Investments In Genome Editing Technologies
S Chandrasegaran February 10, 2018
India is soon poised to become the world’s most populous nation, overtaking China. India faces the critical challenge of producing sufficient food for a growing population living in a changing climate. Substantial research investments have been made to sequence, assemble, and characterize the genomes of major crop plants by other countries, which have led to important discoveries of crop genes and their functions. This knowledge will be valuable in increasing agricultural production by using synthetic biology and genome editing, technological advancements for precise plant engineering. Genome editing and synthetic biology are unprecedented technological breakthroughs, with great potential for crop improvement. Defining gene sequences from diverse species and cultivars has far outpaced our ability to alter those genes in crops. Recent advances in genome engineering (aka genome editing) make it possible to precisely alter DNA sequences in living cells, providing unprecedented control over a plant and animal genetic material.
Potential future crops derived through gene editing and synthetic biology include those that better withstand pests, those that are salt and drought tolerant, that have enhanced nutritional value, and that are able to grow on marginal lands. In many instances, crops with such traits will be created by altering only a few nucleotides among the billions that comprise plant genomes. With the appropriate regulatory structures and oversight in place, crops created through genome editing might prove to be more acceptable to the public than plants that carry foreign DNA in their genomes. Public perception and the performance of the engineered crop varieties will determine the extent to which genome editing and synthetic biology contribute towards securing the world’s food supply.
It is critical for India to make substantial investments in these technologies to make them readily available to indigenous Indian scientists so that they can be part of the upcoming revolution in agriculture. Government of India needs to embrace policies that lift all barriers towards the potential applications of these breakthrough technologies for crop and animal improvement. Such forward thinking policies will not only assure India’s food and economic security, but also to insure that it can compete with other Western nations and China in industrial innovation and production and remain self-reliant.
What is genome editing?
Programmable nucleases, such as zinc finger nucleases (ZFNs), transcription activator like effector nucleases (TALENs) and RNA-guided CRISPR-associated Cas9 nuclease, induce a DNA double-strand break (DSB) at a user-defined genomic site of living cells. Since DSBs are lethal to cells, they are immediately repaired through one of the two evolutionarily conserved pathways: (1) non-homologous end joining (NHEJ), which is error prone; or (2) homology-directed repair (HDR). Using these repair processes (Figure 1), scientists have been able to disrupt specific genes, or correct mutations in disease-causing genes, or insert exogenous DNA sequences at a pre-determined locus within the genome of living cells, which were not possible prior to the creation of programmable nucleases. As a result, the genome editing technologies have revolutionized life sciences research as well as biotechnology and biomedical fields. These disruptive technologies have the potential to have a great impact on agriculture through precise crop engineering, animal husbandry in the near future and on human therapeutics through engineered autologous cell-based therapies in the future.
What is synthetic biology?
Synthetic biology can be broadly defined as the design and construction of novel artificial biological pathways or organisms, or the redesign of existing natural biological systems. It is an emerging discipline where artificially synthesized genetic material from nucleotides, are introduced into an organism. Synthetic biology brings the application of engineering principles to biology; it aims to design and fabricate biological components and systems that do not already exist in the natural world. Synthetic genomics is a sub-discipline of synthetic biology; it refers to the synthetic assembly of complete chromosomal DNA that is designed from natural genomic sequences. The power of these techniques lies in the use of interchangeable and standardized bio-parts to construct complex genetic networks that include sensing, information processing and effector modules and in the creation of redesigned chromosomes and genomes. They have the potential to create complex new organisms with novel biological pathways and genes constructed to user specifications. Synthetic biology has great potential in biotechnology, agriculture and regenerative medicine.
Work done by us in genome editing and synthetic genomics:
I have been very fortunate to be involved in two exciting areas of life sciences research over 30-year career in the Department of Environmental Health Sciences at the Johns Hopkins School of Public Health. First is the creation of zinc finger nucleases (ZFNs), which was a culmination of seven year research effort on the study of FokI restriction endonuclease. Later, in collaboration with Dana Carroll lab, we showed stimulation of gene targeting by a ZFN-induced targeted double-strand break using frog oocytes as a model system, which ushered in the era of genome editing. We have continued this research to date, with the current focus being on the generation and genetic correction of disease-specific human induced pluripotent stem cells for human therapeutics.
Second is the total synthesis of a functional designer eukaryotic (yeast) chromosome III (aka synIII). We have embarked on the creation of a synthetic yeast genome (Sc2.0), in collaboration with Jef Boeke at NYU and several other international collaborators. Our lab reported the creation of the first fully functional synthetic 272-kb synIII yeast chromosome with numerous changes compared to the native chromosome. Currently, our focus is on completing another designer yeast chromosome IX (aka synIX).
