Cas Endonuclease Guide RNA Library for Genome Editing Specificity
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Solution Overview
Problem
Current genome engineering technologies, such as designer zinc finger nucleases and TALENs, face challenges with low specificity and high costs due to the need for redesigning nucleases for each target site, making them time-consuming and expensive, while also being limited in their ability to target multiple positions within an organism's genome effectively.
Innovation Solution
The development of methods for rapid characterization and utilization of Cas endonuclease systems, including the use of randomized Protospacer-Adjacent-Motif (PAM) sequences and guide RNA elements, to create a plasmid DNA library that can be used to identify and modify target sites within a genome, allowing for precise and scalable genome editing.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If designer zinc finger nucleases or TALENs are used for targeted genome modification, then site-specific integration can be achieved, but the systems have low specificity and require redesign for each target site, making them costly and time-consuming
Solution Approach 1:
The CRISPR-Cas9 system employs a universal nuclease (Cas9) that can target multiple genomic locations by simply changing the guide RNA sequence, eliminating the need to redesign the nuclease itself for each target site. This multi-functional approach allows a single Cas9 protein to perform genome editing at numerous different locations throughout the genome.
Solution Approach 2:
The invention changes the targeting parameter from the nuclease sequence (which would require complete redesign) to the guide RNA sequence (which can be quickly synthesized). By modifying only the 20-nucleotide guide sequence while keeping the Cas9 protein constant, the system achieves rapid adaptation to new target sites.
2Measurement precision
If designer zinc finger nucleases or TALENs are used for targeted genome modification, then site-specific integration can be achieved, but these systems are costly and time-consuming to prepare
Solution Approach 1:
The universal Cas9 nuclease can be produced once and reused for multiple target sites, significantly reducing the cost of preparation compared to designing and producing custom nucleases for each target. The system separates the expensive, complex nuclease component from the inexpensive, simple guide RNA component.
Solution Approach 2:
The guide RNA is designed as a disposable, inexpensive component that can be quickly synthesized and discarded after use. Each new target site requires only a new guide RNA synthesis, not a new nuclease production, making the system cost-effective for multiple applications.
3Measurement precision
If conventional genome engineering technologies are used, then targeted modifications can be made, but they are limited in their ability to target multiple positions within the genome effectively
Solution Approach 1:
The Cas9 nuclease serves as a universal platform that can target multiple positions throughout the genome by accepting different guide RNAs. This allows simultaneous or sequential editing of multiple genes or genomic locations using the same nuclease molecule.
Solution Approach 2:
The targeting function is segmented into two independent components: the Cas9 nuclease (which provides the cutting function) and the guide RNA (which provides the targeting function). This segmentation allows the targeting component to be easily changed to reach multiple positions while keeping the nuclease component constant.
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
This approach enables precise and efficient genome editing by directly examining PAM specificity and guide RNA requirements, facilitating targeted modifications across multiple genomic locations with improved specificity and reduced costs, as demonstrated by the successful application in various organisms including plants.
Implementation Method 1
a first single stranded oligonucleotide comprising a target sequence that can be recognized by a guide RNA/Cas endonuclease complex
Implementation Method 2
a guide RNA and a Cas endonuclease protein, wherein said guide RNA and Cas endonuclease protein can form a complex that is capable of introducing a double strand break into the said target sequence
Implementation Method 3
producing a ligation product by ligating the oligoduplex from (c) with a linearized plasmid
Data Source
AI summary
Compositions and methods are provided for rapid characterization of Cas endonuclease systems and the elements comprising such systems, including, but not limiting to, rapid characterization of PAM sequences, guide RNA elements and Cas endonucleases. Type II Cas9 endonuclease systems originating from Brevibacillus laterosporus, Lactobacillus reuteri MIc3, Lactobacillus rossiae DSM 15814, Pediococcus pentosaceus SL4, Lactobacillus nodensis JCM 14932, Sulfurospirillum sp. SCADC, Bifidobacterium thermophilum DSM 20210, Loktanella vestfoldensis, Sphingomonas sanxanigenens NX02, Epilithonimonas tenax DSM 16811, Sporocytophaga myxococcoides are described herein. The present disclosure also describes methods for genome modification of a target sequence in the genome of a cell, for gene editing, and for inserting a polynucleotide of interest into the genome of a cell.