Chromosome engineering in zygotes with CRISPR/Cas9.

Deletions, duplications, and inversions of large genomic regions covering several genes are an important class of disease causing variants in humans. Modeling these structural variants in mice requires multistep processes in ES cells, which has limited their availability. Mutant mice containing small insertions, deletions, and single nucleotide polymorphisms can be reliably generated using CRISPR/Cas9 directly in mouse zygotes. Large structural variants can be generated using CRISPR/Cas9 in ES cells, but it has not been possible to generate these directly in zygotes. We now demonstrate the direct generation of deletions, duplications and inversions of up to one million base pairs by zygote injection

Revealing hidden complexities of genomic rearrangements generated with Cas9.

Modelling human diseases caused by large genomic rearrangements has become more accessible since the utilization of CRISPR/Cas9 in mammalian systems. In a previous study, we showed that genomic rearrangements of up to one million base pairs can be generated by direct injection of CRISPR/Cas9 reagents into mouse zygotes. Although these rearrangements are ascertained by junction PCR, we describe here a variety of anticipated structural changes often involving reintegration of the region demarcated by the gRNAs in the vicinity of the edited locus. We illustrate here some of this diversity detected by high-resolution fibre-FISH and conclude that extensive molecular analysis is required to fully understand the structure of engineered chromosomes generated by Cas9

Isolation of homozygous mutant mouse embryonic stem cells using a dual selection system.

Obtaining random homozygous mutants in mammalian cells for forward genetic studies has always been problematic due to the diploid genome. With one mutation per cell, only one allele of an autosomal gene can be disrupted, and the resulting heterozygous mutant is unlikely to display a phenotype. In cells with a genetic background deficient for the Bloom's syndrome helicase, such heterozygous mutants segregate homozygous daughter cells at a low frequency due to an elevated rate of crossover following mitotic recombination between homologous chromosomes. We constructed DNA vectors that are selectable based on their copy number and used these to isolate these rare homozygous mutant cells independent of their phenotype. We use the piggyBac transposon to limit the initial mutagenesis to one copy per cell, and select for cells that have increased the transposon copy number to two or more. This yields homozygous mutants with two allelic mutations, but also cells that have duplicated the mutant chromosome and become aneuploid during culture. On average, 26% of the copy number gain events occur by the mitotic recombination pathway. We obtained homozygous cells from 40% of the heterozygous mutants tested. This method can provide homozygous mammalian loss-of-function mutants for forward genetic applications

Chromosome localization of microsatellite markers in the shrews of the Sorex araneus group

The extremely high rate of karyotypic evolution that characterizes the shrews of the Sorex araneus group makes this group an exceptionally interesting model for population genetics and evolutionary studies. Here, we attempted to map 46 microsatellite markers at the chromosome arm level using flow-sorted chromosomes from three karyotypically different taxa of the Sorex araneus group (S. granarius and the chromosome races Cordon and Novosibirsk of S. araneus). The most likely localizations were provided for 35 markers, among which 25 were each unambiguously mapped to a single locus on the corresponding chromosomes in the three taxa, covering the three sexual chromosomes (XY1Y2) and nine of the 18 autosomal arms of the S. araneus group. The results provide further evidence for a high degree of conservation in genome organization in the S. araneus group despite the presence of numerous Robertsonian rearrangements. These markers can therefore be used to compare the genetic structure among taxa of the S. araneus group at the chromosome level and to study the role of chromosomal rearrangements in the genetic diversification and speciation process of this grou

Generation and Characterisation of a Pax8-CreERT2 Transgenic Line and a Slc22a6-CreERT2 Knock-In Line for Inducible and Specific Genetic Manipulation of Renal Tubular Epithelial Cells.

