Measuring error rates in genomic perturbation screens: gold standards for human functional genomics

Technological advancement has opened the door to systematic genetics in mammalian cells. Genome‐scale loss‐of‐function screens can assay fitness defects induced by partial gene knockdown, using RNA interference, or complete gene knockout, using new CRISPR techniques. These screens can reveal the basic blueprint required for cellular proliferation. Moreover, comparing healthy to cancerous tissue can uncover genes that are essential only in the tumor; these genes are targets for the development of specific anticancer therapies. Unfortunately, progress in this field has been hampered by off‐target effects of perturbation reagents and poorly quantified error rates in large‐scale screens. To improve the quality of information derived from these screens, and to provide a framework for understanding the capabilities and limitations of CRISPR technology, we derive gold‐standard reference sets of essential and nonessential genes, and provide a Bayesian classifier of gene essentiality that outperforms current methods on both RNAi and CRISPR screens. Our results indicate that CRISPR technology is more sensitive than RNAi and that both techniques have nontrivial false discovery rates that can be mitigated by rigorous analytical methods.     

Counting mutagenized genomes and optimizing genetic screens in Caenorhabditis elegans

In genetic screens, the number of mutagenized gametes examined is an important parameter for evaluating screen progress, the number of genes of a given mutable phenotype, gene size, cost, and labor. Since genetic screens often entail examination of thousands or tens of thousands of animals, strategies for optimizing genetics screens are important for minimizing effort while maximizing the number of mutagenized gametes examined. To date, such strategies have not been described for genetic screens in the nematode Caenorhabditis elegans. Here we review general principles of genetic screens in C. elegans, and use a modified binomial strategy to obtain a general expression for the number of mutagenized gametes examined in a genetic screen. We use this expression to calculate optimal screening parameters for a large range of genetic screen types. In addition, we developed a simple online genetic-screen-optimization tool that can be used independently of this paper. Our results demonstrate that choosing the optimal F2-to-F1 screening ratio can significantly improve screen efficiency.     

Genetic analysis of prokaryotic and eukaryotic DNA-binding proteins in Escherichia coli.

This report describes an Escherichia coli genetic system that permits bacterial genetic methods to be applied to the study of essentially any prokaryotic or eukaryotic site-specific DNA binding protein. It consists of two parts. The first part is a set of tools that facilitate construction of customized E.coli strains bearing single copy lacZYA reporters that are repressed by a specific target protein. The second part is a pair of regulatable protein expression vectors that permit in vivo production of the target protein at levels appropriate for genetic experiments. When expressed in a properly designed reporter strain, the target protein represses the lac genes, resulting in an E.coli phenotype that can be quantitatively measured or exploited in large scale genetic screens or selections. As a result, large plasmid-based libraries of protein genes or pools of mutagenized variants of a given gene may be examined in relatively simple genetic experiments. The strain construction technique is also useful for generating E.coli strains bearing reporters for other types of genetic systems, including repression-based and activation-based systems in which chimeric proteins are used to examine interactions between foreign protein domains.     

Expanding the genetic toolbox for Leptospira species by generation of fluorescent bacteria

Our knowledge of the genetics and molecular basis of the pathogenesis associated with Leptospira, in comparison to those of other bacterial species, is very limited. An improved understanding of pathogenic mechanisms requires reliable genetic tools for functional genetic analysis. Here, we report the expression of gfp and mRFP1 genes under the control of constitutive spirochetal promoters in both saprophytic and pathogenic Leptospira strains. We were able to reliably measure the fluorescence of Leptospira by fluorescence microscopy and a fluorometric microplate reader-based assay. We showed that the expression of the gfp gene had no significant effects on growth in vivo and pathogenicity in L. interrogans. We constructed an expression vector for L. biflexa that contains the lacI repressor, an inducible lac promoter, and gfp as the reporter, demonstrating that the lac system is functional in Leptospira. Green fluorescent protein (GFP) expression was induced by the addition of isopropyl-β-d-thiogalactopyranoside (IPTG) in L. biflexa transformants harboring the expression vector. Finally, we showed that GFP can be used as a reporter to assess promoter activity in different environmental conditions. These results may facilitate further advances for studying the genetics of Leptospira spp.     

