An efficient CRISPR-based strategy to insert small and large fragments of DNA using short homology arms

Abstract

We previously reported a CRISPR-mediated knock-in strategy into introns of Drosophila genes, generating an attP-FRT-SA-T2A-GAL4-polyA-3XP3-EGFP-FRT-attP transgenic library for multiple uses (Lee et al., 2018b). The method relied on double stranded DNA (dsDNA) homology donors with ~1 kb homology arms. Here, we describe three new simpler ways to edit genes in flies. We create single stranded DNA (ssDNA) donors using PCR and add 100 nt of homology on each side of an integration cassette, followed by enzymatic removal of one strand. Using this method, we generated GFP-tagged proteins that mark organelles in S2 cells. We then describe two dsDNA methods using cheap synthesized donors flanked by 100 nt homology arms and gRNA target sites cloned into a plasmid. Upon injection, donor DNA (1 to 5 kb) is released from the plasmid by Cas9. The cassette integrates efficiently and precisely in vivo. The approach is fast, cheap, and scalable.

Use of the CRISPR‐Cas9 System in Drosophila Cultured Cells to Introduce Fluorescent Tags into Endogenous Genes

“…We note that Kanca et al. () report isolation of GFP‐tagged cell lines using the ssDNA Drop‐In approach for some targets expressed at moderate or low levels.…”

“…Example results, including information about molecular and image‐based validation of successful knock‐in events, are presented in Kanca et al. () and Bosch et al. ().…”

“…The efficiency of introduction of tags into Drosophila cells is dramatically improved by introduction of the CRISPR‐Cas9 system as a tool to facilitate insertion or ‘knock in’ of an insertion cassette into a specific locus. Indeed, CRISPR approaches have been successfully used to generate Drosophila cells in which endogenous loci are tagged by GFP fusion, e.g., Bosch, Colbeth, Zirin, & Perrimon, ; Bottcher et al., ; Kanca et al., ; Kunzelmann, Bottcher, Schmidts, & Forstemann, ; Wang et al., .…”

“…The standard protocol involves production of a plasmid donor with ∼500‐ to 1000‐bp homology arms (Housden & Perrimon, ). Alternative approaches, as presented here, can accelerate the CRISPR knock‐in workflow, for example by making it easier to obtain or prepare donor constructs (Bosch et al., ; Kanca et al., ). Specifically, we present, as alternatives to the standard approach, a single‐stranded DNA (ssDNA) “Drop‐In” method based on in vitro synthesis of an ssDNA donor (Kanca et al., ; Basic Protocol 1) and a “CRISPaint”‐based approach that relies on universal donors (Bosch et al., ; Schmid‐Burgk, Honing, Ebert, & Hornung, ; Basic Protocol 2).…”

“…These improve donor production efficiency; however, the size of the ssDNA insertion cassette is limited compared to the standard approach due to the way in which the ssDNA is generated (Support Protocol 2). The specific ssDNA Drop‐In protocol described here corresponds to the research report by Kanca et al., () and is based on insertion of sfGFP as an artificial exon (Basic Protocol 1). With the CRISPaint method (Basic Protocol 2), there are no gene‐specific regions in the donor; however, because the donor plasmid is linearized and integrated in full into the target locus, the CRISPaint method is only useful for C‐terminal tagging.…”