An efficient CRISPR-based strategy to insert small and large fragments of DNA using short homology arms
- Baylor College of Medicine, United States
- Harvard Medical School, United States
- Janelia Research Campus, Howard Hughes Medical Institute, United States
- Howard Hughes Medical Institute, Carnegie Institution for Science, United States
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.
Data availability
All the fly lines and cell lines generated in this manuscript will be made available through Bloomington Drosophila Stock center and Drosophila Genomics Resource Center
Article and author information
Author details
Funding
NIH Office of the Director (R01GM067858)
- Hugo J Bellen
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Copyright
© 2019, Kanca et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
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An efficient CRISPR-based strategy to insert small and large fragments of DNA using short homology arms
eLife 8:e51539.
https://doi.org/10.7554/eLife.51539
Further reading
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