RNA viruses (e.g., Coronaviruses, Flaviviridae, Filoviridae, Orthomyxoviridae, etc.) pose a global threat for human, plant, and animal health. Viral infections are often resistant to existing drugs and there is a critical need for innovative therapeutics that are responsive to the emergence of new viral variants, seasonal outbreaks, or novel viral pandemics. RNA-targeting CRISPR nucleases have the potential to be developed as effective, programmable, and versatile antiviral treatments that also have considerable value for testing gene function during drug development for the design of attenuated genotypes for vaccine development. However, many RNA viruses have evolved strategies to protect their RNA from cytosolic RNA-sensors and ribonucleases by sequestering viral RNA in replication organelles (ROs) that are composed of membranes sourced from specific organelles of the host. For example, replication organelles formed by flavi-, corona- and picornaviruses use membranes from the endoplasmic reticulum (ER) or Golgi apparatus, alphaviruses use plasma- and endo-lysosomal membranes, and nodaviruses use mitochondrial membranes.
The disclosure relates to techniques and systems for specific delivery of RNA-targeting CRISPR-Cas proteins and other effectors to cellular location. The cellular location may be subcellular locations such as viral replication organelles within infected cells of a subject. This delivery approach is generalizable to any protein or ribonucleoprotein complex.
In some aspects, the techniques described herein relate to a method of programmable delivery of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas proteins for subcellular localization, the method including: fusing one or more lipidation motifs to a CRISPR-Cas protein to generate an engineered CRISPR protein, wherein the one or more lipidation motifs are post-translationally modified to anchor the one or more lipidation motifs and the fused CRISPR-Cas protein to a target cellular location; and administering the engineered CRISPR protein to a subject.
In some aspects, the techniques described herein relate to a method, wherein the cellular location is a membrane of a subcellular organelle.
In some aspects, the techniques described herein relate to a method, wherein the CRISPR-Cas protein is a CRISPR-Cas nuclease that cleaves target nucleic acid as part of a treatment regimen for the subject.
In some aspects, the techniques described herein relate to a method, wherein the target nucleic acid is viral nucleic acid.
In some aspects, the techniques described herein relate to a method, wherein the cellular location reduces cellular toxicity of the CRISPR-Cas nuclease.
In some aspects, the techniques described herein relate to a method, wherein the one or more lipidation motifs include a CTIL motif.
In some aspects, the techniques described herein relate to a method, wherein the one or more lipidation motifs include a CVIS motif.
In some aspects, the techniques described herein relate to a method, wherein fusing the one or more lipidation motifs to the CRISPR-Cas protein includes inserting a linker to account for amino acids that affect the efficiency of the post-translational modification.
In some aspects, the techniques described herein relate to a method, wherein the linker is derived from a C-terminal end of OAS1 or ZAP-L proteins.
In some aspects, the techniques described herein relate to an engineered Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas protein, including: a CRISPR-Case protein; and one or more lipidation motifs fused to the CRISPR-Cas protein, the one or more lipidation motifs being post-translationally modified to anchor the one or more lipidation motifs and the fused CRISPR-Cas protein to a target cellular location.
In some aspects, the techniques described herein relate to the engineered CRISPR-Cas protein, further including: a linker to account for amino acids that affect the efficiency of the post-translational modification.
In some aspects, the techniques described herein relate to a method of generating an engineered Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas protein, including: generating a library of CRISPR-Cas proteins including a CRISPR-Cas protein; and contacting one or more lipidation motifs with the library CRISPR-Cas proteins to fuse the one or more of lipidation motifs to the CRISPR-Cas protein, wherein the one or more lipidation motifs are post-translationally modified to anchor the one or more lipidation motifs and the fused CRISPR-Cas protein to a target cellular location.
Features of the present disclosure may be illustrated by way of example and not limited in the following figure(s), in which like numerals indicate like elements, in which:
Disclosed herein are techniques and systems for specific delivery of RNA-targeting CRISPR-Cas proteins and other effectors to viral replication organelles within infected cells. This delivery approach is generalizable to any protein or ribonucleoprotein complex.
