Abstract
Site-specific insertion of DNA into endogenous genes (knock-in) is a powerful method to study gene function. However, traditional methods for knock-in require laborious cloning of long homology arms for homology-directed repair. Here, we report a simplified method in Drosophila melanogaster to insert large DNA elements into any gene using homology-independent repair. This method, known as CRISPaint, employs CRISPR-Cas9 and non-homologous end joining (NHEJ) to linearize and insert donor plasmid DNA into a target genomic cut site. The inclusion of commonly used elements such as GFP on donor plasmids makes them universal, abolishing the need to create gene-specific homology arms and greatly reducing user workload. Using this method, we show robust gene-specific integration of donor plasmids in cultured cells and the fly germ line. Furthermore, we use this method to analyze gene function by fluorescently tagging endogenous proteins, disrupting gene function, and generating reporters of gene expression. Finally, we assemble a collection of donor plasmids for germ line knock-in that contain commonly used insert sequences. This method simplifies the generation of site-specific large DNA insertions in Drosophila cell lines and fly strains, and better enables researchers to dissect gene function in vivo.
Summary We report a new homology-independent genomic knock-in method in Drosophila to insert large DNA elements into any target gene. Using CRISPR-Cas9 and non-homologous end joining (NHEJ), an entire donor plasmid is inserted into the genome without the need for homology arms. This approach eliminates the burden associated with designing and constructing traditional donor plasmids. We demonstrate its usefulness in cultured cells and in vivo to fluorescently tag endogenous proteins, generate reporters of gene expression, and disrupt gene function.
