RESUMEN
CRISPR-Cas greatly facilitated the integration of exogenous sequences into specific loci. However, knockin generation in multicellular animals remains challenging, partially due to the complexity of insertion screening. Here, we describe SEED/Harvest, a method to generate knockins in Drosophila, based on CRISPR-Cas and the single-strand annealing (SSA) repair pathway. In SEED (from "scarless editing by element deletion"), a switchable cassette is first integrated into the target locus. In a subsequent CRISPR-triggered repair event, resolved by SSA, the cassette is seamlessly removed. Germline excision of SEED cassettes allows for fast and robust knockin generation of both fluorescent proteins and short protein tags in tandem. Tissue-specific expression of Cas9 results in somatic cassette excision, conferring spatiotemporal control of protein labeling and the conditional rescue of mutants. Finally, to achieve conditional protein labeling and manipulation of short tag knockins, we developed a genetic toolbox by functionalizing the ALFA nanobody.
RESUMEN
Developmental biology has greatly profited from genetic and reverse genetic approaches to indirectly studying protein function. More recently, nanobodies and other protein binders derived from different synthetic scaffolds have been used to directly dissect protein function. Protein binders have been fused to functional domains, such as to lead to protein degradation, relocalization, visualization, or posttranslational modification of the target protein upon binding. The use of such functionalized protein binders has allowed the study of the proteome during development in an unprecedented manner. In the coming years, the advent of the computational design of protein binders, together with further advances in scaffold engineering and synthetic biology, will fuel the development of novel protein binder-based technologies. Studying the proteome with increased precision will contribute to a better understanding of the immense molecular complexities hidden each step along the way to generate form and function during development.
RESUMEN
The generation of knockins is fundamental to dissect biological systems. SEED/Harvest, a technology based on CRISPR-Cas9, offers a powerful approach for seamless genome editing in Drosophila. Here, we present a protocol to tag any gene in the Drosophila genome using SEED/Harvest technology. We describe knockin design, plasmid preparation, injection, and insertion screening. We then detail procedures for germline harvesting. The technique combines straightforward cloning and robust screening of insertions, while still resulting in scarless gene editing. For complete details on the use and execution of this protocol, please refer to Aguilar et al.1.