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Recent advances in genome editing strategies for balancing growth and defence in sugarcane (Saccharum officinarum).
Sugarcane (Saccharum officinarum ) has gained more attention worldwide in recent decades because of its importance as a bioenergy resource and in producing table sugar. However, the production capabilities of conventional varieties are being challenged by the changing climates, which struggle to meet the escalating demands of the growing global population. Genome editing has emerged as a pivotal field that offers groundbreaking solutions in agriculture and beyond. It includes inserting, removing or replacing DNA in an organism's genome. Various approaches are employed to enhance crop yields and resilience in harsh climates. These techniques include zinc finger nuclease (ZFN), transcription activator-like effector nuclease (TALEN) and clustered regularly interspaced short palindromic repeats/associated protein (CRISPR/Cas). Among these, CRISPR/Cas is one of the most promising and rapidly advancing fields. With the help of these techniques, several crops like rice (Oryza sativa ), tomato (Solanum lycopersicum ), maize (Zea mays ), barley (Hordeum vulgare ) and sugarcane have been improved to be resistant to viral diseases. This review describes recent advances in genome editing with a particular focus on sugarcane and focuses on the advantages and limitations of these approaches while also considering the regulatory and ethical implications across different countries. It also offers insights into future prospects and the application of these approaches in agriculture.
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CRISPR-Cas9-mediated functional dissection of 3'-UTRs.
Many studies using reporter assays have demonstrated that 3' untranslated regions (3'-UTRs) regulate gene expression by controlling mRNA stability and translation. Due to intrinsic limitations of heterologous reporter assays, we sought to develop a gene editing approach to investigate the regulatory activity of 3'-UTRs in their native context. We initially used dual-CRISPR (clustered, regularly interspaced, short palindromic repeats)-Cas9 targeting to delete DNA regions corresponding to nine chemokine 3'-UTRs that destabilized mRNA in a reporter assay. Targeting six chemokine 3'-UTRs increased chemokine mRNA levels as expected. However, targeting CXCL1, CXCL6 and CXCL8 3'-UTRs unexpectedly led to substantial mRNA decreases. Metabolic labeling assays showed that targeting these three 3'-UTRs increased mRNA stability, as predicted by the reporter assay, while also markedly decreasing transcription, demonstrating an unexpected role for 3'-UTR sequences in transcriptional regulation. We further show that CRISPR-Cas9 targeting of specific 3'-UTR elements can be used for modulating gene expression and for highly parallel localization of active 3'-UTR elements in the native context. Our work demonstrates the duality and complexity of 3'-UTR sequences in regulation of gene expression and provides a useful approach for modulating gene expression and for functional annotation of 3'-UTRs in the native context.
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CRISPR-Cas12b enables efficient plant genome engineering.
Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas12b is a newly emerged genome engineering system. Here, we compared Cas12b from Alicyclobacillus acidoterrestris (Aac), Alicyclobacillus acidiphilus (Aa), Bacillus thermoamylovorans (Bth) and Bacillus hisashii (Bh) for genome engineering in rice, an important crop. We found AaCas12b was more efficient than AacCas12b and BthCas12b for targeted mutagenesis, which was further demonstrated in multiplexed genome editing. We also engineered the Cas12b systems for targeted transcriptional repression and activation. Our work establishes Cas12b as the third promising CRISPR system, after Cas9 and Cas12a, for plant genome engineering.
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Safety concerns surrounding AAV and CRISPR therapies in neuromuscular treatment.
Gene therapies, notably those leveraging CRISPR and adeno-associated virus vectors (AAVs), have risen to the forefront of potential treatments for neuromuscular disorders. The recent demise of a Duchenne muscular dystrophy (DMD) patient following a trial utilizing CRISPR transactivation with AAV has cast a spotlight on the potential risks associated with these approaches.1.
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Development of CRISPR-Mediated Nucleic Acid Detection Technologies and Their Applications in the Livestock Industry.
