gRNA Scaffold Screening for Stable CRISPR Multiplexing
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Solution Overview
Problem
Existing CRISPR-based gene drives face issues with significant stretches of homologous repeats that affect generational stability, limiting the effectiveness of simultaneous targeting of multiple DNA sites.
Innovation Solution
A function-screening system and processing-screening system are developed to identify guide RNA (gRNA) scaffold molecules that minimize repetitive sequences, allowing for the construction of minimally repetitive arrays capable of targeting multiple DNA sites efficiently.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If arrays of guides with different spacers are used to target many sites simultaneously, then the effectiveness of CRISPR-based gene drives is improved, but significant stretches of homologous repeats are introduced that affect generational stability
Solution Approach 1:
The gRNA array is segmented into multiple independent gRNA units, each with its own unique spacer sequence targeting a specific DNA site. The scaffold regions are standardized while the spacer regions are diversified, allowing simultaneous targeting of multiple sites without extensive homology between units. This segmentation reduces the overall homogenous repetitive content while maintaining multiplexing capability.
Solution Approach 2:
Different regions of the gRNA array are assigned different properties: the scaffold regions provide structural consistency for Cas protein binding, while the spacer regions provide sequence diversity for specific target recognition. This local differentiation allows the array to function effectively as a multiplexing tool while minimizing the harmful effects of repetitive sequences through localized homology only where necessary for function.
2Adaptability or versatility
If previously available gRNA arrays are used to provide multiplexing capability, then the ability to target multiple sites is improved, but significant stretches of homologous repeats are introduced that affect stability
Solution Approach 1:
The invention changes the sequence parameters of the gRNA array by using standardized scaffold sequences combined with diverse spacer sequences. This parameter optimization allows the array to maintain adaptability for targeting multiple sites while reducing the homology parameter that causes instability. The scaffold regions are kept consistent for functional reliability, while spacer regions are varied to minimize repetitive sequence effects.
3Adaptability or versatility
If gRNA arrays with extensive homology are constructed to enable multiplexing, then the versatility of the system is improved, but the generational stability is reduced
Solution Approach 1:
The gRNA array is divided into modular units where each unit contains a conserved scaffold portion and a variable spacer portion. This segmentation allows the system to maintain versatility through diverse spacers while reducing generational instability by limiting homology to only the essential scaffold regions, thereby minimizing recombination events between array elements.
Solution Approach 2:
The standardized scaffold sequence serves as a universal element that performs the essential function of Cas protein binding and RNA processing across all gRNA units in the array. This universality in the scaffold region provides functional consistency while the variable spacer regions provide target diversity, achieving multi-functionality without requiring extensive homology throughout the entire array.
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 systems enable the synthesis of highly active and evolutionarily stable gRNA arrays that maximize activity and minimize secondary structure, enhancing the efficiency and stability of CRISPR-based gene editing.
Implementation Method 1
the binding of the riboprotein complex to the promoter reduces transcription of the sequence encoding the reporter protein
Implementation Method 2
the reporter protein comprises a fluorescent protein
Data Source
AI summary
The invention relates, in part, to methods and systems with which to identify guide RNAs (gRNAs) and methods and systems with which to prepare, design, and generate gRNAs and minimally repetitive arrays of gRNAs.