptive immune responses.
[0155] Lentiviral systems are also useful for nucleic acid delivery and gene therapy via viral transduction. Lentiviral vectors offer several attractive properties as gene-delivery vehicles, including: (i) sustained gene delivery through stable vector integration into the host cell genome; (ii) the ability to infect both dividing and non-dividing cells; (iii) broad tissue tropisms, including important gene- and cell-therapy-target cell types; (iv) no expression of viral proteins after vector transduction; (v) the ability to deliver complex genetic elements, such as polycistronic or intron-containing sequences; (vi) a potentially safer integration site profile (e.g., by targeting a site for integration that has little or no oncogenic potential); and (vii) a relatively easy system for vector manipulation and production.
Methods of the Disclosure
Methods of targeting nucleic acid molecules
[0156] Provided herein are methods of targeting (e.g., binding to, modifying, detecting, etc.) one or more target nucleic acid molecules (e.g., dsDNA) using a split Casl2a system as described herein. Such methods can include contacting one or more target nucleic acid molecules with a non-naturally occurring or engineered (e.g., does not naturally occur in the cell or system into which it is introduced) split Casl2a complex and a crRNA. In some embodiments, a spacer sequence within the crRNA molecule is not naturally occurring, and has been modified to be complementary to a target nucleic acid molecule. In some embodiments, the target nucleic acid molecule is a DNA molecule, such as a genomic DNA. [0157] In some embodiments, provided herein is a method of conditionally targeting (e.g., binding to, modifying, detecting, etc.) a target nucleic acid in a sample (e.g., a biological sample, such as a cell) comprising introducing into the sample the components of a split Casl2a system as described herein, wherein for two or more components of the split Casl2a system, the expression of each of the components is individually under the control of different inputs to the sample such that targeting of the target nucleic acid by a split Casl2a/crRNA complex only occurs when each of the different inputs have a desired state. In some embodiments, the split Casl2a system comprises (a) nucleic acid encoding two or more effector subunits comprising fragments of a Casl2a protein or variant thereof, wherein the two or more effector subunits are capable of spontaneously assembling to form a split Casl2a complex retaining at least some of the effector activity of the Casl2a protein or variant thereof; and (b) a CRISPR RNA (crRNA) or nucleic acid encoding the crRNA, wherein the crRNA comprises a spacer having sufficient complementary to a target polynucleotide sequence in the target nucleic acid in the cell such that it can hybridize to the target nucleic acid. In some embodiments, the split Casl2a system comprises nucleic acid encoding the first crRNA. In some embodiments, the split Casl2a system further comprises nucleic acid comprising a NOT inducible promoter operably linked to a polynucleotide sequence encoding an anti-CRISPR (Acr) protein capable of inhibiting activity of the split Casl2a complex, wherein a NOT input to the cell is capable of increasing the expression of Acr protein in the cell such that activity of split Cas 12a complex in the cell is inhibited. In some embodiments, the sample is a cell or a composition comprising a cell. In some embodiments, the cell is a mammalian cell, e.g., a human cell.
[0158] Targeting a nucleic acid molecule can include one or more of cutting or nicking the target nucleic acid molecule; modulating the expression of a gene present in the target nucleic acid molecule (such as by regulating transcription of the gene from a target DNA, e.g., to downregulate or upregulate expression of the gene); visualizing, labeling, or detecting the target nucleic acid molecule; binding the target nucleic acid molecule, editing the target nucleic acid molecule, trafficking the target nucleic acid molecule, and masking the target nucleic acid molecule. In some embodiments, modifying the target nucleic acid molecule includes introducing one or more of a nucleobase substitution, a nucleobase deletion, a nucleobase insertion, a break in the target nucleic acid molecule, methylation of the target nucleic acid molecule, and demethylation of the nucleic acid molecule. In some embodiments, such methods are used to treat a disease, such as a disease in a human. In such embodiments, one or more target nucleic acid molecules are associated with the disease.
Methods of generating an output to a sample based on the state of a plurality of inputs to the sample
[0159] In some aspects of the disclosure, a split Casl2a system as described herein is introduced into a sample (e.g., a biological sample, such as a cell) to allow for the conditional generation of an output to the sample, wherein for two or more components of the split Cas 12a system, the expression of each of the components is individually under the control of different inputs to the sample. In some embodiments, the inputs are selected from physical inputs, small molecule inputs, and biologic inputs. In some embodiments, the output comprises modulating gene expression from a target nucleic acid in the sample, or otherwise modifying the target nucleic acid.
[0160] In some embodiments, provided herein is a method of conditionally generating an output to a sample comprising introducing into the sample the components of a split Cas 12a system as described herein, wherein for two or more components of the split Cas 12a system, the expression of each of the components is individually under the control of different inputs to the sample such that an output mediated by a split Cas 12a/ crRNA complex is only generated when each of the different inputs have a desired state. In some embodiments, the split Cas 12a system comprises (a) nucleic acid encoding two or more effector subunits comprising fragments of a Cas 12a protein or variant thereof, wherein the two or more effector subunits are capable of spontaneously assembling to form a split Cas 12a complex retaining at least some of the effector activity of the Cas 12a protein or variant thereof; and (b) a CRISPR RNA (crRNA) or nucleic acid encoding the crRNA, wherein the crRNA comprises a spacer having sufficient complementary to a target polynucleotide sequence in the target nucleic acid in the sample such that it can hybridize to the target nucleic acid. In some embodiments, the split Cas 12a system comprises nucleic acid encoding the first crRNA. In some embodiments, the split Cas 12a system further comprises nucleic acid comprising a NOT inducible promoter operably linked to a polynucleotide sequence encoding an anti-CRISPR (Acr) protein capable of inhibiting activity of the split Cas 12a complex, wherein a NOT input to the sample is capable of increasing the expression of Acr protein in the cell such that activity of split Cas 12a complex in the cell is inhibited. In some embodiments, the sample is a cell or a composition comprising a cell. In some embodiments, the cell is a mammalian cell, e.g., a human cell.
Methods of nucleic acid editing
[0161] In some embodiments, provided herein are methods of conditionally editing a target nucleic acid in a sample using a split Cas 12a system as described herein. The methods generally involve contacting the target nucleic acid with a nuclease-competent split Casl2a/crRNA complex comprising activity that cleaves the target nucleic acid. In some embodiments, the split Casl2a/crRNA complex has nucleobase-editing activity. In some embodiments, the target nucleic acid molecule is a DNA molecule, such as a genomic DNA.
[0162] In some embodiments, provided herein is a method of conditionally editing a target nucleic acid in a sample comprising introducing into the sample the components of a split Cas 12a system as described herein, wherein for two or more components of the split Cas 12a system, the expression of each of the components is individually under the control of different inputs to the sample such that editing of the target nucleic acid by a split Casl2a/crRNA complex only occurs when each of the different inputs have a desired state. In some embodiments, the split Cas 12a system comprises (a) nucleic acid encoding two or more effector subunits comprising fragments of a Cas 12a protein or variant thereof having a nucleic acid-editing effector activity, wherein the two or more effector subunits are capable of spontaneously assembling to form a split Cas 12a complex retaining at least some of the effector activity of the Cas 12a protein or variant thereof; and (b) a CRISPR RNA (crRNA) or nucleic acid encoding the crRNA, wherein the crRNA comprises a spacer having sufficient complementary to a target polynucleotide sequence in the target nucleic acid in the sample such that it can hybridize to the target nucleic acid. In some embodiments, the split Cas 12a system comprises nucleic acid encoding the first crRNA. In some embodiments, the split Cas 12a system further comprises nucleic acid comprising a NOT inducible promoter operably linked to a polynucleotide sequence encoding an anti-CRISPR (Acr) protein capable of inhibiting activity of the split Cas 12a complex, wherein a NOT input to the sample is capable of increasing the expression of Acr protein in the sample such that activity of split Cas 12a complex in the sample is inhibited. In some embodiments, the sample is a cell or a composition comprising a cell. In some embodiments, the cell is a mammalian cell, e.g., a human cell. [0163] In some embodiments, according to any of the methods of conditionally editing a target nucleic acid in a sample described herein employing nucleic acid comprising a polynucleotide sequence encoding an Acr protein, the Acr protein is AcrVAl or a variant thereof. In some embodiments, the AcrVAl or variant thereof comprises the amino acid sequence encoded by SEQ ID NO: 9 or a variant thereof having at least 80% sequence identity to the amino acid sequence encoded by SEQ ID NO: 9.
[0164] In some embodiments, according to any of the methods of conditionally editing a target nucleic acid in a sample described herein, a split Cas 12a complex that retains at least some of the effector activity of a Cas 12a protein or variant thereof retains at least about 5% (such as at least about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or greater) of the effector activity of the Cas 12a protein or variant thereof.
[0165] In some embodiments, according to any of the methods of conditionally editing a target nucleic acid in a sample described herein, the crRNA comprises a spacer sequence selected from the polynucleotide sequence of any one of SEQ ID NOs: 15-43.
[0166] In some embodiments, according to any of the methods of conditionally editing a target nucleic acid in a sample described herein employing nucleic acid comprising an inducible promoter, the inducible promoter is selected from an RRM2 promoter, a hypoxia-inducible promoter, a TRE3G promoter, a CMV5-CuO promoter, and a P2 inducible promoter. In some embodiments, the RRM2 promoter comprises the polynucleotide sequence of SEQ ID NO: 10. In some embodiments, the hypoxia-inducible promoter comprises the polynucleotide sequence of SEQ ID NO: 11. In some embodiments, the TRE3G promoter comprises the polynucleotide sequence of SEQ ID NO: 12. In some embodiments, the CMV5-CuO promoter comprises the polynucleotide sequence of SEQ ID NO: 13. In some embodiments, the P2 inducible promoter comprises the polynucleotide sequence of SEQ ID NO: 14. In some embodiments, where the split Cas 12a system comprises nucleic acid comprising a plurality of inducible promoters, each of the plurality of inducible promoters operably linked to a polynucleotide sequence encoding an effector subunit are different. In some embodiments, where the split Cas 12a system comprises nucleic acid comprising a plurality of inducible promoters, each of the plurality of inducible promoters operably linked to a polynucleotide sequence encoding a crRNA are different. In some embodiments, where the split Cas 12a system comprises nucleic acid comprising a plurality of inducible promoters, at least some (such as at least about any of 2, 3, 4, or more) of the plurality of inducible promoters operably linked to a polynucleotide sequence encoding a crRNA are the same. In some embodiments, where the split Cas 12a system comprises nucleic acid comprising a plurality of inducible promoters, all of the plurality of inducible promoters operably linked to a polynucleotide sequence encoding a crRNA are the same. In some embodiments, where the split Cas 12a system comprises nucleic acid comprising a plurality of inducible promoters, each of the plurality of inducible promoters are different. [0167] In some embodiments, according to any of the methods of conditionally editing a target nucleic acid in a sample described herein, the system is configured such that all components of the Casl2a/crRNA complex are present or capable of being expressed in the sample when the first input and the second input each have a desired state. In some embodiments, the system is configured such that all components of the Casl2a/crRNA complex are present or capable of being expressed in the sample when the first input is present and the second input is present. [0168] In some embodiments, according to any of the methods of conditionally editing a target nucleic acid in a sample described herein, the system comprises (a) nucleic acid encoding a first effector subunit and a second effector subunit of the two or more effector subunits, wherein the nucleic acid comprises a first inducible promoter operably linked to a polynucleotide sequence encoding the first effector subunit and a second inducible promoter operably linked to a polynucleotide sequence encoding the second effector subunit, and the first input is capable of modulating the expression of the first effector subunit in the sample and the second input is capable of modulating the expression of the second effector subunit in the sample; or (b) nucleic acid encoding a first effector subunit of the two or more effector subunits, wherein the nucleic acid comprises a first inducible promoter operably linked to a polynucleotide sequence encoding the first effector subunit; and nucleic acid encoding the first crRNA, wherein the nucleic acid comprises a second inducible promoter operably linked to a polynucleotide sequence encoding the first crRNA, and the first input is capable of modulating the expression of the first effector subunit in the sample and the second input is capable of modulating the expression of the first crRNA in the sample. In some embodiments, modulating the expression of each of the Casl2a/crRNA complex components comprises increasing their expression. [0169] In some embodiments, according to any of the methods of conditionally editing a target nucleic acid in a sample described herein, the expression of a third component of the Casl2a/crRNA complex is under the control of a third input to the sample, and the system is configured such that all components of the Casl2a/crRNA complex are present or capable of being expressed in the sample when the first input, the second input, and the third input each have a desired state. In some embodiments, the system is configured such that all components of the Casl2a/crRNA complex are present or capable of being expressed in the sample when the first input is present, the second input is present, and the third input is present. In some embodiments, the system comprises (a) nucleic acid encoding a first effector subunit, a second effector subunit, and a third effector subunit of the two or more effector subunits, wherein the nucleic acid comprises a first inducible promoter operably linked to a polynucleotide sequence encoding the first effector subunit, a second inducible promoter operably linked to a polynucleotide sequence encoding the second effector subunit, and a third inducible promoter operably linked to a polynucleotide sequence encoding the third effector subunit, and the first input is capable of modulating the expression of the first effector subunit in the sample, the second input is capable of modulating the expression of the second effector subunit in the sample, and the third input is capable of modulating the expression of the third effector subunit in the sample; or (b) nucleic acid encoding a first effector subunit and a second effector subunit of the two or more effector subunits, wherein the nucleic acid comprises a first inducible promoter operably linked to a polynucleotide sequence encoding the first effector subunit and a second inducible promoter operably linked to a polynucleotide sequence encoding the second effector subunit; and nucleic acid encoding the first crRNA, wherein the nucleic acid comprises a third inducible promoter operably linked to a polynucleotide sequence encoding the first crRNA, and the first input is capable of modulating the expression of the first effector subunit in the sample, the second input is capable of modulating the expression of the second effector subunit in the sample, and the third input is capable of modulating the expression of the first crRNA in the sample. In some embodiments, modulating the expression of each of the Casl2a/crRNA complex components comprises increasing their expression. [0170] In some embodiments, according to any of the methods of conditionally editing a target nucleic acid in a sample described herein, the expression of a third component of the Casl2a/crRNA complex is under the control of a third input to the sample and the expression of a fourth component of the Casl2a/crRNA complex is under the control of a fourth input to the sample, and the system is configured such that all components of the Casl2a/crRNA complex are present or capable of being expressed in the sample when the first input, the second input, the third input, and the fourth input each have a desired state. In some embodiments, the system is configured such that all components of the Casl2a/crRNA complex are present or capable of being expressed when the first input is present, the second input is present, the third input is present, and the fourth input is present. In some embodiments, the system comprises nucleic acid encoding a first effector subunit, a second effector subunit, and a third effector subunit of the two or more effector subunits, wherein the nucleic acid comprises a first inducible promoter operably linked to a polynucleotide sequence encoding the first effector subunit, a second inducible promoter operably linked to a polynucleotide sequence encoding the second effector subunit, and a third inducible promoter operably linked to a polynucleotide sequence encoding the third effector subunit, and the first input is capable of modulating the expression of the first effector subunit in the sample, the second input is capable of modulating the expression of the second effector subunit in the sample, and the third input is capable of modulating the expression of the third effector subunit in the sample; and nucleic acid encoding the first crRNA, wherein the nucleic acid comprises a fourth inducible promoter operably linked to a polynucleotide sequence encoding the first crRNA, and the fourth input is capable of modulating the expression of the first crRNA. In some embodiments, modulating the expression of each of the Casl2a/crRNA complex components comprises increasing their expression.
[0171] In some embodiments, according to any of the methods of conditionally editing a target nucleic acid in a sample described herein, the two or more effector subunits comprise a first effector subunit and a second effector subunit, wherein the first effector subunit comprises an N-terminal fragment of the Casl2a protein or variant thereof up to and including a split point amino acid, and the second effector subunit comprises a C-terminal fragment of the Casl2a protein or variant thereof following the split point amino acid. In some embodiments, the split point amino acid corresponds to A406 or G550 with respect to a parental Cas 12a protein amino acid sequence of SEQ ID NO: 1. In some embodiments, the first effector subunit further comprises a first stabilization domain at its C-terminus and the second effector subunit further comprises a second stabilization domain at its N-terminus, wherein the first stabilization domain and the second stabilization domain are capable of interacting with one another to stabilize a complex comprising the first effector subunit and the second effector subunit in the sample.
[0172] In some embodiments, according to any of the methods of conditionally editing a target nucleic acid in a sample described herein, the two or more effector subunits comprise a first effector subunit and a second effector subunit, wherein the Casl2a protein or variant thereof is a fusion protein comprising an effector domain fused to a Casl2a domain, and the first effector subunit comprises the Casl2a domain and the second effector subunit comprises the effector domain. In some embodiments, the first effector subunit further comprises a first stabilization domain at either terminus and the second effector subunit further comprises a second stabilization domain at either terminus, wherein the first stabilization domain and the second stabilization domain are capable of interacting with one another to stabilize a complex comprising the first effector subunit and the second effector subunit in the sample.
[0173] In some embodiments, according to any of the methods of conditionally editing a target nucleic acid in a sample described herein, the two or more effector subunits comprise a first effector subunit, a second effector subunit, and a third effector subunit. In some embodiments, the Casl2a protein or variant thereof is a fusion protein comprising an effector domain fused to a Casl2a domain, and the first effector subunit comprises an N-terminal fragment of the Casl2a domain up to and including a split point amino acid, the second effector subunit comprises a C-terminal fragment of the Casl2a domain following the split point amino acid; and the third effector subunit comprises the effector domain. In some embodiments, the split point amino acid corresponds to A406 or G550 with respect to a parental Cas 12a protein amino acid sequence of SEQ ID NO: 1. In some embodiments, the first effector subunit further comprises a first stabilization domain at its C-terminus and the second effector subunit further comprises a second stabilization domain at its N-terminus, wherein the first stabilization domain and the second stabilization domain are capable of interacting with one another to stabilize a complex comprising the first effector subunit and the second effector subunit in the sample; and/or the third effector subunit further comprises a third stabilization domain at either terminus and (i) the second effector subunit further comprises a fourth stabilization domain at its C-terminus or (ii) the first effector subunit further comprises the fourth stabilization domain at its N-terminus, wherein the third stabilization domain and the fourth stabilization domain are capable of interacting with one another to stabilize a complex comprising the first effector subunit, the second effector subunit, and the third effector subunit in the sample.
[0174] In some embodiments, according to any of the methods of conditionally editing a target nucleic acid in a sample described herein employing a first stabilization domain and a second stabilization domain, the first stabilization domain and the second stabilization domain are derived from subunits of a heterodimerizing binding pair or a split-intein. In some embodiments, where the method further employs a third stabilization domain and a fourth stabilization domain, the third stabilization domain and the fourth stabilization domain are derived from subunits of a heterodimerizing binding pair or a split-intein and are orthogonal to the first stabilization domain and the second stabilization domain. In some embodiments, the first stabilization domain and the second stabilization domain are derived from subunits of a heterodimerizing binding pair and the third stabilization domain and the fourth stabilization domain are derived from subunits of a split-intein; or the first stabilization domain and the second stabilization domain are derived from subunits of a split-intein and the third stabilization domain and the fourth stabilization domain are derived from subunits of a heterodimerizing binding pair. In some embodiments, the heterodimerizing binding pair is a coiled-coil (CC) heterodimer. In some embodiments, the CC heterodimer is a leucine zipper. In some embodiments, the leucine zipper subunits comprise RR12EE345L and EE12RR345L. In some embodiments, RR12EE345L comprises the amino acid sequence encoded by SEQ ID NO: 6. In some embodiments, EE12RR345L comprises the amino acid sequence encoded by SEQ ID NO: 5. In some embodiments, the CC heterodimer is a CC peptide heterodimer ( see Lebar, T., et al. (2020). Nature Chemical Biology, 1-7). In some embodiments, the split-intein subunits comprise DnaE N-intein and C-intein. In some embodiments, DnaE N-intein comprises the amino acid sequence encoded by SEQ ID NO: 7. In some embodiments, DnaE C-intein comprises the amino acid sequence encoded by SEQ ID NO: 8. In some embodiments, the split-intein is gp41-l, gp41-8, NrdJ-1, or IMPDH-1 ( see Dassa, B., et al. (2009). Nucleic acids research, 57(8), 2560-2573). In some embodiments, the split-intein comprises split portions of the Mxe GyrA intein (see Fine, E. J., et al. (2015). Scientific reports, 5, 10777).
