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Overview of guide RNA design tools for CRISPR-Cas9 genome editing technology
Lihua Julie Zhu
Front. Biol.. 2015, 10 (4 ): 289-296.
https://doi.org/10.1007/s11515-015-1366-y
CRISPR-Cas (Clustered, Regularly Interspaced, Short Palindromic Repeats – CRISPR-associated (Cas)) RNA guided endonuclease has emerged as the most effective and widely used genome editing technology, which has become the most exciting and rapidly advancing research field. Efficient genome editing by the CRISPR-Cas9 system has been demonstrated in many species, and several laboratories have established CRISPR-Cas9 as a screening tool for systematic genetic analysis, similar to shRNA screening. At least three companies have been founded to leverage this technology for therapeutic uses. To facilitate the implementation of this technology, many software tools have been developed to identify guide RNAs that effectively target a desired genomic region. Here, I provide an overview of the technology, focusing on guide RNA design principles, available software tools and their strengths and weaknesses.
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A SteMNess perspective of survival motor neuron function: splicing factors in stem cell biology and disease
Stuart J. Grice,Ji-Long Liu
Front. Biol.. 2015, 10 (4 ): 297-309.
https://doi.org/10.1007/s11515-015-1368-9
Genome-wide analyses of metazoan messenger RNA (mRNA) species are unveiling the extensive transcriptional diversity generated by alternative splicing (AS). Research is also beginning to identify the splicing factors and AS events required to maintain the balance between stem cell renewal (i.e stemness properties) and differentiation. One set of proteins at the center of spliceosome biogenesis are the survival motor neuron (SMN) complex constituents, which have a critical role in the assembly of spliceosomal small nuclear ribonucleoproteins (snRNPs) in all cells. In this review we discuss what is currently known about how AS controls pluripotency and cell fate and consider how an increased requirement for splicing factors, including SMN, helps to maintain an enrichment of stem cell-specific AS events. Furthermore, we highlight studies showing that mutations in specific splicing factors can lead to the aberrant development, and cause targeted degeneration of the nervous system. Using SMN as an example, we discuss the perspective of how stem cell-specific changes in splicing factors can lead to developmental defects and the selective degeneration of particular tissues. Finally we consider the expanding role of SMN, and other splicing factors, in the regulation of gene expression in stem cell biology, thereby providing insight into a number of debilitating diseases.
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MicroRNAs in erythropoiesis and red blood cell disorders
Javad Mohammdai-asl,Abolfazl Ramezani,Fatemeh Norozi,Amal Saki Malehi,Ali Amin Asnafi,Mohammad Ali Jalali Far,Seyed Hadi Mousavi,Najmaldin Saki
Front. Biol.. 2015, 10 (4 ): 321-332.
https://doi.org/10.1007/s11515-015-1365-z
MicroRNAs (miRNAs) are 19-24 nucleotide non-coding ribonucleic acids binding DNA or RNA and controlling gene expression via mRNA degradation or its transcription inhibition. Erythropoies is a multi step differentiation process of erythroid progenitors to nucleate red blood cells. Maturation, proliferation and differentiation of red blood cells is affected by erythroid factors, signaling pathways in niche of hematopoietic cells, transcription factors as well as miRNAs. Expression of different types of miRNAs during erythroid development provides a background for the study of these molecules to control erythroid differentiation and maturation as well as their use as diagnostic and prognostic markers to treat erythroid disorders like thalassemia, sickle cell disease and erythrocyte enzyme deficiencies. In this paper, with reference to biosynthesis of miRNAs, their function in normal and anemic erythropoiesis has been investigated. The target molecule of each of these miRNAs has been cited in an attempt to elucidate their role in erythropoiesis.
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Less is more: Nutrient limitation induces cross-talk of nutrient sensing pathways with NAD+ homeostasis and contributes to longevity
Felicia Tsang,Su-Ju Lin
Front. Biol.. 2015, 10 (4 ): 333-357.
