Multiplex CRISPR gRNA Expression via Csy4 Cleavage
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Solution Overview
Problem
Current methods for expressing multiple guide RNAs (gRNAs) in mammalian cells from a single transcript are inefficient, particularly when using RNA polymerase II promoters, which are imprecise for short gRNA expression and restrict the 5' nucleotide identity, limiting the versatility of CRISPR/Cas9 systems for multiplex genome editing.
Innovation Solution
Employing Csy4 endoribonuclease to cleave multiple functional gRNAs encoded on a single longer RNA transcript, flanked by Csy4 cleavage sites, from either RNA polymerase II or III promoters, allowing for precise and efficient production of gRNAs with any desired 5' nucleotide, enabling multiplex genome editing and tissue-specific expression.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If RNA polymerase II promoters are used to express multiple gRNAs from a single transcript, then productivity is improved, but manufacturing precision deteriorates due to imprecise transcription start sites and 5' nucleotide restrictions
Solution Approach 1:
The patent divides the single long RNA transcript into multiple individual gRNA units by introducing Csy4 cleavage sites between them. The Csy4 endoribonuclease specifically recognizes and cleaves at these sites, releasing individual functional gRNAs from the transcript. This segmentation allows the system to maintain the high productivity of Pol II promoter-driven single transcript expression while achieving precise 5' nucleotide control for each individual gRNA, as each gRNA is released with a defined 5' end at the cleavage site.
2Device complexity
If multiple gRNAs are expressed from a single transcript, then device complexity is reduced, but reliability deteriorates due to inefficient processing and 5' nucleotide restrictions
Solution Approach 1:
The patent introduces Csy4 endoribonuclease as an intermediary component that processes the single long transcript into multiple functional gRNAs. This intermediary enzyme specifically recognizes Csy4 cleavage sites inserted between gRNA sequences and catalyzes their release. The addition of this intermediary processing step enables reliable and efficient generation of multiple gRNAs with precise 5' ends from a single transcript, overcoming the limitations of direct Pol II transcription while maintaining simplified device architecture.
3Manufacturing precision
If RNA polymerase III promoters are used for gRNA expression, then manufacturing precision is improved, but adaptability deteriorates due to restricted 5' nucleotide identity requirements
Solution Approach 1:
The patent performs preliminary action by designing the Csy4 cleavage sites with specific sequences that ensure precise cleavage occurs at defined positions. The cleavage sites are engineered to produce gRNAs with exact 5' nucleotide sequences required for Cas9 targeting. This preliminary design of cleavage sites allows the system to achieve Pol III-level precision in 5' nucleotide control while using the more versatile Pol II promoter system, thereby restoring full adaptability for gRNA sequence design without nucleotide restrictions.
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 efficient production and simultaneous expression of multiple gRNAs in mammalian cells, directing Cas9 nuclease to specific targets, overcoming previous limitations in CRISPR/Cas9 multiplex applications and allowing for inducible and tissue-specific gene regulation.
Implementation Method 1
Csy4, an endoribonuclease that recognizes a short RNA hairpin sequence, can be used to cleave out multiple functional gRNAs encoded on a single longer RNA transcript
Data Source
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AI summary
Methods and constructs for the multiplex expression of highly active CRISPR guide RNAs (gRNAs) from RNA Polymerase II and III promoters, optionally in mammalian cells. The present invention is based, at least in part, on the discovery that Csy4, an endoribonuclease that recognizes a short RNA hairpin sequence, can be used to cleave out multiple functional gRNAs encoded on a single longer RNA transcript (produced from an RNA pol II or III promoter) in which the individual gRNAs are separated by Csy4 cleavage sites.