Optimized CRISPR/Cas tools for efficient germline and somatic genome engineering in Drosophila.

BackgroundBarcodes are unique DNA sequence tags that can be used to specifically label individual mutants. The barcode-tagged open reading frame (ORF) haploid deletion mutant collections in the budding yeast Saccharomyces cerevisiae and the fission yeast Schizosaccharomyces pombe allow for high-throughput mutant phenotyping because the relative growth of mutants in a population can be determined by monitoring the proportions of their associated barcodes. While these mutant collections have greatly facilitated genome-wide studies, mutations in essential genes are not present, and the roles of these genes are not as easily studied. To further support genome-scale research in S. pombe, we generated a barcode-tagged fission yeast insertion mutant library that has the potential of generating viable mutations in both essential and non-essential genes and can be easily analyzed using standard molecular biological techniques.ResultsAn insertion vector containing a selectable ura4+ marker and a random barcode was used to generate a collection of 10,000 fission yeast insertion mutants stored individually in 384-well plates and as six pools of mixed mutants. Individual barcodes are flanked by Sfi I recognition sites and can be oligomerized in a unique orientation to facilitate barcode sequencing. Independent genetic screens on a subset of mutants suggest that this library contains a diverse collection of single insertion mutations. We present several approaches to determine insertion sites.ConclusionsThis collection of S. pombe barcode-tagged insertion mutants is well-suited for genome-wide studies. Because insertion mutations may eliminate, reduce or alter the function of essential and non-essential genes, this library will contain strains with a wide range of phenotypes that can be assayed by their associated barcodes. The design of the barcodes in this library allows for barcode sequencing using next generation or standard benchtop cloning approaches.

Essential genes are those genes required for an organism to complete development and survive to birth. There is debate as to whether essential genes play a role in human disease, because if they are critical for survival then mutations in these genes will cause lethality during development, removing individuals carrying these mutations from the population. Yet, essential genes can have diverse mutations that either limit or alter their function in a manner that allows individuals with these mutations to survive. These nonlethal, yet pathological, mutations in essential genes do contribute to human disease. Studies have demonstrated that essential disease genes are highly conserved, participate in many protein–protein interactions and may cause both Mendelian and complex disorders. These results confirm that essential genes are valid candidates as disease loci. Key Concepts: Genetic mutations create different alleles of genes. Hypomorphic alleles retain some gene function. Essential genes are those that are absolutely required for the survival of the organism, and have null alleles with lethal phenotypes. Human disease can be caused by hypomorphic mutations in essential genes. Essential genes do contribute to human nondevelopmental diseases. Essential genes can contribute to Mendelian disease and complex disorders.

CRISPR/Cas9-Mediated Genome Editing Corrects Dystrophin Mutation in Skeletal Muscle Stem Cells in a Mouse Model of Muscle Dystrophy.

The CRISPR/Cas9 gene-editing technology has been widely used in defining gene functions and crop improvement. However, some genes are essential for plant growth and development. Loss-of-function homozygous mutations in essential genes lead to plant death or sterility. Mutations in essential genes need to be maintained and propagated in heterozygous plants. CRISPR/Cas9 technology is highly efficient in generating homozygous or bi-allelic mutations at T0 generation in rice, making it difficult to generate useful genetic materials for essential genes using traditional gene editing technology. In this study, we designed Transgene-Killer CRISPR (TKC)-mediated mismatch-spacer targeting (TKC-M) to efficiently generate heritable heterozygous mutations in essential genes in rice. Leveraging our earlier transgenic offspring self-elimination TKC platform, TKC-M relied on timely self-elimination of Cas9 and engineered gRNA-target mismatches to enrich heritable heterozygous or mosaic incomplete-edited T0 mutants and heterozygous progeny. We found that the sensitivity of targets to spacer mismatch(es) varies. A single-base mismatch at gRNA positions 11 or 17 yielded abundant heritable heterozygotes in sensitive targets. For insensitive targets, dual mismatches at positions 8 and 15 maximised heritable heterozygotes. Co-transformation of rice with TKC vectors carrying gRNA without mismatches (G1), gRNA with a mismatch at position 11 (M11) and M8 + M15 spacers, termed TKC-M Cocktail (TKC-MC) significantly increased the incomplete-edited mutant ratio compared with using G1 alone. This work establishes a technical foundation for generating mutant libraries that cover every single gene in a plant genome and for in-depth research on essential genes.