https://doi.org/10.1007/s11515-015-1367-x
Nutrient sensing pathways and their regulation grant cells control over their metabolism and growth in response to changing nutrients. Factors that regulate nutrient sensing can also modulate longevity. Reduced activity of nutrient sensing pathways such as glucose-sensing PKA, nitrogen-sensing TOR and S6 kinase homolog Sch9 have been linked to increased life span in the yeast, Saccharomyces cerevisiae , and higher eukaryotes. Recently, reduced activity of amino acid sensing SPS pathway was also shown to increase yeast life span. Life span extension by reduced SPS activity requires enhanced NAD+ (nicotinamide adenine dinucleotide, oxidized form) and nicotinamide riboside (NR, a NAD+ precursor) homeostasis. Maintaining adequate NAD+ pools has been shown to play key roles in life span extension, but factors regulating NAD+ metabolism and homeostasis are not completely understood. Recently, NAD+ metabolism was also linked to the phosphate (Pi)-sensing PHO pathway in yeast. Canonical PHO activation requires Pi-starvation. Interestingly, NAD+ depletion without Pi-starvation was sufficient to induce PHO activation, increasing NR production and mobilization. Moreover, SPS signaling appears to function in parallel with PHO signaling components to regulate NR/NAD+ homeostasis. These studies suggest that NAD+ metabolism is likely controlled by and/or coordinated with multiple nutrient sensing pathways. Indeed, cross-regulation of PHO , PKA, TOR and Sch9 pathways was reported to potentially affect NAD+ metabolism; though detailed mechanisms remain unclear. This review discusses yeast longevity-related nutrient sensing pathways and possible mechanisms of life span extension, regulation of NAD+ homeostasis, and cross-talk among nutrient sensing pathways and NAD+ homeostasis.
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Fern spore germination in response to environmental factors
Jinwei Suo,Sixue Chen,Qi Zhao,Lei SHI,Shaojun Dai
Front. Biol.. 2015, 10 (4 ): 358-376.
https://doi.org/10.1007/s11515-015-1342-6
Fern spore germination gives rise to the rhizoid and protonemal cell through asymmetric cell division, and then develops into a gametophyte. Spore germination is also a representative single-cell model for the investigation of nuclear polar movement, asymmetrical cell division, polarity establishment and rhizoid tip-growth. These processes are affected by various environmental factors, such as light, gravity, phytohormones, metal ions, and temperature. Here, we present a catalog of spore germination in response to different environmental factors. They are as follows: (1) Representative modes of light affecting spore germination from different fern species include red light-stimulated and far red light-inhibited spore germination, far red light-uninhibited spore germination, blue light-inhibited spore germination, and spore germination in the dark. The optimal light intensity and illumination time for spore germination are different among various fern species. Light response upon spore germination is initiated from the cell mitosis that regulated by phytochromes (PHYs) and cryptochromes (CRYs). Ac PHY2, Ac CRY3 and/or Ac CRY4 are hypothesized to be involved in spore germination; (2) Gravity and calcium are crucial to early nuclear movement and polarity establishment of spores; (3) Gibberellin and antheridiogen can initiate and promote spore germination in many species, but abscisic acid, jasmonic acid, and ethylene pose only minor effects; (4) Spores can obtain the maximal germination rate in their favorable growth medium. Moreover, metal ions, pH, and spore density in the culture medium also affect spore germination; (5) Most fern spores germinate at 25°C, and an optimal CO2 concentration is necessary for spore germination of certain fern plants. These provide valuable information for understanding fern spore germination in response to environmental factors.
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Functional and structural characterization of missense mutations in PAX6 gene
S. Udhaya Kumar,N. Priyanka,P. Sneha,C. George Priya Doss
Front. Biol.. 2015, 10 (4 ): 377-385.
https://doi.org/10.1007/s11515-015-1346-2
The PAX6 gene belongs to the Paired box (PAX) family of transcription factors that is tissue specific and required for the differentiation and proliferation of cells in embryonic development. PAX6 regulates the pattern formation in early developmental stages. This function of PAX6 protein enables the successful completion of neurogenesis and oculogenesis in most animals such as mice, Drosophila and some other model organisms including humans. A variation in the sequence of PAX6 gene may alter the function and structure of the protein. Such changes can produce adverse effects on functioning of the PAX6 protein which were clinically observed to occur in a broad range of ocular defects such as aniridia in humans. We employed in silico prediction methods such as SIFT, PolyPhen 2; I mutant 3.0, SNAP, SNPs&GO, and PHD-SNP to screen the pathogenic missense mutation in PAX6 and DNA binding sites by BindN and BindN+ . Furthermore, we employed KD4V server to examine the structural and functional modifications that occur in the PAX6 protein as a result of mutation. Based on the results obtained from the in silico prediction methods, we carried out modeling analysis for V53L, I56T, G64V, and I87R to visualize the impact of mutation in structural context.
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