Extended Guide RNA for Base Editing at PAM Positions 18–30
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Solution Overview
Problem
Existing gene-editing technologies for base editing have limitations in the range and efficiency of DNA base changes, particularly outside the conventional 13 to 17 positions from the protospacer adjacent motif (PAM), necessitating an extension of the operational range for more precise and effective editing.
Innovation Solution
The use of extended guide RNA (sgRNA) with additional guanines and nucleotides at the 5' end to expand the range of base editing, incorporating 1 to 10 nucleotides and 1 to 3 guanines, enhancing the efficiency and range of base changes to positions 18 to 30 from the PAM.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Length of moving object
If conventional guide RNA (19-20 nucleotides) is used for base editing, then the base editor operates within positions 13 to 17 from PAM, but the editing range is limited and efficiency outside this range is very low
Solution Approach 1:
The patent applies parameter changes by modifying the guide RNA length from the conventional 19-20 nucleotides to extended lengths of 21-30 nucleotides. This parameter change enables the base editor to operate at positions 18 to 30 from PAM, significantly expanding the editable range while maintaining the fundamental sgRNA structure and function
Solution Approach 2:
The patent extends the guide RNA into an additional dimensional space by adding 1 to 10 nucleotides at the 5' end beyond the conventional length. This dimensional extension allows the base editor to access and edit DNA bases that were previously unreachable, effectively adding a new operational dimension to the base editing system
2Adaptability or versatility
If guide RNA length is extended to positions 18 to 30 from PAM, then the base-editing range is expanded, but the efficiency and precision may be compromised
Solution Approach 1:
The patent applies local quality by adding specific guanines (1 to 3 G residues) at the 5' end of the extended guide RNA. This localized modification enhances the stability and binding affinity of the extended sgRNA, ensuring that even at extended positions 18 to 30 from PAM, the base editor maintains high precision and efficiency through optimized local interactions
3Area of stationary object
If conventional base editing is used, then the operational range is limited to positions 13 to 17 from PAM, but extending the range reduces editing efficiency
Solution Approach 1:
The patent applies preliminary action by pre-extending the guide RNA with 1 to 10 additional nucleotides at the 5' end before the base editing process. This preliminary structural preparation ensures that when the base editor binds to the target DNA, the extended sgRNA is already positioned to access positions 18 to 30 from PAM, maintaining high efficiency across the expanded editing window without requiring additional optimization steps
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
The extended guide RNA significantly increases the efficiency and range of base editing, achieving up to 60 times higher efficiency in certain regions, allowing for more precise genetic modifications in eukaryotic cells and organisms.
Implementation Method 1
an extended guide RNA for base editing hybridizable with a target sequence
Implementation Method 2
changes cytosine (C) to uracil (U) or adenine (A) to hypoxanthine (I) using a deaminase capable of acting on single-stranded DNA
Data Source
AI summary
The present disclosure relates to: an extended guide RNA and a composition for base editing, comprising the same; and a method for base editing and a method for producing genetically modified animals or plants, both methods using the composition for base editing.