Single-Vector Cas3 Cascade Expression for Low-Toxicity DNA Amplification
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Solution Overview
Problem
Existing CRISPR/Cas systems often require multiple vectors for efficient delivery and expression of Cas and Cascade proteins, leading to potential toxicity, mutation, and reduced yield in bacterial and archaeal production strains, as well as inefficiencies in target sequence modification.
Innovation Solution
A single-vector system using a nucleic acid vector with a Cas3 and Cascade protein-encoding sequences under controlled promoters, including attenuated promoters, for coordinated expression in host cells, reducing toxicity and enhancing yield during DNA amplification.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If multiple vectors are used for delivery of Cas and Cascade proteins, then delivery efficiency is improved, but device complexity and potential toxicity increase
Solution Approach 1:
The patent combines multiple Cas protein-encoding sequences (Cas3 and Cascade proteins) into a single nucleic acid vector. This merging approach maintains delivery efficiency while reducing the complexity of using multiple separate vectors, and minimizes potential toxicity associated with multiple vector integrations into the host genome.
Solution Approach 2:
The single nucleic acid vector is designed to perform multiple functions: it encodes both Cas3 endonuclease and Cascade proteins, includes promoter sequences for coordinated expression, and contains origin of replication sequences. This multi-functional design eliminates the need for separate vectors while maintaining comprehensive CRISPR system functionality.
2Quantity of substance
If strong promoters are used for Cas protein expression, then expression level is improved, but toxicity and mutation in production strains increase
Solution Approach 1:
The patent employs inducible promoter sequences that allow dynamic control of Cas protein expression. During the DNA amplification phase in production strains, the promoters remain inactive or at low activity levels, minimizing toxicity and mutation. Upon induction (e.g., by IPTG or other inducers), the promoters activate to produce high levels of Cas proteins when needed, thus resolving the contradiction between expression level and cellular health.
Solution Approach 2:
The expression system operates in periodic phases: first, DNA amplification occurs with minimal Cas protein expression (promoters inactive or repressed); second, upon induction, Cas protein expression is activated for the desired function. This periodic control pattern allows high expression when needed while protecting production strains from continuous toxicity and mutation effects.
3Productivity
If DNA amplification is performed in production strains, then yield of amplified DNA is improved, but toxicity and mutation from Cas expression reduce yield
Solution Approach 1:
The patent designs the system so that DNA amplification occurs first in production strains before Cas protein expression is activated. The promoter sequences are configured to allow DNA replication to proceed with minimal interference from Cas proteins. After sufficient DNA amplification has occurred, induction triggers Cas protein expression for the intended application, thus preserving production strain reliability during the critical amplification phase.
Solution Approach 2:
The inducible promoter system dynamically adjusts Cas protein expression levels based on the growth phase and induction conditions. During logarithmic growth and DNA amplification, expression remains low to maintain production strain stability. Upon induction with appropriate molecules, expression rapidly increases to achieve the desired functional output, thus resolving the contradiction between yield and reliability.
Data Source
AI summary
The invention relates to the production and use of Cas-encoding sequences and vectors comprising these. Aspects of the invention provide products, vectors, delivery vehicles, uses and methods for producing Cas-encoding sequences in bacterial or archaeal cells.