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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- Chromosomes and Gene Expression
We generated a library of ~1000 Drosophila stocks in which we inserted a construct in the intron of genes allowing expression of GAL4 under control of endogenous promoters while arresting transcription with a polyadenylation signal 3’ of the GAL4. This allows numerous applications. First, ~90% of insertions in essential genes cause a severe loss-of-function phenotype, an effective way to mutagenize genes. Interestingly, 12/14 chromosomes engineered through CRISPR do not carry second-site lethal mutations. Second, 26/36 (70%) of lethal insertions tested are rescued with a single UAS-cDNA construct. Third, loss-of-function phenotypes associated with many GAL4 insertions can be reverted by excision with UAS-flippase. Fourth, GAL4 driven UAS-GFP/RFP reports tissue and cell-type specificity of gene expression with high sensitivity. We report the expression of hundreds of genes not previously reported. Finally, inserted cassettes can be replaced with GFP or any DNA. These stocks comprise a powerful resource for assessing gene function.
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- Developmental Biology
- Genetics and Genomics
Repurposing of pleiotropic factors during execution of diverse cellular processes has emerged as a regulatory paradigm. Embryonic development in metazoans is controlled by maternal factors deposited in the egg during oogenesis. Here, we explore maternal role(s) of Caspar (Casp), the Drosophila orthologue of human Fas-associated factor-1 (FAF1) originally implicated in host-defense as a negative regulator of NF-κB signaling. Maternal loss of either Casp or it’s protein partner, transitional endoplasmic reticulum 94 (TER94) leads to partial embryonic lethality correlated with aberrant centrosome behavior, cytoskeletal abnormalities, and defective gastrulation. Although ubiquitously distributed, both proteins are enriched in the primordial germ cells (PGCs), and in keeping with the centrosome problems, mutant embryos display a significant reduction in the PGC count. Moreover, the total number of pole buds is directly proportional to the level of Casp. Consistently, it’s ‘loss’ and ‘gain’ results in respective reduction and increase in the Oskar protein levels, the master determinant of PGC fate. To elucidate this regulatory loop, we analyzed several known components of mid-blastula transition and identify the translational repressor Smaug, a zygotic regulator of germ cell specification, as a potential critical target. We present a detailed structure-function analysis of Casp aimed at understanding its novel involvement during PGC development.
