Multiplexed CRISPR Editing via Modular gRNA-tRNA Assembly
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Solution Overview
Problem
Current methods for multiplexed genetic modification using CRISPR-Cas9 systems face challenges in the modular assembly of plasmids with multiple guide-RNA (gRNA) sequences, leading to plasmid instability and variable expression levels due to the need for multiple independent expression cassettes and the limitations of Golden Gate cloning.
Innovation Solution
A nucleic acid construct and composition featuring a plasmid backbone with type IIs restriction sites, allowing for the modular assembly of gRNA-tRNA sequences, enabling efficient insertion and expression of multiple gRNAs with different tRNA sequences, facilitating stable and efficient multiplexed genetic editing.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If multiple independent expression cassettes are used on a single plasmid for multiplexed CRISPR editing, then multiple gRNAs can be expressed, but plasmid instability increases and expression levels become variable
Solution Approach 1:
The patent merges multiple gRNA expression units into a single polycistronic transcript by removing independent promoters and terminators. Instead, it uses a single U6 promoter to drive transcription of multiple gRNA sequences separated by tRNA sequences, which are processed by host cell tRNA processing systems to release individual gRNAs. This consolidation reduces plasmid complexity and improves stability while maintaining the ability to express multiple gRNAs.
Solution Approach 2:
The patent employs a universal tRNA sequence that serves multiple functions: (1) acts as a spacer between gRNA sequences, (2) serves as a processing signal for the host cell's tRNA processing system to release individual gRNAs, and (3) maintains plasmid stability. This multi-functional element eliminates the need for multiple independent expression cassettes while ensuring stable plasmid maintenance and consistent gRNA expression.
2Adaptability or versatility
If Golden Gate cloning with ligation sites in variable spacer sequences is used for assembling gRNA arrays, then multiplexed editing can be achieved, but modularity is limited since each PCR amplicon can only produce a single assembly
Solution Approach 1:
The patent segments the gRNA array into modular units where each gRNA is separated by a standardized tRNA sequence. The tRNA acts as a universal delimiter and processing signal, allowing PCR amplicons to be assembled in any order without requiring specific ligation sites within the spacer sequences. This segmentation enables flexible, modular assembly where the same tRNA-gRNA units can be recombined in different configurations to create various gRNA arrays.
Solution Approach 2:
The patent introduces tRNA sequences as intermediary elements between gRNA sequences. These tRNA intermediaries serve as universal spacers and processing signals that facilitate modular assembly. Instead of using ligation sites within variable spacers, the tRNA intermediaries allow PCR amplicons to be joined through standard cloning methods, with the host cell's tRNA processing system then releasing the individual gRNAs. This intermediary approach greatly simplifies the assembly process and enhances modularity.
3Adaptability or versatility
If a single tRNA sequence is repeated across the construct for gRNA separation, then polycistronic arrays can be formed, but construct instability increases due to repeated DNA sequences
Solution Approach 1:
The patent applies local quality by using different tRNA sequences at different positions within the polycistronic array. Instead of repeating the same tRNA sequence throughout, the invention employs a variety of tRNA sequences (e.g., tRNA-Ala, tRNA-Gly, tRNA-Leu, tRNA-Ser) separated by flexible linkers. This variation in local sequence composition reduces the formation of secondary structures and repetitive element instability while maintaining the essential function of tRNA processing at each gRNA site.
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 stable and efficient multiplexed genetic editing by allowing for the modular assembly of gRNA-tRNA sequences, improving plasmid stability and expression levels, and enabling simultaneous editing of multiple genomic loci with increased precision and efficiency.
Implementation Method 1
a first type IIs restriction site, a guide RNA (gRNA) and a tRNA sequence with a second type IIs restriction site within the tRNA sequence
Data Source
AI summary
Provided herein are nucleic acid constructs comprising multiple guide RNAs interspersed with tRNA sequence at regular intervals as well as expression vectors and compositions comprising the same. Also provided herein are methods for assembling the nucleic acid constructs comprising multiple guide RNAs interspersed with tRNA sequence at regular intervals in a pooled and/or modular manner. Methods for using the nucleic acid constructs comprising multiple guide RNAs interspersed with tRNA sequence at regular intervals to facilitate multiplexed genomic editing of a host cell comprising said nucleic acid constructs are also provided herein.