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Ends-out gene targeting allows seamless replacement of endogenous genes with engineered DNA fragments by homologous recombination, thus creating designer “genes” in the endogenous locus. Conventional gene targeting in Drosophila involves targeting with the preintegrated donor DNA in the larval primordial germ cells. Here we report gene targeting during oogenesis with lethality inhibitor and CRISPR/Cas (Golic+), which improves on all major steps in such transgene-based gene targeting systems. First, donor DNA is integrated into precharacterized attP sites for efficient flip-out. Second, FLP, I-SceI, and Cas9 are specifically expressed in cystoblasts, which arise continuously from female germline stem cells, thereby providing a continual source of independent targeting events in each offspring. Third, a repressor-based lethality selection is implemented to facilitate screening for correct targeting events. Altogether, Golic+ realizes high-efficiency ends-out gene targeting in ovarian cystoblasts, which can be readily scaled up to achieve high-throughput genome editing.

Site-specific recombinases have been used for two decades to manipulate the structure of animal genomes in highly predictable ways and have become major research tools. However, the small number of recombinases demonstrated to have distinct specificities, low toxicity, and sufficient activity to drive reactions to completion in animals has been a limitation. In this report we show that four recombinases derived from yeast—KD, B2, B3, and R—are highly active and nontoxic in Drosophila and that KD, B2, B3, and the widely used FLP recombinase have distinct target specificities. We also show that the KD and B3 recombinases are active in mice.

A wide variety of biological experiments rely on the ability to express an exogenous gene in a transgenic animal at a defined level and in a spatially and temporally controlled pattern. We describe major improvements of the methods available for achieving this objective in Drosophila melanogaster. We have systematically varied core promoters, UTRs, operator sequences, and transcriptional activating domains used to direct gene expression with the GAL4, LexA, and Split GAL4 transcription factors and the GAL80 transcriptional repressor. The use of site-specific integration allowed us to make quantitative comparisons between different constructs inserted at the same genomic location. We also characterized a set of PhiC31 integration sites for their ability to support transgene expression of both drivers and responders in the nervous system. The increased strength and reliability of these optimized reagents overcome many of the previous limitations of these methods and will facilitate genetic manipulations of greater complexity and sophistication.

We demonstrate the feasibility of generating thousands of transgenic Drosophila melanogaster lines in which the expression of an exogenous gene is reproducibly directed to distinct small subsets of cells in the adult brain. We expect the expression patterns produced by the collection of 5,000 lines that we are currently generating to encompass all neurons in the brain in a variety of intersecting patterns. Overlapping 3-kb DNA fragments from the flanking noncoding and intronic regions of genes thought to have patterned expression in the adult brain were inserted into a defined genomic location by site-specific recombination. These fragments were then assayed for their ability to function as transcriptional enhancers in conjunction with a synthetic core promoter designed to work with a wide variety of enhancer types. An analysis of 44 fragments from four genes found that >80% drive expression patterns in the brain; the observed patterns were, on average, comprised of D. melanogaster genome contains >50,000 enhancers and that multiple enhancers drive distinct subsets of expression of a gene in each tissue and developmental stage. We expect that these lines will be valuable tools for neuroanatomy as well as for the elucidation of neuronal circuits and information flow in the fly brain.

Drosophila melanogaster plays an important role in molecular, genetic, and genomic studies of heredity, development, metabolism, behavior, and human disease. The initial reference genome sequence reported more than a decade ago had a profound impact on progress in Drosophila research, and improving the accuracy and completeness of this sequence continues to be important to further progress. We previously described improvement of the 117-Mb sequence in the euchromatic portion of the genome and 21 Mb in the heterochromatic portion, using a whole-genome shotgun assembly, BAC physical mapping, and clone-based finishing. Here, we report an improved reference sequence of the single-copy and middle-repetitive regions of the genome, produced using cytogenetic mapping to mitotic and polytene chromosomes, clone-based finishing and BAC fingerprint verification, ordering of scaffolds by alignment to cDNA sequences, incorporation of other map and sequence data, and validation by whole-genome optical restriction mapping. These data substantially improve the accuracy and completeness of the reference sequence and the order and orientation of sequence scaffolds into chromosome arm assemblies. Representation of the Y chromosome and other heterochromatic regions is particularly improved. The new 143.9-Mb reference sequence, designated Release 6, effectively exhausts clone-based technologies for mapping and sequencing. Highly repeat-rich regions, including large satellite blocks and functional elements such as the ribosomal RNA genes and the centromeres, are largely inaccessible to current sequencing and assembly methods and remain poorly represented. Further significant improvements will require sequencing technologies that do not depend on molecular cloning and that produce very long reads., This work was supported by NIH grants P50 HG00750 (G.M.R.), R01 HG00747 (G.H.K.), and R01 HG002673 (S.E.C.) and performed under U.S. Department of Energy Contracts DE-AC0376SF00098 and DE-AC02-05CH11231, University of California. I.F.Z. was supported by grant 13-04-40137 from the Russian Federation; E.N.A. was supported by grant 12-04-00874-a from the Russian Federation; P.D. was supported by a grant from the Instituto Pasteur-Fondazione Cenci Bolognetti; A.V. was supported by Ministerio de Economía y Competitividad grant BFU2011-30295-C02-01; and A.B.C. was supported by NIH grant R01 GM064590 and grants from Fundaçao de Amparo a Pesquisa do Estado do Rio de Janeiro (FAPERJ) and the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq).

