Patent Application
20220056097
The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Apr. 5, 2021, is named 079445-003410US-1238452_SL.txt and is 9,957 bytes in size.
The 3-dimensional (3D) spatial organization of polynucleotides within living cells plays an important role in such processes as regulating and maintaining gene expression, genome stability, and cellular function.
There exists a need for systems and methods to carry out the spatial organization of target polynucleotides. The present disclosure addresses this and other needs.
In general, provided herein are systems and methods for programmable polynucleotide re-organization. The systems and methods can couple an actuator moiety with cellular compartment-specific proteins via a chemically inducible system, and can allow efficient, inducible, and dynamic repositioning of polynucleotides, e.g., genomic loci, to particular cellular positions, e.g., the nuclear periphery, Cajal bodies, and PML nuclear bodies. (
In some aspects, a composition comprises: a) a compartment-constituent protein linked to a first dimerization domain; and b) an actuator moiety linked to a scaffold, wherein the scaffold is linked to a second dimerization domain; and wherein the first dimerization domain binds to the second dimerization domain. In some embodiments, the scaffold is linked to at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more second dimerization domains. In some embodiments, the compartment-constituent protein is further linked to a second scaffold, wherein the second scaffold is linked to at least one first dimerization domain. In some embodiments, the second scaffold is linked to at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more first dimerization domains. In some embodiments, the scaffold or the second scaffold is a repeating peptide array. In some embodiments, the scaffold or the second scaffold is a SpyTag, SunTag, split sfGFP, MoonTag, SnoopTag, split sfCherry2, or mNeonGreen2 protein scaffold. In some embodiments, the composition further comprises a ligand, wherein the binding of the first dimerization domain to the second dimerization domain occurs in the presence of the ligand. The composition of any one of claims 1-6, wherein the composition regulates an expression of the target polynucleotide. In some embodiments, the composition increases an expression of the target polynucleotide. In some embodiments, the composition decreases an expression of the target polynucleotide. In some embodiments, the composition regulates an expression of an additional polynucleotide. In some embodiments, the composition increases an expression of an additional polynucleotide. In some embodiments, the composition decreases an expression of an additional polynucleotide. In some embodiments, the additional polynucleotide is a distal gene to the target polynucleotide. In some embodiments, the additional polynucleotide is a proximal gene to the target polynucleotide. In some embodiments, a three-dimensional structure of the complex regulates a gene or a regulatory element of a gene. In some embodiments, the regulatory element is an enhancer or a promoter. In some embodiments, the three-dimensional structure forms a three-dimensional loop, a chromosome boundary, a topologically associating domain, or a gene cluster. In some embodiments, the three-dimensional loop is formed between an enhancer and a promoter. In some embodiments, the composition insulates a target polynucleotide. In some embodiments, the composition insulates a target polynucleotide by separating the target polynucleotide for chromosome protection or manipulation. In some embodiments, the composition traps a target polynucleotide in a spatial region of a compartment. In some embodiments, the spatial region of a compartment manipulates the fate or function of the target polynucleotide or the additional polynucleotide. In some embodiments, the spatial region of a compartment promotes or prevents recombination or mutagenesis, promotes or inhibits gene expression, splicing or translation, promotes or inhibits polynucleotide transport or movement In some embodiments, the composition introduces epigenetic modifications at the target polynucleotide. In some embodiments, the epigenetic modification is DNA methylation, DNA demethylation, histone methylation, histone demethylation, acetylation, deacetylation, phosphorylation, dephosphorylation, ubiquitylation, GlcNAcylation, citrullination, krotonilation, isomerization, or any combination thereof. In some embodiments, the composition repairs a DNA break. In some embodiments, the composition repairs a DNA break by introducing exogenous DNA. In some embodiments, the composition repairs a DNA break by introducing recombination, non-homologous end-joining, or homology-directed repair. In some embodiments, the composition creates an artificial aggregate, wherein the artificial aggregate comprises protein, RNA, DNA, or a combination thereof. In some embodiments, the artificial aggregate proteins, RNAs, DNAs, or a combination thereof are recruited by the compartment-constituent protein. In some embodiments, the target polynucleotide is genomic DNA. In some embodiments, the target polynucleotide is a polynucleotide encoding a gene. In some embodiments, the target polynucleotide is a non-coding polynucleotide. In some embodiments, the target polynucleotide is a tandem repeat region of genomic DNA. In some embodiments, the target polynucleotide is RNA. In some embodiments, the compartment is a Cajal body. In some embodiments, the compartment-constituent protein comprises a protein from a Cajal body. In some embodiments, the compartment-constituent protein comprises coilin, SMN, Gemin 3, SmD1, SmE, or a combination thereof. In some embodiments, the compartment is a nuclear speckle. In some embodiments, the compartment-constituent protein is a protein from a nuclear speckle. In some embodiments, the compartment-constituent protein comprises SC35. In some embodiments, the compartment is a PML body. In some embodiments, the compartment-constituent protein is a protein from a PML body. In some embodiments, the compartment-constituent protein comprises PML, SP100, or a combination thereof. In some embodiments, the compartment is a cytosolic compartment. In some embodiments, the compartment-constituent protein is a protein from a cytosolic compartment. In some embodiments, the compartment is a synthetic cellular phase. In some embodiments, the compartment-constituent protein is a protein from a synthetic cellular phase. In some embodiments, the compartment-constituent protein comprises Rad51, Rad52, RPA, MRN complex, MRX complex, CtlP, Sae2, BLM, Sgs1, BRCA2, exonucleases such as Exo1 or OsExo1, ATM, BRCA2, RAD54, or MDC1. In some embodiments, the synthetic cellular phase facilitates homology directed repair. In some embodiments, the compartment is nuclear heterochromatin. In some embodiments, the compartment-constituent protein is a protein from nuclear heterochromatin. In some embodiments, the compartment-constituent protein comprises HP1α, HP1β, KAP1, KRAB, SUV39H1, or G9a. In some embodiments, the compartment-constituent protein further comprises an oligomerization domain. In some embodiments, the compartment-constituent protein is further linked to a fluorescent protein. In some embodiments, the actuator moiety comprises a binding protein that hybridizes to the target polynucleotide. In some embodiments, the actuator moiety comprises a Cas protein, and wherein the system further comprises: (c) a guide RNA that complexes with the actuator moiety and hybridizes to the target polynucleotide. In some embodiments, the actuator moiety comprises an RNA binding protein complexed with a guide RNA that hybridizes to the target polynucleotide, and wherein the composition further comprises: (c) a Cas protein that complexes with the guide RNA. In some embodiments, the Cas protein substantially lacks DNA cleavage activity. In some embodiments, the Cas protein is a Cas9 protein, a Cas12 protein, a Cas13 protein, a CasX protein, or a CasY protein. In some embodiments, the Cas12 protein is selected from the group consisting of Cas12a, Cas12b, Cas12c, Cas12d, and Cas12e. In some embodiments, the actuator moiety comprises a binding protein that hybridizes to the target polynucleotide, wherein the binding protein is a zinc finger nuclease or a TALE nuclease. In some embodiments, the actuator moiety comprises an Argonaute protein complexed with a guide polynucleotide, wherein the guide polynucleotide is a guide RNA or a guide DNA, and wherein the guide polynucleotide hybridizes to the target polynucleotide. In some embodiments, the actuator moiety is further linked to a fluorescent protein. In some embodiments, the first dimerization domain binds to the second dimerization domain in the presence of a ligand. In some embodiments, the first dimerization domain binds to the ligand and the ligand binds to the second dimerization domain to assemble a dimer. In some embodiments, the ligand is a chemically inducible. In some embodiments, the ligand is abscisic acid. In some embodiments, the dimer is inducible and reversible. In some embodiments, the dimer is a heterodimer. In some embodiments, the dimer is a homodimer. In some embodiments, the first dimerization domain is an ABI domain. In some embodiments, the second dimerization domain is a PYL1 domain. In some embodiments, the first dimerization domain is a PYL1 domain. In some embodiments, the second dimerization domain is an ABI domain. In some embodiments, the compartment-constituent protein linked to the first dimerization domain is a fusion protein. In some embodiments, the actuator moiety linked to the second dimerization domain is a fusion protein. In some embodiments, the compartment-constituent protein linked to the first dimerization domain by a linker. In some embodiments, the actuator moiety linked to the second dimerization domain by a linker.
In some aspects, the method comprises: (a) providing a compartment-constituent protein linked to a first dimerization domain; (b) providing an actuator moiety linked to a second dimerization domain, wherein the actuator moiety and the target polynucleotide form a complex; and (c) assembling a dimer comprising the first dimerization domain and the second dimerization domain of the complex, thereby forming the compartment around the target polynucleotide. In some embodiments, the method further comprises providing a ligand before step (c), wherein the ligand binds the first dimerization domain to the second dimerization domain for assembling the dimer of step (c). In some embodiments, the method further comprises regulating an expression of the target polynucleotide after the formation of the compartment around the target polynucleotide. In some embodiments, the method further comprises increasing an expression of the target polynucleotide after the formation of the compartment around the target polynucleotide compared to before the formation of the compartment around the target polynucleotide. In some embodiments, the method further comprises decreasing an expression of the target polynucleotide after the formation of the compartment around the target polynucleotide compared to before the formation of the compartment around the target polynucleotide. In some embodiments, the method further comprises regulating an expression of an additional polynucleotide after the formation of the compartment around the target polynucleotide. In some embodiments, the method further comprises increasing an expression of an additional polynucleotide after the formation of the compartment around the target polynucleotide compared to before the formation of the compartment around the target polynucleotide. In some embodiments, the method further comprises decreasing an expression of an additional polynucleotide after the formation of the compartment around the target polynucleotide compared to before the formation of the compartment around the target polynucleotide. In some embodiments, the additional polynucleotide is a distal gene to the target polynucleotide. In some embodiments, the additional polynucleotide is a proximal gene to the target polynucleotide. In some embodiments, a three-dimensional structure of the complex regulates a gene or a regulatory element of a gene. In some embodiments, the regulatory element is an enhancer or a promoter. In some embodiments, the three-dimensional structure forms a three-dimensional loop, a chromosome boundary, a topologically associating domain, or a gene cluster. In some embodiments, the three-dimensional loop is formed between an enhancer and a promoter. In some embodiments, the method further comprises insulating a target polynucleotide. In some embodiments, the insulating comprises separating the target polynucleotide for chromosome protection or manipulation. In some embodiments, the method further comprises trapping a target polynucleotide in a spatial region of a compartment. In some embodiments, the method further comprises manipulating the fate or function of the target polynucleotide or the additional polynucleotide in the spatial region or compartment. In some embodiments, the spatial region of a compartment promotes or prevents recombination or mutagenesis, promotes or inhibits gene expression, splicing or translation, promotes or inhibits polynucleotide transport or movement. In some embodiments, the method further comprises introducing epigenetic modifications at the target polynucleotide after the forming of the compartment around the target polynucleotide.
The method of any one of claim 100, wherein the epigenetic modification is DNA methylation, DNA demethylation, histone methylation, histone demethylation, acetylation, deacetylation, phosphorylation, dephosphorylation, ubiquitylation, GlcNAcylation, citrullination, krotonilation, isomerization, or any combination thereof. In some embodiments, the method further comprises repairing a DNA break. In some embodiments, the repairing comprises introducing exogenous DNA. In some embodiments, the introducing comprises recombination, non-homologous end-joining, or homology-directed repair. In some embodiments, the formation of the compartment around the target polynucleotide further comprises creating an artificial aggregate, wherein the artificial aggregate comprises protein, RNA, DNA, or a combination thereof. In some embodiments, the artificial aggregate proteins, RNAs, DNAs, or a combination thereof are recruited by the compartment-constituent protein. In some embodiments, the target polynucleotide is genomic DNA. In some embodiments, the target polynucleotide is polynucleotide encoding a gene. In some embodiments, the target polynucleotide is noncoding polynucleotide. In some embodiments, the target polynucleotide is a tandem repeat region of genomic DNA. In some embodiments, the target polynucleotide is RNA. In some embodiments, the compartment is a Cajal body. In some embodiments, the compartment-constituent protein comprises a protein from a Cajal body. In some embodiments, the compartment-constituent protein comprises coilin, SMN, Gemin 3, SmD1, SmE, or a combination thereof. In some embodiments, the compartment is a nuclear speckle. In some embodiments, the compartment-constituent protein is a protein from a nuclear speckle. In some embodiments, the compartment-constituent protein comprises SC35. In some embodiments, the compartment is a PML body. In some embodiments, the compartment-constituent protein is a protein from a PML body. In some embodiments, the compartment-constituent protein comprises PML, SP100, or a combination thereof. In some embodiments, the compartment is a cytosolic compartment. In some embodiments, the compartment-constituent protein is a protein from a cytosolic compartment. In some embodiments, the compartment is a synthetic cellular phase. In some embodiments, the compartment-constituent protein is a protein from a synthetic cellular phase. In some embodiments, the compartment-constituent protein comprises Rad51, Rad52, RPA, MRN complex, MRX complex, CtlP, Sae2, BLM, Sgs1, BRCA2, exonucleases such as Exo1 or OsExo1, ATM, BRCA2, RAD54, or MDC1. In some embodiments, the synthetic cellular phase facilitates homology directed repair. In some embodiments, the compartment is nuclear heterochromatin. In some embodiments, the compartment-constituent protein is a protein from nuclear heterochromatin. In some embodiments, the compartment-constituent protein comprises HP1α, HP1β, KAP1, KRAB, SUV39H1, or G9a. In some embodiments, the compartment-constituent protein further comprises an oligomerization domain. In some embodiments, the compartment-constituent protein is further linked to a fluorescent protein. In some embodiments, the actuator moiety comprises a binding protein that hybridizes to the target polynucleotide. In some embodiments, the actuator moiety comprises a Cas protein, and wherein the system further comprises: (c) a guide RNA that complexes with the actuator moiety and hybridizes to the target polynucleotide. In some embodiments, the actuator moiety comprises an RNA binding protein complexed with a guide RNA that hybridizes to the target polynucleotide, and wherein the system further comprises: (c) a Cas protein that complexes with the guide RNA. In some embodiments, the Cas protein substantially lacks DNA cleavage activity. In some embodiments, the Cas protein is a Cas9 protein, a Cas12 protein, a Cas13 protein, a CasX protein, or a CasY protein. In some embodiments, the Cas12 protein is selected from the group consisting of Cas12a, Cas12b, Cas12c, Cas12d, and Cas12e. In some embodiments, the actuator moiety comprises a binding protein that hybridizes to the target polynucleotide, wherein the binding protein is a zinc finger nuclease or a TALE nuclease. In some embodiments, the actuator moiety comprises an Argonaute protein complexed with a guide polynucleotide, wherein the guide polynucleotide is a guide RNA or a guide DNA, and wherein the guide polynucleotide hybridizes to the target polynucleotide. In some embodiments, the actuator moiety is further linked to a fluorescent protein. In some embodiments, the actuator moiety is further linked to a scaffold, wherein the actuator is linked to the scaffold and the scaffold is linked to at least one second dimerization domains In some embodiments, the actuator moiety is further linked to a scaffold, wherein the actuator is linked to the scaffold and the scaffold is linked to 2, 3, 4, 5, 6, 7, 8, 9, 10, or more second dimerization domains. In some embodiments, the scaffold is a repeating peptide array. In some embodiments, the scaffold is a SpyTag, SunTag, split sfGFP, MoonTag, SnoopTag, split sfCherry2, or mNeonGreen2 protein scaffold. In some embodiments, the assembling occurs in the presence of a ligand. In some embodiments, the first dimerization domain binds to the ligand and ligand binds to the second dimerization domain to assemble the dimer. In some embodiments, the ligand is a chemically inducible In some embodiments, the ligand is abscisic acid. In some embodiments, the assembling is inducible and reversible. In some embodiments, the dimer is a heterodimer. In some embodiments, the dimer is a homodimer. In some embodiments, the first dimerization domain is an ABI domain. In some embodiments, the second dimerization domain is a PYL1 domain. In some embodiments, the first dimerization domain is a PYL1 domain. In some embodiments, the second dimerization domain is a ABI domain. In some embodiments, the compartment-constituent protein linked to the first dimerization domain is a fusion protein. In some embodiments, the actuator moiety linked to the second dimerization domain is a fusion protein. In some embodiments, the compartment-constituent protein is linked to the first dimerization domain by a linker. In some embodiments, the actuator moiety is linked to the second dimerization domain by a linker.
In some aspects, a method of treating a disease in a subject in need thereof, by administering the composition of any one of the previous embodiments. In some embodiments, the disease is a protein misfolding disease. In some embodiments, the disease is selected from the group consisting of Alzheimer's disease (AD), Parkinson's disease (PD), multisystem atrophy, Huntington's disease (HD), prion diseases, Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD).
In some aspects, a system comprises the composition of any one of the preceding embodiments.
All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.
The novel features of the disclosure are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present disclosure will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings of which:
The 3-dimensional (3D) spatial organization of polynucleotides within living cells can play an important role in such processes as regulating and maintaining gene expression, genome stability, and cellular function. For example, genomic sequences that associate with nuclear lamina or the nuclear periphery often exhibit low transcriptional activity, while those that localize to the nuclear interior often exhibit relatively higher activity. Furthermore, the eukaryotic cell nucleus can contain many membraneless nuclear bodies, such as Cajal bodies, PML bodies, nucleolus and speckles, that can be functionally important in a variety of biological processes. A central goal in genomics and cell biology has been to understand the relationship between genome structure, its organization within various nuclear compartments, and gene expression, but this goal has been constrained by currently available methods.
A correlation between genome organization and cell fate determination has been suggested by numerous studies using microscopy-based imaging (e.g., FISH) and chromosome conformation capture (3C) techniques. For example, during lymphocyte development, the IgH and Igx loci that are positioned at the nuclear periphery in progenitor cells often relocate to nuclear interior in pro-B cells, a process that is synchronous with the activation and rearrangement of immunoglobulin loci. Similarly, the genomic locus of the proneural transcription factor Ascl1 can be located in the nuclear periphery in undifferentiated embryonic stem cells, but can relocate to the nuclear interior during neuronal differentiation. Moreover, 3C-based studies have revealed changes in high-resolution chromatin interactions (e.g., topologically associated domains) during development and disease processes. Altogether, these are powerful methods for mapping genome organization and measuring physical interactions of chromatin elements, they often cannot provide causal links between genome positioning and function and they are unable to measure dynamic changes in living cells.
