• Desirazu N Rao

Articles written in Resonance – Journal of Science Education

• Rewriting the Genome of the Model Eukaryote Saccharomyces cerevisiae

An international consortium of scientists has embarked on the total design and synthesis of all the 16 yeast chromosomes of the laboratory organism, Saccharomyces cerevisiae.Once constructed, the 16 synthetic chromosomes will be consoli-dated into a single yeast strain along with a new 17th yeast chromosome called the “neochromosome” which contains all the tRNA genes, to generate a designer eukaryotic genome, Sc2.0. The key criterion for the stream-lined yeast (Sc2.0) is that it should retain the same cell ﬁtness and phenotype of the wild-type (Sc1.0), but show increased genetic stabil-ity and ﬂexibility to enable future studies. All the 16 syn-thetic yeast chromosomes have been designed using BioStu-dio, an open-source framework that was developed speciﬁ-cally to design and construct chromosome-size fragments in silico. The completely redesigned Sc2.0 genome is a highly modiﬁed version of the S. cerevisiaegenome, with a reduction in the size of ∼1.1 million base pairs, which is about 8% of the native genome. In 2017, the Sc2.0 consortium reported the complete synthesis and assembly of 6.5 individual yeast chromosomes in discrete strains and showed consolidation of 2.5 synthetic chromosomes (synIII/synVI/synIXR) into a sin-gle yeast strain that bodes well for the successful completion of the Sc2.0 genome.

• Genome Editing: Revolution in Life Sciences

Programmable nucleases—ZFNs, TALENs and CRISPR-Cas9—have equipped scientists with an unprecedented ability to modify cells and organisms almost at will, with great implications across life sciences: biology, agriculture, ecology and medicine. Nucleases-based genome editing (aka gene editing) depends on cellular responses to a targeted double-strand break(DSB). The ﬁrst truly targetable reagents were zinc ﬁnger nu-cleases (ZFNs) showing that arbitrary DNA sequences within a mammalian genome, could be addressed by protein engi-neering, ushering in the era of genome editing. ZFNs that are fusions of zinc ﬁnger proteins (ZFPs) and FokI cleavage domain, resulted from the basic research on Type IIs FokI re-striction enzyme, which showed a bipartite structure with a separable DNA-binding domain and a non-speciﬁc cleavage domain. Studies on 3-ﬁnger ZFNs established that the preferred substrates were paired binding sites, which doubled the size of the target recognition sequence from 9 to 18 bp that is large enough to specify a unique genomic locus in plant and mammalian cells, including human cells. Subsequently, a ZFN-induced DSB was shown to stimulate homologous re-combination in frog eggs. Transcription activator-like eﬀec-tor nucleases (TALENs) that are based on bacterial TALEs fused to FokI cleavage domain expanded the capability. ZFNs and TALENs have been successfully used to modify a multi-tude of recalcitrant organisms and cell types that were unap-proachable previously attesting to the success of protein engi-neering, long before the arrival of CRISPR. The recent tech-nique to deliver a targeted DSB to cellular genomes are RNA-guided nucleases as exempliﬁed by the Type II prokaryotic CRISPR-Cas9 system. Unlike ZFNs and TALENs that use protein motifs for DNA sequence recognition, CRISPR-Cas9 depends on RNA-DNA recognition. The advantages of the CRISPR-Cas9 system, which include ease of RNA design for new targets and dependence on a single constant Cas9 protein, have led to its wide adoption by research labs around the world. The 2020 Nobel Prize for Chemistry was awarded to Jennifer Doudna and Emmanualle Charpentier for harnessing CRISPR-Cas9 system to provide a simpliﬁed technique for genome editing. The programmable nucleases have also been shown to cut at oﬀ-target sites with mutagenic consequences, which is a serious concern for human therapeutic applications. Therefore, applications of genome editing technologies to human therapeutics will ultimately depend on risk versus beneﬁt analysis and informed consent

• The Human Microbiome: An Acquired Organ?

A diverse milieu of harmless microbes thrives on the surface of the human body. These human-associated microbes comprise an enormous collection of prokaryotes (archaea and bacteria), eukaryotes (fungi and protozoa), and viruses. The discovery of universal phylogenetic taxonomic molecular markers and the availability of robust deoxyribonucleic acid (DNA) sequencing tools have enabled the identification of several previously unknown human-associated microbes. Consequently, the Human Microbiome Project (HMP)---the first comprehensive survey of the human-associated microbes---has determined the microbial diversity and its abundance in health and disease. HMP study shows that human adults have a similar microbial composition as that of higher taxonomic level (phylum), although uniquely differing from lower taxonomic level (genus and species). Bacteria are the predominant microbial constituent of the human body, and the large intestine (the lower gut), especially, is the most densely populated microbial niche. The human gut is estimated to have over 100 trillion microbes encompassing over 1000 bacterial species, outnumbering the total human body cells by a factor of ten. Gut microbes have a significant impact on human physiology through their role in protection against gut infections, expanding nutrient harvest, educating the infant immune system, modulating drug efficacy, and so forth. The gut microbial communities are collectively recognized as an organ' for their indispensable contribution to health. Gut microbes supplement human biology with numerous functional genes, metabolic pathways, bioactive metabolites, etc. The perturbation of gut microbiota composition has a pathological impact on human physiology. Hence, the ensemble of the microbial genetic material associated with us represents our acquired genome'. Overall, the human-microbial synergistic interaction is an evolutionary amalgamation of three domains of cellular life---Archaea, Bacteria, and Eukarya---mutually co-existing with acellular viruses, and collectively referred to as a `superorganism'.

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