Unlocking the Bottleneck in Forward Genetics Using Whole-Genome Sequencing and Identity by Descent to Isolate Causative Mutations

Forward genetics screens with N-ethyl-N-nitrosourea (ENU) provide a powerful way to illuminate gene function and generate mouse models of human disease; however, the identification of causative mutations remains a limiting step. Current strategies depend on conventional mapping, so the propagation of affected mice requires non-lethal screens; accurate tracking of phenotypes through pedigrees is complex and uncertain; out-crossing can introduce unexpected modifiers; and Sanger sequencing of candidate genes is inefficient. Here we show how these problems can be efficiently overcome using whole-genome sequencing (WGS) to detect the ENU mutations and then identify regions that are identical by descent (IBD) in multiple affected mice. In this strategy, we use a modification of the Lander-Green algorithm to isolate causative recessive and dominant mutations, even at low coverage, on a pure strain background. Analysis of the IBD regions also allows us to calculate the ENU mutation rate (1.54 mutations per Mb) and to model future strategies for genetic screens in mice. The introduction of this approach will accelerate the discovery of causal variants, permit broader and more informative lethal screens to be used, reduce animal costs, and herald a new era for ENU mutagenesis.     

Automated on-chip rapid microscopy, phenotyping and sorting of C. elegans

Microscopy, phenotyping and visual screens are frequently applied to model organisms in combination with genetics. Although widely used, these techniques for multicellular organisms have mostly remained manual and low-throughput. Here we report the complete automation of sample handling, high-resolution microscopy, phenotyping and sorting of Caenorhabditis elegans. The engineered microfluidic system, coupled with customized software, has enabled high-throughput, high-resolution microscopy and sorting with no human intervention and may be combined with any microscopy setup. The microchip is capable of robust local temperature control, self-regulated sample-loading and automatic sample-positioning, while the integrated software performs imaging and classification of worms based on morphological and intensity features. We demonstrate the ability to perform sensitive and quantitative screens based on cellular and subcellular phenotypes with over 95% accuracy per round and a rate of several hundred worms per hour. Screening time can be reduced by orders of magnitude; moreover, screening is completely automated.     

乳糖操纵子介导的半乳糖苷酶在转基因动物中的应用前景

细菌的乳糖操纵子可以在哺乳动物中调控基因的表达,修饰阻抑物基因和操纵基因可调控阻抑物诱导能力及其对操纵基因的亲和力,更好的适应高等动物的内环境。半乳糖苷酶对乳糖操纵子系统有正向调控作用,利用乳糖操纵子在转基因动物中可诱导性调控半乳糖苷酶基因的表达,能有效的提高转基因动物利用半乳糖苷、吸收营养物质的能力。以下从乳糖操纵子的结构功能、乳糖阻抑物功能活性的基因调控、操纵基因的功能以及其在哺乳动物的应用现状四个方面,结合半乳糖苷酶的生理功能和其在转基因动物中应用两个方面进行综述,对乳糖操纵子介导的半乳糖苷酶在转基因动物中的应用效果和前景进行了分析探讨。     

The effect of genomic position on reversion of a lac frameshift mutation (lacIZ33) during non-lethal selection (adaptive mutation)

