RNA-Guided Endonuclease Targeting Precision via Guide RNA Segmentation
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Solution Overview
Problem
Current genome modification technologies, such as zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs), require custom design for each new genomic target, are costly, and prone to off-target effects.
Innovation Solution
Development of RNA-guided endonucleases, specifically derived from CRISPR/Cas systems, which include a nuclear localization signal, a nuclease domain, and a guide RNA interaction domain, allowing for universal targeting of specific nucleotide sequences with reduced off-target effects.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Manufacturing precision
If zinc finger nucleases (ZFNs) or transcription activator-like effector nucleases (TALENs) are used for targeted genome modification, then specific genomic locations can be modified, but custom design is required for each new target which increases cost and time consumption
Solution Approach 1:
The system divides the targeting function into two separate components: a reusable nuclease enzyme (Cas9) and a programmable guide RNA molecule. The guide RNA can be independently designed for different targets without modifying the nuclease, allowing rapid reconfiguration for new genomic targets while maintaining precise targeting capability.
Solution Approach 2:
The Cas9 nuclease serves as a universal platform that can target multiple different genomic locations by simply changing the guide RNA sequence. This single nuclease enzyme can perform the same cutting function across numerous different targets, eliminating the need to design and construct new nucleases for each target site.
2Reliability
If zinc finger nucleases (ZFNs) or transcription activator-like effector nucleases (TALENs) are used, then genome modification can be achieved, but off-target cleavages occur due to specificity limitations
Solution Approach 1:
The system replaces the protein-protein DNA binding mechanism of ZFNs and TALENs with an RNA-DNA hybridization mechanism. The guide RNA base-pairs with the target DNA sequence through complementary base pairing, providing more precise and programmable recognition that reduces off-target binding and cleavage events.
3Manufacturing precision
If custom designed nucleases are prepared for each genomic target, then specific targeting is achieved, but the process becomes costly and time-consuming
Solution Approach 1:
Instead of creating new nuclease proteins for each target, the system uses RNA copies or transcripts that encode the target sequence information. These guide RNA molecules can be rapidly synthesized through transcription from DNA templates or even chemically synthesized, providing a fast and inexpensive way to program the nuclease for different targets.
Data Source
AI summary
The present invention provides RNA-guided endonucleases, which are engineered for expression in eukaryotic cells or embryos, and methods of using the RNA-guided endonuclease for targeted genome modification in in eukaryotic cells or embryos. Also provided are fusion proteins, wherein each fusion protein comprises a CRISPR/Cas-like protein or fragment thereof and an effector domain. The effector domain can be a cleavage domain, an epigenetic modification domain, a transcriptional activation domain, or a transcriptional repressor domain. Also provided are methods for using the fusion proteins to modify a chromosomal sequence or regulate expression of a chromosomal sequence.