Advances and Challenges in Mycobacterial Genetic Engineering: Techniques for Knockout, Knockdown and Overexpression

Genetic engineering has transformed modern agriculture, offering solutions to enhance crop productivity, resilience, and nutritional quality. This review explores the innovations and challenges in crop genetic engineering, focusing on key techniques such as CRISPR-Cas systems, RNA interference, and transgenic methods. These technologies have facilitated the development of stress-resilient crops capable of withstanding drought, salinity, heat, and cold, thereby supporting agricultural sustainability in the face of climate change. Nutrient use efficiency has been improved through genetic modifications that enhance nitrogen and phosphorus uptake, reducing the reliance on chemical fertilizers and minimizing environmental impacts. Additionally, genetic engineering has advanced pest and disease resistance, decreasing the need for chemical pesticides and contributing to environmental conservation. However, the adoption of genetically modified crops is influenced by various socioeconomic factors, including public perception, regulatory frameworks, and intellectual property rights. Ethical concerns regarding biosafety, labeling, and consumer choice persist, necessitating transparent communication and robust risk assessments. Despite technical challenges such as off-target effects and resistance development, innovations like base and prime editing, as well as synthetic biology, offer promising avenues for more precise and efficient genetic modifications. Future research should prioritize the development of climate-resilient and nutritionally enhanced crops, with an emphasis on biofortification to address global micronutrient deficiencies. Integrating digital technologies such as machine learning and big data analytics can accelerate trait discovery and optimize breeding strategies. Moreover, exploring the synergy between genetic engineering and sustainable agricultural practices can promote resilient farming systems that ensure long-term productivity and environmental health. By addressing technical, ethical, and social considerations, genetic engineering can significantly contribute to global food security and sustainability, providing a foundation for future agricultural advancements in an ever-changing world.

In recent years, genetic engineering has witnessed a remarkable shift towards harnessing the potential of microalgae for various applications including enhanced biomass production, biofuel production, wastewater treatment and the synthesis of valuable bioactive compounds. Our previous study has proven that genetic modifications of Chlorella vulgaris Beijer. using random mutagenesis significantly enhanced the lipid content, making it more ideal for biofuel production in C. vulgaris. However, efficient genetic engineering tools are still lacking in their ability to simultaneously augment the overall production of biomass and bioactive compounds. The present review discusses the most recent tools and strategies that are used to engineer microalgal strains, from culturing to modern gene-editing techniques like Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR associated protein 9 (CRISPR-Cas9). Numerous studies have reported that targeted nucleases represent a remarkable advancement in genome manipulation, offering unparalleled precision. A novel variant of CRISPR, known as CRISPRi technique was reported to yield significant outcomes in microalgal species even under non-stressful conditions. Further, to curtail the bottlenecks due to high guanine-cytosine contents of DNA in microalgae, a new approach such as Adaptive Single Guide Assisted Regulation DNA (ASGARD) was explored along with CRISPRi, which yielded higher lipid and protein contents, thus finding indispensable applications in industry. Hence, this review effectively conveys the advantages and disadvantages associated with various genetic engineering tools and the complexity and precision required in genetic modification and the resulting potential for improved biomass, lipid and bioactive compounds productivity in marine microalgal species.

in recent years, genetic engineering has witnessed a remarkable shift towards harnessing the potential of microalgae for various applications including enhanced biomass production, biofuel production, wastewater treatment and the synthesis of valuable bioactive compounds. Our previous study has proven that genetic modifications of <i>Chlorella vulgaris</i> Beijer. using random mutagenesis significantly enhanced the lipid content, making it more ideal for biofuel production in <i>C. vulgaris</i>. However, efficient genetic engineering tools are still lacking in their ability to simultaneously augment the overall production of biomass and bioactive compounds. The present review discusses the most recent tools and strategies that are used to engineer microalgal strains, from culturing to modern gene-editing techniques like Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR associated protein 9 (CRISPR-Cas9). Numerous studies have reported that targeted nucleases represent a remarkable advancement in genome manipulation, offering unparalleled precision. A novel variant of CRISPR, known as CRISPRi technique was reported to yield significant outcomes in microalgal species even under non-stressful conditions. Further, to curtail the bottlenecks due to high guanine-cytosine contents of DNA in microalgae, a new approach such as Adaptive Single Guide Assisted Regulation DNA (ASGARD) was explored along with CRISPRi, which yielded higher lipid and protein contents, thus finding indispensable applications in industry. Hence, this review effectively conveys the advantages and disadvantages associated with various genetic engineering tools and the complexity and precision required in genetic modification and the resulting potential for improved biomass, lipid and bioactive compounds productivity in marine microalgal species.

