Genetically modified crops: current status and future prospects.

MAIN CONCLUSION: While transgenic technology has heralded a new era in crop improvement, several concerns have precluded their widespread acceptance. Alternative technologies, such as cisgenesis and genome-editing may address many of such issues and facilitate the development of genetically engineered crop varieties with multiple favourable traits. Genetic engineering and plant transformation have played a pivotal role in crop improvement via introducing beneficial foreign gene(s) or silencing the expression of endogenous gene(s) in crop plants. Genetically modified crops possess one or more useful traits, such as, herbicide tolerance, insect resistance, abiotic stress tolerance, disease resistance, and nutritional improvement. To date, nearly 525 different transgenic events in 32 crops have been approved for cultivation in different parts of the world. The adoption of transgenic technology has been shown to increase crop yields, reduce pesticide and insecticide use, reduce CO2 emissions, and decrease the cost of crop production. However, widespread adoption of transgenic crops carrying foreign genes faces roadblocks due to concerns of potential toxicity and allergenicity to human beings, potential environmental risks, such as chances of gene flow, adverse effects on non-target organisms, evolution of resistance in weeds and insects etc. These concerns have prompted the adoption of alternative technologies like cisgenesis, intragenesis, and most recently, genome editing. Some of these alternative technologies can be utilized to develop crop plants that are free from any foreign gene hence, it is expected that such crops might achieve higher consumer acceptance as compared to the transgenic crops and would get faster regulatory approvals. In this review, we present a comprehensive update on the current status of the genetically modified (GM) crops under cultivation. We also discuss the issues affecting widespread adoption of transgenic GM crops and comment upon the recent tools and techniques developed to address some of these concerns.

Characterization of genomic deletion efficiency mediated by clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 nuclease system in mammalian cells.

The clustered regularly interspaced short [corrected] palindromic repeats (CRISPR)/CRISPR-associated (Cas) 9 nuclease system has provided a powerful tool for genome engineering. Double strand breaks may trigger nonhomologous end joining repair, leading to frameshift mutations, or homology-directed repair using an extrachromosomal template. Alternatively, genomic deletions may be produced by a pair of double strand breaks. The efficiency of CRISPR/Cas9-mediated genomic deletions has not been systematically explored. Here, we present a methodology for the production of deletions in mammalian cells, ranging from 1.3 kb to greater than 1 Mb. We observed a high frequency of intended genomic deletions. Nondeleted alleles are nonetheless often edited with inversions or small insertion/deletions produced at CRISPR recognition sites. Deleted alleles also typically include small insertion/deletions at predicted deletion junctions. We retrieved cells with biallelic deletion at a frequency exceeding that of probabilistic expectation. We demonstrate an inverse relationship between deletion frequency and deletion size. This work suggests that CRISPR/Cas9 is a robust system to produce a spectrum of genomic deletions to allow investigation of genes and genetic elements.

Chloroplasts evolved an additional layer of translational regulation based on non-AUG start codons for proteins with different turnover rates.

Chloroplasts have evolved from photosynthetic cyanobacteria-like progenitors through endosymbiosis. The chloroplasts of present-day land plants have their own transcription and translation systems that show several similarities with prokaryotic organisms. A remarkable feature of the chloroplast translation system is the use of non-AUG start codons in the protein synthesis of certain genes that are evolutionarily conserved from Algae to angiosperms. However, the biological significance of such use of non-AUG codons is not fully understood. The present study was undertaken to unravel the significance of non-AUG start codons in vivo using the chloroplast genetic engineering approach. For this purpose, stable transplastomic tobacco plants expressing a reporter gene i.e. uidA (GUS) under four different start codons (AUG/UUG/GUG/CUG) were generated and ß-glucuronidase (GUS) expression was compared. To investigate further the role of promoter sequences proximal to the start codon, uidA was expressed under two different chloroplast gene promoters psbA and psbC that use AUG and a non-AUG (GUG) start codons, respectively, and also showed significant differences in the DNA sequence surrounding the start codon. Further, to delineate the role of RNA editing that creates AUG start codon by editing non-AUG codons, if any, which is another important feature of the chloroplast transcription and translation system, transcripts were sequenced. In addition, a proteomic approach was used to identify the translation initiation site(s) of GUS and the N-terminal amino acid encoded when expressed under different non-AUG start codons. The results showed that chloroplasts use non-AUG start codons in combination with the translation initiation site as an additional layer of gene regulation to over-express proteins that are required at high levels due to their high rates of turnover.

Genome-wide transcriptomic analysis of cotton under drought stress reveal significant down-regulation of genes and pathways involved in fibre elongation and up-regulation of defense responsive genes.

Cotton is an important source of natural fibre used in the textile industry and the productivity of the crop is adversely affected by drought stress. High throughput transcriptomic analyses were used to identify genes involved in fibre development. However, not much information is available on cotton genome response in developing fibres under drought stress. In the present study a genome wide transcriptome analysis was carried out to identify differentially expressed genes at various stages of fibre growth under drought stress. Our study identified a number of genes differentially expressed during fibre elongation as compared to other stages. High level up-regulation of genes encoding for enzymes involved in pectin modification and cytoskeleton proteins was observed at fibre initiation stage. While a large number of genes encoding transcription factors (AP2-EREBP, WRKY, NAC and C2H2), osmoprotectants, ion transporters and heat shock proteins and pathways involved in hormone (ABA, ethylene and JA) biosynthesis and signal transduction were up-regulated and genes involved in phenylpropanoid and flavonoid biosynthesis, pentose and glucuronate interconversions and starch and sucrose metabolism pathways were down-regulated during fibre elongation. This study showed that drought has relatively less impact on fibre initiation but has profound effect on fibre elongation by down-regulating important genes involved in cell wall loosening and expansion process. The comprehensive transcriptome analysis under drought stress has provided valuable information on differentially expressed genes and pathways during fibre development that will be useful in developing drought tolerant cotton cultivars without compromising fibre quality.