α‐linolenic acid (αLA) is an essential fatty acid that functions in the maintenance of cell membrane fluidity and the synthesis of eicosanoids. This study examined the effect of an αLA‐enriched feed (camelina meal) on mRNA expression of lipid metabolism genes in somatic cells harvested from milk. Primiparous dairy cows (n=18) were fed a ration containing 0, 3, 6, and 9% camelina meal substituting for canola meal (18:1 cis9) in a 4x4 Latin square design. Somatic cells (primarily leukocytes and epithelial cells) were separated out of milk and total RNA was extracted for gene expression analysis with real time rt‐PCR. Expression of acetyl CoA carboxylase (ACC), fatty acid synthase (FAS), lipoprotein lipase and stearoyl CoA desaturase was increased in milk cells with the 6% diet whereas milk fat percent and yield were decreased. A negative correlation was observed between the concentrations of serum triglycerides and expression of ACC and FAS whereas a positive correlation was observed between the concentrations of serum non‐esterified fatty acids and expression of the same two genes. More research is needed to elucidate the relationship between mRNA expression of lipid metabolism genes in milk somatic cells and serum lipid content. Use of total milk somatic cells may not be an accurate method of measuring mammary epithelial cell mRNA expression. Supported by the United Dairymen of Idaho and NIH‐NRRI grant P20 RR15587.

Two intragenic deletions (exon 18-19 and exon 24) and two point mutations (one missense mutation in exon 21 and one mutation at splice-donor site for exon 13) were detected in the retinoblastoma gene in somatic and tumor cells of patients with hereditary retinoblastoma. Three mutations were located in a domain essential for binding to oncoproteins encoded by DNA tumor viruses (Hu et al., 1990; Huang et al., 1990). One mutation (deletion of exon 24) was outside this domain but it is in the region essential for binding to transcriptional factor E2F, and for suppression of malignant phenotypes (Qian et al., 1992; Qin et al., 1992). A minisatellite-like sequence and short repeated sequences were located at the breakpoint of the deletion of exon 24, suggesting that two deletions on both sides of the minisatellite-like sequence may be generated by a "DNA slippage and misalignment" mechanism. Upon amplification of cDNA by the polymerase chain reaction, no transcript of gene with frameshift mutation (deletion of exon 24) was detected in skin fibroblasts, while transcripts of genes with missense mutations were detected. The results, in combination with previous reports (Dunn et al., 1989; Hashimoto et al., 1991), suggest the instability of transcripts with a premature stop codon or the suppressed expression of alleles with a premature stop codon in the retinoblastoma gene in somatic cells of hereditary patients.

E2F6, a member of the E2F-family of transcription factors, is a retinoblastoma protein-independent transcriptional repressor. E2F6 associates with polycomb group (Pc-G) multiprotein complexes that contain histone H3 methyltransferases, suggesting that E2F6 represses genes by covalent histone modification. However, genes that are repressed by E2F6 via a mechanism that involves histone H3 methylation have not been identified. Using cDNA microarray experiments comparing wild-type and E2f6-/- mouse embryonic fibroblasts, we now found that E2F6 is required to silence the meiosis-specific genes SMC1beta and STAG3 in somatic cells. Re-expression of E2F6 in E2f6-/- cells was sufficient to restore their repression. E2F6 binds in vivo to the promoters of these genes through a conserved binding site. Transcriptional repression of SMC1beta and STAG3 by E2F6 involves multiple mechanisms, including methylation of histone H3 on lysine 9 and lysine 27. Our findings suggest a molecular mechanism for the stable transcriptional silencing of meiotic genes in somatic cells by E2F6.

