CRIMP: a CRISPR/Cas9 insertional mutagenesis protocol and toolkit.

Area of Science:

• Genetics
• Molecular Biology
• Developmental Biology

Background:

• Site-directed insertion is crucial for generating mutant alleles but often suffers from low efficiency and requires extensive customization.
• Existing methods can be inefficient and labor-intensive, limiting their broad applicability in genetic research.

Purpose of the Study:

• To develop a highly efficient and versatile targeted insertional mutagenesis system to overcome the limitations of current methods.
• To create a plasmid toolkit that simplifies and standardizes the generation of null mutant alleles.

Main Methods:

• Development of the CRISPR-mediated random insertion mutagenesis and protein-trapping (CRIMP) system.
• Creation of the CRIMPkit, a collection of 24 ready-to-use plasmid vectors for seamless gene mutagenesis.
• Utilizing fluorescent readouts for efficient selection of mutagenized individuals.

Main Results:

• CRIMP system achieves high frequency of targeted insertion events, enabling very early mutagenesis during the first cell division.
• CRIMPkit facilitates complete disruption of gene expression, generating null alleles without inducing genetic compensation.
• Fluorescent markers allow for easy identification of successfully mutagenized fish and heterozygous/mutant animals.

Conclusions:

• The CRIMP system and CRIMPkit significantly enhance the efficiency and ease of generating and studying mutant lines.
• These tools provide a standardized and customizable approach for rapid gene function analysis in model organisms.
• The ability to generate early-stage mutations facilitates studies on developmental processes and gene function in early embryos.