Use of Membrane Targeting to Increase Efficacy of CRISPR-Based Antivirals
A subset of human antiviral proteins are targeted to cellular membranes and can access viral RNAs within the ROs. Specifically, RNA-sensors of the innate immunity, OAS1 and ZAP-L, encode “lipidation” motifs at the C-terminus (−CAAX motif) which are post-translationally modified to anchor proteins in the cellular membranes. Here, this natural mechanism is re-engineered by fusing lipidation motifs to CRISPR nucleases to anchor them in membranes appropriated by RNA viruses (as illustrated in
Use of Membrane Targeting to Limit Cellular Toxicity of CRISPR Nucleases.
Type VI (i.e., Cas13) and type III (i.e., Cas7-11) CRISPR systems target RNA. To date, only Cas13 have been tested as antiviral tool in cell cultures and as a prophylactic drug in rodent models. Upon target recognition Cas13 activates a multi-turnover non-sequence-specific “collateral nuclease” activity, which can cause cell toxicity. Type III CRISPR systems rely on multisubunit CRISPR RNA (crRNA)-guided complexes that specifically cleave the complementary RNA at six nucleotide intervals. Recently described single protein Type III effector Cas7-11 cuts RNA without showing any collateral activity or cell toxicity. Targeting Cas13 to specific cellular membranes by design may enhance antiviral efficacy and deplete cytosolic levels of the protein, which may limit toxicity due to collateral damage to host RNAs. Compartmentalization of the nuclease may restrict collateral RNA damage to viral replication organelles and facilitates clearing of viral RNA. Alternatively, Cas7-11 nucleases lacking collateral activity may be used to limit nuclease activity to specific sites in viral RNAs.
Targeting Negative-Sense (−) RNA to Increase Efficacy of CRISPR-Based Antivirals.
Replication organelles are the production sites of viral RNAs that are further exported to cytoplasm for translation and packaging into new viral particles. Replication of (+) RNA viruses starts with producing a full-length negative-sense (−) RNA copy that is used as a template to replicate more viral genomes. While nascent (+) RNA copies are exported to cytoplasm, (−) RNA templates remain protected in the ROs. Destroying protected (−) RNA template rather than (+) RNA copies, or combination of both, may increase efficacy of CRISPR-based antivirals. Therefore, a combination of guide RNAs to target CRISPR nucleases fused to lipidation motifs to (−) RNA to efficiently eliminate viral RNA templates and to (+) RNA to degrade synthesized copies.
CRISPR-Csm Cleavage and RNA Ligation Provides a Platform for Flexible and Robust RNA Editing
CRISPR-Csm Cleavage and RNA Ligation Enables Programmed Deletions in RNA Virus Genome.
CRISPR-Csm Cleavage and RNA Ligation Enables Programmed Substitutions in RNA Virus Genome.
Plaque-Purification and Nanopore Sequencing of Edited Viral Clones
The term “engineered”, and similar terms may refer to a deliberate generation of a system that is otherwise non-naturally occurring. Such engineering may include introducing one or more mutations to a genetic sequence, designing a genetic sequence, combining a set of components such as proteins and detection components where such combination does not occur in nature, and/or otherwise generating a non-naturally occurring system to edit nucleic acid such as RNA.
The engineered type III CRISPR complex may include a nuclease that cleaves RNA at specific sites guided by a CRISPR RNA (crRNA) sequence, which may be programmable. The term “programmatic”, “programmable”, and similar terms may refer to modifying a CRISPR complex or components thereof. An example of programmable delivery of RNA-guided CRISPR-Cas effectors (such as CRISPR-Cas proteins) may include fusing one or more lipidation motifs to a CRISPR nuclease to anchor the CRISPR nuclease to membranes appropriated by RNA viruses.
In some instances, a programmable crRNA sequence can be designed to guide the engineered type III CRISPR complex to a specific target portion of interest in the RNA. For example, sequence specific targeting of crRNAs may be performed by designing synthetic spacer sequences. The synthetic spacer sequences may be between 20 and 60 nucleotides long that separate the repeat sequences or end with a self-cleaving ribozyme, such that the crRNA is processed into a short (20-100 nt) crRNA that is incorporated into an assembly of one or more Cas proteins, which together form a ribonucleoprotein complex that stably binds and cleaves RNAs that are complementary to the guide (spacer) sequence. The spacer sequences are designed to be complementary to a target sequence and intentionally designed to avoid complementarity to other “non-target” RNAs. In some examples, crRNA-guides are designed to include a protospacer flanking sequence (PFS) that facilitates binding, cleavage, or cyclic nucleotide synthesis. In other examples, the PFS is any sequence that is not complementary to the 5′ repeat sequence of the crRNA. The programmable crRNA sequence may therefore facilitate specific cleavage of the RNA at one or more specific sites, enabling programmatic RNA editing. One example of an engineered type III CRISPR complex that may be used is a Csm complex, which is a type III-A CRISPR complex that has RNase activity. The Csm complex may be a SthCsm, derived from S. thermophilus.
