Heidelberg CFD CRISPR Library
The Heidelberg CFD CRISPR Library (HD_CFD) is a large-scale collection of transgenic flies for CRISPR-mediated genome engineering in Drosophila.
The Heidelberg CFD CRISPR Library has been generated by the laboratory of Michael Boutros at the German Cancer Research Center (DKFZ) and Heidelberg University and generously donated to the VDRC for distribution to the Drosophila research community.
Full details about the lines can be found in the following eLife publication: A large-scale resource for tissue-specific CRISPR mutagenesis in Drosophila, Port et al (2020).
By selecting the Heidelberg CFD CRISPR Library tools and sgRNA lines most relevant for your experiment, it is possible to generate loss-of-function mutations in essential or non-essential genes in a germline restricted, ubiquitous or tissue specific fashion.
Robust gene knock-outs in a large fraction of cells can be achieved in a strict spatially and temporally controlled manner, by bringing together two sgRNAs targeting the same gene (HD_CFD lines) and Cas9 (HD_CFDtools) under the control of Gal4.
See also https://www.crisprflydesign.org
What are the Heidelberg CFD CRISPR (HD_CFD) lines?
The collection consists primarily of a large-scale library of flies containing transgenic short guide (sg) RNAs (~1600 lines) each expressing two sgRNAs under UAS control. The focus is on targeting kinases, phosphatases and transcription factors, as well as several other genes encoding fly orthologs of genes implicated in human pathologies. Conditional CRISPR mutagenesis using these lines has been shown to be robust across many target genes and can be efficiently employed in various somatic tissues, as well as the germline. This collection has also been shown to be suitable for carrying out large-scale loss of function screens.
What are the Heidelberg CFD CRISPR toolbox (HD_CFDtools) lines?
The Heidelberg CFD CRISPR Library contains a set of toolbox stocks (HD_CFDtools), including a series of UAS-Cas9 lines containing an upstream open reading frame (uORF) of varying length from XS to XXL. These produce a range of expression levels of Cas9 and enable high gene editing activity without detectable toxicity or artefacts that can potentially be caused by high levels of Cas9. The longer the uORF, the lower the Cas9 expression, so the most appropriate Cas9 line to use for an experiment may be carefully selected based on the desired expression level.
To facilitate your experiments further, some lines have already been created to combine UAS-Cas9 with common Gal4 driver lines (e.g. act-, hh-, nub-, ptc-, GMR-, dpp- and vg-Gal4). Such stocks can be crossed to transgenic sgRNA lines to induce conditional CRISPR mutagenesis in Gal4 expressing cells.
Lines for induction of Cas9 expression by FLP-out are also available. Cas9 can be induced in all Gal4 expressing cells or only in a random subset, with the latter approach resulting in fluorescently marked mosaics. Such mosaics can be a powerful method to analyze neighbouring mutant and wildtype cells in the same tissue.
How do I acknowledge use of these lines?
When using lines from the Heidelberg CFD CRISPR Library, please acknowledge Fillip Port and Michael Boutros for providing fly strains, cite “A large-scale resource for tissue-specific CRISPR mutagenesis in Drosophila, Port et al (2020)” https://elifesciences.org/articles/53865, and use the HD-CFD* identifier for each stock in your publications.
Additionally, please acknowledge the VDRC for distributing fly lines. A simple statement is sufficient and can either be placed in the Materials and Methods section or in the Acknowledgements.
Transgenic fly stocks and/or plasmids were obtained from the Vienna Drosophila Resource Center (VDRC, www.vdrc.at).