Advances in genome editing tools make it possible to rapidly develop new cell and gene therapies. Artificial genes and modifications to existing ones can readily be made in a site-specific manner. ...However, the use of gene editing represents a new paradigm for treating disease. Tools are needed to ensure this new class of medicines are safe and efficacious for patients. Genome editing carries risk. These relate to malignant transformation of the target cells by a variety of mechanisms, including the inherent function of the gene editing platforms themselves. Each has the potential to cause genomic instability in the form of breaks at unintended sites in the genome. Off-target editing that activates a proto-oncogene or disrupts a tumor suppressor has the potential to be carcinogenic. Off-target mutagenesis may also contribute to a range of cellular dysfunction.
A variety of genomic tools have been developed to screen for off-target editing. However, each of these methods has acknowledged limitations and none has emerged as a definitive standard. More robust, reliable, reproducible and quantitative methods are needed for testing on and off-target editing during pre-clinical, clinical and post-treatment follow-up. At present an unbiased, standardised and scalable method that can be used across the industry is lacking. Here, we describe the development and characterization of INDUCE-seq to address this.
We describe INDUCE-seq: a cell-based, unbiased, PCR-free platform for the precision measurement of genomic breaks induced by gene editing. A HESI-led public study conducted CRISPR-Cas9-based gene editing of five well-studied genetic targets. Two industry partners, used two cells types and both on and off-target editing was measured using INDUCE-seq. The mutational (editing) outcomes was measured by Duplex Sequencing (DS) with a sensitivity of 0.01%.
We report the reproducibilty of the INDUCE-seq in detecting on and off-target editing within and between experimental replicates by both industry partners. We calculate the sensitivity, specificity, precison and accuracy of the assay. Finally, we report mutation frequencies at these on and off-target sites revealing the performance of INDUCE-seq by showing the correlation between editing-induced breaks and mutation frequency.
We conclude that INDUCE-seq is a reliable and reproducible assay to assess the on and off-target effects of gene editing.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
2.
Biome Mucina, Ladislav
The New phytologist,
April 2019, Volume:
222, Issue:
1
Journal Article
Peer reviewed
Open access
A biome is a key community ecological and biogeographical concept and, as such, has profited from the overall progress of community ecology, punctuated by two major innovations: shifting the focus ...from pure pattern description to understanding functionality, and changing the approach from observational to explanatory and,most importantly, from descriptive to predictive. The functional focus enabled development of mechanistic and function-focused predictive and retrodictive modelling; it also shaped the current understanding of the concept of a biome as a dynamic biological entity having many aspects, with deep roots in the evolutionary past, and which is undergoing change. The evolution of the biome concept was punctuated by three synthetic steps: the first synthesis formulated a solid body of theory explaining the ecological and biogeographical meaning of zonality and collated our knowledge on drivers of vegetation patterns at large spatial scales; the second translated this knowledge into effective mechanistic modelling tools, developing further the link between ecosystem functionality and biogeography; and the third (still in progress) is seeking common ground between large-scale ecological and biogeographic phenomena, using macroecology and macroevolutionary research tools.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NMLJ, NUK, OILJ, PNG, SAZU, SBCE, SBMB, UL, UM, UPUK
ObjectivePublic health research frequently requires the integration of information from different data sources. However, errors in the records and the high computational costs involved make linking ...large administrative databases using record linkage (RL) methodologies a major challenge. We present Tucuxi-BLAST, a versatile tool for probabilistic RL that utilizes a DNA-encoded approach to encrypt, analyze and link massive administrative databases.
Materials and MethodsTucuxi-BLAST encodes the identification records into DNA. BLASTn algorithm is then used to align the sequences between databases. We tested and benchmarked on a simulated database containing records for 300 million individuals and also on four large administrative databases containing real data on Brazilian patients.
ResultsOur method was able to overcome misspellings and typographical errors in administrative databases. In processing the RL of the largest simulated dataset (200k records), the state-of-the art method took 5 days and 7 hours to perform the RL, while Tucuxi-BLAST only took 23 hours. When compared with five existing RL tools applied to a gold-standard dataset from real health-related databases, Tucuxi-BLAST had the highest accuracy and speed.
DiscussionBy repurposing genomic tools, researchers are able to perform subject tracing across multiple large epidemiological databases using a regular laptop.
ConclusionTucuxi-BLAST can improve data-driven medical research and provide a fast and accurate way to link individual information across several administrative databases.
Recent technological advances in the field of genomics offer conservation managers and practitioners new tools to explore for conservation applications. Many of these tools are well developed and ...used by other life science fields, while others are still in development. Considering these technological possibilities, choosing the right tool(s) from the toolbox is crucial and can pose a challenging task. With this in mind, we strive to inspire, inform and illuminate managers and practitioners on how conservation efforts can benefit from the current genomic and biotechnological revolution. With inspirational case studies we show how new technologies can help resolve some of the main conservation challenges, while also informing how implementable the different technologies are. We here focus specifically on small population management, highlight the potential for genetic rescue, and discuss the opportunities in the field of gene editing to help with adaptation to changing environments. In addition, we delineate potential applications of gene drives for controlling invasive species. We illuminate that the genomic toolbox offers added benefit to conservation efforts, but also comes with limitations for the use of these novel emerging techniques.
