CRIMP: a CRISPR/Cas9 insertional mutagenesis protocol and toolkit

• Published in: Nature communications Vol. 15; no. 1; pp. 5011 - 11
• Main Authors: Miles, Lee B., Calcinotto, Vanessa, Oveissi, Sara, Serrano, Rita J., Sonntag, Carmen, Mulia, Orlen, Lee, Clara, Bryson-Richardson, Robert J.
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group UK 12-06-2024; Nature Publishing Group; Nature Portfolio
• Subjects: 13/44; 13/51; 14; 38; 38/35; 42/41; 42/70; 631/1647/1511; 631/208/4041/3196; 64; 64/116; 692/308/1426; Alleles; Animals; Cell division; CRISPR; CRISPR-Cas Systems; Effectiveness; Embryos; Fluorescence; Folding; Gene Editing - methods; Gene expression; Genetic Vectors - genetics; Humanities and Social Sciences; Insertion; Insertional mutagenesis; multidisciplinary; Mutagenesis; Mutagenesis, Insertional - methods; Mutants; Plasmids - genetics; Science; Science (multidisciplinary); Toolkits; Transcription termination; Zebrafish - genetics
• ISSN: 2041-1723; 2041-1723
• DOI: 10.1038/s41467-024-49341-7

Site-directed insertion is a powerful approach for generating mutant alleles, but low efficiency and the need for customisation for each target has limited its application. To overcome this, we developed a highly efficient targeted insertional mutagenesis system, CRIMP, and an associated plasmid toolkit, CRIMPkit, that disrupts native gene expression by inducing complete transcriptional termination, generating null mutant alleles without inducing genetic compensation. The protocol results in a high frequency of integration events and can generate very early targeted insertions, during the first cell division, producing embryos with expression in one or both halves of the body plan. Fluorescent readout of integration events facilitates selection of successfully mutagenized fish and, subsequently, visual identification of heterozygous and mutant animals. Together, these advances greatly improve the efficacy of generating and studying mutant lines. The CRIMPkit contains 24 ready-to-use plasmid vectors to allow easy and complete mutagenesis of any gene in any reading frame without requiring custom sequences, modification, or subcloning.
Site-directed insertion is a powerful approach for generating mutant lines but, historically, technically challenging. Here, the authors demonstrate CRIMP, an optimised protocol and universal toolkit (CRIMPkit), to greatly improve the efficacy of generating and studying mutant lines.