Nuclear compartments have been observed to play an important role in genome organization and function. Nuclear bodies are proposed to assemble through liquid-liquid phase separation, which can be driven by multivalent interactions between proteins and RNAs. De novo nuclear body formation can be nucleated by immobilization of protein or RNA components on chromatin. Among nuclear bodies, Cajal bodies (CBs) can be essential for vertebrate embryogenesis, and can be abundant in tumor cells and neurons. CBs can be marked by a scaffold protein component, Coilin, and can play an important role in small nuclear RNA (snRNA) biogenesis, ribonucleoprotein (RNP) assembly, and telomerase biogenesis. The promyelocytic leukemia (PML) nuclear bodies, marked by a tumor suppressor protein, PML, can be abundant nucleus dot structures that associate with disease processes including tumor and viral infection. However, how the colocalization of nuclear bodies and chromatin can causally affect gene expression, and cellular function remains mostly elusive.
To understand such causal relationships, sequence-specific DNA-protein interactions have been exploited to mediate targeted genomic reorganization. This technique utilizes an array of LacO repeats inserted into a genomic locus, which facilitates tethering of the adjacent genomic sequence to the nuclear periphery when combined with Lad fused to a nuclear membrane protein. Using this technique, several studies have reported that repositioning a gene to the nuclear periphery can lead to gene repression. However, this technique may not suitable for programmable genome targeting, and can be tedious and difficult to implement. For example, creating a stable LacO repeat-containing cell line is a prerequisite for this technique, which already involves many steps such as the random insertion of a large LacO repeat array into the genome, screening for cells containing a single insertion locus, generating stable cell lines, and characterization of the genomic insertion site by FISH. New tools are needed to manipulate the spatial and temporal organization of the genome in a programmable, precise, and targeted manner.
Prokaryotic Class II CRISPR-Cas (Clustered regularly interspaced short palindromic repeats-CRISPR associated) systems can be repurposed as a toolbox (e.g. Cas9 and Cpfl) for gene editing, gene regulation, epigenome editing, chromatin looping, and live-cell genome imaging. Nuclease-deactivated Cas (dCas) proteins coupled with transcriptional effectors or epigenetic modifying domains can allow for regulation of expression of genes adjacent to the single guide RNA (sgRNA) target site. It remains unknown whether the CRISPR-Cas system can be used to mediate genome organization and reposition the location of chromatin DNA relative to various nuclear compartments within mammalian nuclei. In view of the foregoing, there exists a need for alternative systems and methods to carry out the spatial organization of target polynucleotides. The present disclosure addresses this and other needs.
Furthermore, eukaryotic cells are complex structures capable of coordinating numerous biochemical reactions in space and time. Keys to such coordination are both the 3D organization of polynucleotides such as the genome, and the subdivision of intracellular space into functional compartments. Compartmentalization can be achieved by intracellular membranes, which surround organelles and act as physical barriers. In addition, cells have developed sophisticated mechanisms to partition their inner substance in a tightly regulated manner. Recent studies provide compelling evidence that membraneless compartmentalization can be achieved by liquid demixing, a process culminating in liquid-liquid phase separation and the formation of phase boundaries.
The inventors have surprisingly discovered versatile systems and methods that can efficiently control the spatial positioning of polynucleotides relative to the functional compartments, including nuclear compartments such as the nuclear periphery, Cajal bodies, and promyelocytic leukemia (PML) bodies. Additionally, the inventors have discovered versatile systems and methods that can efficiently control the spatial positioning of compartments relative to the polynucleotides. The systems, compositions, and methods can also be useful in generating synthetic phase separations, by forming supramolecular assemblies of proteins, RNA, and/or DNA molecules organized or portioned within a cell. For example, proteins that contribute to the formation of a specific compartment (e.g., a compartment-specific protein or a compartment constituent protein) can be used to form a compartment around a target polynucleotide. The systems and methods disclosed herein can be useful for manipulating the spatiotemporal organization of genomic DNA and RNA components in the nucleus/cytoplasm and for regulating diverse cellular functions. The provided systems, compositions, and methods also can be used for programmable control of spatial genome organization, and for applying this organization to affect polynucleotide regulation and cellular function, and to mediate interacting dynamics between targeted polynucleotides and different cellular compartments. The disclosed systems, compositions, and methods can be used, for example, to achieve the dynamic reorganization of subcellular space as a framework to manipulate pathological protein assembly in diseases including cancer and neurodegeneration. The disclosed systems, compositions, and methods can also be used, for example, to achieve the dynamic reorganization of subcellular space and target polynucleotides to facilitate homology directed repair (HDR), non-homologous end joining (NHEJ), or other methods of polynucleotide repair.
Furthermore, the disclosed systems, compositions, and methods can produce three-dimensional structures including three-dimensional loops, for example, three-dimensional loops between enhancers and promoters, chromosome boundaries, a topologically associating domains, or gene clusters for regulating multiple genes and their regulatory elements. The disclosed systems, compositions, and methods can be used for producing genomic insulators that separate a target region comprising the target polynucleotide for chromosome protection and manipulation. The disclosed systems, compositions, and methods can also be used for localizing polynucleotides within particular types of compartment. For example, the disclosed systems, compositions, and methods, are used to trap a region of polynucleotides comprising the target polynucleotide in a spatial region of compartment or within a compartment that comprises uniquely defined biochemical properties that promote or prevent recombination or mutagenesis, promote or inhibit of gene transcription, splicing, or translation, or promote or inhibit polynucleotide transport and movement. Thus, importantly, the systems, compositions, and methods as described herein can be used to manipulate the fate, function, and properties of target polynucleotide and genomic region comprising the target polynucleotide, which also can be used to manipulate the fate, function, and properties of the cell comprising the target polynucleotide.
The disclosed systems, compositions, and methods can be chemically inducible and reversible, enabling interrogation of real-time dynamics of, for example, chromatin interactions with nuclear compartments in living cells. As further examples, inducible repositioning of genomic loci to the nuclear periphery can allow dissection of mitosis-dependent and -independent relocalization events, interrogation of the relationship between gene position and expression, and understanding of the effects of telomere repositioning on cell growth. The systems, compositions, and methods described herein can mediate rapid de novo formation of Cajal bodies at target chromatin loci and causes significant repression of adjacent endogenous gene expression across long distances (>30 kb). The systems, compositions, and methods described herein can mediate rapid de novo formation of a compartment comprising proteins that facilitate HDR, such as Rad51, Rad52, RPA, MRN complex, MRX complex, CtlP, Sae2, BLM, Sgs1, BRCA2, exonucleases such as Exo1 or OsExo1, ATM, BRCA2, RAD54, or MDC1, at a target polynucleotide that has been cut. The systems, compositions, and methods described herein rapid de novo formation of a compartment that facilitates nuclear heterochromatin formation, such as HP1α, HP1β, KAP1, KRAB, SUV39H1, or G9a. The compartment can comprise proteins that are specific for that particular compartment (compartment-specific proteins) or proteins that are part of that particular compartment (compartment-constituent protein). The provided system, compositions, and methods thus offers a novel platform to investigate large-scale spatial polynucleotide organization and function in a targeted manner and a novel platform for manipulating the fate, function, and properties of a target polynucleotide, the region comprising the target polynucleotide, and the cell comprising the target polynucleotide.
In some embodiments, the use of different sgRNAs allows the system, composition, and method to be programmed to flexibly target different genomic sequences. Target polynucleotide colocalization with a compartment can be triggered through rapid de novo compartment formation, formation of a compartment around a target polynucleotide, or through repositioning target polynucleotide to an existing compartment. The repositioning of genomic loci to the nuclear periphery can be enabled in both mitosis-dependent and -independent manners. Target DNA co-localization with Cajal bodies can be triggered through rapid de novo Cajal body formation or through repositioning target DNA to existing Cajal bodies. Targeting genomic loci to the nuclear periphery or to Cajal bodies using the provided systems and methods can also repress adjacent reporter gene expression. Importantly, colocalization of genomic loci with Cajal bodies also can repress expression of adjacent endogenous genes (>30 kb). Furthermore, the sequestering of telomeres to the nuclear periphery using aspects of the present disclosure can negatively impact cell growth. Targeting genomic loci to a compartment or formation of the compartment comprising proteins that facilitate HDR, such as Rad51, Rad52, RPA, MRN complex, MRX complex, CtlP, Sae2, BLM, Sgs1, BRCA2, exonucleases such as Exo1 or OsExo1, ATM, BRCA2, RAD54, or MDC1, at a target polynucleotide that has been cut can also be used to facilitate HDR of the cut target polynucleotide. Targeting genomic loci to a compartment or formation of the compartment comprising proteins that facilitates nuclear heterochromatin formation, such as HP1α, HP1β, KAP1, KRAB, SUV39H1, or G9a can be used to facilitate transcription regulation of a genomic region comprising the target polynucleotide.
The CRISPR-Cas system has been repurposed as a flexible genome engineering platform, and has been used for applications such as gene editing, transcriptional regulation, epigenetic modifications, DNA looping, and genome imaging. Provided herein are further expansions to the CRISPR-Cas toolbox in the form of a polynucleotide organization system which enables programmable control of targeted polynucleotide positioning within the cellular compartments. In certain aspects, the targeted polynucleotides comprise genomic DNA and the system is referred to as CRISPR-GO (
A major goal in cell biology is the understanding of how genomic interactions with different nuclear compartments affect gene expression, chromatin conformation, and cellular functions. The CRISPR-GO system can efficiently target specific genomic loci to the nuclear periphery, Cajal bodies, and PML bodies, and also holds potential to be expanded to other nuclear compartments such as nucleoli, nuclear pore complexes, and nuclear speckles. Targeting genomic loci to other nuclear compartments can be achieved by coupling CRISPR-GO with different compartment-specific proteins, such as heterochromatin protein 1a (HP1α) (
The provided systems (e.g., CRISPR-GO), compositions, and methods allows programmable re-localization of polynucleotides (e.g., genomic loci) in a precise and targeted manner. The provided systems (e.g., CRISPR-GO), compositions, and methods allows programmable formation of a compartment using compartment constituent protein around a target polynucleotide (e.g., genomic loci) in a precise and targeted manner. The provided systems (e.g., CRISPR-GO), compositions, and methods also allow for control of the local environment of a target polynucleotide by assembly or co-localization of a compartment to a polynucleotide in a precise and targeted manner. For example, the CRISPR-GO system can efficiently target repetitive and non-repetitive chromatin loci located on different chromosomes to nuclear compartments. As another example, the CRISPR-GO system can efficiently assemble a compartment comprising proteins to facilitate HDR to a target repetitive or non-repetitive chromatin loci located on a chromosome. As an additional example, the CRISPR-GO system efficiently forms a compartment, such as a heterochromatin compartment, around target repetitive and non-repetitive chromatin loci located on different chromosomes. The CRISPR-GO system can efficiently assemble a compartment comprising proteins to facilitate nuclear heterochromatin around a target repetitive or non-repetitive chromatin loci located on a chromosome. Unlike the LacI-LacO system, the genomic targets of the CRISPR-GO system can be flexibly defined by the base-pairing interactions between sgRNAs and the target DNA sequence, and simply altering a ˜20 nt region on the sgRNAs allows for the targeting of a different genomic locus. This programmable feature can allow one to use CRISPR-GO to target a variety of genomic elements, including protein-coding genes, non-coding RNA genes, and regulatory elements. In contrast, the LacO-LacI technique is not suitable for programmable genomic targeting, as it can only be performed on well-characterized cell lines containing a highly repetitive LacO array. Creating and characterizing a useful LacO-containing cell line is difficult and laborious. LacO arrays are usually randomly inserted into the genome, after which cells containing a single-copy insertion are selected to build stable cell lines before the precise genome integration sites is characterized by FISH and other methods. In addition, it is possible that integration of a large LacO array in the genome may alter local chromatin conformation. Altogether, the versatility of the systems, compositions, and methods disclosed herein offers a major technological advantage over conventional methods to study cellular organization and to manipulate the fate, function, and properties of a target polynucleotide, a genomic region comprising the target polynucleotide, or the cell comprising the target polynucleotide.
The overall ease of targeting a new locus of polynucleotides or a compartment with the systems, compositions, and methods disclosed herein can facilitate broader studies of the relationship between perturbations in 3D polynucleotide organization and changes in cellular phenotypes. For example, different sgRNA design strategies can be used to target repetitive and non-repetitive genomic loci. Repetitive genomic loci can be easily targeted using a single sgRNA that has multiple targets within a defined genomic region. The human genome has abundant repetitive or repeat-derived sequences, many of which likely have important genome-organization roles. These repetitive sequences are candidates for large-scale screening experiments, opening the door to more high-throughput approaches to study the relationship between genome organization relative to nuclear compartments and cellular phenotype. In addition, non-repetitive genomic loci can be targeted using multiple sgRNAs or using a single sgRNA. To target a non-repetitive locus, a pool of tiling sgRNAs can be used as a starting point.
The provided systems, compositions, and methods can also be useful for studying real-time dynamics of polynucleotide repositioning and the association and dissociation of cellular compartments from specific regions in living cells. In the CRISPR-GO system, genomic loci are targeted to the desired compartments via chemically induced physical interactions between dCas9-bound genomic loci and compartment-specific proteins. Alternatively, a compartment is targeted or assembled at a target polynucleotide via chemically induced physical interactions between dCas9-bound genomic loci and compartment-specific proteins or compartment-constituent proteins. The inducible and reversible feature of CRISPR-GO prevents potential adverse effects from continuously repositioning chromatin DNA to a given nuclear compartment or continuously repositioning a compartment to a target polynucleotide.
As one example, through the combined use of CRISPR-Cas9 live-cell genomic imaging and CRISPR-GO, relocalization of endogenous genomic loci to the nuclear periphery has been shown to occur in both a mitosis-dependent and -independent manner. During mitosis, the nuclear membrane breaks down in prometaphase and then reforms in telophase. The dramatic changes in chromatin and nuclear structure during mitosis could facilitate interactions between genomic loci and the nuclear membrane to create nuclear envelope tethering. During interphase, though chromatin structure remains relatively stable, a genomic locus can still form interactions with the nuclear periphery when it is in close proximity. Nuclear periphery tethering during interphase may rely on proximity between the targeted loci and nuclear periphery, and a genomic locus that is located distal to the nuclear periphery may less likely be tethered through the mitosis-independent manner.
The chemical induction process of some provided embodiments also allows for the investigation of the real-time association between a target polynucleotide locus and cellular compartments in living cells. For example, compared to the relatively slower repositioning to the nuclear periphery (within hours), colocalization between a genomic locus and Cajal bodies occurs at a much faster rate (within minutes), likely because Cajal body components are more diffuse throughout the nucleus. Using the disclosed systems and methods, it has been observed that colocalization between CBs and the target genomic loci could occur in two ways: one is rapid formation of de novo Cajal bodies at the genomic loci, and the other is re-localization of existing CBs with the target genomic loci, a phenomenon which has not been reported before. Previous work has suggested that Cajal bodies are formed by phase separation. The recruiting of nuclear body components (e.g., Coilin for CBs) by CRISPR-GO to targeted genomic loci may generate synthetic phase separation at the target chromatin loci.
The provided compositions, methods, and systems have also been used to observe repression of an adjacent fluorescent reporter gene when repositioning a genomic locus to the nuclear periphery. Previous work reported different effects on gene expression after tethering LacO loci to the nuclear periphery. In particular, earlier studies have observed no change in transcription after LacO repeats were recruited to the nuclear periphery by LacI-Lamin B, and have shown that tethering LacO repeats to nuclear periphery by LacI-Emerin caused repression of adjacent genes. The systems disclosed herein have shown that repositioning the reporter gene to Emerin causes gene repression (˜59%).
The systems, compositions, and methods disclosed herein have also been used to repress both adjacent reporter and endogenous genes after CRISPR-GO-mediated colocalization of a chromatin locus to CBs. Importantly, targeted colocalization of Cajal bodies with endogenous loci represses adjacent gene expression across long distances (>30 kb). This observed gene repression after targeting a genomic locus to CBs has not yet been reported. In contrast, the CRISPRi/a methods function by recruiting transcriptional effectors that mostly affect expression of local genes within a few kilobases around the target site. Thus, the provided methods and systems provide an important new method for regulating polynucleotide expression over a long distance. The methods and systems also provide the ability to control repositioning of target polynucleotides to diverse cellular compartments in a systematic way to investigate cellular effects and program polynucleotide regulation.
The systems, compositions, and methods disclosed herein have also been used to regulate both proximal genes and distal genes after CRISPR-GO-mediated formation of a heterochromatin to a target polynucleotide, such as region on a chromosome comprising tandem repeats. Importantly, targeted formation of heterochromatin with endogenous loci can regulate gene expression across a region comprising a target polynucleotide. This can include regulation of expression of genes that are proximal to the target polynucleotide or distal to the target polynucleotide. Thus, the provided methods and systems provide an important new method for regulating polynucleotide expression over both short and long distances. The methods and systems also provide the ability to control positioning of the formation of the compartment, e.g., heterochromatin compartment, by targeting a specific polynucleotide.
