Seamless knockins in Drosophila via CRISPR-triggered single-strand annealing

Protocol for generating in-frame seamless knockins in Drosophila using the SEED/Harvest technology

“…The selectable marker of SEED cassettes is either 3xP3-dsRED or 3xP3-GFP. Both markers are strongly expressed in different developmental stages, 1 , 2 as well as in adult flies. Flanking the marker, the tag to be inserted is split in two parts, 5′ and 3′, sharing an identical sequence repeat between 100 and 400 base pairs.…”

“…CRISPR-mediated cleavage of these targets induces repair of the SEED cassette via the Single-Strand Annealing pathway (SSA), seamlessly removing the screening marker (see Aguilar et al. for details 1 ). To accelerate this step, transgenic flies are available simultaneously expressing Cas9 and both 1# and 2# gRNAs.…”

“… CRITICAL: If tissue-specific labeling is intended, only SEED cassettes encoding FPs can be used (See Aguilar et al. 1 ). While fluorescent tags result in a truncated fluorescent protein unless rearranged, the shorter peptide tags are always incorporated in both rearranged and non-rearranged cassettes.…”

“…However, for the success of this approach in vivo it is critical to utilize conditionally stable protein binders. 1 In vivo manipulation. The high intracellular stability of nanobodies and other protein binders (engineered scFvs, DARPins, monobodies, etc.)…”

“…However, for the success of this approach in vivo it is critical to utilize conditionally stable protein binders. 1 …”