A major challenge in interpreting genome sequences is understanding how the genome encodes the information that specifies when and where a gene will be expressed. The first step in this process is the identification of regions of the genome that contain regulatory information. In higher eukaryotes, this cis-regulatory information is organized into modular units [cis-regulatory modules (CRMs)] of a few hundred base pairs. A common feature of these cis-regulatory modules is the presence of multiple binding sites for multiple transcription factors. Here, we evaluate the extent to which the tendency for transcription factor binding sites to be clustered can be used as the basis for the computational identification of cis-regulatory modules. By using published DNA binding specificity data for five transcription factors active in the early Drosophila embryo, we identified genomic regions containing unusually high concentrations of predicted binding sites for these factors. A significant fraction of these binding site clusters overlap known CRMs that are regulated by these factors. In addition, many of the remaining clusters are adjacent to genes expressed in a pattern characteristic of genes regulated by these factors. We tested one of the newly identified clusters, mapping upstream of the gap gene giant (gt) , and show that it acts as an enhancer that recapitulates the posterior expression pattern of gt.

In animals, each sequence-specific transcription factor typically binds to thousands of genomic regions in vivo. Our previous studies of 20 transcription factors show that most genomic regions bound at high levels in Drosophila blastoderm embryos are known or probable functional targets, but genomic regions occupied only at low levels have characteristics suggesting that most are not involved in the cis -regulation of transcription. Here we use transgenic reporter gene assays to directly test the transcriptional activity of 104 genomic regions bound at different levels by the 20 transcription factors. Fifteen genomic regions were selected based solely on the DNA occupancy level of the transcription factor Kruppel. Five of the six most highly bound regions drive blastoderm patterns of reporter transcription. In contrast, only one of the nine lowly bound regions drives transcription at this stage and four of them are not detectably active at any stage of embryogenesis. A larger set of 89 genomic regions chosen using criteria designed to identify functional cis -regulatory regions supports the same trend: genomic regions occupied at high levels by transcription factors in vivo drive patterned gene expression, whereas those occupied only at lower levels mostly do not. These results support studies that indicate that the high cellular concentrations of sequence-specific transcription factors drive extensive, low-occupancy, nonfunctional interactions within the accessible portions of the genome.

We established a collection of 7,000 transgenic lines of Drosophila melanogaster. Expression of GAL4 ineach line is controlled by a different, defined fragment of genomic DNA that serves as a transcriptional enhancer. We used confocal microscopy ofdissected nervous systems to determine the expression patterns driven by each fragment in the adult brain and ventral nerve cord. We present image data on 6,650 lines. Using both manual and machine-assisted annotation, we describe the expression patterns in the most useful lines. We illustrate the utility of these data for identifying novel neuronal cell types, revealing brain asymmetry, and describing the nature and extent of neuronal shape stereotypy. The GAL4 lines allow expression of exogenous genes in distinct, small subsets of the adult nervous system. The set of DNA fragments, each driving a documented expression pattern, will facilitate the generation of additional constructs for manipulating neuronal function.

The ability to specify the expression levels of exogenous genes inserted in the genomes of transgenic animals is critical for the success of a wide variety of experimental manipulations. Protein production can be regulated at the level of transcription, mRNA transport, mRNA half-life, or translation efficiency. In this report, we show that several well-characterized sequence elements derived from plant and insect viruses are able to function in Drosophila to increase the apparent translational efficiency of mRNAs by as much as 20-fold. These increases render expression levels sufficient for genetic constructs previously requiring multiple copies to be effective in single copy, including constructs expressing the temperature-sensitive inactivator of neuronal function Shibire ts1 , and for the use of cytoplasmic GFP to image the fine processes of neurons.

