Title

An expanded toolkit for gene tagging based on MiMIC and scarless CRISPR tagging in

Publication Date

8-9-2018

Journal

eLife

DOI

10.7554/eLife.38709

PMID

30091705

PMCID

6095692

Published Open-Access

yes

Keywords

Animals, Clustered Regularly Interspaced Short Palindromic Repeats, Drosophila, Entomology, Gene Targeting, Genes, Reporter, Integrases, Mutagenesis, Insertional

Abstract

We generated two new genetic tools to efficiently tag genes in Drosophila. The first, Double Header (DH) utilizes intronic MiMIC/CRIMIC insertions to generate artificial exons for GFP mediated protein trapping or T2A-GAL4 gene trapping in vivo based on Cre recombinase to avoid embryo injections. DH significantly increases integration efficiency compared to previous strategies and faithfully reports the expression pattern of genes and proteins. The second technique targets genes lacking coding introns using a two-step cassette exchange. First, we replace the endogenous gene with an excisable compact dominant marker using CRISPR making a null allele. Second, the insertion is replaced with a protein::tag cassette. This sequential manipulation allows the generation of numerous tagged alleles or insertion of other DNA fragments that facilitates multiple downstream applications. Both techniques allow precise gene manipulation and facilitate detection of gene expression, protein localization and assessment of protein function, as well as numerous other applications. Research organism: D. melanogaster

Share

COinS