In vivo seamless genetic engineering via CRISPR-triggered single-strand annealing

Abstract

AbstractPrecise genome engineering is essential for both basic and applied research, permitting the manipulation of genes and gene products in predictable ways. The irruption of the CRISPR/Cas technology accelerated the speed and ease by which defined exogenous sequences are integrated into specific loci. To this day, a number of strategies permit gene manipulation. Nevertheless, knock-in generation in multicellular animals remains challenging, partially due to the complexity of insertion screening. Even when achieved, the analysis of protein localization can still be unfeasible in highly packed tissues, where spatial and temporal control of gene labeling would be ideal. Here, we propose an efficient method based on homology-directed repair (HDR) and single-strand annealing (SSA) repair pathways. In this method, HDR mediates the integration of a switchable cassette. Upon a subsequent CRISPR-triggered repair event, resolved by SSA, the cassette is seamlessly removed. By engineering the Hedgehog (Hh) pathway components, we demonstrated fast and robust knock-in generation with both fluorescent proteins and short protein tags in tandem. The use of homology arms as short as 30 base pairs further simplified and cheapened the process. In addition, SSA can be triggered in somatic cells, permitting conditional gene labeling in different tissues. Finally, to achieve conditional labeling and manipulation of proteins tagged with short protein tags, we have further developed a toolbox based on rational engineering and functionalization of the ALFA nanobody.Significance statementCRISPR/Cas9 has revolutionized genome editing. However, seamless editing in multicellular organisms still presents many challenges, mainly derived from insertion screening. The tool we have developed permits fast, robust and cheap gene editing mediated by the SSA repair pathway. This pathway is highly conserved in different animal species; hence the methodology is a promising alternative for gene editing across organisms. We demonstrate that this approach can be used to achieve spatio-temporal control of gene-labeling, mediated by somatic Cas9 expression. This addition to the CRISPR repertoire opens a new avenue for the study of protein function and distribution, alone or in combination with other CRISPR based technologies. We further engineered a nanobody-based toolbox that permits precise manipulation and visualization of the generated knock-ins.