Phage-Assisted Evolution with Dynamic Stringency and Negative Selection

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Solution Overview

Problem

Existing laboratory methods for directed evolution of biomolecules are labor-intensive and limited to a modest number of cycles, hindering the development of molecules with diverse and specific properties, while continuous evolution systems lack generalizability and effective selection strategies.

Innovation Solution

A phage-assisted continuous evolution (PACE) system that allows for continuous mutagenesis, replication, and selection of biomolecules, incorporating stringency modulation and negative selection to evolve molecules with altered properties, using viral vectors and small molecule inducers to control selection pressure.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Measurement precision

If conventional discrete directed evolution cycles are used, then selection precision is improved, but productivity deteriorates due to labor-intensive manual intervention

Engineering Contradiction:

Improveselection precisionVSAvoidevolution cycles per day

Core Design Contradiction:

Measurement precisionVSProductivity

Solution Approach 1:

The system employs phage particles that autonomously perform mutation, replication, and selection functions. The phage display library continuously evolves through self-replication in host cells, with selection occurring automatically based on binding affinity to the target antigen, eliminating the need for manual intervention in each evolution cycle.

Inventive Principle:

Principle #25Self-service

Solution Approach 2:

The evolution process operates continuously rather than in discrete cycles. Phage particles remain in constant circulation, undergoing repeated rounds of mutation and selection simultaneously, allowing dozens of evolution cycles to occur within a single day without interrupting the process for manual harvesting and re-inoculation.

Inventive Principle:

Principle #20Continuity of useful action

2Manufacturing precision

If high selection stringency is applied, then manufacturing precision of desired properties is improved, but adaptability deteriorates as weakly active variants cannot access favorable mutations

Engineering Contradiction:

Improveproperty specificityVSAvoidevolutionary exploration capacity

Core Design Contradiction:

Manufacturing precisionVSAdaptability or versatility

Solution Approach 1:

The selection stringency is made dynamic rather than static. The system automatically adjusts selection pressure based on the evolving phage population's characteristics, allowing weakly active variants to survive initially and accumulate mutations, then progressively increasing stringency as the population evolves toward higher affinity binders.

Inventive Principle:

Principle #15Dynamics

Solution Approach 2:

The system changes selection parameters over time by controlling phage replication conditions, host cell density, and incubation parameters. This temporal variation in selection parameters enables the system to first explore diverse sequence space with lower stringency, then converge on high-specificity binders with increased stringency.

Inventive Principle:

Principle #35Parameter changes

3Productivity

If continuous evolution without negative selection is used, then productivity is improved, but manufacturing precision deteriorates due to accumulation of undesired properties

Engineering Contradiction:

Improveevolution speedVSAvoidproperty specificity

Core Design Contradiction:

ProductivityVSManufacturing precision

Solution Approach 1:

Negative selection is applied proactively to counteract the accumulation of undesired properties before they compromise the evolution process. Phage variants exhibiting off-target binding or unwanted characteristics are selectively removed through competitive binding assays or FACS sorting against control antigens, preventing these variants from dominating the population.

Inventive Principle:

Principle #9Preliminary anti-action

4Measurement precision

If discrete cycles with manual intervention are used, then selection precision is improved, but loss of time increases due to labor-intensive operations

Engineering Contradiction:

Improveselection accuracyVSAvoidtime per evolution cycle

Core Design Contradiction:

Measurement precisionVSLoss of time

Solution Approach 1:

Manual mechanical operations are replaced with automated biological and computational systems. Phage replication, mutation, and selection occur automatically in liquid culture systems, while high-throughput sequencing and bioinformatics algorithms automatically analyze evolution outcomes and guide subsequent rounds, eliminating time-consuming manual harvesting, plating, and screening steps.

Inventive Principle:

Principle #28Mechanics substitution (Replace mechanical system)

Data Source

PatentUS12398390B2Negative selection and stringency modulation in continuous evolution systems

Publication Date: 2025.08.26 PRESIDENT & FELLOWS OF HARVARD COLLEGE

AI summary

Strategies, systems, methods, reagents, and kits for phage-assisted continuous evolution are provided herein. These include strategies, systems, methods, reagents, and kits allowing for stringency modulation to evolve weakly active or inactive biomolecule variants, negative selection of undesired properties, and/or positive selection of desired properties.