Ji-Long Liu

Highly Efficient Targeted Mutagenesis of Drosophila with the CRISPR/Cas9 System

Summary Here, we present a simple and highly efficient method for generating and detecting mutations of any gene in Drosophila melanogaster through the use of the CRISPR/Cas9 system (clustered regularly interspaced palindromic repeats/CRISPR-associated). We show that injection of RNA into the Drosophila embryo can induce highly efficient mutagenesis of desired target genes in up to 88% of injected flies. These mutations can be transmitted through the germline to make stable lines. Our system provides at least a 10-fold improvement in efficiency over previously published reports, enabling wider application of this technique. We also describe a simple and highly sensitive method of detecting mutations in the target gene by high-resolution melt analysis and discuss how the new technology enables the study of gene function.

Efficient gene targeting in Drosophila by direct embryo injection with zinc-finger nucleases.

We report very high gene targeting frequencies in Drosophila by direct embryo injection of mRNAs encoding specific zinc-finger nucleases (ZFNs). Both local mutagenesis via nonhomologous end joining (NHEJ) and targeted gene replacement via homologous recombination (HR) have been achieved in up to 10% of all targets at a given locus. In embryos that are wild type for DNA repair, the products are dominated by NHEJ mutations. In recipients deficient in the NHEJ component, DNA ligase IV, the majority of products arise by HR with a coinjected donor DNA, with no loss of overall efficiency in target modification. We describe the application of the ZFN injection procedure to mutagenesis by NHEJ of 2 new genes in Drosophila melanogaster: coil and pask. Pairs of novel ZFNs designed for targets within those genes led to the production of null mutations at each locus. The injection procedure is much more rapid than earlier approaches and makes possible the generation and recovery of targeted gene alterations at essentially any locus within 2 fly generations.

The Drosophila melanogaster Cajal body

Cajal bodies (CBs) are nuclear organelles that are usually identified by the marker protein p80-coilin. Because no orthologue of coilin is known in Drosophila melanogaster, we identified D. melanogaster CBs using probes for other components that are relatively diagnostic for CBs in vertebrate cells. U85 small CB–specific RNA, U2 small nuclear RNA, the survival of motor neurons protein, and fibrillarin occur together in a nuclear body that is closely associated with the nucleolus. Based on its similarity to CBs in other organisms, we refer to this structure as the D. melanogaster CB. Surprisingly, the D. melanogaster U7 small nuclear RNP resides in a separate nuclear body, which we call the histone locus body (HLB). The HLB is invariably colocalized with the histone gene locus. Thus, canonical CB components are distributed into at least two nuclear bodies in D. melanogaster. The identification of these nuclear bodies now permits a broad range of questions to be asked about CB structure and function in a genetically tractable organism.

Identification and properties of 1,119 candidate lincRNA loci in the Drosophila melanogaster genome.

The functional repertoire of long intergenic noncoding RNA (lincRNA) molecules has begun to be elucidated in mammals. Determining the biological relevance and potential gene regulatory mechanisms of these enigmatic molecules would be expedited in a more tractable model organism, such as Drosophila melanogaster. To this end, we defined a set of 1,119 putative lincRNA genes in D. melanogaster using modENCODE whole transcriptome (RNA-seq) data. A large majority (1.1 of 1.3 Mb; 85%) of these bases were not previously reported by modENCODE as being transcribed. Significant selective constraint on the sequences of these loci predicts that virtually all have sustained functionality across the Drosophila clade. We observe biases in lincRNA genomic locations and expression profiles that are consistent with some of these lincRNAs being involved in the regulation of neighboring protein-coding genes with developmental functions. We identify lincRNAs that may be important in the developing nervous system and in male-specific organs, such as the testes. LincRNA loci were also identified whose positions, relative to nearby protein-coding loci, are equivalent between D. melanogaster and mouse. This study predicts that the genomes of not only vertebrates, such as mammals, but also an invertebrate (fruit fly) harbor large numbers of lincRNA loci. Our findings now permit exploitation of Drosophila genetics for the investigation of lincRNA mechanisms, including lincRNAs with potential functional analogues in mammals.

Freeze-Dried Sperm Fertilization Leads to Full-Term Development in Rabbits

Abstract To date, the laboratory mouse is the only mammal in which freeze-dried spermatozoa have been shown to support full-term development after microinjection into oocytes. Because spermatozoa in mice, unlike in most other mammals, do not contribute centrosomes to zygotes, it is still unknown whether freeze-dried spermatozoa in other mammals are fertile. Rabbit sperm was selected as a model because of its similarity to human sperm (considering the centrosome inheritance pattern). Freeze- drying induces rabbit spermatozoa to undergo dramatic changes, such as immobilization, membrane breaking, and tail fragmentation. Even when considered to be “dead” in the conventional sense, rabbit spermatozoa freeze-dried and stored at ambient temperature for more than 2 yr still have capability comparable to that of fresh spermatozoa to support preimplantation development after injection into oocytes followed by activation. A rabbit kit derived from a freeze-dried spermatozoon was born after transferring 230 sperm-injected oocytes into eight recipients. The results suggest that freeze-drying could be applied to preserve the spermatozoa from most other species, including human. The present study also raises the question of whether rabbit sperm centrosomes survive freeze-drying or are not essential for embryonic development.

CRISPR/Cas9 and genome editing in Drosophila.

