Heritable Genome Research Articles

Targeted Heritable Mutation and Gene Conversion by Cas9-CRISPR in Caenorhabditis elegans

We have achieved targeted heritable genome modification in Caenorhabditis elegans by injecting mRNA of the nuclease Cas9 and Cas9 guide RNAs. This system rapidly creates precise genomic changes, including knockouts and transgene-instructed gene conversion.

Precise and Heritable Genome Editing in Evolutionarily Diverse Nematodes Using TALENs and CRISPR/Cas9 to Engineer Insertions and Deletions

Exploitation of custom-designed nucleases to induce DNA double-strand breaks (DSBs) at genomic locations of choice has transformed our ability to edit genomes, regardless of their complexity. DSBs can trigger either error-prone repair pathways that induce random mutations at the break sites or precise homology-directed repair pathways that generate specific insertions or deletions guided by exogenously supplied DNA. Prior editing strategies using site-specific nucleases to modify the Caenorhabditis elegans genome achieved only the heritable disruption of endogenous loci through random mutagenesis by error-prone repair. Here we report highly effective strategies using TALE nucleases and RNA-guided CRISPR/Cas9 nucleases to induce error-prone repair and homology-directed repair to create heritable, precise insertion, deletion, or substitution of specific DNA sequences at targeted endogenous loci. Our robust strategies are effective across nematode species diverged by 300 million years, including necromenic nematodes (Pristionchus pacificus), male/female species (Caenorhabditis species 9), and hermaphroditic species (C. elegans). Thus, genome-editing tools now exist to transform nonmodel nematode species into genetically tractable model organisms. We demonstrate the utility of our broadly applicable genome-editing strategies by creating reagents generally useful to the nematode community and reagents specifically designed to explore the mechanism and evolution of X chromosome dosage compensation. By developing an efficient pipeline involving germline injection of nuclease mRNAs and single-stranded DNA templates, we engineered precise, heritable nucleotide changes both close to and far from DSBs to gain or lose genetic function, to tag proteins made from endogenous genes, and to excise entire loci through targeted FLP-FRT recombination.

Mining conifers’ mega-genome using rapid and efficient multiplexed high-throughput genotyping-by-sequencing (GBS) SNP discovery platform

Next-generation sequencing (NGS) technologies are revolutionizing both medical and biological research through generation of massive SNP data sets for identifying heritable genome variation underlying key traits, from rare human diseases to important agronomic phenotypes in crop species. We evaluated the performance of genotyping-by-sequencing (GBS), one of the emerging NGS-based platforms, for genotyping two economically important conifer species, lodgepole pine (Pinus contorta) and white spruce (Picea glauca). Both species have very large genomes (>20,000 Mbp), are highly heterozygous, and lack reference sequences. From a small set (six accessions each) of independent replicated DNA samples and a 48-plex read depth, we obtained ~60,000 SNPs per species. After stringent filtering, we obtained 17,765 and 17,845 high-coverage SNPs without missing data for lodgepole pine and white spruce, respectively. Our results demonstrated that GBS is a robust and suitable method for genotyping conifers. The application of GBS to forest tree breeding and genomic selection is discussed.

Heritable and Precise Zebrafish Genome Editing Using a CRISPR-Cas System

We have previously reported a simple and customizable CRISPR (clustered regularly interspaced short palindromic repeats) RNA-guided Cas9 nuclease (RGN) system that can be used to efficiently and robustly introduce somatic indel mutations in endogenous zebrafish genes. Here we demonstrate that RGN-induced mutations are heritable, with efficiencies of germline transmission reaching as high as 100%. In addition, we extend the power of the RGN system by showing that these nucleases can be used with single-stranded oligodeoxynucleotides (ssODNs) to create precise intended sequence modifications, including single nucleotide substitutions. Finally, we describe and validate simple strategies that improve the targeting range of RGNs from 1 in every 128 basepairs (bps) of random DNA sequence to 1 in every 8 bps. Together, these advances expand the utility of the CRISPR-Cas system in the zebrafish beyond somatic indel formation to heritable and precise genome modifications.