Genome-wide transcriptome and proteome analyses of tobacco psaA and psbA deletion mutants.

Photosynthesis in higher land plants is a complex process involving several proteins encoded by both nuclear and chloroplast genomes that require a highly coordinated gene expression. Significant changes in plastid differentiation and biochemical processes are associated with the deletion of chloroplast genes. In this study we report the genome-wide responses caused by the deletion of tobacco psaA and psbA genes coding core components of photosystem I (PSI) and photosystem II (PSII), respectively, generated through a chloroplast genetic engineering approach. Transcriptomic and quantitative proteomic analysis showed the down regulation of specific groups of nuclear and chloroplast genes involved in photosynthesis, energy metabolism and chloroplast biogenesis. Moreover, our data show simultaneous activation of several defense and stress responsive genes including those involved in reactive oxygen species (ROS) scavenging mechanisms. A major finding is the differential transcription of the plastome of deletion mutants: genes known to be transcribed by the plastid encoded polymerase (PEP) were generally down regulated while those transcribed by the nuclear encoded polymerase (NEP) were up regulated, indicating simultaneous activation of multiple signaling pathways in response to disruption of PSI and PSII complexes. The genome wide transcriptomic and proteomic analysis of the ∆psaA and ∆psbA deletion mutants revealed a simultaneous up and down regulation of the specific groups of genes located in nucleus and chloroplasts suggesting a complex circuitry involving both retrograde and anterograde signaling mechanisms responsible for the coordinated expression of nuclear and chloroplast genomes.

Development of a gene-editing approach to restore vision loss in Leber congenital amaurosis type 10.

Leber congenital amaurosis type 10 is a severe retinal dystrophy caused by mutations in the CEP290 gene1,2. We developed EDIT-101, a candidate genome-editing therapeutic, to remove the aberrant splice donor created by the IVS26 mutation in the CEP290 gene and restore normal CEP290 expression. Key to this therapeutic, we identified a pair of Staphylococcus aureus Cas9 guide RNAs that were highly active and specific to the human CEP290 target sequence. In vitro experiments in human cells and retinal explants demonstrated the molecular mechanism of action and nuclease specificity. Subretinal delivery of EDIT-101 in humanized CEP290 mice showed rapid and sustained CEP290 gene editing. A comparable surrogate non-human primate (NHP) vector also achieved productive editing of the NHP CEP290 gene at levels that met the target therapeutic threshold, and demonstrated the ability of CRISPR/Cas9 to edit somatic primate cells in vivo. These results support further development of EDIT-101 for LCA10 and additional CRISPR-based medicines for other inherited retinal disorders.

Use of Nicotiana tabacum transplastomic plants engineered to express a His-tagged CP47 for the isolation of functional photosystem II core complexes.

This work focuses on the development of a molecular tool for purification of Photosystem II (PSII) from Nicotiana tabacum (L.). To this end, the chloroplast psbB gene encoding the CP47 PSII subunit was replaced with an engineered version of the same gene containing a C-terminal His-tag. Molecular analyses assessed the effective integration of the recombinant gene and its expression. Despite not exhibiting any obvious phenotype, the transplastomic plants remained heteroplasmic even after three rounds of regeneration under antibiotic selection. However, the recombinant His-tagged CP47 protein associated in vivo to the other PSII subunits allowing the isolation of a functional PSII core complex, although with low yield of extraction. These results will open up possible perspectives for further spectroscopic and structural studies.

TMEM14C is required for erythroid mitochondrial heme metabolism.

The transport and intracellular trafficking of heme biosynthesis intermediates are crucial for hemoglobin production, which is a critical process in developing red cells. Here, we profiled gene expression in terminally differentiating murine fetal liver-derived erythroid cells to identify regulators of heme metabolism. We determined that TMEM14C, an inner mitochondrial membrane protein that is enriched in vertebrate hematopoietic tissues, is essential for erythropoiesis and heme synthesis in vivo and in cultured erythroid cells. In mice, TMEM14C deficiency resulted in porphyrin accumulation in the fetal liver, erythroid maturation arrest, and embryonic lethality due to profound anemia. Protoporphyrin IX synthesis in TMEM14C-deficient erythroid cells was blocked, leading to an accumulation of porphyrin precursors. The heme synthesis defect in TMEM14C-deficient cells was ameliorated with a protoporphyrin IX analog, indicating that TMEM14C primarily functions in the terminal steps of the heme synthesis pathway. Together, our data demonstrate that TMEM14C facilitates the import of protoporphyrinogen IX into the mitochondrial matrix for heme synthesis and subsequent hemoglobin production. Furthermore, the identification of TMEM14C as a protoporphyrinogen IX importer provides a genetic tool for further exploring erythropoiesis and congenital anemias.