Polycistronic gRNA Arrays via PARA Cloning
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Solution Overview
Problem
The complexity of vector design and construction in multiplexed CRISPR technologies, particularly due to highly repetitive DNA sequences, hinders their widespread adoption in various applications, making efficient multiplexed genome editing and gene regulation challenging.
Innovation Solution
The development of a polycistronic guide RNA (gRNA) array and multiplex CRISPR vectors, utilizing a Prime Assembly of gRNA Arrays (PARA) method for fast cloning of multiple gRNAs into a CRISPR vector, which includes a DNA encoding a polycistronic gRNA array and a destination vector with specific recognition sequences for type IIS restriction enzymes, enabling efficient assembly and expression of multiple gRNAs.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If conventional arrayed multiple individual gRNA expression cassettes or CRISPR arrays are used, then multiplexed CRISPR can achieve gene editing functionality, but vector design and construction becomes complicated due to highly repetitive DNA sequences
Solution Approach 1:
The patent divides the gRNA array construction into modular components: a destination vector with unique restriction sites, gRNA expression cassettes with standardized sequences, and a cloning framework using type IIS restriction enzymes. This segmentation allows each component to be independently designed and assembled, reducing the complexity of handling highly repetitive sequences throughout the entire vector.
Solution Approach 2:
The destination vector is pre-designed with specific restriction sites (e.g., BsaI, BsmBI) and multiple cloning sites positioned before the gRNA array. This preliminary preparation of the vector backbone with predetermined cloning sites simplifies the subsequent insertion of gRNA cassettes, avoiding the need to redesign the entire vector for each multiplexing experiment.
2Ease of manufacture
If traditional multistep modular cloning is used for multiplex gRNA cloning, then some level of optimization is achieved, but the process requires multiple intermediate vectors and remains technically challenging
Solution Approach 1:
The patent merges multiple cloning operations into a single-step Golden Gate assembly process. Multiple gRNA expression cassettes are simultaneously ligated into the destination vector in one reaction using type IIS restriction enzymes, eliminating the need for sequential cloning steps and intermediate vectors that would otherwise be required to build up the array incrementally.
Solution Approach 2:
The destination vector is designed as a universal platform that can accommodate any number of gRNA cassettes (e.g., 2, 4, 6, or more) using the same cloning protocol. The standardized restriction sites and modular cassette design allow the same vector and cloning method to be used across different multiplexing scales, eliminating the need for vector-specific optimization for each cloning scenario.
3Reliability
If synthetic gRNA arrays are used, then higher efficacy of gene disruption is achieved in yeast, Drosophila and plants, but the complicated vector design prevents widespread adoption
Solution Approach 1:
The patent changes the key parameter of restriction enzyme type from traditional Type II (e.g., EcoRI, HindIII) to Type IIS (e.g., BsaI, BsmBI), which cut outside their recognition sites. This parameter change enables the generation of unique overhang sequences that prevent self-ligation and ensure directional cloning, dramatically simplifying the vector design while maintaining synthetic gRNA array efficacy.
Solution Approach 2:
Type IIS restriction enzymes serve as intermediaries that facilitate the assembly of synthetic gRNA arrays by creating compatible overhangs between the destination vector and gRNA cassettes. These enzymes mediate the specific annealing and ligation of modular components, making the otherwise complex assembly process routine and broadly applicable across different organisms and experimental conditions.
Data Source
AI summary
The present disclosure is directed to polycistronic guide RNAs, DNA encoding polycistronic gRNA, multiplex CRISPR vectors, a plurality of component DNA fragments for assembly into a DNA encoding a polycistronic gRNA array, a plurality of primer pairs for making a plurality of component DNA fragments to be assembled into a DNA encoding a polycistronic gRNA, and methods of making multiplex CRISPR vectors. The current disclosure is directed to multiplexed CRISPR technologies that have great potential for pathway engineering and genome editing. In the current disclosure describes efficient assembly of tRNA/Csy4/Ribozyme-based gRNA arrays which can be produced in a quick and effective process.