CRISPR DNA and RNA targeting enzymes and systems

Inventors

Keston-Smith, Elise • Scott, David A. • Cheng, David R. • Yan, Winston X. • Hunnewell, Pratyusha • Carte, Jason

Assignees

Arbor Biotechnologies Inc

Abstract

The disclosure describes novel systems, methods, and compositions for the manipulation of nucleic acids in a targeted fashion. The disclosure describes non-naturally occurring, engineered CRISPR systems, components, and methods for targeted modification of nucleic acids such as DNA. Each system includes one or more protein components and one or more nucleic acid components that together target nucleic acids.

Core Innovation

The invention relates to an engineered, non-naturally occurring CRISPR-associated (Cas) system comprising an RNA guide or a nucleic acid encoding an RNA guide. The RNA guide comprises a direct repeat sequence, a spacer sequence capable of hybridizing to a target nucleic acid in a eukaryotic cell, and a tracrRNA. The system uses a RuvC nuclease domain-containing CRISPR-Cas effector protein, or a nucleic acid encoding the same, that binds to the RNA guide and targets the target nucleic acid complementary to the spacer sequence.

The RuvC nuclease domain-containing effector protein is defined by sequence identity, including an amino acid sequence at least 80% identical to SEQ ID NO: 6. The system provides scope for nucleic-acid targeting and editing of DNA/RNA, including collateral nuclease activity (RNase and DNase), collateral-free target recognition, and PAM/PFS-independent targeting within the described system. It also includes tunable nuclease activity via RuvC-domain motifs and nuclease-inactivating substitutions.

The invention further outlines detection paradigms using labeled reporter/detector nucleic acids with a signal readout. Broad applications include gene regulation, base editing and splicing modulation via fusions, inducible and split/self-activating/self-inactivating designs, and therapeutic and diagnostic uses.

Claims Coverage

The independent claims cover an engineered, non-naturally occurring CRISPR-Cas system built from an RNA guide architecture and a RuvC nuclease domain-containing effector protein defined by sequence identity. The core inventive coverage is centered on guide-directed, eukaryotic-cell targeting of nucleic acids using a Cas effector with sequence identity to SEQ ID NO: 6, with dependent claims further specifying guide/tracrRNA sequences, tighter identity thresholds, and RuvC domain modifications or inactivation.

Across the independent claim, the inventive subject matter is the combination of an RNA guide architecture with a RuvC nuclease domain-containing effector protein that binds the guide and targets the complementary nucleic acid, where the effector is defined by an amino-acid sequence identity threshold relative to SEQ ID NO: 6. Dependent claims further constrain the system by specifying tracrRNA sequence, tightening effector identity thresholds, and adding RuvC-domain substitutions or catalytically inactivating substitutions.

Stated Advantages

Documented Applications