The technology integrates target RNA into DNA genomes by adding short DNA tails to RNA. Direct RNA integration into CRISPR arrays eliminates the use of harsh transfection methods that fragment DNA sequences.
Background
The development of CRISPR-Cas systems, which are widely used for precise genetic modifications, has advanced the field of genome engineering. These systems, evolved by bacterial and archaeal organisms, capture and integrate snippets of nucleic acids (spacers) from invaders (viruses, parasites) into CRISPR arrays, which are then used to guide nucleases (Cas) to destroy the invaders. This system is now being harnessed to edit genes in living organisms with therapeutic ramifications.
While CRISPR-Cas systems are adept at integrating double-stranded DNA (dsDNA) spacers, the mechanisms for integrating RNA-derived spacers are not understood. The challenge is that RNA is inherently less stable than DNA and lacks the necessary 3' deoxynucleotide (dN) tails required for integration by the Cas1/Cas2 complex. Existing approaches have shown that some CRISPR systems can acquire spacers from RNA, but the precise biochemical pathways and the role of associated proteins, such as reverse transcriptases (RTs), in this process are not well-understood. Understanding these mechanisms is crucial for enhancing the versatility and applicability of CRISPR systems, thus advancing genome engineering technologies and developing new biotechnological applications.
Technology description
This patent pending technology uses the RT-Cas1 fusion protein from Marinomonas mediterranea and combines reverse transcriptase (RT) and Cas1 (both components of CRISPR systems), to facilitate the site-specific integration of RNA into DNA genomes. This fusion protein adds short 3'-DNA tails to RNA protospacers, enabling these RNA sequences to be directly integrated into CRISPR arrays as either 3'-dN-RNA/cDNA duplexes or 3'-dN-RNAs.
The reverse transcription of RNA protospacers by this fusion protein occurs through multiple mechanisms: de novo initiation, protein priming with any dNTP, and the use of short exogenous or synthesized DNA oligomer primers. These mechanisms allow the synthesis of cDNAs from various RNA sequences without fixed sequence requirements. The integration of 3'-dN-RNAs or single-stranded DNAs is preferred over duplexes at higher protospacer concentrations, suggesting a potential relevance to spacer acquisition from abundant pathogen RNAs or single-stranded DNA fragments generated by phage-defense nucleases.
Benefits
- Enables site-specific and direct integration of RNA into DNA genomes
- Reveals novel biochemical activities for integrating RNA into DNA
- Potentially relevant for spacer acquisition from abundant pathogen RNAs
- Can synthesize cDNAs from diverse RNA sequences without fixed sequence requirements
- Favored integration of single-stranded DNAs at higher protospacer concentrations
- Significant biotechnological applications
Commercial applications
- Genome engineering
- Prime editing
- Site-specific DNA integration
- Spacer acquisition
- RNA-to-DNA transcription
