CRISPR enzymes and systems
Inventors
Severinov, Konstantin • Zhang, Feng • Wolf, Yuri I. • Shmakov, Sergey • Semenova, Ekaterina • Minakhin, Leonid • Makarova, Kira S. • Koonin, Eugene • Konermann, Silvana • Joung, Julia • Gootenberg, Jonathan S. • Abudayyeh, Omar O. • Lander, Eric S.
Assignees
Skolkovo Institute of Science and Technology • United States, AS REPRESENTED BY SECRETARY Department Health & Human Services • Rutgers State University of New Jersey • Massachusetts Institute of Technology • Broad Institute Inc • Harvard University • US Department of Health and Human Services
Publication Number
US-11060115-B2
Publication Date
2021-07-13
Expiration Date
2036-06-17
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Abstract
The invention provides for systems, methods, and compositions for targeting nucleic acids. In particular, the invention provides non-naturally occurring or engineered RNA-targeting systems comprising a novel RNA-targeting CRISPR effector protein and at least one targeting nucleic acid component like a guide RNA.
Core Innovation
The invention provides for systems, methods, and compositions for targeting nucleic acids, specifically non-naturally occurring or engineered RNA-targeting systems comprising a novel RNA-targeting CRISPR effector protein, C2c2, and at least one targeting nucleic acid component like a guide RNA. C2c2 is characterized as an RNA-guided ribonuclease (RNase) that can be programmed to cleave single-stranded RNA (ssRNA) targets carrying protospacers flanked by a 3' H (non-G) PAM. It contains two conserved HEPN domains whose catalytic residues mediate cleavage; mutation of these residues creates a catalytically inactive RNA-binding protein analogous to dCas9.
The system addresses a need for alternative and robust nucleic acid targeting technologies that are affordable, scalable, amenable to multiple targeting, and employ novel molecular mechanisms for genome and transcriptome engineering. Unlike known DNA-targeting CRISPR-Cas systems, C2c2 utilizes RNA-guided RNA interference, adding to the repertoire of genomic and epigenomic targeting tools.
C2c2's unique features include its single-component effector status, RNA-guided RNase activity dependent on HEPN domains, and capacity for both specific target RNA cleavage and non-specific RNase activity upon priming with cognate target RNA. The invention also elaborates on compositions comprising C2c2 and modified nucleic acid components such as guide RNAs with stem loop secondary structures important for cleavage, and potential modifications like aptamers for protein binding. Methods of delivering C2c2 with guide RNAs to various cells including prokaryotic, eukaryotic, mammalian, and plant cells for modification of target loci and gene expression are detailed.
Claims Coverage
The claims focus on engineered compositions and methods involving a C2c2 effector protein together with nucleic acid components, primarily guide RNAs, to direct sequence-specific RNA targeting and binding in eukaryotic cells.
Engineered composition for eukaryotic RNA targeting
Compositions comprising a Cas polypeptide having two HEPN domains with at least 95% sequence identity to specified sequences, with nuclear localization sequences and codon-optimized polynucleotides, and one or more nucleic acid components capable of forming a CRISPR-Cas complex that binds specifically to an RNA target sequence.
Engineered catalytically inactive C2c2 composition
Compositions with Cas polypeptides comprising mutations (R597A, H602A, R1278A, H1283A) abolishing nuclease activity, together with nucleic acid components forming CRISPR complexes to direct sequence-specific RNA binding without cleavage.
Use of C2c2 effector proteins from specified bacterial genera
Cas polypeptides sourced from defined bacterial genera are incorporated into compositions for RNA-targeting applications.
Compositions with dual direct repeat nucleic acid components
Utilization of nucleic acid components comprising dual direct repeat sequences within the CRISPR complex.
Expression and delivery regulatory elements in polynucleotide compositions
Polynucleotides encoding C2c2 and nucleic acid components operably linked to regulatory elements (promoter or inducible promoter), optionally packaged in one or more vectors including viral vectors such as retroviral, lentiviral, adenoviral, AAV or herpes simplex viral vectors, comprised in delivery systems or vehicles like liposomes or nanoparticles.
Delivery systems and carriers for engineered compositions
The compositions are delivered via delivery vehicles including liposomes, particles, exosomes, microvesicles, gene-gun, or viral vectors, with specified embodiments including viral vector selection for eukaryotic cell expression.
Functional domains attached to inactive C2c2 for gene regulation
Compositions where catalytically inactive C2c2 effectors are fused to or associated with functional domains that modulate transcription or translation through epigenetic or regulatory signals upon target binding.
The claims cover engineered compositions of C2c2 effector proteins, including catalytically inactive mutants, combined with guide RNAs capable of forming CRISPR complexes that target RNA sequences with high specificity. The claims encompass delivery methods, codon optimization, nuclear targeting, and fusion with functional domains to modulate gene expression at RNA level in eukaryotic cells. The compositions originate from defined bacterial genera and utilize modified nucleic acid components including dual direct repeats, and are deliverable via a variety of vectors and delivery vehicles for therapeutic and research applications.
Stated Advantages
Provides non-naturally occurring, robust nucleic acid-targeting systems with novel mechanisms distinct from existing genome editing tools.
Enables RNA-specific targeting and cleavage through a single component, overcoming limitations of DNA-targeting CRISPR systems.
Allows programmability to cleave specific ssRNA targets with high specificity and potential for multiplexing.
Permits generation of catalytically inactive RNA-binding proteins for gene regulation without cleavage.
Facilitates delivery and expression in a wide variety of target cells including eukaryotic, mammalian, prokaryotic and plant cells.
The systems have a distinctive RNA cleavage mechanism via HEPN domains expanding genomic and transcriptomic manipulation capabilities.
Documented Applications
Therapeutic gene or transcriptome editing, gene knockout, knockdown or activation in eukaryotic and prokaryotic cells including mammalian and plant cells.
Development of disease models including genetic and epigenetic conditions for in vitro and in vivo studies.
Use in genome-wide or transcriptome-wide screening approaches to identify gene functions and regulatory elements.
Targeted degradation or modulation of RNA transcripts including mRNA, non-coding RNA, miRNA, viral RNA, and RNA involved in disease conditions such as myotonic dystrophy.
Multiplex RNA targeting for genetic or epigenetic manipulations, RNA interference and RNA detection.
Plant breeding and engineering for traits such as pest resistance, abiotic stress tolerance, biofuel production, and reducing allergenic properties.
Antiviral applications against RNA viruses in prokaryotes and eukaryotes, including targeting phage RNA.
Molecular biology applications including RNA visualization, RNA isolation, fluorescent in situ hybridization, and synthetic biology.
Induction of cell cycle arrest, programmed cell death, growth inhibition and dormancy potentially for therapeutic interventions, such as cancer treatment.
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