The rapid rate of virus transmission and pathogen mutation and evolution highlight the necessity for innovative approaches to the diagnosis and prevention of infectious diseases. Traditional technologies for pathogen detection, mostly PCR-based, involve costly/advanced equipment and skilled personnel and are therefore not feasible in resource-limited areas. Over the years, many promising methods based on clustered regularly interspaced short palindromic repeats and the associated protein systems (CRISPR/Cas), i.e., orthologues of Cas9, Cas12, Cas13 and Cas14, have been reported for nucleic acid detection. CRISPR/Cas effectors can provide one-tube reaction systems, amplification-free strategies, simultaneous multiplex pathogen detection, visual colorimetric detection, and quantitative identification as alternatives to quantitative PCR (qPCR). This review summarizes the current development of CRISPR/Cas-mediated molecular diagnostics, as well as their design software and readout methods, highlighting technical improvements for integrating CRISPR/Cas technologies into on-site applications. It further highlights recent applications of CRISPR/Cas-based nucleic acid detection in livestock industry, including emerging infectious diseases, authenticity and composition of meat/milk products, as well as sex determination of early embryos.
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Orthotopic T-cell receptor replacement in primary human T cells using CRISPR-Cas9-mediated homology-directed repair.
Adoptive T cell therapy using T-cell receptor (TCR)-engineered T cells allows to redirect T cell specificity and to target any antigen of interest. Here, we apply advanced genetic engineering using clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (Cas9) for simultaneous editing of TCR α- and β-chains in primary human T cells. Together with non-virally delivered template DNA, this CRISPR-Cas9-system allows for elimination of the endogenous TCR and orthotopic placement of TCR α- and β-chains. For complete details on the use and execution of this protocol, please refer to Schober et al. (2019) and Müller et al. (2021).
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Trojan Horse-Like Vehicles for CRISPR-Cas Delivery: Engineering Extracellular Vesicles and Virus-Like Particles for Precision Gene Editing in Cystic Fibrosis.
The advent of genome editing has kindled the hope to cure previously uncurable, life-threatening genetic diseases. However, whether this promise can be ultimately fulfilled depends on how efficiently gene editing agents can be delivered to therapeutically relevant cells. Over time, viruses have evolved into sophisticated, versatile, and biocompatible nanomachines that can be engineered to shuttle payloads to specific cell types. Despite the advances in safety and selectivity, the long-term expression of gene editing agents sustained by viral vectors remains a cause for concern. Cell-derived vesicles (CDVs) are gaining traction as elegant alternatives. CDVs encompass extracellular vesicles (EVs), a diverse set of intrinsically biocompatible and low-immunogenic membranous nanoparticles, and virus-like particles (VLPs), bioparticles with virus-like scaffold and envelope structures, but devoid of genetic material. Both EVs and VLPs can efficiently deliver ribonucleoprotein cargo to the target cell cytoplasm, ensuring that the editing machinery is only transiently active in the cell and thereby increasing its safety. In this review, we explore the natural diversity of CDVs and their potential as delivery vectors for the clustered regularly interspaced short palindromic repeats (CRISPR) machinery. We illustrate different strategies for the optimization of CDV cargo loading and retargeting, highlighting the versatility and tunability of these vehicles. Nonetheless, the lack of robust and standardized protocols for CDV production, purification, and quality assessment still hinders their widespread adoption to further CRISPR-based therapies as advanced "living drugs." We believe that a collective, multifaceted effort is urgently needed to address these critical issues and unlock the full potential of genome-editing technologies to yield safe, easy-to-manufacture, and pharmacologically well-defined therapies. Finally, we discuss the current clinical landscape of lung-directed gene therapies for cystic fibrosis and explore how CDVs could drive significant breakthroughs in in vivo gene editing for this disease.
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Differential Divalent Metal Binding by SpyCas9's RuvC Active Site Contributes to Nonspecific DNA Cleavage.
