From Descriptive to Functional Genomics of Leukemias Focusing on Genome Engineering Techniques.
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ABSTRACT:
SUBMITTER: Balla B
PROVIDER: S-EPMC8470190 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:CRISPR-Cas9-based screening with single-guide RNA (sgRNA) libraries has emerged as a revolutionary tool for comprehensive analysis of genetic elements. However, genome-scale sgRNA libraries are currently available only in a few model organisms. The traditional approach is to synthesize thousands to tens of thousands of sgRNAs, which is laborious and expensive. We have developed a simple method, RELATe (restriction/ligation coupled with Agrobacterium-mediated transformation), to generate sgRNA libraries from 10 μg of genomic DNA, targeting over 98% of the protein-coding genes in the human fungal pathogen Cryptococcus neoformans Functional screens identified 142 potential C. neoformans genes contributing to blood-brain barrier penetration. We selected two cryptococcal genes, SFP1 and WDR1, for a proof-of-concept demonstration that RELATe-identified genes are relevant to C. neoformans central nervous system infection. Our RELATe method can be used in many other fungal species and is powerful and cost-effective for genome-wide high-throughput screening for elucidating functional genomics.
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Project description:BackgroundWorldwide, acute leukemia is the most common type of childhood cancer. It is particularly common in the Hispanic populations residing in the United States, Costa Rica, and Mexico City. The objective of this study was to determine the incidence of acute leukemia in children who were diagnosed and treated in public hospitals in Mexico City.MethodsIncluded in this study were those children, under 15 years of age and residents of Mexico City, who were diagnosed in 2006 and 2007 with leukemia, as determined by using the International Classification of Childhood Cancer. The average annual incidence rates (AAIR), and the standardized average annual incidence rates (SAAIR) per million children were calculated. We calculated crude, age- and sex-specific incidence rates and adjusted for age by the direct method with the world population as standard. We determined if there were a correlation between the incidence of acute leukemias in the various boroughs of Mexico City and either the number of agricultural hectares, the average number of persons per household, or the municipal human development index for Mexico (used as a reference of socio-economic level).ResultsAlthough a total of 610 new cases of leukemia were registered during 2006-2007, only 228 fit the criteria for inclusion in this study. The overall SAAIR was 57.6 per million children (95% CI, 46.9-68.3); acute lymphoblastic leukemia (ALL) was the most frequent type of leukemia, constituting 85.1% of the cases (SAAIR: 49.5 per million), followed by acute myeloblastic leukemia at 12.3% (SAAIR: 6.9 per million), and chronic myeloid leukemia at 1.7% (SAAIR: 0.9 per million). The 1-4 years age group had the highest SAAIR for ALL (77.7 per million). For cases of ALL, 73.2% had precursor B-cell immunophenotype (SAAIR: 35.8 per million) and 12.4% had T-cell immunophenotype (SAAIR 6.3 per million). The peak ages for ALL were 2-6 years and 8-10 years. More than half the children (58.8%) were classified as high risk. There was a positive correlation between the average number of persons per household and the incidence of the pre-B immunophenotype (Pearson's r, 0.789; P = 0.02).ConclusionsThe frequency of ALL in Mexico City is among the highest in the world, similar to those found for Hispanics in the United States and in Costa Rica.
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Project description:Main conclusionIn this review, we give an overview of plant sequencing efforts and how this impacts plant functional genomics research. Plant genome sequence information greatly facilitates the studies of plant biology, functional genomics, evolution of genomes and genes, domestication processes, phylogenetic relationships, among many others. More than two decades of sequencing efforts have boosted the number of available sequenced plant genomes. The first plant genome, of Arabidopsis, was published in the year 2000 and currently, 4604 plant genomes from 1482 plant species have been published. Various large sequence initiatives are running, which are planning to produce tens of thousands of sequenced plant genomes in the near future. In this review, we give an overview on the status of sequenced plant genomes and on the use of genome information in different research areas.
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Project description:Over the past decades, genetic advances have allowed a more precise molecular characterization of AML with the identification of novel oncogenes and tumor suppressors as part of a comprehensive AML molecular landscape. Recent advances in genetic sequencing tools also enabled a better understanding of AML leukemogenesis from the preleukemic state to posttherapy relapse. These advances resulted in direct clinical implications with the definition of molecular prognosis classifications, the development of treatment recommendations based on minimal residual disease (MRD) measurement and the discovery of novel targeted therapies, ultimately improving AML patients' overall survival. The more recent development of functional genomic studies, pushed by novel molecular biology technologies (short hairpin RNA (shRNA) and CRISPR-Cas9) and bioinformatics tools design on one hand, along with the engineering of humanized physiologically relevant animal models on the other hand, have opened a new genomics era resulting in a greater knowledge of AML physiopathology. Combining descriptive and functional genomics will undoubtedly open the road for an AML cure within the next decades.
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Project description:In this up-to-date study, we first aimed to highlight the genetic and non-genetic factors associated with respiratory distress syndrome (RDS) while also focusing on the genomic aspect of this condition. Secondly, we discuss the treatment options and the progressing therapies based on RNAs or gene therapy. To fulfill this, our study commences with lung organogenesis, a highly orchestrated procedure guided by an intricate network of conserved signaling pathways that ultimately oversee the processes of patterning, growth, and differentiation. Then, our review focuses on the molecular mechanisms contributing to both normal and abnormal lung growth and development and underscores the connections between genetic and non-genetic factors linked to neonatal RDS, with a particular emphasis on the genomic aspects of this condition and their implications for treatment choices and the advancing therapeutic approaches centered around RNAs or gene therapy.