In a system described by Cairns and Foster, starvation of a particular leaky lac mutant (lacIZ33) in the presence of lactose appears to direct mutation in non-growing cells to sites that allow growth (adaptive mutation). This behaviour requires that the lac operon be located on an F' plasmid. This position effect was investigated by placing the mutant lac operon at many sites in the genome of Salmonella enterica (Typhimurium; LT2) and testing reversion behaviour. Genomic position did not affect reversion during non-selective growth. When lac was at any of 550 chromosomal sites, starvation caused little or no enhancement of reversion. In the 28 strains with the lac on Salmonella's conjugative plasmid (pSLT), selection enhanced reversion strongly, just as seen for strains with lac on an F' plasmid. In 46 strains, the lac operon was inserted within a small chromosomal duplication, and selection stimulated RecA-dependent partial reversion by simple amplification (about 8x) of the mutant lac region. The position of lac on a conjugative plasmid is important to reversion because it allows more frequent gene duplication and amplification. These events are central to growth and reversion under selection because they increase the number of replicating lac alleles within each developing revertant clone.     

Technical approaches for mouse models of human disease

The mouse is the leading organism for disease research. A rich resource of genetic variation occurs naturally in inbred and special strains owing to spontaneous mutations. However, one can also obtain desired gene mutations by using the following processes: targeted mutations that eliminate function in the whole organism or in a specific tissue; forward genetic screens using chemicals or transposons; or the introduction of exogenous transgenes as DNAs, bacterial artificial chromosomes (BACs) or reporter constructs. The mouse is the only mammal that provides such a rich resource of genetic diversity coupled with the potential for extensive genome manipulation, and is therefore a powerful application for modeling human disease. This poster review outlines the major genome manipulations available in the mouse that are used to understand human disease: natural variation, reverse genetics, forward genetics, transgenics and transposons. Each of these applications will be essential for understanding the diversity that is being discovered within the human population.     

Contributions from Caenorhabditis elegans functional genetics to antiparasitic drug target identification and validation: nicotinic acetylcholine receptors, a case study

Following the complete sequencing of the genome of the free-living nematode, Caenorhabditis elegans, in 1998, rapid advances have been made in assigning functions to many genes. Forward and reverse genetics have been used to identify novel components of synaptic transmission as well as determine the key components of antiparasitic drug targets. The nicotinic acetylcholine receptors (nAChRs) are prototypical ligand-gated ion channels. The functions of these transmembrane proteins and the roles of the different members of their extensive subunit families are increasingly well characterised. The simple nervous system of C. elegans possesses one of the largest nicotinic acetylcholine receptor gene families known for any organism and a combination of genetic, microarray, physiological and reporter gene expression studies have added greatly to our understanding of the components of nematode muscle and neuronal nAChR subtypes. Chemistry-to-gene screens have identified five subunits that are components of nAChRs sensitive to the antiparasitic drug, levamisole. A novel, validated target acting downstream of the levamisole-sensitive nAChR has also been identified in such screens. Physiology and molecular biology studies on nAChRs of parasitic nematodes have also identified levamisole-sensitive and insensitive subtypes and further subdivisions are under investigation.     

Novel method for identifying sequence-specific DNA-binding proteins.

We developed a general method for the enrichment and identification of sequence-specific DNA-binding proteins. A well-characterized protein-DNA interaction is used to isolate from crude cellular extracts or fractions thereof proteins which bind to specific DNA sequences; the method is based solely on this binding property of the proteins. The DNA sequence of interest, cloned adjacent to the lac operator DNA segment is incubated with a lac repressor-beta-galactosidase fusion protein which retains full operator and inducer binding properties. The DNA fragment bound to the lac repressor-beta-galactosidase fusion protein is precipitated by the addition of affinity-purified anti-beta-galactosidase immobilized on beads. This forms an affinity matrix for any proteins which might interact specifically with the DNA sequence cloned adjacent to the lac operator. When incubated with cellular extracts in the presence of excess competitor DNA, any protein(s) which specifically binds to the cloned DNA sequence of interest can be cleanly precipitated. When isopropyl-beta-D-thiogalactopyranoside is added, the lac repressor releases the bound DNA, and thus the protein-DNA complex consisting of the specific restriction fragment and any specific binding protein(s) is released, permitting the identification of the protein by standard biochemical techniques. We demonstrate the utility of this method with the lambda repressor, another well-characterized DNA-binding protein, as a model. In addition, with crude preparations of the yeast mitochondrial RNA polymerase, we identified a 70,000-molecular-weight peptide which binds specifically to the promoter region of the yeast mitochondrial 14S rRNA gene.     

