CRISPR-Cas9 Genome Editing via Programmable Guide RNA

The patent applies universality by using a single Cas9 enzyme that can be programmed to target multiple different genomic locations through exchange of guide RNA molecules. This single enzyme system replaces the need for multiple customized protein systems (zinc fingers, TALEs, homing meganucleases), each requiring separate development for different target sites. The guide RNA can be easily redesigned to direct Cas9 to any desired location in the genome, making the system universally applicable across multiple targets without increasing complexity.

The patent employs copying by using RNA molecules as programmable guides that can be easily replicated and modified. Instead of creating custom proteins for each target, the system uses short guide RNA sequences that can be synthesized and exchanged to redirect the same Cas9 enzyme to different genomic locations. This copying approach allows rapid adaptation to new target sites without requiring complex protein engineering.

The patent substitutes a complex mechanical/protein-based targeting system with a simpler RNA-based guidance system. Instead of relying on customized proteins with complex three-dimensional structures that must be engineered for each target, the system uses short RNA sequences that base-pair with target DNA through simple complementary binding. This substitution maintains binding specificity while dramatically simplifying the setup process, as RNA sequences can be designed and synthesized much more easily than custom proteins.

The patent employs disposable guide RNA molecules that can be easily synthesized and replaced to target different genomic locations. Rather than investing in expensive, complex customized protein systems that require extensive engineering and validation for each new target, the system uses inexpensive, easily manufactured guide RNAs that can be quickly exchanged to redirect the same Cas9 enzyme to new targets, making the process both more affordable and easier to set up.

The patent applies segmentation by dividing the genome targeting task into modular components: a constant Cas9 enzyme core and variable guide RNA segments. Each guide RNA can be independently designed to target a specific genomic location, allowing systematic and parallel targeting of multiple positions. This segmentation enables high-throughput approaches where multiple guide RNAs can be used simultaneously or sequentially to catalog genetic factors across the genome, dramatically improving productivity compared to traditional methods that require separate protein engineering for each target.

The patent employs preliminary action by pre-establishing the Cas9 enzyme system with its DNA-cutting capability, and then simply exchanging guide RNA molecules to target different locations. The heavy lifting of creating a functional genome editing system is done once with Cas9, and subsequent targeting of multiple positions requires only the exchange of lightweight guide RNA sequences. This preliminary setup eliminates the need to re-engineer complex protein systems for each new target, enabling rapid and scalable cataloging of genetic factors across multiple genome positions with minimal additional time investment.