Abstract SY25-03: Host and tumor genetic architecture influence clinically relevant oncogenic pathways in neuroblastoma

Abstract Cancer research is at a critical crossroads as genomic technologies have enabled potentially paradigm-shifting discoveries in host genetic alterations that lead to cancer, as well as the major oncogenic drivers that somatically arise and shape the ultimate tumor phenotype. Approaches such as genome-wide association studies (GWAS) to discover heritable susceptibility variants have largely occurred in parallel with tumor profiling efforts designed to discover somatically acquired mutations. We see tremendous opportunity in integrating these efforts under the broad hypothesis that the host's genetic blueprint not only influences susceptibility to cancer, but also significantly influences the cancer phenotype at diagnosis and the pathways upon which the cancer is dependent. By focusing our efforts on the pediatric cancer neuroblastoma, we are directly addressing an important childhood malignancy that continues to exact disproportionate morbidity and mortality. However, we have also shown that this disease also serves as a remarkable model for the study of human cancers in general, and we think that lessons learned here are likely to be more broadly applicable as we are addressing the concept that somatic mutations alone will not define the entire spectrum of oncogenic vulnerabilities in cancer cells. Until recently, the etiology of neuroblastoma was unknown. By studying the 1% of neuroblastoma cases that occur within families, we have discovered ALK and PHOX2B as the two major hereditary predisposition genes.1,2 In parallel, by studying the 99% of cases that occur sporadically in our ongoing GWAS, for which we have now analyzed 4,000 cases and 10,000 controls, we have shown that normal genetic variation at multiple loci influences neuroblastoma susceptibility.3-7 We have several other recently validated associations that are not yet published, including replicated signals in LIN28B and HACE1. Significant lessons from the GWAS to date include: 1) Neuroblastoma is a genetic disease; 2) every signal identified to date is intragenic; 3) associations are enriched in phenotypic subsets; and 4) many of the genes identified by the neuroblastoma GWAS not only influence malignant transformation of a developing neuroblastic cell, but also contribute to the maintenance of the malignant phenotype. For example, SNP variations in the LMO1 gene are highly associated with overexpression of this transcriptional co-factor, and genetic manipulation of expression shows that it functions as an oncogenic driver in a genotype specific manner.7 In other words, our data suggest that subjects harboring LMO1 risk alleles are much more likely to have tumors dependent on LMO1-influenced transcriptional regulation suggesting that oncogene addiction is also hardwired in the heritable genome. In parallel, we and others have shown that somatic activating mutations in the ALK kinase domain occur in ∼10% of sporadic cases.2,8-10 To seek other potential somatically acquired mutations that may be therapeutically relevant in the neuroblastoma coding genome, we have resequenced 99 high-risk neuroblastoma cases using a combination of whole exome, genome and transcriptome sequencing. Tumors contained a median 0.56 non-silent mutations per megabase of coding DNA, one of the lowest rates reported in cancer to date. ALK was the most commonly mutated gene (9%), and no other gene showed mutation frequency rates >5%. These data clearly indicate that somatic mutation in neuroblastoma is relatively rare, leading to the concept that other mechanisms, such as host polymorphic variation or germline mutations in key regulatory regions, play an important role in tumorigenesis. Ongoing work to be discussed at the symposium include efforts to identify and validate rare variants in the host genome that impact neuroblastoma susceptibility, understanding the functional relevance of DNA variation in the developing neuroblastoma and in tumor tissues, completion of our cataloguing of somatic mutations in high-risk disease, and efforts to understand the impact of non-coding DNA alterations in the host in tumor genomes on neuroblastoma clinical phenotype. Efforts to translate these efforts into deliverables for the clinic will be emphasized.

Crosstalk between the DNA damage response pathway and microRNAs.

MicroRNAs (miRNAs) are a family of small, non-coding RNAs that control gene expression at the post-transcriptional level by destabilizing and inhibiting translation of their target messenger RNAs. MiRNAs are involved in the regulation of a number of fundamental biological processes, and their dysregulation is thought to contribute to several disease processes. Emerging evidence suggests that miRNAs also play a critical role in protecting the heritable genome by contributing to the regulation of the DNA damage response. Consequently, much recent investigative effort has been directed towards an improved understanding of how miRNAs are regulated in response to DNA damage. In this review, we discuss the most recent findings regarding the regulation of miRNA expression and the functional roles of miRNAs in the DNA damage response.