In some embodiments, the systems, compositions, and methods disclosed herein are used with endogenous or synthetic oligomerizing proteins that self-aggregate to form an artificial protein/RNA/DNA aggregate, which can possess one or more unique chemical, physical, or biological properties (such as selective diffusion of specific proteins, RNA, or DNA; association or disassociation with other molecules; promotion or inhibition of gene regulation machineries; or promotion or inhibition of DNA recombination or stability machineries). Such an aggregate is a compartment that can be formed around target polyribonucleotide and is referred to herein as a synthetic cellular phases (SCP). These aggregates can have strong effects on gene regulation or polynucleotide repair mechanisms. The formation of these aggregrates can additionally be used for introducing epigenetic modifications to, producing three-dimensional structures, a topologically associating domains, or genomic boundaries comprising the target polynucleotide or an additional polynucleotide (e.g., distal or proximal gene from the target polynucleotide). In some embodiments, a protein, protein domain, RNA, RNA domain, or combination thereof is coupled to a provided system to specifically form a desired SCP around desired chromatin DNA or RNA. For example, proteins that facilitate HDR are used to form a SCP around a target polynucleotide that has been or will be cut by a gene editing technique, such as by CRISPR/Cas, TALENs, ZFNs, or meganucleases, or other polynucleotide cutting mechanism. Exemplary proteins that facilitate HDR can comprise Rad51, Rad52, RPA, MRN complex, MRX complex, CtlP, Sae2, BLM, Sgs1, BRCA2, exonucleases such as Exo1 or OsExo1, ATM, BRCA2, RAD54, or MDC1. Additionally, the SCP that facilitates HDR can comprise a template polynucleotide. For example, proteins that facilitate heterochromatin are used to form a SCP around a target polynucleotide to regulate gene expression of polynucleotides within a genomic region comprising the target polynucleotide. Exemplary proteins that facilitate this heterochromatin SCP are HP1α, HP1β, KAP1, KRAB, SUV39H1, or G9a. In some embodiments, the provided systems, compositions, and methods are useful for manipulating the spatiotemporal organization of genomic DNA and RNA components in the nucleus and/or cytoplasm and for regulating diverse cellular functions.
The systems, compositions, and methods as disclosed herein can be used to control a local environment of a target polynucleotide. This can be accomplished by spatial positioning of proteins to the target polynucleotide. In one aspect, a system is provided for spatial positioning of compartment-constituent protein to a target polynucleotide. The compartment-constituent protein can comprise further oligomerization domains or other domains that recruit additional compartment constituent proteins to the target polynucleotide to form the compartment, such as an SCP. In some embodiments, a method of forming a compartment around a target polynucleotide, the method comprising: (a) providing a compartment-constituent protein linked to a first dimerization domain; (b) providing an actuator moiety linked to a second dimerization domain, wherein the actuator moiety and the target polynucleotide form a complex; and (c) assembling a dimer comprising the first dimerization domain and the second dimerization domain of the complex, thereby forming the compartment around the target polynucleotide. For example, the compartment-constituent protein is a heterochromatin protein. The system can comprise a heterochromatin protein linked to a first dimerization domain. The system further can comprise an actuator moiety that targets the target polynucleotide, wherein the actuator moiety is linked to a second dimerization domain that is capable of assembling into a dimer with the dimerization domain. The assembly of the dimer can result in the positioning of the heterochromatin protein to the target polynucleotide. In some embodiments, the compartment is formed by administering a composition as described herein. A composition can comprise a) a compartment-constituent protein linked to a first dimerization domain; and b) an actuator moiety linked to a second dimerization domain; and wherein the first dimerization domain binds to the second dimerization domain. In some embodiments, the compartment is formed by a composition comprising a) a compartment-constituent protein linked to a first dimerization domain; and b) an actuator moiety linked to a scaffold, wherein the scaffold is linked to a second dimerization domain; and wherein the first dimerization domain binds to the second dimerization domain. In some embodiments, the composition further comprises a ligand, wherein the first dimerization domain and the second dimerization domain bind to the ligand, thereby linking the first dimerization domain to the second dimerization domain. In some embodiments, the ligand is inducible. In some embodiments, the scaffold is linked to at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 second dimerization domains. In some embodiments, the scaffold is linked to more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 second dimerization domains. In some embodiments, the scaffold is linked to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 second dimerization domains. In some embodiments, the compartment-constituent protein is further linked to a second scaffold, wherein the second scaffold is linked to at least one first dimerization domain. In some embodiments, the second scaffold is linked to at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 first dimerization domains. In some embodiments, the second scaffold is linked to more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 first dimerization domains. In some embodiments, the second scaffold is linked to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 second first domains.
In some embodiments, the systems, compositions, and methods comprise an inducible dimerization, wherein the dimerization can be mediated chemically or optogenetically. The dimer can be a homodimer. The dimer can be a heterodimer In some embodiments, the dimerization is mediated by a molecular ligand, such as a chemical inducer. In certain aspects, the dimerization system is selected from an ABA induced ABI/PYL1 dimerization system, a GA induced GA/GAI dimerization system a Rapamycin induced FRB/FKBP dimerization system a TMP-Htag induced HaloTag/DHFR dimerization system, or a dimerization system using an enzyme-catalyzed reaction. Other dimerization systems are also contemplated.
In some embodiments, the targeted polynucleotide of the provided systems and methods comprises DNA, e.g., genomic DNA. In some embodiments, the target polynucleotide comprises RNA, e.g., mRNA, microRNA, siRNA, or non-coding RNA. Actuator moieties and related targeting systems suitable for use with the provided systems and methods include, for example, CRISPR-Cas (including all types of CRISPR, type I, II, III, IV, V, VI, e.g., Cas9, Cas12, Cas13); Argonaute-mediated targeting or zinc finger targeting; TALE (transcription activator-like effectors); LacO-LacI or TetO-TetR; and specific pairs of DNA interacting protein or RNA domains. Cas9 and Cas13 can also target RNA in a sequence-dependent way, and can be used in this way with the provided system to re-localize RNA molecules to different cellular compartments. Cas proteins can lack DNA cleavage activity. The targeting systems can include sequence-specific guide RNAs or guide DNAs.
The actuator moiety can comprise a nuclease (e.g., DNA nuclease and/or RNA nuclease), modified nuclease (e.g., DNA nuclease and/or RNA nuclease) that is nuclease-deficient or has reduced nuclease activity compared to a wild-type nuclease, a derivative thereof, a variant thereof, or a fragment thereof. The actuator moiety can regulate expression or activity of a gene and/or edit the sequence of a nucleic acid (e.g., a gene and/or gene product). In some embodiments, the actuator moiety comprises a DNA nuclease such as an engineered (e.g., programmable or targetable) DNA nuclease to induce genome editing of a target DNA sequence. In some embodiments, the actuator moiety comprises a RNA nuclease such as an engineered (e.g., programmable or targetable) RNA nuclease to induce editing of a target RNA sequence. In some embodiments, the actuator moiety has reduced or minimal nuclease activity. An actuator moiety having reduced or minimal nuclease activity can regulate expression and/or activity of a gene by physical obstruction of a target polynucleotide or recruitment of additional factors effective to suppress or enhance expression of the target polynucleotide. In some embodiments, the actuator moiety comprises a nuclease-null DNA binding protein derived from a DNA nuclease that can induce transcriptional activation or repression of a target DNA sequence. In some embodiments, the actuator moiety comprises a nuclease-null RNA binding protein derived from a RNA nuclease that can induce transcriptional activation or repression of a target RNA sequence. In some embodiments, the actuator moiety is a nucleic acid-guided actuator moiety. In some embodiments, the actuator moiety is a DNA-guided actuator moiety. In some embodiments, the actuator moiety is an RNA-guided actuator moiety. An actuator moiety can regulate expression or activity of a gene and/or edit a nucleic acid sequence, whether exogenous or endogenous.
Any suitable nuclease can be used in an actuator moiety. Suitable nucleases include, but are not limited to, CRISPR-associated (Cas) proteins or Cas nucleases including type I CRISPR-associated (Cas) polypeptides, type II CRISPR-associated (Cas) polypeptides, type III CRISPR-associated (Cas) polypeptides, type IV CRISPR-associated (Cas) polypeptides, type V CRISPR-associated (Cas) polypeptides, and type VI CRISPR-associated (Cas) polypeptides; zinc finger nucleases (ZFN); transcription activator-like effector nucleases (TALEN); meganucleases; RNA-binding proteins (RBP); CRISPR-associated RNA binding proteins; recombinases; flippases; transposases; Argonaute (Ago) proteins (e.g., prokaryotic Argonaute (pAgo), archaeal Argonaute (aAgo), and eukaryotic Argonaute (eAgo)); any derivative thereof; any variant thereof and any fragment thereof.
In some embodiments, the actuator moiety comprises a CRISPR-associated (Cas) protein or a Cas nuclease which functions in a non-naturally occurring CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/Cas (CRISPR-associated) system. In bacteria, this system can provide adaptive immunity against foreign DNA (Barrangou, R., et al, “CRISPR provides acquired resistance against viruses in prokaryotes,” Science (2007) 315: 1709-1712; Makarova, K. S., et al, “Evolution and classification of the CRISPR-Cas systems,” Nat Rev Microbiol (2011) 9:467-477; Garneau, J. E., et al, “The CRISPR/Cas bacterial immune system cleaves bacteriophage and plasmid DNA,” Nature (2010) 468:67-71; Sapranauskas, R., et al, “The Streptococcus thermophilus CRISPR/Cas system provides immunity in Escherichia coli,” Nucleic Acids Res (2011) 39: 9275-9282).
In a wide variety of organisms including diverse mammals, animals, plants, microbes, and yeast, a CRISPR/Cas system (e.g., modified and/or unmodified) can be utilized as a genome engineering tool. A CRISPR/Cas system can comprise a guide nucleic acid such as a guide RNA (gRNA) complexed with a Cas protein for targeted regulation of gene expression and/or activity or nucleic acid editing. An RNA-guided Cas protein (e.g., a Cas nuclease such as a Cas9 nuclease) can specifically bind a target polynucleotide (e.g., DNA) in a sequence-dependent manner. The Cas protein, if possessing nuclease activity, can cleave the DNA (Gasiunas, G., et al, “Cas9-crRNA ribonucleoprotein complex mediates specific DNA cleavage for adaptive immunity in bacteria,” Proc Natl Acad Sci USA (2012) 109: E2579-E286; Jinek, M., et al, “A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity,” Science (2012) 337:816-821; Sternberg, S. H., et al, “DNA interrogation by the CRISPR RNA-guided endonuclease Cas9,” Nature (2014) 507:62; Deltcheva, E., et al, “CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III,” Nature (2011) 471:602-607), and has been widely used for programmable genome editing in a variety of organisms and model systems (Cong, L., et al, “Multiplex genome engineering using CRISPR Cas systems,” Science (2013) 339:819-823; Jiang, W., et al, “RNA-guided editing of bacterial genomes using CRISPR-Cas systems,” Nat. Biotechnol. (2013) 31: 233-239; Sander, J. D. & Joung, J. K, “CRISPR-Cas systems for editing, regulating and targeting genomes,” Nature Biotechnol. (2014) 32:347-355).
In some cases, the Cas protein is mutated and/or modified to yield a nuclease deficient protein or a protein with decreased nuclease activity relative to a wild-type Cas protein. A nuclease deficient protein can retain the ability to bind DNA, but may lack or have reduced nucleic acid cleavage activity. An actuator moiety comprising a Cas nuclease (e.g., retaining wild-type nuclease activity, having reduced nuclease activity, and/or lacking nuclease activity) can function in a CRISPR/Cas system to regulate the level and/or activity of a target gene or protein (e.g., decrease, increase, or elimination). The Cas protein can bind to a target polynucleotide and prevent transcription by physical obstruction or edit a nucleic acid sequence to yield non-functional gene products. A Cas protein can edit a nucleic acid sequence by generating a double-stranded break or single-stranded break in a target polynucleotide. A double-strand break in DNA can result in DNA break repair which allows for the introduction of gene modification(s) (e.g., nucleic acid editing). DNA break repair can occur via non-homologous end joining (NHEJ) or homology-directed repair (HDR). In HDR, a donor DNA repair template or template polynucleotide that contains homology arms flanking sites of the target DNA can be provided.
In some embodiments, the actuator moiety comprises a Cas protein that forms a complex with a guide nucleic acid, such as a guide RNA. In some embodiments, the actuator moiety comprises a Cas protein that forms a complex with a single guide nucleic acid, such as a single guide RNA (sgRNA). In some embodiments, the actuator moiety comprises a RNA-binding protein (RBP) optionally complexed with a guide nucleic acid, such as a guide RNA (e.g., sgRNA), which is able to form a complex with a Cas protein. In some embodiments, the actuator moiety comprises a nuclease-null DNA binding protein derived from a DNA nuclease that can induce transcriptional activation or repression of a target DNA sequence. In some embodiments, the actuator moiety comprises a nuclease-null RNA binding protein derived from a RNA.
Any suitable CRISPR/Cas system can be used. A CRISPR/Cas system can be referred to using a variety of naming systems. Exemplary naming systems are provided in Makarova, K. S. et al, “An updated evolutionary classification of CRISPR-Cas systems,” Nat Rev Microbiol (2015) 13:722-736 and Shmakov, S. et al, “Discovery and Functional Characterization of Diverse Class 2 CRISPR-Cas Systems,” Mol Cell (2015) 60:1-13. A CRISPR/Cas system can be a type I, a type II, a type III, a type IV, a type V, a type VI system, or any other suitable CRISPR/Cas system. A CRISPR/Cas system as used herein can be a Class 1, Class 2, or any other suitably classified CRISPR/Cas system. Class 1 or Class 2 determination can be based upon the genes encoding the effector module. Class 1 systems generally have a multi-subunit crRNA-effector complex, whereas Class 2 systems generally have a single protein, such as Cas9, Cpfl, C2c1, C2c2, C2c3 or a crRNA-effector complex. A Class 1 CRISPR/Cas system can use a complex of multiple Cas proteins to effect regulation. A Class 1 CRISPR/Cas system can comprise, for example, type I (e.g., I, IA, IB, IC, ID, IE, IF, IU), type III (e.g., III, IIIA, IIIB, IIIC, HID), and type IV (e.g., IV, IVA, IVB) CRISPR/Cas type. A Class 2 CRISPR/Cas system can use a single large Cas protein to effect regulation. A Class 2 CRISPR/Cas systems can comprise, for example, type II (e.g., II, IIA, IIB) and type V CRISPR/Cas type. CRISPR systems can be complementary to each other, and/or can lend functional units in trans to facilitate CRISPR locus targeting.
An actuator moiety comprising a Cas protein can be a Class 1 or a Class 2 Cas protein. A Cas protein can be a type I, type II, type III, type IV, type V Cas protein, or type VI Cas protein. A Cas protein can comprise one or more domains. Non-limiting examples of domains include, guide nucleic acid recognition and/or binding domain, nuclease domains (e.g., DNase or RNase domains, RuvC, HNH), DNA binding domain, RNA binding domain, helicase domains, protein-protein interaction domains, and dimerization domains. A guide nucleic acid recognition and/or binding domain can interact with a guide nucleic acid. A nuclease domain can comprise catalytic activity for nucleic acid cleavage. A nuclease domain can lack catalytic activity to prevent nucleic acid cleavage. A Cas protein can be a chimeric Cas protein that is fused to other proteins or polypeptides. A Cas protein can be a chimera of various Cas proteins, for example, comprising domains from different Cas proteins.
Non-limiting examples of Cas proteins include c2c1, C2c2, c2c3, Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas5e (CasD), Cash, Cas6e, Cas6f, Cas7, Cas8a, Cas8a1, Cas8a2, Cas8b, Cas8c, Cas9 (Csn1 or Csx12), Cas10, CaslOd, Cas10, CaslOd, CasF, CasG, CasH, Cpfl, Csy1, Csy2, Csy3, Cse1 (CasA), Cse2 (CasB), Cse3 (CasE), Cse4 (CasC), Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4, and Cul966, and homologs or modified versions thereof.
A Cas protein can be from any suitable organism. Non-limiting examples include Streptococcus pyogenes, Streptococcus thermophilus, Streptococcus sp., Staphylococcus aureus, Nocardiopsis dassonvillei, Streptomyces pristinae spiralis, Streptomyces viridochromo genes, Streptomyces viridochromo genes, Streptosporangiurn roseum, Streptosporangiurn roseum, AlicyclobacHlus acidocaldarius, Bacillus pseudomycoides, Bacillus selenitireducens, Exiguobacterium sibiricum, Lactobacillus delbrueckii, Lactobacillus salivarius, Microscilla marina, Burkholderiales bacterium, Polaromonas naphthalenivorans, Polaromonas sp., Crocosphaera watsonii, Cyanothece sp., Microcystis aeruginosa, Pseudomonas aeruginosa, Synechococcus sp., Acetohalobiurn arabaticum, Ammomfex degensii, Caldicelulosiruptor becscii, Candidatus Desulforudis, Clostridium botulinum, Clostridium difficile, Finegoldia magna, Natranaerobius thermophilus, Pelotomaculum thermopropionicum, Acidithiobacillus caldus, Acidithiobacillus ferrooxidans, Allochromatiurn vinosum, Marinobacter sp., Nitrosococcus halophilus, Nitrosococcus watsoni, Pseudoalteromonas haloplanktis, Ktedonobacter racemifer, Methanohalobiurn evestigatum, Anabaena variabilis, Nodularia spumigena, Nostoc sp., Arthrospira maxima, Arthrospira platensis, Arthrospira sp., Lyngbya sp., Microcoleus chthonoplastes, Oscillatoria sp., Petrotoga mobilis, Thermosipho africanus, Acaryochloris marina, Leptotrichia shahii, and Francisella novicida. In some aspects, the organism is Streptococcus pyogenes (S. pyogenes). In some aspects, the organism is Staphylococcus aureus (S. aureus). In some aspects, the organism is Streptococcus thermophilus (S. thermophilus).