Genetically relevant mouse models need to recapitulate the hallmarks of human disease by permitting spatiotemporal gene targeting. This is especially important for replicating the biology of complex diseases like cancer, where genetic events occur in a sporadic fashion within developed somatic tissues. Though a number of renal tubule targeting mouse lines have been developed their utility for the study of renal disease is limited by lack of inducibility and specificity. In this study we describe the generation and characterisation of two novel mouse lines directing CreERT2 expression to renal tubular epithelia. The Pax8-CreERT2 transgenic line uses the mouse Pax8 promoter to direct expression of CreERT2 to all renal tubular compartments (proximal and distal tubules as well as collecting ducts) whilst the Slc22a6-CreERT2 knock-in line utilises the endogenous mouse Slc22a6 locus to specifically target the epithelium of proximal renal tubules. Both lines show high organ and tissue specificity with no extrarenal activity detected. To establish the utility of these lines for the study of renal cancer biology, Pax8-CreERT2 and Slc22a6-CreERT2 mice were crossed to conditional Vhl knockout mice to induce long-term renal tubule specific Vhl deletion. These models exhibited renal specific activation of the hypoxia inducible factor pathway (a VHL target). Our results establish Pax8-CreERT2 and Slc22a6-CreERT2 mice as valuable tools for the investigation and modelling of complex renal biology and disease.This work was supported by a Cancer Research UK Clinician Scientist Fellowship award (C37839/A12177) to AM. DA, BF, FY are funded by the Wellcome Trust Sanger Institute (grant number WT098051).This is the final version of the article. It first appeared from the Public Library of Science (PLOS) via https://doi.org/10.1371/journal.pone.014805

Neo-sex chromosomes in the black muntjac recapitulate incipient evolution of mammalian sex chromosomes

The nascent neo-sex chromosomes of black muntjacs show that regulatory mutations could accelerate the degeneration of the Y chromosome and contribute to the further evolution of dosage compensation

Multidirectional chromosome painting substantiates the occurrence of extensive genomic reshuffling within Accipitriformes.

BACKGROUND: Previous cross-species painting studies with probes from chicken (Gallus gallus) chromosomes 1-10 and a paint pool of nineteen microchromosomes have revealed that the drastic karyotypic reorganization in Accipitridae is due to extensive synteny disruptions and associations. However, the number of synteny association events and identities of microchromosomes involved in such synteny associations remain undefined, due to the lack of paint probes derived from individual chicken microchromosomes. Moreover, no genome-wide homology map between Accipitridae species and other avian species with atypical karyotype organization has been reported till now, and the karyotype evolution within Accipitriformes remains unclear. RESULTS: To delineate the synteny-conserved segments in Accipitridae, a set of painting probes for the griffon vulture, Gyps fulvus (2n = 66) was generated from flow-sorted chromosomes. Together with previous generated probes from the stone curlew, Burhinus oedicnemus (2n = 42), a Charadriiformes species with atypical karyotype organization, we conducted multidirectional chromosome painting, including reciprocal chromosome painting between B. oedicnemus and G. fulvus and cross-species chromosome painting between B. oedicnemus and two accipitrid species (the Himalayan griffon, G. himalayensis 2n = 66, and the common buzzard, Buteo buteo, 2n = 68). In doing so, genome-wide homology maps between B. oedicnemus and three Accipitridae species were established. From there, a cladistic analysis using chromosomal characters and mapping of chromosomal changes on a consensus molecular phylogeny were conducted in order to search for cytogenetic signatures for different lineages within Accipitriformes. CONCLUSION: Our study confirmed that the genomes of the diurnal birds of prey, especially the genomes of species in Accipitriformes excluding Cathartidae, have been extensively reshuffled when compared to other bird lineages. The chromosomal rearrangements involved include both fusions and fissions. Our chromosome painting data indicated that the Palearctic common buzzard (BBU) shared several common chromosomal rearrangements with some Old World vultures, and was found to be more closely related to other Accipitridae than to Neotropical buteonine raptors from the karyotypic perspective. Using both a chromosome-based cladistic analysis as well as by mapping of chromosomal differences onto a molecular-based phylogenetic tree, we revealed a number of potential cytogenetic signatures that support the clade of Pandionidae (PHA) + Accipitridae. In addition, our cladistic analysis using chromosomal characters appears to support the placement of osprey (PHA) in Accipitridae

Chromosomal evolution and phylogeny in the Nullicauda group (Chiroptera, Phyllostomidae): evidence from multidirectional chromosome painting.