Conditional expression of hairpin constructs in Drosophila is a powerful method to disrupt the activity of single genes with a spatial and temporal resolution that is impossible, or exceedingly difficult, using classical genetic methods. We previously described a method (Ni et al. 2008) whereby RNAi constructs are targeted into the genome by the phiC31-mediated integration approach using Vermilion-AttB-Loxp-Intron-UAS-MCS (VALIUM), a vector that contains vermilion as a selectable marker, an attB sequence to allow for phiC31-targeted integration at genomic attP landing sites, two pentamers of UAS, the hsp70 core promoter, a multiple cloning site, and two introns. As the level of gene activity knockdown associated with transgenic RNAi depends on the level of expression of the hairpin constructs, we generated a number of derivatives of our initial vector, called the “VALIUM” series, to improve the efficiency of the method. Here, we report the results from the systematic analysis of these derivatives and characterize VALIUM10 as the most optimal vector of this series. A critical feature of VALIUM10 is the presence of gypsy insulator sequences that boost dramatically the level of knockdown. We document the efficacy of VALIUM as a vector to analyze the phenotype of genes expressed in the nervous system and have generated a library of 2282 constructs targeting 2043 genes that will be particularly useful for studies of the nervous system as they target, in particular, transcription factors, ion channels, and transporters.

The conditional expression of hairpin constructs in Drosophila melanogaster has emerged in recent years as a method of choice in functional genomic studies. To date, upstream activating site–driven RNA interference constructs have been inserted into the genome randomly using P-element–mediated transformation, which can result in false negatives due to variable expression. To avoid this problem, we have developed a transgenic RNA interference vector based on the phiC31 site-specific integration method.

The fly Drosophila melanogaster is one of the most intensively studied organisms in biology and serves as a model system for the investigation of many developmental and cellular processes common to higher eukaryotes, including humans. We have determined the nucleotide sequence of nearly all of the ∼120-megabase euchromatic portion of the Drosophila genome using a whole-genome shotgun sequencing strategy supported by extensive clone-based sequence and a high-quality bacterial artificial chromosome physical map. Efforts are under way to close the remaining gaps; however, the sequence is of sufficient accuracy and contiguity to be declared substantially complete and to support an initial analysis of genome structure and preliminary gene annotation and interpretation. The genome encodes ∼13,600 genes, somewhat fewer than the smaller Caenorhabditis elegans genome, but with comparable functional diversity.

Background: The identification of sequences that control transcription in metazoans is a major goal of genome analysis. In a previous study, we demonstrated that searching for clusters of predicted transcription factor binding sites could discover active regulatory sequences, and identified 37 regions of the Drosophila melanogaster genome with high densities of predicted binding sites for five transcription factors involved in anterior-posterior embryonic patterning. Nine of these clusters overlapped known enhancers. Here, we report the results of in vivo functional analysis of 27 remaining clusters. Results: We generated transgenic flies carrying each cluster attached to a basal promoter and reporter gene, and assayed embryos for reporter gene expression. Six clusters are enhancers of adjacent genes: giant, fushi tarazu, odd-skipped, nubbin, squeeze and pdm2; three drive expression in patterns unrelated to those of neighboring genes; the remaining 18 do not appear to have enhancer activity. We used the Drosophila pseudoobscura genome to compare patterns of evolution in and around the 15 positive and 18 false-positive predictions. Although conservation of primary sequence cannot distinguish true from false positives, conservation of binding-site clustering accurately discriminates functional binding-site clusters from those with no function. We incorporated conservation of binding-site clustering into a new genome-wide enhancer screen, and predict several hundred new regulatory sequences, including 85 adjacent to genes with embryonic patterns.

Background It is widely accepted that comparative sequence data can aid the functional annotation of genome sequences; however, the most informative species and features of genome evolution for comparison remain to be determined.

Background The Drosophila melanogaster genome was the first metazoan genome to have been sequenced by the whole-genome shotgun (WGS) method. Two issues relating to this achievement were widely debated in the genomics community: how correct is the sequence with respect to base-pair (bp) accuracy and frequency of assembly errors? And, how difficult is it to bring a WGS sequence to the accepted standard for finished sequence? We are now in a position to answer these questions.