FlyBook review on RNAi screening in Drosophila cells and in vivo

Heigwer F, Port F, Boutros M. RNA Interference (RNAi) Screening in Drosophila. Genetics. 2018 Mar;208(3):853-874. PMID: 29487145.

From the abstract: "... RNA interference (RNAi) ... has had an important impact on identifying and characterizing gene function. First discovered in Caenorhabditis elegans, RNAi can be used to silence the expression of genes through introduction of exogenous double-stranded RNA into cells. In Drosophila, RNAi has been applied in cultured cells or in vivo to perturb the function of single genes or to systematically probe gene function on a genome-wide scale. In this review, we will describe the use of RNAi to study gene function in Drosophila with a particular focus on high-throughput screening methods applied in cultured cells. ..."

Drosophila CRISPR protocols series

Thinking fly CRISPR? Series of methods papers out in CSH Protocols.

Housden BE, Perrimon N. Cas9-Mediated Genome Engineering in Drosophila melanogaster. Cold Spring Harb Protoc. 2016 Sep 1;2016(9):pdb.top086843. PMID: 27587786.

Housden BE, Perrimon N. Detection of Indel Mutations in Drosophila by High-Resolution Melt Analysis (HRMA). Cold Spring Harb Protoc. 2016 Sep 1;2016(9):pdb.prot090795. PMID: 27587781.

Housden BE, Perrimon N. Design and Generation of Donor Constructs for Genome Engineering in Drosophila. Cold Spring Harb Protoc. 2016 Sep 1;2016(9):pdb.prot090787. PMID: 27587780.

Housden BE, Hu Y, Perrimon N. Design and Generation of Drosophila Single Guide RNA Expression Constructs. Cold Spring Harb Protoc. 2016 Sep 1;2016(9):pdb.prot090779. PMID: 27587779.

Housden BE, Hu Y, Perrimon N. Design and Generation of Drosophila Single Guide RNA Expression Constructs. Cold Spring Harb Protoc. 2016 Sep 1;2016(9):pdb.prot090779. PMID: 27587779.

From the abstract: "... Here, we provide a step-by-step protocol for designing sgRNA target sites using the Drosophila RNAi Screening Center (DRSC) Find CRISPRs tool (version 2). We also describe the generation of sgRNA expression plasmids for the use in cultured Drosophila cells or in vivo. Finally, we discuss specific design requirements for various genome engineering applications."

Using the white gene as an indicator of CRISPR-based editing

Ge DT, Tipping C, Brodsky MH, Zamore PD. Rapid Screening for CRISPR-Directed Editing of the Drosophila Genome Using white Co-Conversion. G3 (Bethesda). 2016 Aug 19. pii: g3.116.032557. PMID: 27543296.

From the abstract: "... Here, we describe a strategy that reduces the time and effort required to identify flies with targeted genomic changes. The strategy uses editing of the white gene, evidenced by altered eye color, to predict successful editing of an unrelated gene-of-interest. The red eyes of wild-type flies are readily distinguished from white-eyed (end joining-mediated loss of White function) or brown-eyed (recombination-mediated conversion to the whitecoffee allele) mutant flies. ... We find that end joining-mediated mutations often show signatures of microhomology-mediated repair and that recombination-based mutations frequently involve donor plasmid integration at the target locus. Finally, we show that gap repair induced by two guide RNAs more reliably converts the intervening target sequence, whereas the use of Lig4169 mutants to suppress end joining does not improve recombination efficacy."

Gene editing in fly cells--new report from Kunzelmann et al. in G3

Kunzelmann S, Böttcher R, Schmidts I, Förstemann K. A Comprehensive Toolbox for Genome Editing in Cultured Drosophila melanogaster Cells. G3 (Bethesda). 2016 Jun 1;6(6):1777-85. PMID: 27172193

From the abstract: "... Following up on our initial publication, we now describe a considerably simplified, more efficient, and readily scalable experimental workflow for PCR-based genome editing in cultured Drosophila melanogaster cells. Our analysis at the act5C locus suggests that PCR-based homology arms of 60 bp are sufficient to reach targeting efficiencies of up to 80% after selection; extension to 80 bp (PCR) or 500 bp (targeting vector) did not further improve the yield. We have expanded our targeting system to N-terminal epitope tags; this also allows the generation of cell populations with heterologous expression control of the tagged locus via the copper-inducible mtnDE promoter. We present detailed, quantitative data on editing efficiencies for several genomic loci that may serve as positive controls or benchmarks in other laboratories. ..."

