Catalytically Inactive RNA-Guided Endonuclease for SNP Editing
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Solution Overview
Problem
Existing methods for introducing single nucleotide polymorphisms (SNPs) into genomes, such as CRISPR-based approaches, are limited by the need for repair DNA for each target locus and are associated with cytotoxicity due to double-strand breaks.
Innovation Solution
The use of catalytically inactive RNA-guided endonucleases, such as Mad7d, tethered to a nucleobase editing domain in CRISPR-AID technology, which generates SNPs by deaminating exposed nucleobases in a "R loop" without causing double-strand breaks.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Manufacturing precision
If CRISPR-based site-directed mutagenesis is used to introduce target-specific SNPs, then SNP introduction precision is improved, but device complexity increases due to the requirement for repair DNA for every target locus
Solution Approach 1:
The invention extracts and eliminates the requirement for repair DNA by using a catalytically inactive RNA-guided endonuclease that directly deaminates nucleobases in the R loop. This removes the complex repair DNA delivery system needed in traditional CRISPR HDR methods, simplifying the overall mutagenesis approach while maintaining target-specific SNP introduction capability
Solution Approach 2:
The invention introduces an intermediary mechanism where the catalytically inactive endonuclease creates an R loop structure that exposes single-stranded DNA to the nucleobase editing domain. This intermediary R loop formation enables direct base conversion without requiring external repair DNA, bridging the gap between target recognition and SNP introduction
2Manufacturing precision
If CRISPR-based site-directed mutagenesis using RNA-guided endonuclease is used, then SNP introduction precision is improved, but object-affected harmful factors increase due to cytotoxicity from double-strand breaks
Solution Approach 1:
The invention inverts the traditional CRISPR approach by using a catalytically inactive endonuclease that does not create double-strand breaks. Instead of cutting both strands and relying on error-prone NHEJ or HDR, the system uses the R loop structure to enable direct deamination of exposed nucleobases, converting bases in a controlled manner without the harmful cytotoxic effects of DSBs
Solution Approach 2:
The invention converts the potentially harmful R loop structure, which was previously just a byproduct of CRISPR binding, into a beneficial feature. The R loop naturally exposes single-stranded DNA that is highly susceptible to deamination, turning what was merely a structural intermediate into the active site for precise, non-toxic base editing
3Object-affected harmful factors
If CRISPR-AID technology with catalytically inactive endonuclease is used, then object-affected harmful factors are reduced by avoiding double-strand breaks, but productivity decreases due to limited editing window
Solution Approach 1:
The invention applies parameter changes by modifying the endonuclease to be catalytically inactive and fusing it with a nucleobase editing domain. This parameter change transforms the system from one that creates DSBs with limited editing windows to one that enables broader editing through direct deamination of exposed nucleobases in the R loop, improving productivity while maintaining low cytotoxicity
Solution Approach 2:
The invention creates a composite system by fusing the catalytically inactive RNA-guided endonuclease with a nucleobase editing domain (such as AID or APOBEC). This composite structure combines the target-specific binding capability of the endonuclease with the base modification capability of the editing domain, enabling efficient SNP introduction across a broader editing window without the limitations of previous systems
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 allows for efficient and scalable introduction of SNPs with reduced cytotoxicity, enabling the generation of diverse mutants and SNP libraries with improved editing efficiency and a broader editing window compared to existing methods.
Implementation Method 1
an RNA-guided endonuclease such as Cas9 or Cpf1 is directed to a locus of interest using a guide-RNA (gRNA) that has a protospacer region complementary to a DNA sequence in the locus of interest
Implementation Method 2
the nucleobase editing domain is either a cytosine base editor (CBE) that converts C-G base pairs to T-A base pairs or an adenine base editor (ABE) that converts A-T base pairs into G-C base pairs
Data Source
AI summary
The present invention relates to nucleobase editing complexes comprising a) a catalytically inactive RNA-guided endonuclease having a sequence identity of at least 60% to SEQ ID NO: 126 or SEQ ID NO: 155 and b) a nucleobase editing domain, as well as polynucleotides encoding said nucleobase editing complexes, nucleic acid constructs and expression vectors comprising said polynucleotides, host cells comprising said nucleobase editing complexes and/or polynucleotides, and methods for preparing and using said nucleobase editing complexes.