[0175] In some embodiments, according to any of the methods of conditionally editing a target nucleic acid in a sample described herein, the system further comprises one or more additional crRNAs or nucleic acid encoding the one or more additional crRNAs, wherein Casl2a/crRNA complexes formed by association of the one or more additional crRNAs with (I) the Casl2a protein or variant thereof or (II) the two or more effector subunits are capable of generating one or more additional outputs to the sample comprising effector activity of the Casl2a/crRNA complexes at target sites directed by the one or more additional crRNAs. In some embodiments, the system comprises nucleic acid encoding a crRNA array comprising one or more of the first crRNA and the one or more additional crRNAs.
[0176] In some embodiments, according to any of the methods of conditionally editing a target nucleic acid in a sample described herein, the Cas 12a protein or variant thereof is derived from a parental Cas 12a protein from Lachnospiraceae bacterium. In some embodiments, the parental Cas 12a protein from Lachnospiraceae bacterium comprises the amino acid sequence of SEQ ID NO: 1. In some embodiments, the target nucleic acid is a target dsDNA. In some embodiments, a protospacer in the target dsDNA targeted by the crRNA spacer has a 5' PAM selected from 5'-TTTG-3', 5'-TTTA-3', and 5'-TTTC-3'.
[0177] In some embodiments, according to any of the methods of conditionally editing a target nucleic acid in a sample described herein, the Cas 12a protein or variant thereof is a nuclease- competent Cas protein comprising a Cas 12a domain having some, the same, or greater nuclease activity as compared to a parental Cas 12a protein from which it is derived, and the first output comprises cleavage (e.g., double-stranded cleavage) of the first target nucleic acid near the first target polynucleotide sequence. In some embodiments, the Cas 12a domain comprises the amino acid sequence of SEQ ID NO: 1.
[0178] In some embodiments, according to any of the methods of conditionally editing a target nucleic acid in a sample described herein, the Cas 12a protein or variant thereof is a fusion protein comprising an effector domain comprising a polypeptide that can edit a nucleotide fused to a nuclease-deficient dCasl2a domain having reduced or eliminated nuclease activity as compared to a parental Cas 12a protein from which it is derived, and the first output is generated by activity of the effector domain to edit a nucleotide. In some embodiments, the dCasl2a domain comprises a modification of amino acid D832 with respect to SEQ ID NO: 1 that reduces, substantially eliminates, or eliminates nuclease activity of the dCasl2a domain. In some embodiments, the dCasl2a domain comprises a D832A substitution with respect to SEQ ID NO: 1. In some embodiments, the dCasl2a domain comprises the amino acid sequence of SEQ ID NO: 2.
[0179] In some embodiments, according to any of the methods of conditionally editing a target nucleic acid in a sample described herein, the sample is a cell or a composition comprising a cell. In some embodiments, the inputs to the sample are selected from physical inputs, small molecule inputs, and biologic inputs. In some embodiments, the physical inputs are selected from oxygen levels, pH, ion concentrations, temperature, light, mechanical force, ultrasound, shock wave, magnetic field, and electric field; the small molecule inputs are selected from drugs, metabolites, and adjuvants; and/or the biologic inputs are selected from polypeptides, polypeptide derivatives, and nucleic acids.
Methods of modulating transcription
[0180] In some embodiments, provided herein are methods of conditionally modulating transcription of a target nucleic acid in a sample using a split Casl2a system as described herein. The methods generally involve contacting the target nucleic acid with a nuclease- deficient split dCasl2a/crRNA complex comprising activity that modulates the transcription of the target nucleic acid. In some embodiments, the target nucleic acid molecule is a DNA molecule, such as a genomic DNA.
[0181] In some embodiments, the split Casl2a complex of the split Casl2a system has activity that modulates the transcription of target DNA. In some embodiments, a split Casl2a fusion protein or variant thereof comprising an effector subunit comprising a heterologous polypeptide that exhibits the ability to increase or decrease transcription (e.g., transcriptional activator or transcription repressor polypeptides) is used to increase or decrease the transcription of target DNA at a specific location in a target DNA, which is guided by the spacer of the crRNA. Examples of source polypeptides for providing a split Casl2a fusion protein or variant thereof with transcription modulatory activity include, but are not limited to, light-inducible transcription regulators, small molecule/drug-responsive transcription regulators, transcription factors, transcription repressors, etc. In some embodiments, the method is used to control the transcription of a targeted gene-coding RNA (protein-encoding mRNA) and/or a targeted non-coding RNA (e.g., tRNA, rRNA, snoRNA, siRNA, miRNA, long ncRNA. etc.). In some embodiments, the split Casl2a fusion protein or variant has enzymatic activity that modifies a polypeptide associated with DNA (e.g., histone). In some embodiments, the enzymatic activity is methyltransferase activity, demethylase activity, acetyltransferase activity, deacetylase activity, kinase activity, phosphatase activity, ubiquitin ligase activity (e.g., ubiquitination activity), deubiquitinating activity, adenylation activity, deadenylation activity, SUMOylating activity, deSUMOylating activity, ribosylation activity, deribosylation activity, myristoylation activity, demyristoylation activity, glycosylation activity (e.g., from GlcNAc transferase), or deglycosylation activity. The enzymatic activities listed herein catalyze covalent modifications to proteins. Such modifications are known in the art to alter the stability or activity of the target protein (e.g., phosphorylation due to kinase activity can stimulate or silence protein activity depending on the target protein). Histone proteins are known in the art to bind DNA and form complexes known as nucleosomes. Histones can be modified (e.g., by methylation, acetylation, ubiquitination, phosphorylation) to elicit structural changes in the surrounding DNA, thus controlling the accessibility of potentially large portions of DNA to interacting factors such as transcription factors, polymerases, and the like. A single histone can be modified in many different ways and in many different combinations (e.g., trimethylation of lysine 27 of histone 3, H3K27, is associated with DNA regions of repressed transcription while trimethylation of lysine 4 of histone 3, H3K4, is associated with DNA regions of active transcription). Thus, a split Casl2a fusion protein or variant thereof with histone-modifying activity finds use in the site-specific control of chromosomal structure and can be used to alter the histone modification pattern in a selected region of target DNA. Such methods find use in both research and clinical applications.
[0182] In some embodiments, provided herein is a method of conditionally modulating transcription of a target nucleic acid in a sample comprising introducing into the sample the components of a split Casl2a system as described herein, wherein for two or more components of the split Casl2a system, the expression of each of the components is individually under the control of different inputs to the sample such that modulation of transcription of the target nucleic acid by a split Casl2a/crRNA complex only occurs when each of the different inputs have a desired state. In some embodiments, the split Casl2a system comprises (a) nucleic acid encoding two or more effector subunits comprising fragments of a dCasl2a fusion protein or variant thereof comprising an effector domain having a transcription-modulating effector activity, wherein the two or more effector subunits are capable of spontaneously assembling to form a split Casl2a complex retaining at least some of the effector activity of the Casl2a protein or variant thereof; and (b) a CRISPR RNA (crRNA) or nucleic acid encoding the crRNA, wherein the crRNA comprises a spacer having sufficient complementary to a target polynucleotide sequence in the target nucleic acid in the sample such that it can hybridize to the target nucleic acid. In some embodiments, the split Casl2a system comprises nucleic acid encoding the first crRNA. In some embodiments, the split Casl2a system further comprises nucleic acid comprising a NOT inducible promoter operably linked to a polynucleotide sequence encoding an anti-CRISPR (Acr) protein capable of inhibiting activity of the split Casl2a complex, wherein a NOT input to the sample is capable of increasing the expression of Acr protein in the sample such that activity of split Casl2a complex in the sample is inhibited. In some embodiments, the sample is a cell or a composition comprising a cell. In some embodiments, the cell is a mammalian cell, e.g., a human cell.
[0183] In some embodiments, a transcription modulation method described herein allows for conditional modulation (e.g., reduction or increase) of transcription of a target DNA in a cell when each of two or more inputs to the cell have a desired state. For example, in some embodiments, conditional reduction of transcription of a target DNA in a cell, wherein each of the two or more inputs to the cell have the desired state, reduces transcription of the target DNA by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or greater than 90%, compared to the level of transcription of the target DNA in a corresponding cell that does not contain the split Casl2a system or where at least one of the two or more inputs to the corresponding cell does not have the desired state. In some embodiments, conditional reduction of transcription of the target DNA in the cell reduces transcription of the target DNA, but does not substantially reduce transcription of a non- target DNA, e.g., transcription of a non-target DNA is reduced, if at all, by less than 10% compared to the level of transcription of the non-target DNA in the absence of the split Casl2a system or where at least one of the two or more inputs to the cell does not have the desired state. In some embodiments, conditional increase of transcription of a target DNA in a cell, wherein each of the two or more inputs to the cell have the desired state, increases transcription of the target DNA by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or greater than 90%, compared to the level of transcription of the target DNA in a corresponding cell that does not contain the split Cas 12a system or where at least one of the two or more inputs to the corresponding cell does not have the desired state. In some embodiments, conditional increase of transcription of the target DNA in the cell increases transcription of the target DNA, but does not substantially increase transcription of a non-target DNA, e.g., transcription of a non-target DNA is increased, if at all, by less than 10% compared to the level of transcription of the non-target DNA in the absence of the split Cas 12a system or where at least one of the two or more inputs to the cell does not have the desired state.
[0184] In some embodiments, according to any of the methods of conditionally modulating transcription of a target nucleic acid in a sample described herein employing nucleic acid comprising a polynucleotide sequence encoding an Acr protein, the Acr protein is AcrVAl or a variant thereof. In some embodiments, the AcrVAl or variant thereof comprises the amino acid sequence encoded by SEQ ID NO: 9 or a variant thereof having at least 80% sequence identity to the amino acid sequence encoded by SEQ ID NO: 9. [0185] In some embodiments, according to any of the methods of conditionally modulating transcription of a target nucleic acid in a sample described herein, a split Casl2a complex that retains at least some of the effector activity of a Casl2a protein or variant thereof retains at least about 5% (such as at least about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or greater) of the effector activity of the Casl2a protein or variant thereof.
[0186] In some embodiments, according to any of the methods of conditionally modulating transcription of a target nucleic acid in a sample described herein, the crRNA comprises a spacer sequence selected from the polynucleotide sequence of any one of SEQ ID NOs: 15-43. [0187] In some embodiments, according to any of the methods of conditionally modulating transcription of a target nucleic acid in a sample described herein employing nucleic acid comprising an inducible promoter, the inducible promoter is selected from an RRM2 promoter, a hypoxia-inducible promoter, a TRE3G promoter, a CMV5-CuO promoter, and a P2 inducible promoter. In some embodiments, the RRM2 promoter comprises the polynucleotide sequence of SEQ ID NO: 10. In some embodiments, the hypoxia-inducible promoter comprises the polynucleotide sequence of SEQ ID NO: 11. In some embodiments, the TRE3G promoter comprises the polynucleotide sequence of SEQ ID NO: 12. In some embodiments, the CMV5- CuO promoter comprises the polynucleotide sequence of SEQ ID NO: 13. In some embodiments, the P2 inducible promoter comprises the polynucleotide sequence of SEQ ID NO: 14. In some embodiments, where the split Casl2a system comprises nucleic acid comprising a plurality of inducible promoters, each of the plurality of inducible promoters operably linked to a polynucleotide sequence encoding an effector subunit are different. In some embodiments, where the split Casl2a system comprises nucleic acid comprising a plurality of inducible promoters, each of the plurality of inducible promoters operably linked to a polynucleotide sequence encoding a crRNA are different. In some embodiments, where the split Casl2a system comprises nucleic acid comprising a plurality of inducible promoters, at least some (such as at least about any of 2, 3, 4, or more) of the plurality of inducible promoters operably linked to a polynucleotide sequence encoding a crRNA are the same. In some embodiments, where the split Casl2a system comprises nucleic acid comprising a plurality of inducible promoters, all of the plurality of inducible promoters operably linked to a polynucleotide sequence encoding a crRNA are the same. In some embodiments, where the split Cas 12a system comprises nucleic acid comprising a plurality of inducible promoters, each of the plurality of inducible promoters are different.
[0188] In some embodiments, according to any of the methods of conditionally modulating transcription of a target nucleic acid in a sample described herein, the system is configured such that all components of the Casl2a/crRNA complex are present or capable of being expressed in the sample when the first input and the second input each have a desired state. In some embodiments, the system is configured such that all components of the Casl2a/crRNA complex are present or capable of being expressed in the sample when the first input is present and the second input is present.
[0189] In some embodiments, according to any of the methods of conditionally modulating transcription of a target nucleic acid in a sample described herein, the system comprises (a) nucleic acid encoding a first effector subunit and a second effector subunit of the two or more effector subunits, wherein the nucleic acid comprises a first inducible promoter operably linked to a polynucleotide sequence encoding the first effector subunit and a second inducible promoter operably linked to a polynucleotide sequence encoding the second effector subunit, and the first input is capable of modulating the expression of the first effector subunit in the sample and the second input is capable of modulating the expression of the second effector subunit in the sample; or (b) nucleic acid encoding a first effector subunit of the two or more effector subunits, wherein the nucleic acid comprises a first inducible promoter operably linked to a polynucleotide sequence encoding the first effector subunit; and nucleic acid encoding the first crRNA, wherein the nucleic acid comprises a second inducible promoter operably linked to a polynucleotide sequence encoding the first crRNA, and the first input is capable of modulating the expression of the first effector subunit in the sample and the second input is capable of modulating the expression of the first crRNA in the sample. In some embodiments, modulating the expression of each of the Casl2a/crRNA complex components comprises increasing their expression.
[0190] In some embodiments, according to any of the methods of conditionally modulating transcription of a target nucleic acid in a sample described herein, the expression of a third component of the Casl2a/crRNA complex is under the control of a third input to the sample, and the system is configured such that all components of the Casl2a/crRNA complex are present or capable of being expressed in the sample when the first input, the second input, and the third input each have a desired state. In some embodiments, the system is configured such that all components of the Casl2a/crRNA complex are present or capable of being expressed in the sample when the first input is present, the second input is present, and the third input is present. In some embodiments, the system comprises (a) nucleic acid encoding a first effector subunit, a second effector subunit, and a third effector subunit of the two or more effector subunits, wherein the nucleic acid comprises a first inducible promoter operably linked to a polynucleotide sequence encoding the first effector subunit, a second inducible promoter operably linked to a polynucleotide sequence encoding the second effector subunit, and a third inducible promoter operably linked to a polynucleotide sequence encoding the third effector subunit, and the first input is capable of modulating the expression of the first effector subunit in the sample, the second input is capable of modulating the expression of the second effector subunit in the sample, and the third input is capable of modulating the expression of the third effector subunit in the sample; or (b) nucleic acid encoding a first effector subunit and a second effector subunit of the two or more effector subunits, wherein the nucleic acid comprises a first inducible promoter operably linked to a polynucleotide sequence encoding the first effector subunit and a second inducible promoter operably linked to a polynucleotide sequence encoding the second effector subunit; and nucleic acid encoding the first crRNA, wherein the nucleic acid comprises a third inducible promoter operably linked to a polynucleotide sequence encoding the first crRNA, and the first input is capable of modulating the expression of the first effector subunit in the sample, the second input is capable of modulating the expression of the second effector subunit in the sample, and the third input is capable of modulating the expression of the first crRNA in the sample. In some embodiments, modulating the expression of each of the Casl2a/crRNA complex components comprises increasing their expression. [0191] In some embodiments, according to any of the methods of conditionally modulating transcription of a target nucleic acid in a sample described herein, the expression of a third component of the Casl2a/crRNA complex is under the control of a third input to the sample and the expression of a fourth component of the Casl2a/crRNA complex is under the control of a fourth input to the sample, and the system is configured such that all components of the Casl2a/crRNA complex are present or capable of being expressed in the sample when the first input, the second input, the third input, and the fourth input each have a desired state. In some embodiments, the system is configured such that all components of the Casl2a/crRNA complex are present or capable of being expressed when the first input is present, the second input is present, the third input is present, and the fourth input is present. In some embodiments, the system comprises nucleic acid encoding a first effector subunit, a second effector subunit, and a third effector subunit of the two or more effector subunits, wherein the nucleic acid comprises a first inducible promoter operably linked to a polynucleotide sequence encoding the first effector subunit, a second inducible promoter operably linked to a polynucleotide sequence encoding the second effector subunit, and a third inducible promoter operably linked to a polynucleotide sequence encoding the third effector subunit, and the first input is capable of modulating the expression of the first effector subunit in the sample, the second input is capable of modulating the expression of the second effector subunit in the sample, and the third input is capable of modulating the expression of the third effector subunit in the sample; and nucleic acid encoding the first crRNA, wherein the nucleic acid comprises a fourth inducible promoter operably linked to a polynucleotide sequence encoding the first crRNA, and the fourth input is capable of modulating the expression of the first crRNA. In some embodiments, modulating the expression of each of the Casl2a/crRNA complex components comprises increasing their expression.
[0192] In some embodiments, according to any of the methods of conditionally modulating transcription of a target nucleic acid in a sample described herein, the two or more effector subunits comprise a first effector subunit and a second effector subunit, wherein the first effector subunit comprises an N-terminal fragment of the Casl2a protein or variant thereof up to and including a split point amino acid, and the second effector subunit comprises a C- terminal fragment of the Cas 12a protein or variant thereof following the split point amino acid. In some embodiments, the split point amino acid corresponds to A406 or G550 with respect to a parental Cas 12a protein amino acid sequence of SEQ ID NO: 1. In some embodiments, the first effector subunit further comprises a first stabilization domain at its C-terminus and the second effector subunit further comprises a second stabilization domain at its N-terminus, wherein the first stabilization domain and the second stabilization domain are capable of interacting with one another to stabilize a complex comprising the first effector subunit and the second effector subunit in the sample.
[0193] In some embodiments, according to any of the methods of conditionally modulating transcription of a target nucleic acid in a sample described herein, the two or more effector subunits comprise a first effector subunit and a second effector subunit, wherein the Cas 12a protein or variant thereof is a fusion protein comprising an effector domain fused to a Cas 12a domain, and the first effector subunit comprises the Cas 12a domain and the second effector subunit comprises the effector domain. In some embodiments, the first effector subunit further comprises a first stabilization domain at either terminus and the second effector subunit further comprises a second stabilization domain at either terminus, wherein the first stabilization domain and the second stabilization domain are capable of interacting with one another to stabilize a complex comprising the first effector subunit and the second effector subunit in the sample. [0194] In some embodiments, according to any of the methods of conditionally modulating transcription of a target nucleic acid in a sample described herein, the two or more effector subunits comprise a first effector subunit, a second effector subunit, and a third effector subunit. In some embodiments, the Casl2a protein or variant thereof is a fusion protein comprising an effector domain fused to a Casl2a domain, and the first effector subunit comprises an N- terminal fragment of the Cas 12a domain up to and including a split point amino acid, the second effector subunit comprises a C-terminal fragment of the Cas 12a domain following the split point amino acid; and the third effector subunit comprises the effector domain. In some embodiments, the split point amino acid corresponds to A406 or G550 with respect to a parental Casl2a protein amino acid sequence of SEQ ID NO: 1. In some embodiments, the first effector subunit further comprises a first stabilization domain at its C-terminus and the second effector subunit further comprises a second stabilization domain at its N-terminus, wherein the first stabilization domain and the second stabilization domain are capable of interacting with one another to stabilize a complex comprising the first effector subunit and the second effector subunit in the sample; and/or the third effector subunit further comprises a third stabilization domain at either terminus and (i) the second effector subunit further comprises a fourth stabilization domain at its C-terminus or (ii) the first effector subunit further comprises the fourth stabilization domain at its N-terminus, wherein the third stabilization domain and the fourth stabilization domain are capable of interacting with one another to stabilize a complex comprising the first effector subunit, the second effector subunit, and the third effector subunit in the sample.
