Resolving the microalgal gene landscape at the strain level: A novel hybrid transcriptome of Emiliania huxleyi CCMP3266

Microalgae are key ecological players with a complex evolutionary history. Genomic diversity, in addition to limited availability of high-quality genomes, challenge studies that aim to elucidate molecular mechanisms underlying microalgal ecophysiology. Here, we present a novel and comprehensive transcriptomic hybrid approach to generate a reference for genetic analyses, and resolve the microalgal gene landscape at the strain level. The approach is demonstrated for a strain of the coccolithophore microalga Emiliania huxleyi , which is a species complex with considerable genome variability. The investigated strain is commonly studied as a model for algal-bacterial interactions, and was therefore sequenced in the presence of bacteria to elicit the expression of interaction-relevant genes. We applied complementary PacBio Iso-Seq full-length cDNA, and poly(A)-independent Illumina total RNA sequencing, which resulted in a de novo assembled, near complete hybrid transcriptome. In particular, hybrid sequencing improved the reconstruction of long transcripts and increased the recovery of full-length transcript isoforms. To use the resulting hybrid transcriptome as a reference for genetic analyses, we demonstrate a method that collapses the transcriptome into a genome-like dataset, termed “synthetic genome” (sGenome). We used the sGenome as a reference to visually confirm the robustness of the CCMP3266 gene assembly, to conduct differential gene expression analysis, and to characterize novel E. huxleyi genes. The newly-identified genes contribute to our understanding of E. huxleyi genome diversification, and are predicted to play a role in microbial interactions. Our transcriptomic toolkit can be implemented in various microalgae to facilitate mechanistic studies on microalgal diversity and ecology. Importance Microalgae are key players in the ecology and biogeochemistry of our oceans. Efforts to implement genomic and transcriptomic tools in laboratory studies involving microalgae suffer from the lack of published genomes. In the case of coccolithophore microalgae, the problem has long been recognized; the model species Emiliania huxleyi is a species complex with genomes composed of a core, and a large variable portion. To study the role of the variable portion in niche adaptation, and specifically in microbial interactions, strain-specific genetic information is required. Here we present a novel transcriptomic hybrid approach, and generated strain-specific genome-like information. We demonstrate our approach on an E. huxleyi strain that is co-cultivated with bacteria. By constructing a “synthetic genome”, we generated comprehensive gene annotations that enabled accurate analyses of gene expression patterns. Importantly, we unveiled novel genes in the variable portion of E. huxleyi that play putative roles in microbial interactions.

Applications of CRISPR/Cas Technology to Research the Synthetic Genomics of Yeast

The whole genome projects open the prelude to the diversity and complexity of biological genome by generating immense data. For the sake of exploring the riddle of the genome, scientists around the world have dedicated themselves in annotating for these massive data. However, searching for the exact and valuable information is like looking for a needle in a haystack. Advances in gene editing technology have allowed researchers to precisely manipulate the targeted functional genes in the genome by the state-of-the-art gene-editing tools, so as to facilitate the studies involving the fields of biology, agriculture, food industry, medicine, environment and healthcare in a more convenient way. As a sort of pioneer editing devices, the CRISPR/Cas systems having various versatile homologs and variants, now are rapidly giving impetus to the development of synthetic genomics and synthetic biology. Firstly, in the chapter, we will present the classification, structural and functional diversity of CRISPR/Cas systems. Then we will emphasize the applications in synthetic genome of yeast (Saccharomyces cerevisiae) using CRISPR/Cas technology based on year order. Finally, the summary and prospection of synthetic genomics as well as synthetic biotechnology based on CRISPR/Cas systems and their further utilizations in yeast are narrated.

Xenobiology: An expanded semantical review

The definition of “xenobiology” has gradually shifted from the study of the foreign, estranged life forms potentially existing in outer space to the study where the natural and synthetic life are involved. The natural concept of xenobiology governs the unseen, hypothetical life on the outer space, and the hidden life with completely different biochemistry on Earth. The life on the outer space might possess different way to harvest energy from the one on Earth. The hidden life on Earth, or the “Shadow Biosphere” might rose from completely different way of creation and evolution on Earth, which lead to its complete difference from the known biosphere. The newest concept of xenobiology involves synthetic life, built with unnatural base pair of the nucleic acid, with analogous or xeno nucleic acid (XNA), has a synthetic genome which capable of self-replicating or enables the synthetic cell to self-replicate, or even possesses a synthetic physiological pathway. By understanding the broad spectrum of xenobiology, in both natural and synthetic concepts, we can expand our view on how life might develop into a completely estranged system, which is different from anthropocentric view of life available around us on Earth. From these perspectives, we might understand how life evolved by evolving it synthetically.