Next‐generation sequencing applications for wheat crop improvement

Bread wheat (Triticum aestivum; Poaceae) is a crop plant of great importance. It provides nearly 20% of the world's daily food supply measured by calorie intake, similar to that provided by rice. The yield of wheat has doubled over the last 40 years due to a combination of advanced agronomic practice and improved germplasm through selective breeding. More recently, yield growth has been less dramatic, and a significant improvement in wheat production will be required if demand from the growing human population is to be met. Next-generation sequencing (NGS) technologies are revolutionizing biology and can be applied to address critical issues in plant biology. Technologies can produce draft sequences of genomes with a significant reduction to the cost and timeframe of traditional technologies. In addition, NGS technologies can be used to assess gene structure and expression, and importantly, to identify heritable genome variation underlying important agronomic traits. This review provides an overview of the wheat genome and NGS technologies, details some of the problems in applying NGS technology to wheat, and describes how NGS technologies are starting to impact wheat crop improvement.

Abstract CN04-02: Interrogating the heritable genome to achieve the goal of personalized cancer therapeutics.

Abstract CN04-02: Interrogating the heritable genome to achieve the goal of personalized cancer therapeutics.

Unraveling a Connection between DNA Demethylation Repair and Cancer

In this issue of Molecular Cell, Dango and Mosammaparast discover that the human oxidative demethylase ALKBH3 functions in complex with a DNA helicase to eliminate N3-methylcytosine lesions from ssDNA and that specific cancer cell lines are dependent on this activity for proliferation (Dango et al., 2011).

BRCA1 and BRCA2 Variants of Uncertain Significance. Part Two: Medical Management

Genome editing, particularly the use of CRISPR-Cas9-based methodologies, is revolutionizing biology through its impacts on research and the translation of these into applications in biomedicine. Somatic genome editing aimed at treating individuals with disease raises some significant ethical issues, but proposed heritable interventions, through the use of genome editing in gametes or embryos, raise a number of distinct social, ethical and political issues. This review will consider some proposed uses of heritable human genome editing (HHGE) and several of the objections to these that have been raised. Making sense of such proposed uses requires viewing HHGE as an assisted reproductive technology (ART) that, like preimplantation genetic testing (PGT) and mitochondrial replacement techniques (MRT), aims to prevent disease transmission during sexual reproduction, rather than acting as a therapy for an existing individual. Applications beyond the paradigm of disease prevention raise even more difficult scientific and ethical questions. Here, I will discuss various themes that are prominent in discussions of the science and ethics of HHGE, including impacts on human dignity and society, the language of HHGE used for public dialogue and the governance of HHGE.

Protecting the heritable genome: DNA damage response mechanisms in spermatogonial stem cells

Spermatogonial stem cells (SSCs) must maintain the integrity of their genome to prevent reproduction failure and limit the hereditary risk associated with transmission to the progeny. SSCs must therefore have robust response mechanisms to counteract the potentially deleterious effects of DNA damage, with DNA double-strand breaks (DSBs) representing the greatest threat to genomic integrity. Through in vivo analysis of the DNA damage response of SSCs within their physiological tissue context, we aimed to gain insights into the mechanisms by which SSCs preserve genome integrity. After whole-body irradiation of repair-proficient and repair-deficient (DNA-PK- and ATM-deficient) mice, the formation and rejoining of DSBs was analyzed in SSCs of testis compared with somatic cells of other tissues by enumerating γH2AX-, MDC1-, and 53BP1-foci. Caspase-3 and PARP-1 were used as markers for apoptotic cell death. Our results show that DNA damage response mechanisms in SSCs characterized by unique chromatin compositions are markedly different from those of somatic cells. In SSCs lacking compact heterochromatin, histone-associated signaling components of the DNA repair machinery are completely absent and radiation-induced DSBs are rejoined predominantly by DNA-PK-independent pathways, suggesting the existence of alternative repair mechanisms. As a complimentary mechanism characterized by low thresholds for ATM-dependent checkpoint activation, the differentiating progeny, but not the SSCs themselves, promote apoptosis in response to low levels of DNA damage. By evaluating SSCs within their stem cell niche, we show that DNA repair, cell-cycle checkpoints, and apoptosis function together to maintain the integrity of the heritable genome.

One-hit wonders of genomic instability

Recent data show that cells from many cancers exhibit massive chromosome instability. The traditional view is that the gradual accumulation of mutations in genes involved in transcriptional regulation and cell cycle controls results in tumor development. This, however, does not exclude the possibility that some mutations could be more potent than others in destabilizing the genome by targeting both chromosomal integrity and corresponding checkpoint mechanisms simultaneously. Three such examples of "single-hit" lesions potentially leading to heritable genome destabilization are discussed. They include: failure to release sister chromatid cohesion due to the incomplete proteolytic cleavage of cohesin; massive merotelic kinetochore misattachments upon condensin depletion; and chromosome under-replication. In all three cases, cells fail to detect potential chromosomal bridges before anaphase entry, indicating that there is a basic cell cycle requirement to maintain a degree of sister chromatid bridging that is not recognizable as chromosomal damage.

