CRISPR-Cas9 NMT Gene Knockout in Eimeria tenella

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Solution Overview

Problem

Current methods lack a viable approach to knock out the N-myristoyltransferase (NMT) gene from Eimeria tenella, a significant pathogen causing economic losses in livestock, hindering the development of novel anticoccidial drugs and vaccines.

Innovation Solution

A method involving the use of CRISPR/Cas9 technology, where sporozoites of Eimeria tenella are mixed with pCRISPR::EtNMT and pEtNMT::DHFR plasmids, followed by electrotransformation, to successfully knockout the NMT gene, utilizing guide RNA to guide Cas9-mediated DNA cleavage and subsequent repair mechanisms for gene frameshift mutations.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Productivity

If traditional gene knockout methods are used, then the process is time-consuming and inefficient, but CRISPR/Cas9 technology enables precise and rapid gene knockout

Engineering Contradiction:

Improvegene knockout efficiencyVSAvoidmethod complexity

Core Design Contradiction:

ProductivityVSDevice complexity

Solution Approach 1:

The patent replaces traditional mechanical gene knockout methods (such as homologous recombination requiring complex plasmid construction and selection) with CRISPR/Cas9 genome editing technology. The guide RNA directs the Cas9 endonuclease to the target NMT gene sequence, creating precise double-strand breaks that lead to efficient gene knockout through non-homologous end joining (NHEJ) or homology-directed repair (HDR), thereby dramatically improving productivity while maintaining manageable complexity

Inventive Principle:

Principle #28Mechanics substitution (Replace mechanical system)

Solution Approach 2:

The patent introduces guide RNA as an intermediary molecule that bridges the Cas9 endonuclease and the target NMT gene. The guide RNA contains a 20-nucleotide sequence complementary to the target gene, enabling specific recognition and binding. This intermediary mechanism allows precise targeting without complex procedural steps, resolving the contradiction between efficiency and complexity

Inventive Principle:

Principle #24Intermediary (Mediator)

2Reliability

If no gene knockout method is available, then drug target validation is hindered, but CRISPR/Cas9 enables functional studies of NMT gene

Engineering Contradiction:

Improvedrug target validation capabilityVSAvoidmethod feasibility

Core Design Contradiction:

ReliabilityVSEase of manufacture

Solution Approach 1:

The patent performs preliminary action by establishing a reliable CRISPR/Cas9 gene knockout system in E. tenella before conducting drug target validation studies. The system includes pre-designed guide RNA targeting the NMT gene and the Cas9 expression construct, enabling immediate functional studies without needing to develop new methods for each validation experiment, thus improving reliability while maintaining ease of manufacture

Inventive Principle:

Principle #10Preliminary action

Solution Approach 2:

The CRISPR/Cas9 system enables self-service gene knockout capability within the E. tenella research framework. Once the system is established, subsequent gene knockout experiments can be performed by simply designing new guide RNA sequences complementary to target genes, without requiring external specialized services or complex procedural interventions, thereby improving both reliability and ease of manufacture

Inventive Principle:

Principle #25Self-service

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

This approach effectively knocks out the NMT gene, laying a foundation for studying Eimeria tenella's biology and developing vaccines, providing a novel target for drug development and improving disease control in poultry industries.

Implementation Method 1

utilizing guide RNA to guide Cas9-mediated DNA cleavage

Methodology Applied

Scientific EffectCRISPR-Cas9 DNA cleavage:

Implementation Method 2

subjecting a resulting mixture to electrotransformation

Methodology Applied

Scientific EffectElectrotransformation:

Implementation Method 3

subsequent repair mechanisms for gene frameshift mutations

Methodology Applied

Scientific EffectDNA repair and frameshift mutation:

Data Source

PatentUS11866703B2Method for knocking out N-myristoyltransferase (NMT) gene from <i>Eimeria tenella</i>

Publication Date: 2024.01.09 LANZHOU VETERINARY RESEARCH INSTITUTE CHINESE ACADEMY OF AGRICULTURAL SCIENCES(LANZHOU BRANCH CENTER OF CHINA ANIMAL HEALTH & EPIDEMIOLOGY CENTER)

AI summary

The present disclosure provides a method for knocking out an N-myristoyltransferase (NMT) gene from Eimeria tenella, and belongs to the technical field of microorganisms. The method includes: mixing sporozoites of Eimeria tenella with a pCRISPR::EtNMT plasmid and a pEtNMT::DHFR plasmid, and subjecting a resulting mixture to electrotransformation to obtain NMT gene-knockout Eimeria tenella. The method provided by the present disclosure can successfully knock out the NMT gene from Eimeria tenella, which lays a foundation for studying the function of the Eimeria tenella gene and developing a vaccine therefor.