The genetics of phytochrome signalling in Arabidopsis

The application of Arabidopsis genetics to research into the responses of plants to light has enabled rapid recent advances in this field. The plant photoreceptor phytochrome mediates well-defined responses that can be exploited to provide elegant and specific genetic screens. By this means, not only have mutants affecting the phytochromes themselves been isolated, but also mutants affecting the transduction of phytochrome signals. The genes involved in these processes have now begun to be characterized by using this genetic approach to isolate signal transduction components. Most of the components characterized so far are capable of being translocated to the cell nucleus, and they may help to define a new system of regulation of gene expression. This review summarises the ongoing contribution made by genetics to our understanding of light perception and signal transduction by the phytochrome system.     

Making the mouse embryo transparent: identifying developmental malformations using magnetic resonance imaging

Developmental malformations are a major cause of childhood mortality and are typically characterized by lesions that allow survival of the embryo through gestation. The genetics of developmental malformations are powerfully studied by using high-throughput, phenotype-driven screens (e.g., following zebrafish or mouse mutagenesis) or by genotype-driven studies using transgenic or knockout mice. With regard to either approach, the mouse is anatomically and phylogenetically closer to humans than any other genetically tractable model organism. This is particularly important in the cardiovascular and respiratory systems, which have unique mammalian features. The identification of murine models of developmental malformations is, however, hindered by the opacity of the late gestational mouse embryo. In this review, we describe recent advances in magnetic resonance imaging that make it possible to rapidly identify malformations in the developing mouse embryo with high efficiency.     

Functional genomics in Arabidopsis: large-scale insertional mutagenesis complements the genome sequencing project

The ultimate goal of genome research on the model flowering plant Arabidopsis thaliana is the identification of all of the genes and understanding their functions. A major step towards this goal, the genome sequencing project, is nearing completion; however, functional studies of newly discovered genes have not yet kept up to this pace. Recent progress in large-scale insertional mutagenesis opens new possibilities for functional genomics in Arabidopsis. The number of T-DNA and transposon insertion lines from different laboratories will soon represent insertions into most Arabidopsis genes. Vast resources of gene knockouts are becoming available that can be subjected to different types of reverse genetics screens to deduce the functions of the sequenced genes.     

Inverse drug screens: a rapid and inexpensive method for implicating molecular targets

Identification of gene products that function in some specific process of interest is a common goal in developmental biology. Although use of drug compounds to probe biological systems has a very long history in teratology and toxicology, systematic hierarchical drug screening has not been capitalized upon by the developmental biology community. This "chemical genetics" approach can greatly benefit the study of embryonic and regenerative systems, and we have formalized a strategy for using known pharmacological compounds to implicate specific molecular candidates in any chosen biological phenomenon. Taking advantage of a hierarchical structure that can be imposed on drug reagents in a number of fields such as ion transport, neurotransmitter function, metabolism, and cytoskeleton, any assay can be carried out as a binary search algorithm. This inverse drug screen methodology is much more efficient than exhaustive testing of large numbers of drugs, and reveals the identity of a manageable number of specific molecular candidates that can then be validated and targeted using more expensive and specific molecular reagents. Here, we describe the process of this loss-of-function screen and illustrate its use in uncovering novel bioelectrical and serotonergic mechanisms in embryonic patterning. This technique is an inexpensive and rapid complement to existing molecular screening strategies. Moreover, it is applicable to maternal proteins, and model species in which traditional genetic screens are not feasible, significantly extending the opportunities to identify key endogenous players in biological processes.