CRISPR-Cas-mediated tethering recruits the yeast HMR mating-type locus to the nuclear periphery but fails to silence gene expression

To investigate the relationship between genome structure and function, we have developed a programmable CRISPR-Cas system for nuclear peripheral recruitment in yeast. We benchmarked this system at the HMR and GAL2 loci, both well-characterized model systems for localization to the nuclear periphery. Using microscopy and gene silencing assays, we demonstrate that CRISPR-Cas-mediated tethering can recruit the HMR locus but does not silence reporter gene expression. A previously reported Gal4-mediated tethering system does silence gene expression, and we demonstrate that the silencing phenotype has an unexpected dependence on the structure of the protein tether. The CRISPR-Cas system was unable to recruit GAL2 to the nuclear periphery. Our results reveal potential challenges for synthetic genome structure perturbations and suggest that distinct functional effects can arise from subtle structural differences in how genes are recruited to the periphery.

A Synthetic Defective Interfering SARS-CoV-2

Viruses thrive by exploiting the cells they infect but must also produce viral proteins to replicate and infect other cells. As a consequence, they are also susceptible to exploitation by defective versions of themselves that do not produce such proteins. A defective viral genome with deletions in protein-coding genes could still replicate in cells coinfected with full-length viruses, and even replicate faster due to its shorter size, interfering with the replication of the virus. We have created a synthetic defective interfering version of SARS-CoV-2, the virus causing the recent Covid-19 pandemic, assembling parts of the viral genome that do not code for any functional protein but enable it to be replicated and packaged. This synthetic defective genome replicates three times faster than SARS-CoV-2 in coinfected cells, and interferes with it, reducing the viral load of a cell by half in 24 hours. The synthetic genome is transmitted as efficiently as the full-length genome, confirming the location of the putative packaging signal of SARS-CoV-2. A version of such a synthetic construct could be used as a self-promoting antiviral therapy: by enabling replication of the synthetic genome, the virus promotes its own demise. Graphic summary

COVID-19, SARS AND BATS CORONAVIRUSES GENOMES PECULIAR HOMOLOGOUS RNA SEQUENCES

We are facing the worldwide invasion of a new coronavirus. This follows several limited outbreaks of related viruses in various locations in a recent past (SARS, MERS). Although the main current objective of researchers is to bring efficient therapeutic and preventive solutions to the global population, we need also to better understand the origin of the newly coronavirus-induced epidemic in order to avoid future outbreaks. The present molecular appraisal is to study by a bio-infomatic approach the facts relating to the virus and itsprecursors. This article shows how 16 fragments (Env Pol and Integrase genes) from different strains, both diversified and very recent, of the HIV1, HIV2 and SIV retroviruses have high percentage of homology into parts of the genome of COVID_19. Moreover each of these elements is made of 18 or more nucleotides and therefore may have a function. They are called Exogenous Informative Elements (EIE).. Among these EIE, 12 are concentrated in a very small region of the COVID-19 genome, length less than 900 bases, i.e. less than 3% of the total length of this genome. In addition, these EIE are positioned in two functional genes of COVID-19: the orf1ab and S spike genes. Here are the two main facts which contribute to our hypothesis of a partially synthetic genome: A contiguous region representing 2.49% of the whole COVID-19 genome of which 40.99% is made up of 12 diverse fragments originating from various strains of HIV SIV retroviruses. Some of these 12 EIE appear concatenated. Notably, the retroviral part of these regions, which consists of 8 elements from various strainsof HIV1, HIV2 and SIV covers a length of 275 contiguous bases of COVID-19. The cumulative length of these 8 HIV/SIV elements represents 200 bases. Consequently, the HIV SIV density rate of this region of COVID-19 is 200/275 = 72.73%.