[0195] In some embodiments, according to any of the methods of conditionally modulating transcription of a target nucleic acid in a sample described herein employing a first stabilization domain and a second stabilization domain, the first stabilization domain and the second stabilization domain are derived from subunits of a heterodimerizing binding pair or a split- intein. In some embodiments, where the method further employs a third stabilization domain and a fourth stabilization domain, the third stabilization domain and the fourth stabilization domain are derived from subunits of a heterodimerizing binding pair or a split-intein and are orthogonal to the first stabilization domain and the second stabilization domain. In some embodiments, the first stabilization domain and the second stabilization domain are derived from subunits of a heterodimerizing binding pair and the third stabilization domain and the fourth stabilization domain are derived from subunits of a split-intein; or the first stabilization domain and the second stabilization domain are derived from subunits of a split-intein and the third stabilization domain and the fourth stabilization domain are derived from subunits of a heterodimerizing binding pair. In some embodiments, the heterodimerizing binding pair is a coiled-coil (CC) heterodimer. In some embodiments, the CC heterodimer is a leucine zipper. In some embodiments, the leucine zipper subunits comprise RR12EE345L and EE12RR345L. In some embodiments, RR12EE345L comprises the amino acid sequence encoded by SEQ ID NO: 6. In some embodiments, EE12RR345L comprises the amino acid sequence encoded by SEQ ID NO: 5. In some embodiments, the CC heterodimer is a CC peptide heterodimer ( see Lebar, T., et al. (2020). Nature Chemical Biology, 1-7). In some embodiments, the split-intein subunits comprise DnaE N-intein and C-intein. In some embodiments, DnaE N-intein comprises the amino acid sequence encoded by SEQ ID NO: 7. In some embodiments, DnaE C-intein comprises the amino acid sequence encoded by SEQ ID NO: 8. In some embodiments, the split-intein is gp41-l, gp41-8, NrdJ-1, or IMPDH-1 ( see Dassa, B., et al. (2009). Nucleic acids research, 57(8), 2560-2573). In some embodiments, the split-intein comprises split portions of the Mxe GyrA intein (see Fine, E. J., et al. (2015). Scientific reports, 5, 10777).
[0196] In some embodiments, according to any of the methods of conditionally modulating transcription of a target nucleic acid in a sample described herein, the system further comprises one or more additional crRNAs or nucleic acid encoding the one or more additional crRNAs, wherein Casl2a/crRNA complexes formed by association of the one or more additional crRNAs with (I) the Casl2a protein or variant thereof or (II) the two or more effector subunits are capable of generating one or more additional outputs to the sample comprising effector activity of the Casl2a/crRNA complexes at target sites directed by the one or more additional crRNAs. In some embodiments, the system comprises nucleic acid encoding a crRNA array comprising one or more of the first crRNA and the one or more additional crRNAs.
[0197] In some embodiments, according to any of the methods of conditionally modulating transcription of a target nucleic acid in a sample described herein, the Casl2a protein or variant thereof is derived from a parental Casl2a protein from Lachnospiraceae bacterium. In some embodiments, the parental Casl2a protein from Lachnospiraceae bacterium comprises the amino acid sequence of SEQ ID NO: 1. In some embodiments, the target nucleic acid is a target dsDNA. In some embodiments, a protospacer in the target dsDNA targeted by the crRNA spacer has a 5' PAM selected from 5'-TTTG-3', 5'-TTTA-3', and 5'-TTTC-3'.
[0198] In some embodiments, according to any of the methods of conditionally modulating transcription of a target nucleic acid in a sample described herein, the Casl2a protein or variant thereof is a fusion protein comprising an effector domain fused to a nuclease-deficient dCas 12a domain having reduced or eliminated nuclease activity as compared to a parental Casl2a protein from which it is derived, and the first output is generated by activity of the effector domain. In some embodiments, the dCasl2a domain comprises a modification of amino acid D832 with respect to SEQ ID NO: 1 that reduces, substantially eliminates, or eliminates nuclease activity of the dCasl2a domain. In some embodiments, the dCasl2a domain comprises a D832A substitution with respect to SEQ ID NO: 1. In some embodiments, the dCasl2a domain comprises the amino acid sequence of SEQ ID NO: 2. In some embodiments, the effector domain comprises a polypeptide that can (i) activate transcription, (ii) repress transcription, (iii) methylate a nucleic acid, and/or (iv) demethylate a nucleic acid. In some embodiments, the effector domain comprises a transcriptional activation domain, and the first output comprises transcriptional activation of a first gene targeted by the first crRNA. In some embodiments, the transcriptional activation domain is a tripartite VP64-p65-Rta (VPR) transcriptional activation domain or variant thereof. In some embodiments, the effector domain has enzymatic activity that modifies a polypeptide associated with DNA (e.g., histone). In some embodiments, the enzymatic activity is methyltransferase activity, demethylase activity, acetyltransferase activity, deacetylase activity, kinase activity, phosphatase activity, ubiquitin ligase activity (e.g., ubiquitination activity), deubiquitinating activity, adenylation activity, deadenylation activity, SUMOylating activity, deSUMOylating activity, ribosylation activity, deribosylation activity, myristoylation activity, demyristoylation activity, glycosylation activity (e.g., from GlcNAc transferase), or deglycosylation activity.
[0199] In some embodiments, according to any of the methods of conditionally modulating transcription of a target nucleic acid in a sample described herein, the sample is a cell or a composition comprising a cell. In some embodiments, the inputs to the sample are selected from physical inputs, small molecule inputs, and biologic inputs. In some embodiments, the physical inputs are selected from oxygen levels, pH, ion concentrations, temperature, light, mechanical force, ultrasound, shock wave, magnetic field, and electric field; the small molecule inputs are selected from drugs, metabolites, and adjuvants; and/or the biologic inputs are selected from polypeptides, polypeptide derivatives, and nucleic acids.
Methods of evisenome editing
[0200] In some embodiments, provided herein are methods of conditionally editing an epigenome of a target genomic DNA in a cell using a split Casl2a system as described herein. The methods generally involve contacting the target nucleic acid with a nuclease-deficient split dCasl2a/crRNA complex comprising activity that modifies the epigenome of the target genomic DNA.
[0201] In some embodiments, provided herein is a method of conditionally editing an epigenome of a target genomic DNA in a cell using a split Casl2a system as described herein, wherein for two or more components of the split Casl2a system, the expression of each of the components is individually under the control of different inputs to the cell such that editing of the epigenome by a split Cas 12a/ crRNA complex only occurs when each of the different inputs have a desired state. In some embodiments, the split Cas 12a system comprises (a) nucleic acid encoding two or more effector subunits comprising fragments of a dCasl2a fusion protein or variant thereof comprising an effector domain having an epigenetic-editing effector activity, wherein the two or more effector subunits are capable of spontaneously assembling to form a split Cas 12a complex retaining at least some of the effector activity of the Cas 12a protein or variant thereof; and (b) a CRISPR RNA (crRNA) or nucleic acid encoding the crRNA, wherein the crRNA comprises a spacer having sufficient complementary to a target polynucleotide sequence in the target nucleic acid in the cell such that it can hybridize to the target nucleic acid. In some embodiments, the split Cas 12a system comprises nucleic acid encoding the first crRNA. In some embodiments, the split Cas 12a system further comprises nucleic acid comprising a NOT inducible promoter operably linked to a polynucleotide sequence encoding an anti-CRISPR (Acr) protein capable of inhibiting activity of the split Cas 12a complex, wherein a NOT input to the cell is capable of increasing the expression of Acr protein in the cell such that activity of split Cas 12a complex in the cell is inhibited. In some embodiments, the cell is a mammalian cell, e.g., a human cell.
[0202] In some embodiments, according to any of the methods of conditionally editing an epigenome of a target genomic DNA in a cell described herein employing nucleic acid comprising a polynucleotide sequence encoding an Acr protein, the Acr protein is AcrVAl or a variant thereof. In some embodiments, the AcrVAl or variant thereof comprises the amino acid sequence encoded by SEQ ID NO: 9 or a variant thereof having at least 80% sequence identity to the amino acid sequence encoded by SEQ ID NO: 9.
[0203] In some embodiments, according to any of the methods of conditionally editing an epigenome of a target genomic DNA in a cell described herein, a split Cas 12a complex that retains at least some of the effector activity of a Cas 12a protein or variant thereof retains at least about 5% (such as at least about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or greater) of the effector activity of the Cas 12a protein or variant thereof.
[0204] In some embodiments, according to any of the methods of conditionally editing an epigenome of a target genomic DNA in a cell described herein, the crRNA comprises a spacer sequence selected from the polynucleotide sequence of any one of SEQ ID NOs: 15-43.
[0205] In some embodiments, according to any of the methods of conditionally editing an epigenome of a target genomic DNA in a cell described herein employing nucleic acid comprising an inducible promoter, the inducible promoter is selected from an RRM2 promoter, a hypoxia-inducible promoter, a TRE3G promoter, a CMV5-CuO promoter, and a P2 inducible promoter. In some embodiments, the RRM2 promoter comprises the polynucleotide sequence of SEQ ID NO: 10. In some embodiments, the hypoxia-inducible promoter comprises the polynucleotide sequence of SEQ ID NO: 11. In some embodiments, the TRE3G promoter comprises the polynucleotide sequence of SEQ ID NO: 12. In some embodiments, the CMV5- CuO promoter comprises the polynucleotide sequence of SEQ ID NO: 13. In some embodiments, the P2 inducible promoter comprises the polynucleotide sequence of SEQ ID NO: 14. In some embodiments, where the split Casl2a system comprises nucleic acid comprising a plurality of inducible promoters, each of the plurality of inducible promoters operably linked to a polynucleotide sequence encoding an effector subunit are different. In some embodiments, where the split Casl2a system comprises nucleic acid comprising a plurality of inducible promoters, each of the plurality of inducible promoters operably linked to a polynucleotide sequence encoding a crRNA are different. In some embodiments, where the split Casl2a system comprises nucleic acid comprising a plurality of inducible promoters, at least some (such as at least about any of 2, 3, 4, or more) of the plurality of inducible promoters operably linked to a polynucleotide sequence encoding a crRNA are the same. In some embodiments, where the split Casl2a system comprises nucleic acid comprising a plurality of inducible promoters, all of the plurality of inducible promoters operably linked to a polynucleotide sequence encoding a crRNA are the same. In some embodiments, where the split Cas 12a system comprises nucleic acid comprising a plurality of inducible promoters, each of the plurality of inducible promoters are different.
[0206] In some embodiments, according to any of the methods of conditionally editing an epigenome of a target genomic DNA in a cell described herein, the system is configured such that all components of the Casl2a/crRNA complex are present or capable of being expressed in the cell when the first input and the second input each have a desired state. In some embodiments, the system is configured such that all components of the Casl2a/crRNA complex are present or capable of being expressed in the cell when the first input is present and the second input is present. [0207] In some embodiments, according to any of the methods of conditionally editing an epigenome of a target genomic DNA in a cell described herein, the system comprises (a) nucleic acid encoding a first effector subunit and a second effector subunit of the two or more effector subunits, wherein the nucleic acid comprises a first inducible promoter operably linked to a polynucleotide sequence encoding the first effector subunit and a second inducible promoter operably linked to a polynucleotide sequence encoding the second effector subunit, and the first input is capable of modulating the expression of the first effector subunit in the cell and the second input is capable of modulating the expression of the second effector subunit in the cell; or (b) nucleic acid encoding a first effector subunit of the two or more effector subunits, wherein the nucleic acid comprises a first inducible promoter operably linked to a polynucleotide sequence encoding the first effector subunit; and nucleic acid encoding the first crRNA, wherein the nucleic acid comprises a second inducible promoter operably linked to a polynucleotide sequence encoding the first crRNA, and the first input is capable of modulating the expression of the first effector subunit in the cell and the second input is capable of modulating the expression of the first crRNA in the cell. In some embodiments, modulating the expression of each of the Casl2a/crRNA complex components comprises increasing their expression.
[0208] In some embodiments, according to any of the methods of conditionally editing an epigenome of a target genomic DNA in a cell described herein, the expression of a third component of the Casl2a/crRNA complex is under the control of a third input to the cell, and the system is configured such that all components of the Casl2a/crRNA complex are present or capable of being expressed in the cell when the first input, the second input, and the third input each have a desired state. In some embodiments, the system is configured such that all components of the Casl2a/crRNA complex are present or capable of being expressed in the cell when the first input is present, the second input is present, and the third input is present. In some embodiments, the system comprises (a) nucleic acid encoding a first effector subunit, a second effector subunit, and a third effector subunit of the two or more effector subunits, wherein the nucleic acid comprises a first inducible promoter operably linked to a polynucleotide sequence encoding the first effector subunit, a second inducible promoter operably linked to a polynucleotide sequence encoding the second effector subunit, and a third inducible promoter operably linked to a polynucleotide sequence encoding the third effector subunit, and the first input is capable of modulating the expression of the first effector subunit in the cell, the second input is capable of modulating the expression of the second effector subunit in the cell, and the third input is capable of modulating the expression of the third effector subunit in the cell; or (b) nucleic acid encoding a first effector subunit and a second effector subunit of the two or more effector subunits, wherein the nucleic acid comprises a first inducible promoter operably linked to a polynucleotide sequence encoding the first effector subunit and a second inducible promoter operably linked to a polynucleotide sequence encoding the second effector subunit; and nucleic acid encoding the first crRNA, wherein the nucleic acid comprises a third inducible promoter operably linked to a polynucleotide sequence encoding the first crRNA, and the first input is capable of modulating the expression of the first effector subunit in the cell, the second input is capable of modulating the expression of the second effector subunit in the cell, and the third input is capable of modulating the expression of the first crRNA in the cell. In some embodiments, modulating the expression of each of the Casl2a/crRNA complex components comprises increasing their expression.
[0209] In some embodiments, according to any of the methods of conditionally editing an epigenome of a target genomic DNA in a cell described herein, the expression of a third component of the Casl2a/crRNA complex is under the control of a third input to the cell and the expression of a fourth component of the Casl2a/crRNA complex is under the control of a fourth input to the cell, and the system is configured such that all components of the Casl2a/crRNA complex are present or capable of being expressed in the cell when the first input, the second input, the third input, and the fourth input each have a desired state. In some embodiments, the system is configured such that all components of the Casl2a/crRNA complex are present or capable of being expressed when the first input is present, the second input is present, the third input is present, and the fourth input is present. In some embodiments, the system comprises nucleic acid encoding a first effector subunit, a second effector subunit, and a third effector subunit of the two or more effector subunits, wherein the nucleic acid comprises a first inducible promoter operably linked to a polynucleotide sequence encoding the first effector subunit, a second inducible promoter operably linked to a polynucleotide sequence encoding the second effector subunit, and a third inducible promoter operably linked to a polynucleotide sequence encoding the third effector subunit, and the first input is capable of modulating the expression of the first effector subunit in the cell, the second input is capable of modulating the expression of the second effector subunit in the cell, and the third input is capable of modulating the expression of the third effector subunit in the cell; and nucleic acid encoding the first crRNA, wherein the nucleic acid comprises a fourth inducible promoter operably linked to a polynucleotide sequence encoding the first crRNA, and the fourth input is capable of modulating the expression of the first crRNA. In some embodiments, modulating the expression of each of the Casl2a/crRNA complex components comprises increasing their expression.
[0210] In some embodiments, according to any of the methods of conditionally editing an epigenome of a target genomic DNA in a cell described herein, the two or more effector subunits comprise a first effector subunit and a second effector subunit, wherein the first effector subunit comprises an N-terminal fragment of the Casl2a protein or variant thereof up to and including a split point amino acid, and the second effector subunit comprises a C- terminal fragment of the Cas 12a protein or variant thereof following the split point amino acid. In some embodiments, the split point amino acid corresponds to A406 or G550 with respect to a parental Cas 12a protein amino acid sequence of SEQ ID NO: 1. In some embodiments, the first effector subunit further comprises a first stabilization domain at its C-terminus and the second effector subunit further comprises a second stabilization domain at its N-terminus, wherein the first stabilization domain and the second stabilization domain are capable of interacting with one another to stabilize a complex comprising the first effector subunit and the second effector subunit in the cell.
[0211] In some embodiments, according to any of the methods of conditionally editing an epigenome of a target genomic DNA in a cell described herein, the two or more effector subunits comprise a first effector subunit and a second effector subunit, wherein the Cas 12a protein or variant thereof is a fusion protein comprising an effector domain fused to a Cas 12a domain, and the first effector subunit comprises the Cas 12a domain and the second effector subunit comprises the effector domain. In some embodiments, the first effector subunit further comprises a first stabilization domain at either terminus and the second effector subunit further comprises a second stabilization domain at either terminus, wherein the first stabilization domain and the second stabilization domain are capable of interacting with one another to stabilize a complex comprising the first effector subunit and the second effector subunit in the cell.
[0212] In some embodiments, according to any of the methods of conditionally editing an epigenome of a target genomic DNA in a cell described herein, the two or more effector subunits comprise a first effector subunit, a second effector subunit, and a third effector subunit. In some embodiments, the Cas 12a protein or variant thereof is a fusion protein comprising an effector domain fused to a Cas 12a domain, and the first effector subunit comprises an N- terminal fragment of the Cas 12a domain up to and including a split point amino acid, the second effector subunit comprises a C-terminal fragment of the Cast 2a domain following the split point amino acid; and the third effector subunit comprises the effector domain. In some embodiments, the split point amino acid corresponds to A406 or G550 with respect to a parental Casl2a protein amino acid sequence of SEQ ID NO: 1. In some embodiments, the first effector subunit further comprises a first stabilization domain at its C-terminus and the second effector subunit further comprises a second stabilization domain at its N-terminus, wherein the first stabilization domain and the second stabilization domain are capable of interacting with one another to stabilize a complex comprising the first effector subunit and the second effector subunit in the cell; and/or the third effector subunit further comprises a third stabilization domain at either terminus and (i) the second effector subunit further comprises a fourth stabilization domain at its C-terminus or (ii) the first effector subunit further comprises the fourth stabilization domain at its N-terminus, wherein the third stabilization domain and the fourth stabilization domain are capable of interacting with one another to stabilize a complex comprising the first effector subunit, the second effector subunit, and the third effector subunit in the cell.
[0213] In some embodiments, according to any of the methods of conditionally editing an epigenome of a target genomic DNA in a cell described herein employing a first stabilization domain and a second stabilization domain, the first stabilization domain and the second stabilization domain are derived from subunits of a heterodimerizing binding pair or a split- intein. In some embodiments, where the method further employs a third stabilization domain and a fourth stabilization domain, the third stabilization domain and the fourth stabilization domain are derived from subunits of a heterodimerizing binding pair or a split-intein and are orthogonal to the first stabilization domain and the second stabilization domain. In some embodiments, the first stabilization domain and the second stabilization domain are derived from subunits of a heterodimerizing binding pair and the third stabilization domain and the fourth stabilization domain are derived from subunits of a split-intein; or the first stabilization domain and the second stabilization domain are derived from subunits of a split-intein and the third stabilization domain and the fourth stabilization domain are derived from subunits of a heterodimerizing binding pair. In some embodiments, the heterodimerizing binding pair is a coiled-coil (CC) heterodimer. In some embodiments, the CC heterodimer is a leucine zipper. In some embodiments, the leucine zipper subunits comprise RR12EE345L and EE12RR345L. In some embodiments, RR12EE345L comprises the amino acid sequence encoded by SEQ ID NO: 6. In some embodiments, EE12RR345L comprises the amino acid sequence encoded by SEQ ID NO: 5. In some embodiments, the CC heterodimer is a CC peptide heterodimer (see Lebar, T., et al. (2020). Nature Chemical Biology, 1-7). In some embodiments, the split-intein subunits comprise DnaE N-intein and C-intein. In some embodiments, DnaE N-intein comprises the amino acid sequence encoded by SEQ ID NO: 7. In some embodiments, DnaE C-intein comprises the amino acid sequence encoded by SEQ ID NO: 8. In some embodiments, the split-intein is gp41-l, gp41-8, NrdJ-1, or IMPDH-1 ( see Dassa, B., et al. (2009). Nucleic acids research, 57(8), 2560-2573). In some embodiments, the split-intein comprises split portions of the Mxe GyrA intein (see Fine, E. J., et al. (2015). Scientific reports, 5, 10777).
