Poe, A.R., Wang, B., Sapar, M.L., Ji, H., Li, K., Onabajo, T., Fazliyeva, R., Gibbs, M., Qiu, Y., Hu, Y., Han, C. (2019). Robust CRISPR/Cas9-Mediated Tissue-Specific Mutagenesis Reveals Gene Redundancy and Perdurance in Drosophila.  Genetics 211(2): 459--472.

FBrf0241409

Research paper

Tissue-specific loss-of-function (LOF) analysis is essential for characterizing gene function. Here, we present a simple, yet highly efficient, clustered regularly interspaced short palindromic repeats (CRISPR)-mediated tissue-restricted mutagenesis (CRISPR-TRiM) method for ablating gene function in Drosophila This binary system consists of a tissue-specific Cas9 and a ubiquitously expressed multi-guide RNA (gRNA) transgene. We describe convenient toolkits for making enhancer-driven Cas9 lines and multi-gRNAs that are optimized for mutagenizing somatic cells. We demonstrate that insertions or deletions in coding sequences more reliably cause somatic mutations than DNA excisions induced by two gRNAs. We further show that enhancer-driven Cas9 is less cytotoxic yet results in more complete LOF than Gal4-driven Cas9 in larval sensory neurons. Finally, CRISPR-TRiM efficiently unmasks redundant soluble N-ethylmaleimide-sensitive factor attachment protein receptor gene functions in neurons and epidermal cells. Importantly, Cas9 transgenes expressed at different times in the neuronal lineage reveal the extent to which gene products persist in cells after tissue-specific gene knockout. These CRISPR tools can be applied to analyze tissue-specific gene function in many biological processes.

PMC6366929 (PMC) (EuropePMC)