Whole-genome analysis of Fusarium graminearum insertional mutants identifies virulence associated genes and unmasks untagged chromosomal deletions

Background

Identifying pathogen virulence genes required to cause disease is crucial to understand the mechanisms underlying the pathogenic process. Plasmid insertion mutagenesis of fungal protoplasts is frequently used for this purpose in filamentous ascomycetes. Post transformation, the mutant population is screened for loss of virulence to a specific plant or animal host. Identifying the insertion event has previously met with varying degrees of success, from a cleanly disrupted gene with minimal deletion of nucleotides at the insertion point to multiple-copy insertion events and large deletions of chromosomal regions. Currently, extensive mutant collections exist in laboratories globally where it was hitherto impossible to identify all the affected genes.

Results

We used a whole-genome sequencing (WGS) approach using Illumina HiSeq 2000 technology to investigate DNA tag insertion points and chromosomal deletion events in mutagenised, reduced virulence F. graminearum isolates identified in disease tests on wheat (Triticum aestivum). We developed the FindInsertSeq workflow to localise the DNA tag insertions to the nucleotide level. The workflow was tested using four mutants showing evidence of single and multi-copy insertions in DNA blot analysis. FindInsertSeq was able to identify both single and multi-copy concatenation insertion sites. By comparing sequencing coverage, unexpected molecular recombination events such as large tagged and untagged chromosomal deletions, and DNA amplification were observed in three of the analysed mutants. A random data sampling approach revealed the minimum genome coverage required to survey the F. graminearum genome for alterations.

Conclusions

This study demonstrates that whole-genome re-sequencing to 22x fold genome coverage is an efficient tool to characterise single and multi-copy insertion mutants in the filamentous ascomycete Fusarium graminearum. In some cases insertion events are accompanied with large untagged chromosomal deletions while in other cases a straight-forward insertion event could be confirmed. The FindInsertSeq analysis workflow presented in this study enables researchers to efficiently characterise insertion and deletion mutants.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1412-9) contains supplementary material, which is available to authorized users.     

Chromosome-breakage genomic instability and chromothripsis in breast cancer

Background

Chromosomal breakage followed by faulty DNA repair leads to gene amplifications and deletions in cancers. However, the mere assessment of the extent of genomic changes, amplifications and deletions may reduce the complexity of genomic data observed by array comparative genomic hybridization (array CGH). We present here a novel approach to array CGH data analysis, which focuses on putative breakpoints responsible for rearrangements within the genome.

Results

We performed array comparative genomic hybridization in 29 primary tumors from high risk patients with breast cancer. The specimens were flow sorted according to ploidy to increase tumor cell purity prior to array CGH. We describe the number of chromosomal breaks as well as the patterns of breaks on individual chromosomes in each tumor. There were differences in chromosomal breakage patterns between the 3 clinical subtypes of breast cancers, although the highest density of breaks occurred at chromosome 17 in all subtypes, suggesting a particular proclivity of this chromosome for breaks. We also observed chromothripsis affecting various chromosomes in 41% of high risk breast cancers.

Conclusions

Our results provide a new insight into the genomic complexity of breast cancer. Genomic instability dependent on chromosomal breakage events is not stochastic, targeting some chromosomes clearly more than others. We report a much higher percentage of chromothripsis than described previously in other cancers and this suggests that massive genomic rearrangements occurring in a single catastrophic event may shape many breast cancer genomes.

Electronic supplementary material

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Chromosomal deletions and inversions mediated by TALENs and CRISPR/Cas in zebrafish

