Multiplexed Base Editing via dCas9 Deaminase Fusion
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Solution Overview
Problem
Current DNA editing technologies face challenges in achieving large-scale modifications, particularly in mammalian organisms, due to difficulties in delivering multiple guide RNAs and high cytotoxicity associated with genome-wide DNA modifications, which limits the ability to edit hundreds to thousands of loci without inducing toxicity.
Innovation Solution
The development of highly multiplexed base editing methods using a CRISPR-Cas9 system with dead Cas9 (dCas9) fusion proteins and optimal cell conditions, including anti-apoptotic factors and inhibitors of repair mechanisms, to minimize toxicity and enable editing of hundreds to tens of thousands of loci simultaneously.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If multiple guide RNAs are delivered simultaneously for multiplexed genome editing, then the number of editable loci increases, but cytotoxicity increases due to DNA damage sensors activation
Solution Approach 1:
The patent extracts and removes the nuclease activity from the Cas9 protein by using dead Cas9 (dCas9) which lacks DNA cleavage capability. This separation of the targeting function (gRNA-dCas9 complex) from the cutting function eliminates the harmful DNA double-strand breaks that cause cytotoxicity, while preserving the ability to target multiple loci simultaneously through multiplexed gRNA delivery.
Solution Approach 2:
The patent introduces an intermediary mechanism by using dCas9 fused to transcriptional effectors (such as activators or repressors) that mediate gene expression changes without DNA cleavage. This intermediary approach allows multiplexed editing of hundreds to thousands of loci while avoiding the activation of DNA damage sensors and subsequent cytotoxicity.
2Productivity
If genome-wide DNA modifications are performed at hundreds to thousands of loci, then large-scale genome recoding is achieved, but cell viability decreases due to high toxicity
Solution Approach 1:
The patent removes the harmful nuclease activity from the CRISPR system by employing dCas9, enabling large-scale genome modifications at hundreds to thousands of loci without inducing DNA breaks. This extraction of the cutting function while retaining targeting capability allows genome-wide recoding while maintaining cell viability.
Solution Approach 2:
The patent converts the potential harm of DNA breaks into a benefit by using the dCas9-gRNA complex to precisely target and modulate gene expression without cleavage. The system that was originally designed for cutting is repurposed for safe, large-scale transcriptional control, turning a harmful mechanism into a beneficial tool for genome-wide editing with maintained cell viability.
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
These methods enhance cell survival and editing efficiency, allowing for targeted deamination at a much higher number of loci than previously reported, overcoming on-target mutation and toxicity barriers, and facilitating the editing of repetitive elements in human cells.
Implementation Method 1
contacting a nucleic acid molecule with a plurality of fusion proteins, wherein each of the fusion proteins of the plurality comprises (i) a nuclease inactive Cas9 (dCas9) domain and (ii) a deaminase domain
Data Source
AI summary
The present disclosure provides highly multiplexed base editing methods and compositions that minimize the induction of DNA damage sensors in eukaryotic cells and maintain cell viability. The disclosed base editing methods improve the survival of eukaryotic cells after large-scale genome editing. These methods are based upon the discovery that use of a dead Cas9 base editor and optimal cell conditions during and after base editing enhances cells' tolerance to and survival following thousands of edits to the genome. Optimal cell conditions after base editing include the use of a combination of small molecule factors and/or inhibitors. These methods are facilitated by the design and use of tens to hundreds to thousands of gRNAs for guiding the base editor to the target sequences. The disclosed methods are capable of inducing between ten and 300,000 edits to the genome of a eukaryotic cell. Further disclosed are pharmaceutical compositions and compositions of eukaryotic cells comprising fusion proteins and a plurality of unique gRNAs, and a combination of small molecule factors and inhibitors. Also disclosed are kits for the generation of the fusion protein-gRNA complexes described herein.