[0214] In some embodiments, according to any of the methods of conditionally editing an epigenome of a target genomic DNA in a cell described herein, the system further comprises one or more additional crRNAs or nucleic acid encoding the one or more additional crRNAs, wherein Casl2a/crRNA complexes formed by association of the one or more additional crRNAs with (I) the Casl2a protein or variant thereof or (II) the two or more effector subunits are capable of generating one or more additional outputs to the cell comprising effector activity of the Cas 12a/crRNA complexes at target sites directed by the one or more additional crRNAs. In some embodiments, the system comprises nucleic acid encoding a crRNA array comprising one or more of the first crRNA and the one or more additional crRNAs.
[0215] In some embodiments, according to any of the methods of conditionally editing an epigenome of a target genomic DNA in a cell described herein, the Cas 12a protein or variant thereof is derived from a parental Cas 12a protein from Lachnospiraceae bacterium. In some embodiments, the parental Cas 12a protein from Lachnospiraceae bacterium comprises the amino acid sequence of SEQ ID NO: 1. In some embodiments, the target nucleic acid is a target dsDNA. In some embodiments, a protospacer in the target dsDNA targeted by the crRNA spacer has a 5' PAM selected from 5'-TTTG-3', 5'-TTTA-3', and 5'-TTTC-3'.
[0216] In some embodiments, according to any of the methods of conditionally editing an epigenome of a target genomic DNA in a cell described herein, the Cas 12a protein or variant thereof is a fusion protein comprising an effector domain fused to a nuclease-deficient dCas 12a domain having reduced or eliminated nuclease activity as compared to a parental Cas 12a protein from which it is derived, and the first output is generated by activity of the effector domain. In some embodiments, the dCasl2a domain comprises a modification of amino acid D832 with respect to SEQ ID NO: 1 that reduces, substantially eliminates, or eliminates nuclease activity of the dCasl2a domain. In some embodiments, the dCasl2a domain comprises a D832A substitution with respect to SEQ ID NO: 1. In some embodiments, the dCasl2a domain comprises the amino acid sequence of SEQ ID NO: 2. In some embodiments, the effector domain comprises a polypeptide that can (i) methylate a nucleic acid, and/or (ii) demethylate a nucleic acid. In some embodiments, the effector domain has enzymatic activity that modifies a polypeptide associated with DNA (e.g., histone). In some embodiments, the enzymatic activity is methyltransferase activity, demethylase activity, acetyltransferase activity, deacetylase activity, kinase activity, phosphatase activity, ubiquitin ligase activity (e.g., ubiquitination activity), deubiquitinating activity, adenylation activity, deadenylation activity, SUMOylating activity, deSUMOylating activity, ribosylation activity, deribosylation activity, myristoylation activity, demyristoylation activity, glycosylation activity (e.g., from GlcNAc transferase), or deglycosylation activity.
[0217] In some embodiments, according to any of the methods of conditionally editing an epigenome of a target genomic DNA in a cell described herein, the inputs to the cell are selected from physical inputs, small molecule inputs, and biologic inputs. In some embodiments, the physical inputs are selected from oxygen levels, pH, ion concentrations, temperature, light, mechanical force, ultrasound, shock wave, magnetic field, and electric field; the small molecule inputs are selected from drugs, metabolites, and adjuvants; and/or the biologic inputs are selected from polypeptides, polypeptide derivatives, and nucleic acids.
Methods of treating a disease or condition
[0218] In some aspects of the disclosure, a split Casl2a system as described herein is employed to modify target gene expression from a target nucleic acid, or otherwise modify the target nucleic acid, for purposes of treating a disease or condition in a subject. The split Casl2a system components can be incorporated into a variety of formulations. More particularly, the split Casl2a system components of the present disclosure can be formulated into pharmaceutical compositions by combination with appropriate pharmaceutically acceptable carriers or diluents.
[0219] In some embodiments, provided herein are pharmaceutical preparations or compositions comprising components of a Casl2a system including (a) nucleic acid encoding two or more effector subunits comprising fragments of a Casl2a protein or variant thereof, wherein the two or more effector subunits are capable of spontaneously assembling to form a split Casl2a complex retaining at least some of the effector activity of the Casl2a protein or variant thereof; and (b) a CRISPR RNA (crRNA) or nucleic acid encoding the crRNA, wherein the crRNA comprises a spacer having sufficient complementary to a target polynucleotide sequence in a target nucleic acid such that it can hybridize to the target nucleic acid, present in a pharmaceutically acceptable vehicle. In some embodiments, the split Casl2a system comprises nucleic acid encoding the first crRNA. In some embodiments, the split Casl2a system further comprises nucleic acid comprising a NOT inducible promoter operably linked to a polynucleotide sequence encoding an anti-CRISPR (Acr) protein capable of inhibiting activity of the split Casl2a complex, wherein a NOT input to the cell is capable of increasing the expression of Acr protein in the cell such that activity of split Casl2a complex in the cell is inhibited. “Pharmaceutically acceptable vehicles” may be vehicles approved by a regulatory agency of the Federal or a state government or listed in the US Pharmacopeia or other generally recognized pharmacopeia for use in mammals, such as humans. The term “vehicle” refers to a diluent, adjuvant, excipient, or carrier with which a compound of the disclosure is formulated for administration to a mammal. Such pharmaceutical vehicles can be lipids, e.g., liposomes, e.g., liposome dendrimers; liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like, saline; gum acacia, gelatin, starch paste, talc, keratin, colloidal silica, urea, and the like. In addition, auxiliary, stabilizing, thickening, lubricating and coloring agents may be used. Pharmaceutical compositions may be formulated into preparations in solid, semisolid, liquid or gaseous forms, such as tablets, capsules, powders, granules, ointments, solutions, suppositories, injections, inhalants, gels, microspheres, and aerosols. As such, administration of the Casl2a system components can be achieved in various ways, including oral, buccal, rectal, parenteral, intraperitoneal, intradermal, transdermal, intra-tracheal, intraocular, etc., administration. The active agent may be systemic after administration or may be localized by the use of regional administration, intramural administration, or use of an implant that acts to retain the active dose at the site of implantation. The active agent may be formulated for immediate activity or it may be formulated for sustained release.
[0220] Pharmaceutical compositions can include, depending on the formulation desired, pharmaceutically acceptable, non-toxic carriers of diluents, which are defined as vehicles commonly used to formulate pharmaceutical compositions for animal or human administration. The diluent is selected so as not to affect the biological activity of the combination. Examples of such diluents are distilled water, buffered water, physiological saline, PBS, Ringer's solution, dextrose solution, and Hank's solution. In addition, the pharmaceutical composition or formulation can include other carriers, adjuvants, or non-toxic, nontherapeutic, nonimmunogenic stabilizers, excipients and the like. The compositions can also include additional substances to approximate physiological conditions, such as pH adjusting and buffering agents, toxicity adjusting agents, wetting agents and detergents.
[0221] The composition can also include any of a variety of stabilizing agents, such as an antioxidant for example. When the pharmaceutical composition includes a polypeptide, the polypeptide can be complexed with various well-known compounds that enhance the in vivo stability of the polypeptide, or otherwise enhance its pharmacological properties (e.g., increase the half-life of the polypeptide, reduce its toxicity, and/or enhance solubility or uptake). Examples of such modifications or complexing agents include sulfate, gluconate, citrate and phosphate. The nucleic acids or polypeptides of a composition can also be complexed with molecules that enhance their in vivo attributes. Such molecules include, for example, carbohydrates, polyamines, amino acids, other peptides, ions (e.g., sodium, potassium, calcium, magnesium, manganese), and lipids.
[0222] In some embodiments, provided herein is a method of treating a disease or condition in a subject in need thereof, the method comprising: 1) modulating the expression of a target gene in input cells according to any of the methods described herein, thereby producing engineered cells and administering the engineered cells to the subject; or 2) modulating the expression of a target gene in input cells in the subject according to any of the methods described herein, thereby producing engineered cells in the subject. In some embodiments, the input cells of 1) are autologous to the subject. In some embodiments, the input cells of 1) are allogeneic to the subject. In some embodiments, the subject is human.
[0223] In some embodiments, provided herein is a method of treating a disease or condition associated with a disease-associated cell in a subject in need thereof, the method comprising administering to the subject an engineered cell comprising a split Casl2a system as described herein, wherein the engineered cell is capable of mediating killing of the disease-associated cell to treat the disease or condition. In some embodiments, the engineered cell is capable of expressing a target gene, the first target nucleic acid comprises the target gene, and the first output of the system modulates the expression of the target gene in a target cell. In some embodiments, one or more NOT inputs of the split Casl2a system capable of increasing the expression of Acr protein in the engineered cell are associated with off-target cells and not the target cell. In some embodiments, the engineered cell is capable of expressing a target receptor, the first target nucleic acid comprises a polynucleotide sequence encoding the target receptor, and the first output of the system mediates expression of the target receptor. In some embodiments, the target receptor is a chimeric antigen receptor (CAR) targeting the disease- associated cell, the engineered cell is a CAR immune cell, and one or more inputs of the system are associated with the disease-associated cell. In some embodiments, the CAR immune cell is a CAR T cell, a CAR NK cell, or a CAR macrophage cell. In some embodiments, the target receptor is a T cell receptor (TCR) targeting the disease-associated cell, the engineered cell is a T cell, and one or more inputs of the system are associated with the disease-associated cell. In some embodiments, one or more NOT inputs of the system capable of increasing the expression of Acr protein in the cell are associated with healthy cells and not the disease- associated cell. In some embodiments, the engineered cells are autologous to the subject. In some embodiments, the engineered cells are allogeneic to the subject. In some embodiments, the subject is human. The number of administrations of treatment to a subject may vary. In some embodiments, introducing the engineered cells into the subject is a one-time event. In some embodiments, the method further comprises one or more additional administrations of engineered cells.
[0224] In some embodiments, provided herein is a method of treating a disease or condition associated with a disease-associated cell in a subject in need thereof, the method comprising modifying the disease-associated cell in the subject by introduction of a split Casl2a system as described herein to produce an engineered cell, wherein the engineered cell is capable of expressing a target gene, the first target nucleic acid comprises the target gene, one or more inputs of the system are disease-associated inputs characteristic of the disease-associated cell, and the first output of the system modulates expression of the target gene in the engineered cell such that the disease or condition is treated. In some embodiments, the first output of the system modulates expression of the target gene in the engineered cell such that it is rendered benign. In some embodiments, the first output of the system modulates expression of the target gene in the engineered cell such that it is killed. In some embodiments, the disease-associated cell is a cancer cell, and one or more inputs to the cell are cancer-associated inputs characteristic of the cancer cell. In some embodiments, one or more NOT inputs of the system capable of increasing the expression of Acr protein in the engineered cell are associated with non-disease-associated cells and not the disease-associated cell.
[0225] In some embodiments, provided herein is a method of treating a disease or condition associated with a target gene in a target cell in a subject in need thereof, the method comprising modifying the target cell in the subject by introduction of a split Casl2a system as described herein to produce an engineered cell, wherein the engineered cell is capable of expressing the target gene, the first target nucleic acid comprises the target gene, and the first output of the system modulates the expression of the target gene in the target cell, and wherein the expression of the target gene in the engineered cell is modulated such that the disease or condition is treated. In some embodiments, one or more NOT inputs of the split Casl2a system capable of increasing the expression of Acr protein in the engineered cell are associated with off-target cells and not the target cell.
[0226] In some embodiments, according to any of the methods of treating a disease or condition described herein employing nucleic acid comprising a polynucleotide sequence encoding an Acr protein, the Acr protein is AcrVAl or a variant thereof. In some embodiments, the AcrVAl or variant thereof comprises the amino acid sequence encoded by SEQ ID NO: 9 or a variant thereof having at least 80% sequence identity to the amino acid sequence encoded by SEQ ID NO: 9.
[0227] In some embodiments, according to any of the methods of treating a disease or condition described herein, a split Casl2a complex that retains at least some of the effector activity of a Casl2a protein or variant thereof retains at least about 5% (such as at least about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or greater) of the effector activity of the Casl2a protein or variant thereof.
[0228] In some embodiments, according to any of the methods of treating a disease or condition described herein, the crRNA comprises a spacer sequence selected from the polynucleotide sequence of any one of SEQ ID NOs: 15-43.
[0229] In some embodiments, according to any of the methods of treating a disease or condition described herein employing nucleic acid comprising an inducible promoter, the inducible promoter is selected from an RRM2 promoter, a hypoxia-inducible promoter, a TRE3G promoter, a CMV5-CuO promoter, and a P2 inducible promoter. In some embodiments, the RRM2 promoter comprises the polynucleotide sequence of SEQ ID NO: 10. In some embodiments, the hypoxia-inducible promoter comprises the polynucleotide sequence of SEQ ID NO: 11. In some embodiments, the TRE3G promoter comprises the polynucleotide sequence of SEQ ID NO: 12. In some embodiments, the CMV5-CuO promoter comprises the polynucleotide sequence of SEQ ID NO: 13. In some embodiments, the P2 inducible promoter comprises the polynucleotide sequence of SEQ ID NO: 14. In some embodiments, where the split Cas 12a system comprises nucleic acid comprising a plurality of inducible promoters, each of the plurality of inducible promoters operably linked to a polynucleotide sequence encoding an effector subunit are different. In some embodiments, where the split Cas 12a system comprises nucleic acid comprising a plurality of inducible promoters, each of the plurality of inducible promoters operably linked to a polynucleotide sequence encoding a crRNA are different. In some embodiments, where the split Casl2a system comprises nucleic acid comprising a plurality of inducible promoters, at least some (such as at least about any of 2, 3, 4, or more) of the plurality of inducible promoters operably linked to a polynucleotide sequence encoding a crRNA are the same. In some embodiments, where the split Casl2a system comprises nucleic acid comprising a plurality of inducible promoters, all of the plurality of inducible promoters operably linked to a polynucleotide sequence encoding a crRNA are the same. In some embodiments, where the split Casl2a system comprises nucleic acid comprising a plurality of inducible promoters, each of the plurality of inducible promoters are different. [0230] In some embodiments, according to any of the methods of treating a disease or condition described herein, the system is configured such that all components of the Casl2a/crRNA complex are present or capable of being expressed in the sample when the first input and the second input each have a desired state. In some embodiments, the system is configured such that all components of the Casl2a/crRNA complex are present or capable of being expressed in the sample when the first input is present and the second input is present. [0231] In some embodiments, according to any of the methods of treating a disease or condition described herein, the system comprises (a) nucleic acid encoding a first effector subunit and a second effector subunit of the two or more effector subunits, wherein the nucleic acid comprises a first inducible promoter operably linked to a polynucleotide sequence encoding the first effector subunit and a second inducible promoter operably linked to a polynucleotide sequence encoding the second effector subunit, and the first input is capable of modulating the expression of the first effector subunit in the sample and the second input is capable of modulating the expression of the second effector subunit in the sample; or (b) nucleic acid encoding a first effector subunit of the two or more effector subunits, wherein the nucleic acid comprises a first inducible promoter operably linked to a polynucleotide sequence encoding the first effector subunit; and nucleic acid encoding the first crRNA, wherein the nucleic acid comprises a second inducible promoter operably linked to a polynucleotide sequence encoding the first crRNA, and the first input is capable of modulating the expression of the first effector subunit in the sample and the second input is capable of modulating the expression of the first crRNA in the sample. In some embodiments, modulating the expression of each of the Casl2a/crRNA complex components comprises increasing their expression. [0232] In some embodiments, according to any of the methods of treating a disease or condition described herein, the expression of a third component of the Casl2a/crRNA complex is under the control of a third input to the sample, and the system is configured such that all components of the Casl2a/crRNA complex are present or capable of being expressed in the sample when the first input, the second input, and the third input each have a desired state. In some embodiments, the system is configured such that all components of the Casl2a/crRNA complex are present or capable of being expressed in the sample when the first input is present, the second input is present, and the third input is present. In some embodiments, the system comprises (a) nucleic acid encoding a first effector subunit, a second effector subunit, and a third effector subunit of the two or more effector subunits, wherein the nucleic acid comprises a first inducible promoter operably linked to a polynucleotide sequence encoding the first effector subunit, a second inducible promoter operably linked to a polynucleotide sequence encoding the second effector subunit, and a third inducible promoter operably linked to a polynucleotide sequence encoding the third effector subunit, and the first input is capable of modulating the expression of the first effector subunit in the sample, the second input is capable of modulating the expression of the second effector subunit in the sample, and the third input is capable of modulating the expression of the third effector subunit in the sample; or (b) nucleic acid encoding a first effector subunit and a second effector subunit of the two or more effector subunits, wherein the nucleic acid comprises a first inducible promoter operably linked to a polynucleotide sequence encoding the first effector subunit and a second inducible promoter operably linked to a polynucleotide sequence encoding the second effector subunit; and nucleic acid encoding the first crRNA, wherein the nucleic acid comprises a third inducible promoter operably linked to a polynucleotide sequence encoding the first crRNA, and the first input is capable of modulating the expression of the first effector subunit in the sample, the second input is capable of modulating the expression of the second effector subunit in the sample, and the third input is capable of modulating the expression of the first crRNA in the sample. In some embodiments, modulating the expression of each of the Casl2a/crRNA complex components comprises increasing their expression.
[0233] In some embodiments, according to any of the methods of treating a disease or condition described herein, the expression of a third component of the Casl2a/crRNA complex is under the control of a third input to the sample and the expression of a fourth component of the Casl2a/crRNA complex is under the control of a fourth input to the sample, and the system is configured such that all components of the Casl2a/crRNA complex are present or capable of being expressed in the sample when the first input, the second input, the third input, and the fourth input each have a desired state. In some embodiments, the system is configured such that all components of the Casl2a/crRNA complex are present or capable of being expressed when the first input is present, the second input is present, the third input is present, and the fourth input is present. In some embodiments, the system comprises nucleic acid encoding a first effector subunit, a second effector subunit, and a third effector subunit of the two or more effector subunits, wherein the nucleic acid comprises a first inducible promoter operably linked to a polynucleotide sequence encoding the first effector subunit, a second inducible promoter operably linked to a polynucleotide sequence encoding the second effector subunit, and a third inducible promoter operably linked to a polynucleotide sequence encoding the third effector subunit, and the first input is capable of modulating the expression of the first effector subunit in the sample, the second input is capable of modulating the expression of the second effector subunit in the sample, and the third input is capable of modulating the expression of the third effector subunit in the sample; and nucleic acid encoding the first crRNA, wherein the nucleic acid comprises a fourth inducible promoter operably linked to a polynucleotide sequence encoding the first crRNA, and the fourth input is capable of modulating the expression of the first crRNA. In some embodiments, modulating the expression of each of the Casl2a/crRNA complex components comprises increasing their expression.
[0234] In some embodiments, according to any of the methods of treating a disease or condition described herein, the two or more effector subunits comprise a first effector subunit and a second effector subunit, wherein the first effector subunit comprises an N-terminal fragment of the Casl2a protein or variant thereof up to and including a split point amino acid, and the second effector subunit comprises a C-terminal fragment of the Casl2a protein or variant thereof following the split point amino acid. In some embodiments, the split point amino acid corresponds to A406 or G550 with respect to a parental Casl2a protein amino acid sequence of SEQ ID NO: 1. In some embodiments, the first effector subunit further comprises a first stabilization domain at its C-terminus and the second effector subunit further comprises a second stabilization domain at its N-terminus, wherein the first stabilization domain and the second stabilization domain are capable of interacting with one another to stabilize a complex comprising the first effector subunit and the second effector subunit in the sample.
[0235] In some embodiments, according to any of the methods of treating a disease or condition described herein, the two or more effector subunits comprise a first effector subunit and a second effector subunit, wherein the Casl2a protein or variant thereof is a fusion protein comprising an effector domain fused to a Casl2a domain, and the first effector subunit comprises the Casl2a domain and the second effector subunit comprises the effector domain. In some embodiments, the first effector subunit further comprises a first stabilization domain at either terminus and the second effector subunit further comprises a second stabilization domain at either terminus, wherein the first stabilization domain and the second stabilization domain are capable of interacting with one another to stabilize a complex comprising the first effector subunit and the second effector subunit in the sample.
