RNA-GUIDED DNA NUCLEASES AND USES THEREOF

Smart overview of the Invention

The application details advanced systems, methods, and compositions for modifying target DNA sequences within eukaryotic cells. Central to this process is the use of a guide RNA that can hybridize with a target sequence and an RNA-guided DNA nuclease. The invention also includes vectors and vector systems that encode components of a CRISPR complex, along with methods for designing and using these vectors. Additionally, it provides techniques for identifying and validating novel CRISPR systems.

Background

CRISPR systems, found in the genomes of bacteria and archaea, serve as adaptive immune systems that protect against foreign nucleic acids by cleaving them in a sequence-dependent manner. These systems involve CRISPR RNAs (crRNAs) and associated proteins that form complexes to target and cleave invading DNA. The CRISPR/Cas9 complex, for instance, requires a specific DNA sequence known as a protospacer-adjacent motif (PAM) for effective cleavage. The invention leverages these natural mechanisms for precise genome editing.

Recombinant Nucleic Acids and Vectors

The invention encompasses recombinant nucleic acids with heterologous promoters linked to polynucleotides encoding CRISPR enzymes. These enzymes have specific amino acid sequences or high sequence homology with specified sequences. Vectors containing these recombinant nucleic acids are also provided, which can be used in various cell types, including prokaryotic and eukaryotic cells, for transient expression or genome integration.

Methods for DNA Modification

A method for sequence-specific DNA modification involves delivering to a cell a guide RNA specific to the target sequence and a CRISPR enzyme with high sequence homology to certain specified sequences. This method allows for precise modification of target nucleic acids in eukaryotic cells, including plant, algal, and mammalian cells. The system can also modulate transcription selectively by forming a complex with the guide RNA and CRISPR enzyme.

Identification of CRISPR Enzymes

The application describes methods for identifying CRISPR enzymes from bacterial genomes. These methods involve associating polynucleotides encoding CRISPR enzymes with specific CRISPR repeats and Cas proteins within the bacterial genome. The identification process can rely on the presence of Pfam domains and is applicable to a range of bacterial species. This approach helps in discovering new CRISPR enzymes for potential use in genome editing technologies.