CRISPR-Cas and Reverse Transcriptase Editing System
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Solution Overview
Problem
Current base editing tools have limitations such as bystander bases, small editing windows, and limited ability to convert specific nucleotides, restricting their applicability to trait-relevant targets, especially in organisms with low protospacer adjacent motif (PAM) density.
Innovation Solution
A method involving Type V or Type II CRISPR-Cas effector proteins, reverse transcriptases, and extended guide nucleic acids is used to modify target nucleic acids, enhancing editing capabilities by co-localizing these components to facilitate precise nucleotide changes.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If base editing tools are used to change cytosine and adenine residues, then editing efficiency is improved, but the ability to edit thymine or guanine residues is limited
Solution Approach 1:
The patent employs multiple different CRISPR-Cas effector proteins (including Type V and Type II variants) that can target different nucleotide bases. By using a collection of these effectors with different PAM requirements and targeting specificities, the system achieves universal editing capability across all four nucleotide bases (A, T, C, G), rather than being limited to just cytosine and adenine editing.
Solution Approach 2:
The invention changes the parameters of the editing system by introducing effectors with different PAM sequence requirements and spacer lengths. This allows the system to accommodate various target sites throughout the genome, including regions with different nucleotide compositions, thereby expanding the range of editable nucleotides beyond the limitations of conventional base editors.
2Manufacturing precision
If conventional base editing tools are used, then cytosine and adenine conversion is achieved, but bystander base effects occur
Solution Approach 1:
The patent designs guide RNAs with specific spacer lengths and sequences that provide localized precision at the target site. By optimizing the guide RNA structure and using effectors with restricted editing windows, the system confines the editing activity precisely to the intended nucleotide position, preventing off-target modifications to adjacent bystander bases.
3Adaptability or versatility
If base editing tools with limited PAM density are used, then editing capability is restricted, but accessibility to trait-relevant targets is reduced
Solution Approach 1:
The invention employs a diverse array of CRISPR-Cas effector proteins that recognize different PAM sequences. This multi-functional approach ensures that regardless of the PAM density at a given genomic location, there is likely an effector variant available that can bind and edit the target site, thereby maintaining ease of operation across the entire genome.
Solution Approach 2:
The system creates a composite editing platform that combines multiple effector proteins with different PAM specificities. This composite approach allows the system to function effectively across varied genomic landscapes, ensuring accessibility to trait-relevant targets regardless of local PAM density variations.
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 increases the range of possible edits in various organisms, overcoming limitations of existing tools by improving editing efficiency and accuracy.
Implementation Method 1
an extended guide nucleic acid (e.g., extended Type II or Type V CRISPR RNA, extended Type II or Type V CRISPR DNA, extended Type II or Type V crRNA, extended Type II or Type V crDNA)
Implementation Method 2
a reverse transcriptase, and (c) an extended guide nucleic acid
Data Source
AI summary
This invention relates to recombinant nucleic constructs comprising CRISPR-Cas effector proteins, reverse transcriptases and extended guide nucleic acids and methods of use thereof for modifying nucleic acids in plants.