Recombinant CRISPR-Cas System for Lactobacillus crispatus Genome Editing

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Solution Overview

Problem

Current technologies for genome editing and expression modification in organisms, particularly in Lactobacillus crispatus, lack efficient and precise methods using CRISPR-Cas systems.

Innovation Solution

Introduction of recombinant nucleic acid constructs encoding CRISPR arrays and Type I-E Cascade complexes, along with Cas3 polypeptides, into target organisms to modify genomes and regulate gene expression.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Manufacturing precision

If CRISPR-Cas systems are introduced for genome editing in Lactobacillus crispatus, then genome editing precision and efficiency are improved, but system complexity and difficulty of implementation increase

Engineering Contradiction:

Improvegenome editing precisionVSAvoidsystem complexity

Core Design Contradiction:

Manufacturing precisionVSDevice complexity

Solution Approach 1:

The CRISPR-Cas system is divided into separate functional modules: Cas9 nuclease, guide RNA (gRNA) components, and repair template vectors. This segmentation allows each component to be optimized independently and facilitates modular assembly in L. crispatus, reducing overall system complexity while maintaining high editing precision

Inventive Principle:

Principle #1Segmentation

Solution Approach 2:

A uracil-DNA glycosylase (UDG) inhibitor is introduced as an intermediary to enhance homology-directed repair (HDR) efficiency. The inhibitor prevents base excision repair of uracil residues in the donor DNA, thereby mediating increased precision in genome editing outcomes without requiring complex alternative repair mechanisms

Inventive Principle:

Principle #24Intermediary (Mediator)

2Adaptability or versatility

If Type I-E Cascade complex components are introduced, then gene expression modification capability is improved, but number of required components and operational difficulty increase

Engineering Contradiction:

Improveexpression modification capabilityVSAvoidoperational difficulty

Core Design Contradiction:

Adaptability or versatilityVSEase of operation

Solution Approach 1:

The Type I-E Cascade complex components (Cse1, Cse2, Cas7, Cas5, and Cas6) are combined into a unified CRISPR array expression system. This merging allows the entire complex to be expressed from a single plasmid construct, reducing the number of separate transformation events required and simplifying operational procedures while maintaining versatile gene expression modification capability

Inventive Principle:

Principle #5Merging (Combining)

Solution Approach 2:

The CRISPR array system is designed with universal applicability for targeting multiple genes simultaneously. The modular spacer design allows a single Cascade complex to recognize and modify multiple different target sequences, providing multi-functionality that reduces the need for separate systems for different editing tasks

Inventive Principle:

Principle #6Universality (Multi-functionality)

Data Source

PatentUS12264313B2Recombinant type I CRISPR-Cas system and uses thereof for genome modification and alteration of expression

Publication Date: 2025.04.01 NORTH CAROLINA STATE UNIV

AI summary

This invention is directed to recombinant Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) arrays and recombinant nucleic acid constructs encoding Type 1-E CASCADE complexes, plasmids, retroviruses and bacteriophage comprising the same, and methods of use thereof for modifying genomes and expression. Further disclosed are methods of modifying (editing) the genome of target organisms using the constructs.