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Background: Genome-wide saturation mutagenesis and subsequent phenotype-driven screening has been central to a comprehensive understanding of complex biological processes in classical model organisms such as flies, nematodes, and plants. The degree of "saturation" (i.e., the fraction of possible target genes identified) has been shown to be a critical parameter in determining all relevant genes involved in a biological function, without prior knowledge of their products. In mammalian model systems, however, the relatively large scale and labor intensity of experiments have hampered the achievement of actual saturation mutagenesis, especially for recessive traits that require biallelic mutations to manifest detectable phenotypes., Results: By exploiting the recently established haploid mouse embryonic stem cells (ESCs), we present an implementation of almost complete saturation mutagenesis in a mammalian system. The haploid ESCs were mutagenized with the chemical mutagen N-ethyl-N-nitrosourea (ENU) and processed for the screening of mutants defective in various steps of the glycosylphosphatidylinositol-anchor biosynthetic pathway. The resulting 114 independent mutant clones were characterized by a functional complementation assay, and were shown to be defective in any of 20 genes among all 22 known genes essential for this well-characterized pathway. Ten mutants were further validated by whole-exome sequencing. The predominant generation of single-nucleotide substitutions by ENU resulted in a gene mutation rate proportional to the length of the coding sequence, which facilitated the experimental design of saturation mutagenesis screening with the aid of computational simulation., Conclusions: Our study enables mammalian saturation mutagenesis to become a realistic proposition. Computational simulation, combined with a pilot mutagenesis experiment, could serve as a tool for the estimation of the number of genes essential for biological processes such as drug target pathways when a positive selection of mutants is available.

Mouse embryonic stem (ES) cells are an attractive model system for investigating mammalian biology. Their relatively stable genome and high amenability to genome modification enables the generation of large-scale mutant libraries, which can be subsequently used for phenotype-driven genetic screens. While retroviral vectors have traditionally been used to generate insertional mutations in ES cells, their severe distribution-bias in the mammalian genome substantially limits genome-wide mutagenesis. The recent development of the DNA transposon piggyBac offers an efficient and highly versatile alternative for achieving more unbiased mutagenesis. Furthermore, heterozygous mutations created by insertional mutagens can be converted in parallel to homozygosity by using Blm-deficient ES cells, allowing genome-wide loss-of-function screens to be conducted. In this chapter, we describe the principles underpinning genetic screens in mouse ES cells with examples of previously successful screens. Protocols are provided for piggyBac transposon-mediated mutagenesis, production of the corresponding homozygous mutants in a Blm-deficient genetic background, and methods for mapping and validation of mutations recovered from screens of such libraries., (2010 Elsevier Inc. All rights reserved.)

The chief limitation of phenotype-based genetic screening in mammalian systems is the diploid nature of the genome. Cells deficient in the Bloom's syndrome gene (Blm) show an increased rate of loss of heterozygosity. Here we have used a tetracycline-regulated Blm allele (Blm(tet)) to introduce bi-allelic mutations across the genome in mouse embryonic stem (ES) cells. Transient loss of Blm expression induces homologous recombination not only between sister chromatids but also between homologous chromosomes. We considered that the phenotype of ES cells bearing bi-allelic mutations would be maintained after withdrawal of the tetracycline analogue doxycycline. Indeed, a combination of N-ethyl-N-nitrosourea mutagenesis and transient loss of Blm expression enabled us to generate an ES cell library with genome-wide bi-allelic mutations. The library was evaluated by screening for mutants of glycosylphosphatidylinositol-anchor biosynthesis, which involves at least 23 genes distributed throughout the genome. Mutants derived from 12 different genes were obtained and two unknown mutants were simultaneously isolated. Our results indicate that phenotype-based genetic screening with Blm(tet) is very efficient and raises possibilities for identifying gene functions in ES cells.

• CRISPR-Cas9 technology has revolutionised genome-wide mutagenic screens. • Phenotypic screens are now accessible to most laboratories. • Extension of Cas9 functionality permits future deployment of creative screens. Forward genetic screens are a powerful and unbiased approach for uncovering the genetic basis behind a specific phenotype. Genome-wide mutagenesis followed by phenotypic screening represents the ultimate manifestation of this method, directly linking biological phenomena to its corresponding genetic cause. Whilst this has been successful in lower organisms, deployment of genome-wide screens in mammalian systems has been hampered by both limitations of scale and inefficient bi-allelic mutagenesis. CRISPR-Cas9 technology has now largely resolved these issues, whereby delivery of genome-scale gRNA libraries in the presence of gRNA-guided Cas9 endonuclease enables the generation of mutant cell libraries; the perfect platform for performing phenotypic screens. Although the tools are now available for virtually any molecular biology laboratory to conduct such screens, many researchers are daunted by the sheer complexity and scale at which such experiments are performed. This Review will address these concerns, presenting a contextual and practical guide to deploying CRISPR-KO screens in mammalian systems. We will discuss key considerations required in all aspects of screening from initiation to conclusion, which will enable researchers to conduct screens of their own, maximising the potential of this powerful technology. [ABSTRACT FROM AUTHOR]