BACKGROUND: The family Phyllostomidae (Chiroptera) shows wide morphological, molecular and cytogenetic variation; many disagreements regarding its phylogeny and taxonomy remains to be resolved. In this study, we use chromosome painting with whole chromosome probes from the Phyllostomidae Phyllostomus hastatus and Carollia brevicauda to determine the rearrangements among several genera of the Nullicauda group (subfamilies Gliphonycterinae, Carolliinae, Rhinophyllinae and Stenodermatinae). RESULTS: These data, when compared with previously published chromosome homology maps, allow the construction of a phylogeny comparable to those previously obtained by morphological and molecular analysis. Our phylogeny is largely in agreement with that proposed with molecular data, both on relationships between the subfamilies and among genera; it confirms, for instance, that Carollia and Rhinophylla, previously considered as part of the same subfamily are, in fact, distant genera. CONCLUSIONS: The occurrence of the karyotype considered ancestral for this family in several different branches suggests that the diversification of Phyllostomidae into many subfamilies has occurred in a short period of time. Finally, the comparison with published maps using human whole chromosome probes allows us to track some syntenic associations prior to the emergence of this family

Transcription of a protein-coding gene on B chromosomes of the Siberian roe deer (Capreolus pygargus)

BACKGROUND: Most eukaryotic species represent stable karyotypes with a particular diploid number. B chromosomes are additional to standard karyotypes and may vary in size, number and morphology even between cells of the same individual. For many years it was generally believed that B chromosomes found in some plant, animal and fungi species lacked active genes. Recently, molecular cytogenetic studies showed the presence of additional copies of protein-coding genes on B chromosomes. However, the transcriptional activity of these genes remained elusive. We studied karyotypes of the Siberian roe deer (Capreolus pygargus) that possess up to 14 B chromosomes to investigate the presence and expression of genes on supernumerary chromosomes. RESULTS: Here, we describe a 2 Mbp region homologous to cattle chromosome 3 and containing TNNI3K (partial), FPGT, LRRIQ3 and a large gene-sparse segment on B chromosomes of the Siberian roe deer. The presence of the copy of the autosomal region was demonstrated by B-specific cDNA analysis, PCR assisted mapping, cattle bacterial artificial chromosome (BAC) clone localization and quantitative polymerase chain reaction (qPCR). By comparative analysis of B-specific and non-B chromosomal sequences we discovered some B chromosome-specific mutations in protein-coding genes, which further enabled the detection of a FPGT-TNNI3K transcript expressed from duplicated genes located on B chromosomes in roe deer fibroblasts. CONCLUSIONS: Discovery of a large autosomal segment in all B chromosomes of the Siberian roe deer further corroborates the view of an autosomal origin for these elements. Detection of a B-derived transcript in fibroblasts implies that the protein coding sequences located on Bs are not fully inactivated. The origin, evolution and effect on host of B chromosomal genes seem to be similar to autosomal segmental duplications, which reinforces the view that supernumerary chromosomal elements might play an important role in genome evolution

Expansion of the HSFY gene family in pig lineages : HSFY expansion in suids.

BACKGROUND: Amplified gene families on sex chromosomes can harbour genes with important biological functions, especially relating to fertility. The Y-linked heat shock transcription factor (HSFY) family has become amplified on the Y chromosome of the domestic pig (Sus scrofa), in an apparently independent event to an HSFY expansion on the Y chromosome of cattle (Bos taurus). Although the biological functions of HSFY genes are poorly understood, they appear to be involved in gametogenesis in a number of mammalian species, and, in cattle, HSFY gene copy number may correlate with levels of fertility. RESULTS: We have investigated the HSFY family in domestic pig, and other suid species including warthog, bushpig, babirusa and peccaries. The domestic pig contains at least two amplified variants of HSFY, distinguished predominantly by presence or absence of a SINE within the intron. Both these variants are expressed in testis, and both are present in approximately 50 copies each in a single cluster on the short arm of the Y. The longer form has multiple nonsense mutations rendering it likely non-functional, but many of the shorter forms still have coding potential. Other suid species also have these two variants of HSFY, and estimates of copy number suggest the HSFY family may have amplified independently twice during suid evolution. CONCLUSIONS: The HSFY genes have become amplified in multiple species lineages independently. HSFY is predominantly expressed in testis in domestic pig, a pattern conserved with cattle, in which HSFY may play a role in fertility. Further investigation of the potential associations of HSFY with fertility and testis development may be of agricultural interest.We gratefully acknowledge the Wellcome Trust Sanger Institute core teams for fingerprinting, mapping, archiving, library construction, sequence improvement and sequencing and Genus for providing the Duroc boar samples. This work was funded by BBSRC grant BB/F021372/1. The Flow Cytometry and Cytogenetics Core Facilities at the Wellcome Trust Sanger Institute and Sanger investigators are funded by the Wellcome Trust (grant number WT098051)