A Cas protein can be derived from a variety of bacterial species including, but not limited to, Veillonella atypical, Fusobacterium nucleatum, Filifactor alocis, Solobacterium moorei, Coprococcus catus, Treponema denticola, Peptoniphilus duerdenii, Catenibacteriurn mitsuokai, Streptococcus mutans, Listeria innocua, Staphylococcus pseudintermedius, Acidaminococcus intestine, Olsenella uli, Oenococcus kitaharae, Bifidobacterium bifidum, Lactobacillus rhamnosus, Lactobacillus gasseri, Finegoldia magna, Mycoplasma mobile, Mycoplasma gallisepticum, Mycoplasma ovipneumoniae, Mycoplasma canis, Mycoplasma synoviae, Eubacterium rectale, Streptococcus thermophilus, Eubacterium dolichum, Lactobacillus coryniformis subsp. torquens, Ilyobacter polytropus, Ruminococcus albus, Akkermansia mucimphila, Acidothermus cellulolyticus, Bifidobacterium longum, Bifidobacterium dentium, Corynebacterium diphtheria, Elusimicrobiurn minutum, Nitratifractorsalsuginis, Sphaerochaeta globus, Fibrobacter succinogenes subsp. Succinogenes, Bacteroides fragilis, Capnocytophaga ochracea, Rhodopseudomonas palustris, Prevotella micans, Prevotella ruminicola, Flavobacterium columnare, Aminomonas paucivorans, Rhodospirillum rubrum, Candidatus Puniceispirillum marinum, Verminephrobacter eiseniae, Ralstonia syzygii, Dinoroseobacter shibae, Azospirillum, Nitrobacter hamburgensis, Bradyrhizobium, Wolinellasuccinogenes, Campylobacter jejuni subsp. jejuni, Helicobacter mustelae, Bacillus cereus, Acidovorax ebreus, Clostridium perfringens, Parvibaculum lavamentivorans, Roseburia intestinalis, Neisseria meningitidis, Pasteurella multocida subsp. Multocida, Sutterella wadsworthensis, proteobacterium, Legionella pneumophila, Parasutterella excrementihominis, Wolinella succinogenes, and Francisella novicida.
A Cas protein as used herein can be a wildtype or a modified form of a Cas protein. A Cas protein can be an active variant, inactive variant, or fragment of a wild type or modified Cas protein. A Cas protein can comprise an amino acid change such as a deletion, insertion, substitution, variant, mutation, fusion, chimera, or any combination thereof relative to a wild-type version of the Cas protein. A Cas protein can be a polypeptide with at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity or sequence similarity to a wild type exemplary Cas protein. A Cas protein can be a polypeptide with at most about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% sequence identity and/or sequence similarity to a wild type exemplary Cas protein. Variants or fragments can comprise at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity or sequence similarity to a wild type or modified Cas protein or a portion thereof. Variants or fragments can be targeted to a nucleic acid locus in complex with a guide nucleic acid while lacking nucleic acid cleavage activity.
A Cas protein can comprise one or more nuclease domains, such as DNase domains. For example, a Cas9 protein can comprise a RuvC-like nuclease domain and/or an HNH-like 20 nuclease domain. The RuvC and HNH domains can each cut a different strand of double-stranded DNA to make a double-stranded break in the DNA. A Cas protein can comprise only one nuclease domain (e.g., Cpfl comprises RuvC domain but lacks HNH domain).
A Cas protein can comprise an amino acid sequence having at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity or sequence similarity to a nuclease domain (e.g., RuvC domain, HNH domain) of a wild-type Cas protein.
A Cas protein can be modified to optimize regulation of gene expression. A Cas protein can be modified to increase or decrease nucleic acid binding affinity, nucleic acid binding specificity, and/or enzymatic activity. Cas proteins can also be modified to change any other activity or property of the protein, such as stability. For example, one or more nuclease domains of the Cas protein can be modified, deleted, or inactivated, or a Cas protein can be truncated to remove domains that are not essential for the function of the protein or to optimize (e.g., enhance or reduce) the activity of the Cas protein for regulating gene expression.
A Cas protein can be a fusion protein. For example, a Cas protein can be fused to a cleavage domain, an epigenetic modification domain, a transcriptional activation domain, or a transcriptional repressor domain. A Cas protein can also be fused to a heterologous polypeptide providing increased or decreased stability. The fused domain or heterologous polypeptide can be located at the N-terminus, the C-terminus, or internally within the Cas protein.
A Cas protein can be provided in any form. For example, a Cas protein can be provided in the form of a protein, such as a Cas protein alone or complexed with a guide nucleic acid. A Cas protein can be provided in the form of a nucleic acid encoding the Cas protein, such as an RNA (e.g., messenger RNA (mRNA)) or DNA. The nucleic acid encoding the Cas protein can be codon optimized for efficient translation into protein in a particular cell or organism.
Nucleic acids encoding Cas proteins can be stably integrated in the genome of the cell. Nucleic acids encoding Cas proteins can be operably linked to a promoter active in the cell. Nucleic acids encoding Cas proteins can be operably linked to a promoter in an expression construct. Expression constructs can include any nucleic acid constructs capable of directing expression of a gene or other nucleic acid sequence of interest (e.g., a Cas gene) and which can transfer such a nucleic acid sequence of interest to a target cell.
In some embodiments, a Cas protein is a dead Cas protein. A dead Cas protein can be a protein that lacks nucleic acid cleavage activity.
A Cas protein can comprise a modified form of a wild type Cas protein. The modified form of the wild type Cas protein can comprise an amino acid change (e.g., deletion, insertion, or substitution) that reduces the nucleic acid-cleaving activity of the Cas protein. For example, the modified form of the Cas protein can have no more than 90%, no more than 80%, no more than 70%, no more than 60%, no more than 50%, no more than 40%, no more than 30%, no more than 20%, no more than 10%, no more than 5%, or no more than 1% of the nucleic acid-cleaving activity of the wild-type Cas protein (e.g., Cas9 from S. pyogenes). The modified form of Cas protein can have no substantial nucleic acid-cleaving activity. When a Cas protein is a modified form that has no substantial nucleic acid-cleaving activity, it can be referred to as enzymatically inactive and/or “dead” (abbreviated by “d”). A dead Cas protein (e.g., dCas, dCas9) can bind to a target polynucleotide but may not cleave the target polynucleotide. In some aspects, a dead Cas protein is a dead Cas9 protein.
A dCas9 polypeptide can associate with a single guide RNA (sgRNA) to activate or repress transcription of target DNA. sgRNAs can be introduced into cells expressing the engineered chimeric receptor polypeptide. In some cases, such cells contain one or more different sgRNAs that target the same nucleic acid. In other cases, the sgRNAs target different nucleic acids in the cell. The nucleic acids targeted by the guide RNA can be any that are expressed in a cell such as an immune cell. The nucleic acids targeted may be a gene involved in immune cell regulation. In some embodiments, the nucleic acid is associated with cancer. The nucleic acid associated with cancer can be a cell cycle gene, cell response gene, apoptosis gene, or phagocytosis gene. The recombinant guide RNA can be recognized by a CRISPR protein, a nuclease-null CRISPR protein, variants thereof, or derivatives thereof.
Enzymatically inactive can refer to a polypeptide that can bind to a nucleic acid sequence in a polynucleotide in a sequence-specific manner, but may not cleave a target polynucleotide. An enzymatically inactive site-directed polypeptide can comprise an enzymatically inactive domain (e.g. nuclease domain). Enzymatically inactive can refer to no activity. Enzymatically inactive can refer to substantially no activity. Enzymatically inactive can refer to essentially no activity. Enzymatically inactive can refer to an activity no more than 1%, no more than 2%, no more than 3%, no more than 4%, no more than 5%, no more than 6%, no more than 7%, no more than 8%, no more than 9%, or no more than 10% activity compared to a wild-type exemplary activity (e.g., nucleic acid cleaving activity, wild-type Cas9 activity).
One or a plurality of the nuclease domains (e.g., RuvC, HNH) of a Cas protein can be deleted or mutated so that they are no longer functional or comprise reduced nuclease activity. For example, in a Cas protein comprising at least two nuclease domains (e.g., Cas9), if one of the nuclease domains is deleted or mutated, the resulting Cas protein, known as a nickase, can generate a single-strand break at a CRISPR RNA (crRNA) recognition sequence within a double-stranded DNA but not a double-strand break. Such a nickase can cleave the complementary strand or the non-complementary strand, but may not cleave both. If all of the nuclease domains of a Cas protein (e.g., both RuvC and HNH nuclease domains in a Cas9 protein; RuvC nuclease domain in a Cpfl protein) are deleted or mutated, the resulting Cas protein can have a reduced or no ability to cleave both strands of a double-stranded DNA. An example of a mutation that can convert a Cas9 protein into a nickase is a D 10A (aspartate to alanine at position 10 of Cas9) mutation in the RuvC domain of Cas9 from S. pyogenes. H939A (histidine to alanine at amino acid position 839) or H840A (histidine to alanine at amino acid position 840) in the HNH domain of Cas9 from S. pyogenes can convert the Cas9 into a nickase. An example of a mutation that can convert a Cas9 protein into a dead Cas9 is a D10A (aspartate to alanine at position 10 of Cas9) mutation in the RuvC domain and H939A (histidine to alanine at amino acid position 839) or H840A (histidine to alanine at amino acid position 840) in the HNH domain of Cas9 from S. pyogenes.
A dead Cas protein can comprise one or more mutations relative to a wild-type version of the protein. The mutation can result in no more than 90%, no more than 80%, le no more ss than 70%, no more than 60%, no more than 50%, no more than 40%, no more than 30%, no more than 20%, no more than 10%, no more than 5%, or no more than 1% of the nucleic acid-cleaving activity in one or more of the plurality of nucleic acid-cleaving domains of the wild-type Cas protein. The mutation can result in one or more of the plurality of nucleic acid-cleaving domains retaining the ability to cleave the complementary strand of the target nucleic acid but reducing its ability to cleave the non-complementary strand of the target nucleic acid. The mutation can result in one or more of the plurality of nucleic acid-cleaving domains retaining the ability to cleave the non-complementary strand of the target nucleic acid but reducing its ability to cleave the complementary strand of the target nucleic acid. The mutation can result in one or more of the plurality of nucleic acid-cleaving domains lacking the ability to cleave the complementary strand and the non-complementary strand of the target nucleic acid. The residues to be mutated in a nuclease domain can correspond to one or more catalytic residues of the nuclease. For example, residues in the wild type exemplary S. pyogenes Cas9 polypeptide such as Asp10, His840, Asn854 and Asn856 can be mutated to inactivate one or more of the plurality of nucleic acid-cleaving domains (e.g., nuclease domains). The residues to be mutated in a nuclease domain of a Cas protein can correspond to residues Asp10, His840, Asn854 and Asn856 in the wild type S. pyogenes Cas9 polypeptide, for example, as determined by sequence and/or structural alignment.
As non-limiting examples, residues D10, G12, G17, E762, H840, N854, N863, H982, H983, A984, D986, and/or A987 (or the corresponding mutations of any of the Cas proteins) can be mutated. For example, e.g., D 10A, G12A, G17A, E762A, H840A, N854A, N863A, H982A, H983A, A984A, and/or D986A. Mutations other than alanine substitutions can be suitable.
A D1OA mutation can be combined with one or more of H840A, N854A, or N856A mutations to produce a Cas9 protein substantially lacking DNA cleavage activity (e.g., a dead Cas9 protein). A H840A mutation can be combined with one or more of D1OA, N854A, or N856A mutations to produce a site-directed polypeptide substantially lacking DNA cleavage activity. A N854A mutation can be combined with one or more of H840A, D1OA, or N856A mutations to produce a site-directed polypeptide substantially lacking DNA cleavage activity. A N856A mutation can be combined with one or more of H840A, N854A, or D10A mutations to produce a site-directed polypeptide substantially lacking DNA cleavage activity.
In some embodiments, a Cas protein is a Class 2 Cas protein. In some embodiments, a Cas protein is a type II Cas protein. In some embodiments, the Cas protein is a Cas9 protein, a modified version of a Cas9 protein, or derived from a Cas9 protein. For example, a Cas9 protein lacking cleavage activity. In some embodiments, the Cas9 protein is a Cas9 protein from S. pyogenes (e.g., SwissProt accession number Q99ZW2). In some embodiments, the Cas9 protein is a Cas9 from S. aureus (e.g., SwissProt accession number J7RUA5). In some embodiments, the Cas9 protein is a modified version of a Cas9 protein from S. pyogenes or S. aureus. In some embodiments, the Cas9 protein is derived from a Cas9 protein from S. pyogenes or S. aureus. For example, a S. pyogenes or S. aureus Cas9 protein lacking cleavage activity.
Cas9 can generally refer to a polypeptide with at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% sequence identity and/or sequence similarity to a wild type exemplary Cas9 polypeptide (e.g., Cas9 from S. pyogenes). Cas9 can refer to a polypeptide with at most about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% sequence identity and/or sequence similarity to a wild type exemplary Cas9 polypeptide (e.g., from S. pyogenes). Cas9 can refer to the wildtype or a modified form of the Cas9 protein that can comprise an amino acid change such as a deletion, insertion, substitution, variant, mutation, fusion, chimera, or any combination thereof.
In some embodiments, an actuator moiety comprises a “zinc finger nuclease” or “ZFN.” ZFNs refer to a fusion between a cleavage domain, such as a cleavage domain of Fokl, and at least one zinc finger motif (e.g., at least 2, 3, 4, or 5 zinc finger motifs) which can bind polynucleotides such as DNA and RNA. The heterodimerization at certain positions in a polynucleotide of two individual ZFNs in certain orientation and spacing can lead to cleavage of the polynucleotide. For example, a ZFN binding to DNA can induce a double-strand break in the DNA. In order to allow two cleavage domains to dimerize and cleave DNA, two individual ZFNs can bind opposite strands of DNA with their C-termini at a certain distance apart. In some cases, linker sequences between the zinc finger domain and the cleavage domain can require the 5′ edge of each binding site to be separated by about 5-7 base pairs. In some cases, a cleavage domain is fused to the C-terminus of each zinc finger domain. Exemplary ZFNs include, but are not limited to, those described in Urnov et al., Nature Reviews Genetics, 2010, 11:636-646; Gaj et al., Nat Methods, 2012, 9(8):805-7; U.S. Pat. Nos. 6,534,261; 6,607,882; 6,746,838; 6,794,136; 6,824,978; 6,866,997; 6,933,113; 6,979,539; 7,013,219; 7,030,215; 7,220,719; 7,241,573; 7,241,574; 7,585,849; 7,595,376; 6,903,185; 6,479,626; and U.S. Publication Nos. 2003/0232410 and 2009/0203140.
In some embodiments, an actuator moiety comprising a ZFN can generate a double-strand break in a target polynucleotide, such as DNA. A double-strand break in DNA can result in DNA break repair which allows for the introduction of gene modification(s) (e.g., nucleic acid editing). DNA break repair can occur via non-homologous end joining (NHEJ) or homology-directed repair (HDR). In HDR, a donor DNA repair template or template polynucleotide that contains homology arms flanking sites of the target DNA can be provided. In some embodiments, a ZFN is a zinc finger nickase which induces site-specific single-strand DNA breaks or nicks, thus resulting in HDR. Descriptions of zinc finger nickases are found, e.g., in Ramirez et al., Nucl Acids Res, 2012, 40(12):5560-8; Kim et al., Genome Res, 2012, 22(7):1327-33. In some embodiments, a ZFN binds a polynucleotide (e.g., DNA and/or RNA) but is unable to cleave the polynucleotide.
In some embodiments, the cleavage domain of an actuator moiety comprising a ZFN comprises a modified form of a wild type cleavage domain. The modified form of the cleavage domain can comprise an amino acid change (e.g., deletion, insertion, or substitution) that reduces the nucleic acid-cleaving activity of the cleavage domain. For example, the modified form of the cleavage domain can have no more than 90%, no more than than 80%, no more than 70%, no more than 60%, no more than 50%, no more than 40%, no more than 30%, no more than 20%, no more than 10%, no more than 5%, or no more than 1% of the nucleic acid-cleaving activity of the wild-type cleavage domain. The modified form of the cleavage domain can have no substantial nucleic acid-cleaving activity. In some embodiments, the cleavage domain is enzymatically inactive.
In some embodiments, an actuator moiety comprises a “TALEN” or “TAL-effector nuclease.” TALENs refer to engineered transcription activator-like effector nucleases that generally contain a central domain of DNA-binding tandem repeats and a cleavage domain. TALENs can be produced by fusing a TAL effector DNA binding domain to a DNA cleavage domain. In some cases, a DNA-binding tandem repeat comprises 33-35 amino acids in length and contains two hypervariable amino acid residues at positions 12 and 13 that can recognize at least one specific DNA base pair. A transcription activator-like effector (TALE) protein can be fused to a nuclease such as a wild-type or mutated Fokl endonuclease or the catalytic domain of Fokl. Several mutations to Fokl have been made for its use in TALENs, which, for example, improve cleavage specificity or activity. Such TALENs can be engineered to bind any desired DNA sequence. TALENs can be used to generate gene modifications (e.g., nucleic acid sequence editing) by creating a double-strand break in a target DNA sequence, which in turn, undergoes NHEJ or HDR. A double-strand break in DNA can result in DNA break repair which allows for the introduction of gene modification(s) (e.g., nucleic acid editing). DNA break repair can occur via non-homologous end joining (NHEJ) or homology-directed repair (HDR). In HDR, a donor DNA repair template or template polynucleotide that contains homology arms flanking sites of the target DNA can be provided. In some cases, a single-stranded donor DNA repair template is provided to promote HDR. Detailed descriptions of TALENs and their uses for gene editing are found, e.g., in U.S. Pat. Nos. 8,440,431; 8,440,432; 8,450,471; 8,586,363; and U.S. Pat. No. 8,697,853; Scharenberg et al., Curr Gene Ther, 2013, 13(4):291-303; Gaj et al., Nat Methods, 2012, 9(8):805-7; Beurdeley et al., Nat Commun, 2013, 4:1762; and Joung and Sander, Nat Rev Mol Cell Biol, 2013, 14(1):49-55.
In some embodiments, a TALEN is engineered for reduced nuclease activity. In some embodiments, the nuclease domain of a TALEN comprises a modified form of a wild type nuclease domain. The modified form of the nuclease domain can comprise an amino acid change (e.g., deletion, insertion, or substitution) that reduces the nucleic acid-cleaving activity of the nuclease domain. For example, the modified form of the nuclease domain can have no more than 90%, no more than 80%, no more than 70%, no more than 60%, no more than 50%, no more than 40%, no more than 30%, no more than 20%, no more than 10%, no more than 5%, or no more than 1% of the nucleic acid-cleaving activity of the wild-type nuclease domain. The modified form of the nuclease domain can have no substantial nucleic acid-cleaving activity. In some embodiments, the nuclease domain is enzymatically inactive.