The DNA transposon piggyBac is widely used as a tool in mammalian experimental systems for transgenesis, mutagenesis, and genome engineering. We have characterized genome-wide insertion site preferences of piggyBac by sequencing a large set of integration sites arising from transposition from two separate genomic loci and a plasmid donor in mouse embryonic stem cells. We found that piggyBac preferentially integrates locally to the excision site when mobilized from a chromosomal location and identified other nonlocal regions of the genome with elevated insertion frequencies. piggyBac insertions were associated with expressed genes and markers of open chromatin structure and were excluded from heterochromatin. At the nucleotide level, piggyBac prefers to insert into TA-rich regions within a broader GC-rich context. We also found that piggyBac can insert into sites other than its known TTAA insertion site at a low frequency (2%). Such insertions introduce mismatches that are repaired with signatures of host cell repair pathways. Transposons could be mobilized from plasmids with the observed noncanonical flanking regions, indicating that piggyBac could generate point mutations in the genome. [ABSTRACT FROM AUTHOR]

Abstract: We recently reported genome-wide bi-allelic mutagenesis and phenotype-based genetic screening by tetracycline-regulated disruption of the Bloom''s syndrome gene (Blm) in mouse embryonic stem (ES) cells. However, the same approach was hampered in mouse tissues owing to leaky expression of the Blm gene, which is the major obstacle in the tetracycline regulatory system. Here we describe a single-chain reverse tetracycline-controlled trans-silencer (sc rtTS) which reduces leaky expression in the tet-off system. The sc rtTS consists of two silencer moieties linked by a 36 amino acid linker. Although the silencer moiety contained a dimerization domain compatible with the tetracycline-controlled transactivator (tTA), heterodimerization with tTA was prevented because intramolecular self-assembly between linked silencer moieties was preferred. The system was applied to mouse splenic lymphocytes and elevation of sister chromatid exchange, the hallmark of Blm dysfunction, was observed in the presence of doxycycline. A cassette containing both sc rtTS and tTA was introduced into the Blm allele in ES cells and reduction of basal activity was observed upon doxycycline treatment. Our data demonstrate effectiveness of sc rtTS in the tet-off system. Application of sc rtTS in mice may allow us to implement bi-allelic mutagenesis in vivo. [Copyright &y& Elsevier]

Recent consolidation of the whole-genome sequence with genome-wide transcriptome profiling revealed the existence of functional units within the genome in specific chromosomal regions, as seen in the coordinated expression of gene clusters and colocalization of functionally related genes. An efficient region-specific mutagenesis screen would greatly facilitate research in addressing the importance of these clusters. Here we use the 'local hopping' phenomenon of a DNA-type transposon, Sleeping Beauty (SB), for region-specific saturation mutagenesis. A transgenic mouse containing both transposon (acts as a mutagen) and transposase (recognizes and mobilizes the transposon) was bred for germ-cell transposition events, allowing us to generate many mutant mice. All genes within a 4-Mb region of the original donor site were mutated by SB, indicating the potential of this system for functional genomic studies within a specific chromosomal region. [ABSTRACT FROM AUTHOR]

The Sleeping Beauty (SB) transposase is the most active transposase in vertebrate cells, and the SB transposon system has been used as a tool for insertional mutagenesis and gene delivery. Previous studies have indicated that the frequency of chromosomal transposition is considerably higher in mouse germ cells than in mouse embryonic stem cells, suggesting the existence of unknown mechanisms that regulate SB transposition. Here, we demonstrated that CpG methylation of the transposon region enhances SB transposition. The transposition efficiencies of a methylated transposon and an unmethylated transposon which had been targeted in the same genomic loci by recombination-mediated cassette exchange in mouse erythroleukemia cells were compared, and at least a 100-fold increase was observed in the methylated transposon. CpG methylation also enhanced transposition from plasmids into the genome. Chromatin immunoprecipitation assays revealed that histone H3 methylated at lysine-9, a hallmark of condensed heterochromatin, was enriched at the methylated transposon, whereas the unmethylated transposon formed a relaxed euchromatin structure, as evidenced by enrichment of acetylated histone H3 and reporter gene expression. Possible roles of heterochromatin formation in the transposition reaction are discussed. Our findings indicate a novel relationship between CpG methylation and transposon mobilization. [ABSTRACT FROM AUTHOR]

The Sleeping Beauty (SB) transposase reconstructed from salmonid fish has high transposition activity in mammals and has been a useful tool for insertional mutagenesis and gene delivery. However, the transposition efficiency has varied significantly among studies. Our previous study demonstrated that the introduction of methylation into the SB transposon enhanced transposition, suggesting that transposition efficiency is influenced by the epigenetic status of the transposon region. Here, we examined the influence of the chromatin status on SB transposition in mouse embryonic stem cells. Heterochromatin conformation was introduced into the SB transposon by using a tetracycline-controlled transrepressor (tTR) protein, consisting of a tetracycline repressor (TetR) fused to the Kruppel-associated box (KRAB) domain of human KOX1 through tetracycline operator (tetO) sequences. The excision frequency of the SB transposon, which is the first step of the transposition event, was enhanced by approximately 100-fold. SB transposase was found to be colocalized with intense DAPI (4′,6′-diamidino-2-phenylindole) staining and with the HP1 family by biochemical fractionation analyses. Furthermore, chromatin immunoprecipitation analysis revealed that SB transposase was recruited to tTR-induced heterochromatic regions. These data suggest that the high affinity of SB transposase for heterochromatin conformation leads to enhancement of SB transposition efficiency. [ABSTRACT FROM AUTHOR]