Hybrid Guide RNA Multiplex Gene Editing Efficiency
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Solution Overview
Problem
Current CRISPR-based methods for multiplex gene targeting face challenges in editing efficiency, particularly when targeting multiple genetic mutations simultaneously, due to recombination between expression cassettes and competition for enzyme loading, which limits the ability to score phenotypes in the absence of selection.
Innovation Solution
The co-expression of orthologous class II monomeric Cas enzymes, such as Cas9 and Cas12a, together with hybrid guide RNAs (hgRNAs) generated from fusion constructs expressed under a single promoter, enhances editing efficiency by allowing simultaneous targeting of multiple sites, and optimized hgRNAs are designed using deep learning frameworks to improve Cas12a gRNA efficiencies comparable to Cas9 gRNAs.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If multiple expression cassettes are used for multiplex gene targeting, then the ability to target multiple sites is improved, but recombination between cassettes occurs reducing editing efficiency
Solution Approach 1:
The patent merges multiple guide RNA expression cassettes into a single polycistronic transcript under one promoter, eliminating recombination issues between separate cassettes while maintaining the ability to target multiple genomic sites simultaneously
Solution Approach 2:
The invention creates a universal guide RNA architecture where a single transcript can encode multiple functional guide RNAs that direct Cas9 to different target sites, making the system adaptable for multiplex editing without requiring multiple separate expression units
2Adaptability or versatility
If multiple Cas9 guides are co-expressed from multiple promoters, then multiplex targeting capability is improved, but competition for enzyme loading reduces editing efficiency
Solution Approach 1:
The patent combines multiple guide RNA expression units into a single co-transcribed polycistronic RNA molecule, ensuring stoichiometric production of all guide RNAs and eliminating competitive inhibition for Cas9 loading that occurs with separate promoter-driven expressions
3Adaptability or versatility
If traditional CRISPR methods are used for large-scale exon analysis, then the scope of analysis is improved, but the ability to score phenotypes in the absence of selection is limited
Solution Approach 1:
The patent introduces an intermediary readout system using fluorescent reporters and flow cytometry that mediates phenotype detection without requiring selective pressure, enabling reliable scoring of editing outcomes in unscreened cell populations
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 effective large-scale analysis of exon function, identifies genetic interactors, and reveals complex genetic interactions involving paralogous genes, achieving editing efficiencies comparable to the most efficient Cas9 gRNAs and providing insights into critical cell functions.
Implementation Method 1
Cas12a (formerly known as Cpf1) enzymes contain intrinsic RNAse activity and can generate multiple guide (g)RNAs from a single concatemeric guide RNA transcript
Implementation Method 2
The co-expression of orthologous class II monomeric Cas enzymes, such as Cas9 and Cas12a, together with hybrid guide RNAs (hgRNAs) generated from fusion constructs
Data Source
AI summary
A hybrid guide RNA (hgRNA) comprising a proximal spacer, a distal spacer, a type II CRISPR-Cas tracrRNA, and a type V CRISPR-Cas direct repeat. Also provided herein are further multiplexed hgRNAs comprising additional direct repeats and spacers as well as methods of making and using thereof. Libraries comprising said hgRNAs or components thereof, cells, kits and reagents employed in the making or use thereof are also provided.