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-11773412-B2
Publication Date
2023-10-03
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 addresses the pressing need for alternative robust nucleic acid targeting systems and techniques that are affordable, scalable, and amenable to targeting multiple genomic and transcriptomic sites with minimized off-target effects. It provides for comprehensive computational prediction, identification, and engineering of novel Class 2 CRISPR-Cas systems derived from transposons, highlighting the diversity of loci architecture and domain organization in such systems, expanding the genome engineering toolkit with RNA-targeting capabilities.
Claims Coverage
The claims cover non-naturally occurring or engineered compositions and vector systems based on Type VI CRISPR-Cas loci effector proteins, particularly C2c2, and their nucleic acid components, which include guide RNAs capable of forming CRISPR complexes for RNA-targeting with sequence specificity in eukaryotic cells.
RNA-targeting CRISPR-Cas complex of a Type VI Cas polypeptide
The invention provides a composition comprising a Type VI Cas polypeptide with two HEPN domains and one or more nucleic acid components forming a RNA-targeting CRISPR-Cas complex directing sequence-specific binding to a target polynucleotide in eukaryotic cells.
Effector protein C2c2 from diverse bacterial genera
The composition includes C2c2 polypeptides derived from diverse bacterial genera such as Corynebacter, Leptotrichia, and others, or orthologues possessing HEPN domains with conserved catalytic RxxxxH motifs and high sequence identity to known C2c2 sequences.
Codon optimized and mutated effector proteins with functional domains
The C2c2 polypeptide may be codon optimized for eukaryotic expression and can be associated with functional domains like epigenetic modifiers or transcriptional regulators; mutations in HEPN domains (e.g., R597A, H602A, R1278A, H1283A) may modulate nuclease activity.
Versatile nucleic acid components and delivery systems
Nucleic acid components comprise dual direct repeat sequences, lack tracrRNA, and can be delivered via polynucleotide molecules with regulatory elements in vectors including viral vectors, and formulated with liposomes, nanoparticles, and exosomes.
The claims encompass engineered Type VI CRISPR-Cas systems, particularly involving C2c2 polypeptides with two HEPN domains and their guide RNAs, codon optimized for eukaryotic expression, with specified mutations and functional domains, delivered via vectors or particles, enabling sequence-specific RNA targeting and modulation in eukaryotic cells.
Stated Advantages
Provides RNA-targeting CRISPR effector protein systems capable of programmable, sequence-specific RNA cleavage and RNA-binding with potential applications as tools for RNA detection, gene regulation, and transcriptome editing.
Offers affordable, scalable, and robust genome and transcriptome engineering technologies amenable to multiplexed targeting with minimized off-target effects.
Enables novel genome engineering applications through expansion of CRISPR-Cas systems diversity, particularly the inclusion of Type VI RNA-targeting systems.
Documented Applications
Therapeutic editing or modulation of gene expression by targeting RNA transcripts, including allele-specific or mutation-specific knockdown for diseases such as myotonic dystrophy and Huntington's disease.
RNA detection and quantification in cells, including visualization of RNA localization using fluorescently tagged CRISPR components.
Functional genomic screens and multiplexed gene perturbations in prokaryotic and eukaryotic cells for loss- or gain-of-function studies.
Treatment of viral infections by targeting RNA viruses such as MS2 phage, and potentially RNA pathogens including Ebola and Zika viruses.
Induction of programmed cell death, cell dormancy, or cell growth suppression, for example targeting cancer cells or pathogen-infected cells.
Plant and fungal cell engineering including transcriptional activation, repression, and genome editing; improving crops and producing disease resistant or biofuel producing plants.
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