CRISPR Base Editor for Rapid Protein Library Generation

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Solution Overview

Problem

Current gene editing techniques, such as site-saturation mutagenesis and error-prone PCR, are laborious and inefficient for rapidly generating diverse protein libraries, as they require laborious cloning procedures and cannot be rapidly iterated.

Innovation Solution

A CRISPR base editor system comprising a catalytically inactive nuclease, guide RNA, and a MS2 phage coat protein linked to an activation-induced deaminase, integrated with a yeast display system, which introduces targeted mutations into a target protein within yeast cells, enabling rapid and efficient generation of diverse libraries.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Quantity of substance

If traditional mutagenesis techniques (site-saturation mutagenesis and error-prone PCR) are used to generate protein libraries, then sufficient diversity can be introduced, but the process becomes laborious and time-consuming due to required cloning techniques

Engineering Contradiction:

Improveprotein library diversityVSAvoidtime for cloning procedures

Core Design Contradiction:

Quantity of substanceVSLoss of time

Solution Approach 1:

The patent replaces traditional mechanical cloning techniques with an in situ mutagenesis system using CRISPR base editors expressed in yeast cells. The base editors (comprising catalytically inactive nuclease, guide RNA, and activation-induced deaminase) directly introduce mutations into the target protein gene within the yeast genome, eliminating the need for external cloning procedures and enabling rapid library generation.

Inventive Principle:

Principle #28Mechanics substitution (Replace mechanical system)

Solution Approach 2:

The yeast cell itself serves as the platform for generating diversity. The CRISPR base editor system is expressed within the yeast cell, and the cell's own replication machinery facilitates the introduction and propagation of mutations during cell division, making the system self-sufficient without requiring external cloning interventions.

Inventive Principle:

Principle #25Self-service

2Productivity

If traditional cloning techniques are used for mutagenesis, then mutations can be introduced, but rapid iteration is not possible

Engineering Contradiction:

Improverate of library generationVSAvoidcomplexity of cloning procedures

Core Design Contradiction:

ProductivityVSDevice complexity

Solution Approach 1:

The patent substitutes complex mechanical cloning operations with a biological system where CRISPR base editors are expressed in yeast cells. The base editors directly modify the genome in situ, and yeast cell division automatically propagates the mutations, enabling rapid iteration without manual cloning steps.

Inventive Principle:

Principle #28Mechanics substitution (Replace mechanical system)

Solution Approach 2:

The CRISPR base editor components (catalytically inactive nuclease, guide RNA, and activation-induced deaminase) are pre-expressed in the yeast cell before mutation introduction. This preliminary setup allows immediate mutation generation upon activation, significantly accelerating the workflow compared to traditional methods where cloning must be performed after mutation generation.

Inventive Principle:

Principle #10Preliminary action

3Manufacturing precision

If CRISPR base editor system with MCP-AID fusion is used, then targeted DNA diversification is achieved, but the system complexity increases

Engineering Contradiction:

Improveprecision of targeted mutationVSAvoidcomplexity of base editor components

Core Design Contradiction:

Manufacturing precisionVSDevice complexity

Solution Approach 1:

The patent merges the MS2 phage coat protein (MCP) with the activation-induced deaminase (AID) into a fusion protein. This fusion allows the AID to be recruited to specific target sites through MCP's binding to MS2 aptamers in the guide RNA, achieving precise targeted mutation while consolidating multiple functions into a single protein component.

Inventive Principle:

Principle #5Merging (Combining)

Solution Approach 2:

The MS2 aptamer serves as an intermediary element that mediates between the guide RNA targeting sequence and the AID catalytic activity. The MCP binds to the MS2 aptamer, which is positioned at the target site through guide RNA pairing, thereby delivering the AID activity precisely to the intended location for mutation.

Inventive Principle:

Principle #24Intermediary (Mediator)

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

The system enables rapid and targeted DNA diversification in yeast, improving protein function by increasing the mutation rate and facilitating directed evolution experiments, as demonstrated by enhancing antibody affinity through in situ DNA mutagenesis and yeast display.

Implementation Method 1

an activation-induced deaminase (AID)... the AID mutates the target nucleic acid encoding the target protein

Methodology Applied

Scientific EffectDeamination:

Implementation Method 2

the MCP binds the at least one bacteriophage aptamer of the gRNA

Methodology Applied

Scientific EffectProtein-RNA binding:

Implementation Method 3

the catalytically inactive nuclease comprises a dead Cas 9 (dCas9) or a dead Cas12 (dCas12)

Methodology Applied

Scientific EffectCRISPR-Cas binding:

Data Source

PatentUS20240167015A1Enhanced diversifying base editors for directed evolution

Publication Date: 2024.05.23 GEORGIA TECH RES CORP

AI summary

The present disclosure relates gene editing systems, compositions, and methods of use to target proteins for directed evolution.