The CRISPR-based genome editing technology, known as clustered regularly interspaced short palindromic repeats (CRISPR), has sparked renewed interest in gene therapy. This interest is accompanied by the development of single-guide RNAs (sgRNAs), which enable the introduction of desired genetic modifications at the targeted site when used alongside the CRISPR components. However, the efficient delivery of CRISPR/Cas remains a challenge. Successful gene editing relies on the development of a delivery strategy that can effectively deliver the CRISPR cargo to the target site. To overcome this obstacle, researchers have extensively explored non-viral, viral, and physical methods for targeted delivery of CRISPR/Cas9 and a guide RNA (gRNA) into cells and tissues. Among those methods, liposomes offer a promising approach to enhance the delivery of CRISPR/Cas and gRNA. Liposomes facilitate endosomal escape and leverage various stimuli such as light, pH, ultrasound, and environmental cues to provide both spatial and temporal control of cargo release. Thus, the combination of the CRISPR-based system with liposome delivery technology enables precise and efficient genetic modifications in cells and tissues. This approach has numerous applications in basic research, biotechnology, and therapeutic interventions. For instance, it can be employed to correct genetic mutations associated with inherited diseases and other disorders or to modify immune cells to enhance their disease-fighting capabilities. In summary, liposome-based CRISPR genome editing provides a valuable tool for achieving precise and efficient genetic modifications. This review discusses future directions and opportunities to further advance this rapidly evolving field.

Beyond restrictions : Towards biotechnological exploitation of thermophilic clostridia

Highly active and stable biocatalysts are the prerequisite for industrial scale application of the biodesulfurization process. Scientists are making efforts for increasing the desulfurizing activity of native strains by employing various genetic engineering approaches. Nevertheless, the achieved desulfurization rate is lower than the industrial requirements. Thus, there is a dire need to use efficient genetic tools for precise genome editing of desulfurizing bacteria for enhanced efficiency. In comparison to the previously used genetic engineering tools the newly developed CRISPR-Cas is a more efficient and simple genetic tool that has been successfully applied for targeted genome modification of eukaryotes as well as prokaryotes. In this paper, we have reviewed the approaches, previously used to enhance the biodesulfurization rates of the sulfur metabolizing microorganisms and have discussed the potential of CRISPR-Cas systems in engineering desulfurizing biocatalysts. We have also proposed a model to construct competent desulfurizing recombinants involving use of CRISPR-Cas technology. The model can be used to over-express the dsz genes under a constitutive promoter in a suitable heterologous host, to get a steady expression of desulfurization pathway. This may serve as an inducement to develop better performing desulfurizing recombinant strains using CRISPR-Cas systems, which can be helpful in increasing the rate of biodesulfurization in future.

Employment of the CRISPR/Cas9 system to improve cellulase production in Trichoderma reesei

The Broad Institute Scores Another Victory in Its Battle with the University of California over the Patenting of CRIPSR

Genetically modified (GM) crops are now being grown extensively in North and South America and China, although not in Europe. Food produced from these crops has become a part of the normal diet in North and South America and in China, but not in Europe, where contention continues despite the fact that millions of US citizens eat GM soya without any ill effects in a very litigious society, and many Europeans have eaten GM soya while in the US without any adverse consequences. > Why has the British public, who normally so pragmatically welcome scientific advances, resisted the introduction of genetically modified crops? European consumers' continuous and ardent opposition to GM crops and foods has had serious repercussions for plant research, for the commercial development of new crops and, most importantly, for developing countries that could benefit most from GM crops. Several countries in Africa and elsewhere have resisted growing such crops, mainly for fear of being unable to export them to the European market ( The Economist , 2002). It is therefore worthwhile to investigate what actually went wrong in the debate about GM food and crops in Europe and how these foods have earned such a bad name. Such an analysis could not only help to overcome public fears of this technology, but also help scientists and policy makers to address similar concerns in the future, such as the growing debate over nanotechnology. The concerns of European consumers about the potential health and environmental threats of GM crops have resulted in an unprecedented effort to investigate those anxieties and communicate with the wider public, particularly in the UK, where the use of public consultation has been extensively developed. The first of these initiatives was the extensive Farm Scale Evaluations of three GM crops (herbicide‐resistant beet, oil seed rape and maize), whose …

Genome editing has become one of the most promising genetic engineering functional tools for crop traits improvement. As crops are the major staple food consumed globally and hence crop improvement in terms of yield and nutritional quality is requisite to feed a large population. Much work has been done for crop improvement using traditional breeding approaches yet because of the high labour and high time consumption, much faster methods are required. Also, the polyploidy of crops and the complex genome is still a bottleneck for improved trait development. Besides this, a difficult genetic transformation and low transformation efficiency are holding back the production of better crops. However, genome engineering has been proven to be an efficient and much faster biotechnological approach to overcome these challenges. The first-generation tools including zinc finger nuclease (ZFN) and transcription activator-like effector nuclease (TALEN) have been employed to a precise genome manipulation. But, because of complicated protein engineering and being expensive techniques, much simpler tools are required. Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) technology is the recent and revolutionary technique that can create a precise and targeted genetic manipulation in the genome. The recent advances in CRISPR have accelerated genetic engineering even in polyploid crop systems. In this chapter, we provide an insight into the development of Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated protein 9 (CRISPR/Cas9) system as a genome engineering tool and various types of native and modified CRISPR effector proteins and their different application. The major focus of the chapter is the use of CRISPR technology and its advancement for the plant trait modifications.

