In vivoseamless genetic engineering via CRISPR-triggered single-strand annealing

Abstract

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 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.

Heterodimerization-dependent secretion of BMPs in Drosophila

“…To insert the OLLAS:HA-Tag the SEED/Harvest Method (Aguilar et al, 2022) was employed. Briefly, a cassette including 185bp homology arms was synthesized and clone by Genewiz into pUC-GW plasmid.…”

“…In both cases, the PAM sequence of the sgRNA target was changed from GGT to GAT to avoid cutting of the donor template. To insert the OLLAS:HA-Tag the SEED/Harvest Method (Aguilar et al, 2022) was employed. Briefly, a cassette including 185bp homology arms was synthesized and clone by Genewiz into pUC-GW plasmid.…”

“…Briefly, a cassette including 185bp homology arms was synthesized and clone by Genewiz into pUC-GW plasmid. Subsequently, the OLLAS:HA SEED cassette, including the selectable marker was introduced into this vector according to the protocol proposed in: (Aguilar et al, 2022). nos-Cas9 flies (BL 54591) were crossed with P(U6-gbb.gRNA)attP40 males and injected the ssDNA or SEED templates.…”

“…Integration of HA and OLLAS:HA was tested with the following primer pair: 5'-TGCGGCCGCCTGCATAGTCC-3', 5'-CAAGTGGCTGACCGCC-3' Integration of Ollas was tested with the following primer pair: 5'-TGCGGCCGCCTTTACCCATC-3'; 5'-CAAGTGGCTGACCGCC-3' In both cases correct integration resulted in a PCR band of 500bps. SEED stocks were established following the crossing scheme reported in (Aguilar et al 2022). OLLAS-dpp flies were generated as described for HA-dpp in Matsuda et al, 2021. Instead of an HA-tag, a fragment encoding for the OLLAS-tag was inserted between the XhoI and NheI sites in the plasmid pBS-attb-Dpp4.4 for injection.…”

“…The copyright holder for this preprint this version posted August 5, 2022. ; https://doi.org/10.1101/2022.08.04.502599 doi: bioRxiv preprint Supplement 1: Schematic outline of OLLAS-HA double-tag integration in the gbb-locus. To knock-in the OLLAS-HA double tag into the gbb-locus the SEED/Harvest method described in Aguilar et al, 2022 was used. The gRNA and Cas9 were provided by integrated transgenes.…”