Manipulating the Metazoan Mitochondrial Genome with Targeted Restriction Enzymes

High copy number and random segregation confound genetic analysis of the mitochondrial genome. We developed an efficient selection for heritable mitochondrial genome (mtDNA) mutations in Drosophila, thereby enhancing a metazoan model for study of mitochondrial genetics and mutations causing human mitochondrial disease. Targeting a restriction enzyme to mitochondria in the germline compromised fertility, but escaper progeny carried homoplasmic mtDNA mutations lacking the cleavage site. Among mutations eliminating a site in the cytochrome c oxidase gene, mt:CoI(A302T) was healthy, mt:CoI(R301L) was male sterile but otherwise healthy, and mt:CoI(R301S) exhibited a wide range of defects, including growth retardation, neurodegeneration, muscular atrophy, male sterility, and reduced life span. Thus, germline expression of mitochondrial restriction enzymes creates a powerful selection and has allowed direct isolation of mitochondrial mutants in a metazoan.

MicroRNA as bioaptamers in RNP cytokines [ribokines] and codes for epigenetic phenotype alterations and self‐tolerance: Sequence‐defined endogenous redox‐ and metalloregulated, edited and modified “non‐coding” small hairpin RNA

Mammalian miRNA roles were investigated in epigenetic [non-Mendelian] phenotype alterations [EPA] in cells and protein conformations in self-tolerance and discrimination of damaged self- and intruded foreign matter by sustaining and obliterating abnormal hypervascular patterns [Wissler et al., Protides Biol. Fluids 34: 517-536, 1986; Materialwiss. Werkstofftech. 32: 984-1008, 2001; Arch. Surg. 124: 693-698, 1989; Ann. N.Y. Acad. Sci. 961: 292-297, 2002; 991: 333-338, 2003; 1022: 163-184, 2004; FASEB J. 19: A621, 2005; FEBS J. 272-s1: N1-06-4P, 2005; Mol. Biol. Cell Suppl. 12: 149a-150a, 2001; 16: #1459 & 2848, 2005]. Bioactive metalloregulated complexes of entitled RNA bioaptamers [<200 bases] and protein conformers were isolated from cells, supernatants and wound fluids. By Cu ion-structured 5′-CUG-3′ hairpin loops, such RNA fit to binding domains in epigenetic regulator proteins [e.g. angiotropin angio-morphogens], termed K/R3H [K/RxxxH], i.e. -t/s/xK/R/q/nxxxH/y/n/q/e/dx7-9h/xx7-9h/xx5-20K/R/q/n/e/h- with accessory basic [R/K]n, R/K-zipper, SR/K/RS and / or HxxxH/y/n/q segments. By sequence edition, base modification [e.g. to isoguanine], redox- and metalloregulation, i.e. code extensions / alterations, some RNA are not directly retranslatable to heritable genome codes but feedback protein folding to translation and transcription. The results suggest such RNA part of specific clonal recognition in novel EPA, inheritance and cancer escape mechanisms in RNA-operated conformation phase pathway-locked loops. They are comparably effective as antibody [~108], T-cell receptor [~1010] and MHC systems [~1013] but superior in diversity / specificity [estimated repertoire ~1017].

Meiotic behaviours of chromosomes and microtubules in budding yeast: relocalization of centromeres and telomeres during meiotic prophase.

