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 • 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-11421250-B2
Publication Date
2022-08-23
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 systems, methods, and compositions for targeting nucleic acids, particularly 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. The Class 2 type VI single-component CRISPR-Cas effector C2c2 is characterized as an RNA-guided RNase that provides robust interference against RNA phage infection and can be programmed to cleave single-stranded RNA (ssRNA) targets carrying protospacers flanked by a 3' H (non-G) PAM.
The C2c2 effector protein forms a complex with a single crRNA and can be reprogrammed to deplete specific mRNAs in vivo. It has two conserved HEPN domains whose catalytic residues mediate RNA cleavage. Mutation of these residues creates a catalytically inactive programmable RNA-binding protein (dC2c2), analogous to dCas9. LshC2c2 can carry out non-specific RNase activity once primed with the cognate target RNA, broadening understanding of CRISPR-Cas systems and enabling development of RNA-targeting tools.
The problem being solved is the need for new genome and transcriptome engineering technologies that employ novel strategies and molecular mechanisms, which are affordable, easy to set up, scalable, and amenable to targeting multiple positions within eukaryotic genome and transcriptome. Existing techniques like designer zinc fingers, TALEs, or homing meganucleases have limitations, and there is a pressing need for alternative robust systems for targeting nucleic acids.
Claims Coverage
The independent claims cover methods and compositions involving a Type VI Cas polypeptide with two HEPN domains and one or more nucleic acid components forming a complex that binds and modifies a target locus of interest.
Method of modifying target locus using Type VI Cas polypeptide with two HEPN domains
A method comprising delivering to a target locus a non-naturally occurring or engineered composition including a Type VI Cas polypeptide comprising two HEPN domains and one or more nucleic acid components, where the Cas polypeptide forms a complex with the nucleic acid components directing sequence specific binding and modification at the target locus.
Use of guide RNA lacking tracr sequence for targeting RNA
The method employs a guide RNA capable of sequence specific binding to an RNA target without requiring a tracr sequence, enabling RNA targeting and cleavage by the Cas polypeptide.
Targeting disease-associated or disease-specific RNA sequences
Selection of target sequences that are disease associated or disease specific RNAs for modification by the Cas polypeptide complex.
Polynucleotide based delivery using vectors with regulatory elements
Provision of Cas polypeptide and nucleic acid components via polynucleotide molecules operably linked to regulatory elements, optionally including promoters or inducible promoters, packaged in one or more viral vectors including retroviral, lentiviral, adenoviral, adeno-associated or herpes simplex viral vectors.
Delivery via various vehicles including liposomes and viral vectors
Non-naturally occurring or engineered compositions delivered via liposomes, nanoparticles, exosomes, microvesicles, gene-guns, or viral vectors, enabling diverse delivery methods for genome or transcriptome modification.
Modification comprises nucleotide strand break and RNA targeting
Modification includes introduction of nucleotide strand breaks and targeting of RNA molecules, where target loci comprise at least 83% complementary sequences to nucleic acid components guiding the Cas polypeptide.
Use of C2c2 polypeptide orthologs from various bacterial genera
Specifically, C2c2 polypeptides from genera including Corynebacter, Sutterella, Legionella, Treponema, Filifactor, Eubacterium, Streptococcus, Lactobacillus, Mycoplasma, Bacteroides, Flaviivola, Flavobacterium, Sphaerochaeta, Azospirillum, Gluconacetobacter, Neisseria, Roseburia, Porvibaculum, Staphylococcus, Nitratifactor, Campylobacter, Leptotrichia, Rhodobacter, Lachnospiraceae, Carnobacterium, and Paludibacter and their orthologs with HEPN domains are used for RNA targeting applications.
The claims cover methods and compositions utilizing engineered Type VI CRISPR-Cas systems, especially C2c2 proteins with two HEPN domains, complexed with nucleic acid components like guide RNA to bind and modify target RNA loci, delivered via various vectors and vehicles, with modifications that include catalytic site mutations and codon optimization for eukaryotic expression.
Stated Advantages
Provides alternative and robust systems and techniques for targeting nucleic acids with broad applications.
Enables programmable RNA-guided RNase activity for targeted RNA cleavage and transcriptome perturbation.
Offers system amenable to multiplexed targeting and scalable genome and transcriptome editing.
Facilitates development of RNA-targeting tools with distinct modes of action compared to known RNases.
Permits design of programmable RNA-binding proteins for functions such as RNA visualization and transcript capture.
Documented Applications
Targeted RNA cleavage for gene expression modulation and knockdown in prokaryotic and eukaryotic cells.
Interference against RNA phage infection using programmable RNA-guided RNase activity.
Development of RNA-targeting tools for transcriptome perturbation, including multiplexed targeting.
Therapeutic applications involving gene or transcriptome editing or gene therapy.
Use in screening methods, disease models, including modulation of cellular status such as apoptosis or dormancy.
RNA detection and quantification within cells using fluorescently tagged RNA-targeting complexes.
Site-specific transcriptome editing and multiplexed genome engineering.
Genome editing and functional screening in human and non-human cells and organisms in vitro, in vivo and ex vivo.
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