Recent advances in our ability to design DNA binding factors with specificity for desired sequences have resulted in a revolution in genetic engineering, enabling directed changes to the genome to be made relatively easily. Traditional techniques for generating genetic mutations in most organisms have relied on selection from large pools of randomly induced mutations for those of particular interest, or time-consuming gene targeting by homologous recombination. Drosophila melanogaster has always been at the forefront of genetic analysis, and application of these new genome editing techniques to this organism will revolutionise our approach to performing analysis of gene function in the future. We discuss the recent techniques that apply the CRISPR/Cas9 system to Drosophila, highlight potential uses for this technology and speculate upon the future of genome engineering in this model organism.

Coilin Is Essential for Cajal Body Organization in Drosophila melanogaster

Cajal bodies (CBs) are nuclear organelles that occur in a variety of organisms, including vertebrates, insects, and plants. They are most often identified with antibodies against the marker protein coilin. Because the amino acid sequence of coilin is not strongly conserved evolutionarily, coilin orthologues have been difficult to recognize by homology search. Here, we report the identification of Drosophila melanogaster coilin and describe its distribution in tissues of the fly. Surprisingly, we found coilin not only in CBs but also in histone locus bodies (HLBs), calling into question the use of coilin as an exclusive marker for CBs. We analyzed two null mutants in the coilin gene and a piggyBac insertion mutant, which leads to specific loss of coilin from the germline. All three mutants are homozygous viable and fertile. Cells that lack coilin also lack distinct foci of other CB markers, including fibrillarin, the survival motor neuron (SMN) protein, U2 small nuclear RNA (snRNA), U5 snRNA, and the small CB-specific (sca) RNA U85. However, HLBs are not obviously affected in coilin-null flies. Thus, coilin is required for normal CB organization in Drosophila but is not essential for viability or production of functional gametes.

AC1MMYR2, an Inhibitor of Dicer-Mediated Biogenesis of Oncomir miR-21, Reverses Epithelial–Mesenchymal Transition and Suppresses Tumor Growth and Progression

The extensive involvement of miRNAs in cancer pathobiology has opened avenues for drug development based on oncomir inhibition. Dicer is the core enzyme in miRNA processing that cleaves the terminal loop of precursor microRNAs (pre-miRNAs) to generate mature miRNA duplexes. Using the three-dimensional structure of the Dicer binding site on the pre-miR-21 oncomir, we conducted an in silico high-throughput screen for small molecules that block miR-21 maturation. By this method, we identified a specific small-molecule inhibitor of miR-21, termed AC1MMYR2, which blocked the ability of Dicer to process pre-miR-21 to mature miR-21. AC1MMYR2 upregulated expression of PTEN, PDCD4, and RECK and reversed epithelial-mesenchymal transition via the induction of E-cadherin expression and the downregulation of mesenchymal markers, thereby suppressing proliferation, survival, and invasion in glioblastoma, breast cancer, and gastric cancer cells. As a single agent in vivo, AC1MMYR2 repressed tumor growth, invasiveness, and metastasis, increasing overall host survival with no observable tissue cytotoxicity in orthotopic models. Our results offer a novel, high-throughput method to screen for small-molecule inhibitors of miRNA maturation, presenting AC1MMYR2 as a broadly useful candidate antitumor drug.

Intracellular compartmentation of CTP synthase in Drosophila.

Compartmentation is essential for the localization of biological processes within a eukaryotic cell. ATP synthase localizes to organelles such as mitochondria and chloroplasts. By contrast, little is known about the subcellular distribution of CTP synthase, the critical enzyme in the production of CTP, a high-energy molecule similar to ATP. Here I describe the identification of a novel intracellular structure containing CTP synthase, termed the cytoophidium, in Drosophila cells. I find that cytoophidia are present in all major cell types in the ovary and exist in a wide range of tissues such as brain, gut, trachea, testis, accessory gland, salivary gland and lymph gland. In addition, I find CTP synthase-containing cytoophidia in other fruit fly species. The observation of compartmentation of CTP synthase now permits a broad range of questions to be addressed concerning not only the structure and function of cytoophidia but also the organization and regulation of CTP synthesis.

U bodies are cytoplasmic structures that contain uridine-rich small nuclear ribonucleoproteins and associate with P bodies

Uridine-rich small nuclear ribonucleoproteins (U snRNPs) are involved in key steps of pre-mRNA processing in the nucleus of eukaryotic cells. U snRNPs are enriched in the nucleus in discrete organelles that include speckles, Cajal bodies, and histone locus bodies. However, most U snRNPs are assembled in the cytoplasm, not in the nucleus. Despite extensive biochemical information, little is known about the spatial organization of U snRNPs in the cytoplasm. Here we show that U snRNPs in Drosophila are concentrated in discrete cytoplasmic structures, which we call U bodies, because they contain the major U snRNPs. In addition to snRNPs, U bodies contain essential snRNP assembly factors, suggesting that U bodies are sites for assembly or storage of snRNPs before their import into the nucleus. U bodies invariably associate with P bodies, which are involved in RNA surveillance and decay. Genetic disruption of P body components affects the organization of U bodies, suggesting that the two cytoplasmic bodies may cooperate in regulating aspects of snRNP metabolism. The identification of U bodies provides an opportunity to correlate specific biochemical steps of snRNP biogenesis with structural features of the cytoplasm.