In some embodiments, the transcription activator-like effector (TALE) protein is fused to a domain that can modulate transcription and does not comprise a nuclease. In some embodiments, the transcription activator-like effector (TALE) protein is designed to function as a transcriptional activator. In some embodiments, the transcription activator-like effector (TALE) protein is designed to function as a transcriptional repressor. For example, the DNA-binding domain of the transcription activator-like effector (TALE) protein can be fused (e.g., linked) to one or more transcriptional activation domains, or to one or more transcriptional repression domains. Non-limiting examples of a transcriptional activation domain include a herpes simplex VP16 activation domain and a tetrameric repeat of the VP16 activation domain, e.g., a VP64 activation domain. A non-limiting example of a transcriptional repression domain includes a Kruppel-associated box domain.
In some embodiments, an actuator moiety comprises a meganuclease. Meganucleases generally refer to rare-cutting endonucleases or homing endonucleases that can be highly specific. Meganucleases can recognize DNA target sites ranging from at least 12 base pairs in length, e.g., from 12 to 40 base pairs, 12 to 50 base pairs, or 12 to 60 base pairs in length. Meganucleases can be modular DNA-binding nucleases such as any fusion protein comprising at least one catalytic domain of an endonuclease and at least one DNA binding domain or protein specifying a nucleic acid target sequence. The DNA-binding domain can contain at least one motif that recognizes single- or double-stranded DNA. A meganuclease can generate a double-stranded break. A double-strand break in DNA can result in DNA break repair which allows for the introduction of gene modification(s) (e.g., nucleic acid editing). DNA break repair can occur via non-homologous end joining (NHEJ) or homology-directed repair (HDR). In HDR, a donor DNA repair template or template polynucleotide that contains homology arms flanking sites of the target DNA can be provided. The meganuclease can be monomeric or dimeric. In some embodiments, the meganuclease is naturally-occurring (found in nature) or wild-type, and in other instances, the meganuclease is non-natural, artificial, engineered, synthetic, rationally designed, or man-made. In some embodiments, the meganuclease of the present disclosure includes an I-CreI meganuclease, I-CeuI meganuclease, I-Msol meganuclease, I-SceI meganuclease, variants thereof, derivatives thereof, and fragments thereof. Detailed descriptions of useful meganucleases and their application in gene editing are found, e.g., in Silva et al., Curr Gene Ther, 2011, 11(1):11-27; Zaslavoskiy et al., BMC Bioinformatics, 2014, 15:191; Takeuchi et al., Proc Natl Acad Sci USA, 2014, 111(11):4061-4066, and U.S. Pat. Nos. 7,842,489; 7,897,372; 8,021,867; 8,163,514; 8,133,697; 8,021,867; 8,119,361; 8,119,381; 8,124,36; and 8,129,134.
In some embodiments, the nuclease domain of a meganuclease comprises a modified form of a wild type nuclease domain. The modified form of the nuclease domain can comprise an amino acid change (e.g., deletion, insertion, or substitution) that reduces the nucleic acid-cleaving activity of the nuclease domain. For example, the modified form of the nuclease domain can have no more than 90%, no more than 80%, no more than 70%, no more than 60%, no more than 50%, no more than 40%, no more than 30%, no more than 20%, no more than 10%, no more than 5%, or no more than 1% of the nucleic acid-cleaving activity of the wild-type nuclease domain. The modified form of the nuclease domain can have no substantial nucleic acid-cleaving activity. In some embodiments, the nuclease domain is enzymatically inactive. In some embodiments, a meganuclease can bind DNA but cannot cleave the DNA.
In some embodiments, the actuator moiety is fused to one or more transcription repressor domains, activator domains, epigenetic domains, recombinase domains, transposase domains, flippase domains, nickase domains, or any combination thereof. The activator domain can include one or more tandem activation domains located at the carboxyl terminus of the enzyme. In other cases, the actuator moiety includes one or more tandem repressor domains located at the carboxyl terminus of the protein. Non-limiting exemplary activation domains include GAL4, herpes simplex activation domain VP16, VP64 (a tetramer of the herpes simplex activation domain VP16), NF-KB p65 subunit, Epstein-Barr virus R transactivator (Rta) and are described in Chavez et al., Nat Methods, 2015, 12(4):326-328 and U.S. Patent App. Publ. No. 20140068797. Non-limiting exemplary repression domains include the KRAB (Kruppel-associated box) domain of Koxl, the Mad mSIN3 interaction domain (SID), ERF repressor domain (ERD), and are described in Chavez et al., Nat Methods, 2015, 12(4):326-328 and U.S. Patent App. Publ. No. 20140068797. An actuator moiety can also be fused to a heterologous polypeptide providing increased or decreased stability. The fused domain or heterologous polypeptide can be located at the N-terminus, the C-terminus, or internally within the actuator moiety.
An actuator moiety can comprise a heterologous polypeptide for ease of tracking or purification, such as a fluorescent protein, a purification tag, or an epitope tag. Examples of fluorescent proteins include green fluorescent proteins (e.g., GFP, GFP-2, tagGFP, turboGFP, eGFP, Emerald, Azami Green, Monomeric Azami Green, CopGFP, AceGFP, ZsGreenl), yellow fluorescent proteins (e.g., YFP, eYFP, Citrine, Venus, YPet, PhiYFP, ZsYellowl), blue fluorescent proteins (e.g. eBFP, eBFP2, Azurite, mKalamal, GFPuv, Sapphire, T-sapphire), cyan fluorescent proteins (e.g. eCFP, Cerulean, CyPet, AmCyanl, Midoriishi-Cyan), red fluorescent proteins (mKate, mKate2, mPlum, DsRed monomer, mCherry, mRFP1, DsRed-Express, DsRed2, DsRed-Monomer, HcRed-Tandem, HcRedl, AsRed2, eqFP611, mRaspberry, mStrawberry, Jred), orange fluorescent proteins (mOrange, mKO, Kusabira-Orange, Monomeric Kusabira-Orange, mTangerine, tdTomato), and any other suitable fluorescent protein. Examples of tags include glutathione-S-transferase (GST), chitin binding protein (CBP), maltose binding protein, thioredoxin (TRX), poly(NANP), tandem affinity purification (TAP) tag, myc, AcV5, AU1, AUS, E, ECS, E2, FLAG, hemagglutinin (HA), nus, Softag 1, Softag 3, Strep, SBP, Glu-Glu, HSV, KT3, S, SI, T7, V5, VSV-G, histidine (His), biotin carboxyl carrier protein (BCCP), and calmodulin.
In some embodiments, the actuator moiety and the second dimerization domain are linked via a linker. A linker can be any linker known in the art. In some embodiments, the actuator moiety and second dimerization domain are linked as fusion protein. In some embodiments, the compartment-constituent protein and the first dimerization domain are linked via a linker. A linker can be any linker known in the art. In some embodiments, the compartment-constituent protein and the first dimerization domain are linked as fusion protein.
In some embodiments, the target polynucleotide is positioned by the provided systems and methods in an inner nuclear membrane. Compartment specific proteins suitable for targeting the inner nuclear membrane include, but are not limited to, Emerin, Lap2beta, and Lamin B.
In some embodiments, the target polynucleotide is positioned by the provided systems and methods in a Cajal body. In some embodiments, the Cajal body is assembled or positioned by the provided systems and methods at the target polynucleotide. Suitable compartment specific proteins for Cajal bodies for the provided systems and methods include, but are not limited to, Coilin, SMN, Gemin 3, SmD1, and SmE.
In some embodiments, the target polynucleotide is positioned by the provided systems and methods in nuclear speckles. In some embodiments, the nuclear speckle is assembled or positioned by the provided systems and methods at the target polynucleotide. Suitable compartment specific proteins for nuclear speckles for the provided systems and methods include, but are not limited to, SC35.
In some embodiments, the target polynucleotide is positioned by the provided systems and methods in a PML body. In some embodiments, the PML body is assembled or positioned by the provided systems and methods at the target polynucleotide. Suitable compartment specific proteins for PML bodies for the provided systems and methods include, but are not limited to, PML and SP100.
In some embodiments, the target polynucleotide is positioned by the provided systems and methods in a nuclear pore complex. Compartment specific proteins suitable for targeting nuclear pore complexes include, but are not limited to, Nup50, Nup98, Nup53, Nup153, and Nup62.
In some embodiments, the target polynucleotide is positioned by the provided systems and methods in a nucleolus. Compartment specific proteins suitable for targeting the nucleolus include, but are not limited to, nuclear protein B23.
In some embodiments, the target polynucleotide is positioned by the provided systems and methods in a P granule. In some embodiments, P granule is assembled or positioned by the provided systems and methods at the target polynucleotide. Suitable compartment specific proteins for P granules for the provided systems and methods include, but are not limited to, RGG domain proteins, PGL-1 and PGL-3; Dead box proteins, and GLH-1-4.
In some embodiments, the target polynucleotide is positioned by the provided systems and methods in a GW body. In some embodiments, the GW body is assembled or positioned by the provided systems and methods at the target polynucleotide. Suitable compartment specific proteins for GW bodies for the provided systems and methods include, but are not limited to, GW182.
In some embodiments, the target polynucleotide is positioned by the provided systems and methods in a stress granule. In some embodiments, the stress granule is assembled or positioned by the provided systems and methods at the target polynucleotide. Suitable compartment specific proteins for stress granules for the provided systems and methods include, but are not limited to, G3BP (Ras-GAP SH3 binding proteins), TIA-1 (T-cell intracellular antigen), eIF2, and eIF4E.
In some embodiments, the target polynucleotide is positioned by the provided systems and methods in a sponge body. In some embodiments, the sponge body is assembled or positioned by the provided systems and methods at the target polynucleotide. Suitable compartment specific proteins for sponge bodies for the provided systems and methods include, but are not limited to, EXu, Btz, Tral, Cup, eIF4E, Me31B, Yps, Gus, Dcp1/2, Sqd, BicC, Hrb27C, and Bru.
In some embodiments, the target polynucleotide is positioned by the provided systems and methods in a cytoplasmic prion protein induced ribonucleoprotein (CyPrP-RNP) granules. In some embodiments, the CyPrP-RNP is assembled or positioned by the provided systems and methods at the target polynucleotide. Suitable compartment specific proteins for CyPrP-RNP granules for the provided systems and methods include, but are not limited to, Dcpla, DDX6/Rck/p54/Me31B/Dhhl, and Dicer.
In some embodiments, the target polynucleotide is positioned by the provided systems and methods in a U body. In some embodiments, the U body is assembled or positioned by the provided systems and methods at the target polynucleotide. Suitable compartment specific proteins for U bodies for the provided systems and methods include, but are not limited to, uridine-rich small nuclear ribonucleoproteins U1, U2, U4/U6 and U5; LSm1-7; and the survival of motor neurons (SMN) protein.
In some embodiments, the target polynucleotide is positioned by the provided systems and methods in the endoplasmic reticulum. Compartment specific proteins suitable for targeting the endoplasmic reticulum include, but are not limited to, Calreticulin, Calnexin, PDI, GRP 78, and GRP 94.
In some embodiments, the target polynucleotide is positioned by the provided systems and methods in a mitochondrium. Compartment specific proteins suitable for targeting mitochondria include, but are not limited to, HIF1A, PLN, Cox 1, Hexokinase, and TOMM40.
In some embodiments, the target polynucleotide is positioned by the provided systems and methods in the plasma membrane. Compartment specific proteins suitable for targeting the plasma membrane include, but are not limited to, sodium potassium ATPase, CD98, Cadherins, and plasma membrane calcium ATPase (PMCA).
In some embodiments, the target polynucleotide is positioned by the provided systems and methods in golgi. Compartment specific proteins suitable for targeting golgi include, but are not limited to, GM130, MAN2A1, MAN2A2, GLG1, B4GALT1, RCAS1, and GRASP65.
In some embodiments, the target polynucleotide is positioned by the provided systems and methods in a ribosome. In some embodiments, the ribosome is assembled or positioned by the provided systems and methods at the target polynucleotide. Suitable compartment specific proteins for ribosomes for the provided systems and methods include, but are not limited to, AGO2, MTOR, PTEN, RPL26, FBL, and RPS3.
In some embodiments, the target polynucleotide is positioned by the provided systems and methods in a proteasome. In some embodiments, the proteasome is assembled or positioned by the provided systems and methods at the target polynucleotide. Suitable compartment specific proteins for proteasomes for the provided systems and methods include, but are not limited to, PSMA1, PSMBS, PSMC1, PSMD1, and PSMD7.
In some embodiments, the target polynucleotide is positioned by the provided systems and methods in an endosome. Compartment specific proteins suitable for targeting endosomes include, but are not limited to, CFTR, ADRB1, EGFR, IGF2R, AP2S1, CD4, HLA-A, Coveolin, RABS, and ErbB2.
In some embodiments, the target polynucleotide is positioned by the provided systems and methods in a liposome. Compartment specific proteins suitable for targeting liposomes include, but are not limited to, EEA1, LAMTOR2, and LAMTOR4.
In some embodiments, the target polynucleotide is positioned by the provided systems and methods in a synthetic cellular phase. In some embodiments, a synthetic cellular phase is positioned by the provided systems and methods by a target polynucleotide. A synthetic cellular phase can facilitate HDR. Suitable compartment-constituent proteins for facilitating HDR include, but are not limited to Rad51, Rad52, RPA, MRN complex, MRX complex, CtlP, Sae2, BLM, Sgs1, BRCA2, exonucleases such as Exo1 or OsExo1, ATM, BRCA2, RAD54, or MDC1. A synthetic cellular phase can facilitate heterochromatin. Suitable compartment-constituent proteins for facilitating heterochromatin include, but are not limited to, HP1α, HP1(3, KAP1, KRAB, SUV39H1, or G9a.
Other cell compartments that can be targeted with the systems and methods disclosed herein include RNP bodies, mitotic spindles, histone locus bodies, heterochromatin regions, and the cytoskeleton. Additional compartments are also contemplated.
The target polynucleotide can be endogenous or exogenous to the cell compartment to which it is positioned. The target polynucleotide can be endogenous or exogenous to the cell. The target polynucleotide can be human or non-human. The target polynucleotide can be virally derived, a plasmids, a ribonucleoprotein, or a synthesized RNA or DNA strand.
The methods and systems disclosed herein are suitable for use in multiplexed processes in which multiple polynucleotides are repositioned to the same or different cellular compartments.
In some embodiments, the provided systems and methods are used to mediate de novo cellular compartment (e.g., nuclear body) formation at targeted polynucleotide (e.g., genomic) loci, providing a potential method to initiate membraneless organelle formation via liquid-liquid phase separation. Membraneless organelle or compartment assembly can be used to create specific environments around a polynucleotide, such an environment that facilitates polynucleotide repair. For example, a compartment is assembled around a target polynucleotide that has been cut using a gene editing technique, and this compartment comprises a template polynucleotide and Rad51, Rad52, RPA, MRN complex, MRX complex, CtlP, Sae2, BLM, Sgs1, BRCA2, exonucleases such as Exo1 or OsExo1, ATM, BRCA2, RAD54, or MDC1. For example, a compartment is assembled around a target polynucleotide to regulate gene expression, and this compartment comprises HP1α, HP1(3, KAP1, KRAB, SUV39H1, or G9a. Membraneless compartmentalization of the subcellular space occurs by liquid-liquid phase separation. Heterotypic cooperative weak interactions enable rapid rearrangements within liquid compartments. Intrinsically disordered proteins play important roles in phase transitions due to their structural plasticity and prion-like properties. Cells dynamically control the extent and duration of phase transitions. Molecular seeds such as DNA, RNA or poly(ADP-ribose) (PAR) can trigger phase transitions in a stimulus- and context-specific manner. Chaperones, disintegrase machineries, and post-translational modifications cooperate to control phase transitions. A continuum of aggregation propensities exists and cells employ an unanticipated broad range of material states in proteinaceous assemblies. These can progress into pathological aggregates associated with neurodegenerative diseases.
Examples of synthetic phases that can be formed using the systems and methods disclosed herein include, but are not limited to, synthetic PML bodies that can have roles in viral defense and telomere maintenance, synthetic nuclear speckles and paraspeckles that can be stress inducible anti-apoptotic structures, synthetic gems that can be hubs for factors involved in neurodegeneration, synthetic architectural RNAs that can seed nuclear bodies, synthetic nucleoli, synthetic heterochromatin or euchromatin, synthetic histone locus bodies that can be sites of FLASH accumulation and enhance histone mRNA processing, synthetic chromatin packing systems that can involve the use of Xist to silence in cis the whole chromosome, synthetic epigenetic phases, synthetic (cytoplasmic) P bodies, synthetic stress bodies, synthetic germ granules that can generate sexual cells upon meiosis in the developing embryo, synthetic mRNP granules in neurodegenerative disease, synthetic posttranslational modifications (PTM) that can regulate membrane-less organelle structure and dynamics, synthetic IDP (intrinsically disordered proteins) forming aggregates, synthetic prion like domains (PLDs) or RGG-rich low-complexity domains (LCD), and synthetic polynucleotide repair bodies. For example, a synthetic polynucleotide repair body can be a synthetic HDR body that comprises a template polynucleotide and proteins that facilitate HDR such as Rad51, Rad52, RPA, MRN complex, MRX complex, CtlP, Sae2, BLM, Sgs1, BRCA2, exonucleases such as Exo1 or OsExo1, ATM, BRCA2, RAD54, or MDC1. For example, a synthetic gene regulation body can be a synthetic heterochromatin body that comprises protein that facilitate gene regulation such as HP1α, HP1β, KAP1, KRAB, SUV39H1, or G9a. Other non-endogenous protein/RNA aggregates to which polynucleotides can be positioned include β-amyloid bodies, mRNA aggregates, Xist packaging complexes, and others.