Customized TALENs and Cas9/gRNAs have been used for targeted mutagenesis in zebrafish to induce indels into protein-coding genes. However, indels are usually not sufficient to disrupt the function of non-coding genes, gene clusters or regulatory sequences, whereas large genomic deletions or inversions are more desirable for this purpose. By injecting two pairs of TALEN mRNAs or two gRNAs together with Cas9 mRNA targeting distal DNA sites of the same chromosome, we obtained predictable genomic deletions or inversions with sizes ranging from several hundred bases to nearly 1 Mb. We have successfully achieved this type of modifications for 11 chromosomal loci by TALENs and 2 by Cas9/gRNAs with different combinations of gRNA pairs, including clusters of miRNA and protein-coding genes. Seven of eight TALEN-targeted lines transmitted the deletions and one transmitted the inversion through germ line. Our findings indicate that both TALENs and Cas9/gRNAs can be used as an efficient tool to engineer genomes to achieve large deletions or inversions, including fragments covering multiple genes and non-coding sequences. To facilitate the analyses and application of existing ZFN, TALEN and CRISPR/Cas data, we have updated our EENdb database to provide a chromosomal view of all reported engineered endonucleases targeting human and zebrafish genomes.     

PacBio-LITS: a large-insert targeted sequencing method for characterization of human disease-associated chromosomal structural variations

Background

Generation of long (>5 Kb) DNA sequencing reads provides an approach for interrogation of complex regions in the human genome. Currently, large-insert whole genome sequencing (WGS) technologies from Pacific Biosciences (PacBio) enable analysis of chromosomal structural variations (SVs), but the cost to achieve the required sequence coverage across the entire human genome is high.

Results

We developed a method (termed PacBio-LITS) that combines oligonucleotide-based DNA target-capture enrichment technologies with PacBio large-insert library preparation to facilitate SV studies at specific chromosomal regions. PacBio-LITS provides deep sequence coverage at the specified sites at substantially reduced cost compared with PacBio WGS. The efficacy of PacBio-LITS is illustrated by delineating the breakpoint junctions of low copy repeat (LCR)-associated complex structural rearrangements on chr17p11.2 in patients diagnosed with Potocki–Lupski syndrome (PTLS; MIM#610883). We successfully identified previously determined breakpoint junctions in three PTLS cases, and also were able to discover novel junctions in repetitive sequences, including LCR-mediated breakpoints. The new information has enabled us to propose mechanisms for formation of these structural variants.

Conclusions

The new method leverages the cost efficiency of targeted capture-sequencing as well as the mappability and scaffolding capabilities of long sequencing reads generated by the PacBio platform. It is therefore suitable for studying complex SVs, especially those involving LCRs, inversions, and the generation of chimeric Alu elements at the breakpoints. Other genomic research applications, such as haplotype phasing and small insertion and deletion validation could also benefit from this technology.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1370-2) contains supplementary material, which is available to authorized users.     

Coupling the CRISPR/Cas9 System with Lambda Red Recombineering Enables Simplified Chromosomal Gene Replacement in Escherichia coli

To date, most genetic engineering approaches coupling the type II Streptococcus pyogenes clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 system to lambda Red recombineering have involved minor single nucleotide mutations. Here we show that procedures for carrying out more complex chromosomal gene replacements in Escherichia coli can be substantially enhanced through implementation of CRISPR/Cas9 genome editing. We developed a three-plasmid approach that allows not only highly efficient recombination of short single-stranded oligonucleotides but also replacement of multigene chromosomal stretches of DNA with large PCR products. By systematically challenging the proposed system with respect to the magnitude of chromosomal deletion and size of DNA insertion, we demonstrated DNA deletions of up to 19.4 kb, encompassing 19 nonessential chromosomal genes, and insertion of up to 3 kb of heterologous DNA with recombination efficiencies permitting mutant detection by colony PCR screening. Since CRISPR/Cas9-coupled recombineering does not rely on the use of chromosome-encoded antibiotic resistance, or flippase recombination for antibiotic marker recycling, our approach is simpler, less labor-intensive, and allows efficient production of gene replacement mutants that are both markerless and “scar”-less.     

