Monday, September 12, 2016

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.

Tuesday, September 6, 2016

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."

Methods paper -- in vivo RNAi screens

Zhou J, Tong C. Design and Methods of Large-Scale RNA Interference Screens in Drosophila. Methods Mol Biol. 2016;1470:163-9. PMID: 27581292.

From the abstract: "... Here, we discuss methods for the design and performance of a large-scale in vivo RNAi screen in Drosophila. Furthermore, methods for the validation of results and analysis of data will be introduced."

Monday, August 22, 2016

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."

Genome-wide screen in study of heterochromatin factor HP1a

The cell-based RNAi screen described in this study was done using a DRSC library and follow-up reagents. Congratulations, Joel and colleagues!

Swenson JM, Colmenares SU, Strom AR, Costes SV, Karpen GH. The composition and organization of Drosophila heterochromatin are heterogeneous and dynamic. Elife. 2016 Aug 11;5. pii: e16096. PMID: 27514026; PMCID: PMC4981497.

Abstract: "Heterochromatin is enriched for specific epigenetic factors including Heterochromatin Protein 1a (HP1a), and is essential for many organismal functions. To elucidate heterochromatin organization and regulation, we purified Drosophila melanogaster HP1a interactors, and performed a genome-wide RNAi screen to identify genes that impact HP1a levels or localization. The majority of the over four hundred putative HP1a interactors and regulators identified were previously unknown. We found that 13 of 16 tested candidates (83%) are required for gene silencing, providing a substantial increase in the number of identified components that impact heterochromatin properties. Surprisingly, image analysis revealed that although some HP1a interactors and regulators are broadly distributed within the heterochromatin domain, most localize to discrete subdomains that display dynamic localization patterns during the cell cycle. We conclude that heterochromatin composition and architecture is more spatially complex and dynamic than previously suggested, and propose that a network of subdomains regulates diverse heterochromatin functions."

Friday, August 5, 2016

Genome-wide in vivo screen looks at male germline stem cells

Liu Y, Ge Q, Chan B, Liu H, Singh SR, Manley J, Lee J, Weideman AM, Hou G, Hou SX. Whole-animal genome-wide RNAi screen identifies networks regulating male germline stem cells in Drosophila. Nat Commun. 2016 Aug 3;7:12149. PMID: 27484291.

From the abstract: "... To identify genes and pathways that regulate stem-cell fates in the whole organism, we perform a genome-wide transgenic RNAi screen through ubiquitous gene knockdowns, focusing on regulators of adult Drosophila testis germline stem cells (GSCs). Here we identify 530 genes that regulate GSC maintenance and differentiation. Of these, we further knock down 113 selected genes using cell-type-specific Gal4s and find that more than half were external regulators, that is, from the local microenvironment or more distal sources. ..."

Talk of transvection, RNAi, and Gal4-UAS, all in one G3 report