BackgroundEssential genes are critical for the development of all organisms and are associated with many human diseases. These genes have been a difficult category to study prior to the availability of balanced lethal strains. Despite the power of targeted mutagenesis, there are limitations in identifying mutations in essential genes. In this paper, we describe the identification of coding regions for essential genes mutated using forward genetic screens in Caenorhabditis elegans. The lethal mutations described here were isolated and maintained by a wild-type allele on a rescuing duplication.ResultsWe applied whole genome sequencing to identify the causative molecular lesion resulting in lethality in existing C. elegans mutant strains. These strains are balanced and can be easily maintained for subsequent characterization. Our method can be effectively used to analyze mutations in a large number of essential genes. We describe here the identification of 64 essential genes in a region of chromosome I covered by the duplication sDp2. Of these, 42 are nonsense mutations, six are splice signal mutations, one deletion, and 15 are non-synonymous mutations. Many of the essential genes in this region function in cell cycle, transcriptional regulation, and RNA processing.ConclusionsThe essential genes identified here are represented by mutant strains, many of which have more than one mutant allele. The genetic resource can be utilized to further our understanding of essential gene function and will be applicable to the study of C. elegans development, conserved cellular function, and ultimately lead to improved human health.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2164-15-361) contains supplementary material, which is available to authorized users.

Targeted gene mutation by allelic replacement is important for functional genomic analysis and metabolic engineering. However, it is challenging in mutating the essential genes with the traditional method by using a selection marker, since the first step of essential gene knockout will result in a lethal phenotype. Here, we developed a two-end selection marker (Two-ESM) method for site-directed mutation of essential genes in Saccharomyces cerevisiae with the aid of the CRISPR/Cas9 system. With this method, single and double mutations of the essential gene ERG20 (encoding farnesyl diphosphate synthase) in S. cerevisiae were successfully constructed with high efficiencies of 100%. In addition, the Two-ESM method significantly improved the mutation efficiency and simplified the genetic manipulation procedure compared with traditional methods. The genome integration and mutation efficiencies were further improved by dynamic regulation of mutant gene expression and optimization of the integration modules. This Two-ESM method will facilitate the construction of genomic mutations of essential genes for functional genomic analysis and metabolic flux regulation in yeasts. KEY POINTS: • A Two-ESM strategy achieves mutations of essential genes with high efficiency of 100%. • The optimized three-module method improves the integration efficiency by more than three times. • This method will facilitate the functional genomic analysis and metabolic flux regulation.

Three-dimensional (3D) stem cell differentiation cultures recently emerged as a novel model system for investigating human embryonic development and disease progression in vitro, complementing existing animal and two-dimensional (2D) cell culture models. Organoids, the 3D self-organizing structures derived from pluripotent or somatic stem cells, can recapitulate many aspects of structural organization and functionality of their in vivo organ counterparts, thus holding great promise for biomedical research and translational applications. Importantly, faithful recapitulation of disease and development processes relies on the ability to modify the genomic contents in organoid cells. The revolutionary genome engineering technologies, CRISPR/Cas9 in particular, enable investigators to generate various reporter cell lines for prompt validation of specific cell lineages as well as to introduce disease-associated mutations for disease modeling. In this review, we provide historical overviews, and discuss technical considerations, and potential future applications of genome engineering in 3D organoid models.

Cells containing nonsense mutations in essential genes have been isolated in a strain of Escherichia coli that carried the su4(ts) gene which specifies a temperature-sensitive tyrosine transfer ribonucleic acid. Such cells are unable to form colonies at temperatures which inactivate this suppressor transfer ribonucleic acid. A screening procedure for the identification of mutants that carry temperature-sensitive nonsense mutations in essential genes is described, and certain properties of two such mutants are reported.

