194 Oral - Techniques and Technology Session
Saturday April 09, 4:30 PM - 4:45 PM

Authors:
Gustavo Aguilar ¹; Milena Bauer ¹; Alessandra Vigano ¹; Carlos Jiménez-Jiménez ²; Isabel Guerrero ²; Markus Affolter ¹

Affiliations:
1) University of Basel, Switzerland; 2) Centro de Biología Molecular Severo Ochoa (CSIC-UAM), Spain

Keywords:
g. CRISPR/Cas9; g. wing disc

Precise genome engineering is essential for both basic and applied research, permitting the manipulation of genes and gene products in predictable ways. The irruption of CRISPR/Cas technology rocketed the speed and ease by which 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 control of gene labelling would be ideal. Here, we propose a method based on Homologous Directed Repair (HDR) and Single Stranded Annealing (SSA) repair pathways. HDR mediates the integration of a switchable cassette. Upon a subsequent CRISPR-triggered repair event, resolved by SSA, the cassette is seamlessly removed. We named the technique SEED/harvest. By engineering the Hedgehog pathway components, we demonstrated fast and robust knock-in generation with both fluorescent proteins and short protein tags in tandem. The implementation of short homology arms, further simplified and cheapen the process. Seamless knock-in generation can be achieved in as fast as one and a half months.
The use of SEED cassettes is not restricted the germ line. SSA can also be triggered in somatic cells, permitting conditional gene labelling. This is the first time CRISPR is used to trigger endogenous tissue-specific gene labelling. Since the technology is solely based on CRISPR, it can be easily used in combination with other tissue-specific CRISPR tools, as membrane labelling and generation of knock-outs. We provide evidence of cell-type specific endogenous labeling as well as temporal control of gene labeling, mediated by conditional expression of guideRNAs.
While our tools permit control of protein labeling with fluorescent proteins, the in vivo visualization and manipulation of proteins with short tags in tandem still represents a challenge. To complement the SEED technology, we have developed a toolkit based on rational nanobody engineering and functionalization that permits the visualization and manipulation of proteins tagged with short tags.