Heidelberg CFD CRISPR Library
The CRISPRflydesign (CFD) team in the laboratory of Michael Boutros at the German Cancer Research Center (DKFZ) and Heidelberg University have generated a number of large-scale collections of transgenic flies for CRISPR-mediated genome engineering in Drosophila and have generously donated them to the VDRC for distribution to the Drosophila research community:
- HD CFD stocks for Cas9-mediated mutagenesis
- HD CFD stocks for Cas12a-mediated mutagenesis
- HD CFD Base Editing stocks
HD CFD stocks for Cas9-mediated mutagenesis
By selecting the Heidelberg CFD CRISPR Library Cas9 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.
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).
See also https://www.crisprflydesign.org
HD CFD Cas9 sgRNA Stocks (HD-CFD-Cas9)
HDtools Cas9 Stocks (HDtools-Cas9)
What are the Heidelberg CFD CRISPR Cas9 (HD_CFD_Cas9) lines?
- The collection consists primarily of a large-scale library of flies containing transgenic short guide (sg) RNAs (~2000 lines) each expressing two sgRNAs under UAS control.
- The focus is on targeting kinases, phosphatases and transcription factors, as well as 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.
Suggested format:
Transgenic fly stocks and/or plasmids were obtained from the Vienna Drosophila Resource Center (VDRC, www.vdrc.at).
HD CFD stocks for Cas12a-mediated mutagenesis
By selecting the Heidelberg CFD CRISPR Library Cas12a tools and Cas12a 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 four sgRNAs targeting the same gene (HD_CFD Cas12a sgRNA lines) and Cas12a (HDtools-Cas12a) under the control of Gal4.
This highly efficient and specific gene targeting system combines an enhanced variant of Cas12a with quadruple sgRNA arrays. When targeting sites in parallel, sgRNAs can act synergistically to induce deletions between target sites, generating mutations that are more likely to disrupt gene function with undetectable off-target cutting. Direct comparisons with established Cas9-based methods demonstrate superior knockout efficiency of this system across diverse tissues and target genes.
Full details about the lines can be found in the following publication: Enhanced in vivo gene knockout with undetectable off-targets using multiplexed Cas12a sgRNAs, Port et al (2024).
See also https://www.crisprflydesign.org
HD CFD Cas12a sgRNA Stocks (HD-CFD-Cas12a)
HDtools Cas12a Stocks (HDtools-Cas12a)
What are the Heidelberg CFD Cas12a sgRNA (HD_CFD_Cas12a) lines?
- The collection consists primarily of a large-scale Cas12a-based library of flies containing transgenic short guide (sg) RNAs (~850 lines) each line expressing four sgRNAs targeting a single gene under UAS control.
- Multiplexed sgRNAs act synergistically to create deletions between target sites, substantially increasing the fraction of loss-of-function mutations compared to Cas9-based systems.
- 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 is also suitable for carrying out large-scale loss of function screens.
What are the Heidelberg CFD Cas12a toolbox (HDtools-Cas12a) lines?
- The Heidelberg CFD Cas12a toolbox Library contains a set of toolbox stocks (HDtools-Cas12a), for expression of an enhanced variant of the Cas12a CRISPR nuclease, known as Cas12a+. Cas12a+ enables high gene editing activity without detectable toxicity or artefacts and can be expressed either ubiquitously, in the germline or in a spatially/temporally restricted manner.
- To facilitate your experiments further, some lines have already been created to combine UAS-Cas12a with common Gal4 driver lines (e.g. nos-, hh- and pdm2-Gal4). Such stocks can be crossed to transgenic sgRNA lines to induce conditional CRISPR mutagenesis in Gal4 expressing cells.
- Lines for induction of Cas12a expression by FLP-out are also available. Cas12a 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 Cas12a CRISPR Library, please acknowledge Fillip Port and Michael Boutros for providing fly strains, cite “Enhanced in vivo gene knockout with undetectable off-targets using multiplexed Cas12a sgRNAs; Port et al (2024)” https://doi.org/10.1101/2024.11.26.625385.
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.
Suggested format:
Transgenic fly stocks and/or plasmids were obtained from the Vienna Drosophila Resource Center (VDRC, www.vdrc.at).
HD CFD Base Editing stocks
DNA base editors are engineered ribonucleoprotein complexes which, in combination with appropriately designed sgRNAs, can create single base substitutions in DNA. The HD CFD Base Editing stocks are optimized C-to-T base editing (CBE) systems for the generation of precise loss- or gain-of-function alleles in Drosophila.
The predictable editing outcome, high efficiency, and product purity enables near homogeneous induction of precise alleles such as STOP codons or alleles encoding protein variants in vivo. Defined missense variants can be efficiently generated in both soma and germline.
For ubiquitous base editing, base editors have been cloned downstream of the act5c promoter (pAct-dCBEevoAPOBEC1 and pAct-dCBEevoCDA1). Additional lines linking base editor expression to the binary Gal4/UAS and FLP/FRT systems are available, enabling control of base editor activity effectively in time and space.
Full details about the lines can be found in the following publication: A temperature-tolerant CRISPR base editor mediates highly efficient and precise gene editing in Drosophila; Doll et al., Sci Adv (2023)
See also https://www.crisprflydesign.org
HD CFD Base Editing Stocks (HD-CFD-Base Editing)
How do I acknowledge use of these lines?
When using lines from the Heidelberg CFD Base Editing Library, please acknowledge Fillip Port and Michael Boutros for providing fly strains, cite “A temperature-tolerant CRISPR base editor mediates highly efficient and precise gene editing in Drosophila; Doll et al., Sci Adv (2023)” https://www.science.org/doi/10.1126/sciadv.adj1568.
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.
Suggested format:
Transgenic fly stocks and/or plasmids were obtained from the Vienna Drosophila Resource Center (VDRC, www.vdrc.at).