Egg cell-specific promoter-controlled CRISPR/Cas9 efficiently generates homozygous mutants for multiple target genes in Arabidopsis in a single generation

Arabidopsis mutants produced by constitutive overexpression of the CRISPR/Cas9 genome editing system are usually mosaics in the T1 generation. In this study, we used egg cell-specific promoters to drive the expression of Cas9 and obtained non-mosaic T1 mutants for multiple target genes with high efficiency. Comparisons of 12 combinations of eight promoters and two terminators found that the efficiency of the egg cell-specific promoter-controlled CRISPR/Cas9 system depended on the presence of a suitable terminator, and the composite promoter generated by fusing two egg cell-specific promoters resulted in much higher efficiency of mutation in the T1 generation compared with the single promoters.

Electronic supplementary material

The online version of this article (doi:10.1186/s13059-015-0715-0) contains supplementary material, which is available to authorized users.     

Towards accurate characterization of clonal heterogeneity based on structural variation

Background

Recent advances in deep digital sequencing have unveiled an unprecedented degree of clonal heterogeneity within a single tumor DNA sample. Resolving such heterogeneity depends on accurate estimation of fractions of alleles that harbor somatic mutations. Unlike substitutions or small indels, structural variants such as deletions, duplications, inversions and translocations involve segments of DNAs and are potentially more accurate for allele fraction estimations. However, no systematic method exists that can support such analysis.

Results

In this paper, we present a novel maximum-likelihood method that estimates allele fractions of structural variants integratively from various forms of alignment signals. We develop a tool, BreakDown, to estimate the allele fractions of most structural variants including medium size (from 1 kilobase to 1 megabase) deletions and duplications, and balanced inversions and translocations.

Conclusions

Evaluation based on both simulated and real data indicates that our method systematically enables structural variants for clonal heterogeneity analysis and can greatly enhance the characterization of genomically instable tumors.

Electronic supplementary material

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Drug resistant integrase mutants cause aberrant HIV integrations

Background

HIV-1 integrase is the target for three FDA-approved drugs, raltegravir, elvitegravir, and dolutegravir. All three drugs bind at the active site of integrase and block the strand transfer step of integration. We previously showed that sub-optimal doses of the anti-HIV drug raltegravir can cause aberrant HIV integrations that are accompanied by a variety of deletions, duplications, insertions and inversions of the adjacent host sequences.

Results

We show here that a second drug, elvitegravir, also causes similar aberrant integrations. More importantly, we show that at least two of the three clinically relevant drug resistant integrase mutants we tested, N155H and G140S/Q148H, which reduce the enzymatic activity of integrase, can cause the same sorts of aberrant integrations, even in the absence of drugs. In addition, these drug resistant mutants have an elevated IC₅₀ for anti-integrase drugs, and concentrations of the drugs that would be optimal against the WT virus are suboptimal for the mutants.

Conclusions

We previously showed that suboptimal doses of a drug that binds to the HIV enzyme integrase and blocks the integration of a DNA copy of the viral genome into host DNA can cause aberrant integrations that involve rearrangements of the host DNA. We show here that suboptimal doses of a second anti-integrase drug can cause similar aberrant integrations. We also show that drug-resistance mutations in HIV integrase can also cause aberrant integrations, even in the absence of an anti-integrase drug. HIV DNA integrations in the oncogenes BACH2 and MKL2 that do not involve rearrangements of the viral or host DNA can stimulate the proliferation of infected cells. Based on what is known about the association of DNA rearrangements and the activation of oncogenes in human tumors, it is possible that some of the deletions, duplications, insertions, and inversions of the host DNA that accompany aberrant HIV DNA integrations could increase the chances that HIV integrations could lead to the development of a tumor.

     

Characterization of Genomic Deletion Efficiency Mediated by Clustered Regularly Interspaced Palindromic Repeats (CRISPR)/Cas9 Nuclease System in Mammalian Cells

The clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR-associated (Cas) 9 nuclease system has provided a powerful tool for genome engineering. Double strand breaks may trigger nonhomologous end joining repair, leading to frameshift mutations, or homology-directed repair using an extrachromosomal template. Alternatively, genomic deletions may be produced by a pair of double strand breaks. The efficiency of CRISPR/Cas9-mediated genomic deletions has not been systematically explored. Here, we present a methodology for the production of deletions in mammalian cells, ranging from 1.3 kb to greater than 1 Mb. We observed a high frequency of intended genomic deletions. Nondeleted alleles are nonetheless often edited with inversions or small insertion/deletions produced at CRISPR recognition sites. Deleted alleles also typically include small insertion/deletions at predicted deletion junctions. We retrieved cells with biallelic deletion at a frequency exceeding that of probabilistic expectation. We demonstrate an inverse relationship between deletion frequency and deletion size. This work suggests that CRISPR/Cas9 is a robust system to produce a spectrum of genomic deletions to allow investigation of genes and genetic elements.     