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EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
Pastures based on perennial monocotyledonous plants are the principal source of nutrition for ruminant livestock in tropical and subtropical areas across the globe. The
genus comprises important ...species used in pastures, and these mainly include
,
,
, and
. Despite their economic relevance, there is an absence of genomic-level information for these species, and this lack is mainly due to genomic complexity, including polyploidy, high heterozygosity, and genomes with a high repeat content, which hinders advances in molecular approaches to genetic improvement. Next-generation sequencing techniques have enabled the recent release of reference genomes, genetic linkage maps, and transcriptome sequences, and this information helps improve our understanding of the genetic architecture and molecular mechanisms involved in relevant traits, such as the apomictic reproductive mode. However, more concerted research efforts are still needed to characterize germplasm resources and identify molecular markers and genes associated with target traits. In addition, the implementation of genomic selection and gene editing is needed to reduce the breeding time and expenditure. In this review, we highlight the importance and characteristics of the four main species of
used in pastures and discuss the current findings from genetic and genomic studies and research gaps that should be addressed in future research.
Genomic technologies have been increasingly applied in livestock production due to their utility in production management and animal genetic improvement. The current project aimed to develop genomic ...resources for the Canadian bison industry, specifically a parentage verification tool and a subspecies composition tool. Both products stand to help with building and maintaining purebred and crossbred bison populations, and in turn bison conservation and production. The development of this genomic toolkit proceeded in two stages. In the single-nucleotide polymorphism (SNP) discovery and selection stage, raw sequence information from 41 bison samples was analyzed, and approximately 52.5 million candidate biallelic SNPs were discovered from 21 samples with high sequence quality. A set of 19,954 SNPs (2,928 for parentage verification and 17,026 for subspecies composition) were then selected for inclusion on an Axiom myDesign custom array. In the refinement and validation stage, 480 bison were genotyped using the custom SNP panel, and the resulting genotypes were analyzed to further filter SNPs and assess tool performance. In various tests using real and simulated genotypes, the two genomic tools showed excellent performance for their respective tasks. Final SNP sets consisting of 191 SNPs for parentage and 17,018 SNPs for subspecies composition are described. As the first SNP-based genomic toolkit designed for the Canadian bison industry, our results may provide a new opportunity in improving the competitiveness and profitability of the industry in a sustainable manner.
Pearl millet
(L) R. Br. is a hardy cereal crop grown in the arid and semiarid tropics where other cereals are likely to fail to produce economic yields due to drought and heat stresses. Adaptive ...evolution, a form of natural selection shaped the crop to grow and yield satisfactorily with limited moisture supply or under periodic water deficits in the soil. Drought tolerance is a complex polygenic trait that various morphological and physiological responses are controlled by 100s of genes and significantly influenced by the environment. The development of genomic tools will have enormous potential to improve the efficiency and precision of conventional breeding. The apparent independent domestication events, highly outcrossing nature and traditional cultivation in stressful environments maintained tremendous amount of polymorphism in pearl millet. This high polymorphism of the crop has been revealed by genome mapping that in turn stimulated the mapping and tagging of genomic regions controlling important traits such as drought tolerance. Mapping of a major QTL for terminal drought tolerance in independent populations envisaged the prospect for the development of molecular breeding in pearl millet. To accelerate genetic gains for drought tolerance targeted novel approaches such as establishment of marker-trait associations, genomic selection tools, genome sequence and genotyping-by-sequencing are still limited. Development and application of high throughput genomic tools need to be intensified to improve the breeding efficiency of pearl millet to minimize the impact of climate change on its production.
Iron (Fe) deficiency and toxicity are the most widely prevalent soil-related micronutrient disorders in rice (
L.). Progress in rice cultivars with improved tolerance has been hampered by a poor ...understanding of Fe availability in the soil, the transportation mechanism, and associated genetic factors for the tolerance of Fe toxicity soil (FTS) or Fe deficiency soil (FDS) conditions. In the past, through conventional breeding approaches, rice varieties were developed especially suitable for low- and high-pH soils, which indirectly helped the varieties to tolerate FTS and FDS conditions. Rice-Fe interactions in the external environment of soil, internal homeostasis, and transportation have been studied extensively in the past few decades. However, the molecular and physiological mechanisms of Fe uptake and transport need to be characterized in response to the tolerance of morpho-physiological traits under Fe-toxic and -deficient soil conditions, and these traits need to be well integrated into breeding programs. A deeper understanding of the several factors that influence Fe absorption, uptake, and transport from soil to root and above-ground organs under FDS and FTS is needed to develop tolerant rice cultivars with improved grain yield. Therefore, the objective of this review paper is to congregate the different phenotypic screening methodologies for prospecting tolerant rice varieties and their responsible genetic traits, and Fe homeostasis related to all the known quantitative trait loci (QTLs), genes, and transporters, which could offer enormous information to rice breeders and biotechnologists to develop rice cultivars tolerant of Fe toxicity or deficiency. The mechanism of Fe regulation and transport from soil to grain needs to be understood in a systematic manner along with the cascade of metabolomics steps that are involved in the development of rice varieties tolerant of FTS and FDS. Therefore, the integration of breeding with advanced genome sequencing and omics technologies allows for the fine-tuning of tolerant genotypes on the basis of molecular genetics, and the further identification of novel genes and transporters that are related to Fe regulation from FTS and FDS conditions is incredibly important to achieve further success in this aspect.
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