Deleterious mutations inevitably emerge in any evolutionary process and are speculated to decisively influence the structure of the genome. Meiosis, which is thought to play a major role in handling mutations on the population level, recombines chromosomes via non-randomly distributed hot spots for meiotic recombination. In many genomes, various types of genetic elements are distributed in patterns that are currently not well understood. In particular, important (essential) genes are arranged in clusters, which often cannot be explained by a functional relationship of the involved genes. Here we show by computer simulation that essential gene (EG) clustering provides a fitness benefit in handling deleterious mutations in sexual populations with variable levels of inbreeding and outbreeding. We find that recessive lethal mutations enforce a selective pressure towards clustered genome architectures. Our simulations correctly predict (i) the evolution of non-random distributions of meiotic crossovers, (ii) the genome-wide anti-correlation of meiotic crossovers and EG clustering, (iii) the evolution of EG enrichment in pericentromeric regions and (iv) the associated absence of meiotic crossovers (cold centromeres). Our results furthermore predict optimal crossover rates for yeast chromosomes, which match the experimentally determined rates. Using a Saccharomyces cerevisiae conditional mutator strain, we show that haploid lethal phenotypes result predominantly from mutation of single loci and generally do not impair mating, which leads to an accumulation of mutational load following meiosis and mating. We hypothesize that purging of deleterious mutations in essential genes constitutes an important factor driving meiotic crossover. Therefore, the increased robustness of populations to deleterious mutations, which arises from clustered genome architectures, may provide a significant selective force shaping crossover distribution. Our analysis reveals a new aspect of the evolution of genome architectures that complements insights about molecular constraints, such as the interference of pericentromeric crossovers with chromosome segregation.

Computational inference of mutation effects is necessary for genetic studies in which many mutations must be considered as etiologic candidates. Programs such as PolyPhen-2 predict the relative severity of damage caused by missense mutations, but not the actual probability that a mutation will reduce/eliminate protein function. Based on genotype and phenotype data for 116,330 ENU-induced mutations in the Mutagenetix database, we calculate that putative null mutations, and PolyPhen-2-classified “probably damaging”, “possibly damaging”, or “probably benign” mutations have, respectively, 61%, 17%, 9.8%, and 4.5% probabilities of causing phenotypically detectable damage in the homozygous state. We use these probabilities in the estimation of genome saturation and the probability that individual proteins have been adequately tested for function in specific genetic screens. We estimate the proportion of essential autosomal genes in Mus musculus (C57BL/6J) and show that viable mutations in essential genes are more likely to induce phenotype than mutations in non-essential genes.

A number of studies have shown that replication-defective mutant strains of herpes simplex virus (HSV) can induce protective immunity in animal systems against wild-type HSV challenge. However, all of those studies used viruses with single mutations. Because multiple, stable mutations provide optimal levels of safety for live vaccines, we felt that additional mutations needed to be engineered into a candidate vaccine strain for HSV-2 and genital herpes. We therefore isolated an HSV-2 strain with deletion mutations in two viral DNA replication protein genes, UL5 and UL29. The resulting double deletion mutant virus strain, dl5-29, fails to form plaques or to give any detectable single cycle yields in normal monkey or human cells. Nevertheless, dl5-29 expresses nearly the same pattern of gene products as the wild-type virus or the single mutant viruses and induces antibody titers in mice that are equivalent to those induced by single deletion mutant viruses. Therefore, it is feasible to isolate a mutant HSV strain with two mutations in essential genes and with an increased level of safety but which is still highly immunogenic.

Although the phenotypes and functions of nonessential proteins can be studied by deletion of their coding sequences (both gene copies in diploid organisms), essential genes cannot be deleted unless loss of the encoded protein can be bypassed. Bypass is often achieved by supplementation with the product of the enzyme. However, supplementation cannot bypass loss of essential genes such as those encoding enzymes of DNA or RNA synthesis. To study proteins encoded by essential genes that cannot be bypassed, the mutations must be conditional in nature. The mutant cells must be able to grow under a permissive condition, but fail to grow under a different condition, the nonpermissive condition. Several methods have been developed to obtain conditional mutations in essential genes. Mutations that result in proteins abnormally sensitive to high temperatures are called temperature-sensitive (Ts) mutants and are a widely used type of conditional mutation. An alternative to Ts mutants is the "degron" system to target proteins for destruction by cellular proteases. Approaches to conditionally control the functions of proteins encoded by essential genes, plus the advantages and disadvantages of these and other approaches, will be considered.