Effective screen of CRISPR/Cas9-induced mutants in rice by single-strand conformation polymorphism

Key message

A method based on DNA single-strand conformation polymorphism is demonstrated for effective genotyping of CRISPR/Cas9-induced mutants in rice.

Abstract

Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9) has been widely adopted for genome editing in many organisms. A large proportion of mutations generated by CRISPR/Cas9 are very small insertions and deletions (indels), presumably because Cas9 generates blunt-ended double-strand breaks which are subsequently repaired without extensive end-processing. CRISPR/Cas9 is highly effective for targeted mutagenesis in the important crop, rice. For example, homozygous mutant seedlings are commonly recovered from CRISPR/Cas9-treated calli. However, many current mutation detection methods are not very suitable for screening homozygous mutants that typically carry small indels. In this study, we tested a mutation detection method based on single-strand conformational polymorphism (SSCP). We found it can effectively detect small indels in pilot experiments. By applying the SSCP method for CRISRP-Cas9-mediated targeted mutagenesis in rice, we successfully identified multiple mutants of OsROC5 and OsDEP1. In conclusion, the SSCP analysis will be a useful genotyping method for rapid identification of CRISPR/Cas9-induced mutants, including the most desirable homozygous mutants. The method also has high potential for similar applications in other plant species.

     

Unraveling the effect of genomic structural changes in the rhesus macaque - implications for the adaptive role of inversions

Background

By reshuffling genomes, structural genomic reorganizations provide genetic variation on which natural selection can work. Understanding the mechanisms underlying this process has been a long-standing question in evolutionary biology. In this context, our purpose in this study is to characterize the genomic regions involved in structural rearrangements between human and macaque genomes and determine their influence on meiotic recombination as a way to explore the adaptive role of genome shuffling in mammalian evolution.

Results

We first constructed a highly refined map of the structural rearrangements and evolutionary breakpoint regions in the human and rhesus macaque genomes based on orthologous genes and whole-genome sequence alignments. Using two different algorithms, we refined the genomic position of known rearrangements previously reported by cytogenetic approaches and described new putative micro-rearrangements (inversions and indels) in both genomes. A detailed analysis of the rhesus macaque genome showed that evolutionary breakpoints are in gene-rich regions, being enriched in GO terms related to immune system. We also identified defense-response genes within a chromosome inversion fixed in the macaque lineage, underlying the relevance of structural genomic changes in evolutionary and/or adaptation processes. Moreover, by combining in silico and experimental approaches, we studied the recombination pattern of specific chromosomes that have suffered rearrangements between human and macaque lineages.

Conclusions

Our data suggest that adaptive alleles – in this case, genes involved in the immune response – might have been favored by genome rearrangements in the macaque lineage.

Electronic supplementary material

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Transposition of reversed Ac element ends generates novel chimeric genes in maize

The maize Activator/Dissociation (Ac/Ds) elements are members of the hAT (hobo, Ac, and Tam3) superfamily of type II (DNA) transposons that transpose through a “cut-and-paste” mechanism. Previously, we reported that a pair of Ac ends in reversed orientation is capable of undergoing alternative transposition reactions that can generate large-scale chromosomal rearrangements, including deletions and inversions. We show here that rearrangements induced by reversed Ac ends transposition can join the coding and regulatory sequences of two linked paralogous genes to generate a series of chimeric genes, some of which are functional. To our knowledge, this is the first report demonstrating that alternative transposition reactions can recombine gene segments, leading to the creation of new genes.     