[0236] In some embodiments, according to any of the methods of treating a disease or condition described herein, the two or more effector subunits comprise a first effector subunit, a second effector subunit, and a third effector subunit. In some embodiments, the Casl2a protein or variant thereof is a fusion protein comprising an effector domain fused to a Casl2a domain, and the first effector subunit comprises anN-terminal fragment of the Casl2a domain up to and including a split point amino acid, the second effector subunit comprises a C-terminal fragment of the Casl2a domain following the split point amino acid; and the third effector subunit comprises the effector domain. In some embodiments, the split point amino acid corresponds to A406 or G550 with respect to a parental Casl2a protein amino acid sequence of SEQ ID NO: 1. In some embodiments, the first effector subunit further comprises a first stabilization domain at its C-terminus and the second effector subunit further comprises a second stabilization domain at its N-terminus, wherein the first stabilization domain and the second stabilization domain are capable of interacting with one another to stabilize a complex comprising the first effector subunit and the second effector subunit in the sample; and/or the third effector subunit further comprises a third stabilization domain at either terminus and (i) the second effector subunit further comprises a fourth stabilization domain at its C-terminus or (ii) the first effector subunit further comprises the fourth stabilization domain at its N-terminus, wherein the third stabilization domain and the fourth stabilization domain are capable of interacting with one another to stabilize a complex comprising the first effector subunit, the second effector subunit, and the third effector subunit in the sample.
[0237] In some embodiments, according to any of the methods of treating a disease or condition described herein employing a first stabilization domain and a second stabilization domain, the first stabilization domain and the second stabilization domain are derived from subunits of a heterodimerizing binding pair or a split-intein. In some embodiments, where the method further employs a third stabilization domain and a fourth stabilization domain, the third stabilization domain and the fourth stabilization domain are derived from subunits of a heterodimerizing binding pair or a split-intein and are orthogonal to the first stabilization domain and the second stabilization domain. In some embodiments, the first stabilization domain and the second stabilization domain are derived from subunits of a heterodimerizing binding pair and the third stabilization domain and the fourth stabilization domain are derived from subunits of a split-intein; or the first stabilization domain and the second stabilization domain are derived from subunits of a split-intein and the third stabilization domain and the fourth stabilization domain are derived from subunits of a heterodimerizing binding pair. In some embodiments, the heterodimerizing binding pair is a coiled-coil (CC) heterodimer. In some embodiments, the CC heterodimer is a leucine zipper. In some embodiments, the leucine zipper subunits comprise RR12EE345L and EE12RR345L. In some embodiments, RR12EE345L comprises the amino acid sequence encoded by SEQ ID NO: 6. In some embodiments, EE12RR345L comprises the amino acid sequence encoded by SEQ ID NO: 5. In some embodiments, the CC heterodimer is a CC peptide heterodimer ( see Lebar, T., et al. (2020). Nature Chemical Biology, 1-7). In some embodiments, the split-intein subunits comprise DnaE N-intein and C-intein. In some embodiments, DnaE N-intein comprises the amino acid sequence encoded by SEQ ID NO: 7. In some embodiments, DnaE C-intein comprises the amino acid sequence encoded by SEQ ID NO: 8. In some embodiments, the split-intein is gp41 - 1, gp41-8, NrdJ-1, or IMPDH-1 ( see Dassa, B., et al. (2009). Nucleic acids research, 57(8), 2560-2573). In some embodiments, the split-intein comprises split portions of the Mxe GyrA intein (see Fine, E. J., et al. (2015). Scientific reports, 5, 10777).
[0238] In some embodiments, according to any of the methods of treating a disease or condition described herein, the system further comprises one or more additional crRNAs or nucleic acid encoding the one or more additional crRNAs, wherein Casl2a/crRNA complexes formed by association of the one or more additional crRNAs with (I) the Casl2a protein or variant thereof or (II) the two or more effector subunits are capable of generating one or more additional outputs to the sample comprising effector activity of the Casl2a/crRNA complexes at target sites directed by the one or more additional crRNAs. In some embodiments, the system comprises nucleic acid encoding a crRNA array comprising one or more of the first crRNA and the one or more additional crRNAs.
[0239] In some embodiments, according to any of the methods of treating a disease or condition described herein, the Casl2a protein or variant thereof is derived from a parental Casl2a protein from Lachnospiraceae bacterium. In some embodiments, the parental Casl2a protein from Lachnospiraceae bacterium comprises the amino acid sequence of SEQ ID NO: 1. In some embodiments, the target nucleic acid is a target dsDNA. In some embodiments, a protospacer in the target dsDNA targeted by the crRNA spacer has a 5' PAM selected from 5'- TTTG-3', 5'-TTTA-3', and 5'-TTTC-3'. [0240] In some embodiments, according to any of the methods of treating a disease or condition described herein, the Cast 2a protein or variant thereof is a nuclease-competent Cas protein comprising a Cas 12a domain having some, the same, or greater nuclease activity as compared to a parental Cas 12a protein from which it is derived, and the first output comprises cleavage (e.g., double-stranded cleavage) of the first target nucleic acid near the first target polynucleotide sequence. In some embodiments, the Cas 12a domain comprises the amino acid sequence of SEQ ID NO: 1.
[0241] In some embodiments, according to any of the methods of treating a disease or condition described herein, the Cas 12a protein or variant thereof is a fusion protein comprising an effector domain fused to a nuclease-deficient dCasl2a domain having reduced or eliminated nuclease activity as compared to a parental Cas 12a protein from which it is derived, and the first output is generated by activity of the effector domain. In some embodiments, the dCasl2a domain comprises a modification of amino acid D832 with respect to SEQ ID NO: 1 that reduces, substantially eliminates, or eliminates nuclease activity of the dCasl2a domain. In some embodiments, the dCasl2a domain comprises a D832A substitution with respect to SEQ ID NO: 1. In some embodiments, the dCasl2a domain comprises the amino acid sequence of SEQ ID NO: 2. In some embodiments, the effector domain comprises a polypeptide that can (i) cleave a nucleic acid, (ii) edit a nucleotide, (iii) activate transcription, (iv) repress transcription, (v) methylate a nucleic acid, and/or (vi) demethylate a nucleic acid. In some embodiments, the effector domain comprises a transcriptional activation domain, and the first output comprises transcriptional activation of a first gene targeted by the first crRNA. In some embodiments, the transcriptional activation domain is a tripartite VP64-p65-Rta (VPR) transcriptional activation domain or variant thereof.
[0242] In some embodiments, according to any of the methods of treating a disease or condition described herein, the sample is a cell or a composition comprising a cell. In some embodiments, the inputs to the sample are selected from physical inputs, small molecule inputs, and biologic inputs. In some embodiments, the physical inputs are selected from oxygen levels, pH, ion concentrations, temperature, light, mechanical force, ultrasound, shock wave, magnetic field, and electric field; the small molecule inputs are selected from drugs, metabolites, and adjuvants; and/or the biologic inputs are selected from polypeptides, polypeptide derivatives, and nucleic acids.
Methods of generating an engineered cell
[0243] In some aspects of the disclosure, one or more components of a split Cas 12a system are introduced into a cell to generate an engineered cell. In some of these embodiments, components of a split Casl2a system include (a) two or more effector subunits comprising fragments of a Casl2a protein or variant thereof, or nucleic acid encoding the two or more effector subunits, wherein the two or more effector subunits are capable of spontaneously assembling to form a split Casl2a complex retaining at least some of the effector activity of the Casl2a protein or variant thereof; and (b) a first CRISPR RNA (crRNA) or nucleic acid encoding the first crRNA, wherein the first crRNA comprises a first spacer having sufficient complementary to a first target polynucleotide sequence in a first target nucleic acid in the cell such that it can hybridize to the first target nucleic acid. The engineered cells can be incorporated into a variety of formulations. More particularly, the engineered cells of the present disclosure can be formulated into pharmaceutical compositions by combination with appropriate pharmaceutically acceptable carriers or diluents.
[0244] In some embodiments, provided herein is a method of generating an engineered cell comprising introducing into an input cell one or more components of a split Casl2a system as described herein. In some embodiments, the method comprises introducing into the input cell all of the components of the split Casl2a system. In some embodiments, the split Casl2a system comprises (a) nucleic acid encoding two or more effector subunits comprising fragments of a Casl2a protein or variant thereof, wherein the two or more effector subunits are capable of spontaneously assembling to form a split Casl2a complex retaining at least some of the effector activity of the Casl2a protein or variant thereof; and (b) a first CRISPR RNA (crRNA) or nucleic acid encoding the first crRNA, wherein the first crRNA comprises a first spacer having sufficient complementary to a first target polynucleotide sequence in a first target nucleic acid in the cell such that it can hybridize to the first target nucleic acid. In some embodiments, the split Casl2a system comprises nucleic acid encoding the first crRNA. In some embodiments, the split Casl2a system further comprises nucleic acid comprising a NOT inducible promoter operably linked to a polynucleotide sequence encoding an anti-CRISPR (Acr) protein capable of inhibiting activity of the split Casl2a complex, wherein a NOT input to the sample is capable of increasing the expression of Acr protein from the nucleic acid in the sample such that activity of Casl2a complex in the sample is inhibited.
Engineered Cells
[0245] In some embodiments, the split Casl2a systems described herein are used in eukaryotic cells, such as mammalian cells, for example, human cells, to produce engineered cells with modulated expression of a target gene. Any human cell is contemplated for use with the split Casl2a systems disclosed herein.
[0246] In some embodiments, an engineered cell ex vivo or in vitro includes: (a) nucleic acid encoding two or more effector subunits comprising fragments of a Casl2a protein or variant thereof, wherein the two or more effector subunits are capable of spontaneously assembling to form a split Casl2a complex retaining at least some of the effector activity of the Casl2a protein or variant thereof; and (b) a CRISPR RNA (crRNA) or nucleic acid encoding the crRNA, wherein the crRNA comprises a spacer having sufficient complementary to a target polynucleotide sequence in a target nucleic acid in the cell such that it can hybridize to the target nucleic acid.
[0247] In one aspect, some embodiments disclosed herein relate to a method of engineering an input cell that includes introducing into the input cell, such as an animal cell, one or more components of a split Casl2a system as described herein, and selecting or screening for an engineered cell transformed by the one or more system components. The term “engineered cell” refers not only to the particular subject cell but also to the progeny or potential progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein. Techniques for transforming a wide variety of cell are known in the art.
[0248] In a related aspect, some embodiments relate to engineered cells, for example, engineered animal cells that include a heterologous nucleic acid and/or polypeptide as described herein. The nucleic acid can be stably integrated in the host genome, or can be episomally replicating, or present in the engineered cell as a mini-circle expression vector for stable or transient expression.
[0249] In some embodiments, provided herein is an engineered cell, e.g., an isolated engineered cell, prepared by modulating the expression of a target gene in a target nucleic acid or otherwise modifying the target nucleic acid in an input cell according to any of the methods described herein, thereby producing the engineered cell.
[0250] In some embodiments, provided herein is an engineered cell prepared by a method comprising providing to an input cell a split Casl2a system as described herein, wherein for two or more components of the split Casl2a system, the expression of each of the components is individually under the control of different inputs to the engineered cell such that an output mediated by a split Casl2a/crRNA complex is only generated when each of the different inputs have a desired state. In some embodiments, the split Casl2a system comprises (a) nucleic acid encoding two or more effector subunits comprising fragments of a Cast 2a protein or variant thereof, wherein the two or more effector subunits are capable of spontaneously assembling to form a split Casl2a complex retaining at least some of the effector activity of the Casl2a protein or variant thereof; and (b) a CRISPR RNA (crRNA) or nucleic acid encoding the crRNA, wherein the crRNA comprises a spacer having sufficient complementary to a target polynucleotide sequence in the target nucleic acid in the engineered cell such that it can hybridize to the target nucleic acid. In some embodiments, the split Casl2a system comprises nucleic acid encoding the first crRNA. In some embodiments, the split Casl2a system further comprises nucleic acid comprising a NOT inducible promoter operably linked to a polynucleotide sequence encoding an anti-CRISPR (Acr) protein capable of inhibiting activity of the split Casl2a complex, wherein a NOT input to the sample is capable of increasing the expression of Acr protein in the engineered cell such that activity of split Casl2a complex in the engineered cell is inhibited. In some embodiments, the engineered cell is a mammalian cell, e.g., a human cell.
[0251] In some embodiments, according to any of the engineered cells described herein, the engineered cell is capable of expressing a target gene, the first target nucleic acid comprises the target gene, and the first output of the system modulates the expression of the target gene in a target cell. In some embodiments, one or more NOT inputs of the system capable of increasing the expression of Acr protein in the engineered cell are associated with off-target cells and not the target cell.
[0252] In some embodiments, according to any of the engineered cells described herein, the engineered cell is capable of expressing a target receptor, the first target nucleic acid comprises a polynucleotide sequence encoding the target receptor, and the first output of the system mediates expression of the target receptor. In some embodiments, the target receptor is a chimeric antigen receptor (CAR), the engineered cell is a CAR immune cell, and one or more inputs of the system are associated with a target cell targeted by the CAR immune cell. In some embodiments, the CAR immune cell is a CAR T cell, a CAR NK cell, or a CAR macrophage cell. In some embodiments, the target receptor is a T cell receptor (TCR), the engineered cell is a T cell, and one or more inputs of the system are associated with a target cell targeted by the TCR. In some embodiments, one or more NOT inputs of the system capable of increasing the expression of Acr protein in the engineered cell are associated with off-target cells and not the target cell.
[0253] In some embodiments, according to any of the engineered cells described herein, the engineered cell is a disease-associated cell capable of expressing a target gene, the first target nucleic acid comprises the target gene, one or more inputs of the system are disease-associated inputs characteristic of the disease-associated cell, and the first output of the system mediates expression of the target gene in the disease-associated cell. In some embodiments, the first output of the system mediates expression of the target gene in the engineered cell such that it is rendered benign. In some embodiments, the first output of the system mediates expression of the target gene in the engineered cell such that it is killed. In some embodiments, the disease- associated cell is a cancer cell, and one or more inputs to the cell are cancer-associated inputs characteristic of the cancer cell. In some embodiments, one or more NOT inputs of the system capable of increasing the expression of Acr protein in the cell are associated with non-disease- associated cells and not the disease-associated cell.
[0254] All cells suitable to be a target cell as discussed above are also suitable to be an engineered cell. For example, an engineered cell can be prepared from an input cell from any organism, e.g., a bacterial cell, an archaeal cell, a cell of a single-cell eukaryotic organism, a plant cell, an algal cell (e.g., Botryococcus braunii, Chlamydomonas reinhardtii, Nannochloropsis gaditana, Chlorela pyrenoidosa, Sargassum patens C. Agardh, and the like), a fungal cell (e.g., a yeast cell), an animal cell, a cell from an invertebrate animal (e.g., fruit fly, cnidarian, echinoderm, nematode, etc.), a cell from a vertebrate animal (e.g., fish, amphibian, reptile, bird, mammal), a cell from a mammal (e.g., a pig, a cow, a goat, a sheep, a rodent a rat, a mouse, a non-human primate, a human, etc.). In some embodiments, the engineered cell can be prepared from any input cell from a human.
[0255] In some embodiments, an engineered cell is in vitro. In some embodiments, an engineered cell is in vivo. In some embodiments, an engineered cell is a prokaryotic cell or is derived from a prokaryotic cell. In some embodiments, an engineered cell is a bacterial cell or is derived from a bacterial cell. In some embodiments, an engineered cell is an archaeal cell or is derived from an archaeal cell. In some embodiments, an engineered cell is a eukaryotic cell or is derived from a eukaryotic cell. In some embodiments, an engineered cell is a plant cell or is derived from a plant cell. In some embodiments, an engineered cell is an animal cell or is derived from an animal cell. In some embodiments, an engineered cell is an invertebrate cell or is derived from an invertebrate cell. In some embodiments, an engineered cell is a vertebrate cell or is derived from a vertebrate cell. In some embodiments, an engineered cell is a mammalian cell or is derived from a mammalian cell. In some embodiments, an engineered cell is a rodent cell or is derived from a rodent cell. In some embodiments, an engineered cell is a human cell or is derived from a human cell. In some embodiments, the engineered cell is a human cell or is derived from a human cell.
[0256] The present disclosure further provides progeny of an engineered cell, where the progeny can include the same exogenous nucleic acid or polypeptide as the engineered cell from which it was derived. The present disclosure further provides, in some embodiments, a composition comprising an engineered cell.
[0257] In some embodiments, according to any of the engineered cells described herein employing nucleic acid comprising a polynucleotide sequence encoding an Acr protein, the Acr protein is AcrVAl or a variant thereof. In some embodiments, the AcrVAl or variant thereof comprises the amino acid sequence encoded by SEQ ID NO: 9 or a variant thereof having at least 80% sequence identity to the amino acid sequence encoded by SEQ ID NO: 9. [0258] In some embodiments, according to any of the engineered cells described herein, a split Casl2a complex that retains at least some of the effector activity of a Casl2a protein or variant thereof retains at least about 5% (such as at least about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or greater) of the effector activity of the Casl2a protein or variant thereof.
[0259] In some embodiments, according to any of the engineered cells described herein, the crRNA comprises a spacer sequence selected from the polynucleotide sequence of any one of SEQ ID NOs: 15-43.
[0260] In some embodiments, according to any of the engineered cells described herein employing nucleic acid comprising an inducible promoter, the inducible promoter is selected from an RRM2 promoter, a hypoxia-inducible promoter, a TRE3G promoter, a CMV5-CuO promoter, and a P2 inducible promoter. In some embodiments, the RRM2 promoter comprises the polynucleotide sequence of SEQ ID NO: 10. In some embodiments, the hypoxia-inducible promoter comprises the polynucleotide sequence of SEQ ID NO: 11. In some embodiments, the TRE3G promoter comprises the polynucleotide sequence of SEQ ID NO: 12. In some embodiments, the CMV5-CuO promoter comprises the polynucleotide sequence of SEQ ID NO: 13. In some embodiments, the P2 inducible promoter comprises the polynucleotide sequence of SEQ ID NO: 14. In some embodiments, where the split Casl2a system comprises nucleic acid comprising a plurality of inducible promoters, each of the plurality of inducible promoters operably linked to a polynucleotide sequence encoding an effector subunit are different. In some embodiments, where the split Casl2a system comprises nucleic acid comprising a plurality of inducible promoters, each of the plurality of inducible promoters operably linked to a polynucleotide sequence encoding a crRNA are different. In some embodiments, where the split Casl2a system comprises nucleic acid comprising a plurality of inducible promoters, at least some (such as at least about any of 2, 3, 4, or more) of the plurality of inducible promoters operably linked to a polynucleotide sequence encoding a crRNA are the same. In some embodiments, where the split Casl2a system comprises nucleic acid comprising a plurality of inducible promoters, all of the plurality of inducible promoters operably linked to a polynucleotide sequence encoding a crRNA are the same. In some embodiments, where the split Casl2a system comprises nucleic acid comprising a plurality of inducible promoters, each of the plurality of inducible promoters are different.
[0261] In some embodiments, according to any of the engineered cells described herein, the system is configured such that all components of the Casl2a/crRNA complex are present or capable of being expressed in the sample when the first input and the second input each have a desired state. In some embodiments, the system is configured such that all components of the Casl2a/crRNA complex are present or capable of being expressed in the sample when the first input is present and the second input is present.
[0262] In some embodiments, according to any of the engineered cells described herein, the system comprises (a) nucleic acid encoding a first effector subunit and a second effector subunit of the two or more effector subunits, wherein the nucleic acid comprises a first inducible promoter operably linked to a polynucleotide sequence encoding the first effector subunit and a second inducible promoter operably linked to a polynucleotide sequence encoding the second effector subunit, and the first input is capable of modulating the expression of the first effector subunit in the sample and the second input is capable of modulating the expression of the second effector subunit in the sample; or (b) nucleic acid encoding a first effector subunit of the two or more effector subunits, wherein the nucleic acid comprises a first inducible promoter operably linked to a polynucleotide sequence encoding the first effector subunit; and nucleic acid encoding the first crRNA, wherein the nucleic acid comprises a second inducible promoter operably linked to a polynucleotide sequence encoding the first crRNA, and the first input is capable of modulating the expression of the first effector subunit in the sample and the second input is capable of modulating the expression of the first crRNA in the sample. In some embodiments, modulating the expression of each of the Casl2a/crRNA complex components comprises increasing their expression.