Various examples described herein will refer to treatment involving a viral RNA genome. However, any cellular location may be targeted based on the disclosures herein. Target RNA or other target may be obtained from a subject. For example, the target may be an RNA of an organism that infects a host organism. In particular, the target RNA may be an RNA genome of a virus that has infected the subject. In another example, the target RNA may be the RNA of the subject. A subject may refer to an animal, such as a mammalian species (preferably human) or avian (e.g., bird) species, or other organism, such as a plant. More specifically, a subject can be a vertebrate, e.g., a mammal such as a mouse, a primate, a simian or a human. Animals include farm animals, sport animals, and pets. A subject can be a healthy individual, an individual that has symptoms or signs or is suspected of having a disease or a predisposition to the disease, or an individual that is in need of therapy or suspected of needing therapy.
A genetic modification or mutation in the context of an engineered system may refer to an alteration, variant or polymorphism in a nucleic acid that may result in altered or disabled functionality of a corresponding protein. Such alteration, variant or polymorphism can be with respect to a reference genome, the subject or other individual. Variations include one or more single nucleotide variations (SNVs), insertions, deletions, repeats, small insertions, small deletions, small repeats, structural variant junctions, variable length tandem repeats, and/or flanking sequences, CNVs, transversions, gene fusions and other rearrangements may also be considered forms of genetic variation. A variation can be a base change, insertion, deletion, repeat, copy number variation, transversion, or a combination thereof.
A “polynucleotide”, “nucleic acid”, “nucleic acid molecule”, or “oligonucleotide” may each refer to a polymer of nucleosides (including deoxyribonucleosides, ribonucleosides, or analogs thereof) joined by inter-nucleosidic linkages. Typically, a polynucleotide comprises at least three nucleosides. Oligonucleotides often range in size from a few monomeric units, e.g. 3-4, to hundreds of monomeric units. Whenever a polynucleotide is represented by a sequence of letters, such as “AUGCCUG,” it will be understood that the nucleotides are in 5′ to 3′ order from left to right and that “A” denotes adenosine, “C” denotes cytidine, “G” denotes guanosine, and “U” denotes uracil, unless otherwise noted. The letters A, C, G, and U (or “T” denoting thymine in DNA) may be used to refer to the bases themselves, to nucleosides, or to nucleotides comprising the bases, as is standard in the art.
All patent filings, websites, other publications, sequence listings, accession numbers and the like cited above or below are incorporated by reference in their entirety for all purposes to the same extent as if each individual item were specifically and individually indicated to be so incorporated by reference.
If different versions of a sequence are associated with an accession number at different times, the version associated with the accession number at the effective filing date of this application is meant. The effective filing date means the earlier of the actual filing date or filing date of a priority application referring to the accession number if applicable. Likewise, if different versions of a publication, website or the like are published at different times, the version most recently published at the effective filing date of the application is meant unless otherwise indicated. Any feature, step, element, embodiment, or aspect of the disclosure can be used in combination with any other unless specifically indicated otherwise. Although the present disclosure has been described in some detail by way of illustration and example for purposes of clarity and understanding, it will be apparent that certain changes and modifications may be practiced within the scope of the appended claims.
This application claims the benefit of priority of U.S. Provisional Application No. 63/329,952, filed on Apr. 12, 2022, and U.S. Provisional Application No. 63/450,339, filed on Mar. 6, 2023, which are each incorporated by reference in their entireties herein for all purposes.
This invention was made with government support under contract R35GM134867 awarded by the National Institutes of Health. The government has certain rights in the invention.
Number | Date | Country | |
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63329952 | Apr 2022 | US | |
63450339 | Mar 2023 | US |