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.

Resurrecting essential amino acid biosynthesis in a mammalian cell

Major genomic deletions in independent eukaryotic lineages have led to repeated ancestral loss of biosynthesis pathways for nine of the twenty canonical amino acids1. While the evolutionary forces driving these polyphyletic deletion events are not well understood, the consequence is that extant metazoans are unable to produce nine essential amino acids (EAAs). Previous studies have highlighted that EAA biosynthesis tends to be more energetically costly2,3, raising the possibility that these pathways were lost from organisms with access to abundant EAAs in the environment4,5. It is unclear whether present-day metazoans can reaccept these pathways to resurrect biosynthetic capabilities that were lost long ago or whether evolution has rendered EAA pathways incompatible with metazoan metabolism. Here, we report progress on a large-scale synthetic genomics effort to reestablish EAA biosynthetic functionality in a mammalian cell. We designed codon-optimized biosynthesis pathways based on genes mined from Escherichia coli. These pathways were de novo synthesized in 3 kilobase chunks, assembled in yeasto and genomically integrated into a Chinese Hamster Ovary (CHO) cell line. One synthetic pathway produced valine at a sufficient level for cell viability and proliferation, and thus represents a successful example of metazoan EAA biosynthesis restoration. This prototrophic CHO line grows in valine-free medium, and metabolomics using labeled precursors verified de novo biosynthesis of valine. RNA-seq profiling of the valine prototrophic CHO line showed that the synthetic pathway minimally disrupted the cellular transcriptome. Furthermore, valine prototrophic cells exhibited transcriptional signatures associated with rescue from nutritional starvation. This work demonstrates that mammalian metabolism is amenable to restoration of ancient core pathways, thus paving a path for genome-scale efforts to synthetically restore metabolic functions to the metazoan lineage.

EDITORIAL NEED OF OUR TIMES: IMPACT OF GENOME EDITING SCIENCES AND REQUISITE PREPAREDNESS FOR MOLECULAR BIOTERRORISM

The pace of human evolution has accelerated at an unprecedented rate in the last couple of decades. Never ever before the mankind could witness a global hostage situation by a tiny invisible RNA creature. While the global community struggles at large finding plausible solutions in the information replete era, there are seriouslessons to be learnt. The tiny RNA monster has exposed the vulnerabilities of one the considered most intelligent creature posing a question mark about how to strike the intricate balance between preventive approaches and acquiring the postexposure immunity. The rapidly improving genome editing methods along with synthetic genomics has emerged as a double-edged weapon where on one side it opens newer therapeutic avenues to cure disease, but also its malicious use could results in disasters of limitless magnitudes.The delicate boundaries nature may face terrorism in newer clothes at the hands of nano technological tools to modify genome and synthesizing newer life forms. Unstoppable if it becomes can create man-made disasters with issues leading to emergence of black markets for cloning, designer humans ethnic-specific nucleotide editing for worse and possibly much more. The fiction we saw yesterday is today’s science and can lead the human race to point of no return. “He Jiankui affair” is still one of the genome editing dilemma widely criticized for ethical concerns emerging from germ line editing two human embryos for HIV using Cluster RegularlyInterspaced Short Palindromic Repeats (CRISPR) Cas technology.

Building genomes to understand biology

Genetic manipulation is one of the central strategies that biologists use to investigate the molecular underpinnings of life and its diversity. Thus, advances in genetic manipulation usually lead to a deeper understanding of biological systems. During the last decade, the construction of chromosomes, known as synthetic genomics, has emerged as a novel approach to genetic manipulation. By facilitating complex modifications to chromosome content and structure, synthetic genomics opens new opportunities for studying biology through genetic manipulation. Here, we discuss different classes of genetic manipulation that are enabled by synthetic genomics, as well as biological problems they each can help solve.

Genetic Engineering and Synthetic Genomics in Yeast to Understand Life and Boost Biotechnology

The field of genetic engineering was born in 1973 with the “construction of biologically functional bacterial plasmids in vitro”. Since then, a vast number of technologies have been developed allowing large-scale reading and writing of DNA, as well as tools for complex modifications and alterations of the genetic code. Natural genomes can be seen as software version 1.0; synthetic genomics aims to rewrite this software with “build to understand” and “build to apply” philosophies. One of the predominant model organisms is the baker’s yeast Saccharomyces cerevisiae. Its importance ranges from ancient biotechnologies such as baking and brewing, to high-end valuable compound synthesis on industrial scales. This tiny sugar fungus contributed greatly to enabling humankind to reach its current development status. This review discusses recent developments in the field of genetic engineering for budding yeast S. cerevisiae, and its application in biotechnology. The article highlights advances from Sc1.0 to the developments in synthetic genomics paving the way towards Sc2.0. With the synthetic genome of Sc2.0 nearing completion, the article also aims to propose perspectives for potential Sc3.0 and subsequent versions as well as its implications for basic and applied research.