[0263] In some embodiments, according to any of the engineered cells described herein, the expression of a third component of the Casl2a/crRNA complex is under the control of a third input to the sample, and the system is configured such that all components of the Casl2a/crRNA complex are present or capable of being expressed in the sample when the first input, the second input, and the third input each have a desired state. In some embodiments, the system is configured such that all components of the Casl2a/crRNA complex are present or capable of being expressed in the sample when the first input is present, the second input is present, and the third input is present. In some embodiments, the system comprises (a) nucleic acid encoding a first effector subunit, a second effector subunit, and a third effector subunit of the two or more effector subunits, wherein the nucleic acid comprises a first inducible promoter operably linked to a polynucleotide sequence encoding the first effector subunit, a second inducible promoter operably linked to a polynucleotide sequence encoding the second effector subunit, and a third inducible promoter operably linked to a polynucleotide sequence encoding the third effector subunit, and the first input is capable of modulating the expression of the first effector subunit in the sample, the second input is capable of modulating the expression of the second effector subunit in the sample, and the third input is capable of modulating the expression of the third effector subunit in the sample; or (b) nucleic acid encoding a first effector subunit and a second effector subunit of the two or more effector subunits, wherein the nucleic acid comprises a first inducible promoter operably linked to a polynucleotide sequence encoding the first effector subunit and a second inducible promoter operably linked to a polynucleotide sequence encoding the second effector subunit; and nucleic acid encoding the first crRNA, wherein the nucleic acid comprises a third inducible promoter operably linked to a polynucleotide sequence encoding the first crRNA, and the first input is capable of modulating the expression of the first effector subunit in the sample, the second input is capable of modulating the expression of the second effector subunit in the sample, and the third input is capable of modulating the expression of the first crRNA in the sample. In some embodiments, modulating the expression of each of the Casl2a/crRNA complex components comprises increasing their expression.
[0264] In some embodiments, according to any of the engineered cells described herein, the expression of a third component of the Casl2a/crRNA complex is under the control of a third input to the sample and the expression of a fourth component of the Casl2a/crRNA complex is under the control of a fourth input to the sample, and the system is configured such that all components of the Casl2a/crRNA complex are present or capable of being expressed in the sample when the first input, the second input, the third input, and the fourth input each have a desired state. In some embodiments, the system is configured such that all components of the Casl2a/crRNA complex are present or capable of being expressed when the first input is present, the second input is present, the third input is present, and the fourth input is present. In some embodiments, the system comprises nucleic acid encoding a first effector subunit, a second effector subunit, and a third effector subunit of the two or more effector subunits, wherein the nucleic acid comprises a first inducible promoter operably linked to a polynucleotide sequence encoding the first effector subunit, a second inducible promoter operably linked to a polynucleotide sequence encoding the second effector subunit, and a third inducible promoter operably linked to a polynucleotide sequence encoding the third effector subunit, and the first input is capable of modulating the expression of the first effector subunit in the sample, the second input is capable of modulating the expression of the second effector subunit in the sample, and the third input is capable of modulating the expression of the third effector subunit in the sample; and nucleic acid encoding the first crRNA, wherein the nucleic acid comprises a fourth inducible promoter operably linked to a polynucleotide sequence encoding the first crRNA, and the fourth input is capable of modulating the expression of the first crRNA. In some embodiments, modulating the expression of each of the Casl2a/crRNA complex components comprises increasing their expression.
[0265] In some embodiments, according to any of the engineered cells described herein, the two or more effector subunits comprise a first effector subunit and a second effector subunit, wherein the first effector subunit comprises an N-terminal fragment of the Casl2a protein or variant thereof up to and including a split point amino acid, and the second effector subunit comprises a C-terminal fragment of the Casl2a protein or variant thereof following the split point amino acid. In some embodiments, the split point amino acid corresponds to A406 or G550 with respect to a parental Cas 12a protein amino acid sequence of SEQ ID NO: 1. In some embodiments, the first effector subunit further comprises a first stabilization domain at its C- terminus and the second effector subunit further comprises a second stabilization domain at its N-terminus, wherein the first stabilization domain and the second stabilization domain are capable of interacting with one another to stabilize a complex comprising the first effector subunit and the second effector subunit in the sample.
[0266] In some embodiments, according to any of the engineered cells described herein, the two or more effector subunits comprise a first effector subunit and a second effector subunit, wherein the Cas 12a protein or variant thereof is a fusion protein comprising an effector domain fused to a Cas 12a domain, and the first effector subunit comprises the Cas 12a domain and the second effector subunit comprises the effector domain. In some embodiments, the first effector subunit further comprises a first stabilization domain at either terminus and the second effector subunit further comprises a second stabilization domain at either terminus, wherein the first stabilization domain and the second stabilization domain are capable of interacting with one another to stabilize a complex comprising the first effector subunit and the second effector subunit in the sample.
[0267] In some embodiments, according to any of the engineered cells described herein, the two or more effector subunits comprise a first effector subunit, a second effector subunit, and a third effector subunit. In some embodiments, the Casl2a protein or variant thereof is a fusion protein comprising an effector domain fused to a Casl2a domain, and the first effector subunit comprises an N-terminal fragment of the Cas 12a domain up to and including a split point amino acid, the second effector subunit comprises a C-terminal fragment of the Cas 12a domain following the split point amino acid; and the third effector subunit comprises the effector domain. In some embodiments, the split point amino acid corresponds to A406 or G550 with respect to a parental Casl2a protein amino acid sequence of SEQ ID NO: 1. In some embodiments, the first effector subunit further comprises a first stabilization domain at its C- terminus and the second effector subunit further comprises a second stabilization domain at its N-terminus, wherein the first stabilization domain and the second stabilization domain are capable of interacting with one another to stabilize a complex comprising the first effector subunit and the second effector subunit in the sample; and/or the third effector subunit further comprises a third stabilization domain at either terminus and (i) the second effector subunit further comprises a fourth stabilization domain at its C-terminus or (ii) the first effector subunit further comprises the fourth stabilization domain at its N-terminus, wherein the third stabilization domain and the fourth stabilization domain are capable of interacting with one another to stabilize a complex comprising the first effector subunit, the second effector subunit, and the third effector subunit in the sample.
[0268] In some embodiments, according to any of the engineered cells described herein employing a first stabilization domain and a second stabilization domain, the first stabilization domain and the second stabilization domain are derived from subunits of a heterodimerizing binding pair or a split-intein. In some embodiments, where the method further employs a third stabilization domain and a fourth stabilization domain, the third stabilization domain and the fourth stabilization domain are derived from subunits of a heterodimerizing binding pair or a split-intein and are orthogonal to the first stabilization domain and the second stabilization domain. In some embodiments, the first stabilization domain and the second stabilization domain are derived from subunits of a heterodimerizing binding pair and the third stabilization domain and the fourth stabilization domain are derived from subunits of a split-intein; or the first stabilization domain and the second stabilization domain are derived from subunits of a split-intein and the third stabilization domain and the fourth stabilization domain are derived from subunits of a heterodimerizing binding pair. In some embodiments, the heterodimerizing binding pair is a coiled-coil (CC) heterodimer. In some embodiments, the CC heterodimer is a leucine zipper. In some embodiments, the leucine zipper subunits comprise RR12EE345L and EE12RR345L. In some embodiments, RR12EE345L comprises the amino acid sequence encoded by SEQ ID NO: 6. In some embodiments, EE12RR345L comprises the amino acid sequence encoded by SEQ ID NO: 5. In some embodiments, the CC heterodimer is a CC peptide heterodimer ( see Lebar, T., et al. (2020). Nature Chemical Biology, 1-7). In some embodiments, the split-intein subunits comprise DnaE N-intein and C-intein. In some embodiments, DnaE N-intein comprises the amino acid sequence encoded by SEQ ID NO: 7. In some embodiments, DnaE C-intein comprises the amino acid sequence encoded by SEQ ID NO: 8. In some embodiments, the split-intein is gp41-l, gp41-8, NrdJ-1, or IMPDH-1 ( see Dassa, B., et al. (2009). Nucleic acids research, 57(8), 2560-2573). In some embodiments, the split-intein comprises split portions of the Mxe GyrA intein (see Fine, E. J., et al. (2015). Scientific reports, 5, 10777).
[0269] In some embodiments, according to any of the engineered cells described herein, the system further comprises one or more additional crRNAs or nucleic acid encoding the one or more additional crRNAs, wherein Casl2a/crRNA complexes formed by association of the one or more additional crRNAs with (I) the Cas 12a protein or variant thereof or (II) the two or more effector subunits are capable of generating one or more additional outputs to the sample comprising effector activity of the Casl2a/crRNA complexes at target sites directed by the one or more additional crRNAs. In some embodiments, the system comprises nucleic acid encoding a crRNA array comprising one or more of the first crRNA and the one or more additional crRNAs.
[0270] In some embodiments, according to any of the engineered cells described herein, the Cas 12a protein or variant thereof is derived from a parental Cas 12a protein from Lachnospiraceae bacterium. In some embodiments, the parental Cas 12a protein from Lachnospiraceae bacterium comprises the amino acid sequence of SEQ ID NO: 1. In some embodiments, the target nucleic acid is a target dsDNA. In some embodiments, a protospacer in the target dsDNA targeted by the crRNA spacer has a 5' PAM selected from 5'-TTTG-3', 5'- TTTA-3', and 5'-TTTC-3'.
[0271] In some embodiments, according to any of the engineered cells described herein, the Casl2a protein or variant thereof is a nuclease-competent Cas protein comprising a Casl2a domain having some, the same, or greater nuclease activity as compared to a parental Cas 12a protein from which it is derived, and the first output comprises cleavage (e.g., double-stranded cleavage) of the first target nucleic acid near the first target polynucleotide sequence. In some embodiments, the Casl2a domain comprises the amino acid sequence of SEQ ID NO: 1. [0272] In some embodiments, according to any of the engineered cells described herein, the Cas 12a protein or variant thereof is a fusion protein comprising an effector domain fused to a nuclease-deficient dCasl2a domain having reduced or eliminated nuclease activity as compared to a parental Cas 12a protein from which it is derived, and the first output is generated by activity of the effector domain. In some embodiments, the dCasl2a domain comprises a modification of amino acid D832 with respect to SEQ ID NO: 1 that reduces, substantially eliminates, or eliminates nuclease activity of the dCasl2a domain. In some embodiments, the dCasl2a domain comprises a D832A substitution with respect to SEQ ID NO: 1. In some embodiments, the dCasl2a domain comprises the amino acid sequence of SEQ ID NO: 2. In some embodiments, the effector domain comprises a polypeptide that can (i) cleave a nucleic acid, (ii) edit a nucleotide, (iii) activate transcription, (iv) repress transcription, (v) methylate a nucleic acid, and/or (vi) demethylate a nucleic acid. In some embodiments, the effector domain comprises a transcriptional activation domain, and the first output comprises transcriptional activation of a first gene targeted by the first crRNA. In some embodiments, the transcriptional activation domain is a tripartite VP64-p65-Rta (VPR) transcriptional activation domain or variant thereof.
[0273] In some embodiments, according to any of the engineered cells described herein, the inputs to the cell are selected from physical inputs, small molecule inputs, and biologic inputs. In some embodiments, the physical inputs are selected from oxygen levels, pH, ion concentrations, temperature, light, mechanical force, ultrasound, shock wave, magnetic field, and electric field; the small molecule inputs are selected from drugs, metabolites, and adjuvants; and/or the biologic inputs are selected from polypeptides, polypeptide derivatives, and nucleic acids.
Compositions
[0274] In one aspect, some embodiments disclosed herein relate to a composition that includes one or more components of a split Cas 12a system as described herein. In some embodiments, the split Cast 2a system comprises (a) nucleic acid encoding two or more effector subunits comprising fragments of a Casl2a protein or variant thereof, wherein the two or more effector subunits are capable of spontaneously assembling to form a split Casl2a complex retaining at least some of the effector activity of the Casl2a protein or variant thereof; and (b) a CRISPR RNA (crRNA) or nucleic acid encoding the crRNA, wherein the crRNA comprises a spacer having sufficient complementary to a target polynucleotide sequence in a target nucleic acid such that it can hybridize to the target nucleic acid. In some embodiments, the composition is a pharmaceutical composition and further comprises a pharmaceutically acceptable excipient and/or carrier. In some embodiments, the composition is used for carrying out a method of the present disclosure, e.g., a method for site-specific modification of a target DNA; etc.
Kits
[0275] In some embodiments, provided herein are kits for carrying out a method described herein. A kit can include one or more components of a split Cas 12a system as described herein. In some embodiments, the split Cas 12a system comprises (a) nucleic acid encoding two or more effector subunits comprising fragments of a Cas 12a protein or variant thereof, wherein the two or more effector subunits are capable of spontaneously assembling to form a split Cas 12a complex retaining at least some of the effector activity of the Cas 12a protein or variant thereof; and (b) a CRISPR RNA (crRNA) or nucleic acid encoding the crRNA, wherein the crRNA comprises a spacer having sufficient complementary to a target polynucleotide sequence in a target nucleic acid such that it can hybridize to the target nucleic acid.
[0276] A kit as described herein can further include one or more additional reagents, where such additional reagents can be selected from: a buffer for introducing one or more components of a split Cas 12a system into a cell; a dilution buffer; a reconstitution solution; a wash buffer; a control reagent; a control expression vector or polyribonucleotide; a reagent for in vitro production of one or more components of a split Cas 12a system, and the like.
[0277] Components of a kit can be in separate containers; or can be combined in a single container.
[0278] In addition to the above-mentioned components, a kit can further include instructions for using the components of the kit to practice the methods. The instructions for practicing the methods are generally recorded on a suitable recording medium. For example, the instructions may be printed on a substrate, such as paper or plastic, etc. As such, the instructions may be present in the kits as a package insert, in the labeling of the container of the kit or components thereof (e.g., associated with the packaging or sub-packaging) etc. In some embodiments, the instructions are present as an electronic storage data file present on a suitable computer readable storage medium, e.g., CD-ROM, diskette, flash drive, etc. In yet other embodiments, the actual instructions are not present in the kit, but means for obtaining the instructions from a remote source, e.g., via the internet, are provided. An example of this embodiment is a kit that includes a web address where the instructions can be viewed and/or from which the instructions can be downloaded. As with the instructions, this means for obtaining the instructions is recorded on a suitable substrate.
EXAMPLES
[0279] The practice of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology, microbiology, cell biology, biochemistry, nucleic acid chemistry, and immunology, which are well known to those skilled in the art. Such techniques are explained fully in the literature, such as Sambrook, J., & Russell, D. W. (2012). Molecular Cloning: A Laboratory Manual (4th ed.). Cold Spring Harbor, NY : Cold Spring Harbor Laboratory and Sambrook, J., & Russel, D. W. (2001). Molecular Cloning: A Laboratory Manual (3rd ed.). Cold Spring Harbor, NY : Cold Spring Harbor Laboratory (jointly referred to herein as “Sambrook”); Ausubel, F. M. (1987). Current Protocols in Molecular Biology. New York, NY: Wiley (including supplements through 2014); Bollag, D. M. et al. (1996). Protein Methods. New York, NY: Wiley-Liss; Huang, L. et al. (2005). Nonviral Vectors for Gene Therapy. San Diego: Academic Press; Kaplitt, M. G. et al. (1995). Viral Vectors: Gene Therapy and Neuroscience Applications. San Diego, CA: Academic Press; Lefkovits, I. (1997). The Immunology Methods Manual: The Comprehensive Sourcebook of Techniques. San Diego, CA: Academic Press; Doyle, A. et al. (1998). Cell and Tissue Culture: Laboratory Procedures in Biotechnology. New York, NY: Wiley; Mullis, K. B., Ferre, F. & Gibbs, R. (1994). PCR: The Polymerase Chain Reaction. Boston: Birkhauser Publisher; Greenfield, E. A. (2014). Antibodies: A Laboratory Manual (2nd ed.). New York, NY: Cold Spring Harbor Laboratory Press; Beaucage, S. L. et al. (2000). Current Protocols in Nucleic Acid Chemistry. New York, NY : Wiley, (including supplements through 2014); and Makrides, S. C. (2003). Gene Transfer and Expression in Mammalian Cells. Amsterdam, NL: Elsevier Sciences B.V., the disclosures of which are incorporated herein by reference.
[0280] Additional embodiments are disclosed in further detail in the following examples, which are provided by way of illustration and are not in any way intended to limit the scope of this disclosure or the claims. Example 1A: Design and characterization of a split Cas 12a system in mammalian cells [0281] This example demonstrates the design and characterization of a split Casl2a system in mammalian cells, allowing the construction of AND gates.
[0282] The split Casl2a system was based on a nuclease-dead dCasl2a (D832A) from Lachnospiraceae bacterium fused with a tripartite VPR activator for targeted gene activation (Tak, Y.E. et al , 2017. Nature Methods, 14(12), pp.l 163-1166). To minimize the size of the construct, a minimized VPR (miniVPR) was used (Vora, S., et al. (2018). bioRxiv, 298620), which showed comparable activation in a TRE3G-GFP HEK293T reporter cell line (FIGS. 1A and IB, Tables 1-3). A structure-guided approach was used, and five potential sites were selected to split dCasl2a-miniVPR into unstructured surface loops to avoid disruption of native α-helices and b-sheets (FIG. 2A) (Dong, D. et al, 2016. Nature, 532(7600), pp.522-526). To facilitate spontaneous heterodimerization of the split fragments upon co-expression, a pair of leucine zipper domains, RR12EE345L and EE12RR345L, was fused to the split halves (FIG. 2B) (Moll, J.R. et al., 2001. Protein Science, 10(3), pp.649-655). The ability of these split half pairs to reassemble and reconstitute a functional activator was compared, and it was discovered that the split at amino acid 406 resulted in the best performance, with this pair activating GFP to 77% of the full dCasl2a-miniVPR (FIG. 2C).
Table 1: crRNA spacer sequences used in Example 1.
Figure imgf000099_0001
Figure imgf000100_0001
Table 2: Plasmids used in experiments corresponding to indicated FIGS.
Figure imgf000100_0002
Figure imgf000101_0001
Figure imgf000102_0001
Figure imgf000103_0001
Figure imgf000104_0001
Figure imgf000105_0001
Table 3: Fold-changes and statistical analyses related to indicated FIGS.
Figure imgf000105_0002
Figure imgf000106_0001
Figure imgf000107_0001
For ELISA controls below the lower detection limit of 4pg/mL, fold changes are represented as > fold change calculated compared to that lower limit. b.d.l , below detection limit. Statistically significant differences (P<0.05) are bolded.
[0283] It was then tested whether the leucine zipper domains were essential for this spontaneous assembly. Interestingly, without the leucine zipper domain the two 406-split halves could still activate GFP, to 72% of that with the zipper (FIG. 2D). This intrinsic dimerization of the two protein halves, which was then further enhanced by addition of the leucine zippers, may occur via potential interactions between α-helices or scaffolding by the crRNA (FIG. 3A). To investigate the generalizability of split Casl2a architectures, three split sites recently identified for rapamycin-induced dimerization were selected and their ability to spontaneously dimerize using the leucine zipper approach was tested (Nihongaki, Y. et al , 2019. Nature Chemical Biology, pp.1-7). Only one gave strong GFP activation, suggesting efficient design may depend on the selected dimerization domains (FIG. 3B). The 406-split leucine zipper design was selected for further study, with an optimized additional C-terminal nuclear localization signal (NLS) (FIG. 4).
[0284] Next, the performance of the split system was characterized. It was first confirmed that there was no GFP reporter activation from individual N- or C-terminal halves (FIG. 5A and FIG. 5B). This indicates that both protein components are necessary for functional output, thus operating as a 2-input Boolean AND gate. This AND gate was then evaluated for its ability to activate the endogenous CXCR4 gene (FIG. 6), where it showed no significant activation using only the individual halves and strong activation close to that of the full-length activator with both N- and C-terminal halves present (FIG. 5A and FIG. 5B). To further characterize the performance at various expression levels, a stable cell line was generated with the N- and C- terminal halves under the control of cumate and Doxycycline (Dox)-inducible promoters, respectively. The amount of each molecule was titrated and the resulting activation of a P2- GFP reporter was measured (FIGS. 7A, 7B, and 7C) (Nissim, L. et al, 2014. Molecular Cell, 54(4), pp.698-710). Strong GFP activation was found to occur only in the presence of high levels of both inputs (FIG. 7D).