Global detection of DNA repair outcomes induced by CRISPR–Cas9

CRISPR–Cas9 generates double-stranded DNA breaks (DSBs) to activate cellular DNA repair pathways for genome editing. The repair of DSBs leads to small insertions or deletions (indels) and other complex byproducts, including large deletions and chromosomal translocations. Indels are well understood to disrupt target genes, while the other deleterious byproducts remain elusive. We developed a new in silico analysis pipeline for the previously described primer-extension-mediated sequencing assay to comprehensively characterize CRISPR–Cas9-induced DSB repair outcomes in human or mouse cells. We identified tremendous deleterious DSB repair byproducts of CRISPR–Cas9 editing, including large deletions, vector integrations, and chromosomal translocations. We further elucidated the important roles of microhomology, chromosomal interaction, recurrent DSBs, and DSB repair pathways in the generation of these byproducts. Our findings provide an extra dimension for genome editing safety besides off-targets. And caution should be exercised to avoid not only off-target damages but also deleterious DSB repair byproducts during genome editing.     

Large chromosomal deletions and heritable small genetic changes induced by CRISPR/Cas9 in rice

The Cas9/sgRNA of the CRISPR/Cas system has emerged as a robust technology for targeted gene editing in various organisms, including plants, where Cas9/sgRNA-mediated small deletions/insertions at single cleavage sites have been reported in transient and stable transformations, although genetic transmission of edits has been reported only in Arabidopsis and rice. Large chromosomal excision between two remote nuclease-targeted loci has been reported only in a few non-plant species. Here we report in rice Cas9/sgRNA-induced large chromosomal segment deletions, the inheritance of genome edits in multiple generations and construction of a set of facile vectors for high-efficiency, multiplex gene targeting. Four sugar efflux transporter genes were modified in rice at high efficiency; the most efficient system yielding 87–100% editing in T0 transgenic plants, all with di-allelic edits. Furthermore, genetic crosses segregating Cas9/sgRNA transgenes away from edited genes yielded several genome-edited but transgene-free rice plants. We also demonstrated proof-of-efficiency of Cas9/sgRNAs in producing large chromosomal deletions (115–245 kb) involving three different clusters of genes in rice protoplasts and verification of deletions of two clusters in regenerated T0 generation plants. Together, these data demonstrate the power of our Cas9/sgRNA platform for targeted gene/genome editing in rice and other crops, enabling both basic research and agricultural applications.     

Development of a genome editing technique using the CRISPR/Cas9 system in the industrial filamentous fungus <Emphasis Type="Italic">Aspergillus oryzae</Emphasis>

Objectives

To develop a genome editing method using the CRISPR/Cas9 system in Aspergillus oryzae, the industrial filamentous fungus used in Japanese traditional fermentation and for the production of enzymes and heterologous proteins.

Results

To develop the CRISPR/Cas9 system as a genome editing technique for A. oryzae, we constructed plasmids expressing the gene encoding Cas9 nuclease and single guide RNAs for the mutagenesis of target genes. We introduced these into an A. oryzae strain and obtained transformants containing mutations within each target gene that exhibited expected phenotypes. The mutational rates ranged from 10 to 20 %, and 1 bp deletions or insertions were the most commonly induced mutations.

Conclusions

We developed a functional and versatile genome editing method using the CRISPR/Cas9 system in A. oryzae. This technique will contribute to the use of efficient targeted mutagenesis in many A. oryzae industrial strains.

     

Structural and sequence diversity of the transposon Galileo in the Drosophila willistoni genome

Background

Galileo is one of three members of the P superfamily of DNA transposons. It was originally discovered in Drosophila buzzatii, in which three segregating chromosomal inversions were shown to have been generated by ectopic recombination between Galileo copies. Subsequently, Galileo was identified in six of 12 sequenced Drosophila genomes, indicating its widespread distribution within this genus. Galileo is strikingly abundant in Drosophila willistoni, a neotropical species that is highly polymorphic for chromosomal inversions, suggesting a role for this transposon in the evolution of its genome.