Shooting for the moon: Genome editing for pig heart xenotransplantation

Cas9, the RNA-guided DNA endonuclease from the CRISPR-Cas (clustered regularly interspaced short palindromic repeat-CRISPR-associated) system, has been adapted for genome editing and gene regulation in multiple model organisms. Here we characterize a Cas9 ortholog from Streptococcus thermophilus LMG18311 (LMG18311 Cas9). In vitro reconstitution of this system confirms that LMG18311 Cas9 together with a trans-activating RNA (tracrRNA) and a CRISPR RNA (crRNA) cleaves double-stranded DNA with a specificity dictated by the sequence of the crRNA. Cleavage requires not only complementarity between crRNA and target but also the presence of a short motif called the PAM. Here we determine the sequence requirements of the PAM for LMG18311 Cas9. We also show that both the efficiency of DNA target cleavage and the location of the cleavage sites vary based on the position of the PAM sequence.

Genetic tools have revolutionized mustard improvement strategies, offering innovative avenues to enhance its agronomic traits and quality parameters. CRISPR technology, exemplified by research at the National Institute of Plant Genome Research in India, has enabled precise manipulation of glucosinolate levels in mustard plants, improving mustard oil quality. The Barnase-barstar gene system has facilitated the development of mustard hybrids, increasing yield and resilience. Additionally, Microsatellites have emerged as indispensable tools for understanding genetic relationships within mustard populations. These genetic tools hold promise for addressing agronomic challenges and meeting market demands in mustard cultivation. However, their deployment raises ethical, regulatory, and socio-economic considerations that require careful consideration. Responsible stewardship and transparent deployment of these technologies are essential to realize their full potential in enhancing mustard crops and ensuring a sustainable future for food production. In conclusion, genetic engineering offers exciting avenues for mustard improvement, with CRISPR, the Barnase-barstar gene system, and Microsatellites playing pivotal roles in enhancing crop quality, yield potential, and resilience. As mustard continues to play a crucial role in global agriculture and food security, the responsible utilization of these genetic tools holds promise for meeting the evolving needs of farmers and consumers worldwide. Furthermore, the paper briefly discusses the application of these genetic tools in enhancing Dhara Mustard, a popular variety in Indian agriculture, emphasizing its potential impact on addressing agricultural challenges and meeting consumer demands.

Microbial lipids are sustainable feedstock for the production of oleochemicals and biodiesel. Oleaginous yeasts have recently been proposed as alternative lipid producers to plants and animals to promote sustainability in the chemical and fuel industries. The oleaginous yeast Lipomyces starkeyi has great industrial potential as an excellent lipid producer. However, improvement of its lipid productivity is essential for the cost-effective production of oleochemicals and fuels. Genetic and metabolic engineering of L. starkeyi via gene manipulation techniques may result in improvements in lipid production and our understanding of the mechanisms behind lipid biosynthesis pathways. We previously described an integrative transformation system using a drug-resistant marker for L. starkeyi. However, gene-targeting frequencies were very low because non-homologous recombination is probably predominant in L. starkeyi. Genetic engineering tools for L. starkeyi have not been sufficiently developed. In this study, we describe a new genetic tool and its application in L. starkeyi. To develop a highly efficient gene-targeting system for L. starkeyi, we constructed a series of mutants by disrupting genes for LsKu70p, LsKu80p, and/or LsLig4p, which share homology with other yeasts Ku70p, Ku80p, and Lig4p, respectively, being involved in non-homologous end-joining pathway. Deletion of the LsLIG4 gene dramatically improved the homologous recombination efficiency (80.0%) at the LsURA3 locus compared with that in the wild-type strain (1.4%), when 2000-bp homologous flanking regions were used. The homologous recombination efficiencies of the double mutant ∆l sku70∆lslig4 and the triple mutant ∆lsku70∆lsku80∆lslig4 were also markedly enhanced. Therefore, the L. starkeyi ∆lslig4 background strains have promise as efficient recipient strains for genetic and metabolic engineering approaches in this yeast.

Development of efficient strategies for the production of genetically modified pigs