[0285] To demonstrate the multiplexing abilities of the split dCasl2a system, two endogenous immunostimulatory genes, IFNg and IL2, were simultaneously activated (FIGS. 8A-8C). As the dCas 12a protein can process crRNAs from a longer transcript, a single U6-driven array was generated with a crRNA for IFNg and a pair of crRNAs for IL2, interspaced with direct repeats (DRs). Strong activation of both genes was observed, as measured by ELISA (enzyme-linked immunosorbent assay), only when cells were co-transfected with both halves (FIG. 8D). This multiplexed capability allows for expansion to multiple outputs using a single crRNA transcript.
[0286] It was also determined whether the 406-split design with catalytically active Casl2a could be implemented for logic-gated gene editing (FIG. 9A). Cleavage efficiency at the CXCR4 locus was measured using the T7E1 assay, and efficient cleavage of the CXCR4 locus was observed only when both halves were co-transfected (FIG. 9B). The ability to use split Casl2a to edit the genome expands the potential output of generated circuits, enabling logic gating for sophisticated control over gene editing or genome recording.
Example IB: Construction of multi-input logic using split dCas 12a
[0287] This example demonstrates the design and characterization of a split Casl2a system in mammalian cells with multiple-input logic.
[0288] Many applications require detection and integration of more than two inputs, which is challenging for traditional methods. Here, the system is expanded to detect multiple inputs. Two approaches were taken to build 3-input Boolean AND gates — first, modulating expression of the crRNA using an inducible RNA polymerase II (Pol II) promoter; and second, engineering the dCasl2-miniVPR using inteins for a 3 -way protein split.
[0289] Unlike Cas9, Casl2a can process crRNAs from a Pol II promoter-driven transcript (FIG. 10A) (Zhong, G. et al., 2017. Nature Chemical Biology, 13(8), pp.839-841; Campa, C.C. et al, 2019. Nature Methods, pp.1-7). The ability to express crRNAs under the wide variety of inducible Pol II promoters, rather than being limited to a constitutive Pol III promoter like U6, provides another means of controlling the split dCasl2a system activity. It was verified that constitutive CAG and Dox-inducible TRE3G Pol II crRNA cassettes could efficiently activate IFNg (FIGS. 10B-D). Next, the TRE3G-driven crRNA cassette was combined with the split dCasl2a-miniVPR, where it was observed that high IFNg activation occurred only in the presence of the N-terminal and C-terminal halves and added Dox, suggesting this circuit is a Boolean 3-input AND gate (FIG. 10E).
[0290] As an alternative method to construct a 3-input AND gate, a 3-way split of dCasl2a- miniVPR was created by using the 406 leucine zipper split further to separate the fused activator using an orthogonal intein system (Truong, D.-J.J. et al., 2015. Nucleic Acids Research, 43(13), pp.6450-6458). The DnaEN-intein and C-intein domains were placed in the linker between dCasl2a and miniVPR, and it was verified that this 3 -way split protein could reconstitute and activate reporter gene expression, to 43% of the full-length dCasl2al l6 miniVPR (FIG. 11A and 1 IB). The 3 -way split system was then used to generate a tight 3- input Boolean AND gate (FIGS. 11C and 1 ID).
[0291] Some applications require the absence of certain signals as circuit inputs. For Cas-based systems, the natural anti-CRISPR (Acr) proteins can inhibit Cas activity and have been incorporated in dCas9-based circuitry (Nakamura, M. et al, 2019. Nature Communications, 10(1), p.194; Rauch, B.J. et al, 2017. Cell, 168(1-2), p.150-158. elO). AcrVAl, which was reported to inhibit FbCasl2a-mediated DNA cleavage (Watters, K.E. et al., 2018. Science, 362(6411), pp.236-239), was tested, and it was observed that it could also inhibit dCasl2a- mediated gene activation (FIG. 12A and 12B). AcrVAl was used with the split dCas 12a system to generate “A AND B AND (NOT C)” logic gating, and target gene activation was observed only in the presence of the N- and C-terminal halves and the absence of AcrVAl (FIGS. 12C and 12D).
[0292] Next, to construct a 4-input AND gate, the TRE3G-driven crRNA was combined with the 3 -way split dCasl2a-miniVPR protein (FIG. 13). The ability of the system to activate endogenous IFNg was evaluated, and target activation above the detection threshold was observed only in the presence of all three protein components and Dox. It is believed that this is the first synthetic 4-input AND gate to control endogenous gene expression in mammalian cells. These results together establish that the split dCasl2a systems described herein are modular and can be configured for implementing multi-input logic gates. Example 1C: Synthetic split dCas12a systems for cancer-specific activation of therapeutic genes
[0293] This example demonstrates the design and characterization of an exemplary split Casl2a system in mammalian cells for detection and integration of cancer-specific inputs for output of therapeutic gene activation.
[0294] The split dCasl2a system architecture was used to construct a proof-of-concept anti- tumoral circuit with biologically relevant inputs and outputs. Cancer cells exhibit a number of altered genetic signatures and receive distinctive external signals from the surrounding tumor microenvironment (TME) (Hanahan & Weinberg 2011). However, individual signatures are rarely unique and may also be present at varying levels in non-cancerous tissues. Multi-input sensing and integration can provide the control to ensure specificity of therapeutic programs. As a demonstration, a split dCasl2a was expressed with elements under the control of promoters responsive to intracellular and extracellular signals indicative of breast cancer, and the system was used to activate endogenous cytokines (FIG. 14A).
[0295] The N-terminal dCasl2a fragment was placed under the control of an endogenous promoter for ribonucleotide reductase subunit 2 (RRM2). RRM2 converts ribonucleotides to deoxyribonucleotides, catalyzing the rate limiting step for DNA synthesis, and has been reported to be overexpressed in breast cancer (Y un, H J. et al, 2008. Experimental & molecular medicine, 40(3), pp.345-53). Following transduction of this construct into MDA-MB-231 human breast cancer cells and MCF10A normal breast cells, the resulting expression was significantly higher in the cancer cells, allowing RRM2 promoter activity to be used as a first tumor input (FIG. 14B). The C-terminal dCasl2a fragment was placed under the control of a synthetic hypoxia-responsive promoter, which is activated when lack of oxygen in the TME stabilizes cellular HIFlα (Ede, C. et al., 2016. ACS Synthetic Biology, 5(5), pp.395-404). Following transduction of this construct into MDA-MB-231 cells, induced expression was observed upon adding hypoxia agonist DMOG (FIG. 14C).
[0296] The AND-gate performance of the system was evaluated in MDA-MB-231 cells, where a crRNA for the activation of IFNg was introduced, providing the therapeutic output. This immunostimulatory cytokine can remodel the immunosuppressive TME but has systemic toxicity concerns, thus its used as a therapeutic output would benefit from localized tumor- specific expression (Pitt, J.M. et al., 2016. Annals of Oncology, 27(8), pp.1482-1492). The system displayed integrative behavior, showing high IFNγ activation only upon sensing the presence of both hypoxia and intracellular RRM2p activity (FIG. 14D). As a control, the system was tested in MCF10A cells, and no detectable expression of IFNγ was observed (FIG. 14E). This indicates the utility of the split dCasl2a systems described herein in integrating two tumor signatures and generating a therapeutic output.
[0297] To demonstrate the programmability of our split dCas 12a system AND gate, the crRNA was substituted with one capable of activating IL2, a key cytokine for T cell expansion (Rosenberg 2014). Similar to the previous system targeting IFNγ, IL2 activation and secretion was only observed in the presence of both tumor signals in MDA-MB- 231 cells (FIG. 14F). This suggests the split dCasl2a systems described herein are able to drive activation of diverse therapeutic targets upon recognition of specified combinations of inputs.
[0298] The ability to detect more than two inputs greatly expands the power of therapeutic circuits. A 3-input AND gate was generated in MDA-MB-231 cells by placing the crRNA under the control of the TRE3G promoter. Secretion of IFNg above the detection limit was observed only when both of two tumor-relevant signals as well as Dox were present (FIG. 14G). Additionally, U6-driven crlFNγ was used in conjunction with a Dox-inducible AcrVAl protein to generate “A AND B AND (NOT C)” logic, and minimal IFNg secretion was observed upon addition of Dox (FIG. 14H). These two exemplary split dCasl2a systems demonstrate the scalability of the split dCasl2a architecture to build multi- input therapeutic circuits, where in this example an orthogonal input (Dox) could either turn on or turn off circuit behavior to provide additional control. The third component could alternatively be controlled by additional tumor signatures or by signals characteristic of healthy tissue, as could additional components, to build a variety of cancer-targeting systems.
Discussion
[0299] In Examples 1A-1C, the development and repurposing of a split dCasl2a system for implementing versatile, robust, and scalable logic gates is described. It was shown that a simpler 2-input system can be combined with inducible crRNAs and additional split strategies to generate higher-order AND gates (FIG. 15). The availability of anti-CRISPR systems provides a means of implementing “NOT” logic. The exemplified systems behaved robustly in carrying out Boolean logic operations, with a high dynamic range between ON and OFF states. Extending to biologically relevant inputs and outputs, it was demonstrated that elements of the systems can be linked to tumor-relevant promoters, allowing for the detection of combinations of tumor cues for logic integration to control therapeutic targets.
[0300] The systems described herein can serve as a scaffold for future modular engineering of logic gates with diverse inputs and outputs. There are many well-characterized tissue-specific and disease-relevant promoters that can be used to limit split protein expression to cells of interest. In addition to transcription- level control, the split dCasl2a components could be incorporated into systems that control protein localization in response to soluble or cell-surface signals (Kipniss, N.H. et al, 2017. Nature Communications, 8(1), p.2212; Chung, H.K. et al., 2019. Science (New York, N.Y.), 364(6439), p.eaat6982; Morsut, L., et al. (2016). Cell, 164(4), 780-791). Incorporation of cleavable degradation tags at the protein level could expand to sensing of endogenous or viral proteases (Gao, X.J. et al, 2018. Science (New York, N.Y.), 361(6408), pp.1252-1258).
[0301] In terms of outputs, the facile programmability of the dCasl2a systems could be leveraged to effectively activate various endogenous or exogenous gene programs. Activation of secreted factors, including cytokines or antibodies, could be used for localized delivery of cell-manufactured therapeutics. Modulation of targets like transcription factors or noncoding RNAs could change cell behavior or differentiation. Alternatively, changing the fused activator to a repressor, such as KRAB, could expand the output to include repression of endogenous gene targets. Beyond transcription- level control, Casl2a-mediated gene editing or base editing systems could be used to permanently alter the genome, for example, for protein knockout, lineage tracing, or genome recording upon encountering combinations of cellular inputs. [0302] Genome engineering tools are emerging as an avenue to enrich mammalian synthetic biology approaches for treatment of complex diseases. While the performance of the split dCasl2a platform was demonstrated in cancer cells, it can easily be implemented in other therapeutically relevant cell types, including, for example, immune cells and stem cells, to create novel sensor-computation-actuator systems with a variety of inputs and outputs for more effective and safer cell engineering and cell therapy.
Materials and Methods Cell Culture
[0303] HEK293T cells (Clontech) and MDA-MB-231 cells (Sigma- Aldrich) were cultured in DMEM + GlutaMAX (Thermo Fisher) supplemented with 10% Tet System Approved FBS (Clontech) and lOOU/mL of penicillin and streptomycin (Life Technologies). MCF10A cells were a gift from Mingyu Chung (Tobias Meyer lab) and were cultured in MEGM Bullet Kit Growth Media (Fisher) supplemented with lOOng/mL Choleratoxin (Sigma). Cells were maintained at 37°C and 5% C02 and passaged using standard cell culture techniques. Cells were not tested for mycoplasma contamination. For transient transfection of HEK293Ts, cells were seeded the day before transfection at lxl 05 cells/mL. Transient transfections were performed using 3pL of TransIT-LTl transfection reagent (Mirus) per pg of plasmid. Cells were analyzed 2 or 3 days post transfection, as indicated. F or crRNA testing, 25 Ong of dCas 12a construct and 250ng of crRNA plasmid were transfected into 24-well plates. For other activation experiments, 166ng of each plasmid were transfected into 24-well plates. For comparisons with split versus full length activator, 166ng of empty non-coding pUC 19 plasmid were added to keep total transfected DNA amount per well consistent. For P2 -reporter experiments, 250ng each of crP2 plasmid and P2-GFP plasmid were transfected immediately following stimulation into the Dox/cumate inducible stable cell in 24-well plates. For 3- and 4- input AND gate using Dox-inducible crRNA, DNA amount was increased to 250ng per plasmid in a 24-well plate.
Stable cell line generation
[0304] Stable MDA-MB-231 and MCF10A cell lines were generated using lentiviral transduction. For lentiviral production, F1EK293T cells were transfected with 1.51 pg of pHR vector with construct of interest, 1.32 pg of dR8.91 and 165 ng ofpMD2 with 7.5 pL ofMirus TransIT-LTl (for 6 well plate format). One day post transfection media was replaced with fresh media, and two days post transfection lentivirus was harvested and filtered through a 0.45 pm polyvinylidene fluoride filter (Millipore). Following filtration, 1 volume lentivirus was mixed with 4 volumes Lentivirus Precipitation Solution (Alstem) and refrigerated overnight. Cells were seeded for transduction at 1 x 105 cells/well of a 12-well plate. The next day, lentivirus was pelleted at 1500xg for 30 minutes at 4°C and resuspended in 1/100 of original volume before transduction of target cells. Transduced cells were selected for using 200 pg/mL Zeocin (Thermofisher) and 2 pg/mL Puromycin (Gold Biotech).
[0305] The stable F1EK293T cell line for Dox/cumate dose-response was generated as follows. The cumate-inducible construct was introduced by lentivirus transduction, as described above, and selected for using 2 pg/mL Puromycin. The Dox-inducible construct was introduced using the PiggyBac system. Briefly, 250ng of the construct of interest was transfected with lOOng of PiggyBac Transposase vector in cells in 12-well plate. Cells were expanded for 3 days then selected for with 400 pg/mL Zeocin. Following both antibiotic selections, cells were stimulated with both 60 pg/mL cumate (System Biosciences) and lOOOng/mL Dox (Gold Biotech) and sorted for the mCherry+/BFP+ cell population using the SONY SF1800 Cell Sorter to select for cells that were able to successfully turn on both constructs in response to stimulation.
Plasmid cloning
[0306] Standard molecular cloning techniques were used to assemble constructs in this paper. Nuclease-dead (D832A) dCasl2a from Lachnospiraceae bacterium and its crRNA backbone were modified from plasmids obtained from Addgene (#104567 and # 78742) (Tak, Y.E. et al, 2017. Nature Methods, 14(12), pp.l 163-1166; Kleinstiver, B.P. et al, 2016. Nature Biotechnology, 34(8), pp.869-874). The leucine zipper was amplified from plasmids obtained from Addgene (#15304 and #15305) (Luan, H. et al., 2006. Neuron, 52(3), pp.425-436). The P2 reporter was modified from a plasmid obtained from Addgene (#55198) (Nissim, L. et al., 2014. Molecular Cell, 54(4), pp.698-710). The RRM2 promoter was amplified from HEK293T genomic DNA extracted using the DNeasy Blood & Tissue kit (Qiagen). The intein and AcrVAl DNA was cloned from ordered gBlocks (Integrated DNA Technologies). Plasmids were cloned using InFusion (Takara Bio) and Stellar Competent cells (Takara Bio). DNA sequences and plasmids used can be found in Table 2 and the Sequence Listing. The crRNA plasmids were cloned using ligation cloning. Oligos were annealed and phosphorylated with PNK (NEB) and inserted into backbone using T4 DNA Ligase (NEB). The scaffold used (N’s denote spacer sequence): 5’-
AATTTCTACTAAGTGTAGATNNNNNNNNNNNNNNNNNNNNNNN-3’. Spacer sequences for all crRNAs can be found in Table 1.
Flow cytometry
[0307] Cells were dissociated using 0.05% Trypsin-EDTA (Life Technologies), resuspended in PBS+10% FBS, and analyzed for fluorescence using a CytoFLEX S flow cytometer (Beckman Coulter). 10,000 cells from the population of interest (for most experiments, mCherry+ and BFP+ gated based on non-transfected control) were collected for each sample and analyzed using FlowJo. For CXCR4 expression analysis, cells were stained with APC- labeled CXCR4 antibody (BioLegend #306510) prior to analysis.
ELISA
[0308] Supernatants from cell cultures were harvested 2 or 3 days post tran sfecti on/stim ul ati on as indicated, and samples were stored at -80°C. Secreted proteins were measured using the ELISA MAX Deluxe kits (Biolegend). Absorbance was measured at 450 nm and 570 nm using a Synergy HI plate reader (BioTek), and protein concentrations were calculated by standard curves fitted to a power law.
Stimulation
[0309] For Dox and cumate dose-response curves, stable cells were seeded on day 1, media was swapped for fresh media with desired concentration of Dox (Gold Biotech) or Cumate (System Biosciences) on day 2, and mCherry or BFP (respectively) were analyzed by flow cytometry on day 3. For the purposes of plotting on the log axis, the “0” condition was plotted at one order of magnitude lower than the lowest concentration. For titration experiments with P2 reporter activation, stable cells were seeded on day 1 , on day 2 cells were transfected with 250ng each of crP2 and P2-GFP plasmids immediately following swapping of cell culture media with appropriate stimulant concentrations, and cells were analyzed by flow cytometry on day 4. For other Dox stimulation experiments in F1EK293T cells, cells were seeded on day 1 and on day 2 immediately before transfection media was swapped for fresh media with Dox at 1 OOng/mL. F or stimulation experiments in MDA-MB-231 and MCF 10A, cells that had been transduced with the hypoxia-inducible-C term and with or without the RRM2p-N term construct were seeded on day 1 at 1x105 cells/mL, media with 0.5 mM hypoxia agonist DMOG (Cayman) in DMSO was added on day 2, and cells were analyzed on day 4. For the three-input MDA-MB-231 experiments, dox-inducible constructs were also transduced and Dox was also added on day 2 at 1 OOng/mL and cells were analyzed on day 4.
Editing Experiments
[0310] A total of 2 μg of DNA (666 ng/plasmid) was transfected in F1EK293T cells seeded the previous day in a 12 well plate. Three days post transfection, cells were sorted for the presence of mCherry using the SONY SF1800 Cell Sorter. Genomic DNA was isolated using the DNeasy Blood&Tissue Kit (Qiagen), and the target locus was amplified using PCR (Kapa Biosystems) (Primers: F, 5 ’ -CCGCTTGGGGGAGGAGGTGC-3 ’ , R, 5’-
CCAGAAGGGAAGCGTGATGACAAAGAG-3’) and assessed via T7E1 endonuclease activity (NEB). The T7E1 gel was analyzed using ImageJ, and the indel % was calculated using the formula Indel % = 100 x (1 — fl — /cut), where fiut is the fraction 487 of PCR product cleaved.
Quantification and Statistical Analysis
[0311] Data are displayed as individual points, with sample size indicated in figure legends. No randomization or blinding was performed. Sample sizes used are consistent with those used by similar gene regulation studies. Statistical analysis was performed using Prism 7 (Graphpad). To account for unequal variance among conditions, Welch’s two-sided t-test was performed when comparing two conditions, and Welch’s ANOVA with Games-Flowell post hoc tests was performed when comparing more than two conditions with each other.
[0312] While particular alternatives of the present disclosure have been disclosed, it is to be understood that various modifications and combinations are possible and are contemplated within the true spirit and scope of the appended claims. There is no intention, therefore, of limitations to the exact abstract and disclosure herein presented.