To protect against mobile genetic elements (MGEs), some bacteria and archaea have clustered regularly interspaced short palindromic repeats-CRISPR associated (CRISPR-Cas) adaptive immune systems. CRISPR RNAs (crRNAs) bound to Cas nucleases hybridize to MGEs based on sequence complementarity to guide the nucleases to cleave the MGEs. This programmable DNA cleavage has been harnessed for gene editing. Safety concerns include off-target and guide RNA (gRNA)-free DNA cleavages, both of which are observed in the Cas nuclease commonly used for gene editing, Streptococcus pyogenes Cas9 (SpyCas9). We developed a SpyCas9 variant (SpyCas9H982A) devoid of gRNA-free DNA cleavage activity that is more selective for on-target cleavage. The H982A substitution in the metal-dependent RuvC active site reduces Mn2+-dependent gRNA-free DNA cleavage by ∼167-fold. Mechanistic molecular dynamics analysis shows that Mn2+, but not Mg2+, produces a gRNA-free DNA cleavage competent state that is disrupted by the H982A substitution. Our study demonstrates the feasibility of modulating cation:protein interactions to engineer safer gene editing tools.
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Engineering cold resilience: implementing gene editing tools for plant cold stress tolerance.
This paper highlights the need for innovative approaches to enhance cold tolerance. It underscores how genome-editing tools can deepen our understanding of genes involved in cold stress. Cold stress is a significant abiotic factor in high-altitude regions, adversely affecting plant growth and limiting crop productivity. Plants have evolved various mechanisms in response to low temperatures that enable resistance at both physiological and molecular levels during chilling and freezing stress. Several cold-inducible genes have been isolated and characterized, with most playing key roles in providing tolerance against low-temperature stress. However, many plants fail to survive at low temperatures due to the absence of cold acclimatization mechanisms. Conventional breeding techniques, such as inter-specific or inter-genic hybridization, have had limited effectiveness in enhancing the cold resistance of essential crops. Thus, it is crucial to develop crops with improved adaptability, high yields and resistance to cold stress using advanced genomic approaches. The current availability of gene editing tools offers the opportunity to introduce targeted modifications in plant genomes efficiently, thereby developing cold-tolerant varieties. This review discusses advancements in gene editing tools, including zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9)/Cas12a(Cpf1), prime editing (PE) and retron library recombineering (RLR). We focus specifically on the CRISPR/Cas system, which has garnered significant attention in recent years as a groundbreaking tool for genome editing across various species. These techniques will enhance our understanding of molecular interactions under low-temperature stress response and highlight the progress of genome editing in designing future climate-resilient crops.
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i-GONAD: A method for generating genome-edited animals without ex vivo handling of embryos.
The recent development of genome editing technologies has enabled the creation of genome-edited animals, with alterations at the desired target locus. The clustered regularly interspaced short palindromic repeats (CRISPR) system is widely used for this purpose because it is simpler and more efficient than other genome editing technologies. The conventional methods for creation of genome-edited animals involve ex vivo handling of embryos (zygotes) for microinjection or in vitro electroporation. However, this process is laborious and time-consuming, and relatively large numbers of animals are used. Furthermore, these methods require specialized skills for handling embryos. In 2015, we reported a novel method for the creation of genome-edited animals without ex vivo handling of embryos. The technology known as Genome-editing via Oviductal Nucleic Acids Delivery (GONAD) involved intraoviductal instillation of genome editing components into a pregnant female and subsequent in vivo electroporation of an entire oviduct. The genome editing components present in the oviductal lumen are transferred to preimplantation embryos in situ for introducing insertion or deletion (indel) mutations at the desired loci. This technology was further improved by optimizing several parameters to develop improved GONAD (i-GONAD) for the efficient generation of mutant or knock-in animals. In this review, we discuss the historical background, potential applications, advantages, and future challenges of GONAD/i-GONAD technology.
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