Combined CRISPR-RNAi approach

On my desk to read today? This report from former Perrimonian and DRSC screener R. Neumuller:

Wissel S, Kieser A, Yasugi T, Duchek P, Roitinger E, Gokcezade JF, Steinmann V, Gaul U, Mechtler K, Förstemann K, Knoblich JA, Neumüller RA. A Combination of CRISPR/Cas9 and Standardized RNAi as a Versatile Platform for the Characterization of Gene Function. G3 (Bethesda). 2016 Jun 8. pii: g3.116.028571. PMID: 27280787.

From the abstract: "... miGFPi combines CRISPR/Cas9 mediated tagging of genes at their endogenous locus with an immunotag and an exogenous 21 nucleotide RNAi effector sequence with the use of a single reagent, highly validated RNAi line targeting this sequence. We demonstrate the utility and time effectiveness of this method ... In addition, we show that miGFPi serves as a powerful technique to functionally characterize individual isoforms of a gene. ..."

Report of CRISPR-Cas modification of the emerging pest Drosophila suzukii

Interesting. Chance meeting of #blueberry farmer in #Maine brought #Drosophila suzukii #pest issue to life for me. https://t.co/MCDHHKIHV7

— Stephanie Mohr (@smohrfly) January 2, 2016

Review by Venken and colleagues on genome engineering in flies

Venken KJ, Sarrion-Perdigones A, Vandeventer PJ, Abel NS, Christiansen AE, Hoffman KL. Genome engineering: Drosophila melanogaster and beyond. Wiley Interdiscip Rev Dev Biol. 2015 Oct 8. PMID: 26447401.

From the abstract: "Here, we summarize different ways to perform precise inheritable genome engineering using integrases, recombinases, and DNA nucleases in the D. melanogaster."

New at the DRSC website--protocols for single-cell cloning and stable transfection of Drosophila cells

Two new step-by-step protocols available at the DRSC website:

• Single-cell cloning of Drosophila cells
• Making stably transfected Drosophila cells

And check out these related publications and resources:

Housden BE, Lin S, Perrimon N. Cas9-based genome editing in Drosophila. Methods Enzymol. 2014;546:415-39. PMID: 25398351.

Housden BE, Valvezan AJ, Kelley C, Sopko R, Hu Y, Roesel C,
Lin S, Buckner M, Tao R, Yilmazel B, Mohr SE, Manning BD, Perrimon N. Identification of potential drug targets for tuberous sclerosis complex by synthetic screens combining CRISPR-based knockouts with RNAi. Sci Signal. 2015 Sep 8;8(393):rs9. PMID: 26350902.

Santos MG, Jorge SA, Brillet K, Pereira CA. Improving heterologous protein expression in transfected Drosophila S2 cells as assessed by EGFP expression. Cytotechnology. 2007 May;54(1):15-24. PMID: 19003014; PMCID: PMC2267513.

Have a qPCR machine but no software for high-resolution melt analysis following CRISPR modification? Check out the HRMA online tool.

CRISPR + RNAi screening in Drosophila cells points to potential new drug targets for treatment of tuberous sclerosis complex (TSC)

Housden BE, Valvezan AJ, Kelley C, Sopko R, Hu Y, Roesel C, Lin S, Buckner M, Tao R, Yilmazel B, Mohr SE, Manning BD, Perrimon N. Identification of potential drug targets for tuberous sclerosis complex by synthetic screens combining CRISPR-based knockouts with RNAi. Sci Signal. 2015 Sep 8;8(393):rs9. PMID: 26350902.