The controlled positioning of polynucleotides or compartments as describe herein can be used to regulate, modify, or influence, for example, DNA interaction with RNA Polymerases, transcription factors, pioneer factors, mediators, DNA looping molecules, and other DNA associated proteins; epigenetic modification marks or euchromatin/heterochromatin modulating enzymes (e.g., HP1); chromatin compactness and other biophysics/biochemical properties; gene editing, including recombination, NHEJ, or HDR; genome stability and cancer; DNA repair processes; and mRNA metabolism through splicing, degradation, translation, methylation, localization, and interaction with other chaperons and RNA-binding proteins.
The methods, compositions, and systems disclosed herein can be used to establish inducible and reversible disease models to understand disease mechanism. For example, the provided systems and methods can be used to investigate diseases caused by protein/RNA misfolding or aggregations. Proteome imbalances are associated with aging and often involve abundant proteins that exceed solubility and tend to form intracellular and extracellular aggregates. Aging is a risk factor for the onset of several protein misfolding disorders (PMDs), particularly for progressive neurodegeneration. Protein aggregation is the primary hallmark of neurodegeneration, including amyloid beta (Ab) and tau aggregation in Alzheimer's disease (AD), intracellular alpha-synuclein aggregates in Parkinson's disease (PD) and multisystem atrophy, polyQ-driven protein aggregates in Huntington's disease (HD), PrPSc in prion diseases, and TDP-43 and FET protein aggregates in amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), just to list a few examples. Although the chemical nature and the (patho)physiological topology of the proteins involved in plaque formation differ, the principles that govern their aggregation appear surprisingly similar, and the provided methods and systems can be used to position target polynucleotides at these plaques or aggregates.
The systems, compositions, and methods disclosed herein can be used to control cell differentiation by repositioning key drivers genes into different nuclear compartments. The systems and methods can be used to enhance antibody production by controlling the recombination rate at the endogenous VD(J) locus. The systems and methods can be used for mitigating Alzheimer's by eliminating the formation of misfolding protein bodies.
The systems, compositions, and methods disclosed herein can be used to co-localize or assemble a compartment at a target polynucleotide or gene locus. By repositioning or forming the compartment, the location of the target polynucleotide or gene locus is maintained within the cell, and thus allowing for the compartment to impact the target polynucleotide or gene locus without more broadly impacting the position of the chromatin and the functions associated with the positioning of the chromatin. The systems and methods can be used enhance or facilitate polynucleotide repair mechanisms, such as HDR, recombination, or NHEJ, by assembling compartments comprising proteins that facilitate the polynucleotide repair mechanism around a target polynucleotide.
The systems, compositions, and methods disclosed herein are broadly applicable in all kingdoms of life, including plants, bacteria, archaea, yeast, fishes, insects, birds, mammals, mice, pigs, and humans. The systems and methods can be used in living whole organisms or in tissue or cells.
The systems, compositions, and methods disclosed herein can be used to form compartments around a target polynucleotide that can be used for regulating expression of (e.g., increasing or decreasing), introducing epigenetic modifications to, producing three-dimensional structures, a topologically associating domains, or genomic boundaries comprising the target polynucleotide or an additional polynucleotide (e.g., distal or proximal gene from the target polynucleotide).
The following embodiments recite non-limiting permutations of combinations of features disclosed herein. Other permutations of combinations of features are also contemplated. In particular, each of these numbered embodiments is contemplated as depending from or relating to every previous or subsequent numbered embodiment, independent of their order as listed. 1. A system for controlling the spatial and temporal positioning of a target polynucleotide in a compartment of a cell, the system comprising: (a) a compartment-specific protein linked to a first dimerization domain; and (b) an actuator moiety that targets the target polynucleotide, wherein the actuator moiety is linked to a second dimerization domain that is capable of assembling into a dimer with the first dimerization domain. 2. The system of embodiment 1, wherein the target polynucleotide comprises genomic DNA. 3. The system of embodiments 1, wherein the target polynucleotide comprises RNA. 4. The system of any one of embodiments 1-3, wherein the actuator moiety comprises a Cas protein, and wherein the system further comprises: (c) a guide RNA that complexes with the actuator moiety and hybridizes to the target polynucleotide. 5. The system of any one of embodiments 1-3, wherein the actuator moiety comprises an RNA binding protein complexed with a guide RNA that hybridizes to the target polynucleotide, and wherein the system further comprises: (c) a Cas protein that complexes with the guide RNA. 6. The system of embodiment 4 or 5, wherein the Cas protein substantially lacks DNA cleavage activity. 7. The system of any one of embodiments 4-6, wherein the Cas protein is a Cas9 protein, a Cas12 protein, a Cas13 protein, a CasX protein, or a CasY protein. 8. The system of embodiment 7, wherein the Cas12 protein is selected from the group consisting of Cas12a, Cas12b, Cas12c, Cas12d, and Cas12e. 9. The system of any one of embodiments 1-3, wherein the actuator moiety comprises a binding protein that hybridizes to the target polynucleotide, wherein the binding protein is a zinc finger nuclease or a TALE nuclease. 10. The system of any one of embodiments 1-3, wherein the actuator moiety comprises an Argonaute protein complexed with a guide polynucleotide, wherein the guide polynucleotide is a guide RNA or a guide DNA, and wherein the guide polynucleotide hybridizes to the target polynucleotide. 11. The system of any one of embodiments 1-10, wherein the compartment is a nuclear compartment. 12. The system of embodiment 11, wherein the nuclear compartment comprises an inner nuclear membrane. 13. The system of embodiment 12, wherein the compartment-specific protein comprises Emerin, Lap2beta, Lamin B, or a combination thereof 14. The system of embodiment 11, wherein the nuclear compartment comprises a Cajal body. 15. The system of embodiment 14, wherein the compartment-specific protein comprises coilin, SMN, Gemin 3, SmD1, SmE, or a combination thereof 16. The system of embodiment 11, wherein the nuclear compartment comprises a nuclear speckle. 17. The system of embodiment 16, wherein the compartment-specific protein comprises SC35. 18. The system of embodiment 11, wherein the nuclear compartment comprises a PML body. 19. The system of embodiment 18, wherein the compartment-specific protein comprises PML, SP100, or a combination thereof 20. The system of embodiment 11, wherein the nuclear compartment comprises a nuclear core complex. 21. The system of embodiment 20, wherein the compartment-specific protein comprises Nup50, Nup98, Nup53, Nup153, Nup62, or a combination thereof. 22. The system of embodiment 11, wherein the nuclear compartment comprises a nucleolus. 23. The system of embodiment 22, wherein the compartment-specific protein comprises nucleolar protein B23. 24. The system of any one of embodiments 1-10, wherein the compartment is a cytosolic compartment. 25. The system of any one of embodiments 1-24, wherein the compartment-specific protein is further linked to a fluorescent protein. 26. The system of any one of embodiments 1-25, wherein the actuator moiety is further linked to a fluorescent protein. 27. The system of any one of embodiments 1-27, wherein the first dimerization domain and the second dimerization domain assemble to form a dimer only in the presence of a ligand. 28. The system of embodiment 27, wherein the first dimerization domain and the second dimerization domain each bind to the ligand in the presence of the ligand. 29. The system of embodiment 27 or 28, wherein the ligand is a chemical inducer. 30. A method of controlling the spatial and temporal positioning of a target polynucleotide in a compartment of a cell, the method comprising: (a) providing a compartment-specific protein linked to a first dimerization domain; (b) providing an actuator moiety linked to a second dimerization domain; (c) forming a complex comprising the actuator moiety and the target polynucleotide; and (d) assembling a dimer comprising the first dimerization domain and the second dimerization domain, thereby positioning the target polynucleotide in the compartment. 31. The method of embodiment 30, wherein the target polynucleotide is not endogenous to the compartment. 32. The method of embodiment 30 or 31, wherein the positioning of the target polynucleotide comprises regulating the expression of the target polynucleotide. 33. The method of embodiment 32, wherein the regulating comprises decreasing the expression of the target polynucleotide. 34. The method of embodiment 32, wherein the regulating comprises increasing the expression of the target polynucleotide. 35. The method of any one of embodiments 30-34, wherein the positioning of the target polynucleotide further comprises regulating the expression of one or more additional polynucleotides endogenous to the compartment. 36. The method of any one of embodiments 30-35, wherein the positioning of the target polynucleotide further comprises creating one or more additional compartments within the cell. 37. The method of any one of embodiments 30-36, wherein the positioning of the target polynucleotide further comprises repairing a DNA break. 38. The method of embodiment 41, wherein the repairing comprises introducing exogenous DNA. 39. The method of embodiment 42, wherein the introducing comprises recombination, non-homologous end-joining, or homology-directed repair. 40. The method of any one of embodiments 30-39, wherein the positioning of the target polynucleotide further comprises creating an artificial aggregate, wherein the artificial aggregate comprises protein, RNA, DNA, or a combination thereof 41. The method of any one of embodiments 30-40, wherein the target polynucleotide comprises genomic DNA. 42. The method of any one of embodiments 30-40, wherein the target polynucleotide comprises RNA. 43. The method of any one of embodiments 30-42, wherein the actuator moiety comprises a Cas protein, and wherein the system further comprises: (c) a guide RNA that complexes with the actuator moiety and hybridizes to the target polynucleotide. 44. The method of any one of embodiments 30-42, wherein the actuator moiety comprises an RNA binding protein complexed with a guide RNA that hybridizes to the target polynucleotide, and wherein the system further comprises: (c) a Cas protein that complexes with the guide RNA. 45. The method of embodiment 43 or 44, wherein the Cas protein substantially lacks DNA cleavage activity. 46. The method of any one of embodiments 43-45, wherein the Cas protein is a Cas9 protein, a Cas12 protein, a Cas13 protein, a CasX protein, or a CasY protein. 47. The method of embodiment 46, wherein the Cas12 protein is selected from the group consisting of Cas12a, Cas12b, Cas12c, Cas12d, and Cas12e. 48. The method of any one of embodiments 30-42, wherein the actuator moiety comprises a binding protein that hybridizes to the target polynucleotide, wherein the binding protein is a zinc finger nuclease or a TALE nuclease. 49. The method of any one of embodiments 30-42, wherein the actuator moiety comprises an Argonaute protein complexed with a guide polynucleotide, wherein the guide polynucleotide is a guide RNA or a guide DNA, and wherein the guide polynucleotide hybridizes to the target polynucleotide. 50. The method of any one of embodiments 30-49, wherein the compartment is a nuclear compartment. 51. The method of embodiment 50, wherein the nuclear compartment comprises an inner nuclear membrane. 52. The method of embodiment 51, wherein the compartment-specific protein comprises Emerin, Lap2beta, Lamin B, or a combination thereof 53. The method of embodiment 50, wherein the nuclear compartment comprises a Cajal body. 54. The method of embodiment 53, wherein the compartment-specific protein comprises coilin, SMN, Gemin 3, SmD1, SmE, or a combination thereof 55. The method of embodiment 50, wherein the nuclear compartment comprises a nuclear speckle. 56. The method of embodiment 55, wherein the compartment-specific protein comprises SC35. 57. The method of embodiment 50, wherein the nuclear compartment comprises a PML body. 58. The method of embodiment 57, wherein the compartment-specific protein comprises PML, SP100, or a combination thereof 59. The method of embodiment 50, wherein the nuclear compartment comprises a nuclear core complex. 60. The method of embodiment 59, wherein the compartment-specific protein comprises Nup50, Nup98, Nup53, Nup153, Nup62, or a combination thereof 61. The method of embodiment 50, wherein the nuclear compartment comprises a nucleolus. 62. The method of embodiment 61, wherein the compartment-specific protein comprises nucleolar protein B23. 63. The method of any one of embodiments 30-49, wherein the compartment is a cytosolic compartment. 64. The method of any one of embodiments 30-63, wherein the compartment-specific protein is further linked to a fluorescent protein. 65. The method of any one of embodiments 30-64, wherein the actuator moiety is further linked to a fluorescent protein. 66. The method of any one of embodiments 30-65, wherein the assembling occurs only in the presence of a ligand. 67. The method of embodiment 66, wherein the first dimerization domain and the second dimerization domain each bind to the ligand in the presence of the ligand. 68. The method of embodiment 66 or 67, wherein the ligand is a chemical inducer. 69. A method of controlling the spatial and temporal positioning of a compartment of a cell to a target polynucleotide, the method comprising: (a) providing a compartment-constituent protein linked to a first dimerization domain; (b) providing an actuator moiety linked to a second dimerization domain; (c) forming a complex comprising the actuator moiety and the target polynucleotide; and (d) assembling a dimer comprising the first dimerization domain and the second dimerization domain, thereby positioning the compartment around the target polynucleotide. 70. The method of embodiment 69, wherein the target polynucleotide is not endogenous to the compartment. 71. The method of embodiment 69 or 70, wherein the positioning of the compartment comprises regulating the expression of the target polynucleotide. 72. The method of embodiment 71, wherein the regulating comprises decreasing the expression of the target polynucleotide. 73. The method of embodiment 71, wherein the regulating comprises increasing the expression of the target polynucleotide. 74. The method of any one of embodiments 69-75, wherein the positioning of the compartment further comprises regulating the expression of one or more additional polynucleotides endogenous to the compartment. 75. The method of any one of embodiments 69-74, wherein the positioning of the compartment further comprises repairing a DNA break. 76. The method of any one of embodiment 75, wherein the repairing comprises introducing exogenous DNA. 77. The method of embodiment 76, wherein the introducing comprises recombination, non-homologous end-joining, or homology-directed repair. 78. The method of any one of embodiments 69-77, wherein the positioning of the target polynucleotide further comprises creating an artificial aggregate, wherein the artificial aggregate comprises protein, RNA, DNA, or a combination thereof 79. The method of any one of embodiments 69-78, wherein the target polynucleotide comprises genomic DNA. 80. The method of any one of embodiments 69-78, wherein the target polynucleotide comprises RNA. 81. The method of any one of embodiments 69-80, wherein the actuator moiety comprises a Cas protein, and wherein the system further comprises: (c) a guide RNA that complexes with the actuator moiety and hybridizes to the target polynucleotide. 82. The method of any one of embodiments 69-80, wherein the actuator moiety comprises an RNA binding protein complexed with a guide RNA that hybridizes to the target polynucleotide, and wherein the system further comprises: (c) a Cas protein that complexes with the guide RNA. 83. The method of embodiment 81 or 82, wherein the Cas protein substantially lacks DNA cleavage activity. 84. The method of any one of embodiments 81-83, wherein the Cas protein is a Cas9 protein, a Cas12 protein, a Cas13 protein, a CasX protein, or a CasY protein. 85. The method of embodiment 84, wherein the Cas12 protein is selected from the group consisting of Cas12a, Cas12b, Cas12c, Cas12d, and Cas12e. 86. The method of any one of embodiments 69-81, wherein the actuator moiety comprises a binding protein that hybridizes to the target polynucleotide, wherein the binding protein is a zinc finger nuclease or a TALE nuclease. 87. The method of any one of embodiments 69-81, wherein the actuator moiety comprises an Argonaute protein complexed with a guide polynucleotide, wherein the guide polynucleotide is a guide RNA or a guide DNA, and wherein the guide polynucleotide hybridizes to the target polynucleotide. 88. The method of any one of embodiments 69-87, wherein the compartment comprises a Cajal body. 89. The method of embodiment 88, wherein the compartment-constituent protein comprises coilin, SMN, Gemin 3, SmD1, SmE, or a combination thereof 90. The method of any one of embodiments 69-87, wherein the compartment comprises a nuclear speckle. 91. The method of embodiment 90, wherein the compartment-constituent protein comprises SC35. 92. The method of any one of embodiments 69-87, wherein the compartment comprises a PML body. 93. The method of embodiment 93, wherein the compartment-constituent protein comprises PML, SP100, or a combination thereof 94. The method of any one of embodiment 69-87, wherein the compartment is a cytosolic compartment. 95. The method of any one of embodiments 69-87, wherein the compartment is a synthetic cellular phase. 96. The method of embodiment 95, wherein the synthetic cellular phase facilitates homology directed repair. 97. The method of embodiment 96, wherein the compartment-constituent protein comprises Rad51, Rad52, RPA, MRN complex, MRX complex, CO, Sae2, BLM, Sgs1, BRCA2, exonucleases such as Exo1 or OsExo1, ATM, BRCA2, RAD54, or MDC1. 98. The method of any one of embodiments 69-97, wherein the compartment-constituent protein is further linked to a fluorescent protein. 99. The method of any one of embodiments 69-98, wherein the actuator moiety is further linked to a fluorescent protein. 100. The method of any one of embodiments 69-99, wherein the assembling occurs only in the presence of a ligand. 101. The method of embodiment 100, wherein the first dimerization domain and the second dimerization domain each bind to the ligand in the presence of the ligand. 102. The method of embodiment 100 or 101, wherein the ligand is a chemical inducer.
The following examples are given for the purpose of illustrating various embodiments of the disclosure and are not meant to limit the present disclosure in any fashion. The present examples, along with the methods described herein are presently representative of preferred embodiments, are exemplary, and are not intended as limitations on the scope of the disclosure.