WUHAN COVID-19 SYNTHETIC ORIGINS AND EVOLUTION

The main result of this updated release is the formal proof that 2019-nCoV coronavirus is partially a SYNTHETIC genome. We proof the CONCENTRATION in a small région of wuhan New genome (300bp) of 3 different régions from HIV1 ENVELOPPE gene and 3 others from HIV2 and SIV (ENV and POL RT). All this is remarkable and bears the mark of a desire for organization of a human nature: LOGIC, SYMETRIES. In this article, we demonstrate also that there is a kind of global human hosts adaptation strategy of SARS viruses as well as a strategy of global evolution of the genomes of the different strains of SARS which have emerged, mainly in China, between years 2003 first SARS genomes and the last 2019 COVID-19 Wuhan seafood market pneumonia virus isolate Wuhan-Hu-1, complete genome. This global strategy, this temporal link, is materialized in our demonstration by highlighting stationary numerical waves controlling the entire sequence of their genomes. Curiously, these digital waves characterizing the 9 SARS genomes studied here are characteristic whole numbers: the "Fibonacci numbers", omnipresent in the forms of Nature, and which our research for several decades has shown strong links with the proportions of nucleotides in DNA. Here we demonstrate that the complexity and fractal multiplicity of these Fibonacci numerical waves increases over the years of the emergence of new SARS strains. We suggest that this increase in the overall organization of the SARS genomes over the years reflects a better adaptation of SARS genomes to the human host. The question of a link with pathogenicity remains open. However, we believe that this overall strategy for the evolution of the SARS genomes ensures greater unity, consistency and integrity of the genome. Finally, we ask ourselves the question of a possible artificial origin of this genome, in particular because of the presence of fragments of HIV1, HIV2 and SIV retroviruses.

Jean claude Perez § Luc Montagnier - COVID-19, SARS and Bats Coronaviruses Genomes Unexpected Exogenous RNA Sequences

We are facing the worldwide invasion of a new coronavirus. This follows several limited outbreaks of related viruses in various locations in a recent past (SARS, MERS). Although the main objective of researchers is to bring efficient therapeutic and preventive solutions to the global population, we need also to better understand the origin of the newly coronavirus-induced epidemic in order to avoid future outbreaks. The present molecular appraisal is to study by a bio-infomatic approach the facts relating to the virus and its precursors. This article shows how 16 fragments (Env Pol and Integrase genes) from different strains, both diversified and very recent, of the HIV1, HIV2 and SIV retroviruses most likely are present into the genome of COVID-19. Among these fragments, 12 are concentrated in a very small region of the COVID-19 genome, length less than 900bases, i.e. less than 3% of the total length of this genome. In addition, these footprints are positioned in 2 functional genes of COVID-19: the orf1ab and S spike genes. To sum up, here are the two main facts which contribute to our hypothesis of a partially synthetic genome: A contiguous region representing 2.49% of the whole COVID-19 genome of which 40.99% is made up of 12 diverse fragments originating from various strains of HIV SIV retroviruses. On the other hand, these 12 fragments some of which appear concatenated. Notably, the retroviral part of these regions, which consists of 8 elements from various strains HIV1, HIV2 and SIV covers a length of 275 contiguous bases of COVID-19. The cumulative length of these 8 HIV SIV elements represents 200 bases. Consequently, the HIV SIV density rate of this region of COVID-19 is 200/275 = 72.73%, which is considerable s made of. Moreover each of these elements is made of 18 or more nucleotides and therefore may have function. They are called Exogenous Informative Elements. A major part of these 16 EIE already existed in the first SARS genomes as early as 2003. However, we demonstrate how and why a new region including 4 HIV1 HIV2 Exogenous Informative Elements radically distinguishes all COVID-19 strains from all SARS and Bat strains. We then gather facts about the possible origins of COVID_19. We have particularly analyzed this small region of 225 bases common to COVID_19 and batRaTG13 but totally absent in all SARS strains. Then, we discuss the case of bat genomes presumed to be at the origin of COVID_19. In the strain of bat RaTG13 coronavirus isolated in 2013, then sequenced in 2020, the homology profile for HIV1 Kenya 2008 fragment is identical to that of COVID_19. Finally, we have studied the most recent genetic evolution of the COVID_19 strains involved in the world epidemic. We found a significant occurrence of mutations and deletions in the 225b region.On sampling genomes, we finally show that this 225b key region of each genome, rich in EIE, evolves much faster than the corresponding whole genome.The comparative analysis of the SPIKES genes of COVID_19 and Bat RaTG13demonstrates two abnormal facts: on the one hand, the insertion of 4 contiguous amino acids in the middle of SPIKE, on the other hand, an abnormal distribution of synonymous codons in the second half of SPIKE. Finally the insertion in this region of an EIE coming from a Plasmodium Yoelii gene is demonstrated, but above all seems to explain the "strategy" pursued by having "artificially" modified the ratio of synonym codons / non-synonymous codons in this same region of 1770 COVID_19 SPIKE nucleotides.