From the abstract: "The tuberous sclerosis complex (TSC) family of tumor suppressors, TSC1 and TSC2, function together in an evolutionarily conserved protein complex ... Mutation or aberrant inhibition of the TSC complex is common in various human tumor syndromes and cancers. The discovery of novel therapeutic strategies to selectively target cells with functional loss of this complex is therefore of clinical relevance ... We developed a CRISPR-based method to generate homogeneous mutant Drosophila cell lines. By combining TSC1 or TSC2 mutant cell lines with RNAi screens against all kinases and phosphatases, we identified synthetic interactions with TSC1 and TSC2. Individual knockdown of three candidate genes ... reduced the population growth rate of Drosophila cells lacking either TSC1 or TSC2 but not that of wild-type cells ... [and] had similar growth-inhibiting effects in mammalian TSC2-deficient cell lines, including human tumor-derived cells, illustrating the power of this cross-species screening strategy to identify potential drug targets."

CRISPR activation in vivo in Drosophila

Things are looking up: #CRISPR activation in vivo in #Drosophila. Early online in #Genetics. http://t.co/zojqKxmf2G

— Stephanie Mohr (@smohrfly) August 6, 2015

CRISPR, gene drives and safety

Two recent papers speak to CRISPR, gene drives and safety:



Akbari et al. 2015 Safeguarding gene drive experiments in the laboratory. in Early online in Policy Forum section of Science.  

 Abstract: "Multiple strategies are needed to ensure safe gene drive experiments."

Unckless et al. 2015 Modeling the Manipulation of Natural Populations by the Mutagenic Chain Reaction Early online at Genetics.

From the Discussion section:  "... there are conditions in which accidental introductions of a single individual can lead to fixation of the MCR allele ..."

Drosophila CRISPR library reported by Bassett et al

Bassett AR, Kong L, Liu JL. A Genome-Wide CRISPR Library for High-Throughput Genetic Screening in Drosophila Cells. J Genet Genomics. 2015 Jun 20;42(6):301-9. PMID: 26165496.

C. elegans study points to pol theta involvement in CRISPR mediated events

From our worm colleagues--article of possible interest to folks working with CRISPR system:

van Schendel R, Roerink SF, Portegijs V, van den Heuvel S, Tijsterman M. Polymerase Θ is a key driver of genome evolution and of CRISPR/Cas9-mediated mutagenesis. Nat Commun. 2015 Jun 16;6:7394. PMID: 26077599.

From the abstract: "... Here, we demonstrate that the A-family polymerase theta (POLQ) is a major driver of inheritable genomic alterations in Caenorhabditis elegans. Unlike somatic cells, which use non-homologous end joining (NHEJ) to repair DNA transposon-induced DSBs, germ cells use polymerase theta-mediated end joining, a conceptually simple repair mechanism requiring only one nucleotide as a template for repair. Also CRISPR/Cas9-induced genomic changes are exclusively generated through polymerase theta-mediated end joining, refuting a previously assumed requirement for NHEJ in their formation. ... "

CRISPRs in flies -- two papers

Protocol recently published:

Gratz SJ, Harrison MM, Wildonger J, O'Connor-Giles KM. Precise Genome Editing of Drosophila with CRISPR RNA-Guided Cas9. Methods Mol Biol. 2015;1311:335-48. PMID: 25981484.

And early online at G3:

Systematic Evaluation of Drosophila CRISPR Tools Reveals Safe and Robust Alternatives to Autonomous Gene Drives in Basic Research

• Fillip Port,
• Nadine Muschalik,
• and Simon L. Bullock

G3 g3.115.019083; Early Online May 20, 2015, doi:10.1534/g3.115.019083

Report of improved CRISPR-based transcriptional activation approach includes work in Drosophila cultured cells

Chavez A, Scheiman J, Vora S, Pruitt BW, Tuttle M, P R Iyer E, Lin S, Kiani S, Guzman CD, Wiegand DJ, Ter-Ovanesyan D, Braff JL, Davidsohn N, Housden BE, Perrimon N, Weiss R, Aach J, Collins JJ, Church GM. Highly efficient Cas9-mediated transcriptional programming. Nat Methods. 2015 Apr;12(4):326-8. PMID: 25730490.

