A Rapid and Facile Pipeline for Generating Genomic Point Mutants in <em>C. elegans</em> Using CRISPR/Cas9 Ribonucleoproteins

Harriet Prior; Lauren MacConnachie; Jose L. Martinez; Georgina C.B. Nicholl; Asim A. Beg

doi:10.3791/57518

Un rapide et Facile de Pipeline pour générer des Mutants de Point génomique dans c. elegans

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Genetics

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JoVE Journal Genetics

A Rapid and Facile Pipeline for Generating Genomic Point Mutants in C. elegans Using CRISPR/Cas9 Ribonucleoproteins

Un rapide et Facile de Pipeline pour générer des Mutants de Point génomique dans c. elegans en utilisant CRISPR/Cas9 ribonucléoprotéines

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08:37 min

April 30, 2018

DOI: 10.3791/57518-v

Harriet Prior*¹, Lauren MacConnachie*¹, Jose L. Martinez¹, Georgina C.B. Nicholl¹, Asim A. Beg¹

¹Department of Pharmacology,University of Michigan

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Overview

This article presents a method for engineering the genome of C. elegans using CRISPR-Cas9 ribonucleoproteins and homology dependent repair templates. This technique allows for the rapid generation of genomic point mutations, facilitating the functional study of genetic variance in the context of endogenous regulatory control.

Key Study Components

Area of Science

Neuroscience
Genetics
CRISPR Technology

Background

C. elegans is a model organism widely used in genetic studies.
Understanding genetic variance is crucial for insights into neurodegenerative diseases.
CRISPR-Cas9 technology enables precise genome editing.
Homology directed repair templates are essential for generating specific mutations.

Purpose of Study

To generate genomic point mutations in C. elegans.
To investigate the pathological consequences of genetic variance.
To enhance the understanding of neurodegenerative diseases.

Methods Used

Utilization of CRISPR-Cas9 ribonucleoproteins.
Application of single stranded oligonucleotide homology directed repair templates.
Selection of sgRNA targets based on specific genomic sequences.
Rapid generation of mutations for functional studies.

Main Results

Successful generation of genomic point mutations in C. elegans.
Facilitated functional studies of genetic variance.
Demonstrated the advantages of avoiding protein overexpression.
Provided a protocol for sgRNA target selection.

Conclusions

This method offers a rapid approach to genome editing in C. elegans.
It aids in understanding the genetic basis of neurodegenerative diseases.
The technique can be applied to various genetic studies.

Frequently Asked Questions

What is the main advantage of using CRISPR-Cas9 in C. elegans?

The main advantage is the rapid generation of genomic point mutations, allowing for functional studies without the complications of protein overexpression.

How does this method contribute to neuroscience research?

It helps in determining the pathological consequences of genetic variance associated with neurodegenerative diseases.

What are homology directed repair templates?

They are single stranded oligonucleotides used to guide the repair of DNA during the genome editing process.

Can this method be used for other organisms?

While this method is specifically designed for C. elegans, similar CRISPR techniques can be adapted for other organisms.

What is the role of sgRNA in this process?

sgRNA is used to guide the CRISPR-Cas9 complex to the specific genomic location for editing.

Is there a specific protocol for selecting sgRNA targets?

Yes, the protocol involves inputting a sequence flanking the desired edit into a designated webpage for target selection.

Nous présentons ici une méthode pour l’ingénieur le génome de c. elegans en utilisant CRISPR-Cas9 ribonucléoprotéines et modèles de réparation dépendant d’homologie.

L’objectif global de cette procédure est de générer des mutations génomiques ponctuelles chez C. Elegans en utilisant des modèles de réparation dirigée par homologie oligonucléotidique simple brin et des ribonucléoprotéines CRISPR/Cas9. Cette méthode peut aider à répondre à des questions clés dans le domaine des neurosciences, telles que la détermination des conséquences pathologiques de la variance génétique associée aux maladies neurodégénératives. Le principal avantage de cette technique est que des mutations ponctuelles génomiques peuvent être générées rapidement, ce qui permet l’étude fonctionnelle de la variance génétique dans le contexte d’un contrôle régulateur endogène, tout en évitant les mises en garde de la surexpression des protéines.

Pour effectuer la sélection de la cible de l’ARNsg, ouvrez la page Web illustrée ici. Entrez environ 60 paires de bases de séquence flanquant le montage souhaité qui vous intéresse. Après avoir sélectionné le génome de C.Elegans et le motif adjacent protospacer selon le protocole de texte, cliquez sur Soumettre.

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