Developing new methods to manipulate fly genes offers a strong driving force to better understand their function. The main goal of our initiative is to develop a strategy that allows multiple manipulations of a fly gene in
an integrated fashion using a single tool. The strategy that we developed utilizes CRISPR and is named CRIMIC (CRISPR-Mediated Integration Cassette). In simple terms, a CRIMIC insertion can allow us to
- mutate a gene;
- assess where the gene is expressed;
- determine the subcellular localization of the encoded protein;
- identify proteins that interact with the encoded protein;
- remove the protein in any tissue of interest in a temporally controlled and reversible manner;
- create tagged clones of wild type and mutant tissue;
- and perform a structure function analysis of the protein.
Some of these features are shown in Figure 1.
In the Gene Disruption Project, our lab, in collaboration with Norbert Perrimon (Harvard) and Allan Spradling (Carnegie), creates the reagents needed to implement the above mentioned technologies for most genes. Currently,
more than 22,000 stocks created in our labs are distributed by the Bloomington Drosophila Stock Center (BDSC) worldwide. These stocks collectively allow manipulations of more than 70% of all fly genes. Nearly 900,000 copies of these stocks have been distributed, proving the popularity and utility of our resources.
Over the past few years, we have tagged more than 2,000 genes with the multifunctional tags described above (see the Gene Disruption Project database). We are in the process of inserting these very versatile tags in thousands of additional genes. These insertions integrate a GAL4 gene into a gene of interest and these GAL4 insertions allow us, for example, to drive UAS-human cDNAs to assess if human cDNAs can rescue the fly loss of function mutation. To this end we are generating a collection of fly transgenic stocks that carry UAS-human cDNAs in collaboration with Sue Celniker (LBNL), Shinya Yamamoto (BCM) and Toshiyuki Takano-Shimizu (Kyoto Stock Center).
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