To implement an inducible CRISPR-mediated chromatin repositioning system, two chemical-inducible heterodimerization systems were tested. The first was an abscisic acid (ABA) inducible ABI/PYL1 system, and the second was a TMP-Htag (Trimethoprim-Haloligand) inducible DHFR/HaloTag system. For both systems, the Streptococcus pyogenes dCas9 (D10A & H840A) protein was fused to one heterodimer, and an inner nuclear envelope (NE) protein, Emerin, was fused to the cognate heterodimer (
To test if the ABA-inducible CRISPR-GO system was able to alter the position of chromosomes, an endogenous locus on Chromosome 3 (Chr3) was targeted. An sgRNA targeting a highly repetitive (˜500×) region within Chromosome 3 (3q29) was lentivirally transduced into the U2OS cell line that stably expresses ABI-BFP-dCas9 and PYL1-GFP-Emerin (
After 2 days of ABA treatment, significantly increased tethering of targeted Chr3 locus to the nuclear periphery was observed as compared to cells without ABA treatment (
In addition to the Chr3:q29 locus, repositioning other highly repetitive endogenous genomic loci, including Chr13 locus and telomeres, to the nuclear periphery was tested. Using an sgRNA targeting repetitive region (˜350× repeats) on Chromosome 13q34 (Chr13) (
A synthetically integrated LacO array located at Chromosome 1p36 was also targeted in a U2OS 2-6-3 reporter cell line previously used for studying chromosome repositioning (
The efficiency of the CRISPR-GO system in repositioning less repetitive (<100 repeats) sequences was next tested. A genomic region on Chr7 q36.3 containing ˜71 sgRNA-targetable repeats and a genomic region on ChrX p21.2 containing ˜15 sgRNA-targetable repeats were chosen as targets (
Though repetitive sequences are abundantly present in human genome, it is of further interest if the CRISPR-GO system enabled repositioning non-repetitive genomic loci. The non-repetitive gene XIST located at ChrX q13.2, was first targeted and 13 sgRNAs tiling the XIST genomic region were designed (
Whether a single sgRNA targeting a non-repetitive region is sufficient to re-reposition a genomic locus was also tested. Using a single sgRNA (sgCXCR4-1) targeting the CXCR4 locus at Chr2, the percentage of periphery localized CXCR4 loci increased from 20% (n=241) to 50% (n=425, p<0.0001), and the percentage of cells containing periphery-localized loci increased from 52% (n=69) to 85% (n=131, p<0.0001) (
One advantage of the provided systems and methods is the ability to easily switch on or off polynucleotide re-positioning by adding or removing a chemical inducer to the culture medium. Chemical induction and removal experiments were performed to study the dynamics and reversibility of the ABA-inducible CRISPR-GO system (
The CRISPR-GO system was used to target the endogenous Chr3 locus. CRISPR-GO cells containing Chr3-targeting sgRNAs were synchronized and arrested in the S phase by serum starvation and Hydroxyurea (HU) treatment and then treated with ABA for chemical induction (
Mitosis-independent periphery repositioning has not yet been reported to our knowledge. To probe whether a genomic locus can be tethered to the nuclear periphery during interphase, live-cell CRISPR-Cas9 imaging was used to track the dynamics of nuclear periphery tethering. The dynamic process of endogenous Chr3 loci becoming tethered to nuclear periphery during interphase was detected. In the representative example shown in
The short-time movement kinetics of genomic loci after genomic tethering was studied by combining the CRISPR-GO system with CRISPR-Cas9 imaging in living cells. The short-term dynamics of Chr3 loci tethered at the nuclear periphery were examined as compared to 30 untethered loci (
Whether the CRISPR-GO system can mediate colocalization of chromatin loci with membraneless nuclear bodies was next tested. Genomic loci were chosen to recruit to Cajal 20 bodies (CBs). To do this, a Cajal body-targeting CRISPR-GO system was designed by fusing PYL1 with Coilin, a marker of Cajal bodies. PYL1-GFP-Coilin and ABI-dCas9 were introduced into U2OS cells via lentiviral transduction (
Using an sgRNA targeting the LacO sequence, we visualized the spatial positioning of the LacO array using 3D-FISH and the location of CBs by GFP-Coilin after 2 days of ABA treatment (
To target endogenous genomic loci to CBs, the Chr3:q29-targeting sgRNA was introduced into U2OS cells expressing the Cajal body-targeting CRISPR-GO system. Significant colocalization was observed between the Chr3 loci (visualized with CRISPR-Cas9 imaging) and CBs (visualized with GFP-Coilin) 24 hours after ABA treatment (
Whether CRISPR-GO could mediate colocalization of chromatin loci with PML nuclear bodies was also tested. To do this, a PML body-targeting CRISPR-GO system was designed by fusing PYL1 with the PML gene, the scaffold protein of PML bodies. To target endogenous genomic loci to PML bodies, the Chr3:q29-targeting sgRNA was introduced into cells expressing both PYL1-GFP-PML and ABI-dCas9, the positioning of Chr3 loci was visualized by CRISPR-Cas9 imaging and the position of PML bodies was visualized by GFP-PML (
Chemical induction and removal experiments were performed to study the dynamics and reversibility of the CRISPR-GO mediated chromatin colocalization with CBs. Using the LacO locus inserted at Chr1:p36 in U2OS 2-6-3 cells as an example, we observed that the association between LacO loci and GFP-Coilin-marked CBs occurred rapidly: within 30 minutes after ABA addition, the percentage of LacO loci that colocalized with CBs increased from 2.6% (n=78) to 89% (n=85, p<0.0001) (
In cells pretreated with ABA for 1 day, ABA removal was observed to lead to dissociation of CBs from LacO loci. After ABA removal, the percentage of the targeted LacO loci that colocalized with CBs decreased from 89% (n=85) to 22% (n=60, p<0.0001) after 6 hours and further decreased to 4.6% (n=45, p<0.0001) after 24 hours (
To further characterize the dynamics of CRISPR-GO-mediated association of Cajal bodies with targeted genomic loci, time-lapse microscopic imaging of individual cells was performed before and after ABA treatment. Theoretically, colocalization between a genomic locus and nuclear bodies could occur through de novo formation of a nuclear body at the genomic locus, or through repositioning the genomic locus to an existing nuclear body. Previous reports using the LacO-Lacl tethering system suggest that Cajal bodies form de novo at the targeted DNA site.
Using the CRISPR-GO system to target LacO loci to Cajal bodies, rapid (within 20 minutes) de novo CBs formation was observed at the LacO locus in most analyzed cells after addition of ABA (
Interestingly, dynamic repositioning of the targeted chromatin locus with an existing CB was observed if the two were initially spatially close to each other. For example, in a cell where an existing CB was adjacent to a LacO locus without ABA treatment (
Previous studies offer different evidence about the effect that genomic relocalization to the nuclear periphery has on gene expression. Some studies showed that tethering LacO repeats to the nuclear periphery using LacI-Emerin or LacI-Lap2β caused repression of adjacent gene. Other studies showed re-localization of the LacO array to the nuclear periphery using a LacI-Lamin B1 fusion protein in the U2OS 2-6-3 cells, but observed no obvious changes in adjacent gene expression. CRISPR-GO offers another way to study this question, since it is much easier to test the effects of recruiting different chromosome loci to the nuclear periphery.
Whether CRISPR-GO-mediated repositioning of the LacO array to the nuclear periphery could influence gene expression was examined. The LacO locus in the U2OS 2-6-3 cells is located upstream of a Doxycycline (Dox)-inducible TRE (Tetracycline responsive element)-CMV promoter that drives expression of a CFP reporter (
Whether repositioning endogenous genomic loci to the nuclear periphery could alter gene expression was next tested. The Chr3, XIST, and CXCR4 loci were repositioned to the nuclear periphery individually, and RT-qPCR was performed to detect changes in adjacent gene expression (Chr3: ACAP2 & PPP1R2; CXCR4; XIST). Surprisingly, no evidence of gene expression change was seen for these genes (e.g., ACAP2 & PPP1R2 in
Whether colocalization of LacO loci to CBs using the CRISPR-GO system in the U2OS 2-6-3 cell line was sufficient to influence adjacent gene expression was next tested (
Whether colocalizing an endogenous genomic locus to CBs could alter adjacent gene expression was next tested. The CRISPR-GO system was used to induce colocalization of Chr3:q29 with CBs (
The CRISPR-GO system was used to investigate how telomere reorganization to nuclear compartments affected cellular phenotype. Among all genomic loci tested, the dynamics of telomeres are the best studied and are shown to be associated with the nuclear periphery and CBs at certain stages of the cell cycle. Given the important role of telomeres for genome integrity, their interactions with nuclear compartments may have functional implications. For example, during the cell cycle, telomeres are dynamically tethered to the nuclear envelope when the nuclear membrane reassembles in post-mitotic cells, and then relocate to the interior of the nucleus during the G1 phase, where they remain for the rest of cell cycle. The cycle of telomere tethering and untethering to the nuclear envelope may be important for chromatin organization and the cell cycle/viability.
To test this, the CRISPR-GO system was used to disrupt the telomere untethering process during the cell cycle and retain telomeres to the nuclear compartments during interphase (
pHR-SFFV-PYL1-sfGFP-Emerin was cloned by replacing scFv sequence in pHR-SFFV-scFv-sfGFP plasmid (Tanenbaum et al., 2014) with PYL1 and inserting Emerin after sfGFP. Emerin (encoded by the EMD gene) was cloned from Emerin pEGFP-C1 (637), a gift from Eric Schirmer (Zuleger et al., 2011) (Addgene plasmid 61993). pHR-SFFV-PYL1-sfGFP-Coilin was cloned by replacing Emerin in pHR-SFFV-PYL1-sfGFP-Emerin plasmid with Coilin. Coilin was cloned from pEGFP-Coilin (Addgene plasmid 36906), a gift from Dr. Greg Matera. pHR-PGK-PYL1-sfGFP-Coilin was cloned by replacing SFFV promoter in pHR-SFFV-PYL1-sfGFP-Coilin plasmid with PGK promoter. pHR-TRE3G-PYL1-sfGFP-PML or pHR-TRE3G-10 PYL1-sfGFP-HP1α was cloned by replacing PGK promoter with TRE3G promoter, and replacing Coilin with PML or HP1α in the pHR-PGK-PYL1-sfGFP-Coilin plasmid. PML was cloned from pLPC-Flag-PML-IV (addgene plasmid 62804), a gift from Gerardo Ferbeyre (Vernier et al., 2011). HP1α was cloned from GFP-HP1α (Addgene plasmid 17652), a gift from Tom Misteli (Cheutin et al., 2003).
pHR-SFFV-ABI-tagBFP-dCas9 was described before (Gao et al., 2016). pHR-SFFV-ABI-tagBFP-dCas9 was cloned by replacing SFFV promotor with PGK promoter pHR-SFFV-ABI-tagBFP-dCas9. pHR-PGK-ABI-dCas9-P2A-Cherry, or pHR-PGK-ABI-dCas9-P2A-Puro was cloned by replacing SFFV with PGK promoter, deleting tagBFP and adding P2A-mCherry or P2A-Puro in dCas9 pHR-SFFV-ABI-tagBFP-dCas9. ABI and PYL1 were cloned from Addgene plasmid 38247 (Liang et al., 2011), a gift from Dr. J. Crabtree, Stanford.
pHR-TRE3G-dCas9-HaloTag was cloned by replacing SunTag10-P2A-mCherry with HaloTag in the plasmid pHR-TRE3G-dCas9-HA-SunTag10-P2A-mCherry (Tanenbaum et al., 2014). pHR-TRE3G-dCas9-EGFP-HaloTag was cloned by inserting HaloTag after EGFP in pHR-TRE3G-dCas9-EGFP (Chen et al., 2013). pHR-SFFV-DHFR-mCherry-Emerin was cloned by replacing PYL1-sfGFP sequence in pHR-SFFV-PYL1-sfGFP-Emerin with mCherry-DHFR. HaloTag and mCherry-DHFR was cloned from pERB221, gift from David Chenoweth & Michael Lampson (Ballister et al., 2014) (Addgene plasmid 61502).
All sgRNAs were cloned into pHR-U6-sgTel-CMV-puro-P2A-mCherry vector after removing P2A-mCherry (Chen et al., 2013). TRF1-mCherry was cloned into pHR-U6-sgTel-CMV-puro-P2A-mCherry vector in place of mCherry. TRF1 was cloned from pLPC-NFLAG TRF1, a gift from Dr. Titia de Lange (Smogorzewska and de Lange, 2002) (Addgene plasmid #16058).
The U2OS (human bone osteosarcoma epithelial, female) cells and Hela cells (female) were cultured in DMEM with GlutaMAX (Life Technologies) in 10% Tet-system-approved FBS (Life Technologies). U2OS 2-6-3 cell line was a gift from Dr. David L. Spector in Cold Spring Harbor Laboratory and were cultured in the same condition (Kumaran and Spector, 2008). All cells were cultured at 37° C. and 5% CO2 in a humidified incubator.
To create stable CRISPR-GO cell lines targeting endogenous loci to nuclear compartments, U2OS cells were plated into 24-well plates 1 day ahead to reach 50% confluency, and then transduced by lentivirus mixture. Cells transduced by lentivirus expressing PYL1-sfGFP-Emerin, PYL1-sfGFP-Coilin, PYL1-sfGFP-PML, or PYL1-sfGFP-HP1α and ABI-tagBFP-dCas9 were sorted by fluorescence activated cell sorting (FACS) at Stanford shared FACS facility for cells that are BFP and GFP positive to create stable cell lines. For nuclear periphery tethering, cells of high BFP and GFP expression level was selected. For other nuclear compartment tethering, cells of high BFP and GFP expression level was selected. After transducing CRISPR-GO cell lines with lentivirus expressing targeting sgRNAs, sgRNA-positive cells were selected with puromycin at 2 μg/ml.
To target LacO loci in the U2OS 2-6-3 cell lines (Kumaran and Spector, 2008), cells were transduced by lentivirus mixture containing PYL1-sfGFP-Emerin or PYL1-sfGFP-Coilin and ABI-dCas9-P2A-mCherry. Cells containing PYL1-sfGFP-Coilin and ABI-dCas9-P2A-mCherry were sorted for GFP and mCherry positive cells to created stable cell lines. SgRNAs positive cells were selected with puromycin at 2 μg/ml.
To quantify the efficacy of LacO nuclear periphery repositioning by CRISPR imaging, U2OS 2-6-3 cells were transduced with lentivirus coding ABI-dCas9-P2A-Puro instead of ABI-dCas9-P2A-mCherry, and were selected with puromycin at 2 μg/ml.
The efficacy of CRISPR-GO system targeting different chromosomal regions in U2OS cells was tested. Both repetitive regions and non-repetitive genes were tested (
To produce lentivirus, HEK293T cells were transiently transfected with pHR constructs of interest, and packaging plasmids pCMV-dR8.91, and PMD2.G. Lentivirus was collected 72 hours after transfection by filtering supernatant through 0.45 μm filters. When necessary, virus supernatant can be concentrated using Lenti-X concentrator at 4° C. overnight, and centrifuged at 1500 g for 30 min at 4° C. to collect virus pellet. The pellets are suspended in cold culture medium, directly added into cells or frozen down in −80° C.
CRISPR imaging was performed to visualize the localization of Chr3, Chr13 and LacO loci in living cells (
Other genomic loci are labeled by DNA FISH in fixed cells. Cells were grown in ibidi chamber slides with a removable 12 well silicone chamber, and fixed with 4% PFA for 20 minutes. Lac 0, Chr7 and ChrX loci were labeled using synthesized fluorescent nucleotide probes (Integrated DNA Technologies, Redwood City, Calif.) according to a FISH protocol described (Takei et al., 2017). LacO loci were labeled with the Alexa Fluor 647 labeled FISH probe 5′-TTGTTATCCGCTCACAATTCCACATGTGGCCACAAA-3′ (SEQ ID NO: 40) at 10 nM concentration. Chr7 loci were labeled by Cy3 labeled FISH probe 5′-Cy3-CCCACACTCTCACCATAAGAGC-3′ (SEQ ID NO: 41) at 200 nM, and ChrX loci were labeled by 5-Cy3-TTGCCTTGTGCCTTGCCTTGC-3′ (SEQ ID NO: 42) at 200 nM. The CXCR4 FISH probe was purchased from Empire Genomics. The PTEN and XIST FISH probes were purchased from Cell Line Genetics. FISH was performed according merchandiser's protocols.
To detect co-localization between Cajal body markers and targeted LacO loci, U2OS 2-6-3 cells expressing a low level of PYL1-sfGFP-Coilin were transfected with lentivirus coding PGK-ABI-dCas9-P2A-Puro and sgLacO on day 0, treated with puromycin and 3 mM ABA on day 1, and fixed on day 2 after 20 hours of ABA treatment. FISH was performed in fixed samples to detect LacO loci using Alexa Fluor 647 labeled FISH probe, and then immunostaining was performed using mouse monoclonal anti-SMN, anti-Fibrillarin and anti-Gemin2 antibody, and Donkey anti-mouse Alex Fluor 594 secondary antibody.
To detect co-localization between PML body markers and targeted Chr3 loci, U2OS cells expressing PYL1-sfGFP-Coilin and PGK-ABI-dCas9 were transfected with lentivirus coding dCas9-HaloTag (for CRISPR imaging) and sgChr3 on day 0, treated with puromycin and 3 mM ABA on day 1, stained by JF549-HaloTag and fixed in 4% paraformaldehyde (PFA) in Day 3. Immunostaining was performed in fixed samples with rabbit polyclonal anti-SP100, and Donkey anti-rabbit Alex Fluor 647 secondary antibody.
For immunostaining, the fixed samples permeabilized in the permeabilization buffer (PBS, 1% Triton-X100) for 15 min, blocked in blocking buffer (PBS, 0.3% Triton-X 100, 5% Donkey normal Serum) for 1 hour, incubated with the primary antibody diluted in the blocking buffer overnight at 4° C., washed in PBS three times, then incubated with the secondary antibody at room temperature for 1-2 hours, and washed four times in PBS.
For re-localization experiments, U2OS cells containing chemical-inducible re-localization systems and sgRNAs are treated by abscisic acid (ABA, Sigma-Aldrich, A1049) at 3 mM for 2 days before imaging or fixation.
For the time-course chemical induction experiment targeting Chr3 to nuclear periphery, U2OS cells containing CRISPR-GO and CRISPR imaging systems and sgRNAs targeting Chr3 were treated with or without 3 mM ABA, stained by JF549-HaloTag, and fixed at different time points. For the time-course reversal experiment, the Chr3-targeting U2OS cells were pre-treated with 3 mM ABA for 2 days, washed five times, and switched to medium without ABA. Cells were stained by JF549-HaloTag ligand for CRISPR imaging and fixed in 4% paraformaldehyde for 20 min at different time points.
For the time-course chemical induction experiment targeting LacO to Cajal body, U2OS 2-6-3 cells expressing a low level of PYL1-sfGFP-Coilin were transfected with lentivirus coding PGK-ABI-BFP-dCas9 and sgLacO on day 0, treated with puromycin on day 1, treated with or without 3 mM ABA on day 2 and fixed after 30 minutes of ABA treatments. For the time-course reversal experiment, cells were pre-treated with 3 mM ABA for 2 days, washed five times, and switched to medium without ABA. Cells were fixed in 4% paraformaldehyde for 20 min at different time points.