Results

We carried out a detailed characterization of all Galileo copies present in the D. willistoni genome. A total of 191 copies, including 133 with two terminal inverted repeats (TIRs), were classified according to structure in six groups. The TIRs exhibited remarkable variation in their length and structure compared to the most complete copy. Three copies showed extended TIRs due to internal tandem repeats, the insertion of other transposable elements (TEs), or the incorporation of non-TIR sequences into the TIRs. Phylogenetic analyses of the transposase (TPase)-encoding and TIR segments yielded two divergent clades, which we termed Galileo subfamilies V and W. Target-site duplications (TSDs) in D. willistoni Galileo copies were 7- or 8-bp in length, with the consensus sequence GTATTAC. Analysis of the region around the TSDs revealed a target site motif (TSM) with a 15-bp palindrome that may give rise to a stem-loop secondary structure.

Conclusions

There is a remarkable abundance and diversity of Galileo copies in the D. willistoni genome, although no functional copies were found. The TIRs in particular have a dynamic structure and extend in different ways, but their ends (required for transposition) are more conserved than the rest of the element. The D. willistoni genome harbors two Galileo subfamilies (V and W) that diverged ~9 million years ago and may have descended from an ancestral element in the genome. Galileo shows a significant insertion preference for a 15-bp palindromic TSM.

Electronic supplementary material

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A transgenic system for generation of transposon Ac/Ds-induced chromosome rearrangements in rice

The maize Activator (Ac)/Dissociation (Ds) transposable element system has been used in a variety of plants for insertional mutagenesis. Ac/Ds elements can also generate genome rearrangements via alternative transposition reactions which involve the termini of closely linked transposons. Here, we introduced a transgene containing reverse-oriented Ac/Ds termini together with an Ac transposase gene into rice (Oryza sativa ssp. japonica cv. Nipponbare). Among the transgenic progeny, we identified and characterized 25 independent genome rearrangements at three different chromosomal loci. The rearrangements include chromosomal deletions and inversions and one translocation. Most of the deletions occurred within the T-DNA region, but two cases showed the loss of 72 kilobase pairs (kb) and 79 kb of rice genomic DNA flanking the transgene. In addition to deletions, we obtained chromosomal inversions ranging in size from less than 10 kb (within the transgene DNA) to over 1 million base pairs (Mb). For 11 inversions, we cloned and sequenced both inversion breakpoints; in all 11 cases, the inversion junctions contained the typical 8 base pairs (bp) Ac/Ds target site duplications, confirming their origin as transposition products. Together, our results indicate that alternative Ac/Ds transposition can be an efficient tool for functional genomics and chromosomal manipulation in rice.     

Inversions and deletions of the Salmonella chromosome generated by the translocatable tetracycline resistance element Tn10

Spontaneous tetraoyoline-sensitive derivatives of a Tn10 insertion in the hisG gene of Salmonella typhimurium were isolated and subjected to genetic analysis. All 123 of the drug-sensitive derivatives characterized have undergone stable alterations in the Tn10 element itself; over half of the derivatives have also undergone major alterations of neighboring regions of the Salmonella chromosome. These chromosomal rearrangements are of two types: inversions and deletions. Any single inversion or deletion affects a contiguous stretch of chromosomal material extending from the site of the original Tn10 element either leftward or rightward.The genetic properties of deletion and inversion derivatives suggest that these chromosomal alterations are promoted by the Tn10 element itself. The role of translocatable elements in promoting chromosomal deletions is well documented; the ability of an element to promote inversions of chromosomal material has not previously been reported. Possible analogies between the 1400-base-pair inverted repetition at the end of Tn10 and the small insertion sequence IS1 predict particular structures for Tn10-promoted deletions. A structural explanation or model for Tn10-promoted inversions is presented. The observation that Tn10 promotes the formation of inversions suggests that such elements could play a previously unanticipated role in promoting chromosomal inversions during evolution of prokaryotic organisms. Generally applicable genetic methods for the identification and characterization of chromosomal inversions are described.