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Optimization of CRISPR/Cas9 genome editing for loss-of-function in the early chick embryo.
The advent of CRISPR/Cas9 has made genome editing possible in virtually any organism, including those not previously amenable to genetic manipulations. Here, we present an optimization of CRISPR/Cas9 for application to early avian embryos with improved efficiency via a three-fold strategy. First, we employed Cas9 protein flanked with two nuclear localization signal sequences for improved nuclear localization. Second, we used a modified guide RNA (gRNA) scaffold that obviates premature termination of transcription and unstable Cas9-gRNA interactions. Third, we used a chick-specific U6 promoter that yields 4-fold higher gRNA expression than the previously utilized human U6. For rapid screening of gRNAs for in vivo applications, we also generated a chicken fibroblast cell line that constitutively expresses Cas9. As proof of principle, we performed electroporation-based loss-of-function studies in the early chick embryo to knock out Pax7 and Sox10, key transcription factors with known functions in neural crest development. The results show that CRISPR/Cas9-mediated deletion causes loss of their respective proteins and transcripts, as well as predicted downstream targets. Taken together, the results reveal the utility of this optimized CRISPR/Cas9 method for targeted gene knockout in chicken embryos in a manner that is reproducible, robust and specific.
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Prokaryotic Argonautes for in vivo biotechnology and molecular diagnostics.
Prokaryotic Argonautes (pAgos) are an emerging class of programmable endonucleases that are believed to be more flexible than existing CRISPR-Cas systems and have significant potential for biotechnology. Current applications of pAgos include a myriad of molecular diagnostics and in vitro DNA assembly tools. However, efforts have historically been centered on thermophilic pAgo variants. To enable in vivo biotechnological applications such as gene editing, focus has shifted to pAgos from mesophilic organisms. We discuss what is known of pAgos, how they are being developed for various applications, and strategies to overcome current challenges to in vivo applications in prokaryotes and eukaryotes.
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Ultrasound-Mediated CRISPR Delivery and Sonogenetic Modulation for Precision Therapeutics.
The development of CRISPR-Cas9 technology has transformed gene editing with precise mutations in DNA targeted to treat genetic disorders yet various challenges such as immunogenicity, off-target editing, and lack of temporal control after delivery, making it difficult for therapeutic agents to reach deep tissues like brain, and cardiac tissues. Ultrasound has emerged as a promising solution by influencing sonogenetic modulation and delivery of CRISPR tools in deep tissues for targeted gene editing. Sonogenetic editing has revolutionized the technological advancement of CRISPR-editing and among multiple strategies, ultrasound-mediated delivery of CRISPR systems has enabled non-invasive and controlled genome editing through sonoporation, and upregulation of stimuli-responsive genes. Focused ultrasound combined with microbubbles has demonstrated CRISPR delivery to brain tissue with ∼80% higher efficiency than traditional strategies, allowing precise and advanced treatment of neurodegenerative diseases. In cancer applications, ultrasound has enabled complete tumor eradication via sonodynamic therapy, achieving 64% tumor regression via CAR T-cell therapy. Significant advances in gene therapy for cardiovascular diseases, obesity, and related diseases were also demonstrated recently. Collectively, the review elaborates potential of ultrasound-CRISPR technologies as a powerful tool that enables targeted, and image-guided genetic interventions, capable of advancing the outcomes of gene therapy as a widespread clinical practice.
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An Efficient CRISPR/Cas12e System for Genome Editing in Sinorhizobium meliloti.
CRISPR/Cas systems have been widely used in the precise and traceless genetic engineering of bacteria. Sinorhizobium meliloti 320 (SM320) is a Gram-negative bacterium with a low efficiency of homologous recombination but a strong ability to produce vitamin B12. Here, a CRISPR/Cas12e-based genome engineering toolkit, CRISPR/Cas12eGET, was constructed in SM320. The expression level of CRISPR/Cas12e was tuned through promoter optimization and the use of a low copy plasmid to adjust Cas12e cutting activity to the low homologous recombination efficiency of SM320, resulting in improved transformation and precision editing efficiencies. Furthermore, the accuracy of CRISPR/Cas12eGET was improved by deleting the ku gene involved in NHEJ repair in SM320. This advance will be useful for metabolic engineering and basic research on SM320, and it further provides a platform to develop the CRISPR/Cas system in strains where the efficiency of homologous recombination is low.
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From the first human gene-editing to the birth of three-parent baby.
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Synergizing gene editing and cellular agriculture for a sustainable and healthy food future.
The intersection of gene editing and cellular agriculture is transforming food production by offering sustainable, ethical alternatives to conventional agriculture. Cellular agriculture uses tissue engineering and fermentation technologies to produce animal-free food, whereas gene editing tools like CRISPR-Cas9 optimize cellular efficiency, nutritional value, and sustainability. While some researchers emphasize the environmental and food security benefits, others raise concerns about high costs, regulatory challenges, and consumer acceptance. This paper critically examines existing literature, compares breakthroughs and controversies, and provides an expert perspective on the challenges and opportunities in gene-edited cellular agriculture. By tackling key scientific, economic, regulatory, and ethical issues, this article presents a roadmap for responsibly advancing these technologies and integrating them into global food systems. To our knowledge, this is the first work to explore how gene editing and cellular agriculture can be synergized to advance sustainability, food security, and global health.
