Complementary Strand CRISPR Base Editing System
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Solution Overview
Problem
Current CRISPR-associated base editors are limited in their ability to make edits on the target strand and have restricted editing windows due to their dependence on specific PAM motifs and positions, which restricts the range of edits that can be made.
Innovation Solution
Development of a CRISPR-based editing system that includes a guide ribonucleic acid (gRNA) complementary to a nucleic acid strand and a cleavage-deficient Cas nuclease fused with a deaminase, allowing for A to G and C to T modifications on the complementary strand, expanding the editing range by targeting the non-displaced strand.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If base editors are designed to edit the non-target strand, then editing efficiency is improved, but the editing window is restricted due to PAM motif dependence
Solution Approach 1:
The patent inverts the conventional base editing approach by targeting the complementary strand instead of the non-target strand. This inversion allows the deaminase to access and edit bases on the complementary strand that would otherwise be inaccessible, thereby expanding the editing window while maintaining high editing efficiency through the same molecular mechanism.
Solution Approach 2:
The patent extends base editing capability to a new dimension by enabling edits on both strands of the DNA double helix. Conventional base editors only edit the non-target strand, but this invention adds the capability to edit the complementary strand as well, effectively doubling the accessible editing space and providing greater versatility.
2Manufacturing precision
If conventional base editors are used, then C to T editing is achieved on the non-target strand, but editing on the target strand is not possible
Solution Approach 1:
The patent creates a universal base editing system that can edit both strands of DNA. By designing the base editor to target the complementary strand while maintaining the ability to edit the non-target strand, the system achieves multi-functionality, allowing researchers to choose the optimal editing approach for each specific application regardless of strand orientation or PAM motif location.
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 efficient A to G and C to T editing on the complementary strand, expanding the range of edits that can be made, as it allows for modifications on both the complementary and non-complementary strands of the target DNA, enhancing the precision and versatility of genome editing.
Implementation Method 1
the cytidine deaminase catalyzes the deamination of cytidine to uridine (C to U) in the displaced non-target strand
Implementation Method 2
After gRNA-guided recognition, the catalytically inactive variant of Cas9 (D10A and H840A) also known as dead Cas9 (dCas9), which is unable to cleave dsDNA, targets and unwinds its dsDNA target
Data Source
AI summary
The invention relates to methods for clustered regularly interspaced short palindromic repeat (CRISPR)-mediated A to G and/or C to T editing of the guide-complementary strand of a double stranded target DNA. The invention further relates to a nucleotide molecule encoding said CRISPR-mediated base editing system, to an expression vector comprising the nucleotide molecule, and to a cell comprising the expression vector. The invention further relates to an isolated Cas nuclease that allows A to G and/or C to T editing of the complementary strand of a double stranded target nucleic acid.