Cytidine Deaminase Evolution via PACE Systems
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Solution Overview
Problem
Current gene editing technologies, such as NHEJ and HDR, suffer from low efficiency and unwanted gene alterations, and traditional methods for improving protein expression are time-consuming and inefficient, particularly for cytidine deaminase proteins like APOBEC1 that express poorly in E. coli and localize to insoluble fractions.
Innovation Solution
The use of Phage-Assisted Continuous Evolution (PACE) and Soluble Expression PACE (SE-PACE) systems to evolve cytidine deaminase proteins, optimizing their stability and solubility while maintaining function, by introducing specific mutations and using recombinant expression constructs to enhance selection of properly folded proteins.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If traditional directed evolution approaches are used to improve protein function, then protein function can be improved, but time and effort requirements increase substantially
Solution Approach 1:
The PACE system implements continuous evolution by maintaining a constant flow of host cells through the lagoon, allowing phage vectors to replicate and mutate continuously without discrete interruption. This continuous process enables simultaneous improvement of multiple protein properties (solubility, stability, function) over extended periods, resolving the contradiction between achieving reliable protein function and the time required for evolution.
Solution Approach 2:
The system performs preliminary selection for soluble expression by incorporating a soluble expression selection mechanism at the outset of the evolution process. This preliminary action filters out insoluble variants early, ensuring that subsequent evolution rounds focus on improving protein function within the subset of soluble variants, thereby reducing total evolution time while maintaining functional improvement.
2Productivity
If traditional directed evolution approaches are used to improve protein expression, then protein expression can be improved, but substantial time and effort are required
Solution Approach 1:
The continuous flow system allows constant selection pressure for soluble expression, with each incoming host cell representing an opportunity for soluble variant selection. This continuous action accelerates the enrichment of soluble expression variants compared to discrete batch evolution methods, improving protein expression productivity while reducing evolution time.
Solution Approach 2:
The system incorporates feedback through the soluble expression selection mechanism, where the presence or absence of soluble protein directly influences phage replication success. This feedback loop continuously guides the evolution toward variants with improved soluble expression, enabling rapid enrichment of high-expression variants without substantial time investment.
3Ease of manufacture
If cytidine deaminase proteins are expressed in E. coli, then recombinant production is achieved, but proteins undergo proteolysis or misfold into inclusion bodies
Solution Approach 1:
The system employs self-service through autonomous selection, where the E. coli host cells themselves perform the selection function by preferentially supporting replication of phage vectors that produce soluble proteins. The host's natural protein folding and quality control mechanisms serve the evolution process, eliminating the need for external intervention while simultaneously improving solubility and preventing inclusion body formation.
Solution Approach 2:
The system induces mutations in the cytidine deaminase protein sequence that alter biophysical parameters such as folding stability and solubility. These parameter changes are selected for through the soluble expression mechanism, resulting in variants that maintain recombinant production capability while achieving improved solubility and preventing proteolysis or inclusion body formation.
Data Source
AI summary
Some aspects of this disclosure relate to strategies, systems, methods, compositions, and kits that are useful for production (e.g., evolution) of cytidine deaminase protein variants that are characterized by increased soluble expression and/or stability relative to the wild-type cytidine deaminase protein from which they are evolved. In some embodiments, evolved cytidine deaminase variants described by the disclosure are useful for incorporation into targeted nucleic acid editing proteins, for example in fusion proteins with a Cas9 domain or variant thereof.