base-editing-mediated directed protein evolution

Stages

1. 1.
   Comparative assessment of five inducible degradation systems(broad_screen)

   This stage identifies the strongest starting degradation system for further engineering.

   Selection: Comparison across degradation efficiency, basal degradation, target recovery after ligand washout, and ligand impact.

2. 2.
   Base-editing-mediated mutagenesis of OsTIR1(library_build)

   This stage creates candidate OsTIR1 variants for directed evolution.

   Selection: Generate OsTIR1 variant diversity to address basal degradation and slow recovery limitations of AID 2.0.

3. 3.
   Several rounds of functional selection and screening(selection)

   This stage enriches for improved OsTIR1 variants after mutagenesis.

   Selection: Functional performance of OsTIR1 variants in the degron system.

4. 4.
   Resulting improved degron system characterization(confirmatory_validation)

   This stage confirms that the evolved system improves on the starting AID 2.0 tradeoffs.

   Selection: Demonstrate effective depletion with minimal basal degradation and faster recovery after washout in the resulting system.

Steps

1. 1.
   Benchmark five inducible degradation systems across shared performance criteria

   Identify the most robust starting system for further engineering.

   A comparative benchmark is performed first so the engineering campaign starts from the strongest available platform rather than mutating an inferior baseline.

2. 2.
   Mutagenize OsTIR1 by base editingengineering method

   Create OsTIR1 variant diversity to overcome AID 2.0 limitations.

   Mutagenesis follows system selection because the authors first identify AID 2.0 as the best baseline and then engineer its limiting component.

3. 3.
   Perform several rounds of functional selection and screening on OsTIR1 variantsengineered variants under selection

   Enrich for gain-of-function OsTIR1 variants with improved degron performance.

   Selection and screening occur after mutagenesis because variant diversity must first be generated before improved variants can be enriched.

4. 4.
   Characterize the resulting AID 2.1 system for depletion, basal degradation, and recoveryresulting engineered degron system

   Confirm that the evolved system improves the key tradeoffs observed in AID 2.0.

   Final characterization is done after variant selection to verify that the evolved system delivers the intended performance improvements in the finished tool.