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CRISPR-based metabolic editing: Next-generation metabolic engineering in plants.
Plants generate many secondary metabolites, so called phyto-metabolites, which can be used as toxins, dyes, drugs, and insecticides in bio-warfare plus bio-terrorism, industry, medicine, and agriculture, respectively. To 2013, the first generation metabolic engineering approaches like miRNA-based manipulation were widely adopted by researchers in biosciences. However, the discovery of the clustered regularly interspaced short palindromic repeat (CRISPR) genome editing system revolutionized metabolic engineering due to its unique features so that scientists could manipulate the biosynthetic pathways of phyto-metabolites through approaches like miRNA-mediated CRISPR-Cas9. According to the increasing importance of the genome editing in plant sciences, we discussed the current findings on CRISPR-based manipulation of phyto-metabolites in plants, especially medicinal ones, and suggested the ideas to phyto-metabolic editing.
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Editing a Stomatal Developmental Gene in Rice with CRISPR/Cpf1.
CRISPR has arguably been the fastest growing genome editing tool so far. CRISPR/Cas9 (Cas9) has been proved to be efficient and precise in genome editing. However Cas9 has certain limitations. CRISPR/Cpf1 (Cpf1) has been discovered as an alternate approach that can overcome some of those limitations. Cpf1 allows targeting in AT-rich region, creating a staggered cleavage, and cutting at the distal end to the PAM (Protospacer Adjacent Motif) regions. We have successfully tested the efficiency of Cpf1 system in rice using OsEPFL9 which is a developmental gene known to regulate the stomatal density in leaf. Regulation of stomatal density and patterning is an important factor in regulating plant physiology, especially in improving the plant water use efficiency. We targeted the Exon1 of OsEPFL9 and the knockout lines were studied for several generations for establishment of stabilized editing, as well as transmission and segregation of edits through generations. The usage of Cpf1 as a genome editing tool to manipulate stomatal patterning may further help us gain more insight of the physiology of rice in stress conditions.
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Assessing off-target effects in CRISPR/Cas9: challenges and strategies for precision DNA editing.
The emergence of CRISPR/Cas9 technology has transformed the landscape of gene editing, allowing for precise alterations in DNA that hold great promise for research and potential therapies. However, a significant concern is the occurrence of off-target effects, which can lead to unintended genetic modifications with potentially harmful consequences. This paper explores the nature of off-target effects in CRISPR/Cas9, discussing how they arise and their implications for the reliability of gene editing. We identify the challenges faced in detecting and predicting these off-target interactions, including limitations in current detection techniques and the complexities of cellular biology. We present strategies aimed at minimizing off-target effects, such as careful design of guide RNAs, the use of computational tools for prediction, and improved delivery methods. Through a review of case studies, we highlight successful cases where off-target activity has been significantly reduced, offering insights into best practices for enhancing the accuracy of CRISPR/Cas9 applications. Moreover, we provide a comparative overview of Cas9, Cas12, and Cas13 systems, emphasizing their distinct target specificities, mechanisms of action, and off-target profiles. This comparison offers a broader understanding of how alternative CRISPR effectors may be leveraged to improve genome and transcriptome editing precision. This study underscores the importance of continued research to address the challenges of off-target effects, ultimately supporting the development of safer and more effective gene editing methods for clinical use.
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Viral-Based Gene Editing System for Nutritional Improvement of Fructan Content in Lettuce.
Lettuce is a globally cultivated and consumed leafy crop. Here we developed an efficient tobacco rattle virus (TRV)-based guide RNA (gRNA) delivery system for CRISPR/Cas editing in the commercial lettuce cultivar 'Noga'. Plants stably expressing Cas9 were inoculated with TRV vectors carrying gRNAs targeting five nutrient-associated genes. The system achieved an average editing efficiency of 48.7%, with up to 78.9% of regenerated plantlets showing independent mutations. This approach eliminates the need for antibiotic selection, simplifying tissue culture processes. The system supports diverse applications, including Cas12a editing and large-fragment deletions using dual gRNA sets. Targeting the fructan 1-exohydrolase 2 (1-FEH2) gene produced knockout lines with significant increases in prebiotic dietary fibre fructan content, up to 5.2-fold, and an average rise in the degree of polymerisation by 2.15 units compared with controls. Combining 1-FEH1 and 1-FEH2 knockouts did not further increase fructan levels, revealing 1-FEH2 as the predominant isozyme in lettuce. RT-qPCR analysis showed reduced expression of the upstream biosynthetic enzyme sucrose:sucrose 1-fructosyl transferase (1-SST), suggesting potential feedback inhibition in fructan metabolism. This TRV-based gene editing approach, utilised here to increase fructan content, could be applied to improve other valuable traits in lettuce, and may inspire similar systems to enhance nutritional content of crops.
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