From the abstract: "... modest levels of gene activation have limited potential applications. We describe an improved transcriptional regulator obtained through the rational design of a tripartite activator, VP64-p65-Rta (VPR), fused to nuclease-null Cas9. ..."

Drosophila S2R+ cultured cells are among the systems in which this approach was successfully tested. See Results and Supplementary Fig. 9.

C. elegans report presents simple rule for effective CRISPR sgRNA design

Farboud B, Meyer BJ. Dramatic Enhancement of Genome Editing by CRISPR/Cas9 Through Improved Guide RNA Design. Genetics. 2015 Feb 18. PMID: 25695951.

From the abstract: "... Here we describe and validate a strategy for Caenorhabditis elegans ... The key innovation was to design guide RNAs with a GG motif at the 3' end of their target-specific sequences. All guides designed using this simple principle induced a high frequency of targeted mutagenesis via nonhomologous end joining (NHEJ) and a high frequency of precise DNA integration from exogenous DNA templates via homology-directed repair (HDR). Related guide RNAs having the GG motif shifted by only three nucleotides showed severely reduced or no genome editing. ..."

Will this prove true in Drosophila as well? There are certainly guide designs reported to be effective in flies that break the GG rule. And it is hard to believe that the design algorithms developed by various groups would not have picked this up. Nevertheless, it's tempting to give it a try.

Efficient fly CRISPR with limited risk? Preprint from Port and colleagues compares sgRNA transgenic and MCR approaches

Interested to efficiently generate knockout flies without the potential risks presented by the mutagenic chain reaction (MCR) approach? Check out a new pre-print from Port et al.

Port, Muschalik and Bullock (preprint) CRISPR with independent transgenes is a safe and robust alternative to autonomous gene drives in basic research.

From the abstract:  "... Here, we systematically evaluate available CRISPR/Cas reagents and experimental designs in Drosophila. Our findings allow evidence-based choices of Cas9 sources and strategies for generating knock-in alleles. We perform gene editing at a large number of target sites using a highly active Cas9 line and a collection of transgenic gRNA strains. The vast majority of target sites can be mutated with remarkable efficiency using these tools. We contrast our method to recently developed autonomous gene drive technology for genome engineering (Gantz & Bier, 2015) and conclude that optimised CRISPR with independent transgenes is as efficient, more versatile and does not represent a biosafety risk."

"Trojan exon" variation on the theme of MiMIC and CRISPR insertion strategies

Diao F, Ironfield H, Luan H, Diao F, Shropshire WC, Ewer J, Marr E, Potter CJ, Landgraf M, White BH. Plug-and-Play Genetic Access to Drosophila Cell Types using Exchangeable Exon Cassettes. Cell Rep. 2015 Mar 3;10(8):1410-21. PMID: 25732830.

From the abstract: "... we introduce a simple, versatile method for achieving cell-type-specific expression of transgenes ... Our method couples the expression of a transgene to that of a native gene expressed in the cells of interest ... We demonstrate the efficacy of this approach in Drosophila ... We also introduce an exchangeable, MiMIC-like Trojan exon construct that can be targeted to coding introns using the Crispr/Cas system."

The cut that keeps on cutting: CRISPR-Cas mutagenic chain reaction approach

Gantz and Bier (2015) The mutagenic chain reaction: A method for converting heterozygous to homozygous mutations. Science early online DOI: 10.1126/science.aaa5945


Abstract: "An organism with a single recessive loss-of-function allele will typically have a wild-type phenotype while individuals homozygous for two copies of the allele will display a mutant phenotype. Here, we develop a method that we refer to as the mutagenic chain reaction (MCR), which is based on the CRISPR/Cas9 genome editing system for generating autocatalytic mutations to generate homozygous loss-of-function mutations. We demonstrate in Drosophila that MCR mutations efficiently spread from their chromosome of origin to the homologous chromosome thereby converting heterozygous mutations to homozygosity in the vast majority of somatic and germline cells. MCR technology should have broad applications in diverse organisms."

See also Science magazine's news coverage of the report.