COVID-19, SARS and Bats Coronaviruses Genomes Unexpected Exogeneous RNA Sequences

We human are facing the worldwide invasion of a new coronavirus. This follows several limited outbreaks of related viruses in various locations in a recent past (SARS, MERS). Although the main objective of researchers is to bring efficient therapeutic and preventive solutions to the global population, we need also to better understand the origin of the newly coronavirus-induced epidemic in order to avoid future new outbreaks. The present molecular appraisal is to study by a bio-infomatic approach the facts relating to the virus and its precursors. This article demonstrates how 16 « Exogeneous Informative Elements » fragments (Env Pol and Integrase genes) from different strains, both diversified and very recent, of the HIV1, HIV2 and SIV retroviruses most likely are present into the genome of COVID-19. Among these fingerprints, 12 of them would be concentrated in a very small region of the genome COVID-19 of length less than 900bases, i.e. less than 3% of the total length of this genome. In addition, these footprints are positioned in 2 functional genes of COVID-19: the orf1ab and S spike genes.To sum up, here are the two main facts which contribute to our hypothesis of a partially synthetic genome: A contiguous region representing 2.49% of the whole COVID-19 genome is 40.99% made up of 12 diverse Exogeneous Informative Elements (EIE) fragments originating from various strains of HIV SIV retroviruses. On the other hand, these 12 Exogeneous Informative Elements, some of them appear concatenated, that is to say placed side by side in the genome of COVID-19, and this despite natures, strains, and years of emergence all different. Notably, the retroviral part of these regions, which consists of 8 motifs from various strains HIV1, HIV2 and SIV, covers a length of 275 contiguous bases of COVID-19. The cumulative length of these 8 HIV SIV motifs represents 200 bases. Consequently, the HIV SIV density rate of this region of COVID-19 is 200/275 = 72.73%, which is considerable.A major part of these 16 EIE Elements already existed in the first SARS genomes as early as 2003. However, we demonstrate how and why a new region including 4 HIV1 HIV2 Exogeneous Informative Elements radically distinguishes all COVID-19 strains from all SARS and Bat strains. Particularly, we will be interested in the Bat RaTG13 strain whose genomic proximity to COVID-19 will be thoroughly analyzed. Then, we gather facts about the possible origins of COVID_19, we have particularly analyze this small region of 225 bases common to COVID_19 and batRaTG13 but totally absent in all SARS strains. Then, we discuss the case of bat genomes presumed to be at the origin of COVID_19. In the strain of bat RaTG13 bat coronavirus isolated in 2013, then sequenced in 2020, the homology profile for HIV1 Kenya 2008 fingerprint is identical to that of COVID_19. (collected end december 2019, then sequenced in 2020). Finally, we have studied the most recent genetic evolution of the COVID_19 strains involved in the world epidemic. We found an astoneshing occurrence of mutations and deletions in the 225b region.On sampling genomes, we finally show that this 225b key region of each genome, rich in "EIE", evolves much faster than the corresponding whole genome.

Wuhan nCoV-2019 SARS Coronaviruses Genomics Fractal Metastructures Evolution and Origins

Wuhan nCoV-2019 SARS Coronaviruses Genomics Fractal Metastructures Evolution and Origins “Where there is matter, there is geometry.” Johannes Kepler Jean-claude PEREZ, PhD Maths § Computer Science Bordeaux University, RETIRED interdisciplinary researcher (IBM Emeritus, IBM European Research Center on Artificial Intelligence), 7 avenue de terre-rouge F33127 Martignas Bordeaux metropole France, phone 33 0781181112 [email protected] ABSTRACT : The main result of this updated release is the formal proof that 2019-nCoV coronavirus is partially a SYNTHETIC genome. We proof the CONCENTRATION in a small région of wuhan New genome of 3 different régions from HIV1 ENVELOPPE GENE. In this article, we demonstrate that there is a kind of global human hosts adaptation strategy of SARS viruses as well as a strategy of global evolution of the genomes of the different strains of SARS which have emerged, mainly in China, between years 2003 first SARS genomes and the last 2020 nCoV-2019 Wuhan seafood market pneumonia virus isolate Wuhan-Hu-1, complete genome. This global strategy, this temporal link, is materialized in our demonstration by highlighting stationary numerical waves controlling the entire sequence of their genomes. Curiously, these digital waves characterizing the 9 SARS genomes studied here are characteristic whole numbers: the "Fibonacci numbers", omnipresent in the forms of Nature, and which our research for several decades has shown strong links with the proportions of nucleotides in DNA. Here we demonstrate that the complexity and fractal multiplicity of these Fibonacci numerical waves increases over the years of the emergence of new sArs strains. We suggest that this increase in the overall organization of the SARS genomes over the years reflects a better adaptation of SARS genomes to the human host. The question of a link with pathogenicity remains open. However, we believe that this overall strategy for the evolution of the SARS genomes ensures greater unity, consistency and integrity of the genome. Finally, we ask ourselves the question of a possible artificial origin of this genome, in particular because of the presence of fragments of HIV1 retrovirus. KEYWORDS : SARS, Wuhan nCoV-2019, Fibonacci numbers, Fractal genome, Numerical stationary periodic waves, HIV1, synthetic genomes.