Project 1: Genome Engineering using Programmable Nucleases
Our lab originally showed that FokI, a type IIs endonuclease, is comprised of two separable protein domains: a sequence- specific DNA binding and non-specific nuclease domain. We then reported the creation of custom zinc finger nucleases (ZFNs). Later, in collaboration with Dana Carroll’s lab in Utah, we showed stimulation of gene targeting by a ZFN-induced targeted double-strand break, using frog oocytes as a model. Recently, we have shown generation and genetic correction of human pluripotent stem cells using designer ZFNs/TALENs. Our contribution includes the application of ZFN/TALEN/Cas9 technology for targeted modification of human induced pluripotent stem cells. Recently, we reported the generation of precisely targeted genetically well-defined disease-specific hiPSCs using TALENs. Our current focus is on genetic engineering of patient-specific hiPSCs to achieve functional disease correction of monogenic diseases either by targeted genome editing (i.e. gene correction) of the defective gene or by targeted insertion of wild-type therapeutic gene to the CCR5 locus of patient-specific hiPSCs (Ramalingam et al 2013; 2014). The precisely targeted genetically well-defined disease-specific hiPSCs will be very valuable for disease modeling and for drug discovery by screening small compound libraries against the disease-specific hiPSCs. The ZFN/TALEN/Cas9-mediated approach is widely applicable to a variety of other mammalian cells as well and to generate various animal disease models to study and treat human disease in the future. We are currently conducting research to develop a hiPSC-derived HSPCs-based therapy as a curative alternative to the expensive non-curative GCase enzyme replacement therapy (ERT) to treat type 1 GD.
Chandrasegaran S and Carroll D. (2016). Origins of Programmable Nucleases for Genome Engineering. J. Mol. Biol. 428: 963-989. PMID: 26506267
Ramalingam S, Annaluru N, Kandavelou K and Chandrasegaran S. (2014) TALEN-mediated generation and genetic correction of disease-specific human induced pluripotent stem cells. Current Gene Therapy 14: 461-472. PMID: 25245091
Ramalingam S, London V, Kandavelou K, Cebotaru L, Guggino W, Civin CI and Chandrasegaran S. (2013). Generation and genetic engineering of human induced pluripotent stem cells using designed zinc finger nucleases. Stem Cells and Development 22: 595-610. PMID: 22931452
Bibikova M, Carroll D, Segal DJ, Trautman JK, Smith J, Kim YG and Chandrasegaran S. (2001) Stimulation of homologous recombination through targeted cleavage by chimeric nucleases. Mol. Cell Biol. 21: 289-297. PMID: 11113203
Smith J, Bibikova M, Whitby FG, Reddy AR, Chandrasegaran S and Carroll D. (2000) Requirements for double-strand cleavage by chimeric restriction enzymes with zinc finger DNA-recognition domains. Nucleic Acids Res 28: 3361-3369. PMID: 10954606
Kim YG, Cha J and Chandrasegaran S (1996) Hybrid restriction enzymes: zinc finger fusions to Fok I cleavage domain. Proc Natl Acad Sci USA 93: 1156-1160. PMID: 8577732
Kim Y-G and Chandrasegaran S (1994) Chimeric restriction endonuclease. Proc Natl Acad Sci USA 91: 883-887. PMID: 7905633
Li L, Wu LP and Chandrasegaran S. (1992) Functional domains in Fok I restriction endonuclease. Proc Natl Acad Sci USA 89: 4275‑4279. PMID: 1584761
Project 2: Creation of a synthetic yeast (Sc2.0)
In a 2014 Science paper, our lab reported a synthetic designer version of yeast Saccharomyces cerevisiae chromosome III (synIII) with numerous changes, including a built-in recombination system (SCRaMbLE) for inducing genome alterations of the synIII strain [Annaluru et al 2014; 2015; Richardson et al 2016]. The design changes had no impact on cell fitness and phenotype, suggesting plasticity of the yeast genome to the changes introduced. The Sc2.0 consortium, which comprises of a group of international scientists, have recently reported synthesis of five more yeast chromosomes in April, 2017. The ultimate goal of Sc2.0 consortium is to create a designer synthetic yeast genome. Our lab is currently working to complete the synthetic yeast chromosome IX (synIX). The final streamlined minimal yeast genome would serve as a valuable ‘chassis’ organism for the industrial production of biochemical and biological products, including nutraceuticals.
Richardson SM, Mitchell LA, Stracquadanio G, Yang K, Dymond JS, et al. (2017) Design of a synthetic yeast genome. Science 355: 1040-1044. PMID: 28280199
Annaluru N, Ramalingam S and Chandrasegaran S. (2015) Rewriting the blueprint of life by synthetic genomics and genome engineering. Genome Biology 16: 125-136. PMID: 26076868
Annaluru N, Müller H, Mitchell L, Ramalingam S, et al. (2014) Total synthesis of a functional designer eukaryotic chromosome. Science 344: 55-58. PMID: 24674868
Dymond J, Richardson S, Coombes C, Muller H, Narayana A, Blake W, Wu J, Dai J, Lindstrom D, Boeke A, Gottschling D, Chandrasegaran S, Bader J and Boeke J. (2011) Synthetic chromosome arms function in yeast and generate phenotypic diversity by design. Nature 477: 471-476. PMID: 21918511