Meiosis is a process of universal importance in eukaryotic organisms, generating variation in the heritable haploid genome by recombination and re-assortment of chromosomes. The intranuclear movement of chromosomes is expected to achieve pairing and recombination of homologous chromosomes during meiosis. Meiosis in the budding yeast Saccharomyces cerevisiae has been extensively studied, both genetically and by molecular biology; here we report cytological observations of meiotic chromosomal events in this organism. Using fluorescence microscopy, we have examined the behaviour of chromosomes and microtubules during meiosis in S. cerevisiae. We first observed the dynamic behaviour of nuclei in living cells using jellyfish green fluorescent protein (GFP) fused with nucleoplasmin, a Xenopus oocyte nuclear protein. The characterization of nuclear movement in living cells was extended by an analysis of chromosomes and microtubules in fixed specimens. In addition, the nuclear localization of centromeres and telomeres was determined by indirect immunofluorescence microscopy in synchronous populations of meiotic cells. While telomeres remain in clusters of 5-8 throughout meiosis, centromeres change their nuclear localization dramatically during the progression of meiosis: centromeres are first clustered at a single site near the spindle-pole body before the induction of meiosis, and become scattered during the meiotic prophase. Our observations have demonstrated that nuclear and cytoskeletal reorganization take place with meiosis in S. cerevisiae. In particular, the distinct relocalization of centromeres during meiosis indicates a considerable movement of chromosomes within the meiotic prophase nucleus.

The Revised “Points to Consider” Document

Human Gene TherapyVol. 1, No. 1 News and CommentsThe Revised “Points to Consider” DocumentPublished Online:12 Jun 2008https://doi.org/10.1089/hum.1990.1.1-93AboutSectionsPDF/EPUB Permissions & CitationsPermissionsDownload CitationsTrack CitationsAdd to favorites Back To Publication ShareShare onFacebookTwitterLinked InRedditEmail FiguresReferencesRelatedDetailsCited byEmerging treatment for congenital adrenal hyperplasiaCurrent Opinion in Endocrinology, Diabetes & Obesity, Vol. 29, No. 3Governing Heritable Human Genome Editing: A Textual History and a Proposal for the Future LeRoy Walters, Robert M. Cook-Deegan, and Eli Y. Adashi16 August 2021 | The CRISPR Journal, Vol. 4, No. 4Pathways toward the future: points to consider for oncofertility oversight11 December 2012 | Journal of Cancer Survivorship, Vol. 7, No. 1FDA comments on phase I clinical trials without vector biodistribution dataNature Genetics, Vol. 22, No. 4The Application of Genetic Knowledge: Ethical and Policy ImplicationsAACN Clinical Issues: Advanced Practice in Acute and Critical Care, Vol. 9, No. 4Aspekte der somatischen Gentherapie in der Herz- und KreislaufmedizinGene therapy: regulatory issues and international approaches to regulationCurrent Opinion in Biotechnology, Vol. 8, No. 3The Ethics of Somatic and Germline Gene TherapyAnnals of the New York Academy of Sciences, Vol. 816, No. 1 Adolescent GySafety and regulation at the leading edge of biomedical biotechnologyCurrent Opinion in Biotechnology, Vol. 7, No. 3Gene therapy for head and neck cancerHead & Neck, Vol. 17, No. 6Ethical and regulatory issues in gene therapyBritish Journal of Urology, Vol. 76Guidance on Making Proposals to Conduct Gene Therapy Research on Human Subjects Report of the United Kingdom Health Ministers' Gene Therapy Advisory Committee19 March 2008 | Human Gene Therapy, Vol. 6, No. 3Human cancer and gene therapyAnnals of Hematology, Vol. 69, No. 6Improved Methods of Retroviral Vector Transduction and Production for Gene Therapy Hitoshi Kotani, Perry B. Newton, Shuyuan Zhang, Yawen L. Chiang, Edward Otto, Linda Weaver, R. Michael Blaese, W. French Anderson, and Gerard J. McGarrity19 March 2008 | Human Gene Therapy, Vol. 5, No. 1Hepatic gene therapy: Present and futureHepatology, Vol. 18, No. 5Gene Therapy of Cancer11 June 2009 | Cancer Investigation, Vol. 11, No. 6Human gene therapy comes of ageNature, Vol. 357, No. 6378Introduction and Abstracts from Journal of Medicine and Philosophy Thematic Issue on Germ-Line Gene Therapy Eric T. Juengst13 June 2008 | Human Gene Therapy, Vol. 3, No. 1The NIH “Points to Consider” and the Limits of Human Gene Therapy Eric T. Juengst12 June 2008 | Human Gene Therapy, Vol. 1, No. 4 Volume 1Issue 1Apr 1990 InformationCopyright 1990, Mary Ann Liebert, Inc.To cite this article:The Revised “Points to Consider” Document.Human Gene Therapy.Apr 1990.93-103.http://doi.org/10.1089/hum.1990.1.1-93Published in Volume: 1 Issue 1: June 12, 2008PDF download