Claims

CLAIMS WHAT IS CLAIMED IS:
1. A system for conditionally generating an output to a sample comprising the system, the system comprising:
(a) (i) a Cas 12a protein or variant thereof having an effector activity, or nucleic acid encoding the Cas 12a protein or variant thereof; or
(ii) two or more effector subunits comprising fragments of the Cas 12a protein or variant thereof, or nucleic acid encoding the two or more effector subunits, wherein the two or more effector subunits are capable of spontaneously assembling to form a split Cas 12a complex in the sample, and the split Cas 12a complex retains at least some of the effector activity of the Cas 12a protein or variant thereof; and
(b) a first CRISPR RNA (crRNA) or nucleic acid encoding the first crRNA, wherein the first crRNA comprises a first spacer having sufficient complementary to a first target polynucleotide sequence in a first target nucleic acid in the sample such that it can hybridize to the first target nucleic acid, wherein a Casl2a/crRNA complex formed in the sample by association of the first crRNA with (I) the Cas 12a protein or variant thereof or (II) the two or more effector subunits is capable of generating a first output to the sample comprising the effector activity of the Casl2a/crRNA complex at the first target nucleic acid, and wherein the expression of a first component of the Cas 12a/crRNA complex is under the control of a first input to the sample and the expression of a second component of the Casl2a/crRNA complex is under the control of a second input to the sample.
2. The system of claim 1, further comprising nucleic acid comprising a NOT inducible promoter operably linked to a polynucleotide sequence encoding an anti-CRISPR (Acr) protein capable of inhibiting activity of the split Cas 12a complex in the sample, wherein a NOT input to the sample is capable of increasing the expression of Acr protein from the nucleic acid in the sample such that activity of split Cas 12a complex in the sample is inhibited.
3. The system of claim 1 or 2, configured such that all components of the Casl2a/crRNA complex are present or capable of being expressed in the sample when the first input and the second input each have a desired state.
4. The system of claim 3, configured such that all components of the Casl2a/crRNA complex are present or capable of being expressed in the sample when the first input is present and the second input is present.
5. The system of claim 3 or 4, comprising (a) nucleic acid encoding the Cast 2a protein or variant thereof, wherein the nucleic acid encoding the Casl2a protein or variant thereof comprises a first inducible promoter operably linked to a polynucleotide sequence encoding the Cas 12a protein or variant thereof, and the first input is capable of modulating the expression of the Cas 12a protein or variant thereof in the sample; and
(b) nucleic acid encoding the first crRNA, wherein the nucleic acid encoding the first crRNA comprises a second inducible promoter operably linked to a polynucleotide sequence encoding the first crRNA, and the second input is capable of modulating the expression of the first crRNA in the sample.
6. The system of claim 5, wherein modulating the expression of each of the Casl2a/crRNA complex components comprises increasing their expression.
7. The system of claim 3 or 4, comprising
(a) nucleic acid encoding a first effector subunit and a second effector subunit of the two or more effector subunits, wherein the nucleic acid comprises a first inducible promoter operably linked to a polynucleotide sequence encoding the first effector subunit and a second inducible promoter operably linked to a polynucleotide sequence encoding the second effector subunit, and the first input is capable of modulating the expression of the first effector subunit in the sample and the second input is capable of modulating the expression of the second effector subunit in the sample; or
(b) nucleic acid encoding a first effector subunit of the two or more effector subunits, wherein the nucleic acid comprises a first inducible promoter operably linked to a polynucleotide sequence encoding the first effector subunit; and nucleic acid encoding the first crRNA, wherein the nucleic acid comprises a second inducible promoter operably linked to a polynucleotide sequence encoding the first crRNA, and the first input is capable of modulating the expression of the first effector subunit in the sample and the second input is capable of modulating the expression of the first crRNA in the sample.
8. The system of claim 7, wherein modulating the expression of each of the Casl2a/crRNA complex components comprises increasing their expression.
9. The system of claim 1, wherein the expression of a third component of the Casl2a/crRNA complex is under the control of a third input to the sample, and the system is configured such that all components of the Casl2a/crRNA complex are present or capable of being expressed in the sample when the first input, the second input, and the third input each have a desired state.
10. The system of claim 9, configured such that all components of the Casl2a/crRNA complex are present or capable of being expressed in the sample when the first input is present, the second input is present, and the third input is present.
11. The system of claim 9 or 10, comprising
(a) nucleic acid encoding a first effector subunit, a second effector subunit, and a third effector subunit of the two or more effector subunits, wherein the nucleic acid comprises a first inducible promoter operably linked to a polynucleotide sequence encoding the first effector subunit, a second inducible promoter operably linked to a polynucleotide sequence encoding the second effector subunit, and a third inducible promoter operably linked to a polynucleotide sequence encoding the third effector subunit, and the first input is capable of modulating the expression of the first effector subunit in the sample, the second input is capable of modulating the expression of the second effector subunit in the sample, and the third input is capable of modulating the expression of the third effector subunit in the sample; or
(b) nucleic acid encoding a first effector subunit and a second effector subunit of the two or more effector subunits, wherein the nucleic acid comprises a first inducible promoter operably linked to a polynucleotide sequence encoding the first effector subunit and a second inducible promoter operably linked to a polynucleotide sequence encoding the second effector subunit; and nucleic acid encoding the first crRNA, wherein the nucleic acid comprises a third inducible promoter operably linked to a polynucleotide sequence encoding the first crRNA, and the first input is capable of modulating the expression of the first effector subunit in the sample, the second input is capable of modulating the expression of the second effector subunit in the sample, and the third input is capable of modulating the expression of the first crRNA in the sample.
12. The system of claim 11, wherein modulating the expression of each of the Casl2a/crRNA complex components comprises increasing their expression.
13. The system of claim 1, wherein the expression of a third component of the Casl2a/crRNA complex is under the control of a third input to the sample and the expression of a fourth component of the Casl2a/crRNA complex is under the control of a fourth input to the sample, and the system is configured such that all components of the Casl2a/crRNA complex are present or capable of being expressed in the sample when the first input, the second input, the third input, and the fourth input each have a desired state.
14. The system of claim 13, configured such that all components of the Casl2a/crRNA complex are present or capable of being expressed when the first input is present, the second input is present, the third input is present, and the fourth input is present.
15. The system of claim 13 or 14, comprising
(a) nucleic acid encoding a first effector subunit, a second effector subunit, and a third effector subunit of the two or more effector subunits, wherein the nucleic acid comprises a first inducible promoter operably linked to a polynucleotide sequence encoding the first effector subunit, a second inducible promoter operably linked to a polynucleotide sequence encoding the second effector subunit, and a third inducible promoter operably linked to a polynucleotide sequence encoding the third effector subunit, and the first input is capable of modulating the expression of the first effector subunit in the sample, the second input is capable of modulating the expression of the second effector subunit in the sample, and the third input is capable of modulating the expression of the third effector subunit in the sample; and
(b) nucleic acid encoding the first crRNA, wherein the nucleic acid comprises a fourth inducible promoter operably linked to a polynucleotide sequence encoding the first crRNA, and the fourth input is capable of modulating the expression of the first crRNA.
16. The system of claim 15, wherein modulating the expression of each of the Casl2a/crRNA complex components comprises increasing their expression.
17. The system of any one of claims 1-12, wherein the two or more effector subunits consist of a first effector subunit and a second effector subunit.
18. The system of claim 17, wherein the first effector subunit comprises an N-terminal fragment of the Casl2a protein or variant thereof up to and including a split point amino acid, and the second effector subunit comprises a C-terminal fragment of the Casl2a protein or variant thereof following the split point amino acid.
19. The system of claim 18, wherein the split point amino acid corresponds to A406 or G550 with respect to a parental Casl2a protein amino acid sequence of SEQ ID NO: 1.
20. The system of claim 18 or 19, wherein the first effector subunit further comprises a first stabilization domain at its C-terminus and the second effector subunit further comprises a second stabilization domain at its N-terminus, wherein the first stabilization domain and the second stabilization domain are capable of interacting with one another to stabilize a complex comprising the first effector subunit and the second effector subunit in the sample.
21. The system of claim 17, wherein the Cas 12a protein or variant thereof is a fusion protein comprising an effector domain fused to a Cas 12a domain, and the first effector subunit comprises the Cas 12a domain and the second effector subunit comprises the effector domain.
22. The system of claim 21, wherein the first effector subunit further comprises a first stabilization domain at either terminus and the second effector subunit further comprises a second stabilization domain at either terminus, wherein the first stabilization domain and the second stabilization domain are capable of interacting with one another to stabilize a complex comprising the first effector subunit and the second effector subunit in the sample.
23. The system of claim 20 or 22, wherein the first stabilization domain and the second stabilization domain are derived from subunits of a heterodimerizing binding pair or a split- intein.
24. The system of claim 23, wherein the first stabilization domain and the second stabilization domain are derived from subunits of a leucine zipper.
25. The system of claim 24, wherein the leucine zipper subunits comprise RR12EE345L and EE12RR345L.
26. The system of claim 23, wherein the first stabilization domain and the second stabilization domain are derived from subunits of a split-intein, and the split-intein subunits comprise DnaE N-intein and C-intein.
27. The system of any one of claims 1-16, wherein the two or more effector subunits consist of a first effector subunit, a second effector subunit, and a third effector subunit.
28. The system of claim 27, wherein the Cas 12a protein or variant thereof is a fusion protein comprising an effector domain fused to a Cas 12a domain, and
(a) the first effector subunit comprises an N-terminal fragment of the Cas 12a domain up to and including a split point amino acid,
(b) the second effector subunit comprises a C-terminal fragment of the Cas 12a domain following the split point amino acid; and
(c) the third effector subunit comprises the effector domain.
29. The system of claim 28, wherein the split point amino acid corresponds to A406 or G550 with respect to a parental Casl2a protein amino acid sequence of SEQ ID NO: 1.
30. The system of claim 28 or 29, wherein
(a) the first effector subunit further comprises a first stabilization domain at its C- terminus and the second effector subunit further comprises a second stabilization domain at its N-terminus, wherein the first stabilization domain and the second stabilization domain are capable of interacting with one another to stabilize a complex comprising the first effector subunit and the second effector subunit in the sample; and/or
(b) the third effector subunit further comprises a third stabilization domain at either terminus and (i) the second effector subunit further comprises a fourth stabilization domain at its C-terminus or (ii) the first effector subunit further comprises the fourth stabilization domain at its N-terminus, wherein the third stabilization domain and the fourth stabilization domain are capable of interacting with one another to stabilize a complex comprising the first effector subunit, the second effector subunit, and the third effector subunit in the sample.
31. The system of claim 30, wherein the first stabilization domain and the second stabilization domain are derived from subunits of a heterodimerizing binding pair or a split- intein, and the third stabilization domain and the fourth stabilization domain are derived from subunits of a heterodimerizing binding pair or a split-intein and are orthogonal to the first stabilization domain and the second stabilization domain.
32. The system of claim 31, wherein the heterodimerizing binding pair is a leucine zipper.
33. The system of claim 32, wherein
(a) the first stabilization domain and the second stabilization domain are derived from subunits of a leucine zipper and the third stabilization domain and the fourth stabilization domain are derived from subunits of a split-intein; or
(b) the first stabilization domain and the second stabilization domain are derived from subunits of a split-intein and the third stabilization domain and the fourth stabilization domain are derived from subunits of a leucine zipper.
34. The system of claim 32 or 33, wherein the leucine zipper subunits comprise RR12EE345L and EE12RR345L and/or the split-intein subunits comprise DnaE N-intein and C-intein.
35. The system of any one of claims 1 to 34, comprising one or more additional crRNAs or nucleic acid encoding the one or more additional crRNAs, wherein Casl2a/crRNA complexes formed by association of the one or more additional crRNAs with (I) the Casl2a protein or variant thereof or (II) the two or more effector subunits are capable of generating one or more additional outputs to the sample comprising effector activity of the Casl2a/crRNA complexes at target sites directed by the one or more additional crRNAs.
36. The system of claim 35, comprising nucleic acid encoding a crRNA array comprising one or more of the first crRNA and the one or more additional crRNAs.
37. The system of any one of claims 1 to 36, wherein the Casl2a protein or variant thereof is derived from a parental Casl2a protein from Lachnospiraceae bacterium comprising the amino acid sequence of SEQ ID NO: 1.
38. The system of any one of claims 1 to 37, wherein the target nucleic acid is a target dsDNA.
39. The system of claim 38, wherein a protospacer in the target dsDNA targeted by the crRNA spacer has a 5' PAM selected from 5'-TTTG-3', 5'-TTTA-3', and 5'-TTTC-3'.
40. The system of any one of claims 1 to 39, wherein the Casl2a protein or variant thereof is a nuclease- competent Cas protein comprising a Casl2a domain having some, the same, or greater nuclease activity as compared to a parental Cas 12a protein from which it is derived, and the first output comprises cleavage of the first target nucleic acid near the first target polynucleotide sequence.
41. The system of claim 42, wherein the Cas 12a domain comprises the amino acid sequence of SEQ ID NO: 1.
42. The system of any one of claims 1 to 41, wherein the Cas 12a protein or variant thereof is a fusion protein comprising an effector domain fused to a nuclease-deficient dCas 12a domain having reduced or eliminated nuclease activity as compared to a parental Cas 12a protein from which it is derived, and the first output is generated by activity of the effector domain.
43. The system of claim 42, wherein the dCasl2a domain comprises a modification of amino acid D832 with respect to SEQ ID NO: 1 that reduces, substantially eliminates, or eliminates nuclease activity of the dCasl2a domain.
44. The system of claim 43, wherein the dCasl2a domain comprises a D832A substitution with respect to SEQ ID NO: 1.
45. The system of claim 44, wherein the dCasl2a domain comprises the amino acid sequence of SEQ ID NO: 2.
46. The system of any one of claims 42 to 45, wherein the effector domain comprises a polypeptide that can (i) cleave a nucleic acid, (ii) edit a nucleotide, (iii) activate transcription, (iv) repress transcription, (v) methylate a nucleic acid, and/or (vi) demethylate a nucleic acid.
47. The system of claim 46, wherein the effector domain comprises a transcriptional activation domain, and the first output comprises transcriptional activation of a first gene targeted by the first crRNA.
48. The system of claim 47, wherein the transcriptional activation domain is a tripartite VP64-p65-Rta (VPR) transcriptional activation domain or variant thereof.
49. The system of claim 47, wherein targeting of the genomic DNA by the Casl2a system is capable of enhancing transcription of the mRNA.
50. The system of any one of claims 1 to 50, wherein the sample is a cell or a composition comprising a cell.
51. The system of claim 50, wherein the inputs to the sample are selected from physical inputs, small molecule inputs, and biologic inputs.
52. The system of claim 51 , wherein
(a) the physical inputs are selected from oxygen levels, pH, ion concentrations, temperature, light, mechanical force, ultrasound, shock wave, magnetic field, and electric field;
(b) the small molecule inputs are selected from drugs, metabolites, and adjuvants; and/or
(c) the biologic inputs are selected from polypeptides, polypeptide derivatives, and nucleic acids.
53. An engineered cell prepared by introducing the system of any one of claims 1 to 52 into an input cell.
54. The engineered cell of claim 53, wherein the engineered cell is capable of expressing a target gene, the first target nucleic acid comprises the target gene, and the first output of the system modulates the expression of the target gene in the engineered cell.
55. The engineered cell of claim 54, wherein the input cell is a target cell, and one or more NOT inputs of the system capable of increasing the expression of Acr protein in the engineered cell are associated with off-target cells and not the target cell.
56. The engineered cell of claim 54 or 55, wherein the target gene encodes a target receptor.
57. The engineered cell of claim 56, wherein the input cell is a target immune cell, the target receptor is a chimeric antigen receptor (CAR) targeting a disease-associated cell, one or more inputs of the system are associated with the disease-associated cell, and the first output of the system increases the expression of the CAR in the engineered cell.
58. The engineered cell of claim 57, wherein the engineered cell is a CAR T cell, a CAR NK cell, or a CAR macrophage cell.
59. The engineered cell of claim 56, wherein the input cell is a target T cell, the target receptor is a T cell receptor (TCR) targeting a disease-associated cell, one or more inputs of the system are associated with the disease-associated cell, and the first output of the system increases the expression of the TCR in the engineered cell.
60. The engineered cell of any one of claims 57 to 59, wherein one or more NOT inputs of the system capable of increasing the expression of Acr protein in the engineered cell are associated with healthy cells and not the disease-associated cell.
61. The engineered cell of claim 54 or 55, wherein the input cell is a target disease- associated cell, and one or more inputs of the system are associated with the disease-associated cell.
62. The engineered cell of claim 61 , wherein the first output of the system modulates the expression of the target gene in the engineered cell such that it is rendered benign.
63. The engineered cell of claim 61, wherein the first output of the system modulates the expression of the target gene in the engineered cell such that it is killed.
64. The engineered cell of any one of claims 61 to 63, wherein the target disease-associated cell is a cancer cell, and one or more inputs to the cell are cancer-associated inputs characteristic of the cancer cell.
65. The engineered cell of any one of claims 61 to 64, wherein one or more NOT inputs of the system capable of increasing the expression of Acr protein in the engineered cell are associated with healthy cells and not the disease-associated cell.
66. A method for conditionally generating an output to a sample, the method comprising providing to the sample the system of any one of claims 1 to 52, thereby generating an output to the sample mediated by a split Casl2a/crRNA complex when all components of the split Casl2a/crRNA complex are expressed.
67. The method of claim 66, wherein the output comprises targeting of a target nucleic acid by the split Casl2a/crRNA complex.
68. The method of claim 66, wherein the Casl2a protein or variant thereof has a nucleic acid-editing effector activity and the output comprises editing of a target nucleic acid by the split Casl2a/crRNA complex.
69. The method of claim 66, wherein the Casl2a protein or variant thereof is a dCasl2a fusion protein or variant thereof comprising an effector domain having a transcription- modulating effector activity and the output comprises modulation of transcription of a target nucleic acid by the split Casl2a/crRNA complex.
70. The method of claim 66, wherein the Casl2a protein or variant thereof is a dCasl2a fusion protein or variant thereof comprising an effector domain having an epigenetic-editing effector activity and the output comprises editing of the epigenome of a target genomic DNA by the split Casl2a/crRNA complex.
71. The method of any one of claims 66 to 70, wherein the sample is a cell or a composition comprising a cell.
72. The method of any one of claims 66 to 71 , wherein the inputs to the sample are selected from physical inputs, small molecule inputs, and biologic inputs.
73. The method of claim 72, wherein
(a) the physical inputs are selected from oxygen levels, pH, ion concentrations, temperature, light, mechanical force, ultrasound, shock wave, magnetic field, and electric field;
(b) the small molecule inputs are selected from drugs, metabolites, and adjuvants; and/or
(c) the biologic inputs are selected from polypeptides, polypeptide derivatives, and nucleic acids.
74. A method for treating a disease or condition associated with a target gene in a subject in need thereof, the method comprising: modifying a target cell in the subject to produce an engineered cell according to claim 54 or 55, wherein the expression of the target gene in the engineered cell is modulated such that the disease or condition is treated.
75. A method for treating a disease or condition associated with a disease-associated cell in a subject in need thereof, the method comprising: providing to the subject an engineered cell according to any one of claims 53 to 60, wherein the engineered cell is capable of mediating killing of the disease-associated cell to treat the disease or condition.
76. A method for treating a disease or condition associated with a disease-associated cell in a subject in need thereof, the method comprising: modifying the disease-associated cell in the subject to produce an engineered cell according to any one of claims 61 to 65, wherein the expression of the target gene in the engineered cell is modulated such that the disease or condition is treated.
77. A kit comprising one or more components of the system of any one of claims 1 to 52.
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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2019034784A1 (en) * 2017-08-18 2019-02-21 Universität Heidelberg Use of anti-crispr polypeptides for specific activation of cas nucleases
WO2019126762A2 (en) * 2017-12-22 2019-06-27 The Broad Institute, Inc. Cas12a systems, methods, and compositions for targeted rna base editing

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2019034784A1 (en) * 2017-08-18 2019-02-21 Universität Heidelberg Use of anti-crispr polypeptides for specific activation of cas nucleases
WO2019126762A2 (en) * 2017-12-22 2019-06-27 The Broad Institute, Inc. Cas12a systems, methods, and compositions for targeted rna base editing

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