CRISPR/Cas9 RNP Multiplex Knockout for CHO Cells
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Solution Overview
Problem
Current CRISPR/Cas9 multiplex knockout strategies for CHO cells are inefficient due to variable editing efficiencies of gRNA sequences, resource-intensive screening processes, and unstable integration of Cas9 DNA, leading to lengthy and costly processes for achieving multiplex knockouts in therapeutic protein production.
Innovation Solution
The method involves forming ribonucleoprotein complexes (RNPs) by combining guide RNAs (gRNAs) with Cas9 protein to achieve high indel formation at target loci, followed by serial transfection of cells until desired edit percentages are reached, and isolating cells through single-cell cloning, optimizing RNP-to-cell ratios and gRNA types for efficient gene editing.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If DNA plasmids are used to deliver Cas9 and gRNA for gene editing, then gene knockout can be achieved, but the process becomes lengthy and expensive due to synthesis, cloning, and screening requirements
Solution Approach 1:
The patent pre-assembles CRISPR/Cas9 ribonucleoprotein complexes (RNPs) in vitro before delivery to cells. The guide RNA and Cas9 protein are combined and prepared in advance with optimized sequences, eliminating the need for time-consuming plasmid synthesis, cloning, and screening steps. This preliminary preparation of functional editing complexes ready for immediate cellular delivery directly reduces process time while maintaining knockout efficiency.
Solution Approach 2:
The patent extracts and separates the Cas9 protein from its DNA plasmid carrier, delivering only the essential ribonucleoprotein complex (Cas9 + gRNA) to cells. This extraction eliminates unnecessary DNA synthesis and cloning steps, focusing delivery on the minimal functional components needed for gene editing, thereby reducing process complexity and time.
2Adaptability or versatility
If multiple gRNA plasmids are synthesized for multiplex knockout, then multiple target loci can be edited, but the process becomes expensive and complex
Solution Approach 1:
The patent merges multiple gRNA sequences into a single delivery system by combining several guide RNAs with Cas9 protein to form a multiplex ribonucleoprotein complex. This single complex can simultaneously target multiple loci, eliminating the need for separate plasmid synthesis and transfection for each target. The merging of multiple editing functions into one unified delivery vehicle reduces process complexity while maintaining multiplex capability.
Solution Approach 2:
The patent creates a universal CRISPR/Cas9 RNP platform that can be configured to target any number of loci by simply changing the guide RNA sequences. This multi-functional system allows the same Cas9 protein to perform multiple knockout functions simultaneously, providing adaptability across different target genes without requiring separate optimized protocols for each target.
3Stability of the object's composition
If Cas9 DNA is integrated into host genome, then stable expression can be achieved, but undesirable outcomes occur for therapeutic protein production
Solution Approach 1:
The patent uses transient, non-integrating CRISPR/Cas9 ribonucleoprotein complexes that perform their editing function and then degrade naturally without permanent genomic integration. These disposable RNP complexes deliver the editing capability temporarily, achieving stable gene knockout without the harmful long-term effects of Cas9 DNA integration, such as insertional mutagenesis or sustained Cas9 expression that could affect therapeutic protein production.
4Reliability
If traditional screening methods are used to identify edited cells, then knockout can be confirmed, but the process lacks speed and sensitivity
Solution Approach 1:
The patent replaces traditional mechanical screening methods (Western blotting, T7 endonuclease assays, size-based PCR analysis) with next-generation sequencing technology. NGS provides rapid, high-sensitivity detection of indel formation at edited loci, simultaneously achieving high accuracy in knockout confirmation and high throughput screening of multiple cells and targets, thus improving both reliability and productivity.
Data Source
AI summary
The present disclosure relates to modified mammalian cells having reduced or eliminated expression of certain cellular proteins, CRISPR/Cas9 multiplex knockout strategies for making such cells, and methods of using such cells, e.g., in the context of cell-based therapy or as host cells in the production of a product of interest.