CRISPR enzymes and systems
Inventors
Koonin, Eugene • Zhang, Feng • Wolf, Yuri I. • Shmakov, Sergey • Severinov, Konstantin • Semenova, Ekaterina • Minakhin, Leonid • Makarova, Kira S. • Konermann, Silvana • Joung, Julia • Gootenberg, Jonathan S. • Abudayyeh, Omar O.
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-11180751-B2
Publication Date
2021-11-23
Expiration Date
2036-06-17
Interested in licensing this patent?
MTEC can help explore whether this patent might be available for licensing for your application.
Abstract
The invention provides for systems, methods, and compositions for targeting nucleic acids. In particular, the invention provides non-naturally occurring or engineered DNA or RNA-targeting systems comprising a novel DNA or RNA-targeting CRISPR effector protein and at least one targeting nucleic acid component like a guide RNA.
Core Innovation
The invention provides non-naturally occurring or engineered DNA or RNA-targeting CRISPR effector proteins and systems. These include Type V and VI CRISPR-Cas loci effector proteins, particularly C2c1 or C2c3 proteins as Type V effectors and C2c2 as a Type VI effector. The DNA or RNA-targeting systems are composed of the effector protein and nucleic acid components, such as guide RNAs, that form a complex which binds specifically to target nucleic acid sequences and induces modifications such as strand breaks at the target locus.
The problem being addressed is the need for alternative and robust genome engineering technologies that employ novel molecular mechanisms. Current genome-editing tools like zinc fingers or TALEs have limitations in affordability, scalability, and amenability to target multiple genomic sites within eukaryotes. Additionally, CRISPR-Cas systems show extreme diversity, necessitating the discovery and characterization of novel effector proteins to enhance genome engineering tools.
The invention details the identification and characterization of novel Class 2 CRISPR-Cas systems including C2c1, C2c3, and C2c2 proteins. These proteins possess distinct domain architectures, such as the presence of RuvC nuclease domains but the absence of HNH domains in Type V effectors, and two HEPN domains in Type VI effectors suggesting RNA targeting. The invention also provides methods for delivering and using these effector protein-nucleic acid complexes to modify genetic loci of interest in vitro and in vivo across a wide range of cell types, including prokaryotic and eukaryotic cells.
Claims Coverage
The independent claims primarily cover engineered Type V CRISPR-Cas compositions and methods including effector proteins with one or more catalytic RuvC motifs but lacking HNH motifs, and their use with nucleic acid components featuring guide and tracrRNA sequences for specific targeting and modification of nucleic acid sequences.
Engineered Type V Cas polypeptides with RuvC nuclease motifs and guide RNA complexes
Compositions comprising Type V Cas polypeptides that have one or more catalytic motifs of a RuvC nuclease domain, lack an HNH domain, and bind nucleic acid components including a heterologous guide sequence and optionally a tracrRNA sequence, forming complexes capable of site-specific targeting of nucleic acids.
Effector polypeptide domain architecture and species origin
Type V Cas polypeptides that comprise arginine rich clusters that do not bind zinc or comprise zinc finger regions with at least one Zn-binding cysteine. Particularly, the C2c1 or C2c3 polypeptides; C2c1 variants derived from specific bacterial genera and species including Alicyclobacillus and Bacillus species.
Reduced nuclease activity and linkage to functional domains
Type V Cas polypeptides may comprise mutations in catalytically active domains resulting in reduced or abolished nuclease activity compared to wild type versions, and may be linked to heterologous functional domains including nuclear localization signals and transcriptional activation or repression domains.
Vectors and expression systems encoding engineered Type V compositions
Vectors encoding said Type V Cas polypeptides and nucleic acid components, optionally comprising regulatory elements including inducible promoters, and being codon optimized for expression in eukaryotic cells are covered.
Methods of modifying target nucleic acid sequences using engineered Type V Cas compositions
Methods including delivering engineered Type V Cas polypeptide complexes with nucleic acid components to target loci comprising DNA to induce modifications such as strand breaks, with cleavage typically distal to target PAM sequences.
The claims cover engineered Type V CRISPR-Cas compositions featuring Cas polypeptides with RuvC nuclease motifs but lacking HNH domains, core DNA-targeting systems including specific nucleic acid components like guide and tracrRNAs, and methods utilizing these complexes for site-specific nucleic acid modifications, with supporting embodiments encompassing species origin, domain architecture, mutagenesis reducing nuclease activity, linkage to diverse functional domains, and codon-optimized vectors for eukaryotic expression.
Stated Advantages
The invention provides novel genome engineering technologies employing alternative and robust CRISPR effector proteins expanding the repertoire beyond existing genome-editing tools.
The CRISPR systems and compositions enable high specificity for target nucleic acids with minimized or abolished off-target effects.
The identified effector proteins and nuclease complexes are amenable to application in a broad range of cell types, including prokaryotic and eukaryotic cells, with potential for multiplexed targeting.
The invention offers scalable, affordable, and versatile genome-editing tools suitable for in vitro and in vivo applications, including therapeutic genome editing.
Documented Applications
Genome-editing in prokaryotic and eukaryotic cells to modify DNA or RNA sequences at target loci, including inducing strand breaks, deletions, insertions, and gene regulation.
Therapeutic gene editing including correction of mutations associated with genetic disorders such as Sickle Cell Anemia, Beta Thalassemia, Hemophilia B, SCID-X1, adrenoleukodystrophy (ALD), Fanconi anemia, cystic fibrosis, Huntington's disease, Alzheimer's disease, retinal disorders, and viral infections including HBV and HIV.
Agricultural applications including generating plants with modified traits such as pest or disease resistance, herbicide tolerance, drought tolerance, nutritional enhancement, and male sterility.
Development of animal models and cell lines with targeted genomic modifications for research, drug screening, functional genomics, and disease modeling.
RNA-targeted applications including mRNA targeting in type VI systems, potentially for antiviral and RNA expression modulation treatments.
Adoptive immune cell therapies involving targeted genetic modification of T cells and hematopoietic stem cells for cancer immunotherapy and treatment of immunodeficiency disorders.
Applications in multiplexed genome engineering, saturation mutagenesis, and large-scale genetic screening.
Interested in licensing this patent?