To dissect mitosis-dependence effect of genomic re-localization, U2OS cells containing CRISPR-GO and CRISPR imaging systems and sgRNAs targeting Chr3 were used for this experiment. On day −3, cells were starved in 0.5% FBS in medium for 2 days. On day −1, cells were switched to normal growth medium with 10% FBS and treated with 2 mM hydroxyurea (HU) for G1/S phase blockage for 1 day. On day 0, while keeping the HU treatment, cells were treated with or without ABA. Control cells were treated in the same way but without HU. Cells were stained by JF549-HaloTag for CRISPR imaging and fixed in 4% paraformaldehyde 24 h or 48 h after ABA treatment.
With the exception of
For long-term live cell imaging shown in
To visualize the dynamics of chromatin-Cajal body association in individual cells (
Image processing was performed in Fiji (image J) (Schindelin et al., 2012) or MetaMorph (Molecular devices, CA). A single microscope plane showing maximum fluorescence of labeled genomic loci, or the average of two/three adjacent Z planes showing maximum loci fluorescence are shown in the drawings herein. Some images were processed using the “smooth” function in Fiji to reduce noises for visualization only.
Line scan was performed using the “Analyze/Plot Profile” function in Fiji, analyzed in Excel and plotted in GraphPad Prism (Version 7.00 for Mac OS, GraphPad Software, La Jolla Calif. USA, www.graphpad.com). Fluorescence intensity at each point along the line were normalized relative to the maximum (=1) and the minimum (=0) fluorescence intensity along the line.
Linear tracing to evaluate co-localization of target proteins on cells (
To determine the peripheral recruitment efficacy in living U2OS cells, Chr3, Chr13 and Chr1/Lac0 loci are labeled by CRISPR imaging and telomeres are labeled by TRF1-mCherry, while the nuclear membrane is labeled by PYL1-sfGFP-Emerin. After scanning Z-stacks of confocal planes, the position of each labeled locus is viewed in slice viewer (NIS element viewer) to determine its position in XY, XZ and YZ planes. Without double counting any loci, the loci were categorized into three categories: loci located directly in the nucleus periphery that co-localize with PYL1-GFP-Emerin in XY, YZ and YZ planes, loci that do not co-localize with PYL1-GFP-Emerin, and loci that co-localize with internal PYL1-GFP-Emerin not at nuclear periphery (in rare cases). The number of loci in each category was recorded for each individual cell. Only loci of the first category that co-localize with PYL1-GFP-Emerin at the nuclear envelope were counted as nuclear periphery positioned loci. Cells containing at least one nuclear periphery positioned loci were quantified.
To determine peripheral recruitment efficacy in fixed U2OS cells (e.g., Chr7, ChrX, PTEN, CXCR4, XIST), targeted genomic loci are labeled by FISH and the nucleus are stained by DAPI. After scanning Z-stacks of confocal planes, the position of each labeled locus is viewed in 3D space to determine its position in XY, XZ and YZ planes. A genomic locus that located at the edge of nucleus (DAPI) in 3D space is categorized as a periphery-located locus. Otherwise it is considered as an internal-located locus. The number of loci in each category was recorded for each individual cell. Cells containing at least one nuclear periphery positioned loci were also quantified.
To detrmine the Cajal body co-localizing efficacy in fixed U2OS 2-6-3 cells, targeted LacO loci were labeled by FISH, nuclei were stained by Hoechst 33342, and Cajal bodies were labeled by PYL1-GFP-Coilin. After scanning Z-stacks of confocal planes, we identified the position of each LacO locus in 3D space. Without double counting, the loci were categorized two categories: loci that co-localize with PYL1-GFP-Coilin, and loci that do not co-localize with PYL1-GFP-Coilin. The number of loci in each category was recorded for each individual cell. Cells containing at least one Cajal body-co-localized loci were also quantified.
For quantification of CFP-SKL expression, U2OS 2-6-3 cells containing ABI-dCas9-P2A-mCherry and PYL1-sfGFP-Emerin or PYL1-sfGFP-Coilin were transduced with sgRNA targeting lacO loci or non-targeting sgRNAs, treated with ABA at 3 mM for 2 days and then induced with doxycycline at 50 ng/ml for 40 hours (nuclear periphery tethering) or 24 hours (Cajal body tethering). After the treatment, U2OS 2-6-3 cells were dissociated using 0.25% Trypsin EDTA (Life Technologies) and analyzed by flow cytometry on CytoFlex S (Beckman Coulter Life Sciences) using 405-nm, 488-nm and 561-nm lasers. At least 8,000 cells were analyzed for each sample. Cells were gated for positive dCas9 (mCherry) and Emerin (GFP) expression. CFP-SKL fluorescence was detected using the 405 nm laser and 450/45 filter. To quantify relative fluorescence, the average total fluorescence of untreated (without Dox and ABA) cells is set to 0, while the average total fluorescence of doxycycline induced cells (with Dox only) is set to 1. Technical replicates in 3 independent experiments are reported.
Real-time RT-PCR were performed to determine the expression change in PPP1R2 TFRC and ACAP2 gene adjacent to targeted Chr3 loci after genomic re-organization. For each sample, total RNAs were isolated using RNeasy Plus Mini Kit (Qiagen Cat 74134) and cDNAs were synthesized using the iScript cDNA Synthesis Kit (BioRad, Cat 1708890), according to manufacturer's protocols. Quantitative PCR was performed using the PrimePCR assay with the SYBR Green Master Mix (BioRad), and run on Biorad CFX384 real-time system (C1000 Touch Thermal Cycler), according to manufacturer's instructions. Cq values was used to quantify gene expression. The relative expression of the PPP1R2, TFRC, and ACAP2 genes was normalized to GAPDH control. To calculate the relative mRNA expression level, the relative expression of each treatment was normalized by setting the average value in non-ABA treated samples as 1. Replicates in 3 experiments are reported.
Cell viability assay was performed using Alamar blue cell viability reagents (ThermoFisher Scientific), which measures the metabolic activity of the cells. For each condition, 100 μl cells treated with and without ABA were seeded at equal concentration (500-1000 cells/well) in the same 96-well plate. At the time of detection, 10 μl of Alamar blue reagents were added to each well and the plates were incubated at 37° C. for 1 hour. After that, the fluorescent intensity was measured in the Synergy H1 microplate reader (Biotek Inc.) using the excitation wavelength at 540 nm and the emission wavelength at 585 nm. Average fluorescent intensity of wells containing only 100 μl culture medium (with and without ABA) was used as blanks. For each well, the relative fluorescent intensity is calculated by subtracting background (average intensity of blank wells) from its raw fluorescent intensity. To calculate the relative cell viability, the relative florescent intensity in each well was normalized by setting the average value in non-ABA treated wells as 1. Replicates in 3 experiments are reported.
To quantify how telomere nuclear periphery tethering affect cell cycle progression, U2OS cells containing nuclear periphery tethering system were treated with lentivirus mixtures coding sgTelomere and TRF1-mcherry, or lentivirus coding a non-targeting sgRNA. Telomere tethering was confirmed by microscopy after 2 days of ABA treatment. After 3 day of ABA treatment, control and treated cells were dissociated using 0.25% Trypsin EDTA, with stained Hoechst 33342 at 1:1000 dilution for 1 h, and analyzed by flow cytometry on CytoFlex S (Beckman Coulter Life Sciences) using 405-nm lasers. At least 20,000 cells were analyzed for each sample. Cell cycle analysis was performed using FlowJO.
The software Tandem Repeats Finder (Benson, 1999) was used to identify all tandem repeats of 14-nucleotides or longer sequences from the human genome (hg38). Regions that contain ten or more identical tandem repeats were defined a “repetitive sequence cluster.” These repetitive sequence clusters were to each human chromosome. Distances between the repetitive sequence clusters and genes were calculated using the BEDTools suite.
Genomic loci tracking was performed using the TrackMate plugin (Tinevez et al., 2017) in Fiji. For tracking genomic loci, the estimated blob diameter was set between 0.5-1. Linking max distance was set to 2 and gap closing distance was set to 3 μm and gap closing max frame was set to 2. Position of each locus (xt, yt) at different time point (t) were measured, analyzed in Excel and plotted in GraphPad Prism 7. The movement step (dx, dy) was calculated by subtracting the position of a previous time point from the new position: dxt=xt−xt-1 & dyt=yt−yt-1, where (xt,yt) is position of a locus at time t, while (xt-1, yt-1) is the position of the locus at the previous time point (t−1). Step distance=√((xt−xt-1)2+(yt−yt-1)2) is calculated as how far a locus move away from its position at the previous time point.
To compare step distances, 1696 step distances of 19 interior-localized Chr3 loci and 1669 step distances of 14 periphery-localized Chr3 loci were analyzed. The two-side t-test with unequal variance was performed. Histogram were analyzed using Histogram function in Excel and plotted in in GraphPad Prism 7.
For quantification of re-localization efficacy (
Although the foregoing invention has been described in some detail by way of illustration and example for purpose of clarity of understanding, one of skill in the art will appreciate that certain changes and modifications may be practiced within the scope of the appended claims. In addition, each reference provided herein is incorporated by reference in its entirety to the same extent as if each reference was individually incorporated by reference.
To implement an inducible CRISPR-mediated chromatin repositioning system to facilitate homology directed repair (HDR), a chemical-inducible heterodimerization system is tested. This system is an abscisic acid (ABA) inducible ABI/PYL1 system. The Streptococcus pyogenes dCas9 (D10A & H840A) protein is fused to one heterodimer, and various proteins that facilitate HDR, such as Rad51, Rad52, and the MRN complex, are fused to the cognate heterodimer. A template polynucleotide to be used for HDR is also fused to the cognate heterodimer. U2OS human bone osteosarcoma epithelial cell lines are created using lentiviral transduction that stably expressed the dimerization system. In these cell lines, spatial re-localization of HDR fusion proteins to the ABI-BFP-dCas9 protein is caused by addition of ABA, due to its dimerization with PYL1-GFP-HDR proteins. A CRISPR/Cas9 complex that generates a double stranded break in the target polynucleotide of the ABI-BFP-dCas9 protein is introduced into the cells, and a double stranded break in the target polynucleotide is generated. The double stranded break in the target polynucleotide is preferentially repaired by HDR.
This example shows the generation of heterochromatin domains at a targeted genomic locus. An inducible dCas9 and a heterochromatin protein are fused to complementary pairs of heterodimerization domains, which assemble in the presence of the chemical inducer.
In order to generate high concentrations of heterochromatin proteins at target sites, an inducible ABI-PYL1 heterodimer system is used.
This example shows the dynamic recruitment of engineered heterochromatin protein HP1α to a target site on Chromosome 3. In order to generate heterochromatin protein HP1α to the target sites, an inducible ABI-PYL1 was used to effect dimerization of the protein complex upon addition of abscisic acid. dCas9-HaloTag, ABI-BFP-dCas9, PYL1-sfGFP-HP1α, and sgChr3q29 were stably integrated into U2OS human osteosarcoma cells by lentiviral transduction (sgChr3q29: SEQ ID NO: 1; target PAM sequence for sgChr3q29: TGG (SEQ ID NO: 37)).
Upon treatment with ABA, PYL1-sfGFP-HP1α formed strongly fluorescent foci at the Chr3q29 locus. Time lapse confocal microscopy revealed that PYL1-sfGFP-HP1α recruitment and foci formation occurred as early as 1-10 min after ABA addition.
Furthermore, expression of distal genes on Chr3q29 was repressed by recruitment of PYL1-sfGFP-HP1α. Expression of three genes on Chr3q29 at between 35 kb and 575 kb flanking the tandem repeat target site were quantified by RT-qPCR after 4 days treatment with either 100 μM ABA or DMSO vehicle.
Furthermore, free-floating HP1α is recruited to synthetic HP1α foci. Natural heterochromatin recruits heterochromatin protein 1 (HP1) family proteins HP and HP1(3.
Linear fluorescence intensity tracing was used to confirm the co-localization of mCherry-HP1α with synthetic HP1α foci.
HP1β is an ortholog of HP1α and also was enriched within natural heterochromatin. The enrichment of HP1β was tested by immunofluorescence and fluorescence tracing. Cells were immunostained with HP1β primary and AlexaFluor647 secondary antibodies after 2 days of 100 μM ABA treatment.
Local chromatin density does not increase at synthetic HP1α foci.
Another key marker of HP1α-associated natural heterochromatin is the post-translational trimethylation of lysine 9 on the tail of histone 3 (H3K9me3). Cells were immunostained with H3K9me3 primary and AlexaFluor647 secondary antibodies after 2 or 5 days of 100 uM ABA treatment.
The presence of KAP1, a co-repressor scaffold protein that binds both HP1α and other chromatin modifying proteins, at synthetic HP1α foci was also assayed using immunostaining. Cells were immunostained with KAP1 primary and AlexaFluor647 secondary antibodies after 2 days of 100 μM ABA treatment
To determine whether the repression resulting from HP1α is due to the function of any particular domain within HP1α, mutant HP1α in which the chromodomain is removed, e.g. HP1α (CSD), and in which the chromoshadow domain is rendered incapable of homodimerizing, e.g. HP1α (I165E), were tested against full length wild-type HP1α for repression on Chr3q29. Only full length wild-type HP1α was capable of repressing the three tested distal genes as measured by RT-qPCR.
To understand how the heterochromatin properties of the HP1α(I165E) and HP1α(CSD) mutants differ from the full length wild-type protein, their abilities to recruit free-floating mCherry-HP1α were examined. While the HP1α(CSD) was still capable of recruiting mCherry-HP1α to synthetic HP1α foci, the HP1α(I165E) mutant lost this capacity.
While the HP1α(CSD) mutant was still competent for recruiting free-floating HP1α to synthetic HP1α foci, the removal of the N-terminus and the chromodomain impairs its ability to localize to natural heterochromatin sites (using mCherry-HP1α subnuclear localization as a proxy).
To determine whether the incomplete heterochromatin marks from synthetic HP1α was due to impaired HP1α function as a result of fusing additional protein domains to its N-terminus, the ability for indirectly recruiting unencumbered, endogenous HP1α was tested. In this alternative strategy, HP1α in the inducible system was replaced with the KRAB domain commonly used for CRISPR interference (CRISPRi). KRAB acts as a recruitment signal for the co-repressor scaffold protein KAP1, which in turn recruits endogenous HP1α (Ecco et. al., Development 2017).
The alternative KRAB heterochromatin system also demonstrated the ability to recruit free-floating HP1α to synthetic heterochromatin foci, though at seemingly lower levels than for the original synthetic HP1α system. Immunofluorescence analysis showed that HP1α localization is weakly enriched at KRAB-tethered foci.
Unlike with the original synthetic HP1α system, the KRAB system was enriched for KAP1 at the synthetic heterochromatin foci.
Despite recruiting both HP1α and KAP1 to synthetic foci, KRAB was still unable to increase H3K9me3 abundance at the Chr3q29 target site.
Similarly, local chromatin/histone density, as measured by H2B-mCherry fluorescence, nor DNA density, as measured by SiR-DNA staining intensity, was increased at KRAB-based synthetic foci.
The KRAB and HP1α systems were also tested at an alternative genomic context on Chr1p36. This target site contains 36 copies of a tandem repeat region, with closer proximity to flanking genes. The transcriptional start site of the nearest gene, DVL1, lies 0.9 kb away from the tandem repeat site. In this site, RT-qPCR after 5 days ABA treatment showed KRAB was able to strongly repress DVL1 expression but exerted no notable repression of distal genes INTS11 and CPTP. On the other hand, HP1α completely failed to repress any of the three genes.
Gene expression at the Chr1p36 target site was affected when both KRAB and HP1α were recruited to the same site.
Likewise, when the same experiment was performed for the Chr3q29 test site, the distal gene repression effect observed for HP1α alone was diluted out when KRAB was co-recruited the tandem repeat site.
To better understand why H3K9me3 enrichment was not observed with either synthetic HP1α or KRAB-based heterochromatin, the effector in the inducible system was replaced with SUV39H1, a human histone methyltransferase that enzymatically deposits H3K9me3 marks. If the locus was amenable to H3K9me3 and confocal microscopy had sufficient resolving power, enrichment of H3K9me3 would be expected to be observed when SUV39H1 is recruited to the Chr3q29 site.
Additional histone methyltransferase variants were also tested. Unlike the full length SUV39H1, the catalytic domain variant of SUV3H1, in which the first 76 amino acids of the protein (containing the HP1-interacting domain and the chromodomain) was removed and failed to deposit H3K9me3 marks at the Chr3q29 target site.
A second human histone methyltransferase, G9a was also tested. While G9a directly catalyzes H3K9me1 and H3K9me2 deposition, its presence at a locus is associated with increased H3K9me3, possibly via indirect mechanisms such as generation of H3K9me2 substrates to facilitate further modification to H3K9me3 or through complex formation with SUV39H1. No enrichment of H3K9me3 was observed when full-length G9a was fused to the inducible system and recruited to Chr3q29.
Lastly, catalytic domain of G9a alone was tested, in which the first 829 amino acids were deleted. This variant has previously been shown in vitro to retain enzymatic activity. However, removal of the first 829 amino acids of G9a caused the fusion protein to localize to the cytoplasm instead of nucleus, likely due to removal of the native nuclear localization sequence within G9a.
Despite enriching for H3K9me3 marks at the Chr3q29 target locus, recruitment of the full length SUV39H1 protein did not visibly enrich for endogenous HP1α at the target locus as assayed by immunostaining.
The present application is a continuation of International Application No. PCT/US2019/055976, filed Oct. 11, 2019, which claims priority to and benefit from U.S. Provisional Application No. 62/744,504, filed Oct. 11, 2018, the entire contents of which are herein incorporated by reference.
| Number | Date | Country | |
|---|---|---|---|
| 62744504 | Oct 2018 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | PCT/US2019/055976 | Oct 2019 | US |
| Child | 17222851 | US |