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Project description:The complete 1,751,377-bp sequence of the genome of the thermophilic archaeon Methanobacterium thermoautotrophicum deltaH has been determined by a whole-genome shotgun sequencing approach. A total of 1,855 open reading frames (ORFs) have been identified that appear to encode polypeptides, 844 (46%) of which have been assigned putative functions based on their similarities to database sequences with assigned functions. A total of 514 (28%) of the ORF-encoded polypeptides are related to sequences with unknown functions, and 496 (27%) have little or no homology to sequences in public databases. Comparisons with Eucarya-, Bacteria-, and Archaea-specific databases reveal that 1,013 of the putative gene products (54%) are most similar to polypeptide sequences described previously for other organisms in the domain Archaea. Comparisons with the Methanococcus jannaschii genome data underline the extensive divergence that has occurred between these two methanogens; only 352 (19%) of M. thermoautotrophicum ORFs encode sequences that are >50% identical to M. jannaschii polypeptides, and there is little conservation in the relative locations of orthologous genes. When the M. thermoautotrophicum ORFs are compared to sequences from only the eucaryal and bacterial domains, 786 (42%) are more similar to bacterial sequences and 241 (13%) are more similar to eucaryal sequences. The bacterial domain-like gene products include the majority of those predicted to be involved in cofactor and small molecule biosyntheses, intermediary metabolism, transport, nitrogen fixation, regulatory functions, and interactions with the environment. Most proteins predicted to be involved in DNA metabolism, transcription, and translation are more similar to eucaryal sequences. Gene structure and organization have features that are typical of the Bacteria, including genes that encode polypeptides closely related to eucaryal proteins. There are 24 polypeptides that could form two-component sensor kinase-response regulator systems and homologs of the bacterial Hsp70-response proteins DnaK and DnaJ, which are notably absent in M. jannaschii. DNA replication initiation and chromosome packaging in M. thermoautotrophicum are predicted to have eucaryal features, based on the presence of two Cdc6 homologs and three histones; however, the presence of an ftsZ gene indicates a bacterial type of cell division initiation. The DNA polymerases include an X-family repair type and an unusual archaeal B type formed by two separate polypeptides. The DNA-dependent RNA polymerase (RNAP) subunits A', A", B', B" and H are encoded in a typical archaeal RNAP operon, although a second A' subunit-encoding gene is present at a remote location. There are two rRNA operons, and 39 tRNA genes are dispersed around the genome, although most of these occur in clusters. Three of the tRNA genes have introns, including the tRNAPro (GGG) gene, which contains a second intron at an unprecedented location. There is no selenocysteinyl-tRNA gene nor evidence for classically organized IS elements, prophages, or plasmids. The genome contains one intein and two extended repeats (3.6 and 8.6 kb) that are members of a family with 18 representatives in the M. jannaschii genome.
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Project description:The ?-glucosidase inhibitor acarbose is produced in large-scale by strains derived from Actinoplanes sp. SE50 and used widely for the treatment of type-2 diabetes. Compared with the wild-type SE50, a high-yield derivative Actinoplanes sp. SE50/110 shows 2-fold and 3-7-fold improvement of acarbose yield and acb cluster transcription, respectively. The genome of SE50 was fully sequenced and compared with that of SE50/110, and 11 SNVs and 4 InDels, affecting 8 CDSs, were identified in SE50/110. The 8 CDSs were individually inactivated in SE50. Deletions of ACWT_4325 (encoding alcohol dehydrogenase) resulted in increases of acarbose yield by 25% from 1.87 to 2.34?g/L, acetyl-CoA concentration by 52.7%, and PEP concentration by 22.7%. Meanwhile, deletion of ACWT_7629 (encoding elongation factor G) caused improvements of acarbose yield by 36% from 1.87 to 2.54?g/L, transcription of acb cluster, and ppGpp concentration to 2.2 folds. Combined deletions of ACWT_4325 and ACWT_7629 resulted in further improvement of acarbose to 2.83?g/L (i.e. 76% of SE50/110), suggesting that the metabolic perturbation and improved transcription of acb cluster caused by these two mutations contribute substantially to the acarbose overproduction. Enforced application of similar strategies was performed to manipulate SE50/110, resulting in a further increase of acarbose titer from 3.73 to 4.21?g/L. Therefore, the comparative genomics approach combined with functional verification not only revealed the acarbose overproduction mechanisms, but also guided further engineering of its high-yield producers.
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Project description:Next-generation sequencing approaches have fundamentally changed the types of questions that can be asked about gene function and regulation. With the goal of approaching truly genome-wide quantifications of all the interaction partners and downstream effects of particular genes, these quantitative assays have allowed for an unprecedented level of detail in exploring biological interactions. However, many challenges remain in our ability to accurately describe and quantify the interactions that take place in those hard to reach and extremely repetitive regions of our genome comprised mostly of transposable elements (TEs). Tools dedicated to TE-derived sequences have lagged behind, making the inclusion of these sequences in genome-wide analyses difficult. Recent improvements, both computational and experimental, allow for the better inclusion of TE sequences in genomic assays and a renewed appreciation for the importance of TE biology. This review will discuss the recent improvements that have been made in the computational analysis of TE-derived sequences as well as the areas where such analysis still proves difficult. This article is part of a discussion meeting issue 'Crossroads between transposons and gene regulation'.
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