CRISPR Nuclease Identification via Segmented Mutation Screening
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Current methods for identifying functional CRISPR enzymes are inefficient due to the haphazard and combined nature of mutations, making it difficult to assess specific effects and requiring a more methodical, rapid, and high-throughput workflow to identify functional activity in multiple putative enzyme sequences.
Innovation Solution
The development of reaction mixtures and enzyme systems comprising nucleic acid-guided CRISPR nucleases with at least 85% amino acid homology to specific sequences, paired with guide nucleic acids having direct repeat sequences, to form functional complexes that can cleave substrates with high specificity.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If directed evolution is used to engineer CRISPR enzymes, then functional enzymes can be identified, but the haphazard and combined nature of mutations makes it difficult to assess specific effects and complicates further engineering efforts
Solution Approach 1:
The patent segments the CRISPR enzyme evaluation process into independent components: (1) systematic mutation generation at specific positions, (2) individual assessment of each mutant's functional activity, and (3) evaluation of specific effects without combining multiple mutations simultaneously. This segmentation allows methodical evaluation of each mutation's contribution to enzyme function, resolving the complexity issue while maintaining reliable functional identification.
2Productivity
If high throughput screening is designed to identify enzymes with very specific activities, then functional enzymes can be identified, but the complexity of assessing specific effects in multi-component complexes increases
Solution Approach 1:
The patent extracts and evaluates specific functional activities from the complex CRISPR enzyme system by focusing on individual mutation effects and their specific contributions to enzyme function. This extraction allows high-throughput screening to assess specific effects independently, maintaining both speed and accuracy without the complexity of evaluating all possible combination effects simultaneously.
3Productivity
If multiple putative enzyme sequences are evaluated simultaneously, then productivity increases, but the ability to assess specific effects accurately decreases
Solution Approach 1:
The patent applies local quality by evaluating each putative enzyme sequence and its specific mutations individually rather than grouping them together. Each sequence is assessed for its specific functional characteristics and effects, allowing accurate measurement of specific effects while maintaining high throughput through systematic evaluation of multiple sequences using the same methodical approach.
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 the rapid and high-throughput identification of functional CRISPR enzyme activities, improving the efficiency of engineering and exploration by ensuring specific effects can be accurately assessed and utilized.
Implementation Method 1
a nucleic acid-guided nuclease (CRISPR nuclease) having at least 85% amino acid homology to any one of SEQ ID NOs:1-93; a guide nucleic acid (gRNA) for complexing with the nucleic acid-guided CRISPR nuclease
Data Source
AI summary
The present disclosure relates to novel CRISPR enzymes, reaction mixtures, methods of using the same in the detection of microorganisms, such as FluA, FluB, amongst others. The disclosure particularly relates to the discovery of novel functional CRISPR enzyme complexes, particularly systems displaying trans cleavage activity.